Biomedical subjects

Ling Gao

Publications and source records attributed to Ling Gao.

50 records · Page 3Linked to original sources

PubMed2003

Mechanisms for the formation of isoprostane endoperoxides from arachidonic acid. "Dioxetane" intermediate versus beta-fragmentation of peroxyl radicals.

The isoprostanes are a class of autoxidation products generated from arachidonic acid (or its esters) by a free radical initiated process. The potent biological activity of these compounds has been attracting intense research interest since they were detected in humans as well as animal models in the early 1990s. The measurement of these compounds has been regarded as one of the most useful non-invasive biomarkers for oxidative stress status. Two mechanisms for the formation of these compounds have been proposed. In the first mechanism, a peroxyl radical undergoes successive 5-exo cyclizations analogous to the enzymatic mechanism proposed for prostaglandin biosynthesis. The second mechanism starts with a 4-exo cyclization of a peroxyl radical leading to an intermediate dioxetane, a mechanism that has also been proposed for prostaglandin biosynthesis as well as for the formation of 4-hydroxy nonenal (HNE). Autoxidation of cholesteryl-15-HpETE under free radical conditions provides Type IV isoprostanes. The "dioxetane" mechanism for isoprostane generation from 15-HpETE requires that optically pure products are formed from an optically pure reactant, whereas an alternate mechanism for the process involving beta-fragmentation of the 15-peroxyl would give racemic isoprostane products. We have carried out a test of the mechanism based upon these stereochemical requirements. The results of analysis of the product mixture derived from autoxidation of optically pure Ch-15-HpETE by atmospheric pressure chemical ionization-mass spectrometry coupled with chiral high performance liquid chromatography indicate that the major isoprostane diastereomers are formed as a racemic mixture. These experimental results are consistent with a mechanism for isoprostane formation involving beta-fragmentation of the 15-peroxyl radical followed by re-addition of oxygen to form the 11-HPETE peroxyl, and they exclude a mechanism proceeding through the formation of a dioxetane intermediate.

Arachidonic Acid↗

PubMed2003

The photosensitiser xanthurenic acid is not present in normal human lenses.

UV light has often been investigated as a risk factor for the most common cause of blindness, human age-related cataract. One mechanism whereby UV light could induce cataract is via the action of photosensitisers. In this regard, xanthurenic acid has recently been highlighted since it has been reported to be present in the human lens and, in model studies, it markedly enhances the photo-oxidation of proteins by wavelengths of light that penetrate the cornea. In this study we used HPLC and mass spectrometry to examine whether xanthurenic acid is indeed present in human lenses and, if so, the effect of age on its lenticular concentration. Xanthurenic acid could be formed artefactually by incubation of 3-hydroxykynurenine (3OHKyn) yellow, a known autoxidation product of the lenticular UV filter, 3OHKyn, in the presence of air and light, however, it could not be detected in any human lenses studied. Therefore, it appears unlikely that xanthurenic acid plays a role in lens aging or human cataract.

Adolescent↗

PubMed2003

Rocuronium infusion requirements and plasma concentrations at constant levels of neuromuscular paralysis during three phases of liver transplantation.

STUDY OBJECTIVE: To develop a rapid online test of graft liver function during liver transplantation. DESIGN: Prospective, observational study. SETTING: University hospital transplant unit. PATIENTS: 17 adult patients with end-stage liver disease who underwent liver transplantation surgery. INTERVENTIONS: Rocuronium infusion dose requirements and plasma concentrations to maintain constant levels of neuromuscular paralysis during three phases of liver transplantation and their relationship with early postoperative liver function tests were studied. MEASUREMENTS: Infusion dose requirements of rocuronium, assay of rocuronium plasma concentrations using gas chromatography-mass spectrometry, and intensity of neuromuscular blockade were measured. MAIN RESULTS: A 24% decrease in rocuronium infusion requirements was observed during the an-hepatic phase. Rocuronium requirement during the neohepatic phase was increased only modestly or remained unchanged in 14 of the 16 patients who had normal graft function in the immediate postoperative period. Rocuronium plasma concentrations for maintaining constant levels of paralysis were significantly lower during the neohepatic phase than during the paleohepatic and anhepatic phases, indicating that there is likely to be a change in pharmacodynamics during this phase. Significant reduction in rocuronium infusion requirements during the neohepatic phase was observed in the only patient who had poor graft function in the early postoperative stage, suggesting that the reduced infusion requirement to maintain a constant neuromuscular paralysis may be related to the functional state of the graft liver after reperfusion. CONCLUSIONS: A significant reduction in rocuronium infusion requirement during the neohepatic phase may be suggestive of impaired organ function after reperfusion of the graft liver. Rocuronium may serve as a potential online indicator of graft liver function during liver transplantation by measurement of its infusion requirements during transplantation.

Adolescent↗

PubMed2003

luxS and arcB control aerobic growth of Actinobacillus actinomycetemcomitans under iron limitation.

LuxS is responsible for the production of autoinducer 2 (AI-2), which functions in Vibrio harveyi as a quorum-sensing signal that controls the cell density-dependent expression of the lux operon. In nonluminescent organisms, the physiologic role of AI-2 is not clear. We report that inactivation of luxS in Actinobacillus actinomycetemcomitans JP2 results in reduced growth of the mutant, but not the wild-type organism, under aerobic, iron-limited conditions. Stunted cultures of the luxS mutant A. actinomycetemcomitans JP2-12 grew to high cell density when subcultured under iron-replete conditions. In addition, the mutant strain grew to high cell density under iron limitation after transformation with a plasmid containing a functional copy of luxS. Results of real-time PCR showed that A. actinomycetemcomitans JP2-12 exhibited significantly reduced expression of afuA (eightfold), fecBCDE (10-fold), and ftnAB (>50-fold), which encode a periplasmic ferric transport protein, a putative ferric citrate transporter, and ferritin, respectively. The expressions of putative receptors for transferrin, hemoglobin, and hemophore binding protein were also reduced at more modest levels (two- to threefold). In contrast, expressions of sidD and frpB (encoding putative siderophore receptors) were increased 10- and 3-fold, respectively, in the luxS mutant. To better understand the mechanism of the AI-2 response, the A. actinomycetemcomitans genome was searched for homologs of the V. harveyi signal transduction proteins, LuxP, LuxQ, LuxU, and LuxO. Interestingly, ArcB was found to be most similar to LuxQ sensor/kinase. To determine whether arcB plays a role in the response of A. actinomycetemcomitans to AI-2, an arcB-deficient mutant was constructed. The isogenic arcB mutant grew poorly under anaerobic conditions but grew normally under aerobic iron-replete conditions. However, the arcB mutant failed to grow aerobically under iron limitation, and reverse transcriptase PCR showed that inactivation of arcB resulted in decreased expression of afuA and ftnAB. Thus, isogenic luxS and arcB mutants of A. actinomycetemcomitans exhibit similar phenotypes when cultured aerobically under iron limitation, and both mutants exhibit reduced expression of a common set of genes involved in the transport and storage of iron. These results suggest that LuxS and ArcB may act in concert to control the adaptation of A. actinomycetemcomitans to iron-limiting conditions and its growth under such conditions.

Aerobiosis↗

PubMed2003

Positive and negative cis-acting regulatory sequences control expression of leukotoxin in Actinobacillus actinomycetemcomitans 652.

Integration of IS1301 into an AT-rich inverted repeat located upstream of the ltx operon was previously shown to confer a hyperleukotoxic phenotype in Actinobacillus actinomycetemcomitans IS1 (T. He, T. Nishihara, D. R. Demuth, and I. Ishikawa, J. Periodontol. 70:1261-1268, 1999), but the mechanism leading to increased leukotoxin production was not determined. We show that an IS1 ltx promoter::lacZ reporter construct expresses 12-fold higher levels of beta-galactosidase activity than a reporter containing the ltx promoter from A. actinomycetemcomitans 652, suggesting that IS1301 increases transcription of the ltx operon. Examination of the IS1301 sequence identified a potential outwardly directed promoter. However, site-specific mutagenesis of the -35 element of the putative promoter had no effect on the transcriptional activity of the IS1 reporter construct. Furthermore, reverse transcriptase PCR and real-time PCR experiments did not detect a transcript that was initiated within IS1301. These results suggest that increased expression of leukotoxin in strain IS1 does not arise from an outwardly directed IS1301 promoter. To determine how IS1301 alters transcriptional regulation of the ltx operon, cis-acting sequences that regulate leukotoxin expression were identified. The AT-rich sequence that resides downstream from the site of IS1301 insertion was shown to function as a positive cis-acting regulator of leukotoxin expression. This sequence resembles an UP element in its location, AT-rich content, and activity and is homologous to the consensus UP element sequence. In addition, a negative cis-acting sequence was identified upstream from the site of IS1301 insertion, and deletion of this region increased promoter activity by fourfold. Mobility shift experiments showed that this region bound to a protein(s) in extracts from A. actinomycetemcomitans 652. The specific sequences required for this interaction were localized to a 26-nucleotide segment of the ltx promoter that resides 17 bp upstream from the site of IS1301 insertion. Together, these results suggest that positive and negative cis-acting sequences regulate leukotoxin expression and that IS1301 may increase transcription of the ltx operon in A. actinomycetemcomitans IS1 by displacing a negative cis-acting regulator approximately 900 bp upstream from the basal elements of the ltx promoter.

Actinobacillus Infections↗

PubMed2003

Captopril inhibits glucose accumulation in retinal cells in diabetes.

PURPOSE: Clinical studies have detected an unexpected inhibition of diabetic retinopathy by angiotensin-converting enzyme (ACE) inhibitors, but the mechanism for this action is unclear. In light of evidence indicating that the severity of hyperglycemia is a major initiating factor in the pathogenesis of the retinopathy, this study was conducted to examine the effect of ACE inhibitors on glucose accumulation in retinas of diabetic rats. METHODS: Rats were made experimentally diabetic by injection of streptozotocin and treated with captopril (25 mg/kg body weight per day) or atenolol (10 mg/kg body weight per day) for 8 weeks. Bovine retinal endothelial cells were cultured in medium containing either 5.5 or 25 mM glucose and treated with different concentrations of captopril or lisinopril for 5 days. Glucose content in retinas and cultured cells was measured by spectrometry. Expression of glucose transporter (GLUT)-1 in retinas and cultured cells was determined by Western blot analysis. Glucose uptake was performed by using 3-O-methyl-D-[(3)H] glucose. RESULTS: Treatment of rats with captopril inhibited the diabetes-induced accumulation of glucose in the retina by 48% (P < 0.01) compared with the diabetic control, but atenolol had no significant effect. Similarly, captopril and lisinopril significantly inhibited intracellular accumulation of glucose in primary bovine retinal endothelial cells cultured in an elevated glucose concentration. These data indicate that the captopril-induced inhibition of glucose accumulation observed in retinal tissue of diabetic rats was not due to reduction in blood pressure or in vascular permeability. Although it had no effect on the expression of the GLUT1 in retinas and cultured retinal endothelial cells, captopril at a concentration of 2 mM significantly inhibited the high-glucose-induced increase in GLUT1-mediated glucose transport in cultured retinal endothelial cells by 67% +/- 10%. CONCLUSIONS: Inhibition of glucose accumulation within retinal cells probably contributes at least in part to the observed inhibition of diabetic retinopathy by ACE inhibitors.

Adrenergic beta-Antagonists↗

PubMed2003

[Study on the gene knockout model mice of Duchenne muscular dystrophy].

OBJECTIVE: With a view to further experimental studies on Duchenne muscular dystrophy (DMD), we investigated the motor function, serum creatine kinase (CK) level and pathological characteristics of muscular tissue in C57, mdx, dystrophin/utrophin gene double knockout (dko)mice. METHODS: Gene identification was performed for the filial generation of heterozygote of utrophin gene knockout of mdx mice for acquiring mdx and dko mice. The motor function, serum CK level, and the pathology of muscular tissue of C57, mdx and dko mice were compared. RESULTS: There were significant differences in all detecting aspects between C57 mouse and dko mouse. In the aspects of serum CK level and pathology of muscular tissue, there were differences between C57 mouse and mdx mouse; however, no significant difference in motor function was observed between them. CONCLUSION: Compared with mdx mouse, dko mouse is in a more serious illness state which is nearer to the natural state of DMD. Therefore, dko mouse is a more ideal model for studying the praxiology of DMD.

Animals↗

PubMed2002

Potent suppression of viral infectivity by the peptides that inhibit multimerization of human immunodeficiency virus type 1 (HIV-1) Vif proteins.

Virion infectivity factor (Vif) is essential for the replication of human immunodeficiency virus type 1 (HIV-1) in vivo, but its function remains uncertain. Recently, we have shown that Vif proteins are able to form multimers, including dimers, trimers, or tetramers. Because the multimerization of Vif proteins is required for Vif function in the viral life cycle, we propose that it could be a novel target for anti-HIV-1 therapeutics. Through a phage peptide display method, we have identified a set of 12-mer peptides containing a PXP motif that binds to HIV-1 Vif protein. These proline-enriched peptides potently inhibited the Vif-Vif interaction in vitro. We have also screened a set of synthesized Vif peptides (15-mer), which covers all the amino acids of the HIV-1 Vif protein sequence, for their ability to inhibit the Vif-Vif interaction in vitro. We demonstrated that Vif-derived proline-enriched peptides that contain the (161)PPLP(164) domain are able to inhibit the Vif-Vif interaction. Conversely, the deletion of the (161)PPLP(164) domain of Vif protein will significantly impair the capability of Vif proteins to interact with each other, indicating that the (161)PPLP(164) domain plays a key role in Vif multimerization. All these results demonstrate that the proline-enriched peptides block the multimerization of Vif through interfering with the polyproline interfaces of Vif formed by (161)PPLP(164) domain. Moreover, these peptides which inhibit the Vif-Vif interaction in vitro potently inhibit HIV-1 replication in the "nonpermissive" T-cells. We propose that this study starts a novel strategy to develop structural diverse inhibitors of Vif such as peptidomimetics or small organic molecules.

Amino Acid Sequence↗

PubMed2002

Formation of highly reactive A-ring and J-ring isoprostane-like compounds (A4/J4-neuroprostanes) in vivo from docosahexaenoic acid.

Free radical-initiated oxidant injury and lipid peroxidation have been implicated in a number of neural disorders. Docosahexaenoic acid is the most abundant unsaturated fatty acid in the central nervous system. We have shown previously that this 22-carbon fatty acid can yield, upon oxidation, isoprostane-like compounds termed neuroprostanes, with E/D-type prostane rings (E(4)/D(4)-neuroprostanes). Eicosanoids with E/D-type prostane rings are unstable and dehydrate to cyclopentenone-containing compounds possessing A-type and J-type prostane rings, respectively. We thus explored whether cyclopentenone neuroprostanes (A(4)/J(4)-neuroprostanes) are formed from the dehydration of E(4)/D(4)-neuroprostanes. Indeed, oxidation of docosahexaenoic acid in vitro increased levels of putative A(4)/J(4)-neuroprostanes 64-fold from 88 +/- 43 to 5463 +/- 2579 ng/mg docosahexaenoic acid. Chemical approaches and liquid chromatography/electrospray ionization tandem mass spectrometry definitively identified them as A(4)/J(4)-neuroprostanes. We subsequently showed these compounds are formed in significant amounts from a biological source, rat brain synaptosomes. A(4)/J(4)-neuroprostanes increased 13-fold, from a basal level of 89 +/- 72 ng/mg protein to 1187 +/- 217 ng/mg (n = 4), upon oxidation. We also detected these compounds in very large amounts in fresh brain tissue from rats at levels of 97 +/- 25 ng/g brain tissue (n = 3) and from humans at levels of 98 +/- 26 ng/g brain tissue (n = 5), quantities that are nearly an order of magnitude higher than other classes of neuroprostanes. Because of the fact that A(4)/J(4)-neuroprostanes contain highly reactive cyclopentenone ring structures, it would be predicted that they readily undergo Michael addition with glutathione and adduct covalently to proteins. Indeed, incubation of A(4)/J(4)-neuroprostanes in vitro with excess glutathione resulted in the formation of large amounts of adducts. Thus, these studies have identified novel, highly reactive A/J-ring isoprostane-like compounds that are derived from docosahexaenoic acid in vivo.

Animals↗

PubMed2002

[Effect of cilostazol on adhesion molecules of STZ-induced diabetic rats].

OBJECTIVE: To investigate the effect of cilostazol on the expression of CD(54), CD(106), CD(62P) in kidney, aorta, surface of platelet, and mononuclear cell of blood in STZ-induced diabetic rats. METHODS: Rats were randomly divided into four groups: normal control group (n = 8), diabetes control group (n = 8), insulin group (n = 7 subcutaneous injection of insulin, 1 approximately 4 U * d(-1)), and cilostazol group (n = 7, gastric infusion of cilostazol, 18 mg * kg(-1) * d(-1)). Sciatic nerve conductive velocity and level of CD(54)/CD(62p) on the surface of mononuclear cell/platelet were examined by flow cytometry. Immunohistochemistry was used to examine the expression of CD(54) and/or CD(106) in kidney and aorta. RESULTS: The sciatic nerve conductive velocity was 20.3 +/- 2.2 m * s(-1) and 28.9 +/- 7.9 m * s(-1) in diabetic rats and cilostazol group respectively (P < 0.05). The mononuclear cell surface expression of CDa-a(c)54 was 69.1 +/- 14.9% and 25.9 +/- 8.6% in diabetic group and cilostazol group respectively (P < 0.05). The expression of CD(54)/CD(106) in kidney and aorta was decreased. The expression of CD(62p) on the surface of platelets was 40.4 +/- 8.7% in diabetic group and 28.5 +/- 3.7% in cilostazol group (P > 0.05). CONCLUSION: Cilostazol treatment delays the development of diabetic pathological change in kidney, aorta and sciatic nerve and decreases the expression of CD(54) and CD(106).

Animals↗

PubMed2002

The cyclopentenone product of lipid peroxidation, 15-A(2t)-isoprostane (8-isoprostaglandin A(2)), is efficiently conjugated with glutathione by human and rat glutathione transferase A4-4.

Glutathione transferases (GSTs) are a large family of enzymes that can be divided into different classes based on structure. There has been considerable interest in the ability of GSTs to conjugate and inactivate endogenously derived reactive lipid peroxidation products that contain alpha,beta-unsaturated carbonyl moieties such as 4-hydroxyalkenals. One enzyme with prominent activity toward these substrates is human GST A4-4. Recently, we described a novel series of compounds termed A(2)/J(2)-isoprostanes (IsoPs) that are formed endogenously in humans from the free radical-initiated peroxidation of arachidonic acid. These compounds contain alpha,beta-unsaturated carbonyl groups and have structures similar to cyclooxygenase-derived PGA(2) and PGJ(2). Because of their chemical reactivity, these compounds may mediate tissue injury associated with oxidant stress. Herein, we report that the A-ring IsoP 15-A(2t)-IsoP (8-iso-PGA(2)) is efficiently conjugated to glutathione (GSH) by human GST A4-4 with a k(cat)/K(m) value of >200 s(-)(1) mM(-)(1). The k(cat)/K(m) value for conjugation of 15-A(2t)-IsoP by the homologous rat GST A4-4 is >2000 s(-)(1) mM(-)(1). Similar high enzyme activities were observed when PGA(2) was used as a substrate. In contrast, the human GSTs A1-1, M1-1, M2-2, P1-1, and T1-1 and rat GST T2-2 did not significantly metabolize 15-A(2t)-IsoP. These studies have therefore defined a potentially important route by which cyclopentenone IsoPs are metabolized that may serve as a mechanism for the inactivation of these highly reactive compounds.

Animals↗

PubMed2002

Determination of domperidone in human plasma by LC-MS and its pharmacokinetics in healthy Chinese volunteers.

AIM: To determine the concentration of domperidone in human plasma and investigate its pharmacokinetics in Chinese male volunteers following oral administration of a single dose of the medicine 20 mg. METHODS: Plasma samples were processed by liquid-liquid extraction and a liquid chromatographic-mass spectrometric assay was developed for the determination of domperidone in human plasma. RESULTS: Assay linearity was obtained in the range of 0.52 - 154.5 microg/L (r = 0.9999). The recovery of domperidone from human plasma was more than 75 %. The intraday and interday relative standard deviations (RSD) for the lowest concentration examined (0.52 microg/L) were 9.4 % and 7.6 %, respectively. The method was utilized to determine the concentration of domperidone in Chinese male volunteers. The concentration-time curve was fitted to a two-compartment model. Its main pharmacokinetic parameters were: Tmax = (0.8 +/- 0.7) h, Cmax = (50 +/- 32) microg/L, T1/2 = (7.8 +/- 1.6) h. CONCLUSION: The method described in this report was of high sensitivity and selectivity for accurate determination of the plasma concentration of domperidone in human.

Adult↗

PubMed2002

Morphological changes in canine lungs perfused with modified Euro-Collins solution.

OBJECTIVE: To investigate the structural changes in canine lungs perfused with modified Euro-Collins solution (mECS), thus providing insight into the preservation of the donor lung tissue for transplantation. METHODS: Six dogs were anesthetized and pulmonary perfusion with mECS (4 degrees Celsius) was performed before the lungs were isolated thereafter and stored at 4 to 8 degrees Celsius for 30, 60, 120, 180, 240 min respectively, after which tissue samples of the donor lung were taken for morphological observation with both light and transmission electron microscope. RESULTS: Immediately after reperfusion, the pulmonary tissues exhibited clear and intact structures,showing that mitochondrion swelled slightly in the alveolar epithelial cells (AECs). Mild edema occurred in the alveolar wall and the tissues around the veins at 30 min after preservation, which exacerbated to dilapidation of the alveolar wall and obvious tumefaction of the mitochondrion in the AECs at 60 min. At 180 min, rupture of alveolar wall and emergence of the alveolus was observed at 120 min, and a few pulmonary bullae were formed, the AECs (I) of which presented vacuolar changes in the cytoplasma and mitochondrion, with disappearance of the tiny villus of the AECs (II) at 240 min. CONCLUSION: Within 4 h of preservation in mECS, the pulmonary tissues do not undergo obvious changes as signs of injury, which, however, may not be the case after longer preservation.

Animals↗

PubMed1997

The Properties of Serum GPI-PLD.

The purified glycosylphosphatidylinositol phospholipase D (GPI-PLD) is a single-polypeptide with a molecular weight of about 1OO kD. However, the enzyme is eluted in the fraction of 500 kD when human serum undergoes gel filtration chromatography. To study the natural state of GPI-PLD might help to visualize its physiological function. By using gel filtration, hydrophobic column chromatography and ultracentrifugation, and analyzing the concentration of phospholipids, triglycerides and cholesterol, we found that GPI-PLD did not exist as polymer of polypeptids in serum, but combined with the serum lipids to form a complex of lipids and proteins. As a result, it is present in a density zone similar to HDL after ultracentrifugation, this complex exists in serum separately from the subfractions of HDL which are abundant in Apo-A1. In addition it could bind on the heparin-Sepharose affinity chromatography column as Apo-E.

Journal Article↗