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M Yang

Publications and source records attributed to M Yang.

At least 307 records · Page 17Linked to original sources
PubMed1997

Identification of residues in the class II-associated Ii peptide (CLIP) region of invariant chain that affect efficiency of MHC class II-mediated antigen presentation in an allele-dependent manner.

Invariant chain (Ii) associates with class II MHC molecules and is crucial for Ag presentation by class II molecules. A general explanation for how invariant chain (Ii) associates with polymorphic MHC class II molecules has been suggested by the crystallographic structure of CLIP (class II-associated Ii peptide) complexed with an HLA class II molecule, HLA-DR3. We show here that methionine residues at positions 93 and 99 in Ii are important in MHC class II-mediated Ag presentation, but function in an allele-dependent manner. Introduction of a Met-->Ala mutation at position 99 in Ii (M99AIi) impaired presentation of peptides derived from exogenous proteins by I-Ad and I-Au class II molecules. Mutating Met-->Ala in Ii at position 93 (M93AIi) abrogated presentation by I-Au molecules, but not by I-Ad. Impaired Ag presentation was associated with conformationally altered expression of I-A molecules on the surface of cells expressing mutated Ii. Cell surface CLIP staining and immunoprecipitation studies showed that both I-Ad and I-Au molecules were associated with an increased abundance of Ii peptides, CLIP, in cells expressing mutated Ii. These results show that methionine 93 and methionine 99 play an important physiologic role in Ii association with class II molecules by regulating release of CLIP from class II in the endocytic compartments to allow binding of cognate peptides.

Alanine↗
PubMed1997

Alanine-scanning mutagenesis of Bacillus subtilis trp RNA-binding attenuation protein (TRAP) reveals residues involved in tryptophan binding and RNA binding.

In Bacillus subtilis, expression of the trp genes is negatively regulated by an RNA binding protein called TRAP (trp RNA-binding Attenuation Protein), which is activated to bind RNA by binding l-tryptophan. TRAP contains 11 identical subunits assembled in a symmetric ring. We have used alanine-scanning mutagenesis to analyze the functions of surface amino acid residues of TRAP. The in vivo regulatory activity of each mutant TRAP was analyzed in a B. subtilis reporter strain containing a trpE'-'lacZ fusion. Mutant TRAP proteins with defective in vivo regulatory activities were characterized in vitro by measuring their tryptophan binding and RNA binding activities. Most of the mutant proteins with altered tryptophan binding, either affinity or cooperativity, contained substituted residues located on two loops formed by residues 25 to 33 and residues 49 to 52, as well as on the beta-strand and beta-turn contiguous with these loops. Substitution of three residues (Lys37, Lys56 and Arg58) with alanine resulted in significant decreases in the RNA binding activity of TRAP without altering tryptophan binding. Structural analysis shows that these three residues are directly aligned on the outer edge of TRAP. Further mutagenic analysis of these three residues revealed that only lysine or arginine residues at positions 37 or 58 allow proper TRAP function, whereas at position 56, only lysine is functional. Residue Asn20 is the only other residue in TRAP that is located on the line formed by residues 37, 56 and 58, and virtually any amino acid residue is functional at position 20. We propose that RNA wraps around TRAP by interacting with residues Lys37, Lys56 and Arg58.

Alanine↗
PubMed1997

A novel matrix metalloproteinase gene (XMMP) encoding vitronectin-like motifs is transiently expressed in Xenopus laevis early embryo development.

To study the role of matrix metalloproteinases (MMPs) in early vertebrate development, we cloned cDNAs for six different MMPs from the frog Xenopus laevis embryos at different stages of development and describe here a novel MMP called XMMP. Xenopus XMMP has 604 amino acids including a putative signal peptide of 22 residues. At the carboxyl-terminal end of the propeptide, XMMP has a 37-amino acid-long insertion domain containing a segment that is 38% identical with a rat vitronectin sequence between residues 108-135. Following this domain is an RRKR motif, a putative cleavage site for intracellular activation by furin proteinases. XMMP lacks a proline-rich linker peptide, or hinge region, typically found in other MMPs between the catalytic domain and carboxyl-terminal "hemopexin/vitronectin-like" domain. In XMMP, the carboxyl-terminal domain is composed of four tandem repeats that are 21-33% identical to a sequence (residues 213-264) encoded by vitronectin exon-5. Interestingly, XMMP gene is transiently expressed during Xenopus embryo development. XMMP mRNA of 3.0 kilobase pairs was undetected in the blastula stage embryo, induced in gastrula embryo, expressed in neurula embryo, and then down-regulated in pretailbud embryo. In comparison, other Xenopus MMP genes that we have cloned show a different developmental regulation. In blastula embryo, the only MMP gene expressed was found to be 92-kDa type IV collagenase, which was also expressed in the gastrula, neurula, and pretailbud embryos. Expression of stromelysin-1, stromelysin-3, and two different membrane type-MMPs was first detected in the neurula and pretailbud embryos. These results suggest that MMPs and the novel XMMP reported here play a role in Xenopus early development.

Amino Acid Sequence↗
PubMed1997

Functional domains are specified to single-cell resolution in a Drosophila epithelium.

Specification of pattern is fundamental to the development of a multicellular organism. The Malpighian (renal) tubule of Drosophila melanogaster is a simple epithelium that proliferates under the direction of a single tip cell into three morphologically distinct domains. However, systematic analysis of a panel of over 700 P[GAL4] enhancer trap lines reveals unexpected richness for such an apparently simple tissue. Using numerical analysis, it was possible formally to reconcile apparently similar or complementary expression domains and thus to define at least five genetically defined domains and multiple cell types. Remarkably, the positions of domain boundaries and the numbers of both principal and secondary ("stellate") cell types within each domain are reproducible to near single-cell precision between individual animals. Domains of physiological function were also mapped using transport or expression assays. Invariably, they respect the boundaries defined by enhancer activity. These genetic domains can also be visualized in vivo, both in transgenic and wild-type flies, providing an "identified cell" system for epithelial physiology. Building upon recent advances in Drosophila Malpighian tubule physiology, the present study confirms this tissue as a singular model for integrative physiology.

Animals↗
PubMed1997

First intracellular loop of the human cholecystokinin-A receptor is essential for cyclic AMP signaling in transfected HEK-293 cells.

Cholecystokinin (CCK)-A and CCK-B receptors are highly homologous members of the seven transmembrane domain G-protein-coupled receptor superfamily. Genes of both receptors contain five exons and share a similar exon-intron organization. To determine the structural basis of CCK-A receptor (CCK-AR) functionally coupled to Gs, a series of chimeric mutants were constructed by replacing exons of human CCK-B receptor (CCK-BR), from the second to the fifth (last) exon, with human CCK-AR counterparts. Binding and signal transduction properties of wild-type and chimeric receptors were examined in stably transfected HEK-293 cells. Chimeric receptors that maintained high affinity binding to CCK exhibited dose-dependent increases in intracellular calcium mobilization similar to both wild-type receptors. However, only the wild-type CCK-AR and chimeric mutants containing the second exon of CCK-AR were able to mediate significantly greater increases in intracellular cAMP content and adenylyl cyclase activity compared with wild-type CCK-BR. A CCK-BR mutant was further constructed by replacing five amino acids, Gly-Leu-Ser-Arg-(Arg)-Leu, in the first intracellular loop with the corresponding five CCK-AR specific amino acids, Ile-Arg-Asn-Lys-(Arg)-Met. The resultant receptor maintained high affinity binding to both CCK and gastrin and dose-dependent calcium responses similar to wild-type CCK-BR. However, this first intracellular loop mutant also gained positive cAMP responses to both sulfated CCK-8 and gastrin-17 with EC50 values of 8.5 +/- 1 nM and 23 +/- 7 nM, respectively. These data suggest that the first intracellular loop of CCK-AR is essential for coupling to Gs and activation of adenylyl cyclase signal transduction cascade.

Adenylyl Cyclases↗
PubMed1997

Altering the reaction coordinate of the ATP sulfurylase-GTPase reaction.

ATP sulfurylase, isolated from Escherichia coli K-12, catalyzes and couples two reactions: the hydrolysis of GTP and the synthesis of APS (adenosine 5'-phosphosulfate). Its GTPase activity is regulated in response to ligand binding at the APS-forming active site. In particular, AMP mimics an intermediate-like form of the enzyme that increases the k(cat) for GTP hydrolysis 180-fold. Using equilibrium and pre-steady-state methods, we have determined the relative Gibbs energies for many of the ground and transition states in the GTPase catalytic cycle, in the presence and absence of AMP. GTP and AMP energetically interact throughout the substrate branch of the reaction coordinate; however, once bond breaking occurs, communication between nucleotides ceases. Stopped-flow experiments, using the fluorescent nucleotides 2'-deoxy-mant-GTP and -GDP, indicate that the binding of AMP fosters a conformation of the enzyme that hinders the addition of 2'-deoxy-mant-GTP into the active site without affecting its escaping tendency. These results explain the effects of AMP on the equilibrium binding of the 2'-deoxy-mant-GTP. The second-order rate constants for the binding of 2'-deoxy-mant-GTP or -GDP, approximately 1 x 10(-6) M(-1) s(-1), are 2-3 orders of magnitude less than expected for simple diffusion models, and the binding progress curves appear biphasic. These findings suggest the presence of an intermediate(s) in the binding reactions. The Gibbs energy changes that occur in the reaction coordinate upon binding of AMP clearly show that the catalytic effect of AMP is due primarily to its -3.1 kcal/mol stabilization of the rate-limiting transition state.

Adenosine Monophosphate↗
PubMed1997

The transmembrane domain of a carboxyl-terminal anchored protein determines localization to the endoplasmic reticulum.

UBC6 is a C-terminal membrane-anchored (type IV) protein, native to Saccharomyces cerevisiae, where it is found in the endoplasmic reticulum. When expressed in mammalian cells, this novel ubiquitin-conjugating enzyme also localizes to the endoplasmic reticulum. UBC6 lacks a lumenal domain and contains no known endoplasmic reticulum retention signals. Analysis of chimeric proteins in which the cytosolic domain of UBC is linked to a heterologous transmembrane domain, or in which the UBC6 transmembrane domain is appended to an unrelated soluble protein, led to the determination that the transmembrane domain of UBC6 plays a dominant role in its compartmental localization. The basis for the transmembrane domain-mediated subcellular targeting of UBC6 was evaluated by lengthening the wild type UBC6 hydrophobic segment from 17 to 21 amino acids, which resulted in re-targeting to the Golgi complex. A further increase in length to 26 amino acids allowed this modified protein to traverse the secretory pathway and gain expression at the plasma membrane. These findings are consistent with models in which, in the absence of dominant cytosolic or lumenal targeting determinants, proteins may be sorted within the secretory pathway based on interactions between their transmembrane domains and the surrounding lipid bilayer.

Amino Acid Sequence↗
PubMed1997

GAL4 enhancer traps expressed in the embryo, larval brain, imaginal discs, and ovary of Drosophila.

We have screened a collection of approximately 400 GAL4 enhancer trap lines for useful patterns of expression in the embryo, larval brain, imaginal discs, and ovary using a UAS-lacZ reporter construct. Although similar patterns of expression have previously been reported in the original P[lacZ] enhancer trap screens, these lines are useful for directing ectopic expression of genes in discrete patterns during these stages. In addition, we have identified some unique patterns of expression that have not been previously reported.

Animals↗
PubMed1997

Apolipoprotein E-associated risk for Alzheimer's disease in the African-American population is genotype dependent.

As a part of our ongoing study on Alzheimer's disease (AD) in elderly African Americans, we obtained clinical assessment and apolipoprotein E (ApoE) genotype data on 288 individuals (including 60 with AD). The ApoE epsilon4 allele frequency was significantly increased in AD patients compared with controls. The age-adjusted odds ratio (OR) for AD in epsilon4 homozygotes was 4.83 (95% confidence interval [CI], 1.71-13.64) compared with the epsilon3/epsilon3 genotype, but the OR for AD with the epsilon3/epsilon4 genotype did not reach significance (1.20; 95% CI, 0.58-2.45). These findings suggest that the association between ApoE epsilon4 and AD is weaker in African Americans than in whites.

Aged↗
PubMed1997

Development of insulitis and diabetes in B cell-deficient NOD mice.

Insulin-dependent diabetes mellitus (IDDM) is believed to be an autoimmune disease that results from autoimmune destruction of the insulin-secreting beta-cells of the pancreas. In addition to a lymphocytic infiltration (insulitis) of the islets, patients with IDDM have autoantibodies directed against the components of the islet cells. Several beta-cell proteins have been identified as candidate autoantigens. The non-obese diabetic (NOD) mouse is a murine model for spontaneous IDDM. It is generally accepted that IDDM in patients and NOD mice results from the T lymphocyte-mediated destruction of beta-cells. However, the direct role of B lymphocytes in the disease process has not yet been clarified. To test directly the role of B cells in IDDM, we have generated B cell-deficient NOD mice by backcrossing the microMT-/- B cell 'knockout mice' onto the NOD background. The mice had no evidence of functional B cells as determined by flow cytometry and antibody production. We show that two out of seven of these mice developed insulitis and diabetes. These results suggest that despite an absence of B cells some NOD mice can still develop insulitis and diabetes.

Animals↗
PubMed1997

Rotational Analyses of the Laser Induced Fluorescence Excitation Spectra of Jet-Cooled CF3O and CF3S

The high resolution, rotationally resolved laser induced fluorescence spectra for the A2A1 left and right arrow X2E transitions of CF3O and CF3S were recorded. In addition to the origins, the symmetric vibrational band 301 and the nontotally symmetric bands 501 and 601 were analyzed for CF3O, while for CF3S the symmetric bands 101, 201, and 301, as well as the nontotally symmetric bands 601, 301501, and 301601, were analyzed. Copyright 1997 Academic Press. Copyright 1997Academic Press

Journal Article↗
PubMed1997

Is alpha1D-adrenoceptor protein detectable in rat tissues?

We have used the alpha1D-adrenoceptor selective antagonist, BMY 7378, the alpha1D-selective agonists, adrenaline and phenylephrine, the alpha1A-selective antagonists, (+)-niguldipine, SB 216469 and WB4101, and the non-subtype-selective alpha1-adrenoceptor antagonist, nemonapride, to investigate the presence of alpha1D-adrenoceptors in rat tissues at the protein level. Radioligand binding studies using [3H]prazosin as the radioligand were performed in three tissues containing alpha1D-adrenoceptor mRNA, spleen, cerebral cortex and kidney, and in comparison in one tissue not containing alpha1D-adrenoceptor mRNA, liver. Cerebral cortex and kidney were also studied upon alpha1B-adrenoceptor inactivation by chloroethylclonidine treatment (10 microM, 30 min, 37 degrees C). Experiments with cloned rat alpha1-adrenoceptor subtypes transiently expressed in COS cells confirmed the known selectivity of the investigated drugs for alpha1-adrenoceptor subtypes or the lack thereof of nemonapride. Accordingly nemonapride had steep and monophasic competition curves in all native and chloroethylclonidine-treated tissues. BMY 7378 also had steep and monophasic competition curves and low affinity in all native tissues. In contrast, adrenaline and phenylephrine (in the presence of 100 microM GTP) had monophasic competition curves of low affinity in liver and spleen but biphasic competition curves in cerebral cortex and kidney. Following chloroethylclonidine treatment competition curves for adrenaline, phenylephrine, (+)-niguldipine, SB 216469 and WB 4101 remained biphasic in cerebral cortex and kidney while those for nemonapride remained monophasic. We conclude that alpha1D-adrenoceptors are not readily detectable at the protein level in a variety of rat tissues where their mRNA is expressed. The biphasic competition curves of some agonists and antagonists in chloroethylclonidine-treated rat tissues do not represent alpha1D-adrenoceptors and are not readily explained by the present alpha1A/alpha1B/alpha1D-adrenoceptor classification.

Adrenergic alpha-Agonists↗
PubMed1997

Oxidation of bovine beta-casein by hypochlorite.

We recently observed two 2,4-dinitrophenylhydrazine (DNPH)-reactive proteins of 40 and 120 kDa in the bronchoalveolar lavage fluids of rats exposed to >95% O(2) for 48 h. The N-terminal sequences of these proteins were both identical over 16 amino acids with rat beta-casein, which, in addition to its more common association with milk, is produced by cytotoxic T-lymphocytes, and has been found to have proinflammatory properties. Because of the inflammatory response that accompanies hyperoxic lung injury, we investigated the oxidation of bovine beta-casein by HOCl. Following exposure to HOCl at 4 degrees C for 15 min, derivatization with DNPH, washing, and digestion with trypsin, the resultant peptides were separated by reverse-phase HPLC. One peptide isolated from a peak absorbing at 365 nm was identified as AVP(Y*)PQR, corresponding to amino acids 177-183 of bovine beta-casein. Analysis of the peptide by both electrospray and matrix assisted laser desorption ionization (MALDI) mass spectrometry identified a molecular ion MH+ of 1008.5 Da, which represents an increase of 178 Da from the calculated monoisotopic MH+ of the unmodified peptide of 830.45 Da. Daughter ion spectra of the doubly charged parent ion of the peptide further support the oxidation of the tyrosine to the quinone methide, with subsequent conversion to the corresponding hydrazone with DNPH. A second pair of products were identified as arising from oxidation of Y(193) within the tryptic peptide constituted by amino acids 184-202, and the corresponding chymotryptic cleavage side product, 191-202. Exposure of beta-casein to increasing amounts of HOCl revealed that M and Y residues were the most susceptible, although bovine beta-casein contains no C, and a single W, which would not be detected by our methods. The approach described in the present report can be used to evaluate the contributions of distinct mechanisms of oxidation in other experimental or pathological models.

Amino Acid Sequence↗
PubMed1997

Oxidative modifications of apoB-100 by exposure of low density lipoproteins to HOCL in vitro.

Although the products of oxidation of the lipid components of LDL have been studied extensively, much less is known about the specific products of oxidative modification of the apoprotein. We reacted native LDL and LDL that had been treated with HOCl with 2,4-dinitrophenylhydrazine (DNPH), delipidated and trypsinized the protein, and analyzed the products by HPLC. Although tryptic digests of native LDL and LDL oxidized by limited quantities of HOCl showed similar patterns by HPLC with detection at 220 nm, oxidized LDL showed several discrete peaks at 365 nm, which is characteristic of hydrazones formed with aldehydes and ketones, commonly termed protein carbonyls. Native LDl showed no peaks in the chromatograms at 365 nm. Peptides absorbing at 365 nm were isolated by HPLC and characterized. In most cases, the probable sites of modification on the peptides could be implied by failure of an anticipated amino acid to appear in the expected sequence. Of the 14 peptides isolated and characterized to date, eight peptides contained Cys residues. In other peptides, Lys, Trp, and Met were identified as amino acid residues apparently modified by HOCl treatment of LDL. Thirteen of the peptides identified are from trypsin-releasable peptides located on the surface of unoxidized native LDL. Our studies suggest a selective process of modification of apoB-100 by HOCl and the approaches used in the present studies should be useful for the characterization of the mechanisms of oxidation of this and other proteins.

Amino Acid Sequence↗
PubMed1997

Detection of micrometastatic prostate cancer cells in the bone marrow of patients with prostate cancer.

Thirty-five patients with prostate cancer were examined for micrometastases to the bone marrow using reverse transcription-polymerase chain reaction (RT-PCR) with primers specific for the prostate-specific antigen (PSA) gene. Of nine patients with bone metastases detectable by bone scan imaging, five patients had PSA mRNA expression in the bone marrow detectable by RT-PCR. Of 26 patients with negative bone scan findings, seven patients had PSA mRNA expression detectable in the bone marrow. RT-PCR could detect micrometastatic prostate cancer cells in the bone marrow that were not detectable by bone scan imaging. Of 16 patients with a serum PSA concentration of 25 ng ml(-1) or greater, only nine (56.3%) had bone metastases detected by bone scans. Of the remaining seven patients, five had micrometastases to the bone marrow detected by RT-PCR. Overall, 14 of 16 patients (87.5%) with a serum PSA concentration of 25 ng ml(-1) or greater had metastatic bone diseases including bone marrow micrometastases. Of 19 patients with a serum PSA concentration of less than 25 ng ml(-1), two (10.5%) had only micrometastatic disease detected by RT-PCR. A significant correlation was observed between the incidence of bone involvement and the serum PSA concentration. This study suggests that RT-PCR will potentially develop into a relevant tool to assess bone involvement including bone marrow micrometastases and establish a precise correlation between serum PSA concentration and metastatic bone disease in patients with prostate cancer.

Bone Marrow↗
PubMed1997

Vaccination with a multi-epitopic recombinant allergen induces specific immune deviation via T-cell anergy.

Prophylactic vaccination has recently emerged as a major paradigm toward the prevention and therapy of allergies and asthma; however, the immunological basis of this approach remains to be elucidated. We examined the potential and mechanism of prophylaxis of allergic response in B6D2F1 mice with a multi-epitopic recombinant allergen, rKBG8.3 (MERA-8.3), which represents a major group of allergens of grass pollens, used herein as a model of MERA vaccine. Vaccination (subcutaneous) with soluble MERA-8.3, prior to immunization with the MERA-8.3 in alum, led to suppression of the IgE antibody response and a concomitant increase in IgG2a antibody response specific to the MERA-8.3 in a dose-dependent manner. Analysis of cytokine patterns in spleen and lymph node cells revealed a marked decrease of interleukin-2 (IL-2) and IL-4 production and to a lesser extent a decrease of interferon-gamma (IFN-gamma) synthesis, resulting in an increased ratio of IFN-gamma: IL-4 in vaccinated-immunized mice compared with untreated-immunized control mice. Furthermore, splenocytes of mice treated with the MERA-8.3 alone proliferated to MERA-8.3 in vitro with reduced capacity compared with the splenocytes of MERA-8.3-alum immunized mice, owing to a markedly reduced level of IL-2 production in the former. Collectively, these results suggest that vaccination with the MERA-8.3 induces T-cell anergy, which is pivotal to deviation of specific immunity from Th2- to Th1-like, and may serve as an important approach to prevention and therapy of allergic disorders.

Allergens↗