Crystal structures of the state 1 conformations of the GTP-bound H-Ras protein and its oncogenic G12V and Q61L mutants

GTP-bound Ras adopts two interconverting conformations, "inactive" state 1 and "active" state 2. However, the tertiary structure of wild-type (WT) state 1 remains unsolved. Here we solve the state 1 crystal structures of H-Ras WT together with its oncogenic G12V and Q61L mutants. They assume open structures characterized by impaired interactions of both Thr-35 in switch I and Gly-60 in switch II with the γ-phosphate of GTP and possess two surface pockets of mutually different shapes unseen in state 2, a potential target for selective inhibitor development. Furthermore, they provide a structural basis for the low GTPase activity of state 1.     

The SI strain of measles virus derived from a patient with subacute sclerosing panencephalitis possesses typical genome alterations and unique amino acid changes that modulate receptor specificity and reduce membrane fusion activity

Subacute sclerosing panencephalitis (SSPE) is a fatal sequela associated with measles and is caused by persistent infection of the brain with measles virus (MV). The SI strain was isolated in 1976 from a patient with SSPE and shows neurovirulence in animals. Genome nucleotide sequence analyses showed that the SI strain genome possesses typical genome alterations for SSPE-derived strains, namely, accumulated amino acid substitutions in the M protein and cytoplasmic tail truncation of the F protein. Through the establishment of an efficient reverse genetics system, a recombinant SI strain expressing a green fluorescent protein (rSI-AcGFP) was generated. The infection of various cell types with rSI-AcGFP was evaluated by fluorescence microscopy. rSI-AcGFP exhibited limited syncytium-forming activity and spread poorly in cells. Analyses using a recombinant MV possessing a chimeric genome between those of the SI strain and a wild-type MV strain indicated that the membrane-associated protein genes (M, F, and H) were responsible for the altered growth phenotype of the SI strain. Functional analyses of viral glycoproteins showed that the F protein of the SI strain exhibited reduced fusion activity because of an E300G substitution and that the H protein of the SI strain used CD46 efficiently but used the original MV receptors on immune and epithelial cells poorly because of L482F, S546G, and F555L substitutions. The data obtained in the present study provide a new platform for analyses of SSPE-derived strains as well as a clear example of an SSPE-derived strain that exhibits altered receptor specificity and limited fusion activity.     

Radical scavenging activity of dicaffeoyloxycyclohexanes: contribution of an intramolecular interaction of two caffeoyl residues

Six regio- and stereoisomers of dicaffeoyloxycyclohexanes and 2,4-di-O-caffeoyl-1,6-anhydro-beta-D-glucose were synthesized as model compounds of dicaffeoylquinic acids, and their radical scavenging activity was evaluated by DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt) radical scavenging tests. Both DPPH and ABTS radical scavenging reactions of these compounds consisted of two different steps. In the first step, catechol moieties of the caffeoyl residues were rapidly converted to o-quinone structures and no significant difference in the reactivity was observed among the tested compounds. In the second step, however, the rate of the reaction increased as the intramolecular distance of the two caffeoyl residues decreased. A novel intramolecular coupling product, which could scavenge additional radicals, was isolated from the reaction mixture of trans-1,2-dicaffeoyloxycyclohexane and DPPH radical. The result suggests that the second step of the radical scavenging reaction is arising from an intramolecular interaction between the two caffeoquinone residues to regenerate catechol structures, and that the closer their distance is, the more rapidly they react. The radical scavenging activity of natural dicaffeoylquinic acids in a biological aqueous system might also depend on the positions of caffeoyl ester groups.     

A repeating unit of the DYZ1 family on the human Y chromosome consists of segments with partial male-specificity

An average-sized human Y chromosome contains about 3,000 copies of the repeating DNA family DYZ1. A major repeating unit of the family, pHY10, has been cloned and an entire 3,564-bp sequence has already been determined by Nakahori et al. (1986). In the present study, pHY10 was divided into six consecutive segments, A to F, which were independently amplified by the PCR technique to see if they were strictly male-specific. pHY10 appears to consist of segments of various male-specificity. The B segment was apparently male-specific; however, the use of additional techniques (Southern-blot analysis or second PCR amplification in combination with the standard PCR) revealed homologous sequences in some females. None of the six segments of pHY10 may be male-specific in a strict sense. Different segments appear to be conserved during evolution to different extents. The 323-bp E segment appears to be the least conserved and to be responsible for the generation of most variations within the DYZ1 family.     

Rad50 is involved in MMS-induced recombination between homologous chromosomes in mitotic cells

The structural maintenance of chromosomes (SMC) family proteins (Smc1-Smc6) typically consist of two coiled-coil domains, an amino-terminal head domain, and a carboxyl-terminal tail domain. Rad50, a component of the Mre11/Rad50/Xrs2 (MRX) complex, has a similar domain structure to the SMC proteins. In Saccharomyces cerevisiae, the MRX complex appears to be essential for recombination between homologous chromosomes in meiotic cells, but not in cells undergoing vegetative growth. Here we provide for the first time evidence that Rad50, like Smc6, is required for the induction of recombination between homologous chromosomes in cells in the vegetative growth state upon exposure to methyl methanesulfonate. However, UV-induced recombination between homologous chromosomes is intact in both rad50 and smc6-56 mutant cells.     

ROCK inhibitor Y-27632 attenuates stellate cell contraction and portal pressure increase induced by endothelin-1

By using a selective ROCK inhibitor Y-27632, the role of Rho-ROCK signaling in the function of hepatic stellate cells in culture was studied. Stellate cells maintained the "star-like" configuration of the quiescent stage in the presence of Y-27632, while the expression of smooth muscle alpha-actin and PDGF receptor beta was not affected by the agent. Serum-stimulated migration of the cells was significantly suppressed by Y-27632. The contraction of stellate cells induced by 5 nM endothelin-1 was attenuated by the agent in a dose-dependent manner. Formation of F-actin stress fibers and phosphorylation of myosin light chain was apparently reduced by Y-27632 even under the stimulation with endothelin-1. On the other hand, ex vivo liver perfusion experiment revealed that endothelin-1 (2 nM)-induced increase of portal vein constriction was almost completely inhibited by 20 microM Y-27632 with a concomitant improvement of hepatocyte degeneration. These results suggest that ROCK is one of the key regulators of stellate cell motility and that the clinical application of ROCK inhibitors such as Y-27632 should be considered in the reduction of portal hypertension in liver fibrosis and cirrhosis.     

High-efficiency bypass of DNA damage by human DNA polymerase Q

Endogenous DNA damage arises frequently, particularly apurinic (AP) sites. These must be dealt with by cells in order to avoid genotoxic effects. DNA polymerase theta; is a newly identified enzyme encoded by the human POLQ gene. We find that POLQ has an exceptional ability to bypass an AP site, inserting A with 22% of the efficiency of a normal template, and continuing extension as avidly as with a normally paired base. POLQ preferentially incorporates A opposite an AP site and strongly disfavors C. On nondamaged templates, POLQ makes frequent errors, incorporating G or T opposite T about 1% of the time. This very low fidelity distinguishes POLQ from other A-family polymerases. POLQ has three sequence insertions between conserved motifs in its catalytic site. One insert of approximately 22 residues into the tip of the polymerase thumb subdomain is predicted to confer considerable flexibility and additional DNA contacts to affect enzyme fidelity. POLQ is the only known enzyme that efficiently carries out both the insertion and extension steps for bypass of AP sites, commonly formed as endogenous genomic lesions.