Translational readthrough of a termination codon in the yeast mitochondrial mRNA VAR1 as a result of mutation in the release factor mRF1

Yeast mitochondrial DNA codes for eight major polypeptides. Translation of he mitochondrially encoded polypeptides in strains with mutated mitochondrial release factor, mRF1, was found to result in the synthesis of a novel protein, V2. Different mrf1 alleles were associated with different efficiency of V2p synthesis. Translation of V2p was enhanced by paromomycin. Comparative analysis of peptides resulting from protease digestion indicated that V2p is a derivative of Var1p. According to our hypothesis, V2p represents a readthrough product of the natural stop codon in VAR1 mRNA.     

Solution structure of conformationally restricted vasopressin analogues

In recent years, a massive effort has been directed towards designing potent and selective antagonists of neurohypophyseal hormones substituted at position 3. Modification of vasopressin at position 3 with 4,4'-biphenylalanine results in pharmacologically inactive analogues. Chemically, this substitution appears to vary only slightly from those previously made by us (1-Nal or 2-Nal), which afforded potent agonists of V(2) receptors. In this situation, it seemed worthwhile to study the structure of the analogues with 4,4'-biphenylalanine (BPhe) at position 3 in aqueous solution using NMR spectroscopy and total conformational analysis. This contribution is part of extensive studies aimed at understanding spatial structures of 3-substituted [Arg(8)]vasopressin analogues of different pharmacological properties. NMR data were used to calculate 3D structures for all the analogues using two methods, EDMC with the ECEPP/3 force field, and molecular dynamic with the simulated annealing (SA) algorithm. The structures obtained by the first method show a better fit between the NMR spectral evidence and the calculation for all the peptides.     

A simple method for selection of trypsin chromogenic substrates using combinatorial chemistry approach

A tetrapeptide combinatorial library, considered as chromogenic substrates of bovine beta-trypsin, was synthesized by the solid phase method. The peptides contain an analog of p-nitroanilide, obtained by attaching 5-amino-2-nitrobenzoic acid (Anb(5,2)) to the C-termini. Deconvolution of the peptide library, performed in solution using an iterative method, yielded four efficient trypsin substrates. The most active one, Phe-Val-Pro-Arg-Anb(5,2)-NH(2), appeared to be 125-fold more active than Bz-D,L-Arg-pNA (BAPNA) used as a reference compound. The reported method of designing trypsin chromogenic substrate libraries is straightforward. Such p-nitroanilides may be useful for the investigation of any protease substrate specificity.     

A study for minisatellitic markers of Conidiobolus coronatus' pathogenicity to Galleria mellonella larvae

A selected panel of 13 colonies of entomopathogenic fungus Conidiobolus coronatus representing 6 variants of pathogenicity to Galleria mellonella larvae (ranged from 100 to 10% of efficiency), derived from single spores, were tested for the presence of hypervariable loci in their genomes by hybridization with Jeffreys' human minisatellite probe 33.6. The probe produced informative fingerprints and revealed slight differences among colonies analyzed. Up to 20 variable bands per colony were recognized in the size range of 2-20 kb. The band sharing within groups with the same pathogenicity ranged from 0.966 to 0.800. The genetic distance between different variants ranged from 0.026 to 0.282. A few characteristic bands for high and low pathogenicity to the larvae were found.     

Methylotrophic extremophilic yeast Trichosporon sp.: a soil-derived isolate with potential applications in environmental biotechnology

A yeast isolate revealing unique enzymatic activities and substrate-dependent polymorphism was obtained from autochthonous microflora of soil heavily polluted with oily slurries. By means of standard yeast identification procedures the strain was identified as Trichosporon cutaneum. Further molecular PCR product analyses of ribosomal DNA confirmed the identity of the isolate with the genus Trichosporon. As it grew on methanol as a sole carbon source, the strain appeared to be methylotrophic. Furthermore, it was also able to utilize formaldehyde. A multi-substrate growth potential was shown with several other carbon sources: glucose, glycerol, ethanol as well as petroleum derivatives and phenol. Optimum growth temperature was determined at 25 degrees C, and strong inhibition of growth at 37 degrees C together with the original soil habitat indicated lack of pathogenicity in warm-blooded animals and humans. The unusually high tolerance to xenobiotics such as diesel oil (>30 g/l), methanol (50 g/l), phenol (2 g/l) and formaldehyde (7.5 g/l) proved that the isolate was an extremophilic organism. With high-density cultures, formaldehyde was totally removed at initial concentrations up to 7.5 g/l within 24 h, which is the highest biodegradation capability ever reported. Partial biodegradation of methanol (13 g/l) and diesel fuel (20 g/l) was also observed. Enzymatic studies revealed atypical methylotrophic pathway reactions, lacking alcohol oxidase, as compared with the conventional methylotroph Hansenula polymorpha. However, the activities of glutathione-dependent formaldehyde dehydrogenase, formaldehyde reductase, formate dehydrogenase and unspecific aldehyde dehydrogenase(s) were present. An additional glutathione-dependent aldehyde dehydrogenase activity was also detected. Metabolic and biochemical characteristics of the isolated yeast open up new possibilities for environmental biotechnology. Some potential applications in soil bioremediation and wastewater decontamination are discussed.     

Association of model peptides and dehydropeptides: N-acetyl-L-butyrine and (Z)-dehydrobutyrine N',N'-dimethylamides

These comparative studies on the aggregation behaviour of Ac-(Z)-DeltaAbu-NMe(2) and Ac-L-Abu-NMe(2) in carbon tetrachloride were performed by the analysis of their FTIR spectra and by theoretical calculations. The percentage of the monomeric form (alpha) decreased as concentration increased and this occurred to a higher degree for the (Z)-DeltaAbu derivative than for its saturated analogue. The dimerization constant K(D), calculated on the basis of the intensity of the monomer and associate bands in the nu(s)(N-H) vibration region, is by three orders of magnitude larger for Ac-(Z)-DeltaAbu-NMe(2) than for Ac-L-Abu-NMe(2). The obtained dimer geometries of the dehydro- compound were calculated by the B3LYP/6-31+G** method.     

Conformational properties of N-acetyl-N-methyl-alpha,beta-dehydroalanine N'-methylamide

The conformational properties of Ac-Delta(Me)Ala-NHMe (N-acetyl-N-methyl-alpha,beta-dehydroalanine N'-methylamide), as the simplest model of N-methyl-alpha,beta-dehydroamino acids, was examined with theoretical methods and in comparison with Ac-DeltaAla-NHMe and Ac-DeltaAla-NMe(2). The N-terminal amide of the Delta(Me)Ala residue easily adopts the configuration cis and the torsion angles phi, psi are highly flexible. The Delta(Me)Ala residue is a conformational flexibilizer as compared to the parent DeltaAla, which is a conformational stiffener. This seems to be the reason why Delta(Me)Ala is found in small natural cyclic peptides, where it ensures the conformational flexibility necessary for biological action.     

Synthetic peptides mimicking antigenic epitope of Helicobacter pylori urease

Short peptides resembling the Helicobacter pylori urease antigen (UreB F8 Ser-Ile-Lys-Glu-Asp-Val-Gln-Phe) with deleted aspartic acid and glutamic acid residues, anchored through a triazine linker via the N-terminal moiety to cellulose plate were prepared. The peptides were used for binding of antibodies from sera of patients with medically confirmed atherosclerosis. Recognition of the peptides was also tested with anti-Jack beans urease antibodies. The important role of a Gly-Gly spacer separating the peptides from the cellulose support was shown. Different patterns of binding of antibodies from H. pylori infected patients and anti-Jack bean urease antibodies were observed only in the case of pentapeptides. The peptide Gly-Gly-Leu-Val-Phe-Lys-Thr was recognized by most of the tested sera.     

Genetic structure is influenced by environmental barriers: empirical evidence from the common voleMicrotus arvalis populations

One of the major challenges in population biology is the identification of barriers to gene flow and/or secondary contacts between differentiated entities. The level of genetic differentiation among eight populations of the common voleMicrotus arvalis (Pallas, 1779) around the Biebrza Wetlands, NE Poland was examined by analyzing seven microsatellite loci for 140 voles and testing for the presence of barriers to gene flow. Overall population differentiation was moderate and significant (F _ST = 0.081,p < 0.001) and there was no correlation between geographical and genetic distances among populations. We found a relatively high level of genetic variability within the populations studied. This could be explained by male bias in dispersal, a phenomenon recently found inM. arvalis. Patterns of genetic structure visualized in synthetic genetic maps showed clear gradients along a southeast-northwest axis across the study area, as well as the presence of a potential barrier to dispersal. The position of a barrier to gene flow identified using Monmonier’s maximum difference algorithm likely corresponds to humid habitats of the Biebrza Wetlands. These results suggest that the presence of environmental barriers to gene flow and drift may be responsible for the observed spatial genetic structure ofM. arvalis in the Biebrza Valley. Institute of Biology, University of Białystok, OEwierkowa 20 B, 15-950 Białystok, Poland,