Dimerization of the ATRIP protein through the coiled-coil motif and its implication to the maintenance of stalled replication forks

ATR (ATM and Rad3-related), a PI kinase-related kinase (PIKK), has been implicated in the DNA structure checkpoint in mammalian cells. ATR associates with its partner protein ATRIP to form a functional complex in the nucleus. In this study, we investigated the role of the ATRIP coiled-coil domain in ATR-mediated processes. The coiled-coil domain of human ATRIP contributes to self-dimerization in vivo, which is important for the stable translocation of the ATR-ATRIP complex to nuclear foci that are formed after exposure to genotoxic stress. The expression of dimerization-defective ATRIP diminishes the maintenance of replication forks during treatment with replication inhibitors. By contrast, it does not compromise the G2/M checkpoint after IR-induced DNA damage. These results show that there are two critical functions of ATR-ATRIP after the exposure to genotoxic stress: maintenance of the integrity of replication machinery and execution of cell cycle arrest, which are separable and are achieved via distinct mechanisms. The former function may involve the concentrated localization of ATR to damaged sites for which the ATRIP coiled-coil motif is critical.     

Modulation by NaCl of atrial natriuretic peptide receptor levels and cyclic GMP responsiveness to atrial natriuretic peptide of cultured vascular endothelial cells.

Type C atrial natriuretic peptide (ANP) receptor levels in cultured vascular endothelial cells were found to be very sensitive to NaCl and shown to be inversely related to the magnitude of ANP-induced cGMP response of the cells. Endothelial cells from bovine carotid artery were subcultured in Eagle's minimum essential medium supplemented with 10% fetal bovine serum (MEM-FBS) and in MEM-FBS plus 25 and 50 mM NaCl. Determination, after several passages, of ANP receptor levels in these cells by 125I-ANP binding assay and affinity labeling revealed a marked reduction in the number of type C receptor in the NaCl-treated cells, whereas type A receptor density was not affected. RNase protection assay to estimate the levels of type C receptor mRNA indicated that the reduction occurred at a pre-translational level. In spite of the decrease in type C receptor number and no significant change in type A receptor (i.e. particulate guanylate cyclase) levels, cGMP response of the NaCl-treated cells to ANP was greatly exaggerated; this sensitization was also observed in membrane preparations. Simple masking of type C ANP receptor with C-ANF (des-[Gln18,Ser19,Gly20,Leu21,Gly22]ANP), a ring-deleted ANP analog, did not produce any sensitization of the cGMP response to ANP; therefore, the above phenomenon cannot simply be explained by the clearance function of the type C receptor. Although whether the type C receptor depletion is directly related to the sensitization of the type A receptor/cyclase is not known, the phenomenon reported and characterized here will serve as a useful basis for elucidating ANP receptor regulation and activation.     

Development of microsatellite markers in the toxic dinoflagellate Alexandrium tamarense (Dinophyceae)

Outbreaks of paralytic shellfish poisoning caused by the toxic dinoflagellate Alexandrium tamarense (Dinophyceae) are currently a serious problem from an economic and food hygiene point of view throughout the world. We isolated 13 polymorphic microsatellite loci from this species. These loci provided microsatellite markers with high polymorphism ranging from four to 15 alleles per locus and gene diversity between 0.632 and 0.974. The markers are available for more detailed investigations of genetic structure and gene flow of A. tamarense populations.     

Dissociation kinetics of 19 base paired oligonucleotide-DNA duplexes containing different single mismatched base pairs.

The dissociation kinetics of 19 base paired oligonucleotide-DNA duplex containing a various single mismatched base pair are studied on dried agarose gels. The kinetics of the dissociation are first order under our experimental conditions. The incorporation of a single mismatched base pair destabilizes the DNA duplexes to some extent, the amount depending on the nature of the mismatched base pair. G-T and G-A mismatches slightly destabilize a duplex, while A-A, T-T, C-T and C-A mismatches significantly destabilize it. The activation energy for the overall dissociation processes for these oligonucleotide-DNA duplexes containing 19 base pairs is 52 +/- 2 Kcal mol-1 as determined from the slope of Arrhenius plot.     

Limited synthesis of labile hemoglobin A1 in vivo and in vitro by the preexisting hemoglobin A1 in diabetic subjects

The synthesis of labile hemoglobin A1 in vivo was studied in subjects with non-insulin dependent diabetes mellitus, impaired and normal glucose tolerance. The labile hemoglobin A1 index defined as delta labile hemoglobin A1 divided by delta plasma glucose at 30 min after oral glucose load, representing the rate of labile hemoglobin A1 synthesis in vivo, was low in diabetic subjects and high in normal subjects, showing an inverse correlation with the amount of preexisting hemoglobin A1. The study on the synthesis of labile hemoglobin A1 in vitro showed a lower initial rate of synthesis and a smaller increase in labile hemoglobin A1 at saturation in red blood cells from diabetic subjects with a relatively large amount of preexisting hemoglobin A1, as opposed to red blood cells from normal subjects. Although the further study is necessary in which delta plasma glucose levels are kept relatively constant in each of 3 groups by glucose-clamp methods, our data suggest that the synthesis of labile hemoglobin A1 is limited in vivo and in vitro in diabetic subjects by the preexisting hemoglobin A1 due to the saturability of its synthesis.     

ESA Hyporesponsiveness Is Associated with Adverse Events in Maintenance Hemodialysis (MHD) Patients,But Not with Iron Storage

Objective

It has been reported that hyporesponsiveness to erythropoiesis-stimulating agent (ESA) is associated with adverse events in patients on maintenance hemodialysis (MHD). However, it has not been determined whether higher iron storage is associated with an improved response, including better survival, to ESA.

Design and Method

We measured serum ferritin, hemoglobin (Hb), and transferrin saturation (TSAT) levels every three months for two years in 1,095 MHD patients. The weekly dose of ESA to Hb ratio was also calculated as an index of ESA responsiveness (ERI).

Results

A significant correlation (p<0.001, R = 0.89) between ferritin and Hb was only observed in the patients with ferritin levels <50 ng/mL. High-dose (≥50 mg/week) intravenous iron administration, female sex, low serum albumin, and angiotensin-converting enzyme inhibitor/angiotensin receptor blocker use were significant predictors of a high ERI value (>280); however, serum ferritin and TSAT levels did not predict a higher ERI. In the time-dependent Cox hazard model, the risk for a composite event in the patients with a high ERI (≥280) and a high ferritin level (≥100 ng/mL) was significantly greater (hazard ratio [HR], 2.09, P = 0.033) than that for patients with a high ERI and a low ferritin (<100 ng/mL) level.

Conclusion

Hb was dependent upon ferritin levels in patients with ferritin levels <50 ng/mL but not in patients with ferritin levels ≥50 ng/mL. Patients with hyporesponsiveness to ESA had a greater risk of composite events, but ERI was unrelated to iron storage.     

Prolipoprotein signal peptidase of Escherichia coli requires a cysteine residue at the cleavage site

A signal peptidase specifically required for the secretion of the lipoprotein of the Escherichia coli outer membrane cleaves off the signal peptide at the bond between a glycine and a cysteine residue. This cysteine residue was altered to a glycine residue by guided site-specific mutagenesis using a synthetic oligonucleotide and a plasmid carrying an inducible lipoprotein gene. The induction of mutant lipoprotein production was lethal to the cells. A large amount of the prolipoprotein was accumulated in the outer membrane fraction. No protein of the size of the mature lipoprotein was detected. These results indicate that the prolipoprotein signal peptidase requires a glyceride modified cysteine residue at the cleavage site.     

Culture Shapes Efficiency of Facial Age Judgments

Background

Cultural differences in socialization can lead to characteristic differences in how we perceive the world. Consistent with this influence of differential experience, our perception of faces (e.g., preference, recognition ability) is shaped by our previous experience with different groups of individuals.

Methodology/Principal Findings

Here, we examined whether cultural differences in social practices influence our perception of faces. Japanese, Chinese, and Asian-Canadian young adults made relative age judgments (i.e., which of these two faces is older?) for East Asian faces. Cross-cultural differences in the emphasis on respect for older individuals was reflected in participants'' latency in facial age judgments for middle-age adult faces—with the Japanese young adults performing the fastest, followed by the Chinese, then the Asian-Canadians. In addition, consistent with the differential behavioural and linguistic markers used in the Japanese culture when interacting with individuals younger than oneself, only the Japanese young adults showed an advantage in judging the relative age of children''s faces.

Conclusions/Significance

Our results show that different sociocultural practices shape our efficiency in processing facial age information. The impact of culture may potentially calibrate other aspects of face processing.     

Isolation of Crystalline Quinolinate Phosphoribosyltransferase from Alcaligenes eutrophus nov. subsp. quinoiinicus and Its Behavior on Polyacrylamide Gel Disc Electrophoresis

Quinolinate phosphoribosyltransferase (EC 2.4.2.19) was purified and crystallized from cell-free extracts of Alcaligenes eutrophus nov. subsp. quinoiinicus, IAM 12305 which was isolated in our laboratory from soil. The enzyme was labile in the cold, and all purification steps were performed at room temperature (10~15°C). The crystalline enzyme was certified to be homogeneous by ultracentrifugal analysis and starch-gel electrophoresis. On polyacrylamide gel disc electrophoresis, the crystalline enzyme showed a multiple profile, but it showed a single band by addition of a certain amount of glycerol and 2-mercaptoethanol. The adding effect of these compounds was discussed.