Biomedical subjects

Zhi-Wei Chen

Publications and source records attributed to Zhi-Wei Chen.

14 recordsLinked to original sources

PubMed2005

SARS coronavirus spike polypeptide DNA vaccine priming with recombinant spike polypeptide from Escherichia coli as booster induces high titer of neutralizing antibody against SARS coronavirus.

Different forms of SARS coronavirus (SARS-CoV) spike protein-based vaccines for generation of neutralizing antibody response against SARS-CoV were compared using a mouse model. High IgG levels were detected in mice immunized with intraperitoneal (i.p.) recombinant spike polypeptide generated by Escherichia coli (S-peptide), mice primed with intramuscular (i.m.) tPA-optimize800 DNA vaccine (tPA-S-DNA) and boosted with i.p. S-peptide, mice primed with i.m. CTLA4HingeSARS800 DNA vaccine (CTLA4-S-DNA) and boosted with i.p. S-peptide, mice primed with oral live-attenuated Salmonella typhimurium (Salmonella-S-DNA-control) and boosted with i.p. S-peptide, mice primed with oral live-attenuated S. typhimurium that contained tPA-optimize800 DNA vaccine (Salmonella-tPA-S-DNA) and boosted with i.p. S-peptide, and mice primed with oral live-attenuated S. typhimurium that contained CTLA4HingeSARS800 DNA vaccine (Salmonella-tPA-S-DNA) and boosted with i.p. S-peptide. No statistical significant difference was observed among the Th1/Th2 index among these six groups of mice with high IgG levels. Sera of all six mice immunized with i.p. S-peptide, i.m. DNA vaccine control and oral Salmonella-S-DNA-control showed no neutralizing antibody against SARS-CoV. Sera of the mice immunized with i.m. tPA-S-DNA, i.m. CTLA4-S-DNA, oral Salmonella-S-DNA-control boosted with i.p. S-peptide, oral Salmonella-tPA-S-DNA, oral Salmonella-tPA-S-DNA boosted with i.p S-peptide, oral Salmonella-CTLA4-S-DNA and oral Salmonella-CTLA4-S-DNA boosted with i.p. S-peptide showed neutralizing antibody titers of <1:20-1:160. Sera of all the mice immunized with i.m. tPA-S-DNA boosted with i.p. S-peptide and i.m. CTLA4-S-DNA boosted with i.p. S-peptide showed neutralizing antibody titers of >or=1:1280. The present observation may have major practical value, such as immunization of civet cats, since production of recombinant proteins from E. coli is far less expensive than production of recombinant proteins using eukaryotic systems.

Administration, Oral↗

PubMed2005

Molecular cloning and structural analysis of quinohemoprotein alcohol dehydrogenase ADH-IIG from Pseudomonas putida HK5.

Depending on the alcohols used as growth substrates, Pseudomonas putida HK5 produces two distinct quinohemoprotein alcohol dehydrogenases, ADH-IIB and ADH-IIG, both of which contain pyrroloquinoline quinone (PQQ) and heme c as the prosthetic groups but show different substrate specificities, especially for diol substrates. Molecular cloning of the gene of ADH-IIB and its crystal structure are already reported. Here, molecular cloning of the gene, qgdA, and solution of the three-dimensional structure of ADH-IIG are reported. The enzyme consists of 718 amino acid residues including a signal sequence of 29 amino acid residues. The PQQ domain is highly homologous to other quinoproteins, especially to quinohemoproteins. The crystal structure of ADH-IIG, determined at 2.2A resolution, shows that the overall structure and the amino acid residues involved in PQQ binding are quite similar to ADH-IIB and to another quinohemoprotein ADH, qhEDH from Comamonas testosteroni. However, the lengths of the linker regions connecting the PQQ and the cytochrome domains are different from each other, leading to a significant difference in orientation of the cytochrome domain with respect to the PQQ domain. Apart from ADH-IIB and qhEDH, ADH-IIG has an extra 12-residue helix within loop 3 in the PQQ domain and an extra 3(10) helix in the C terminus of the cytochrome domain, and both helices appear parallel and linked by a hydrogen bond. The amino acid residues contacting substrate/product in the crystal structures are also different among them. In the crystal structure of ADH-IIG with 1,2-propanediol, one of the hydroxyl groups of the substrate forms a hydrogen bond with O5 of PQQ and OD1 of Asp300, and the other interacts with a water molecule and with NE2 of Trp386, the corresponding residue of which is not found in ADH-IIB and qhEDH, and might be the residue responsible for making ADH-IIG prefer diol substrates.

Alcohol Oxidoreductases↗

PubMed2005

Cloning, expression and crystallization of heterotetrameric sarcosine oxidase from Pseudomonas maltophilia.

Heterotetrameric sarcosine oxidase (TSOX) is a complex bifunctional enzyme that catalyzes the oxidation of the methyl group in sarcosine (N-methylglycine) and transfer of the oxidized methyl group into the one-carbon metabolic pool. In addition to four different subunits, TSOX contains three coenzymes (FAD, FMN, and NAD) and a binding site for tetrahydrofolate, the coenzyme acceptor of the oxidized methyl group from sarcosine. Based on preliminary success in crystallization of the natural enzyme, the genes encoding the subunits for TSOX from Pseudomonas maltophilia (pTSOX) were cloned by functional screening of a genomic library. Recombinant enzyme exhibiting the same specific activity as natural pTSOX could not be isolated using a similar or identical purification procedure. This difficulty was overcome by affinity purification of recombinant pTSOX containing a C-terminal (His)(6) tag on the subunit (gamma) encoded by soxG, the gene located at the 3' end of the pTSOX operon. Affinity-purified pTSOX could not be crystallized, a problem traced to microheterogeneity in the recombinant enzyme where about half of the FMN is present in a modified form that is not found in the natural enzyme and may be a biosynthetic intermediate. The modified flavin was eliminated by expression of the recombinant enzyme in the presence of sarcosine, the same reagent used to induce expression of the natural enzyme. Homogenous recombinant pTSOX was isolated from cells grown in the presence of sarcosine by chromatography on affinity and hydrophobic interaction matrices. High quality crystals that diffract to 1.85 A resolution have been obtained.

Amino Acid Sequence↗

PubMed2005

A novel, potent dual inhibitor of the leukocyte proteases cathepsin G and chymase: molecular mechanisms and anti-inflammatory activity in vivo.

Certain leukocytes release serine proteases that sustain inflammatory processes and cause disease conditions, such as asthma and chronic obstructive pulmonary disease. We identified beta-ketophosphonate 1 (JNJ-10311795; RWJ-355871) as a novel, potent dual inhibitor of neutrophil cathepsin G (K(i) = 38 nm) and mast cell chymase (K(i) = 2.3 nm). The x-ray crystal structures of 1 complexed with human cathepsin G (1.85 A) and human chymase (1.90 A) reveal the molecular basis of the dual inhibition. Ligand 1 occupies the S(1) and S(2) subsites of cathepsin G and chymase similarly, with the 2-naphthyl in S(1), the 1-naphthyl in S(2), and the phosphonate group in a complex network of hydrogen bonds. Surprisingly, however, the carboxamido-N-(naphthalene-2-carboxyl)piperidine group is found to bind in two distinct conformations. In cathepsin G, this group occupies the hydrophobic S(3)/S(4) subsites, whereas in chymase, it does not; rather, it folds onto the 1-naphthyl group of the inhibitor itself. Compound 1 exhibited noteworthy anti-inflammatory activity in rats for glycogen-induced peritonitis and lipopolysaccharide-induced airway inflammation. In addition to a marked reduction in neutrophil influx, 1 reversed increases in inflammatory mediators interleukin-1alpha, interleukin-1beta, tissue necrosis factor-alpha, and monocyte chemotactic protein-1 in the glycogen model and reversed increases in airway nitric oxide levels in the lipopolysaccharide model. These findings demonstrate that it is possible to inhibit both cathepsin G and chymase with a single molecule and suggest an exciting opportunity in the treatment of asthma and chronic obstructive pulmonary disease.

Acute Disease↗

PubMed2005

Data classification with radial basis function networks based on a novel kernel density estimation algorithm.

This paper presents a novel learning algorithm for efficient construction of the radial basis function (RBF) networks that can deliver the same level of accuracy as the support vector machines (SVMs) in data classification applications. The proposed learning algorithm works by constructing one RBF subnetwork to approximate the probability density function of each class of objects in the training data set. With respect to algorithm design, the main distinction of the proposed learning algorithm is the novel kernel density estimation algorithm that features an average time complexity of O(n log n), where n is the number of samples in the training data set. One important advantage of the proposed learning algorithm, in comparison with the SVM, is that the proposed learning algorithm generally takes far less time to construct a data classifier with an optimized parameter setting. This feature is of significance for many contemporary applications, in particular, for those applications in which new objects are continuously added into an already large database. Another desirable feature of the proposed learning algorithm is that the RBF networks constructed are capable of carrying out data classification with more than two classes of objects in one single run. In other words, unlike with the SVM, there is no need to resort to mechanisms such as one-against-one or one-against-all for handling datasets with more than two classes of objects. The comparison with SVM is of particular interest, because it has been shown in a number of recent studies that SVM generally are able to deliver higher classification accuracy than the other existing data classification algorithms. As the proposed learning algorithm is instance-based, the data reduction issue is also addressed in this paper. One interesting observation in this regard is that, for all three data sets used in data reduction experiments, the number of training samples remaining after a naive data reduction mechanism is applied is quite close to the number of support vectors identified by the SVM software. This paper also compares the performance of the RBF networks constructed with the proposed learning algorithm and those constructed with a conventional cluster-based learning algorithm. The most interesting observation learned is that, with respect to data classification, the distributions of training samples near the boundaries between different classes of objects carry more crucial information than the distributions of samples in the inner parts of the clusters.

Algorithms↗

PubMed2005

Key role of cysteine residues in catalysis and subcellular localization of sulfur oxygenase-reductase of Acidianus tengchongensis.

Analysis of known sulfur oxygenase-reductases (SORs) and the SOR-like sequences identified from public databases indicated that they all possess three cysteine residues within two conserved motifs (V-G-P-K-V-C(31) and C(101)-X-X-C(104); numbering according to the Acidianus tengchongensis numbering system). The thio-modifying reagent N-ethylmaleimide and Zn(2+) strongly inhibited the activities of the SORs of A. tengchongensis, suggesting that cysteine residues are important. Site-directed mutagenesis was used to construct four mutant SORs with cysteines replaced by serine or alanine. The purified mutant proteins were investigated in parallel with the wild-type SOR. Replacement of any cysteine reduced SOR activity by 98.4 to 100%, indicating that all the cysteine residues are crucial to SOR activities. Circular-dichroism and fluorescence spectrum analyses revealed that the wild-type and mutant SORs have similar structures and that none of them form any disulfide bond. Thus, it is proposed that three cysteine residues, C(31) and C(101)-X-X-C(104), in the conserved domains constitute the putative binding and catalytic sites of SOR. Furthermore, enzymatic activity assays of the subcellular fractions and immune electron microscopy indicated that SOR is not only present in the cytoplasm but also associated with the cytoplasmic membrane of A. tengchongensis. The membrane-associated SOR activity was colocalized with the activities of sulfite:acceptor oxidoreductase and thiosulfate:acceptor oxidoreductase. We tentatively propose that these enzymes are located in close proximity on the membrane to catalyze sulfur oxidation in A. tengchongensis.

Acidianus↗

PubMed2004

The anticoagulant thrombin mutant W215A/E217A has a collapsed primary specificity pocket.

The thrombin mutant W215A/E217A features a drastically impaired catalytic activity toward chromogenic and natural substrates but efficiently activates the anticoagulant protein C in the presence of thrombomodulin. As the remarkable anticoagulant properties of this mutant continue to be unraveled in preclinical studies, we solved the x-ray crystal structures of its free form and its complex with the active site inhibitor H-d-Phe-Pro-Arg-CH(2)Cl (PPACK). The PPACK-bound structure of W215A/E217A is identical to the structure of the PPACK-bound slow form of thrombin. On the other hand, the structure of the free form reveals a collapse of the 215-217 strand that crushes the primary specificity pocket. The collapse results from abrogation of the stacking interaction between Phe-227 and Trp-215 and the polar interactions of Glu-217 with Thr-172 and Lys-224. Other notable changes are a rotation of the carboxylate group of Asp-189, breakage of the H-bond between the catalytic residues Ser-195 and His-57, breakage of the ion pair between Asp-222 and Arg-187, and significant disorder in the 186- and 220-loops that define the Na(+) site. These findings explain the impaired catalytic activity of W215A/E217A and demonstrate that the analysis of the molecular basis of substrate recognition by thrombin and other proteases requires crystallization of both the free and bound forms of the enzyme.

Anticoagulants↗

PubMed2004

Molecular dissection of Na+ binding to thrombin.

Na(+) binding near the primary specificity pocket of thrombin promotes the procoagulant, prothrombotic, and signaling functions of the enzyme. The effect is mediated allosterically by a communication between the Na(+) site and regions involved in substrate recognition. Using a panel of 78 Ala mutants of thrombin, we have mapped the allosteric core of residues that are energetically linked to Na(+) binding. These residues are Asp-189, Glu-217, Asp-222, and Tyr-225, all in close proximity to the bound Na(+). Among these residues, Asp-189 shares with Asp-221 the important function of transducing Na(+) binding into enhanced catalytic activity. None of the residues of exosite I, exosite II, or the 60-loop plays a significant role in Na(+) binding and allosteric transduction. X-ray crystal structures of the Na(+)-free (slow) and Na(+)-bound (fast) forms of thrombin, free or bound to the active site inhibitor H-d-Phe-Pro-Arg-chloromethyl-ketone, document the conformational changes induced by Na(+) binding. The slow --> fast transition results in formation of the Arg-187:Asp-222 ion pair, optimal orientation of Asp-189 and Ser-195 for substrate binding, and a significant shift of the side chain of Glu-192 linked to a rearrangement of the network of water molecules that connect the bound Na(+) to Ser-195 in the active site. The changes in the water network and the allosteric core explain the thermodynamic signatures linked to Na(+) binding and the mechanism of thrombin activation by Na(+). The role of the water network uncovered in this study establishes a new paradigm for the allosteric regulation of thrombin and other Na(+)-activated enzymes involved in blood coagulation and the immune response.

Allosteric Site↗

PubMed2004

Separation and characterization of different signals from intermolecular three-spin orders in solution NMR.

In this paper, signals originating from a pure specific coherence of intermolecular three-spin orders were separated and characterized experimentally in highly polarized two-component spin systems. A modified CRAZED sequence with selective radio-frequency excitation was designed to separate the small signals from the strong conventional single-spin single-quantum signals. General theoretical expressions of the pulse sequence with arbitrary flip angle pulses were derived using dipolar field treatment. The expressions were used to predict the relaxation and diffusion properties and optimal experimental parameters such as flip angles. For the first time, relaxation and diffusion properties of pure intermolecular single-quantum, double-quantum, and triple-quantum coherences of three-spin orders were characterized and analyzed in one-dimensional experiments. All experimental observations are in excellent agreement with the theoretical predictions. The theoretical results show that the quantum-mechanical treatment leads to exactly the same predictions as the dipolar field treatment. The quantitative study of intermolecular multiple-quantum coherences of three-spin orders presented herein provides a better understanding of their mechanisms.

Journal Article↗

PubMed2004

[Plant retrotransposons and their application].

Retrotransposons are one of the most unstable genetic elements in the plant kingdom, they have the potential to dramatically affect gene function and host genome structure. The current status of their types and structure, expression regulation, transposition, and evolution are reviewed. Their potential as genetic tools are also discussed.

Gene Expression Regulation, Plant↗

PubMed2004

[Study on the distribution of human immunodeficiency virus-1 subtypes in different regions of China and mother-to-child transmission].

OBJECTIVE: To study the distribution of human immunodeficiency virus (HIV)-1 genotypes in major prevalent regions of China and to illustrate the relationship between HIV-1 subtypes and mother-to-child transmission in a retrospective cohort. METHODS: HIV-1 gag p17 and env C2-V4 region were amplified by nested-polymerase chain reaction (nPCR) and the sequences were obtained by sequencing gag nPCR products or clones of env gene. RESULTS: 60 HIV-1 positive individuals were subject to typing for gag p17 and 69 for env C2-V4 region. Single clade was only found in Henan (subtype B') and Xinjiang (subtype C), and subtypes C and E were demonstrated in Yunnan. These regions represented most of the HIV-1 infections in China. Multiple subtypes (A, B, C, E, etc.) were found in Beijing and Shanghai, where HIV infections were still in low level. The sequences of subtype C were less diversive in Xinjiang (p17: 0.0192 +/- 0.0078, C2-V4: 0.0455 +/- 0.0145) than in Yunnan (p17: 0.0279 +/- 0.0102, C2-V4: 0.0482 +/- 0.0171), but all of them clustered in "C" branch in phylogenetic trees. Trafficking of subtype C from Yunnan to Xinjiang was found but had already been reported by others. Compared to subtype C, subtype E was quite divergent (p17: 0.0473 +/- 0.0105, C2-V4: 0.1114 +/- 0.0112) in Yunnan, but no recombination was found in the C2-V4 region of env gene. Highe divergence of subtype B' was found in Henan and the peripheral provinces (p17: 0.0381 +/- 0.0101, C2-V4: 0.0691 +/- 0.0166), which might be attributed to the early epidemics of HIV-1 in these areas (early 1990's). In maternal-child cohort, subtypes B (7/21), C (11/21), E (1/21) and undefined types (2/21) were identified in non-transmitting HIV-1 positive mothers, while only subtype B (7/11) and C (4/11) appeared in transmitting HIV-1 positive mothers. The rate of transmission was 53.8% (7/13) in mothers infected with subtype B and 30.8% (4/13) in those infected with subtype C, but with no significant difference (P = 0.196). The imbalancing distribution of subtypes might be explained by the fact that transfusion or illegal blood would increased mother-to-child transmission on HIV-1 and most of mothers with clade B were infected by illegal blood transfusion in this cohort. In addition, most of the maternal-child pair's sequences clustered in gag or env phylogenetic trees but only a few did disperse among the unrelated patients because children were older (>/= 4 years). CONCLUSION: The characteristics of HIV-1 clade's distribution differed over most parts of China but no difference was demonstrated between subtype B and C in mother-to-child transmission on HIV-1.

Child, Preschool↗

PubMed2003

Purification and properties of the sulfur oxygenase/reductase from the acidothermophilic archaeon, Acidianus strain S5.

The sulfur oxygenase/reductase (SOR) of Acidianus strain S5 was purified and characterized after expressing the SOR gene in a recombinant strain of Escherichia coli. The N-terminal sequence of the purified SOR protein was the same as the deduced amino acid sequence from previously cloned SOR genes. Enzymatic studies indicated that the SOR catalyzed the conversion of elemental sulfur (S(o)) to sulfite, thiosulfate, and sulfide. The optimal pH and temperature were 5.0 and 70 degrees C, respectively. Comparison of this SOR and that of A. ambivalens revealed several differences between these two SORs. The most striking difference is that the SOR of Acidianus S5 had maximal activity at acidic pH. By application of anti-SOR serum and the Western blot technique, it was found that SOR proteins existed in A. brierleyi and in Acidianus S5 cells cultivated with thiosulfate as the sole energy source, indicating that SOR may also play a role in thiosulfate metabolism.

Acidianus↗

PubMed2003

[Analysis of yellow peach germplasm revealed by RAPD markers].

Genomic DNAs of 37 cultivars of yellow peach (Amgdalus persica) were amplified by RAPD (Randomly Amplified Polymorphic DNA) with 22 primers selected from 200 arbitrary 10 decamer primers. Phylogenetic relationship was analyzed through cluster of bands in 180 amplified sites. Molecular checking index and conservation of key germplasm were put forward according to special bands.

English Abstract↗

PubMed2002

Monomeric sarcosine oxidase: role of histidine 269 in catalysis.

Conservative mutation of His269 (to Asn, Ala, or Gln) does not-significantly affect the expression of monomeric sarcosine oxidase (MSOX), covalent flavinylation, the physicochemical properties of bound FAD, or the overall protein structure. Turnover with sarcosine and the limiting rate of the reductive half-reaction with L-proline at pH 8.0 are, however, nearly 2 orders of magnitude slower than that with with wild-type MSOX. The crystal structure of the His269Asn complex with pyrrole-2-carboxylate shows that the pyrrole ring of the inhibitor is displaced as compared with wild-type MSOX. The His269 mutants all form charge-transfer complexes with pyrrole-2-carboxylate or methylthioacetate, but the charge-transfer bands are shifted to shorter wavelengths (higher energy) as compared with wild-type MSOX. Both wild-type MSOX and the His269Asn mutant bind the zwitterionic form of L-proline. The E(ox).L-proline complex formed with the His269Asn mutant or wild-type MSOX contains an ionizable group (pK(a) = 8.0) that is required for conversion of the zwitterionic L-proline to the reactive anionic form, indicating that His269 is not the active-site base. We propose that the change in ligand orientation observed upon mutation of His269 results in a less than optimal overlap of the highest occupied orbital of the ligand with the lowest unoccupied orbital of the flavin. The postulated effect on orbital overlap may account for the increased energy of charge-transfer bands and the slower rates of electron transfer observed for mutant enzyme complexes with charge-transfer ligands and substrates, respectively.

Base Sequence↗