Modulation of Intracellular Calcium Levels by Calcium Lactate Affects Colon Cancer Cell Motility through Calcium-Dependent Calpain

Cancer cell motility is a key phenomenon regulating invasion and metastasis. Focal adhesion kinase (FAK) plays a major role in cellular adhesion and metastasis of various cancers. The relationship between dietary supplementation of calcium and colon cancer has been extensively investigated. However, the effect of calcium (Ca²⁺) supplementation on calpain-FAK-motility is not clearly understood. We sought to identify the mechanism of FAK cleavage through Ca²⁺ bound lactate (CaLa), its downstream signaling and role in the motility of human colon cancer cells. We found that treating HCT116 and HT-29 cells with CaLa immediately increased the intracellular Ca²⁺ (iCa²⁺) levels for a prolonged period of time. Ca²⁺ influx induced cleavage of FAK into an N-terminal FAK (FERM domain) in a dose-dependent manner. Phosphorylated FAK (p-FAK) was also cleaved in to its p-N-terminal FAK. CaLa increased colon cancer cells motility. Calpeptin, a calpain inhibitor, reversed the effects of CaLa on FAK and pFAK cleavage in both cancer cell lines. The cleaved FAK translocates into the nucleus and modulates p53 stability through MDM2-associated ubiquitination. CaLa-induced Ca²⁺ influx increased the motility of colon cancer cells was mediated by calpain activity through FAK and pFAK protein destabilization. In conclusion, these results suggest that careful consideration may be given in deciding dietary Ca²⁺ supplementation to patient undergoing treatment for metastatic cancer.     

Simultaneous saccharification and protein enrichment fermentation of sugar beet pulp

Summary A product with 40 % protein content was obtained from sugar beet pulp (1.25–2.0 mm) in 48 h one stage (simultaneous) saccharification/fermentation process under optimized conditions using a specific enzyme mixture andCandida tropicalis strain, also saving about 40 % enzymes in comparison to a 2-stage process.     

Selection of preculture conditions for solid state fermentation of sugar beet pulp

Summary For the protein upgrading of sugar-beet pulp in solid state fermentation byTrichoderma reesei andFusarium oxysporum, serveral conditions were studied to prepare an economical preculture for large scale process. The best performance was shown by a preculture obtained in 24 h from 1.5 % molasses solution at pH 4.5–5.0 with 1.0 % milled beet pulp.     

Interrelation of active oxygen species,membrane damage and altered calcium functions

Incubation of freshly isolated rat liver mitochondria in the presence of oxygen free radical generating hypoxanthine —xanthine oxidase system led to swelling of mitochondria as measured by the change in optical density, which was reversed by the addition of superoxide dismutase. O₂ ^– in the presence of CaCl₂ enhanced the peroxidative decomposition of mitochondrial membrane lipids along with swelling of the organelle. Free radical generation led to enhancement of monoamine oxidase activity while glutathione peroxidase and cytochrome c oxidase were inhibited. Tertbutyl hydroperoxide (t-BHP) caused mitochondrial swelling through oxidative stress. Incorporation of ruthenium red, which is a Ca²⁺ transport blocker, during assay abolished peroxidative membrane damage and swelling. Dithiothreitol (DTT) accorded protection against t-BHP induced mitochondrial swelling. The above in vitro data suggest a possible interrelationship of active oxygen species, membrane damage and calcium dynamics.     

Changes in glucose metabolism from discrete regions of rat brain and its relationship to reproductive failure during experimental diabetes

This study reports the effects of alloxan induced diabetes on glucose metabolism enzymes viz. Hexokinase, Lactate dehydrogenase, and Glucose-6-phosphate dehydrogenase from discrete brain regions. Enzymes activity was assayed from hypothalamic areas such as medial preoptic area and median eminence-arcuate region which have gonadotropin releasing hormone cell bodies and their terminals, respectively and other brain regions like septum, amygdala, hippocampus, and thalamus. In all the areas studied, induction of diabetes resulted in a significant decrease in particulate bound HK activity, whereas soluble HK, LDH and G6PDH activity showed increase at 3, 8, 15 and 28 days intervals. Insulin treatment of diabetic rats led to recovery in enzyme activity. Blood glucose levels increased significantly after induction of diabetes and recovery was seen after insulin treatment. The present results suggest that altered cerebral glucose metabolism may also be responsible for reproductive failure observed in diabetic rats. (Mol Cell Biochem141: 97–102, 1994)     

Increase in Lysolecithin Content of Brain Microsomes in Phenobarbitone-Administered Rats and Its Relation to Lipid Peroxidation

Administration of phenobarbitone caused a marked increase in the capacity of rat brain microsomes to produce thiobarbituric acid-reactive substances in vitro. Enzymatic peroxidation of lipids was more affected than the nonenzymatic processes occurring in heat-inactivated preparations. Analysis of the phospholipid profile showed a drastic decrease in phosphatidylcholine and total phospholipid contents in the exposed animals, but about a fivefold increase in the lysophosphatidylcholine fraction. Data for in vivo incorporation of [14C]choline showed a similar pattern of high radioactivity in lysolecithin. The increase in lipid peroxidation could be related to the higher level of lysolecithin and the accompanying structural and functional changes in microsomes resulting from the neurotoxic effects of phenobarbitone.     

Ubiquitin‐Activated Interaction Traps (UBAITs) identify E3 ligase binding partners

We describe a new class of reagents for identifying substrates, adaptors, and regulators of HECT and RING E3s. UBAITs (Ub iquitin‐A ctivated I nteraction T raps) are E3‐ubiquitin fusion proteins and, in an E1‐ and E2‐dependent manner, the C‐terminal ubiquitin moiety forms an amide linkage to proteins that interact with the E3, enabling covalent co‐purification of the E3 with partner proteins. We designed UBAITs for both HECT (Rsp5, Itch) and RING (Psh1, RNF126, RNF168) E3s. For HECT E3s, trapping of interacting proteins occurred in vitro either through an E3 thioester‐linked lariat intermediate or through an E2 thioester intermediate, and both WT and active‐site mutant UBAITs trapped known interacting proteins in yeast and human cells. Yeast Psh1 and human RNF126 and RNF168 UBAITs also trapped known interacting proteins when expressed in cells. Human RNF168 is a key mediator of ubiquitin signaling that promotes DNA double‐strand break repair. Using the RNF168 UBAIT, we identify H2AZ—a histone protein involved in DNA repair—as a new target of this E3 ligase. These results demonstrate that UBAITs represent powerful tools for profiling a wide range of ubiquitin ligases.     

An amperometric biosensor based on laccase immobilized onto MnO2NPs/cMWCNT/PANI modified Au electrode

A method is described for construction of an amperometric biosensor for detection of phenolic compounds based on covalent immobilization of laccase (Lac) onto manganese dioxide nanoparticles (MnO(2)NPs) decorated carboxylated multiwalled carbon nanotubes (cMWCNTs)/PANI composite electrodeposited onto a gold (Au) electrode through N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS) chemistry. The modified electrode was characterized by scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The biosensor showed optimum response at pH 5.5 (0.1M sodium acetate buffer) and 35°C, when operated at 0.3 V vs. Ag/AgCl. Linear range, response time, detection limit were 0.1-10 μM (lower concentration range) and 10-500 μM (higher concentration range), 4s and 0.04 μM, respectively. Biosensor measured total phenolic content in tea leaves extract. The enzyme electrode was used 150 times over a period of 5 months.