Genome-wide identification,classification, and expression profiling of serine esterases and other esterase-related proteins in the tobacco hornworm,Manduca sexta

Serine esterases (SEs) are hydrolases that catalyze the conversion of carboxylic esters into acids and alcohols. Lipases and carboxylesterases constitute two major groups of SEs. Although over a hundred of insect genomes are known, systematic identification and classification of SEs are rarely performed, likely due to large size and complex composition of the gene family in each species. Considering their key roles in lipid metabolism and other physiological processes, we have categorized 144 M. sexta SEs and SE homologs (SEHs), 114 of which contain a motif of GXSXG. Multiple sequence alignment and phylogenetic tree analysis have revealed 39 neutral lipases (NLs), 3 neutral lipase homologs (NLHs), 11 acidic lipases (ALs), 3 acidic lipase homologs (ALHs), a lipase-3, a triglyceride lipase, a monoglyceride lipase, a hormone-sensitive lipase, and a GDSL lipase. Eighty-three carboxylesterase genes encode 29 α-esterases (AEs), 12 AEHs (e.g., SEH4-1–3), 20 feruloyl esterases (FEs), 2 FEHs, 2 β-esterases (BEs), 2 integument esterases (IEs), 1 IEH, 4 juvenile hormone esterases, 2 acetylcholinesterases, gliotactin, 6 neuroligins, neurotactin, and an uncharacteristic esterase homolog. In addition to these GXSXG proteins, we have identified 26 phospholipases and 13 thioesterases. Expression profiling of these genes in specific tissues and stages has provided insights into their functions including digestion, detoxification, hormone processing, neurotransmission, reproduction, and developmental regulation. In summary, we have established a framework of information on SEs and related proteins in M. sexta to stimulate their research in the model species and comparative investigations in agricultural pests or disease vectors.     

A short-chain carbonyl reductase mutant is an efficient catalyst in the production of (R)-1,3-butanediol

R-1,3-butanediol (R-1,3-BDO) is an important chiral intermediate of penem and carbapenem synthesis. Among the different synthesis methods to obtain pure enantiomer R-1,3-BDO, oxidation–reduction cascades catalysed by enzymes are promising strategies for its production. Dehydrogenases have been used for the reduction step, but the enantio-selectivity is not high enough for further organic synthesis efforts. Here, a short-chain carbonyl reductase (LnRCR) was evaluated for the reduction step and developed via protein engineering. After docking result analysis with the substrate 4-hydroxy-2-butanone (4H2B), residues were selected for virtual mutagenesis, their substrate-binding energies were compared, and four sites were selected for saturation mutagenesis. High-throughput screening helped identify a Ser154Lys mutant which increased the catalytic efficiency by 115% compared to the parent enzyme. Computer-aided simulations indicated that after single residue replacement, movements in two flexible areas (VTDPAF and SVGFANK) facilitated the volumetric compression of the 4H2B-binding pocket. The number of hydrogen bonds between the stabilized 4H2B-binding pocket of the mutant enzyme and substrate was higher (from four to six) than the wild-type enzyme, while the substrate-binding energy was decreased (from −17.0 kJ/mol to −29.1 kJ/mol). Consequently, the catalytic efficiency increased by approximately 115% and enantio-selectivity increased from 95% to 99%. Our findings indicate that compact and stable substrate-binding pockets are critical for enzyme catalysis. Lastly, the utilization of a microbe expressing the Ser154Lys mutant enzyme was proven to be a robust process to conduct the oxidation–reduction cascade at larger scales.     

红原鸡与家鸡的亲缘关系研究

对中国红原鸡滇地亚种和海南亚种与我国茶花鸡,泰和鸡和寿光鸡等地方鸡种以及芦花鸡,洛岛红等外国鸡种进行了血型（３个位点,１３个等位基因）,蛋白质（酶）多态（５个位点,１１个等位基因）和ＤＮＡ指纹分析,结果表明,红原鸡与茶花鸡（原始型品种）的亲缘关系较近;与泰和鸡,寿光鸡,芦花鸡,洛岛红（进化型品种）的亲缘关系较远,呈红原鸡－茶花鸡－泰和鸡,寿光鸡或芦花鸡,洛岛红这样一个进化阶梯,以上结果与国外资料（     

关中小麦品种产量构成因素的相互关系和产量育种目标

对关中地区不同年代及当前大面积推广的小麦品种（系）的产量及其构成因素进行了遗传分析和通径分析。结果表明：陕西关中地区近期小麦品种的产量育种目标的趋势是在提高现有穗粒数的基础上,保持现有的穗数（７００万／ｈｍ＾２）及千粒重（３８ｇ左右）,以求在提高品种产量潜力的同时,保持或增加品种的稳定性能;或是大幅度提高穗粒数（３５粒左右）,适当提高千粒重（４０ｇ左右）,适当降低穗数（６００万／ｈｍ＾２左右）,以     

Conservation of an intact human immunodeficiency virus type 1 vif gene in vitro and in vivo.

Replication of vif-negative human immunodeficiency virus type 1 (HIV-1) is attenuated in certain cell lines and highly impaired in peripheral blood lymphocytes in vitro. To determine whether intact vif is positively selected during natural HIV-1 infection and to determine vif sequence variability, we employed PCR amplification, cloning, and sequencing to investigate the vif region of replicating virus in short-term-passage HIV-1 primary isolates from five asymptomatic individuals and from five persons with AIDS. A total of 46 vif clones were obtained and analyzed. Recombinant proviruses were constructed from selected vif clones from one patient and found to be fully infectious. We found that 38 of the 46 clones sequenced carried open vif reading frames and that there was a low degree of heterogeneity of vif genes within isolates from the same individual and among isolates from different donors. The cysteines previously found to be essential for vif protein function were conserved in all clones. A phylogenetic tree constructed from all available vif nucleotide sequences resulted in a virus grouping similar to those of gag and env. Direct sequencing of vif amplified by PCR from uncultured lymphocytes of 15 individuals at various stages of progression toward AIDS demonstrated vif open reading frames in 13 of 15 samples tested. There was no obvious correlation between disease status and the presence of an intact vif within this sample group at the time of sample procurement. The conservation of the vif open reading frame in vitro and in vivo and its limited variability following virus transmission in vitro are consistent with a role for vif in natural HIV-1 infection.     

Aberrant Gag protein composition of a human immunodeficiency virus type 1 vif mutant produced in primary lymphocytes.

Productive, spreading infection of peripheral blood lymphocytes (PBL) with human immunodeficiency virus type 1 (HIV-1) requires the viral protein Vif. To study the requirement for vif in this system, we infected PBL with a phenotypically complemented HIV-1 clone mutated in vif. Progeny virus was produced which was noninfectious in PBL but replicated in SupT1 cells. Analysis of metabolically labeled proteins of sedimentable extracellular particles made in PBL by radioimmunoprecipitation with either serum from a patient with AIDS or a monoclonal antibody reactive with HIV-1 Gag proteins revealed that vif-negative but not wild-type particles carry higher levels of p55, p41, and p38 Gag-specific proteins compared with those of p24. Similar results were obtained with sucrose-purified virions. Our data indicate that vif plays a role in Gag protein processing or in incorporation of processed Gag products into mature virions. The presence of unprocessed precursor Gag polyprotein (Pr55gag) and other Gag processing intermediates in PBL-derived vif-negative extracellular particles may contribute to the reduced infectivity of this virus.     

The Impact of Chlorophyll-Retention Mutations,d1d2 and cyt-G1, during Embryogeny in Soybean

The ultrastructural, physiological, and molecular changes in developing and mature seeds were monitored in a control line (Glycine max [L.] Merr., cv Clark) that exhibited seed degreening and two mutant lines (d1d2 and cyt-G1) that retained chlorophyll upon seed maturation. Ultrastructural studies showed that the control line had no internal membranes, whereas stacked thylakoid membranes were detected in the green seed from the mutant lines. Pigment analyses indicated that total chlorophyll was lowest in the mature seeds of the control line. Mature d1d2 and cyt-G1 seed had elevated Chl a and Chl b levels, respectively. In both control and mutant lines, Lhcb1, Lhcb2, and RbcS mRNAs were abundant in embryos prior to cotyledon filling, declined after the onset of storage protein accumulation, and were barely detectable or undetectable in all later stages of seed development. Therefore, the chlorophyll-retention phenotype must be a result of the alteration of a process that occurs after translation of photosynthesis-related mRNAs to stabilize apoprotein and pigment levels. Furthermore, different elements controlling either the synthesis or turnover of Chl a and Chl b must be impaired in the d1d2 and cyt-G1 lines. No reproducible differences in total leaf, embryonic, and chloroplast protein profiles and plastid DNAs could be correlated with the mutations that induced chlorophyll retention.     

Ras transformation results in an elevated level of cyclin D1 and acceleration of G1 progression in NIH 3T3 cells.

Ectopic overexpression of v-H-Ras protein in NIH 3T3 cells resulted in cellular transformation and an acceleration of G1 progression of these cells. A shortened G1 phase was found to be associated with an increased level of cyclin D1 but not cyclin E protein. Using an antisense blocking method, reduced synthesis of cyclin D1 in v-H-Ras transformants resulted in a slower G1 progression rate of these cells. Although constitutive overexpression of cyclin D1 in NIH 3T3 cells accelerated G1 progression, cells remained untransformed. Furthermore, inhibition of cyclin D1 synthesis greatly impaired the soft-agar cloning efficiency of v-H-Ras transformants. These results suggest that increased expression of cyclin D1 is necessary but not sufficient for the transforming activity of v-H-Ras. Similar effect on cell cycle progression was also observed in Raf-transformed cells. In addition to cyclin D1, cyclin E protein was found to be elevated in Src transformants. This may account for the further shortening of the G1 phase of these cells. Activation of an additional Ras-independent pathway was suggested to be responsible for the further acceleration of the G1 phase in Src transformants.     

Production of bacterial cellulose by Acetobacter xylinumwith an air-lift reactor

Bacterial cellulose was produced by Acetobacter xylinum subsp. surcrofermentans BPR2001 in a 50 liter air-lift reactor using fructose as the main carbon source. When air was supplied, the production of the cellulose was only 2.3 g/l in 80 h but when O -fortified air was supplied, the cellulose concentration increased to 5.63 g/l in 28 h and the productivity of the cellulose in an air-lift reactor with O -fortified air supply was comparable to that in a mechanically agitated jar fermenter.