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Biomedical subjects

G R Cunningham

Publications and source records attributed to G R Cunningham.

At least 37 records · Page 2Linked to original sources
PubMed1988

Monoclonal antibodies against the androgen receptor: recognition of human and other mammalian androgen receptors.

Monoclonal antibodies against the androgen receptor (AR) will provide useful probes for elucidating both the structure and function of this important regulatory protein. Recently, human autoimmune anti-AR sera have been described. The purpose of the current work was to immortalize lymphocytes from the blood of patients with high titer anti-AR antibodies and to produce monoclonal antibodies against the receptor in vitro. Human serum samples (10 microliters) were incubated in high ionic strength buffer (400 mM KCl) for 16 h at 0 C with [3H]Mibolerone-labeled cytosol (100-200 fmol AR) from Dunning tumors. Receptor-antibody complexes were precipitated with goat antihuman immunoglobulin (Ig) antibody. From our 1005 serum samples examined, 5 specimens were detected which precipitated greater than 40% of the AR. These antibodies recognized the AR from human, rat, mouse, dog, steer, chicken, and hamster, but did not recognize estrogen, progesterone, or glucocorticoid receptors. By sucrose gradient analysis in high salt (0.4 M KCl) 1 of the antisera shifted the 4.4S monomeric receptor to 8S, and the others shifted the receptor to 18S. However, all of the antibodies were shown to be IgG class by immunoprecipitation with class-specific second antibodies. Peripheral blood lymphocytes donated by these patients were isolated by histopaque density gradient sedimentation, activated in vitro, transformed with Epstein-Barr virus, and seeded into 96-well plates. From 263 million human lymphocytes plated in 96-well dishes, 1215 wells gave rise to Epstein-Barr virus-transformed lymphoblastoid cells, and 8 of these wells were determined to be anti-AR positive. Cells from 2 of the positive wells were cloned and designated CB54 and UA67, both of which secreted IgG class antibodies against the AR. These 2 monoclonal antibodies have been shown to be highly specific for the AR and to cross-react with the AR from human, rat, and hamster. Studies with the monomeric form of the AR and its proteolytic fragment using sucrose density gradients have suggested that the 2 antibodies recognize different epitopes on the monomeric AR molecule. Furthermore, by Western blot analysis the antibodies have identified the AR as an 118K protein on a sodium dodecyl sulfate gel, which is consistent with our previous findings of the mol wt of the AR.

Animals↗
PubMed1987

Acid phosphatase activity: a marker of androgen action in prostate explant cultures.

Acid phosphatase activity in rat ventral prostate explants has been assayed to determine if this parameter could serve as a specific and quantitative marker of androgen action in this in vitro model. Dihydrotestosterone (10 nM) caused an absolute increase in both total (42.5 +/- 2.9 vs control 27.1 +/- 4.0 nmoles p-nitrophenol generated in 30 min/micrograms DNA, P less than .01) and tartrate-resistant acid phosphatase activity (34.1 +/- 1.5 vs control 17.2 +/- 2.8 U/micrograms DNA, P less than .05), and this effect was maximal on the 4th day of culture. This was the time when explant weight and DNA content tended to fall or only to be maintained by androgen. Similar changes were observed with the potent synthetic androgen, mibolerone. The addition of either the antiandrogen cyproterone acetate or flutamide in a 100-fold excess to that of androgen caused significant inhibition in acid phosphatase activity. No significant change was observed at low concentrations of estradiol or progesterone, and only minimal and inconsistent increases in activity were noted at high concentrations. No increase was noted when cortisol, cyproterone acetate, or flutamide was added to the media. We conclude that measurement of acid phosphatase activity in cultured explants of rat ventral prostate provides a biochemical marker of androgenicity that is more specific than measurement of [3H]-thymidine incorporation.

Acid Phosphatase↗
PubMed1987

Successful immunosuppressive therapy in a patient with autoantibodies to insulin receptors and immune complex glomerulonephritis.

Altered mental status, acanthosis nigricans, immune complex glomerulonephritis with nephrotic syndrome, fasting hypoglycemia, and postprandial hyperglycemia associated with anti-insulin receptor antibodies (type B insulin resistance) developed in a 43-year-old black woman who initially was treated for diabetes mellitus. Her HLA phenotype was A2, A29, Bw45(w6), B13(w4), Cw3, DR1 (DQw1). Her serum contained immune complexes, low complement levels, and antibodies that bound to the glomerular mesangium of mouse kidney. All clinical and serologic abnormalities resolved with combination cyclophosphamide and glucocorticoid treatment, and low doses of these agents have maintained the remission for more than a year.

Adult↗
PubMed1986

Age dependency of androgen and estrogen effects on incorporation of (3H)-thymidine by rat prostates in organ culture.

Organ culture of the rat ventral prostate has been evaluated as a model for studying the biological effects of androgen agonists, androgen antagonists, and estrogens. Explants were cultured for up to 8 days, and incorporation of (3H)-thymidine and (3H)-uridine by the explants was measured. Dihydrotestosterone (DHT) increased the incorporation of (3H)-thymidine/microgram DNA when compared with the untreated controls at 4 days, P less than .05; and at 6 days, P less than .01. Enhanced uptake in explants from 6 to 8-week old rats also was observed with 10 nM estradiol (P less than .05) and 10 nM cyproterone acetate (P less than .02). DHT (10 nM) caused greater enhancement of uptake in explants from 3-week-old rats than in explants from 6- or 12-week-old rats. In contrast, estradiol (E2) increased incorporation only in prostates from the 6-week-old rats. Since both DHT and E2 can enhance (3H)-thymidine uptake even though they are associated with strikingly different effects on prostate morphology, it suggests that their effects on (3H)-thymidine incorporation are mediated by different cells.

Adenocarcinoma↗
PubMed1983

Characterization of steroid binding specificity of the androgen receptor in human foreskin fibroblasts.

Our objective was to evaluate a convenient in vitro model for measuring steroid affinities to the human androgen receptor. The ability of unlabeled analogues of dihydrotestosterone (DHT) to compete with [3H]DHT for binding to the receptor in human fibroblasts was measured and expressed relative to DHT. The C-3 ketone group and the planar configuration of the A and B rings were critical for binding. Absence of the 10 beta-methyl group increased affinity of the androstane compounds for the receptor. The 17 beta-hydroxyl group was also essential for high affinity binding and addition of a 17 alpha-methyl group enhanced binding. Binding of steroids with a delta 4 double bond was consistently less than that of the 5 alpha-reduced steroids. This was true of both the androstene and estrene series. We conclude that human foreskin fibroblasts offer a useful model for in vitro studies characterizing the effects of steroid structural modifications on binding to the human androgen receptor.

Binding, Competitive↗
PubMed1983

Plasma growth hormone during sleep in young and aged men.

Plasma growth hormone measured at 20-min intervals across the night via indwelling venous cannula revealed a significant reduction in 16 healthy aged men as compared with 14 healthy young men. The decrease in growth hormone was entirely confined to the first 3 hours of the night (M +/- SEM for the integrated level was 5.0 +/- 1.2 and 21 +/- 4.2 ng . hr/ml). Growth hormone across the latter part of the night did not differ. Day and evening growth hormone levels measured hourly in five of the aged and nine of the young men failed to reveal an age effect. Growth hormone release is known to be associated with sleep onset, and particularly with slow wave sleep, stage 3 and 4, which was reduced in amount in these aged men (9.9 +/- 1.9 and 22.8 +/- 2.3% of time in bed for aged and young groups, respectively). Sleep stage 3 and 4 correlated significantly with growth hormone level (R = .463, p less than .01) for young and aged men combined, but not for either age group alone, indicating that growth hormone does not have a direct, simple relationship with slow wave sleep.

Adult↗
PubMed1981

Steroid structural requirements for high affinity binding to human sex steroid binding protein (SBP).

The sex steroid binding protein (SBP) which binds androgens circulating in the blood of man has been examined to determine the structural requirements for high affinity binding. SBP was purified partially and the ability of each of more than 150 steroids to compete with [3H]dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one) for binding to SBP was assessed. Binding was enhanced by reduction of the delta 4 double bond to 5 alpha-dihydro, addition of a methyl group at C-4 and in one case unsaturation at C-14,15. Affinity was always reduced by modifications of the C-17 beta hydroxy. Binding was also severely decreased by deletion of the keto moiety at C-3; however, relatively high affinity was retained by an alcohol or an unsubstituted pyrazole group at C-3. Certain alpha surface substitutions such as 17 alpha-ethinyl had limited effects on binding; whereas, other modifications such as 7 alpha-methyl or 17 alpha-methyl caused significant reduction in binding. Most modifications at C-2, 6, 9 or 11 also impaired affinity, and the 5 beta steroids had reduced affinity.

Androgen-Binding Protein↗
PubMed1979

Differences in steroid specificity for rat androgen binding protein and the cytoplasmic receptor.

Two proteins in the rat, androgen binding protein (ABP) and the cytoplasmic receptor (CR), have high affinity and limited capacity for binding androgens. To determine the structural requirements for binding with high affinity, each protein was partially purified and the ability of over 100 steroids to compete with [3H]dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one) for binding sites was assessed. The results indicate marked differences in the steroid specificities of the two proteins. Some alterations of dihydrotestosterone at C-2 or C-2 and C-3 increase binding to ABP two to four-fold. Similarly, the affinity of 17 beta-hydroxy-7 alpha-methyl-4-estren-3-one for ABP increases two-fold when a double bond is created at C-14. Addition of a methyl group in the alpha position at C-7 or C-17, or an ethinyl group at C-17 cause little change in affinity; however, modifications at C-11 and C-17 beta, and deletion of the methyl group at C-10 significantly impair binding to ABP. Binding to the CR is maintained or increased by deletion of the methyl group at C-10. Binding is lessened by modifications at C-3 and C-17 beta. Most alterations at C-2, C-7, C-11, and C-17 alpha have only minor effects on binding to the CR. These studies should provide a molecular basis for predicting the effects of specific structural modifications. When some modifications at C-2 or C-2 and C-3 are combined with changes at C-17 beta, the resulting steroids retain very high affinity for ABP and very limited binding to the CR. Such steroids may provide a means for assessing the function of ABP.

Androgens↗
PubMed1978

Mechanisms for the testicular hypertrophy which follows hemicastration.

The hormonal and testicular effects of hemicastration have been examined using rodent models. When rats were hemicastrated at 5 days of age, significant testicular hypertrophy was noted within 5 days. Hypertrophy decreased as the age at hemicastration approached 20 days and did not occur in rats 45 days of age or older. The changes in testicular weight were associated with significant (P less than 0.001) increases in serum FSH values from 10-20 days, but no significant alterations in the intratesticular concentration of testosterone occurred. Hemicastration also caused significant hypertrophy in testes depleted of germ cells. This was reflected by a substantial increase in testicular protein and DNA when compared to intact controls at 10, 15, and 20 days of age; however, the concentration of protein/microgram DNA was increased only at 15 days. These data indicate that 1) changes in serum FSH and not intratesticular testosterone are associated with the testicular hypertrophy which follows unilateral castration of immature animals; 2, a significant proportion of the hypertrophy can be attributed to non-germinal cells, the Sertoli cell being the prime candidate;3) the increase in testicular weight is primarily the result of an increase in cell number.

Animals↗
PubMed1978

The mechanism of ovulation inhibition by triamcinolone acetonide.

A single dose of 25 mg triamcinolone acetonide, when given on day 1 or 2 of the menstrual cycle, inhibits ovulation. To examine the mechanism of this action, daily determinations of plasm FSH, LH, estrone plus estradiol (E1 + E2), and progestins were performed. Some subjects also received a single dose of LH-RH or hCG on cycle day 15 or clomiphene citrate on days 5-9. Triamcinolone acetonide itself caused variable suppression of plasma estrogens, loss of the mid-cycle gonadotropin surge, and a deficient or absent rise in plasma progestins. Impaired secretion of estrogen did not seem to be due to low gonadotropin levels. FSH and LH responses to LH-RH were adequate in relation to prevailing estrogen levels. Four of six women treated with clomiphene responded with plasma progestin levels which exceed 8 ng/ml. Triamcinolone acetonide seems to affect the hypothalamic-pituitary-ovarian axis mainly by hypothalamic suppression and possibly by a direct effect on the ovary as well.

Adult↗
PubMed1978

Isolated deficiency of adrenocorticotropin (ACTH) and lipotropins (LPHs).

A patient with hypoadrenocorticism was found to have low basal plasma concentrations of ACTH and lipotropins and deficient responses of these hormones to insulin-induced hypoglycemia and lysine vasopressin. The adequacy of secretion of other anterior pituitary hormones was assessed either directly, by measuring their concentration in plasma, or indirectly, by assessing end organ function, under basal and stimulated conditions. The responses of gonadotropins to LRH and of PRL and TSH to TRH were normal. The etiology of this rare condition of isolated deficiency of ACTH and lipotropins remains to be elucidated.

17-Ketosteroids↗