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Biomedical subjects

J R Dave

Publications and source records attributed to J R Dave.

At least 55 records · Page 3Linked to original sources
PubMed1986

Modulation of prolactin binding sites in vitro by membrane fluidizers. IV. Differential effects on plasma membrane and Golgi fractions of male prostate and female liver in the rat.

In vitro treatment of crude particulate fractions of male rat ventral prostate and female rat liver with membrane fluidizers (aliphatic alcohols) has been previously reported by us to increase prolactin (PRL) receptor levels, presumably by unmasking cryptic prolactin receptors. The objective of this study was to determine if similar in vitro treatment of purified plasma membrane- and Golgi-rich fractions of male rat prostate and female rat liver with ethanol produced differential effects on prolactin binding in these two subcellular fractions. The degree of fluidization was monitored by a fluorescence polarization method using 1,6-diphenylhexatriene. 125I-PRL specific binding to Golgi-rich fractions of male ventral prostate and female liver was approximately 4-fold higher than that observed in plasma membrane-rich fractions. The microviscosity parameter, inversely related to lipid fluidity, was consistently lower in Golgi-rich fractions than that in plasma membrane-rich fractions in both prostate and liver. In vitro ethanol treatment of prostatic and hepatic plasma membrane fractions produced a dose-related increase and then decline in prolactin binding and a maximal (60-75%) increase in prolactin binding was observed at 4.8% and 2.0% ethanol in prostatic and hepatic membranes, respectively. This in vitro treatment also produced a significant increase in apparent lipid fluidity of plasma membrane-rich fractions of prostate gland and liver. However, similar in vitro ethanol treatment of Golgi fractions of both prostate gland and liver exhibited little increase in prolactin binding without changing microviscosity. Our observations are consistent with the direct relationship between membrane fluidity and prolactin receptor levels. The changes in prostatic and hepatic plasma membrane fractions following in vitro ethanol treatment suggest that prolactin receptors located on the plasma membranes may be modulated (via membrane lipid microviscosity changes) in vivo to a greater extent by various physiological agents than those located within the Golgi fraction.

Animals↗
PubMed1986

Demonstration that corticotropin-releasing factor binding to rat peripheral tissues is modulated by glucocorticoid treatment in vivo and in vitro.

In a recent study we reported the presence of specific binding sites for corticotropin-releasing factor (CRF) in peripheral tissues of the rat (Endocrinology, 116, 2151, 1985). The objective of this study was to determine if CRF binding to peripheral tissues was modified following adrenalectomy and glucocorticoid replacement therapy. Adult male rats were adrenalectomized and CRF binding to liver, spleen and testicular membranes was determined at 5, 7 or 14 days following adrenalectomy. An additional group of adrenalectomized rats received subcutaneous injections of dexamethasone (75 micrograms/day) for 14 days. Adrenalectomy of rats for 14 days increased CRF binding to liver, kidney, testis, spleen and ventral prostate by approximately 65%-125% above sham-control values. CRF binding to membrane preparations obtained from the pancreas of sham-operated rats was undetectable; however, adrenalectomy produced detectable CRF binding in this tissue. Adrenalectomy produced a time-related increase in CRF binding to ventral prostate, spleen and liver tissue. Administration of dexamethasone to adrenalectomized animals prevented increased CRF binding to peripheral tissues observed following adrenalectomy alone. In vitro dexamethasone treatment of prostatic or hepatic homogenates from adrenalectomized rats resulted in a dose-related decrease in CRF binding activity. However, similar in vitro treatment of prostatic or hepatic homogenate with progesterone exhibited no significant effects on CRF binding. Our results suggest that glucocorticoids may be a regulator of peripheral CRF receptors.

Adrenalectomy↗
PubMed1986

A new enzyme-linked fluorescence assay (ELFA) for use with peroxidase-antibody conjugates: a comparison with ELISA for the quantitation of IgM antibodies to hepatitis B core antigen.

A new enzyme-linked fluorescence assay (ELFA) suitable for use with peroxidase-antibody conjugates is described. The substrate for the assay is p-hydroxyphenylacetic acid, the fluorescent product of which is stable and unaffected by light. The assay compared favourably with a standard ELISA for the quantitation of IgM antibodies to hepatitis B core antigen.

Enzyme-Linked Immunosorbent Assay↗
PubMed1986

Ethanol exposure decreases pituitary corticotropin-releasing factor binding, adenylate cyclase activity, proopiomelanocortin biosynthesis, and plasma beta-endorphin levels in the rat.

Animals exposed continuously for 14 days to ethanol vapor in an inhalation chamber at sufficient ethanol vapor concentration to maintain blood ethanol levels from 100-250 mg/100 ml exhibited approximately 36% lower corticotropin-releasing factor binding and 24% lower adenylate cyclase activity in anterior (AL) and neurointermediate lobe (NIL) membranes of the pituitary gland compared to controls not treated with ethanol. To determine the effect of chronic ethanol exposure on proopiomelanocortin (POMC) biosynthesis, the levels of POMC mRNA in the AL and NIL were quantified by Northern blot and slot blot techniques. Ethanol treatment for 1, 7, or 14 days produced a time-related decrease in POMC mRNA levels, relative to total RNA levels, in both the AL and NIL. Ethanol treatment caused a greater reduction in NIL POMC mRNA than in AL POMC mRNA. Exposure to ethanol vapors for 14 days decreased immunoreactive beta-endorphin in plasma by approximately 82%. The observed reduction of immunoreactive beta-endorphin in plasma after long term exposure of rats to ethanol may be related to the alcohol-mediated decrease in corticotropin-releasing factor binding and adenylate cyclase activity, which, in turn, leads to decreased intracellular POMC levels through reduced production of POMC mRNA in the AL and NIL of the rat pituitary gland.

Adenylyl Cyclases↗
PubMed1985

Modulation of prolactin binding sites in vitro by membrane fluidizers. Effects on male prostatic and female hepatic membranes in alcohol-fed rats.

The objectives of this study were (i) to determine if in vivo administration of ethanol to rats produced changes in apparent lipid fluidity and prolactin binding capacity of male prostatic and female hepatic membranes and (ii) to compare the effects of membrane fluidizers (aliphatic alcohols) in vitro on prolactin binding of prostatic and hepatic membranes in control and alcohol-fed animals. In vitro ethanol has been shown by us previously to increase prolactin receptor levels presumably by unmasking cryptic prolactin receptors. The degree of fluidization was monitored by a fluorescence polarization method using 1,6-diphenylhexatriene. Adult male and female rats were given either water or 4% ethanol as the sole source of drinking fluid for a period of 6 weeks. No significant changes in plasma prolactin were observed between control and ethanol-treated groups of either sex. However, the microviscosity parameter, inversely related to lipid fluidity, was increased approx. 34% and 40%, respectively, in male prostatic and female rat hepatic membranes after ethanol feeding. Furthermore, 125I-prolactin binding capacity was decreased approx. 30% and 26%, respectively, in prostatic and hepatic membranes of alcohol fed animals. In vitro treatment with aliphatic alcohols had no effect on either microviscosity or prolactin binding in hepatic or prostatic membranes from ethanol-fed rats, but both fluidized and increased prolactin binding in the same membrane preparations from control rats. Our observations are consistent with the direct relationship between membrane fluidity and prolactin receptor levels. The changes in prostatic and hepatic membranes after alcohol feeding, namely decreased prolactin receptor levels, decreased fluidity and increased resistance to the fluidizing effects of in vitro aliphatic alcohols may reflect a fundamental membrane defect.

1-Butanol↗
PubMed1985

Endocrine-mediated parallel changes in hepatic glucocorticoid and prolactin receptors.

The objective of this study was to determine whether in female rat liver any relationship existed between prolactin and glucocorticoid receptors after hormonal manipulation. Bromocryptine (CB-154) treatment of adult SD female rats (80-100 days old) for 48 h decreased prolactin binding to hepatic membranes 49% and dexamethasone binding in hepatic cytosol 40% below control values. Administration of rat prolactin along with bromocriptine prevented these changes. In another study, prolactin binding to hepatic membranes increased 53% and dexamethasone binding in hepatic cytosol increased 113% above sham-control values, 3 days after adrenalectomy. On the other hand, hydrocortisone treatment of sham-operated rats reduced prolactin binding by 57% and dexamethasone binding by 76%. Scatchard analyses of the prolactin or dexamethasone binding data indicated that these manipulations changed the number of prolactin or dexamethasone binding sites rather than their apparent affinity constants. In vitro treatment of rat whole liver homogenate with various doses (10(-9) - 10(-5) M) of dexamethasone and corticosterone for 15 min at 22 degrees C resulted in a dose-dependent decrease in prolactin binding activity. However, direct addition of dexamethasone to a hepatic 15 000 X g to 100 000 X g membrane preparation exhibited no significant effects on prolactin binding. In conclusion, these studies show that (a) there is a parallel in vivo modulation of rat liver prolactin and glucocorticoid receptors under various experimental conditions and (b) in vitro exposure of whole liver homogenate to glucocorticoids inhibits the prolactin binding activity.

Adrenalectomy↗
PubMed1985

Molecular cloning of mouse mammary gland kappa-casein: comparison with rat kappa-casein and rat and human gamma-fibrinogen.

A cDNA clone for kappa-casein mRNA from the lactating mouse mammary gland was isolated and its nucleotide sequence determined. Analysis of the deduced amino acid sequence revealed a precursor protein with a 21-amino-acid signal sequence and a mature protein of 160 amino acids, the mature mouse protein being 3 amino acids longer than the rat kappa-casein. Northern blot analysis of the lactating rat and mouse mammary gland showed a specific mRNA for rat kappa-casein and two distinct mRNAs for mouse kappa-casein. This result is explained by the presence of two putative polyadenylation sites in mouse kappa-casein cDNA, whereas rat kappa-casein cDNA has only one polyadenylation site. Comparison of the nucleotide sequence and of the deduced amino acid sequence of kappa-casein from mouse with that of the rat showed 85% homology between the two sequences. However, when amino acid sequences of kappa-casein from rat and mouse were compared with ovine kappa-casein, only a 45% homology was observed. Amino acid sequences of kappa-casein from rat, mouse, and sheep were 36.53% homologous with rat and human gamma-fibrinogen. The extent of homology was similar (32%) when nucleotide sequences of corresponding cDNAs were compared. The stretches of homology existing at different regions between the two proteins were more confined toward the amino-terminal half of gamma-fibrinogen. However, when nucleotide sequences were compared, mouse kappa-casein cDNA showed homology only with the second half of the rat gamma-fibrinogen cDNA, i.e., between nucleotides 661-1135. The homology with the human gamma-fibrinogen cDNA spanned over two regions, one between nucleotides 1-328 and the second between nucleotides 591-726.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1985

Prolactin increases lipid fluidity and prolactin binding of rat prostatic membranes.

The objective of these studies was to determine whether prolactin could modify the lipid fluidity of rat ventral and dorsolateral prostate membranes and subsequently modify the availability of prolactin receptors. Additional studies were also undertaken to determine the effects of prolactin on serum lipid fluidity. Adult male rats were injected with 0, 100, or 400 micrograms ovine prolactin/day subcutaneously for a period of 5 days. Serum and prostatic membrane lipid fluidity was measured by a fluorescence polarization method using a lipid probe 1,6-diphenylhexatriene. Prolactin binding in dextran-coated charcoal-pretreated prostatic membranes was determined by radioreceptor assay. This pretreatment has been reported by us to remove the endogenous substances that interfere with prolactin binding assay (J. R. Dave and R. J. Witorsch, Endocrinology 111: 2144-2146, 1982). Prolactin binding increased by approximately 44 and 72% in dorsolateral prostate and 16 and 39% in ventral prostate in 100- and 400-micrograms groups, respectively. Membrane fluidity increased by approximately 16 and 19% in dorsolateral prostate and 10 and 13% in ventral prostate in 100- and 400-micrograms groups, respectively. Serum lipid fluidity increased 50 and 79% in 100- and 400-micrograms groups, respectively.

Animals↗
PubMed1985

Corticotropin-releasing factor binding to peripheral tissue and activation of the adenylate cyclase-adenosine 3',5'-monophosphate system.

Specific binding sites for rat corticotropin-releasing factor (rCRF) are present in rat adrenal medulla, ventral prostate, spleen, liver, kidney, and testis and bovine chromaffin cells in culture. Maximal binding of [125I]rCRF occurred within 25 min at 4 C and was saturable. Scatchard analysis of rCRF binding to rat adrenal membranes and bovine chromaffin cells revealed the existence of two classes of binding sites. One class had a relatively higher apparent affinity and lower number of binding sites, whereas the other class had a relatively lower affinity and higher number of binding sites. CRF induced a dose-related increase in rat adrenal membrane adenylate cyclase activity and cAMP levels in bovine chromaffin cells. Nanomolar concentrations of rCRF maximally stimulated adenylate cyclase activity in rat adrenal membranes and maximally increased cAMP levels in bovine chromaffin cells to 86% and 130% above control values, respectively. The demonstration of specific CRF-binding sites in a variety of peripheral tissues and the finding that activation of specific CRF-binding sites in adrenal tissue stimulates the adenylate cyclase-cAMP system suggest that CRF may have an important regulatory role in various peripheral tissues.

Adenylyl Cyclases↗
PubMed1985

Evidence that beta-endorphin binds to specific receptors in rat peripheral tissues and stimulates the adenylate cyclase-adenosine 3',5'-monophosphate system.

With the use of [125I]acetyl human beta-endorphin (Ac-hBE), specific binding sites for beta-endorphin (BE) were identified in the liver, kidney, adrenal, spleen, and testis of adult male rats, whereas specific BE-binding sites were not present in the ventral prostate or pancreas. In those tissues containing specific BE-binding sites, microsomal membranes (15,000-100,000 X g pellet) exhibited higher BE-binding capacity than the crude homogenate (125-100,000 X g pellet). The binding of BE was saturable, and maximal, specific binding was achieved with a 60-min incubation at 22 C. Furthermore, optimal BE binding was dependent on the presence of magnesium chloride. Scatchard analysis of BE binding to hepatic membranes revealed the existence of two classes of binding sites. One class had an apparent Ka of 0.019 X 10(9) M-1 and a lower number of binding sites (9.1 pmol BE/mg protein), whereas the other class had a lower affinity (apparent Ka of 0.0006 X 10(9) M-1) and a higher number of binding sites (159 pmol/mg protein). Specific BE binding to hepatic membranes was inhibited (80-100%) by rat AcBE-(1-27) and -(1-31), nonacetylated rat BE-(1-31), and human beta-lipotropin. At substantially higher peptide concentrations (greater than 10(-5) M), gamma-endorphin, met-enkephalin, or leu-enkephalin inhibited BE binding by 20-40%. In addition, opiate receptor-binding drugs, such as morphine and naloxone, at 10(-5) M did not alter BE binding to hepatic membranes. Incubation of hepatic membranes with BE induced a dose-related increase in membrane adenylate cyclase activity, and 0.5 X 10(-10) M BE resulted in a maximal enhancement of adenylate cyclase activity to 148% above control values. Water-deprived or salt-loaded male rats with chronically lowered immunoreactive plasma BE exhibited substantially increased BE binding to adrenal and kidney tissue. Specific binding sites for BE occur in a variety of peripheral tissues, and alterations of circulating BE result in changes in the capacity of certain peripheral tissues to bind BE. Finally, occupancy of specific BE-binding sites in peripheral tissue stimulates the adenylate cyclase-cAMP system, which suggests that the peripheral actions of circulating BE may be mediated via this system.

Adenylyl Cyclases↗
PubMed1984

Modulation of prolactin binding sites in vitro by membrane fluidizers. II. Age-dependent effects on rat ventral prostatic membranes.

The objectives of this study were to determine (i) if the age-related changes in 125I-labeled ovine prolactin specific binding of rat ventral prostate was correlated with changes in membrane lipid microviscosity and (ii) if membrane fluidizers produced age-dependent effects on prolactin binding of prostatic membranes. The degree of fluidization was monitored by a fluorescence polarization method using 1,6-diphenylhexatriene. Membrane preparations of ventral prostate glands obtained from immature (24-25 days old), young-adult (80-90 days old) and aged (550-610 days old) male rats were used for prolactin binding and membrane lipid microviscosity measurements. Relative to immature rats, prostatic prolactin binding decreased approximately 50% in young-adult rats and 75% in aged rats. Membrane lipid microviscosity, relative to immature rats, was increased 72% in young-adult rats and 140% in aged rats. Prostatic membranes obtained from immature animals exhibited no significant effects of in vitro alcohol treatment on prolactin binding, whereas, those obtained from aged animals exhibited maximal increase in prolactin binding. The value of the microviscosity parameter, after in vitro alcohol exposure, exhibited no significant changes in immature animals, whereas, this parameter was decreased approximately 15% in young-adults and approximately 30% in aged animals. These data suggest that in vitro fluidization of prostatic membrane exhibits an age-dependent modification of prolactin binding.

1-Propanol↗
PubMed1983

Modulation of prolactin binding sites in vitro by membrane fluidizers. I. Effects on adult rat ventral prostatic membranes.

The objective of this study was to determine if aliphatic alcohols, known fluidizers of certain membranes, could increase in vitro the apparent fluidity and prolactin binding capacity of membrane preparations obtained from ventral prostate glands of adult male rats. The degree of fluidization was monitored by a fluorescence polarization method using 1,6-diphenylhexatriene. Membrane preparations were either incubated with varying concentrations of ethanol, 1-propanol or 1-butanol and 125I-oPRL overnight at room temperature or were exposed to the alcohols for 15 min at room temperature and washed prior to the overnight incubation with ligand. Regardless of the conditions of incubation, alcohol exposure produced the same effects, a dose-dependent elevation and then decline in specific prolactin binding. Butanol produced a maximal 37-42% increase in prolactin binding at a concentration of 1.0%, propanol produced a maximal 40-56% increase in prolactin binding at a concentration of 3.8%, and ethanol produced a maximal 54-77% increase in prolactin binding at concentration of 4.8%. Scatchard analysis of the oPRL binding of ventral prostatic membranes indicated that the in vitro treatment of these membrane fluidizers increased the number of oPRL binding sites rather than the apparent affinity constant. The value of the microviscosity parameter decreased by 10-13%, 13-15% and 21-25%, after a 15 min exposure of prostatic membranes to 1.0% butanol, 3.8% propanol and 4.8% ethanol, respectively. These data suggest that in vitro fluidization of prostatic membrane modifies prolactin binding capacity and are consistent with in vivo prostatic prolactin receptor level-membrane fluidity relationships observed in earlier studies.

1-Butanol↗
PubMed1983

Indomethacin decreases both luteinizing hormone binding and fluidity of testicular microsomal membranes in rat.

The objective of these studies was to examine the effects of in vivo treatment with indomethacin, a prostaglandin synthesis inhibitor, on luteinizing hormone receptors and fluidity in microsomal membrane preparations of rat testis. Adult male rats were treated with vehicle or either of two doses of indomethacin (3.8 micrograms/g body weight or 7.5 micrograms/g body weight) every 8 h for 1, 2 and 3 days. Indomethacin treatment decreased human luteinizing hormone (hLH) binding in rat testis in a time-dependent fashion. The lower dose of indomethacin decreased hLH binding by 19%, 35% and 50%, respectively, after 1, 2 and 3 days of treatment. The higher dose of indomethacin decreased hLH binding at these respective times by 21%, 44% and 56%. Scatchard analysis of the hLH binding of testicular membrane preparations indicated that indomethacin treatment decreased the number of hLH binding sites rather than the apparent affinity constant. Testicular membrane fluidity was estimated by a fluorescence polarization technique. The effects of indomethacin on apparent fluidity of microsomal membrane paralleled that of LH binding. The values of apparent microviscosity, inversely related to the fluidity, also increased in a time-dependent fashion. These simultaneous effects of indomethacin on testicular LH binding and fluidity suggest that these phenomena are interrelated and that prostaglandins are involved in the maintenance of LH receptors in vivo.

Animals↗
PubMed1983

Membrane microviscosity is increased in the erythrocytes of patients with adrenoleukodystrophy and adrenomyeloneuropathy.

Adrenoleukodystrophy (ALD) and adrenomyeloneuropathy (AMN) are related X-linked disorders characterized by adrenal, gonadal, and nervous system dysfunction. While the pathologic finding common to these tissues appears to be the accumulation of excessive amounts of very long chain fatty acids, the mechanism leading to functional impairment in these tissues is unclear. Measurements of fluorescence polarization (P), using the lipid probe diphenylhexatriene, demonstrate a highly significant increase in the microviscosity of erythrocyte membranes in affected patients (P = 0.286 +/- 0.012) vs. normals (P = 0.239 +/- 0.020). Analyses of these membranes by gas-liquid chromatography revealed 1.9-, 1.6-, and 1.3-fold increases above normal values in the C25:0, C26:0, and C27:0 fatty acids, respectively. These observations are compatible with previously obtained data in animals that correlate membrane microviscosity with the number of hormone receptors in target tissues. The present data support the thesis that a decrease in responsiveness to trophic hormones in ALD and AMN is secondary to changes in the membrane microviscosity of the target tissues and suggest a mechanism by which adrenal and gonadal failure occur in such patients.

Adrenal Insufficiency↗
PubMed1983

Prolactin-binding capacity, prostaglandin synthesis and fluidity of murine hepatic membranes are modified during pregnancy and lactation.

The objective of these studies was to determine whether the changes observed in prolactin-binding capacity of mouse liver microsomal membranes during pregnancy and lactation correlated with those observed in the fluidity and prostaglandin (PG)-synthesizing activity of these membrane preparations. Prolactin-binding capacity increased with the advance of pregnancy to reach a peak at 16 days of gestation and declined thereafter to non-pregnant, non-lactating (NPNL) levels. Membrane microviscosity, studied by fluorescence polarization, was significantly decreased throughout early gestation but returned to NPNL levels by day 20 of gestation and remained unchanged thereafter. The amount of PGE synthesized in vitro by these membranes increased during gestation to reach a peak at 12 days of gestation but declined thereafter to below NPNL levels on the day of parturition and returned to NPNL levels by day 20 of lactation. Synthesis of PGF2 alpha remained at a higher level from day 12 to day 18 followed by a decline in activity at parturition to NPNL levels. These changes and other data suggest an interrelationship of receptor levels, fluidity and PG synthesis during pregnancy. Such modifications in local PG synthesis may influence the fluidity of these membranes, which ultimately play a significant role in maximal exposure of membrane receptors during pregnancy.

Animals↗