Uronema marinum: identification and biochemical characterization of phosphatidylcholine-hydrolyzing phospholipase C

Phosphatidylcholine (PC)-specific phospholipase D (PC-PLD) and phosphatidylcholine (PC)-specific phospholipase C (PC-PLC) activities have been detected in Uronema marinum. Partial purification of PC-PLC revealed that two distinct forms of PC-PLC (named as mPC-PLC and cPC-PLC) were existed in membrane and cytosol fractions. The two PC-PLC enzymes showed the preferential hydrolyzing activity for PC with specific activity of 50.4 for mPC-PLC and 28.3 pmol/min/mg for cPC-PLC, but did not hydrolyze phosphatidylinositol or phosphatidylethanolamine. However, the biochemical characteristics and physiological roles of both enzymes were somewhat different. mPC-PLC had a pH optimum in the acidic region at around, pH 6.0, and required approximately 0.4 mM Ca2+ and 2.5 mM Mg2+ for maximal activity. cPC-PLC had a pH optimum in the neutral region at around, pH 7.0, and required 1.6 mM Ca2+ and 2.5 mM Mg2+ for maximal activity. cPC-PLC, but not mPC-PLC, showed a dose-dependent inhibitory effect on the luminal-enhanced chemiluminescence (CL) responses and the viability of zymosan-stimulated phagocytes of olive flounder, indicating that cPC-PLC may contribute to the parasite evasion against the host immune response. Our results suggest that U. marinum contains PC-PLD as well as two enzymatically distinct PC-PLC enzymes, and that mPC-PLC may play a role in the intercellular multiplication of U. marinum and cPC-PLC acts as a virulence factor, serving to actively disrupt the host defense mechanisms.     

Molecular and expression characterization of a nanos1 homologue in Chinese sturgeon, Acipenser sinensis

The nanos gene family was essential for germ line development in diverse organisms. In the present study, the full-length cDNA of a nanos1 homologue in A. sinensis, Asnanos1, was isolated and characterized. The cDNA sequence of Asnanos1 was 1489 base pairs (bp) in length and encoded a peptide of 228 amino acid residues. Multiple sequence alignment showed that the zinc-finger motifs of Nanos1 were highly conserved in vertebrates. By RT-PCR analysis, Asnanos1 mRNAs were ubiquitously detected in all tissues examined except for the fat, including liver, spleen, heart, ovary, kidney, muscle, intestines, pituitary, hypothalamus, telencephalon, midbrain, cerebellum, and medulla oblongata. Moreover, a specific polyclonal antibody was prepared from the in vitro expressed partial AsNanos1 protein. Western blot analysis revealed that the tissue expression pattern of AsNanos1 was not completely coincided with that of its mRNAs, which was not found in fat, muscle and intestines. Additionally, by immunofluoresence localization, it was observed that AsNanos1 protein was in the cytoplasm of primary oocytes and spermatocytes. The presented results indicated that the expression pattern of Asnanos1 was differential conservation and divergence among diverse species.     

AMPK promotes survival and adipogenesis of ischemia-challenged ADSCs in an autophagy-dependent manner

Some studies have shown that transplanted fat tissues usually cannot survive for long if adipose-derived stem cells (ADSCs) are removed from the tissues in advance. It is more meaningful to explore the mechanism mediating survival and differentiation of ADSCs in the transplanted microenvironment. AMP-activated protein kinase (AMPK) has been shown to be one of the energy receptors that regulate many aspects of cellular metabolism. AMPK activation has been implicated in models of adult ischemic injury, but the mechanism and the regulating effects of AMPK on survival and adipogenesis of transplanted ADSCs are still little known. In this study, we simulated the transplanted microenvironment using oxygen-glucose deprivation (OGD) to test the survival and adipogenesis of ADSCs. We found that OGD treatment triggered significant apoptosis and promoted autophagy. Simultaneously, OGD hindered the differentiation of ADSCs into mature adipocytes. After inhibiting AMPK, the OGD-induced apoptosis rate increased but autophagy was inhibited. The adipogenesis level also decreased. To show that the effects of AMPK on apoptosis and adipogenesis were autophagy-dependent, we pre-inhibited or pre-promoted autophagy with siATG7 or rapamycin while blocking AMPK. We found that inhibiting or improving autophagy exacerbated or alleviated the role of AMPK prohibition in apoptosis and adipogenesis. Furthermore, we showed that AMPK inhibition significantly lowered ULK1 activity but promoted mTOR activity, so that to inhibit autophagy. Our study shows that AMPK plays a protective role in maintaining survival and adipogenesis of OGD-challenged ADSCs partly by positively regulating autophagy. AMPK positively regulates autophagy by inhibiting mTOR but promoting ULK1 activity in OGD condition.     

三卡因浓度对斑马鱼心脏手术模型麻醉的影响

目的:探讨斑马鱼麻醉过程中三卡因浓度对其心脏手术后生存率的影响。方法:以经典实验方法中的三卡因(MS-222)浓度150 mg/L为对照,分别用浓度为20 mg/L、40 mg/L、60 mg/L、80 mg/L、100 mg/L、150 mg/L、200 mg/L的MS-222麻醉液麻醉,观察斑马鱼术后行为及术后生存率的差异。结果:随着MS-222浓度的提高,斑马鱼表现为入麻时间的逐渐减少、苏醒时间的逐渐增加。MS-222麻醉斑马鱼成鱼的有效浓度为40~160 mg/L,在此浓度范围内,鱼体能够在3 min内达到可施行手术操作的麻醉状态(Ⅱ或Ⅲ期),10 min内可苏醒恢复;随着浓度的增大,呼吸频率的下降速率增大;MS-222浓度为40 mg/L时斑马鱼的术后存活率最高(98.3%)。结论:当MS-222浓度为40 mg/L时,斑马鱼入麻及苏醒较为平稳,术后斑马鱼复苏时间较短,且保持了较高的术后生存率。在实验操作或者生产应用时推荐MS-222麻醉药液最佳麻醉浓度为40 mg/L,所对应的麻醉时间约为15 min左右。     

不同麻醉方法对降低草鱼捕捉应激的作用

正应激(Stress)指生物受到外源或内源刺激后内稳态改变而产生的一系列反应~[1]。在鱼类养殖中,各种操作对鱼产生的惊扰及环境变化均会导致鱼类应激~[2,3]。应激使鱼类代谢强度增加,免疫和生长性能下降~[4,5]。有关人类和哺乳动物的应激研究已积累了大量的资料,相比之下,鱼类应激方面     

Plasma membrane vesicles prepared from unadhered monocytes: Characterization of calcium transport and the calcium ATPase

We have purified unadhered human monocytes in sufficient quantities to prepare monocyte plasma membrane vesicles and study vesicular calcium transport. Monocytes were isolated from plateletpheresis residues by counterflow centrifugal elutriation. By combining this source and procedure, 7 x 10(8) monocytes of over 90% purity were obtained. The membranes, isolated on a sucrose step gradient, had an 18-fold enrichment in Na,K-ATPase, a 29-fold diminution of succinate dehydrogenase activity and were vesicular on transmission electron micrographs. The membrane vesicles loaded with oxalate accumulated calcium only in the presence of Mg and ATP. Calcium uptake did not occur if ATP was replaced by any of five nucleotide phosphates or if Mg was omitted. Calcium transport had a maximal velocity of 4 pmoles calcium/micrograms vesicle protein/min and a Km for calcium of 0.53 microM. The ionophore A23187 completely inhibited calcium accumulation while 5 mM sodium cyanide and 10 microM ouabain had no effect. A calcium-activated ATPase was present in the same plasma membrane vesicles. The calcium ATPase had a maximal velocity of 18.0 pmoles calcium/micrograms vesicle protein/min and a Km for calcium of 0.60 microM. Calcium-activated ATPase activity was absent if Mg was omitted or if (gamma - 32P) GTP replaced (gamma - 32P) ATP. Monocyte plasma membranes that were stripped of endogenous calmodulin by EGTA treatment showed a reduced level of calcium uptake and calcium ATPase activity. The addition of exogenous calmodulin restored the transport activity to that of unstripped monocyte plasma membranes. Thus, monocyte plasma membrane vesicles contain a highly specific, ATP-dependent calcium transport system and a calcium-ATPase with similar high calcium affinities.     

The role of ‘Norin 10’ dwarfing genes in photosynthetic and respiratory activity of wheat leaves

Summary A comparative analysis of eight cultivars of spring wheat (Triticum aestivum) classified by height as tall (T), semi-dwarf (D₁), dwarf (D₂) and very dwarf (D₃) was conducted to study their efficiency of oxygen exchange during photosynthesis and dark respiration. Two cultivars were included in each height group.Cultivars carrying Norin 10 dwarfing genes (D₁, D₂ and D₃) were found to have a significantly higher photosynthetic rate per unit leaf area than talls (T) that lack these genes. Among the Norin gene carriers, dwarf group (D₂) was most efficient, followed by very dwarf (D₃) and semi-dwarf (D₁).Photosynthetic rate and respiratory rate were found to have a positive relationship.     

Production of large multienzyme complex by aerobic thermophilic fungus Chaetomium sp. nov. MS-017 grown on palm oil mill fibre

AIMS: A novel xylanolytic multienzyme complex of the aerobic thermophilic fungus Chaetomium sp. nov. MS-017 was produced on palm oil mill fibre (POMF) and partially characterized. METHODS AND RESULTS: The assay of the extracellular enzymes of Chaetomium sp. nov. MS-017 on POMF in solid-state fermentation revealed cellulolytic, pectinolytic and extremely high xylanolytic activities. The protein was purified by Sephadex G-200 column chromatography. The SDS-PAGE demonstrated that the purified protein is a complex with at least five xylanolytic, four cellulolytic and eight pectinolytic components. The characterization of the complex at various temperatures showed that the reactivity and stability of the complex are not lost up to 60 degrees C. In addition, the complex was very stable in a wide range of pH (3-9) and at high concentrations (10 mm) of cations and EDTA. The major products of xylan hydrolysis by the purified complex were determined to be xylobiose and xylotriose by thin-layer chromatography. CONCLUSION: Chaetomium sp. nov. MS-017 preferentially produces a xylanolytic multienzyme complex on POMF in solid-state fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the xylanolytic multienzyme complex produced by an aerobic thermophilic fungus.