Natural variations at position 93 of the invariant Vα24-Jα18 α chain of human iNKT-cell TCRs strongly impact on CD1d binding

Human invariant natural killer T (NKT) cell TCRs bind to CD1d via an "invariant" Vα24-Jα18 chain (iNKTα) paired to semi-invariant Vβ11 chains (iNKTβ). Single-amino acid variations at position 93 (p93) of iNKTα, immediately upstream of the "invariant" CDR3α region, have been reported in a substantial proportion of human iNKT-cell clones (4-30%). Although p93, a serine in most human iNKT-cell TCRs, makes no contact with CD1d, it could affect CD1d binding by altering the conformation of the crucial CDR3α loop. By generating recombinant refolded iNKT-cell TCRs, we show that natural single-nucleotide variations in iNKTα, translating to serine, threonine, asparagine or isoleucine at p93, exert a powerful effect on CD1d binding, with up to 28-fold differences in affinity between these variants. This effect was observed with CD1d loaded with either the artificial α-galactosylceramide antigens KRN7000 or OCH, or the endogenous glycolipid β-galactosylceramide, and its importance for autoreactive recognition of endogenous lipids was demonstrated by the binding of variant iNKT-cell TCR tetramers to cell surface expressed CD1d. The serine-containing variant showed the strongest CD1d binding, offering an explanation for its predominance in vivo. Complementary molecular dynamics modeling studies were consistent with an impact of p93 on the conformation of the CDR3α loop.

Study Of The Binding Of Substrate By The Phe557Val Mutant Soybean Lipoxygenase-1

Lipoxygenases are a class of enzymes which consist of non-heme iron dioxygenases that are produced by fungi, plants, and mammals and catalyze the oxygenation of polyunsaturated fatty acid substrates to unsaturated fatty acid hydroperoxide products. The unsaturated fatty acid hydroperoxide products are stereo- and regiospecific. One such lipoxygenase, soybean lipoxygenase-1 (SBLO-1), catalyzes the conversion of linoleate to 13-hydroperoxy-9(Z),11(E)-octadecadienoate (13-HPOD) and a small amount of 9-hydroperoxy-10(E),12(Z)-octadecadienoate (9-HPOD). Although the structure of SBLO-1 is known and it is the most widely studied lipoxygenase, how it binds to substrate is still poorly understood. Two competing binding hypotheses that have been used to understand and explain the binding are the head first binding model and the tail first binding model. The head first binding model predicts linoleate binds with its polar carboxylate group in the binding pocket and the methyl terminus at the surface of the binding pocket. The tail first binding model predicts that linoleate binds with its methyl terminus end in the binding pocket and the polar carboxylate group at the surface of the binding pocket. Both binding models have been used in the explanation of previous work. In previous work the replacement of phenylalanine with valine has been performed to produce the phe557val mutant SBLO-1. The mutant SBLO-1 was then used in the enzymatic oxygenation of linoleate. With this mutant, the amount of 9-HPOD that is formed increases. This result has been interpreted using the head-first binding model in which the smaller valine residue allows linoleate to bind with the polar carboxylate group of linoleate interacting with arginine-707. The work presented in this thesis confirms the regiochemical results of the previous work and further tests the head-first binding model. If head-first binding occurs, the 9-HPOD is expected to have primarily S configuration. Utilizing chiral-phase HPLC, it was found that the 9-HPOD produced by the phe557val mutant SBLO-1 is primarily S, consistent with head-first binding. The head-first binding model was also tested using linoleyl dimethylamine (LDMA), which has been shown to be a good substrate for SBLO-1 at pH 7.0, where LDMA is thought to be positively charged. This model predicts that less of the 9-peroxide should be produced with this substrate. Through the use of gas chromatography/mass spectrometry, it was found that the conversion of LDMA by the phe557val mutant SBLO-1 resulted in the formation of a 46:54 mixture of the 13-peroxide:9-peroxide. The higher amount of 9-peroxide is the opposite of what is expected for the currently proposed model suggesting that the proposed model may not be entirely correct. The results thus far have been consistent with reverse binding but not with the proposed interaction of the polar end of the substrate with arginine-707.

Increased expression of the auxiliary beta2-subunit of ventricular L-type Ca2+ channels leads to single-channel activity characteristic of heart failure

BACKGROUND: Increased activity of single ventricular L-type Ca(2+)-channels (L-VDCC) is a hallmark in human heart failure. Recent findings suggest differential modulation by several auxiliary beta-subunits as a possible explanation. METHODS AND RESULTS: By molecular and functional analyses of human and murine ventricles, we find that enhanced L-VDCC activity is accompanied by altered expression pattern of auxiliary L-VDCC beta-subunit gene products. In HEK293-cells we show differential modulation of single L-VDCC activity by coexpression of several human cardiac beta-subunits: Unlike beta(1) or beta(3) isoforms, beta(2a) and beta(2b) induce a high-activity channel behavior typical of failing myocytes. In accordance, beta(2)-subunit mRNA and protein are up-regulated in failing human myocardium. In a model of heart failure we find that mice overexpressing the human cardiac Ca(V)1.2 also reveal increased single-channel activity and sarcolemmal beta(2) expression when entering into the maladaptive stage of heart failure. Interestingly, these animals, when still young and non-failing ("Adaptive Phase"), reveal the opposite phenotype, viz: reduced single-channel activity accompanied by lowered beta(2) expression. Additional evidence for the cause-effect relationship between beta(2)-subunit expression and single L-VDCC activity is provided by newly engineered, double-transgenic mice bearing both constitutive Ca(V)1.2 and inducible beta(2) cardiac overexpression. Here in non-failing hearts induction of beta(2)-subunit overexpression mimicked the increase of single L-VDCC activity observed in murine and human chronic heart failure. CONCLUSIONS: Our study presents evidence of the pathobiochemical relevance of beta(2)-subunits for the electrophysiological phenotype of cardiac L-VDCC and thus provides an explanation for the single L-VDCC gating observed in human and murine heart failure.

An fMRI-study of locally oriented perception in autism: altered early visual processing of the block design test

Autism has been associated with enhanced local processing on visual tasks. Originally, this was based on findings that individuals with autism exhibited peak performance on the block design test (BDT) from the Wechsler Intelligence Scales. In autism, the neurofunctional correlates of local bias on this test have not yet been established, although there is evidence of alterations in the early visual cortex. Functional MRI was used to analyze hemodynamic responses in the striate and extrastriate visual cortex during BDT performance and a color counting control task in subjects with autism compared to healthy controls. In autism, BDT processing was accompanied by low blood oxygenation level-dependent signal changes in the right ventral quadrant of V2. Findings indicate that, in autism, locally oriented processing of the BDT is associated with altered responses of angle and grating-selective neurons, that contribute to shape representation, figure-ground, and gestalt organization. The findings favor a low-level explanation of BDT performance in autism.

Executive Reorganization: Building a State Government That Worked for People -- Tom Harrison, Diana Dowling & Sheena Wilson “In the Crucible of Change”

One of the primary accomplishments of Governor Forrest Anderson in 1969-71 was the reorganization of the Executive Branch of Montana government, something that had been attempted six different times between 1919 and 1962 as state government had grown from twenty agencies to almost 200 uncontrolled boards, bureaus and commissions. The chaotic structure of the executive branch disempowered governors of both parties and empowered the private corporations and organizations that were the power structure of Montana. With remarkable political acumen, Governor Anderson figured out how to get that near impossible job done. Central to his efforts was the creation of an Executive Reorganization Commission, including eight legislators and the Governor, the adoption of a Constitutional Amendment that limited the executive branch to no more than twenty departments under the Governor, and the timely completion of a massive research effort to delineate the actual structure of the twenty departments. That story is told in this episode by three major players in the effort, all involved directly with the Executive Reorganization Commission: Tom Harrison, Diana Dowling and Sheena Wilson. Their recollections reflect an insider’s perspective of this significant accomplishment that helped change Montana “In the Crucible of Change.” Tom Harrison is a former Republican State Representative and State Senator from Helena, who was a member of the Executive Reorganization Commission. As Majority Leader in the Montana House of Representatives in 1971, he was the primary sponsor of the House’s executive reorganization bill and helped shepherd the Senate’s version to passage. Harrison was the Republican candidate for Attorney General in 1976 after which he practiced private law for 3 more decades. He served in the Montana Army National Guard for almost 34 years, rising to the rank of Colonel in the position of Judge Advocate General. He was a founding Director of Federal Defenders of Montana (legal representation for indigents accused within the Federal Judicial System); appointed Chairman of the original Montana State Fund (workers' compensation insurance) by Gov. Stephens; served as President of the Montana Trial Lawyers Association, Helena Kiwanis Club and St. Peter's Community Hospital Foundation, as well as Chairman and Director of AAA MountainWest; and was a founder, first Chairman and Director of the Valley Bank of Helena for over 25 years. Diana Dowling was an attorney for the Executive Reorganization Commission and helped draft the legislation that was passed. She also worked for Governor Forrest Anderson and for the 1972 Constitutional Convention where she prepared and directed publication of official explanation of the new Constitution that was mailed to all Montana voters. Diana was Executive Director of the Montana Bar Association and for 20 years held various legal positions with the Montana Legislative Council. For 12 years she was a commissioner on the National Conference of Commissioners on Uniform State Laws and for 7 years was a member of Montana State Board of Bar Examiners. Diana was the first director of the Montana Lottery, an adjunct professor at both Carroll College and the UM Law School, and an administrative officer for Falcon Press Publishing Co. Diana is currently - and intends to continue being - a perpetual college student. Sheena Wilson came fresh out of the University of Montana to become a Research Assistant for the Executive Reorganization Commission. Later she worked for seven years as a field representative in Idaho and Montana for the Mountain Plains Family Education Program, for thirteen years with Congressman Pat Williams as Executive Assistant in Washington and Field Assistant here in Montana, owned and managed a Helena restaurant for seven years, worked as Executive Assistant for State Auditor John Morrison and was Deputy Chief of Staff for Governor Brian Schweitzer his full 8 years in the Governorship. Though currently “retired”, Sheena serves on the Montana Board of Investments, the Public Employees Retirement Board and the Capitol Complex Advisory Council and is a partner in a dry-land wheat farm in Teton County that was homesteaded by her great uncle.

Social Structural Effects on the Level and Development of the Individual Experience of Anomie in the German Population

Can one observe an increasing level of individual lack of orientation because of rapid social change in modern societies? This question is examined using data from a representative longitudinal survey in Germany conducted in 2002–04. The study examines the role of education, age, sex, region (east/west), and political orientation for the explanation of anomia and its development. First we present the different sources of anomie in modern societies, based on the theoretical foundations of Durkheim and Merton, and introduce the different definitions of anomia, including our own cognitive version. Then we deduce several hypotheses from the theory, which we test by means of longitudinal data for the period 2002–04 in Germany using the latent growth curve model as our statistical method. The empirical findings show that all the sociodemographic variables, including political orientation, are strong predictors of the initial level of anomia. Regarding the development of anomia over time (2002–04), only the region (west) has a significant impact. In particular, the results of a multi-group analysis show that western German people with a right-wing political orientation become more anomic over this period. The article concludes with some theoretical implications.

The Social Dynamics of Communal Violence in India

Contrasting strands of explanation of the motives underlying collective action, as either culturally determined, as an attempt at compensation, point towards an understanding of identity politics as a reaction to given conditions. They pay little attention to the social dynamics that evolve in relation to the conflict within a group, and the possible motivation that can ensue from these. This article analyses the mobilisation among Hindu-nationalist organisations. Rather than seeking their attraction in their discursive outputs and the possible answers they might give in times of change, the contention is that they are to be sought in the specific internal dynamics and the possibilities they create within their historical context. These specific opportunities for action are inherent firstly in the mode of operation relying on participation and involvement, on their direct intervention, their localness and accessibility. Moreover, the dichotomisation inherent in violence makes possible the integration of different interests and different discontents under one banner and therefore contributes to the project of unification undertaken by Hindu-nationalism.

Effects of two traits of the ecological state equation on our understanding of species coexistence and ecosystem services

Species coexistence has been a fundamental issue to understand ecosystem functioning since the beginnings of ecology as a science. The search of a reliable and all-encompassing explanation for this issue has become a complex goal with several apparently opposing trends. On the other side, seemingly unconnected with species coexistence, an ecological state equation based on the inverse correlation between an indicator of dispersal that fits gamma distribution and species diversity has been recently developed. This article explores two factors, whose effects are inconspicuous in such an equation at the first sight, that are used to develop an alternative general theoretical background in order to provide a better understanding of species coexistence. Our main outcomes are: (i) the fit of dispersal and diversity values to gamma distribution is an important factor that promotes species coexistence mainly due to the right-skewed character of gamma distribution; (ii) the opposite correlation between species diversity and dispersal implies that any increase of diversity is equivalent to a route of “ecological cooling” whose maximum limit should be constrained by the influence of the third law of thermodynamics; this is in agreement with the well-known asymptotic trend of diversity values in space and time; (iii) there are plausible empirical and theoretical ways to apply physical principles to explain important ecological processes; (iv) the gap between theoretical and empirical ecology in those cases where species diversity is paradoxically high could be narrowed by a wave model of species coexistence based on the concurrency of local equilibrium states. In such a model, competitive exclusion has a limited but indispensable role in harmonious coexistence with functional redundancy. We analyze several literature references as well as ecological and evolutionary examples that support our approach, reinforcing the meaning equivalence between important physical and ecological principles.

14-3-3SIGMA NEGATIVELY REGULATES THE STABILITY AND SUBCELLULAR LOCALIZATION OF COP1

Mammalian constitutive photomorphogenic 1 (COP1), a p53 E3 ubiquitin ligase, is a key negative regulator for p53. DNA damage leads to the translocation of COP1 to the cytoplasm, but the underlying mechanism remains unknown. We discovered that 14-3-3σ controlled COP1 subcellular localization and protein stability. Investigation of the underlying mechanism suggested that, upon DNA damage, 14-3-3σ bound to phosphorylated COP1 at S387, resulting in COP1 translocation to the cytoplasm and cytoplasmic COP1 ubiquitination and proteasomal degradation. 14-3-3σ targeted COP1 for degradation to prevent COP1-mediated p53 degradation, p53 ubiquitination, and p53 transcription repression. COP1 expression promoted cell proliferation, cell transformation, and tumor progression, attesting to its role in cancer promotion. 14-3-3σ negatively regulated COP1 function and prevented tumor growth in cancer xenografts. COP1 protein levels were inversely correlated with 14-3-3σ protein levels in human breast and pancreatic cancer specimens. Together, these results define a novel, detailed mechanism for the posttranslational regulation of COP1 upon DNA damage and provide a mechanistic explanation of the correlation of COP1 overexpression with 14-3-3σ downregulation during tumorigenesis.

THE ROLE AND MECHANISM OF THE HOMEOBOX GENE DLX4 IN TRANSFORMING GROWTH FACTOR-B RESISTANCE IN CANCER

Transforming growth factor-b (TGF-b) is a cytokine that plays essential roles in regulating embryonic development and tissue homeostasis. In normal cells, TGF-b exerts an anti-proliferative effect. TGF-b inhibits cell growth by controlling a cytostatic program that includes activation of the cyclin-dependent kinase inhibitors p15Ink4B and p21WAF1/Cip1 and repression of c-myc. In contrast to normal cells, many tumors are resistant to the anti-proliferative effect of TGF-b. In several types of tumors, particularly those of gastrointestinal origin, resistance to the anti-proliferative effect of TGF-b has been attributed to TGF-b receptor or Smad mutations. However, these mutations are absent from many other types of tumors that are resistant to TGF-b-mediated growth inhibition. The transcription factor encoded by the homeobox patterning gene DLX4 is overexpressed in a wide range of malignancies. In this study, I demonstrated that DLX4 blocks the anti-proliferative effect of TGF-b by disabling key transcriptional control mechanisms of the TGF-b cytostatic program. Specifically, DLX4 blocked the ability of TGF-b to induce expression of p15Ink4B and p21WAF1/Cip1 by directly binding to Smad4 and to Sp1. Binding of DLX4 to Smad4 prevented Smad4 from forming transcriptional complexes with Smad2 and Smad3, whereas binding of DLX4 to Sp1 inhibited DNA-binding activity of Sp1. In addition, DLX4 induced expression of c-myc, a repressor of p15Ink4B and p21WAF1/Cip1 transcription, independently of TGF-b signaling. The ability of DLX4 to counteract key transcriptional control mechanisms of the TGF-b cytostatic program could explain in part the resistance of tumors to the anti-proliferative effect of TGF-b. This study provides a molecular explanation as to why tumors are resistant to the anti-proliferative effect of TGF-b in the absence of mutations in the TGF-b signaling pathway. Furthermore, this study also provides insights into how aberrant activation of a developmental patterning gene promotes tumor pathogenesis.

Chromosomal organization and evolutionary conservation of the human chromosome 19q linkage group containing three DNA repair genes

A series of human-rodent somatic cell hybrids were investigated by Southern blot analysis for the presence or absence of twenty-six molecular markers and three isozyme loci from human chromosome 19. Based on the co-retention of these markers in the various independent hybrid clones containing portions of human chromosome 19 and on pulsed field mapping, chromosome 19 is divided into twenty ordered regions. The most likely marker order for the chromosome is: (LDLR, C3)-(cen-MANNB)-D19S7-PEPD-D19S9-GPI-TGF$ \beta$-(CYP2A, NCA, CGM2, BCKAD)-PSG1a-(D19S8, XRCC1)-(D19S19, ATP1A3)-(D19S37, APOC2)-CKMM-ERCC2-ERCC1-(D19S62, D19S51)-D19S6-D19S50-D19S22-(CGB, FTL)-qter.^ The region of 19q between the proximal marker D19S7 and the distal gene coding for the beta subunit of chorionic gonadotropin (CGB) is about 37 Mb in size and covers about 37 cM genetic distance. The ration of genetic to physical distance on 19q is therefore very close to the genomic average OF 1 cM/Mb. Estimates of physical distances for intervals between chromosome 19 markers were calculated using a mapping function which estimates distances based on the number of breaks in hybrid clone panels. The consensus genetic distances between individual markers (established at HBM10) were compared to these estimates of physical distances. The close agreement between the two estimates suggested that spontaneously broken hybrids are as appropriate for this type of study as radiation hybrids.^ All three DNA repair genes located on chromosome 19 were found to have homologues on Chinese hamster chromosome 9, which is hemizygous in CHO cells, providing an explanation for the apparent ease with which mutations at these loci were identified in CHO cells. Homologues of CKMM and TGF$\beta$ (from human chromosome 19q) and a mini-satellite DNA specific to the distal region of human chromosome 19q were also mapped to Chinese hamster 9. Markers from 19p did not map to this hamster chromosome. Thus the q-arm of chromosome 19, at least between the genes PEPD and ERCC1, appears to be a linkage group which is conserved intact between humans and Chinese hamsters. ^

The role of the bed nucleus of the stria terminalis in stress: A behavioral, neurophysiological and neuropharmacological study

During the fifty-five years since the origin of the modern concept of stress, a variety of neurochemical, physiological, behavioral and pathological data have been collected in order to define stress and catalogue the components of the stress response. Over the last twenty-five years, as interest in the neural mechanisms underlying the stress response grew, most of the studies have focused on the hypothalamus and major limbic structures such as the amygdala or on nuclei involved in neurochemical changes observed during stress. There are other CNS sites, such as the bed nucleus of the stria terminalis (BNST), that neuroanatomical and neurochemical studies suggest may be involved in stress, but these sites have rarely been studied. Four experiments were performed for this dissertation, the goal of which was to examine the BNST to determine its role in the regulation of the stress response. The first experiment demonstrated that electrical stimulation of BNST was sufficient to produce stress-like behaviors. The second experiment demonstrated that single BNST neurons altered their firing rate in response to both a noxious somatosensory stimulus such as tail pinch and electrical stimulation of the amygdala (AmygS). The third experiment showed that the opioid, cholinergic, and noradrenergic systems, three neurotransmitter systems implicated in the control of the stress response, were effective in altering the firing rate of BNST neurons. The fourth experiment demonstrated that the cholinergic effects were mediated via muscarinic receptors and showed that the effects of AmygS were not mediated via cholinergic pathways. Collectively, these findings provide a possible explanation for the nonspecificity in causation of stress and the invariability of the stress response and suggest a neurochemical basis for its pharmacological control. ^

Positive effect of the yellow morph on female reproductive success in the flower colour polymorphic Iris lutescens (Iridaceae), a deceptive species

The deceptive Iris lutescens (Iridaceae) shows a heritable and striking flower colour polymorphism, with both yellow- and purple-flowered individuals growing sympatrically. Deceptive species with flower colour polymorphism are mainly described in the family Orchidaceae and rarely found in other families. To explain the maintenance of flower colour polymorphism in I.lutescens, we investigated female reproductive success in natural populations of southern France, at both population and local scales (within populations). Female reproductive success was positively correlated with yellow morph frequency, at both the population scale and the local scale. Therefore, we failed to observe negative frequency-dependent selection (NFDS), a mechanism commonly invoked to explain flower colour polymorphism in deceptive plant species. Flower size and local flower density could also affect female reproductive success in natural populations. Pollinator behaviour could explain the positive effect of the yellow morph, and our results suggest that flower colour polymorphism might not persist in I.lutescens, but alternative explanations not linked to pollinator behaviour are discussed. In particular, NFDS, although an appealingly simple explanation previously demonstrated in orchids, may not always contribute to maintaining flower colour polymorphism, even in deceptive species.

Passionate thinkers feel better: Self-control capacity as mediator of the relationship between need for cognition and affective adjustment

The present study tested a possible explanation for the positive relationship between the motivation to engage in cognitive endeavors (need for cognition, NFC) and indicators of affective adjustment (e.g., higher self-esteem, lower depression) that has been demonstrated in previous studies. We suggest that dispositional self-control capacity mediates this relationship, since NFC has been found to be related to self-control capacity, and self-control capacity is crucial for adjustment. NFC, dispositional self-control capacity, self-esteem, habitual depressive mood, and tendency to respond in a socially desirable manner were measured among 150 university students via self-report. Regression analyses and Sobel tests revealed that self-control capacity was a potential mediator of the positive relationship between NFC and affective adjustment. The findings were robust in terms of social desirability.

"Let's Talk Cash": Cantons' Interests and the Reform of Swiss Federalism

In 2004 the Swiss people accepted a new equalization scheme and a new distribution of competences between the federal state and the cantons. It was argued that the reform was successful because of the capacity of veto-players to overcome their interests and adopt a ‘problem-solving’ interaction mode. We propose a different interpretation and argue that distributive issues and the accommodation of actors' interests crucially mattered. We identify three mechanisms that contribute to a successful reform, i.e. package-deals, side-payments and the downsizing of the reform. Our in-depth, mainly qualitative study of both the content of the reform and related decision-making process supports the pertinence of these strategies for the explanation of the successful reform of Swiss federalism.