Structure and binding analysis of Polyporus squamosus lectin in complex with the Neu5Ac{alpha}2-6Gal{beta}1-4GlcNAc human-type influenza receptor

Glycan chains that terminate in sialic acid (Neu5Ac) are frequently the receptors targeted by pathogens for initial adhesion. Carbohydrate-binding proteins (lectins) with specificity for Neu5Ac are particularly useful in the detection and isolation of sialylated glycoconjugates, such as those associated with pathogen adhesion as well as those characteristic of several diseases including cancer. Structural studies of lectins are essential in order to understand the origin of their specificity, which is particularly important when employing such reagents as diagnostic tools. Here, we report a crystallographic and molecular dynamics (MD) analysis of a lectin from Polyporus squamosus (PSL) that is specific for glycans terminating with the sequence Neu5Acα2-6Galβ. Because of its importance as a histological reagent, the PSL structure was solved (to 1.7??) in complex with a trisaccharide, whose sequence (Neu5Acα2-6Galβ1-4GlcNAc) is exploited by influenza A hemagglutinin for viral adhesion to human tissue. The structural data illuminate the origin of the high specificity of PSL for the Neu5Acα2-6Gal sequence. Theoretical binding free energies derived from the MD data confirm the key interactions identified crystallographically and provide additional insight into the relative contributions from each amino acid, as well as estimates of the importance of entropic and enthalpic contributions to binding.     

The C. elegans PRMT-3 possesses a type III protein arginine methyltransferase activity

Protein arginine methylation is a common post-translational modification in eukaryotes that is catalyzed by a family of the protein arginine methyltransferases (PRMTs). PRMTs are classified into three types: type I and type II add asymmetrically and symmetrically dimethyl groups to arginine, respectively, while type III adds solely monomethyl group to arginine. However, although the enzymatic activity of type I and type II PRMTs have been reported, the substrate specificity and the methylation activity of type III PRMTs still remains unknown. Here, we report the characterization of Caenorhabditis elegans PRMT-2 and PRMT-3, both of which are highly homologous to human PRMT7. We find that these two PRMTs can bind to S-adenosyl methionine (SAM), but only PRMT-3 has methyltransferase activity for histone H2A depending on its SAM-binding domain. Importantly, thin-layer chromatographic analysis demonstrates that PRMT-3 catalyzes the formation of monomethylated, but not dimethylated arginine. Our study thus identifies the first type III PRMT in C. elegans and provides a means to elucidate the physiological significance of arginine monomethylation in multicellular organisms.     

Predicting the Phenotypic Values of Physiological Traits Using SNP Genotype and Gene Expression Data in Mice

Predicting phenotypes using genome-wide genetic variation and gene expression data is useful in several fields, such as human biology and medicine, as well as in crop and livestock breeding. However, for phenotype prediction using gene expression data for mammals, studies remain scarce, as the available data on gene expression profiling are currently limited. By integrating a few sources of relevant data that are available in mice, this study investigated the accuracy of phenotype prediction for several physiological traits. Gene expression data from two tissues as well as single nucleotide polymorphisms (SNPs) were used. For the studied traits, the variance of the effects of the expression levels was more likely to differ among the genes than were the effects of SNPs. For the glucose concentration, the total cholesterol amount, and the total tidal volume, the accuracy by cross validation tended to be higher when the gene expression data rather than the SNP genotype data were used, and a statistically significant increase in the accuracy was obtained when the gene expression data from the liver were used alone or jointly with the SNP genotype data. For these traits, there were no additional gains in accuracy from using the gene expression data of both the liver and lung compared to that of individual use. The accuracy of prediction using genes that were selected differently was examined; the use of genes with a higher tissue specificity tended to result in an accuracy that was similar to or greater than that associated with the use of all of the available genes for traits such as the glucose concentration and total cholesterol amount. Although relatively few animals were evaluated, the current results suggest that gene expression levels could be used as explanatory variables. However, further studies are essential to confirm our findings using additional animal samples.     

Two classes of continuous cell lines established from Syrian hamster 9 day gestation embryos: Preneoplastic cells and progenitor cells

Summary Primary cultures of 9-d-gestation Syrian hamster embryo (E9) cells are distinct from primary cultures of later gestational age in terms of their growth and differentiation. First, primary E9 cell cultures express multiple mesenchymal differentiation lineages (e.g., adipocyte, myoblast) only rarely seen in cultures of 13-d-gestation fetal (F13) cells. Second, although most primary E9 cultures have a limited in vitro proliferative life span and exhibit cellular senescence similar to primary cultures of F13 cells, E9 cultures seem to have higher frequency of escape from senescence and conversion to continuous cell lines compared to F13 cells. Moreover, this frequency can be further increased 4- to 5-fold by continuous exposure of the E9 cells to tumor promoters or epidermal growth factor. Eleven continuous cell lines have been isolated from unreated, promoter-treated, or epidermal growth factor-treated primary E9 cultures. Seven of these are neoplastic or preneoplastic. However, the remaining four do not show any evidence of being in neoplastic progression and three of these continue to express the same differentiated phenotype observed in ther parental primary cell cultures. These studies were supported in part by grants from the National Institutes of Health (AG 01998), Bethesda, MD, and the U.S. Department of Energy (DE-A-C02-76-EVO-3280), Washington, DC.     

Genome-wide identification of chromatin-enriched RNA reveals that unspliced dentin matrix protein-1 mRNA regulates cell proliferation in squamous cell carcinoma

Chromatin-enriched noncoding RNAs (ncRNAs) have emerged as key molecules in epigenetic processes by interacting with chromatin-associated proteins. Recently, protein-coding mRNA genes have been reported to be chromatin-tethered, similar with ncRNA. However, very little is known about whether chromatin-enriched mRNA is involved in the chromatin modification process. Here, we comprehensively examined chromatin-enriched RNA in squamous cell carcinoma (SQCC) cells by RNA subcellular localization analysis, which was a combination of RNA fractionation and RNA-seq. We identified 11 mRNAs as highly chromatin-enriched RNAs. Among these, we focused on the dentin matrix protein-1 (DMP-1) gene because its expression in SQCC cells has not been reported. Furthermore, we clarified that DMP-1 mRNA was retained in chromatin in its unspliced form in SQCC in vitro and in vivo. As the inhibition of the unspliced DMP-1 mRNA (unspDMP-1) expression resulted in decreased cellular proliferation in SQCC cells, we performed ChIP-qPCR to identify cell cycle-related genes whose expression was epigenetically modified by unspDMP-1, and found that the CDKN1B promoter became active in SQCC cells by inhibiting unspDMP-1 expression. This result was further validated by the increased CDKN1B gene expression in the cells treated with siRNA for unspDMP-1 and by restoration of the decreased cellular proliferation rate by simultaneously inhibiting CDKN1B expression in SQCC cells. Further, to examine whether unspDMP-1 was able to associate with the CDKN1B promoter region, SQCC cells stably expressing PP7-mCherry fusion protein were transiently transfected with the unspDMP-1 fused to 24 repeats of the PP7 RNA stem loop (unspDMP-1-24xPP7) and we found that unspDMP-1-24xPP7 was efficiently precipitated with the antibody against mCherry and was significantly enriched in the CDKN1B promoter region. Thus, unspDMP-1 is a novel chromatin-enriched RNA that epigenetically regulates cellular proliferation of SQCC.     

Utilization of Alcohols by Rhodopseudomonas sp. No. 7 Isolated from n-Propanol–Enrichment Cultures

A purple non-sulfur bacterium, Rhodopseudomonas sp. No. 7, was isolated from n-propanol–enrichment cultures under anaerobic-light conditions. Strain No. 7 can produce hydrogen from alcohols. The rate of hydrogen production from n-propanol was 34 μl/hr/mg dry cells. Strain No. 7 showed multiplication by budding and the best growth on n-propanol among other organic compounds tested. But its growth on n-propanol was poor under aerobic-dark conditions. NAD-linked alcohol dehydrogenase, NAD-linked aldehyde dehydrogenase, acyl-CoA synthetase and malate synthetase were found in strain No. 7. These enzymes were constitutive. On the other hand, isocitrate lyase was induced in cells grown on ethanol but not on n-propanol. No activity of phenazine methosulfate-linked alcohol dehydrogenase was detected in strain No. 7.     

Nalidixic acid-resistant mutations of the gyrB gene of Escherichia coli

Summary DNA fragments of 3.4 kb containing the gyrB gene were cloned from Escherichia coli KL-16 and from spontaneous nalidixic acid-resistant mutants. The mutations (nal-24 and nal-31) had been determined to be in the gyrB gene by transduction analysis. Nucleotide sequence analysis of the cloned DNA fragments revealed that nal-24 was a G to A transition at the first base of the 426th codon of the gyrB gene, resulting in an amino acid change from aspartic acid to asparagine, and nal-31 was an A to G transition at the first base of the 447th codon, resulting in an amino acid change from lysine to glutamic acid. This indicates that mutations in the gyrB gene are responsible for nalidixic acid resistance.     

Isolation,functional characterization and stress responses of raffinose synthase genes in sugar beet

Raffinose (sucrosylgalactoside oligosaccharide) is a water soluble carbohydrate and accumulates in response to abiotic stresses in plants. Plant raffinose synthases are poorly characterized, and the genes involved in raffinose biosynthesis are unknown in sugar beet. Here, we report the isolation of two genes encoding raffinose synthase (BvRS1 and BvRS2) as well as a gene encoding galactinol synthase (BvGolS1) from sugar beet. BvRS1 and BvRS2 show high homologies to Arabidopsis raffinose synthase AtRS5. BvRS1 and BvGolS1 were expressed in Escherichia coli. Crude extracts showed the activities of raffinose synthase and galactinol synthase. The K _m values of BvRS1 for galactinol and sucrose and the K _m values of BvGolS1 for UDP-galactose and myo-inositol were determined. The expression levels of BvRS1 were significantly higher than that of BvRS2. The mRNA for BvRS1 was rapidly induced by cold stress whereas the mRNA for BvRS2 was slowly induced by cold and salt stresses. These data suggest that BvRS1 and BvRS2 encode raffinose synthase genes responsible to cold and salt stress, respectively.     

Coordinated ciliary beating requires Odf2-mediated polarization of basal bodies via basal feet

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Highlights? Mice expressing truncated Odf2 cough and sneeze due to primary ciliary dyskinesia ? Full-length Odf2 is needed for the formation of basal body-associated basal feet ? In the absence of basal feet, basal bodies fail to align with planar polarity cues ? Polarization of basal bodies by Odf2 is required for coordinated ciliary beating