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Biomedical subjects

H W Sun

Publications and source records attributed to H W Sun.

16 recordsLinked to original sources
PubMed2005

Rapid non-genomic inhibitory effects of glucocorticoids on human neutrophil degranulation.

BACKGROUND: Glucocorticoids acting as anti-inflammatory or immunosuppressive drugs have been shown to exert most of their effects genomically. Recent findings suggest that non-genomic activity might be relatively more important in mediating the therapeutic effects of high-dose pulsed glucocorticoid. However, few non-genomic anti-inflammatory effects were reported, much less non-genomic mechanisms. OBJECTIVE: This study was performed to investigate the nongenomic effects of glucocorticoids on human neutrophil degranulation. METHODS: Purified human neutrophils were pretreated with 6 alpha-methylprednisolone or hydrocortisone for 5 min, and then primed with N-formyl-methionyl-leucyl-phenylalanine (fMLP) (10(-6) M) or phorbol myristate acetate (PMA) (50 ng/ml) in the presence of cytochalasin B. The release of two markers of neutrophil granules, lactoferrin and myeloperoxidase, was measured by ELISA and enzymology methods respectively. RESULTS: Both 6 alpha-methylprednisolone (10(-5)-10(-4) M) and hydrocortisone (10(-4) M) showed significant inhibitory effects on neutrophil degranulation within 5 min after fMLP administration. For PMA stimulated degranulation, 6 alpha-methylprednisolone (10(-4) M) showed significant inhibitory effects (p < 0.01), while hydrocortisone (10(-4) M) only showed an inhibitory tendency (P > 0.05). Neither RU486 (10(-5) M) nor cycloheximide (10(-4) M) could alter the inhibitory effects of glucocorticoids. CONCLUSION: Our results demonstrate that megadoses of glucocorticoids exert rapid inhibitory effects on human neutrophil degranulation at the cellular level via a new mechanism that is independent of corticosteroid type II receptor occupation or protein synthesis. We infer that these effects may be very important when glucocorticoids act as anti-inflammatory drugs during pulse therapy.

Cell Degranulation↗
PubMed2001

[The design and evaluation of horizontal pipe mini-flame atomization and ionization synchronous detector in GC/AAS].

Gas chromatography/atomic absorption spectroscopy(GC/AAS) is a good method for the species analysis of organometallic compounds. But the traditional atomizers are not very suitable for this technology and all of them response only to one kind of signal--the concentration of the metallic atoms of the ground state. They can not give any information about the organic group of organometallic compounds and organic compounds which coexist with the former. For GC/AAS we want to design and manufacture a new kind of detector which is much more sensitive and has a much smaller dead volume and will sensitively and synchronously response to the atomization signal of organometallic compound and ionization signal of organic compound. The authors have noticed that the atomization of organometallic compound and ionization of organic compound have been existing in the same hydrogen flame. The question is how to gain and exchange and output the two signals which are completely different in characters. For this purpose we designed and manufactured a new type of horizontal pipe mini-flame atomization and ionization synchronous detector. The key part is a T type glass tube (80 mm x 13 mm x 10 mm i.d.) which covers horizontally on the jet of the mini-flame atomizer and a long pipe stainless steel collector (70 mm x 9.5 mm o.d. x 9 mm i.d.) is tightly inserted in the tube. The light beam of the hollow cathode lamp passes through the hydrogen flame along the axial center of the glass tube and the ground state metallic atoms in the flame diffuses to both ends of the tube along the axial center of the glass tube too. This process enriches the depth of the light absorption and then increases the sensitivity of atomization. At the same time the long pipe collector can enrich the collection efficiency of the ionization and increases the sensitivity and widens the linear range. So the detector can synchronously and sensitively detect the organometallic and organic compounds. The detection limit for atomization of the organometallic compound(diethylmercury) is 2.5 x 10(-11) g.s-1 and for ionization of the organic compound (benzene) is 1.0 x 10(-11) g.s-1. The linear range is 7.0 x 10(2) for atomization of diethylmercury (r = 0.9989) and 2.5 x 10(5) for ionization of benzene (r = 0.9992). The selectivity of the detector to the organic solvent under the optimum operating condition of atomization is 10(4)-10(7). The relative standard deviation (RSD, n = 11) of the peak area in the optimum operating conditions of atomization for the organometallic compound (diethylmercury) and of ionization for the organic compound (benzene) is 1.8% and 1.5% respectively. Under the same operating condition, the atomization and the ionization can not reach the optimum performance at the same time. If the atomization is in the optimum performance state, the performance for ionization is like a chromatographic thermal conductivity detector (TCD). The optimum performance of the atomization and ionization can be quickly adjusted by changing the flow rates of the corresponding gases such as H2, N2 and air.

Benzene↗
PubMed1998

Cloning, characterization, and expression in Escherichia coli of three creatine kinase muscle isoenzyme cDNAs from carp (Cyprinus carpio) striated muscle.

In vertebrates, the creatine kinase isoenzyme family consists of four types of isoforms: cytosolic muscle type (M-CK), cytosolic brain type (B-CK), mitochondrial ubiquitous, acidic type (Miu-CK), and mitochondrial sarcomeric, basic type (Mis-CK). Until recently, the existence of more than one subisoform of CK isoenzyme has been demonstrated only in fishes by starch gel electrophoresis. We report herein the isolation of three full-length cDNAs that correspond to three closely related creatine kinase M-CK genes from common carp (Cyprinus carpio), designated the M1-CK, M2-CK, and M3-CK genes. Using oligonucleotide probes that correspond to the same region but with the most variable sequences, different restricted genomic hybridization patterns have been obtained. These Southern blot results indicate that the three cDNAs come from different genes. Northern blot analysis using probes that correspond to the 3'-untranslated regions further show that all three subisoforms are expressed specifically in carp muscle. The deduced amino acid sequences of these three subisoforms of carp M-CK show about 85% identity to mammalian M-CK isoenzyme. Finally, the three cDNAs have been expressed in Escherichia coli with a molecular mass of approximately 43,000 Da, and these recombinant proteins exhibit creatine kinase activity. All of these data suggest that the M-CK isoenzymes have at least three subisoforms in carp.

Amino Acid Sequence↗
PubMed1998

Direct link between cytokine activity and a catalytic site for macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF) is a secreted protein that activates macrophages, neutrophils and T cells, and is implicated in sepsis, adult respiratory distress syndrome and rheumatoid arthritis. The mechanism of MIF function, however, is unknown. The three-dimensional structure of MIF is unlike that of any other cytokine, but bears striking resemblance to three microbial enzymes, two of which possess an N-terminal proline that serves as a catalytic base. Human MIF also possesses an N-terminal proline (Pro-1) that is invariant among all known homologues. Multiple sequence alignment of these MIF homologues reveals additional invariant residues that span the entire polypeptide but are in close proximity to the N-terminal proline in the folded protein. We find that p-hydroxyphenylpyruvate, a catalytic substrate of MIF, binds to the N-terminal region and interacts with Pro-1. Mutation of Pro-1 to a glycine substantially reduces the catalytic and cytokine activity of MIF. We suggest that the underlying biological activity of MIF may be based on an enzymatic reaction. The identification of the active site should facilitate the development of structure-based inhibitors.

Amino Acid Sequence↗
PubMed1998

Macrophage migration inhibitory factor interactions with glutathione and S-hexylglutathione.

Macrophage migration inhibitory factor (MIF) has been reported to interact with glutathione and S-hexylglutathione and to possess glutathione S-transferase activity. However, contrary to these reports, a recent NMR study concluded that MIF shows no affinity for glutathione. Re-examination of the glutathione-MIF interactions indicates that the reported increase in fluorescence upon addition of glutathione is because of pH-induced unfolding of the protein and not to any direct interactions. Circular dichroism shows that MIF remains folded from pH 4.5-7.5 but is 50% unfolded at pH 2.9 +/- 0.2. The reported increase in fluorescence can be achieved by acid titration. Under strongly buffered conditions, no fluorescence change is observed upon addition of glutathione. In contrast to the results with glutathione, MIF binds S-hexylglutathione with a Kd of 2.5 +/- 0.6 mM. Using NMR spectroscopy, a binding site which clusters around the N-terminal proline was identified. These data indicate that the binding site for S-hexylglutathione is the same as the catalytic site for the dopachrome tautomerase activity of MIF. Consequently, the binding of S-hexylglutathione as well as hexanethiol inhibits this catalytic activity.

Binding Sites↗
PubMed1996

Crystal structure at 2.6-A resolution of human macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF) was the first cytokine to be described, but for 30 years its role in the immune response remained enigmatic. In recent studies, MIF has been found to be a novel pituitary hormone and the first protein identified to be released from immune cells on glucocorticoid stimulation. Once secreted, MIF counterregulates the immunosuppressive effects of steroids and thus acts as a critical component of the immune system to control both local and systemic immune responses. We report herein the x-ray crystal structure of human MIF to 2.6 angstrom resolution. The protein is a trimer of identical subunits. Each monomer contains two antiparallel alpha-helices that pack against a four-stranded beta-sheet. The monomer has an additional two beta-strands that interact with the beta-sheets of adjacent subunits to form the interface between monomers. The three beta-sheets are arranged to form a barrel containing a solvent-accessible channel that runs through the center of the protein along a molecular 3-fold axis. Electrostatic potential maps reveal that the channel has a positive potential, suggesting that it binds negatively charged molecules. The elucidated structure for MIF is unique among cytokines or hormonal mediators, and suggests that this counterregulator of glucocorticoid action participates in novel ligand-receptor interactions.

Amino Acid Sequence↗
PubMed1996

The subunit structure of human macrophage migration inhibitory factor: evidence for a trimer.

The subunit structure of human macrophage migration inhibitory factor (MIF) has been studied by preliminary X-ray analysis of wild-type and selenomethionine-MIF and dynamic light scattering. Crystal form I of MIF belongs to space group P2(1)2(1)2(1) and is grown from 2 M ammonium sulfate at pH 8.5. A native data set has been collected to 2.4 A resolution. Self-rotation studies and Van values indicate that three molecules per asymmetric unit are present. A data set to 2.8 A resolution has been collected for crystal form II, which belongs to space group P3(1)21 or P3(2)21 and grows from 2 M ammonium sulfate, 2% polyethylene glycol (average molecular mass 400) 0.1 M HEPES, pH 7.5. Three, four, five or six monomers in the asymmetric unit are consistent with Van values for this crystal form. Analysis of crystal form II containing selenomethionine-MIF indicates nine selenium sites are present per asymmetric unit. Dynamic light scattering of MIF suggests that the major form of the protein in solution is a trimer. The results of these studies are in contrast to previous reports indicating that MIF is a monomer or dimer. The subunit arrangement of MIF is similar to that of tumor necrosis factor and suggests that signal transduction might require trimerization of receptor subunits.

Crystallization↗
PubMed1995

Salvaging recombinants from low-efficiency ligase reactions for more efficient subcloning.

Certain types of ligase reactions can be problematic, such as those involving PCR products, blunt-ends and multiple DNA inserts. A simple PCR-based strategy was developed to overcome cloning difficulties with these inefficient ligase reactions. After an initial ligase reaction, primers complementary to the vector are utilized to amplify the DNA fragment from (the few) successful recombinants in the ligation mixture. This DNA fragment is processed for use in a more conventional and straightforward ligase reaction. We demonstrate the potential of the technique by applying it to a variety of difficult ligase reactions.

Animals↗
PubMed1993

Inversion of the substrate specificity of yeast alcohol dehydrogenase.

The relationship between the size of the substrate binding pocket and the catalytic reactivities with varied alcohols was studied with the Saccharomyces cerevisiae alcohol dehydrogenase I (ScADH) and compared with the liver enzymes from horse (EqADH, EE isoenzyme) and monkey (MmADH alpha, alpha-isoenzyme). The yeast enzyme is most active with ethanol, and its activity decreases as the size of the alcohol is increased, whereas the activities of the liver enzymes increase with larger alcohols. The substrate pocket in ScADH was enlarged by single substitutions of Thr-48 to Ser (T48S), Trp-57 to Met (W57M), and Trp-93 to Ala (W93A), and a double change, T48S:W93A, and a triple, T48S:W57M:W93A. The T48S enzyme has the same pattern of activity (V/K) as wild-type ScADH for linear primary alcohols. The W57M enzymes have lowered reactivity with primary and secondary alcohols. The W93A and T48S:W93A enzymes resemble MmADH alpha in having an inverted specificity pattern for primary alcohols, being 3- and 10-fold more active on hexanol and 350- and 540-fold less active on ethanol, and are as reactive as the liver enzymes with long chain primary alcohols. The three Ala-93 enzymes also acquired weak activity on branched chain alcohols and cyclohexanol.

Aldehyde Dehydrogenase↗
PubMed1992

Progressive sequence alignment and molecular evolution of the Zn-containing alcohol dehydrogenase family.

Sequences of 47 members of the Zn-containing alcohol dehydrogenase (ADH) family were aligned progressively, and an evolutionary tree with detailed branch order and branch lengths was produced. The alignment shows that only 9 amino acid residues (of 374 in the horse liver ADH sequence) are conserved in this family; these include eight Gly and one Val with structural roles. Three residues that bind the catalytic Zn and modulate its electrostatic environment are conserved in 45 members. Asp 223, which determines specificity for NAD, is found in all but the two NADP-dependent enzymes, which have Gly or Ala. Ser or Thr 48, which makes a hydrogen bond to the substrate, is present in 46 members. The four Cys ligands for the structural zinc are conserved except in zeta-crystallin, the sorbitol dehydrogenases, and two bacterial enzymes. Analysis of the evolutionary tree gives estimates of the times of divergence for different animal ADHs. The human class II (pi) and class III (chi) ADHs probably diverged about 630 million years ago, and the newly identified human ADH6 appeared about 520 million years ago, implying that these classes of enzymes may exist or have existed in all vertebrates. The human class I ADH isoenzymes (alpha, beta, and gamma) diverged about 80 million years ago, suggesting that these isoenzymes may exist or have existed in all primates. Analysis of branch lengths shows that these plant ADHs are more conserved than the animal ones and that class III ADHs are more conserved than class I ADHs. The rate of acceptance of point mutations (PAM units) shows that selection pressure has existed for ADHs, implying that these enzymes play definite metabolic roles.

Alcohol Dehydrogenase↗
PubMed1990

Study of normal and cancerous urine using photoacoustic spectroscopy.

Photoacoustic spectroscopy (PAS) is a valuable technique, increasingly applied in clinical and biological analysis. Light absorption properties of urine and body tissue can be obtained more easily by the PAS technique because of its high sensitivity, than by the conventional optical techniques. By observing their absorption properties it is possible to diagnose some diseases and in this study we have used PAS to assess cancerous changes in urine and tissue. Three groups were studied: normal individuals; patients with cancer; and patients with cancer and subsequent inflammation; and the characteristic light absorption peaks obtained for a certain band (240-420 nm). These results were compared with the clinical diagnosis and proved consistent and reliable for the diagnosis of urinary tract cancer subsequently confirmed by operation. This method will probably be a useful tool for the early clinical diagnosis of cancer of the urinary system.

Diagnosis, Computer-Assisted↗
PubMedDate not supplied

Cytotoxicity of glutaraldehyde and formaldehyde in relation to time of exposure and concentration.

The interacting effects of time of exposure and concentration as factors in cytotoxicity were compared for glutaraldehyde and formaldehyde. Cells from a human fibroblast cell line (WI-38) grown to confluence in 24-well trays were exposed to a range of concentrations of each agent, for periods of 4 to 24 hr. Cytotoxicity was measured by its effects on mitochondrial dehydrogenase activity, as assayed biochemically. Cytotoxic effects of formaldehyde occurred over a narrow concentration range from nontoxic to maximally toxic, and the range was little affected by time of exposure. In contrast, glutaraldehyde exerted its effect over a wider concentration range, and longer exposure times were necessary for maximal toxicity. The data suggest that long contact times of glutaraldehyde with dental pulp are necessary for maximum fixation. While 19% formaldehyde appeared to be more toxic than 2.5% glutaraldehyde in terms of serial dilution, little difference in cytotoxicity was observed when the data were calculated in terms of molar concentrations of the two agents.

Cell Line↗