A cDNA encoding a human ortholog of mouse DNA helicase B, which may play a role in DNA replication, has been cloned and expressed as a recombinant protein. The predicted human DNA helicase B (HDHB) protein contains conserved helicase motifs (superfamily 1) that are strikingly similar to those of bacterial recD and T4 dda proteins. The HDHB gene is expressed at low levels in liver, spleen, kidney, and brain and at higher levels in testis and thymus. Purified recombinant HDHB hydrolyzed ATP and dATP in the presence of singlestranded DNA, displayed robust 5-3 DNA helicase activity, and interacted physically and functionally with DNA polymerase ␣-primase. HDHB proteins with mutations in the Walker A or B motif lacked ATPase and helicase activity but retained the ability to interact with DNA polymerase ␣-primase, suggesting that the mutants might be dominant over endogenous HDHB in human cells. When purified HDHB protein was microinjected into the nucleus of cells in early G 1 , the mutant proteins inhibited DNA synthesis, whereas the wild type protein had no effect. Injection of wild type or mutant protein into cells at G 1 /S did not prevent DNA synthesis. The results suggest that HDHB function is required for S phase entry.DNA helicases are an abundant class of DNA metabolic enzymes, surpassing even the DNA polymerases in number and complexity, as well as in their resistance to experimental efforts to elucidate their functions. Although prokaryotic and viral DNA helicases are comparatively well studied, eukaryotic DNA helicases remain poorly understood. The 134 helicaserelated genes encoded by Saccharomyces cerevisiae constitute more than 2% of the genome, but physiological functions of few of them are known (1). A better understanding of DNA replication, repair, and recombination pathways and the interplay among them in eukaryotic cells will depend on elucidation of the DNA helicases involved and their roles in each pathway.SV40 T antigen, a multifunctional viral protein, has served as a paradigm for a replicative helicase in eukaryotes (2, 3). It assembles on the viral origin of DNA replication, unwinds the parental strands, and directs the assembly of the cellular DNA polymerase ␣-primase (pol-prim) 1 (4) and replication protein A (RPA) (5) on the DNA, mediating the synthesis of the first RNA primers. A cellular DNA helicase, mouse DNA helicase B, was reported to share with T antigen the capacity to load pol-prim on RPA-coated single-stranded DNA and activate RNA primer synthesis (6, 7). Moreover, in a mutant derivative of FM3A mouse mammary carcinoma cells that express a thermolabile mutant of murine DNA helicase B, the onset of DNA replication was blocked at the non-permissive temperature (8), consistent with a possible role of the helicase in initiation of DNA replication. A cDNA encoding mouse DNA helicase B was recently cloned and characterized as a member of helicase superfamily 1 (9), which includes several well studied prokaryotic helicases, e.g. Escherichia coli uvrD/Helicase II, rep, recB(CD), and Bac...

Abstract. Terminally differentiated adult injured cardiac myocytes have been used for various animal models of heart failure. It has recently been shown that isoproterenol induces injury in rat neonatal cardiac myocytes via a β-adrenergic pathway, suggesting that it might be one of the factors involved in myocardial cell injury in heart failure in vivo. In the study, silibinin, a plant flavanoid from milk thistle was first evaluated for its protective effect against β-adrenergic agonist isoproterenol-induced injury in cultured rat neonatal cardiac myocytes. The viability, activation of lactate dehydrogenase (LDH), and content of maleic dialdehyde (MDA) were chosen for measuring the degree of cardiac myocytes injury. As a result, silibinin protected isoproterenol-treated rat cardiac myocytes from death and significantly decreased LDH release and MDA production. Silibinin increased superoxide dismutase activity, decreased [Ca 2+ ] i , and increased mitochondrial membrane potential (∆Ψ). Furthermore, the release of pro-apoptotic cytochrome c from mitochondria was reduced by silibinin. Silibinin increased the expression of anti-apoptotic Bcl-2 family protein Bcl-2, and up-regulation of SIRT1 inhibited the translocation of Bax from cytoplasm to mitochondria, which caused mitochondrial dysfunction and cell injury. These results demonstrate that silibinin protects against isoproterenol-induced cardiac myocytes injury through resuming mitochondrial function and regulating the expression of SIRT1 and Bcl-2 family members.

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Caloric restriction (CR) is known to effectively elongate mammalian life-spans. The compound 2-deoxy-D-glucose (2DG), which is often used as an inhibitor of glucose utilization, is a mimetic agent of CR. In this study, we examined the changes of telomerase and Werner's syndrome RecQ (WRN) helicase after treatment with 2DG, because of the involvement of recQ helicase in the regulation of telomeres. Interestingly, 2DG treatment increased the expression of WRN protein in accordance with induction of its promoter activity and gene expression. Furthermore, the activation of telomerase was observed after 2DG treatment, whereas it resulted in the reduction of cell proliferation. These results suggest that 2DG could up-regulate telomere maintenance factors accompanied with suppression of proliferation.

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