Hirudin (HV) is known as the most potent and specific inhibitor of thrombin. Although hirudin has many advantages , it has the bleeding side effect and this is the great shortage of hiudin for clinical application. In order to alleviate bleeding side effect of hirudin, fusion protein, named as FHV (fusion hirudin linked with FXa recognition peptide) was designed. The fusion protein gene ( fhv) was cloned into plasmid pPIC9K. FHV engineered Pichia pastoris containing high copies was chosen for fermentation and purification at 30 L fermentor scale, finally, FHV with purity of above 97% was obtained. To investigate the function of FHV in vivo, mouse tail thrombosis model was used. In the mice thrombus tail model induced by carrageenan, FHV decreased the length of tail thrombus significantly, similar to that of HV control, and had no obvious effects on the TT, PT and APTT. In conclusion, FHV is constructed and expressed in yeast. FHV fusion proteins is obtained by fermentation and purification. FHV has antithrombotic effects not influencing IT, PT and APTT after administration immediately in animal models. Therefore, FHV is a promising anticoagulant and antithrombotic drug.

OBJECTIVETo compare the effect of transumbilical single-site single-port with that of transumbilical single-site double-port laparoscopic varicocelectomy in the treatment of varicocele in adolescents.

METHODSWe randomly assigned 80 varicocele patients aged 10 - 16 years to two groups of equal number to receive transumbilical single-site single-port and single-site double-port laparoscopic varicocelectomy, respectively. We compared the operation time, postoperative hospital stay, incisional pain, complications and satisfaction with the abdominal cosmetic outcomes between the two groups.

RESULTSAll the operations were successfully performed. The double-port group showed a significantly higher score on the Visual Analogue Scale than the single-port group (4.8 +/- 1.4 vs 3.6 +/- 1.1, t = -4.986, P < 0.01), but there were no significant differences between the two groups in the operation time ([29.8 +/- 4.2] vs [31.2 +/- 4.6] min, t = 1.383, P = 0.171), postoperative hospital stay ([1.95 +/- 0.7] vs [1.82 +/- 0.8] d, t = -0.784, P = 0.436), complications (0 vs 0) and scores on the satisfaction with abdominal cosmetic outcomes (4.6 +/- 0.6 vs 4.8 +/- 0.5, t = 1.253, P = 0.214). No recurrence, umbilical hernia, hydrocele and orchiatrophy were found in the two groups of patients at 6 months after operation, and no visible scar was observed on the abdominal surface.

CONCLUSIONWith strict surgical indications, single-site single-port and single-site double-port laparoscopic varicocelectomies have similar clinical effects in the treatment of varicocele, which leave no scar on the abdominal surface. Single-site double-port laparoscopy needs no special instruments and therefore is worthier of wide clinical application.

Adolescent ; Child ; Humans ; Laparoscopy ; methods ; Length of Stay ; Male ; Operative Time ; Umbilicus ; surgery ; Varicocele ; surgery

Objective To investigate the effects of advanced glycation end-products(AGEPs)on the function of normal keratinocytes in vitro so as to explore the role of AGEPs in impaired wound healing. Methods Normal rat keratinocytes were incubated with different concentrations of AGEPs.After 48 hours of culturing,the cell proliferation rates were measured by MTT colorimetric determination.The cell cycle distributions and apoptosis were analyzed with flow cytometry,and the migration was investigated by 24-well fluorimetric cell migration assay kit by exposing to 100?g/ml AGEPs.Nuclear extracts from these cells were examined for binding of nucleotides containing NF-?B consensus by immunocytochemistry and EMSA in vitro.Results The proliferations of normal keratinocytes were significantly arrested and many cells were induced to early apoptosis compared with control ones(P＜0.05)by exposing to AGEPs for 48 hours. Meanwhile AGEPs also irritated keratinocytes migration compared with control ones(P＜0.05).Inhibiting the activation of NF-?B could partly recover the proliferation of keratinocytes,reverse apoptosis and attenu- ate migration.Conclusion AGEPs are correlated with the migration,proliferation and apoptosis of kera- tinocytes by NF-?B.

Objective To investigate and analyze a food borne disease event caused by Vibrio parahaemolyticus (VP) which happened in a company in Shanghai, and to explore the significance of laboratory testing technology in event traceability analysis, then making suggestions on key directions for food-borne disease prevention. Methods On the basis of epidemiological and hygienic investigation, the virulence genes and molecular typing techniques were used for the VP strains detected in the incident. Results A total of 65 patients were consistent with the case definition.The restaurant had no food business license, and its employees had no health certificate.VP was detected in anal swabs of 5 patients and 2 employees, and the PFGE map showed the same. Conclusion The event is suspiciously caused by food contamination from restaurant employees during food processing, assembly or transportation.It is suggested that the management should be improved of all aspects of the restaurant after cooking, and restaurants providing takeaway should be strengthened.

This study was aimed to investigate the expression of CD96 on bone marrow mononuclear cells (BMMNC) from 91 patients with acute leukemia, and the results were analyzed with clinical pathological data. Flow cytometry was used to detect CD96 molecule on the bone marrow mononuclear cell surface of 91 newly diagnosed patients with acute leukemia, and 15 healthy adults were served as normal controls. The results showed that the average rate of CD96(+) expression on BMMNC (CD45(+) CD34(+) CD19(+)) of 21 patients with B-ALL was (17.41 ± 27.97)%, the average rate of CD96(+) expression on stem cells (CD45(+)CD34(+)CD7(+)) of 11 patients with T-ALL was (46.98 ± 45.55)%, the average rate of CD96(+) expression on BMMNC (CD45(+)CD34(+)CD38(-)) of 59 patients with AML was (16.69 ± 25.08)%, while the average rate of CD96(+) on BMMNC of healthy adult controls was (0.52 ± 1.84)%, there was significant difference in average rate of CD96(+) expression between above-mentioned patients and healthy adult controls (p < 0.05). Otherwise the average rate of CD96(+) on BMMNC after treatment showed no statistical difference between patient group with CR (1.68 ± 2.31) and healthy controls, but demonstrated statistical difference between patients without CR and healthy controls (p > 0.05). The leukocyte count, hemoglobin level and platelet count in CD96(+) group had no obvious difference from CD96(-) ones (p > 0.05). No change found in the field of molecular biology and cytogenetic between these 2 groups. It is concluded that CD96 expression is different in different types of leukemia. The positive expression of CD96 on bone marrow hematopoietic stem cells in patients with acute leukemia may be associated with primary drug resistance, relapse and progression. The CD96 on BMMNC of acute leukemias can be a helpful prognostic indicator in treatment response assessment.
Acute Disease ; Antigens, CD ; metabolism ; Bone Marrow Cells ; metabolism ; Case-Control Studies ; Flow Cytometry ; Humans ; Leukemia ; metabolism ; Leukemia, Myeloid, Acute ; metabolism

The study was purposed to investigate the role of extracellular signal-regulated kinase (ERK) pathway in the differentiation of human MDS cell lines SKM-1 induced by sodium butyrate (NaB), and to elucidate the molecular mechanism of differentiation in SKM-1 cells induced by NaB. The expression levels of total ERK and phosphorylated-ERK were determined by Western blot. The effect of NaB in combination with the ERK inhibitor PD98059 on the proliferation/differentiation of SKM-1 cells was studied, and then the expression levels of the P21 and HDAC protein were detected by Western blot. The results showed that the expression level of phosphorylated ERK was down-regulated by the 1 mmol/L NaB, and the level of total ERK had not changed. NaB or combination of the MEK inhibitor PD98059 with NaB could increase the differentiation of the SKM-1 cells and up-regulated the levels of the P21 and HDAC protein, but the effect of combination of NaB with PD98059 was higher than that of NaB alone. It is concluded that the inhibition of ERK may be involved in sodium butyrate inducing differentiation in SKM-1 cells.
Butyrates ; pharmacology ; Cell Transformation, Neoplastic ; drug effects ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Humans ; Myelodysplastic Syndromes ; enzymology ; pathology ; Tumor Cells, Cultured

OBJECTIVETo explore the effect of glutathione (GSH) and sodium selenite on the metabolism of arsenic in the liver, kidney and blood of mice exposed to iAsIII through drinking water.

METHODSThe mice were randomly divided into control, arsenic, GSH and sodium selenite group, respectively. And each group had eight mice and the mice were exposed to 50 mg/L arsenite by drinking water for 4 weeks. Mice were intraperitoneally injected with GSH (600 mg/kg) and sodium selenite (1 mg/kg) for seven days from the beginning of the fourth week. At the end of the fourth week, liver, kidney and blood were sampled to assess the concentrations of inorganic arsenic (iAs), monomethylarsenic acid (MMA), dimethylarsenic acid (DMA) by hydride generation trapping by ultra-hypothermia coupled with atomic absorption spectrometry.

RESULTSThe liver DMA (233.76 +/- 60.63 ng/g) concentration in GSH group was significantly higher than the arsenic group (218.36 +/- 42.71 ng/g). The concentration of DMA (88.52 +/- 30.86 ng/g) and total arsenic (TAs) (162.32 +/- 49.45 ng/g) in blood of GSH group was significantly higher than those [(45.32 +/- 12.19 ng/g), (108.51 +/- 18.00 ng/g), respectively] of arsenic groups(q values were 3.06, 6.40, 10.72 respectively, P < 0.05). The primary methylated index (PMI) (0.65 +/- 0.050) and secondary methylated index (SMI) (0.55 +/- 0.050) in liver sample of GSH group were significantly higher than those (0.58 +/- 0.056, 0.44 +/- 0. 093) in arsenic group. In blood samples, the PMI (0.85 +/- 0.066) in GSH group was significantly higher than that (0.54 +/- 0.113) in arsenic group (q values were 3.75, 5.26, 4.21 respectively, P < 0.05). However, no significant difference was identified between sodium selenite and arsenic groups in liver, kidney or blood samples. And no significant difference was detected in kidney samples among all arsenic exposing groups.

CONCLUSIONExogenous GSH could promote the methylated metabolism of iAsIII, but sodium selenite showed no significant effects.

Animals ; Arsenic ; analysis ; metabolism ; Arsenic Poisoning ; metabolism ; Environmental Exposure ; Female ; Glutathione ; pharmacology ; Male ; Mice ; Mice, Inbred Strains ; Sodium Selenite ; pharmacology ; Water Supply

OBJECTIVETo fabricate polyvinyl alcohol (PVA)/chitosan hybrid nanofibrous scaffolds owning the similar physiological structure of ECM, and to observe its biodegradation behavior in vivo and in vitro.

METHODS(1) The PVA nanofibrous scaffold and PVA/chitosan hybrid nanofibrous scaffold were fabricated by electrospinning technique, and then they were crosslinked by glutaraldehyde vapor method. The morphology of both scaffolds was observed by scanning electron microscope (SEM). (2) Biodegradation experiment in vitro: the samples of two scaffolds with size of 2 cm x 2 cm were placed into phosphate-buffer saline (PBS) fluid under 37.0 degrees C water for incubation, and then they were dried to observe morphologic changes under SEM on post incubation day (PID) 3, 7, and 14. (3) Biodegradation experiment in vivo: 48 Wistar rats were divided into PVA group and PVA/chitosan group according to the random number table, with 24 rats in each group. PVA or PVA/chitosan nanofibrous scaffold was implanted into subcutaneous tissue on both sides of back in rats of both groups, with 4 scaffolds in each rat. The scaffold samples were harvested to observe morphologic changes with HE staining on post operation day (POD) 3, 7, 14, and 28.

RESULTS(1) After crosslinking, the surface of fibers in PVA and PVA/chitosan hybrid nanofibrous scaffolds were smooth, and the diameters of fibers were similar, ranging from 200 to 300 nm, with high porosity. (2) Biodegradation experiment in vitro showed that the morphologic changes in fiber was respectively swelling, dissolution, fusion in PVA nanofibrous scaffold on PID 3, 7, 14, and that in PVA/chitosan hybrid nanofibrous scaffold was respectively swelling, dissolution and fragmentation, and disappearance. (3) Biodegradation experiment in vivo showed that the morphologic changes in scaffold structure was respectively loosening, fuzziness of edges, degradation, and disappearance in PVA group and PVA/chitosan group on POD 3, 7, 14, 28.

CONCLUSIONSPVA/chitosan hybrid nanofibrous scaffolds can be prepared with electrospinning technique, and it has an appropriate biodegradation rate compatible with tissue reconstruction after crosslinking.

Animals ; Biocompatible Materials ; Cells, Cultured ; Chitosan ; chemical synthesis ; chemistry ; Materials Testing ; Polyvinyl Alcohol ; chemical synthesis ; chemistry ; Rats ; Rats, Wistar ; Tissue Engineering ; methods ; Tissue Scaffolds

OBJECTIVETo investigate the possible mechanism of L-arginine supplementation on the angiogenesis of burn wounds in diabetic rats.

METHODSOne hundred male Sprague-Dawley (SD) rats were used in the study and were randomly divided into A (scalding control, n = 25), B (scalding of the rats with diabetes, n = 25), C (L-glycine control, n = 25) and D (L-arginine supplementation, n = 25) groups. Diabetes was produced by intra-peritoneal injection of streptozotocin (STZ) in B, C and D groups. The rats in C and D groups were gavaged with L-glycine and L-arginine in dose of 200 mg.kg(-1).d(-1), respectively. The glucose content of the back skin tissue was determined for five rats in each group eight weeks after STZ administration. Deep partial thickness scalding of 20% TBSA was engendered on the back in the other 80 rats. The wound area, wound healing rate, and microvascular density with CD34 immunohistochemistry staining were determined on 3rd, 7th, 14th, and 21st post scalding days (PSDs), In addition, the amount of nitric oxide (NO) released from the wound tissue and the tissue contents of vascular endothelial growth factor (VEGF) and transforming growth factor beta1 (TGF-beta1) from wound were determined at the above time points.

RESULTSCompared to those in group B, the wound healing rate in group D increased significantly since the 7th PSD [(44.10 +/- 3.50)%, P < 0.05], and the wound MVD value was increased significantly at all postburn time points. Furthermore, the levels of VEGF, NO and TGF-beta1 in the wound tissue was also increased significantly, while the glucose content in the cutaneous tissue was decreased to (1.380 +/- 0.120) mg/g.

CONCLUSIONL-arginine supplementation could be beneficial to the angiogenesis in the burn wound of the rats with diabetes, as well as to wound healing by increasing the synthesis and the release of VEGF, NO and TGF-beta1 from burn wound and by decreasing the glucose content in the cutaneous tissue of diabetic rats.

Animals ; Arginine ; therapeutic use ; Blood Glucose ; metabolism ; Burns ; metabolism ; therapy ; Diabetes Mellitus, Experimental ; metabolism ; therapy ; Male ; Neovascularization, Physiologic ; drug effects ; Nitric Oxide ; metabolism ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor beta1 ; metabolism ; Vascular Endothelial Growth Factor A ; metabolism ; Wound Healing ; physiology

BACKGROUNDThe myocardial ATP sensitive potassium channel (K(ATP) channel) has been known for more than two decades, the properties of this channel have been intensively investigated, especially the myocardial protection effect by opening this channel. Numerous studies, including hypothermic, using K(ATP) agonists to achieve a hyperpolarizing cardioplegic arrest, have shown a better myocardial protection than potassium arrest. However, there is no evidence showing that K(ATP) channel could be opened by its agonists under profound hypothermia. We investigated the effect of temperature on activation of myocardial K(ATP) channel by nicorandil.

METHODSIsolated ventricular myocytes were obtained by collagenase digestion of the hearts of guinea pigs and stored in KB solution at 4 degrees C. With a steady ground current, the myocytes were perfused with 1 mmol/L nicorandil until a steady IK(ATP) occurred. Then the cells were perfused with 1 mmol/L nicorandil plus 1 micromol/L glybenclamide. Currents signals were recorded on whole cells using patch clamp technique at several temperatures. The temperature of the bath solution around myocytes was monitored and was controlled at 4 degrees C, 10 degrees C, 20 degrees C, 25 degrees C and 35 degrees C respectively. About 10 cells were tested at each temperature, the cells were considered useful only when the outward current could be induced by nicorandil and blocked by glybenclamide. All data were analyzed using Graphpad PRISM 3.0 (Graphpad, San Diego, CA, USA). Nonlinear curve fitting was done in Clampfit (Axon) or Sigmaplot (SPSS).

RESULTSAt 4 degrees C, 10 degrees C, 20 degrees C, 25 degrees C and 35 degrees C, the time needed to open the myocardial K(ATP) channel was (81.0 +/- 0) minutes, (50.5 +/- 11.7) minutes, (28.8 +/- 2.3) minutes, (9.4 +/- 10.2) minutes and (2.3 +/- 1.0) minutes respectively (P = 0.003). The linear relationship between temperature and time needed to open the channel was y (min) = (4348.790 - 124.277x)/60, where y (min) is time needed to open K(ATP) channel, x is temperature, correlation coefficient r = -0.942 (P = 0.00), regression coefficient b = -124.277 (P = 0.00). The current densities among different temperatures were statistically different (P = 0.022), the current density was greater after the activation of K(ATP) channel at higher temperatures. The lower the temperature, the fewer cells in which K(ATP) channels could be opened. At 4 degrees C, only one cell in which the K(ATP) channel could be opened, took a quite long time (81 minutes) and the I-V curve was quite untypical.

CONCLUSIONSK(ATP) channel activated by nicorandil is temperature dependent and the temperature linearly related to time needed to open K(ATP) channel; the lower the temperature, the longer the time needed to open channel and the smaller the current density. At profound hypothermia, it is difficult to activate K(ATP) channels.

Adenosine Triphosphate ; pharmacology ; Animals ; Female ; Glyburide ; pharmacology ; Guinea Pigs ; Heart Ventricles ; Male ; Myocytes, Cardiac ; metabolism ; Nicorandil ; pharmacology ; Patch-Clamp Techniques ; Potassium Channels ; drug effects ; physiology ; Temperature