Peculiarities of dopamine receptors on the membrane of multipolar spinal cord neurons of the brook lamprey <Emphasis Type="Italic">Lampetra planeri</Emphasis>

On isolated multipolar neurons of spinal cord of amniocoete (larva of the brook lamprey Lampetra planeri) by the patch-clamp method in configuration “the whole cell,” a modulating effect of dopamine on potential-activated Na⁺ currents was studied. Application of dopamine (10 μM) was shown to produce a complex action on the sodium current amplitude. In some cases a decrease of the amplitude, on average, by 13.5 ± 2.2% was found, while in others—an increase, on average, by 8.6 ± 6.1%. The modulation dopamine effect was not accompanied by any changes either of the threshold of the current appearance or of resistance of neuronal cell membranes. Pharmacological analysis with use of dopamine agonist has shown that the agonist of D1-receptors (−)-SKF-38393 (10 μM) decreases the Na+ current amplitude, whereas the agonist of D2-receptors (−)-quinpirole (10 μM) can produce in different cells both an increase, by 30.7 ± 17.0%, and a decrease, by 13.2 ± 3.1%, of the Na+ current amplitude. The obtained data indicate the existence of D1-and D2-receptors on the membrane of multipolar spinal neurons of the amniocoete (larva of the brook lamprey). Study of action of antagonists has shown that the antagonist of D1-receptors (+)-SCH-23390 (10 μM) does not affect action of the agonist of D1-receptors (−)-SKF-38393 (10 μM); the antagonist of D2-receptors (−)-sulpiride (10 μM) blocks completely effects both of the agonist of D1-receptors (−)-SKF-38393 (10 μM) and of the agonist of D2-receptors (−)-quinpirole (10 μM). The antagonist of D1-receptors (+)-SCH-23390 (10 μM) produced no effect on action of the agonist of D1-receptors (−)-SKF-38393 (10 μM). The obtained data indicate peculiarities of dopamine receptors of Cyclostomata as compared with those in mammals. Original Russian Text ? A. A. Bukinich, E. A. Tsvetkov, and N. P. Vesselkin, 2007, published in Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, 2007, Vol. 43, No. 1, pp. 39–45.     

Rational design of a multiepitope vaccine encoding T-lymphocyte epitopes for treatment of chronic hepatitis B virus infections

Protein sequences from multiple hepatitis B virus (HBV) isolates were analyzed for the presence of amino acid motifs characteristic of cytotoxic T-lymphocyte (CTL) and helper T-lymphocyte (HTL) epitopes with the goal of identifying conserved epitopes suitable for use in a therapeutic vaccine. Specifically, sequences bearing HLA-A1, -A2, -A3, -A24, -B7, and -DR supertype binding motifs were identified, synthesized as peptides, and tested for binding to soluble HLA. The immunogenicity of peptides that bound with moderate to high affinity subsequently was assessed using HLA transgenic mice (CTL) and HLA cross-reacting H-2^bxd (BALB/c × C57BL/6J) mice (HTL). Through this process, 30 CTL and 16 HTL epitopes were selected as a set that would be the most useful for vaccine design, based on epitope conservation among HBV sequences and HLA-based predicted population coverage in diverse ethnic groups. A plasmid DNA-based vaccine encoding the epitopes as a single gene product, with each epitope separated by spacer residues to enhance appropriate epitope processing, was designed. Immunogenicity testing in mice demonstrated the induction of multiple CTL and HTL responses. Furthermore, as a complementary approach, mass spectrometry allowed the identification of correctly processed and major histocompatibility complex-presented epitopes from human cells transfected with the DNA plasmid. A heterologous prime-boost immunization with the plasmid DNA and a recombinant MVA gave further enhancement of the immune responses. Thus, a multiepitope therapeutic vaccine candidate capable of stimulating those cellular immune responses thought to be essential for controlling and clearing HBV infection was successfully designed and evaluated in vitro and in HLA transgenic mice.     

HysTag--a novel proteomic quantification tool applied to differential display analysis of membrane proteins from distinct areas of mouse brain

A novel isotopically labeled cysteine-tagging and complexity-reducing reagent, called HysTag, has been synthesized and used for quantitative proteomics of proteins from enriched plasma membrane preparations from mouse fore- and hindbrain. The reagent is a 10-mer derivatized peptide, H(2)N-(His)(6)-Ala-Arg-Ala-Cys(2-thiopyridyl disulfide)-CO(2)H, which consists of four functional elements: i) an affinity ligand (His(6)-tag), ii) a tryptic cleavage site (-Arg-Ala-), iii) Ala-9 residue that contains four (d(4)) or no (d(0)) deuterium atoms, and iv) a thiol-reactive group (2-thiopyridyl disulfide). For differential analysis cysteine residues in the compared samples are modified using either (d(4)) or (d(0)) reagent. The HysTag peptide is preserved in Lys-C digestion of proteins and allows charge-based selection of cysteine-containing peptides, whereas subsequent tryptic digestion reduces the labeling group to a di-peptide, which does not hinder effective fragmentation. Furthermore, we found that tagged peptides containing Ala-d(4) co-elute with their d(0)-labeled counterparts. To demonstrate effectiveness of the reagent, a differential analysis of mouse forebrain versus hindbrain plasma membranes was performed. Enriched plasma membrane fractions were partially denatured, reduced, and reacted with the reagent. Digestion with endoproteinase Lys-C was carried out on nonsolubilized membranes. The membranes were sedimented by ultra centrifugation, and the tagged peptides were isolated by Ni(2+) affinity or cation-exchange chromatography. Finally, the tagged peptides were cleaved with trypsin to release the histidine tag (residues 1-8 of the reagent) followed by liquid chromatography tandem mass spectroscopy for relative protein quantification and identification. A total of 355 unique proteins were identified, among which 281 could be quantified. Among a large majority of proteins with ratios close to one, a few proteins with significant quantitative changes were retrieved. The HysTag offers advantages compared with the isotope-coded affinity tag reagent, because the HysTag reagent is easy to synthesize, economical due to use of deuterium instead of (13)C isotope label, and allows robust purification and flexibility through the affinity tag, which can be extended to different peptide functionalities.     

Benthic decomposition of Zostera marina roots: a controlled laboratory experiment

The initial benthic decomposition of Zostera marina roots was studied in a controlled flow-through chamber experiment for 23 days. Sediment chambers without added roots served as controls. The inflowing and outflowing artificial seawater (ASW) was analyzed for O₂, ΣCO₂, urea-N, NH₄⁺ and NO₂⁻+NO₃⁻. Sediment profiles of Eh, particulate organic carbon (POC) and nitrogen, dissolved organic nitrogen (DON), dissolved free amino acids (DFAA), urea-N, NH₄⁺, DFAA and urea turnover rates, sulfate reduction and counts of total anaerobic heterotrophic bacteria and different functional groups were determined. Fluxes of O₂, ΣCO₂, urea-N and NH₄⁺ were stimulated during root decomposition compared to the unamended control. There were indications of stimulated bacterial growth based on counts of total anaerobic heterotrophic bacteria, anaerobic phosphatase utilizers, ammonifyers and sulfate reducers. Independent estimates of nitrogen and carbon incorporation into bacterial biomass during root decomposition indicate that a major fraction of the nitrogen for microbial growth was mobilized from the indigenous particulate organic nitrogen (PON) pool, whereas the energy source for bacterial growth was mainly obtained from the added eelgrass roots. Most of the nitrogen mineralized during root decomposition was incorporated into the bacterial biomass resulting in a low efflux of urea-N and inorganic nitrogen from the sediment to the water column.     

Urea production by Thiosphaera pantotropha and by anaerobic enrichment cultures from marine sediments

Abstract The production of urea by Thiosphaera pantotropha was studied. Batch cultures were grown on acetate as energy source and with NO₃⁻ or O₂ as terminal electron acceptor. Urea accumulated in the media during exponential growth in aerobic and anaerobic cultures of T. pantotropha . Urea production continued after the cells had entered the stationary growth phase. Bacterial ability to produce urea was supported by studies of cultures enriched for denitrifying, sulphate-reducing and fermenting bacteria. The results implied that urea production was common among bacteria normally considered to be important in marine sediments.     

PURIFICATION OF PHOSPHATE-DEPENDENT PIG BRAIN GLUTAMINASE

Abstract— A procedure for preparing highly purified phosphate-activated glutaminase (EC 3.5.1.2, L-glutamine amidohydrolase) from pig brain is described. The main steps consist of extraction with acetone, followed by sodium sulphate fractionation of the solubilized acetone powder. Thereafter, solubilization by dialysis against a buffer containing tris-HC1, mercaptoethanol, and EDTA, followed by precipitation with phosphate-borate, is repeated twice. The final preparation contains no impurities which can be detected by polyacrylamide gel electrophoresis, isoelectric focusing, and sedimentation equilibrium centrifugation. By the latter method, molecular weight is determined to be 187,000. By polyacrylamide gel electrophoresis in sodium dodecyl sulphate, one protein band with molecular weight 64,000 is found.     

Rapid quantitative assay for acaricidal effects on Sarcoptes scabiei var. suis and Otodectes cynotis

Brimer et al. (Vet. Parasitol. 51: 123-135, 1993 and 59: 249-255, 1995) developed a migration assay for acaricidal effect of acetylcholinesterase inhibitors and macrocyclic lactones utilising Sarcoptes scabiei var. suis mites. In contrast to many others, this assay is fully quantitative but quite time-consuming. The aim of the present investigation was to modify this assay to become faster and simpler. As a result accurate determinations can now be obtained within 6h, as opposed to 24h. Furthermore it was demonstrated that also Otodectes cynotis mites can be used with only minor modifications of the procedures. The cholinesterase inhibitor diazinon and the formamide amitraz were used as acaricides. Thus, the mite migration assay now has been proven useful for acaricidal compounds belonging to three chemical groups with different modes of action, namely organophosphorous cholinesterase inhibitors, macrocyclic lactones acting on the glutamanergic/GABAegic motoneurons, and formamide inhibitors of the octopamine systems of arthropods.     

A Study on the Effects of Tactical Drenchings with a Benzimidazole against Ostertagiasis in Calves

In the present study tactical fenbendazole treatments against ostertagiasis in calves were given at short intervals towards the end of the grazing season. This prevented clinical disease and had moderate effects on parasitic loads in animals that stayed on the same pasture throughout the season. The feasibility of tactical treatments is discussed in relation to various strategical control measures.