bluuebunny/crossref_metadata_2025_split huggingface dataset:
The cytosolic SycE and SycH chaperones of Yersinia protect the region of YopE and YopH involved in translocation across eukaryotic cell membranes
Authors: Woestyn Sophie , Sory Marie‐Paule , Boland Anne , Lequenne Olivier , Cornelis Guy R. 
Published: October 2006

Summary Yersinia adhering at the surface of eukaryotic cells secrete a set of proteins called Yops. This secretion which occurs via a type III secretion pathway is immediately followed by the injection of some Yops into the cytosol of eukaryotic cells. Translocation of YopE and YopH across the eukaryotic cell membranes requires the presence of the translocators YopB and YopD. YopE and YopH are modular proteins composed of an N‐terminal secretion signal, an internalization domain, and an effector domain. Secretion of YopE and YopH requires the presence of the specific cytosolic chaperones SycE and SycH, respectively. In this work, we have mapped the regions of YopE and YopH that are involved in binding of their cognate chaperone. There is only one Syc‐binding domain in YopE (residues 15–50) and YopH (residues 20–70). This domain is localized immediately after the secretion signal and it corresponds to the internalization domain. Removal of this bifunctional domain did not affect secretion of YopE and YopH and even suppressed the need for the chaperone in the secretion process. Thus SycE and SycH are not secretion pilots. Instead, we propose that they prevent intrabacterial interaction of YopE and YopH with proteins involved in translocation of these Yops across eukaryotic cell membranes.
A novel DnaJ‐like protein in Escherichia coli inserts into the cytoplasmic membrane with a Type III topology
Authors: Clarke David J. , Jacq A. , Holland I. B. 
Published: October 2006

Summary We describe a novel Escherichia coli protein, DjlA, containing a highly conserved J‐region motif, which is present in the DnaJ protein chaperone family and required for interaction with DnaK. Remarkably, DjlA is shown to be a membrane protein, localized to the inner membrane with the unusual Type III topology (N‐out, C‐in). Thus, DjlA appears to present an extremely short N‐terminus to the periplasm and has a single transmembrane domain (TMD) and a large cytoplasmic domain containing the C‐terminal J‐region. Analysis of the TMD of DjIA and recently identified homologues in Coxiella burnetti and Haemophilus influenzae revealed a striking pattern of conserved glycines (or rarely alanine), with a four‐residue spacing. This motif, predicted to form a spiral groove in the TMD, is more marked than a repeating glycine motif, implicated in the dimerization of TMDs of some eukaryotic proteins. This feature of DjlA could represent a promiscuous docking mechanism for interaction with a variety of membrane proteins. DjlA null mutants can be isolated but these appear rapidly to accumulate suppressors to correct envelope and growth defects. Moderate (10‐fold) overproduction of DjlA suppresses a mutation in FtsZ but markedly perturbs cell division and cell‐envelope growth in minimal medium. We propose that DjlA plays a role in the correct assembly, activity and/or maintenance of a number of membrane proteins, including two‐component signal‐transduction systems.
Developmentally regulated synthesis of an unusually small, basic peptide by Coxiella burnetii
Authors: Heinzen R. A. , Howe D. , Mallavia L. P. , Rockey D. D. , Hackstadt T. 
Published: October 2006

Summary Coxiella burnetii undergoes a poorly defined developmental cycle within phagolysosomes of eukaryotic host cells. Two distinct developmental forms are part of this cycle: a small‐cell variant (SCV) and large‐cell variant (LCV). Ultrastructurally, the SCV is distinguished from the LCV by its smaller size and condensed chromatin. At a molecular level, little is known about morphogenesis in C. burnetii, and no proteins specific to the SCV have been identified. Preparative isoelectric focusing was conducted to purify basic proteins possibly involved in SCV chromatin structure. A predominant protein of low Mr was present in the most basic fraction, eluting with a pH of approx. 11. Degenerate deoxyoligonucleotides corresponding to the N‐terminal sequence of this protein were used to recover a cosmid clone from a C. burnetii genomic library. Nucleotide sequencing of insert DNA revealed an open reading frame designated scvA (small‐cell‐variant protein A) with coding potential for a 30 amino acid protein (ScvA) with a predicted Mr of 3610. ScvA is 46% arginine plus 46% glutamine with a predicted pi of 12.6. SDS‐PAGE and silver staining of lysates of SCV and LCV purified by caesium chloride‐equilibrium density centrifugation revealed a number of proteins unique to each cell type. Immunoblot analysis with ScvA antiserum demonstrated the presence of ScvA only in the SCV. By immunoelectron microscopy, ScvA antiserum labelled only the SCV, with the label concentrated on the condensed nucleoid. In addition, ScvA bound double‐stranded DNA in gel mobility‐shift assays. A 66% reduction in the mean number of gold particles per Coxiella cell was observed at 12 h post‐infection when compared with the starting inoculum. Collectively, these data suggest that synthesis of ScvA is developmentally regulated, and that the protein may serve a structural or functional role as an integral component of the SCV chromatin. Moreover, degradation of this protein may be a necessary prerequisite for morphogenesis from SCV to LCV.
An elongation factor Tu (EF‐Tu) resistant to the EF‐Tu inhibitor GE2270 in the producing organism Planobispora rosea
Authors: Sosio Margherita , Amati Giuseppe , Cappellano Carmela , Sarubbi Edoardo , Monti Federica , Donadio Stefano 
Published: October 2006

Summary Using a cell‐free protein‐synthesis system, we have established that the elongation factor (EF) Tu (EF‐Tu) of the actinomycete Planobispora rosea, the producer of the thiazolyl peptide GE2270, a specific EF‐Tu inhibitor, is highly resistant to its own antibiotic, while it is completely inhibited by kirromycin, which is another inhibitor of this factor. P. rosea was found to possess a single tuf gene, located between fus and rpsJ, encoding other components of the protein‐synthesis machinery. The P. rosea tuf gene was expressed as a translations! fusion to maIE in Escherichia coli, and the resulting EF‐Tu with an N‐terminal Gly‐Met extension was able to promote poly(U)‐directed poly(Phe) synthesis in cell‐free systems. This activity was not affected by GE2270, and the recombinant protein was incapable of binding the antibiotic, indicating that the P. rosea EF‐Tu is intrinsically resistant to this inhibitor. Inspection of the translated tuf sequence revealed a number of amino acid substitutions in highly conserved positions. These residues, which are likely to be involved in conferring GE2270 resistance, map in EF‐Tu domain II, as do the only two known mutations conferring resistance to this class of thiazolyl peptides in Bacillus subtilis.
Regulated expression of the dinR and recA genes during competence development and SOS induction in Bacillus subtilis
Authors: Haijema Bert Jan , van Sinderen Douwe , Winterling Kevin , Kooistra Jan , Venema Gerard , Hamoen Leendert Willem 
Published: October 2006

SummaryIt has been hypothesized that the dinR gene product of Bacillus subtilis acts as a repressor of the SOS regulon by binding to DNA sequences located upstream of SOS genes, including dinR and recA. Following activation as a result of DNA damage, RecA is believed to catalyse DinR‐autocleavage, thus derepressing the SOS regulon. The present results support this hypothesis: a dinR insertion mutation caused a high, constitutive expression of both dinR and recA, which could not be further elevated by SOS‐induction. In addition, gel‐retardation assays demonstrated a direct interaction between the dinR gene product and the recA and dinR promoter regions. Epistatic interactions and gel‐retardation assays demonstrated that the previously reported competence‐specific expression of recA directly depended upon the gene product of comK, the competence transcription factor. These data demonstrate the existence of a direct regulatory link between the competence signal‐transduction pathway and the SOS regulon.
Preparation, Characterization and Epitope Mapping of Monoclonal Antibodies Specific to Human Mast Cell Carboxypeptidase
Authors: Zhou Y. , Chen Z. , He S. 
Published: October 2006

AbstractHuman mast cell carboxypeptidase (hMC‐CP) is a unique product of mast cells. Unlike tryptase and chymase, its potential function and expression in diseased conditions remain largely unknown. To develop an assay for hMC‐CP, the recombinant fusion protein of hMC‐CP and purified native skin hMC‐CP was prepared, and two novel monoclonal antibodies against hMC‐CP named CCP1 (IgG1 isotype) and CCP2 (IgM isotype) were raised in the present study. Epitope analysis shows that CCP1 and CCP2 antibodies recognize epitopes located in the region of amino acids 112–202 of hMC‐CP, and hydrophilicity analysis implies that epitopes might be located in the amino acid residues 123–134 and 165–177. Furthermore, using a competition enzyme‐linked immunosorbent assay, it was shown that the epitope recognized by CCP1 is close to that recognized by CCP2 or the two antibodies partially share the same epitope. Flow cytometry analysis shows that basophilic leukemia cell line KU812 reacts with both CCP1 and CCP2 antibodies, suggesting that this cell line expresses hMC‐CP. In conclusion, although the two antibodies possess different isotypes, they may partially share the same epitope. These two antibodies will be valuable tools for the development of an assay to detect the levels of hMC‐CP in the biological fluids in man.
B‐cell CD25 Expression in Murine Primary and Secondary Lymphoid Tissue
Authors: Amu S. , Gjertsson I. , Tarkowski A. , Brisslert M. 
Published: October 2006

AbstractB cells are in analogy with T cells capable of expressing functional IL‐2 receptors. IL‐2R α‐chain (CD25) positive T cells have been studied in detail but not much is known about CD25 positive B cells. The aim of this study was to examine the phenotypic properties of the CD25 expressing B cells collected from different lymphoid organs in mice. Samples were stained for various cell surface markers and analysed using flow cytometry. We found that approximately 49% of B cells in bone marrow, 16% in peritoneal cavity, 2% in spleen and 1% in lymph nodes express CD25. In contrast, CD25 expressing B cells were not found in the blood or in Peyer's patches. Phenotypic characterization showed that CD25+ B cells in spleen, lymph nodes and peritoneal cavity have higher expression of AA4.1, CD5, CD69, CD80, CD86, CD122, CD132, IgA, IgG and IgM on their surface in comparison with CD25− B cells. In contrast, expression of IgD and IA‐IE was lower on CD25+ B cells in spleen and lymph nodes. In bone marrow, the expression of CD5, CD80, CD86, CD122, CD132, IgA, IgD and IgM was lower, while the expression of AA4.1, IgG and IA‐IE was increased on CD25+ B cells compared with CD25− B cells. In conclusion, our results indicate that B cells expressing CD25 are phenotypically distinctly different from those that are CD25 negative. Our findings suggest that CD25+ B cells are more prone to efficient antigen presentation and display a more mature phenotype.
Dendritic Cells Therapy Confers a Protective Microenvironment in Murine Pregnancy
Authors: Miranda S. , Litwin S. , Barrientos G. , Szereday L. , Chuluyan E. , Bartho J. S. , Arck P. C. , Blois S. M. 
Published: October 2006

AbstractThe fetal–placental unit is a semi‐allograft and immunological recognition of pregnancy, together with the subsequent response of the maternal immune system, is necessary for a successful pregnancy. Dendritic cells (DC) show a biological plasticity that confers them special characteristics regulating both immunity and tolerance. Therapy employing DC proved to diminish the abortion in the DBA/2J‐mated CBA/J females; however, the underlying mechanisms remain unknown. Here, we evaluated whether DC therapy influences the presence of immunoregulatory populations of cells at the fetal–maternal interface. To address this hypothesis, we analysed the pregnancy‐protective CD8, γδ cell populations as well as transforming growth factor (TGF)‐β1 and progesterone‐induced blocking factor (PIBF) expression at the fetal–maternal interface from abortion‐prone female mice that had previously received adoptive transfer of syngeneic DC. Syngeneic DC therapy induced an increase in the number of CD8 and γδ cells. Additionally, an upregulation of TGF‐β1 and PIBF expression could be detected after DC transfer. We suggest that DC therapy differentially upregulates a regulatory/protective population of cells at the fetal–maternal interface. It is reasonable to assure that this mechanism would be responsible for the lower abortion rate.
IMMORTALITY, HUMAN NATURE, THE VALUE OF LIFE AND THE VALUE OF LIFE EXTENSION
Authors: HORROBIN STEVEN 
Published: October 2006

ABSTRACTThe emerging discourse concerning the desirability of intervention in senescence to achieve radical life extension for persons has featured some striking blurring in traditional liberal and conservative commitments and positions. This affords an opportunity for re‐evaluation of these same. The canonical conservative view of the intrinsic value of life is re‐examined and found primarily to involve a denial of human prerogative, rather than an active underwriting of the value of life extension. A critique is offered of an attempted argument against aging intervention from a proto‐conservative worry about a purported threat to human nature. Immortality is found to be a red herring, but a revealing one. Further, the classic liberal view is examined and found wanting in terms of the gravity of its own commitment to, and fullness of its account of the value of life, and the value of life extension. An analysis of the liberal conception of personhood is proposed that both defines persons necessarily as processes, and demonstrates the inalienable quality of the value of life extension to persons so defined.
BIOETHICS AND DEMOCRACY: COMPETING ROLES OF NATIONAL BIOETHICS ORGANISATIONS
Authors: DODDS SUSAN , THOMSON COLIN 
Published: October 2006

ABSTRACTIn establishing National Bioethics Organisations (NBOs), liberal democracies seek to acknowledge the diversity of strongly held ethical positions and the imperative to engage in public debate about important bioethical decisions. NBOs are typically given a range of responsibilities, including contributing to and stimulating public debate; providing expert opinion on relevant issues for policy deliberations; and developing public policy. The state is now found to have an interest in areas previously thought to be a matter of individual choice. NBOs can provide one way of opening up public debate to allow the diversity of views to be heard in a manner that is well‐informed, articulate and responsive to both expert and ‘lay’ public views. We draw on debates in political theory about democratic decision‐making and on the policy making roles of some key NBOs. We are particularly interested in examining the capacity of NBOs to meet the democratic ideal of effective participation by the public, or citizenry, especially by those who are directly affected by the policies, in the development of effective public policy. We provide a basic framework for policy development involving NBOs that can begin to meet this ideal, a process of ‘contested deliberation’.
Gender‐specific development of nonverbal behaviours and mild depression in adolescence
Authors: Van Beek Yolanda , Van Dolderen Marlies S. M. , Demon Dubas Judith J. S. 
Published: October 2006

Background:  Individual differences in depressive symptoms have been linked with social skill deficits in adults and children, yet empirical studies on adolescents are lacking. The present research examines age and gender differences in nonverbal behaviour between mildly depressed and nondepressed (pre‐) adolescents during conversations with an adult (study 1) and a same‐aged peer (study 2). Both studies also examine whether conversation partners respond differently to mildly depressed versus nondepressed (pre)adolescents.Methods:  Study 1 reports on observations of conversations of 9–15‐year‐old children (n = 122) with a female adult partner. Study 2 reports findings of observations of 12–17‐year‐old adolescents (n = 154) in conversation with same‐age, same‐sex peers.Results:  Both studies show gender and/or age effects in gazing, smiling and backchannel behaviours that indicate that as adolescents mature they increasingly behave according to gender‐specific display rules. While talking to an adult, depressed (pre‐)adolescents and the adult partner differed in backchannel behaviours. While talking to peers, only depressed adolescent girls showed less gazing towards the partner during listening. Moreover, adolescents smiled less often towards depressed than nondepressed partners.Conclusions:  Gender‐specific development of nonverbal behaviour may help to understand the development of gender differences in depression in adolescence. Females who fail to exhibit other‐oriented social skills may be particularly at risk for depressive symptoms.
Annotation: What electrical brain activity tells us about brain function that other techniques cannot tell us – a child psychiatric perspective
Authors: Banaschewski Tobias , Brandeis Daniel 
Published: October 2006

Background:  Monitoring brain processes in real time requires genuine subsecond resolution to follow the typical timing and frequency of neural events. Non‐invasive recordings of electric (EEG/ERP) and magnetic (MEG) fields provide this time resolution. They directly measure neural activations associated with a wide variety of brain states and processes, even during sleep or in infants. Mapping and source estimation can localise these time‐varying activation patterns inside the brain. Methods: Recent EEG/ERP research on brain functions in the domains of attention and executive functioning, perception, memory, language, emotion and motor processing in ADHD, autism, childhood‐onset schizophrenia, Tourette syndrome, specific language disorder and developmental dyslexia, anxiety, obsessive‐compulsive disorder, and depression is reviewed.Results:  Over the past two decades, electrophysiology has substantially contributed to the understanding of brain functions during normal development, and psychiatric conditions of children and adolescents. Its time resolution has been important to measure covert processes, and to distinguish cause and effect.Conclusions:  In the future, EEG/ERP parameters will increasingly characterise the interplay of neural states and information processing. They are particularly promising tools for multilevel investigations of etiological pathways and potential predictors of clinical treatment response.
Solubilization of Serotonin1a and Serotonin1b Binding Sites from Bovine Brain
Authors: Asarch Kenneth B. , Shih Jean C. 
Published: October 2006

Abstract:Serotonin, (5‐hydroxytryptamine1, 5‐HT1) binding sites have been solubilized from bovine brain cortex using a mixture of 0.1 % Nonidet P‐40 and 0.3% digitonin in a low‐salt buffer containing 0.1 % ascorbic acid. The affinity of [3H]5‐HT for the soluble cortical binding sites (2.1 nM) is identical to its affinity at membrane‐bound binding sites (2.1 nM). [3H]8‐Hydroxy‐2‐(di‐n‐propylamino)tetraIin ([3H]DPAT), a selective 5‐HT1a, radioligand, also binds to soluble cortical binding sites with high affinity (1.8 nM) comparable with its affinity in the crude membranes (1.7 nM). A significant correlation exists in the rank order potency of serotonergic agents for [3H]5‐HT binding and for [3H]DPAT binding to crude and soluble membranes. The density of [3H]DPAT binding sites relative to the [3H]5‐HT sites in the solubilized cortical membranes (35%) corresponds well with the proportion of 5‐HT1a sites in the crude membranes determined by spiperone displacement (33%), suggesting that both the 5‐HT1a and 5‐HT1b binding sites have been cosolubilized. [3H]5‐HT binding in the soluble preparations was inhibited by GTP, suggesting that a receptor complex may have been solubilized. [3H]Spiperone‐specific binding was not detectable in this preparation, suggesting that 5‐HT2 sites were not cosolubilized.
Histamine H1‐Receptor in Heart: Unique Electrophoretic Mobility and Autoradiographic Localization
Authors: Ruat M. , Bouthenet M. L. , Schwartz J.‐C. , Ganellin C. R. 
Published: October 2006

AbstractHistamine H1‐receptors, visualized in the guinea pig heart by autoradiography using [125I]iodoboipyramine as a specific probe, are abundant in the nodal tissue and cardiac vessels but also occur heterogeneously in the myocardium. Following photoaffinity labeling with [125I]iodoazidophenpyramine and electrophoresis, the ligand binding domain of the heart H1‐receptor was shown to be present on a major 68‐kDa and a less abundant 54‐ to 58‐kDa protein. The 68‐kDa protein displayed a molecular size higher in heart than in all other tissues (56 kDa). This indicates the existence of at least two isoforms of the H1‐receptor; the cardiac isoform, however, was pharmacologically indistinguishable from the common isoform studied in cerebellar membranes using available ligands. Its distinct electrophoretic properties suggest that the cardiac isoform may have a unique function.
A Nerve Growth Factor‐Dependent Protein Kinase That Phosphorylates Microtubule‐Associated Proteins In Vitro: Possible Involvement of Its Activity in the Outgrowth of Neurites from PC12 Cells
Authors: Sano Mamoru , Nishiyama Kaoru , Kitajima Satoko 
Published: October 2006

AbstractWe have established a subline of PC12 cells (PC12D) that extend neurites very quickly in response not only to nerve growth factor (NGF) but also to cyclic AMP (cAMP) in the same way as primed PC12 cells (NGF‐pretreated cells). When phosphorylation of brain microtubule proteins by extracts of these cells was monitored, two distinct kinase activities were found to be increased [from three‐ to eightfold in terms of phosphorylation of microtubule‐associated protein (MAP) 2] by a brief exposure of cells to NGF or to dibutyryl cAMP(dbcAMP). The effect of the combined stimulation with both NGF and dbcAMP was additive in terms of the phosphorylation of MAP2. The apparent molecular mass of the kinase activated by dbcAMP was 40 kDa, and this kinase appears to be cAMP‐dependent protein kinase. The molecular mass of the kinase activated by NGF was 50 kDa. The latter was activated to a measurable extent after 5 min of exposure of cells to NGF; it required Mg2+ for activity but not Mn2+ or Ca2+. This kinase appears to be distinct from previously reported kinases in PC12 cells, and it has been designated as NGF‐dependent MAP kinase, although its physiological substrates are not known at present. An inhibitor of protein kinases, K‐252a, selectively inhibited the outgrowth of neurites from PC12D cells in response to NGF but not to dbcAMP. When this inhibitor was added to the incubation medium of cells exposed simultaneously to NGF or dbcAMP, the increase in activity of the NGF‐dependent MAP kinase was selectively abolished. We isolated several mutant clones of PC12D cells that were deficient in the ability to induce neurites in response to either of the two stimulators. In these variant cells, the activity of the relevant protein kinase was decreased, in parallel with the deficiency in the neurite response to NGF or dbcAMP. These observations suggest that the NGF‐dependent MAP kinase may play an important role in the outgrowth of neurites from PC12 cells in response to NGF.