Influence of Antioxidants on the Peroxidative Swelling of Mitochondria in vitro

To clarify the role of prooxidative processes during in vitro swelling of freshly isolated rat liver mitochondria, the influence of different antioxidants and free-radical scavengers was tested. Ascorbate below 10 mmol/L without externally added Fe²⁺ acted as a prooxidant and enhanced swelling. Higher concentrations in the presence of Fe²⁺ showed antioxidant properties and a decrease in swelling and lipid peroxidation. Swelling was abolished by -tocopherol and reduced to 50% by butylated hydroxytoluene. Glutathione supplementation decreased both swelling and lipid peroxidation. Oxidized glutathione caused swelling without any effect on peroxidation. Hydrogen peroxide, cumene hydroperoxide and t-butyl hydroperoxide caused progressive decreases in glutathione and reduced niacinamide coenzyme levels, suggesting prooxidative changes. Dithiothreitol was found to abolish this effect. Thus, antioxidants reverse superoxide-induced mito chondrial swelling and lipid peroxidation in vitro.     

Molecular markers for resistance to Heterodera glycines in advanced soybean germplasm

Germplasm line J87-233 is resistant to soybean cyst nematode (SCN) races 1, 2, 3, 5 and moderately resistant to race 14 with resistance derived from 3 primitive sources, Peking, PI 88788 and PI 90763. F_2:3 progeny of J87-233 and SCN-susceptible Hutcheson cross were evaluated for response to SCN races 1, 2, 3, 5 and 14. Linkage groups (LG) A, B, F, G, J, M, N, S were tested with 215 genomic clones and 45 decamers for parental genotypes. QTL for race 1 and QTL for race 3 were detected on LG A2, the region of BLT65V and SCAR 548/563_{1100/1025,975.} The cluster analysis of 12 soybean cultivars and 38 plant introductions confirmed association of SCAR_{1100/1025,975} with resistance to races 1 and 3, and suggested possible DNA rearrangements that might give rise to new resistance specificities in the region. The highly significant association of K69T marker with SCN race 1 resistance in conjunction with its location, 18.5 cM from the reported QTL, exemplifies the importance of the QTL locus on LG G and suggests expansion of the linkage map in the LG G-terminal region. Detected interaction between loci on LG A2 and LG G, and also with loci on LG F and LG M, may play a significant role in the genotype-specific response to SCN. Identification of two major regions on LG A2 and LG G for SCN resistance shows their applicability to advanced germplasm, however, transmission of molecular marker alleles indicates that applied markers are not yet reliable in revealing all possible recombination events in breeding for SCN resistance.     

Structural and kinetic analysis of drug resistant mutants of HIV-1 protease.

Mutants of HIV-1 protease that are commonly selected on exposure to different drugs, V82S, G48V, N88D and L90M, showed reduced catalytic activity compared to the wild-type protease on cleavage site peptides, CA-p2, p6pol-PR and PR-RT, critical for viral maturation. Mutant V82S is the least active (2-20% of wild-type protease), mutants N88D, R8Q, and L90M exhibit activities ranging from 20 to 40% and G48V from 50 to 80% of the wild-type activity. In contrast, D30N is variable in its activity on different substrates (10-110% of wild-type), with the PR-RT site being the most affected. Mutants K45I and M46L, usually selected in combination with other mutations, showed activities that are similar to (60-110%) or greater than (110-530%) wild-type, respectively. No direct relationship was observed between catalytic activity, inhibition, and structural stability. The mutants D30N and V82S were similar to wild-type protease in their stability toward urea denaturation, while R8Q, G48V, and L90M showed 1.5 to 2.7-fold decreased stability, and N88D and K45I showed 1.6 to 1.7-fold increased stability. The crystal structures of R8Q, K45I and L90M mutants complexed with a CA-p2 analog inhibitor were determined at 2.0, 1.55 and 1.88 A resolution, respectively, and compared to the wild-type structure. The intersubunit hydrophobic contacts observed in the crystal structures are in good agreement with the relative structural stability of the mutant proteases. All these results suggest that viral resistance does not arise by a single mechanism.     

Ceramide synthase 4 and de novo production of ceramides with specific N-acyl chain lengths are involved in glucolipotoxicity-induced apoptosis of INS-1 β-cells

Pancreatic β-cell apoptosis induced by palmitate requires high glucose concentrations. Ceramides have been suggested to be important mediators of glucolipotoxicity-induced β-cell apoptosis. In INS-1 β-cells, 0.4 mM palmitate with 5 mM glucose increased the levels of dihydrosphingosine and dihydroceramides, two lipid intermediates in the de novo biosynthesis of ceramides, without inducing apoptosis. Increasing glucose concentrations to 30 mM amplified palmitate-induced accumulation of dihydrosphingosine and the formation of (dihydro)ceramides. Of note, glucolipotoxicity specifically induced the formation of C(18:0), C(22:0) and C(24:1) (dihydro)ceramide molecular species, which was associated with the up-regulation of CerS4 (ceramide synthase 4) levels. Fumonisin-B1, a ceramide synthase inhibitor, partially blocked apoptosis induced by glucolipotoxicity. In contrast, apoptosis was potentiated in the presence of D,L-threo-1-phenyl-2-palmitoylamino-3-morpholinopropan-1-ol, an inhibitor of glucosylceramide synthase. Moreover, overexpression of CerS4 amplified ceramide production and apoptosis induced by palmitate with 30 mM glucose, whereas down-regulation of CerS4 by siRNA (short interfering RNA) reduced apoptosis. CerS4 also potentiates ceramide accumulation and apoptosis induced by another saturated fatty acid: stearate. Collectively, our results suggest that glucolipotoxicity induces β-cell apoptosis through a dual mechanism involving de novo ceramide biosynthesis and the formation of ceramides with specific N-acyl chain lengths rather than an overall increase in ceramide content.     

Perspective on the host response to human metapneumovirus infection: what can we learn from respiratory syncytial virus infections?

Human metapneumovirus (HMPV) is a recently discovered pathogen first identified in respiratory specimens from young children suffering from clinical respiratory syndromes ranging from mild to severe lower respiratory tract illness. HMPV has worldwide prevalence, and is a leading cause of respiratory tract infection in the first years of life, with a spectrum of disease similar to respiratory syncytial virus (RSV). The disease burden associated with HMPV infection has not been fully elucidated; however, studies indicate that HMPV may cause upper or lower respiratory tract illness in patients between ages 2 months and 87 years, may co-circulate with RSV, and HMPV infection may be associated with asthma exacerbation. The mechanisms and effector pathways contributing to immunity or disease pathogenesis following infection are not fully understood; however, given the clinical significance of HMPV, there is a need for a fundamental understanding of the immune and pathophysiological processes that occur following infection to provide the foundation necessary for the development of effective vaccine or therapeutic intervention strategies. This review provides a current perspective on the processes associated with HMPV infection, immunity, and disease pathogenesis.     

Structural predictions of the binding site architecture for monoclonal antibody NC6.8 using computer-aided molecular modeling, ligand binding, and spectroscopy.

Monoclonal antibody NC6.8 binds the superpotent sweetener ligand N-(p-cyanophenyl)-N'-(diphenylmethyl) guanidineacetic acid with high affinity (Kd = 53 nM). Using computer-aided molecular modeling and several experimental techniques, such as competitive ligand binding, absorbance spectroscopy, and fluorescence spectroscopy, we have predicted the structure of the variable domain fragment (Fv) and identified the key residues in the combining site of the antibody. We have identified nine specific amino acids as being involved in ligand recognition and complexation. Most notable are H:33W, which is responsible for ligand-induced tryptophan fluorescence quenching, H:56R, which forms a salt bridge with the carboxylate moiety of the ligand, and L:34H, which, deep in the binding site, interacts with the cyanophenyl portion of the ligand. Two residues located deep in the putative binding pocket, H:35E and H:50E, provide the negatively charged potential for interaction with the protonated aryl nitrogen and the positive guanidinium group. These modeling predictions were made before the solution of high-resolution structures of the native Fab (2.6 A) and the Fab-ligand complex (2.2 A). Comparisons between the theoretical model and experimental native and liganded Fab structures are made.     

Modulatory effect of sesamol on DOCA-salt-induced oxidative stress in uninephrectomized hypertensive rats

The present study was undertaken to investigate the antihypertensive and antioxidant effects of sesamol on uninephrectomized deoxycorticosterone acetate (DOCA)-salt-induced hypertensive rats. Hypertension was induced in surgically single-kidney-removed (left) adult male albino Wistar rats, weighing 180–200 g, by injecting DOCA (25 mg/kg BW) subcutaneously twice a week for 6 weeks, with saline instead of tap water for drinking. Rats were treated with three different doses of sesamol (50, 100 and 200 mg/kg BW) post-orally by gavage daily for 6 weeks. Hypertension was revealed by increased systolic and diastolic blood pressure and the toxicity of DOCA-salt was determined using hepatic marker enzymes, aspartate aminotransferase, alanine aminotransferase, alkaline phospatase and gamma-glutamyl transpeptidase; and, lipid peroxidative markers, thiobarbituric acid reactive substances, lipid hydroperoxides and conjugated dienes were assayed. The activities of enzymatic antioxidants, superoxide dismutase, catalase and glutathione peroxidase and the levels of non-enzymatic antioxidants (vitamin C, vitamin E and reduced glutathione) were evaluated in erythrocytes, plasma and tissues. Post-oral administration of sesamol at the dosage of 50 mg/kg BW remarkably decreased systolic and diastolic blood pressure, hepatic marker enzyme activities and lipid peroxidation products and also enhanced the antioxidant activity. The biochemical observations were also supported by histopathological examinations of the rat liver, kidney and heart sections. These results suggest that sesamol possesses antihypertensive and antioxidant effects.     

Protective effect of n-3 polyunsaturated fatty acids concentrate on isoproterenol-induced myocardial infarction in rats

The protective effect of PUFA concentrate prepared from fish oil on isoproterenol-induced myocardial infarction in male albino rats was investigated with respect to changes in the levels of diagnostic marker enzymes, cholesterol, triglycerides, free fatty acids, phospholipids, reduced glutathione (GSH) and lipid peroxides (LPO). Administration of PUFA concentrate significantly prevented the isoproterenol-induced elevation in the levels of plasma diagnostic marker enzymes (ALT [93.5%], AST [95.6%], LDH [94.7%] and CPK [96.1%]). PUFA concentrate feeding exerted a significant antilipidemic effect against isoproterenol-induced myocardial infarction by reducing the levels of lipid components in plasma (cholesterol [71.5%], triglycerides [79.7%] and free fatty acids [70.7%] and heart tissue (cholesterol [81.4%], triglycerides [76.3%] and free fatty acids [78.6%]). A tendency to prevent the isoproterenol-induced phospholipids depletion (74.4%) in the myocardium of experimental rats was also observed. The level of lipid peroxidation was also found to be significantly lower in PUFA treated animals (2.72+/-0.15nmol/ml in plasma; 1.18+/-0.08nmol/mg protein in heart tissue) as compared to that of isoproterenol-injected groups (5.77+/-0.43nmol/ml in plasma; 2.14+/-0.15nmol/mg protein in heart tissue) of rats. Also the level of reduced GSH significantly higher in the heart tissue of PUFA administered experimental rats (5.65+/-0.98 microg/g) as compared to myocardial infarction induced control rats (2.39+/-0.18 microg/g). The results of the present study indicate that the overall cardioprotective effect of PUFA concentrate is probably related to its ability to inhibit lipid accumulation by its hypolipidaemic property.     

Comparison of Effects of p53 Null and Gain-of-Function Mutations on Salivary Tumors in MMTV-Hras Transgenic Mice

p53 is an important tumor suppressor gene which is mutated in ~50% of all human cancers. Some of these mutants appear to have acquired novel functions beyond merely losing wild-type functions. To investigate these gain-of-function effects in vivo, we generated mice of three different genotypes: MMTV-Hras/p53^+/+, MMTV-Hras/p53^-/-, and MMTV-Hras/p53^R172H/R172H. Salivary tumors from these mice were characterized with regard to age of tumor onset, tumor growth rates, cell cycle distribution, apoptotic levels, tumor histopathology, as well as response to doxorubicin treatment. Microarray analysis was also performed to profile gene expression. The MMTV-Hras/p53^-/- and MMTV-Hras/p53^R172H/R172H mice displayed similar properties with regard to age of tumor onset, tumor growth rates, tumor histopathology, and response to doxorubicin, while both groups were clearly distinct from the MMTV-Hras/p53^+/+ mice by these measurements. In addition, the gene expression profiles of the MMTV-Hras/p53^-/- and MMTV-Hras/p53^R172H/R172H tumors were tightly clustered, and clearly distinct from the profiles of the MMTV-Hras/p53^+/+ tumors. Only a small group of genes showing differential expression between the MMTV-Hras/p53^-/- and MMTV-Hras/p53^R172H/R172H tumors, that did not appear to be regulated by wild-type p53, were identified. Taken together, these results indicate that in this MMTV-Hras-driven salivary tumor model, the major effect of the p53 R172H mutant is due to the loss of wild-type p53 function, with little or no gain-of-function effect on tumorigenesis, which may be explained by the tissue- and tumor type-specific properties of this gain-of-function mutant of p53.