Biomedical subjects

L Yu

Publications and source records attributed to L Yu.

At least 145 records · Page 8Linked to original sources

PubMed2000

Molecular cloning and mapping of the brain-abundant B1gamma subunit of protein phosphatase 2A, PPP2R2C, to human chromosome 4p16.

Protein phosphatase 2A (PP2A) is one kind of serine/ threonine protein phosphatase regulating mainly cell growth and division. It comprises three subunits, A, B, and C. The B-subunit is involved in enzyme activity and substrate specificity. The B-subunit family is of great diversity and is divided into three classes, the B1, B2, and B3 subfamilies. Until now, two members of the B1 subfamily, B1alpha and B1beta, have been identified in human. In this report, the third member of the sub-family, B1gamma, was identified, and its cDNA was isolated from a human brain cDNA library. This novel cDNA is 4,120 bp in length and contains an open reading frame (nt 55-1,398) encoding 447 amino acid residues. The putative protein shares 81 and 85% identity with B1alpha (PPP2R2A) and B1beta (PPP2R2B), respectively, and was named PPP2R2C for its high level of homology to the other two isoforms. One remarkable characteristic of this novel gene is that it is highly expressed in brain with a 4.7-kb transcript while it is nearly undetectable in other tissues. In addition, the PPP2R2C gene was localized to human chromosome 4p16 between markers D4S2925 and D4S3007 with 5.45 cR (LOD > 14) and 2.63 cR (LOD > 15) RH distance, respectively, by radiation hybrid panel mapping.

Amino Acid Sequence↗

PubMed2000

Adsorption of PBTCA on Alumina Surfaces and Its Influence on the Fractal Characteristics of Sediments.

The adsorption of 2-phosphonobutane-1,2,4-tricarboxylic acid (PBTCA) on Al(2)O(3) powder has been studied as a function of pH and concentration. The adsorption density of PBTCA is found to decrease with an increase of pH. Zeta potential measurements show that the addition of PBTCA results in a dramatic increase in the absolute zeta potential, as well as a shift of the isoelectric point to the more acidic region. PBTCA considerably enhances the stability of the alumina suspension via an electrostatic mechanism. The surface properties of alumina suspensions are examined by using Auger electron microscopy and FTIR analysis. Chemical interactions take place at the solid/water interface by forming complexes between Al(3+) ions and PBTCA. The dispersing ability of PBTCA is believed to be related to its high adsorption ability and the high number of dissociable protons. The morphology of the sediments is observed with scanning electron microscopy. It is found that the sediment surfaces exhibit fractal characteristics. The fractal dimension values of sediments are correlated with PBTCA concentration in the experimental range. Copyright 2000 Academic Press.

Journal Article↗

PubMed2000

Molecular cloning, expression characterization, and mapping of a novel putative inhibitor of rho GTPase activity, RTKN, to D2S145-D2S286.

The Rho proteins are a class of small molecular GTPases that regulate multiple fundamental cellular processes by mediating the G-protein-coupled receptor signaling pathway. Rhotekin, which is one of the downstream target molecules of Rho with a Rho binding motif class I domain, can inhibit endogenous or RhoGAP-stimulating Rho GTPase activity to regulate the signaling pathway. Here, a novel human cDNA containing an intact open reading frame that encodes 544 amino acids has been identified. As this putative protein shares 84. 6% amino acid identity with mouse Rhotekin, and has a tandem Rho binding domain class 1 and Pleckstrin homology domain, it was regarded as a human homologue of the mouse Rhotekin and assigned a symbol of RTKN. With the human Rhotekin cDNA as a probe, Northern hybridization revealed that a 4.0-kb transcript was expressed at a high level in prostate and at a middle level in 13 of 16 tissues examined, but it cannot be detected in liver and lung tissues. Meanwhile, a 2.4-kb transcript was expressed at a middle level in prostate and another 3.0-kb transcript in kidney. In addition, the RTKN gene was localized to chromosome 2p13 between markers D2S145 at 6.94 cR (LOD > 12) and D2S286 at 8.12 cR (LOD > 9.7) by radiation hybrid panel mapping. Compared with BAC clone AC005041 sequence, there were 12 exons for the RTKN gene and it spanned a 16.5-kb genomic region.

Amino Acid Motifs↗

PubMed2000

Magnetic-field effects on two-Leg heisenberg antiferromagnetic ladders: thermodynamic properties

Using the recently developed transfer-matrix renormalization group method, we have studied the thermodynamic properties of two-leg antiferromagnetic ladders in a magnetic field. Based on different magnetization behaviors, we found a disordered spin liquid, the Luttinger liquid, spin-polarized phases, and a classical regime. Our calculations in the Luttinger liquid regime suggest that both the divergence of the NMR relaxation rate and the anomalous specific heat behavior observed on Cu2(C5H12N2)2Cl4 are due to a quasi-one-dimensional effect rather than three-dimensional ordering.

Journal Article↗

PubMed2000

Subunit IV of cytochrome bc1 complex from Rhodobacter sphaeroides. Localization of regions essential for interaction with the three-subunit core complex.

Recombinant subunit IV mutants which identify the regions essential for restoration of bc(1) activity to the three-subunit core complex of Rhodobacter sphaeroides were generated and characterized. Four C-terminal truncated mutants: IV(1-109), IV(1-85), IV(1-76), and IV(1-40) had 100, 0, 0, and 0% of reconstitutive activity of the wild-type IV, indicating that residues 86-109 are essential. IV(1-109) is associated with the core complex in the same manner as the wild-type IV while mutants IV(1-85), IV(1-76), and IV(1-40) do not associate with the core complex, indicating that subunit IV requires its transmembrane helix region (residues 86-109) for assembly into the bc(1) complex. Since GST-IV(86-109) fusion protein has little reconstitutive activity, some region(s) in residues 1-85 are required for bc(1) activity restoration after subunit IV is incorporated into the complex through the transmembrane helix, presumably by interaction with cytochrome b in the core complex. The interacting regions are identified as residues 41-53 and 77-85, since mutants IV(21-109), IV(41-109), IV(54-109), and IV(77-109) had 95, 98, 53, and 53% of the reconstitutive activity of the wild-type IV. These two interacting regions are on the cytoplasmic side of the chromatophore membrane and closed to the DE loop and helix G of cytochrome b, respectively.

Amino Acid Sequence↗

PubMed2000

Processing and maturation of flavocytochrome b558 include incorporation of heme as a prerequisite for heterodimer assembly.

The phagocyte NADPH-dependent oxidase generates superoxide by reducing molecular oxygen through a transmembrane heterodimer known as flavocytochrome b(558) (flavocytochrome b). We investigated the biosynthesis of flavocytochrome b subunits gp91(phox) and p22(phox) to elucidate features of flavocytochrome b processing in myeloid cells. Although the gp91(phox) precursor, gp65, was processed to gp91(phox) within 4-8 h of chase, unassembled gp65 and p22(phox) monomers were degraded by the cytosolic proteasome. gp65 associated with p22(phox) post-translationally, within 1-4 h of chase, but prior to its modification in the Golgi complex. Moreover, p22(phox) coprecipitated with unglycosylated gp91(phox) primary translation product made in the presence of tunicamycin, suggesting that heterodimer formation does not require glycosylation. Blocking heme synthesis with succinyl acetone completely inhibited heterodimer formation, although biogenesis of gp65 and p22(phox) was unaffected. In succinyl acetone-treated cells, p22(phox) and gp65 were degraded completely by 8 h of chase, a process mediated by the cytosolic proteasome. Taken together, these data suggest that the formation of the gp65-p22(phox) heterodimer is relatively inefficient and that acquisition of heme by gp65 precedes and is required for its association with p22(phox), a process that requires neither the addition of N-linked oligosaccharides nor modification in the Golgi complex.

Cytochrome b Group↗

PubMed2000

Use of a photoactivated ruthenium dimer complex to measure electron transfer between the Rieske iron-sulfur protein and cytochrome c(1) in the cytochrome bc(1) complex.

Electron transfer between the Rieske iron-sulfur protein (Fe(2)S(2)) and cytochrome c(1) was studied using the ruthenium dimer, Ru(2)D, to either photoreduce or photooxidize cytochrome c(1) within 1 micros. Ru(2)D has a charge of +4, which allows it to bind with high affinity to the cytochrome bc(1) complex. Flash photolysis of a solution containing beef cytochrome bc(1), Ru(2)D, and a sacrificial donor resulted in reduction of cytochrome c(1) within 1 micros, followed by electron transfer from cytochrome c(1) to Fe(2)S(2) with a rate constant of 90,000 s(-1). Flash photolysis of reduced beef bc(1), Ru(2)D, and a sacrificial acceptor resulted in oxidation of cytochrome c(1) within 1 micros, followed by electron transfer from Fe(2)S(2) to cytochrome c(1) with a rate constant of 16,000 s(-1). Oxidant-induced reduction of cytochrome b(H) was observed with a rate constant of 250 s(-1) in the presence of antimycin A. Electron transfer from Fe(2)S(2) to cytochrome c(1) within the Rhodobacter sphaeroides cyt bc(1) complex was found to have a rate constant of 60,000 s(-1) at 25 degrees C, while reduction of cytochrome b(H) occurred with a rate constant of 1000 s(-1). Double mutation of Ala-46 and Ala-48 in the neck region of the Rieske protein to prolines resulted in a decrease in the rate constants for both cyt c(1) and cyt b(H) reduction to 25 s(-1), indicating that a conformational change in the Rieske protein has become rate-limiting.

Amino Acid Substitution↗

PubMed2000

An alternatively spliced transcript of the rat nociceptin receptor ORL1 gene encodes a truncated receptor.

Opioid receptor-like protein ORL1, the receptor for the neuropeptide nociceptin (also named orphanin FQ), has two alternatively spliced isoforms in the rat. This alternative splicing event is generated by retaining of intron 3, 81 bases in length, in the mRNA region encoding the second extracellular loop of ORL1. A full-length rat ORL1 receptor has 367 amino acid residues. However, as revealed by sequencing of rat ORL1 genomic DNA and cDNA, the insertion of the unspliced intron 3 brings in an in-frame stop codon and, therefore, creates a truncated open-reading frame encoding only the N-terminal half of ORL1 (from the N-terminus to an alternate extracellular tail C-terminal to the fourth transmembrane domain). The two alternatively spliced transcripts are differentially expressed in tissues. In transfected mammalian cells, the full-length ORL1 displays high-affinity and selective binding for nociceptin, and inhibits the production of cyclic AMP. In contrast, the truncated ORL1 binds nociceptin and other opioid peptides very poorly and non-selectively (affinity in micromolar range), and it does not mediate any inhibitory effects on cyclic AMP production. Apparently, this truncated ORL1 does not function as a receptor for nociceptin or other ligands tested. Such alternative splicing to create a truncated ORL1 receptor might be an endogenous mechanism to negatively regulate nociceptin/ORL1 functions.

Alternative Splicing↗

PubMed2000

Refusing to twist: demonstration of a line hexatic phase in DNA liquid crystals

We report conclusive high resolution small angle x-ray scattering evidence that long DNA fragments form an untwisted line hexatic phase between the cholesteric and the crystalline phases. The line hexatic phase is a liquid-crystalline phase with long-range hexagonal bond-orientational order, long-range nematic order, but liquidlike, i.e., short-range, positional order. So far, it has not been seen in any other three dimensional system. By line-shape analysis of x-ray scattering data we found that positional order decreases when the line hexatic phase is compressed. We suggest that such anomalous behavior is a result of the chiral nature of DNA molecules.

Journal Article↗

PubMed2000

Decreased serum concentrations of nitric oxide metabolites among Chinese in an endemic area of chronic arsenic poisoning in inner Mongolia.

Prolonged exposure to arsenic results in peripheral and cardiovascular manifestations, as does impaired production of endothelial nitric oxide (NO). In vitro studies have indicated that endothelial cells undergo damage by arsenic. However, no information has been available on the relationship between NO synthesis and chronic arsenic poisoning in humans. The present study was designed to reveal this question. The subjects were 33 habitants who continued to drink well water containing high concentrations of inorganic arsenic (mean value = 0.41 microg/ml) for about 18 years in Inner Mongolia, China, and 10 other people who lived in this area but exposed to minimal concentrations of arsenic (mean value = 0.02 microg/ml) were employed as controls. Mean blood concentration of total arsenic was six times higher in exposed subjects than controls; 42.1 vs. 7.3 ng/ml, p <.001. Mean serum concentration of nitrite/nitrate, stable metabolites of endogenous NO, was lower in arsenic-exposed subjects than in controls: 24.7 vs. 51.6 microM, p<.001. In total samples, an inverse correlation with serum nitrite/nitrate levels was strong for blood inorganic arsenic (r = -0.52, p <.001) and less strong for its metabolites, monomethyl arsenic (r = -0.45, p<.005) and dimethyl arsenic (r = -0.37, p<.05). Furthermore, serum nitrite/nitrate concentration was significantly correlated with nonprotein sulfhydryl level in whole blood (r = 0.58, p<.001). In an in vitro study, we demonstrated that inorganic arsenite or arsenate suppresses the activity of endothelial NO synthase in human umbilical vein endothelial cells. These results suggest that long-term exposure to arsenic by drinking well water possibly reduces NO production in endothelial cells, resulting in a decrease in reduced nitrite/nitrate concentrations. Peripheral vascular disorders caused by arsenic may be attributable in part to impairment of NO production in vivo.

Adolescent↗

PubMed2000

Cloning, characterization and mapping of the human ATP5E gene, identification of pseudogene ATP5EP1, and definition of the ATP5E motif.

A cDNA encoding the epsilon subunit of human ATP synthase, ATP5E, was isolated from heart, skeletal muscle and spleen cDNA libraries respectively. Its genome structure was characterized as comprising three exons and two introns within a stretch of 5 kb, according to the genomic sequence AL109840. The gene was mapped to human chromosome 20q13.3 between marker D20S173 and 20qter using the radiation hybrid GB4 panel. Northern blot analysis showed that the ATP5E gene was expressed as a single 0.6 kb transcript in all 16 human tissues tested, with a high level present in heart and skeletal muscle. A new conserved motif composed of 24 residues, termed the ATP5E motif [W(R/K)X(5)YX(2)(Y/F)X(3)(C/A)X(4)RX(3)K], was defined on the basis of sequences of ATP synthase epsilon subunits from ten different organisms. In addition, a pseudogene ATP5EP1 was also identified on the basis of genomic sequence AC004066, localized on human chromosome 4q25. By analysing these results combined with the Southern blot patterns of human DNA hybridized with bovine ATP5E cDNA reported previously [Vinas, Powell, Runswick, Iacobazzi and Walker (1990) Biochem. J. 265, 321-326], we provide evidence of yet further homologous sequences (either gene or pseudogene) of ATP5E, in addition to ATP5E and ATP5EP1 in the human genome.

Amino Acid Sequence↗

PubMed2000

Definition of the interaction domain for cytochrome c on the cytochrome bc(1) complex. Steady-state and rapid kinetic analysis of electron transfer between cytochrome c and Rhodobacter sphaeroides cytochrome bc(1) surface mutants.

The interaction domain for cytochrome c on the cytochrome bc(1) complex was studied using a series of Rhodobacter sphaeroides cytochrome bc(1) mutants in which acidic residues on the surface of cytochrome c(1) were substituted with neutral or basic residues. Intracomplex electron transfer was studied using a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine 72 (Ru-72-Cc). Flash photolysis of a 1:1 complex between Ru-72-Cc and cytochrome bc(1) at low ionic strength resulted in electron transfer from photoreduced heme c to cytochrome c(1) with a rate constant of k(et) = 6 x 10(4) s(-1). Compared with the wild-type enzyme, the mutants substituted at Glu-74, Glu-101, Asp-102, Glu-104, Asp-109, Glu-162, Glu-163, and Glu-168 have significantly lower k(et) values as well as significantly higher equilibrium dissociation constants and steady-state K(m) values. Mutations at acidic residues 56, 79, 82, 83, 97, 98, 213, 214, 217, 220, and 223 have no significant effect on either rapid kinetics or steady-state kinetics. These studies indicate that acidic residues on opposite sides of the heme crevice of cytochrome c(1) are involved in binding positively charged cytochrome c. These acidic residues on the intramembrane surface of cytochrome c(1) direct the diffusion and binding of cytochrome c from the intramembrane space.

Amino Acid Sequence↗

PubMed2000

Antinociceptive effects of calcitonin gene-related peptide injected into periaqueductal grey of rats with mononeuropathy.

Intra-periaqueductal grey (PAG) injection of calcitonin gene-related peptide (CGRP) induced dose-dependent increases in hindpaw withdrawal latency (HWL) to thermal and mechanical stimulation in rats with mononeuropathy. CGRP-induced increases in HWLs were blocked by intra-PAG injection of the CGRP antagonist CGRP8-37. The results demonstrated that CGRP and CGRP receptors in PAG play an important role in antinociception in rats with mononeuropathy.

Analgesia↗

PubMed2000

Antinociceptive effects induced by intra-periaqueductal grey administration of neuropeptide Y in rats.

Hindpaw withdrawal latency (HWL) to thermal and mechanical stimulation increased dose-dependently after intra-periaqueductal grey (PAG) injection of neuropeptide Y (NPY). Furthermore, the NPY-induced increases in HWLs were attenuated by intra-PAG injection of the Y1 receptor antagonist NPY28-36. The results demonstrated that NPY plays an important role in antinociception in PAG, in which Y1 receptor is involved in.

Analgesia↗

PubMed2000

pH-induced intramolecular electron transfer between the iron-sulfur protein and cytochrome c(1) in bovine cytochrome bc(1) complex.

Structural analysis of the bc(1) complex suggests that the extra membrane domain of iron-sulfur protein (ISP) undergoes substantial movement during the catalytic cycle. Binding of Qo site inhibitors to this complex affects the mobility of ISP. Taking advantage of the difference in the pH dependence of the redox midpoint potentials of cytochrome c(1) and ISP, we have measured electron transfer between the [2Fe-2S] cluster and heme c(1) in native and inhibitor-treated partially reduced cytochrome bc(1) complexes. The rate of the pH-induced cytochrome c(1) reduction can be estimated by conventional stopped-flow techniques (t1/2, 1-2 ms), whereas the rate of cytochrome c(1) oxidation is too high for stopped-flow measurement. These results suggest that oxidized ISP has a higher mobility than reduced ISP and that the movement of reduced ISP may require an energy input from another component. In the 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT)-inhibited complex, the rate of cytochrome c(1) reduction is greatly decreased to a t1/2 of approximately 2.8 s. An even lower rate is observed with the stigmatellin-treated complex. These results support the idea that UHDBT and stigmatellin arrest the [2Fe-2S] cluster at a fixed position, 31 A from heme c(1), making electron transfer very slow.

Animals↗

PubMed2000

The Chk1 protein kinase and the Cdc25C regulatory pathways are targets of the anticancer agent UCN-01.

A checkpoint operating in the G(2) phase of the cell cycle prevents entry into mitosis in the presence of DNA damage. UCN-01, a protein kinase inhibitor currently undergoing clinical trials for cancer treatment, abrogates G(2) checkpoint function and sensitizes p53-defective cancer cells to DNA-damaging agents. In most species, the G(2) checkpoint prevents the Cdc25 phosphatase from removing inhibitory phosphate groups from the mitosis-promoting kinase Cdc2. This is accomplished by maintaining Cdc25 in a phosphorylated form that binds 14-3-3 proteins. The checkpoint kinases, Chk1 and Cds1, are proposed to regulate the interactions between human Cdc25C and 14-3-3 proteins by phosphorylating Cdc25C on serine 216. 14-3-3 proteins, in turn, function to keep Cdc25C out of the nucleus. Here we report that UCN-01 caused loss of both serine 216 phosphorylation and 14-3-3 binding to Cdc25C in DNA-damaged cells. In addition, UCN-01 potently inhibited the ability of Chk1 to phosphorylate Cdc25C in vitro. In contrast, Cds1 was refractory to inhibition by UCN-01 in vitro, and Cds1 was still phosphorylated in irradiated cells treated with UCN-01. Thus, neither Cds1 nor kinases upstream of Cds1, such as ataxia telangiectasia-mutated, are targets of UCN-01 action in vivo. Taken together our results identify the Chk1 kinase and the Cdc25C pathway as potential targets of G(2) checkpoint abrogation by UCN-01.

Alkaloids↗

PubMed2000

Early expression of antiinsulin autoantibodies of humans and the NOD mouse: evidence for early determination of subsequent diabetes.

With the development of an insulin autoantibody (IAA) assay performed in 96-well filtration plates, we have evaluated prospectively the development of IAA in NOD mice (from 4 weeks of age) and children (from 7 to 10 months of age) at genetic risk for the development of type 1 diabetes. NOD mice had heterogeneous expression of IAA despite being inbred. IAA reached a peak between 8 and 16 weeks and then declined. IAA expression by NOD mice at 8 weeks of age was strongly associated with early development of diabetes, which occurred at 16-18 weeks of age (NOD mice IAA(+) at 8 weeks: 83% (5/6) diabetic by 18 weeks versus 11% (1/9) of IAA negative at 8 weeks; P <.01). In man, IAA was frequently present as early as 9 months of age, the first sampling time. Of five children found to have persistent IAA before 1 year of age, four have progressed to diabetes (all before 3.5 years of age) and the fifth is currently less than age 2. Of the 929 children not expressing persistent IAA before age 1, only one has progressed to diabetes to date (age onset 3), and this child expressed IAA at his second visit (age 1.1). In new onset patients, the highest levels of IAA correlated with an earlier age of diabetes onset. Our data suggest that the program for developing diabetes of NOD mice and humans is relatively "fixed" early in life and, for NOD mice, a high risk of early development of diabetes is often determined by 8 weeks of age. With such early determination of high risk of progression to diabetes, immunologic therapies in humans may need to be tested in children before the development of IAA for maximal efficacy.

Age Factors↗

PubMed2000

Expression, purification, and characterization of deglycosylated human pro-prostate-specific antigen.

Wild-type and deglycosylated forms of human prostate-specific antigen were expressed in Chinese hamster ovary (CHO) cells as zymogens. ProPSA was collected from conditioned medium and purified using a single cation-exchange chromatographic step for the deglycosylated form and cation-exchange followed by gel filtration chromatography for the wild-type form. Recombinant wild-type proPSA produced in CHO cells has an average MW of 34.5 kDa, whereas the deglycosylated proPSA has a MW of 32.4 kDa. Both forms of proPSA were activated in vitro and the kinetic properties measured for the deglycosylated PSA are very similar to those of the wild-type recombinant PSA and the native PSA isolated from seminal fluid. These results suggest that deglycosylated PSA is likely to be very similar to native PSA with respect to its three-dimensional structure and will provide a homogeneous protein preparation necessary for X-ray crystallographic analysis.

Animals↗