Mitochondrial DNA in the aquatic fungus Allomyces

Summary The mitochondrial DNA from seven species of the aquatic phycomycete Allomyces has been isolated and characterized by restriction enzyme analysis. Comparison of the mitochondrial DNA restriction enzyme fragmentation patterns showed pronounced differences not only among species but also among four isolates of A. arbuscula. The mitochondrial DNAs range in size from 39 kbp in A. neo-moniliformis to 56 kbp in A. macrogynus.A physical map of the mitochondrial DNA of Allomyces arbuscula strain Costa Rica 21 has been constructed. The genome is circular and has a size of 49.2 kbp. The genes coding for the small and large ribosomal RNAs, cytochrome oxidase subunits 1, 2, and 3, apocytochrome b, and ATPase subunits 6 and 9 were localized in the mitochondria) DNA by heterologous hybridization with specific mitochondria) gene probes from Saccaromyces cerevisiae and Neurospora crassa. Comparison of the gene map of the closely related species Blastocladiella emersonii with that of A. arbuscula indicates a similar gene order in the two organisms.     

Physical activity and plasma interleukin-6 in humans – effect of intensity of exercise

The present study included data from three marathon races to investigate the hypothesis that a relationship exists between running intensity and elevated concentrations of interleukin (IL)-6 in plasma. The study included a total of 53 subjects whose mean age was 30.6 [95% confidence interval (CI) 1.4] years, mean body mass 77.7 (95%CI 2.0) kg, mean maximal oxygen uptake (V˙O_2max) 59.3 (95%CI 1.4) ml · min⁻¹ · kg⁻¹, and who had participated in the Copenhagen Marathons of 1996, 1997 or 1998, achieving a mean running time of 206 (95%CI 7) min. Running intensity was calculated as running speed divided by V˙O_2max. The concentration of IL-6 in plasma peaked immediately after the run. There was a negative correlation between peak IL-6 concentration and running time (r=−0.30, P < 0.05) and a positive correlation between peak IL-6 concentration and running intensity (r=0.32, P < 0.05). The IL-1 receptor antagonist (IL-1ra) plasma concentration peaked 1.5 h after the run and there was a positive correlation between the peak plasma concentrations of IL-6 and IL-1ra (r=0.39, P < 0.01). Creatine kinase (CK) plasma concentration peaked on the 1st day after the run, but no association was found between peak concentrations of IL-6 and CK. In conclusion, the results confirmed the hypothesized association between plasma IL-6 concentration and running intensity, but did not confirm the previous finding of a connection between IL-6 plasma concentration and muscle damage. Accepted: 6 August 2000     

Down-regulation of natural killer cell activity by autologous polymorphonuclear leucocytes

It has been recently reported that neutrophils are involved in the regulation of NK cell activity. However, the mechanism of such regulation is unclear. The present study was designed to investigate the mechanisms involved in the regulation of NK cytotoxicity by human neutrophils. The role of indomethacin, an anti-inflammatory drug, in this interaction was studied. NK cells were purified from peripheral blood obtained from normal individuals. NK cell cytotoxicity was tested on K 562 cell line by Cr release assay. Autologous neutrophils obtained from peripheral blood were stimulated by opsonized zymosan either in the presence or absence of indomethacin. The role of neutrophil supernatant containing oxygen radicals and prostaglandins on NK cytotoxicity was examined. It was shown that supernatants from stimulated neutrophils significantly inhibited (P less than 0.05) the autologous NK cell cytotoxicity. The presence of indomethacin in the in vitro reaction mixture, or given orally to donors, partially or completely abolished the inhibitory effect of neutrophil supernatant. Indomethacin inhibited prostaglandin E2 release, and luminol-enhanced, myeloperoxidase-mediated chemiluminescence of activated PMN. Diafiltration of neutrophil supernatant showed that the inhibitory activity was present in the fraction containing molecules lower than 5,000 daltons. In conclusion, our findings indicate that down-regulation of NK cytotoxicity is mediated by prostaglandins produced by stimulated neutrophils and possibly by oxygen radicals.     

Inhibition of human natural killer cell activity by Pseudomonas aeruginosa alkaline protease and elastase.

The present study was designed to examine the effect of Pseudomonas aeruginosa alkaline protease (AP) and elastase (Ela) on human natural killer (NK) cell activity in vitro. AP and Ela were found to inhibit NK cell function. Addition of alpha interferon and interleukin-2 did not abolish this inhibition of NK cell activity. Adhesion of effector to target cells was studied in a single-cell agarose assay of monocyte-depleted NK-cell-enriched cell populations. AP and Ela were shown to inhibit effector/target cell conjugate formation. Furthermore, AP and Ela inhibited the binding of the monoclonal antibody Leu-11, which reacts with the Fc receptor of NK cells. The inhibition of NK cell binding to the target cell by P. aeruginosa proteases is most likely due to proteolytic cleavage of the surface receptors involved in the binding of the effector cell to the target cell.     

Detection of Campylobacter spp. in chicken fecal samples by real-time PCR

A real-time PCR assay for detecting thermophilic Campylobacter spp. directly in chicken feces has been developed. DNA was isolated from fecal material by using magnetic beads followed by PCR with a prealiquoted PCR mixture, which had been stored at -18 degrees C. Campylobacter could be detected in less than 4 h, with a detection limit of 100 to 150 CFU/ml, in a fecal suspension. A bacterial internal control was added before DNA extraction to control both DNA isolation and the presence of PCR inhibitors in the samples. The assay was performed on 111 swab samples from a Danish surveillance program and compared to conventional culturing using selective enrichment. There was no statistically significant difference in performance between real-time PCR and culture by selective enrichment, and the diagnostic specificity was 0.96 with an agreement of 0.92. Therefore, the assay should be useful for screening poultry flocks for the presence of Campylobacter.     

Characterization of a monoclonal anti-Bw4 antibody (Tü109): Evidence for similar epitopes on the Bw4 and Bw6 antigens

The production and serologic, as well as immunochemical properties of a cytotoxic murine IgG monoclonal antibody (Tü109) that precipitates HLA-class I molecules, are described. In the microcytotoxicity assay Tü109 supernatant was demonstrated on a panel of 424 HLA-ABC, -DR, -DQ, -MT typed normal Caucasian blood donors to define an epitope on HLA-B locus molecules in great association with the supertypic specificity Bw4. Reactivity of supernatant showed MHC linked inheritance of the Tü109 determinant and discriminated the HLA-Bw4/Bw6 associated HLA-B locus split antigens. Weak or lack of binding on lymphocytes from some HLA-Bw4 heterozygous individuals, particularly typing for HLA-Bw44, appeared to be due to qualitative and/or quantitative variations of HLA-B locus molecules on the cell surface. With Tü109 ascites fluid, however, extra-reactivity on all HLA-Bw6⁺ cells was demonstrated. Preferential binding of supernatant to HLA-Bw4, but reactivity of ascites fluid with HLA-Bw6⁺ molecules in addition, was furthermore confirmed by IEF analysis of antigens immunoprecipitated with Tü109 from cell lysates. Thus the antibody may help to analyze the evolutionary relationship of the diallelic specificities Bw4 and Bw6.     

Expression of interleukin-15 in human skeletal muscle – effect of exercise and muscle fibre type composition

The cytokine interleukin-15 (IL-15) has been demonstrated to have anabolic effects in cell culture systems. We tested the hypothesis that IL-15 is predominantly expressed by type 2 skeletal muscle fibres, and that resistance exercise regulates IL-15 expression in muscle. Triceps brachii, vastus lateralis quadriceps and soleus muscle biopsies were obtained from normally physically active, healthy, young male volunteers ( n = 14), because these muscles are characterized by having different fibre-type compositions. In addition, healthy, normally physically active male subjects ( n = 8) not involved in any kind of resistance exercise underwent a heavy resistance exercise protocol that stimulated the vastus lateralis muscle and biopsies were obtained from this muscle pre-exercise as well as 6, 24 and 48 h post-exercise. IL-15 mRNA levels were twofold higher in the triceps (type 2 fibre dominance) compared with the soleus muscle (type 1 fibre dominance), but Western blotting and immunohistochemistry revealed that muscle IL-15 protein content did not differ between triceps brachii, quadriceps and soleus muscles. Following resistance exercise, IL-15 mRNA levels were up-regulated twofold at 24 h of recovery without any changes in muscle IL-15 protein content or plasma IL-15 at any of the investigated time points. In conclusion, IL-15 mRNA level is enhanced in skeletal muscles dominated by type 2 fibres and resistance exercise induces increased muscular IL-15 mRNA levels. IL-15 mRNA levels in skeletal muscle were not paralleled by similar changes in muscular IL-15 protein expression suggesting that muscle IL-15 may exist in a translationally inactive pool.     

Exercise-induced metallothionein expression in human skeletal muscle fibres

Exercise induces free oxygen radicals that cause oxidative stress, and metallothioneins (MTs) are increased in states of oxidative stress and possess anti-apoptotic effects. We therefore studied expression of the antioxidant factors metallothionein I and II (MT-I + II) in muscle biopsies obtained in response to 3 h of bicycle exercise performed by healthy men and in resting controls. Both MT-I + II proteins and MT-II mRNA expression increased significantly in both type I and II muscle fibres after exercise. Moreover, 24 h after exercise the levels of MT-II mRNA and MT-I + II proteins were still highly increased and the MT-II mRNA expression reached a 15-fold increase. As expected, immunohistochemical detection of malondialdehyde (MDA) and nitrotyrosine (NITT) showed that formation of free radicals and oxidative stress were clearly increased in exercising muscle peaking shortly after the end of exercise in both type I and II muscle fibres. This is the first report demonstrating that MT-I + II are significantly induced in human skeletal muscle fibres following exercise. As MT-I + II are antioxidant factors that protect various tissues during pathological conditions, the MT-I + II increases post exercise may represent a mechanism whereby contracting muscle fibres are protected against cellular stress and injury.     

Glial Fibrillary Acidic Protein Expression in Pleomorphic Adenoma of Salivary Gland: An Immunoelectron Microscopic Study

Previous immunocytochemical studies of pleomorphic adenomas have demonstrated consistent labeling with glial fibrillary acidic protein (GFAP). Cross-reactivity with other intermediate filaments of similar structure and chemical composition has been suggested to account for this seemingly inappropriate pattern of immunoreactivity. To investigate further this phenomenon, we examined five pleomorphic adenomas by immunoelectron microscopy. Ultrastructural features were similar to those described by other investigators, with ductal epithelium being surrounded by myoepithelial cells and modified cells becoming detached to form the isolated stellate and spindle cells of the stroma. As part of this process, many neoplastic myoepithelial cells appeared to lose their specialized ultrastructural features, assuming a rather undifferentiated appearance. Single and double immunoelectron microscopic labeling showed vimentin filaments in all these neoplastic myoepithelial cells. In contrast, GFAP filaments were identified only in the most undifferentiated cells. Such restriction of GFAP filaments to an ultrastructurally definable subset of neoplastic cells provides strong evidence against nonspecific staining due to cross-reactivity. Given the previously described coexpression of vimentin and GFAP by neoplastic cartilage, it appears likely that this immunophenotype in neoplastic myoepithelial cells reflects early chondroid differentiation.     

The Compliance Curve for the Flow Limiting Segments of the Airway

Maximum effort flow-static recoil curves were obtained in 5 healthy subjects breathing air, He/O₂, and SF₆/O₂ mixtures. In 4 of them maximum effort flows corresponded to really maximal flows and their curves were transformed into compliance curves for the flow limiting segments of the airway and analyzed from the point of view of a previously presented lung model (Pedersen and Nielsen 1976). The results showed, that viscosity dependent pressure losses from the alveoli to the flow limiting segments were minimal for air and SF₆/O₂, but not for He/O₂. When viscosity dependent pressure losses could be neglected, then expiration of gases of different densities gave almost identical compliance curves for the flow limiting segments. This supported the applicability of the model. The calculated compliance curves for the flow limiting segments were compared with data from the literature, and the findings indicated that flow limitation during expirations with just maximal flows throughout began in the extrapulmonary airways and moved upstream during the expiration.