Similar integration but different stability of Alus and LINEs in the human genome

Alus and LINEs (LINE1) are widespread classes of repeats that are very unevenly distributed in the human genome. The majority of GC-poor LINEs reside in the GC-poor isochores whereas GC-rich Alus are mostly present in GC-rich isochores. The discovery that LINES and Alus share similar target site duplication and a common AT-rich insertion site specificity raised the question as to why these two families of repeats show such a different distribution in the genome. This problem was investigated here by studying the isochore distributions of subfamilies of LINES and Alus characterized by different degrees of divergence from the consensus sequences, and of Alus, LINEs and pseudogenes located on chromosomes 21 and 22. Young Alus are more frequent in the GC-poor part of the genome than old Alus. This suggests that the gradual accumulation of Alus in GC-rich isochores has occurred because of their higher stability in compositionally matching chromosomal regions. Densities of Alus and LINEs increase and decrease, respectively, with increasing GC levels, except for the telomeric regions of the analyzed chromosomes. In addition to LINEs, processed pseudogenes are also more frequent in GC-poor isochores. Finally, the present results on Alu and LINE stability/exclusion predict significant losses of Alu DNA from the GC-poor isochores during evolution, a phenomenon apparently due to negative selection against sequences that differ from the isochore composition.     

Functional specialization of the epithelium in the mesonephric tubules

An electron microscopy study was aimed to correlate structural differentiation of the epithelium in mesonephric proximal tubules (PT) with the expression of membrane activities of alkaline phosphatase (AP) and 5'-nucleotidase (AMP). Tissue samples of mesonephros were taken from 5 to 16 days old chick embryos. Both enzymes were detected with cerium technique, Mayahara modification of lead capture method was used also for localization of AP. Control incubation was performed with levamisole. The formation of absorptive apparatus was characterized by the differentiation of PT epithelium. Activities of AP and AMP appeared to increase rapidly with the differentiation of epithelium. Reaction products of AP and AMP were detected on brush border as well as on membranes of tubular invaginations, transport tubules and endocytotic vacuoles. The basolateral cell surfaces of epithelium were projected in short interdigitating microvilli and the expression of AP and AMP activities on their membranes suggested the transport role of this structural specialization.     

Findings of mycobacteria in insectivores and small rodents

The organs of 30 insectivorous mammals and 62 rodents from areas inhabited by people or livestock where cattle paratuberculosis or mycobacterial infections of swine had been found to occur were examined by cultivation during the monitoring of occurrence and spread of mycobacterioses in cattle and swine. Mycobacteria were found in the organs of 3 insectivores (10%) and 6 rodents (9.7%). Mycobacterium chelonae was isolated from the organs of the lesser white-toothed shrew (Crocidura suaveolens) and the common vole (Microtus arvalis), and M. vaccae and M. avium subsp. avium (IS901+, serotype 1) from the organs of the common shrew (Sorex araneus). M. avium subsp. avium (IS901+, serotype 1) was also isolated from the organs of the yellow-necked mouse (Apodemus flavicollis). Slow-growing mycobacteria of group III (according to Runyon) were isolated from the organs of the mouse (Mus musculus sensu lato) and the yellow-necked mouse (A. flavicollis). These findings had no connection with the epizootological situation in the nearby livestock. M. fortuitum was isolated from the organs of the common vole (M. arvalis) caught in a field within easy reach of a swine breeding herd. M. fortuitum was also identified in the lymph nodes and droppings of this swine herd, as well as in the straw, scrapings from the floor of stalls, troughs and banisters, as well as from larvae and imagoes of dipterous insects. These results demonstrate the possibility that insectivores and small rodents can spread the causative agents of mycobacteria in wild and domestic animals.     

Metabolic profiling of a potential antifungal drug, 3-(4-bromophenyl)-5-acetoxymethyl-2,5-dihydrofuran-2-one, in mouse urine using high-performance liquid chromatography with UV photodiode-array and mass spectrometric detection

3-(4-bromophenyl)-5-acetyloxymethyl-2,5-dihydrofuran-2-one (LNO-18-22) is a representative member of a novel group of potential antifungal drugs, derived from a natural 3,5-disubstituted butenolide, (-)incrustoporine, as a lead structure. This lipophilic compound is characterized by high in vitro antifungal activity and low acute toxicity. For the purpose of in vivo studies, a new bioanalytical high-performance liquid chromatographic method with UV photodiode-array and mass spectrometric detection (HPLC-PDA-MS), involving a direct injection of diluted mouse urine was developed and used in the evaluation of the metabolic profiling of this drug candidate. The separation of LNO-18-22 and its phase I metabolites was performed in 37 min on a 125 mmx4 mm chromatographic column with Purospher RP-18e using an acetonitrile-water gradient elution. Scan mode of UV detection (195-380 nm) was employed for the identification of the parent compound and its biotransformation products in the biomatrix. Finally, the identity of LNO-18-22 and its metabolites was confirmed using HPLC-MS analyses of the eluate. These experiments demonstrated the power of a comprehensive analytical approach based on the combination of xenobiochemical methods and the results from tandem HPLC-PDA-MS (chromatographic behaviour, UV and MS spectra of native metabolites versus synthetic standards). The chemical structures of five phase I LNO-18-22 metabolites and one phase II metabolite were elucidated in the mouse urine, with two of these metabolites having very unexpected structures.     

Technical note monitoring native vegetation on a dumpsite of PCB-contaminated soil

Composition of native vegetation on a polychlorinated biphenyls (PCB)-contaminated soil dumpsite at Lhenice, South Bohemia (Czech Republic), was determined and species variability in the accumulation of PCBs in plant biomass was investigated. Soil stripping contaminated by PCBs originated at a factory producing electrical transformers that mostly used the commercial PCB mixture Delor 103 and 106. The PCB content of soil in the most contaminated part of the dumpsite reached 153 mg kg(-1) dry soil. Low diversity of plant species was found on the dumpsite. Results showed three grass species, Festuca arundinacea Schreb., Phalaroides arundinacea (L.) Rauschert., and Calamagrostis epigeios (L.) Roth., to be the major components of the vegetation and confirmed their high tolerance toward PCB contamination. The highest content of PCB in plant biomass--813.2 microg kg(-1) dry biomass--was determined in Festuca aboveground biomass. For phytoextraction purposes especially, Festuca can be recommended due to its high biomass yield, but its bioconcentration factor was very low (0.006). Tripleurospermum maritimum (L.) Sch. Bip. and Cirsium arvense (L.) Scop. grew mainly at the margins of the most contaminated part of the dumpsite. The PCB content determined in their aboveground biomass-278.7 and 289.5 microg kg(-1) dry biomass, respectively--was nonsignificantly lower compared to grass species Phalaroides and Calamagrostis. Salix (Salix viminalis L. and Salix caprea L.) was monitored among plant species composition at this site as a representative of woody species.     

Isolation, biochemical characterization and crystallization of the p15gag proteinase of myeloblastosis associated virus expressed in E. coli.

1. The p15gag proteinase responsible for the processing of the polyprotein precursor of the myeloblastosis associated virus was obtained by a recombinant technique in an E. coli expression system. The massive expression of the intentionally truncated precursor (Pr25lac-delta gag) was accompanied by its structurally correct processing. 2. Three procedures for the purification of the recombinant proteinase from both the cytoplasmic fraction and the inclusion bodies were developed. 3. The purified proteinase was compared with the authentic proteinase isolated from MAV virions by N-terminal sequence analysis and amino acid analysis, molecular weight determination, reverse-phase HPLC and FPLC elution profiles, electrophoretic mobility and isoelectric point determination, and activity assays with proteins and synthetic substrates. The identity of both enzymes was shown. 3. Contrary to reported data, the amino acid sequence of the p15gag proteinase differs from the sequence of the homologous Rous sarcoma virus proteinase in one residue only, as follows from cDNA sequencing. 4. Crystallization of the proteinase from a citrate-phosphate buffer at pH 5.6 afforded hexagonal crystals which diffracted well as 2.3 A without deterioration.     

Molecular Mechanisms Leading to Neuroprotection/Ischemic Tolerance: Effect of Preconditioning on the Stress Reaction of Endoplasmic Reticulum

Ischemic tolerance can be developed by prior ischemic non-injurious stimulus preconditioning. The molecular mechanisms underlying ischemic tolerance are not yet fully understood. The purpose of this study is to evaluate the effect of preconditioning/preischemia on ischemic brain injury. We examined the endoplasmic reticulum stress response (unfolded protein response (UPR)) by measuring the mRNA and protein levels of specific genes such as ATF6, GRP78, and XBP1 after 15 min 4-VO ischemia and different times of reperfusion (1, 3, and 24 h). The data from the group of naïve ischemic rats were compared with data from the group of preconditioned animals. The results of the experiments showed significant changes in the gene expression at the mRNA level in the all ischemic/reperfusion phases. The influence of preischemia on protein level of XBP was significant in later ischemic times and at 3 h, the reperfusion reached 230% of the controls. The protein levels of GRP78 in preischemic animals showed a significant increase in ischemic and reperfusion times. They exceeded to 50% levels of corresponding naïve ischemic/reperfusion groups. Preconditioning also induced remarkable changes in the levels of ATF6 protein in the ischemic phase (about 170%). The levels of ATF6 remained elevated in earlier reperfusion times (37 and 62%, respectively) and persisted significantly elevated after 24 h of reperfusion. This data suggest that preconditioning paradigm (preischemia) underlies its neuroprotective effect by the attenuation of ER stress response after acute ischemic/reperfusion insult.     

Biologically Active Metabolites Produced by the Basidiomycete Quambalaria cyanescens

Four strains of the fungus Quambalaria cyanescens (Basidiomycota: Microstromatales), were used for the determination of secondary metabolites production and their antimicrobial and biological activities. A new naphthoquinone named quambalarine A, (S)-(+)-3-(5-ethyl-tetrahydrofuran-2-yliden)-5,7,8-trihydroxy-2-oxo-1,4-naphthoquinone (1), together with two known naphthoquinones, 3-hexanoyl-2,5,7,8-tetrahydroxy-1,4-naphthoquinone (named here as quambalarine B, 2) and mompain, 2,5,7,8-tetrahydroxy-1,4-naphthoquinone (3) were isolated. Their structures were determined by single-crystal X-ray diffraction crystallography, NMR and MS spectrometry. Quambalarine A (1) had a broad antifungal and antibacterial activity and is able inhibit growth of human pathogenic fungus Aspergillus fumigatus and fungi co-occurring with Q. cyanescens in bark beetle galleries including insect pathogenic species Beauveria bassiana. Quambalarine B (2) was active against several fungi and mompain mainly against bacteria. The biological activity against human-derived cell lines was selective towards mitochondria (2 and 3); after long-term incubation with 2, mitochondria were undetectable using a mitochondrial probe. A similar effect on mitochondria was observed also for environmental competitors of Q. cyanescens from the genus Geosmithia.     

Changes in cell adhesivity and cytoskeleton‐related proteins during imatinib‐induced apoptosis of leukemic JURL‐MK1 cells

The fusion protein Bcr–Abl, which is the molecular cause of chronic myelogenous leukemia (CML) interacts in multiple points with signaling pathways regulating the cellular adhesivity and cytoskeleton architecture and dynamics. We explored the effects of imatinib mesylate, an inhibitor of Bcr–Abl protein used in front‐line CML therapy, on the adhesivity of JURL‐MK1 cells to fibronectin and searched for underlying changes in the cell proteome. As imatinib induces apoptosis of JURL‐MK1 cells, we used three different caspase inhibitors to discriminate between direct consequences of Bcr–Abl inhibition and secondary changes related to the apoptosis. Imatinib treatment caused a transient increase in JURL‐MK1 cell adhesivity to fibronectin, possibly due to the switch off of Bcr–Abl activity. Subsequently, we observed a number of changes including a decrease in cell adhesivity, F‐actin decomposition, reduction of integrin β1, CD44, and paxillin expression levels and a marked increase in cofilin phophorylation at Ser3. These events were generally related to the proceeding apoptosis but they differed in their sensitivity to the individual caspase inhibitors. J. Cell. Biochem. 111: 1413–1425, 2010. © 2010 Wiley‐Liss, Inc.