The endothelial-specific regulatory mutation, Mvwf1, is a common mouse founder allele

Mvwf1 is a cis-regulatory mutation previously identified in the RIIIS/J mouse strain that causes a unique tissue-specific switch in the expression of an N-acetylgalactosaminyltransferase, B4GALNT2, from intestinal epithelium to vascular endothelium. Vascular B4galnt2 expression results in aberrant glycosylation of von Willebrand Factor (VWF) and accelerated VWF clearance from plasma. We now report that 13 inbred mouse strains share the Mvwf1 tissue-specific switch and low VWF phenotype, including five wild-derived strains. Genomic sequencing identified a highly conserved 97-kb Mvwf1 haplotype block shared by these strains that encompasses a 30-kb region of high nucleotide sequence divergence from C57BL6/J flanking B4galnt2 exon 1. The analysis of a series of bacterial artificial chromosome (BAC) transgenes containing B4galnt2 derived from the RIIIS/J or C57BL6/J inbred mouse strains demonstrates that the corresponding sequences are sufficient to confer the vessel (RIIIS/J) or intestine (C57BL6/J)-specific expression patterns. Taken together, our data suggest that the region responsible for the Mvwf1 regulatory switch lies within an approximately 30-kb genomic interval upstream of the B4galnt2 gene. The observation that Mvwf1 is present in multiple wild-derived strains suggests that this locus may be retained in wild mouse populations due to positive selection. Similar selective pressures could contribute to the high prevalence of von Willebrand disease in humans

Comparative functional analysis of aquaporins/glyceroporins in mammals and anurans

Maintenance of fluid homeostasis is critical to establishing and maintaining normal physiology. The landmark discovery of membrane water channels (aquaporins; AQPs) ushered in a new area in osmoregulatory biology that has drawn from and contributed to diverse branches of biology, from molecular biology and genomics to systems biology and evolution, and from microbial and plant biology to animal and translational physiology. As a result, the study of AQPs provides a unique and integrated backdrop for exploring the relationships between genes and genome systems, the regulation of gene expression, and the physiologic consequences of genetic variation. The wide species distribution of AQP family members and the evolutionary conservation of the family indicate that the control of membrane water flux is a critical biological process. AQP function and regulation is proving to be central to many of the pathways involved in individual physiologic systems in both mammals and anurans. In mammals, AQPs are essential to normal secretory and absorptive functions of the eye, lung, salivary gland, sweat glands, gastrointestinal tract, and kidney. In urinary, respiratory, and gastrointestinal systems, AQPs are required for proper urine concentration, fluid reabsorption, and glandular secretions. In anurans, AQPs are important in mediating physiologic responses to changes in the external environment, including those that occur during metamorphosis and adaptation from an aquatic to terrestrial environment and thermal acclimation in anticipation of freezing. Therefore, an understanding of AQP function and regulation is an important aspect of an integrated approach to basic biological research

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Studies on the effects of saccharin on synaptic transmission in the hippocampus

Saccharin has been shown to block long-term potentiation (LTP) of the hippocampal CA1 neuronal excitatory postsynaptic potentials (EPSPs) (Chirwa, 1988; Morishita et. al.. 1992). The mechanisms involved in this action were, however, unknown. The present electrophysiological investigation on guineapig hippocampal slices was undertaken to determine whether saccharin interfered with LTP by modulating the excitatory and the inhibitory synaptic transmission or the excitability of the CA1b, neurons. Application of 10 mM saccharin for 10 minutes did not alter the field or intracellular EPSPs in CA1b neurons elicited by low frequency stimulation of the stratum radiatum but prevented LTP of the EPSPs following a brief tetanic stimulation of the afferents. A post-tetanic application of saccharin did not prevent LTP from developing, indicating that the induction and not the maintenance of LTP was blocked by the drug. This agent also inhibited LTP induced by pairing sustained postsynaptic depolarization with low frequency activation of the stratum radiatum. Saccharin, at the concentrations that blocked LTP, did not alter the membrane potential or input resistance of the neurons. Since the induction of LTP appears to require the activation of N-methyl- D-aspartate (NMDA) receptors (Collingridge et. al.. 1983) and is modulated by activation of the A and B subtypes of y-aminobutyric acid (GABA) receptors (Wigstrom and Gustafsson, 1988; Davies et. al.. 1991). a variety of intracellular experiments were conducted to determine whether the blockade of LTP by saccharin was the result of the drug acting on these receptor systems. The depolarization of CAlb neurons produced by a tetanic stimulation i n normal medium or by brief applications of NMDA in either a Mg2+-free medium or a Mg2+-free medium containing 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a drug that antagonizes non-NMDA glutamate receptors, was not significantly altered in the presence of saccharin. Moreover, the slope and the height of the intracellular EPSP evoked in a Mg2+-free medium containing CNQX as well as in a normal medium containing 2-amino-5-phosphonovalerate (APV), a drug that antagonizes NMDA receptors, were also not significantly altered by the drug. These results suggested that saccharin blocked the induction of LTP by mechanisms that did not involve a blockade of the NMDA and the non-NMDA glutamate receptors. "Input-output" (I-O) curves constructed from the EPSPs and the inhibitory postsynaptic potentials (IPSPs) revealed that saccharin selectively increased the height of the IPSP. Pharmacological separation of the IPSP into its GABA receptor-mediated fast, and GABAg receptor-mediated slow components revealed that saccharin significantly increased the duration and height of the fast IPSP but decreased the height of the slow IPSP. In neurons injected with QX-314 (to block the postsynaptic GABAB receptor-mediated IPSPs), paired-pulse depression of the fast IPSP evoked in a CNQX and APV containing medium was not significantly altered in the presence of saccharin, suggesting that the drug did not interfere with the presynaptic GABAB receptors. Saccharin prevented LTP of the field and intracellular EPSP when the fast IPSP was blocked by picrotoxinin, suggesting that the alteration of the fast IPSP by saccharin was not responsible for the ability of the drug to block LTP. Taken togther, the results from the present study suggest that saccharin blocks the induction of hippocampal LTP at a step beyond the activation of the NMDA and non-NMDA glutamate receptors. Actions of this agent on the GABAA and GABAB receptor-mediated responses also appear not to be responsible for its LTP-blocking action. It is possible that saccharin might have interfered with LTP-inducing growth-related substances (Chirwa and Sastiy, 1986; Morishita et. al.. 1992; Sastiy et. al.. 1988a; Sastiy et. al.. 1988b; Xle et. al.. 1991) or with intracellular facilitators of LTP.Medicine, Faculty ofAnesthesiology, Pharmacology and Therapeutics, Department ofGraduat

Studies on GABAergic synaptic transmission in neurons of the deep cerebellar nuclei

In the cerebellum, the corticonuclear projection subserves as the major efferent pathway for the cerebellar cortical networks. This pathway, consists of a direct axonal projection from the Purkinje cells to the neurons of the deep cerebellar nuclei (DCN). It has been demonstrated both in vivo and in vitro that stimulation of the Purkinje cell axons exerts a powerful inhibitory influence on DCN neurons mediated by the neurotransmitter, ƴ-aminobutyric acid (GABA). However, despite the wealth of anatomical and biochemical information, few electrophysiological studies have been done to characterize GABAergic synaptic transmission in DCN neurons. For example, it is not clear whether synaptic release of GABA activates pre- or postsynaptic GABAB receptors despite the finding that GABAB binding sites are present in the DCN. Furthermore, although GABAergic transmission in the DCN exhibits paired-pulse, frequency-dependent, as well as long-term depressions, the mechanisms underlying these plasticity's are yet to be resolved. In the present study, both perforated and whole-cell patch clamp recording techniques were utilized to determine whether preand postsynaptic GABAB receptors are present in the DCN and to test if endogenous release of GABA can activate either of the receptors. In addition, the contribution of GABAB receptors to paired-pulse and frequency-dependent depression of the deep nuclear inhibitory postsynaptic current (IPSC) was also assessed. Finally, experiments were conducted to investigate the properties of a tetanic stimulation-induced deep nuclear long-term depression (LTD) of the IPSC and to examine the role of Ca^2+ and protein phosphatases as potential mediators of the sustained depression. The results of the studies indicated that postsynaptic GABAB receptors are present on the membrane of DCN neurons. Activation of these receptors produces a G-protein-dependent response similar to that observed in other central neurons. In addition, presynaptic GABAB receptors are also present in the DCN. Activation of these receptors produces a suppression of deep nuclear IPSCs. However, deep nuclear preand postsynaptic GABAB receptors were found not to be activated by endogenous release of GABA. Furthermore, these receptors appear not to be involved in pairedpulse and frequency-dependent depressions of the IPSC. In voltage-clamped DCN neurons, LTD of the IPSC was induced reliably if the LTD-inducing train was delivered under current-clamp conditions where the membrane potential was allowed to fluctuate. Using this protocol in subsequent experiments, it was found that currents elicited by iontophoretic applications of THIP, a GABAA agonist, also exhibited LTD following a tetanic stimulation of the input. It was also demonstrated that LTD can be induced heterosynaptically. Furthermore, activation of the IPSC during the train was not required for LTD to occur. However, postsynaptic Ca^2+ accumulations via influx though A/-methyl-D-aspartate receptor-gated channels and/or voltage-gated Ca^2+ channels appear to play an important role in the generation of LTD. Moreover, protein phosphatase activity appears to be necessary for the induction of the depression. It is concluded that postsynaptic mechanisms contribute to LTD of GABAergic transmission in neurons of the DCN. Bhagavatula R. Sastry, Ph.D., Research Supervisor.Pharmaceutical Sciences, Faculty ofGraduat