Biomedical subjects

D A Walsh

Publications and source records attributed to D A Walsh.

At least 73 records · Page 4Linked to original sources

PubMed1993

Caffeine does not increase synthesis of heat shock proteins in rat embryos.

Caffeine exposure in utero in rats is known to result in intrauterine growth retardation and lowered birth weight as well as changes to behaviour and brain biochemistry. We have investigated whether caffeine's embryotoxicity is a result of the events associated with increased hsp synthesis, i.e., disruption to normal protein synthesis. Caffeine (30 mg/kg) was administered orally to pregnant rats as single or repeated doses. Embryos were removed 3 h after dosing on gestation day (GD) 9, 10, 11 and 12 and total embryonic protein and RNA analysed. There was no change in the mRNA or protein levels of hsp 88, 71/73, and 25 after acute or chronic treatment. To separate the direct effect of caffeine from those mediated through the mother, whole rat embryo culture was used. Caffeine (50 micrograms/ml) for 90 min did not increase hsp 88, 73 or 25 mRNA levels in 9.5, 10.5 and 11.5 GD cultured embryos. We conclude that in vivo or in vitro treatment of 9-12 GD rat embryos with moderate to high doses of caffeine does not increase the synthesis of the major mammalian hsps. Hence, hsp induction is unlikely to play a role in the embryotoxic actions of caffeine.

Animals↗

PubMed1993

Localization and characterization of neuropeptide Y binding sites in porcine and human colon.

1. We have used quantitative receptor autoradiography to investigate the localization and characteristics of binding sites for 125Iodine-Bolton Hunter-labelled human neuropeptide Y ([125I]-BH-NPY) in porcine and human colon, and compared the binding characteristics with those found in porcine spleen. 2. Saturable, specific, high affinity [125I]-BH-NPY binding was localized to myenteric ganglia in porcine and human colons, and to submucosal ganglia in porcine colon. 3. Specific [125I]-BH-NPY binding to porcine myenteric ganglia was reversible in the presence of guanosine 5'-O-(3-thiotriphosphate) and was inhibited by related peptides with the rank order of potency; porcine NPY = human NPY = peptide tyrosine tyrosine (PYY) >> pancreatic polypeptide. 4. The Y2 selective analogue, NPY (13-36), competed for [125I]-BH-NPY binding to porcine myenteric ganglia with greater potency than the Y1 selective analogue, [Leu31, Pro34] NPY, the difference being small, but significant. 5. The characteristics of [125I]-BH-NPY binding to porcine myenteric ganglia were similar to those observed concurrently to porcine splenic red pulp. 6. The small difference in inhibitory potencies between NPY(13-36) and [Leu31, Pro34]NPY observed in this study in comparison with previous studies was not explained by differential ligand depletion during incubations, but may be due to differences in methodology between binding studies performed on tissue sections and on membranes. 7. We conclude that specific [25I]-BH-NPY binding sites are present in the myenteric and submucosal ganglia of the colon and that these sites may act as functional receptors by which NPY and PYY modulate colonic motility and electrolyte transport.

Animals↗

PubMed1993

Microvascular substance P binding to normal and inflamed rat and human synovium.

The regulatory peptide substance P has been implicated in the development and persistence of inflammatory synovitis. The authors used quantitative in vitro receptor autoradiography to compare synovial binding of 125Iodine-Bolton Hunter-labeled substance P ([125I]BH-SP) in rats and humans and between uniflamed and persistently inflamed synovium. [125I]BH-SP binding to microvascular endothelium paralleled the distribution of substance P-immunoreactive nerves and had characteristics of the neurokinin (NK) 1 class of tachykinin receptor. Specific binding was inhibited by the selective NK1 receptor antagonist, FK888, and the dual NK1/NK2 receptor antagonist FK224, with Hill coefficients near unity. FK888 was > 1000 times and FK224 > 10 times more potent at inhibiting binding in human compared with rat synovium. Synovium from patients and rats with chronic arthritis contained heterogeneously distributed inflammatory cell infiltrates. For the 10 microvessels with the densest [125I]BH-SP binding in each section, no significant differences in binding density, affinity, or Ki values for substance P, FK888 or FK224 were found between synovium from naive and monoarthritic rats, nor between that from patients with rheumatoid arthritis or osteoarthritis. However, in both rat and human specimens, microscopic examination suggested that microvascular [125I]BH-SP binding in intensely infiltrated regions of synovium was less dense than in adjacent, less infiltrated areas. It was concluded that NK1 receptors are similarly distributed in rat and human synovium but show major differences in selectivity for antagonists such as FK888. NK1 receptors in synovium may mediate proinflammatory actions of locally released substance P; defective neurovascular regulation may contribute to the persistence of chronic arthritis.

Adult↗

PubMed1993

Neuropeptide degrading enzymes in normal and inflamed human synovium.

Regulatory peptides, including neuropeptides, are metabolized by membrane-bound peptidases. We have localized the membrane peptidases angiotensin-converting enzyme (ACE), dipeptidyl peptidase IV (DPPIV), and aminopeptidase M (APM) in normal and inflamed human synovium by immunohistochemistry and enzyme histochemistry. ACE was localized to endothelial cells of all vessels, whereas endothelial DPPIV and APM were restricted to veins and capillaries of some cases. Perivascular spindle-shaped cells stained positively for APM, but rarely for ACE and DPPIV. Synovial lining cells were universally positive for APM but rarely for ACE or DPPIV, whereas a subintimal layer of spindle-shaped cells was frequently positive for all three enzymes, particularly APM. Staining for each enzyme was also observed on some stromal cells. Inflamed synovium displayed increased cellularity with corresponding increases in membrane peptidase staining. APM-positive, perivascular spindle-shaped cells and DPPIV-positive lymphocytes frequently bore HLA class II antigens. This distribution of membrane peptidases supports the hypothesis of a functional compartmentalization of vascular peptidergic systems, with the activities of vasoactive peptides localized to their sites of release. Furthermore, metabolic pathways for regulatory peptides may vary between different structures within human synovium, and these enzymes may be of different relative importance in normal and inflamed tissues. The implied regional control of regulatory peptide activity by membrane peptidases suggests novel potential approaches to the pharmacological manipulation of inflammation by specific enzyme inhibitors.

Adolescent↗

PubMed1993

Species and tissue specificity of vasoactive regulatory peptides.

Peptide regulatory systems are increasingly being implicated in inflammatory processes through their actions on vascular function. Peptides released by sensory nerves (substance P and calcitonin gene-related peptide) and autonomic nerves (neuropeptide Y), or generated by endothelium (angiotensin II) or injured tissues (bradykinin), regulate vascular tone, permeability and proliferation. Peptide systems comprise multiple components responsible for generation, release and metabolism of peptides, as well as receptor expression, combinations of which act together to produce a particular biological effect. Each of these components may vary between species, between tissues and during disease, and the particular combination of these components contributes to the specificity of peptide regulation of tissue function. Understanding the species and tissue specificity of peptide-induced responses is important in developing animal models of human disease and in developing drugs for subsequent use in man. In this paper we review some of the evidence for and implications of species and tissue specificity of vasoactive peptide systems.

Animals↗

PubMed1992

Autophosphorylation of the catalytic subunit of cAMP-dependent protein kinase.

The catalytic subunit of cAMP-dependent protein kinase contains two stable phosphorylation sites, Thr-197 and Ser-338 (Shoji, S., Titani, K., Demaille, J. G., and Fischer, E. H. (1979) J. Biol. Chem. 254, 6211-6214). Thr-197 is very resistant to dephosphorylation and thus cannot typically be autophosphorylated in vitro once the stable subunit is formed. Ser-338 is slowly dephosphorylated and can be rephosphorylated autocatalytically. In addition to these two stable phosphorylation sites, a new site of autophosphorylation, Ser-10, was identified. Phosphorylation at Ser-10 does not have a major effect on activity, and phosphates from Ser-10 or Ser-338 are not transferred to physiological substrates such as the type II regulatory subunit. Autophosphorylation at Ser-10 is associated with one of the two major isoelectric variants of the catalytic subunit. The form having the more acidic pI can be autophosphorylated at Ser-10 while the more basic form of the catalytic subunit cannot. Phosphorylation at Ser-10 does not account for the two isoenzyme forms. Since the reason for two isoelectric variants of the catalytic subunit is still unknown, it is not possible to provide a structural basis for the difference in accessibility of Ser-10 to phosphorylation. Either Ser-10 is not accessible in the more basic form of the catalytic subunit or some other type of post- or cotranslational modification causes Ser-10 to be a poor substrate. Whether the myristoyl group at the amino-terminal Gly is important for Ser-10 autophosphorylation remains to be established. The isoenzyme forms of the catalytic subunit do not correspond to the gene products coded for by the C alpha and C beta genes.

Adenosine Triphosphate↗

PubMed1992

Analysis by mutagenesis of the ATP binding site of the gamma subunit of skeletal muscle phosphorylase kinase expressed using a baculovirus system.

Active gamma subunit of skeletal muscle phosphorylase kinase has been obtained by expression of the rat soleus cDNA in a baculovirus system. The protein exhibited the expected pH 6.8/8.2 activity ratio of 0.6, and its activity was insensitive to Ca2+ addition, indicating that it was free gamma subunit and not a gamma subunit-calmodulin complex. It was stimulated approximately 2-fold by Ca(2+)-calmodulin addition, demonstrating that it had retained high-affinity calmodulin binding. By site-directed mutagenesis, we have examined the role of six of the amino acids that constitute the consensus ATP binding site of the protein kinase, which in the gamma subunit is represented by the sequence 26Gly.Arg.Gly.Val.Ser.Ser.Val.Val33. Changes were evaluated by the kinetic determination of the dissociation constants of gamma-ATP, gamma-ADP, gamma-AMP.PCP, and gamma-phosphorylase and the maximum catalytic activity. The mutants Ser26-gamma, Ser29-gamma, Phe30-gamma, and Gly31-gamma each exhibited an essentially identical dissociation constant for gamma subunit phosphorylase, indicating that these mutations had not caused a global alteration in the protein structure but were limited to changes in the nucleotide binding site domain. Substitution of either Val33 (by Gly) or Gly28 (by Ser), two of the most conserved residues in all protein kinases, resulted in enzyme with marginally detectable activity. In noted contrast, the Ser26 mutant, which substituted the first glycine of the consensus glycine trio motif, and which is also very highly conserved, retained at least 25% of the enzymatic activity. The Gly31 substitution, which restored a glycine to a position characteristic for most protein kinases, had little overall effect upon the maximum rate of catalysis. Restoration of Ser30 to the more typical phenylalanine, which is present in most protein kinases, had minimal effect on catalysis. These data provide the first direct evaluation of the roles that different residues play within this consensus glycine trio/valine motif of the protein kinases, which up to now have only been surmised to be of importance because of their conservation. Two unexpected findings are that for one residue that is very conserved (Gly26) there is some flexibility of substitution not apparent from the evolutionary conservation and that a second quite conserved residue in protein kinases (equivalent to Gly at position 31) does not produce a protein optimized for nucleotide binding.

Adenosine Triphosphate↗

PubMed1992

Coordinated expression of phosphorylase kinase subunits in regenerating skeletal muscle.

The developmental expression of the alpha, beta, and gamma subunits of skeletal muscle phosphorylase kinase has been examined in regenerating muscle. Rat extensor digitorum longus (EDL) muscles, treated with bupivacaine, promptly undergo a rapid degeneration of the muscle, followed by regeneration and recovery of essentially normal morphology and physiology by 3-4 weeks post-treatment (Hall-Craggs, E. C. B., and Seyan, H. S. (1975) Exp. Neurol. 46, 345-354). Phosphorylase kinase activity dropped to approximately 10% of control within 3 days of bupivacaine treatment and remained at this low level for several days but had attained at least 60% of normal levels by day 21. The pH 6.8/8.2 activity ratio was unusually high during the period of low activity, suggesting that the catalytic activity was not under normal regulation at this time. The subunit mRNAs were readily detected in control EDL but were undetectable at day 3 post-bupivacaine treatment. Very small amounts of message for all three subunits were evident by day 6 and began to approach normal levels by day 12-15. The mRNA for both the alpha and alpha' subunits of phosphorylase kinase exhibited a similar pattern of recovery, as did also the mRNA for phosphorylase. In contrast to both phosphorylase kinase and phosphorylase, actin mRNA exhibited a quite a different pattern, with a nearly full recovery of message levels by day 6 post-bupivacaine. These data indicate that synthesis of phosphorylase and the alpha, beta, and gamma subunits of phosphorylase kinase appears to be coordinately regulated at the level of message accumulation and that the expression of phosphorylase kinase activity is likely to be also regulated post-transcriptionally.

Amino Acid Sequence↗

PubMed1992

A kinetic re-interpretation of the regulation of rabbit skeletal-muscle phosphorylase kinase activity by Ca2+ and phosphorylation.

The regulation of phosphorylase kinase has been proposed to occur physiologically under conditions of zero-order ultrasensitivity [Meinke & Edstrom (1991) J. Biol. Chem. 266, 2259-2266]. This is also one of the conditions that recent theoretical approaches have indicated to be essential in order for an interconvertible enzyme cascade to generate a sensitive response to an effector [Cardenas & Cornish-Bowden (1989) Biochem. J. 257, 339-345]. In contrast, all published kinetic data to date have strongly suggested that activation of phosphorylase kinase by Ca2+ or phosphorylation is attributable solely to a change in affinity for phosphorylase, with no effect on the Vmax. of the reaction. In this study an attempt is made to resolve this conflict. Findings suggest that changes in Vmax. can fully account for the activation of phosphorylase kinase by the physiological mechanisms of cyclic AMP-dependent phosphorylation and increase in Ca2+ concentration.

Animals↗

PubMed1992

Structural basis for the low affinities of yeast cAMP-dependent and mammalian cGMP-dependent protein kinases for protein kinase inhibitor peptides.

Affinities of the catalytic subunit (C1) of Saccharomyces cerevisiae cAMP-dependent protein kinase and of mammalian cGMP-dependent protein kinase were determined for the protein kinase inhibitor (PKI) peptide PKI(6-22)amide and seven analogues. These analogues contained structural alterations in the N-terminal alpha-helix, the C-terminal pseudosubstrate portion, or the central connecting region of the PKI peptide. In all cases, the PKI peptides were appreciably less active as inhibitors of yeast C1 than of mammalian C alpha subunit. Ki values ranged from 5- to 290-fold higher for the yeast enzyme than for its mammalian counterpart. Consistent with these results, yeast C1 exhibited a higher Km for the peptide substrate Kemptide. All of the PKI peptides were even less active against the mammalian cGMP-dependent protein kinase than toward yeast cAMP-dependent protein kinase, and Kemptide was a poorer substrate for the former enzyme. Alignment of amino acid sequences of these homologous protein kinases around residues in the active site of mammalian C alpha subunit known to interact with determinants in the PKI peptide [Knighton, D. R., Zheng, J., Ten Eyck, L. F., Xuong, N-h, Taylor, S. S., & Sowadski, J. M. (1991) Science 253, 414-420] provides a structural basis for the inherently lower affinities of yeast C1 and cGMP-dependent protein kinase for binding peptide inhibitors and substrates. Both yeast cAMP-dependent and mammalian cGMP-dependent protein kinases are missing two of the three acidic residues that interact with arginine-18 in the pseudosubstrate portion of PKI. Further, the cGMP-dependent protein kinase appears to completely lack the hydrophobic/aromatic pocket that recognizes the important phenylalanine-10 residue in the N-terminus of the PKI peptide, and binding of the inhibitor by the yeast protein kinase at this site appears to be partially compromised.

Amino Acid Sequence↗

PubMed1992

Solution structure of phosphorylase kinase studied using small-angle X-ray and neutron scattering.

Small-angle X-ray and neutron scattering have been used to characterize the solution structure of rabbit skeletal phosphorylase kinase. The radius of gyration of the unactivated holoenzyme determined from neutron scattering is 94 A, and its maximum dimension is approximately 275-295 A. A planar model has been constructed that is in general agreement with the dimensions of the transmission electron microscope images of negatively stained phosphorylase kinase and that gives values for the radius of gyration, maximum linear dimension, and a pair distribution function for the structure that are consistent with the scattering data.

Animals↗

PubMed1992

High and intermediate affinity calmodulin binding domains of the alpha and beta subunits of phosphorylase kinase and their potential role in phosphorylation-dependent activation of the holoenzyme.

Phosphorylase kinase is a calcium-regulated multimeric enzyme of composition (alpha beta gamma delta)4, which contains calmodulin as the integral delta subunit and also is activated further by addition of extrinsic calmodulin. Previous studies by Dasgupta, M., Honeycutt, T., and Blumenthal, D.K. ((1989) J. Biol. Chem. 264, 17156-17163) have identified gamma 302-326 and gamma 342-366 as two calmodulin binding regions. Using peptides that were synthesized based on alpha and beta primary structure and that were predicted to contain the basic amphiphilic alpha-helix motif thought important for calmodulin binding, four additional potential calmodulin binding domains have now been identified: one of high affinity, beta 770-794; two of intermediate affinity, beta 5-28 and beta 920-946; and one with marginally low affinity, alpha 1070-1093. Peptide beta 770-794 was of higher calmodulin affinity than either gamma 302-326 or gamma 342-366; it was of higher affinity than the model synthetic peptide IV defined by O'Neil, K.T., and DeGrado, W.F. ((1990) Trends Biochem. Sci. 15, 59-64); and it is currently the most potent calmodulin-binding peptide so far described. Correlated with their affinity for calmodulin, all six phosphorylase kinase-derived peptides and several other established calmodulin-binding peptides inhibited phosphorylase kinase previously activated by cAMP-dependent phosphorylation, reducing its activity to the level of the nonactivated enzyme. However, these peptides did not inhibit (and some peptides slightly activated) the nonphosphorylated enzyme. Even in the presence of these peptides both activated and nonactivated enzyme remained fully Ca(2+)-dependent. The beta 770-794 peptide has at least a 5-fold greater calmodulin binding affinity than the holo-phosphorylase kinase. This, and its higher affinity for calmodulin than either of the sites on the gamma subunit, raises the possibility that in the native enzyme it may be involved in binding the intrinsic delta subunit. Further, inhibition of activated but not nonactivated enzyme by calmodulin-binding peptides would suggest that the phosphorylation-dependent activation of phosphorylase kinase may be mediated by changes in the binding interactions of the intrinsic calmodulin delta subunit.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1992

Autoradiographic localization and analysis of endothelin-1 binding sites in human synovial tissue.

OBJECTIVE: To determine the localization of endothelin binding sites and immunoreactivity in human synovial tissues. METHODS: Quantitative in vitro autoradiographic and immunohistochemical techniques were used to localize and characterize 125I-labeled endothelin-1 (125I-ET-1) binding sites and endothelin-like immunoreactivity in sections of rheumatoid, osteoarthritic, and normal synovium. RESULTS: Specific 125I-ET-1-binding sites, characteristic of the ETA receptor, were localized to the media of synovial blood vessels in all 3 groups. No difference was found in vascular binding site density in rheumatoid and osteoarthritic synovium. Endothelin-like immunoreactivity was localized to endothelial cells in blood vessels displaying 125I-ET-1 binding sites. CONCLUSION: We conclude that endothelin may act locally, modulating synovial perfusion and exacerbating hypoxia in chronic arthritis.

Aged↗

PubMed1992

Substrate diversity of the cAMP-dependent protein kinase: regulation based upon multiple binding interactions.

The proposition is forwarded that the cAMP-dependent protein kinase is one of quite a small class of enzymes wherein differential modes of binding of its multiple substrates make an important contribution to the end physiological response. It is postulated that a variety of different substrate affinities may have evolved in order to regulate the order of substrate phosphorylation. The recent elucidation of the protein's three-dimensional structure provides the opening to test this as a new concept of cellular regulation.

Amino Acid Sequence↗

PubMed1992

Spatial cognition and neighborhood use: the relationship in older adults.

Measures of spatial cognition, neighborhood knowledge, and neighborhood use from the research described by Walsh, Krauss, and Regnier (1981) were examined to determine if laboratory tests of spatial cognition were significant predictors of older adults' use of their neighborhoods. The overall results show that laboratory-based measures of spatial cognition and subjects' knowledge of their neighborhoods are both significant predictors of their use of neighborhood goods and services. The ability to learn and remember the location and orientation of objects, when contextual cues were not provided at test, was more predictive of the participants' neighborhood use than either the participants' number of years in the neighborhood or their mobility. Spatial memory as measured by the Educational Testing Service Building Memory task predicted neighborhood knowledge, which was predictive of neighborhood use.

Activities of Daily Living↗

PubMed1992

The effects of calcitonin gene-related peptide on formation of intra-articular oedema by inflammatory mediators.

1. The temporal and quantitative effects of inflammatory mediators on plasma extravasation in the rat knee were investigated by use of a perfusion technique. 2. Intra-articular perfusion of substance P (SP), bradykinin or histamine over a 5 min test period produced rapid-onset and prolonged plasma extravasation in a dose-dependent fashion. The rank order of potency was bradykinin greater than SP greater than histamine. 3. Calcitonin gene-related peptide (CGRP) did not induce plasma extravasation but enhanced substance P-induced plasma extravasation in a dose-dependent fashion. A 5 min co-perfusion of the two agents produced short-term enhancement lasting 10 min while continuous co-perfusion produced enhancement for the duration of the perfusion. 4. A 5 min perfusion of CGRP enhanced plasma extravasation when co-perfused with bradykinin but not histamine. However, when CGRP and histamine were continuously co-perfused over a 20 min test period, an enhanced response was apparent. 5. The results indicate that intra-articular perfusion of CGRP enhances synovial plasma extravasation induced by agents that increase vascular permeability, but suggest that the response is not uniform and is critically dependent on the duration of perfusion within the joint.

Animals↗

PubMed1992

Localisation and characterisation of substance P binding to human synovial tissue in rheumatoid arthritis.

The neuropeptide substance P is found in perivascular and free unmyelinated nerve fibres in human synovial tissue. Quantitative receptor autoradiography was used to show specific, high affinity (Kd = 0.75 (0.21), nmol/l (mean (standard error of the mean)), low capacity (Bmax = 27.8 (7.9) amol/mm2) binding sites for substance P Bolton Hunter-labelled with iodine-125 localised to vascular endothelial cells in human synovial tissue. The binding could be saturated, was reversible, and was dependent on the magnesium concentration. Unlabelled substance P and neurokinin A competitively inhibited specific binding with 50% inhibition at concentrations of 1.25 (0.21) and 175 (29) nmol/l respectively. Neurokinin B (mumol/l) and calcitonin gene related peptide (1 mumol/l) did not inhibit binding. These binding sites show characteristics of the neurokinin 1 tachykinin receptor subtype. This provides further evidence that substance P may play a part in the vascular control of human synovium and may influence inflammatory processes in joints.

Adult↗

PubMed1992

The alpha- and beta-isoforms of the inhibitor protein of the 3',5'-cyclic adenosine monophosphate-dependent protein kinase: characteristics and tissue- and developmental-specific expression.

The inhibitor protein (PKI) of the cAMP-dependent protein kinase was first characterized from rabbit skeletal muscle. More recently a form of PKI was isolated and cloned from rat testis which shares relatively limited amino acid sequence with the rabbit skeletal muscle form. We have now isolated a cDNA from rat brain which encodes a protein corresponding to the rabbit skeletal muscle PKI. This establishes the presence of the "skeletal muscle" and "testis" proteins in the same species and therefore that they clearly represent distinct isoforms. We have also demonstrated that the isoform from testis, like the skeletal muscle isoform, is specific for the cAMP-dependent protein kinase and that it is able to inhibit this enzyme when expressed in cultured JEG-3 cells. Both forms contain the five specific amino acid recognition determinants which have been shown to be required for high affinity binding to the protein kinase catalytic site, although there is some noted lack of conservation of codons used for these residues. Overall, the two rat isoforms are only 41% identical at the amino acid level and 46% at the level of coding nucleotides. We propose that the rabbit skeletal muscle and rat testis forms be designated PKI alpha and PKI beta, respectively. Using Northern blot analysis, we have examined the tissue distribution of the two forms in the rat and their relative expression during development. In the adult rat, mRNA of the PKI alpha species is highest in muscle (both skeletal and cardiac) and brain (cortex and cerebellum).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗