Apolar distal pocket mutants of yeast cytochrome c peroxidase: Hydrogen peroxide reactivity and cyanide binding of the TriAla,TriVal, and TriLeu variants

Three yeast cytochrome c peroxidase (CcP) variants with apolar distal heme pockets have been constructed. The CcP variants have Arg48, Trp51, and His52 mutated to either all alanines, CcP(triAla), all valines, CcP(triVal), or all leucines, CcP(triLeu). The triple mutants have detectable enzymatic activity at pH 6 but the activity is less than 0.02% that of wild-type CcP. The activity loss is primarily due to the decreased rate of reaction between the triple mutants and H₂O₂ compared to wild-type CcP. Spectroscopic properties and cyanide binding characteristics of the triple mutants have been investigated over the pH stability region of CcP, pH 4 to 8. The absorption spectra indicate that the CcP triple mutants have hemes that are predominantly five-coordinate, high-spin at pH 5 and six-coordinate, low-spin at pH 8. Cyanide binding to the triple mutants is biphasic indicating that the triple mutants have two slowly-exchanging conformational states with different cyanide affinities. The binding affinity for cyanide is reduced at least two orders of magnitude in the triple mutants compared to wild-type CcP and the rate of cyanide binding is reduced by four to five orders of magnitude. Correlation of the reaction rates of CcP and 12 distal pocket mutants with H₂O₂ and HCN suggests that both reactions require ionization of the reactants within the distal heme pocket allowing the anion to bind the heme iron. Distal pocket features that promote substrate ionization (basic residues involved in base-catalyzed substrate ionization or polar residues that can stabilize substrate anions) increase the overall rate of reaction with H₂O₂ and HCN while features that inhibit substrate ionization slow the reactions.     

Excess Cardiovascular Risk Burden in Jamaican Women Does Not Influence Predicted 10-Year CVD Risk Profiles of Jamaica Adults: An Analysis of the 2007/08 Jamaica Health and Lifestyle Survey

Background

Black Caribbean women have a higher burden of cardiovascular disease (CVD) risk factors than their male counterparts. Whether this results in a difference in incident cardiovascular events is unknown. The aim of this study was to estimate the 10 year World Health Organization/International Society for Hypertension (WHO/ISH) CVD risk score for Jamaica and explore the effect of sex as well as obesity, physical activity and socioeconomic status on these estimates.

Methods and Findings

Data from 40–74 year old participants in the 2007/08 Jamaica Health and Lifestyle Survey were used. Trained interviewers administered questionnaires and measured anthropometrics, blood pressure, fasting glucose and cholesterol. Education and occupation were used to assess socioeconomic status. The Americas B tables were used to estimate the WHO/ISH 10 year CVD risk scores for the population. Weighted prevalence estimates were calculated. Data from 1,432 (450 men, 982 women) participants were analysed, after excluding those with self-reported heart attack and stroke. The women had a higher prevalence of diabetes (19%W;12%M), hypertension (49%W;47%M), hypercholesterolemia (25%W;11%M), obesity (46%W;15%M) and physical inactivity (59%W;29%M). More men smoked (6%W;31%M). There was good agreement between the 10-year cardiovascular risk estimates whether or not cholesterol measurements were utilized for calculation (kappa –0.61). While 90% had a 10 year WHO/ISH CVD risk of less than 10%, approximately 2% of the population or 14,000 persons had a 10 year WHO/ISH CVD risk of ≥30%. As expected CVD risk increased with age but there was no sex difference in CVD risk distribution despite women having a greater risk factor burden. Women with low socioeconomic status had the most adverse CVD risk profile.

Conclusion

Despite women having a higher prevalence of CVD risk factors there was no sex difference in 10-year WHO/ISH CVD risk in Jamaican adults.     

In vitro evolution of RNA aptamers recognizing carcinogenic aromatic amines

The modification of cellular DNA by environmental substances is thought to be a crucial event in chemical induced carcinogenesis. Among the environmental carcinogens, aromatic amines are known for the fact that they can induce several types of cancers through the formation of so-called DNA adducts. We took advantage of the potential of the SELEX method to select for highly specific RNA ligands that recognize specific genotoxic aromatic amines. The aromatic amine 4,4'-methylenedianiline (MDA) was used as a target. Following in vitro selection, we obtained specific MDA-binding RNA molecules based on an affinity chromatography assay. These results open the possibility of using the SELEX technique to generate RNA molecules as diagnostic tools for the detection of DNA damaging compounds and ultimately DNA adducts.     

Workshop--Predicting the structure of biological molecules

This April, in Cambridge (UK), principal investigators from the Mathematical Biology Group of the Medical Research Council's National Institute of Medical Research organized a workshop in structural bioinformatics at the Centre for Mathematical Sciences. Bioinformatics researchers of several nationalities from labs around the country presented and discussed their computational work in biomolecular structure prediction and analysis, and in protein evolution. The meeting was intensive and lively and gave attendees an overview of the healthy state of protein bioinformatics in the UK.     

Age-Related Reference Intervals of the Main Biochemical and Hematological Parameters in C57BL/6J, 129SV/EV and C3H/HeJ Mouse Strains

Background

Although the mouse is the animal model most widely used to study the pathogenesis and treatment of human diseases, reference values for biochemical parameters are scanty or lacking for the most frequently used strains. We therefore evaluated these parameters in the C57BL/6J, 129SV/EV and C3H/HeJ mice.

Methodology/Principal Findings

We measured by dry chemistry 26 analytes relative to electrolyte balance, lipoprotein metabolism, and muscle/heart, liver, kidney and pancreas functions, and by automated blood counter 5 hematological parameters in 30 animals (15 male and 15 female) of each mouse strain at three age ranges: 1–2 months, 3–8 months and 9–12 months. Whole blood was collected from the retro-orbital sinus. We used quality control procedures to investigate analytical imprecision and inaccuracy. Reference values were calculated by non parametric methods (median and 2.5^th and 97.5^th percentiles). The Mann-Whitney and Kruskal-Wallis tests were used for between-group comparisons. Median levels of GLU, LDH, Chol and BUN were higher, and LPS, AST, ALP and CHE were lower in males than in females (p range: 0.05–0.001). Inter-strain differences were observed for: (1) GLU, t-Bil, K⁺, Ca⁺⁺, PO₄ ⁻ (p<0.05) and for TAG, Chol, AST, Fe⁺⁺ (p<0.001) in 4–8 month-old animals; (2) for CK, Crea, Mg⁺⁺, Na⁺⁺, K⁺, Cl⁻ (p<0.05) and BUN (p<0.001) in 2- and in 10–12 month-old mice; and (3) for WBC, RBC, HGB, HCT and PLT (p<0.05) during the 1 year life span.

Conclusion/Significance

Our results indicate that metabolic variations in C57BL/6J, 129SV/EV and C3H/HeJ mice after therapeutic intervention should be evaluated against gender- and age-dependent reference intervals.     

Both linear and discontinuous ribosome scanning are used for translation initiation from bicistronic human immunodeficiency virus type 1 env mRNAs

Human immunodeficiency virus type 1 (HIV-1) generates 16 alternatively spliced isoforms of env mRNA that contain the same overlapping open reading frames for Vpu and Env proteins but differ in their 5' untranslated regions (UTR). A subset of env mRNAs carry the extra upstream Rev initiation codon in the 5' UTR. We explored the effect of the alternative UTR on the translation of Vpu and Env proteins from authentic env mRNAs expressed from cDNA constructs. Vpu expression from the subset of env mRNA isoforms with exons containing an upstream Rev AUG codon was minimal. However, every env mRNA isoform expressed similar levels of Env protein. Mutations that removed, altered the strength of, or introduced upstream AUG codons dramatically altered Vpu expression but had little impact on the consistent expression of Env. These data show that the different isoforms of env mRNA are not redundant but instead regulate Vpu production in HIV-1-infected cells. Furthermore, while the initiation of Vpu translation conforms to the leaky ribosome-scanning model, the consistent Env synthesis infers a novel, discontinuous ribosome-scanning mechanism to translate Env.     

Synthesis, liposomal preparation, and in vitro toxicity of two novel dodecaborate cluster lipids for boron neutron capture therapy

A new class of lipids, containing the closo-dodecaborate cluster, has been synthesized. Two lipids, S-(N, N-(2-dimyristoyloxyethyl)acetamido)thioundecahydro-closo-dodecaborate (2-) (B-6-14) and S-(N, N-(2-dipalmitoyloxyethyl)acetamido)thioundecahydro-closo-dodecaborate (2-) (B-6-16) are described. Both of them have a double-tailed lipophilic part and a headgroup carrying two negative charges. Differential scanning calorimetry shows that B-6-14 and B-6-16 bilayers have main phase transition temperatures of 18.8 and 37.9 degrees C, respectively. Above the transition temperature of 18.8 degrees C, B-6-14 can form liposomal vesicles, representing the first boron-containing lipid with this capability. Upon cooling below the transition temperature, stiff bilayers are formed. When incorporated into liposomal formulations with equimolar amounts of distearoyl phosphatidylcholine (DSPC) and cholesterol, stable liposomes are obtained. The zeta-potential measurements indicate that both B-6-14- and B-6-16-containing vesicles are negatively charged, with the most negative potential described of any liposome so far. The liposomes are of high potential value as transporters of boron to tumor cells in treatments based on boron neutron capture therapy (BNCT). Liposomes prepared from B-6-14 were slightly less toxic in V79 Chinese hamster cells (IC50 5.6 mM) than unformulated Na2B12H11SH (IC50 3.9 mM), while liposomes prepared from B-6-16 were not toxic even at 30 mM.     

Sex steroids,ANGELS and osteoporosis

Osteoporosis is characterized by reduced bone density and strength. Bone mass peaks between age 30 and 40 and then declines. This can be accelerated by factors including menopause and insufficient dietary calcium. Hormone replacement therapy (HRT) is currently the standard treatment for osteoporosis. However, growing concern over potential side effects of HRT has driven a search for alternative therapies. A recent report 1 reveals a potential alternative to HRT: a gender-neutral synthetic steroid that increases bone mass and strength without affecting reproductive organs. This compound acts via a novel extranuclear sex steroid receptor signaling mechanism that has important implications for nuclear receptor biology and human health.