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Y Xu

Publications and source records attributed to Y Xu.

At least 649 records · Page 36Linked to original sources
PubMed1998

Catalysis in human hypoxanthine-guanine phosphoribosyltransferase: Asp 137 acts as a general acid/base.

Hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) catalyzes the reversible formation of IMP and GMP from their respective bases hypoxanthine (Hx) and guanine (Gua) and the phosphoribosyl donor 5-phosphoribosyl-1-pyrophosphate (PRPP). The net formation and cleavage of the nucleosidic bond requires removal/addition of a proton at the purine moiety, allowing enzymic catalysis to reduce the energy barrier associated with the reaction. The pH profile of kcat for IMP pyrophosphorolysis revealed an essential acidic group with pKa of 7.9 whereas those for IMP or GMP formation indicated involvement of essential basic groups. Based on the crystal structure of human HGPRTase, protonation/deprotonation is likely to occur at N7 of the purine ring, and Lys 165 or Asp 137 are each candidates for the general base/acid. We have constructed, purified, and kinetically characterized two mutant HGPRTases to test this hypothesis. D137N displayed an 18-fold decrease in kcat for nucleotide formation with Hx as substrate, a 275-fold decrease in kcat with Gua, and a 500-fold decrease in kcat for IMP pyrophosphorolysis. D137N also showed lower KD values for nucleotides and PRPP. The pH profiles of kcat for D137N were severely altered. In contrast to D137N, the kcat for K165Q was decreased only 2-fold in the forward reaction and was slightly increased in the reverse reaction. The Km and KD values showed that K165Q interacts with substrates more weakly than does the wild-type enzyme. Pre-steady-state experiments with K165Q indicated that the phosphoribosyl transfer step was fast in the forward reaction, as observed with the wild type. In contrast, D137N showed slower phosphoribosyl transfer chemistry, although guanine (3000-fold reduction) was affected much more than hypoxanthine (32-fold reduction). In conclusion, Asp137 acts as a general catalytic acid/base for HGPRTase and Lys165 makes ground-state interactions with substrates.

Amino Acid Substitution↗
PubMed1998

The crystal structure of bikunin from the inter-alpha-inhibitor complex: a serine protease inhibitor with two Kunitz domains.

Bikunin is a serine protease inhibitor found in the blood serum and urine of humans and other animals. Its sequence shows internal repetition, suggesting that it contains two domains that resemble bovine pancreatic trypsin inhibitor (BPTI). A fragment of bikunin has been crystallised, its structure solved and subsequently refined against 2.5 A data. The two BPTI-like domains pack closely together and are related by an approximate 60 degrees rotation combined with a translation. These domains are very similar to each other and other proteins with this fold. The largest variations occur in the loops responsible for protease recognition. The loops of the first domain are unobstructed by the remaining protein. However, the loops of the second domain are close to the first domain and it is possible that protease binding may be affected or, in some cases, abolished by the presence of the first domain. Thus, cleavage of the two domains could alter the substrate specificity of domain II. Bikunin has a hydrophobic patch close to the N terminus of domain I, which is the most likely site for cell-surface receptor binding. In addition, there is a basic patch at one end of domain II that may be responsible for the inhibition of calcium oxalate crystallization in urine.

Amino Acid Sequence↗
PubMed1998

Selective determination of a group of organic compounds in complex sample matrixes by LC/MIMS with on-line immunoaffinity extraction.

An on-line immunoaffinity extraction with liquid chromatography/membrane introduction mass spectrometry (IAE/LC/MIMS) method for the determination of BTEX compounds in complex sample matrixes is described. This method uses an immunoaffinity column (1 mm i.d. x 20 mm) for on-line sample cleanup and enrichment, a 5-micron C18 trapping column (2 mm i.d. x 20 mm) for analyte focusing, a 3-micron C18 analytical column (3.2 mm i.d. x 100 mm) for separation, and a membrane introduction mass spectrometer for quantitation. The immunoaffinity column was evaluated in terms of binding capacity, recovery, and enrichment factor. The method was optimized for the determination of BTEX compounds in a mixture of 30 volatile organic compounds, which showed no matrix interference and a dramatic improvement of the detection limit over that of the LC/MIMS method (up to 474-fold). This method was also used for the determination of BTEX compounds in several gasoline-contaminated water samples, and the results were compared with the EPA reference methods.

Antibodies↗
PubMed1998

Removal of anti-porcine natural antibodies from human and nonhuman primate plasma in vitro and in vivo by a Galalpha1-3Galbeta1-4betaGlc-X immunoaffinity column.

BACKGROUND: Natural antibodies (NAbs) against a terminal alpha1-3 galactosyl (alphaGal) epitope have been identified as the major human anti-pig NAbs. METHODS AND RESULTS: We used two synthetic alphaGal trisaccharides--type 6 (alphaGal6) and type 2(alphaGal2)--linked to an inert matrix to remove NAbs from human plasma in vitro. Flow cytometry indicated that an average of 85% of the NAb binding activity was depleted by adsorption with alphaGal6. By measuring the binding of NAbs to pig peripheral blood mononuclear cells and bone marrow cells, we demonstrated that alphaGal6 was more effective than alphaGal2 in removing NAbs, and the combination of alphaGal6 + alphaGal2 did not further increase removal of NAbs. The specificity of the removal of NAbs (IgM and IgG) reactive with the alphaGal epitope by alphaGal6 matrix was shown by enzyme-linked immunosorbent assay. In vivo studies in nonhuman primates compared plasma perfusion through a alphaGal6 immunoaffinity column with hemoperfusion through a pig liver for changes in blood pressure, hematocrit, platelets, and NAb adsorption. CONCLUSIONS: Both methods reduced the level of anti-pig IgM and IgG xenoreactive antibodies to nearly background, but column perfusion caused less hypotension and reduction in platelets than liver perfusion. Four pig kidneys transplanted into monkeys after column perfusion did not undergo hyperacute rejection, remaining functional for 2-10 days, with a mean functional period of 7 days, demonstrating that a pig kidney can support renal function in a primate.

Animals↗
PubMed1998

Creation of a fully functional human chimeric DNA repair protein. Combining O6-methylguanine DNA methyltransferase (MGMT) and AP endonuclease (APE/redox effector factor 1 (Ref 1)) DNA repair proteins.

A dose-limiting toxicity of certain chemotherapeutic alkylating agents is their toxic effects on nontarget tissues such as the bone marrow. To overcome the myelosuppression observed by chemotherapeutic alkylating agents, one approach is to increase the level of DNA repair proteins in hematopoietic stem and progenitor cells. Toward this goal, we have constructed a human fusion protein consisting of O6-methylguanine DNA methyltransferase coupled with an apurinic endonuclease, resulting in a fully functional protein for both O6-methylguanine and apurinic/apyrimidinic (AP) site repair as determined by biochemical analysis. The chimeric protein protected AP endonuclease-deficient Escherichia coli cells against methyl methanesulfonate and hydrogen peroxide (H2O2) damage. A retroviral construct expressing the chimeric protein also protected HeLa cells against 1,3-bis(2-chloroethyl)-1-nitrosourea and methyl methanesulfonate cytotoxicity either when these agents were used separately or in combination. Moreover, as predicted from previous analysis, truncating the amino 150 amino acids of the apurinic endonuclease portion of the O6-methylguanine DNA methyltransferase-apurinic endonuclease protein resulted in the retention of O6-methylguanine DNA methyltransferase activity but loss of all AP endonuclease activity. These results demonstrate that the fusion of O6-methylguanine DNA methyltransferase and apurinic endonuclease proteins into a combined single repair protein can result in a fully functional protein retaining the repair activities of the individual repair proteins. These and other related constructs may be useful for protection of sensitive tissues and, therefore, are candidate constructs to be tested in preclinical models of chemotherapy toxicity.

Antineoplastic Agents↗
PubMed1998

Syntaxin 7, a novel syntaxin member associated with the early endosomal compartment.

Members of the syntaxin family are key molecules involved in diverse vesicle docking/fusion events. We report here the molecular, biochemical, and cell biological characterizations of a novel member (syntaxin 7) of the syntaxin family. Syntaxin 7 is structurally related to all known syntaxins. Within a 79-residue region preceding the C-terminal hydrophobic tail, syntaxin 7 is 35, 34, 34, 34, 25, and 19% identical to syntaxins 1, 2, 3, 4, 5, and 6, respectively. Northern blot analysis showed that syntaxin 7 is widely expressed. Indirect immunofluorescence microscopy revealed that syntaxin 7 is primarily associated with the early endosome. In vitro binding assays established that syntaxin 7 in membrane extracts interacts with immobilized recombinant alpha-soluble N-ethylmaleimide-sensitive factor attachment proteins fused to glutathione S-transferase. Our results highlight the general importance of members of the syntaxin family in protein trafficking and provide new avenues for future functional and mechanistic studies of this first endosomal syntaxin as well as the endocytotic pathway.

Amino Acid Sequence↗
PubMed1998

Tumor cell apoptosis, lymphocyte recruitment and tumor vascular changes are induced by low temperature, long duration (fever-like) whole body hyperthermia.

A single treatment of low-temperature, long-duration, whole-body hyperthermia of either severe combined immunodeficient (SCID) mice bearing human breast tumor xenografts or Balb/c mice bearing syngeneic tumors for 6-8 hr can cause a temporary reduction of tumor volume and/or a growth delay. In both animal model systems, this inhibition is correlated with the appearance of large numbers of apoptotic tumor cells. Because this type of mild heat exposure, comparable to a common fever, is not itself directly cytotoxic, other explanations for the observed tumor cell death were considered. Our data support the hypothesis that this hyperthermia protocol stimulates some component(s) of the immune response, which results in increased antitumor activity. In support of this hypothesis, increased numbers of lymphocyte-like cells, macrophages, and granulocytes are observed in the tumor vasculature and in the tumor stroma immediately following this mild hyperthermia exposure. In Balb/c mice, an infiltrate persists in the tumor for at least 2 weeks. Using the SCID mouse/human tumor system, we found that both host natural killer (NK) cells and injected human NK cells were increased at the site of tumor following hyperthermia treatment. Experiments using anti-asialo-GM1 antibodies indicate that the tumor cell apoptosis seen in the SCID mouse appears to be due largely to the activity of NK cells, although additional roles for other immunoeffector cells and cytokines appear likely in the immunologically complete Balb/c model. Another interrelated hypothesis is that immunoeffector cells may have greater access to the interior of the tumor because we have observed that this treatment causes an obvious expansion in the diameter of blood vessels within the tumor and an increase in nucleated blood cells within the vessels, which persists as long as 2 weeks after treatment. Further study of the mechanisms by which mild hyperthermia exerts antitumor activity could result in this treatment protocol being used as an effective, nontoxic adjuvant to immunotherapy and/or other cancer therapies.

Adipose Tissue↗
PubMed1998

Studies on the animal model of traumatic pseudoaneurysm.

Sixty-nine cases of pseudoaneurysm were made in 72 femoral arteries of 54 Japanese white rabbits, with a successful rate of 95.8%. Colored Doppler's ultrasonic imaging, CT, MRI, arterial angiography, anatomic dissection, and histologic observation were carried out at different times after the operation. The mechanism for the formation of pseudoaneurysm was discussed and the effects of colored Doppler's ultrasonic imaging, CT, MRI, and arterial angiography in the diagnosis of pseudoaneurysm were evaluated.

Aneurysm, False↗
PubMed1998

Influence of dioxin and metal-contaminated sediment on phase I and II biotransformation enzymes in silver crucian carp.

Several biochemical responses were measured in silver crucian carp (Carassius auratus gibelio) after exposure to sediments obtained from contaminated Ya-Er Lake, No. 1 pond, and an unpolluted reference site, Honglian Lake. After 1 week of exposure, a significant induction of the phase I biotransformation enzyme (ethoxylresorufin-o-deethylase, EROD) was found (83-fold of control), whereas the phase II biotransformation enzyme (glutathione S-transferase, GST) exhibited a slight, but significant induction (1.4-fold of control) after 4 weeks of exposure. The level of cellular glutathione in the liver was also slightly elevated after 4 weeks of exposure. The delayed response of GST to the contaminants indicates that the phase I and phase II biotransformation enzymes are regulated differently in fish. The results suggest that EROD is a sensitive bioindicator to assess the toxicity of dioxin-contamined sediment in the laboratory.

Animals↗
PubMed1998

Glutathione S-transferase activity in aquatic macrophytes with emphasis on habitat dependence.

Glutathione S-transferase activity of both the microsomal and soluble fractions was determined in a variety of aquatic macrophytes. The examined enzyme extract was prepared from a combination of leaves and shoots. Four different model substrates were used. The highest conjugation rate was obtained for 1-iodo-2,4-dinitrobenzene, followed by 1-chloro-2,4-dinitrobenzene, p-nitrobenzoyl chloride, and 1,2-dichloro-4-nitrobenzene. Comparison of several samples of Nuphar lutea L. from two different lake areas revealed increased glutathione S-transferase activity in plants from the site contaminated with polyaromatic hydrocarbons.

Environment↗
PubMed1998

Phorbol 12-myristate 13-acetate stimulates lysophosphatidic acid secretion from ovarian and cervical cancer cells but not from breast or leukemia cells.

Lysophosphatidic acid (LPA) is present in ascites from patients with ovarian cancer. It stimulates calcium release and growth of ovarian cancer cells both in vitro and in vivo. Recently, we found that LPA levels were significantly elevated in plasma from patients with ovarian cancer and other gynecological cancers. In contrast, LPA levels were not elevated in patients with breast cancer and leukemias. In view of this, we investigated whether gynecological cancer cells could produce LPA. LPA was extracted from the supernatant of cells cultured in vitro and purified by thin layer chromatography. After hydrolysis and transmethylation, the fatty acid derivatives were analyzed by gas chromatography. We found that the phorbol ester, phorbol 12-myristate 13-acetate (PMA), significantly stimulated the production of LPA in ovarian and cervical cancer cells. In contrast, a small or negligible amount of LPA was produced in breast cancer and leukemia cells upon PMA stimulation. This cell type specificity correlates with our values of plasma LPA, suggesting that gynecological tumor cells may be an important source of the elevated LPA noted in the plasma of patients with these cancers. The cytosolic PLA2 (cPLA2)/Ca2+-independent PLA2 (iPLA2) inhibitor, AACOCF3, inhibited 75.6% PMA-stimulated LPA secretion in ovarian cancer cells, suggesting a cPLA2/iPLA2 activity was involved in LPA production from ovarian cancer cells.

Breast Neoplasms↗
PubMed1998

Rotational Spectra of NeCO2 Isotopomers.

Rotational spectra of seven isotopomers of the weakly bound complex NeCO2, i.e., NeCO2, 22NeCO2, Ne13CO2, 22Ne13CO2, Ne18O13CO, Ne17OCO, and 22Ne17OCO, were measured using a pulsed molecular beam Fourier transform microwave spectrometer. Rotational and centrifugal distortion constants as well as the nuclear quadrupole coupling constants of 17O in Ne17OCO and 22Ne17OCO were determined. The obtained constants were used to derive structural and force field information. The parameters obtained are discussed, and compared with those of other, previously measured, rare gas-CO2 complexes. The 17O nuclear quadrupole coupling constants were analyzed and interpreted in terms of the angular anisotropy of the intermolecular potential energy surface. Copyright 1998 Academic Press.

Journal Article↗
PubMed1998

Role of oxidative stress in the selective toxicity of dieldrin in the mouse liver.

Dieldrin, an organochlorine insecticide, induces hepatic tumors in mice but not in rats. Although the mechanism(s) responsible for this species specificity is not fully understood, accumulating evidence indicates that oxidative stress may be involved. This study examined the association of dieldrin-induced hepatic DNA synthesis with the modulation of biomarkers of oxidative damage to lipids (malondialdehyde [MDA]) and DNA (8-hydroxy-2-deoxyguanosine [oh8dG]), in male B6C3F1 mice and F344 rats fed dieldrin (0.1, 1.0, or 10 mg/kg diet) for 7, 14, 28, and 90 days. The nonenzymatic components of the antioxidant defense system (ascorbic acid, glutathione, and alpha-tocopherol) were also examined. Increased urinary MDA was observed in mice fed 0.1, 1.0, or 10 mg dieldrin/kg diet for 7, 14, 28, and 90 days; while increased hepatic MDA was seen only after 7 days in mice fed 0.1, 1.0, or 10 mg dieldrin/kg diet and after 14 days in mice fed 10 mg/kg diet. In rats, dieldrin had no effect on either hepatic MDA or urine MDA levels after 7, 14, and 28 days of treatment. A dose-dependent increase in urinary MDA was observed in rats at the 90-day sampling time. The only significant elevation in urinary or hepatic oh8dG content was limited to urinary oh8dG in mice fed 10 mg/kg dieldrin diet for 14 days. Dietary dieldrin produced sustained decreases in hepatic and serum alpha-tocopherol and sustained elevations in hepatic ascorbic acid in both mice and rats. Rats, however, possessed a three- to four-fold higher content of endogenous or basal (control) hepatic alpha-tocopherol; and, even when fed 10 mg dieldrin/kg diet, the levels of hepatic alpha-tocopherol were maintained at higher levels than those of mice fed control diet. In both rats and mice fed dieldrin, transient (14 and 28 days on diet) elevations in hepatic glutathione were observed. These data support the hypothesis that the species specificity of dieldrin-induced hepatotoxicity may be related to dieldrin's ability to induce oxidative stress in the liver of mice, but not in rats. Only in mice fed dieldrin was a temporal association of increases in hepatic MDA content and hepatic DNA synthesis seen, suggesting that oxidative damage (shown by increased lipid peroxidation) may be involved in early events in dieldrin-induced hepatocarcinogenesis. Rats may be protected from dieldrin-induced oxidative stress by a more effective antioxidant defense system, characterized by higher basal levels of hepatic alpha-tocopherol and ascorbic acid than that seen in the mouse.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1998

Induction of oxidative stress in rat brain by acrylonitrile (ACN).

Chronic treatment with acrylonitrile (ACN) has been shown to produce a dose-related increase in glial cell tumors (astrocytomas) in rats. The mechanism(s) for ACN-induced carcinogenicity remains unclear. While ACN has been reported to induce DNA damage in a number of short-term systems, evidence for a genotoxic mechanism of tumor induction is the brain is not strong. Other toxic mechanisms appear to participate in the induction of tumor or induce the astrocytomas solely. In particular, nongenotoxic mechanisms of carcinogen induction have been implicated in this ACN-induced carcinogenic effect in the rat brain. One major pathway of ACN metabolism is through glutathione (GSH) conjugation. Extensive utilization and depletion of GSH, an important intracellular antioxidant, by ACN may lead to cellular oxidative stress. The present study examined the ability of ACN to induce oxidative stress in male Sprague-Dawley rats. Rats were administered ACN at concentrations of 0, 5, 10, 100, or 200 ppm in the drinking water and sampled after 14, 28, or 90 days of continuous treatment. Oxidative DNA damage indicated by the presence of 8-hydroxy-2'-deoxyguanosine (OH8dG) and lipid peroxidation indicated by the presence of malondialdehyde (MDA), a lipid peroxidation product, in rat brains and livers were examined. The levels of reactive oxygen species (ROS) were also determined in different rat tissues. Both the levels of nonenzymatic antioxidants (GSH, vitamin E) and the activities of enzymatic antioxidants (catalase, superoxide dismutase, glutathione peroxidase) in rat brains and livers were measured. Increased levels of OH8dG, MDA, and ROS were found in the brains of ACN-treated rats. Decreased levels of GSH and activities of catalase and SOD were also observed in the brains of ACN-treated rats compared to the control group. Interestingly, there were no changes of these indicators of oxidative stress in the livers of ACN-treated rats. Rat liver is not a target for ACN-induced carcinogenesis. These data indicate that ACN selectively induces oxidative stress in rat brain at doses that produce carcinogenesis in chronic treatment studies.

3,4-Methylenedioxyamphetamine↗
PubMed1998

The regulating effects of protein kinase C on the tone of guinea-pig trachea and human lobus bronchi.

The regulating effects of protein kinase C (PKC) on the tone of guinea-pig trachea and human lobus bronchi were investigated by measuring the tone of isolated tracheal and bronchial strips. The effects of PKC on the tone of guinea-pig tracheal and human lobus bronchi were observed and compared. The results showed that: (1) PKC activator PMA induced concentration-dependent relaxation in guinea-pig isolated trachea strips. This relaxation was completely ablated by the pretreatment with 5 x 10(-6) mol/L Ro31-8220 which is a PKC-specific inhibitor, but was not affected by the removal of epithelium (EP), or by the pretreatment with propranolol (beta-receptor blocker) or atropin (M-receptor blocker); (2) PMA led to concentration-dependent contraction in human lobus bronchi. This contractile response was completely depressed by the pretreatment with 5 x 10(-6) mol/L Ro31-8220 and was partly inhibited by 1 x 10(-5) mol/L isoptin (Ca(2+)-antagonist), but was not significantly affected by propranolol or atropin. It is concluded that PKC is involved in the regulation of airway smooth muscle tone. The regulating effects may vary in different animals.

Animals↗
PubMed1998

Mineral catalysis of a potentially prebiotic aldol condensation.

Minerals may have played a significant role in chemical evolution. In the course of investigating the chemistry of phosphonoacetaldehyde (PAL), an analogue of glycolaldehyde phosphate, we have observed a striking case of catalysis by the layered hydroxide mineral hydrotalcite ([Mg2Al(OH)6][Cl.nH2O]). In neutral or moderately basic aqueous solutions, PAL is unreactive even at a concentration of 0.1 M. In the presence of a large excess of NaOH (2 M), the compound undergoes aldol condensation to produce a dimer containing a C3-C4 double-bond. In dilute neutral solutions and in the presence of the mineral, however, condensation takes place rapidly, to produce a dimer which is almost exclusively the C2-C3 unsaturated product.

Acetaldehyde↗
PubMed1998

Colocalization of collagen overexpression and inflammatory cell infiltration in the two-kidney one-clip rat model from the early days of hypertension onward.

In the first 6 days of hypertension, infiltrated mononuclear cells were colocalized with collagen (I) mRNA-overexpressing fibroblasts in the adventitial area of unclipped kidney. The number of adventitial infiltrated mononuclear cells was correlated with adventitial collagen (I) surface expansion. After 22 days of hypertension no collagen (I) mRNA-overexpressing fibroblasts or any increase in collagen area or mononuclear cell infiltration was observed. In the interstitium of unclipped kidney, collagen (I) mRNA overexpression, collagen (I) expansion and mononuclear cell infiltration began later, from the 7th day of hypertension, and kept increasing. In the clipped kidney, after expansion in the first 6 days of hypertension, the adventitial collagen remained stable. These results suggest that in the unclipped kidney fibroblastic activation begins within the first 6 days of hypertension in the adventitial area, but is transient, and fibrosis then spreads in the interstitium. Mononuclear cell infiltration is colocalized and correlated with adventitial and interstitial fibrosis. In the first 6 days, hypertension is not the only cause of fibrosis; the same level of adventitial fibrosis is detected in the nonhypertensive clipped kidney. All observed pathological phenomena could be detected within the first 3 days of hypertension.

Animals↗