Involvement of proliferating cell nuclear antigen (cyclin) in DNA replication in living cells.

Proliferating cell nuclear antigen (PCNA) (also called cyclin) is known to stimulate the activity of DNA polymerase delta but not the other DNA polymerases in vitro. We injected a human autoimmune antibody against PCNA into unfertilized eggs of Xenopus laevis and examined the effects of this antibody on the replication of injected plasmid DNA as well as egg chromosomes. The anti-PCNA antibody inhibited plasmid replication by up to 67%, demonstrating that PCNA is involved in plasmid replication in living cells. This result further implies that DNA polymerase delta is necessary for plasmid replication in vivo. Anti-PCNA antibody alone did not block plasmid replication completely, but the residual replication was abolished by coinjection of a monoclonal antibody against DNA polymerase alpha. Anti-DNA polymerase alpha alone inhibited plasmid replication by 63%. Thus, DNA polymerase alpha is also required for plasmid replication in this system. In similar studies on the replication of egg chromosomes, the inhibition by anti-PCNA antibody was only 30%, while anti-DNA polymerase alpha antibody blocked 73% of replication. We concluded that the replication machineries of chromosomes and plasmid differ in their relative content of DNA polymerase delta. In addition, we obtained evidence through the use of phenylbutyl deoxyguanosine, an inhibitor of DNA polymerase alpha, that the structure of DNA polymerase alpha holoenzyme for chromosome replication is significantly different from that for plasmid replication.     

Phosphorylation of 4EBP by oral leucine administration was suppressed in the skeletal muscle of PGC-1α knockout mice

Leucine is known to increase mTOR-mediated phosphorylation of 4EBP. In this study, leucine was administered to skeletal muscle-PGC-1α knockout mice. We observed attenuated 4EBP phosphorylation in the skeletal muscle, but not in the liver, of the PGC-1α knockout mice. These data suggest that skeletal muscle-PGC-1α is important for leucine-mediated mTOR activation and protein biosynthesis.     

Nectin4 is an epithelial cell receptor for canine distemper virus and involved in neurovirulence

Canine distemper virus (CDV) uses signaling lymphocyte activation molecule (SLAM), expressed on immune cells, as a receptor. However, epithelial and neural cells are also affected by CDV in vivo. Wild-type CDV strains showed efficient replication with syncytia in Vero cells expressing dog nectin4, and the infection was blocked by an anti-nectin4 antibody. In dogs with distemper, CDV antigen was preferentially detected in nectin4-positive neurons and epithelial cells, suggesting that nectin4 is an epithelial cell receptor for CDV and also involved in its neurovirulence.     

Growth and nitrogen use in Xanthium canadense grown in an open or in a dense stand

Plants develop branches profusely when grown solitarily, while less so when grown in a dense stand. Such changes in architecture are associated with changes in dry mass allocation and nitrogen use. Here, we studied what traits in plant growth and nitrogen use were influenced by different light climates in the stand. Annual plants (Xanthium canadense) were grown solitarily or in a dense stand. Dry mass growth was analyzed as the product of the net assimilation rate (NAR) and leaf area (LA). Nitrogen use efficiency (NUE) was analyzed as the product of nitrogen productivity (NP) and the mean residence time (MRT) of nitrogen. These growth variables were further factorized into their components. Solitary plants maintained a high NAR, whereas plants in the dense stand decreased the NAR due to mutual shading. Plants in the dense stand developed a larger LA with a higher specific leaf area than solitary plants. Solitary plants had higher NUE due to higher NP. A temporal increase in NUE was attributed to the increase in MRT of nitrogen. Light climate was different between solitary and dense-stand plants, but they took up a comparable amount of nitrogen and used it differently in response to the given light climate. NUE was thus demonstrated to be a useful tool for analyzing the mechanism leading to different N use in plant growth.     

Simultaneous determination of residual veterinary drugs in bovine, porcine, and chicken muscle using liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A simple and rapid method using liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/MS/MS) for the simultaneous determination of 130 veterinary drugs and their metabolites in bovine, porcine, and chicken muscle was developed. The drugs (1 to 10 ng/g, in muscle) were extracted from bovine, porcine, or chicken muscles with acetonitrile-methanol (95:5, v/v), and the extracts were delipidated with n-hexane saturated with acetonitrile. The extracts were evaporated, dissolved with methanol, analyzed by liquid chromatography with gradient elution on a C18 column, and determined by electrospray ionization tandem mass spectrometry. The detection limits ranged from 0.03 to 3 ng/g. The quantitation limits ranged from 0.1 to 10 ng/g. One hundred eleven, 122, and 123 drugs from bovine, porcine, and chicken muscle respectively showed recoveries between 70 and 110%.     

Crystal structure of hypothetical protein TTHB192 from Thermus thermophilus HB8 reveals a new protein family with an RNA recognition motif-like domain

We have determined the crystal structure of hypothetical protein TTHB192 from Thermus thermophilus HB8 at 1.9 A resolution. This protein is a member of the Escherichia coli ygcH sequence family, which contains approximately 15 sequence homologs of bacterial origin. These homologs have a high isoelectric point. The crystal structure reveals that TTHB192 consists of two independently folded domains, and that each domain exhibits a ferredoxin-like fold with a four-stranded antiparallel beta-sheet packed on one side by alpha-helices. These two tandem domains face each other to generate a beta-sheet platform. TTHB192 displays overall structural similarity to Sex-lethal protein and poly(A)-binding protein fragments. These proteins have RNA binding activity which is supported by a beta-sheet platform formed by two tandem repeats of an RNA recognition motif domain with signature sequence motifs on the beta-sheet surface. Although TTHB192 does not have the same signature sequence motif as the RNA recognition motif domain, the presence of an evolutionarily conserved basic patch on the beta-sheet platform could be functionally relevant for nucleic acid-binding. This report shows that TTHB192 and its sequence homologs adopt an RNA recognition motif-like domain and provides the first testable functional hypothesis for this protein family.