Ectoine Production by Halomonas boliviensis: Optimization Using Response Surface Methodology

Two cultivation steps were used for production of biomass and ectoine by Halomonas boliviensis, respectively. The optimization of some nutrient parameters in each step was investigated by using response surface methodology. Twenty and 12 experiments were performed to attain optimal conditions for biomass and ectoine production, respectively. The model predicted a maximum biomass concentration of 3.34 g/L on optimization of NH₄Cl, K₂HPO₄, and MgSO₄•7H₂O concentrations during the first cultivation, while a maximum ectoine concentration of 1.27 g/L was predicted on optimizing NaCl and monosodium glutamate concentrations in the second cultivation. The experimental values obtained (3.36 g biomass/L and 1.25 g ectoine/L) were in good agreement with the predicted values. The optimized conditions were also used for two-step 1.5-L fed-batch fermentations. In the first step, biomass concentration of 28.7 g/L was obtained while in the second step biomass concentration increased to 63 g/L. Ectoine concentration of 9.2 g/L was obtained, and the overall ectoine productivity was 6.3 g/L/day, being among the highest reported so far.     

Consequences of cps mutation of Klebsiella pneumoniae on 1,3-propanediol fermentation

The filtration in 1,3-propanediol (1,3-PD) downstream process is influenced by the large amounts of capsular polysaccharides (CPS) produced by Klebsiella pneumoniae CGMCC 1.6366. The morphological and fermentation properties were investigated with the CPS-deficient mutant K. pneumoniae CGMCC 1.6366 CPS. Similar biomass was obtained with CGMCC 1.6366, and the mutant strain in batch cultures indicating the cell growth was slightly inhibited by CPS defection. The viscosity of fermentation broth by mutant strain decreased by 27.45%. The flux with ceramic membrane filter was enhanced from 168.12 to 303.6 l h⁻¹ m⁻², exhibiting the great importance for downstream processing of 1,3-PD fermentation. The products spectrum of mutant isolate changed remarkably regarding to the concentration of fermentation products. The synthesis of important 1,3-PD and 2,3-butanediol was enhanced from 9.73 and 4.06 g l⁻¹ to 10.37 and 4.77 g l⁻¹ in batch cultures. The noncapsuled K. pneumoniae provided higher 1,3-PD yield of 0.54 mol mol⁻¹ than that of encapsuled wild parent in batch cultures. The fed-batch fermentation of mutant strain resulted in 1,3-PD concentration, yield, and productivity of 78.13 g l⁻¹, 0.53 mol mol⁻¹, and 1.95 g l⁻¹ h⁻¹, respectively.     

Simulated Microgravity Induces the Proliferative Inhibition and Morphological Changes in Porcine Granulosa Cells

Astronauts are always faced with serious health problems during prolonged spaceflights. Previous studies have shown that weightlessness significantly affects the physiological function of female astronauts, including a change in reproductive hormones and ovarian cells, such as granulosa and theca cells. However, the effects of microgravity on these cells have not been well characterized, especially in granulosa cells. This study aimed to investigate the effects of simulated microgravity (SMG) on the proliferation and morphology of porcine granulosa cells (pGCs). pGC proliferation from the SMG group was inhibited, demonstrated by the reduced O.D. value and cell density in the WST-1 assay and cell number counting. SMG-induced pGCs exhibited an increased ratio of cells in the G0/G1 phase and a decreased ratio of cells in the S and G2/M phase. Western blot analysis indicated a down-regulation of cyclin D1, cyclin-dependent kinase 4 (cdk4), and cyclin-dependent kinase 6 (cdk6), leading to the prevention of the G1-S transition and inducing the arrest phase. pGCs under the SMG condition showed an increase in nuclear area. This caused a reduction in nuclear shape value in pGCs under the SMG condition. SMG-induced pGCs exhibited different morphologies, including fibroblast-like shape, rhomboid shape, and pebble-like shape. These results revealed that SMG inhibited proliferation and induced morphological changes in pGCs.     

The E3 ubiquitin ligase activity of Trip12 is essential for mouse embryogenesis

Protein ubiquitination is a post-translational protein modification that regulates many biological conditions. Trip12 is a HECT-type E3 ubiquitin ligase that ubiquitinates ARF and APP-BP1. However, the significance of Trip12 in vivo is largely unknown. Here we show that the ubiquitin ligase activity of Trip12 is indispensable for mouse embryogenesis. A homozygous mutation in Trip12 (Trip12(mt/mt)) that disrupts the ubiquitin ligase activity resulted in embryonic lethality in the middle stage of development. Trip12(mt/mt) embryos exhibited growth arrest and increased expression of the negative cell cycle regulator p16. In contrast, Trip12(mt/mt) ES cells were viable. They had decreased proliferation, but maintained both the undifferentiated state and the ability to differentiate. Trip12(mt/mt) ES cells had increased levels of the BAF57 protein (a component of the SWI/SNF chromatin remodeling complex) and altered gene expression patterns. These data suggest that Trip12 is involved in global gene expression and plays an important role in mouse development.     

Codon Optimization Enhances Protein Expression of Bombyx mori Nucleopolyhedrovirus DNA Polymerase in E. coli

Bombyx mori nucleopolyhedrovirus (BmNPV) is a major viral agent that causes deadly grasserie disease in silkworms, while BmNPV DNA polymerase (BmNPV-pol), encoded by ORF53 gene, plays a central role in viral DNA replication. Efficacy studies of BmNPV-POL are limited because of poor heterologous protein expression in E. coli. Here, we redesigned the BmNPV-pol to preferentially match codon frequencies of E. coli without altering the amino acid sequence. Following de novo synthesis, codon-optimized BmNPV-pol (co-BmNPV-pol) gene was cloned into pET32a and pGEX-4T-2 vector. The expression of co-BmNPV-POL in E. coli was significantly increased when BmNPV-POL was fused with GST protein rather than a His-tag. The co-BmNPV-POL fusion proteins were isolated using GST affinity chromatography and Mono Q iron exchange chromatography. Protein purity and identity were confirmed by western blot and MALDI-TOF analyses. The biological activity of purified proteins was measured on a poly(dA)/oligo(dT) primer/template. The specific polymerasing activity of the recombinant BmNPV-POL was 6,329 units/mg at optimal conditions. Thus, a large amount of purified protein as a soluble form with high activity would provide many benefits for the functional research and application of BmNPV-POL.     

鲥鱼的驯养与生物学研究Ⅰ.O+龄幼鱼的生长与食性

本文主要描述池塘养殖条件下鲥鱼幼鱼的生长与食性。1)1987-1989年8月至11月幼鱼生长迅速,快于同期珠江天然个体的生长速度;2)体长26.6-94.9mm的幼鱼主要摄食浮游动物,轮虫和浮游植物出现率随个体增大而减少。食物共有18个属种,摄食频度为100%;3)幼鱼的平均饱满指数为75.70‰。各类食物重量百分比的顺序为:桡足类>枝角类>无节幼体>虾类溞状幼体>轮虫>藻类。     

Elongated mouse chromosomes suitable for enhanced molecular cytogenetics

Characterization of genetic disorders in humans and animal models requires identification of chromosomal aberrations. However, identifying fine deletions or insertion in metaphase chromosomes has been always a challenge due to limitations of resolution. In this study we developed a rapid method for chromosome elongation using two different intercalating agents: ethidium bromide and 5-bromo-2′-deoxyuridine (BrdU), together with a short-term mitotic block using colcemid. About 70% of the chromosomes from cells that underwent this elongation procedure reached three times longer than those prepared from control cells. FISH experiments using elongated chromosomes revealed a duplicated region of chromosome 11 that was not visible in cells prepared with conventional methods.     

Double chip protein arrays using recombinant single-chain Fv antibody fragments

Protein arrays permit the parallel analysis of many different markers in a small sample volume. However, the problem of cross-reactivity limits the degree of multiplexing in parallel sandwich immunoassays (using monoclonal antibodies (mAbs)), meaning antibodies must be prescreened in order to reduce false positives. In contrast, we use a second chip surface for the local application of detection antibodies, thereby efficiently eliminating antibody cross-reactions. Here, we illustrate the potential advantages of using single-chain Fv fragments rather than mAbs as capture and detection molecules with this double chip technology.