Bleeding complications following percutaneous coronary intervention associate with increased mortality. However, the underlying molecular mechanisms are insufficiently understood. Platelet recruitment and activation at sites of vascular injury depends on the function of integrin adhesion receptors. Besides GPIIbIIIa as the most abundant integrin receptor, platelets relevantly express ß1 integrins. Experimental evidence from in vivo studies suggests a significant role of ß1 integrins in primary haemostasis. However, little is known about the clinical impact of genetic alterations of the β1 subunit, which might contribute to bleeding complications in patients. In this study, we performed DNA sequencing of patients suffering from bleeding complications after coronary artery stenting according to TIMI or BARC classification. We isolated DNA samples from 741 patients out of a cohort from 14,160 patients recruited in seven randomized clinical trials between June 2000 and May 2011. Subsequently, Sanger sequencing was performed covering the β1 integrin cytoplasmic activation domain (exon16) and its non-coding upstream region. Out of 764 patients suffering from bleeding complications, 741 DNA samples were successfully sequenced. Genotype variation was detected for SNP rs2153875 located within the non-coding upstream region with following allele frequency in study population: CC (7.3%), CA (35%) and AA (57.8%), which is similar to a general population cohort. Further, genotype variation in SNP rs2153875 do not associate with the frequency of TIMI or BARC classified access or non-access site bleedings. Genotype variations of the β1 integrin activation domain do not associate with bleeding risk after PCI.
Ladderanes are hydrocarbon chains with three or five linearly concatenated cyclobutane rings that are uniquely produced as membrane lipid components by anammox (anaerobic ammonia-oxidizing) bacteria. By virtue of their angle and torsional strain, ladderanes are unusually energetic compounds, and if produced biochemically by engineered microbes, could serve as renewable, high-energy-density jet fuel components. The biochemistry and genetics underlying the ladderane biosynthetic pathway are unknown, however, previous studies have identified a pool of 34 candidate genes from the anammox bacterium, Kuenenia stuttgartiensis, some or all of which may be involved with ladderane fatty acid biosynthesis. The goal of the present study was to establish a systematic means of testing the candidate genes from K. stuttgartiensis for involvement in ladderane biosynthesis through heterologous expression in E. coli under anaerobic conditions. This study describes an efficient means of assembly of synthesized, codon-optimized candidate ladderane biosynthesis genes in synthetic operons that allows for changes to regulatory element sequences, as well as modular assembly of multiple operons for simultaneous heterologous expression in E. coli (or potentially other microbial hosts). We also describe in vivo functional tests of putative anammox homologs of the phytoene desaturase CrtI, which plays an important role in the hypothesized ladderane pathway, and a method for soluble purification of one of these enzymes. This study is, to our knowledge, the first experimental effort focusing on the role of specific anammox genes in the production of ladderanes, and lays the foundation for future efforts toward determination of the ladderane biosynthetic pathway. Our substantial, but far from comprehensive, efforts at elucidating the ladderane biosynthetic pathway were not successful. We invite the scientific community to take advantage of the considerable synthetic biology resources and experimental results developed in this study to elucidate the biosynthetic pathway that produces unique and intriguing ladderane lipids. 相似文献
In our previous work we found considerable accumulation of early light-inducible proteins (ELIPs) in barley during adaptation
to combined high light and cold stress, an accumulation which occurred preferentially in the apical part of the leaves (M.-H.
Montané et al., 1997, Planta 202: 293–302). Here we studied, under the same conditions, the effect of adaptation on the composition
of thylakoid membrane proteins and pigments, particularly xanthophylls and chlorophyll, and their distribution within the
barley leaf. It was observed that high light fluxes appeared to favour the trimerization of the light-harvesting complex of
photosystem II (LHC II) whereas cold appeared to favour the monomers of LHC II. High light, cold or the combination of both
factors had only a small effect on the protein composition of the thylakoid membranes except for the proteins of LHC II which
were found to decrease under high light to a greater extent at 25 °C than at 5 °C. The total xanthophyll-cycle carotenoid
content increased linearly with cellular development, the highest amount being observed in the apical part of the leaf. Cold
and high light acted synergistically to induce less than a doubling in the amount of total xanthophylls, while chlorophylls
a and b remained nearly constant. The fraction consisting of antheraxanthin plus zeaxanthin was up to 4- to 5-fold higher at 5 °C
than at 25 °C. As determined previously (Montané et al. 1997), the same conditions caused a 15-fold increase in the accumulation
of ELIPs. Consequently, neither the distribution of total xanthophylls nor that of antheraxanthin plus zeaxanthin along the
leaf followed the same pattern as ELIP. Thus, the accumulation of xanthophylls cannot be stoichiometrically correlated with
that of ELIPs. Using electrophoresis in the presence of decylmaltoside, we could demonstrate for the first time that ELIPs
of 13.5 kDa are contained in high-molecular-mass complexes of >100 kDa, which are located in the unstacked stroma lamellar
region of the thylakoid membranes.
Received: 6 April 1998 / Accepted: 26 January 1999 相似文献
Leaf development involves many complex genetic interactions,signals between adjacent cells or between more distant tissues and consequent changes in cell fate.This review describes three stages in leaf development where regulation by small RNAs have been used to modulate gene expression patterns. 相似文献
Amino acid oxidation is known to affect the structure, activity, and rate of degradation of proteins. Methionine oxidation is one of the several chemical degradation pathways for recombinant antibodies. In this study, we have identified for the first time a solvent accessible tryptophan residue (Trp-32) in the complementary-determining region (CDR) of a recombinant IgG1 antibody susceptible to oxidation under real-time storage and elevated temperature conditions. The degree of light chain Trp-32 oxidation was found to be higher than the oxidation level of the conserved heavy chain Met-429 and the heavy chain Met-107 of the recombinant IgG1 antibody HER2, which have already been identified as being solvent accessible and sensitive to chemical oxidation. In order to reduce the time for simultaneous identification and functional evaluation of potential methionine and tryptophan oxidation sites, a test system employing tert-butylhydroperoxide (TBHP) and quantitative LC-MS was developed. The optimized oxidizing conditions allowed us to specifically oxidize the solvent accessible methionine and tryptophan residues that displayed significant oxidation in the real-time stability and elevated temperature study. The achieved degree of tryptophan oxidation was adequate to identify the functional consequence of the tryptophan oxidation by binding studies. In summary, the here presented approach of employing TBHP as oxidizing reagent combined with quantitative LC-MS and binding studies greatly facilitates the efficient identification and functional evaluation of methionine and tryptophan oxidation sites in the CDR of recombinant antibodies. 相似文献
Successful metabolic engineering relies on methodologies that aid assembly and optimization of novel pathways in microbes. Many different factors may contribute to pathway performance, and problems due to mRNA abundance, protein abundance, or enzymatic activity may not be evident by monitoring product titers. To this end, synthetic biologists and metabolic engineers utilize a variety of analytical methods to identify the parts of the pathway that limit production. In this study, targeted proteomics, via selected-reaction monitoring (SRM) mass spectrometry, was used to measure protein levels in Escherichia coli strains engineered to produce the sesquiterpene, amorpha-4,11-diene. From this analysis, two mevalonate pathway proteins, mevalonate kinase (MK) and phosphomevalonate kinase (PMK) from Saccharomyces cerevisiae, were identified as potential bottlenecks. Codon-optimization of the genes encoding MK and PMK and expression from a stronger promoter led to significantly improved MK and PMK protein levels and over three-fold improved final amorpha-4,11-diene titer (>500 mg/L). 相似文献
Transmissible spongiform encephalopathy diseases are untreatable, uniformly fatal degenerative syndromes of the central nervous
system that can be transmitted both within as well as between species. The bovine spongiform encephalopathy (BSE) epidemic
and the emergence of a new human variant of Creutzfeldt-Jakob disease (vCJD), have profoundly influenced beef production processes
as well as blood donation and surgical procedures. Simple, robust and cost effective diagnostic screening and surveillance
tools are needed for both the preclinical and clinical stages of TSE disease in order to minimize both the economic costs
and zoonotic risk of BSE and to further reduce the risk of secondary vCJD. 相似文献