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Homodimer
Selected AbstractsDiannexin, a Novel Annexin V Homodimer, Protects Rat Liver Transplants Against Cold Ischemia-Reperfusion InjuryAMERICAN JOURNAL OF TRANSPLANTATION, Issue 11 2007X.-D. Shen Ischemia/reperfusion injury (IRI) remains an important problem in clinical transplantation. Following ischemia, phosphatidylserine (PS) translocates to surfaces of endothelial cells (ECs) and promotes the early attachment of leukocytes/platelets, impairing microvascular blood flow. Diannexin, a 73 KD homodimer of human annexin V, binds to PS, prevents attachment of leukocytes/platelets to EC, and maintains sinusoidal blood flow. This study analyzes whether Diannexin treatment can prevent cold IRI in liver transplantation. Rat livers were stored at 4°C in UW solution for 24 h, and then transplanted orthotopically (OLT) into syngeneic recipients. Diannexin (200 ,g/kg) was infused into: (i) donor livers after recovering and before reperfusion, (ii) OLT recipients at reperfusion and day +2. Controls consisted of untreated OLTs. Both Diannexin regimens increased OLT survival from 40% to 100%, depressed sALT levels, and decreased hepatic histological injury. Diannexin treatment decreased TNF-,, IL-1,, IP-10 expression, diminished expression of P-selectin, endothelial ICAM-1, and attenuated OLT infiltration by macrophages, CD4 cells and PMNs. Diannexin increased expression of HO-1/Bcl-2/Bcl-xl, and reduced Caspase-3/TUNEL+ apoptotic cells. Thus, by modulating leukocyte/platelet trafficking and EC activation in OLTs, Diannexin suppressed vascular inflammatory responses and decreased apoptosis. Diannexin deserves further exploration as a novel agent to attenuate IRI, and thereby improve OLT function/increase organ donor pool. [source] Immune modulation of HLA-G dimer in maternal-fetal interfaceEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 7 2007Kimiko Kuroki Abstract HLA-G is a non-classical human MHC class I molecule, which has several characteristics distinct from classical MHC, such as low polymorphism and restricted tissue distribution. HLA-G is expressed on placenta, thymus and some tumors. At the maternal-fetal interface, trophoblasts do not express major classical MHC class I molecules (MHCI), HLA-A and -B, to prevent normal T cell responses. Instead, HLA-G is expressed and can suppress a wide range of immune responses by binding to inhibitory immune cell surface receptors, such as leukocyte Ig-like receptor (LILR) B1 and LILRB2. HLA-G exists in various forms, including ,2m-associated or -free disulfide-linked dimers that can be expressed either at the cell surface or in soluble form. However, until recently the physiological role of these different molecular forms has been unclear. In this issue of the European Journal of Immunology, one article demonstrates that the disulfide-linked homodimer of ,2m-associated HLA-G is the major fraction expressed by trophoblast cells. The HLA-G dimer modulates the function of LILRB1-expressing antigen-presenting cells by principally binding to LILRB1. On the other hand, another recent report showed that ,2m-free disulfide-linked HLA-G dimers are produced by villous cytotrophoblast cells. Taken together, these results provide strong evidence in support of the hypothesis that HLA-G dimers play a role in immune suppression at the maternal-fetal interface. Further in-depth investigation will help to clarify the precise mechanism of HLA-G receptor recognition and signaling in vivo and the role of these interactions in successful reproduction. See accompanying article: http://dx.doi.org/10.1002/eji.200737089 [source] Humanin antagonists: mutants that interfere with dimerization inhibit neuroprotection by HumaninEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 9 2004Yuichi Hashimoto Abstract The 24-residue peptide Humanin (HN) protects neuronal cells from insults of various Alzheimer's disease (AD) genes and A, by forming a homodimer. We have previously shown that P3A, S7A, C8A, L9A, L12A, T13A, S14A and P19A mutations nullify the neuroprotective function of HN [Yamagishi, Y., Hashimoto, Y., Niikura, T. & Nishimoto, I. (2003) Peptides, 24, 585,595]. Here we examined whether any of these ,null' mutants could function as dominant-negative mutants. Homodimerization-defective mutants, P3A-, L12A-, S14A- and P19A-HN, specifically blocked neuroprotection by HN, but not by activity-dependent neurotrophic factor. Furthermore, insertion of S7A, the mutation that blocks the homodimerization of HN, but not insertion of G5A abolished the antagonizing function of L12A-HN. While L12A-HN and G5A/L12A-HN actually inhibited HN homodimerization, S7A/L12A-HN had no effect. These data indicate that P3A-, L12A-, S14A- and P19A-HN function as HN antagonists by forming an inactive dimer with HN. This study provides a novel insight into the understanding of the in vivo function of HN, as well as into the development of clinically applicable HN neutralizers. [source] Proteolytic activation and function of the cytokine Spätzle in the innate immune response of a lepidopteran insect, Manduca sextaFEBS JOURNAL, Issue 1 2010Chunju An The innate immune response of insects includes induced expression of genes encoding a variety of antimicrobial peptides. The signaling pathways that stimulate this gene expression have been well characterized by genetic analysis in Drosophila melanogaster, but are not well understood in most other insect species. One such pathway involves proteolytic activation of a cytokine called Spätzle, which functions in dorsal,ventral patterning in early embryonic development and in the antimicrobial immune response in larvae and adults. We have investigated the function of Spätzle in a lepidopteran insect, Manduca sexta, in which hemolymph proteinases activated during immune responses have been characterized biochemically. Two cDNA isoforms for M. sexta Spätzle-1 differ because of alternative splicing, resulting in a 10 amino acid residue insertion in the pro-region of proSpätzle-1B that is not present in proSpätzle-1A. The proSpätzle-1A cDNA encodes a 32.7 kDa polypeptide that is 23% and 44% identical to D. melanogaster and Bombyx mori Spätzle-1, respectively. Recombinant proSpätzle-1A was a disulfide-linked homodimer. M. sexta hemolymph proteinase 8 cleaved proSpätzle-1A to release Spätzle-C108, a dimer of the C-terminal 108 residue cystine-knot domain. Injection of Spätzle-C108, but not proSpätzle-1A, into larvae stimulated expression of several antimicrobial peptides and proteins, including attacin-1, cecropin-6, moricin, lysozyme, and the immunoglobulin domain protein hemolin, but did not significantly affect the expression of two bacteria-inducible pattern recognition proteins, immulectin-2 and ,-1,3-glucan recognition protein-2. The results of this and other recent studies support a model for a pathway in which the clip-domain proteinase pro-hemolymph proteinase 6 becomes activated in plasma upon exposure to Gram-negative or Gram-positive bacteria or to ,-1,3-glucan. Hemolymph proteinase 6 then activates pro-hemolymph proteinase 8, which in turn activates Spätzle-1. The resulting Spätzle-C108 dimer is likely to function as a ligand to activate a Toll pathway in M. sexta as a response to a wide variety of microbial challenges, stimulating a broad response to infection. Structured digital abstract ,,MINT-7295125: Spätzle 1A (uniprotkb:C8BMD1) and Spätzle 1A (uniprotkb:C8BMD1) bind (MI:0407) by comigration in gel electrophoresis (MI:0807) [source] Physicochemical properties and distinct DNA binding capacity of the repressor of temperate Staphylococcus aureus phage ,11FEBS JOURNAL, Issue 7 2009Tridib Ganguly The repressor protein and cognate operator DNA of any temperate Staphylococcus aureus phage have not been investigated in depth, despite having the potential to enrich the molecular biology of the staphylococcal system. In the present study, using the extremely pure repressor of temperate Staphylococcus aureus phage ,11 (CI), we demonstrate that CI is composed of ,-helix and ,-sheet to a substantial extent at room temperature, possesses two domains, unfolds at temperatures above 39 °C and binds to two sites in the ,11 cI - cro intergenic region with variable affinity. The above CI binding sites harbor two homologous 15 bp inverted repeats (O1 and O2), which are spaced 18 bp apart. Several guanine bases located in and around O1 and O2 demonstrate interaction with CI, indicating that these 15 bp sites are used as operators for repressor binding. CI interacted with O1 and O2 in a cooperative manner and was found to bind to operator DNA as a homodimer. Interestingly, CI did not show appreciable binding to another homologous 15 bp site (O3) that was located in the same primary immunity region as O1 and O2. Taken together, these results suggest that ,11 CI and the ,11 CI,operator complex resemble significantly those of the lambdoid phages at the structural level. The mode of action of ,11 CI, however, may be distinct from that of the repressor proteins of , and related phages. [source] Structure,activity relationships of wheat flavone O -methyltransferase , a homodimer of convenienceFEBS JOURNAL, Issue 9 2008Jack A. Kornblatt Wheat flavone O -methyltransferase catalyzes three sequential methylations of the flavone tricetin. Like other flavonoid O -methyltransferases, the protein is a homodimer. We demonstrate, using analytical ultracentrifugation, that perchlorate dissociates the dimer into monomers. The resulting monomers retain all their catalytic capacity, including the ability to catalyze the three successive methylations. We show, using isothermal titration calorimetry, that the binding constant for S -adenosyl- l -methionine does not change significantly as the protein dissociates. The second substrate, tricetin, binds to the dimers but could not be tested with the monomers. CD, UV and fluorescence spectroscopy show that there are substantial changes in the structure of the protein as it dissociates. The fact that there are differences between the monomers and dimers even as the monomers maintain activity may be the result of the very low catalytic capacity of this enzyme. Maximal turnover numbers for the dimers and monomers are only about 6,7 per minute. Even though the binding pockets for S -adenosyl- l -methionine, tricetin, selgin and tricin are intact, selection of a catalytically competent structure may be a very slow step during catalysis. [source] Native and subunit molecular mass and quarternary structure of the hemoglobin from the primitive branchiopod crustacean Triops cancriformisFEBS JOURNAL, Issue 17 2006Morgane Rousselot Many branchiopod crustaceans are endowed with extracellular, high-molecular-weight hemoglobins whose exact structural characteristics have remained a matter of conjecture. By using a broad spectrum of techniques, we provide precise and coherent information on the hemoglobin of one of the phylogenetically ,oldest' extant branchiopods, the tadpole shrimp Triops cancriformis. The hemoglobin dissociated under reducing conditions into two subunits, designated TcHbA and TcHbB, with masses of 35 775 ± 4 and 36 055 ± 4 Da, respectively, determined by ESI-MS. Nonreducing conditions showed only two disulfide-bridged dimers, a homodimer of TcHbA, designated D1 (71 548 ± 5 Da), and the heterodimer D2 (71 828 ± 5 Da). Carbamidomethylation of free SH groups revealed the presence of three cysteines per subunit and indicated one intrasubunit and one intersubunit disulfide bridge. Ultracentrifugation and light-scattering experiments under nondenaturating conditions yielded mass estimates that suggested an uneven number of 17 subunits forming the native hemoglobin. This unrealistic number resulted from the presence of two size classes (16-mer and 18-mer), which were recognized by native PAGE and Ferguson plot analysis. ESI-MS revealed three hemoglobin isoforms with masses of 588.1 kDa, 662.0 kDa, and 665.0 kDa. The 16-mer and the smaller 18-mer species are supposed to be composed of TcHbA only, given the dominance of this subunit type in SDS/PAGE. Transmission electron microscopy of negatively stained specimens showed a population of compact molecules with geometrical extensions of 14, 16 and 9 nm. The proposed stoichiometric model of quarternary structure provides the missing link to achieve a mechanistic understanding of the structure,function relationships among the multimeric arthropodan hemoglobins. [source] Fission yeast decaprenyl diphosphate synthase consists of Dps1 and the newly characterized Dlp1 protein in a novel heterotetrameric structureFEBS JOURNAL, Issue 20 2003Ryoichi Saiki The analysis of the structure and function of long chain-producing polyprenyl diphosphate synthase, which synthesizes the side chain of ubiquinone, has largely focused on the prokaryotic enzymes, and little is known about the eukaryotic counterparts. Here we show that decaprenyl diphosphate synthase from Schizosaccharomyces pombe is comprised of a novel protein named Dlp1 acting in partnership with Dps1. Dps1 is highly homologous to other prenyl diphosphate synthases but Dlp1 shares only weak homology with Dps1. We showed that the two proteins must be present simultaneously in Escherichia coli transformants before ubiquinone-10, which is produced by S. pombe but not by E. coli, is generated. Furthermore, the two proteins were shown to form a heterotetrameric complex. This is unlike the prokaryotic counterparts, which are homodimers. The deletion mutant of dlp1 lacked the enzymatic activity of decaprenyl diphosphate synthase, did not produce ubiquinone-10 and had the typical ubiquinone-deficient S. pombe phenotypes, namely hypersensitivity to hydrogen peroxide, the need for antioxidants for growth on minimal medium and an elevated production of H2S. Both the dps1 (formerly dps) and dlp1 mutants could generate ubiquinone when they were transformed with a bacterial decaprenyl diphosphate synthase, which functions in its host as a homodimer. This indicates that both dps1 and dlp1 are required for the S. pombe enzymatic activity. Thus, decaprenyl diphosphate from a eukaryotic origin has a heterotetrameric structure that is not found in prokaryotes. [source] Characterization of a Saccharomyces cerevisiae NADP(H)-dependent alcohol dehydrogenase (ADHVII), a member of the cinnamyl alcohol dehydrogenase familyFEBS JOURNAL, Issue 22 2002Carol Larroy A new NADP(H)-dependent alcohol dehydrogenase (the YCR105W gene product, ADHVII) has been identified in Saccharomyces cerevisiae. The enzyme has been purified to homogeneity and found to be a homodimer of 40 kDa subunits and a pI of 6.2,6.4. ADHVII shows a broad substrate specificity similar to the recently characterized ADHVI (64% identity), although they show some differences in kinetic properties. ADHVI and ADHVII are the only members of the cinnamyl alcohol dehydrogenase family in yeast. Simultaneous deletion of ADH6 and ADH7 was not lethal for the yeast. Both enzymes could participate in the synthesis of fusel alcohols, ligninolysis and NADP(H) homeostasis. [source] Molecular modeling of the dimeric structure of human lipoprotein lipase and functional studies of the carboxyl-terminal domainFEBS JOURNAL, Issue 18 2002Yoko Kobayashi Lipoprotein lipase (LPL) plays a key role in lipid metabolism. Molecular modeling of dimeric LPL was carried out using insight ii based upon the crystal structures of human, porcine, and horse pancreatic lipase. The dimeric model reveals a saddle-shaped structure and the key heparin-binding residues in the amino-terminal domain located on the top of this saddle. The models of two dimeric conformations , a closed, inactive form and an open, active form , differ with respect to how surface-loop positions affect substrate access to the catalytic site. In the closed form, the surface loop covers the catalytic site, which becomes inaccessible to solvent. Large conformational changes in the open form, especially in the loop and carboxyl-terminal domain, allow substrate access to the active site. To dissect the structure,function relationships of the LPL carboxyl-terminal domain, several residues predicted by the model structure to be essential for the functions of heparin binding and substrate recognition were mutagenized. Arg405 plays an important role in heparin binding in the active dimer. Lys413/Lys414 or Lys414 regulates heparin affinity in both monomeric and dimeric forms. To evaluate the prediction that LPL forms a homodimer in a ,head-to-tail' orientation, two inactive LPL mutants , a catalytic site mutant (S132T) and a substrate-recognition mutant (W390A/W393A/W394A) , were cotransfected into COS7 cells. Lipase activity could be recovered only when heterodimerization occurred in a head-to-tail orientation. After cotransfection, 50% of the wild-type lipase activity was recovered, indicating that lipase activity is determined by the interaction between the catalytic site on one subunit and the substrate-recognition site on the other. [source] Critical role of C/EBP, and C/EBP, factors in the stimulation of the cyclooxygenase-2 gene transcription by interleukin-1, in articular chondrocytesFEBS JOURNAL, Issue 23 2000Béatrice Thomas The activity of the [,831; +103] promoter of the human cyclooxygenase-2 gene in cultured rabbit chondrocytes is stimulated 2.9 ± 0.3-fold by interleukin-1, and this stimulation depends on [,132; ,124] C/EBP binding-and [,223; ,214] NF-,B binding-sites. The C/EBP, and C/EBP, factors bind to the [,132; ,124] sequence. The [,61; ,53] sequence is also recognized by C/EBP, and C/EBP, as well as USF. Mutation of the whole [,61; ,53] sequence abolished the stimulation of transcription but single mutations of the C/EBP or USF site did not alter the activity of the promoter, suggesting that the factors bound to the proximal [,61; ,53] sequence interact with different members of the general transcription machinery. The [,223; ,214] site binds only the p50/p50 homodimer and a non-rel-related protein, but not the transcriptionally active heterodimer p50/p65. The p50/p50 homodimer could interact with the C/EBP family members bound to the [,132; ,124] sequence for full stimulation of the COX-2 transcription by interleukin-1, in chondrocytes. By contrast, the [,448; ,449] sequence binds with a low affinity both the p50/p50 homodimeric and p50/p65 heterodimeric forms of NF-,B but has no role in the regulation of the human COX-2 promoter in chondrocytes. [source] Characterization of a nif-regulated flavoprotein (FprA) from Rhodobacter capsulatusFEBS JOURNAL, Issue 3 20002S] ferredoxin, Redox properties, molecular interaction with a [2Fe A flavoprotein from Rhodobacter capsulatus was purified as a recombinant (His)6 -tag fusion from an Escherichia coli clone over-expressing the fprA structural gene. The FprA protein is a homodimer containing one molecule of FMN per 48-kDa monomer. Reduction of the flavoprotein by dithionite showed biphasic kinetics, starting with a fast step of semiquinone (SQ) formation, and followed by a slow reduction of the SQ. This SQ was in the anionic form as shown by EPR and optical spectroscopies. Spectrophotometric titration gave a midpoint redox potential for the oxidized/SQ couple of Em1 = +20 mV (pH 8.0), whereas the SQ/hydroquinone couple could not be titrated due to the thermodynamic instability of SQ associated with its slow reduction process. The inability to detect the intermediate form, SQ, upon oxidative titration confirmed this instability and led to an estimate of Em2 , Em1 of > 80 mV. The reduction of SQ by dithionite was significantly accelerated when the [2Fe,2S] ferredoxin FdIV was used as redox mediator. The midpoint redox potential of this ferredoxin was determined to be ,275 ± 2 mV at pH 7.5, consistent with FdIV serving as electron donor to FprA in vivo. FdIV and FprA were found to cross-react when incubated together with the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, giving a covalent complex with an Mr of , 60 000. Formation of this complex was unaffected by the redox states of the two proteins. Other [2Fe,2S] ferredoxins, including FdV and FdVI from R. capsulatus, were ineffective as electron carriers to FprA, and cross-reacted poorly with the flavoprotein. The possible function of FprA with regard to nitrogen fixation was investigated using an fprA -deleted mutant. Although nitrogenase activity was significantly reduced in the mutant compared with the wild-type strain, nitrogen fixation was apparently unaffected by the fprA deletion even under iron limitation or microaerobic conditions. [source] Glucose-6-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima: expression of the g6pd gene and characterization of an extremely thermophilic enzymeFEMS MICROBIOLOGY LETTERS, Issue 2 2002Thomas Hansen Abstract The gene (open reading frame Tm1155, g6pd) encoding glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49) of the hyperthermophilic bacterium Thermotoga maritima was cloned and functionally expressed in Escherichia coli. The purified recombinant enzyme is a homodimer with an apparent molecular mass of 95 kDa composed of 60-kDa subunits. Rate dependence (at 80°C) on glucose-6-phosphate and NADP+ followed Michaelis,Menten kinetics with apparent Km values of 0.15 mM and 0.03 mM, respectively; apparent Vmax values were about 20 U mg,1. The enzyme also reduced NAD+ (apparent Km 12 mM, Vmax 12 U mg,1). The 1000-fold higher catalytic activity (kcat/Km) with NADP+ over NAD+ defines the G6PD as NADP+ specific in vivo. G6PD activity was competitively inhibited by NADPH with a Ki value of 0.11 mM. With a temperature optimum of 92°C the enzyme is the most thermoactive G6PD described. [source] Functionally important structural elements of the cyanobacterial clock-related protein PexGENES TO CELLS, Issue 1 2009Shunsuke Kurosawa Pex, a clock-related protein involved in the input pathway of the cyanobacterial circadian clock system, suppresses the expression of clock gene kaiA and lengthens the circadian period. Here, we determined the crystal structure of Anabaena Pex (AnaPex; Anabaena sp. strain PCC 7120) and Synechococcus Pex (SynPex; Synechococcus sp. strain PCC 7942). Pex is a homodimer that forms a winged-helix structure. Using the DNase I protection and electrophoresis mobility shift assays on a Synechococcus kaiA upstream region, we identified a minimal 25-bp sequence that contained an imperfectly inverted repeat sequence as the Pex-binding sequence. Based on crystal structure, we predicted the amino acid residues essential for Pex's DNA-binding activity and examined the effects of various Ala-substitutions in the ,3 helix and wing region of Pex on in vitro DNA-binding activity and in vivo rhythm functions. Mutant AnaPex proteins carrying a substitution in the wing region displayed no specific DNA-binding activity, whereas those carrying a substitution in the ,3 helix did display specific binding activity. But the latter were less thermostable than wild-type AnaPex and their in vitro functions were defective. We concluded that Pex binds a kaiA upstream DNA sequence via its wing region and that its ,3 helix is probably important to its stability. [source] Actin filaments-stabilizing and -bundling activities of cofilin-phosphatase Slingshot-1GENES TO CELLS, Issue 5 2007Souichi Kurita Slingshot-1 (SSH1) is known to regulate actin filament dynamics by dephosphorylating and activating cofilin, an actin-depolymerizing factor. SSH1 binds to filamentous (F-) actin through its multiple F-actin-binding sites and its cofilin-phosphatase activity is enhanced by binding to F-actin. In this study, we demonstrate that SSH1 has F-actin-stabilizing and -bundling activities. In vitro actin depolymerization assays revealed that SSH1 suppressed spontaneous and cofilin-induced actin depolymerization in a dose-dependent manner. SSH1 inhibited F-actin binding and severing activities of cofilin. Low-speed centrifugation assays combined with fluorescence and electron microscopic analysis revealed that SSH1 has F-actin-bundling activity, independently of its cofilin-phosphatase activity. Deletion of N- or C-terminal regions of SSH1 significantly reduced its F-actin-stabilizing and -bundling activities, indicating that both regions are critical for these functions. As SSH1 does not form a homodimer, it probably bundles F-actin through its multiple F-actin-binding sites. Knockdown of SSH1 expression by RNA interference significantly suppressed stress fiber formation in C2C12 myoblast cells, indicating a role for SSH1 in stress fiber formation or stabilization in cells. SSH1 thus has the potential to regulate actin filament dynamics and organization in cells via F-actin-stabilizing and -bundling activities, in addition to its ability to dephosphorylate cofilin. [source] Novel heat shock protein HspQ stimulates the degradation of mutant DnaA protein in Escherichia coliGENES TO CELLS, Issue 12 2004Toh-ru Shimuta Escherichia coli DnaA protein initiates chromosomal replication and is an important regulatory target during the replication cycle. In this study, a suppressor mutation isolated by transposon mutagenesis was found to allow growth of the temperature-sensitive dnaA508 and dnaA167 mutants at 40 °C. The suppressor consists of a transposon insertion in a previously annotated ORF, here termed hspQ, a novel heat shock gene whose promoter is recognized by the major heat shock sigma factor ,32. Expression of hspQ on a pBR322 derivative inhibits growth of the dnaA508 and dnaA167 mutants at 30 °C, whereas growth of dnaA46 and other dnaA mutants is insensitive to changes in the level of hspQ. Cellular DnaA508 protein is degraded rapidly at elevated temperature, but hspQ disruption impedes this process. In contrast, DnaA46 protein is rapidly degraded in an hspQ -independent manner. Gel-filtration and chemical cross-linking experiments suggest that HspQ forms a stable homodimer in solution and can form homomultimers consisting of about four monomers. Heat-shock induced proteases such as Clp contain homomultimers of subunit proteins. We propose that HspQ is a new factor involved in the quality control of proteins and that it functions by excluding denatured proteins. [source] An architectural perspective on signaling by the pre-, ,, and ,, T cell receptorsIMMUNOLOGICAL REVIEWS, Issue 1 2003Sandra M. Hayes Summary: The T cell antigen receptor (TCR) is a multimeric complex composed of an antigen-binding clonotypic heterodimer and a signal transducing complex consisting of the CD3 dimers (CD3,, and CD3,,) and a TCR-, homodimer. In all jawed vertebrates there are two T cell lineages, ,, and ,,, distinguished by the clonotypic subunits contained within their TCRs (TCR-, and -, or TCR-, and -,, respectively). A third receptor complex, the preTCR, is only expressed on immature T cells. The preTCR, which contains the invariant pre-T, (pT,) chain in lieu of TCR-,, plays a critical role in the early development of ,, lineage cells. The subunit composition of the signal transducing complexes of the pre-, ,,- and ,,TCRs was previously thought to be identical. However, recent data demonstrate that there are significant differences in the signal transducing complexes of these three TCRs. For example, ,,TCRs contain both CD3,, and CD3,, dimers, whereas ,,TCRs contain only CD3,, dimers. Moreover, preTCR function appears to be unaffected in the absence of CD3,, suggesting that CD3,, dimers are dispensable for pre-TCR assembly. In this review, we summarize current data relating to the subunit composition of the pre-, ,,- and ,,TCRs and discuss how these structural differences may impact receptor signaling and ,,/,, lineage determination. [source] Expression of CD8, identifies a distinct subset of effector memory CD4+ T lymphocytesIMMUNOLOGY, Issue 2 2006Iole Macchia Summary Circulating CD4+ CD8+ T lymphocytes have been described in the peripheral blood of humans and several animal species. However, the origin and functional properties of these cells remain poorly understood. In the present study, we evaluated the frequency, phenotype and function of peripheral CD4+ CD8+ T cells in rhesus macaques. Two distinct populations of CD4+ CD8+ T cells were identified: the dominant one was CD4hi CD8lo and expressed the CD8,, homodimer, while the minor population was CD4lo CD8hi and expressed the CD8,, heterodimer. The majority of CD4hi CD8,lo T cells exhibited an activated effector/memory phenotype (CCR5lo CD7, CD28, HLA-DR+) and expressed relatively high levels of granzyme B. Intracellular cytokine staining assays demonstrated that the frequency of cytomegalovirus-specific T cells was enriched five-fold in CD4hi CD8,lo T cells compared to single-positive CD4+ T cells, whereas no consistent enrichment was observed for simian immunodeficiency virus (SIV)-specific T cells. Cross-sectional studies of SIV-infected animals demonstrated that the frequency of CD4hi CD8,lo T cells was lower in wild-type SIV-infected animals compared to uninfected controls, although prospective studies of SIV-infected animals demonstrated depletion of CD4hi CD8,lo lymphocytes only in a subset of animals. Taken together, these data suggest that CD4+ T cells expressing CD8, represent an effector/memory subset of CD4+ T cells and that this cell population can be depleted during the course of SIV infection. [source] Cloning, expression and localization of a trypsin-like serine protease in the spruce budworm, Choristoneura fumiferanaINSECT SCIENCE, Issue 6 2009Wen-Ying He Abstract, A trypsin-like molting-related serine protease cDNA (CfMRSP) was cloned from the spruce budworm, Choristoneura fumiferana. The full-length CfMRSP complementary DNA (cDNA) encoded a 43 kDa protein that contained a trypsin-like serine protease catalytic domain, but no clip domain. The C-terminal extension contained five cystein residues, which may allow the protein to form a homodimer through interchain disulfide bonds and regulate the activity of CfMRSP. Phylogenetic tree analysis showed that CfMRSP clusters with lepidopteran homologues such as serine protease 1 of Lonomia obliqua, hemolymph proteinase 20 (HP20), pattern recognition serine proteinase precursor (ProHP14) and a trypsin-like protein of Manduca sexta. Northern blot analysis of developmental expression of CfMRSP indicated that its transcripts were found primarily in the epidermis and were produced during all of the tested stadia, from 4th instar larvae to pupae, but increased levels of CfMRSP transcripts were always found after each molt. A high level of the protein was found in the epidermis by immunohistochemistry analysis. Altogether these data suggest that CfMRSP plays a role in the epidermis during molting and metamorphosis. [source] A Mu-class glutathione S -transferase from gills of the marine shrimp Litopenaeus vannamei: Purification and characterizationJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 2 2007Carmen A. Contreras-Vergara Abstract Glutathione S -transferases (GSTs) are a family of detoxifying enzymes that catalyze the conjugation of glutathione (GSH) to electrophiles, thereby increasing the solubility of GSH and aiding its excretion from the cell. In this study, a glutatione S -transferase from the gills of the marine shrimp Litopenaeus vannamei was purified by affinity chromatography using a glutathione,agarose affinity column. GST was purified to homogeneity as judged by reducing SDS-PAGE and zymograms. This enzyme is a homodimer composed of ,25-kDa subunits and identified as a Mu-class GST based on its activity against 1-chloro-2,4-dinitrobenzene (CDNB) and internal peptide sequence. The specific activity of purified GST was 440.12 ,mol/(min mg), and the Km values for CDNB and GSH are very similar (390 and 335 ,M, respectively). The intersecting pattern of the initial velocities of this enzyme in the Lineweaver,Burke plot is consistent with a sequential steady-state kinetic mechanism. The high specific activity of shrimp GST may be related to a highly effective detoxification mechanism necessary in gills since they are exposed to the external and frequently contaminated environment. © 2007 Wiley Periodicals, Inc. J Biochem Mol Toxicol 21:62,67, 2007; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20162 [source] Diverse effects of Stat1 on the regulation of hsp90, gene under heat shock,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 4 2007Xue-song Chen Abstract Stat1 has been known as a regulator of gene expression and a mediator of IFN, signaling in mammalian cells, while its effect in a heat shock response remains unclear. We used RNAi knockdown, point mutations, ChIP and promoter activity assays to study the effect of Stat1 on the heat-shock induction of the hsp90, gene under heat shock conditions. We found that Stat1 regulates the heat shock induction of its target genes, the hsp90, gene in a heat shock response while the constitutive activity of the gene remains unaffected. The result of Stat1 in complex with Stat3 and HSF1 that bound at the GAS to lead a moderate heat shock induction was designated as an "intrinsic" induction of the hsp90, gene. Additionally a reduced or an elevated level of heat shock induction was also controlled by the Stat1 on hsp90,. These diverse effects on the hsp90, gene were a "reduced" induction with over-expressed Stat1 elicited by transfection of wild-type Stat1 or IFN, treatment, bound at the GAS as homodimer; and an "enhanced" heat shock induction with a mutation-mediated prohibition of Stat1/GAS binding. In conclusion, the status and efficacy of Stat1 bound at the GAS of its target gene are pivotal in determining the impact of Stat1 under heat shock. The results provided the first evidence on the tumor suppressor Stat1 that it could play diverse roles on its target genes under heat shock that also shed lights on patients with fever or under thermotherapy. J. Cell. Biochem. 102: 1059,1066, 2007. © 2007 Wiley-Liss, Inc. [source] Withania somnifera (Ashwagandha): a Novel Source of L-asparaginaseJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 2 2009Vishal P. Oza Abstract Different parts of plant species belonging to Solanaceae and Fabaceae families were screened for L-asparaginase enzyme (E.C.3.5.1.1.). Among 34 plant species screened for L-asparaginase enzyme, Withania somnifera L. was identified as a potential source of the enzyme on the basis of high specific activity of the enzyme. The enzyme was purified and characterized from W. somnifera, a popular medicinal plant in South East Asia and Southern Europe. Purification was carried out by a combination of protein precipitation with ammonium sulfate as well as Sephadex-gel filtration. The purified enzyme is a homodimer, with a molecular mass of 72 ± 0.5 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresisand size exclusion chromatography. The enzyme has a pH optimum of 8.5 and an optimum temperature of 37 °C. The Km value for the enzyme is 6.1 × 10,2 mmol/L. This is the first report for L-asparaginase from W. somnifera, a traditionally used Indian medicinal plant. [source] Interleukin-12 P40 induces the expression of TNF-, in microglia and macrophagesJOURNAL OF NEUROCHEMISTRY, Issue 2002M. Jana Recently, it has been found that overproduction of IL-12 can be dangerous to the host as it is involved in the pathogenesis of a number of autoimmune inflammatory diseases such as multiple sclerosis. It is composed of two different subunits , p40 and p35. Expression of p40 mRNA but not that of p35 mRNA in excessive amount in the CNS of patients with Multiple Sclerosis (MS) suggests that IL-12 p40 may have a role in the pathogenesis of the disease. The present study was undertaken to explore the role of p40 in the expression of TNF-, in microglia. Interestingly, we have found that IL-12 p70, p402 (the p40 homodimer) and p40 (the p40 monomer) dose-dependently induced the production of TNF-, in BV-2 microglial cells. This induction of TNF-, production was accompanied by an induction of TNF-, mRNA. In addition to BV-2 glial cells, p70, p402 and p40 also induced the production of TNF-, in mouse primary microglia and peritoneal macrophages. Since the activation of both NF-,B and C/EBPb is important for the expression of TNF-, in microglial cells, we investigated the effect of p40 on the activation of NF-,B as well as C/EBPb. Activation of NF-,B as well as C/EBPb by p40 and inhibition of p40-induced expression of TNF-, by Dp65, a dominant-negative mutant of p65, and DC/EBPb, a dominant-negative mutant of C/EBPb, suggests that p40 induces the expression of TNF-, through the activation of NF-,B and C/EBPb. This study delineates a novel role of IL-12 p40 in inducing the expression of TNF-, in microglial cells which may participate in the pathogenesis of neuroinflammatory diseases. Acknowledgements:, This study was supported by NIH grants (NS39940 and AG19487). [source] TrkB dimerization during development of the prefrontal cortex of the macaqueJOURNAL OF NEUROSCIENCE RESEARCH, Issue 5 2001Koji Ohira Abstract To date, two subtypes of TrkB, a BDNF receptor, have been described. One is full-length TrkB (TK+), which has a tyrosine kinase-containing intracellular domain. The other is truncated TrkB (TK,), which has a short intracellular domain lacking the tyrosine kinase. In this study, we investigated the dimerization of TrkB subtypes in the developing monkey prefrontal cortex by means of cross-linking. At embryonic day 120, the TK+/TK+ and the 100 kDa/100 kDa homodimers were observed with BDNF stimulation. At the newborn stage, the TK+/TK+ and the TK,/TK, homodimers were observed with BDNF stimulation. At the adult stage, the TK,/TK, homodimer and the TK+/TK, heterodimer were formed by BDNF stimulation. The levels of all dimers increased in proportion to the concentration of BDNF. Moreover, the dimers were clearly formed within 5 min of treatment with BDNF. BDNF and NT-4/5 induced the dimers, whereas NT-3 formed slight dimers but NGF did not. Furthermore, anti-BDNF antibody inhibited the TrkB dimerization. Moreover, the intercellular binding proteins of TrkB were not cross-linked by BS3. Therefore, these results suggest that the change in dimerization among TrkB subtypes occurs during development of the monkey prefrontal cortex. J. Neurosci. Res. 65:463,469, 2001. © 2001 Wiley-Liss, Inc. [source] Characterisation of a haemagglutinin from Hokkaido red bean (Phaseolus vulgaris cv. Hokkaido red bean)JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 1 2010Jack H Wong Abstract BACKGROUND: A haemagglutinin was purified from Japanese Hokkaido red beans (Phaseolus vulgaris cv. Hokkaido red bean) with a procedure that included three chromatographic media. RESULTS: Haemagglutinating activity was adsorbed on DEAE cellulose, Affi-gel blue gel and Mono S. The pure haemagglutinin was a homodimer and each subunit was around 30 kDa in molecular mass. The haemagglutinating activity of this agglutinin could not be inhibited by a variety of simple sugars at 200 mmol L,1 concentration including ,- L -fucose, D(+)-galactose, D(+)-glucose, D(+)-glucosamine, D(,)galactosamine, galacturonic acid, (+)-lactose, D(+)-melibose, L(,)-mannose, D(+)-mannose, D -mannosamine, D(+)-raffinose, L -rhamnose, (+)-xylose and galacturonic acid. The haemagglutinating activity was fully retained at pH 4,11 and at 0,80 °C, but was completely lost at extreme pH values (0,2 and 13,14) and at very high temperatures (90 °C and 100 °C). The haemagglutinin exhibited a weak mitogenic activity toward mouse splenocytes, a stronger anti-proliferative activity than Con A toward HepG2 (human hepatoma) cells and inhibited >80% of HIV-1 reverse transcriptase inhibitory activity at 3.3 µmol L,1. It was devoid of anti-fungal activity. CONCLUSION: Hokkaido red bean haemagglutinin possesses a potent anti-proliferative effect on HepG2 cells. Copyright © 2009 Society of Chemical Industry [source] Structural and functional features of factor XIJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 2009D. GAILANI Summary., Factor XI (FXI) has structural and mechanistic features that distinguish it from other coagulation proteases. A relatively recent addition to vertebrate plasma coagulation, FXI is a homodimer, with each subunit containing four apple domains and a protease domain. The apple domains form a disk structure with binding sites for platelets, high molecular weight kininogen, and the substrate factor IX (FIX). FXI is converted to the active protease FXIa by cleavage of the Arg369,Ile370 bond on each subunit. This converts the catalytic domains to the active forms, and unmasks exosites on the apple domains required for FIX binding. FXI activation by factor XIIa or thrombin proceeds through an intermediate with only one activated submit (1/2-FXIa). 1/2-FXIa activates FIX in a similar manner to FXIa. While the importance of the homodimeric structure of FXI is not certain, it may represent a strategy for binding to FIX and a platelet surface simultaneously. [source] Mutations in the signature motif in MutS affect ATP-induced clamp formation and mismatch repairMOLECULAR MICROBIOLOGY, Issue 6 2008Samir Acharya Summary MutS protein dimer recognizes and co-ordinates repair of DNA mismatches. Mismatch recognition by the N-terminal mismatch recognition domain and subsequent downstream signalling by MutS appear coupled to the C-terminal ATP catalytic site, Walker box, through nucleotide-mediated conformational transitions. Details of this co-ordination are not understood. The focus of this study is a conserved loop in Escherichia coli MutS that is predicted to mediate cross-talk between the two ATP catalytic sites in MutS homodimer. Mutagenesis was employed to assess the role of this loop in regulating MutS function. All mutants displayed mismatch repair defects in vivo. Biochemical characterization further revealed defects in ATP binding, ATP hydrolysis as well as effective mismatch recognition. The kinetics of initial burst of ATP hydrolysis was similar to wild type but the magnitude of the burst was reduced for the mutants. Given its proximity to the ATP bound in the opposing monomer in the crystal and its potential analogy with signature motif of ABC transporters, the results strongly suggest that the loop co-ordinates ATP binding/hydrolysis in trans by the two catalytic sites. Importantly, our data reveal that the loop plays a direct role in co-ordinating conformational changes involved in long-range communication between Walker box and mismatch recognition domains. [source] Reciprocal regulation of the mouse protamine genes by the orphan nuclear receptor germ cell nuclear factor and CREM,MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2004Geoffrey C. Hummelke Abstract Germ cell nuclear factor (GCNF) is a member of the nuclear receptor superfamily, which is expressed in the adult predominantly in the male and female germ cells. In the male, GCNF is expressed in spermatogenic cells. GCNF binds as a homodimer to direct repeat response elements of the consensus half-site sequence, AGGTCA, with 0 bp spacing (DR0). Using this information, a search of genomic databases was performed to identify candidate GCNF responsive, spermatogenic-specific, genes that contain DR0 sequences. The mouse protamine genes are the strongest candidates identified to date, as they are post-meiotically expressed in round spermatids and contain DR0 elements in their proximal promoters. Previous work has shown that both recombinant and endogenous GCNF bind to DR0 elements in the mouse protamine 1 and 2 (Prm 1 and Prm 2) promoters with high affinity and specificity. The present work shows that in transient transfection assays in GC-1 and JEG-3 cells, co-transfection of a GCNF-VP16 expression plasmid with reporter plasmids containing either the wild type Prm 1 or Prm 2 promoter established that GCNF-VP16 is able to regulate transcription from both promoters in a DR0-dependent manner. Wild type GCNF, in contrast, acts as a repressor of basal transcription on both the Prm 1 and Prm 2 promoters in a DR0-dependent manner. Furthermore, CREM, activation of these promoters is also repressed by wild-type GCNF, indicating that GCNF also acts as a repressor of activated transcription. GCNF therefore defines a novel nuclear receptor-signaling pathway that may regulate a subset of genes involved in the terminal differentiation process of spermatogenesis, exemplified by the protamines. Mol. Reprod. Dev. 68: 394,407, 2004. © 2004 Wiley-Liss, Inc. [source] Molecular mechanism of monocyte predominant infiltration in chronic inflammation: Mediation by a novel monocyte chemotactic factor, S19 ribosomal protein dimerPATHOLOGY INTERNATIONAL, Issue 11 2000Tetsuro Yamamoto A novel monocyte chemotactic factor, a cross-linked homodimer of S19 ribosomal protein (RP S19) was initially isolated from a rheumatoid arthritis synovial lesion. The RP S19 dimer causes the monocyte specific chemotaxis in vitro and the monocyte predominant infiltration in vivo, via its agonistic and antagonistic effects on the C5a receptors of monocytes and polymorphonuclear leukocytes, respectively. The agonistic effect is attributed to the similarity of regional structures between RP S19 and C5a, the complement C5-derived leukocyte chemotactic factor, although overall homology of the amino acid sequence between these molecules is only 4%. The antagonistic effect depends upon the C-terminal portion of RP S19. The RP S19 dimer is produced and released by apoptotic cells, and this dimer recruits monocytes from the circulation to the apoptotic lesion. The infiltrated monocytes/macrophages engulf the apoptotic cells, translocate to regional lymph nodes via lymphatics and present the antigenic information of the apoptotic cells to the T cell repertoire. In this manner, the apoptotic cell clearance system connects to the acquired immune system. The innate and acquired immune mechanisms, mediated by the RP S19 dimer, participate in the pathology of inveterate chronic inflammation such as rheumatoid arthritis. [source] Signal Transfer in Haloarchaeal Sensory Rhodopsin, Transducer Complexes,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2008Jun Sasaki Membrane-inserted complexes consisting of two photochemically reactive sensory rhodopsin (SR) subunits flanking a homodimer of a transducing protein subunit (Htr) are used by halophilic archaea for sensing light gradients to modulate their swimming behavior (phototaxis). The SR,Htr complexes extend into the cytoplasm where the Htr subunits bind a his-kinase that controls a phosphorylation system that regulates the flagellar motors. This review focuses on current progress primarily on the mechanism of signal relay within the SRII,HtrII complexes from Natronomonas pharaonis and Halobacterium salinarum. The recent elucidation of a photoactive site steric trigger crucial for signal relay, advances in understanding the role of proton transfer from the chromophore to the protein in SRII activation, and the localization of signal relay to the membrane-embedded portion of the SRII,HtrII interface, are beginning to produce a clear picture of the signal transfer process. The SR,Htr complexes offer unprecedented opportunities to resolve first examples of the chemistry of signal relay between membrane proteins at the atomic level, which would provide a major contribution to the general understanding of dynamic interactions between integral membrane proteins. [source] | ||||||||||||||||