Purification and bioactivity of exendin-4, a peptide analogue of GLP-1, expressed in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Exendin-4, a peptide analogue of glucagon-like peptide-1 (GLP-1), has been developed for treatment of type 2 diabetes. Herein, the secretive exendin-4 fusion protein, expressed by methanol induction in Pichia pastoris system, was purified to homogeneity by chromatography followed by enterokinase cleavage of the fusion protein and subsequent purification of the recombinant exendin-4. Purity of the recombinant exendin-4 was 95.6%. Bioactivity assay revealed that it had glucose-lowering and insulin-releasing action in vivo.     

Evaluating cell-surface expression and measuring activation of mammalian odorant receptors in heterologous cells

A fundamental question in olfaction is which odorant receptors (ORs) are activated by a given odorant. A major roadblock to investigating odorant-OR relationships in mammals has been the inability to express ORs in heterologous cells suitable for screening active ligands for ORs. The discovery of the receptor-transporting protein family has facilitated the effective cell-surface expression of ORs in heterologous cells. The establishment of a robust heterologous expression system for mammalian ORs facilitates the high-throughput 'deorphanization' of these receptors by matching them to their cognate ligands. This protocol details the method used for evaluating the cell-surface expression and measuring the functional activation of ORs of transiently expressed mammalian ORs in HEK293T cells. The stages of OR cell-surface expression include cell culture preparation, transfer of cells, transfection, immunocytochemistry or flow cytometry, odorant stimulation and luciferase assay. This protocol can be completed in a period of 3 d from the transfer of cells to cell-surface expression detection and/or measurement of functional activation.     

Sucrose exposure in early life alters adult motivation and weight gain

The cause of the current increase in obesity in westernized nations is poorly understood but is frequently attributed to a 'thrifty genotype,' an evolutionary predisposition to store calories in times of plenty to protect against future scarcity. In modern, industrialized environments that provide a ready, uninterrupted supply of energy-rich foods at low cost, this genetic predisposition is hypothesized to lead to obesity. Children are also exposed to this 'obesogenic' environment; however, whether such early dietary experience has developmental effects and contributes to adult vulnerability to obesity is unknown. Using mice, we tested the hypothesis that dietary experience during childhood and adolescence affects adult obesity risk. We gave mice unlimited or no access to sucrose for a short period post-weaning and measured sucrose-seeking, food consumption, and weight gain in adulthood. Unlimited access to sucrose early in life reduced sucrose-seeking when work was required to obtain it. When high-sugar/high-fat dietary options were made freely-available, however, the sucrose-exposed mice gained more weight than mice without early sucrose exposure. These results suggest that early, unlimited exposure to sucrose reduces motivation to acquire sucrose but promotes weight gain in adulthood when the cost of acquiring palatable, energy dense foods is low. This study demonstrates that early post-weaning experience can modify the expression of a 'thrifty genotype' and alter an adult animal's response to its environment, a finding consistent with evidence of pre- and peri-natal programming of adult obesity risk by maternal nutritional status. Our findings suggest the window for developmental effects of diet may extend into childhood, an observation with potentially important implications for both research and public policy in addressing the rising incidence of obesity.     

GABA/progesterone-induced polyphosphoinositide (PPI) breakdown and its role in the acrosome reaction of guinea pig spermatozoain vitro

To investigate whether GABA/progesterone (P₄) stimulates PPI breakdown and its role in the acrosome reaction (AR), spermatozoa of guinea pig were preincubated in MCM-LCa²⁺ for 5.5 h and then labeled with [³²P]pi for 1 h. Samples were washed through a three-step gradient Percoll, adjusted to 5×10⁷ cells/mL and exposed to 2 mmol/L Ca²⁺, 5 ?mol/L GABA, 10 ?mol/L P₄ and other agents. Lipids were separated by t.l.c. and radioactivity in spots determined by scintillation counting. The AR was assessed by phase-contrast microscopy. The results showed that (i) when spermatozoa were treated with GABA, ³²P-label diminished rapidly in phosphatidylinositol 4, 5-bisphosphate (PIP₂), phosphatidylinositol 4-phosphate (PIP), and increased in phosphatidic acid (PA). The loss of label from PPI was almost completed by 10 min. The time-course of the AR was much slower than PPI when spermatozoa reached a maximal response by 15 min; (ii) the pattern of PPI hydrolysis and stimulation of AR was similar for the three agonists tested;their potency followed the order A23187＞progesterone≥GABA; (iii) GABA-induced PIP₂ hydrolysis and rise in PA and the AR were prevented by inclusion of 10 mmol/L neomycin; (iv) the loss of PIP2 labeling and the increase in PA labeling abolished when spermatozoa were exposed to EGTA or Ca²⁺ channel blocker. These results indicate that GABA or P₄^-induced PPI breakdown is an important and essential event in the series of changes to membrane fusion during the AR of guinea pig spermatozoa and this effect is mediated via calcium by activation of phosphatidylinositol-specific phospholipase C.     

溶菌酶对瘤胃体外发酵、甲烷生成及微生物菌群结构的影响

【目的】通过体外静态模拟瘤胃发酵法研究溶菌酶对瘤胃发酵、甲烷生成及微生物菌群结构的影响。【方法】采用单因素多水平试验设计,溶菌酶添加水平分别为0(L-0,对照组)、0.1 mg/100 m L(L-0.1)、1 mg/100 m L(L-1)、10 mg/100 m L(L-10)和100 mg/100 m L(L-100),定时测定产气量和甲烷产量,培养24 h后,发酵液用于发酵参数和微生物菌群数量的q PCR测定,其中L-0、L-1和L-100三个组发酵液同时进行16S r RNA基因Illumina高通量测序。【结果】与对照组相比,低剂量溶菌酶添加(L-0.1组)不影响甲烷产量、氨氮浓度、干物质消失率、有机物消失率和总挥发性脂肪酸等瘤胃发酵参数(P0.05);随着剂量提高,L-1处理组甲烷产量、氨氮浓度显著降低(P0.05),丙酸浓度显著增加(P0.05),并且干物质消失率、有机物消失率和总挥发性脂肪酸不受影响(P0.05);而较高剂量组(L-10和L-100组)虽然甲烷产量显著降低,丙酸浓度显著增加(P0.05),但干物质消失率和有机物消失率也显著降低(P0.05)。q PCR结果显示高剂量组(L-100组)总菌、原虫、甲烷菌数量与对照组相比显著降低(P0.05),而L-0.1、L-1和L-10组总菌、真菌和原虫数量与对照组相比均无显著变化(P0.05)。高通量测序主成分分析(PCA)显示对照组与溶菌酶添加组间瘤胃细菌组成的明显区分,说明添加溶菌酶显著改变了瘤胃细菌菌群结构。溶菌酶通过增加月形单胞菌和琥珀酸弧菌等丙酸生成菌的相对丰度,使更多的氢被用于生成丙酸,导致甲烷产量降低;溶菌酶可抑制普雷沃氏菌和拟杆菌属等蛋白降解菌的生长,进而减少蛋白质过度降解,降低氨氮浓度。【结论】添加适宜浓度(1 mg/100 m L)的溶菌酶可通过调控瘤胃微生态改变瘤胃发酵模式,降低瘤胃甲烷和氨的生成,短期内并不影响饲料消化。     

微生物帮助烟草抗旱的机理及其应用

干旱是中国烟草种植业面临的较为严重的非生物胁迫.很多与植物共生或联合的根际微生物能帮助植物避旱和耐旱.微生物能通过菌丝吸水并转运到植物,通过产生植物激素或改变植物内源激素的平衡来促进根发育和伸长,或诱导叶片关闭气孔,促进根吸水和减少叶片散失水分来避旱.微生物能通过调整不同激素介导的信号通路,诱导植物产生系统抗逆性,促进植物细胞产生渗透保护剂、抗氧化物和活性氧清除剂而耐旱.微生物还能帮助植物吸收营养,以支持植物在干旱胁迫下的代谢和生长.本文关注丛枝菌根真菌、模式内生真菌印度梨形孢和根际促植物生长细菌帮助烟草和番茄等植物抗旱的机理,探讨如何在烟草育苗和栽培中应用有益微生物来帮助烟草抗旱.     

Ectopic expression of <Emphasis Type="Italic">OsMADS1</Emphasis> caused dwarfism and spikelet alteration in rice

In rice, an E-class gene, OsMADS1, acts to specify the identities of the lemma and palea. In this study, the OsMADS1 gene with a CaMV35S promoter was transformed into a japonica cultivar, Zhonghua 11. All transgenic plants successfully showed similar phenotypes, including dwarfism, distorted panicles, decreased numbers of branches and spikelets, and elongated sterile lemma. Histological analysis showed that the elongated sterile lemma developed with silicified epidermal and sclerenchymal cells, which were lacking in the wild-type sterile lemma, suggesting that the elongated sterile lemma had assumed the identity of the lemma or palea. Some marker genes were subjected to a detailed analysis of the distribution of their expression among the lemma, palea and sterile lemma. DROOPING LEAF (DL) and OsMADS6 genes were only expressed in the normal lemma or palea, respectively. In the elongated sterile lemma, a high level of DL gene expression was detected, while no expression of OsMADS6 was found, implying that the sterile lemma transformed into the lemma but not the palea. These results provide clues to elucidate the mechanism of evolution from lemma to sterile lemma in rice. qPCR analysis also suggested that the ectopic expression of OsMADS1 induced abnormal brassinosteroid and gibberellin acid activation, and then resulted in developmental defects in the stem and panicle.     

Mutation breeding of high avermectin B<Subscript>1a</Subscript>-producing strain by the combination of high energy carbon heavy ion irradiation and sodium nitrite mutagenesis based on high throughput screening

Microbial mutation breeding has been widely used because it is one of the most efficient and practical breeding strategies in the fermentation industry. However, different mutagenesis methods cause various degrees of DNA damage to individual microorganisms, which lead to diverse characteristics of the mutants. In this study, the effects of four different mutagenesis methods on the mutation breeding of Streptomyces avermitilis for improving avermectin B1a production were investigated with an optimized liquid microtiter plate (MTP) culture system. First, an effective and feasible MTP system for mutant strain screening was evaluated through the optimization of the oxygen transfer rate and rapid titer determination. Then, high energy carbon heavy ion irradiation, diethyl sulfate, ultraviolet- (UV) irradiation combined with lithium chloride, and sodium nitrite were used as the mutagens for mutation breeding, respectively. Results showed that carbon heavy ion irradiation had the advantages of possessing the highest positive mutation rate and mean-production of positive mutant strains in the first generation. Sodium nitrite treatment resulted in mutant strains with better inherited stability than the other three methods. Through the combined treatment of carbon heavy ion irradiation and sodium nitrite treatment, an inheritstable mutant S. avermitilis S-233 with high avermectin B_1a production was successfully obtained. The fermentation verification in a 500-liter (L) bioreactor demonstrated that the avermectin B_1a produced by mutant S. avermitilis S-233 reached 6818 μg/mL, which was 23.8% higher than that of parent strains.     

Segregation distortion and genome-wide digenic interactions affect transmission of introgressed chromatin from wild cotton species

Key message

This study reports transmission genetics of chromosomal segments into Gossypium hirsutum from its most distant euploid relative, Gossypium mustelinum . Mutilocus interactions and structural rearrangements affect introgression and segregation of donor chromatin.

Abstract

Wild allotetraploid relatives of cotton are a rich source of genetic diversity that can be used in genetic improvement, but linkage drag and non-Mendelian transmission genetics are prevalent in interspecific crosses. These problems necessitate knowledge of transmission patterns of chromatin from wild donor species in cultivated recipient species. From an interspecific cross, Gossypium hirsutum × Gossypium mustelinum, we studied G. mustelinum (the most distant tetraploid relative of Upland cotton) allele retention in 35 BC₃F₁ plants and segregation patterns in BC₃F₂ populations totaling 3202 individuals, using 216 DNA marker loci. The average retention of donor alleles across BC₃F₁ plants was higher than expected and the average frequency of G. mustelinum alleles in BC₃F₂ segregating families was less than expected. Despite surprisingly high retention of G. mustelinum alleles in BC₃F₁, 46 genomic regions showed no introgression. Regions on chromosomes 3 and 15 lacking introgression were closely associated with possible small inversions previously reported. Nonlinear two-locus interactions are abundant among loci with single-locus segregation distortion, and among loci originating from one of the two subgenomes. Comparison of the present results with those of prior studies indicates different permeability of Upland cotton for donor chromatin from different allotetraploid relatives. Different contributions of subgenomes to two-locus interactions suggest different fates of subgenomes in the evolution of allotetraploid cottons. Transmission genetics of G. hirsutum × G. mustelinum crosses reveals allelic interactions, constraints on fixation and selection of donor alleles, and challenges with retention of introgressed chromatin for crop improvement.