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At least 991 records · Page 55Linked to original sources
PubMed2001

Proximal protein tyrosine kinases in immunoreceptor signaling.

Immunoreceptor engagement results in the sequential activation of several classes of protein tyrosine kinases, including the Src and Syk/Zap-70 families. Recent progress has been made in our understanding of the regulation and function of these molecules. First, it was revealed that membrane compartmentation of protein tyrosine kinases may be essential for their proper biological function. Second, Src family kinases were found to act not only as positive regulators, but also as inhibitors of cell activation. Third, it was appreciated that Csk, a potent inhibitor of Src kinases, is regulated by an assortment of protein-protein interactions. Fourth, differences in the regulation of Syk and Zap-70 were observed, suggesting significant distinctions in the purpose of these two kinases in immunoreceptor signaling. And fifth, it was suggested that proximal kinases implicated in immunoreceptor-mediated signal transduction may be regulated by protein degradation via binding to c-Cbl, a ubiquitin ligase.

Animals↗
PubMed1996

Regulation of antigen receptor signal transduction by protein tyrosine kinases.

The past two years have seen further clarification of the early events occurring in antigen receptor signal transduction that are mediated by the immunoreceptor tyrosine-based activation motif (ITAM). The ITAM was shown to be a specific binding site for the ZAP-70/Syk protein tyrosine kinases and the structure of this complex was solved. In addition, possible mechanisms of activation and functions for these kinases were reported. Lastly, genetic studies established the critical importance of these kinases in antigen-receptor signaling and lymphocyte development.

Animals↗
PubMed1999

T cell antigen-receptor signal transduction.

During the past year, major progress has been made in understanding proximal TCR signal-transduction events. Cbl has been identified as a negative regulator of kinases from the ZAP-70/Syk family. Studies on LAT, SLP-76, Itk and Vav have revealed their role in the activation of Ras and phospholipase-Cgamma1-Ca2+ signalling pathways. TCR-induced cytoskeletal changes involve signalling through SLP-76-Vav-Nck to activate effectors of the Rho-family of GTPases. Finally, glycolipid-enriched microdomains play a crucial role in T cell activation.

Adaptor Proteins, Signal Transducing↗
PubMed1997

T cell antigen receptor signal transduction.

The T cell antigen receptor (TCR) initiates signal transduction by activating multiple cytoplasmic protein tyrosine kinases (PTKs). Considerable progress in the field of TCR signal transduction has been made in three areas recently: first, in understanding the structure and function of the PTK ZAP-70; second, in the elucidation of the function of the substrates and pathways downstream of the PTKs; and third, in the identification of molecules that negatively regulate TCR signalling.

Animals↗
PubMed2003

Profiling genes expressed in human fetal cartilage using 13,155 expressed sequence tags.

OBJECTIVE: To analyze the gene expression profile of human fetal cartilage by expressed sequence tags (ESTs). METHODS: A human fetal cartilage (8-12 weeks) cDNA library was constructed using the lambda ZAP Express vector. ESTs were obtained by partial sequencing of cDNA clones. The basic local alignment search tool algorithm was used to compare all generated ESTs to known sequences. RESULTS: A total of 13,155 ESTs were analyzed, of which 8696 ESTs (66.1%) matched known genes, 53 ESTs (0.4%) were putatively novel (with no match) and the rest matched other ESTs, genomic DNA and repetitive sequences. Importantly, we identified 2448 unique known genes through non-redundancy analysis of the known gene matches, which were then functionally categorized. The tissue specificity of this library was reflected by its EST profile of the extracellular matrix (ECM) proteins. Collagens were the major transcripts, representing 68.5% of the ECM proteins. Proteoglycans were the second most abundant, constituting 9.5%. Collagen type II was the most abundant gene of all. Glypican 3, decorin and aggrecan were the major transcripts of proteoglycans. Many genes involved in cartilage development were identified, such as insulin-like growth factor-II, its receptor and binding proteins, connective tissue growth factor and fibroblast growth factors. Proteases and their regulatory factors were also identified, including matrix metalloprotease 2 and tissue inhibitor of metalloproteinase 1. CONCLUSIONS: The EST approach is an effective way of characterizing the genes expressed in cartilage. These data represent the most extensive molecular information on human fetal cartilage to date. The availability of this information will serve as a basis for further research to identify genes that are essential in cartilage development.

Cartilage, Articular↗
PubMed1996

Sequential involvement of Lck and SHP-1 with MHC-recognizing receptors on NK cells inhibits FcR-initiated tyrosine kinase activation.

Recognition of major histocompatibility (MHC) class I complexes on target cells by killer cell inhibitory receptors (KIR) blocks natural killer (NK) and T cell cytotoxic function. The inhibitory effect of KIR ligation requires the phosphotyrosine-dependent association of KIR with the cytoplasmic SH2-containing protein tyrosine phosphatase SHP-1. Using a somatic genetic model, we first define a requirement for the Src family protein tyrosine kinase (PTK) Lck in mediating KIR tyrosine phosphorylation. We then investigate how KIR ligation interrupts PTK-dependent NK cell activation signals. Specifically, we show that KIR ligation inhibits the Fc receptor (FcR)-induced tyrosine phosphorylation of the FcR-associated zeta signaling chain, the PTK ZAP-70, and phospholipase C gamma. Overexpression of catalytically inactive SHP-1 (acting as a dominant negative) restores the tyrosine phosphorylation of these signaling events and reverses KIR-mediated inhibition of NK cell cytotoxic function. These results suggest sequential roles for Lck and SHP-1 in the inhibition of PTK following MHC recognition by NK cells.

Amino Acid Sequence↗
PubMed2001

Involvement of SHP-1 tyrosine phosphatase in TCR-mediated signaling pathways in lipid rafts.

To elucidate the process of TCR-mediated signaling pathways in lipid rafts, we constructed a chimeric molecule that localizes activated SHP-1 to rafts. Raft targeting of activated SHP-1 in Jurkat-derived transfectants completely inhibited the expression of CD69 and transcriptional factors after TCR cross-linking. Whereas the inducible tyrosine phosphorylation of TCR zeta and ZAP-70 and the kinase activity of Lck were intact, phosphorylated LAT was rapidly dephosphorylated by raft targeting of activated SHP-1, leading to defects in LAT activation and subsequent downstream signaling events. Intriguingly, recruitment of endogenous SHP-1 to rafts and its association with LAT were dramatically increased after TCR engagement, suggesting that SHP-1 is involved in raft-mediated T cell activation.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Key disulfide bonds in an insect hormone binding protein: cDNA cloning of a juvenile hormone binding protein of Heliothis virescens and ligand binding by native and mutant forms.

The hemolymph juvenile hormone binding protein (JHBP) from the early fifth instar larvae of Heliothis virescens (Lepidoptera, Noctuidae) has been purified, and three cDNA clones for this protein have been isolated from a fat body cDNA library constructed in bacteriophage lambda ZAP XR. The deduced amino acid sequence of the full-length clone predicts a mature protein consisting of 224 residues, a molecular mass of 24,976 Da, and a pI of 5.29. Comparison of the amino acid sequence to that of the previously described JHBP from Manduca sexta shows 51% overall identity with highly conserved N- and C-terminal regions. One of the three clones bound photoactivatable analogs of juvenile hormones with much lower affinity than the other two. This clone had Phe150 in place of the expected Cys150 conserved in other JHBP clones. The F150C mutant of this clone regained native binding affinity. For native Hvir-JHBP, the affinity for [3H]JH I was lower under reducing conditions (87 nM) relative to a 40 nM affinity under nonreducing conditions. The importance of pairs of Cys residues was addressed by preparing Cys to Ala mutants at each site. Expressed proteins were tested for binding affinity by photoaffinity labeling with tritium-labeled JH analogs and by binding assays using (10R,11S)-[3H]JH I. Curiously, the C150A mutant retained full activity, implying that the aberrant C150F was dysfunctional due to steric hindrance rather than to a missing disulfide linkage. Likewise, C29A and C194A had binding affinities unchanged from that of the full-length wild-type clone.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1991

Mouse pulmonary cytochrome P-450 naphthalene hydroxylase: cDNA cloning, sequence, and expression in Saccharomyces cerevisiae.

We have isolated a cDNA clone, Nah-2, encoding the cytochrome P-450Nah (naphthalene hydroxylase) from a mouse lung lambda ZAP cDNA library using anti-cytochrome P-450Nah IgG as a probe. This same antibody selectively blocked [Nagata, K., Martin, B.M., Gillette, J.R., & Sasame, H.A. (1990) Drug Metab. Dispos. 18, 557-564] the cytochrome P-450 in mouse lung microsomes that catalyzed the conversion of naphthalene to (1R,2S)-naphthalene 1,2-oxide, which has been postulated as a causative agent in the naphthalene-induced tissue-specific necrosis of Clara cells in mouse lung. The toxic effect is seen in mouse and not in rat. The cDNA encodes a polypeptide of 491 amino acids with a molecular mass of 50 kDa. Northern blot analysis with an Nah-2-specific probe revealed that the mRNA is expressed in a species- and tissue-specific manner, present only in mouse lung and liver and not in that of rat. The mRNA encoding Nah-2 is constitutively expressed and is not induced by either phenobarbital, pyrazole, pregnenolone 16 alpha-carbonitrile, or 3-methylcholanthrene. Comparative amino acid sequence analyses with other documented members of the P-450 gene superfamily revealed that this encoded protein is in the IIF subfamily. To analyze its substrate specificity, the cDNA was inserted into the vector, pAAH5, and expressed in the Saccharomyces cerevisiae strain, AH22. The presence of cytochrome P-450Nah in the microsomes isolated from transformed cells and analyzed by Western blot was confirmed by immunocomplexing product with anti-cytochrome P450Nah IgG. Furthermore, activity toward naphthalene in the microsomes from the transformed cells established that this clone encodes a naphthalene hydroxylase.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1988

Conservation of primary structure in the lipoyl-bearing and dihydrolipoyl dehydrogenase binding domains of mammalian branched-chain alpha-keto acid dehydrogenase complex: molecular cloning of human and bovine transacylase (E2) cDNAs.

The subunit structures and conservation of the dihydrolipoyl transacylase (E2) components of bovine and human branched-chain alpha-keto acid dehydrogenase complexes were investigated by Western blotting, peptide sequencing, and cDNA cloning methods. Rabbit antiserum prepared against the sodium dodecyl sulfate (SDS) denaturated bovine E2 subunit recognized the inner E2 core, and the first hinge region of the E2 chain, but failed to react with the lipoyl-bearing domain as determined by Western blot analysis. The lack of antigenicity in the lipoyl-bearing domain was confirmed with antibodies directed against the native E2 component. A human E2 cDNA (1.6 kb) was isolated from a human liver cDNA library in lambda gt11 with a combination of the above anti-native and anti-SDS-denatured E2 immunoglobulin G's as a probe. The fidelity of the human E2 cDNA was established by nucleotide sequencing which showed the determined peptide sequences of the amino terminus and tryptic fragments of bovine E2. A bovine E2 cDNA (0.7 kb) was also isolated from a bovine liver cDNA library in lambda ZAP with the human E2 cDNA as a probe. Northern blot analysis using the human E2 cDNA probe showed that E2 mRNAs in bovine liver and human kidney mesangial cells are 3.3 and 4.6 kb in size, respectively. Primary structures derived from human and bovine E2 cDNAs show leader sequences including the initiator methionine and the homologous mature peptides consisting of complete lipoyl-bearing and dihydrolipoyl dehydrogenase (E3) binding domains and two hinge regions. In addition, the human E2 cDNA contains a portion of the inner E2 core sequence, a 3'-untranslated region, and a poly(A+) tail. Deduced amino acid sequences of the mammalian E2's were compared with those of Escherichia coli transacetylase and transsuccinylase and bovine kidney transacetylase. The results indicate a high degree of conservation in the sequence flanking the lipoyl-attachment site and in the E3-binding domain. Models are presented to discuss implications for the conserved structure-function relationship in the lipoyl-bearing and E3-binding domains of alpha-keto acid dehydrogenase complexes.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed2003

NF-kappaB induction by bisperoxovanadium compounds requires CD45, p36(LAT), PKC, and IKK activity and exhibits kinetics of activation comparable to those of TCR/CD28 coengagement.

We have previously shown that bisperoxovanadium (bpV) phosphotyrosyl phosphatase inhibitors can potently activate NF-kappaB. We have already determined that p56(lck), ZAP-70, SLP-76, capacitative entry of calcium, and calcium-regulated effectors are important in bpV-induced NF-kappaB activation. In this study, we evaluated whether other signal transducers previously reported in NF-kappaB induction by T cell activating stimuli are also activated by bpV compounds. Nuclear translocation of NF-kappaB was evaluated in cell lines deficient for either CD45 or p36(LAT) to assess the role of these signal transducers in bpV-mediated NF-kappaB activation. A deficiency of either protein greatly reduced the extent of NF-kappaB nuclear translocation following bpV treatment. Isoform-specific PKC inhibitors were then used to show that bpV compounds activate NF-kappaB through both calcium-sensitive and -insensitive PKC isoforms. The implication of the IkappaB-kinase complex was then investigated through the use of an IkappaBalpha-specific kinase assay and plasmids expressing catalytically inactive forms of IKKalpha and IKKbeta. Upstream kinases involved in IKK complex activation such as TPL-2/COT, NIK, and IKKepsilon were also shown to play an important role in bpV-mediated NF-kappaB activation. Finally, reporter gene transcriptional assays and gel shift assays were performed to compare the kinetics of activation of NF-kappaB by bpV with those of antigenic and TNFalpha stimulation. We demonstrate, both in Jurkat cells and in primary T cells, that bpV-mediated NF-kappaB activation kinetics are comparable to those of an antigenic stimulation but occur much slower than the kinetics seen upon TNFalpha treatment.

CD28 Antigens↗
PubMed2005

Tyrosine phosphorylation of adaptor protein 3BP2 induces T cell receptor-mediated activation of transcription factor.

Molecular adaptors/scaffolds have indispensable roles in the activation of lymphocytes. In this report, we have demonstrated the role of tyrosine phosphorylation of an adaptor protein 3BP2 (c-Abl-SH3 domain binding protein-2, also known as SH3BP2) in T cell receptor (TCR)-mediated activation of transcription factor. Short interfering RNA for 3BP2 suppresses the expression level of endogenous 3BP2 and inhibits TCR-mediated activation of interleukin (IL)-2 promoter and nuclear factor of activated T cells (NFAT) element. Engagement of TCR induces tyrosine phosphorylation and lipid raft translocation of 3BP2. The overexpression studies reveal that substitution of 3BP2-Tyr(183), Tyr(446), or Arg(486) in the SH2 domain suppresses TCR-mediated activation of NFAT. Point mutations of 3BP2 cannot affect the translocation of 3BP2 into the lipid raft. Phosphorylation of Tyr(183) is required for the interaction with Vav1, the guanine nucleotide exchanging factor of Rac1. In fact, overexpression of dominant-negative form of Rac1 inhibits TCR-mediated activation of NFAT. Phosphorylation of Tyr(446) recruits the SH2 domain of Lck for the optimal activation of transcription factors. Furthermore, point mutation of Arg(486) in the 3BP2-SH2 domain that couples ZAP-70 to LAT dramatically reduces NFAT activation. These results suggest that the site-directed functions of 3BP2 induce the activation of transcription factors.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Selective Itk inhibitors block T-cell activation and murine lung inflammation.

Nonreceptor protein tyrosine kinases including Lck, ZAP-70, and Itk play essential roles in T-cell receptor (TCR) signaling. Gene knockout studies have revealed that mice lacking these individual kinases exhibit various degrees of immunodeficiency; however, highly selective small molecule inhibitors of these kinases as potential immunosuppressive agents have not been identified. Here we discovered two novel compounds, BMS-488516 and BMS-509744, that potently and selectively inhibit Itk kinase activity. The compounds reduce TCR-induced functions including PLCgamma1 tyrosine phosphorylation, calcium mobilization, IL-2 secretion, and T-cell proliferation in vitro in both human and mouse cells. The inhibitors suppress the production of IL-2 induced by anti-TCR antibody administered to mice. BMS-509744 also significantly diminishes lung inflammation in a mouse model of ovalbumin-induced allergy/asthma. Our findings represent the first description of selective inhibitors to probe human Itk function and its associated pathway, and support the hypothesis that Itk is a therapeutic target for immunosuppressive and inflammatory diseases.

Animals↗
PubMed1998

Ro 09-2210 exhibits potent anti-proliferative effects on activated T cells by selectively blocking MKK activity.

By using high throughput screening of microbial broths, we have identified a compound, designated Ro 09-2210, which is able to block anti-CD3 induced peripheral blood T cell activation with an IC50 = 40 nM. Ro 09-2210 was also able to block antigen-induced IL-2 secretion with an IC50 = 30 nM, but was considerably less potent at blocking Ca2+ flux stimulated by anti-CD3 treatment. To determine the mechanism of action of Ro 09-2210, we set up a transient expression system in Jurkat T cells using a variety of reporter gene constructs and showed effective inhibition of phorbol ester/ionomycin-induced NF-AT activation and anti-CD3 induced NF-AT with IC50 = 7.7 and 10 nM, respectively. Ro 09-2210 was also able to inhibit phorbol ester/ionomycin-induced activation of AP1 with IC50 = <10 nM. We further showed that Ro 09-2210 was unable to inhibit c-jun induced expression of AP1-dependent reporter constructs (IC50 > 500 nM), but was able to potently inhibit ras-induced AP1 activation (IC50 = 20 nM). This suggested that Ro 09-2210 was inhibiting an activator of AP-1 which was upstream of c-jun and downstream of ras signaling. To investigate further, we then purified a number of different kinases, including PKC, PhK, ZAP-70, ERK, and MEK 1 (a MKK), and showed that Ro 09-2210 was a selective inhibitor of MEK1 in vitro (IC50 = 59 nM).

DNA-Binding Proteins↗
PubMed2004

History-dependent sorption in humic acids and a lignite in the context of a polymer model for natural organic matter.

We examined sorption of two apolar compounds in three samples of macromolecular natural organic matter (NOM) in order to test whether history-dependent ("irreversible") behaviors, including sorption hysteresis and the conditioning effect, agree with a pore deformation/creation hypothesis applicable to the glassy organic solid state as proposed in the polymer literature. The compounds are 1,2,4-trichlorobenzene (TCB) and naphthalene (Naph). The NOM samples are a soil humic acid (H-HA), an Al3+-exchanged form of the same humic acid (Al-HA), and a low-rank coal (Beulah-Zap lignite, BZL). The HAs, at least, are believed free of environmental black carbon. The degree of nonlinearity in the isotherm and the ratio of hole-filling to solid-phase dissolution increased in the order of hardness (stiffness) of the solid: H-HA < Al-HA < BZL. Independent of solid, solutes show a 14-18 kJ/mol preference for hole "sites" as compared to dissolution "sites", which we attribute to the free energy needed in the dissolution domain to create a cavity to accommodate the solute. All solids exhibited hysteresis and the conditioning effect, which refers to enhanced re-sorption after pretreatment with a conditioning agent (in this case, chlorobenzene). Conditioning the sample results in increased sorption and increased contribution of hole-filling relative to dissolution. The effects of original hole population, matrix stiffness, and solute concentration on the hysteresis index and on the magnitude of the conditioning effect are consistent with a pore-deformation mechanism as the underlying cause of sorption irreversibility. This mechanism involves concurrent processes of irreversible hole expansion and the creation of new holes by the incoming sorbate (or conditioning agent). The results show that nonlinear and irreversible behavior may be expected for macromolecular forms of NOM that are in a glassy state and emphasize the case that NOM is not a passive sorbent but may be physically altered by the sorbate.

Adsorption↗
PubMed2005

An isotope exchange technique to assess mechanisms of sorption hysteresis applied to naphthalene in kerogenous organic matter.

The sorption of organic compounds to natural sorbents is often found to show hysteresis. The objective of this study was to develop an experimental technique based on the use of 14C isotopes to distinguish hysteresis due to experimental artifacts from true hysteresis due to thermodynamically irreversible processes. The study was also designed to investigate causation of true hysteresis (irreversible sorption). The technique determines the rates and the degree of isotope exchange (IE) on equilibrated sorption and desorption points at different constant bulk chemical concentrations. The technique was applied to the sorption of naphthalene (NAPH) on Beulah-Zap lignite, a low rank reference coal composed mainly of kerogen. Sorption of bulk was found to be reversible below 10(-5) g L(-1), but irreversible above 10(-4) g L(-1). Complete isotope exchange on sorption and desorption points that defined an irreversible cycle demonstrated that hysteresis was true. A comparison of normalized uptake and release kinetics of labeled and bulk NAPH at different concentrations revealed slow structural deformation processes of the sorbent during bulk sorption and desorption. This is taken as corroborating evidence for the pore deformation hypothesis of hysteresis in which incoming sorbate molecules induce quasi-reversible changes in the organic matter that lead to different pathways for sorption and desorption. Although unable to rule it out completely, the data demonstrate that physical entrapment of sorbate molecules plays a minor, if any, role to the observed hysteresis in this system.

Adsorption↗
PubMed1999

Improvement of cattle productivity through rapid alleviation of African animal trypanosomosis by integrated disease management practices in the agropastoral zone of Yalé, Burkina Faso.

Investigations to identify the causes of high mortalities in cattle in the agropastoral zone (ZAP) of Yalé started in March 1993. African animal trypanosomosis (AAT) was found to be the major constraint, with incidence rates exceeding 30%, justifying a tsetse control programme, which started in March/ April 1994. The treatment of all cattle at bimonthly intervals with deltamethrin 1% pour on and the display of 1500 insecticide impregnated targets during the 6 months of the dry season each year helped to reduce the tsetse populations (Glossina tachinoides and G. morsitans submorsitans) by more than 90%. In less than 7 months, the incidence of AAT dropped below 5% and remained there throughout the intervention until June 1996, in spite of an increase to 3 months in the interval between the treatments. Mean PCV values increased significantly from 26.5-30.9%, before, to 30.7-36.3% during the intervention. The improvement in the overall health resulted in a resumption in fertility and milk production, allowing the sale of dairy products in Léo, thus creating a gross income of about $US3/day for the Fulani women.

Animals↗
PubMed2001

Farmers' perceptions of the impacts of tsetse and trypanosomosis control on livestock production: evidence from southern Burkina Faso.

Interviews with all the households in the Agropastoral Zone (ZAP) of Yalé, southern Burkina Faso, were conducted in 1994 and again in 1997 to assess the impacts of a tsetse control programme implemented from 1994, using insecticide-impregnated targets and pour-on treatments of all cattle with deltamethrin 1%. In the absence of health and productivity monitoring, data were collected in single-visit surveys in order to generate quantitative estimates of relevant reproductive performance variables for cattle and to assess changes in the inputs used and outputs produced. The results indicate a 25% increase in herd size and an increase in the number of oxen from 0.1 to 1.1 per household; a reduction in mortality from 63.1% to 7.1% and reductions in the rates of abortions and stillbirth of 55.9% and 51.3%, respectively; and an increase in the rate of live births of 57.6% and in the milk yield from 0.2 to 2.2 litres/cow per day in the dry season. These results show the dramatic impacts that trypanosomosis control can have on Zebu cattle exposed to high tsetse challenge. Well-designed surveys can be a cost-effective way to obtain estimates of productivity impacts that can be used to simulate projections of herd growth and meat and milk production in herd models. While there are many confounding factors associated with farmers' perception of a gain in productivity, these estimates form a useful alternative to subjective assessments in modelling the economic benefits of tsetse and trypanosomosis control.

Agriculture↗