Interaction between Escherichia coli DNA polymerase IV and single-stranded DNA-binding protein is required for DNA synthesis on SSB-coated DNA

DNA polymerase IV (Pol IV) is one of three translesion polymerases in Escherichia coli. A mass spectrometry study revealed that single-stranded DNA-binding protein (SSB) in lysates prepared from exponentially-growing cells has a strong affinity for column-immobilized Pol IV. We found that purified SSB binds directly to Pol IV in a pull-down assay, whereas SSBΔC8, a mutant protein lacking the C-terminal tail, failed to interact with Pol IV. These results show that the interaction between Pol IV and SSB is mediated by the C-terminal tail of SSB. When polymerase activity was tested on an SSBΔC8-coated template, we observed a strong inhibition of Pol IV activity. Competition experiments using a synthetic peptide containing the amino acid sequence of SSB tail revealed that the chain-elongating capacity of Pol IV was greatly impaired when the interaction between Pol IV and SSB tail was inhibited. These results demonstrate that Pol IV requires the interaction with the C-terminal tail of SSB to replicate DNA efficiently when the template ssDNA is covered with SSB. We speculate that at the primer/template junction, Pol IV interacts with the tail of the nearest SSB tetramer on the template, and that this interaction allows the polymerase to travel along the template while disassembling SSB.     

Overexpression of serine hydroxymethyltransferase from halotolerant cyanobacterium in Escherichia coli results in increased accumulation of choline precursors and enhanced salinity tolerance

A real-time PCR procedure targeting the gene of the molecular cochaperon DnaJ (dnaJ) was developed for specific detection of strains belonging to the Enterobacter cloacae group. The inclusivity and exclusivity of the real-time PCR assay were assessed with seven reference strains of E.?cloacae, 12 other Enterobacter species and 41 non-Enterobacter strains. Inclusivity as well as exclusivity of the duplex real-time PCR was 100%. In contrast, resolution of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was inadequate for delineation of Enterobacter asburiae, Enterobacter hormaechei, Enterobacter kobei and Enterobacter ludwigii from E.?cloacae. Eleven of 56 (20%) clinical isolates of the E.?cloacae group could not be clearly identified as a certain species using MALDI-TOF MS. In summary, the combination of MALDI-TOF MS with the E.?cloacae-specific duplex real-time PCR is an appropriate method for identification of the six species of the E.?cloacae complex.     

Increased age of transformed mouse neural progenitor/stem cells recapitulates age‐dependent clinical features of human glioma malignancy

Increasing age is the most robust predictor of greater malignancy and treatment resistance in human gliomas. However, the adverse association of clinical course with aging is rarely considered in animal glioma models, impeding delineation of the relative importance of organismal versus progenitor cell aging in the genesis of glioma malignancy. To address this limitation, we implanted transformed neural stem/progenitor cells (NSPCs), the presumed cells of glioma origin, from 3‐ and 18‐month‐old mice into 3‐ and 20‐month host animals. Transplantation with progenitors from older animals resulted in significantly shorter (P ≤ 0.0001) median survival in both 3‐month (37.5 vs. 83 days) and 20‐month (38 vs. 67 days) hosts, indicating that age‐dependent changes intrinsic to NSPCs rather than host animal age accounted for greater malignancy. Subsequent analyses revealed that increased invasiveness, genomic instability, resistance to therapeutic agents, and tolerance to hypoxic stress accompanied aging in transformed NSPCs. Greater tolerance to hypoxia in older progenitor cells, as evidenced by elevated HIF‐1 promoter reporter activity and hypoxia response gene (HRG) expression, mirrors the upregulation of HRGs in cohorts of older vs. younger glioma patients revealed by analysis of gene expression databases, suggesting that differential response to hypoxic stress may underlie age‐dependent differences in invasion, genomic instability, and treatment resistance. Our study provides strong evidence that progenitor cell aging is responsible for promoting the hallmarks of age‐dependent glioma malignancy and that consideration of progenitor aging will facilitate development of physiologically and clinically relevant animal models of human gliomas.     

A food-derived synergist of NGF signaling: identification of protein tyrosine phosphatase 1B as a key regulator of NGF receptor-initiated signal transduction

Neurotrophins, such as the nerve growth factor (NGF), play an essential role in the growth, development, survival and functional maintenance of neurons in the central and peripheral systems. They also prevent neuronal cell death under various stressful conditions, such as ischemia and neurodegenerative disorders. NGF induces cell differentiation and neurite outgrowth by binding with and activating the NGF receptor tyrosine kinase followed by activation of a variety of signaling cascades. We have investigated the NGF-dependent neuritogenesis enhancer potential of a food-derived small molecule contained in Brassica vegetables and identified the protein tyrosine phosphatase (PTP) 1B as a key regulator of the NGF receptor-initiated signal transduction. Based on an extensive screening of Brassica vegetable extracts for the neuritogenic-promoting activity in the rat pheochromocytoma cell line PC12, we found the Japanese horseradish, wasabi (Wasabia japonica, syn. Eutrema wasabi), as the richest source and identified 6-methylsulfinylhexyl isothiocyanate (6-HITC), an analogue of sulforaphane isolated from broccoli, as one of the major neuritogenic enhancers in the wasabi. 6-HITC strongly enhanced the neurite outgrowth and neurofilament expression elicited by a low-concentration of NGF that alone was insufficient to induce neuronal differentiation. 6-HITC also facilitated the sustained-phosphorylation of the extracellular signal-regulated kinase and the autophosphorylation of the NGF receptor TrkA. It was found that PTP1B act as a phosphatase capable of dephosphorylating Tyr-490 of TrkA and was inactivated by 6-HITC in a redox-dependent manner. The identification of PTP1B as a regulator of NGF signaling may provide new clues about the chemoprotective potential of food components, such as isothiocyanates.     

Involvement of multidrug resistance-associated protein 2 (ABCC2/Mrp2) in biliary excretion of micafungin in rats

The drug transporter, multidrug resistance-associated protein 2 (ABCC2/Mrp2), is known to play important roles in excretion of various drugs. In the present study, we investigated whether Mrp2 is involved in the transport of micafungin, a newly developed antifungal agent. When Sprague-Dawley rats received an intravenous injection of micafungin (1 mg/kg) in combination with cyclosporine, the cyclosporine significantly delayed the disappearance of micafungin from plasma and decreased the systemic clearance and volume of distribution at steady-state of micafungin to 54% and 65% of the corresponding control values, respectively. When Sprague-Dawley rats received a constant-rate infusion of micafungin, cyclosporine significantly decreased the steady-state biliary clearance of micafungin (~80%). A significant decrease in the biliary clearance of micafungin (~60%) was observed in Eisai hyperbilirubinemic rats, which have a hereditary deficiency in Mrp2. The present findings at least suggest that Mrp2 is involved mainly in the hepatobiliary excretion of micafungin in rats.     

MURC, a muscle-restricted coiled-coil protein that modulates the Rho/ROCK pathway, induces cardiac dysfunction and conduction disturbance

We identified a novel muscle-restricted putative coiled-coil protein, MURC, which is evolutionarily conserved from frog to human. MURC was localized to the cytoplasm with accumulation in the Z-line of the sarcomere in the murine adult heart. MURC mRNA expression in the heart increased during the developmental process from the embryonic stage to adulthood. In response to pressure overload, MURC mRNA expression increased in the hypertrophied heart. Using the yeast two-hybrid system, we identified the serum deprivation response (SDPR) protein, a phosphatidylserine-binding protein, as a MURC-binding protein. MURC induced activation of the RhoA/ROCK pathway, which modulated serum response factor-mediated atrial natriuretic peptide (ANP) expression and myofibrillar organization. SDPR augmented MURC-induced transactivation of the ANP promoter in cardiomyocytes, and RNA interference of SDPR attenuated the action of MURC on the ANP promoter. Transgenic mice expressing cardiac-specific MURC (Tg-MURC) exhibited cardiac contractile dysfunction and atrioventricular (AV) conduction disturbances with atrial chamber enlargement, reduced thickness of the ventricular wall, and interstitial fibrosis. Spontaneous episodes of atrial fibrillation and AV block were observed in Tg-MURC mice. These findings indicate that MURC modulates RhoA signaling and that MURC plays an important role in the development of cardiac dysfunction and conduction disturbance with increased vulnerability to atrial arrhythmias.     

Allorecognition mechanisms during ascidian fertilization

Ascidians (primitive chordates) are hermaphroditic animals, releasing sperm and eggs nearly simultaneously. But, many ascidians, including Ciona intestinalis and Halocynthia roretzi, show self-sterility or preference for cross-fertilization rather than self-fertilization. The molecular mechanisms underlying this allorecognition process are only poorly understood. We recently identified the genes responsible for self-incompatibility in C. intestinalis by a positional cloning: sperm-borne polycystin 1-like receptor, referred to as s-Themis, and its fibrinogen-like ligand called v-Themis on the vitelline coat (VC) are highly polymorphic and appear to be responsible for allorecognition in the fertilization of C. intestinalis. In H. roretzi, on the other hand, we revealed that HrVC70, a 70-kDa main component of the VC consisting of 12 epidermal-growth-factor (EGF)-like repeats, is a candidate allorecognition protein, since the attachment of this protein to the VC during oocyte maturation and its detachment by weak acid are closely linked to the gain and the loss of self-sterility, respectively, and also since nonself-sperm rather than self-sperm efficiently bound to HrVC70-agarose. As a binding partner of HrVC70, a 35-kDa GPI-anchored glycoprotein in sperm lipid rafts, referred to as HrUrabin, was identified: HrUrabin appears to play a key role in allorecognizable sperm binding to HrVC70 during fertilization. In the present review, we describe the current progress on the molecular bases of allorecognition, or self-incompatibility, during ascidian fertilization, by considering the SI systems in another organisms including fungies and flowering plants.