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D Xu

Publications and source records attributed to D Xu.

At least 271 records · Page 15Linked to original sources
PubMed1997

Protein binding versus protein folding: the role of hydrophilic bridges in protein associations.

The role of hydrophilic bridges between charged, or polar, atoms in protein associations has been examined from two perspectives. First, statistical analysis has been carried out on 21 data sets to determine the relationship between the binding free energy and the structure of the protein complexes. We find that the number of hydrophilic bridges across the binding interface shows a strong positive correlation with the free energy; second, the electrostatic contribution of salt bridges to binding has been assessed by a continuum electrostatics calculation. In contrast to protein folding, we find that salt bridges across the binding interface can significantly stabilize complexes in some cases. The different contributions of hydrophilic bridges to folding and to binding arise from the different environments to which the involved hydrophilic groups are exposed before and after the bridges are formed. These groups are more solvated in a denatured protein before folding than on the surface of the combining proteins before binding. After binding, they are buried in an environment whose residual composition can be much more hydrophilic than the one after folding. As a result, the desolvation cost of a hydrophilic pair is lower, and the favorable interactions between the hydrophilic pair and its surrounding residues are generally stronger in binding than in folding. These results complement our recent finding that while hydrophobic effect in protein-protein interfaces is significant, it is not as strong as that observed in the interior of monomers. Taken together, these studies suggest that while the types of forces in protein-protein interaction and in protein folding are similar, their relative contributions differ. Hence, association of protein monomers which do not undergo significant conformational change upon binding differs from protein folding, implying that conclusions (e.g. statistics, energetics) drawn from investigating folding may not apply directly to binding, and vice versa.

Binding Sites↗
PubMed1997

Bacterial translocation is inhibited in inducible nitric oxide synthase knockout mice after endotoxin challenge but not in a model of bacterial overgrowth.

BACKGROUND: Studies have shown that nitric oxide (NO) and NO synthase (NOS) inhibitors injure and protect organs after endotoxin (lipopolysaccharide [LPS]) challenge. OBJECTIVE: To test the hypothesis that LPS-induced gut injury and bacterial translocation (BT) are mediated through activation of inducible NOS (iNOS). DESIGN: A randomized, controlled study using genetically altered, iNOS gene knockout mice. SETTING: University research laboratory. METHODS: Forty-five wild-type (iNOS+/+) or homozygous mutant (iNOS-/-) mice weighing 25 to 35 g were challenged with Escherichia coli LPS or saline (10 mg/ kg) intraperitoneally (n = 8/group). In a second set of experiments, a bacterial overgrowth model of BT (E coli monoassociation) was tested (n = 6-7/group). The mesenteric lymph nodes and cecums were cultured, and liver, ileal, and blood nitrite and nitrate levels measured 24 hours after LPS or E coli monoassociation. RESULTS: After LPS challenge, 87.5% of the iNOS+/+ mice but 0% of the iNOS-/- mice had BT to their mesenteric lymph nodes (P < .01; chi 2 analysis). Nitrite and nitrate levels of the liver, ileum, and blood were higher in the iNOS+/+ mice (P < .05). In the E coli overgrowth model, BT to mesenteric lymph nodes occurred in 100% of iNOS-/- and iNOS+/+ mice. CONCLUSIONS: In this limited study, LPS-induced BT did not occur in iNOS-deficient mice, suggesting that LPS induction of increased iNOS activity is necessary for LPS-induced BT to occur. In contrast, iNOS activation does not seem to be necessary in a bacterial overgrowth model of BT.

Animals↗
PubMed1997

Structural motifs at protein-protein interfaces: protein cores versus two-state and three-state model complexes.

The general similarity in the forces governing protein folding and protein-protein associations has led us to examine the similarity in the architectural motifs between the interfaces and the monomers. We have carried out extensive, all-against-all structural comparisons between the single-chain protein structural dataset and the interface dataset, derived both from all protein-protein complexes in the structural database and from interfaces generated via an automated crystal symmetry operation. We show that despite the absence of chain connections, the global features of the architectural motifs, present in monomers, recur in the interfaces, a reflection of the limited set of the folding patterns. However, although similarity has been observed, the details of the architectural motifs vary. In particular, the extent of the similarity correlates with the consideration of how the interface has been formed. Interfaces derived from two-state model complexes, where the chains fold cooperatively, display a considerable similarity to architectures in protein cores, as judged by the quality of their geometric superposition. On the other hand, the three-state model interfaces, representing binding of already folded molecules, manifest a larger variability and resemble the monomer architecture only in general outline. The origin of the difference between the monomers and the three-state model interfaces can be understood in terms of the different nature of the folding and the binding that are involved. Whereas in the former all degrees of freedom are available to the backbone to maximize favorable interactions, in rigid body, three-state model binding, only six degrees of freedom are allowed. Hence, residue or atom pair-wise potentials derived from protein-protein associations are expected to be less accurate, substantially increasing the number of computationally acceptable alternate binding modes (Finkelstein et al., 1995).

Chemical Phenomena↗
PubMed1997

Analysis of human IL-2/IL-2 receptor beta chain interactions: monoclonal antibody H2-8 and new IL-2 mutants define the critical role of alpha helix-A of IL-2.

Interleukin 2 (IL-2) interacts with a receptor (IL-2R) composed of three subunits (IL-2R alpha, IL-2R beta and IL-2R gamma). IL-2R beta plays a critical role in signal transduction. An anti-human IL-2 mAb (H2-8) produced after immunization with peptide 1-30 of IL-2 was found to recognize the region occupied by Asp20, at the exposed interface between alpha-helices A and C. Muteins at position 17 and 20 are not recognized by mAb H2-8. mAb H2-8 specifically inhibits the IL-2 proliferation of TS1beta cells which are dependent on the expression of human IL-2R beta chain for IL-2 proliferation. Substitution at internal position Leu17 demonstrates that this position is essential for IL-2 binding and IL-2 bioactivity. New IL-2 mutants at position Asp20 have been analysed. Substitutions Asp --> Asn, Asp --> Lys, Asp --> Leu, show a correlation between diminished affinity for IL-2 receptor and reduced bioactivity measured on TS1beta cells. Mutein Asp Arg lose affinity for IL-2R and bioactivity simultaneously. Furthermore, during the course of the study we have found that mutein Asp20 --> Leu is an IL-2 antagonist. The biological effects of mAb H2-8 and the properties of new mutants at positions 17 and 20 demonstrate that this region of alpha helix-A is involved in IL-2-IL-2R beta interactions.

Animals↗
PubMed1997

Kinetics of irreversible inhibition of yeast alcohol dehydrogenase during modification by 4,4'-dithiodipyridine.

The course of inactivation of yeast alcohol dehydrogenase (YADH) using 4,4'-dithiodipyridine (DSDP) has been studied in this paper. The results show that the reaction mechanism between DSDP and YADH is a competitive, complexing inhibition. The microscopic constants for the inactivation of the free enzyme and the enzyme-substrate complex were determined. The presence of the substrate NAD+ offers strong protection for this enzyme against inactivation by DSDP. The above results suggest that two Cys residues are essential for activity and are situated at the active site. These essential Cys residues should be Cys-46 and Cys-174 which are ligands to the catalytic zinc ion. Another Cys residue, which can be modified by DSDP, is non-essential for activity of the enzyme.

Alcohol Dehydrogenase↗
PubMed1997

Epitope characterization of malondialdehyde-acetaldehyde adducts using an enzyme-linked immunosorbent assay.

Malondialdehyde (MDA) and acetaldehyde react together with proteins in a synergistic manner and form hybrid protein adducts, designated as MAA adducts. In a previous study, a polyclonal antibody specific for MAA-protein adducts was used in an immunoassay to detect the presence of MAA adducts in livers of ethanol-fed rats. In the present study, the specific epitope recognized by the antibody was defined and the chemistry of MAA adduct formation was further characterized. When several synthetic analogs were tested for their ability to inhibit antibody binding in a competitive ELISA, the results indicated that the major determinant of antibody binding was a highly fluorescent cyclic adduct composed of two molecules of MDA and one of acetaldehyde. The structure of this adduct was shown to be a 4-methyl-1,4-dihydropyridine-3,5-dicarbaldehyde derivative of an amino group of a protein. Examination of MAA adduct formation with a variety of proteins indicated that in addition to this specific fluorescent adduct, MAA adducts were also comprised of other nonfluorescent products. The amount of fluorescent epitopes present on a given protein was the major determinant of antibody binding as assessed in a competitive ELISA, although the efficiency of inhibition of antibody binding by these fluorescent epitopes on MAA-adducted proteins varied depending upon the particular protein. However, when these MAA-adducted proteins were hydrolyzed with Pronase, the concentration of these modified proteins necessary to achieve 50% inhibition of antibody binding in a competitive ELISA fell into a much narrower range of values, indicating that protein hydrolysis equalized the accessibility of the antibody to bind the epitope on these various derivatized proteins. In summary, a cyclic fluorescent adduct of defined structure has been identified as the epitope recognized by our MAA adduct antibody. In addition to this specific adduct, MAA adducts are also comprised of other nonfluorescent products.

Acetaldehyde↗
PubMed1997

The essential tryptophan residues of pig kidney aminoacylase.

The tryptophan residues in pig kidney aminoacylase (N-acylamino acid amido hydrolase, EC 3.5.1.14) have been modified by N-bromosuccinimide (NBS) at low pH. The modification of eight tryptophan residues as measured by spectrophotometric and spectrofluorimetric methods leads to complete loss of enzymatic activity. The decreases in absorption at 280 nm and fluorescence emission at 337 nm indicate the modification of tryptophan residues. Both the inactivation and tryptophan residual modification are monophasic, first-order reactions. Quantitative treatment of the data (Tsou, C. L., Sci. Sin., 1962, 11, 1535-1558) shows that among the tryptophan residues modified, two are essential for aminoacylase catalytic activity. Kördel and Schneider (Hoppe-Seyler's Physiol. Chem. 1976, 357, 1109-1115) reported that the modification of tryptophan residues led to inactivation of aminoacylase, and suggested that tryptophan residues are essential for enzymatic activity. We have now shown that eight tryptophan residues can be modified by N-bromosuccinimide and that two of them are essential for the catalytic activity of this enzyme.

Amidohydrolases↗
PubMed1997

Electron microscopic observations on the normal development of Trichinella spiralis from muscle larvae to adult worms in BALB/c mice with emphasis on the body wall, genital organs and gastrointestinal organs.

Ultrastructural changes during the development of Trichinella spiralis from muscle larvae to adult worms are described with emphasis on the body wall, genital organs and gastrointestinal organs. The cuticle of the body wall and hindgut underwent the first molting at 14 h post-infection (PI). The esophagus cuticle never molted. The hypodermal gland was formed in the lateral cords by 30 h PI. The genital primordium developed to either male or female genitals, accomplishing their sexual maturity and fertilization by 3 days PI. Glycogen disappeared during the development.

Animals↗
PubMed1997

Electron microscopic observations of the stichosome during the normal development of Trichinella spiralis from muscle larvae to adult worms in BALB/c mice.

The exocrine granules of the stichosome of Trichinella spiralis contain excretory and secretory (ES) products that may alter host cell physiology in such a way that T.spiralis can establish parasitism in the host [1,2]. The stichosome is the most intriguing but still mysterious exocrine organ. This paper describes ultrastructural changes of the stichosome during the normal development from muscle larvae to adult worms. Stichocyte granules of the muscle larva stage were excreted by 14 h post-infection (PI). Then the stichosome synthesized a new type of granules, which disappeared from the stichosome by 30 h PI. These transient granules were morphologically different from granules of muscle larva and adult stages.

Animals↗
PubMed1997

Prediction of protein supersecondary structures based on the artificial neural network method.

The sequence patterns of 11 types of frequently occurring connecting peptides, which lead to a classification of supersecondary motifs, were studied. A database of protein supersecondary motifs was set up. An artificial neural network method, i.e. the back propagation neural network, was applied to the predictions of the supersecondary motifs from protein sequences. The prediction correctness ratios are higher than 70%, and many of them vary from 75 to 82%. These results are useful for the further study of the relationship between the structure and function of proteins. It may also provide some important information about protein design and the prediction of protein tertiary structure.

Amino Acid Sequence↗
PubMed1997

Hydrogen bonds and salt bridges across protein-protein interfaces.

To understand further, and to utilize, the interactions across protein-protein interfaces, we carried out an analysis of the hydrogen bonds and of the salt bridges in a collection of 319 non-redundant protein-protein interfaces derived from high-quality X-ray structures. We found that the geometry of the hydrogen bonds across protein interfaces is generally less optimal and has a wider distribution than typically observed within the chains. This difference originates from the more hydrophilic side chains buried in the binding interface than in the folded monomer interior. Protein folding differs from protein binding. Whereas in folding practically all degrees of freedom are available to the chain to attain its optimal configuration, this is not the case for rigid binding, where the protein molecules are already folded, with only six degrees of translational and rotational freedom available to the chains to achieve their most favorable bound configuration. These constraints enforce many polar/charged residues buried in the interface to form weak hydrogen bonds with protein atoms, rather than strongly hydrogen bonding to the solvent. Since interfacial hydrogen bonds are weaker than the intra-chain ones to compete with the binding of water, more water molecules are involved in bridging hydrogen bond networks across the protein interface than in the protein interior. Interfacial water molecules both mediate non-complementary donor-donor or acceptor-acceptor pairs, and connect non-optimally oriented donor-acceptor pairs. These differences between the interfacial hydrogen bonding patterns and the intra-chain ones further substantiate the notion that protein complexes formed by rigid binding may be far away from the global minimum conformations. Moreover, we summarize the pattern of charge complementarity and of the conservation of hydrogen bond network across binding interfaces. We further illustrate the utility of this study in understanding the specificity of protein-protein associations, and hence in docking prediction and molecular (inhibitor) design.

Crystallography, X-Ray↗
PubMed1997

Role of bacterial adherence and the mucus barrier on bacterial translocation: effects of protein malnutrition and endotoxin in rats.

OBJECTIVE: The purpose of the study was to investigate the potential relations between mucosal bacterial adherence, intestinal mucus and mucin content, and bacterial translocation. SUMMARY BACKGROUND DATA: The attachment of bacteria to mucosal surfaces is the initial event in the pathogenesis of most bacterial infections that originate at mucosal surfaces, such as the gut. The intestinal mucus layer appears to function as a defensive barrier limiting micro-organisms present in the intestinal lumen from colonizing enterocytes. Consequently, studies focusing on the biology of bacterial adherence to the intestinal mucosa likely are to be important in clarifying the pathogenesis of gut origin sepsis. METHODS: To explore the relations between intestinal bacterial adherence, mucus bacterial binding, and bacterial translocation, two models were used. One (protein malnutrition) in which profound alterations in intestinal morphology occurs in the absence of significant translocation and one (endotoxin challenge) in which bacterial translocation occurs and intestinal morphology is relatively normal. RESULTS: Protein malnutrition was not associated with bacterial translocation and measurement of enteroadherent, mucosally associated bacterial population levels documented that the total number of gram-negative enteric bacilli adherent to the ileum and cecum was less in the protein-malnourished rats than in the normally nourished animals (p < 0.01). Furthermore, there was an inverse relation between the duration of protein malnutrition and bacterial adherence to the intestinal mucosa (r = 0.62, p < 0.002). In contrast, after endotoxin challenge, the level of enteroadherent bacteria was increased and bacterial translocation was observed. The binding of Escherichia coli to immobilized ileal mucus in vitro was decreased significantly in protein-malnourished rats, whereas E. coli binding to insoluble ileal mucus was increased in the rats receiving endotoxin. CONCLUSIONS: This study indicates that the adherence of bacteria to the intestinal mucosal surface is an important factor in bacterial translocation, that intestinal mucus modulates bacterial adherence, and that increased levels of mucosally associated bacteria are associated with a loss intestinal barrier function to bacteria.

Animals↗
PubMed1997

Association of the brain natriuretic peptide gene with blood pressure and heart weight in the rat.

1. Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are important in the control of body fluid homeostasis, blood pressure (BP) regulation and vascular remodelling. The genes for these peptides may, therefore, be involved in the pathogenesis of genetic hypertension. We have previously described a quantitative trait locus (QTL) for BP in the ANP gene region on rat chromosome 5. We have now assessed the possibility that this QTL lies at the closely linked BNP locus. 2. Intra-arterial BP and heart weight were measured in 12-week-old (n = 207) and 24-week-old (n = 88) F2 rats derived from crosses between Wistar-Kyoto normotensive rats and spontaneously hypertensive rats. We designed polymerase chain reaction primers to amplify a microsatellite in the BNP gene from genomic DNA. Analysis of variance was used for cosegregation analysis. Linkage mapping and localization of QTL was performed using the Mapmaker computer package. 3. A significant correlation was found between genotype for the BNP gene and systolic BP (P < 0.001) in 12-week-old rats. The ANP gene, but not the BNP gene, was associated with systolic BP in 24 week rats. There was no segregation of heart weight with BNP genotype at 12 or 24 weeks of age. The BNP gene mapped approximately 20 cM from the ANP gene in our rat hybrids, away from the previously described QTL. There was evidence for a second BP locus near to but distinct from the BNP gene. 4. These results suggest that BP QTL are present in the natriuretic peptide gene region but that the ANP and BNP genes themselves have no major effect on BP in this cross.

Animals↗
PubMed1997

Vaccine efficacy of Salmonella strains expressing glycoprotein 63 with different promoters.

The development of Salmonella vaccine vectors has been hindered by both the requirement for multiple doses to induce immune responses and a lack of plasmid stability. Direct comparisons of different promoter systems with the same antigen are necessary to address these important issues. We have previously described an AroA- AroD- deletion mutant of Salmonella typhimurium (GID101) which expresses the gene encoding the Leishmania major promastigote surface glycoprotein gp63 (GID101). While this construct provided significant protection against L. major challenge to highly susceptible BALB/c mice, this required at least two oral doses. We report here the use of two different inducible promoters, the nirB and osmC promoters, to improve vaccine efficacy. These constructs (termed GID105 and GID106, respectively) expressed gp63 in vitro under inducible conditions and colonized BALB/c mice after oral administration. GID105 demonstrated greater plasmid stability in vitro and in vivo than did either GID106 or GID101, which expresses gp63 constitutively. Spleen and lymph node cells from mice immunized with a single oral dose of GID105 proliferated in vitro in response to L. major and secreted gamma interferon, whereas cells from mice given the other constructs did not. Mice immunized with a single oral dose of GID1O5 or GID106 developed significantly smaller lesions upon challenge with L. major, whereas mice administered GID101 did not. Mice administered GID105 also showed considerable resistance to Leishmania donovani infection. These data provide a direct comparison of promoter systems and demonstrate that the use of inducible promoters such as the nirB promoter allows a considerable improvement over the previous vaccine construct in terms of protection against infection.

Animals↗
PubMed1997

A novel phospholipase C- and cAMP-independent positive inotropic mechanism via a P2 purinoceptor.

Although ATP, acting through a P2 purinoceptor, can stimulate a pronounced positive inotropic effect in cardiac ventricular myocytes, the receptor-effector mechanism that underlies this stimulatory cardiac action is not well understood. The objectives of the present study were to develop the cultured chick embryo ventricular myocytes as a novel model for the cardiac P2 purinoceptor and to determine the mechanism underlying its positive inotropic effect. ATP caused an 89 +/- 8.9% (n = 14 cells) increase in the myocyte contractility, with an efficacy and potency order of ATP > ADP > AMP >> adenosine. 2-Methylthio-ATP (2-MeS-ATP) but not alpha,beta-methylene-ATP was able to stimulate myocyte contractility, with a maximal increase of 54 +/- 2.6% (n = 11 cells). Although UTP potently stimulates phosphoinositide hydrolysis, it had an only modest positive inotropic effect (27 +/- 7% maximal increase; n = 8 cells). In contrast to previous suggestions, the 2-MeS-ATP-stimulated positive inotropic response does not require the action of phospholipase C (PLC), such as that of the inositol phosphates; the UTP effect on contractility appears to be mediated via the 2-MeS-ATP-sensitive P2 receptor. The PLC inhibitor U-73122 had no effect on the 2-MeS-ATP-stimulated increase in contractility, providing further evidence against a role for PLC in the inotropic effect of 2-MeS-ATP. An adenosine 3',5'-cyclic monophosphate-independent Ca2+ entry-stimulating mechanism appears to underlie a direct coupling of the receptor to stimulation of the myocyte contractility. This new PLC- and adenosine 3',5'-cyclic monophosphate-independent positive inotropic mechanism represents a target for developing novel positive inotropic therapeutics.

Adenosine↗
PubMed1997

[Prevention and treatment of seasonal asthmatic patients by combined invigorating kidney for preventing asthma tablets and beclomethasone dipropinate].

OBJECTIVE: To evaluate the efficacy of combined Invigorating Kidney for Preventing Asthma (IKPA) tablets and beclomethasone dipropinate (BDP). METHODS: Fifty seven seasonal asthmatic patients were studied. They were randomly assigned to either of the two groups: the treated group (n = 32) treated with the IKPA tablets and inhalated BDP, the control group (n = 25) inhalated BDP singly. Measurements of serum eosinophil cationic protein (ECP), soluble IL-2 receptor (SIL-2), total eosinophil counts, spirometry and methacholine challenge were performed before and after treatment. RESULTS: After 3 months' treatment and 6 months' observation the authors observed a significant improvement in both groups. The treated group had better clinical efficacy and the ECP level had reduced significantly, FEV1 increased greatly than the control group. CONCLUSIONS: The two drugs influenced the function of eosinophils and and T lymphocytes and this might contribute to the efficacy.

Adolescent↗