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1.
Identification of polypeptides encoded in open reading frame 1b of the putative polymerase gene of the murine coronavirus mouse hepatitis virus A59. 总被引:11,自引:9,他引:2 下载免费PDF全文
M R Denison P W Zoltick J L Leibowitz C J Pachuk S R Weiss 《Journal of virology》1991,65(6):3076-3082
The polypeptides encoded in open reading frame (ORF) 1b of the mouse hepatitis virus A59 putative polymerase gene of RNA 1 were identified in the products of in vitro translation of genome RNA. Two antisera directed against fusion proteins containing sequences encoded in portions of the 3'-terminal 2.0 kb of ORF 1b were used to immunoprecipitate p90, p74, p53, p44, and p32 polypeptides. These polypeptides were clearly different in electrophoretic mobility, antiserum reactivity, and partial protease digestion pattern from viral structural proteins and from polypeptides encoded in the 5' end of ORF 1a, previously identified by in vitro translation. The largest of these polypeptides had partial protease digestion patterns similar to those of polypeptides generated by in vitro translation of a synthetic mRNA derived from the 3' end of ORF 1b. The polypeptides encoded in ORF 1b accumulated more slowly during in vitro translation than polypeptides encoded in ORF 1a. This is consistent with the hypothesis that translation of gene A initiates at the 5' end of ORF 1a and that translation of ORF 1b occurs following a frameshift at the ORF 1a-ORF 1b junction. The use of in vitro translation of genome RNA and immunoprecipitation with antisera directed against various regions of the polypeptides encoded in gene A should make it possible to study synthesis and processing of the putative coronavirus polymerase. 相似文献
2.
Maintenance of the cellobiose utilization genes of Escherichia coli in a cryptic state 总被引:6,自引:1,他引:5
The genes for cellobiose utilization are normally cryptic in Escherichia
coli. The cellobiose system was used as a model to understand the process
by which silent genes are maintained in microbial populations. Previously
reported was (1) the isolation of a mutant strain that expresses the
cellobiose-utilization (Cel) genes and (2) that expression of those genes
allows utilization of three beta- glucoside sugars: cellobiose, arbutin,
and salicin. The Cel gene cluster has now been cloned from that mutant
strain. In the course of locating the Cel genes within the cloned DNA
segment, it was discovered that inactivation of the Cel-encoded hydrolase
rendered the host strain sensitive to all three beta-glucosides as potent
inhibitors. This sensitivity arises from the accumulation of the
phosphorylated beta- glucosides. Because even the fully active genes
conferred some degree of beta-glucoside sensitivity, the effects of
cellobiose on a series of five Cel+ mutants of independent origin were
investigated. Although each of those strains utilizes cellobiose as a sole
carbon and energy source, cellobiose also acts as a potent inhibitor that
reduces the growth rate on glycerol 2.5-16.5-fold. On the other hand,
wild-type strains that cannot utilize cellobiose are not inhibited. The
observation that the same compound can serve either as a nutrient or as an
inhibitor suggests that, under most conditions in which cellobiose will be
present together with other resources, there is a strong selective
advantage to having the cryptic (Cel0) allele. In those environments in
which cellobiose is the sole, or the best, resource, mutants that express
the genes (Cel+) will have a strong selective advantage. It is suggested
that temporal alternation between these two conditions is a major factor in
the maintenance of these genes in E. coli populations. This alternation of
environments and fitnesses was predicted by the model for cryptic-gene
maintenance that was previously published.
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3.
A prokaryotic vector, pGE374, containing the recA and lacZ genes, out-of-frame, was used for the expression of cDNA derived from the putative polymerase-encoding gene of the coronavirus mouse hepatitis virus strain A59 (MHV-A59). The pGE374/viral recombinant vector generates a tripartite bacterial/viral protein composed of a segment of the RecA protein at the N terminus, the coronaviral sequences in the middle, and an enzymatically active beta-galactosidase at the C terminus. Rabbits immunized with such recombinant proteins generated antibodies to the MHV-A59 portion of the tripartite protein. Because the MHV-A59 polymerase proteins have been difficult to identify during infection, we used a novel method to demonstrate the viral specificity of the antiserum. The viral cDNA was excised from the expression vector, and transferred to a pGem vector, downstream from and in-frame with a portion of the cat gene. This construct contained a bacteriophage RNA polymerase promoter that enabled the cell-free synthesis of a fusion protein that was used to verify that antibodies were generated to the expressed viral DNA. This strategy was shown to successfully result in the specific generation of antibodies to the encoded information of the viral cDNA. Furthermore, this method has general applicability in the generation and characterization of antibodies directed against proteins encoded in cDNAs. 相似文献
4.
5.
Elisabeth APM Romme Piet Geusens Willem F Lems Erica PA Rutten Frank WJM Smeenk Joop PW van den Bergh Peter ThW van Hal Emiel FM Wouters 《Respiratory research》2015,16(1)
Although osteoporosis and its related fractures are common in patients with COPD, patients at high risk of fracture are poorly identified, and consequently, undertreated. Since there are no fracture prevention guidelines available that focus on COPD patients, we developed a clinical approach to improve the identification and treatment of COPD patients at high risk of fracture. We organised a round-table discussion with 8 clinical experts in the field of COPD and fracture prevention in the Netherlands in December 2013. The clinical experts presented a review of the literature on COPD, osteoporosis and fracture prevention. Based on the Dutch fracture prevention guideline, they developed a 5-step clinical approach for fracture prevention in COPD. Thereby, they took into account both classical risk factors for fracture (low body mass index, older age, personal and family history of fracture, immobility, smoking, alcohol intake, use of glucocorticoids and increased fall risk) and COPD-specific risk factors for fracture (severe airflow obstruction, pulmonary exacerbations and oxygen therapy). Severe COPD (defined as postbronchodilator FEV1 < 50% predicted) was added as COPD-specific risk factor to the list of classical risk factors for fracture. The 5-step clinical approach starts with case finding using clinical risk factors, followed by risk evaluation (dual energy X-ray absorptiometry and imaging of the spine), differential diagnosis, treatment and follow-up. This systematic clinical approach, which is evidence-based and easy-to-use in daily practice by pulmonologists, should contribute to optimise fracture prevention in COPD patients at high risk of fracture. 相似文献
6.
M A Oleshansky J M Zoltick R H Herman E H Mougey J L Meyerhoff 《European journal of applied physiology and occupational physiology》1990,59(6):405-410
Neuroendocrine and sympathoadrenal responses to exhaustive graded treadmill exercise were examined in 17 male subjects of varying degrees of fitness. The mean duration of exercise to exhaustion was 15.2 +/- 0.7 (+/- SE) min. Exercise duration was inversely correlated with baseline heart rate (P less than 0.05). Compared to standing baseline values, mean plasma norepinephrine and epinephrine levels increased 339% and 301%, respectively, in an integrated 2-min blood sample collected immediately after completion of exercise. Mean adrenocorticotrophic hormone (ACTH), beta-endorphin (beta-EP), beta-lipotropin (beta-LPH), and prolactin levels increased 282%, 720%, 372%, and 211%, respectively, in an integrated 4-min blood sample beginning 2 min after completion of exercise. Cortisol levels increased 183% in the sample collected 17-21 min after exercise. The magnitude of these neuroendocrine responses to exercise was similar among individuals at the same relative intensity of exhaustive exercise, regardless of the duration of exercise. The exercise-induced increases of the pro-opiomelanocortin (POMC)-derived peptides, ACTH, beta-EP, and beta-LPH, were highly correlated with each other (P values less than 0.001), and were correlated with prolactin increases, (P values less than 0.05). During a 20-min recovery period after exercise, changes in heart rate, ACTH, and beta-LPH levels were correlated with duration of exercise, (P less than 0.01, P less than 0.03, and P less than 0.03, respectively).(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
7.
Fredrick M Mobegi Sacha AFT van Hijum Peter Burghout Hester J Bootsma Stefan PW de Vries Christa E van der Gaast-de Jongh Elles Simonetti Jeroen D Langereis Peter WM Hermans Marien I de Jonge Aldert Zomer 《BMC genomics》2014,15(1)
Background
Bacterial respiratory tract infections, mainly caused by Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis are among the leading causes of global mortality and morbidity. Increased resistance of these pathogens to existing antibiotics necessitates the search for novel targets to develop potent antimicrobials.Result
Here, we report a proof of concept study for the reliable identification of potential drug targets in these human respiratory pathogens by combining high-density transposon mutagenesis, high-throughput sequencing, and integrative genomics. Approximately 20% of all genes in these three species were essential for growth and viability, including 128 essential and conserved genes, part of 47 metabolic pathways. By comparing these essential genes to the human genome, and a database of genes from commensal human gut microbiota, we identified and excluded potential drug targets in respiratory tract pathogens that will have off-target effects in the host, or disrupt the natural host microbiota. We propose 249 potential drug targets, 67 of which are targets for 75 FDA-approved antimicrobials and 35 other researched small molecule inhibitors. Two out of four selected novel targets were experimentally validated, proofing the concept.Conclusion
Here we have pioneered an attempt in systematically combining the power of high-density transposon mutagenesis, high-throughput sequencing, and integrative genomics to discover potential drug targets at genome-scale. By circumventing the time-consuming and expensive laboratory screens traditionally used to select potential drug targets, our approach provides an attractive alternative that could accelerate the much needed discovery of novel antimicrobials.Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-958) contains supplementary material, which is available to authorized users. 相似文献8.
Gene and domain duplication in the chordate Otx gene family: insights from amphioxus Otx 总被引:6,自引:1,他引:5
We report the genomic organization and deduced protein sequence of a
cephalochordate member of the Otx homeobox gene family (AmphiOtx) and show
its probable single-copy state in the genome. We also present molecular
phylogenetic analysis indicating that there was single ancestral Otx gene
in the first chordates which was duplicated in the vertebrate lineage after
it had split from the lineage leading to the cephalochordates. Duplication
of a C-terminal protein domain has occurred specifically in the vertebrate
lineage, strengthening the case for a single Otx gene in an ancestral
chordate whose gene structure has been retained in an extant
cephalochordate. Comparative analysis of protein sequences and published
gene expression patterns suggest that the ancestral chordate Otx gene had
roles in patterning the anterior mesendoderm and central nervous system.
These roles were elaborated following Otx gene duplication in vertebrates,
accompanied by regulatory and structural divergence, particularly of Otx1
descendant genes.
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9.
Humoral immunity to adeno-associated virus type 2 vectors following administration to murine and nonhuman primate muscle 总被引:9,自引:0,他引:9 下载免费PDF全文
Chirmule N Xiao W Truneh A Schnell MA Hughes JV Zoltick P Wilson JM 《Journal of virology》2000,74(5):2420-2425
Adeno-associated virus (AAV) is being developed as a vector capable of conferring long-term gene expression, which is useful in the treatment of chronic diseases. In most therapeutic applications, it is necessary to readminister the vector. This study characterizes the humoral immune response to AAV capsid proteins following intramuscular injection and its impact on vector readministration. Studies of mice and rhesus monkeys demonstrated the formation of neutralizing antibodies to AAV capsid proteins that persisted for over 1 year and then diminished, but this did not prevent the efficacy of vector readministration. More-detailed studies strongly suggested that the B-cell response was T cell dependent. This was further evaluated with a blocking antibody to human CD4, primatized for clinical trials, in a biologically compatible mouse in which the endogenous murine CD4 gene was functionally replaced with the human counterpart. Transient pharmacologic inhibition of CD4 T cells with CD4 antibody prevented an antivector response long after the effects of the CD4 antibody diminished; readministration of vector without diminution of gene expression was possible. Our studies suggest that truly durable transgene expression (i.e., prolonged genetic engraftment together with vector readministration) is possible with AAV in skeletal muscle, although it will be necessary to transiently inhibit CD4 T-cell function to avoid the activation of memory B cells. 相似文献
10.
IB Masters MM Eastburn PW Francis R Wootton PV Zimmerman RS Ware AB Chang 《Respiratory research》2005,6(1):16