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Biomedical subjects

G Luo

Publications and source records attributed to G Luo.

At least 109 records · Page 6Linked to original sources
PubMed1997

Pharmacology of ABT-491, a highly potent platelet-activating factor receptor antagonist.

ABT-491 (4-ethynyl-N, N-dimethyl-3-[3-fluoro-4-[(2-methyl-1H-imidazo-[4,5-c]pyridin-1-yl)methy l]benzoyl]-1H- indole-1-carboxamide hydrochloride) is a novel PAF (platelet-activating factor) receptor antagonist with a K(i) for inhibiting PAF binding to human platelets of 0.6 nM. Binding kinetics of ABT-491 to the PAF receptor is consistent with a relatively slow off-rate of the antagonist when compared to PAF. Inhibition of PAF binding is selective and is correlated with functional antagonism of PAF-mediated cellular responses (Ca2+ mobilization, priming, and degranulation). Administration of ABT-491 in vivo leads to potent inhibition of PAF-induced inflammatory responses (increased vascular permeability, hypotension, and edema) and PAF-induced lethality. Oral potency (ED50) was between 0.03 and 0.4 mg/kg in rat, mouse, and guinea-pig. When administered intravenously in these species, ABT-491 exhibited ED50 values between 0.005 and 0.016 mg/kg. An oral dose of 0.5 mg/kg in rat provided > 50% protection for 8 h against cutaneous PAF challenge. ABT-491 administered orally was also effective in inhibiting lipopolysaccharide-induced hypotension (ED50 = 0.04 mg/kg), gastrointestinal damage (0.05 mg/kg, 79% inhibition), and lethality (1 mg/kg, 85% vs. 57% survival). The potency of this novel antagonist suggests that ABT-491 will be useful in the treatment of PAF-mediated diseases.

Acute Disease↗
PubMed1997

Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein.

Calcification of the extracellular matrix (ECM) can be physiological or pathological. Physiological calcification occurs in bone when the soft ECM is converted into a rigid material capable of sustaining mechanical force; pathological calcification can occur in arteries and cartilage and other soft tissues. No molecular determinant regulating ECM calcification has yet been identified. A candidate molecule is matrix GLA protein (Mgp), a mineral-binding ECM protein synthesized by vascular smooth-muscle cells and chondrocytes, two cell types that produce an uncalcified ECM. Mice that lack Mgp develop to term but die within two months as a result of arterial calcification which leads to blood-vessel rupture. Chondrocytes that elaborate a typical cartilage matrix can be seen in the affected arteries. Mgp-deficient mice additionally exhibit inappropriate calcification of various cartilages, including the growth plate, which eventually leads to short stature, osteopenia and fractures. These results indicate that ECM calcification must be actively inhibited in soft tissues. To our knowledge, Mgp is the first inhibitor of calcification of arteries and cartilage to be characterized in vivo.

Animals↗
PubMed1997

Purification and characterization of bimodular and trimodular derivatives of the erythromycin polyketide synthase.

Modular polyketide synthases (PKSs), such as the 6-deoxyerythronolide B synthase (DEBS), catalyze the biosynthesis of structurally complex and medicinally important natural products. DEBS is a dimeric protein complex that consists of three large multidomain polypeptide chains, DEBS 1, DEBS 2, and DEBS 3. In turn, each polypeptide includes two modules, where one module is responsible for a single round of condensation and associated reduction reactions. A hybrid protein comprised of the first two modules of DEBS fused to a thioesterase domain (DEBS 1 + TE) was purified to homogeneity in a fully active form (Kcat = 4.8 min-1). Synthesis of the anticipated triketide lactone required the presence of (2RS)-methylmalonyl-CoA and NADPH. When available, propionyl-CoA is the preferred source of primer units. However, in its absence the enzyme can derive primer units via decarboxylation of a methylmalonyl extender. The two subunits of an engineered trimodular derivative of DEBS, DEBS 1 and module 3 of DEBS 2 linked to the TE domain (module 3 + TE), were also individually purified and reconstituted to produce the expected tetraketide lactone in vitro (Kcat = 0.23 min-1). The considerably lower specific activity of this trimodular PKS relative to its bimodular counterpart presumably reflects inefficient association between DEBS 1 and module 3 + TE. As expected, module 3 + TE could be efficiently cross-linked as a homodimer. In contrast, no cross-links were detectable between modules 2 and 3, even though biosynthesis of the tetraketide requires transient interactions to occur between these two modules. Since module 3 only contains the minimal set of active sites required in a module (a ketosynthase, an acyltransferase, and an acyl carrier protein domain) and is the first active unimodular protein to be purified to homogeneity, it represents an attractive target for future biophysical and structural studies.

Cell-Free System↗
PubMed1997

Complete cDNA sequence and tissue localization of N-RAP, a novel nebulin-related protein of striated muscle.

We have cloned and sequenced the full-length cDNA of N-RAP, a novel nebulin-related protein, from mouse skeletal muscle. The N-RAP message is specifically expressed in skeletal and cardiac muscle, but is not detected by Northern blot in non-muscle tissues. The full-length N-RAP cDNA contains an open reading frame of 3,525 base pairs which is predicted to encode a protein of 133 kDa. A 587 amino acid region near the C-terminus is 45% identical to the actin binding region of human nebulin, containing more than 2 complete 245 residue nebulin super repeats. The N-terminus contains the consensus sequence of a cysteine-rich LIM domain, which may function in mediating protein-protein interactions. These data suggest that the encoded protein may link actin filaments to some other proteins or structure. We expressed full-length N-RAP in Escherichia coli, as well as the nebulin-like super repeat region of N-RAP (N-RAP-SR) and the region between the LIM domain and N-RAP-SR (N-RAP-IB). An anti-N-RAP antibody raised against a 30 amino acid peptide corresponding to sequence from N-RAP-IB detected recombinant N-RAP and N-RAP-IB, but failed to detect N-RAP-SR. This antibody specifically identified a 185 kDa band as N-RAP on immunoblots of mouse skeletal and cardiac muscle proteins. In an assay of actin binding to electrophoresed and blotted proteins, we detected significant actin binding to expressed nebulin super repeats and N-RAP-SR, but only a trace amount of binding to N-RAP-IB. In immunofluorescence experiments, N-RAP was found to be localized at the myotendinous junction in mouse skeletal muscle and at the intercalated disc in cardiac muscle. Based on its domain organization, actin binding properties, and tissue localization, we propose that N-RAP plays a role in anchoring the terminal actin filaments in the myofibril to the membrane and may be important in transmitting tension from the myofibrils to the extracellular matrix.

Actins↗
PubMed1997

Inhibition of influenza viral polymerases by minimal viral RNA decoys.

All gene segments of influenza virus share a common feature at their respective termini. Both the 5'- and 3'-terminal sequences are highly conserved and possess partial inverted complementarity. This allows for the formation of a double-stranded duplex, which plays a major role in transcription, replication and packaging of the viral genome. In vitro studies have shown that the viral polymerase binds to short RNA molecules containing these termini. In this study, attempts were made to test whether mini-RNA decoys containing either or both termini can inhibit the activity of the viral polymerase in vivo. RNA molecules containing either the 5' or the 3' noncoding sequences were unable to inhibit NS-CAT RNA replication, while mini-RNA decoys consisting of both the 5' and 3' noncoding sequences of vRNA or cRNA were able to efficiently inhibit the activity of the viral polymerases expressed from vaccinia virus vectors.

Base Sequence↗
PubMed1997

Molecular mechanism underlying the action of a novel fusion inhibitor of influenza A virus.

In the initial stages of influenza virus infection, the hemagglutinin (HA) protein of influenza virus mediates both adsorption and penetration of the virus into the host cell. Recently, we identified and characterized BMY-27709 as an inhibitor of the H1 and H2 subtypes of influenza A virus that specifically inhibits the HA function necessary for virus-cell membrane fusion (G.-X. Luo, R. Colonno, and M. Krystal, Virology 226:66-76, 1996). Studies presented herein show that the inhibition is mediated through specific interaction with the HA protein. This binding represses the low-pH-induced conformational change of the HA protein which is a prerequisite for membrane fusion. In an attempt to define the binding pocket within the HA molecule, a number of drug-resistant viruses have been isolated and characterized. Sequence analyses of the HA gene of these drug-resistant viruses mapped amino acid changes responsible for drug resistance to a region located near the amino terminus of HA2. In addition, we have identified inactive analogs of BMY-27709 which are able to compete out the inhibitory activity of BMY-27709. This finding suggests that inhibition of the HA-mediated membrane fusion by this class of compounds is not solely the result of binding within the HA molecule but requires specific interactions.

Animals↗
PubMed1997

Growth hormone-induced tyrosyl phosphorylation and deoxyribonucleic acid binding activity of Stat5A and Stat5B.

GH is known to activate JAK2 tyrosine kinase and members of the Stat family of transcription factors, including Stats 1, 3, and 5. The recent observation that at least two Stat5 proteins (Stat5A and Stat5B) exist in mouse and human, raises the question of whether GH activates both Stat5A and Stat5B and, if so, whether the requirements for activation are the same. An initial report investigating this issue demonstrated GH-dependent activation of Stat5A but not Stat5B. In this paper, we demonstrate (in COS cells expressing rat GH receptor (rGHR) and either Stat5A or Stat5B, 3T3-F442A fibroblasts, and CHO cells expressing rGHR) that GH induces tyrosyl phosphorylation of both Stat5A and Stat5B. Similar time courses of phosphorylation were observed for the two proteins. Interestingly, the pattern of observed bands differs for the two forms of Stat5. Two closely migrating Stat5A bands can be detected in cells treated with or without GH. Both of these bands become tyrosyl phosphorylated in response to GH. Three species of Stat5B are observed in untreated cells. An additional, more slowly migrating Stat5B band, appears upon treatment with GH. The three more slower migrating Stat5B bands observed in response to GH contain phosphorylated tyrosyl residues. We further demonstrate that GH induces binding of Stat5A and Stat5B, as well as Stat1, to the GAS-like element in the beta-casein promoter. We and others have demonstrated previously that specific regions of GHR are required for GH-dependent activation of what is here identified as Stat5B. To gain insight into the mechanism by which GH promotes tyrosyl phosphorylation of Stat5A, GH-dependent tyrosyl phosphorylation of Stat5A was examined in CHO cells expressing truncated and mutated rGHR. The results indicate that Stat5A and Stat5B require the same regions of rGHR for maximal activation by GH: the C-terminal half of the cytoplasmic domain; tyrosines 333 and/or 338 in the N-terminal half of the cytoplasmic domain; and the regions required for JAK2 activation. To dissect further the mechanism by which GH activates Stat5A and B, the requirement for JAK2 in GH-dependent Stat5 tyrosyl phosphorylation was assessed using JAK2-deficient cells expressing GHR (gamma2A-GHR) and the wild-type parental cell line expressing GHR (2C4-GHR). GH-induced tyrosyl phosphorylation of Stat5B in 2C4-GHR cells but not in the JAK2 deficient, gamma2A-GHR cells, indicating that JAK2 is required for GH-dependent tyrosyl phosphorylation of Stat5B. Western blotting revealed that Stat5A is not expressed in this cell type. Taken together, these findings suggest that: 1) GH activates both Stat5A and Stat5B in several cell types; 2) the pattern of bands observed differs for Stat5A and Stat5B; 3) GH-dependent tyrosyl phosphorylation of Stat5A requires specific regions of GHR, and these requirements are the same as for Stat5B; and 4) JAK2 kinase is required for GH-dependent tyrosyl phosphorylation of Stat5B and, most likely, Stat5A.

Animals↗
PubMed1997

[Effect of hypoxia on maximal myocardial blood flow in right ventricle].

In order to study the changes of coronary reserve capacity, the effects of hypoxia on hemodynamics and maximal myocardial blood flow in right ventricle were observed. Rats wre divided into 3 groups:normoxic group (control), acute hypoxic group and chronic hypoxic group. Maxmimal myocardial blood flow in the right ventricle was measured with 99 m Tc radiolabelled from RBC during adenosine infusion. The results showed that cardiac output, PaO2 and oxygen delivery were decreased during acute hypoxia, but myocardial blood flow in right ventricle was increased, as compared with the control group. There were no significant change of maximal myocardial blood flow in the right ventricle in acute hypoxic rats. Hematocrit and blood viscosity at different shear rate and RV weight index were augmented, whereas oxygen delivery and myocardial blood flow became normal during chronic hypoxia. Maximal blood flow in RV was significantly decreased whereas arterial wall thickness and collagen in arterial adventitia were increased in chronic hypoxic rats. The above results suggest that the decreased coronary reserve might beresulted from the increasedblood viscosity, arterial wall thickness and collagen in arterial adventitia and right ventricular hypertrophy.

Animals↗
PubMed1997

[Effect of interleukin-1, interleukin-6 on the intercellular communication of rat thyroid FRTL-5 cells].

We observed the effects of thyrotropin (TSH), interleukin (IL)-1 beta and IL-6 on the intercellular communication of FRTL-5 cells with fluorenscence redistribution after photobleaching (FRAP) analysis. FRTL-5 cells were cultured and exposed to the different concentrations of TSH, IL-1 beta and IL-6 for 12 hours. The mean fluorescence recovery rate (MFRR, %/min) of the cells labelled with carboxyfluocein diacetate (CFDA) after photobleaching was measured with laser scanning cytometry. The MFRR (%/min) of the cells after exposure to TSH was 0.445 +/- 0.033 at the control group, 0.679 +/- 0.054 at the group of 0.1 U/L, 0.950 +/- 0.073 at the group of 1 U/L, and 0.799 +/- 0.082 at the group of 5 U/L, respectively (F = 11.44, P < 0.01). The indicated that TSH could enhance the intercellular communication of FRTL-5 cells. The MFRR after exposure to IL-1 beta was 0.564 +/- 0.032 at the control group, 0.485 +/- 0.042 at the group of 10(3) U/L, 0.445 +/- 0.043 at the group of 10(4) U/L and 0.405 +/- 0.029 at the group of 10(5) U/L, respectively (F = 3.58, P < 0.01). The suggested that IL-1 beta could inhibite the intercellular communication of FRTL-5 cells. IL-6 had no obvious effect on the intercellular communication of FRTL-5 cells.

Animals↗
PubMed1997

[Local electroretinogram and pattern visual evoked potential in maculopathies].

OBJECTIVES: To observe the changes of local electroretinogram (LERG) and pattern visual evoked potential (PVEP) in maculopathies and to evaluate the value of both LERG and PVEP in clinical application. METHODS: LERGs at 5 degrees, 10 degrees and 15 degrees macular region were recorded from 27 normal subjects (54 eyes). The LERG and PVEP were recorded from 25 patients (35 eyes) with maculopathies. RESULTS: In the patients with maculopathies, the amplitudes of LERG a wave and b wave at 5 degrees, 10 degrees and 15 degrees macular region were significantly lowered (P < 0.01), the mean value of P1 latency was prolonged and that of N1-P1 amplitude of VEP was lowered at 36.5', 73' and 146' in the comparison with the control group. In approximately the same stimulated area (15 degrees LERG and 14.8 degrees x 19.0 degrees PVEP), the abnormal rate of LERG was 62.2%, and the abnormal rates of PVEPs were 48.5% (146'), 54.5% (73') and 48.5% (36.5') respectively, no significant difference in the abnormal rate between LERG and each value of PVEP (P > 0.05) being found. CONCLUSION: The abnormal rates of LERG and PVEP in maculopathies are close. Because LERG is not influenced by visual pathway diseases and visual acuity, LERG is more effective than PVEP and is a direct way in measuring macular function.

Adult↗
PubMed1997

[Lecture of principles and applications of capillary electrophoresis (V) Applications of capillary electrophoresis in pharmaceutical analysis and clinical chemistry].

A review with 68 references is presented on the application and new developments of CE in pharmaceutical analysis, clinical chemistry and single cell analysis etc. The pharmaceutical analysis includes main component assay, determination of drug-related impurities, stoichiometric determination and quantitative determinations. The Chinese herb analysis includes determinations of main components and prescriptions of Chinese herb. The chiral separation includes separation mechanism and some new chiral selectors. The application of CE in clinical chemistry includes determinations of serum and protein, monitoring of drug metabolism, etc. Single cell analysis includes determinations of component in neurocells and human erythrocyte.

Animals↗
PubMed1997

[Selective extraction of organotin from biological sample by supercritical fluid extraction].

The preliminary separation and concentration of organotin compounds from the matrix of biological samples is necessary, due to the very low content of organotin and the fat included in samples will largely interfere the analysis of organotins. In this work, supercritical fluid extraction (SFE) for the preliminary separation and concentration of organotin has been studied. Pure CO2 was used as mobile phase. The extractives can be used to supercritical fluid chromatographic (SFC) analysis of organotins. The model sample was prepared by adding diphenyltin (DPT) to soya powder. The effects of the pressure and temperature on SFE were investigated. A small amount of fat was also extracted together with organotins. Extract was injected into SFC system, in which a Kapcell Pak ODS column was used for separation of organotin and fat. The effect of pressure on the extraction percentage of DPT and fat was investigated at 40 degrees C. At lower pressure (10-15 MPa), a maximum extraction efficiency (63%) was attained for DPT, while the fat is extracted only 28.3%. The increase in temperature is advantageous for the selective extraction of DPT. However, the extraction efficiency of DPT decreased as the extraction temperature was increased. When a lower pressure (10 MPa) and a higher temperature (50 degrees C) were used, the organotin (DPT) was extracted up to a maximum (59.9%) within 30 min, while fat was extracted only 4.9%.

Chromatography, Supercritical Fluid↗
PubMed1997

[High performance liquid chromatographic analysis of organic acids in foods].

Because of the speed, selectivity, reliability, and simple sample preparation high performance liquid chromatography (HPLC) is the preferred method for simultaneous separation and determination of organic acids in foods. Ion exchange chromatography, ion exclusion chromtography and reversed phase HPLC are the most common chromatographic modes for analysis of organic acids in foods. The development and application of the three chromatographic modes are reviewed with 54 references. Ion exchange chromatography is most satisfactory for analysis of organic acids in foods, because organic acids are not interfered by other organic compounds, and inorganic anions in foods are determined simultaneously with organic acids.

Carboxylic Acids↗
PubMed1997

[Study on separating characteristics in capillary reversed-phase electrochromatography].

Capillary Electrochromatography (CEC) driven by electroosmotic flow is a new technique using fused silica capillaries packed with HPLC-type stationary phase. The analytes are separated by normal chromatographic partition between a mobile phase and a stationary phase. By using electroosmotic flow driving mobile phase, CEC promises theoretical plate efficiencies for HPLC. In this paper, some separating characteristics in capillary electrochromatography were studied by carrying out the separation of some neutral compounds on capillary column with 75microm i.d. x 30cm/37cm packed with ODS particles (3microm). The mobile phase was acetonitrile and water, which contains certain concentration of electrolyte. Samples were injected electrokinetically into the separating column. On-line UV detector was used. The Joule heating effect in packed CEC was lower than that in CZE, and a good linear relationship between the elecrtoosmotic flow (EOF) velocity and applied voltage was obtained as shown in Fig. 1. The influence of electrolyte and acetonitrile concentrations on the electroosmotic flow was studied, as shown in Fig. 2 and 5. The EOF was monitored by using thiourea as a marker. The EOF will decrease when the electrolyte concentration increases. The higher the acetonitrile concentration was, the lower the EOF was. The effect of linear velocity on column efficiencies was also investigated. The column effeciencies less than 2. 0 reduced plate height were obtained. The highest efficiency obtained in this work was 236000 plate per meter for a retained solute, benzene. It was found that the concentrations of acetonitrile and electrolyte were important experimental variables in performance of capillary electrochromatography.

English Abstract↗
PubMed1997

[Evaluation of capillary chromatographic columns packed by electrokinetic packing method].

In this paper, a method for electrokinetic packing capillary columns is reported. A higher column effeciency was obtained by performing electrochromatography on electrokinetic packing columns. The highest column efficiency in number of theoretical plate per meter was more than 200000, corresponding to reduced plate height less than 2. The reproducibilities of the same column in different intervals and different columns prepared from the same or different batches were compared. The relative standard deviations of the number of theoretical plate and retention time were less than 10% and 8%, respectively. The results indicated that high column efficiency and good reproducibility can be obtained on these new capillary packed columns.

English Abstract↗
PubMed1997

Molecular characterization of the murine Hif-1 alpha locus.

Hypoxia inducible factor 1 alpha (HIF-1 alpha) is a basic helix-loop-helix-PAS (bHLH-PAS) transcription factor that mediates certain cellular responses to low oxygen tension, iron chelators, Co2+, Ni2+, Mg2+, and low intracellular glucose concentration. Upon exposure to the above conditions, HIF-1 alpha is upregulated and heterodimerizes with the Ah receptor nuclear translocator (ARNT, also known as HIF-1 beta), the heterodimeric complex binds TACGTG-containing genomic enhancer elements, and activates transcription of target genes. As a first step in developing genetic models to study the biology related to cellular hypoxia, we have cloned the murine HIF-1 alpha cDNA, determined the tissue-specific expression of its mRNA, functionally analyzed its protein product, and characterized its promoter and its genomic structure. A comparison between the murine and human HIF-1 alpha protein sequence reveals 95%, 99%, and 83% identity in the bHLH, PAS, and variable domains, respectively. RNAse protection assays demonstrate that in adult mice, the mHIF-1 alpha mRNA is expressed at high levels in kidney, heart, brain, thymus, and placenta, with moderate expression in liver, spleen, testis, and lung and much lower expression in skeletal muscle testis. Northern blot analysis indicates that the mRNA of the murine HIF-1 alpha is transcribed in two forms, a major 4-kb species and a minor 5-kb species; both are present in all tissues examined. The Hif-1 alpha promoter is GC rich, does not have a TATA element near its transcriptional start site, and does not respond to hypoxia or Co2+. The mHIF-1 alpha structural gene is composed of 15 exons. The splice junction sites within the bHLH and the PAS domains of HIF-1 alpha gene are highly conserved with respect to a number of previously characterized members of the bHLH-PAS superfamily. However, unlike other bHLH-PAS genes, where the variable domain is encoded by 2 exons, the variable region of the mHIF-1 alpha gene is encoded by 7 exons. Furthermore, most of these splice junction sites in the variable region are conserved with that of HIF-2 alpha, a recently cloned hypoxia-responsive bHLH-PAS protein (also known as MOP2, EPAS1, and HLF). These data suggest that HIF-1 alpha, along with HIF-2 alpha, represents a new subclass of the bHLH-PAS superfamily.

Amino Acid Sequence↗
PubMed1996

6-deoxyerythronolide B synthase 1 is specifically acylated by a diketide intermediate at the beta-ketoacyl-acyl carrier protein synthase domain of module 2.

We have used 6-deoxyerythronolide B synthase (DEBS) as a model system to investigate molecular recognition by a modular polyketide synthase (PKS). DEBS consists of three proteins (DEBS1, -2, and -3) that biosynthesize the polyketide skeleton of the antibiotic erythromycin from propionyl-CoA and methylmalonyl-CoA. Active sites within these multifunctional proteins are organized into biosynthetic "modules", each of which catalyzes a discrete round of polyketide chain elongation and adjusts the appropriate level of beta-ketoacylthioester reduction. Using DEBS1, we demonstrate that there is a substantial degree of molecular recognition in the processing of the natural diketide chain elongation intermediate. Exogenously added (2S,3R)-2-methyl-3-hydroxypentanoic acid N-acetylcysteamine thioester is exclusively recognized by its cognate beta-ketoacyl-acyl carrier protein synthase domain in module 2 (KS2). Labeled diketide specifically acylated DEBS1 in crude protein extracts and limited proteolysis localized the binding to module 2. The precise site of acylation in DEBS1 was established by the finding that a Cys2200 Ala mutant of DEBS1, lacking the KS2 active-site cysteine, did not undergo acylation by the diketide. Pretreatment of the wild-type protein with the beta-ketoacyl-ACP synthase inhibitor cerulenin also blocked acylation. These results indicate that in addition to the purely organizational consequences resulting from the order of active-site domains, the programming of polyketide biosynthesis by modular PKSs involves a substantial level of molecular recognition. This conclusion has important implications for the use of PKSs to rationally design novel polyketides.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗