The effect of immobilization on body fluid volume in the rat

Total body water (TBW) and extracellular volume (EVC) were measured simultaneously with³H and⁸²Br in male albino rats after two, four and eight weeks of immobilization. On the basis of these measurements the intracellular fluid volume (ICV) was calculated. It was found that immobilization caused no changes in the volume and distribution of body fluids.     

Multi-locus sequence typing (MLST) of non-fermentative Gram-negative bacilli isolated from bloodstream infections in southern Poland

Non-fermentative Gram-negative bacilli are now one of the most important causes of severe infections in Polish hospitals. Acinetobacter species are serious concern because of the high prevalence of multi-drug resistance among strains. Resistance profiles for 53 Gram-negative non-fermentative blood isolates were done. MLST was carried out using 44 strains representing the most commonly isolated species: A. baumannii, P. aeruginosa, and S. maltophilia. MLST revealed that all 22 A. baumannii belonged to sequence type (ST) 2. The P. aeruginosa isolates belonged to 10 different STs. Four S. maltophilia isolates matched STs present in the database (ST4, ST15, ST116, ST142), seven isolates showing novel sequence types. Among P. aeruginosa and S. maltophilia PFGE confirmed the genetical variety of strains.     

Genetic composition of interspecific potato somatic hybrids and autofused 4<Emphasis Type="Italic">x</Emphasis> plants evaluated by DArT and cytoplasmic DNA markers

Key message

Using DArT analysis, we demonstrated that all Solanum × michoacanum (+) S. tuberosum somatic hybrids contained all parental chromosomes. However, from 13.9 to 29.6 % of the markers from both parents were lost in the hybrids.

Abstract

Somatic hybrids are an interesting material for research of nucleus-cytoplasm interaction and sources of new nuclear and cytoplasmic combinations. Analyses of genomes of somatic hybrids are essential for studies on genome compatibility between species, its evolution and are important for their efficient exploitation. Diversity array technology (DArT) permits analysis of the composition of nuclear DNA of somatic hybrids. The nuclear genome compositions of 97 Solanum × michoacanum (+) S. tuberosum [mch (+) tbr] somatic hybrids from five fusion combinations and 11 autofused 4x mch were analyzed for the first time based on DArT markers. Out of 5358 DArT markers generated in a single assay, greater than 2000 markers were polymorphic between parents, of which more than 1500 have a known chromosomal location on potato genetic or physical map. DArT markers were distributed along the entire length of 12 chromosomes. We noticed elimination of markers of wild and tbr fusion components. The nuclear genome of individual somatic hybrids was diversified. Mch is a source of resistance to Phytophthora infestans. From 97 mch (+) tbr somatic hybrids, two hybrids and all 11 autofused 4x mch were resistant to P. infestans. The analysis of the structure of particular hybrids’ chromosomes indicated the presence of markers from both parental genomes as well as missing markers spread along the full length of the chromosome. Markers specific to chloroplast DNA and mitochondrial DNA were used for analysis of changes within the organellar genomes of somatic hybrids. Random and non-random segregations of organellar DNA were noted.

     

Usefulness of real-time PCR in long-term follow-up of follicular lymphoma patients

The aim of this study was to evaluate the usefulness of quantitative real-time PCR (RQ-PCR) for the monitoring of molecular remission in follicular lymphoma (FL) patients during long-term follow-up. RQ-PCR by the use of TaqMan detection system is a sensitive tool to monitor minimal residual disease (MRD) in FL through amplification of the t(14;18) fusion gene during and post-therapy. In most cases the breakpoint region occurs within the major breakpoint region (MBR). Among 75 patients diagnosed with FL, cells harboring the fusion gene BCL2/JH were found in peripheral blood of 31 patients (41%). We further monitored 30 of these patients in a period varying from 6 months to 5 years by RQ-PCR. In our study the level indicating the possibility of the presence of MRD was established at more than five t(14;18)-positive cells in the background of 83,000 normal cells. The results of this work also confirmed that the presence of MRD detected by RQ-PCR is an indication for careful observation of patients because of a higher risk of disease recurrence.     

Engineered resistance against proteinases

Exogenous proteinase inhibitors are valuable and economically interesting protective biotechnological tools. We examined whether small proteinase inhibitors when fused to a selected target protein can protect the target from proteolytic degradation without simultaneously affecting the function and activity of the target domain. Two proteinase inhibitors were studied: a Kazal-type silk proteinase inhibitor (SPI2) from Galleria mellonella, and the Cucurbita maxima trypsin inhibitor I (CMTI I). Both inhibitors target serine proteinases, are small proteins with a compact structure stabilized by a network of disulfide bridges, and are expressed as free polypeptides in their natural surroundings. Four constructs were prepared: the gene for either of the inhibitors was ligated to the 5' end of the DNA encoding one or the other of two selected target proteins, the coat protein (CP) of Potato potyvirus Y or the Escherichia coli beta-glucuronidase (GUS). CMTI I fused to the target proteins strongly hampered their functions. Moreover, the inhibitory activity of CMTI I was retained only when it was fused to the CP. In contrast, when fused to SPI2, specific features and functions of both target proteins were retained and the inhibitory activity of SPI2 was fully preserved. Measuring proteolysis in the presence or absence of either inhibitor, we demonstrated that proteinase inhibitors can protect target proteins used either free or as a fusion domain. Interestingly, their inhibitory efficiency was superior to that of a commercial inhibitor of serine proteinases, AEBSF.     

Yolk protein is expressed in the insect testis and interacts with sperm

Background  

Male and female gametes follow diverse developmental pathways dictated by their distinct roles in fertilization. While oocytes of oviparous animals accumulate yolk in the cytoplasm, spermatozoa slough off most of their cytoplasm in the process of individualization. Mammalian spermatozoa released from the testis undergo extensive modifications in the seminal ducts involving a variety of glycoproteins. Ultrastructural studies suggest that glycoproteins are involved in sperm maturation in insects; however, their characterization at the molecular level is lacking. We reported previously that the circadian clock controls sperm release and maturation in several insect species. In the moth, Spodoptera littoralis, the secretion of glycoproteins into the seminal fluid occurs in a daily rhythmic pattern. The purpose of this study was to characterize seminal fluid glycoproteins in this species and elucidate their role in the process of sperm maturation.     

LTHREADER: prediction of extracellular ligand-receptor interactions in cytokines using localized threading

Identification of extracellular ligand-receptor interactions is important for drug design and the treatment of diseases. Difficulties in detecting these interactions using high-throughput experimental techniques motivate the development of computational prediction methods. We propose a novel threading algorithm, LTHREADER, which generates accurate local sequence-structure interface alignments and integrates various statistical scores and experimental binding data to predict interactions within ligand-receptor families. LTHREADER uses a profile of secondary structure and solvent accessibility predictions with residue contact maps to guide and constrain alignments. Using a decision tree classifier and low-throughput experimental data for training, it combines information inferred from statistical interaction potentials, energy functions, correlated mutations, and conserved residue pairs to predict interactions. We apply our method to cytokines, which play a central role in the development of many diseases including cancer and inflammatory and autoimmune disorders. We tested our approach on two representative families from different structural classes (all-alpha and all-beta proteins) of cytokines. In comparison with the state-of-the-art threader RAPTOR, LTHREADER generates on average 20% more accurate alignments of interacting residues. Furthermore, in cross-validation tests, LTHREADER correctly predicts experimentally confirmed interactions for a common binding mode within the 4-helical long-chain cytokine family with 75% sensitivity and 86% specificity with 40% gain in sensitivity compared to RAPTOR. For the TNF-like family our method achieves 70% sensitivity with 55% specificity with 70% gain in sensitivity. LTHREADER combines information from multiple complex templates when such data are available. When only one solved structure is available, a localized PSI-BLAST approach also outperforms standard threading methods with 25%-50% improvements in sensitivity.     

Genotoxicity of alpha-asarone analogues

The role of cholesterol in the formation of atherosclerotic lesions during hypercholesterolemia has been confirmed. alpha-Asarone is a substance of a potent hypolipidemic activity which is isolated from plants. We previously described the synthesis of several alpha-asarone analogues exhibiting hypolipidemic and antiplatelet activity. Genotoxic activity of four selected alpha-asarone analogues was theoretically evaluated based on quantum-mechanical method for calculation of enthalpy of carbocations formation (DeltaH(R)) according to the Testa's method. In the present paper, we evaluated the mutagenic and genotoxic activity of alpha-asarone isomers 2-5 based on the reference Ames test and micronucleus test. Results obtained in the study show that tested isomers were non-mutagenic, however, they exhibited growing cytotoxic activity. Relationship between the heat of formation of their putative metabolic intermediates and mutagenic/genotoxic activity was not confirmed.     

Synthesis, alpha 1-adrenoceptor antagonist activity, and SAR study of novel arylpiperazine derivatives of phenytoin

In the search for new antiarrhythmic agents, some active 2-methoxyphenylpiperazine derivatives of phenytoin were obtained as a chemical modification of compound AZ-99 (3-ethyl-1-[2-hydroxy-3-(4-phenylpiperazin-1-yl)-propyl]-2,4-dioxo-5,5-diphenylimidazolidine). These compounds possessed structural properties similar to those of alpha(1)-adrenoceptor antagonists. In the present study, the affinities of the 2-methoxyphenylpiperazine derivatives (1a-3a) for alpha(1)- and alpha(2)-adrenoceptors were evaluated using radioligand ([(3)H]prazosin, [(3)H]clonidine) binding assays. In the next step, a new series of phenylpiperazine derivatives of phenytoin (4a-16a) containing 2-methoxyphenyl-, 2-ethoxyphenyl-, 2-pyridyl- or 2-furoylpiperazine moiety, as well as, various ester or alkyl substituents at 3-position of hydantoin ring were synthesized. The newly synthesized compounds were tested for their affinity to alpha(1)- and alpha(2)-adrenoceptors. They have shown affinities for alpha(1)-adrenoceptors at nanomolar to submicromolar range. Some compounds were moderately selective ligands of alpha(1)-adrenoceptors. Selected compounds (3a-5a, 7a, 13a, 14a) were also evaluated for their alpha(1)-adrenoceptor antagonistic properties in functional bioassays. A SAR study indicated that the most active compounds contain 2-alkoxyphenylpiperazine moieties and methyl or 2-methylpropionate substituent at 3-N position in hydantoin. The exchange of 2-alkoxyphenyl moiety into 2-furoyl or 2-pyridyl group significantly decreased affinities for alpha(1)-adrenoceptors. Molecular modelling results obtained using conformational analysis CONFLEX and PM5 method for geometry optimization, allowed for comparison of the spatial properties of tested compounds with pharmacophore model created by Barbaro et al. for the ideal alpha(1)-adrenoceptor antagonist.     

Morphine and HIV-Tat increase microglial-free radical production and oxidative stress: possible role in cytokine regulation

Opiate abuse alters the progression of human immunodeficiency virus and may increase the risk of neuroAIDS. As neuroAIDS is associated with altered microglial reactivity, the combined effects of human immunodeficiency virus-Tat and morphine were determined in cultured microglia. Specifically, experiments determined the effects of Tat and morphine on microglial-free radical production and oxidative stress, and on cytokine release. Data show that combined Tat and morphine cause early and synergistic increases in reactive oxygen species, with concomitant increases in protein oxidation. Furthermore, combined Tat and morphine, but not Tat or morphine alone, cause reversible decreases in proteasome activity. The effects of morphine on free radical production and oxidative stress are prevented by pre-treatment with naloxone, illustrating the important role of opioid receptor activation in these phenomena. While Tat is well known to induce cytokine release from cultured microglia, morphine decreases Tat-induced release of the cytokines tumor necrosis factor-α and interleukin-6, as well as the chemokine monocyte chemoattractant protein-1 (MCP-1). Finally, experiments using the reversible proteasome inhibitor MG115 show that temporary, non-cytotoxic decreases in proteasome activity increase protein oxidation and decrease tumor necrosis factor-α, interleukin-6, and MCP-1 release from microglia. Taken together, these data suggest that oxidative stress and proteasome inhibition may be involved in the immunomodulatory properties of opioid receptor activation in microglia.