Bacillus subtilis ZC-7 was obtained by implantation with N+ ions beam to B.subtilis AS1.398,and compared with the AS1.398 neutral protease,the enzyme activity of ZC-7 neutral protease was about 1 timeshigher in previous research.A neutral protease was purified from the culture of B.Subtilis ZC-7 by the procedures including amoninium sulfate precipitation,ultrafiltration,DEAE-Sepharose Fast Flow chromatography and Sephadex G-75 chromatography.By multi-step purification,the ZC-7 neutral protease was purified to 78.5 folds and its yield was 27.7%,at last,the specific activity of ZC-7 neutral protease was up to 4.1?105U/mg.Analysed by SDS-PAGE,the purified protease has shown a molecular mass of about 42kDa.The Km for casein hydrolysis was 3.67?10-3?g/ml and the Vmax was 12.21?g/min.The optimum pH and temperature forhydrolysis of casein were 7.0 and 55℃,respectively.This protease was stable up to 40℃ within the pH range of 6.5 and 8.0.EDTA,isopropanol and alcohol nearly inhibited its activity while some ions such as Ca2+,Mg2+,Fe3+ can improve its activity.In addition,it could resist 1 mol/L H2O2.

Renal transplant (RT) recipients have a high risk of developing cardiovascular diseases.However,the effects of renal transplantation on the development of arteriosclerosis have been controversial.The carotid intima-media thickness (CIMT) and diameter (CD) are important indicators of vascular remodeling and arteriosclerosis.In this study,31 patients with hemodialysis (HD),31 RT recipients and 84 age-and gender-matched control subjects were enrolled.Their CIMT and CD were measured by ultrasonic radiofrequency tracking,and the linear regression models and Z test were used to identify the progression of arteriosclerosis and the risk factors.Compared with HD group,RT group had significantly lower CIMT and CD.CIMT was found to be associated with age,body weight,resistance index and diastolic velocity,while CD was associated significantly with age,body weight,pulsatility index,end diastolic velocity and diastolic blood pressure (DBP),respectively.The correlation curves between CIMT and age showed the slopes of curves were decreased successively in control,RT and HD groups,and the curves between CD and age showed the slopes were decreased in order of RT ＞ control ＞ HD groups.It was concluded that CIMT and CD were significantly correlated with age in RT and moderately with age in HD patients.RT could reduce the progress of arteriosclerosis in patients with end-stage renal disease.

Objective To investigate the performance of triple rule-out with(M-slice spiral CT in the combined examination of pulmonary artery,thoracic aorta and coronary artery for patients with acute chest pain.Methods Seventy patients who presented with acute chest pain were included in the study.All of the patients underwent retrospective ECG-gated 64-slice computed tomography triple rule-out examination to evaluate the pulmonary arteries,thoracic aorta and coronary arteries.Multi-planar reconstruction(MPR), maximum intensity projection(MIP),curved-planar reconstruction(CPR)and volume rendering(VR) were used to display pulmonary arteries,thoracic aorta and coronary arteries.We evaluated the image quality of coronary artery and the enhancement of the pulmonary artery and thoracic aorta to estimate if the examination can fulfill the clinical demand for the differential diagnosis of acute chest pain.Results The mean scan time was(8.5?1.0)s,and the dose of contrast medium injected was 100 ml.There were 95.7%(67/70)of patients whose CT values detected in the pulmonary artery and thoracic aorta after enhancement were ≥200 HU.The image quality of 85.8%(720/839)coronary segments was classified as excellent,8.6%(72/839)as good,and 5.6%(47/839)as poor.There were 20 cases with coronary stenoses≥50%,2 cases with pulmonary embolism,and 2 cases with aortic dissection.Conclusion The triple ride-out examination with 64-slice spiral CT could depict pulmonary artery,thoracic aorta,and coronary artery in 8 s with good image quality.It has great potential in the etiological diagnosis for the patients with acute chest pain.

OBJECTIVETo study the feasibility and early results of radical resection of esophageal carcinoma using single-port thoracoscopy combined with laparoscopy.

METHODSFrom March 2010 to December 2010, 6 patients with esophageal carcinoma underwent radical resection by single-port thoracoscopy combined with laparoscopy in the General Hospital of People's Liberation Army. With the patients at a supine position, laparoscopy was performed to complete stomach mobilization and abdominal lymph node dissection. Thoracoscopy was then carried out with the patients lying on the left to mobilize the esophagus and dissect thoracic lymph nodes. Finally, the stomach was pulled into the thoracic cavity via the hiatus of the diaphragm to construct a tube-like stomach, which was then anastomosed to the esophagus using the OrVil system.

RESULTSNo patient was converted to open surgery during the operation. The total operative time ranged from 200 to 320 min. The mean laparoscopic time was 75(range, 45-90) min, and the mean thoracoscopic time 160(120-240) min. The mean intraoperative blood loss was 220(160-300) ml. The mean lymph node retrieval was 12(9-18). No anastomotic fistula, chylothorax, lung infection were found postoperatively.

CONCLUSIONAfter esophageal resection using single-port thoracoscopic and laparoscopy, reconstruction using OrVil system is safe and feasible.

Aged ; Esophageal Neoplasms ; surgery ; Esophagectomy ; methods ; Female ; Humans ; Laparoscopy ; methods ; Male ; Middle Aged ; Thoracoscopy ; methods

OBJECTIVETo study the molecular genetic mechanism of mucopolysaccharidosis type IV A(MPS IV A), and reveal the relationship between the genotype and phenotype, and provide a basis for prenatal gene diagnosis in the future.

METHODSA preliminary diagnosis was made by qualitative detection of urinary glycosaminoglycans of the suspected MPS IV A proband. Then, mutation detection was performed on the proband and her family members with PCR and direct sequencing of the PCR products. After a novel c.1567T to G mutation was detected, Xsp I restriction enzyme digestion and amplification refractory mutation system (ARMS) fast specific identification were established to analyze the sequences of exon 14 in GALNS gene, including 110 randomly selected healthy controls, the proband and other pedigree members. At the same time, bioinformatic approaches for protein secondary, tertiary structure prediction were applied to identify the novel pathologic mutation.

RESULTSThe proband's urine GAGs test was a weak positive(± ), and a c.1567T to G heterozygous termination codon mutation in exon 14 and a c.374C to T heterozygous missense mutation in exon 4 were found. The proband was compound heterozygous of the two mutations, so was her younger sister. Her mother was a carrier with only a c.1567T to G heterozygous mutation in exon 14. Her father had a heterozygous mutation of c.374C to T in exon 4. After Xsp I restriction enzyme digestion, healthy controls had three bands including 28 bp, 120 bp and 399 bp, while the proband and her mother had four bands consisting of 28 bp, 120 bp, 148 bp and 399 bp. For amplification by ARMS specific primers, it was negative for the controls, while it was positive for the proband and the carrier. The results of protein secondary and tertiary structure prediction showed that the c.1567T to G mutation located in the stop codon, resulted in stop codon (TAG) changing to glutamic acid (GAG), with the peptide chain extending 92 amino acid residues, and secondary and tertiary protein structure change, which were not found in the controls. The result of enzyme assay showed that the activity of GALNS enzyme in the affected child was 8.3 nmol/17h/mg pr, which was obviously lower than the normal value (the normal range is 41.9-92.1 nmol/17h/mg pr).

CONCLUSIONThese results illustrate that the c.1567 T to G is a novel pathologic mutation, which is the main cause of the disease in this family.

Amino Acid Sequence ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; Child ; Chondroitinsulfatases ; chemistry ; genetics ; metabolism ; Female ; Genotype ; Humans ; Infant ; Molecular Sequence Data ; Mucopolysaccharidosis IV ; enzymology ; genetics ; Mutation ; genetics ; Pedigree ; Protein Conformation ; Sequence Alignment

Recently many reports have described that recombinant baculovirus could serve as a new gene transfer vehicle for mammalian cells with many unique advantages. In this study, the constructed recombinant baculovirus BacV-CMV-EGFPA containing the enhanced green fluorescent protein (eGFP) gene driven by CMV promoter was used to explore the feasibility of improving the efficiencies of transduction experiment in CV-1 cells by centrifugal method. Refer to the centrifugal transduction protocol of recombinant lentivirus, CV-1 cells were incubated with the culture supematant of Sf-21 cells infected by BacV-CMV-ECFPA (moi = 30) and then centrifuged at 600g for 1 h at RT, reporter gene transfer and expression efficiencies were analyzed by flow cytometry (FCM) 48h post transduction. Results showed that centrifugal method can achieve higher gene delivery and expression efficiencies than transduction by simple virus-cell mixing for 4h at 27 t with least impairment to cell viability. The centrifugal transduction protocol was further optimized by testing different centrifugal times, post-centrifugation incubation times and surrounding solutions. We found that centrifugation at 600g for 1 h at RT is sufficient to achieve the highest transduction efficiencies in target cells and PBS is more suitable than other surrounding solutions. Compared with previous protocol in which tranduction occurs for 4 - 8h at 27 degrees C, centrifugal method developed in this study could achieve more higher transduction efficiencies in more shorter time. Nine different mammalian cell lines (CV-1, 293FT, HepG2, 293T, CHO, C127, MT4, H9, Molt-4) were used to investigate the feasibility of delivering exogenous genes into different mammalian cells with the BacV-CMV-EGFPA supernatant (moi = 30) at 600g for 1h in PBS surrounding solution at RT. Results showed that most mammalian cell lines used in this study could be effectively transduced with recombinant baculoviruses by centrifugal method, and more higher and satisfactory transduction efficiencies could be achieved in primate adherent culture cells than in suspended culture cells. These results show that the baculovirus centrifugal transduction protocol have notable advantages: more rapid, efficient and nontoxic, and could be easily used in daily common experiments.
Animals ; Baculoviridae ; genetics ; CHO Cells ; Cell Line ; Cell Line, Tumor ; Centrifugation ; methods ; Cricetinae ; Cricetulus ; Feasibility Studies ; Flow Cytometry ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Hep G2 Cells ; Humans ; Microscopy, Fluorescence ; Recombinant Fusion Proteins ; genetics ; metabolism ; Reproducibility of Results ; Spodoptera ; Transduction, Genetic ; methods

OBJECTIVETo identify potential mutation in a Chinese family featuring X-linked alpha thalassemia/mental retardation syndrome (ATR-X).

METHODSBased on clinical symptoms and inheritance pattern, linkage analysis of X chromosome short tandem repeats (X-STR) loci was carried out to locate the candidate gene. Subsequently, sequences of exons and exon-intron boundaries of the candidate gene were amplified with polymerase chain reaction (PCR). Potential mutations were detected by direct DNA sequencing. All patients were also analyzed for the trait of thalassemia.

RESULTSLinkage analysis indicated the candidate gene to be ATRX. Subsequently, a homozygous missense mutation c.736C>T (p.R246C) was found in exon 9 of ATRX in all of the 3 patients. And a heterozygous mutation c.736C>T (p.R246C) was also identified in the patient's mother and grandmother. Similar mutations were not detected in other members of the family. Alpha thalassemia was detected in the proband and another patient, whose genotypes were determined as -α(3.7)/αα and --(sea)/αα, respectively.

CONCLUSIONMissense mutation of c.736C>T in ATRX gene is a mutation hotspot, and p.R246C may disturb the function of ATRX-DNMT3-DNMT3L domain (ADD), which may be responsible for the disease in this family.

Asian Continental Ancestry Group ; genetics ; Child, Preschool ; DNA Helicases ; genetics ; DNA Mutational Analysis ; methods ; Female ; Humans ; Male ; Mental Retardation, X-Linked ; genetics ; Mutation, Missense ; Nuclear Proteins ; genetics ; Pedigree ; X-linked Nuclear Protein ; alpha-Thalassemia ; genetics

OBJECTIVETo explore whether normal platelet contains tissue factor (TF), and the significance of platelet-associated TF (PATF).

METHODSPlatelets were isolated by Sepharose 2B gel column. ELISA was used to detect the TF content in the lysates of washed platelets. Procoagulant activity of PATF was measured by one stage clotting time assay. The mRNA of TF was detected by reverse transcription polymerase chain reaction (RT-PCR).

RESULTSA certain amount of TF antigen (16.37 +/- 6.39) ng/L was detected in the washed-platelet lysates. Upon activation by collagen, platelets released TF and caused a marked increase in TF level in plasma (P <0.05). Resting platelets had no TF procoagulant activity, while procoagulant activity of platelets activated by collagen increased significantly, which could be blocked by TF McAb and poor VII plasma. TF mRNA could not be detected in washed platelets. TF content in platelets from patients with coronary heart disease was significantly higher than that from normal controls (P < 0.05). Resting platelets from the patients showed a higher procoagulant activity, which could be inhibited by TF McAb.

CONCLUSIONPlatelets contain TF and the latter released by activated platelet was functionally active. Platelet itself might not synthesize TF. Protein content and procoagulant activity of PATF in patients with coronary heart disease were higher than that in controls. All these indicate that platelet may be involved in coagulation and thrombosis by releasing TF.

Adolescent ; Adult ; Blood Platelets ; chemistry ; Coronary Artery Disease ; metabolism ; physiopathology ; Female ; Humans ; Male ; Middle Aged ; Platelet Activation ; Thromboplastin ; metabolism ; physiology

OBJECTIVETo sum up the experience in administering oral tadalafil on alternate days for the treatment of erectile dysfunction (ED) that fails to respond to on-demand medication.

METHODSWe retrospectively analyzed the clinical data of 15 cases of ED treated with oral tadalafil on alternate days from September 2010 to March 2012. All the patients had failed to respond to on-demand medication of sildenafil previously.

RESULTSAfter 4 weeks of tadalafil treatment, 11 (73.3%) of the cases were remarkably improved, with significant difference in IIEF-5 scores before and after treatment (P < 0.05). Transient adverse reactions were observed in the other 4 cases, including mild headache in 2, slight backache in 1, and facial flush in 1.

CONCLUSIONOral tadalafil on alternate days is safe and effective in the treatment of ED that fails to respond to on-demand medication of sildenafil.

Administration, Oral ; Adult ; Carbolines ; administration & dosage ; therapeutic use ; Erectile Dysfunction ; drug therapy ; Humans ; Male ; Phosphodiesterase Inhibitors ; administration & dosage ; therapeutic use ; Piperazines ; therapeutic use ; Purines ; therapeutic use ; Retrospective Studies ; Sildenafil Citrate ; Sulfones ; therapeutic use ; Tadalafil ; Treatment Failure ; Treatment Outcome

OBJECTIVETo assess the effect of a liquid embolic agent 2-poly-hydroxyethyl -methacrylate (2-P-HEMA) for renal artery embolization in rabbits.

METHODSThe precipitation time of different concentrations (2%, 3.5%, 5%, 6.5%, 8% and 9.5%) of 2-P-HEMA dissolved in different solutions (ethanol, ethanol/iobitridol, and ethanol/Bi2O3) were determined in flowing water. The mixtures of 2-P-HEMA (2%, 5%, and 8%) with ethanol/ Bi2O3 were injected into the renal arteries of the rabbits, and the artery-embolizing effects were assessed using angiography at 2 and 12 weeks after the injection, with also macroscopic and microscopic examination of the embolized kidneys.

RESULTSThe mixtures of 2-P-HEMA and ethanol formed flocculent precipitation a few seconds after injection into flowing water, and the precipitation time showed no significant variations with the concentration of 2-P-HEMA in the mixture. Low and moderate concentrations of 2-P-HEMA could pass through the microcatheter smoothly with little injection resistance, and resulted in complete occlusion of the renal arteries without adhesion to the microcatheter. Angiography at 2 and 12 weeks detected no recanalization of the occluded renal arteries. Macroscopically, the lumen of the renal arteries was found to be occluded by the embolic agents, and deep penetration of the embolic agents into the glomerular arteries was observed microscopically. The mixture containing high-concentration 2-P-HEMA was difficult to deliver through the microcatheter due to high injection resistance.

CONCLUSION2-P-HEMA can be rapidly precipitated after injection into flowing water, and allows complete embolization of the renal arteries of rabbits at proper concentrations, suggesting its great potential as an endovascular liquid embolic agent.

Animals ; Embolization, Therapeutic ; methods ; Female ; Male ; Polyhydroxyethyl Methacrylate ; Rabbits ; Radiography ; Random Allocation ; Renal Artery ; diagnostic imaging ; pathology