AIM:To observed the protective effect of diminazene aceturate ( DIZE) , an angiotensin-converting enzyme 2 (ACE2) activator, on diabetic nephropathy (DN) rats.METHODS:Male Wistar rats (n=30) were randomly divided into normal control (NC) group, DN group and DIZE group (each group consisted of 10 rats).The rats in DN group and DIZE group were induced by intraperitoneal injection of streptozotocin at dose of 65 mg/kg.After 12 weeks, the rats in DIZE group and DN group received subcutaneous injection of DIZE (15 mg· kg -1 · d-1 ) or vehicle for 4 weeks. The samples of blood and urine were collected at week 16, and ratio of kidney weight to body weight (KW/BW), plasma glucose (GLU), 24 h urinary protein (24UP) and serum creatinine (SCr) were measured.The renal pathological changes in each group were observed by periodic acid-Schiff ( PAS) staining and immunohistochemistry .The levels of AngⅡ and Ang-(1-7) in the plasma, and TGF-β1 and VCAM-1 in the renal tissues were measured by ELISA .The mRNA and protein levels of collagen I and FN were determined by quantification real-time PCR and immunohistochemistry .The effects of DIZE on the expression of ACE2 in DN rats were determined by Western blot .RESULTS:DIZE remarkably increased the expression of ACE2 and Ang-(1-7) in DN rats.Compared with NC group , the GLU, KW/BW, 24UP, SCr, and the ex-pression of collagen I , FN, TGF-β1 and VCAM-1 in DN group and DIZE group were increased .However , after treatment of the DN rats with DIZE, these indicators were decreased except the KW/BW.The GLU showed no significant change . CONCLUSION:DIZE raised the activity of ACE2 and increased the expression of Ang-(1-7), thus alleviating fibrosis and inflammation in the kidney and having therapeutic potential for diabetic nephropathy .

OBJECTIVE:To investigate the effect of Wuzhi soft capsule on the pharmacokinetics of sirolimus in rats. METH-ODS:Wistar rats were randomly divided into 5 groups,with 6 rats in each group. They were given very low-dose,low-dose,me-dium-dose and high-dose of Wuzhi soft capsule(67,134,268 and 536 mg/kg)ig or blank solvent,respectively;and then given si-rolimus 0.4 mg/kg. The blood 100 μl was sampled by capillary eyes before giving sirolimus(0 h)and 0.5,0.75,1,1.5,2,2.5, 3,4,6,8,12,24 and 36 h after giving sirolimus and put into edetic acid anticoagulation tube. Blood concentration of sirolimus was assayed by LC-MS/MS. The pharmacokinetic parameter was calculated by DAS 2.0 software using non-compartment model. RESULTS:The pharmacokinetic parameters of sirolimus in very low-dose,low-dose,medium-dose and high-dose groups and blank solvent group were cmax of(6.79±1.15),(17.40±3.13),(21.24±3.14),(22.06±4.82),(2.80±0.72)ng/ml;t1/2 of(11.01± 0.82),(12.20±2.02),(12.28±2.38),(12.36±0.73),(10.59±0.69)h;AUC0-∞ of(85.79±15.26),(162.18±41.75),(273.12± 73.69),(268.79±28.46),(36.72±3.01)ng·h/ml,respectively. Compared with blank solvent group,cmax and AUC0-∞ of sirolimus were all increased in very low-dose,low-dose,medium-dose and high-dose groups,but t1/2 changed slightly. CONCLUSIONS:Wu-zhi soft capsule can substantially enhance the absorption of sirolimus in rats.

BACKGROUND: That the nerve growth factor(NGF) is capable of treating peripheral nerve injury has been broadly acknowledged. But it is not sure whether it is able to pass through blood brain barrier(BBB) to act on central nerve system. In this study, the NGF encapsulated in liposome was compared with NGF alone in their abilities of passing through BBB.OBJECTIVE: To compare the dedicated distribution of NGF in different forms using single photon emission computerized tomography(SPECT).DESIGN: It is a randomized controlled study with New Zealand rabbits as subject.SETTING: An affiliated hospital of Nanjing Medical College.MATERIALS:The trial was conducted in Nanjing Senke Medical Company and the Nuclear Medicine Department of the First Hospital of Nanjing Medical College from June 2003 to May 2004. The subjects were 19 New Zealand rabbits of either sex, weighting (2.0 ±0. 2) kg, from Anlimo Technology Company. [99Tcm] -NGF(labeling rate 98.9%, purity 99.7% )was made by Senke Medical Company. Liposome was provided by Shengyang Pharmaceutical College. Urethine solution(200 g/L) was from Pharmacy Department of NanJing Medical Collage.METHODS: [99Tcm]-NGF was encapsulated in liposome and was treated as the following: The liposomesA containing 1.48 × 108Bq[99Tcm] -NGF were injected into rabbits and its distribution percentage was analyzed with SPECT. The same amounts of[99Tcm] -NGF and[99Tcm] -NGF-ordinary liposomesB were treated in the same way.MAIN OUTCOME MEASURES: The ratio of concentration and radiation percentage of NGF in brain to those in the whole body.RESULTS: The[99Tcm]-NGF encapsulated in self-made liposomeA presented high radiation in the brain. But[99Tcm] -NGF alone was almost completely excreted through urinary system and[99Tcm] -NGF encapsuled in ordinary liposomeB was mostly phagocytized by liver reticuloendothelial system.CONCLUSION: The self-made NGF-liposomeA is brain-dedicated, which set a basis for drugs to pass through BBB.

To clone cDNA of human leptin gene and obtain leptin protein for future study on leptin binding proteins. The cDNA of human leptin with 6 x his-tag was cloned by over-hang extension PCR protocol using human genomic DNA as template, and subcloned into in vitro expression vector pIVEX2.3MCS, and the fusion protein was expressed in vitro by Rapid Translation System (RTS) (RTS500 cycle primer Kit and RTS500 ProteoMaster of Roche company). The apparent molecular weight(19.46 kD) and the immuno-specificity of the fusion protein were confirmed by SDS-PAGE and Western blot, and the expressed fusion protein stayed mainly in the supernatant of the reaction mixture in soluble form. This work provides us solid basis for further study on new leptin-associated proteins.
Blotting, Western ; Cloning, Molecular ; DNA, Complementary ; genetics ; Electrophoresis, Polyacrylamide Gel ; Humans ; Leptin ; chemistry ; genetics ; metabolism ; Molecular Weight ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; chemistry ; genetics ; metabolism

To identify SJCHGC01743 gene of Schistosoma japonicum and evaluate the potential of the recombinant protein as a new vaccine candidate for schistosomiasis, polymerase chain reaction (PCR) technique was used to amplify the cDNA of the gene and real-time RT-PCR was used to analyze the transcription profiles of SJCHGC01743 at different development stages. Recombinant plasmid was successfully constructed and transformed into competent Escherichia coli BL21 (DE3). Then the recombinant protein was expressed, purified and emulsified with ISA206 adjuvant to immunize BALB/c mice for three times. The immunogenicity was confirmed by Western blotting and tissue localization was detected by indirect immunofluorescent assay. The specific antibody level was detected by ELISA. The immunoprotection of rSjOST48 was evaluated by the reduction in worm and egg counts in mice. A cDNA with 1 248 nucleotides was isolated from 28-day-old schistosomes cDNAs by PCR. Sequence analysis revealed that SJCHGC01743 was a 48-kDa subunit of the oligosaccharyltransferase complex (OST48) and named as SjOST48. Real-time PCR analysis indicated that this gene was expressed in all investigated stages and had the highest expression level in 28 d worms, the level of gene transcription in female worms was significantly higher than that of male worms. Then recombinant plasmid pET28a(+)-SjOST48 was successfully constructed and expressed in E. coli BL21 (DE3). Western blotting analysis showed that rSjOST48 had good immunogenicity. Indirect immunofluorescent analysis revealed that SjOST48 was mainly distributed on the tegument of the worms. The result of ELISA indicated that the rSjOST48 vaccinated group could induce a significant increase in the level of specific IgG, IgG1 and IgG2a. An immunoprotection experiment showed that the vaccination of rSjOST48 in mice induced 32.62% (P < 0.05) reduction in the numbers of worms and 57.61% (P < 0.01) in eggs in liver, compared with that of the control group. This study provides the foundation for proceeding further research on the biological function of SjOST48 and screening new vaccine candidates for schistosomiasis.
Animals ; Antibodies, Helminth ; blood ; Cloning, Molecular ; DNA, Complementary ; Escherichia coli ; Female ; Genes, Helminth ; Helminth Proteins ; genetics ; immunology ; Immunoglobulin G ; blood ; Male ; Mice ; Mice, Inbred BALB C ; Real-Time Polymerase Chain Reaction ; Recombinant Proteins ; immunology ; Schistosoma japonicum ; genetics ; Schistosomiasis japonica ; prevention & control ; Vaccination

OBJECTIVETo study the function of F10 gene, a novel hydaditiform mole-related gene.

METHODSA549 cell line was transfected with the F10 gene of forward or reverse sequence or with the empty vector, respectively. The cellular mRNA was extracted after 24 h of transfection to screen for the differentially expressed genes among the 3 transfected and the control cells using differential display-polymerase chain reaction (ddPCR).

RESULTSThe bands representing differentially expressed genes were amplified from the cells, and the products were linked to T-Vector for sequence analysis. Several genes were screened by Blasting and their expressions were confirmed by fluorescent quantitative PCR.

CONCLUSIONF10 gene is functionally related to cell proliferation and apoptosis.

Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Epithelial Cells ; metabolism ; Female ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Humans ; Hydatidiform Mole ; genetics ; Hydatidiform Mole, Invasive ; genetics ; Lung Neoplasms ; genetics ; pathology ; Oncogenes ; genetics ; Pregnancy ; Transfection ; Uterine Neoplasms ; genetics

The aim of the present study is to explore the mechanism of estrogen on regulating cardiac function disorder by adjusting the stimulating adenylate cyclase G α protein (Gαs)-cycle adenosine monophosphate (cAMP) signal pathway. Adult female rats were randomly divided into five groups: sham group, ovariectomized group (OVX), OVX and 17β-estradiol given group (OVX+E₂), OVX and isoprenaline injected group (OVX+ISO), OVX and 17β-estradiol, isoprenaline injected group (OVX+E₂+ISO). Rats were ovariectomized, and two weeks later, OVX+E₂group was injected with E₂, OVX+ISO group was injected with ISO, OVX+E₂+ISO group was injected with E₂and ISO. Another four weeks later, the hemodynamic parameters were monitored by carotid artery intubation: left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), maximal differentials of left ventricular developed pressure (+dp/dt(max)), and minimal differentials of left ventricular developed pressure (-dp/dt(max)). Brain natriuretic peptide (BNP) and cAMP concentration in plasma were determined; Gα(s) protein expression in myocardium was determined. The results showed that the hemodynamic parameters, the concentration of BNP and cAMP in plasma had no significant changes after ovariectomy compared with sham group. But after isoprenaline injection in ovariectomized rats, LVSP and +dp/dt(max) declined (P < 0.01), LVEDP and -dp/dt(max) elevated (P < 0.01); plasma BNP concentration increased (P < 0.01); plasma cAMP concentration decreased (P < 0.01), compared with OVX group. Further estrogen supplements improved the heart function treated by isoprenaline: LVSP and +dp/dt(max) elevated (P < 0.01), LVEDP and -dp/dtmax declined (P < 0.05, P < 0.01); the plasma BNP concentration decreased (P < 0.01); the plasma cAMP concentration increased (P < 0.01). Estrogen had no significant influence on Gαs protein expression. The results suggest that estrogen can alleviate myocardial injury and regulate cardiac function disorder by increasing cAMP level, finally improved the excessive suppression of myocardium.
Animals ; Cyclic AMP ; blood ; Estradiol ; pharmacology ; Estrogens ; pharmacology ; Female ; GTP-Binding Protein alpha Subunits, Gs ; metabolism ; Hemodynamics ; Isoproterenol ; adverse effects ; Myocardium ; pathology ; Natriuretic Peptide, Brain ; blood ; Ovariectomy ; Rats ; Signal Transduction

OBJECTIVETo explore the feasibility of inducing fungal infection by Cryptococcus neoformans aerosol inhalation in immunosuppressed Balb/c mice.

METHODSTwenty-four Balb/c mice were randomized into cyclophosphamide (CTX) group and control group (n=12). The mice in CTX group were subject to inhalation of Cryptococcus neoformans aerosol prepared using a ultrasonic nebulizer 4 days after intraperitoneal CTX injection, and the control mice received the inhalation only. The leukocyte count and changes in appetite, body weight, and behaviors of the mice were observed after the treatments. On days 3 and 7 after the inoculation, the mice were sacrificed to analyze the fungal counts in brain and lung tissue homogenates and examine the pulmonary pathologies.

RESULTSCTX injection caused lowered appetite and body weight loss in the mice, and significantly reduced the leukocyte counts (2.77∓0.45 vs 8.26∓0.56, P<0.05). At days 3 and 7 after inoculation, the Cryptococci load in the lungs increased obviously in CTX group with a colony-forming unit (CFU) of the lungs of 271.67∓122.22 and 41.67∓0.28, respectively, significantly higher than that in the control group (60.00∓43.36 and 3.00∓5.30, respectively, P<0.05). In CTX group, the CFU was 10.17∓5.42 and 9.17∓6.34 in the peripheral blood and 6.83∓4.92 and 11.00∓5.44 in the brain tissue at days 3 and 7, respectively, whereas no Cryptococci was detected in the peripheral blood or in the brain tissue in the control group. Pathological examination of the lungs revealed destruction of normal alveolar structure in CTX group, with numerous infiltrating inflammatory cells and visible yeast.

CONCLUSIONInhalation of Cryptococcus neoformans aerosol can cause fungal infection in the lungs of immunosuppressed Balb/c mice.

Aerosols ; Animals ; Brain ; microbiology ; Colony Count, Microbial ; Cryptococcosis ; microbiology ; Cryptococcus neoformans ; growth & development ; Disease Models, Animal ; Immunocompromised Host ; Inhalation ; Lung Diseases, Fungal ; microbiology ; Male ; Mice ; Mice, Inbred BALB C ; Ultrasonics

Radiation sensitive protein 23 (RAD23) is a nucleotide excision repair (NER) protein that plays an important role in Ubiquitin-proteasome pathway (UPP). Schistosoma japonicum radiation sensitive protein23 (SjRAD23) cDNA sequences were amplified by PCR and cloned into pET28a (+) vector to construct recombinant expression plasmid pET28a(+)-SjRAD23. The recombinant protein was expressed as both inclusion bodies and the supernatant in Escherichia coli BL21 (DE3) cell. Immunofluorescence observation shows that SjRAD23 was mainly distributed on the tegument surface of the worms. ELISA assay reveals that specific IgG, IgG1 and IgG2a antibodies could be detected in the sera of rSjRAD23 immunized mice. Western blotting analysis shows that the recombinant SjRAD23 could be recognized by serum specific to soluble adult worm antigen of S. japonicum. BALB/c mice vaccinated with rSjRAD23 combined with 206 adjuvant revealed 35.94% worm reduction and 40.59% liver egg reduction when compared with that of the adjuvant control
Animals ; Antibodies, Helminth ; blood ; Blotting, Western ; Cloning, Molecular ; DNA Repair Enzymes ; genetics ; metabolism ; DNA, Complementary ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; Genetic Vectors ; Helminth Proteins ; genetics ; immunology ; Immunoglobulin G ; blood ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; genetics ; immunology ; Schistosoma japonicum ; genetics ; metabolism ; Schistosomiasis japonica ; prevention & control ; Vaccines ; immunology

OBJECTIVETo investigate the pain features of temporomandibular disorders (TMD) by qualitatively and quantitatively analyzing patient multi-dimension sensations.

METHODSTwo hundred and fifty patients with painful TMD from January to December 2005 were included, short-form mcgill pain questionnaire (SF-MPQ) and visual analog scale (VAS) were administered to assess patients' sensation, affection and intensity of pain. The data were analyzed by SAS 8.0 software.

RESULTSAll patients were assigned to three groups including 139 joint pain, 47 muscle pain and 64 both joint and muscle pain group. In joint pain group, the total number of descriptors was 250, 1.80 in average; in muscle pain group, the total number was 99, 2.11 in average; in both joint and muscle pain group, the total number was 107, 1.64 in average. The sensitive descriptors most frequently chosen in all three groups were "aching", "heavy", and "tender". "Tiring-exhausting" and "sickening" were high frequency descriptors in affective items. There were higher affective scores in the muscle pain group than in others. Muscle pain group had a higher VAS score at rest than the other two (P < 0.05), but had a lower VAS score during function than the other two (P < 0.001). All three groups usually had no pain at rest, and complained a slight to moderate pain during function.

CONCLUSIONSTMD pain was generally slight to moderate; "aching", "heavy", and "tender" were the most frequently sensitive descriptors, while unpleasant feelings were described as "tiring-exhausting" and "sickening"; pain generally occurred or exacerbated during mandibular function; compared to joint pain, muscle pain had its own features.

Adult ; Facial Pain ; epidemiology ; etiology ; Female ; Humans ; Male ; Middle Aged ; Pain ; epidemiology ; etiology ; Pain Measurement ; Surveys and Questionnaires ; Temporomandibular Joint Disorders ; complications ; epidemiology ; Young Adult