To determine whether Smac/DIABLO (second mitochondrial activator of caspases/direct inhibitor of apoptosis protein-binding protein of low isoelectric point [PI]) and XIAP (X-chromosome-linked inhibitor of apoptosis protein) serve to regulate neuronal apoptosis following seizures, we investigated seizure-induced changes in caspase-9, Smac/DIABLO and XIAP protein expression and the in vivo effect of caspase-9 inhibition. Animals received unilateral intra-amygdaloid injection of kainic acid (0.5 microg) to induce seizures for 1 h. The seizures were then terminated by diazepam (30 mg/kg). Animals were killed 0, 2, 4, 8, 24 or 72 h following diazepam administration. The apoptotic and surviving neurons in hippocampus were observed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and cresyl violet staining, the expression of Smac/DIABLO, XIAP and caspase-9 was detected with immunofluorescence and western blot. The results showed that the levels of XIAP and the 46-kDa proenzyme form of caspase-9 were unaffected by the seizures. The expression of Smac increased at 2 h and the 37-kD cleaved fragment of caspase-9 was detected at 4 h, TUNEL-positive neurons appeared at 8 h and reached maximal at 24 h following seizure cessation within the ipsilateral (the same side as the intra-amygdaloid injection of kainic acid) CA3 subfield of the hippocampus. Intracerebroventricular infusion of caspase-9 inhibitor z-LEHD-fluoromethyl ketone (z-LEHD-fmk) significantly decreased TUNEL-positive neurons and increased the number of surviving cells. Caspase-9 immunoreactivity increased and Smac/DIABLO, XIAP immunoreactivity became extensive within the ipsilateral CA3 neurons. TUNEL-positive neurons and the alterations of the expression of Smac/DIABLO and XIAP within the ipsilateral CA3 were not detected within the contralateral hippocampus. These results suggest that seizures lead the translocation of Smac/DIABLO into the cytosol, the activation of caspase-9 and the change of subcellular locoalization of XIAP. These changes may play a role in the brain damage induced by seizures. Caspase-9 is possibly a potential therapeutic target in the treatment of brain injury associated with seizures.
Amygdala ; physiology ; Animals ; Caspase 9 ; Caspases ; biosynthesis ; genetics ; Complement Membrane Attack Complex ; Complement System Proteins ; Glycoproteins ; biosynthesis ; genetics ; Hippocampus ; metabolism ; Kainic Acid ; Limbic System ; Male ; Microinjections ; Protein Biosynthesis ; Proteins ; genetics ; Rats ; Rats, Sprague-Dawley ; Seizures ; chemically induced ; metabolism ; X-Linked Inhibitor of Apoptosis Protein

Department of Pediatrics, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China. zhuchuanlong@jsph.org.cn.
Adolescent ; Child ; Female ; Glycyrrhizic Acid ; therapeutic use ; Humans ; Male ; Non-alcoholic Fatty Liver Disease ; drug therapy ; Tablets

Objective To investigate the polymorphism of human cytomegalovirus (HCMV) UL144 gene of the low passage clinical isolates in Guangzhou and explore the role of UL144 gene in HCMV pathogenicity. Methods The clinical isolates of HCMV were obtained from the urine sample collected from those infants with intra-uterus HCMV infection in Guangzhou. The virus genome DNA was extracted. According to the genome sequence of Toledo, primers for UL144 gene were designed and used to amplify the complete open reading frames (ORF) of the UL144 gene in our 3 different clinical isolates. These ORFs of the UL144 gene were cloned into pMD18-T vector and their sequences were confirmed by sequencing. Bioinformatics methods were used subsequently to analyze the polymorphisms of these genes in different stains. Results Three HCMV low passage clinical isolates were successfully isolated, named D2, D3 and D52. As shown by PCR, all of these three strains contained UL144 ORF region. Three complete ORFs were amplified in total and their sequences were submitted to GenBank (Accession No.: DQ180368, DQ180382 and DQ180355). In D2, D3 and D52 isolates, their UL144 ORFs consisted of 531 nucleotides. DNA sequences were quite conservative,all variability were base substitution, and the amino acid sequences were high conservative, the rate of amino acid variability was 1.1%. There were no additional or deleted sites of posttranslational modification of UL144 protein in all clinical isolates. There were some differences in the secondary structure among different isolates. The isoelectric point of UL144 protein of all clinical isolates was 8.97. Conclusions All DNA and deduced amino acid sequences of UL144 gene share great similarity among Guangzhou HCMV clinical strains regardless of their polymorphism. It implies that maybe UL144 gene plays an important role in congenital infection.

The objective of study was to explore the role of vascular endothelial growth factor (VEGF) and its receptor-2 in pathogenesis of acute myeloid leukemia. The acute myeloid leukemia model was established on 20 mice with severe combined immunodeficiency (SCID) transplanted by HL-60 cells. The mice were divided into the normal control and test group randomly. The expression of VEGF was detected by enzyme linked immunosorbent assay (ELISA). The expression of VEGFR-2 mRNA was detected by RT-PCR. The results showed that the establishment of acute myeloid leukemia model was succeeded on all SCID mice by HL-60 cell transplantation. The expressions of VEGF and VEGFR-2 mRNAs could be determined on all mice. As compared with the normal control group, the expressions of VEGF and VEGFR-2 mRNAs in the test group significantly increased, but gradually increased during the course of disease. It is concluded that the abnormal expressions of VEGF and VEGFR-2 exist in mice with acute myeloid leukemia, which may be involved in the pathogenesis of AML.
Animals ; Bone Marrow ; pathology ; HL-60 Cells ; Humans ; Mice ; Mice, SCID ; Neoplasm Transplantation ; Vascular Endothelial Growth Factor A ; metabolism ; Vascular Endothelial Growth Factor Receptor-2 ; metabolism

OBJECTIVESTo investigate whether the human PC-3 cell infected with recombinant Ad-PTEN and Ad-p27Kip1 can steadily produce PTEN and p27Kip1 protein and change the biologic behaviors such as cell proliferation, cell cycle and apoptosis. The synergistic effect of PTEN and p27Kip1 on the therapy for prostate cancer has also been investigated.

METHODSWe constructed recombinant adenovirus vector of human tumor suppressor gene PTEN and p27Kip1. The viral titer was examined by plaque assay and the mRNA and protein expressions of PTEN and p27Kip1 in human prostate cancer cell line PC-3 infected with Ad-PTEN and Ad-p27Kip1 were determined by RT-PCR and Western blot respectively. MTT assay was used to determine the effect of PTEN and p27Kip1 on growth and proliferation of PC-3 cell; the change of cell cycle and apoptosis was examined by flow cytometry, and to compare between the combined therapy group and single gene therapy group.

RESULTSThe viral titers of Ad-PTEN and Ad-p27Kip1 were 1.8 x 10(7) pfu/ml and 1.2 x 10(9) pfu/ml respectively. After infected by adenovirus, it had been verified that the mRNA and protein expression of PTEN and p27Kip1 were steady in human PC-3 cell. Ad-PTEN and Ad-p27 Kip1 inhibited the growth and proliferation of PC-3 cells. The progression of cell cycle of PC-3 cell was arrested in G(0)-G(1) phase, meanwhile the apoptosis rate of PC-3 was also affected after Ad-PTEN or/and Ad-p27 Kip1 infected. There was significant difference between combined therapy group and single gene therapy group.

CONCLUSIONThe recombinant Ad-PTEN and Ad-p27Kip1 vector were constructed successfully and the expression of specific PTEN and p27Kip1 was high, steadily in PC-3 cell line. These results suggested that combination of PTEN with p27Kip1 has an application value in treatment of prostate cancer in future.

Adenoviridae ; genetics ; Apoptosis ; drug effects ; Cell Division ; drug effects ; Cell Line, Tumor ; Chromosomes, Human, Pair 10 ; genetics ; Cyclin-Dependent Kinase Inhibitor p27 ; Gene Deletion ; Genetic Therapy ; Genetic Vectors ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; pharmacology ; Male ; PTEN Phosphohydrolase ; genetics ; pharmacology ; Prostatic Neoplasms ; genetics ; physiopathology ; therapy ; Transfection

Objective To detect the resistance index and esterase activity of each generation of DDVP-resistant Culex mosquitoes and analyze the relationship between insecticide resistance and esterase. Methods WHO bioassay and micro-plate measurement were used for the detection. Results The resistance index increased to 12.17 after 43 generations' insecticide selection compared to 1.00 as sensitive isolate. The nonspecific esterase(NSE) activity of the mosquitoes became strengthened with the extension of the generations, and the individual frequency of those with OD values no less than 0.9 increased gradually, consistent basically to the bioassay. The AChE average inhibition rate decreased with the extended generation and increased resistance, and the individual frequency of those with inhibition rate less than 30% became strengthened with the extension of generations, showing a positive correlation. Conclusion The activity of NSE and AChE shows a correlation with DDVP resistance.

Objective To evaluate the chemopreventive effects of boswellic acid and curcumin on 7,12-dimethyl benzanthracene (DMBA)-induced oral carcinogenesis in the hamster cheek pouch model.Methods Male Syrian golden hamsters(6-8 weeks old,80-130 g in weight) were randomly divided into seven groups,with group A serving as the untreated negative control.The left cheek pouch of the remaining hamsters was topically treated with 0.5％ DMBA in mineral oil three times a week for 6 weeks.They were then randomized to six groups with group B serving as a positive control and receiving no further treatment.Groups C-G were treated topically with 5,10 mg/L boswellic acid,5,10 μ moL/L curcumin,or the combination of 5 mg/L boswellic acid and 5 μmnol/L curcumin three times per week for 18 weeks.The animals were injected with bromodeoxyuridine intraperitoneally at 50 mg/kg 2 h prior to killing.At the 25 th week all the hamsters were sacrificed and cheek pouch tissue was harvested.One half of the tissue was snap frozen in liquid nitrogen for analysis of arachidonic acid metabolites,and the other half was fixed in 10％ phosphate-buffered saline(PBS)-buffered formalin for histopathological examination.Results Six-weeks of DMBA followed by 18-weeks of topical application of boswellic acid and curcumin,both boswellic acid (5,10 mg/L) and curcumin (5,10 μmol/L) significantly inhibited the incidence from 93.8％ to 73.9％ (P＞0.05),numbers from 2.19 ± 0.98 to 1.13 ± 0.81 (P ＜ 0.01) and size of visible tumors.Microscopically the incidence of squamous cell carcinoma and BrdU index were also significantly suppressed by boswellic acid and curcumin.Conclusions Both boswellic acid and curcumin were effective in preventing oral carcinogenesis in DMBA-induced hamster cheek pouch model.

The purpose of the present study was to explore the seizure-induced changes in Bad (Bcl-2-associated death protein), 14-3-3, phosphoBad, Bcl-2 and Bcl-XL expression in the rat model of focal limbic seizure. Unilateral intra-amygdaloid injection of kainic acid (KA) was made to induce seizure. Electroencephalogram (EEG) and regional cerebral flow (r-CBF) were monitored continuously. Diazepam (30 mg/kg) was administered to terminate the seizure. The apoptotic and surviving neurons in the hippocampus were observed by terminal deoxynucleotidyl transferrase-mediated dUTP nick end labeling (TUNEL) and cresyl violet staining, the expression of Bad, 14-3-3, phosphoBad, Bcl-2 and Bcl-XL were detected with immunofluorescence, Western blot and immunoprecipitation. The results showed that TUNEL-positive neurons appeared at 8 h and reached maximum at 24 h following seizure cessation within the ipsilateral CA3 subfield of the hippocampus. Seizure induced the dephosphorylation of Bad and the dissociation of Bad from its chaperone protein 14-3-3 and subsequent dimerization of Bad with Bcl-XL. The expression of phosphoBad decreased and Bcl-2 increased. There was little change in r-CBF after the seizure. These results suggest that seizure leads to a dephosphorylation of Bad and an upregulation of Bcl-2. Dephosphorylation of Bad may be injurious while the upregulation of Bcl-2 may be protective to the brain damage induced by seizures, but not related with r-CBF.
Amygdala ; physiology ; Animals ; Epilepsies, Partial ; chemically induced ; metabolism ; Hippocampus ; metabolism ; Kainic Acid ; Male ; Microinjections ; Phosphorylation ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; Rats ; Up-Regulation ; bcl-Associated Death Protein ; metabolism

OBJECTIVETo establish transplanted models of VX2 tongue carcinoma in rabbits by three methods and compare these models.

METHODSAfter establishment of VX2 tumor-bearing rabbits, 72 New-Zealand white rabbits were randomly divided into 3 groups. Intact tumour tissue, modified tumour cell suspension, tumour cell suspension were respectively injected into the middle-third lateral border of the tongues of rabbits in 3 groups to induce transplanted VX2 tongue carcinoma. The histological features, the tumour-take rates and the metastasis rates of the 3 models were observed.

RESULTSThe tumour-take rate of 3 models were 83.3%, 91.7% and 33.3% respectively; the lymph node metastasis rates were 71.4%, 100.0% and 37.5% respectively; the lung metastasis rates were 35.7%, 81.3% and 0 respectively. The histological features of the transplanted VX2 tongue carcinoma of 3 models were all consistent with those of moderately differentiated carcinoma.

CONCLUSIONThe biological properties of the transplanted VX2 tongue carcinoma of 3 models is much alike to tongue carcinoma in humans. The model established with modified tumor cell suspension is considered to be more suitable for tongue cancer study.

Animals ; Carcinoma ; Disease Models, Animal ; Humans ; Lymphatic Metastasis ; Neoplasm Transplantation ; Rabbits ; Tongue Neoplasms

OBJECTIVETo evaluate stress changes of intervertebral space and adjacent intervertebral space after artificial disc replacement with angles.

METHODSArtificial disc replacement with angles were designed according to existing data. Axial pressure, flexion/extension, lateral bending and torsion loading were applied on finite element models of normal cervical discs on C4,5 segments, C4,5 segments with 0 degrees artificial cervical discs and C4,5 segments with 10 degrees artificial cervical discs, then stress changes of C4,5 space was observed. The same loadings were applied on finite element models of normal cervical discs on C4-C6 segments, C4,5 segments with 0 degrees, C4,5 segments with 10 degrees, then stress changes of replaced segments space and adjacent segment space were observed.

RESULTSFor C4,5 segments, 80 MPa/0 degrees artificial discs and 80 MPa/10 degrees artificial discs had the similar equivalent shear stress (Se), and were both larger than that of normal discs, when lateral bending were performed, 80 MPa/0 degrees artificial discs were closed to normal discs when axial pressure and flexion/extension were carried out, while 80 MPa/10 degrees artificial discs had a larger Se than that of normal ones,when torsion loading were applied, Szx/Szy stress of 80 MPa/0 degrees and 80 MPa/10 degrees artificial discs were closed to normal ones. For C4-C6 segments, the axial pressure, flexion/extension and lateral bending of C5,6 were all lower than normal discs after C4,5 discs were replaced by 80 MPa/10 degrees artificial discs, while Szx/Szy of torsion loading were closed to normal ones.

CONCLUSIONArtificial discs with 10 degrees have less influences on stress of adjacent intervertebral space and closer to mechanical property after being implanted into intervertebral space.

Cervical Vertebrae ; surgery ; Humans ; Stress, Mechanical ; Total Disc Replacement ; methods