bluuebunny/crossref_metadata_2025_split huggingface dataset:
Kinetics of thrombomodulin release and endothelial cell injury by neutrophil‐derived proteases and oxygen radicals
Authors: Boehme Michael W. J. , Galle Peter , Stremmel Wolfgang 
Published: March 2003

SummaryThrombomodulin is a transmembranous glycoprotein of endothelial cells. In vitro it is a marker of endothelial cell injury. In vivo the levels of serum thrombomodulin are regarded as a parameter of activity in vasculitides. The latter are pathophysiologically determined by neutrophil‐derived inflammation and endothelial cell injury caused by secretion of proteases and hydrogen peroxide. It was the objective of this study to determine whether thrombomodulin is only a late marker of advanced endothelial cell injury or whether it indicates also earlier stages of cell alterations. Over 24 hr endothelial cell cultures were incubated with hydrogen peroxide or the neutrophil proteases proteinase‐3, elastase and cathepsin G. The time‐dependent increase of thrombomodulin in the supernatant was determined by enzyme‐linked immunosorbent assay and immunoblot. In addition the viability (eosin, tetrazolium dye assay), detachment (crystal‐violet assay), and apoptosis (4′,6‐diamine‐2′‐phenylindole‐dihydrochloride assay) of the respective endothelial cells were determined for adherent and non‐adherent cells. A rapid thrombomodulin increase was found under all experimental conditions. The additional immunoblotting analysis showed the pattern of proteolytic cleavage caused by the protease reactivity. In case of hydrogen peroxide the thrombomodulin increase was closely correlated with the loss of cell viability and lysis. The incubation of endothelial cells with the different proteases resulted in a time‐dependent detachment of primarily viable cells. In addition to cell necrosis apoptotic cell death was found in the subgroup of detached endothelial cells after prolonged incubation over 24 hr with proteinase‐3 (23%), elastase (31%), and cathepsin G (19%). In contrast, still adhering cells did not show any signs of necrosis or apoptosis. In summary these studies confirm in vitro that soluble thrombomodulin is not only a parameter of advanced endothelial cell destruction itself but also in addition an early marker of initial endothelial cell membrane changes induced by neutrophil derived proteases and oxygen radicals.
The haemopoietic growth factor, Flt3L, alters the immune response induced by transcutaneous immunization
Authors: Baca‐Estrada Maria E. , Ewen Catherine , Mahony Donna , Babiuk Lorne A. , Wilkie Darryl , Foldvari Marianna 
Published: March 2003

SummaryTopical application of antigen induces antigen‐specific humoral and cellular immune responses. In this study we examined whether expansion of dendritic cells (DC) by Flt3 ligand (Flt3L) treatment influences the induction of immune responses following transcutaneous immunization. Mice were treated intraperitoneally with Flt3L or phosphate‐buffered saline (PBS) and immunized transcutaneously with hen egg lysozyme (HEL). Flt3L‐treated mice developed lower HEL‐specific cellular and humoral immune responses than PBS‐treated mice. However, in the presence of cholera toxin (CT), a potent adjuvant for mucosal and transcutaneous immunization, Flt3L‐treated mice developed significantly higher cellular and humoral immune responses to HEL when compared to PBS‐treated mice. We assessed whether the immunomodulatory effects of CT were a result of activation of epidermal dendritic cells (Langerhans' cells; LC). Our results indicate that within 8–12 hr of topical application of CT, epidermal LC cells lose their dendritic morphology and become rounder in appearance. In addition, we observed enhanced expression of major histocompatibility complex (MHC) class II, and of adhesion molecules CD11c and intracellular adhesion molecule‐1 (ICAM‐1). Our observations support the concept that the state of activation of DC in the skin is central to the regulation of immune responses. This information is relevant to the design of effective transcutaneous vaccination strategies.
Flavoxobin, a serine protease from Trimeresurus flavoviridis (habu snake) venom, independently cleaves Arg726‐Ser727 of human C3 and acts as a novel, heterologous C3 convertase
Authors: Yamamoto Chieko , Tsuru Daisuke , Oda‐Ueda Naoko , Ohno Motonori , Hattori Shosaku , Kim Sung‐Teh 
Published: March 2003

SummaryWe have recently shown that crude Trimeresurus flavoviridis (habu snake) venom has a strong capability for activating the human alternative complement system. To identify the active component, the crude venom was fractionated and purified by serial chromatography using Sephadex G‐100, CM‐cellulose C‐52, diethylaminoethyl‐Toyopearl 650M, and Butyl‐Toyopearl, and the active fractions were evaluated by the C3a‐releasing and soluble membrane attack complex‐forming activities. Two peak fractions with the highest activities were detected after gel filtration and ion exchange chromatography, and the first fraction was purified to homogeneity. The homogeneous protein was examined for its N‐terminal amino acid sequence by Edman degradation. The determined sequence of 25 amino acids completely coincided with that of a previously reported serine protease with coagulant activity, flavoxobin, purified from the same snake venom. To elucidate the molecular mechanism of the complement activation, the reactive products of the mixture of the purified human C3 and flavoxobin were examined by sodium dodecyl sulphate–polyacrylamide gel electrophoresis. The digesting pattern revealed that flavoxobin cleaves the α chain of the C3 molecule into two fragments. The N‐terminal amino acid sequences for the remnant fragments of C3 disclosed that flavoxobin severs the human C3 at the Arg726‐Ser727 site to form C3b and C3a the way C3bBb, the human alternative C3 convertase, does. In conclusion, flavoxobin acts as a novel, heterologous C3 convertase that independently cleaves human C3 and kick‐starts the complement cascade.
G1/S cell cycle arrest provoked in human T cells by antibody to CD26
Authors: Ohnuma Kei , Ishii Tomonori , Iwata Satoshi , Hosono Osamu , Kawasaki Hiroshi , Uchiyama Masahiko , Tanaka Hirotoshi , Yamochi Tadanori , Dang Nam H. , Morimoto Chikao 
Published: March 2003

SummaryCD26 is T cell costimulatory molecule with dipeptidyl peptidase IV (DPPIV) enzyme activity located in its extracellular region. The expression of CD26 is enhanced after activation of T cells, while it is preferentially expressed on a subset of CD4+ memory T cells in the resting state. In this paper, we demonstrate that binding of the soluble anti‐CD26 monoclonal antibody (mAb) 1F7 inhibits human T‐cell growth and proliferation in both CD26‐transfected Jurkat T‐cell lines and human T‐cell clones by inducing G1/S arrest, which is associated with enhancement of p21Cip1 expression. This effect depends on the DPPIV enzyme activity of the CD26 molecule. Moreover, we show that expression of p21Cip1 after treatment with the anti‐CD26 mAb 1F7 appears to be induced through activation of extracellular signal‐regulated kinase (ERK) pathway. These data thus suggest that anti‐CD26 treatment may have potential use in the clinical setting involving activated T cell dysregulation, including autoimmune disorders and graft‐vs.‐host disease.
Opposing roles of activator protein‐1 and CCAAT/enhancer binding protein β in the regulation of inducible granulysin gene expression in a human monocytic cell line, THP‐1
Authors: Kida Yutaka , Shimizu Takashi , Kuwano Koichi 
Published: March 2003

SummaryWe previously reported that inducible granulysin gene expression in a human monocytic cell line, THP‐1 is dominantly dependent on transcription factor activator protein‐1 (AP‐1). Here, we further examined the precise regulatory mechanisms underlying granulysin gene expression using THP‐1 cells treated with Acholeplasma laidlawii. Transfection of reporter gene constructs into THP‐1 cells indicated that the presence of a positive regulatory element(s) is located from −329 to −85 base pairs, containing two distinct AP‐1 binding sites and one nuclear factor‐κB (NF‐κB) binding site. Deletion or mutation of the NF‐κB binding site failed to affect inducible promoter activity, whereas deletion or mutation of both the AP‐1 binding sites abrogated the promoter activity. Interestingly, deletion of the putative CCAAT/enhancer binding protein β (C/EBPβ) binding site upstream of the positive regulatory element induced the augmentation of granulysin promoter activity. Electrophoretic mobility shift assays demonstrated that nuclear extract prepared from A. laidlawii‐treated THP‐1 cells generated a specific binding to oligonucleotides, including AP‐1, C/EBPβ, and NF‐κB element. Furthermore, over‐expression of liver‐enriched transcriptional activator protein, a subunit of C/EBPβ, augmented A. laidlawii‐induced granulysin promoter activity, whereas over‐expression of liver‐enriched transcriptional inhibitory protein inhibited the promoter activity. NF‐κB p50 homodimer had no transactivation property, although it bound to the NF‐κB site. These results indicate that AP‐1 and C/EBPβ, but not NF‐κB participate in the regulation of inducible granulysin gene expression in THP‐1 cells. Moreover, the Toll‐like receptor 2‐dependent signalling pathway may be involved in A. laidlawii‐induced transactivation of the granulysin promoter. Thus, these results suggest that the gene expression of granulysin in macrophages would be exquisitely regulated by positive and negative transcription factors when microbial invasion occurs.
B‐cell repertoire specific for an unfolded self‐determinant of mouse lysozyme escape tolerance and dominantly participate in the autoantibody response
Authors: Tsujihata Yoshiyuki , Mizukami Yousuke , Ueda Tadashi , So Takanori , Imoto Taiji 
Published: March 2003

SummaryWe previously found that autoantibodies against mouse lysozyme (ML) were strongly induced in normal BALB/c mice when immunized with mutant ML that has triple mutations rendering the dominant T‐cell epitope of hen egg lysozyme (HEL), HEL 107–116. As T cells specific for HEL 107–116 were primed in these mice, the anti‐ML immunoglobulin G (IgG) responses would be the result of collaborations between autoreactive B cells specific for ML and T cells specific for HEL 107–116. Serum IgG responses against ML were dominantly focused on the ML 14–69 region, indicating that B cells responding to the epitope escape tolerance. In the present study, we prepared several monoclonal antibodies (mAbs) specific for ML 14–69 and examined their antigen specificities in detail, to characterize the nature of the remaining B‐cell repertoire specific for ML. mAbs specific for ML 14–69 interacted weakly with soluble, native ML, but the interactions were strengthened by denaturation of ML. The apparent affinity constants between these mAbs and ML showed an increase, ranging from six‐ to 80‐fold, by denaturation of ML. Therefore, these mAbs were more specific for the denatured determinant than for the determinant in the native structure. These results indicate that a substantial number of autoreactive B cells, specific for the unfolded conformation of ML, escape tolerance and are dominantly involved in the autoantibody response to ML. Our finding provides important information to understand the naturally occurring autoreactive B‐cell repertoire in normal mice.
Respiratory syncytial virus infection and virus‐induced inflammation are modified by contaminants of indoor air
Authors: Foster Serene , Bedford Kirk J. , Gould Melanie E. L. , Coward William R. , Hewitt Colin R. A. 
Published: March 2003

SummaryThe airway epithelium is the first cellular component of the lung to be encountered by the particles and pathogens present in inhaled air. In addition to its role as a physical barrier, the immunological activity of the airway epithelium is an essential part of the pulmonary immune system. This means that the symptoms of lung diseases that involve immunological mechanisms are frequently exacerbated by infection of the airway epithelium with respiratory viruses. The virus‐induced enhancement of immunological activity in infected epithelial cells is well characterized. However, the effects that contaminants of inhaled air have upon the infectivity and replication of respiratory viruses and the inflammation they cause, are comparatively unknown. In this study, we have shown that pre‐exposure of airway epithelial cells to bacterial lipopolysaccharides or a proteolytically active house dust mite allergen, is able to, respectively, inhibit or enhance the level of cellular infection with respiratory syncytial virus and similarly alter virus‐induced expression of the inflammatory chemokine interleukin‐8. These results suggest that respiratory syncytial virus infection and the inflammation caused by respiratory syncytial virus may be modified by the biologically active contaminants of indoor air.
Roles for CD40, B7 and major histocompatibility complex in induction of enhanced immunity by cryptococcal polysaccharide‐pulsed antigen‐presenting cells
Authors: Blackstock Rebecca 
Published: March 2003

SummaryImmunization of mice with activated antigen‐presenting cells (APC) pulsed ex vivo with cryptococcal capsular polysaccharide, a glucuronoxylomannan (GXM‐APC) results in prolongation of survival and delayed‐type hypersensitivity (DTH) responsiveness following infection with Cryptococcus neoformans (NU‐2). GXM‐APC has both non‐specific and GXM‐specific effects that influence the immune responses that develop in mice after infection with NU‐2. Type 1 cytokine responses are augmented after immunization with APC alone, while GXM must be present for the vaccine to influence survival and DTH reactions. This investigation evaluated the role that major histocompatibility complex (MHC) and co‐stimulatory molecules play in the non‐specific and GXM‐specific responses induced by GXM‐APC. APC from CD40 knockout mice were as effective as wild‐type APC for the induction of non‐specific and GXM‐specific responses. Blocking activity of B7‐1 and B7‐2 by treatment of immunized mice with monoclonal antibodies specific for these molecules just before and for 6 days following GXM‐APC immunization decreased the splenic interferon‐γ response of mice subsequently infected with NU‐2, but only in mice that were treated with both antibodies. These antibody treatments had no effect on DTH reactivity in similarly treated animals. MHC class I molecules were not involved in the antigen non‐specific or GXM‐specific activities of the vaccine. MHC class II molecules were not required for augmentation of type 1 cytokine responses but were needed for induction of the GXM‐specific response that regulates the expression of DTH reactivity. This investigation has shown that an MHC class II‐restricted, GXM‐specific response is responsible for altering DTH responsiveness which is the correlate of immunity in this model.
Dismantling the silos: extracting new value from IT investments in public administration
Authors: Bannister Frank 
Published: March 2003

Abstract.The drive for information technology‐led organizational and operational change in public administration has lagged behind that in the private sector. For good reasons, central public administrations are conservative by nature. Most public administrations are bureaucracies and bureaucracies tend to resist change. Nevertheless the pressure to obtain better value from public administration information technology investments is growing and the debate as to how to achieve this is increasingly important. Part of this debate is concerned with how best to confront the formidable and specific challenges faced by the sector. These include cultural, structural, resource and technical problems as well as a legacy of isolated developments which do not interrelate. The difficulties are compounded by the problems of evaluation generally in public administration – problems which are reflected in the evaluation of public sector information systems. This drive for change is reviewed and discussed in the context of the Irish civil service, in which there is a growing awareness of the strategic importance of breaking down specialized vertical systems and providing an integrated service to the citizen. A new approach to the problem, based on adapting the concept of business objects, is suggested.
Two‐dimensional gel analysis of midgut proteins of Anopheles stephensi lines with different susceptibility to Plasmodium falciparum infection
Authors: Prévot G. I. , Laurent‐Winter C. , Feldmann A. M. , Rodhain F. , Bourgouin C. 
Published: March 2003

AbstractLittle is known about the composition of the mosquito midgut which plays a central role in the development and subsequent transmission of malaria parasites. As a first step towards the characterization of mosquito midgut molecules involved in the transmission of malaria parasites, we analysed two‐dimensional gel electrophoresis patterns of the midgut proteins of sugar‐fed and blood‐fed Anopheles stephensi lines of different susceptibility to P. falciparum infection. Two lines fully susceptible and one line (Pb3‐9A) of reduced susceptibility were used. In the refractory line ookinetes do develop but are only inefficiently transformed into oocysts (Feldmann & Ponnudurai, 1989). The protein profiles of midguts from all sugar‐fed mosquito lines were similar. However, after blood feeding, the midgut of the fully susceptible lines contained proteins not found in the midgut of line Pb3‐9A. Twenty‐nine such proteins were detected and are candidates for involvement in the interaction between the mosquito midgut and P. falciparum.
Genetic differentiation in the yellow fever virus vector, Aedes simpsoni complex, in Africa: Sequence variation in the ribosomal DNA internal transcribed spacers of anthropophilic and non‐anthropophilic populations
Authors: Mukwaya L. G. , Kayondo J. K. , Crabtree M. B. , Savage H. M. , Biggerstaff B. J. , Miller B. R. 
Published: March 2003

AbstractMosquitoes of the Aedes simpsoni complex are important vectors of yellow fever virus in Africa. We examined the ribosomal DNA sequence divergence in the internal transcribed spacer regions (ITS‐1 and ITS‐2) for populations of mosquitoes that were determined to be anthropophilic or non‐anthropophilic in their bloodmeal host preference. A neighbour‐joining tree produced two clades: one contained all of the individual mosquitoes from anthropophilic populations and the other contained all of the individual mosquitoes from non‐anthropophilic populations. There was no segregation of the taxa within each of the two clades based on geographical origin. The data suggest the exisf′tence of two distinct species of Ae. simpsoni s.l. in Uganda that correlates with their host blood‐feeding preference. The current taxonomic status of the complex is discussed in relation to these findings.
Cloning of a catalytic subunit of cAMP‐dependent protein kinase from the honeybee (Apis mellifera) and its localization in the brain
Authors: Eisenhardt D. , Fiala A. , Braun P. , Rosenboom H. , Kress H. , Ebert P. R. , Menzel R. 
Published: March 2003

AbstractIn the honeybee the cAMP‐dependent signal transduction cascade has been implicated in processes underlying learning and memory. The cAMP‐dependent protein kinase (PKA) is the major mediator of cAMP action. To characterize the PKA system in the honeybee brain we cloned a homologue of a PKA catalytic subunit from the honeybee. The deduced amino acid sequence shows 80–94% identity with catalytic subunits of PKA from Drosophila melanogaster, Aplysia californica and mammals. The corresponding gene is predominantly expressed in the mushroom bodies, a structure that is involved in learning and memory processes. However, expression can also be found in the antennal and optic lobes. The level of expression varies within all three neuropiles.
Leveraging genomic databases: from an Aedes albopictus mosquito cell line to the malaria vector Anopheles gambiae via the Drosophila genome project
Authors: Eccleston E. D. , Gerenday Anna , Fallon Ann M. 
Published: March 2003

AbstractAn important justification for genome sequencing efforts is the anticipation that data from model organisms will provide a framework for the more rapid analysis of other, less studied genomes. In this investigation, we sequenced an internal region of 25 amino acids from a 52 kDa protein that was differentially expressed in 20‐hydroxyecdysone‐treated Aedes albopictus cells in culture. Within the GenBank non‐mouse and non‐human expressed sequence tag (EST) database, this ‘Aedes peptide’ uncovered a putative homology to hypothetical translation products from Anopheles gambiae, Caenorhabditis elegans and Drosophila melanogaster. The hypothetical translation product from D. melanogaster, which included 462 amino acids, uncovered five expressed sequence tags (ESTs) from the malaria vector, Anopheles gambiae. When the Anopheles ESTs were aligned against the hypothetical Drosophila protein, we found that in aggregate they covered 324 amino acids, with gaps measuring 19, 30, and 87 amino acids. To approximate the complete amino acid sequence, gaps between translation products from Anopheles ESTs were replaced with corresponding amino acids from Drosophila to arrive at a calculated mass of 51 104 and a pI of 5.84 for the mosquito protein, consistent with the position of the Ae. albopictus protein on two‐dimensional polyacrylamide gels. Finally, tandem mass spectrometry of a tryptic digest of the 52 kDa Ae. albopictus protein revealed 33 peptides with masses within 1 Dalton of those predicted from an in silico digestion of the reconstructed Anophleles protein. In addition to providing the first direct evidence that a hypothetical protein in Drosophila is in fact translated, this analysis provides a general approach for maximizing recovery, from existing databases, of information that can facilitate prioritization of efforts among several candidate proteins.
Transgenic Drosophila reveals a functional in vivo receptor for the Bacillus thuringiensis toxin Cry1Ac1
Authors: Gill Michael , Ellar David 
Published: March 2003

AbstractThe bacterium Bacillus thuringiensis synthesizes toxins (δ‐endotoxins) that are highly specific for insects. Once ingested, the activated form of the toxin binds to a specific receptor(s) located on the midgut epithelial cells, inserts into the membrane causing the formation of leakage pores and eventual death of the susceptible insect larvae. Manduca sexta larvae are highly susceptible to Cry1Ac1, a toxin that is believed to bind M. sexta Aminopeptidase N, a glycoprotein located on the apical membrane. However, the binding data obtained to date only support the interaction of Cry1Ac1 with APN in vitro. To explore the in vivo role of APN, we have utilized the GAL4 enhancer trap technique to drive the expression of M. sexta APN in both midgut and mesodermal tissues of Cry1Ac1 insensitive Drosophila larvae. Transgenic Drosophila fed the toxin were now killed, demonstrating that APN can function as a receptor for Cry1Ac1 in vivo.