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1.
An amino acid analysis method using a commercially available analyzer that accurately quantitates protein-derived amino acids in the 10-100 pmol range is described. The method utilizes the robotic capability of the analyzer's autosampler to perform precolumn derivatization of both primary and secondary amino acids with o-phthalaldehyde and 9-fluorenylmethyl chloroformate, respectively. The derivatized amino acids are then separated on a C-18 reverse-phase amino acid column and quantitated in a single run by fluorescence detection. The characterization of beta-lactoglobulin and two tryptic peptides from the bacterial enzyme diaminopimelic acid epimerase is used to demonstrate the sensitivity and utility of this method. 相似文献
2.
Shibin Gao Carlos Martinez Debra J. Skinner Alan F. Krivanek Jonathan H. Crouch Yunbi Xu 《Molecular breeding : new strategies in plant improvement》2008,22(3):477-494
Leaf collection from the field, labeling and tracking back to the source plants after genotyping are rate limiting steps in
leaf DNA-based genotyping. In this study, an optimized genotyping method using endosperm DNA sampled from single maize seeds
was developed, which can be used to replace leaf DNA-based genotyping for both genetic studies and breeding applications.
A similar approach is likely to be suitable for all plants with relatively large seeds. Part of the endosperm was excised
from imbibed maize seeds and DNA extracted in 96-tube plates using individuals from eight F2 populations and seven inbreds. The quality of the resultant DNA was functionally comparable to DNA extracted from leaf tissue.
Extraction from 30 mg of endosperm yields 3–10 μg DNA, which is sufficient for analysis of 200–400 agarose-gel PCR-based markers,
with the potential for several million chip-based SNP marker analyses. By comparing endosperm DNA and leaf DNA for individuals
from an F2 population, genotyping errors caused by pericarp contamination and hetero-fertilization were found to average 3.8 and 0.6%,
respectively. Endosperm sampling did not affect germination rates under controlled conditions, although under normal field
conditions the germination rate, seedling establishment, and growth vigor were significantly lower than that of non-sampled
controls for some genotypes. However, careful field management can compensate for these effects. Seed DNA-based genotyping
lowered costs by 24.6% compared to leaf DNA-based genotyping due to reduced field plantings and labor costs. A substantial
advantage of this approach is that it can be used to select desirable genotypes before planting. As such it provides an opportunity
for dramatic improvements in the efficiency and selective gain of breeding systems based on optimum combinations of marker-assisted
selection and phenotypic selection within and between generations. 相似文献
3.
Abstract Several different species of freshwater Bryozoa, belonging to the genera Plumatella, Rumarcanella and Fredericella, were detected within the Northern Mallee Pipeline (NMP) system in Victoria, Australia, that required definitive identification. These organisms produce asexual buds called statoblasts, with valves composed of sclerotised chitin that bear minute micro-ornamentations of considerable taxonomical significance. Imaging and analysis of these distinctive micro-ornamentations using scanning electron microscopy (SEM) is often employed for species identification. Meticulous preparation of statoblast samples is therefore required that necessitates the removal of adhering debris, dehydration and drying—whilst mitigating specimen damage and distortion. This technical note describes an approach whereby each of these three steps have been individually designed to be as benign as possible, using mild detergent/sonication to remove debris, a gradual and gentle dehydration procedure using ethanol, and critical point drying. For the overall process, these methods are chosen to optimise control and to minimise the use of harsh and hazardous chemicals. 相似文献
4.
Michel Batista Fabricio K Marchini Paola AF Celedon Stenio P Fragoso Christian M Probst Henrique Preti Luiz S Ozaki Gregory A Buck Samuel Goldenberg Marco A Krieger 《BMC microbiology》2010,10(1):259
Background
The three trypanosomatids pathogenic to men, Trypanosoma cruzi, Trypanosoma brucei and Leishmania major, are etiological agents of Chagas disease, African sleeping sickness and cutaneous leishmaniasis, respectively. The complete sequencing of these trypanosomatid genomes represented a breakthrough in the understanding of these organisms. Genome sequencing is a step towards solving the parasite biology puzzle, as there are a high percentage of genes encoding proteins without functional annotation. Also, technical limitations in protein expression in heterologous systems reinforce the evident need for the development of a high-throughput reverse genetics platform. Ideally, such platform would lead to efficient cloning and compatibility with various approaches. Thus, we aimed to construct a highly efficient cloning platform compatible with plasmid vectors that are suitable for various approaches. 相似文献5.
Haumann M Barra M Loja P Löscher S Krivanek R Grundmeier A Andreasson LE Dau H 《Biochemistry》2006,45(43):13101-13107
Chloride is an important cofactor in photosynthetic water oxidation. It can be replaced by bromide with retention of the oxygen-evolving activity of photosystem II (PSII). Binding of bromide to the Mn(4)Ca complex of PSII in its dark-stable S(1) state was studied by X-ray absorption spectroscopy (XAS) at the Br K-edge in Cl(-)-depleted and Br(-)-substituted PSII membrane particles from spinach. The XAS spectra exclude the presence of metal ions in the first and second coordination spheres of Br(-). EXAFS analysis provided tentative evidence of at least one metal ion, which may be manganese or calcium, at a distance of approximately 5 A to Br(-). The native Cl(-) ion may bind at a similar distance. Accordingly, water oxidation may not require binding of a halide directly to the metal ions of the Mn complex in its S(1) state. 相似文献
6.
Salame MY More RS Verheye S Leimbach ME Iii SB Chronos NA 《International journal of cardiovascular interventions》1999,2(4):207-215
Glycoprotein IIb/IIIa receptor inhibitors represent a relatively new therapeutic approach in the field of antiplatelet therapy. Following the development of abciximab a number of small molecule GPIIb/IIIa inhibitors have been introduced such as tirofiban and eptifibatide. In this fast-moving field the interventional cardiologist needs a framework to guide decision-making for the individual patient. This review covers the efficacy and safety data from the clinical trials of GPIIb/IIIa inhibitors in the context of patients undergoing percutaneous coronary intervention for unstable angina/non-Q-wave myocardial infarction. There is an increasing body of evidence to support the efficacy of GPIIb/IIIa inhibitors in reducing the risk of adverse ischemic events in high and low risk patients undergoing percutaneous coronary intervention. A number of unresolved efficacy and safety issues remain, including the duration of treatment before and after intervention; whether a reduction in the heparin dose would further decrease the risk of hemorrhage without affecting the periprocedural thrombotic rate in patients undergoing PTCA with adjunctive GPIIb/IIIa inhibitors; and the cost-effectiveness of this therapy. When a thorough analysis of cost-effectiveness has been made, it will be easier to advocate the widespread use of these agents in all patients undergoing coronary intervention. 相似文献
7.
Krivanek AF Famula TR Tenscher A Walker MA 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2005,111(1):110-119
The inheritance of resistance to Xylella fastidiosa (Xf), the bacterium which causes Pierces disease (PD) in grapevines, was evaluated within a factorial mating design consisting of 16 full-sib families with resistance derived from Vitis arizonica interspecific hybrids. Measurements of disease progression under greenhouse conditions were based on quantitative assessment of Xf populations in stem tissues and on three phenotypic scores: leaf scorch, a cane maturation index (CMI) and an index that incorporated shoot stunting into the cane maturation index (CMSSI). Measurement of bacterial populations yielded the highest broad-sense heritability for resistance on a genotype mean basis (0.97), indicating that this measure of resistance was the least effected by environmental variation. Narrow-sense heritability of PD resistance was moderately high and measured 0.52, 0.60, 0.63 and 0.37 for Xf populations, CMI scores, CMSSI scores and leaf scorch values, respectively. Complex segregation analysis using the computer program Statistical Analysis for Genetic Epidemiology (sage) strongly indicated the existence of a major gene for PD resistance, which accounted for 91% of the total genetic variance. Conversion of the quantitative data into qualitative resistance levels and evaluation via a chi-square analysis showed that 15 of the 16 families segregated in accordance with a single gene hypothesis with a dominant allele controlling PD resistance. These data indicate that the trait should be relatively easy to pass on from parents to progeny in a breeding program for the development of PD-resistant grape cultivars, particularly when selection is based on cane maturation scores or stem Xf populations. 相似文献
8.
The Mn4Ca complex bound to photosystem II (PSII) is the active site of photosynthetic water oxidation. Its assembly involves binding and light-driven oxidation of manganese, a process denoted as photoactivation. The disassembly of the Mn complex is a thermally activated process involving distinct intermediates. Starting from intermediate states of the disassembly, which was initiated by a temperature jump to 47 degrees C, we photoactivated PSII membrane particles and monitored the activity recovery by O2 polarography and delayed chlorophyll fluorescence measurements. Oxidation state and structural features of the formed intermediates of the Mn complex were assayed by X-ray absorption spectroscopy at the Mn K-edge. The photoactivation time courses, which exhibit a lag phase characteristic of intermediate formation only when starting with the apo-PSII, suggest that within approximately 5 min of photoactivation of apo-PSII, a binuclear Mn complex is formed. It is proposed that a MnIII2(di-mu-oxo) complex is a key intermediate both in the disassembly and in the assembly reaction paths. 相似文献
9.
Expression of recombinant diaminopimelate epimerase in Escherichia coli. Isolation and inhibition with an irreversible inhibitor 总被引:1,自引:0,他引:1
W Higgins C Tardif C Richaud M A Krivanek A Cardin 《European journal of biochemistry》1989,186(1-2):137-143
Recombinant diaminopimelate epimerase is overproduced to give 1% of soluble protein when grown under the appropriate conditions in Escherichia coli. This compares with 0.02% of the constitutive level of wild-type enzyme. A new purification procedure now yields milligram quantities of homogeneous enzyme of high specific activity (192 U/mg). This has enabled sufficient amounts of enzyme both to compare with wild-type enzyme and to enable active site modification studies to be performed. Incubation of the enzyme with 2-(4-amino-4-carboxybutyl)-2-aziridine-carboxylic acid (AZIDAP), results in time-dependent irreversible inhibition. Tryptic digestion of the inactivated enzyme and peptide-mapping show that AZIDAP is specifically and covalently bound to the enzyme at a unique peptide. Determination of the amino acid sequence of this peptide and comparison with the sequence deduced from the DNA sequence of the dapF gene shows that Cys73 is labelled. Finally based on limited sequence similarities around this cysteine and active-site cysteines of proline racemase and 1-hydroxyproline 2-epimerase, together with mechanistic considerations, we propose that all three non-pyridoxal-phosphate-containing racemases/epimerases derive from a common evolutionary origin. 相似文献
10.
Lewis RN Liu F Krivanek R Rybar P Hianik T Flach CR Mendelsohn R Chen Y Mant CT Hodges RS McElhaney RN 《Biochemistry》2007,46(4):1042-1054
The effects of the hydrophobicity and the distribution of hydrophobic residues on the surfaces of some designed alpha-helical transmembrane peptides (acetyl-K2-L(m)-A(n)-K2-amide, where m + n = 24) on their solution behavior and interactions with phospholipids were examined. We find that although these peptides exhibit strong alpha-helix forming propensities in water, membrane-mimetic media, and lipid model membranes, the stability of the helices decreases as the Leu content decreases. Also, their binding to reversed phase high-performance liquid chromatography columns is largely determined by their hydrophobicity and generally decreases with decreases in the Leu/Ala ratio. However, the retention of these peptides by such columns is also affected by the distribution of hydrophobic residues on their helical surfaces, being further enhanced when peptide helical hydrophobic moments are increased by clustering hydrophobic residues on one side of the helix. This clustering of hydrophobic residues also increases peptide propensity for self-aggregation in aqueous media and enhances partitioning of the peptide into lipid bilayer membranes. We also find that the peptides LA3LA2 [acetyl-K2-(LAAALAA)3LAA-K2-amide] and particularly LA6 [acetyl-K2-(LAAAAAA)3LAA-K2-amide] associate less strongly with and perturb the thermotropic phase behavior of phosphatidylcholine bilayers much less than peptides with higher L/A ratios. These results are consistent with free energies calculated for the partitioning of these peptides between water and phospholipid bilayers, which suggest that LA3LA2 has an equal tendency to partition into water and into the hydrophobic core of phospholipid model membranes, whereas LA6 should strongly prefer the aqueous phase. We conclude that for alpha-helical peptides of this type, Leu/Ala ratios of greater than 7/17 are required for stable transmembrane associations with phospholipid bilayers. 相似文献