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L Pinsky

Publications and source records attributed to L Pinsky.

At least 109 records · Page 6Linked to original sources
PubMed1983

Regulation of the androgen receptor by androgen in normal and androgen-resistant genital skin fibroblasts.

Normal genital skin fibroblast (GSF) monolayers incubated with serum-free medium containing 3 nM [3H]-5 alpha-dihydrotestosterone (DHT) at 37 degrees C for 20 h have about 35% more specific DHT-binding than replicates incubated in serum-free medium with [3H]-DHT for only 1 h to saturate basal specific androgen-receptor activity. If, after 19 h, spent medium is replaced by fresh medium with 3 nM [3H]-DHT for 1 h, specific DHT binding is 85% more than basal. The acquisition of increased binding is temperature dependent (37 greater than 27 degrees C) and cycloheximide (2 microM) suppressible. The increased binding activity is considered to represent an augmentation of androgen receptor concentration because it has the same equilibrium dissociation constant (KD approximately 0.5 nM), rate constant of dissociation (k-1 approximately 6 x 10(-3) min-1) and ligand specificity as basal androgen-receptor activity, and because basal DHT-binding activity is stable in cells preincubated in androgen-free or serum-free medium alone for up to 72 h before assay. Prolonged incubation with methyltrienolone (R1881), a nonmetabolizable synthetic androgen, causes a greater, more persistent increment of androgen receptor activity than does equimolar DHT. The fibroblasts from two subjects with receptor-positive, partial androgen resistance lose their basal receptor activity during prolonged incubation with DHT, but augment it normally with R1881. This suggests that defective DHT metabolism is somehow involved in the pathogenesis of their androgen resistance.

Cycloheximide↗
PubMed1983

The dissociation of testosterone- and 5 alpha-dihydrotestosterone-receptor complexes formed within cultured human genital skin fibroblasts.

We have studied the rate and character with which testosterone (T) and 5 alpha-dihydrotestosterone (DHT) dissociate from the androgen receptor both within intact cultured genital skin fibroblasts of a subject with 5 alpha-reductase deficiency and after the androgen-receptor complexes have been extracted from the cells. Within the cells, the kinetics of the dissociative process for each hormone was first-order, but T dissociated four times faster than DHT. An Arrhenius plot of the variation of the dissociation rate constants with temperature for T was linear and yielded an activation energy of 28 kcal/mol. This value is identical with the one previously determined for activated DHT-receptor complexes. T-receptor complexes extracted from the cells dissociated with complex kinetics: at 37 degrees C the rate constants of the "fast" and "slow" components were 40 and 14 X 10(-3) min-1, respectively. In contrast, DHT-receptor complexes extracted from the cells dissociated with first-order kinetics and at a rate identical to that observed within cells, except after exposure to pyridoxal 5'-phosphate (5 mM) or concentration by Amicon (B-15) filtration, when their dissociation kinetics became complex. We interpret these data to mean that, within the cells, both T- and DHT-receptor complexes exist predominantly in the activated state whereas, when extracted from the cells, DHT-receptor complexes remain activated, unless perturbed, while T-receptor complexes become unstable spontaneously, probably by reverting to a preactivated state.

Cells, Cultured↗
PubMed1983

Possible implications of the comparative dissociative behavior of testosterone- and 5 alpha-dihydrotestosterone-receptor complexes formed by human skin fibroblasts.

We have studied the dissociative behavior of 5 alpha-dihydrotestosterone (DHT)- or testosterone (T)-receptor complexes extracted from normal and 5 alpha-reductase-deficient human genital skin fibroblasts, respectively. The rate at which each hormone dissociates from the androgen receptor is temperature-dependent (29-40 degrees C), but DHT does so in a first-order (monophasic) manner, while T yields complex (biphasic) dissociation kinetics. Prior exposure to 0.1 M sodium thiocyanate induces DHT-receptor complexes to dissociate with complex kinetics. The temperature dependence of these complex dissociation profiles can be simulated identically by a kinetic model involving three conformational states of the androgen-receptor complex. Neither the rate constants of the rapidly- or slowly-dissociating components of T-receptor dissociation, nor the magnitude of the latter are affected by sodium molybdate (5 mM). Similarly, neither sodium molybdate nor potassium chloride (0.1-0.3 M) alters the complex dissociative behavior of DHT-receptor complexes that is induced by 0.1 M sodium thiocyanate.

Dihydrotestosterone↗
PubMed1983

Regulation of the androgen receptor in human genital skin fibroblasts, with a review of sex steroid receptor regulation by homologous and heterologous steroids.

When normal human genital skin fibroblasts are cultured for 3 days with 2-3 nM methyltrienolone (R1881, a synthetic nonmetabolizable androgen), they augment their specific androgen-receptor activity (two- to four-fold) with a time pattern that is always most rapid in the first 24 h, usually peaks by 48 h, and often declines, sometimes substantially, in the third 24-h interval. The time pattern is highly reproducible within experiments on confluent monolayers and is not influenced by the presence or absence of 10% fetal calf serum in the medium, the temperature (37 vs. 40 degrees C) at which the incubation is conducted, or the basal activity (up to 40 fmol/mg protein) that is saturated by incubation for 30-45 min at 37 degrees C. It does appear to be influenced by an unusually rapid increase in the first 24 h and by nonconfluent density of the monolayers, but these factors do not explain the considerable interexperimental variation, within and among cell lines, under nominally identical conditions. The time pattern is interpreted to represent an initial phase of "up-regulation" that is followed by one of compensatory adjustment, despite a constant level of unmetabolized ligand. A review of sex steroid receptor regulation by homologous and heterologous sex steroids reveals numerous examples of apparently homologous regulatory behavior.

Animals↗
PubMed1982

Defective activation of androgen-receptor complexes: a marker of androgen insensitivity.

Partially-purified 5 alpha-dihydrotestosterone-receptor (DHT-R) complexes, extracted from normal genital skin fibrolasts (GSF) previously labelled with [3H]DHT, dissociate with monophasic kinetics and dissociation rate constants (k-2) of 10, 6, 3 and 2 x 10(-3) min-1 at 40, 37, 32 and 29 degrees C, respectively. An Arrhenius plot yields an activation energy of 28 kcal/mole. We studied 2 subjects who have constitutional androgen insensitivity (AI) despite a normal level of specific DHT-R activity in their GSF. Subject 1 has complete AI and unambiguous female external genitalia; subject 2 has partial AI and had ambiguous external genitalia at birth. In contrast to normal, the DHT-R complexes extracted from the GSF of these 2 subjects dissociate with biphasic kinetics. At 37 degrees C the k-1 of their early ('fast') component is 21 +/- 0.4(+/-SEM) x 10(-3) min-1(n = 7), while that of their late ('slow') component (k-2) is 7.8 +/- 0.3 x 10(-3) min-1 (n = 7). The latter value is very similar to the single k-2 (6.1 +/- 0.1 x 10(-3) min-1, n = 9) of the DHT-R complexes extracted from normal fibroblasts. When dissociation of DHT-R complexes is studied with intact fibroblasts, monophasic kinetics are observed for both the normal and mutant subjects. A k-1 of 18 x 10(-3) min-1 was previously observed for both mutant subjects at 37 degrees C (normal: K-2, 5.9 +/- 0.3 x 10(-3) min-1, n = 15). At 40 degrees C subject 1 has a rate constant of 25 while that of subject 2 is 50 x 10(-3) min-1(normal: 10 x 10(-3) min-1). An Arrhenius plot of the results from subject 1 yields an activation energy of 18 kcal/mole. The 2 sets of data suggest that inability of DHT-R complexes to transform from a rapidly dissociating to a slowly-dissociating form within intact target cells is a marker of genetic mutations that alter the androgen receptor and thereby cause certain types of partial of complete AI.

Androgen-Insensitivity Syndrome↗
PubMed1982

Sodium thiocyanate: a probe for the conformations of the androgen-receptor complex.

In the presence of sodium thiocyanate (NaCNS), partially purified 5 alpha-dihydrotestosterone-receptor (DHT-R) complexes extracted from normal genital skin fibroblasts (GSF) dissociate with complex (biphasic) kinetics. The rate constant of the 'fast' component and the magnitude of the 'slow' component vary with temperature (29-37 degrees C) and NaCNS concentration (0.1-0.4 M). Equimolar sodium bromide is much less effective; potassium chloride up to 1 M has no effect. DHT-R complexes from the GSF of a subject with partial androgen insensitivity (PAI) yield biphasic dissociation profiles that differ from normal and are influenced by NaCNS. Together with the temperature-dependent, first-order (monophasic) dissociative behavior of normal DHT-R complexes in the absence of NaCNS (Kaufman et al., 1982), the foregoing data have been used to construct a kinetic model involving the dissociation of DHT from 3 conformationally related forms of the androgen-receptor complex: (1) dysactivated; (2) preactivated; (3) activated.

Dihydrotestosterone↗
PubMed1979

Complete androgen insensitivity with a normal amount of 5 alpha-dihydrotestosterone-binding activity in labium majus skin fibroblasts.

We have studied labium majus skin fibroblast strains from six unrelated, previously unreported, patients with complete androgen insensitivity (CAI). Four have markedly reduced specific 5 alpha-dihydrotestosterone-binding (DHT-binding) activity (receptor-negative). The other two (receptor-positive) have normal activities in whole-cell homogenates, the 100,000g supernatant of cell sonicates prepared in 0.4 MKCl, purified whole nuclei, and the nuclear fraction that resists extraction with 0.4 MKCl. In one of the two, the DHT-binding macromolecules in the cytosol and 0.4 M KCl-extractable nuclear fraction have normal molecular sieving profiles and binding activities. One of the receptor-positive patients has maternally related affected relatives in three successive genetations. Appreciable unexplained interexperimental variation of the DHT-binding activities in genital skin fibroblast strains demands that multiple assays be performed on a strain before its receptor status is classified quantitatively. In our experience to date with genital skin fibroblasts, four of 13 propositi with CAI have been receptor-positive.

Adolescent↗
PubMed1978

5alpha-reductase activity of genital and nongenital skin fibroblasts from patients with 5alpha-reductase deficiency, androgen insensitivity, or unknown forms of male pseudohermaphroditism.

Serially subcultured fibroblast strains from genital (foreskin, labium majus) skin, as a group, have considerably more steroid (testosterone) 5alpha-reductase activity than those form nongenital skin. Foreskin strains varied 40-fold and labial strains even more. Labial strains overlapped nongenital strains of either sex more frequently than did prepuce strains. The activity of foreskin strains from two siblings with proven 5alpha-reductase deficiency was clearly lower than that of any of 18 control stains. The comparative behavior of the various strain types indicates that labial and nongenital strains should not be used to support a clinical suspicion of male pseudohermaphroditism due to 5alpha-reductase deficiency. The activities of labial strains from patients with complete androgen insensitivity (testicular feminization) - five with the receptor-negative variety and two with the receptor-positive type - were as variable as those of control labial strains. The decreased 5alpha-reductase activity observed in fresh skin slices of some patients is probably and expression of their functional estrogen/androgen imbalance in vivo.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1978

The syndromology of anorectal malformation (atresia, stenosis, ectopia).

The syndromes, associations, and developmental field defects that include anorectal dysgenesis (atresia, stenosis, ectopia) as a principal or facultative sign are discussed. Most of these disorders are identifiable by their genetic or teratogenic etiology, their distinctive phenotype, or both. Their precise diagnosis is crucial for estimation of recurrence risk and other aspects of reproductive counseling, and it is essential for classificatory progress. The "VACTERL association" should not be used to label a patient with anorectal malformation and other anomalies except by exclusion; this rule is particularly relevant when the patient lacks tracheoesophageal malformation. The degree (or variety) of anorectal malformation that occurs in a given pattern of multiple congenital anomalies may be inconstant. Furthermore, anorectal malformation may be a solitary expression of a familial syndrome.

Abnormalities, Multiple↗
PubMed1978

Some properties of the specific androgen-binding activities in cultured human genital skin fibroblasts.

Specific 5 alpha-dihydrotestosterone (DHT)-binding activity in the cytosol (C) and 0.4 M KCl-extractable nuclear fraction (N) of cultured human fibroblast cell strains developed from preputial (n = 12) and labium majus (n = 12) skin were analyzed by gel exclusion chromatography, sucrose gradient sedimentation, and thermostability. Both fractions had activities that were excluded from Sephacryl S-200 columns; another component (mol wt, 20,000) was present in the N fraction. The C was more thermostable than a homologous N activity, and addition to the former of KCl to 0.4 M had no effect. There was large, overlapping variation in thermostability of the C and N activities among strains from either site, sister strains developed from a single skin biopsy, and even among serial subcultures within a strain; likewise, the variable sedimentability of the C (4-7S) and N (3.2-5.9S) activities prevented their consistent discrimination. Each type of variation occurred despite excellent intraexperimental replication. The thermostability of a given N activity varied directly with its sedimentation coefficient. By cluster analysis, the data relating thermostability of a given N activity with the percentage of 0.4 M KCl-resistant nuclear activity segregated into two populations; within each population these two measurements were related inversely. We suggest that these coordinate behaviors of the N activity reflect intrinsic properties of the androgen-receptor system in normal genital skin fibroblasts which may be useful for defining qualitative aberrations of the system in receptor-positive forms of congenital androgen insensitivity.

Adolescent↗
PubMed1977

Male pseudohermaphroditism: diagnosis in cell culture.

Testicular feminization is a classic form of complete male pseudohermaphroditism. The individuals have a normal XY karyotype but unambiguously female external genitalia. They have congenital complete insensitivity to androgen due to an X-linked mutation. In four patients (from tow families with several affected members) with the typical phenotype of testicular feminization, a severe deficit of specific androgen-binding activity was detected in cultured fibroblasts from labium majus skin. Measurement of this activity in genital skin fibroblasts improves the differential diagnosis in patients with complete or imcomplete male pseudohermaphroditism before puberty.

Adolescent↗