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.     

Diacylglycerol specifically blocks spontaneous integration of membrane proteins and allows detection of a factor-assisted integration

We recently found that the spontaneous integration of M13 procoat is blocked by diacylglycerol (DAG) (Nishiyama, K., Ikegami, A., Moser, M., Schiltz, E., Tokuda, H., and Muller, M. (2006) J. Biol. Chem. 281, 35667-35676). Here, we demonstrate that the spontaneous integration of Pf3 coat, another membrane protein that has been thought to be integrated spontaneously into liposomes, can be blocked by DAG at physiological concentrations. Moreover, the spontaneous integration of the membrane potential-independent version of Pf3 coat (3L-Pf3 coat), which is independent of YidC, was also blocked by DAG. To clarify the mechanism by which DAG blocks spontaneous integration, we examined lipid compounds similar to DAG and DAG derivatives. The blockage of spontaneous integration was specific to DAG, as fatty acids, monoacylglycerol, and phosphatidic acids were not effective for the blockage. When the acyl chains in DAG were shortened even to octanoyl residues, it still blocked spontaneous integration, whereas diheptanoylglycerol did not block it at all. Triacylglycerol was more effective than DAG. However, the lipid A-derivative-dependent integration of M13 procoat could not be reconstituted when triacylglycerol was included in the liposomes. On the other hand, when DAG was included in the liposomes, we found that the integration of 3L-Pf3 coat was strictly dependent on the lipid A-derived integration factor. We propose that the bulky structure of DAG rather than changes in membrane curvature is essential for the blockage of spontaneous integration. We also demonstrated that the blockage of spontaneous integration by DAG is also operative in native membrane vesicles.     

Development of heterogeneous base catalysts for biodiesel production

Investigations were conducted on heterogeneous base catalysts for the transesterification of oil aimed at effective production of biodiesel. Thirteen different kinds of metal oxides containing calcium, barium, magnesium, or lanthanum were prepared as catalysts. Their catalytic activities were tested for transesterification at 60 degrees C with a 6:1 molar ratio of methanol to oil and a reaction time of 10h. The calcium-containing catalysts - CaTiO3, CaMnO3, Ca2Fe2O5, CaZrO3, and CaO-CeO2 - showed high activities and approximately 90% yields of methyl ester. Furthermore, catalytic durability tests were performed by repeating the transesterification reaction several times with the calcium-containing catalysts recovered from the previous reaction mixture. It was found that CaZrO3 and CaO-CeO2 show high durability and have the potential to be used in biodiesel production processes as heterogeneous base catalysts.     

On the reflex coactivation of ankle flexor and extensor muscles induced by a sudden drop of support surface during walking in humans.

Recent studies have revealed that the stretch reflex responses of both ankle flexor and extensor muscles are coaugmented in the early stance phase of human walking, suggesting that these coaugmented reflex responses contribute to secure foot stabilization around the heel strike. To test whether the reflex responses mediated by the stretch reflex pathway are actually induced in both the ankle flexor and extensor muscles when the supportive surface is suddenly destabilized, we investigated the electromyographic (EMG) responses induced after a sudden drop of the supportive surface at the early stance phase of human walking. While subjects walked on a walkway, the specially designed movable supportive surface was unexpectedly dropped 10 mm during the early stance phase. The results showed that short-latency reflex EMG responses after the impact of the drop (<50 ms) were consistently observed in both the ankle flexor and extensor muscles in the perturbed leg. Of particular interest was that a distinct response appeared in the tibialis anterior muscle, although this muscle showed little background EMG activity during the stance phase. These results indicated that the reflex activities in the ankle muscles certainly acted when the supportive surface was unexpectedly destabilized just after the heel strike during walking. These reflex responses were most probably mediated by the facilitated stretch reflex pathways of the ankle muscles at the early stance phase and were suggested to be relevant to secure stabilization around the ankle joint during human walking.     

Quantitative analysis of human tissue-specific differences in methylation

Tissue-specific differentially methylated regions (tDMRs) have been identified and implicated for their indispensable involvement in mammalian development and tissue differentiation. In this report, a quantitative DNA methylation analysis was performed for 13 human orthologous regions of recently confirmed mouse tDMRs by using Sequenom Mass Array, by which bisulfite-treated fragments are quantitatively detected using time of flight mass spectroscopy analysis. Eight regions were shown as tDMRs in various tissues from three independent individuals. Testis DNA samples from eight individuals were also analyzed for methylation. Interestingly, there is evidence that the DNA methylation level is divergent among individuals. DNA methylation levels of five testis-specific DMRs were significantly inversely correlated with the number of spermatocytes. However, a positive correlation was seen at tDMRs located near the TRIM38 and CASZ1 genes. Our results indicate that tDMRs are conserved between mouse and human and may have an important role in regulating tissue function, differentiation, and aging.     

DDB accumulates at DNA damage sites immediately after UV irradiation and directly stimulates nucleotide excision repair.

Damaged DNA-binding protein, DDB, is a heterodimer of p127 and p48 with a high specificity for binding to several types of DNA damage. Mutations in the p48 gene that cause the loss of DDB activity were found in a subset of xeroderma pigmentosum complementation group E (XP-E) patients and have linked to the deficiency in global genomic repair of cyclobutane pyrimidine dimers (CPDs) in these cells. Here we show that with a highly defined system of purified repair factors, DDB can greatly stimulate the excision reaction reconstituted with XPA, RPA, XPC.HR23B, TFIIH, XPF.ERCC1 and XPG, up to 17-fold for CPDs and approximately 2-fold for (6-4) photoproducts (6-4PPs), indicating that no additional factor is required for the stimulation by DDB. Transfection of the p48 cDNA into an SV40-transformed human cell line, WI38VA13, was found to enhance DDB activity and the in vivo removal of CPDs and 6-4PPs. Furthermore, the combined technique of recently developed micropore UV irradiation and immunostaining revealed that p48 (probably in the form of DDB heterodimer) accumulates at locally damaged DNA sites immediately after UV irradiation, and this accumulation is also observed in XP-A and XP-C cells expressing exogenous p48. These results suggest that DDB can rapidly translocate to the damaged DNA sites independent of functional XPA and XPC proteins and directly enhance the excision reaction by core repair factors.     

Update of MAGEST: Maboya Gene Expression patterns and Sequence Tags

MAGEST is a database for maternal gene expression information for an ascidian, Halocynthia roretzi. The ascidian has become an animal model in developmental biological research because it shows a simple developmental process, and belongs to one of the chordate groups. Various data are deposited into the MAGEST database, e.g. the 3′- and 5′-tag sequences from the fertilized egg cDNA library, the results of similarity searches against GenBank and the expression data from whole mount in situ hybridization. Over the last 2 years, the data retrieval systems have been improved in several aspects, and the tag sequence entries have increased to over 20 000 clones. Additionally, we constructed a database, translated MAGEST, for the amino acid fragment sequences predicted from the EST data sets. Using this information comprehensively, we should obtain new information on gene functions. The MAGEST database is accessible via the Internet at http://www.genome.ad.jp/magest/.