Hepatitis C virus (HCV) genotyping by annealing reverse transcription-PCR products with genotype-specific capture probes

The genotype of the hepatitis C virus (HCV) strain infecting a given patient is an important predictive factor for the clinical outcome of chronic liver disease and its response to anti-viral therapeutic agents. We herein sought to develop a new easy, sensitive and accurate HCV genotyping method using annealing genotype-specific capture probes (AGSCP) in an automation-friendly 96-well plate format. The validation of our new AGSCP was performed using the Standard HCV Genotype Panel. We then used both our AGSCP and the commercially available INNO-LiPA assay to analyze the HCV genotypes from 111 Korean patients. Discordant results were analyzed by direct sequencing. AGSCP successfully genotyped the standard panel. The genotypes of 111 patient samples were also obtained successfully by AGSCP and INNO-LiPA. We observed a high concordance rate (93 matched samples, 83.8%) between the two assays. Sequencing analysis of the 18 discordant results revealed that the AGSCP had correctly identified 12 samples, whereas the INNO-LiPA had correctly identified only 6. These results collectively indicate that AGSCP assay is a convenient and sensitive method for large-scale genotyping, and it may be a promising tool for the determination of HCV and other genotypes in clinical settings.     

The identification of a novel <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> dsRNA virus and determination of the distribution of viruses in mushroom spores

Double-stranded RNAs and virus particles were identified in Pleurotus ostreatus strain Shin-Nong in Korea. Isometric virus particles with a diameter of 33 nm were purified, which are similar to other Pleurotus viruses reported previously. This strain contains 5 dsRNAs, 8.0, 2.5, 2.4, 2.0, and 1.8 kb in size. The virus particles contain 2 dsRNAs, designated RNA-1 (2.5 kb), and RNA-2 (2.4 kb) which is a typical pattern of Partitiviridae. A non-encapsidated dsRNA of about 8.0 kb also was identified. Partial cDNA from RNA-1 was cloned, and sequence analysis revealed that this gene codes for RdRp. The comparison of the sequence from partial cDNA clone showed 35% amino acid homology with the C-terminal end of the RdRp gene of Helicobasidum mompa virus and Rosalinia necatrix virus. Specific primers designed from the partial sequences successfully amplified RT-PCR product from the infected mycelium and a single spore culture. We used these primers to determine the pattern of distribution of viruses in spores. Of the 96 different single spore cultures generated from Shin-Nong strain, a specific RT-PCR product was identified in 25 cultures, indicating that about 26% of basidiospores contain viruses.     

Plant growth promotion and Penicillium citrinum

Background

Lamivudine is an oral nucleoside analogue widely used for the treatment of chronic hepatitis B. The main limitation of lamivudine use is the selection of resistant mutations that increases with time of utilization. Hepatitis B virus (HBV) isolates have been classified into eight genotypes (A to H) with distinct geographical distributions. HBV genotypes may also influence pathogenic properties and therapeutic features. Here, we analyzed the HBV genotype distribution and the nature and frequency of lamivudine resistant mutations among 36 patients submitted to lamivudine treatment for 12 to 84 months.

Results

Half of the patients were homosexual men. Only 4/36 (11%) patients were HBV DNA negative. As expected for a Brazilian group, genotypes A (24/32 positive individuals, 75%), D (3/32, 9.3%) and F (1/32, 3%) were present. One sample was from genotype C, which is a genotype rarely found in Brazil. Three samples were from genotype G, which had not been previously detected in Brazil. Lamivudine resistance mutations were identified in 20/32 (62%) HBV DNA positive samples. Mean HBV loads of patients with and without lamivudine resistance mutations were not very different (2.7 × 10⁷ and 6.9 × 10⁷ copies/mL, respectively). Fifteen patients showed the L180M/M204V lamivudine resistant double mutation. The triple mutant rt173V/180M/204V, which acts as a vaccine escape mutant, was found in two individuals. The three isolates of genotype G were entirely sequenced. All three showed the double mutation L180M/M204V and displayed a large genetic divergence when compared with other full-length genotype G isolates.

Conclusion

A high (55%) proportion of patients submitted to long term lamivudine therapy displayed resistant mutations, with elevated viral load. The potential of transmission of such HBV mutants should be monitored. The identification of genotypes C and G, rarely detected in South America, seems to indicate a genotype distribution different to that observed in non treated patients. Disparities in routes of transmission (genotype G seems to be linked to homosexual behavior) and in pathogenic properties (genotype C is very aggressive) among HBV genotypes may explain the presence of rare genotypes in the present work.     

ortho-dihydroxyisoflavone derivatives from aged Doenjang (Korean fermented soypaste) and its radical scavenging activity

One new ortho-dihydroxyisoflavone, 7,3',4'-trihydroxyisoflavone (2), and two known ortho-dihydroxyisoflavone derivatives were isolated from 5-year-old Doenjang (Korean fermented soypaste), and evaluated as potent antioxidant by comparing with other known isoflavones. 7,8,4'-Trihydroxyisoflavone (1), 7,3',4'-trihydroxyisoflavone (2), and 6,7,4'-trihydroxyisoflavone (3) inhibited DPPH (Diphenyl-1-picryl hydrazyl) formation by 50% at a concentration of 21.5+/-0.2, 28.7+/-0.4 and 32.6+/-0.6 (IC(50)), respectively, whereas three isoflavones showed weak DPPH radical scavenging activity. In xanthine oxidase (XO) system, in which both inhibition of xanthine oxidase and superoxide scavenging effect were measured in one assay. Compound 1 (IC(50)= 6.6+/-0.4 microM) and 2 (IC(50)=16.8+/-1.2 microM) show significant inhibitory activity and greater effect than allopurinol. But, compound 3 and other isoflavones showed lower inhibition activity. This study shows that the position of hydroxyl substituent at the aromatic ring of isoflavone plays an important role in radical scavenging effect.     

Activation profiles of human kallikrein-related peptidases by proteases of the thrombostasis axis

The human kallikrein-related peptidases (KLKs) comprise 15 members (KLK1-15) and are the single largest family of serine proteases. The KLKs are utilized, or proposed, as clinically important biomarkers and therapeutic targets of interest in cancer and neurodegenerative disease. All KLKs appear to be secreted as inactive pro-forms (pro-KLKs) that are activated extracellularly by specific proteolytic release of their N-terminal pro-peptide. This processing is a key step in the regulation of KLK function. Much recent work has been devoted to elucidating the potential for activation cascades between members of the KLK family, with physiologically relevant KLK regulatory cascades now described in skin desquamation and semen liquefaction. Despite this expanding knowledge of KLK regulation, details regarding the potential for functional intersection of KLKs with other regulatory proteases are essentially unknown. To elucidate such interaction potential, we have characterized the ability of proteases associated with thrombostasis to hydrolyze the pro-peptide sequences of the KLK family using a previously described pro-KLK fusion protein system. A subset of positive hydrolysis results were subsequently quantified with proteolytic assays using intact recombinant pro-KLK proteins. Pro-KLK6 and 14 can be activated by both plasmin and uPA, with plasmin being the best activator of pro-KLK6 identified to date. Pro-KLK11 and 12 can be activated by a broad-spectrum of thrombostasis proteases, with thrombin exhibiting a high degree of selectivity for pro-KLK12. The results show that proteases of the thrombostasis family can efficiently activate specific pro-KLKs, demonstrating the potential for important regulatory interactions between these two major protease families.     

Cellular functions of GEF-H1, a microtubule-regulated Rho-GEF: is altered GEF-H1 activity a crucial determinant of disease pathogenesis?

The Rho guanine nucleotide exchange factor GEF-H1 is uniquely regulated by microtubule binding and is crucial in coupling microtubule dynamics to Rho-GTPase activation in a variety of normal biological situations. Here, we review the roles of GEF-H1 in epithelial barrier permeability, cell motility and polarization, dendritic spine morphology, antigen presentation, leukemic cell differentiation, cell cycle regulation, and cancer. GEF-H1 might also contribute to pathophysiological signaling involved in leukemias, and in cancers associated with mutated p53 tumor suppressor gene, epithelial and endothelial cell dysfunction, infectious disease, and cardiac hypertrophy. We suggest that GEF-H1 could be a novel therapeutic target in multiple human diseases.     

Design of new potent and selective secretory phospholipase A2 inhibitors. Part 5: synthesis and biological activity of 1-alkyl-4-[4,5-dihydro-1,2,4-[4H]-oxadiazol-5-one-3-ylmethylbenz-4'-yl(oyl)] piperazines

Among the different PLA(2)s identified to date, the group IIA secretory PLA(2) (sPLA(2) GIIA) is implied in diverse pathological conditions. In this work we describe the synthesis, inhibitory activities, and structure-activity relationships (SAR) of a new class of substituted piperazine derivatives. The in vitro fluorimetric assay using two groups of enzymes, GIB and GIIA, revealed several compounds as highly potent inhibitors (IC(50)=0.1 microM). The in vivo activity assessed by ip or per os administration in a carrageenan-induced edema test in rats showed that two compounds proved to be as potent as indomethacin (10 mg/kg).     

Mind bomb 1-expressing intermediate progenitors generate notch signaling to maintain radial glial cells

Notch signaling is critical for the stemness of radial glial cells (RGCs) during embryonic neurogenesis. Although Notch-signal-receiving events in RGCs have been well characterized, the signal-sending mechanism by the adjacent cells is poorly understood. Here, we report that conditional inactivation of mind bomb-1 (mib1), an essential component for Notch ligand endocytosis, in mice using the nestin and hGFAP promoters resulted in complete loss of Notch activation, which leads to depletion of RGCs, and premature differentiation into intermediate progenitors (IPs) and finally neurons, which were reverted by the introduction of active Notch1. Interestingly, Mib1 expression is restricted in the migrating IPs and newborn neurons, but not in RGCs. Moreover, sorted Mib1+ IPs and neurons can send the Notch signal to neighboring cells. Our results reveal that not only newborn neurons but also IPs are essential Notch-ligand-presenting cells for maintaining RGC stemness during both symmetric and asymmetric divisions.