Fibrolase is a non-hemorrhagic zinc metalloproteinase isolated from southern copperhead snake venom (Agkistrodon contortrix contortrix) and is capable of degrading fibrin clots resulting from purified fibrinogen or from blood plasma. Alfimeprase, a truncated form of fibrolase, as a clinical agent was successfully completed PhaseII clinical trials.The cDNA of alfimeprase was amplified by recursive PCR, digested with BamHI and HindII, and cloned into pET43.1a, pMALp2X and pMALc2X vectors to generate fusions with NusA, MBP and sMBP(with signal peptide), respectively. Nus/alfimeprase was expressed in soluble form by co-expressing with chaperone FkpA and inducing with1mmol/L IPTG. The fusion protein accounted for about 25 % of total protein following cell lysis. Alfimeprase was successfully purifiesd by Ni-NTA affinity chromatography and cleaved by enterokinase. The results demonstrate the fibrinolytic activity of recombinant alfimeprase using fibrin plate assays and fibrinogen hydrolysis.

Alfimeprase(ALF)is a recombinantly modified variant of non-hemorrhagic zinc metalloproteinase fibrolase.The target gene alf was obtained from the clone vector p43-alf and cloned into the Pichia pastoris expression vector pPICZ? A.Through high efficiency transformation and Zeocin selection,the recombinant strains of pPICZ?A-alf /GS115 were isolated.In order to achieve a high level expression of recombinant Alfimeprase(rALF),optimization of pH value,methanol daily addition concentration,cell density and methanol induction time points were carried out,and the production of rALF reached up to 425 mg/L.By His?Bind chromatography,the purity of secreted rALF was as high as 95 %.SDS-PAGE and Western blot analysis show that rALF has a molecular weight of about 24 kDa and is bound specifically to anti-His?tag monoclonal antibody.Activity identification results of the modified fibrin plate method demonstrate that the secreted rALF has high fibrinolytic activity.Thus sets up an optimized expression system for ALF,which will play an important role in its further studies and industrial production.

OBJECTIVETo study the pathological features of uveal melanoma and to evaluate their influence on patients' prognosis.

METHODSParaffin embedded uveal melanoma tissues of 115 cases were examined using routine pathologic methods. Three histological types were classified according to the modified Callender system and patients were followed clinically. The data were done regression and survival analysis by SPSS statistic soft.

RESULTSThe patient with epithelial cell type, mixed type, and spindle cell type uveal melanoma have different life times, the average life time is 35.6 +/- 21.5 months, 63.7 +/- 37.0 months, 69.5 +/- 36.5 months in turn, patients with epithelial uveal melanoma had shorter survival time than other two types. The survival time was negatively related to the largest diameter of contact area with the sclera, the largest height and the depth of tumor invasion to the sclera.

CONCLUSIONSEpithelial uveal melanoma is more malignant than the other two types. Histological classification of this tumor combined with other pathologic features can indicate the patient's prognosis.

Eye Neoplasms ; mortality ; pathology ; Humans ; Melanoma ; mortality ; pathology ; Prognosis ; Survival Rate ; Uveal Neoplasms ; mortality ; pathology

OBJECTIVETo screen the FANCA gene mutation and explore the FANCA protein function in Fanconi anemia (FA) patients.

METHODSFANCA protein expression and its interaction with FANCF were analyzed using Western blot and immunoprecipitation in 3 cases of FA-A. Genomic DNA was used for MLPA analysis followed by sequencing.

RESULTSFANCA protein was undetectable and FANCA and FANCF protein interaction was impaired in these 3 cases of FA-A. Each case of FA-A contained biallelic pathogenic mutations in FANCA gene.

CONCLUSIONSNo functional FANCA protein was found in these 3 cases of FA-A, and intragenic deletion, frame shift and splice site mutation were the major pathogenic mutations found in FANCA gene.

Cell Line ; DNA Mutational Analysis ; Fanconi Anemia ; genetics ; metabolism ; Fanconi Anemia Complementation Group A Protein ; genetics ; metabolism ; Humans ; Mutation

OBJECTIVETo study the effect of interferon-gamma (IFN-gamma) on the proliferation of mesangial cells (MsC) and transforming growth factor (TGF)-beta/Smad signal pathway, the mRNA and protein expression of matrix metalloproteinase-2 (MMP-2) and tissue inhibitors of matrix metalloproteinase-2 (TIMP-2), and to provide an experimental basis for IFN-gamma treatment of renal fibrosis.

METHODSCultured MsC were treated with IFN-gamma at different concentrations and the proliferation of MsC was examined by MTT. Protein and RNA samples were extracted from MsC at 0, 0.5, 1, 2, 4, 6, 12, 24 h after treated by 100 IU/ml IFN-gamma. The mRNA and protein expression of Smad3, Smad7, MMP-2 and TIMP-2 were analyzed by real-time RT-PCR and Western blot, respectively.

RESULTSThe expression of Smad7 mRNA and protein were promptly elevated at 0.5 hour after the IFN-gamma treatment and lasted for 6 hours, but the proliferation of MsC was not altered. The elevated expression of Smad3, MMP2 mRNA and proteins persisted after 6 hours, whereas the expression of TIMP-2 mRNA and protein decreased.

CONCLUSIONThe therapeutic effect of IFN-gamma of renal fibrosis may be mediated by TGF-beta/smads signal pathway through up-regulation of MMP-2 expression, coupled with down-regulation of TIMP-2 expression.

Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Interferon-gamma ; pharmacology ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; Mesangial Cells ; cytology ; metabolism ; RNA, Messenger ; metabolism ; Rats ; Recombinant Proteins ; Signal Transduction ; Smad3 Protein ; genetics ; metabolism ; Smad7 Protein ; genetics ; metabolism ; Tissue Inhibitor of Metalloproteinase-2 ; genetics ; metabolism ; Transforming Growth Factor beta ; metabolism

Esophageal and Gastric Varices ; drug therapy ; etiology ; Female ; Gastrointestinal Agents ; therapeutic use ; Gastrointestinal Hemorrhage ; drug therapy ; etiology ; Glucagon ; blood ; Humans ; Liver Cirrhosis ; complications ; Male ; Middle Aged ; Nitric Oxide ; blood ; Octreotide ; therapeutic use ; Somatostatin ; analogs & derivatives

BACKGROUNDThe high incidence of neuropsychologic deficits after cardiac surgery, including cognitive dysfunction and mood status, has significantly influenced the prognosis, outcome of treatment and long-term quality of life of patients. With a circadian secretion pattern, melatonin and cortisol are capable of modulating the human physiological processes and neuropsychological status, whereas disorder of their secretion pattern may lead to many diseases. However, it is unclear whether neuroendocrine variations are related to the neuropsychologic status in patients undergoing coronary artery bypass grafting (CABG).

METHODSForty male patients scheduled for CABG with hypothermic cardiopulmonary bypass (CPB) (n = 20) or off-pump coronary artery bypass (OPCAB) (n = 20) were studied. Blood samples were taken intraoperatively at specific time-points and every 3 hours within the first postoperative 24 hours to determine plasma concentrations of melatonin and cortisol. A neuropsychologic test battery including depression and anxiety was administered preoperatively and 7 to 10 days postoperatively. Statistical methods included the nonparametric analysis, multiple linear regression and cosinor analysis.

RESULTSThe patients in the CPB group exhibited more severe neuropsychologic deficits and more anxious than those in the OPCAB group after surgery. In both groups, patients were more depressed postoperatively than preoperatively and recovered 3 months after surgery. Depression and anxiety were correlated with some factors of cognitive dysfunctions. In the postoperative 24 hours, 2 patients in the CPB group, and 6 patients in the OPCAB group showed a circadian rhythm of melatonin secretion. As for cortisol secretion, there were 3 patients in the CPB group and 7 in the OPCAB group respectively. Parameters of circadian rhythm of melatonin in the CPB group and those of secretion rhythm of cortisol in both groups were correlated with depression and some neuropsychologic tests.

CONCLUSIONSThe incidence of neuropsychological deficits was higher in patients receiving CABG with CPB than in those without CPB. The status of mood may contribute to the perioperative cognitive dysfunctions. The disordered circadian rhythm of melatonin secretion in patients undergoing CABG with CPB and the disordered cortisol secretion may correlate directly or indirectly through mood with neuropsychological deficits.

Cardiopulmonary Bypass ; adverse effects ; Circadian Rhythm ; physiology ; Cognition Disorders ; etiology ; Coronary Artery Bypass ; adverse effects ; Humans ; Hydrocortisone ; blood ; secretion ; Male ; Melatonin ; blood ; secretion ; Middle Aged ; Neuropsychological Tests ; Postoperative Complications ; etiology

BACKGROUNDTherapeutic hypercapnia (TH) has been demonstrated to protect several organs ischemia-reperfusion injury. The study aimed to investigate the effects of therapeutic hypercapnia on hepatic ischemia-reperfusion injury (HIRI).

METHODSThirty adult male Wistar rats weighing (250+/-20) g were randomized into 3 groups (n=10 in each), group C (control group), group A (hypercapnia group) and group B (CO2 preconditioning group). A segmental ischemia of the liver was induced by interrupting the blood vessels including the bile duct to the median and left lateral lobes for 60 minutes and all the animals were sacrificed after 240 minutes observation period of reperfusion. Mean arterial pressure (MAP) and the blood gases were measured before ischemia (baseline) and at 30, 60, 120, 180 and 240 minutes after reperfusion. Arterial blood samples were obtained for determination of serum levels of TNF-alpha, IL-10, serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT). The histopathology of liver tissues was evaluated by light microscopy. The NF-kappaB expression and apoptotic hepatocytes were respectively determined by immunohistochemistry and TUNEL assay.

RESULTSThe serum levels of liver enzymes and TNF-alpha were significantly decreased while the IL-10 level was significantly increased in groups A and B than in group C (P<0.05), and group B surpassed group A (P<0.05). The histopathological scores, the NF-kappaB immunohistochemical score (IHS) and apoptotic index were significantly lower in groups A and B than in group C (P<0.05), and the decrease in group B was more obvious than in group A (P<0.05).

CONCLUSIONTherapeutic hypercapnia attenuates ischemia-reperfusion injury to the liver. Moreover, the effects of CO2 preconditioning are outstandingly notable.

Animals ; Apoptosis ; drug effects ; Carbon Dioxide ; therapeutic use ; Hemodynamics ; drug effects ; Immunohistochemistry ; In Situ Nick-End Labeling ; Inflammation ; drug therapy ; metabolism ; Interleukin-10 ; blood ; Liver ; drug effects ; metabolism ; pathology ; Male ; NF-kappa B ; metabolism ; Random Allocation ; Rats ; Rats, Wistar ; Reperfusion Injury ; blood ; drug therapy ; metabolism ; Tumor Necrosis Factor-alpha ; blood

OBJECTIVETo compare the efficacy and safety of remifentanil with fentanyl used for intraoperative anesthesia.

METHODSFifty-four patients undergoing modified radical mastectomy or total hysterectomy were randomly assigned to remifentanil group or fentanyl group with 27 cases in each group. Anesthesia was induced with propofol (2 mg/kg) and either remifentanil (2 micrograms/kg) or fentanyl (2.5 micrograms/kg), and was maintained with inhalation of nitrous oxide in oxygen (2:1) and a continuous infusion of either remifentanil (0.2 microgram.kg-1.min-1) or fentanyl (0.03 microgram.kg-1.min-1). Depth of anesthesia, hemodynamic changes, recovery profile of anesthesia, postoperative analgesia and adverse reactions were observed.

RESULTSThe number of patients exhibited light depth of anesthesia during tracheal intubation and maintenance in the remifentanil group was significantly fewer than that in the fentanyl group (P < 0.05). Hemodynamic changes during intubation, skin incision, maintenance of anesthesia and extubation in the remifentanil group were significantly smaller than those in the fentanyl group (P < 0.05, P < 0.01). The time to opening eyes on command and the time for extubation after surgery were comparable between the two groups. More patients in the remifentanil group required bolus injection of morphine for postoperative pain relief than those in the fentanyl group (P < 0.05). There was no significant difference between the two groups in the aspect of adverse reactions.

CONCLUSIONThe anesthetic and analgesic effects of remifentanil are more potent than those of fentanyl. Remifentanil can offer superior intraoperative hemodynamic stability compared with fentanyl without compromising recovery from anesthesia.

Adolescent ; Adult ; Anesthetics, Intravenous ; therapeutic use ; Breast Neoplasms ; physiopathology ; surgery ; Female ; Fentanyl ; therapeutic use ; Hemodynamics ; drug effects ; Humans ; Hysterectomy ; Mastectomy, Modified Radical ; Middle Aged ; Pain, Postoperative ; prevention & control ; Piperidines ; therapeutic use

OBJECTIVETo study the anti-endotoxin activity and mechanism of F022 from Radix Isatidis.

METHODThe production of TNF-alpha and IL-6 of murine peritoneal macrophages stimulated by LPS was measured by ELISA. The temperature in rabbits was tested after i.v. administration of LPS. The lethality of BCG-primed mice was induced by LPS.

RESULTIf F022 was added to macrophages culture simultaneously with LPS or 1 h before addition of LPS, production of TNF-alpha and IL-6 by macrophages was remarkably inhibited in vitro. F022 inhibited the fever induced by LPS in rabbits and protected BCG-primed mice from LPS induced lethality if given before administration of LPS.

CONCLUSIONThe anti-endotoxin effect of F022 may inhibit LPS binding to its receptor, and it may be a LPS receptor antagonist.

Animals ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Female ; Fever ; chemically induced ; drug therapy ; Interleukin-6 ; secretion ; Isatis ; chemistry ; Lipopolysaccharides ; antagonists & inhibitors ; Macrophages, Peritoneal ; secretion ; Male ; Mice ; Mice, Inbred C57BL ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry ; Rabbits ; Tumor Necrosis Factor-alpha ; secretion