Effect of environmental factors on biofilm formation by clinical<Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> isolates

The influence of environmental factors (temperature, aerobiosis-anaerobiosis, static-dynamic conditions, pH) was determined on biofilm formation by 51 S. maltophilia clinical isolates. The strains produced more biofilm at 32 degrees C than at 37 or 18 degrees C. Aerobic and 6% CO2 atmosphere yielded comparable biofilm amounts, higher than under anaerobic conditions. Biofilm production was not affected by static vs. agitated culture conditions. Biofilm production at pH 7.5 and 8.5 was comparable but significantly higher than at pH 5.5. The capacity of individual strains to form biofilm and thus contribute to the severity of some diseases is influenced by host traits and environmental conditions at the site of infection, and play an important role in the pathogenesis of biomaterial-related disease caused by S. maltophilia.     

Legumain/asparaginyl endopeptidase controls extracellular matrix remodeling through the degradation of fibronectin in mouse renal proximal tubular cells

Legumain/asparaginyl endopeptidase (EC 3.4.22.34) is a novel cysteine protease that is abundantly expressed in the late endosomes and lysosomes of renal proximal tubular cells. Recently, emerging evidence has indicated that legumain might play an important role in control of extracellular matrix turnover in various pathological conditions such as tumor growth/metastasis and progression of atherosclerosis. We initially found that purified legumain can directly degrade fibronectin, one of the main components of the extracellular matrix, in vitro. Therefore, we examined the effect of legumain on fibronectin degradation in cultured mouse renal proximal tubular cells. Fibronectin processing can be inhibited by chloroquine, an inhibitor of lysosomal degradation, and can be enhanced by the overexpression of legumain, indicating that fibronectin degradation occurs in the presence of legumain in lysosomes from renal proximal tubular cells. Furthermore, in legumain-deficient mice, unilateral ureteral obstruction (UUO)-induced renal interstitial protein accumulation of fibronectin and renal interstitial fibrosis were markedly enhanced. These findings indicate that legumain might have an important role in extracellular matrix remodeling via the degradation of fibronectin in renal proximal tubular cells.     

Novel mutation assay with high sensitivity based on direct measurement of genomic DNA alterations: comparable results to the Ames test

Almost all of the methodologies developed to date to assay the potential mutagenicity of chemical substances are based on detection of altered phenotypic traits. The alternative approach of directly screening the whole genome for mutations is not feasible because of the logistics of carrying out mass sequencing of genes. Here we describe a novel and highly sensitive mutation assay, which we term the 'genome profiling-based mutation assay' (GPMA) that directly detects mutations generated in genomic DNA. We used GPMA to detect mutations caused by known mutagens such as AF2 and ethidium bromide even at concentrations of 30 ppb. The number of mutations detected was dependent on the number of generations in culture and the concentrations of the mutagens. Almost complete agreement was observed between GPMA and the Ames test in the discrimination of mutagens (63 out of 64). Owing to the high sensitivity of GPMA, the effects of long-term and low-dose exposures and the influence of chemicals of low solubility can also be screened. Thus, genotype-based GPMA can complement the Ames test, which is the standard technology in this field and is based on phenotypic traits.     

Snapin, a new regulator of receptor signaling, augments alpha1A-adrenoceptor-operated calcium influx through TRPC6

Activation of G(q)-protein-coupled receptors, including the alpha(1A)-adrenoceptor (alpha(1A)-AR), causes a sustained Ca(2+) influx via receptor-operated Ca(2+) (ROC) channels, following the transient release of intracellular Ca(2+). Transient receptor potential canonical (TRPC) channel is one of the candidate proteins constituting the ROC channels, but the precise mechanism linking receptor activation to increased influx of Ca(2+) via TRPCs is not yet fully understood. We identified Snapin as a protein interacting with the C terminus of the alpha(1A)-AR. In receptor-expressing PC12 cells, co-transfection of Snapin augmented alpha(1A)-AR-stimulated sustained increases in intracellular Ca(2+) ([Ca(2+)](i)) via ROC channels. By altering the Snapin binding C-terminal domain of the alpha(1A)-AR or by reducing cellular Snapin with short interfering RNA, the sustained increase in [Ca(2+)](i) in Snapin-alpha(1A)-AR co-expressing PC12 cells was attenuated. Snapin co-immunoprecipitated with TRPC6 and alpha(1A)-AR, and these interactions were augmented upon alpha(1A)-AR activation, increasing the recruitment of TRPC6 to the cell surface. Our data suggest a new receptor-operated signaling mechanism where Snapin links the alpha(1A)-AR to TRPC6, augmenting Ca(2+) influx via ROC channels.     

Growth rate,health and welfare in a dairy herd with natural suckling until 6–8 weeks of age: a case report

Over a period of two years, growth rate and health were measured for dairy calves allowed to suckle their mothers up to 6–8 weeks of age. Thirty-one calves were weighted weekly, and the mean daily growth rate was 1.2 ± 0.03 kg from birth up to 13 weeks of age. Illness in calves and young stock was not observed. In the cows, the mean incidences of ketosis, displaced abomasum, puerperal paresis, mastitis, teat injury and retained placenta were 0, 0, 8, 22, 1 and 1%, respectively, during a 6-year period. The mean daily gain of 56 growing bulls was 1.4 kg when slaughtered at 15 months of age, which is higher than the mean daily gain of 0.95 kg in the population. Probiotics, hormones and vaccines were not used, and antibiotics were only used for treating illness. The present study indicates many advantages and few problems when dairy calves are penned together with the cows and allowed natural feeding up to 6–8 weeks of age. This production system was easy to manage, preferred by the farmer, and may satisfy the public concern regarding the practice of immediate separation of cow and calf in commercial milk production.     

<Emphasis Type="Italic">Mycoplasma alkalescens</Emphasis> demonstrated in bronchoalveolar lavage of cattle in Denmark

Mycoplasma alkalescens is an arginine-metabolizing mycoplasma, which has been found in association with mastitis and arthritis in cattle. Routine bacteriological examination of 17 bronchoalveolar lavage samples from calves with pneumonia in a single herd in Denmark, identified M. alkalescens in eight samples. The organism was found as a sole bacterilogical findings in five of the samples as well as in combination with Mannheimia haemolytica, Haemophilus somni and Salmonella Dublin. This is the first report of isolation of M. alkalescens in Denmark.