Biomedical subjects

D N Wang

Publications and source records attributed to D N Wang.

At least 19 recordsLinked to original sources

PubMed2001

High-yield expression and functional analysis of Escherichia coli glycerol-3-phosphate transporter.

The glycerol-3-phosphate (G3P) transporter, GlpT, from Escherichia coli mediates G3P and inorganic phosphate exchange across the bacterial inner membrane. It possesses 12 transmembrane alpha-helices and is a member of the Major Facilitator Superfamily. Here we report overexpression, purification, and characterization of GlpT. Extensive optimization applied to the DNA construct and cell culture has led to a protocol yielding approximately 1.8 mg of the transporter protein per liter of E. coli culture. After purification, this protein binds substrates in detergent solution, as measured by tryptophan fluorescence quenching, and its dissociation constants for G3P, glycerol-2-phosphate, and inorganic phosphate at neutral pH are 3.64, 0.34, and 9.18 microM, respectively. It also shows transport activity upon reconstitution into proteoliposomes. The phosphate efflux rate of the transporter in the presence of G3P is measured to be 29 micromol min(-1) mg(-1) at pH 7.0 and 37 degrees C, corresponding to 24 mol of phosphate s(-1) (mol of protein)(-1). In addition, the glycerol-3-phosphate transporter is monomeric and stable over a wide pH range and in the presence of a variety of detergents. This preparation of GlpT provides ideal material for biochemical, biophysical, and structural studies of the glycerol-3-phosphate transporter.

Biological Transport↗

PubMed2001

Monomeric state and ligand binding of recombinant GABA transporter from Escherichia coli.

The gamma-aminobutyric acid (GABA) transporter from Escherichia coli was homologously overexpressed and purified to homogeneity with a yield of 1.0 mg per liter culture. The purification procedure consists of a cobalt affinity column, proteolytic cleavage of His- and myc-tags, and size-exclusion chromatography. The purified transporter exists as a monomer in FOS-Choline 12 detergent, with a Stokes radius of 45 A for the protein-detergent complex. In detergent solution the protein binds substrates, as indicated by tryptophan fluorescence quenching. Its dissociation constants (K(d)) for GABA, muscimol and nipecotic acid are 13.8, 13.3 and 27.9 microM, respectively. This protein preparation provides ideal starting materials for future biochemical, biophysical and structural studies of the GABA transporter.

Bacterial Proteins↗

PubMed2001

Purification and characterization of human erythrocyte glucose transporter in decylmaltoside detergent solution.

The facilitative glucose transporter from human erythrocyte membrane, Glut1, was purified by a novel method. The nonionic detergent decylmaltoside was selected for solubilization on the basis of its efficiency to extract Glut1 from the erythrocyte membrane and its ability to maintain the protein in a monodisperse state. A positive, anion-exchange chromatography protocol produced a Glut1 preparation of 95% purity with little copurified lipid. This protein preparation exhibited cytochalasin B binding in detergent solution, as measured by tryptophan fluorescence quenching. The transporter existed as a monomer in decylmaltoside, with a Stokes radius of 50 A and a molecular mass of 147 kDa for the protein-detergent complex. We screened detergent, pH, additive, and lipid and have found conditions to maintain Glut1 monodispersity for 8 days at 25 degrees C or over 5 weeks at 4 degrees C. This Glut1 preparation represents the best available material for two- and three-dimensional crystallization trials of the human glucose transporter protein.

Chromatography, Ion Exchange↗

PubMed2001

[The new antiendotoxin strategies].

Lipopolysaccharide(LPS) is important in the pathogenesis of sepsis infected by gram-negative bacteria in humans, and part or all of this pathophysiology is mediated by a complex secondary inflammatory response. Administration of purified LPS or a variety of LPS-free recombinant mediators can reproduce much of the pathophysiology of gram-negative sepsis. There has been an explosion of new information over the last several years regarding the interactions of LPS with proteins and cells. Blocking sepsis at the bacterial toxin level is appealing because it is upstream, so that the secondary pathologic inflammatory cascade may be limited or prevented. A variety of antiendotoxin strategies have been proposed. Therapies under investigation include agents that bind and neutralize LPS, agents or systems that enhance LPS clearance, and agents that inhibit LPS interaction with serum elements or cellular receptors.

Acute-Phase Proteins↗

PubMed2000

Interlaboratory study of identification and quantitation of multiresidue pyrethroids in agricultural products by gas chromatography-mass spectrometry.

This paper deals with the different GC-MS analytical conditions adopted by four laboratories in an attempt to confirm the accuracy of the GC-electron-capture detection (ECD) analytical results during the international collaborative study for the establishment of the AOAC Official Method 998. 01. What is especially noted is that two laboratories have conducted comparative analysis of the respective 12 blind samples with both methods of GC-ECD and GC-MS, and the analytical results of the two methods turn out to be basically identical. This fully demonstrates that GC-MS is not only an effective confirmation tool in the analysis of the pyrethroid residues but also of sufficient sensitivity regarding the maximum residue limit of determination prescribed by FAO/WHO. Moreover, its selectivity is better than GC-ECD.

Gas Chromatography-Mass Spectrometry↗

PubMed2000

Trimeric ring-like structure of ArsA ATPase.

ArsA protein is the soluble subunit of the Ars anion pump in the Escherichia coli membrane which extrudes arsenite or antimonite from the cytoplasm. The molecular weight of the subunit is 63 kDa. In the cell it hydrolyzes ATP, and the energy released is used by the membrane-bound subunit ArsB to transport the substrates across the membrane. We have obtained two-dimensional crystals of ArsA in the presence of arsenite on negatively-charged lipid monolayer composed of DMPS and DOPC. These crystals have been studied using electron microscopy of negatively-stained specimens followed by image processing. The projection map obtained at 2.4 nm resolution reveals a ring-like structure with threefold symmetry. Many molecular assemblies with the same ring-shape and dimensions were also seen dispersed on electron microscopy grids, prepared directly from purified ArsA protein solution. Size-exclusion chromatography of the protein sample with arsenite present revealed that the majority of the protein particles in solution have a molecular weight of about 180 kDa. Based on these experiments, we conclude that in solution the ArsA ATPase with substrate bound is mainly in a trimeric form.

Adenosine Triphosphatases↗

PubMed2000

[Cloning of the zot gene of Vibrio cholerae and its expression in Escherichia coli].

The zot gene encoding Zonula occludens toxin was amplified from classic Vibrio cholerae genomic DNA by PCR. The result of sequencing indicated that zot gene encodes 399 amino acid residues. The sequence of zot gene was a little bit different from that of reported including 14 nucleotides and four amino acid residues. The expression plasmid pET-ZOT was constructed by inserting zot gene into plasmid pET-28a(+) containing the T7 promoter. The expression plasmid was induced into E. coli BL21 (DE3) and expression strain BLZOT was selected. SDS-PAGE analysis revealed that the ZOT protein was expressed and accumulated up to above 15% of bacterial soluble protein after induced by IPTG. A protein of 47 kD was expressed as including body. Western blot analysis revealed that the expressed protein was ZOT.

Base Sequence↗

PubMed1996

[Effect of new zhengtian pill on 5-hydroxytryptamine content in platelet and plasma of migraine patients].

Changes of 5-hydroxytryptamine (5-HT) content in platelet and plasma before and after New Zhengtian Pill (NZTP) therapy in migraine patients were assayed with fluorospectrophotometry. Results showed that during the period of attack, the platelet 5-HT level of patients in comparing with normal control group, was increased (P < 0.05) but the plasma 5-HT level decreased (P < 0.01) and the ratio of the platelet and plasma 5-HT was abnormal P < 0.01. While during the intermittent period the 5-HT was increased both in platelet and plasma and the ratio of intra- and extra-platelet 5-HT was normal. Also, in comparing with before treatment, the post-treatmental plasma levels of 5-HT in patients with or without acute attack were all increased, the above-mentioned 5-HT ratio was also resumed obviously (P < 0.05). This study revealed that both the plasma 5-HT and platelet 5-HT levels and their ratio of patients in acute attack period were abnormal, which might be the pathogenetic basis of migraine. It suggested the mechanism of NZTP in treating migraine might be through its action on absorption, releasing and metabolism of 5-HT by platelet and maintaining a physiologic balance in them.

Adolescent↗

PubMed1995

Purification and two-dimensional crystallization of bacterial cytochrome oxidases.

A novel strategy which employes chromatography on an immobilized metal ion has been developed for the purification of bacterial cytochrome c and quinol oxidases. Many bacterial oxidase complexes appear to have a natural affinity to bind to the chelated copper ion. A combination of three different chromatographic principles (anion exchange, metal-affinity and gel filtration) makes an effective tool chest for the preparation of homogeneous and protein-chemically pure bacterial oxidases. These preparations have been used for two-dimensional crystallization. Until now, crystals have been obtained using the Paracococcus denitrificans and Rhodobacter sphaeroides cytochrome aa3 and the Escherichia coli cytochrome bo. The crystals diffract to approximately 2.5 nm in negative stain and have potential for further structural studies.

Bacteria↗

PubMed1995

[Study on ultrastructure of platelets in migraine patients with blood stasis type].

To study the relationship between ultrastructure of platelets and migraine patients with blood stasis type (MBS). The morphologic appearance and aggregation of platelets in MBS patients were examined before and after treatment with Tongbike oral liquor (20 cases in treated group and.10 in control). The results showed that the MBS patients' platelets appeared irregular, easy to aggregate and adhere to red cells, which was more severe when ADP was added, but improved after treated with Chinese medicinal herbs. According to the results it was assumed that platelets from MBS patients were in an abnormal activated state, the abnormal morphology and functional disturbance might be the pathologic basis of MBS. Tongbike oral liquor could markedly improve the morphologic and functional state of platelets, which was relevant to the inhibition of platelet aggregation and the inducing of platelet depolymerization.

Adolescent↗

PubMed1994

Three-dimensional map of the dimeric membrane domain of the human erythrocyte anion exchanger, Band 3.

The electroneutral exchange of chloride and bicarbonate across the human erythrocyte membrane is facilitated by Band 3, a 911 amino acid glycoprotein consisting of a 43 kDa N-terminal cytosolic domain that binds the cytoskeleton, haemoglobin and glycolytic enzymes and a 52 kDa C-terminal membrane domain that mediates anion transport. Electron microscopy and three-dimensional image reconstruction of negatively stained two-dimensional crystals of the dimeric membrane domain revealed a U-shaped structure with dimensions of 60 x 110 A, and a thickness of 80 A. The structure is open on the top and at the sides, with the monomers in close contact at the base. The basal domain is 40 A thick and probably spans the lipid bilayer. The upper part of the dimer consists of two elongated protrusions measuring 25 x 80 A in projection, with a thickness of 40 A. The protrusions form the sides of a canyon, enclosing a wide space that narrows down and converges into a depression at the centre of the dimer on the top of the basal domain. This depression may represent the opening to a transport channel located at the dimer interface. Based on the available protein-chemical data, the two protrusions face the cytosolic side of the membrane and they appear to be dynamic.

Amino Acid Sequence↗

PubMed1994

Band 3 protein: structure, flexibility and function.

The electroneutral exchange of chloride and bicarbonate across the human erythrocyte membrane is facilitated by Band 3, a 911 amino acid glycoprotein. The 43 kDa amino-terminal cytosolic domain binds the cytoskeleton, haemoglobin and glycolytic enzymes. The 52 kDa carboxyl-terminal membrane domain mediates anion transport. The protein is a functional dimer, in which the two subunits probably interact with one another by an allosteric mechanism. It is proposed that the link between the mobile cytoplasmic and the membrane-spanning domains of the protein is flexible, based on recent biochemical, biophysical and structural data. This explains the long-standing puzzle that attachment to the cytoskeletal spectrin and actin does not appear to restrict the rotational movement of the Band 3 protein in the erythrocyte membrane. In the Band 3 isoform from the Southeast Asian Ovalocytes (SAO) this link is altered, resulting a tighter attachment of the cytoskeleton to the plasma membrane and a more rigid red blood cell.

Allosteric Regulation↗

PubMed1994

Atomic model of plant light-harvesting complex by electron crystallography.

The structure of the light-harvesting chlorophyll a/b-protein complex, an integral membrane protein, has been determined at 3.4 A resolution by electron crystallography of two-dimensional crystals. Two of the three membrane-spanning alpha-helices are held together by ion pairs formed by charged residues that also serve as chlorophyll ligands. In the centre of the complex, chlorophyll a is in close contact with chlorophyll b for rapid energy transfer, and with two carotenoids that prevent the formation of toxic singlet oxygen.

Amino Acid Sequence↗

PubMed1993

Lipid-protein interactions in crystals of plant light-harvesting complex.

Two different thylakoid lipids are specifically associated with the light-harvesting complex of photosystem II (LHC-II). Digalactosyl diacyl glycerol (DGDG) binds to the isolated complex but can be removed by mild detergent treatment and anion-exchange chromatography. Removal of this lipid renders the complex unable to form two-dimensional or three-dimensional crystals. The ability to crystallize is completely restored by addition of pure DGDG, at a ratio of about four molecules per polypeptide for three-dimensional crystals, suggesting several binding sites at the periphery of the trimeric complex. Two-dimensional crystals of purified protein grown in the presence of DGDG are more highly ordered than those obtained from the unfractionated complex. The other lipid, phosphatidyl glycerol (PG), binds more firmly and cannot be removed with non-ionic detergent. Complete delipidation of LHC-II can be achieved either with phospholipase or by proteolytic cleavage of 49 amino acid residues at the N terminus. Both treatments dissociate the native, trimeric complex into monomers. This indicates that PG is directly involved in the formation of trimers, which are a prerequisite for two-dimensional and three-dimensional crystallization. Both lipids are therefore present in two-dimensional and three-dimensional crystals and have distinct roles in the structure of the complex.

Chromatography, Ion Exchange↗

PubMed1993

Two-dimensional structure of the membrane domain of human band 3, the anion transport protein of the erythrocyte membrane.

The membrane domain of human erythrocyte Band 3 protein (M(r) 52,000) was reconstituted with lipids into two-dimensional crystals in the form of sheets or tubes. Crystalline sheets were monolayers with six-fold symmetry (layer group p6, a = b = 170 A, gamma = 60 degrees), whereas the symmetry of the tubular crystals was p2 (a = 104 A, b = 63 A, gamma = 104 degrees). Electron image analysis of negatively stained specimens yielded projection maps of the protein at 20 A resolution. Maps derived from both crystal forms show that the membrane domain is a dimer of two monomers related by two-fold symmetry, with each monomer consisting of three subdomains. In the dimer, two subdomains of each monomer form a roughly rectangular core (40 x 50 A in projection), surrounding a central depression. The third subdomain of the monomer measures approximately 15 x 25 A in projection and appears to be connected to the other two by a flexible link. We propose that the central depression may represent the channel for anion transport while the third subdomain appears not to be directly involved in channel formation.

Anion Exchange Protein 1, Erythrocyte↗

PubMed1992

Long-distance restriction mapping of the proximal long arm of human chromosome 21 with Not I linking clones.

Human chromosome 21 is the smallest of the 22 autosomes and 2 sex chromosomes. Hybridization of the human repetitive sequence Alu to pulsed-field gel-fractionated Not I-digested genomic DNA from a human-mouse hybrid cell line containing chromosome 21 as the sole human component identified chromosome 21 Not I restriction fragments. A Not I restriction map of regions of the chromosome was constructed, by identifying neighboring Alu bands with Not I linking clones. This approach simplifies the task of physical mapping and avoids ambiguities in Not I fragment assignments that arise from gel-to-gel mobility variations. A contiguous map was constructed with six Not I linking clones that covers at least the proximal one-third of the long arm of chromosome 21 and spans 20 megabases. A more detailed restriction map revealed 11 likely CpG islands in this region and localized 11 additional DNA markers.

Chromosomes, Human, Pair 21↗

PubMed1991

Three-dimensional structure of plant light-harvesting complex determined by electron crystallography.

The structure of the light-harvesting chlorophyll a/b-protein complex, a membrane protein serving as the major antenna of solar energy in plant photosynthesis, has been determined at 6 A resolution by electron crystallography. Within the complex, three membrane-spanning alpha helices and 15 chlorophyll molecules are resolved. There is an intramolecular diad relating two of the alpha helices and some of the chlorophylls. The spacing of the chlorophylls suggests energy transfer by delocalized exciton coupling and Förster mechanisms.

Chlorophyll↗

PubMed1991

High-resolution electron crystallography of light-harvesting chlorophyll a/b-protein complex in three different media.

Large two-dimensional crystals of the light-harvesting chlorophyll a/b-protein complex (LHC-II) from the photosynthetic membrane of pea chloroplasts were grown by a new method from detergent solution. The structure of these crystals was examined by electron crystallography, using three different media to preserve high-resolution detail: vitrified water, glucose and tannin. The crystals diffracted electrons to at least 3.2 A resolution in all three media. R-factors between the three data sets of electron diffraction amplitudes ranged from 6.4% to 14.3%. Fourier difference maps were generated and compared to a projection map of the complex at 3.4 A resolution. No significant differences were found, proving that all three media preserved the native structure of LHC-II at high resolution. The probability of recording high-quality electron diffraction patterns with tannin was 90%. With glucose and water this probability was lower by a factor of 10 to 20, suggesting that tannin may be preferable as a preserving medium for sensitive biological specimens.

Chlorophyll↗