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Biomedical subjects

L R Jones

Publications and source records attributed to L R Jones.

At least 19 recordsLinked to original sources
PubMed1992

Phospholamban expressed in slow-twitch and chronically stimulated fast-twitch muscles minimally affects calcium affinity of sarcoplasmic reticulum Ca(2+)-ATPase.

Chronic excitation, at 2 Hz for 6-7 weeks, of the predominantly fast-twitch canine latissimus dorsi muscle promoted the expression of phospholamban, a protein found in sarcoplasmic reticulum (SR) from slow-twitch and cardiac muscle but not in fast-twitch muscle. At the same time that phospholamban was expressed, there was a switch from the fast-twitch (SERCA1) to the slow-twitch (SERCA2a) Ca(2+)-ATPase isoform. Antibodies against Ca(2+)-ATPase (SERCA2a) and phospholamban were used to assess the relative amounts of the slow-twitch/cardiac isoform of the Ca(2+)-ATPase and phospholamban, which were found to be virtually the same in SR vesicles from the slow-twitch muscle, vastus intermedius; cardiac muscle; and the chronically stimulated fast-twitch muscle, latissimus dorsi. The phospholamban monoclonal antibody 2D12 was added to SR vesicles to evaluate the regulatory effect of phospholamban on calcium uptake. The antibody produced a strong stimulation of calcium uptake into cardiac SR vesicles, by increasing the apparent affinity of the Ca2+ pump for calcium by 2.8-fold. In the SR from the conditioned latissimus dorsi, however, the phospholamban antibody produced only a marginal effect on Ca2+ pump calcium affinity. These different effects of phospholamban on calcium uptake suggest that phospholamban is not tightly coupled to the Ca(2+)-ATPase in SR vesicles from slow-twitch muscles and that phospholamban may have some other function in slow-twitch and chronically stimulated fast-twitch muscle.

Animals↗
PubMed1992

Phospholemman expression induces a hyperpolarization-activated chloride current in Xenopus oocytes.

A new type of chloride channel has been identified by functional expression of phospholemman, a 72-amino acid cardiac sarcolemmal protein with a single transmembrane domain. Xenopus oocytes injected with phospholemman RNA developed a chloride-selective current, which was activated by hyperpolarizing pulses. The current activated very slowly with a pronounced sigmoidal delay, did not inactivate, and increased in amplitude with trains of pulses, depolarized holding potentials, and low extracellular pH. Point mutations within the single transmembrane region abolished the sigmoidal delay of expressed currents. Phospholemman appears to be the smallest plasma membrane channel protein yet known. The structure is dissimilar to any chloride channel described thus far.

Amino Acid Sequence↗
PubMed1992

Cardiac-specific phosphorylation site for multifunctional Ca2+/calmodulin-dependent protein kinase is conserved in the brain ryanodine receptor.

An antiserum raised against the region of the cardiac ryanodine receptor (residues 2805-2819) containing the phosphorylation site for multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) was used to identify the brain ryanodine receptor. This antiserum, which is cardiac isoform-specific, immunoprecipitated greater than 90% of the [3H]ryanodine receptor binding sites solubilized from guinea pig brain membranes. The immunoprecipitated brain receptor exhibited the characteristic cardiac-type mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The brain ryanodine receptor, like the cardiac ryanodine receptor, was a substrate for CaM kinase. Affinity-purified, site-specific antibodies completely blocked phosphorylation of both brain and cardiac receptors by CaM kinase, and two-dimensional peptide mapping identified the same major 32P-labeled peptide in receptors from both tissues. 125I-Labeled receptors also gave the same peptide maps. These results confirm that mammalian brain expresses the cardiac isoform of the ryanodine receptor. Furthermore, the unique CaM kinase phosphorylation site, which has been shown to regulate Ca2+ channel activity, is conserved.

Animals↗
PubMed1992

Effects of total body irradiation on salivary gland function and caries-associated oral microflora in bone marrow transplant patients.

Forty-one cancer patients who received chemoradiotherapy conditioning with bone marrow transplantation were examined for changes in salivary gland function and caries-associated oral microflora. Salivary flow rates (stimulated and unstimulated) and Streptococcus mutans levels decreased after pretransplant cytoreductive therapy and posttransplant prophylactic antibiotic therapy. Normal levels returned with time after the patients left the protected environment. Lactobacillus counts were not affected. Chronic graft-versus-host disease did not significantly influence saliva production. Results indicate that irradiation is probably the major factor responsible for the transient xerostomia.

Administration, Topical↗
PubMed1992

Estimating milk, fat, and protein lactation curves with a test day model.

Test day models were used to estimate lactation curves for milk, fat, protein, fat percentage, and protein percentage and to study the influence of age, season, and herd productivity on Holstein lactation curves. Random effects of lactation within herd and fixed effects of herd test date were absorbed. Fixed effects of cow's age on test day and either DIM (57 divisions) by parity (1, 2, greater than or equal to 3) class or season of calving (winter or summer) by DIM by parity class were estimated. Lactation curves for yield traits derived from DIM solutions were flatter for first versus later lactation, even without addition of age effects. Differences between lactation curves for the two seasons were slight, suggesting that most observed seasonal differences are caused by seasonal productivity accounted for by herd test date effects. At peak, winter calving cows yielded slightly more milk of similar fat percentage but of lower protein percentage than those calving in summer. Data were also partitioned into nine subsets based on rolling herd milk and fat percentage. Lactation curves for yield traits, but not percentage traits, varied with rolling herd milk. Lactation curves for fat yield and percentage varied with rolling herd fat percentage.

Age Factors↗
PubMed1991

Unique phosphorylation site on the cardiac ryanodine receptor regulates calcium channel activity.

Ryanodine receptors have recently been shown to be the Ca2+ release channels of sarcoplasmic reticulum in both cardiac muscle and skeletal muscle. Several regulatory sites are postulated to exist on these receptors, but to date, none have been definitively identified. In the work described here, we localize one of these sites by showing that the cardiac isoform of the ryanodine receptor is a preferred substrate for multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase). Phosphorylation by CaM kinase occurs at a single site encompassing serine 2809. Antibodies generated to this site react only with the cardiac isoform of the ryanodine receptor, and immunoprecipitate only cardiac [3H]ryanodine-binding sites. When cardiac junctional sarcoplasmic reticulum vesicles or partially purified ryanodine receptors are fused with planar bilayers, phosphorylation at this site activates the Ca2+ channel. In tissues expressing the cardiac isoform of the ryanodine receptor, such as heart and brain, phosphorylation of the Ca2+ release channel by CaM kinase may provide a unique mechanism for regulating intracellular Ca2+ release.

Amino Acid Sequence↗
PubMed1991

Purification and complete sequence determination of the major plasma membrane substrate for cAMP-dependent protein kinase and protein kinase C in myocardium.

A protein of apparent Mr = 15,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is the major plasma membrane substrate for cAMP-dependent protein kinase (PK-A) and protein kinase C (PK-C) in several different tissues. In the work described here, we purified, cloned, and sequenced the canine cardiac sarcolemmal "15-kDa protein." The amino terminus of the purified protein was not blocked, allowing determination of 50 consecutive residues by standard Edman degradation. Overlapping proteolytic phosphopeptides yielded 22 additional residues at the carboxyl terminus. Dideoxy sequencing of the full-length cDNA confirmed that the 15-kDa protein contains 72 amino acids, plus a 20-residue signal sequence. The mature protein has a calculated Mr = 8409. There is one hydrophobic membrane-spanning segment composed of residues 18-37. The acidic amino-terminal end (residues 1-17) of the protein is oriented extracellularly, whereas the basic carboxyl-terminal end (residues 38-72) projects into the cytoplasm. The positively charged carboxyl terminus contains the phosphorylation sites for PK-A and PK-C. In the transmembrane region, the 15-kDa protein exhibits 52% amino acid identity with the "gamma" subunit of Na,K-ATPase. High stringency Northern blot analysis revealed that 15-kDa mRNA is present in heart, skeletal muscle, smooth muscle, and liver but absent from brain and kidney. We propose the name "phospholemman" for the 15-kDa protein, which denotes the protein's location within the plasma membrane and its characteristic multisite phosphorylation.

Amino Acid Sequence↗
PubMed1991

Identification of the major protein phosphatases in mammalian cardiac muscle which dephosphorylate phospholamban.

The protein phosphatases which dephosphorylate native, sarcoplasmic reticulum (SR)-associated phospholamban were studied in cardiac muscle extracts and in a Triton fraction prepared by detergent extraction of myofibrils, the latter fraction containing 70-80% of the SR-associated proteins present in the tissue. At physiological concentrations of free Mg2+ (1 mM), protein phosphatase 1 (PP1) accounted for approximately 70% of the total phospholamban phosphatase activity in these fractions towards either Ser-16 (the residue labelled by cAMP-dependent protein kinase, PK-A) or Thr-17 (the residue phosphorylated by an SR-associated Ca2+/calmodulin-dependent protein kinase). Protein phosphatase 2A (PP2A) and protein phosphatase 2C (PP2C) accounted for the remainder of the activity. A major form of cardiac PP1, present in comparable amounts in both the extract and Triton fraction, was similar, if not identical, to skeletal muscle protein phosphatase 1G (PP1G), which is composed of the PP1 catalytic (C) subunit complexed to a G subunit of approximately 160 kDa, responsible for targeting PP1 to both the SR and glycogen particles of skeletal muscle. This conclusion was based on immunoblotting experiments using antibody to the G subunit, ability to bind to glycogen and the release of PP1 activity from glycogen upon incubation with PK-A and MgATP. PP1 accounted for approximately 90% of the phospholamban (Ser-16 or Thr-17) phosphatase activity in the material sedimented by centrifugation at 45,000 x g, a fraction prepared from cardiac extracts which is enriched in SR membranes. The G subunit in this fraction could be solubilised by Triton X-100, but not with 0.5 M NaCl or digestion with alpha-amylase, indicating that it is bound to membranes and not to glycogen. By analogy with the situation in skeletal muscle, the PK-A catalysed phosphorylation of the G subunit, with ensuing release of the C subunit from the SR, may prevent PP1 from dephosphorylating SR-bound substrates and represent one of the mechanisms by which adrenalin increases the phosphorylation of cardiac phospholamban (Ser-16 and Thr-17) in vivo. Hearts left in situ post mortem lose 85-95% of their PP1 activity within 20-30 min. This remarkable disappearance of PP1 may partly explain why the importance of this enzyme in cardiac muscle metabolism has not been recognized previously.

Animals↗
PubMed1991

Syntheses of tolrestat analogues containing additional substituents in the ring and their evaluation as aldose reductase inhibitors. Identification of potent, orally active 2-fluoro derivatives.

A series of aldose reductase inhibitors were prepared which were analogues of the potent, orally active inhibitor tolrestat (1). These compounds (5, 7, 9, and 10) have an extra substituent on one of the unoccupied positions on the naphthalene ring of 1. Primary amide prodrugs of several members from the series 5 and 7, namely 6 and 8, respectively, were also prepared. These compounds were evaluated in two in vitro systems: an isolated enzyme preparation from bovine lens to assess their intrinsic inhibitory activity and an isolated sciatic nerve assay to determine their ability to penetrate membranes of nerve tissue. These compounds were also evaluated in vivo as inhibitors of galactitol accumulation in the lens, sciatic nerve, and diaphragm of galactose-fed rats. In general, compounds in series 5, 7, 9, and 10 were potent inhibitors of bovine lens aldose reductase. 2-Halo-substituted analogues from the series 5, 7, and 9 exhibited high activity in the nerve of the 4-day-galactose-fed rat, and in several instances, the primary amide prodrug 8 enhanced the in vivo potency of the respective carboxylic acid 7. Two 2-fluoro-derivatives, 8a and 9a, had especially high activity in vivo and were chosen for additional studies. These compounds were found to be approximately equipotent to tolrestat in the sciatic nerve of the galactose-fed rat and the STZ rat, as judged by their ED50's in these assays. Although primary amide analogue 8a did not have intrinsic inhibitory activity toward aldose reductase, it was metabolized to an active form in vivo and also in vitro within the sciatic nerve.

Administration, Oral↗
PubMed1991

Phospholamban mediates the beta-adrenergic-enhanced Ca2+ uptake in mammalian ventricular myocytes.

To probe the molecular mechanism responsible for the relaxant effect of catecholamines in heart muscle, we studied the effect of a monoclonal antibody (2D12) against phospholamban in intact whole cell clamped guinea pig ventricular myocytes, in which intracellular Ca2+ transient and Ca2+ current were simultaneously measured. The antibody stimulated Ca2+ uptake in guinea pig ventricular sarcoplasmic reticular vesicles, shifting the apparent dissociation constant for activation by Ca2+ from 200 to 60 nM. The stimulatory effect of the antibody could be mimicked by the catalytic subunit of adenosine 3',5'-cyclic monophosphate-dependent kinase and could be blocked by phospholamban peptide 2-25. Dialysis of ventricular myocytes with the antibody enhanced the rate of uptake of Ca2+ and significantly suppressed the ability of isoproterenol to enhance the rate of uptake and release of Ca2+ by depolarizing pulses. These data suggest that not only is phosphorylation of phospholamban crucial in sequestration of Ca2+ by the sarcoplasmic reticulum, but that this process may account for the catecholamine-enhanced rate of Ca2+ uptake release in heart muscle.

Adrenergic beta-Agonists↗
PubMed1979

Mg2+ and ATP effects on K+ activation of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum.

ATP and the divalent cations Mg2+ and Ca2+ regulated K+ stimulation of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum vesicles. Millimolar concentrations of total ATP increased the K+-stimulated ATPase activity of the Ca2+ pump by two mechanisms. First, ATP chelated free Mg2+ and, at low ionized Mg2+ concentrations, K+ was shown to be a potent activator of ATP hydrolysis. In the absence of K+ ionized Mg2+ activated the enzyme half-maximally at approximately 1 mM, whereas in the presence of K+ the concentration of ionized Mg2+ required for half-maximal activation was reduced at least 20-fold. Second MgATP apparently interacted directly with the enzyme at a low affinity nucleotide site to facilitate K+-stimulation. With a saturating concentration of ionized Mg2+, stimulation by K+ was 2-fold, but only when the MgATP concentration was greater than 2 mM. Hill plots showed that K+ increased the concentration of MgATP required for half-maximal enzymic activation approx. 3-fold. Activation of K+-stimulated ATPase activity by Ca2+ was maximal at an ionized Ca2+ concentration of approx. 1 microM. At very high concentrations of either Ca2+ or Mg2+, basal Ca2+-dependent ATPase activity persisted, but the enzymic response to K+ was completely inhibited. The results provide further evidence that the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum has distinct sites for monovalent cations, which in turn interact allosterically with other regulatory sites on the enzyme.

Adenosine Triphosphatases↗
PubMed1979

Separation of vesicles of cardiac sarcolemma from vesicles of cardiac sarcoplasmic reticulum. Comparative biochemical analysis of component activities.

Sarcolemmal and sarcoplasmic reticulum membrane vesicle fractions were isolated from cardiac microsomes. Separation of sarcolemmal and sarcoplasmic reticulum membrane markers was documented by a combination of correlative assay and centrifugation techniques. To facilitate the separation, the crude microsomes were incubated in the presence of ATP, Ca2+, and oxalate to increase the density of the sarcoplasmic reticulum vesicles. After sucrose gradient centrifugation, the densest subfraction (sarcoplasmic reticulum) contained the highest (K+,Ca2+)-ATPase activity and virtually no (Na2+,K+)-ATPase activity, even when latent (Na+,K+)-ATPase activity was unmasked. In addition, the sarcoplasmic reticulum fraction contained no significant sialic acid, beta receptor binding activity, or adenylate cyclase activity. Sarcolemmal membrane fractions were of low buoyant density. Preparations most enriched in sarcolemmal vesicles contained the highest level of all the other parameters and only about 10% of the (K+,Ca2+)-ATPase activity of the sarcoplasmic reticulum fraction. The results suggest that (Na+,K+)-ATPase, sialic acid, beta-adrenergic receptors, and adenylate cyclase can be entirely accounted for by the sarcolemmal content of cardiac microsomes. Gel electrophoresis of the sarcolemmal and sarcoplasmic reticulum membrane fractions showed distinct bands. Membrane proteins exclusive to each of the fractions were also demonstrated by phosphorylation. Cyclic AMP stimulated phosphorylation by [gamma-32P]ATP of two proteins of apparent Mr = 20,000 and 7,000 that were concentrated in sarcoplasmic reticulum, but the stimulation was markedly dependent on the presence of added soluble cyclic AMP-dependent protein kinase. Cyclic AMP also stimulated phosphorylation of membrane proteins in sarcolemma, but this phosphorylation was mediated by an endogenous protein kinase activity. The apparent molecular weights of these phosphorylated proteins were 165,000, 90,000, 56,000, 24,000, and 11,000. The results suggest that sarcolemma may contain an integral enzyme complex, not present in sarcoplasmic reticulum, that contains beta-adrenergic receptors, adenylate cyclase, cyclic AMP-dependent protein kinase, and several substrates of the protein kinase.

Animals↗
PubMed1979

Psychogenic urinary retention.

Six cases (5 females and 1 male) of unexplained urinary retention are reported. The diagnosis of psychogenic urinary retention was considered after negative medical, urologic, urodynamic, and neurologic evaluations. This diagnosis was then confirmed by psychiatric consultation. Urinary retention was managed by combining biofeedback-monitored relaxation and specific imagery techniques with intermittent self-catheterization on a fixed schedule. Psychotherapy, pharmacotherapy, and behavior therapy were also utilized with good to excellent short-term results.

Adolescent↗
PubMed1979

Calcium handling by cardiac sarcoplasmic reticulum.

Over the past ten years knowledge of the biochemistry of cardiac sarcoplasmic reticulum vesicles has been considerably extended. In almost all respects the Ca2+ pump of cardiac sarcoplasmic reticulum shows striking similarities to the Ca2+ pump of skeletal muscle sarcoplasmic reticulum. On the other hand, Ca2+ release mechanisms seem to be more complex in cardiac than in skeletal sarcoplasmic reticulum. Future research undoubtedly will involve characterizing the function of the many additional proteins present in cardiac sarcoplasmic reticulum, and determining possible roles for these proteins in regulating Ca2+ uptake and Ca2+ release by these membranes.

Animals↗
PubMed1979

Ryanodine: its alterations of cat papillary muscle contractile state and responsiveness to inotropic interventions and a suggested mechanism of action.

Cat right ventricular papillary muscles responded biphasically to cumulative additions of ryanodine. A progressive and pronounced negative inotropic effect was observed with low to intermediate ryanodine concentrations (5 nM-1 muM) while a rebound or reversal of these initial changes back toward pre-drug values was obtained as the ryanodine concentration was further increased to 100 muM. Active force development (DF), the rate of force development (dF/dt), as well as the rate of relaxation all exhibited these bidirectional changes. In contrast, time to peak force underwent only a progressive prolongation over the entire concentration range tested. This response pattern was observed with both normal and K+-depolarized (isoproterenol- or dibutyryl cAMP-restored) preparations. The response to a single addition of 100 muM ryanodine, in the presence of 2.5 mM Ca++ mimicked both the qualitative and quantitative aspects of the cumulative concentration response curve. In the presence of 5.0 mM Ca++ the high concentration of ryanodine no longer caused depression but instead caused only a slowly developing, monophasic increase in DF. Ryanodine also changed the response of ventricular muscle to other inotropic interventions. Ryanodine (1 muM; 2.5 mM Ca++) abolished the normal increase in dF/dt following either paired electrical stimulation (PES) or 50 mOsM mannitol, but not that in response to a doubling of the stimulation rate (0.2--0.4 Hz). After ryanodine exposure, the potentiation of developed force by PES was shifted from the first (regular) to the second (premature) contraction, producing a summation-like waveform. Prior addition of the calcium channel antagonist D600 (1 muM) did not alter ryanodine-induced changes in PES. Caffeine (1 mM) produced alterations in the responses to PES and hyperosmolarity which were similar to those observed with ryanodine. In the presence of high concentrations of both ryanodine (100 muM) and calcium (5 mM) both the transient and steady-state responses to a doubling of the stimulation rate (0.2--0.4 Hz) were markedly depressed, whereas the decrease in DF or dF/dt normally accompanying a reduction in the rate of stimulation was attenuated. The data obtained in the present study are consistent with a functional inhibition of sarcoplasmic reticular calcium release by ryanodine.

Alkaloids↗