Histone H1 and chromatin interactions in human fibroblast nuclei after H1 depletion and reconstitution with H1 subfractions.

BACKGROUND: Linker histones constitute a family of lysine-rich proteins associated with nucleosome core particles and linker DNA in eukaryotic chromatin. In permeabilized cells, they can be extracted from nuclei by using salt concentration in the range of 0.3 to 0.7 M. Although other nuclear proteins are also extracted at 0.7 M salt, the remaining nucleus represents a template that is relatively intact. METHODS: A cytochemical method was used to study the affinity of reconstituted linker histones for chromatin in situ in cultured human fibroblasts. We also investigated their ability to condense chromatin by using DNA-specific osmium ammine staining for electron microscopy. RESULTS: Permeabilized and H1-depleted fibroblast nuclei were suitable for the study of linker histone-chromatin interactions after reconstitution with purified linker histone subfractions. Our results showed that exogenous linker histones bind to chromatin with lower affinity than the native ones. We detected no significant differences between the main H1 and H1 degrees histone fractions with respect to their affinity for chromatin or in their ability to condense chromatin. CONCLUSIONS: Linker histone interactions with chromatin are controlled also by mechanisms independent of linker histone subtype composition.     

L-Tenuazonic Acid,a New Inhibitor of Paenibacillus Larvae

A search for bioactive compounds, inhibitors of Paenibacillus larvae, the causal agent of American foulbrood, a honeybees' disease, was carried on. Extracts of two fungal strains, Alternaria brassicicola and Alternaria raphani, isolated from pollen collected from beehives, exhibited a specific inhibitory activity against this bacterium. From these extracts and by means of chromatographic steps and bioassay-guided fractionation, three tetramic acids were isolated. The compounds were identified by spectroscopic methods and the absolute stereochemistry was chemically determined. L-Tenuazonic acid was shown to be responsible for the antibiotic activity. This compound showed a MIC of 32 μg/ml, comparable with that of oxytetracycline, an antibiotic currently used for the prevention of American foulbrood. This revised version was published online in July 2006 with corrections to the Cover Date.     

In vivo 5-flourouracil-induced apoptosis on murine thymocytes: involvement of FAS,Bax and Caspase3

Apoptosis is a highly regulated and programmed cell breakdown process characterized by numerous changes. It was reported as the major mechanism of anticancer drug-induced cells death. Unfortunately, many of these drugs are non-specific and cause severe side effects. The effects of 5-fluorouracil (5-FU) on the apoptotic events in normal murine thymus were evaluated using an in vivo model. A single dose of 5-FU (150 mg/kg ip) was injected to CF-1 mice. A multiparametric analysis of thymic weight, cellularity, viability, architectural organization, apoptosis, DNA fragmentation, and the expression of several apoptotic proteins was evaluated in 10 days time-course study post-5-FU dosing. Total organ weights, thymocyte counts, and cell viabilities diminished drastically from the second day. The thymus architecture assessed through electron scanning microscopy revealed deep alterations and the lost of cell-cell contact between the first and the third days. DNA fragmentation and apoptotic indexes (May Grünwald Giemsa staining, double fluorescent dyes, and TdT-mediated dUTP nick-end labeling assay) revealed that cell death was maximal on the second day (three times over control). Furthermore, the pro-apoptotic proteins FAS and Bax were strongly up-regulated during the first 2 days. The aforementioned morphological and biochemical changes were also accompanied within the same period by caspase 3 activation. This study revealed that in vivo apoptosis in normal thymus after 5-FU administration is related to FAS, Bax, and Caspase 3 co-expressions under the current experimental conditions, these findings, therefore, contribute to a new insight into the molecular mechanisms involved during 5-FU administration upon the thymus and the possible events committed in the lymphophenia associated with chemotherapy.     

Passive leg movement enhances interstitial VEGF protein, endothelial cell proliferation, and eNOS mRNA content in human skeletal muscle

The present study used passive limb movement as an experimental model to study the effect of increased blood flow and passive stretch, without enhanced metabolic demand, in young healthy male subjects. The model used was 90 min of passive movement of the leg leading to a 2.8-fold increase (P < 0.05) in blood flow without a significant enhancement in oxygen uptake. Muscle interstitial fluid was sampled with microdialysis technique and analyzed for vascular endothelial growth factor (VEGF) protein and for the effect on endothelial cell proliferation. Biopsies obtained from the musculus vastus lateralis were analyzed for mRNA content of VEGF, endothelial nitric oxide synthase (eNOS), and matrix metalloproteinase-2 (MMP-2). The passive leg movement caused an increase (P < 0.05) in interstitial VEGF protein concentration above rest (73 +/- 21 vs. 344 +/- 83 pg/ml). Addition of muscle dialysate to cultured endothelial cells revealed that dialysate obtained during leg movement induced a 3.2-fold higher proliferation rate (P < 0.05) than dialysate obtained at rest. Passive movement also enhanced (P < 0.05) the eNOS mRNA level fourfold above resting levels. VEGF mRNA and MMP-2 mRNA levels were unaffected. The results show that a session of passive leg movement, elevating blood flow and causing passive stretch, augments the interstitial concentrations of VEGF, the proliferative effect of interstitial fluid, and eNOS mRNA content in muscle tissue. We propose that enhanced blood flow and passive stretch are positive physiological stimulators of factors associated with capillary growth in human muscle.     

Serum Selenium Concentrations Correlate Significantly with Inflammatory Biomarker High-sensitive CRP Levels in Hungarian Gestational Diabetic and Healthy Pregnant Women at Mid-pregnancy

Selenium is an essential trace element and a component of various enzymes with antioxidant functions. High-sensitive C-reactive protein (hsCRP) is an early indicator of increased lipid peroxidation. The serum selenium concentration, lipid parameters, and hsCRP values of gestational diabetic pregnant women (GD), control pregnant women (CP), and healthy nonpregnant controls (HC) were compared. Blood was taken between the 24th and the 28th week of pregnancy when the oral glucose tolerance test was performed. Selenium concentration was determined by atomic absorption spectrometry after hydride generation. HsCRP was measured by immunturbidimetry. HC had significantly higher serum selenium concentrations than GD and CP women (HC = 77.4 ± 14.82, GD = 51.7 ± 11.62, and CP = 40.5 ± 8.03 μg/l, respectively). HsCRP values of both GD and nondiabetic pregnant women were significantly higher compared to controls. Significant negative correlations were found between serum selenium and total cholesterol, low-density lipoprotein cholesterol, and hsCRP values indicating that low selenium levels are associated with increased lipid peroxidation. Serum selenium concentrations of Hungarian pregnant women are low compared to internationally published data.     

Decolourisation of mushroom farm wastewater by <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>

Mushroom production on coffee pulp as substrate generates an intense black residual liquid, which requires suitable treatment. In the present study, Pleurotus ostreatus growth in wastewater from mushroom farm was evaluated as a potential biological treatment process for decolourisation as well as to obtain biomass (liquid inoculum). Culture medium components affecting mycelial growth were determined, evaluating colour removal. Laccase activity was monitored during the process. P. ostreatus was able to grow in non diluted WCP. Highest biomass yield was obtained when glucose (10 g/l) was added. The addition of this carbon source was necessary for efficient decolourisation. Agitation of the culture improved biodegradation of WCP as well as fungal biomass production. Laccase and manganese-independent peroxidase activities were detected during fungal treatment of the WCP by P. ostreatus CCEBI 3024. The laccase enzyme showed good correlation with colour loss. Both wastewater colour and pollution load (as chemical oxygen demand) decreased more than 50% after 10 days of culture. Phenols were reduced by 92%.     

Intermediate filament-like proteins in bacteria and a cytoskeletal function in Streptomyces

Actin and tubulin cytoskeletons are conserved and widespread in bacteria. A strikingly intermediate filament (IF)-like cytoskeleton, composed of crescentin, is also present in Caulobacter crescentus and determines its specific cell shape. However, the broader significance of this finding remained obscure, because crescentin appeared to be unique to Caulobacter. Here we demonstrate that IF-like function is probably a more widespread phenomenon in bacteria. First, we show that 21 genomes of 26 phylogenetically diverse species encoded uncharacterized proteins with a central segmented coiled coil rod domain, which we regarded as a key structural feature of IF proteins and crescentin. Experimental studies of three in silico predicted candidates from Mycobacterium and other actinomycetes revealed a common IF-like property to spontaneously assemble into filaments in vitro. Furthermore, the IF-like protein FilP formed cytoskeletal structures in the model actinomycete Streptomyces coelicolor and was needed for normal growth and morphogenesis. Atomic force microscopy of living cells revealed that the FilP cytoskeleton contributed to mechanical fitness of the hyphae, thus closely resembling the function of metazoan IF. Together, the bioinformatic and experimental data suggest that an IF-like protein architecture is a versatile design that is generally present in bacteria and utilized to perform diverse cytoskeletal tasks.     

Synthesis and in silico biological activity evaluation of new N-substituted pyrazolo-oxazin-2-one systems

Cyclisation of pyrazolo-beta-enaminones 3 readily obtained from 4-aceto acetyl pyrazol 2 with triphosgene led to the formation of N-substituted pyrazolo-1,3-oxazin-2-ones 4 in good yields. Estimation of pharmacotherapeutic potential, possible molecule mechanisms of action, toxic/side effects and interaction with drug-metabolizing enzymes were made for synthesised compounds on the basis of prediction of activity spectra for substances (PASS) prediction results and their analysis by PharmaExpert software. COX inhibition predicted by PASS was confirmed by experimental evaluation.