Engineering and characterization of human manganese superoxide dismutase mutants with high activity and low product inhibition

Human manganese superoxide dismutase is a mitochondrial metalloenzyme that is involved in protecting aerobic organisms against superoxide toxicity, and has been implicated in slowing tumor growth. Unfortunately, this enzyme exhibits strong product inhibition, which limits its potential biomedical applications. Previous efforts to alleviate human manganese superoxide dismutase product inhibition utilized rational protein design and site-directed mutagenesis. These efforts led to variants of human manganese superoxide dismutase at residue 143 with dramatically reduced product inhibition, but also reduced catalytic activity and efficiency. Here, we report the use of a directed evolution approach to engineer two variants of the Q143A human manganese superoxide dismutase mutant enzyme with improved catalytic activity and efficiency. Two separate activity-restoring mutations were found--C140S and N73S--that increase the catalytic efficiency of the parent Q143A human manganese superoxide dismutase enzyme by up to five-fold while maintaining low product inhibition. Interestingly, C140S is a context-dependent mutation, and the C140S-Q143A human manganese superoxide dismutase did not follow Michaelis-Menten kinetics. The re-engineered human manganese superoxide dismutase mutants should be useful for biomedical applications, and our kinetic and structural studies also provide new insights into the structure-function relationships of human manganese superoxide dismutase.     

Statin therapy lowers muscle sympathetic nerve activity and oxidative stress in patients with heart failure

Despite standard drug therapy, sympathetic nerve activity (SNA) remains high in heart failure (HF) patients making the sympathetic nervous system a primary drug target in the treatment of HF. Studies in rabbits with pacing-induced HF have demonstrated that statins reduce resting SNA, in part, due to reductions in reactive oxygen species (ROS). Whether these findings can be extended to the clinical setting of human HF remains unclear. We first performed a study in seven statin-na?ve HF patients (56 ± 2 yr; ejection fraction: 31 ± 4%) to determine if 1 mo of simvastatin (40 mg/day) reduces muscle SNA (MSNA). Next, to control for possible placebo effects and determine the effect of simvastatin on ROS, a double-blinded, placebo-controlled crossover design study was performed in six additional HF patients (51 ± 3 yr; ejection fraction: 22 ± 4%), and MSNA, ROS, and superoxide were measured. We tested the hypothesis that statin therapy decreases resting MSNA in HF patients and this would be associated with reductions in ROS. In study 1, simvastatin reduced resting MSNA (75 ± 5 baseline vs. 65 ± 5 statin bursts/100 heartbeats; P < 0.05). Likewise, in study 2, simvastatin also decreased resting MSNA (59 ± 5 placebo vs. 45 ± 6 statin bursts/100 heartbeats; P < 0.05). In addition, statin therapy significantly reduced total ROS and superoxide. As expected, cholesterol was reduced after simvastatin. Collectively, these findings indicate that short-term statin therapy concomitantly reduces resting MSNA and total ROS and superoxide in HF patients. Thus, in addition to lowering cholesterol, statins may also be beneficial in reducing sympathetic overactivity and oxidative stress in HF patients.     

Amelioration of Cancer Stem Cells in Macrophage Colony Stimulating Factor-Expressing U87MG-Human Glioblastoma upon 5-Fluorouracil Therapy

Macrophage colony stimulating factor (MCSF) regulates growth, proliferation and differentiation of haematopoietic cell lineages. Many cancers are known to secrete high level of MCSF, which recruit macrophages into the tumour micro-environment, supporting tumour growth. Herein, we report the cloning of MCSF and subsequent generation of U87MG expressing MCSF stable cell line (U87-MCSF). Cytotoxicity of anti-cancer drug 5-fluorouracil (5-FU) was evaluated on both U87MG and U87-MCSF cells. Interestingly, the proliferation of U87-MCSF cells was less (p<0.001) than that of U87MG cells alone, after treatment with 5-FU. Significant decrease in expression levels of cyclin E and A2 quantified by real time PCR analysis corroborated the reduced proliferation of 5-FU treated U87-MCSF cells. However, JC-1 staining did not reveal any apoptosis upon 5-FU treatment. Notch-1 upregulation induced a possible epithelial-mesenchymal transition in U87-MCSF cells, which accounted for an increase in the proportion of CD24^high/CD44^less cancer stem cells in U87-MCSF cells after 5-FU treatment. The elevated resistance of U87-MCSF cells towards 5-FU was due to the increase in the expressions (10.2 and 6 fold) of ABCB1 and mdm2, respectively. Furthermore, increase in expressions of ABCG1, mdm2 and CD24 was also observed in U87MG cells after prolonged incubation with 5-FU. Our studies provided mechanistic insights into drug resistance of U87MG cells and also described the pivotal role played by MCSF in augmenting the resistance of U87MG cells to 5-FU.     

Utility of Eucalyptus tereticornis (Smith) bark and Desulfotomaculum nigrificans for the remediation of acid mine drainage

The efficacy of the bark of Eucalyptus tereticornis (Smith) as an adsorbent for the removal of metal ions and sulphate from acid mine water was assessed. About 96% of Fe, 75% of Zn, 92% of Cu and 41% of sulphate removal was achieved from the acid mine water of pH 2.3 with a concomitant increase in pH value by about two units after interaction with the tree bark, under appropriate conditions. The adsorption isotherms adhered to Freundlich and Langmuir relationships and were exothermic in nature. The free energy of the adsorption process was found to be negative attesting to the feasibility of the reaction. The adsorption kinetics followed the first-order Lagergren rate equation. The filtrate obtained after treatment with E. tereticornis (Sm) bark was found to contain essential elements like potassium, magnesium, calcium, sodium and phosphate apart from carbon which served as a successful growth medium for the sulphate reducing bacteria (SRB) namely Desulfotomaculum nigrificans. Bacterial growth studies showed that about 57% and 72% of sulphate reduction could be achieved at initial pH values of 4.1 and 5.5 respectively of the acid mine water. Pretreatment of the acid mine water with tree bark followed by bioremoval using Dsm. nigrificans resulted in about 75% and 84% respectively of sulphate reduction at pH 4.1 and 5.5, cumulatively by biosorption and bioreduction. The mechanisms of metal ion removal using tree bark and sulphate reduction using Dsm. nigrificans are discussed.     

Adaptive mechanisms to compensate for overnutrition-induced cardiovascular abnormalities

In conditions of overnutrition, cardiac cells must cope with a multitude of extracellular signals generated by changes in nutrient load (glucose, amino acids, and lipids) and the hormonal milieu [increased insulin (INS), ANG II, and adverse cytokine/adipokine profile]. Herein, we review the diverse compensatory/adaptive mechanisms that counter the deleterious effects of excess nutrients and growth factors. We largely focus the discussion on evidence obtained from Zucker obese (ZO) and Zucker diabetic fatty (ZDF) rats, which are useful models to evaluate adaptive and maladaptive metabolic, structural, and functional cardiac remodeling. One adaptive mechanism present in the INS-resistant ZO, but absent in the diabetic ZDF heart, involves an interaction between the nutrient sensor kinase mammalian target of rapamycin complex 1 (mTORC1) and ANG II-type 2 receptor (AT2R). Recent evidence supports a cardioprotective role for the AT2R; for example, suppression of AT2R activation interferes with antihypertrophic/antifibrotic effects of AT1R blockade, and AT2R agonism improves cardiac structure and function. We propose a scenario, whereby mTORC1-signaling-mediated increase in AT2R expression in the INS-resistant ZO heart is a cardioprotective adaptation to overnutrition. In contrast to the ZO rat, heart tissues of ZDF rats do not show activation of mTORC1. We posit that such a lack of activation of the mTOR?AT2R integrative pathway in cardiac tissue under conditions of obesity-induced diabetes may be a metabolic switch associated with INS deficiency and clinical diabetes.     

Mapping quantitative trait loci controlling oil content,oleic acid and linoleic acid content in sunflower (<Emphasis Type="Italic">Helianthus annuus</Emphasis> L.)

Sunflower oil with high oleic acid content is in great demand due to its nutritional as well as industrial benefits. The trait is mainly controlled by dominant alleles at a major gene, Ol, with other modifiers. The objectives of this research were to map the oil content, oleic acid and linoleic acid content in sunflower seeds. An F2 mapping population from cytoplasmic male-sterile line COSF 7A (33–35 % oleic acid) and high oleic acid inbred line HO 5–13 (88–90 % oleic acid) was developed and phenotyped for oil content, oleic acid and linoleic acid content at the F2 seed level. High phenotypic and genotypic coefficients of variation were recorded for oleic acid and linoleic acid content. High heritability and high genetic advance as percent of mean was recorded for oleic acid and linoleic acid content. This indicated the presence of the additive type of gene action controlling the traits oleic acid content and linoleic acid content. The Ol gene was mapped to linkage group (LG) 14 and tightly linked to the marker HO_Fsp_b. In addition, two more quantitative trait loci (QTLs) for oleic acid content were identified in LG8 and LG9. Two QTLs for oil content and two QTLs for linoleic acid content were also identified. All these QTLs explained over 10 % of phenotypic variation. A study was conducted with 13 genotypes differing in oil quality as well as quantity over three seasons to assess the reliability of the identified QTLs over seasons. It resulted in the identification of two potential QTLs for oleic acid as well as linoleic acid content with the markers ORS 762 and HO_Fsp_b. These markers explained more than 57.6–66.6 % of phenotypic variation. Hence it can be concluded that these markers/QTLs would be useful in the marker-assisted selection breeding programme to improve oil quality. The present study also indicated the presence of at least two other genomic regions controlling oleic and linoleic acid content in sunflower.     

Autocatalytic cleavage of ADAMTS-4 (Aggrecanase-1) reveals multiple glycosaminoglycan-binding sites

ADAMTS-4, also referred to as aggrecanase-1, is a glutamyl endopeptidase capable of generating catabolic fragments of aggrecan analogous to those released from articular cartilage during degenerative joint diseases such as osteoarthritis. Efficient aggrecanase activity requires the presence of sulfated glycosaminoglycans (GAGs) attached to the aggrecan core protein, implying the contribution of substrate recognition/binding site(s) to ADAMTS-4 activity. In the present study, we demonstrate that full-length ADAMTS-4 (M(r) approximately 68,000) undergoes autocatalytic C-terminal truncation to generate two discrete isoforms (M(r) approximately 53,000 and M(r) approximately 40,000), which exhibit a marked reduction in affinity of binding to sulfated GAGs. C-terminal sequencing and mass analyses revealed that the GAG-binding thrombospondin type I motif was retained following autocatalysis, indicating that sites present in the C-terminal cysteine (cys)-rich and/or spacer domains also effect binding of full-length ADAMTS-4 to sulfated GAGs. Binding-competition experiments conducted using native and deglycosylated aggrecan provided direct evidence for interaction of the ADAMTS-4 cysteine-rich/spacer domains with aggrecan GAGs. Furthermore, synthetic peptides mimicking putative (consensus) GAG-binding sequences located within the ADAMTS-4 cysteine-rich and spacer domains competitively blocked binding of sulfated GAGs to full-length ADAMTS-4, thereby identifying multiple GAG-binding sites, which may contribute to the regulation of ADAMTS-4 function.