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Biomedical subjects

D Feldman

Publications and source records attributed to D Feldman.

At least 145 records · Page 8Linked to original sources
PubMed1991

The effect of progesterone upon first trimester trophoblastic cell differentiation and human chorionic gonadotrophin secretion.

The effect of progesterone (P) upon first trimester placental secretion of human chorionic gonadotrophin (HCG) and cellular differentiation was studied using both static and kinetic methods. At 1 microM, P inhibited spontaneous episodic secretion of HCG when given in short pulses (1-4 min) to placental explants in superfusion. Both HCG pulse frequency and amplitude were reduced. At 0.1-0.01 microM P concentrations, the effect of HCG secretion was milder. P also blocked the maximally effective concentration 100 pM of gonadotrophin releasing hormone (GnRH) analogue, a known HCG stimulant, when given together with it for 1 min. This inhibitory effect lasted for 1 h after P administration. Progesterone at 1 microM, added daily for 1 week blocked HCG secretion by isolated trophoblastic cells in static culture. This inhibitory effect lasted until the fifth day. No effect on differentiation and long-term viability was noticed in P-treated cells. Incubation with 0.1-1.0 microM P did not affect HCG secretion by explants after 24 h. In contrast, the effect of 1 microM cortisol or 1 nM oestradiol was stimulatory. In conclusion, P exerts both a rapid and delayed inhibitory effect upon HCG secretion and production. It may do so by counteracting the stimulatory effect of endogenous GnRH on gonadotrophin secretion by the placenta.

Cell Differentiation↗
PubMed1991

Ultrastructural effects of UVB radiation and subsequent retinoic acid treatment on the skin of hairless mice.

The ultrastructure of hairless mouse skin exposed to UVB radiation and followed by retinoic acid treatment was studied to identify alterations induced in both epidermis and dermis. Female mice were irradiated 3 times weekly for 5-6 months; a group of these mice was then treated topically 3 times weekly for 10 weeks with either 25 micrograms all-trans-retinoic acid dissolved in acetone or with acetone alone. Age-matched, unexposed, untreated mice served as controls. Cutaneous changes induced by UVB radiation included keratinocyte mitochondrial inclusions often accompanied by damaged cristae, duplication of basement membrane, increased number of dermal fibroblasts, inflammatory cells and elastic fibers, and abnormal elastic fibers. Subsequent retinoic acid treatment resulted in more prominent mitochondrial inclusions which sometimes coalesced to form irregular contoured bodies. Also observed were lipid droplets in the stratum corneum, glycogen deposits in keratinocytes and granular material in dilated keratinocyte endoplasmic reticulum. Poorly differentiated epidermis with necrotic or apoptotic cells was present in some specimens. Elastic fibers were fewer and usually morphologically normal. Skin exposed to UVB and treated with vehicle appeared similar to control except for the presence of excess basement membrane and occasional small mitochondrial inclusions. Because of the heightened concern regarding UV radiation-induced damage to the human skin and the current topical use of retinoids, the cutaneous changes described are considered worthy of attention.

Animals↗
PubMed1991

Activation of protein kinase-C inhibits vitamin D receptor gene expression.

The abundance of 1,25-dihydroxyvitamin D3 receptors (VDR) in cultured cells has been shown to vary in direct relation to the rate of cell proliferation. This study examines the question of whether the growth-factor mediated up-regulation of VDR is due to direct modulation of VDR gene expression or is secondary to the stimulation of cell cycle events. Mitogenic agents, such as basic fibroblast growth factor and phorbol esters, were found to cause significant decreases in VDR abundance, while substantially stimulating proliferation of NIH-3T3 cells. Potent phorbol esters, such as phorbol myristate acetate (PMA) and phorbol-12,13-dibutyrate, whose biological actions have been shown to be mediated through the activation of protein kinase-C, down-regulated VDR in a time- and dose-dependent manner. An inactive phorbol ester, 4 alpha-phorbol-12,13-didecanoate, which does not activate protein kinase-C, did not alter VDR levels. Desensitization of protein kinase-C by prolonged exposure of cells to phorbol esters eliminated the PMA-mediated down-regulation of VDR. Staurosporine, an inhibitor of protein kinase-C, blocked the actions of PMA. Oleoyl acetyl glycerol, a synthetic diacyl glycerol, and A23187, a calcium ionophore, were both able to suppress VDR abundance alone and were additive in combination. The results suggest that activation of the protein kinase-C pathway and elevation of intracellular Ca2+ lead to significant down-regulation of VDR. The inhibitory effect of PMA appears to be exerted at the level of VDR mRNA expression. Northern blot analysis revealed significant decreases in steady state levels of VDR mRNA species that qualitatively corresponded to the decrease in VDR protein concentration seen on a Western blot.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaloids↗
PubMed1991

Effects of dexmedetomidine, a novel imidazole sedative-anesthetic agent, on adrenal steroidogenesis: in vivo and in vitro studies.

Inhibition of steroidogenesis may be produced perioperatively by imidazole compounds, such as the hypnotic agent etomidate, with potentially serious consequences for patient morbidity and mortality. Dexmedetomidine, ([+]4-[1-[2,3-dimethylphenyl]-ethyl]-1H-imidazole), another imidazole compound with anesthetic like properties, is now being used perioperatively. Therefore, we investigated the effects of dexmedetomidine on steroidogenesis as well as on binding to glucocorticoid receptors in a series of in vitro and in vivo animal studies. The effect of dexmedetomidine, 10(-8)-10(-3) M, on adrenocorticotrophic hormone (ACTH) stimulated release of corticosterone was assessed in isolated rat adrenal cells. To characterize dexmedetomidine interactions with the glucocorticoid receptor, dexmedetomidine's ability to compete for [3H]dexmethasone binding sites was studied in renal tubular cells. The effect of dexmedetomidine, 80 micrograms/kg subcutaneously, on ACTH-stimulated release of cortisol was studied in separate cohorts of dogs at various time intervals during and after anesthesia was given. To compare the inhibitory effects of etomidate and dexmedetomidine on steroidogenesis, ACTH-stimulated release of cortisol was studied in dogs treated with anesthetic doses of either dexmedetomidine (80 micrograms/kg IV) or etomidate (1 mg/kg IV). Finally, dogs were given dexmedetomidine by continuous subcutaneous infusion for 7 days at sedative doses after which their cortisol response to ACTH was determined. At dexmedetomidine concentrations greater than 10(-7) M, a dose-dependent inhibition of corticosterone release was detected in response to ACTH stimulation in vitro. At these high dexmedetomidine concentrations, [3H]dexamethasone binding was not affected. In the in vivo dog experiments, basal cortisol levels decreased and the cortisol response to ACTH was blunted 3 h after dexmedetomidine administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Cortex↗
PubMed1991

Genetic defects of the 1,25-dihydroxyvitamin D3 receptor.

Target organ resistance to steroid hormone action is known to produce clinical disorders ranging from testicular feminization in the case of androgen resistance to hypocalcemic vitamin D-resistant ricets (HVDRR) in the case of 1,25-dihydroxyvitamin D3. The etiologic basis of these disorders is thought to be genetic mutations in the gene encoding receptors for these hormones. We investigated this possibility by analyzing the vitamin D receptor (VDR) protein, mRNA, and DNA from patients with HVDRR. This autosomal recessive disease of children is characterized by early onset rickets, hypocalcemia, hyperparathyroidism, and elevated levels of 1,25-(OH)2D3. Cells from patients fall into three general classes of molecular defects: (i) decreased or absent hormone binding; (ii) decreased affinity of VDR for DNA, or; (iii) defective nuclear translocation or retention. Analysis of the DNA and/or mRNA from these cells has identified missense mutations in the DNA binding (zinc finger) domain and a nonsense mutation in the steroid binding domain of VDR. The mutations were individually recreated in wild type VDR and the expressed mutant protein behaved biochemically identically to the patient receptor. Further studies have shown that the receptor is unable to interact with the specific hormone response element (HRE) of the osteocalcin gene and activate appropriate transcription. Rapid diagnostic genotyping of these mutations is possible with either restriction digestion or allele-specific oligonucleotide hybridization. Analysis of these naturally occurring, disease producing mutations of a gene regulatory protein should provide insight into the key amino acid residues of the protein and the mechanism by which steroids modulate gene transcription.

Amino Acid Sequence↗
PubMed1991

Experimental and clinical applications of fibrin glue.

A 2-year experience with laboratory and clinical applications of fibrin glue is presented. An autologous technique, which eliminates the danger of multidonor preparations, has been developed in our blood bank. While one can obtain different fibrinogen concentrations from the same amount of a patient's blood, in vitro mechanical testing demonstrated that at higher fibrinogen concentrations there is an increase in shear adhesive strength. Evaluation of skin-graft take in 16 Sprague-Dawley rats did not demonstrate significant differences in healing when adhesive use was compared with suture technique. In a clinical study, four different groups of patients (facial burns, hand burns, difficult graft sites, and miscellaneous surgical applications) benefited from autologous or single-donor fibrin glue for a total of 82 cases. There are several distinct advantages to the use of fibrin adhesive: The autologous technique eliminates the risk of transmissible viral diseases (AIDS, hepatitis); it can be used as a sealant in the treatment of seromas, dural leaks, and lymphoceles; and it improves hemostasis and early graft adherence. Face and hands are resurfaced with sheet grafts in a single procedure, obtaining a better aesthetic result with complete graft take and immediate start of physical therapy. Neither sutures nor pressure dressings are required. The minimal postoperative care associated with early return to normal activities seems to increase the satisfaction of patients and nurse personnel.

Adolescent↗
PubMed1990

N-methyl-D-aspartate-evoked calcium uptake by kitten visual cortex maintained in vitro.

As a functional measure of NMDA receptor effectiveness in kitten striate cortex, the uptake of 45Ca by visual cortical slices was measured after 2 minute bath applications of N-methyl-D-aspartate (NMDA). Significant Ca uptake occurred in response to 12.5-100 microM NMDA in slices prepared from visual cortex of normal animals aged 28-48 days. Basal uptake (in the absence of NMDA) was increased and evoked uptake was decreased in visual cortical slices prepared from age-matched dark-reared animals. Four days of binocular deprivation in otherwise normally reared animals had no effect on basal uptake, but significantly lowered NMDA-evoked Ca uptake at agonist concentrations greater than 25 microM. These data suggest that even brief manipulations of sensory experience are sufficient to alter visual cortical calcium regulation.

Animals↗
PubMed1990

Mitochondrial inclusions in keratinocytes of hairless mouse skin exposed to UVB radiation.

Mitochondrial inclusions were observed in keratinocytes during an ultrastructural investigation of the skin of hairless mice exposed to UVB radiation. Mice were irradiated 3 times a week for 5-6 months with sunlamps at individual doses seldom exceeding 0.06 J/cm2. Strips of dorsal skin were processed for electron microscopic examination; blocks were sectioned to include both epidermis and dermis. Mitochondrial inclusions were observed in keratinocytes of the basal, spinous and granular layers. They were spherical in shape and of moderate and homogeneous electron density. Mitochondria toward the upper regions of the epidermis were swollen and had fragmented cristae; mitochondria in the lower areas of the epidermis usually contained smaller and less dense inclusions and intact or partially disrupted cristae. Because mitochondria are essential in providing the energy for cellular function, keratinocyte mitochondrial damage induced by UVB radiation may have serious pathological consequences. Possible mechanisms involved in mitochondrial inclusion formation are suggested.

Animals↗
PubMed1990

The molecular basis of hereditary 1,25-dihydroxyvitamin D3 resistant rickets in seven related families.

Hereditary 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] resistant rickets (HVDRR) is an autosomal recessive disease caused by target organ resistance to the action of 1,25(OH)2D3, the active form of the hormone. The defect in target cells is heterogenous and commonly appears to be a mutation in the gene encoding the vitamin D receptor (VDR). We have studied cultured skin fibroblasts and Epstein-Barr virus transformed lymphoblasts of seven family branches of an extended kindred having eight children affected with HVDRR. We have previously shown that cells from three affected children in this group contain an "ochre" nonsense mutation coding for a premature stop codon in exon 7 within the steroid-binding domain of the VDR gene. In the current studies, we found that cells from affected children failed to bind [3H]1,25(OH)2D3 and had undetectable levels of VDR as determined by immunoblots using an anti-VDR monoclonal antibody. Measurement of VDR mRNA by hybridization to a human VDR cDNA probe showed undetectable or decreased abundance of steady-state VDR mRNA. Parents, expected to be obligate heterozygotes, showed approximately half the normal levels of [3H]1,25(OH)2D3 binding, VDR protein, and mRNA. The mutation at nucleotide 970 (counting from the mRNA CAP site) results in the conversion of GTAC to GTAA, which eliminates an Rsa I restriction enzyme site and facilitates identification of the mutation. We found that polymerase chain reaction (PCR) amplification of exons 7 and 8 from family members and subsequent Rsa I digestion allows detection of the specific genotype of the individuals. When Rsa I digests of PCR-amplified DNA are subjected to polyacrylamide gel electrophoresis, children with HVDRR exhibit a homozygous banding pattern with loss of an Rsa I site. Parents exhibit a heterozygotic DNA pattern with detection of both normal and mutant alleles. In summary, our data show that the genetic abnormality is a point mutation within the steroid-binding domain of the VDR in all seven related families with HVDRR. Analysis of restriction fragment length polymorphism at the 970 locus of PCR-amplified DNA fragments can be used to diagnose this mutation in both affected children and parents carrying the disease.

Blotting, Northern↗
PubMed1990

The dual effect of epidermal growth factor upon human chorionic gonadotropin secretion by the first trimester placenta in vitro.

Epidermal growth factor (EGF) is believed to play an important role in the regulation of placental function. We have examined the effect of EGF upon first trimester (7-10 gestational weeks) placental hCG secretion and cellular differentiation using both static (explants and isolated cells) and kinetic (superfusion of explants) culture methods. In superfused explants, short (1-4 min) pulses of EGF increased both the rate and amplitude of spontaneous pulsatility of hCG. The frequency increased from 3/h to 5/h, and the amplitude increased compared to the control channels as calculated by the area under the curve. This effect was dose dependent and the concentration of 50 ng/ml, which was the lowest dose tested, was the most effective. In explants cultured for 24 h, EGF caused a 2-fold increase in hCG secretion, compared to control, P less than 0.05. In two different dispersed trophoblastic cell cultures, EGF added daily for the first week caused a 180% increase in hCG secretion, P less than 0.05. However, according to morphological criteria, i.e. light microscopy and vital staining, no significant effect upon the rate of differentiation to syncytiotrophoblast was observed in long-term cultures of one of these preparations. In conclusion, EGF plays a dual trophic role in stimulating hCG secretion in the first trimester. However, this effect is not dependent on cellular differentiation.

Cell Differentiation↗
PubMed1989

Mutant vitamin D receptors which confer hereditary resistance to 1,25-dihydroxyvitamin D3 in humans are transcriptionally inactive in vitro.

Hereditary 1,25-dihydroxyvitamin D3-resistant rickets is a human syndrome that arises as a result of heterogeneous molecular defects in the vitamin D3 receptor. Recent studies have identified single unique point mutations within the second or third exons that encode the DNA-binding domain of the vitamin D receptor (VDR) gene in two families with this syndrome. In the experiments reported here, these mutations were introduced into the normal VDR cDNA by site-directed mutagenesis and the mutant products evaluated for hormone, nuclear, and DNA-binding characteristics. Each mutant VDR was expressed in COS-1 cells at equivalent levels, and saturation analysis of cell cytosol revealed normal affinity for the 1,25-dihydroxyvitamin D3 hormone. Incubation of transfected cells with radiolabeled hormone followed by lysis and extraction suggests a lowered salt dependence for solubilization of the mutant VDR. Concomitantly, mutant receptors exhibited reduced affinity for immobilized calf thymus DNA. While cotransfection of the wild type receptor together with a vitamin D-inducible (osteocalcin) chloramphenicol acetyltransferase reporter gene construction in CV-1 cells resulted in strong induction by 1,25-dihydroxyvitamin D3, neither mutant receptor was capable of directing significant activity either as a function of receptor or hormone concentration. These data suggest that the unique point mutations identified in each of these two families are responsible not only for the phenotype originally ascribed to the abnormal receptor but also severely compromise each protein's ability to activate transcription.

Animals↗
PubMed1989

Steroid metabolism as a mechanism of escape from progesterone-mediated growth inhibition in Trichophyton mentagrophytes.

It has been shown by us and others that progesterone inhibits the growth of Trichophyton mentagrophytes and that the organism escapes from this inhibition over time. We report here studies which show that escape from growth inhibition is related to the enzymatic transformation of progesterone to polar metabolites. Isolation and identification of the progesterone metabolites confirm the production of 15 alpha-hydroxyprogesterone. In addition, three other metabolites were isolated. Two of these were determined to be 1-dehydroprogesterone and 11 alpha-hydroxyprogesterone. The third metabolite was a 1-dehydro-hydroxyprogesterone, but the location of the hydroxyl group could not be determined unequivocally. Studies using authentic 15 alpha-hydroxyprogesterone, 1-dehydroprogesterone, and 11 alpha-hydroxyprogesterone reveal that these derivatives are significantly less inhibitory to the growth of T. mentagrophytes than progesterone. Pretreatment of organisms with progesterone augments the rate of metabolism and enhances escape. We have described previously a progesterone-binding protein (PBP) in cytoplasmic extracts of T. mentagrophytes and hypothesized that progesterone mediates growth inhibition by binding to the PBP of this organism. The relative binding affinity that progesterone and its metabolites display for PBP correlates with the relative growth inhibitory potency of these compounds. These results suggest that metabolism of progesterone to more polar and less inhibitory compounds, which exhibit lower affinity for PBP, is the mechanism of escape from progesterone-mediated inhibition of growth in this organism.

Binding, Competitive↗
PubMed1989

Sequence and expression of human myosin alkali light chain isoforms.

In order to initiate the study of the functional differences between myosin alkali light chain isoforms and to investigate the mechanisms of their differential expression, we have isolated cDNA clones for two human alkali light chain isoforms. Here we report DNA sequence and RNA blotting analyses that demonstrate that these cDNAs represent transcripts encoding human MLC3F and MLC1Sb. The sequence of the human MLC1Sb cDNA offers the first fully characterized example of a slow-fiber skeletal muscle alkali light chain isoform from any species. The sequence analysis of these two cDNAs allows an examination of evolutionarily conserved features of mammalian alkali light chain genes. Examination of the genomic organization of the human alkali light chain isoform genes revealed that, in contrast with some strains of mice, both are single copy genes. RNA blot analysis conclusively demonstrates that the human skeletal muscle MLC1Sb gene is also expressed in the heart ventricle but not the atria. In addition, we examined the expression of alkali light chain isoforms during the in vitro differentiation of a variety of human and rodent myogenic cells and found striking variation in the pattern of alkali light chain isoform gene expression in different myogenic cells.

Amino Acid Sequence↗
PubMed1989

An ochre mutation in the vitamin D receptor gene causes hereditary 1,25-dihydroxyvitamin D3-resistant rickets in three families.

Hereditary 1,25-dihydroxyvitamin D3-resistant rickets is a rare autosomal-recessive disease resulting from target-organ resistance to the action of the active hormonal form of vitamin D. Four affected children from three related families with the classical syndrome of hereditary 1,25-dihydroxyvitamin D3-resistant rickets and the absence of detectable binding to the vitamin D receptor (VDR) in cultured fibroblasts or lymphoblasts were examined for genetic abnormalities in the VDR gene. Genomic DNA from Epstein-Barr virus-transformed lymphoblasts of eight family members was isolated and amplified by polymerase chain reaction techniques. Amplified fragments containing the eight structural exons encoding the VDR protein were sequenced. The DNA from all affected children exhibited a single C----A base substitution within exon 7 at nucleotide 970 that resulted in the conversion of the normal codon for tyrosine (TAC) into a premature termination codon (TAA) at amino acid 292. This mutation causes a truncation of the VDR protein thereby deleting a large portion of the steroid hormone binding domain (amino acids 292-424). Although the affected children were all homozygotic for the mutation, the four parents tested all exhibited both wild-type and mutant alleles, indicating a heterozygous state. The functional consequences of this mutation were confirmed after expression of the recreated mutant VDR cDNA in mammalian cells. Recreated mutant receptor exhibited no specific 1,25-[3H]dihydroxyvitamin D3 binding and failed to activate a cotransfected VDR promoter-reporter gene construct. Thus these findings identify an ochre mutation in a human steroid hormone receptor in patients with hereditary 1,25-dihydroxyvitamin D3-resistant rickets.

Amino Acid Sequence↗
PubMed1989

Influence of oestradiol on protein expression and methionine utilization during morphogenesis of Paracoccidioides brasiliensis.

The temporal sequence of cytosolic protein expression during phase transition of Paracoccidioides brasiliensis was examined. Electrophoretic analysis of cytosol proteins by one-dimensional SDS-PAGE revealed numerous differences between the mycelial and yeast forms as well as alterations induced by 17 beta-oestradiol. Using either protein staining or fluorography of [35S]methionine-labelled proteins 30 phase-specific bands were detected, 12 mycelial-associated bands (range 30 to 140 kDa) and 18 yeast-associated bands (range 22 to 127 kDa). In cells undergoing mycelial to yeast transition after a shift from 25 degrees C to 37 degrees C, the protein patterns showed a temporal progression toward the yeast profile with the accumulation of yeast bands prior to observable morphogenesis. Five novel protein bands (range 23 to 50 kDa) were detected by silver staining during transition. Treatment of temperature-shifted mycelial cultures with 2.6 x 10(-7) M-oestradiol altered observed profiles; 4 of 12 mycelial-associated bands were maintained whereas the appearance of the 5 novel transition bands and 9 of 18 yeast-associated bands was blocked or delayed. Analysis of [35S]methionine-labelled proteins revealed that oestradiol induced label uptake by mycelial cells, blocked the synthesis of a 92 kDa yeast-specific band 72 h into transition, and diminished label incorporation 120 h into transition. In conjunction with these steroid-induced alterations of protein expression, little or no morphological transformation occurred. These results support our hypothesis that, analogous to mammalian steroid receptor action, the functional responses of P. brasiliensis to oestradiol are related to regulation of protein expression, presumably mediated via a specific binding protein-ligand complex.

Cytosol↗