PubMed Health⌕ Search

Biomedical subjects

D Feldman

Publications and source records attributed to D Feldman.

At least 37 records · Page 2Linked to original sources
PubMed1997

1,25-dihydroxyvitamin D3 induction of nerve growth factor in L929 mouse fibroblasts: effect of vitamin D receptor regulation and potency of vitamin D3 analogs.

Decreased nerve growth factor (NGF) synthesis in the hippocampus and reduced nerve growth factor receptor immunoreactivity in CH1-4 basal forebrain areas have been implicated in neurodegeneration. Vitamin D receptors (VDR) have been located in brain areas affected by neurodegenerative diseases. 1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3], the active form of vitamin D, has been shown to induce NGF in L929 mouse fibroblasts and rat hippocampus. In the present study we analyzed the VDR in L929 cells, which we used as a model system. We studied the regulation of VDR abundance and the ability of 1,25-(OH)2D3 to induce NGF synthesis. Scatchard analysis of [3H]1,25-(OH)2D3 binding showed the VDR concentration to be 173 fmol/mg protein and the affinity to be 0.12 nM. VDR was localized to nuclei of L929 cells by immunocytochemistry. Treatment of cells with forskolin (FSK; 50 microM), which activates the cAMP-protein kinase A pathway, resulted in an 8- to 10-fold up-regulation of VDR by 6 h, and VDR remained elevated at 24 h, as we have reported for other cells. NGF secretion was measured in serum-free conditioned medium using a double sided enzyme-linked immunosorbent assay. 1,25-(OH)2D3 treatment (0.1 pM to 10 nM) for 24 h increased the NGF concentration 2- to 3-fold, an effect that plateaued at 1 nM 1,25-(OH)2D3. VDR up-regulation by FSK pretreatment augmented the NGF response to 1,25-(OH)2D3 2-fold compared to that in vehicle-pretreated cells for a total 6-fold increase compared to basal NGF levels. The vitamin D analogs EB-1089 and 22-oxacalcitriol, which have been found to be less calcemic than 1,25-(OH)2D3, also induced NGF synthesis. The effects of these analogs were further enhanced by prior up-regulation of VDR with FSK. In conclusion, we have characterized the VDR in L929 cells and shown that 1,25-(OH)2D3 and its less calcemic analogs induce NGF. Furthermore, up-regulation of VDR abundance enhanced NGF induction. These effects of 1,25-(OH)2D3 and its analogs via VDR to regulate NGF synthesis may have significance for the eventual treatment of neurodegenerative diseases that are caused by decreased NGF production.

Animals↗
PubMed1997

1alpha,25-dihydroxyvitamin D3 actions in LNCaP human prostate cancer cells are androgen-dependent.

We and others have recently shown that 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] significantly inhibits cell proliferation and increases secretion of prostate-specific antigen (PSA) in LNCaP cells, an androgen-responsive human prostate cancer cell line. The present study was designed to investigate the possible interactions between 1,25-(OH)2D3 and androgens in the regulation of LNCaP cellular function. LNCaP cell growth was dose-dependently inhibited by 1,25-(OH)2D3 (60% inhibition at 10 nM) when cells were cultured in medium supplemented with FBS (FBS medium). 1,25-(OH)2D3-treated cells showed a 5-fold increase in PSA secretion, similar to the increase seen in dihydrotestosterone (DHT)-treated cells. In combination, 1,25-(OH)2D3 and DHT synergistically enhanced PSA secretion 22-fold. This synergistic effect was even greater when cells were cultured in medium supplemented with charcoal-stripped serum (CSS medium), where endogenous steroids are substantially depleted. Under these conditions, 1,25-(OH)2D3 and DHT together stimulated PSA secretion up to 50-fold over the untreated control. Radioligand binding assays and Western blot analyses showed that the androgen receptor (AR) content was increased significantly by 1,25-(OH)2D3 at 48 h. Furthermore, the steady-state mRNA level of AR was up-regulated approximately 2-fold by 1,25-(OH)2D3 at 24 h. When cells were grown in CSS medium, 1,25-(OH)2D3 alone no longer inhibited cell growth or induced PSA secretion. Titration experiments revealed that the addition of DHT at 1 nM to the medium restored the antiproliferative activity of 1,25-(OH)2D3. Conversely, an antiandrogen, Casodex, completely blocked 1,25-(OH)2D3 antiproliferative and PSA stimulation activities when cells were cultured in FBS medium. In conclusion, these results demonstrate that the antiproliferative and PSA induction activities of 1,25-(OH)2D3 in LNCaP cells are dependent upon androgen action and that AR up-regulation by 1,25-(OH)2D3 likely contributes to the synergistic actions of 1,25-(OH)2D3 and DHT in these cells.

Analysis of Variance↗
PubMed1997

Analysis of vitamin D analog-induced heterodimerization of vitamin D receptor with retinoid X receptor using the yeast two-hybrid system.

Several synthetic analogs of 1 alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] are potent inducers of cellular differentiation and inhibitors of cell growth, yet they are less calcemic than 1,25-(OH)2D3 itself. The mechanisms by which these vitamin D analogs elicit a different profile of cellular activities than 1,25-(OH)2D3 are not fully understood. We propose that the analogs bind to the vitamin D receptor (VDR) to produce a conformational change that is more or less constrained than that induced by 1,25-(OH)2D3. This conformational change determines the extent of the VDR-retinoid X receptor (RXR) heterodimerization which, in turn, determines the interaction with other factors that specify the selectivity and magnitude of gene transactivation. We used the yeast two-hybrid system to evaluate a series of six vitamin D analogs for their ability to induce VDR-RXR heterodimerization. The VDR-RXR interaction was elicited by the analogs in a concentration-dependent manner. To evaluate how this activity compared with other known steps in 1,25-(OH)2D3 action, we also measured the ability of the same six analogs to bind to VDR, to enhance the binding of VDR-RXR to DNA, to transactivate a vitamin D-response element-reporter construct, and to inhibit proliferation in mammalian cells. Our results indicate that, for most analogs, the level of transcriptional activation correlates well with the strength of VDR-RXR heterodimerization in intact cells. We conclude that the yeast two-hybrid system provides a useful means to investigate heterodimerization potency and that this property contributes significantly to the overall pattern of analog activity. The yeast two-hybrid system, being an intact cell assay and easy to perform, may be a useful supplement to the conventional assays employed to screen vitamin D analogs.

Animals↗
PubMed1997

Revamping mental health care in Israel: from the Netanyahu Commission to National Health Insurance Law.

This paper describes the basis for the reform in mental health care system in Israel as presented in the report of the Netanyahu Commission (State Commission for Investigation of Functioning and Efficiency of the Health Care System in Israel, 1990) and the report of the State Comptroller's office (State Comptroller, 1991). These reports pointed to seven major problem areas in the mental health care system: (1) segregation of mental health and general health care systems, (2) variations in availability of services across the country, (3) conflict of interests within Ministry of Health which provides services and oversight, (4) overuse of hospital based care and under use of community based care, (5) reliance on hospitals for custodial care, (6) lack of appreciation of mental health service needs of non-severely mentally ill, and (7) lack of regional service planning. The article describes these problems and the proposed solutions.

Health Care Reform↗
PubMed1997

Vitamin D receptor polymorphisms, bone mineral density, and bone metabolism in postmenopausal Mexican-American women.

Common polymorphisms in the vitamin D receptor (VDR) gene have been shown to correlate with bone mineral density (BMD). However, attempts to replicate the original findings in other populations have yielded variable results. These disparities may reflect ethnic or environmental differences in the expression of the VDR effect upon BMD. We examined a relatively ethnically homogeneous group of 103 healthy postmenopausal Caucasian women of Mexican descent living in Northern California. We determined the VDR genotype and measured the BMD at the lumbar spine and femoral neck by dual-energy X-ray absorptiometry, as well as several biochemical indices of mineral metabolism. The prevalence of the BB genotype, associated in previous studies with the lowest BMD, was 8% and highly linked to the tt genotype. Absolute and age-adjusted BMD at both hip and spine showed a trend toward lower BMD in the BB, AA, and tt genotypes, but this trend did not achieve statistical significance. There were no consistent intergroup differences in change in BMD over 2 years of follow-up, nor in mean serum concentrations of 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, osteocalcin, or total urinary pyridinolines. Intact parathyroid hormone concentrations were significantly higher in subjects with the AA genotype, with a trend toward higher values in those with the BB and tt genotypes as well. Our data suggest that there may be a decrease in BMD associated with the B, A, and t alleles, but the intergroup difference in BMD is 0.2-0.5 standard deviations (SD) at the lumbar spine and 0.3 SD at the femoral neck, decreases that are smaller than previously reported. Given the relatively low prevalence of the BB/tt genotype in Mexican-American Caucasians, a larger sample would be required to detect a significant association between VDR alleles and differences in BMD of the magnitude suggested by our data. We conclude that a genotype effect of this magnitude, if present, would be clinically relevant, but the impact on BMD is too small to detect with statistical significance in a study of this size.

Aged↗
PubMed1997

The vitamin D receptor start codon polymorphism (FokI) and bone mineral density in premenopausal American black and white women.

This study examines the association between bone mineral density (BMD) and a start codon polymorphism (SCP) at the translation initiation site of the vitamin D receptor (VDR) gene. The thymine/cytosine (T/C) polymorphism in the first of two start (ATG) codons can be detected by a restriction fragment length polymorphism (RFLP) using the endonuclease FokI, which recognizes ATG as part of its restriction site. F indicates absence of the first ATG and a VDR that is shorter by three amino acids. The FokI genotype was determined in 154 premenopausal American women (72 black and 82 white) who were 20-40 years old. BMD of the total body, femoral neck, and lumbar spine were measured by dual-energy X-ray absorptiometry. The distribution of the SCP genotypes differed significantly by race (p < 0.001): 4% of blacks versus 18% of whites were ff homozygous and 65% of blacks versus 37% of whites were FF homozygous. There was no statistically significant interaction between race and SCP genotype in analyses of BMD at any skeletal site. In the group as a whole, the ff women had femoral neck BMD that was 7.4% lower than that of the FF women. The ff white women had total body BMD values that were 4.3% lower and femoral neck values that were 12.1% lower than FF white women. Total body and femoral neck BMD did not differ significantly by genotype in black women, and spine BMD did not differ by genotype in either race. Addition of the SCP genotype to analysis of covariance models comparing BMD of the black and white women reduced estimated differences in femoral neck BMD between the two groups by about 35%. In conclusion, the SCP polymorphism, detected with the endonuclease FokI, appears to influence peak bone density, particularly at the femoral neck. Racial differences in its distribution may explain some of the racial difference in femoral neck BMD.

Adult↗
PubMed1997

Juvenile fibromyalgia: clinical and polysomnographic aspects.

OBJECTIVE: To identify the child-mother diagnostic correlation in fibromyalgia (FM), to study sleep disturbance in juvenile FM, and to compare clinical aspects and sleep disorders between these groups. METHODS: We studied 34 children with confirmed FM aged 11 +/- 1 years, 10 children with diffuse pain, and 17 age and sex matched asymptomatic controls. The respective 61 mothers were included: 34 asymptomatic and 27 with FM. All participants were subjected to clinical evaluation, a sleep questionnaire, and nocturnal polysomnography, preceded by a night of adaptation. Sleep scoring was done visually and a computerized analysis was performed for alpha, theta, and delta waves in slow wave sleep (SWS). RESULTS: A significant predominance of mothers with FM was observed in the group of children with FM (71%) compared to children with diffuse pain (30%) and asymptomatic children (0%). According to the sleep questionnaire, the complaints of superficial sleep and nonrestorative sleep were more prominent in mothers with FM than in children with FM, whereas motor agitation during sleep was more frequent in the children with FM. Polysomnographic anomalies were also more prominent in mothers with FM than in children with FM in terms of decrease in sleep efficiency, increase of number of arousals during sleep, and alpha intrusion in SWS. Both FM groups presented an increased alpha + theta time/delta time index during SWS compared to respective controls, and mothers with FM also showed an increase in alpha time/delta time index during SWS, compared to asymptomatic mothers. A correlation was found between alpha + theta time/delta time index during SWS and intensity of clinical manifestations of pain and sleep anomalies in children and their mothers. CONCLUSION: Significant concordance was observed regarding FM diagnosis in children and their mothers. Sleep complaints and polysomnography findings were less prominent in affected children compared to mothers with FM. In addition, we observed a significant correlation between polysomnographic indexes, sleep anomalies, and pain manifestations in children and their mothers.

Adult↗
PubMed1996

Syndrome of lipoatrophic diabetes, vitamin D resistant rickets, and persistent Müllerian ducts in a Turkish boy born to consanguineous parents.

Congenital lipodystrophy (MIM 269700), persistent Müllerian ducts (MIM 261550), and vitamin D resistant rickets (MIM 277440) were observed in an 8 1/2-year-old boy born to consanguineous parents. Measurements of hormone sensitive lipase activity from a sample of the suprapubic fat depot were normal. Although the insulin receptor appeared normal (including autophosphorylation), insulin action, assessed by induction of total mRNA, was decreased. The vitamin D receptor was normal in size and amount, with a slight decrease in affinity for 1,25(OH)2D3. Induction of 24-hydroxylase, used as a measure of responsiveness to 1,25(OH)2D3, was only mildly defective. Assessment of anti-Müllerian hormone (AMH) failed to show any abnormalities explaining the persistent Müllerian ducts. We speculate that a defect in general hormone action common to 1,25(OH)2D3, insulin, and AMH may exist in this patient although we can not exclude the unlikely possibility that he is homozygous for two or three individually rare mutations.

Adult↗
PubMed1996

Parathyroid hormone--related protein (PTHrP) is an epidermal growth factor-regulated secretory product of human prostatic epithelial cells.

Parathyroid hormone-related protein (PTHrP) has previously been shown to be expressed in human prostatic tissue and in prostatic cancer cell lines. In the present study, PTHrP immunoreactivity was detected in the glandular epithelium of normal prostate and benign prostatic hyperplasia (BPH), as well as in prostatic adenocarcinoma (CaP). Epithelial cell cultures derived from normal, BPH, and CaP tissues were also stained by antibodies against PTHrP, and northern analysis revealed multiple transcripts of PTHrP in the cellular RNA. PTHrP (1-34) was measurable by radioimmunoassay (RIA) in media conditioned by the prostatic epithelial cell cultures, and PTHrP accumulated in conditioned media during a 72 hr time course. Addition of complete growth medium to starved cells resulted in increased PTHrP mRNA levels by 1 hr, with maximal stimulation at 8-24 hr. Several individual factors contained in the complete growth medium were tested for their ability to regulate PTHrP expression. Epidermal growth factor (EGF) was the major inducer of PTHrP expression, while cholera toxin, bovine pituitary extract, hydrocortisone, and insulin had minimal or no effect on PTHrP transcript levels. Since each of these factors is growth stimulatory, the unique ability of EGF to induce PTHrP is apparently unrelated to mitogenicity. 1,25-Dihydroxyvitamin D3[1,25(OH)2D3], an inhibitor of PTHrP expression in several other cell types, had no effect on steady-state levels of PTHrP mRNA expressed by epithelial cells in complete growth medium, although prostate cells have vitamin D receptors and are responsive to 1,25(OH)2D3 in other ways. Our results indicate that PTHrP expression is not confined to the neuroendocrine cells of the human prostate and that our culture system can be used as a model to investigate the role of PTHrP in the prostate.

Adenocarcinoma↗
PubMed1996

The presence of a polymorphism at the translation initiation site of the vitamin D receptor gene is associated with low bone mineral density in postmenopausal Mexican-American women.

We examined the association of bone mineral density (BMD) with a polymorphism in the gene encoding the vitamin D receptor (VDR) that causes a change in the predicted protein sequence. The polymorphism results from a C-to-T transition and creates an initiation codon (ATG) three codons proximal to a downstream start site. The polymorphism can be defined by a restriction fragment length polymorphism (RFLP) using the restriction endonuclease FokI. The presence of a FokI site, designated f, allows protein translation to initiate from the first ATG. The allele lacking the site (designated F), initiates from a second ATG site. Thus, translation products from these alleles are predicted to differ by three amino acids with the f variant elongated. In a group of 100 postmenopausal Mexican-American Caucasian women, subjects with the ff genotype (15% of the study population) had a 12.8% lower BMD at the lumbar spine than FF subjects (37% of the population) (p = 0.01). Heterozygote (Ff) subjects (48% of the population) had an intermediate BMD. This association between BMD and genotype was not apparent at the femoral neck or forearm. Over a 2-year follow-up period, a decrease in BMD at the femoral neck was greater in ff compared with FF subjects (-4.7% vs. -0.5%, p = 0.005). This trend was not apparent at the lumbar spine or forearm. There were no differences between genotype groups in measurements of 25-hydroxyvitamin D (25(OH)D), calcitriol, parathyroid hormone (PTH), osteocalcin, or urinary pyridinolines. We conclude that the FokI polymorphism of the VDR gene correlates significantly with decreased BMD at the lumbar spine and with an increased rate of bone loss at the hip in ff subjects. We emphasize that these initial data should be interpreted with caution but that the utility of this polymorphism as a genetic marker to determine BMD and osteoporosis risk warrants further study in larger populations with subjects of diverse ethnic backgrounds.

Aged↗
PubMed1996

Telling patients about the diagnosis of HIV infection.

This study used a questionnaire to examine how patients in the HIV/AIDS Clinic at a Department of Veterans Affairs hospital were told of their diagnosis, by whom, and to what degree they were given emotional and educational support. Nearly 17% of patients were informed by someone not in the health professions (often military personnel), and 27% of patients were notified in a nonprivate setting. Forty-seven percent indicated they received little or no educational support at the time of diagnosis, while 39% received little or no emotional support. Educational and emotional support for patients at the time of HIV diagnosis may be lacking.

Adult↗
PubMed1996

Primary health care physicians' treatment of psychosocial problems: implications for social work.

This study explores the extent to which primary care physicians serve as gatekeepers for the treatment of psychosocial problems and the extent to which they have contact with social workers. We also attempted to identify physician variables related to gatekeeping and amount of contact with social workers. Data from a national study of the role of primary care physicians in Israel were used. About one-third of the physicians had regular contact with social workers, one-quarter had contact less often, and about one-third almost never had contact. Variables associated with gatekeeping for psychosocial problems included having a large caseload, specializing in family medicine, practicing in a rural location, and being under age 55. Variables associated with social work contact were affiliation with the largest health maintenance organization and specialization in family medicine.

Adolescent↗
PubMed1996

An innovative CME program in cardiology for primary care practitioners.

The authors describe an innovative continuing medical education (CME) program they developed to improve the ability of community practitioners to manage common cardiology problems. The program includes an objective structured clinical examination (OSCE) with nine standardized patient (SP) stations. The SPs are trained to use checklists to assess the examinees' clinical skills, and to enter the checklist data directly into computers located within each of the examining rooms. Checklists cover the participants' knowledge of cardiology, and their interviewing, physical-examination, and counseling skills. The computer immediately generates detailed individual reports, which include a skills report, reflecting performance of core skills across all nine stations with group means for comparison, and an omissions report, listing items missed in each case for each participant and, again, comparing the results with group means. Participants review and discuss their performances and then discuss identified learning issues with a cardiologist. The cardiologist also reviews state-of-the-art diagnosis and treatment for the cases presented in the OSCE. Participants have reported having a high regard for the program and have indicated that the program has left them better prepared to deal with clinical cardiology issues.

Attitude of Health Personnel↗
PubMed1996

A novel mutation in the deoxyribonucleic acid-binding domain of the vitamin D receptor causes hereditary 1,25-dihydroxyvitamin D-resistant rickets.

Mutations in the vitamin D receptor (VDR) result in hereditary 1,25-dihydroxyvitamin D3-resistant rickets (HVDRR), an autosomal recessive disease caused by target organ resistance to the action of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. In this study, we investigated the molecular basis of HVDRR in a child from Saudi Arabia who was previously shown to be resistant to 1,25-(OH)2D3 action, but whose cultured skin fibroblasts exhibited normal [3H]1,25-(OH)2D3 binding. Using the PCR, exons 2 and 3 of the VDR gene that encode the DNA-binding region of the receptor were amplified and sequenced. A novel point mutation at nucleotide 252 in exon 2 of the VDR was identified. This missense mutation (GGC to GAC) resulted in the conversion of glycine to aspartic acid at amino acid position 46 (G46D), located at the base of the first zinc finger. This single base change was introduced into wild-type VDR complementary DNA by site-directed mutagenesis, and the mutant VDR was then expressed in COS-1 cells. The expressed mutant VDR displayed a normal binding affinity (Kd = 1.2 x 10(-10) mol/L) for [3H]1,25-(OH)2D3 as determined by Scatchard analysis. However, the mutant VDR was shown to have reduced binding affinity for DNA by DNA-cellulose chromatography. In COS-7 cells cotransfected with a vitamin D response element-chloramphenicol acetyltransferase reporter construct and the mutant VDR complementary DNA expression vector, the mutant VDR was unable to activate gene transcription in cells treated with up to 100 nmol/L 1,25-(OH)2D3. Restriction fragment length polymorphism analysis using MwoI restriction digests of exon 2 demonstrated that the affected child is homozygous for the mutation, whereas the child's father is heterozygous and a carrier of the defective allele. In conclusion, a new mutation was identified in exon 2 of the VDR gene. This mutation, which occurs in the first zinc finger of the DNA-binding domain of the receptor, blocks 1,25-(OH)2D3 action and leads to the syndrome of HVDRR.

Base Sequence↗
PubMed1995

Vitamin D and prostate cancer.

Our findings demonstrate the presence of VDR in various human prostate cancer cell lines and in primary cultures derived from normal, BPH and prostate cancer. In addition, 1,25-D induced several bioresponses in these cells including growth inhibition and PSA stimulation. Based on examples in many different malignant cells as well as our data in prostate cells, that vitamin D is anti-proliferative and promotes cellular maturation, it seem clear that vitamin D must be viewed as an important cellular modulator of growth and differentiation if addition to its classical role as regulator of calcium homeostasis. In this respect, vitamin D has the potential to have beneficial actions on various malignancies including prostate cancer. Its ultimate role in prostate cancer remains to be determined, but 1,25-D may prove useful in chemoprevention and/or differentiation therapy. We believe the data currently available provide the basis for an optimistic view on the possible use of vitamin D to treat prostate cancer in patients and that further investigation is clearly warranted to better define its potential therapeutic utility.

Calcitriol↗
PubMed1995

Detecting psychological distress among patients attending secondary health care clinics. Self-report and physician rating.

A study was conducted to determine the prevalence of psychological distress, as reported by patients and their physicians, in orthopedic, neurology, dermatology, and ophthalmology clinics; to study their accuracy in detecting psychological distress; and to determine if there is any connection among psychological distress, accuracy of detecting distress, and use of mental health and primary health care physicians' prognosis for the somatic complaints. Five hundred and fifty-six patients, ages 18-21, responded to the Psychiatric Epidemiology Research Interview Demoralization Scale (PERI-D), a measure of psychological distress, and to questions about their mental health and use of mental health and primary health services. Physicians, who were blind to patients' responses, were asked to what extent they thought the cause of patients' complaints was physical and to what extent they thought it was psychological in nature, and to prognosticate. Based on the PERI-D, about 25% of patients were distressed, this was less for females than males and varied between clinics. Based on self-reporting, about 14% of patients (males and females) were distressed. Based on physician reporting, about 17% (males less) were distressed. Physicians identified 35% of the PERI-D-distressed cases and 79% of nondistressed cases. About 66% of patients identified their distress and 83% their lack of distress. Increased use of primary health care and mental health care was related to distress. The prognosis was negatively related to distress. Based on this study, there is a need for more attention to psychological distress among secondary health care patients. Patients' ability to identify their distress suggests the importance of involving the patient in the diagnostic process. Correct detection of distress alone does not appear to decrease the use of primary medical and mental health services.

Adolescent↗
PubMed1995

Oestrogen-binding protein in Candida albicans: antibody development and cellular localization by electron immunocytochemistry.

Candida albicans, the most common fungal pathogen of humans, possesses an oestrogen (estrogen)-binding protein (EBP) that binds oestrogens with high affinity and specificity. The gene that encodes the EBP (CaEBP1) has been cloned and sequenced and shown to be structurally related to the old yellow enzyme from Saccharomyces cerevisiae. Here, we report the purification and the subcellular localization of the EBP from C. albicans. Using ion-exchange chromatography and an oestradiol affinity column, the EBP was purified from a strain of C. albicans (strain 422) which was selected because it constitutively expressed elevated levels of the binding protein. The purified protein displayed a subunit molecular mass of approximately 46 kDa when examined by denaturing gel electrophoresis, which is consistent with the size estimated from the sequence of the cloned CaEBP1 gene. An immunoaffinity column, prepared using a polyclonal antisera generated against EBP, depleted the oestrogen-binding activity from C. albicans cell extracts. Western blot analysis showed that the antisera specifically recognized the EBP from C. albicans. The antibodies also recognized the protein when the cloned CaEBP1 gene was expressed in S. cerevisiae and did not cross react with S. cerevisiae proteins. Using electron microscopy and antigen detection by immunogold staining, the EBP appeared to be primarily associated with vacuoles. However, when overexpressed in S. cerevisiae, the EBP was found diffusely throughout the cell. In conclusion, the EBP has been purified from C. albicans and antibodies generated against the protein were used to demonstrate that EBP is found associated with vacuoles in C. albicans.

Blotting, Western↗