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Biomedical subjects

Y Luo

Publications and source records attributed to Y Luo.

At least 325 records · Page 18Linked to original sources
PubMed1996

Molecular modeling and deletion mutagenesis implicate the nuclear translocation sequence in structural integrity of fibroblast growth factor-1.

The sequence NYKKPKL in the NH2 terminus of fibroblast growth factor (FGF)-1 has been proposed to affect the long term activities of FGF-1 through its function as a nuclear translocation signal or its role in stabilization of the structure required to sustain binding and activation of the transmembrane receptor kinase. A dynamic molecular model of FGF-1 docked into a duplex of the FGF receptor ectodomain and a hexadecameric heparin chain suggests that the NYKKPKL sequence does not directly interact with heparin or the receptor, but rather the lysine-leucine residues within the sequence indirectly stabilize a major receptor-binding domain. Concurrent with a marked increase in dependence on exogenous heparin for optimal activity, sequential deletion of residues in the NYKKPKL sequence in FGF-1 resulted in a progressive loss of thermal stability, resistance to protease, mitogenic activity, and affinity for the transmembrane receptor. The largest change resulted from deletion of the entire sequence through the lysine-leucine residues. In the presence of sufficiently high concentrations of heparin, the deletion mutants exhibited mitogenic activity equal to wild-type FGF-1. The results confirm that a primary role of the NYKKPKL sequence domain is to maintain the structural integrity of FGF-1 required for optimal binding to and activation of the heparan sulfate-transmembrane receptor complex.

3T3 Cells↗
PubMed1996

Physiological levels of beta-amyloid peptide promote PC12 cell proliferation.

Alzheimer's beta-amyloid peptide (A beta) is normally present at a subnanomolar (225-625 pM) concentration in body fluids and in the medium of cultured cells. The potential actions of physiologic levels of A beta are being investigated. We have recently shown that nanomolar doses of A beta can stimulate tyrosine phosphorylation and activate phosphatidylinositol-3-kinase in neuronal cells. Here we show evidence that A beta at nanomolar levels promotes cell growth determined by [3H]thymidine incorporation, protein content and cell counts. Physiological levels of A beta peptides, including 1-40, 1-42, 25-35, all promote growth of PC12 cells in low serum medium with doubling times of 71 h to around 30 h. Although the promotion of cell proliferation can be detected at nanomolar levels of A beta, it's potency is less than that of serum. This suggests that A beta may normally play a neurotrophic/mitogenic role in neuronal biology.

Amyloid beta-Peptides↗
PubMed1996

The B-cell-specific transcription coactivator OCA-B/OBF-1/Bob-1 is essential for normal production of immunoglobulin isotypes.

OCA-B was initially identified as a B-cell-restricted coactivator that functions with octamer binding transcription factors (Oct-1 and Oct-2) to mediate efficient cell type-specific transcription of immunoglobulin promoters in vitro. Subsequent cloning studies led to identification of the coactivator as a single polypeptide, designated either as OCA-B (ref. 3), OBF-1 (ref. 4) or Bob-1 (ref. 5). OCA-B itself does not bind to DNA directly, but interacts with either Oct-1 or Oct-2 to potentiate transcriptional activation. To determine the biological role of OCA-B, we generated OCA-B-deficient mice by gene targeting. Mice lacking OCA-B undergo normal antigen-independent, B-cell differentiation, including appropriate expression of both immunoglobulin genes and other early B-cell-restricted genes. However, antigen-dependent maturation of B cells is greatly affected. The proliferative response to surface IgM crosslinking is impaired, and there is a severe deficiency in the production of secondary immunoglobulin isotypes including IgG1, IgG2a, IgG2b, IgG3, IgA and IgE in OCA-B-deficient B cells. This defect is not due to a failure of the isotype switching process, but rather to reduced levels of transcription from normally switched immunoglobulin heavy-chain loci. In accord with the defective isotype production, germinal centre formation is absent in these mutant mice.

Animals↗
PubMed1996

Fe2+, Fe3+, and oxygen react with DNA-derived radicals formed during iron-mediated Fenton reactions.

Oxidative DNA damage is decreased by the presence of O2 during Fe(2+)-mediated Fenton reactions when H2O2 is in excess. During these reactions, the presence of DNA increases H2O2 consumption relative to Fe2+ consumption under anaerobic conditions, but decreases H2O2 consumption relative to Fe2+ consumption under aerobic conditions. The pseudobimolecular rate constant of H2O2 consumption is the same under both conditions, however, indicating that the presence of DNA affects the oxidation and/or reduction of the iron pool. To understand the basis of these effects, DNA was replaced with ethanol as a model compound. Computer simulations of Fe2+ and H2O2 consumption were experimentally verified and allowed identification of the predominant reactions leading to the changes in stoichiometry. Based upon these results and upon qualitative and quantitative differences in DNA damages between aerobic and anaerobic conditions, it was concluded that, in the presence of DNA, Fe3+ is reduced by some DNA radicals. However, if O2 is present, these radicals react instead with O2 and the product of these reactions can then oxidize Fe2+. Mechanisms proposed for the alteration by O2 of products from dC- and dG-containing substrates after exposure to Fe and H2O2 fit these general schemes. These results provide another distinction between DNA damage caused by ionizing radiation and that caused by Fenton reactions.

Acetaldehyde↗
PubMed1996

ErbB receptor activation, cell morphology changes, and apoptosis induced by anti-Her2 monoclonal antibodies.

A panel of mAbs were generated against the purified soluble form of erbB2/Her2 receptor, corresponding to the extracellular region of the receptor, and examined for their ability to mimic the receptor ligand. Some of the mAbs strongly induced tyrosine phosphorylation of 180-185 kDa proteins, including not only Her2 but also Her3 and Her4 receptors, when they were expressed on the surface of breast cancer cells. These mAbs do not cross-react with Her3 or Her4 as demonstrated by competition study. Receptor phosphorylation was also observed with the cell lines transfected with Her2 or a chimeric receptor consisting of the extracellular domain of Her2 and the transmembrane and cytoplasmic domains of epidermal growth factor receptor. Selected mAbs were tested for their ability to change cell morphology, and one specific mAb, mAb74, induced cell morphology changes and apoptosis.

Animals↗
PubMed1996

Beta-chemokine TCA3 binds to and activates rat vascular smooth muscle cells.

The present study compares the activity of TCA3 with other beta-chemokines (macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, and monocyte chemoattractant protein (MCP)-1) on rat vascular smooth muscle cells. TCA3, MIP-1 alpha, and MCP-1 (but not MIP-1 beta) treatment stimulates chemotaxis of vascular smooth muscle cells. TCA3-mediated chemotactic responses are sensitive to treatment with pertussis toxin, suggesting that G alpha-i proteins are involved in TCA3 signaling of smooth muscle. In addition, TCA3, MIP-1 alpha, and MCP-1 increase vascular smooth muscle cell adhesiveness to type III collagen. In contrast, stimulation with TCA3, but not other beta-chemokines, induces proliferation of vascular smooth muscle cells. TCA3 receptors were identified on rat vascular smooth muscle cells by direct binding of radiolabeled ligand. TCA3 binds to this receptor with high affinity (3 nM). Rat vascular smooth muscle cells display approximately 75,000 binding sites/cell. Competitive inhibition studies indicated that murine MIP-1 alpha, murine MCP-1, and human RANTES are weak partial competitors of TCA3 binding, demonstrating the existence of a unique receptor for TCA3. Murine MIP-1 beta, which fails to stimulate any biologic functions in vascular smooth muscle cells, also does not inhibit TCA3 binding. The combined data demonstrate that TCA3 and other beta-chemokines can modulate vascular smooth muscle cell function.

Animals↗
PubMed1996

Oxidative damage to DNA constituents by iron-mediated fenton reactions. The deoxycytidine family.

Damage by iron-mediated Fenton reactions under aerobic or anaerobic conditions to deoxycytidine, deoxycytidine-5'-monophosphate, d-CpC, d-CpCpC, and dCMP residues in DNA resulted in at least 26 distinguishable products. Of these, 24 were identified by high performance liquid chromatography retention times, radiolabeling, UV absorption spectra, chemical synthesis, fast atom bombardment mass spectrometry, high resolution fast atom bombardment mass spectrometry, and/or NMR. The nature of the products was qualitatively similar for each substrate except for d-CpC (and possibly d-CpCpC) under anaerobic conditions for which 5-hydroxy-deoxycytidine was uniquely present and 1-carbamoyl-1-carboxy-4-(2-deoxy-beta-D-erythropentofuranosyl) glycinamide was uniquely absent. Damage to dC, d-CpC, and d-CpCpC but not to dCMP or DNA was largely quenched by ethanol, indicating that iron is strongly associated only with dCMP and DNA. The presence of oxygen had little effect with dC or dCMP but had quantitative and qualitative effects with d-CpC and a significantly quantitative but not a qualitative effect with DNA. NADH could drive the Fenton reaction to cause damage to the dC family in vitro, consistent with a previous proposal that NADH was the reducing agent for the Fenton reaction in vivo (Imlay, J.A., and Linn, S. (1988) Science 240, 1302-1309). Finally, the damage spectrum of the dC family by the Fenton reaction is compared with that by ionizing radiation and chemical mechanisms leading to the formation of the 24 identified products are proposed.

Chromatography, High Pressure Liquid↗
PubMed1996

Oxidative damage to DNA constituents by iron-mediated fenton reactions. The deoxyguanosine family.

2'-Deoxyguanosine, 3'-dGMP, 5'-dGMP, d-GpG, or double-stranded DNA were exposed to H2O2 in the presence of Fe2+ under anaerobic conditions or under aerobic conditions in the presence of Fe3+, Fe2+, Fe2+/NADH, or Fe3+/NADH with and without ethanol. The products were enzymatically digested to nucleosides, separated by high performance liquid chromatography (HPLC), quantified, and characterized by HPLC retention time, radiolabeling, UV absorbance spectrometry, NMR, and mass spectrometry. 20 products, constituting 78-81% of the damage, were distinguished from aerobic reactions of Fe2+/H2O2 with dG and dGMP, 16 of which were identified. The product spectra from dG, 3'-dGMP, and 5'-dGMP differ from one another, and the spectrum of the 5' nucleoside of d-GpG differs from that of the 3' nucleoside. 7, 8-Dihydro-8-oxo-2'-deoxyguanosine is the most abundant DNA-bound product aside from abasic sites, and its formation was more closely analyzed. Both NADH, which can reduce Fe3+, and ethanol, which can scavenge some free radicals, change the product profiles. Damage enhancement by NADH follows the sequence dG < d-GpG < 3'-dGMP < 5'-dGMP < DNA; the reverse sequence is observed for ethanol quenching. This sequence of susceptibility and the product differences seen for the 3' and 5' phosphate may reflect localization of iron and the damaging radicals upon the substrate.

Chromatography, High Pressure Liquid↗
PubMed1996

Mouse astrocytes respond to the chemokines MCP-1 and KC, but reverse transcriptase-polymerase chain reaction does not detect mRNA for the KC or new MCP-1 receptor.

Previous studies demonstrated the involvement of astrocytes in the development of astrogliosis, a condition in which these cells undergo proliferation and hypertrophy. To examine whether astrocytes could migrate into lesions, we tested the influence of the murine chemokines MCP-1, KC, TCA3, and MIP-1 beta on migration of cultured neonatal mouse astrocytes. Subnanomolar concentrations of MCP-1 and KC were active chemoattractants indicating that these molecules were effective at physiologic concentrations. Specificity of MCP-1 was demonstrated by antibody inhibition and by the finding that the chemokine MIP-1 beta failed to induce astrocyte migration. The migratory responses were sensitive to pertussis toxin; this finding is consistent with involvement of G protein-coupled receptors. To examine the receptors for these chemokines further, we cloned the mouse homolog of the human MCP-1 receptor from a mouse peritoneal exudate cell cDNA library. The gene had 78% nucleotide sequence homology with the human MCP-1 receptor (the nucleotide sequence of clone 1 encoding the mouse MCP-1 receptor can be obtained from the GenBank database, accession number U56819). However, reverse transcriptase-polymerase chain reaction (RT-PCR) failed to detect message for either the MCP-1 or KC receptors in astrocytes. The combined data suggest that mouse astrocytes use novel receptors to recognize these chemokines.

Animals↗
PubMed1996

GDNF-induced activation of the ret protein tyrosine kinase is mediated by GDNFR-alpha, a novel receptor for GDNF.

We report the expression cloning and characterization of GDNFR-alpha, a novel glycosylphosphatidylinositol-linked cell surface receptor for glial cell line-derived neurotrophic factor (GDNF). GDNFR-alpha binds GDNF specifically and mediates activation of the Ret protein-tyrosine kinase (PTK). Treatment of Neuro-2a cells expressing GDNFR-alpha with GDNF rapidly stimulates Ret autophosphorylation. Ret is also activated by treatment with a combination of GDNF and soluble GDNFR-alpha in cells lacking GDNFR-alpha, and this effect is blocked by a soluble Ret-Fc fusion protein. Ret activation by GDNF was also observed in cultured embryonic rat spinal cord motor neurons, a cell type that responds to GDNF in vivo. A model for the stepwise formation of a GDNF signal-transducing complex including GDNF, GDNFR-alpha, and the Ret PTK is proposed.

Amino Acid Sequence↗
PubMed1996

Purification, identification, and properties of a Saccharomyces cerevisiae oleate-activated upstream activating sequence-binding protein that is involved in the activation of POX1.

Peroxisomes have a central function in lipid metabolism, and it is well established that these organelles are inducible by many compounds including fatty acids. Peroxisomes are the sole site for the beta-oxidation of fatty acids in yeast. The first and rate-limiting enzyme of this cycle is fatty acyl-CoA oxidase. The gene encoding this enzyme in Saccharomyces cerevisiae (POX1) undergoes a complex regulation that is dependent on the growth environment. When this yeast is grown in medium containing oleic acid as the main carbon source, peroxisomes are induced and POX1 expression is activated. When cells are grown in the presence of glucose, the expression of POX1 mRNA is repressed, whereas growth on a carbon source such as glycerol or raffinose causes derepression. This rigorous regulation is brought about by the complex interactions between trans-acting factors and cis-elements in the POX1 promoter. Previously, we characterized regulatory elements in the promoter region of POX1 that are involved in the repression and activation of this gene (Wang, T., Luo, Y., and Small, G. M. (1994) J. Biol. Chem. 269, 24480-24485). In this study we have purified and identified an oleate-activated transcription factor (Oaf1p) that binds to the activating sequence (UAS1) in the POX1 gene. The protein has a predicted molecular mass of approximately 118 kDa.

Acyl-CoA Oxidase↗
PubMed1996

Structure of human salivary alpha-amylase at 1.6 A resolution: implications for its role in the oral cavity.

Salivary alpha-amylase, a major component of human saliva, plays a role in the initial digestion of starch and may be involved in the colonization of bacteria involved in early dental plaque formation. The three-dimensional atomic structure of salivary amylase has been determined to understand the structure-function relationships of this enzyme. This structure was refined to an R value of 18.4% with 496 amino-acid residues, one calcium ion, one chloride ion and 170 water molecules. Salivary amylase folds into a multidomain structure consisting of three domains, A, B and C. Domain A has a (beta/alpha)(8-) barrel structure, domain B has no definite topology and domain C has a Greek-key barrel structure. The Ca(2+) ion is bound to Asnl00, Arg158, Asp167, His201 and three water molecules. The Cl(-) ion is bound to Arg195, Asn298 and Arg337 and one water molecule. The highly mobile glycine-rich loop 304-310 may act as a gateway for substrate binding and be involved in a 'trap-release' mechanism in the hydrolysis of substrates. Strategic placement of calcium and chloride ions, as well as histidine and tryptophan residues may play a role in differentiating between the glycone and aglycone ends of the polysaccharide substrates. Salivary amylase also possesses a suitable site for binding to enamel surfaces and provides potential sites for the binding of bacterial adhesins.

Journal Article↗
PubMed1996

Determination of dihydroetorphine in biological fluids by gas chromatography-mass spectrometry using selected-ion monitoring.

A method for the determination of dihydroetorphine hydrochloride, a powerful anaesthetic and analgesic drug, in biological fluids by GC-MS with selected-ion monitoring using etorphine as internal standard was established. Dihydroetorphine was extracted from human blood and urine with dichloromethane and then derivatized with N-heptafluorobutyrylimidazole after concentration to dryness. A dihydroetorphine monoheptafluorobutyl derivative was formed which showed good behavior on GC-MS with electronic-impact ionization. The main fragment, m/z 522, which is the base peak, was selected as the ion for quantitation and the corresponding ion, m/z 520, was selected for monitoring the internal standard, etorphine. The recoveries and coefficients of variation of the whole procedure were determined with five controlled dihydroetorphine-free urine and plasma samples spiked with different concentrations of dihydroetorphine. The concentration of dihydroetorphine for quantitation was in the range 1-20 ng/ml for urine and 2.5-250 ng/ml for plasma. The correlation coefficients of the standard curves are sufficient to determine the dihydroetorphine. The accuracy for quantitation of dihydroetorphine in urine and plasma is less than 10.6%.

Analgesics, Opioid↗
PubMed1996

The phosphatase inhibitor calyculin antagonizes the rapid initiation of apoptosis by photodynamic therapy.

DNA fragmentation and internucleosomal cleavage were rapidly initiated after lysosomal photodamage to murine leukemia cells, with apoptotic chromatin and DNA 'ladders' detected within 30 min after irradiation. Apoptosis was inhibited by concurrent exposure of cells to a 10 nM concentration of the serine-threonine phosphatase inhibitor calyculin A and promoted by a serine/threonine kinase inhibitor. These results indicate that a late stage in apoptosis requires serine/threonine dephosphorylation.

Animals↗
PubMed1996

Exclusion of linkage between RET and neuronal intestinal dysplasia type B.

Neuronal Intestinal Dysplasia type B (NID B) is a complex alteration of the enteric nervous system belonging to the group of intestinal dysganglionoses which may involve rectum, colon, and small intestine. Second only to Hirschsprung disease (HSCR), NID B is one of the most frequent causes of chronic constipation and pseudo-obstructive intestinal dysmotility. Since NID B is often associated with HSCR and point mutations in the RET proto-oncogene have been identified in HSCR patients, we analyzed two NID B pedigrees to investigate if RET mutations might cause also the NID B phenotype. Linkage analysis demonstrated that the NID B locus is not linked to RET in the pedigrees analyzed. Further genetic analyses will possibly improve the understanding of the cause and facilitate diagnostic procedures in NID B.

Drosophila Proteins↗
PubMed1996

Thermodynamic cycles as probes of structure in unfolded proteins.

The relationship between structure and stability has been investigated for the folded forms and the unfolded forms of iso-2 cytochrome c and a variant protein with a stability-enhancing mutation, N52I iso-2. Differential scanning calorimetry has been used to measure the reversible unfolding transitions for the proteins in both heme oxidation states. Reduction potentials have been measured as a function of temperature for the folded forms of the proteins. The combination of measurements of thermal stability and reduction potential gives three sides of a thermodynamic cycle and allows prediction of the reduction potential of the thermally unfolded state. The free energies of electron binding for the thermally unfolded proteins differ from those expected for a fully unfolded protein, suggesting that residual structure modulates the reduction potential. At temperatures near 50 degrees C the N52I mutation has a small but significant effect on oxidation state-sensitive structure in the thermally unfolded protein. Inspection of the high-resolution X-ray crystallographic structures of iso-2 and N52I iso-2 shows that the effects of the N52I mutation and oxidation state on native protein stability are correlated with changes in the mobility of specific polypeptide chain segments and with altered hydrogen bonding involving a conserved water molecule. However, there is no clear explanation of oxidation state or mutation-induced differences in stability of the proteins in terms of observed changes in structure and mobility of the folded forms of the proteins alone.

Cytochrome c Group↗
PubMed1996

Beta-chemokine TCA3 binds to mesangial cells and induces adhesion, chemotaxis, and proliferation.

Specific receptors for the beta-chemokine TCA3 have been identified on mouse monocyte/macrophage cell lines and on mouse mesangial cells. Using Scatchard plot analysis with 125I-labeled TCA3, a single high-affinity receptor (3-4 nM) was identified. Cells of the monocyte lineage express 1,400 to 8,600 TCA3 binding sites, while mesangial cells display 40,000 to 49,000 sites/cell. Competitive inhibition studies indicated that the TCA3 receptor is unique, although MCP-1, IL-8, and RANTES were weak competitors of TCA3 binding. We also established the functional activity of TCA3 and other chemokines on primary cultures of mouse mesangial cells. TCA3 treatment induces increased mesangial cell adhesiveness to fibronectin. TCA3 is also a chemoattractant for mesangial cells. In addition, TCA3 treatment stimulates [3H]thymidine uptake by mesangial cells. The combined results indicate that TCA3 and other chemokines interact with a broader range of target cells than previously considered.

Animals↗