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Biomedical subjects

Y Tu

Publications and source records attributed to Y Tu.

At least 127 records · Page 7Linked to original sources
PubMed1996

[The PCR amplification, cloning, sequencing, expression in E. coli of gene encoding endoflagella subunit protein (fla B) from Leptospira interrogans serovar lai].

A pair of oligonucleotide primers were designed by ourselves to amplify the endoflagella gene of L. interrogans serovar lai. A fragment about 840 bp was generated with PCR and inserted into plasmid pUC8 after the fragment and pUC8 were digested respectively with Bam HI and Pst I. A recombinant plasmid (designated as pLF1) was obtained. SDS-PAGE analysis indicated that a 33 kd was expressed in E. coli JM103 harboring pLF1 and the expression level of the protein was 11% of total bacterial soluble proteins. Western blot analysis showed that the protein band could be recognized by the antiserum against the endoflagella (Axiall filament) of Leptospira interrogans serovar lai. Nucleotide seguence data showed an open reading frame encoding 282 aminoacids residues, corresponding to a protein of molecular weight 33.6 kd. The G + C content of endoflagella subunit protein gene was 48 mol%. Therefore, the G + C content of the leptospiral fla B Gene is significantly higher than the reported 39 mol% G + C content of leptospiral genome of L.interrogans serovar lai but similar to the G + C of the Treponema pallidum genome. Comparison of the deduced endoflagellar subunit protein (fla B) amino acid sequence with flagellins from other bacteria revealed a high level of identity with the Treponema pallidum fla B proteins. Immunization/protection experiment was performed on the model of BALB/c mice and showed that the survival rate in the group JM103-pLF1 was higher than that in the group JM103-pUC8, but statistically the difference between them was significant (P < 0.05) and pLF1 did not induce significant levels of agglutinating antibodies against L.interrogans serovar lai.

Amino Acid Sequence↗
PubMed1996

[Research on the recombinant plasmid pDJH2 of L. interrogans serovar lai: sequencing and alignment with other known bacterial Omp sequence].

The Leptospira whole cell vaccine (LWCV) currently used in China is safe and effective, out the immunity following vaccination with two doses of the fluid medium vaccine is of low order. The duration of immunity conferred by this vaccine is rather short, six months or at most one year. Therefore, it is necessary to develop new generation vaccines against Leptospirosis for the developing world. In this paper we report the sequencing of the insert fragment of pDJH2 from genomic DNA of L. interrogans sevovar lai strain 017 and its alignment with other bacterial omp sequences. A genomic library of Leptospira interrogaans serovar lai strain 017 was constructed with the plasmid vector pUC18. A recombinant plasmid designated pJDH2 was screened from the genomic library. Inserted fragment of pDH2 is 1.9 kb by gel electrophoresis. Immunization/protection was studied in BALB/c mice model. The results showed highly significant difference between pDJH2 and pUC18 (control). Inserted fragment of pDJH2 DNA sequencing was performed by Dr Yan Zhengxin (Max-Planck-Institut for Biology. Tubingen, Germany). Insert fragment was cloned into pBluescript II KS-(stratagene) and sequenced by using AB1 (Applied Bio Systems, Model 373A). Two open reading frames of 565 and 662 nucleotides were identified. There were identifiable initiation codons, terminators, Shine-Dalgano ribosome combining site, Pribnow boxes and Sextama boxes within the 2 sequenced regions. Nucleotide sequences were analysed using Gene Work, a suit of computer program developed by Department of Biochemistry St. Jude Children's Research Hospital Memphis. U.S.A. The results of formatted alignment showed the predicted nucleotide sequence of ORF1 of the serovar lai had significant similarity with ORF2 (49.36%). L. kirschneri ompL1 (49.26%), Borrelia burgdoferi omp (48.97%), Treponema phagedenis omp (47.3%); Salmonella typhimurium ompC(46.87%), Yersinia enterocolitica ompH (46.7%), Leptospira borgpeterseni pfap (46.3%), and Serratia marcescens omp (43.3%). The close relationship of the pDJH2 ORF1 and ORF2 nucleotide sequences from Leptospira kirschneri ompL 1 is apparent. Whether the recombinant pDJH2 will prove useful for vaccine development remains to be tested.

Animals↗
PubMed1996

[Subclones of fragment DNA recombinant rpDJH2 of L. interrogans serovar lai strain 017 and it's expression of high level in E. coli].

Fragment of 1.9 kb recombinant DNA of pDJH2 was linked with vectors pT7-7 and pRSETs. Then they were transformed into E. coli JM109 (DE3) respectively. Expression of subclones was achieved in E. coli JM109 (DE3) with IPTG inducement. SDS-PAGE showed that the molecular weights of products were 68kd and 23 kd respectively. The amount of production seemed to be higher than that of the outer membrane proteins of L. interrogans serovar strain 017 in nature. Immunoblotting of pDJt and pDJrB2 (both are subclones) with the specific antiserum of anti-OMP of L. interrogans serovar lai strain 017 and the experiment of initiative immuno-protection in guinea pigs showed both protein-68 kd and 23 kd might be the antigens of immuno-protection on the outer membrane of L. interrogans serovar lai strain 017.

Animals↗
PubMed1995

Sensitivity to inhibition by N-ethylmaleimide: a property of nitrobenzylthioinosine-sensitive equilibrative nucleoside transporter of murine myeloma cells.

Murine myeloma SP2/0-Ag14 cells possess both nitrobenzylthioinosine (NBMPR)-sensitive and NBMPR-insensitive equilibrative uridine transport systems. No Na(+)-dependent uridine transport system was detected. The NBMPR-insensitive transport system is similarly insensitive to inhibition by dilazep and dipyridamole. Dose-response curve for the inhibition of equilibrative uridine transport by N-ethylmaleimide (NEM), a sulfhydryl reagent, in these cells was biphasic. About 30-40% of the uridine transport was inhibited by NEM at IC50 value of 0.15 mM. The other 60-70% of the transport activity remained insensitive to NEM at concentration as high as 3 mM. The decrease in NBMPR-sensitive uridine transport in the presence of 0.3 mM NEM was due to a 3-fold decrease in transport affinity. Apparent Km values of 500 and 1600 microM and Vmax values of 13 and 12 microM/s were obtained for untreated and NEM-treated cells, respectively. NEM (0.3 mM) has little effect on the Km of NBMPR-insensitive transporter, with apparent Km values of 100 and 110 microM and Vmax values of 3.0 and 2.5 microM/s for untreated and NEM-treated cells, respectively. High sensitivity of NBMPR-sensitive transporter to NEM inhibition was also observed in HL-60 and MCF-7 cells. Decrease in specific 3H-NBMPR equilibrium binding affinity in myeloma cells was observed after treatment with 0.3 mM NEM. Apparent Kd values of 0.32 and 2.3 nM with Bmax values of 48,000 and 44,000 sites/cell were obtained for untreated and NEM-treated cells, respectively. NBMPR, dilazep and dipyridamole at 30 microM, and uridine at 10 mM failed to protect the NBMPR-sensitive transporter against NEM inhibition. It is possible that a critical sulfhydryl residue is closed to substrate binding/transporting site of the NBMPR-sensitive transporter. NEM, a sulfhydryl reagent containing an activated double bond, hinders the affinity of this transporter by forming a stable thiol ether bond with the reactive residue.

Animals↗
PubMed1995

Cardiac allograft survival in mice deficient in intercellular adhesion molecule-1.

BACKGROUND: Intercellular adhesion molecule-1 (ICAM-1, CD54) is a cell adhesion molecule that interacts with the leukocyte beta 2 integrins, lymphocyte function-associated antigen-1, and macrophage antigen-1. ICAM-1 is postulated to play a key role in several cell-cell interactions that are important in allograft rejection, including antigen presentation, transendothelial migration of leukocytes, and leukocyte-medicated myocyte injury. METHODS AND RESULTS: Mice homozygous for a gene-targeted mutation of ICAM-1 were used in two different cardiac transplant models to further define the role of ICAM-1 in the process of allograft rejection. In the first model, hearts from newborn mice were implanted in the ear pinnae of H-2-incompatible recipients. In the second model, intra-abdominal transplantation by direct vascular anastomosis was performed. Time to rejection was defined by the loss of pulsatile activity assessed by visual inspection in the ear model or by cessation of palpable cardiac impulse in the abdominal model. Allograft survival did not differ significantly between control groups that express normal levels of ICAM-1 and those groups using ICAM-1-deficient mutants as either donors or recipients. Histological examination of rejection of both normal and mutant (ICAM-1-deficient) cardiac allografts revealed similar patterns of infiltration of mononuclear and granulocytic leukocytes and myocyte necrosis. Immunostaining with anti-ICAM-1 antibodies showed ICAM-1-positive infiltrating cells in both mutant (ICAM-1-deficient) and normal allografts, with the graft endothelium negative for ICAM-1 staining in the mutant allografts. CONCLUSIONS: The absence of surface expression of ICAM-1 in the donor allograft or recipient is insufficient to produce a significant impact on cardiac allograft survival. This study highlights the need to understand more precisely the mechanism of action whereby monoclonal antibodies to ICAM-1 prolong cardiac allograft survival before new therapeutic strategies based on gene transfer technology or small molecule inhibitors are developed. Mutant mice with targeted mutations in cell adhesion molecules provide powerful tools to study the complex role that cell adhesion molecules play in the cellular interactions between donor graft tissue and the recipient that culminate in graft rejection.

Animals↗
PubMed1995

The synergistic effects of cyclosporine, sirolimus, and brequinar on heart allograft survival in mice.

The effects of cyclosporine (CsA), sirolimus (RAPA), and/or brequinar (BQR) were examined in a vascularized heterotopic heart transplant model in mice. Untreated C3H (H-2k) recipients reject C57 BL/10 (H-2b) heart allografts at a mean survival time (MST) of 7.7 +/- 1.4 days. A 7-d intravenous (i.v.) infusion by osmotic pump of CsA at doses of 5.0, 10.0, or 20.0 mg/kg extended heart allograft survival to 9.8 +/- 1.3 d (NS), 15.0 +/- 5.1 d (P < 0.01) or 15.0 +/- 1.9 d (P < 0.01), respectively. RAPA delivered i.v. for 7 d at a dose of 0.1 mg/kg produced an MST of 13.0 +/- 7.5 d; 0.2 mg/kg, 20.0 +/- 10.9 d; and 0.4 mg/kg, 15.8 +/- 4.1 d (all P < 0.01). A 7-d alternate-day (q.o.d.) course of oral gavage with BQR (0.5, 1.0, or 2.0 mg/kg) produced survivals of 12.0 +/- 2.4 d, 17.6 +/- 3.4 d, and 20.0 +/- 4.1 d, respectively (all P < 0.01). The combination of 2.5 mg/kg CsA with 0.05 mg/kg RAPA extended graft survival to 18.2 +/- 2.9 d (P < 0.01), and 5.0 mg/kg CsA with 0.1 mg/kg RAPA prolonged survival to 23.0 +/- 9.0 d (P < 0.01). These combinations represent synergistic interactions based upon combination index (CI) values of 0.1-0.6. Although 7-d courses of 0.5 mg/kg CsA (7.3 +/- 1.0 d; NS), 0.01 mg/kg RAPA (7.6 +/- 0.9 d; NS), or 0.125 mg/kg BQR (7.6 +/- 0.9 d; NS) were individually ineffective, the triple-drug combination prolonged the MST to 64.6 +/- 32.7 d (P < 0.005; CI = 0.001), with 2/5 grafts beating for more than 100 d. Similar results were produced by 14-day therapy in the BALB/c (H-2d) to C3H combination.

Animals↗
PubMed1995

Interleukin-6 inhibits apoptosis of malignant plasma cells.

Multiple myeloma (MM) is a slow-growing malignancy whose plasma cells express the BCL-2 antiapoptosis gene. It is also associated with high levels of interleukin-6 (IL-6), a cytokine that prevents programmed cell death (PCD) in other target cell types. We thus investigated the ability of MM cells to undergo PCD and the possible regulatory effects of IL-6. Four MM cell lines underwent PCD when exposed to serum starvation, doxorubicin (dox), etoposide (VP-16), or dexamethasone (dex). Apoptosis was confirmed by morphologic criteria and/or detection of endonucleosomal DNA fragmentation. The concentrations of dox, VP-16, and dex required for PCD were at least 10-fold greater than that required to inhibit proliferation. Addition of IL-6 (but not IL-1 beta, IL-4, IL-7, or IL-10) inhibited PCD of 8226 targets induced by serum starvation or dexamethasone in a concentration-dependent fashion. In contrast, it had no effect on PCD induced by dox or VP-16. Exposure of targets to IL-6 did not increase BCL-2 expression (it actually consistently decreased expression), suggesting IL-6's protection against apoptosis was not mediated by direct effects on BCL-2. Targets protected from PCD by IL-6 were still sensitive to serum starvation and dex-induced cytostasis, but, after reculturing in drug-free complete media, they reinitiated normal proliferation. These data suggest that high levels of IL-6 may contribute to expansion of myeloma clones by inhibiting apoptotic death.

Apoptosis↗
PubMed1995

Atypical apoptotic cell death induced in L929 targets by exposure to tumor necrosis factor.

The mechanism by which tumor necrosis factor (TNF) induces cytotoxicity of murine fibroblasts was investigated. Electrophoresis of DNA extracted from TNF-treated L929 targets showed fragmentation of DNA into a ladder-like pattern, typical of cells dying by apoptosis. Morphologic analysis also indicated apoptotic cell death, demonstrating clumping and crescentic condensation of chromatin. In contrast, chromatin condensation and ladder-like DNA fragmentation were not detected in L929 targets dying by necrosis from exposure to heat, repeated cycles of freeze-thaw, and sodium azide. Chromatin condensation was an early event, detected as early as 6 h of incubation. However, DNA fragmentation (assayed by double-stranded fragmentation assay and gel electrophoresis), as well as the apoptotic changes detected by Hoechst fluorescence, both occurred later and did not precede TNF cytotoxicity (membrane permeabilization detected by trypan blue or propidium iodide staining). This atypical pattern of apoptosis was a characteristic of L929 target cells rather than a generalized cytotoxic response to TNF because TNF-treated squamous cancer cells showed typical features of apoptosis (DNA fragmentation before cytotoxicity) and etoposide-treated L929 cells demonstrated the same atypical kinetics as TNF-treated cells. Zinc significantly inhibited TNF cytotoxicity as well as DNA fragmentation of L929. However, because DNA fragmentation occurred belatedly in TNF-treated targets, lagging behind cytotoxicity, the protection by zinc against TNF appears mediated by events that occur before the ultimate endonuclease-induced cleavage of DNA into small fragments.

Animals↗
PubMed1995

Transmembrane Ca2+ gradient-mediated phosphatidylcholine modulating sarcoplasmic reticulum C(a2+)-ATPase.

The sarcoplasmic reticulum (SR) C(a2+)-ATPase was purified and reconstituted into the sealed phospholipids vesicles with or without transmembrane Ca2+ gradient. The role of phospholipids, especially phosphatidylcholine (PC), in the modulation of C(a2+)-ATPase by transmembrane Ca2+ gradient was investigated. The results are as follows. (i) Incubated with phospholipids, the enzyme activity of the delipidated C(a2+)-ATPase is inhibited by Ca2+ and the highest inhibition is observed in the presence of PC. (ii) When there exists a transmembrane Ca2+ gradient (higher Ca2+ concentration inside vesicles, 1,000 mumol/L:50 mumol/L, similar to the physiological condition), the inhibition of C(a2+)-ATPase by transmembrane Ca2+ gradient can be only observed in the vesicles containing PC:PE, but not in those containing PS:PE or PG:PE. The highest inhibition is obtained at a 50:50 molar ratio of PC:PE (iii) By comparing the effects of PC differing in acyl chains, higher inhibition of C(a2+)-ATPase is observed in vesicles containing DPPC:PE and DOPC:PE, while no inhibition in DMPC:PE vesicles (iv) If the transmembrane Ca2+ gradient is in the inverse direction, the enzyme activity of C(a2+)-ATPase is inhibited whenever reconstituted with acidic or neutral phospholipids.

Animals↗
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↗
PubMed1994

Blocking of heart allograft rejection by intercellular adhesion molecule-1 antisense oligonucleotides alone or in combination with other immunosuppressive modalities.

Intercellular adhesion molecule-1 (ICAM-1) binds circulating leukocytes through interactions with beta 2 integrins, LFA-1, and macrophage Ag-1. The phosphorothioate antisense oligodeoxynucleotide, IP-3082, specific for ICAM-1 mRNA inhibited ICAM-1, but not vascular cell adhesion molecule-1, mRNA induction and expression of ICAM-1 molecules by mouse endothelioma cells. Scrambled control oligonucleotides were ineffective. Untreated C3H (H-2k) mice rejected C57BL/10 (H-2b) heart allografts with a mean survival time of 7.7 +/- 1.4 days. Administration i.v. of IP-3082 by a 7-day osmotic pump prolonged the survival of heart allografts in a dose-dependent fashion: 1.25 mg/kg, to 11 +/- 0 days; 2.5 mg/kg, to 12 +/- 2.7 days; 5 mg/kg, to 14.1 +/- 2.7 days; and 10 mg/kg, to 15.3 +/- 5.8 days (all p < 0.01). Control IP-1082 (10 mg/kg) was ineffective (7 +/- 0.8 days). Although 7-day anti-LFA-1 mAb (50 micrograms/day; i.p.) prolonged allograft survival to 14.1 +/- 2.7 days, the addition of IP-3082 (5.0 mg/kg x 7 days) induced donor-specific transplantation tolerance (> 150 days). Furthermore, IP-3082 (5 mg/kg x 7 days) acted synergistically with antilymphocyte serum, rapamycin, and brequinar, but not cyclosporin A: a single antilymphocyte serum (0.2 ml) i.p. injection alone prolonged graft survival to 10 +/- 0.5 days (p < 0.01) and in combination with IP-3082 (5 mg/kg), to 32.2 +/- 8.3 days (p < 0.001); rapamycin (0.1 mg/kg x 7 days; i.v.) alone prolonged survival to 13 +/- 7.5 days (p < 0.01), and with IP-3082, to 32.4 +/- 8.9 days (p < 0.03); brequinar (0.5 mg/kg x 7 days; oral gavage) alone to 12 +/- 2.4 days (p < 0.05), and with IP-3082 (5 mg/kg), to 38.8 +/- 30.2 days (p < 0.01); in contrast, cyclosporin A (5 mg/kg x 7 days; i.v.) alone produced graft survival of 9.8 +/- 1.3 days (p < 0.1 and in combination with IP-3082 (5 mg/kg), produced survival of 7.8 +/- 3.5 days (NS). Thus, antisense oligonucleotides may proffer a selective gene-targeted immunosuppressive therapy for organ transplantation.

Animals↗
PubMed1994

The effect of L-glutamate on the afferent resting activity in the cephalopod statocyst.

The effects of bath application of L-glutamate and of excitatory amino acid agonists and antagonists on the resting activity of afferent crista fibers were studied in isolated preparations of the statocyst of the cuttlefish, Sepia officinalis. L-Glutamate (threshold 10(-5) M) and its agonists quisqualate and kainate (thresholds 10(-6) M) increased the resting activity in a dose-dependent manner. Glutamine (threshold 10(-5) M) was also excitatory, while D-glutamate had no effect. Also, no obvious excitatory effects were seen for NMDA and L-aspartate, nor was any antagonistic effect seen for the selective NMDA-receptor antagonist D-2-amino-5-phosphonovaleric acid (D-AP-5). The spider toxin Argiotoxin636 (threshold 10(-11) M), 2-amino-4-phosphonobutyric acid (AP-4), glutamic acid diethyl ester (GDEE), gamma-D-glutamylaminomethyl-sulfonic acid (GAMS), and kynurenic acid decreased the resting activity and effectively blocked or reversed the effect of L-glutamate and its non-NMDA agonists. Preliminary experiments with statocysts from the squid Sepioteuthis lessoniana and the octopod Octopus bimaculoides gave comparable results. All data show that in cephalopod statocysts L-glutamate, via non-NMDA receptors, has an excitatory effect on the activity of afferent fibers, an effect consistent with its possible function as a hair cell transmitter.

Action Potentials↗