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Biomedical subjects

E Shaw

Publications and source records attributed to E Shaw.

At least 91 records · Page 5Linked to original sources
PubMed1990

Lysis of trypanosomes by peptidyl fluoromethyl ketones.

Peptidyl fluoromethyl ketones, improved reagents for inactivating cysteinyl and serine proteinases, have provided unexpected results when applied to intact trypanosomes. A lethal effect was observed but limited to the infectious phase of the parasitic growth cycle. Since the inhibitors are known only to act on proteases, the result implies the existence of a protease of critical importance during the infectious phase. A labelled inhibitor, Cbz-Ala-[3H]Phe-CH2F, indicated that the killing effect correlated with the labelling of a 68 kd protein in the trypomastigotes which we deduce is an essential protease.

Animals↗
PubMed1990

Pseudoarginine: synthesis and properties of derivatives of delta-(1-imidazolyl)norvaline.

An analogue of arginine has been synthesized in which an imidazole ring occupies the position of the guanidino group of the natural amino acid. It was expected that peptides containing this amino acid when protonated might bind at enzymic sites specific for arginine, but that the pK of the imidazole ring, near 7, would facilitate entry of such peptides into cells, in contrast to peptides containing arginine. Other analogues of arginine can be visualized with a low side-chain pK, including isomers of the imidazole derivative which is the subject of this paper. These are viewed as 'pseudoarginines'. Our initial observations concern the properties of delta-(1-imidazolyl)norvaline in which a ring nitrogen atom is attached to norvaline, which thus becomes comparable to the guanidino delta-nitrogen. Its synthesis is described along with several derivatives examined as substrates or inhibitors. Potential ligands containing delta-(1-imidazolyl)norvaline (ImNva) did not give evidence of interaction with trypsin or plasma kallikrein, serine proteinases which bind arginine derivatives. However, clostripain, a bacterial cysteine proteinase specific for arginine, was readily inactivated by Cbz-Phe-ImNva-CH2F and the rate of inactivation showed an acid pH-dependence not observed, for example, in the inactivation of clostripain by Bz-Phe-LysCH2F.

Arginine↗
PubMed1990

Functional sites of glia-derived nexin (GDN): importance of the site reacting with the protease.

Glia-derived nexin (GDN) is a 43-kDa serine protease inhibitor with neurite promoting activity in mouse neuroblastoma cells (Guenther et al., 1985). In chick sympathetic neurons, GDN but not hirudin and synthetic peptide inhibitors promoted neurite outgrowth (Zurn et al., 1988). Thus, it was considered that the protease inhibitory activity cannot account for the total biological activity of GDN. We show here that synthetic peptide inhibitors with thrombin specificity mimic GDN at similar concentrations in neuroblastoma cells. Limited proteolysis of GDN with elastase causes a cleavage between sites P1 and P2, corresponding to residues Ala-344-Arg-345 of the molecule. The resulting fragments still copurify on heparin-Sepharose, but the protease inhibitor activity of GDN and the GDN neurite promoting activity are lost. The results confirm the necessity of an intact reactive site for the biological activity of GDN.

Amino Acid Sequence↗
PubMed1990

Inhibition of intraerythrocytic development of Plasmodium falciparum by proteinase inhibitors.

A group of inactivators of cysteinyl proteinases which function by covalent bond formation have been examined for their ability to inhibit the development of Plasmodium falciparum within red blood cells. The most effective of these caused inactivation of the parasite near 10(-8) M concentration. The range of inhibitory action varied with peptide structure in a manner characteristic of affinity labels for proteinases suggesting that the target of inhibition was an unidentified proteinase, probably of the cysteinyl type, but different from cathepsins B and L.

Animals↗
PubMed1990

Cysteinyl proteinases and their selective inactivation.

The affinity-labeling of cysteinyl proteinases may now be carried out with a number of peptide-derived reagents with selectivity, particularly for reactions carried out in vitro. These reagents have been described with emphasis on their selectivity for cysteine proteinases and lack of action on serine proteinases, the most likely source of side reactions among proteinases. Perhaps a crucial feature of this selectivity is an enzyme-promoted activation due to initial formation of a hemiketal, which may destabilize the reagent. Prominent among the reagent types that have this class selectivity are the peptidyl diazomethyl ketones, the acyloxymethyl ketones, the peptidylmethyl sulfonium salts, and peptidyl oxides analogous to E-64. The need for specific inhibitors capable of inactivating the target enzyme in intact cells and animals is inevitably pushing the biochemical application of these inhibitors into more complex molecular environments where the possibilities of competing reactions are greatly increased. In dealing with the current state and potential developments for the in vivo use of affinity-labeling reagents of cysteine proteinases, the presently known variety of cysteinyl proteinases had to be considered. Therefore this chapter has, at the same time, attempted to survey these proteinases with respect to specificity and gene family. The continual discovery of new proteinases will increase the complexity of this picture. At present the lysosomal cysteine proteinases cathepsins B and L and the cytoplasmic calcium-dependent proteinases are reasonable goals for a fairly complete metabolic clarification. The ability of investigators to inactivate individual members of this family in vivo, possibly without complications due to concurrent inactivation of serine proteinases by improvements in reagent specificity, is increasing. Among the cysteine proteinases, at least those of the papain super family, hydrophobic interactions in the S2 and S3 subsites are important and some specificity has been achieved by taking advantage of topographical differences among members of this group. Some of this has probably involved surface differences removed from the regions involved in proteolytic action. The emerging cysteine proteinases include some which, in contrast to the papain family, have a pronounced specificity in S1 for the binding of basic side chains, familiar in the trypsin family of serine proteinases. At least a potential conflict with serine proteinases can be avoided by choice of a covalent bonding mechanism. The departing group region, has not been exploited. As a sole contributor to binding, this region may be rather limited as a source of specificity.(ABSTRACT TRUNCATED AT 400 WORDS)

Affinity Labels↗
PubMed1990

Inhibition of endoproteolytic cleavage of cytomegalovirus (HCMV) glycoprotein B by palmitoyl-peptidyl-chloromethyl ketone.

Endoproteolytic cleavage of glycoprotein B (gB) of human cytomegalovirus (HCMV) is inhibited by palmitoylated peptidyl-chloromethyl ketone (palFAKR-CEK) at concentrations above 30 microM. Inhibitor treatment of HCMV-infected human fibroblasts neither interfered with exposure of gB on the plasma membrane, detected by surface membrane immunostaining, nor reduced production of intracellular infectious viral progeny. Release of infectious virus, on the other hand, was impaired.

Amino Acid Chloromethyl Ketones↗
PubMed1990

Pharmacokinetics of once daily intra-peritoneal aztreonam and vancomycin in the treatment of CAPD peritonitis.

The pharmacokinetics of aztreonam and vancomycin were studied in six adult patients who developed peritonitis during Continuous Ambulatory Peritoneal Dialysis. Aztreonam 3 g was added to the first exchange in each 24 h period with vancomycin 500 mg on the first day and 250 mg on each subsequent day (provided the pre-dose serum vancomycin concentration was less than 10 mg/l) for a total of ten days. Aztreonam and vancomycin concentrations were measured in the serum and dialysate at 1, 2, 6, 12, 18 and 24 h after the initial dose and pre-dose on days 5 and 10. By the end of the ten day study all patients had recovered clinically and had a normal dialysate. For aztreonam, the mean peak serum concentration was 84.3 mg/l (range 59.6-102.8), the mean 24 h pre-dose serum concentration was 23.0 mg/l (range 8.6-39.2) and the mean 24 h pre-dose dialysate concentration was 15.5 mg/l (range 5.0-32.0). For vancomycin, the mean peak serum concentration was 10.2 mg/l (range 8.1-12.6), the mean 24 h pre-dose concentration was 5.5 mg/l (range 3.6-6.4), and the mean 24 h pre-dose dialysate concentration was 3.4 mg/l (range 2.4-4.6). In the treatment of CAPD peritonitis, once daily intra-peritoneal administration of aztreonam 3 g with vancomycin 500 mg initially and 250 mg on subsequent days (serum concentrations permitting) provides concentrations of antibiotic in both the serum and dialysate throughout the 24 h period in excess of the inhibitory concentrations of those organisms most frequently encountered in this condition.

Aztreonam↗
PubMed1990

A comparative study of intravenous ciprofloxacin and benzylpenicillin versus netilmicin and piperacillin for the empirical treatment of fever in neutropenic patients.

We examined the efficacy of ciprofloxacin as an empirical treatment for fever in 97 neutropenic patients in a randomized study of ciprofloxacin and benzylpenicillin versus netilmicin and piperacillin. Benzylpenicillin was included because of evidence of in-vitro resistance to ciprofloxacin in some streptococci. Clinical response rate was similar in the two groups (46% resolution for ciprofloxacin/benzylpenicillin and 52% for netilmicin/piperacillin). Microbiological assessment revealed more pathogens eradicated by ciprofloxacin and benzylpenicillin (66%) and fewer persisting (3%) than in patients receiving netilmicin and piperacillin (52% and 13% respectively). Staphylococcus epidermidis was the commonest pathogen, accounting for 38% of all isolates and 30% of all treatment failures. There were no treatment failures or superinfections due to streptococci. More therapy-related adverse reactions were seen in patients on netilmicin and piperacillin (28%) compared with those on ciprofloxacin and benzylpenicillin (10%). The combination of ciprofloxacin and benzylpenicillin is as effective as a standard regimen of netilmicin and piperacillin, with fewer adverse effects, and is highly attractive as empirical therapy for the febrile, neutropenic host. The inclusion of benzylpenicillin prevents streptococcal-associated treatment failure.

Adult↗
PubMed1990

Incidence of leukemia in occupations with potential electromagnetic field exposure in United States Navy personnel.

Leukemia is the fourth most commonly occurring cancer in the United States population between the ages of 17 and 34 years, an age group heavily represented in the US Navy. Historical computerized military career records maintained at the Naval Health Research Center, San Diego, California, were used to determine person-years at risk (total, 4,072,502 person-years) by demographic characteristics and occupation for active-duty naval personnel during 1974-1984. Computerized inpatient medical records were searched for first hospitalizations for leukemia. Cases of leukemia (n = 102) were verified by using pathology reports or Navy Medical Board or Physical Evaluation Board findings. For comparisons, age-adjusted incidence rates and standardized incidence ratios were calculated by using rates for the US population provided by the Surveillance, Epidemiology, and End Results program of the National Cancer Institute. The overall age-adjusted incidence rate of leukemia in active-duty naval personnel was found to be very close to that of the Surveillance, Epidemiology, and End Results program population (6.0 vs. 6.5 per 100,000 person-years). Only one occupation, electrician's mate, emerged with a borderline statistically significant excess risk of leukemia (standardized incidence ratio compared with the Surveillance, Epidemiology, and End Results program population = 2.4, 95% confidence interval 1.0-5.0). This finding is intriguing in the light of several studies showing an excess risk of leukemia associated with exposure to electromagnetic fields.

Adolescent↗
PubMed1990

Specific labeling of cysteine proteinases in pathogenic and nonpathogenic Entamoeba histolytica.

Growth of Entamoeba histolytica trophozoites was inhibited by 50% at low concentrations (2.0 micrograms/ml) of the diazopeptidyl inhibitor benzyloxycarbonyl-leucyl-L-tyrosyldiazomethane (Z-L-Leu-L-Tyr-CHN2). Iodination of the tyrosine residue lowered the growth inhibitory efficacy of the diazopeptidyl inhibitor (50% inhibition, approximately 10 micrograms/ml). However, even at this concentration, practically all of the cysteine proteinase activity of the cells was irreversibly inactivated as shown by fluorescence microscopy with the dipeptide substrate L-Arg-L-Arg-4-methoxy-beta-napthylamide or colorimetrically with azocasein as the substrate. Growth of trophozoites of E. histolytica from various strains, including both pathogenic and nonpathogenic zymodemes, was similarly inhibited. The concentration of inhibitor required to inactivate the proteinase activity of nonpathogenic cells was lower. Lysates from trophozoites grown in the presence of sublethal concentrations of 125I-labeled protease inhibitor (10 micrograms/ml) showed as many as eight radioactive bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (molecular sizes, 73, 68, 56, 40, 39, 35, 29, and 27 kilodaltons). Two of these bands (molecular sizes, 29 and 27 kilodaltons) could be seen in gels of the cytoplasmic fraction, whereas the high-molecular-size bands were mostly associated with the membrane fraction. The radioactive bands in pathogenic and nonpathogenic strains were very similar with only minor differences. The results obtained show that E. histolytica cells, irrespective of their pathogenicity, possess a number of cysteine proteinases of similar molecular sizes which are vital for cell growth.

Affinity Labels↗
PubMed1989

Intravenous ciprofloxacin as empirical treatment of febrile neutropenic patients.

A randomized study of treatment with ciprofloxacin combined with benzylpenicillin (CB) versus a standard regimen of netilmicin combined with piperacillin (NP) as first-line empiric therapy was conducted in febrile neutropenic patients. Ninety-six patients were evaluable for determination of efficacy: 50 patients received CB and 46 patients received NP. There was no significant difference between the two groups in terms of age or primary diagnosis. Overall clinical response rate at the end of therapy was 66 percent for CB and 65 percent for NP. Microbiologic assessment revealed more pathogens eradicated by CB (64 percent) and fewer persisting (4 percent) than in the NP group (52 percent eradicated, 13 percent persisting). Only 10 percent of patients in the CB group had treatment-related adverse reactions as opposed to 28 percent of the NP-treated patients; these were predominantly renal impairment and were likely to have been due to the aminoglycoside. Staphylococcus epidermidis was the most commonly isolated pathogen, accounting for 38 percent of all isolates and 30 percent of all patients in whom treatment failed. Although streptococci accounted for 18 percent of the isolated pathogens, no treatment failures or superinfections were due to these organisms. This indicates an advantage of combining ciprofloxacin with benzylpenicillin. We conclude that the CB regimen is as effective as the NP treatment and is associated with fewer side effects.

Adult↗
PubMed1989

The inactivation of the cysteinyl exopeptidases cathepsin H and C by affinity-labelling reagents.

An attempt has been made to extend to the cysteinyl exopeptidases cathepsins H and C affinity-labelling approaches shown to be effective with cysteinyl endopeptidases such as cathepsins B and L and the calcium-activated proteinase. This involved the preparation of amino acid and dipeptide derivatives with unblocked N-termini to satisfy the aminopeptidase and dipeptidyl aminopeptidase characteristics of cathepsins H and C respectively. For covalent reactivity, the possibilities examined included diazomethanes (-CHN2), fluoromethanes (-CH2F) and dimethylsulphonium salt [-CH2S+(CH3)2]. A dipeptidylfluoromethane with a free amino group could not be prepared, perhaps due to inherent instability. Cathepsin H was inactivated by 1 microM-H2N-Phe-CH2F (the 'H2N' indicates a free unblocked amino group) (k2 = 1878 M-1.s-1); this reagent was without effect on cathepsins C and B, even at 100-fold this concentration. Analogous selectivity was shown by H2N-Ser(OBzl)-CHN2 and H2N-Phe-CH2S+(CH3)2, members of other classes of covalently binding reagents. For cathepsin C the dipeptide derivatives H2N-Gly-Phe-CHN2 and H2N-Phe-Ala-CH2S+(CH3)2 caused rapid inactivation near 10(-7) M. Higher concentrations inactivated cathepsins H and B, but the rates were slower by two to three orders of magnitude than for cathepsin C.

Affinity Labels↗
PubMed1989

Inhibition of cancer procoagulant by peptidyl diazomethyl ketones and peptidyl sulfonium salts.

Cancer procoagulant (CP) is a cysteine proteinase from cancer cells that initiates blood coagulation. Members of two classes of unique and highly specific cysteine proteinase inhibitors, peptidyl diazomethyl ketones (PDK) and peptidyl sulfonium salts (PSS), were studied to determine whether or not they inhibited CP. The inhibitors did not impair the activity of the coagulation system. There was a differential inhibitory effect of the 6 PDK and 2 PSS inhibitors, influenced by the amino acid composition or sequence of the peptide moiety, that suggests differences in structural features of the active site of CP and papain. CP was inhibited by both classes of inhibitors.

Animals↗
PubMed1989

The inhibition of proinsulin-processing endopeptidase activities by active-site-directed peptides.

Inhibitor studies were performed on the two endopeptidase activities involved in proinsulin conversion in isolated insulin secretory granules [Davidson, Rhodes & Hutton (1988) Nature (London) 333, 93-96]. The active-site-directed peptides L-alanyl-L-arginyl-L-arginylmethyldimethylsulphonium and L-alanyl-L-lysyl-L-arginylmethyldimethylsulphonium inhibited these activities in accordance with the observed cleavage pattern, suggesting that the primary amino acid sequence of the dibasic site was an important determinant of the endopeptidase substrate specificities.

Animals↗
PubMed1989

The properties of peptidyl diazoethanes and chloroethanes as protease inactivators.

Earlier work has demonstrated the irreversible inactivation of serine and cysteine proteinases by peptides with a C-terminal chloromethyl ketone group. With a C-terminal diazomethyl ketone, on the other hand, peptides become reagents specific for cysteine proteinases. We have now synthesized and examined the properties of reagents with an additional methyl side chain near the reactive grouping with the goal of diminishing side reactions in a cellular environment. Derivatives of neutral amino acids as well as of lysine and arginine have been prepared. The chloroethyl ketones are about 60% less reactive to chemical nucleophiles than the chloromethyl ketones. However, the susceptibilities of the proteases examined varied remarkably. Cathepsins B and L of the papain family of cysteine proteinases were much less susceptible (about 2 orders of magnitude less) to both peptidyl diazoethyl and chloroethyl ketones. In marked contrast, clostripain, a cysteine proteinase of a separate family was decisively more susceptible to chloroethyl ketones. The serine proteinases showed a drop in susceptibility to the chloroethyl ketones generally, and this was similar to the drop in chemical reactivity in proceeding from the chloromethyl to the chloroethyl ketone.

Amino Acid Chloromethyl Ketones↗
PubMed1989

Leishmania amazonensis: involvement of cysteine proteinases in the killing of isolated amastigotes by L-leucine methyl ester.

L-leucine-methyl ester (Leu-OMe) kills Leishmania mexicana amazonensis amastigotes by a mechanism which requires proteolytic cleavage of the ester. N-Benzyloxycarbonyl-phenylalanyl-alanyl diazomethane (Z-Phe-AlaCHN2), a specific and irreversible inhibitor of cysteine proteinases, was used to characterize the enzymes involved in parasite destruction. It was shown that (1) amastigotes preincubated with micromolar concentrations of Z-Phe-AlaCHN2 survived challenge with Leu-OMe concentrations lethal to control parasites; (2) the proteolytic activity of 25- to 33-kDa cysteine proteinases in parasite lysates subjected to electrophoresis in gelatin-containing acrylamide gels was selectively inhibited in parasites pretreated with Z-Phe-AlaCHN2 and chased in inhibitor-free medium; and (3) cysteine proteinase activity was also inhibited in gels incubated with amino acid and dipeptide esters, possibly because the compounds were acting either as substrates (e.g., Leu-Leu-OMe) or as inhibitors (e.g., Ile-OMe) of the enzyme. The results support the involvement of low molecular weight cysteine proteinases in the destruction of amastigotes by Leu-OMe. Characterization of the structure and substrate specificity of the enzymes may permit the rational development of more selectively leishmanicidal amino acid derivatives.

Animals↗
PubMed1989

Inhibition of proteolytic activation of influenza virus hemagglutinin by specific peptidyl chloroalkyl ketones.

Lysates of cultured cells have been analyzed for arginine-specific endoproteases using peptidyl-p-nitroanilides as chromogenic substrates. The enzymes present in MDBK, MDCK, VERO, BHK, and chick embryo cells required lysine-arginine or arginine-arginine pairs as cleavage sites, whereas chorioallantoic membrane cells contained, in addition, an activity that could cleave at a single arginine. The effect of peptidyl chloroalkyl ketones on the activation of the fowl plague virus hemagglutinin by the proteases specific for paired basic residues has been investigated. When virions containing uncleaved hemagglutinin were incubated with lysates of uninfected cells, cleavage was completely inhibited by peptidyl chloroalkyl ketones containing paired basic residues at a concentration of 1 mM. In contrast a compound containing a single arginine had no inhibitory activity. When dibasic peptidyl chloroalkyl ketones were added to infected cell cultures, cleavage of hemagglutinin and multiple cycles of virus replication were inhibited at 10 mM. However, a 100- to 200-fold increase of the inhibitory activity in intact cells could be achieved by N-terminal acylation. These studies suggest a potential role of peptidyl chloroalkyl ketones as antiviral agents.

Amino Acid Chloromethyl Ketones↗