bluuebunny/crossref_metadata_2025_split huggingface dataset:
Glucocorticoid Enhances the Neurotoxic Actions of Quinolinic Acid in the Striatum in a Cell‐Specific Manner
Authors: Ngai L.‐Y. , Herbert J. 
Published: June 2005

AbstractThis study demonstrates that corticosterone can exacerbate the damaging effects of infused quinolinic acid (QA) on the dorsal striatum. Adult adrenalectomised male rats were pretreated subcutaneously with graded doses of corticosterone (0, 0.5, 2, 5, 20 and 40 mg/kg/day) for 2 days and then received a unilateral infusion of QA (45 nmol) (under Isoflurane/N2O anaesthesia) into the dorsal striatum. A control infusion (vehicle) was made into the striatum on the other side. Corticosterone treatment was continued and they were killed 7 days later. Plasma corticosterone was measured by radioimmunoassay, and thymus weights were used as an integrated measure of glucocorticoid activity. Lesion volumes were measured on neuronal nuclei stained sections, dopamine and cyclic AMP‐regulated phosphoprotein 32 (DARRP‐32) was used to assess medium spiny neurone survival, NADPH‐diaphorase histochemistry to assess medium aspiny neurones and, finally, choline acetyltransferase to assess large aspiny neurones. Adrenalectomised rats showed smaller lesions than control (sham‐operated) rats, suggesting significant protection. Increasing doses of corticosterone resulted in larger lesions up to an apparent ceiling effect at higher doses; there was no evidence of a U‐shaped dose–response. There was a differential effect of both QA and corticosterone on the cell populations of the striatum. Medium spiny neurones were most vulnerable to the effects of QA and to the exacerbating actions of corticosterone. Medium aspiny neurones were equally vulnerable to QA but corticosterone had no additional effect. Large aspiny neurones were relatively less sensitive to QA and there was no additional action of administered corticosterone. These results show that corticosterone has a selective neuroendangering action within the striatum, but there is no evidence for a protective action of glucocorticoids at lower doses.
Characterization of Chlamydia MurC‐Ddl, a fusion protein exhibiting D‐alanyl‐D‐alanine ligase activity involved in peptidoglycan synthesis and D‐cycloserine sensitivity
Authors: McCoy Andrea J. , Maurelli Anthony T. 
Published: June 2005

SummaryRecent characterization of chlamydial genes encoding functional peptidoglycan (PG)‐synthesis proteins suggests that the Chlamydiaceae possess the ability to synthesize PG yet biochemical evidence for the synthesis of PG has yet to be demonstrated. The presence of D‐amino acids in PG is a hallmark of bacteria. Chlamydiaceae do not appear to encode amino acid racemases however, a D‐alanyl‐D‐alanine (D‐Ala‐D‐Ala) ligase homologue (Ddl) is encoded in the genome. Thus, we undertook a genetics‐based approach to demonstrate and characterize the D‐Ala‐D‐Ala ligase activity of chlamydial Ddl, a protein encoded as a fusion with MurC. The full‐length murC‐ddl fusion gene from Chlamydia trachomatis serovar L2 was cloned and placed under the control of the arabinose‐inducible ara promoter and transformed into a D‐Ala‐D‐Ala ligase auxotroph of Escherichia coli possessing deletions of both the ddlA and ddlB genes. Viability of the E. coliΔddlAΔddlB mutant in the absence of exogenous D‐Ala‐D‐Ala dipeptide became dependent on the expression of the chlamydial murC‐ddl thus demonstrating functional ligase activity. Domain mapping of the full‐length fusion protein and site‐directed mutagenesis of the MurC domain revealed that the structure of the full fusion protein but not MurC enzymatic activity was required for ligase activity in vivo. Recombinant MurC‐Ddl exhibited substrate specificity for D‐Ala. Chlamydia growth is inhibited by D‐cycloserine (DCS) and in vitro analysis provided evidence for the chlamydial MurC‐Ddl as the target for DCS sensitivity. In vivo sensitivity to DCS could be reversed by addition of exogenous D‐Ala and D‐Ala‐D‐Ala. Together, these findings further support our hypothesis that PG is synthesized by members of the Chlamydiaceae family and suggest that D‐amino acids, specifically D‐Ala, are present in chlamydial PG.
Genetic analysis of the Legionella pneumophila DotB ATPase reveals a role in type IV secretion system protein export
Authors: Sexton Jessica A. , Yeo Hye‐Jeong , Vogel Joseph P. 
Published: June 2005

SummaryThe pulmonary pathogen Legionella pneumophila uses the Dot/Icm type IV secretion system (T4SS) to replicate inside host cells. This apparatus translocates proteins into macrophages to alter their endocytic pathway and enable bacterial growth. Although the secretion ATPase DotB is critical for T4SS function, its specific role in type IV secretion remains undefined. Due to similarity to the VirB11 and PilT ATPases, DotB has been proposed to play a role in assembly of the T4SS, retraction of pili and/or export of substrates. With the goal of understanding the protein's function(s), we isolated and characterized 30 dotB alleles using a variety of phenotypic and biochemical assays. Twenty‐four of these alleles possess several dot/icm mutant phenotypes, including a complete lack of intracellular replication, plasmid mobilization and contact‐dependent cytotoxicity. These 24 non‐functional alleles fall into three classes: those with a known biochemical defect, those with a predicted enzymatic defect and those with an unknown defect. Six other alleles display partial activity in dot/icm phenotypic assays, thus constituting a fourth class. Two mutants in this class are unable to export a subset of T4SS substrates, providing the first evidence for a DotB function in substrate export and suggesting a possible role in substrate selection.
The Aspergillus nidulans sldIRAD50 gene interacts with bimEAPC1, a homologue of an anaphase‐promoting complex subunit
Authors: Malavazi Iran , Lima Joel Fernandes , Fagundes Márcia Regina von Zeska Kress , Efimov Vladimir P. , Goldman Maria Helena de Souza , Goldman Gustavo Henrique 
Published: June 2005

SummaryThe Mre11–Rad50–Nbs1 protein complex has emerged as a central component in the human cellular DNA damage response, and recent observations suggest that these proteins are at least partially responsible for the linking of DNA damage detection to DNA repair and cell cycle checkpoint functions. We have identified Aspergillus nidulans sldI1444D mutant in a screen for dynein synthetic lethals. The sldIRAD50 gene was cloned by complementation of the sporulation deficiency phenotype of this mutant. A transversion G→C at the position 2509 (Ala‐692‐Pro amino acid change) in the sldI1444D mutant causes sensitivity to several DNA‐damaging agents. The mutation sldI1 occurs at the CXXC hinge domain of Rad50. We have deleted part of the coiled‐coil and few amino acids of the Rad50–Mre11 interaction region and assessed several phenotypic traits in this deletion strain. Besides sensitivity to a number of DNA‐damaging agents, this deletion strain is also impaired in the DNA replication checkpoint response, and in ascospore viability. There is no delay of the S‐phase when germlings of both sldI RAD50 and mreAMRE11 inactivation strains were exposed to the DNA damage caused by bleomycin. Transformation experiments and Southern blot analysis indicate homologous recombination is dependent on scaANBS1 function in the Mre11 complex. There are epistatic and synergistic interactions between sldI RAD50 and bimEAPC1 at S‐phase checkpoints and response to hydroxyurea and UV light. Our results suggest a possible novel feature of the Mre11 complex in A. nidulans, i.e. a relationship with bimE APC1.
Identification of the membrane penetrating domain of Vibrio cholerae cytolysin as a β‐barrel structure
Authors: Valeva Angela , Walev Iwan , Boukhallouk Fatima , Wassenaar Trudy M. , Heinz Natalia , Hedderich Julia , Lautwein Sina , Möcking Moritz , Weis Silvia , Zitzer Alexander , Bhakdi Sucharit 
Published: June 2005

Summary Vibrio cholerae cytolysin (VCC) is an oligomerizing pore‐forming toxin that is related to cytolysins of many other Gram‐negative organisms. VCC contains six cysteine residues, of which two were found to be present in free sulphydryl form. The positions of two intramolecular disulphide bonds were mapped, and one was shown to be essential for correct folding of protoxin. Mutations were created in which the two free cysteines were deleted, so that single cysteine substitution mutants could be generated for site‐specific labelling. Employment of polarity‐sensitive fluorophores identified amino acid side‐chains that formed part of the pore‐forming domain of VCC. The sequence commenced at residue 311, and was deduced to form a β‐barrel in the assembled oligomer with the subsequent odd‐numbered residues facing the lipid bilayer and even‐numbered residues facing the lumen. Pro328/Lys329 were tentatively identified as the position at which the sequence turns back into the membrane and where the antiparallel β‐strand commences. This was deduced from fluorimetric analyses combined with experiments in which the pore was reversibly occluded by derivatization of sulphydryl groups with a bulky moiety. Our data support computer‐based predictions that the membrane‐permeabilizing amino acid sequence of VCC is homologous to the β‐barrel‐forming sequence of staphylococcal cytolysins and identify the β‐barrel as a membrane‐perforating structure that is highly conserved in evolution.
Memories of Biology, Mathematics and Ivan
Authors: Henry C. 
Published: June 2005

AbstractThe plaque assay years at Pittsburgh that included efforts to optimize the detection for single immunoglobulin M (IgM) and IgG antibody‐producing cells are described. Sensitivity of the assay and precision of the plaque count was established, and a series of response curves were obtained that enabled to calculate the frequency of precursors and to identify the target of the antigen. If one ignored a number of anomalies of the curves and made the simple assumption that a single hit by antigen on a pre‐committed precursor yielded a clone of antibody‐forming cells, estimates of some 700 target cells (per mouse spleen) were obtained, figures not too far from the number that Ivan would later find.Years at the Paul Ehrlich Institute and visits to the Basel Institute for Immunology are described.
Pittsburgh 1962/63 Revisited: Too Many Antibodies, Too Few Ribosomes?
Authors: Staehelin T. 
Published: June 2005

AbstractJerne's success in developing the plaque‐forming cell methodology for determining the number of specific antibody‐producing lymphocytes during the immune response has prompted scientists to calculate the number of antibody molecules produced by a single cell. In this article, calculations of the rate of antibody production are made, and a special attention is called to the number of ribosomes in a cell, the rate of protein chain elongation and various other parameters. The generally accepted notion of 2000 IgM molecules or some 15,000 IgG molecules per second (per cell) remains an acceptable estimate. The article describes the work of Ivan Lefkovits and the interaction among research groups leading to quantitative evaluations of the components of the immune system.
From Poisson Distribution to Interleukins
Authors: Aarden L. 
Published: June 2005

AbstractIn the mid‐1970s, Ivan Lefkovits and co‐workers introduced limiting dilution analysis of T and B cells as a method to quantify antigen‐specific T and B cells. In this study, I describe how my co‐operation with Ivan on the subject led not only to heated discussions on the validity of the approach but also to the identification of interleukin‐2 as T‐cell‐derived factor, capable of replacing T‐cell help required for the activation of B cells.
Importance of Counting in Biology
Authors: Pink J. R. L. 
Published: June 2005

AbstractIn this study, analysis of biological problems through structural and quantitative approaches is described. Classical genetics on major histocompatibility complex in avian species has been evaluated. Experiments showing that antibodies from various individuals were generally distinct have led to further analysis of immunoglobulin light chain diversity by means of O'Farrell's 2D gel technique. The transition of the research towards vaccine development has been described.   ‘It's not the voting that's democracy, it's the counting.'  – Tom Stoppard (Czech‐born dramatist), Jumpers (1972) act 1.
Dissection of Systems, Cell Populations and Molecules
Authors: Benner R. , Khan N. A. 
Published: June 2005

AbstractThis paper summarizes studies on antibody formation in the bone marrow and the suppressive effects of intravenous immunization with allogeneic blood cells on T‐cell function in mice. The latter studies were extended by employing the limiting dilution culture system developed in Ivan Lefkovits' laboratory and implemented in collaboration with Lucien Aarden. Thereby, the functional data were complemented with frequencies of alloantigen‐activated helper (Th) and suppressor T cells after intravenous alloimmunization. These results led the Rotterdam group to studies on the prevention of rejection of the foetal ‘allograft’. Th cells are central in foetal allograft rejection and pregnancy success. Characteristic for human pregnancy is the production of the glycoprotein chorionic gonadotropin (hCG) hormone. The in vivo liberated peptide fragments originating from nicking of the sequence MTRVLQGVLPALPQ in the β‐chain of hCG were considered for their immunoregulating capacity related to pregnancy success. These peptides – prepared synthetically – (MTR, MTRV, LQG, LQGV, VLPALP and others) indeed showed a remarkable spectrum of biological effects (e.g. modulation of angiogenesis, inhibition of septic shock syndrome, prevention of diabetes and reduction of ischaemia‐reperfusion damage). The paper interprets and generalizes these findings and projects them into various research directions, especially towards the proteomics framework studies built up in Ivan Lefkovits' laboratory in the nineties. During the time period, when Ivan spent a mini‐sabbatical in Rotterdam (months after closing down the BII) more detailed discussions were intiated. This paper is meant to keep the discussions between the involved research groups going on.
Mouse Hepatitis Virus 3 Binding to Macrophages Correlates with Resistance to Experimental Infection
Authors: Pereira C. A. , Moreira C. , Tsuhako M. H. , De Franco M. T. 
Published: June 2005

AbstractMouse hepatitis virus 3 (MHV3) infection of A/J and BALB/c mice has been used as a model of resistance/susceptibility. A/J mice recover from a mild disease after 4–6 days of infection and the BALB/c mice develop an acute hepatitis and die after 3–4 days of infection. In view of studying the MHV3 binding to cells or cell extracts, we performed an enzyme‐linked immunosorbent assay‐like virus‐binding assay, preparing microplates with L929 cells, A/J or BALB/c mouse macrophages and also with proteins extracted from these cells. Higher MHV3 bindings were observed to proteins of BALB/c macrophages than to the A/J ones. The interferon‐γ (IFN‐γ) activation led to a reduction of MHV3 binding only to proteins of resistant A/J mouse macrophages. Our experiments contribute to the hypothesis that IFN‐γ activation of macrophages plays an important role against MHV3 infection by downregulating the expression of viral receptors.
Post‐transplant upregulation of chemokine messenger RNA in non‐human primate recipients of intraportal pig islet xenografts
Authors: Hårdstedt Maria , Finnegan Colleen P. , Kirchhof Nicole , Hyland Kendra A. , Wijkstrom Martin , Murtaugh Michael P. , Hering Bernhard J. 
Published: June 2005

Abstract:  Background:  We have previously shown that pig‐to‐primate intraportal islet xenografts reverse diabetes, escape hyperacute rejection, and undergo acute cellular rejection in non‐immunosuppressed recipients. To gain a better understanding of mechanisms contributing to xenoislet rejection in non‐human primates we examined gene expression in livers bearing islet xenografts in the first 72 h after transplantation.Methods:  Liver specimens were collected at sacrifice from seven non‐immunosuppressed rhesus macaques at 12, 24, 48 and 72 h after intraportal porcine islet transplantation. Following total RNA extraction, mRNA was quantified using SYBR green real‐time reverse transcription polymerase chain reaction (RT‐PCR) for species‐specific immune response genes. Data were analyzed using comparative cycle threshold (Ct) analysis, adjusted for specific primer‐efficiencies and normalized to cyclophilin expression.Results:  Porcine insulin mRNA was detected in all liver samples. Cluster analysis revealed differential gene expression patterns at 12 and 24 h (early) compared with at 48 and 72 h (late) post‐transplant. Gene expression patterns were associated with histological findings of predominantly neutrophils and only a few lymphocytes at 12 and 24 h and an increasing number of lymphocytes and macrophages at 48 and 72 h. Transcript levels of CXCR3 and its ligands, interferon‐inducible protein 10 (IP‐10) and monokine induced by IFN‐γ (Mig), significantly increased between early and late time points together with expression of MIP‐1α, regulated on activation normal T expressed and secreted protein (RANTES) and MCP‐1. CCR5 showed only a marginal, non‐significant increase. Fas ligand, perforin and granzyme B transcripts were all elevated at 48 and 72 h post‐transplant.Conclusions:  Our data suggest that CXCR3, with ligands IP‐10 and Mig, is involved in T cell recruitment in acute islet xenograft rejection in non‐human primates. Upregulation of RANTES and MIP‐1α transcripts in the absence of a significant CCR5 increase suggests a possible involvement of other chemokine receptors. MCP‐1 expression is associated with T cell and macrophage infiltration. Elevated cytotoxic effector molecule expression (Fas ligand, perforin, granzyme B) indicates T‐cell mediated graft destruction by cytotoxic and cytolytic mechanisms within 48 to 72 h after transplantation. These results identify the CXCR3‐mediated chemoattractant pathway as an immunosuppressive target in pig‐to‐primate islet xenotransplantation.
Genetic and functional evaluation of the level of inbreeding of the Westran pig: a herd with potential for use in xenotransplantation
Authors: O'Connell Philip J. , Hawthorne Wayne J. , Simond Denbigh , Chapman Jeremy R. , Chen Yizhou , Patel Anita T. , Walters Stacey N. , Burgess Jane , Weston Lyanne , Stokes Rebecca A. , Moran Chris , Allen Richard 
Published: June 2005

Abstract:  Background:  The Westran pig has been purposely inbred for use in xenotransplantation. The herd originated in the wild from a limited gene pool and has been inbred by repeated full‐sib matings for nine generations.Methods:  The aim of this study was to evaluate the level of inbreeding by functional assays, such as bi‐directional MLR and reciprocal skin grafts between herd members, and by genetic analysis using highly polymorphic genetic markers to calculate the level of inbreeding.Results:  The MLR between herd members were non‐reactive whereas there was a prompt response to third party pig lymphocytes, indicative of a normal immune responsiveness in Westran pigs but isogenicity of the major histocompatibility complex. Skin grafts between male siblings or female sibling skin grafts on male recipients showed prolonged survival but with few exceptions did not survive beyond 100 days suggesting that by the fifth generation the Westran herd was still mismatched at minor histocompatibility antigens. This level of functional inbreeding was confirmed by microsatellite analysis of highly polymorphic markers, which showed that 52 of 53 chromosomally dispersed markers were fixed by the ninth generation. This level of fixation was consistent with 19 to 20 generations of full‐sibling inbreeding. The calculated inbreeding coefficient at generation 10 was 0.98159.Conclusions:  This analysis confirms that the Westran pig is highly inbred and we propose that analysis of chromosomally dispersed highly polymorphic markers is an accurate and reproducible method for assessing the level of inbreeding of a pig herd.
Method for evaluating quality of cultured neonatal pig Sertoli cells
Authors: Valdés‐González Rafael , Silva‐Torres Lourdes , Ramírez‐González Briceyda , Ormsby Christopher E , Terán‐Ortiz Luis , Ayala‐Sumuano Jorge‐Tonatiuh 
Published: June 2005

Abstract:  Background:  Sertoli cells (SC) in the testis secrete factors that nourish and immunoprotect developing spermatozoa, which have made them the focus of studies that aim to generate localized tolerance, particularly for transplantation and perhaps autoimmunity. Several methods have been described to isolate these cells, which include a two‐step enzymatic digestion with limited assessment of the culture. Here we describe a one‐step method, and a series of tests for determining purity, viability, and function of the cultured cells.Methods:  We isolated SC from neonatal pigs using Liberase HI digestion. Viability and apoptosis of cultured cells were measured by flow cytometry with propidium iodide and annexin, respectively. Specific identification of the Sertoli type was made by immunodetection of Sox9, vimentin, and Mullerian inhibiting substance. Moreover, for functionality we were able to detect clusterin in the cultured cells by Western blot.Results:  Our isolation method had a yield and purity similar to previous reports measured with two‐step methods. Viability was 95.22 ± 0.57% and apoptotic cells were 10.5 ± 0.32% after 48 h in culture. At 7 days, practically all cells expressed Sox9, Mullerian inhibiting substance, clusterin, and vimentin.Conclusions:  We describe an alternative strategy for preparing and identifying cultured SC for further assays of metabolic activity or in transplantation models. Establishing a one‐step Liberase‐digestion method for isolation, evaluating viability and apoptosis by more sensitive methods, and detecting specific markers in culture can help to evaluate the quality of cultured cells. Specific cell markers for identifying SC may be critical when identifying SC outside the testis, in contrast with vimentin which is useful only for in situ cells.