HCV RNA in serum of asymptomatic blood donors involved in post-transfusion hepatitis (PTH).

A group of blood donors involved in post-transfusion hepatitis was investigated for the presence of the anti-HCV antibody and of HCV RNA as a more direct infection marker. RNA was extracted from serum, reverse transcribed and amplified using primers which belonged to the non structural region. The amplified product of the PCR reaction was 582 base pairs. Seven (25.9%) of the 27 blood donors examined were found anti-HCV-positive by ELISA; five (71.4%) of these were HCV RNA positive. Among the 20 anti-HCV-negative blood donors, four (20.0%) were HCV RNA positive. ALT levels were below 45 UI/l in 18 donors, while the other nine had ALTs over the limit accepted for transfusion. The anti-HCV-negative HCV RNA-positive blood donors had normal ALTs. Our study offers a direct explanation for the substantial proportion of residual cases of anti-HCV-positive post-transfusion hepatitis and suggests the necessity of creating a register of blood donors who have at some time presented blood enzyme abnormalities and for whom second level investigations such as HCV RNA should be used.     

Rapid induction of apoptosis in rat liver by cycloheximide.

A single administration of the inhibitor of protein synthesis cycloheximide results in the occurrence of apoptosis in rat liver. The presence of intracellular apoptotic bodies was detected as early as 2 hours after treatment. No evidence of cell necrosis could be observed by histologic and biochemical analysis. Apoptosis was followed by an increased expression of testosterone-repressed prostate message-2 RNA, a gene whose activity has been associated to apoptotic cell death in involuting rat prostate. The finding of in vivo induction of apoptosis in nonproliferating cells by an inhibitor of protein synthesis, together with the rapidity and synchrony in the occurrence of cell death make this model potentially useful for the analysis of the kinetics of the apoptotic cycle and in exploring some of the mechanisms of regulation of genes possibly involved in this type of cell death.     

Characterization of estrogen receptor from human liver

Characterization of the estrogen receptor in cytosol from human male liver was undertaken to further understanding of the molecular basis of estrogen action in this tissue. By analysis of estrogen binding data of crude cytosol, saturable estrogen binding showed a Kd = 4.7 X 10(-10) M. High levels of nonsaturable binding were also detected. The estrogen-binding activities detected could be distinguished by their steroid specificity, hydrodynamic parameters, ionic properties, and sensitivity to proteolytic attack. Our findings also confirmed that the moderate-affinity estrogen binders found in rodent liver cannot be detected in human tissue. We concluded that the properties of estrogen receptor of human liver cytosol allow its separation from nonsaturable estrogen-binding components.     

Comparison between TaqMan and LightCycler technologies for quantification of minimal residual disease by using immunoglobulin and T-cell receptor genes consensus probes.

Quantification of residual leukemic cells at early time points during therapy can reliably predict the outcome in children with acute lymphoblastic leukemia (ALL). Recently, semiquantitative minimal residual disease (MRD) detection assays such as dot-blot hybridization have been replaced by real-time quantitative PCR. We tested the flexibility of the two most used real-time PCR machines: the SDS 7700 or 'TaqMan' (TM) (Applied Biosystems) and the LightCycler (LC) (Roche) instruments. Clonal T-cell receptor and immunoglobulin gene rearrangements were used for MRD detection with germline hydrolyzation probes and clone-specific primers. Sensitivity tests for 65 clonal gene rearrangements and MRD quantification in 90 bone marrow samples during therapy of 49 children with ALL at diagnosis or relapse were performed with both machines. Both real-time PCR systems provided specific results for MRD quantification in all follow-up samples. In conclusion, we were able to demonstrate that TM and LC real-time PCR technologies produce similar MRD quantification results and that the quantification assays can be easily transferred from one detection system to the other. Using the same detection format, both techniques can be applied in combination in multicenter MRD studies.     

Allylation of isatin-derived N-Boc-hydrazones followed by Pd-catalyzed carboamination reaction: an entry to 3-spiro-pyrazolidyl-oxindoles

The indium-mediated allylation of novel 3-(2-Boc-hydrazono)indolin-2-one derivatives, followed by a palladium-catalysed carboamination reaction, is described to afford unprecedented spirocyclic oxindoles in good yields. The method provides an efficient access to both cis and trans diastereoisomers of highly functionalized compounds, bearing an N-Boc, 5-substituted pyrazolidine ring at the C3-oxindole spiro junction. The versatility of the method is fully demonstrated starting from a series of substituted isatins and employing a variety of aryl halides in the key cyclization step.

The indium-mediated allylation of novel 3-(2-Boc-hydrazono)indolin-2-one derivatives, followed by a palladium-catalysed carboamination reaction, is described to afford unprecedented spirocyclic oxindoles in good yields.