Human Zona Pellucida(ZP), which is a complex matrix surrounding oocytes,is comprised of three immunologically distinct glycoproteins(hZP1, hZP2 and hZP3). Because hZP3 possesses the sperm receptor activity and the acrosome-inducing activity, it has long been used as a candidate antigen to develop an immunocontraceptive vaccine. However, a large amount of native hZP3 protein is unavailable. It is an effective way to express hZP3 protein directly in vitro. Nevertheless, it had been reported that the rhZP3 protein produced in Pichia pastoris was not secreted but accumulated in the cells and could only be purified after being solubilized by strong denaturants. More unfortunately, after purification the final product required 6mol/L urea to maintain solubility. An improved project was advanced with the aim to express secreted and soluble rhZP3 protein in yeast. In this study, the fragment of hZP3 cDNA coding for aa 23 - 408, which the N-terminal leader was removed and most of the C-terminal transmembrane-like domain was reserved, was amplified by two PCR primers including EcoR I and Not I sites respectively and a His6 codon cassette was added to 5'-terminal. The hZP3 insert was incorporated into expression vector pPIC9K. The resulting recombinant yeast expression vector was designated pPIC9K-rhZP3. Linearized pPIC9K-rhZP3 was transformed into Pichia pastoris. After G418 selection, the recombinant Pichia pastoris strains were identified by PCR and the rhZP3 was expressed following the manufacturer' s protocol. Following induction with methanol, the rhZP3 protein was secreted and dissolved into the culture supernatant. SDS-PAGE and Western blot analyses showed that the apparent molecular weight of the expressed rhPZ3 proteins in yeast was smaller and a little size heterogeneity than native ones; after purified with Ni-chelating affinity chromatography, the final product's apparent molecular weight was about 32 - 34KD and their yield more than 20mg/L. We supposed that the C-terminal transmembrane-like domain be useful for secretion of rhZP3 into the culture supernatant and the expressed rhZP3 protein be incompletely digested by proteinases of Pichia into shorter fragments which all were glycosylated inhomogeneously. Fortunately, the fragments of rhZP3 protein can be recognized in Western blot by the polyclonal antibodies to porcine ZP3 which has showed a cross-reactivity with human ZP in vitro. It will be expected that the rhZP3 protein expressed in Pichia pastoris not only has immunogencity, say, it can rise antibodies in vivo to prevent spermatozoa-ovum binding, but also does not contain ovarian factors that might be the cause of undesired side effects, e.g. ovaritis and can be used as a safe immunogen in human antifertility vaccine research.
Blotting, Western ; Chromatography, Affinity ; Egg Proteins ; genetics ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Genetic Vectors ; genetics ; Humans ; Membrane Glycoproteins ; genetics ; metabolism ; Pichia ; genetics ; metabolism ; Polymerase Chain Reaction ; Receptors, Cell Surface ; genetics ; metabolism ; Recombinant Proteins ; genetics ; isolation & purification ; metabolism ; Zona Pellucida Glycoproteins

OBJECTIVETo investigate the possibility of adipose-derived stromal cells (ADSCs) transfeced by adenovirus containing human bone morphogenetic protein-2 (Ad-hBMP-2) gene and their osteogenic potential.

METHODSADSCs were obtained from inguinal fat tissue of 4 weeks old SD rats. After exposure to adenovirus containing green fluorescent protein(Ad-GFP), fluorescent microscope was used to observe gene transfection effect once 12 hours. After transfected with Ad-hBMP-2, cytochemistry, immmucytochemistry and Western blot were used to examine the expression of alkaline phosphatase (ALP), osteocalcin (OC) and hBMP-2.

RESULTSAfter exposed to Ad-GFP 12 hours, 52% ADSCs were observed being transfected and 48 hours later reached 95%. The double number time belonged after transfecting with Ad-hBMP-2, and cytochemistry, immucytochemistry and Western blot examines indicated positive results of ALP, OC, hBMP-2 after 48 hours.

CONCLUSIONAdipose tissue contains abundant ADSCs which could be transfected as gene vectors by adenovirus, ADSCs transfected with Ad-hBMP-2 can convert to ostoeblasts, and can act as a kind of seed cells for osteo-tissue engineering.

Adenoviridae ; Adipocytes ; Adipose Tissue ; Animals ; Bone Morphogenetic Protein 2 ; Cells, Cultured ; Genetic Vectors ; Humans ; Rats ; Rats, Sprague-Dawley ; Stromal Cells ; Tissue Engineering ; Transfection

OBJECTIVETo investigate the rehabilitation effect of electroacupuncture (EA) at early stage on hemiplegia in the postoperative patient of hypertensive hemorrhage.

METHODSSeventy-two cases of postoperative patient of hypertensive hemorrhage were divided into an EA group (n = 42) treated with EA at Quchi (LI 11), Shousanli (LI 10), Hegu (LI 4), Zusanli (ST 36), etc., from 1-3 days after operation, twice each day; and a control group (n = 30) with functional exercises. Their rehabilitation effects were compared after treatment of one month.

RESULTSThe rehabilitation effect in the EA group was significant better than that in the control group (P < 0.01).

CONCLUSIONEA at early stage has good recovery effect for the patient of hemiplegia after operation of hypertensive hemorrhage.

Acupuncture Points ; Electroacupuncture ; Hemorrhage ; Humans ; Hypertension ; Postoperative Period

OBJECTIVETo investigate the role of inhibitor of DNA binding-1 (Id-1) gene in adenoid cystic carcinoma cell growth and invasion behavior.

METHODSWith salivary adenoid cystic carcinoma cell lines ACC-M and ACC-2, dedected Id-1 gene expression was screened with immunofluorescence assay. After Id-1 mRNA knocking-down using small interfering RNA, RT-PCR and Western blot were used to detect the different expressions before and after interference, and the growth of cells before and after interference was deceted using the MTT assay, and the cell invasion ability was checked with the use of Transwell chamber assay.

RESULTSId-1 were both expressed in the ACC-M and ACC-2, and the expression in ACC-M was higher than that in ACC-2. After Id-1 RNA interference, the growth and invasiveness of ACC-M and ACC-2 were inhibited with the restrained degree in ACC-M much stronger than that in the ACC-2.

CONCLUSIONIn view of the important role of Id-1 in the behavior of growth and invasion in ACC cell, interfering the expression of Id-1 gene is expected to be a novel and effective means for the treatment of adenoid cystic carcinoma.

Carcinoma, Adenoid Cystic ; Cell Line, Tumor ; Cell Proliferation ; DNA ; DNA-Binding Proteins ; Gene Silencing ; Humans ; RNA, Messenger ; Salivary Gland Neoplasms

OBJECTIVETo investigate the prevalence of diabetes and prediabetes and their association with the risk for coronary heart disease (CHD) in elderly residents in Haizhu District of Guangzhou.

METHODSStratified random sampling was employed to select a total of 1800 resident aged 50 years or older in the region. The fasting fingertip blood glucose>5.6 mmol/L was used as the criterion for the initial screening. The data were collected from qualified subjects via scheduled questionnaire surveys, blood collection and testing, and physical examination. The subjects were divided into the 3 groups, namely normal blood glucose, prediabetes, and diabetes groups. The combination rates of the relevant risk factors (hypertension, hyperlipemia, obesity, and central obesity) were compared among the groups by Framingham Heart Study to predict the occurrence of CHD in 10 years.

RESULTSThe incidence was 11.00% for prediabetes and 7.56% for diabetes in the elderly residents in Haizhu District. The occurrence of hypertension, hyperlipemia, obesity, and central obesity was significant higher in the prediabetes and diabetes group than in the normal blood glucose group, and showed no significant differences between the former two groups. The 10-year risks for CHD were markedly higher in both the prediabetes and diabetes groups than in the normal blood glucose group, but similar between the former two groups.

CONCLUSIONElderly patients with prediabetes and diabetes have significantly increased 10-year risk for CHD in comparison with those with normal blood glucose, but the risk is similar between the former two groups, indicating a close association of IGR (impaired fasting glucose+ impaired fasting glucose) with CHD. Early control of blood glucose is essential to the prevention and control of CHD.

Aged ; Blood Glucose ; analysis ; China ; epidemiology ; Coronary Disease ; blood ; epidemiology ; Cross-Sectional Studies ; Diabetes Mellitus, Type 2 ; epidemiology ; Female ; Humans ; Male ; Middle Aged ; Prediabetic State ; epidemiology ; Prevalence ; Risk Factors ; Sampling Studies

OBJECTIVETo investigate the effects and related mechanisms of Tumstatin 185-191 as a single agent or in combination with cisplatin on proliferation and apoptosis in a cisplatin-resistant human lung adenocarcinoma cell line A549-DDP cells.

METHODSA549-DDP cells were treated with Tumstatin185-191 and cisplatin at varying concentrations. Cell viability was assessed by a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. 50% inhibiting concentration (IC(50)) values of the chemotherapeutic drugs were analyzed by MTT assay. Cell apoptosis was measured by flow cytometry. The activation of Akt and ERK was evaluated by Western blotting.

RESULTSTumstatin185-191 inhibited the proliferation of A549-DDP cells and its IC(50) value was 80.25 micromol/L. After cotreatment with 20 micromol/L Tum185-191, the IC(50) value of cisplatin in A549-DDP cells reduced from 77.16 micromol/L to 57.97 micromol/L, the reverse index was 1.33, while with 40 micromol/L Tumstatin185-191 the IC(50) was reduced from 77.16 to 26.40 micromol/L and the reverse index was 2.92. The early apoptosis rate was 19.5% +/- 1.1% in the cotreatment group, while 13.3% +/- 1.5% in cisplatin group and 10.2% +/- 2.0% in Tum185-191 group (F = 4.09, P < 0.05). The levels of phospho-Akt (p-Akt) and phospho-ERK (p-ERK) in the A549-DDP cells were remarkably lower after treatment with Tumstatin 185-191. The Tumstatin 185-191 treatment alone or in combination with cisplatin had a similar effect on the protein levels of p-Akt and p-ERK in A549-DDP cells.

CONCLUSIONOur data suggest that Tumstatin185-191 may promote apoptosis, downregulate proliferation and partly reverse the drug resistance of A549-DDP cells to cisplatin. The effects induced by Tum185-191 may be mediated through inactivation of the Akt and ERK pathways.

Adenocarcinoma ; pathology ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Autoantigens ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cisplatin ; pharmacology ; Collagen Type IV ; pharmacology ; Drug Resistance, Neoplasm ; Drug Synergism ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Humans ; Lung Neoplasms ; pathology ; Peptide Fragments ; pharmacology ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; metabolism

OBJECTIVETo investigate the effect of farnesoid-X-receptor (FXR) antagonist Z-guggulsterone in an in vivo high-fat fed apolipoprotein E knockout (ApoE(-/-)) mice model of myocardial ischemia/reperfusion (I/R).

METHODSMale ApoE(-/-) mice were randomly divided into three groups: standard ApoE(-/-) group (fed with standard mouse diet for 12 weeks before myocardial I/R procedure, n = 18), high-fat ApoE(-/-) group (fed with high-fat mouse diet for 12 weeks before myocardial I/R procedure, n = 22), and high-fat ApoE(-/-) + FXR antagonist group(fed with high-fat mouse diet for 12 weeks and received FXR antagonist Z-Guggulsterone 30 minutes before myocardial I/R procedure, n = 17). The expression of FXR was detected by real-time quantitative-PCR. Myocardial infarct size was determined by Evans blue/TTC double staining methods. Myocardial apoptosis was determined by in situ TUNEL technique. Markers of the mitochondrial-mediated apoptotic pathway (cytochrome c release, caspase-9 activity, and BAX and BCL-2 levels), endoplasmic reticulum stress apoptotic pathway (caspase-12 activity and CHOP level), and death receptor apoptotic pathway (caspase-8 activity, and Fas and FasL levels) were also measured.

RESULTFXR expression (3.7-fold higher, P < 0.01), myocardial infarct size [(62.1 ± 7.0)% vs. (33.8 ± 5.8)%, P < 0.01] and myocardial apoptosis index[ (36.8 ± 5.7)% vs. (17.2 ± 3.8)%, P < 0.01]were all significantly higher in high-fat ApoE(-/-) group than those in standard ApoE(-/-) group. Compared with high-fat ApoE(-/-) group, myocardial infarct size [(24.4 ± 4.7)% vs. (62.1 ± 7.0)%, P < 0.01] and myocardial apoptosis index [(13.8 ± 2.7)% vs. (36.8 ± 5.7)%, P < 0.01] were significantly reduced in high-fat ApoE(-/-) + FXR antagonist group. Moreover, levels of mitochondrial-mediated apoptotic pathway markers (cytochrome c release, caspase-9 activity, and BAX/BCL-2 levels) and endoplasmic reticulum stress apoptotic pathway markers (caspase-12 activity and CHOP level) were significantly lower in high-fat ApoE(-/-) + FXR antagonist group than those in high-fat ApoE(-/-) group (all P < 0.01). Levels of death receptor apoptotic pathway markers (caspase-8 activity, and Fas and FasL levels) were similar between high-fat ApoE(-/-) group and high-fat ApoE(-/-) + FXR antagonist group.

CONCLUSIONFXR antagonist alleviates myocardial reperfusion injury in cholesterol-fed ApoE(-/-) mice via inhibition of the mitochondrial-mediated and endoplasmic-reticulum stress pathway.

Animals ; Apolipoproteins E ; genetics ; Apoptosis ; drug effects ; Caspase 9 ; metabolism ; Cholesterol, Dietary ; administration & dosage ; Cytochromes c ; metabolism ; Disease Models, Animal ; Endoplasmic Reticulum Stress ; Male ; Mice ; Mice, Knockout ; Myocardial Reperfusion Injury ; metabolism ; pathology ; prevention & control ; Pregnenediones ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Receptors, Cytoplasmic and Nuclear ; antagonists & inhibitors ; metabolism ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo study the histological changes in livers of chronic hepatitis B (CHB) patients with persistently normal serum ALT levels (PNAL).

METHODS274 CHB patients who had percutaneous liver biopsies and had a detectable viral load (lower limit of detection is 10(3) copies/ml) in our department between October 2003 and February 2007 were included in this study. Among these patients, 139 had PNAL, group A, (with at least 3 normal serum ALT levels, with intervals of more than two months over a period of 12 or more months before the biopsy). The other 135 patients, group B, had abnormal serum ALT levels during the same period. The histological changes in the livers of the two groups of patients were compared.

RESULTSSixty-six (47.5%) patients with PNAL had normal liver histology, but significant pathohistological changes such as significant necroinflammation, fibrosis and/or cirrhosis were found in 33 (23.7%) patients. Thirteen (9.4%) had established cirrhosis. When compared to patients within (0-0.75)x upper limit of normal (ULN) ALT, patients within (0.76-1.00)x ULN ALT had higher scores of histological changes (43.5% vs. 19.8%, P < 0.05). In the PNAL group, scores of histological changes increased sharply in parallel with an age increase of older than 40 yrs. However neither viral loads nor HBeAg statuses of the PNAL patients had any predictive meaning to the scores of the histological findings.

CONCLUSIONS23.7% of our CHB patients with PNAL, regardless of what their HBeAg statuses or viral load levels were, had significant liver pathohistological changes. Liver biopsies should be considered in CHB patients with PNAL, especially those older than 40 yrs and with a higher ALT within (0.76-1) x ULN.

Adolescent ; Adult ; Aged ; Alanine Transaminase ; blood ; Female ; Hepatitis B e Antigens ; blood ; Hepatitis B, Chronic ; blood ; pathology ; physiopathology ; Humans ; Liver ; pathology ; Male ; Middle Aged ; Young Adult

Plant active components characterized of many different structures and activities on multiple targets, have made them to be the important sources of inhibitors on HIV-1. For finding leading compounds with new structure against HIV-1, three key HIV-1 replicative enzymes (reverse transcriptase, protease and integrase) were used as screening models. The in vitro activities of 45 plant derived components isolated from Schisandraceae, Rutaceae and Ranunculaceae were reported. Within twelve triterpene components isolated, eight compounds were found to inhibit HIV-1 protease, in these eight active compounds, kadsuranic acid A (7) and nigranoic acid (8), inhibited both HIV-1 protease and integrase; Among fifteen lignans, meso-dihydroguaiaretic acid (15) and kadsurarin (16) were active on HIV-1 reverse transcriptase, and 4, 4-di(4-hydroxy-3-methoxyphenly)-2, 3-dimethylbutanol (13) active on HIV-1 integrase. All of the six alkaloids, seven flavones, and five others compounds were not active or only with low activities against HIV-1 replicative enzymes. Further studies of the triterpene components showing strong inhibitory activities on HIV-1 were warranted.
Alkaloids ; chemistry ; isolation & purification ; pharmacology ; Anti-HIV Agents ; chemistry ; isolation & purification ; pharmacology ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; pharmacology ; Flavones ; chemistry ; isolation & purification ; pharmacology ; Guaiacol ; analogs & derivatives ; chemistry ; isolation & purification ; pharmacology ; HIV Integrase ; drug effects ; HIV Protease ; drug effects ; HIV Reverse Transcriptase ; antagonists & inhibitors ; Lignans ; chemistry ; isolation & purification ; pharmacology ; Plants, Medicinal ; chemistry ; Ranunculaceae ; chemistry ; Rutaceae ; chemistry ; Schisandraceae ; chemistry ; Triterpenes ; chemistry ; isolation & purification ; pharmacology

OBJECTIVETo investigate the proper time of cryo-preserving tracheal allograft so as to minimize its antigenicity.

METHODSOn a dog model, this study was carried out by allografting a tracheal into a muscular flap formed with sternocephalic muscle and sternohyoid--sternothyroid muscle. The tracheal was treated with cryopreservation in defferent intervals. The viability of the graft was evaluated by the examination of fiberoptic bronchoscopy, histopathology and microangiography. The blood flow of the tracheal mucous was measured with a blood flowmeter and the survival area was decided in the calculation of the percentage.

RESULTSThere are no significant differences in the mucous membrane appearance and the mucosal blood flow one week after the surgery among the non-cryopreservation group and the groups treated with cryopreservation in 1 day, 2 weeks, 4 weeks, 6 weeks and 8 weeks. The graft was found to start necrosis 2 weeks after the transplantation with the infiltration of mononuclear cells examined under light microscope in almost all of the groups, especially in the non-cryopreservation group and the groups treated with cryopreservation in 1 day, 2 weeks. However, there was no significant difference among the autograft group and the allograft groups cryopreservated in 6 weeks and 8 weeks, and the infiltration of the mononuclear cells was not found in these groups either.

CONCLUSIONThe antigenicity of the tracheal allografts could be significantly decreased by the treatment of cryopreservation over 6 weeks.

Animals ; Bronchoscopes ; Cryopreservation ; methods ; Dogs ; Flowmeters ; Models, Animal ; Respiratory Mucosa ; blood supply ; pathology ; Trachea ; immunology ; pathology ; transplantation ; Transplantation, Homologous