Probing the active site tightness of DNA polymerase in subangstrom increments

We describe the use of a series of gradually expanded thymine nucleobase analogs in probing steric effects in DNA polymerase efficiency and fidelity. In these nonpolar compounds, the base size was increased incrementally over a 1.0-A range by use of variably sized atoms (H, F, Cl, Br, and I) to replace the oxygen molecules of thymine. Kinetics studies with DNA Pol I (Klenow fragment, exonuclease-deficient) in vitro showed that replication efficiency opposite adenine increased through the series, reaching a peak at the chlorinated compound. Efficiency then dropped markedly as a steric tightness limit was apparently reached. Importantly, fidelity also followed this trend, with the fidelity maximum at dichlorotoluene, the largest compound that fits without apparent repulsion. The fidelity at this point approached that of wild-type thymine. Surprisingly, the maximum fidelity and efficiency was found at a base pair size significantly larger than the natural size. Parallel bypass and mutagenesis experiments were then carried out in vivo with a bacterial assay for replication. The cellular results were virtually the same as those seen in solution. The results provide direct evidence for the importance of a tight steric fit on DNA replication fidelity. In addition, the results suggest that even high-fidelity replicative enzymes have more steric room than necessary, possibly to allow for an evolutionarily advantageous mutation rate.     

The chemistry and biology of aflatoxin B(1): from mutational spectrometry to carcinogenesis

Dietary exposure to aflatoxin B(1) (AFB(1)) is associated with an increased incidence of hepatocellular carcinoma (HCC), especially in populations in which exposure to hepatitis B virus (HBV) is a common occurrence. Most HCC samples from people living where HBV is prevalent have one striking mutational hotspot: a GC-->TA transversion at the third position of codon 249 of the p53 gene. In this review, the chemical reaction of an electrophilic derivative of aflatoxin with specific DNA sequences is examined, along with the types of mutations caused by AFB(1) and the sequence context dependence of those mutations. An attempt is made to assign the source of these mutations to specific chemical forms of AFB(1)-DNA damage. In addition, epidemiological and experimental data are examined regarding the synergistic effects of AFB(1) and HBV on HCC formation and the predominance of one hotspot GC-->TA transversion in the p53 gene of affected individuals.     

The role of recombination signal sequences in the preferential joining by deletion in DH-JH recombination and in the ordered rearrangement of the IgH locus

The bias favoring deletion over inversion in DH-JH rearrangement has been known for years, but the underlying mechanism has yet to be fully defined. It has been suggested that the ratio of deletion/inversion is determined by the combined effect of two factors: (i) the relative strengths of 5' and 3' recombination signal sequences (RSS) of a DH segment, and (ii) the efficiency with which the deletional product (one joint) forms relative to the inversional product (two joints). In this study, we analyzed for the first time the effect of factor 1 alone on the biased 3' RSS utilization in DH-JH joining by using deletional plasmids in an extrachromosomal substrate V(D)J recombination assay. It was found that the 3' RSS and associated coding end (12 bp) mediate recombination more efficiently than the 5' RSS/coding end DH-JH plasmids. These results demonstrate that the effect of the RSS/coding end alone can account, at least partially, for the predominant deletion in DH-JH recombination. The potential effect of the relative strength of RSS and associated coding end on the ordered rearrangement of DH-JH followed by VH to DH-JH was also assessed. When recombination frequencies of D-->J (3' DH to J3) were compared with frequencies of V-- >D (VHPJ14 to 3' DH or VHOX2 to 3' DH), it was found that V-->D joining was, if anything, more efficient than D-->J joining. Therefore, if all three segments were accessible, RSS/coding end effects would not contribute to the ordered rearrangement of the IgH locus.      

高三尖杉酯碱酰胺的结构测定

三尖杉树皮粗提取物中的一个新生物碱—高三尖杉酯碱酰胺(homoharringtonamide)的结构,经质谱—质谱分析,初步建议为16。类似的酰胺类生物碱,例如三尖杉碱酰胺(cephalotaxamide,6)、11-羟基三尖杉碱酰胺(11-hydroxycephalotaxamide,9)、三尖杉酯碱酰胺(harfingtonamide,14)或异三尖杉酯碱酰胺(isoharringtonamide,15)也可能存在,后三者(9,14,15)尚未见报道。     

Rare detection of hepatitis B and hepatitis C virus genomes by polymerase chain reaction in seronegative donors with elevated alanine aminotransferase

BACKGROUND: Since screening for antibody to hepatitis C virus (HCV) was introduced in 1990, posttransfusion hepatitis has been reduced to nearly background levels. This has led to reconsideration of the value of testing donated blood for elevated alanine aminotransferase (ALT). The contribution of ALT testing in detecting seronegative infection was evaluated by the performance of polymerase chain reaction (PCR) for hepatitis B virus (HBV) or HCV in plasma from ALT-elevated blood units. STUDY DESIGN AND METHODS: Testing was performed on 375 units of plasma, derived from an equivalent of 47,500 blood donations, with a highly sensitive hemi-nested PCR procedure. Using a triplet of primers directed at the conserved regions of HBV DNA and 5'-noncoding regions of HCV RNA, the hemi-nested PCR assay can reliably amplify 10 viral molecules to levels detectable in ethidium bromide-stained agarose gels. Pools of plasma from groups of four donors were screened with hemi-nested PCR. For any reactive pools, the plasma from individual donors was retested twice on different aliquots. RESULTS: Two of 375 units, both with midrange ALT elevation, were repeatedly reactive in hemi-nested PCR (one each for HBV DNA and HCV RNA). However, samples from the two suspect donors tested 9 and 5 months later revealed no seroconversion, elevated ALT, or viral genomes in hemi-nested PCR. CONCLUSION: The lack of confirmed HBV or HCV infection in this study representing an estimated 47,500 voluntary blood donations suggests that routine ALT testing for further prevention of posttransfusion hepatitis after exclusion of HBV- and/or HCV-seropositive blood may be superfluous.     

T-cell death by apoptosis in vertically human immunodeficiency virus- infected children coincides with expansion of CD8+/interleukin-2 receptor-/HLA-DR+ T cells: sign of a possible role for herpes viruses as cofactors?

One mechanism proposed to play a role in T-cell depletion in human immunodeficiency virus (HIV) infection is apoptosis (activation-induced cell death). We assessed whether apoptosis is related to activation of T cells in vivo and its possible triggers. DNA was extracted from peripheral blood mononuclear cells (PBMC) taken from 16 vertically HIV- infected children and 9 HIV-negative children born to HIV-positive mothers (controls) and tested by agarose gel electrophoresis for the presence of DNA fragments specific for apoptosis. Signs of apoptosis were found on in vitro culture of PBMC from 12 of 16 HIV-infected children, but not in PBMC from the nine controls. Eleven of the 12 HIV- infected children with apoptosis showed an elevated (> 15%) proportion of CD3+/HLA-DR+ cells. This was due to an increased proportion of CD8+/HLA-DR+ cells, as shown in 7 of 7 further tested patients. In none of the probands an increased (> 5%) proportion of IL-2 receptor expressing CD3+ cells was found. T cells undergoing apoptosis were preferentially of the CD8+ phenotype. Expansion of circulating CD8+/interleukin-2 receptor (IL-2R)-/HLA-DR+ T cells is known to occur during active infection with herpes viruses. To investigate the possible role of herpes viral coinfections for apoptosis in HIV infection, we focused on Epstein-Barr virus (EBV) as an example for a herpes virus usually acquired during childhood. In 10 of 12 patients with apoptosis, we found increased levels of EBV genome in PBMC and/or tissues, indicating active EBV replication. By contrast, no increased burden of EBV was found in the four HIV-infected patients without apoptosis or in the controls. Our data indicate that in children the occurrence of apoptosis in HIV infection is closely related to activation of CD8+ T cells. Furthermore, primoinfection with or reactivation of herpes viruses, such as EBV, may substantially contribute to such T-cell activation and the ensuing apoptosis. Additional studies are warranted to evaluate the contribution of herpes virus-triggered apoptosis to the T-cell loss leading to the acquired immunodeficiency syndrome.     

Intraoperative cell salvage with autologous transfusion in liver transplantation

Liver transplant(LT) is the primary treatment for patients with end-stage liver disease. About 25000 LTs are performed annually in the world. The potential for intraoperative bleeding is quite variable. However, massive bleeding is common and requires blood transfusion. Allogeneic blood transfusion has an immunosuppressive effect and an impact on recipient survival, in addition to the risk of transmission of viral infections and transfusion errors, among others.Techniques to prevent excessive bleeding or to use autologous blood have been proposed to minimize the negative effects of allogeneic blood transfusion.Intraoperative reinfusion of autologous blood is possible through previous selfdonation or blood collected during the operation. However, LT does not normally allow autologous transfusion by prior self-donation. Hence, using autologous blood collected intraoperatively is the most feasible option. The use of intraoperative blood salvage autotransfusion(IBSA) minimizes the perioperative use of allogeneic blood, preventing negative transfusion effects without negatively impacting other clinical outcomes. The use of IBSA in patients with cancer is still a matter of debate due to the theoretical risk of reinfusion of tumor cells. However, studies have demonstrated the safety of IBSA in several surgical procedures, including LT for hepatocellular carcinoma. Considering the literature available to date, we can state that IBSA should be routinely used in LT, both in patients with cancer and in patients with benign diseases.