Proteomic identification of E6AP as a molecular target of tamoxifen in MCF7 cells

Tamoxifen (Tam) is most widely used selective estrogen receptor modulator (SERM) for treatment of hormone-responsive breast cancer. Despite being regularly used in clinical therapy for breast cancer since 1971, the mechanism of Tam action remains largely unclear. In order to gain insights into Tam-mediated antibreast cancer actions, we applied 2DE and MS based proteomics approach to identify target proteins of Tam. We identified E6-associated protein, i.e. E6AP (UBE3A) among others to be regulated by Tam that otherwise is upregulated in breast tumors. We confirmed our 2DE finding by immunoblotting and further show that Tam leads to inhibition of E6AP expression presumably by promoting its autoubiquitination, which is coupled with nuclear export and subsequent proteasome-mediated degradation. Furthermore, we show that Tam- and siE6AP-mediated inhibition of E6AP leads to enhanced G0-G1 growth arrest and apoptosis, which is also evident from significant upregulation of cytochrome-c, Bax, p21, and PARP cleavage. Taken together, our data suggest that, Tam-targeted E6AP inhibition is in fact required for Tam-mediated antibreast cancer actions. Thus, E6AP may be a therapeutic target in breast cancer.     

Diarrhea,Stimulation and Growth Predict Neurodevelopment in Young North Indian Children

Background and Objective

Infants and young children in low to middle-income countries are at risk for adverse neurodevelopment due to multiple risk factors. In this study, we sought to identify stimulation and learning opportunities, growth, and burden of respiratory infections and diarrhea as predictors for neurodevelopment.

Methods

We visited 422 North Indian children 6 to 30 months old weekly for six months. Childhood illnesses were assessed biweekly. At end study, we assessed neurodevelopment using the Ages and Stages Questionnaire 3^rd ed. (ASQ-3) and gathered information on stimulation and learning opportunities. We identified predictors for ASQ-3 scores in multiple linear and logistic regression models.

Results

We were able to explain 30.5% of the variation in the total ASQ-3 score by the identified predictors. When adjusting for child characteristics and annual family income, stimulation and learning opportunities explained most of the variation by 25.1%. Height for age (standardized beta: 0.12, p<.05) and weight for height z-scores (std. beta: 0.09, p<.05) were positively associated with the total ASQ-3 score, while number of days with diarrhea was negatively associated with these scores (std. beta: -0.13, p<0.01).

Conclusion

Our results support the importance of early child stimulation and general nutrition for child development. Our study also suggests that diarrhea is an additional risk factor for adverse neurodevelopment in vulnerable children.     

Polymorphisms in CaSR and CLDN14 Genes Associated with Increased Risk of Kidney Stone Disease in Patients from the Eastern Part of India

Kidney stone disease (KSD) is a major clinical problem imposing a large burden for both healthcare and economy globally. In India, the prevalence of kidney stone disease is rapidly increasing. This study aimed to evaluate the association between genetic defects in vitamin D receptor (VDR), calcium sensing receptor (CaSR) and claudin 14 (CLDN14) genes and kidney stone disease in patients from eastern India. We enrolled 200 consecutive kidney stone patients (age 18–60 years) (cases) and their corresponding sex and age matched 200 normal individuals (controls). To identify genetic variants responsible for KSD, we performed sequence analysis of VDR, CaSR and CLDN14 genes. Four non-synonymous (rs1801725, rs1042636, rs1801726 and rs2228570), one synonymous (rs219780) and three intronic single nucleotide polymorphisms (SNPs) (rs731236, rs219777 and rs219778) were identified. Genotype and allele frequency analysis of these SNPs revealed that, rs1801725 (Ala986Ser), rs1042636 (Arg990Gly) of CaSR gene and rs219778, rs219780 (Thr229Thr) of CLDN14 gene were significantly associated with KSD. Serum calcium levels were significantly higher in subjects carrying 986Ser allele and calcium excretion was higher in subjects bearing 990Gly allele. In conclusion, rs1801725, rs1042636, rs219778 and rs219780 SNPs were associated with kidney stone risk in patients from the eastern part of India.     

Hysteresis in coral reefs under macroalgal toxicity and overfishing

Macroalgae and corals compete for the available space in coral reef ecosystems.While herbivorous reef fish play a beneficial role in decreasing the growth of macroalgae, macroalgal toxicity and overfishing of herbivores leads to proliferation of macroalgae. The abundance of macroalgae changes the community structure towards a macroalgae-dominated reef ecosystem. We investigate coral-macroalgal phase shifts by means of a continuous time model in a food chain. Conditions for local asymptotic stability of steady states are derived. It is observed that in the presence of macroalgal toxicity and overfishing, the system exhibits hysteresis through saddle-node bifurcation and transcritical bifurcation. We examine the effects of time lags in the liberation of toxins by macroalgae and the recovery of algal turf in response to grazing of herbivores on macroalgae by performing equilibrium and stability analyses of delay-differential forms of the ODE model. Computer simulations have been carried out to illustrate the different analytical results.

Electronic Supplementary Material

The online version of this article (doi:10.1007/s10867-014-9371-y) contains supplementary material, which is available to authorized users.     

Melatonin rhythms in the pineal and non-pineal tissues and their physiological implications in subtropical fish

Abstract

Melatonin (N-acetyl-5-methoxy tryptamine), following discovery from the extracts of bovine pineal gland, has been detected in the pineal as well as several extra-pineal tissues/organs of different vertebrates including fish. The unique feature of melatonin in the pineal gland is its rhythmic biosynthesis and release in blood in synchronization with the environmental light-–dark cycle. Accordingly, melatonin produced in the pineal of an animal living in a changing environment is implicated to the regulation of seasonal reproduction by acting as a hormone at one or more levels of hypothalamo-hypophyseal-gonadal axis. Additionally, melatonin is known to act as a potent free-radical scavenger or antioxidant to influence maturation of oocytes. However, possible relationship between extra-pineal melatonin and seasonality of reproduction in any animal remains enigmatic. Perhaps, carp is the only known animal in which temporal patterns of melatonin levels in the serum as well as in the extracts of pineal, retina, ovary, gut, and liver have been studied in relation to the reproductive events in an annual cycle. The purpose of current review is to bring those fascinating, and arguably most important data together to underline their significance in the control of seasonal reproduction in subtropical fish in general and in carp in particular.     

Improved Culture Media for Embryogenic Callus Generation in Sorghum [<i>Sorghum bicolor</i> (L.) Moench]

Many attempts on optimization of sorghum [Sorghum bicolor (L.) Moench] tissue culture induction media have been made, but the culture system remains with some bottlenecks compared to that of other crops. This study aimed at assessing the suitability of various induction media to produce embryogenic callus (yellow and friable) with high induction rates and reduced phenolic exudation. The six culture medium modifications: 3 based on Murashige and Skoog (MS) medium and one each based on Chu N6, Gamborg B5 and 190-2 media respectively were applied in the culture of mature embryos from 10 sorghum genotypes. Although there was a genotype influence on the attainment of a yellow callus, friability of the callus was determined to be dependent on the culture medium and not the genotype. Half strength MS medium with 0.2 mg/l 2,4-D with 2.8 g/l Gelrite® as the gelling agent modified with 1.0 g/l KH₂PO₄, 1.0 g/l L-proline, 1.0 g/l L-asparagine and 0.16 mg/l CuSO₄·5H₂O (type E) was found to be the most effective resulting in about 60% yellow coloured callus induction with 25% friability. Addition of CuSO₄·5H₂O, KH₂PO₄, L-proline and L-asparagine significantly reduced the phenolic production. Half strength MS medium was observed to contribute to quality callus production when compared to full strength MS media modified with the compounds. The half strength MS medium was also observed to suppress phenolic production. Medium 190-2 produced the highest regeneration frequency (40%) among the 3-regeneration media tested. The results provide information on a suitable sorghum callus induction medium necessary for embryogenesis.     

Curcumin stably interacts with DNA hairpin through minor groove binding and demonstrates enhanced cytotoxicity in combination with FdU nucleotides

We report, based on biophysical studies and molecular mechanical calculations that curcumin binds DNA hairpin in the minor groove adjacent to the loop region forming a stable complex. UV–Vis and fluorescence spectroscopy indicated interaction of curcumin with DNA hairpin. In this novel binding motif, two ? H of curcumin heptadiene chain are closely positioned to the A₁₆-H8 and A₁₇-H8, while G₁₂-H8 is located in the close proximity of curcumin α H. Molecular dynamics (MD) simulations suggest, the complex is stabilized by noncovalent forces including; π-π stacking, H-bonding and hydrophobic interactions. Nuclear magnetic resonance (NMR) spectroscopy in combination with molecular dynamics simulations indicated curcumin is bound in the minor groove, while circular dichroism (CD) spectra suggested minute enhancement in base stacking and a little change in DNA helicity, without significant conformational change of DNA hairpin structure. The DNA:curcumin complex formed with FdU nucleotides rather than Thymidine, demonstrated enhanced cytotoxicity towards oral cancer cells relative to the only FdU substituted hairpin. Fluorescence co-localization demonstrated stability of the complex in biologically relevant conditions, including its cellular uptake. Acridine orange/EtBr staining further confirmed the enhanced cytotoxic effects of the complex, suggesting apoptosis as mode of cell death. Thus, curcumin can be noncovalently complexed to small DNA hairpin for cellular delivery and the complex showed increased cytotoxicity in combination with FdU nucleotides, demonstrating its potential for advanced cancer therapy.     

Designing novel inhibitors against falcipain-2 of Plasmodium falciparum

Coumarin containing pyrazoline derivatives have been synthesized and tested as inhibitors of in vitro development of a chloroquine-sensitive (MRC-02) and chloroquine-resistant (RKL-2) strain of Plasmodium falciparum and in vivo Plasmodium berghei malaria. Docking study was also done on cysteine protease falcipain-2 which showed that the binding pose of C-14 molecule and epoxysuccinate, inhibitor of falcipain-2, binds in the similar pattern. The most active antimalarial compound was 3-(1-benzoyl-5-(4-flurophenyl)-4,5-dihydro-1H-pyrazol-3yl)-7-(diethyamino)-2H-chromen-2-one C-14, with an IC₅₀ of 4.21?µg/ml provided complete protection to the infected mice at 24?mg/kg X 4?days respectively.     

Induction of endothelial apoptosis by 4-hydroxyhexenal.

Lipid peroxidation and its products such as 4-hydroxy-2-nonenal (HNE) and 4-hydroxyhexenal (HHE) are known to affect redox balance during aging and various degenerative processes, including vascular dysfunction. Deterioration of the endothelial cells that line the vascular wall is known to be an underlying cause of vascular dysfunction. At present, little is known about the mechanism by which HHE induces endothelial cell death (i.e. apoptosis), although HNE-induced apoptotic cell death has been reported. The aim of this study was to determine whether apoptosis induced by HHE in endothelial cells involves peroxynitrite (ONOO(-)). Our results show that in endothelial cells HHE triggers apoptotic cell death by inducing apoptotic Bax coupled with a decrease in anti-apoptotic Bcl-2. Results show that HHE induces reactive oxygen species (ROS), nitric oxide, and ONOO(-) generation, leading to redox imbalance. Furthermore, the antioxidant N-acetyl cysteine, ROS scavenger, and penicillamine, an ONOO(-) scavenger, were found to block HHE-mediated apoptosis. We used confocal laser microscopy to estimate the ability of these inhibitors to attenuate HHE-induced intracellular ONOO(-) levels thus confirming the oxidative mediation of apoptosis in endothelial cells. These findings strongly suggest that accumulated HHE triggers reactive species-mediated endothelial apoptosis, leading to vascular dysfunction as well as vascular aging. During aging, increased lipid peroxidation and its associated production of HHE may exacerbate the weakened redox balance, leading to various chronic degenerative processes including vascular dysfunction.