bluuebunny/crossref_metadata_2025 huggingface dataset:
NFκB activation by Fas is mediated through FADD, caspase-8, and RIP and is inhibited by FLIP
Authors: Kreuz Sebastian , Siegmund Daniela , Rumpf Jost-Julian , Samel Dierk , Leverkus Martin , Janssen Ottmar , Häcker Georg , Dittrich-Breiholz Oliver , Kracht Michael , Scheurich Peter , Wajant Harald
Published: August 2004

Fas (APO-1/CD95) is the prototypic death receptor, and the molecular mechanisms of Fas-induced apoptosis are comparably well understood. Here, we show that Fas activates NFκB via a pathway involving RIP, FADD, and caspase-8. Remarkably, the enzymatic activity of the latter was dispensable for Fas-induced NFκB signaling pointing to a scaffolding-related function of caspase-8 in nonapoptotic Fas signaling. NFκB was activated by overexpressed FLIPL and FLIPS in a cell type–specific manner. However, in the context of Fas signaling both isoforms blocked FasL-induced NFκB activation. Moreover, down-regulation of both endogenous FLIP isoforms or of endogenous FLIPL alone was sufficient to enhance FasL-induced expression of the NFκB target gene IL8. As NFκB signaling is inhibited during apoptosis, FasL-induced NFκB activation was most prominent in cells that were protected by Bcl2 expression or caspase inhibitors and expressed no or minute amounts of FLIP. Thus, protection against Fas-induced apoptosis in a FLIP-independent manner converted a proapoptotic Fas signal into an inflammatory NFκB-related response.
β-Catenin is required for endothelial-mesenchymal transformation during heart cushion development in the mouse
Authors: Liebner Stefan , Cattelino Anna , Gallini Radiosa , Rudini Noemi , Iurlaro Monica , Piccolo Stefano , Dejana Elisabetta
Published: August 2004

During heart development endocardial cells within the atrio-ventricular (AV) region undergo TGFβ-dependent epithelial-mesenchymal transformation (EMT) and invade the underlying cardiac jelly. This process gives rise to the endocardial cushions from which AV valves and part of the septum originate. In this paper we show that in mouse embryos and in AV explants TGFβ induction of endocardial EMT is strongly inhibited in mice deficient for endothelial β-catenin, leading to a lack of heart cushion formation. Using a Wnt-signaling reporter mouse strain, we demonstrated in vivo and ex vivo that EMT in heart cushion is accompanied by activation of β-catenin/TCF/Lef transcriptional activity. In cultured endothelial cells, TGFβ2 induces α-smooth muscle actin (αSMA) expression. This process was strongly reduced in β-catenin null cells, although TGFβ2 induced smad phosphorylation was unchanged. These data demonstrate an involvement of β-catenin/TCF/Lef transcriptional activity in heart cushion formation, and suggest an interaction between TGFβ and Wnt-signaling pathways in the induction of endothelial-mesenchymal transformation.
Mouse centric and pericentric satellite repeats form distinct functional heterochromatin
Authors: Guenatri Mounia , Bailly Delphine , Maison Christèle , Almouzni Geneviève
Published: August 2004

Heterochromatin is thought to play a critical role for centromeric function. However, the respective contributions of the distinct repetitive sequences found in these regions, such as minor and major satellites in the mouse, have remained largely unsolved. We show that these centric and pericentric repeats on the chromosomes have distinct heterochromatic characteristics in the nucleus. Major satellites from different chromosomes form clusters associated with heterochromatin protein 1α, whereas minor satellites are individual entities associated with centromeric proteins. Both regions contain methylated histone H3 (Me-K9 H3) but show different micrococcal nuclease sensitivities. A dinucleosome repeating unit is found specifically associated with major satellites. These domains replicate asynchronously, and chromatid cohesion is sustained for a longer time in major satellites compared with minor satellites. Such prolonged cohesion in major satellites is lost in the absence of Suv39h histone methyltransferases. Thus, we define functionally independent centromeric subdomains, which spatio-temporal isolation is proposed to be important for centromeric cohesion and dissociation during chromosome segregation.
Prevention and Treatment of Experimental Autoimmune Encephalomyelitis by Soluble CD83
Authors: Zinser Elisabeth , Lechmann Matthias , Golka Antje , Lutz Manfred B. , Steinkasserer Alexander
Published: August 2004

CD83 is up-regulated on the surface of dendritic cells (DCs) during maturation and has been widely used as a marker for mature DCs. Recently, we reported the recombinant expression of the extracellular immunoglobulin domain of human CD83 (hCD83ext). Using this soluble form of CD83, allogeneic as well as specific cytotoxic T lymphocyte proliferation could be blocked in vitro. Here we report the functional analysis of soluble CD83 in vivo, using murine experimental autoimmune encephalomyelitis (EAE) as a model. Strikingly, only three injections of soluble CD83 prevented the paralysis associated with EAE almost completely. In addition, even when the EAE was induced a second time, CD83-treated mice were protected, indicating a long-lasting suppressive effect. Furthermore, soluble CD83 strongly reduced the paralysis in different therapeutic settings. Most important, even when the treatment was delayed until the disease symptoms were fully established, soluble CD83 clearly reduced the paralyses. In addition, also when EAE was induced a second time, soluble CD83-treated animals showed reduced disease symptoms. Finally, hCD83ext treatment almost completely reduced leukocyte infiltration in the brain and in the spinal cord. In summary, this work strongly supports an immunosuppressive role of soluble CD83, thereby indicating its therapeutic potential in the regulation of immune disorders in vivo.
Mesothelin-specific CD8+ T Cell Responses Provide Evidence of In Vivo Cross-Priming by Antigen-Presenting Cells in Vaccinated Pancreatic Cancer Patients
Authors: Thomas Amy Morck , Santarsiero Lynn M. , Lutz Eric R. , Armstrong Todd D. , Chen Yi-Cheng , Huang Lan-Qing , Laheru Daniel A. , Goggins Michael , Hruban Ralph H. , Jaffee Elizabeth M.
Published: August 2004

Tumor-specific CD8+ T cells can potentially be activated by two distinct mechanisms of major histocompatibility complex class I–restricted antigen presentation as follows: direct presentation by tumor cells themselves or indirect presentation by professional antigen-presenting cells (APCs). However, controversy still exists as to whether indirect presentation (the cross-priming mechanism) can contribute to effective in vivo priming of tumor-specific CD8+ T cells that are capable of eradicating cancer in patients. A clinical trial of vaccination with granulocyte macrophage–colony stimulating factor–transduced pancreatic cancer lines was designed to test whether cross-presentation by locally recruited APCs can activate pancreatic tumor-specific CD8+ T cells. Previously, we reported postvaccination delayed-type hypersensitivity (DTH) responses to autologous tumor in 3 out of 14 treated patients. Mesothelin is an antigen demonstrated previously by gene expression profiling to be up-regulated in most pancreatic cancers. We report here the consistent induction of CD8+ T cell responses to multiple HLA-A2, A3, and A24-restricted mesothelin epitopes exclusively in the three patients with vaccine-induced DTH responses. Importantly, neither of the vaccinating pancreatic cancer cell lines expressed HLA-A2, A3, or A24. These results provide the first direct evidence that CD8 T cell responses can be generated via cross-presentation by an immunotherapy approach designed to recruit APCs to the vaccination site.
Highly Selective Escape from KSHV-mediated Host mRNA Shutoff and Its Implications for Viral Pathogenesis
Authors: Glaunsinger Britt , Ganem Don
Published: August 2004

During Kaposi's sarcoma (KS)–associated herpesvirus (KSHV) lytic infection, many virus-encoded signaling molecules (e.g., viral G protein–coupled receptor [vGPCR]) are produced that can induce host gene expression in transiently transfected cells, and roles for such induced host genes have been posited in KS pathogenesis. However, we have recently found that host gene expression is strongly inhibited by 10–12 h after lytic reactivation of KSHV, raising the question of whether and to what extent de novo host gene expression induced by viral signaling molecules can proceed during the lytic cycle. Here, we show by microarray analysis that expression of most vGPCR target genes is drastically curtailed by this host shutoff. However, rare cellular genes can escape the host shutoff and are potently up-regulated during lytic KSHV growth. Prominent among these is human interleukin-6, whose striking induction may contribute to the overexpression of this cytokine in several disease states linked to KSHV infection.
Intracellular Triggering of Fas Aggregation and Recruitment of Apoptotic Molecules into Fas-enriched Rafts in Selective Tumor Cell Apoptosis
Authors: Gajate Consuelo , del Canto-Jañez Esther , Acuña A. Ulises , Amat-Guerri Francisco , Geijo Emilio , Santos-Beneit Antonio M. , Veldman Robert J. , Mollinedo Faustino
Published: August 2004

We have discovered a new and specific cell-killing mechanism mediated by the selective uptake of the antitumor drug 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3, Edelfosine) into lipid rafts of tumor cells, followed by its coaggregation with Fas death receptor (also known as APO-1 or CD95) and recruitment of apoptotic molecules into Fas-enriched rafts. Drug sensitivity was dependent on drug uptake and Fas expression, regardless of the presence of other major death receptors, such as tumor necrosis factor (TNF) receptor 1 or TNF-related apoptosis-inducing ligand R2/DR5 in the target cell. Drug microinjection experiments in Fas-deficient and Fas-transfected cells unable to incorporate exogenous ET-18-OCH3 demonstrated that Fas was intracellularly activated. Partial deletion of the Fas intracellular domain prevented apoptosis. Unlike normal lymphocytes, leukemic T cells incorporated ET-18-OCH3 into rafts coaggregating with Fas and underwent apoptosis. Fas-associated death domain protein, procaspase-8, procaspase-10, c-Jun amino-terminal kinase, and Bid were recruited into rafts, linking Fas and mitochondrial signaling routes. Clustering of rafts was necessary but not sufficient for ET-18-OCH3–mediated cell death, with Fas being required as the apoptosis trigger. ET-18-OCH3–mediated apoptosis did not require sphingomyelinase activation. Normal cells, including human and rat hepatocytes, did not incorporate ET-18-OCH3 and were spared. This mechanism represents the first selective activation of Fas in tumor cells. Our data set a framework for the development of more targeted therapies leading to intracellular Fas activation and recruitment of downstream signaling molecules into Fas-enriched rafts.
Tumor Necrosis Factor (TNF) Receptor Shedding Controls Thresholds of Innate Immune Activation That Balance Opposing TNF Functions in Infectious and Inflammatory Diseases
Authors: Xanthoulea Sofia , Pasparakis Manolis , Kousteni Stavroula , Brakebusch Cord , Wallach David , Bauer Jan , Lassmann Hans , Kollias George
Published: August 2004

Tumor necrosis factor (TNF) is a potent cytokine exerting critical functions in the activation and regulation of immune and inflammatory responses. Due to its pleiotropic activities, the amplitude and duration of TNF function must be tightly regulated. One of the mechanisms that may have evolved to modulate TNF function is the proteolytic cleavage of its cell surface receptors. In humans, mutations affecting shedding of the p55TNF receptor (R) have been linked with the development of the TNFR-associated periodic syndromes, disorders characterized by recurrent fever attacks and localized inflammation. Here we show that knock-in mice expressing a mutated nonsheddable p55TNFR develop Toll-like receptor–dependent innate immune hyperreactivity, which renders their immune system more efficient at controlling intracellular bacterial infections. Notably, gain of function for antibacterial host defenses ensues at the cost of disbalanced inflammatory reactions that lead to pathology. Mutant mice exhibit spontaneous hepatitis, enhanced susceptibility to endotoxic shock, exacerbated TNF-dependent arthritis, and experimental autoimmune encephalomyelitis. These results introduce a new concept for receptor shedding as a mechanism setting up thresholds of cytokine function to balance resistance and susceptibility to disease. Assessment of p55TNFR shedding may thus be of prognostic value in infectious, inflammatory, and autoimmune diseases.
Wavelets with composite dilations
Authors: Guo Kanghui , Labate Demetrio , Lim Wang-Q , Weiss Guido , Wilson Edward
Published: August 2004

A wavelet with composite dilations is a function generating an orthonormal basis or a Parseval frame for

L
2

(

R

n

)

L^2({\mathbb R}^n)

under the action of lattice translations and dilations by products of elements drawn from non-commuting matrix sets

A
A

and

B
B

. Typically, the members of

B
B

are shear matrices (all eigenvalues are one), while the members of

A
A

are matrices expanding or contracting on a proper subspace of

R

n

{\mathbb R}^n

. These wavelets are of interest in applications because of their tendency to produce “long, narrow” window functions well suited to edge detection. In this paper, we discuss the remarkable extent to which the theory of wavelets with composite dilations parallels the theory of classical wavelets, and present several examples of such systems.
Chandler in a Larger Frame: Markets, Transaction Costs, and Organizational Form in History
Authors: Langlois Richard N.
Published: August 2004

In 1977, when Alfred D. Chandler's pathbreaking bookThe Visible Handappeared, the large, vertically integrated, “Chandlerian” corporation had dominated the organizational landscape for nearly a century. In some interpretations, possibly including Chandler's own,The Visible Handand subsequent works constitute a triumphalist account of the rise of that organizational form: the large, vertically integrated firm arose and prospered because of its inherent superiority, in all times and places, to more decentralized, market-oriented production arrangements. A quarter century later, however, the Chandlerian firm no longer dominates the landscape. It is under siege from a panoply of decentralized and market-like forms that often resemble some of the “inferior” nineteenth-century structures that the managerial enterprise had replaced.
Against Whig History
Authors: Lamoreaux Naomi R. , Raff Daniel M. G. , Temin Peter
Published: August 2004

By the end of the twentieth century, it had become clear that the grand synthesis laid out by Alfred D. Chandler, Jr., inThe Visible Hand, Scale and Scope, and other writings was badly in need of revision. It was in need of revision because the type of enterprise that Chandler took to be the acme of capitalist economic organization–the large, vertically integrated, horizontally diversified, managerially directed corporation–was clearly in retreat. This development cast into doubt not only the substantive content of Chandler’s interpretation but also its methodological underpinnings.
Neither Modularity nor Relational Contracting: Inter-Firm Collaboration in the New Economy
Authors: Sabel Charles F. , Zeitlin Jonathan
Published: August 2004

In a series of recent essays, including their contributions to this symposium, Richard N. Langlois and Naomi R. Lamoreaux, Daniel M. G. Raff, and Peter Temin (hereafter LRT) present interesting but contradictory views of the decentralized or vertically disintegrated post-Chandlerian economy from whose vantage point they seek to reconceptualize business history. Starting from an orientation that uneasily combines Adam Smith’s ideas about the division of labor with organizational learning, Langlois sees the current situation as dominated by the modularization of production. This modularization and the arm’s-length transactions it facilitates create a world reminiscent of the antebellum United States, although today’s highthroughput differentiated exchanges are underpinned by a set of market-supporting institutions, notably standard interfaces or design rules. Starting from an orientation toward Oliver Williamson and the minimization of coordination costs, LRT in contrast see a world of collaborators joined by long-term, largely informal relations of a distinct type reducible neither to markets nor hierarchies.
The End of Managerial Ideology: From Corporate Social Responsibility to Corporate Social Indifference
Authors: Englander Ernie , Kaufman Allen
Published: August 2004

During the 1990s, U.S. managerial capitalism underwent a profound transformation from a technocratic to a “proprietary” form. In the technocratic era, managers had functioned as teams to sustain the firm and to promote social welfare by satisfying the demands of competing stakeholders. In the new proprietary era, corporate bureaucratic teams broke up into tournaments in which managers competed for advancement toward the CEO prize. The reward system of the new era depended heavily on stock options that were accompanied by downside risk protection. The tournaments turned managers into a special class of shareholders who sought to maximize their individual utility functions even if deviating from the firm's best interest. Once this new regime became established, managers discarded their technocratic, stakeholder creed and adopted a property rights ideology, originally elaborated in academia by financial agency theorists. Managers hardly noticed (or cared) they were capturing a disproportionate share of the new wealth being generated in the U.S. economy. When critics brought this fact to light, managers replied like well-schooled economists: markets worked efficiently. Whether they worked fairly was a question they did not address.
The German Octopus: The British Metal Corporation and the Next War, 1914–1939
Authors: Ball Simon
Published: August 2004

After World War I, the nonferrous metals trade played a critical role in Britain's preparations for a future European war. Yet it has attracted little attention. The British Metal Corporation (BMC) was formed at the end of the Great War as a state-sponsored corporation to conduct the development of the nonferrous metals industry on behalf of the British state. This article uses the papers of the BMC to explore the politics of a strategically vital trade, the functioning of the British state's institutional memory, and the role of business in appeasement and rearmament. It concludes that the state-sponsored corporation was, on balance, an effective strategic instrument. Although politics, trade, and strategy proved difficult to reconcile with one another, the pursuit of profit did not dictate business attitudes toward Nazi Germany in the 1930s.
Corporate Governance and the Eclectic Paradigm: The Investment Motives of Philips in Taiwan in the 1960s
Authors: van der Putten Frans-Paul
Published: August 2004

Today Philips is one of the largest foreign investors in East Asia. The foundation for the company's East Asian position was laid by investment in Taiwan in the 1960s and early 1970s, at a time when this country and the rest of the region were still largely ignored by other European investors. This article investigates the motives for Philips to play a pioneering role among European firms in large-scale investing in Taiwan. The author uses the traditional method for direct investment analysis, known as Dunning's eclectic paradigm. He also addresses shortcomings in this method regarding individual company behavior, by adding a corporate governance analysis that is based on Freeman's stakeholder theory. The resulting analysis shows that, although locational factors constituted the main precondition to direct investment by Philips, the crucial reason Philips made its early investments in Taiwan was its chief executive officer's enthusiasm for this particular project.
The Candida albicans pH-regulated KER1 gene encodes a lysine/glutamic-acid-rich plasma-membrane protein that is involved in cell aggregation
Authors: Galán Amparo , Casanova Manuel , Murgui Amelia , MacCallum Donna M. , Odds Frank C. , Gow Neil A. R. , Martínez José P.
Published: August 2004

Immunoscreening of aCandida albicanscDNA library with a polyclonal germ-tube-specific antibody (pAb anti-gt) resulted in the isolation of a gene encoding a lysine/glutamic-acid-rich protein, which was consequently designatedKER1. The nucleotide and deduced amino acid sequences of this gene displayed no significant homology with any other known sequence.KER1encodes a 134 kDa lysine (14·5 %)/glutamic acid (16·7 %) protein (Ker1p) that contains two potential transmembrane segments.KER1was expressed in a pH-conditional manner, with maximal expression at alkaline pH and lower expression at pH 4·0, and was regulated byRIM101. A Δker1/Δker1null mutant grew normally but was hyperflocculant under germ-tube-inducing conditions, yet this behaviour was also observed in stationary-phase cells grown under other incubation conditions. Western blotting analysis of different subcellular fractions, using as a probe a monospecific polyclonal antibody raised against a highly antigenic domain of Ker1p (pAb anti-Ker1p), revealed the presence of a 134 kDa band in the purified plasma-membrane fraction from the wild-type strain that was absent in the homologous preparation from Δker1/Δker1mutant. The pattern of cell-wall protein and mannoprotein species released by digestion withβ-glucanases, reactive towards pAbs anti-gt and anti-Ker1p, as well as against concanavalin A, was also different in the Δker1/Δker1mutant. Mutant strains also displayed an increased cell-surface hydrophobicity and sensitivity to Congo red and Calcofluor white. Overall, these findings indicate that the mutant strain was affected in cell-wall composition and/or structure. The fact that theker1mutant had attenuated virulence in systemic mouse infections suggests that this surface protein is also important in host–fungus interactions.
Biofilms promote altruism
Authors: Kreft Jan-Ulrich
Published: August 2004

The origin of altruism is a fundamental problem in evolution, and the maintenance of biodiversity is a fundamental problem in ecology. These two problems combine with the fundamental microbiological question of whether it is always advantageous for a unicellular organism to grow as fast as possible. The common basis for these three themes is a trade-off between growth rate and growth yield, which in turn is based on irreversible thermodynamics. The trade-off creates an evolutionary alternative between two strategies: high growth yield at low growth rate versus high growth rate at low growth yield. High growth yield at low growth rate is a case of an altruistic strategy because it increases the fitness of the group by using resources economically at the cost of decreased fitness, or growth rate, of the individual. The group-beneficial behaviour is advantageous in the long term, whereas the high growth rate strategy is advantageous in the short term. Coexistence of species requires differences between their niches, and niche space is typically divided into four ‘axes' (time, space, resources, predators). This neglects survival strategies based on cooperation, which extend the possibilities of coexistence, arguing for the inclusion of cooperation as the fifth ‘axis’. Here, individual-based model simulations show that spatial structure, as in, for example, biofilms, is necessary for the origin and maintenance of this ‘primitive’ altruistic strategy and that the common belief that growth rate but not yield decides the outcome of competition is based on chemostat models and experiments. This evolutionary perspective on life in biofilms can explain long-known biofilm characteristics, such as the structural organization into microcolonies, the often-observed lack of mixing among microcolonies, and the shedding of single cells, as promoting the origin and maintenance of the altruistic strategy. Whereas biofilms enrich altruists, enrichment cultures, microbiology's paradigm for isolating bacteria into pure culture, select for highest growth rate.
Expression of mptC of Listeria monocytogenes induces sensitivity to class IIa bacteriocins in Lactococcus lactis
Authors: Ramnath Manilduth , Arous Safia , Gravesen Anne , Hastings John W. , Héchard Yann
Published: August 2004

Sensitivity to class IIa bacteriocins from lactic acid bacteria was recently associated with the mannose phosphotransferase system (PTS) permease,, inListeria monocytogenes. To assess the involvement of this protein complex in class IIa bacteriocin activity, themptACDoperon, encoding, was heterologously expressed in an insensitive species, namelyLactococcus lactis, using the NICE double plasmid system. Upon induction of the cloned operon, the recombinantLc. lactisbecame sensitive to leucocin A. Pediocin PA-1 and enterocin A also showed inhibitory activity againstLc. lactiscultures expressingmptACD. Furthermore, the role of the three genes of themptACDoperon was investigated. Derivative plasmids containing various combinations of these three genes were made from the parentalmptACDplasmid by divergent PCR. The results showed that expression ofmptCalone is sufficient to confer sensitivity to class IIa bacteriocins inLc. lactis.
Topological and deletion analysis of CorS, a Pseudomonas syringae sensor kinase
Authors: Smirnova Angela V. , Ullrich Matthias S.
Published: August 2004

A modified two-component regulatory system consisting of two response regulators, CorR and CorP, and the histidine protein kinase CorS, regulates the thermoresponsive production of the phytotoxin coronatine (COR) in Pseudomonas syringae PG4180. COR is produced at the virulence-promoting temperature of 18 °C, but not at 28 °C, the optimal growth temperature of PG4180. Assuming that the highly hydrophobic N-terminus of CorS might be involved in temperature-signal perception, the membrane topology of CorS was determined using translational phoA and lacZ fusions, leading to a topological model for CorS with six transmembrane domains (TMDs). Interestingly, three PhoA fusions located downstream of the sixth TMD showed a thermoresponsive phenotype. Enzymic activity, immunoblot, and protease-sensitivity assays were performed to localize the CorS derivatives, to analyse the expression level of hybrid proteins and to examine the model. In-frame deletions of the last four, or all six TMDs gave rise to non-functional CorS. The results indicated that the transmembrane region is important for CorS to function as a temperature sensor, and that the membrane topology of CorS might be involved in signal perception.
The novel extracellular Streptomyces reticuli haem-binding protein HbpS influences the production of the catalase-peroxidase CpeB
Authors: Lucana Darío Ortiz de Orué , Schaa Tanja , Schrempf Hildgund
Published: August 2004

The Gram-positive soil bacterium and cellulose degraderStreptomyces reticulisynthesizes the mycelium-associated enzyme CpeB, which displays haem-dependent catalase and peroxidase activity, as well as haem-independent manganese-peroxidase activity. Downstream of thecpeBgene, a so far unknown gene was identified. The new gene and its mutated derivatives were cloned inEscherichia colias well as inStreptomyces lividansand a gene-disruption mutant within the chromosome of the originalS. reticulihost was constructed, comparative physiological, biochemical and immunological studies then allowed the deduction of the following characteristics of the novel gene product. (i) The protein was found extracellularly; the substitution of twin arginines within the signal peptide abolished its secretion. (ii) The highly purified protein interacted specifically with haem and hence was designated HbpS (haem-binding protein ofStreptomyces). (iii) HbpS contained three histidine residues surrounded by hydrophobic amino acids; one of them was located within the motif LX3THLX10AA, which is related to the motif within the yeast cytochromecperoxidase LX2THLX10AA whose histidine residue interacts with haem. (iv) The addition of haemin (Fe3+oxidized form of haem) to theStreptomycescultures led to enhanced levels of HbpS which correlated with increased haemin-resistance. (v) The presence of HbpS increased synthesis of the highly active catalase-peroxidase CpeB containing haem. In this process HbpS could act as a chaperone that binds haem and then delivers it to the mycelium-associated CpeB; HbpS could also interact with membrane-associated proteins involved in a signal transduction cascade regulating the expression ofcpeB. (vi) HbpS shared varying degrees of amino acid identities with bacterial proteins of so far unknown function. This report contributes to the elucidation of the biological function of these proteins.
Green fluorescent protein (GFP) as a vital marker for pathogenic development of the dermatophyte Trichophyton mentagrophytes
Authors: Kaufman Gil , Horwitz Benjamin A. , Hadar Ruthi , Ullmann Yehuda , Berdicevsky Israela
Published: August 2004

Skin infections by dermatophytes of the genus Trichophyton are widespread, but methods to investigate the molecular basis of pathogenicity are only starting to be developed. The initial stages of growth on the host can only be studied by electron microscopy, which requires fixing the tissue. This paper shows that restriction-enzyme-mediated integration (REMI) provides stable expression of the green fluorescent protein (GFP) in a clinical isolate of Trichophyton mentagrophytes. Under control of a constitutively active fungal promoter, GFP renders the hyphae fluorescent both in culture and in a recently developed model using human skin explants. Stages of infection and penetration into the skin layers were visualized by confocal microscopy. The stages of infection can thus be followed using GFP as a vital marker, and this method will also provide, for the first time, a means to follow gene expression during infection of skin by dermatophyte fungi.
Identification of two new genes involved in twitching motility in Pseudomonas aeruginosa
Authors: Shan Zhiying , Xu Haijin , Shi Xingqi , Yu Yan , Yao Hongming , Zhang Xiuming , Bai Yanling , Gao Caichang , Saris Per E. J. , Qiao Mingqiang
Published: August 2004

Mu transposition complexes were used for transposon mutagenesis ofPseudomonas aeruginosastrain PA68. Mu DNA transposition complexes were assembled with MuA transposase and an artificial mini-Mu transposonin vitro, and introduced intoPseudomonas aeruginosaby electroporation. Eight mutants deficient in twitching motility were isolated. Southern blotting confirmed that the insertions had occurred as single events. DNA sequencing of the region flanking the insertion in the twitching-motility mutants revealed that the mini-Mu transposon had inserted into six different genes,PAO171,PA1822,PAO413,PA4959,PA4551andPA5040. Four of these have previously been proven to be needed for twitching motility, whereas thePA1822andPA0171genes have not previously been shown to be required for twitching motility. The twitching-motility defect in thePA1822mutant was partially complemented by providing thePA1822genein trans, and the defect in thePA0171mutant was fully complemented whenPA0171was provided. APA0171mutant and aPA1822mutant were constructed by gene replacement in theP. aeruginosaPAO1 strain. These mutants were deficient in twitching motility, showing that both thePA1822and thePA0171gene are involved in twitching motility.
Identification and transcriptional organization of a gene cluster involved in biosynthesis and transport of acinetobactin, a siderophore produced by Acinetobacter baumannii ATCC 19606T
Authors: Mihara Kazutoshi , Tanabe Tomotaka , Yamakawa Yoshiko , Funahashi Tatsuya , Nakao Hiroshi , Narimatsu Shizuo , Yamamoto Shigeo
Published: August 2004

In order to assimilate iron,Acinetobacter baumanniiATCC 19606Tproduces a siderophore named acinetobactin (Ab) that is composed of equimolar quantities of 2,3-dihydroxybenzoic acid (DHBA),l-threonine andN-hydroxyhistamine. Application of the Fur titration assay system toA. baumanniigenomic libraries, followed by further cloning of the regions surrounding the candidate genes, led to the identification of the Ab cluster, which harbours the genetic determinants necessary for the biosynthesis and transport of the siderophore. However, anentAhomologue essential for DHBA biosynthesis was not found in this cluster. Functions of potential biosynthetic genes inferred by homology studies suggested that the precursors, DHBA,l-threonine andN-hydroxyhistamine, are linked in steps resembling those of bacterial non-ribosomal peptide synthesis to form Ab. Genes responsible for the two-step biosynthesis ofN-hydroxyhistamine from histidine were also identified in this cluster. Their genetic organization suggests that five genes involved in the transport system of ferric Ab into the cell cytosol form an operon. Construction of disruptants of some selected genes followed by phenotypic analysis supported their predicted biological functions. Interestingly, three additional genes probably involved in the intracellular release of iron from ferric Ab and the secretion of nascent Ab are contained in this cluster. Primer extension and RT-PCR analyses suggested that the Ab cluster, which includes 18 genes, is organized in seven transcriptional units originating from respective Fur-regulated promoter-operator regions.
Controlled expression of CluA in Lactococcus lactis and its role in conjugation
Authors: Stentz Régis , Jury Karen , Eaton Tracy , Parker Mary , Narbad Arjan , Gasson Mike , Shearman Claire
Published: August 2004

CluA is a 136 kDa surface-bound protein encoded by the chromosomally located sex factor of Lactococcus lactis MG1363 and is associated with cell aggregation linked to high-frequency transfer of the sex factor. To further investigate the involvement of CluA in these phenomena, the cluA gene was cloned on a plasmid, downstream from the lactococcal nisA promoter. In a sex-factor-negative MG1363 derivative, nisin-controlled CluA expression resulted in aggregation, despite the absence of the other genes of the sex factor. Therefore, CluA is the only sex factor component responsible for aggregation. The direct involvement of CluA in the establishment of cell-to-cell contact for aggregate formation was observed by electron microscopy using immunogold-labelled CluA antibodies. Inactivation of cluA in an MG1363 background led to a dramatic decrease in sex factor conjugation frequency compared to the parental strain. Increasing levels of CluA expressed in trans in the cluA-inactivated donor strain facilitated a gradual restoration of conjugation frequency, reaching that of the parental strain. In conclusion, CluA is essential for efficient sex factor transfer in conjugation of L. lactis.
AlgR functions in algC expression and virulence in Pseudomonas syringae pv. syringae
Authors: Peñaloza-Vázquez Alejandro , Fakhr Mohamed K. , Bailey Ana M. , Bender Carol L.
Published: August 2004

Pseudomonas syringaepv. syringae strain FF5 is a phytopathogen associated with a rapid dieback on ornamental pear trees.P. syringaeand the human pathogenPseudomonas aeruginosaproduce the exopolysaccharide alginate, a copolymer of mannuronic and guluronic acid. InP. aeruginosa, the response regulator AlgR (AlgR1) is required for transcription ofalgCandalgD, which encode key enzymes in the alginate biosynthetic pathway. InP. syringaeFF5, however,algRis not required for the activation ofalgD. Interestingly,algRmutants ofP. syringaeremain nonmucoid, indicating an undefined role for this response regulator in alginate biosynthesis. In the current study, thealgCpromoter region was cloned fromP. syringaepv. syringae strain FF5, and sequence analysis of thealgCpromoter indicated the presence of potential binding sites for AlgR andσ54, the alternative sigma factor encoded byrpoN. ThealgCpromoter fromP. syringaeFF5 (PsalgC) was cloned upstream of a promoterless glucuronidase gene (uidA), and thePsalgC–uidAtranscriptional fusion was used to monitoralgCexpression in strains FF5.32 (algRmutant ofP. syringaeFF5) and PG4180.K2 (rpoNmutant ofP. syringaepv. glycinea PG4180). Expression of thePsalgC–uidAfusion was fourfold lower in both thealgRandrpoNmutants as compared to respective wild-type strains, indicating that both AlgR andσ54are required for full activation ofalgCtranscription inP. syringaepv. syringae. AlgR fromP. syringaewas successfully overproduced inEscherichia colias a C-terminal translational fusion to the maltose-binding protein (MBP). Gel shift experiments indicated that MBP–AlgR binds strongly to thealgCpromoter region. Biological assays demonstrated that thealgRmutant was significantly impaired in both pathogenicity and epiphytic fitness as compared to the wild-type strain. These results, along with the gene expression studies, indicate that AlgR has a positive role in the activation ofalgCinP. syringaeand contributes to both virulence and epiphytic fitness. Furthermore, the symptoms observed with wild-typeP. syringaeFF5 suggest that this strain can move systemically in leaf tissue, and that a functional copy ofalgRis required for systemic movement.
N-terminal polyubiquitination and degradation of the Arf tumor suppressor
Authors: Kuo Mei-Ling , den Besten Willem , Bertwistle David , Roussel Martine F. , Sherr Charles J.
Published: August 2004

Unknown mechanisms govern degradation of the p19Arf tumor suppressor, an activator of p53 and inhibitor of ribosomal RNA processing. Kinetic metabolic labeling of cells with [3H]-leucine indicated that p19Arf is a relatively stable protein (half-life ∼6 h) whose degradation depends upon the ubiquitin–proteasome pathway. Although p19Arf binds to the Mdm2 E3 ubiquitin protein ligase to activate p53, neither of these molecules regulates p19Arf turnover. In contrast, the nucleolar protein nucleophosmin/B23, which binds to p19Arf with high stoichiometry, retards its turnover, and Arf mutants that do not efficiently associate with nucleophosmin/B23 are unstable and functionally impaired. Mouse p19Arf, although highly basic (22% arginine content), contains only a single lysine residue absent from human p14ARF, and substitution of arginine for lysine in mouse p19Arf had no effect on its rate of degradation. Mouse p19Arf (either wild-type or lacking lysine) and human p14ARF undergo N-terminal polyubiquitination, a process that has not as yet been documented in naturally occurring lysine-less proteins. Re-engineering of the p19Arf N terminus to provide consensus sequences for N-acetylation limited Arf ubiquitination and decelerated its turnover.
The X-ray structure of the papillomavirus helicase in complex with its molecular matchmaker E2
Authors: Abbate Eric A. , Berger James M. , Botchan Michael R.
Published: August 2004

DNA replication of the papillomaviruses is specified by cooperative binding of two proteins to the ori site: the enhancer E2 and the viral initiator E1, a distant member of the AAA+ family of proteins. Formation of this prereplication complex is an essential step toward the construction of a functional, multimeric E1 helicase and DNA melting. To understand how E2 interacts with E1 to regulate this process, we have solved the X-ray structure of a complex containing the HPV18 E2 activation domain bound to the helicase domain of E1. Modeling the monomers of E1 to a hexameric helicase shows that E2 blocks hexamerization of E1 by shielding a region of the E1 oligomerization surface and stabilizing a conformation of E1 that is incompatible with ATP binding. Further biochemical experiments and structural analysis show that ATP is an allosteric effector of the dissociation of E2 from E1. Our data provide the first molecular insights into how a protein can regulate the assembly of an oligomeric AAA+ complex and explain at a structural level why E2, after playing a matchmaker role by guiding E1 to the DNA, must dissociate for subsequent steps of initiation to occur. Building on previously proposed ideas, we discuss how our data advance current models for the conversion of E1 in the prereplication complex to a hexameric helicase assembly.
Role of the Escherichia coli RecQ DNA helicase in SOS signaling and genome stabilization at stalled replication forks
Authors: Hishida Takashi , Han Yong-Woon , Shibata Tatsuya , Kubota Yoshino , Ishino Yoshizumi , Iwasaki Hiroshi , Shinagawa Hideo
Published: August 2004

The RecQ protein family is a highly conserved group of DNA helicases that play roles in maintaining genomic stability. In this study, we present biochemical and genetic evidence that Escherichia coli RecQ processes stalled replication forks and participates in SOS signaling. Cells that carry dnaE486, a mutation in the DNA polymerase III α-catalytic subunit, induce an RecA-dependent SOS response and become highly filamented at the semirestrictive temperature (38°C). An recQ mutation suppresses the induction of SOS response and the filamentation in the dnaE486 mutant at 38°C, causing appearance of a high proportion of anucleate cells. In vitro, RecQ binds and unwinds forked DNA substrates with a gap on the leading strand more efficiently than those with a gap on the lagging strand or Holliday junction DNA. RecQ unwinds the template duplex ahead of the fork, and then the lagging strand is unwound. Consequently, this process generates a single-stranded DNA (ssDNA) gap on the lagging strand adjacent to a replication fork. These results suggest that RecQ functions to generate an initiating signal that can recruit RecA for SOS induction and recombination at stalled replication forks, which are required for the cell cycle checkpoint and resumption of DNA replication.
BMP signaling mediated by ALK2 in the visceral endoderm is necessary for the generation of primordial germ cells in the mouse embryo
Authors: de Sousa Lopes Susana M. Chuva , Roelen Bernard A.J. , Monteiro Rui M. , Emmens Roul , Lin Herbert Y. , Li En , Lawson Kirstie A. , Mummery Christine L.
Published: August 2004

Deletion of various bone morphogenetic proteins (BMPs) and their downstream Smads in mice have clearly shown that BMP signaling is essential for the formation of primordial germ cells (PGCs). However, the molecular mechanism through which this takes place is still unclear. Here, we demonstrate that BMP4 produced in the extraembryonic ectoderm signals through ALK2, a type I BMP receptor, in the visceral endoderm (VE) to induce formation of PGCs from the epiblast. Firstly, embryonic day 5.5–6.0 (E5.5–E6.0) embryos cultured on fibronectin formed PGCs in the presence of VE, but not in its absence. Secondly, Alk2-deficient embryos completely lacked PGCs and the heterozygotes had reduced numbers, resembling Bmp4-deficient phenotypes. Thirdly, expression of constitutively active ALK2 in the VE, but not in the epiblast, was sufficient to rescue the PGC phenotype in Bmp4-deficient embryos. In addition, we show that the requirement for the VE at E5.5–E6.0 can be replaced by culturing embryos stripped of VE on STO cells, indicating that STO cells provide or transduce signals necessary for PGC formation that are normally transmitted by the VE. We propose a model in which direct signaling to proximal epiblast is supplemented by an obligatory indirect BMP-dependent signal via the VE.
Comparative Analysis of Gene Expression for Convergent Evolution of Camera Eye Between Octopus and Human
Authors: Ogura Atsushi , Ikeo Kazuho , Gojobori Takashi
Published: August 2004

Although the camera eye of the octopus is very similar to that of humans, phylogenetic and embryological analyses have suggested that their camera eyes have been acquired independently. It has been known as a typical example of convergent evolution. To study the molecular basis of convergent evolution of camera eyes, we conducted a comparative analysis of gene expression in octopus and human camera eyes. We sequenced 16,432 ESTs of the octopus eye, leading to 1052 nonredundant genes that have matches in the protein database. Comparing these 1052 genes with 13,303 already-known ESTs of the human eye, 729 (69.3%) genes were commonly expressed between the human and octopus eyes. On the contrary, when we compared octopus eye ESTs with human connective tissue ESTs, the expression similarity was quite low. To trace the evolutionary changes that are potentially responsible for camera eye formation, we also compared octopus-eye ESTs with the completed genome sequences of other organisms. We found that 1019 out of the 1052 genes had already existed at the common ancestor of bilateria, and 875 genes were conserved between humans and octopuses. It suggests that a larger number of conserved genes and their similar gene expression may be responsible for the convergent evolution of the camera eye.
The Use of MPSS for Whole-Genome Transcriptional Analysis in Arabidopsis
Authors: Meyers Blake C. , Tej Shivakundan Singh , Vu Tam H. , Haudenschild Christian D. , Agrawal Vikas , Edberg Steve B. , Ghazal Hassan , Decola Shannon
Published: August 2004

We have generated 36,991,173 17-base sequence “signatures” representing transcripts from the model plant Arabidopsis. These data were derived by massively parallel signature sequencing (MPSS) from 14 libraries and comprised 268,132 distinct sequences. Comparable data were also obtained with 20-base signatures. We developed a method for handling these data and for comparing these signatures to the annotated Arabidopsis genome. As part of this procedure, 858,019 potential or “genomic” signatures were extracted from the Arabidopsis genome and classified based on the position and orientation of the signatures relative to annotated genes. A comparison of genomic and expressed signatures matched 67,735 signatures predicted to be derived from distinct transcripts and expressed at significant levels. Expressed signatures were derived from the sense strand of at least 19,088 of 29,084 annotated genes. A comparison of the genomic and expression signatures demonstrated that ∼7.7% of genomic signatures were underrepresented in the expression data. These genomic signatures contained one of 20 four-base words that were consistently associated with reduced MPSS abundances. More than 89% of the sum of the expressed signature abundances matched the Arabidopsis genome, and many of the unmatched signatures found in high abundances were predicted to match to previously uncharacterized transcripts.
Fosmid-Based Physical Mapping of the Histoplasma capsulatum Genome
Authors: Magrini Vincent , Warren Wesley C. , Wallis John , Goldman William E. , Xu Jian , Mardis Elaine R. , McPherson John D.
Published: August 2004

A fosmid library representing 10-fold coverage of the Histoplasma capsulatum G217B genome was used to construct a restriction-based physical map. The data obtained from three restriction endonuclease fingerprints, generated from each clone using BamHI, HindIII, and PstI endonucleases, were combined and used in FPC for automatic and manual contig assembly builds. Concomitantly, a whole-genome shotgun (WGS) sequencing of paired-end reads from plasmids and fosmids were assembled with PCAP, providing a predicted genome size of up to 43.5 Mbp and 17% repetitive DNA. Fosmid paired-end sequences in the WGS assembly provide anchoring information to the physical map and result in joining of existing physical map contigs into 84 clusters containing 9551 fosmid clones. Here, we detail mapping the Histoplasma capsulatum genome comprehensively in fosmids, resulting in an efficient paradigm for de novo sequencing that uses a map-assisted whole genome shotgun approach.