Disruption of the putative splice acceptor site for SIV(mac239)Vif reveals tight control of SIV splicing and impaired replication in Vif non-permissive cells

Vif is dispensable for simian immunodeficiency virus (SIV) replication in some cells, termed permissive (i.e., CEM-SS), but not in others, termed non-permissive (i.e., H9, CEMx174, and peripheral blood lymphocytes). Non-permissive cells express the RNA editing enzyme, APOBEC3G. To determine whether vif mRNA could be alternatively spliced, a mutation altering the putative vif splice acceptor site (SA1) was introduced into SIV(mac239) (SIV(Deltavif-SA)). Despite three consensus splice acceptor sites nearby SA1, SIV(Deltavif-SA) did not efficiently generate alternatively spliced vif mRNA. SIV(Deltavif-SA) was growth attenuated in CEMx174 and H9 cells but not in CEM-SS cells. Following SIV(Deltavif-SA), but not SIV(mac239), infection in either H9 or CEMx174 cells viral cDNA contained numerous G to A mutations; no such differences were observed in CEM-SS cells. This pattern is consistent with mutations generated by APOBEC3G in the absence of Vif. Therefore, efficient splicing of SIV vif mRNA is tightly controlled and requires the SA1 site.     

p53, Ki-67, and serum alpha feto-protein as predictors of hepatocellular carcinoma recurrence in liver transplant patients.

Patients with hepatocellular carcinoma who undergo orthotopic liver transplantation (OLT) are at risk for post-transplant tumor recurrence. The aim of this study was to evaluate whether expression of p53 and Ki-67 in hepatocellular carcinoma lesions present in explanted liver tissue was associated with time to tumor recurrence after OLT. Subjects consisted of 20 consecutive patients who underwent OLT and were found to have hepatocellular carcinoma in the liver explant. Immunostaining for p53 and Ki-67 was performed by standard methods. The presence of nuclear immunostaining in >10% of the tumor tissue was considered positive. Time to recurrence of hepatocellular carcinoma after OLT was compared between patients with positive and negative immunostaining by the log rank test. Multivariate analysis was performed using a Cox regression model to control for potentially confounding clinical factors. Time to post-transplant hepatocellular carcinoma recurrence was significantly more rapid in p53+ (P=0.0007) and Ki-67+ cases (P=0.001). These associations remained significant in multivariate analysis. Furthermore, time to recurrent hepatocellular carcinoma was significantly shorter in patients with a serum alpha feto-protein (AFP) level >or=100 ng/ml at time of diagnosis, compared to those with an AFP level <100 ng/ml (P=0.003). In conclusion, expression of p53 and Ki-67 in hepatocellular carcinoma lesions, and a serum AFP level >or=100 ng/ml were associated with more rapid recurrence of hepatocellular carcinoma after OLT. Identification of patients at risk for early post-transplant recurrence could be used to guide surveillance and adjuvant treatment strategies.     

Exposure of the Rh0(D) antigen on the surface and cytoplasmic domains of the red cell membrane

Inside-out (IO) and right-side-out (RO) vesicles derived form human red blood cells were tested for their ability to bind 125I-labelled IgG anti-RHO(D). The binding of anti-RHO(D) to RO vesicles from RHO(D)-positive cells was quantitatively similar to that exhibited by intact cells when compared on a membrane surface area basis. There was no significant binding of labelled antibody to IO vesicles from RhO(D)-positive cells or to either RO or IO vesicles derived from RhO(D)-negative cells. The RhO(D) antigen was immunologically accessible on only the plasma side of the membrane in RhO(D)-positive red cells, as has been shown for blood group antigens defined by carbohydrate determinants. No immunologically reactive RhO(D) antigen was present on either RO or IO vesicles derived from RHO(D)-negative red cells.     

Laboratory infection of the mosquito,Toxorhynchites brevipalpis (Diptera,Culicidae), with bluetongue virus

Summary The use ofToxorhynchites brevipalpis as a system for the propagation and isolation of bluetongue virus (BTV) was investigated.BTV was found to multiply inT. brevipalpis after infection by intrathoracic inoculation. Virus concentrations of up to 6.9 log₁₀ TCID₅₀ per mosquito were found within 7 days of infection and were maintained for at least 6 days. Virus could be detected by an indirect fluorescent antibody test applied to head and thorax tissue smears. These results are comparable to those obtained after inoculation ofCulicoides variipennis with the same virus.Comparison ofT. brevipalpis and baby hamster kidney (BHK) cells as systems for isolation of BTV showed that there was little difference in sensitivity between the two systems for the stock BTV used. Field samples were not available for test. It was concluded that the use ofT. brevipalpis as an isolation system for BTV would have no apparent advantage if BHK cells were available.With 1 Figure     

A phase II trial of temozolomide and IFN-alpha in patients with advanced renal cell carcinoma.

The combination of temozolomide (TEM) and interferon-alpha (IFN-alpha) previously demonstrated a 30% response rate in metastatic melanoma. A single institution, phase II trial evaluating the efficacy of TEM/IFN in patients with advanced renal cell carcinoma (RCC) was conducted. Safety and tumor response were the main outcomes. Eligible patients received 200 mg/m(2)/day TEM orally on days 1-5 every 28 days, with IFN 2.5 million U/m(2)/day subcutaneously (s.c.) three alternate days/week for days 1-15 first cycle, then 5 million U/m(2)/day s.c. 3 alternate days/week throughout each 28-day cycle. Efficacy was evaluated every 8 weeks, and dose-limiting toxicities (DLTs) were treated with dose reductions of the culprit drug. Sixteen patients (ages 37-67) were initially enrolled. Of the 14 evaluable patients, there was one minor response. Best response was stable disease, with 7 patients remaining on study for > or =6 months. Five were alive for more than 2 years, and 2 remain alive at 45 and 50 months after enrollment. DLTs included TEM-induced myelosuppression and IFN-induced fever/chills. Other toxicities were mild to moderate (grades 1-3). The combination of TEM/IFN proved quite tolerable. This regimen appears inactive in terms of response in this population with poor prognosis, but the patients with stable disease > or =6 months remain of interest.     

Inhibition of cellular responses to insulin in a rat liver cell line. A role for PKC in insulin resistance

The initial response of liver cells to insulin is mediated through exocytosis of Cl⁻ channel-containing vesicles and a subsequent opening of plasma membrane Cl⁻ channels. Intracellular accumulation of fatty acids leads to profound defects in metabolism, and is closely associated with insulin resistance. It is not known whether the activity of Cl⁻ channels is altered in insulin resistance and by which mechanisms. We studied the effects of fatty acid accumulation on Cl⁻ channel opening in a model liver cell line. Overnight treatment with amiodarone increased the fat content by ∼2-fold, and the rates of gluconeogenesis by ∼5-fold. The ability of insulin to suppress gluconeogenesis was markedly reduced indicating that amiodarone treatment induces insulin resistance. Western blot analysis showed that these cells express the same number of insulin receptors as control cells. However, insulin failed to activate exocytosis and Cl⁻ channel opening. These inhibitory effects were mimicked in control cells by exposures to arachidonic acid (15 μ m ). Further studies demonstrated that fatty acids stimulate the PKC activity, and inhibition of PKC partially restored exocytosis and Cl⁻ channel opening in insulin-resistant cells. Accordingly, activation of PKC with PMA in control cells potently inhibited the insulin responses. These results suggest that stimulation of PKC activity in insulin resistance contributes to the inhibition of cellular responses to insulin in liver cells.     

Analysis of the roles of bluetongue virus outer capsid proteins VP2 and VP5 in determination of virus serotype

Analyses of reassortant and parental strains of BTV serotypes 3 and 10, in serum neutralization tests, confirmed the major role of outer capsid protein VP2 in determination of virus serotype and its involvement in serum neutralization. However, a reassortant BTV strain (R70), containing protein VP5 derived from BTV 3 and VP2 derived from BTV 10, cross-neutralized with both parental virus strains (BTV 3 and BTV 10). It is concluded that VP5 also plays some part in serotype determination of these virus isolates, as analyzed by serum-neutralization, but its role may be less significant than that of VP2.