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PubMed2001

Relatedness of Streptococcus suis isolates of various serotypes and clinical backgrounds as evaluated by macrorestriction analysis and expression of potential virulence traits.

We evaluated the genetic diversity of Streptococcus suis isolates of different serotypes by macrorestriction analysis and elucidated possible relationships between the genetic background, expression of potential virulence traits, and source of isolation. Virulence traits included expression of serotype-specific polysaccharides, muramidase-released protein (MRP), extracellular protein factor (EF), hemolysin activity, and adherence to epithelial cells. Macrorestriction analysis of streptococcal DNA digested with restriction enzymes SmaI and ApaI allowed differentiation of single isolates that could be assigned to four major clusters, named A1, A2, B1, and B2. Comparison of the genotypic and phenotypic features of the isolates with their source of isolation showed that (i) the S. suis population examined, which originated mainly from German pigs, exhibited a genetic diversity and phenotypic patterns comparable to those found for isolates from other European countries; (ii) certain phenotypic features, such as the presence of capsular antigens of serotypes 2, 1, and 9, expression of MRP and EF, and hemolysin activity (and in particular, combinations of these features), were strongly associated with the clinical background of meningitis and septicemia; and (iii) isolates from pigs with meningitis and septicemia showed a significantly higher degree of genetic homogeneity compared to that for isolates from pigs with pneumonia and healthy pigs. Since the former isolates are considered highly virulent, this supports the theory of a clonal relationship among highly virulent strains.

Animals↗
PubMed2006

Proteins of the Rpf (resuscitation promoting factor) family are peptidoglycan hydrolases.

The secreted Micrococcus luteus protein, Rpf, is required for successful resuscitation of dormant "non-culturable" M. luteus cells and for growth stimulation in poor media. The biochemical mechanism of Rpf action remained unknown. Theoretical predictions of Rpf domain architecture and organization, together with a recent NMR analysis of the protein structure, indicate that the conserved Rpf domain has a lysozyme-like fold. In the present study, we found that both the secreted native protein and the recombinant protein lyse crude preparations of M. luteus cell walls. They also hydrolyze 4-methylumbelliferyl-beta-D-N,N',N''-triacetylchitotrioside, a synthetic substrate for peptidoglycan muramidases, with optimum activity at pH 6. The Rpf protein also has weak proteolytic activity against N-CBZ-Gly-Gly-Arg-beta-naphthylamide, a substrate for trypsin-like enzymes. Rpf activity towards 4-methylumbelliferyl-beta-D-N,N',N''-triacetylchitotrioside was reduced when the glutamate residue at position 54, invariant for all Rpf family proteins and presumably involved in catalysis, was altered. The same amino acid substitution resulted in impaired resuscitation activity of Rpf. The data indicate that Rpf is a peptidoglycan-hydrolyzing enzyme, and strongly suggest that this specific activity is responsible for its growth promotion and resuscitation activity. A possible mechanism of Rpf-mediated resuscitation is discussed.

Bacterial Proteins↗
PubMed1984

Immunohistochemical demonstration of leucocyte elastase in human tissues.

A polyclonal antiserum, produced in sheep and reactive against purified human leucocyte elastase, has been applied to paraffin sections from a range of human tissues by means of the indirect immunoperoxidase method. Striking and consistent results have been obtained. Normal or inflammatory granulocytes were intensely positive, the reaction being "blocked" by pure elastase. Activity was not seen in other sites, including histiocytic reticulum and lymphoid cells, although some weaker reaction was present in gastric and ileal lining epithelium. Strong reactivity was also seen in the cells of acute and chronic myeloid leukaemia and in extramedullary haemopoiesis. The advantages of this technique are compared with those for muramidase (lysozyme), alpha 1-antitrypsin, and naphthol-AS-D-chloroacetate esterase.

Granulocytes↗
PubMed1991

Chronic myelomonocytic leukaemia associated with T cell receptor delta gene rearrangement.

Morphological, immunophenotypic, and genetic analyses were carried out on peripheral blood, bone marrow, and pharyngeal biopsy material from a patient with chronic myelomonocytic leukaemia (CMML). Morphological analysis of bone marrow was diagnostic of CMML; immunophenotypic analysis of peripheral blood and bone marrow were negative for B and T cell antigens, and immunochemistry performed on the pharyngeal extramedullary infiltrate showed the presence of large monocytoid cells which stained positively for muramidase. Genotypic analysis, however, showed clonal rearrangement of the T cell receptor (TCR) delta chain gene, a marker of T cell or, less commonly, B cell lymphoid neoplasms. Other TCR genes, beta and gamma, were germline in all tissues examined. TCR delta is rearranged in precursor B cell and most T lymphoid neoplasms. A small proportion of cases (10%) of acute myeloid leukaemia (AML) also show rearrangement of the TCR delta gene. To date TCR delta rearrangement has not been described in CMML. The aberrant TCR delta rearrangement shown in this patient with CMML provides further evidence of the clonal nature of this disorder.

Bone Marrow↗
PubMed2000

Histiocytic lymphoma presenting as a testicular tumour and terminating in acute monoblastic leukaemia.

A 58 year old man presented in 1995 with a swollen testicle. After orchidectomy, a diagnosis of poorly differentiated lymphoma was made. Lymphoid, epithelial, and seminoma markers were all negative. Six months later he developed a buccal lesion, which was biopsied and reported as a high grade non-Hodgkin's lymphoma. It responded completely to chemotherapy but within a year he developed a forearm swelling, which was biopsied and imprints made before fixation of the material. Immunocytochemistry on the imprints showed positivity with antibodies to CD4, CD68, and muramidase, and the non-specific esterase cytochemical stain was strongly positive, leading to a diagnosis of true histiocytic lymphoma. Despite further treatment, the patient entered a terminal acute leukaemic phase, the blasts marking as monoblasts. Review of all the biopsies, including molecular investigations and further immunohistochemistry studies performed retrospectively on the original biopsy, demonstrated that this was the same malignant cell line throughout, and we conclude that this is a case of histiocytic lymphoma, initially presenting as a testicular tumour and terminating in acute monoblastic leukaemia. A diagnosis of histiocytic lymphoma should be considered when lymphoid markers are negative in an apparent lymphoma, but should not be made without recourse to appropriate immunophenotypic and molecular studies.

Disease Progression↗
PubMed1985

Isolation and characterization of the peptidoglycans from selected gram-positive and gram-negative periodontal pathogens.

The peptidoglycans from several Gram-negative and Gram-positive periodontal pathogens were isolated, purified, and characterized both morphologically and chemically. In addition, the effects of the mureolytic enzymes, lysozyme, M-1 N-acetyl-muramidase, and the AM-3 endopeptidase, on the peptidoglycans were examined. These enzymes were found to be highly effective in the degradation of the purified peptidoglycans; however, a Bacteroides capillus peptidoglycan-protein complex exhibited a greater resistance to these enzymes. Morphologically, the peptidoglycans consisted of large saccular sheets which, when viewed by scanning electron microscopy, contained numerous holes and tears. Chemically, the peptidoglycans consisted of muramic acid, glucosamine, alanine, glutamic acid, and meso-diaminopimelic acid (DAP). One Bacteroides species, Bacteroides gingivalis strain W, contained glycine and LL-DAP, suggestive of an indirectly cross-linked A3 gamma peptidoglycan.

Actinomyces↗
PubMed1985

Multivacuolated cells in human cartilage canals.

Light and electron microscopic examination of cartilage canals in 39 infants (from fetuses to 2 years of age) revealed the presence of multivacuolated cells in the connective tissue of the canals. These cells showed an irregular and electron-dense nucleus and cytoplasm; the latter was almost filled by large, irregular, membrane-bound vacuoles without or with hardly apparent content. Histochemistry revealed a weakly positive stain with periodic-acid-Schiff, oil-red-o and muramidase techniques in these cells which seem to be degenerated macrophages.

Bone and Bones↗
PubMed1976

Characterization of blast cells in acute nonlymphoid leukemias by consecutive cytochemical reactions.

Blast cells of 12 acute nonlymphoid leukemias have been investigated by consecutive application on the same preparation of three cytochemical reactions: alpha-naphthylacetate esterase, AS-D-choroacetate esterase and PAS. A fourth reaction (alpha-naphthylbutyrate esterase) was added in some preparations for a more detailed definition of the monocytoid-oriented cells. The cytochemical techniques were further combined with NaF inhibition and with the cytobacterial test for muramidase. The combined cytochemical approach allowed the identification of most of the blast cells, according to an empirically determined positivity to one or more reactions, and proved to be particularly valuable in classifying the cells after chemotherapy.

Acetates↗
PubMed1987

Autologous blood perfusion for myocardial protection during coronary angioplasty: a feasibility study.

During coronary angioplasty, inflation of the balloon within the coronary artery produces transient arterial occlusion and frequently results in myocardial ischemia. Delivery of oxygenated autologous blood to the myocardium at risk during inflation may help mitigate this ischemia. Accordingly, we investigated the feasibility and safety of infusing blood through the central lumen of a dilatation catheter around the guidewire using both a model in vitro and clinical trials. In the tests in vitro, fresh blood was infused at flow rates up to 120 ml/min. Hemolysis was minimal at flow rates of 60 ml/min or less (less than or equal to 0.92 +/- 0.18%), but increased exponentially at higher rates (13.64 +/- 2.37% at 120 ml/min, p less than .002). A similar pattern was observed for potassium release. Platelet and leukocyte counts did not vary significantly, and beta-thromboglobulin and muramidase remained at control levels. Although mean erythrocyte volume did not change, erythrocyte histograms and light microscopy demonstrated a subpopulation of red cell fragments averaging 25 to 40 fl in size at higher rates. A randomized, crossover clinical trial was next performed by delivery of blood perfusion at 60 ml/min to 15 patients undergoing coronary angioplasty. Levels of plasma hemoglobin, beta-thromboglobulin, lactate dehydrogenase, and potassium remained constant before and after the perfusion and the control inflations. The maximum pain score was significantly lower with the perfusion inflation (4.1 +/- 0.8 vs 6.0 +/- 0.9, p less than .003). Relative to baseline, the maximum ST segment elevation during the perfusion inflation (0.5 +/- 0.3 mm) was nearly one-fourth that during the control inflation (1.9 +/- 0.6 mm, p less than .02). Thus, myocardial protection with oxygenated autologous blood perfusion at rates of 60 ml/min appears to be a safe and effective technique that may permit increased inflation time and extend the range of coronary angioplasty to include individuals at high risk for the procedure.

Angioplasty, Balloon↗
PubMed1971

Biochemical and biophysical aspects of human platelet adhesion to collagen fibers.

A method has been developed for measuring the adhesion of platelets to purified collagen fibers obtained from bovine tendon. This method differs from others in that: (a) platelet adhesion is measured in the absence of platelet aggregation; (b) platelet-rich plasma collected in ACD (acid citrate dextrose) or EDTA, or washed platelets can be employed; (c) adherent platelets are enumerated directly; (d) erythrocytes and leukocytes do not adhere. Washed platelets suspended in human Ringer solution exhibit negligible adhesion (at the platelet concentrations employed) in contrast to washed platelets suspended in plasma. Addition of purified human fibrinogen (95% clottable, 2-4 mg/ml) to human Ringer solution completely restores the ability of washed platelets to adhere to collagen fibers. Albumin (fatty acid free, 50 mg/ml) is also capable of restoring adhesion. Albumin and seven other proteins at concentrations of 5-10 mg/ml, with varying molecular weights, isoelectric points, and frictional coefficients are incapable of supporting the adhesion of washed platelets. The proteins tested were human globulin, hexokinase, hemoglobin, cytochrome-C, insulin, thyroglobulin, and muramidase. Platelet adhesion is proportional to both platelet concentration and fibrinogen concentration, but is independent of temperature or glycogen stores. Modification of fibrinogen by acylation of amino groups or removal of sialic acid has no effect on its ability to support platelet adhesion. Degradation of fibrinogen with purified plasmin results in decreased support of platelet adhesion. This accompanied formation of early breakdown products with clottability ranging from 84-0%. Formation of fibrinogen degradation products was monitored by SDS-polyacrylamide gel electrophoresis of the corresponding fibrins after reduction of disulfide bonds (a method capable of distinguishing alpha-, beta- and gamma-chains). Decreased support of platelet adhesion is associated with the disappearance of intact alpha- chains and early modification of the beta-chains. Purified proteinpolysaccharide macromolecules obtained from bovine nasal and humeral cartilage, and from nucleosus pulposus are as effective as fibrinogen on a weight basis and ten to thirty times more effective on a molar basis in supporting platelet adhesion. The purified mucopolysaccharide side chains: chondroitin-4-sulfate, chondroitin-6-sulfate, and keratan-sulfate are incapable of supporting platelet adhesion.

Adsorption↗
PubMed1987

Diagnostic immunohistochemistry of canine round cell tumors.

Sixty-five canine skin neoplasms studied using immunocytochemistry, included 22 histiocytomas, 18 amelanotic melanomas, 14 cutaneous lymphosarcomas, six mast cell tumors, and five transmissible venereal tumors. Formalin-fixed, paraffin-embedded sections were stained using the avidin-biotin-peroxidase complex (ABC) immunoperoxidase technique for reactivity with S-100 protein, kappa and lambda immunoglobulin light chains, alpha-1-antitrypsin, alpha-1-antichymotrypsin, leukocyte common antigen (LCA), neuron-specific enolase, keratin, cytokeratin, muramidase, and vimentin. Detection of S-100, kappa and lambda light chains, neuron-specific enolase, and vimentin were most useful for screening these neoplasms. None of the markers examined was consistent in staining histiocytomas. While reactivity of S-100 (ten cases) and neuron-specific enolase (ten cases) was detected in some amelanotic melanomas, lambda light chain immunoglobulin (eight cases) was relatively consistent in cutaneous lymphomas. Mast cell neoplasms reacted with avidin and, therefore, were positive, even on negative control sections. Vimentin reacted strongly on all amelanotic melanomas and transmissible venereal tumors examined. These antibodies are helpful adjuncts in the differential diagnosis of canine skin tumors.

Animals↗
PubMed1990

Vitamin E and selenium in gentamicin nephrotoxicity.

1. The sequence of renal cellular membrane damage induced by gentamicin was studied in the rat by using the release of alkaline phosphatase, acid phosphatase, muramidase and protein from renal cells as indices of renal damage. 2. The protective effect of a combination of vitamin E and selenium against renal damage was also investigated. 3. Gentamicin (60 mg kg-1 body weight) was nephrotoxic within 12 h of the first dose. 4. The plasma membrane of the renal tubules is damaged before the lysosomal membrane is affected. 5. A combination of vitamin E (1 mg g-1 body weight) and selenium (4 x 10(-3) mg g-1 body weight) attenuates the renal damage induced by gentamicin. Results suggest synergism between vitamin E and selenium in attenuating the renal damage. The possible mechanism of attenuation is discussed. 6. Vitamin E and selenium may have anti-diuretic potential.

Animals↗
PubMed2003

Cheese whey-induced high-cell-density production of recombinant proteins in Escherichia coli.

BACKGROUND: Use of lactose-rich concentrates from dairy processes for the induction of recombinant gene's expression has not received much attention although they are interesting low cost substrates for production of recombinant enzymes. Applicability of dairy waste for induction of recombinant genes in Escherichia coli was studied. Clones expressing Lactobacillus phage muramidase and Lactobacillus alcohol dehydrogenase were used for the experiments. RESULTS: Shake flask cultivations in mineral salt medium showed that cheese whey or deproteinised whey induced gene expression as efficiently as IPTG (isopropyl-beta-D-thiogalactopyranoside) or pure lactose. Addition of yeast extract or proteolytically degraded whey proteins did not improve the recombinant protein yield. In contrast, addition of yeast extract to the well-balanced mineral salt medium decreased the product yield. Feeding with glycerol provided sufficient amount of easily assimilable carbon source during the induction period without preventing lactose intake and induction by lactose. High-cell-density fed-batch cultivations showed that product yields comparable to IPTG-induction can be achieved by feeding bacteria with a mixture of glycerol and concentrated whey permeate during the induction. CONCLUSION: Whey and concentrated whey permeate can be applied as an alternative inducer in recombinant high-cell-density fed-batch fermentations. The yield of the recombinant product was comparable to fermentations induced by IPTG. In low-cell-density shake flask experiments the yield was higher with whey or whey permeate than with IPTG.

Journal Article↗
PubMed2001

Isolation of a new lytic enzyme for hiochi bacteria and other lactic acid bacteria.

A microorganism producing a lytic enzyme preparation that could rapidly lyse bacterial cells such as hiochi bacteria and other lactic acid bacteria was screened. The microorganism was identified as Streptomyces fulvissimus. The enzyme produced by this organism lysed boil-denatured cells quicker than intact cells of hiochi bacteria. A mutant strain of S. fulvissimus producing the enzyme exhibiting high activity against intact cells of hiochi bacteria was screened on plates, containing intact cells inactivated with UV irradiation. The optimal pH for lytic activity against intact cells of the hiochi bacterium Lactobacillus casei S-4 was from 3.5 to 4.0, and the optimum temperature was close to 50 degrees C. This enzyme activity was stable between pH 3.5 and pH 8.0 and up to 60 degrees C. The enzyme exhibits N-acetyl glucosaminidase and muramidase activities. The effects of adjusting the pH and using different inducers for enzyme production were investigated. Chitin was the most effective inducer of enzyme production. Intact DNA was easily isolated from the cells of many lactic acid bacteria following lysis with the enzyme. It is thought that this enzyme will be a good biotechnological tool.

Journal Article↗
PubMed1993

Ultrastructural observations on a variant of acute promyelocytic leukemia.

A patient with acute leukemia is presented in whom the leukemic cells, as seen by light microscopy were typical promyelocytes. The cells had normal or slightly invaginated nuclei with typical cytoplasmic granules and the diagnosis was confirmed by cytochemistry. The clinical course was rapid and the patient died of disseminated intravascular coagulation and urosepsis within a few days of diagnosis. However, electron microscopic examination showed cells with extremely convoluted and lobulated nuclei with nuclear pockets and cytoplasmic bridges as well as the complete absence of cytoplasmic granules in the majority of the cells. Furthermore, the urine lysozyme (muramidase) was elevated. These findings suggest that the leukemia in this patient may be classified as a hypogranular variant of acute promyelocytic leukemia (APL), with monocytoid ultrastructural appearances.

Biomarkers, Tumor↗
PubMed1986

Giant-cell myocarditis in a newborn with congenital herpes simplex virus (HSV) infection: an immunohistochemical study on the origin of the giant cells.

Giant-cell myocarditis is a rare inflammatory disorder characterized by degeneration and necrosis of myocardial fibers and presence of chronic inflammatory infiltrates associated with multinucleated giant cells forming a granulomatous inflammatory reaction. The etiology of giant-cell myocarditis is unknown. Many conditions have been reported as associated with this phenomenon such as fungi, virus, sarcoidosis, and hypersensitivity or autoimmune reactions. We are reporting a case of giant-cell myocarditis discovered in a newborn with congenital herpetic sepsis. The myogenic origin of the giant-cells of this case is supported by the positivity for desmin and myoglobin and negativity for muramidase and alpha-1-antichymotrypsin after immunoperoxidase procedure. The presence of Herpes simplex virus type II was confirmed by indirect immunoperoxidase reaction in most of the viscera including the heart, but is not considered a factor in the production of giant cells.

Female↗
PubMed2006

An analysis of the effectiveness of heat-killed lactic acid bacteria in alleviating allergic diseases.

Allergic diseases are reported to be caused by a skew in the balance between T helper type 1 and 2 cells. Because some lactic acid bacteria have been shown to stimulate IL-12 (p70) production, which in turn shifts the balance between the T helper type 1 and 2 cell response from the latter to the former, they have the potential to either prevent or ameliorate disease conditions or both. They have therefore been extensively studied in the recent past for their probiotic activities. Nevertheless, much less information is available concerning the microbial factors that determine the strain-dependent ability to affect the production of cytokines. The objectives of our study were first to select potentially probiotic lactobacilli that strongly stimulate cytokine production in vitro, and then to determine whether the selected Lactobacillus strains could suppress antigen-specific IgE production in vivo by using allergic model animals. Finally, our investigation was extended to analyze which bacterial components were responsible for the observed biological activity. Twenty strains of heat-killed lactobacilli isolated from humans were screened for their stimulatory activity for the production of IL-12 (p70) by murine splenocytes in vitro. The results showed that some strains of Lactobacillus plantarum and Lactobacillus gasseri had a higher stimulatory activity for IL-12 (p70) production than the other lactobacilli tested; however, this effect was strain dependent rather than species dependent. Oral administration of the heat-killed strains that showed higher stimulatory activity for IL-12 (p70) production tended to reduce the serum antigen-specific IgE levels in ovalbumin-sensitized BALB/c mice compared with the controls. Among the lactobacilli tested, L. gasseri OLL2809 showed the highest activity in reducing the level of antigen-specific IgE. Furthermore, the stimulatory activity for IL-12 (p70) production was found to be reduced after treating the lactobacilli with N-acetyl-muramidase and to be positively correlated with the amount of peptidoglycan in the cells. The present data suggest that L. gasseri OLL2809 is a good candidate for potential probiotics in terms of either the prevention or amelioration of allergic diseases or both. In addition, the strain-dependent stimulatory activity for IL-12 (p70) production was found to be due, at least in part, to the amount of peptidoglycan present in the cells.

Animals↗
PubMed1994

Insulin reverses the protection given by diabetes against gentamicin nephrotoxicity in the rat.

Rats with untreated diabetes mellitus are protected from gentamicin-induced nephrotoxicity. In order to evaluate the role of hyperglycemia, glycosuria, and polyuria in this phenomenon, miniosmotic pumps filled with insulin were implanted for 15 days in seven female Sprague-Dawley rats with streptozotocin-induced diabetes mellitus. Plasma glucose levels were successfully maintained under 126 mg/dl. To serve as the control group, eight age-matched diabetic (plasma glucose > 400 mg/dl) rats had miniosmotic pumps placed delivering only Ringer's solution. Six days after placement of the pumps, gentamicin (40 mg/Kg/day) was administered to all animals for 9 days. The insulin-treated diabetic rats exhibited clear signs of nephrotoxicity by Day 6 of gentamicin, whereas the diabetic control group remained free from any functional or morphological evidence of proximal tubular damage throughout the 9 days of the aminoglycoside administration. At the end of the experiment, the creatinine clearance in the insulin-treated diabetic group was 45% lower than in the untreated diabetic group (P < 0.005). In addition, there was a rise in plasma creatinine (P < 0.02), muramidase appeared in the urine, and mild patchy acute tubular necrosis of the renal cortex was observed by light microscopic examination. The insulin-treated group also accumulated more gentamicin in the renal cortex than the untreated animals (P < 0.005). It is concluded that protection against the nephrotoxic effects of gentamicin is a feature of untreated experimental diabetes mellitus in the rat and that correction of the hyperglycemic state with insulin reverses this resistance.

Animals↗