Biomedical subjects

T B Miller

Publications and source records attributed to T B Miller.

At least 37 records · Page 2Linked to original sources

PubMed1989

Amino-terminal sequence analysis of rat heart and muscle glycogen synthase: homology to the rabbit enzyme and the implications for hormonal control.

Glycogen synthase was purified from rat heart and muscle and electroblotted from sodium dodecyl sulfate polyacrylamide gels to polyvinylidene difluoride, and the NH2-terminal amino acid sequence was determined. The NH2-terminal amino acid sequence of the enzymes was identical. Further, phosphorylation site 2, a major cyclic AMP-dependent protein kinase recognition site in the rabbit muscle isozyme, is conserved in the rat isozymes suggesting that it serves an important function in hormonal regulation. However, two potentially important differences were observed. Threonine-5 and valine-8 of the rabbit muscle enzyme are serine and methionine residues, respectively, in the rat isozyme, the latter being critical in the analysis of phosphopeptides produced by cyanogen bromide cleavage. These variations may provide a partial explanation for previously observed differences in rat and rabbit phosphopeptide maps.

Amino Acid Sequence↗

PubMed1989

Phosphorylase synthesis in diabetic hepatocytes and cardiomyocytes.

Whereas total cardiac glycogen phosphorylase activity appears to be unaffected by severe insulin deficiency, a diabetes-induced decreased in hepatic glycogen phosphorylase activity has been demonstrated by our laboratory and others using liver extracts, isolated perfused liver, and cultured hepatocytes. The loss of activity in diabetic liver can be correlated with a drop in protein levels. Using primary cultures of cells from normal and diabetic rats and phosphorylase specific antibodies, we found a corresponding decrease in phosphorylase synthesis in diabetic hepatocytes cultured for 2 days in a serum-free, chemically defined medium. When hepatocytes are cultured in the presence of insulin, triiodothyronine, and cortisol, there is a significant recovery in the rate of phosphorylase synthesis after 3 days. Over the 3-day time period, there is no significant difference in the rate of phosphorylase degradation in normal compared with diabetic hepatocytes. Total protein synthesis in both hepatocytes and cardiomyocytes is unaffected by diabetes, as is phosphorylase synthesis in cultured cardiomyocytes.

Animals↗

PubMed1988

Human liver phosphatase 2A: cDNA and amino acid sequence of two catalytic subunit isotypes.

Two cDNA clones were isolated from a human liver library that encode two phosphatase 2A catalytic subunits. The two cDNAs differed in eight amino acids (97% identity) with three nonconservative substitutions. All of the amino acid substitutions were clustered in the amino-terminal domain of the protein. Amino acid sequence of one human liver clone (HL-14) was identical to the rabbit skeletal muscle phosphatase 2A cDNA (with 97% nucleotide identity). The second human liver clone (HL-1) is encoded by a separate gene, and RNA gel blot analysis indicates that both mRNAs are expressed similarly in several human clonal cell lines. Sequence comparison with phosphatase 1 and 2A indicates highly divergent amino acid sequences at the amino and carboxyl termini of the proteins and identifies six highly conserved regions between the two proteins that are predicted to be important for phosphatase enzymatic activity.

Amino Acid Sequence↗

PubMed1987

Phosphorylation of rat heart glycogen synthase: studies in cardiomyocytes and in vitro phosphorylations with cAMP-dependent kinase and protein phosphatase-1.

The phosphorylation of glycogen synthase has been studied in freshly isolated adult rat cardiomyocytes. Six peaks of 32P-labeled tryptic peptides are recovered via C-18 high performance liquid chromatography (HPLC) when synthase is immunoprecipitated from 32P-labeled cardiomyocytes and digested with trypsin. When epinephrine treated cells are used as a source of enzyme, the same HPLC profile is obtained with a dramatic enhancement of 32P recovered in two of the HPLC peaks. In vitro phosphorylation of rat heart synthase by cAMP-dependent protein kinase stimulates the conversion of synthase from the I to the D form and results in the recovery of the same tryptic peptides from the C-18 as is the case for synthase derived from cardiomyocytes. Treatment of cAMP-dependent kinase phosphorylated synthase with protein phosphatase-1 leads to a reactivation of the enzyme and a dephosphorylation of the same tryptic peptides that are selectively phosphorylated in epinephrine treated cardiomyocytes. These results are discussed in relation to hormonal control of glycogen metabolism in cardiac tissue.

Animals↗

PubMed1987

Regulation of glycogen metabolism in primary cultures of rat hepatocytes. Restoration of acute effects of glucose in cells from diabetic rats involves protein synthesis.

Defective acute regulation of hepatic glycogen synthase by glucose and insulin, caused by severe insulin deficiency, can be corrected in adult rat hepatocytes in primary culture by inclusion of insulin, triiodothyronine, and cortisol in a chemically defined serum-free culture medium over a 3-day period (Miller, T. B., Jr., Garnache, A. K., Cruz, J., McPherson, R. K., and Wolleben, C. (1986) J. Biol. Chem. 261, 785-790). Using primary cultures of hepatocytes isolated from normal and diabetic rats in the same serum-free chemically defined medium, the present study addresses the effects of cycloheximide and actinomycin D on the chronic actions of insulin, triiodothyronine, and cortisol to facilitate the direct effects of glucose on the short-term activation of glycogen synthase. The short-term presence (1 h) of the protein synthesis blockers had no effect on acute activation of glycogen synthase by glucose in primary hepatocyte cultures from normal rats. Normal cells maintained in the presence of cycloheximide or actinomycin D for 2 and 3 days exhibited unimpaired responsiveness to glucose activation of synthase. The protein synthesis inhibitors were effective at blocking the restoration of glucose activation of synthase in diabetic cells in media which restored the activation in their absence. Restoration of glycogen synthase phosphatase activity by insulin, triiodothyronine, and cortisol in primary cultures of diabetic hepatocytes was also blocked by cycloheximide or actinomycin D. These data clearly demonstrate that restoration of acute glycogen synthase activation by glucose and restoration of glycogen synthase phosphatase activity in primary cultures of hepatocytes from adult diabetic rats are dependent upon the synthesis of new protein.

Animals↗

PubMed1987

Use of adult rat cardiomyocytes to study cardiac glycogen metabolism.

The use of adult rat cardiomyocytes to model cardiac glycogen metabolism was investigated by monitoring the response of glycogen phosphorylase and glycogen synthase to epinephrine and insulin treatment. Cardiomyocytes derived from normal rats respond to epinephrine in the range of 1 X 10(-7) to 5.5 X 10(-6) M epinephrine with an increase in the percent of phosphorylase in the AMP-independent form from 11.5 to 24.8%. In the same cells, insulin in the range of 10(-9) to 10(-7) M increased the glucose 6-phosphate independent form of glycogen synthase from 30.5 to 40.5%. Cells derived from alloxan-diabetic hearts exhibit a hypersensitive phosphorylase activation and a refractile synthase inactivation in response to epinephrine treatment. This pattern is similar to that recorded using perfused heart preparations. The data presented suggests that adult rat cardiomyocytes represent a valid model of glycogen metabolism in both the normal and alloxan-diabetic rat.

Alloxan↗

PubMed1987

Sequence homologies between type 1 and type 2A protein phosphatases.

The Mr = 33,000 catalytic fragment of rabbit skeletal muscle type 1 protein phosphatase was digested with trypsin after reduction and alkylation. The resulting peptides were isolated, subjected to automated Edman degradation, and their sequences compared to the deduced peptide sequence of the bovine type 2A protein phosphatase cDNA. Of 10 tryptic peptides from the type 1 phosphatase that were sequenced, nine showed a high degree of homology with the type 2A phosphatase. This provides the first direct sequence comparison suggesting that the type 1 and type 2 protein phosphatases, distinguished functionally by their substrate specificities and sensitivity to inhibitors, make up part of a family of closely related gene products with similar structures.

Amino Acid Sequence↗

PubMed1986

Isolation of partial cDNAs for rat liver and muscle glycogen phosphorylase isozymes.

cDNA clones for the rat liver and muscle glycogen phosphorylase isozymes have been isolated using isozyme-selective antibodies and libraries prepared in the expression vector, lambda gt11. A 1.2 kb cDNA coding for the carboxy-terminal domain of rat liver phosphorylase was found to have 82% homology with the amino acid sequence of rabbit muscle phosphorylase. Limited sequencing of rat muscle phosphorylase cDNA indicated a 95% homology with the rabbit muscle enzyme. The rat liver clone has eight additional amino acid residues at the COOH-terminus compared to the rat muscle clone. Furthermore, 17 of 26 (65%) residues between amino acids 815-840 differ between liver and muscle isozymes. The similarity in enzymatic properties and conservation of structure except at the COOH-terminus suggest that the liver and muscle phosphorylase isozymes do not exist in order to have significant differences in the regulation of glycogen breakdown in the two tissues. Rather, the phosphorylase isozymes probably evolved for tissue-specific transcriptional regulation of the genes in liver and muscle.

Animals↗

PubMed1986

Regulation of glycogen metabolism in primary cultures of rat hepatocytes. Restoration of acute effects of insulin and glucose in cells from diabetic rats.

Defects in the deposition of glycogen and the regulation of glycogen synthesis in the livers of severely insulin-deficient rats can be reversed, in vivo, within hours of insulin administration. Using primary cultures of hepatocytes isolated from normal and diabetic rats in a serum-free chemically defined medium, the present study addresses the chronic action of insulin to facilitate the direct effects of insulin and glucose on the short term regulation of the enzymes controlling glycogen metabolism. Primary cultures were maintained in the presence of insulin, triiodothyronine, and cortisol for 1-3 days. On day 1 in alloxan diabetic cultures, 10(-7) M insulin did not acutely activate glycogen synthase over a period of 15 min or 1 h, whereas insulin acutely activated synthase in cultures of normal hepatocytes. By day 3 in hepatocytes isolated from alloxan diabetic rats, insulin effected an approximate 30% increase in per cent synthase I within 15 min as was also the case for normal cells. The acute effect of insulin on synthase activation was independent of changes in phosphorylase alpha. Whereas glycogen synthase phosphatase activity could not be shown to be acutely affected by insulin, the total activity in diabetic cells was restored to normal control values over the 3-day culture period. The acute effect of 30 mM glucose to activate glycogen synthase in cultured hepatocytes from normal rats after 1 day of culture was missing in hepatocytes isolated from either alloxan or spontaneously diabetic (BB/W) rats. After 3 days in culture, glucose produced a 50% increase in glycogen synthase activity during a 10-min period under the same conditions. These studies clearly demonstrate that insulin acts in a chronic manner in concert with thyroid hormones and steroids to facilitate acute regulation of hepatic glycogen synthesis by both insulin and glucose.

Animals↗

PubMed1986

Intracranial hypertension and cerebrospinal fluid production in dogs: effects of furosemide.

A method using chronically prepared, anesthetized dogs was devised for studying the effects of treatments of intracranial hypertension induced by applying a reversible extradural mass lesion while simultaneously measuring production of cerebrospinal fluid. This was measured with a ventricular-cisternal perfusion technique in which the rate of cisternal outflow could be controlled by a pump and matched to the inflow, allowing intracranial pressure to fluctuate despite simultaneous measurement of cerebrospinal fluid formation. Elevations of intracranial pressure to the range 20 to 35 Torr were induced and maintained during perfusion, but elevations above 35 Torr would not permit continued perfusion. At normal intracranial pressure, 10 Torr or less, rates of cerebrospinal fluid formation were the same whether the outflow controlling pump or free outflow was used. Formation of cerebrospinal fluid decreased progressively as intracranial pressure increased above 20 Torr. It also decreased with time after the start of perfusion during the course of 5 h, but returned to the initial range during the control phase of subsequent experiments in the same animal. Furosemide, 3 mg kg-1, i.v., had no significant effect on rate of formation but did induce a small decrease in ICP in time-controlled experiments in which i.v. fluid replacement limited net fluid losses to 20 ml kg-1 with no change in mean arterial or central venous pressures.

Animals↗

PubMed1986

Teaching skills.

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Faculty, Medical↗

PubMed1986

Characterization of a Mannitol-Utilizing, Nitrogen-Fixing Bradyrhizobium japonicum USDA 110 Derivative.

We have isolated a colonial derivative of Bradyrhizobium japonicum USDA 110 (designated MN-110) that is both mannitol utilizing and N(2) fixing. Derivative MN-110 showed growth on mannitol and glucose similar to that of non-N(2)-fixing, mannitol-utilizing L2-110. Derivative MN-110 showed high constitutive and induced d-mannitol dehydrogenase activity (similar to L2-110) relative to N(2)-fixing, non-mannitol-utilizing I-110. Hybridization to EcoRI and HindIII total DNA digests with cloned USDA 110 nif DK and nif H genes revealed similar patterns for non-N(2)-fixing mannitol-utilizing derivative L1-110 and derivative MN-110. Symbiotic tests with soybean cultivars Ransom and Lee indicate MN-110 to be a superior N(2)-fixing derivative compared with derivative I-110 and the parent strain USDA 110. However, these differences were not revealed when comparing 28-day-old soybean-B. japonicum associations but were apparent in 49-day-old associations. It was apparent from this work that mannitol utilization was not necessarily correlated to symbiotic effectiveness in B. japonicum and that gene rearrangements were not responsible for differences in N(2) fixation between L1-110 or L2-110 and MN-110.

Journal Article↗

PubMed1986

Effects on canine prostatic secretion of some drugs that modify ion transport.

The effects of acetazolamide, amiloride, furosemide, ouabain and theophylline on nerve-induced prostatic secretion were determined in pentobarbital-anesthetized dogs. In one set of experiments the glands were exposed to normal saline or drug-containing saline between periods of nerve-induced secretion by connecting a urethral cannula for 30-min periods to a buret filled with normal saline or drug-containing saline at a height of 100 cm above the gland and by allowing retrograde penetration of these solutions into the gland. Secretion in response to stimulation of the hypogastric nerves at 20 Hz was markedly inhibited by intraurethral exposure to 0.1 and 1 mM ouabain but not to 10 mM furosemide, 3 mM amiloride or 30 mM theophylline; responses to stimulation at 2 Hz were potentiated by 30 mM theophylline. In other experiments, secretion in response to stimulation at 20 Hz was inhibited by i.v. administration of 10 to 30 mg/kg of furosemide but not 2 to 8 mg/kg of amiloride or 18.5 to 25 mg/kg of acetazolamide. Because active Cl- transport is a major process in canine prostatic fluid formation and because secretion of this fluid was selectively modified by these drugs in the same manner as is Na+,K+-coupled active Cl- transport by a variety of other epithelia, these epithelia appear to share a common Cl- transporting system.

Acetazolamide↗

PubMed1985

Immunologic identification of a glycogen synthase 93,000-dalton subunit from rat heart and liver.

A polyclonal sheep antibody to rat heart glycogen synthase has been used for immunoblot analysis and immunoprecipitation of both rat heart and liver synthase. The purified antibody completely inhibits glycogen synthase activity in rat heart preparations and specifically blots to a 93-kDa band in the 10,000 X g supernatants of both heart and liver homogenates. Immunoprecipitation of in vitro translation products from rat heart or liver poly(A+) RNA yields a unique band with a molecular mass of 93 kDa. Thus the subunit molecular mass of active glycogen synthase in rat heart is 93 kDa. In rat liver at least one form of glycogen synthase also appears to have a molecular mass of 93 kDa. Protocols used to purify rat liver synthase yield a subunit of 80-87 kDa, which retains activity, but which is no longer recognized by the antibody. This suggests that 1) a specific antigenic sequence has been proteolytically removed from the NH2 or COOH terminus of the protein, or 2) that limited proteolysis has led to a conformational change in the enzyme such that the antibody binding site is no longer recognized. Either or both of these possibilities represent a significant alteration in the enzyme due to proteolysis. In vitro studies using synthase preparations having molecular masses less than 93 kDa must be interpreted with caution due to possible structural changes which occur during purification which may alter the regulation or covalent modification of synthase.

Animals↗

PubMed1984

Insulin regulation of glycogen synthase phosphatase in primary cultures of hepatocytes.

Activation of glycogen synthase in the perfused rat liver is defective in severely diabetic rats. In the present study, activation of glycogen synthase by glucose and increased incorporation of [14C]glucose into glycogen by insulin are defective in hepatocytes isolated from alloxan diabetic rats. Acute activation of glycogen synthase in hepatocytes isolated from diabetic rats was restored by treatment of the rats with insulin in vivo. Restoration of synthase activation was not achieved by incubation of hepatocytes in the presence of insulin in vitro for up to 12 h. When isolated hepatocytes from diabetic rats were placed in primary culture in a serum-free defined medium over a 3-day period, glycogen synthesis was partially restored by cortisol and triiodothyronine and dramatically increased by insulin. Concomitant with restoration of [14C]glycogen synthesis was an insulin-mediated increase in glycogen synthase I and synthase phosphatase activity. Restoration of regulation of glycogen synthesis in primary cultures of hepatocytes from diabetic rats by insulin required the presence of cortisol and triiodothyronine. Primary cultures of hepatocytes from normal rats did not require triiodothyronine for insulin to effect glycogenesis over a 3-day period. These data demonstrate that insulin acts in a chronic manner in concert with other hormones to control synthase phosphatase activity, an effect which may be influencing acute control of hepatic glycogen synthesis.

Animals↗

PubMed1984

Flow microfluorometry analysis of alterations in T-lymphocyte subsets during murine listeriosis.

C57BL/6 mice were infected intravenously with 6 X 10(3) Listeria monocytogenes organisms. As early as day 3 of infection, there was a marked reduction in the number of lymphocytes recovered from the peripheral blood, bone marrow, and thymuses of infected animals. Concomitantly, there was an increase in the number of splenic lymphocytes. By day 14, both the total and differential cell counts were similar in both infected and normal animals. Flow microfluorometric studies comparing the Thy-1.2, Lyt-1, Lyt-2, and surface immunoglobulin (SIg) phenotypes of lymphocytes from normal and infected mice were performed. Between days 3 and 5, there was a decrease in the percentage of Thy-1.2+ cells in the spleens of L. monocytogenes-infected animals. Conversely, the percentages of Lyt-1+, Lyt-2+, and SIg+ cells remained constant. At day 7 of infection, the percentage of Thy-1.2+ splenocytes was within normal limits, and at day 10, the percentage of Thy-1.2+ cells was elevated slightly. The absolute numbers of Thy-1.2+ cells were comparable in both infected and normal animals at early stages (days 3 to 5) of L. monocytogenes infection, but there was a marked elevation of Thy-1.2+ splenocytes at days 7 to 14 of infection. Lyt-1+, Lyt-2+, and SIg+ splenocytes increased in absolute numbers as early as day 3 of infection and were still elevated at day 14. Adrenalectomy before infection had no effect on the results obtained, suggesting that these changes were not mediated by endogenous steroids.

Adrenalectomy↗

PubMed1984

Phosphorylase activation hypersensitivity in hearts of diabetic rats.

A hypersensitivity of glycogen phosphorylase activation by epinephrine and glucagon has been demonstrated in isolated perfused working and non-working hearts from diabetic rats. Accumulation of tissue cAMP and activation of cAMP-dependent protein kinase in response to epinephrine and glucagon were no greater and usually less in hearts of diabetic than of normal rats. Insulin deficiency was not associated with greater changes in epinephrine-induced activation of glycogen phosphorylase kinase than that observed in normal hearts. Perfusion of hearts with subphysiological concentrations of calcium (0.83 mM) partially reversed the diabetes-related hypersensitivity of phosphorylase activation by epinephrine. The phosphorylase activation hypersensitivity to epinephrine was completely reversed by adrenalectomizing diabetic rats 5 days before heart perfusion, an effect potentially caused by steroid-induced changes in cardiac calcium metabolism. These data are consistent with the hypothesis that phosphorylase activation by phosphorylase kinase is allosterically increased in the diabetic due to a diabetes-related increase in free intracellular calcium concentrations.

Animals↗

PubMed1984

Effects of vasoactive intestinal peptide on canine prostatic contraction and secretion.

Vasoactive intestinal peptide (VIP)-like immunoreactivity was found in intrinsic autonomic ganglion cells and nerve fibers located at the surface and within the canine prostate. In anesthetized dogs, porcine VIP (100-3,000 ng/kg iv) decreased arterial pressure and increased heart rate but did not result in the release of fluid from the prostate, indicating that VIP neither contracted glandular smooth muscle to expel fluid nor provoked secretion. Intravenous infusions of VIP at 10, 50, 100, and/or 200 ng X kg-1 X min-1 produced dose-related decreases in arterial pressure, increases in heart rate, and potentiation of the secretory response to the intravenous administration of pilocarpine and to electrical stimulation of the hypogastric nerves at 2 but not at 20 Hz. VIP at 1 microgram/ml neither contracted isolated strips of prostate nor modified their contraction by norepinephrine. It is unlikely that VIP mediates hypogastric nerve-induced prostatic contraction or secretion in the dog, but VIP may serve as a neuromodulator of nerve-induced secretion.

Animals↗