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

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 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

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 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 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

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