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At least 1,009 records · Page 56Linked to original sources
PubMed1997

Regulation of two loblolly pine (Pinus taeda L.) isocitrate lyase genes in megagametophytes of mature and stratified seeds and during postgerminative growth.

Two full-length cDNAs encoding the glyoxysomal enzyme isocitrate lyase (ICL) were isolated from a lambda ZAP cDNA library prepared from megagametophyte mRNAs extracted from seeds imbibed at 30 degrees C for 8 days. The cDNAs, designated Ptbs ICL 8 and Ptbs ICL 12, have open reading frames of 1740 and 1719 bp, with deduced amino acid sequences of 580 and 573 residues, respectively. The predicted amino acid sequences of Ptbs ICL 8 and Ptbs ICL 12 exhibit a 79% identity with each other, and have a greater than 75% identity with ICLs from various angiosperm species. The C-termini of Ptbs ICL 8 and Ptbs ICL 12 terminate with the tripeptide Ser-Arg-Met and Ala-Arg-Met, respectively, both being conserved variants of the type 1 peroxisomal targeting signal. RNA blot and slot analysis revealed that Ptbs ICL 8 and Ptbs ICL 12 mRNAs were present at low levels in the megagametophyte of the mature and stratified seeds, and that the level of both transcripts increased markedly upon seed germination. Protein blot analysis indicated that the steady-state level of ICL was low in the mature and stratified seed, then increased rapidly upon seed germination, peaking at around 8-10 days after imbibition (DAI). Changes in the level of ICL activity in cell-free extracts was similar to the steady-state protein content with the exception that ICL activity was not detected in megagametophyte extracts of mature or stratified seeds. From 10-12 DAI when the megagametophyte tissue senesced, ICL activity decreased rapidly to near undetectable levels. In contrast, steady-state levels of ICL protein and mRNA remained relatively constant during megagametophyte senescence. In vivo synthesis of ICL protein was measured to shed light on these differences. ICL immunoselected from [(35)S]-methionine labelled proteins indicated that ICL was synthesized at very low levels during megagametophyte senescence. Together, the results show that loblolly pine ICL gene expression is complex. While temporal regulation appears to be primarily transcriptional, it also involves a number of post-transcriptional processes including at least one translational and/or post-translational mechanism.

Amino Acid Sequence↗
PubMed2000

Regulation of NADPH-cytochrome P450 reductase expressed during Douglas-fir germination and seedling development.

NADH-cytochrome P450 is a key enzyme that transfers electrons from NADPH to the cytochrome P450 family of enzymes. To begin to determine the regulation of CPR gene expression and enzyme activity in Douglas-fir a full-length cDNA was isolated from a seedling lambda ZAP cDNA library and the ORF was used to develop a synthetic CPR-peptide-based antiserum. Northern blot analysis indicated CPR expression was regulated both developmentally prior to seed maturation and during germination, and differentially in the cotyledons, radicle and megagametophyte of seed and seedling tissues. The CPR-peptide antiserum detected a single CPR in seed and seedling microsomes analyzed by western blot of two-dimensional SDS-polyacrylamide gels. In microsomal extracts from whole seeds and seedlings, the amount of CPR protein remained constant while NADPH:cytochrome c reductase activity increased during stratification, germination and early seedling development. In contrast to cotyledons and megagametophyte, the level of CPR protein detected in radicles was higher than expected when compared to the amount of CPR transcript.

Amino Acid Sequence↗
PubMed2001

Analysis of candidate antagonists of IAP-mediated caspase inhibition using yeast reconstituted with the mammalian Apaf-1-activated apoptosis mechanism.

We have reconstituted the Apaf-1-activated apoptosis mechanism in Sacchromyces cerevisiae such that the presence of a constitutively active form of Apaf-1 together with both Caspase-9 and Caspase-3 results in yeast death. This system is a good model of the Apaf-1-activated pathway in mammalian cells: MIHA (XIAP/hILP), and to a lesser degree MIHB (c-IAP1/HIAP2) and MIHC (c-IAP-2/HIAP1) can inhibit caspases in this system, and protection by IAPs (inhibitor of apoptosis) can be abrogated by coexpression of the Drosophila pro-apoptotic proteins HID and GRIM or the mammalian protein DIABLO/Smac. Using this system we demonstrate that unlike DIABLO/Smac, other proteins which interact with mammalian IAPs (TAB-1, Zap-1, Traf-1 and Traf-2) do not act to antagonise IAP- mediated caspase inhibition.

Animals↗
PubMed2004

Role of GM3-enriched microdomains in signal transduction regulation in T lymphocytes.

Gangliosides, sialic acid containing glycosphigolipids, are ubiquitous constituents of cell plasma membranes. Each cell type shows a peculiar ganglioside expression pattern. In human T lymphocytes monosialoganglioside GM3 represents the main ganglioside constituent of cell plasma membrane where it is concentrated in glycosphingolipid-enriched microdomains (GEM). The presence of tyrosine kinase receptors, mono- (Ras, Rap) and heterotrimeric G proteins, Src-like tyrosine kinases (lck, lyn, fyn), PKC isozymes, glycosylphosphatidylinositol (GPI)-anchored proteins and, after T cell activation, the Syk-family kinase Zap-70, prompts these portions of the plasma membrane to be considered as "glycosignaling domains." In particular, during T cell activation and/or other dynamic functions of the cell, such as apoptosis, key signaling molecules are recruited to these microdomains, where they strictly interact with GM3. The association of transducer proteins with GM3 in microdomains suggests that this ganglioside is the main marker of GEM in human lymphocytes and is a component of a cell plasma membrane multimolecular signaling complex involved in cell-cell interaction, signal transduction, and cell activation.

Apoptosis↗
PubMed2004

[Chronic lymphocytic leukemia--the old and the new].

Chronic Lymphocytic Leukemia (CLL) is the most frequent lymphoproliferative disease and leukaemia in western countries. CLL occurs more frequently in men than women, the median age at diagnosis is 65 years. CLL is defined as a persisting chronic lymphocytosis > 5 G/l with classical morphological features (small lymphocytic cells with round nuclei, dense chromatin and small cytoplasmic rim) and a classical immunophenotype (CD5+, CD19+, CD20+, CD23+); however, deviations from classical morphology are frequent. In cases with classical diagnostic features in the peripheral blood, a bone marrow biopsy is not necessary for diagnosis. Prognostic features comprise the stage of the disease according to the Rai or Binet systems, laboratory markers such as LDH, beta-2-microglobulin, lymphocyte doubling time and CD38 expression by flow cytometry (and ZAP-70 expression if available) as well as the status on IgV(H) hypermutations and cytogenetic analysis. Up to 2/3 of patients do not need treatment at the time of diagnosis and can initially be followed using a watch and wait strategy. If therapy becomes necessary, initial standard treatment still is chlorambucil. Later, purine analogues and Alemtuzumab are treatment options for refractory or relapsing disease. Therapies with antibodies such as Alemtuzumab and Rituximab in combination with purine analogues are currently under clinical investigation. With recurrent or atypical infections, hypogammaglobulinemia should be searched for and immunoglobulins should be substituted if necessary. However, their prophylactic use is not recommended.

Aged↗
PubMed2000

Novel immunodeficiency data servers.

The Internet contains scientific information in increasing amounts. It is possible to obtain the latest information, and Web services can easily be maintained and updated. We have set up three Internet services on immunodeficiencies. Immunodeficiency-related mutation infor mation is available in immunodeficiency mutation databases (IDbases). Currently 14 registries are distributed, including information about Bloom syndrome (BLMbase), X-linked agammaglobulinemia (BTKbase), X-linked and autosomal recessive chronic granulomatous diseases (CYBBbase for X-linked CGD, CYBAbase for p22(phox) deficiency, NCF1base for p47(phox) deficiency, NCF2base for p67(phox) deficiency), CD3gamma and CD3epsilon deficiencies (CD3Gbase, CD3Ebase), X-linked hyper-IgM syndrome (CD40Lbase), T-B+ severe combined immunodeficiency (JAK3base), V(D)J recombination defects (RAG1base, RAG2base), X-linked lymphoproliferative syndrome (SH2D1Abase), and ZAP-70 deficiency (ZAP70base). Information on laboratories analysing the genetic defects is collected to IDdiagnostics registry. Due to the rareness of immunodeficiencies there are very few laboratories performing genetic diagnostics. Such laboratories are listed in IDdiagnostics and physicians can use the registry to find a suitable laboratory for their diagnostic needs. Immunodeficiency Resource (IDR) is a comprehensive integrated knowledge base for all the information on immunode ficiencies, including clinical, biochemical, genetic, structural and computational data and analyses. All three services are available at http: //www.uta.fi/imt/bioinfo/.

Artificial Intelligence↗
PubMed2002

The regulation of actin remodeling during T-cell-APC conjugate formation.

The T-cell cytoskeleton is intimately involved in determining the efficiency and fidelity of the immune response. During T-cell interactions with antigen-presenting cells (APCs), dynamic remodeling of the actin cytoskeleton is particularly important for stabilizing long-lived integrin-dependent adhesive interactions. In addition, actin remodeling is important for facilitating the sustained signaling required for full T-cell activation. Although the relationship between T-cell signaling and cytoskeletal remodeling is complex, new molecular genetic tools are making it possible to investigate individual molecular interactions in the context of bona fide conjugate formation. We describe here the progress from our laboratory toward defining the pathways required for actin remodeling during conjugate formation. Our studies show that engagement of T-cell receptor (TCR) and leukocyte functional antigen-1 (LFA-1) leads to distinct effects on the remodeling of individual cytoskeletal elements. Downstream of TCR, we find that p56Lck (Lck) plays a critical role in integrin-dependent adhesion independent of its ability to activate zeta-associated protein of 70 kDa (ZAP-70). TCR engagement also results in the assembly of a signaling complex that facilitates the activation of Wiskott-Aldrich syndrome protein (WASP) by colocalization with Cdc42-GTP. These events, together with other parallel actin regulatory pathways, induce localized actin polymerization at the site of APC binding.

Actins↗
PubMed2003

Negative feedback of T cell activation through inhibitory adapters and costimulatory receptors.

Antigen recognition by the T cell receptor (TCR) complex induces the formation of a TCR signalosome by recruiting various signaling molecules, generating the recognition signals for T cell activation. The activation status and functional outcome are positively and negatively regulated by dynamic organization of the signalosome and by costimulation signals. We have studied the negative regulation of T cell activation, particularly through inhibitory adapters and costimulation receptors that are little expressed in resting cells but are induced upon T cell activation. We described Grb-associated binder 2 (Gab2) and cytotoxic T lymphocyte antigen-4 (CTLA-4) as a representative inhibitory adapter and a negative costimulation receptor, respectively, both of which exhibit negative feedback. Gab2 functions as a signal branch for activation vs. inhibition, as phosphorylation of either Src homology 2 (SH2) domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) or Gab2 by zeta-associated protein of 70 kDa (ZAP-70) determines the fate of the response. As a professional inhibitory receptor, CTLA-4 inhibits T cell response by competition of ligand binding with positive costimulator receptor CD28, and also induces inhibitory signaling. The trafficking and the cell surface expression of CTLA-4 are dynamically regulated and induced. CTLA-4 is accumulated in lysosomes and secreted to the T cell-APC contact site upon TCR stimulation. As T cell activation proceeds, these inhibitory adapters and costimulation receptors are induced and suppress/regulate the responses as negative feedback.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Cbl-b regulates the CD28 dependence of T-cell activation.

Whereas co-stimulation of the T-cell antigen receptor (TCR) and CD28 triggers T-cell activation, stimulation of the TCR alone may result in an anergic state or T-cell deletion, both possible mechanisms of tolerance induction. Here we show that T cells that are deficient in the adaptor molecule Cbl-b (ref. 3) do not require CD28 engagement for interleukin-2 production, and that the Cbl-b-null mutation (Cbl-b(-/-)) fully restores T-cell-dependent antibody responses in CD28-/- mice. The main TCR signalling pathways, such as tyrosine kinases Zap-70 and Lck, Ras/mitogen-activated kinases, phospholipase Cgamma-1 and Ca2+ mobilization, were not affected in Cbl-b(-/-) T cells. In contrast, the activation of Vav, a guanine nucleotide exchange factor for Rac1/Rho/CDC42, was significantly enhanced. Our findings indicate that Cbl-b may influence the CD28 dependence of T-cell activation by selectively suppressing TCR-mediated Vav activation. Mice deficient in Cbl-b are highly susceptible to experimental autoimmune encephalomyelitis, suggesting that the dysregulation of signalling pathways modulated by Cbl-b may also contribute to human autoimmune diseases such as multiple sclerosis.

Adaptor Proteins, Signal Transducing↗
PubMed2000

A motif in the alphabeta T-cell receptor controls positive selection by modulating ERK activity.

Positive selection allows thymocytes that recognize an individual's own major histocompatibility complex (self-MHC) molecules to survive and differentiate, whereas negative selection removes overtly self-reactive thymocytes. Although both forms of thymic selection are mediated by the alphabeta T-cell receptor (TCR) and require self-MHC recognition, an important question is whether they are controlled by distinct signalling cascades. We have shown that mutation of an essential motif within the TCR alpha-chain-connecting peptide domain (alpha-CPM) profoundly affects positive but not negative selection. Using transgenic mice expressing a mutant alpha-CPM TCR we examined the contribution of several mitogen-activated protein kinase (MAPK) cascades to thymic selection. Here we show that in thymocytes expressing a mutant alpha-CPM receptor, a positively selecting peptide failed to activate the extracellular signal-regulated kinase (ERK), although other MAPK cascades were induced normally. The defect in ERK activation was associated with impaired recruitment of the activated tyrosine kinases Lck and ZAP-70, phosphorylated forms of the TCR component CD3zeta and the adaptor protein LAT to detergent-insoluble glycolipid-enriched microdomains (DIGs). Therefore, an intact DIG-associated signalosome is essential for sustained ERK activation, which leads to positive selection.

Amino Acid Motifs↗
PubMed2000

Different initiation of pre-TCR and gammadeltaTCR signalling.

Lineage choice is of great interest in developmental biology. In the immune system, the alphabeta and gammadelta lineages of T lymphocytes diverge during the course of the beta-, gamma- and delta-chain rearrangement of T-cell receptor (TCR) genes that takes place within the same precursor cell and which results in the formation of the gammadeltaTCR or pre-TCR proteins. The pre-TCR consists of the TCRbeta chain covalently linked to the pre-TCRalpha protein, which is present in immature but not in mature T cells which instead express the TCRalpha chain. Animals deficient in pre-TCRalpha have few alphabeta lineage cells but an increased number of gammadelta T cells. These gammadelta T cells exhibit more extensive TCRbeta rearrangement than gammadelta T cells from wild-type mice. These observations are consistent with the idea that different signals emanating from the gammadeltaTCR and pre-TCR instruct lineage commitment. Here we show, by using confocal microscopy and biochemistry to analyse the initiation of signalling, that the pre-TCR but not the gammadeltaTCR colocalizes with the p56lck Src kinase into glycolipid-enriched membrane domains (rafts) apparently without any need for ligation. This results in the phosphorylation of CD3epsilon and Zap-70 signal transducing molecules. The results indicate clear differences between pre-TCR and gammadeltaTCR signalling.

Animals↗
PubMed1995

Syk tyrosine kinase required for mouse viability and B-cell development.

The Syk cytoplasmic protein-tyrosine kinase has two amino-terminal SH2 domains and a carboxy-terminal catalytic domain. Syk, and its close relative ZAP-70, are apparently pivotal in coupling antigen- and Fc-receptors to downstream signalling events. Syk associates with activated Fc receptors, the T cell receptor complex and the B-cell antigen-receptor complex (BCR) in immature and mature B lymphocytes. On receptor activation, the tandem SH2 domains of Syk bind dual phosphotyrosine sites in the conserved ITAM motifs of receptor signalling chains, such as the immunoglobulin alpha and beta-chains of the BCR, leading to Syk activation. Here we have investigated Syk function in vivo by generating a mouse strain with a targeted mutation in the syk gene. Homozygous syk mutants suffered severe haemorrhaging as embryos and died perinatally, indicating that Syk has a critical role in maintaining vascular integrity or in wound healing during embryogenesis. Analysis of syk-/- lymphoid cells showed that the syk mutation impaired the differentiation of B-lineage cells, apparently by disrupting signalling from the pre-BCR complex and thereby preventing the clonal expansion, and further maturation, of pre-B cells.

Animals↗
PubMed1997

Phosphotyrosine-dependent activation of Rac-1 GDP/GTP exchange by the vav proto-oncogene product.

The oncogenic protein Vav harbours a complex array of structural motifs, including leucine-rich, Dbl-homology, pleckstrin-homology, zinc-finger, SH2 and SH3 domains. Upon stimulation by antigens or mitogens, Vav becomes phosphorylated on key tyrosine residues and associates with other signalling proteins, including the mitogen receptors Zap-70 (ref. 6), Vap-1 (ref. 5) and Slp-76 (ref. 7). Disruption of the vav locus by homologous recombination causes severe defects in signalling by primary antigen receptors, leading to abnormal lymphocyte proliferation and lymphopenia. Despite the importance of Vav cell signalling, the function of this protein remains unknown. Here we show that tyrosine-phosphorylated Vav, but not the non-phosphorylated protein, catalyses GDP/GTP exchange on Rac-1, a protein implicated in cell proliferation and cytoskeletal organization, causing this GTPase to switch from its inactive to its active state. Transfection experiments also show that phosphorylation of Vav on tyrosine residues leads to nucleotide exchange on Rac-1 in vivo and stimulates c-Jun kinase, a downstream element in the signalling pathway involving this GTPase. Our results have identified a function for Vav and define a mechanism in which engaged membrane receptors activate its signalling pathway.

Animals↗
PubMed2000

The 21- and 23-kD forms of TCR zeta are generated by specific ITAM phosphorylations.

The T cell receptor (TCR) zeta subunit contains three immunoreceptor tyrosine-based activation motifs (ITAMs) that translate effective extracellular ligand binding into intracellular signals by becoming phosphorylated into 21- and 23-kD forms. We report here that the 21-kD form of TCR zeta is generated by phosphorylation of the tyrosines in the second and third ITAMs, whereas the 23-kD form is formed by the additional phosphorylation of the membrane-proximal ITAM tyrosines. The stable formation of the 21- and 23-kD species requires the binding of the tandem SH2 domains of ZAP-70. We also report that TCR-mediated signaling processes can proceed independently of either the 21- or 23-kD species of TCR zeta.

Amino Acid Sequence↗
PubMed1995

Lack of serological specificity of recombinant heat shock protein of Leishmania donovani.

In order to identify a specific recombinant antigen of Leishmania donovani with potential use for diagnosis, a cDNA library was constructed in lambda ZAP II expression vector. On screening the cDNA library using pooled sera from Indian patients with kala azar, 20 antibody reactive clones were identified. These were subcloned into pBluescript phagemid by an in vivo excision procedure. The molecular weights of the expressed recombinant proteins varied from 15 to 70 kDa and the cDNA insert sizes varied from 0.5 kb to the largest size of approximately 2.0 kb which was designated as the E2b clone. The nucleotide sequencing revealed that 50% of the clones had sequence homology to the heat shock protein gene of L. donovani. The serological studies conducted with the kala azar positive sera and sera from healthy laboratory workers using the recombinant protein from the E2b clone and having sequence homology to Ldhsp 70, indicated that although all the kala azar sera was positive, 12 of 20 healthy individuals also showed antibodies against the recombinant hsp70, indicating that this antigen is not suitable for serological diagnosis of kala azar.

Animals↗
PubMed1997

The role of CD45 and CD45-associated molecules in T cell activation.

CD45 (lymphocyte common antigen) is a receptor-linked protein tyrosine phosphatase that is expressed on all leucocytes, and which plays a crucial role in the function of these cells. On T cells the extracellular domain of CD45 is expressed in several different isoforms, and the particular isoform(s) expressed depends on the particular subpopulation of cell, their state of maturation, and whether or not they have previously been exposed to antigen. It has been established that the expression of CD45 is essential for the activation of T cells via the TCR, and that different CD45 isoforms display a different ability to support T cell activation. Although the tyrosine phosphatase activity of the intracellular region of CD45 has been shown to be crucial for supporting signal transduction from the TCR, the nature of the ligands for the different isoforms of CD45 have been elusive. Moreover, the precise mechanism by which potential ligands may regulate CD45 function is unclear. Interestingly, in T cells CD45 has been shown to associate with numerous molecules, both membrane associated and intracellular; these include components of the TCR-CD3 complex and CD4/CD8. In addition, CD45 is reported to associate with several intracellular protein tyrosine kinases including p56lck and p59fyn of the src family, and ZAP-70 of the Syk family, and with numerous proteins of 29-34 kDa. These CD45-associated molecules may play an important role in regulating CD45 tyrosine phosphatase activity and function. However, although the role of some of the CD45-associated molecules (e.g. CD45-AP and LPAP) has become better understood in recent years, the role of others still remains obscure. This review aims to summarize recent findings on the role of CD45 and CD45-associated molecules in T cell activation, and to highlight issues that seem relevant to ongoing research in this area.

Animals↗
PubMed1983

Symptoms and activation of granulocytes and complement with two dialysis membranes.

Complement (C) activation, neutropenia, and mild pulmonary dysfunction attend hemodialysis (HD) with cellophane [for example, cuprophan (Cu)] membranes. While usually asymptomatic, these phenomena may cause distress in patients with cardiopulmonary disease, and "start-up" symptoms of HD might be mediated by C-stimulated granulocytes (PMNs). Cellulose acetate (CA) hemodialysis membranes have been devised and claimed more blood compatible than Cu. In a blinded series of HD patients, pruritus, fatigue, and sense of well-being were each scored statistically more favorably by the patients during HD with CA than during HD with Cu (P less than 0.05). Postulating that less C activation might underlie the benefit, we showed that neutropenia was less severe with CA (nadir 77.6% of initial count, +/- 4 SEM) than with Cu (38.3% +/- 2.9; P less than 0.01). In vitro, incubation of CA membranes with plasma led to less C3 conversion (20% vs. 40%), less PMN aggregating activity (5.9 ZAP units vs. 36.3) and less decrement in CH50 (6.5% vs. 22%) than like incubations of Cu. C activation was also less potent in vivo: During HD plasma C3a rose from a mean 401 ng/ml to a peak 6,325 in patients on Cu dialyzers, but from 426 to only 3,637 in patients on CA devices (P less than 0.05). Time-course studies suggested CA was initially as potent an activator as Cu but rapidly lost ability to activate C, possibly because of saturation of C3b binding sites. As an index of PMN activation, we also assayed plasma lactoferrin and found levels significantly higher during Cu than CA dialysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Anaphylatoxins↗
PubMed2006

Neuron-mediated generation of regulatory T cells from encephalitogenic T cells suppresses EAE.

Neurons have been neglected as cells with a major immune-regulatory function because they do not express major histocompatibility complex class II. Our data show that neurons are highly immune regulatory, having a crucial role in governing T-cell response and central nervous system (CNS) inflammation. Neurons induce the proliferation of activated CD4+ T cells through B7-CD28 and transforming growth factor (TGF)-beta1-TGF-beta receptor signaling pathways, resulting in amplification of T-cell receptor signaling through phosphorylated ZAP-70, interleukin (IL)-2 and IL-9. The interaction between neurons and T cells results in the conversion of encephalitogenic T cells to CD25+ TGF-beta1+ CTLA-4+ FoxP3+ T regulatory (Treg) cells that suppress encephalitogenic T cells and inhibit experimental autoimmune encephalomyelitis. Suppression is dependent on cytotoxic T lymphocyte antigen (CTLA)-4 but not TGF-beta1. Autocrine action of TGF-beta1, however, is important for the proliferative arrest of Treg cells. Blocking the B7 and TGF-beta pathways prevents the CNS-specific generation of Treg cells. These findings show that generation of neuron-dependent Treg cells in the CNS is instrumental in regulating CNS inflammation.

Animals↗