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1.
Vaccinia virus, the prototypic poxvirus, efficiently and faithfully replicates its ~200-kb DNA genome within the cytoplasm of infected cells. This intracellular localization dictates that vaccinia virus encodes most, if not all, of its own DNA replication machinery. Included in the repertoire of viral replication proteins is the I3 protein, which binds to single-stranded DNA (ssDNA) with great specificity and stability and has been presumed to be the replicative ssDNA binding protein (SSB). We substantiate here that I3 colocalizes with bromodeoxyuridine (BrdU)-labeled nascent viral genomes and that these genomes accumulate in cytoplasmic factories that are delimited by membranes derived from the endoplasmic reticulum. Moreover, we report on a structure/function analysis of I3 involving the isolation and characterization of 10 clustered charge-to-alanine mutants. These mutants were analyzed for their biochemical properties (self-interaction and DNA binding) and biological competence. Three of the mutant proteins, encoded by the I3 alleles I3-4, -5, and -7, were deficient in self-interaction and unable to support virus viability, strongly suggesting that the multimerization of I3 is biologically significant. Mutant I3-5 was also deficient in DNA binding. Additionally, we demonstrate that small interfering RNA (siRNA)-mediated depletion of I3 causes a significant decrease in the accumulation of progeny genomes and that this reduction diminishes the yield of infectious virus.  相似文献   
2.
The gene encoding the Enn protein (enn) of the M untypeable group A streptococcal (GAS) strain 64/14 was amplified by polymerase chain reaction, cloned into the expression vector pJLA602 and expressed in Escherichia coli DH5α. Unlike other GAS–Enn proteins, which exhibit IgA-binding activity, the recombinant Enn enn64/14 protein reacted preferentially with human IgG3. The 1050 bp open reading frame comprising the enn64/14 gene was completely sequenced. The region of the gene encoding the signal peptide and the C-terminus exhibited >95% homology to corresponding sections of other enn genes. The region of enn64/74 encoding the N-terminus of the mature Enn protein was found to be highly homologous to the corresponding section of the gene encoding the M-like protein of GAS serotype M9 (emmL9). The reoombinant protein encoded by emmL9 was found to react with all four human IgG subclasses. About 30% of the 1152bp open reading frame of emmiL9 encoding the N-terminus was found to display >90% homology to the corresponding section of enn64/14 but was <50% homologous in the remainder of the gene sequence. The functional analysis of the subcloned N-terminal section of emmL9 demonstrated a polypeptide exhibiting selective binding to human IgG3. These findings suggested that enn64/14 was a hybrid gene formed by recombination of an enn gene and an emmL9 gene. The putative recombinational event could have Involved a set of flanking 7bp direct repeats. Since enn64/14 and emmL9 are genes from different phylogenetic lineages of GAS, this report provides evidence that intergenomic recombinations between different types of GAS genes can occur and could lead to hybrid proteins with unique Ig-binding characteristics.  相似文献   
3.
Experimental and computational studies have shown that cellular membranes deform to stabilize the inclusion of transmembrane (TM) proteins harboring charge. Recent analysis suggests that membrane bending helps to expose charged and polar residues to the aqueous environment and polar head groups. We previously used elasticity theory to identify membrane distortions that minimize the insertion of charged TM peptides into the membrane. Here, we extend our work by showing that it also provides a novel, computationally efficient method for exploring the energetics of ion and small peptide penetration into membranes. First, we show that the continuum method accurately reproduces energy profiles and membrane shapes generated from molecular simulations of bare ion permeation at a fraction of the computational cost. Next, we demonstrate that the dependence of the ion insertion energy on the membrane thickness arises primarily from the elastic properties of the membrane. Moreover, the continuum model readily provides a free energy decomposition into components not easily determined from molecular dynamics. Finally, we show that the energetics of membrane deformation strongly depend on membrane patch size both for ions and peptides. This dependence is particularly strong for peptides based on simulations of a known amphipathic, membrane binding peptide from the human pathogen Toxoplasma gondii. In total, we address shortcomings and advantages that arise from using a variety of computational methods in distinct biological contexts.  相似文献   
4.
E L Lipman  D R Offord  M H Boyle 《CMAJ》1997,156(5):639-645
OBJECTIVE: To examine the sociodemographic, physical and mental health characteristics of single mothers in Ontario. DESIGN: Cross-sectional. SETTING: Ontario. PARTICIPANTS: Ontario residents aged 15 years or older who participated in the Ontario Health Supplement survey conducted between December 1990 and April 1991; of 9953 eligible participants, 1540 were mothers with at least 1 dependent child (less than 16 years of age). OUTCOME MEASURES: Prevalence rates of sociodemographic, physical and mental health characteristics. RESULTS: Single mothers were significantly more likely than the mothers in 2-parent families to be poor, to be 25 years of age or less, to have mental health problems (dissatisfaction with multiple aspects of life, affective disorder ever and 1 or more psychiatric disorders in the past year or ever) and to use mental health services. When compared by income level, poor single mothers had a higher prevalence of all mental health outcomes measured; the difference was significant for anxiety disorder in the past year or ever and for 1 or more psychiatric disorders in the past year or ever. In a logistic regression analysis, single-mother status was found to have the strongest independent effect on predicting mental health morbidity and utilization of mental health services; the next strongest was low income. CONCLUSIONS: Single mothers are more likely to be poor, to have an affective disorder and to use mental health services than mothers in 2-parent families. The risk of mental health problems is especially pronounced among poor single mothers. Further studies are needed to determine which aspects of single motherhood, apart from economic status, affect mental health outcomes.  相似文献   
5.
ABSTRACT: BACKGROUND: Plant biotechnology can be leveraged to produce food, fuel, medicine, and materials. Standardized methods advocated by the synthetic biology community can accelerate the plant design cycle, ultimately making plant engineering more widely accessible to bioengineers who can contribute diverse creative input to the design process. RESULTS: This paper presents work done largely by undergraduate students participating in the 2010 International Genetically Engineered Machines (iGEM) competition. Described here is a framework for engineering the model plant Arabidopsis thaliana with standardized, BioBrick compatible vectors and parts available through the Registry of Standard Biological Parts (www.partsregistry.org). This system was used to engineer a proof-of-concept plant that exogenously expresses the taste-inverting protein miraculin. CONCLUSIONS: Our work is intended to encourage future iGEM teams and other synthetic biologists to use plants as a genetic chassis. Our workflow simplifies the use of standardized parts in plant systems, allowing the construction and expression of heterologous genes in plants within the timeframe allotted for typical iGEM projects.  相似文献   
6.
Antibody responses in mice immunized by a single gene gun inoculation of plasmid expressing the influenza virus H1 hemagglutinin and in mice immunized by a sublethal H1 influenza virus infection have been compared. Both immunizations raised long-lived serum responses that were associated with the localization of antibody-secreting cells (ASC) to the bone marrow. However, the kinetics of these responses were 4 to 8 weeks slower in the DNA-immunized than in the infection-primed mice. Following a gene gun booster, the presence of ASC in the inguinal lymph nodes, but not in other lymph nodes, revealed gene gun responses being initiated in the nodes that drain the skin target site. Both pre- and postchallenge, the DNA-immunized mice had 5- to 10-times-lower levels of antibody and ASC than the infection-primed mice.  相似文献   
7.
The levels of adenosine, the adenine nucleotides AMP, ADP, ATP, as well as NAD, NADP, protein and chlorophyll were determined in young barley plants of which the primary leaves were infected by Erysiphe graminis DC. f. sp. hordei MARCHAL. The largest changes of these metabolite levels, compared to the non-infected control, occurred in the infected leaf and to a lesser degree also in the roots and in healthy younger leaves. The increase in the levels of most metabolites in the primary leaf revealed the sink property of this infected tissue and possible stress or defence reactions of the host, whereas the reductions in the roots showed the impaired supply of this natural sink organ due to the infection. Changes in the healthy leaves were most pronounced in the tertiary leaf and may reflect metabolic stimulation in that healthy organ. The changes of the adenosine pool, a precursor of the adenine nucleotides, were discussed in terms of translocation and its possible role as a precursor for fungal purine nucleotide synthesis.  相似文献   
8.
BackgroundDiffuse large B-cell lymphoma (DLBCL) is an aggressive disease, with 30% to 40% of patients failing to be cured with available primary therapy. microRNAs (miRNAs) are RNA molecules that attenuate expression of their mRNA targets. To characterize the DLBCL miRNome, we sequenced miRNAs from 92 DLBCL and 15 benign centroblast fresh frozen samples and from 140 DLBCL formalin-fixed, paraffin-embedded tissue samples for validation.ResultsWe identify known and candidate novel miRNAs, 25 of which are associated with survival independently of cell-of-origin and International Prognostic Index scores, which are established indicators of outcome. Of these 25 miRNAs, six miRNAs are significantly associated with survival in our validation cohort. Abundant expression of miR-28-5p, miR-214-5p, miR-339-3p, and miR-5586-5p is associated with superior outcome, while abundant expression of miR-324-5p and NOVELM00203M is associated with inferior outcome. Comparison of DLBCL miRNA-seq expression profiles with those from other cancer types identifies miRNAs that were more abundant in B-cell contexts. Unsupervised clustering of miRNAs identifies two clusters of patients that have distinct differences in their outcomes. Our integrative miRNA and mRNA expression analyses reveal that miRNAs increased in abundance in DLBCL appear to regulate the expression of genes involved in metabolism, cell cycle, and protein modification. Additionally, these miRNAs, including one candidate novel miRNA, miR-10393-3p, appear to target chromatin modification genes that are frequent targets of somatic mutation in non-Hodgkin lymphomas.ConclusionsOur comprehensive sequence analysis of the DLBCL miRNome identifies candidate novel miRNAs and miRNAs associated with survival, reinforces results from previous mutational analyses, and reveals regulatory networks of significance for lymphomagenesis.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0568-y) contains supplementary material, which is available to authorized users.  相似文献   
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