Expression of a novel recombinant stem cell factor/macrophage colony-stimulating factor fusion protein in baculovirus-infected insect cells

A novel human stem cell factor (SCF)/macrophage colony-stimulating factor (M-CSF) fusion protein gene was constructed, in which the coding regions of human SCF cDNA (1-165aa) and the truncated M-CSF cDNA (1-149aa) were connected by a linker sequence encoding a short peptide GGGGSGGGGSGG. The SCF/M-CSF gene was cloned into baculovirus transfer vector pVL1392 under the control of polyhedrin promoter and expressed in the Sf9 cells (Spodoptera frugiperda). SDS-PAGE and Western blot analysis showed that the purified fusion protein was a homodimer with a molecular weight about 84kDa under non-reducing conditions or a monomer about 42kDa under reducing conditions. The specific activity of rhSCF/M-CSF was 17 times as high as that of monomeric rhSCF to stimulate the proliferation of TF-1 cell. The results of macrophages colony-forming (CFU-M) assay performed with human bone marrow mononuclear cells demonstrated that rhSCF/M-CSF was more potent in promoting CFU-M than the equimolar of SCF, M-CSF or that of two cytokines mixture.     

Cloning and heterologous expression of a bacteriocin sakacin P from <Emphasis Type="Italic">Lactobacillus sakei</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Sakacin P, a bacteriocin from Lactobacillus sakei, shows strong activity against food-borne pathogens such as Listeria monocytogenes. In L. sakei, the structural gene (sppA) encoding sakacin P is controlled by a strict regulatory mechanism, and the quantity of secreted sakacin P is limited. In this study, the sppA gene was synthesized by splicing overlap extension PCR and cloned into Escherichia coli. After the induction with isopropyl-β-d-thiogalactopyranoside, the recombinant sakacin P was successfully expressed. The collected cells were sonicated, and the activity was detected by agar diffusion method. The results also showed that the low-temperature induction can improve the activity of sakacin P.     

Role of Malic Enzyme during Fatty Acid Synthesis in the Oleaginous Fungus Mortierella alpina

The generation of NADPH by malic enzyme (ME) was postulated to be a rate-limiting step during fatty acid synthesis in oleaginous fungi, based primarily on the results from research focusing on ME in Mucor circinelloides. This hypothesis is challenged by a recent study showing that leucine metabolism, rather than ME, is critical for fatty acid synthesis in M. circinelloides. To clarify this, the gene encoding ME isoform E from Mortierella alpina was homologously expressed. ME overexpression increased the fatty acid content by 30% compared to that for a control. Our results suggest that ME may not be the sole rate-limiting enzyme, but does play a role, during fatty acid synthesis in oleaginous fungi.     

Role of Ca2+/Calmodulin-dependent Protein Kinase II in Drosophila Photoreceptors

Ca²⁺ modulates the visual response in both vertebrates and invertebrates. In Drosophila photoreceptors, an increase of cytoplasmic Ca²⁺ mimics light adaptation. Little is known regarding the mechanism, however. We explored the role of the sole Drosophila Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) to mediate light adaptation. CaMKII has been implicated in the phosphorylation of arrestin 2 (Arr2). However, the functional significance of Arr2 phosphorylation remains debatable. We identified retinal CaMKII by anti-CaMKII antibodies and by its Ca²⁺-dependent autophosphorylation. Moreover, we show that phosphorylation of CaMKII is greatly enhanced by okadaic acid, and indeed, purified PP2A catalyzes the dephosphorylation of CaMKII. Significantly, we demonstrate that anti-CaMKII antibodies co-immunoprecipitate, and CaMKII fusion proteins pull down the catalytic subunit of PP2A from fly extracts, indicating that PP2A interacts with CaMKII to form a protein complex. To investigate the function of CaMKII in photoreceptors, we show that suppression of CaMKII in transgenic flies affects light adaptation and increases prolonged depolarizing afterpotential amplitude, whereas a reduced PP2A activity brings about reduced prolonged depolarizing afterpotential amplitude. Taken together, we conclude that CaMKII is involved in the negative regulation of the visual response affecting light adaptation, possibly by catalyzing phosphorylation of Arr2. Moreover, the CaMKII activity appears tightly regulated by the co-localized PP2A.Visual transduction is the process that converts the signal of light (photons) into a change of membrane potential in photoreceptors (see Ref. 1 for review). Visual signaling is initiated upon the activation of rhodopsins by light: light switches on rhodopsin to generate metarhodopsin, which activates the heterotrimeric G_q in Drosophila (2). Subsequently, the GTP-bound Gα_q subunit activates phospholipase Cβ4 encoded by the norpA (no receptor potential A) gene (3). Phospholipase Cβ4 catalyzes the breakdown of phosphoinositol 4,5-bisphosphate to generate diacylglycerol, which or its metabolite has been implicated in gating the transient receptor potential (TRP)² and TRP-like channels (4, 5). TRP is the major Ca²⁺ channel that mediates the light-dependent depolarization response leading to an increase of cytosolic Ca²⁺ in photoreceptors. The rise of intracellular Ca²⁺ modulates several aspects of the visual response including activation, deactivation, and light adaptation (6). For example, Ca²⁺ together with diacylglycerol activates a classical protein kinase C, eye-PKC, which is critical for the negative regulation of visual signaling by modulating deactivation and light adaptation (7–11).Light adaptation is the process by which photoreceptors adjust the visual sensitivity in response to ambient background light by down-regulating rhodopsin-mediated signaling. Light adaptation can be arbitrarily subdivided into long term and short term adaptation and may involve multiple regulations to reduce the efficiency of rhodopsin, G protein, or cation channels. For example, translocation of both G_q (12, 13) and TRP-like channels (14, 15) out of the visual organelle may contribute to long term adaptation in Drosophila. In contrast, short term adaptation may be orchestrated by modulating the activity of signaling proteins by protein kinases. Hardie and co-workers (16) demonstrated that an increase of cytoplasmic [Ca²⁺] mimicked light adaptation, leading to inhibition of the light-induced current. These authors also showed that light adaptation is independent of eye-PKC. Thus the effect of cytoplasmic Ca²⁺ to control light adaptation is likely mediated via calmodulin and CaMKII. The contribution of CaMKII to light adaptation has not been explored.CaMKII is a multimeric Ca²⁺/calmodulin-dependent protein kinase that modulates diverse signaling processes (17). Drosophila contains one CaMKII gene (18) that gives rise to at least four protein isoforms (19). These CaMKII isoforms share over 85% sequence identities with the α isoform of vertebrate CaMKII. For insights into the in vivo physiological role of CaMKII, Griffith et al. (20) generated transgenic flies (ala) expressing an inhibitory domain of the rat CaMKII under the control of a heat shock promoter, hsp70. They demonstrated that, upon heat shock treatment, the overexpression of the inhibitory peptide resulted in a suppression of the endogenous CaMKII activity in the transgenic flies (20). It has been shown that inhibition of CaMKII affects learning and memory (20) and neuronal functions (21–24). In photoreceptors, CaMKII has been implicated in the phosphorylation of the major visual arrestin, Arr2 (25, 26). However, how phosphorylation of Arr2 by CaMKII modifies the visual signaling remains to be elucidated.Here we report the biochemical and electrophysiological analyses of CaMKII in Drosophila retina. We demonstrate that suppression of CaMKII in ala¹ transgenic flies leads to a phenotype indicative of defective light adaptation. The ala¹ flies also display greater visual response, suggesting a defect in Arr2. These results support the notion that CaMKII plays a role in the negative regulation of the visual response. Our biochemical analyses demonstrate that dephosphorylation of CaMKII is mediated by protein phosphatase 2A (PP2A). Importantly, we show that PP2A interacts with CaMKII, indicating that CaMKII forms a stable protein complex with PP2A to ensure a tight regulation of the kinase activity. Thus a partial loss of function in PP2A would elevate the CaMKII activity. Indeed, we show that mts heterozygotes display reduced prolonged depolarizing potential (PDA) amplitude. This PDA phenotype strongly suggests that Arr2 becomes more effective to terminate the visual signaling in mts flies. Together, our findings indicate that the ability of Arr2 to terminate metarhodopsin is increased upon phosphorylation by CaMKII, and the retinal CaMKII activity is regulated by PP2A.     

Molecular phylogeny and maternal progenitor implication in the genus Kengyilia (Triticeae: Poaceae): Evidence from COXII intron sequences

Kengyilia is a perennial genus distributing in central and western Asia. Here, the levels of nucleotide diversity for COXII intron were obtained. The estimates of nucleotide diversity for different genome constitution ranged from θ = 0.00082 and π = 0.00082 for St genome species to π = 0.01227 and θ = 0.01229 for P genome species. Employing COXII intron sequences, the phylogenetic relationships within Kengyilia and between Kengyilia genus and its closely related genera were examined. The Maximum Parsimony analysis demonstrated that Kengyilia species were positioned into two clades corresponding to different maternal genomic donor. Kengyilia stenachyra, Kengyilia grandiglumis, Kengyilia hirsuta, Kengyilia melanthera, Kengyilia thoroldiana, Kengyilia alatavica and Kengyilia zhaosuensis were related to species of Agropyron, while Kengyilia kokonorica, Kengyilia rigidula, Kengyilia nana, Kengyilia mutica, Kengyilia longiglumis, Kengyilia laxiflora and Kengyilia gobicola were close to species of Roegneria and Pseudoroegneria. In addition, other three species of Kengyilia, such as Kengyilia batalinii, Kengyilia tahelacana and Kengyilia kaschgarica, were related to Douglasdeweya deweyi, Pseudoroegneria strigosa and Roegneria tibetica. This result indicated that there had been two phylogenetically divergent maternal donors within Kengyilia. Our new finding will help to understand the evolutionary history of the genus Kengyilia.     

组学技术在产油微生物中的应用

微生物油脂是未来燃料和食品用油的重要潜在资源。近年来,随着系统生物学技术的快速发展,从全局角度理解产油微生物生理代谢及脂质积累的特征成为研究热点。组学技术作为系统生物学研究的重要工具被广泛用于揭示产油微生物脂质高效生产的机制研究中,这为产油微生物理性遗传改造和发酵过程控制提供了基础。文中对组学技术在产油微生物中的应用概况进行了综述,介绍了产油微生物组学分析常用的样品前处理及数据分析方法,综述了包括基因组、转录组、蛋白(修饰)组及代谢(脂质)组等在内的多种组学技术,以及组学数据基础上的数学模型在揭示产油微生物脂质高效生产机制中的研究,并对未来发展和应用进行了展望。     

GH42家族保守氨基酸位点累积突变对Geobacillus stearothermophilus来源β-半乳糖苷酶BgaB催化活性的影响

【背景】β-半乳糖苷酶转糖苷活性弱,产物低聚半乳糖(galactooligosaccharides, GOS)易被水解,致其催化得率普遍较低。【目的】以GH42家族Geobacillus stearothermophilus来源β-半乳糖苷酶BgaB为对象,探讨家族保守氨基酸位点突变对β-半乳糖苷酶BgaB催化活性的影响。【方法】在单点突变体功能研究基础上,采用定点突变与化学修饰相结合的方法,对保守氨基酸位点E303与F341进行累积突变。【结果】与野生型酶相比,所构建双点突变体Ox-E303C/F341S水解活性降低为30%;GOS最大得率由0.75%提高到19.50%。【结论】家族保守氨基酸位点累积突变能够使单点突变体功能得到共同进化,降低β-半乳糖苷酶水解活性和底物抑制作用,能够提高其转糖苷催化活性。     

Physiological and biochemical responses to light and temperature stress in free-living conchocelis of Neopyropia katadae (Bangiales,Rhodophyta)

Journal of Applied Phycology - Neopyropia katadae is one of the important economic seaweeds in China. A mass of free-living conchocelis is needed in the commercial cultivation of N. katadae. This...