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Biomedical subjects

E Shaw

Publications and source records attributed to E Shaw.

At least 55 records · Page 3Linked to original sources
PubMed1994

Inhibition of growth of human TE2 and C-33A cells by the cell-permeant calpain inhibitor benzyloxycarbonyl-Leu-Leu-Tyr diazomethyl ketone.

Calpains are Ca(2+)-requiring, nonlysosomal proteases which are thought to participate in some aspects of intracellular Ca(2+)-signal transduction. However, their exact physiologic function has not yet been established. Addition of the cell-permeant, irreversible calpain inhibitor, ZLLY-CHN2, to human TE2 or C-33A cells inhibited growth, as assessed either by mitochondrial MTT reductase activity or by direct cell counting. Inhibition of growth produced by a 24-h exposure to 50 microM ZLLY-CHN2 was reversed upon substituting growth medium without inhibitor. Homogenates produced from cells cultured in the presence of ZLLY-CHN2 displayed decreased calpain and Ca(2+)-independent proteolytic activities. Protein immunoblot analysis showed that cell cultures which had lost 80% of their calpain activity still retained full calpain immunoreactivity. Therefore, inhibition by ZLLY-CHN2 appeared to result in accumulation of irreversibly inactivated calpain within the cells. Homogenates from cells cultured in the presence of 20 or 50 microM ZLVG-CHN2, a cell-permeant inhibitor with little activity against calpains, had decreased Ca(2+)-independent proteolytic activity, but demonstrated no decrease in calpain activity. ZLVG-CHN2 did not inhibit cell growth under these conditions. Growth of Saccharomyces cerevisiae cells, which do not appear to express calpain-like proteases, was not inhibited by including 50 microM ZLLY-CHN2 in the culture medium. These results indicate that calpains participate in the social regulation of cell growth in multicellular organisms.

Calcium-Binding Proteins↗
PubMed1994

Processing of viral glycoproteins by the subtilisin-like endoprotease furin and its inhibition by specific peptidylchloroalkylketones.

The spike glycoproteins of many enveloped viruses are proteolytically cleaved at the carboxytermini of sequences containing the basic motif R-X-K/R-R. Cleavage is often necessary for the fusion capacity of the glycoproteins and, thus, for virus infectivity. Among these viruses are pathogenic avian influenza viruses, human parainfluenza virus, human cytomegalovirus, and human immunodeficiency virus; it has been demonstrated that these viruses can be activated by furin. Indigenous furin has been identified in T-lymphocytes, which are host cells for HIV. Furin has been localized in the TGN and on the surface of cells after vectorial expression. Peptidylchloroalkylketones have been designed that inhibit with high specificity cleavage and fusion activity of viral glycoproteins, as well as virus replication.

Amino Acid Chloromethyl Ketones↗
PubMed1994

Regulatory circuits involved with pH-regulated gene expression in Salmonella typhimurium.

Salmonella typhimurium encounters a variety of acid conditions during both its natural and pathogenic existence. The ability of this organism to respond transcriptionally to low pH is an area of active interest but little knowledge. As part of an ongoing investigation of low-pH adaptation, 18 pH-controlled lacZ operon fusions in Salmonella typhimurium have been identified (15 in this study) and categorized into at least 11 different loci. They include iroA (at 57 min), aciA (99 min), aciB (90-93 min), aciD (ompC, 45 min), aciJ, aciK (33-36 min), aniC (93 min), anil (33-36 min), hyd (59 min), cadA (54 min) and aniG (63 min). All but two were induced by low pH. One of the exceptions, the iron-regulated iroA locus, was induced at high pH. The unusual aciA locus was induced by low pH under semiaerobic conditions but high pH under aerobic conditions. Most of the other aci genes were expressed best under anaerobic conditions. Many of these genes exhibited strict co-inducer requirements for small molecules to be expressed in minimal medium. These included iron for iroA, tyrosine for aniC, I and aciK, mannose for aniG, formate for hyd, lysine for cadA, and unknown components of complex medium for aciA, aciB and aciD. Six regulatory circuits were revealed involving at least five regulatory loci (fur, oxrG, earAB, earC and ompR). As part of the adaptive response to low pH, S. typhimurium will induce an acid protection system called the acid tolerance response (ATR). As has been shown for fur mutations, the oxrG regulatory mutation interfered with the normal induction of this system.

Anaerobiosis↗
PubMed1994

Proteolytic cleavage of wild type and mutants of the F protein of human parainfluenza virus type 3 by two subtilisin-like endoproteases, furin and Kex2.

The fusion (F) protein of human parainfluenza virus type 3 contains the tribasic cleavage site R-T-K-R, which was altered by site-directed mutagenesis. Wild-type F protein and various mutants were expressed by recombinant vaccinia viruses. The endogenous endoprotease present in CV-1 cells cleaves F variants containing the furin recognition motif R-X-K/R-R but not variants containing the dibasic site K-R or a single R at the cleavage site. A similar cleavage pattern was obtained when the subtilisin-like endoproteases Kex2 and furin were coexpressed with the wild type and mutants of the F protein. Peptidylchloromethylketone inhibitors mimicking basic cleavage sites prevent cleavage of the precursor Fo by the endogenous protease only when the furin-specific motif is present in the peptidyl portion. The data support the concept that furin is a cellular protease responsible for the activation of the F protein of human parainfluenza virus type 3.

Amino Acid Sequence↗
PubMed1993

Reaction of proteasomes with peptidylchloromethanes and peptidyldiazomethanes.

The multicatalytic endopeptidase complex (proteasome) has multiple distinct peptidase activities. These activities have often been referred to as 'chymotrypsin-like', 'trypsin-like' and 'peptidylglutamyl-peptide hydrolase' activities according to the type of residue in the P1 position, although it is now clear that mammalian proteasomes have at least five distinct catalytic sites. In the present study, potential affinity-labelling reagents (peptidylchloromethanes, peptidyldiazomethanes, a peptidylfluoromethane and peptidylsulphonium salts) containing hydrophobic, basic or acidic amino acid residues in the P1 position have been tested for inhibition of the different activities of the rat liver proteinase complex. The results show that individual peptidase activities of proteasomes can be inhibited by a variety of peptidylchloromethanes and peptidyldiazomethanes. Although the rate of inactivation of proteasomes by even the most effective peptidylchloromethanes and peptidyldiazomethanes are often quite slow (k(obs)/[I] in the range 0.1-10 M-1 x s-1) compared with the reaction of similar compounds with some other proteinases, the results provide useful information concerning the specificity of the distinct catalytic centres of proteasomes, and some selective affinity-labelling reagents have been identified. Tyr-Gly-Arg-chloromethane was found to be a useful inhibitor of trypsin-like activity. Inhibition of the other peptidase activities was often incomplete, even after repeated addition of inhibitor, and it proved to be difficult to predict the effect of different reagents. For example, Cbz-Tyr-Ala-Glu-chloromethane was found to inhibit 'chymotrypsin-like' activity (assayed with Ala-Ala-Phe-7-amino-4-methylcoumarin or succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin), while the best inhibitors of 'peptidylglutamyl-peptide hydrolase' activities (assayed with benzyloxycarbonyl-Leu-Leu-Glu beta-naphthylamide) were peptidyldiazomethanes containing hydrophobic amino acid residues. These results suggest that the original nomenclature of proteasome activities is misleading, because the residue in the P1 position is not the only determinant of specificity.

Affinity Labels↗
PubMed1993

Radiation Therapy Oncology Group: radiosurgery quality assurance guidelines.

A multidisciplinary Radiation Therapy Oncology Group (RTOG) task force has developed quality assurance guidelines for radiosurgery. The purpose of the guidelines are fourfold: (1) To ensure that participating institutions have the proper equipment and appropriate technique(s) to administer radiosurgery; (2) to outline a standard data set for each treated patient to assess protocol compliance; (3) to define minor and major deviations in protocol treatment; and (4) to set forth clinical data necessary to determine treatment efficacy, including failure patterns, and treatment toxicity. These guidelines are being implemented into active and developing radiosurgery protocols.

Brain Neoplasms↗
PubMed1993

The affinity-labelling of cathepsin S with peptidyl diazomethyl ketones. Comparison with the inhibition of cathepsin L and calpain.

Since peptidyl diazomethyl ketones are useful irreversible inhibitors for inactivating cysteinyl proteinases in vitro and in vivo and in order to reveal their role, we set out to obtain selective and effective reagents for cathepsin S. A number of such derivatives with hydrophobic amino acid residues, such as valine, leucine and tryptophane in positions adjacent to the primary specificity site were synthesized and these provided inhibitors rapidly acting at high dilution. For example, 1 nM Z-Leu-Leu-Nle-CHN2 inactivates cathepsin S with k2nd = 4.6 x 10(6) M-1 x s-1 at pH 6.5, 25 degrees C. Similarities to the specificities of cathepsin L and calpain were evident. However, Z-Val-Val-NleCHN2 is over 300 times more effective in inactivating S than L. On the other hand, Z-Phe-Tyr(t-Bu)CHN2 is about 10(4) more effective against L than S. Reagents are thus now available for a clear discrimination between these proteases.

Amino Acid Sequence↗
PubMed1993

The synthesis of inhibitors for processing proteinases and their action on the Kex2 proteinase of yeast.

Peptidyl chloromethane and sulphonium salts containing multiple Arg and Lys residues were synthesized as potential inhibitors of prohormone and pro-protein processing proteinases. The potencies of these compounds were assayed by measuring the kinetics of inactivation of the yeast Kex2 proteinase, the prototype of a growing family of eukaryotic precursor processing proteinases. The most potent inhibitor, Pro-Nvl-Tyr-Lys-Arg-chloromethane, was based on cleavage sites in the natural Kex2 substrate pro-alpha-factor. This inhibitor exhibited a Ki of 3.7 nM and a second-order inactivation rate constant (k2/Ki) of 1.3 x 10(7) M-1.s-1 comparable with the value of kcat./Km obtained with Kex2 for the corresponding peptidyl methylcoumarinylamide substrate. The enzyme exhibited sensitivity to the other peptidyl chloromethanes over a range of concentrations, depending on peptide sequence and alpha-amino decanoylation, but was completely resistant to peptidyl sulphonium salts. Kinetics of inactivation by these new inhibitors of a set of 'control' proteinases, including members of both the trypsin and subtilisin families, underscored the apparent specificity of the compounds most active against Kex2 proteinase.

Amino Acid Chloromethyl Ketones↗
PubMed1993

Synthesis of oligopeptide chloromethanes to investigate extended binding regions of proteinases: application to the interaction of fibrinogen with thrombin.

A method was established for the synthesis of oligopeptide chloromethanes which should be useful in the study of serine and cysteine proteinases with extended binding sites. The method involved condensation of an N-terminal peptide fragment obtained by solid-phase synthesis with a C-terminal peptide chloromethane synthesized by solution-phase chemistry. By using this procedure, oligopeptide chloromethanes of up to 16 residues were synthesized. These chloromethanes were based on the sequence of fibrinopeptide A. By using oligopeptide chloromethanes of different length, it was possible to show that the residues Asp7-Phe8-Leu9 play a crucial role in the recognition of fibrinopeptide A by thrombin. In contrast, the residues Ala1-Asp2-Ser3-Gly4-Glu5-Gly6 seem to play a minor role. Substitution of valine for Gly12, which occurs in a dysfibrinogenaemia, markedly decreased the rate of inactivation of thrombin by the oligopeptide chloromethane. The results are discussed in terms of the recently published structure of the complex between human thrombin and a chloromethane inhibitor based on fibrinopeptide A.

Amino Acid Sequence↗
PubMed1993

Affinity labelling of the Ca(2+)-activated neutral proteinase (calpain) in intact human platelets.

Two irreversible calpain inhibitors, benzyloxycarbonyl (Cbz)-Leu-Leu-Tyr-Ch2F and Cbz-Leu-Leu-Tyr-CHN2, were shown earlier [Anagli, Hagmann and Shaw (1991) Biochem. J. 274, 497-502] to penetrate intact platelets and to inactivate calpain. This permitted an evaluation of certain functions attributed to this proteinase. For example, in platelets pretreated with these inhibitors, talin and actin-binding protein were protected from subsequent degradation when the Ca2+ level was raised. On the other hand, additional properties of stimulated platelets attributed to calpain remained unaffected by this treatment, and such hypotheses may be dismissed. Radioiodinated inhibitors permitted confirmation of the labelling of calpain by the procedures used. Although Cbz-Leu-Leu-Tyr-CHN2 is more effective in vitro than the corresponding fluoromethyl ketone, we now show that the latter penetrates more readily. These two inhibitors, and two additional ones, t-butyloxycarbonyl-Val-Lys(Cbz)-Leu-Tyr- CHN2 and Cbz-Leu-Tyr-CH2F, have been radioiodinated to permit a comparison of their intracellular labelling patterns in activated platelets. Calpain is the major target of all four inhibitors. Although they are closely related peptide structures, variations with respect to the labelling of additional proteins were observed. These were minor in the case of the peptidyl diazomethyl ketones, but were major in the case of the fluoromethyl ketones. However, in contrast to calpain, this labelling was neither time-dependent nor Ca(2+)-dependent. Radiolabelling and cellular fractionation studies were used to localize active calpain during platelet activation. Calpain appears to be activated in the cytosol and translocated to the membrane or cytoskeletal sites.

Affinity Labels↗
PubMed1993

Cross-reactivity between B. burgdorferi and other spirochetes affects specificity of serotests for detection of antibodies to the Lyme disease agent in dogs.

Western immunoblots, the kinetics-based enzyme-linked immunosorbent assay (KELA), and the microagglutination test were used to evaluate cross-reactivity among antibodies to serovars of Leptospira interrogans (leptospiral serovars), and B. burgdorferi from naturally infected dogs, and to Serpulina (Treponema) hyodysenteriae from vaccinated rabbits. Whole-cell lysates from Borrelia spp., leptospiral serovars, and Serpulina spp. were used for SDS-PAGE, western blots, and KELA. Crossreactivity occurred between the antibodies to B. burgdorferi and leptospiral serovars when tested on the heterologous antigens. Antibodies to leptospiral serovars tended to cross-react more strongly with antigens of B. burgdorferi spp. than did antibodies to B. burgdorferi when tested against antigens of leptospiral serovars. The antibodies against B. burgdorferi showed a lesser degree of cross-reactivity to the antigens of S. hyodysenteriae and S. innocens than they did to leptospiral serovars. We conclude that cross-reactivity occurs between B. burgdorferi and leptospiral serovars. Validation and interpretation of ELISA tests for detection of antibody activity to whole cell lysates of the Lyme agent must take this cross-reactivity into consideration. Conversely, dogs infected with the Lyme agent do not show significant cross-reactivity in the microagglutination test for antibody to the leptospiral serovars.

Agglutination Tests↗
PubMed1993

The Gloucestershire Aneurysm Screening Programme: the first 2 years' experience.

5337 men aged 65 were invited for ultrasound examination of the abdominal aorta between September 1990 and October 1992 as part of a General Practice based aneurysm screening programme covering the County of Gloucestershire. 4232 (79%) attended. 3877 (91.6%) had aortic diameters equal to or less than 2.5 cm. 302 (7.1%) had aortic diameters in the range 2.6-3.9 cm and 53 (1.3%) had a diameter of 4.0 cm or greater. Mean aortic diameter was 2.04 cm (S.D. 0.50 cm). The practicalities, costs and benefits of a community based aneurysm screening programme are discussed.

Aged↗
PubMed1993

Familial abdominal aortic aneurysm: prevalence and implications for screening.

The high prevalence of abdominal aortic aneurysm (AAA) in men over the age of 65 has led to the establishment of ultrasound screening programmes for this group. The apparent familial tendency towards AAA formation suggests that relatives of aneurysm patients may form another subpopulation in whom screening is appropriate. The first degree relatives of 100 consecutive aneurysm patients were identified. Of 110 relatives over 50 years of age, two were known to have had AAA and ultrasound scans were performed on 74, providing information on aortic size for 76 relatives (69%). No further aortic aneurysms (antero-posterior diameter > or = 4.0 cm) were found on scanning. However, nine relatives were demonstrated to have aortic dilatation (2.5-3.9 cm). Aortic dilatation was observed in 21% of male first degree relatives over 50 years of age, affecting 27% of sons and 17% of brothers. Only 4% of the sisters and none of the daughters were found to have aortic dilatation. The prevalence of aortic enlargement seems to be sufficiently high amongst male first degree relatives of AAA patients over 50 years of age to justify aortic screening.

Aged↗
PubMed1992

Inhibition of furin-mediated cleavage activation of HIV-1 glycoprotein gp160.

The envelope glycoprotein of human immunodeficiency virus (HIV) initiates infection by mediating fusion of the viral envelope with the cell membrane. Fusion activity requires proteolytic cleavage of the gp160 protein into gp120 and gp41 at a site containing several arginine and lysine residues. Activation at basic cleavage sites is observed with many membrane proteins of cellular and viral origin. We have recently found that the enzyme activating the haemagglutinin of fowl plague virus (FPV), an avian influenza virus, is furin. Furin, a subtilisin-like eukaryotic endoprotease, has a substrate specificity for the consensus amino-acid sequence Arg-X-Lys/Arg-Arg at the cleavage site. We show here that the glycoprotein of HIV-1, which has the same protease recognition motif as the FPV haemagglutinin, is also activated by furin.

Amino Acid Chloromethyl Ketones↗
PubMed1992

Peptidyldiazomethanes. A novel mechanism of interaction with prolyl endopeptidase.

Peptidyldiazomethanes with proline in the P1 position were found to be competitive slow-binding inhibitors of prolyl endopeptidase. Progress-curve experiments monitoring the increase in the degree of inhibition with time indicated that the kinetic mechanism involved an initial complex that isomerized to form a tighter complex. Reversibility of the inhibited complex was demonstrated by monitoring the regain of enzyme activity after removal of free inhibitor and dilution into an assay containing competing substrate. The kinetics of the reversal of inhibition indicated a more complicated inhibitory mechanism involving more than one pathway for reversal of the tight complex. A slow-binding mechanism of inhibition has not been previously observed with peptidyldiazomethanes. Incorporation of [3H]Ac-Ala-Ala-Pro-diazomethane into prolyl endopeptidase was observed after denaturation of the inhibited complex. The peptide labelled with [3H]Ac-Ala-Ala-Pro-diazomethane was isolated and found to contain the active-site serine residue.

Amino Acid Sequence↗
PubMed1992

Characterisation of a cysteine protease from bloodstream forms of Trypanosoma congolense.

A cysteine protease (trypanopain-Tc) with cathepsin-L-like properties has been purified from Trypanosoma congolense. The enzyme has an apparent molecular mass of 31-32 kDa by SDS/PAGE and 66 kDa by gel chromatography. It has a pI 7.4 and a high affinity for concanavalin A. Trypanopain-Tc catalyses the limited proteolysis of a variety of protein substrates such as fibrinogen, serum albumin and trypanosome variant-surface glycoprotein. It has minimal or no activity against casein or elastin. A variety of peptidyl amidomethylcoumarins and peptidyl diazomethanes were used to test the specificity of trypanopain-Tc. The better substrates had Arg or Lys in P1 and hydrophobic amino acids in P2 and P3. The best substrate found for trypanopain-Tc was Z-Phe-Arg-NHMec (Z, benzyloxycarbonyl; NHMec, 7-amido-4-methylcoumarin). The kinetic constants for the hydrolysis of Z-Phe-Arg-NHMec were kcat = 17.4 s-1, Km = 4.4 microM, kcat/Km = 4.0 microM-1.s-1, which are very similar to those of cathepsin L with this substrate. The specific substrates for cathepsin B (Z-Arg-Arg-NHMec) and cathepsin H (Arg-NHMec) were not hydrolysed by trypanopain-Tc under the conditions tested. The pH optimum of trypanopain-Tc against Z-Phe-Arg-NHMec was pH 6.0 but it showed a broad peak of activity extending well into the alkaline region. The enzyme was activated by low-molecular-mass thiol compounds and inhibited by cystatin, L-trans-epoxysuccinyl-4-guanidinobutane (E-64) and a variety of peptidyl diazomethanes. The most effective diazomethane inhibitors (Z-Leu-Leu-Met-CHN2, Z-Leu-Met-CHN2 and Z-Leu-Lys-CHN2, were inhibitory at nanomolar concentrations and were trypanocidal in vitro after 24-48 h incubation in greater than or equal to 20 microM [inhibitor]. However, it is not clear whether the trypanocidal activity of these inhibitors is a consequence of the inhibition of trypanopains or of some other essential proteolytic activities within the parasites.

Amino Acid Sequence↗