Restriction endonucleases are important molecular biology tools for DNA recombination.Because of the cleavage of DNA,their recombinant expression is difficult with low yields and complicated purification processes.In commercial productions,the technology that uses specific methylases to protect host DNA from digestion of the expressed restriction enzymes was cumbersome and practically limited.For solving this problem the expression of restriction enzyme Not Ⅰ was performed by using the DNA methylase M.Sss Ⅰ derived from Spiroplasma sp.MQ1 which specifically kept CpG sequence methylated.The methylated DNA was protected from the cutting of Not Ⅰ whose recognition sequence contained CpG.The gene of methylase M.Sss Ⅰ was introduced into Escherichia coli ER2566 and constitutively expressed,resulting in the CpG methylation pattern of the host DNA.Restriction enzyme Not Ⅰ was successfully expressed in this E.coli strain.Furthermore,by adding a purification tag to one terminus of the enzyme,recombinant Not Ⅰ was prepared as a highly purified and active product through two simple Ni-affinity and anion exchange chromatography steps.This expression system can be applied for the preparation of a series of restriction enzymes with CpG in their recognition sequences.

In order to investigate the affinity of aptamers to Bacillus anthracis spore, a custom synthesized 78 mer random DNA library was subjected to 15 rounds of selection against spores of vaccine strain A.16R by using SELEX method. The selected aptamers were cloned and sequenced. Macaw 2.05 and DNAsis 2.5 package were employed to analyze the conserved sequences and second structure of the aptamers, respectively. Affinities of aptamers to the spores were visualized by biotin streptavidin horseradish peroxidase system. The results showed that affinities of the aptamers were different. The highest OD at 450nm was 1.2, and the lowest was 0.25. The second structure analysis revealed possible stem loops for binding to the spores. The conserved sequences, AGGGG, CCCCG, GGGTT and ACACT, were found and the aptamers having same conserved sequence demonstrated similar affinity to the spores.

The primary structure of rhFK506-Binding Protein (FKBP) 12 was studied with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) by measuring its molecular weight and tryptic peptide mass fingerprinting.The calculated molecular weight of rhFKBP12 was 11819.5 and the measured value was 11818.0. The relative error was only 0.01%. The measured peptide mass fingrprinting of rhFKBP12was identical with that deduced from its amino acid sequence. It proved that the primary structure of rhFKBP12 was correct and there was no amino acid deletion, mutation and modification in its expression, refolding andpurification.

Objective To improve the awareness,diagnosis and treatment of pneumocystis carinii pneumonia (PCP) after renal transplantation.Methods A retrospective review was performed in 28 patients who underwent renal transplantation and developed PCP afterwards.The main clinical manifestations were fever(28 cases),nonproductive cough(28 cases),chest distress (12 cases).Occurrences of PCP were described 1.5 to 7 months after the renal transplantation.There were 10 patients treated with tacrolimus (FK506 2-6 rag/d,FK506 concentration 4-10 ng/ml) and 18 patients treated with cyclosporine (CsA 200-500 mg/d,CsA trough level:150-250 ng/ml) based immunosuppressive regimen.Anti-CD_(25)~+ monoclonal antibody (anti-CDCD_(25)~+mAb) was used in 10 cases for immune induction before operation while single steroid in 18 cases.Creatinine of patients with PCP was 70 to 106 μmol/L.CD_4~+ lymphocyte counts of the peripheral blood were 245±32/μl before PCP treatment and 536±25/μl after recovery.The most abnormal chest radiological findings were bilateral patchy ground-glass opacity.All the patients were diagnosed with PCP by bronchoalveolar lavage.Treatment was performed by reducing immunosuppressive agents and giving SMZco.Nineteen patients who had a PaP2 less than 70 mm Hg were given intravenous small-dose steroid.Results All the patients recovered from PCP 2 to 3 weeks after treatment.One patient experienced recurrence half year later.Five patients with higher creatinine after treatment recovered to normal levels after stopping the treatment of SMZco.No significant differences were seen in PCP patients treated with CsA and FK506,P＞0.05.The similar results were observed in use of anti-CDCD_(25)~+ mAb and single steroid,P＞0.05.Significant differences were observed in PCP patient peripheral blood CD_4~+ lymphocyte counts before and after treatment (P=0.001).Conclusions Patients who have fever,cough and hypoxia,chest imaging showing bilateral lung interstitial inflammation,might be PCP patients in the early post-renal transplantation period.Effective treatment should be performed by reducing immunosuppressive agents and giving SMZco.

Japanese encephalitis virus (JEV) is a single-stranded and positive-sense RNA, which has a single ORF (open reading frame), encoding a polyprotein precursor. Non-structural protein 3 (NS3) plays an important role in processing the polyprotein precursor and has become an important drug target of flavivirus. In this study, NS2BH-NS3 gene was amplified by PCR and subcloned to the prokaryotic expression plasmid, resulting pET30a-NS2BH-NS3. The fusion protein was expressed in Escherichia coli BL21 (DE3) in soluble form after induction by Isopropyl beta-D-1-Thiogalactopyranoside (IPTG). The recombinant protein was purified by Ni-NTA affinity column. Then a fluorescence resonance energy transfer (FRET) method was used to determine enzymatic activity and the assay conditions were optimized. After screening 113 compounds, we found two compounds inhibiting the activity of NS2BH-NS3. This study provides a convenient and cost-effective method for screening of JEV NS3 protease inhibitor.
Encephalitis Virus, Japanese ; enzymology ; Escherichia coli ; metabolism ; High-Throughput Screening Assays ; Protease Inhibitors ; chemistry ; RNA Helicases ; metabolism ; Recombinant Fusion Proteins ; metabolism ; Serine Endopeptidases ; metabolism ; Viral Nonstructural Proteins ; metabolism

AIM: To study the changes of serum autoantibodies against ? 1- adrenergic and M 2-muscarinic receptors in obstructive sleep apnea syndrome (OSAS) patients with chronic obstructive pulmonary disease (COPD), that is, overlap syndrome (OS). METHOD: Serum autoantibodies against ? 1 and M 2 receptors in 26 cases with OS, 32 with OSAS, 30 with COPD and 28 normal subjects were determined by using enzyme-linked immunosorbent assay(ELISA). RESULTS: The positive rates and titer of ? 1 and M 2 receptor autoantibodies are significantly increased in OS group (92.2%,57.7% and 1∶98, 1∶67), compared to OSAS (71.9%, 40.6% and 1∶83, 1∶30) or COPD group (70.0%, 36.7% and 1∶79, 1∶28) (P

Objective To investigate the relationship between the disorder of learning and memory in lead rats and the argnine vasopressin (AVP) and the somatotatin (SS) of hippocampus.Methods Sixty healthy 2-month old Sprague-Dawley rats were randomly divided into normal control group and lead group. Normal control group was given deionized water and the lead group was given 0.1% lead acetate deionized water for 3 months to make chronic lead rat. Learning and memory ability was tested by the Y-maze test. The content of AVP and SS in hippocampus were tested by the immunohistochemical method. Results The learning memory ability and the content of AVP and SS in hippocampus CA1 section of the lead group were degraded obviously than the control group(all P0.05)in hippocampus CA3 section of the lead group were degraded also.Conclusion The disorder of the learning and memory of the lead exposure rats may be due to the decrease of the content of AVP and SS in hippocampus.

Objective To investigate the factors related with plagiocephaly and brachycephaly. Methods 239 infants with plagiocephaly and brachycephaly were investigated, and analyzed with univariate analyses and multivariate Logistic regression analysis. Results The factors, such as gestational age birth (OR=0.636, P<0.001), birth weight (OR=0.095, P<0.001), time of hospitalization (OR=1.307, P<0.001), preterm birth (OR=2.649, P<0.001), stay in newborn intensive care unit (OR=4.456, P<0.001), change the position (OR=0.046, P<0.001), accepted early intervention guidance (OR=0.054, P<0.001), were significantly related with plagiocephaly and brachycephaly. Conclusion Preterm birth, low birth weight, and newborn complications are the risk factors for plagiocephaly and brachycephaly, while change the position and early intervention may prevent it.

AIM: To observe the clinical effects of intravitreal injection of triamcinolone acetonide ( TA ) before vitrectomy for retinal detachment associated with choroidal detachment. · METHODS: Totally 23 cases ( 23 eyes ) of retinal detachment associated with choroidal detachment in our hospital were treated by intravitreal injection of TA 4-5d before 23-Gauge micro-invasive vitrectomy combined with silicone oil injection. All the cases were followed up between 6 to 9mo. The anatomic retinal reattachment, visual acuity, intraocular pressure and postoperative complications were observed and analyzed. ·RESULTS: After the surgery, the visual acuity of all patients were improved, with 9 eyes better than 0. 3 (39%), and 18 eyes better than 0. 05 (78%). The BCVA at 1wk, 1 and 3mo and last follow up were different compared with before operations (P<0. 05). The mean intraocular pressure was 4. 02±1. 47mmHg before injection, 13.69±4. 68mmHg before operation (P<0. 05), and17.72±5.87 mmHg after operation (P<0.05). The retina of all patients treated were reattached 2wk post-operatively. The retinal reattachment rate after the primary surgery and the secondary surgery was 87% and 100%, respectively. Post-operative complications included 7 eyes of transient high intraocular pressure, occurred during 12-14d after operations and returned to normal after less glucocorticoid eye drops and giving IOP lowering drugs. There were no intraocular hemorrhage, iatrogenic retinal breaks, infections, or lens injuries. · CONCLUSION: Intravitreal injection of TA before vitrectomy for retinal detachment associated with choroidal could improve the clinical effects, and decrease the difficulty of surgery while the injection itself is pretty safe.

Objective:The purpose of this study is to investigate the expression of tumor stem cell marker aldehyde dehydrogE-nase 1 (ALDH1) in breast cancer and its clinical significance. Methods:The expression of ALDH1 protein was examined by immunohistochemical staining in 92 breast cancer tissues. The correlation analysis and diseasE-free survival analysis of patients was evaluated based on the clinical follow-up data. Results:Expression of ALDH1 protein had a significant correlation with progesterone receptor (PR) and cerb-B2 (P<0.05), but had no significant correlation with age, tumor size, clinical staging, and lymph node metastasis (P>0.05). The 2-year diseasE-free survival rate of AlDH1-positive patients was lower than that of ALDH1-negative patients (P<0.05). ALDH1-positive patients, who received CEF regimen chemotherapy and hormone therapy, had lower 2-year diseasE-free survival rate than that of ALDH1-negative patients (P<0.05). Conclusion:The decreased diseasE-free survival rate of ALDH1-positive patients is related with drug resistance. ALDH1 could be used as an independent factor for predicting the prognosis of breast cancer.