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P Lachance

Publications and source records attributed to P Lachance.

17 recordsLinked to original sources
PubMed2001

Substrate specificity of the streptococcal cysteine protease.

The streptococcal pyrogenic exotoxin B (SpeB) is an important factor in mediating Streptococcus pyogenes infections. SpeB is the zymogen of the streptococcal cysteine protease (SCP), of which relatively little is known regarding substrate specificity. To investigate this aspect of SCP function, a series of internally quenched fluorescent substrates was designed based on the cleavage sites identified in the autocatalytic processing of SpeB to mature SCP. The best substrates for SCP contain three amino acids in the nonprimed position (i.e. AIK in P(3)-P(2)-P(1)). Varying the length of the substrate on the primed side of the scissile bond has a relatively lower effect on activity. The highest activity (k(cat)/K(M) = 2.8 +/- 0.6 (10(5) x m(-1)s(-1)) is observed for the pentamer 3-aminobenzoic acid-AIKAG-3-nitrotyrosine, which spans subsites S(3) to S(2)' on the enzyme. High pressure liquid chromatography and mass spectrometry analyses show that the substrates are cleaved at the site predicted from the autoprocessing experiments. These results show that SCP can display an important level of endopeptidase activity. Substitutions at position P(2) of the substrate clearly indicate that the S(2) subsite of SCP can readily accommodate substrates containing a hydrophobic residue at that position and that some topological preference exists for that subsite. Substitutions in positions P(3), P(1), and P(1)' had little or no effect on SCP activity. The substrate specificity outlined in this work further supports the similarity between SCP and the cysteine proteases of the papain family. From the data regarding the identified or proposed natural substrates for SCP, it appears that this substrate specificity profile may also apply to the processing of mammalian and streptococcal protein targets by SCP.

Binding Sites↗
PubMed2001

Cathepsins X and B can be differentiated through their respective mono- and dipeptidyl carboxypeptidase activities.

Several new cysteine proteases of the papain family have been discovered in the past few years. To help in the assignment of physiological roles and in the design of specific inhibitors, a clear picture of the specificities of these enzymes is needed. One of these novel enzymes, cathepsin X, displays a unique specificity, cleaving single amino acid residues at the C-terminus of substrates very efficiently. In this study, the carboxypeptidase activities and substrate specificity of cathepsins X and B have been investigated in detail and compared. Using quenched fluorogenic substrates and HPLC measurements, it was shown that cathepsin X preferentially cleaves substrates through a monopeptidyl carboxypeptidase pathway, while cathepsin B displays a preference for the dipeptidyl pathway. The preference for one or the other pathway is about the same for both enzymes, i.e., approximately 2 orders of magnitude, a result supported by molecular modeling of enzyme-substrate complexes. Cleavage of a C-terminal dipeptide of a substrate by cathepsin X can become more important under conditions that preclude efficient monopeptidyl carboxypeptidase activity, e.g., nonoptimal interactions in subsites S(2)-S(1). These results confirm that cathepsin X is designed to function as a monopeptidyl carboxypeptidase. Contrary to a recent report [Klemencic, I., et al. (2000) Eur. J. Biochem. 267, 5404-5412], it is shown that cathepsins X and B do not share similar activity profiles, and that reagents are available to clearly distinguish the two enzymes. In particular, CA074 was found to inactivate cathepsin B at least 34000-fold more efficiently than cathepsin X. The insights obtained from this and previous studies have been used to produce an inhibitor designed to exploit the unique structural features responsible for the carboxypeptidase activity of cathepsin X. Although of moderate potency, this E-64 derivative is the first reported example of a cathepsin X-specific inhibitor.

Cathepsin B↗
PubMed2001

Cathepsins X and B display distinct activity profiles that can be exploited for inhibitor design.

The carboxypeptidase and endopeptidase activities of cathepsins X and B, as well as their inhibition by E-64 derivatives, have been investigated in detail and compared. The results clearly demonstrate that cathepsins X and B do not share similar activity profiles against substrates and inhibitors. Using quenched fluorogenic substrates, we show that cathepsin X preferentially cleaves substrates through a monopeptidyl carboxypeptidase pathway, while cathepsin B displays a preference for the dipeptidyl pathway. The preference for one or the other pathway is about the same for both enzymes, i. e. approximately 2 orders of magnitude. Cleavage of a C-terminal dipeptide of a substrate by cathepsin X can be observed under conditions that preclude efficient monopeptidyl carboxypeptidase activity. In addition, an inhibitor designed to exploit the unique structural features responsible for the carboxypeptidase activity of cathepsin X has been synthesized and tested against cathepsins X, B and L. Although of moderate potency, this E-64 derivative is the first reported example of a cathepsin X-specific inhibitor. By comparison, CA074 was found to inactivate cathepsin B at least 34000-fold more efficiently than cathepsin X.

Cathepsin B↗
PubMed1999

An unusual case of carbon monoxide poisoning.

Carbon monoxide, a gas originating from incomplete combustion of carbon-based fuels, is an important cause of human deaths. In this paper, we describe an unusual carbon monoxide poisoning in a dwelling without obvious sources of combustion gases, for which two adults had to be treated in a hyperbaric chamber. Carbon monoxide readings were taken in the house and in the neighboring homes. Methane gas and nitrogen oxide levels were also monitored in the house air. Soil samples were collected around the house and tested for hydrocarbon residues. The investigation revealed the presence of a pocket of carbon monoxide under the foundation of the house. The first readings revealed carbon monoxide levels of 500 ppm in the basement. The contamination lasted for a week. The investigation indicated that the probable source of contamination was the use of explosives at a nearby rain sewer construction site. The use of explosives in a residential area can constitute a major source of carbon monoxide for the neighboring populations. This must be investigated, and public health authorities, primary-care physicians, governmental authorities, and users and manufacturers of explosives must be made aware of this problem.

Adult↗
PubMed1998

Golf injuries. An overview.

Over the years, golf has become an increasingly popular sport, attracting new players of almost all ages and socioeconomic groups. Golf is practised by up to 10 to 20% of the overall adult population in many countries. Beyond the enjoyment of the sport itself, the health-related benefits of the exercise involved in walking up to 10 km and of relaxing in a pleasant natural environment are often reported to be the main motives for adhering to this activity by recreational golfers. Golf is considered to be a moderate risk activity for sports injury; however, excessive time spent golfing and technical deficiencies lead to overuse injuries. These are the 2 main causes of injuries among golfers, and each has specific differences in the pattern in which they occur in professional and amateur golfers. Golf injuries originate either from overuse or from a traumatic origin and primarily affect the elbow, wrist, shoulder and the dorsolumbar sites. Professional and weekend golfers, although showing a similar overall anatomical distribution of injuries by body segment, tend to present differences in the ranking of injury occurrence by anatomical site; these differences can be explained by their playing habits and the biomechanical characteristics of their golf swing. Many of these injuries can be prevented by a preseason, and year-round, sport-specific conditioning programme including: (i) muscular strengthening, flexibility and aerobic exercise components; (ii) a short, practical, pre-game warm-up routine; and (iii) the adjustment of an individual's golf swing to meet their physical capacities and limitations through properly supervised golf lessons. Finally, the correct selection of golf equipment and an awareness of the environmental conditions and etiquette of golf can also contribute to making golf a safe and enjoyable lifetime activity.

Adult↗
PubMed1996

Contribution to activity of histidine-aromatic, amide-aromatic, and aromatic-aromatic interactions in the extended catalytic site of cysteine proteinases.

Within the papain family of cysteine proteinases few other residues in addition to the catalytic triad, Cys25-His159-Asn175 (papain numbering) are completely conserved [Berti & Storer (1995) J. Mol. Biol. 246, 273-283]. One such residue is tryptophan 177 which participates in a Trp-His-type interaction with the catalytic His159. In all enzymes of this class for which a three-dimensional structure has been reported, an additional highly conserved tryptophan, Trp181, also interacts with Trp177 via an aromatic-aromatic interaction in which the planes of the indole rings are essentially perpendicular. Also, both indole rings participate as pseudo-hydrogen bond acceptors in interactions with the two side chain amide protons of Asn175. Clearly, the proximity of Trp177 and Trp181 to the catalytic triad residues His159 and Asn175 and their network of interactions points to potential contributions of these aromatic residues to catalysis. In this paper, using cathepsin S, a naturally occurring variant that has a phenylalanine residue at position 181, we report the kinetic characterization of mutants of residues 175, 177, and 181. The results are interpreted in terms of the side chain contributions to catalytic activity and thiolate-imidazolium ion-pair stability. For example, the side chain of Asn175 has a major influence on the ion-pair stability presumably through its hydrogen bond to His159. The magnitude of this effect is modulated by Trp177, which shields the His159-Asn175 hydrogen bond from solvent. The His159-Trp177 interaction also contributes significantly to ion-pair stability; however, Trp181 and its interactions with Asn175 and Trp177 do not influence ion-pair stability to a significant degree. The observation that certain mutations at positions 177 and 181 result in a reduction of kcat/Km but do not appear to influence ion-pair stability probably reflects the contributions of these residues to substrate binding.

Amino Acid Sequence↗
PubMed1994

Engineering the S2 subsite specificity of human cathepsin S to a cathepsin L- and cathepsin B-like specificity.

The primary specificity of papain-like proteinases is largely determined by S2-P2 site interactions. According to the three-dimensional structure of a papain-inhibitor complex, the S2 subsite is defined by residues 67, 68, 133, 157, 160, and 205, with residues 133, 157, and 205 integrated into the wall and bottom of the side chain binding cavity. The S2 binding site specificity of this enzyme has been altered to mimic that of cathepsin B or L by the application of site-directed mutagenesis at these latter three positions in the cathepsin S sequence. The replacement of Gly-133 in cathepsin S by an alanine residue that is normally found at this position in both cathepsin B and L results in a pattern of specificity toward hydrophobic residues in P2 that is very similar to that of cathepsin B and L. The replacement of other cathepsin S S2 subsite residues with their cathepsin L equivalents (mutants Val-157-->Leu, Phe-205-->Ala) does not significantly change the specificity of cathepsin S. Cathepsin B is distinguished from both cathepsin L and S by its ability to efficiently hydrolyze substrates containing a basic P2 residue. A single mutation in position 205 of cathepsin S (Phe-205-->Glu) results in a change of specificity toward that of cathepsin B, i.e. the second-order rate constant for the hydrolysis of the cathepsin B-specific substrate benzyloxycarbonyl-Arg-Arg-4-methyl-7-coumaryl-amide is increased 77-fold for this mutant compared with the wild-type enzyme. A cathepsin S double mutant Gly-133-->Ala/Phe-205-->Glu is characterized by somewhat improved kinetic parameters compared with the Phe-205-->Glu single mutant. The hydrolysis rate of the benzyloxy-carbonyl-Arg-Arg-4-methyl-7-coumarylamide substrate by this double mutant is 130-fold higher than that of the wild-type enzyme. As with cathepsin B, the activities of the Phe-205-->Glu single and the Gly-133-->Ala/Phen-205-->Glu double mutants of cathepsin S toward the dibasic substrate is modulated by an additional ionizable group with a pKa of 5.7.

Alanine↗
PubMed1994

E64 [trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane] analogues as inhibitors of cysteine proteinases: investigation of S2 subsite interactions.

A number of epoxysuccinyl amino acid benzyl esters (HO-Eps-AA-OBzl) and benzyl amides (HO-Eps-AA-NHBzl) (where AA represents amino acid) were synthesized as analogues of E64, a naturally occurring inhibitor of cysteine proteinases. These inhibitors were designed to evaluate if selectivity for cathepsin B could be achieved by varying the amino acid on the basis of known substrate specificity. Contrary to the situation with substrates, it was found that variation of the amino acid in the E64 analogues does not lead to major changes in the kinetic parameter kinac./Ki and that the specificity of these analogues does not parallel that observed for substrates. This is particularly true in the case of the benzyl ester derivatives where the deviation from substrate-like behaviour is more important than with the benzyl amide derivatives. The results suggest that the amide proton of the benzyl amide group in HO-Eps-AA-NHBzl interacts in the S2 subsite in both cathepsin B and papain and contributes to increase the potency of these inhibitors. The kinetic data also suggest that differences in the orientation of the C alpha-C beta bond of the side chain in the S2 subsite of the enzyme might explain the differences between substrate and E64 analogue specificities. This hypothesis is supported by the fact that the order of inactivation rates with chloromethane inhibitors (which are believed to be good models of enzyme-substrate interactions) is indeed very similar to that observed with the corresponding amidomethylcoumarin substrates. In conclusion, the information available from S2-P2 interactions with substrates cannot be used to enhance the selectivity of the E64 analogues in a rational manner.

Cathepsin B↗
PubMed1993

Epoxysuccinyl dipeptides as selective inhibitors of cathepsin B.

Epoxysuccinyl dipeptide analogs of E-64 (R-EpsLeuPro-R') (Figure 1) have been synthesized with the carboxylate group on the epoxide ring either free (R = OH) or converted to an ester or an amide (R = EtO or i-BuNH) and with the C-terminal amino acid proline either blocked (R' = OBzl) or free (R' = OH). These compounds were used to investigate the recently reported selectivity of this type of inhibitor for the lysosomal cysteine protease cathepsin B. It was shown that derivatization of the carboxylate on the epoxide ring confers selectivity for cathepsin B over papain only when it is combined to a dipeptidyl moiety with a free negatively charged C-terminal residue. It is proposed that this selectivity reflects interactions with histidine residues on a loop located in the primed subsites of cathepsin B which provides a positively charged anchor for the C-terminal carboxylate group of the inhibitor. The primed subsite loop of cathepsin B is not found in other cysteine proteases of the papain family and offers a unique template for designing selectivity in cysteine protease inhibitors.

Cathepsin B↗
PubMed1993

Functional expression of human cathepsin S in Saccharomyces cerevisiae. Purification and characterization of the recombinant enzyme.

A cDNA encoding the human lysosomal cysteine proteinase cathepsin S precursor has been expressed in yeast using the pVT100-U expression vector containing the alpha-factor promoter. The procathepsin S gene was expressed either as a fusion protein with the pre-region or with the prepro-region of the yeast alpha-factor precursor gene. Following in vitro processing both constructs gave an identical active mature enzyme with a molecular weight of 24,000. After prolonged cultivation of the cells the recombinant protein is also found as an active proteinase in the culture supernatant. The precursor can be activated in vitro at pH 4.5 and 40 degrees C under reducing conditions. The in vitro activated enzyme has a 6-amino acid NH2-terminal extension when compared with the native bovine enzyme. The purified enzyme displays a bell-shaped pH activity profile with a pH optimum of 6.5 and pK values of 4.5 and 7.8. The isoelectric point of the recombinant human cathepsin S is between 8.3 and 8.6 and about 1.5 pH units higher than for the bovine enzyme. The kinetic data for several synthetic substrates and inhibitors reveal a preference for smaller amino acid residues in the binding subsites S2 and S3 of cathepsin S. Like the bovine enzyme, the recombinant human cathepsin S is characterized by a broader range of pH stability (pH 5-7.5) than cathepsins B and L.

Base Sequence↗
PubMed1992

Resonance Raman spectroscopic and kinetic consequences of a nitrogen ... sulphur enzyme-substrate contact in a series of dithioacylpapains.

The resonance Raman (RR) spectroscopic, conformational, and kinetic properties of six dithioacylpapain intermediates have been examined. Five of the intermediates are of the form N-(methyloxycarbonyl)-X-glycine-C(= S)S-papain, where X is L-phenyl-alanine, D-phenylalanine, glycine, L-phenylglycine, or D-phenylglycine. The sixth intermediate is N-phenylacetyl-glycine-C(= S)S-papain. Throughout the series there is an approximately 50-fold variation in kcat, the rate constant for deacylation, and a 1750-fold variation in kcat/KM. Existing RR spectra structure correlations allow us to define the torsional angles in the NH-CH2-C(= S)-S-CH2-CH fragment of the functioning intermediates. The values of these angles for each bound substrate appear to be very similar, with the substrates assuming a B-type conformer such that the nitrogen atom of the P1 glycine residue is cis to the thiol sulphur atom of cysteine-25. For each intermediate, the C(= S)S-CH2CH torsional angle is approximately -90 degrees, whereas for the SCH2-CH torisonal angle the cysteine-25 thiol sulphur (S) and cysteine-25 C alpha hydrogen (H) atoms are approximately trans. The three acyl-enzymes with the lowest catalytic rate constants, viz. N-(methyloxycarbonyl)-glycine-glycine-, N-(methyloxycarbonyl)-L-phenylglycine-glycine-, or N-(phenylacetyl)-glycine-dithioacylpapains, have atypical RR spectra in that they show a feature of medium intensity in the 1,085-cm-1 region. This band is sensitive to NH to ND exchange of the P1 glycine residues' (-NH-) function and, thus, the corresponding mode involves an excursion of the NH hydrogen. It is hypothesized that the high intensity is due to a particularly strong interaction between the P1 glycine nitrogen atom and the thiol sulphur of cysteine-25, which also has the effect of retarding deacylation, because the nitrogen . . . sulphur contact has to be broken in the rate-determining step.

Amino Acid Sequence↗
PubMed1992

Molecular cloning and characterization of PEA3, a new member of the Ets oncogene family that is differentially expressed in mouse embryonic cells.

The PEA3 motif, first recognized in the polyomavirus enhancer, is an oncogene, serum growth factor, and phorbol ester-responsive element. An activity capable of binding to this sequence, termed PEA3 (polyomavirus enhancer activator 3), was identified in mouse 3T6 cell nuclear extracts. We have cloned cDNAs that encode PEA3 from a mouse FM3A cell cDNA library. A continuous open reading frame in the longest cDNA predicts a 555-amino-acid protein with a calculated molecular mass of 61 kD. Recombinant PEA3 binds to DNA with the same sequence specificity as that endogenous to FM3A cells and activates transcription through the PEA3 motif in HeLa cells. Deletion mapping of the protein revealed that the DNA-binding domain is located within a stretch of 102 amino acids near the carboxyl terminus. This region shares extensive sequence similarity with the ETS domain, a conserved protein sequence common to all ets gene family members. PEA3 is encoded by a 2.4-kb mRNA that is expressed to differing extents in fibroblastic and epithelial cell lines but not in hematopoietic cell lines. In the mouse, PEA3 expression is highly restricted; only the epididymis and the brain contain readily detectable amounts of its mRNA. Interestingly, the amount of PEA3 mRNA is down-regulated during retinoic acid-induced differentiation of mouse embryonic cell lines. These findings suggest that PEA3 plays a regulatory role during mouse embryogenesis.

Amino Acid Sequence↗
PubMed1989

Effects of exercise-rest cycles on energy balance in rats.

The present study investigated the nutritional energetics of rats exercised either intermittently or continually. Male rats were divided into three groups: a sedentary group, an intermittently trained group, and a continually trained group. Continually trained rats were exercised every day for a period of 8 wk. Over the same period, intermittently trained rats were exercised for 4 wk, each week being followed by a week of rest. Rats were trained on a rodent treadmill at a moderate intensity. Carcasses were analyzed for energy, fat, and protein contents. Brown adipose tissue (BAT) thermogenesis was assessed by measuring mitochondrial guanosine 5'-diphosphate binding. Energy intake was lower in both intermittently and continually trained rats than in sedentary animals. The weight, fat, and protein gains were, in continually trained rats, significantly lower than in sedentary animals. Similarly, intermittently trained rats had lower gains than sedentary animals, although the difference between the two groups was not as marked as the difference between sedentary and continually trained animals. Energy expenditure, which represents the difference between energy intake and energy gain, was less than sedentary controls in both intermittently and continually trained rats. The low expenditure in trained groups did not, however, relate to changes in facultative BAT thermogenesis.A low energy expenditure on components such as basal metabolic rate and obligatory diet-induced thermogenesis would most likely account for the difference in expenditure between sedentary and trained rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue, Brown↗
PubMed1988

Occupational asthma from reactivity to an alkaline hydrolysis derivative of gluten.

A 29-year-old female subject had been working for 13 years in a company producing biscuits. She was exposed intermittently to an alkaline hydrolysis wheat gluten derivative (AHGD) that was incorporated into marshmallows. Five years before being referred, she started reporting rhinoconjunctivitis and asthmatic symptoms immediately after handling AHGD. When she was first assessed, she had avoided contact with AHGD for 1 year, and she was asymptomatic. Normal spirometry and bronchial responsiveness to histamine (provocative concentration causing a 20% fall in FEV1, 22 mg/ml) were obtained. There was a positive allergy skin test to ragweed pollen and to AHGD at 0.01 mg/ml. Skin tests with individual extracts of cereals, including wheat, were negative. Inhalation challenge with AHGD for 15 seconds caused immediate rhinoconjunctivitis and bronchoconstriction (maximum fall of 40% in FEV1 30 minutes after the exposure) with complete recovery 2 hours later. There was no reaction thereafter, and the provocative concentration of histamine causing a 20% fall in FEV1 was not significantly changed (13.1 mg/ml) 9 hours after the challenge. Inhalation challenge with wheat flour containing native gluten for 30 minutes did not cause any symptoms or significant change in FEV1. IgE RAST counts were greater with the AHGD than with gluten and the whole wheat extract; the opposite was generally found with sera from individuals with baker's asthma and wheat food allergy. With the serum of the affected worker, AHGD completely inhibited the corresponding RAST, but gluten did not. With wheat food allergy, AHGD was relatively inactive inhibition of the wheat and gluten RAST, but gluten was effective.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1985

Production of RNA-dependent haemolysin by Haemophilus pleuropneumoniae.

Five strains of Haemophilus pleuropneumoniae, out of eight strains tested, produced extracellular haemolysin(s) when grown in liquid culture in the presence, but not in the absence, of RNA. The haemolysin produced by the neotype strain was unstable, heat labile, and sensitive to degradation by pronase, trypsin, and chymotrypsin; moreover, trypan blue treated haemolysin preparations were less effective at causing erythrocyte lysis than were untreated preparations. Following growth in the absence of RNA, washed suspensions of the neotype strain produced extracellular haemolysin when incubated in the presence of RNA, glucose, and casein acid hydrolysate; extracellular haemolysin could not be detected if the incubation mixture contained chloramphenicol. The haemolysin produced by washed bacterial suspensions was similar to that produced by growing cultures in that it was unstable, heat labile, and sensitive to inactivation by the same complement of enzymes. Erythrocyte lysis induced by either haemolysin preparation was preceded by a prelytic phase, the duration of which was dependent upon haemolysin concentration and the initial temperature of the haemolysin--erythrocyte mixture. It is concluded that the haemolysin(s) produced by the neotype strain of H. pleuropneumoniae is distinct from, but closely related to both streptolysin S and the haemolysin produced by Treponema hyodysenteriae.

Animals↗
PubMed1967

[Evaluation of a new cholecystokinetic agent].

Boyden's test meal (egg yolk beaten with cream), used during cholecystography, remains in the stomach for a long time and could interfere with the radiological findings on barium meal examination performed after cholecystography.A new preparation based on corn oil emulsion (G.P. Prep) was evaluated in 33 patients with and without symptoms of gallbladder disease. The criteria used in the evaluation of results were three: reduction in the gallbladder dimensions, variations in the cholecystovertebral angle, and visualization of the extrahepatic bile ducts.The gallbladder dimensions were determined before and after contraction with the aid of a metallic perforated ruler (Colcher's) placed at the estimated level of the gallbladder at the time of exposure.

Adult↗