Structural and functional analysis of Saccharomyces cerevisiae Mob1

The Mob proteins function as activator subunits for the Dbf2/Dbf20 family of protein kinases. Human and Xenopus Mob1 protein structures corresponding to the most conserved C-terminal core, but lacking the variable N-terminal region, have been reported and provide a framework for understanding the mechanism of Dbf2/Dbf20 regulation. Here, we report the 2.0 A X-ray crystal structure of Saccharomyces cerevisiae Mob1 containing both the conserved C-terminal core and the variable N-terminal region. Within the N-terminal region, three novel structural elements are observed; namely, an alpha-helix denoted H0, a strand-like element denoted S0 and a short beta strand denoted S-1. Helix H0 associates in an intermolecular manner with a second Mob1 molecule to form a Mob1 homodimer. Strand S0 binds to the core domain in an intramolecular manner across a putative Dbf2 binding site mapped by Mob1 temperature-sensitive alleles and NMR binding experiments. In vivo functional analysis demonstrates that Mob1 mutants that target helix H0 or its reciprocal binding site are biologically compromised. The N-terminal region of Mob1 thus contains structural elements that are functionally important.     

Allosteric potentiators of the metabotropic glutamate receptor 2 (mGlu2). Part 2: 4-thiopyridyl acetophenones as non-tetrazole containing mGlu2 receptor potentiators

We have identified and synthesized a series of 4-thiopyridyl acetophenones as positive allosteric potentiators of the metabotropic glutamate receptor 2. Structure-activity relationship studies directed toward replacement of the tetrazole in the initial lead led to the discovery of 16 (EC(50)=340 nM), which showed improved brain penetration over the initial lead.     

Temperature regulation and microhabitat choice by free-ranging Galapagos fur seal pups (Arctocephalus galapagoensis)

Summary Behavioural activity, and core and surface temperatures of 4 unrestrained Galapagos fur seals were recorded in the natural habitat during their first weeks of life. Climatic variables were registered simultaneously. Pup behaviours were divided into bouts of resting (55% of total time), sucking (23%) and other activities (22%). Pups maintained a constant body temperature from their first day. Core temperature (T _e ) was 37.7° C±0.3° C (x ± SD) over 39 pup-days and 8 pup-nights. Skin temperature was correlated with T _c , but flipper temperature was not. No daily T _c rhythm was detected. Microclimate data were used to calculate operative temperature T _e . Environmental temperatures can be very high, with T _e above T _c 6–9 h a day for animals exposed to the sun, but below it in the shade. T _c is about 22° C at night. Pups avoid overheating mainly by withdrawing into the shade and reducing activity to a minimum during the hot hours of the day. Sun-exposed pups could be active at any time during the day if they had access to water, which was usually around high tide.     

Up-regulation of endogenous RGS2 mediates cross-desensitization between Gs and Gq signaling in osteoblasts

Regulator of G protein signaling (RGS) proteins limit G protein signals. In this study, we investigated the role of RGS2 in the control of G protein signaling cascades in osteoblasts, the cells responsible for bone formation. Expression of RGS2 was up-regulated in primary cultures of mouse calvarial osteoblasts by parathyroid hormone-related peptide (PTHrP)-(1-34), which stimulates G(s) signaling. RGS2 was also up-regulated by extracellular ATP, which selectively activates G(q), as well as by forskolin and phorbol myristate acetate, which activate targets downstream of G(s) and G(q), respectively. To assess the role of endogenous RGS2, we characterized G(s) and G(q) signaling in osteoblasts derived from wild type and rgs2(-/-) mice. Under control conditions, nucleotide-stimulated calcium release, endothelin-stimulated accumulation of inositol phosphates, and PTHrP-stimulated cAMP accumulation were equivalent in osteoblasts isolated from wild type and rgs2(-/-) mice. Thus, basal levels of endogenous RGS2 do not appear to regulate G(s) or G(q) signaling in osteoblasts. Interestingly, forskolin treatment of wild type but not rgs2(-/-) osteoblasts suppressed both endothelin-stimulated accumulation of inositol phosphates and nucleotide-stimulated calcium release, indicating that up-regulation of RGS2 by G(s) signaling desensitizes G(q) signals. Furthermore, pretreatment with ATP suppressed PTHrP-dependent cAMP accumulation in wild type but not rgs2(-/-) osteoblasts, implying that up-regulation of RGS2 by G(q) signaling desensitizes G(s) signals. Our findings demonstrate that endogenously expressed RGS2 can limit G(s) signaling. Moreover, up-regulation of RGS2 contributes to cross-desensitization of G(s)- and G(q)-coupled signals.     

The microevolution of the Galápagos marine iguana Amblyrhynchus cristatus assessed by nuclear and mitochondrial genetic analyses

Marine iguanas may have inhabited the Galápagos archipelago and its former, now sunken islands for more than 10 million years (Myr). It is therefore surprising that morphological and immunological data indicate little evolutionary divergence within the genus. We utilized mitochondrial DNA (mtDNA) sequence analyses and nuclear DNA fingerprinting to re-evaluate the level and pattern of genetic differentiation among 22 marine iguana populations from throughout the archipelago. Both genetic marker systems detect a low level of within-genus divergence, but they show contrasting levels of geographical subdivision among the populations. The mitochondrial gene pools of populations from different regions of the archipelago are isolated, and the mtDNA pattern appears to follow the sequence in which the islands were colonized by marine iguanas. Conversely, the nuclear DNA study indicates substantial interpopulational gene exchange, and the geographical distribution of the nuclear markers seems to be determined by isolation by distance among the populations. The natural history of marine iguanas suggests that the contrasting nuclear and mitochondrial DNA patterns result from an asymmetric migration behaviour of the two sexes, with higher (active and passive) interisland dispersal for males than females. Separate genetic analyses for the sexes appear to support this hypophesis. Based on these findings, a scenario is proposed that explains the marine iguanas' low genetic divergence, notwithstanding their long evolutionary history in the Galápagos archipelago.     

Spacing behaviour and its implications for the mating system of a precocial small mammal: an almost asocial cavyCavia magna ?

Socioecological models for small mammals attempt to explain the causal relations between the spatiotemporal distributions of food resources, females and males. We tested their predictions for a wild population of Cavia magna, a grazing, precocial rodent, by analysing spacing behaviour in relation to various demographic features. Between May 1999 and January 2001 we collected capture-recapture data on 309 individuals and monitored 55 females and 49 males by radiotelemetry in periodically inundated wetland in Uruguay. Cavies showed a nonstationary use of space: monthly home ranges drifted over the whole study site. Female home ranges overlapped with those of several others. Females were randomly distributed and we found no evidence for socially mediated reproductive synchrony. Males ranged over larger areas than females, showing even less site fidelity, and also overlapped with several rivals. This basic spacing system remained stable over a wide range of densities and sex ratios. Independent of sex, animals used overlap zones randomly with respect to each other. Significant dynamic (spatiotemporal) interaction was most frequent between males and females. However, interaction analyses revealed no evidence for stable social bonds between animals, regardless of sex. We suggest that unpredictable female locations prevent males monopolizing females spatially. Because females are solitary, males could monopolize only one female by maintaining close proximity, rendering a roaming mating tactic more successful. Our findings point to a solitary ‘social’ system and overlap promiscuity as the likely mating system for the C. magna population studied. Copyright 2003 Published by Elsevier Ltd on behalf of The Association for the Study of Animal Behaviour.      

Molecular regulation of autophagy and apoptosis during ischemic and non-ischemic cardiomyopathy

A significant understanding of the genetic signaling pathways governing the extrinsic and intrinsic apoptotic pathways has been established. In recent years, the role of apoptosis in the heart during ischemic and non-ischemic cardiomyopathies has been under investigation and reported to contribute to ventricular remodeling and heart failure. Autophagy has been recently characterized as an essential cellular process in the heart, but whether autophagy functions as a pro-death or pro-survival program during disease conditions is still not completely understood. The mitochondrial death protein Bnip3 has been implicated in both apoptosis and autophagy, and its role in both processes is also discussed.