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At least 937 records · Page 52Linked to original sources
PubMed1995

Bacterial expression and characterization of a cDNA for human liver estrogen sulfotransferase.

A distinct human estrogen sulfotransferase (hEST-1) cDNA has been isolated from a human liver lambda Zap cDNA library using a PCR procedure. The enzymatically active protein has been expressed in two bacterial expression systems and the kinetic and immunologic properties of the enzyme have been characterized. The full-length cDNA for hEST-1 is 994 base pairs in length and encodes a 294 amino acid protein with a calculated molecular mass of 35,123 Da. Purified hEST-1 migrated with an apparent molecular mass of 35,000 Da during SDS-polyacrylamide gel electrophoresis. Immunoblot analysis of hEST-1 expressed in E. coli with a rabbit anti-hEST-1 antibody yields a band of approximately 35,000 Da. The anti-hEST-1 antibody also detects a single band in human liver and jejunum cytosol which migrates with the same molecular mass as expressed hEST-1. There was also no cross-reactivity of hEST-1 with rabbit anti-hP-PST or rabbit anti-hDHEA-ST antibodies upon immunoblot analysis. hEST-1 was expressed in bacteria and purified to homogeneity. Expressed hEST-1 activity has a significantly greater affinity for estrogen sulfation than that found for the other human STs which conjugate estrogens. hEST-1 maximally sulfates beta-estradiol and estrone at concentrations of 20 nM. hEST-1 also sulfates dehydroepiandrosterone, pregnenolone, ethinylestradiol, and 1-naphthol, at significantly higher concentrations; however, cortisol, testosterone and dopamine are not sulfated. The results presented in this paper describe the expression and characterization of a human EST distinct from other human STs which sulfate estrogens. The high affinity of hEST-1 for estrogens indicates that this ST may be important in both the metabolism of estrogens and in the regulation of their activities.

Amino Acid Sequence↗
PubMed1995

CMP-KDO synthetase: overproduction and application to the synthesis of CMP-KDO and analogs.

CTP:CMP-3-deoxy-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase, EC 2.7.7.38) has been cloned and overexpressed in Escherichia coli. The structure gene was amplified from the total DNA of E. coli K-235 through the primer-directed polymerase chain reaction. The gene was then cloned into lambda ZAP vector at the EcoRI and XbaI restriction sites and overexpressed in E. coli Sure strain at a level approximately 400 times as much as that produced in the host strain. Application of the enzyme to the synthesis of cytidine 5'-monophospho-3-deoxy-D-manno-2-octulosonic acid (CMP-KDO) and analogs was studied. Of several KDO analogs tested, 5-fluoro-2-keto-3,5-dideoxyoctulosonic acid (5-FKDO) was found to be a good substrate of the enzyme, and the product (CMP-5-FKDO) was prepared and characterized, representing the first stable CMP-KDO analog prepared enzymatically to date. The natural enzyme product, CMP-KDO, was however quite unstable (t1/2 = 19 min, in 50 mM MgCl2, 0.2 M Tris buffer, pH 9.0). A mechanism for the decomposition of CMP-KDO involving the hydrogen bonding interactions between the OH groups of C-5 and C-7 (and/or C-8) and the phosphate oxygens was proposed.

Base Sequence↗
PubMed2004

Tyrosine kinase inhibitors: a new approach for asthma.

The pathogenesis of allergic asthma involves the interplay of inflammatory cells and airway-resident cells, and of their secreted mediators including cytokines, chemokines, growth factors and inflammatory mediators. Receptor tyrosine kinases are important for the pathogenesis of airway remodeling. Activation of epidermal growth factor (EGF) receptor kinase and platelet-derived growth factor (PDGF) receptor kinase leads to hyperplasia of airway smooth muscle cells, epithelial cells and goblet cells. Stimulation of non-receptor tyrosine kinases (e.g. Lyn, Lck, Syk, ZAP-70, Fyn, Btk, Itk) is the earliest detectable signaling response upon antigen-induced immunoreceptor activation in inflammatory cells. Cytokine receptor dimerization upon ligand stimulation induces activation of Janus tyrosine kinases (JAKs), leading to recruitment and phosphorylation of signal transducer and activator of transcription (STAT) for selective gene expression regulation. Activation of chemokine receptors can trigger JAK-STAT pathway, Lck, Fyn, Lyn, Fgr, and Syk/Zap-70 to induce chemotaxis of inflammatory cells. Inhibitors of tyrosine kinases have been shown in vitro to block growth factor-induced hyperplasia of airway-resident cells; antigen-induced inflammatory cell activation and cytokine synthesis; cytokine-mediated pro-inflammatory gene expression in inflammatory and airway cells; and chemokine-induced chemotaxis of inflammatory cells. Recently, anti-inflammatory effects of tyrosine kinase inhibitors (e.g. genistein, tyrphostin AG213, piceatannol, tyrphostin AG490, WHI-P97, WHI-P131, Syk antisense) in animal models of allergic asthma have been reported. Therefore, development of inhibitors of tyrosine kinases can be a very attractive strategy for the treatment of asthma.

Animals↗
PubMed2003

Splice variant in TCRzeta links T cell receptor signaling to a G-protein-related signaling pathway.

The T cell receptor zeta chain is required for efficient receptor expression and contributes to T cell receptor-mediated activation of ZAP-70 and PLC-gamma1 as well as other signaling functions. A splice variant of zeta has been described which contains a 3bp insert coding for a glutamine in the cytoplasmic domain. The variant, here designated zeta-Q, is abundant, comprising 20-50% of zeta transcripts in humans, and production of the two isoforms is conserved among distantly related vertebrate species. Analysis of the peptide region in which the insert occurs reveals an unexpected homology with G-protein gamma chains. Transfection studies suggest that disruption in the alignment of three conserved prolines by the insertion of an extra glutamine impairs TCR-mediated PLC activation. Experiments with human lymphocytes suggest that zeta-Q message undergoes upregulation following cellular activation. Our data suggest that regulation of the relative levels of these two transcripts is related to an ancient mechanism which functions to raise the number of receptors required to produce cellular activation during the course of prolonged cellular stimulation, perhaps through a G-protein-related pathway.

Alternative Splicing↗
PubMed2006

Stromal-cell-derived factor-1/CXCL12-induced chemotaxis of a T cell line involves intracellular signaling through Cbl and Cbl-b and their regulation by Src kinases and CD45.

Stromal-cell-derived factor-1alpha (SDF-1alpha/CXCL12) is a potent chemoattractant for T cells. We report that Cbl family members, Cbl and Cbl-b, are tyrosine-phosphorylated after SDF-1alpha/CXCL12 stimulation of Jurkat T cells. Enhanced phosphorylation of Cbl and Cbl-b was regulated by src family kinases, and perhaps Fyn. Activated Cbl and Cbl-b interacted with Crk-L, Zap-70, Nck, PLC-gamma and Fyb after SDF-1alpha/CXCL12 stimulation, implicating association of these proteins in SDF-1alpha/CXCL12 actions. SDF-1alpha/CXCL12 did not induce tyrosine phosphorylation of Cbl or Cbl-b in Lck-deficient T cell line J.CaM1.6 or CD45-deficient T cell line J45.01. Thus, Lck Src kinase and tyrosine phosphatase CD45 are likely involved in regulating activation of Cbl family members. A functional role for Cbl and Cbl-b in migration was demonstrated by the decrease in SDF-1/CXCL12-induced migration in a T cell line in which transfected small interfering RNA for Cbl and Cbl-b decreased expression of Cbl and Cbl-b, but not MAPK activity. SDF-1alpha/CXCL12-induced chemotaxis was greatly reduced in the CD45-deficient T cell line. Our results implicate CD45, Cbl, Cbl-b, src kinases and potentially other associated proteins as mediators of SDF-1alpha/CXCL12-induced cell migration of Jurkat T cells.

Adolescent↗
PubMed2005

Synthetic studies on novel Syk inhibitors. Part 1: Synthesis and structure-activity relationships of pyrimidine-5-carboxamide derivatives.

Spleen tyrosine kinase (Syk) is a non-receptor-type tyrosine kinase which mediates diverse responses in haematopoietic cells. Therefore, Syk is an attractive therapeutic target, and in a study of Syk inhibitors as potentially new therapeutic agents, we discovered the 4-anilinopyrimidine-5-carboxamides. Enzyme screening indicated that an aminoethylamino moiety at the 2-position of the pyrimidine ring was important for Syk inhibitory activity, and an investigation of the substituents at the 4-position revealed that an anilino moiety substituted at the meta position was preferred. These compounds showed high selectivity for Syk, compared to other kinases, such as ZAP-70, c-Src, and PKC, and exhibited good inhibitory activities against 5-HT release from RBL-cells. Among them, compound 9a inhibited the passive cutaneous anaphylaxis reaction in mice, with an ID50 of 13 mg/kg following subcutaneous administration. These results suggest that our compounds are worthy of further evaluation as new anti-allergic agents.

Amides↗
PubMed2005

Commensal microbiota alter the abundance and TCR responsiveness of splenic naïve CD4+ T lymphocytes.

The epidemiologic risk of certain systemic immunologic diseases is affected by commensal or environmental microbiota, but the cellular basis of the "hygiene hypothesis" is poorly understood. In this study, we demonstrate that composition of the commensal microbiota affects the functional state of the peripheral naïve (CD62L(hi)CD44(lo)) T lymphocyte populations. Restricted flora (RF) mice (stably colonized with excess nonpathogenic Clostridium sp., and changes in other bacterial and fungal taxa) were distinguished after the neonatal period by a progressive deficiency in absolute numbers of naïve CD4+ and CD8+ T lymphocytes. SPF and RF mice had comparable levels of memory CD4+ and CD8+ T cells. This phenotype was attributable to the altered levels of certain commensals and their products, since germ-free mice had normal absolute numbers of splenic CD4+ and CD8+ T cells and their respective naïve and memory subsets. The naïve CD4+ T cell subset was functionally distinguished in RF mice versus SPF mice by TCR hyperresponsiveness, pro-inflammatory cytokine production, and increased activation-induced cell death. Biochemically, these traits were associated with higher basal phosphorylation of the TCR signaling proteins ZAP-70, Lck, and LAT. These findings indicate that enteric microbial products, through unknown cellular circuitry, influence steps in CD4 T cell differentiation moderating basal TCR signaling and immune responsiveness.

Animals↗
PubMed2005

Inhibitors of the tyrosine kinase signaling cascade for asthma.

The pathogenesis of allergic asthma involves the interplay of inflammatory cells and resident airway cells, and of their secreted mediators including cytokines, chemokines, growth factors and inflammatory mediators. Tyrosine kinase signaling cascades play a critical role in the pathogenesis of allergic airway inflammation. Receptor tyrosine kinases (e.g. epidermal growth factor receptor [EGFR] and platelet-derived growth factor receptor) are important for the pathogenesis of airway remodeling. Stimulation of non-receptor tyrosine kinases (e.g. Lyn, Lck, Syk, ZAP-70, Btk, Itk and JAK) is the earliest detectable signaling response upon activation of immune receptors (T cell receptor, B cell receptor and FCepsilonR1), cytokine receptors and chemokine receptors in inflammatory cells. Activation of tyrosine kinases invokes multiple downstream signaling pathways, including phosphoinositide 3-kinase (PI3K), mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-kappaB), leading to cell differentiation, survival, proliferation, degranulation and chemotaxis. Inhibitors targeted at different enzyme molecules of the tyrosine kinase signaling cascade might afford therapeutic potential for asthma. Anti-inflammatory effects of pharmacological agents targeted at tyrosine kinases, Syk, Itk, signal transducer and activator of transcription-1, NF-kappaB, GATA3, EGFR, PI3K, MEK1/2, p38 MAPK and JNK have been reported in animal models of allergic airway inflammation. Therefore, development of inhibitors targeted at the tyrosine kinase signaling cascade is an attractive strategy for the treatment of asthma.

Animals↗
PubMed2004

Tyrosine phosphorylation-dependent activation of NFkappaB is compromised in T cells from the elderly.

NFkappaB induction and gene regulation are compromised in T lymphocytes during aging. This has been attributed to altered proteasomal function resulting in decreased ubiquitin-mediated degradation of IkappaBalpha. However, little is known about the impact of aging on the mechanisms that lead to the release of active NFkappaB employing pro-oxidant pathways. Oxidant-mediated activation of NFkappaB has been previously shown to involve proteasome independent mechanisms and hence may be an important alternate conduit to the induction of this central transcription factor in aging. Employing H(2)O(2) and pervanadate we not only demonstrate lowered tyrosine phosphorylation of IkappaBalpha, but also compromised induction of nuclear NFkappaB in T cells from the elderly. Lowered tyrosine phosphorylation of IkappaBalpha may be due to a decrease in activity of p56(lck) and ZAP-70, since treatment with piceatannol, an inhibitor of syk and src family kinases, mimics age associated decline in tyrosine phosphorylation of IkappaBalpha in T cells from young donors. Thus, alternate pathways of NFkappaB induction are also impaired in T cells from the elderly and may underlie immune-deficit accompanying aging.

Adult↗
PubMed2005

Molecular cloning and characterization of a cellulase gene from a symbiotic protist of the lower termite, Coptotermes formosanus.

The endo-beta-1,4-glucanase gene was cloned from a cDNA library constructed from the mixed population of symbiotic protists in the hindgut of the lower termite, Coptotermes formosanus, using the lambda ZAP II vector. The recombinant phage library was screened for cellulolytic activity by the Congo red staining procedure. The nucleotide sequence comprised 941 nucleotides including a polyA tail sequence and showed high sequence similarity with endoglucanase genes belonging to glycosyl hydrolase family 5. Determination of the 5' end of the cellulase gene using the 5'RACE method showed that the full-length cDNA comprised a 921-bp ORF, encoding a putative 33,620 Da protein. The organismal source of this cellulase gene was identified using PCR with gene-specific primers and whole-cell in situ hybridization as the smallest symbiotic hypermastigote protist, Spirotrichonympha leidyi. The optimal pH and temperature of the cellulase heterologously expressed in Escherichia coli were 5.8-6.0 and 70 degrees C, respectively. The Km and Vmax values on carboxymethyl cellulose (CMC) substrate were 1.90 mg/ml and 148.2 units/mg protein, respectively.

Amino Acid Sequence↗
PubMed2004

Molecular genetics and its clinical relevance.

Molecular genetic methods such as fluorescence in situ hybridization and DNA sequencing have greatly improved our understanding of pathogenic events and prognostic markers in chronic lymphocytic leukemia (CLL). There are genomic aberrations detected in over 80% of CLL cases, and genes potentially involved in the pathogenesis were identified with ATM in a subset of cases with 11q deletion and p53 in cases with 17p13 deletion. Genetic subgroups with distinct clinical features have been identified, such as 11q deletion, which is associated with marked lymphadenopathy and rapid disease progression, whereas 17p deletion predicts for treatment failure with alkylating agents, fludarabine, and short survival times. There is mutation status of the VH genes that allows the separation into patients with long (mutated VH) or short (unmutated VH) survival times. V-gene usage, VDJ structure, and gene expression differences in the two subgroups allow insights into differential pathogenic mechanisms and provide further prognostic information (V3-21 usage, ZAP-70 expression). The VH mutation status and genomic abnormalities have been shown to be of independent prognostic value in multivariate analysis, seem to allow outcome predication irrespective of the clinical stage, and may therefore allow a risk assessment for individual patients early in the course of their disease.

Chromosome Aberrations↗
PubMed2005

ITAM-based signaling beyond the adaptive immune response.

Classical immunoreceptors like lymphocyte antigen receptors and Fc-receptors (FcR) are central players of the adaptive immune response. These receptors utilize a common signal transduction mechanism, which relies on immunoreceptor tyrosine-based activation motifs (ITAMs) present in the receptor complex. Upon ligand binding to the receptors, tyrosines within the ITAM sequence are phosphorylated by Src-family kinases, leading to an SH2-domain mediated recruitment and activation of the Syk or the related ZAP-70 tyrosine kinase. These kinases then initiate further downstream signaling events. Here we review recent evidence indicating that components of this ITAM-based signaling machinery are also present in a number of non-lymphoid or even non-immune cell types and they participate in diverse biological functions beyond the adaptive immune response, including innate immune mechanisms, platelet activation, bone resorption or tumor development. These results suggest that the ITAM-based signaling paradigm has much wider implications than previously anticipated.

Animals↗
PubMed2005

CAML is a p56Lck-interacting protein that is required for thymocyte development.

Calcium modulating cyclophilin ligand (CAML) is a ubiquitously expressed protein implicated in T cell signaling, although its mechanism and physiologic role in the immune system are unknown. We show here that CAML is essential for peripheral T cell development. Inactivation of CAML in mouse thymocytes lowered the numbers of double-positive and single-positive thymocytes, concomitant with reduced positive and enhanced negative selection. We found that CAML interacts with p56Lck and appears to regulate subcellular localization of the kinase in both resting and T cell receptor (TCR)-stimulated cells. CAML-deficient cells displayed enhanced p56lck and ZAP-70 phosphorylation and increased IL2 production and cell death after TCR stimulation, suggesting that CAML may act as a negative regulator of p56lck. Our data establish a novel role for CAML as an essential mediator of T cell survival during thymopoiesis and indicate that its loss deregulates p56Lck signaling.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Impaired activation and localization of LAT in anergic T cells as a consequence of a selective palmitoylation defect.

The molecular basis of T cell anergy is not completely understood. We show that in antigen-primed anergic murine CD4(+) T cells the linker for activation of T cells (LAT) is hypophosphorylated upon CD3/CD28 restimulation. Signaling events downstream of LAT (PLCgamma1 phosphorylation and p85 [PI3-K] association) were impaired, whereas upstream events (CD3zeta and ZAP-70 phosphorylation) remained intact. LAT recruitment to the immunological synapse and its localization in detergent-resistant membrane (DRM) fractions were defective in anergic T cells. These defects resulted from impaired palmitoylation of LAT and were selective since the DRM localization and palmitoylation of Fyn were intact. This LAT defect was independent of Cbl-b and did not reflect enhanced LAT degradation. These results identify LAT as the most upstream target of anergy induction; moreover, they suggest that regulation of the amount of LAT in the immunological synapse and DRM by posttranslational palmitoylation contributes to the induction of T cell anergy.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Endothelial nitric oxide synthase regulates T cell receptor signaling at the immunological synapse.

The role of nitric oxide (NO) in T cells remains controversial, and the origin and localization of endogenous NO and whether it regulates lymphocyte activation are unclear. We show here that, within minutes of binding to antigen, T cells produce NO via endothelial nitric oxide synthase (eNOS). This process required increased intracellular Ca2+ and phosphoinositide3-kinase activity. By using an eNOS-green fluorescent fusion protein and fluorescent probes to detect NO, we show that eNOS translocates with the Golgi apparatus to the immune synapse of T helper cells engaged with antigen-presenting cells (APC), where it was fully activated. Overexpression of eNOS prevented the central coalescence of CD3 at the T cell-APC contact site, which was accompanied by increased phosphorylation of CD3zeta chain, ZAP-70, and extracellular signal-regulated kinases and increased IFN-gamma synthesis, but reduced production of IL-2. Therefore, eNOS-derived NO selectively potentiates T cell receptor signaling to antigen at the immunological synapse.

Animals↗
PubMed2005

Suppression of CD4+ T cell activation by a novel inhibitor of Src family kinases.

The Src family kinases Lck and Fyn play an important role in T cell development and function. We have synthesized a novel small molecule, A-420983, which inhibits Lck and Fyn, as well as other Src family kinases, but has selectivity with respect to non-Src family kinases. A-420983 completely inhibited antigen-stimulated production of IFN-gamma and IL-4 by mouse Th1 and Th2 cells, respectively. Antigen-induced T cell proliferation was also blocked by treatment with A-420983. In contrast, IL-15-induced proliferation was unaffected by A-420983, suggesting that TCR-independent pathways of T cell activation were not impaired. When mice were dosed orally, A-420983 inhibited TCR-mediated c-jun and ZAP-70 phosphorylation in CD4+ T cells and suppressed the disease course of established EAE. Treatment with A-420983 for 7 days resulted in a block in thymocyte development at the CD4- CD8- stage, consistent with inhibition of Lck and Fyn in vivo. These results demonstrate that a small molecule inhibitor of Lck and Fyn can block TCR-induced T cell activation in vitro and in vivo. Furthermore, CNS demyelination mediated by activated encephalitogenic CD4+ T cells is dependent upon the kinase activity of these Src family members. We conclude that inhibition of Src family kinases may represent a promising strategy for the treatment of T cell-mediated disorders.

Animals↗
PubMed2004

Novel concepts and treatments for autoimmune disease: ten focal points.

Understanding the development of autoimmunity is a crucial step toward improving the management, not only of autoimmune diseases, but also of tumors and primary immunodeficiency syndromes. The rapid expansion of knowledge on autoimmunity is fueling the development of a novel approach known as targeted immunotherapy. The present review will concentrate on ten areas where major advances have been achieved: 1) early regulation of B-cell mediated autoimmunity; 2) thymic regulation of tolerance to tissue-restricted antigens via the transcription factor AIRE; 3) role for a population of regulatory T cells (CD4+ CD25+ Tregs) with unique effects; 4) major role for dendritic cells in the development of autoimmunity in conditions such as lupus; 5) role for T cells in autoimmune diseases; 6) role for T cells in rheumatoid arthritis, with new data from a murine model of spontaneous arthritis related to a ZAP-70 mutation; 7) role for the environment via innate immunity, in particular mediated by the toll-like receptors (TLR); identification of new autoantigens with the description of sense-antisense peptides (e.g., proteinase 3-complementary proteinase 3); the immunosenescence concept, which suggests that some autoimmune diseases may be related to premature aging of the immune system; 10) identification of new immunotherapy targets, including costimulation pathway molecules (CD28, CTLA4), original activation systems (BAFF/BLyS), and receptors such as TLRs. These exciting insights into the pathogenic mechanisms underlying immune dysfunction will play a key role in advancing the field of immunorheumatology.

Autoimmune Diseases↗
PubMed2005

Characterization of cDNA encoding a human sperm membrane protein sharing homology with Huntington protein.

In our earlier study, we have reported the identification and characterization of a 57-kDa sperm membrane protein from human spermatozoa. The protein was found to be localized on the acrosome in acrosome intact spermatozoa and shifted to equatorial region after acrosomal induction. Further, we demonstrated that it plays a critical role in sperm--egg binding and fusion. Since the protein was found to be either absent or poorly expressed on the spermatozoa from infertile men, we designated it as Fertility Associated Sperm Antigen (FASA). A human epididymal cDNA library in Lambda-ZAP vector was screened with a polyclonal antibody raised against FASA. A clone was obtained with an insert of approximately 1.9 kb cDNA. The sequence showed 99% homology with a part of the human chromosome 11. EST database showed that a portion of 1.9 kb gene has 87% homology to gene encoding Huntington disease protein (HDP). The mutated form of this protein is responsible for Huntington's disease (HD) and is found in the brain cells of HD patients.

Amino Acid Sequence↗