Biomedical subjects

B Dai

Publications and source records attributed to B Dai.

At least 55 records · Page 3Linked to original sources

PubMed1995

Novel gastrin receptors mediate mitogenic effects of gastrin and processing intermediates of gastrin on Swiss 3T3 fibroblasts. Absence of detectable cholecystokinin (CCK)-A and CCK-B receptors.

We have reported previously mitogenic effects of gastrin on several immortalized and neoplastic cell lines, including Swiss 3T3 fibroblasts. Receptor subtypes, cholecystokinin (CCK)-A and CCK-B, for a closely related peptide, cholecystokinin, were recently cloned. These studies were undertaken to investigate if CCK-A- and CCK-B receptors were perhaps mediating the mitogenic effects of gastrin on Swiss 3T3 cells. Receptor antagonists that inhibit the biological effects and binding of peptides to the CCK-A (L-364,718 (L18)) and CCK-B (L-365,260 (L60)) receptors were ineffective toward inhibiting the binding and proliferative effects of gastrin on Swiss 3T3 cells. Radiolabeled L18 and L60 demonstrated no binding to the cells, indicating that CCK-A and CCK-B receptors may be absent on Swiss 3T3 cells. Radiolabeled CCK-8, gastrin, L18, and L60, on the other hand, demonstrated specific binding to a pancreatic cancer cell line (AR42J cells) (used as a positive control). In cross-linking studies the molecular mass of the major band of gastrin receptors (GR) on Swiss 3T3 cells was determined to be approximately 45 kDa. The mitogenic potency of 0.1-1.0 nM gastrin-like peptides on Swiss 3T3 cells was in the order of G1-17 > or = G1-17-Gly > G5-17 > or = G5-17-Gly > G2-17 > CCK-8-Gly > or = G1-17-Lys > or = CCK-8. The relative binding affinity of the peptides (based on the dose-dependent inhibition of binding of 125I-G1-17 to Swiss 3T3 cells) was similar to the relative mitogenic potency of the peptides as given above. Furthermore, G1-17-Gly was equally effective as G1-17 in displacing the binding of 125I-G1-17 to the 45-kDa GR from the Swiss 3T3 cells. Based on these studies it became evident that the novel gastrin preferring GR, expressed by Swiss 3T3 cells, binds and mediates the mitogenic effects of not only the mature (amidated) forms of gastrin-like peptides but also binds and mediates the mitogenic effects of glycine-extended forms of gastrin-like peptides. Possible mRNA expression of CCK-A and CCK-B receptor subtypes by gastrin-responsive rodent intestinal and fibroblast cell lines (Swiss 3T3, IEC-6, CA) was measured by the methods of Northern blot analysis and reverse transcriptase-polymerase chain reaction. mRNA from rat pancreas, AR42J cells, and rat antrum served as positive controls.(ABSTRACT TRUNCATED AT 400 WORDS)

3T3 Cells↗

PubMed1995

[Identification of pathogenic and nonpathogenic leptospires by recombinant DNA probe].

A gene bank of L. interrogans serovar lai strain 017 was constructed with plasmid vector pUC 18. Recombinant plasmids designated pDJ 6 and pDJ 8 were screened from the gene bank. Inserted fragments of them are 1.9kb and 2.2kb respectively. Diglabelled 1.9kb inserted fragment of pDJ 6. Results showed that the probe had hybridization with pathogenic leptospires, but it did not have hybridization with nonpathogenic leptospires such as L. biflexa strain Patoc I and Leptonema illini. The probe did not have hybridization with nonhomologous DNA (e.g. human leucocyte and E. coli JM 103), either. So, the recombinant probe is a good tool for distinguishing and identifying genus, species, pathogenic leptospires and nonpathogenic leptospires.

DNA Probes↗

PubMed1995

[Homology of the recombinant DNA of plasmid pDJH2 with the recombinant DNA probe of L. alstoni and analysis of its expression in Escherichia coli].

Dig-labeled recombinant DNA probe of L. alstoni which contains the entire structural OmpL1 gene was hybridized with the recombinant DNA of the plasmid named pDJH2 of the gene library of L. interrogans serovar lai strain 017. The result showed a high degree of homology among them; expression of recombinant DNA of pDJH2 was achieved by beta-D-galactosidase (IPTG) induction in E. coli. The molecular weight of this product is 68kd. Then they were treated with proteinase K and subjected to SDS-PAGE. The results showed it is a protein in nature. Using the specific monoclonal antibody E4B7D5 on immunoblotting and specific polyclonal antibody on dot-ELISA assay, we investigated the immune reaction and noticed that protein 68kd might be an antigen in character. E. coli which contains the recombinant plasmid pDJH2 were injected into BALB/c mice. Then the mice were challenged by leptospires of the strong virulence strain 017, but all the infected mice survived. In this paper, we first report the expression of recombinant DNA of L. interrogans serovar lai strain 017 in E. coli when injected with IPTG, and immunoprotection of BALB/c mice which were injected with the expression against the infection of L. interrogans serovar lai strain 017. pDJH2 may be the first recombinant for which the gene has been cloned and its expression product 68kd may be the immunoprotective antigens.

Animals↗

PubMed1995

[Homology study of leptospires in China with the OmpL1 gene].

Southern hybridization analysis with the OmpL1 gene was performed. The results of probing EcoRI-restricted genomic DNA from 18 strains in China showed that the homology fragments of OmpL1 gene were presented in 12 pathogenic Leptospira strains: sero-group Icterohaemorrhagiae (2 strains), Canicola, Ballum, Pyrogenes, Autumnalis, Australis, Pomona, Grip-potyphosa, Hebdomadis, Bataviae, and Sejroe, but they were not presented in the nonpathogenic Leptospira biflexa strain Patoc I, Leptonema illini strain 3055 and pathogenic Leptospira Serogroup Javanica, Tarassovi, Manhao and Mini.

Bacterial Outer Membrane Proteins↗

PubMed1995

[Construction of genomic library of L. interrogans serovar lai and preliminary study of recombinant plasmid pDC38].

A genomic library, consisting of approximate 12 000 recombinants, has been constructed for the Leptospira interrogans serovar lai in E. coli. using pUC18 as the vector. Hybridization analysis with the DNA fragment containing OmpL1 gene was performed and 10 positive clones were screened from the genomic library. One of the positive clones, designated pDC38, showed hybridization signal with the DNA of 7 serovars 8 strains of pathogenic leptospires, but not with the DNA of nonpathogenic leptospires (L. biflexa), Leptonama illini, E. coli., and 2 strains of L. interrongans.

DNA, Recombinant↗

PubMed1994

Episomal expression of sense and antisense insulin-like growth factor (IGF)-binding protein-4 complementary DNA alters the mitogenic response of a human colon cancer cell line (HT-29) by mechanisms that are independent of and dependent upon IGF-I.

HT-29 cells express and secrete insulin-like growth factor (IGF)-II and only one of the six IGF-binding proteins, IGFBP-4. In the present study, the physiological role of endogenous IGFBP-4 in regulating the growth response of HT-29 cells to exogenous and endogenous IGFs was examined. Both the basal and the IGF-stimulated growth of HT-29 cells was significantly increased over control values in the presence of IGFBP-4 antibody, suggesting that endogenous IGFBP-4 is a potent inhibitor of the mitogenic effects of endogenous and exogenous IGFs. In order to further confirm the inhibitory role of endogenous IGFBP-4, sense and antisense complementary DNA fragments of human IGFBP-4 were ligated into an episomal mammalian expression vector (pCEP4). Restriction mapping and Southern blot analysis were used to confirm directional cloning of the IGFBP-4 complementary DNA fragments in the sense and antisense directions in the pCEP4 vectors. HT-29 cells were transfected with either the control (no insert, C-P), sense (S-P), or antisense (AS-P) vectors and subjected to hygromycin selection. The functional nature of the transfectants was confirmed by measuring IGFBP-4 concentrations in the conditioned media (CM) of 10(7) cells by ligand and immunoblot analysis. IGFBP-4 concentrations were 7.4 +/- 1.7-fold higher in the CM of S-P cells compared to that in the CM of C-P cells, while IGFBP-4 concentrations in the CM of AS-P cells were significantly lower than those present in the CM of C-P cells. Both the basal and the IGF-I-stimulated growth of the AS-P cells was significantly higher than that of the C-P and S-P cells. The basal (non-stimulated) and the IGF-I-stimulated growth of the S-P cells was not significantly different from that of the C-P cells, suggesting that overexpression of IGFBP-4 was not inhibitory to the growth of the HT-29 cells. The basal growth of the S-P and C-P cells was significantly increased in the presence of IGFBP-4 antibody, once again suggesting that endogenous IGFBP-4 was a potent inhibitor of autocrine effects of endogenous factors (IGF-II). Addition of IGFBP-4 antibody had no significant effect on the basal growth of the AS-P cells, confirming that the difference between the growth response of the AS-P, C-P, and S-P cells was largely contributed by the difference in the endogenous secretion of IGFBP-4 by the cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies↗

PubMed1994

Gastrin gene expression in human colon cancer cells measured by a simple competitive PCR method.

Gastrin is mitogenic for several colon cancers and is postulated as an autocrine growth factor for colon cancer cells. In the present study we report the development of a simple competitive polymerase chain reaction (PCR) method for measuring relative abundance of gastrin gene expression in colon cancer cells. Primers flanking exons 2 and 3 of the gastrin gene were utilized for co-amplification of cDNA and genomic DNA. The amplification of genomic DNA was distinguished from that of cDNA by the presence of the 130 bp intron sequence which was resolved by electrophoresis on agarose gels. A standard reaction of competitive PCR, using known concentrations of genomic DNA and cDNA, was first established. The steady state levels of gastrin mRNA were next quantitated in three human colon cancer cell lines (HCT-116, Colo-205 and DLD-1) by competitive PCR. Gastrin mRNA levels in these cell lines ranged from approximately 0.1 to 1.0 fmoles/mg total RNA (approximately 2-25 copies of gastrin mRNA per cell). Thus low to moderate levels of gastrin were expressed by human colon cancer cell lines which may function as autocrine growth factors for colon cancers.

Base Sequence↗

PubMed1994

Incomplete processing of progastrin expressed by human colon cancer cells: role of noncarboxyamidated gastrins.

Gastrin is mitogenic for several colon cancers. To assess a possible autocrine role of gastrin in colon cancers, we examined human colon cancer cell lines for expression of gastrin mRNA and various forms of gastrin. Gastrin mRNA was not detected in the majority of colon cancer cell lines by Northern hybridization but was detected in all human colon cancer lines by the sensitive method of reverse transcriptase-polymerase chain reaction (PCR). Gastrin mRNA was quantitated by the competitive PCR method. The majority of cell lines expressed very low levels of gastrin mRNA (< 1-5 copies/cell); only one cell line expressed > 20 copies/cell. The mature carboxyamidated form of gastrin was not detected in any of the cell lines by radioimmunoassay or immunocytochemistry. Results suggested that either gastrin mRNA expressed by colon cancer cells was altered (mutated) or posttranslational processing of progastrin was incomplete. Gastrin cDNA from all the colon cancer cell lines had an identical sequence to the published sequence of human gastrin cDNA. Specific antibodies against precursor forms of gastrin were used, and significant concentrations of nonamidated (glycine-extended) and prepro forms of gastrin were measured in tumor extracts of representative colon cancer cell lines. The presence of precursor forms of gastrin suggested a lack of one or more of the processing enzymes and/or cofactors. Significant concentrations of the processing enzyme (peptidylglycine alpha-amidating monooxygenase) were detected in colon cancer cells by immunocytochemistry. Therefore, lack of other cofactors or enzymes may be contributing to incomplete processing of precursor forms of gastrin, which merits further investigation. Since low levels of gastrin mRNA were expressed by the majority of human colon cancer cell lines and progastrin was incompletely processed, it seems unlikely that gastrin can function as a viable autocrine growth factor for colon cancer cells. High concentrations of glycine-extended gastrin-17 (GG) (> 10(-6) M) were mitogenic for a gastrin-responsive human colon cancer (DLD-1) cell line in vitro. It remains to be seen if GG or other precursor forms of gastrin are similarly mitogenic in vivo, which may then lend credibility to a possible autocrine role of gastrinlike peptides in colon cancers.

Base Sequence↗

PubMed1994

Expression of IGF-II and IGF-binding proteins by colon cancer cells in relation to growth response to IGFs.

We previously reported that even though virtually all human colon cancers were positive for IGF-I receptors, only 50% responded to growth effects of insulin-like growth factor (IGF)-I (1-100 nM). The present studies were undertaken to determine whether expression and secretion of IGFs (IGF-I, IGF-II) and IGF-binding proteins (BPs; 1-6) were perhaps different in IGF-responsive (COLO 205, COLO 320, Caco-2) and IGF-nonresponsive (HCT 116, HT-29, DLD-1) cells. Several bands (2.0-6.0 kb) of IGF-II mRNA transcripts were detected in all the cell lines; none expressed IGF-I. Significant concentrations of IGF-II (0.2-0.9 ng/10(6) cells) were measured in the conditioned media (CM) of the cells. All cell lines expressed BP2 and/or BP4 mRNA and secreted BP4 (24 kDa) and/or BP2 (32.5 kDa); BP1 was not detected in any cell line. Interestingly, BP3 mRNA was measured only in the responsive cell lines. The relative concentration of total BPs tended to be higher in the CM of nonresponsive cells. Interestingly, a large concentration of 44- to 48-kDa BP (BP3?) was associated with the membranes of only the responsive cell lines. Our present studies thus demonstrate that human colon cancers do not secrete IGF-I and BP1. Of all the IGF-related factors examined, the quantity and the type of BPs expressed by the human colon cancer cell lines (especially BP2, BP4, and BP3) may significantly dictate the growth response of the cells to exogenous IGF-I.

Animals↗

PubMed1994

[16S rRNA reverse transcription-polymerase chain reaction of leptospires].

We amplified the leptospiral RNAs of Leptospira interrogans serovar lai strain Lai and L. biflexa serovar patoc strain Patoc I, extracted by the method of SiO2-high concentration salt solution (8 mol/L GuHCl) absorption, with the 16S rRNA gene primers of Leptospira interrogans by reverse transcription-polymerase chain reaction (RT-PCR). It demonstrated that the detecting sensitivity by naked eye after electrophoresis could be 100 times higher when we amplified DNA and RNA of leptospires at the same time by RT-PCR.

Base Sequence↗

PubMed1994

Identification of pathogenic leptospires by recombinant DNA probes.

Early diagnosis of leptospirosis of pulmonary diffuse hemorrhage type (PDH) is of crucial importance in saving patients. To develop a sensitive and specific method for diagnosis, a genomic library of the main pathogen of PDH, L. interrogans serovar lai strain 017, was constructed with the plasmid vector pUC9. Recombinant plasmids which have homologous fragments of pathogenic leptospires were screened from the bank. A recombinant plasmid, designated pCX7, could detect 1.7 kb fragment of strain 017, 9.0 kb of strain 601 and 30.0 kb of strain Hebdomadis, respectively, without cross hybridization with nonpathogenic leptospires such as L. biflexa strain Patoc I and Leptonema illini. The recombinant plasmid pCX7 could detect pathogenic leptospires which are the main pathogens endemic to Sichuan Province.

Blotting, Southern↗

PubMed1994

[Specificity of the recombinant DNA pCX7 and its restriction enzyme map].

The recombinant DNA pCX7 derived from L. interrogans serovar lai strain 017 was applied for hybridization with 17 strains (8 serogroups of L. interrogans, L. biflexa strain Patoc I and Leptonema strain 3055); meanwhile 15 restriction endonucleases were used for analysis of the restriction enzyme map of pCX7. The result showed that the recombinant DNA probe is specific to species L. interrogans and its homology with DNA from different leptospires. A restriction enzyme map of pCX7 was constructed. Unique site for Bgl II and 3 sites for Bst BI were located within the cloned fragment (1.7 kb).

DNA, Bacterial↗

PubMed1994

[Analysis on early serum samples from patients with leptospirosis by polymerase chain reaction].

Fifty-five serum samples from patients with leptospirosis treated by the method of high SiO2 concentration salt solution absorption were analysed with the variable sequences of the 16S rRNA genes of Leptospria interrogans as primers. It is shown that the 16S rRNA gene primer is useful for the clinical detection and the epidemic investigation of leptospirosis, and the detecting effect by PCR is superior to that by the blood culture and MAT (P < 0.01).

Genes, Bacterial↗

PubMed1993

[PCR amplification of the leptospiral DNAs from different genus and species with the variable sequences of 16S rRNA gene].

We designed a pair of primers from the variable regions (V2 and V4) of 16S rRNA gene of Leptospira interrogans, i. e. PI: 5'GGG AAC CTA ATA CTG GAT GG; PII: 5' ACA TAG TTT CAA GTG GAG GC, and amplified the leptospiral DNAs from different genus and species. When denaturing with 55 degrees C, all DNAs of L. interrogans had the same products not only in length but also with Kpn I-digested pattern. The DNA of L. biflexa could be amplified with a c. a. 280 bp-band but not digested by Kpn I, while the DNAs of Leptonema and other control bacteria had no amplification. In addition, the products of L. interrogans spp. could be hybridized with the PCR product of L. interrogans serovar lai strain Lai labelled with 32P, while the product of L. biflexa had no hybridization. It proved that the 16S rRNA gene primers is useful for the classification and detection of leptospires.

Base Sequence↗

PubMed1993

[Antigen analysis of McAb E4B7D5 directed against outer envelope of Leptospira interrogans serovar lai by SDS-PAGE and immunoblot].

McAb E4B7D5 was prepared by hybridoma technology in BALB/c mice immunized to outer envelope of Leptospira interrogans serovar lai. This McAb agglutinated specifically with all the 13 serovars of Icterohaemorrhagiae serogroup in MAT test at high titres and protected the guinea pigs against the attack of virulent strain (017) of serovar lai. SDS-PAGE and immunoblot were used to analyse the reaction of the outer envelopes of the five strains of Leptospira (Leptospira interrogans Icterohaemorrhagiae serogroup, serovar lai, 017 and 601 stains; L. interrogans Hebdomadis serogroup, hebdomadis serovar strain 156; L. interrogans Australis serogroup australis serovar strain 620; biflexa leptospira patoc serovar strain Patoc I; with McAb E4B7D5. Results indicated that this McAb E4B7D5 recognized specifically 34.5 kd and 39.5 kd outer envelope proteins of serovar lai, strain 017 and 601, so it might be an immunoprotective McAb directed against Leptospira interrogans serovar lai. It was suggested that 34.5 kd and 39.5 kd outer envelope proteins of serovar lai recognized specifically by McAb E4B7D5 might be immunoprotective antigens.

Animals↗

PubMed1993

[Restriction endonuclease analysis of leptospiral DNA from different serogroup and serovar].

The DNAs of different serogroups and serovars of leptospires were analyzed with seven kinds of restriction endonuclease (Bam HI, Bgl II, Cfo I, Eco RI, Hind III, Kpn I and Pst I). The results showed that there are significant differences among the leptospiral DNAs of different serovars, especially in the high molecular-weight region, but the patterns of different strains belonging to the same serovar are similar. In addition, Cfo I and Eco RI are more effective for differentiating leptospires.

DNA Restriction Enzymes↗

PubMed1993

[Amplified 23S rRNA gene of 52 strains of Leptospira and detection of leptospiral DNA in 55 patients by PCR].

Based upon the polymerase chain reaction (PCR), We have developed a sensitive assay for Leptospira interrogans, the agent of leptospirosis. DNA amplification was carried out using primer A: 5'GATCTAATTCGCTGTAGCAGG3' and primer B: 5'ACTTTCACCCTCTATGGTCGG3'. After 30 cycles of amplification, the product could be detected by agarose gel electrophoresis. A segment (124 bp) was amplified in all strains of L. interrogans including 20 Serogroups, 49 Serovars tested, but it was not detected in Patoc I strain, Serovar patoc of Leptospira biflexa and 3055 strain of Leptonema illini (both of which are nonpathogenic). All of the serum (first time) which proved either by blood culture or MAT showed that positive rates were 100%. Leptospiral infection in humans and some domestic animals leads to one or more of a variety of manifestation and persists through a considerable duration of time. However it is relatively difficult to demonstrate the presence of leptospires in serum. The serum (second time) obtained from 55 patients with leptospirosis (6-60 days after on set) showed that PCR positive rates were 74.55% (41/55). The PCR positive rates for healthy subjects were 15% (3/20), P < 0.001. The diagnosis of leptospirosis by using PCR may become a significant addition to the routine laboratory diagnosis and a valuable technique for the investigation of leptospirosis pathogenesis.

Base Sequence↗

PubMed1993

[Parietal prosthetic mesh in the treatment of large incisional hernias].

The repair of abdominal wall deficiencies has been simplified by the introduction of synthetic prostheses. The aim of this work was to describe the treatment of very large incisional abdominal hernias with a technique using a prosthetic mesh anchoring to the fibro-aponeurotic layers without closure the fascia. The procedure described has been used in 32 cases between 1974 and 1991 (15 male, 17 female). The median age was 62 years. All the patients were reexamined (median time: 4 years, range: 6 months-14 years). There was no operative mortality. Six patients had a subcutaneous seroma treated by multiple punctions. Three patients presented in the postoperative time a respiratory failure which necessitated artificial ventilation during 7 to 15 day. Two patients were reoperated, 6 months and 3 years after the first incisional hernia operation (1 cutaneous fistula, 1 hernia recurrence). Extremely large incisional hernias can be closed safely and effectively using this technique, without mortality and with a low morbidity.

Aged↗