Biomedical subjects
Publications and source records attributed to J Yeh.
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Our objective is to test the hypothesis that inhibition of mitogen-activated protein (MAP) kinase kinase (MEK) with PD98059 in human luteinized granulosa cells will block epidermal growth (EGF)-stimulated MAP kinase activity and induce apoptosis. Luteinized granulosa cells from human in vitro fertilization aspirates were cultured and treated with the following: (1) vehicle; (2) PD98059; (3) EGF; (4) PD98059 + EGF. Treatment with PD98059 suppressed MAP kinase activity, inhibited MAP kinase phosphorylation by Western blot analysis, blocked nuclear translocation of phosphorylated MAP kinase by confocal microscopy, and increased percentages of subdiploid apoptotic nuclei by flow cytometry. Our data are the first evidence that a relationship may exist between the MAP kinase pathway and control of apoptosis in human luteinized granulosa cells. These results support the hypothesis that suppression of the MAP kinase pathway may lead to apoptosis in these cells.
OBJECTIVE: To assess the expression of messenger RNA (mRNA) for estrogen receptors alpha and beta in the vaginal walls and uterosacral ligaments of premenopausal and postmenopausal women. DESIGN: In vitro experiment. SETTING: Academic research environment. PATIENT(S): Sixteen consecutively seen women who underwent hysterectomy. INTERVENTION(S): Samples of anterior vaginal walls and uterosacral ligaments were obtained during hysterectomy. MAIN OUTCOME MEASURE(S): Reverse transcriptase-polymerase chain reaction analysis for estrogen receptor mRNA. RESULT(S): Messenger RNA transcripts for estrogen receptor alpha were present in all samples of vaginal walls (16/16) and uterosacral ligaments (16/16). Estrogen receptor beta mRNA was detected in all samples of vaginal walls from premenopausal women (12/12) but in none of those from postmenopausal women (0/4). Estrogen receptor beta mRNA was found in most samples of uterosacral ligaments from premenopausal women (10/12) and in some of those from postmenopausal women (2/4). CONCLUSION(S): Estrogen receptors alpha and beta were expressed in the vaginal walls and uterosacral ligaments of premenopausal and postmenopausal women. Estrogen receptor beta was absent from the vaginal walls of postmenopausal women.
OBJECTIVE: To investigate the role of transforming growth factor-beta (TGF-beta) in the regulation of human fetal ovarian development. DESIGN: Reverse transcription-polymerase chain reaction and comparative immunohistochemical analysis of the localization and staining intensity of TGF-beta1, TGF-beta2, and TGF-beta3, and of their receptors. SETTING: Academic research environment. PATIENT(S): Human fetal ovaries were obtained from terminated normal intact pregnancies at 11-24 weeks' gestation. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Messenger RNA analysis and protein expression of TGF-beta isoforms and their receptors in human fetal ovaries at 11-24 weeks of gestational age. RESULT(S): Messenger RNAs for the three TGF-beta isoforms and the two TGF-beta receptors were demonstrated in all the developmental ages studied: 11, 14, 18, 20, and 22 weeks of gestation. During the first trimester, immunohistochemical analysis for TGF-beta1, TGF-beta2, and TGF-beta receptor type I revealed homogeneous light staining of the ovary. Staining for TGF-beta3 and TGF-beta receptor type II was predominantly in the oocytes. During the second trimester, staining for all three TGF-beta isoforms and both receptors was predominantly in the oocytes. In addition, for receptor types I and II, staining was observed in the pregranulosa cells. CONCLUSION(S): Our findings support the hypothesis that expression of the TGF-beta system changes from the first to the second trimester of fetal development and may have an autocrine and/or paracrine regulatory role during ovarian development.
The regulation of casein gene expression by both PRL and glucocorticoids has been a well studied paradigm for understanding how the signaling pathways regulated by these two hormones interact in the nucleus. Previous studies have demonstrated that the downstream effectors of these pathways, signal transducer and activator of transcription 5 (STAT5) and the glucocorticoid receptor (GR), are associated via protein-protein interactions and act synergistically to enhance beta-casein gene transcription. Indirect immunofluorescence microscopy was used to demonstrate that PRL-activated STAT5 can translocate GR into the nucleus, and that ligand-bound GR can translocate STAT5 into the nucleus. This provided further support of an interaction between the two proteins. To better understand the mechanism of transcriptional synergy between STAT5 and GR, experiments were performed in cells transiently transfected with STAT5 alone or with STAT5 and GR. GR cotransfection enhanced the DNA-binding activity of STAT5 without affecting STAT5 protein levels. The enhancement of STAT5 DNA binding by GR resulted in the formation of a complex that exhibited prolonged DNA binding after PRL treatment. This was correlated with increased STAT5 tyrosine phosphorylation, suggesting that GR enhances STAT5 DNA binding by modulating the rate of STAT5 dephosphorylation. In contrast, cotransfection of the estrogen receptor resulted in an overall decrease in STAT5 tyrosine phosphorylation, without changing the kinetics of dephosphorylation. Enhancement of STAT5 activity by GR is, therefore, one component of the transcriptional synergy exhibited by STAT5 and GR at the beta-casein promoter and is an example of how transcription factors at a composite response element may modulate each other's activity.
The reasons for the rapidly increasing prevalence of diabetes (NIDDM) among Alaskan Eskimos are only partly understood. This study examines the association of fatty acid metabolism in 68 Alaskan Eskimos with NIDDM or impaired glucose tolerance (IGT) and 386 with normal glucose tolerance > 24 years old. The prevalence of NIDDM was 12% and IGT was 18% in those > 54 years of age and in those < 55 years of age was 3.7% and 3.0%, respectively. Those with abnormal glucose tolerance had lower concentrations of some omega-3 fatty acids (FAs 18:3 omega-3, 20:5 omega-3) and some omega-6 FAs (18:3 omega-6, 20:3 omega-6, 22:4 omega-6) and higher concentrations of palmitic acid (16:0) and oleic acid (18:1 omega-9) than the normo-glycemic participants. These data provide evidence that glucose intolerance and insulin resistance are associated with a deviation from a traditional diet of fish and marine mammals (high in omega-3 FAs and low in saturated fats) to commercial foods (low in omega-3 FAs and high in saturated fats). The low plasma concentrations of the long-chain omega-6 FAs in the glucose impaired may reflect a defect in desaturase activity.
Lesch-Nyhan syndrome is a hereditary disorder of purine metabolism causing overproduction of uric acid and neurological problems including spasticity, choreoathetosis, mental retardation, and compulsive self-mutilation. The syndrome is caused by a defect in the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT), which converts guanine and hypoxanthine to the nucleotides GMP and IMP. There is evidence that the neurological problems are due to an adverse effect of the HPRT deficiency on the survival and/or development of dopaminergic neurons, specifically. Here we report that HPRT-deficient PC12 mutants that have a normal or near normal dopamine content (55-97% of that of wild-type cells) fail to undergo neuronal differentiation induced by nerve growth factor (NGF) when the de novo pathway of purine synthesis is partially inhibited. However, nerve growth factor-induced differentiation is near normal under these conditions in PC12 HPRT-deficient mutants containing much lower dopamine levels (<8% of that of wild type cells), indicating a neurotoxic effect of the endogenous dopamine in the mutants. The degree of inhibition of the de novo pathway of purine synthesis was the same in both classes of HPRT-deficient mutants. Expression of BCl-2 in a PC12 mutant that has a normal dopamine content allowed partial NGF-induced differentiation suggesting that the apoptotic pathway might be involved in the failure of differentiation when the de novo pathway of purine synthesis is partially inhibited.
Community mortality surveillance for 1984-1988 was conducted by researchers of the Strong Heart Study, which examined the incidence, prevalence, and risk factors of cardiovascular disease in three American Indian populations, aged 45-74 years, in Arizona, Oklahoma, and South/North Dakota. All-cause and cardiovascular disease mortality rates were determined through the use of death certificate data. Cardiovascular disease deaths were confirmed by independent systematic review of medical records. In all three populations, men had higher all-cause and cardiovascular disease mortality rates than did women. Oklahoma exhibited slightly lower 5-year, age-adjusted, all-cause mortality (96/1,000) than did Arizona (107/1,000) and South/North Dakota (114/1,000). The leading cause of death among both sexes in Oklahoma and in South/North Dakota was cardiovascular disease. Diabetes mellitus led among Arizona women. The other major causes of death were cancer, liver disease including cirrhosis, and injury. When compared with the rates in each state, average annual all-cause mortality rates were higher for the American Indian populations in almost every age group. The all-cause annual mortality rates in the three Indian populations were close to rates in the US black population and higher than the rates of the entire US population and of US whites. This trend was amplified in the 45- to 64-year age group. Only in the 65- to 74-year age group did mortality rates in the Indian population approach those of the US population. Cardiovascular disease mortality rates were close to the US averages in Arizona and Oklahoma, but they were more than two times higher in South/North Dakota among those between 45 and 64 years of age. Thus, American Indians in Arizona, Oklahoma, and South/North Dakota exhibit high all-cause mortality rates. In particular, the South/North Dakota population cardiovascular disease death rate appears to present a potential target for community-based programs to intervene on known risk factors to promote healthy lifestyles.
Cadaveric osseoligamentous lower cervical spines (C2-C7) are often used in the investigation of spinal biomechanics in vitro. Surprisingly, however, the techniques of harvesting at postmortem and preparation of cadaveric osseoligamentous lower cervical spine for biomechanical testing have not been described in detail. We describe a simple and effective method that can be readily integrated into the routine autopsy procedure. Points on the avoidance of disfiguring the cadaver and damaging the spinal osseoligamentous structures are highlighted.
OBJECTIVE: We sought to test the hypothesis that apoptosis is a method of remodeling in second-trimester fetal heart development. We also hypothesized that hearts from fetuses with Down syndrome would have different levels of apoptosis than would control hearts, associated with their abnormal heart development. STUDY DESIGN: We obtained hearts from fetuses between 14 and 23 weeks' gestation with Down syndrome without anomalies (n = 10) and with the atrioventricular canal defect (n = 5). These hearts were compared with control hearts without anomalies (n = 5). Hearts were subjected to in situ end-labeling of deoxyribonucleic acid to test for evidence of apoptosis. The apoptotic indices were compared by anatomic location. RESULTS: Apoptotic nuclei were observed in each anatomic location in every category. The apoptotic indices were significantly lower in the atrial myocardial tissues of fetuses with Down syndrome than in control preparations (P < .05). The apoptotic index did not differ significantly in the atrial septum, ventricular myocardium, or ventricular septum. CONCLUSIONS: Apoptosis occurs in human fetal hearts during the second trimester. The different levels observed in our study suggest a different remodeling process in hearts of fetuses with Down syndrome than in hearts of control fetuses. Further study is needed to determine whether the different level of apoptosis associated with Down syndrome is due to the abnormal karyotype or to the presence of an anomaly.
OBJECTIVE: To determine if apoptosis is involved in development of the human fetal mullerian tract and regression of the uterine septum and to localize Bcl-2. a protein involved with regulating apoptosis. DESIGN: Descriptive controlled study. SETTING: Tertiary academic medical center. PATIENT(S): Eight human fetal uteri from 12 to 21 weeks' gestation. INTERVENTION(S): Immunohistochemistry using a monoclonal antibody for Bcl-2. MAIN OUTCOME MEASURE(S): Immunostaining. RESULTS: Bcl-2 was localized in endometrial cells, tubal muscularis and epithelium, and myometrial edges. It was absent from the septum of 4 uteri. CONCLUSIONS: The presence of Bcl-2 suggests that development of the human fetal müllerian tract involves apoptosis. Bcl-2 may protect the fetal endometrium from apoptosis as it continues to grow. The superior, inferior, and lateral myometrium as well as the tubal epithelium and muscularis also may represent active growth zones that are protected from apoptosis. The notable absence of staining for Bcl-2 in the embryonal uterine septum may indicate lack of protection from apoptosis in this area. This finding supports our hypothesis that apoptosis may be a mechanism by which the uterine septum regresses.
OBJECTIVE: To investigate protein and messenger RNA expression products for a subset of apoptosis-related genes in human granulosa cells. DESIGN: In vitro experiment. SETTING: Department of Obstetrics, Gynecology, and Reproductive Biology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts. PATIENT(S): Women undergoing oocyte retrieval for IVF after ovulation induction with gonadotropins. INTERVENTION(S): Granulosa cells were isolated from follicular aspirates after oocyte removal. MAIN OUTCOME MEASURE(S): Reverse transcription polymerase chain reaction and Western blotting for apoptosis-related gene products. RESULT(S): The expression of caspase-1, caspase-3, DNA fragmentation factor, and apoptotic protease activating factor-1, which are intermediate molecules in phylogenetically conserved apoptotic pathways, was demonstrated in granulosa cells from patients undergoing IVF. Moreover, proforms, but not activated enzymes, for both caspase-1 and caspase-3 were observed. CONCLUSION(S): Granulosa cells from patients undergoing IVF have intrinsic apoptotic machinery that could be activated for tissue remodeling.
OBJECTIVE: The objectives of this study were to determine the prevalence of diabetes and impaired glucose tolerance (IGT) in three Alaskan Eskimo populations, using standardized diagnostic criteria, and to evaluate family history and obesity as risk factors. RESEARCH DESIGN AND METHODS: This cross-sectional study involved men and women > or = 25 years of age from three Eskimo ethnic groups (Siberian Yupik, Central Yupik, and Inupiat) residing in northwestern Alaska. Glucose tolerance status was defined by World Health Organization criteria and was based on a 75-g oral glucose tolerance test. Data on age, family history of diabetes, and degree of Eskimo ancestry were obtained from a personal interview. Obesity was assessed using BMI. RESULTS: A total of 454 of 899 (50.5%) eligible participants were examined for diabetic status (239 Siberian Yupik, 106 Central Yupik, and 109 Inupiat participants). The prevalence of diabetes was more than twice as high among the Siberian Yupik (9.6%) as among the Central Yupik (2.8%) and Inupiat participants (3.7%). Diabetes was more prevalent in women than men (8.8 vs. 4.2%). IGT was found in an additional 11.7% of the women and 4.7% of the men. The combined prevalence of diabetes and IGT in the population > or = 55 years of age was 30.4% (diabetes 12.0%, IGT 18.4%). Of the people identified with diabetes, 47% had not been previously diagnosed. Age-specific prevalences were similar to those found in U.S. whites in the National Health and Nutrition Examination Survey II. After adjustment for age, family history of diabetes was associated with diabetes in study participants with an odds ratio of 4.4, while obesity was associated with diabetes with an odds ratio of 2.6. CONCLUSIONS: These prevalences of diabetes are the highest yet reported among Eskimo populations. Obesity and family history of diabetes are associated with increased odds of developing diabetes. These data underscore the need to further examine risk factors and to design effective interventions.
This study investigates the efficiency of community-based youth services in the Commonwealth of Virginia using data envelopment analysis (DEA). Efficiency was calculated based on resources available, the demand of services, and the service units produced. Results demonstrated the extent of inefficiency in community-based youth programs and how much efficiency can be improved relative to comparison with peer groups. The differences between large and small communities, urban and rural communities, and rich and poor communities were analyzed. There was no significant difference between each type for all three categories.
The factors regulating modifications of terminal beta-Gal residues in lactosaminyl (Gal beta 1-->4GlcNAc beta-->R) units in intact glycoproteins are not well understood. To examine these factors, rat liver alpha 2,3 sialyltransferase (alpha 2,3ST) and alpha 2,6 sialyltransferase (alpha 2,6ST) and the murine alpha 1,3 galactosyltransferase (alpha 1,3GT) were incubated with a variety of well-defined desialylated glycoproteins and with glycoproteins in extracts of the Lec2 mutant CHO cells. Lec2 cells constitutively synthesize nonsialylated glycoproteins with terminal lactosaminyl sequences. The results demonstrate that each enzyme displays preferences for glycoprotein acceptors and in the types of N-glycans recognized. The alpha 2,3ST, in contrast to the alpha 2,6ST and alpha 1,3GT, prefers more branched N-glycans compared to diantennary N-glycans. However, only the alpha 1,3GT is able to efficiently modify polylactosamines (3Gal beta 1-->4GlcNAc beta 1-->)n in N-glycans. Glycopeptides were also prepared by proteolysis of Lec2 glycoproteins and tested as acceptors compared to intact Lec2 glycoproteins. The alpha 2,6ST and alpha 1,3GT utilized intact glycoproteins and glycopeptides with a 2-fold preference for the former over the latter. In contrast, the alpha 2,3ST showed a 20-fold preference for intact glycoproteins over glycopeptides. These results demonstrate that each of these terminal glycosyltransferases differentially recognizes glycans and glycoprotein acceptors, and that the alpha 2,3ST requires peptide features for efficient utilization of branched N-glycan acceptors.
Apoptosis is a process of single-cell deletion requiring active participation of the cell in its own demise. First described in 1972, it is now known to play a major role in embryogenesis, tissue homeostasis and neoplasia. Apoptosis can be initiated when DNA damage occurs causing the cell to pause in its reproductive cycle. If the DNA damage is beyond repair, the cell proceeds to apoptotic cell death. When the genetic mechanism(s) involved in the pathway of apoptosis is altered, the cell does not die. Further mutations occur by proliferation and such multiple mutational events can lead to a malignant phenotype and cancer growth. The tumour suppressor gene p53 causes a DNA-damaged cell to rest and attempt repair. If damage is irreparable, p53 levels will continue to increase, initiating apoptosis. Mutation of p53, found in approximately 50% of cancers, can stop the apoptotic process. Increased bcl-2 expression, an apoptosis inhibitor, also plays a role in cellular transformation and cancer growth. Its altered expression occurs in the presence of oncogene expression. This paper reviews the role of apoptosis in malignant transformation, cancer growth, and response to therapy for gynaecological cancers. For cervical cancer and its precursors, data on apoptotic index, bcl-2 and Bax expression are presented and discussed in relationship to human papillomavirus expression. In ovarian epithelial malignancies, the role that apoptosis plays in chemotherapeutic responses is reviewed. The data for endometrial cancer are currently limited to apoptotic index.
We have used a fluorometric assay to determine the relative amounts of polymerized actin (F-actin) in wild-type and profilin mutant yeast cells. Our results indicate that profilin plays a role in maintaining normal F-actin levels in response to shifts to high temperature. Cells lacking profilin display a greater drop in F-actin levels upon such temperature shifts, and are slower to recover to initial F-actin levels than are wild-type cells. Interestingly, shifts to cold temperatures result in rapid increases of F-actin levels in wild-type and profilin null cells. We have further determined that shifting to high-osmolarity growth conditions causes a relatively slow decrease in F-actin levels in wild-type cells, and a small but rapid increase in the F-actin levels in profilin null cells. Profilin null cells contain normal concentrations of F-actin while growing exponentially at room temperature, indicating that profilin is not essential for maintaining F-actin concentrations during steady-state growth. Our data suggest that actin is inherently unstable in vivo at high temperatures, and that profilin helps to maintain actin in its filamentous state at these temperatures, perhaps by stimulating actin polymerization in a proper temporal and spatial fashion.
Treatment of mouse teratocarcinoma F9 cells with all-trans-retinoic acid (RA) causes a 9-fold increase in steady-state levels of mRNA for UDP-Gal:beta-D-Gal alpha1,3-galactosyltransferase (alpha1,3GT) beginning at 36 h. Enzyme activity rises in a similar fashion, which also parallels the induction of laminin and type IV collagen. Nuclear run-on assays indicate that this increase in alpha1,3GT in RA-treated F9 cells, like that of type IV collagen, is transcriptionally regulated. Differentiation also results in increased secretion of soluble alpha1,3GT activity into the growth media. The major alpha-galactosylated glycoprotein present in the media of RA-treated F9 cells, but not of untreated cells, was identified as laminin. Differentiation of F9 cells is accompanied by an increase in alpha-galactosylation of membrane glycoproteins and a decrease in expression of the stage-specific embryonic antigen, SSEA-1 (also known as the Lewis X antigen or LeX), which has the structure Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-R. However, flow cytometric analyses with specific antibodies and lectins, following treatment of cells with alpha-galactosidase, demonstrate that differentiated cells contain LeX antigens that are masked by alpha-galactosylation. Thus, RA induces alpha1,3GT at the transcriptional level, resulting in major alterations in the surface phenotype of the cells and masking of LeX antigens.