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Biomedical subjects

N Guo

Publications and source records attributed to N Guo.

At least 37 records · Page 2Linked to original sources
PubMed2000

Enhanced antiviral and opsonic activity of a human mannose-binding lectin and surfactant protein D chimera.

The carbohydrate recognition domains (CRDs) of human serum mannose-binding lectin (MBL) and pulmonary surfactant protein D (SP-D) have distinctive monosaccharide-binding properties, and their N-terminal and collagen domains have very different quaternary structures. We produced a chimeric protein containing the N terminus and collagen domain of human SP-D and the neck region and CRD of human MBL (SP-D/MBLneck+CRD) to create a novel human collectin. The chimera bound to influenza A virus (IAV), inhibited IAV hemagglutination activity and infectivity, and induced aggregation of viral particles to a much greater extent than MBL. Furthermore, SP-D/MBLneck+CRD caused much greater increases in neutrophil uptake of, and respiratory burst responses to, IAV than MBL. These results indicate that pathogen interactions mediated by the MBL CRD are strongly influenced by the N-terminal and collagen-domain backbone to which it is attached. The presence of the CRD of MBL in the chimera resulted in altered monosaccharide binding properties compared with SP-D. As a result, the chimera caused greater aggregation and neutralization of IAV than SP-D. Distinctive functional properties of collectin collagenous domains and CRDs can be exploited to generate novel human collectins with potential for therapy of influenza.

Animals↗
PubMed2000

Rem2, a new member of the Rem/Rad/Gem/Kir family of Ras-related GTPases.

Here we report the molecular cloning and biochemical characterization of Rem2 (for Rem, Rad and Gem-related 2), a novel GTP-binding protein identified on the basis of its homology with the Rem, Rad, Gem and Kir (RGK) family of Ras-related small GTP-binding proteins. Rem2 mRNA was detected in rat brain and kidney, making it the first member of the RGK family to be expressed at relatively high levels in neuronal tissues. Recombinant Rem2 binds GTP saturably and exhibits a low intrinsic rate of GTP hydrolysis. Surprisingly, the guanine nucleotide dissociation constants for both Rem2 and Rem are significantly different than the majority of the Ras-related GTPases, displaying higher dissociation rates for GTP than GDP. Localization studies with green fluorescent protein (GFP)-tagged recombinant protein fusions indicate that Rem2 has a punctate, plasma membrane localization. Deletion of the C-terminal seven amino acid residues that are conserved in all RGK family members did not affect the cellular distribution of the GFP fusion protein, whereas a larger deletion, including much of the polybasic region of the Rem2 C-terminus, resulted in its redistribution to the cytosol. Thus Rem2 is a GTPase of the RGK family with distinctive biochemical properties and possessing a novel cellular localization signal, consistent with its having a unique role in cell physiology.

Amino Acid Sequence↗
PubMed2000

Characterization and gene structure of a novel retinoblastoma-protein-associated protein similar to the transcription regulator TFII-I.

Retinoblastoma protein (Rb) is an important regulator of vertebrate cell cycle and development. It functions through a direct interaction with protein factors involved in cell cycle progression and differentiation. In the present study we characterized a novel Rb-associated protein, Cream1, which bound to Rb specifically through a C-terminal region. Cream1 contained 959 amino acid residues and migrated as a protein of approx. 120 kDa on SDS/PAGE. It was a widely expressed nuclear protein with a nuclear localization signal resembling that of the large T antigen of simian virus 40. Its primary sequence was characteristic of five direct repeats that were similar to, but distinct from, those of TFII-I, a multifunctional transcription regulator. Three additional regions were also highly conserved in both proteins. Cream1 exhibited an activation activity that was attributed to its N-terminal portion when assayed in yeast. Its relationship with the muscle-enhancer-binding protein MusTRD1 further suggests a role in regulating gene expression. The structural gene, CREAM1, contained 27 exons and spanned more than 150 kb. It was located at human chromosome 7q11.23 in a region deleted for Williams' syndrome, a neurodevelopmental disease with multisystem abnormalities, implying its involvement in certain disorders. Taken together, our results suggest that Cream1 might serve as a positive transcription regulator under the control of Rb.

Amino Acid Sequence↗
PubMed2000

A novel DNA damage checkpoint involving post-transcriptional regulation of cyclin A expression.

The intracellular metabolism of many carcinogenic polycyclic aryl hydrocarbons (PAHs, typified by the ubiquitous pollutant benzo[a]pyrene or B[a]P) generates electrophilic products that react covalently with genomic DNA. Cells that acquire PAH-induced DNA damage undergo growth arrest in a p53-independent manner (Vaziri, C., and Faller, D. V. (1997) J. Biol. Chem. 272, 2762-2769). In this report we have investigated the molecular basis of PAH-induced cell cycle arrest. Mitogenic signaling events involving cyclins D and E, Rb phosphorylation, and transcriptional activation of E2F-responsive genes (including cyclin E and cyclin A) were unaffected in cells containing PAH-damaged DNA. However, PAH-induced growth arrest was associated with post-transcriptional decreases in cyclin A expression. Mitogen-induced expression of cyclin B, an event that is temporally distal to cyclin A expression, was also inhibited in PAH-treated cells. The PAH-induced cell cycle block was transient, and arrested cells resumed DNA synthesis after a prolonged ( approximately 20 h) delay. Resumption of DNA synthesis in PAH-treated cells occurred concomitant with elevated expression of cyclins A and B. PAH-induced cell cycle arrest was overcome by ectopically expressed cyclin A (encoded by a recombinant adenovirus in transiently infected cells). Overall, our results suggest the existence of a DNA damage checkpoint pathway that arrests cell cycle progression via post-transcriptional control of cyclin A expression.

3T3 Cells↗
PubMed2000

Thrombospondin-1 promotes alpha3beta1 integrin-mediated adhesion and neurite-like outgrowth and inhibits proliferation of small cell lung carcinoma cells.

Although human small cell lung carcinoma (SCLC) cell lines are typically anchorage-independent and do not attach on most extracellular matrix proteins, OH-1, and several other SCLC cell lines attached on substrates coated with thrombospondin-1 (TSP1). SCLC cells grew long-term as adherent cells on a TSP1-coated substrate. Adhesion of SCLC cells on TSP1 was inhibited by heparin, function-blocking antibodies recognizing alpha3 or beta1 integrin subunits, and by soluble alpha3beta1 integrin ligands. SCLC cells extended neurite-like processes on a TSP1 substrate, which was also mediated by alpha3beta1 integrin. Process formation on a TSP1 substrate was specifically stimulated by epidermal growth factor and somatostatin. Adhesion on TSP1 weakly inhibited SCLC cell proliferation, but this inhibition was strongly enhanced in the presence of epidermal growth factor. TSP1 and an alpha3beta1 integrin-binding peptide from TSP1 also inhibited proliferation when added in solution. High-affinity binding of 125I-labeled TSP1 to OH-1 cells was heparin-dependent and may be mediated by sulfated glycolipids, which are the major sulfated glycoconjugates synthesized by these cells. Synthesis or secretion of TSP1 by SCLC cells could not be detected. On the basis of these results, the alpha3beta1 integrin and sulfated glycolipids cooperate to mediate adhesion of SCLC cells on TSP1. Interaction with TSP1 through this integrin inhibits growth and induces neurotypic differentiation, which suggests that this response to TSP1 may be exploited to inhibit the progression of SCLC.

Carcinoma, Small Cell↗
PubMed2000

Single-chain antibodies against human insulin-like growth factor I receptor: expression, purification, and effect on tumor growth.

Insulin-like growth factors (IGF) I and II are potent mitogens for a variety of cancer cells. The proliferative and anti-apoptotic actions of IGF are mediated by the IGF-I receptor (IGF-IR), to which both IGF-I and IGF-II bind with high affinity. To investigate the mitogenic and anti-apoptotic activities of IGF-IR and to achieve better inhibition of IGF-IR function, single-chain antibodies against human IGF-IR (alphaIGF-IR scFvs) were constructed and expressed. IgG cDNA encoding variable regions of light and heavy chains (VL and VH) from mouse IgG were cloned from a hybridoma producing the 1H7 alphaIGF-IR monoclonal antibody [Li et al., Biochem Biophys Res Commun 196: 92-98 (1993)]. The splice-overlap extension polymerase chain reaction was used to assemble a gene encoding the alphaIGF-IR scFv, including the N-terminal signal peptide, VL, linker peptide, VH, and C-terminal DYKD tag. Two types of soluble alphaIGF-IR scFvs, a prototype alphaIGF-IR scFv and its alternative type alphaIGF-IR scFv-Fc, were constructed and expressed in murine myeloma cells. alphaIGF-IR scFv-Fc, containing the human IgG1 Fc domain, was stably expressed in NS0 myeloma cells, using a glutamine synthase selection system, and purified from the conditioned medium of stable clones by protein-A--agarose chromatography. Levels of alphaIGF-IR scFv-Fc expression ranged from 40 mg/l to 100 mg/l conditioned medium. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis under reducing and nonreducing conditions indicated that alphaIGF-IR scFv-Fc is a dimeric antibody. alphaIGF-IR scFv-Fc retained general characteristics of the parental 1H7 monoclonal antibody except that its binding affinity for IGF-IR was estimated to be approximately 10(8) M(-1), which was one-order of magnitude lower than that of 1H7 monoclonal antibody. Injection of alphaIGF-IR scFv-Fc (500 microg/mouse, twice a week) significantly suppressed MCF-7 tumor growth in athymic mice. These results suggest that the alphaIGF-IR scFv-Fc is a first-generation recombinant alphaIGF-IR for the potential development of future alphaIGF-IR therapeutics.

Animals↗
PubMed2000

Trehalose expression confers desiccation tolerance on human cells.

Many organisms that withstand desiccation express the disaccharide trehalose. We have now expressed the otsA and otsB genes of Escherichia coli, which encode trehalose biosynthetic enzymes, in human primary fibroblasts using a recombinant adenovirus vector. Infected cells produced increased amounts of trehalose with increasing multiplicity of infection (MOI). Human primary fibroblasts expressing trehalose could be maintained in the dry state for up to five days. Fourier transform infrared spectroscopy indicated that dry, but viable, human cells contained no detectable water. This study shows that mammalian cells can be engineered to retain viability in the absence of water.

Bacterial Proteins↗
PubMed2000

A quantitative reverse transcription and polymerase chain reaction assay for human IGF-II allows direct comparison of IGF-II mRNA levels in cancerous breast, bladder, and prostate tissues.

Previously, we showed by in situ hybridization that insulin-like growth factor (IGF)-II is upregulated in approximately 50% of prostate, breast, and bladder tumours. In this study, a quantitative competitive reverse transcription and polymerase chain reaction (QC RT-PCR) assay was established and used to quantify human IGF-II mRNA levels in cells and tissues. In this QC RT-PCR assay, a competitor IGF-II RNA, prepared from a newly constructed plasmid encoding the human IGF-II sequence with a 110-bp fragment inserted, was added to RNA samples prior to RT-PCR. The human IGF-II specific QC RT-PCR assay has allowed us to readily compare the levels of IGF-II mRNA in human tissues and cultured cells. Consistent with our previous observations by in situ hybridization, IGF-II mRNA was up-regulated in 50% of cancerous breast tissues examined as compared to the matching benign tissues, and IGF-II mRNA levels were higher in bladder tumours than breast and prostate tumours. In summary, we present here quantitative data confirming that a subclass of breast cancer samples has elevated levels of IGF-II transcripts by the new competitive RT-PCR assay.

Breast Neoplasms↗
PubMed2000

Activation-induced nuclear translocation of RING3.

RING3 is a novel protein kinase linked to human leukaemia. Its Drosophila homologue female sterile homeotic is a developmental regulator that interacts genetically with trithorax, a human homologue of which is also associated with leukaemia. The RING3 structure contains two mutually related bromodomains that probably assist in the remodelling of chromatin and thereby affect transcription. Consistent with this hypothesis, a RING3-like protein has been identified in the mouse Mediator complex, where it is associated with transcription factors. We show that, whilst RING3 is constitutively localised to the nucleus of exponentially growing HeLa cells, it is delocalised throughout serum-starved fibroblasts. We use immunostaining and confocal microscopy to demonstrate that RING3 translocates to the fibroblast nucleus upon serum stimulation. After translocation, RING3 participates in nuclear protein complexes that include E2F proteins; it transactivates the promoters of several important mammalian cell cycle genes that are dependent on E2F, including dihydrofolate reductase, cyclin D1, cyclin A and cyclin E. We use site-directed mutagenesis of a putative nuclear localisation motif to show that the activation-induced nuclear localisation and consequent transcriptional activity of RING3 depends on a monopartite, classical nuclear localisation sequence. These observations refine and extend the mechanism by which RING3 contributes to E2F-regulated cell cycle progression. Deregulation of this mechanism may be leukaemogenic.

Animals↗
PubMed2000

Assessment of autonomic function in patients with acute myocardial infarction or diabetes mellitus by heart rate variability, ventricular late potential and QT dispersion.

To compare the efficacy and sensitivity of heart rate variability (HRV), QT dispersion (QTd) and ventricular late potential (VLP) examination in judging autonomic function. Thirty three patients with acute myocardial infarction (AMI) and 33 patients with diabetes mellitus (DM), all of whom were diagnosed with autonomic neuropathy determined by a standard test of cardiovascular autonomic function, were examined by HRV (timing domain methods), QTd and VLP. Thirty three normal individuals served as controls. The mean SD of the normal R-R interval (SDNN) in both the AMI and DM groups was significantly less than that in the control group (p< 0.01); and of course, the QTd of these groups was significantly greater than that of the controls (p< 0.01). The VLP positive rate of the AMI and DM groups were much higher than that of the control group (p< 0.001). SDNN was shown to be significantly negatively correlated to QTd (r= -0.45); and significantly negatively correlated to VLP (r= -0.47); QTd was shown to be positively, though not significantly, correlated to VLP (r=0.48). QTd could be looked as sieving index; HRV could be looked as routine examination of cardiovascular autonomic function, especially SDNN; the combination of HRV and VLP could improve the accuracy of diagnosis.

Adult↗
PubMed2000

RING3 kinase transactivates promoters of cell cycle regulatory genes through E2F.

RING3 is a novel, nuclear-localized, serine-threonine kinase that has elevated activity in human leukemias. RING3 transforms NIH/3T3 cells and is activated by mitogenic signals, all of which suggest that it may play a role in cell cycle-responsive transcription. We tested this hypothesis with transient transfection of RING3 into fibroblasts and assayed transactivation of the promoters of cyclin D11 cyclin A, cyclin E, and dihydrofolate reductase (dhfr) genes. RING3 transactivates these promoters in a manner dependent on ras signaling. A kinase-deficient point mutant of RING3 does not transactivate. Mutational analysis of the dhfr promoter reveals that transactivation also depends on the presence of a functional E2F binding site. Furthermore, ectopic expression of Rb protein, a negative regulator of E2F activity, suppresses the RING3-dependent transactivation of this promoter. Consistent with a potential role of E2F in RING3-dependent transcription, anti-RING3 immunoaffinity chromatography or recombinant RING3 protein affinity chromatography of nuclear extracts copurified a protein complex that contains E2F-1 and E2F-2. These data suggest that RING3 is a potentially important regulator of E2F-dependent cell cycle genes.

3T3 Cells↗
PubMed2000

[Detection of TT virus DNA by polymerase chain reaction-microplate hybridization].

OBJECTIVE: To establish a method for the detection of TT virus (TTV) DNA by polymerase chain reaction (PCR)-microplate hybridization and to find out various influential factors of the assay. METHODS: TTV DNA extracted by guanidinium thiocyanate was amplified by PCR, the PCR primers were pre-labeled with biotin. By conjugation of biotin with streptavidin, the amplified products were coated to the microplate well, and hybridized with FITC- probe simultaneously. After being reacted with Anti-Fluorescein POD, the products that contained FITC probe were coloured by TMB-H2O2 and then measured the optical density using a microplate reader at 450 nm wavelength. RESULTS: The optimal concentration of NaOH was 0.3 mol/L, and the optimal time of hybridization and enzymatic reaction was 60 minutes and 45 minutes, respectively. The results also showed that the positive rate of TTV infection in normal population, hepatitis A-G and hepatitis non A-G was 13.6%, 20.3% and 28.1%, respectively. CONCLUSIONS: Comparing the PCR-microplate hybridization with the electrophoresis assay, the former is simple, rapid, sensitive and specific and is expected to be a new technique instead of electrophoresis assay as a routine method for TTV DNA detection.

DNA Virus Infections↗
PubMed1999

The peptide bond between E292-A293 of Escherichia coli leucyl-tRNA synthetase is essential for its activity.

Escherichia coli leucyl-tRNA synthetase (LeuRS) is a class I aminoacyl-tRNA synthetase that contains a large connecting polypeptide (CP1) inserted into its nucleotide binding fold, or active site. In this study, purified leucyl-tRNA synthetase was found to be cleaved between E292 and A293 in its CP1 domain. SDS-PAGE analysis showed peptides of 63 and 34 kDa in addition to the native 97.3 kDa synthetase. By internal complementation, the two peptides could form a 97.3 kDa complex similar to the native LeuRS. This complex could support the ATP approximately PP(i) exchange activity of LeuRS, but could not complement for aminoacylation. To study the function of the region around the bond of E292 and A293, four pairs of peptides resulting from different cleavage sites in CP1 were reconstituted in vivo. With the exception of the enzyme assembled from the E292-A293 cleavage site, all the reassembled LeuRSs catalyzed the aminoacylation of tRNA(Leu). Although the E292-A293-cleaved LeuRS could not catalyze aminoacylation, fluorescence titration revealed that its tRNA binding ability was almost identical to that of wild-type LeuRS. These results suggest that the region around E292-A293 may be responsible for maintaining the proper conformation of LeuRS required for the tRNA charging activity.

Acylation↗
PubMed1999

Discrimination of tRNALeu isoacceptors by the insertion mutant of Escherichia coli leucyl-tRNA synthetase.

A variant (LeuRS-A) of Escherichia coli leucyl-tRNA synthetase (LeuRS) carrying a 40-residue duplication in its connective peptide 1 (CP1) has a 3-fold lower specificity for than for, whereas wild-type LeuRS has the same specificity for these two isoacceptors. The replacement of the acceptor stem of with yields a chimeric tRNA(Leu) for which wild-type LeuRS has the same specificity as it does for the two normal isoacceptors mentioned, but for which LeuRS-A has a reduced specificity similar to that for, indicating a difference between these two acceptor stems. LeuRS-A is slightly less stable than the native enzyme. Wild-type LeuRS and LeuRS-A have almost same K(d) value for their interaction with as determined by fluorescence quenching. No difference was detected between these two proteins by CD and fluorescence spectroscopy. These results show that LeuRS-A can discriminate between the two isoacceptors of tRNA(Leu).

Acylation↗
PubMed1999

[Prophylaxis and treatment of cytomegalovirus infection in allogeneic bone marrow transplantation].

OBJECTIVE: To assess the efficacy and safety of Cymevene (ganciclovir) in the prophylaxis and treatment of cytomegalovirus (CMV) infection after allogeneic bone marrow transplantation (allo-BMT). METHODS: Sixty patients with leukemia undergoing allo-BMT were observed. Cymevene was given prophylactically to 20 patients at a dose of 5 mg/kg twice daily from day -9 to day -2 and restarted when the ANC > or = 1.0 x 10(9)/L and BPC > or = 30 x 10(9)/L at a daily dose of 5 mg/kg for 5 days every week until day +100. In diagnosed symptomatic CMV infections, the dose of Cymevene was 5 mg/kg twice daily for 2-3 weeks followed by 5 mg/kg twice a day for 5 days every week for an additional 2-3 weeks. RESULTS: The incidence of active CMV infection was 0 in the prophylaxis-treated patients and 25% in the controls (P < 0.05). In diagnosed CMV diseases, marked improvement was noted in 17 of 20 patients (85%) treated with Cymevene. The most common adverse events of Cymevene were leukopenia and thrombocytopenia, but were reversible. CONCLUSION: Cymevene is effective and safe for the prophylaxis and treatment of CMV infections after allo-BMT.

Acute Disease↗
PubMed1999

[ABO-incompatible allogeneic bone marrow transplantation].

OBJECTIVE: To explore the immunohematological problems in ABO-incompatible allogeneic bone marrow transplantation (allo-BMT). METHODS: Among 250 HLA-matched allo-BMTs, 61 were performed between ABO incompatible siblings. Out of these 61 BMTs, 38 were major ABO-incompatible and 23, minor. Control group included 61 HLA-matched Allo-BMTs with ABO compatible grafts. The major ABO incompatible patients received marrow grafts depleted of erythrocytes by hydroxyethyl starch (HES) sedimentation. The minor or bidirectional received marrow grafts depleted of plasma to reduce anti-A or anti-B agglutinins. ABO antigen and antibody in the recipients sera were determined once a week. RESULTS: ABO-incompatible grafts for BMT had no adverse effect on engraftment, recovery of platelets, incidence of GVHD or survival. No recipient developed clinically hemolysis during or after marrow infusion. In ABO major compatible group, the onset of erythropoiesis after BMT was delayed and more RBC transfusions were required. Five of 10 recipients with blood group "O" in this group developed pure red cell aplasia (PRCA), lasting 7-24 months. Two of them recovered without therapeutic intervention. In the PRCA patients, the RBC antibody titers remained high for a longer time after BMT than in those whose erythropoiesis reconstituted timely. Serious morbidity related to ABO incompatibility did not occur. CONCLUSION: ABO-incompatible allo-BMT is fairly safe if there is indication, however, PRCA may develop in isolated cases.

ABO Blood-Group System↗
PubMed1999

[Allogeneic bone marrow transplantation for chronic myeloid leukemia: 118 cases analysis].

OBJECTIVE: To analyze the outcome of allogeneic bone marrow transplantation (allo-BMT) performed for chronic myeloid leukemia (CML) patients. METHODS: One hundred and eighteen CML patients received allo-BMT with conditioning regimens of TBI or modified BU/CY. The pre-BMT situations were, 91 cases in first chronic phase (CP1), 19 in accelerating phase (AP), 2 in blast crisis (BC) and 6 in advanced CP (> CP2). RESULTS: One hundred and nine patients were successfully engrafted. The probabilities of 5 year survival for CP1 and AP patients were 69.6% and 51.0%, and 5 year relapse rates were 3.2% and 12.5%, respectively. CONCLUSIONS: Disease stage, conditioning regimen and splenomegaly did not affect the time for engraftment. For patients in CP1, splenomegaly was positively correlated with relapse. There was no difference between the effect of the two conditioning regimens on the prognosis.

Adolescent↗