Objective The present study was designed to investigate the efficiency of simvastatin therapy for experimental pul‐monary hypertension (PH) in rat ,and the effects on the number and function of circulating endothelial progenitor cells (EPC) . Methods Twenty four Sprague Dawley rats were divided into 3 groups randomly :model group ,treatment group and control group , 8 rats in each group .Rats were treated with a single subcutaneous injection of monocrotaline to induce PH (PBS used as control) . The rats in the experimental group were administrated with simvastatin 3 days following subcutaneous injection of monocrotaline .In the 21st day ,the number of circulating EPC ,the right ventricle systolic pressure of rat ,pulmonary vascular structural changes and the quantity of cultured EPC were measured .At the same time ,EPC in each group were cultured in vitro ,then the ability of prolif‐eration and function were analyzed and compared .Results The number of circulating EPC in model group was decreased signifi‐cantly compared to both control and model groups (P< 0 .01) .Compared with model group ,simvastatin treatment markedly de‐creased the RVSP and the ratio of media thickness to eternal diameter (P<0 .01) ,but the ratio of vessel area to total arterial area (VA/TAA) was definitively increased(P<0 .01) .After 7 days of culture in vitro ,both the output of EPC and the ability of prolif‐eration ,conglutination and migration of EPC in treatment group were up -regulated compared with those in model group (P<0 .01) .Conclusion This study confirmed that simvastatin effectively treat experimental PH through improving quantity and func‐tion of circulating EPC .

Objective To examine neuroinflammation,oxidative damage and neuron loss in the contralateral parie-tal lobecortex of TBI model mice, and to investigate effects of berberine chloride on such secondary damage.Methods TBI model was established by a weight-drop hitting device and mice in berberine group were administered intragastrically with berberine chloride (50mg/kg.day) for 21 days.Immunofluorescence staining was used to assess activity of microglia and astrocyte.Immunohistochemistry was used to assess DNA oxidative damage, neuron loss and expression of COX-2 and iN-OS.Results Activation of microglia and astrocyte, expressions of COX-2 and iNOS and DNA oxidative damage were ob-viously increased by TBI,(19.82 ±1.88)and(16.96 ±1.69)、(13.79 ±4.32)and(8.67 ±0.96)、(27.86 ±5.38) and (16.00 ±7.59)、(31.92 ±6.57)and(24.79 ±2.78)respectively (P<0.01 or P<0.05).Activation of microglia and ex-pressions of COX-2 and iNOS were significantly suppressed by berberine ,(15.49 ±1.88)and(19.82 ±1.88)、(16.83 ± 7.89)and(27.86 ±5.38)、(26.25 ±2.41)and(31.92 ±6.57) respectively(P<0.01 or P<0.05).There was no differ-ence in neuron loss among three groups, (49.05 ±4.38),(48.56 ±3.56)and (47.75 ±4.14) respectively (P>0.05). Conclusions TBI can cause neuroinflammation and oxidative damage but not neuron loss in the contralateral parietal lobe cortex.Berberine chloride can significantly suppress neuroinflammtion in the contralateral parietal lobe cortex after TBI.

Objective To study the effects of granisetron hydrochloride on vasovagal syncope(VVS) in rabbits.Methods Twenty-four healthy New Zealand rabbits were divided stochastically into control group and intervention group,12 in each group. The control group was injected intravenously with normal saline. The intervention group was injected intravenously with granisetron hydrchloiride.Rabbit VVS models were established,each was taken at 4 points in time in the bloodletting process:T1,T2,T3,T4,to compare the bloodletting time,the concentration of 5-hydroxytryptamine(5-HT) in T2,T3,T4 and the total blood volume between the groups,and monitor the heart rate, blood pressure during the entire process.Results 1.The time of intervention group in T2,T3,T4 was longer than the time of control group obviously(P

Interleukin-1 receptor antagonist (IL-1ra), a member of IL-1 family, is a naturally occurring IL-1 inhibitor as "receptor antagonist", which blocks biological responses mediated by IL-1. Recombinant human IL-1ra (rhIL-1ra, Kineret) was introduced in clinical trials involving patients with RA. Between 2001 to approximately 2002, rhIL-1 ra was approved by the US Food and Drug Administration and the European Agency for the Evaluation of Medicine Procedure. Unfortunately, 10,000 to 100,000-fold excess amounts of IL-1ra are needed to relieve disease because minimal IL-1 can induce complete biological responses, and the dosage of 100 to approximately 150mg/day in a RA patient is so big that it greatly influence patients' physical, psychological and economical situation. In this study, IL-1ra mutants were established by site-specific mutagenesis to improve its stability. The sites of mutagenesis included R6 K7-AA,R93 K94-AA and K97 R98-AA. IL-1ra and its mutants were expressed in E. coli BL21 (DE3) using pTIG-Trx expressing system with the induction of IPTG. The recombinant proteins were purified by Ni2+ chelate chromatography and Sephadex G75 gel filtration chromatography. The activity of mutants is as high as IL-1ra. We characterized the pharmacokinetic profile of IL-1ra and its mutants. The third mutant's half life is 2.26 times than wt IL-1ra. The study has provided some approaches and experience for further research to improve the metabolism stability of IL-1ra.
Animals ; Escherichia coli ; genetics ; metabolism ; Female ; Humans ; Interleukin 1 Receptor Antagonist Protein ; biosynthesis ; genetics ; pharmacokinetics ; Mutagenesis, Site-Directed ; methods ; Mutant Proteins ; biosynthesis ; pharmacokinetics ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacokinetics

BACKGROUND: The association of genetic variation in the IRAK-1 gene with sepsis outcome has been proved. However, few studies have addressed the impact of the IRAK-4 gene variants on sepsis risk. This study aimed to determine whether the polymorphisms in the IRAK-4 gene are associated with susceptibility to and prognosis of severe sepsis in the Chinese Han ethnic population.METHODS: In this case-control study, 192 patients with severe sepsis hospitalized in the emergency department of Zhongshan Hospital from February 2006 to December 2009 and 192 healthy volunteers were enrolled. Exclusion criteria included metastatic tumors, autoimmune diseases, AIDS or treatment with immunosuppressive drugs. This study was approved by the ethical committee of Zhongshan Hospital, Fudan University. Sepsis patients were divided into a survival group (n=124) and a non-survival group (n=68) according to the 30-day mortality. Primer 3 software was used to design PCR and sequencing primers. Genomic DNA was extracted from peripheral blood mononuclear cells. Seven tagSNPs in IRAK-4 were selected according to the data of the Chinese Han population in Beijing from the Hapmap project and genotyped by direct sequencing. The chi-square test was used to evaluate the differences in genotype and allele frequencies between the two groups.RESULTS: The distributions of all tagSNPs were consistent with Hardy-Weinberg equilibrium. The allele and genotype frequencies of rs4251545 (G/A) were significantly different between the severe sepsis and healthy control groups (P=0.015, P=0.035, respectively). Carriers of the rs4251545A had a higher risk for severe sepsis compared with carriers of the rs4251545G (OR=1.69, 95％ CI: 1.10-2.58). The allele and genotype frequencies of all SNPs were not significantly different between the survival group and non-survival group.CONCLUSION: These findings indicate that the variants in IRAK-4 are significantly associated with susceptibility to severe sepsis in the Chinese Han ethnic population.

Calcitonin gene-related peptide (CGRP) play a key role in some physiological and pathological progresses. The latest studies indicate that CGRP might involve in some disease progress and has a close relation with wound healing. It is significant to further investigate and then apply it to clinical diagnosis and therapy as well as forensic pathology.
Animals ; Calcitonin Gene-Related Peptide/physiology* ; Forensic Medicine ; Humans ; Receptors, Calcitonin Gene-Related Peptide/physiology* ; Wound Healing

Human transferrin receptor (TfR) was isolated from homogenates of placental tissues by affinity chromatography on transferrin-Sepharose, and then used to screen human scFv against it from a fully-synthesized phage scFv library. After verifying the specificity, gene fragment of one of the selected scFv was inserted into the plasmid pET22b(+) and transformed into E. coli BL21(DE3) . Expression of scFv in transformant was induced with 0.5mmol/L IPTG. ELISA assay on HeLa cells showed that scFv protein could recognize and bind to TfR on the surface of HeLa cells. The scFv was purified by one-step affinity chromatography with Ni+ -NTA agarose, and injected into Kunming mouse via tail veins. This scFv was detected in brain tissues 1h later by capillary depletion method, which indicates that scFv protein can permeate through the blood brain barrier by mediation of the TfR receptor. Our works lay the foundation for the treatment of tumors and central nervous system diseases.
Animals ; Antibodies, Anti-Idiotypic ; genetics ; isolation & purification ; metabolism ; Escherichia coli ; genetics ; metabolism ; HeLa Cells ; Humans ; Immunoglobulin Fragments ; biosynthesis ; genetics ; immunology ; Immunoglobulin Variable Region ; biosynthesis ; genetics ; immunology ; Mice ; Receptors, Transferrin ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; genetics ; Transferrin ; metabolism

To compare the DAAO expression level in different Pichia pastoris host strains, the gene encoding DAAO from Trigonopsis variabilis was cloned into plasmid pPIC3.5k and then transformed into P. pastoris GS115 and KM71 respectively. The positive transformants PDK13 (MutS) and PD27 (Mut+) were obtained by PCR analysis. Their optimal and different expression conditions were investigated. To compare with PD27, PDK13 was determined to poss a slower consumption of methanol, a longer induction time, a lower oxygen request and apparently higher expression of DAAO. The highest expression levels were reached up to 2700, 2500 IU/L in shaking flask and 10140, 8463.5 IU/L in fermentor respectively. The over-expression of DAAO can meet its large demand for production of 7-ACA, alpha-keto acid and L-amino acid. In addition, the phenylpyruvate and L-phenylalanine were obtained by crude DAAO reacting with DL-phenylalanine at 37 degrees C for 3h.
D-Amino-Acid Oxidase ; genetics ; Fermentation ; Methanol ; metabolism ; Phenylalanine ; metabolism ; Pichia ; genetics ; Polymerase Chain Reaction

BACKGROUNDInhibition of tumor growth by endostatin has been shown to be an effective strategy in cancer therapy in mice. However, its widespread application has been hampered by difficulties in a large-scale production of the recombinant endostatin protein, rapid loss bioactivity of the protein, and the cumbersome daily administration. These limitations could be resolved by in vivo delivery and expression of the endostatin gene. In this study, we observed the effect and advantage of endostatin gene therapy mediated by a recombinant adenoviral vector (Ad/hEndo) on the growth of hepatocellular carcinoma BEL-7402 xenografted tumors, comparison with recombinant endostatin protein.

METHODSHepatocellular carcinoma BEL-7402 cells were inoculated subcutaneously in the flank of Balb/c nude mice. Nine days after tumor cell inoculation, animals were given a cycle of four courses of intra-tumoral injections of Ad/hEndo of 5 x 10(8) pfu (low-dose group) and 1 x 10(9) pfu (high-dose group) at intervals of six days, respectively. Recombinant human endostatin protein (rhEndo) was administrated daily subcutaneously at a dose of 10 mg.kg(-1).d(-1) at a site nearby the tumor for ten days. The expression of endostatin mRNA in tumor tissue was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) after Ad/hEndo injection. Dynamic changes of concentration of endostatin protein in tumor tissue were quantitated by enzyme-linked immunosorbent assay (ELISA).

RESULTSAfter 4 courses of treatment, the tumor growth rates of high-dose treated group with 1 x 10(9) pfu of Ad/hEndo were inhibited by 42.26% compared with the Ad/LacZ control group (P = 0.001) and by 46.26% compared with the NIH buffer control group (P = 0.003), respectively. However, in this study, Ad/hEndo at low dose of 5 x 10(8) pfu failed to demonstrate significant inhibition of tumor growth, compared with control groups. After daily administration of recombinant human endostatin protein (rhEndo) for 9 days, the ratio of T/C (rhEndo group versus PBS group) was less than 47%. However, two days after rhEndo treatment ceased, the ratio of T/C was more than 50%. The peak of expression of endostatin mRNA in tumor tissue was at 2 or 3 days after administration intratumorally with Ad/hEndo of 1 x 10(9) pfu and gradually dropped undetectable by day 7. Dynamic analysis of endostatin concentration in tumor tissue showed that the highest level of mRNA is up at the third day after injection, and dropped to basal level three weeks later.

CONCLUSIONSEndostatin gene therapy mediated by a recombinant adenoviral vector had significantly inhibited the growth of hepatocellular carcinoma BEL-7402 xenografted tumors at a high dose of 1 x 10(9) pfu compared with other groups. The analysis of dynamic expression of endostatin in vivo indicated that Ad/hEndo had acquired a high-level, relatively long-term expression in vivo and bioactivity capability.

Adenoviridae ; genetics ; Animals ; Cell Line, Tumor ; Endostatins ; analysis ; genetics ; therapeutic use ; Genetic Therapy ; Humans ; Liver Neoplasms, Experimental ; therapy ; Mice ; Mice, Nude ; Neoplasm Transplantation ; RNA, Messenger ; analysis ; Recombinant Proteins ; therapeutic use ; Transplantation, Heterologous

OBJECTIVETo analyse the plasmid-mediated SHV type beta-lactamases-encoding genes sequence and to identify its subtype of multiple-drug-resistant acinetobacter baumannii isolated from Huzhou district, Zhejiang province, China.

METHODSSixty strains of acinetobacter baumannii were isolated from hospitalized patients between Jul, 2000 and Dec, 2002. Susceptibility of antimicrobial agents and confirmatory tests for Extended-spectrum beta-lactamases (ESBLs) were tested by microdilute method. SHV type beta-lactamases-encoding genes were tested by polymerase chain reaction (PCR). SHV sequences of acinetobacter baumannii HZ02 and HZ10 strains were detected by ABI automated sequencer and were analysed to compare with SHV genes that had been published in GenBank.

RESULTSEighteen (30.0%) strains of acinetobacter baumannii isolated between Jun, 2001 and Jan, 2002 were carrying SHV beta-lactamases resistant gene of plasmids. Detected SHV sequences of acinetobacter baumannii HZ02 strain and HZ10 strain had 825 and 833 nucleotides respectively and had the same gene sequence as the gene encoding SHV-12 subtype of ESBLs discovered in Switzerland.

CONCLUSIONSThirty percentage of the clinically isolated acinetobacter baumannii were carrying SHV type (extended-spectrum) beta-lactamases resistant gene of plasmids and causing an outbreak in hospital and was discovered to have carried the strains of SHV-12 subtype producing ESBLs gene in acinetobacter baumannii which was the first reported case in the world.

Acinetobacter Infections ; epidemiology ; microbiology ; Acinetobacter baumannii ; enzymology ; genetics ; isolation & purification ; Amino Acid Sequence ; Base Sequence ; China ; epidemiology ; DNA, Bacterial ; genetics ; isolation & purification ; Drug Resistance, Multiple ; Humans ; Molecular Epidemiology ; Molecular Sequence Data ; Sequence Analysis, DNA ; beta-Lactamases ; classification ; genetics