Application of cation-exchange membranes for characterisation and imaging ammonia-oxidising bacteria in soils

A new approach, in which ammonia-oxidizing bacteria (AOB) are entrapped from soil onto cation-exchange membranes, was applied to identify terrestrial AOB by fluorescence in situ hybridization (FISH). An experimental hot spot of ammonia oxidation was developed by establishing a gradient of ammonium substrate (200 to <20 mg NH4+-N l(-1)) diffused through the cation-exchange membranes incubated in soil for 6 months. By this approach we were able to characterise and image indigenous AOB populations growing in heavily oil-polluted soil using FISH and sequence analysis of PCR-amplified 16S rRNA genes, respectively. The FISH results revealed that Nitrosospira-like AOB were dominant on the ammonium-enriched membranes incubated in the soil. Fourteen unique Nitrosospira-like 16S rRNA gene sequences belonging to clusters 2 and 3 were recovered from the soil-incubated membranes and from the soil, suggesting the importance of Nitrosospira-like AOB in the oil-polluted landfarming soil.     

Severe acute respiratory syndrome coronavirus fails to activate cytokine-mediated innate immune responses in cultured human monocyte-derived dendritic cells

Activation of host innate immune responses was studied in severe acute respiratory syndrome coronavirus (SCV)-infected human A549 lung epithelial cells, macrophages, and dendritic cells (DCs). In all cell types, SCV-specific subgenomic mRNAs were seen, whereas no expression of SCV proteins was found. No induction of cytokine genes (alpha interferon [IFN-alpha], IFN-beta, interleukin-28A/B [IL-28A/B], IL-29, tumor necrosis factor alpha, CCL5, or CXCL10) or IFN-alpha/beta-induced MxA gene was seen in SCV-infected A549 cells, macrophages, or DCs. SCV also failed to induce DC maturation (CD86 expression) or enhance major histocompatibility complex class II expression. Our data strongly suggest that SCV fails to activate host cell cytokine gene expression in human macrophages and DCs.     

Properties of hemagglutination by Prevotella melaninogenica

Although Prevotella melaninogenica belongs to the commensal oral microbiota, some strains possess putative virulence factors. For example, we have previously described fimbriated, hemagglutinating strains of P. melaninogenica, isolated from patients with periodontal disease. The aim of this investigation was to compare some chemical and physical properties of hemagglutination (HA) of P. melaninogenica with those of other pigmented gram-negative anaerobes. HA of 13 P. melaninogenica strains proved to be considerably weaker than that of the major periodontal pathogen, Porphyromonas gingivalis. Vigorous shaking reduced HA of shaken cells but the shaken supernatant had the same hemagglutinating activity as non-shaken cells. The hemagglutinating agent on P. melaninogenica seemed to be a protein, which can be separated from the cell and binds to lactose-, galactose-, and raffinose-containing carbohydrates on the erythrocytes. Adherence to epithelial cells did not differ significantly between the hemagglutinating and non-hemagglutinating strains of P. melaninogenica. Although P. melaninogenica is able to agglutinate erythrocytes, this potential virulence factor is of a considerably lower magnitude than that of major periodontal pathogens.     

Cranefly (Diptera: Tipuloidea) fauna of a boreal mire system in relation to mire trophic status: implications for conservation and bioassessment

Craneflies (Diptera Tipuloidea) are a typical but poorly known insect group in various moist environments, such as mires. The area of natural mires has strongly decreased in Finland, and there is an urgent need to study and describe the fauna of mires and to determine whether different mire categories support different assemblages of craneflies that might have indicator value. Craneflies were studied using Malaise traps in the Kauhaneva mire system in minerotrophic and ombrotrophic sites, the former subdivided into meso- and oligotrophic sites. A total of 29 cranefly species were recorded. Species richness was highest in mesotrophic sites while the number of species was equally low in oligo- and ombrotrophic sites. Phylidorea squalens, Erioptera flavata, Pedicia rivosa and Tricyphona immaculata were identified as indicators for mesotrophic sites, but no indicators were found for oligo- or ombrotrophic sites. No differences between the species composition of minerotrophic (meso- and oligotrophic combined) and ombrotrophic sites were detected, but when three classes of trophic status were compared, a statistical difference was found. Cranefly species richness in Kauhaneva was low compared to pristine spring habitats. Our results imply, that a focus towards conservation and restoration of mire types with high trophic status would benefit also the conservation of cranefly diversity in the boreal ecoregion. Bioassesments and ecological surveys of craneflies should be designed to cover adequately all trophic status classes within a mire, and especially the mire types with highest trophic status. We also review the distribution and ecology of some potentially regionally threatened cranefly species.     

Autoantibodies against oxidized LDL are associated with severe chest pain attacks in patients with coronary heart disease

Autoantibodies against oxidized low-density lipoprotein (oxLDL) predict the progression of atherosclerosis. Several studies have shown that oxLDL is present in atherosclerotic lesions and that several factors present in active atherosclerotic plaques can oxidatively modify LDL. Oxidation of LDL induces production of autoantibodies against oxLDL (oxLDLab) that can be measured using an EIA test. Our aim was to see whether oxLDLab are associated with severe chest pain attacks in coronary heart disease (CHD) patients. Patients having two- or three-vessel CHD, as assessed by coronary angiography, and their siblings were recruited into the study (n = 568, mean age 55.8 years, range 29.3–83.2 years). Nondiabetic patients having a history of severe chest pain attacks had significantly higher oxLDLab levels (0.611 ± 0.56) than those who did not have a history of severe chest pain attacks (0.487 ± 0.40) (p = 0.027), even though age, cholesterol level, body mass index, and blood pressure were similar in both groups. However, no difference was found in oxLDLab levels in diabetic patients with or without a history of severe chest pain attacks. Increased levels of oxLDL autoantibodies are associated with severe chest pain attacks in nondiabetic patients with CHD.     

Induction of CD69 expression and Th1 cytokines release from human peripheral blood lymphocytes after in vitro stimulation with Alloiococcus otitidis and three middle ear pathogens

Alloiococcus otitidis is a recently discovered pathogen of otitis media. However, only a limited number of studies are available about the pathogenic and immunological role of A. otitidis. The aim of this study was to investigate the activation and the cytokine production of human peripheral blood lymphocytes at the early immune response after stimulation with A. otitidis. After stimulation of whole human peripheral blood lymphocytes for 18 h with whole killed A. otitidis or the three major middle ear pathogens (Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis), the expression of CD69 and the production of cytokines were analyzed. The expression of CD69 on T cells and B cells was dose-dependently enhanced after stimulation with A. otitidis. The release of interleukin (IL)-12 was induced after stimulation with A. otitidis, whereas the release of IL-4 was not induced after stimulation with A. otitidis. In addition, the release of interferon (IFN)-gamma was induced after stimulation with A. otitidis. Although the release of IFN-gamma started within 18 h after stimulation with A. otitidis, intracellular production of IFN-gamma was not observed in either CD4+ T cells or CD8+ T cells within 18 h upon stimulation. The patterns of CD69 expression and T helper-type 1 (Th1)-promoting cytokines production were similarly shown when human peripheral blood lymphocytes were stimulated with the other three major pathogens. Our results suggest that A. otitidis has sufficient immunogenic potential to modulate a host immune response, like the other three major middle ear pathogens, and also suggest that the immunogenicity of A. otitidis is very similar, at the early immune response, to that of the three major middle ear pathogens.     

Effects of acid sphingomyelinase deficiency on male germ cell development and programmed cell death

Deficiency of acid sphingomyelinase (ASM), an enzyme responsible for producing a pro-apoptotic second messenger ceramide, has previously been shown to promote the survival of fetal mouse oocytes in vivo and to protect oocytes from chemotherapy-induced apoptosis in vitro. Here we investigated the effects of ASM deficiency on testicular germ cell development and on the ability of germ cells to undergo apoptosis. At the age of 20 weeks, ASM knock-out (ASMKO) sperm concentrations were comparable with wild-type (WT) sperm concentrations, whereas sperm motility was seriously affected. ASMKO testes contained significantly elevated levels of sphingomyelin at the age of 8 weeks as detected by high-performance, thin-layer chromatography. Electron microscopy revealed that the testes started to accumulate pathological vesicles in Sertoli cells and in the interstitium at the age of 21 days. Irradiation of WT and ASMKO mice did not elevate intratesticular ceramide levels at 16 h after irradiation. In situ end labeling of apoptotic cells also showed a similar degree of cell death in both groups. After a 21-day recovery period, the numbers of primary spermatocytes and spermatogonia at G2 as well as spermatids were essentially the same in the WT and ASMKO testes, as detected by flow cytometry. In serum-free cultures both ASMKO and WT germ cells showed a significant increase in the level of ceramide, as well as massive apoptosis. In conclusion, ASM is required for maintenance of normal sphingomyelin levels in the testis and for normal sperm motility, but not for testicular ceramide production or for the ability of the germ cells to undergo apoptosis.     

Physical changes of β-sitosterol crystals in oily suspensions during heating

The aim of this research was to describe the thermal behavior of β-sitosterol crystals in oil-suspensions with a focus on the role of water during heating. The suspensions were prepared by recrystallization in order to achieve a microcrystalline particle size. The structural changes together with the mechanical properties of the suspensions during heating were studied by using variable temperature X-ray powder diffractometry (VT-XRPD), differential scanning calorimetry (DSC), and dynamic mechanical analysis (DMA). Hydrated β-sitosterol crystals in an oil-suspension, dehydrated, despite the composition of the suspensions, at low temperatures. At high β-sitosterol concentration, the monohydrate crystal form changed partially to a hemihydrated form, and when only a small amount of water was initially incorporated, the hemihydrate crystal form dehydrated to a mostly anhydrate crystal form. The released water, which was immiscible in the surrounding oil, caused the recrystallization of hydrated β-sitosterol during cooling. This procedure indicated a reversible dehydration process. Structural and thermal analysis of β-sitosterol crystals in suspensions, together with mechanical analysis made it possible to understand various physical changes during heating. Published: October 19, 2005     

Mechano-acoustic determination of Young's modulus of articular cartilage

The compressive stiffness of an elastic material is traditionally characterized by its Young's modulus. Young's modulus of articular cartilage can be directly measured using unconfined compression geometry by assuming the cartilage to be homogeneous and isotropic. In isotropic materials, Young's modulus can also be determined acoustically by the measurement of sound speed and density of the material. In the present study, acoustic and mechanical techniques, feasible for in vivo measurements, were investigated to quantify the static and dynamic compressive stiffness of bovine articular cartilage in situ. Ultrasound reflection from the cartilage surface, as well as the dynamic modulus were determined with the recently developed ultrasound indentation instrument and compared with the reference mechanical and ultrasound speed measurements in unconfined compression (n=72). In addition, the applicability of manual creep measurements with the ultrasound indentation instrument was evaluated both experimentally and numerically. Our experimental results indicated that the sound speed could predict 47% and 53% of the variation in the Young's modulus and dynamic modulus of cartilage, respectively. The dynamic modulus, as determined manually with the ultrasound indentation instrument, showed significant linear correlations with the reference Young's modulus (r(2)=0.445, p<0.01, n=70) and dynamic modulus (r(2)=0.779, p<0.01, n=70) of the cartilage. Numerical analyses indicated that the creep measurements, conducted manually with the ultrasound indentation instrument, were sensitive to changes in Young's modulus and permeability of the tissue, and were significantly influenced by the tissue thickness. We conclude that acoustic parameters, i.e. ultrasound speed and reflection, are indicative to the intrinsic mechanical properties of the articular cartilage. Ultrasound indentation instrument, when further developed, provides an applicable tool for the in vivo detection of cartilage mechano-acoustic properties. These techniques could promote the diagnostics of osteoarthrosis.     

Lucigenin and coelenterazine as superoxide probes in mitochondrial and bacterial membranes

The chemiluminescent superoxide indicators lucigenin and coelenterazine were compared in rat liver submitochondrial particles and cytoplasmic membranes from Paracoccus denitrificans. Qualitative monitoring is possible with both probes, but quantitative work with lucigenin is hampered by its dependence on one-electron reduction before the photon-emitting reaction. Therefore, calibration of measurements on complex I, capable of efficient lucigenin prereduction with reduced nicotinamide adenine dinucleotide, against xanthine oxidase, which in the presence of hypoxanthine is not able to reduce the probe to a significant rate compared to complex I, may give results in error by one order of magnitude. Coelenterazine, although susceptible of storage-dependent high background chemiluminescence, does not require prereduction and is thus a more reliable probe.