NS3 Peptide, a novel potent hepatitis C virus NS3 helicase inhibitor: its mechanism of action and antiviral activity in the replicon system.

Hepatitis C virus (HCV) chronic infections represent one of the major and still unresolved health problems because of low efficiency and high cost of current therapy. Therefore, our studies centered on a viral protein, the NS3 helicase, whose activity is indispensable for replication of the viral RNA, and on its peptide inhibitor that corresponds to a highly conserved arginine-rich sequence of domain 2 of the helicase. The NS3 peptide (p14) was expressed in bacteria.

Regulation of sugar catabolism in Lactococcus lactis.

The increasing number of genomic and post-genomic studies on Gram-positive organisms and especially on lactic acid bacteria brings a lot of information on sugar catabolism in these bacteria. Like for many other bacteria, glucose is the most preferred source of carbon and energy for Lactococcus lactis. Other carbon sources can induce their own utilization in the absence of well-metabolized sugar.

Putative type IV secretion genes in Bacillus anthracis.

Although the physiology of Bacillus anthracis, the causative agent of anthrax, has been studied extensively, we still do not know how toxins are dispatched from the bacterial cell. Here, by means of distant homology and genome context analyses, we identify genes encoding putative type IV secretion system-related elements on the B. anthracis plasmids pXO1 and pXO2 and in the chromosome.

In vitro DNA binding of purified CcpA protein from Lactococcus lactis IL1403.

During this study His-tagged CcpA protein purified under native conditions to obtain a biologically active protein was used for molecular analysis of CcpA-dependent regulation. Using electrophoretic mobility shift assays it was demonstrated that CcpA of L. lactis can bind DNA in the absence of the HPr-Ser-P corepressor and exhibits DNA-binding affinity for nucleotide sequences lacking cre sites.

Infection of tobacco with different Pseudomonas syringae pathovars leads to distinct morphotypes of programmed cell death.

Tobacco plants (Nicotiana tabacum cv. Xanthi-nc) infiltrated with either of two pathovars of Pseudomonas syringae- an avirulent strain of P. syringae pv.

Precise bacterial polyprenol length control fails in Saccharomyces cerevisiae.

A comparison of amino acid sequences of yeast Rer2p and Srt1p Z-prenyltransferases shows that the spatial organization of their substrate tunnels agrees with that determined by X-ray for the E. coli undecaprenyl diphosphate synthase (UPPs). The observed trend in the maxima of product length distribution shifted from C(55) in UPPs to C(80) in Rer2p and to C(110) in Srt1p.

Searching for a new anti-HCV therapy: synthesis and properties of tropolone derivatives.

Hepatitis C virus (HCV) is considered one of the most dangerous pathogens since about 3% of the world population is HCV-infected and the virus is a major cause of hepatitis, cirrhosis, and liver carcinoma. A need for a more efficient therapy prompted us to investigate new class of compounds, such as tropolone derivatives that possess antiviral, antibacterial, and antifungal activities. To synthesize bromo- and morpholinomethyl-analogues of tropolone, the previously reported methods were modified.

Interplay between the cis-prenyltransferases and polyprenol reductase in the yeast Saccharomyces cerevisiae.

Dolichol formation is examined in three Saccharomyces cerevisiae strains with mutations in the ERG20 gene encoding farnesyl diphosphate synthase (mevalonic acid pathway) and/or the ERG9 gene encoding squalene synthase (sterol synthesis pathway) differing in the amount and chain length of the polyisoprenoids synthesized. Our results suggest that the activities of two yeast cis-prenyltransferases Rer2p and Srt1p and polyprenol reductase are not co-regulated and that reductase may be the rate-limiting enzyme in dolichol synthesis if the amount of polyisoprenoids synthesized exceeds a certain level. We demonstrate that reductase preferentially acts on typical polyprenols with 13-18 isoprene residues but can reduce much longer polyprenols with even 32 isoprene residues.

Virus-like particles of potato leafroll virus as potential carrier system for nucleic acids.

Potato leafroll virus is a member of the polerovirus genus. The isometric virion is formed by a coat protein encapsidating single-stranded, positive-sense, mono-partite genomic RNA with covalently attached viral protein at the 5' end. The coat protein of the virus exists in two forms: i) a 23 kDa protein, the product of the coat protein gene, and ii) a 78 kDa protein, the product of the coat protein gene and an additional open reading frame expressed by read-through of the coat protein gene stop codon.

Protein production and secretion in an Aspergillus nidulans mutant impaired in glycosylation.

O-glycosylation has been considered a limiting factor in protein secretion in filamentous fungi. Overexpression of the yeast DPM1 gene encoding dolichylphosphate mannose synthase (DPMS) in an Aspergillus nidulans mutant (BWB26A) deficient in O-glycosylation caused an increase in the number of secretory vesicles and changes in protein secretion. However, the secretory proteins, primarily O-mannosylated glucoamylase and N-glycosylated invertase, were mainly trapped in the periplasmic space.

Direct fluorometric measurement of hepatitis C virus helicase activity.

The non-structural protein 3 (NS3) of hepatitis C virus (HCV) is a highly promising target for anti-HCV therapy because of its multiple enzymatic activities, such as RNA-stimulated nucleoside triphosphatase, RNA helicase and serine protease. The helicase domain of NS3 as well as domain 2 of the helicase were expressed in a baculovirus system to obtain in high yield active proteins for prospective studies of complexes of the helicase with its inhibitors. A novel direct fluorometric test of helicase activity with a quenched DNA substrate, 3' labeled with a Cy3 dye and 5' labeled with a Black Hole Quencher, was developed and optimal reaction conditions established.

Somatic hybrids Solanum nigrum (+) S. tuberosum: morphological assessment and verification of hybridity.

Somatic hybrids between the cultivated potato diploid hybrid clone, ZEL-1136, and hexaploid non-tuber-bearing wild species Solanum nigrum L. exhibiting resistance to Phytophthora infestans were regenerated after PEG-mediated fusion of mesophyll protoplasts. The objective was to transfer the late-blight resistance genes from the wild species into plants of the cultivated potato clone.

The ORFO product of Potato leafroll virus is indispensable for virus accumulation.

Using a cDNA expression cassette in combination with agroinoculation of potato leaf discs we have investigated the role the protein encoded by ORF0 of Potato leafroll virus (PLRV) and have shown its importance for virus accumulation. Two mutations introduced into ORF0 by site-directed mutagenesis prevented expression of the corresponding protein and completely abolished virus accumulation in plant cells. They did not, however, affect translation of ORF1 and ORF2.

Mutational analysis of the proteinase function of Potato leafroll virus.

cDNA expression vectors of Potato leafroll virus (PLRV) were used to analyse specific mutations in the proteinase and replicase domains of the proteins encoded by ORF1 and ORF2. Agrobacterium-mediated DNA transfer was used to introduce a PLRV RNA expression unit, controlled by the 35S promoter of Cauliflower mosaic virus, into potato leaf cells. Expression of unmodified PLRV cDNA led to the replication of viral genomic and subgenomic RNAs and accumulation of the viral capsid protein, whereas alteration of amino acids GDD513-515 of the replicase to VHD abolished PLRV replication.

A novel family of longer chain length dolichols present in oleate-induced yeast Saccharomyces cerevisiae.

The typical size of the yeast dolichol family ranges from 14 to 19 isoprene units D((14-19)) with dolichol(16) being the dominating species. Induction of peroxisome proliferation by growing the cells in medium containing oleate as carbon source induces the synthesis of an additional family of longer dolichols D((19-24)) with D(21) being the most prominent. This phenomenon is abolished in the peroxisome biogenesis deficient strain in which the PEX1 gene (encoding Pex1p peroxin) has been disrupted.

The role of ERG20 gene (encoding yeast farnesyl diphosphate synthase) mutation in long dolichol formation. Molecular modeling of FPP synthase.

The yeast Saccharomyces cerevisiae strain LB332 bearing a mutation in the ERG20 gene encoding farnesyl diphosphate synthase (FPPS) synthesizes significantly longer dolichols than the wild type strain FL100 (14-31 and 14-19 isoprene units, respectively). The measurement of the short chain prenyl alcohols excreted into the medium shows that increased amounts of geraniol, dimethylallyl and isopentenyl alcohols but not farnesol are synthesized by the mutant strain. The wild type FPPS synthesizes farnesyl diphosphate (FPP) as the only product.

Spectroscopic detection of photoproducts in lecithin model system after 8-methoxypsoralen plus UV-A treatment.

Photoreactions of 8-methoxypsoralen (8-MOP) in the presence of synthetic lecithins esterified at the beta-position with linoleic/oleic and gamma-palmitic acid (PCd2/d1pal) have been studied. Following UV-A (320-400 nm) irradiation, the photoproducts separated by thin-layer chromatography are analysed by UV absorption spectroscopy, nuclear magnetic resonance and mass spectroscopy. The new isolated products are lecithin double cyclobutane adducts, PC-(8-MOP)2, fatty acid-8-MOP split adducts from phosphatidylcholine and lecithin adducts with photo-oxidized 8-MOP.

Products of S cerevisiae cis-prenyltransferase activity in vitro.

Products of cis-prenyltransferase activity, the first committed enzyme of the dolichol biosynthetic pathway, have been characterized in Saccharomyces cerevisiae. The evidence based on the results of ion exchange, HPTLC chromatography and acid phosphatase digestion has been presented indicating that the final product of the enzyme action in vitro is free polyprenol and not polyprenol mono- or diphosphate. On the other hand, the results of HPLC analysis confirmed that in vivo yeast accumulate alpha-saturated polyprenols (dolichols).