Distinct requirements for Hfe in basal and induced hepcidin levels in iron overload and inflammation

Hepcidin is a negative regulator of iron absorption produced mainly by the liver in response to changes in iron stores and inflammation, and its levels have been shown to regulate the intestinal basolateral iron transporter ferroportin1 (Fp1). Hereditary hemochromatosis patients and Hfe-deficient mice show inappropriate expression of hepcidin but, in apparent contradiction, still retain the ability to regulate iron absorption in response to alterations of iron metabolism. To further understand the molecular relationships among Hfe, hepcidin, and Fp1, we investigated hepcidin and Fp1 regulation in Hfe-deficient mice (Hfe-/- and beta2m-/-) in response to iron deprivation, iron loading, and acute inflammation. We found that whereas basal hepcidin levels were manifestly dependent on the presence of Hfe and on the mouse background, all Hfe-deficient mice were still able to regulate hepcidin in situations of altered iron homeostasis. In the liver, Fp1 was modulated in opposite directions by iron and LPS, and its regulation in Hfe-deficient mice was similar to that observed in wild-type mice. In addition, we found that iron-deprived mice were able to mount a robust response after LPS challenge and that Toll-like receptor 4 (TLR-4)-deficient mice fail to regulate hepcidin expression in response to LPS. In conclusion, these results suggest that although Hfe is necessary for the establishment of hepcidin basal levels, it is dispensable for hepcidin regulation through both the iron-sensing and inflammatory pathways, and hepatic Fp1 regulation is largely independent of hepcidin and Hfe. The inflammatory pathway overrides the iron-sensing pathway and is TLR-4 dependent.     

Cerebral inflammation contributes to encephalopathy and brain edema in acute liver failure: protective effect of minocycline

Encephalopathy and brain edema are serious complications of acute liver failure (ALF). The precise pathophysiologic mechanisms responsible have not been fully elucidated but it has been recently proposed that microglia‐derived proinflammatory cytokines are involved. In the present study we evaluated the role of microglial activation and the protective effect of the anti‐inflammatory drug minocycline in the pathogenesis of hepatic encephalopathy and brain edema in rats with ALF resulting from hepatic devascularisation. ALF rats were killed 6 h after hepatic artery ligation before the onset of neurological symptoms and at coma stages of encephalopathy along with their appropriate sham‐operated controls and in parallel with minocycline‐treated ALF rats. Increased OX‐42 and OX‐6 immunoreactivities confirming microglial activation were accompanied by increased expression of interleukins (IL‐1β, IL‐6) and tumor necrosis factor‐alpha (TNF‐α) in the frontal cortex at coma stage of encephalopathy in ALF rats compared with sham‐operated controls. Minocycline treatment prevented both microglial activation as well as the up‐regulation of IL‐1β, ΙL‐6 and TNF‐α mRNA and protein expression with a concomitant attenuation of the progression of encephalopathy and brain edema. These results offer the first direct evidence for central proinflammatory mechanisms in the pathogenesis of brain edema and its complications in ALF and suggest that anti‐inflammatory agents may be beneficial in these patients.     

Study of human SP-A,SP-B and SP-D loci: allele frequencies,linkage disequilibrium and heterozygosity in different races and ethnic groups

Background  

SP-A, SP-B, and SP-D are pulmonary surfactant proteins. Several linkage and association studies have been done using these genes as markers to locate pulmonary disease susceptibility genes, but few have studied the markers systematically in different ethnic groups. Here we studied eight markers in SP-A, SP-B, and SP-D genes in seven ethnic groups from three races (Caucasian, Black and Hispanic). We measured the similarity of the marker distribution among the ethnic groups in order to see whether people in different ethnic groups or races could be mixed together for linkage and association studies. To evaluate the usefulness of these markers, we estimated the informativeness of each marker loci in the seven ethnic groups by assessing their heterozygosity and PIC values. We also conducted linkage disequilibrium (LD) analysis to identify associated marker loci and to estimate the haplotype frequencies in each of the seven ethnic groups in an attempt to find valuable haplotypes so that the level of polymorphism of the "markers" could be increased.     

SP-A1 and SP-A2 variants differentially enhance association of Pseudomonas aeruginosa with rat alveolar macrophages

Chronic airway inflammation caused by Pseudomonas aeruginosa is an important feature of cystic fibrosis (CF). Surfactant protein A (SP-A) enhances phagocytosis of P. aeruginosa. Two genes, SP-A1 and SP-A2, encode human SP-A. We hypothesized that genetically determined differences in the activity of SP-A1 and SP-A2 gene products exist. To test this, we studied association of a nonmucoid P. aeruginosa strain (ATCC 39018) with rat alveolar macrophages in the presence or absence of insect cell-expressed human SP-A variants. We used two trios, each consisting of SP-A1, SP-A2, and their coexpressed SP-A1/SP-A2 variants. We tested the 6A(2) and 6A(4) alleles (for SP-A1), the 1A(0) and 1A alleles (for SP-A2), and their respective coexpressed SP-A1/SP-A2 gene products. After incubation of alveolar macrophages with P. aeruginosa in the presence of the SP-A variants at 37 degrees C for 1 h, the cell association of bacteria was assessed by light microscopy analysis. We found 1) depending on SP-A concentration and variant, SP-A2 variants significantly increased the cell association more than the SP-A1 variants (the phagocytic index for SP-A1 was approximately 52-95% of the SP-A2 activity); 2) coexpressed variants at certain concentrations were more active than single gene products; and 3) the phagocytic index for SP-A variants was approximately 18-41% of the human SP-A from bronchoalveolar lavage. We conclude that human SP-A variants in vitro enhance association of P. aeruginosa with rat alveolar macrophages differentially and in a concentration-dependent manner, with SP-A2 variants having a higher activity compared with SP-A1 variants.     

mTORC1 Regulates Flagellin-Induced Inflammatory Response in Macrophages

Bacterial flagellin triggers inflammatory responses. Phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) regulate the production of pro- and anti-inflammatory cytokines that are induced by extrinsic antigens, but the function of mTORC1 in flagellin-induced inflammatory response is unknown. The purpose of this study was to examine the role and the mechanism of PI3K/Akt/mTOR pathway in flagellin-induced cytokine expression in mouse macrophages. We observed that flagellin upregulated TNF-α time- and dose-dependently. Flagellin stimulated rapid (<15 min) PI3K/Akt/mTOR phosphorylation that was mediated by TLR5. Inhibition of PI3K with {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and wortmannin, and of mTORC1 with rapamycin decreased flagellin-induced TNF-α and IL-6 expression and cell proliferation. The activation of NF-κB p65 and STAT3 was regulated by mTORC1 via degradation of IκBα and phosphorylation of STAT3 in response to flagellin, respectively. Thus, the PI3K/Akt/mTORC1 pathway regulates the innate immune response to bacterial flagellin. Rapamycin is potential therapy that can regulate host defense against pathogenic infections.     

Silica-bonded N-propyl sulfamic acid used as a heterogeneous catalyst for transesterification of soybean oil with methanol

The transesterification of soybean oil with methanol was carried out, to produce biodiesel, over silica-bonded N-propyl sulfamic acid in a heterogeneous manner. Results showed that a maximum conversion of 90.5% was achieved using a 1:20 M ratio of soybean oil to methanol and a catalyst amount of 7.5 wt.% at 423 K for 60 h. It was found that the free fatty acid (FFA) and water present in the feedstock had no significant influence on the catalytic activity to the transesterification reaction. Besides, the catalyst also showed activities towards the esterification reaction of FFAs, in terms of the FFA conversion of 95.6% at 423 K for 30 h. Furthermore, the catalyst could be recovered with a better reusability.     

Endothelial progenitor cells as a possible component of stem cell niche to promote self-renewal of mesenchymal stem cells

Stem cells dwell at the “stem cell niche” to accomplish a series of biological processes. The composition of the niche should be determined because the insufficient understanding of this feature limits the development in the study of stem cells. We showed in our study on mesenchymal stem cells (MSCs) that the MSCs first neighbored to CD31⁺ cells, which proved to be endothelial progenitor cells (EPCs), and formed a group of cell colony before they exerted their biological functions. It was further proved that EPCs have close interactions with MSCs and promoted the self-renewal of the MSCs in vitro and in vivo. Together with these achievements, we hypothesized that EPCs may be a possible biological component of the MSC stem cell niche and affect the biological processes of MSCs.     

构建LEF1基因毛囊特异表达载体并稳定转染胎儿成纤维细胞

旨在构建内蒙古白绒山羊(Capra hircus)淋巴样增强因子-1(Lymphoid enhancer factor,LEF1)基因真核表达载体并转染胎儿成纤维细胞,获得稳定表达红色荧光蛋白及毛囊特异性表达LEF1的转基因细胞克隆。以pCDsRed2载体为基本骨架将LEF1基因亚克隆到KAP6-1启动子下游,连接红色荧光蛋白表达元件,构建LEF1基因毛囊特异表达载体pCDsRed-KL。外源表达载体以lipofectamineTM2000介导转染胎儿成纤维细胞,通过G418筛选获得稳定转染的细胞克隆。PCR鉴定外源基因在细胞基因组中的整合。测序显示构建的表达载体pCDsRed-KL序列中,LEF1基因正确连接在KAP6-1启动子下游,顺序连接CMV启动子和红色荧光蛋白基因,载体构建正确。脂质体介导的稳定转染效率约为14.0%,经G418筛选得到高效表达红色荧光蛋白转基因细胞克隆。PCR检测显示外源KAP6-1启动子和LEF1基因整合到胎儿成纤维细胞基因组中。     

内蒙古白绒山羊IGF-IR基因cDNA克隆及组织表达特异性分析

旨在克隆内蒙古白绒山羊IGF-IR基因并分析其基本表达模式.采用RT-PCR克隆基因,将得到的IGF-IR基因cDNA片段的核苷酸序列及其编码的氨基酸序列进行生物信息学分析.半定量RT-PCR进行组织特异性表达检测.获得了内蒙古白绒山羊IGF-IR基因3’端编码区2118 bp的cDNA序列(JN200823),编码705个氨基酸残基.核苷酸序列与牛的IGF-IR( XM606794.3)基因同源性为98％,相应的氨基酸序列同源性为99％.SMART分析表明,推导出的编码蛋白具有跨膜域,酪氨酸激酶催化域.半定量RT-PCR检测表明,IGF-IR基因在绒山羊脑、胰腺、肝、肾组织中均有表达.