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Y Yang

Publications and source records attributed to Y Yang.

At least 1,189 records · Page 66Linked to original sources
PubMed1994

Secondary structure of neutrophil-activating peptide-2 determined by 1H-nuclear magnetic resonance spectroscopy.

Neutrophil-activating protein-2 (NAP-2) is a 72 residue protein demonstrating a range of proinflammatory activities. The solution structure of monomeric NAP-2 has been investigated by two-dimensional 1H-n.m.r. spectroscopy. Sequence-specific proton resonance assignments have been made and secondary structural elements have been identified on the basis of nuclear Overhauser data, coupling constants and amide hydrogen/deuteron exchange. The NAP-2 monomer consists of a triple-stranded anti-parallel beta-sheet arranged in a 'Greek key' and a C-terminal helix (residues 59-70) and is very similar to that found in the n.m.r. solution conformation of dimeric interleukin-8 and the crystal structure of tetrameric bovine platelet factor-4. Results are discussed in terms of heparin binding and neutrophil-activation properties of NAP-2.

Amino Acid Sequence↗
PubMed1994

Insulin-induced activation of glycerol-3-phosphate acyltransferase by a chiro-inositol-containing insulin mediator is defective in adipocytes of insulin-resistant, type II diabetic, Goto-Kakizaki rats.

Type II diabetic Goto-Kakizaki (GK) rats were insulin-resistant in euglycemic-hyperinsulinemic clamp studies. We therefore examined insulin signaling systems in control Wistar and diabetic GK rats. Glycerol-3-phosphate acyltransferase (G3PAT), which is activated by headgroup mediators released from glycosyl-phosphatidylinositol (GPI), was activated by insulin in intact and cell-free adipocyte preparations of control, but not diabetic, rats. A specific chiro-inositol-containing inositol phosphoglycan (IPG) mediator, prepared from beef liver, bypassed this defect and comparably activated G3PAT in cell-free adipocyte preparations of both diabetic GK and control rats. A myo-inositol-containing IPG mediator did not activate G3PAT. Relative to control adipocytes, labeling of GPI by [3H]glucosamine was diminished by 50% and insulin failed to stimulate GPI hydrolysis in GK adipocytes. In contrast to GPI-dependent G3PAT activation, insulin-stimulated hexose transport was intact in adipocytes and soleus and gastrocnemius muscles of the GK rat, as was insulin-induced activation of mitogen-activated protein kinase and protein kinase C. We conclude that (i) chiro-inositol-containing IPG mediator activates G3PAT during insulin action, (ii) diabetic GK rats have a defect in synthesizing or releasing functional chiro-inositol-containing IPG, and (iii) defective IPG-regulated intracellular glucose metabolism contributes importantly to insulin resistance in diabetic GK rats.

Adipocytes↗
PubMed1994

Broad tuning for spatial frequency of neural mechanisms underlying visual perception of coherent motion.

Neural events underlying perception of coherent motion are generally believed to be hierarchical: information about local motion is registered by spatio-temporal coincidence detectors whose outputs are cooperatively integrated at a subsequent stage. There is disagreement, however, concerning the spatial scale of the neural filters underlying these operations. According to one class of models, motion registration is initially accomplished in parallel at multiple spatial scales, with filters tuned to lower spatial frequencies responsive to larger motion displacements than filters tuned to higher frequencies. According to another scheme, motion analysis involves a single, broadly tuned spatial filter, with optimal displacement dependent on spacing of local elements. Here we use a masking procedure to measure the extent to which dynamic noise depicted at one spatial scale interferes with detection of coherent motion conveyed by image features at another spatial scale. Our results indicate that a single filter, broadly tuned for spatial frequency, is mediating detection of coherent motion. This finding dovetails with known physiological properties of neurons at an intermediate stage of motion processing.

Fourier Analysis↗
PubMed1994

Transgenic mice carrying the erythropoietin gene promoter linked to lacZ express the reporter in proximal convoluted tubule cells after hypoxia.

Results on the localization of erythropoietin (Epo) synthesis in renal cells have been contradictory, implicating either interstitial or tubular cells. We fused the lacZ gene to a 7-kb DNA fragment of the mouse Epo gene encompassing a portion of the first intron, the first exon, and a 6-kb sequence of the 5'-flanking region. Transgenic mice carrying this construct show a low level of specific expression of the lacZ gene in proximal convoluted tubule (PCT) cells. Without hypoxia, no significant expression was detected in the liver. Hypoxia induced a large degree of lacZ expression, mainly in kidney PCT cells and to a lesser degree in liver. However, anemia induced lacZ expression in both kidney and liver. These findings indicate that, under these conditions, Epo is expressed in tubular cells, specifically PCT cells.

Anemia↗
PubMed1994

Nucleotide binding of the C-terminal domains of the major histocompatibility complex-encoded transporter expressed in Drosophila melanogaster cells.

The C-terminal domains of the mouse transporter associated with antigen processing (TAP) were expressed as soluble proteins in Drosophila melanogaster cells and labeled by [alpha-32P]8-azido-ATP after UV-irradiation. The relative potencies of the nucleotides in preventing azido-ATP labeling were in the order of ATP > GTP > CTP > ITP > UTP for both the TAP1 and TAP2 C-terminal domains, suggesting ATP to be the natural substrate of the transporter. Our data provide the first evidence that the individual C-terminal domain of either TAP1 or TAP2 can be expressed as a functional ATP-binding protein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Subunit association and structural analysis of platelet basic protein and related proteins investigated by 1H NMR spectroscopy and circular dichroism.

Platelet basic protein (PBP) (94 residues) is naturally processed via N-terminal cleavage to yield connective tissue activating peptide-III (85 residues), beta-thromboglobulin (81 residues), and neutrophil activating peptide-2 (70 residues). Chemical cross-linking and gel filtration data indicate that each homolog can form dimers and tetramers. Subunit association equilibria for dimer (KD) and tetramer (KT) formation have been derived for each species from 1H NMR (600 MHz) spectral analysis of slowly exchanging (NMR time scale) monomer- dimer-tetramer aggregation state populations. In general, raising the pH from about pH 3.5 to pH 6 increases KD by two to three orders in magnitude and decreases KT by some 50-fold. Ionic strength effects also suggest that intersubunit electrostatic interactions are critical to subunit association. Subunit stabilization can be ranked proportional to N-terminal chain length: platelet basic protein > connective tissue activating peptide-III > beta-thromboglobulin > neutrophil activating peptide-2. Under more physiologic conditions, PBP family monomers are favored at normal cytokine protein concentrations and may form the biologically active state. CD and NMR data indicate conservation of alpha-helix and anti-parallel beta-sheet structure among PBP-related species and support the idea that the extended N terminus folds over and masks the neutrophil activation domain and is part of the intersubunit binding domain.

Amino Acid Sequence↗
PubMed1994

Dosage compensation in Drosophila: the X-chromosomal binding of MSL-1 and MLE is dependent on Sxl activity.

In Drosophila, dosage compensation, i.e. the equalization of levels of X-linked gene products in the two sexes, is achieved by the hypertranscription of most X-linked genes in males relative to females. The products of at least four genes, collectively termed male-specific lethal (msl) genes, are required for this process and, at least in the case of three of them, mediate this function through an association with the X chromosome in males. We have studied some of the parameters that affect the association of the msl-1 gene product and found that its presence is dependent on the wild-type function of the other three genes, leading to the conclusion that these gene products contribute to the formation of a multi-subunit complex. Furthermore, the X-chromosomal association of the msl-1 and mle gene products is negatively correlated with the level of function of the master regulatory gene Sxl and can assume either a mosaic or a uniform distribution in the tissues of mutant XX individuals. Surprisingly, we also found that the association of these two msl gene products with the two X chromosomes in females of certain mutant genotypes does not result in the hypertranscription of X-linked genes or in any apparent reduction in viability.

Animals↗
PubMed1994

Displacement of housekeeping proteasome subunits by MHC-encoded LMPs: a newly discovered mechanism for modulating the multicatalytic proteinase complex.

The degradation of cytoplasmic antigens to peptides presented by class I MHC molecules is thought to be mediated by the ubiquitin/proteasome pathway. Support for this view came from our observation that the subunit composition of proteasomes can be changed by interferon-gamma (IFN-gamma) treatment. Thereby two subunits, LMP2 and LMP7, which are encoded in the MHC class II region, are incorporated into the proteasomal complex, whereas other subunits disappear. In the experiments reported in this communication we studied the subunit changes occurring in cell lines where the expression of LMP2 or LMP7 can be regulated individually either by IFN-gamma induction or by applying a new system to control the expression of transfected LMPs. In both situations LMP2 induction leads exclusively to the disappearance of housekeeping subunit 2, whereas LMP7 affects only subunit 10. Subunit 2 was found to be 76% homologous to LMP2. Since incorporation of LMP2 into the proteasomal complex prevents processing of the subunit 2 precursor, we conclude that LMP2 displaces subunit 2 during assembly. Subunit displacement is most likely a general mechanism to modulate the catalytic activity of the proteasomal complex without changing its structure. Furthermore, the controlled incorporation of transfected subunits into the complex offers a new approach to study proteasome function in vivo.

Amino Acid Sequence↗
PubMed1994

Allele-specific expression of a variant-specific surface protein (VSP) of Giardia lamblia.

The surfaces of Giardia lamblia trophozoites demonstrate variable expression of a set of cysteine-rich surface proteins, called variant-specific surface proteins (VSP). The cloned Giardia line, WBA6, expresses a 170 kD VSP (VSPA6 or CRP170) which contains approximately 18 to 23 copies of a 65 amino acid repeat. We have cloned the expressed vspA6 gene containing 23 repeats from a genomic library as well as copies of the vspA6 gene with only 8 or 9 repeats from both WBA6 and from WB1269, a cloned line derived from WBA6 which has lost the expressed copy of the gene. The recombinant clones containing the genes with only 8 or 9 repeats have 8 nucleotide substitutions in the coding region. All the recombinant clones map to the same chromosomal location, yet RNA sequencing and comparison with the transcript size indicate that only the clone with 23 repeats contains a gene producing a stable transcript. The most likely interpretation of these data is that G.lamblia trophozoites contain multiple alleles of the vspA6 gene of which only one is expressed.

Alleles↗
PubMed1994

Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy.

An important limitation that has emerged in the use of adenoviruses for gene therapy has been loss of recombinant gene expression that occurs concurrent with the development of pathology in the organ expressing the transgene. We have used liver-directed approaches to gene therapy in mice to study mechanisms that underlie the problems with transient expression and pathology that have characterized in vivo applications of first-generation recombinant adenoviruses (i.e., those deleted of E1a and E1b). Our data are consistent with the following hypothesis. Cells harboring the recombinant viral genome express the transgene as desired; however, low-level expression of viral genes also occurs. A virus-specific cellular immune response is stimulated that leads to destruction of the genetically modified hepatocytes, massive hepatitis, and repopulation of the liver with nontransgene-containing hepatocytes. These findings suggest approaches for improving recombinant adenoviruses that are based on further crippling the virus to limit expression of nondeleted viral genes.

Adenovirus E1A Proteins↗
PubMed1994

In vivo regulation of the assembly and intracellular transport of class I major histocompatibility complex molecules.

Using H-2Kb-transfected Balb/c 3T3 cells which generate "empty" H-2Kb molecules devoid of antigenic peptides, we show that peptide availability determines the stability of class I molecules and dictates the overall intracellular transport rate of the class I complexes. Our data also indicate that chaperonin-like proteins are involved in class I assembly. Using Drosophila cells transfected with H-2Kb and murine beta 2-microglobulin, we show that one possible candidate, calnexin, associates with class I molecules prior to peptide acquisition. These data suggest that both peptide supply and assembly proteins dictate cell surface expression of class I major histocompatibility complex molecules and ultimately influence T cell recognition. The role of beta 2-microglobulin in class I assembly is also discussed.

3T3 Cells↗
PubMed1994

Peptide length and sequence specificity of the mouse TAP1/TAP2 translocator.

The transporter associated with antigen processing (TAP) delivers peptides to the lumen of the endoplasmic reticulum in an adenosine triphosphate (ATP) dependent fashion for presentation by major histocompatibility complex class I molecules. We show that the mouse TAP translocator (H-2b haplotype) selects peptides based on a minimal size of nine residues, and on the presence of a hydrophobic COOH-terminal amino acid. The preponderance of COOH-terminal hydrophobic amino acids in peptides capable of binding to mouse class I molecules thus fits remarkably well with the specificity of the TAP translocator. In addition to transport in the lumenal direction, efflux of peptide in the cytosolic direction is observed in an ATP- and temperature-dependent manner. By maintaining a low peptide concentration at the site of class I assembly, this efflux mechanism may ensure that class I molecules are loaded preferentially with high affinity peptides.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Human osteosarcoma (U-2 OS) cells express both insulin-like growth factor-I (IGF-I) receptors and insulin-like growth factor-II/mannose-6-phosphate (IGF-II/M6P) receptors and synthesize IGF-II: autocrine growth stimulation by IGF-II via the IGF-I receptor.

UNLABELLED: Recently, insulin-like growth factor-I and -II (IGF-I and -II) have been implicated in the growth promotion of tumors in vivo and tumor cells in vitro. We have studied the human osteosarcoma cell line U-2 OS in order 1) to gain more insight into the growth promoting actions of the IGFs and 2) to establish an in vitro tissue culture model of IGF action in human tumor cells. Specific binding of 125I-IGF-I and 125I-IGF-II to IGF-I receptors and IGF-II/mannose-6-phosphate (M6P) receptors on U-2 OS cells was demonstrated in competitive binding experiments and in affinity crosslinking experiments. Western blotting of cell extracts confirmed the expression of the IGF-II/M6P receptor. In addition, in Northern blotting experiments using total RNA from U-2 OS cells IGF-I receptor RNA of 11 kb and IGF-II/M6P receptor RNA of approximately 9 kb were detected. Solution hybridization experiments confirmed the presence of IGF-I receptor and IGF-II/M6P receptor RNA. In a subset of experiments DNA synthesis was measured as 3H-thymidine uptake into cellular DNA of U-2 OS cells. Normal rat serum stimulated DNA synthesis maximally. IGF-I-deficient serum from hypophysectomized rats as well as IGF-I or IGF-II without serum were approximately twofold and tenfold, respectively, less potent than serum in stimulating 3H-thymidine uptake. The concentrations of IGF-I and IGF-II needed for half maximal stimulation of DNA synthesis corresponded well with the respective concentrations required for half maximal inhibition of 125I-IGF-I binding to U-2 OS cells. The anti-IGF-I receptor antibody alpha IR3 blocked the IGF-I and IGF-II stimulated increase of 3H-thymidine uptake. In addition, basal DNA synthesis was partially inhibited by the anti-IGF-I receptor antibody. These data suggest that U-2 OS cells synthesize and secrete IGF-like peptides. Northern blotting experiments confirmed that U-2 OS cells express an IGF-II RNA species of 5.3 kb but no IGF-I transcripts. In a series of RNase protection assays, protected RNA fragments were detected with an IGF-II riboprobe. Also, cell-conditioned medium from U-2 OS cells contained 1-2 ng/ml IGF-II immunoreactivity as measured in an IGF-binding protein blocked IGF-II radioimmunoassay. IN CONCLUSION: 1) U-2 OS cells express IGF-I and IGF-II/M6P receptors. 2) U-2 OS tumor cells respond to the addition of exogenous IGF-I and IGF-II with an increase of DNA synthesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Two-step selective formation of three disulfide bridges in the synthesis of the C-terminal epidermal growth factor-like domain in human blood coagulation factor IX.

The 45-residue C-terminal EGF-like domain in human blood coagulation factor IX has been synthesized by a 2-step method to form selectively 3 disulfide bridges. Four out of 6 cysteines are blocked with either trityl or 4-methyl-benzyl, and the remaining 2 cysteines are blocked with acetamidomethyl (Acm). In the first step, 4 free cysteinyl thiols are released concurrently with the removal of all protecting groups except Acm and are oxidized to form 1 of the 3 possible isomers containing 2 pairs of disulfides. In the second step, iodine is used to remove the Acm groups to yield the third disulfide bridge. This approach reduces the number of possible disulfide bridging patterns from 15 to 3. To determine the optimal protecting group strategy, 3 peptides are synthesized, each with Acm blocking 1 of the 3 pairs of cysteines involved in disulfide bridges: Cys5 to Cys16 (Cys 1-3), Cys12 to Cys26 (Cys 2-4), or Cys28 to Cys41 (Cys 5-6). Only the peptide having the Cys 2-4 pair blocked with Acm forms the desired disulfide isomer (Cys 1-3/5-6) in high yield after the first step folding, as identified by proteolytic digestion in conjunction with mass spectrometric peptide mapping. Thus, the choice of which pair of cysteines to block with Acm is critically important. In the case of EGF-like peptides, it is better to place the Acm blocking groups on one of the pairs of cysteines involved in the crossing of disulfide bonds.

Amino Acid Sequence↗