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R W Wallace

Publications and source records attributed to R W Wallace.

At least 37 records · Page 2Linked to original sources
PubMed1987

The bioflavonoid quercetin inhibits neutrophil degranulation, superoxide production, and the phosphorylation of specific neutrophil proteins.

Quercetin, a C-kinase antagonist, inhibits neutrophil degranulation and superoxide production induced by f-met-leu-phe, solid phase IgG, zymosan treated serum and a phorbol ester (PMA). Quercetin is more effective in inhibiting degranulation (IC50 = 20 uM) than superoxide production (IC50 = 80 microM). Neutrophil activation by PMA is accompanied by the phosphorylation of neutrophil proteins of 205, 170, 130, 91, 77, 67, 56, 47, 39, 34, 27, and 20 kilodaltons; quercetin also inhibits the phosphorylation of these proteins. Dose-response studies indicated that phosphorylation of the 67 kilodalton protein was particularly sensitive to inhibition by quercetin at concentrations that also inhibit neutrophil degranulation and superoxide production. These results suggest that phosphorylation of the 67 kilodalton protein may be an important intracellular reaction associated with neutrophil activation.

Adult↗
PubMed1987

Altered binding of 125I-labeled calmodulin to a 46.5-kilodalton protein in skin fibroblasts cultured from patients with cystic fibrosis.

The levels of calmodulin and calmodulin-binding proteins have been determined in cultured skin fibroblasts from patients with cystic fibrosis (CF) and age- and sex-matched controls. Calmodulin ranged from 0.20 to 0.76 microgram/mg protein; there was no difference between calmodulin concentration in fibroblasts from CF patients and controls. Calmodulin-binding proteins of 230, 212, 204, 164, 139, 70, 59, 46.5, and 41 kD were identified. A protein with a mobility identical to the 59-kD calmodulin-binding protein was labeled by antiserum against calmodulin-dependent phosphatase. Although Ca2+/calmodulin-dependent phosphatase activity was detected, there was no different in activity between control and CF fibroblasts or in the level of phosphatase protein as determined by radioimmunoassay. Lower amounts of 125I-calmodulin were bound to the 46.5-kD calmodulin-binding protein in CF fibroblasts as compared with controls. The 46.5-kD calmodulin-binding protein may be reduced in CF fibroblasts or its structure may be altered resulting in a reduced binding capacity and/or affinity for calmodulin and perhaps reflecting, either directly or indirectly, the genetic defect responsible for cystic fibrosis.

Calmodulin↗
PubMed1986

Protein phosphorylation and oocyte maturation. II. Inhibition of starfish oocyte maturation by intracellular microinjection of protein phosphatases 1 and 2A and alkaline phosphatase.

Oocyte maturation (meiosis re-initiation) in starfish is induced by the natural hormone 1-methyladenine (1-MeAde). Following hormonal stimulation of the oocyte, an intracellular Maturation Promoting Factor (MPF) appears in the cytoplasm which triggers nuclear envelope breakdown and maturation divisions. Microinjection of pure preparations of the catalytic subunits of protein phosphatases 1 and 2A inhibits 1-MeAde-induced maturation in a dose-dependent manner. Calmodulin-dependent protein phosphatase 2B is inefficient. Maturation induced by mimetics of 1-MeAde, such as dithiothreitol (DTT), methylglyoxal-bis(guanylhydrazone) (MGBG), 8-hydroxyeicosatetraenoic acid (8 HETE) and arachidonic acid (AA) is also inhibited by these protein phosphatases. In all cases inhibition can be reversed by increasing the concentration of 1-Me-Ade or of mimetic. Alkaline phosphatase also inhibits maturation in a dose-dependent way and in a reversible manner. Microinjection of protein phosphatase is still effective when preformed long after the end of the hormone-dependent period, and can even be effective a few minutes before the breakdown of the nuclear envelope. No detectable MPF activity is found in 1-MeAde-treated phosphatase-injected oocytes. However, microinjection of phosphatase 2A simultaneously with MPF (obtained from 1-MeAde-treated donors) does not result in inhibition. These results constitute direct evidence for the necessity of an elevated level of phosphorylated proteins for MPF activity and maturation. The mode of action of 1-MeAde in inducing starfish oocyte maturation is discussed in relation to protein phosphorylation.

Adenine↗
PubMed1985

Calmodulin antagonists elevate the levels of 32P-labeled polyphosphoinositides in human platelets.

The calmodulin antagonists trifluoperazine, chlorpromazine, perphenazine, promazine, tamoxifen and the naphthalene sulfonamide derivatives W7 and W13 increased the level of 32P-incorporation into human platelet PIP and PIP2. Various drugs with poor anti-calmodulin activity were ineffective. The increase in 32P-PIP and 32P-PIP2 required micromolar concentrations of trifluoperazine and was time-dependent, reaching half-maximal within two minutes of the addition of the drug. These results indicate that the calmodulin antagonists perturb polyphosphoinositide metabolism, probably at the level of the PI- and PIP-kinases and/or the PIP2- and PIP-phosphomonoesterases.

Blood Platelets↗
PubMed1985

Characterization of a calmodulin-dependent protein phosphatase from human platelets.

A calmodulin-dependent protein phosphatase has been identified in human platelets by its cross-reactivity with an antibody developed against a bovine brain calmodulin-dependent protein phosphatase and by its calmodulin-stimulated dephosphorylation of 32P-labeled substrates. The platelet enzyme was partially purified to separate it from calmodulin and calmodulin-independent phosphatases. The partially purified enzyme was stimulated by calmodulin, requiring 15 nM calmodulin for half-maximal activation. Calmodulin increased the Vmax of the phosphatase, with no significant effect on its Km. The enzyme was stimulated irreversibly and made calmodulin-independent by limited proteolysis. The optimal pH for the phosphatase was 7.5. After partial purification, phosphatase activity was significantly increased in the presence of Mn2+ and Ca2+ over that observed in the presence of Ca2+ alone. The enzyme effectively dephosphorylated casein, histone, protamine, and platelet actin. The holophosphatase was estimated to have a molecular weight of 76,900 as determined by sedimentation on sucrose gradients. Immunoblotting techniques using an antibody against the brain phosphatase suggests that the enzyme consists of 2 subunits of 60,000 and 16,500 daltons; the 60,000-dalton subunit co-migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a 60,000-dalton calmodulin-binding protein in the platelet suggesting that it is the calmodulin-binding subunit of the enzyme. The identification of a calmodulin-dependent protein phosphatase in human platelets suggests a role for Ca2+-dependent dephosphorylation in platelet activation.

Animals↗
PubMed1985

Calmodulin accelerates the rate of polymerization of human platelet actin and alters the structural characteristics of actin filaments.

Calmodulin stimulated the rate of Mg2+-induced polymerization of human platelet actin. The stimulatory effect was due to an increase in the nucleation phase of the reaction; there was no effect on the steady-state viscosity. The calmodulin antagonist trifluoperazine blocked the stimulatory effect of calmodulin. Addition of EGTA to the reaction mixture also stimulated the rate of actin polymerization; however, the effect of calmodulin on actin polymerization is not due to Ca2+ chelation, as is presumed to be the case for EGTA. Electron microscopy revealed structural differences in the filaments prepared in the presence of calmodulin as compared to those prepared with trifluoperazine. In the presence of calmodulin, the filaments were thicker, suggesting that they consisted of multiple actin polymers. In addition, numerous projections were present perpendicular to the filaments, as well as localized areas of filament bundling. It was not possible to demonstrate a direct interaction between calmodulin and actin, which raises the possibility that the calmodulin effect may be indirect through a calmodulin-binding protein or calmodulin-dependent enzyme. Regardless of whether calmodulin is acting directly or indirectly, these results provide evidence that calmodulin may play a regulatory role in either the polymerization of actin or in determining the structural characteristics of actin filaments.

Actins↗
PubMed1984

Polyamines stimulate D-glucose transport in isolated renal brush-border membrane vesicles.

Polyamines are natural constituents of most living organisms. However, their function in normal or pathological conditions is not fully understood. We have investigated in vitro effects of polyamines on characteristic properties of isolated renal brush-border membrane vesicles in order to determine whether polyamines have a regulatory role in membrane transport processes. The polyamines putrescine, spermidine and spermine were found to stimulate D-glucose uptake. Diffusional L-glucose uptake was not altered, indicating that the polyamines affected the active transport of D-glucose, rather than inducing nonspecific changes in membrane lipid properties. The amiloride-sensitive Na+ /H + exchange was slightly inhibited by polyamines while Mg2+ -ATPase activity was stimulated. The polyamine effects could not be explained solely by the polycationic properties of these agents, since polycationic polypeptides had an opposite effect. For example, lysozyme was found to inhibit D-glucose transport. Spermine was incorporated into the trichloroacetic acid-insoluble fraction of brush-border membrane proteins. Results indicated that this incorporation process consisted of at least two components: a Ca2+ -independent component and a Ca2+ -dependent component, possibly as a result of transglutaminase activity which was present in the isolated renal brush-border membranes. By using SDS-polyacrylamide gel electrophoresis in conjunction with fluorography, [3H]spermine was shown to be incorporated into several brush-border membrane proteins, mainly the 57 kDa, 74 kDa, 100 kDa, a heavy molecular weight band (greater than 200 kDa) and a low molecular weight band (less than 10 kDa). Our results suggest that the polyamine effects on membrane function may be due to a covalent modification of membrane proteins, possibly via a transglutaminase-mediated incorporation of polyamines or to the crosslinking of membrane proteins.

Adenosine Triphosphatases↗
PubMed1983

Geriatric medical education: a project in faculty development.

To increase the competence of physicians to practice and teach geriatric medicine, the Office of Geriatric Medicine at Case Western Reserve University School of Medicine in Cleveland, Ohio, offered an 11-week course to interested physicians on the faculty. This course, "Medical Management of the Aged--A State of the Art Symposium for Medical Faculty," was conducted as part of an overall plan of the Office to enhance faculty and curriculum development in geriatrics within the School. The weekly sessions consisted of clinical and research information presented by recognized experts in the field of geriatric medicine. Of the 52 formal registrants, 79 per cent were physicians and were from such fields of specialization as internal medicine, family medicine, and psychiatry. The mean attendance was 37. An overview and evaluation of the geriatric faculty development program is given.

Curriculum↗
PubMed1982

Calcium binding domains of calmodulin. Sequence of fill as determined with terbium luminescence.

Terbium, a trivalent lanthanide, effectively substituted for Ca2+ in calmodulin as judged by several criteria: intrinsic fluorescence spectra, altered mobilities on polyacrylamide gel electrophoresis, formation of a stable complex with troponin I or calcineurin, and stimulation of phosphodiesterase. Calmodulin harbors four Ca2+ binding domains; domains I and II contain no tyrosine, whereas domains III and IV each have one tyrosine. The binding of Tb3+ to calmodulin was followed by the increase of Tb3+ fluorescence at 545 nm upon binding to calmodulin. This fluorescence was elicited either by exciting Tb3+ directly at 222 nm or by exciting the calmodulin tyrosine at 280 nm with resulting energy transfer from tyrosine to Tb3+. Fluorescence generated by direct excitation measures binding of Tb3+ to any of the Ca2+ binding domains, whereas energy transfer through indirect excitation is effective only when Tb3+ is within 5 A of tyrosine, indicating that Tb3+ necessarily occupies a Ca2+ binding domain that contains tyrosine. A judicious use of the direct and indirect excitation could reveal the sequence of fill of the binding domains. Our results suggest these domains are filled in the following sequence: 1) domain I or II; 2) domains III and IV; and 3) domain II or I that has not been filled initially.

Animals↗
PubMed1982

Factorial analysis of the MMPI profiles of low back pain patients.

To examine the interrelationships of MMPI scale scores among low back pain patients, 50 profiles were scored on the traditional 13 scales, Hanvik's Lb scale and seven subscales derived from Scales 1 and 3. The profiles were randomly divided into two equal samples and factor analyzed. Comparison of the two samples yielded three common factors. The first factor measured severity of the symptoms, the second assessed the level of anger and aggression and the third assessed the psychogenic components of the syndrome. The implications of the factors are discussed.

Back Pain↗
PubMed1981

cAMP renders Ca2+-dependent phosphodiesterase refractory to inhibition by a calmodulin-binding protein (calcineurin).

Calmodulin is a ubiquitous, multifunctional, Ca2+-dependent regulatory protein, controlling a wide variety of Ca2+-mediated reactions. The versatility of calmodulin raises the question of how it exerts specificity at the molecular level. Cyclic nucleotide phosphodiesterase consists of multiple forms, one of which requires calmodulin for full activity. Calcineurin, a calmodulin-binding protein, inhibits the calmodulin-stimulated phosphodiesterase activity by competing with the enzyme for calmodulin. In this report, we present experiments which indicate that, although calcineurin potentially inhibits calmodulin-supported enzyme activity, its effectiveness as an inhibitor depends on the level of cAMP. In the presence of elevated levels of cAMP, the affinity of calmodulin for phosphodiesterase increased markedly, but that for calcineurin was not altered. Thus, the enzyme became relatively refractory to inhibition by calcineurin. This finding suggests that an increase of cellular cAMP could lead to a condition favorable to its own hydrolysis and that this phenomenon might represent an example of molecular specificity in calmodulin-regulated reactions.

Animals↗
PubMed1981

Immunofluorescent localization of cyclic GMP, calmodulin and cyclic GMP-dependent protein kinase in the choroid plexus.

An immunofluorescent technique has demonstrated that tissue-bound pools of cyclic GMP, calmodulin and cyclic GMP-dependent protein kinase are localized within the cytoplasm of the epithelial cells of the choroid plexus. In all other cell types and regions of the central nervous system previously examined, however, these molecules have shown contrasting immunofluorescent localization. These results suggest that the interaction in the choroid plexus may be related to the specialized physiological functions of the neuroglial epithelial cell.

Animals↗
PubMed1981

Calmodulin activates NAD kinase of sea urchin eggs: an early event of fertilization.

NAD kinase, one of the first enzymes activated after fertilization of sea urchin eggs, is regulated by Ca2+ and calmodulin in vitro. The evidence is the requirement for low amounts of Ca2+ (Kd for Ca2+ of 4 x 10(-7) M) and the dissociation of a heat-stable activator from the enzyme which is similar to calmodulin on the basis of radioimmunoassay, activation of bovine brain phosphodiesterase and coelectrophoresis of a major protein of the activator fraction with bovine calmodulin. Also, the calcium stimulation of the enzyme is prevented by trifluoperazine, an inhibitor of calmodulin-associated reactions. In vivo studies show that the enzyme is activated by artificial parthenogenesis regimes that increase cytosolic Ca2+, but not by ammonia activation which only partially activates eggs and bypasses the Ca2+-rise step. These in vitro and in vivo studies indicate that calmodulin is part of the linkage between the rise in Ca2+ at fertilization and the turning on of egg metabolism.

Animals↗