Potentiation of tumour promotion by topical application of argemone oil/isolated sanguinarine alkaloid in a model of mouse skin carcinogenesis

Several incidences of adverse effects on human health have been reported in many countries, due to consumption of edible oil adulterated with argemone oil (AO). The clinical manifestation of the disease is commonly referred to as epidemic dropsy. Our prior studies have shown that AO and isolated sanguinarine alkaloid (SANG) possess genotoxic and tumour initiating activity. In this study, the effect of AO/SANG was investigated on the development of tumour formation in mice using 7,12-dimethylbenz (a) anthracene (DMBA) initiated followed by tetradecanoyl phorbol acetate (TPA)-promoted skin tumour protocol. Single application of AO (300 μl) or SANG (4.5 μmol) when used during initiation phase in DMBA/TPA group did not reveal substantial difference in tumourigenic response. However, twice weekly application of AO (100 μl) or SANG (1.5 μmol) during promotion phase (25 weeks) resulted in enhanced tumourigenic response by ≥30% in DMBA/TPA treated group along with significant decrease in dermal tyrosinase (45–49%), histidase (30–32%), superoxide dismutase (53–56%), catalase (41%), GSH reductase (37–40%) and GSH-peroxidase activity (29–33%) compared to control. Furthermore, significant decrease of epidermal GSH (64–66%) content and enhanced formation of lipid peroxides (96–121%) was noticed following AO or SANG treatment during promotion phase to DMBA/TPA induced animals indicating the modified pro-oxidant status in skin. Although dermal biochemical parameters were also altered by AO or SANG when used during initiation phase in DMBA/TPA treated animals, nonetheless, the response in these parameters were relatively more when AO or SANG were used during promotion phase in DMBA/TPA treated animals. These results clearly suggest that AO and SANG have the ability to enhance the tumourigenic response, which may have relevance to its carcinogenic potential.     

Epidermal growth factor receptor and notch pathways participate in the tumor suppressor function of gamma-secretase

Gamma-secretase, a unique aspartyl protease, is required for the regulated intramembrane proteolysis of Notch and APP, pathways that are implicated, respectively, in the pathogenesis of cancer and Alzheimer disease. However, the mechanism whereby reduction of gamma-secretase causes tumors such as squamous cell carcinoma (SCC) remains poorly understood. Here, we demonstrate that gamma-secretase functions in epithelia as a tumor suppressor in an enzyme activity-dependent manner. Notch signaling is down-regulated and epidermal growth factor receptor (EGFR) is activated in SCC caused by genetic reduction of gamma-secretase. Moreover, the level of EGFR is inversely correlated with the level of gamma-secretase in fibroblasts, suggesting that the up-regulation of EGFR stimulates hyperproliferation in epithelia of mice with genetic reduction of gamma-secretase. Supporting this notion is our finding that the proliferative response of fibroblasts lacking gamma-secretase activity is more sensitive when challenged by either EGF or an inhibitor of EGFR as ompared with wild type cells. Interestingly, the up-regulation of EGFR is independent of Notch signaling, suggesting that the EGFR pathway functions in parallel with Notch in the tumorigenesis of SCC. Collectively, our results establish a novel mechanism linking the EGFR pathway to the tumor suppressor role of gamma-secretase and that mice with genetic reduction of gamma-secretase represent an excellent rodent model for clarifying pathogenesis of SCC and for testing therapeutic strategy to ameliorate this type of human cancer.     

A New Target for Amyloid Beta Toxicity Validated by Standard and High-Throughput Electrophysiology

Background

Soluble oligomers of amyloid beta (Aβ) are considered to be one of the major contributing factors to the development of Alzheimer''s disease. Most therapeutic development studies have focused on toxicity directly at the synapse.

Methodology/Principal Findings

Patch clamp studies detailed here have demonstrated that soluble Aβ can also cause functional toxicity, namely it inhibits spontaneous firing of hippocampal neurons without significant cell death at low concentrations. This toxicity will eventually lead to the loss of the synapse as well, but may precede this loss by a considerable amount of time. In a key technological advance we have reproduced these results utilizing a fast and simple method based on extracellular electrophysiological recording of the temporal electrical activity of cultured hippocampal neurons using multielectrode arrays (MEAs) at low concentrations of Aβ (1–42). We have also shown that this functional deficit can be reversed through use of curcumin, an inhibitor of Aβ oligomerization, using both analysis methods.

Conclusions/Significance

The MEA recording method utilized here is non-invasive, thus long term chronic measurements are possible and it does not require precise positioning of electrodes, thus it is ideal for functional screens. Even more significantly, we believe we have now identified a new target for drug development for AD based on functional toxicity of hippocampal neurons that could treat neurodegenerative diseases prior to the development of mild cognitive impairment.     

Threonine-insensitive Homoserine Dehydrogenase from Soybean: GENOMIC ORGANIZATION, KINETIC MECHANISM, AND IN VIVO ACTIVITY*

Aspartate kinase (AK) and homoserine dehydrogenase (HSD) function as key regulatory enzymes at branch points in the aspartate amino acid pathway and are feedback-inhibited by threonine. In plants the biochemical features of AK and bifunctional AK-HSD enzymes have been characterized, but the molecular properties of the monofunctional HSD remain unexamined. To investigate the role of HSD, we have cloned the cDNA and gene encoding the monofunctional HSD (GmHSD) from soybean. Using heterologously expressed and purified GmHSD, initial velocity and product inhibition studies support an ordered bi bi kinetic mechanism in which nicotinamide cofactor binds first and leaves last in the reaction sequence. Threonine inhibition of GmHSD occurs at concentrations (K_i = 160–240 mm) more than 1000-fold above physiological levels. This is in contrast to the two AK-HSD isoforms in soybean that are sensitive to threonine inhibition (K_i∼150 μm). In addition, GmHSD is not inhibited by other aspartate-derived amino acids. The ratio of threonine-resistant to threonine-sensitive HSD activity in soybean tissues varies and likely reflects different demands for amino acid biosynthesis. This is the first cloning and detailed biochemical characterization of a monofunctional feedback-insensitive HSD from any plant. Threonine-resistant HSD offers a useful biotechnology tool for manipulating the aspartate amino acid pathway to increase threonine and methionine production in plants for improved nutritional content.     

AFLP-Based Genetic Diversity Assessment of Commercially Important Tea Germplasm in India

India has a large repository of important tea accessions and, therefore, plays a major role in improving production and quality of tea across the world. Using seven AFLP primer combinations, we analyzed 123 commercially important tea accessions representing major populations in India. The overall genetic similarity recorded was 51%. No significant differences were recorded in average genetic similarity among tea populations cultivated in various geographic regions (northwest 0.60, northeast and south both 0.59). UPGMA cluster analysis grouped the tea accessions according to geographic locations, with a bias toward China or Assam/Cambod types. Cluster analysis results were congruent with principal component analysis. Further, analysis of molecular variance detected a high level of genetic variation (85%) within and limited genetic variation (15%) among the populations, suggesting their origin from a similar genetic pool.     

High level expression of soybean trypsin inhibitor gene in transgenic tobacco plants failed to confer resistance against damage caused byHelicoverpa armigera

Helicoverpa armigera is a major pest of many tropical crop plants. Soybean trypsin inhibitor (SBTI) was highly effective against the proteolytic activity of gut extract of the insect. SBTI was also inhibitory to insect growth when present in artificial diet. The gene coding for SBTI was cloned from soybean (Glycine max, CVBirsa) and transferred to tobacco plants for constitutive expression. Young larvae ofH. armigera, fed on the leaves of the transgenic tobacco plants expressing high level of SBTI, however, maintained normal growth and development. The results suggest that in certain cases the trypsin inhibitor gene(s) may not be suitable candidates for developing insect resistant transgenic plants.     

Mechanisms of curcumin-induced apoptosis of Ehrlich's ascites carcinoma cells.

Curcumin, the active ingredient from the spice turmeric (Curcuma longa Linn), is a potent antioxidant and anti-inflammatory agent. It has been recently demonstrated to possess discrete chemopreventive activities. However, the molecular mechanisms underlying such anticancer properties of curcumin still remain unrealized, although it has been postulated that induction of apoptosis in cancer cells might be a probable explanation. In the current study, curcumin was found to decrease the Ehrlich's ascites carcinoma (EAC) cell number by the induction of apoptosis in the tumor cells as evident from flow-cytometric analysis of cell cycle phase distribution of nuclear DNA and oligonucleosomal fragmentation. Probing further into the molecular signals leading to apoptosis of EAC cells, we observed that curcumin is causing tumor cell death by the up-regulation of the proto-oncoprotein Bax, release of cytochrome c from the mitochondria, and activation of caspase-3. The status of Bcl-2 remains unchanged in EAC, which would signify that curcumin is bypassing the Bcl-2 checkpoint and overriding its protective effect on apoptosis.     

Studies on the interaction of anesthetic steroids with phosphatidylcholine using 2H and 13C solid state NMR

The effects of the anesthetic steroid alphaxalone and its inactive analog delta 16-alphaxalone on model phospholipid membranes were studied using 13C and 2H solid-state nuclear magnetic resonance spectroscopy. Aqueous multilamellar dispersions of dipalmitoylphosphatidylcholine (DPPC) with specific 13C and 2H labels as endogenous probes at the carbonyl and the C-7 methylene groups, respectively, of the sn-2 chain were used to study the conformational and dynamical properties of the bilayer as a function of temperature. There were no significant changes between the 13C and 2H spectra of the DPPC preparation containing the inactive steroid and that of DPPC with no drug. However, the physiologically active steroid produces significant spectral 2H and 13C changes. These changes include a reduction of the main phase transition temperature and a broadening of that transition. Alphaxalone also increases the relative number of gauche conformers in the liquid-crystalline phase of DPPC and increases the rate of axial diffusion in both the gel and liquid-crystalline phase. The thermotropic properties of the above preparations, as monitored by differential scanning calorimetry, were congruent with the spectroscopic data.     

Fatty acid-induced angiogenesis in first trimester placental trophoblast cells: Possible roles of cellular fatty acid-binding proteins

Angiogenesis is involved in the growth of new blood vessels from the existing one. Consequently, angiogenesis plays an indispensable role in tissue growth and repair including early placentation processes. Besides angiogenic growth factors (vascular endothelial growth factor (VEGF), angiopoietin-like 4 (ANGPTL4), placental growth factor (PlGF), platelet derived growth factor (PDGF), fibroblast growth factors (FGF)), dietary fatty acids (c>16) also directly or indirectly modulate angiogenic processes in tumors and other cell systems. Usually n − 3 fatty acids inhibit whereas n − 6 fatty acids stimulate angiogenesis in tumors and other cells. Contrary to this, docosahexaenoic acid, 22:6n − 3 (DHA) and other fatty acids including conjugated linoleic acid stimulate angiogenesis in placental first trimester cells. In addition to the stimulation of expression of major angiogenic factors such as VEGF and ANGPTL4, fatty acids also stimulate expression of intracellular fatty acid-binding proteins (FABPs) FABP-4 and FABP-3 those are known to directly modulate angiogenesis. Emerging data indicate that FABPs may be involved in the angiogenesis process. This paper reviews the fatty acid mediated angiogenesis process and the involvement of their binding proteins in these processes.     

Survivability of parthenogenetic embryos following in vivo transfer in naturally synchronized Capra hircus

The present study was conducted to see the in vivo developmental potency of caprine parthenogenetic embryos generated in a modified way. The good quality caprine oocytes were matured in presence of cytochalasin B (CCB) and then activated by 7% ethanol followed by 2 mM 6-dimethyl amino purine (6-DMAP) and embryo development was recorded. Early stage parthenogenetic embryos (two to four cells) were surgically transferred in recipients (10). The pregnancy diagnosis was done by nonreturn to oestrus, ultrasonography (USG), and progesterone estimation. The levels of progesterone were above normal values (1 ng/ml) of pregnancy and fall below the level of pregnancy just before retuned to oestrus. Progesterone profile revealed that out of ten recipients (G1–G10), four goats (G1, G2, G3, and G5) returned to oestrus after 43?±?7.29 (Mean?±?SE) d of embryo transfer and six goats (G4, G6, G7, G8, G9, and G10) did not return to cycle even after 70 d of embryo transfer. In three recipients (G4, G5, and G6), the USG on day 40 revealed that there was fluid filled uterine body with solid fetus-like structure. These might be dead fetus and had started resorption. The progesterone profile also corroborated the assumption of pregnancy in these animals. Authors believe that this may be the first report on in vivo diploid parthenogenetic embryo development in caprine species.