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.

Objective Brucine and strychnine monomer reference substance as extremely toxic substance had potential threat during transportation and utilization. In this study we investigated the homogeneity, stability and assignment accuracy of the mixture reference solution of strychnine and brucine, so as to provide reference for the quality control of extremely toxic chemical reference substances for Chinese medicine. Methods Following the assay in Chinese Pharmacopoeia volume I (2015), we prepared the mixture reference solution of brucine and strychnine, and investigated the solvents and the concentration of mixutre reference solution. The stability test lasted for 12 months. F-test was used for heterogeneity assay. Three researchers were involved for collaboration. Results Methanol and chloroform solution were selected as the solvents for the stability test. Results showed the difference was not statistically significant among various mixture solutions. The results of value assignment were 0.14 mg/mL for strychnine (sR = 0.5％)and 0.10 mg/mL for brucine (sR = 1.0％). The stability of mixture solution were better under the conditions of methanol solution at 4 ℃ or -20 ℃. Conclusion The results provide a possible way to develop the mixture solution in place of the monomer reference, and the mixture reference solution is expected for the quality control in the slices of Semen Strychni and its compound preparations.

BACKGROUNDHigh peritoneal transport status was previously thought to be a poor prognostic factor in peritoneal dialysis (PD) patients. However, its effect on diabetic nephropathy PD patients is unclear in consideration of the adverse impact of diabetes itself. The purpose of this study was to investigate the influence of peritoneal transport characteristics on nutritional status and clinical outcome in diabetic nephropathy patients on PD.

METHODSOne hundred and two diabetic nephropathy patients on PD were enrolled in this observational cohort study. According to the initial peritoneal equilibration test result, patients were divided into two groups: Higher transport group (HT, including high and high average transport) and lower transport group (LT, including low and low-average transport). Demographic characteristics, biochemical data, dialysis adequacy, and nutritional status were evaluated. Clinical outcomes were compared. Risk factors for death-censored technique failure and mortality were analyzed.

RESULTSCompared with LT group (n = 37), serum albumin was significantly lower and the incidence of malnutrition by subjective global assessment was significantly higher in HT group (n = 65) (P < 0.05). Kaplan-Meier analyses showed that death-censored technique failure and mortality were significantly increased in HT group compared with that in LT group. On multivariate Cox analyses, higher peritoneal transport status and lower residual renal function (RRF) were independent predictors of death-censored technique failure when adjusted for serum albumin and total weekly urea clearance (Kt/V). Independent predictors of mortality were advanced age, anemia, hypoalbuminemia, and lower RRF, but not higher peritoneal transport status.

CONCLUSIONSHigher peritoneal transport status has an adverse influence on nutrition for diabetic nephropathy patients on PD. Higher peritoneal transport status is a significant independent risk factor for death-censored technique failure, but not for mortality in diabetic nephropathy patients on PD.

Aged ; Aged, 80 and over ; Biological Transport ; Cohort Studies ; Diabetic Nephropathies ; metabolism ; therapy ; Female ; Humans ; Kidney ; metabolism ; pathology ; Male ; Middle Aged ; Nutritional Status ; Peritoneal Dialysis

Small intestinal obstruction is a common complication of primary gastrointestinal cancer or metastatic cancers.Patients with this condition are often poor candidates for surgical bypasses,and placement of self-expanding metal stent (SEMS) can be technically challenging.In this study,we examined the feasibility of combined application of single-balloon enteroscope (SBE) and colonoscope for SEMS placement in patients with malignant small intestinal obstruction.Thirty-four patients were enrolled in this study,among which 22 patients received SEMS placement by using SBE and colonoscope,while the other 12 patients received conservative medical treatment.The patients were followed up for one year.Stent placernent was technically feasible in 95.5％ (21/22).Clinical improvement was achieved in 86.4％ (19/22).For the 19 clinical success cases,the average time of benefits from a gastric outlet obstruction scoring system (GOOSS) increase ≥1 was 111.9±89.5 days.For the 12 patients receiving conservative medical treatment,no significant improvement in GOOSS score was observed.Moreover,a significant increase of Short-Form-36 health survey score was observed in the 19 patients at time of 30 days after stent placement.By Kaplan-Meier analysis,a significant survival improvement was observed in patients with successful SEMS placement,compared with patients receiving conservative medical treatment.Taken together,combined use of SBE and colonoscope makes endoscopic stent placement feasible in patients with malignant small intestinal obstruction,and patients can benefit from it in terms of prolonged survival and improved quality of life.

OBJECTIVETo investigate the influence of the individual genotype differences of DC-SIGN and DC-SIGNR on the mother-to-neonate intrauterine infection of HBV.

METHODSThe genotypes of the gene DC-SIGN and DC-SIGNR in the pregnant women with HBV positive were detected by PCR and agarose gel electrophoresis. The significant difference of gene diversity of DC-SIGN and DC-SIGNR was analyzed by chi-square test.

RESULTS(1) All of 29 cases in intrauterine infection group were 7/7 DC-SIGN genotype. In the non-intrauterine infection group, 7/5 genotype were observed in 2 of 54 cases, and the other 52 cases were 7/7 genotype. The two groups was no significant difference (P = 0.54). (2) 29 cases of intrauterine infection group was observed 4 genotypes of DC-SIGNR such as 7/7, 7/5, 9/7 and 6/5, the genotype frequencies were 0.3793, 0.3448, 0.2414 and 0.0345 respectively. 54 cases of non-intrauterine infection group was found 6 genotypes such as 7/7, 7/5, 9/5, 9/7, 7/6 and 6/5, genotype frequencies were 0.5186, 0.1481, 0.0926, 0.1852, 0.0370 and 0.0185 respectively. The distribution of 7/5 genotype in the intrauterine infection group (29 cases) and the non-intrauterine infection group (54 cases) was statistically significant (P = 0.038) , and no significant difference was found in other genotypes between the two groups (P > 0.05).

CONCLUSIONThe gene DC-SIGN showed relatively little variation in the pregnant women infected with HBV. On the countrary, there were multiple genotypes of the gene DC-SIGNR in these women, and the genotype "7/5" of DC-SIGNR might be one of the susceptibility genes associated with intrauterine infection.

Cell Adhesion Molecules ; genetics ; metabolism ; Female ; Genetic Predisposition to Disease ; Genetic Variation ; Hepatitis B ; genetics ; transmission ; virology ; Hepatitis B virus ; genetics ; isolation & purification ; Humans ; Infant, Newborn ; Infectious Disease Transmission, Vertical ; Lectins, C-Type ; genetics ; metabolism ; Male ; Polymorphism, Genetic ; Pregnancy ; Pregnancy Complications, Infectious ; genetics ; virology ; Receptors, Cell Surface ; genetics ; metabolism

OBJECTIVETo explore the effects of different hepatic inflow occlusion methods on liver regeneration in rats after partial hepatectomy (PH).

METHODSMale Wistar-Furth rats were randomly assigned to three groups: control group, underwent 68% hepatectomy alone; occlusion of portal triad (OPT) group, subjected to occlusion of portal triad under portal blood bypass; and occlusion of portal vein (OPV) group, subjected to occlusion of portal vein under portal blood bypass. Blood flow was occluded for 20, 30, and 40 minutes before 68% hepatectomy. According to the 7-day survival of each group, a same occlusion time T was set. Each group was divided into two subgroups (n = 8), in which animals were killed 3 and 7 days later. Liver regeneration was calculated as a percent of initial liver weight. Immunohistochemistry for proliferating cell nuclear antigen (PCNA) and Ki-67 was performed to quantify proliferating cells. In addition, functional liver volume represented by 99Tc(m)-GSA radioactivity was assessed.

RESULTSThe safe tolerance limit time was 30 minutes for OPT group and 40 minutes for OPV group. At 3 days after PH, no significant difference was observed in the regeneration rate of each group (P > 0.05). However, liver radioactive activity, PCNA labeling index, and Ki-67 index of OPV group was significantly higher than those of OPT group (P < 0.05); the latter were similar to those of control group (P > 0.05). At 7 days after PH, no significant difference was observed in all indexes among three groups (P > 0.05).

CONCLUSIONCompared with Pringle maneuver, preserving the hepatic artery flow during portal triad blood inflow occlusion can promote remnant liver regeneration early after PH.

Animals ; Hepatectomy ; methods ; Liver ; blood supply ; surgery ; Liver Regeneration ; physiology ; Male ; Postoperative Period ; Rats ; Rats, Wistar

OBJECTIVETo illuminate the preliminary genotype and allele frequency distribution of D6S477 and the other four short tandem repeat(STR) loci in Chinese Han population in Qingdao area and to probe the possibility of their genetic application.

METHODSTwo hundred ACD-blood specimens were collected from the unrelated individuals in Qingdao. The DNA samples were extracted with Chelex method and were amplified by polymerase chain reaction technique. The PCR products were analyzed by polyacrylamide gel electrophoresis and displayed using silver staining.

RESULTSThe authors obtained the allele frequency distribution and preliminary genotype of D6S477, D9S1118, D18S865, D19S400 and D20S161 STR loci. No deviation from Hardy-Weinberg equilibrium was observed in the five loci.

CONCLUSIONAll the five loci have higher chance of exclusion and discriminating power, and they will be useful markers for researches in genetics.

Asian Continental Ancestry Group ; genetics ; China ; Gene Frequency ; Genotype ; Humans ; Linkage Disequilibrium ; Microsatellite Repeats ; genetics ; Polymerase Chain Reaction ; Polymorphism, Genetic

OBJECTIVETo assess the efficacy and safety of damage control surgery with abdominal packing in non-trauma patients with severe abdominal hemorrhage.

METHODSA retrospective review of consecutive non-trauma patients who underwent abdominal packing to control severe abdominal hemorrhage between February 2002 and February 2007 were performed. The demographics, physiological parameters, surgical indications and procedures, mortality, morbidity and volumes of resuscitation were retrieved. The observed mortality was compared to those calculated from the Physiological and Operative Severity Score for the Enumeration of Mortality and Morbidity (POSSUM) and Portsmouth Predictor Equation (P-POSSUM) scores.

RESULTSA total of 26 non-trauma patients were included in this study, with a mean age of (42.6 +/- 15.8) years (range, 18 - 72 years). The most common etiologies associated with the severe hemorrhage was necrotizing pancreatitis (11 cases), intestinal fistula (5 cases) and tumor (4 cases). Of the patients, 24 cases (92.4%) achieved hemostasis by simple packing, 1 achieved hemostasis by using packing and angiographic embolization, and the other one failed and died. The mean intra-operative blood loss during the initial procedure was 1253.8 ml. The physiological parameters which improved significantly after rewarming and resuscitation in ICU phase included: body temperature, systolic blood pressure, heart rate, arterial pH, base excess, hemoglobin, hematocrit, prothrombin time, and international normalized ratio. The mean duration of packing was 4.3 days. The mean length of SICU stay and hospital stay was 40.5 and 67.4 days, respectively. Mortality rate predicted by POSSUM and P-POSSUM was 77.7% and 63.4%, respectively. Seven patients (26.9%) died after operation, brought an observed mortality rate significantly lower than predicted (P = 0.001 and 0.025, respectively). The most common complications included pneumonia (57.7%), bacteremia (50.0%), and re-bleeding (26.9%).

CONCLUSIONSDamage control laparotomy with packing is an effective procedure in the management of severe non-trauma abdominal hemorrhage, it can prevent the aggravation of "lethal triad" characterized by hypothermia, coagulopathy and acidosis. Appropriate application of the technique in strictly selected patients can result in a lower mortality rate.

Adolescent ; Adult ; Aged ; Bandages ; Female ; Hemoperitoneum ; therapy ; Hemostatic Techniques ; Humans ; Male ; Middle Aged ; Prognosis ; Retrospective Studies ; Treatment Outcome ; Young Adult

OBJECTIVETo investigate the effect of a continuous infusion of low-dose dexmedetomidine on patient-controlled analgesia (PCA) with fentanyl in elderly patients after total hip replacement.

METHODSForty patients (ASA I-II) aged 66-81 years after total hip replacement were randomized equally into the control and test groups. The patients in the test group received continuous infusion of dexmedetomidine at the rate of 0.2 µg·kg(-1)·h(-1) from the beginning to the end of PCA with fentanyl after the surgery, while those in the control group received normal saline. The cumulative fentanyl dose, VAS pain scores and Ramsay sedation score were recorded at 0, 4, 8, 12 and 24 h after the surgery.

RESULTSAll the patients in the two groups reported good pain relief and none needed additional fentanyl. The VAS pain score was significantly lower (P<0.05 or 0.01), while the Ramsay sedation scores higher (P<0.05) in the test group than in the control group. The cumulative fentanyl dose was significantly lower in the test group (P<0.05 or 0.01). The incidence of such adverse effects as nausea and vomiting was significantly lower in the test group (P<0.05).

CONCLUSIONPCA with fentanyl combined with low-dose dexmedetomidine infusion is safe for elderly patients, and can decrease fentanyl consumption and improve the effect of PCA with fentanyl.

Aged ; Aged, 80 and over ; Analgesia, Patient-Controlled ; methods ; Arthroplasty, Replacement, Hip ; Dexmedetomidine ; administration & dosage ; therapeutic use ; Female ; Fentanyl ; administration & dosage ; therapeutic use ; Humans ; Infusions, Intravenous ; Male ; Pain, Postoperative ; drug therapy

The aim of the present study was to investigate the effects of quercetin on colon contractility and voltage-dependent Ca(2+) channels in the single smooth muscle cell isolated from the proximal colon of guinea-pig and to clarify whether its effect on L-type Ca(2+) current (I(Ca,L)) would be related to its myorelaxing properties. Colon smooth muscle strips were used to take contractile tension recordings. Smooth muscle cells were freshly isolated from the proximal colon of guinea-pig by means of papain treatment. I(Ba,L) (barium instead of calcium as current carrier) was measured by using whole-cell patch-clamp techniques. The results showed that quercetin relaxed colon muscle strips in a concentration-dependent manner and antagonized the contractile effect of acetylcholine and neostigmine. Preincubation with indomethcin [cyclooxygenase (COX) inhibitor] and methylene blue [guanylate cyclase (GC) inhibitor] significantly attenuated the relaxing effect of quercetin, respectively. Quercetin increased I(Ba,L) in a concentration- [EC(50)= (7.59+/-0.38) mumol/L] and voltage-dependent pattern, and shifted the maximum of the current-voltage curve by 10 mV in the depolarizing direction without modifying the threshold potential for Ca(2+) influx. Quercetin shifted the steady-state inactivation curve toward more positive potentials by approximately 3.75 mV without affecting the slope of activation and inactivation curve. H-89 (PKA inhibitor) abolished quercetin-induced I(Ba,L) increase, while cAMP enhanced the quercetin-induced I(Ba,L) increase. The patch-clamp results proved that quercetin increased I(Ba,L) via PKA pathway. It is therefore suggested that the relaxing effect of quercetin attributes to the interaction of GC and COX stimulation, as well as the antagonism effect on acetylcholine, which hierarchically prevails over the increase in the Ca(2+) influx to be expected from I(Ca,L) stimulation.
Animals ; Calcium Channels, L-Type ; metabolism ; Cells, Cultured ; Colon ; drug effects ; Guinea Pigs ; Muscle Contraction ; Myocytes, Smooth Muscle ; drug effects ; Patch-Clamp Techniques ; Quercetin ; pharmacology