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Transforming growth factor-beta1 (TGFbeta1) induces a mesenchyme-like cell shape in some epithelial cell types. To clarify the role of TGFbeta1 in the morphological regulation of thyrocytes, we performed collagen gel culture of porcine thyrocytes with serum-free medium. TGFbeta1-nontreated cells organized follicles. In contrast, the cells treated with 10 ng/ml TGFbeta1 became spindle shaped, i.e. they resembled mesenchymal fibroblasts, and did not form follicles. To characterize the spindle-shaped cells, we examined the fine structures and expression of thyroglobulin (Tg) and cytoskeletal proteins using electron microscopy, immunohistochemistry, and immunoblotting. TGFbeta1-nontreated cells had microvilli at the apical side facing follicle lumen and had basal lamina at the basal side in contact with collagen gel. TGFbeta1-treated cells showed both microvilli and basal lamina at the basal side. TGFbeta1-nontreated cells expressed Tg, whereas TGFbeta1-treated cells showed no expression. TGFbeta1-nontreated cells barely expressed vimentin, but they expressed enough cytokeratin. TGFbeta1-treated cells extensively displayed vimentin along with the change in shape to become spindle-like and retained a decreased expression of cytokeratin. TSH (10 mU/ml) did not essentially influence any TGFbeta1 effects on the cells. These results indicate that TGFbeta1 induces a mesenchyme-like cell shape accompanied by cytoskeletal molecular change and the loss of both epithelial polarization and a function in thyrocytes, and that it results in inhibiting thyroid folliculogenesis with or without TSH. |
Fibroblasts from different regions of the human body exhibit substantial phenotypic diversity, some of which relates to the capacity for cross-talk with cells of the immune system. We examine, for the first time, thyroid fibroblast biology in culture. Thyroid explants were placed in culture, and fibroblasts were outgrown and serially passaged. These fibroblasts take on a morphology in culture resembling cells from other anatomic regions. When treated with PGE2, they assume a stellate morphology similar to that of prostanoid-treated orbital fibroblasts. The ganglioside profile exhibited by these cells is distinct from that observed previously in orbital and dermal fibroblasts. They uniformly express Thy-1, a surface glycoprotein. Messenger RNA encoding CD40, a surface receptor found on bone marrow-derived cells, and CD40 protein were expressed constitutively at low levels. Interferon-gamma (500 U/ml) treatment for 48-72 h resulted in high levels of surface HLA-DR and CD40 display. When CD40 is engaged with CD40 ligand (CD40L), nuclear factor-kappaB binding activity is up-regulated as is interleukin (IL)-6 and IL-8 expression. IL-1beta treatment up-regulates the expression of IL-1alpha, IL-1beta, and PGE2. These observations suggest that thyroid fibroblasts possess the molecular machinery necessary for cross-talk with immunocompetent cells such as lymphocytes and mast cells through the CD40/CD40L complex, as well as through classic cytokine networks, and to participate potentially in the inflammatory response of the thyroid gland. |
Adrenomedullin (AM) is a multifunctional peptide involved in a variety of physiological functions, including growth regulation and antimicrobial activity. We have determined by immunohistochemistry and in situ hybridization that AM and its receptor are present in all the epithelial cells of the normal skin, including keratinocytes of the epidermis and hair follicles, as well as cells of the glands and secretory ducts. We also have detected AM in the sweat, by RIA. In addition, AM and its receptor were found in skin tumors of different histologies. The presence of AM and its receptor in normal and neoplastic skin was confirmed by RT-PCR and Western blot analysis performed on cell extracts from human skin cell lines. Radiolabeled AM bound to specific sites in cultured cells with a Kd of 9 nM. This binding was blocked by the addition of cold AM but not by related peptides such as AM 22-52, pro-AM 20 N-terminal peptide, calcitonin gene-related peptide, calcitonin gene-related peptide 8-37, or amylin. Finally, exposure to synthetic AM resulted in an increase of thymidine intake by skin cells. These results implicate AM as a potential player in skin defense against infectious microorganisms and as a possible autocrine growth factor in normal skin physiology and tumor development. |
The present study compares the effects of tamoxifen and EM-800, both administered at the oral daily dose of 100 microg for 6 months, on the uterus, vagina, and mammary gland in the mouse at histopathological examination. Treatment of intact animals with EM-800 resulted in uterine and vaginal atrophy even greater than that achieved after ovariectomy, while the developmental growth of the mammary gland was completely blocked and serum LH was increased. In ovariectomized animals, treatment with EM-800 decreased uterine and vaginal wt below the values observed in control ovariectomized mice while no significant change was observed on serum LH, thus indicating the lack of estrogenic activity of EM-800. Tamoxifen, on the other hand, showed a stimulatory estrogenic-like action on the mouse uterus in both intact and ovariectomized animals, thus resulting in moderate to severe endometrial hyperplasia. These morphological changes were accompanied by a marked stimulation of both the estrogenic and androgenic 17beta-hydroxysteroid dehydrogenase as well as 5alpha-reductase uterine activities. The histological atrophic changes observed in the vagina after tamoxifen treatment were less pronounced than those seen after treatment with EM-800. The agonistic estrogen-like action of tamoxifen was also illustrated by the suppression of serum LH levels in ovariectomized animals. A marked stimulation of the ovarian stroma, accompanied by a significant reduction in folliculogenic activity, was observed after EM-800 or tamoxifen administration, although the interstitial ovarian hyperplasia was more pronounced after EM-800 treatment. While both antiestrogens blocked the developmental growth of the mammary gland, EM-800 showed more potent antiestrogenic activity than tamoxifen. The highly potent and specific antiestrogenic activity of EM-800 suggests that this compound could improve the therapy of breast cancer while avoiding the undesirable stimulation of the endometrium. |
The primate brain was thought to contain only the GnRH known as mammalian GnRH (mGnRH). This study investigates whether a second form of GnRH exists within the primate brain. We found that brain extracts from adult stumptail and rhesus monkeys contained two forms of GnRH that were similar to mGnRH and chicken GnRH-II (cGnRH-II) based on the elution position of the peptides from HPLC and on cross-reactivity with antisera that are specific to mammalian or chicken GnRH-II in RIAs. The fetal brain of rhesus monkeys also contained mGnRH and a cGnRH-II-like peptide by the same criteria. Immunocytochemistry with a cGnRH-II-specific antiserum in adult and fetal rhesus monkeys showed immunopositive neurons generally scattered in the periaqueductal region of the midbrain, with a few positive cells in the posterior basal hypothalamus. Neurons immunopositive for cGnRH-II were fewer in number and smaller in size, with less defined nuclei and thinner neurites compared with those for mGnRH. Administration of synthetic cGnRH-II to adult rhesus monkeys resulted in a significant increase in the plasma LH concentration during the luteal phase of the menstrual cycle, but not during the midfollicular phase. We conclude that the primate brain contains mGnRH and a cGnRH-II-like molecule, although the function of the latter is unknown. |
The conversion of insulin-like growth factor-I (IGF-I) to the biologically more active des (1-3) IGF-I variant is catalyzed by a ubiquitous protease. This proteolytic activity is inhibited by human alpha1-antitrypsin and soy-bean trypsin inhibitor and is up-regulated in serum and tissue extracts of hypophysectomized rats. These observations lead us to investigate whether the growth hormone regulated, serine protease inhibitor, Spi 2.1 was able to inhibit the des (1-3) IGF-I generating protease. Dihydrofolate reductase deficient Chinese hamster ovary (CHO(dhfr-ve)) cells were transfected with a rat Spi 2.1 expression vector containing the dhfr and neomycin resistance gene. Stable transfectants were selected using G418 and amplified using methotrexate. Conditioned medium from Spi 2.1 transfected CHO cells potently inhibited proteolytic activity directed against a synthetic hexa-peptide with a sequence identical to the N-terminal of IGF-I. In contrast conditioned medium from wild-type CHO cells had little effect. Based upon these observations we suggest that our previous finding of enhanced des (1-3) IGF-I generating protease activity in growth hormone deficient rats may be, at least partly explained by reduced levels of Spi 2.1. Furthermore, we propose that the regulation of the generation of des (1-3) IGF-I may be an additional potential site of growth hormone regulation of IGF-I action. |
Leptin, the product of the ob gene, is a recently discovered hormone secreted by adipocytes that regulates food intake and energy expenditure. The site of action of leptin is likely to be the hypothalamus, since this area is important in the control of food intake and leptin receptor mRNA is particularly abundant in this area. In order to further unravel the mechanisms by which leptin acts, we have studied the effect of leptin on in vitro somatostatin synthesis and secretion. Leptin administration to fetal rat neurones in monolayer culture led to a time dependent decrease in basal somatostatin secretion and somatostatin mRNA levels, the maximal effect being observed with 6x10(-8) M leptin after 24 h incubation. Furthermore, leptin completely blunted 10(-7) M Neuropeptide Y-induced increase in somatostatin secretion and somatostatin mRNA levels as well as 10(-3) M (Bu)2-cAMP and 10(-6) M A23187-induced somatostatin secretion. Finally, leptin (3x10(-8) M M) also inhibited low glucose (1.1 mM) induced-somatostatin secretion in perifused adult hypothalami. This data indicates that leptin can influence the neuroendocrine system by regulating hypothalamic somatostatin gene expression. |
Oxytocin is an acute stimulus of prostaglandin (PG) F2alpha secretion from the ovine uterine endometrium. The high level of PGF2alpha secretion induced by oxytocin and the short half-life of the prostaglandin synthase-2 (PGHS-2) enzyme implies that synthesis of PGHS-2 may be essential at this time. The objective of this study was to determine if the increase in PGF2alpha secretion induced by oxytocin is associated with an increase in PGHS-2 mRNA. In experiment 1, oxytocin induced a rapid increase in serum concentration of 13,14-dihydro-15-keto-prostaglandin F2alpha (the stable metabolite of PGF2alpha; PGFM) that was detected within 7.5 min (P < 0.05) and peaked at 25 min post injection. This was associated with an unusually rapid increase in the concentration of PGHS-2 mRNA at 25 min post oxytocin injection (P < 0.05). Endometrial concentrations of PGHS-2 mRNA returned to basal levels at 90 min post injection. Experiment 2 was conducted to further characterize the time course of induction of PGHS-2 mRNA following oxytocin administration. Oxytocin induced a rapid increase in serum concentrations of PGFM. As in experiment 1, an increase in concentrations of PGHS-2 mRNA was detected at 25 min after oxytocin (P = 0.06). Concentrations of PGHS-2 mRNA were intermediate at 40 min and returned to basal levels at 60 min post injection. Thus, there is a rapid increase in endometrial concentrations of PGHS-2 mRNA following oxytocin stimulation of PGF2alpha secretion. This increase in PGHS-2 mRNA may be required to maintain PGHS-2 enzyme levels during pulsatile secretion of PGF2alpha at luteolysis. |
Neurons in the hypothalamus of estrogen receptor alpha-knockout (ER alphaKO) mice have been shown to concentrate radiolabeled estrogen and estrogen treatment regulates the expression of progesterone receptor mRNA. The purpose of the present study was to utilize in situ hybridization histochemistry to determine the anatomical distribution of ER beta mRNA in ER alphaKO mouse forebrain. The results of these studies revealed an extensive distribution of ER beta mRNA in the hypothalamic regions including medial preoptic area, suprachiasmatic nucleus, paraventricular nucleus, dorsomedial nucleus, medial tuberal nucleus, and the premammillary nuclei. Additional labeled perikarya were also detected in the glomerular layer of the olfactory bulb; tenia tecta; anterior septum; bed nucleus of the stria terminalis; medial, basolateral and cortical nuclei of the amygdala; cerebral and entorhinal cortex; the septohippocampal nucleus; Ammon's horn of the hippocampus and the dorsal raphe. The results of these in situ hybridization histochemical studies have provided novel information about the distribution of ER beta mRNA in the ER alphaKO mouse forebrain. In addition, these morphological data provides evidence that estrogen may exert its actions in the ER alphaKO mouse brain via ER beta and thereby maintain organizational and activational effects. |
Our objective was to evaluate the on-going European pilot project for breast cancer screening in Navarra, Spain, and to predict the effects and costs of the programme in the long run. Observed results in Navarra, consisting of more than 100,000 screens, were compared with expected results. A microsimulation screening analysis model was used that included demographical, epidemiological and screening characteristics of Navarra. Alternative assumptions on epidemiological and screening characteristics were also addressed. The observed detection rate (5.9 per 1,000 screened women) in the first round was 18% higher than expected; the observed rate in the subsequent round (2.9) was 17% lower than expected. Longer pre-clinical durations, lower sensitivity or the existence of a high-risk group in Navarra could not satisfactorily explain the first and second round results together. Nevertheless, the programme will have an important health benefit for the women involved, due to an important trend in incidence in recent years and the relatively unfavourable clinical stage distribution in Navarra. The proportion T2+ cancers that will be prevented after 10 years of screening amounts to 36%. The annual mortality reduction in steady state is expected to range between 17% (if the observed rates in the second round indicate real screening performance) to 23% (if the first round indicates real performance). Our results demonstrate that a high detection rate in the first round is insufficient to evaluate the quality of a programme. Interval cancer rates, results of the subsequent round and size distributions are also crucial indicators of the quality of the screening programme and should be analysed in their specific context. |
Experiments were performed to compare the ability of ocular and skin melanoma cells to stimulate T cells. Primary melanoma cell lines were obtained from a series of patients with either eye or skin melanoma. The ability of tumor cells to stimulate T cells in the absence of exogenous growth factors was assessed in mixed-lymphocyte tumor cell cultures in which allogeneic lymphocytes were stimulated with irradiated ocular or skin melanoma cells. Expression of HLA class I and class II on tumor cells, in the presence or absence of IFN-gamma, was determined by flow cytometry. The ability of tumor cells to inhibit T-cell proliferation was determined by adding various concentrations of irradiated tumor cells to standard mixed-lymphocyte cultures. Our results indicate that primary skin melanoma cells induce vigorous proliferation of allo-antigen-specific T cells. By contrast, ocular melanoma cells failed to induce significant T-cell proliferation. The failure of ocular melanoma cells to stimulate lymphocyte proliferation was not due to low levels of either class I or class II on tumor cells since tumor cells treated with IFN-gamma expressed high levels of class I and class II but still failed to induce lymphocyte proliferation. Ocular melanoma cells inhibited lymphocyte proliferation, as shown by experiments in which a small number of tumor cells prevented proliferation of T cells in mixed-lymphocyte cultures. Inhibition of lymphocyte proliferation required cell-to-cell contact, and supernatants from tumor cell cultures did not prevent lymphocyte proliferation. Moreover, the ability of ocular melanoma cells to inhibit T-cell proliferation was lost when tumor cells migrated from the eye and formed hepatic metastases. We conclude that there is a fundamental difference in the immunogenicity of ocular and skin melanoma cells. Ocular melanomas, but not primary skin melanomas, are poorly immunogenic tumors that inhibit T-cell proliferation. Our results imply that the immunogenicity of melanoma cells is altered when they develop within the unique ocular micro-environment. |
Hormones are involved in the regulation of intercellular communication, and gap junction intercellular communication may play an important role in the prevention of endometrial cancer. We have investigated changes in the expression of the gap junction proteins connexin 26 (Cx26) and Cx32 in human endometrial glandular epithelium during the reproductive cycle as well as the influence of hormone replacement therapy. Frozen sections from 71 endometrial tissue samples (53 taken from women who had undergone hysterectomy during the menstrual cycle, 3 early pregnancy deciduae and 15 from menopausal women, some of whom were receiving estrogen alone or estrogen plus progesterone) were analyzed by immunofluorescence and confocal laser scanning microscopy. Cx26 and Cx32 were expressed weakly in the proliferative phase, markedly during ovulation and most strongly in the mid-secretory phase; by the late secretory phase, they decreased drastically. Cx26 and Cx32 also were expressed in early pregnancy. Women who had received estrogen and progesterone expressed the Cxs, but those who had received estrogen only or no therapy did not. These results were confirmed by Western blot analysis. They indicate that expression of Cx26 and Cx32 is correlated with cell differentiation and with the glandular function of the endometrial epithelium and suggest that expression of Cxs is controlled by serum progesterone. |
A displacement assay with tamoxifen, based on the relative binding affinity of tamoxifen and estradiol for the estrogen receptor (ER), was proposed in 1990 as prognostic indicator for breast-cancer patients. Validation of its predictive results in relation to the outcome of 73 patients with ER+ tumors is analyzed. ER, progesterone receptor (PgR) determinations and other conventional prognostic factors in relation to the displacement assay, were considered. Displacement assay results allowed ER+ tumors to be grouped as displaceable (D) or weakly displaceable (WD), with the implication that D tumors should respond better to tamoxifen (Tam) administration. Survival and disease-free interval curves showed highly significant differences between patients with ER+ D and ER+ WD tumors. For survival, including all tumor stages, 73.9% of patients were alive at 9 years after surgery in the group with D tumors and 37.0% in the group with WD tumors (p < 0.005); relative contribution of the different stages is analyzed. Addition of axillary-node number increased the prognostic significance of displacement categories for survival and disease-free interval. PgR determination as another ER functional expression failed to show significant differences for survival and disease-free interval between ER+ PgR+ and ER+ PgR- tumors. Thus, results from the displacement assay and from PgR determinations reflect 2 independent ER functional expressions. Displacement assay data appear as reliable prognostic indicators of breast-cancer outcome, and contribute to more appropriate treatment decisions in this pathology. |
Occupational exposure to gasoline has been identified in several studies as a risk factor for renal-cell cancer. Cases of renal-cell cancer with and without work-related exposure to gasoline or gasoline and diesel fuel were studied for the presence of mutations in the tumour-suppressor gene p53 (n = 23 exposed and 30 non-exposed cases studied) and ras oncogene (n = 30 exposed and 36 non-exposed cases studied). An average cumulative exposure was estimated at 10 ppm-years benzene among the exposed. Three p53 mutations were detected by denaturing-gradient gel electrophoresis (DGGE) among the 23 exposed cases (3/23, 13%). Of the non-exposed referent cases, 4 had a mutation (4/30, 13%). All but one of the cases with a p53 mutation had smoked. A ras gene (K-ras or N-ras) mutation was found in 3 (3/66, 4.5%) cases, all of whom were smoker referents. We conclude that p53 and ras mutations are infrequent in renal-cell cancer associated with occupational exposure to gasoline. However, the majority of the mutations (6/7 for p53, and 3/3 for ras genes) were seen in smokers. |
We conducted a large, sigmoidoscopy-based case-control study to examine the relation of intake of macronutrients, meat, and fiber to occurrence of adenomas of the large bowel. Cases were subjects diagnosed for the first time with one or more histologically confirmed adenomas. Controls had no polyps of any type at sigmoidoscopy, had no history of polyps, and were individually matched to cases by gender, age, date of sigmoidoscopy, and Kaiser Center. The response rate was 84% for cases and 82% for controls. Complete dietary data for 488 matched pairs were available. All odds ratios are from matched analyses adjusted for energy. We observed positive associations with risk of adenomas for calories, animal fat, saturated fat, red meat, and the ratio of red meat to poultry and fish. Protective effects were observed for vegetable protein, carbohydrates, and dietary fiber. The fiber effects diminished after adjusting for fruits and vegetables. Results after mutually adjusting for the effects of saturated fat, fiber and the ratio of red meat to chicken and fish suggest that each of these variables has an effect on risk of adenomas that is independent of the other 2 exposures. |
A national population-based case-control study was used to assess the influence on breast cancer risk of a family history of the disease and the possibility of an interaction with reproductive risk factors. A total of 891 women aged 25-54 years with a first diagnosis of breast cancer and 1,864 control subjects randomly selected from the electoral rolls were interviewed. Age-adjusted relative risks (RR) of breast cancer were similar for mothers (RR = 2.3) and sisters (RR = 2.7) but somewhat higher for first-degree (RR = 2.6) than for second-degree (RR = 1.7) relatives. Cases reporting a first- or second-degree relative with breast cancer were no more likely to be diagnosed at an early age than those with no family history. With regard to the age at diagnosis of the relative, the RR was higher if breast cancer had been diagnosed before the age of 45 years than later; this was true for first-degree as well as for second-degree relatives. In women with no family history, the falling RRs with increasing age at menarche reflected the usual pattern, but no such trend was apparent in those reporting a mother or sister with breast cancer. For age at first full-term pregnancy, parity, breast-feeding, menopausal status, infertility, history of benign breast disease and body mass index, no evidence was seen of effect modification by a family history of breast cancer. Mothers of cases had about twice the cumulative rate of breast cancer as mothers of controls, a similar difference being seen between sisters of cases and sisters of controls. |
Samples of normal esophageal squamous epithelium (n = 10), severe squamous cell dysplasia (n = 22), carcinoma in situ (n = 15), invasive squamous cell carcinoma (n = 172), lymph-node metastasis (n = 21) and 2 permanent esophageal squamous cell carcinoma cell lines were analyzed immunohistochemically for Bax expression using a polyclonal anti-Bax antibody. Immunostaining was evaluated according to a score system (0-8 points) based on the percentage of positive tumor cells and the relative immunostaining intensity. Cytoplasmatic staining for Bax protein was found uniformly in all cell layers of the normal esophageal squamous epithelium. In contrast, a gradual loss of immunoreactivity for Bax was found in a fraction of pre-neoplastic and neoplastic lesions. Upon comparison of the amount of Bax expression between the different types of lesion, however, no significant differences were found between severe squamous cell dysplasias, carcinomas in situ, invasive carcinomas and lymph-node metastases. In both esophageal carcinoma cell lines, immunoreactivity for Bax was found and confirmed by means of Northern blot analysis. In invasive carcinomas, Bax immunoreactivity was inversely correlated with Bcl-2 expression (p = 0.0243) and decreased continuously with decreasing tumor differentiation (p = 0.0011). No correlation was found between Bax expression and the following parameters: depth of invasion, nodal status and tumor size. Bax expression had no influence on the post-operative survival of esophageal cancer patients. |
Post-transplant lymphoproliferative disease (PTLD) is a major cause of death and disease in transplant patients. We describe 4 cases with histologically confirmed malignant lymphoma arising in the Birmingham liver transplant programme between 1982 and 1995. One was an EBV-positive diffuse large B-cell lymphoma, 2 were EBV-positive Burkitt's lymphomas and the 4th was an EBV-negative Burkitt's lymphoma. Immunohistochemistry revealed expression of the EBV-encoded latent membrane protein LMP1 and of the BZLF1 trans-activator protein in 2 cases each, whereas the virus-encoded nuclear antigen EBNA2 was not detectable. All available post-transplant biopsies from the 3 patients with EBV-associated lymphoma were then studied to test whether the detection of EBV-positive cells in liver allograft biopsies could be used to identify patients at risk for the development of PTLD. Two patients showed infrequent EBV-positive cells in liver allograft biopsies up to 14 months before the occurrence of lymphoma and a marked increase in the number of such cells at the time of lymphoma diagnosis. Multiple biopsies from the 3rd patient did not reveal any EBV-carrying cells in the entire post-transplant period. Our results demonstrate a low incidence of PTLD in the Birmingham liver transplant programme. The PTLDs were morphologically high-grade malignant lymphomas. Only 3 cases were associated with EBV infection, and these showed heterogeneous patterns of EBV latent protein expression. Our results also suggest that the examination of liver allograft biopsies using EBER in situ hybridisation is not an appropriate method for identifying patients at risk of developing PTLD. |
Renal oncocytomas reveal a considerable (cyto)genetic heterogeneity. At least 2 genetic subsets are currently recognized, characterized by (1) translocations involving breakpoint 11q13 and (2) the combined loss of chromosomes 1 and X/Y. We present a case of oncocytoma revealing a 3-way translocation involving breakpoint 11q13, a der(1)t(1;8) and an add(19). The der(1) resulted in loss of chromosome 1 sequences. Using fluorescence in situ hybridization, the 11q13 breakpoint of the present case proved to be slightly different from the one observed previously in 3 cases of renal oncocytoma. Whether the 11q13 breakpoint observed in our case resides in or near another gene remains to be elucidated. |
The relationship between various micronutrients and colorectal cancer risk was investigated using data from a case-control study conducted between January 1992 and June 1996 in Italy. Cases were 1,953 incident, histologically confirmed colorectal cancers (1,225 of the colon and 728 of the rectum), admitted to the major teaching and general hospitals in the study areas, and 4,154 controls with no history of cancer, admitted to hospitals in the same catchment areas for acute, non-neoplastic diseases unrelated to the digestive tract and requiring no long-term modifications of the diet. Dietary habits were investigated using a validated food-frequency questionnaire. Odds ratio (ORs) were computed after allowance for age, sex and other potential confounding factors, including physical activity, total energy and fibre intake. For most micronutrients, ORs were below unity with increasing quintile of intake. The most consistent protective effects were for carotene, riboflavin and vitamin C (Multivariate ORs from the continuous model, with unit set as the difference between the upper cut-point of the 4th quintile and that of the 1st one, were 0.65, 0.73 and 0.80, respectively). Inverse relationships were observed also for calcium and vitamin D (ORs of 0.85 and 0.93, respectively). When the combined effect of calcium and vitamin D and selected anti-oxidants was considered, the OR reached 0.46 in subjects reporting high calcium/vitamin D and high anti-oxidant intake compared to those reporting low intake of both groups of micronutrients. Most results were apparently stronger for colon cancer and among females. Our results provide further support for a protective effect of several micronutrients on colorectal cancer risk and some indications for a specific and stronger effect of selected anti-oxidants. |
The CDKN2A gene encodes p16 (CDKN2A), a cell-cycle inhibitor protein which prevents inappropriate cell cycling and, hence, proliferation. Germ-line mutations in CDKN2A predispose to the familial atypical multiple-mole melanoma (FAMMM) syndrome but also have been seen in rare families in which only 1 or 2 individuals are affected by cutaneous malignant melanoma (CMM). We therefore sequenced exons 1alpha and 2 of CDKN2A using lymphocyte DNA isolated from index cases from 67 families with cancers at multiple sites, where the patterns of cancer did not resemble those attributable to known genes such as hMLH1, hMLH2, BRCA1, BRCA2, TP53 or other cancer susceptibility genes. We found one mutation, a mis-sense mutation resulting in a methionine to isoleucine change at codon 53 (M531) of exon 2. The individual tested had developed 2 CMMs but had no dysplastic nevi and lacked a family history of dysplastic nevi or CMM. Other family members had been diagnosed with oral cancer (2 persons), bladder cancer (1 person) and possibly gall-bladder cancer. While this mutation has been reported in Australian and North American melanoma kindreds, we did not observe it in 618 chromosomes from Scottish and Canadian controls. Functional studies revealed that the CDKN2A variant carrying the M531 change was unable to bind effectively to CDK4, showing that this mutation is of pathological significance. Our results have confirmed that CDKN2A mutations are not limited to FAMMM kindreds but also demonstrate that multi-site cancer families without melanoma are very unlikely to contain CDKN2A mutations. |
The growth of human lung cancer cells is regulated positively and negatively by a variety of growth factors through autocrine as well as paracrine mechanisms. In the present report, we studied the differential role and expression of a neuropolypeptide growth factor in 26 lung cancer cell lines. Expression of the heparin-binding growth-associated molecule (HB-GAM) in 12 small cell lung cancer (SCLC) cell lines was compared to that in 14 non-small cell lung cancer (NSCLC) cell lines. HB-GAM mRNA was expressed in 9 of 12 SCLC and 3 of 14 NSCLC cell lines as determined by RT-PCR analyses. Normal human bronchial epithelial cells were used as negative controls. All cell lines which expressed HB-GAM mRNA produced HB-GAM protein as well. Western blot analysis showed that the tumor cells secreted HB-GAM into the media. HB-GAM, purified from lung cancer cell lines, exerted biological activity on fibroblasts, endothelial cells and SW13 cells as determined by thymidine incorporation and soft agar cloning assays. In addition, the biological activity of HB-GAM was blocked by a specific antibody in a dose-dependent way. Our findings suggest that HB-GAM may serve as a marker for SCLC cell lines and that it may function as a paracrine growth factor in human lung cancer. HB-GAM may be a further member of the network of growth factors involved in proliferation, angiogenesis and metastasis of lung tumors. |
Progressive deregulation of the cell-division cycle is thought to contribute to the establishment and progression of neoplasia. Previously, we have documented the in vivo inactivation of p16INK4A, an inhibitor of G1 cyclin-dependent kinases, in squamous cell carcinomas of the head and neck region. In the present study, we extend these findings by examining the expression and functional activity of cyclin-dependent kinases (CDKs) and their regulatory subunits using a model system of cell lines derived from squamous cell carcinomas. Increased activity of CDK4 and 6 was universal in tumor cells compared with normal keratinocytes, reflecting over-expression of either or both kinases. In contrast to other studies, over-expression of cyclin D1, a regulatory subunit of CDK4 and 6, was not observed. Increased activity of CDK2 was less frequent and was related to over-expression of cyclin A and/or E. All tumor cell lines showed increased expression of proliferating cell nuclear antigen compared to normal keratinocytes. Four SCC cell lines, including one tumor-metastasis pair derived from a single patient, failed to express the p15INK4B transcript. Western blot analysis of cell lysates revealed normal or reduced levels of p27KIP1 in tumor cells compared to normal keratinocytes. However, failure to express wild-type p53 was not reflected by lower levels of p21WAF1. Our data suggest that cell-cycle deregulation is likely to occur by multiple mechanisms during the genesis of head and neck squamous cell carcinomas. Furthermore, p16INK4A is likely to be the primary target for inactivation on chromosome 9p21 in these tumors as p15INK4B loss occurs less frequently. |
Primary lymphomatous effusions are represented by cases of non-Hodgkin's lymphoma (NHL) which grow in liquid phase in the serous body cavities in the absence of solid tumour masses. Based on morphologic, immunophenotypic, virologic and genotypic features, primary lymphomatous effusions are distinguished into body cavity-based lymphoma (BCBL), Burkitt's lymphoma (BL) and immunoblastic large-cell lymphoma. The histogenesis and pathogenesis of primary lymphomatous effusions are virtually unclarified. In this study, we have established 2 cell lines (CRO-AP/1 and CRO-AP/2) representative of 2 distinct categories of primary lymphomatous effusion, BCBL (CRO-AP/2) and BL (CRO-AP/1). Both CRO-AP/1 and CRO-AP/2 carry monoclonal re-arrangements of the immunoglobulin genes which are identical to those of the respective parental tumours. Consistent with its BCBL origin, CRO-AP/2 is characterised by a non-B, non-T phenotype and harbours infection by HHV-8 (approx. 100 viral copies/cell) and Epstein-Barr virus. Conversely, CRO-AP/1 expresses several B cell-associated antigens, lacks HHV-8 infection and carries the genetic hallmark of BL, i.e., a chromosomal breakpoint of band 8q24. CRO-AP/1 and CRO-AP/2 may be valuable for the biologic characterization of primary lymphomatous effusions, particularly since the number of available cell lines derived from such lymphomatous effusions is extremely limited. |
Proton magnetic resonance spectroscopy (1H MRS) and DNA flow cytometry were used to monitor the effects of the cationic lipophilic phosphonium salt and potential antineoplastic agent tetraphenylphosphonium chloride (TPP) on the transformed human breast cell line HBL-100. TPP treatment for 48 hr was cytostatic at low concentrations and cytotoxic at higher concentrations with an IC50 of 55 microM as measured by Trypan blue exclusion. At micromolar concentrations, TPP caused a significant increase in the methylene MR signal arising from mobile lipid as measured by the ratio of the lipid CH2 peak height to either the CH3 peak height (internal referencing) or the peak height for p-aminobenzoic acid (PABA) as an external reference in a co-axial capillary within the sample. Over the same concentration range, TPP caused a slowing of passage through S phase as demonstrated by a significant depletion of cells in G2/M phase with a concurrent but non-significant increase in cells in S. Time-dependent increases in MR-visible lipid were observed with 2 microM TPP treatment, and the removal of TPP from the culture medium caused no significant reduction in mobile lipid. Two-dimensional 1H-1H COSY spectra of TPP-treated HBL-100 cells revealed concentration-dependent increases in cross-peak volume ratios arising from lipid acyl chains relative to both internal (lysine, polyamines) and external (PABA) standards. Increases in choline and glycerophosphocholine cross-peak volume ratios were observed, indicating that the catabolism or rearrangement of phospholipids may be responsible for the observed MR-visible lipid increases. |
The novel anti-estrogen EM-800 and medroxyprogesterone acetate (MPA) inhibit estrone (E1)-stimulated growth of dimethylbenz[a]anthracene (DMBA)-induced mammary tumors in a rat model. After 65 days, ovariectomy (OVX) decreased total tumor area to 9.6 +/- 3.9% of initial size, while E1 (1.0 microg, s.c., twice daily) stimulated tumor growth to 225 +/- 40.9% of initial size. Daily oral administration of 2.5 mg/kg body weight of EM-800 completely abolished E1-stimulated tumor growth. A low daily dose of EM-800 (0.25 mg/kg body weight) or MPA (1 mg, s.c., twice daily) used alone partially reversed the stimulatory effect of E1 on the growth of DMBA-induced tumors. The combination of both compounds, however, caused a more potent inhibitory effect than each compound used alone. A high dose of EM-800 completely or almost completely inhibited the E1-stimulated vaginal and uterine weights, respectively. The same dose of EM-800 completely reversed the inhibitory effect of E1 on serum luteinizing hormone levels. Uterine, vaginal and tumoral estrogen and progesterone receptor levels were reduced markedly following treatment with EM-800. Our data show that the combination of the pure anti-estrogen EM-800 with the androgenic compound MPA achieves greater inhibition of the growth of DMBA-induced mammary carcinoma than that achieved by each compound used alone. |
Chronic hepatitis B virus infection as well as consumption of food contaminated with the mycotoxin aflatoxin B1 are considered to be 2 major risk factors for the development of primary liver cancer in humans. Furthermore, epidemiological surveys indicate that hepatitis B virus and aflatoxin B1 might act synergistically to induce primary liver cancer. In the present study, we have tested the hypothesis that the metabolic activation of aflatoxin B1 to aflatoxin B1-8,9-epoxide, the ultimate mutagenic and carcinogenic mycotoxin metabolite, is enhanced in an experimental model of chronic hepatitis using woodchucks, chronically infected with the woodchuck hepatitis virus. Woodchuck liver microsomes were incubated with radiolabeled aflatoxin B1, the resulting aflatoxin B1-8,9-epoxide was trapped as a glutathione conjugate and its formation rate was determined by a reversed-phase HPLC analysis. In woodchuck hepatitis virus-positive woodchucks, activation of aflatoxin B1 to aflatoxin B1-8,9-epoxide was reduced when compared to woodchuck hepatitis virus-free animals, and the extent of the reduction was dependent on the severity of the hepatitis. Hence, at least in woodchucks, a chronic hepadnaviral infection does not lead to an enhanced activation of aflatoxin B1. |
We investigated the role of p53 and of the Bcl-2 family proteins in the apoptotic response of a panel of testicular tumour cell lines (NT2, NCCIT, S2 and 2102 EP). The p53 gene status and the capacity of the p53 protein to transactivate the p21/WAF/CIP gene were determined, and we examined the correlation between p53 status and the susceptibility to cisplatin-induced apoptosis. In contrast to wild-type p53-containing NT2 and 2102 EP cells, NCCIT (mutant p53) and S2 (no p53 protein) cells were shown to be p53-transactivation defective. However, NCCIT and S2 cells with non-functional p53 were readily triggered into apoptosis by cisplatin, whereas p53-transactivation competent 2102 EP cells failed to undergo cisplatin-induced apoptosis. The defective apoptotic pathway in 2102 EP cells was reflected by a 4-fold decreased sensitivity to cisplatin in the MTT assay. We further demonstrated that the p53-independent differential cisplatin sensitivity among the testicular germ cell tumour (TGCT) cell lines was not due to differences in cellular cisplatin accumulation or DNA platination. The pattern of endogenous expression levels of Bax, Bcl-2, Bcl-x and Bak, which was not modulated by cisplatin treatment, demonstrated that these Bcl-2 family proteins are not involved in drug-induced apoptosis in the TGCT cell lines. Our results suggest a lack of correlation between cisplatin-induced apoptosis, p53 status and expression of Bcl-2 family proteins in our panel of TGCT cell lines. We conclude that the cisplatin-induced apoptotic pathway in TGCT cell lines might be p53-independent and is probably not associated with differences in the Bcl-2/Bax rheostat. |
Confocal laser microspectrofluorometry was used to investigate restoration of nuclear pirarubicin (THP-DOX) accumulation and sensitivity by verapamil, quinine and S9788 in 2 variants of the Chinese hamster ovary cell lines LR73, selected for resistance to doxorubicin (LR73D) or transfected with the mdr1 gene (LR73R). The 2 resistant cell lines present a multidrug-resistance phenotype (MDR). Verapamil and S9788, which interact with P-glycoprotein (P-gp), were able to restore nuclear THP-DOX accumulation in LR73R and LR73D cells to a level equivalent to that in sensitive cells. On the other hand, quinine was unable to increase nuclear THP-DOX accumulation significantly even at a concentration of 50 microM. All modulators completely restored THP-DOX sensitivity in resistant cell lines. Our results also show that verapamil and S9788 allow high nuclear drug accumulation, whereas quinine did not affect nuclear accumulation. The effect of quinine on the mdr1 gene expression was determined by the use of reverse transcription coupled with polymerase chain reaction. After a 2 hr treatment with 20 microM of quinine, mdr1 gene expression increased slightly. |
In order to determine the effects of single unsaturated fatty acids (UFAs) or combinations on establishment of lung metastatic colonies, UFAs were administered orally to CDF1 mice bearing s.c. implants of the highly metastatic colon carcinoma 26. Oleic acid (OA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) demonstrated significant inhibition. In the case of DHA, this inhibitory potential was markedly reduced by co-administration of linoleic acid (LA) or EPA. Furthermore, while tumor cells treated with DHA showed a very low potential for lung colony formation when injected i.v., this again being partially reversed by co-administration of EPA. UFAs were found to be well absorbed into tumor tissues after oral administration, causing marked changes in relative levels, the arachidonic acid (AA) content, in particular, being markedly decreased by treatment with DHA or EPA, but not with DHA plus EPA or with DHA plus LA. Investigation of the gelatinolytic activity of the 57-kDa and 92-kDa isoforms of type-IV collagenase (MMP-2 and MMP-9, respectively) showed a clear reduction in the former by treatment with OA, while DHA, but not DHA plus LA or EPA, caused a decrease in the 92-kDa isoform, which was well correlated with AA content in tumor tissues (r = 0.900, p < 0.001). These results suggest that inhibition of metastasis due to treatment with OA and DHA might be due to depressed type-IV collagenase activity. |
Levels of S100beta, a calcium-binding protein found in astrocytes, were measured using a sandwich ELISA in the cerebrospinal fluid (CSF) of patients with frontotemporal dementia and Alzheimer's disease and compared with controls. Mean CSF S100beta concentrations were significantly raised in patients with frontotemporal dementia when compared with healthy controls (0.49 +/- 0.28 vs. 0.22 +/- 0.08 ng/ml, P < 0.001). There was no correlation between age at disease onset, disease severity or length of illness. The increased concentration of CSF S100beta seen in frontotemporal dementia may reflect the marked astrocytosis seen in this condition. |
Amyloid precursor protein (APP) is known to have neurotrophic effects but little information is available on the signaling pathways activated by APP. Since neurotrophic factors activate tyrosine phosphorylation signaling pathway in general, we investigated whether or not APP activates tyrosine phosphorylation pathway. Alpha-secretase derived APP (sAPP alpha) increased the number of neurites per cell and enhanced tyrosine phosphorylation levels on distinct 125 and 200 kDa protein bands. The APP3 19-335 17-mer peptide, which has been reported to be responsible for the neurotrophic effect of sAPP alpha [Jin, L.-W., Ninomiya, H., Roch, J.-M., Schubert, D., Masliah, E., Otero, D.A.C. and Saitoh, T., J. Neurosci., 14 (1994) 5461-5470], increased neurite extension as well as tyrosine phosphorylation on 125 and 200 kDa proteins in a similar manner to sAPP alpha. Both effects were blocked by an antagonist peptide to 17-mer ERMSQ (APP329-333). These results indicate that the 17-mer domain of APP induces tyrosine phosphorylation on distinct proteins under the condition that induces neurite extension. |
The leukocyte adhesion molecule L-selectin plays a key role in the initial steps of transendothelial migration of T cells and monocytes. In this study we investigated the role of L-selectin in the pathogenesis of experimental autoimmune neuritis (EAN) an animal model of the Guillain-Barré syndrome. EAN was induced in Lewis rats by sensitization with peripheral nerve myelin. Treatment with HRL3, a monoclonal antibody to L-selectin, efficiently suppressed clinical signs of EAN. Histological examination of the peripheral nervous system (PNS) revealed a marked reduction of inflammatory infiltrates and demyelination during treatment with HRL3. We conclude that L-selectin-dependent mechanisms are of pathophysiological relevance in EAN. Modulation of L-selectin in vivo could be a novel therapeutic approach to autoimmune diseases of the PNS. |
The present work studied the effect of a calcium channel blocker (nimodipine) on rat behavioural changes and brain lesions observed after seizures induced by high doses of pilocarpine (400 mg/kg, s.c.; P400), and the association of lithium (3 mEq/kg, i.p., daily during 7 days) plus pilocarpine (a single dose of 15 mg/kg, s.c.) administered 24 h after the last injection of lithium. In the P400 model, nimodipine (5 or 10 mg/kg, i.p.) inhibited convulsions, status epilepticus, and significantly decreased the percentage of death and cerebral changes (Mann-Whitney, P = 0.0057). In the lithium-pilocarpine (Li-Pi) induced seizures, nimodipine even increased convulsive action and did not interfere with brain lesions. The results suggested that a calcium channel mechanism is involved in the P400 induced seizures, and that there is a difference in the physiopathology of epileptic seizures and brain damage induced by either P400 and Li-Pi models. |
The presenilin-1 (PS-1) and amyloid precursor protein (APP) genes carry mutations which co-segregate with early-onset familial Alzheimer's disease. The APP and PS-1 gene products may be involved in the aetiology of the more common late onset form of Alzheimer's disease, where increasing age is a major risk factor. To investigate whether age affected mRNA expression of these genes, we quantified PS-1, total APP, APP containing the kunitz-type protease inhibitor (KPI) domain and amyloid precursor-like protein 2 (APLP2) mRNAs in post-mortem superior frontal cortices from 23 control subjects aged 38 to 89 years using solution hybridisation-RNase protection assays. PS-1, total APP, APP KPI and APLP2 mRNA levels were unchanged over this age range. PS-1 was the least abundant mRNA, at approximately 7% of total APP, the most highly expressed mRNA studied (10.8 copies/pg total RNA). The proportion of total APP encoding the KPI domain (approximately 52%) was unaffected by age. APLP2 mRNA was present at approximately 29% of the total APP mRNA level. Significant positive correlations were present between total APP, APP KPI and APLP2 mRNA levels. These results indicate that the increased prevalence of Alzheimer's disease cannot be attributed to alterations in cortical PS-1, APP and APLP2 mRNA levels or APP KPI splicing during aging. |
Positron emission tomography (PET) data were obtained from subjects performing a synchronization task (target duration 2700 ms). A conjunction analysis was run to identify areas prominently activated both in this task and in a temporal generalization task (target duration 700 ms) used previously. The common pattern of activation included the right prefrontal, inferior parietal and anterior cingulate cortex, the left putamen and the left cerebellar hemisphere. These areas are assumed to play a major role in time processing, in relation to attention and memory mechanisms. |
Paradoxical sleep deprivation was performed on rats using platform technique to investigate the oxidative process associated with it. Levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), total glutathione (GSH) and malondialdehyde production were measured in brain of rats under control conditions (C) and those on single large platforms (SLP), multiple large platforms (MLP), single small platforms (SSP) and multiple small platforms (MSP) groups. SOD, CAT and GPx brain activity and malondialdehyde production were not modified by any of the procedures. Brain GSH, however, was significantly reduced in both SSP and SLP groups. These results suggest that paradoxical sleep deprivation per se is not associated with oxidative damage. The observed alterations could be attributed to factors such as immobilization and social isolation present in the single platform techniques. |
Oxygen free radicals are postulated to participate in the pathogenesis of ischemic brain injury. The present study investigated the response of the endogenous antioxidant enzyme, superoxide dismutase (SOD), in a model of transient focal ischemia in the rat neocortex. SOD activity was increased significantly in the penumbra region at 6-24 h postischemia, while no significant changes in SOD activity were observed in either the core region or striatum. These results indicate that endogenous antioxidant activity is differentially affected by the intensity of ischemic challenge and suggest that the regional effects of oxygen free radicals may vary substantially following ischemia-reperfusion. |
We report an association between a language deficit following brain lesion and a new strategy in temporal integration. Patients with different brain lesions mentally grouped sequences of identical acoustic stimuli generated at various frequencies. They were asked while listening to the stimuli to accentuate every second, third or other stimulus to create an individual rhythmic pattern. After each sequence patients reported how many stimuli they had united into a perceptual unit. The integration interval was defined as the number of reported stimuli multiplied by the temporal interval between two successive stimuli. Results indicate different integration strategies depending on the lesion site, i.e. Broca's aphasics behaving differently than all other patient groups. At lower frequencies they showed longer, at higher frequencies they displayed shorter integration. From this observation we conclude that the Broca's patients acquired a new strategy because of the lesion; they relied on mental counting and less on automatic temporal integration, which is usually the case. |
The present study was designed to observe the effect of orphanin FQ (OFQ, also known as 'nociceptin'), a newly-discovered neuropeptide, on pain behavior and morphine analgesia evaluated by formalin test in rats. It was found that intracerebroventricular (i.c.v.) injection of 0.1 microg OFQ had no effect on formalin-induced pain behavior; but 1, 5, 10 or 20 microg OFQ produced prolonged lifting, licking, biting or shaking of the affected paw with higher pain scoring in dose dependent manner. Repeated i.c.v. injection of antisense olignucleotide (ASO) complementary to OFQ receptor but not mismatch olignucleotide (MSO) resulted in the decrease of pain behavior; in such circumstances, OFQ showed no enhancing effect on formalin nociception. OFQ (0.1 or 1 microg, i.c.v.) significantly attenuated morphine analgesia and ASO could validly antagonize the effect of it. Pretreatment with MSO had no such effect. The present results suggest that OFQ enhances the pain behavior of rat and antagonizes morphine analgesia in formalin test. |
The present study was designed to further investigate the effects of intrinsic or extrinsic influences on the development of the efferent connectivity of frontal neocortical neurons. The lateral or medial parts of the frontal neocortex of embryonic (E) day 16 fetuses were grafted into homo- (medial-to-medial) or heterotopic (lateral-to-medial) position in the medial part of the left frontal cortex of newborn hosts. Three to four months after grafting, a retrograde neurotracer was injected into the dorsomedial or ventrolateral quadrant of the left caudate-putamen (CPU). The ensuing retrograde labeling in the transplants was then compared to that found in an equivalent cortical area in control animals. Medial-to-medial transplants developed a striatal projection whose mediolateral organization conforms to that of the projection arising from the medial part of the intact frontal cortex. The mediolateral distribution of the projection arising from lateral-to-medial transplants was not fundamentally different from that originating from medial-to-medial transplants, a finding which stands in marked contrast with what was found recently [7] with medial-to-lateral transplants. These results indicate that inside the frontal cortex, different subregions are not totally interchangeable, at least in terms of development of efferent connectivity. |
Phosphorylation of the retinoblastoma protein (RB) was observed during apoptosis of B50 neuroblastoma cells following induction by dibutyryl cAMP, after differentiation into neurons, or by cycloheximide during proliferation. A weak but distinct increase in a RB and histone H1 kinase activity was detected at the time of RB phosphorylation. However, the RB kinase appeared to correspond to neither p34cdc2 kinase, CDK2 nor CDK5 because it was not inhibited by butyrolactone I, an inhibitor for them. Expression of CDK4 and 6 along with several cyclins also did not coincide with the appearance of phosphorylated RB in the apoptotic process. |
Regional cerebral metabolic rate of glucose (rCMRglc) was studied in 19 patients with a clinical diagnosis of dementia with Lewy bodies (DLB) and 19 patients with a clinical diagnosis of Alzheimer's disease (AD), using [18F]fluorodeoxyglucose ([18F]FDG) and positron emission tomography (PET). The two groups were matched with age, gender, disease duration and severity of cognitive disturbances. In 'dementia with Lewy bodies' (DLB) patients, when compared with AD patients, significant rCMRglc decreases were distributed in the temporo-parieto-occipital association cortices and the cerebellar hemispheres. In contrast, the medial temporal and cingulate rCMRglc were significantly lower in AD patients than those in DLB patients. These different regional emphases of glucose hypometabolism are consistent with the pathological and neurochemical differences between DLB and AD and explain the different clinical features of the two diseases. |
Glial fibrillary acidic protein (GFAP), a biochemical marker of astrocytes and glial reaction, was quantified by immunoblotting in different brain areas from 33 non-demented patients with a Mini Mental State Examination score above 26 and aged from 12 to 98 years. An increase of GFAP with age was first found in the hippocampus and then in the entorhinal cortex. In both regions, GFAP amounts were correlated with age (r = 0.768). In the isocortex, the increase of GFAP as a function of age was also significant (r = 0.672), but less than for the hippocampal region. GFAP levels increased dramatically after the age of 65 years, and more especially in the hippocampal formation. This glial reaction was observed in aged controls that do not show cognitive impairment and the neuropathological hallmarks of Alzheimer's disease. |
We initiated the present work in order to determine if the Ala53Thr mutation of the alpha-synuclein gene previously described by Polymeropoulos et al. [Science, 276 (1997) 2045-2047] could be detected in Spanish early onset Parkinson's disease (PD) patients. Thirty-four PD patients were evaluated. Of these, 13 were considered early onset patients (six familial and seven sporadic) and were included in the genetic study. We detected the presence of genetic anticipation in four kindreds with early onset PD members. The Ala53Thr mutation of the alpha-synuclein gene was absent in all patients. The results do not support a role for this mutation in our patients with early onset PD and, in agreement with the results previously reported, indicate that the Ala53Thr mutation of the alpha-synuclein gene is a rare cause of PD. |
Using nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase (ND) histochemistry, this study reports a wide distribution of ND activity in the hypothalamus, and for the first time in the median eminence (ME), the neural lobe (NL) and in the pars distalis (PD) of the frog, Rana esculenta. Perikarya are ND-active within the nucleus preopticus (NPO), the nucleus preopticus periventricularis (NPP), located around the preoptic recess (PR), the suprachiasmatic nucleus (SCN) and within five infundibular nuclei. Several ND-positive neurons of the nucleus infundibularis ventralis are cerebrospinal fluid-contacting in nature, while a few occupy a subependymal region. The infundibulum shows a thick sheet-like fiber plexus which receives fibers not only from its ND-active neurons, but also from the anterior and central thalamic nuclei. The ME, NL and most cells of the posterodorsal PD are ND-positive. The pituitary function may be mediated by nitric oxide through modulating the secretion of hormone-releasing factors of the hypothalamus. Possible functional significance of the ND-stained hypothalamic areas is discussed. |
The ability to detect unusual and novel events is an important prerequisite for storage of information in memory. Non-tonal novel sounds that deviate from an ongoing auditory environment elicit a positive event-related potential (ERP) component, the so-called novel P3. Though there is converging evidence on the neuronal network engaged in novelty detection, little attention has been paid to the properties of novel sounds, such as their typicality or relationship to mental concepts. Here we report the ERPs evoked by two types of generically novel stimuli, namely identifiable (meaningful) and non-identifiable (non-meaningful) novel sounds. The ERP analysis revealed a novel P3 for both types of sounds. However, when subjects actively attended to the stimuli only identifiable novel sounds evoked a right-lateralized negativity (N4) that peaked shortly after the novel P3. We conclude that novelty detection not only includes the registration of deviancy but also fast access and identification of related semantic concepts. |
This study investigated the effects of sera from amyotrophic lateral sclerosis (ALS) patients on high voltage activated (HVA) Ca2+ current in mice dorsal root ganglion (DRG) cells using whole-cell voltage-clamp method. Mice were injected with sera from healthy adults, from patients with other neurological diseases, and from patients with the sporadic form of ALS, for a period of 3 days. Sera from five of six ALS patients reduced HVA Ca2+ current amplitude. The peak Ca2+ current was significantly reduced by ALS sera while the sera from healthy adults and patients with other diseases did not alter Ca2+ current. The inactivation kinetics was altered by ALS sera, and the half-inactivation voltage shifted to more negative potential in ALS group. These results suggest that sporadic ALS serum factors may exert interactions with the HVA Ca2+ channel in DRG cells to reduce the Ca2+ current. |
This study investigates the antinociception caused by intradermal (i.d) or intracerebroventricular (i.c.v.) injection of the capsaicin receptor antagonist capsazepine (CPZ), and ruthenium red (RR) (a cation-selective antagonist coupled to vanilloid receptor of capsaicin), on the chemical nociception caused by i.d. injection of formalin (FM) and capsaicin (CAP) into the mouse paw. The i.d. injection of either CPZ or RR in association with FM or CAP, inhibited the early phase, and to a lesser extent the late phase, of the FM, as well as CAP-induced nociception. Given i.c.v., both CPZ and RR caused discrete antinociception in the FM (both phases), while producing graded inhibition of CAP. The actions of CPZ and RR were insensitive to i.p. injection of naloxone (5 mg/kg). These results indicate that i.d. injection of CPZ and RR produce marked antinociception in chemical models of neurogenic pain induced by CAP and FM in mice. However, administered by supraspinal site, both CPZ and RR were inactive in inhibiting FM, but prevented, in a graded manner, CAP-induced algesic response, suggesting the participation of distinct mechanisms in the nociception induced by FM and CAP. Thus, vanilloid selective antagonists seem to be useful tools for investigating the nociception elicited by CAP and FM. |
Antioxidants may delay or prevent neural diseases. Depletion of the non-enzymatic antioxidant, glutathione, in a mouse model was produced by inhibiting its rate-limiting enzyme, gamma-glutamylcysteine synthetase, for 7 weeks. Ileum and colon were obtained from treated and control (saline) mice. Glutathione levels and nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity were determined by spectrophotometric assays; vasoactive intestinal peptide (VIP) levels were measured by radioimmunoassay. Glutathione levels were higher in ileum than colon. Colonic glutathione was decreased in treated mice compared to controls; there were no differences in ileal glutathione levels. VIP was decreased in ileum compared to controls, while NADPH diaphorase activity was decreased in colon compared to controls. In this chronic mouse model, glutathione appeared to regulate expression of enteric inhibitory nerve cell products. |
An endogenous MPTP-like dopaminergic neurotoxin, N-methyl(R)salsolinol, increases in the parkinsonian cerebrospinal fluid and accumulates in the human nigro-striatum. An N-methyltransferase specific for (R)salsolinol was found in human brain with optimal pH at 7.0 and 8.5. The correlation of the enzyme activity with the level of N-methyl(R)salsolinol and its oxidation product, 1,2-dimethyl-6,7-dihydroxyisoquinolinium ion was examined in the brain regions. Neutral N-methyltransferase activity in the striatum was found to correlate with the level of the endogenous MPP+-like isoquinolinium ion in the substantia nigra (P < 0.001). Considering that this neutral N-methyltransferase activity increases in parkinsonian lymphocytes, the enzyme may be an endogenous factor in the pathogenesis of Parkinson's disease. |
We examined whether or not nitration of tyrosine residues takes place in the ischemic and postischemic reperfused brain. The nitration of tyrosine residues to produce nitrotyrosine is a sensitive marker elicited by peroxynitrite, a powerful oxidant formed by the reaction of nitric oxide (NO) with superoxide. Mongolian gerbils were subjected to 60 min ischemia induced by occlusion of the right common carotid artery (ischemia group), to 30 min recirculation following 60 min ischemia (reperfusion group) or to sham surgery (sham group). Immunohistochemical staining with polyclonal anti-nitrotyrosine antibody revealed the widespread and distinct occurrence of nitrotyrosine in cortical neurons on the reperfused side of the brain in the reperfusion group, while only partial or weak immunoreactivity was noted on the contralateral side. On the other hand, nitrotyrosine was not detected in the brain of the ischemia and sham groups. These findings suggest that nitration of tyrosine residues in various proteins may be closely associated with reperfusion injury of the brain. |
The crucian carp (Carassius carassius) and freshwater turtles (Trachemys scripta) are among the very few vertebrates that can survive extended periods of anoxia. The major problem for an anoxic brain is energy deficiency. In the brain, the Na+/K+-ATPase is the single most ATP consuming enzyme, being responsible for maintaining ion gradients. We here show that the Na+/K+-ATPase activity in the turtle brain is reduced by 31% in telencephalon and by 34% in cerebellum after 24 h of anoxia. Both changes were reversed upon reoxygenation. By contrast, the Na+/K+-ATPase activities were maintained in the anoxic crucian carp brain. These results support the notion that crucian carp and turtles use divergent strategies for anoxic survival. The fall in Na+/K+-ATPase activities displayed by the turtle is likely to be related to the strong depression of brain electric and metabolic activity utilized as an anoxic survival strategy by this species. |
There is electrophysiological evidence of a functional role for gamma-aminobutyric acid (GABA) and GABA transporters (GATs) in the neonatal rat optic nerve, and that they are down-regulated during development. The results of the present study demonstrate directly by reverse transcription-polymerase chain reaction (RT-PCR) that the mRNAs encoding for GAT-1, -2 and -3 are expressed in the optic nerves of both neonatal (5 day old) and adult rats. The results support a role for GABA in the developing rat optic nerve, a typical white matter tract which contains axons and glia, but neither neuronal cell bodies nor synapses. Significantly, the persistence of GAT mRNAs suggests an enduring function for both GABA and glial uptake mechanisms in the adult optic nerve. |
Understanding the determinants of maternal mortality is a complex task, in part because maternal deaths are influenced by many different categories of events or conditions. Biology, economics, culture, demography and the distribution and effectiveness of health services all contribute. Conceptual frameworks have made important contributions to our understanding of the determinants of other, equally complex events, such as fertility and child survival. Also referred to as 'proximate determinants frameworks', such models are useful because they identify the specific mechanisms through which social, economic and cultural factors lead to the event of interest. Our model identifies the precise sequence of events that lead to maternal death (pregnancy, complication and death) and specifies categories of intermediate factors and distant factors that directly affect one or more of these events. When the world literature on maternal mortality was analyzed in light of the causal pathways laid out in the conceptual framework, it became clear that some pathways are more amenable to intervention and change than others. Implications for strategies, programs and monitoring and evaluation are discussed. |
The projects of the PMM Network are based on a strategic model that focuses sharply on the interval between the obvious onset of a serious obstetric complication and the provision of emergency obstetric care (EmOC). The reason for this is that most of these complications cannot be predicted or prevented, but they can be successfully treated. The implications of this model for program design are profound. The emphasis is on improving the accessibility, quality and utilization of EmOC for women who develop such complications, rather than on having contact with all pregnant women. |
Since 1988, the Prevention of Maternal Mortality (PMM) Network has developed, implemented and evaluated projects that focus directly on prevention of maternal deaths. The Network, which consists of 11 multidisciplinary teams in West Africa and one at Columbia University, grew from discussions between the Carnegie Corporation of New York and researchers at Columbia School of Public Health. Its goals are: to strengthen capacities in developing countries; to provide program models for preventing maternal deaths; and to inform policymakers about the importance of maternal mortality. This paper describes the development and functioning of the Network. The initial steps included identifying interested partners in Africa and encouraging them to form multidisciplinary teams. Each African team received two grants: one to perform a needs assessment and then another to develop and implement projects based on the results. The Columbia team provided technical assistance in a variety of ways, including site visits, workshops and correspondence. Teams tested program models and reported findings both to local policymakers and in international fora. Collaboration with government and community leaders helped facilitate progress at all stages. At the PMM Network Results Conference in 1996, the teams decided to continue their work by forming the Regional PMM (RPMM) Network, an entirely African entity. |
PRELIMINARY STUDIES Research at Ahmadu Bello University Teaching Hospital (ABUTH), Zaria, Nigeria, showed delay in treating women with obstetric complications and highlighted multiple contributing factors. INTERVENTIONS In response, a surgical theater was restored to working order, the maternity ward renovated, resident physicians trained in obstetrics and an emergency drug pack system instituted. A system of blood donation from families of women attending antenatal clinics was introduced. Later, community interventions focused on improving access and reducing delay in seeking care. RESULTS Mean admission-to-treatment interval was reduced by 57%, from 3.7 h in 1990 to 1.6 h in 1995. The proportion of women treated in less than 30 min increased from 39% in mid-1993 to 87% in late 1995. Case fatality rate (CFR) among women with major obstetric complications fell from 14% in 1990 to 11% in 1995. The annual number of women with complications seen, however, declined from 326 in 1990 to 65 in 1995. COSTS Cost of material improvements was approximately US$135,000, of which 65% was provided by government. An additional $8000 per year in new staff salaries was paid by the government. CONCLUSIONS Hospital obstetric services can be improved and government can be mobilized to contribute. Treatment delay and obstetric CFR can be reduced. Deteriorating economic conditions, however, may diminish utilization of services despite improvements. |
PRELIMINARY STUDIES Facility reviews and focus group discussions revealed several factors at the district hospital contributing to maternal deaths in Ekpoma District, Nigeria. INTERVENTIONS In response, the necessary equipment for the operating theater, labor suite and laboratory were repaired or purchased. A blood bank and standby generator were repaired. Drugs and consumable material were purchased and a revolving fund established. Refresher courses were held for medical officers, nursing staff and laboratory technicians. At a later stage, community interventions focused on improving access and reducing delay in seeking care. RESULTS The number of cesarean sections performed increased from zero in 1990-1991 to between seven and 13/year in the period 1992-1995. The number of women with major obstetric complications seen at the hospital increased from seven in 1990 (5% of obstetric admissions) to a high of 29 (20% of obstetric admissions) in 1993. These gains were not sustained, however. In 1995, only 12 women with complications (9% of obstetric admissions) were seen. COSTS The cost of improvements was approximately US $12,800, of which 41% was paid by the government and the rest by the project. CONCLUSIONS Improving obstetric care at the district hospital can increase use by women with complications. However, sociopolitical and economic problems can hamper success. |
PRELIMINARY STUDIES A facility review and focus group discussions revealed poor capacity to manage obstetric complications. INTERVENTIONS In response, a physician with obstetric skills was posted, and a second physician was trained. Courses in life-saving obstetric skills were held for nurses and midwives. An unused operating theater was made functional with simple modifications. A generator and blood bank were installed. Drugs and supplies were made available through a revolving fund. Subsequently, community interventions focused on improving utilization. RESULTS The number of women seeking treatment for major obstetric complications at the district hospital increased from 31 in 1990 to 98 in 1995, while the case fatality rate (CFR) among these women dropped from 32% to 5%. Cesarean sections increased from two in 1990 to 38 in 1995. In 1995, 444 abortion-related procedures were performed--almost all of them for unwanted pregnancy--compared with only 22 in 1990. COSTS The cost of material improvements and training was approximately US$39,000, of which 46% was from project funds, 41% from non-governmental organizations and 13% from government. CONCLUSIONS Women with obstetric complications will seek hospital care if services are available. Government hospital services can be improved by building on existing resources. Obstetric CFR can be dramatically reduced. The need for safe abortion services, which are currently illegal in Sierra Leone, is demonstrated. |
PRELIMINARY STUDIES A 1991 inventory at the State Hospital, Ota, in Ogun State, Nigeria, showed inadequate surgical equipment, drugs, blood and power supply. A time-motion study indicated substantial delays in receiving obstetric care. INTERVENTIONS In 1994, medical officers and midwives were given refresher courses in emergency obstetric skills. In 1995, the surgical theater, labor ward and laboratory were provided with the necessary supplies and equipment. A reliable electrical supply was set up, but problems were encountered in establishing blood services. Subsequent community interventions focused on improving access and reducing delay in seeking care. RESULTS The annual number of women with complications seen, which had been declining--from 123 in 1992 to 55 in 1994--increased to 91 in 1995. Case fatality rate (CFR) due to major direct obstetric complications did not change appreciably, i.e. it was 6.6% in 1995, as compared with 7.3%, 8.3% and 7.3% for the years 1992-1994, respectively. COSTS The cost of hospital improvements was approximately US $46,000. CONCLUSIONS The facility improvements were completed only recently in mid-1995. It is hoped that improved services will result in reductions in CFR and motivate more women with complications to seek hospital care, despite difficult economic conditions prevailing in Nigeria. |
PRELIMINARY STUDIES An inventory at Birnin Kebbi Specialist Hospital in Nigeria revealed poor capacity in emergency obstetric care and excessive delays. Focus group discussions among community members emphasized poor services as a reason for not seeking care. INTERVENTIONS During 1992-1993, several specialist obstetricians visited the hospital. They provided training to general physicians and midwives and care to patients, and stayed approximately five weeks each. An obstetric first-aid box with essential drugs and supplies was introduced. Midwives were trained in recognition and management of obstetric complications. Subsequent community interventions focused on improving access and reducing delay in seeking care. RESULTS The number of cesarean sections performed increased from 101 in 1990 to 131 in 1995. The case fatality rate among women with complications dropped from 22% to 5% over the same period. The number of women with complications seeking treatment in the hospital increased from 200 in 1990 to 227 in 1994, and then declined to 152 in 1995. COSTS The cost of improvements at the hospital was approximately US$12,300, with 10% contributed from government sources. CONCLUSION Improving hospital obstetric services is feasible and affordable. Better services, however, may not be sufficient to reverse declining hospital utilization related to worsening economic conditions. |
PRELIMINARY STUDIES Inventory and observations at Juaben Teaching Health Center (JTHC) revealed an inability to treat obstetric complications. Women with complications needed to be referred to other institutions, resulting in delays. INTERVENTIONS During 1993 and 1994, an operating theater and blood bank were established and equipped, the maternity refurbished, and a revolving drug fund created. A physician was posted and trained in obstetrics, and midwives were trained in life-saving skills. A running water supply was established. Subsequent community interventions focused on improving access and reducing the delay in seeking care. RESULTS The number of women with complications coming for care increased almost three-fold, from 26 in 1993 to 73 in 1995 and the proportion of these who were referred for treatment dropped from 42 to 14%. Surgical obstetric procedures performed at JTHC increased from 23 in 1993 to 90 in 1995. Midwives performed 32% of manual removals, 58% of vacuum extractions and 98% of episiotomy repairs. No deaths occurred among the women treated. COSTS The cost of improvements was approximately US $30,000, mostly for equipment and supplies. Forty percent came from project funds, 36% from non-governmental organizations (NGOs), 15% from government and 9% from community members. The salary of the new physician cost an additional $4700 annually. CONCLUSIONS Modest improvements can increase the provision and utilization of emergency obstetric care. Collaboration with NGOs, government and the community can be beneficial. |
PRELIMINARY STUDIES Focus group discussions in Akwapim South District, Ghana, highlighted a lack of accessible health facilities as an important factor contributing to maternal deaths. INTERVENTIONS In 1991, a health center was established by rennovating an abandoned warehouse. The center was equipped with beds, a refrigerator, a safe water supply, drugs and supplies. With the posting of a community health nurse from the Ministry of Health (MOH), it began functioning as a maternal and child health/family planning clinic. A senior nurse-midwife was posted in 1992 and obstetric services were offered. Starting in 1994, community interventions focused on reducing the delay in seeking care. RESULTS An average of nine women with major obstetric complications were seen each 12-month period between 1992 and 1995. Many minor complications and non-obstetric ailments were also treated. COSTS The material costs of establishing the health center amounted to US $12,550: 47% came from the community, 43% came from non-governmental organizations (NGOs), 7% from PMM and 3% from MOH. The costs of staff salaries were paid by MOH. CONCLUSIONS It is possible to mobilize communities, government and NGOs to help provide emergency obstetric services. If emergency obstetric services are available, women will use them, even before the launching of community information and education campaigns. |
PRELIMINARY STUDIES Facility review at the secondary hospital in Makarfi revealed a lack of drugs and skilled personnel and delays in treating and referring women with obstetric complications. INTERVENTIONS In 1994, maternity facilities were renovated, a revolving drug fund was introduced, midwives were trained and an ambulance was restored to service. Attempts to secure a physician with skills in treating obstetric emergencies were unsuccessful. Prior to these activities, obstetric services at the referral hospital were improved. Community interventions focused on improving utilization by women with complications. RESULTS Between 1990 and 1995, substantial increases occurred in antenatal attendance (2517 to 5565 per year) and deliveries (325 to 1952 per year). The number of women with complications seeking care at this facility, however, dropped from 85 in 1990 to 28 in 1995. Referrals to higher level facilities increased from four in 1990 to 17 in 1995. COSTS The cost of the interventions was approximately US $32,000. Ninety-eight percent was paid by the government and 2% by PMM. CONCLUSIONS Improving the quality of maternity services can increase utilization by women with uncomplicated pregnancies. However, utilization of emergency services appears to be influenced by other factors, such as the ability to treat obstetric complications and prevailing economic conditions. |
PRELIMINARY STUDIES Of 11 facilities providing obstetric services in the Njikoka Local Government Area, four were private, for-profit institutions. Focus group discussions in seven communities revealed a preference for private facilities due to flexible payment schedules, proximity, reliable availability of a medical doctor and poor quality government services. Each of the private facilities had one doctor and one midwife and the bulk of patient care was performed by health aides with no formal midwifery training. INTERVENTIONS In 1992, 15 aides from the private facilities were trained in the recognition and management of obstetric complications. The training consisted of one week of classroom instruction and two weeks of practical training in local missionary hospitals. RESULTS Improvements were assessed by a written test. The percent of trainees obtaining a passing test mark increased from 33% (pre-training) to 61% (post-classroom) to 77% (post-practicum). COSTS The cost of this intervention was approximately US $18,000. CONCLUSIONS Auxiliaries' skills can be improved with classroom and practical training. The involvement of private sector institutions is important where they provide a substantial proportion of emergency obstetric services. However, maintaining improvements requires sustained efforts. |
PRELIMINARY STUDIES Focus group studies and facility reviews revealed poorly equipped and under-utilized emergency obstetric facilities in two rural communities in Cross River State, Nigeria. INTERUENTIONS: Beginning in late 1992, state and local government leaders were contacted to sensitize them to the problem of maternal mortality and enlist their cooperation in activities to upgrade obstetric services. RESULTS A maternal health unit was created in the State Ministry of Health. Government officials were instrumental in upgrading hospital and polyclinic facilities, correcting staff shortages, repairing an ambulance, establishing a blood bank, granting leave to midwives for participation in skills workshops and facilitating dissemination of information. COSTS Nineteen visits to government officials required approximately 33 person-days of the team's time. Transportation, communication and per diems were also paid. Government contributed a considerable amount in staff time and provided venues for workshops. CONCLUSIONS A great deal can be achieved to improve emergency obstetric services through collaboration with government. Support and cooperation of government leaders can also enhance sustainability of interventions. |
PRELIMINARY STUDIES A study of institutions in Cross River State, Nigeria, revealed poor storage facilities and inadequate availability of blood. Focus group discussions highlighted people's fears and misconceptions about blood donations as important factors contributing to the problem. INTERVENTIONS Blood facilities were upgraded in the teaching hospital and a secondary institution. Fifteen community mobilization sessions to improve attitudes and stimulate voluntary blood donation were conducted over three months in 1994. Non-cash incentives were offered, including certificates of honor, free blood screening and assurance of priority if a family member needed blood. RESULTS Blood donations to the teaching hospital maternity increased from 40 pints per month in 1991 to 81 in 1994. However, national strikes interrupted service and the increase was not sustained. At the secondary facility, blood transfusions increased from 14 in 1991 to 35 in 1995, with 100% of donations from volunteers. COSTS The cost of community mobilization for blood donation was US $17,531. CONCLUSIONS Increasing voluntary blood donations through non-cash incentives and community mobilization is possible. No conclusions may be drawn regarding sustainability, however. |
PRELIMINARY STUDIES A situation analysis of Bo Government Hospital found there was no functioning blood bank. Focus group discussions revealed negative perceptions of community members regarding donation and transfusion of blood. INTERVENTIONS In late 1992, the hospital laboratory was refurbished: a refrigerator, freezer, reagents and other equipment and supplies were provided and technicians were trained. Consequently, small quantities of blood could be stored and rapidly transfused. Other improvements to obstetric services in the hospital were undertaken at the same time. Afterwards, community education to encourage blood donation was done. RESULTS The number of units of blood drawn increased from 304 to 501 (65%) from 1992 to 1993 (the first intervention year); the number actually transfused increased from 296 to 452 (53%). By the third intervention year (1995), the annual numbers of units drawn and transfused were 469 and 422, respectively. Case fatality rates for major obstetric complications were lower in all post-intervention years (10% in 1993, 7% in 1994, 10% in 1995) as compared to 1992 (13%). COSTS Cost of improvements to blood services in the hospital was nearly US $10,000, of which 77% was used to purchase supplies and equipment. CONCLUSIONS Even in settings with irregular electrical supply, storage of small quantities of blood is possible. Ready availability of blood may contribute to improved quality of obstetric care and improved survival among patients. |
PRELIMINARY STUDIES A facility review at Enugu-Ukwu General Hospital in Anambra State revealed limited blood transfusion and no blood storage capabilities. Focus groups indicated fears and misconceptions in the population regarding risks of blood donation and transfusion. INTERVENTIONS In 1994, a blood bank was established, including a refrigerator, backup generator, reagents and supplies. Refresher training was provided to the laboratory technologist. A public education campaign was launched one year later to encourage blood donation and dispel fears of transfusion. RESULTS Voluntary blood donations in the hospital increased from zero units before the program to 15 in 1995. Transfusions increased from three in 1993 to 17 in 1995. Eight of the 17 were for obstetric cases. No donations or transfusions occurred until six months after the establishment of the blood bank. Problems encountered in obtaining the cooperation of hospital management may partly explain the delayed response. COSTS The cost of establishing the blood bank was US $8800: 51% material costs and 42% training. CONCLUSIONS Improving the availability of blood at small hospitals need not be very expensive. Community education activities may increase blood donation, but sustained efforts are likely to be required. Ministry of Health (MOH) involvement is important to the success of interventions in government hospitals. |
PRELIMINARY STUDIES Focus group discussions and health facility inventories in a rural district hospital indicated a shortage of basic drugs and supplies. INTERVENTIONS The project procured obstetric drugs and supplies through a commercial source outside normal government channels. They were managed through the standard district system and made available at prices calculated to cover costs and an 85% mark-up. Obstetric emergency drug packs were created to ensure 24-h availability. Patients from the study area unable to pay at time of treatment were given credit and followed up. RESULTS No shortages of obstetric drugs occurred after interventions were introduced. Prices of drugs to obstetric patients ranged from 46% to 68% of the prices charged by the government hospital and private pharmacies. Of 26 obstetric patients from the study area who received drugs in 1993, 12 paid in full and nine paid in part. The total amount billed during 1993 was US $2537, of which $1451 was recovered. COSTS Improvements cost approximately $17,500, of which 83% was used for the initial supply of drugs. CONCLUSIONS Cost recovery systems for drugs can help maintain consistent drug supplies at health facilities. Careful pricing is critical. For the system to be sustainable, the mark-up must cover defaulters. |
PRELIMINARY STUDIES Data on obstetric complications are the basis of monitoring maternal mortality interventions in the PMM Network. A review of recordkeeping procedures at 10 facilities in the study area revealed that information on obstetric complications was often inconsistent or missing. Some hospital records were not designed to collect such information at all. INTERVENTIONS In 1992, registers at facilities were revised to collect information on complications and time of treatment. Doctors, nurses, midwives and clerks were trained to record, compile and analyze data. Monitoring and supervisory mechanisms were also set up. RESULTS Recordkeeping has improved. Data collection and analysis have been regular and timely. Doctors have begun using the data for morning meetings. Nurses and midwives compile monthly summaries of data showing complications by type. Two other districts outside the research area have adopted the reporting system and it is possible that facilities in the whole region will follow suit. COSTS The cost of improving recordkeeping at the 10 healthcare facilities was approximately US $2543, with 85% coming from project funds. CONCLUSIONS Existing recordkeeping systems can be modified to collect data necessary to monitor maternal mortality interventions. Staff training and monitoring visits are important to success. |
PRELIMINARY STUDIES Focus group discussions revealed poor roads, few vehicles, and high transportation costs as major causes of delay in deciding to seek and in reaching emergency obstetric care. INTERVENTIONS Beginning in September 1992, a four-wheel drive vehicle was posted at Bo Government Hospital (BGH). Motorbikes to summon the vehicle were posted at the eight project-area primary health units (PHUs). Problems with the motorbike system (accidents, breakdowns) led to the installation of a radio system linking the hospital, PHUs and the referral vehicle. These interventions were complemented by community education activities and earlier improvements in the health facilities. RESULTS The number of women with major obstetric complications arriving at BGH from the project area increased from 0.9 to 2.6 per month, while case fatality rate dropped from 20% to 10%. In the post-intervention period, approximately half of women with complications from the project area utilizing BGH came by project vehicle. The mean time from the vehicle being called by the PHU to the patient's arrival at BGH was 3.1 h. Case fatality rate did not differ by whether or not women came by project vehicle. COSTS Cost of the transport and communication intervention was approximately US $75,000, including: vehicle, $27,500; radios, $12,500; motorbikes, $27,000. CONCLUSIONS Improvements in transport can help greater numbers of women with complications reach hospitals and may improve their chances of survival. |
PRELIMINARY STUDIES Focus group discussions with community members in Nsawam District, Ghana, identified poor roads, scarce transport and exorbitant fees for emergency transport as barriers to reaching the district hospital for treatment of an obstetric complication. INTERVENTIONS To minimize delay in the event of a complication, a maternity waiting home (MWH) was established in Nsawam in 1994. One ward of an abandoned hospital was renovated and furnished for this purpose. The objective was to encourage women at high risk of obstetric complications to move to the MWH so they could be transferred to the hospital when labor began. RESULTS Of 25 women referred to the MWH by health personnel over 12 months, only one complied, for one night. Focus group discussions with community members and hospital staff later revealed that cost and hardship of staying away from home, absence of health personnel, distance from hospital, desolate surroundings and lack of perceived need were reasons for poor utilization. COSTS The intervention cost approximately US $10,500, shared approximately equally between the project and government. The main government contribution was the building. CONCLUSIONS It is important to consult potential users not only to identify problems, but also to identify appropriate solutions. Careful 'market research' should be done before launching interventions. |
PRELIMINARY STUDIES Focus group discussions and a village case study in Kebbi State revealed delay in the transport of women with obstetric complications. Among contributing factors identified were shortages of vehicles and fuel, and unwillingness of drivers to transport women at affordable fares. INTERVENTIONS The cooperation of the local transport workers union was enlisted to address the situation. In 1993, drivers were sensitized and trained and a revolving emergency fuel fund was established. Prior to these activities, emergency obstetric services at nearby facilities had been upgraded. RESULTS Over two years, 29 women with obstetric complications were transported. Of these, only one died. Mean cost of transport to patients was US $5.89. Mean time from the onset of complications to treatment was 9 h. Substantial numbers of non-obstetric patients in need of emergency care were also transported. Although defaulting eventually resulted in depletion of the fuel fund, the reimbursement system had become sufficiently well-established that most drivers no longer requested funds in advance. COSTS Cost of the transport intervention was US $268, with 72% coming from project funds. CONCLUSIONS Improving transport to emergency care does not necessarily require ambulances. Commercial transport owners and communities can be mobilized to provide affordable emergency transport for women with complications. |
PRELIMINARY STUDIES Twenty-one focus groups and a survey in two rural communities revealed socioeconomic and cultural barriers to utilization of emergency obstetric services. INTERVENTIONS To facilitate the use of services, 20 community educators were trained and an education campaign was conducted beginning in 1994. Educational activities emphasized the need for women with obstetric complications to use obstetric services at two local health facilities and one teaching hospital. Communities were also mobilized to set up loan and transport programs. RESULTS Awareness of obstetric complications increased in both communities and for all complications: increases ranged from 5% (for obstructed labor) to 63% (for hemorrhage). Fourteen of 39 project communities established new loan programs (six communities already had them). Loans were granted in only nine communities. Transport systems were established in nine communities. Referrals to the teaching hospital of women with major obstetric complications from two health facilities in the study area increased from three in 1990 to 11 in 1995 in one community and from four to eight in the other. COSTS The cost of the mobilization activities was approximately US $6500. CONCLUSION Community education and mobilization can help increase awareness of obstetric complications. |
PRELIMINARY STUDIES Focus group discussions and a mini-survey in Kebbi State, Nigeria, revealed poor knowledge of obstetric complications, lack of confidence in health services, and cultural barriers to seeking care. INTERVENTIONS After upgrading services in emergency obstetric facilities, the team began community education to encourage utilization. Messages focused on recognition and need for prompt treatment of complications, and addressed men's role as decision-makers. Beginning in 1992, messages were disseminated through weekly meetings with community opinion leaders, video shows, posters and handbills. RESULTS A post-intervention mini-survey showed knowledge gains of over 30% among women and men. The increase was greatest (59% increase among women and 55% among men) on the need for prompt care for women with obstetric complications. However, utilization of emergency obstetric services did not increase. At Maiyama Maternity Center, referrals declined from 18 in 1992 to four in 1995. At Jega Health Center, referrals remained relatively stable at 20-30/year, but the number of women with major obstetric complications treated declined from 234 in 1992 to 136 in 1995. At Birnin Kebbi Specialist Hospital, the referral center, the number of women with complications treated declined from 200 in 1990 to 152 in 1995. COSTS The cost of community education was approximately US $9500, of which 15% was contributed by the community. CONCLUSIONS Increased awareness of the signs of obstetric complications and the need for prompt treatment among community women and men did not result in greater utilization of emergency obstetric services at the facilities studied. |
PRELIMINARY STUDIES Focus group studies in the Ashanti region showed that people avoided utilizing health facilities because of lack of confidence in the services and concern about the availability of drugs and supplies, among other reasons. INTERVENTIONS After services at the health center were upgraded, community education activities began in early 1994. These activities were carried out through existing mechanisms--e.g. Ministry of Health (MOH) outreach workers and village health workers, public health nurses and midwives, and village health committees. They addressed a variety of audiences, including women's and church groups, emphasizing early recognition and treatment of obstetric complications, and the improved availability of services. RESULTS The number of women with obstetric complications admitted to the health center rose from 26 in 1993 to 73 in 1995. It was the impression of the health center staff that women were also coming for treatment more promptly. COSTS The cost of this intervention was US$1950. This was mostly project funds, with the government and community together contributing approximately one-fifth. CONCLUSIONS Once services are available, community education and information activities can enhance utilization. The cost of such activities can be reduced, and sustainability promoted, by involving MOH personnel and community groups. |
PRELIMINARY STUDIES An appraisal of government health facilities documented generally poor conditions, a shortage of drugs and supplies, and inadequate skills among staff. Focus group discussions in the community revealed a lack of knowledge about obstetric complications and difficulty in reaching health facilities. INTERVENTIONS Improvements were made in services at primary health units (PHUs) through May 1993. Community motivators were trained in June and provided with bicycles and raingear in September 1993. Among their duties were community education, formation of village action groups for emergency transport and facilitation of referral for women with obstetric complications. RESULTS Between August 1992 and December 1995, women with major complications seen at the PHUs increased from nine to 16 per month, with fluctuations due to disruptions in services and civil strife. The proportion of women with complications who were referred by the community motivators diminished over time, dropping from 24% to 1% over the first two years (1993-1994) and then recovering somewhat to 10% in the final period of observation. COSTS The cost of this intervention was approximately US$5082, with about half coming from the project and the rest from the government (44%) and the community (8%). CONCLUSIONS The project improved utilization of the PHUs by women with complications, indicating that once services are of acceptable quality, many women will use them. The contribution of the motivators was positive, though smaller than expected. |
PRELIMINARY STUDIES Focus group discussions revealed delay in seeking and reaching emergency care as a factor contributing to maternal mortality in Njikoka Local Government Area. INTERVENTIONS After obstetric services at local hospitals had been upgraded, 48 community contact persons were recruited. They were trained to improve community awareness of obstetric complications and to facilitate referral of women with complications. They were responsible for establishing links with pregnant women, improving access to transport, and mobilizing the community to donate blood. RESULTS Over 18 months, community contact persons referred 18 women with major obstetric complications for treatment at health facilities. Fifty-two pregnant women were referred for other reasons. A total of 129 women were assisted by the community contact persons. This number declined sharply over time, from 60 in the first half of 1994 to 16 in the first half of 1995. COSTS The community contact persons intervention cost approximately US $635, with 64% coming from project funds, 25% from government and 11% from the community. CONCLUSIONS Community contact persons can perform a valuable role in facilitating referral of women with obstetric complications and supporting health education activities. |
PRELIMINARY STUDIES Focus group discussions revealed that a lack of funds often contributed to a delay for women receiving treatment for obstetric complications. INTERVENTIONS Improvements were made in health facilities and transport, then, beginning in 1992, meetings were held to mobilize communities to establish emergency loan funds. Per capita levies were set and repayment was enforced by the most paramount chief of the area. Funds were managed by existing village development committees and loans were granted to women who could not pay hospital bills immediately. RESULTS Of the six chiefdoms contacted, two successfully established loan funds. Utilization of Bo Government Hospital by women with complications from the two chiefdoms with loan funds increased from five in 1992 to 12 in 1993. Utilization from other chiefdoms remained basically unchanged. Of women from loan fund chiefdoms, half paid their hospital bills in full and one-third paid in part. COSTS The cost of community mobilization was about US $472. CONCLUSIONS The establishment of loan funds depended on strong community leadership and required substantial mobilization efforts. Where community loan funds are established, utilization of emergency obstetric care may increase. |
PRELIMINARY STUDIES Focus group discussions in the community identified difficulties in paying for transport as a major barrier to seeking and reaching emergency care for obstetric complications. INTERVENTIONS After emergency obstetric services in local health facilities had been upgraded, the clans in Ekpoma were mobilized in 1995 to set up emergency loan funds for women with complications. Funds were managed entirely by the clans, with ongoing monitoring and supervision by project staff. Two percent simple interest was charged. RESULTS Of the 13 clans contacted, 12 successfully launched loan funds. Total donations amounted to US$793, of which four-fifths were contributed by the community. In the 1st year of the operation, 456 women/families requested loans (ranging from US$7 to US$15), and 380 (83%) were granted. Three-hundred and fifty-four (93%) loans were repaid in full. In addition to being used for transport, loans were used to help pay for drugs, blood and hospital fees. COSTS The cost of establishing the loan fund was US$1360, including initial donations to the loan funds. The PMM project paid 55% of the total. CONCLUSIONS With relatively little outside financial input, communities can set up and administer loan funds for emergency obstetric transport and care. However, sustaining the funds over the long term requires continuing effort and involvement with the communities. |
PRELIMINARY STUDIES Focus group discussions and a community survey indicated that inadequate funds and transport caused delays in deciding to seek emergency obstetric care and in reaching facilities. INTERVENTIONS Following improvements in the quality of obstetric services, a community loan program was established in early 1995. Community members determined its features: compulsory contributions; community administration; loans for obstetric complications only; no interest; a 6-month grace period; and 24-month repayment. A transport system was also established, in which private vehicle drivers agreed to respond to calls for emergency transport and charge a set fee. RESULTS The equivalent of US $20,500 was collected from 81 annual and 2273 one-time contributors. Eighteen loans were approved in 9 months. Repayment data are not yet available. For the transport system, 23 drivers pledged permanent participation and 58 pledged to take part in 6-month rotations. They transported 18 women. COSTS The cost of these interventions was $3409 for the loan fund and $2272 for the transport system. Sixty percent of the cost was paid by the community and the rest by the PMM project. CONCLUSIONS Community-managed loan and transport systems for women with obstetric emergencies can be established and may contribute to reducing delay in obtaining emergency obstetric care. |
One element of the operations research carried out by the Prevention of Maternal Mortality (PMM) teams in West Africa was a test of the indicators they used to monitor and evaluate their efforts. A small number of process indicators directly related to the PMM interventions in the health facilities and in the communities was selected and monitored. An examination of the teams' experiences in gathering and using these indicators shows: (i) that they are extremely useful for project design and management; (ii) that the necessary data are obtainable; (iii) that staff need training to gather, interpret and use the data; (iv) that monitoring and evaluation systems must be designed so that managers will use the information; and (v) that data on the costs of interventions can be tracked and are useful for evaluation and replication. |
For eight years, the Prevention of Maternal Mortality (PMM) Network in West Africa designed, implemented and evaluated projects to reduce barriers to care for women with obstetric complications. Many valuable lessons were learned concerning program development and implementation. A multidisciplinary approach enabled the Network to address the various aspects of the problem; collaboration between teams encouraged the sharing of resources and experiences; and collaboration with government and communities enhanced project sustainability. Capacity building through long-term, systematic technical support resulted in a Network of proficient researchers. Existing human and material resources were utilized to enhance the feasibility and sustainability of projects--a strategy that could be adopted in other developing countries. Cost-tracking was used as a management and evaluation tool. The collective experience of the Network offers guidance for program planners and researchers working on maternal mortality in developing countries. |
The Prevention of Maternal Mortality (PMM) Network designed and tested projects for reducing maternal deaths. The focus was on improving the availability, quality and utilization of emergency obstetric care (EmOC) for women with serious complications. Teams' projects included interventions in health facilities (to improve skills and services and reduce delays in treatment) and in communities (to address lack of transport, funds and information concerning obstetric complications). The teams' results, reported in this volume, offer several lessons for program planners. Despite difficult conditions in the project countries, the teams demonstrated that it is almost always possible to make improvements in the delivery of EmOC. Their work shows that EmOC can be improved not only by concentrating on hospitals and physicians, but also by focusing on peripheral facilities and other qualified staff. The teams' findings regarding utilization of EmOC suggest that more people utilize services when they know them to be functioning well. Community efforts, including education and mobilization, have a role to play in improving utilization once services are in place. Improving EmOC need not be costly, because in many areas the necessary facilities exist and staff are already in place. |
The experience of the PMM Network offers several insights for donors seeking to work on maternal mortality, particularly in Africa where the risk of maternal death is highest. First, the projects provide evidence that multidisciplinary operations research can result in the development and implementation of successful, relatively low-cost interventions. In addition, the work of PMM highlights the need for greater donor funding to build and support partnerships--to include researchers, communities and policymakers--that enhance sustainability of efforts. Finally, the work of the teams confirms the benefits of donor commitment to building local capacity. |
Up to now, the carcinogenicity of a substance, i.e., its cancer-causing effect, has been usually determined with the help of extensive animal testing. The in ovo carcinogenicity assay (IOCA) has been suggested as a rapid and inexpensive, non-animal, method for carcinogenicity testing and for experimental studies on mechanisms of carcinogenesis. The substance to be tested is injected into a fertilized ovum. No later than four days before the hatching date, the embryos are released, and the liver is removed for identifying the effect of hepatocarcinogens. Histological, cytological and molecular biological alterations of the embryonic liver have been induced with a variety of chemical carcinogens. After sufficiently high doses of hepatocarcinogens, tubular structures predominate in the liver and replace the normal trabecular pattern. The cell and nuclear size of the hepatocytes in embryonic liver is severely increased after exposure to chemical carcinogens over a wide dose range including doses that fail to elicit cytotoxicity in the embryonic liver. |
We have cloned and characterized a human gene encoding TP2 (telomerase-associated protein 2), a protein with similarity to reverse transcriptases and the catalytic telomerase subunits from Saccharomyces cerevisiae and Euplotes aediculatus. Indirect immunofluorescence revealed that TP2 was localized to the nucleus. Using antibodies to endogenous and epitope-tagged TP2, we found that TP2 was associated specifically with human telomerase activity and the recently identified telomerase-associated protein TP1. Mutation of conserved residues within the reverse transcriptase domain of TP2 severely reduced associated telomerase activity. These results suggest that telomerase is an evolutionarily conserved multisubunit complex composed of both structural and catalytic subunits. |
Promoter-proximal pausing during transcriptional elongation is an important way of regulating many diverse loci, including the human hsp70 gene. Pausing of RNA polymerase can be enhanced by chromatin structure. We demonstrate that activation of hsp70 leads to disruption of transcribed chromatin in front of RNA polymerase. In vivo, disruption of chromatin in the first 400 bp of the transcribed region of hsp70 following heat shock is resistant to the transcriptional inhibitor alpha-amanitin. Disruption of chromatin farther downstream also occurs following activation but is sensitive to alpha-amanitin, suggesting that polymerase movement is needed to disrupt distal portions of the hsp70 gene. In vitro, disruption of transcribed chromatin is dependent on the presence of the human heat shock factor 1 (HSF1) activation domains. These experiments demonstrate that HSF1 can direct disruption of chromatin in transcribed regions. We suggest that this is one of the mechanisms used by HSF1 to facilitate transcriptional elongation. |
Upon infection of human cells, the herpes simplex virus protein VP16 associates with the endogenous cell-proliferation factor HCF. VP16 can also associate with HCFs from invertebrates, suggesting that VP16 mimics a cellular protein whose interaction with HCF has been conserved. Here, we show that VP16 mimics the human basic leucine-zipper protein LZIP, which, through association with HCF, may control cell-cycle progression. VP16 and LZIP share a tetrapeptide motif-D/EHXY-used to associate with human HCF. The LZIP-related Drosophila protein BBF-2/dCREB-A contains this HCF-binding motif, indicating that the LZIP-HCF interaction has been conserved during metazoan evolution. |
In mice, the imprinted Igf2 gene (expressed from the paternal allele), which encodes a growth-promoting factor (IGF-II), is linked closely to the reciprocally imprinted H19 locus on chromosome 7. Also imprinted (expressed from the maternal allele) is the Igf2r gene on chromsome 17 encoding the type 2 IGF receptor that is involved in degradation of excess IGF-II. Double mutant embryos carrying a deletion around the H19 region and also a targeted Igf2r allele, both inherited maternally, have extremely high levels of IGF-II (7- and 11-fold higher than normal in tissues and serum, respectively) as a result of biallelic Igf2 expression (imprint relaxation by deletion of H19-associated sequence) in combination with lack of the IGF2R-mediated IGF-II turnover. This excess of IGF-II causes somatic overgrowth, visceromegaly, placentomegaly, omphalocele, and cardiac and adrenal defects, which are also features of the Beckwith-Wiedemann syndrome (BWS), a genetically complex human disorder associated with chromosomal abnormalities in the 11p15.5 region where the IGF2 gene resides. In addition, the double mutant mouse embryos exhibit skeletal defects and cleft palate, which are manifestations observed frequently in the Simpson-Golabi-Behmel syndrome, another overgrowth disorder overlapping phenotypically, but not genetically, with BWS. |
Previous studies suggest that plectin, a versatile cytoskeletal linker protein, has an important role in maintaining the structural integrity of diverse cells and tissues. To establish plectin's function in a living organism, we have disrupted its gene in mice. Plectin (-/-) mice died 2-3 days after birth exhibiting skin blistering caused by degeneration of keratinocytes. Ultrastructurally, hemidesmosomes and desmosomes appeared unaffected. In plectin-deficient mice, however, hemidesmosomes were found to be significantly reduced in number and apparently their mechanical stability was altered. The skin phenotype of these mice was similar to that of patients suffering from epidermolysis bullosa simplex (EBS)-MD, a hereditary skin blistering disease with muscular dystrophy, caused by defects in the plectin gene. In addition, plectin (-/-) mice revealed abnormalities reminiscent of minicore myopathies in skeletal muscle and disintegration of intercalated discs in heart. Our results clearly demonstrate a general role of plectin in the reinforcement of mechanically stressed cells. Plectin (-/-) mice will provide a useful tool for the study of EBS-MD, and possibly other types of plectin-related myopathies involving skeletal and cardiac muscle, in an organism amenable to genetic manipulation. |
Upon ligand binding, the receptors of the TGFbeta family phosphorylate Smad proteins, which then move into the nucleus where they activate transcription. To carry out this function, the receptor-activated Smads 1 and 2 require association with the product of deleted in pancreatic carcinoma, locus 4 (DPC4), Smad4. We investigated the step at which Smad4 is required for transcriptional activation. Smad4 is not required for nuclear translocation of Smads 1 or 2, or for association of Smad2 with a DNA binding partner, the winged helix protein FAST-1. Receptor-activated Smad2 takes Smad4 into the nucleus where they form a complex with FAST-1 that requires these three components to activate transcription. Smad4 contributes two functions: Through its amino-terminal domain, Smad4 promotes binding of the Smad2/Smad4/FAST-1 complex to DNA; through its carboxy-terminal domain, Smad4 provides an activation function required for Smad1 or Smad2 to stimulate transcription. The dual function of Smad4 in transcriptional activation underscores its central role in TGFbeta signaling. |
The induction of neurite outgrowth by NGF is a transcription-dependent process in PC12 cells, but the transcription factors that mediate this process are not known. Here we show that the bHLH transcriptional repressor HES-1 is a mediator of this process. Inactivation of endogenous HES-1 by forced expression of a dominant-negative protein induces neurite outgrowth in the absence of NGF and increases response to NGF. In contrast, expression of additional wild-type HES-1 protein represses and delays response to NGF. Endogenous HES-1 DNA-binding activity is post-translationally inhibited during NGF signaling in vivo, and phosphorylation of PKC consensus sites in the HES-1 DNA-binding domain inhibits DNA binding by purified HES-1 in vitro. Mutation of these sites generates a constitutively active protein that strongly and persistently blocks response to NGF. These results suggest that post-translational inhibition of HES-1 is both essential for and partially mediates the induction of neurite outgrowth by NGF signaling. |
Mutations that influence lin-12 activity in Caenorhabditis elegans may identify conserved factors that regulate the activity of lin-12/Notch proteins. We describe genetic evidence indicating that sel-10 is a negative regulator of lin-12/Notch-mediated signaling in C. elegans. Sequence analysis shows that SEL-10 is a member of the CDC4 family of proteins and has a potential human ortholog. Coimmunoprecipitation data indicate that C. elegans SEL-10 complexes with LIN-12 and with murine Notch4. We propose that SEL-10 promotes the ubiquitin-mediated turnover of LIN-12/Notch proteins, and discuss potential roles for the regulation of lin-12/Notch activity by sel-10 in cell fate decisions and tumorigenesis. |
The Arabidopsis gai mutant allele confers a reduction in gibberellin (GA) responsiveness. Here we report the molecular cloning of GAI and a closely related gene GRS. The predicted GAI (wild-type) and gai (mutant) proteins differ only by the deletion of a 17-amino-acid segment from within the amino-terminal region. GAI and GRS contain nuclear localization signals, a region of homology to a putative transcription factor, and motifs characteristic of transcriptional coactivators. Genetic analysis indicates that GAI is a repressor of GA responses, that GA can release this repression, and that gai is a mutant repressor that is relatively resistant to the effects of GA. Mutations at SPY and GAR2 suppress the gai phenotype, indicating the involvement of GAI, SPY, and GAR2 in a signaling pathway that regulates GA responses negatively. The existence of this pathway suggests that GA modulates plant growth through derepression rather than through simple stimulation. |
This study explores signal transduction pathways that function during mating and infection in the opportunistic, human fungal pathogen Cryptococcus neoformans. The gene encoding a G-protein alpha subunit homolog, GPA1, was disrupted by homologous recombination. The gpa1 mutant strain was viable but exhibited a defect in mating in response to nitrogen starvation. Additionally, the gpa1 mutant strain failed to induce two well-established virulence factors-melanin synthesis, in response to glucose starvation; and capsule production, in response to iron limitation. As a consequence, virulence of the gpa1 mutant strain was significantly attenuated in an animal model of cryptococcal meningitis. Reintroduction of the wild-type GPA1 gene complemented the gpa1 mutant phenotypes and restored mating, melanin and capsule production, and virulence. Similarly, exogenous cAMP also suppressed the gpa1 mutant phenotypes, restoring mating and production of melanin and capsule. These observations support a model in which GPA1 has a role in sensing diverse environmental signals required for mating and virulence by regulating cAMP metabolism in C. neoformans. |
Genetic analysis was applied to identify novel genes involved in G protein-linked pathways controlling development. Using restriction enzyme-mediated integration (REMI), we have identified a new gene, Pianissimo (PiaA), involved in cAMP signaling in Dictyostelium discoideum. PiaA encodes a 130-kD cytosolic protein required for chemoattractant receptor and G protein-mediated activation of the 12 transmembrane domain adenylyl cyclase. In piaA- null mutants, neither chemoattractant stimulation of intact cells nor GTPgammaS treatment of lysates activates the enzyme; constitutive expression of PiaA reverses these defects. Cytosols of wild-type cells that contain Pia protein reconstitute the GTPgammaS stimulation of adenylyl cyclase activity in piaA- lysates, indicating that Pia is directly involved in the activation. Pia and CRAC, a previously identified cytosolic regulator, are both essential for activation of the enzyme as lysates of crac- piaA- double mutants require both proteins for reconstitution. Homologs of PiaA are found in Saccharomyces cerevisiae and Schizosaccaromyces pombe; disruption of the S. cerevisiae homolog results in lethality. We propose that homologs of Pia and similar modes of regulation of these ubiquitous G protein-linked pathways are likely to exist in higher eukaryotes. |
African trypanosomes such as Trypanosoma brucei undergo antigenic variation in the bloodstream of their mammalian hosts by regularly changing the variant surface glycoprotein (VSG) gene expressed. The transcribed VSG gene is invariably located in a telomeric expression site. There are multiple expression sites and one way to change the VSG gene expressed is by activating a new site and inactivating the previously active one. The mechanisms that control expression site switching are unknown, but have been suggested to involve epigenetic regulation. We have found previously that VSG genes in silent (but not active) expression sites contain modified restriction endonuclease cleavage sites, and we have presented circumstantial evidence indicating that this is attributable to the presence of a novel modified base beta-D-glucosyl-hydroxymethyluracil, or J. To directly test this, we have generated antisera that specifically recognize J-containing DNA and have used these to determine the precise location of this modified thymine in the telomeric VSG expression sites. By anti J-DNA immunoprecipitations, we found that J is present in telomeric VSG genes in silenced expression sites and not in actively transcribed telomeric VSG genes. J was absent from inactive chromosome-internal VSG genes. DNA modification was also found at the boundaries of expression sites. In the long 50-bp repeat arrays upstream of the promoter and in the telomeric repeat arrays downstream of the VSG gene, J was found both in silent and active expression sites. This suggests that silencing results in a gradient of modification spreading from repetitive DNA flanks into the neighboring expression site sequences. In this paper, we discuss the possible role of J in silencing of expression sites. |
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