Proteomic analysis of endogenous nitrotryptophan-containing proteins in rat hippocampus and cerebellum

Nitration of tryptophan residues is a novel post-translational modification. In the present study, we examined whether NO₂Trp (nitrotryptophan)-containing proteins are produced in the hippocampus and cerebellum of the adult rat under physiological conditions in vivo. Using Western blot analysis with anti-6-NO₂Trp-specific antibody, we found many similar immunoreactive spots in the protein extracts from both regions. These spots were subsequently subjected to trypsin digestion and LC-ESI-MS/MS (LC-electrospray ionization-tandem MS) analysis. We identified several cytoskeletal proteins and glycolytic enzymes as NO₂Trp-containing proteins and determined the position of nitrated tryptophan residues with significant ion score levels (P<0.05) in several proteins in both regions. We also observed that the total amount of NO₂Trp-containing proteins in the cerebellum was significantly greater than that in the hippocampus (P<0.05). Moreover, IP (immunoprecipitation) assays using anti-aldolase C antibody showed that the relative intensity of immunostaining for NO₂Trp over aldolase C was much higher in cerebellum than in hippocampus. The amounts of nNOS (neuronal nitric oxide synthase) and eNOS (endothelial nitric oxide synthase) were much greater in cerebellum than in hippocampus. This is the first evidence of several specific sites of nitrated tryptophan in proteins under physiological conditions in vivo.     

Mutational analysis of the stability of the H2A and H2B histone monomers

The eukaryotic histone heterodimer H2A-H2B folds through an obligatory dimeric intermediate that forms in a nearly diffusion-limited association reaction in the stopped-flow dead time. It is unclear whether there is partial folding of the isolated monomers before association. To address the possible contributions of structure in the monomers to the rapid association, we characterized H2A and H2B monomers in the absence of their heterodimeric partner. By far-UV circular dichroism, the H2A and H2B monomers are 15% and 31% helical, respectively—significantly less than observed in X-ray crystal structures. Acrylamide quenching of the intrinsic Tyr fluorescence was indicative of tertiary structure. The H2A and H2B monomers exhibit free energies of unfolding of 2.5 and 2.9 kcal mol^− 1, respectively; at 10 μM, the sum of the stability of the monomers is ∼ 60% of the stability of the native dimer. The helical content, stability, and m values indicate that H2B has a more stable, compact structure than H2A. The monomer m values are larger than expected for the extended histone fold motif, suggesting that the monomers adopt an overly collapsed structure. Stopped-flow refolding—initiated from urea-denatured monomers or the partially folded monomers populated at low denaturant concentrations—yielded essentially identical rates, indicating that monomer folding is productive in the rapid association and folding of the heterodimer. A series of Ala and Gly mutations were introduced into H2A and H2B to probe the importance of helix propensity on the structure and stability of the monomers. The mutational studies show that the central α-helix of the histone fold, which makes extensive intermonomer contacts, is structured in H2B but only partially folded in H2A.     

Probing the role of aromatic residues at the secondary saccharide-binding sites of human salivary alpha-amylase in substrate hydrolysis and bacterial binding

Human salivary α-amylase (HSAmy) has three distinct functions relevant to oral health: (1) hydrolysis of starch, (2) binding to hydroxyapatite (HA), and (3) binding to bacteria (e.g., viridans streptococci). Although the active site of HSAmy for starch hydrolysis is well-characterized, the regions responsible for bacterial binding are yet to be defined. Since HSAmy possesses several secondary saccharide-binding sites in which aromatic residues are prominently located, we hypothesized that one or more of the secondary saccharide-binding sites harboring the aromatic residues may play an important role in bacterial binding. To test this hypothesis, the aromatic residues at five secondary binding sites were mutated to alanine to generate six mutants representing either single (W203A, Y276A, and W284A), double (Y276A/W284A and W316A/W388A), or multiple [W134A/W203A/Y276A/W284A/W316A/W388A; human salivary α-amylase aromatic residue multiple mutant (HSAmy-ar)] mutations. The crystal structure of HSAmy-ar as an acarbose complex was determined at a resolution of 1.5 Å and compared with the existing wild-type acarbose complex. The wild-type and the mutant enzymes were characterized for their abilities to exhibit enzyme activity, starch-binding activity, HA-binding activity, and bacterial binding activity. Our results clearly showed that (1) mutation of aromatic residues does not alter the overall conformation of the molecule; (2) single or double mutants showed either moderate or minimal changes in both starch-binding activity and bacterial binding activity, whereas HSAmy-ar showed significant reduction in these activities; (3) starch-hydrolytic activity was reduced by 10-fold in HSAmy-ar; (4) oligosaccharide-hydrolytic activity was reduced in all mutants, but the action pattern was similar to that of the wild-type enzyme; and (5) HA binding was unaffected in HSAmy-ar. These results clearly show that the aromatic residues at the secondary saccharide-binding sites in HSAmy play a critical role in bacterial binding and in starch-hydrolytic functions of HSAmy.     

Rapid incorporation of functional rhodopsin into nanoscale apolipoprotein bound bilayer (NABB) particles

Human apolipoprotein A-I (apo A-I) and its engineered constructs form discoidal lipid bilayers upon interaction with lipids in vitro. We now report the cloning, expression, and purification of apo A-I derived from zebrafish (Danio rerio), which combines with phospholipids to form similar discoidal bilayers and may prove to be superior to human apo A-I constructs for rapid reconstitution of seven-transmembrane helix receptors into nanoscale apolipoprotein bound bilayers (NABBs). We characterized NABBs by gel-filtration chromatography, native polyacrylamide gradient gel electrophoresis, UV-visible photobleaching difference spectroscopy, and fluorescence spectroscopy. We used electron microscopy to determine the stoichiometry and orientation of rhodopsin (rho)-containing NABBs prepared under various conditions and correlated stability and signaling efficiency of rho in NABBs with either one or two receptors. We discovered that the specific activity of G protein coupling for single rhos sequestered in individual NABBs was nearly identical with that of two rhos per NABB under conditions where stoichiometry and orientation could be inferred by electron microscopy imaging. Thermal stability of rho in NABBs was superior to that of rho in various commonly used detergents. We conclude that the NABB system using engineered zebrafish apo A-I is a native-like membrane mimetic system for G-protein-coupled receptors and discuss strategies for rapid incorporation of expressed membrane proteins into NABBs.     

The integrated use of chemical insecticides and the entomopathogenic nematode,Steinernema carpocapsae (Nematoda: Steinernematidae), for the control of sweetpotato whitefly,Bemisia tabaci (Hemiptera: Aleyrodidae)

The integration of chemical insecticides and infective juveniles of the entomopathogenic nematode Steinernema carpocapsae (Wesier) (Nematoda: Steinernematidae), to control second instars of the sweetpotato whitefly, Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae) was investigated. Using a sand bioassay, the effects of direct exposure of S. carpocapsae for 24 h to field rate dilutions of four insecticides (spiromesifen, thiacloprid, imidacloprid and pymetrozine) on infectivity to Galleria mellonella larvae were tested. Although all chemicals tested, except spiromesifen, produced acceptable nematode infectivity rates, they were all significantly less than the water control. The effect of insecticide treatment (dry residues of spiromesifen, thiacloprid and pymetrozine and soil drench of imidacloprid) on the efficacy of the nematode against B. tabaci was also investigated. Nematodes in combination with thiacloprid and spiromesifen gave higher B. tabaci mortality (86.5% and 94.3% respectively) compared to using nematodes alone (75.2%) on tomato plants. There was no significant difference in B. tabaci mortality when using the chemicals imidacloprid, pymetrozine and spiromesifen in conjunction with nematodes compared to using the chemicals alone. However, using thiacloprid in combination with the nematodes produced significantly higher B. tabaci mortality than using the chemical alone. The integration of S. carpocapsae and these chemical agents into current integrated pest management programmes for the control of B. tabaci is discussed.     

Backbone dynamics of alamethicin bound to lipid membranes: spin-echo electron paramagnetic resonance of TOAC-spin labels

Alamethicin F50/5 is a hydrophobic peptide that is devoid of charged residues and that induces voltage-dependent ion channels in lipid membranes. The peptide backbone is likely to be involved in the ion conduction pathway. Electron spin-echo spectroscopy of alamethicin F50/5 analogs in which a selected Aib residue (at position n = 1, 8, or 16) is replaced by the TOAC amino-acid spin label was used to study torsional dynamics of the peptide backbone in association with phosphatidylcholine bilayer membranes. Rapid librational motions of limited angular amplitude were observed at each of the three TOAC sites by recording echo-detected spectra as a function of echo delay time, 2τ. Simulation of the time-resolved spectra, combined with conventional EPR measurements of the librational amplitude, shows that torsional fluctuations of the peptide backbone take place on the subnanosecond to nanosecond timescale, with little temperature dependence. Associated fluctuations in polar fields from the peptide could facilitate ion permeation.     

Reactivity of asparagine residue at the active site of the D105N mutant of fluoroacetate dehalogenase from Moraxella sp. B

Fluoroacetate dehalogenase from Moraxella sp. B (FAc-DEX) catalyzes cleavage of the carbon–fluorine bond of fluoroacetate, whose dissociation energy is among the highest found in natural products. Asp105 functions as the catalytic nucleophile that attacks the α-carbon atom of the substrate to displace the fluorine atom. In spite of the essential role of Asp105, we found that site-directed mutagenesis to replace Asp105 by Asn does not result in total inactivation of the enzyme. The activity of the mutant enzyme increased in a time- and temperature-dependent manner. We analyzed the enzyme by ion-spray mass spectrometry and found that the reactivation was caused by the hydrolytic deamidation of Asn105 to generate the wild-type enzyme. Unlike Asn10 of the l-2-haloacid dehalogenase (L-DEX YL) D10N mutant, Asn105 of the fluoroacetate dehalogenase D105N mutant did not function as a nucleophile to catalyze the dehalogenation.     

On residues in the disallowed region of the Ramachandran map.

An analysis of the occurrence of nonglycyl residues in conformations disallowed in the Ramachandran plot is presented. Ser, Asn, Thr, and Cys have the highest propensities to exhibit such conformations, and the branched aliphatic residues the lowest. Residues cluster in five regions and there are some trends in the types of residues and their side-chain conformations (chi(1)) occupying these. Majority of the residues are found at the edge of helices and strands and in short loops, and are involved in different types of weak, stabilizing interactions. A structural motif has been identified where a residue in disallowed conformation occurs as the first residue of a short 3(10)-helix. On the basis of the types of neighboring residues, the location in the three-dimensional structure and accessibility, there are similarities with the occurrence of cis peptide bonds in protein structures.     

Peripheral-zone treatments for plum curculio management: validation in commercial apple orchards

A field trial was conducted using an oil formulation of the entomopathogenic fungus, Metarhizium flavoviride Gams & Rozyspal (Deuteromycotina: Hyphomycetes), to control the variegated grasshopper, Zonocerus variegatus L. (Orthoptera: Pyrgomorphidae). The influence of dispersal and secondary pick-up from the spray residue on mortality was assessed by sampling insects from the treated plots at intervals and monitoring disease levels during subsequent incubation in the laboratory. The cumulative mortality curves showed the pathogen to be highly effective, even in samples taken 8 days after spraying. A model was developed to link the influence of mortality, incubation, secondary pick-up of spores and insect dispersal, on the shape of these cumulative mortality curves. The model proved useful for data received from an experiment using small plot sizes, where insect dispersal is a big problem. The spray residue had an important influence on overall insect mortality. The model also indicated a difference in speed of kill between field and laboratory samples, suggesting an incubation delay in the field.     

Chemically-Synthesized Gene Encoding Modified Human Superoxpde Dismutase: Its Construction,Expression and Properties of the Product

The gene encoding modified human superoxide dismutax (h-SOD) with 153 amino acid residues was constructed by chemical synthesis using the phosphoramidite method. The gene was designed so as to use bacterial codons for expression in prokaryotes and to introduce several unique restriction sites for further mutagenesis by the cassette exchange method. The distance between Shine-Dalgarno sequence and initiation codon was adjusted to maximum expression by using synthesized oligonucleotide. In addition, Cys 6 of h-SOD was changed to Ala to improve instability of native h-SOD.

Synthesized structural gene of h-SOD was expressed in E. coli after induction of isopropyl β-D-thiogalactoside by inserting the gene into the expression vector pKK223–3 having tac promoter. The gene that has 10 base pairs between Shine-Dalgarno sequence and initiation codøn showed the most efficient expression. The gene produced three active SOD isomers as revealed by chromatofocusing.

The main isomer was purified to homogeneity and characterized. The h-SOD-Ala⁶ showed similar properties to those of native h-SOD with respect to molecular weight, subunit structure, absorption spectrum. but the modified SOD was more resistant to heat denaturation than was native h-SOD; half-denaturing temperature was shifted by 10°C. Thus. the exchange of Cys 6 to Ala of h-SOD increased a stability of the enzyme.