Preparation of amino-acid-type selective isotope labeling of protein expressed in Pichia pastoris

We report the culture conditions for successful amino-acid-type selective (AATS) isotope labeling of protein expressed in Pichia pastoris (P. pastoris). Rhodostomin (Rho), a six disulfide-bonded protein expressed in P. pastoris with the correct fold, was used to optimize the culture conditions. The concentrations of [alpha-15N] selective amino acid, nonlabeled amino acids, and ammonium chloride, as well as induction time, were optimized to avoid scrambling and to increase the incorporation rate and protein yield. The optimized protocol was successfully applied to produce AATS isotope-labeled Rho. The labeling of [alpha-15N]Cys has a 50% incorporation rate, and all 12 cysteine resonances were observed in HSQC spectrum. The labeling of [alpha-15N]Leu, -Lys, and -Met amino acids has an incorporation rate greater than 65%, and the expected number of resonances in the HSQC spectra were observed. In contrast, the labeling of [alpha-15N]Asp and -Gly amino acids has a low incorporation rate and the scrambling problem. In addition, the culture condition was successfully applied to label dendroaspin (Den), a four disulfide-bonded protein expressed in P. pastoris. Therefore, the described condition should be generally applicable to other proteins produced in the P. pastoris expression system. This is the first report to present a protocol for AATS isotope labeling of protein expressed in P. pastoris for NMR study.     

Effect of population size on the estimation of QTL: a test using resistance to barley stripe rust

The limited population sizes used in many quantitative trait locus (QTL) detection experiments can lead to underestimation of QTL number, overestimation of QTL effects, and failure to quantify QTL interactions. We used the barley/barley stripe rust pathosystem to evaluate the effect of population size on the estimation of QTL parameters. We generated a large (n=409) population of doubled haploid lines derived from the cross of two inbred lines, BCD47 and Baronesse. This population was evaluated for barley stripe rust severity in the Toluca Valley, Mexico, and in Washington State, USA, under field conditions. BCD47 was the principal donor of resistance QTL alleles, but the susceptible parent also contributed some resistance alleles. The major QTL, located on the long arm of chromosome 4H, close to the Mlo gene, accounted for up to 34% of the phenotypic variance. Subpopulations of different sizes were generated using three methods—resampling, selective genotyping, and selective phenotyping—to evaluate the effect of population size on the estimation of QTL parameters. In all cases, the number of QTL detected increased with population size. QTL with large effects were detected even in small populations, but QTL with small effects were detected only by increasing population size. Selective genotyping and/or selective phenotyping approaches could be effective strategies for reducing the costs associated with conducting QTL analysis in large populations. The method of choice will depend on the relative costs of genotyping versus phenotyping. Electronic Supplementary Material Supplementary material is available for this article at     

Structure and expression of mouse alpha 1-acid glycoprotein gene-3 (AGP-3).

The genome of Mus domesticus has multiple genes of the alpha 1-acid glycoprotein (AGP). Two cDNA clones were identified corresponding to AGP-1 and AGP-2. Moreover, two alleles of AGP-1 exist in inbred mice. The genomic DNA of the AGP-2 gene has been cloned and studied. Here we report the genomic organization of three M. domesticus AGP genes, the sequence analysis of the AGP-3 genomic DNA, and the expression of the AGP-3 gene. The major structural differences between AGP-2 and AGP-3 genes are located in introns 1 and 5. The low level of AGP-3 mRNA can be detected by the polymerase chain reaction (PCR). The molecular basis of the low level expression of AGP-3 and the possible classification of AGP-3 as a pseudogene are discussed.     

Tandem mass spectrometry identifies proteins phosphorylated by cyclic AMP-dependent protein kinase when sea urchin sperm undergo the acrosome reaction

The exocytotic acrosome reaction (AR), which is required for fertilization, occurs when sea urchin sperm contact the egg jelly (EJ) layer. Among other physiological changes, increases in adenylyl cyclase activity, cAMP and cAMP-dependent protein kinase (PKA) activity occur coincident with the AR. By using inhibitors of PKA, a permeable analog of cAMP and the phosphodiesterase inhibitor IBMX, we show that PKA activity is required for AR induction by EJ. A minimum of six sperm proteins are phosphorylated by PKA upon exposure to EJ, as detected by a PKA substrate-specific antibody. The phosphorylation of these proteins and the percentage of acrosome reacted sperm can be regulated by PKA modulators. The fucose sulfate polymer (FSP), a major component of EJ, is the molecule that triggers sperm PKA activation. Extracellular Ca(2+) is required for PKA activation. Six sperm proteins phosphorylated by PKA were identified by tandem mass spectrometry (MS/MS) utilizing the emerging sea urchin genome. Based on their identities and localizations in sperm head and flagellum, the putative functions of these proteins in sperm physiology and AR induction are discussed.     

PNAS-4 expression and its relationship to p53 in colorectal cancer

PNAS-4 is a novel pro-apoptotic protein activated during the early response to DNA damage; however, the molecular mechanisms and pathways regulating PNAS-4 expression in tumors are not well understood. We hypothesized that PNAS-4 is a p53 down-stream target gene and designed this study. We searched online for putative p53-binding sites in the entire PNAS-4 gene and did not find any corresponding information. In HCT116 colon cancer cells, after being transfected with small interfering RNA to silence p53, the expressions of PNAS-4 and other known p53 target gene (Apaf1, Bax, Fas and Dr5) were determined by real-time PCR. We found that PNAS-4 was up-regulated while Apaf1, Bax, Fas and Dr5 were down-regulated. We then examined the expression of PNAS-4 and p53 mutation in colorectal cancer patients. PNAS-4 expressed both in colorectal cancers and normal tissues, but compared with paired control, PNAS-4 was up-regulated in cancers (P = 0.018). PNAS-4 overexpression ratios were correlated to the p53 mutant status (P = 0.001). The mean PNAS-4 expression levels of p53 mutant homozygote group and heterozygote group were higher than that of p53 wild type group (P = 0.013). The expression ratios of PNAS-4 (every sample in relative to its paired normal mucosa) were different between negative lymph node metastasis (66% up-regulated, 34% down-regulated) and positive metastasis (42% up-regulated, 58% down-regulated). Taken together, these findings suggested that PNAS-4 was not a p53 target, but overexpression of PNAS-4 was correlated to p53 inactivity in colorectal cancer.     

HIV-1 glycoprotein 41 ectodomain induces activation of the CD74 protein-mediated extracellular signal-regulated kinase/mitogen-activated protein kinase pathway to enhance viral infection

Besides mediating the viral entry process, the human immunodeficiency virus (HIV-1) envelope protein gp41 can bind to many host cell components and regulate cell functions. Using a yeast two-hybrid system, we screened a human bone marrow cDNA library and identified a novel gp41-binding protein, CD74 (the MHC class II-associated invariant chain). Here, we report possible biological effects mediated by interaction between gp41 and CD74. We found that HIV-1 gp41 could bind directly to host CD74 in HIV-1-infected cells, and the peptide 6358 derived from gp41 loop region (aa 597-611) could effectively block the gp41-CD74 interaction. As a result of this binding, recombinant soluble gp41 and gp41 peptide 6358 activated the CD74-mediated ERK/MAPK pathway and significantly enhanced HIV-1 infection in vitro. Conversely, the enhancing effect could be suppressed by the recombinant CD74 extracellular domain. These results reveal a novel mechanism underlying gp41 mediation of HIV-1 infection and replication.     

分批发酵生产谷氨酰胺转氨酶的温度控制策略

微生物谷氨酰胺转氨酶 (Ｍｉｃｒｏｂｉａｌｔｒａｎｓｇｌｕｔａｍｉｎａｓｅ ,简称ＭＴＧ ,ＥＣ2 3 2 13)由于能催化许多食品中蛋白质的交联反应 ,改善各种蛋白质的功能性质 ,在食品工业具有广泛的应用潜力[1] ,因而引起了人们的极大兴趣。谷氨酰胺转氨酶的生产通常采用从豚鼠肝脏或组织中提取 ,由于豚鼠肝脏或组织来源稀少 ,谷氨酰胺转胺酶的分离纯化过程复杂 ,因而价格昂贵。 2 0世纪 80年代末 ,Ａｎｄｏ和Ｍｏｔｏｋｉ等人[2 ,3 ] 首先报道了利用微生物发酵法生产谷氨酰胺转胺酶的结果 ;近年来 ,Ｇｅｒｂｅｒ等人[4 ] 对其下游技术进…     

Effects of Fragmentation of Evergreen Broad-leaved Forests on Genetic Diversity of Ardisia Crenata var. Bicolor (Myrsinaceae)

Due to the long generation times and high densities, dominant tree species usually did not respond consistently with theoretical predictions to the recent fragmentation. Genetic structures of shrubs and herbs, especially those with low densities, may be more sensitive to forest fragmentation. We studied the genetic structure of a self-compatible subshrub, Ardisia crenata var. bicolor (Myrsinaceae) in a recently fragmented landscape. Ten RAPD primers used for analysis generated a total of 76 bands. We found that A. c. var. bicolor had relatively low species-level (P₉₅ = 63.2%; H = 0.106; Shannon diversity index (SI) = 0.246) and within-population diversity (P₉₅ = 5.3−46.1%; H = 0.026−0.175; SI = 0.032−0.253), and significant population differentiation (G_ST = 0.445). Significantly positive relationships were found between measures of diversity (P₉₅, H and SI) and the log of estimated population size. No significant relationship was observed between Nei's genetic distance and spatial distance of pairwise populations, indicating no isolation-by-distance. Given most species of forests are shrubs and herbs with short generation times, our observation indicated that distinct genetic consequences of recent fragmentation may be expected for quite a number of plant species.