Antioxidant and antimutagenic activity of mannan neoglycoconjugates: mannan-human serum albumin and mannan-penicillin G acylase

The antioxidant and antimutagenic activity of the yeast cell-wall mannan and mannan conjugates--in particular the mannan of Saccharomyces cerevisiae (M-S.c.) and conjugates of mannan S. cerevisiae with human serum albumin (M-HSA1, M-HSA2) and the microbial enzyme penicillin G acylase (M-PGA)--were evaluated in vitro in the unicellular flagellate Euglena gracilis exposed to the genotoxic agents ofloxacin and acridine orange (AO). M-S.c., M-HSA1, M-HSA2 and M-PGA show a statistically significant, concentration-dependent protective antigenotoxic activity against both compounds. M-PGA was the most efficient inhibitor of ofloxacin- and AO-induced chloroplast DNA damage, whereas M-HSA2 and M-HSA1 were less effective and M-S.c. had the lowest antigenotoxic activity. It is suggested that different mechanisms may be involved in their protective effect--antioxidant activity in the case of ofloxacin-induced DNA damage and direct adsorption of AO on mannan conjugates as possible mechanisms of protection, based on spectrophotometric measurements. The important characteristics of yeast cell-wall mannans and mannan conjugates, such as their high water solubility, their broad spectrum of biological activity, low toxicity, stability and their antimutagenic effects via different modes of action, appear to be promising features for their practical application as antioxidants and antimutagenic agents.     

Calorie restriction reduces oxidative stress by SIRT3-mediated SOD2 activation

A major cause of aging and numerous diseases is thought to be cumulative oxidative stress, resulting from the production of reactive oxygen species (ROS) during respiration. Calorie restriction (CR), the most robust intervention to extend life span and ameliorate various diseases in mammals, reduces oxidative stress and damage. However, the underlying mechanism is unknown. Here, we show that the protective effects of CR on oxidative stress and damage are diminished in mice lacking SIRT3, a mitochondrial deacetylase. SIRT3 reduces cellular ROS levels dependent on superoxide dismutase 2 (SOD2), a major mitochondrial antioxidant enzyme. SIRT3 deacetylates two critical lysine residues on SOD2 and promotes its antioxidative activity. Importantly, the ability of SOD2 to reduce cellular ROS and promote oxidative stress resistance is greatly enhanced by SIRT3. Our studies identify a defense program that CR provokes to reduce oxidative stress and suggest approaches to combat aging and oxidative stress-related diseases.     

Essential role for the d-Asb11 cul5 Box domain for proper notch signaling and neural cell fate decisions in vivo

ECS (Elongin BC-Cul2/Cul5-SOCS-box protein) ubiquitin ligases recruit substrates to E2 ubiquitin-conjugating enzymes through a SOCS-box protein substrate receptor, an Elongin BC adaptor and a cullin (Cul2 or Cul5) scaffold which interacts with the RING protein. In vitro studies have shown that the conserved amino acid sequence of the cullin box in SOCS-box proteins is required for complex formation and function. However, the in vivo importance of cullin boxes has not been addressed. To explore the biological functions of the cullin box domain of ankyrin repeat and SOCS-box containing protein 11 (d-Asb11), a key mediator of canonical Delta-Notch signaling, we isolated a zebrafish mutant lacking the Cul5 box (Asb11(Cul)). We found that homozygous zebrafish mutants for this allele were defective in Notch signaling as indicated by the impaired expression of Notch target genes. Importantly, asb11(Cul) fish were not capable to degrade the Notch ligand DeltaA during embryogenesis, a process essential for the initiation of Notch signaling during neurogenesis. Accordingly, proper cell fate specification within the neurogenic regions of the zebrafish embryo was impaired. In addition, Asb11(Cul) mRNA was defective in the ability to transactivate a her4::gfp reporter DNA when injected in embryos. Thus, our study reporting the generation and the characterization of a metazoan organism mutant in the conserved cullin binding domain of the SOCS-box demonstrates a hitherto unrecognized importance of the SOCS-box domain for the function of this class of cullin-RING ubiquitin ligases and establishes that the d-Asb11 cullin box is required for both canonical Notch signaling and proper neurogenesis.     

Nanoscale Imaging of Epidermal Growth Factor Receptor Clustering: EFFECTS OF INHIBITORS*

The development of some solid tumors is associated with overexpression of the epidermal growth factor receptor (EGFR) and often correlates with poor prognosis. Near field scanning optical microscopy, a technique with subdiffraction-limited optical resolution, was used to examine the influence of two inhibitors (the chimeric 225 antibody and tyrosine phosphorylation inhibitor AG1478) on the nanoscale clustering of EGFR in HeLa cells. The EGFR is organized in small clusters, average diameter of 150 nm, on the plasma membrane for both control and EGF-treated cells. The numbers of receptors in individual clusters vary from as few as one or two proteins to greater than 100. Both inhibitors yield an increased cluster density and an increase in the fraction of clusters with smaller diameters and fewer receptors. Exposure to AG1478 also decreases the fraction of EGFR that colocalizes with both rafts and caveolae. EGF stimulation results in a significant loss of the full-length EGFR from the plasma membrane with the concomitant appearance of low molecular mass proteolytic products. By contrast, AG1478 reduces the level of EGFR degradation. Changes in receptor clustering provide one mechanism for regulating EGFR signaling and are relevant to the design of strategies for therapeutic interventions based on modulating EGFR signaling.     

Selection for low or high primary dormancy in Lolium rigidum Gaud seeds results in constitutive differences in stress protein expression and peroxidase activity

Seed dormancy in wild Lolium rigidum Gaud (annual ryegrass) populations is highly variable and not well characterized at the biochemical level. To identify some of the determinants of dormancy level in these seeds, the proteomes of subpopulations selected for low and high levels of primary dormancy were compared by two-dimensional polyacrylamide gel electrophoresis of extracts from mature, dry seeds. High-dormancy seeds showed higher expression of small heat shock proteins, enolase, and glyoxalase I than the low-dormancy seeds. The functional relevance of these differences in protein expression was confirmed by the fact that high-dormancy seeds were more tolerant to high temperatures imposed at imbibition and had consistently higher glyoxalase I activity over 0-42 d dark stratification. Higher expression of a putative glutathione peroxidase in low-dormancy seeds was not accompanied by higher activity, but these seeds had a slightly more oxidized glutathione pool and higher total peroxidase activity. Overall, these biochemical and physiological differences suggest that L. rigidum seeds selected for low dormancy are more prepared for rapid germination via peroxidase-mediated cell wall weakening, whilst seeds selected for high dormancy are constitutively prepared to survive environmental stresses, even in the absence of stress during seed development.     

Effects of ramipril and valsartan on proteinuria and renal function in patients with nondiabetic proteinuria

The renin-angiotensin system is involved in the progression of chronic renal disease of both diabetic and nondiabetic origin. The angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers have been demonstrated to reduce urinary protein excretion and attenuate the development of renal injury. This prospective, randomized, 12-month study assessed the effects of ramipril (N = 23) vs. valsartan (N = 22) vs. combination of ramipril and valsartan (N = 26) on proteinuria, renal function and metabolic profile in 71 patients with nondiabetic proteinuria with normal or slightly impaired renal function. Monotherapy with ramipril or valsartan and combination of these two drugs significantly reduced proteinuria, serum creatinine, cholesterol and triglycerides as well as systolic and diastolic arterial blood pressure. There was no significant difference among three study groups according to reduction of arterial blood pressure, serum cholesterol and triglycerides. At one year, a significant reduction in serum creatinine was recorded in all three study groups, whereas at 3 and 6 months a statistically significant reduction in serum creatinine was only observed in patients on combination therapy. In addition, at 3 months the reduction of proteinuria was significantly greater in patients on combination therapy than in those on either monotherapy. These results indicated the combination therapy with angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers to be more efficacious than either monotherapy in reducing proteinuria and serum creatinine level in the first 3 (proteinuria and serum creatinine) or 6 (serum creatinine) months of treatment.     

The pattern of physical comorbidity and the psychosocial determinants of depression: a prospective cohort study on a representative sample of family practice attendees in Slovenia

Objectives This study aims to present the patterns of physical comorbidity in depressed patients and factors strongly associated with depression in a representative sample of Slovenian family practice attendees.Methods Medical data was obtained for 911 general practice attendees. Of them, 221 (24.3%) were diagnosed as depressed. The depressive states of the subjects were evaluated using the Composite International Diagnostic Interview (CIDI). Physical comorbidity was assessed with a questionnaire covering the most common health problems in the Slovenian adult population. Several psycho-social factors were also analysed.Results Those variables significantly related to ICD depression were included in multivariate binary logistic regression analysis, adjusted by age, gender and education. The calculation included the chi-square, odds ratio (OR) with confidence interval (95% CI) and P-value. A P-value < 0.05 was marked as statistically significant.Conclusions There was no significant difference in the number of concurrent chronic diseases in depressed and non-depressed subjects. The risk of depression was increased by the presence of several concomitant factors. The burden of somatic co-morbidity was shown to be smaller than the impact of psychosocial determinants, which also acted as protective factors: the feeling of safety at home and the absence of problems in intimate relationships. The abuse of alcohol and drugs by a family member and current poor financial situation were strongly associated with depression. The impact of concurrent incontinence and chronic bowel disease was also important, though somewhat weaker.     

A reverse genetics approach to study feline infectious peritonitis

Feline infectious peritonitis (FIP) is a lethal immunopathological disease caused by feline coronaviruses (FCoVs). Here, we describe a reverse genetics approach to study FIP by assessing the pathogenicity of recombinant type I and type II and chimeric type I/type II FCoVs. All recombinant FCoVs established productive infection in cats, and recombinant type II FCoV (strain 79-1146) induced FIP. Virus sequence analyses from FIP-diseased cats revealed that the 3c gene stop codon of strain 79-1146 has changed to restore a full-length open reading frame (ORF).