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.

To study the protective effect of astragalus saponin extracts (AS) on kidneys of diabetic rats. Totally 32 diabetic rats induced by streptozotocin (STZ) were divided into AS high and low dose groups, the positive control group and the model group (DM group) and orally administered with 50 mg x- kg(-1) x d(-1) AS 200, 25 mg x kg(-1) x d(-1) valsartan, 10 mL x kg(-1) x d(1) physiological saline, respectively. Another 8 healthy rats were collected in the normal control group (NC group, physiological saline 10 mL x kg(-1). d(-1)). All rats were treated for consecutively 6 weeks. After the administration, the body weight was measured every week, the concentration of blood glucose was monitored on week 2, 4 and 6. The total urine and total urinary protein (U-TP) in 24 h were measured by the metabolic cage method on week 6; At the end of week 6, blood samples were collected from hearts to detect blood urea nitrogen (BUN), serum creatinine (Scr), uric acid (UA) , total cholesterol (CH) triglyceride (TG) by biochemical methods. Kidneys were collect to calculate the kidney hypertrophy index and observe the pathological sections. The laboratory results show that in the DM group, the blood glucose, metabolic cost in 24 h, kidney hypertrophy index, U-TP, BUN, Scr, UA, TG were significantly higher than that in the NC group (P < 0.01, P < 0.05) , with significant pathological changes; After the intervention with AS, the metabolic value in 24 h, kidney hypertrophy index, U-TP, BUN, Scr, UA, TG were significantly lower in the high dose group (P < 0.01, P < 0.05), and the kidney hypertrophy index, BUN, Scr, UA, TG in the low dose group were also significantly lower (P < 0.05), with slight reduction in renal pathological changes in both groups. In conclusion, Astragalus saponin extracts have a certain protective effect on kidneys of diabetic rats.
Animals ; Astragalus Plant ; chemistry ; Blood Glucose ; metabolism ; Blood Urea Nitrogen ; Diabetic Nephropathies ; metabolism ; prevention & control ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Kidney ; drug effects ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Saponins ; administration & dosage ; Uric Acid ; metabolism

OBJECTIVETo distinguish female and male plants of Gynostemma pentaphyllum quickly and accurately in the stage of seedling and to understand the relation between sex differentiation and endogenous plant hormone.

METHODStained methods of BTB and enzyme linked immuno-sorbent Assay were used.

RESULT10 Hours after being dyed, the liquid extract of female plants turned into yellow, while extract of male ones turned green. There were obvious differences on iPAs and GA(1 + 3) contents but no differences on IAA and ABA contents between male and female plants during the stages of seedling and vegetative growth.

CONCLUSIONBTB can be used for distinguishing female and male plants and there were a certain relation between sex and endogenous plant hormones.

Gibberellins ; metabolism ; Gynostemma ; growth & development ; metabolism ; Plant Growth Regulators ; metabolism ; Plant Leaves ; growth & development ; metabolism ; Plants, Medicinal ; growth & development ; metabolism ; Sex Factors

OBJECTIVETo evaluate protein loss in critically ill patients with acute renal failure during continuous veno-venous hemofiltration (CVVH) and analysis the major factor impacting protein clearance.

METHODSA analysis was carried out in eighteen (twelve male and six female) sepsis or severe acute pancreatitis patients with acute renal failure from September 2008 to September 2009. The average age was 45 years (39 - 62 years). CVVH was conducted for 24 h in all patients. Effluent volume, blood speed, ultrafiltration rate and transmembrane pressure (TMP) were 4000 ml/h, (277 ± 89) ml/h, (179 ± 4) ml/min and (173 ± 48) mm Hg (1 mm Hg = 0.133 kPa) respectively. Blood samples were collected before and after filtration in order to detect protein concentration. Ultrafiltrate was obtained hourly to measure protein concentration and calculate protein loss during session.

RESULTSMean protein concentration was (231 ± 67) mg/L and protein loss was (22 ± 6) g/d in ultrafiltrate samples. The difference in serum protein level during hemofiltration was not significant [(56 ± 6) g/L vs. (55 ± 10) g/L, P > 0.05], while there was a weak, but statistically significant correlation between the ultrafiltrate protein concentration and the corresponding value for serum protein (r = 0.481, P < 0.05). However, there was a strong and statistically significant correlation between the ultrafiltrate protein concentration and the TMP (r = 0.564, P < 0.01). Stepwise multiple regression analysis showed that TMP and serum protein concentration played a pivotal role in ultrafiltrate protein loss.

CONCLUSIONSIn addition to renal replacement therapy, serum protein would be cleared through hemofilter during CVVH. TMP and serum protein concentration are the main factors that affect protein loss in ultrafiltrate. As a result, it is necessary to take account of the protein loss in ultrafiltrate when setting nutritional schedule.

Acute Kidney Injury ; therapy ; Adult ; Blood Proteins ; deficiency ; Critical Illness ; Female ; Hemofiltration ; adverse effects ; Humans ; Male ; Malnutrition ; etiology ; Middle Aged

Objective To observe the dynamic changes of plasma matrix metalloproteinases (MMPs) and investigate the effect of early or delayed percutaneous coronary intervention (PCI) in the presence or absence cilostazol on left ventricle (LV) remodeling in patients with non-ST elevation myocardial infarction (NSTEMI). Methods One hundred and sixty-four patients undergoing PCI with NSTEMI were randomized to early PCI (PCI within 24 h) group or delayed PCI group (PCI after 36 h), and patients in both group were further assigned to cilostazol or no cilostazol group. Plasma MMP-2 and MMP-9concentrations were measured at 2, 4 days and 2 and 4 weeks after PCI. Left ventricular end-diastolic volume ( LVEDV ), left ventricle ejection fraction ( LVEF), left ventricle posterior wall ( LVPW ) and interventricular septum (IVS) were measured by echocardiography at baseline and 1 year after PCI. Results MMP-2 concentration at 2 weeks after PCI is higher than that at 2, 4 days and 4 weeks after PCI. MMP-9concentration at 4 days is higher than that at 2 days, 2 weeks and 4 weeks after PCI. MMP-2 and MMP-9were significantly lower in cilostazol group compared with that in non-cilostazol group at 4 days, 2 weeks and 4 weeks after NSTEMI ( all P ＜ 0. 05 ). Changes of LVEDV and LVEF were significantly less in cilostazol group and early PCI group than that in no cilostazol group and delay PCI group (P＜0.05 or P＜0. 01) at 1year after NSTEMI. Conclusions Early PCI and Cilostazol use are associated with less LV remodeling in patients with NSTEMI. Cilostazol attenuated LV remodeling possibly by reducing concentration of MMP-2and MMP-9 after PCI.

OBJECTIVETo explore the mechanism of reinforcing function of moxibustion to spleen-stomach.

METHODSForty healthy Sprague Dawley rats were randomly divided into 4 groups: group A (blank group), group B (model group), group C (moxibustion group) and group D (herbs group). The rat model of spleen-deficiency was established by intragastric administration with 200% Dahuang (Rhubarb) infusion. The rats in group A and B, and D served as the blank control, model, and Sijunzi decoction group respectively, while those in group C received moxibustion at "Zusanli" (ST 36), "Zhongwan" (CV 12), "Guanyuan" (CV 4), "Pishu" (BL 20) and "Weishu" (BL 21), etc. The common symptoms and intestinal propulsive rate were observed. The content of I-xylose in serum was detected by phloroglucinol method. Colorimetry method was used to detected content of ATP in jejunum tissues.

RESULTSCompared with group A, the symptom score in group B was increased significantly (both P < 0.01), while the intestinal propulsive rates, the content of D-xylose in serum and ATP in jejunum tissues were decreased significantly (P < 0.05, P < 0.01). Compared with group B, the symptom score in group C and D was decreased significantly (both P < 0.01), while the intestinal propulsive rates, the content of D-xylose in serum and ATP in jejunum tissues were increased significantly (P < 0.05, P < 0.01). There were no significant difference between group C and D (P > 0.05).

CONCLUSIONMoxibustion at "Zusanli" (ST 36) etc. could relieve symptoms of spleen-deficiency, enhance motility and absorption functions of small intestine and improve metabolism of small intestine. The efficacy is equal to administration of Sijunzi decoction.

Adenosine Triphosphate ; metabolism ; Animals ; Female ; Humans ; Intestinal Absorption ; Intestine, Small ; metabolism ; physiopathology ; Male ; Moxibustion ; Rats ; Rats, Sprague-Dawley ; Spleen ; physiopathology ; Splenic Diseases ; metabolism ; physiopathology ; therapy

OBJECTIVE: To explore the effects of NGX6 on the transcriptional activation of beta-catenin/TCF/LEF in Wnt/beta-catenin signal pathway, and to identify the role of NGX6 in Wnt signal pathway. METHODS: The eukaryotic expression vector pcDNA3.1(+)-beta-catenin (WT) was constructed. pcDNA3.1(+)-beta-catenin (WT) and pCMV-myc-NGX6 were cotransfected to COS-7 and the transcriptional activity of TCF/LEF was detected by TCF-4 luciferase report system. Without extro-genous beta-catenin, pCMV-myc-NGX6 was transfected alone to COS-7 and colon cancer cell line SW620, and the transcriptional activity of TCF/LEF was detected by TCF-4 luciferase report system, and then the expression of nucleus beta-catenin and TCF-4 was detected by Western blot. RESULTS: The eukaryotic expression vector pcDNA3.1(+)-beta-catenin (WT) was successfully constructed. The activation of TCF-4 luciferase report gene in the cotransfection group in COS-7 was less than that in NGX6 alone transfection group (P<0.05). The activation of TCF-4 luciferase report gene in NGX6 alone transfection group without extro-genous beta-catenin was less than that in pCMV-myc transfection group in COS-7 and SW620. The expression of beta-catenin and TCF-4 was decreased after the NGX6 transfection in COS-7 and SW620 cells. CONCLUSION NGX6 can inhibit the transcriptional activation of beta-catenin/TCF/LEF in Wnt signal pathway by its negative regulation in the nuclear translocation of beta-catenin.
Animals ; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors ; genetics ; COS Cells ; Cell Line, Tumor ; Chlorocebus aethiops ; Genes, Reporter ; Humans ; Membrane Proteins ; genetics ; Transcription Factor 4 ; Transcription Factors ; genetics ; Transcriptional Activation ; Transfection ; Tumor Suppressor Proteins ; genetics ; Wnt Signaling Pathway ; beta Catenin ; genetics ; metabolism

Human metapneumovirus (hMPV) is a recently identified respiratory virus more like human respiratory syncytial virus in clinical symptoms. Matrix protein (M) is one of the most important structural proteins. For further studying of hMPV, the full length of M genes from the recombinant plasmid pUCm-M1816 and pUCmM1817 were cloned by PCR and sub-cloned into the pET30a(+) vector, which is a prokaryotic expression vector, after dual-enzyme digestion with Bam HI and Xho I. The positive recombinated plasmids were transformed into E. coli BL21 (DE3) and expressed under the inducing of IPTG. Target proteins were characterized by SDS-PAGE and Western blotting. In this article, we' ve successfully constructed the recombinated plasmids pET30a-M1816 and pET30a-M1817 which have correct open reading frames confirmed by dual-enzyme digestion analysis and sequencing. The fusion proteins with 6 x His-N were highly produced after inducing by 1mmol/ L IPTG at 37 degrees C. A unique protein band with approximate 27.6 kD was characterized by SDS-PAGE. Most of the target protein existed in inclusion body. Western blot analysis showed that the target protein has specific binding reaction to rabbit antiserum against polypeptides of the matrix protein of hMPV. So the M genes were highly expressed in the prokaryotic system and the expressed M proteins have specific antigenic activities. It can be used for further studying of hMPV infections in Beijing.
Animals ; Antigens, Viral ; genetics ; immunology ; metabolism ; Blotting, Western ; China ; Gene Expression ; Genetic Vectors ; genetics ; Humans ; Immune Sera ; immunology ; Metapneumovirus ; genetics ; immunology ; metabolism ; Plasmids ; genetics ; Prokaryotic Cells ; metabolism ; Rabbits ; Species Specificity ; Viral Structural Proteins ; genetics ; immunology ; metabolism

OBJECTIVETo investigate the effect of Nutrition MCT and Nutrition MF enteral nutrition on nutritional status of patients after major abdominal operation.

METHODSIn a double- blinded and randomized cross- cover study, Nutrition MCT and Nutrition MF enteral nutrition were fed in patients when the gut function restored after operation. The total calorie was 104.6 kJ(25 kcal) x kg(-1) x d(-1) and the period of full dose of enteral nutrition was 5 days. The blood samples were collected before operation,before enteral nutrition and the sixth day after full dose of enteral nutrition for the measurement of pre- albumin, total protein,albumin, transferrin and triglyceride. The urine, stool and drainage fluid were collected to analyze nitrogen balance.

RESULTSThe plasma protein and fat were obviously dropped in patients after abdominal operation and improved after the enteral nutrition support in two groups. However, the pre- albumin level increased more in patients of Nutrition MCT than Nutrition MF.

CONCLUSIONNutrition MCT can obviously improve the nutritional status of patients after major abdominal operation.

Abdomen ; surgery ; Adult ; Aged ; Cross-Over Studies ; Double-Blind Method ; Enteral Nutrition ; methods ; Female ; Humans ; Male ; Middle Aged ; Nutritional Status ; Proteins ; therapeutic use ; Triglycerides ; therapeutic use

This paper aims to summarize the achievements during the implementation process of good agricultural practice (GAP) in Chinese Materia Medica (CMM), and on basis of analyzing the existing problems of GAP, to propose further implementation of GAP in TCM growing. Since the launch of GAP in CMM growing ten years ago, it has acquired great achievements, including: (1) The promulgation of a series of measures for the administration of the GAP approval in the CMM growing; (2) The expanded planting area of CMM; (3) The increased awareness of standardized CMM growing among farmers and enterprises; (4) The establishment of GAP implementation bases for CMM growing; (5) The improvement of theory and methodology for CMM growing; (6) The development of a large group of experts and scholars in GAP approval for CMM production. The problems existing in the production include: (1) A deep understanding of GAP and its certification is still needed; (2) The distribution of the certification base is not reasonable; (3) The geo-economics effect and the backward farming practices are thought to be the bottlenecks in the standardization of CMM growing and the scale production of CMM; (4) Low comparative effectiveness limits the development of the GAP; (5) The base of breeding improved variety is blank; (6) The immature of the cultivation technique lead to the risk of production process; (7) The degradation of soil microbial and the continuous cropping obstacle restrict the sustainable development of the GAP base. To further promote the health and orderly GAP in the CMM growing, the authors propose: (1) To change the mode of production; (2) To establish a sound standard system so as to ensure quality products for fair prices; (3) To fully consider the geo-economic culture and vigorously promote the definite cultivating of traditional Chinese medicinal materials; (4) To strengthen the transformation and generalization of basic researches and achievements, in order to provide technical support for the CMM production; (5) To deepen the understanding of GAP, to vigorously promote ecological planting and precision agriculture, in order to overcome the continuous cropping obstacle. The authors think that despite the fact that we are still facing with a huge array of management and technological problems, the GAP in the CMM growing has already enjoyed widespread support and showed great potential. In the future, with people's deeper understanding of GAP and the great progress of the science and technology, the GAP will constantly be fused with the theory, methodology and technology in the modern agriculture like precision agriculture, eco-agriculture and etc.
Agriculture ; economics ; methods ; standards ; trends ; China ; Drugs, Chinese Herbal ; analysis ; economics ; standards ; Humans ; Materia Medica ; chemistry ; economics ; standards ; Plants, Medicinal ; chemistry ; growth & development