OBJECTIVETo develop a human ovarian carcinoma SKOV3 model in severe combined immunodeficiency (SCID) mouse and to study its biologic characteristics.

METHODSHuman ovarian carcinoma SKOV3 cells were injected intraperitoneally into female SCID mouse to establish a transplantation model of human ovarian carcinoma. The biological characteristics, metastasis and morphology of transplanted tumors were studied.

RESULTAll tumors grew progressively with no sign of regression. The tumor cells spread around the peritoneal cavity and mainly on the diaphragm, mesentery, peritoneum and around the liver, which was confirmed by histopathology. The morphology, growth pattern and CA125 secretion of primary culture of transplanted cells remained as same as those of ovarian carcinoma cell line SKOV3.

CONCLUSIONAn intraperitoneal transplantation model of human ovarian carcinoma SKOV3 in SCID mice has been developed successfully, which can simulate the biological behavior of peritoneal metastasis of human ovarian carcinoma.

Animals ; Disease Models, Animal ; Female ; Humans ; Mice ; Mice, SCID ; Neoplasm Transplantation ; Ovarian Neoplasms ; pathology ; ultrastructure ; Peritoneal Neoplasms ; secondary ; Transplantation, Heterologous

OBJECTIVETo explore the dynamic change in biochemical markers of bone turnover in preterm infants and the effect of early parenteral calcium supply.

METHODSForty preterm infants were divided into parenteral calcium supply group and control group. Blood and urine samples were collected at 24 h and 11 d after birth. Serum osteocalcin (OC) was measured with ELISA, serum carboxyterminal telopeptide type I collagen (ICTP) with radioimmunoassay, serum alkaline phosphatase (AKP), calcium, phosphate, and urine calcium, phosphate, creatinine with automatic biochemical analyzer. Blood samples were also collected from 22 term infants as control. Calcium gluconate (10%, 4 ml/kg x d(-1)) was administered intravenously in parenteral calcium supply group.

RESULTAt 24 h, serum AKP, ICTP [(147.86+/- 44.87)IU, (57.36+/- 6.34)micro g/L] in preterm infants were significantly higher than those [(147.86+/- 44.87)IU, (57.36+/- 6.34)micro g/L] in term infants, and negatively correlated with gestational age and birth weight (r =-0.528, P<0.01; -0.614, P< 0.01), but serum OC [(648.77+/- 238.89) nmol/L] in preterm infants was lower than that [(851.68+/- 238.69)nmol/L] of term infants, and positively correlated with gestational age and birth weight (r=0.359, P< 0.05; 0.376, P< 0.01). At 11 day, serum OC [947.25+/- 335.47)nmol/L] in preterm infants was markedly elevated and reached the level of term infants [(941.65+/- 297.28)nmol/L], but serum ICTP [(65.44+/- 6.24)micro g/L] in preterm infants was higher than that [(57.10+/- 3.48)micro g/L] in term infants all along. Serum AKP [(246.00+/-66.64)IU] in parenteral calcium supply group was higher than that [(206.53+/- 53.9)IU] in the control group. There were no significantly differences in serum OC and ICTP between parenteral calcium supply group and the control group. Calcium in serum and urine was elevated, phosphate in serum and urine was reduced in the parenteral calcium supply group. Urine analysis and kidney ultrasounds were normal.

CONCLUSIONThere is active bone formation and bone resorption in preterms as compared with terms. Alone parenteral calcium supply during early life can not increase formation of bone protein or decrease degradation of bone collagen, but can elevate serum calcium and urine calcium levels. Hematuria and renal calcification were not found in short duration.

Alkaline Phosphatase ; blood ; Bone and Bones ; metabolism ; Calcium ; administration & dosage ; blood ; urine ; Collagen Type I ; Female ; Humans ; Infant, Newborn ; Infant, Premature ; Injections, Intravenous ; Male ; Osteocalcin ; blood ; Peptide Fragments ; blood ; Peptides ; Procollagen ; blood

OBJECTIVETo investigate the distribution of G894T mutation of the endothelial nitric oxide synthase (eNOS) gene in Chinese Han nationality.

METHODSG894T mutation of the eNOS gene of 108 unrelated healthy individuals was studied by means of polymerase chain reaction/restriction fragment length polymorphism (PCR-RFLP) analysis.

RESULTAll subjects were genotyped for eNOS gene; the frequencies of the GG, GT and TT genotypes were 0.9095, 0.0883 and 0.0021, respectively. In the Han nationality, the frequency of the GT, TT genotypes was lower than that in Japanese and Caucasian of UK (P< 0.05).

CONCLUSIONThese results suggest that there are significant differences in G894T mutation of eNOS gene between the Chinese Han nationality and other ethnic populations.

Asian Continental Ancestry Group ; genetics ; China ; ethnology ; Genotype ; Humans ; Mutation ; Nitric Oxide Synthase ; genetics ; Nitric Oxide Synthase Type III ; Polymerase Chain Reaction

OBJECTIVETo construct a chimeric SEA-hPLAP-1 cDNA with gene splicing by overlap extension.

METHODSThe SEA gene and a DNA fragment encoding the signal for GPI-anchor attachment of hPLAP -1 were amplified by PCR. The two amplified gene sequence was annealed to form a chimeric GPI- anchored SEA molecule with gene splicing by overlap extension. The resulting chimera was cloned in pGEM-T vector and verified by sequencing analysis.

RESULTA chimeric SEA-hPLAP-1 cDNA was successfully constructed with gene splicing by overlap extension.

CONCLUSIONGene splicing by overlap extension is a successful specific PCR technique for gene recombination.

Alkaline Phosphatase ; Base Sequence ; Enterotoxins ; genetics ; GPI-Linked Proteins ; Isoenzymes ; genetics ; Molecular Sequence Data ; Polymerase Chain Reaction ; RNA Splicing ; Recombinant Fusion Proteins ; genetics

OBJECTIVETo determine the genome structure of human telomeric repeat binding factor 1 (TERF1) and its pseudogenes.

METHODSSequences were obtained from GenBank and analyzed using the BLAST program and other relevant biology program (Sequencher, DNA Strider and Autoassembler, etc) to determine the genome and pseudogenome structure of TERF1. PCR and sequencing were performed to verify the results.

RESULTTERF1 gene which mapped to 8q13 was divided into 10 exons. It had four processed pseudogenes located on chromosome 13, 18, 21 and X respectively (Psi TERF1-13 Psi TERF1-18 Psi TERF1-21 and Psi TERF1-X ). They were entire intronless TERF1 genes which lacked some exons. Three homologous fragments of at least 60 kb on the flanking region of Psi TERF1-13, Psi TERF1-18 and Psi TERF1-21, respectively were noted.

CONCLUSIONTERF1 gene has 10 exons. It has four processed pseudogenes which are located on chromosome 13, 18, 21, and X, respectively. Large homologous fragments that belong to the recently duplicated segments are transchromosomal duplications.

Chromosome Mapping ; Genetic Structures ; Humans ; Pseudogenes ; Telomeric Repeat Binding Protein 1 ; genetics

OBJECTIVETo establish a HepG2 cell line stably expressing the human cytochrome P450 1A2 and to study its metabolic activity.

METHODSThe human wild-type CYP1A2 cDNA was subcloned into a mammalian expression vector pREP9. A transgenic cell line was established by transfecting the recombinant plasmid of pREP9-CYP1A2 to HepG2 cells. The expression of CYP1A2 mRNA was validated by RT PCR. The metabolic activation of HepG2 CYP1A2 cells on aflatoxin B1 (AFB1) was assayed by cytotoxicity test.

RESULTThe HepG2-CYP1A2 cells expressed CYP1A2 mRNA and could increase the cytotoxicity to AFB1 in comparison with that of wild type HepG2 cells.

CONCLUSIONThe established HepG2-CYP1A2 can express the mRNA and has the metabolic activity to AFB1. The cell line may be useful for testing the toxicity and metabolism of xenobiotics, which might possibly be activated or metabolized by CYP1A2.

Aflatoxin B1 ; metabolism ; Biotransformation ; Cell Line ; Cytochrome P-450 CYP1A2 ; genetics ; metabolism ; DNA, Complementary ; chemistry ; Humans ; RNA, Messenger ; analysis

OBJECTIVETo investigate the function of POLH(polymerase eta) through establishment of the POLH gene-blocked cell line FL-POLH(-).

METHODSA mammalian expression vector expressing antisense POLH gene fragment pMAMneo-amp-POLHA (-) was constructed by cloning the 1473 - 2131 fragment of POLH gene into the mammalian expression vector pMAMneo-amp(-) in antisense orientation. The FL cells were transfected with this antisense RNA expressing vector and selected by G418. The mutation assay was conducted using the shuttle plasmid pZ189.

RESULTThe spontaneous mutation frequency of SupF tRNA gene in the plasmid replicated in the FL-POLH(-) was 13.5 x 10(-4), while it was 4.9x10(-4) and 3.7x10(-4) in the control cells FL and FL-M, respectively. The nontargeted mutation frequency of SupF tRNA gene decreased in the plasmid replicated in these cell lines pretreated with MNNG.

CONCLUSIONPOLH plays an important role in maintenance of genetic stability and genesis of nontargeted mutation.

Antisense Elements (Genetics) ; pharmacology ; Cell Line ; DNA-Directed DNA Polymerase ; genetics ; physiology ; Methylnitronitrosoguanidine ; toxicity ; Mutagenesis

OBJECTIVETo understand the up regulatory mechanism of human REV3 gene induced by the chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).

METHODSBioinformatic analysis of human REV3 gene promoter region was based on BLAST alignment, promoter prediction software and recognition of transcriptional factor binding sites. Cloning of human REV3 gene promoter region was performed by nested PCR. Response of human REV3 gene promoter to the chemical carcinogen MNNG was measured by transient transfection assay based on the dual luciferase reporter assay system.

RESULTBioinformatic analysis showed that human REV3 gene promoter region was located on chromosome 6 PAC clone RP3-415N12, and that the hypothetical promoter region contained promoter sequences, rich CpG islands, and putative recognition sites for several transcriptional factors, including AP-1/c-Jun/c-Fos, AP-2, STAT, CREBP, and NF-kappaB. Reconstructed reporter plasmid pGL3- 2582 was established by inserting 2582 nucleotides from the promoter region into the luciferase reporter vector pGL3-Basic. Transient transfection assay showed the hypothetical REV3 promoter region had promoter function, and it responded to MNNG treatment (P<0.01).

CONCLUSIONHuman mutator REV3 gene promoter region has been successfully cloned. The response of REV3 promoter region to MNNG suggests that REV3 gene can be regulated at transcriptional level under conditions of genotoxic stress.

Base Sequence ; Binding Sites ; Cloning, Molecular ; Computational Biology ; DNA-Directed DNA Polymerase ; genetics ; Humans ; Methylnitronitrosoguanidine ; toxicity ; Molecular Sequence Data ; Polymerase Chain Reaction ; Promoter Regions, Genetic ; Saccharomyces cerevisiae Proteins ; genetics

OBJECTIVETo investigate the effect of MNNG on some of the transcription factors such as NF- kappaB, CREB, AP-1 and c-Myc.

METHODSThe activities of these transcription factors were measured by transient transfection assay of SEAP vectors.

RESULTThe expressions of AP-1, CREB and NF- kappaB driven reporter genes were elevated for about 1.3, 1.4 and 1.3 times in MNNG-treated cells, respectively, as compared to untreated controls. However, the exposure of MNNG had no effect on the activity of c-Myc.

CONCLUSIONThe activation of certain transcription factors might be involved in the process of untargeted mutation induced by low concentration of MNNG treatment.

Animals ; Cercopithecus aethiops ; Cyclic AMP Response Element-Binding Protein ; drug effects ; Methylnitronitrosoguanidine ; toxicity ; Mutation ; NF-kappa B ; drug effects ; Proto-Oncogene Proteins c-myc ; drug effects ; Transcription Factor AP-1 ; drug effects ; Transcription Factors ; drug effects ; Vero Cells

OBJECTIVETo study the effect of MNNG on inducement of non-targeted mutation and activation of several cellular signal transduction pathways, and to determine whether the activation of these signaling pathways was dependent on the DNA-damage.

METHODSVero cells were enucleated by discontinuous density centrifugation. The PKA activities were measured by enzyme-linked immunosorbent assay. The status of cell membrane receptors was studied with immunofluorescent staining and confocal microscopy.

RESULTIn enucleated cytoplasts, MNNG-treatment increased PKA activity for about 2.3-fold in accordance with the 2.7-fold up-regulation of PKA activity in whole vero cells exposed to MNNG. The clustering of cell surface receptors of epidermal growth factor and tumor necrosis factor alpha was also observed in cells exposed to MNNG; this phenomenon was also found in enucleated cells.

CONCLUSIONThe results indicate that the initiation of signal cascades induced by low concentration of MNNG might be associated with its interaction with cell surface receptors and/or direct activation of related signal proteins but not its DNA damage.

Animals ; Cell Nucleus ; physiology ; Cercopithecus aethiops ; Cyclic AMP-Dependent Protein Kinases ; metabolism ; DNA Damage ; Enzyme Activation ; drug effects ; Methylnitronitrosoguanidine ; toxicity ; Receptor, Epidermal Growth Factor ; drug effects ; metabolism ; Receptors, Tumor Necrosis Factor ; drug effects ; metabolism ; Signal Transduction ; drug effects ; Vero Cells