Revisiting the mouse mitochondrial DNA sequence

The existence of reliable mtDNA reference sequences for each species is of great relevance in a variety of fields, from phylogenetic and population genetics studies to pathogenetic determination of mtDNA variants in humans or in animal models of mtDNA-linked diseases. We present compelling evidence for the existence of sequencing errors on the current mouse mtDNA reference sequence. This includes the deletion of a full codon in two genes, the substitution of one amino acid on five occasions and also the involvement of tRNA and rRNA genes. The conclusions are supported by: (i) the re-sequencing of the original cell line used by Bibb and Clayton, the LA9 cell line, (ii) the sequencing of a second L-derivative clone (L929), and (iii) the comparison with 12 other mtDNA sequences from live mice, 10 of them maternally related with the mouse from which the L cells were generated. Two of the latest sequences are reported for the first time in this study (Balb/cJ and C57BL/6J). In addition, we found that both the LA9 and L929 mtDNAs also contain private clone polymorphic variants that, at least in the case of L929, promote functional impairment of the oxidative phosphorylation system. Conse quently, the mtDNA of the strain used for the mouse genome project (C57BL/6J) is proposed as the new standard for the mouse mtDNA sequence.     

NMR study on the interaction between RPA and DNA decamer containing cis-syn cyclobutane pyrimidine dimer in the presence of XPA: implication for damage verification and strand-specific dual incision in nucleotide excision repair

In mammalian cells, nucleotide excision repair (NER) is the major pathway for the removal of bulky DNA adducts. Many of the key NER proteins are members of the XP family (XPA, XPB, etc.), which was named on the basis of its association with the disorder xerodoma pigmentosum. Human replication protein A (RPA), the ubiquitous single-stranded DNA-binding protein, is another of the essential proteins for NER. RPA stimulates the interaction of XPA with damaged DNA by forming an RPA–XPA complex on damaged DNA sites. Binding of RPA to the undamaged DNA strand is most important during NER, because XPA, which directs the excision nucleases XPG and XPF, must bind to the damaged strand. In this study, nuclear magnetic resonance (NMR) spectroscopy was used to assess the binding of the tandem high affinity DNA-binding domains, RPA-AB, and of the isolated domain RPA-A, to normal DNA and damaged DNA containing the cyclobutane pyrimidine dimer (CPD) lesion. Both RPA-A and RPA-AB were found to bind non- specifically to both strands of normal and CPD- containing DNA duplexes. There were no differences observed when binding to normal DNA duplex was examined in the presence of the minimal DNA-binding domain of XPA (XPA-MBD). However, there is a drastic difference for CPD-damaged DNA duplex as both RPA-A and RPA-AB bind specifically to the undamaged strand. The strand-specific binding of RPA and XPA to the damaged duplex DNA shows that RPA and XPA play crucial roles in damage verification and guiding cleavage of damaged DNA during NER.     

Mutational analysis of BRAF and K-ras in gastric cancers: absence of BRAF mutations in gastric cancers

Recently, BRAF mutations were found in a variety of human cancers. Interestingly, the most common of BRAF mutation (V599E) has not been identified in tumors with K-ras mutations. Whereas the majority of human cancer types has been screened for BRAF mutations, no detailed studies on gastric cancers have been investigated. Thus, we decided to investigate the incidence of BRAF mutations in gastric cancers, and the relationship between BRAF and K-ras mutations in such cancers. Three non-pathogenic BRAF polymorphisms and seven K-ras missense mutations were found in 66 gastric cancers and 16 gastric cancer cell lines. Although only 9% of our gastric cancer panels had K-ras mutations, the incidence of BRAF mutations was not high. Thus, BRAF mutations, which are present in a variety of other human cancers, do not seem to be involved in gastric cancer development.     

Exercise improves postischemic function in aging hearts

Exercise improves cardioprotection against ischemia-reperfusion in young animals but has not been investigated in older animals, which represent the population most likely to suffer an ischemic event. Therefore, we sought to determine the effects of aging on exercise-induced cardioprotection. Young, middle-aged, and old (4, 12, and 21 mo old) male Fischer 344 rats ran 60 min at 70-75% of maximum oxygen consumption. Twenty-four hours postexercise, isolated perfused working hearts underwent 22.5 min of global ischemia and then 30 min of recovery (reperfusion). Compared with sedentary rats (n = 8-9 rats/group), recovery of function (cardiac output x systolic pressure) improved after exercise (n = 9 rats/group) by 40% at 4 mo, 78% at 12 mo, and 59% at 21 mo. Exercise increased inducible heat shock protein 70 expression 105% at 4 mo but only 27% at 12 mo and 24% at 21 mo. Catalase activity progressively increased with age (P < 0.05) and was increased by exercise at 4 mo (26%) and 21 mo (19%). Manganese superoxide dismutase activity was increased by exercise only at 21 mo (45%). No exercise-related change in any antioxidant enzyme was observed at 12 mo. We conclude that exercise can enhance cardioprotection regardless of age, but the cardioprotective protein phenotype changes with age.     

Caveolin-1 null mice develop cardiac hypertrophy with hyperactivation of p42/44 MAP kinase in cardiac fibroblasts

Recently, development ofa caveolin-1-deficient (Cav-1 null) mouse model has allowed thedetailed analysis of caveolin-1's function in the context of awhole animal. Interestingly, we now report that the hearts ofCav-1 null mice are markedly abnormal, despite the fact that caveolin-1is not expressed in cardiac myocytes. However, caveolin-1 is abundantlyexpressed in the nonmyocytic cells of the heart, i.e., cardiacfibroblasts and endothelia. Quantitative imaging studies of Cav-1 nullhearts demonstrate a significantly enlarged right ventricular cavityand a thickened left ventricular wall with decreased systolic function.Histological analysis reveals myocyte hypertrophy withinterstitial/perivascular fibrosis. Because caveolin-1 is thought toact as a negative regulator of the p42/44 MAP kinase cascade, weperformed Western blot analysis with phospho-specific antibodies thatonly recognize activated ERK1/2. As predicted, the p42/44 MAP kinasecascade is hyperactivated in Cav-1 null heart tissue (i.e.,interstitial fibrotic lesions) and isolated cardiac fibroblasts. Inaddition, endothelial and inducible nitric oxide synthase levels aredramatically upregulated. Thus loss of caveolin-1 expression drivesp42/44 MAP kinase activation and cardiac hypertrophy.

     

Synthesis of 6-formyl-pyridine-2-carboxylate derivatives and their telomerase inhibitory activities

Twenty-one pyridine-2-carboxylate derivatives were prepared by the coupling of 6-formyl-2-carboxylic acid with the corresponding phenol, thiophenol, and aniline, substituted with various functional groups. Among them, the 3,4-dichlorothiophenol ester (9p) showed the highest in vitro telomerase inhibitory activity and quite significant in vivo tumor suppression activity.     

PLD1 regulates mTOR signaling and mediates Cdc42 activation of S6K1

BACKGROUND: The mammalian target of rapamycin (mTOR) regulates cell growth and proliferation via the downstream targets ribosomal S6 kinase 1 (S6K1) and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1). We have identified phosphatidic acid (PA) as a mediator of mitogenic activation of mTOR signaling. In this study, we set out to test the hypotheses that phospholipase D 1 (PLD1) is an upstream regulator of mTOR and that the previously reported S6K1 activation by Cdc42 is mediated by PLD1. RESULTS: Overexpression of wild-type PLD1 increased S6K1 activity in serum-stimulated cells, whereas a catalytically inactive PLD1 exerted a dominant-negative effect on S6K1. More importantly, eliminating endogenous PLD1 by RNAi led to drastic inhibition of serum-stimulated S6K1 activation and 4E-BP1 hyperphosphorylation in both HEK293 and COS-7 cells. Knockdown of PLD1 also resulted in reduced cell size, suggesting a critical role for PLD1 in cell growth control. Using a rapamycin-resistant S6K1 mutant, Cdc42's action was demonstrated to be through the mTOR pathway. When Cdc42 was mutated in a region specifically required for PLD1 activation, its ability to activate S6K1 in the presence of serum was hindered. However, when exogenous PA was used as a stimulus, the PLD1-inactive Cdc42 mutant behaved similarly to the wild-type protein. CONCLUSIONS: Our observations reveal the involvement of PLD1 in mTOR signaling and cell size control, and provide a molecular mechanism for Cdc42 activation of S6K1. A new cascade is proposed to connect mitogenic signals to mTOR through Cdc42, PLD1, and PA.     

A Rapid and Simple PCR-based Method for Analysis of Transgenic Fish using a Restricted Amount of Fin Tissue

The protocol described in this paper offers a simple and rapid method for PCR analysis of transgenes using a restricted amount of fin tissue from small-sized transgenic fish. A simple preparation of fin lysate using a buffer containing a low concentration of an ionic detergent, SDS (0.01%), followed by neutralization with a second buffer containing higher concentrations of non-ionic detergents NP40 (2%) and Tween 20 (2%) consistently provides a reliable quantity of high-quality DNA template for PCR amplification of transgenes. Based on this protocol, transgenic fish can be clearly distinguished from non-transgenic fish using PCR in a rapid and reproducible manner. Tedious DNA purifications are avoided while fidelity of amplification and efficient identification of transgenic fish are maintained.