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M M Compton

Publications and source records attributed to M M Compton.

At least 37 records · Page 2Linked to original sources
PubMed1991

Identification and characterization of glucocorticoid-regulated nuclease(s) in lymphoid cells undergoing apoptosis.

Apoptosis is a physiological process by which selected cells are deleted from a population in response to specific regulatory signals. A hallmark of apoptosis is the internucleosomal degradation of DNA prior to cell death. We are studying glucocorticoid-induced lymphocytolysis as a model system for apoptosis within the immune system. In rat thymocytes, the internucleosomal DNA cleavage which occurs following glucocorticoid treatment is both time- and dose-dependent, and is blocked by the glucocorticoid antagonist RU 486, indicating that this effect is mediated by the glucocorticoid receptor. Similar experiments using glucocorticoid-responsive (wt) and glucocorticoid-resistant (nt-) S49.1 lymphoma cell lines confirm that internucleosomal DNA degradation and cell death are glucocorticoid receptor-mediated events and thus reflect the direct effects of glucocorticoids on lymphocytes. In an effort to identify the nuclease(s) responsible for the DNA degradation, we have developed two assays to detect nucleases whose activity is altered by glucocorticoid treatment. The first assay involves electrophoresing extracts of nuclear protein from control and glucocorticoid-treated lymphoid cells into SDS-polyacrylamide gels containing [32P]DNA within the gel matrix. This assay is used to estimate the molecular mass of the nuclease, based on the observed in situ nuclease activity. The second assay uses HeLa nuclei as a substrate to detect internucleosomal cleavage activity present in nuclear extracts of control and glucocorticoid-treated lymphoid cells. Using these assays we have identified a novel Ca2+, Mg(2+)-dependent nuclease with an apparent molecular weight of 18 kDa in both S49 wt cells and rat thymocytes treated with glucocorticoids. Furthermore, nuclear extracts of glucocorticoid-treated, but not control, rat thymocytes and S49 wt cells were capable of cleaving HeLa chromatin at internucleosomal sites. In an effort to determine the identity of the nuclease capable of internucleosomal cleavage of DNA, nuclear extracts from dex-treated rat thymocytes were fractionated by gel filtration chromatography under non-denaturing conditions, and the fractions were analyzed using the [32P]DNA SDS-PAGE and HeLa nuclei assays. When analyzed under native conditions, the 18 kDa nuclease described previously appears to exist as a congruent to 25 kDa protein which may be part of a high molecular weight complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Development of an apoptosis endonuclease assay.

A biochemical hallmark of cells undergoing programmed cell death, or apopotosis, is the endonucleolytic cleavage of genomic DNA at internucleosomal sites. To study further the nuclease involved in this process, an assay system was developed to measure internucleosomal DNA degradation. Micrococcal nuclease (MNase), a bacterial enzyme that cleaves chromatin at internucleosomal intervals, was used to validate the assay procedure. Thymocyte nuclear proteins obtained from glucocorticoid-treated chickens, a source of internucleosomal DNA-degrading activity, were incubated with chicken red blood cell nuclei, and genomic DNA was subsequently extracted and analyzed by agarose gel electrophoresis. Generation of internucleosomal DNA degradation products by the thymocyte protein extract required ATP and was both time and protein concentration dependent. This nuclease activity could be inhibited by EDTA, EGTA, alkylating agents, or heat denaturation. Addition of purified proteinases, RNases, or other types of nucleases to the assay failed to generate discrete internucleosomal lengths of DNA, thus confirming the nuclease specificity of this assay. On the basis of these data, we believe that this assay system will be instrumental in isolating and characterizing the nuclease(s) associated with apoptosis.

Adenosine Triphosphate↗
PubMed1991

Activation of thymocyte deoxyribonucleic acid degradation by endogenous glucocorticoids.

Immature lymphocytes in the thymus gland are killed by treatment with exogenous glucocorticoids. This steroid-mediated lymphocytolysis is preceded by numerous alterations in lymphocyte metabolism, including a DNA-degrading process in which the genome is cleaved at internucleosomal intervals. To date, this process has only been characterized by treating lymphocytes in vitro with glucocorticoids or by exogenous treatment of whole animals with adrenal steroids. To determine whether thymocyte DNA degradation could be activated by endogenous glucocorticoids, 4-wk-old chicks were treated with porcine adrenocorticotropic hormone (ACTH). This procedure elevated serum corticosterone levels approximately 80-fold within 2 h of hormone treatment. Following ACTH administration, thymocyte DNA was isolated and analyzed by agarose gel electrophoresis. The ACTH activated a DNA-degrading process that generated internucleosomal fragments of DNA identical in size to those observed following exogenous treatment with synthetic or naturally occurring glucocorticoids. Furthermore, this response could be inhibited by the glucocorticoid antagonist RU486 (17 beta-hydroxy-11 beta, 4-dimethylaminophenyl-17 alpha-propynl-estra-4,9,diene-3-one), indicating that adrenal steroids activate this process via the glucocorticoid receptor. These results demonstrate that lymphocyte DNA degradation does not result solely from exogenous glucocorticoid treatment; moreover, endogenous glucocorticoids can mediate this process and may thereby play an important role in thymic gland function.

Adrenocorticotropic Hormone↗
PubMed1990

Glucocorticoid-mediated activation of DNA degradation in avian lymphocytes.

Little information is known about the molecular mechanism of programmed cell death in the avian species. In the current study we have analyzed this process in chickens using a glucocorticoid-lymphocyte model system. Three-week-old male broiler chicks were treated in vivo with the synthetic glucocorticoid, dexamethasone. Following this treatment genomic DNA was isolated from thymocytes and analyzed by agarose gel electrophoresis. Dexamethasone activated a DNA degrading process in which the genome was specifically cleaved at internucleosomal intervals. This steroid-induced response occurred prior to thymocyte cell death and was time and glucocorticoid dose dependent, as well as tissue and steroid specific. Only the glucocorticoid class of steroid hormones could elicit this response and DNA degradation was only detectable in lymphoid tissues that contained immature lymphocytes. Internucleosomal DNA degradation could also be elicited via administration of adrenocorticotrophic hormone, a treatment that elevates endogenous glucocorticoids. Based on these data, glucocorticoid-activated DNA degradation of the avian thymocyte genome appears to be a steroid receptor-mediated process which involves the activation of an endogenous nuclease that cleaves the genome at internucleosomal sites. Degradation of the thymocyte genome occurs prior to cell death and may represent an initial event in a cascade of hormone-mediated processes that culminate in a type of cellular suicide referred to as programmed cell death.

Animals↗
PubMed1990

Glucocorticoid activation of deoxyribonucleic acid degradation in bursal lymphocytes.

Treatment of animals with exogenous adrenal steroids or elevation of endogenous glucocorticoids results in a profound involution of lymphoid tissue. In rodent species, this lymphoinvolution is accompanied by lymphocyte cell death and extensive degradation of the genome prior to lymphocytolysis. In the present study, this process was investigated in the bursa of Fabricius of domestic fowl. Four-wk-old chicks were treated with a single injection of dexamethasone, and bursal regression and cell viability were monitored over a 72-h period. Following hormone treatment, DNA was extracted from bursal lymphocytes and analyzed by agarose gel electrophoresis. Dexamethasone treatment resulted in a rapid regression of bursal tissue that could be detected as soon as 6 h posttreatment, but lymphocyte viability was not altered until 24 h afterward. The DNA isolated from bursal lymphocytes of glucocorticoid-treated birds appeared to be degraded at internucleosomal sites and generated a "ladder" of discrete DNA fragments when analyzed by agarose gel electrophoresis. This form of hormone-induced cell death, referred to as programmed cell death, may play a key role in glucocorticoid-mediated immunosuppression.

Animals↗
PubMed1988

Analysis of glucocorticoid actions on rat thymocyte deoxyribonucleic acid by fluorescence-activated flow cytometry.

Glucocorticoids induce thymocyte cell death via mechanisms that occur in concert with internucleosomal DNA cleavage. To further study this process at the single cell level, glucocorticoid effects on rat thymocyte DNA were analyzed using fluorescence-activated flow cytometry. Thymocytes were isolated from adrenalectomized rats, treated in vitro with steroid hormones, stained with 0.1 mM acridine orange, and analyzed by flow cytometry. After 5 h of treatment with 10(-7) M dexamethasone a population of thymocytes with reduced acridine orange binding was observed. Thymocyte viability after glucocorticoid treatment was greater than 95%, indicating that the observed changes in dye binding occurred before cell death. Induction of this subpopulation of thymocytes was both time and steroid concentration dependent, being detectable within 2 h of in vitro steroid treatment. This response to glucocorticoids was blocked by the glucocorticoid receptor antagonist RU 486, indicating a steroid receptor-mediated process. Furthermore, both actinomycin D and cycloheximide blocked the appearance of this glucocorticoid effect, thus demonstrating a dependence upon RNA and protein biosynthesis, respectively. Incubation of thymocytes with inhibitors of DNA repair (aminobenzamide or methylnicotinamide) also diminished acridine orange binding to thymocytes. The effects of these DNA repair inhibitors and glucocorticoids were additive, suggesting independent mechanisms of action. Dexamethasone treatment (10(-7) M) of cells that fail to die in response to glucocorticoids (HeLa S3) had no effect on dye binding. These findings are consistent with the concept that glucocorticoids induce a lysis gene product that alters the thymocyte genome and thus leads to cell death.

Acridine Orange↗
PubMed1987

Identification of a glucocorticoid-induced nuclease in thymocytes. A potential "lysis gene" product.

Glucocorticoids initiate a cytolytic process in lymphoid cells that is characteristic of programmed cell death. In vivo treatment of adrenalectomized rats with glucocorticoids results in the rapid degradation of the thymocyte genome at internucleosomal sites. This DNA degradation occurs prior to cell death, and considerable evidence indicates that this nucleolytic event is central to the initiation of lymphocytolysis. To further characterize this process, we have searched for the gene products in thymocytes which may be responsible for steroid-induced DNA degradation. Adrenalectomized rats were treated in vivo with dexamethasone or a vehicle control; nuclear thymocyte proteins were extracted with 0.6 M NaCl and analyzed for protein content or nuclease activity on sodium dodecyl sulfatepolyacrylamide gels containing calf thymus DNA. Glucocorticoid treatment resulted in the induction of two major protein families, a 30-32-kDa protein doublet and a series of 3-4 proteins of 12-19 kDa, both of which express prominent DNase activity. Induction of the lower molecular weight nucleases increased with time after steroid treatment and paralleled the time course of glucocorticoid-mediated DNA degradation. Nuclease induction was blocked by the glucocorticoid antagonist RU 486, indicating a steroid receptor-mediated process. When nuclei from glucocorticoid-resistant cells were incubated with nuclear extracts from glucocorticoid-treated rats, the DNA was cleaved at internucleosomal sites, whereas extracts from vehicle-treated animals were virtually inactive. Based on these findings we propose that glucocorticoids, acting via a receptor-mediated pathway, induce a nucleolytic "lysis gene" product(s) responsible for lymphocytolysis.

DNA↗
PubMed1987

Glucocorticoid action on the immune system.

Glucocorticoids have profound effects on immune function that are mediated, in part, by steroid-induced cell death. Our studies have been aimed at identifying the mechanism of this lymphocytolytic process using the rat thymocyte as a model system. Administration of glucocorticoids in vivo resulted in internucleosomal cleavage of the lymphocyte genome that was detectable within 2 h of treatment and increased with time after hormone administration. Six h after steroid treatment greater than 50% of the genome was degraded, yet cell viability remained greater than 90% indicating that this event preceded cell death. Furthermore, this process appeared to be mediated by the glucocorticoid receptor since the antagonist RU 486 blocked glucocorticoid-mediated DNA degradation. To further characterize this lymphocytolysis we have analyzed glucocorticoid-treated thymocytes for nucleases. Two families of nuclear proteins have been identified, a 30-32 kDa doublet and a series of 3-4 proteins that are 12-19 kDa, both of which are induced by glucocorticoid treatment (137 +/- 6% and 342 +/- 24%, respectively) and have prominent nuclease activity. These nucleases can also be induced in vitro indicating that glucocorticoids act directly on thymocytes to mediate this response. Moreover, this nuclease induction, like glucocorticoid-mediated DNA degradation, could be blocked by RU 486. Based on these findings we propose a working model of glucocorticoid-mediated lymphocytolysis in which these steroids, acting via a receptor mediated process, induce the expression of a lysis gene product (nuclease) which degrades the genome and results in cell death.

Animals↗
PubMed1986

Proteolytic modification of rat prolactin by subcellular fractions of the lactating rat mammary gland.

The current study explored prolactin proteolysis by rat lactating mammary gland. 125I-labelled rat prolactin was incubated with tissue fractions of lactating mammary gland and the extent of prolactin degradation and fragment formation was visualized and densitometrically quantitated from autoradiographs derived from SDS-polyacrylamide gel electrophoresis under reducing conditions. At pH 4.5, the 25 000 X g pellet of mammary gland converted intact prolactin (23 kDa band) to proteolytic fragments (8-16 kDa bands) in a time- and tissue concentration-dependent fashion similar to that reported previously for rat ventral prostate. The prolactin-degrading and -fragmenting activity in lactating mammary gland was 5-10-times that observed for ventral prostate, the most active male tissue. This activity at acid pH was also demonstrable in other fractions of mammary gland but appeared to predominate in the cytosol. The above activities in mammary gland virtually disappeared at pH 7.4, appeared sensitive to aspartate and sulfhydryl proteinase inhibitors, and insensitive to serine and metalloenzyme proteinase inhibitors. The distribution of this activity could not be correlated with a particular enzyme marker. These characteristics of mammary gland activity differed significantly from those reported previously for prostate. When electrophoresis was conducted under non-reducing conditions, prolactin proteolysis in prostate and mammary gland was primarily associated with the formation of a more slowly migrating product (24 kDa band) with little spontaneous 8-16 kDa fragment formation. Re-electrophoresis of the 24 kDa band under reducing conditions resulted in the appearance of the 8 and 16 kDa fragments. In conclusion, prolactin is proteolytically modified by prostate and lactating mammary gland to a variant of intact hormone (24 kDa band) with a cleavage site in its large loop, by two or more widely distributed, acid-dependent proteinases. Lactating mammary gland, the principal target for prolactin, has the capacity to cleave the hormone in its loop at rates higher than any other tissue examined to date.

Animals↗
PubMed1986

Rapid in vivo effects of glucocorticoids on the integrity of rat lymphocyte genomic deoxyribonucleic acid.

Glucocorticoid-induced lymphocytolysis has been studied for many years; however, the mechanism of lymphoid cell death has not been elucidated. In this study we have investigated the effects of glucocorticoids on the lymphocyte genome using the rat thymocyte model. Adrenalectomized rats were injected ip with dexamethasone (DEX) and killed thereafter. The thymus gland was removed, and DNA was extracted from isolated thymocytes and then separated electrophoretically on 1.8% agarose gels. Administration of glucocorticoids in vivo resulted in the cleavage of lymphocyte DNA at internucleosomal intervals. Genomic DNA separated on agarose gels from DEX-treated rats appeared as a ladder of DNA fragments which were multiples of about 180 base pairs, while DNA from control rats appeared as a single high mol wt band. This cleavage of thymocyte DNA was a rapid effect of adrenal steroid treatment and occurred before cell death. Thymocyte DNA fragmentation increased with time after DEX treatment and the dose of half-maximal response in vivo was estimated to be about 1.8 X 10(-8) M. Internucleosomal cleavage of DNA was only observed in lymphoid tissues (thymus and spleen), but not in other glucocorticoid-sensitive tissues (kidney, liver, heart, brain, or testis). Treatment of rats with estrogen, androgen, or progestin failed to elicit thymocyte DNA degradation. Glucocorticoid-induced DNA cleavage was partly inhibited by the glucocorticoid antagonist RU 486 (17 beta-hydroxy-11 beta,4-dimethylaminophenyl-17 alpha-propynl-estra-4,9-diene-3-one). These findings suggest that glucocorticoids activate, via a receptor-mediated process, an endonuclease-like activity in lymphoid tissues which cleaves the lymphocyte genome at internucleosomal sites. Activation of this nuclease by glucocorticoids precedes lymphocytolysis and may represent the hormonal regulation of programmed cell death.

Adrenalectomy↗
PubMed1984

Proteolytic degradation and modification of rat prolactin by subcellular fractions of the rat ventral prostate gland.

We previously showed that at pH 7.4, the cytosol and a low speed pellet (3300 X g) of rat ventral prostate degraded rat PRL, whereas a high speed pellet (25,000 X g) was inactive. The current study further explores PRL degradation by ventral prostatic tissue. Enzyme markers indicated that the low speed pellet was mitochondria enriched (cytochrome c oxidase), and the high speed pellet was lysosome enriched (acid phosphatase), although there was considerable cross-contamination between the two fractions. Proteolysis of hormone was examined by incubating 125I-labeled rat iodo-PRL with tissue fractions, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and radioautography to identify products and quantify the extent of PRL degradation. Acidification of the incubation medium (pH 4-5) to optimize lysosomal protease activities inhibited PRL degradation in cytosol and activated the process in both pellets consistent with the distribution of acid phosphatase. Under acidic incubation conditions, both low and high speed sediments produced the same electrophoretic pattern of PRL degradation, indicating the generation of peptide fragments from the hormone with a molecular mass of approximately 16,000-8,000 daltons. Cytosol (pH 7.4) also produced peptide fragments in this weight range, although at much lower rates and in relatively lower proportions. Peptide fragment formation from PRL by the low speed pellet (pH 7.4) was minimal. On a protein basis, PRL degradation by the high speed sediment (pH 4.5) was 3-10 times that by the low speed sediment (pH 7.4). Enzyme inhibitor analysis indicated differences in each of the cell fractions with regard to the types of proteases involved in PRL degradation. When compared with other tissues of the male rat, ventral prostate was the most active in degrading PRL and generating peptide fragments. In addition, kidney, spleen, and lung were among the more active tissues, whereas liver, was deferens, and dorsolateral prostate were relatively less active in degrading hormone. Our studies indicate that qualitatively different processes degraded PRL in each of the active subcellular fractions and that the generation of peptide fragments from PRL predominates in a lysosome-rich fraction of ventral prostate. The possible significance of subcellular variations in PRL processing and the generation of peptide fragments of the hormone by peripheral tissue are discussed.

Animals↗
PubMed1983

Proteolytic fragmentation of rat prolactin by the rat ventral prostate gland.

Homogenates of rat ventral prostate diminished the immunoreactivity of rat prolactin (PRL) in a fashion dependent on time and tissue concentration, suggestive of hormone degradation. Direct evidence of PRL proteolysis was demonstrated by subjecting ventral prostate homogenates and cell fractions that were incubated with 125I-labeled rat PRL to SDS-PAGE and radioautography. Heat-labile PRL proteolysis predominated in the crude homogenate, the 3,300g pellet and the 100,000g (100 K g) supernatant of rat ventral prostate. The degradation of PRL by the 100 K g supernatant led to the formation of two stable peptide fragments weighing approximately 16,000 and 12,000 daltons. PRL proteolysis was negligible in 100 K g supernatants of the liver, salivary gland, testis, epididymis, vas deferens, seminal vesicle, and dorsolateral prostates of male rats. On the other hand, 100 K g supernatants of rat spleen, lung, and kidney did degrade rat PRL, although the patterns of peptide fragment formation differed from that of ventral prostate. Enzyme inhibitor analysis suggested that PRL degradation by the 100 K g supernatants of rat ventral prostate was due to sulfhydryl and serine proteases and not due to metalloenzyme or aspartate proteases. The functional significance of PRL fragmentation by rat ventral prostate cytosol remains to be determined.

Animals↗
PubMed1981

Storage and evacuation of spermatozoa from the uterovaginal sperm-host glands in domestic fowl.

Autoradiography with 3H-labeled spermatozoa was utilized to study spermatozoal oviductal interrelationships. Results demonstrated that sperm displacement from the uterovaginal sperm-host glands did not occur in domestic fowl. Furthermore, there was no indication that phagocytosis of labeled spermatozoa by sperm gland epithelium occurred. Associated studies demonstrated that extensive head-to-head agglutination occurred between freshly ejaculated spermatozoa, but the ability to agglutinate was lost as the sperm cells aged. On the basis of these observations, it was proposed that agglutination may be the basic mechanism controlling sperm storage and release from the uterovaginal sperm-host glands. A working model to this effect was presented.

Animals↗
PubMed1981

The effects of claw removal on growth rate, gonadal steroids, and stress response in cage reared pullets.

The purpose of this study was to investigate the effects of claw removal on young pullets grown in wire cages. Body weight, feed consumption, stress response, and gonadal steroid date were collected to assess the effects of claw removal and growing pullets. Claw removal posed an initial physiological insult upon chicks that resulted in a reduction in body weight gain and reduced feed consumption during the early grow-out period. However, by 20 weeks of age there were no significant differences between the body weights of intact and declawed pullets. No significant differences in plasma corticosterone, progesterone, or estradiol were observed, nor were there any significant differences in mortality or antibody response during the course of the 20-week study. Nevertheless, several declawed pullets demonstrated relatively precocious egg laying, suggesting that claw removal enhanced sexual maturity.

Animals↗
PubMed1979

Emptying of the uterovaginal sperm storage glands in the absence of ovulation and oviposition in the domestic hen.

Domestic hens were treated with pregnant mare serum to study the temporal pattern of sperm gland emptying in the absence of ovulation and oviposition. After a cessation of ovulation and oviposition, the hens were artificially inseminated with .034 ml of pooled semen. Subsequent histological examination of the uterovaginal sperm glands on days 1, 6, and 12 post-insemination indicated that the temporal rate of sperm-gland emptying was not significantly different from that of control birds which were laying normally. This observation suggests that ovulation and oviposition, along with their assoicated humoral factors, have a minimal effect on the emptying of the uterovaginal sperm storage glands.

Animals↗
PubMed1979

A histological examination of the uterovaginal sperm storage glands in the domestic hen following an insemination with variable semen dosages.

The uterovaginal sperm storage glands of the domestic hen were examined histologically and scored for filling rates 24 hr after an insemination. Two semen dosages (.023 and .047 ml) were employed and a comparison was made between the large and small semen dose. No significant differences were found between the two groups, suggesting that only a specific number of spermatozoa are capable of entering the uterovaginal sperm storage glands during a given time interval.

Animals↗
PubMed1978

The filling and emptying of the uterovaginal sperm-host glands in the domestic hen.

In a series of three replicated experiments, dwarf hens were inseminated with semen of one phenotype (normal or dwarf) and then reinseminated 4 hr later with semen of the other phenotype. All eggs were pedigree-hatched for two weeks following the inseminations. Subsequent phenotypic determinations of the progeny demonstrated a preponderance of offspring from the latter insemination. The results suggested that spermatozoa sequentially filled the uterovaginal sperm-host glands which produced a permanent stratification of sperm cells, and mixing did not occur within a gland. Emptying of the host-glands appeared to follow a pattern of slow superficial leakage where spermatozoa from the second insemination were the first spermatozoa released, giving rise to the large number of progeny phenotypes from the second insemination. In addition, progeny phenotypes from the second insemination were predominant throughout the fertility period suggesting that the storage capacity of the uterovaginal sperm-host glands can exceed the functional life span of stored spermatozoa following a normal insemination.

Animals↗