Dual effect of cannabinoid CB<Subscript>1</Subscript> receptor stimulation on a vanilloid VR1 receptor-mediated response

Cannabinoid CB1 receptors and vanilloid VR1 receptors are co-localized to some extent in sensory neurons of the spinal cord and dorsal root ganglia. In this study, we over-expressed both receptor types in human embryonic kidney (HEK)-293 cells and investigated the effect of the CB1 agonist HU-210 on the VR1-mediated increase in intracellular Ca2+ ([Ca2+]i), a well-known response of the prototypical VR1 agonist capsaicin. After a 5-min pre-treatment, HU-210 (0.1 microM) significantly enhanced the effect of several concentrations of capsaicin on [Ca2+]i in HEK-293 cells over-expressing both rat CB1 and human VR1 (CB1-VR1-HEK cells), but not in cells over-expressing only human VR1 (VR1-HEK cells). This effect was blocked by the CB1 receptor antagonist SR141716A (0.5 microM), and by phosphoinositide-3-kinase and phospholipase C inhibitors. The endogenous agonist of CB1 and VR1 receptors, anandamide, was more efficacious in inducing a VR1-mediated stimulation of [Ca2+]i in CB1-VR1-HEK cells than in VR1-HEK cells, and part of its effect on the former cells was blocked by SR141716A (0.5 microM). Pre-treatment of CB1-VR1-HEK cells with forskolin, an adenylate cyclase activator, enhanced the capsaicin effect on [Ca2+]i. HU-210, which in the same cells inhibits forskolin-induced enhancement of cAMP levels, blocked the stimulatory effect of forskolin on capsaicin. Our data suggest that in cells co-expressing both CB1 and VR1 receptors, pre-treatment with CB1 agonists inhibits or stimulates VR1 gating by capsaicin depending on whether or not cAMP-mediated signalling has been concomitantly activated.     

Glycoconjugates in sperm function and gamete interactions: how much sugar does it take to sweet-talk the egg?

Glycoconjugates in the mammalian reproductive tract are critical components of the molecular mechanisms that control sperm maturation, sperm transport and gamete interactions. In the oviduct of many species, sperm transport and maturation are regulated by protein-carbohydrate interactions that form a sperm reservoir. Subsequently, gamete interactions are mediated by the binding of lectin-like sperm proteins with carbohydrate moieties on the zona pellucida. The sperm glycocalyx is extensively modified during sperm transport and maturation. Multiple functions have been proposed for this dense carbohydrate layer overlying the sperm plasmalemma, and sperm-surface carbohydrates have been implicated in immune-mediated human infertility. The structure and function of glycoconjugates in the oviductal sperm reservoir, the zona pellucida, and on the sperm surface are reviewed.     

Human mitochondrial tRNAs in health and disease

The human mitochondrial genome encodes 13 proteins, all subunits of the respiratory chain complexes and thus involved in energy metabolism. These genes are translated by 22 transfer RNAs (tRNAs), also encoded by the mitochondrial genome, which form the minimal set required for reading all codons. Human mitochondrial tRNAs gained interest with the rapid discovery of correlations between point mutations in their genes and various neuromuscular and neurodegenerative disorders. In this review, emerging fundamental knowledge on the structure/function relationships of these particular tRNAs and an overview of the large variety of mechanisms within translation, affected by mutations, are summarized. Also, initial results on wide-ranging molecular consequences of mutations outside the frame of mitochondrial translation are highlighted. While knowledge of mitochondrial tRNAs in both health and disease increases, deciphering the intricate network of events leading different genotypes to the variety of phenotypes requires further investigation using adapted model systems.Received 3 December 2002; received after revision 14 January 2003; accepted 27 January 2003     

A cryo-electron microscopic study of ribosome-bound termination factor RF2

Protein synthesis takes place on the ribosome, where genetic information carried by messenger RNA is translated into a sequence of amino acids. This process is terminated when a stop codon moves into the ribosomal decoding centre (DC) and is recognized by a class-1 release factor (RF). RFs have a conserved GGQ amino-acid motif, which is crucial for peptide release and is believed to interact directly with the peptidyl-transferase centre (PTC) of the 50S ribosomal subunit. Another conserved motif of RFs (SPF in RF2) has been proposed to interact directly with stop codons in the DC of the 30S subunit. The distance between the DC and PTC is approximately 73 A. However, in the X-ray structure of RF2, SPF and GGQ are only 23 A apart, indicating that they cannot be at DC and PTC simultaneously. Here we show that RF2 is in an open conformation when bound to the ribosome, allowing GGQ to reach the PTC while still allowing SPF-stop-codon interaction. The results indicate new interpretations of accuracy in termination, and have implications for how the presence of a stop codon in the DC is signalled to PTC.     

A natural allele of Nxf1 suppresses retrovirus insertional mutations

Endogenous retroviruses have shaped the evolution of mammalian genomes. Host genes that control the effects of retrovirus insertions are therefore of great interest. The modifier-of-vibrator-1 locus (Mvb1) controls levels of correctly processed mRNA from genes mutated by endogenous retrovirus insertions into introns, including the Pitpn(vb) tremor mutation and the Eya1(BOR) model of human branchiootorenal syndrome. Positional complementation cloning identifies Mvb1 as the nuclear export factor Nxf1, providing an unexpected link between the mRNA export receptor and pre-mRNA processing. Population structure of the suppressive allele in wild Mus musculus castaneus suggests selective advantage. A congenic Mvb1(CAST) allele is a useful tool for modifying gene expression from existing mutations and could be used to manipulate engineered mutations containing retroviral elements.     

An epi-allelic series of p53 hypomorphs created by stable RNAi produces distinct tumor phenotypes in vivo

The application of RNA interference (RNAi) to mammalian systems has the potential to revolutionize genetics and produce novel therapies. Here we investigate whether RNAi applied to a well-characterized gene can stably suppress gene expression in hematopoietic stem cells and produce detectable phenotypes in mice. Deletion of the Trp53 tumor suppressor gene greatly accelerates Myc-induced lymphomagenesis, resulting in highly disseminated disease. To determine whether RNAi suppression of Trp53 could produce a similar phenotype, we introduced several Trp53 short hairpin RNAs (shRNAs) into hematopoietic stem cells derived from E(mu)-Myc transgenic mice, and monitored tumor onset and overall pathology in lethally irradiated recipients. Different Trp53 shRNAs produced distinct phenotypes in vivo, ranging from benign lymphoid hyperplasias to highly disseminated lymphomas that paralleled Trp53-/- lymphomagenesis in the E(mu)-Myc mouse. In all cases, the severity and type of disease correlated with the extent to which specific shRNAs inhibited p53 activity. Therefore, RNAi can stably suppress gene expression in stem cells and reconstituted organs derived from those cells. In addition, intrinsic differences between individual shRNA expression vectors targeting the same gene can be used to create an 'epi-allelic series' for dissecting gene function in vivo.