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T Dick

Publications and source records attributed to T Dick.

At least 37 records · Page 2Linked to original sources
PubMed1999

The nature of the MHC class I peptide loading complex.

Peptide binding to major histocompatibility complex (MHC) class I molecules occurs in the endoplasmic reticulum (ER). Efficient peptide binding requires a number of components in addition to the MHC class I-beta 2 microglobulin dimer (beta 2m). These include the two subunits of the transporter associated with antigen presentation (TAP1 and TAP2), which are essential for introducing peptides into the ER from the cytosol, and tapasin, an MHC-encoded membrane protein. Prior to peptide binding, MHC class I-beta 2m dimers form part of a large multisubunit ER complex which includes TAP and tapasin. In addition to these specialized components two soluble 'house-keeping' proteins, the chaperone calreticulin and the thiol oxidoreductase ERp57, are also components of this complex. Our current understanding of the nature and function of the MHC class I peptide loading complex is the topic of this review.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Oxygen depletion-induced dormancy in Mycobacterium bovis BCG.

Gradual depletion of oxygen causes the shift-down of aerobic growing Mycobacterium bovis BCG to an anaerobic synchronized state of nonreplicating persistence. The persistent culture shows induction of glycine dehydrogenase and alpha-crystallin-like protein and is sensitive to metronidazole.

Amino Acid Oxidoreductases↗
PubMed1998

Increased alanine dehydrogenase activity during dormancy in Mycobacterium smegmatis.

The aerobic fast-growing Mycobacterium smegmatis has, like its slow-growing pathogenic counterpart M. tuberculosis, the capability to adapt to anaerobiosis by shifting down to a drug resistant dormant state. Here, we report the identification of the first enzyme, L-alanine dehydrogenase, whose specific activity is increased during dormancy development in M. smegmatis. This mycobacterial enzyme activity was previously identified as the 40-kDa antigen in M. tuberculosis and shows a preference for the reductive amination of pyruvate to alanine at physiological pH. The determination of the temporal profile of alanine dehydrogenase activity during dormancy development showed that the activity stayed at a low baseline level during the initial aerobic exponential growth phase (0.7 mU mg-1 min-1). After termination of aerobic growth, alanine dehydrogenase activity increased rapidly 5-fold. As oxygen becomes more and more limiting, the enzyme activity declined until it reached a level about two-third that of the peak value. The strong induction immediately after deflection from aerobic growth suggests that alanine might be required for the adaptation from aerobic growth to anaerobic dormancy. As alanine synthesis is coupled to NADH oxidation, we propose that the induction of alanine dehydrogenase activity might also support the maintenance of the NAD pool when oxygen as a terminal electron acceptor becomes limiting.

Adaptation, Physiological↗
PubMed1998

Oxygen depletion induced dormancy in Mycobacterium smegmatis.

We report here that the physiological behaviour of the fast growing saprophytic Mycobacterium smegmatis under in vitro oxygen-depletion and reactivation conditions is strikingly similar to the characteristics shown by the slow growing pathogenic M. tuberculosis. M. smegmatis died rapidly when shifted abruptly from aerobic to anaerobic conditions. In contrast to the lethal shock of abrupt oxygen depletion, the slow depletion through a self generated oxygen gradient permitted an adaptation to a persistent state which showed increased resistance against the bactericidal effects of anaerobiosis. The anaerobic persistent culture did not synthesise DNA and showed synchronised division upon reactivation in oxygen rich medium, indicating that the persistent bacilli are uniformly arrested at a defined stage of the cell cycle. Upon reactivation the persistent culture started synthesising DNA only after the first cell division, suggesting that the persistent cells contain two chromosomes. Furthermore, the persistent culture developed sensitivity to metronidazole and resistance against ofloxacin. These results suggest that M. smegmatis might be useful as a fast growing non-pathogenic model for comparative molecular analyses of mycobacterial dormancy.

Aerobiosis↗
PubMed1998

Upregulation of a histone-like protein in dormant Mycobacterium smegmatis.

The aerobic saprophyte Mycobacterium smegmatis, like its pathogenic counterpart M. tuberculosis, has the ability to adapt to anaerobiosis by shifting down to a dormant state. Here, we report the identification and molecular genetic characterisation of the first dormancy-induced protein in M. smegmatis. Comparative SDS-polyacrylamide gel electrophoresis of protein extracts of aerobically growing and dormant anaerobic M. smegmatis cultures revealed the upregulation of a 27-kDa protein in the dormant state. Peptide sequencing showed that the induced protein is a homologue of the histone-like protein H1p, predicted by the M. tuberculosis genome project. The corresponding hlp gene was cloned from M. smegmatis and sequenced. Disruption of the hlp gene eliminated the histone-like protein but did not affect the viability of the dormant culture.

Amino Acid Sequence↗
PubMed1997

Drosophila DPP2C1, a novel member of the protein phosphatase 2C (PP2C) family.

We report the molecular cloning, chromosome mapping and developmental transcription pattern of a putative serine/threonine protein phosphatase 2C (PP2C), DPP2C1, from Drosophila melanogaster. The 6-kb transcript of this first Drosophila PP2C gene encodes a 1428-aa deduced protein. The DPP2C1 protein contains a approximately 330-aa PP2C-like catalytic domain flanked by extensive N- and C-terminal sequences showing no similarities to other PP2Cs. The dpp2c1 gene maps to 4E1-2 on the X chromosome, 1.5 kb upstream of the ddlc1 gene. Northern blot analyses showed that dpp2c1 transcription is developmentally regulated, accumulating maximally during early (0-6 h) and late (12-24 h) embryogensis. The presented molecular characterisation provides the basis for a genetic dissection of DPP2C1 function.

Amino Acid Sequence↗
PubMed1996

Functionally active T cell receptor/CD3 complexes are present at the surface of cloned cytotoxic T cells without fluorescence-immunological detectability.

The cytotoxic T cell clone 10BK.1 is activated in response to the ovalbumin peptide OVA257-264 in a major histocompatibility complex class I-restricted manner. Following activation 10BK.1 cells proliferate, secrete lymphokines, and kill syn- and allogeneic target cells. Using immunofluorescence analysis we detected CD8, LFA-1, and ICAM-1 on the surface of 10BK.1 cells, but no CD3 or T cell receptor (TCR). In contrast, the proliferative response of 10BK.1 cells to antigen was efficiently blocked by soluble antibodies directed at CD3 epsilon or TCR alpha beta, but not by antibodies directed at TCR gamma delta. In addition, lysis of target cells was blocked by F(ab')2 fragments of antibodies directed at CD3 epsilon, and 10BK.1 cells proliferate in response to immobilized anti-CD3 epsilon or anti-TCR alpha beta antibodies. Furthermore, 10BK.1 cells lyse hybridoma cells that secrete antibodies directed at CD3 epsilon or TCR alpha beta, but not TCR gamma delta. These results demonstrate that functionally active molecules of the TCR/CD3 complex exist on the surface of 10BK.1 cells, obviously in low amounts, undetectable by immunofluorescence. In contrast, using permeabilized cells, we found high cytoplasmatic expression of TCR alpha beta, CD3 epsilon, and zeta-chain in 1OBK.1 cells, indicating that the low level of TCR/CD3 expression on the surface is not a consequence of a reduced synthesis of these molecules, but that the transport of these molecules to the surface is reduced. Our data demonstrate that the absence of TCR/CD3 complexes on the surface of cells, as detected by immunofluorescence, does not warrant the conclusion that these complexes are also functionally absent from the surface of these cells.

Animals↗
PubMed1996

Molecular and genetic characterization of SLC1, a putative Saccharomyces cerevisiae homolog of the metazoan cytoplasmic dynein light chain 1.

Cytoplasmic dynein is a multisubunit, microtubule-dependent motor enzyme that has been proposed to function in a variety of intracellular movements. As part of an effort to understand the evolution and the biological roles of cytoplasmic dynein, we have identified the first non-metazoan dynein light chain 1, SLC1, in the yeast Saccharomyces cerevisiae. The amino acid sequence of the SLC1 protein is similar to those of the human, Drosophila and Caenorhabditis cytoplasmic dynein light chains 1. The SLC1 gene lies adjacent to the YAP2 (= CAD1) transcription unit. The SLC1 coding sequence is split by two introns and its mRNA is detectable throughout the cell cycle. Tetrad analysis of heterozygotes harboring a TRP insertion in the SLC1 coding region indicate that SLC1 function is not essential for cell viability. Furthermore, we demonstrate that double mutants, defective for SLC1 and the kinesin-related CIN8 genes are non-lethal. The redundancy of SLC1 function in yeast contrasts with the cell death caused by loss-of-function mutations in the dynein light chain 1 gene in Drosophila melanogaster.

Acyltransferases↗
PubMed1996

Strong association of dermatomyositis-specific Mi-2 autoantibodies with a tryptophan at position 9 of the HLA-DR beta chain.

OBJECTIVE: To characterize the clinical and immunogenetic features of patients with Mi-2 autoantibodies. METHODS: Eighteen adult white patients with Mi-2 antibodies were clinically characterized and compared with 41 Mi-2-negative dermatomyositis (DM) patients. HLA class I and class II typing for DRB alleles was done by microcytotoxicity assay and for DQA and DQB alleles by polymerase chain reaction-based oligotyping. RESULTS: Seventeen of the 18 Mi-2-positive patients had DM. Symptoms of scleroderma, lung involvement, and arthritis were less common in this group than in the Mi-2-negative DM patients; the V-sign rash and nailfold involvement were found more frequently. Mi-2 antibodies were strongly associated with HLA-DR7 (88% versus 24% in healthy controls), HLA-DQA1*0201 (86% versus 23%), and DR7 "homozygosity" (31% versus 0%). A tryptophan residue at position 9 of the HLA-DR beta chain was present in all Mi-2-positive patients (100% versus 62%; homozygous in 81% versus 15%). CONCLUSION: Our results reemphasize the specificity of Mi-2 antibodies for DM, and extend previous reports that Mi-2 antibody production is associated with certain HLA class II antigens. We propose beta 9-Trp as a candidate epitope on the HLA-DR beta chain as a prerequisite for this type of autoimmune response.

Adenosine Triphosphatases↗
PubMed1996

Cytoplasmic dynein (ddlc1) mutations cause morphogenetic defects and apoptotic cell death in Drosophila melanogaster.

We report the molecular and genetic characterization of the cytoplasmic dynein light-chain gene, ddlc1, from Drosophila melanogaster. ddlc1 encodes the first cytoplasmic dynein light chain identified, and its genetic analysis represents the first in vivo characterization of cytoplasmic dynein function in higher eucaryotes. The ddlc1 gene maps to 4E1-2 and encodes an 89-amino-acid polypeptide with a high similarity to the axonemal 8-kDa outer-arm dynein light chain from Chlamydomonas flagella. Developmental Northern (RNA) blot analysis and ovary and embryo RNA in situ hybridizations indicate that the ddlc1 gene is expressed ubiquitously. Anti-DDLC1 antibody analyses show that the DDLC1 protein is localized in the cytoplasm. P-element-induced partial-loss-of-function mutations cause pleiotropic morphogenetic defects in bristle and wing development, as well as in oogenesis, and hence result in female sterility. The morphological abnormalities found in the ovaries are always associated with a loss of cellular shape and structure, as visualized by a disorganization of the actin cytoskeleton. Total-loss-of-function mutations cause lethality. A large proportion of mutant animals degenerate during embryogenesis, and the dying cells show morphological changes characteristic of apoptosis, namely, cell and nuclear condensation and fragmentation, as well as DNA degradation. Cloning of the human homolog of the ddlc1 gene, hdlc1, demonstrates that the dynein light-chain 1 is highly conserved in flies and humans. Northern blot analysis and epitope tagging show that the hdlc1 gene is ubiquitously expressed and that the human dynein light chain 1 is localized in the cytoplasm. hdlc1 maps to 14q24.

Alleles↗
PubMed1995

On the functional overlap between two Drosophila POU homeo domain genes and the cell fate specification of a CNS neural precursor.

The approximately 200 distinct neurons comprising each hemisegment of the Drosophila embryonic CNS are derived from a stereotypic array of approximately 30 progenitor stem cells, called neuroblasts (NBs). Each NB undergoes repeated asymmetric divisions to produce several smaller ganglion mother cells (GMCs), each of which, in turn, divides to produce two neurons and/or glia cells. To understand the process by which cell type diversity is generated in the CNS, we are focusing on identifying genes that affect cell identity in the NB4-2 lineage from which the RP2 motoneuron is derived. We show here that within the early part of the NB4-2 lineage, two closely linked and structurally related POU homeo domain genes, pdm-2 (dPOU28) and pdm-1 (dPOU19), both encode proteins that accumulate to high levels only in the first GMC (GMC4-2a) and not in its progeny, the RP2 motoneuron. Our results from the genetic and developmental analysis of pdm-1 and pdm-2 demonstrate that these genes are not required for the birth of GMC4-2a; however, they are both involved in specifying the identity of GMC4-2a and, ultimately, in the genesis of RP2 neurons, with pdm-2 being the more dominant player in this process. In mutant animals where both pdm-1 and pdm-2 functions are removed, GMC4-2a fails to express markers consistent with a GMC4-2a identity and no mature (Eve protein expressing) RP2 neurons are produced. We demonstrate that in some mutant combinations in which no mature RP2 neurons are produced, some GMC4-2a cells can nevertheless divide. Hence, the failure of the POU mutants to produce mature RP2 neurons is not attributable to a block in GMC4-2a cell division per se but, rather, because the GMC4-2a cells fail to acquire their correct cellular identity.

Animals↗
PubMed1995

Clinical relevance and HLA association of autoantibodies against the nucleolus organizer region (NOR-90).

OBJECTIVE: NOR-90 autoantibodies directed against the nucleolus organizer region (NOR) have been described as rare scleroderma associated antibodies. We studied the clinical features of patients with NOR-90 antibodies as well as their HLA phenotype. METHODS: NOR-90 antibodies were detected by indirect immunofluorescence assay using HEp-2 cells, by chromosome spreads as a substrate and in addition by Western blot analysis with HeLa-S3 nucleolar extract. HLA antigens of the NOR-90 antibody positive patients were typed with the standard NIH complement dependent microcytotoxicity test. RESULTS: Nine sera selected by means of the indirect immunofluorescence revealed a typical double band pattern of about 90 kDa identical with the pattern of 2 NOR-90 reference sera by Western blot analysis. Only one patient positive for NOR-90 antibodies suffered from systemic sclerosis (limited cutaneous scleroderma). The other patients with NOR-90 antibodies showed no signs of systemic sclerosis. All patients with NOR-90 antibodies were women and 8 of 9 patients (89 versus 13% of healthy controls, Pcorr < 0.001) were positive for the HLA-DR1 allele. CONCLUSION: In contrast to the first report on NOR-90 antibodies we demonstrated no association of these antibodies with systemic sclerosis; however, we found strong evidence for an immunogenetic background of NOR-90 antibody formation.

Adult↗
PubMed1994

The antigen self-presentation function of the cytotoxic T-cell clone 10BK.1 depends on reciprocal peptide presentation.

Cells of the cytotoxic T-cell clone 10BK.1 proliferate in response to ovalbumin (OVA) not only in the presence, but also in the absence, of antigen-presenting cells. This response is the consequence of reciprocal antigen presentation by two 10BK.1 cells. At the single-cell level, we found antigen-induced changes in the cytosolic Ca2+ concentration ([Ca2+]i) only in 10BK.1 cells that had contact with another 10BK.1 cell. This finding suggests that a single cell is unable to present the antigen to itself, and that two 10BK.1 cells are necessary and adequate to present the antigen to each other. Our data indicate that 10BK.1 cells are not capable of processing the OVA molecule. The naturally processed peptide OVA257-264 is a very potent antigen for these cells. A corresponding peptide seems to be present not only in preparations of native OVA, but also in preparations of the commonly used peptide OVA258-276. This 19mer peptide does not bind with high affinity to the relevant Kb class I molecule.

Animals↗
PubMed1993

Manifestation of the MHC-unrestricted killing potential of a cytotoxic T cell clone requires activation in response to MHC-restricted self-presentation of antigen.

10BK.1 T clone cells of (B10 x B10.BR)F1 genotype specifically react to peptides of OVA, frequently encountered in OVA preparations, and to the synthetic peptide OVA257-264 in an H-2Kb-restricted manner; the T clone cells produce lymphokines and proliferate in the absence of added APC, suggesting self-presentation of the Ag by the T cells. In accordance with their CD8+ CD4- phenotype 10BK.1 cells exhibit cytotoxic capacity. When the target cells were pretreated with OVA257-264 only cells bearing H-2Kb molecules were lysed. However, when the relevant OVA peptide was present during the 4-h 51Cr release assay 10BK.1 cells lysed target cells expressing H-2Kb molecules as well as target cells lacking H-2Kb elements. Likewise, 10BK.1 cells pretreated with OVA257-264 for 1 h and washed extensively to remove residual Ag were able to kill syngeneic and allogeneic target cells. Killing of allogeneic targets in the presence of Ag was inhibited by antibodies directed at H-2Kb. Allogeneic target cells were not killed when 10BK.1 cells were stimulated by peptide-pulsed syngeneic cells. Triggering of the TCR/CD3 complex of 10BK.1 cells was involved during the activation phase but not during the lytic phase. IL-2 did not participate in the MHC-unrestricted killing event. To explain the observed MHC-unrestricted cytotoxicity of 10BK.1 cells, the following model is proposed. In an initial step 10BK.1 cells present the relevant OVA peptide to one another in a H-2Kb-restricted fashion. The cells are thereby triggered to mobilize the lytic machinery. In a second step sensitized 10BK.1 cells lyse allogeneic target cells. Thus, the MHC-unrestricted cytotoxicity can be dissected into an MHC-restricted phase of 10BK.1 cell activation and an MHC-unrestricted lytic phase. Cytotoxic T cells with Ag self-presentation functions may account for tissue damage during bacterial infections.

Animals↗
PubMed1993

An ovalbumin peptide-specific cytotoxic T cell clone with antigen self-presentation capacity uses two distinct mechanisms to kill target cells.

Cloned 10BK.1 T cells with specificity for the ovalbumin peptide OVA257-264 are representative of a novel cell type within the CD8+ subset of T cells. In the presence and in the absence of added antigen presenting cells these T cells react toward antigen (Ag) by proliferation and lymphokine production. These data suggest self-presentation of the Ag by 10BK.1 cells. Here we present evidence that 10BK.1 cells exhibit cytotoxic activity that involves two different cytotoxic effector mechanisms. (i) One mechanism is fast killing activity, apparent within 4 hr. Constitutive mouse T cell-specific proteinase-1 (MTSP-1) activity, constitutive expression of MTSP-1 RNA, increased by Ag challenge, and Ag-inducible perforin RNA expression were observed. Electron microscopic dense granules of the CTL were oriented toward Ag-pulsed target cells. The fast form of cytotoxicity was triggered by Ag recognition and by contact with IL-2. (ii) The other mechanism is slow cytolytic activity, manifested within 2 days. This activity was contained in the supernatant of 10BK.1 cells after Ag activation. It was inhibited by monoclonal anti-TNF antibodies and therefore presumably represents TNF alpha/beta. Cytotoxic T cells capable of antigen self-presentation may be responsible for tissue damage during bacterial and viral infections.

Animals↗
PubMed1993

The role of a Drosophila POU homeo domain gene in the specification of neural precursor cell identity in the developing embryonic central nervous system.

The Drosophila embryonic central nervous system (CNS) is derived from a stereotypic array of progenitor stem cells called neuroblasts (NBs). Each of the approximately 25 NBs per hemisegment undergoes repeated asymmetric divisions to produce, on average, 5-10 smaller ganglion mother cells (GMCs); each GMC, in turn, divides to produce two neurons. We demonstrate that the protein product encoded by a POU homeo domain gene (dPOU28/pdm-2) is expressed in the cell nuclei of a subset of NBs and GMCs. In the wild-type animal, GMC-1 is the only identified cell in the NB4-2 lineage that expresses dPOU28 protein to a high level, and it divides to produce the RP2 neuron and a second cell of unknown fate. Our results suggest that the presence of ectopically induced dPOU28/pdm-2 protein in the progeny of GMC-1 is sufficient to cause both of these cells to adopt their parental GMC-1 cell fate, leading to duplication of the RP2 neuron (and its sister cell) on the basis of both immunological and morphological criteria. These observations clearly implicate a role for dPOU28/pdm-2 in the specification of GMC-1 cell identity in the NB4-2 lineage and possibly in the specification of cell fate in other NB lineages in the developing embryonic CNS.

Animals↗