粪产碱杆菌青霉素G酰化酶的分离纯化及酶学性质

采用DEAE-Sepharose阴离子交换层析和Butyl-Sepharose CL 4B疏水层析,从重组大肠杆菌DH5a菌体中分离纯化得到粪产碱杆菌青霉素G酰化酶(AfPGA).经纯化,AfPGA纯度较粗酶提高50.6倍、比活达到212.7 U/mg,经HPLC测定纯度为92.8%,收率为78%.理化性质分析表明:AfPGA含有2个亚基(α亚基和β亚基,其Mw分别为2.90×104和5.92×104;AfP-GA等电点约为7.9～8.0,最适pH约为10.0,并在pH>7.0时表现出了稳定的催化活力.     

粪产碱杆菌青霉素酰化酶在枯草芽孢杆菌中的表达优化

在5L发酵罐中进行了表达粪产碱杆菌青霉素G酰化酶的重组枯草杆菌WB600(pMA5)的发酵研究。实验表明，发酵液中的葡萄糖浓度和菌体产酶能力密切相关，维持低葡萄糖水平，可以提高枯草杆菌WB600(pMA5)青霉素G酰化酶的合成能力。采用混合碳源进行发酵，在生长阶段流加葡萄糖，并维持低浓度，在发酵12h菌体浓度达到约13．8g／L时停止流加葡萄糖，使茵体利用发酵液中的可溶性淀粉作为碳源，避免葡萄糖的代谢副产物对茵体产酶能力的抑制，青霉素G酰化酶的表达水平从原来的55u／L提高到582u／L。     

粪产碱杆菌来源的青霉素G酰化酶拆分制备（S)-2-氨基-4-苯基丁氨酸

粪产碱杆菌来源的青霉素G 酰化酶（Alcaligenes faecalis penicillin G acylase ,AfPGA）可以对映选择性地水解N苯乙酰苯基丁氨酸制备（S）-2-氨基-4-苯基丁氨酸。反应体系的pH及温度对酶的对映选择性影响不显著。研究表明,水饱和的乙酸乙酯体系要优于传统的水相体系,该体系显著提高了底物N苯乙酰化苯基丁氨酸的溶解度,并可简化产物的分离过程。当底物的转化率为48.6%时,（S）2氨基4苯基丁氨酸的光学纯度（e.e.值）为99.8%。     

固定化青霉素酰化酶在光敏感可再生两水相体系中催化青霉素G为6-APA

在一种光敏感可再生高聚物(PNBC 300000)与葡聚糖20000(Dextran 20000)形成的两水相体系中进行固定化青霉素酰化酶的相转移催化青霉素G产生6-APA的反应。在这个两水相体系中,当pH为7.8,底物浓度为62 mmol/L,反应温度为20℃,在50 mmol/L KCl存在下,6-APA的分配系数可达8.4。催化动力学显示,达到平衡的时间近6 h,PG(Na)转化率约82.6%。3批半连续反应转化率为60%~70%,较相近条件下的单水相反应得率提高近20%。在两水相中,底物及产物主要分配在上相,固定化酶分配在下相,底物青霉素G进入下相经酶催化产生的6-APA及苯乙酸又转入上相,从而解除了青霉素酰化酶催化反应的底物及产物抑制作用,达到提高产物得率的效果。形成两水相的高聚物通过488 nm的激光照射可实现循环利用,高聚物的光照回收率在95%~98%。     

聚丙烯酸载体用于青霉素酰化酶的固定

以反应性单体丙烯酸和交联剂二乙烯基苯,以石油醚为致孔剂,通过悬浮聚合制备固定化酶的载体,并用于对青霉素酰化酶的固定。研究了丙烯酸与二乙烯基苯以不同摩尔比对青霉素酰化酶固定活性的影响,以及悬浮聚合时水油相比例的不同所合成的载体对固定化酶性能的影响。当丙烯酸和二乙烯基苯摩尔比为84.2:4时合成的载体固定青霉素酰化酶的酶活为2784U/g,而水油相比为2.75:1（丙烯酸和二乙烯基苯摩洋比为84.2:5）时固定青霉素酰化酶活达到2183U/g。固定青霉素酰化酶可使青霉素转化,得到半合成青霉素的中间体6-氨基青霉烷酸,由此可制成高效、广谱、服用方便的新青霉素。     

聚甲基丙烯酸缩水甘油酯固定化青霉素酰化酶性质的研究

应用反相悬浮技术合成了珠状甲基丙烯酸缩水甘油酯 - N ,N′-亚甲基双 (丙烯酰胺 )共聚物 ,并将巨大芽孢杆菌 ( Bacillus megaterium)青霉素酰化酶共价偶联到甲基丙烯酸缩水甘油酯共聚物载体上 ,制成固定化青霉素酰化酶 ,其表观活性为 3 71 U/g(干重 ) ,水解青霉素 G钾盐的最适温度为 47°C,最适 p H为 8.5 ,在 p H4.5～ 9.0 ,温度 40°C以下时酶的活性稳定 ,表观米氏常数 Km为 1 .3 3× 1 0 -2 mol/L ,最大反应速度 vmax为 1 .2 7× 1 0 -5mol/min,固定化酶在 4°C冰箱保存 3 5 d,再水解 w=0 .0 2的青霉素 G钾盐溶液 ,重复使用 3 0次 ,保留酶活性 88.1 %。     

医院内携带的葡萄球菌对8种抗生素的敏感度

测定216株葡萄球菌(126株金葡菌,90株凝固酶阴性葡萄球菌)对8种临床常用抗生素药敏。结果为:医院内携带的金葡菌对青霉素G、红霉素、林可霉素耐药率分别为76.42%、61.36%、27.26%,对头孢噻啶、利福霉素敏感性较好。医院内表葡菌对青霉素G、红霉素、林可霉素耐药率分别达65.29%、58.82%、50%。故治疗医院内金葡菌、表葡菌感染时要选用有效药物。医院内携带的金葡菌产酶率81.40%,CNS产酶率71.11%,绝大多数产酶株对青霉素G耐药,头孢噻啶对产酶株有良好的抗菌活性。     

青霉素酰化酶研究——Ⅰ.抑制作用,纯化和性质

丙酮、乙醇和苯丙氨酸是大肠杆菌青霉素酰化酶的竞争性抑制剂,分别抑制模拟底物3-苯乙酰胺基6-硝基苯甲酸水解的活力。青霉素酰化酶粗酶液经硫酸铵盐析,pH5沉淀,SE-Sephadex C50层析,苯丙氨酸-Sepharose 4B疏水层析和DEAE-Sephadex A25层析可得纯酶,聚丙烯酰胺凝胶梯度电泳呈现一条带,比活为27.5u/mg蛋白。纯化总活力回收率为28.1%。青霉素酰化酶分子量约为90000,有两个亚基,分子量分别为70000和20000,其最适温度为40℃,最适pH为8.0,催化青霉素G水解的米氏常数为2.94mmol/L。     

在可回用两水相体系中进行青霉素酰化酶的相转移催化裂解青霉素G为6-APA

在光敏感可回用高聚物PNBC与pH敏感型可回用高聚物PADB形成的两水相体系中进行同定化青霉素酰化酶的相转移催化青霉素G产生6-APA的反应.在这个两水相体系中,通过优化,在1%NaCl存在下,6-APA的分配系数可达5.78.催化动力学显示,达平衡的时间近7 h,反应最高得率约85.3%(pH 7.8,20℃).较相近条件下的单水相反应得率提高近20%.在两水相中,底物及产物主要分配在上相,固定化酶分配在下相,底物青霉素G进入下相经酶催化产生的6-APA及苯乙酸义转入上相,从而解除了青霉素酰化酶催化反应的底物及产物抑制作用,达到提高产物得率的效果.此外,采用周定化酶较固定化细胞效率高,占用下相体积小,较游离酶稳定性高,且完全单侧分配在下相.因此,在两水相中进行固定化酶的催化反应具有明显的优越性.形成两水相的高聚物PNBC通过488 nm的激光照射或经滤光的450nm光源照射,pH敏感型成相聚合物PADB可实现循环利用,高聚物的回收率在95%～98%之间.     

本院230株肠球菌的鉴定及耐药性分析

目的 研究尿路感染中肠球菌的鉴定及耐药性.方法 常规分离菌株并鉴定,药敏试验采用K-B法,并进行高水平耐庆大霉素的肠球菌筛选;用头孢硝噻吩纸片法检测β-内酰胺酶.结果 230株肠球菌中,粪肠球菌123株(53.5%),屎肠球菌73株(31.7%),鸟肠球菌34株(14.8%),对青霉素和红霉素具有较高的耐药性,对呋喃妥因、万古霉素仍保持很好的敏感性.检出高水平耐庆大霉素的肠球菌168株(73.0%).β-内酰胺酶粪肠球菌产酶率4.9%,屎肠球菌产酶率8.2%,鸟肠球菌产酶率5.9%.结论 导致尿路感染的肠球菌中,常见的是粪肠球菌、屎肠球菌、鸟肠球菌.临床分离的肠球菌属不仅要鉴定到种并要尽早进行药敏试验,以便合理的应用抗生素治疗.     

Value of D-Dimers in patients with acute aortic dissection

Objective: To evaluatel the value of D-dimers in patients with acute aortic dissection (AAD). Methods: This study consisted of 16 patients with AAD and 27 non-AAD patients. Serum D-dimets were measured by Sta-Liatest D-DI immunoturbidimetric assay. Results: D-dimer level was higher (P ＜ 0.001) in patients with AAD(7.91 ± 5.52 μg/ml) than that in non- AAD group(1.57±1.24 μg/ml). D-dimer was positive (＞0.4 μg/ml) in all patients with AAD and in 10 control group patients (37%). Among patients with acute AAD, D-dimers tended to be higher in Stanford A than in Stanford B (8.67 ± 4.31 μg/ml vs. 3.24±1.27 μg/ml, P ＜0.01). D-dimer values tended to be higher in more extended disease(3.84 ± 1.65 μg/ml, 8.57 ± 3.58 μg/ml and 11.87 ± 5.69 μg/ml in thoracic aorta, thoracic and abdominal aorta, thoracic and abdominal aorta and iliacal arteries, respectively, P ＜ 0.05 for both 8.57 ± 3.58 and 11.87 ± 5.69 vs. 3.84 ± 1.65 ). Including the control group into the analysis, we found a sensitivity of 100%, a negative predictive value of 100%, and a specificity of 66% and a positive predictive value of 64% for D-dimer in diagnosis of AAD in our patients with suspected AAD. Conclusion: D-dimer was elevated in patients with AAD. A negative D-dimer test result could be useful in excluding AAD.     

Research of combined liver-kidney transplantation model in rats

Objective： To set up a simple and reliable rat model of combined liver-kidney transplantation. Methods： SD rats served as both donors and recipients. 4℃ sodium lactate Ringer＇s was infused from portal veins to donated livers,and from abdominal aorta to donated kidneys, respectively. Anastomosis of the portal vein and the inferior vena cava （IVC） inferior to the right kidney between the graft and the recipient was performed by a double cuff method, then the superior hepatic vena cava with suture. A patch of donated renal artery was anastomosed to the recipient abdominal aorta. The urethra and bile duct were reconstructed with a simple inside bracket. Results： Among 65 cases of combined liver-kidney transplantation, the success rate in the late 40 cases was 77.5%. The function of the grafted liver and kidney remained normal. Conclusion： This rat model of combined liver-kidney transplantation can be established in common laboratory conditions with high success rate and meet the needs of renal transplantation experiment.     

Development on Adjustable Calibration Marker for Shock Wave Focus

Shock wave lithotripsy （SWL） is a treatment of choice for upper urinary stones. However, this procedure is inappropriate for obese patients because the focus is often unable to reach the target owing to the limited focal distance in shock wave source. Although treating such patients in a blast path may increase the application length of shock wave source, it＇s difficult to find this path on the lithotripter monitor. For this reason, we invented an adjustable calibration marker in order to set an effective focus in the shock wave hath.     

Study on the TCM Pathogenesis of Angiopathic Complications of Type II Diabetes

Excess production of reactive oxygen species(ROS)of mitochondrion mediated by hyperglycemia is the common pathogenesis of angiopathic complications of diabetes.TCM holds that the damp from the dysfunction of spleen.kidney and liver is the causative factor of complications of diabetes.This is similar to the mechanism of Ros resulting in angiopathic complications of diabetes.When the angiopathic complications of type II diabetes mellitus(T2DM)are difierentiated as caused by turbid damp in TCM can be explained as ROS.Since the obstruction of pathogenic damp in channels and collaterals is said to be the main pathogenesis,the treating principle should be dissolving the damp to remove the obstruction.     

Leptin, Ghrelin and TNF-α before and after Hypo-caloric Traditional Chinese Diet and Auricular Acupuncture

INTRODUCTION Obesity is a complex emergent problem, which can be possibly solved not only by the diet but also by the life style and promotion of a constant physical exercise. 1, 2 No doubt careful attentions must be given to the nutritional condition of obese people, the dietary habits, the somatic build (i.e. distribution of fat mass) and the organic functions linked to formation of the fat mass. All the parameters should be constantly monitored before, during and after a diet treatment. 3, 4, 5     

BLOOD PRESSURE CHANGE WITH AGE IN SALT-SENSITIVE TEENAGERS

Objective To observe blood pressure change with age in salt-sensitive teenagers whose salt sensitivity were determined by repeated testing.Methods Salt sensitivity was determined through intravenous infusion of normal saline combined with volume-depletion by oral diuretic furosemide in 55 teenagers. After five years, salt sensitivity was re-examined and subject blood pressure was followed up. Blood pressure changes in salt-sensitive teenagers were compared to that of non-salt sensitive teenagers over five years.Results After 5 years, the repetition rate of salt sensitivity determined by intravenous saline loading is 92.7%. In teenagers with salt sensitivity on the baseline, both the systolic blood pressure increments and increment rates were much higher than non-salt sensitive teenagers (12.7±12.1 mmHg vs. 2.8±5.2 mmHg, P＜ 0.01; 12.2%± 12.0% vs. 2.5% ±4.4%, P＜ 0.001,respectively). There was a similar trend for diastolic blood pressure (8.4 ± 6.4 mmHg vs. 3.7 ± 6.4 mmHg, P = 0.052; 13.2% ±10.6 % vs. 6.8%± 10.1%, P = 0.053, respectively).Conclusions Salt sensitivity determined by intravenous saline loading showed good reproducibility. Blood pressure increments with age were much higher in salt-sensitive teenagers than non-salt sensitive teenagers, especially in terms of systolic blood pressure.     

Relationship between Dysglycemia and Carotid Atherosclerosis in Tibetan Population

People with dysglycemia are at high risk for atherosclerotic diseases. This study aims at investigating the atherosclerotic vascular damage in dysglycemia and its metabolic origin in Tibetan population.     

APPLICATION OF LORNOXICAM TO PATIENT-CONTROLLED ANALGESIA IN PATIENTS UNDERGOING ABDOMINAL SURGERIES

FOR anesthesiologis s ,treatingpostoperativepainhas alwaysbeen a problem.Althoughopioidshave been provedtobe effective,theirsideeffectscouldnotbeignored.With thedevelopmentofscienceand pharmacology,many drugs with aspectsof satisfactoryanalgesicefficacyand couldbe welltoleratedby patientshave been developed.And lornoxicamisone of them, which isa non-steroidalanti-inflammatorydrug (NSAID ), with analgesic, anti-infl-ammatory,andantipyreticproperties.Itseliminationhalf-time(3 to 5 hours) isle…     

CLINICAL SIGNIFICANCE OF SERUM CYTOKINES IL-1β,sIL-2R,IL-6,TNF-α,AND IFN-v IN ACUTE CORONARY SYNDROME

Objectives To explore serum cytokines levels (including IL-1 β, sIL-2R, IL-6, TNF-α, and IFN-v) and their significance in patients with acute coronary syndrome (ACS) and the subsequent follow-ups, with attempt to estimate the role of various serum inflammatory markers in the diagnosis and assessment of ACS.Methods The study population include 40 patients with acute myocardial infarction (AMI), 40 patients with unstable angina pectoris (UAP), and 40 controls. Among the 80 patients, 60 patients attended a follow up 4 months later. Serum inflammatory markers including IL-1 β, sIL-2R, IL-6, TNF-α, and IFN-v were measured by enzyme linked immunosorbent assay.Results Serum IL- 1 β, sIL-2R, IL-6, TNF-α were significantly higher in AMI group or UAP group compared to the control group and became significantly lower 4 months later in the follow-up patients. Serum levels of IFN-v shows no significant difference between AMI group or UAP group and controls, also showing no significant change when measured in follow up patients. There was no correlation between serum creatine kinase-MB isoenzyme levels and serum inflammatory markers either in UAP or AMI group. Furthermore, when divided into two subgroups using Wagner's QRS scoring system in the AMI group, there is no difference of each serum inflammatory marker between ≤ 6 scores group and ＞ 6 scores group.Conclusion Serum levels of certain inflammatory markers may have some diagnostic value for ACS, and can be a useful marker reflecting disease stability.     

Possible Spread of adenovirus type 3 from poultry to humans ：indirect evidence from an outbreak in China

Objective:To explore the epidemiology and etiology for an outbreak of acute respiratory tract infection that occurred in one county of Jiangsu Province, China 2004. Methods: Only cases meeting the case definition were included in the study. We reviewed the medical records of the cases who were admitted to the local hospitals, interviewed cases by a standard questionnaire, and then described the epidemiotogic features and analyzed risk factors by means of a case-control study. We collected pharyngeal swab specimens and sent them to different laboratories for isolation and culture. The laboratory used different detection methods such as DIP, PCR, electron microscope examination and microneutralization assay, to identify and then type the positive specimens. Results:A total of 871 cases were reported during the period from April 18 to July 4,2004. The distribution of onset times presented two peaks, one in late May and another in middle June. The epidemic occurred mainly in the elementary and junior high schools in ten townships of one county, and the mean age of the cases was 12 years (range 7 months to 18 years). The course of the disease was acute, and was characterized by fever accompanied with sore throat and tonsillitis. The WBC count of cases was normal or elevated. The mean duration of illness was 5 days (range 2 to 12 days). No fatalities from illness were reported. A case-control study indicated that the possible risk factors were close contact with a case and/or poultry before onset and sharing of towels among members of the family. The typical CPE was observed through inoculating pharyngeal swab specimens into the HEP-2 cell cultures in different laboratories. An infection of adenovirus type 3 was verified by detecting positive specimens in different methods. Conclusion:This investigation demonstrated that the acute respiratory infection in cases was caused by adenovirus type 3. Cases occurred in over 70 schools in ten townships in 2004, and the route of transmission was possibly close contact with cases or droplet transmission.