Complete Genome Sequence of Human Respiratory Syncytial Virus Genotype A with a 72-Nucleotide Duplication in the Attachment Protein G Gene

The complete genome sequence of human respiratory syncytial virus genotype A (HRSV-A) with a 72-nucleotide duplication in the C-terminal part of the attachment protein G gene was determined and analyzed. The genome was 15,277 bp in length, and 0.46 to 6.03% variations were identified at the nucleotide level compared with the previously reported complete genome of HRSV-A. Characterization of the genome will improve understanding of the diversity of the HRSV-A major antigens and enable an in-depth analysis of its genetics.     

Epitope spreading to citrullinated antigens in mouse models of autoimmune arthritis and demyelination

Introduction  

Anti-citrullinated protein antibodies have a diagnostic role in rheumatoid arthritis (RA); however, little is known about their origins and contribution to pathogenesis. Citrullination is the post-translational conversion of arginine to citrulline by peptidyl arginine deiminase, and increased citrullination of proteins is observed in the joint tissue in RA and in brain tissue in multiple sclerosis (MS).     

In vivo post-translational modifications of recombinant mussel adhesive protein in insect cells

Mussel adhesive proteins (MAPs) have been suggested as promising bioadhesives for diverse application fields, including medical uses. Previously, we successfully constructed and produced a new type of functional recombinant MAP, fp-151, in a prokaryotic Escherichia coli expression system. Even though the E. coli-derived MAP showed several excellent features, such as high production yield and efficient purification, in vitro enzymatic modification is required to convert tyrosine residues to l-3,4-dihydroxyphenyl alanine (dopa) molecules for its adhesive ability, due to the intrinsic inability of E. coli to undergo post-translational modification. In this work, we produced a soluble recombinant MAP in insect Sf9 cells, which are widely used as an effective and convenient eukaryotic expression system for eukaryotic foreign proteins. Importantly, we found that insect-derived MAP contained converted dopa residues by in vivo post-translational modification. In addition, insect-derived MAP also had other post-translational modifications including phosphorylation of serine and hydroxylation of proline that originally occurred in some natural MAPs. To our knowledge, this is the first report on in vivo post-translational modifications of MAP containing dopa and other modified amino acid residues.     

Intrinsically bent DNA in the promoter regions of the yeast GAAL1-10 and GAL80 genes

Circular permutation analysis has detected fairly strong sites of intrinsic DNA bending on the promoter regions of the yeast GAL1-10 and GAL80 genes. These bends lie in functionally suggestive locations. On the promoter of the GAL1-10 structural genes, strong bends bracket nucleosome B, which lies between the UAS(G) and the GAL1 TATA. These intrinsic bends could help position nucleosome B. Nucleosome B plus two other promoter nucleosomes protect the TATA and start site elements in the inactive state of expression but are completely disrupted (removed) when GAL1-10 expression is induced. The strongest intrinsic bend ( approximately 70 degrees ) lies at the downstream edge of nucleosome B; this places it approximately 30 base pairs upstream of the GAL1 TATA, a position that could allow it to be involved in GAL1 activation in several ways, including the recruitment of a yeast HMG protein that is required for the normally robust level of GAL1 expression in the induced state (Paull, T., Carey, M., and Johnson, R. (1996) Genes Dev. 10, 2769-2781). On the regulatory gene GAL80, the single bend lies in the non-nucleosomal hypersensitive region, between a GAL80-specific far upstream promoter element and the more gene-proximal promoter elements. GAL80 promoter region nucleosomes contain no intrinsically bent DNA.     

Importance of homodimerization for the in vivo function of yeast RNA triphosphatase

Saccharomyces cerevisiae RNA triphosphatase Cet1 is an essential component of the yeast mRNA capping apparatus. The active site of Cet1 resides within a topologically closed hydrophilic beta-barrel (the triphosphate tunnel) that is supported by a globular hydrophobic core. The homodimeric quaternary structure of Cet1 is formed by a network of contacts between the partner protomers. By studying the effects of alanine-cluster mutations, we highlight the contributions of two separate facets of the crystallographic dimer interface to Cet1 function in vivo. One essential facet of the interface entails hydrophobic cross-dimer interactions of Cys(330) and Val(331) and a cross-dimer hydrogen bond of Asp(280) with the backbone amide of Gln(329). The second functionally relevant dimer interface involves hydrophobic side-chain interactions of Phe(272) and Leu(273). Ala-cluster mutations involving these residues elicited lethal or severe temperature-sensitive phenotypes that were suppressed completely by fusion of the mutated triphosphatases to the guanylyltransferase domain of mammalian capping enzyme. The recombinant D279A-D280A and F272A-L273A proteins retained phosphohydrolase activity but sedimented as monomers. These results indicate that a disruption of the dimer interface is uniquely deleterious when the yeast RNA triphosphatase must function in concert with the endogenous yeast guanylyltransferase. We also identify key residue pairs in the hydrophobic core of the Cet1 protomer that support the active site tunnel and stabilize the triphosphatase in vivo.     

Patterns of selection: stress resistance and energy storage in density-dependent populations of Drosophila melanogaster

Populations of Drosophila melanogaster subjected to extreme larval (CU) or adult (UC) densities for multiple generations were assayed for a variety of life history characters. When reared under either crowded or uncrowded larval conditions, populations which had been selected to tolerate the limitation of resources imposed by extreme larval (CU) crowding, exhibited greater starvation resistance and lipid content than did populations which do not routinely undergo larval density-dependent regulation. Previous studies have shown that the CU populations do not show a correlated increase in longevity; as has been generally observed for these characteristics in age-structured populations of D. melanogaster. This suggests that density-dependent natural selection may not always shape life histories of the same characteristic in the same direction that age-specific selection does.     

Bioactive constituents from gum guggul (Commiphora wightii)

Bioactivity-directed fractionation and purification afforded cytotoxic components of Commiphora wightii. The exudates of C. wightii were extracted with EtOAc and the extract was subjected to repeated column chromatography. A fraction showing cytotoxic activity was characterized as a mixture of two ferulates with an unusual skeleton by spectral and chemical methods, including by NMR, GC-MS and chemical derivatization. This fraction also showed moderate scavenging effect against 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals.     

Cluster growth by mitosis

A stochastic mitosis growth model is introduced. The colony enlarge by random mitosis of a mother cell which is replaced by two daughter cells. Unlike growth models based on a grid, the present model is isotropic. Using simulation of colonies of 10,000 cells, it is found that the density is constant and the boundary is fractal.     

Effects of ethanol on lipid bilayers with and without cholesterol: the distearoylphosphatidylcholine system

Differential scanning calorimetry (DSC) and fluorescence spectroscopy are useful techniques for investigating the phase transitions of phospholipid bilayers. In this study, these methods have been extended to determine the effects of ethanol on DSPC and DSPC/2 mol.% cholesterol bilayers. The biphasic effect of the main transition was observed on the DSC heating scans above 0.60 M ethanol. In addition, the concentration at which the biphasic effect occurs is not significantly changed in the presence of 2 mol.% cholesterol. For the fluorescence studies, 1,6-diphenyl-1,3,5-hexatriene (DPH) has been incorporated into the bilayer to monitor the phase transitions through the displacement of DPH. This fluorescent probe is used to directly determine the onset of interdigitation in the bilayer systems as indicated by a large decrease in the DPH fluorescence intensity. The addition of cholesterol lowered and broadened the transition temperatures of the phosphatidylcholine (PC) system. However, 2 mol.% cholesterol did not have a significant effect on the induction of the interdigitated phase in DSPC as observed from the small difference in ethanol threshold concentration for the two systems. This suggests that DSPC forms a more stable interdigitated gel phase than other PCs with shorter acyl chains.     

Molecular dynamics simulations of various coronavirus main proteinases

In this study, two homology models (denoted as MproST and MproSH) of main proteinase (Mpro) from the novel coronavirus associated with severe acute respiratory syndrome (SARS-CoV) were constructed based on the crystal structures of Mpro from transmissible gastroenteritis coronavirus (TGEV) (MproT) and human coronavirus HcoV-229E (MproH), respectively. Both MproST and MproSH exhibit similar folds as their respective template proteins. These homology models reveal three distinct functional domains as well as an intervening loop connecting domains II and III as found in both template proteins. A catalytic cleft containing the substrate binding sites S1 and S2 between domains I and II are also observed. S2 undergoes more significant structural fluctuation than S1 during the 400 ps molecular dynamics simulations because it is located at the open mouth of the catalytic cleft, while S1 is situated in the very bottom of this cleft. The thermal unfolding of these proteins begins at domain III, where the structure is least conserved among these proteins. Mpro may still maintain its proteolytic activity while it is partially unfolded. The electrostatic interaction between Arg40 and Asp186 plays an important role in maintaining the structural integrity of both S1 and S2.