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Biomedical subjects

G Gao

Publications and source records attributed to G Gao.

At least 19 recordsLinked to original sources
PubMed2000

NMR structure of tissue inhibitor of metalloproteinases-1 implicates localized induced fit in recognition of matrix metalloproteinases.

A high quality solution structure of the matrix metalloproteinase inhibitory N-terminal domain of recombinant human tissue inhibitor of metalloproteinases-1 (N-TIMP-1) has been determined. For the rigidly packed residues, the average RMSD to the mean structure is 0. 57 A for the backbone atoms and 1.00 A for all heavy atoms. Comparison of the solution structure of free N-TIMP-1 with the crystal structure of TIMP-1 bound to the catalytic domain of MMP-3 ( Gomis-R]uth et al., 1997 ) shows that the structural core of the beta barrel flanked by helices is nearly unchanged by the association with MMP-3, evident from a backbone RMSD of 1.15 A. However, clear differences in the conformation of the MMP-binding ridge of free and MMP-bound TIMP-1 suggest induced fit throughout the ridge. The MMP-dependent conformational changes in the ridge include a dramatic bending of AB loop residues Glu28 through Leu34, moderate hinge bending of the CD-loop about residues Ala65 and Cys70, and modest bending of the Cys1 through Pro6 segment. A large number of interresidue Nuclear Overhauser enhancements (NOEs) augmented by stereospecific assignments, torsion restraints, and dipolar couplings (an average of 18 non-trivial restraints per residue) engender confidence in these structural inferences. A tight cluster of three lysine residues and one arginine residue atop beta-strands A and B, and identical among TIMP sequences, form the heart of a highly conserved electropositive patch that may interact with anionic components of the extracellular matrix.

Catalytic Domain↗
PubMed2000

1,1-Dimethylallylcoumarins potently suppress both lipopolysaccharide- and interferon-gamma-induced nitric oxide generation in mouse macrophage RAW 264.7 cells.

We investigated the suppressive effects of 16 coumarin-related compounds on both lipopolysaccharide (LPS)- and interferon (IFN)-gamma-induced nitric oxide (NO) generation in a mouse macrophage cell line, RAW 264.7. Notably, coumarins possessing prenyl unit(s) were found to be highly active, a tendency consistent with our previous study. Among the coumarins tested, 1,1-dimethylallylcoumarins showed the highest inhibitory activity. Western blotting analysis revealed that they inhibited NO generation by suppressing inducible NO synthase (iNOS) protein expression. Our ongoing studies suggest that coumarins are prominent natural compounds that attenuate excessive and prolonged NO generation at inflammatory sites.

Animals↗
PubMed1999

Cyclic nucleotide phosphodiesterases (PDE) 3 and 4 in normal, malignant, and HTLV-I transformed human lymphocytes.

Intracellular cyclic AMP, determined in part by cyclic nucleotide phosphodiesterases (PDEs), regulates proliferation and immune functions in lymphoid cells. Total PDE, PDE3, and PDE4 activities were measured in phytohemagglutinin (PHA)-activated peripheral blood mononuclear cells (PBMC-PHA), normal natural killer (NK) cells, Jurkat and Kit225-K6 leukemic T-cells, T-cell lines transformed with human T-lymphotropic virus (HTLV)-I (a retrovirus that causes adult T-cell leukemia/lymphoma) and HTLV-II (a nonpathogenic retrovirus), normal B-cells, and B-cells transformed with Epstein-Barr virus (EBV). All cells exhibited PDE3 and PDE4 activities but in different proportions. In EBV-transformed B cells, PDE4 was much higher than PDE3. HTLV-I+ T-cells differed significantly from other T-lymphocyte-derived cells in also having a higher proportion of PDE4 activities, which apparently were not related to selective induction of any one PDE4 mRNA (judged by reverse transcription-polymerase chain reaction) or expression of the HTLV-I regulatory protein Tax. In MJ cells (an HTLV-I+ T-cell line), Jurkat cells, and PBMC-PHA cells, the tyrosine kinase inhibitor herbimycin A strongly inhibited PDE activity. Growth of MJ cells was inhibited by herbimycin A and a protein kinase C (PKC) inhibitor, and was arrested in G1 by rolipram, a specific PDE4 inhibitor. Proliferation of several HTLV-I+ T-cell lines, PBMC-PHA, and Jurkat cells was inhibited differentially by forskolin (which activates adenylyl cyclase), the selective PDE inhibitors cilostamide and rolipram, and the nonselective PDE inhibitors pentoxifylline and isobutyl methylxanthine. These results suggest that PDE4 isoforms may be functionally up-regulated in HTLV-I+ T-cells and may contribute to the virus-induced proliferation, and that PDEs could be therapeutic targets in immune/inflammatory and neoplastic diseases.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1999

IL-3 and IL-4 activate cyclic nucleotide phosphodiesterases 3 (PDE3) and 4 (PDE4) by different mechanisms in FDCP2 myeloid cells.

In FDCP2 myeloid cells, IL-4 activated cyclic nucleotide phosphodiesterases PDE3 and PDE4, whereas IL-3, granulocyte-macrophage CSF (GM-CSF), and phorbol ester (PMA) selectively activated PDE4. IL-4 (not IL-3 or GM-CSF) induced tyrosine phosphorylation of insulin-receptor substrate-2 (IRS-2) and its association with phosphatidylinositol 3-kinase (PI3-K). TNF-alpha, AG-490 (Janus kinase inhibitor), and wortmannin (PI3-K inhibitor) inhibited activation of PDE3 and PDE4 by IL-4. TNF-alpha also blocked IL-4-induced tyrosine phosphorylation of IRS-2, but not of STAT6. AG-490 and wortmannin, not TNF-alpha, inhibited activation of PDE4 by IL-3. These results suggested that IL-4-induced activation of PDE3 and PDE4 was downstream of IRS-2/PI3-K, not STAT6, and that inhibition of tyrosine phosphorylation of IRS molecules might be one mechnism whereby TNF-alpha could selectively regulate activities of cytokines that utilized IRS proteins as signal transducers. RO31-7549 (protein kinase C (PKC) inhibitor) inhibited activation of PDE4 by PMA. IL-4, IL-3, and GM-CSF activated mitogen-activated protein (MAP) kinase and protein kinase B via PI3-K signals; PMA activated only MAP kinase via PKC signals. The MAP kinase kinase (MEK-1) inhibitor PD98059 inhibited IL-4-, IL-3-, and PMA-induced activation of MAP kinase and PDE4, but not IL-4-induced activation of PDE3. In FDCP2 cells transfected with constitutively activated MEK, MAP kinase and PDE4, not PDE3, were activated. Thus, in FDCP2 cells, PDE4 can be activated by overlapping MAP kinase-dependent pathways involving PI3-K (IL-4, IL-3, GM-CSF) or PKC (PMA), but selective activation of PDE3 by IL-4 is MAP kinase independent (but perhaps IRS-2/PI3-K dependent).

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1999

Novel organization and sequences of five genes encoding all six enzymes for de novo pyrimidine biosynthesis in Trypanosoma cruzi.

A 25 kb segment of genomic DNA from Trypanosoma cruzi, the causative agent of Chagas' disease, was sequenced. It contains five genes, pyr1, pyr2, pyr3, pyr4, and pyr6-5, encoding all six enzymes involved in de novo pyrimidine biosynthesis, glutamine-dependent carbamoyl-phosphate synthetase, aspartate carbamoyltransferase, dihydroorotase, dihydroorotate dehydrogenase, and orotidine-5'-phosphate decarboxylase linked with orotate phosphoribosyltransferase, respectively. The pyr genes constitute a polycistronic transcription unit on an 800 kb chromosomal DNA in the order of pyr1, pyr3, pyr6-5, pyr2, and pyr4 from the 5' terminus, with intervening sequences of 2.2, 0.4, 8.1, and 0.8 kb. The amino acid sequences deduced from the trypanosomatid pyr genes, except for pyr6, showed closer similarities to mammalian and yeast sequences, and less similarity to archaeal and bacterial sequences. The last two enzymes encoded by a single gene, pyr6-5, are covalently linked in the order opposite to mammalian pyr5-6, and possess a putative glycosomal targeting signal tripeptide, serine-lysine-leucine, at the C terminus. The calculated isoelectric points of 9.3 and 9.9 are also diagnostic of the glycosomal localization of these enzymes. We conclude that the T. cruzi pyr gene organization represents an early progenitor in de novo pyrimidine biosynthesis in eukaryotic lineage, and that the independent pyr genes may have evolved before the gene fusion events that resulted in the three mammalian-type genes, pyr1-3-2, pyr4, and pyr5-6, for UMP synthesis. Peculiarities in the trypanosomatid pyr6-5 gene product are discussed.

Amino Acid Sequence↗
PubMed1999

Regulated delivery of therapeutic proteins after in vivo somatic cell gene transfer.

Stable delivery of a therapeutic protein under pharmacologic control was achieved through in vivo somatic gene transfer. This system was based on the expression of two chimeric, human-derived proteins that were reconstituted by rapamycin into a transcription factor complex. A mixture of two adeno-associated virus vectors, one expressing the transcription factor chimeras and one containing erythropoietin (Epo) under the control of a promoter responsive to the transcription factor, was injected into skeletal muscle of immune-competent mice. Administration of rapamycin resulted in 200-fold induction of plasma Epo. Stable engraftment of this humanized system in immune-competent mice was achieved for 6 months with similar results for at least 3 months in a rhesus monkey.

Animals↗
PubMed1999

Identification of coumarins from the fruit of Citrus hystrix DC as inhibitors of nitric oxide generation in mouse macrophage RAW 264.7 cells.

Three known coumarins have been isolated from Citrus hystrix DC as inhibitors of both lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma)-induced nitric oxide (NO) generation in RAW 264.7 cells. The inhibitory activity of bergamottin (IC(50) = 14 microM) was comparable to that of N-(iminoethyl)-L-ornithine (L-NIO) (IC(50) = 7. 9 microM), whereas oxypeucedanin and 5-[(6',7'-dihydroxy-3', 7'-dimethyl-2-octenyl)oxy]psoralen, structurally different from bergamottin only in their side-chain moieties, were notably less active. Using 21 coumarins, we structurally classified various types of coumarins into groups A-C: (A) bearing an isoprenyl (IP) or a geranyl (GR) group, highly active; (B) bearing an IP group cyclized to a coumarin ring, moderately active; (C) bearing an IP group modified with hydroxyl group(s) and/or having other functional groups except for the IP, completely inactive. Cellular uptake studies suggested that coumarins in group C are inactive because of poor permeability to the cell membrane.

Animals↗
PubMed1999

Somatic cell mutants resistant to retrovirus replication: intracellular blocks during the early stages of infection.

To identify cellular functions involved in the early phase of the retroviral life cycle, somatic cell mutants were isolated after selection for resistance to infection. Rat2 fibroblasts were treated with chemical mutagens, and individual virus-resistant clones were recovered after selection for resistance to infection. Two clones were characterized in detail. Both mutant lines were resistant to infection by both ecotropic and amphotropic murine viruses, as well as by human immunodeficiency virus type 1 pseudotypes. One clone showed a strong block to reverse transcription of the retroviral RNA, including formation of the earliest DNA products. The second clone showed normal levels of viral DNA synthesis but did not allow formation of the circular DNAs normally found in the nucleus. Cell fractionation showed that the viral preintegration complex was present in a form that could not be extracted under conditions that readily extracted the complex from wild-type cells. The results suggest that the DNA was trapped in a nonproductive state and excluded from the nucleus of the infected cell. The properties of these two mutant lines suggest that host gene products play important roles both before and after reverse transcription.

Animals↗
PubMed1999

Pneumolysin, a protein toxin of Streptococcus pneumoniae, induces nitric oxide production from macrophages.

Nitric oxide (NO) production by inducible NO synthase (iNOS) during inflammation is an essential element of antimicrobial immunity but can also contribute to host-induced tissue damage. Under conditions of bacterial sepsis, large amounts of NO are produced, causing hypotension, a critical pathological feature of septic shock. In sepsis caused by gram-positive organisms, the bacterial factors contributing to host NO production are poorly characterized. We show that a soluble toxin of Streptococcus pneumoniae, pneumolysin (Pln), is a key component initiating NO production from macrophages. In contrast to wild-type bacteria, a mutant of S. pneumoniae lacking Pln failed to elicit NO production from murine macrophages. Purified recombinant Pln induced NO production at low concentrations and independently of exogenous gamma interferon (IFN-gamma) priming of RAW 264.7 macrophages. However, IFN-gamma was essential for Pln-induced NO production, since primary macrophages from mice lacking the IFN-gamma receptor or interferon regulatory factor 1, a transcription factor essential for iNOS expression, failed to produce NO when stimulated with Pln. In addition, Pln acts as an agonist of tumor necrosis factor alpha and interleukin 6 production in macrophages. The properties of Pln, previously identified as a pore-forming hemolysin, also include a role as a general inflammatory agonist.

Animals↗
PubMed1999

Gene therapy vectors based on adeno-associated virus type 1.

The complete sequence of adeno-associated virus type 1 (AAV-1) was defined. Its genome of 4,718 nucleotides demonstrates high homology with those of other AAV serotypes, including AAV-6, which appears to have arisen from homologous recombination between AAV-1 and AAV-2. Analysis of sera from nonhuman and human primates for neutralizing antibodies (NAB) against AAV-1 and AAV-2 revealed the following. (i) NAB to AAV-1 are more common than NAB to AAV-2 in nonhuman primates, while the reverse is true in humans; and (ii) sera from 36% of nonhuman primates neutralized AAV-1 but not AAV-2, while sera from 8% of humans neutralized AAV-2 but not AAV-1. An infectious clone of AAV-1 was isolated from a replicated monomer form, and vectors were created with AAV-2 inverted terminal repeats and AAV-1 Rep and Cap functions. Both AAV-1- and AAV-2-based vectors transduced murine liver and muscle in vivo; AAV-1 was more efficient for muscle, while AAV-2 transduced liver more efficiently. Strong NAB responses were detected for each vector administered to murine skeletal muscle; these responses prevented readministration of the same serotype but did not substantially cross-neutralize the other serotype. Similar results were observed in the context of liver-directed gene transfer, except for a significant, but incomplete, neutralization of AAV-1 from a previous treatment with AAV-2. Vectors based on AAV-1 may be preferred in some applications of human gene therapy.

Adolescent↗
PubMed1998

Carbamoyl-phosphate synthetase II in kinetoplastids.

Genes for carbamoyl-phosphate synthetase II (CPS II), the first enzyme of de novo pyrimidine biosynthesis, were cloned from kinetoplastids, Trypanosoma cruzi and Leishmania mexicana. T. cruzi CPS II gene encodes a protein of 1524 amino acids that encompasses the glutaminase and CPS domains, but incorporates neither aspartate carbamoyltransferase nor dihydroorotase. The residue corresponding to lysine 993 of Escherichia coli CPS, a residue that characterizes the CPS inhibited by UMP and that is replaced by tryptophan in those inhibited by UTP, is in kinetoplastids a hydrophilic glutamine, in line with the preferential inhibition by UDP of kinetoplastid CPS II.

Amino Acid Sequence↗
PubMed1998

A study of multiple biomarkers in coke oven workers--a cross-sectional study in China.

We conducted a cross-sectional molecular epidemiological study of coke oven workers exposed to the established carcinogen polycyclic aromatic hydrocarbons (PAHs) to evaluate the relationships between both traditional 'exposure markers' and a series of biomarkers, including urinary 1-hydroxypyrene as a marker of internal dose, leukocyte aromatic DNA adducts as markers of biologically effective dose, serum p53 protein as a response marker and genetic polymorphisms of cytochrome P4501A1 and glutathione S-transferase MI as susceptibility markers. Twenty-five male subjects each were randomly selected from the top, middle and bottom work areas of the oven, and the control plant. They were matched for age and smoking status. The mean levels of PAH exposure, monitored by stationary and personal samplers, and of worker urinary 1-hydroxypyrene differed significantly between the top, middle and bottom of the oven and control work areas. The highest stationary and personal PAH concentrations and 1-hydroxypyrene levels were demonstrated at the top work area. Good correlations were found between the stationary PAH levels, personal PAH levels and urinary 1-hydroxypyrene levels. No positive correlations were demonstrated between aromatic DNA adduct levels and current or cumulative PAH exposure dose. In the presence of genetic polymorphisms of cytochrome P4501A1, a positive correlation was demonstrated between aromatic DNA adducts and urinary 1-hydroxypyrene levels. There was also a significant correlation between serum p53 protein levels and the cumulated benzo[a]pyrene exposure dose. Although these biomarkers have certain limitations, they are applicable to cancer epidemiology, and may contribute to our understanding of the mechanisms of carcinogenesis.

Adult↗
PubMed1998

Replication defect of moloney murine leukemia virus with a mutant reverse transcriptase that can incorporate ribonucleotides and deoxyribonucleotides.

Reverse transcriptase (RT) plays a critical role in retrovirus replication, directing the synthesis of a double- stranded DNA copy of the viral RNA genome. We have previously described a mutant RT of the Moloney murine leukemia virus in which F155 was replaced by valine, and we demonstrated that this substitution allowed the enzyme to incorporate ribonucleotides to form RNA while still retaining its normal ability to incorporate deoxyribonucleotides to form DNA. When introduced into the viral genome, this mutation rendered the virus incapable of replication. Characterization of the mutant virus revealed that the enzyme was still active and able to synthesize minus-strand strong stop DNA and some longer products but failed to make full-length minus-strand DNA. We propose that the failure of the enzyme to complete DNA synthesis in vivo resulted from its ability to incorporate ribonucleotides into the products, which served as inhibitors for DNA synthesis. We also tested seven other amino acid residues for their abilities to substitute for F155 in virus replication; of these, only tyrosine could support virus replication. In an attempt to select for second-site suppressor mutations, the F155V mutant was subjected to random mutagenesis and was used as a parent for the isolation of revertant viruses. Two independent revertants were found to have changed the valine residue at position 155 back to the wild- type phenylalanine. These results suggest that an aromatic ring at this position is important for virus replication.

3T3 Cells↗
PubMed1998

[Microsurgical repair of defects of soft tissue and infected wounds of extremities].

A study was carried out to observe the application of microsurgical technique in the repair defects of soft tissue and infected wounds of extremities. Eighty-three patients with soft tissue defects and infected wounds of extremities were treated by either transferring of vascularized cutaneous flap or transplantation of myocutaneous flap with vascular anastomosis. The result showed that eighty-three patients had gained success after a follow-up of 6 months to 4 years. It was concluded that soft tissue defects and infected wounds of extremities should be repaired as early as possible. Selecting the donor flap near the recipient site was of first choice. The method used for repair should be simple and easily applicable rather these very complicated one. The success depended on the correct treatment of local conditions, resonable design of donor flap and close monitoring after operation.

Adolescent↗
PubMed1997

Differential expression of cyclic nucleotide phosphodiesterase 3 and 4 activities in human T cell clones specific for myelin basic protein.

Little is known concerning the relative distribution and function of the different cyclic nucleotide phosphodiesterases (PDEs) in lymphocytes. Recent reports, however, have indicated that specific PDE4 inhibitors were effective in treatment of experimental allergic encephalomyelitis, an animal model of multiple sclerosis. The therapeutic effect of PDE4 inhibitors is thought to be related to inhibition of autoreactive CD4+ T cells specific for myelin basic protein (MBP) or other myelin proteins. Human autoreactive CD4+ T lymphocyte clones (TCC), specific for the immunodominant MBP epitope (amino acids 83-99), contain PDE3 and PDE4, two PDEs that exhibit a high affinity for cAMP. Amplification of TCC mRNA by reverse transcription-PCR indicated that TCC PDE3 mRNA was of the PDE3B, not PDE3A, subtype. Different TCC contained different proportions of PDE3 and PDE4, and their activities increased during Ag (MBP) stimulation. Specific PDE3 (cilostamide) and PDE4 (rolipram) inhibitors suppressed [3H]thymidine incorporation in TCC. Since it is believed that many autoimmune diseases are at least partially mediated by autoreactive CD4+ T cells, these observations may have important implications not only for the treatment of multiple sclerosis but also for other autoimmune diseases.

3',5'-Cyclic-AMP Phosphodiesterases↗