Effect of molybdate ions on methanation of simulated and natural waste-waters

Summary Sulphate in concentrations of 500 and 1000 mg SO₄-S/l did not inhibit methanation of synthetic waste-water (acetate + methanol + glucose) by sludge from a digester treating neutral spent sulphite process effluents. The role of sulphate reducers in the conversion of those substrates was minor although sulphate-reducing bacteria were present with a viable count similar to that of methane-producing bacteria in the sludge. Neutral spent sulphite liquor was partially converted to methane (40% of chemical oxygen demand) under these conditions.Molybdate (20 mM) inhibited methanation of both synthetic waste-water and neutral spent sulphite liquor. Acetate accumulated in glucose plus molybdate media. Molybdate had a direct inhibitory effect on enriched acetoclastic methane-producing bacteria. Molybdate was bacteriocidic to sulphate-reducing bacteria and bacteriostatic to methane-producing bacteria.     

Comparative study of potential virulence factors in human pathogenic and saprophytic Trichoderma longibrachiatum strains

Potential virulence factors of 9 saprophytic and 12 clinical Trichoderma longibrachiatum strains were examined in the present study, in order to compare their capacity to cause infection in humans. All of the strains were able to grow at temperatures up to 40 degrees C and at pH values ranging from 2.0 to 9.0. Carbon and nitrogen source utilization experiments revealed that all of the strains were able to utilize a series of basic amino acids both as sole carbon and nitrogen sources. The MIC values of the tested antifungal drugs were found to be 0.016-8 microg/ml for amphotericin B, 64-256 microg/ml for fluconazole, 0.5-32 microg/ml for itraconazole and 0.008-1 microg/ml for ketoconazole in the case of the examined isolates. Metabolites of the strains inhibited the growth of different bacteria, furthermore, compounds produced by three clinical isolates reduced the motility of boar spermatozoa, indicating their toxicity to mammalian cells as well. On the whole, there were no significant differences in the examined features between strains derived from clinical or soil samples. The question, however, whether all environmental Trichoderma longibrachiatum strains have the capacity to cause infections or not, remains still unanswered.     

Biological effects of Trichoderma harzianum peptaibols on mammalian cells

Trichoderma species isolated from water-damaged buildings were screened for toxicity by using boar sperm cells as indicator cells. The crude methanolic cell extract from Trichoderma harzianum strain ES39 inhibited the boar sperm cell motility at a low exposure concentration (50% effective concentration, 1 to 5 microg [dry weight] ml of extended boar semen(-1)). The same exposure concentration depleted the boar sperm cells of NADH(2). Inspection of the exposed boar sperm cells by transmission electron microscopy revealed damage to the plasma membrane. By using the black lipid membrane technique, it was shown that the semipurified metabolites (eluted from a SepPak C(18) cartridge) of T. harzianum strain ES39 induced voltage-dependent conductivity. The high-performance liquid chromatography-purified metabolites of T. harzianum strain ES39 dissipated the mitochondrial membrane potential (Deltapsi(m)) of human lung epithelial carcinoma cells (cell line A549). The semipurified metabolites (eluted from a SepPak C(18) cartridge) of T. harzianum strain ES39 were analyzed by mass spectrometry (MS). Matrix-assisted laser desorption ionization and nanoflow electrospray ionization MS revealed five major peptaibols, each of which contained 18 residues and had a mass ranging from 1,719 to 1,775 Da. Their partial amino acid sequences were determined by collision-induced dissociation tandem MS.     

Immune cell phenotype and functional defects in Netherton syndrome

Background

Netherton syndrome (NS) is a rare life-threatening syndrome caused by SPINK5 mutations leading to a skin barrier defect and a severe atopic diathesis. NS patients are prone to bacterial infections, but the understanding of the underlying immune deficiency is incomplete.

Results

We analyzed blood lymphocyte phenotypes and function in relation to clinical infections in 11 Finnish NS patients, aged 3 to 17?years, and healthy age-matched controls. The proportion of B cells (CD19⁺) and naïve B cells (CD27^?, IgD⁺) were high while memory B cells (CD27⁺) and switched memory B cells (CD27⁺IgM^?IgD^?), crucial for the secondary response to pathogens, was below or in the lowest quartile of the reference values in 8/11 (73%) and 9/11 (82%) patients, respectively. The proportion of activated non-differentiated B cells (CD21^low, CD38l^ow) was below or in the lowest quartile of the reference values in 10/11 (91%) patients. Despite normal T cell counts, the proportion of naïve CD4⁺ T cells was reduced significantly and the proportion of CD8⁺ T central memory significantly elevated. An increased proportion of CD57⁺ CD8⁺ T cells indicated increased differentiation potential of the T cells. The proportion of cytotoxic NK cells was elevated in NS patients in phenotypic analysis based on CD56DIM, CD16⁺ and CD27^? NK cells but in functional analysis, decreased expression of CD107a/b indicated impaired cytotoxicity.The T and NK cell phenotype seen in NS patients also significantly differed from that of age-matched atopic dermatitis (AD) patients, indicating a distinctive profile in NS. The frequency of skin infections correlated with the proportion of CD62L⁺ T cells, naïve CD4⁺ and CD27⁺ CD8⁺ T cells and with activated B cells. Clinically beneficial intravenous immunoglobulin therapy (IVIG) increased naïve T cells and terminal differentiated effector memory CD8⁺ cells and decreased the proportion of activated B cells and plasmablasts in three patients studied.

Conclusions

This study shows novel quantitative and functional aberrations in several lymphocyte subpopulations, which correlate with the frequency of infections in patients with Netherton syndrome. IVIG therapy normalized some dysbalancies and was clinically beneficial.

     

Contact-dependent inhibition of angiogenesis by cardiac fibroblasts in three-dimensional fibrin gels in vitro: implications for microvascular network remodeling and coronary collateral formation

Angiogenesis and coronary artery collateral formation can improve blood flow and thereby prevent myocardial ischemia. The role of perivascular fibroblasts in neovascularization remains incompletely understood. Here we investigated the effects of epicardial and myocardial fibroblasts on angiogenesis in vitro by using a serum-free microcarrier-based fibrin gel angiogenesis system. To clearly distinguish between different cell types, we either stained endothelial cells or fibroblasts in the living with 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine-perchlorate (DiI). In cocultures, low numbers of heart fibroblasts stimulated endothelial sprouting, and capillary growth was also induced by fibroblast-conditioned media, indicating a paracrine mechanism. Capillary formation was decreased by increasing the density of fibroblasts in the cocultures, indicating contact-dependent inhibition. Using time-lapse studies, it turned out that close contacts between fibroblasts and endothelial cells resulted in rapid retraction of endothelial cells or, rarely, in cell death. Depending on the local ratio of fibroblasts to endothelial cell numbers, fibroblasts determined the location of capillary growth and the size of developing capillaries and thereby contributed to capillary network remodeling. In contrast to primary heart fibroblasts, NIH 3T3 fibroblasts did not display contact-dependent inhibition of endothelial sprouts. NIH fibroblasts were frequently seen in close association with endothelial capillaries, resembling pericytes. Contact-dependent inhibition of angiogenesis by epicardial fibroblasts could not be reversed by addition of neutralizing anti-TGF-β1 antibodies, by addition of serum, of medium conditioned by hypoxic tumor cells or myocardium, by various cytokines or by growing cocultures under hypoxic conditions. Our results implicate a pivotal role of periendothelial mesenchymal cells for the regulation of microvascular network remodeling and collateral formation. Received: 15 September 1997 / Accepted: 6 April 1998     

Gene deletions in X-linked muscular dystrophy

Of the approximately 170 families with X-linked muscular dystrophy of the Duchenne (DMD) and Becker (BMD) type in Finland, we have studied 90 unrelated patients for intragenic deletions by using the cDNA probes described by Koenig et al. Forty-five patients (50%) had molecular deletions of one or several of the 65 exon-containing HindIII fragments. In six deletion cases junction fragments of altered size were seen. Thirty-eight (84%) of the 45 deletions were detected using only two (1–2a and 8) of the six cDNA subclones. Using a wheelchair age of 12 years to distinguish between DMD and BMD, we found that the proportions of patients with deletions were similar. Deletions were equally common in familial and sporadic disease. BMD was more commonly caused by deletions in the 5' end of the gene than was DMD. In at least three instances deletions of similar type resulted in diseases of similar severity. Of 14 patients with mental retardation seven had deletions; six of these comprised exons contained in probe 8. We conclude that cDNA hybridization studies provide a powerful diagnostic tool in DMD and BMD and that they promise to produce better insights into molecular-clinical correlations.     

Peripheral blood leukocytes of cows with subclinical endometritis show an altered cellular composition and gene expression

Subclinical endometritis (SCE) is an important postpartum disease in dairy cows, but conventional cytobrush diagnosis often gives imprecise results. The aim of this study was to analyze disease-associated changes in peripheral blood as potential diagnostic parameters. Cellular subpopulations of blood leukocytes from cows with or without SCE (45–55 days postpartum) were flow-cytometrically quantified. Gene expression of whole blood leukocytes was assessed by PAXgene analysis. Subclinical endometritis cows showed significantly higher number of blood mononuclear cells and neutrophils. Among mononuclear cells, numbers of B-cells, NK-cells, and CD172a-positive monocytes were significantly elevated. Compared with non-SCE cows, blood leukocytes of SCE cows significantly expressed higher copy numbers of CXCL8, TNF, and IL12. To test whether circulating plasma factors are responsible for these changes, leukocytes, polymorphonuclear cells, and monocyte subpopulations (classical, intermediate, nonclassical) of healthy cows were stimulated with plasma of SCE and non-SCE cows. Although gene expression of whole leukocytes and polymorphonuclear cells remained unaltered, plasma from SCE animals significantly elevated expressed messenger RNA copy numbers of CXCL8, CXCL1, and IL1B in intermediate monocytes. In conclusion, elevated number of selected mononuclear subpopulations in peripheral blood and enhanced expression of distinct genes encoding for inflammatory mediators in blood leukocytes reflect the subclinical uterine inflammatory process in cows. Whether the observed changes in the periphery of SCE cows are the consequence of the uterine inflammatory process, or whether they affect the pathogenesis of the disease is currently unknown.     

16S rRNA targeted sandwich hybridization method for direct quantification of mycobacteria in soils

Boreal soils have been suspected reservoirs of infectious environmental mycobacteria. Detection of these bacteria in the environment is hampered by their slow growth. We applied a quantitative sandwich hybridization approach for direct detection of mycobacterial 16S rRNA in soil without a nucleic acid amplification step. The numbers of mycobacterial 16S rRNA molecules found in the soil indicated the presence of up to 10(7) to 10(8) mycobacterial cells per gram of soil. These numbers exceed by factor of 10 to 100 x the previous estimates of mycobacteria in soil based on culture methods. When real-time PCR with mycobacteria targeting primers was used to estimate the number of 16S rDNA copies in soil, one copy of 16S rDNA was detected per 10(4) copies of 16S rRNA. This is close to the number of 16S rRNA molecules detected per cell by the same method in laboratory pure cultures of M. chlorophenolicum. Therefore a major part of the mycobacterial DNA in the studied soils may thus have represented metabolically active cells. The 16S rRNA sandwich hybridization method described in this paper offers a culture independent solution for tracking environmental reservoirs of viable and potentially infectious mycobacteria.     

The thioredoxin system in aging muscle: key role of mitochondrial thioredoxin reductase in the protective effects of caloric restriction?

Cellular redox balance is maintained by various antioxidative systems. Among those is the thioredoxin system, consisting of thioredoxin, thioredoxin reductase, and NADPH. In the present study, we examined the effects of caloric restriction (2 mo) on the expression of the cytosolic and mitochondrial thioredoxin system in skeletal muscle and heart of senescent and young rats. Mitochondrial thioredoxin reductase (TrxR2) is significantly reduced in aging skeletal and cardiac muscle and renormalized after caloric restriction, while the cytosolic isoform remains unchanged. Thioredoxins (mitochondrial Trx2, cytosolic Trx1) are not influenced by caloric restriction. In skeletal and cardiac muscle of young rats, caloric restriction has no effect on the expression of thioredoxins or thioredoxin reductases. Enforced reduction of TrxR2 (small interfering RNA) in myoblasts under exposure to ceramide or TNF-alpha causes a dramatic enhancement of nucleosomal DNA cleavage, caspase 9 activation, and mitochondrial reactive oxygen species release, together with reduced cell viability, while this TrxR2 reduction is without effect in unstimulated myoblasts under basal conditions. Oxidative stress in vitro (H2O2 in C2C12 myoblasts and myotubes) results in different changes: TrxR2, Trx2, and Trx1 are induced without alterations in the cytosolic thioredoxin reductase isoforms. Thus aging is associated with a TrxR2 reduction in skeletal muscle and heart, which enhances susceptibility to apoptotic stimuli but is renormalized after short-term caloric restriction. Exogenous oxidative stress does not result in these age-related changes of TrxR2.     

The incidence and survival of acute de novo leukemias in Estonia and in a well-defined region of western Sweden during 1997–2001: A survey of patients aged ≥65 years

Background: In a recently published retrospective population-based study over three 5-year periods (1982–1996) we investigated the outcome for de novo acute leukemia (AL) patients aged ≥65 years at diagnosis in Estonia (a country that had been occupied by the Soviet Union over 5 decades) and in the so-called Western Swedish Health Care Region. The age-standardized yearly incidence rates regarding the total number of de novo AL was 5.3/100 000 inhabitant for Estonia and 8.0 for Sweden, this difference being statistically significant merely as regards acute myeloid leukemia (AML). The relative survival for the total cohort of de novo AL as well as for de novo AML was significantly longer (p < 0.001) for Swedish as compared to Estonian patients. Methods: In view of the miserable outcome for the Estonian patients we decided to prospectively compare the results for incidence and outcome of de novo AL between the two countries. Results: The present report covers the first 5-year period comprising 1997–2001 and deals only with patients aged ≥65 years at diagnosis. The age-adjusted annual incidence rates for de novo AML were lower in Estonia (6.4/100 000) than in Sweden (9.2/100 000) but not significantly so. The present results also show that the outcome for the Estonian AML patients had improved considerably over the study period; thus, at no time point, i.e., at 1, 3 and 5 years did relative survival between the two countries differ significantly. Conclusion: Yet, as compared to the Swedish cohort relative survival for the Estonian patients did still not reach an acceptable level.