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1.
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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2.
Bergum PW Cruikshank A Maki SL Kelly CR Ruf W Vlasuk GP 《The Journal of biological chemistry》2001,276(13):10063-10071
Recombinant nematode anticoagulant protein c2 (rNAPc2) is a potent, factor Xa (fXa)-dependent small protein inhibitor of factor VIIa-tissue factor (fVIIa.TF), which binds to a site on fXa that is distinct from the catalytic center (exo-site). In the present study, the role of other fX derivatives in presenting rNAPc2 to fVIIa.TF is investigated. Catalytically active and active site blocked fXa, as well as a plasma-derived and an activation-resistant mutant of zymogen fX bound to rNAPc2 with comparable affinities (K(D) = 1-10 nm), and similarly supported the inhibition of fVIIa.TF (K(i)* = approximately 10 pm). The roles of phospholipid membrane composition in the inhibition of fVIIa.TF by rNAPc2 were investigated using TF that was either detergent-solubilized (TF(S)), or reconstituted into membranes, containing phosphatidylcholine (TF(PC)) or a mixture of phosphatidylcholine and phosphatidylserine (TF(PCPS)). In the absence of the fX derivative, inhibition of fVIIa.TF was similar for all three conditions (K(i) approximately 1 microm), whereas the addition of the fX derivative increased the respective inhibition by 35-, 150-, or 100,000-fold for TF(S), TF(PC), and TF(PCPS). The removal of the gamma-carboxyglutamic acid-containing domain from the fX derivative did not affect the binding to rNAPc2, but abolished the effect of factor Xa as a scaffold for the inhibition of fVIIa.TF by rNAPc2. The overall anticoagulant potency of rNAPc2, therefore, results from a coordinated recognition of an exo-site on fX/fXa and of the active site of fVIIa, both of which are properly positioned in the ternary fVIIa.TF.fX(a) complex assembled on an appropriate phospholipid surface. 相似文献
3.
Tully TP Bergum JS Schwarz SR Durand SC Howell JM Patel RN Cino PM 《Journal of industrial microbiology & biotechnology》2007,34(3):193-202
BMS-353645, also known as sordarin, was of interest based on its activity against pathogenic fungi. The objective of these
studies was to provide high quality starting substrate for chemical modification aimed at further improving biological activity,
with particular interest in the inhibition of Aspergillus. In the work presented here, Design of Experiments, or DOE, was successfully combined with traditional approaches to significantly
improve sordarin yields in fermentation flasks. Overall, yields were increased 25-fold from <100 μg/g to as high as 2,609 μg/g
in flasks through the use of various medium and conduction changes supplemented with DOE. The improved process was then successfully
scaled to pilot plant tanks with the best batch producing 2,389 μg/g sordarin at the 250-l scale. 相似文献
4.
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
5.
Niranjan Kottala Admassu Abebe Omar Sprockel James Bergum Faranak Nikfar Alberto M. Cuiti?o 《AAPS PharmSciTech》2012,13(4):1236-1242
Bilayer tableting technology has gained popularity in recent times, as bilayer tablets offer several advantages over conventional tablets. There is a dearth of knowledge on the impact of material properties and process conditions on the performance of bilayer tablets. This paper takes a statistical approach to develop a model that will determine the effect of the material properties and bilayer compression process parameters on the bonding strength and mode of breakage of bilayer tablets. Experiments were carried out at pilot scale to simulate the commercial manufacturing conditions. As part of this endeavor, a seven-factor half-fraction factorial (27−1) design was executed to study the effect of bilayer tablet compression process factors on the bonding strength of bilayer tablets. Factors studied in this work include: material properties (plastic and brittle), layer ratio, dwell time, layer sequence, first- and second-layer forces, and lubricant concentration. Bilayer tablets manufactured in this study were tested using the axial tester, as it considers both the interfacial and individual layer bonding strengths. Responses of the experiments were analyzed using PROC GLM of SAS (SAS Institute Inc, Cary, North Carolina). A model was fit using all the responses to determine the significant interactions (p < 0.05). The results of this study indicated that nature of materials played a critical role on the strength of bilayer compacts and also on mode of fracture. Bilayer tablets made with brittle materials in both the layers are strongest, and fracture occurred in the first layer indicating that interface is stronger than layers. Significant interactions were observed between the selected factors and these results will provide an insight into the interplay of material properties, process parameters, and lubricant concentration on the bonding strength and mode of breakage of bilayer tablets.
Electronic supplementary material
The online version of this article (doi:10.1208/s12249-012-9845-9) contains supplementary material, which is available to authorized users.KEY WORDS: axial tester, bilayer tablets, DOE, interfacial strength, process parameters 相似文献6.
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7.
8.
N. Theriault Hatzenbuhler E. F. Rehberg J. B. Carter D. P. Palermo E. V. Hubert P. W. Bergum 《Nucleosides, nucleotides & nucleic acids》2013,32(5-6):991-994
Abstract We have designed and constructed a cassette tPA gene with unique restriction sites in the interdomain regions. Each domain was chemoenzymatically assembled, cloned and sequenced. We have constructed and expressed the full-length molecule and some deletion mutants. 相似文献
9.
Nematode anticoagulant protein c2 reveals a site on factor Xa that is important for macromolecular substrate binding to human prothrombinase 总被引:2,自引:0,他引:2
Buddai SK Toulokhonova L Bergum PW Vlasuk GP Krishnaswamy S 《The Journal of biological chemistry》2002,277(29):26689-26698
The binding of recombinant nematode anticoagulant protein c2 (NAPc2) to either factor X or Xa is a requisite step in the pathway for the potent inhibition of VIIa tissue factor. We have used NAPc2 as a tight binding probe of human Xa to investigate protein substrate recognition by the human prothrombinase complex. NAPc2 binds with high affinity (K(d) approximately 1 nm) to both X and Xa in a way that does not require or occlude the active site of the enzyme. In contrast, NAPc2 is a tight binding, competitive inhibitor of protein substrate cleavage by human Xa incorporated into prothrombinase with saturating concentrations of membranes and Va. By fluorescence binding studies we show that NAPc2 does not interfere with the assembly of human prothrombinase. These are properties expected of an inhibitor that blocks protein substrate recognition by targeting extended macromolecular recognition sites (exosites) on the enzyme complex. A weaker interaction (K(d) = 260-500 nm) observed between NAPc2 and bovine X was restored to a high affinity one in a recombinant chimeric bovine X derivative containing 25 residues from the COOH terminus of the proteinase domain of human X. This region implicated in binding NAPc2 is spatially adjacent to a site previously identified as a potential exosite. Despite the weaker interaction with bovine Xa, NAPc2 was a tight binding competitive inhibitor of protein substrate cleavage by bovine prothrombinase as well. Extended enzymic surfaces elucidated with exosite-directed probes, such as NAPc2, may define a unique region of factor Xa that is modulated following its assembly into prothrombinase and in turn determines the binding specificity of the enzyme complex for its protein substrate. 相似文献
10.
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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