Minimizing the overlap problem in protein NMR: a computational framework for precision amino acid labeling

MOTIVATION: Recent advances in cell-free protein expression systems allow specific labeling of proteins with amino acids containing stable isotopes ((15)N, (13) C and (2)H), an important feature for protein structure determination by nuclear magnetic resonance (NMR) spectroscopy. Given this labeling ability, we present a mathematical optimization framework for designing a set of protein isotopomers, or labeling schedules, to reduce the congestion in the NMR spectra. The labeling schedules, which are derived by the optimization of a cost function, are tailored to a specific protein and NMR experiment. RESULTS: For 2D (15)N-(1)H HSQC experiments, we can produce an exact solution using a dynamic programming algorithm in under 2 h on a standard desktop machine. Applying the method to a standard benchmark protein, calmodulin, we are able to reduce the number of overlaps in the 500 MHz HSQC spectrum from 10 to 1 using four samples with a true cost function, and 10 to 4 if the cost function is derived from statistical estimates. On a set of 448 curated proteins from the BMRB database, we are able to reduce the relative percent congestion by 84.9% in their HSQC spectra using only four samples. Our method can be applied in a high-throughput manner on a proteomic scale using the server we developed. On a 100-node cluster, optimal schedules can be computed for every protein coded for in the human genome in less than a month. AVAILABILITY: A server for creating labeling schedules for (15)N-(1)H HSQC experiments as well as results for each of the individual 448 proteins used in the test set is available at http://nmr.proteomics.ics.uci.edu.     

Simultaneous determination of thiamphenicol, florfenicol and florfenicol amine in eggs by reversed-phase high-performance liquid chromatography with fluorescence detection

A specific, sensitive and widely applicable reversed-phase high-performance liquid chromatography with fluorescence detection (RP-HPLC-FLD) method was developed for the simultaneous determination of thiamphenicol (TAP), florfenicol (FF) and florfenicol amine (FFA) in eggs. Samples were extracted with ethyl acetate-acetonitrile-ammonium hydroxide (49:49:2, v/v), defatted with hexane, followed by RP-HPLC-FLD determination. Liquid chromatography was performed on a 5 μm LiChrospher C(18) column using a mobile phase composed of acetonitrile (A), 0.01 M sodium dihydrogen phosphate containing 0.005 M sodium dodecyl sulfate and 0.1% triethylamine, adjusted to pH 4.8 by 85% phosphoric acid (B) (A:B, 35:65 v/v), at a flow rate of 1.0 mL/min. The fluorescence detector of HPLC was set at 224 nm for excitation wavelength and 290 nm for emission wavelength. Limits of detection (LODs) were 1.5 μg/kg for TAP and FF, 0.5 μg/kg for FFA in eggs; limits of quantitation (LOQs) were 5 μg/kg for TAP and FF, 2 μg/kg for FFA in eggs. Linear calibration curves were obtained over concentration ranges of 0.025-5.0 μg/mL for TAP with determination coefficients of 0.9997, 0.01-10.0 μg/mL for FF with determination coefficients of 0.9997 and 0.0025-2.50 μg/mL for FFA with determination coefficients of 0.9998, respectively. The recovery values ranged from 86.4% to 93.8% for TAP, 87.4% to 92.3% for FF and from 89.0% to 95.2% for FFA. The corresponding intra-day and inter-day variation (relative standard deviation, R.S.D.) found to be less than 6.7% and 10.8%, respectively.     

Changes in germinability,lipid peroxidation,and antioxidant enzyme activities in pear stock (<Emphasis Type="Italic">Pyrus betulaefolia</Emphasis> Bge.) seeds during room- and low-temperature storage

The aim of this study was to determine if loss of germinability in Pyrus betulaefolia seeds stored at 4°C and at room temperature is associated with a loss of membrane lipid peroxidation or changes in antioxidant enzyme activities. The results indicated that germination percentage clearly decreased when seeds were stored at room temperature rather than at 4°C from 6 to 12 months. Room-temperature storage of the pear stock seed for 12 months decreased germination to 15.52%, but germination percentage was not changed when seed was stored at 4°C for 12 months. MDA, a marker for membrane lipid peroxidation, increased significantly under room-temperature storage conditions. Antioxidant enzyme (SOD, POD, and CAT) activities were a good indicator of germination percentage in pear stock seeds. Antioxidant enzyme activities of pear stock seeds at 4°C were higher than antioxidant enzyme activities in seeds stored at room temperature from 6 to 12 months. Antioxidant enzyme activities of the pear stock seed decreased markedly under conditions of room-temperature storage from 6 to 12 months. The results of this study showed that long-term room-temperature storage was detrimental for maintaining the vigor of P. betulaefolia seeds. The mechanisms responsible for this outcome are a higher level of membrane lipid peroxidation and a lower level of activity of antioxidant enzymes.     

Genomic polymorphism of the pandemic A (H1N1) influenza viruses correlates with viral replication, virulence, and pathogenicity in vitro and in vivo

The novel pandemic A (H1N1) virus was first identified in Mexico in April 2009 and quickly spread worldwide. Like all influenzas, the H1N1 strain-specific properties of replication, virulence, and pathogenicity are a result of the particular genomic sequence and concerted expression of multiple genes. Thus, specific mutations may support increased virulence and may be useful as biomarkers of potential threat to human health. We performed comparative genomic analysis of ten strains of the 2009 pandemic A (H1N1) influenza viruses to determine whether genotypes associated with clinical phenotypes, which ranged from mild to severe illness and up to lethal. Virus replication capacity was tested for each strain in vitro using cultured epithelial cells, while virulence and pathogenicity were investigated in vivo using the BALB/c mouse model. The results indicated that A/Sichuan/1/2009 strain had significantly higher replication ability and virulence than the other strains, and five unique non-synonymous mutations were identified in important gene-encoding sequences. These mutations led to amino acid substitutions in HA (L32I), PA (A343T), PB1 (K353R and T566A), and PB2 (T471M), and may be critical molecular determinants for replication, virulence, and pathogenicity. Our results suggested that the replication capacity in vitro and virulence in vivo of the 2009 pandemic A (H1N1) viruses were not associated with the clinical phenotypes. This study offers new insights into the transmission and evolution of the 2009 pandemic A (H1N1) virus.     

Replacement of nonmuscle myosin II-B with II-A rescues brain but not cardiac defects in mice

The purpose of these studies was to learn whether one isoform of nonmuscle myosin II, specifically nonmuscle myosin II-A, could functionally replace a second one, nonmuscle myosin II-B, in mice. To accomplish this, we used homologous recombination to ablate nonmuscle myosin heavy chain (NMHC) II-B by inserting cDNA encoding green fluorescent protein (GFP)-NMHC II-A into the first coding exon of the Myh10 gene, thereby placing GFP-NMHC II-A under control of the endogenous II-B promoter. Similar to B(-)/B(-) mice, most B(a*)/B(a*) mice died late in embryonic development with structural cardiac defects and impaired cytokinesis of the cardiac myocytes. However, unlike B(-)/B(-) mice, 15 B(a*)/B(a*) mice of 172 F2 generation mice survived embryonic lethality but developed a dilated cardiomyopathy as adults. Surprisingly none of the B(a*)/B(a*) mice showed evidence for hydrocephalus that is always found in B(-)/B(-) mice. Rescue of this defect was due to proper localization and function of GFP-NMHC II-A in place of NMHC II-B in a cell-cell adhesion complex in the cells lining the spinal canal. Restoration of the integrity and adhesion of these cells prevents protrusion of the underlying cells into the spinal canal where they block circulation of the cerebral spinal fluid. However, abnormal migration of facial and pontine neurons found in NMHC II-B mutant and ablated mice persisted in B(a*)/B(a*) mice. Thus, although NMHC II-A can substitute for NMHC II-B to maintain integrity of the spinal canal, NMHC II-B plays an isoform-specific role during cytokinesis in cardiac myocytes and in migration of the facial and pontine neurons.     

Association of MT-ND5 gene variation with mitochondrial respiratory control ratio and NADH dehydrogenase activity in Tibet chicken embryos

NADH dehydrogenase (complex I) couples the oxidation of NADH for the reduction of ubiquinone with the generation of a proton gradient that can be used for the synthesis of ATP. We have found a missense mutation in the MT-ND5 subunit of NADH dehydrogenase in the Tibet chicken breed. In the present study, the mitochondrial respiratory control ratio (RCR) and NADH dehydrogenase activity in Tibet chicken embryonic brain with different genotypes were measured. Significant differences between animals carrying mitochondria with the EF493865.1:m.1627A vs. EF493865.1:m.1627C alleles were observed for RCR and enzyme activity.     

氟化物对桑蚕幼虫中肠ATP酶活性影响的电镜细胞化学定位

氟化物对桑蚕幼虫中肠ＡＴＰ酶活性影响的电镜细胞化学定位陈玉银,包焕盛,吴玉澄（浙江农业大学杭州３１００Ｎ）桑蚕Ｂｏｍｂｙｘｍｏｒｉ（Ｌ．）氟中毒导致蚕茧严重减产是目前蚕业界最为关切的问题,有关桑蚕幼虫氟中毒的机理研究也从各方面展开［１１］。已知桑蚕氟...     

人结肠癌(CCA)单克隆抗体及其识别抗原的分析(简报)

正常细胞转化成癌细胞后,其表型发生了一系列不同于正常细胞的变化,成为肿瘤细胞的标志。Gold和Freeman(1965)用人结肠癌组织的抽提物免疫兔,发现有些用人正常结肠组织吸收后的抗血清能够与肿瘤组织和胚胎肠道抽提物起反应,但不与正常组织抽提物起反应,由于这种抗原最初被发现在胚胎组织,故名为癌胚抗原(embryonic carcinoma antigen,简称CEA)。用敏感的放射免疫或免疫酶标方     

Silicon-mediated alleviation of Mn toxicity in Cucumis sativus in relation to activities of superoxide dismutase and ascorbate peroxidase

The effects of exogenous silicon (Si) on plant growth, activities of superoxide dismutase (SOD), guaiacol peroxidase (GPX), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), glutathione reductase (GR) and catalase, and concentrations of ascorbate and glutathione were investigated in cucumber (Cucumis sativus L.) plants treated with excess manganese (Mn) (600 microM). Compared with the treatment of normal Mn (10 microM), excess Mn significantly increased H2O2 concentration and lipid peroxidation indicated by accumulation of thiobarbituric acid reactive substances. The leaves showed apparent symptoms of Mn toxicity and the plant growth was significantly inhibited by excess Mn. The addition of Si significantly decreased lipid peroxidation caused by excess Mn, inhibited the appearance of Mn toxicity symptoms, and improved plant growth. This alleviation of Mn toxicity by Si was related to a significant increase in the activities of SOD, APX, DHAR and GR and the concentrations of ascorbate and glutathione.