IMMUNOLOGY, Issue 1 2006
Katherine S. Koch
Summary Embryonic mouse STO (S, SIM; T, 6-thioguanine resistant; O, ouabain resistant) and 3(8)21-enhanced green fluorescent protein (EGFP) cell lines exhibit long-term survival and hepatic progenitor cell behaviour after xenogeneic engraftment in non-immunosuppressed inbred rats, and were previously designated major histocompatibility complex (MHC) class I- and class II-negative lines. To determine the molecular basis for undetectable MHC determinants, the expression and haplotype of H-2K, H-2D, H-2L and I-A proteins were reassessed by reverse transcriptase,polymerase chain reaction (RT-PCR), cDNA sequencing, RNA hybridization, immunoblotting, quantitative RT-PCR (QPCR), immunocytochemistry and flow cytometry. To detect cell differentiation (CD) surface antigens characteristic of stem cells, apoptotic regulation or adaptive immunity that might facilitate progenitor cell status or immune privilege, flow cytometry was also used to screen untreated and cytokine [interferon (IFN)-,]-treated cultures. Despite prior PCR genotyping analyses suggestive of H-2q haplotypes in STO, 3(8)21-EGFP and parental 3(8)21 cells, all three lines expressed H-2K cDNA sequences identical to those of d-haplotype BALB/c mice, as well as constitutive and cytokine-inducible H-2Kd determinants. In contrast, apart from H-2Ld[LOW] display in 3(8)21 cells, H-2Dd, H-2Ld and I-Ad determinants were undetectable. All three lines expressed constitutive and cytokine-inducible CD34; however, except for inducible CD117[LOW] expression in 3(8)21 cells, no expression of CD45, CD117, CD62L, CD80, CD86, CD90·1 or CD95L/CD178 was observed. Constitutive and cytokine-inducible CD95[LOW] expression was detected in STO and 3(8)21 cells, but not in 3(8)21-EGFP cells. MHC (class I+[LOW]/class II,) and CD (CD34+/CD80,/CD86,/CD95L,) expression patterns in STO and STO cell-derived progenitor cells resemble patterns reported for human embryonic stem cell lines. Whether these patterns reflect associations with mechanisms that are regulatory of immune privilege or functional tissue-specific plasticity is unknown. [source]

Nitric oxide in inflammatory bowel disease: a universal messenger in an unsolved puzzle

IMMUNOLOGY, Issue 4 2004
George Kolios
Summary In recent years, nitric oxide (NO), a gas previously considered to be a potentially toxic chemical, has been established as a diffusible universal messenger that mediates cell,cell communication throughout the body. Constitutive and inducible NO production regulate numerous essential functions of the gastrointestinal mucosa, such as maintenance of adequate perfusion, regulation of microvascular and epithelial permeability, and regulation of the immune response. Up-regulation of the production of NO via expression of inducible nitric oxide synthase (iNOS) represents part of a prompt intestinal antibacterial response; however, NO has also been associated with the initiation and maintenance of inflammation in human inflammatory bowel disease (IBD). Recent studies on animal models of experimental IBD have shown that constitutive and inducible NO production seems to be beneficial during acute colitis, but sustained up-regulation of NO is detrimental. This fact is also supported by studies on mice genetically deficient in various NOS isoforms. However, the mechanism by which NO proceeds from being an indispensable homeostatic regulator to a harmful destructor remains unknown. Furthermore, extrapolation of data from animal colitis models to human IBD is questionable. The purpose of this review is to update our knowledge about the role of this universal mediator and the enzymes that generate it in the pathogenesis of IBD. [source]

Association of the toll-like receptor 2 A-16934T promoter polymorphism with severe atopic dermatitis

ALLERGY, Issue 11 2009
D.-Y. Oh
Background:, Atopic dermatitis (AD) is a chronic inflammatory skin disease with a multifactorial pathogenesis and increasing incidence in the Western world. A genetically determined defective function of pattern recognition receptors such as toll-like receptors (TLRs) has been proposed as a candidate mechanism in the pathogenesis of AD. Aim:, To study the impact of genetic predisposition of five genes encoding for pattern recognition-related molecules for the phenotype of AD. Methods:, We examined nine different single-nucleotide polymorphism (SNP) frequencies in the genes encoding TLR1, -2, -4, -9 and the adapter molecule TIRAP by PCR with subsequent melting curve analysis in a case/control cohort of 136 adult AD patients and 129 age and gender matched non-atopic, healthy individuals. TLR2-expression and -function in cells from genotyped individuals were analysed. Results:, For the SNPs examined, similar genotype frequencies were found in both groups. In a subgroup of patients suffering from severe AD (SCORAD >50), a significantly increased representation of the A-allele in position ,16934 of the tlr2 gene was present (P = 0.004). Constitutive tlr2 mRNA expression in peripheral monocytes was independent of this tlr2 promoter SNP. Stimulation assays indicated that IL-6, but not TNF-, secretion following TLR2 stimulation is reduced in homozygous tlr2 -16934-A allele carriers. Conclusion:, These data indicate that TLR2 is relevant for the phenotype of severe AD in adults. [source]

Relative contributions of nine genes in the pathway of histidine biosynthesis to the control of free histidine concentrations in Arabidopsis thaliana

PLANT BIOTECHNOLOGY JOURNAL, Issue 6 2009
Jonathan D. Rees
Summary Despite the functional importance of histidine (His) as an essential amino acid in proteins and as a metal-coordinating ligand, comparatively little is known about the regulation of its biosynthesis in plants and the potential for metabolic engineering of this pathway. To investigate the contribution of different steps in the pathway to overall control of His biosynthesis, nine His biosynthetic genes were individually over-expressed in Arabidopsis thaliana to determine their effects on free amino acid pools. Constitutive, CaMV 35S -driven over-expression of the cDNAs encoding either isoform of ATP-phosphoribosyltransferase (ATP-PRT), the first enzyme in the pathway, was sufficient to increase the pool of free His by up to 42-fold in shoot tissue of Arabidopsis, with negligible effect on any other amino acid. In contrast, over-expression of cDNAs for seven other enzymes in the biosynthetic pathway had no effect on His content, suggesting that control of the pool of free His resides largely with ATP-PRT activity. Over-expression of ATP-PRT and increased His content had a negative pleiotropic effect on plant biomass production in 35S:PRT1 lines, but this effect was not observed in 35S:PRT2 lines. In the presence of 100 µM Ni, which was inhibitory to wild-type plants, a strong positive correlation was observed between free His content and biomass production, indicating that the metabolic cost of His overproduction was outweighed by the benefit of increased tolerance to Ni. His-overproducing plants also displayed somewhat elevated tolerance to Co and Zn, but not to Cd or Cu, indicating chemical selectivity in intracellular metal binding by His. [source]

A Revision of the Constitutive and Epistemic Coherence Theories in Law

RATIO JURIS, Issue 2 2001
Veronica Rodriguez-Blanco
This paper analyses and criticizes Joseph Raz's attacks on coherentist theories. It is argued that Raz's characterisation of epistemic coherence theories is too narrow and that his criticism of constitutive coherence theories is based on a conceptual mistake in his own description. The study is an indirect argument to rethink coherence theories of law and adjudication within a more powerful framework than that propounded by Raz. [source]

New insights into pigmentary pathways and skin cancer

BRITISH JOURNAL OF DERMATOLOGY, Issue 1 2010
A.J. Miller
Summary The ability of cells to respond to and to mitigate environmental stress is crucial for their survival. Constitutive and facultative pigmentation have evolved in order for human skin to contend with high levels of terrestrial ultraviolet radiation (UVR). When this melanin ,shield' is compromised, individuals are exposed to increased skin cancer risk. The purpose of this review is to discuss new insights into the genetic basis of phenotypic risk factors for skin cancer, their connection to pigmentation and tanning, the precise molecular connections linking UVR to the tanning response, and potential methods of modulating pigmentation that avoid genotoxic damage. Highly translational implications of this research include a scientific basis on which to counsel patients regarding the carcinogenicity of UVR exposure related to tanning and potential new tanning agents that may actually protect against skin cancer by circumventing the need for UVR exposure. [source]

Mediation, power, and cultural difference

CONFLICT RESOLUTION QUARTERLY, Issue 3 2003
Morgan Brigg
In Western mediation practice, conflict and violence are typically seen as destructive and unhelpful ways of being, and this does not allow for the constitutive and productive role of conflict in many non-Western traditions. The playing out of these assumptions in mediation practice effects an operation of power that is particularly significant in intercultural mediations. Explicit and implicit mediator techniques lead disputants in intercultural mediations to behave in ways consistent with the goals of mediation and Western norms around conflict and selfhood. The specificity of this analysis means that the findings are indicative and explorative rather than comprehensive. Nevertheless, the results highlight the need to consider ways in which researchers and mediators can begin to mitigate this operation of power and respond to cultural difference in ethical ways. [source]

COSMOPOLITANISM, REMEDIATION, AND THE GHOST WORLD OF BOLLYWOOD

CULTURAL ANTHROPOLOGY, Issue 1 2010
DAVID NOVAK
ABSTRACT This essay considers the process of remediation in two North American reproductions of the song-and-dance sequence Jaan Pehechaan Ho from the 1965 "Bollywood" film Gumnaam. The song was used in the opening sequence of the 2001 U.S. independent film Ghost World as a familiar-but-strange object of ironic bewilderment and fantasy for its alienated teenage protagonist Enid. But a decade before Ghost World's release, Jaan Pehechaan Ho had already become the lynchpin of a complex debate about cultural appropriation and multicultural identity for an "alternative" audience in the United States. I illustrate this through an ethnographic analysis of a 1994 videotape of the Heavenly Ten Stems, an experimental rock band in San Francisco, whose performance of the song was disrupted by a group of activists who perceived their reproduction as a mockery. How is Bollywood film song, often itself a kitschy send-up of American popular culture, remediated differently for different projects of reception? How do these cycles of appropriation create overlapping conditions for new identities,whether national, diasporic, or "alternative",within the context of transcultural media consumption? In drawing out the "ghost world" of Bollywood's juxtapositions, I argue that the process of remediation produces more than just new forms and meanings of media, but is constitutive of the cosmopolitan subjects formed in its global circulations. [source]

AN ACOUSTIC REGISTER, TENACIOUS IMAGES, AND CONGOLESE SCENES OF RAPE AND REPETITION

CULTURAL ANTHROPOLOGY, Issue 2 2008
NANCY ROSE HUNT
ABSTRACT This article argues for the importance of rewriting the conventional atrocity narrative about violence in King Leopold's Congo Free State in relation to the present, the ongoing war-related humanitarianism and sexual violence in the DRC. The central idea is to push beyond the shock and tenacity of the visual, the ubiquitous mutilation photographs that tend to blot out all else; and instead seek weaker, more fragile acoustic traces in a diverse archive with Congolese words and sounds. This sensory, nonspectral mode of parsing the archive tells us something new about the immediacy of violence, its duration in memory, and the bodily and reproductive effects of sexually torturing women. The sound of twisted laughter convulsed around forms of sexual violence that were constitutive of reproductive ruination during the rubber regime in Leopold's Congo. The work of strategically tethering the past to the present should not be about forging historicist links across time but about locating repetitions and difference, including differences among humanitarian modes and strategies in the early 20th and the early 21st centuries. [source]

Technologies of the Voice: FM Radio, Telephone, and the Nepali Diaspora in Kathmandu

CULTURAL ANTHROPOLOGY, Issue 3 2006
Laura Kunreuther
Through the public broadcast of intimate telephone conversations between Nepalis abroad and those in Kathmandu, the diaspora is made "present" in Kathmandu. On these commercial FM programs, the voice is viewed as a key sign of emotional directness, authenticity, and intimacy. Simultaneously, the figure of the voice has been central in discussions about the promises (and failures) of democracy and transparent governance. These two seemingly distinct formations of voice are mutually constitutive. Sentimental discourse about the voice reiterates modern neoliberal discourse about democracy and vice versa. Both are crucial to the formation of an urban Nepali subject in this political moment, which is deeply shaped by the figure of the diaspora. [source]

Plant profilin isovariants are distinctly regulated in vegetative and reproductive tissues

CYTOSKELETON, Issue 1 2002
Muthugapatti K. Kandasamy
Abstract Profilin is a low-molecular weight, actin monomer-binding protein that regulates the organization of actin cytoskeleton in eukaryotes, including higher plants. Unlike the simple human or yeast systems, the model plant Arabidopsis has an ancient and highly divergent multi-gene family encoding five distinct profilin isovariants. Here we compare and characterize the regulation of these profilins in different organs and during microspore development using isovariant-specific monoclonal antibodies. We show that PRF1, PRF2, and PRF3 are constitutive, being strongly expressed in all vegetative tissues at various stages of development. These profilin isovariants are also predominant in ovules and microspores at the early stages of microsporogenesis. In contrast, PRF4 and PRF5 are late pollen-specific and are not detectable in other cell types of the plant body including microspores and root hairs. Immunocytochemical studies at the subcellular level reveal that both the constitutive and pollen-specific profilins are abundant in the cytoplasm. In vegetative cell types, such as root apical cells, profilins showed localization to nuclei in addition to the cytoplasmic staining. The functional diversity of profilin isovariants is discussed in light of their spatio-temporal regulation during vegetative development, pollen maturation, and pollen tube growth. Cell Motil. Cytoskeleton 52:22,32, 2002. © 2002 Wiley-Liss, Inc. [source]

In vivo dynamics of CNS sensory arbor formation: A time-lapse study in the embryonic leech

DEVELOPMENTAL NEUROBIOLOGY, Issue 1 2003
Michael W. Baker
Abstract In the embryo of the leech Hirudo medicinalis, afferent projections of peripheral sensory neurons travel along common nerve tracts to the CNS, where they defasciculate, branch, and arborize into separate, modality-specific synaptic laminae. Previous studies have shown that this process requires, at least in part, the constitutive and then modality-specific glycosylations of tractin, a leech L1 homologue. We report here on the dynamics of growth of these projections as obtained by examining the morphology of single growing dye-filled sensory afferents as a function of time. Using 2-photon laser-scanning microscopy of the intact developing embryo, we obtained images of individual sensory projections at 3 to 30 min intervals, over several hours of growth, and at different stages of development. The time-lapse series of images revealed a highly dynamic and maturation-state-dependent pattern of growth. Upon entering the CNS, the growth cone-tipped primary axon sprouted numerous long filopodial processes, many of which appeared to undergo repeated cycles of extension and retraction. The growth cone was transformed into a sensory arbor through the formation of secondary branches that extended within the ganglionic neuropil along the anterior-posterior axis of the CNS. Numerous tertiary and quaternary processes grew from these branches and also displayed cycles of extension and retraction. The motility of these higher-order branches changed with age, with younger afferents displaying higher densities and greater motility than older, more mature sensory arbors. Finally, coincident with a reduction in higher order projections was the appearance of concavolar structures on the secondary processes. Rows of these indentations suggest the formation of presynaptic en-passant specializations accompanying the developmental onset of synapse formation. © 2003 Wiley Periodicals, Inc. J Neurobiol 56: 41,53, 2003 [source]

Behavioural activity levels and expression of stress proteins under predation risk in two damselfly species

ECOLOGICAL ENTOMOLOGY, Issue 3 2009
STEFANIE SLOS
Abstract 1.,It has become apparent that predators may strongly decrease prey fitness without direct contact with the prey, as they induce the development of defence systems that limit the availability of energy for growth and reproduction. Recent studies suggest that stress proteins may help prey organisms deal with this stress. The pattern is not general, however, and little is known about species differences in physiological traits in coping with predator stress, and covariation of physiological with other antipredator traits. 2.,To explore these issues, we quantified levels of constitutive and fish-induced stress proteins (Hsp60 and Hsp70) and anti-predator behaviours in larvae of two damselfly species that differ in lifestyle. Both stress proteins were fixed at higher levels in Erythromma najas, which has a slow lifestyle, than in Lestes sponsa, which has a fast lifestyle. Similarly, anti-predator behaviours were fixed at safer levels in E. najas than in L. sponsa. 3.,These results suggest that stress proteins may be part of anti-predator syndromes of damselfly larvae, and there may be trait co-specialisation between stress proteins and behavioural anti-predator traits. Studies formally testing these hypotheses in more species may prove rewarding in advancing our understanding of the functional integration of physiological anti-predator traits in relation to the prey's lifestyle. [source]

An ecological cost of plant defence: attractiveness of bitter cucumber plants to natural enemies of herbivores

ECOLOGY LETTERS, Issue 3 2002
Anurag A. Agrawal
Abstract Plants produce defences that act directly on herbivores and indirectly via the attraction of natural enemies of herbivores. We examined the pleiotropic effects of direct chemical defence production on indirect defence employing near-isogenic varieties of cucumber plants (Cucumis sativus) that differ qualitatively in the production of terpenoid cucurbitacins, the most bitter compounds known. In release,recapture experiments conducted in greenhouse common gardens, blind predatory mites were attracted to plants infested by herbivorous mites. Infested sweet plants (lacking cucurbitacins), however, attracted 37% more predatory mites than infested bitter plants (that produce constitutive and inducible cucurbitacins). Analysis of the headspace of plants revealed that production of cucurbitacins was genetically correlated with large increases in the qualitative and quantitative spectrum of volatile compounds produced by plants, including induced production of (E,)-,-ocimene (3E,)-4,8-dimethyl-1,3,7-nonatriene, (E,E)-,-farnesene, and methyl salicylate, all known to be attractants of predators. Nevertheless, plants that produced cucurbitacins were less attractive to predatory mites than plants that lacked cucurbitacins and predators were also half as fecund on these bitter plants. Thus, we provide novel evidence for an ecological trade-off between direct and indirect plant defence. This cost of defence is mediated by the effects of cucurbitacins on predator fecundity and potentially by the production of volatile compounds that may be repellent to predators. [source]

The Quest for Distinction: A Reappraisal of the Rural Labor Process in Kheda District (Gujarat), India,

ECONOMIC GEOGRAPHY, Issue 2 2000
Vinay Gidwani
Abstract: In this article I examine how the rural labor process is constitutive of social identity, particularly status, by harnessing empirical evidence from Kheda District, Gujarat, and other parts of India. Emphasis is on the labor practices of the dominant Lewa Patel caste, and only secondarily on the practices of other caste groups. My central claim is that the labor process is a primary arena in which the quest for social distinction occurs and that the primary source of distinction is the ability to withdraw family labor power from the commoditized labor circuit. In this paper I seek to deepen conventional understandings of the labor process within economic geography, agrarian studies, and mainstream economics. [source]

Coevolution of antibiotic production and counter-resistance in soil bacteria

ENVIRONMENTAL MICROBIOLOGY, Issue 3 2010
Paris Laskaris
Summary We present evidence for the coexistence and coevolution of antibiotic resistance and biosynthesis genes in soil bacteria. The distribution of the streptomycin (strA) and viomycin (vph) resistance genes was examined in Streptomyces isolates. strA and vph were found either within a biosynthetic gene cluster or independently. Streptomyces griseus strains possessing the streptomycin cluster formed part of a clonal complex. All S. griseus strains possessing solely strA belonged to two clades; both were closely related to the streptomycin producers. Other more distantly related S. griseus strains did not contain strA. S. griseus strains with only vph also formed two clades, but they were more distantly related to the producers and to one another. The expression of the strA gene was constitutive in a resistance-only strain whereas streptomycin producers showed peak strA expression in late log phase that correlates with the switch on of streptomycin biosynthesis. While there is evidence that antibiotics have diverse roles in nature, our data clearly support the coevolution of resistance in the presence of antibiotic biosynthetic capability within closely related soil dwelling bacteria. This reinforces the view that, for some antibiotics at least, the primary role is one of antibiosis during competition in soil for resources. [source]

Regulation of erythropoietin production

EUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 2005
K.-U. Eckardt
Abstract The glycoprotein hormone erythropoietin (EPO) is an essential growth and survival factor for erythroid progenitor cells, and the rate of red blood cell production is normally determined by the serum EPO concentration. EPO production is inversely related to oxygen availability, so that an effective feedback loop is established, which controls erythropoiesis. Since recombinant EPO became available as an effective therapeutic agent, significant progress has also been made in understanding the basis of this feedback control. The main determinant of EPO synthesis is the transcriptional activity of its gene in liver and kidneys, which is related to local oxygen tensions. This control is achieved by hypoxia-inducible transcription factors (HIF), consisting of a constitutive ,-subunit and one of two alternative oxygen-regulated HIF, subunits (HIF-1, and HIF-2,). In the presence of oxygen (normoxia) the HIF, subunits are hydroxylated, which targets them for proteasomal degradation. Under hypoxia, because of the lack of molecular oxygen, HIF cannot be hydroxylated and is thereby stabilized. Although HIF-1, was the first transcription factor identified through its ability to bind to an enhancer sequence of the EPO gene, more recent evidence suggests that HIF-2, is responsible for the regulation of EPO. Although EPO is a prime example for an oxygen- regulated gene, the role of the HIF system goes far beyond the regulation of EPO, because it operates widely in almost all cells and controls a broad transcriptional response to hypoxia, including genes involved in cell metabolism, angiogenesis and vascular tone. Further evidence suggests that apart from its effect as an erythropoietic hormone EPO acts as a paracrine, tissue-protective protein in the brain and possibly also in other organs. [source]

Viral infection and Toll-like receptor agonists induce a differential expression of type,I and , interferons in human plasmacytoid and monocyte-derived dendritic cells

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 3 2004
Eliana
Abstract In humans, the type,I interferon (IFN) family consists of 13 IFN-, subtypes, IFN-, and IFN-o the newly discovered IFN-like family consists of IFN-,1, -,2 and -,3. We have investigated the expression of type,I and , IFN genes following virus infections or Toll-like receptor (TLR) triggering in monocyte-derived DC (MDDC) and plasmacytoid DC (pDC). We found thatall IFN-,, -,, -o and -, subtypes are expressed in influenza-virus-infected MDDC or pDC. Conversely, differential type,I IFN gene transcription was induced in MDDC and pDC stimulated by specific TLR agonists. TLR-9 stimulation by CpG DNA induced the expression of all IFN-,, -,, -o and -, subtypes in pDC, whereas TLR-4 stimulation by LPS, or TLR-3 stimulation bypoly I:C, induced only IFN-, and IFN-, gene expression in MDDC. The expression pattern of IFN regulatory factor (IRF)-5 and IRF-7 in MDDC and pDC was also determined. IRF-5 was constitutively expressed in the two DC subsets whereas IRF-7 was constitutive in pDC but its expression was induced along MDDC maturation. Overall, our data indicate that the coordinated expression of IFN-, with IFN-, would be of crucial importance for the maturation of DC. [source]

p38 MAPK is a critical regulator of the constitutive and the ,4,integrin-regulated expression of IL-6 in human normal thymic epithelial cells

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 11 2003
Fabrizio Mainiero
Abstract Cytokines and adhesion receptors are key mediators in the dialog occurring between thymic epithelial cells (TEC) and thymocytes and regulating T,cell maturation and epithelial embryonic differentiation. Among cytokines, IL-6 can be critical in the thymus, fostering proliferation, differentiation and/or survival of both TEC and thymocytes. We have previously reported in human normal TEC that clustering of the laminin receptor ,6,4 integrin induced by thymocyte contact or monoclonal antibody-mediated cross-linking regulates IL-6 gene expression via activation of NF-,B and NF-IL6 transactivators. Here we show that ,6,4 integrin activates p38 mitogen-activated protein kinase (MAPK) and that p38 is essential for IL-6 gene expression. In fact, ,4 cross-linking activated p38 and extracellular signal-regulated kinase (ERK) MAPK, Rac1, p21-activated protein kinase,1 (PAK1) and MAPK kinases (MKK),3/MKK6. However, pharmacological blockade of p38 or ERK demonstrated that p38 inhibition abrogated both basal and ,4,integrin-induced production of IL-6 preventing NF-,B and NF-IL6 activation, whereas ERK inhibition reduced IL-6 production, hampering only NF-,B activation. Overall, our results indicate that p38 MAPK and ,6,4,integrin, expressed by TEC throughout their life, are critical regulators of the intrathymic availability of a cytokine controlling fate and functions of cells governing development and maintenance of thymic architecture and immune responses. [source]

Long-term depression activates transcription of immediate early transcription factor genes: involvement of serum response factor/Elk-1

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2006
Antje Lindecke
Abstract Long-term depression (LTD) is one of the paradigms used in vivo or ex vivo for studying memory formation. In order to identify genes with potential relevance for memory formation we used mouse organotypic hippocampal slice cultures in which chemical LTD was induced by applications of 3,5-dihydroxyphenylglycine (DHPG). The induction of chemical LTD was robust, as monitored electrophysiologically. Gene expression analysis after chemical LTD induction was performed using cDNA microarrays containing >7000 probes. The DHPG-induced expression of immediate early genes (c-fos, junB, egr1 and nr4a1) was subsequently verified by TaqMan polymerase chain reaction. Bioinformatic analysis suggested a common regulator element [serum response factor (SRF)/Elk-1 binding sites] within the promoter region of these genes. Indeed, here we could show a DHPG-dependent binding of SRF at the SRF response element (SRE) site within the promoter region of c-fos and junB. However, SRF binding to egr1 promoter sites was constitutive. The phosphorylation of the ternary complex factor Elk-1 and its localization in the nucleus of hippocampal neurones after DHPG treatment was shown by immunofluorescence using a phosphospecific antibody. We suggest that LTD leads to SRF/Elk-1-regulated gene expression of immediate early transcription factors, which could in turn promote a second broader wave of gene expression. [source]

Interaction with calmodulin is important for the secretion of thimet oligopeptidase following stimulation

FEBS JOURNAL, Issue 16 2009
Lilian C. Russo
Thimet oligopeptidase (EC 3.4.24.15; EP24.15) was originally described as a neuropeptide-metabolizing enzyme, highly expressed in the brain, kidneys and neuroendocrine tissue. EP24.15 lacks a typical signal peptide sequence for entry into the secretory pathway and is secreted by cells via an unconventional and unknown mechanism. In this study, we identified a novel calcium-dependent interaction between EP24.15 and calmodulin, which is important for the stimulated, but not constitutive, secretion of EP24.15. We demonstrated that, in vitro, EP24.15 and calmodulin physically interact only in the presence of Ca2+, with an estimated Kd value of 0.52 ,m. Confocal microscopy confirmed that EP24.15 colocalizes with calmodulin in the cytosol of resting HEK293 cells. This colocalization markedly increases when cells are treated with either the calcium ionophore A23187 or the protein kinase A activator forskolin. Overexpression of calmodulin in HEK293 cells is sufficient to greatly increase the A23187-stimulated secretion of EP24.15, which can be inhibited by the calmodulin inhibitor calmidazolium. The specific inhibition of protein kinase A with KT5720 reduces the A23187-stimulated secretion of EP24.15 and inhibits the synergistic effects of forskolin with A23187. Treatment with calmidazolium and KT5720 nearly abolishes the stimulatory effects of A23187 on EP24.15 secretion. Together, these data suggest that the interaction between EP24.15 and calmodulin is regulated within cells and is important for the stimulated secretion of EP24.15 from HEK293 cells. Structured digital abstract ,,MINT-7148420: EP24.15 (uniprotkb:P52888) and Calmodulin (uniprotkb:P62161) bind (MI:0407) by surface plasmon resonance (MI:0107) ,,MINT-7148437: EP24.15 (uniprotkb:P52888) and Calmodulin (uniprotkb:P62158) colocalize (MI:0403) by surface plasmon resonance (MI:0107) ,,MINT-7148406: Calmodulin (uniprotkb:P62161) binds (MI:0407) to EP24.15 (uniprotkb:P52888) by pull down (MI:0096) [source]

Suppression of urokinase receptor expression by bikunin is associated with inhibition of upstream targets of extracellular signal-regulated kinase-dependent cascade

FEBS JOURNAL, Issue 16 2002
Hiroshi Kobayashi
Our laboratory showed that bikunin, a Kunitz-type protease inhibitor, suppresses 4,-phorbol 12-myristate 13-acetate (PMA)- or tumor necrosis factor-alpha (TNF,)-induced urokinase-type plasminogen activator (uPA) expression in different cell types. In addition to its effects on protease inhibition, bikunin could be modulating other cellular events associated with the metastatic cascade. To test this hypothesis, we examined whether bikunin was able to suppress the expression of uPA receptor (uPAR) mRNA and protein in a human chondrosarcoma cell line, HCS-2/8, and two human ovarian cancer cell lines, HOC-I and HRA. The present study showed that (a) bikunin suppresses the expression of constitutive and PMA-induced uPAR mRNA and protein in a variety of cell types; (b) an extracellular signal-regulated kinase (ERK) activation system is necessary for the PMA-induced increase in uPAR expression, as PD098059 and U0126, which prevent the activation of MEK1, reduce the uPAR expression; (c) bikunin markedly suppresses PMA-induced phosphorylation of ERK1/2 at the concentration that prevents uPAR expression, but does not reduce total ERK1/2 antigen level; (d) bikunin has no ability to inhibit overexpression of uPAR in cells treated with sodium vanadate; and (e) we further studied the inhibition of uPAR expression by stable transfection of HRA cells with bikunin gene, demonstrating that bikunin secretion is necessary for inhibition of uPAR expression. We conclude that bikunin downregulates constitutive and PMA-stimulated uPAR mRNA and protein possibly through suppression of upstream targets of the ERK-dependent cascade, independent of whether cells were treated with exogenous bikunin or transfected with bikunin gene. [source]

Expression of penicillin G acylase from the cloned pac gene of Escherichia coli ATCC11105

FEBS JOURNAL, Issue 5 2001
Effects of pacR, temperature
The structural gene pac in Eschericia coli ATCC11105 encodes penicillin G acylase (PGA). Within the pac gene, there is a regulatory gene pacR, which is transcribed in the opposite direction. Site-directed mutagenesis was performed at base 1045 of pac by replacing a T with a C. This substitution did not alter the amino-acid sequence of PGA, but changed the translation start codon of pacR from AUG to GUG. The expression of the mutant pacR decreased dramatically and the lacZ transcriptional fusion analysis showed that GUG was an extremely poor initiation codon for pacR. The pacR mutation caused PGA expression to be constitutive rather than inductive in two strains (E. coli A56, DH10B). The pac inducer phenylacetic acid (PAA) gave significant induction of PGA production at a concentration of 0.2% in wild type, but PAA at this concentration inhibited both cell growth and PGA production in the pacR mutated strains. The temperature-dependent expression character of pac is preserved in the pacR translation-initiation mutant and the optimum temperature of PGA production was 22 °C in both wild type and mutant. At a higher temperature of 37 °C, the PGA precursor polypeptide could not be matured into subunits and formed inclusion bodies, as revealed by western blot analysis. Our investigations confirmed the hypothesis of pacR-mediated PAA induction for PGA expression and clarified the inhibitory effect of high temperature upon the post-translational processing of the PGA precursor polypeptide. [source]

Kinetic evidences for facilitation of peptide channelling by the proteasome activator PA28

FEBS JOURNAL, Issue 20 2000
Ralf Stohwasser
The activation kinetics of constitutive and IFN,-stimulated 20S proteasomes obtained with homomeric (recPA28,, recPA28,) and heteromeric (recPA28,,) forms of recombinant 11S regulator PA28 was analysed by means of kinetic modelling. The activation curves obtained with increasing concentrations of the individual PA28 subunits (RecP28,/RecP28,/RecP28,+ RecP28,) exhibit biphasic characteristics which can be attributed to a low-level activation by PA28 monomers and full proteasome activation by assembled activator complexes. The dissociation constants do not reveal significant differences between the constitutive and the immunoproteasome. Intriguingly, the affinity of the proteasome towards the recPA28,, complex is about two orders of magnitude higher than towards the homomeric PA28, and PA28, complexes. Striking similarities can been revealed in the way how PA28 mediates the kinetics of latent proteasomes with respect to three different fluorogenic peptides probing the chymotrypsin-like, trypsin-like and peptidylglutamyl-peptide hydrolyzing like activity: (a) positive cooperativity disappears as indicated by a lack of sigmoid initial parts of the kinetic curves, (b) substrate affinity is increased, whereby (c), the maximal activity remains virtually constant. As these kinetic features are independent of the peptide substrates, we conclude that PA28 exerts its activating influence on the proteasome by enhancing the uptake (and release) of shorter peptides. [source]

Multiple roles of PPAR, in brown adipose tissue under constitutive and cold conditions

GENES TO CELLS, Issue 2 2010
Makiko Komatsu
Peroxisome proliferator-activated receptor , (PPAR,) is a member of the nuclear receptor family, regulating fatty acid degradation in many organs. Two-dimensional SDS-PAGE of brown adipose tissue (BAT) from PPAR,-null mice produced a higher-density spot. Proteomic analysis indicated that the protein was pyruvate dehydrogenase , (PDH,). To observe PDH, regulation in BAT, the organ was stimulated by long-term cold exposure, and the activities of associated enzymes were investigated. Histological and biochemical analyses of BAT showed a significant decrease in the triglyceride content in wild-type mice and some degree of decrease in PPAR,-null mice on cold exposure. Analyses of molecules related to glucose metabolism showed that the expression of PDH, is under PPAR,-specific regulation, and that glucose degradation ability may decrease on cold exposure. In contrast, analyses of molecules related to fatty acid metabolism showed that numerous PPAR,/, target molecules are induced on cold exposure, and that fatty acid degradation ability in wild-type mice is markedly enhanced and also increases to same degree in PPAR,-null mice on cold exposure. Thus, this study proposes novel and multiple roles of PPAR, in BAT. [source]

Clock-dependent and independent transcriptional control of the two isoforms from the mouse Ror,gene

GENES TO CELLS, Issue 12 2008
Valérie Mongrain
Accumulating evidence indicate that molecular mechanisms generating circadian rhythms display some degree of tissue-specificity. More specifically, distinct patterns of expression for nuclear receptors of the ROR family indicate that the transcriptional control of the clock gene Bmal1 differs among tissues. This study aims to investigate the expression of Ror,isoforms (Ror, and Ror,t) and characterize the molecular mechanisms underlying their tissue-specific expression. The expression of Ror, isoforms was assessed in mouse liver, muscle, thymus and testis throughout 24 h using quantitative RT-PCR. Although the expression of Ror, was rhythmic in the liver and thymus, it was constitutively expressed in muscle and testis. In contrast, the expression of Ror,t was constitutive in all four tissues. Furthermore, rhythmic expression of Ror, was impaired in Clock mutant mice whereas the mutation had no effect on Ror,t expression. In line with these findings, luciferase assays revealed that transcription of the Ror, promoter is clock-controlled whereas that of Ror,t promoter is essentially clock-independent. Our results provide insights into the molecular mechanisms that lead to differential expression of Ror, and Ror,t and are suggestive of a framework that might account for tissue-specific circadian regulation. [source]

Role for CXCR2 and CXCL1 on glia in multiple sclerosis

GLIA, Issue 1 2006
Kakuri M. Omari
Abstract As part of a need to understand myelin repair mechanisms, molecular pathways underlying oligodendrocyte behavior and central nervous system (CNS) remyelination are currently key topics in multiple sclerosis (MS). In the present study, we report expression of a chemoattractant receptor of the immune system, the chemokine receptor, CXCR2, on normal and proliferating oligodendrocytes in active MS lesions. Proliferating oligodendrocytes were occasionally associated with reactive astrocytes positive for CXCL1 (GRO-,), the ligand for CXCR2. CXCL1 expression was not seen on astrocytes in control and normal CNS tissue, while CXCR2 expression was constitutive on oligodendrocytes. At the functional level, following stimulation with the proinflammatory cytokine, interleukin-1, (IL-1,), we found high-level synthesis of CXCL1 by human fetal astrocytes in vitro. In contrast, human oligodendrocytes in culture expressed the receptor, CXCR2, constitutively. We propose that the concurrence of CXCR2 on oligodendrocytes and induced CXCL1 on hypertrophic astrocytes in MS provides a novel mechanism for recruitment of oligodendrocytes to areas of damage, an essential prerequisite for lesion repair in this devastating human condition. © 2005 Wiley-Liss, Inc. [source]

Principles for Public Management Practice: From Dichotomies to Interdependence

GOVERNANCE, Issue 3 2001
Martha S. Feldman
In this essay we explore the relationship between management practices and a basic governance dilemma: how to manage flexibly and accountably. The challenge is both practical and theoretical. Managers must respond flexibly to the changing demands and expectations of the public and the ever-changing nature of public problems, yet they must do so in a manner that provides accountability to the public and political overseers. A dichotomous approach to the study of leadership as management action and the governance structures within which managers operate has inhibited the search for a public management theory that reconciles the dilemma. Emphasis upon managers as leaders typically focuses on the flexible actions managers might take to overcome structural "barriers," while emphasis upon governance structures typically focuses on the essential role of structure in ensuring accountability and restraining or motivating particular management efforts. The practicing manager, however, cannot deal with these aspects of the work separately. Managers must attend to demands for both flexible leadership action and structures that promise accountability. Anecdotal evidence provides illustrations of some of the ways that managers can integrate these demands. We suggest that these efforts point to an alternative theoretical framework that understands action and structure as mutually constitutive, creating a dynamic tension in which attention to one requires attention to the other. [source]

Enhanced expression of B7-1, B7-2, and intercellular adhesion molecule 1 in sinusoidal endothelial cells by warm ischemia/reperfusion injury in rat liver

HEPATOLOGY, Issue 4 2001
Naosuke Kojima
To elucidate a role of costimulatory molecule and cell adhesion molecule in hepatic ischemia/reperfusion injury, we examined an alteration in B7-1 (CD80), B7-2 (CD86), and intercellular adhesion molecule 1 (ICAM-1; CD54) expression in the rat liver after warm ischemia/reperfusion injury. To induce hepatic warm ischemia in a rat model, both portal vein and hepatic artery entering the left-lateral and median lobes were occluded by clamping for 30 minutes or 60 minutes, and then reperfused for 24 hours. B7-1, B7-2, and ICAM-1 expressions in the liver were analyzed by immunofluorescence staining and real-time reverse transcription polymerase chain reaction (RT-PCR). Although B7-1 and B7-2 expressions were at very low levels in the liver tissues from normal or sham-operated control rats, both B7-1 and B7-2 expressions were enhanced at protein and messenger RNA (mRNA) levels in the affected, left lobes after warm ischemia/reperfusion. ICAM-1 protein and mRNA were constitutively expressed in the liver of normal and sham-operated control rats, and further up-regulated after warm ischemia/reperfusion. Localization of increased B7-1, B7-2, and ICAM-1 proteins, as well as von Willebrand factor as a marker protein for endothelial cells, was confined by immunofluorescence staining to sinusoidal endothelial cells in hepatic lobules. Data from quantitative real-time RT-PCR analysis revealed that B7-1 and B7-2 mRNA levels were elevated in hepatic lobes after warm ischemia/reperfusion (5.13- and 52.9-fold increase, respectively), whereas ICAM-1 mRNA expression was rather constitutive but further enhanced by warm ischemia/reperfusion (4.24-fold increase). These results suggest that hepatic sinusoidal endothelial cells play a pivotal role as antigen-presenting cells by expressing B7-1 and B7-2 in warm hepatic ischemia/reperfusion injury, and that B7-1 and/or B7-2 might be the primary target to prevent early rejection and inflammatory reactions after hepatic ischemia/reperfusion injury associated with liver transplantation. [source]

Nabobs Revisited: A Cultural History of British Imperialism and the Indian Question in Late-Eighteenth-Century Britain

HISTORY COMPASS (ELECTRONIC), Issue 4 2006
Tillman W. Nechtman
Studies of the late eighteenth-century British empire in India have long used the figure of the nabob to personify political debates collectively known as "the India question." These nabobs, employees of the East India Company, were (and continue to be) represented as rapacious villains. This article will revisit the history of nabobs to offer a cultural history of British imperialism in late eighteenth-century India. It will argue that nabobs were representative figures in the political debates surrounding imperialism in South Asia because they were hybrid figures who made Britain's empire more real to domestic British observers. It will argue that the nabobs' hybrid identity hinged on the collection of material artifacts they brought back to Britain from India. Nabobs stood at the boundary between nation and empire, and they suggested the frontier was permeable. They exposed the degree to which the projects of building a nation and an empire were mutually constitutive. [source]