Biomedical subjects

B L Clarke

Publications and source records attributed to B L Clarke.

At least 19 recordsLinked to original sources

PubMed1991

beta-Phosphorothioate analogs of nucleotide sugars are resistant to hydrolytic degradation and utilized efficiently by glycosyltransferases.

The alpha- and beta-phosphorothioate analogs of UDP-Gal and UDP-Glc, in which a sulfur is exchanged for a non-bridging oxygen at one of the phosphate groups, have been synthesized and tested for their resistance to enzymatic degradation and for their usefulness in glycosyltransferase reactions. The alpha analogs were found to be no more resistant to hydrolysis than the native nucleotide sugars, but as previously reported (R. B. Marchase et al. (1987) Biochim. Biophys. Acta 916: 157) the beta S analogs were approximately 10 times more resistant. The beta S analog and native UDP-Glc were found to have comparable Km's when used in assays for glucosylphosphoryl dolichol synthase with rat liver and hen oviduct microsomes, although the apparent Vmax of the reaction was about twofold higher for the analog, presumably due to its resistance to degradation. Partially purified 4 beta-galactosyltransferase exhibited a Vmax with (beta S)UDP-Gal that was only slightly lower than that with UDP-Gal and a Km that was slightly increased. The effectiveness of the analog was especially apparent in assays for 4 beta-galactosyltransferase on intact sperm and in rat liver homogenates, in which hydrolysis of the normal substrate was very rapid and net incorporation was at least 4 times greater with the beta S analog in each system.

Animals↗

PubMed1991

A monoclonal anti-peptide antibody mimics adrenocorticotropic hormone activity.

A monoclonal antibody that specifically recognizes the adrenocorticotropic receptor (ACTH) on rat adrenal cells was tested for hormonal activity. The antibody behaved as an agonist based on three different biological activities associated with ACTH. An antibody concentration of 16 micrograms/ml stimulated isolated rat adrenal cells to secrete 800 ng/10(4) cells of corticosterone with a concomitant 10-fold increase of cAMP to 30 pmol/10(5) cells. Antibody concentrations above 16 micrograms/ml inhibited mitotic activity in mouse Y-1 adrenal cells. A radio-immunoassay using an anti-ACTH antibody showed that the monoclonal anti-adrenocorticotropic receptor antibody and ACTH are antigenically related. These findings suggest that the anti-receptor antibody recognizes the ligand binding domain of the ACTH receptor.

Adrenal Cortex↗

PubMed1990

Modulation of IgM secretion and H chain mRNA expression in CH12.LX.C4.5F5 B cells by adrenocorticotropic hormone.

The murine B cell line CH12.LX.C4.5F5 (CH12 (5F5) expresses adrenocorticotropin (ACTH) receptors, which can modulate IgM secretion by these cells. Interestingly, the response to ACTH was concentration dependent, inducing IgM secretion at subnanomolar amounts and suppressing secretion at micromolar amounts. With the use of an enzyme-linking immunospot assay it was possible to demonstrate that the ACTH-induced increase in IgM secretion by CH12 (5F5) cells was caused at least in part by an increase in the number of cells secreting IgM. CH12 (5F5) cells activated with suboptimal concentrations of LPS demonstrated a similar biphasic response. ACTH at concentrations of 10(-13) to 10(-9) M augmented IgM secretion in LPS-activated cells as much as sixfold, whereas 10(-6) M ACTH slightly decreased LPS-induced IgM secretion. At the mRNA level, subnanomolar concentrations of ACTH increased microH chain mRNA expression up to twofold in unstimulated or LPS-stimulated CH12 (5F5) cells. Taken together, these studies show that physiologically relevant concentrations of ACTH can interact directly with receptors on these B lymphocytes to enhance IgM secretion and microH chain mRNA expression. Although ACTH does increase intracellular cAMP levels in CH12 (5F5) B cells, it is unlikely that the induction of this second messenger pathway is by itself responsible for the ACTH induced B cell differentiation. The concentration of ACTH necessary to stimulate significant intracellular cAMP increases was 10- to 100-fold higher than that required to increase IgM secretion. Furthermore, CH12 (5F5) cells treated with varying concentrations of 8-bromo cAMP or cholera toxin were inhibited in their ability to secrete IgM. These results strongly suggest that the enhancing effects of ACTH on CH12 (5F5) IgM secretion are via mechanisms independent of those mediated by cAMP.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1990

A monoclonal anti-peptide antibody recognizes the adrenocorticotropic receptor.

We have produced a monoclonal antibody that specifically recognizes the adrenocorticotropic receptor on rat adrenal cells. The immunogen was designed from an RNA sequence complementary to the mRNA coding for ACTH1-24. This complementary peptide, termed HTCA, has been shown to specifically bind ACTH and was proposed to mimic the ACTH binding site of the hormone receptor. The monoclonal anti-HTCA antibody recognized a restricted domain of the HTCA peptide, bound to Y-1 adrenal cells with a KD of 1.8 nM, and blocked the binding of 125I-ACTH to rat adrenal cells. These findings show that anti-HTCA competes with ACTH for binding to the ACTH receptor.

Adrenal Medulla↗

PubMed1990

The surface activity of the same subpopulation of galactosyl receptors on isolated rat hepatocytes is modulated by colchicine, monensin, ATP depletion, and chloroquine.

In this study, we characterized and compared the ligand-independent loss of surface galactosyl (Gal) receptor activity on isolated rat hepatocytes treated with monensin, chloroquine, microtubule depolymerizing agents, or NaN3 and NaF at 37 degrees C. Freshly isolated hepatocytes exhibit predominately one subset of surface Gal receptors, termed State 1 receptors (Weigel, P. H., Clarke, B. L., and Oka, J. A. (1986) Biochem. Biophys. Res. Commun. 140, 43-50). During equilibration at 37 degrees C, these cells also express a second subset of Gal receptors at the surface, termed State 2 receptors, and routinely double their total surface Gal receptor activity. Following equilibration at 37 degrees C and then inhibitor treatment, hepatocytes bound 40-60% less 125I-asialoorosomucoid (ASOR) at 4 degrees C than did untreated cells. Treated cells maintained a basal nonmodulated level of surface receptor activity regardless of temperature, perturbant concentration, or incubation time. Loss of surface Gal receptor activity on cells treated with multiple inhibitors simultaneously or sequentially was not additive. Thus, all treatments affected the same subpopulation of surface Gal receptors. None of these inhibitors decreased surface State 1 Gal receptor activity, but all prevented the normal appearance of State 2 Gal receptors on freshly isolated cells during incubation at 37 degrees C. The endocytic capability of residual surface State 1 Gal receptors on inhibitor-treated cells varied depending on the inhibitor. Hepatocytes treated first at 24 degrees C or with colchicine at 37 degrees C internalized greater than 85% of surface-bound 125I-ASOR. In contrast, monensin- or chloroquine-treated cells internalized approximately 50% of surface-bound 125I-ASOR. Azide-treated cells internalized less than 20% of surface-bound 125I-ASOR. We conclude that only surface State 2 Gal receptor activity is sensitive to these various perturbants. State 1 Gal receptor activity is not modulated. These data are consistent with the conclusion that only State 2 Gal receptors constitutively recycle.

Adenosine Triphosphate↗

PubMed1990

Steroidogenic activity of a peptide specified by the reversed sequence of corticotropin mRNA.

The molecular recognition theory predicts that a reversed (3'----5') reading of an mRNA should yield a peptide that is structurally and functionally similar to that specified in the 5'----3' direction. We tested this idea by synthesizing a corticotropin (ACTH) analogue using a reverse reading of bovine mRNA for ACTH-(1-24). This peptide, designated ACTH-3'----5', had a similar hydropathic profile to native ACTH-5'----3' but had only 30% sequence homology and eight different charge substitutions. ACTH-3'----5' specifically bound to the surface of mouse Y-1 adrenal cells and to polyclonal anti-ACTH antibody. Additionally, ACTH-3'----5' stimulated cAMP synthesis and steroidogenesis in adrenal cells. These findings show that ACTH-3'----5' mimics the corticotropic properties of native ACTH, thereby further validating the molecular recognition theory.

Adrenal Glands↗

PubMed1989

Differential effects of leupeptin, monensin and colchicine on ligand degradation mediated by the two asialoglycoprotein receptor pathways in isolated rat hepatocytes.

We have shown that degradation of asialo-orosomucoid (ASOR) in isolated rat hepatocytes occurs by two different intracellular pathways [Clarke, Oka & Weigel (1987) J. Biol. Chem. 262, 17384-17392] mediated by two subpopulations of cell surface galactosyl (Gal) receptors, designated State 1 or State 2 receptors. In the present study, several inhibitors were tested for their effects on ligand degradation by the State 1 or State 2 pathway. Leupeptin, monensin and chloroquine completely inhibited degradation of 125I-labelled ASOR in both pathways. Dose-response studies showed, however, that the State 2 pathway was more sensitive to leupeptin or monensin than the State 1 pathway. No differences were observed with chloroquine. For example, the onset of inhibition in the State 2 and State 1 pathways occurred at about 0.05 and 0.3 microM-leupeptin respectively, a 6-fold difference. At 3.5 microM-monensin, 125I-ASOR degradation in the State 2 pathway was completely blocked, whereas degradation in the State 1 pathway was essentially unaffected. Colchicine was observed to give the largest differential sensitivity between the two pathways. The State 2 degradation pathway was about 30-fold more sensitive to colchicine than the State 1 pathway. Lumicolchicine had no affect. The onset of inhibition of the rate of 125I-ASOR degradation in the State 2 and State 1 pathways occurred at approximately 0.1 and 3.0 microM-colchicine respectively. At very high concentrations (greater than 0.1 mM), the State 1 pathway could be completely inhibited. We conclude that intracellular 125I-ASOR processing or delivery to degradative compartments in both the State 1 and State 2 Gal receptor pathways requires low pH. Ligand delivery to the degradative compartment does not require microtubules in the State 1 pathway, consistent with the very rapid onset of degradation in this pathway. The State 2 degradation pathway does require microtubules.

Animals↗

PubMed1989

Differential expression of functional adrenocorticotropic hormone receptors by subpopulations of lymphocytes.

In an effort to investigate the presence of adrenocorticotropic hormone (ACTH) receptors on rat lymphocytes, cells were separated by a panning procedure into T and B cell populations. By using the radiolabeled ACTH agonist, (125I-Tyr23) phenylalanine2-norleucine4-ACTH1-24, substantial numbers of ACTH binding sites were detected on T and B lymphocytes, but not on thymocytes. Scatchard analysis revealed two types of binding sites on each cell population, one with Kd1 = 0.088 +/- 0.025 nM and one with Kd2 = 4.2 +/- 0.6 nM; however, the absolute number of binding sites per cell was different. B lymphocytes expressed approximately three times the number of Kd1 binding sites per cell when compared with T lymphocytes. However, ACTH receptor expression by these cell populations was not static as suggested by the ability to induce receptor expression via mitogens. B or T cells and thymocytes stimulated with the mitogens LPS or Con A, respectively, substantially increased their number of Kd1 binding sites per cell (approximately three-fold). Even more dramatic increases in Kd1 receptor expression (approximately 100-fold) were observed when comparing "normal" and stimulated thymocytes. To demonstrate that these ACTH binding sites were in fact functional, cAMP levels were measured in lymphocytes 10 min after exposure to varying concentrations of ACTH. Dose-dependent increases in cAMP levels were observed, with significant stimulation occurring with as little as 0.1 nM ACTH added. Taken together, these studies demonstrate the presence of functional ACTH receptors on normal, rat T and B lymphocytes.

Adrenocorticotropic Hormone↗

PubMed1989

Comparative studies on mannosylphosphoryl dolichol and glucosylphosphoryl dolichol synthases.

Factors affecting the synthesis of mannosylphosphoryl dolichol and glucosylphosphoryl dolichol hen oviduct microsomes were compared in order to gain insight into the properties of their respective synthases. A stabilized form of mannosylphosphoryl dolichol synthase, but not glucosylphosphoryl dolichol synthase, was released from microsomes by freezing the membranes after exposure to the detergent CHAPSO. The activation energy for mannosylphosphoryl dolichol synthesis in membranes was 9.4 glucosylphosphoryl dolichol synthesis in membranes had a similar activation energy, 8.1 kcal/mol, but below 18 degrees C the value was 16.7 kcal/mol. Tryptic digestion of sealed microsomes preferentially inactivated mannosylphosphoryl dolichol synthase; however, both synthases were equally inactivated in detergent-permeabilized microsomes. Periodate-oxidized UDP-Glc was used to probe the topological orientation of glucosylphosphoryl dolichol synthase in rat liver microsomes. Sealed microsomes treated with oxidized UDP-Glc were inactive in synthesis of glucosylphosphoryl dolichol. However, when these treated microsomes were permeabilized, glucosylphosphoryl dolichol synthase activity was readily detected. From these studies we conclude that although mannosyl- and glucosylphosphoryl dolichol synthases catalyze chemically similar reactions in the endoplasmic reticulum, they differ in several respects. These differences were interpreted in terms of a topological model in which the active sites of the two enzymes reside on opposite faces of the endoplasmic reticulum, with that of the glucosyl lipid synthase facing the lumen and that of the mannosyl lipid synthase facing the cytosol.

Animals↗

PubMed1987

Degradation of asialoglycoproteins mediated by the galactosyl receptor system in isolated hepatocytes. Evidence for two parallel pathways.

The ability of rat hepatocytes to degrade internalized surface-bound 125I-asialoorosomucoid (ASOR) was determined by measuring the appearance of acid-soluble radioactivity at 37 degrees C. The degradation kinetics were biphasic in cells previously equilibrated at 37 degrees C for 1 h or cultured for 24 h. Degradation began immediately and was linear for at least 20 min after which the rate increased to a steady state value 3-4 times greater than the initial rate. We previously showed that hepatocytes have two functionally distinct populations of galactosyl receptors that mediate ligand dissociation by two kinetically different pathways (Weigel, P. H., Clarke, B. L., and Oka, J. A. (1986) Biochem. Biophys. Res. Commun. 140, 43-50). The activity of one receptor population, designated State 2 galactosyl receptors, can be reversibly modulated by incubating cells between 22 and 37 degrees C and is not expressed on the surface of freshly isolated cells. When 125I-ASOR was prebound to freshly isolated cells at 4 degrees C and degradation was assessed subsequently at 37 degrees C, the kinetics were monophasic, not biphasic. Degradation of the surface-bound 125I-ASOR began immediately and was greater than 90% complete by 6 h. Freshly isolated cells were incubated at temperatures between 22 and 37 degrees C, chilled to 4 degrees C, allowed to pre-bind 125I-ASOR, and then incubated at 37 degrees C. As the State 2 galactosyl receptor population increased, the kinetics of degradation became progressively more biphasic and the rate of the delayed degradation process increased. This effect could be reversed in cells in culture or in suspension by down-modulating surface receptor activity at temperatures below 37 degrees C; only the degradation process appearing after a 20-min lag was affected. Degradation in both pathways is an apparent first order process with identical rate constants (kappa = 0.006 min-1, t1/2 = 116 min). We conclude that there are two separate pathways by which asialoglycoproteins are degraded. The major "classic" pathway mediated by State 2 galactosyl receptors occurs after a 20-min lag and the minor pathway mediated by State 1 galactosyl receptors begins immediately with no detectable lag.

Animals↗

PubMed1987

Altered glucagon release in biotin deficiency.

Classical biotin deficiency in young chickens is characterized by a reduction in the activity of liver biotin-dependent enzymes which thus impairs gluconeogenesis during periods of food withdrawal. Because the normal bird maintains an elevated fasting blood glucose level, the ability of the classically biotin-deficient animal to resist changes induced by fasting has not been established. Whether the release of insulin and/or glucagon is affected by biotin deficiency has not been investigated and so determining the importance of these hormones in maintaining fasting glucose levels as well as their ability to respond to a challenge is the objective of the present study. Experimental animals (15-week-old cockerels) were fed a biotin-deficient diet for 9 weeks while control animals (n = 8) were pair-fed biotin-supplemented diets. Before fasting, concentrations of plasma glucose, insulin, and glucagon were not different between the two groups. After 72 h of fasting basal glucose levels remained the same in both groups and concentrations of plasma insulin were equally suppressed but concentrations of glucagon were significantly elevated in biotin-deficient animals (P less than 0.025). Ten minutes after an iv glucose challenge the magnitude of the increase in plasma insulin concentrations was equivalent for both groups of animals whereas the magnitude of the decline in plasma glucagon concentrations was greater for biotin-deficient birds. Twenty minutes after a protein challenge (purified casein diet) the levels of plasma glucagon in both groups were maximally increased although the concentrations remained elevated only in control animals. In conclusion the release of insulin and glucagon is not impaired in biotin-deficient animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

The hepatic galactosyl receptor system: two different ligand dissociation pathways are mediated by distinct receptor populations.

After internalization of 125I-asialo-orosomucoid (ASOR) by isolated rat hepatocytes, ligand dissociates by two kinetically distinct pathways (Oka and Weigel, J. Biol. Chem. 257, 10,253, 1983). These slow and fast dissociation pathways correspond to two functionally different subpopulations of cell surface galactosyl receptors designated, respectively, State 1 and State 2 receptors. Freshly isolated cells or cells equilibrated below 24 degrees C express only State 1 receptors. Cells equilibrated at 37 degrees C express both State 1 and State 2 receptors. Ligand dissociation after internalization of surface-bound 125I-ASOR was measured using the permeabilizing detergent, digitonin. The slow dissociation pathway was mediated by State 1 receptors and was the only pathway expressed by cells which were freshly isolated or had been equilibrated at 24 degrees C. State 2 receptors are expressed at temperatures above about 20 degrees C, and both the fast and slow dissociation pathways occurred in cells equilibrated at 37 degrees C. State 2 receptors therefore mediate the rapid dissociation pathway. Dissociation and subsequent degradation of specifically bound ligand routed in either pathway were complete, respectively, within 3 and 6 hrs.

Animals↗

PubMed1986

Regulation of glucokinase activity in the domestic fowl.

The factors which regulate soluble and particulate glucokinase and hexokinase activity in the liver of domestic chickens has been investigated. Pretreatment with oral administration (via tube feeding) of glucose plus injection of insulin resulted in a significant increase in the activity of soluble (p less than 0.01) and particulate (p less than 0.01) glucokinase activity whereas fasting for 48 hours reduced glucokinase and hexokinase activity (p less than 0.01) in the particulate fraction only. Treatment of fed chickens for 2 weeks with thyroxine (50 micrograms: i.m. daily) plus triiodothyronine (50 micrograms) resulted in a marginal decrease (NS) in soluble glucokinase activity but significantly increased soluble hexokinase (p less than 0.05) activity. Thyroidectomized animals showed a decline in both soluble glucokinase (p less than 0.01) and hexokinase (p less than 0.025) activity. There was no effect of thyroid hormone manipulation on particulate glucokinase activity although there was a significant reduction in particulate hexokinase activity (p less than 0.05) in thyroidectomized birds. These data establish a physiological role for the glucokinase enzyme activity in avian carbohydrate metabolism and suggest that in contrast with the mammal, the particulate fraction is the more physiologically important enzyme.

Animals↗

PubMed1985

Recycling of the asialoglycoprotein receptor in isolated rat hepatocytes. ATP depletion blocks receptor recycling but not a single round of endocytosis.

Continuous endocytosis of 125I-asialo-orosomucoid (ASOR) mediated by the galactosyl receptor in rat hepatocytes is a cyclic process. 125I-ASOR-receptor complexes are internalized, processed, and the ligand is degraded while the receptor is returned to the cell surface for reutilization. Since a true cycle has a thermodynamic requirement for the input of external energy, we examined the effects of changes in intracellular ATP levels on the function of the receptor cycle. Hepatocytes were depleted of ATP to various extents prior to endocytosis by incubating cells at 15 degrees C in the presence of 2 mM NaF and 0-20 mM NaN3. A luciferase-luciferin bioluminescence assay was used to quantitate the amount of cellular ATP. ATP-depleted cells were allowed to bind 125I-ASOR at 0 degrees C, washed through discontinuous Percoll gradients, and only viable cells were isolated and incubated at 37 degrees C to initiate a synchronous single round of endocytosis. The extent of internalization of this surface-bound 125I-ASOR was unaffected by an ATP depletion to less than 1% of the control level. The rate of internalization of surface-bound ligand was unaffected until the ATP levels decreased to 30% or less; at greater than 98% ATP depletion the initial rate decreased by a maximum of 55% and the kinetics became biphasic. In contrast, continuous endocytosis in the presence of excess ASOR was inhibited by only a 25% decline in cellular ATP content and demonstrated a very sharp threshold response to changing ATP levels. Continuous endocytosis, which requires receptor recycling, was completely inhibited when the total cellular ATP level decreased by only 40%. We conclude that the internalization phase of endocytosis is not dependent on ATP but that the processing and/or externalization phases of the complete receptor cycle are either directly or indirectly dependent on ATP and very sensitive to changes in cellular ATP content.

Adenosine Triphosphate↗

PubMed1971