Targeted overexpression of inducible 6-phosphofructo-2-kinase in adipose tissue increases fat deposition but protects against diet-induced insulin resistance and inflammatory responses

Increasing evidence demonstrates the dissociation of fat deposition, the inflammatory response, and insulin resistance in the development of obesity-related metabolic diseases. As a regulatory enzyme of glycolysis, inducible 6-phosphofructo-2-kinase (iPFK2, encoded by PFKFB3) protects against diet-induced adipose tissue inflammatory response and systemic insulin resistance independently of adiposity. Using aP2-PFKFB3 transgenic (Tg) mice, we explored the ability of targeted adipocyte PFKFB3/iPFK2 overexpression to modulate diet-induced inflammatory responses and insulin resistance arising from fat deposition in both adipose and liver tissues. Compared with wild-type littermates (controls) on a high fat diet (HFD), Tg mice exhibited increased adiposity, decreased adipose inflammatory response, and improved insulin sensitivity. In a parallel pattern, HFD-fed Tg mice showed increased hepatic steatosis, decreased liver inflammatory response, and improved liver insulin sensitivity compared with controls. In both adipose and liver tissues, increased fat deposition was associated with lipid profile alterations characterized by an increase in palmitoleate. Additionally, plasma lipid profiles also displayed an increase in palmitoleate in HFD-Tg mice compared with controls. In cultured 3T3-L1 adipocytes, overexpression of PFKFB3/iPFK2 recapitulated metabolic and inflammatory changes observed in adipose tissue of Tg mice. Upon treatment with conditioned medium from iPFK2-overexpressing adipocytes, mouse primary hepatocytes displayed metabolic and inflammatory responses that were similar to those observed in livers of Tg mice. Together, these data demonstrate a unique role for PFKFB3/iPFK2 in adipocytes with regard to diet-induced inflammatory responses in both adipose and liver tissues.     

Expressions and purification of a mature form of recombinant human Chemerin in Escherichia coli

Chemerin is a novel chemokine that binds to the G protein-coupled receptor (GPCR) ChemR23, also known as chemokine-like receptor 1 (CMKLR1). It is secreted as a precursor and executes pro-inflammatory functions when the last six amino acids are removed from its C-terminus by serine proteases. After maturation, Chemerin attracts dendritic cells and macrophages through binding to ChemR23. We report a new method for expression and purification of mature recombinant human Chemerin (rhChemerin) using a prokaryotic system. After being expressed in bacteria, rhChemerin in inclusion bodies was denatured using 6 M guanidine chloride. Soluble rhChemerin was prepared by the protein-specific renaturation solution under defined conditions. It was subsequently purified using ion-exchange columns to more than 95% purity with endotoxin level <1.0 EU/μg. We further demonstrated its biological activities for attracting migration of human dendritic cells and murine macrophages in vitro using established chemotaxis assays.     

Systems Mapping for Hematopoietic Progenitor Cell Heterogeneity

Cells with the same genotype growing under the same conditions can show different phenotypes, which is known as “population heterogeneity”. The heterogeneity of hematopoietic progenitor cells has an effect on their differentiation potential and lineage choices. However, the genetic mechanisms governing population heterogeneity remain unclear. Here, we present a statistical model for mapping the quantitative trait locus (QTL) that affects hematopoietic cell heterogeneity. This strategy, termed systems mapping, integrates a system of differential equations into the framework for systems mapping, allowing hypotheses regarding the interplay between genetic actions and cell heterogeneity to be tested. A simulation approach based on cell heterogeneity dynamics has been designed to test the statistical properties of the model. This model not only considers the traditional QTLs, but also indicates the methylated QTLs that can illustrate non-genetic individual differences. It has significant implications for probing the molecular, genetic and epigenetic mechanisms of hematopoietic progenitor cell heterogeneity.     

Phosphatidylserine is involved in the ferrichrome-induced plasma membrane trafficking of Arn1 in Saccharomyces cerevisiae

Arn1 is an integral membrane protein that mediates the uptake of ferrichrome, an important nutritional source of iron, in Saccharomyces cerevisiae. In the absence of ferrichrome, Arn1p is sorted directly from the trans-Golgi network to the vacuolar lumen for degradation. In the presence of low levels of ferrichrome, the siderophore binds to a receptor domain on Arn1, triggering the redistribution of Arn1 to the plasma membrane. When extracellular ferrichrome levels are high, Arn1 cycles between the plasma membrane and intracellular vesicles. To further understand the mechanisms of trafficking of Arn1p, we screened 4580 viable yeast deletion mutants for mislocalization of Arn1-GFP using synthetic genetic array technology. We identified over 100 genes required for trans-Golgi network-to-vacuole trafficking of Arn1-GFP and only two genes, SER1 and SER2, required for the ferrichrome-induced plasma membrane trafficking of Arn1-GFP. SER1 and SER2 encode two enzymes of the major serine biosynthetic pathway, and the Arn1 trafficking defect in the ser1Δ strain was corrected with supplemental serine or glycine. Plasma membrane trafficking of Hxt3, a structurally related glucose transporter, was unaffected by SER1 deletion. Serine is required for the synthesis of multiple cellular components, including purines, sphingolipids, and phospholipids, but of these only phosphatidylserine corrected the Arn1 trafficking defects of the ser1Δ strain. Strains with defects in phospholipid synthesis also exhibited alterations in Arn1p trafficking, indicating that the intracellular trafficking of some transporters is dependent on the phospholipid composition of the cellular membranes.     

A major QTL for resistance to Gibberella stalk rot in maize

Fusarium graminearum Schwabe, the conidial form of Gibberella zeae, is the causal fungal pathogen responsible for Gibberella stalk rot of maize. Using a BC₁F₁ backcross mapping population derived from a cross between ‘1145’ (donor parent, completely resistant) and ‘Y331’ (recurrent parent, highly susceptible), two quantitative trait loci (QTLs), qRfg1 and qRfg2, conferring resistance to Gibberella stalk rot have been detected. The major QTL qRfg1 was further confirmed in the double haploid, F₂, BC₂F₁, and BC₃F₁ populations. Within a qRfg1 confidence interval, single/low-copy bacterial artificial chromosome sequences, anchored expressed sequence tags, and insertion/deletion polymorphisms, were exploited to develop 59 markers to saturate the qRfg1 region. A step by step narrowing-down strategy was adopted to pursue fine mapping of the qRfg1 locus. Recombinants within the qRfg1 region, screened from each backcross generation, were backcrossed to ‘Y331’ to produce the next backcross progenies. These progenies were individually genotyped and evaluated for resistance to Gibberella stalk rot. Significant (or no significant) difference in resistance reactions between homozygous and heterozygous genotypes in backcross progeny suggested presence (or absence) of qRfg1 in ‘1145’ donor fragments. The phenotypes were compared to sizes of donor fragments among recombinants to delimit the qRfg1 region. Sequential fine mapping of BC₄F₁ to BC₆F₁ generations enabled us to progressively refine the qRfg1 locus to a ~500-kb interval flanked by the markers SSR334 and SSR58. Meanwhile, resistance of qRfg1 to Gibberella stalk rot was also investigated in BC₃F₁ to BC₆F₁ generations. Once introgressed into the ‘Y331’ genome, the qRfg1 locus could steadily enhance the frequency of resistant plants by 32–43%. Hence, the qRfg1 locus was capable of improving maize resistance to Gibberella stalk rot.     

表达SLTEC保护性抗原的重组猪霍乱沙门氏菌C500株的构建及生物学特性

摘要:【目的】 利用平衡致死系统构建表达产类志贺氏毒素大肠杆菌(Shiga-like toxin Escherichia coli , SLTEC)保护性抗原的减毒猪霍乱沙门氏菌。【方法】 构建表达SLT-IIeB－FedF的重组质粒 ,再将其电转入终宿主菌减毒猪霍乱沙门氏菌ΔasdC500株中构建成口服活疫苗株 ,经聚丙烯酰胺凝胶电泳检测SLT-IIeB－FedF融合蛋白的表达情况,并观察重组菌体外培养的稳定性。【结果】 　利用宿主-载体平衡致死系统构建了表达SLTEC保护性抗原的重组减毒猪霍乱沙门氏菌     

Medium‐Chain Oil Reduces Fat Mass and Down‐regulates Expression of Adipogenic Genes in Rats

Objective: To test the hypothesis that adipose tissue could be one of the primary targets through which medium‐chain fatty acids (MCFAs) exert their metabolic influence. Research Methods and Procedures: Sprague‐Dawley rats were fed a control high‐fat diet compared with an isocaloric diet rich in medium‐chain triglycerides (MCTs). We determined the effects of MCTs on body fat mass, plasma leptin and lipid levels, acyl chain composition of adipose triglycerides and phospholipids, adipose tissue lipoprotein lipase activity, and the expression of key adipogenic genes. Tissue triglyceride content was measured in heart and gastrocnemius muscle, and whole body insulin sensitivity and glucose tolerance were also measured. The effects of MCFAs on lipoprotein lipase activity and adipogenic gene expression were also assessed in vitro using cultured adipose tissue explants or 3T3‐L1 adipocytes. Results: MCT‐fed animals had smaller fat pads, and they contained a considerable amount of MCFAs in both triglycerides and phospholipids. A number of key adipogenic genes were down‐regulated, including peroxisome proliferator activated receptor γ and CCAAT/enhancer binding protein α and their downstream metabolic target genes. We also found reduced adipose tissue lipoprotein lipase activity and improved insulin sensitivity and glucose tolerance in MCT‐fed animals. Analogous effects of MCFAs on adipogenic genes were found in cultured rat adipose tissue explants and 3T3‐L1 adipocytes. Discussion: These results suggest that direct inhibitory effects of MCFAs on adiposity may play an important role in the regulation of body fat development.     

Rab39, a novel Golgi-associated Rab GTPase from human dendritic cells involved in cellular endocytosis

Rab GTPases are Ras-like small molecular weight GTP binding proteins that are involved in various steps along the exocytic and endocytic pathways. Here we report that Rab39, a novel Rab protein, is a Golgi-associated protein involved in endocytosis of HeLa cells. Full-length cDNA of Rab39 contains 1251bp with an open reading frame (ORF) of 636bp, which is predicted to encode a 211 aa protein. By blast analysis of Rab39 cDNA and protein sequence with homologues, we find that Rab39 may be a short variant of Rab34. Rab39 contains conserved motifs involved in phosphate/guanosine binding and a microbody C-terminal targeting signal. RT-PCR analysis indicates that Rab39 is mainly detected in epithelial cell lines, and Northern blot analysis shows that Rab39 is expressed ubiquitously in human tissues. By using FITC-BSA as an endocytic tracer, we show that Rab39 can facilitate endocytosis in HeLa cells when expressed either transiently or stably. Confocal microscopy examination of Rab39 subcellular localization suggests that Rab39 is associated with Golgi-associated organelles. Our findings demonstrate that Rab39 is a novel Rab GTPase involved in cellular endocytosis.