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Biomedical subjects

R W Wallace

Publications and source records attributed to R W Wallace.

At least 19 recordsLinked to original sources
PubMed1998

The Human Genome Diversity Project: medical benefits versus ethical concerns.

By the year 2005 the entire human genome should have been sequenced and the genes identified. But the resulting genomic sequence, although a marvelous accomplishment, will be a composite of just a handful of individuals selected at random. The Human Genome Diversity Project was proposed as a means to overcome these limitations by obtaining genetic information from many diverse populations of the world. This would give medical geneticists a handle on the variations in susceptibility to disease among different populations, as well as being of anthropological value. But would such a project risk exploiting the indigenous populations involved?

Bioethics↗
PubMed1997

Drugs from the sea: harvesting the results of aeons of chemical evolution.

Despite the recent development of combinatorial chemistry for the rapid generation of thousands of new chemicals, the pharmaceutical industry still looks to the natural environment to uncover novel compounds that extend the boundaries of our chemical imagination. The terrestrial environment has been mined for such compounds for many years with great success. Now the world's oceans are beginning to yield hidden treasures. Many of these chemicals are structurally complex, stretching the prowess of modern organic chemists to their limits as they attempt to mimic the synthetic versatility of nature. In the future, these marine compounds are likely to yield entirely new classes of drugs that will be a valuable contribution to our ability to treat human disease.

Animals↗
PubMed1997

DNA on a chip: serving up the genome for diagnostics and research.

It is expected that by the year 2003 the entire human genome will be sequenced, providing us with new insight into human disease. The amount of human sequence information that is already available (only a few percent of the total) is, however, already much greater than can be routinely evaluated with existing, commonly used diagnostic technology. New technology based on attaching DNA to a chip for parallel hybridization analysis is generating much interest in both the basic research and the clinical diagnostic community. It will increase, by orders of magnitude, our ability to evaluate an individual's genetic heritage and to conduct basic genetic research. The new technology will be a major advance in screening for genetic diseases, but the ability to treat these newly understood genetic defects will lag significantly behind the new-found diagnostic capabilities.

BRCA2 Protein↗
PubMed1993

Purification and properties of a human platelet inositol 1,4,5-trisphosphate 3-kinase.

An inositol 1,4,5-trisphosphate 3-kinase (Ins(1,4,5)P3 3-kinase) has been purified 943-fold from a 30,000g human platelet extract and has a specific activity of 283 nmol/min/mg protein and an apparent Km for inositol 1,4,5-trisphosphate of 0.76 microM; the optimal pH for the enzymatic activity was 7.2. Under both denaturing and nondenaturing conditions, the kinase preparation contained two polypeptides, both of which exhibited Ca2+/calmodulin-dependent Ins(1,4,5)P3 3-kinase activity. In the presence and absence of calmodulin, Ins(1,4,5)P3 3-kinase exhibited a biphasic response to Ca2+, being stimulated between 10(-7) and 10(-6) M Ca2+ and inhibited when the Ca2+ level was further increased. Ins(1,4,5)P3 3-kinase was stimulated by calmodulin approximately 10-fold, requiring 55 nM calmodulin for a half-maximal effect. Calmodulin stimulation was immediately reversed upon chelation of Ca2+ by ethylene glycol bis (beta-amino-ethyl ether) N,N'-tetraacetic acid consistent with a mechanism of activation involving a direct interaction of calmodulin with Ins(1,4,5)P3 3-kinase. Since we have previously shown that Ins(1,4,5)P3 3-kinase can also be phosphorylated and consequently inactivated by protein kinase C in vitro (Lin, A. N., Barnes, S., and Wallace, R. W., 1990, Biochem. Biophys. Res. Commun. 170, 1369-1376), Ins(1,4,5)P3 3-kinase appears to be a key enzyme in the inositol phosphate signaling pathway and as such may play an important role in human platelet function.

Animals↗
PubMed1992

Intercellular adhesion molecule-1 gene expression in human endothelial cells. Differential regulation by tumor necrosis factor-alpha and phorbol myristate acetate.

Intercellular adhesion molecule-1 (ICAM-1) is an inducible glycoprotein expressed on the surface of inflamed endothelium which mediates in part the extravasation of granulocytes into sites of infection or injury. ICAM-1 mRNA is not detected in unstimulated human umbilical vein endothelial cells (HUVECs), but accumulates transiently following tumor necrosis factor-alpha (TNF-alpha) or phorbol myristate acetate (PMA) treatment with maximal steady state levels occurring at 2 or 4 h, respectively. Pretreating HUVECs with PMA for 72 h down-regulates protein kinase C and inhibits the subsequent induction of ICAM-1 mRNA by PMA, but does not affect TNF-alpha-induced message accumulation. Nuclear run-on assays showed that the ICAM-1 gene is transcribed under basal conditions in HUVECs, and that TNF-alpha stimulates transcriptional activity 3- to 4-fold within 30 min of treatment. In contrast, PMA has little effect on ICAM-1 gene transcription up to 4 h following stimulation. Message stability studies established that ICAM-1 mRNA induced by PMA has a longer half-life than the TNF-alpha-induced message. These results suggest that PMA acts through protein kinase C to up-regulate ICAM-1 expression primarily at a post-transcriptional level by stabilizing ICAM-1 mRNA, whereas TNF-alpha transcriptionally regulates ICAM-1 gene expression through an undefined, protein kinase C-independent pathway.

Antigens, CD↗
PubMed1992

Discriminatory effects of protein kinase inhibitors and calcium ionophore on endothelial ICAM-1 induction.

Intercellular adhesion molecule 1 (ICAM-1) is a proinflammatory adhesion glycoprotein induced by cytokines such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) as well as lipopolysaccharide (LPS). Little is known, however, concerning the intracellular regulatory mechanisms that modulate ICAM-1 expression in endothelial cells. We probed the involvement of protein kinase function and intracellular calcium ion upon ICAM-1 expression of human umbilical vein endothelial cells activated alternatively by TNF-alpha, IL-1 beta, LPS, or phorbol 12-myristate 13-acetate (PMA). Methodologies for the detection of ICAM-1 included both enzyme-linked immunosorbent assay and immunoprecipitation from biosynthetically labeled cells. The protein kinase inhibitor H-7 blocked induction of ICAM-1 by all of the activators; nonlinear regression analysis revealed 50% inhibitory concentration (IC50) values of 6-10 microM. Another kinase inhibitor, HA1004, did not block expression of the adhesion molecule at concentrations up to 50 microM. In contrast, the kinase inhibitor staurosporine dose dependently inhibited ICAM-1 expression triggered by PMA (IC50 67 +/- 4 nM) but, at similar concentrations, did not inhibit ICAM-1 expression induced by the other inflammatory stimuli. The divalent cation ionophore ionomycin (0.5 microM) interacted synergistically with PMA but not with cytokines or LPS in upregulating ICAM-1. We conclude from these data that although PMA-induced ICAM-1 expression may be triggered through activation of protein kinase C, ICAM-1 induction by IL-1 beta, TNF-alpha, or LPS may involve distinct regulatory pathway(s).

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Induction of ICAM-1 by TNF-alpha, IL-1 beta, and LPS in human endothelial cells after downregulation of PKC.

The intercellular adhesion molecule 1 (ICAM-1) is induced on endothelial cells by tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and lipopolysaccharide (LPS). We have reported the sensitivity of cytokine-induced ICAM-1 expression to protein kinase inhibitors, including inhibitors of protein kinase C (PKC) [C. L. Myers, S. N. Desai, J. Schembri-King, G. L. Letts, and R. W. Wallace. Am. J. Physiol. 262 (Cell Physiol. 31): C365-C373, 1992]. To directly investigate the role of PKC in ICAM-1 induction, we downregulated PKC by pretreatment of human umbilical vein endothelial cells with phorbol 12-myristate 13-acetate (PMA) and assessed ICAM-1 protein and mRNA induction elicited by subsequent exposure to inflammatory stimuli. PMA treatment results in ICAM-1 protein induction that declines to basal levels by 3 days. Western blots of endothelial cell lysates reveal a nearly complete loss of immunologically reactive PKC. Subsequent activation with cytokine or LPS leads to reinduction of ICAM-1 protein and mRNA; however, the cells no longer produced substantial amounts of ICAM-1 protein or mRNA in response to PMA stimulation. Cross desensitization is observed with phorbol dibutyrate, while 4 alpha-phorbol has no desensitizing effect. The data indicate that PKC activation, while capable of inducing ICAM-1 expression, is not essential for ICAM-1 induction by the inflammatory mediators TNF-alpha, IL-1 beta, or LPS.

Cell Adhesion Molecules↗
PubMed1991

The cytoplasmic domain of the integrin lymphocyte function-associated antigen 1 beta subunit: sites required for binding to intercellular adhesion molecule 1 and the phorbol ester-stimulated phosphorylation site.

We have defined the regions of the cytoplasmic domain of the leukocyte integrin lymphocyte function-associated antigen 1 (LFA-1) that are required for active binding of its extracellular domain to intercellular adhesion molecule 1 (ICAM-1). The NH2-terminal 28 amino acids in the cytoplasmic domain are dispensable, but a segment of 5 amino acids including three contiguous threonines (758-760) and Phe 766 in the COOH-terminal third of the cytoplasmic domain are required for binding to ICAM-1. Mutation and phosphoamino acid analysis show that Ser 756 is the major residue phosphorylated in response to phorbol ester. Furthermore, multiple mutations demonstrate that serine phosphorylation can be dissociated from phorbol ester-stimulated binding of LFA-1 to ICAM-1. The sites we have defined are previously unremarked, are well conserved in the beta 1, beta 3, and beta 7 integrin subunits, and may be of broad importance in regulating adhesiveness of integrins.

Amino Acid Sequence↗
PubMed1990

Phosphorylation by protein kinase C inactivates an inositol 1,4,5-trisphosphate 3-kinase purified from human platelets.

An inositol 1,4,5-trisphosphate 3-kinase purified from human platelets contains two major components, 53 and 36 kDa polypeptides. Each polypeptide expresses Ca2+/calmodulin-dependent enzymatic activity and is phosphorylated by an unidentified protein kinase in the enzyme preparation. The 36-kDa polypeptide may be further phosphorylated on serine residues by protein kinase C to a stoichiometry of 0.8 mole phosphate per mole of protein. Phosphorylation of the 36-kDa component is correlated with inhibition of the kinase activity; the inhibitory effect is dependent upon Ca2+ and phosphatidylserine/diolein and may be blocked by a selective peptide inhibitor of protein kinase C. Phosphorylation by protein kinase C decreases the Vmax of the enzyme from 160 to 28 nmol/mg/min; the Km (0.76 microM) is not altered. These data suggest that protein kinase C may negatively regulate inositol 1,4,5-trisphosphate 3-kinase activity in the human platelet.

Amino Acid Sequence↗
PubMed1990

A cystic fibrosis pancreatic adenocarcinoma cell line.

We established a pancreatic adenocarcinoma cell line (CFPAC-1) from a patient with cystic fibrosis (CF) and assessed some of its properties. The cells show epithelial morphology and express cytokeratin and oncofetal antigens characteristic of pancreatic duct cells. Basal and stimulated levels of cAMP and cAMP-dependent protein kinase and the biophysical properties of single Cl- channels in CFPAC-1 are similar to those of airway and sweat gland primary cultures and Cl(-)-secreting epithelial cell lines. Anion transport and single Cl- channel activity was stimulated by Ca2+ ionophores but not by forskolin, cAMP analogs, or phosphodiesterase inhibitors. The cells express the CF gene and manifest the most common CF mutation, deletion of three nucleotides resulting in a phenylalanine-508 deletion. These properties have been stable through greater than 80 passages (24 months), suggesting that CFPAC-1 can serve as a continuous cell line that displays the CF defect.

Adenocarcinoma↗
PubMed1989

Platelet glycoprotein Ib beta is phosphorylated on serine 166 by cyclic AMP-dependent protein kinase.

Platelet responses are inhibited by agents such as prostaglandin E1 that increase the cytoplasmic concentration of cyclic AMP. Inhibition is thought to result from phosphorylation of specific proteins. One protein that becomes phosphorylated is glycoprotein (GP) Ib beta, a component of the GP Ib.IX complex. We have suggested that phosphorylation of GP Ib beta inhibits the collagen-induced polymerization of actin. The aim of the present study was to identify the amino acid(s) in GP Ib beta that is phosphorylated. Purified GP Ib.IX complex was phosphorylated by the catalytic subunit of purified bovine cyclic AMP-dependent protein kinase in the presence of [gamma-32P]ATP. Phosphoamino acid analysis showed that in GP Ib beta, [32P]phosphate was incorporated only into serine and was in a single tryptic peptide. Amino acid sequencing showed that this peptide was from the cytoplasmic domain of GP Ib beta and encompassed residues 161-175. A single serine residue, serine 166, contained the radiolabel. To determine whether the same residue was phosphorylated in intact platelets, GP Ib beta was isolated from 32P-labeled platelets before or after their exposure to prostaglandin E1. In both cases, radiolabel was present in phosphoserine and was in a single tryptic peptide. This peptide was the same as that which was phosphorylated in the purified GP Ib.IX complex, as shown by its identical mobility on two-dimensional tryptic maps, the presence of a positively charged residue in the fourth position, and the presence of the radiolabel in the sixth position of the peptide. This study shows that when cyclic AMP concentrations rise in platelets, the cytoplasmic domain of GP Ib beta is phosphorylated on serine 166, probably by cyclic AMP-dependent protein kinase. We suggest that phosphorylation of this residue may contribute to the inhibitory actions of cyclic AMP by inhibiting collagen-induced polymerization of actin.

Adult↗
PubMed1989

Calmodulin and protein kinase C antagonists also inhibit the Ca2+-dependent protein protease, calpain I.

The calmodulin and C-kinase antagonists melittin, calmidazolium, N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide (W7), and trifluoperazine (TFP) also inhibit the activity of the human erythrocyte Ca2+-dependent protease, calpain I. W-5, the nonchlorinated derivative of W-7, was ineffective as an inhibitor of calpain I just as it is for calmodulin and protein kinase C. Dose response studies provided the following IC50 values: melittin, 2.6 microM; calmidazolium, 6.2 microM; trifluoperazine, 130 microM; W-7, 251 microM. These IC50 values indicate that the compounds have affinities 10 to 600 fold less for calpain I than for calmodulin; however, the affinities of the inhibitory compounds are comparable for calpain I and protein kinase C. Kinetic analysis indicates that the compounds are competitive inhibitors of calpain I with respect to substrate.

Bee Venoms↗
PubMed1988

Activation of a calmodulin-dependent phosphatase by a Ca2+-dependent protease.

A calmodulin-dependent protein phosphatase (calcineurin) was converted to an active, calmodulin-independent form by a Ca2+-dependent protease (calpain I). Proteolysis could be blocked by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, leupeptin, or N-ethylmaleimide, but other protease inhibitors such as phenylmethanesulfonyl fluoride, aprotinin, benzamidine, diisopropyl fluorophosphate, and trypsin inhibitor were ineffective. Phosphatase proteolyzed in the absence of calmodulin was insensitive to Ca2+ or Ca2+/calmodulin; the activity of the proteolyzed enzyme was greater than the Ca2+/calmodulin-stimulated activity of the unproteolyzed enzyme. Proteolysis of the phosphatase in the presence of calmodulin proceeded at a more rapid rate than in its absence, and the proteolyzed enzyme retained a small degree of sensitivity to Ca2+/calmodulin, being further stimulated some 15-20%. Proteolytic stimulation of phosphatase activity was accompanied by degradation of the 60-kilodalton (kDa) subunit; the 19-kDa subunit was not degraded. In the absence of calmodulin, the 60-kDa subunit was sequentially degraded to 58- and 45-kDa fragments; the 45-kDa fragment was incapable of binding 125I-calmodulin. In the presence of calmodulin, the 60-kDa subunit was proteolyzed to fragments of 58, 55 (2), and 48 kDa, all of which retained some ability to bind calmodulin. These data, coupled with our previous report that the human platelet calmodulin-binding proteins undergo Ca2+-dependent proteolysis upon platelet activation [Wallace, R. W., Tallant, E. A., & McManus, M. C. (1987) Biochemistry 26, 2766-2773], suggest that the Ca2+-dependent protease may have a role in the platelet as an irreversible activator of certain Ca2+/calmodulin-dependent reactions.

Animals↗
PubMed1987

Bryostatins mimic the effects of phorbol esters in intact human platelets.

Bryostatin-7 induces aggregation of human platelets and the phosphorylation of specific platelet proteins. Both the rate and extent of aggregation are similar to that induced by the tumor promoter phorbol ester 12-myristate 13-acetate (PMA); however, the rate of response is markedly reduced compared to that induced by thrombin. The addition of bryostatin-7 to 32P-labeled platelets results in a time-dependent incorporation of 32P into proteins of 20, 47 and 250 kDa; proteins of similar molecular mass are phosphorylated in response to the addition of thrombin or PMA. The time courses and dose responses of the phosphorylations induced by bryostatin-7 are similar to those found with PMA. In addition, bryostatin-7 increases the level of 32P incorporation into platelet polyphosphoinositides, which also occurs in response to PMA. These results suggest that, in intact human platelets, bryostatin-7 mimics the phorbol ester tumor promoter by directly activating protein kinase C.

Blood Platelets↗
PubMed1987

Human platelet calmodulin-binding proteins: identification and Ca2+-dependent proteolysis upon platelet activation.

Calmodulin-binding proteins have been identified in human platelets by using Western blotting techniques and 125I-calmodulin. Ten distinct proteins of 245, 225, 175, 150, 90, 82 (2), 60, and 41 (2) kilodaltons (kDa) bound 125I-calmodulin in a Ca2+-dependent manner; the binding was blocked by ethylene glycol bis(beta-aminoethyl ether)-N,N,N'N'-tetraacetic acid (EGTA), trifluoperazine, and nonradiolabeled calmodulin. Proteins of 225 and 90 kDa were labeled by antisera against myosin light chain kinase; 60- and 82-kDa proteins were labeled by antisera against the calmodulin-dependent phosphatase and caldesmon, respectively. The remaining calmodulin-binding proteins have not been identified. Calmodulin-binding proteins were degraded upon addition of Ca2+ to a platelet homogenate; the degradation could be blocked by either EGTA, leupeptin, or N-ethylmaleimide which suggests that the degradation was due to a Ca2+-dependent protease. Activation of intact platelets by thrombin, adenosine 5'-diphosphate, and collagen under conditions which promote platelet aggregation (i.e., stirring with extracellular Ca2+) also resulted in limited proteolysis of calmodulin-binding proteins including those labeled with antisera against myosin light chain kinase and the calmodulin-dependent phosphatase. Activation by the Ca2+ ionophores A23187 and ionomycin also promoted degradation of the calmodulin-binding proteins in the presence of extracellular Ca2+; however, degradation in response to the ionophores did not require stirring of the platelet suspension to promote aggregation. Many Ca2+/calmodulin-regulated enzymes are irreversibly activated in vitro by limited proteolysis. Our data indicate that limited proteolysis of Ca2+/calmodulin-regulated enzymes also occurs in the intact platelet and suggest that the proteolysis is triggered by an influx of extracellular Ca2+ associated with platelet aggregation.

Adenosine Diphosphate↗
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↗