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E Kessler

Publications and source records attributed to E Kessler.

At least 37 records · Page 2Linked to original sources
PubMed1996

Bone morphogenetic protein-1: the type I procollagen C-proteinase.

Bone morphogenetic proteins (BMPs) are bone-derived factors capable of inducing ectopic bone formation. Unlike other BMPs, BMP-1 is not like transforming growth factor-beta (TGF-beta), but it is the prototype of a family of putative proteases implicated in pattern formation during development in diverse organisms. Although some members of this group, such as Drosophila tolloid (TLD), are postulated to activate TGF-beta-like proteins, actual substrates are unknown. Procollagen C-proteinase (PCP) cleaves the COOH-propeptides of procollagens I, II, and III to yield the major fibrous components of vertebrate extracellular matrix. Here it is shown that BMP-1 and PCP are identical. This demonstration of enzymatic activity for a BMP-1/TLD-like protein links an enzyme involved in matrix deposition to genes involved in pattern formation.

Amino Acid Sequence↗
PubMed1996

Prevalence of latex-specific IgE antibodies in hospital personnel.

BACKGROUND: Rubber latex hypersensitivity is an important concern for health care workers. PURPOSE: The Center for Devices and Radiological Health, in collaboration with the Consumer Product Safety Commission, conducted a multicenter study of the prevalence of latex-specific IgE antibodies among United States hospital personnel. MATERIALS AND METHODS: Nine hospitals participated in the cross-sectional study. A total of 504 hospital personnel completed questionnaires that provided an array of demographic, occupational, and clinical information, including a history, if any, of allergies and the use of latex and nonlatex gloves. More than three-quarters (76.5%) of the participants were tested for total IgE and latex specific IgE. RESULTS: A total of 21 (5.5%, 95% CI = 3%-7%) of the tested participants were positive for the presence of latex specific IgE antibodies, defined as a latex IgE level of > or = 0.6 ng/mL. Latex specific IgE antibodies were more prevalent in participants who reported tachycardia, palpitations, flushing, or wheezing associated with latex gloves (Odds Ratio = 10.2, 95% CI = 3.7-28.6). CONCLUSION: The study's results suggest that the prevalence of latex-specific IgE antibodies among hospital personnel is appreciable and these personnel and their health care providers should be aware of this entity.

Adult↗
PubMed1996

A substitution at His-120 in the LasA protease of Pseudomonas aeruginosa blocks enzymatic activity without affecting propeptide processing or extracellular secretion.

The LasA protease of Pseudomonas aeruginosa can degrade elastin and is an important contributor to the pathogenesis of this organism. LasA (20 kDa) is a member of the beta-lytic endopeptidase family of extracellular bacterial proteases, and it shows high-level staphylolytic activity. We sequenced the lasA gene from strain FRD1 and overexpressed it in Escherichia coli. The lasA gene encodes a precursor, known as pre-proLasA, of 45,582 Da. Amino-terminal sequence analysis allowed the identification of the signal peptidase cleavage site and revealed that the 31-amino-acid signal peptide was removed in E. coli. The remaining proLasA (42 kDa) did not undergo autoproteolytic processing and showed little staphylolytic activity. However, it was readily processed to a 20-kDa active staphylolytic protease by incubation with trypsin or with the culture filtrate of a P. aeruginosa lasAdelta mutant. Thus, removal of the propeptide (22 kDa) was required to convert proLasA into an active protease. Although LasA protease was critical for staphylolytic activity, other proteases like elastase were found to enhance staphylolysis. Under the control of an inducible trc promoter, lasA was overexpressed in P. aeruginosa and the processing intermediates were examined. Compared with wild-type cells, the overproducing cells accumulated more 42-kDa proLasA species, and the culture supernatants of the overproducing cells showed increased levels of active 20-kDa LasA protease. Small amounts of a 25-kDa extracellular LasA-related protein, which could represent a potential processing intermediate, were also observed. To better understand the structure-function relationships in LasA protease, we tested whether His-120-X-His-122 in the mature portion of LasA plays a role in activity. This motif and surrounding sequences are conserved in the related beta-lytic protease of Achromobacter lyticus. Oligonucleotide-directed mutagenesis was used to change His-120 to Ala-120, thus forming the lasA5 allele. The product of lasA5 expressed from the chromosome of P. aeruginosa was processed to a stable, secreted 20-kDa protein (designated LasA-H120A) which was devoid of staphylolytic activity. This suggests that His-120 is essential for LasA activity and favors the possibility that proLasA processing and secretion in P. aeruginosa can proceed via mechanisms which do not involve autoproteolysis.

Amino Acid Sequence↗
PubMed1995

The elastase propeptide functions as an intramolecular chaperone required for elastase activity and secretion in Pseudomonas aeruginosa.

Several proteases are secreted by Pseudomonas aeruginosa including elastase, an abundantly secreted neutral zinc-metalloprotease. Elastase (encoded by lasB) is first synthesized with a relatively large propeptide (18 kDa) domain. Here, we present evidence that this propeptide functions as an intramolecular chaperone (IMC) essential for proper maturation of elastase into a hydrolytically active enzyme. An altered elastase allele (lasB6) that encoded an elastase precursor with a precise propeptide deletion was expressed in Escherichia coli, and disrupted cells contained only inactive elastase. However, co-expression of an allele (lasB7) expressing the propeptide as an independent, non-covalently linked protein rescued about one-third of the hydrolytic activity when compared with that obtained with wild-type lasB. Thus, the propeptide was essential for elastase activity and so defined elastase as an IMC-containing protease. We examined the possibility that the propeptide of elastase also plays a role in the localization of the mature protein past the outer bacterial membrane. Expression of lasB6 in P. aeruginosa (lasB delta) in the absence of the propeptide resulted in production of inactive elastase that accumulated within the cell and was not secreted to the culture medium. When lasB7 co-expressed the non-covalently linked propeptide in the same cell with lasB6, efficient secretion was restored and active elastase was then found in the supernatant. Thus, the propeptide was needed for secretion of the mature protein as well as enzymatic activity. This chaperone-like activity of the propeptide appears to involve a direct interaction between the mature and propeptide sequences, and evidence for this was obtained by demonstrating that the non-covalently attached 18 kDa propeptide was co-precipitated with elastase using elastase antibodies. These results are consistent with a hypothesis that the propeptide domain acts as an IMC to control both enzymatic activity and competence for secretion.

Base Sequence↗
PubMed1994

Type I procollagen COOH-terminal proteinase enhancer protein: identification, primary structure, and chromosomal localization of the cognate human gene (PCOLCE).

Type I procollagen COOH-terminal proteinase (C-proteinase) enhancer, a glycoprotein that binds to the COOH-terminal propeptide of type I procollagen and enhances procollagen C-proteinase activity, was purified from mouse fibroblast culture media. Partial amino acid sequences obtained from proteolytic fragments were found to have identity with the deduced amino acid sequence of a cDNA clone of unknown function, previously isolated from a mouse astrocyte library. Sequences of mouse enhancer cDNA, obtained in the present study, predict a approximately 50-kDa, 468-amino acid protein that differs from the 43-kDa, 402-amino acid protein predicted by the previously reported astrocyte-derived clone. Human cDNAs encode an enhancer of 449 amino acids. Previous biochemical studies have found the mouse enhancer as a 55-kDa form, which is readily processed to 36- and 34-kDa forms, retaining full C-proteinase enhancing activity and the ability to bind the COOH-terminal propeptide. Data presented here show the 36-kDa form to correspond to the amino-terminal portion of the 55-kDa protein. This is the most conserved region between mouse and human enhancers, comprising two domains with homology to domains found in a number of proteases and proteins with developmental functions. Such domains are thought to mediate interactions between proteins. Mouse enhancer RNA is shown to be at highest levels in collagen-rich tissues, especially tendon. The human enhancer gene, PCOLCE, is localized to 7q21.3-->q22, the same chromosomal region containing the type I collagen alpha 2 chain gene, COL1A2.

Amino Acid Sequence↗
PubMed1994

The propeptide of Pseudomonas aeruginosa elastase acts an elastase inhibitor.

Elastase, an extracellular protease of Pseudomonas aeruginosa, is synthesized as a preproenzyme containing a large amino-terminal propeptide. The propeptide is cleaved within the periplasm to form a noncovalent complex with the elastase moiety. The propeptide-elastase complex was purified from the cell extract of P. aeruginosa by affinity chromatography on Gly3-D-Phe-Sepharose. The purified fraction was proteolytically inactive and contained the propeptide-elastase complex as the major protein component. Activation by limited proteolysis with trypsin was associated with the disappearance of the propeptide. To correlate individual proteins in the preparation with proteolytic activity, the purified fraction was subjected to polyacrylamide gel electrophoresis under nondenaturing conditions and subsequent incubation of the separation gel over a skim milk-agarose-indicator gel. Clearing zones due to proteolysis were produced either by mature elastase (control) or the free processed periplasmic enzyme, a low level of which was present in the purified propeptide-elastase complex preparation. No clearing was evident with the propeptide-elastase complex, indicating inhibition by the bound propeptide. Proteolytic activity of mature elastase was inhibited by various Pseudomonas cell fractions. This inhibition was abolished by antipropeptide antibodies, and, as evident from immunoblotting analysis, was consistent with propeptide presence in the effective fraction, whole cell extract, cytosol, and one of the two periplasmic fractions obtained upon conversion of P. aeruginosa cells to spheroplasts. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electro-blotting of the various cell fractions onto nitrocellulose membranes followed by incubation of the membranes with elastase and subsequent probing with antielastase antibodies revealed elastase propeptide binding. This binding of mature elastase to the propeptide was prevented by antibodies to the propeptide but not by inhibitors of elastase activity. Thermolysin, a neutral metalloprotease homologous to elastase, was not recognized by the elastase propeptide. In addition, propeptide containing P. aeruginosa fractions that were inhibitory to elastase had no effect on thermolysin activity. We conclude that elastase propeptide functions as an elastase inhibitor. Inhibition is specific, effective against both periplasmic and mature elastase, and depends on enzyme propeptide binding.

Amino Acid Sequence↗
PubMed1994

Intraduct carcinoma of the breast (duct carcinoma in situ).

A retrospective study of non-invasive ductal carcinoma in situ in a series of women is presented. The epidemiology, clinical characteristics and results in 65 patients treated by mastectomy or breast-conserving surgery, with or without radiation therapy, are reported. The significant recurrence rate and high proportion of invasive cancers among patients with recurrences are noted. Recurrences were more frequent in patients with larger tumours and those with comedo-type intraduct carcinomas. Implications and guidelines for therapy are presented.

Adult↗
PubMed1993

Secreted LasA of Pseudomonas aeruginosa is a staphylolytic protease.

Full expression of the elastolytic phenotype of Pseudomonas aeruginosa depends on LasA, an extracellular protease with restricted specificity whose mode of action on elastin and biological role is not understood. LasA exhibits amino acid sequence homology to some bacteriolytic proteases and shares several physicochemical properties with the staphylolytic protease of P. aeruginosa. This led us to examine whether the two proteases are the same. Production of LasA and staphylolytic protease by prototrophic and lasA mutant strains of P. aeruginosa was investigated. The two prototrophic strains examined, PAO1 and FRD2, exhibited extracellular staphylolytic activity and secreted LasA. LasA mutants, PAO-E64 lasA1 (Ts), FRD2128 delta lasA, and FRD244 lasA::mTn10, did not exhibit staphylolytic activity. A low level of the LasA protein was detected in the culture filtrate of the temperature-sensitive lasA mutant PAO-E64, but none was detectable in those of the deletion and insertion mutants, FRD2128, and FRD244, respectively. The staphylolytic protease was purified from the culture filtrate of P. aeruginosa strain FRD2 by DEAE-cellulose chromatography. The purified enzyme hydrolyzed pentaglycine into the respective di- and tripeptides and reacted specifically with antibodies against a synthetic peptide identical in sequence to positions 77-98 in LasA. The amino-terminal sequence of the first 15 amino acid residues of the staphylolytic protease was found to be identical with that of the secreted LasA. These results clearly indicate that LasA is a staphylolytic protease. In addition to lysing staphylococci, it may enhance elastolysis by cleaving Gly-Gly bonds, which are abundant in elastin.

Amino Acid Sequence↗
PubMed1992

Identification of cleavage sites involved in proteolytic processing of Pseudomonas aeruginosa preproelastase.

The extracellular elastase (33 kDa) of Pseudomonas aeruginosa is synthesized as a 53.6 kDa preproenzyme containing a long, N-terminal propeptide. The free propeptide and the elastase precursor generated upon propeptide removal were isolated from P. aeruginosa cells and subjected to N-terminal amino acid sequence analysis. The results identified Ala-174 and Ala+1 as the amino terminal residues of the propeptide and the elastase precursor, respectively, indicating that: (1) the signal peptide consists of 23 amino acid residues and its molecular weight is 2.4 kDa, (2) the propeptide contains 174 amino acid residues and is of 18.1 kDa molecular weight, and (3) no additional N-terminal proteolytic cleavage is required for elastase maturation.

Amino Acid Sequence↗
PubMed1992

Exogenous nitric oxide stress on endothelial cells and macrophages.

Bovine endothelial cells (ECs, P1) and lipopolysaccharide/gamma-interferon-induced mouse macrophages (MMs) were incubated in the presence of SIN-1 and C 3754 (1 microM to 1 mM), sydnonimine metabolites of the antianginal predrugs molsidomine and pirsidomine, respectively up to 48 h. No change of the endogenous nitric oxide output from MMs and A23187- or adenosine triphosphate-stimulated ECs was found by means of the methemoglobin method. Data indicate that downregulation of the nitric oxide (NO) synthase is not obvious within the intact cells under exogenous NO stress supplied by high concentrations of the spontaneous NO donors. Cytosolic MM NO synthase extracts, however, revealed reduction in the enzymic [3H]arginine turnover to [3H]citrulline by SIN-1, but not by C 3786, the pharmacologically active metabolite of pirsidomine.

Amino Acid Oxidoreductases↗
PubMed1992

Unusual renal deposit in mixed connective tissue disease.

We describe a patient with clinical signs of mixed connective tissue disease who developed nephrotic syndrome. The kidney biopsy revealed glomerular and vascular deposits that stained positive with Congo red and showed a green birefringence. The Congo red positivity became negative in sections treated with potassium permanganate. Peroxidase-antiperoxidase staining with anti-AA antibodies was strongly positive. Ultrastructurally, although the deposits were similar to amyloid, they were about twice the size of amyloid fibrils. To our knowledge, the deposition of an amyloidlike material that shows the histochemical and immunohistochemical features of amyloid AA but is lacking the distinctive ultrastructural characteristics of amyloid fibrils has not been described.

Amyloid↗
PubMed1991

Substitution of active-site His-223 in Pseudomonas aeruginosa elastase and expression of the mutated lasB alleles in Escherichia coli show evidence for autoproteolytic processing of proelastase.

The neutral metalloprotease elastase is one of the major proteins secreted into the culture medium by many Pseudomonas aeruginosa strains. Encoded by the lasB gene, the 33-kDa elastase is initially synthesized as a 53-kDa preproenzyme which is processed to the mature form via a 51-kDa proelastase intermediate. To facilitate studies on proteolytic processing of elastase precursors and on secretion, we developed systems for overexpression of lasB in Escherichia coli under the control of the inducible T7 and tac promoters. Although the 51-kDa proelastase form was detectable in E. coli under inducible conditions, most of the elastase produced under these conditions was found in an enzymatically active 33-kDa form. The amino-terminal sequence of the first 15 amino acid residues of this 33-kDa elastase species was identical to that of the mature P. aeruginosa enzyme, suggesting that processing was autocatalytic. To test this possibility, the codon in lasB encoding His-223, a presumed active-site residue, was changed to encode Asp-223 (lasB1) and Tyr-223 (lasB2). The effects of these mutations on enzyme activity and processing were examined. No proteolytic or elastolytic activities were detected in extracts of E. coli cells containing the lasB mutant alleles. Overexpression of the mutated lasB genes in E. coli resulted in the accumulation of the corresponding 51-kDa proelastase species. These were processed in vitro to the respective 33-kDa forms by incubation with exogenous purified elastase, without an increase in proteolytic activity. Molecular modeling studies suggest that the mutations have little or no effect on the conformation of the mutant elastases. In addition, wild-type elastase and the mutant proelastases were localized to the periplasm of E. coli. The present results confirm that His-223 is essential for elastase activity and provide evidence for autoproteolytic processing of proelastase.

Alleles↗
PubMed1990

Analysis of site-directed mutations in human pro-alpha 2(I) collagen which block cleavage by the C-proteinase.

We have used site-directed mutagenesis to obtain human pro alpha 2(I) cDNAs containing novel mutations designed to inhibit cleavage at the C-proteinase site. Deletion of six relatively conserved amino acids which surround the cleavage site did not interfere with assembly of the triple helix in transfected rat cells, but blocked cleavage of the constituent mutated chains by endogenous C-proteinase. Substitution for a conserved Asp, which forms part of the Ala-Asp bond cleaved by C-proteinase, also blocked cleavage by endogenous C-proteinase. The conserved Asp is, therefore, a necessary component of the C-proteinase cleavage site. Incubation in vitro with a purified mouse C-proteinase, confirmed both mutations to be resistant to cleavage by high concentrations of the physiologically relevant enzyme. Mutant pro alpha 2(I) chains, resistant to cleavage by C-proteinase in culture media, were processed in cell layers by a different protease which cleaved telopeptide domains. Naturally occurring mutations at the C-proteinase site have not been described in human patients. The mutations characterized here, further define the C-proteinase cleavage site and provide reagents which may be informative when introduced into transgenic mice.

Bone Morphogenetic Protein 1↗
PubMed1990

Procollagen type I C-proteinase enhancer is a naturally occurring connective tissue glycoprotein.

Using antibodies to the procollagen C-proteinase enhancer of mouse fibroblast culture medium, we have screened by immunoblotting extracts of several post natal mouse and rat tissues for the presence of the enhancer antigen. All rodent connective tissues were relatively rich in enhancer; lower amounts were found in skeletal muscle and heart and essentially no enhancer was detected in kidney, liver or brain. The amounts of enhancer in mouse tendon and calvaria extracts were age related, with highest amounts in 11 and 19 d tendons and in 1 d calvaria-the times of rapid growth of these organs. The results suggest that procollagen C-proteinase enhancer is a specific connective tissue glycoprotein that is likely to regulate procollagen processing in vivo.

Animals↗
PubMed1990

Free proximal trisomy 21 in the mother and malformation syndrome in the son.

We report on a new case of duplication of the proximal part of the long arm of chromosome 21. The proposita presents normal mental development, no trisomy 21 manifestations; on the contrary, she had a few monosomy 21-like stigmata. She gave birth to a severely malformed infant with a pattern of malformations suggesting a partial 21-monosomy syndrome, but with a 46,XY normal karyotype in his peripheral blood lymphocytes. The findings are explained in the following way: the infant probably had originally a 47,XY,+21q- karyotype like his mother. Post zygotic nondisjunctional events produced a prevalent 46,XY,21q- line responsible for the severe malformations and the normal 46,XY line found in his blood lymphocytes.

Abnormalities, Multiple↗
PubMed1990

Preferential uptake of a water-soluble phthalocyanine by atherosclerotic plaques in rabbits.

This study is the first demonstration of preferential accumulation of a water soluble phthalocyanine dye in atheromatous plaques in the rabbit. Two groups of rabbits with diet-induced atheromatous plaques were killed 4 and 24 h following intravenous administration of copper phthalocyanine tetrasulfonate. Uptake of the dye by plaque-containing and normal appearing aortae was evaluated macroscopically and quantitatively by extraction of the dye from the tissues. The concentration of the dye in the atheromatous plaques was 2.6 and 1.7 times higher than in the normal vessel wall at 4 and 24 h, respectively. The concentration of the dye in normal appearing aortae in the 2 study groups was similar to that of aortae of control rabbits which were fed a normal diet and exposed to the dye for the same time periods. We conclude that copper phthalocyanine accumulates preferentially in atheromatous plaques in rabbits. These findings provide a basis for the utilization of phthalocyanines for plaque identification and for photodynamic therapy of atherosclerosis.

Animals↗