Biomedical subjects

J Fu

Publications and source records attributed to J Fu.

At least 145 records · Page 8Linked to original sources

PubMed1999

[New coleopterancidai strains of Bacillus thuringiensis and production of coleopterancide].

Two coleopterancidal strains of Bacillus thuringiensis, YM-03 and SHQ11-10, were isolated from soil in China. H-Serotype of YM-03 was H8a8b, and H-Serotype of SHQ11-10 has not been identified. Both of them produced flat bipyramid-like crystals which consisted of 68-70 kD and 65 kD protein respectively. Bioassay indicated that these two Bt strains were very active on the larva of Leptinotarsa decemlineata and Plagioder aversicolora. They grew prosperously in fermentation. Powder of YM-03 product was high effect against colorado potato beetle in the field experiment.

Animals↗

PubMed1998

[Expression of human SRY gene and the DNA-binding property of its product].

OBJECTIVE: To investigate the role which human SRY gene plays in the regulation of the downstream gene. METHODS: The fragment of SRY (sex determinating region on the Y chromosome) HMG domain was cloned into the expressing vector pET-15b. The hSRY gene recombinant plasmid-pETSY was transformed and expressed in E.coliBL21. The target protein was purified by pET His. Tag system. The DNA-binding retardation test and its competitive reaction were conducted between SRY protein and the fragment of Mullerian inhibiting substance (MIS) promoter. RESULTS: The molecular weight of the expressed hSRY protein was shown to be approximately 21kD. The specific DNA binding property of SRY protein to the fragment of MIS was confirmed in the retardation test and its competitive reaction. CONCLUSION: The results suggest that the product of SRY gene can bind the MIS promoter region and may initiate the transcription of MIS.

DNA-Binding Proteins↗

PubMed1998

DAD1 is required for the function and the structural integrity of the oligosaccharyltransferase complex.

Asparagine-linked glycosylation is a highly conserved protein modification reaction that occurs in all eukaryotic organisms. The oligosaccharyltransferase (OST), which has its active site exposed on the luminal face of the endoplasmic reticulum (ER), catalyzes the transfer of preassembled high mannose oligosaccharides onto certain asparagine residues of nascent polypeptides. The mammalian OST complex was initially thought to be composed of three transmembrane proteins, ribophorin I (RI), ribophorin II (RII), and OST48. Most recently, a small integral membrane protein of 12 kDa called DAD1 has been identified as an additional member of the mammalian OST complex. A point mutation in the DAD1 gene is responsible for the temperature-sensitive phenotype of a baby hamster kidney-derived cell line (tsBN7) that undergoes apoptosis at the non-permissive temperature. Furthermore, the mutant protein DAD1 is not detectable in tsBN7 cells 6 h after shifting the cells to the non-permissive temperature. This temperature-sensitive cell line offered unique opportunities to study the effects caused by the loss of one OST subunit on the other three subunits and also on N-linked glycosylation. Western blot analysis of cell lysates showed that after 6 h at the non-permissive temperature, steady-state levels of the ribophorins were reduced by about 50%, and OST48 was barely detectable. On the other hand, steady-state levels of other components of the rough ER, such as the alpha-subunits of the TRAP (translocon-associated membrane protein) and the Sec61 complex, which are components of the translocation apparatus, are not affected by the instability of the OST subunits. Furthermore, N-glycosylation of the ribophorins was seriously affected 6 h after shifting the cells to the non-permissive temperature, and after 12 h they were synthesized only in the non-glycosylated form. As may be expected, this defect in the OST complex at the non-permissive temperature caused also the underglycosylation of a secretory glycoprotein. We concluded that degradation of DAD1 at the non-permissive temperature not only affects the stability of OST48 and the ribophorins but also results in the functional inactivation of the OST complex.

Animals↗

PubMed1998

A human SPT3-TAFII31-GCN5-L acetylase complex distinct from transcription factor IID.

In yeast, SPT3 is a component of the multiprotein SPT-ADA-GCN5 acetyltransferase (SAGA) complex that integrates proteins with transcription coactivator/adaptor functions (ADAs and GCN5), histone acetyltransferase activity (GCN5), and core promoter-selective functions (SPTs) involving interactions with the TATA-binding protein (TBP). In particular, yeast SPT3 has been shown to interact directly with TBP. Here we report the molecular cloning of a cDNA encoding a human homologue of yeast SPT3. Amino acid sequence comparisons between human SPT3 (hSPT3) and its counterparts in different yeast species reveal three highly conserved domains, with the most conserved 92-amino acid N-terminal domain being 25% identical with human TAFII18. Despite the significant sequence similarity with TAFII18, native hSPT3 is not a bona fide TAFII because it is not associated in vivo either with human TBP/TFIID or with a TFIID-related TBP-free TAFII complex. However, we present evidence that hSPT3 is associated in vivo with TAFII31 and the recently described longer form of human GCN5 (hGCN5-L) in a novel human complex that has histone acetyltransferase activity. We propose that the human SPT3-TAFII31-GCN5-L acetyltransferase (STAGA) complex is a likely homologue of the yeast SAGA complex.

Acetyltransferases↗

PubMed1998

The five cysteine residues located in the active site region of bovine aspartyl (asparaginyl) beta-hydroxylase are not essential for catalysis.

In previous chemical modification studies on bovine aspartyl (asparaginyl) beta-hydroxylase, cysteines were implicated as critical catalytic residues. Using site-directed mutagenesis, the five cysteine residues located in a highly conserved region of the enzyme identified as the active site were individually mutated to alanine. Substitutions at cysteine 637, 644, 656, 681, and 696 resulted in active mutant enzymes indicating that these residues are not required for catalysis.

Alanine↗

PubMed1998

Repeated tertiary fold of RNA polymerase II and implications for DNA binding.

X-ray diffraction data from two forms of yeast RNA polymerase II crystals indicate that the two largest subunits of the polymerase, Rpb1 and Rpb2, may have similar folds, as is suggested by secondary structure predictions. DNA may bind between the two subunits with its 2-fold axis aligned to a pseudo 2-fold axis of the protein.

Amino Acid Sequence↗

PubMed1998

Recombinant dengue virus type 1 NS3 protein exhibits specific viral RNA binding and NTPase activity regulated by the NS5 protein.

The full-length dengue virus NS3 protein has been successfully expressed as a 94-kDa GST fusion protein in Escherichia coli. Treatment of the purified fusion protein with thrombin released a 68-kDa protein which is the expected molecular mass for the DEN1 NS3 protein. The identity of this protein was confirmed by Western blotting using dengue virus antisera. Two related activities of the recombinant NS3 protein were characterized, which were the binding of the protein to the 3'-noncoding region of the dengue virus RNA genome and NTPase activity. We demonstrated using a band shift assay that the DEN1 NS3 protein could form a complex with the stem-loop structure in the 3'-noncoding region (3'-NCR), although sites outside the stem-loop may also participate in binding. Using various unlabeled homopolymeric and heteropolymeric RNAs as competitors for binding, it was further shown that the DEN1 NS3 protein exhibits preferential binding to a 94-nt RNA transcript from the 3'-NCR of the dengue virus. The NTPase activity of the recombinant DEN1 NS3 protein was characterized using a thin-layer chromatography assay. We found that the DEN1 NS3 protein possesses some aspects of NTPase activity, which are distinct from those found in other flaviviruses. Although the NS3 protein was able to utilize all four ribonucleoside triphosphates as its substrates, the NS3 protein showed a distinct preference for purine triphosphates (i.e., ATP and GTP). The addition of poly(U) did not stimulate NTPase activity in DEN1 NS3 protein, which contrasts with the reports for other flaviviral NS3 proteins. However, NTPase activity was specifically stimulated by the viral NS5 protein, which was manifested by a more than twofold increase in the rate of ATP hydrolysis and a 25% increase in the yield of ADP at the end of a 120-min reaction. These data suggest that the NTPase activity of the NS3 protein may be regulated by the viral NS5 protein during virus replication.

Adenosine Triphosphatases↗

PubMed1998

Genotoxicity of nitric oxide produced from sodium nitroprusside.

Induction of mutation and micronucleus (MN) formation by nitric oxide (NO) was investigated in mammalian cells using sodium nitroprusside (SNP) as a drug donor of NO. Results showed that the concentration of NO2- in the tested solution rose according to time- and concentration-exposure to SNP. The treatment of SNP (0.5-8 micromol/ml with S9 or 2-8 micromol/ml without S9) induced a concentration-dependent increase in the mutation frequency at the gpt gene locus in g12 cells and caused a 13- (-S9) to 25- (+S9) fold increase above the background level at the highest concentration. A statistically significant increase in the number of micronucleated binucleated cells (MNBN) was also observed in treated groups. MNBN per thousand, MN per thousand and the proportion of the multiple micronuleated cells increased in a concentration-dependent manner in the concentration range of SNP (0.5-4 micromol/ml with S9 or 2-8 micromol/ml without S9). Our results indicate that SNP, an NO releasing drug, is genotoxic in g12 cells.

Cell Line↗

PubMed1998

Toward the phylogeny of the family Lacertidae: implications from mitochondrial DNA 12S and 16S gene sequences (Reptilia: Squamata).

A phylogeny of the family Lacertidae was derived from mtDNA gene sequence data. Seventeen species, representing 16 currently recognized genera and subgenera, were included in the analysis. A total of 954 bp was obtained and aligned from 12S and 16S partial gene sequences. A preferred tree was selected based on weighted parsimony and functional ingroup and outgroup analyses. Decay analysis, bootstrapping, and permutation tail probability were used to evaluate support for the recovered nodes. The genus Gallotia was resolved as the basal taxon and the sister group of all remaining lacertids. Takydromus branched off next. All African lacertids grouped together and formed a monophyletic clade with the Eurasian genera Eremias and Ophisops. The remaining Eurasian lacertids sequentially branched off near the base of the tree in a "comb-like" fashion. The basal position of Gallotia and the monophyly of African lacertids are consistent with previous hypotheses. The European-origin hypothesis of lacertids is favored, and the distribution of lacertids in Africa is likely a Miocene dispersal event. Most of the extant European lacertids probably arose after the Eocene. The classification of the family needs to be revised.

Animals↗

PubMed1998

The parthenogenetic rock lizard lacerta unisexualis: An example of limited genetic polymorphism

Protein electrophoresis of Lacerta unisexualis from three populations found that 21 of 36 allozyme loci were homozygous, while 14 expressed fixed heterozygotes and one locus was variable. Three clones were detected at the locus Cat-A. Two individuals represent two rare clones while all others form a common clone. Our favored explanation is the mutation of a preexisting common clone rather than multiple origins.

Journal Article↗

PubMed1998

Association of distal chromosome 2q with IDDM in Japanese subjects.

An insulin-dependent diabetes mellitus (IDDM)-susceptibility gene (IDDM13) has recently been mapped to a region of distal chromosome 2q, which is syntenic to the region of mouse chromosome 1 containing a murine susceptibility gene for IDDM, Idd5. To determine the contribution of this region to IDDM disease susceptibility further and to narrow the region for positional cloning of susceptibility genes, we have studied the association of distal chromosome 2q with IDDM in the genetically distinct Japanese population. A 137 mobility unit (mu) allele at D2S137 locus was significantly associated with IDDM (odds ratio 1.92, p = 0.0016). Other markers, D2S301 and D2S143, located in the same region were not associated with IDDM, indicating that IDDM13 is in linkage disequilibrium with D2S137, but not with D2S301 or D2S143. The association of D2S137 with IDDM was observed in patients lacking one of two high risk HLA alleles, DQBI*0303 and DQBI*0401, but not in patients with either of these alleles. The frequency of high risk HLA alleles was significantly lower in patients with the susceptible allele at D2S137, suggesting that IDDMI3 contributes to IDDM susceptibility in subjects without high risk genotypes at IDDM1. Demonstration of allelic association of D2S137 with IDDM localizes IDDM13 in the close vicinity (<2 centiMorgans) of D2S137, greatly facilitating fine structure mapping and positional cloning of IDDM13.

Adolescent↗

PubMed1998

Hybrid bilayer membranes in air and water: infrared spectroscopy and neutron reflectivity studies.

In this report we describe the fabrication and characterization of a phospholipid/alkanethiol hybrid bilayer membrane in air. The bilayer is formed by the interaction of phospholipid with the hydrophobic surface of a self-assembled alkanethiol monolayer on gold. We have characterized the resulting hybrid bilayer membrane in air using atomic force microscopy, spectroscopic ellipsometry, and reflection-absorption infrared spectroscopy. These analyses indicate that the phospholipid added is one monolayer thick, is continuous, and exhibits molecular order which is similar to that observed for phospholipid/phospholipid model membranes. The hybrid bilayer prepared in air has also been re-introduced to water and characterized using neutron reflectivity and impedance spectroscopy. Impedance data indicate that when moved from air to water, hybrid bilayers exhibit a dielectric constant and thickness that is essentially equivalent to hybrid bilayers prepared in situ by adding phospholipid vesicles to alkanethiol monolayers in water. Neutron scattering from these samples was collected out to a wave vector transfer of 0.25 A(-1), and provided a sensitivity to changes in total layer thickness on the order of 1-2 A. The data confirm that the acyl chain region of the phospholipid layer is consistent with that observed for phospholipid-phospholipid bilayers, but suggest greater hydration of the phospholipid headgroups of HBMs than has been reported in studies of lipid multilayers.

Lipid Bilayers↗

PubMed1998

Higher levels of thymidylate synthase gene expression are observed in pulmonary as compared with hepatic metastases of colorectal adenocarcinoma.

PURPOSE: It has been observed previously that the pulmonary metastases of colorectal adenocarcinoma are less responsive to therapy with fluorouracil (FUra) as compared with other sites of metastasis (liver, local). To investigate the basis of this chemoresistance, the levels of thymidylate synthase (TS) mRNA and protein were measured, as TS expression has been shown to be predictive of response to therapy in colorectal cancer. MATERIALS AND METHODS: Tumors were obtained from 19 patients with metastatic colorectal cancer (12 hepatic and seven pulmonary). TS expression was measured by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and TS protein levels were measured by Western blotting. The presence of TS amplification was assessed by Southern blotting. Levels of p53 protein were determined using immunohistochemistry. RESULTS: TS mRNA expression was shown to be significantly higher in the pulmonary metastases (mean TS/beta-actin ratio, 19.7; n = 7) as compared with the hepatic metastases (mean TS/beta-actin ratio, 4.7; n = 11) of colorectal cancer. Lower TS expression was observed in patients with hepatic metastases who had received prior FUra versus patients who had not been treated. High levels of TS expression in some samples was associated with low-level (two to three gene copies) increases in TS gene copy numbers and this was observed more frequently in the pulmonary metastatic samples. The increased gene copy numbers occurred both in samples with wild-type p53 and those with mutant p53 tumor-suppressor gene as determined by immunohistochemistry. CONCLUSION: High levels of TS enzyme may be the basis of the lack of response of pulmonary metastases to FUra treatment.

Adenocarcinoma↗

PubMed1998

Effect of interleukin-6 on the growth of human lung cancer cell line.

OBJECTIVE: To investigate the effect of interleukin-6 (IL-6) on the growth of human lung cancer in vivo as well as in vitro. METHODS: To examine the mRNA level of IL-6 receptor (IL-6R) in high-metastatic human lung giant cell carcinoma cell line PG by means of reverse transcription polymerase chain reaction (RT-PCR). To assess the existence of IL-6 receptor complex (including IL-6R and gp130) with the treatment of PG cells by use of recombinant human IL-6 (rhIL-6), recombinant human oncostatin M (rhOSM), and recombinant human leukemia inhibitory factor (rhLIF), respectively. To detect the expression of IL-6 by Northern blotting hybridization and bioactive assay. To identify the effect of IL-6 secreted by PG cells by use of IL-6 and IL-6R antisense oligodeoxynucleotides (ODNs), and specific neutralizing antibody to IL-6. To document the influence of IL-6 on PG cells growth in vivo through the strategy of the transfection of expression vector inserted antisense IL-6 cDNA. RESULTS: RT-PCR analysis revealed that PG cells expressed IL-6R mRNA. Any one of the recombinant cytokine IL-6, OSM and LIF stimulated the growth of PG cells in vitro in a concentration-dependent manner. These results demonstrated IL-6 receptor complex exist in PG cells. At the same time, PG cells expressed IL-6 mRNA and secreted bioactive IL-6. Both IL-6 antisense ODNs and IL-6R ODNs inhibited PG cells proliferation. Treatment of PG cells with IL-6 antibodies reduced the growth of PG cells in vitro. PG cells transfected with IL-6 antisense expression vector showed a decreased growth in nude mice. CONCLUSION: IL-6 functions as an autocrine growth stimulator for PG cells in vivo as well as in vitro.

Animals↗

PubMed1998

[Telomerase activity in liver cancer and the adjacent tissue].

OBJECTIVE: To investigate the possibility of using telomerase as a tumor marker for human primary hepatocellular carcinomas (HCC). METHODS: Human liver specimens, comprising 33 HCC and adjacent peritumoral tissues, 4 hepatic metastases and the adjacent peritumoral tissues, 6 benign liver lesions and 6 normal liver tissues, were examined for telomerase activity by TRAP assay based on PCR. RESULTS: Thirty of 33 HCC and 9 of 33 adjacent tissue specimens were positive for telomerase activity with a positive rate of 90.9% and 27.3%, respectively. Four hepatic metastatic cancer and 2 adjacent tissues were positive. Telomerase activity was negative in 6 nomal liver tissues and 5 of 6 benign liver lesions. There was no correlation between the clinical stage of HCC and their telomerase activity. CONCLUSION: Positive telomerase activity occurs in the majority of HCC examined. It can be used as a tumor marker in the diagnosis of HCC.

Biomarkers, Tumor↗

PubMed1998

[Efficient gene transfer mediated by retrovirus vector in leukemic cells].

OBJECTIVE: To establish an efficient and safe gene transfer system mediated by retrovirus for providing an experimental basis of gene therapy in human leukemia. METHODS: The retrovirus vector pLXSN, carrying the NeoR, was transferred into amphotropic packaging cell line GP + envAm12 by liposome method or by transduction. Retrovirus with higher titer was used to infect human leukemic cell lines NB4, U937 and THP-1. The efficiency of gene transduction was tesed on colonies formed by K562 cells after gene transduction. RESULTS: The titer of DOSPER liposome transfected GP + envAm12 pool on NIH3T3 cells was 8.0 x 10(5) CFU/ml while that of producer pool infected with mouse retrovirus was 1.6 x 10(7) CFU/ml. Integration of NeoR into human leukemic cells was confirmed by PCR. By both nested PCR and NeoR rescue assay, no helper virus was found. NeoR gene transfer with the efficiency as high as 93.3 to 100% in K562 cells was verified by semi-nested PCR on the colonies after seven days' culture. CONCLUSION: The efficiency and safety of the gene transfer system may provide an optimal experimental system in gene therapy for human leukemia.

Animals↗

PubMed1998

[Interleukin-12 gene transfection into murine B16 melanoma cells suppresses tumorigenicity and decreases metastatic potential].

OBJECTIVE: To study the tumorigenicity and metastasis of poorly immunogenic murine B16 melanoma cells transfected with murine interleukin-12 (IL-12) gene. METHODS: Two recombinant vectors containing the full length cDNA of p40 or p35 subunit of IL-12 were constructed. They were cotransfected into B16 melanoma cells by LipofectAMIN method. The expressions of p40 and p35 mRNAs were analyzed by means of reverse transcription polymerase chain reaction (RT-PCR). The secretion of bioactive IL-12 was detected by bioassay (measuring the proliferative response of PMA-activated murine splenocytes). To determine the effects of IL-12 secreted by genetically engineered B16 cells (B16T-IL12) on tumorigenesis and metastasis, the cells were inoculated s.c. or i.v. into C57BL/6 mice. RESULTS: B16T-IL12 cells expressed both p40 and p35 mRNAs, also produced bioactive IL-12. Only 70% of the mice injected with B16T-IL12 cells developed palpable tumors. The emergence of palpable tumors in the mice inoculated with B16T-IL12 cells was significantly delayed (P < 0.01), the growth rate of the tumors was obviously decreased (P < 0.01), and the survival time of tumor-bearing mice was prolonged substantially (P < 0.01), compared with the control B16 or B16Tneo cells. The incidence of experimental pulmonary metastasis of B16T-IL12 cells was inhibited, and the number of pulmonary metastases was reduced markedly, in contrast to the i.v. inoculation of B16 cells (P < 0.01). CONCLUSION: Transfection of IL-12 gene into B16 cells results in the inhibition of tumorigenesis and in the suppression of experimental pulmonary metastasis.

Animals↗

PubMed1998

[Human tumor xenografted into SCID mice and human immune function reconstitution].

OBJECTIVE: In order to observe (1) The behavior of growth and metastasis of PG and PGPTS7 in SCID mice and human immune function reconstituted mice; (2) The ability of interleukin-6 autosecreted from PGTS7 in enhancing the anti-tumor activity of the peripheral blood lymphocytes (PBL). METHODS: Xenografting of PG and PGTS7 into the subepithelial space of the SCID mice, and in some of these animals, human PBL were administrated simultaneously into the peritoneal cavities. The latent period, taken rate, growth speed, volume of the grafted tumor, incidences of metastasis in lungs and lymph nodes and serum level of human immunoglobulin of the immunity reconstituted mice were examined. RESULTS: Although tumor growth had been detected in all the experimental animals, the latent period of grafted PGTS7 was postponed and the volume of tumor mass as well as the incidence of lymph node metastasis all became lower in the immunity reconstituted mice accompanied simultaneously with a higher serum level of human immunoglobulin (HIg). CONCLUSIONS: (1) The SCID mice are good as an appropriated hosts in studying the behavior of growth and metastasis of PG and PGTS7. (2) IL-6 autosecreted from PGTS7 stimulates the proliferation and promotes the activating of PBL. It seems also able to enhance the liberation of human immunoglobulin: to suppress the growth of tumor cells and to reduced the rate of lymph node metastasis.

Animals↗