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Biomedical subjects

R A Murphy

Publications and source records attributed to R A Murphy.

At least 19 recordsLinked to original sources
PubMed1995

Genistein, a tyrosine kinase inhibitor, reduces Ca2+ mobilization in swine carotid media.

DiSalvo and colleagues (Biochem. Biophys. Res. Commun. 190: 968-974, 1993) found that tyrosine kinase inhibitors reduced force at constant Ca2+ concentrations in permeabilized mesenteric arterioles. These data suggest that tyrosine kinase activation could regulate Ca2+ sensitivity in intact vascular smooth muscle. We tested this hypothesis by examining the effects of the tyrosine kinase inhibitor genistein on intracellular Ca2+ concentration ([Ca2+]i), myosin regulatory light chain (MRLC) phosphorylation, and isometric stress in intact swine carotid media tissues. Pretreatment with 30 microM genistein attenuated histamine-induced increases in [Ca2+]i (estimated using the photoprotein aequorin), MRLC phosphorylation, and stress. The genistein-dependent decrease in [Ca2+]i quantitatively accounted for the decrease in MRLC phosphorylation and stress. There was no measurable change in the Ca2+ dependence of MRLC phosphorylation or the dependence of force on MRLC phosphorylation. Genistein inhibited contractions independently of the source of activator Ca2+. These data suggest that tyrosine kinase(s) may influence force development in the intact swine carotid media by altering [Ca2+]i rather than modulating the Ca2+ sensitivity of MRLC phosphorylation.

Aequorin↗
PubMed1995

Technical note: detection and quantification of supplemental fungal beta-glucanase activity in animal feed.

Selected hydrolytic enzymes are added to animal feeds in order to degrade specific antinutritional factors and(or) to increase availability of certain components of feedstuffs to the animal. A method is described that allows detection and quantification of beta-glucanase activity in complex feedstuffs. The method is based on radial diffusion of an enzyme-containing feed extract through an agar gel in which lichenan substrate (a relatively inexpensive glucan of mixed beta 1-->4 and beta 1-->3 linkages) has been dissolved. A linear relationship between the diameter of the zone of substrate hydrolyzed and the log of enzyme activity present was observed. The assay described is technically straightforward and requires no specialized equipment. At typical commercial inclusion levels (1 kg/t), the activity of a supplemental beta-glucanase, added to feed in a commercial mill was determined by averaging several measurements, with a precision of +/- 4%, variation between individual readings of +/- 11.3% (SD), and recovery of 109%. By using high-concentration feed extracts, the method was sensitive enough to detect background and(or) supplemental beta-glucanase activities as low as .05 kg/t supplement equivalent. This method allows consumers, producers, and regulatory authorities to measure the activity of beta-glucanase in feed at commercial inclusion levels and, hence, study the effects of processes such as pelleting and extrusion on such supplements.

Animal Feed↗
PubMed1994

Dependence of ATP consumption on cross-bridge phosphorylation in swine carotid smooth muscle.

1. Ca(2+)-dependent phosphorylation of the myosin regulatory light chain (MRLC) initiates cross-bridge cycling and contraction in smooth muscle. A four-state cross-bridge model, in which Ca(2+)-dependent phosphorylation is the only proposed regulatory mechanism, can predict the mechanical output of the swine carotid media. Our aims were to determine whether ATP consumption rates and the economy of force maintenance are regulated functions of MRLC phosphorylation as predicted by the model. 2. Steady-state force and oxygen consumption were measured in medial rings of swine carotid arteries activated with depolarizing solutions and agents capable of maintaining a wide range of steady-state myoplasmic Ca2+ and MRLC phosphorylation levels. 3. Suprabasal ATP consumption increased almost linearly with MRLC phosphorylation and exhibited a hyperbolic increase with active stress, as predicted. 4. The economy of stress maintenance fell with increases in suprabasal phosphorylation. 5. In absolute terms the energetic cost of covalent regulation by cross-bridge phosphorylation was small, although it may be a significant fraction of the ATP consumption associated with contraction.

Adenosine Triphosphate↗
PubMed1994

Regulation of tissue-specific expression of alternative peripheral myelin protein-22 (PMP22) gene transcripts by two promoters.

Mutations affecting the peripheral myelin protein-22 (PMP22) gene have been shown to be associated with inherited peripheral neuropathies. To provide the molecular basis for the analysis of such mutations, we have cloned and characterized the human PMP22 gene. It spans approximately 40 kilobases and contains four coding exons. Detailed analysis of its 5'-flanking region suggested the presence of two alternatively transcribed, but untranslated exons. Mapping of separate PMP22 mRNA transcription initiation sites to each of these exons indicates that PMP22 expression is regulated by two alternatively used promoters. In support of this hypothesis, both putative promoter sequences demonstrated the ability to drive expression of reporter genes in transfection experiments. Furthermore, the structures of the 5'-portions of the PMP22 genes appear to be identical in rat and human, supporting the biological significance of the observed arrangement of regulatory regions. The relative expression of the alternative PMP22 transcripts is tissue-specific, and high levels of the exon 1A-containing transcript are tightly coupled to myelin formation. In contrast, exon 1B-containing transcripts are predominant in non-neural tissues and in growth-arrested primary fibroblasts. Interestingly, although a strong upregulation of PMP22 mRNA was observed in cultured Schwann cells in the presence of the adenylate cyclase activator forskolin under various culture conditions, the regulation of the different PMP22 mRNA species did not mimic the regulation that occurs during myelin formation in vivo. The observed regulation of the PMP22 gene by a complex molecular mechanism is consistent with the proposed dual role of PMP22 in neural and non-neural tissue.

Alternative Splicing↗
PubMed1994

What is special about smooth muscle? The significance of covalent crossbridge regulation.

Knowledge of the molecular mechanisms involved in chemomechanical transduction and the regulatory elements determining contraction and relaxation are largely derived from cross-striated muscle. However, it has become clear that vertebrate smooth muscles have features lacking in striated muscle. These derive from regulated crossbridge cycling rates observed as variable shortening velocities and ATP consumption rates. The special properties of smooth muscle are associated with differences in the physiological roles of muscle in the walls of hollow organs and reflect a common molecular motor governed by different regulatory mechanisms. A remarkably simple scheme involving Ca(2+)-dependent phosphorylation of crossbridges in smooth muscle can predict much of the mechanical and energetics behavior characterizing the muscle of hollow organs. Nevertheless, many unresolved issues are identified that are the focus of current research efforts.

Animals↗
PubMed1994

Cyclic nucleotide dependent relaxation in vascular smooth muscle.

Although not without controversy, the mechanisms inducing contraction of vascular smooth muscle are relatively well defined. There is a stimulus-induced increase in myoplasmic [Ca2+] with activation of myosin light chain kinase by the Ca(2+)-calmodulin complex, phosphorylation of the 20-kDa regulatory light chain of myosin, with subsequent cross-bridge cycling and force development. Ca(2+)-dependent phosphorylation of the myosin regulatory light chain appears to be the primary mechanism responsible for regulating stress in vascular smooth muscle. The relationship between myoplasmic [Ca2+] and myosin phosphorylation (i.e., the calcium sensitivity of phosphorylation) is regulated. It is higher with agonist stimulation than in tissues depolarized with high potassium solutions or after skinning procedures. The relationship between myosin phosphorylation and stress appears to be invariant with physiologic stimulation. This suggests that cross-bridge phosphorylation normally determines contraction. The mechanisms of relaxation are less well defined. In the most simple scheme, reduction of myoplasmic [Ca2+] with a fall in myosin light chain kinase activity would suffice to account for dephosphorylation of the regulatory light chain and relaxation. However, other mechanisms have been implicated in cyclic nucleotide dependent relaxation in vascular and other smooth muscle tissues. The current hypotheses of the mechanism of cyclic nucleotide dependent relaxation in vascular smooth muscle are reviewed.

Animals↗
PubMed1994

Energetics of crossbridge phosphorylation and contraction in vascular smooth muscle.

Ca(2+)-dependent crossbridge phosphorylation is the primary mechanism governing crossbridge cycling in smooth muscle. A four-state crossbridge model in which phosphorylation is the only proposed regulatory mechanism was successful in predicting the mechanical properties of the swine carotid media including latch (sustained force with reduced crossbridge cycling). This model also predicts that the ATP consumption of crossbridge phosphorylation is approximately equal to that of crossbridge cycling and that ATP consumption will rise hyperbolically with increases in steady-state force. This review shows these predictions to be consistent with the available energetics data for the carotid media. The absolute energetic cost of covalent regulation is modest and less than the energy savings associated with latch. However, covalent regulation should reduce the total mechanical efficiency of smooth muscle relative to striated muscle.

Animals↗
PubMed1993

Immunocytochemical localization of sodium channels in an insect central nervous system using a site-directed antibody.

Antibodies to channel proteins and specific peptide sequences have been previously used to localize voltage-activated sodium channels in the rat brain. Here we describe the first localization of sodium channels in an insect nervous system using a site-directed antibody. The mesothoracic ganglion of the cockroach was stained with an antibody to the highly conserved SP19 sequence. Antibody labelling was visualized by light microscopy using the avidin/biotin method on wax sections, and transmission electron microscopy of immunogold-labelled thin sections. Central ganglia of insects contain clearly separated regions of cell bodies, synaptic neuropil, axon tracts, and nerves. Antibody staining by light microscopy was limited to neurons, and was intense in axons throughout the ganglion and nerves. Staining was also strong in the cytoplasm, but not the nuclei, of many neuronal cell bodies. Neuropil regions were relatively lightly labelled. These findings can be correlated with the known electrophysiology of the ganglion. Electron microscopy detected sodium channels in areas surrounding axons, probably including axon membranes and enveloping glial cell membranes. Axonal mitochondria were also heavily labelled, suggesting a sodium channel transport function for these organelles.

Amino Acid Sequence↗
PubMed1993

Models of the mechanism for crossbridge attachment in smooth muscle.

The mechanism responsible for formation of attached, dephosphorylated crossbridges (latchbridges) in smooth muscle is controversial. Myosin light chain phosphorylation may be obligatory for crossbridge attachment; if this were the case, latchbridges would arise solely by dephosphorylation of attached, phosphorylated crossbridges. Alternatively, the presence of attached crossbridges could induce cooperative activation by allowing dephosphorylated crossbridges to attach to the thin filament. We evaluated whether four-state models based on dephosphorylation and/or cooperativity-regulated attachment could quantitatively predict smooth muscle contractile behaviour. Five quantitative models for transitions between crossbridge states were developed. Mechanisms for latchbridge formation included: (1) dephosphorylation, (2) cooperativity-regulated attachment dependent only on attached, phosphorylated crossbridges, (3) cooperativity-regulated attachment dependent on all attached crossbridges, (4) dephosphorylation and cooperativity-regulated attachment dependent only on attached, phosphorylated crossbridges, and (5) dephosphorylation and cooperativity-regulated attachment dependent on all attached crossbridges. All five models approximated the time course of contraction and the dependence of steady-state stress on myosin phosphorylation in the swine carotid artery. In the two models that had cooperative attachment regulated by all attached crossbridges, small increases in the rate constant for cooperativity-regulated attachment resulted in positive feedback and irreversible contraction. We suggest that a number of four-state crossbridge models can predict contractile behaviour in arterial smooth muscle. Potentially, latchbridges could be formed by both dephosphorylation and cooperativity-regulated attachment. If cooperativity-regulated latchbridge attachment does exist in smooth muscle, we suggest that it should be dependent only on the number of phosphorylated crossbridges rather than all attached crossbridges.

Actomyosin↗
PubMed1993

Expression of myosin regulatory light-chain isoforms and regulation of phosphorylation in smooth muscle.

Our objectives were to 1) determine how growth state and cell density affect the expression of the smooth muscle (SM) and nonmuscle (NM) isoforms of the 20-kDa myosin regulatory light chains (MLC20) in cultured rat aortic smooth muscle cells (SMC) and 2) to determine whether angiotensin II stimulates differential phosphorylation of SM and NM MLC20 isoforms in an effort to assess whether the SM and NM isoforms may subserve different cellular functions. The results demonstrated that changes in the SM MLC20 isoform content were inversely correlated with cell growth but independent of cell density. MLC20 phosphorylation levels were 20.8 +/- 2.9 and 19.4 +/- 3.7% for SM and NM isoforms, respectively, in unstimulated, substrate-attached SMC. Angiotensin II transiently elevated phosphorylation levels of both the SM and NM MLC20 isoforms to 60-70%. No differences in either the magnitude or the kinetics of phosphorylation were observed for the SM vs. NM isoforms. Forskolin, 3-isobutyl-1-methylxanthine, or isoproterenol treatment led to parallel dephosphorylation of the SM- and NM-specific isoforms followed by depolymerization of stress fibers and cell arborization. The studies provide evidence that growth arrest of cultured SMC enhances expression of cell-specific/-selective proteins characteristic of differentiated SM. However, there was no evidence for differential phosphorylation changes of SM and NM MLC20 isoforms in response to activating or relaxing agents as expected if these isoforms subserve different cellular functions.

Animals↗
PubMed1993

Immunological relationships of NGF, BDNF, and NT-3: recognition and functional inhibition by antibodies to NGF.

Polyclonal antibodies raised against mouse 2.5S NGF (mNGF) and against synthetic peptides made from hydrophilic portions of mNGF have been used to compare the immunological properties of mNGF, human recombinant brain-derived neurotrophic factor (hrBDNF), and human recombinant neurotrophin-3 (hrNT-3). Affinity-isolated antibodies raised against intact mNGF reacted with all three neurotrophins when tested by ELISA and totally or partially blocked the bioactivities of the proteins in survival assays of embryonic chicken sensory and sympathetic neurons. On Western blots, mNGF antibodies reacted with all three neurotrophins but less well with hrBDNF and hrNT-3 than with mNGF. Antibodies to hydrophilic peptides within NGF (amino acids 23-35, 59-67, 69-79, and 91-100) showed partial reactivity with some but not all of the neurotrophins when tested by ELISA and on Western blots. The peptide antibodies were also selectively effective in reducing the survival-promoting activity of the neurotrophins on sensory neurons. Results show that mNGF, hrBDNF, and hrNT-3 are immunologically related proteins and that mNGF antibodies react also with other members of the neurotrophin family.

Amino Acid Sequence↗
PubMed1992

The nerve growth factor receptor: a multicomponent system that mediates the actions of the neurotrophin family of proteins.

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin 3 (NT-3) are members of a family of structurally related proteins termed neurotrophins that promote the growth and survival of neurons in the central and peripheral nervous systems. Each of these proteins bind to at least two membrane receptors. One is the low affinity nerve growth factor receptor (p75), which binds each member of the neurotrophin family. The other is one of a family of tyrosine kinase receptors--trkA binds only NGF, the related trkB receptor binds BDNF and NT-3, and trkC binds NT-3 alone. This article reviews kinetic and biochemical information on p75 and its relationship to the trk gene products.

Animals↗
PubMed1992

Adenosine 5'-triphosphate consumption by smooth muscle as predicted by the coupled four-state crossbridge model.

We have proposed a four-state crossbridge model to explain contraction and the latch state in arterial smooth muscle. Ca(2+)-dependent crossbridge phosphorylation was the only postulated regulatory mechanism and the latchbridge (a dephosphorylated, attached crossbridge) was the only novel element in the model. In this study, we used the model to predict rates of ATP consumption by crossbridge phosphorylation (JPhos) and cycling (JCycle) during isometric and isotonic contractions in arterial smooth muscle; then we compared model predictions with experimental data. The model predicted that JPhos and JCycle were similar in magnitude in isometric contractions, and both increased almost linearly with myosin phosphorylation. The predicted relationship between isometric stress and ATP consumption was quasihyperbolic, but approximately linear when myosin phosphorylation was below 35%, in agreement with most of the available data. Muscle shortening increased the predicted values of JCycle up to 3.7-fold depending on shortening velocity and the level of myosin phosphorylation. The predicted maximum work output per ATP was 7.4-7.8 kJ/mol ATP and was relatively insensitive to changes in myosin phosphorylation. The predicted increase in JCycle with shortening was in agreement with available data, but the model prediction that work output per ATP was insensitive to changes in myosin phosphorylation was unexpected and remains to be tested in future experiments.

Adenosine Triphosphate↗
PubMed1992

Steady-state dependence of stress on cross-bridge phosphorylation in the swine carotid media.

Tonic contractions of the swine carotid media are typically characterized by initial transients in myoplasmic [Ca2+] and cross-bridge phosphorylation followed by force maintenance with reduced intracellular [Ca2+] and cross-bridge phosphorylation ("latch"). The presence of effective mechanisms in the carotid media to limit steady-state myoplasmic [Ca2+] and cross-bridge phosphorylation to modest increases over resting values has limited experimental attempts to determine the dependence of active stress (force/tissue cross-sectional area) on cross-bridge phosphorylation. In this study, we employed stimulation protocols that combined effective contractile agonists with inhibitors of Ca2+ extrusion or sequestration to achieve high steady-state levels of cross-bridge phosphorylation (up to 60%). Increases in cross-bridge phosphorylation from 30 to 60% were not associated with significant increases in stress in agreement with the predictions of Hai and Murphy [Am. J. Physiol. 254 (Cell Physiol. 23): C99-C106, 1988] four-state cross-bridge model for the carotid media. Thus cross-bridge phosphorylation may suffice to determine force generation in vascular smooth muscle if both phosphorylated and dephosphorylated attached cross bridges (or latch bridges) contribute to active stress.

Animals↗
PubMed1992

Nitrovasodilators relax arterial smooth muscle by decreasing [Ca2+]i and uncoupling stress from myosin phosphorylation.

Elevations in guanosine 3',5'-cyclic monophosphate concentration ([cGMP]) are proposed to induce arterial smooth muscle relaxation by either 1) decreasing myoplasmic [Ca2+] ([Ca2+]i), 2) decreasing the [Ca2+]i sensitivity of phosphorylation, or 3) uncoupling force from myosin phosphorylation. We evaluated the importance of each of these mechanisms by measuring changes in [cGMP], aequorin- and fura-2-estimated [Ca2+]i, myosin light chain phosphorylation, and stress in histamine-stimulated swine carotid arteries. In tissues submaximally stimulated with 3 microM histamine, nitroprusside (NP) induced a proportional decrease in myoplasmic [Ca2+] and myosin phosphorylation, suggesting that the relaxation was at least partially induced by decreases in [Ca2+]i without a change in the [Ca2+]i sensitivity of phosphorylation. In tissues maximally stimulated with 10 microM histamine, NP and nitroglycerin produced significant relaxations that were not associated with significant sustained reductions in [Ca2+]i or myosin phosphorylation. With both submaximal and maximal histamine stimulation, nitrovasodilators produced more substantial relaxation than that expected from the nitrovasodilator-induced reduction in myosin phosphorylation. These results suggest that nitrovasodilators relax histamine-stimulated swine arterial smooth muscle by at least two mechanisms: 1) reducing [Ca2+]i, an effect observed in submaximally stimulated tissues, and 2) uncoupling of stress from myosin phosphorylation.

Animals↗
PubMed1992

Evaluation of the cariogenic potential of the intense natural sweeteners stevioside and rebaudioside A.

Stevioside and rebaudioside A, two intense natural sweeteners, that are constituents of the South American plant Stevia rebaudiana, were tested for cariogenicity in albino Sprague-Dawley rats. Sixty rat pups colonized with Streptococcus sobrinus were divided into four groups and fed stevioside, rebaudioside A or sucrose added to basal diet 2000 as follows: group 1, 30% sucrose; group 2, 0.5% stevioside; group 3, 0.5% rebaudioside A, and group 4, no addition. All four groups were sacrificed after 5 weeks. S. sobrinus counts were made and caries was evaluated according to Keyes' technique. There were no differences in food and water intake and weight gains between the four groups. There were significant differences in sulcal caries scores (p < 0.02) and S. sobrinus counts (p < 0.05) between group 1 and the other three groups. There were no significant differences between the stevioside, rebaudioside A and no-addition groups. It was concluded that neither stevioside nor rebaudioside A is cariogenic under the conditions of this study.

Animals↗
PubMed1992

Spatiotemporal increases in epidermal growth factor receptors following peripheral nerve injury.

Non-neuronal cells of peripheral nerve respond to axonal injury with a series of cellular changes that facilitate neuronal regeneration. To characterize the potential role of the epidermal growth factor (EGF) family of proteins in this response, we monitored the expression of EGF receptor mRNA and protein in the injured rat sciatic nerve. EGF receptor mRNA is synthesized in both primary cultured fibroblasts and Schwann cells, and Schwann cells express EGF receptor-like immunoreactivity. In situ hybridization and immunocytochemistry revealed that EGF receptor mRNA and protein are expressed in Schwann cells and fibroblasts of the sciatic nerve in vivo, and that receptor levels increase following nerve injury. Thirty-six hours postlesion, EGF receptors were expressed in gradients along the nerve both proximal and distal to the lesion, with the highest levels localized adjacent to the transection site. By 72 hr, receptor levels were maintained in a gradient in the proximal segment, but were uniformly increased throughout the portions of the distal segment that were analyzed. These changes were similar to those observed for low-affinity NGF receptor mRNA and protein, with transection causing increased expression in both Schwann cells and fibroblasts. Northern blots confirmed that primary cultured fibroblasts express low-affinity NGF receptor mRNA. To determine whether spatiotemporal gradients were a general characteristic of the nerve injury response, we monitored expression of the mRNA encoding the major myelin protein P0. Levels of P0 mRNA decreased initially in cells immediately adjacent to the transection site and, by 72 hr, were uniformly decreased throughout the distal segment. These data suggest that members of the EGF family of proteins may play a role in the peripheral nerve response to injury, and demonstrate a generalized gradient of cellular responses that commence at the transection site and progress distally in the nerve in the absence of intact axons.

Animals↗