A new wood-degrading fungus Monodictys asperospera(Cooke & Massee) Ellis with a high level of laccase production was chosen to study.This laccase was purified by ammonium sulfate precipitation,DEAE-cellulose and sephacryl S-300.Purification of about 8.1 fold was achieved with an overall yield of 5.7%.Its molecular weight was estimated to be about 77 kD.The optimum temperature and pH of the lac-case activity were 55?C and 6.0,respectively.Kinetic studies of the laccase showed that the Km and the Vmax for using syringaldazine as substrate was 0.163 mmol/L and 0.194 mmol/(L.min),respectively.The carbo-hydrate content was 18.14%.In addition,it was found that laccase activity was significantly inhibited by Cu2+.

PCR method was used to introduce the code sequence of Factor Xa cleavage site to the 5′ end of cecropin CMIV mutant gene X, then the gene was cloned into the expression vector pGEX-KG, and was highly expressed in E. coli BL21 by IPTG induction. The fusion protein was purified by affinity-chromatography and was cleaved by Factor Xa. Cecropin X with antibacterial activity was obtained after purified by ion-exchange chromatography.

OBJECTIVETo investigate the effects of adenovirus-mediated human tissue kallikrein (Ad-hKLK1) gene transfer on platelet-derived growth factor-BB (PDGF-BB)-induced migration of vascular smooth muscle cells from spontaneously hypertensive rats (VSMC(SHR)).

METHODSA bicistronic recombinant adenovirus vector (Ad-hKLK1) carrying the target hKLK1 gene and the reporter gene EGFP was constructed. VSMCs isolated from the thoracic aorta of male SHR were passaged, and the quiescent VSMC(SHR) in passages 3-6 seeded in 6-well plates were treated with Ad-hKLK1 and control virus. Human PDGF-BB or icatibant Hoe140, a BK B2 antagonistat, was used as the chemoattractant and placed in the bottom chamber of the Boyden chamber. The mRNA expressions of bradykinin B1 receptor and B2 receptor were detected by RT-PCR in VSMC(SHR).

RESULTShKLK1 gene transfer significantly inhibited PDGF-BB-induced migration of VSMC(SHR), with the peak inhibition rate of 34.6% (P<0.001). PDGF-BB significantly increased the mRNA expression of B2 receptor but not B1 receptor in VSMC(SHR).

CONCLUSIONShKLK1 gene transfer can inhibit the migration of VSMC(SHR) induced by PDGF-BB, and the inhibitory effects may be not mediated by bradykinin B2 receptor.

Adenoviridae ; genetics ; metabolism ; Animals ; Aorta, Thoracic ; cytology ; Cell Movement ; drug effects ; genetics ; Cells, Cultured ; Gene Transfer Techniques ; Humans ; Hypertension ; pathology ; Male ; Muscle, Smooth, Vascular ; cytology ; Platelet-Derived Growth Factor ; pharmacology ; Proto-Oncogene Proteins c-sis ; Rats ; Rats, Inbred SHR ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Tissue Kallikreins ; biosynthesis ; genetics

OBJECTIVE: To screen the differentially expressed gene profile from the smooth muscles in the fundus uterus at the active stage of labor, and to provide candidate genes for picking out the drug targets related to uterine contraction. METHODS: Differentially expressed genes of uterine smooth muscles in the corpus from pro and post spontaneous parturition and those induced by oxytocin,as well as those from the corpus and the lower portion spontaneous parturition,were scanned respectively by human full-length genetic cDNA microarray with 8064 probe sets. Semi-quantitative RT-PCR was applied to testify the expression of voltage dependent calcium channel-L subtype (CACNA). The differentially expressed genes in the structure and function of the drug targets were picked out by bio-informatics to serve as candidate drug targets related to uterine contraction. RESULTS: The expressions of 29 genes were upregulated in fundus smooth muscles from the pro and post natural parturition, the pro and post inductive parturition of oxytocin, and the natural parturition. The expression of CACNA gene in RT-PCR was in accordance with that in the microarray. Among the 29 genes, neuromedin B receptor (NMBR) gene and neuropeptide Y (NPY) gene were the genes which not only had the targets of uterine contracted medicine, but also could contract the uterine. The differential expression ratios of NMBR in the above 3 types of uterine myometrium were 6.9,11.3, and 9.0, respectively while those of NPY were 6.0,29.8, and 2.9 respectively. CONCLUSION NMBR, whose expression in the uterine smooth muscles is always up-regulated at different parturition conditions, is likely to be an ideal candidate target of uterotonic drugs.
Calcium Channels ; genetics ; Drug Evaluation, Preclinical ; Female ; Gene Expression ; Gene Expression Profiling ; Humans ; Myometrium ; drug effects ; Neuropeptide Y ; genetics ; Oligonucleotide Array Sequence Analysis ; Pregnancy ; Receptors, Bombesin ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Uterine Contraction ; drug effects

Objective Tissue kallikrein cleaves kininogen substrate to produce vasoactive kinin peptides that have been implicated in the proliferation of vascular smooth muscle cells. We investigated the effects of adenovirus-mediated human tissue kallikerin(Ad-hKLK1)gene delivery on the proliferation of vascular smooth muscle cells of SHR(VSMCsSHR)induced by platelet derived growth factor-BB(PDGF-BB). Methods Primary VSMCsSHR were isolated and cultured from thoracic aorta of male SHR. The VSMCsSHR proliferation induced by PDGF-BB was accessed by cell counting and methyl thiazolyl tetrazoliuin(MTT). Western blot was used to determine the protein expression of hKLK1, the cycle-independent kinase inhibitors p27Kip1 and p21Cip1 . The mRNA expressions of bradykinin B1 receptor and B2 receptor were detected by RT-PCR in VSMCsSHR. Results Proliferation of VSMCsSHR induced by PDGF-BB was significantly inhibited post transfection of Ad-hKLK1(20 - 100 MOI)in a MOI-dependent manner. The peak inhibition titer of Ad-hKLK1 was 100 MOI with peak inhibition rate of 39.3%(cell counting, n = 3,P ＜0.01), 30.2%(MTT, n-3 ,P ＜ 0.01)and 36.4%(peak stunning rate of cell-cycle in phase G0/G1). The inhibitory effects of proliferation and cell-cycle caused by hKLK1 gene delivery could be abolished by Hoe140, a bradykinin B2 receptor antagonist. The protein expression of p27Kip1 and p21Cip1 increased significantly after the hKLK1 gene delivery, whereas Hoe140 nearly completely blocked these effects(n = 3,P ＜0.001 ,respectively). PDGF-BB also significantly upregulated the mRNA expression of B2 receptor but not B1 receptor in VSMCsSHR. Conclusion The hKLK1 gene delivery could inhibit PDGF-BB induced proliferation in VSMCsSHR through Bradykinin B2 receptor and up-regulate expression of p27Kip1 and p21Cip1.

Objective To investigate the effects of atorvastatin on advanced glycation end products (AGE) induced monocyte chemoattractant protein-1(MCP-1) expression in human umbilical vein endothelial cells(HUVECs)and whether this effect could be linked to peroxisome proliferator-activated receptor-γ (PPAR-γ) and nuclear factor-κB(NF-κB).Methods Grouping: (1)Blank control group;(2)BSA group;(3)AGE group:cells were incubated with different concentrations of AGE(10-4,10-3, 10-2 and 10-1g/L)for 24 hours; (4)AGE+Atorvastatin group: cells were incubated with different concentrations of atorvastatin(0.1,1,10 μmol/L)for 1 hour,then incubated with AGE (10-1 g/L) for 24 hours; (5)PPAR-γ agonist(15 d-PGJ2)group: cells were incubated with 15 d-PGJ2(10 μmol/L)for 1 hour,then incubated with AGE (10-1g/L) for 24 hours;(6)PPAR-γ inhibitor(GW9662)group:cells were incubated with GW9662(5000 nmol/L)for 1 hour,then incubated with atorvastatin (1 μmol/L)and AGE (10-1g/L) for 24 hours. Collagenase was used to isolate the endothelial cell from human umbilical vein;RT-PCR was performed to examine the mRNA expression of MCP-1 and PPAR-γ;Western blot was performed to detect NF-κB p65 protein.Results (1) The expression of MCP-1 mRNA was increased in proportion with increasing concentrations of AGEs which could be blocked by atorvastatin in a dose-dependent manner. (2) AGE(10-1g/L)significantly downregulated the expression of PPAR-γ mRNA(0.22±0.08 vs. 0.69±0.09, P<0.01) while upregulated the expression of phospho-NF-κB p65 protein (0.78±0.06 vs. 0.31±0.01,P<0.01) and nonphospho-NF-κB p65 protein (1.61±0.16 vs. 0.59±0.14,P<0.01) comparaed with the control group which could be significantly attenuated by atorvastatin. (3) PPAR-γ agonist decreased the expression of phospho-NF-κB p65 protein (0.21±0.01 vs. 0.78±0.06, P<0.01),nonphospho-NF-κB p65 protein (0.67±0.14 vs. 1.61±0.16,P<0.01)and MCP-1 mRNA (0.17±0.02 vs. 0.93±0.12, P<0.01)compared with AGE(10-1g/L)group. (4) PPAR-γ inhibitor antagonized the effect of atorvastatin on the expression of phospho-NF-κB p65 protein, nonphospho-NF-κB p65 protein and MCP-1 mRNA stimulated by AGE in HUVECs(P<0.01).Conclusion The anti-inflammatory properties of atorvastatin in AGE stimulated HUVECs may partly be attributed to the effect on upregulation of PPAR-γ and downregulation of NF-κB signaling pathway.

Adult ; Female ; Hepatitis B, Chronic ; complications ; physiopathology ; Humans ; Liver ; physiopathology ; Liver Function Tests ; Male ; Retrospective Studies ; Severe Acute Respiratory Syndrome ; complications ; physiopathology

The treatment strategy for blast injuries is closely linked to the clinical outcome of blast injury casualties. However, the application of military surgery experience to blast injuries caused by production safety accidents is relatively uncommon. In this study, the authors present 2 cases of blast injuries caused by one gas explosion, both cases involved individuals of the same age and gender and experienced similar degree of injury. The authors highlight the importance of using a military surgery treatment strategy, specifically emphasizing the need to understand the concept of damage control and disposal. It is recommended that relevant training in this area should be strengthened to improve the clinical treatment of such injuries. This study provides a valuable reference for healthcare professionals dealing with blast injuries.

Objective To identification and analysis Aichi virus from diarrhea and normal children in Lanzhou, and discuss the relationship between Aichi virus and Infant Diarrhea. Methods According to the literature published data, Using RT-PCR method to amplified Aichi virus 3CD fragment and the positive products were sequenced and determined, and made the alignment analysis between the nucleotide sequences of the amplified fragment with the known sequence of this virus. Results There was 1 case detection of Aichi virus in the 46 hospitalized children with diarrhea and 299 children with diarrhea out-patients specifically, Overall detection rate was 0.06% , and there was no Aichi virus was detected in normal control children. 2 viral 3CD gene and the known reference strains of nucleotide sequences were 97% , while phylogenetic analysis showed that genotype of 2 viral belongs to the B. Conclusions There existed B Genotype of Aichi virus in China, and more research is needed to clarified the etiology and epidemiology of Aichi virus characteristics.

Objective To investigate the effects of human tissue kallikrein 1 (Ad-hKLK1) gene delivery on the neointima formation in carotid arteries of spontaneously hypertensive rats (SHRs). Methods Carotid artery restenosis was induced in male SHR rats by balloon-injury. Rats were randomly assigned into 4 groups: Sham-operated (n = 6); Angioplasty (phosphate buffered solution 50 μl, n = 8); Vector virus (control virus, 1×10~9 IU in 50 μl, n =8) and Ad-hKLK1 (Ad-hKLK1, 1×10~9 IU in 50 μl, n =8). Rats were sacrificed 4 weeks later. The wall-to-lumen area ratio and intima/media ratio in carotid artery were assessed by image analysis in HE stained sections. The mRNA bradykinin receptor(B1R and B2R) expressions were detected by RT-PCR. The protein expression of the cycle-independent kinase inhibitors p27Kip1 and p21Cip1 were determined by Western blot analysis. Results Wall-to-lumen area ratio reduced 35.6% and intima/media ratio reduced 38.8% in Ad-hKLK1 treated SHRs compared to angioplasty group (all P <0.001). The expression of p27Kip1 and p21Cip1 increased significantly in Ad-hKLK1 treated SHRs compared with angioplasty rats (all P <0.001). The mRNA expression of B2R was significantly upregulated in angioplasty rats compared with sham-operated rats (P <0.05) while mRNA expression of B1R was similar between the 2 groups. Conclusion hKLK1 gene delivery may effectively reduce neointimal formation via downregulating bradykinin B2R and up-regulating the expressions of p27Kip1, p21Cip1 signaling pathways in carotid arteries of SHRs after balloon injury.