Topology of a human equilibrative, nitrobenzylthioinosine (NBMPR)-sensitive nucleoside transporter (hENT1) implicated in the cellular uptake of adenosine and anti-cancer drugs

The human equilibrative nucleoside transporter hENT1, the first identified member of the ENT family of integral membrane proteins, is the primary mechanism for the cellular uptake of physiologic nucleosides, including adenosine, and many anti-cancer nucleoside drugs. We have produced recombinant hENT1 in Xenopus oocytes and used native and engineered N-glycosylation sites in combination with immunological approaches to experimentally define the membrane architecture of this prototypic nucleoside transporter. hENT1 (456 amino acid residues) is shown to contain 11 transmembrane helical segments with an amino terminus that is intracellular and a carboxyl terminus that is extracellular. Transmembrane helices are linked by short hydrophilic regions, except for a large glycosylated extracellular loop between transmembrane helices 1 and 2 and a large central cytoplasmic loop between transmembrane helices 6 and 7. Sequence analyses suggest that this membrane topology is common to all mammalian, insect, nematode, protozoan, yeast, and plant members of the ENT protein family.     

Expression of the gene for large subunit of m-calpain is elevated in skeletal muscle from Duchenne muscular dystrophy patients

Calpain is an intracellular nonlysosomal protease involved in essential regulatory or processing functions of the cell, mediated by physiological concentrations of Ca²⁺. However, in an environment of abnormal intracellular calcium, such as that seen in Duchenne muscular dystrophy (DMD), calpain is suggested to cause degeneration of muscle owing to enhanced activity. To test whether the reported increase in calpain activity in DMD results fromde novo synthesis of the protease, we have assessed the quantitative changes in mRNA specific for m-calpain. mRNA isolated from DMD and control muscle was analysed by dot blot hybridization using a cDNA probe for the large subunit of m-calpain. Compared to control a four-fold increase in specific mRNA was observed in dystrophic muscle. This enhanced expression of the m-calpain gene in dystrophic condition suggests that the reported increase in m-calpain activity results fromde novo synthesis of protease and underlines the important role of m-calpain in DMD.     

Warfarin administration reduces synthesis of sulfatides and other sphingolipids in mouse brain

The modulation of phosphosphingolipid synthesis by vitamin K depletion has been observed in the vitamin K-dependent microorganism, Bacteriodes levii. When cultured briefly without the vitamin, a reduction occurred in the activity of the first enzyme of the sphingolipid pathway, 3-ketodihydrosphingosine synthase. In this report, 16-day-old mice were treated with the vitamin K antagonist, warfarin. Brain microsomes from these animals showed a 19% reduction in synthase activity. Mice treated with warfarin for 2 weeks showed a major reduction in sulfatide level (42%), with a lesser degree or no reduction in levels of gangliosides and cerebrosides. In further experiments, mice were treated with warfarin for 2 weeks and a group was then injected with vitamin K1 (aquamephyton) for 3 days. Enzyme activity returned to a normal level within 2-3 days. Sulfatide levels had increased 33% in the vitamin K-injected group and ganglioside levels also increased, where levels of cerebrosides and sphingomyelin declined. Sulfatide synthesis determined by [35S] sulfate incorporation, showed a 52% increase in incorporation following administration of vitamin K for 3 days. These results suggest a role for vitamin K in the biosynthesis of sulfatides and other sphingolipids in brain. This putative role could be by post-translational protein modification analogous to the role of vitamin K in other systems.     

Regulatory characteristics of phosphoenolpyruvate carboxylase from the extreme thermophile, Thermus aquaticus.

Phosphoenolpyruvate carboxylase from the extremely thermophilic bacterium, Thermus aquaticus YT-1, exhibits a virtually absolute requirement for acetyl CoA and there is strong positive cooperativity in the interaction of this activator with the enzyme. Several tricarboxylic acid cycle intermediates inhibit the enzyme. These findings suggest an anaplerotic role for the enzyme and an allosteric modulation of its activity by acetyl CoA and tricarboxylic acid cycle intermediates.     

Phagodeterrence and insecticidal activity of Hyptis suaveolens (Poit.) against four important lepidopteran pests

This study was carried out to evaluate the antifeedant and larvicidal activities of Hyptis suaveolens Poit. (Lamiaceae) leaves crude extracts and their fractions against four lepidopteran pests namely Helicoverpa armigera (Hbn.), Spodoptera litura (Fab.), Earias vittella (Fab.) and Leucinodes orbonalis (G.). Hexane, chloroform and ethyl acetate extracts were tested by leaf disc and fruit disc no-choice methods at 1% and 1,000?ppm concentrations for crude and fractions, respectively. Ethyl acetate extract of H. suaveolens exhibited the maximum antifeedant and insecticidal activity at 1% concentration against all the tested insects. It was subjected to fractionation using silica column chromatography with different combinations of hexane and ethyl acetate used as mobile phase. Among the 15 fractions obtained, fraction 2 showed the maximum antifeedant and insecticidal activity against all tested insects at 1000?ppm concentration. Preliminary phytochemical analysis of fraction 2 showed the presence of terpenoids and alkaloids. H. suaveolens could be considered as a safe and eco-friendly insecticide for lepidopteron pest management.     

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed “enteroids” when derived from small intestine and “colonoids” when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.