The nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is considered to possess RNA-dependent RNA polymerase (RdRp) activity and to play an essential role for the viral replication. In this study, we expressed the NS5B protein of 65 kd by a recombinant baculovirus. With the highly purified NS5B protein, we established an in vitro system for RdRp activity by using poly(A) as a template and a 15-mer oligo(U) (oligo(U) 15 ) as a primer. Optimal conditions of temperature and pH for primer-dependent polymerase activity of the NS5B were 32ЊC and pH 8. Hepatitis C virus (HCV) is the major etiologic agent of both posttransfusion and sporadic non-A, non-B hepatitis throughout the world. 1-3 HCV infection often leads to persistence, resulting in chronic hepatitis, and develops into hepatocellular carcinoma. Although the incidence of infection with HCV was drastically decreased by introduction of an efficient blood screening system, 1% to 2% of the worldwide population are already infected with HCV. Furthermore, seroepidemiological studies indicated that there is a clear correlation between infection with HCV and the development of hepatocellular carcinoma. It is therefore very important to develop anti-HCV agents with different antiviral mechanisms for the treatment of liver diseases associated with HCV infection. A number of other drugs with a broad spectrum of antiviral activity have been given to patients with hepatitis C. 4 To date, however, few convincing positive results have been obtained.HCV is a positive-stranded RNA virus with the genome size of approximately 9,400 bases and contains a large open reading frame encoding a precursor polyprotein of about 3,000 amino acids. [5][6][7][8] From their deduced amino acid sequence, HCV was shown to be related to pestiviruses and distantly related to flaviviruses. [8][9][10][11] Recent studies revealed that this polyprotein was cleaved into at least 9 mature proteins by host cellular signalases and viral coded proteinases. 12,13 However, because of the lack of an efficient in vitro cell culture system to grow the virus, little is known about the mechanisms of the replication of HCV. Synthesis of both replicative intermediate minus-strand HCV RNA and progeny positive-strand HCV RNA is thought to occur via an RNA-dependent RNA polymerase (RdRp) reaction. The nonstructural 5B (NS5B) protein of 65 kd is located at the C-terminal region of the HCV polyprotein and shares its sequence with those of other proteins of animal and plant viruses and bacteriophages possessing RdRp activity. 14,15

Although initial rituximab-containing chemotherapies achieve high response rates, indolent B-cell non-Hodgkin lymphoma (B-NHL), such as follicular lymphoma (FL), is still incurable. Therefore, new effective agents with novel mechanisms are anticipated. In this multicentre phase II study, patients with relapsed/refractory indolent B-NHL and mantle cell lymphoma (MCL) received vorinostat 200 mg twice daily for 14 consecutive days in a 21-d cycle until disease progression or unacceptable toxicity occurred. The primary endpoint was overall response rate (ORR) in FL patients and safety and tolerability in all patients. Secondary endpoints included progression-free survival (PFS). Fifty-six eligible patients were enrolled; 50 patients (39 with FL, seven with other B-NHL, and four with MCL) were evaluable for ORR, and 40 patients had received rituximab-containing prior chemotherapeutic regimens. For the 39 patients with FL, the ORR was 49% [95% confidence interval (CI): 32·4, 65·2] and the median PFS was 20 months (95% CI: 11·2, 29·7). Major toxicities were manageable grade 3/4 thrombocytopenia and neutropenia. Vorinostat offers sustained antitumour activity in patients with relapsed or refractory FL with an acceptable safety profile. Further investigation of vorinostat for clinical efficacy is warranted.

Aims/IntroductionDental pulp stem cells (DPSCs) are thought to be an attractive candidate for cell therapy. We recently reported that the transplantation of DPSCs increased nerve conduction velocity and nerve blood flow in diabetic rats. In the present study, we investigated the immunomodulatory effects of DPSC transplantation on diabetic peripheral nerves.Materials and Methods DPSCs were isolated from the dental pulp of Sprague–Dawley rats and expanded in culture. Eight weeks after the streptozotocin injection, DPSCs were transplanted into the unilateral hindlimb skeletal muscles. Four weeks after DPSC transplantation, neurophysiological measurements, inflammatory gene expressions and the number of CD68‐positive cells in sciatic nerves were assessed. To confirm the immunomodulatory effects of DPSCs, the effects of DPSC‐conditioned media on lipopolysaccharide‐stimulated murine macrophage RAW264.7 cells were investigated.ResultsDiabetic rats showed significant delays in sciatic nerve conduction velocities and decreased sciatic nerve blood flow, all of which were ameliorated by DPSC transplantation. The number of CD68‐positive monocytes/macrophages and the gene expressions of M1 macrophage‐expressed cytokines, tumor necrosis factor‐α and interleukin‐1β, were increased in the sciatic nerves of the diabetic rats. DPSC transplantation significantly decreased monocytes/macrophages and tumor necrosis factor‐α messenger ribonucleic acid expression, and increased the gene expression of the M2 macrophage marker, CD206, in the sciatic nerves of the diabetic rats. The in vitro study showed that DPSC‐conditioned media significantly increased the gene expressions of interleukin‐10 and CD206 in lipopolysaccharide‐stimulated RAW264.7 cells.ConclusionsThese results suggest that DPSC transplantation promoted macrophages polarization towards anti‐inflammatory M2 phenotypes, which might be one of the therapeutic mechanisms for diabetic polyneuropathy.

IL-35 and IL-17, but not IL-27, may play important roles in the pathogenesis of periodontitis.

We cloned a gene, bexA, that codes for a multidrug efflux transporter from the chromosomal DNA of Bacteroides thetaiotaomicron ATCC 29741 by using an Escherichia coli ⌬acrAB ⌬acrEF mutant as a host. Although the initial recombinant construct contained other open reading frames, the presence of bexA alone was sufficient to confer to the E. coli host elevated levels of resistance to norfloxacin, ciprofloxacin, and ethidium bromide. Disruption of bexA in B. thetaiotaomicron made the strain more susceptible to norfloxacin, ciprofloxacin, and ethidium bromide, showing that this gene is expressed in this organism and functions as a multidrug efflux pump. The deduced BexA protein sequence was homologous to the protein sequence of Vibrio parahaemolyticus NorM, a multidrug efflux transporter, and thus, BexA belongs to the multidrug and toxic compound extrusion (MATE) family.Members of the Bacteroides fragilis group are anaerobic bacteria of the highest clinical relevance, and B. fragilis and Bacteroides thetaiotaomicron are often isolated from patients with suppurative anaerobic infections. They are gram-negative, obligately anaerobic organisms with a broad spectrum of recognized resistance to antimicrobial agents (23), including aminoglycosides, most of the penicillins and cephalosporins, and fluoroquinolones (except for a few recently developed compounds such as trovafloxacin and clinafloxacin) (2, 3).Active multidrug efflux processes, usually involving secondary transporters belonging to the major facilitator superfamily, small multidrug resistance family, and resistance-nodulationdivision (RND) superfamily, are now known to be important, especially in the baseline or intrinsic resistance of many bacteria to antimicrobial agents (16,21). More recently, a new family, the multidrug and toxic compound extrusion (MATE) family, has been discovered (4), but its contribution to drug resistance has been known only for a few isolated cases (12, 13).We found previously that at least a portion of the remarkable norfloxacin resistance (MICs, 16 to 32 g/ml) of B. fragilis was attributed to active efflux of this agent (12). To elucidate the efflux mechanism, we attempted to clone the genes responsible for norfloxacin efflux from the chromosomal DNAs of B. fragilis and B. thetaiotaomicron using as the host an Escherichia coli mutant strain with defects in multidrug efflux pumps. Here we report on the cloning and sequencing of a gene, bexA, that is involved in the efflux of norfloxacin, ciprofloxacin, and ethidium bromide from B. thetaiotaomicron. Interestingly, the BexA transporter is a member of the MATE family. MATERIALS AND METHODSBacterial strains and growth conditions. B. fragilis ATCC 25285 and B. thetaiotaomicron ATCC 29741 5482 and were grown in general anaerobic GAM broth (Nissui, Tokyo, Japan) and supplemented Trypticase soy broth (sTSB) (12) in an anaerobic chamber.For cloning, two host strains were used. They were E. coli DH5␣ [K-12 supE44 ⌬lacU169 (80 lacZ⌬M15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1] (26) and AG102AX [K-...