Oxygen dependent electron transfer in the cytochrome bc1 complex

The effect of molecular oxygen on the electron transfer activity of the cytochrome bc₁ complex was investigated by determining the activity of the complex under the aerobic and anaerobic conditions. Molecular oxygen increases the activity of Rhodobacter sphaeroides bc₁ complex up to 82%, depending on the intactness of the complex. Since oxygen enhances the reduction rate of heme b_L, but shows no effect on the reduction rate of heme b_H, the effect of oxygen in the electron transfer sequence of the cytochrome bc₁ complex is at the step of heme b_L reduction during bifurcated oxidation of ubiquinol.     

Fifteen weeks of dietary n-3 polyunsaturated fatty acid deprivation increase turnover of n-6 docosapentaenoic acid in rat-brain phospholipids

Docosapentaenoic acid (DPAn-6, 22:5n-6) is an n-6 polyunsaturated fatty acid (PUFA) whose brain concentration can be increased in rodents by dietary n-3 PUFA deficiency, which may contribute to their behavioral dysfunction. We used our in vivo intravenous infusion method to see if brain DPAn-6 turnover and metabolism also were altered with deprivation. We studied male rats that had been fed for 15weeks post-weaning an n-3 PUFA adequate diet containing 4.6% alpha-linolenic acid (α-LNA, 18:3n-3) or a deficient diet (0.2% α-LNA), each lacking docosahexaenoic acid (22:6n-3) and arachidonic acid (AA, 20:4n-6). [1-(14)C]DPAn-6 was infused intravenously for 5min in unanesthetized rats, after which the brain underwent high-energy microwaving, and then was analyzed. The n-3 PUFA deficient compared with adequate diet increased DPAn-6 and decreased DHA concentrations in plasma and brain, while minimally changing brain AA concentration. Incorporation rates of unesterified DPAn-6 from plasma into individual brain phospholipids were increased 5.2-7.7 fold, while turnover rates were increased 2.1-4.7 fold. The observations suggest that increased metabolism and brain concentrations of DPAn-6 and its metabolites, together with a reduced brain DHA concentration, contribute to behavioral and functional abnormalities reported with dietary n-3 PUFA deprivation in rodents. (196 words).     

Genetic Variation Analysis of Mugil cephalus in China Sea Based on Mitochondrial COI Gene Sequences

In this study, genetic diversity and population genetic structure of flathead grey mullet, Mugil cephalus, among four China Sea populations were investigated by COI sequences. All the populations studied had high values of haplotype and nucleotide diversity, except for the Yellow Sea population. In the phylogenetic tree, these haplotypes clustered in two groups, one for the populations from the Bohai and East China seas, and the other from the Yellow and South China seas. Analysis of molecular variance indicated that the northern populations (Bohai and East China) had lower genetic divergence (0.0725, P > 0.05) than that of the southern population (South China) (0.4530-0.6827, P < 0.001), suggesting that two distinct genetic groups exist in Chinese waters. Tests of neutral evolution and mismatch distribution indicated that no historical demographic expansion occurred in these populations. The results provide new information for genetic assessment, fishery management, and conservation of this species.     

RAGE binds C1q and enhances C1q-mediated phagocytosis

RAGE, the multiligand receptor of the immunoglobulin superfamily of cell surface molecules, is implicated in innate and adaptive immunity. Complement component C1q serves roles in complement activation and antibody-independent opsonization. Using soluble forms of RAGE (sRAGE) and RAGE-expressing cells, we determined that RAGE is a native C1q globular domain receptor. Direct C1q-sRAGE interaction was demonstrated with surface plasmon resonance (SPR), with minimum K(d) 5.6 μM, and stronger binding affinity seen in ELISA-like experiments involving multivalent binding. Pull-down experiments suggested formation of a receptor complex of RAGE and Mac-1 to further enhance affinity for C1q. C1q induced U937 cell adhesion and phagocytosis was inhibited by antibodies to RAGE or Mac-1. These data link C1q and RAGE to the recruitment of leukocytes and phagocytosis of C1q-coated material.     

Suppression of rat Frizzled-2 attenuates hypoxia/reoxygenation-induced Ca2+ accumulation in rat H9c2 cells

Growing evidence suggests that Ca(2+) overload is one of the major contributors of myocardial ischemia/reperfusion-induced injury. Since Frizzled-2 receptor, a seven transmembrane protein, transduces downstream signaling by specialized binding of Wnt5a to increase intracellular Ca(2+) release, this work aimed to investigate the effect of Frizzled-2 on Ca(2+) accumulation in H9c2 cells, which were subjected to hypoxia/reoxygenation to mimic myocardial ischemia/reperfusion. After exposing H9c2 cells to hypoxia/reoxygenation, we observed higher expression of Frizzled-2 and Wnt5a as compared to control group cells. Hypoxia/reoxygenation-induced intracellular Ca(2+) accumulation approached that of cells transfected with frizzled-2 plasmid. In cells treated with RNAi specifically designed against frizzled-2, intracellular Ca(2+) in both hypoxia/reoxygenation-treated cells and plasmid-treated cells were decreased. Rats that underwent ischemia/reperfusion injury exhibited increased intracellular Ca(2+) with high expression levels of Frizzled-2 and Wnt5a as compared to the sham group. Our data indicates that upon binding to Wnt5a, increased Frizzled-2 expression after hypoxia/reoxygenation treatment activated intracellular calcium release in H9c2 cells. Our findings provide a new perspective in understanding calcium overload in myocardial ischemia/reperfusion.     

犬细小病毒NS1 非结构蛋白可诱导细胞凋亡

【目的】研究犬细小病毒(Canine parvovirus,CPV)非结构蛋白NS1在CPV引起宿主细胞凋亡中的作用,初步探讨CPV引起细胞凋亡的机制。【方法】首先采用PCR方法从犬细小病毒基因组中扩增NS1编码基因,然后利用pcDNA3.1A质粒构建NS1真核表达载体pcDNA-NS1,并通过HEK293FT细胞瞬时表达NS1重组蛋白,用Western-blot检测以确定重组NS1蛋白能否在真核细胞中表达。然后用CPV感染和用pcDNA-NS1表达载体转染F81宿主细胞,通过AnnexinV/PI双染法检测磷脂酰丝氨酸外翻和通过化学发光法检测caspase-3/7活性,分析感染CPV或转染NS1基因对F81宿主细胞凋亡的影响。【结果】结果表明,本实验扩增的NS1基因序列与GenBank的序列一致,构建的表达载体结构正确,并能够介导NS1基因在真核细胞中表达。感染CPV和转染NS1基因均能诱导F81细胞膜磷脂酰丝氨酸外翻和明显提高细胞内caspase-3/7的活性,表明CPV和NS1蛋白均能引起细胞的凋亡。【结论】CPV诱导宿主细胞凋亡与其编码的NS1非结构蛋白有关。     

Resolvin d1 and resolvin d2 govern local inflammatory tone in obese fat

The unprecedented increase in the prevalence of obesity and obesity-related disorders is causally linked to a chronic state of low-grade inflammation in adipose tissue. Timely resolution of inflammation and return of this tissue to homeostasis are key to reducing obesity-induced metabolic dysfunctions. In this study, with inflamed adipose, we investigated the biosynthesis, conversion, and actions of Resolvins D1 (RvD1, 7S,8R,17S-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid) and D2 (RvD2, 7S,16R,17S-trihydroxy-4Z,8E,10Z,12E,14E,19Z-docosahexaenoic acid), potent anti-inflammatory and proresolving lipid mediators (LMs), and their ability to regulate monocyte interactions with adipocytes. Lipid mediator-metabololipidomics identified RvD1 and RvD2 from endogenous sources in human and mouse adipose tissues. We also identified proresolving receptors (i.e., ALX/FPR2, ChemR23, and GPR32) in these tissues. Compared with lean tissue, obese adipose showed a deficit of these endogenous anti-inflammatory signals. With inflamed obese adipose tissue, RvD1 and RvD2 each rescued impaired expression and secretion of adiponectin in a time- and concentration-dependent manner as well as decreasing proinflammatory adipokine production including leptin, TNF-α, IL-6, and IL-1β. RvD1 and RvD2 each reduced MCP-1 and leukotriene B(4)-stimulated monocyte adhesion to adipocytes and their transadipose migration. Adipose tissue rapidly converted both resolvins (Rvs) to novel oxo-Rvs. RvD2 was enzymatically converted to 7-oxo-RvD2 as its major metabolic route that retained adipose-directed RvD2 actions. These results indicate, in adipose, D-series Rvs (RvD1 and RvD2) are potent proresolving mediators that counteract both local adipokine production and monocyte accumulation in obesity-induced adipose inflammation.     

Telomerase Activity Is Sensitive to Subtle Perturbations of the TLC1 Pseudoknot 3′ Stem and Tertiary Structure

Pseudoknot formation in the core region of the telomerase RNA has been demonstrated to be important for telomerase activity in vertebrates, ciliates, and yeast. Characterization of the Saccharomyces cerevisiae telomerase RNA (TLC1) pseudoknot identified tertiary structural interactions that are also important for telomerase activity, as previously observed for the Kluyveromyces lactis and human telomerase RNA pseudoknots. In addition, the contributions of backbone ribose 2′-OH groups in the pseudoknot to telomerase catalysis were investigated previously, using 2′-OH (ribose) to 2′-H (deoxyribose) or 2′-O-methyl substitutions in the stem 2 helix, and it was proposed that one or more 2′-OH groups from the stem 2 sequences at or near the triple helix participate in telomerase catalysis. Based on these studies and investigations of the structural and thermodynamic properties of the TLC1 RNA pseudoknot region, we have examined the structural and thermodynamic perturbations of the 2′-O-methyl and 2′-H substituted pseudoknots, using UV-monitored thermal denaturation, native gel electrophoresis, and circular dichroism spectroscopy. Our results demonstrate the presence of A-form helical geometry perturbations in the backbone sugar substituted pseudoknots, show a correlation between thermodynamic stability and telomerase activity, and are consistent with the identification of the U809 ribose 2′-OH as a potential contributor to telomerase activity.     

Injection of duck recombinant follistatin fusion protein into duck muscle tissues stimulates satellite cell proliferation and muscle fiber hypertrophy

Follistatin (FST) can inhibit the expression of myostatin, which is a predominant inhibitor of muscle development. The potential application of myostatin-based technology has been prompted in different ways in agriculture. We previously constructed an expression vector of duck FST and isolated the FST fusion protein. After the protein was purified and refolded, it was added to the medium of duck myoblasts cultured in vitro. The results show that the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide value of the myoblasts in the duck FST treatment group is higher than that in the control group, which indicates that the duck FST fusion protein exhibits the biological activities that can accelerate myoblast proliferation. To further investigate the roles of duck FST on muscle development, we injected the protein into the duck muscle tissues in vivo. The results show that both the duck muscle fiber cross-sectional area and the satellite cell activation frequency are influenced more in the FST treatment group than they are in the control group. In addition to these phenomena, expression of MyoD and Myf5 were increased, and the expression of myostatin was decreased. Together, these results suggest the potential for using duck FST fusion protein to inhibit myostatin activity and subsequently to enhance muscle growth in vivo. The mechanism by which FST regulates muscle development in the duck is similar to that in mammals and fishes.