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Biomedical subjects

A F Clark

Publications and source records attributed to A F Clark.

At least 73 records · Page 4Linked to original sources
PubMed1989

Anterior chamber-associated immune deviation induced by soluble antigens.

Immune responses to cellular antigens placed in the anterior chamber of the eye are deviant: antibodies and cytotoxic T cells are generated, but delayed hypersensitivity is impaired. To determine whether a similar pattern of unusual reactivity would be induced by soluble antigens placed in this privileged site, we have examined the systemic immune responses of mice to anterior chamber injections of bovine serum albumin and bovine retinal S antigen--both soluble molecules. Recipients of intraocular injections of these antigens without adjuvant developed no detectable systemic immune response. When BSA was mixed with complete or incomplete Freund's adjuvant and injected into the anterior chamber, recipients produced serum specific antibodies; however, they displayed impaired delayed hypersensitivity. Anterior chamber recipients of soluble antigens subsequently proved refractory to the development of delayed hypersensitivity when immunogenic doses of the same antigens were placed subcutaneously. Moreover, the inability to mount delayed hypersensitivity could be adoptively transferred with spleen cells from animals that had previously received intraocular injections of bovine albumin or S antigen. It is concluded that soluble antigens, as well as surface membrane-bound antigens, are capable of inducing anterior chamber-associated immune deviation (ACAID). The possibility is discussed that the capacity of soluble retinal S antigen to induce ACAID may be pertinent to the maintenance of self-tolerance to this autologous, intraocular molecule.

Animals↗
PubMed1989

Ocular injection of retinal S antigen: suppression of autoimmune uveitis.

Uveoretinitis induced in Lewis rats by foot pad injections of retinal soluble (S) antigen is a model system used to study the immunopathogenesis of the autoimmune uveoretinitides in which delayed hypersensitivity plays a dominant role. Since down-regulation of delayed hypersensitivity can be achieved by injection of antigens into the anterior chamber of the eye, we have examined the potential of intracameral S antigen to mitigate experimental autoimmune uveitis. The results of these studies reveal that pretreatment of Lewis rats with S antigen injected into the anterior chamber markedly reduces the incidence and expression of experimental autoimmune uveitis.

Animals↗
PubMed1988

Androgen-independent epithelial cells of the rat ventral prostate.

Androgen-independent cell lines have been clonally selected from primary cultures of androgen-dependent epithelial cells from the rat ventral prostate. These rapidly dividing epithelial-like cells (RDE) have altered morphology and adherence characteristics. Unlike normal prostate epithelial cells, the RDE cell lines do not require androgens for cell division or cell survival. In the presence of physiological concentrations of testosterone, the isoelectric focusing patterns of prostatic acid phosphatases are abnormal in these RDE cells, and the prostate steroid-binding protein genes are not expressed. The loss of androgen dependence is not due to the inability of RDE cells to metabolize testosterone to 5 alpha-dihydrotestosterone, the active androgen, since the RDE cell lines metabolize testosterone in a manner similar to normal androgen-dependent epithelial cells. When RDE cells are grown on collagen matrices, the cells assume ductlike structures, similar to prostatic acini, although PSBP gene expression is not induced. When seeded into soft agar these cell lines form distinct foci, suggesting that they are potentially tumorigenic.

Acid Phosphatase↗
PubMed1988

Effect of a 4-methyl-4-aza steroid on androgen metabolism by rat ventral prostate epithelial and stromal cell cultures: selective inhibition of 5 alpha-reductase activity.

The effect of a potent steroid metabolic inhibitor, 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one (DMAA), on androgen metabolism was investigated in primary monolayer cultures of rat ventral prostate epithelial and stromal cells. Using testosterone (T) as substrate, 5 alpha-reductase (5 alpha-R) activity in both cell types was inhibited by greater than 98% at an inhibitor concentration of 1000 nM. The concentrations required to produce a 50% inhibition (IC50) were 7.4 and 9.0 nM for epithelial and stromal cells, respectively. To examine the specificity of this compound, its effect on other steroid-metabolic enzymes was examined. DMAA at a concentration of 1,000 nM had no effect on 3 alpha-hydroxysteroid oxidase (3 alpha-HSORox), 3-ketosteroid reductase (3 alpha-HSORred), and 6/7-hydroxylase (6/7-HSH) activities in both cell types; 17 beta-hydroxysteroid oxidase (17 beta-HSORox) activity, located primarily in epithelial cells, also was not influenced by DMAA. In contrast, epithelial 3 beta-hydroxysteroid oxidase (3 beta-HSORox) and 3-ketosteroid reductase (3 beta-HSORred) activities were inhibited by 65% (P less than .001) and 58% (P greater than .05), respectively, albeit the latter result was not statistically significant. Stromal 3 beta-HSORox and 3 beta-HSORred activities were negligible; hence the effect of the inhibitor of these enzymes could not be assessed. In conclusion, DMAA is a relatively selective and potent inhibitor of 5 alpha-R activity in primary cultures of rat ventral prostate epithelial and stromal cells and should be a useful compound for antagonizing androgen-mediated actions in the prostate and other androgen target tissues.

5-alpha Reductase Inhibitors↗
PubMed1988

Estrogen metabolism by primary cultures of rat ventral prostate epithelial and stromal cells.

Estrogen metabolism was examined in primary cultures of rat ventral prostate epithelial and stromal cells developed from young (approximately 3 weeks old) animals. Supraphysiologic concentrations (50 nM) of tritium-labelled estradiol (E2) and estrone (E1) were incubated separately with each cell type and the metabolites formed were measured at selected time points over a 24 h period. The metabolites were analyzed using high performance liquid chromatography. Epithelial cells exhibited an equal capability to interconvert E2 and E1 thus demonstrating the presence of similar oxidative and reductive activities for 17 beta-hydroxysteroid oxidoreductase (17 beta-HSOR) [0.45 and 0.40 pmol/3 h/microgram DNA respectively]. In contrast, stromal cells showed a 6-fold lower rate of oxidation of E2 to E1 (0.08 pmol/3 h/microgram DNA) but exhibited an approx 5-fold higher rate of reduction of E1 to E2 (1.81 pmol/3 h/microgram DNA). Estriol (E3) formation from either substrate was not detected in the two cell types. The results demonstrate that rat ventral prostate epithelial cells have similar capabilities to form or remove biologically active E2. In contrast, prostate stromal cells exhibited a preferential capability to form and possibly maintain high levels of biologically active E2. These findings are discussed with reference to the actions of estrogens on prostate epithelial-stromal cellular interactions.

17-Hydroxysteroid Dehydrogenases↗
PubMed1988

Androgen metabolism and regulation of rat ventral prostate growth and acid phosphatase during sexual maturation.

Androgen metabolism and the regulation of rat ventral prostate cell proliferation and secretory function were examined during sexual maturation. Changes in acid phosphatase (AP) characteristics were measured as a marker of androgen-dependent prostatic secretory function. In immature (21-day-old) rats, total AP activity per cell was low (14.2 +/- 1.3 mol p-nitrophenol phosphate hydrolysed/h per mg DNA); it increased threefold as the weight, protein and DNA contents of the prostate increased to adult (65-day) levels. This corresponded with significant (P less than 0.001) increases in the staining intensities of three of the four bands of secretory AP on isoelectric focusing gels. The extent of inhibition of AP by tartrate decreased at the same time. Secretory AP is known to be relatively tartrate-resistant. The changes in AP activity occurred after prostatic 5 alpha-dihydrotestosterone (5 alpha-DHT) levels increased from 4.6 +/- 0.7 pmol/mg DNA (21 days) to reach a peak of 17.6 +/- 2.3 pmol/mg DNA at 58 days. Prostatic 5 alpha-DHT concentrations were always higher than testosterone levels. Prostatic 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-Adiol) levels were lower than 5 alpha-DHT levels except on day 58 when levels peaked dramatically at 26.2 +/- 5.5 pmol/mg DNA. Changes in prostatic 5 alpha-DHT and 3 alpha-Adiol levels corresponded with changes in 5 alpha-reductase and 3 alpha-hydroxysteroid oxidoreductase (3 alpha-HSOR) activities. The oxidative reaction of 3 alpha-HSOR was approximately fourfold higher than the reductive reaction, indicating a preference for the formation of 5 alpha-DHT. The plasma levels of testosterone, 5 alpha-DHT and 3 alpha-Adiol cannot account for their respective prostatic levels, indicating the importance of the steroid-metabolizing enzymes in regulating intracellular androgen levels. Changes in the AP characteristics could be correlated with the androgen status of the prostate.

Acid Phosphatase↗
PubMed1988

Induction of anterior chamber associated immune deviation in rats receiving intracameral injections of retinal S antigen.

The injection of a variety of antigens, including transplantation antigens, haptens, and viral glycoproteins, into the anterior chamber of eyes of mice produces a characteristic spectrum of immune responses in which suppressed cell mediated immunity dominates. This phenomenon is called Anterior Chamber Associated Immune Deviation (ACAID). Having recently demonstrated that soluble antigens also can induce ACAID in mice, we examined the possibility that an important soluble ocular antigen, retinal S antigen (S Ag) may be capable of inducing ACAID in rats. We have found that injection of S Ag with adjuvant into the anterior chamber of eyes of adult Lewis rats evokes neither a humoral nor a cell mediated immune response that can be detected. However, animals that received S Ag intracamerally in this manner did respond to a subsequent immunogenic dose of S Ag in CFA injected into the hind foot pads, by producing serum anti S Ag antibodies. Importantly, these rats failed to display antigen-specific delayed hypersensitivity. Moreover, lymphoid cells from anterior chamber pre-treated animals, when adoptively transferred, inhibited the development of S Ag-specific delayed hypersensitivity in naive recipients indicating that an active suppression mechanism had been generated. Thus, a soluble antigen injected intracamerally in adult Lewis rats can generate ACAID. The possibility that ACAID may be relevant to the development of S Ag induced uveitis is discussed.

Animals↗
PubMed1987

Mania following head injury. A report of two cases and a review of the literature.

Secondary mania has been described in association with a variety of physical conditions. While there have been a number of reports of mania occurring in individuals with intracranial cerebral lesions, there have been few reporting its occurrence in association with non-penetrating cerebral trauma. Two further cases of mania following non-penetrating head injury and the efficacy of ECT in its management are reported, and a brief review of the literature relating to the subject is given.

Aged↗
PubMed1986

Disproportionate reduction of actin synthesis in hearts of starved rats.

We examined the synthesis of proteins in rat myocardium after starvation. Rates of total protein synthesis in myofibrillar and nonmyofibrillar fractions of myocardium of starved animals were reduced similarly (to 70-80% of the rates in hearts of fed animals, p less than 0.002), but rates of synthesis of some individual proteins were affected discoordinately. Radiolabeled proteins from atrial and ventricular explants, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed that starvation for 2 days reduced the rate of cardiac actin synthesis to 26-38% of control levels, while the rate of myosin heavy chain synthesis in the same hearts was only moderately reduced (74-80% of control levels). This starvation-induced reduction in actin synthesis could be accounted for at least in part by disproportionately decreased levels of actin mRNA in starved hearts, as revealed by Northern blot hybridization and by in vitro translation analysis. The dramatic decrease in cardiac actin synthesis was rapidly reversible, and actin synthesis returned to normal after a single day of refeeding. The selective reduction of actin synthesis after starvation was specific for the heart: rates of myosin heavy chain and actin synthesis in skeletal muscles (soleus and extensor digitorum longus) were coordinately reduced in response to starvation. To our knowledge, this is the first example of such dramatic discoordinate regulation of myofibrillar protein synthesis in response to a physiological stimulus.

Actins↗
PubMed1986

Fractionation and quantification of calcium-dependent proteinase activity from small tissue samples.

Almost all mammalian tissues contain a cytoplasmic Ca2+-dependent proteolytic system consisting of two different proteinases (CDP I and CDP II) and an endogenous inhibitor specific for these proteinases. It was difficult to determine the relative activities of CDP I and CDP II directly and accurately without extensive purification, requiring relatively large amounts of tissue. We developed a simple technique based on Reactive Red-agarose affinity chromatography for quantitatively measuring CDP II activities in small (less than 100 mg) tissue samples. This technique is rapid, sensitive and highly reproducible. CDP II activities can be quantified in numerous tissue samples in a single day. Using this method, we analysed the components of the CDP system in various rat tissues and demonstrated quantitative differences in CDP II activities as well as relative differences in the amounts of CDP-inhibitor activity among the various tissues. This technique should prove useful in the efforts to define the currently unknown physiological function(s) of the Ca2+-dependent proteolytic system by allowing comparison of CDP activities in tissues under diverse conditions of protein metabolism.

Animals↗
PubMed1986

Androgen metabolism and actions in rat ventral prostate epithelial and stromal cell cultures.

The rat ventral prostate requires androgens for normal development, growth, and function. To investigate the relationship between androgen metabolism and its effects in the prostate and to examine differences between the epithelial and stromal cells, we have established a system of primary cell cultures of immature rat ventral prostate cells. Cultures of both cell types after reaching confluency (6-7 days) actively metabolized 3H-labelled testosterone (T), 5 alpha-dihydrotestosterone (5 alpha-DHT), 5 alpha-androstane-3 alpha,17 beta-diol, and 5 alpha-androstane-3 beta,17 beta-diol. The epithelial cells actively reduced T to 5 alpha-DHT and formed significant amounts of 5 alpha-androstane-3,17-dione from T, 5 alpha-DHT, and 5 alpha-androstane-3 alpha,17 beta-diol. All substrates were converted to significant amounts of C19O3 metabolites. The stromal cells also metabolized all substrates, but very little 5 alpha-androstane-3,17-dione was formed. The metabolism studies indicate that both cell types have delta 4-5 alpha-reductase, 3 alpha- and 3 beta-hydroxysteroid oxidoreductase and hydroxylase activities. The epithelial cells have significant 17 beta-hydroxysteroid oxidoreductase activity. The epithelial cells cultures grown in the presence of T have higher acid phosphatase (AP) contents (demonstrated histochemically and by biochemical assay). Tartrate inhibition studies indicate that the epithelial cells grown in the presence of T are making secretory AP. Stromal cell AP is not influenced by T. The results indicate that the cultured cells maintain differentiated prostatic functions: ability to metabolize androgens and, in the case of the epithelial cells, synthesize secretory AP.

Acid Phosphatase↗
PubMed1986

Effects of glucocorticoid treatment on cardiac protein synthesis and degradation.

We treated rats with dexamethasone (DEX, 1 mg . kg-1 . day-1) and examined the effects of this glucocorticoid on heart protein metabolism using atrial explant and Langendorff perfusion preparations. Fasted rats treated with DEX for 2 days had significantly lower body weights (92% of control, P less than 0.001) and larger hearts (106% of control, P less than 0.005) than fasted control animals. Protein and RNA concentrations remained constant. In atrial explants, DEX treatment produced a 19% increase in protein synthesis (P less than 0.001) and a 13% increase in protein degradation (P less than 0.002). In Langendorff-perfused hearts, DEX treatment caused a 36% increase in protein synthesis (P less than 0.02), while protein degradation was 8% above control (P greater than 0.05). Thus, in contrast to their catabolic effects on skeletal muscle, glucocorticoids are anabolic on the heart. The increased accumulation of total cardiac protein during early glucocorticoid administration is mediated entirely via increased rates of synthesis.

Animals↗
PubMed1985

Inter-laboratory quality control of estrogen and progesterone receptor assays in breast cancer tissue using lyophilised cytosols.

In 1981 a quality control (QC) program for estrogen and progesterone receptor assays was organized among six laboratories in Ontario, Canada. Twenty-three vials of lyophilised cytosol prepared from human breast tumor tissues were analysed by each laboratory over a two-year period. Samples of each batch of QC material were analysed at least twice: either in the same batch or on separate occasions. The present study demonstrates the stability of the QC material, defines the relative accuracy of the receptor assays, and provides estimates of within-batch and between-batch precision of the receptor assays.

Breast Neoplasms↗
PubMed1985

Effects of tamoxifen on testosterone metabolism in postmenopausal women with breast cancer.

Testosterone is a known estrogen precursor especially in postmenopausal women. Tamoxifen, an anti-estrogen, is used in the treatment of women with breast cancer in whom metastatic disease has been demonstrated. The action of Tamoxifen is thought to be to occupy the intracellular estrogen receptor sites in target tissues and thus block the action of the biologically active estrogen, estradiol. Effects of Tamoxifen on the production and metabolism of hormones have been postulated. We studied the kinetics of testosterone metabolism by the constant infusion of 3H-testosterone in six postmenopausal women with breast cancer prior to and during Tamoxifen therapy. The Tamoxifen did not produce any significant change in the metabolic clearance rate, the plasma concentration or the calculated blood production rate of testosterone. The only significant alteration in the conversion ratio of testosterone to metabolites was the reduction (p less than 0.02) in conversion to 5 alpha-dihydrotestosterone. A significant reduction in the plasma concentrations (p less than 0.05) of dehydroepiandrosterone and of luteinizing hormone (p less than 0.02) was found. Other steroid and peptide hormones did not show any significant changes. We conclude that Tamoxifen therapy has very little effect on the kinetics of testosterone metabolism in postmenopausal women with metastatic breast cancer.

Aged↗
PubMed1984

A protein activator of the plasma membrane Ca++-ATPase of heart sarcolemma.

A detergent extract of dog or beef heart sarcolemmal vesicles was prepared and found to have a stimulatory effect on the Ca++-ATPase of plasma membranes from human erythrocyte and cardiac sarcolemma. A procedure is described which enriches the activating fraction. The protein nature of the preparation is illustrated by its sensitivity to boiling and to the proteolytic enzyme(s) trypsin and chymotrypsin. SDS polyacrylamide gels indicate that the protein(s) involved have a molecular weight of 56 and 60 kDa. The sarcolemmal activator can stimulate the Ca++-ATPase activity of the isolated enzyme more than 100% in the presence of saturating amounts of calmodulin. The activation is calcium dependent, being greatest at approximately 10 microns Ca++, free, but does not change the Km for Ca++. A possible physiological role for the activator is discussed.

Animals↗
PubMed1984

Dehydroepiandrosterone sulfate: kinetics of metabolism in normal young men and women.

The constant infusion technique was used to study the kinetics of dehydroepiandrosterone sulfate (DHEAS) metabolism in normal young men and women. The metabolic clearance rates (MCR) (means +/- SEM) for normal young men and women were 15.2 +/- 1.7 1/24 h (8.2 +/- 0.7 1/m2/24 h) and 11.8 +/- 0.8 1/24 h (7.3 +/- 0.4 1/m2/24 h) respectively. Coupled with the plasma levels of 5.07 +/- 1.95 and 4.02 +/- 0.57 mumole/L the resulting blood production rates were 76.7 +/- 25.7 and 48.0 +/- 9.4 mumole/24 h for men and women respectively. The conversion ratios for the conversion of DHEAS to dehydroepiandrosterone (DHEA) were 0.006 for men and 0.004 for women. Because of the high metabolic clearance rates for DHEA relative to those for DHEAS and the high production rates of DHEAS most of the DHEA produced per day can arise from DHEAS.

Adult↗