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M R Walters

Publications and source records attributed to M R Walters.

At least 37 records · Page 2Linked to original sources
PubMed1999

Case-stimulated learning within endocrine physiology lectures: an approach applicable to other disciplines.

The problem of presenting endocrine physiology lectures in a format that interests medical students was addressed. Incorporating truncated case-stimulated learning sessions into the lectures has proven to be a successful solution to this issue, while also providing continuity between topics in the lecture block. This method of providing a direct clinical link for basic physiological concepts and providing a more active learning experience is adaptable to most basic science disciplines.

Education, Medical, Undergraduate↗
PubMed1998

Metronidazole may inhibit intestinal colonization with Clostridium difficile.

PURPOSE: Antibiotics suppress normal gut flora, allowing overgrowth of acquired or native Clostridium difficile, with release of toxins that cause mucosal inflammation. Oral metronidazole is used to treat antibiotic-associated colitis (pseudomembranous colitis). This study was designed to determine whether oral metronidazole, as part of preoperative bowel preparation, prevents or decreases incidence of antibiotic-associated colitis after elective colonic and rectal procedures. METHODS: Eighty-two patients (40 men) were prospectively, randomly assigned to receive one of two oral antibiotic regimens before colorectal surgery. All patients underwent mechanical bowel preparation with polyethylene glycol-electrolyte lavage solution before administration of oral antibiotics. Group 1 (n = 42) patients received three doses (1 g/dose) of neomycin and erythromycin. Group 2 (n = 40) patients received three doses (1 g/dose) of neomycin and metronidazole. Both groups received one preoperative and three postoperative doses of intravenous cefotetan (2 g/dose). Both groups had stool samples tested for C. difficile toxin in the preoperative and postoperative periods by enzyme-linked immunoabsorbent assay or by tissue culture cytotoxicity. Patients with preoperative stool studies positive for C. difficile were excluded from the study. RESULTS: Treatment groups were not different for age, gender, or surgical procedure. Mean age +/- 1 standard deviation was 67.6 +/- 13.6 (range, 34-94) years in Group 1 and 62.1 +/- 13.5 (range, 35-84) years in Group 2 (P = 0.069). Mean length of hospital stay +/- 1 standard deviation was 9.76 +/- 4.9 (range, 4-28) days for Group 1 and 8.05 +/- 2.6 (range, 3-14) days for Group 2 (P = 0.053). Five patients in Group 1 (neomycin and erythromycin) and one patient in Group 2 (neomycin and metronidazole) had positive stool studies for C. difficile. Relative risk of colonization with C. difficile in Group 1 was 4.76 times that in Group 2 (95 percent confidence interval, 0.581, 39). This difference was not statistically significant (P = 0.202). There were no significant differences in C. difficile colonization rates with respect to age, length of stay, or gender. CONCLUSIONS: This study suggests that there may be a clinical association between use of metronidazole preoperatively and inhibition of intestinal colonization by C. difficile in this patient population undergoing colonic and rectal surgery.

Adult↗
PubMed1998

Reliability and validity testing of the Michigan Hand Outcomes Questionnaire.

In this study, psychometric principles were used to develop an outcomes questionnaire capable of measuring health state domains important to patients with hand disorders. These domains were hypothesized to include (1) overall hand function, (2) activities of daily living (ADL), (3) pain, (4) work performance, (5) aesthetics, and (6) patient satisfaction with hand function. An initial pool of 100 questions was pilot-tested for clarity in 20 patients; following factor analysis, the number of questions was reduced to a 37-item Michigan Hand Outcomes Questionnaire (MHQ). The MHQ, along with the Short Form-12, a generic health status outcomes questionnaire, was then administered to 200 consecutive patients at a university-based hand surgery clinic and was subjected to reliability and validity testing. The mean time required to complete the questionnaire was 10 minutes (range, 7-20 minutes). Factor analysis supported the 6 hypothesized scales. Test-retest reliability using Spearman's correlation demonstrated substantial agreement, ranging from 0.81 for the aesthetics scale to 0.97 for the ADL scale. In testing for internal consistency, Cronbach's alphas ranged from 0.86 for the pain scale to 0.97 for the ADL scale (values >0.7 for Cronbach's alpha are considered a good internal consistency). Correlation between scales gave evidence of construct validity. In comparing similar scales in the MHQ and the Short Form-12, a moderate correlation (range, 0.54-0.79) for the ADL, work performance, and pain scales was found. In evaluating the discriminate validity of the aesthetics scale, a significant difference (p = .0012) was found between the aesthetics scores for patients with carpal tunnel syndrome and patients with rheumatoid arthritis. The MHQ is a reliable and valid instrument for measuring hand outcomes. It can be used in a clinic setting with minimal burden to patients. The questions in the MHQ have undergone rigorous psychometric testing, and the MHQ is a promising instrument for evaluation of outcomes following hand surgery.

Factor Analysis, Statistical↗
PubMed1998

Endoscopic versus open carpal tunnel release: a cost-effectiveness analysis.

Endoscopic carpal tunnel release is a controversial procedure used in the treatment of carpal tunnel syndrome. Although endoscopic carpal tunnel release is associated with less incisional pain and faster recovery time than the open carpal tunnel release, opponents of endoscopic carpal tunnel release suggest that its benefits are outweighed by its higher complication rates from median nerve transection and transient numbness of the fingers. Because of the huge economic and social impact of carpal tunnel syndrome in this country, we performed a cost-effectiveness analysis comparing endoscopic carpal tunnel release and open carpal tunnel release using guidelines established by the Panel on Cost-Effectiveness in Health and Medicine of the U.S. Public Health Service. A decision analytic model was used to measure differences in cost and effectiveness--expressed as quality-adjusted life-years (QALYs)--between endoscopic carpal tunnel release and open carpal tunnel release. The societal perspective was chosen, and probabilities for various outcomes for the two procedures were obtained from published randomized-controlled trials. Cost data were derived from the Medicare Resource-Based Relative Value Units published in the Federal Register. QALYs were obtained from two groups of health care providers using a utility-assessment questionnaire. Using probabilities for various outcomes from the two published randomized-controlled trials comparing endoscopic carpal tunnel release and open carpal tunnel release, we constructed a decision tree to derive both the cost and the QALYs for the two procedures. The incremental cost difference between endoscopic carpal tunnel release and open carpal tunnel release was $46, using Medicare cost and probabilities of various outcomes derived from a study by Brown et al. in 1993. We calculated QALYs for five age groups--25, 35, 45, 55, 65--assuming a life expectancy of 75 years. The marginal effectiveness (QALY of endoscopic carpal tunnel release minus QALY of open carpal tunnel release) ranged from 0.235 QALY for the 25-year-old age group to 0.066 QALY for the 65-year-old age group, giving a cost-effectiveness ratio of $195/QALY and $693/QALY, respectively. When compared with other accepted medical interventions such as breast cancer screening ($4836/QALY) and exercise to prevent coronary heart disease ($13,508/QALY), endoscopic carpal tunnel release seems to be cost-effective. However, our sensitivity analysis indicated that the cost-effectiveness ratio was very sensitive to a major complication such as median nerve injury. For endoscopic carpal tunnel release to be a cost-effective procedure, the incidence of median nerve injury must be one percentage point less for endoscopic carpal tunnel release than for open carpal tunnel release. Based on the data from the randomized-controlled trials, endoscopic carpal tunnel release seems to be a cost-effective procedure; however, before it can be recommended, greater emphasis must be given to the training of surgeons in this new technique, so that major complications such as median nerve injuries can be avoided. In addition, future studies must better define the actual incidence of nerve injuries for both endoscopic carpal tunnel release and open carpal tunnel release in the community setting.

Adult↗
PubMed1998

Homologous up-regulation of vitamin D receptors is tissue specific in the rat.

1,25-dihydroxyvitamin D3 (1,25(OH)2D3) receptors (VDR) are expressed in multiple tissues within the body. VDR levels are increased by 1,25(OH)2D3 in intestine and kidney and in numerous cell models. The ability of 1,25(OH)2D3 to affect VDR levels in other target tissues in vivo was studied by assessing VDR levels by the 3H-1,25(OH)2D3 binding assay under varied physiological conditions in the rat. When compared with vitamin D-deficient (-D) controls, rats raised on a normal vitamin D-sufficient (+D) diet showed elevated VDR levels in kidney (391 +/- 53 vs. 913 +/- 76 fmol/g of tissue;p < 0.05), but not in testis, heart, or lung. Up-regulation of the VDR also occurred in kidney of +D rats 1 day after a single 100-ng dose of 1,25(OH)2D3 (454 +/- 43 vs. 746 +/- 113 fmol/mg of DNA; p < 0.05), but no changes were seen in intestine, testis, or lung. Because 1,25(OH)2D3-induced hypercalcemia may independently affect VDR regulation, 1,25(OH)2D3 was infused into -D rats, and normocalcemia was maintained by reduced dietary calcium intake. In this model, the renal VDR was again up-regulated (446 +/- 115 vs. 778 +/- 58 fmol/mg of DNA; p < 0.05), but VDR levels in testis and lung were unaffected. Scatchard analysis and tests of 1,25(OH)2D3 dose (1-100 ng/day for 7 days) and temporal (100 ng/day for 1-7 days) responsiveness further supported the tissue-specific nature of the homologous VDR regulation. Assay of VDR levels by L-1-tosylamido-2-phenylethyl chloromethyl ketone-3H-1,25(OH)2D3 exchange assay ruled out differences in endogenous 1,25(OH)2D3 occupancy as the basis for the observed differences in VDR regulation. Finally, coidentity of the VDR-like sites in kidney versus testis was confirmed by competitive binding analysis comparing their relative affinities for 25(OH)D3 versus 1,25(OH)2D3 (30.5 +/- 6.4 vs. 35.6 +/- 3.6 in kidney and testis, respectively) and by immunoblot analysis using a highly specific monoclonal anti-rat VDR antibody. Thus, under a wide variety of experimental conditions, homologous up-regulation of the VDR occurs in the rat kidney in vivo, but not in several other target tissues which do not regulate plasma calcium homeostasis. Moreover, this differential VDR regulation did not result from secondary changes in plasma calcium, from differential 1,25(OH)2D3 responsiveness in the various tissues, nor from differences in endogenous 1,25(OH)2D3 occupancy of the VDR. These studies thus establish that, in contrast to observations in vitro, the widely described phenomenon of homologous VDR up-regulation in kidney and intestine is not a universal property of 1,25(OH)2D3 target tissues in vivo in the rat.

Animals↗
PubMed1998

A quantitative analysis of knee joint kinematics during the sidestep cutting maneuver. Implications for non-contact anterior cruciate ligament injury.

This study accurately quantifies the knee joint kinematics associated with sidestep cutting maneuvers in vivo. These data were subsequently evaluated to determine the relationship between sidestep cutting and non-contact anterior cruciate ligament (ACL) injury. Sixteen male subjects, proficient in the sidestep cutting maneuver, had knee joint kinematic data recorded during the stance phase of straight line running and sidestep cutting. Cutting speeds (5.5 msec-1 to 7.0 msec-1) and angles (30 degrees to 55 degrees) were chosen to reflect the sporting context. All subjects underwent medical screening prior to testing to ensure data represented joint biomechanics of healthy, ACL intact individuals. The temporal three dimensional positions of externally mounted (skin) markers were submitted to a custom software package (JTMOTION), which described in clinically meaningful terms, movements for the three rotational degrees of freedom at the knee joint. While mean patterns of motion for the three clinical knee rotations compared favorably between running and cutting data, sidestepping induced significantly (p < 0.01) greater maximum knee joint rotations during stance. Knee joint rotational ranges were also found to be significantly (p < 0.01) larger during cutting when compared to running. Increases in knee joint kinematics during cutting however, were not of a great enough magnitude to alone elicit spontaneous non-contact ACL injury, falling well within "safe" ranges of knee motion. Significant increases in inter-trial variability for the three rotations were observed in some subjects for sidestepping compared to running. It was concluded that a lack of consistency in knee joint biomechanics between cutting maneuvers increased the risk of ACL injury with the performance of an abnormal and potentially hazardous sidestep being more likely. The risk of non-contact ACL injury during cutting maneuvers was suggested to increase further when an individual displayed these "atypical" joint biomechanics in conjunction with specific joint structures, levels of experience and conditioning, and ligament morphologies.

Adult↗
PubMed1997

Altered phosphorylation of a 91-kDa protein in particulate fractions of rat kidney after protracted 1,25-dihydroxyvitamin D3 or estrogen treatment.

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] treatment in vitamin D-deficient (-D) rats results in a dose-dependent decrease in phosphorylation of a 91-kDa protein (PP-D91) in particulate fractions of the kidney. This recently reported 1,25(OH)2D3 effect was examined in detail herein. In contrast to the pattern expected of a rapid signal transduction event, time course (4 h-7 days) experiments demonstrated that PP-D91 phosphorylation was not decreased until 3-5 days 1,25(OH)2D3 treatment, resulting in a 61 +/- 3% (P < 0.01, n = 3) decrease in PP-D91 phosphorylation by 7 days. These effects paralleled increases in plasma calcium from 9.3 +/- 0.6 to 13.9 +/- 0.7 mg/dl after 0 vs 7 days 1,25(OH)2D3 treatment, respectively. Subcellular fractionation demonstrated that the renal PP-D91 was predominantly localized and 1,25(OH)2D3-regulated in crude mitochondrial and microsomal fractions. Further, PP-D91 was present and 1,25(OH)2D3-regulated in enriched preparations of both proximal and distal renal tubule segments. Tissue distribution studies demonstrated that the PP-D91 was predominantly present and 1,25(OH)2D3 regulated in the kidney, although low levels of a vitamin D-independent phosphorylated band of similar size were observed in the lung and heart. In contrast to 1,25(OH)2D3, estradiol-17B treatment (1 mg/day x 7 day) significantly (P < 0.01) increased PP-D91 phosphorylation in kidney of both -D and +D rats (increased 118.5 +/- 10.6 and 81.9 +/- 6.3%, respectively). Phosphoamino acid analysis after PP-D91 phosphorylation, isolation, and proteolysis indicated that these hormones alter 32P incorporation into phosphoserine residues. In conclusion, the 1,25(OH)2D3 effect to reduce PP-D91 phosphorylation in particulate fractions of the rat kidney is a protracted, tissue-specific effect which parallels elevated plasma calcium levels in this model. Moreover, renal PP-D91 phosphorylation is differentially regulated by 1,25(OH)2D3 vs E2 treatment and occurs on phosphoserine residues. The parallel between decreased PP-D91 phosphorylation and 1,25(OH)2D3-induced hypercalcemia may suggest a role for PP-D91 in the renal response to hypervitaminosis D.

Animals↗
PubMed1997

The dynamics of bimanual circle drawing.

A bimanual circle drawing task was employed to elucidate the dynamics of intralimb and interlimb coordination. Right-handed subjects were required to produce circles with both hands in either a symmetrical (mirror) mode (i.e. one hand moving clockwise, the other counter-clockwise) or in an asymmetrical mode (i.e. both hands moving clockwise or counter-clockwise). The frequency of movement was scaled by an auditory metronome from 1.50 Hz to 3.25 Hz in 8 (8-sec) steps. In the asymmetrical mode, distortions of the movement trajectories, transient departures from the target pattern of coordination, and phase wandering were evidence as movement frequency was increased. These features suggested loss of stability. Deviations from circular trajectories were most prominent for movements of the left hand. Transient departures from the required mode of coordination were also largely precipitated by the left hand. The results are discussed with reference to manual asymmetries and mechanisms of interlimb and intersegmental coordination.

Adult↗
PubMed1996

On the crisis in biomedical education: is there an overproduction of biomedical PhDs?

The United States is the world leader in biomedical science (BMS) education and research. This preeminence is reflected in superior medical education, the attraction of U.S. educational institutions to foreign visitors seeking advanced training, and a high rate of transfer of knowledge between basic biomedical research and the delivery of health care at the bedside. The foundation for this excellence and leadership has been the research carried out by MD and PhD biomedical scientists. It has been suggested that there is now an oversupply of BMS PhDs, and thus that BMS PhD programs should be downsized. Full examination of the issues involved, including a case study of doctoral graduates and postdoctoral fellows at Tulane Medical Center, leads the authors to conclude that a biomedical PhD "glut" does not exist at the present time, that downsizing training programs would have a serious, long-term negative impact on biomedical research, and that medical school administrators and faculty should resist attempts to reduce biomedical research and training at the local and national level. However, times have changed and training programs must evolve to adapt to the technologic changes occurring in the workplace. Alternatives, such as new alliances with industry, must be sought to compensate for decreased resources at federal and institutional levels; new and innovative curricula must be developed to prepare biomedical scientists for nonacademic, as well as academic, job opportunities in the twenty-first century; and medical center administrators and faculties must work together to increase the visibility of BMS and stress its critical relationship to the research base of the nation.

Academic Medical Centers↗
PubMed1996

Identification of environmental chemicals with estrogenic activity using a combination of in vitro assays.

Environmental chemicals that function as estrogens have been suggested to be associated with an increase in disease and dysfunctions in animals and humans. To characterize chemicals that may act as estrogens in humans, we have compared three in vitro assays which measure aspects of human estrogen receptor (hER)-mediated estrogenicity. Chemicals were first tested for estrogen-associated transcriptional activity in the yeast estrogen screen (YES). This was created by expressing hER and two estrogen response elements linked to the lacZ gene in yeast. Second, chemicals that were tested in YES were then assayed for direct interaction with hER in a competition binding assay. Third, chemicals were tested in the estrogen-responsive MCF-7 human breast cancer cell line transiently transfected with a plasmid containing two estrogen response elements linked to the luciferase gene. Together, these assays have identified two metabolites of DDT, o,p'-DDD and p,p'-DDD, that have estrogenic activity. Interestingly, previous studies had reported that the DDD metabolites were nonestrogenic in whole animal models. Alachlor, the most frequently used herbicide in the United States, cis-nonachlor, and trans-nonachlor displayed weak estrogenic activity in the combined assays. The antifungal agent benomyl had no estrogenic activity. We propose that a combination of in vitro assays can be used in conjunction with whole animal models for a more complete characterization of chemicals with estrogenic activity.

Binding, Competitive↗
PubMed1995

Protracted 1,25-dihydroxyvitamin D treatment stimulates multiple calmodulin binding proteins in rat kidney.

Previous studies demonstrated that 1,25-dihydroxyvitamin D [1,25-(OH)2D] treatment in vivo stimulates [125I]calmodulin (CaM) binding to several proteins (detected by [125I]CaM gel overlay) in cytosol preparations from rat kidney. This study establishes the sizes of the principal stimulated forms and physiological aspects of their stimulation by the hormone. Densitometric analysis of the 1,25-(OH)2D-stimulated [125I]CaM binding activities demonstrated induction of two major bands, M(r) = 110 +/- 2.4 and 94 +/- 1.2 K. This analysis also revealed induction of a previously existing band at 150 +/- 2.7 K and induction of a 74 +/- 1.1 K band. 1,25-(OH)2D-induction of the [125I]CaM binding activities (CaMBP-Ds) was observed in both vitamin D-deficient and normal vitamin D-sufficient rats. The [125I]CaM binding activities were abolished by incubation with 1000-fold excess CaM, but not calbindin-D28, troponin C, parvalbumin, or alpha-lactalbumin. 1,25-(OH)2D induction of the [125I]CaM binding activities exhibited a graded dose response at 5-100 ng/day, and 5-7 days treatment was required for strong induction. The [125I]CaM binding activities in the kidney exhibited differential subcellular distributions: 150 K CaMBPs were present in crude preparations of nuclei, microsomes, and mitochondria; a 110 K CaMBP was present in the microsomal preparation; and the 94 and 74 K CaMBPs were restricted to the cytosol. 1,25-(OH)2D treatment resulted in the induction of the microsomal 110 K CaMBP and possibly the nuclear (but not in mitochondrial or microsomal) 150 K CaMBPs. In conclusion, there are at least four 1,25-(OH)2D-induced [125I]CaM binding activities in the rat kidney, with some variations in subcellular distribution. Moreover, their pattern of induction suggests that 1,25-(OH)2D regulation of the [125I]CaM binding activities is not a part of the immediate 1,25-(OH)2D signal transduction pathway, but rather may result from altered genomic activity after hormone treatment.

Animals↗
PubMed1994

1,25-Dihydroxyvitamin D3 effects in rat kidney: regulation of protein phosphorylation.

This study tested the hypothesis that 1,25-dihydroxyvitamin D3 treatment alters the pattern of protein phosphorylation in rat kidney. Treatment with 1,25-dihydroxyvitamin D3 (100 ng/day s.c. x 7d) in vitamin D-deficient rats markedly decreased phosphorylation of a particulate protein (91 +/- 0.6 kDa, n = 9) and moderately increased phosphorylation of a cytosolic protein (108 +/- 0.8 kDa, n = 9) in the kidney. The decreased phosphorylation of the 91-kDa particulate protein showed a graded dose response (0-200 ng/day), as did the more moderate increase in phosphorylation of the 108-kDa cytosolic protein. In conclusion, this study has provided evidence that 1,25-dihydroxyvitamin D3 plays an important role in the regulation of protein phosphorylation in rat kidney and adds these biochemical events to the growing list of 1,25-dihydroxyvitamin D3 effects in this poorly understood target tissue.

Animals↗
PubMed1993

Dexamethasone downregulates vitamin D receptors in rat kidney, unmasking a high affinity binding site.

Dexamethasone exerted tissue-specific effects on rat vitamin D receptor levels: upregulation of receptors in intestine, downregulation in kidney, and no effect on receptors in testis, heart, and lung. Scatchard analysis showed selective downregulation of the low affinity receptor site (Kd = 0.9 nM) in kidney, thus unmasking a high affinity binding site (Kd = 0.05 nM). The single low affinity site in intestine (Kd = 0.3-0.7 nM) was upregulated, while the single high affinity site in testis (Kd = 0.07-0.09 nM) was not changed. These results demonstrate the existence of two types of nuclear binding sites in rat kidney and establish that there are similarities between the high affinity binding site in the rat kidney and the single high affinity receptor site in the testis.

Animals↗
PubMed1992

1,25-Dihydroxyvitamin D effects in the kidney: induction of calmodulin binding proteins.

A marked induction of 125I-calmodulin binding proteins (185kDa and 115kDa) occurred in the rat kidney in response to treatment with 1,25-dihydroxyvitamin D3 (100 ng/day s.c. x 7d). These 125I-calmodulin binding proteins, measured by the gel overlay procedure, exhibited calcium dependence and were abolished in the presence of excess unlabelled calmodulin. The response was tissue specific: there was no change in 125I-calmodulin binding in rat testis, heart, and brain and only a modest elevation of binding to one calmodulin binding protein in the intestinal mucosa. These results are particularly important in suggesting that the calmodulin signal transduction mechanism may, via changes in its acceptor proteins, participate in mediating some biological effects of 1,25-dihydroxyvitamin D3.

Animals↗
PubMed1992

Specific 1,25-dihydroxyvitamin D3 binding sites in choroid plexus.

Quantitative autoradiographic analysis of [3H] 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) binding in vitamin D deficient mice provided evidence for high levels of specific binding in choroid plexus and, to a lesser extent, ventral hippocampus. Sucrose gradient analysis yielded a 3-4S peak of specific [3H]1,25(OH)2D3 binding in bovine choroid plexus, but not amygdala or hippocampus. Scatchard analysis of [3H]1,25(OH)2D3 binding in bovine choroid plexus yielded KD = 0.23 +/- 0.06 nM and Nmax = 43.5 +/- 0 fmol/g tissue (n = 5). This result indicates the presence of significant receptor-like [3H]1,25(OH)2D3 binding sites in the choroid plexus and, thus, suggests roles for this hormone in regulating the entry of calcium into the brain and/or in the central regulation of calcium homeostasis.

Animals↗
PubMed1992

Absence of calbindin-D28 expression in nonclassical 1,25-dihydroxyvitamin D targets: analysis by polymerase chain reaction.

CaBP-D28 mRNA expression in rat heart, testis, and lung was assessed by polymerase chain reaction (PCR). The animal model used was the hyperinduced vitamin D-treated rat (100 ng 1,25-dihydroxyvitamin D subcutaneously, daily for 7 days). For the PCR studies, two pairs of 20 mer oligonucleotide primers (designated 1-4 according to their position on the coding strand, but with primers 3 and 4 in reverse orientation) derived from the rat CaBP-D28 cDNA sequence were tested in various combinations. Optimal conditions were established using a 1:100 dilution of cDNA from normal rat kidney. Bands of the predicted sizes of 869 (1, 3), 994 (1, 4), 725 (2, 3), and 850 (2, 4) nucleotide base pairs resulted, but with varying intensities: 2,4 approximately 1,3 > 1,4 > 2,3. Repeat PCR (recycling after 1:100 dilution and readdition of reagents and primers with at least one different primer) provided strong additional amplification, particularly with the 1,4/2,4 combination. Under these conditions, mixing experiments showed that CaBP-D28 transcripts were detectable at 10(-7)- to 10(-9)-fold lower levels of expression than in D+ kidney. When RNA was isolated and cDNA generated from test tissues from 4 individual vitamin D-stimulated (D+) and vitamin D-deficient (D-) rats, repeat PCR (1,4/2,4 primer combination) provided no evidence of significant CaBP-D28 mRNA expression in the nonclassic target tissues, in contrast to strong bands in both the D- kidney (undiluted) and D+ kidney (1:100 dilution) preparations.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Physiological effects of 1,25-dihydroxyvitamin D3 in TM4 Sertoli cell line.

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] receptors have been previously described in Sertoli cells. This study was performed to assess biological activity of the receptor in the mouse Sertoli cell line TM4. A 2-h preincubation with 0.01-25 nM 1,25(OH)2D3 resulted in a dose-dependent rapid uptake of 45Ca2+ within 5 min of addition of the isotope to the cells (27 +/- 8%, n = 4 experiments; P less than 0.05). This response was specific for 1,25(OH)2D3, in that it was not induced by 25-hydroxyvitamin D3, estradiol, cortisol, R 5020 (promegestone), or testosterone. However, a combination of testosterone and 1,25(OH)2D3 inhibited uptake by 23 +/- 8% (n = 3 experiments, P less than 0.01). That the mechanism responsible for 1,25(OH)2D3-stimulated uptake may involve 1,25(OH)2D3 receptor interaction is supported by the observation that cycloheximide inhibited the response. Conversely, there was no detectable change in uptake by 1,25(OH)2D3-treated cells after 24-h incubation with 0.1-5 nM 1,25(OH)2D3. Increased levels of DNA and protein content also resulted from a 2-h incubation with the steroid and were sustained up to 24 h without a concomitant increase in cell number or a detectable change in cell morphology. The presence of specific 1,25(OH)2D3 receptor-like binding sites was demonstrated by sucrose gradient analysis and hydroxylapatite assay. These data demonstrate that 1,25(OH)2D3 may play an important role in testicular function through regulation of receptor-mediated events.

Animals↗