Reactions of sperm whale myoglobin with hydrogen peroxide. Effects of distal pocket mutations on the formation and stability of the ferryl intermediate

Distal pocket mutants of sperm whale oxymyoglobin (oxy-Mb) were reacted with a 2.5-fold excess of hydrogen peroxide (HOOH) in phosphate buffer at pH 7.0, 37 degreesC. We describe a mechanism composed of three distinct steps: 1) initial oxidation of oxy- to ferryl-Mb, 2) autoreduction of the ferryl intermediate to ferric metmyoglobin (metMb), and 3) reaction of metMb with an additional HOOH molecule to regenerate the ferryl intermediate creating a pseudoperoxidase catalytic cycle. Mutation of Leu-29(B10) to Phe slows the initial oxidation reaction 3-fold but has little effect on the rate of ferryl reduction to ferric met-aquo-myoglobin. In contrast, the Val-68(E11) to Phe mutation causes a small, 60% increase in the initial oxidation reaction and a much larger 2. 5-fold increase in the rate of autoreduction. Double insertion of Phe at both the B10- and E11-positions (L29F/V68F) produces a mutant with oxidation characteristics of both single mutants, slow initial oxidation, and rapid autoreduction, but an extraordinarily high affinity for O2. Replacing His-64(E7) with Gln produces 3-4-fold increases in both processes. Combining the mutation H64Q with L29F results in a myoglobin with enhanced resistance to metMb formation in the absence of antioxidant enzymes (i.e. catalase and superoxide dismutase) due to its own high pseudoperoxidase activity, which rapidly removes any HOOH produced in the initial stages of autoxidation. This double substitution occurs naturally in the myoglobin of Asian elephants, and similar multiple replacements have been used to reduce selectively the rate of nitric oxide (NO)-induced oxidation of both recombinant MbO2 and HbO2 blood substitute prototypes without altering O2 affinity.     

Dynamic equilibrium unfolding pathway of human tumor necrosis factor-alpha induced by guanidine hydrochloride

The dynamic equilibrium unfolding pathway of human tumor necrosis factor-alpha (TNF-alpha) during denaturation at different guanidine hydrochloride (GdnHCl) concentrations (0-4.2 M) was investigated by steady-state fluorescence spectroscopy, potassium iodide (KI) fluorescence quenching, far-UV circular dichroism (CD), picosecond time-resolved fluorescence lifetime, and anisotropy decay measurements. We utilized the intrinsic fluorescence of Trp-28 and Trp-114 to characterize the conformational changes involved in the equilibrium unfolding pathway. The detailed unfolding pathway under equilibrium conditions was discussed with respect to motional dynamics and partially folded structures. At 0-0.9 M [GdnHCl], the rotational correlation times of 22-25 ns were obtained from fluorescence anisotropy decay measurements and assigned to those of trimeric states by hydrodynamic calculation. In this range, the solvent accessibility of Trp residues increased with increasing [GdnHCl], suggesting the slight expansion of the trimeric structure. At 1.2-2.1 M [GdnHCl], the enhanced solvent accessibility and the rotational degree of freedom of Trp residues were observed, implying the loosening of the internal structure. In this [GdnHCl] region, TNF-alpha was thought to be in soluble aggregates having distinct conformational characteristics from a native (N) or fully unfolded state (U). At 4.2 M [GdnHCl], TNF-alpha unfolded to a U-state. From these results, the equilibrium unfolding pathway of TNF-alpha, trimeric and all beta-sheet protein, could not be viewed from the simple two state model (N-->U).     

Phase IIa safety and immunogenicity of a therapeutic vaccine, TA-GW, in persons with genital warts

A fusion protein vaccine consisting of human papillomavirus 6 L2E7 with Alhydrogel was developed for the treatment of genital warts. Twenty-seven subjects with genital warts received 3 immunizations over 4 weeks in an open-label study. The vaccine was well-tolerated, and all subjects made serum IgG antibodies, predominantly IgG1, against L2E7. Nineteen of 25 tested persons made antigen-specific T cell proliferative responses to L2E7, and peripheral blood mononuclear cells when cultured with L2E7 in vitro produced both interferon-gamma and interleukin (IL)-5, although IL-5 predominated after the final vaccination. Five subjects completely cleared warts within 8 weeks. Subjects whose warts were not cleared by 8 weeks were offered conventional therapy. Recurrence of warts was not seen in any of the 13 persons whose warts cleared by vaccine alone or with conventional therapy. While these preliminary results of the use of this therapeutic immunogen are encouraging, proof of efficacy will require randomized double-blind trials.     

Novel Peptide‐Heterocycle Hybrids: Synthesis and Preliminary Studies on Calpain Inhibition

New peptidic compounds, having peptide chains linked to bi‐ and tricyclic heterocycles (peptide‐heterocycle hybrids), have been synthesized. The heterocyclic components are derivatives of partially reduced isoquinoline and pyrido[1,2‐b]isoquinoline bearing α,β‐unsaturated carbonyl functionalities. The heterocyclic compounds have been used as acylating agents in coupling reactions with short N‐unprotected peptides. Based on our interest on potential calpain inhibitors, we have used short (2–4 amino acids) peptides with hydrophobic amino acids of the two enantiomeric series. We report preliminary studies on the inhibition of calpain, with some compounds having IC₅₀ values in the nanomolar range.     

Single step low temperature synthesis of gadolinium gallium garnet nanopowders

Solution combustion synthesis of single-phase gadolinium gallium oxide(Gd3Ga5O12,GGG)nanopowders,by a fuel mixture approach using urea and glycine at a low temperature of 500oC,was being reported for the first time.Based on the fact that urea and glycine are good fuels for gallium oxide and gadolinium oxide synthesis,the fuel mixture composition was obtained,which could lead to direct phase pure cubic Gd3Ga5O12 formation without any subsequent calcination step.Combustion was carried out in furnace pre-heated at 500oC.Thermogravimetric analysis(TGA)of combustion product showed negligible mass loss indicating direct formation of GGG powder.Fourier transform infrared(FTIR)spectrum of combusted product showed peak characteristic of GGG in case of mixed fuel.X-ray diffraction(XRD)confirmed formation of phase pure GGG at 500°C in preheated furnace.Very fine,well dispersed nanometric particles of size range of 50-100 nm were obtained,being uniform and close to spherical morphology as observed by transmission electron microscope(TEM).     

Does gender moderate the relationship between driver aggression and its risk factors?

Aim

The current study assessed gender as a potential moderator of the relationship between self-reported driver aggression and various demographic variables, general and driving-related risk factors.

Methods

Using data from a general-population telephone survey conducted from July 2002 through June 2005, two approaches to binary logistic regression were adopted. Based on the full dataset (n = 6259), the initial analysis was a hierarchical-entry regression examining self-reported driver aggression in the last 12 months. All demographic variables (i.e., gender, age, income, education, marital status), general risk factors (i.e., psychological distress, binge drinking, cannabis use), and driving-related risk factors (i.e., driving exposure, stressful driving, exposure to busy roads, driving after drinking, driving after cannabis use) were entered in the first block, and all two-way interactions with gender were entered stepwise in the second block. The subsequent analysis involved dividing the sample by gender and conducting logistic regressions with main effects only for males (n = 2921) and females (n = 3338) separately.

Results

Although the prevalence of driver aggression in the current sample was slightly higher among males (38.5%) than females (32.9%), the difference was small, and gender did not enter as a significant predictor of driver aggression in the overall logistic regression. In that analysis, difficulty with social functioning and being older were associated with a reduced risk of driver aggression. Marital status and education were unrelated to aggression, and all other variables were associated with an increased risk of aggression. Gender was found to moderate the relationships between driver aggression and only three variables: income, psychological distress, and driving exposure. Separate analyses on the male and female sub-samples also found differences in the predictive value of income and driving exposure; however, the difference for psychological distress could not be detected using this separate regression approach. The secondary analysis also identified slight differences in the predictive value of four of the risk factors, where the odds ratios for both males and females were in the same direction but only one of the two was statistically significant.

Conclusions

The results demonstrate the importance of conducting the gender analysis using both regression approaches. With few exceptions, factors that were predictive of driver aggression were generally the same for both male and female drivers.     

Verification of some building materials as gamma-ray shields

The shielding properties for gamma rays of a few low Z materials were investigated. The values of the mass attenuation coefficient, equivalent atomic number, effective atomic number, exposure buildup factor and energy absorption buildup factor were calculated and used to estimate the shielding effectiveness of the samples under investigation. It has been observed that the shielding effectiveness of a sample is directly related to its effective atomic number. The shielding character of any sample is a function of the incident photon energy. Good shielding behaviour has been verified in soil samples in the photon energy region of 0.015-0.30 MeV and of dolomite in 3-15 MeV. The results have been shown graphically with more useful conclusions.