3.Inducement, Purification and Characterization of?-mannanase from Armillariella tabescens EJLY2098
Dong-Sheng YAO ; Xiao-Kui HUANG ; Da-Ling LIU ; Chun-Fang XIE ; Rong HU ;
China Biotechnology 2006;0(07):-
Armillariella tabescens EJLY2098 was capable of secreting p-mannanase by konjac inducement. A 34 orthogonal design was applied to determine the optimum medium of inducing mannanase by Armillariella tabescens EJLY2098. The results suggested that Armillariella tabescens EJLY2098 secreted the high-activity enzyme in the optimum medium, which was composed of 2% konjac, 1% peptone, 25% potato juice,0.3% KH2PO4,15% MgSO4?7H2O, 0.01% VitB1. Purified by DEAE-anion exchange chromatography, two eluting peaks (P1 and P2) with the p-mannanase activity were obtained, and one of them (named?-mannanase P2) was a single band by the SDS-PAGE, and the molecular weight of?-mannanase P2 was 78. 9kDa. The isoelectric point of?-mannanase P2 was estimated to be 4.0-4. 1. The optimum activity for the enzyme was found at 60℃and pH4. 0 - 6. 0, and the enzyme was stable between pH4. 5 - 6. 0. The activity of?-mannanase P2 were enhanced by Na+ and Ba2+ . This?-mannanase can be used in feed industy. a new fungi secreting?-mannanase was obtained, providing an important base for cloning mannanase gene and constructing recombin microbe expressing?-mannanase .
4.Expression of the immune inhibitors of human B cell activating factor belonging to the tumor necrosis factor family and evaluation of immune response in vaccinated mice
Hui-Guang GAO ; Feng-Tian HE ; Rong-Fen LI ; Qing JI ; Gang HUANG ; Da-Qiang HU ; Li ZHANG ; Wei GONG ; Ying HU
Chinese Journal of Rheumatology 2001;0(05):-
Objective To prepare T-helper cell(Th)epitnpe-modified human soluble B cell activating factor belonging to the tumor necrosis factor family(BAFF)mutants and evaluate their immune response in vaccinated mice.Methods Recombinant cDNAs were cloned and ligated into the prokaryotie expression vec- tor pQE-80L respectively.The recombinant proteins were induced to express by IPTG in E.coli DH5?and purified with Ni-NTA chromatography.BALB/c mice were immunized with recombinant proteins respectively and the titres of the antibodies that were cross-reactive with BAFF in sera were analyzed by ELISA.Inhibiting ability of the antibodies in sera was analyzed by MTT assay.Results The recombinant proteins were highly expressed in E.coli DH5?.After purification,the purity of recombinant proteins was more than 90%.BALB/c mice immunized with recombinant proteins produced high levels of BAFF-specific antibodies.Cell proliferation assay showed that the sera of immunized mice could inhibit the proliferation-inducing activity of recombinant sBAFF and natural sBAFF.Conclusion The immune inhibitors of human BAFF which can induce polyclonal antibodies that are cross-reactive with BAFF are successfully prepared.These results may provide the basis for further study of their therapeutic effects.
5.hPARP1 genetic polymorphism in southern Chinese Han and Miao populations.
Huan-wen TANG ; Hai-rong LIANG ; Zhi-xiong ZHUANG ; Da-lin HU
Chinese Journal of Medical Genetics 2006;23(5):578-580
OBJECTIVETo study hPARP1 genetic polymorphism in southern Chinese Han and Miao populations.
METHODSBlood samples from 187 and 210 southern healthy Han and Miao populations were collected. The mutations of exons 12,13,16 and 17 of hPARP1 gene were investigated by PCR-single-strand conformation polymorphism(SSCP).
RESULTSFragments of 253 bp,313 bp,175 bp,362 bp within exons 12,13,16 and 17 respectively of hPARP1 gene were amplified by multiple PCR. An SSCP variant in exons 12,13,16 and 17 of PARP1 gene in 187 healthy Han and 210 healthy Miao individuals was identified. Seven single-base substitutions compared with the sequence of PARP1 gene were identified: a T to C transition in exon 12 (Phe548Ser), a G to T transition in exon 13 (Ala683Ser), a G to T transition in exon 16 (Asp798Tyr), and a A to G transition in exon 17 (His808Arg).
CONCLUSIONThere were polymorphism sites in exons 12,13,16,17 of hPARP1 gene in southern Chinese Han and Miao populations; these results may be useful for the establishment of PARP1 genotyping, and these newly described PARP1 alleles would be advantageous indicators for population studies.
Adult ; Alleles ; China ; Exons ; genetics ; Female ; Gene Frequency ; Genotype ; Humans ; Male ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerases ; genetics ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide ; genetics ; Polymorphism, Single-Stranded Conformational
6.Association between systemic inflammation and autoimmunity parameters and plasma lipid in patients with rheumatoid arthritis.
Chao XUE ; Wen-ling LIU ; Yi-hong SUN ; Rong-jing DING ; Da-yi HU
Chinese Journal of Cardiology 2011;39(10):941-945
OBJECTIVEThe purpose of this study was to observe the association between inflammation status/autoimmune antibodies and plasma lipid in patients with rheumatoid arthritis (RA).
METHODSA total of 402 RA patients were admitted into our hospital during January 2008 to March 2009 and 225 RA patients who met the inclusion criteria were selected to perform a full lipid profile examination including total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and triglyceride (TG). Erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), rheumatoid factor (RF), anti-cyclic citrullinated peptide (anti-CCP), anti-keratin antibody (AKA), anti-perinuclear factor autoantibody (APF) and complement (C) were also evaluated. Atherogenic index of plasma (AIP) was calculate by the formula Log (TG/HDL-C).
RESULTS(1) There were 12.9%, 10.2% and 14.2% patients with elevated TC, LDL-C and TC respectively, patients with reduced HDL-C accounted for 43.6%. (2) C(3) was higher in elevated TC group than normal TC group (P < 0.05). ESR and CRP were significantly higher in decreased HDL-C group than in normal HDL-C group (P < 0.05). CRP, C(3) and C(4) were significantly higher in elevated LDL-C group than in normal LDL-C group (P < 0.05). (3) Multiple stepwise regression analysis showed that C(3) was positively correlated with TC (R(2) = 0.067, P < 0.05). Both ESR and CRP were negative correlated with HDL-C (R(2) = 0.202, P < 0.05). CRP and anti-CCP were positively correlated with LDL-C (R(2) = 0.129, P < 0.05). ESR and C(4) were positively correlated with AIP (R(2) = 0.046, P < 0.05).
CONCLUSIONThis study showed that rheumatoid arthritis is associated with an abnormal lipid profile, especially in patients with increased inflammation markers and autoimmune antibodies. Moreover, ESR and C(4) were predictors of increased AIP in this cohort.
Aged ; Arthritis, Rheumatoid ; blood ; immunology ; physiopathology ; Autoantibodies ; blood ; Autoimmunity ; C-Reactive Protein ; analysis ; Cholesterol, HDL ; blood ; Cholesterol, LDL ; blood ; Complement System Proteins ; Humans ; Inflammation ; Lipids ; blood ; Lipoproteins, HDL ; blood ; Triglycerides ; blood
7.Study of mouse marrow cells differentiation into a hepatocyte lineage in vitro.
Yi-Ming ZHOU ; Da-Rong HU ; Peng YAO ; Gong-Ren FAN
Chinese Journal of Hepatology 2004;12(12):722-725
OBJECTIVETo explore whether bone marrow stem cells (MSCs) from adult mice can be induced to differentiate into hepatocytes by hepatocyte growth factor (HGF) alone and the time phase characteristics in the differentiation progress.
METHODSAdult mouse MSCs were treated with or without 100 ng/ml HGF, on days 0, 7, 14, 21, and 28. The morphologic characteristics of the cells were examined; the albumin (ALB), AFP mRNA was analyzed sub-quantively using reverse transcription polymerase chain reaction (RT-PCR) and immumohistochemistry techniques. The expression of ALB, AFP and CK19 were detected by using anti-ALB, AFP and CK19 antibodies.
RESULTSFreshly isolated adult mouse MSCs expressed ALB and AFP mRNA weakly; in the group without HGF, no ALB mRNA was detected on day 7. The expression of AFP mRNA was reduced significantly on day 7, and could not be detected anymore after day 14. In the HGF treated group, ALB mRNA was not detected on day 7, but the positive lane appeared again on day 14, and the expression of ALB mRNA was increased on day 21 but reduced in the following days. The AFP mRNA was positive at all times, however it tended to decrease after day 14 in the HGF treated groups. The result of immumohistochemistry was consistent with that of RT-PCR, and CK19 was always negative.
CONCLUSIONAdult mouse MSCs can be induced into hepatocyte differentiation in vitro. The optimal time for the induction was 2 to 3 weeks.
Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Hepatocyte Growth Factor ; pharmacology ; Hepatocytes ; cytology ; Male ; Mice ; Stem Cells ; cytology ; Time Factors
9.Mitogenic effects of growth and differentiation factors on rat liver stem cell WB-F344 in vitro.
Peng YAO ; Yi-qiong ZHAN ; Wang-xiang XU ; Chang-yan LI ; Xiao-ming YANG ; Da-rong HU
Chinese Journal of Hepatology 2003;11(1):33-36
OBJECTIVETo explore the relationship between the proliferation, differentiation of rat hepatic stem like cell line WB-F344 and cytokines in vitro.
METHODS(3)H thymidine labelling of new synthesized DNA was used to examine the mitogenic responsiveness of WB-F344 cells to cytokines, western blot was used to study the expression of cytokines receptors on hepatic stem cells, and apoptotic cells were detected by Flow cytometry.
RESULTSWB-F344 cells showed a proliferative response to the cytokines of hepatocyte growth factor (HGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), Insulin at the dose of 80 ng/ml, and the relative cpm values are 982.95, 906.32, 863.98 and 968.67 respectively, while non response to interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) at the same dose, and an inhibition or apoptosis response to transforming growth factor-beta (TGF-beta) at 80 ng/ml with a 26.89% apoptotic rate. Western blot showed that there were HGF, EGF, FGF, TGF-beta receptors expressed on WB-F344 cells. When WB-F344 cells were cultured in the differential system (DMEM, 10% Fcs, HGF 10 to approximately 50 ng/ml, EGF 20 ng/ml, Insulin 1 microg/ml, Dex 1 micromol/L), the cells could differentiated into hepatocytes. In addition, HGF could scattered WB-F344 cells.
CONCLUSIONThe proliferation and differentiation of liver stem cells are regulated by various cytokines which may play an important role when liver is damaged seriously.
Animals ; Cell Differentiation ; drug effects ; Cell Division ; drug effects ; Cytokines ; pharmacology ; Epidermal Growth Factor ; pharmacology ; Hepatocyte Growth Factor ; pharmacology ; Insulin ; pharmacology ; Liver ; cytology ; Rats ; Rats, Inbred F344 ; Stem Cells ; cytology ; drug effects ; Transforming Growth Factor beta ; pharmacology
10.An experiment study and clinical observation of the influence of free-skin-grafted scrotal avulsion injuries on spermatogenesis.
Guang-feng SUN ; Da-li WANG ; Zai-rong WEI ; Zhi-jun LUO ; Kai-yu NIE ; Wen-hu JIN
Chinese Journal of Surgery 2012;50(1):45-48
OBJECTIVETo explore the influence of Free-skin-grafted penoscrotal avulsion injuries on spermatogenesis.
METHODSForty-two male New Zealand albino rabbits during child-bearing period were divided into the experimental group (n = 24) and the control group (n = 18) using random digits table, and 24 female rabbits with reproductive history were used for mating experiment. The experimental group animal's scrotum skin were excised, and the split skin from abdominal region was used to repair the skin defect of scrotum. The control group did not any processing. Six rabbits were randomly chosen respectively in control group and on the 3rd and 8th weekend after the model was successfully established in experimental group. The testicular surface temperature was measured in the eighteen rabbits using the method of burying thermometer, then the testicular biopsy were performed for hematoxylin-eosin (HE) staining. On the 8(th) weekend after the model was successfully established in experimental group, matched-pair feed was performed in the other 12 rabbits respectively in experimental group and in control group. Observation of corresponding mother rabbit fertility. Three patients of penoscrotal avulsion injuries were treated using split skin grafts, and the information of sex life and the quality of sperm were obtained by follow up.
RESULTSThe testicular surface temperature was similar on the 3rd and 8th weekend after the model was successfully established in experimental group [(36.15 ± 0.24)°C, (36.77 ± 0.42)°C] with that of the control group. Testis tissue (HE) staining showed the tier of spermatogenic cells was rule arrangement and lot of mature sperms were found in the convoluted seminiferous tubules in control group. The tier of spermatogenic cells was diminished and disposed derangement, the spermatozoa were not seen on the 3(th) weekend of the experiment group. The tier of spermatogenic cells was increased and some spermatozoa were seen on the 8th weekend of the experiment group. Male and female matched-pair feed showed the experimental group conception rate 8/12, and 4.1 ± 3.2 rabbit babies were born averagely, while that of was 12/12 and 6.0 ± 1.3 in control group (P > 0.05). The skin grafts there were some contracture in early stage (1 - 2 months) when the skin grafts applied to repair the avulsing scrotum in three patients. But the skin grafts became loose with downward sagging and there were the good cosmetic result in one year, and without any contracture. The sperm quality was normal after the skin grafts applied to repair the avulsing scrotum in the late stage.
CONCLUSIONSThe skin grafting is little arrest the testicle spermatogenesis in the three methods (skin flap reconstruction scrotum, testicle buried, split skin grafting) that have usually been used to repair scrotum skin lose. For a young male, the best treatment for penoscrotal avulsion injuries is free skin grafting, while skin flaps are not recommended for reconstructing the scrotum.
Adult ; Animals ; Female ; Follow-Up Studies ; Humans ; Male ; Rabbits ; Scrotum ; injuries ; surgery ; Skin Transplantation ; methods ; Spermatogenesis ; Surgical Flaps