The complete coding sequences of Eya gene family was amplified by standard PCR fromhuman tissues or cells cDNA library.The product of PCR was cloned into the eukaryotic expression vector pcDNA3-FLAG,generating pcDNA3-FLAG-Eya1～4.Thenhuman embryo kidney 293T cells were transfected with the recombinant plasmids and the expression of Eya genes were identified by Western blot.Transcriptional assay using a reporter containing myogenin enhance factor indicated that expression of Eya cooperation with Six in 293T cells affected the Myogenin gene expression.The expression vectors of Eya genes were constructed and confirmed by restriction enzyme digestion and DNA sequence analysis.Transcriptional assay using a reporter containing myogenin enhance factor indicated that expression of Eya in coordination with Six in 293T cells stimulated the Myogenin gene expression.Eya proteins are transcriptional activator of Six and can improve the activity of myogenin promoter.

OBJECTIVETo analyze the forces of rotational wall vessel (RWV) bioreactor on small tissue pieces or microcarrier particles and to determine the tracks of microcarrier particles in RWV bioreactor.

METHODSThe motion of the microcarrier in the rotating wall vessel (RWV) bioreactor with both the inner and outer cylinders rotating was modeled by numerical simulation.

RESULTSThe continuous trajectory of microcarrier particles, including the possible collision with the wall was obtained. An expression between the minimum rotational speed difference of the inner and outer cylinders and the microcarrier particle or aggregate radius could avoid collisions with either wall. The range of microcarrier radius or tissue size, which could be safely cultured in the RWV bioreactor, in terms of shear stress level, was determined.

CONCLUSIONThe model works well in describing the trajectory of a heavier microcarrier particle in rotating wall vessel.

Bioreactors ; Computer Simulation ; Microspheres ; Motion ; Porosity ; Rheology ; Rotation ; Stress, Mechanical ; Tissue Engineering ; methods

Objective To investigate the 16S rRNA methylase genes and Aminoglycoside modifying enzymes(AMEs)genes in Enterobacter cloacae isolated from the People's Liberation Army 98th Hospital,Huzhou district,Zhejiang province,China.Methods 40 strains of Enterobacter cloacae were isolated from the inpatients between September,2003 and November,2004.5 kinds of 16S rRNA methylase gene (including armA,rmtA,rmtB,rmtC and rmtD)and 9 kinds of AMEs gene[including aac(3)-Ⅰ,aac(3)-Ⅱ,aac(3)-Ⅲ,aac(3)-Ⅳ,aac(6')-Ⅰ b,aac(6')-Ⅱ,ant(3'')-Ⅰ,ant(2'')-Ⅰ and aph(3')-Ⅵ]were analyzed by PCR and verificated by DNA sequencing.Results In 40 strains of Enterobacter cloacae,the positive rates of genes of rmtB,aac(3)-Ⅱ,aac(6')-Ⅰ b,ant(3'')-Ⅰ,ant(2'')-Ⅰ and aph(3')-Ⅵ were 12.5%(5/40),27.5%(11/40),72.5%(29/40),32.5%(13/40),5.0%(2/40)and 5.0%(2/40),respectively.8 kinds of the rest of genes were all tested negative.The total positive rate of AMEs gene was 85.0%(34/40).Among 29 strains of Enterobacter cloacae that the aac(6')-Ⅰ b gene was positive,through PCR and verification by DNA sequencing,7 strains(24.1%)were confirmed to take the aac(6')-Ⅰ b-cr(the GenBank register number:EF375620,EU159121)alone,18 strains(62.1%)were confirmed to take the aac(6')-Ⅰ b-Suzhou(EU085533)alone,3 strains(10.3%)were confirmed to take both aac (6')-Ⅰ b-Suzhou and aac(6')-Ⅰ b-cr while only 1(3.4%)was aac(6')-Ⅰ b(the classical type).Conclusion There was lower positive rate of 16S rRNA methylase gene but very high AMEs genotypes in Enterobacter cloacae isolated from inpatients and the finding of rmtB gene was reported for the first time in the world.At least 5 kinds of AMEs gene existed in Enterobacter cloacae were isolated and they were the new host of both gene of aac(6')-Ⅰ b-cr and aac(6')-Ⅰ b-Suzhou,with aac(6')-Ⅰ b-Suzhou gene was the predominance subtype in aac(6')-Ⅰ b.

OBJECTIVETo screen and analyze the important associated genes in different stages of gastric cancer.

METHODSUsing suppression subtractive hybridization (SSH) to screen differentially expressed genes; detecting the expression of genes in different stages of gastric cancer with dot blot hybridization; and verifying the results with semi-quantitative reverse transcriptase-polymerase chain reaction(RT-PCR).

RESULTSTwenty-six differentially expressed gene fragments were obtained by means of SSH. Among them,24 were known genes, 1 was a new expressed sequence tags(EST), and 1 was a hypothetical gene. The results of dot blot hybridization demonstrated that the expressions of Annexin A2, RPS29, RPS12 etc. in dysplasia were higher than those in normal mucosa; the expressions of RPS12 etc.in early cancer were higher than those in normal mucosa;the expressions of cytochromosome C oxidase II, ferritin light chain, RPS12 etc. in advanced gastric cancer and lymph node metastases were consistently higher than those in normal mucosa. The expression of proteasome 26S subunit gene in advanced gastric cancer was higher than that in normal mucosa. The expression of RPS12 was consistently higher in different stages of gastric cancer. It was demonstrated by RT-PCR that the expression of RPS12 in gastric cancer was higher than that in normal mucosa.

CONCLUSIONThe authors have identified some important genes that might be involved in the carcinogenesis and progression of gastric cancer, and RPS12 may play more important roles in gastric cancer.

Gene Expression Profiling ; methods ; Gene Expression Regulation, Neoplastic ; Genetic Testing ; methods ; Humans ; Nucleic Acid Hybridization ; methods ; Reverse Transcriptase Polymerase Chain Reaction ; Stomach Neoplasms ; diagnosis ; genetics

OBJECTIVETo analyze the early and long-term results after mitral valve replacement for rheumatic valvular disease by using home-made tilting disc valve, and the determinant factors involved and subsequent therapies.

METHODSOne hundred and five patients, including 31 patients with rheumatic mitral stenosis, 92 patients with mixed mitral stenosis and regurgitation, and 2 patients with bacterial endocarditis, underwent prosthetic mitral valve replacement with home-made tilting disc valve from September 1978 to June 1982. Three patients had a history of mitral commissurotomy, and 5 patients had concomitant functional tricuspid regurgitation. All patients were operated on under cardiopulmonary bypass with implantation of 25 - 29 mm size home-made tilting disc valve prosthesis. The associated functional tricuspid lesions were treated at the same time with modified DeVega's valvuloplasty or Kays bicuspidate valvuloplasty.

RESULTSEleven patients died during the hospital stay with an early operative mortality of 8.8%. The major causes of the early death were low cardiac output syndrome (4 patients), respiratory failure (2), acute renal failure (2), extrinsic prosthesis dysfunction (1), ventricular arrhythmia (1), and left ventricular rupture (1). Ninety-eight survivors were followed up (total 1,162.2 years) for mean duration of 12.8 years. Eighty-nine patients (78%) survived over 10 years after operation, 58 (51%) over 15 years, and 55 (48%) over 20 years. There were 16 late deaths due to heart failure, anticoagulation related bleeding, thromboembolism and recurrence of rheumatic fever. The survival rates at 10 and 20 years were 82.3% and 51.1% respectively. Among the patients who survived over 20 years, 37 patients had the cardiac functional status returned to Class II, 13 Class III, and Class IV.

CONCLUSIONSSevere post-rheumatic valve deformity may occur in younger patients in China. Long-acting penicillin regimen given for 3 - 5 years for the prevention of rheumatic fever relapse is advocated. A low intensity anticoagulant regimen after mitral valve replacement is advisable in lowering the incidence of anticoagulant related bleeding, while optimizing sufficient protection against thromboembolic complication. Proper operative timing (e.g. when the patient is in sinus rhythm and in NYHA functional class II) is of great importance in achieving satisfied long-term results.

Adolescent ; Adult ; Anticoagulants ; administration & dosage ; adverse effects ; therapeutic use ; Female ; Follow-Up Studies ; Heart Valve Diseases ; etiology ; surgery ; Heart Valve Prosthesis Implantation ; instrumentation ; methods ; mortality ; Hemorrhage ; chemically induced ; prevention & control ; Humans ; Male ; Middle Aged ; Mitral Valve ; surgery ; Postoperative Complications ; prevention & control ; Retrospective Studies ; Rheumatic Heart Disease ; complications ; Secondary Prevention ; Survival Analysis ; Survival Rate ; Time Factors ; Treatment Outcome ; Young Adult

OBJECTIVETo elucidate the early and long-term results of surgical treatment for complex infective endocarditis with prosthetic valve replacement.

METHODSFifty-seven patients of complex native valve endocarditis, including 25 cases of aortic valve, 16 of mitral valve and 16 of double valves, who underwent operative interventions with prosthetic valve replacement between December 1988 and June 2002, were analyzed retrospectively. Intraoperative findings demonstrated aortic annular abscesses (n = 19), root abscesses (n = 4), mitral posterior annular abscesses (n = 11), myocardial abscesses (n = 6), massive leaflet destruction (n = 32) and valvular vegetations (n = 55). Complex reconstruction of the aortic and mitral annulus was required in 35 patients. Associated procedures included Bentall's procedure (n = 4), aortic valve replacement (n = 21), mitral valve replacement (n = 16) and double valve replacements (n = 16).

RESULTSThe operative mortality was 11%. Complications included low cardiac output syndrome, recurrence of endocarditis, multiple organ failure, ventricular arrhythmia, bleeding, mediastinal infection, respiratory insufficiency and heart block. Follow-up was 100% complete at a mean of 5.93 years. There were five late deaths (3 prosthetic valve endocarditis, 2 valve-related). The NYHA functional status recovered to Class I in 17 patients, Class II in 27 and Class III in 2 at 1 year follow-up. Kaplan-Meier analysis showed the 5-year actuarial freedom from reoperation was (84 +/- 3)%, and actuarial survivorship at 5 years was (61 +/- 9)%.

CONCLUSIONSUrgent or even emergency operation is advocated for complex infective endocarditis. Proper intraoperative reconstruction of the aortic and mitral annulus and optimized perioperative management, especially the strategy for prevention of recurrent endocarditis, are of great importance in achieving satisfied early and long-term clinical outcomes.

Adolescent ; Adult ; Aged ; Aortic Valve ; surgery ; Bioprosthesis ; Debridement ; methods ; Endocarditis, Bacterial ; surgery ; Female ; Follow-Up Studies ; Heart Valve Prosthesis ; Heart Valve Prosthesis Implantation ; Humans ; Male ; Middle Aged ; Mitral Valve ; surgery ; Retrospective Studies

OBJECTIVETo summarize the clinical characteristics, diagnosis and surgical in-treatment results of congenital coronary artery fistulas (CAF) in adults.

METHODSFourteen patients (8 men, 6 women), aged from 18 to 60 years with a mean of 32 +/- 13 years, underwent surgical correction of CAF between March 1985 and April 2002. Eleven of the 14 patients (78.57%) were symptomatic. The diagnosis of CAF was made by echocardiography or angiocardiography preoperatively. The fistulae originated from the right, left and double coronary arteries in 10 (71%), 3 (21%) and 1 (7%) patient(s), respectively. The fistulae drained into the right ventricle (8 patients), left ventricle (4), right atrium (1) and pulmonary artery (1), respectively. The diameter of fistulae ranged from 0.30 to 1.80 cm with a mean of (1.16 +/- 0.49) cm. There were 6 CAF patients associated with coronary artery aneurysms and 4 CAF patients with other coexisting cardiac defects. The distal fistulae were closed in 10 patients with cardiopulmonary bypass (CPB) and 4 patients without CPB. The coexisting defects were corrected simultaneously.

RESULTSThere was no early and late death. One patient had low cardiac output syndrome and cured during early postoperative period. Twelve patients (85.71%) were followed up for a mean period of 3.35 +/- 4.28 years without myocardial ischemia or infarction and recurrent fistulae. Heart function was improved to NYHA functional class I in 11 patients and class II in 1 patient.

CONCLUSIONSAll adult patients with CAF who have demonstrable hemodynamic and cardiovascular morphological changes should be surgically treated as early as possible. The appropriate surgical management and reliable myocardial protection are key points of good surgical results.

Adolescent ; Adult ; Coronary Vessel Anomalies ; diagnosis ; surgery ; Female ; Fistula ; congenital ; diagnosis ; surgery ; Humans ; Male ; Middle Aged

BACKGROUNDTranscription factor Pokemon, a central regulation gene of the important tumor suppressor ARF gene, exerted its activity by acting upstream of many tumor-suppressing genes and proto-oncogenes. Its expression in non-small cell lung cancer (NSCLC) and its clinical significance remains unclear. The aim of this study was to investigate the expression of Pokemon in NSCLC and to explore its correlation with the clinical pathological characteristics and its influence on patients' prognosis.

METHODSFifty-five cases of NSCLC were involved in this study. The expression of Pokemon in the tumor tissue, the corresponding tumor adjacent tissue and the surrounding tissue was detected via reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, with the aim of investigating the correlation between the expression of Pokemon in tumor tissue of NSCLC and its clinical pathological characteristics. Moreover, a prognostic analysis was carried out based upon the immunohistochemical (IHC) detection of the expression of Pokemon gene in archival tumor specimens (5 years ago) of 62 cases of NSCLC.

RESULTSStatistical significance of the expression of Pokemon mRNA and protein was determined in the tumor tissue, the tumor adjacent tissue and the surrounding tissue (P<0.05). The expression of Pokemon was determined not to be associated with the patients' sex, age, smoking condition, tumor differentiation degree, histology and lymph node metastasis condition. However, its relationship with TNM staging was established (P<0.05). Furthermore, it was shown that the survival rate of patients with negative Pokemon expression was significantly higher than that of those with positive Pokemon expression (P=0.004), therefore, the expression of Pokemon is believed to be an independent factor affecting prognosis (P=0.034).

CONCLUSIONPokemon was over-expressed in NSCLC tissue and the expression of Pokemon might be of clinical significance in non-small cell lung cancer prognostic evaluation.

Adult ; Aged ; Carcinoma, Non-Small-Cell Lung ; metabolism ; mortality ; Female ; Humans ; Immunohistochemistry ; Lung Neoplasms ; metabolism ; mortality ; Male ; Middle Aged ; RNA, Messenger ; analysis ; Transcription Factors ; analysis ; genetics

OBJECTIVETo screen hepatocellular carcinoma (HCC) autoantibodies as diagnostic biomarkers or therapy targets by serologic proteome analysis (SERPA).

METHODSTotal proteins extracted from human HCC cell line HCCLM3 were separated by two-dimensional electrophoresis (2-DE) and then transferred onto PVDF membranes, which were subsequently incubated with sera from HCC, hepatitis B virus (HBV) infected patients or healthy volunteers. All immuno-reactive protein spots on blot films were matched to those on 2-DE gel maps by image analysis and identified by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS/MS).

RESULTS2-DE gel maps of HCCLM3 and corresponding blot films of good quality and reproducibility were established. The number of spots on HCCLM3 2-DE reference gel totaled 603 and those on HCC, HBV and healthy sera blotted films were 70.75+/-24.25, 68.5+/-23.44 and 41.38+/-15.05, respectively. Blot films of HCC and HBV groups had more spots than those of the healthy group (P < 0.05) while no significance was found between films of HCC and HBV groups. By identification, those HCC autoantibodies could be classified as nuclear proteins, cytoskeleton proteins, heat shock proteins and metabolic enzymes.

CONCLUSIONSerological proteome analysis is a high throughput technique for screening tumor autoantibodies. Those newly identified HCC associated tumor antigens and corresponding autoantibodies can be used in the early diagnosis or immuno-therapy of HCC.

Antibodies, Neoplasm ; analysis ; Autoantibodies ; analysis ; Carcinoma, Hepatocellular ; immunology ; Electrophoresis, Gel, Two-Dimensional ; Humans ; Liver Neoplasms ; immunology ; Proteomics ; methods ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Tumor Cells, Cultured

OBJECTIVETo explore the differences of PDGF and EGF expression in the wound healing between fatal and adult.

METHODSWith the established animal model of fetal scarless healing and the adult samples, an immunohistochemical technique was used to evaluate the expression of PDGF and EGF in the normal adult skin, normal fetal skin, and the process of their wound healing.

RESULTS1. The expression of the PDGF was not found in the fetal skin, but a mild amount of the PDGF was shown in the epidermis and the upper dermal layer 12 hours and 1 day after the wounding process. In the normal adult skin, expression of PDGF was shown in the dermal fibroblasts, macrophagocytes and blood capillaries, and a strong expression was presented during its wound healing process. 2. In the fetal skin, the expression of the EGF was seen in the epidermis, hair follicles, sebaceous glands and sweat glands, but there were no markedly changes during the wound healing. In the adult skin, a positive stain of the EGF was shown in the basal layer of the epidermis while the mild stain in hair follicles and sweat glands. The level of the expression became gradually decreasing with the time going in the wounded adult skin.

CONCLUSIONThe different expression of growth factors between fetal and adult skin in wound healing may be one of the important reasons that the fetal wound could produce scarless healing.

Adult ; Epidermal Growth Factor ; metabolism ; Epidermis ; metabolism ; Fetus ; Fibroblasts ; metabolism ; Hair Follicle ; metabolism ; Humans ; Platelet-Derived Growth Factor ; metabolism ; Skin ; injuries ; metabolism ; Sweat Glands ; metabolism ; Wound Healing