Biomedical subjects

L Yu

Publications and source records attributed to L Yu.

At least 145 records · Page 8Linked to original sources

PubMed2000

Human glial cell line-derived neurotrophic factor receptor alpha 4 is the receptor for persephin and is predominantly expressed in normal and malignant thyroid medullary cells.

Glial cell line-derived neurotrophic factor (GDNF) family ligands signal through receptor complex consisting of a glycosylphosphatidylinositol-linked GDNF family receptor (GFR) alpha subunit and the transmembrane receptor tyrosine kinase RET. The inherited cancer syndrome multiple endocrine neoplasia type 2 (MEN2), associated with different mutations in RET, is characterized by medullary thyroid carcinoma. GDNF signals via GFRalpha1, neurturin via GFRalpha2, artemin via GFRalpha3, whereas the mammalian GFRalpha receptor for persephin (PSPN) is unknown. Here we characterize the human GFRalpha4 as the ligand-binding subunit required together with RET for PSPN signaling. Human and mouse GFRalpha4 lack the first Cys-rich domain characteristic of other GFRalpha receptors. Unlabeled PSPN displaces (125)I-PSPN from GFRA4-transfected cells, which express endogenous Ret. PSPN can be specifically cross-linked to mammalian GFRalpha4 and Ret, and is able to promote autophosphorylation of Ret in GFRA4-transfected cells. PSPN, but not other GDNF family ligands, promotes the survival of cultured sympathetic neurons microinjected with GFRA4. We identified different splice forms of human GFRA4 mRNA encoding for two glycosylphosphatidylinositol-linked and one putative soluble isoform that were predominantly expressed in the thyroid gland. Overlapping expression of RET and GFRA4 but not other GFRA mRNAs in normal and malignant thyroid medullary cells suggests that GFRalpha4 may restrict the MEN2 syndrome to these cells.

Animals↗

PubMed2000

Confirmation of the involvement of protein domain movement during the catalytic cycle of the cytochrome bc1 complex by the formation of an intersubunit disulfide bond between cytochrome b and the iron-sulfur protein.

To study the essentiality of head domain movement of the Rieske iron-sulfur protein (ISP) during bc(1) catalysis, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc(1) complexes with three pairs of cysteines engineered (one cysteine each) on the interface between cytochrome b and ISP, A185C(cytb)/K70C(ISP), I326C(cytb)/G165C(ISP), and T386C(cytb)/K164C(ISP), were generated and characterized. Formation of an intersubunit disulfide bond between cytochrome b and ISP is detected in membrane (intracytoplasmic membrane and air-aged chromatophore), and purified bc(1) complex was prepared from the A185C(cytb)/K70C(ISP) mutant cells. Formation of the intersubunit disulfide bond in this cysteine pair mutant complex is concurrent with the loss of its bc(1) activity. Reduction of this disulfide bond by beta-mercaptoethanol restores activity, indicating that mobility of the head domain of ISP is functionally important in the cytochrome bc(1) complex. The rate of intramolecular electron transfer, between 2Fe2S and heme c(1), in the A185C(cytb)/K70C(ISP) mutant complex is much lower than that in the wild type or in their respective single cysteine mutant complexes, indicating that formation of an intersubunit disulfide bond between cytochrome b and ISP arrests the head domain of ISP in the "fixed state" position, which is too far for electron transfer to heme c(1).

Amino Acid Substitution↗

PubMed2000

Molecular cloning, genomic organization, and mapping of PRKAG2, a heart abundant gamma2 subunit of 5'-AMP-activated protein kinase, to human chromosome 7q36.

5'-AMP-activated protein kinase (AMPK) acts as a major regulator of cellular ATP levels and protects cells against stresses that cause ATP depletion. AMPK is a protein heterotrimer composed of a catalytic alpha subunit and two regulatory subunits, beta and gamma. In the present study, a homologue of the AMPK gamma1-subunit cDNA with an open reading frame encoding 328 amino acids was identified. The putative protein sequence is about 76% identical to the 331-amino-acid gamma1 subunit and also has four consecutive cystathionine-beta-synthase (CBS) domains, a characteristic structure of AMPK gamma subunits from various species. This cDNA (tentatively termed PRKAG2-b) is identical to a recently reported cDNA (tentatively termed PRKAG2-a) of human AMPK gamma subunits except in their 5'-end regions, suggesting that these two cDNAs are two different transcripts of the same gene. To determine the expression pattern of the gene, two probes, one from the 3'-UTR of PRKAG2-b and the other from the 5'- unique region of PRKAG2-a, were used to hybridize MTN membranes. Three transcripts (3.8, 3.0, and 2.4 kb) were observed when the first probe was used, whereas only 3.8- and 3.0-kb transcripts were seen when the second probe was used. Thus, the PRKAG2-b corresponded to the 2.4-kb transcript, which is ubiquitously expressed except in liver and thymus. The highest level was detected in heart, while abundant expression also existed in placenta and testis. The expression pattern of PRKAG2-b is completely different from those of PRKAG2-a and PRKAG1, whose expression patterns were also determined in the current study. The PRKAG2 gene was located to human chromosome 7q36 between markers D7S2439 and D7S2462 by radiation hybrid mapping. The genomic organization of PRKAG2-b was identified by comparing its cDNA sequence with two genomic sequences AC006358 and AC006966, which showed that PRKAG2-b spanned an approximately 80-kb region and was composed of 12 exons.

AMP-Activated Protein Kinases↗

PubMed2000

Cloning and characterization of human VPS35 and mouse Vps35 and mapping of VPS35 to human chromosome 16q13-q21.

Maintenance of different organelles in eukaryotic cells depends on sorting proteins, which ensure the proper delivery of organelle-specific proteins. The studies on yeast (Saccharomyces cerevisiae) VPS35, a hydrophilic membrane protein having a direct role in the retrieval of cargo proteins, suggest a mechanism underlying a possible lysosomal protein-sorting pathway in mammalian cells. Here, we report the isolation of human and mouse VPS35 cDNAs, which are 3208 and 3186 bp in length, respectively. The deduced proteins of the two cDNAs, which are both composed of 796 amino acids and share 99% identity, show homology to yeast VPS35 and other VPS35 homologues of various sources ranging from Schizosaccharomyces pombe to Drosophila melanogaster (31-56% identity and 49-71% similarity), especially in their amino- and carboxyl-termini. The conservation of VPS35 suggests that the function of this class of protein is important. The results of Northern hybridization of human VPS35 in 16 tissues showed that one transcript of 3.6 kb was highly expressed in brain, heart, testis, ovary, small intestine, spleen, skeletal muscle, and placenta and expressed at moderate or low levels in other tissues. Another transcript of 3.0 kb was also expressed with proportionally lower levels than the 3.6-kb transcript in all the tissues except that the 3.0-kb transcript was not detected in brain. Mouse Vps35 was widely expressed as a 3.4-kb transcript. In addition, human VPS35 was assigned to human chromosome 16q13-q21 by radiation hybrid mapping.

Amino Acid Sequence↗

PubMed2000

Accumulation of dietary cholesterol in sitosterolemia caused by mutations in adjacent ABC transporters.

In healthy individuals, acute changes in cholesterol intake produce modest changes in plasma cholesterol levels. A striking exception occurs in sitosterolemia, an autosomal recessive disorder characterized by increased intestinal absorption and decreased biliary excretion of dietary sterols, hypercholesterolemia, and premature coronary atherosclerosis. We identified seven different mutations in two adjacent, oppositely oriented genes that encode new members of the adenosine triphosphate (ATP)-binding cassette (ABC) transporter family (six mutations in ABCG8 and one in ABCG5) in nine patients with sitosterolemia. The two genes are expressed at highest levels in liver and intestine and, in mice, cholesterol feeding up-regulates expressions of both genes. These data suggest that ABCG5 and ABCG8 normally cooperate to limit intestinal absorption and to promote biliary excretion of sterols, and that mutated forms of these transporters predispose to sterol accumulation and atherosclerosis.

ATP Binding Cassette Transporter, Subfamily G, Mem↗

PubMed2000

Effects of galanin on wide-dynamic range neuron activity in the spinal dorsal horn of rats with sciatic nerve ligation.

Galanin is a 29-amino acid peptide with a suggested role in nociception. The effect of galanin on wide-dynamic range neuron discharge frequency in rats with nerve ligation, used as a model of neurogenic pain, was investigated by extracellular recording methods. Seven to 14 days after sciatic nerve ligation, 0.1, 0.5 or 1 nmol of galanin was administered directly on the dorsal surface of the L3-L5 spinal cord of rats with sciatic nerve ligation. It was found that galanin inhibited the activity of wide-dynamic range neurons dose-dependently, an effect was more pronounced in sciatic nerve ligated rats than intact rats. Furthermore, when 1 nmol of galantide, the galanin antagonist, was administered on the dorsal surface of the L3-L5 spinal cord, the wide-dynamic range neuron discharge frequency increased significantly. The results suggest that galanin plays an important role in the modulation of presumed nociception in mononeuropathy.

Animals↗

PubMed2000

Involvement of neuropeptide Y and Y1 receptor in antinociception in nucleus raphe magnus of rats.

The nociceptive response latencies increased significantly after intra-nucleus raphe magnus administration of 0.1 or 0.4 nmol of neuropeptide Y, but not 0.04 nmol, in rats. The neuropeptide Y-induced increases in hindpaw withdrawal latency were reversed by following injection of 0.42 nmol of the Y1 antagonist, NPY(28-36). The results indicate that NPY plays an antinociceptive role in nucleus raphe magnus in rats, which is mediated by the Y1 receptor. Furthermore, the neuropeptide Y-induced increases in hindpaw withdrawal latency were attenuated by following intra-nucleus raphe magnus injection of 6 nmol of the opioid antagonist naloxone, indicating that there is an interaction between NPY and opioids in nucleus raphe magnus.

Animals↗

PubMed2000

Reconstitution of mitochondrial processing peptidase from the core proteins (subunits I and II) of bovine heart mitochondrial cytochrome bc(1) complex.

Mature core I and core II proteins of the bovine heart mitochondrial cytochrome bc(1) complex were individually overexpressed in Escherichia coli as soluble proteins using the expression vector pET-I and pET-II, respectively. Purified recombinant core I and core II alone show no mitochondrial processing peptidase (MPP) activity. When these two proteins are mixed together, MPP activity is observed. Maximum activity is obtained when the molar ratio of these two core proteins reaches 1. This indicates that only the two core subunits of thebc(1) complex are needed for MPP activity. The properties of reconstituted MPP are similar to those of Triton X-100-activated MPP in the bovine bc(1) complex. When Rieske iron-sulfur protein precursor is used as substrate for reconstituted MPP, the processing activity stops when the amount of product formation (subunit IX) equals the amount of reconstituted MPP used in the system. Addition of Triton X-100 to the product-inhibited reaction mixture restores MPP activity, indicating that Triton X-100 dissociates bound subunit IX from the active site of reconstituted MPP. The aromatic group, rather than the hydroxyl group, at Tyr(57) of core I is essential for reconstitutive activity.

Amino Acid Sequence↗

PubMed2000

Crystal structure of human cathepsin V.

Cathepsin V is a lysosomal cysteine protease that is expressed in the thymus, testis and corneal epithelium. We have determined the 1.6 A resolution crystal structure of human cathepsin V associated with an irreversible vinyl sulfone inhibitor. The fold of this enzyme is similar to the fold adopted by other members of the papain superfamily of cysteine proteases. This study provides a framework for understanding the structural basis for cathepsin V's activity and will aid in the design of inhibitors of this enzyme. A comparison of cathepsin V's active site with the active sites of related proteases revealed a number of differences, especially in the S2 and S3 subsites, that could be exploited in identifying specific cathepsin V inhibitors or in identifying inhibitors of other cysteine proteases that would be selective against cathepsin V.

Binding Sites↗

PubMed2000

Effect of oral CDP-choline on plasma choline and uridine levels in humans.

Twelve mildly hypertensive but otherwise normal fasting subjects received each of four treatments in random order: CDP-choline (citicoline; 500, 2000, and 4000 mg) or a placebo orally at 8:00 a.m. on four different treatment days. Eleven plasma samples from each subject, obtained just prior to treatment (8:00 a.m.) and 1-12 hr thereafter, were assayed for choline, cytidine, and uridine. Fasting terminated at noon with consumption of a light lunch that contained about 100 mg choline. Plasma choline exhibited dose-related increases in peak values and areas under the curves (AUCs), remaining significantly elevated, after each of the three doses, for 5, 8, and 10 hr, respectively. Plasma uridine was elevated significantly for 5-6 hr after all three doses, increasing by as much as 70-90% after the 500 mg dose, and by 100-120% after the 2000 mg dose. No further increase was noted when the dose was raised from 2000 to 4000 mg. Plasma cytidine was not reliably detectable, since it was less than twice blank, or less than 100 nM, at all of the doses. Uridine is known to enter the brain and to be converted to UTP; moreover, we found that uridine was converted directly to CTP in neuron-derived PC-12 cells. Hence, it seems likely that the circulating substrates through which oral citicoline increases membrane phosphatide synthesis in the brains of humans involve uridine and choline, and not cytidine and choline as in rats.

Administration, Oral↗

PubMed2000

A self-assembled light-harvesting array of seven porphyrins in a wheel and spoke architecture.

[reaction: see text]A shape-persistent cyclic array of six zinc porphyrins provides an effective host for a dipyridyl-substituted free base porphyrin, yielding a self-assembled structure for studies of light harvesting. Energy transfer occurs essentially quantitatively from uncoordinated to pyridyl-coordinated zinc porphyrins in the cyclic array. Energy transfer from the coordinated zinc porphyrin to the guest free base porphyrin is less efficient (phitrans approximately 40%) and is attributed to a Förster through-space process.

Chemical Phenomena↗

PubMed2000

Protein fold recognition by total alignment probability.

We present a protein fold-recognition method that uses a comprehensive statistical interpretation of structural Hidden Markov Models (HMMs). The structure/fold recognition is done by summing the probabilities of all sequence-to-structure alignments. The optimal alignment can be defined as the most probable, but suboptimal alignments may have comparable probabilities. These suboptimal alignments can be interpreted as optimal alignments to the "other" structures from the ensemble or optimal alignments under minor fluctuations in the scoring function. Summing probabilities for all alignments gives a complete estimate of sequence-model compatibility. In the case of HMMs that produce a sequence, this reflects the fact that due to our indifference to exactly how the HMM produced the sequence, we should sum over all possibilities. We have built a set of structural HMMs for 188 protein structures and have compared two methods for identifying the structure compatible with a sequence: by the optimal alignment probability and by the total probability. Fold recognition by total probability was 40% more accurate than fold recognition by the optimal alignment probability. Proteins 2000;40:451-462.

Algorithms↗

PubMed2000

Human galectin-3 is a novel chemoattractant for monocytes and macrophages.

Galectin-3 is a beta-galactoside-binding protein implicated in diverse biological processes. We found that galectin-3 induced human monocyte migration in vitro in a dose-dependent manner, and it was chemotactic at high concentrations (1.0 microM) but chemokinetic at low concentrations (10-100 nM). Galectin-3-induced monocyte migration was inhibited by its specific mAb and was blocked by lactose and a C-terminal domain fragment of the protein, indicating that both the N-terminal and C-terminal domains of galectin-3 are involved in this activity. Pertussis toxin (PTX) almost completely blocked monocyte migration induced by high concentrations of galectin-3. Galectin-3 caused a Ca2+ influx in monocytes at high, but not low, concentrations, and both lactose and PTX inhibited this response. There was no cross-desensitization between galectin-3 and any of the monocyte-reactive chemokines examined, including monocyte chemotactic protein-1, macrophage inflammatory protein-1alpha, and stromal cell-derived factor-1alpha. Cultured human macrophages and alveolar macrophages also migrated toward galectin-3, but not monocyte chemotactic protein-1. Finally, galectin-3 was found to cause monocyte accumulation in vivo in mouse air pouches. These results indicate that galectin-3 is a novel chemoattractant for monocytes and macrophages and suggest that the effect is mediated at least in part through a PTX-sensitive (G protein-coupled) pathway.

Animals↗

PubMed2000

High-gain harmonic-generation free-electron laser

A high-gain harmonic-generation free-electron laser is demonstrated. Our approach uses a laser-seeded free-electron laser to produce amplified, longitudinally coherent, Fourier transform-limited output at a harmonic of the seed laser. A seed carbon dioxide laser at a wavelength of 10.6 micrometers produced saturated, amplified free-electron laser output at the second-harmonic wavelength, 5.3 micrometers. The experiment verifies the theoretical foundation for the technique and prepares the way for the application of this technique in the vacuum ultraviolet region of the spectrum, with the ultimate goal of extending the approach to provide an intense, highly coherent source of hard x-rays.

Journal Article↗

PubMed2000

Cloning and characterization of human DDX24 and mouse Ddx24, two novel putative DEAD-Box proteins, and mapping DDX24 to human chromosome 14q32.

DEAD-box proteins are a large group of putative RNA helicases that exist ubiquitously in organisms ranging from bacteria to humans. They are likely to play important roles in many different RNA metabolic processes. In this paper, we report the cloning of human DDX24, a putative DEAD-box protein, and its ortholog, Ddx24 in mouse. The deduced proteins encoded by these two cDNAs share 78.7% identity at the amino acid level and possess all the well-conserved motifs of DEAD-box proteins. However, little homology can be found between them and other DEAD-box proteins, even in their core region (identity <40%). Northern blot analysis showed that a 3.0-kb transcript of human DDX24 exists ubiquitously in the 16 human tissues examined and was most abundant in heart and brain, but with lowest levels in thymus and small intestine. The mouse Ddx24, whose transcript is 4.0 kb, was also expressed widely in 10 tissues tested with the highest level in heart and testis. By radiation hybrid mapping, the human DDX24 gene was localized to human chromosome 14q32 between the markers D14S81 and D14S265. Moreover, the gene structure of DDX24 was determined by comparing its cDNA and genomic sequence from BAC R-1089B7, which showed that the gene spanned a 30-kb region and consisted of at least nine exons.

Amino Acid Sequence↗

PubMed2000

A repressor sequence in the juxtamembrane domain of Flt-1 (VEGFR-1) constitutively inhibits vascular endothelial growth factor-dependent phosphatidylinositol 3'-kinase activation and endothelial cell migration.

Vascular endothelial growth factor (VEGF) has two highly homologous tyrosine kinase receptors: Flt-1 (VEGFR-1) and KDR (VEGFR-2). KDR is strongly phosphorylated on tyrosines and can transmit mitogenic and motogenic signals following VEGF binding, while Flt-1 is markedly less effective in mediating such functions. To dissect the regions that account for the differences between the two receptors, we generated a series of chimeric Flt-1-KDR molecules. We found that the juxtamembrane region of Flt-1 prevents key signaling functions. When the juxtamembrane region of Flt-1 is replaced by that of KDR, Flt-1 becomes competent to mediate endothelial cell migration and phosphatidylinositol 3'-kinase activation in response to VEGF. Further mutational analysis shows that a short divergent sequence is responsible for such repressor function. However, mutant Flt-1 receptors lacking this sequence do not transmit effective proliferative signals, suggesting that this receptor function is regulated separately. These results define a novel functional domain that serves to repress Flt-1 activity in endothelial cells.

Amino Acid Sequence↗

PubMed2000

Cloning and mapping of human PKIB and PKIG, and comparison of tissue expression patterns of three members of the protein kinase inhibitor family, including PKIA.

Two novel members of the human cAMP-dependent protein kinase inhibitor (PKI) gene family, PKIB and PKIG, were cloned. The deduced proteins showed 70% and 90% identity with mouse PKIbeta and PKIgamma respectively. Both the already identified pseudosubstrate site and leucine-rich nuclear export signal motifs were defined from the 11 PKIs of different species. The PKIB and PKIG genes were mapped respectively to chromosome 6q21-22.1, using a radiation hybrid GB4 panel, and to chromosome 20q13.12-13.13, using a Stanford G3 panel. Northern-blot analysis of three PKI isoforms, including the PKIA identified previously, revealed significant differences in their expression patterns. PKIB had two transcripts of 1.9 kb and 1.4 kb. The former transcript was abundant in both placenta and brain and the latter was expressed most abundantly in placenta, highly in brain, heart, liver, pancreas, moderately in kidney, skeletal muscle and colon, and very little in the other eight tissues tested. PKIG was widely expressed as a 1.5-kb transcript with the highest level in heart, hardly detectable in thymus and peripheral blood leucocytes and was moderately expressed in the other tissues, with slightly different levels. However, PKIA was specifically expressed as two transcripts of 3.3 kb and 1.5 kb in heart and skeletal muscle. The distinct expression patterns of the three PKIs suggest that their roles in various tissues are probably different.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Localization of linear B-cell epitopes on infectious bronchitis virus nucleocapsid protein.

The nucleocapsid (N) protein of many viruses is highly conserved, immunogenic, and abundantly expressed during infection. These features make it a suitable candidate for diagnostic applications. The nucleocapsid protein of infectious bronchitis virus (IBV) was dissected into 12 fragments and expressed in Escherichia coli. Sera against Australia T, China Ch5, Singapore P4, USA M41 and China T3 isolates were used to study the conservation and localization of the antigenic region on the IBV nucleocapsid protein. Our results show linear immunodominant epitopes, which were found in three fragments covering amino acid residues 175-241, 310-370 and 360-409.

Animals↗