Biomedical subjects

Yong Xia

Publications and source records attributed to Yong Xia.

10 recordsLinked to original sources

PubMed2006

Proteasome inhibition down-regulates endothelial nitric-oxide synthase phosphorylation and function.

Endothelial nitric-oxide synthase (eNOS) function is fundamentally modulated by protein phosphorylation. In particular, phosphorylation of serine 1179 (bovine)/1177 (human) by Akt has been shown to be the central mechanism of eNOS regulation. Here we revealed a novel role of proteasome in controlling eNOS serine 1179 phosphorylation and function. Rather than affecting eNOS turnover, proteasomal inhibition specifically dephosphorylated eNOS serine 1179, leading to decreased enzymatic activity. Blocking protein phosphatase 2A (PP2A) by okadaic acid or PP2A knockdown restored eNOS serine 1179 phosphorylation and activity in proteasome-inhibited cells. Although total PP2A expression and activity in cells were not affected by proteasome inhibitors, proteasomal inhibition induced PP2A ubiquitination and ubiquitinated PP2A translocated from cytosol to membrane. Further biochemical analyses demonstrated that eNOS associated with PP2A on cell membranes. Proteasomal inhibition markedly enhanced PP2A association to eNOS, and this increase of PP2A dephosphorylated eNOS and its upstream kinase Akt. Taken together, these studies identified a novel pathway in which proteasome modulates eNOS phosphorylation by inducing intracellular PP2A translocation.

Animals↗

PubMed2006

PIN inhibits nitric oxide and superoxide production from purified neuronal nitric oxide synthase.

A protein inhibitor of neuronal nitric oxide synthase (nNOS) was identified and designated as PIN. PIN was reported to inhibit nNOS activity in cell lysates through disruption of enzyme dimerization. However, there has been lack of direct characterization of the effect of PIN on NO production from purified nNOS. Furthermore, nNOS also generates superoxide (.O(2)(-)) at low levels of L-arginine. It is unknown whether PIN affects .O(2)(-) generation from nNOS. Therefore, we performed direct measurements of the effects of PIN on NO and .O(2)(-) generation from purified nNOS using electron paramagnetic resonance spin trapping techniques. nNOS was isolated by affinity chromatography and a fusion protein CBP-PIN was used to probe the effect of PIN. While the tag CBP did not affect nNOS activity, CBP-PIN caused a dose-dependent inhibition on both NO and L-citrulline production. In the absence of L-arginine, strong .O(2)(-) generation was observed from nNOS, and this was blocked by CBP-PIN in a dose-dependent manner. With low-temperature polyacrylamide gel electrophoresis, neither CBP nor CBP-PIN was found to affect nNOS dimerization. Thus, these results suggested that PIN not only inhibits NO but also .O(2)(-) production from nNOS, and this is through a mechanism other than decomposition of nNOS dimers.

Animals↗

PubMed2006

Calcineurin regulates myocardial function during acute endotoxemia.

RATIONALE: Cyclosporin A (CsA) is known to preserve cardiac contractile function during endotoxemia, but the mechanism is unclear. Increased nitric oxide (NO) production and altered mitochondrial function are implicated as mechanisms contributing to sepsis-induced cardiac dysfunction, and CsA has the capacity to reduce NO production and inhibit mitochondrial dysfunction relating to the mitochondrial permeability transition (MPT). OBJECTIVES: We hypothesized that CsA would protect against endotoxin-mediated cardiac contractile dysfunction by attenuating NO production and preserving mitochondrial function. METHODS: Left ventricular function was measured continuously over 4 h in cats assigned as follows: control animals (n = 7); LPS alone (3 mg/kg, n = 8); and CsA (6 mg/kg, n = 7), a calcineurin inhibitor that blocks the MPT, or tacrolimus (FK506, 0.1 mg/kg, n = 7), a calcineurin inhibitor lacking MPT activity, followed in 30 min by LPS. Myocardial tissue was then analyzed for NO synthase-2 expression, tissue nitration, protein carbonylation, and mitochondrial morphology and function. MEASUREMENTS AND MAIN RESULTS: LPS treatment resulted in impaired left ventricular contractility, altered mitochondrial morphology and function, and increased protein nitration. As hypothesized, CsA pretreatment normalized cardiac performance and mitochondrial respiration and reduced myocardial protein nitration. Unexpectedly, FK506 pretreatment had similar effects, normalizing both cardiac and mitochondrial parameters. However, CsA and FK506 pretreatments markedly increased protein carbonylation in the myocardium despite elevated manganese superoxide dismutase activity during endotoxemia. CONCLUSIONS: Our data indicate that calcineurin is a critical regulator of mitochondrial respiration, tissue nitration, protein carbonylation, and contractile function in the heart during acute endotoxemia.

Animals↗

PubMed2006

Morphology-based multifractal estimation for texture segmentation.

Multifractal analysis is becoming more and more popular in image segmentation community, in which the box-counting based multifractal dimension estimations are most commonly used. However, in spite of its computational efficiency, the regular partition scheme used by various box-counting methods intrinsically produces less accurate results. In this paper, a novel multifractal estimation algorithm based on mathematical morphology is proposed and a set of new multifractal descriptors, namely the local morphological multifractal exponents is defined to characterize the local scaling properties of textures. A series of cubic structure elements and an iterative dilation scheme are utilized so that the computational complexity of the morphological operations can be tremendously reduced. Both the proposed algorithm and the box-counting based methods have been applied to the segmentation of texture mosaics and real images. The comparison results demonstrate that the morphological multifractal estimation can differentiate texture images more effectively and provide more robust segmentations.

Algorithms↗

PubMed2005

Roles of 3-phosphoinositide-dependent kinase 1 in the regulation of endothelial nitric-oxide synthase phosphorylation and function by heat shock protein 90.

The 90-kDa heat shock protein (Hsp90) plays an important role in endothelial nitric-oxide synthase (eNOS) regulation. Besides acting as an allosteric enhancer, Hsp90 was shown to serve as a module recruiting Akt to phosphorylate the serine 1179/1177 (bovine/human) residue of eNOS. Akt is activated by the phosphorylation of 3-phosphoinositide-dependent kinase 1 (PDK1). Whether PDK1 is involved in the actions of Hsp90 on eNOS phosphorylation and function remains unknown. To address this issue, we treated bovine eNOS stably transfected human embryonic kidney 293 cells with Hsp90 inhibitors and determined the alterations of phospho-eNOS, Akt, and PDK1. Both geldanamycin and radicicol, two structurally different Hsp90 inhibitors, selectively reduced serine 1179-phosphorylated eNOS, leading to decreased enzyme activity. In Hsp90-inhibited cells, eNOS-associated phospho-Akt was decreased, but the total amount of Akt associated with eNOS remained the same. Further studies showed that Hsp90 inhibition dramatically depleted intracellular PDK1. Proteasome but not caspase blockade prevented the loss of PDK1 caused by Hsp90 inhibition. Silencing the PDK1 gene by small interfering RNA was sufficient to induce reduction of phospho-Akt and consequent loss of serine 1179-phosphorylated eNOS. Moreover, overexpression of PDK1, but not Akt, reversed Hsp90 inhibition-induced loss of eNOS serine 1179 phosphorylation and salvaged enzymatic activity. Thus, in addition to functioning as a module to recruit Akt to eNOS, Hsp90 also critically stabilized PDK1 by preventing it from proteasomal degradation. Inhibition of Hsp90 function resulted in PDK1 depletion and thus triggered a cascade of Akt deactivation, loss of eNOS serine 1179 phosphorylation, and decrease of enzyme function.

3-Phosphoinositide-Dependent Protein Kinases↗

PubMed2004

Endogenous methylarginines modulate superoxide as well as nitric oxide generation from neuronal nitric-oxide synthase: differences in the effects of monomethyl- and dimethylarginines in the presence and absence of tetrahydrobiopterin.

The endogenous methylarginines asymmetric dimethylarginine (ADMA) and N(G)-monomethyl-L-arginine (L-NMMA) regulate nitric oxide (NO) production from neuronal NO synthase (nNOS). Under conditions of L-arginine or tetrahydrobiopterin (BH(4)) depletion, nNOS also generates superoxide, O(2)(.); however, the effects of methylarginines on this O(2)(.) generation are poorly understood. Therefore, we measured the dose-dependent effects of ADMA and L-NMMA on the rate and amount of O(2)(.) production from nNOS under conditions of L-arginine and/or BH(4) depletion, using electron paramagnetic resonance spin trapping. In the absence of L-arginine, ADMA (1 microm) inhibited O(2)(.) generation by approximately 60% from a rate of 56 to 23 nmol/mg/min, whereas L-NMMA (0.1-100 microm) had no effect. L-Arginine markedly decreased the observed O(2)(.) adduct formation; however, O(2)(.) generation from the enzyme still occurs at a low rate (12.1 nmol/mg/min). This O(2)(.) leak is NOS-derived as it is not seen in the absence of calcium and calmodulin and demonstrates that O(2)(.) generation from NOS occurs even when normal substrate/ cofactor levels are present. Under conditions of BH(4) depletion, ADMA had no effect on O(2)(.), whereas L-NMMA increased O(2)(.) production almost 3-fold. This O(2)(.) generation was >90% inhibited by imidazole, indicating that it occurred at the heme center. Thus, methylarginines can profoundly shift the balance of NO and O(2)(.) generation from nNOS. These observations have important implications with regard to the therapeutic use of methylarginine-NOS inhibitors in the treatment of disease.

Animals↗

PubMed2004

Antimutagenicity of propolis against some mutagens in vivo and in vitro.

OBJECTIVE: To evaluate the antimutagenicity of propolis in vivo and in vitro. METHODS: Salmonella typhimurium strains TA98 and TA100 were used as a test model in vitro against a direct mutagen DMC and an indirect mutagen 2AF with or without S9 mix, and MN formation of mice bone marrow cell and CAs induction of mice testicle cell were applied as a test model in vivo against two mutagens CP and MMC. RESULTS: The present study clearly demonstrated that propolis could inhibit mutagenicity of both DMC and 2AF directly in a dose-dependent manner, and significant antimutagenic effects (P < 0.05) were obtained in TA98 strain at 2000 and 3000 microg/plate. It also could inhibit mutagenicity of both DMC and 2AF to TA98 strain in a dose-dependent manner, with significant antimutagenic effects (P < 0.05) appeared at 1000, 2000, and 3000 microg/plate. The results of antimutagenicity test in vivo revealed that propolis could inhibit MN formation significantly (P < 0.05) at the doses of 45.0 and 135.0 mg/kg b. w., and decrease the frequency of chromosome aberrants and chromosome aberrant cells significantly (P < 0.05) only at the dose of 135.0 mg/kg b. w. CONCLUSION: The propolis is a good inhibitor for mutagencity of DMC and 2AF in vitro, as well as for CP and MMC in vivo.

Air Pollutants↗

PubMed2003

Heat shock protein 90 as an endogenous protein enhancer of inducible nitric-oxide synthase.

Nitric oxide (NO) generated by inducible NO synthase (iNOS) plays crucial roles in inflammation and host defense. With an intrinsically bound calmodulin, iNOS is fully active once expressed in cells. Thus, regulation of NO production from iNOS was thought to primarily occur at the enzyme transcriptional level. Here we show that NO synthesis from iNOS can be profoundly modulated by heat shock protein 90 (hsp90) through protein-protein interaction. To study whether hsp90 affects iNOS function, recombinant murine iNOS was purified from an Escherichia coli expression system by affinity chromatography. Hsp90, at physiological concentrations (10-500 nm), dose-dependently increased iNOS activity. This was a specific effect because neither denatured hsp90 nor irrelevant bovine serum albumin affected iNOS function. Overexpression of hsp90 enhanced NO production in iNOS-transfected cells. On the contrary, hsp90 inhibition dramatically decreased NO formation from iNOS in macrophages. Co-immunoprecipitation studies showed that hsp90 and iNOS associated with each other in cells. Overexpression of iNOS resulted in NO-mediated cellular injury. Hsp90 inhibition markedly attenuated NO formation and prevented cellular injury. These results demonstrated that hsp90 is an allosteric enhancer of iNOS. iNOS is coupled with hsp90 in cells, and this coupling facilitates NO synthesis. In light of the critical role of hsp90 in iNOS-mediated cytotoxic action, modulating the interaction between hsp90 and iNOS may be a new approach to intervene inflammation and immune response.

Allosteric Site↗

PubMed2002

Inhibition of superoxide generation from neuronal nitric oxide synthase by heat shock protein 90: implications in NOS regulation.

Besides NO, neuronal NO synthase (nNOS) also produces superoxide (O(2)(-.) at low levels of L-arginine. Recently, heat shock protein 90 (hsp90) was shown to facilitate NO synthesis from eNOS and nNOS. However, the effect of hsp90 on the O(2)(-.) generation from NOS has not been determined yet. The interrelationship between its effects on O(2)(-.) and NO generation from NOS is also unclear. Therefore, we performed electron paramagnetic resonance measurements of O(2)(-.) generation from nNOS to study the effect of hsp90. Purified rat nNOS generated strong O(2)(-.) signals in the absence of L-arginine. In contrast to its effect on NO synthesis, hsp90 dose-dependently inhibited O(2)(-.) generation from nNOS with an IC(50) of 658 nM. This inhibition was not due to O(2)(-.) scavenging because hsp90 did not affect the O(2)(-.) generated by xanthine oxidase. At lower levels of L-arginine where marked O(2)(-.) generation occurred, hsp90 caused a more dramatic enhancement of NO synthesis from nNOS as compared to that under normal L-arginine. Significant O(2)(-.) production was detected from nNOS even at intracellular levels of L-arginine. Adding hsp90 prevented this O(2)(-.) production, leading to enhanced nNOS activity. Thus, these results demonstrated that hsp90 directly inhibited O(2)(-.) generation from nNOS. Inhibition of O(2)(-.) generation may be an important mechanism by which hsp90 enhances NO synthesis from NOS.

Animals↗

PubMed2002

[Determination of 7-aminoflunitrazepam, the major metabolite of flunitrazepam in urine by high performance thin-layer chromatography].

A rapid and highly sensitive method, based on high performance thin-layer chromatography, is described for the qualitative and semiquantitative determination of 7-aminoflunitrazepam, the main metabolite of flunitrazepam in human urine. 7-Aminoflunitrazepam in specimen was extracted by solid phase extraction using GDX-403 porous polymer bead as sorbent and ethyl ether as eluant. Fluorescamine was used as a reagent to produce fluorescent product on the plate. The limit of detection was 5 micrograms/L and the limit of quantitation was 15 micrograms/L for 7-aminoflunitrazepam in urine. The method can be successfully used for measuring 7-aminoflunitrazepam in urine samples of the subjects excreted over a 48 h period after receiving 1 mg flunitrazepam orally. The method is applicable to drug examination for the cases of drug-facilitated robbery and rape.

Chromatography, Thin Layer↗