Partial genome sequence of murine gammaherpesvirus 72 and its analysis.

Murine gammaherpesvirus 68 (MHV-68)-infected mouse is a well known model for studies of Epstein-Barr virus (EBV)-related lymphoproliferative diseases (LPD). Murine gammaherpesvirus 72 (MHV-72) has been considered a close relative of MHV-68 but its replication in murine mammary gland cells and kinetics of infection of mice were found to be different. Pathological studies of a long-term-infection of mice revealed a similar or higher malignancy development rate in MHV-72-infected mice as compared with that of MHV-68. Previous comparison of MHV-72 with MHV-68 revealed their diversity in M3, MK3, and M7 genes encoding the chemokine-binding protein, immune evasion protein and glycoprotein 150, respectively. In this study, a portion (22,899 bp) of MHV-72 genome sequence was determined, analyzed and compared with that of MHV-68. Nucleotide sequences of 13 structural and 6 non-structural genes of MHV-72 and deduced amino acid sequences revealed their identity to those of MHV-72 except for differences in 9 nucleotides and 8 amino acids, occurring in 5 genes and their proteins. Due to these differences, 4 structural proteins encoded by ORF20, ORF26, ORF48, and ORF52, respectively, and a non-structural protein encoded by ORF4, all of MHV-72, are predicted to have altered hydrophilicity and surface exposure in comparison with their MHV-68 counterparts. These differences obviously contribute to some different pathogenetical features of these viruses and could explain the reduced immunogenicity of MHV-72 in relation to MHV-68, allowing MHV-72 to escape the host immune surveillance.

Effects of herbal preparation on libido and semen quality in boars.

The objective of this study was to investigate the effects of a preparation from herbal extracts (PHE) on libido and semen quality in breeding artificial insemination boars. Ten fertile boars were divided into control and experimental groups according to significant difference of libido. There were no differences in semen quality between groups. Animals were fed a commercial feeding mixture for boars. The feeding mixture for the experimental group was enriched with PHE, which was prepared from Eurycoma longifolia, Tribulus terrestris and Leuzea carthamoides. Duration of the experiment was 10 weeks. Samples of ejaculate were collected weekly. Libido was evaluated according to a scale of 0-5 points. Semen volume, sperm motility, percentage of viable spermatozoa, sperm concentration, morphologically abnormal spermatozoa, daily sperm production and sperm survival were assessed. Amounts of mineral components and free amino acids were analysed in seminal plasma. Significant differences were found in these parameters: libido (4.05 ± 0.22 vs 3.48 ± 0.78; p < 0.001), semen volume (331.75 ± 61.91 vs 263.13 ± 87.17 g; p < 0.001), sperm concentration (386.25 ± 107.95 vs 487.25 ± 165.50 × 10(3) /mm(3); p < 0.01), morphologically abnormal spermatozoa (15.94 ± 11.08 vs 20.88 ± 9.19%; p < 0.001) and Mg concentration (28.36 ± 11.59 vs 20.27 ± 13.93 mm; p < 0.05). The experimental group's libido was increased by 20% in comparison with the beginning of the experiment. Results of this study showed positive effect of PHE on libido and some parameters of boar semen quality.

Small proteoglycans in human diabetic nephropathy: discrepancy between glomerular expression and protein accumulation of decorin, biglycan, lumican, and fibromodulin.

Small leucine-rich proteoglycans (SLRPs), for example, decorin, biglycan, fibromodulin, and lumican, are extracellular matrix organizers and binding partners of TGF-b. Decorin is also involved in growth control and angiogenesis. Hence, these proteoglycans are likely of importance in the pathogenesis of diabetic glomerulosclerosis. In normal kidney, SLRPs were preferentially expressed in the tubulointerstitium. Weak expression occurred in the mesangial matrix. Biglycan was expressed by glomerular endothelial cells and, together with fibromodulin, by distal tubular cells and in collecting ducts. In all stages of diabetic nephropathy, there was a marked up-regulation of the proteoglycans in tubulointerstitium and glomeruli. Decorin and lumican became expressed in tubuli. However, in glomeruli, overexpression was not mirrored by local proteoglycan accumulation except in advanced nephropathy. In severe glomerulosclerosis, increased decorin concentrations were found in plasma and urine, and urinary TGF-b/decorin complexes could be demonstrated indirectly. The failure to detect an increased glomerular proteoglycan quantity during the development of nephropathy could be explained by assuming that they are secreted into the mesangial matrix, but cleared via the vasculature or the urinary tract, in part as complexes with TGF-b. They could thereby counteract the vicious circle being characterized by increased TGF-b production and increased matrix deposition in diabetic nephropathy.

The bystander effect mediated by the new murine gammaherpesvirus 72--thymidine kinase/5'-fluoro-2'-deoxyuridine (MHV72-TK/5-FUdR) system in vitro.

To investigate the potential of murine gamma-herpesvirus 72 thymidine kinase (MHV-72-TK) to act as a suicide gene, we used a mammalian expression vector on rat fibroblastoid cells deficient in the cellular TK gene. Substrate specificity was assessed in vitro in cells with stable expression of MHV-72-TK. The Herpes simplex virus 1-TK (HSV-1-TK) was used as a reference suicide gene. Unlike HSV-1-TK modified cells, which were sensitive to ganciclovir (GCV) (IC50=9.7 microM), cells modified by MHV-72-TK did not show sensitivity to this drug. The use of 3'-azido-3'-deoxythymidine (AZT) and (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) did not affect the growth of cells expressing either MHV-72-TK or HSV-1-TK in the range of concentration used for AZT (0-375 microM) and for BVDU (0-50 microM). In contrast, 5'-fluoro-2'-deoxyuridine (5-FUdR) was extremely cytotoxic and effectively killed MHV-72-TK expressing cells (IC50 value 2.1 microM). This value was 16 times lower than that required to kill cells expressing HSV1-TK. To test whether the bystander effect between two heterologous cell types could be mediated by the MHV-72-TK/5-FUdR system in vitro, cells expressing MHV-72-TK were co-cultured with the tumour fibroblastoid cell line NAD for 48 hours before the drug (10.8 microM) was added. The cell mixtures contained various ratios of cells expressing MHV-72-TK (0 to 50% of total cells). Only 1% of MHV-72-TK-expressing cells were needed to enhance mouse tumour cell killing and to decrease the survival rate to 25.6%. The bystander effect was more pronounced when 10% of cells expressing MHV-72-TK were used, decreasing survival to 17.4%. In parallel, the same concentration of 5-FUdR dose only marginally inhibited tumour cell growth in the absence of exogenous TK activity (84% survival). These results demonstrate the efficiency of MHV-72-TK as a suicide gene when 5-FUdR is used as a prodrug. When sequenced, MHV-72-TK proved to be identical to MHV-68 strain TK.

DNA-protective activity of new ribonucleotide reductase inhibitors.

The DNA-protective activity of hydroxyurea (HU) and novel ribonucleotide reductase (RR) inhibitors amidox (AX), didox (DX) and trimidox (TX) was examined using hydrogen peroxide as the DNA-damaging agent. The exposure of superspiralized plasmid DNA molecules (pBR 322) to H2O2 under precisely defined in vitro conditions initiates a change in DNA topology (DNA from I relaxes to DNA form II). This electrophoretically monitored change in the plasmid DNA topology is related to the induction of ss-DNA breaks and corresponds with DNA exposition to free radicals. The inhibition of DNA relaxation (the prevention of DNA damage induced by hydrogen peroxide) depended on the free radical scavenging capacity of the drugs investigated. HU exerted DNA protective activity at a concentration of 4 mM, AX at concentration of 1 microM, TX at a concentration of 5 microM and DX at a concentration of 25 microM (the free radical scavenging activity increases from HU to AX in following manner: HU << DX < TX < AX). It can be concluded that the new synthetic RR-inhibitor AX which is being investigated at the preclinical level as a potential anti-cancer drug possess the highest capacity for scavenging of free radicals.

Genotoxic properties of the newly synthesized antineoplastic agents amidox, didox and trimidox.

Toxic and genotoxic effects of three polyhydroxy-substituted benzohydroxamates (amidox, didox and trimidox), having antineoplastic activities by the mechanism of the ribonucleotid reductase activity inhibition, were evaluated by reverse mutation assay on Salmonella typhimurium strains TA97, TA98, TA100, TA102. While amidox did not exhert any toxic effect, didox and trimidox were toxic. The toxicity of the test chemicals was dependent on the structure of their molecule and the repair capacity of the test strains. Trimidox exhibited the highest toxicity, and it was proved as a direct-acting frameshift mutagen. Its mutagenic effect was increased after a metabolic activation. Amidox and didox can be classified as frameshift promutagens.