Delta6-fatty acid desaturase is a membrane-bound enzyme, which is rate-limiting for the biosynthesis of polyunsaturated fatty acids. A cDNA sequence putatively encoding a delta6-fatty acid desaturase was isolated from Rhizopus arrhizus NK300037 using RT-PCR and RACE methods in our previous work. Sequence and function analysis indicated that this sequence was a novel delta6-fatty acid desaturase gene which had an open reading frame of 1377bp coding 458 amino acids of 52kD. The methylotrophic yeast Pichia pastoris, has been developed into a highly successful system for the production of a variety of heterologous proteins during the past 20 years. In this work, the Rhizopus arrhizus delta6-fatty acid desaturase gene (RAD6) was subcloned into expression vector pPIC3.5K to generate a recombinant plasmid pPICRAD6, which was subsequently transformed into Pichia pastoris strain GS115 for heterologous expression by electroporation method. Total fatty acids were extracted from the induced cells and methylated. The resultant fatty acid methyl esters (FAME) were analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). Fatty acids analysis showed that the coding product introduced a new double bond at delta6 position of appropriate fatty acid substrates including C16:1, C17:1, C18:1, linoleic acid and alpha-linolenic acid without chain length specificity of fatty acids. Furthermore, modification of sequence flanking AUG codon of this delta6-fatty acid desaturase gene increased the expression of target gene in P. pastoris. All of these results suggest that P. pastoris is an optimal expression system of delta6-fatty acid desaturase gene.
Cloning, Molecular ; Electroporation ; Fungal Proteins ; biosynthesis ; genetics ; Genetic Vectors ; Linoleic Acid ; metabolism ; Linoleoyl-CoA Desaturase ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Rhizopus ; enzymology ; genetics

OBJECTIVETo observe the association between plasma endothelin (ET) concentration and blood pressure level in essential hypertensive (EH) patients with or without complications and possible impact of statins on ET concentration.

METHODSFrom Sep 2007 to Mar 2009, 149 patients with EH were analyzed [44 EH, 40 EH complicated by left ventricular hypertrophy (EH-LVH), 36 EH complicated by atrial fibrillation (EH-AF), and 29 EH complicated by lacunar infarction (EH-LI)], 30 healthy persons were selected as controls. EH patients were randomly divided into routine treatment group (calcium antagonists, ACEI, diuretics, beta-receptor blocker for 8 weeks) and simvastatin intervention group (routine treatment + simvastatin 40 mg/d for 8 weeks), plasma ET concentrations before and after drug intervention were measured.

RESULTS(1) ET concentration was higher in EH group than that in control group [(71.42 +/- 6.62) pg/ml vs. (45.52 +/- 8.28) pg/ml, P < 0.01]. ET concentration was higher in EH-LVH group, EH-AF group and EH-LI group than that in EH group [(97.67 +/- 10.53) pg/ml, (102.15 +/- 12.96) pg/ml, (103.49 +/- 9.91) pg/ml vs. (71.42 +/- 6.62) pg/ml, P <0.01]. The degrees of elevated blood pressure was positively correlated with ET concentrations(all P < 0.001). (2) The left atrial diameters of EH-AF group were positively correlated with ET concentration (r = 0.684, P < 0.001). The left ventricular mass index of EH-LVH group were positively correlated with ET concentration (r = 0.545, P < 0.001). (3) The percentages of class 3 hypertension in EH-LVH group, EH-AF group and EH-LI group were higher than that in EH group (57.5%, 50.0%, 62.1% vs. 25.0%, all P < 0.05). (4) Blood pressure in class 3 hypertension patient treated with simvastatin decreased more significantly than that in routine treatment group (P < 0.05). (5) ET concentration of class 2 hypertension patient treated with simvastatin decreased significantly than that in routine treatment group (P < 0.05). ET concentrations of class 3 hypertension patient treated with simvastatin and routine treatment patient decreased significantly after treatment (P < 0.05), and the former was lower (P < 0.05).

CONCLUSIONThe level of ET were positively correlated with the severity of EH. Simvastatin could decrease the ET levels of patients with EH and blood pressure levels of patients with class 3 hypertension. It suggested that therapeutic alliance of antihypertensive drugs and statins could be benefit to patients with EH.

Aged ; Aged, 80 and over ; Antihypertensive Agents ; therapeutic use ; Blood Pressure ; Endothelins ; blood ; Female ; Humans ; Hydroxymethylglutaryl-CoA Reductase Inhibitors ; therapeutic use ; Hypertension ; blood ; complications ; drug therapy ; Male ; Middle Aged ; Simvastatin ; therapeutic use

Objective To observe the protective effects of a mixture for protecting liver and supplementing stomach (保肝益胃合剂) on rat acute liver damage induced by carbon tetrachloride (CCl_4). Methods The model of rat acute liver damage was established by injection of CCl_4 2 ml/kg into the abdominal cavity.The rat models were treated respectively by the mixture for protecting liver and supplementing stomach 30 g?kg~(-1)?d~(-1),the polyene phosphatide acid radical choline capsule [Yi Shanfu (易善复), 180 mg?kg~(-1)?d~(-1)],the glycyrrhizic acid diaminogen capsule [Gan Lixin (甘利欣),30 mg?kg~(-1)?d~(-1)] infused into the stomach.The activities of serum alanine transaminase (ALT) and aspartate transaminase (AST) were detected.In the mean time,the liver pathological changes were observed,the degree of liver cell necrosis was evaluated,and the rat mortality was noted in various groups of treatment.Results The values of ALT,AST and the score of liver cell necrosis in the group treated with the mixture for protecting liver and supplementing stomach [(1.168?1.066) kU/L,(1.845?2.212) kU/L,(0.56?0.53) score] were significantly lower than those in the model group [(4.982?3.502) kU/L,(7.030?3.616) kU/L, (1.38?0.92) scores],and all the differences being statistically significant (all P

Adult ; Hepatitis B ; prevention & control ; Humans ; Immunity, Active ; Liver Transplantation ; immunology ; methods ; Male ; Postoperative Complications ; prevention & control

Adult ; Antigens, CD34 ; Antiviral Agents ; therapeutic use ; Bone Marrow Cells ; virology ; DNA, Viral ; blood ; Female ; Hepatitis B virus ; genetics ; physiology ; Humans ; Immunoglobulins ; therapeutic use ; Lamivudine ; therapeutic use ; Liver Transplantation ; Male ; Middle Aged ; Monocytes ; Postoperative Period ; Virus Replication

BACKGROUNDThe relationship between inflammatory markers and the characteristics of coronary atherosclerosis plaques is uncertain. The aim of the present study was to evaluate the relationship between the characteristics of coronary atherosclerosis plaques and inflammatory markers such as high sensitivity C-reactive proteins (Hs-CRP) and interleukin-6 (IL-6).

METHODSAll patients suspected of having coronary heart disease (CHD) underwent Siemens 64-slice CT angiography (64-SCTA) to distinguish the quality of plaque of coronary artery lesions. Blood samples were taken to measure levels of serum Hs-CRP and IL-6 in different plaque groups and the control group and compared with the value of 64-SCTA for detection of coronary artery plaque.

RESULTSThe sensitivity of detecting coronary artery plaque by 64-SCTA was 87.4%, the specificity was 87.1%, the positive predictive value was 82.2%, and the negative predictive value was 91.0%. Comparing the levels of serum Hs-CRP and IL-6 among plaque groups, the mean levels of serum Hs-CRP and IL-6 in three plaque groups were significantly higher than those in the control group (P < 0.01). The mean levels of serum Hs-CRP and IL-6 in the soft plaque group and mixed plaque group were significantly higher than those in hard plaque group (P < 0.01). Plaque burden in the soft plaque group and mixed plaque group was significantly higher than in the hard plaque group (P < 0.01), but there was no statistical difference between the soft plaque group and mixed plaque group (P = 0.246). There was a negative correlation between the CT scale and Hs-CRP and IL-6 levels in the soft plaque group (r = -0.621, P < 0.01, and r = -0.593, P < 0.01 respectively). There was a positive correlation between the plaque burden and Hs-CRP and IL-6 levels in the soft plaque group (r = 0.579, P < 0.05 and r = 0.429, P < 0.05 respectively).

CONCLUSIONS64-SCTA is an effective way to distinguish the different quality of coronary atherosclerosis plaque. Serum Hs-CRP and IL-6 levels can be considered as the indexes to judge the degree of CHD and may reflect the activity of plaque in CHD patients. Thus it is important for clinical diagnosis and risk evaluation of acute coronary syndrome (ACS) patients.

Aged ; C-Reactive Protein ; metabolism ; Coronary Artery Disease ; blood ; diagnostic imaging ; metabolism ; Female ; Humans ; Interleukin-6 ; blood ; metabolism ; Male ; Middle Aged ; Tomography, Spiral Computed

OBJECTIVETo research the clinical feasibility of emergency right lobe adult-to-adult live-donor liver transplantation in treating acute liver failure following severe hepatitis.

METHODSConsecutive ten severe hepatitis patients (4 acute-on-chronic severe hepatitis and 6 acute severe hepatitis; 9 caused by HBV and 1 with drug-induced acute liver failure) underwent emergency right lobe adult-to-adult live-donor liver transplantation in our hospital from April 2007 to December 2007. The +/- s of model for end-stage liver disease score was 33.22 +/- 6.55. The outcomes of these recipients were prospectively analyzed.

RESULTSAmong them, 8 ABO blood group were identical and 2 compatible. One was Rh sub-group negative. Except 2 recipients died (1 acute renal failure caused by veno cava thrombosis, 1 liver graft lose caused by hepatic artery thrombosis), the rest of recipients (80%) and all donors were safe. The mean graft-to-recipient weight ratio was (1.19 +/- 0.14)%, and graft volume to recipient estimated standard liver volume ratio was (65.13 +/- 8.75)%. Right lobe grafts with middle hepatic vein (MHV) 3 cases, without MHV 4 cases, without MHV but followed by V and VIII hepatic vein outflow reconstruction 3 cases. Encouraging outcome was achieved in this group of recipient: elevated serum creatinine, serum endotoxin, decreased serum prothrombin activity (PTA) and total bilirubin returned to normal about on postoperative day (POD) 3, POD 7, POD 14 and POD 28, respectively.

CONCLUSIONSOutcomes of emergency right lobe adult-to-adult live-donor liver transplantation for acute hepatic failure following severe hepatitis are fairly encouraging and acceptable. emergency right lobe adult-to-adult live-donor liver transplantation is an effective and life-saving modality for acute liver failure following severe hepatitis.

Adult ; Female ; Follow-Up Studies ; Hepatitis ; complications ; Humans ; Liver Failure, Acute ; etiology ; surgery ; Liver Transplantation ; methods ; Living Donors ; Male ; Middle Aged ; Prospective Studies ; Treatment Outcome

OBJECTIVE: To investigate the changes of incidence and prognosis of epithelial ovarian cancer in thirty years in Taiwan. METHODS: The databases of women with epithelial ovarian cancer during the period from 1979 to 2008 were retrieved from the National Cancer Registration System of Taiwan. The incidence and prognosis of these patients were analyzed. RESULTS: Totally 9,491 patients were included in the study. The age-adjusted incidences of epithelial ovarian cancer were 1.01, 1.37, 2.37, 3.24, 4.18, and 6.33 per 100,000 person-years, respectively, in every 5-year period from 1979 to 2008. The age-specific incidence rates increased especially in serous, endometrioid and clear cell carcinoma, and the age of diagnosis decreased from sixty to fifty years old in the three decades. Patients with mucinous, endometrioid, or clear cell carcinoma had better long-term survival than patients with serous carcinoma (log rank test, p<0.001). Patients with undifferentiated carcinoma or carcinosarcoma had poorer survival than those with serous carcinoma (log rank test, p<0.001). The mortality risk of age at diagnosis of 30-39 was significantly higher than that of age of 70 years or more (test for trend, p<0.001). The mortality risk decreased from the period of 1996-1999 (hazard ratio [HR], 0.90; p=0.054) to the period after 2000 (HR, 0.74; p<0.001) as compared with that from the period of 1991-1995. CONCLUSION: An increasing incidence and decreasing age of diagnosis in epithelial ovarian cancer patients were noted. Histological type, age of diagnosis, and treatment period were important prognostic factors for epithelial ovarian carcinoma.
Carcinoma ; Carcinosarcoma ; Female ; Humans ; Incidence ; Mucins ; Neoplasms, Glandular and Epithelial ; Ovarian Neoplasms ; Prognosis ; Taiwan

OBJECTIVESTo study the active immunity response of liver transplant patients for HBV-related diseases after hepatitis B virus (HBV) vaccine immunization and to investigate the factors that influence the effectiveness of the vaccination in order to find measures to increase its success.

METHODSThirteen patients who had liver transplants because of HBV-related end-stage liver diseases received hepatitis B virus immunoglobulin and lamivudine for an average of 38 months (range 27-77 months). They received double intramuscular doses (40 microg) of a recombinant vaccine at months 0, 1, 2 and 6. The anti-HBs titers were tested regularly at months 1, 2, 3, 6 and 7.

RESULTSSeven of the 13 patients (53.8%) developed higher serum titers of anti-HBs compared with their titers prior to the vaccinations, 2 patients of the 13 (15.4%) developed an increase by 100 U/L and in 4 patients (30.8%) their base levels were doubled. Those responding patients were followed-up for another 8 months after the fourth vaccination, and only 1 patient among them had a decrease of the anti-HBs titers below the level prior to the vaccination.

CONCLUSIONHepatitis B vaccine immunization can be used to enhance the active immunity against HBV in patients who had liver transplants for HBV-related diseases.

Adult ; Female ; Hepatitis B ; immunology ; Hepatitis B Vaccines ; therapeutic use ; Hepatitis B virus ; Humans ; Immunity, Active ; Liver Diseases ; immunology ; virology ; Liver Transplantation ; Male ; Middle Aged ; Postoperative Period

OBJECTIVETo observe the morphological and molecular biological peculiarities of the experimental autoimmune prostatitis (EAP) rat model made by SC purified prostate protein twice with immune adjuvant.

METHODSMale rats were intradermally immunized with a saline extract of male rat prostate glands (RPG) in Freund's complete adjuvant (FCA) and Pertussis-Diphtheria-Tetanus vaccine 0.5 ml i.p. at the 0 and 30th day, and the concentrations of the extract were respectively 5 mg/ml, 10 mg/ml and 15 mg/ml. At the 45th day, the rats were sacrificed and the morphological and molecular biological changes of the prostate specimens were observed to determine the effective concentration of RPG for a successful model.

RESULTSThe expression of inflammation genes such as TNF-alpha, IL-1beta, IL-2 and iNOS obviously increased in the high-dosage model group; LM, EM and in situ hybridization revealed appearant chronic inflammation response, but this was not the case in the other two dosage groups.

CONCLUSION15 mg/ml RPG mixed with FCA (1:1) 1.0 ml SC with Pertussis-Diphtheria-Tetanus vaccine 0.5 ml i.p. was an effective dosage for the successful model in our experiment.

Animals ; Autoimmune Diseases ; immunology ; metabolism ; pathology ; Diphtheria-Tetanus-Pertussis Vaccine ; administration & dosage ; Disease Models, Animal ; Freund's Adjuvant ; administration & dosage ; Injections, Intraperitoneal ; Male ; Prostate ; metabolism ; pathology ; Prostatitis ; immunology ; metabolism ; pathology ; Proteins ; administration & dosage ; Rats ; Rats, Wistar