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

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

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

Animals ; Apoptosis ; drug effects ; Cell Line ; Cell Proliferation ; Curcuma ; Extracellular Matrix ; metabolism ; Hepatic Stellate Cells ; drug effects ; pathology ; Plant Extracts ; pharmacology ; Plant Oils ; pharmacology ; Rats

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

OBJECTIVETo investigate perioperative patterns of melatonin and cortisol secretion rhyme in patients undergoing coronary artery bypass grafting surgery.

METHODSEleven male patients scheduled for elective coronary artery bypass grafting surgery (CABG) under hypothermic cardiopulmonary bypass (CPB) were enrolled in the study. Anesthesia was induced and maintained with propofol (3 mg.kg-1.h-1) and supplemented with fentanyl (15 micrograms/kg). Blood samples were taken during surgery at specific time-points and every 3 h in the immediate postoperative period and postoperative day 2 and day 3. Plasma melatonin and cortisol levels were measured by radioimmunoassay and enzyme-linked immunosorbent assay respectively.

RESULTSDuring surgery, plasma melatonin levels were below the minimum sensitivity level but low levels, without circadian variation, were measured during the immediate postoperative period. During postoperative day 2 and day 3, circadian secretion patterns of melatonin were present in 10 patients and showed an inverse correlation with light intensity exposed (r = -0.480, P = 0.01). Plasma cortisol levels in the immediate postoperative period were significantly higher than those before induction of anesthesia (P < 0.01). During postoperative day 2 and day 3, only 3 patients regained circadian secretion of cortisol.

CONCLUSIONSIt is concluded that melatonin and cortisol secretion are disrupted during cardiac surgery and in the immediate postoperative period.

Cardiopulmonary Bypass ; Circadian Rhythm ; Coronary Artery Bypass ; Coronary Disease ; physiopathology ; surgery ; Humans ; Hydrocortisone ; secretion ; Intraoperative Period ; Male ; Melatonin ; secretion ; Middle Aged ; Monitoring, Intraoperative ; Postoperative Period

OBJECTIVETo evaluate the residual paralysis after a single intubating dose of rocuronium and its effect of residual paralysis after a single dose of rocuronium on the postoperative pulmonary function of patients undergoing laparoscopic gynecological surgeries.

METHODSSixty American Society of Anesthesiologists (ASA) I - II patients undergoing laparoscopic gynecological surgeries were randomly divided into rocuronium (R) group (n = 30) and rocuronium + neostigmine (R + N) group (n = 30).All patients received midazolam (0.02 mg/kg), fentanyl (1 microg/kg), propofol(1.5-2 mg/kg), and rocuronium (0.6 mg/kg) to facilitate tracheal intubation and no more relaxant thereafter. Anesthesia was maintained with isoflurane and nitrous oxide in oxygen (N(2)O:O(2) = 1:1). At the end of the procedure, neuromuscular blockade was not reversed in R group, while antagonism was accomplished with neostigmine (0.04 mg/kg) and atropine (0.02 mg/kg) in R + N group. Immediately after tracheal extubation and on arrival in the PACU, the train-of-four (TOF) ratio at the adductor pollicis of all patients were measured using acceleromyography. Forced vital capacity (FVC), forced expiratory volume in one second (FEV(1)), and peak expiratory flow rate (PEFR) of all patients were measured using spirometry before surgery, after administration of midazolam and fentanyl, immediately after tracheal extubation, on arrival in the PACU, and after the TOF ratio recovered to 1.0. The TOF ratio and pulmonary function between two groups were compared.

RESULTSImmediately after tracheal extubation and on arrival in the PACU, the mean TOF ratio in R group was significantly lower than that in R + N group (P < 0.05). The mean time to achieve TOF ratio of 0.9 and 1.0 in R group was significantly longer than in R + N group (P < 0.05). Immediately after tracheal extubation and on arrival in the PACU, FVC, FEV(1), and PEFR were significantly lower in R group than in R + N group (P < 0.05). FVC, FEV(1), and PEFR after administration of midazolam and fentanyl and after TOF ratio recovered to 1.0 were significantly lower than the baseline values in all patients (P < 0.01).

CONCLUSIONSAfter a single intubating dose of rocuronium, residual paralysis exists in the majority of patients undergoing laparoscopic gynecological surgeries. The pulmonary function is impaired after the surgery, even after recovery of TOF ratio to 1.0.

Adolescent ; Adult ; Airway Extubation ; Androstanols ; administration & dosage ; adverse effects ; Female ; Gynecologic Surgical Procedures ; Humans ; Intubation, Intratracheal ; Laparoscopy ; Middle Aged ; Neuromuscular Nondepolarizing Agents ; administration & dosage ; adverse effects ; Paralysis ; chemically induced ; Postoperative Period ; Respiratory Function Tests ; Young Adult

OBJECTIVETo explore the application of caffeine-halothane contracture test (CHCT) in the confirmation of malignant hyperthermia (MH).

METHODSOne patient who underwent radical gastrectomy presented with clinical manifestations of MH during routine intravenous-inhalation anesthesia process. Isoflurane inhalation and the operation were ceased immediately and emergency management approaches such as physical cooling therapy were taken. Meanwhile, the levels of serum creatine kinase (CK), serum myoglobin, and urinary myoglobin were examined and rectus abdominis was taken and then CHCT was performed to confirm the clinical diagnosis. Total genome was extracted from the patient and then exons 2-18, 39-46, and 90-104 of ryanodine receptor 1 (RYR1) gene were screened to detect mutations using DNA sequencing technique.

RESULTSThe patient was diagnosed as MH episode by clinical characteristics and postoperatively continuous elevation of the levels of CK, serum myoglobin, and urinary myoglobin (30 times higher than normal level). Despite halothane test was negative, the diagnosis of MH was verified by the positive result of caffeine test. DNA sequencing of RYR1 gene of the patient revealed c. 6724C > T (p. T 2 206M).

CONCLUSIONCHCT can be used to confirm the diagnosis of MH.

Anesthetics, Inhalation ; therapeutic use ; Caffeine ; Creatine Kinase ; blood ; Enzyme-Linked Immunosorbent Assay ; Halothane ; Humans ; Isoflurane ; therapeutic use ; Malignant Hyperthermia ; blood ; diagnosis ; genetics ; Muscle, Skeletal ; drug effects ; physiopathology ; Myoglobin ; blood ; Ryanodine Receptor Calcium Release Channel ; genetics