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PubMed1993

Influence of hydration states on the acute nephrotoxic effect of gentamicin in the rat.

The influence of acute water loading and water deprivation on gentamicin-induced nephrotoxicity was examined in rats by administering the drug (50 mg kg-1) into a group of hydrated rats and another 24 h-dehydrated group. Renal damage was assessed by measuring urinary enzymes, protein, urine flow rate and cell excretion in urine. Water loading alone caused significant alterations (2-5-fold) of the parameters under observation while these parameters were either lower or of comparable values to the untreated controls in water-deprived rats. Gentamicin administration further resulted in the elevation of all parameters in water-loaded rats. However, elevated alkaline phosphatase (P < 0.01), similar excretion of muramidase and decreased excretion of N-acetyl-beta-D-glucosaminidase compared to the controls was observed in water-deprived rats given gentamicin. Cells excreted in the urine were mainly renal tubular and squamous epithelial types in the control and all treated rats. These cells contain between 2 and 10% of the total enzyme activity in the urine of untreated-control rats. Increases in the percentage of these enzymes were observed as follows: in water-loaded (12-24%); water-deprived (4-26%); gentamicin alone (16-27%); water-loaded rats given gentamicin (11-30%); and water-deprived rats receiving gentamicin (8-15%). These observations demonstrate that the toxicity of gentamicin could be aggravated by both water loading and water deprivation. Water load alone is also associated with physiopathological changes in the kidney cells.

Animals↗
PubMed1988

Bacteriophages of lactobacilli.

Lactobacilli are members of the bacterial flora of lactic starter cultures used to generate lactic acid fermentation in a number of animal or plant products used as human or animals foods. They can be affected by phage outbreaks, which can result in faulty and depreciated products. Two groups of phages specific of Lactobacillus casei have been thoroughly studied. 1. The first group is represented by phage PL-1. This phage behaves as lytic in its usual host L. casei ATCC 27092, but can lysogenize another strain, L. casei ATCC 334. Bacterial receptors of this phage are located in a cell-wall polysaccharide and rhamnose is the main component of the receptors. Ca2+ and adenosine triphosphate (ATP) are indispensable to ensure the injection of the phage DNA into the bacterial cell. The phage DNA is double-stranded, mostly linear, but with cohesive ends which enables it to be circularized. The vegetative growth of PL-1 proceeds according to the classical mode. Cell lysis is produced by an N-acetyl-muramidase at the end of vegetative growth. 2. The second group is represented by the temperate phage phi FSW of L. casei ATCC27139. It has been shown how virulent phages originate from this temperate phage in Japanese dairy plants. The lysogenic state of phi FSW can be altered either by point mutations or by the insertion of a mobile genetic element called ISL 1, which comes from the bacterial chromosome. This is the first transposable element that has been described in lactobacilli. Lysogeny appears to be widespread among lactobacilli since one study showed that 27% of 148 strains studied, representing 15 species, produced phage particles after induction by mitomycin C. Similarly, 23 out of 30 strains of Lactobacillus salivarius are lysogenic and produce, after induction by mitomycin C, temperate phages, killer particles, or defective phages. Temperate phages have also been found in 10 out of 105 strains of Lactobacillus bulgaricus or Lactobacillus lactis after induction by mitomycin C. Phages so far studied of the latter 2 and closely related lactobacilli, either temperate or isolated as lytic, may be divided into 4 unrelated groups called a, b, c and d. Most of these phages are found in group a and an unquestionable relationship has already been shown between lytic phages and temperate phages that belong to this group. Lytic phage LL-H of L. lactis LL 23, isolated in Finland, is one of the most representative of those of group a and has been extensively studied on the molecular level.

Bacteriophages↗
PubMed1989

Endochitinase from Aspergillus nidulans implicated in the autolysis of its cell wall.

An endochitinase from centrifuged autolyzed cultures of Aspergillus nidulans has been purified 100 times. The enzyme has Mw 27,000, pI of 4.8 units, pH optimum around 5 pH units. It is unstable at temperature greater than 70 degrees C and does not have a cation requirement. It is inhibited by Hg2+, Cu2+, Ca2+ and Ag+ and it does not have muramidase activity. The enzyme depolymerizes chitin rapidly with production of high molecular weight polysaccharides, and then slowly degrades these with production of N,N'-diacetylchitobiose. The enzyme hydrolyzes N,N',N''-triacetylchitotriose with production of N,N'-diacetylchitobiose and N-acetylglucosamine and this hydrolysis is inhibited by other chitin oligomers and N-acetylglucosamine. This enzyme hydrolyzes in the same way the chitin obtained from the cell wall of Aspergillus nidulans.

Aspergillus nidulans↗
PubMed1996

Cloning, sequence analysis, and expression of the genes encoding lytic functions of Bacteriophage phi g1e.

The lysis genes of a Lactobacillus phage phi g1e were cloned, sequenced, and expressed in Escherichia coli. Nucleotide sequencing of a 3813-bp phi g1e DNA revealed five successive open reading frames (ORF), Rorf50, Rorf118, hol, and lys and Rorf175, in the same DNA strand. By comparative analysis of the DNA sequence, the putative hol product (holin) has an estimated molecular weight is 14.2 kDa, and contains two potential transmembrane helices and highly charged N- and C-termini, resembling predicted holins (which are thought to be a cytoplasmic membrane-disrupting protein) encoded by other phages such as mv1 from Lactobacillus bulgaricus, phi adh from Lactobacillus gasseri, as well as monocins from Listeria. On the other hand, the putative phi g1e lys product (lysin) of 48.4 kDa shows significant similarity with presumed muramidase, known as a cell wall peptidoglycandegrading enzyme, encoded by the Lactobacillus phage mv1 and phi adh, the Lactococcus lactis phage phi LC3, and the Streptococcus pneumoniae phages Cp-1, Cp-7 and Cp-9. When expressed in E. coli, the phi g1e lysin and/or holin decreased the cell turbidity significantly, suggesting that the phi g1e hol-lys system is involved in cytolytic process.

Amino Acid Sequence↗
PubMed1993

Discrimination between virulent and nonvirulent Streptococcus suis type 2 strains by enzyme-linked immunosorbent assay.

Discrimination between virulent and nonvirulent strains of Streptococcus suis type 2 will allow proper diagnosis of diseased pigs and the identification of carrier pigs. To discriminate between virulent and nonvirulent strains, we developed two double antibody sandwich (DAS) enzyme-linked immunosorbent assays (ELISA) using specific monoclonal antibodies directed against two virulence markers of S. suis type 2. One mAb was directed against the 136-kilodalton (kDa) cell-wall-associated protein, designated muramidase-released-protein (MRP). The other mAb was directed against a 110-kDa extracellular protein, provisionally called extracellular factor (EF). We examined 179 strains of S. suis serotype 2, 22 strains of S. suis serotypes 1 to 22, 22 other streptococci, 20 other bacterial strains, and one yeast. The ELISA results were almost identical with western blot analysis of these strains. Visual readings of the two DAS-ELISAs were enough to discriminate accurately between the three phenotypes of S. suis type 2. We concluded that the two DAS-ELISAs are reliable, rapid, and simple assays to identify virulent strains of S. suis type 2.

Animals↗
PubMed1990

Covalent linkage between the capsular polysaccharide and the cell wall peptidoglycan of Streptococcus pneumoniae revealed by immunochemical methods.

The attachment of capsular polysaccharide to Streptococcus pneumoniae was examined using monoclonal and polyclonal antibodies. Among the strains examined, the capsular polysaccharide of types 2, 4, 6A, 6B, 7F, 8, 14, 19F and 23F was bound to the pneumococci whereas that of a type 3 strain was not. Sequential treatment with 2% SDS at 100 degrees C, pronase, and EDTA did not dissociate the capsular polysaccharide from the pneumococci. Treatment of the cells with mutanolysin, a muramidase that degrades the cell wall peptidoglycan of pneumococci and other streptococci, released both the capsular and the cell wall C-polysaccharide (C-Ps). Type 6A capsular polysaccharide released from cell walls by mutanolysin treatment, was fractionated by high performance liquid chromatography and examined by immunoelectrophoresis. It was found to be bound to both the C-Ps and the peptidoglycan. The bond between the capsular polysaccharide and the peptidoglycan has not yet been identified but is probably covalent, as the two components could not be dissociated after boiling in SDS. Based on our studies with type 6A, we propose that capsular polysaccharide and C-Ps of the pneumococcus are linked to the peptidoglycan at different sites and, thereby, indirectly to each other. Studies in mice showed that the peptidoglycan enhanced the serum antibody response to C-Ps but not to type 6A polysaccharide.

Amino Acids↗
PubMedDate not supplied

Murein chemistry of cell division in Escherichia coli.

The length distribution of the glycan strands of murein has been analysed with a novel method in filamentous and spherical cells of Escherichia coli, as well as during septum formation and cell separation. A shift to the longer glycan strands was observed in the murein of furazlocillin-induced filaments. In contrast, shorter glycan strands were increased in the murein of mecillinam-induced spherical cells. During septum formation in a chain-forming envA mutant that is defective in the splitting process of the septum, a shift to the shorter glycan strands was detected that was not seen in wild type E. coli cells. It is concluded that septum-specific murein structures of rather short glycan strands are released during splitting of the septum. This intermediate material remains present in the septum of the envA mutant. The splitting process of the septum was investigated by analysing the murein during penicillin-induced bacteriolysis, which is known to take place by strictly localized murein degradation in the equatorial zone of the cell. No changes in the length distribution of the glycan strands could be detected during penicillin-induced lysis, with the exception of an increase in disaccharides, the shortest glycan strands possible. This is explained by the action of exo-muramidases progressively digesting glycan strands, leaving disaccharide units covalently linked to the remaining murein at the sites of murein cross-linkage. It is proposed that this "zipper-like" mechanism represents the normal cutting process of the septum during cell separation.

Cell Division↗
PubMed1994

'Holy' proteins. II: The soluble lytic transglycosylase.

Enzymes involved in the metabolism of the bacterial cell wall peptidoglycan are excellent targets for antibiotics. Penicillins and related beta-lactam antibiotics inhibit the enzymes that act on the peptide cross-links of the peptidoglycan. The X-ray structure of the transglycosylase revealed a two-layered ring of alpha-helices in a right-handed superhelical arrangement with a separate catalytic domain on top, which resembles the fold of goose-type lysozyme. Three sequence motifs were found that characterize the catalytic and substrate-binding sites in the enzyme. These motifs are present in a broad family of muramidases and chitinases.

Amino Acid Sequence↗
PubMedDate not supplied

Central granular cell odontogenic tumor: immunohistochemistry and ultrastructure.

Central granular cell odontogenic tumors are rare, with only 30 cases having been reported. The tumors usually occur in the mandibular molar area and are seen as localized painless swellings in patients older than 40 years. We report an additional case that occurred in the posterior mandible of an elderly black woman. All reported cases of this tumor are benign, and cure is effected by localized surgical excision. Ultrastructurally, the cells contain numerous lysosomes and phagocytic vacuoles. Immunohistochemically, the granular cells were positive for vimentin, CD68, muramidase, carcinogenic embryonic antigen, and bcl-2. These features support a mesenchymal origin with a possible histiocytic lineage for the granular cells. Awareness of the occurrence of this neoplasm is important to promote detection and differentiation from other intraoral granular cell lesions.

Aged↗
PubMed2004

Inhibition of membrane-bound lytic transglycosylase B by NAG-thiazoline.

The lytic transglycosylases cleave the bacterial cell wall heteropolymer peptidoglycan with the same specificity as the muramidases (lysozymes), between the N-acetylmuramic acid and N-acetylglucosamine residues, with the concomitant formation of a 1,6-anhydromuramoyl residue. The putative catalytic residue in the family 3 lytic transglycosylase from Pseudomonas aeruginosa, Glu162 as identified by sequence alignment to the homologous enzyme from Escherichia coli, was replaced with both Ala and Asp by site-directed mutagenesis. Neither mutant enzyme differed structurally from the wild-type enzyme, as judged by CD spectroscopy, but both were enzymatically inactive confirming the essential role of Glu162 in the mechanism of action of this lytic transglycosylase. The beta-hexosaminidase inhibitor NAG-thiazoline was shown to inhibit the activity of lytic transglycosylase activity, thus providing the first direct evidence that the formation of the 1,6-anhydromuramoyl residue may proceed through an oxazolinium ion intermediate involving anchimeric assistance. Using surface plasmon resonance and difference absorbance spectroscopy, Kd values of 1.8 and 1.4 mM, respectively, were determined for NAG thiazoline, while its parent compound N-acetylglucosamine neither inhibited nor appeared to bind the lytic transglycosylase with any significant affinity.

Acetylglucosamine↗
PubMed2005

Molecular properties of the two-component cell lysis system encoded by prophage phigaY of Lactobacillus gasseri JCM 1131T: cloning, sequencing, and expression in Escherichia coli.

Shotgun cloning of the Lactobacillus gasseri JCM 1131T whole DNA yielded two recombinant plasmids, p118gaY1 and p118gaY2, which directed cell lysis activity. Sequencing analysis revealed that the two plasmids carried almost identical inserted genes in following orders (truncated genes, in parentheses): in p118gaY1, (orf149)-orf92-holgaY-lysgaY-orf35-attL-(mnaAgaY1); in p118gaY2, (orfXgaY1)-orf169-orf149-orf92-holgaY-lysgaY-orf35-attP-(intgaY). The lysgaY-encoded protein (designated as LysgaY, 33.7 kDa) showed significant homology with putative muramidases (peptidoglycan-degrading enzyme) of the Lactobacillus phage phiadh, Lj965, Lj928, LL-H, mv4, and mv1. By zymogram analysis, LysgaY overproduced in Escherichia coli exhibited lytic activity towards 17 Gram-positive bacterial strains, including lactobacilli, lactococci, and staphylococci. The holgaY-encoded protein (15.7 kDa) contained three potential transmembrane helices, resembling putative holins (cytoplasmic membrane-disrupting protein) of Lj928 and Lj965. On the other hand, another clone p118gaYR obtained by EcoRI-shotgun cloning carried the (ptsCgaY1)-attR-(intgaY) genes. Three sequences, attL, attP, and attR, had a 47-bp common (core) sequence, and the core of attR was located in 3' region of a potential tRNA(Arg) gene. These results suggested that (i) attL and attR are phage-host junctions, left- and right-arms, respectively, (ii) attP is a phage attachment site, and (iii) intgaY is an integrase gene for phage integration and/or excision. After mitomycin C-induction, phage particles were demonstrated by electron microscopy. The prophage (phigaY) is somewhat leaky in the host, and has the two-component lysis system (HolgaY-LysgaY), closely resembling that of Lj928 as well as Lj965.

Amino Acid Sequence↗
PubMed2004

Bactericidal cationic peptides can also function as bacteriolysis-inducing agents mimicking beta-lactam antibiotics?; it is enigmatic why this concept is consistently disregarded.

Although there is a general consensus that highly cationic peptides kill bacteria primarily by injuring their membranes, an additional hypothesis is proposed suggesting that a large variety of cationic peptides might also render bacteria non viable by activating their autolytic wall enzymes - muramidases (a "Trojan Horse" phenomenon), resulting in bacteriolysis. This group of cationic peptides includes: lysozyme, lactoferrin, neutrophil-derived permeability increasing peptides, defensins, elastase, cathepsin G, and secretory phopholipase A2. In this respect, cationic peptides mimic the bactericidal/bacteriolytic effects exerted by of beta-lactam antibiotics. Bacteriolysis results in a massive release of the pro-inflammatory cell-wall components, endotoxin (LPS), lipoteichoic acid (LTA) and peptidoglycan (PPG), which if not effectively controlled, can trigger the coagulation and complement cascades, the release from phagocytes of inflammatory cytokines, reactive oxygen and nitrogen species, and proteinases. Synergism (a "cross-talk") among such agonists released following bacteriolysis, is probably the main cause for septic shock and multiple organ failure. It is proposed that a use of bacteriolysis-inducing antibiotics should be avoided in bacteremic patients and particularly in those patients already suspected of developing shock symptoms as these might further enhance bacteriolysis and the release of LPS, LTA and PPG. Furthermore, in additonal to the supportive regimen exercised in intensive care settings, a use of non bacteriolysis-inducing antibiotics when combined with highly sulfated compounds (e.g. heparin, and other clinically certified polysufates) should be considered instead, as these might prevent the activation of the microbial own autolytic systems induced either by highly cationic peptides released by activated phagocytes or by the highly bacteriolytic beta-lactams. Polysulfates might also depress the deleterious effects of the complement cascade and the use of combinations among anti-oxidants ( N-acetyl cysteine), proteinase inhibitors and phospholipids might prove effective to depress the synergistic cytotoxic effects induced by inflammatory agonists. Also, a use of gamma globulin enriched either in anti-LPS or in anti-LTA activities might serve to prevent the binding of these toxins to receptors upon macrophage which upon activation generate inflammatory cytokines. Thus, a use of "cocktails" of anti-inflammatory agents might replace the unsuccessful use of single antagonists proven in scores of clinical trials of sepsis to by ineffective in prolonging the lives of patients. It is enigmatic why the concept, and the publications which support a role for cationic peptides also as potent inducers of bacteriolysis, an arch evil and a deleterious phenomenon which undoubtedly plays a pivotal role in the pathophysiology of post-infectious sequelae, has been consistently disregarded.

Anti-Bacterial Agents↗
PubMed2006

Virulence-associated gene profiling of Streptococcus suis isolates by PCR.

Definition of virulent Streptococcus suis strains is controversial. One successful approach for identification of virulent European strains is differentiation of capsular serotypes (or the corresponding cps types) and subsequent detection of virulence-associated factors, namely the extracellular factor (EF, epf), the muramidase-released protein (MRP, mrp) and the hemolysin suilysin (SLY, sly). In this work we present a novel multiplex PCR (MP-PCR) and an mrp variant PCR for identification and characterization of virulent S. suis strains. These new methods were used to identify association of disease with particular profiles of virulence-associated genes. The MP-PCR allowed identification of S. suis through detection of the housekeeping gene gdh, differentiation of four cps types (1, 2, 7 and 9), and detection of epf, mrp, sly and arcA (arginine deiminase from S. suis). Furthermore, this study describes the first PCR assay for differentiation of at least six mrp variants. Expression of the corresponding size variants of MRP was shown for four of the six mrp variants, but was undetectable for the two larger mrp variants in the particular strains investigated. The results of this study suggest that cps7 strains are associated with pneumonia and that variation of mrp is very pronounced among these strains. Gene profiles of invasive, pneumonia and carrier S. suis isolates by combination of PCR assays allowed differentiation of 24 different genotypes among cps1, 2, 7 and 9 strains. Forty-five percent of the invasive S. suis diseases investigated in this study were caused by only two of these genotypes, namely cps2/mrp+/epf+/sly+ and cps9/mrp(*)/epf-/sly+. Thus, this study demonstrates for the first time a uniform profile of the particular virulence-associated genes for the vast majority of the investigated invasive cps9 strains.

Animals↗
PubMed1983

Immunoassay of enzymes--an overview.

Immunochemical techniques with enzymes as the antigen have grown in frequency during the last few years. These techniques have allowed evaluation of enzymes in the presence of endogenous inhibitors. Among those enzymes measured by immunochemical techniques and which have found diagnostic application, mention will be made of alkaline phosphatase (with particular reference to the intestinal, placental, and Regan isoenzymes), lactate dehydrogenase (in which renewed interest has developed due to techniques for specifically measuring the LD-1 isoenzyme), aspartate aminotransferase (of which the cytosolic and mitochondrial forms can now be independently measured by immunochemical techniques), acid phosphatase (for which a specific immunochemical assay for the prostatic enzyme has been widely introduced in diagnostic laboratories), and creatine kinase (for which a variety of immunochemical techniques to measure the M- and B-subunits are now part of standard laboratory assays). Other enzymes which will be discussed in this review include phosphohexose isomerase, amylase, ribonuclease, and lysozyme (muramidase). Finally, the use of enzymes, particularly asparaginase, in the chemotherapy of cancer will be outlined.

Animals↗
PubMed1991

An immunohistochemical study of spontaneous histiocytic tumours in the rat.

Immunocytochemistry was used to examine 18 cases of rat fibrous histiocytic tumours (11 malignant; seven benign). The diagnosis was made by light microscopic criteria and all cases were categorized as pleomorphic-storiform. A selection of polyclonal antibodies to histiocytic, muscle, neural and mesenchymal antigens was used. Fifteen tumours were positive with alpha 1-antitrypsin, four with alpha 1-chymotrypsin, ten with muramidase, five with desmin, 15 with neuron-specific enolase, 14 with S100, one with glial fibrillary acid protein and 12 with vimentin. Many tumours expressed several antigens, highlighting the confusion which has arisen with regard to the histiogenesis of fibrous histiocytic tumours in man, and supporting the concept of differentiation from a primitive mesenchymal common precursor able to differentiate in several directions.

Animals↗
PubMed1981

Nonlymphoid hematopoietic malignant disease of the lymph nodes.

The extension of myeloproliferative malignant tumors to lymph nodes was studied in seven excision biopsies and 27 autopsies in which lymph nodes were obtained from all nodal regions. We observed the proliferation of poorly, partially, and well differentiated neoplastic cells with a predominance of immature cells of the granulocytic series and fewer cells of the erythroid and megakaryocytic cells lines. Disturbances of the lymph node architecture consisted of invasion by abnormal cells of the trabecular stroma, disrupting the reticulin mesh and extending toward the pericapsular area. In the lymph node a loose network of thin and thick fibers replaced the original framework. Generalized lymph node involvement at autopsy showed preservation of the architecture, both in cases with total replacement of the lymphoid population by abnormal cells and in cases with involvement by single cells or small groups of cells. Partial involvement occurred chiefly in the medullary zone, probably the result of hematogenous dissemination from the vascular plexus in medullary cords. Further abnormal cell development was linked to trabecular infiltration. The extensive involvement seen at autopsy took place by lymphatic dissemination. In lymph nodes studied at autopsy an abnormal immunoblastic reaction was observed. At first these abnormal cells were suspected of representing the myeloproliferative malignant disease, but the application of special staining techniques revealed their polyclonal cytoplasmic immunoglobulins. The chloroacetate esterase stain and the immunohistochemical stains for muramidase and hemoglobulin were especially useful in demonstrating that myeloproliferative diseases develop in the environment found in lymph nodes.

Adult↗
PubMed1987

Recurrent "syncytial variant" of Hodgkin's disease: an immunohistologic diagnosis.

A case of recurrent Hodgkin's disease of the "sarcomatoid" or "syncytial variant" type was seen that occurred as an extension from the mediastinum to a previously uninvolved extranodal site (breast) and pericardium after treatment of classical nodular sclerosing Hodgkin's disease based in the lymph nodes. This histologic variant was composed of sheets of large, undifferentiated neoplastic cells with few, if any, diagnostic features of nodular sclerosing Hodgkin's disease. For this reason, the differential diagnosis of this variant was difficult and included non-Hodgkin's lymphoma (peripheral T-cell lymphoma), Ki-1-positive lymphoma, medullary carcinoma, metastatic carcinoma, melanoma, and granulocytic sarcoma. Immunologic analysis by immunoperoxidase technique showed a phenotype consistent with "syncytial variant" Hodgkin's disease: Leu-M1+, Ki-1+, IL-2+, HLA-DR+, T11-, pan B-, K-, lambda-, cytokeratin-, S-100-, muramidase-.

Adult↗
PubMed1999

Amplification of fluorescently labelled DNA within gram-positive and acid-fast bacteria.

Representative organisms from a variety of Gram-positive genera were subjected to varying regimes in order to optimise the intracellular amplification of DNA. The bacteria were subjected to treatments with paraformaldehyde, muramidases and mild acid hydrolysis to discover which regime made each organism permeable to the amplification reagents yet allowed retention of the fluorescein-labelled amplified products within the cell. Scanning electron micrographs were used to corroborate the effectiveness of the treatments, as seen by fluorescent photomicrographs, with the damage caused to the bacterial walls. A combination of mutanolysin and lysozyme was found most effective for Bacillus cereus, whereas permeabilisation of Streptomyces coelicolor, Lactococcus lactis and Clostridium sporogenes was most effective when exposed to lysozyme only. Surprisingly, direct amplification with no pre-treatment gave the brightest fluorescence in Mycobacterium phlei. Comparing the techniques of whole cell PCR, primed in situ labelling (PRINS), and cycle PRINS showed that under the conditions used the strongest intensity of fluorescence was obtained with in situ PCR; only L. lactis and M. phlei produced signals with cycle PRINS, fluorescence was not seen for any of the organisms with PRINS.

DNA, Bacterial↗