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Effects of leptin on porcine primary adiocytes lipolysis and mRNA expression of key lipolytic enzymes.

Chinese Journal of Biotechnology.2008;24(9):1613-1619.

Leptin, a cytokine predominantly secreted from fat tissue, plays an important role in regulating organism energy balance. Leptin can stimulate lipolysis, but the mechanism is unclear. In order to study the molecular mechanism of leptin stimulating lipolysis, we systemically studied the mRNA expression of key lipolytic enzymes. Morphological observation, Oil Red O staining and RT-PCR were used to identify pig primary adipocytes; commercial kits were used to measure the glycerol and FFA release; Semiquantitative RT-PCR was used to detect the mRNA expression of key lipolytic enzymes. The results showed that 100 nmol/L leptin up-regulated the mRNA expression of ATGL, TGH-2, HSL, MGL and LPL (P<0.01), but down-regulated the Perilipin mRNA expression (P<0.01). At the same time, leptin promoted the glycerol release in a dose dependent manner (P<0.01), but had no effect on the FFA release (P>0.05). These indicate that leptin may mainly stimulate lipolysis in pig primary adipocytes by up-regulating the expression of ATGL, MGL, LPL and down-regulating the expression of Perilipin. The unchanged FFA release may be resulted from Leptin promoting UCPs mRNA expression and increasing FFA expenditure.
Adipocytes ; cytology ; enzymology ; metabolism ; Animals ; Animals, Newborn ; Cells, Cultured ; Leptin ; pharmacology ; Lipase ; genetics ; metabolism ; Lipolysis ; drug effects ; Male ; Monoacylglycerol Lipases ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Swine

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Influences of phosphate deficiency in the medium on growth, activities of antioxidant enzymes and utilization of nitrogen resource in Cucumis sativus hairy roots.

Yue ZHANG ; Heping SHI

Chinese Journal of Biotechnology.2008;24(9):1604-1612.

Effects of phosphorus deficiency in the medium on growth and morphology and activities of SOD and POD, utilization of nitrogen and calcium in the medium during liquid culture of Cucumis sativus hairy roots were investigated. The results showed that C. sativus hairy roots can not grow in the medium without addition of any phosphorus. When cultured into the medium with different Pi concentrations, the growth of C. sativus hairy roots was significantly inhibited with the decreasing of Pi concentration in the medium, its main roots became thinner and longer, the number of its lateral roots was decreased and its lateral roots became shorter and smaller. Compared to the medium with full strength phosphorus, the content of soluble proteins in C. sativus hairy roots cultured under Pi deficiency was significantly lower than that with standard full-length Pi, while POD and SOD activities in C. sativus hairy roots were significantly stimulated. Compared to the control (without addition of any phosphorus in the medium), the activities of POD and SOD in C. sativus hairy roots cultured in the medium with different concentration of Pi were lower than the control. When C. sativus hairy roots were cultured into medium with different Pi concentrations, the medium conductivity was gradually decreased with time and with direct proportion of the initial Pi concentration of the medium; NH4(+)-N and NO3(-)-N of the medium was gradually absorbed and utilized, at day 15, NH4(+)-N of the medium was nearly used up but its NO3(-)-N was not used up until cultured for 30 days. Pi deficiency in the medium could decrease the consumption rate of NO3(-)-N and inhibited the absorption and utilization of calcium of the medium by C. sativus hairy roots. Proper enhancement of Pi concentration could stimulate absorption and consumption of calcium of the medium.
Calcium ; metabolism ; Cucumis sativus ; growth & development ; metabolism ; Culture Media ; Nitrogen ; metabolism ; Phosphates ; pharmacology ; Plant Roots ; growth & development ; metabolism ; Superoxide Dismutase ; metabolism ; Tissue Culture Techniques

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Breeding of Actinobacillus succiniogenes mutants with improved succinate production based on metabolic flux analysis.

Lijun PAN ; Xingjiang LI ; Shaotong JIANG ; Zhaojun WEI ; Xiaohui CHEN ; Licheng CAI ; Hefeng WANG ; Jijun JIANG

Chinese Journal of Biotechnology.2008;24(9):1595-1603.

It is very important to obtain high yield mutant strains on the base of metabolic flux analysis of Actinobacillus succinogenes S.JST for the industrial bioconversion of succinic acid. The metabolic pathway was analized at first and the flux of the metabolic networks was calculated by matrix. In order to decrease acetic acid flux, the strains mutated by soft X-ray of synchronous radiation were screened on the plates with high concentration of fluoroacetic acid. For decreasing the metabolic flux of ethanol the site-directed mutagenesis was carried out for the reduction of alcohol dehydrogenase(Adh) specific activity. Then the enzyme activity determination and the gene sequence analysis of the mutant strain was compared with those of the parent strain. Metabolic flux analysis of the parent strain indicated that the flux of succinic acid was 1.78(mmol/g/h) and that the flux of acetic acid and ethanol were 0.60 (mmol/g/h) and 1.04( mmol/g/h), respectively. Meanwhile the metabolic pathway analysis showed that the ethanol metabolism enhanced the lacking of H electron donor during the synthesis of succinic acid and that the succinic acid flux was weakened by the metabolism of byproducts ethanol and acetic acid. Compared with the parent strain, the acetic acid flux of anti-fluoroacetic mutant strain S.JST1 was 0.024 (mmol/g/h), decreasing by 96%. Then the enzyme determination showed that the specific activity unit of phosphotransacetylase(Pta) decreased from 602 to 74 and a mutated site was founded in the pta gene of the mutant strain S.JST1. Compared with that of the parent strain S.JST1 the ethanol flux of adh-site-directed mutant strain S.JST2 was 0.020 (mmol/g/h), decreasing by 98%. Then the enzyme determination showed that the specific activity unit of Adh decreased from 585 to 62 and the yield of end product succinic acid was 65.7 (g/L). The interdiction of Adh and Pta decreased the metabolism of byproducts and the H electron donor was well balanced, thus the succinic acid flux was strengthened by the redundant carbon flux from these byproducts. The mutant strain S.JST2 obtained in this paper deserves being extended to application of industrial fermentation.
Actinobacillus ; enzymology ; genetics ; growth & development ; Alcohol Dehydrogenase ; metabolism ; Metabolic Networks and Pathways ; genetics ; Mutagenesis, Site-Directed ; Mutation ; Phosphate Acetyltransferase ; metabolism ; Succinic Acid ; metabolism

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Hematopoietic repopulating ability of human CD34+ cells and CD34- cells in NOD/SCID mice.

Huili JIN ; Haibo CAI ; Shi YANG ; Wensong TAN

Chinese Journal of Biotechnology.2008;24(9):1588-1594.

The hematopoietic repopulating ability of fresh and cultured CD34+ cells and CD34- cells derived from cord blood were compared by nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse model. Fresh CD34+ cells and CD34- cells were isolated from fresh cord blood. Cultured CD34+ cells and CD34- cells were separated from cultured mononuclear cells (MNC). We transplanted these cells into sublethally irradiated NOD/SCID mice via the tail vein and sacrificed surviving mice after 6 weeks. The peripheral blood, spleen and bone marrow from each mouse were harvested for flow cytometry, colony-forming cells and human Alu sequences analyses. The proportions of CD45+ cells and human multilineage hematopoietic cells in NOD/SCID mice received CD34+ cells were close to that in the mice received both CD34+ cells and CD34- cells, while it was significantly higher than that in the mice received CD34- cells. Six weeks after transplantation, all the mice injected with cultured CD34- cells dead. The survival rate of mice injected with cultured CD34+ cells was 66.7%. All of the mice injected with both cultured CD34- and CD34+ cells survived. Moreover, CD45+ cells could be detected in all surviving mice, and human CD34, CD3, CD19, CD33 and CD71 antigen also could be detected on these CD45+ cells. The results showed that both fresh and cultured CD34+ cells had the capability of engraftment and hematopoiesis reconstitution, but CD34- cells hadn't the ability. However, CD34- cells had assistant effect on the hematopoietic repopulating ability of CD34+ cells.
Animals ; Cells, Cultured ; Cord Blood Stem Cell Transplantation ; Embryonic Stem Cells ; cytology ; Fetal Blood ; cytology ; Hematopoiesis ; physiology ; Humans ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Transplantation, Heterologous

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Differentiation of human amniotic fluid stem cells into cardiomyocytes through embryonic body formation.

Han WANG ; Shuai CHEN ; Xiang CHENG ; Zhongying DOU ; Huayan WANG

Chinese Journal of Biotechnology.2008;24(9):1582-1587.

To isolate human amniotic fluid stem cells (hASCs) and induce hASCs into cardiomyocytes after forming the embryonic bodies. We cultivated hASCs isolated from the amniotic fluid continually for over 42 passages. The biological characteristics of hASCs were detected by immunocytochemistry, RT-PCR and flow cytometer, hASCs at 10-15th passage were suspension cultured to form embryonic bodies that were induced to cardiomyocytes. Fibroblastoid-type hASCs were obtained. Immunocytochemistry, RT-PCR and flow cytometry analysis demonstrated that hASCs were positive for some specific makers of the embryonic stem cell. hASCs could form embryonic bodies that were alkaline-phosphatase positive and expressed fgf5, zeta-globin and alpha-fetoprotein. The embryonic bodies could differentiate into cardiomyocytes showing alpha-actin positive and Tbx5, Nkx2.5, GATA4 and alpha-MHC positive. We conclued that hASCs obtained from human amniotic fluid could differentiate into cardiomyocytes through the formation of embryonic bodies.
Amniotic Fluid ; cytology ; Cell Differentiation ; physiology ; Cells, Cultured ; Embryo, Mammalian ; Embryonic Stem Cells ; cytology ; Humans ; Myocytes, Cardiac ; cytology

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Construction and application of chimeric infectious clones of porcine reproductive and respiratory syndrome virus.

Xiangjian LI ; Jianwu ZHANG ; Jian LÜ ; Dandan YU ; Huochun YAO ; Shishan YUAN

Chinese Journal of Biotechnology.2008;24(9):1573-1581.

In recent years, mass outbreaks of highly pathogenic (HP) porcine reproductive and respiratory syndrome virus (PRRSV) have spread all over the Chinese swine industry. Based on the first infectious cDNA clone of HP PRRSV strain pJX143 and that of an attenuated PRRSV, pAPRRS, constructed in our group, we constructed several chimeric clones with various substitutions of structural protein genes (ORF4-7) and 3' UTR between attenuated pAPRRS and virulent pJX143.Upon transfection of MA-104 cultured cells, all chimeric constructs pSX12, p5NX12, and p56N12 were rescued. The rescued viruses maintained the similar virological properties, based on the results of the growth curve of the rescued viruses. To test if the chimeric viruses can be used as a vaccine candidate, vSX12 and v56N12 vaccinated pigs were challenged with the HP PRRSV JX143 strain. As a result, the vSX12 vaccinated pigs were all seroconverted by 14-day-post vaccination, while v56N12 vaccinated pigs showed poor antibody response. Upon challenge, the vSX12-vaccinated group showed no signs of clinical PRRS syndrome, and virema period was shorten to 6 days post-challenge. Our results demonstrated that 1) vSX12 chimeric virus is a good vaccine candidate; 2) the virulence determinants of HP PRRSV probably located in coding regions other than ORF3-7 and 3' UTR, as our chimeric viruses were proved to be attenuated.
Animals ; Cloning, Molecular ; Open Reading Frames ; Porcine Reproductive and Respiratory Syndrome ; virology ; Porcine respiratory and reproductive syndrome virus ; genetics ; immunology ; Recombinant Fusion Proteins ; genetics ; immunology ; Recombination, Genetic ; genetics ; Swine ; Vaccines, Attenuated ; immunology ; Viral Envelope Proteins ; Viral Proteins ; biosynthesis ; genetics ; Viral Vaccines ; immunology

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Construction of eukaryotic express vector of duck interleukin 18 gene and identification of bioactivity of its expressed protein.

Hongying CHEN ; Xinsheng LI ; Baoan CUI ; Ping'an XIA ; Hongying ZHANG ; Mingfan YANG

Chinese Journal of Biotechnology.2008;24(9):1568-1572.

Duck IL-18 gene was amplified from plasmid pGEM-DuIL-18 by PCR. The PCR product digested with Pst I and Xho I was inserted into eukaryotic express vector pcDNA3.1(+) to generate an recombinant expression plasmid pcDNA3.1/DuIL-18 (pDuIL-18), and transformed into Escherichia coli JM109. The recombinant colonies were identified by restriction enzyme digestion, PCR and sequencing. DNA sequence confirmed the correct sequence of the recombinant eukaryotic expression plasmid pDuIL-18 in the reading frame and the ligation part. After the transfection of pDuIL-18 into Cos7 cells, duck IL-18 mRNA was expressed in Cos7 cell. The SDS-PAGE analysis showed that the expressed duck IL-18 protein had molecular weight of 23 000 D. The results of methyl thiazolyl tetrazolium (MTT) assay showed that duck IL-18 protein expressed in Cos7 cell could induce significantly transformation of duck T lymphocytes. Immunoenhancement effect of recombinant expression plasmid pDuIL18 on avian influenza vaccine was observed by proliferation response of the T lymphocytes from spleen. It can obviously enhance the cell-mediated immune response.
Animals ; COS Cells ; Cell Proliferation ; Cercopithecus aethiops ; Ducks ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Immunity, Cellular ; Interleukin-18 ; genetics ; immunology ; Recombinant Proteins ; genetics ; immunology ; T-Lymphocytes ; immunology ; Transfection

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Cloning, expression and immunity of pilA gene and ompC gene from avian pathogenic Escherichia coli.

Shan YU ; Qian ZHANG ; Xiaoxi SHUI ; Zhouliang YU ; Baohua ZHAO

Chinese Journal of Biotechnology.2008;24(9):1561-1567.

In order to amplify pilA gene and ompC gene of avian pathogenic Escherichia coli (APEC) strain, two pairs of primers were designed according to the GenBank sequences, and a 549 bp pilA gene and a 1104 bp ompC gene were obtained by PCR separately. Sequence analysis indicated that the homology of the nucleotide sequence of AEPC strain to those other reference strains was 98.18% of the pilA gene and 97.28% of the ompC gene. Two expression plasmids pETpilA and pETompC were constructed by inserting pilA gene and ompC gene into the prokaryotic expression vector pET-28a. The two plasmids were transformated into E. coli BL21 separately and two recombinant strains BL21 (pETpilA) and BL21 (pETompC) were obtained. The type 1 fimbraie and the out membrane protein were highly expressed when the recombinant strain BL21 (pETpilA) and BL21 (pETompC) were induced by IPTG Two specific proteins were detected by SDS-PAGE and immunogenicity of the expressed protein was confirmed by Western blotting and ELISA. The expressed fimbraie and OmpC were transformed into vaccine. The protective immune response was proved after the mice were immunized with the two vaccines. The results showed that the recombinant strain BL21 (pETpilA) and BL21 (pETompC) could be as candidate vaccine to provide protective immune response against AEPC infection.
Animals ; Cloning, Molecular ; Escherichia coli ; genetics ; immunology ; metabolism ; Escherichia coli Proteins ; genetics ; immunology ; metabolism ; Escherichia coli Vaccines ; immunology ; Fimbriae Proteins ; genetics ; immunology ; metabolism ; Gene Expression Regulation, Bacterial ; Genes, Bacterial ; Mice ; Porins ; genetics ; immunology ; metabolism ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism

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Comparison of antiviral activity between FeIFN-omega and FeIFN-alpha.

Hongbin WANG ; Xiaojuan JIA ; Limin YANG ; Lei SUN ; Hongning WANG ; Wenjun LIU

Chinese Journal of Biotechnology.2008;24(9):1556-1560.

Both IFN-omega and IFN-alpha belong to type I interferon and have antiviral, antiproliferative, immunomodulatory activities, but their bioactivities are usually different. FeIFN-omega gene was amplified by PCR. FeIFN-alpha gene was synthesized based on the published sequences of GenBank. Then the two types of feline interferon genes were subcloned into the pET-His vector, and expressed in Escherichia coli Rosetta (DE3). Recombinant interferons were purified by affinity chromatography with immobilized nickel chelating NTA (Ni-NTA) and their antiviral activity was estimated according to the ability of IFNs to inhibit the cytopathic effects (CPE) of virus on cells. Results showed that the antiviral activities against various viruses of FeIFN-omega were higher than those of FeIFN-alpha. Against H9N2 subtype avian influenza virus (AIV) and canine distemper virus (CDV), the antiviral activities of FeIFN-omega were 160 folds and 4 folds higher than those of FeIFN-alpha.
Animals ; Antiviral Agents ; pharmacology ; Cats ; Distemper Virus, Canine ; drug effects ; Influenza A Virus, H9N2 Subtype ; drug effects ; Interferon Type I ; biosynthesis ; genetics ; pharmacology ; Interferon-alpha ; biosynthesis ; genetics ; pharmacology

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Cloning, expressing and characterizing of a phosphoglycerate mutase gene of Schistosoma japonncum.

Yan ZHOU ; Jiaojiao LIN ; Lixiao YAO ; Xinzhi WANG ; Yaojun SHI ; Ke LU ; JinMing LIU ; Zhiqiang FU ; Lihong TAO

Chinese Journal of Biotechnology.2008;24(9):1550-1555.

Phosphoglycerate mutase (PGAM) is a key enzyme in glycolytic pathways. With PCR technique based on an EST identified in our lab, a novel gene named SjPGAM (GenBank Accession No. EU374631) was cloned. Sequence analysis revealed that the ORF of SjPGAM gene contained 753 nucleotides, encoding 250 amino acids, and the molecular weight was about 28.26 kD. Real-time PCR analysis showed that the mRNA level of SjPGAM was much higher in the 14 days and 19 days schistosomula than other stages, suggesting that the gene was a schistosomula stage differential expression gene. The SjPGAM cDNA fragment was subcloned into an expression vector pET-28a (+) and transformed into Escherichia coli BL21 cells. In the presence of IPTG, the 31 kD fusion protein was expressed in included bodies. Western blotting revealed that the fusion protein could be recognized by the rabbit serum anti-Schistosoma japonicum adult worm antigen preparation. The study provides important basis for investigating the mechanism of the PGAM in the glycolytic pathways of Schistosoma japonnicum.
Animals ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Male ; Phosphoglycerate Mutase ; genetics ; immunology ; RNA, Messenger ; genetics ; metabolism ; Rabbits ; Recombinant Proteins ; genetics ; metabolism ; Schistosoma japonicum ; enzymology ; genetics ; Schistosomiasis japonica ; immunology ; parasitology

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