Antioxidant protein 2 prevents methemoglobin formation in erythrocyte hemolysates.

Antioxidant protein 2 (AOP2) is a member of a family of thiol-specific antioxidants, recently renamed peroxiredoxins, that evolved as part of an elaborate system to counteract and control detrimental effects of oxygen radicals. AOP2 is found in endothelial cells, erythrocytes, monocytes, T and B cells, but not in granulocytes. AOP2 was found solely in the cytoplasm and was not associated with the nuclear or membrane fractions; neither was it detectable in plasma. Further experiments focused on the function of AOP2 in erythrocytes where it is closely associated with the hemoglobin complex, particularly with the heme. An investigation of the mechanism of this interaction demonstrated that the conserved cysteine-47 in AOP2 seems to play a role in AOP2-heme interactions. Recombinant AOP2 prevented induced as well as noninduced methemoglobin formation in erythrocyte hemolysates, indicating its antioxidant properties. We conclude that AOP2 is part of a sophisticated system developed to protect and support erythrocytes in their many physiological functions.     

Assessment of DNA damage and repair in adults consuming allyl isothiocyanate or Brassica vegetables

Allyl isothiocyanate (AITC) is a dietary component with possible anticancer effects, though much information about AITC and cancer has been obtained from cell studies. To investigate the effect of AITC on DNA integrity in vivo, a crossover study was conducted. Adults (n= 46) consumed AITC, AITC-rich vegetables [mustard and cabbage (M/C)] or a control treatment with a controlled diet for 10 days each. On day 11, volunteers provided blood and urine before and after consuming treatments. Volunteers were characterized for genotype for GSTM1 and GSTT1 (glutathione S-transferases) and XPD (DNA repair). DNA integrity in peripheral blood mononuclear cells was assessed by single-cell gel electrophoresis. Urine was analyzed for 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG) and creatinine. Ten-day intake of neither AITC nor M/C resulted in statistically significant differences in DNA strand breaks [least squares mean (LSmean) % DNA in tail±S.E.M.: 4.8±0.6 for control, 5.7±0.7 for AITC, 5.3±0.6 for M/C] or urinary 8-oxodG (LSmean μg 8-oxodG/g creatinine±S.E.M.: 2.95±0.09 for control, 2.88±0.09 for AITC, 3.06±0.09 for M/C). Both AITC and M/C increased DNA strand breaks 3 h postconsumption (LSmean % DNA in tail±S.E.M.: 3.2±0.7 for control, 8.3±1.7 for AITC, 8.0±1.7 for M/C), and this difference disappeared at 6 h (4.2±0.9 for control, 5.7±1.2 for AITC, 5.5±1.2 for M/C). Genotypes for GSTM1, GSTT1 and XPD were not associated with treatment effects. In summary, DNA damage appeared to be induced in the short term by AITC and AITC-rich products, but that damage disappeared quickly, and neither AITC nor AITC-rich products affected DNA base excision repair.     

Antigenic and Immunogenic Characteristics of Nuclear and Membrane-Associated Simian Virus 40 Tumor Antigen

Antisera were prepared in syngeneic hosts against subcellular fractions of simian virus 40 (SV40)-transformed cells (MoalphaPM, MoalphaNuc), glutaraldehydefixed SV40-transformed cells (HaalphaH-50-G, MoalphaVLM-G), and electrophoretically purified denatured SV40 tumor antigen (T-ag) (RaalphaT). Immune sera were also collected from animals bearing tumors induced by SV40-transformed cells (HaalphaT, MoalphaT, HAF) and from SV40-immunized animals that had rejected a transplant of SV40-transformed cells (HaalphaS, MoalphaS). Immunological reagents prepared against cell surface (MoalphaPM, HaalphaS, MoalphaS, HaalphaH-50-G, MoalphaVLM-G) reacted exclusively with the surface of SV40-transformed cells by indirect immunofluorescence or protein A surface antigen radioimmunoassay. Immunological reagents prepared against the nuclear fraction (MoalphaNuc) or whole-cell determinants (HaalphaT, MoalphaT, HAF, RaalphaT) reacted with both the nuclei and surface of SV40-transformed or -infected cells. All reagents were capable of immunoprecipitating 96,000-molecular weight large T-ag from solubilized whole cell extracts of SV40-transformed cells. The exclusive surface reactivity of HaalphaS exhibited in immunofluorescence tests was abolished by solubilization of subcellular fractions, which then allowed immunoprecipitation of T-ag by HaalphaS from both nuclear and plasma membrane preparations. Specificity was established by the fact that all T-reactive reagents failed to react in serological tests against chemically transformed mouse cells, and sera from mice bearing transplants chemically transformed mouse cells (MoalphaDMBA-2) failed to react with SV40-transformed mouse or hamster cells. Reagents demonstrating positive surface immunofluorescence and protein A radioimmunoassay reactions against SV40-transformed cells were capable of blocking the surface binding of RaalphaT to SV40-transformed cells in a double-antibody surface antigen radioimmunoassay. This blocking ability demonstrated directly that a component specificity of each surface-reactive reagent is directed against SV40 T-ag. A model is presented which postulates that the differential detection of T-ag by the various serological reagents is a reflection of immunogenic and antigenic differences between T-ag polypeptides localized in nuclei and plasma membranes.     

A billion-fold range in acidity for the solvent-exposed amides of Pyrococcus furiosus rubredoxin

The exchange rates of the static solvent-accessible amide hydrogens of Pyrococcus furiosus rubredoxin range from near the diffusion-limited rate to a billion-fold slower for the non-hydrogen-bonded Val 38 (eubacterial numbering). Hydrogen exchange directly monitors the kinetic acidity of the peptide nitrogen. Electrostatic solvation free energies were calculated by Poisson-Boltzmann methods for the individual peptide anions that form during the hydroxide-catalyzed exchange reaction to examine how well the predicted thermodynamic acidities match the experimentally determined kinetic acidities. With the exception of the Ile 12 amide, the differential exchange rate constant for each solvent-exposed amide proton that is not hydrogen bonded to a backbone carbonyl can be predicted within a factor of 6 (10 (0.78)) root-mean-square deviation (rmsd) using the CHARMM22 electrostatic parameter set and an internal dielectric value of 3. Under equivalent conditions, the PARSE parameter set yields a larger rmsd value of 1.28 pH units, while the AMBER parm99 parameter set resulted in a considerably poorer correlation. Either increasing the internal dielectric value to 4 or reducing it to a value of 2 significantly degrades the quality of the prediction. Assigning the excess charge of the peptide anion equally between the peptide nitrogen and the carbonyl oxygen also reduces the correlation to the experimental data. These continuum electrostatic calculations were further analyzed to characterize the specific structural elements that appear to be responsible for the wide range of peptide acidities observed for these solvent-exposed amides. The striking heterogeneity in the potential at sites along the protein-solvent interface should prove germane to the ongoing challenge of quantifying the contribution that electrostatic interactions make to the catalytic acceleration achieved by enzymes.     

Cyclin-dependent kinase 4 signaling acts as a molecular switch between syngenic differentiation and neural transdifferentiation in human mesenchymal stem cells

Multipotent mesenchymal stem/stromal cells (MSCs) are capable of differentiating into a variety of cell types from different germ layers. However, the molecular and biochemical mechanisms underlying the transdifferentiation of MSCs into specific cell types still need to be elucidated. In this study, we unexpectedly found that treatment of human adipose- and bone marrow-derived MSCs with cyclin-dependent kinase (CDK) inhibitor, in particular CDK4 inhibitor, selectively led to transdifferentiation into neural cells with a high frequency. Specifically, targeted inhibition of CDK4 expression using recombinant adenovial shRNA induced the neural transdifferentiation of human MSCs. However, the inhibition of CDK4 activity attenuated the syngenic differentiation of human adipose-derived MSCs. Importantly, the forced regulation of CDK4 activity showed reciprocal reversibility between neural differentiation and dedifferentiation of human MSCs. Together, these results provide novel molecular evidence underlying the neural transdifferentiation of human MSCs; in addition, CDK4 signaling appears to act as a molecular switch from syngenic differentiation to neural transdifferentiation of human MSCs.     

Induction of apoptosis and activation of NF-kappaB by CD95 require different signalling thresholds

Mutations in the death domain of the death receptor CD95 (APO-1/Fas) cause lymphoproliferation and autoimmune disease in both lpr(cg) mice and in patients with autoimmune lymphoproliferative syndrome (ALPS) type Ia. By testing lymphocytes from ALPS type Ia patients, comparing heterozygous with homozygous lpr(cg) mice and coexpressing wild-type and mutant CD95 receptors, we demonstrate that induction of apoptosis requires two wild-type alleles of CD95. By contrast, nuclear factor-kappaB (NF-kappaB) can be fully activated in cells expressing both a mutant and a wild-type CD95 allele, suggesting different thresholds to activate the two signalling pathways. This was confirmed by testing lymphocytes from heterozygous lpr mice, which showed reduced sensitivity to CD95-mediated apoptosis but normal activation of NF-kappaB when compared with wild-type mice. Mutations in CD95 may eliminate the tumour-suppressive function of CD95, at the same time allowing induction of survival or proliferative pathways, which could contribute to the increased risk for lymphoma seen in ALPS type Ia patients.     

Patient Retention,Clinical Outcomes and Attrition-Associated Factors of HIV-Infected Patients Enrolled in Zimbabwe's National Antiretroviral Therapy Programme, 2007–2010

Background

Since establishment of Zimbabwe''s National Antiretroviral Therapy (ART) Programme in 2004, ART provision has expanded from <5,000 to 369,431 adults by 2011. However, patient outcomes are unexplored.

Objective

To determine improvement in health status, retention and factors associated with attrition among HIV-infected patients on ART.

Methods

A retrospective review of abstracted patient records of adults ≥15 years who initiated ART from 2007 to 2009 was done. Frequencies and medians were calculated for rates of retention in care and changes in key health status outcomes at 6, 12, 24 and 36 months respectively. Cox proportional hazards models were used to determine factors associated with attrition.

Results

Of the 3,919 patients, 64% were female, 86% were either WHO clinical stage III or IV. Rates of patient retention at 6, 12, 24 and 36 months were 90.7%, 78.1%, 68.8% and 64.4%, respectively. After ART initiation, median weight gains at 6, 12, and 24 months were 3, 4.5, and 5.0 kgs whilst median CD4+ cell count gains at 6, 12 and 24 months were 122, 157 and 279 cells/µL respectively. Factors associated with an increased risk of attrition included male gender (AHR 1.2; 95% CI, 1.1–1.4), baseline WHO stage IV (AHR 1.7; 95% CI, 1.1–2.6), lower baseline body weight (AHR 2.0; 95% CI, 1.4–2. 8) and accessing care from higher level healthcare facilities (AHR 3.5; 95% 1.1–11.2).

Conclusions

Our findings with regard to retention as well as clinical and immunological improvements following uptake of ART, are similar to what has been found in other settings. Factors influencing attrition also mirror those found in other parts of sub-Saharan Africa. These findings suggest the need to strengthen earlier diagnosis and treatment to further improve treatment outcomes. Whilst decentralisation improves ART coverage it should be coupled with strategies aimed at improving patient retention.