Histology and tissue chemistry of tidemark separation in hamsters.
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Adult articular cartilage is divided by the tidemark into a deep calcified layer and a more superficial uncalcified layer. Histologic examination of articular cartilage from the knee joint of golden Syrian hamsters 123 days of age or older revealed defects at the tidemark in the tibia. Defects ranged from small separations of the calcified and uncalcified layers along the tidemark to progressively larger defects apparently formed by dissolution. These larger defects appeared as cavities in the noncalcified cartilage, had smooth rather than rough edges, frequently contained coalesced debris, and often resulted in a bulge in the articular surface. Occasionally, these large defects broke through the articular surface. Defects were not observed in tibial cartilage of younger (<90 days old) hamsters or in femoral cartilage from hamsters of any age. Exercise neither protected against nor increased the severity of the defects. Collagen cross-linking by pyridinoline was examined as a function of age and increased from 1,090 to 3,062 micromoles of pyridinoline/mole of hydroxyproline over the period of 1-9 months of age but was not correlated with defect formation. With increasing age, these focal tidemark defects could lead to osteoarthrosis-like cartilage lesions. (+info)
Two-component transcriptional regulation of N-acyl-homoserine lactone production in Pseudomonas aureofaciens.
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Production of phenazine antibiotics by the biological control bacterium Pseudomonas aureofaciens 30-84 is regulated in part by the PhzI/PhzR N-acyl-homoserine lactone (AHL) response system (L. S. Pierson III, V. D. Keppenne, and D. W. Wood, J. Bacteriol. 176:3966-3974, 1994; D. W. Wood and L. S. Pierson III, Gene 168:49-53, 1996). Two mutants, 30-84W and 30-84.A2, were isolated and were found to be deficient in the production of phenazine, protease, hydrogen cyanide (HCN), and the AHL signal N-hexanoyl-homoserine lactone. These mutants were not complemented by phzI, phzR, or the phenazine biosynthetic genes (phzFABCD) (L. S. Pierson III, T. Gaffney, S. Lam, and F. Gong, FEMS Microbiol. Lett. 134:299-307, 1995). A 2.2-kb region of the 30-84 chromosome which fully restored production of all of these compounds in strain 30-84W was identified. Nucleotide sequence analysis of this region revealed a single open reading frame encoding a predicted 213-amino-acid protein which is very similar to the global response regulator GacA. Strain 30-84.A2 was not complemented by gacA or any cosmid from a genomic library of strain 30-84 but was complemented by gacS (formerly lemA) homologs from Pseudomonas fluorescens Pf-5 (N. Corbel and J. E. Loper, J. Bacteriol. 177:6230-6236, 1995) and Pseudomonas syringae pv. syringae B728a (E. M. Hrabek and D. K. Willis, J. Bacteriol. 174:3011-3020, 1992). Transcription of phzR was not altered in either mutant; however, phzI transcription was eliminated in strains 30-84W and 30-84.A2. These results indicated that the GacS/GacA two-component signal transduction system of P. aureofaciens 30-84 controls the production of AHL required for phenazine production by mediating the transcription of phzI. Addition of exogenous AHL did not complement either mutant for phenazine production, indicating that the GacS/GacA global regulatory system controls phenazine production at multiple levels. Our results reveal for the first time a mechanism by which a two-component regulatory system and an AHL-mediated regulatory system interact. (+info)
Validity of histopathological grading of articular cartilage from osteoarthritic knee joints.
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OBJECTIVES: To determine the validity of the histological-histochemical grading system (HHGS) for osteoarthritic (OA) articular cartilage. METHODS: Human articular cartilage was obtained from macroscopically normal (n = 13) and OA (n = 21) knee joints. Sections of central and peripheral regions of normal samples were produced. Sections of regions containing severe, moderate, and mild OA changes were produced from each OA sample. A total of 89 sections were graded by means of the HHGS (0-14) twice by three observers. RESULTS: Average scores for regions designated severe (8.64) and moderate (5.83) OA were less than the expected (10-14 and 6-9, respectively) according to the HHGS, whereas average scores for the region designated mild (5.29) OA and central and peripheral regions (2.19) of normal cartilage were higher than expected (2-5 and 0-1, respectively). The HHGS was capable of differentiating between articular cartilage from macroscopically normal and OA joints and between the region designated severe OA and other regions. However, the HHGS did not adequately differentiate between regions designated mild and moderate OA. Values for sensitivity, specificity, and efficiency for all regions varied considerably. CONCLUSION: The HHGS is valid for normal and severe OA cartilage, but does not permit distinction between mild and moderate OA changes in articular cartilage. (+info)
Antimycobacterial activities of riminophenazines.
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Riminophenazines were specifically developed as drugs active against Mycobacterium tuberculosis but extensive research over several decades has shown that these compounds are also active against many other mycobacterial infections, particularly those caused by Mycobacterium leprae and the Mycobacterium avium complex (MAC). Clofazimine, the lead compound in this series, is included in the regimens that are approved by the WHO for the treatment of leprosy and has contributed significantly to the control of that disease, particularly that caused by dapsone-resistant bacteria. Despite early problems, clofazimine has shown clinical efficacy in tuberculosis, in particular that caused by multiple drug resistant strains. Clofazimine does not induce resistance and also inhibits emergence of resistance to isoniazid in M. tuberculosis. The efficacy of clofazimine against MAC is more varied and the availability of better drugs has limited its use. Newer riminophenazines, such as B746 and B4157, not only showed increased anti-mycobacterial activity but also produced less skin pigmentation, which is the main drawback of this group of compounds. The most important virtues of riminophenazines, such as intracellular accumulation in mononuclear phagocytic cells, anti-inflammatory activity, a low incidence of drug resistance and slow metabolic elimination, make them attractive candidates for the treatment of mycobacterial infections. It is essential, however, to investigate the newer analogues clinically, while continuing the pursuit of alternate candidates that demonstrate higher anti-mycobacterial activity and lower rates of skin pigmentation. (+info)
Novel reactions involved in energy conservation by methanogenic archaea.
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Methanogenic archaea of the order Methanosarcinales which utilize C(1) compounds such as methanol, methylamines or H(2)+CO(2), employ two novel membrane-bound electron transport systems generating an electrochemical proton gradient: the H(2):heterodisulfide oxidoreductase and the F(420)H(2):heterodisulfide oxidoreductase. The systems are composed of the heterodisulfide reductase and either a membrane-bound hydrogenase or a F(420)H(2) dehydrogenase which is functionally homologous to the proton-translocating NADH dehydrogenase. Cytochromes and the novel electron carrier methanophenazine are also involved. In addition, the methyl-H(4)MPT:HS-CoM methyltransferase is bioenergetically relevant. The enzyme couples methyl group transfer with the translocation of sodium ions and seems to be present in all methanogens. The proton-translocating systems with the participation of cytochromes and methanophenazine have been found so far only in the Methanosarcinales. (+info)
Exposure of N-formyl-L-methionyl-L-leucyl-L-phenylalanine-activated human neutrophils to the Pseudomonas aeruginosa-derived pigment 1-hydroxyphenazine is associated with impaired calcium efflux and potentiation of primary granule enzyme release.
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The effects of pathologically relevant concentrations (0.38 to 12.5 microM) of the proinflammatory, Pseudomonas aeruginosa-derived pigment 1-hydroxyphenazine (1-hp) on Ca2+ metabolism and intracellular cyclic AMP (cAMP) in N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP; 1 microM)-activated human neutrophils, as well as on the release of myeloperoxidase (MPO) and elastase from these cells, have been investigated in vitro. Ca2+ fluxes were measured by the combination of a fura-2/AM-based spectrofluorimetric method and radiometric procedures, which together enable distinction between net efflux and influx of the cation, while radioimmunoassay and colorimetric methods were used to measure cAMP and granule enzymes, respectively. Coincubation of neutrophils with 1-hp did not affect intracellular cAMP levels or the FMLP-activated release of Ca2+ from intracellular stores but did retard the subsequent decline in the chemoattractant-induced increase in the concentration of cytosolic free Ca2+. These effects of 1-hp on the clearance of Ca2+ from the cytosol of activated neutrophils were associated with decreased efflux of the cation from the cells and increased release of MPO and elastase, while the delayed store-operated influx of the cation into the cells was unaffected by the pigment. The plasma membrane Ca2+-ATPase rather than a Na+-Ca2+ exchanger appeared to be the primary target of 1-hp. These observations suggest that the proinflammatory interactions of 1-hp with activated human neutrophils are a consequence of interference with the efflux of cytosolic Ca2+ from these cells. (+info)
Possible mechanism of hepatocyte injury induced by diphenylamine and its structurally related nonsteroidal anti-inflammatory drugs.
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Diphenylamine is a common structure of nonsteroidal anti-inflammatory drugs (NSAIDs) to uncouple mitochondrial oxidative phosphorylation and to cause a decrease in hepatocellular ATP content and hepatocyte injury. The mechanism for acute cell injury induced by diphenylamine and its structurally related NSAIDs was investigated with rat liver mitochondria and freshly isolated hepatocytes, focusing on the relation to the uncoupling of oxidative phosphorylation. Incubation of mitochondria with diphenylamine as well as mefenamic acid and diclofenac caused pseudoenergetic mitochondrial swelling, indicating that these compounds induce mitochondrial membrane permeability transition. Diphenylamine also caused changes in safranine-binding spectra to mitochondria that was energized by succinate oxidation. This spectral shift indicates the loss of mitochondrial membrane potentials, which is known as one of the characteristics for uncouplers of oxidative phosphorylation, and also was caused by mefenamic acid and diclofenac. Incubation of hepatocytes with mefenamic acid, diclofenac, and diphenylamine diminished cellular ATP content, followed by leakage of lactose dehydrogenase from hepatocytes. Fructose, a low K(m) substrate for glycolysis, partially protected against the ATP depletion and hepatocyte injury induced by these compounds. Further addition of oligomycin, which blocks ATPase, pronounced the protection against cell injury. These results suggested that decreases in cellular ATP content, mainly caused by uncoupling of mitochondrial oxidative phosphorylation, were responsible for acute hepatocyte injury induced by diphenylamine and structurally related NSAIDs. (+info)
Gram stain of bronchoalveolar lavage fluid in the early diagnosis of ventilator-associated pneumonia.
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To assess the usefulness of the Gram stain in the early diagnosis of ventilator-associated pneumonia (VAP), we performed 146 protected specimen brushings (PSB) and bronchoalveolar lavages (BAL) in 118 patients suspected of having nosocomial pneumonia. Gram stain and counts of infected cells were performed in all samples from BAL fluid. A final diagnosis of pneumonia was established in 51 patients and there was no infection in 95 cases. A threshold of 2% of infected cells was used to distinguish between VAP and the group without VAP (sensitivity 86.3%, specificity 78.9%, positive predictive value 68.7% and negative predictive value 91.4%); there was good agreement with the final diagnosis (kappa statistic 0.616; concordance 81.5%). Regarding detection of bacteria using the Gram stain, we found a sensitivity of 90.2%, specificity 73.7%, positive predictive value 64.8% and negative predictive value 93.3%; there was moderate agreement with the final diagnosis (kappa statistic 0.586; concordance 79.4%). In the VAP group, we analysed the degree of qualitative agreement between Gram stain and PSB quantitative cultures: the correlation was complete in 51% (26 of 51 VAP), partial in 39.2% (20 of 51 VAP) and there was no correlation in 9.8% (five of 51 VAP). We conclude that the Gram stain is useful for rapid diagnosis of VAP but unreliable for early adaptation of empiric therapy. (+info)