Biomedical subjects

J Wallach

Publications and source records attributed to J Wallach.

At least 37 records · Page 2Linked to original sources

PubMed1998

Quantitation and enzymatic activity of the alkaline protease from Pseudomonas aeruginosa in culture supernatants from clinical strains.

Alkaline protease from Pseudomonas aeruginosa (E.C. 3.4.24.40), also called pseudomonal serralysin, plays an important role in the infection bacterial process. The alkaline protease concentration and activity in the culture supernatants of 23 Pseudomonas aeruginosa strains from patients suffering from cystic fibrosis were determined by using an ELISA procedure and a specific enzymatic activity test. No correlation was demonstrated between the activity and the concentration of alkaline protease. This could be explained by the presence of an inactive precursor and a partial hydrolysis of the enzyme, confirmed by SDS-PAGE and Western blotting in the supernatants. The activity test is the only one allowing a quantification of the presence of an active form of alkaline protease.

Blotting, Western↗

PubMed1998

Biological effects of elastin peptides.

Atherosclerosis is clearly one of the most life-threatening diseases and a major cause of morbidity and mortality in industrialized countries. Typical arterial lesions contain both cells originating from the blood (monocytes/macrophages) and locally-recruited smooth muscle cells. The structure of the artery is profoundly disrupted. Degradation of arterial elastin fibers results in loss of elasticity, and several elastin peptides are released that can interact with various cells via an increasingly well-characterized elastin receptor. Elastin receptor-mediated reactions that are of obvious physiologic importance include vasodilating effects and induction of mesenchymal cell adhesion to elastin fibers. Other effects are potentially harmful, such as increased elastase production, free radical release, induction of LDL oxidation, and stimulation of endogenous cholesterol production. These deleterious effects become predominant during aging as a result of chronic exposure of the elastin receptor to circulating elastin peptides. This review describes the results of recent investigations into the biological effects of elastin peptides.

Animals↗

PubMed1997

Capillary electrophoresis in the assay of the hydrolysis of glycine-containing peptides by a protease from Pseudomonas aeruginosa.

A rapid and simple capillary zone electrophoresis (CZE) method for measuring the activity of a minor protease from Pseudomonas aeruginosa is described. When glycine-containing oligopeptides were used as substrates, it was possible to separate and quantify substrate and products. Moreover oligopeptide hydrolysates were analysed by fast atom bombardment mass spectrometry providing the sites of splitting of the substrates. By comparison with CZE calibration curves constructed with reference peptides, the initial rates of hydrolysis were calculated. The method, validated for pentaglycine hydrolysis, was also used for the analysis of reaction mixtures and for monitoring the enzymic hydrolysis of various peptides in order to investigate enzyme specificity.

Electrophoresis, Capillary↗

PubMed1997

LasA, alkaline protease and elastase in clinical strains of Pseudomonas aeruginosa: quantification by immunochemical methods.

Thirty Pseudomonas aeruginosa strains were isolated from the sputa of cystic fibrosis patients. In each culture supernatant, the amount of three exoproteases (LasA, alkaline protease and elastase) was determined using immunochemical procedures. These assays used selected peptide-MAP (multiple antigen peptide) strategy as antigen for animal immunisation. The method appeared to be reproducible, simple, sensitive and specific without cross-reactivity between the antisera. The resulting values differed from one strain to another mostly for elastase production. Despite the fact that four genes (lasA, lasB, lasR and rhlR) were shown to be necessary for full elastolytic activity, it was obvious that if LasA was not secreted in a naturally non-elastase-producing strain, in return in an elastase-producing strain, there were no apparent relationships between LasA and elastase production and between LasA and alkaline protease secretion. Furthermore, in vitro, the secretion of the three exoproteases seemed to be independent of the mucoid or non-mucoid phenotype of the bacteria.

Amino Acid Sequence↗

PubMed1996

Cloning, sequencing and overexpression in Escherichia coli of the alginatelyase-encoding aly gene of Pseudomonas alginovora: identification of three classes of alginate lyases.

A gene of Pseudomonas alginovora, called aly, has been cloned in Escherichia coli using a battery of PCR techniques and sequenced. It encodes a 210-amino-acid alginate lyase (EC 4.2.2.3), Aly, in the form of a 233-amino-acid precursor. P. alginovora Aly has been overproduced in E. coli with a His-tag sequence fused at the C-terminal end under conditions in which the formation of inclusion bodies is avoided. His-tagged P. alginovora Aly has the same enzymic properties as the wild-type enzyme and has the specificity of a mannuronate lyase. It can be purified in a one-step procedure by affinity chromatography on Ni(2+)-nitriloacetate resin. The yield is of 5 mg of enzyme per litre of culture. The amplification factor is 12.5 compared with the level of production by wild-type P. alginovora. The six alginate lyases of known primary structure fall into three distinct classes, one of which comprises the pair P. alginovora Aly and Klebsiella pneumoniae Aly.

Amino Acid Sequence↗

PubMed1995

Effect of the lipopeptide antibiotic, iturin A, on morphology and membrane ultrastructure of yeast cells.

The effects of iturin A, at fungicidal concentrations, on yeast cells were studied by scanning electron microscopy and by transmission electron microscopy. A depression, observed in each iturin A-treated cell, was the consequence of the release of electrolytes and other cytoplasmic components. Iturin A passes through the cell wall and disrupts the plasma membrane with the formation of small vesicles and the aggregation of intramembranous particles. Moreover, iturin A passes through the plasma membrane and interacts with the nuclear membrane and probably with membranes of other cytoplasmic organelles.

Anti-Bacterial Agents↗

PubMed1995

Growth stimulation of human skin fibroblasts by elastin-derived peptides.

Elastin-derived peptides (kappa-elastin: kappa E, mean molecular mass: 75 kDa), either coated onto plastic dishes or added to culture media (0.26 to 1.33 nM) stimulated the growth of human skin fibroblasts (HSF) strains obtained from different donors and tested at different cell passages (4 to 12). Coated 44.4 micrograms/cm2 insoluble elastin (iE) exhibited the same action; coated iE or kappa E significantly modifies the HSF morphology: after 5-6 days of culture, HSF are more elongated, and at preconfluence state, formation of HSF clusters surrounding iE were observed. Increased 3H thymidine incorporation and proliferative effect of HSF by kappa E (1.3 to 2.2 fold as compared to control cells) was observed after a lag phase period which raised with initial HSF density. Optimal proliferative effect was obtained at kappa E 8.5 10(-10) M, a value close to the dissociation constant (kD = 2.7 10(-10) M) of kappa E to HSF. Valine-glycine-valine-alanine-proline-glycine (VGVAPG), but not valine-glycine-valine (VGV) or Valine-glycine-valine-valine-glycine-alanine (VGVVGA) also significantly stimulated, optimally at 7.0 10(-10) M, HSF proliferation. It was concluded that the stimulatory influence of elastin derived peptides on HSF proliferation was mediated through a binding to plasmalemmal receptor of HSF.

Adult↗

PubMed1995

Effects of synthesized elastin peptides on human leukocytes.

It is well demonstrated that various peptides derived from elastin are biologically active. The hexapeptide (Val-Gly-Val-Ala-Pro-Gly; VI) as well as elastin peptides were demonstrated to be chemotactic for fibroblasts, while kappa-elastin had marked biological effects on human PMNLs. The aim of our present work was to synthesize various elastin peptides and compare their action to that of kappa-elastin and this hexapeptide. The results indicate that the hexapeptide (Val-Gly-Val-Ala-Pro-Gly) and the two other synthesized hexapeptides (Pro-Gly-Val-Gly-Val-Ala; III and Val-Gly-Val-Gly-Val-Ala; IV) had very similar and specific effects on intracellular free calcium metabolism, on superoxide anion production and elastase release. The other peptides had no effects on these parameters, except a tripeptide (Val-Gly-Val; V) on superoxide anion production. Moreover, the effect of the hexapeptides (III and VI) could be abolished by Pertussis toxin preincubation. All peptides had very similar stimulating effects on H2O2 production and myeloperoxidase release. We conclude that most probably the peptide size and conformation, as well as peptide composition play a role in the biological effects of these peptides, through specific receptors on PMNLs surface.

Amino Acid Sequence↗

PubMed1994

[Ala4]surfactin, a novel isoform from Bacillus subtilis studied by mass and NMR spectroscopies.

When Bacillus subtilis S 499 was grown on a culture medium containing L-alanine as nitrogen source, a mixture of surfactins was obtained. Suitable chromatographic conditions allowed the separation of isoforms. Among these compounds, a new variant of surfactin was isolated and its structure was established by chemical and spectrometric methods, especially by NMR spectrometry. It contains a peptide sequence which differs from that of standard surfactin by the replacement of the L-valine residue by L-alanine residue in position 4. The folding mode of [Ala4]surfactin as deduced from NMR results was compared with that of standard surfactin and the structure/properties relationship issuing from the study of this new isoform is discussed.

Alanine↗

PubMed1994

Laboratory diagnosis of factitious disorders.

Self-induced factitious disorders are defined and distinguished from conditions they may resemble. Review of the literature since 1965 indicates much more frequent reporting in recent years, but most health care providers are still not sufficiently aware of the common factitious disorder. Up to 5% of physician-patient encounters may be because of factitious disorders, but these are only suspected when the workup leads to contradictory findings. Laboratory tests are often the only definitive diagnostic method, and clinicians may not be familiar with current technologies. Some clinical clues are listed; heightened awareness and the need for early diagnosis are emphasized. Discordant laboratory results should raise the possibility of a factitious disorder. Sophisticated laboratory tests that can accurately assay very small amounts of specific hormones or foreign substances in body fluids facilitate the diagnosis. The primary physician can now confirm an initial clinical diagnosis of factitious disorder promptly and directly rather than only by exclusion.

Clinical Laboratory Techniques↗

PubMed1994

Purification and characterization of an alkaline elastase from Myxococcus xanthus.

An extracellular elastase, termed Myxococcus xanthus alkaline protease 1 (MAP1), has been purified from M. xanthus DK1622 culture supernatants by a combination of ion-exchange and affinity chromatographies. It consists of a single peptide chain of 39 kDa. The elastolytic activity was totally suppressed by 10 mM 1,10-phenanthroline and the enzyme may then be classified as a metalloprotease. Its pH optimum was estimated to be 8.2 with both elastin-orcein and succinyl-Ala3 p-nitroanilide as substrates. Despite its low pI (5.2), MAP1 was adsorbed on elastin at 80%, a result which privileges hydrophobic interactions between MAP1 and elastin rather than salt bridges, as for known basic elastases. About 80% of the original amidasic and elastolytic activities were conserved after a 30-min prior incubation of the enzyme at 40 degrees C; however, 70% of the amidasic activity is measured, instead of 15% for the elastolytic activity, after 30 min at 50 degrees C. Thermal denaturation at this temperature may prevent adsorption of the enzyme on elastin without any important change of the elastase structure. MAP1 readily hydrolyzes the Gly23-Phe24 bond in the oxidized insulin B chain; the peptide bonds Ala14-Leu15, Leu15-Tyr16, Phe24-Phe25, Phe25-Tyr26 are also cleaved, suggesting a primary specificity of the enzyme for hydrophobic or aromatic residues at the first amino acid towards the C-terminus from the cleavage site (P'1 position) [Schechter, I. & Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 157-162]. This hypothesis is consistent with the fact that Ala2-Phe-Ala and Ala3-Phe-Ala are hydrolyzed even though tri-alanine to hexa-alanine oligomers are not. The evidence of an elastase with the same molecular mass and pI as MAP1 is given during fruiting body development in submerged culture of M. xanthus. The fact that aromatic amino acids have been found to be the most representative of A-signal [Kuspa, A., Plamann, L. & Kaiser, D. (1992) J. Bacteriol. 174, 3319-3326] is consistent with the hypothesis that, regarding its specificity, MAP1 is likely to play a role in development of myxobacteria.

Amino Acid Sequence↗

PubMed1994

Diversity in the processing events at the N-terminus of type-V collagen.

The processing of human collagen type-V chains was studied using anti-peptide polyclonal antibodies raised against peptide sequences at the N-terminal non-triple-helical region of pro-alpha 1(V) and pro-alpha 2(V) chains. The anti-peptide polyclonal antibody raised against positions 48-57 of the N-terminal alpha 2(V) sequence recognized the mature form of the human alpha 2(V) chain extracted without any proteolytic treatment from several tissues in the presence of a mixture of protease inhibitors. It also recognized the pro-alpha 2(V) and pN-alpha 2(V) collagen chains secreted in the cell-culture media of the rhabdomyosarcoma A204 cell line. The pN-alpha 2(V) collagen chain from this cell line migrated during electrophoresis with the alpha 2(V) chain obtained from tissues. This demonstrates that the alpha 2(V) chain in tissues is incompletely processed and is present as the pN-alpha 2(V) collagen chain which lacks the C-propeptide. In comparison, an anti-peptide polyclonal antibody raised against residues at positions 284-299 of the N-terminal alpha 1(V) human sequence failed to recognize the mature form of the alpha 1(V) chain while it reacted with the pN-alpha 1(V) collagen chain form. These results suggest that the alpha 1(V) chain undergoes a processing event in the N-terminal region that involves the removal of at least the first 284 residues. Amino acid sequence analysis was performed on cyanogen-bromide-generated or trypsin-generated peptides of the two electrophoretic bands obtained for the tissue form of collagen V. The slower-migrating band corresponding to the intact alpha 1(V) chain gave, as expected, only sequences corresponding to the alpha 1(V) chain. However, the band previously considered to be the intact alpha 2(V) chain also gave sequences for the alpha 1(V) chain in addition to the alpha 2(V) chain. This result indicates the presence in tissue extracts of a further processed form of alpha 1(V) chain which migrates with the intact alpha 2(V) chain. On further analysis, we observed that the two bands of the tissue form of collagen V occurred in a 1:1 ratio whereas, after the pepsin digestion to remove non-collagenous regions, two bands were observed with an alpha 1(V)/alpha 2(V) chain ratio of 3:1. These results indicate that the alpha 1(V) chain exists in an additional stoichiometry, different from [alpha 1(V)]2 alpha 2(V).(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1994

Molecular cloning and expression of a novel type V adenylyl cyclase from rabbit myocardium.

A cDNA of a novel form of type V adenylyl cyclase has been cloned from rabbit myocardium using oligonucleotide probes derived from peptides that were produced by enzymatic cleavage of purified heart cyclase. A corresponding mRNA (6 kb) has been detected in rabbit myocardial tissue by Northern blot analysis. The cDNA encodes a protein of 1,264 amino acids exhibiting 12 putative membrane-spanning regions in its hydrophilicity profile. Sequence comparison to two other previously published type V adenylyl cyclase reveals amino-terminal domains of different length and low correlative homology, whereas the rest of the sequences is almost identical. The nonconserved amino-terminal region of the subtype consists of 214 amino acids and exceeds the length of the others by 40 and 80 residues, respectively. Its presence in membrane preparations from different tissues has been confirmed immunologically using an antibody directed against a synthetic peptide. The cloned adenylyl cyclase was functionally expressed in COS-1 cells to attain an enzymatic activity 3.5- to 14-fold above control in the presence of forskolin.

Adenylyl Cyclases↗

PubMed1994

Elastolytic activity of MAP1, a protease from Myxococcus xanthus.

MAP1, a protease isolated from Myxococcus xanthus, is demonstrated to be an elastase. Although its elastolytic activity is lower than that of other well-known elastases, the size distribution of solubilized elastin peptides is similar. MAP1 as pancreatic elastase releases peptides with molecular weights higher than 10,000. However, the specificity of MAP1 is different since this enzyme cannot hydrolyze alanine oligomers as do pancreatic and Pseudomonas aeruginosa elastases.

Animals↗

PubMed1993

Peptide synthesis catalysed by Pseudomonas aeruginosa elastase.

Pseudomonas aeruginosa elastase was used for peptide-bond synthesis with benzyloxycarbonylalanine and amino acid amides as nucleophilic substrates. Dipeptide-bond synthesis was observed only for hydrophobic amino acid amides. The rate of peptide synthesis, measured by h.p.l.c., was in the decreasing order: Phe > Leu > Tyr > Val, Ile > Ala, which is consistent with the decreasing order of hydrolysis rates of the corresponding tetrapeptides Ala-Ala-Xaa-Ala. In contrast with thermolysin, Ps. aeruginosa elastase permits the synthesis of tyrosine-containing peptides with tyrosine in the P'1 position. Furthermore, the rates of synthesis for other hydrophobic amino acid amides are higher with elastase than with thermolysin.

Amino Acid Sequence↗

PubMed1992

Identification of a small epitope in domain Ib of Pseudomonas aeruginosa exotoxin A that elicits enzyme-neutralizing antibodies.

A peptide corresponding to amino acids 392-404 of the amino acid sequence of Pseudomonas aeruginosa exotoxin A (the last 13 amino acids of domain Ib) was synthesized and coupled to thyroglobulin. The conjugate induced an antiserum in rabbits with high antibody titer against native toxin as measured by ELISA, and this antiserum was highly efficient in inhibiting the ADP-ribosyltransferase activity of exotoxin A. These data corroborate the potential importance of amino acids 400-404 in the enzymatic mechanism of exotoxin A.

ADP Ribose Transferases↗

PubMed1991

Human brucellosis: immunoblotting analysis of three Brucella abortus antigenic fractions allows the detection of components of diagnostic importance.

Results indicating that analysis of the immune humoral response of brucellosis patients by immunoblotting provides useful information for the characterization of antigenic fractions of possible diagnostic importance in human brucellosis are presented. Sera of 90 patients were obtained: 23 suffering from chronic brucellosis, 20 from acute brucellosis and 47 belonging to the group of serologically positive individuals (SPI) without clinical evidence of active infection at the time of examination, and 35 healthy volunteers. They were tested against three antigenic fractions: cytoplasmic (CYT), outer membrane (OM) and inner membrane (IM). These fractions, which include virtually all the bacterial protein components, were prepared from Brucella abortus 1119/3 strain by detergent solubilization, enzymatic digestion and ultracentrifugation. Results obtained with these fractions showed the existence of antigens that permit the detection of brucellosis patients and their differentiation from SPI patients, with very high sensitivity.

Antigens, Bacterial↗

PubMed1989

Micropreparative separation of peptides derived from sodium dodecyl sulphate-solubilized proteins.

A systematic investigation of the influence of the detergent sodium dodecyl sulphate (SDS) on micropreparative peptide separations on microbore reversed-phase high-performance liquid chromatographic columns is reported. A tryptic digest of bovine serum albumin and a mixture of synthetic peptides were used to monitor the separation behaviour of a 1.6 mm I.D. Nucleosil C18 column in the presence of various amounts of SDS. The data demonstrate that even traces of SDS in the sample reduce the separation efficiency and peptide recovery. An extraction method is presented which reduces the SDS content in peptide mixtures below the critical concentration without affecting significantly the recovery of individual peptides. After acidification of the sample, the detergent is extracted into heptane-isoamyl alcohol (4:1, v/v). In combination with chemical or enzymatic fragmentation techniques, this extraction method facilitates the sequence analysis of minute amounts of SDS-solubilized hydrophobic proteins. The applicability of the method is demonstrated on the example of the integral membrane protein bacteriorhodopsin.

Amino Acid Sequence↗