Molecular insights into the association of obesity with breast cancer risk: relevance to xenobiotic metabolism and CpG island methylation of tumor suppressor genes

Obesity, genetic polymorphisms of xenobiotic metabolic pathway, hypermethylation of tumor suppressor genes, and hypomethylation of proapoptotic genes are known to be independent risk factors for breast cancer. The objective of this study is to evaluate the combined effect of these environmental, genetic, and epigenetic risk factors on the susceptibility to breast cancer. PCR–RFLP and multiplex PCR were used for the genetic analysis of six variants of xenobiotic metabolic pathway. Methylation-specific PCR was used for the epigenetic analysis of four genetic loci. Multifactor dimensionality reduction analysis revealed a significant interaction between the body mass index (BMI) and catechol-O-methyl transferase H108L variant alone or in combination with cytochrome P450 (CYP) 1A1m1 variant. Women with “Luminal A” breast cancer phenotype had higher BMI compared to other phenotypes and healthy controls. There was no association between the BMI and tumor grade. The post-menopausal obese women exhibited lower glutathione levels. BMI showed a positive association with the methylation of extracellular superoxide dismutase (r = 0.21, p < 0.05), Ras-association (RalGDS/AF-6) domain family member 1 (RASSF1A) (r = 0.31, p < 0.001), and breast cancer type 1 susceptibility protein (r = 0.19, p < 0.05); and inverse association with methylation of BNIP3 (r = ?0.48, p < 0.0001). To conclude based on these results, obesity increases the breast cancer susceptibility by two possible mechanisms: (i) by interacting with xenobiotic genetic polymorphisms in inducing increased oxidative DNA damage and (ii) by altering the methylome of several tumor suppressor genes.     

Adaptive developmental plasticity in methylene tetrahydrofolate reductase (MTHFR) C677T polymorphism limits its frequency in South Indians

Methylene tetrahydrofolate reductase (MTHFR) C677T polymorphism shows considerable heterogeneity in its distribution in humans worldwide. The current study was conducted to investigate whether this polymorphism exhibited adaptive developmental plasticity in the control of the TT-genotype frequency. We screened 1,818 South Indian subjects (895 males and 923 females) for MTHFR C677T polymorphism using PCR–restriction fragment length polymorphism approach. MTHFR 677T-allele frequency in males and females was 9.1 and 11.0 %, respectively. Compared to females, males had lower frequency of TT-genotype [odds ratio 0.31, 95 % confidence interval (CI) 0.08–1.01]. The frequency of MTHFR 677T-allele was highest in the age group of 20–40 years and it gradually decreased from 40–60 to 60–80 years (P _trend <0.0001). MTHFR 677TT-genotype was associated with 7.02-folds (95 % CI: 2.12–25.63, P < 0.0001) cumulative risk for recurrent pregnancy loss (RPL), neural tube defects (NTDs) and deep vein thrombosis (DVT). Linear regression model suggested that male gender exhibited increased homocysteine levels by 9.35 μmol/L while each MTHFR 677T-allele contributed to 4.63 μmol/L increase in homocysteine. Plasma homocysteine showed inverse correlation with dietary folate (r = ?0.17, P < 0.0001), B₂ (r = ?0.14, P < 0.0001) and B₆ (r = ?0.07, P = 0.03). Examination of the spontaneously aborted fetuses (n = 35) showed no significant association of fetal genotype on its in utero viability. From the current study, it was concluded that C677T seemed to have acquired adaptive developmental plasticity among South Indians due to environmental influences thus contributing to hyperhomocysteinemia and its associated complications such as RPL, NTDs, DVT, etc.     

PURIFICATION AND CHARACTERIZATION OF β‐GLUCOSIDASE FROM GREATER WAX MOTH Galleria mellonella L. (LEPIDOPTERA: PYRALIDAE)

The greater wax moth, Galleria mellonella, is one of the most ruinous pests of honeycomb in the world. Beta‐glucosidases are a type of digestive enzymes that hydrolytically catalyzes the beta‐glycosidic linkage of glycosides. Characterization of the beta‐glucosidase in G. mellonella could be a significant stage for a better comprehending of its role and establishing a safe and effective control procedure primarily against G. mellonella and also some other insect pests. Laboratory reared final instar stage larvae were randomly selected and homogenized for beta‐glucosidase activity assay and subsequent analysis. The enzyme was purified to apparent homogeneity by salting out with ammonium sulfate and using sepharose‐4B‐l ‐tyrosine‐1‐naphthylamine hydrophobic interaction chromatography. The purification was 58‐fold with an overall enzyme yield of 29%. The molecular mass of the protein was estimated as ca. 42 kDa. The purified beta‐glucosidase was effectively active on para/ortho‐nitrophenyl‐beta‐d ‐glucopyranosides (p‐/o‐NPG) with Km values of 0.37 and 1.9 mM and Vmax values of 625 and 189 U/mg, respectively. It also exhibits different levels of activity against para‐nitrophenyl‐β‐d ‐fucopyranoside (p‐NPF), para/ortho‐nitrophenyl β‐d ‐galactopyranosides (p‐/o‐NPGal) and p‐nitrophenyl 1‐thio‐β‐d ‐glucopyranoside. The enzyme was competitively inhibited by beta‐gluconolactone and also was very tolerant to glucose against p‐NPG as substrate. The Ki and IC₅₀ values of δ‐gluconolactone were determined as 0.021 and 0.08 mM while the enzyme was more tolerant to glucose inhibition with IC50 value of 213.13 mM for p‐NPG.     

Ionic Liquid–Sulfolane Composite Electrolytes for High‐Performance and Stable Dye‐Sensitized Solar Cells

Ionic liquid electrolytes are prepared using sulfolane as a plasticizer for eutectic melts to realize highly stable and efficiently performing dye‐sensitized solar cells (DSCs) in hot climate conditions. Variations in the viscosity of the formulations with sulfolane content are measured and performance in DSCs is investigated using the ruthenium dye C106 as a sensitizer. A power conversion efficiency (PCE) of 8.2% is achieved under standard reporting conditions. Apart from lowering the viscosity, the addition of sulfolane induces a negative shift of the TiO₂ conduction band edge. Strikingly the device performance increases to 8.4% at 50 °C due to higher short circuit photocurrent and fill factor, over‐compensating the loss in open circuit voltage with increasing temperature. The PCE increases also upon decreasing the light intensity of the solar simulator, reaching up to 9% at 50 mW cm^?2. Devices based on these new electrolyte formulations show excellent stability during light soaking for 2320 h under full sunlight at 60 °C and also during a 1065 h long heat stress at 80 °C in the dark. A detailed investigation provides important information about the factors affecting the principal photovoltaic parameters during the aging process and the first results from a series of outdoor measurements are reported.     

Evidence for the presence of somatic mitochondrial DNA mutations in right atrial appendage tissues of coronary artery disease patients

Coronary artery disease (CAD) is a multifactorial disease with the underlying involvement of environment, life style and nuclear genetics. However, the role of extranuclear genetic material in terms of somatically acquired mutations in mitochondrial tRNA and protein coding genes in the initiation or progression of CAD is not well defined. Hence, in the present study, right atrial appendage tissues and matched blood samples of 150 CAD patients were screened for mutations in nucleotide regions encompassing the Cytochrome c oxidase subunit II (MT-CO2), tRNA lysine (MT-TK), ATP synthase F0 subunit 8 (MT-ATP8) and Cytochrome b (MT-CYB) genes of mitochondrial DNA. We have found 9 different somatic mutations in 6 % of the CAD patients. Out of these mutations, 4 each were localized in MT-TK gene (T8324A, A8326G, A8331G and A8344G) and MT-CYB genes (T15062C, C15238A, T15378G and C15491G) in addition to one mutation in non-coding region 7 (A8270T) of mitochondrial genome. In addition, we noticed that majority (85.3 %) of CAD patients showed double repeats of germ-line “CCCCCTCTA” intergenic sequence between MT-CO2 and MT-TK genes. Our in-silico investigations of missense mutations revealed that they may alter the free energy and stability of polypeptide chains of MT-CYB protein of complex III of mitochondrial respiratory chain. Based on our study findings, we hypothesize that the somatically acquired variations in MT-TK and MT-CYB genes may negatively impact the energy metabolism of cardiomyocytes in right atrial appendage tissues and contribute in the cardiac dysfunction among CAD patients. In conclusion, our findings may be likely to have potential implications in understanding the disease pathophysiology, diagnosis as well as for the better therapeutic management of CAD patients.     

Two-state reactivity mechanisms of hydroxylation and epoxidation by cytochrome P-450 revealed by theory

Recent computational studies of alkane hydroxylation and alkene epoxidation by a model active species of the enzyme cytochrome P-450 reveal a two-state reactivity (TSR) scenario in which the information content of the product distribution is determined jointly by two states. TSR is used to reconcile the dilemma of the consensus 'rebound mechanism' of alkane hydroxylation, which emerged from experimental studies of ultra-fast radical clocks. The dilemma, stated succinctly as 'radicals are both present and absent and the rebound mechanism is both right and wrong', is simply understood once one is cognizant that the mechanism operates by two states, one low-spin (LS) the other high-spin (HS). In both states, bond activation proceeds in a manner akin to the rebound mechanism, but the LS mechanism is effectively concerted, whereas the HS is stepwise with incursion of radical intermediates.     

Indirect regeneration of the Cancer bush (<Emphasis Type="Italic">Sutherlandia frutescens</Emphasis> L.) and detection of <Emphasis Type="SmallCaps">l</Emphasis>-canavanine in <Emphasis Type="Italic">in vitro</Emphasis> plantlets using NMR

The present study reports a simple protocol for indirect shoot organogenesis and plant regeneration of Sutherlandia using rachis and stem segments. Different concentrations (0.0–68.08 μmol l⁻¹) of thidiazuron (TDZ) were used for callus induction and shoot organogenesis. The highest percentage of callus formation (97.5%) and the highest percentage of explants forming shoots (88.8%) were obtained from rachis explants cultured onto Murashige and Skoog (MS) medium (Murashige and Skoog, Physiol. Plant. 15:473–495, 1962) supplemented with 45.41 μmol l⁻¹ TDZ. Scanning electron microscopy demonstrated the early development of adventitious shoots derived from callus cultures. Shoot clusters were further developed and grown in MS hormone-free medium. The presence of l-canavanine was determined by thin-layer chromatography and confirmed after column fractionation using silica gel and nuclear magnetic resonance spectroscopy. Individual shoots were rooted on different concentrations and combinations of MS salt strength and IBA. Half-strength MS salt medium supplemented with 24.6 μmol l⁻¹ IBA was optimal for root induction in which 78% of shoots were rooted. The in vitro plants were successfully acclimatized in a growth chamber with a 90% survival rate.     

On the identity and reactivity patterns of the “second oxidant” of the T252A mutant of cytochrome P450cam in the oxidation of 5-methylenenylcamphor

Density functional calculations show that in the absence of Compound I, the primary oxidant species of P450, the precursor species, Compound 0 (FeOOH), can effect double bond activation of 5-methylenylcamphor (1). The mechanism is initiated by homolytic cleavage of the O–O bond and formation of an OH radical bound to the Compound II species by hydrogen bonding interactions. Subsequently, the so-formed OH radical can either activate the double bond of 1 or attack the meso position of the heme en route to heme degradation. The calculations show that double bond activation is preferred over attack on the heme. Past the double bond activation, the intermediate can either lead to epoxidation or to a glycol formation. The glycol formation is predicted to be preferred, although in the P450_cam pocket the competition may be closer. Therefore, in the absence of Compound I, Compound 0 will be capable of epoxidizing double bonds. Previous studies [E. Derat, D. Kumar, H. Hirao, S. Shaik, J. Am. Chem. Soc. 128 (2006) 473–484] showed that in the case of a substrate that can undergo only C–H activation, the bound OH prefers heme degradation over hydrogen abstraction. Since the epoxidation barrier for Compound I is much smaller than that of Compound 0 (12.8 vs. 18.9 kcal/mol), when Compound I is present in the cycle, Compound 0 will be silent. As such, our mechanism explains lucidly why T252A P450_cam can epoxidize olefins like 5-methylenylcamphor but is ineffective in camphor hydroxylation [S. Jin, T.M. Makris, T. A. Bryson, S.G. Sligar, J.H. Dawson, J. Am. Chem. Soc. 125 (2003) 3406–3407]. Our calculations show that the glycol formation is a marker reaction of Compound 0 with 5-methylenylcamphor. If this product can be found in T252A P450_cam or in similar mutants of other P450 isozymes, this will constitute a more definitive proof for the action of Cpd 0 in P450 enzymes.     

Evaluation of adaptogenic activity profile of herbal preparation

The herbal formulation, AVM is a proprietary formula that consists of extracts of herbs that have been used in Indian traditional medicine to promote physical and mental health, improve defense mechanisms of the body and enhance longevity. AVM (500 and 1000 mg/kg) was tested for its adaptogenic activity by determining antistress, anabolic and immunomodulatory effects. In antistress activity, pretreatment with AVM significantly attenuated the changes in ascorbic acid (from blood and adrenal), cortisol (from plasma and adrenal) and adrenal gland weights induced due to restrain stress (physical immobilization). Its antistress effect at 1000 mg/kg was comparable to that of diazepam (5 mg/kg) treated group. Leucopenia, and anemia induced by cyclophosphamide (CYP) was shown to reduce significantly by AVM. Treatment of AVM + CYP had increased spleen and thymus weights significantly as compared to CYP alone treated group. The anabolic activity was evaluated by weight gain of the levator ani muscle, ventral prostrate gland and seminal vesicles in rats as compared to untreated control.     

Role of quercetin (a natural herbal compound) in allergy and inflammation

An investigated flavonoid, quercetin, is reviewed in this article. Quercetin is a bioflavonoid found in red wine, grapefruit, onions, apples, black tea, and, in lesser amounts, in leafy green vegetables and beans. Quercetin has an antioxidant and anti-inflammatory activity and prevents cancer. Quercitin inhibits the growth of certain malignant cells in vitro, and histamine and most cyclin-dependent kinases and also displays unique anticancer properties. Quercetin is a natural compound that blocks substances involved in allergies and is able to act as an inhibitor of mast cell secretion, causes a decrease in the release of tryptase, MCP-1 and IL-6 and the down-regulation of histidine decarboxylase (HDC) mRNA from few mast cell lines. Quercetin is a safe, natural therapy that may be used as primary therapy or in conjunction with conventional methods.