Characterization of the activity of fatty-acid epoxide hydrolase in seeds of castor bean (Ricinus communis L.)

Epoxide hydrolase (EC 3.3.2.3) activity was measured with [1-¹⁴C]cis-9,10-epoxystearic acid as the substrate. Homogenates were prepared from the endosperm tissue of germinating seeds of castor bean (Ricinus communis L. zanzibariensis). The activity of fatty-acid epoxide hydrolase was characterized with respect to dependence on time, amount of protein, pH and temperature. Analyses of enzyme distribution in endosperm, cotyledons, root and hypocotyl showed the highest total activity in the endosperm, less in the cotyledons and low activity in the root and hypocotyl. The specific activity was similar for cotyledons and endosperm. Analysis of the temporal expression of the enzyme in the endosperm during germination revealed high activity already in the imbibed seed. Activity was maximal between days four to six and then decreased at the end of one week. Subcellular fractionation of endosperm revealed a dual distribution of activity between the glyoxysomal and the cytosolic fractions.     

Chronic instrumentation and longterm investigation in the fetal and maternal baboon: tether system, conditioning procedures and surgical techniques

A tether system, conditioning procedures and surgical techniques were designed to maintain chronic catheters and electrodes in the pregnant baboon and her fetus. The tether system was comprised of a lightweight metal backpack containing catheters and electrodes, couplers, pressure transducers and electrical cabling. The backpack was held snugly in place by shoulder and body straps. A flexible metal tether connected the pack to a ball bearing assembly mounted on the top of the animal's home cage. Attached to the assembly were two infusion pumps, fluid reservoir and slip ring electrical connector. The entire system rotated freely with the movements of the animal; thus, the instrumentation and connectors were secure while access was maintained for continuous physiologic recording and intravascular infusion or intermittent blood sampling with minimal physical restraint. Animals were conditioned to accept the system prior to pregnancy and animals who demonstrated tolerance were bred. An initial group of 10 pregnant animals were sham tethered during pregnancy at 102 +/- 7 days with term gestation estimated at 180 days. Surgical procedures were done at 136 +/- 4 days with placement of catheters in the maternal femoral artery and vein, fetal carotid artery jugular vein and trachea, amniotic fluid cavity, and electrodes for fetal electrocardiogram and electroencephalogram. The mean fetal survival time was 9.3 (range 0 to 29) days. The major complications which led to early delivery were placental abruption and rupture of amniotic membranes. With ultrasonic localization of the placenta and determination of fetal position before surgery, these complications may be avoided.(ABSTRACT TRUNCATED AT 250 WORDS)     

Y chromosome haplotype distribution of brown bears (Ursus arctos) in Northern Europe provides insight into population history and recovery

High‐resolution, male‐inherited Y‐chromosomal markers are a useful tool for population genetic analyses of wildlife species, but to date have only been applied in this context to relatively few species besides humans. Using nine Y‐chromosomal STRs and three Y‐chromosomal single nucleotide polymorphism markers (Y‐SNPs), we studied whether male gene flow was important for the recent recovery of the brown bear (Ursus arctos) in Northern Europe, where the species declined dramatically in numbers and geographical distribution during the last centuries but is expanding now. We found 36 haplotypes in 443 male extant brown bears from Sweden, Norway, Finland and northwestern Russia. In 14 individuals from southern Norway from 1780 to 1920, we found two Y chromosome haplotypes present in the extant population as well as four Y chromosome haplotypes not present among the modern samples. Our results suggested major differences in genetic connectivity, diversity and structure between the eastern and the western populations in Northern Europe. In the west, our results indicated that the recovered population originated from only four male lineages, displaying pronounced spatial structuring suggestive of large‐scale population size increase under limited male gene flow within the western subpopulation. In the east, we found a contrasting pattern, with high haplotype diversity and admixture. This first population genetic analysis of male brown bears shows conclusively that male gene flow was not the main force of population recovery.     

Root responses and nitrogen acquisition by<Emphasis Type="Italic"> Artemisia tridentata</Emphasis> and<Emphasis Type="Italic"> Agropyron desertorum</Emphasis> following small summer rainfall events

Resources in the Great Basin of western North America often occur in pulses, and plant species must rapidly respond to temporary increases in water and nutrients during the growing season. A field study was conducted to evaluate below ground responses of Artemisia tridentata and Agropyron desertorum, common Great Basin shrub and grass species, respectively, to simulated 5-mm (typical summer rain) and 15-mm (large summer rain) summer rainfall events. The simulated rainfall was labeled with K(15)NO(3) so that timing of plant nitrogen uptake could be monitored. In addition, soil NH(4)(+) and NO(3)(-) concentrations and physiological uptake capacities for NO(3)(-) and NH(4)(+) were determined before and after the rainfall events. Root growth in the top 15 cm of soil was monitored using a minirhizotron system. Surprisingly, there was no difference in the amount of labeled N acquired in response to the two rainfall amounts by either species during the 7-day sample period. However, there were differences between species in the timing of labeled N uptake. The N label was detected in above ground tissue of Agropyron within 1 h of the simulated rainfall events, but not until 24 h after the rainfall in Artemisia. For both Agropyron and Artemisia, root uptake capacity was similarly affected by the 5-mm and 15-mm rainfall. There was, however, a greater increase in uptake capacity for NH(4)(+) than for NO(3)(-), and the 15-mm event resulted in a longer response. No root growth occurred in either species in response to either rainfall event during this 8-day period. The results of this study indicate that these species are capable of utilizing nitrogen pulses following even small summer rainfall events during the most stressful period of the summer and further emphasize the importance of small precipitation events in arid systems.     

Cover-Encodings of Fitness Landscapes

The traditional way of tackling discrete optimization problems is by using local search on suitably defined cost or fitness landscapes. Such approaches are however limited by the slowing down that occurs when the local minima that are a feature of the typically rugged landscapes encountered arrest the progress of the search process. Another way of tackling optimization problems is by the use of heuristic approximations to estimate a global cost minimum. Here, we present a combination of these two approaches by using cover-encoding maps which map processes from a larger search space to subsets of the original search space. The key idea is to construct cover-encoding maps with the help of suitable heuristics that single out near-optimal solutions and result in landscapes on the larger search space that no longer exhibit trapping local minima. We present cover-encoding maps for the problems of the traveling salesman, number partitioning, maximum matching and maximum clique; the practical feasibility of our method is demonstrated by simulations of adaptive walks on the corresponding encoded landscapes which find the global minima for these problems.     

Large-scale mutagenesis in p19(ARF)- and p53-deficient mice identifies cancer genes and their collaborative networks

p53 and p19(ARF) are tumor suppressors frequently mutated in human tumors. In a high-throughput screen in mice for mutations collaborating with either p53 or p19(ARF) deficiency, we identified 10,806 retroviral insertion sites, implicating over 300 loci in tumorigenesis. This dataset reveals 20 genes that are specifically mutated in either p19(ARF)-deficient, p53-deficient or wild-type mice (including Flt3, mmu-mir-106a-363, Smg6, and Ccnd3), as well as networks of significant collaborative and mutually exclusive interactions between cancer genes. Furthermore, we found candidate tumor suppressor genes, as well as distinct clusters of insertions within genes like Flt3 and Notch1 that induce mutants with different spectra of genetic interactions. Cross species comparative analysis with aCGH data of human cancer cell lines revealed known and candidate oncogenes (Mmp13, Slamf6, and Rreb1) and tumor suppressors (Wwox and Arfrp2). This dataset should prove to be a rich resource for the study of genetic interactions that underlie tumorigenesis.     

IR-inducible clusterin gene expression: a protein with potential roles in ionizing radiation-induced adaptive responses, genomic instability, and bystander effects

Clusterin (CLU) plays numerous roles in mammalian cells after stress. A review of the recent literature strongly suggests potential roles for CLU proteins in low dose ionizing radiation (IR)-inducible adaptive responses, bystander effects, and delayed death and genomic instability. Its most striking and evident feature is the inducibility of the CLU promoter after low, as well as high, doses of IR. Two major forms of CLU, secreted (sCLU) and nuclear (nCLU), possess opposite functions in cellular responses to IR: sCLU is cytoprotective, whereas nCLU (a byproduct of alternative splicing) is a pro-death factor. Recent studies from our laboratory and others demonstrated that down-regulation of sCLU by specific siRNA increased cytotoxic responses to chemotherapy and IR. sCLU was induced after low non-toxic doses of IR (0.02-0.5 Gy) in human cultured cells and in mice in vivo. The low dose inducibility of this survival protein suggests a possible role for sCLU in radiation adaptive responses, characterized by increased cell radioresistance after exposure to low adapting IR doses. Although it is still unclear whether the adaptive response is beneficial or not to cells, survival of damaged cells after IR may lead to genomic instability in the descendants of surviving cells. Recent studies indicate a link between sCLU accumulation and cancer incidence, as well as aging, supporting involvement of the protein in the development of genomic instability. Secreted after IR, sCLU may also alter intracellular communication due to its ability to bind cell surface receptors, such as the TGF-beta receptors (types I and II). This interference with signaling pathways may contribute to IR-induced bystander effects. We hypothesize that activation of the TGF-beta signaling pathway, which often occurs after IR exposure, can in turn activate the CLU promoter. TGF-beta and IR-inducible de novo synthesized sCLU may then bind the TGF-beta receptors and suppress downstream growth arrest signaling. This complicated negative feedback regulation most certainly depends on the cellular microenvironment, but undoubtedly represents a potential link between IR-induced adaptive responses, genomic instability and bystander effects. Further elucidation of clusterin protein functions in IR responses are clearly warranted.     

Insulin responsiveness of glucose transporter 4 in 3T3-L1 cells depends on the presence of sortilin

Insulin-dependent translocation of glucose transporter 4 (Glut4) to the plasma membrane of fat and skeletal muscle cells plays the key role in postprandial clearance of blood glucose. Glut4 represents the major cell-specific component of the insulin-responsive vesicles (IRVs). It is not clear, however, whether the presence of Glut4 in the IRVs is essential for their ability to respond to insulin stimulation. We prepared two lines of 3T3-L1 cells with low and high expression of myc₇-Glut4 and studied its translocation to the plasma membrane upon insulin stimulation, using fluorescence-assisted cell sorting and cell surface biotinylation. In undifferentiated 3T3-L1 preadipocytes, translocation of myc₇-Glut4 was low regardless of its expression levels. Coexpression of sortilin increased targeting of myc₇-Glut4 to the IRVs, and its insulin responsiveness rose to the maximal levels observed in fully differentiated adipocytes. Sortilin ectopically expressed in undifferentiated cells was translocated to the plasma membrane regardless of the presence or absence of myc₇-Glut4. AS160/TBC1D4 is expressed at low levels in preadipocytes but is induced in differentiation and provides an additional mechanism for the intracellular retention and insulin-stimulated release of Glut4.Adipocytes, skeletal muscle cells, and some neurons respond to insulin stimulation by translocating intracellular glucose transporter 4 (Glut4) to the plasma membrane. In all these cells, the insulin-responsive pool of Glut4 is localized in small membrane vesicles, the insulin-responsive vesicles (IRVs; Kandror and Pilch, 2011 ; Bogan, 2012 ). The protein composition of these vesicles has been largely characterized (Kandror and Pilch, 2011 ; Bogan, 2012 ). The IRVs consist predominantly of Glut4, insulin-responsive aminopeptidase (IRAP), sortilin, low-density-lipoprotein receptor–related protein 1 (LRP1), SCAMPs, and VAMP2. Glut4, IRAP, and sortilin physically interact with each other, which might be important for the biogenesis of the IRVs (Shi and Kandror, 2007 ; Shi et al., 2008 ). In addition, the IRVs compartmentalize recycling receptors, such as the transferrin receptor and the IGF2/mannose 6-phosphate receptor, although it is not clear whether these receptors represent obligatory vesicular components or their presence in the IRVs is explained by mass action (Pilch, 2008 ), inefficient sorting, or other reasons.Deciphering of the protein composition of the IRVs is important because it is likely to explain their unique functional property: translocation to the plasma membrane in response to insulin stimulation. Even if we presume that IRV trafficking is controlled by loosely associated peripheral membrane proteins, the latter should still somehow recognize the core vesicular components that create the “biochemical individuality” of this compartment. In spite of our knowledge of the IRV protein composition, however, the identity of the protein(s) that confer insulin sensitivity to these vesicles is unknown.Insulin responsiveness of the IRVs was associated with either IRAP or Glut4. Thus it was shown that Glut4 interacted with the intracellular anchor TUG (Bogan et al., 2003 , 2012 ), whereas IRAP associated with other proteins implemented in the regulation of Glut4 translocation, such as AS160 (Larance et al., 2005 ; Peck et al., 2006 ), p115 (Hosaka et al., 2005 ), tankyrase (Yeh et al., 2007 ), and several others (reviewed in Bogan, 2012 ). Results of these studies, or at least their interpretations, are not necessarily consistent with each other, as the existence of multiple independent anchors for the IRVs is, although possible, unlikely.Ablation of the individual IRV proteins has also led to controversial data. Thus knockout of IRAP decreases total protein levels of Glut4 but does not affect its translocation in the mouse model (Keller et al., 2002 ). On the contrary, knockdown of IRAP in 3T3-L1 adipocytes has a strong inhibitory effect on translocation of Glut4 (Yeh et al., 2007 ). In yet another study, knockdown of IRAP in 3T3-L1 adipocytes did not affect insulin-stimulated translocation of Glut4 but increased its plasma membrane content under basal conditions (Jordens et al., 2010 ). By the same token, total or partial ablation of Glut4 had various effects on expression levels, intracellular localization, and translocation of IRAP (Jiang et al., 2001 ; Abel et al., 2004 ; Carvalho et al., 2004 ; Gross et al., 2004 ; Yeh et al., 2007 ). Knockdown of either sortilin or LRP1 decreased protein levels of Glut4 (Shi and Kandror, 2005 ; Jedrychowski et al., 2010 ).One model that might explain these complicated and somewhat inconsistent results is that depletion of either major integral protein of the IRVs disrupts the network of interactions between vesicular proteins and thus decreases the efficiency of protein sorting into the IRVs (Kandror and Pilch, 2011 ). Correspondingly, the remaining IRV components that cannot be faithfully compartmentalized in the vesicles are either degraded (Jiang et al., 2001 ; Keller et al., 2002 ; Abel et al., 2004 ; Carvalho et al., 2004 ; Shi and Kandror, 2005 ; Yeh et al., 2007 ; Jedrychowski et al., 2010 ) or mistargeted (Jiang et al., 2001 ; Jordens et al., 2010 ), depending on experimental conditions and types of cells used in these studies. In other words, knockdown of any major IRV component may decrease vesicle formation along with insulin responsiveness. Thus, in spite of a large body of literature, the identity of protein(s) that confer insulin responsiveness to the IRVs is unknown.Here we used a gain-of-function approach to address this question. Specifically, we attempted to “build” functional IRVs in undifferentiated 3T3-L1 preadipocytes by forced expression of the relevant proteins. Undifferentiated preadipocytes do not express Glut4 or sortilin and lack IRVs (ElJack et al., 1999 ; Shi and Kandror, 2005 ; Shi et al., 2008 ). Correspondingly, IRAP, which is expressed in these cells, shows low insulin response (Ross et al., 1998 ; Shi et al., 2008 ). We found that ectopic expression of increasing amounts of Glut4 in undifferentiated preadipocytes does not lead to its marked translocation to the plasma membrane upon insulin stimulation. On the contrary, sortilin expressed in undifferentiated preadipocytes was localized in the IRVs and was translocated to the plasma membrane in response to insulin stimulation. Moreover, upon coexpression with Glut4, sortilin dramatically increased its insulin responsiveness to the levels observed in fully differentiated adipocytes. Thus sortilin may represent the key component of the IRVs, which is responsible not only for the formation of vesicles (Shi and Kandror, 2005 ; Ariga et al., 2008 ; Hatakeyama and Kanzaki, 2011 ), but also for their insulin responsiveness. It is worth noting that sortilin levels are significantly decreased in obese and diabetic humans and mice (Kaddai et al., 2009 ). We thus suggest that sortilin may be a novel and important target in the fight against insulin resistance and diabetes.Our experiments also demonstrate that undifferentiated preadipocytes lack a mechanism for the full intracellular retention of Glut4 that can be achieved by ectopic expression of AS160/TBC1D4.