Bipartite 3'-cis-acting signal for replication in yeast 23 S RNA virus and its repair

23 S RNA narnavirus is a persistent positive strand RNA virus found in Saccharomyces cerevisiae. The viral genome is small (2.9 kb) and only encodes its RNA-dependent RNA polymerase. Recently, we have succeeded in generating 23 S RNA virus from an expression vector containing the entire viral cDNA sequence. Using this in vivo launching system, we analyzed the 3'-cis-acting signals for replication. The 3'-non-coding region of 23 S RNA contains two cis-elements. One is a stretch of 4 Cs at the 3' end, and the other is a mismatched pair in a stem-loop structure that partially overlaps the terminal 4 Cs. In the latter element, the loop or stem sequence is not important but the stem structure with the mismatch pair is essential. The mismatched bases should be purines. Any combination of purines at the mismatch pair bestowed capability of replication on the RNA, whereas converting it to a single bulge at either side of the stem abolished the activity. The terminal and penultimate Cs at the 3' end could be eliminated or modified to other nucleotides in the launching plasmid without affecting virus generation. However, the viruses generated regained or restored these Cs at the 3' terminus. Considering the importance of the viral 3' ends in RNA replication, these results suggest that this 3' end repair may contribute to the persistence of 23 S RNA virus in yeast by maintaining the genomic RNA termini intact. We discuss possible mechanisms for this 3' end repair in vivo.     

Enhancement of serum- and platelet-derived growth factor-induced cell proliferation by Necl-5/Tage4/poliovirus receptor/CD155 through the Ras-Raf-MEK-ERK signaling

Necl-5/Tage4/poliovirus receptor/CD155 has been shown to be the poliovirus receptor and to be up-regulated in rodent and human carcinoma. We have found previously that mouse Necl-5 regulates cell motility. We show here that mouse Necl-5 is furthermore involved in the regulation of cell proliferation. Studies using a specific antibody against Necl-5 and a dominant negative mutant of Necl-5 revealed that Necl-5 enhanced the serum-induced proliferation of NIH3T3, Swiss3T3, and mouse embryonic fibroblast cells. Necl-5 enhanced the serum-induced activation of the Ras-Raf-MEK-ERK signaling, up-regulated cyclins D2 and E, and down-regulated p27(Kip1), eventually shortening the period of the G(0)/G(1) phase of the cell cycle in NIH3T3 cells. Necl-5 similarly enhanced the platelet-derived growth factor-induced activation of the Ras-Raf-MEK-ERK signaling and shortened the period of the G(0)/G(1) phase of the cell cycle in NIH3T3 cells. Necl-5 acted downstream of the platelet-derived growth factor receptor and upstream of Ras. Moreover, up-regulated Necl-5 was involved at least partly in the enhanced proliferation of transformed cells including NIH3T3 cells transformed by an oncogenic Ras or v-Src. These results indicate that Necl-5 plays roles not only in cell motility but also in cell proliferation.     

DiaA, a novel DnaA-binding protein, ensures the timely initiation of Escherichia coli chromosome replication

The DnaA protein is the initiator of Escherichia coli chromosomal replication. In this study, we identify a novel DnaA-associating protein, DiaA, that is required for the timely initiation of replication during the cell cycle. DiaA promotes the growth of specific temperature-sensitive dnaA mutants and ensures stable minichromosome maintenance, whereas DiaA does not decrease the cellular DnaA content. A diaA::Tn5 mutation suppresses the cold-sensitive growth of an overinitiation type dnaA mutant independently of SeqA, a negative modulator of initiation. Flow cytometry analyses revealed that the timing of replication initiation is disrupted in the diaA mutant cells as well as wild-type cells with pBR322 expressing the diaA gene. Gel filtration and chemical cross-linking experiments showed that purified DiaA forms a stable homodimer. Immunoblotting analysis indicated that a single cell contains about 280 DiaA dimers. DiaA stimulates minichromosome replication in an in vitro system especially when the level of DnaA included is limited. Moreover, specific and direct binding between DnaA and DiaA was observed, which requires a DnaA N-terminal region. DiaA binds to both ATP- and ADP-bound forms of DnaA with a similar affinity. Thus, we conclude that DiaA is a novel DnaA-associating factor that is crucial to ensure the timely initiation of chromosomal replication.     

Aliphatic acid amides of the fruits of Zanthoxylum piperitum

Six aliphatic acid amides (1-6) were isolated from the pericarp of Zanthoxylum piperitum fruits. MS and NMR spectroscopic investigation revealed that these compounds have a ketone and/or hydroxyl group(s) in the unsaturated aliphatic acid moiety of the structure of the amides. Combinations 3-4 and 5-6 are stereoisomers in terms of the relative configurations of their two asymmetric carbons.     

Angiotensin II type 1 receptor blocker ameliorates overproduction and accumulation of triglyceride in the liver of Zucker fatty rats

The effects of angiotensin II type 1 receptor blocker (ARB) on triglyceride (TG) metabolism associated with insulin resistance were explored in Zucker fatty (ZF) rats. Olmesartan medoxomil, a newly developed ARB, was given as a 0.01% drinking solution ad libitum to ZF and Zucker lean (ZL) rats for 4 wk. Olmesartan lowered blood pressure in both strains to the same extent. ZF rats had a markedly low insulin sensitivity index (SI) and glucose effectiveness (SG), together with significantly increased glucose levels. Olmesartan treatment substantially elevated both SI and SG. The ZF rats were hyperlipidemic, with plasma TG levels sixfold higher than those of the ZL rats. Olmesartan remarkably decreased the plasma free fatty acid level in the ZF rats, but it did not exert a significant effect on the plasma TG level. The TG secretion rate assessed by the Triton WR-1339 technique was almost six times higher in the ZF than in the ZL rats, and olmesartan treatment suppressed this TG overproduction by one-half. The TG content in the liver was ten times higher in the ZF than in the ZL rats, and olmesartan halved this high hepatic TG content without affecting the cholesterol content. The fatty liver developed in the ZF rats was ameliorated by olmesartan treatment. Olmesartan treatment had no significant effects on TG metabolism or insulin sensitivity in the ZL rats. Taken in sum, ARB improves the overproduction and accumulation of TG in the liver associated with insulin resistance, and it does so through mechanisms independent of its hypotensive action.     

First case of aplastic anemia in a Japanese child with a homozygous missense mutation in the <Emphasis Type="Italic">NBS1</Emphasis> gene (I171V) associated with genomic instability

The NBS1 gene is strongly linked to several factors involved in genome integrity. Functional disruption of NBS1 could therefore induce genomic instability and carcinogenesis. Four children with acute lymphoblastic leukemia have been reported to be heterozygous for a germline and/or somatic missense mutation in NBS1, leading to the I171V substitution. We screened healthy controls and pediatric patients with hematological malignancies and aplastic anemia (AA) for the presence of I171V. Of the 62 patients, one individual with AA was confirmed to harbor a homozygous I171V mutation. Genetic analysis of NBS1 in this patient and her healthy parents indicated that she inherited the germline I171V mutation from her father and the wild-type allele from her mother, and that the second I171V hit occurred on the wild-type allele early in embryonic development. Furthermore, cytogenetic analysis of lymphoblastic cell lines from the patient indicated a remarkable increase in numerical and structural chromosomal aberrations in the absence of clastogens, suggesting that she potentially carried genomic instability. This is the first report of AA with a homozygous I171V mutation. We hypothesize that NBS1 may play an important role in the pathogenesis of AA.H. Shimada and K. Shimizu contributed equally to this work     

A comparison of oxidized LDL-induced collagen secretion by graft and aortic SMCs: role of PDGF

Smooth muscle cells (SMCs) from prosthetic vascular grafts constitutively secrete higher levels of collagen than aortic SMCs. Lipid oxidation products accumulate in grafts, and we postulated that they stimulate SMC production of collagen. The effect of oxidized low-density lipoprotein (oxLDL) on type I collagen secretion by aortic and graft SMCs was compared. SMCs isolated from the canine thoracic aorta or Dacron thoracoabdominal grafts (n = 10) were incubated with native LDL or oxLDL (0-400 microg cholesterol/ml) for 72 h. Type I collagen in the conditioned medium was measured by ELISA. OxLDL increased collagen production by graft SMCs from 4.1 +/- 0.3 to 11.0 +/- 0.4 ng/microg DNA and by aortic SMCs from 2.3 +/- 0.1 to 3.5 +/- 0.2 ng/microg DNA. Native LDL had little effect. LY-83583, a superoxide generator, stimulated a dramatic increase in collagen secretion by graft SMCs and a smaller but significant elevation by aortic SMCs. OxLDL has been shown to increase PDGF production by graft SMCs, and PDGF can stimulate collagen production. Anti-PDGF antibody inhibited the increase in collagen production by graft SMCs that was stimulated by oxLDL, implicating PDGF as one mechanism of oxLDL-induced collagen production. Lipid oxidation products that accumulate in prosthetic vascular grafts can cause an oxidative stress that stimulates PDGF production by graft SMCs that in turn stimulates collagen production, contributing to the progression of intimal hyperplasia.     

Crystal structures of acid blue and alkaline purple forms of bacteriorhodopsin

Bacteriorhodopsin, a light-driven proton pump found in the purple membrane of Halobacterium salinarum, exhibits purple at neutral pH but its color is sensitive to pH. Here, structures are reported for an acid blue form and an alkaline purple form of wild-type bacteriorhodopsin. When the P622 crystal prepared at pH 5.2 was acidified with sulfuric acid, its color turned to blue with a pKa of 3.5 and a Hill coefficient of 2. Diffraction data at pH 2-5 indicated that the purple-to-blue transition accompanies a large structural change in the proton release channel; i.e. the extracellular half of helix C moves towards helix G, narrowing the proton release channel and expelling a water molecule from a micro-cavity in the vicinity of the retinal Schiff base. In this respect, the acid-induced structural change resembles the structural change observed upon formation of the M intermediate. But, the acid blue form contains a sulfate ion in a site(s) near Arg82 that is created by re-orientations of the carboxyl groups of Glu194 and Glu204, residues comprising the proton release complex. This result suggests that proton uptake by the proton release complex evokes the anion binding, which in turn induces protonation of Asp85, a key residue regulating the absorption spectrum of the chromophore. Interestingly, a pronounced structural change in the proton release complex was also observed at high pH; i.e. re-orientation of Glu194 towards Tyr83 was found to take place at around pH 10. This alkaline transition is suggested to be accompanied by proton release from the proton release complex and responsible for rapid formation of the M intermediate at high pH.     

Expression patterns of genes encoding carbohydrate-metabolizing enzymes and their relationship to grain filling in rice (Oryza sativa L.): comparison of caryopses located at different positions in a panicle

In rice, caryopses located at the base of the panicle have a lower growth rate than those at the tip of the panicle. The former and latter types of caryopses are called inferior and superior caryopses, respectively. Taking the different growth rate into consideration, sugar status and the expression of genes encoding carbohydrate-metabolizing enzymes in inferior caryopses were compared with those in superior caryopses. During the first 5 d after flowering, superior caryopses elongated rapidly, but inferior caryopses did not. At this phase, inferior caryopses had a low ratio of hexose to sucrose, high activity of acid invertase and the absence of the expression of the genes encoding the above enzymes except for two isoforms of cell wall invertase, OsCIN4 and INV1, in comparison with superior caryopses. At the start of caryopsis elongation in both superior and inferior caryopses, the hexose/sucrose ratio increased accompanied by gene expression of vacuolar invertase (INV3), sucrose synthase (RSus1) and ADP-glucose pyrophosphorylase (AGP-L2: D50317). Furthermore, the genes related to endospermal starch accumulation were expressed highly with the decrease in the hexose/sucrose ratio after its peak. Based on the comparison of superior and inferior caryopses, the possible mechanism of grain filling in rice is discussed.