CXCL14 Displays Antimicrobial Activity against Respiratory Tract Bacteria and Contributes to Clearance of Streptococcus pneumoniae Pulmonary Infection

CXCL14 is a chemokine with an atypical, yet highly conserved, primary structure characterized by a short N terminus and high sequence identity between human and mouse. Although it induces chemotaxis of monocytic cells at high concentrations, its physiological role in leukocyte trafficking remains elusive. In contrast, several studies have demonstrated that CXCL14 is a broad-spectrum antimicrobial peptide that is expressed abundantly and constitutively in epithelial tissues. In this study, we further explored the antimicrobial properties of CXCL14 against respiratory pathogens in vitro and in vivo. We found that CXCL14 potently killed Pseudomonas aeruginosa, Streptococcus mitis, and Streptococcus pneumoniae in a dose-dependent manner in part through membrane depolarization and rupture. By performing structure-activity studies, we found that the activity against Gram-negative bacteria was largely associated with the N-terminal peptide CXCL141-13. Interestingly, the central part of the molecule representing the β-sheet also maintained ∼62% killing activity and was sufficient to induce chemotaxis of THP-1 cells. The C-terminal α-helix of CXCL14 had neither antimicrobial nor chemotactic effect. To investigate a physiological function for CXCL14 in innate immunity in vivo, we infected CXCL14-deficient mice with lung pathogens and we found that CXCL14 contributed to enhanced clearance of Streptococcus pneumoniae, but not Pseudomonas aeruginosa. Our comprehensive studies reflect the complex bactericidal mechanisms of CXCL14, and we propose that different structural features are relevant for the killing of Gram-negative and Gram-positive bacteria. Taken together, our studies show that evolutionary-conserved features of CXCL14 are important for constitutive antimicrobial defenses against pneumonia.

O PODER DE ZEFERINA NO QUILOMBO DO URUBU UMA RECONSTRUÇÃO HISTÓRICA POLÍTICO-SOCIAL

Os ideais de liberdade exigiram do povo negro diferenciadas práticas para romper com o sistema escravista. Eram as rebeliões em navios, os atos de infanticídio, os justiçamentos dos feitores, as revoltas, além de participações em movimentos libertários e formações de quilombos. Dentre estas formas de organização, o quilombo foi fenômeno essencial nos mais de 300 anos de escravismo no Brasil. Em cada região existiam quilombos, pois para a população negra, cativa ou não, esse era o melhor meio de alcançar a liberdade, um meio coletivo para enfrentar o sistema. O Quilombo do Urubu representou a insistência em garantir a condição humana que o regime escravista negava, sobretudo às mulheres, aos homens e às crianças negras. Essa era uma força que saía de suas entranhas como grito de liberdade, configurada nas fugas em busca de um lugar que lhes assegurasse aproximação de uma vida digna e que pudessem orgulhar-sedo seu porte físico e da sua cultura. Todo esse desprendimento, além de uma força física, exigia um completo conhecimento histórico e espiritual, resguardado pela religiosidade que fortalecia seus espíritos para lutar contra toda negação de humanidade do século XIX no subúrbio da capital baiana. A líder Zeferina, inconformada com a exclusão social de seu povo negro, e entusiasmada pelo poder de herança de ancestralidade, pelo conhecimento de raiz da cultura matrilinear angolana, pelo profundo conhecimento histórico de resistência da rainha Nzinga Mbandi e pela tradição de quilombolas e guerreiras, viveu e lutou pelo sonho de liberdade. Hoje, a chama desse poder é mantida acesa na caminhada de celebração do 20 de novembro pela comunidade de Pirajá e arredores, enquanto referencial de resistência negra na luta contra as exclusões sociais vigentes.(AU)

Bcl-XL interacts with Apaf-1 and inhibits Apaf-1-dependent caspase-9 activation

Recent studies indicate that Caenorhabditis elegans CED-4 interacts with and promotes the activation of the death protease CED-3, and that this activation is inhibited by CED-9. Here we show that a mammalian homolog of CED-4, Apaf-1, can associate with several death proteases, including caspase-4, caspase-8, caspase-9, and nematode CED-3 in mammalian cells. The interaction with caspase-9 was mediated by the N-terminal CED-4-like domain of Apaf-1. Expression of Apaf-1 enhanced the killing activity of caspase-9 that required the CED-4-like domain of Apaf-1. Furthermore, Apaf-1 promoted the processing and activation of caspase-9 in vivo. Bcl-XL, an antiapoptotic member of the Bcl-2 family, was shown to physically interact with Apaf-1 and caspase-9 in mammalian cells. The association of Apaf-1 with Bcl-XL was mediated through both its CED-4-like domain and the C-terminal domain containing WD-40 repeats. Expression of Bcl-XL inhibited the association of Apaf-1 with caspase-9 in mammalian cells. Significantly, recombinant Bcl-XL purified from Escherichia coli or insect cells inhibited Apaf-1-dependent processing of caspase-9. Furthermore, Bcl-XL failed to inhibit caspase-9 processing mediated by a constitutively active Apaf-1 mutant, suggesting that Bcl-XL regulates caspase-9 through Apaf-1. These experiments demonstrate that Bcl-XL associates with caspase-9 and Apaf-1, and show that Bcl-XL inhibits the maturation of caspase-9 mediated by Apaf-1, a process that is evolutionarily conserved from nematodes to humans.

CLARP, a death effector domain-containing protein interacts with caspase-8 and regulates apoptosis

We have identified and characterized CLARP, a caspase-like apoptosis-regulatory protein. Sequence analysis revealed that human CLARP contains two amino-terminal death effector domains fused to a carboxyl-terminal caspase-like domain. The structure and amino acid sequence of CLARP resemble those of caspase-8, caspase-10, and DCP2, a Drosophila melanogaster protein identified in this study. Unlike caspase-8, caspase-10, and DCP2, however, two important residues predicted to be involved in catalysis were lost in the caspase-like domain of CLARP. Analysis with fluorogenic substrates for caspase activity confirmed that CLARP is catalytically inactive. CLARP was found to interact with caspase-8 but not with FADD/MORT-1, an upstream death effector domain-containing protein of the Fas and tumor necrosis factor receptor 1 signaling pathway. Expression of CLARP induced apoptosis, which was blocked by the viral caspase inhibitor p35, dominant negative mutant caspase-8, and the synthetic caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethylketone (zVAD-fmk). Moreover, CLARP augmented the killing ability of caspase-8 and FADD/MORT-1 in mammalian cells. The human clarp gene maps to 2q33. Thus, CLARP represents a regulator of the upstream caspase-8, which may play a role in apoptosis during tissue development and homeostasis.

Proteasome regulation of activation-induced T cell death

Lactacystin, a microbial metabolite that inhibits protease activity only in the proteasome, was used to study the role of the proteasome in the activation-induced cell death (AICD) of T cells. Lactacystin induces DNA fragmentation and apoptosis in a T cell hybridoma (DO.11.10) in a dose-dependent manner. Between 1 and 10 μM, the mildly cytotoxic lactacystin inhibited the AICD of DO.11.10 cells cultured in anti-CD3-coated wells. Degradation of IκBβ and the translocation of the NF-κB (p50/RelA) into the nucleus, which occurred at 1.5 hr after anti-CD3 activation, were inhibited by lactacystin. Lactacystin did not inhibit the expression of nuclear transcription factor Oct-1. The activation-induced expression of the immediate–early gene, Nur77, and the T cell death genes, CD95 (Fas) and CD95 ligand (FasL), were inhibited. Functional expression of FasL cytotoxicity and the increase of cell surface Fas were also inhibited. Lactacystin must be added within 2 hr of activation to efficiently block AICD. In addition, lactacystin failed to inhibit the killing of DO.11.10 by FasL-expressing allo-specific cytotoxic effector cells. These observations strongly suggest a direct link between the proteasome-dependent degradation of IκBβ and the AICD that occurs through activation of the FasL gene and up-regulation of the Fas gene.

A cytotoxic ribonuclease which specifically cleaves four isoaccepting arginine tRNAs at their anticodon loops

Colicin D has long been thought to stop protein synthesis in infected Escherichia coli cells by inactivating ribosomes, just like colicin E3. Here, we show that colicin D specifically cleaves tRNAsArg including four isoaccepting molecules both in vivo and in vitro. The cleavage occurs in vitro between positions 38 and 39 in an anticodon loop with a 2′,3′-cyclic phosphate end, and is inhibited by a specific immunity protein. Consistent with the cleavage of tRNAsArg, the RNA fraction of colicin-treated cells significantly reduced the amino acid-accepting activity only for arginine. Furthermore, we generated a single mutation of histidine in the C-terminal possible catalytic domain, which caused the loss of the killing activity in vivo together with the tRNAArg-cleaving activity both in vivo and in vitro. These findings show that colicin D directly cleaves cytoplasmic tRNAsArg, which leads to impairment of protein synthesis and cell death. Recently, we found that colicin E5 stops protein synthesis by cleaving the anticodons of specific tRNAs for Tyr, His, Asn, and Asp. Despite these apparently similar actions on tRNAs and cells, colicins D and E5 not only exhibit no sequence homology but also have different molecular mechanisms as to both substrate recognition and catalytic reaction.

Characterization of a Gene for Spinach CAP160 and Expression of Two Spinach Cold-Acclimation Proteins in Tobacco1

The cDNA sequence for CAP160, an acidic protein previously linked with cold acclimation in spinach (Spinacia oleracea L.), was characterized and found to encode a novel acidic protein of 780 amino acids having very limited homology to a pair of Arabidopsis thaliana stress-regulated proteins, rd29A and rd29B. The lack of similarity in the structural organization of the spinach and Arabidopsis genes highlights the absence of a high degree of conservation of this cold-stress gene across taxonomic boundaries. The protein has several unique motifs that may relate to its function during cold stress. Expression of the CAP160 mRNA was increased by low-temperature exposure and water stress in a manner consistent with a probable function during stresses that involve dehydration. The coding sequences for CAP160 and CAP85, another spinach cold-stress protein, were introduced into tobacco (Nicotiana tabacum) under the control of the 35S promoter using Agrobacterium tumefaciens-based transformation. Tobacco plants expressing the proteins individually or coexpressing both proteins were evaluated for relative freezing-stress tolerance. The killing temperature for 50% of the cells of the transgenic plants was not different from that of the wild-type plants. As determined by a more sensitive time/temperature kinetic study, plants expressing the spinach proteins had slightly lower levels of electrolyte leakage than wild-type plants, indicative of a small reduction of freezing-stress injury. Clearly, the heterologous expression of two cold-stress proteins had no profound influence on stress tolerance, a result that is consistent with the quantitative nature of cold-stress-tolerance traits.

Coordinate regulation of Bacillus subtilis peroxide stress genes by hydrogen peroxide and metal ions.

The Bacillus subtilis mrgA gene encodes an abundant DNA-binding protein that protects cells against the lethal effects of H2O2. Transcription of mrgA is induced by H2O2 or by entry into stationary phase when manganese and iron levels are low. We have selected for strains derepressed for transcription of mrgA in the presence of Mn(II). The resulting cis-acting mutants define an operator site just upstream of the mrgA promoter. Similar sequences flank the promoters for the catalase gene, katA, and the heme biosynthesis operon, hemAXCDBL. Like mrgA, transcription of the katA and hem genes is repressed by Mn(II), which thereby potentiates the killing action of H2O2. We identified two classes of trans-acting mutants derepressed for mrgA transcription in the presence of Mn(II): some exhibit a coordinate derepression of MrgA, catalase, heme biosynthesis, and alkyl hydroperoxide reductase and are H2O2 resistant, while others have reduced catalase activity and are H2O2 sensitive. These data indicate that the peroxide stress response of B. subtilis is regulated by a repressor that senses both metal ion levels and H2O2.

Editorial wild oats,

My first literary venture. -- Journalism in Tennessee. -- Nicodemus Dodge -- printer. -- Mr. Bloke's item. -- How I edited an agricultural paper. -- The Killing of Julius Caesar localized.

Effector function of leucocytes from susceptible and resistant mice against distinct isolates of Candida albicans

Neutrophils and macrophages were generated in vitro from mice that display either high or low tissue susceptibilities to Candida albicans infection and their ability to phagocytose and kill three isolates of the yeast with different virulence characteristics was evaluated. In the absence of opsonization, phagocytosis by BALB/c and CBA/CaH neutrophils was comparable, but the killing was very poor. Opsonization with normal serum slightly decreased phagocytosis, but it had markedly different effects on killing, either enhancing or inhibiting candidacidal activity, depending on the combination of yeast isolate and mouse strain. In contrast, BALB/c macrophages showed high levels of phagocytosis and killing of both unopsonized yeasts and opsonized yeasts; whereas killing of unopsonized yeasts by CBA/CaH macrophages was poor, it was markedly enhanced by opsonization.

O PODER DE ZEFERINA NO QUILOMBO DO URUBU UMA RECONSTRUÇÃO HISTÓRICA POLÍTICO-SOCIAL

Os ideais de liberdade exigiram do povo negro diferenciadas práticas para romper com o sistema escravista. Eram as rebeliões em navios, os atos de infanticídio, os justiçamentos dos feitores, as revoltas, além de participações em movimentos libertários e formações de quilombos. Dentre estas formas de organização, o quilombo foi fenômeno essencial nos mais de 300 anos de escravismo no Brasil. Em cada região existiam quilombos, pois para a população negra, cativa ou não, esse era o melhor meio de alcançar a liberdade, um meio coletivo para enfrentar o sistema. O Quilombo do Urubu representou a insistência em garantir a condição humana que o regime escravista negava, sobretudo às mulheres, aos homens e às crianças negras. Essa era uma força que saía de suas entranhas como grito de liberdade, configurada nas fugas em busca de um lugar que lhes assegurasse aproximação de uma vida digna e que pudessem orgulhar-sedo seu porte físico e da sua cultura. Todo esse desprendimento, além de uma força física, exigia um completo conhecimento histórico e espiritual, resguardado pela religiosidade que fortalecia seus espíritos para lutar contra toda negação de humanidade do século XIX no subúrbio da capital baiana. A líder Zeferina, inconformada com a exclusão social de seu povo negro, e entusiasmada pelo poder de herança de ancestralidade, pelo conhecimento de raiz da cultura matrilinear angolana, pelo profundo conhecimento histórico de resistência da rainha Nzinga Mbandi e pela tradição de quilombolas e guerreiras, viveu e lutou pelo sonho de liberdade. Hoje, a chama desse poder é mantida acesa na caminhada de celebração do 20 de novembro pela comunidade de Pirajá e arredores, enquanto referencial de resistência negra na luta contra as exclusões sociais vigentes.(AU)

El terror en los orígenes del totalitarismo y la política de la muerte

Con Los orígenes del totalitarismo Hannah Arendt propuso identificar el mal radical que los regímenes totalitarios efectuaron durante la primera mitad del siglo XX. La política de la muerte de Hit­ler y Stalin llegó a ser un flagelo hacia la humanidad y causó una gran intimidación que no escatimó la violencia y la aniquila­ción total de sus opositores. La política de la muerte significa no solo exterminar la vida desde un aspecto físico sino también desde un aspecto político, demostrando la capacidad de aislar al ser humano de su espontaneidad en la esfera pública y de su innegable pluralidad en los asuntos humanos. Las ideologías políticas que antecedieron a los regímenes totalitarios influyeron en la constitución de los partidos nacionalsocialista y bolchevismo, basando su discurso y ejecución mediante aconteci­mientos históricos que no fueron ajenos a su erección. La política de la muerte fue la determinación de todo un aparato estatal para acabar con la diversidad de los hom­bres y sumergirlos en la política macabra de los regímenes totalitarios, una política basada en la muerte.

Manos manchadas de sangre: los orígenes cristianos del mito antijudío del crimen ritual

La acusación antijudía del crimen o asesinato ritual gozó de una enorme popularidad en la Edad Media. A mediados del siglo XII, surgió de forma “espontánea” en diferentes lugares de la Europa medieval y dio lugar a infinidad de variantes. Su versión definitiva fue la del asesinato, preferentemente por crucifixión, de un niño cristiano a manos de los judíos con el fin de incorporar su sangre al pan ázimo. Sin embargo, su origen más remoto debe buscarse en la Antigüedad. De hecho, su más incipiente desarrollo puede encontrarse ya a comienzos del siglo V en el historiador cristiano Sócrates (Hist. Eccl., VII, 16), quien cuenta que hacia el año 415, en Inmestar (Siria), con motivo de las celebraciones de la fiesta de Purim, los judíos, embriagados por el vino, amarraron a un niño cristiano a una cruz y lo asesinaron. El relato de este hecho monstruoso plantea algunas dudas acerca de lo acontecido. Seguramente la historia sea falsa y se sitúe en el contexto de una ley del Codex Theodosianus del año 408 (XVI, 8, 18) que prohibía insultar a la Cruz durante la celebración de dicha festividad judía.

Killing our darling: why we need to let go of the entrepreneurial opportunity construct

We support Shane and Venkataraman’s (2000) basic idea of an “entrepreneurship nexus” where characteristics of the actor as well as those of the “opportunity” they work on influence action and outcomes in the creation of new economic activities. However, a review of the literature reveals that minimal progress has been made on the core issues pertaining to the nexus idea. We argue that this is rooted in fundamental and insurmountable problems with the “opportunity” construct itself, and demonstrate the state of confusion in the literature caused by inconsistent use of the construct within and across works and authors. As an alternative, we suggest the admittedly subjective notion of New Venture as a more workable alternative. We provide a comprehensive definition and explanation of this construct, and take steps towards improved conceptualization and operationalization of its subdimensions. With some further work on these conceptualizations and operationalizations it will be possible to implement a comprehensive research program that can finally deliver on the promise outlined by Shane and Venkataraman (2000).

Virus resistance and gene silencing : killing the messenger

On occasion, virus-derived transgenes in plants can be poorly expressed and yet provide excellent virus resistance, and transgene constructs designed to supplement the expression of endogenous genes can have the effect of co-suppressing themselves and the endogenous genes. These two phenomena appear to result from the same post-transcriptional silencing mechanism, which operates by targeted-RNA degradation. Recent research into RNA-mediated virus resistance and co-suppression has provided insights into the interactions between plant viruses and their hosts, and spawned several models to explain the phenomenon.