SMN is essential for the biogenesis of U7 snRNP and 3′-end formation of histone mRNAs

PubMed Central

Tisdale, Sarah; Lotti, Francesco; Saieva, Luciano; Van Meerbeke, James P.; Crawford, Thomas O.; Sumner, Charlotte J.; Mentis, George Z.; Pellizzoni, Livio

2013-01-01

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by a deficiency in the survival motor neuron (SMN) protein. SMN mediates the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs) and possibly other RNPs. Here we investigated SMN requirement for the biogenesis and function of U7—an snRNP specialized in the 3′-end formation of replication-dependent histone mRNAs that normally are not polyadenylated. We show that SMN deficiency impairs U7 snRNP assembly and decreases U7 levels in mammalian cells. The SMN-dependent U7 reduction affects endonucleolytic cleavage of histone mRNAs leading to abnormal accumulation of 3′-extended and polyadenylated transcripts, followed by downstream changes in histone gene expression. Importantly, SMN deficiency induces defects of histone mRNA 3′-end formation in both SMA mice and human patients. These findings demonstrate that SMN is essential for U7 biogenesis and histone mRNA processing in vivo, and identify a novel RNA pathway disrupted in SMA. PMID:24332368

Evidence that C9ORF72 Dipeptide Repeat Proteins Associate with U2 snRNP to Cause Mis-splicing in ALS/FTD Patients.

PubMed

Yin, Shanye; Lopez-Gonzalez, Rodrigo; Kunz, Ryan C; Gangopadhyay, Jaya; Borufka, Carl; Gygi, Steven P; Gao, Fen-Biao; Reed, Robin

2017-06-13

Hexanucleotide repeat expansion in the C9ORF72 gene results in production of dipeptide repeat (DPR) proteins that may disrupt pre-mRNA splicing in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients. At present, the mechanisms underlying this mis-splicing are not understood. Here, we show that addition of proline-arginine (PR) and glycine-arginine (GR) toxic DPR peptides to nuclear extracts blocks spliceosome assembly and splicing, but not other types of RNA processing. Proteomic and biochemical analyses identified the U2 small nuclear ribonucleoprotein particle (snRNP) as a major interactor of PR and GR peptides. In addition, U2 snRNP, but not other splicing factors, mislocalizes from the nucleus to the cytoplasm both in C9ORF72 patient induced pluripotent stem cell (iPSC)-derived motor neurons and in HeLa cells treated with the toxic peptides. Bioinformatic studies support a specific role for U2-snRNP-dependent mis-splicing in C9ORF72 patient brains. Together, our data indicate that DPR-mediated dysfunction of U2 snRNP could account for as much as ∼44% of the mis-spliced cassette exons in C9ORF72 patient brains. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Two Routes to Genetic Suppression of RNA Trimethylguanosine Cap Deficiency via C-Terminal Truncation of U1 snRNP Subunit Snp1 or Overexpression of RNA Polymerase Subunit Rpo26.

PubMed

Qiu, Zhicheng R; Schwer, Beate; Shuman, Stewart

2015-04-24

The trimethylguanosine (TMG) caps of small nuclear (sn) RNAs are synthesized by the enzyme Tgs1 via sequential methyl additions to the N2 atom of the m(7)G cap. Whereas TMG caps are inessential for Saccharomyces cerevisiae vegetative growth at 25° to 37°, tgs1∆ cells that lack TMG caps fail to thrive at 18°. The cold-sensitive defect correlates with ectopic stoichiometric association of nuclear cap-binding complex (CBC) with the residual m(7)G cap of the U1 snRNA and is suppressed fully by Cbc2 mutations that weaken cap binding. Here, we show that normal growth of tgs1∆ cells at 18° is also restored by a C-terminal deletion of 77 amino acids from the Snp1 subunit of yeast U1 snRNP. These results underscore the U1 snRNP as a focal point for TMG cap function in vivo. Casting a broader net, we conducted a dosage suppressor screen for genes that allowed survival of tgs1∆ cells at 18°. We thereby recovered RPO26 (encoding a shared subunit of all three nuclear RNA polymerases) and RPO31 (encoding the largest subunit of RNA polymerase III) as moderate and weak suppressors of tgs1∆ cold sensitivity, respectively. A structure-guided mutagenesis of Rpo26, using rpo26∆ complementation and tgs1∆ suppression as activity readouts, defined Rpo26-(78-155) as a minimized functional domain. Alanine scanning identified Glu89, Glu124, Arg135, and Arg136 as essential for rpo26∆ complementation. The E124A and R135A alleles retained tgs1∆ suppressor activity, thereby establishing a separation-of-function. These results illuminate the structure activity profile of an essential RNA polymerase component. Copyright © 2015 Qiu et al.

Differential immunoglobulin class-mediated responses to components of the U1 small nuclear ribonucleoprotein particle in systemic lupus erythematosus and mixed connective tissue disease.

PubMed

Mesa, A; Somarelli, J A; Wu, W; Martinez, L; Blom, M B; Greidinger, E L; Herrera, R J

2013-11-01

The objective of this paper is to determine whether patients with systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) possess differential IgM- and IgG-specific reactivity against peptides from the U1 small nuclear ribonucleoprotein particle (U1 snRNP). The IgM- and IgG-mediated responses against 15 peptides from subunits of the U1 snRNP were assessed by indirect enzyme linked immunosorbent assays (ELISAs) in sera from patients with SLE and MCTD and healthy individuals (n = 81, 41, and 31, respectively). Additionally, 42 laboratory tests and 40 clinical symptoms were evaluated to uncover potential differences. Binomial logistic regression analyses (BLR) were performed to construct models to support the independent nature of SLE and MCTD. Receiver operating characteristic (ROC) curves corroborated the classification power of the models. We analyzed IgM and IgG anti-U1 snRNP titers to classify SLE and MCTD patients. IgG anti-U1 snRNP reactivity segregates SLE and MCTD from nondisease controls with an accuracy of 94.1% while IgM-specific anti-U1 snRNP responses distinguish SLE from MCTD patients with an accuracy of 71.3%. Comparison of the IgG and IgM anti-U1 snRNP approach with clinical tests used for diagnosing SLE and MCTD revealed that our method is the best classification tool of those analyzed (p ≤ 0.0001). Our IgM anti-U1 snRNP system along with lab tests and symptoms provide additional molecular and clinical evidence to support the hypothesis that SLE and MCTD may be distinct syndromes.

U1 small nuclear RNA variants differentially form ribonucleoprotein particles in vitro.

PubMed

Somarelli, Jason A; Mesa, Annia; Rodriguez, Carol E; Sharma, Shalini; Herrera, Rene J

2014-04-25

The U1 small nuclear (sn)RNA participates in splicing of pre-mRNAs by recognizing and binding to 5' splice sites at exon/intron boundaries. U1 snRNAs associate with 5' splice sites in the form of ribonucleoprotein particles (snRNPs) that are comprised of the U1 snRNA and 10 core components, including U1A, U1-70K, U1C and the 'Smith antigen', or Sm, heptamer. The U1 snRNA is highly conserved across a wide range of taxa; however, a number of reports have identified the presence of expressed U1-like snRNAs in multiple species, including humans. While numerous U1-like molecules have been shown to be expressed, it is unclear whether these variant snRNAs have the capacity to form snRNPs and participate in splicing. The purpose of the present study was to further characterize biochemically the ability of previously identified human U1-like variants to form snRNPs and bind to U1 snRNP proteins. A bioinformatics analysis provided support for the existence of multiple expressed variants. In vitro gel shift assays, competition assays, and immunoprecipitations (IPs) revealed that the variants formed high molecular weight assemblies to varying degrees and associated with core U1 snRNP proteins to a lesser extent than the canonical U1 snRNA. Together, these data suggest that the human U1 snRNA variants analyzed here are unable to efficiently bind U1 snRNP proteins. The current work provides additional biochemical insights into the ability of the variants to assemble into snRNPs. Copyright © 2014 Elsevier B.V. All rights reserved.

U1snRNP-mediated suppression of polyadenylation in conjunction with the RNA structure controls poly (A) site selection in foamy viruses

PubMed Central

2013-01-01

Background During reverse transcription, retroviruses duplicate the long terminal repeats (LTRs). These identical LTRs carry both promoter regions and functional polyadenylation sites. To express full-length transcripts, retroviruses have to suppress polyadenylation in the 5′LTR and activate polyadenylation in the 3′LTR. Foamy viruses have a unique LTR structure with respect to the location of the major splice donor (MSD), which is located upstream of the polyadenylation signal. Results Here, we describe the mechanisms of foamy viruses regulating polyadenylation. We show that binding of the U1 small nuclear ribonucleoprotein (U1snRNP) to the MSD suppresses polyadenylation at the 5′LTR. In contrast, polyadenylation at the 3′LTR is achieved by adoption of a different RNA structure at the MSD region, which blocks U1snRNP binding and furthers RNA cleavage and subsequent polyadenylation. Conclusion Recently, it was shown that U1snRNP is able to suppress the usage of intronic cryptic polyadenylation sites in the cellular genome. Foamy viruses take advantage of this surveillance mechanism to suppress premature polyadenylation at the 5’end of their RNA. At the 3’end, Foamy viruses use a secondary structure to presumably block access of U1snRNP and thereby activate polyadenylation at the end of the genome. Our data reveal a contribution of U1snRNP to cellular polyadenylation site selection and to the regulation of gene expression. PMID:23718736

Functional Cus1p Is Found with Hsh155p in a Multiprotein Splicing Factor Associated with U2 snRNA

PubMed Central

Pauling, Michelle Haynes; McPheeters, David S.; Ares, Manuel

2000-01-01

To explore the dynamics of snRNP structure and function, we have studied Cus1p, identified as a suppressor of U2 snRNA mutations in budding yeast. Cus1p is homologous to human SAP145, a protein present in the 17S form of the human U2 snRNP. Here, we define the Cus1p amino acids required for function in yeast. The segment of Cus1p required for binding to Hsh49p, a homolog of human SAP49, is contained within an essential region of Cus1p. Antibodies against Cus1p coimmunoprecipitate U2 snRNA, as well as Hsh155p, a protein homologous to human SAP155. Biochemical fractionation of splicing extracts and reconstitution of heat-inactivated splicing extracts from strains carrying a temperature-sensitive allele of CUS1 indicate that Cus1p and Hsh155p reside in a functional, high-salt-stable complex that is salt-dissociable from U2 snRNA. We propose that Cus1p, Hsh49p, and Hsh155p exist in a stable protein complex which can exchange with a core U2 snRNP and which is necessary for U2 snRNP function in prespliceosome assembly. The Cus1p complex shares functional as well as structural similarities with human SF3b. PMID:10688664

Discovery of native autoantigens via antigen surrogate technology: application to type 1 diabetes.

PubMed

Doran, Todd M; Simanski, Scott; Kodadek, Thomas

2015-02-20

A fundamental goal in understanding the mechanisms of autoimmune disease is the characterization of autoantigens that are targeted by autoreactive antibodies and T cells. Unfortunately, the identification of autoantigens is a difficult problem. We have begun to explore a novel route to the discovery of autoantibody/autoantigen pairs that involves comparative screening of combinatorial libraries of unnatural, synthetic molecules for compounds that bind antibodies present at much higher levels in the serum of individuals with a given autoimmune disease than in the serum of control individuals. We have shown that this approach can yield "antigen surrogates" capable of capturing disease-specific autoantibodies from serum. In this report, we demonstrate that the synthetic antigen surrogates can be used to affinity purify the autoantibodies from serum and that these antibodies can then be used to identify their cognate autoantigen in an appropriate tissue lysate. Specifically, we report the discovery of a peptoid able to bind autoantibodies present in about one-third of nonobese diabetic (NOD) mice. The peptoid-binding autoantibodies were highly enriched through peptoid affinity chromatography and employed to probe mouse pancreatic and brain lysates. This resulted in identification of murine GAD65 as the native autoantigen. GAD65 is a known humoral autoantigen in human type 1 diabetes mellitus (T1DM), but its existence in mice had been controversial. This study demonstrates the potential of this chemical approach for the unbiased identification of autoantigen/autoantibody complexes.

Modeling of the U1 snRNP assembly pathway in alternative splicing in human cells using Petri nets.

PubMed

Kielbassa, J; Bortfeldt, R; Schuster, S; Koch, I

2009-02-01

The investigation of spliceosomal processes is currently a topic of intense research in molecular biology. In the molecular mechanism of alternative splicing, a multi-protein-RNA complex - the spliceosome - plays a crucial role. To understand the biological processes of alternative splicing, it is essential to comprehend the biogenesis of the spliceosome. In this paper, we propose the first abstract model of the regulatory assembly pathway of the human spliceosomal subunit U1. Using Petri nets, we describe its highly ordered assembly that takes place in a stepwise manner. Petri net theory represents a mathematical formalism to model and analyze systems with concurrent processes at different abstraction levels with the possibility to combine them into a uniform description language. There exist many approaches to determine static and dynamic properties of Petri nets, which can be applied to analyze biochemical systems. In addition, Petri net tools usually provide intuitively understandable graphical network representations, which facilitate the dialog between experimentalists and theoreticians. Our Petri net model covers binding, transport, signaling, and covalent modification processes. Through the computation of structural and behavioral Petri net properties and their interpretation in biological terms, we validate our model and use it to get a better understanding of the complex processes of the assembly pathway. We can explain the basic network behavior, using minimal T-invariants which represent special pathways through the network. We find linear as well as cyclic pathways. We determine the P-invariants that represent conserved moieties in a network. The simulation of the net demonstrates the importance of the stability of complexes during the maturation pathway. We can show that complexes that dissociate too fast, hinder the formation of the complete U1 snRNP.

Sm protein methylation is dispensable for snRNP assembly in Drosophila melanogaster.

PubMed

Gonsalvez, Graydon B; Praveen, Kavita; Hicks, Amanda J; Tian, Liping; Matera, A Gregory

2008-05-01

Sm proteins form stable ribonucleoprotein (RNP) complexes with small nuclear (sn)RNAs and are core components of the eukaryotic spliceosome. In vivo, the assembly of Sm proteins onto snRNAs requires the survival motor neurons (SMN) complex. Several reports have shown that SMN protein binds with high affinity to symmetric dimethylarginine (sDMA) residues present on the C-terminal tails of SmB, SmD1, and SmD3. This post-translational modification is thought to play a crucial role in snRNP assembly. In human cells, two distinct protein arginine methyltransferases (PRMT5 and PRMT7) are required for snRNP biogenesis. However, in Drosophila, loss of Dart5 (the fruit fly PRMT5 ortholog) has little effect on snRNP assembly, and homozygous mutants are completely viable. To resolve these apparent differences, we examined this topic in detail and found that Drosophila Sm proteins are also methylated by two methyltransferases, Dart5/PRMT5 and Dart7/PRMT7. Unlike dart5, we found that dart7 is an essential gene. However, the lethality associated with loss of Dart7 protein is apparently unrelated to defects in snRNP assembly. To conclusively test the requirement for sDMA modification of Sm proteins in Drosophila snRNP assembly, we constructed a fly strain that exclusively expresses an isoform of SmD1 that cannot be sDMA modified. Interestingly, these flies were viable, and snRNP assays revealed no defects in comparison to wild type. In contrast, dart5 mutants displayed a strong synthetic lethal phenotype in the presence of a hypomorphic Smn mutation. We therefore conclude that dart5 is required for viability when SMN is limiting.

The nuclear cap-binding complex interacts with the U4/U6·U5 tri-snRNP and promotes spliceosome assembly in mammalian cells

PubMed Central

Pabis, Marta; Neufeld, Noa; Steiner, Michaela C.; Bojic, Teodora; Shav-Tal, Yaron; Neugebauer, Karla M.

2013-01-01

The nuclear cap-binding complex (CBC) binds to the 7-methyl guanosine cap present on every RNA polymerase II transcript. CBC has been implicated in many aspects of RNA biogenesis; in addition to roles in miRNA biogenesis, nonsense-mediated decay, 3′-end formation, and snRNA export from the nucleus, CBC promotes pre-mRNA splicing. An unresolved question is how CBC participates in splicing. To investigate CBC’s role in splicing, we used mass spectrometry to identify proteins that copurify with mammalian CBC. Numerous components of spliceosomal snRNPs were specifically detected. Among these, three U4/U6·U5 snRNP proteins (hBrr2, hPrp4, and hPrp31) copurified with CBC in an RNA-independent fashion, suggesting that a significant fraction of CBC forms a complex with the U4/U6·U5 snRNP and that the activity of CBC might be associated with snRNP recruitment to pre-mRNA. To test this possibility, CBC was depleted from HeLa cells by RNAi. Chromatin immunoprecipitation and live-cell imaging assays revealed decreased cotranscriptional accumulation of U4/U6·U5 snRNPs on active transcription units, consistent with a requirement for CBC in cotranscriptional spliceosome assembly. Surprisingly, recruitment of U1 and U2 snRNPs was also affected, indicating that RNA-mediated interactions between CBC and snRNPs contribute to splicing. On the other hand, CBC depletion did not impair snRNP biogenesis, ruling out the possibility that decreased snRNP recruitment was due to changes in nuclear snRNP concentration. Taken together, the data support a model whereby CBC promotes pre-mRNA splicing through a network of interactions with and among spliceosomal snRNPs during cotranscriptional spliceosome assembly. PMID:23793891

Autoantigenicity of nucleolar complexes.

PubMed

Welting, Tim J M; Raijmakers, Reinout; Pruijn, Ger J M

2003-10-01

Autoantibodies targeting nucleolar autoantigens (ANoA) are most frequently found in sera from patients with systemic sclerosis (SSc, also designated scleroderma) or with SSc overlap syndromes. During the last decade an extensive number of nucleolar components have been identified and this allowed a more detailed analysis of the identity of nucleolar autoantigens. This review intends to give an overview of the molecular composition of the major (families of) autoantigenic nucleolar complexes, to provide some insight into their functions and to summarise the data concerning their autoantigenicity.

Reconstitution of a functional 7SK snRNP

PubMed Central

Brogie, John E.

2017-01-01

Abstract The 7SK small nuclear ribonucleoprotein (snRNP) plays a central role in RNA polymerase II elongation control by regulating the availability of active P-TEFb. We optimized conditions for analyzing 7SK RNA by SHAPE and demonstrated a hysteretic effect of magnesium on 7SK folding dynamics including a 7SK GAUC motif switch. We also found evidence that the 5΄ end pairs alternatively with two different regions of 7SK giving rise to open and closed forms that dictate the state of the 7SK motif. We then used recombinant P-TEFb, HEXIM1, LARP7 and MEPCE to reconstruct a functional 7SK snRNP in vitro. Stably associated P-TEFb was highly inhibited, but could still be released and activated by HIV-1 Tat. Notably, P-TEFb association with both in vitro-reconstituted and cellular snRNPs led to similar changes in SHAPE reactivities, confirming that 7SK undergoes a P-TEFb-dependent structural change. We determined that the xRRM of LARP7 binds to the 3΄ stem loop of 7SK and inhibits the methyltransferase activity of MEPCE through a C-terminal MEPCE interaction domain (MID). Inhibition of MEPCE is dependent on the structure of the 3΄ stem loop and the closed form of 7SK RNA. This study provides important insights into intramolecular interactions within the 7SK snRNP. PMID:28431135

Retinal glycoprotein enrichment by concanavalin a enabled identification of novel membrane autoantigen synaptotagmin-1 in equine recurrent uveitis.

PubMed

Swadzba, Margarete E; Hauck, Stefanie M; Naim, Hassan Y; Amann, Barbara; Deeg, Cornelia A

2012-01-01

Complete knowledge of autoantigen spectra is crucial for understanding pathomechanisms of autoimmune diseases like equine recurrent uveitis (ERU), a spontaneous model for human autoimmune uveitis. While several ERU autoantigens were identified previously, no membrane protein was found so far. As there is a great overlap between glycoproteins and membrane proteins, the aim of this study was to test whether pre-enrichment of retinal glycoproteins by ConA affinity is an effective tool to detect autoantigen candidates among membrane proteins. In 1D Western blots, the glycoprotein preparation allowed detection of IgG reactions to low abundant proteins in sera of ERU patients. Synaptotagmin-1, a Ca2+-sensing protein in synaptic vesicles, was identified as autoantigen candidate from the pre-enriched glycoprotein fraction by mass spectrometry and was validated as a highly prevalent autoantigen by enzyme-linked immunosorbent assay. Analysis of Syt1 expression in retinas of ERU cases showed a downregulation in the majority of ERU affected retinas to 24%. Results pointed to a dysregulation of retinal neurotransmitter release in ERU. Identification of synaptotagmin-1, the first cell membrane associated autoantigen in this spontaneous autoimmune disease, demonstrated that examination of tissue fractions can lead to the discovery of previously undetected novel autoantigens. Further experiments will address its role in ERU pathology.

PP2B and PP1α cooperatively disrupt 7SK snRNP to release P-TEFb for transcription in response to Ca2+ signaling

PubMed Central

Chen, Ruichuan; Liu, Min; Li, Huan; Xue, Yuhua; Ramey, Wanichaya N.; He, Nanhai; Ai, Nanping; Luo, Haohong; Zhu, Ying; Zhou, Nan; Zhou, Qiang

2008-01-01

The positive transcription elongation factor b (P-TEFb), consisting of Cdk9 and cyclin T, stimulates RNA polymerase II elongation and cotranscriptional pre-mRNA processing. To accommodate different growth conditions and transcriptional demands, a reservoir of P-TEFb is kept in an inactive state in the multisubunit 7SK snRNP. Under certain stress or disease conditions, P-TEFb is released to activate transcription, although the signaling pathway(s) that controls this is largely unknown. Here, through analyzing the UV- or hexamethylene bisacetamide (HMBA)-induced release of P-TEFb from 7SK snRNP, an essential role for the calcium ion (Ca2+)–calmodulin–protein phosphatase 2B (PP2B) signaling pathway is revealed. However, Ca2+ signaling alone is insufficient, and PP2B must act sequentially and cooperatively with protein phosphatase 1α (PP1α) to disrupt 7SK snRNP. Activated by UV/HMBA and facilitated by a PP2B-induced conformational change in 7SK snRNP, PP1α releases P-TEFb through dephosphorylating phospho-Thr186 in the Cdk9 T-loop. This event is also necessary for the subsequent recruitment of P-TEFb by the bromodomain protein Brd4 to the preinitiation complex, where Cdk9 remains unphosphorylated and inactive until after the synthesis of a short RNA. Thus, through cooperatively dephosphorylating Cdk9 in response to Ca2+ signaling, PP2B and PP1α alter the P-TEFb functional equilibrium through releasing P-TEFb from 7SK snRNP for transcription. PMID:18483222

Localization of the human 64kD autoantigen D1 to myofibrils in a subset of extraocular muscle fibers

NASA Technical Reports Server (NTRS)

Conley, C. A.; Fowler, V. M.

1999-01-01

PURPOSE. To evaluate the tissue-specific expression pattern of the 64kD human autoantigen D1, a tropomodulin-related protein that may be involved in thyroid-associated ophthalmopathy. METHODS. Recombinant 64kD human autoantigen D1 was generated in a bacterial expression system and used to immunize rabbits. Specific antibodies were affinity-purified and used for Western blots on normal and hyperthyroid rat and rabbit tissue, and immunofluorescence localization on cryosections of rat tissue. RESULTS. Anti-64kD human autoantigen D1 antibodies recognize specifically a approximately 70kD polypeptide in western blots of extraocular muscle, sternothyroid muscle, and smooth muscle. Immunofluorescence staining demonstrates that the 64kD human autoantigen D1 localizes to myofibrils in slow fibers from rat extraocular and sternothyroid muscle. The level of this protein is not altered in extraocular muscles from hyperthyroid rabbits. CONCLUSIONS. The 64kD human autoantigen D1 is expressed in slow fibers of extraocular and sternothyroid muscles as a component of myofibrils, and is not upregulated in conditions of hyperthyroidism.

Immunological cross-reactivity to multiple autoantigens in patients with liver kidney microsomal type 1 autoimmune hepatitis.

PubMed

Choudhuri, K; Gregorio, G V; Mieli-Vergani, G; Vergani, D

1998-11-01

We describe two patients with liver kidney microsomal antibody type 1 (LKM1)-positive autoimmune hepatitis (AIH) with associated endocrinopathies. The first patient had insulin-dependent diabetes (IDDM), and the second patient had Addison's disease and hypoparathyroidism, and is also positive for islet cell antibodies, without overt diabetes. To account for the existence of multiple endocrinopathy in these patients, we investigated whether there is sequence similarity between the target of LKM1 antibodies, cytochrome P4502D6 (CYP2D6), and other human proteins, and if so, whether this structural similarity produces a detectable cross-reactive immune response. Our database search identified two proteins, carboxypeptidase H, an autoantigen in insulin-dependent diabetes, and 21-hydroxylase, the major autoantigen in Addison's disease, that share sequence similarity to the second major LKM1 epitope on CYP2D6. We tested the reactivity of sera from these patients to the homologous regions of the three autoantigens using an enzyme-linked immunosorbent assay (ELISA). The cut-off for positivity was established by testing sera from 22 healthy children. To determine the significance of reactivity to the peptide homologues of the three autoantigens, we investigated 16 additional patients with LKM1 AIH and 20 children with chronic hepatitis B virus infection as pathological controls. We found that reactivity to the second major epitope of CYP2D6 is significantly associated with reactivity to the homologous regions of carboxypeptidase H (CPH) and 21-hydroxylase (21-OHase) in patients with LKM1 AIH, and that this simultaneous recognition is cross-reactive. We suggest that a cross-reactive immune response between homologous autoantigens may contribute to the development of multiple endocrinopathies in LKM1 AIH.

Recurrent LRP1-SNRNP25 and KCNMB4-CCND3 fusion genes promote tumor cell motility in human osteosarcoma.

PubMed

Yang, Jilong; Annala, Matti; Ji, Ping; Wang, Guowen; Zheng, Hong; Codgell, David; Du, Xiaoling; Fang, Zhiwei; Sun, Baocun; Nykter, Matti; Chen, Kexin; Zhang, Wei

2014-10-10

The identification of fusion genes such as SYT-SSX1/SSX2, PAX3-FOXO1, TPM3/TPM4-ALK and EWS-FLI1 in human sarcomas has provided important insight into the diagnosis and targeted therapy of sarcomas. No recurrent fusion has been reported in human osteosarcoma. Transcriptome sequencing was used to characterize the gene fusions and mutations in 11 human osteosarcomas. Nine of 11 samples were found to harbor genetic inactivating alterations in the TP53 pathway. Two recurrent fusion genes associated with the 12q locus, LRP1-SNRNP25 and KCNMB4-CCND3, were identified and validated by RT-PCR, Sanger sequencing and fluorescence in situ hybridization, and were found to be osteosarcoma specific in a validation cohort of 240 other sarcomas. Expression of LRP1-SNRNP25 fusion gene promoted SAOS-2 osteosarcoma cell migration and invasion. Expression of KCNMB4-CCND3 fusion gene promoted SAOS-2 cell migration. Our study represents the first whole transcriptome analysis of untreated human osteosarcoma. Our discovery of two osteosarcoma specific fusion genes associated with osteosarcoma cellular motility highlights the heterogeneity of osteosarcoma and provides opportunities for new treatment modalities.

Design of a Type-1 Diabetes Vaccine Candidate Using Edible Plants Expressing a Major Autoantigen

PubMed Central

Bertini, Edoardo; Merlin, Matilde; Gecchele, Elisa; Puggia, Andrea; Brozzetti, Annalisa; Commisso, Mauro; Falorni, Alberto; Bini, Vittorio; Klymyuk, Victor; Pezzotti, Mario; Avesani, Linda

2018-01-01

Type-1 diabetes (T1D) is a metabolic disease involving the autoimmune destruction of insulin-producing pancreatic beta cells. It is often diagnosed by the detection of autoantibodies, typically those recognizing insulin itself or the 65-kDa isoform of glutamic acid decarboxylase (GAD65). Oral insulin can be used to induce systemic immunological tolerance and thus prevent or delay the onset of T1D, suggesting that combination treatments with other autoantigens such as GAD65 could be even more successful. GAD65 has induced oral tolerance and prevented T1D in preclinical studies but it is difficult to produce in sufficient quantities for clinical testing. Here we combined edible plant systems, namely spinach (Spinacia oleracea cv Industra) and red beet (Beta vulgaris cv Moulin Rouge), with the magnICON® expression system to develop a safe, cost-effective and environmentally sustainable platform for the large-scale production of GAD65. The superior red beet platform was extensively characterized in terms of recombinant protein yields and bioequivalence to wild-type plants, and the product was tested for its ability to resist simulated gastric digestion. Our results indicate that red beet plants are suitable for the production of a candidate oral vaccine based on GAD65 for the future preclinical and clinical testing of T1D immunotherapy approaches. PMID:29765386

Structural Basis for Recognition and Sequestration of UUUOH 3 ' Temini of Nascent RNA Polymerase III Transcripts by La, a Rheumatic Disease Autoantigen

DOE Office of Scientific and Technical Information (OSTI.GOV)

Teplova,M.; Yuan, Y.; Phan, A.

2006-01-01

The nuclear phosphoprotein La was identified as an autoantigen in patients with systemic lupus erythematosus and Sjogren's syndrome. La binds to and protects the UUUOH 3' terminii of nascent RNA polymerase III transcripts from exonuclease digestion. We report the 1.85 Angstroms crystal structure of the N-terminal domain of human La, consisting of La and RRM1 motifs, bound to r(U1-G2-C3-U4-G5-U6-U7-U8-U9OH). The U7-U8-U9OH 3' end, in a splayed-apart orientation, is sequestered within a basic and aromatic amino acid-lined cleft between the La and RRM1 motifs. The specificity-determining U8 residue bridges both motifs, in part through unprecedented targeting of the {beta} sheet edge,more » rather than the anticipated face, of the RRM1 motif. Our structural observations, supported by mutation studies of both La and RNA components, illustrate the principles behind RNA sequestration by a rheumatic disease autoantigen, whereby the UUUOH 3' ends of nascent RNA transcripts are protected during downstream processing and maturation events.« less

Structural basis for recognition and sequestration of UUU(OH) 3' temini of nascent RNA polymerase III transcripts by La, a rheumatic disease autoantigen.

PubMed

Teplova, Marianna; Yuan, Yu-Ren; Phan, Anh Tuân; Malinina, Lucy; Ilin, Serge; Teplov, Alexei; Patel, Dinshaw J

2006-01-06

The nuclear phosphoprotein La was identified as an autoantigen in patients with systemic lupus erythematosus and Sjogren's syndrome. La binds to and protects the UUU(OH) 3' terminii of nascent RNA polymerase III transcripts from exonuclease digestion. We report the 1.85 angstroms crystal structure of the N-terminal domain of human La, consisting of La and RRM1 motifs, bound to r(U1-G2-C3-U4-G5-U6-U7-U8-U9OH). The U7-U8-U9OH 3' end, in a splayed-apart orientation, is sequestered within a basic and aromatic amino acid-lined cleft between the La and RRM1 motifs. The specificity-determining U8 residue bridges both motifs, in part through unprecedented targeting of the beta sheet edge, rather than the anticipated face, of the RRM1 motif. Our structural observations, supported by mutation studies of both La and RNA components, illustrate the principles behind RNA sequestration by a rheumatic disease autoantigen, whereby the UUU(OH) 3' ends of nascent RNA transcripts are protected during downstream processing and maturation events.

R2TP/Prefoldin-like component RUVBL1/RUVBL2 directly interacts with ZNHIT2 to regulate assembly of U5 small nuclear ribonucleoprotein

PubMed Central

Cloutier, Philippe; Poitras, Christian; Durand, Mathieu; Hekmat, Omid; Fiola-Masson, Émilie; Bouchard, Annie; Faubert, Denis; Chabot, Benoit; Coulombe, Benoit

2017-01-01

The R2TP/Prefoldin-like (R2TP/PFDL) complex has emerged as a cochaperone complex involved in the assembly of a number of critical protein complexes including snoRNPs, nuclear RNA polymerases and PIKK-containing complexes. Here we report on the use of multiple target affinity purification coupled to mass spectrometry to identify two additional complexes that interact with R2TP/PFDL: the TSC1–TSC2 complex and the U5 small nuclear ribonucleoprotein (snRNP). The interaction between R2TP/PFDL and the U5 snRNP is mostly mediated by the previously uncharacterized factor ZNHIT2. A more general function for the zinc-finger HIT domain in binding RUVBL2 is exposed. Disruption of ZNHIT2 and RUVBL2 expression impacts the protein composition of the U5 snRNP suggesting a function for these proteins in promoting the assembly of the ribonucleoprotein. A possible implication of R2TP/PFDL as a major effector of stress-, energy- and nutrient-sensing pathways that regulate anabolic processes through the regulation of its chaperoning activity is discussed. PMID:28561026

Rrp6p, the yeast homologue of the human PM-Scl 100-kDa autoantigen, is essential for efficient 5.8 S rRNA 3' end formation.

PubMed

Briggs, M W; Burkard, K T; Butler, J S

1998-05-22

The eukaryotic 25 S, 18 S, and 5.8 S rRNAs are synthesized as a single transcript with two internal transcribed spacers (ITS1 and ITS2), which are removed by endo- and exoribonucleolytic steps to produce mature rRNA. Genetic selection for suppressors of a polyadenylation defect yielded two cold-sensitive alleles of a gene that we named RRP6 (ribosomal RNA processing). Molecular cloning of RRP6 revealed its homology to a 100-kDa human, nucleolar PM-Scl autoantigen and to Escherichia coli RNase D, a 3'-5' exoribonuclease. Recessive mutations in rrp6 result in the accumulation of a novel 5. 8 S rRNA processing intermediate, called 5.8 S*, which has normal 5' ends, but retains approximately 30 nucleotides of ITS2. Pulse-chase analysis of 5.8 S rRNA processing in an rrp6- strain revealed a precursor-product relationship between 5.8 S* and 5.8 S rRNAs, suggesting that Rrp6p plays a role in the removal of the last 30 nucleotides of ITS2 from 5.8 S precursors. A portion of 5.8 S* rRNA assembles into 60 S ribosomes which form polyribosomes, suggesting that they function in protein synthesis. These findings indicate that Rrp6p plays a role in 5.8 S rRNA 3' end formation, and they identify a functional intermediate in the rRNA processing pathway.

The 7SK snRNP associates with the little elongation complex to promote snRNA gene expression.

PubMed

Egloff, Sylvain; Vitali, Patrice; Tellier, Michael; Raffel, Raoul; Murphy, Shona; Kiss, Tamás

2017-04-03

The 7SK small nuclear RNP (snRNP), composed of the 7SK small nuclear RNA (snRNA), MePCE, and Larp7, regulates the mRNA elongation capacity of RNA polymerase II (RNAPII) through controlling the nuclear activity of positive transcription elongation factor b (P-TEFb). Here, we demonstrate that the human 7SK snRNP also functions as a canonical transcription factor that, in collaboration with the little elongation complex (LEC) comprising ELL, Ice1, Ice2, and ZC3H8, promotes transcription of RNAPII-specific spliceosomal snRNA and small nucleolar RNA (snoRNA) genes. The 7SK snRNA specifically associates with a fraction of RNAPII hyperphosphorylated at Ser5 and Ser7, which is a hallmark of RNAPII engaged in snRNA synthesis. Chromatin immunoprecipitation (ChIP) and chromatin isolation by RNA purification (ChIRP) experiments revealed enrichments for all components of the 7SK snRNP on RNAPII-specific sn/snoRNA genes. Depletion of 7SK snRNA or Larp7 disrupts LEC integrity, inhibits RNAPII recruitment to RNAPII-specific sn/snoRNA genes, and reduces nascent snRNA and snoRNA synthesis. Thus, through controlling both mRNA elongation and sn/snoRNA synthesis, the 7SK snRNP is a key regulator of nuclear RNA production by RNAPII. © 2017 The Authors.

Analysis of anticentromere autoantibodies using cloned autoantigen CENP-B.

PubMed

Earnshaw, W C; Machlin, P S; Bordwell, B J; Rothfield, N F; Cleveland, D W

1987-07-01

A cDNA clone encoding CENP-B, the 80-kDa human centromere autoantigen, was used to construct a panel of hybrid proteins containing four different regions of CENP-B. These have allowed us to identify three independent epitopes on CENP-B that are targets of autoantibodies. Two of these are recognized concurrently in greater than or equal to 90% of patient sera containing anticentromere autoantibodies (ACA), conclusively demonstrating that this autoimmune response is polyclonal. When present and previous data are combined, ACA are shown to recognize at least five independent epitopes on CENP-B. A radioimmunoassay based on cloned CENP-B has demonstrated that sera from greater than or equal to 96% of patients with ACA recognize the cloned antigen, thus defining a region of the protein that is recognized by virtually all patients with ACA. These findings have significant implications for models that seek to explain the origin of ACA and for the future detection of this group of autoantibodies in the clinical setting.

The dermatomyositis-specific autoantigen Mi2 is a component of a complex containing histone deacetylase and nucleosome remodeling activities.

PubMed

Zhang, Y; LeRoy, G; Seelig, H P; Lane, W S; Reinberg, D

1998-10-16

Histone acetylation and deacetylation were found to be catalyzed by structurally distinct, multisubunit complexes that mediate, respectively, activation and repression of transcription. ATP-dependent nucleosome remodeling, mediated by different multisubunit complexes, was thought to be involved only in transcription activation. Here we report the isolation of a protein complex that contains both histone deacetylation and ATP-dependent nucleosome remodeling activities. The complex contains the histone deacetylases HDAC1/2, histone-binding proteins, the dermatomyositis-specific autoantigen Mi2beta, a polypeptide related to the metastasis-associated protein 1, and a novel polypeptide of 32 kDa. Patients with dermatomyositis have a high rate of malignancy. The finding that Mi2beta exists in a complex containing histone deacetylase and nucleosome remodeling activities suggests a role for chromatin reorganization in cancer metastasis.

Autoantigen-specific B-cell depletion overcomes failed immune tolerance in type 1 diabetes.

PubMed

Henry, Rachel A; Kendall, Peggy L; Thomas, James W

2012-08-01

Eliminating autoantigen-specific B cells is an attractive alternative to global B-cell depletion for autoimmune disease treatment. To identify the potential for targeting a key autoimmune B-cell specificity in type 1 diabetes, insulin-binding B cells were tracked within a polyclonal repertoire using heavy chain B-cell receptor (BCR) transgenic (VH125Tg) mice. Insulin-specific B cells are rare in the periphery of nonautoimmune VH125Tg/C57BL/6 mice and WT/NOD autoimmune mice, whereas they clearly populate 1% of mature B-cell subsets in VH125Tg/NOD mice. Autoantigen upregulates CD86 in anti-insulin B cells, suggesting they are competent to interact with T cells. Endogenous insulin occupies anti-insulin BCR beginning with antigen commitment in bone marrow parenchyma, as identified by a second anti-insulin monoclonal antibody. Administration of this monoclonal antibody selectively eliminates insulin-reactive B cells in vivo and prevents disease in WT/NOD mice. Unexpectedly, developing B cells are less amenable to depletion, despite increased BCR sensitivity. These findings exemplify how a critical type 1 diabetes B-cell specificity escapes immune tolerance checkpoints. Disease liability is corrected by eliminating this B-cell specificity, providing proof of concept for a novel therapeutic approach for autoimmune disease.

Analysis of anticentromere autoantibodies using cloned autoantigen CENP-B.

PubMed Central

Earnshaw, W C; Machlin, P S; Bordwell, B J; Rothfield, N F; Cleveland, D W

1987-01-01

A cDNA clone encoding CENP-B, the 80-kDa human centromere autoantigen, was used to construct a panel of hybrid proteins containing four different regions of CENP-B. These have allowed us to identify three independent epitopes on CENP-B that are targets of autoantibodies. Two of these are recognized concurrently in greater than or equal to 90% of patient sera containing anticentromere autoantibodies (ACA), conclusively demonstrating that this autoimmune response is polyclonal. When present and previous data are combined, ACA are shown to recognize at least five independent epitopes on CENP-B. A radioimmunoassay based on cloned CENP-B has demonstrated that sera from greater than or equal to 96% of patients with ACA recognize the cloned antigen, thus defining a region of the protein that is recognized by virtually all patients with ACA. These findings have significant implications for models that seek to explain the origin of ACA and for the future detection of this group of autoantibodies in the clinical setting. Images PMID:2440036

Identification and functional validation of novel autoantigens in equine uveitis.

PubMed

Deeg, Cornelia A; Pompetzki, Dirk; Raith, Albert J; Hauck, Stefanie M; Amann, Barbara; Suppmann, Sabine; Goebel, Thomas W F; Olazabal, Ursula; Gerhards, Hartmut; Reese, Sven; Stangassinger, Manfred; Kaspers, Bernd; Ueffing, Marius

2006-08-01

The development, progression, and recurrence of autoimmune diseases are frequently driven by a group of participatory autoantigens. We identified and characterized novel autoantigens by analyzing the autoantibody binding pattern from horses affected by spontaneous equine recurrent uveitis to the retinal proteome. Cellular retinaldehyde-binding protein (cRALBP) had not been described previously as autoantigen, but subsequent characterization in equine recurrent uveitis horses revealed B and T cell autoreactivity to this protein and established a link to epitope spreading. We further immunized healthy rats and horses with cRALBP and observed uveitis in both species with typical tissue lesions at cRALBP expression sites. The autoantibody profiling outlined here could be used in various autoimmune diseases to detect autoantigens involved in the dynamic spreading cascade or serve as predictive markers.

Protein composition of catalytically active U7-dependent processing complexes assembled on histone pre-mRNA containing biotin and a photo-cleavable linker

PubMed Central

Skrajna, Aleksandra; Yang, Xiao-cui; Dadlez, Michał; Marzluff, William F; Dominski, Zbigniew

2018-01-01

Abstract 3′ end cleavage of metazoan replication-dependent histone pre-mRNAs requires the multi-subunit holo-U7 snRNP and the stem–loop binding protein (SLBP). The exact composition of the U7 snRNP and details of SLBP function in processing remain unclear. To identify components of the U7 snRNP in an unbiased manner, we developed a novel approach for purifying processing complexes from Drosophila and mouse nuclear extracts. In this method, catalytically active processing complexes are assembled in vitro on a cleavage-resistant histone pre-mRNA containing biotin and a photo-sensitive linker, and eluted from streptavidin beads by UV irradiation for direct analysis by mass spectrometry. In the purified processing complexes, Drosophila and mouse U7 snRNP have a remarkably similar composition, always being associated with CPSF73, CPSF100, symplekin and CstF64. Many other proteins previously implicated in the U7-dependent processing are not present. Drosophila U7 snRNP bound to histone pre-mRNA in the absence of SLBP contains the same subset of polyadenylation factors but is catalytically inactive and addition of recombinant SLBP is sufficient to trigger cleavage. This result suggests that Drosophila SLBP promotes a structural rearrangement of the processing complex, resulting in juxtaposition of the CPSF73 endonuclease with the cleavage site in the pre-mRNA substrate. PMID:29529248

Author Correction: CryoEM structure of Saccharomyces cerevisiae U1 snRNP offers insight into alternative splicing.

PubMed

Li, Xueni; Liu, Shiheng; Jiang, Jiansen; Zhang, Lingdi; Espinosa, Sara; Hill, Ryan C; Hansen, Kirk C; Zhou, Z Hong; Zhao, Rui

2018-04-11

The originally published version of this Article contained several errors in Figure 2, panel a: the basepair register in SL3-4 of yeast U1 snRNA was depicted incorrectly; the basepair for A287-U295 in yeast U1 snRNA was erroneously present; basepairs for U84-G119, G309-U532, A288-U295 and U289-A294 in yeast U1 snRNA were missing; the bulging nucleotide in SL3 of human U1 snRNA was depicted as G instead of C; and the dashed boxes defining the 5' ss binding site and Sm site in both human and yeast snRNAs were not drawn accurately. These have now been corrected in both the PDF and HTML versions of the Article.

Mercuric chloride induces autoantibodies against U3 small nuclear ribonucleoprotein in susceptible mice.

PubMed Central

Reuter, R; Tessars, G; Vohr, H W; Gleichmann, E; Lührmann, R

1989-01-01

Autoantibodies to nucleolar components are a common serological feature of patients suffering from scleroderma, a collagen vascular autoimmune disease. While animal models, which spontaneously develop abundant anti-nucleolar antibodies, have not yet been described, high titers of such antibodies may be induced by treating susceptible strains of mice with mercuric chloride. We have identified the nucleolar autoantigen against which the HgCl2-induced IgG autoantibodies from mice of strain B10.S are directed. It is a protein with an apparent molecular mass of 36 kDa and a pI value of approximately 8.6, which is associated with the nucleolar small nuclear RNA U3, and by these criteria must be identical with a polypeptide called fibrillarin. It is striking that scleroderma patients spontaneously produce autoantibodies against the same U3 ribonucleoprotein (RNP). The HgCl2-induced murine and the scleroderma-specific human anti-U3 RNP autoantibodies were indistinguishable in their reactivities toward fibrillarin. They further resemble each other insofar as both recognize epitopes on the 36-kDa protein, which have been highly conserved throughout evolution. Our results provide a basis to investigate at the molecular level whether similar immunoregulatory dysfunctions may lead to the preferential anti-U3 RNP autoantibody production in the animal model and in scleroderma patients. Images PMID:2521387

CRALBP is a highly prevalent autoantigen for human autoimmune uveitis.

PubMed

Deeg, Cornelia A; Raith, Albert J; Amann, Barbara; Crabb, John W; Thurau, Stephan R; Hauck, Stefanie M; Ueffing, Marius; Wildner, Gerhild; Stangassinger, Manfred

2007-01-01

Cellular retinaldehyde binding protein (CRALBP) is an autoantigen in spontaneous equine recurrent uveitis. In order to test whether CRALBP contributes to human autoimmune uveitis, the specificity of antibodies from human uveitis patient's sera was first evaluated in two-dimensional (2D) Western blot analysis. Subsequent identification of the immunoreactive proteins by mass spectrometry resulted in the identification of CRALBP as a putative autoantigen. Additionally, sera from human uveitis and control patients were by Western blot using purified human recombinant CRALBP. Anti-CRALBP autoantibodies occur more frequently (P<.01) in human uveitis patients than in normal controls. Thirty out of 56 tested uveitis patient's sera contained autoantibodies reactive against CRALBP, compared to only four out of 23 normal control subjects. The presence of CRALBP autoantibodies in 54% of tested uveitis patients supports CRALBP as a possible autoantigen in human autoimmune uveitis.

Vitiligo: How do oxidative stress-induced autoantigens trigger autoimmunity?

PubMed

Xie, Heng; Zhou, Fubo; Liu, Ling; Zhu, Guannan; Li, Qiang; Li, Chunying; Gao, Tianwen

2016-01-01

Vitiligo is a common depigmentation disorder characterized by a loss of functional melanocytes and melanin from epidermis, in which the autoantigens and subsequent autoimmunity caused by oxidative stress play significant roles according to hypotheses. Various factors lead to reactive oxygen species (ROS) overproduction in the melanocytes of vitiligo: the exogenous and endogenous stimuli that cause ROS production, low levels of enzymatic and non-enzymatic antioxidants, disturbed antioxidant pathways and polymorphisms of ROS-associated genes. These factors synergistically contribute to the accumulation of ROS in melanocytes, finally leading to melanocyte damage and the production of autoantigens through the following ways: apoptosis, accumulation of misfolded peptides and cytokines induced by endoplasmic reticulum stress as well as the sustained unfolded protein response, and an 'eat me' signal for phagocytic cells triggered by calreticulin. Subsequently, autoantigens presentation and dendritic cells maturation occurred mediated by the release of antigen-containing exosomes, adenosine triphosphate and melanosomal autophagy. With the involvement of inducible heat shock protein 70, cellular immunity targeting autoantigens takes the essential place in the destruction of melanocytes, which eventually results in vitiligo. Several treatments, such as narrow band ultraviolet, quercetin and α-melanophore-stimulating hormone, are reported to be able to lower ROS thereby achieving repigmentation in vitiligo. In therapies targeting autoimmunity, restore of regulatory T cells is absorbing attention, in which narrow band ultraviolet also plays a role. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Automated microfluidic assay system for autoantibodies found in autoimmune diseases using a photoimmobilized autoantigen microarray.

PubMed

Matsudaira, Takahiro; Tsuzuki, Saki; Wada, Akira; Suwa, Akira; Kohsaka, Hitoshi; Tomida, Maiko; Ito, Yoshihiro

2008-01-01

Autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, and autoimmune diabetes are characterized by the production of autoantibodies that serve as useful diagnostic markers, surrogate markers, and prognostic factors. We devised an in vitro system to detect these clinically pivotal autoantibodies using a photoimmobilized autoantigen microarray. Photoimmobilization was useful for preparing the autoantigen microarray, where autoantigens are covalently immobilized on a plate, because it does not require specific functional groups of the autoantigens and any organic material can be immobilized by a radical reaction induced by photoirradiation. Here, we prepared the microarray using a very convenient method. Aqueous solutions of each autoantigen were mixed with a polymer of poly(ethylene glycol) methacrylate and a photoreactive crosslinker, and the mixtures were microspotted on a plate and dried in air. Finally, the plate was irradiated with an ultraviolet lamp to obtain immobilization. In the assay, patient serum was added to the microarray plate. Antigen-specific IgG adsorbed on the microspotted autoantigen was detected by peroxidase-conjugated anti-IgG antibody. The chemical luminescence intensities of the substrate decomposed by the peroxidase were detected with a sensitive CCD camera. All autoantigens were immobilized stably by this method and used to screen antigen-specific IgG. In addition, the plate was covered with a polydimethylsiloxane sheet containing microchannels and automated measurement was carried out.

Scleroderma Autoantigens Are Uniquely Fragmented by Metal-catalyzed Oxidation Reactions: Implications for Pathogenesis

PubMed Central

Casciola-Rosen, Livia; Wigley, Fredrick; Rosen, Antony

1997-01-01

The observation that revelation of immunocryptic epitopes in self antigens may initiate the autoimmune response has prompted the search for processes which induce novel fragmentation of autoantigens as potential initiators of autoimmunity. The reversible ischemia reperfusion which characterizes scleroderma has focused attention on reactive oxygen species as molecules which might induce autoantigen fragmentation. We demonstrate that several of the autoantigens targeted in diffuse scleroderma are uniquely susceptible to cleavage by reactive oxygen species, in a metal-dependent manner. Multiple features of the fragmentation reaction and its inhibition indicate that these autoantigens possess metal-binding sites, which focus metal-catalyzed oxidation reactions (and consequent fragmentation) to specific regions of the antigens. These data suggest that the autoantibody response in scleroderma is the immune marker of unique protein fragmentation, induced by ischemia reperfusion in the presence of appropriate metals, and focus attention on abnormal metal status as a potential pathogenic principle in this disease. PMID:8996243

Identification of TPIT and other novel autoantigens in lymphocytic hypophysitis; immunoscreening of a pituitary cDNA library and development of immunoprecipitation assays

PubMed Central

Smith, Casey Jo Anne; Bensing, Sophie; Burns, Christine; Robinson, Phillip J; Kasperlik-Zaluska, Anna A; Scott, Rodney J; Kämpe, Olle; Crock, Patricia A

2012-01-01

Background Lymphocytic hypophysitis is an organ-specific autoimmune disease of the pituitary gland. A specific and sensitive serological test currently does not exist to aid in the diagnosis. Objective To identify target autoantigens in lymphocytic hypophysitis and develop a diagnostic assay for these proteins. Design/methods A pituitary cDNA expression library was immunoscreened using sera from four patients with lymphocytic hypophysitis. Relevant cDNA clones from screening, along with previously identified autoantigens pituitary gland-specific factor 1a and 2 (PGSF1a and PGSF2) and neuron-specific enolase (NSE) were tested in an in vitro transcription and translation immunoprecipitation assay. The corticotroph-specific transcription factor, TPIT, was investigated separately as a candidate autoantigen. Results Significantly positive autoantibody reactivity against TPIT was found in 9/86 hypophysitis patients vs 1/90 controls (P=0.018). The reactivity against TPIT was not specific for lymphocytic hypophysitis with autoantibodies detectable in the sera from patients with other autoimmune endocrine diseases. Autoantibodies were also detected against chromodomain-helicase-DNA binding protein 8, presynaptic cytomatrix protein (piccolo), Ca2+-dependent secretion activator, PGSF2 and NSE in serum samples from patients with lymphocytic hypophysitis, but at a frequency that did not differ from healthy controls. Importantly, 8/86 patients with lymphocytic hypophysitis had autoantibodies against any two autoantigens in comparison with 0/90 controls (P=0.0093). Conclusions TPIT, a corticotroph-specific transcription factor, was identified as a target autoantigen in 10.5% of patients with lymphocytic hypophysitis. Further autoantigens related to vesicle processing were also identified as potential autoantigens with different immunoreactivity patterns in patients and controls. PMID:22193973

Structural requirements for protein-catalyzed annealing of U4 and U6 RNAs during di-snRNP assembly

PubMed Central

Didychuk, Allison L.; Montemayor, Eric J.; Brow, David A.; Butcher, Samuel E.

2016-01-01

Base-pairing of U4 and U6 snRNAs during di-snRNP assembly requires large-scale remodeling of RNA structure that is chaperoned by the U6 snRNP protein Prp24. We investigated the mechanism of U4/U6 annealing in vitro using an assay that enables visualization of ribonucleoprotein complexes and faithfully recapitulates known in vivo determinants for the process. We find that annealing, but not U6 RNA binding, is highly dependent on the electropositive character of a 20 Å-wide groove on the surface of Prp24. During annealing, we observe the formation of a stable ternary complex between U4 and U6 RNAs and Prp24, indicating that displacement of Prp24 in vivo requires additional factors. Mutations that stabilize the U6 ‘telestem’ helix increase annealing rates by up to 15-fold, suggesting that telestem formation is rate-limiting for U4/U6 pairing. The Lsm2–8 complex, which binds adjacent to the telestem at the 3′ end of U6, provides a comparable rate enhancement. Collectively, these data identify domains of the U6 snRNP that are critical for one of the first steps in assembly of the megaDalton U4/U6.U5 tri-snRNP complex, and lead to a dynamic model for U4/U6 pairing that involves a striking degree of evolved cooperativity between protein and RNA. PMID:26673715

Protein arginine methylation of Npl3 promotes splicing of the SUS1 intron harboring non-consensus 5' splice site and branch site.

PubMed

Muddukrishna, Bhavana; Jackson, Christopher A; Yu, Michael C

2017-06-01

Protein arginine methylation occurs on spliceosomal components and spliceosome-associated proteins, but how this modification contributes to their function in pre-mRNA splicing remains sparse. Here we provide evidence that protein arginine methylation of the yeast SR-/hnRNP-like protein Npl3 plays a role in facilitating efficient splicing of the SUS1 intron that harbors a non-consensus 5' splice site and branch site. In yeast cells lacking the major protein arginine methyltransferase HMT1, we observed a change in the co-transcriptional recruitment of the U1 snRNP subunit Snp1 and Npl3 to pre-mRNAs harboring both consensus (ECM33 and ASC1) and non-consensus (SUS1) 5' splice site and branch site. Using an Npl3 mutant that phenocopies wild-type Npl3 when expressed in Δhmt1 cells, we showed that the arginine methylation of Npl3 is responsible for this. Examination of pre-mRNA splicing efficiency in these mutants reveals the requirement of Npl3 methylation for the efficient splicing of SUS1 intron 1, but not of ECM33 or ASC1. Changing the 5' splice site and branch site in SUS1 intron 1 to the consensus form restored splicing efficiency in an Hmt1-independent manner. Results from biochemical studies show that methylation of Npl3 promotes its optimal association with the U1 snRNP through its association with the U1 snRNP subunit Mud1. Based on these data, we propose a model in which Hmt1, via arginine methylation of Npl3, facilitates U1 snRNP engagement with the pre-mRNA to promote usage of non-consensus splice sites by the splicing machinery. Published by Elsevier B.V.

Autoantigens targeted in scleroderma patients with vascular disease are enriched in endothelial lineage cells

PubMed Central

McMahan, Zsuzsanna H.; Cottrell, Tricia R.; Wigley, Fredrick M.; Antiochos, Brendan; Zambidis, Elias T.; Park, Tea Soon; Halushka, Marc K.; Gutierrez-Alamillo, Laura; Cimbro, Raffaello; Rosen, Antony; Casciola-Rosen, Livia

2016-01-01

Objective Scleroderma patients with autoantibodies to centromere proteins (CENPs) and/or interferon-inducible protein 16 (IFI16) are at increased risk of severe vascular complications. We set out to define whether these autoantigens are enriched in cells of the vasculature. Methods Successive stages of embryoid bodies (EBs) as well as vascular progenitors were used to evaluate the expression of scleroderma autoantigens IFI16 and CENP by immunoblotting. CD31 was included to mark early blood vessels. IFI16 and CD31 expression were defined in skin paraffin sections from scleroderma patients and from healthy controls. IFI16 expression was determined by flow cytometry in circulating endothelial cells (CECs) and circulating progenitor cells (CPCs). Results Expression of CENP-A, IFI16 and CD31 was enriched in EBs at days 10 and 12 of differentiation, and particularly in cultures enriched in vascular progenitors (IFI16, CD31, CENPs A and-B). This pattern was distinct from that of comparator autoantigens. Immunohistochemical staining of skin paraffin sections showed enrichment of IFI16 in CD31-positive vascular endothelial cells in biopsies from scleroderma patients and normal controls. Flow cytometry analysis revealed IFI16 expression in CPCs, but minimal expression in CECs. Conclusion Expression of scleroderma autoantigens IFI16 and CENPs, which are associated with severe vascular disease, is increased in vascular progenitors and mature endothelial cells. High level, lineage-enriched expression of autoantigens may explain the striking association between clinical phenotypes and the immune targeting of specific autoantigens. PMID:27159521

Major retinal autoantigens remain stably expressed during all stages of spontaneous uveitis.

PubMed

Deeg, Cornelia A; Hauck, Stefanie M; Amann, Barbara; Kremmer, Elisabeth; Stangassinger, Manfred; Ueffing, Marius

2007-07-01

Equine recurrent uveitis (ERU) is a valuable model for autoimmune diseases, since it develops frequently and occurs spontaneously. We investigated the overall expression level of three major retinal autoantigens in normal retinas and various ERU stages. Analysis of retinal proteomes of both, healthy and diseased retinas revealed an almost unaffected expression of IRBP, S-antigen and cRALBP in ERU cases. Validation of these findings with western blots and immunohistochemistry confirmed constant to increased expression of these autoantigens, although loss of their physiological expression sites within retina is evident. In contrast to stable expression of autoantigens, rhodopsin, the major component of phototransduction in photoreceptors, disappeared from destructed retinas. These results explain persistent uveitic attacks even in severely damaged eyes and draw the attention to further investigations of biological pathways and regulations in autoimmune target tissues.

Identification of autoantigens in body fluids by combining pull-downs and organic precipitations of intact immune complexes with quantitative label-free mass spectrometry.

PubMed

Merl, Juliane; Deeg, Cornelia A; Swadzba, Margarete E; Ueffing, Marius; Hauck, Stefanie M

2013-12-06

Most autoimmune diseases are multifactorial diseases and are caused by the immunological reaction against a number of autoantigens. Key for understanding autoimmune pathologies is the knowledge of the targeted autoantigens, both initially and during disease progression. We present an approach for autoantigen identification based on isolation of intact autoantibody-antigen complexes from body fluids. After organic precipitation of high molecular weight proteins and free immunoglobulins, released autoantigens were identified by quantitative label-free liquid chromatography mass spectrometry. We confirmed feasibility of target enrichment and identification from highly complex body fluid proteomes by spiking of a predefined antibody-antigen complex at low level of abundance. As a proof of principle, we studied the blinding disease autoimmune uveitis, which is caused by autoreactive T-cells attacking the inner eye and is accompanied by autoantibodies. We identified three novel autoantigens in the spontaneous animal model equine recurrent uveitis (secreted acidic phosphoprotein osteopontin, extracellular matrix protein 1, and metalloproteinase inhibitor 2) and confirmed the presence of the corresponding autoantibodies in 15-25% of patient samples by enzyme-linked immunosorbent assay. Thus, this workflow led to the identification of novel autoantigens in autoimmune uveitis and may provide a versatile and useful tool to identify autoantigens in other autoimmune diseases in the future.

Immunological Tolerance to Muscle Autoantigens Involves Peripheral Deletion of Autoreactive CD8+ T Cells

PubMed Central

Franck, Emilie; Bonneau, Carole; Jean, Laetitia; Henry, Jean-Paul; Lacoume, Yann; Salvetti, Anna; Boyer, Olivier; Adriouch, Sahil

2012-01-01

Muscle potentially represents the most abundant source of autoantigens of the body and can be targeted by a variety of severe autoimmune diseases. Yet, the mechanisms of immunological tolerance toward muscle autoantigens remain mostly unknown. We investigated this issue in transgenic SM-Ova mice that express an ovalbumin (Ova) neo-autoantigen specifically in skeletal muscle. We previously reported that antigen specific CD4+ T cell are immunologically ignorant to endogenous Ova in this model but can be stimulated upon immunization. In contrast, Ova-specific CD8+ T cells were suspected to be either unresponsive to Ova challenge or functionally defective. We now extend our investigations on the mechanisms governing CD8+ tolerance in SM-Ova mice. We show herein that Ova-specific CD8+ T cells are not detected upon challenge with strongly immunogenic Ova vaccines even after depletion of regulatory T cells. Ova-specific CD8+ T cells from OT-I mice adoptively transferred to SM-Ova mice started to proliferate in vivo, acquired CD69 and PD-1 but subsequently down-regulated Bcl-2 and disappeared from the periphery, suggesting a mechanism of peripheral deletion. Peripheral deletion of endogenous Ova-specific cells was formally demonstrated in chimeric SM-Ova mice engrafted with bone marrow cells containing T cell precursors from OT-I TCR-transgenic mice. Thus, the present findings demonstrate that immunological tolerance to muscle autoantigens involves peripheral deletion of autoreactive CD8+ T cells. PMID:22570714

Defining essential elements and genetic interactions of the yeast Lsm2-8 ring and demonstration that essentiality of Lsm2-8 is bypassed via overexpression of U6 snRNA or the U6 snRNP subunit Prp24.

PubMed

Roth, Allen J; Shuman, Stewart; Schwer, Beate

2018-06-01

A seven-subunit Lsm2-8 protein ring assembles on the U-rich 3' end of the U6 snRNA. A structure-guided mutational analysis of the Saccharomyces cerevisiae Lsm2-8 ring affords new insights to structure-function relations and genetic interactions of the Lsm subunits. Alanine scanning of 39 amino acids comprising the RNA-binding sites or intersubunit interfaces of Lsm2, Lsm3, Lsm4, Lsm5, and Lsm8 identified only one instance of lethality (Lsm3-R69A) and one severe growth defect (Lsm2-R63A), both involving amino acids that bind the 3'-terminal UUU trinucleotide. All other Ala mutations were benign with respect to vegetative growth. Tests of 235 pairwise combinations of benign Lsm mutants identified six instances of inter-Lsm synthetic lethality and 45 cases of nonlethal synthetic growth defects. Thus, Lsm2-8 ring function is buffered by a network of internal genetic redundancies. A salient finding was that otherwise lethal single-gene deletions lsm2 Δ, lsm3 Δ, lsm4 Δ, lsm5 , and lsm8 Δ were rescued by overexpression of U6 snRNA from a high-copy plasmid. Moreover, U6 overexpression rescued myriad lsm Δ lsm Δ double-deletions and lsm Δ lsm Δ lsm Δ triple-deletions. We find that U6 overexpression also rescues a lethal deletion of the U6 snRNP protein subunit Prp24 and that Prp24 overexpression bypasses the essentiality of the U6-associated Lsm subunits. Our results indicate that abetting U6 snRNA is the only essential function of the yeast Lsm2-8 proteins. © 2018 Roth et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Leiomodins: larger members of the tropomodulin (Tmod) gene family

NASA Technical Reports Server (NTRS)

Conley, C. A.; Fritz-Six, K. L.; Almenar-Queralt, A.; Fowler, V. M.

2001-01-01

The 64-kDa autoantigen D1 or 1D, first identified as a potential autoantigen in Graves' disease, is similar to the tropomodulin (Tmod) family of actin filament pointed end-capping proteins. A novel gene with significant similarity to the 64-kDa human autoantigen D1 has been cloned from both humans and mice, and the genomic sequences of both genes have been identified. These genes form a subfamily closely related to the Tmods and are here named the Leiomodins (Lmods). Both Lmod genes display a conserved intron-exon structure, as do three Tmod genes, but the intron-exon structure of the Lmods and the Tmods is divergent. mRNA expression analysis indicates that the gene formerly known as the 64-kDa autoantigen D1 is most highly expressed in a variety of human tissues that contain smooth muscle, earning it the name smooth muscle Leiomodin (SM-Lmod; HGMW-approved symbol LMOD1). Transcripts encoding the novel Lmod gene are present exclusively in fetal and adult heart and adult skeletal muscle, and it is here named cardiac Leiomodin (C-Lmod; HGMW-approved symbol LMOD2). Human C-Lmod is located near the hypertrophic cardiomyopathy locus CMH6 on human chromosome 7q3, potentially implicating it in this disease. Our data demonstrate that the Lmods are evolutionarily related and display tissue-specific patterns of expression distinct from, but overlapping with, the expression of Tmod isoforms. Copyright 2001 Academic Press.

Identification of human cytochrome P450s as autoantigens.

PubMed

Manns, M P; Johnson, E F

1991-01-01

Antimicrosomal antibodies in inflammatory liver diseases all seem to be directed against members of the cytochrome P450 family of proteins. These autoantigens seem to be genetically polymorphic, the autoantibodies are inhibitory, and the autoepitopes are generally conserved among species. Anti-P450 autoantibodies share these characteristics with other autoantibodies, for example, antinuclear antibodies in systemic lupus erythematosus. The identification of P450s as human autoantigens is clinically important. Diagnostic tests will be developed on the basis of cloned antigen, facilitating a better diagnosis of drug-induced and idiopathic autoimmune hepatitis. It is unknown what triggers autoantibody production against cytochrome P450 proteins. Furthermore, their pathogenetic role and thus their involvement in tissue destruction is unclear. In this context LKM1 autoantibodies may serve as a model. Although LKM1 antibodies are inhibitory, all LKM1 antibody-positive patients tested so far are extensive metabolizers for drug metabolism mediated by P450IID6 and express this protein in their livers. Thus, the inhibitory LKM1 autoantibody does not sufficiently penetrate through the intact liver cell membrane to inhibit enzyme function in vivo. Presumably, tissue destruction in autoimmune hepatitis is mediated by liver-infiltrating T lymphocytes. T lymphocytes have been cloned from liver tissue that specifically proliferate in the presence of recombinant cytochrome P450IID6. The construction of overlapping cDNA subclones is also valuable to identify immunodominant B cell as well as relevant T cell epitopes.

Endoplasmic reticulum stress causes autophagy and apoptosis leading to cellular redistribution of the autoantigens Ro/Sjögren's syndrome-related antigen A (SSA) and La/SSB in salivary gland epithelial cells.

PubMed

Katsiougiannis, S; Tenta, R; Skopouli, F N

2015-08-01

The aim of this study was to examine the levels of endoplasmic reticulum (ER) stress in minor salivary glands, to investigate the interplay between ER stress-induced autophagy and apoptosis in human salivary gland (HSG) cells and to test the effect of ER stress-induced apoptosis on the cellular redistribution of the two major Sjögren's syndrome (SS) autoantigens Ro/Sjögren's syndrome-related antigen A (SSA) and La/Sjögren's syndrome-related antigen B (SSB). Minor salivary gland biopsies from SS patients and sicca controls were examined by immunohistochemistry for the expression of 78 kDa glucose-regulated protein/binding immunoglobulin protein (GRP78/BiP) as an indicator of unfolded protein response (UPR). HSG cells were treated with thapsigargin (TG) and cell viability, autophagy and apoptosis were assessed. Immunoblot was applied to detect the conversion of LC3I to LC3II and the protein levels of GRP78/BiP and X-box binding protein-1 (XBP-1). Apoptosis was evaluated by a single-stranded DNA enzyme-linked immunosorbent assay (ELISA). Ro/SSA and La/SSB localization was visualized using immunofluorescence. GRP78/BiP was expressed by acinar and ductal epithelial cells in salivary glands of patients and sicca controls. TG treatment induced autophagy, as indicated by enhanced protein expression of LC3II. The protein levels of UPR marker XBP-1 were increased after TG treatment, while GRP78/BiP levels were decreased. TG treatment resulted in induction of HSG apoptosis. Ro/SSA and La/SSB autoantigens were localized predominantly to the cytoplasm in resting cells, while they were redistributed to cell membrane and blebs in the apoptotic cells. In conclusion, ER stress is activated in minor salivary gland epithelial cells from SS patients and controls. ER stress-induced apoptosis in HSG cells leads to cell surface and apoptotic blebs relocalization of Ro/SSA and La/SSB autoantigens. © 2015 British Society for Immunology.

Autoantigen-specific regulatory T cells induced in patients with Type 1 Diabetes Mellitus by Insulin B-chain immunotherapy

PubMed Central

Orban, Tihamer; Farkas, Klara; Jalahej, Heyam; Kis, Janos; Treszl, Andras; Falk, Ben; Reijonen, Helena; Wolfsdorf, Joseph; Ricker, Alyne; Matthews, Jeffrey B.; Tchao, Nadio; Sayre, Peter; Bianchine, Pete

2009-01-01

There is a growing body of evidence to suggest that the autoimmunity observed in type 1 diabetes mellitus (T1DM) is the result of an imbalance between autoaggressive and regulatory cell subsets. Therapeutics that supplement or enhance the existing regulatory subset are therefore a much sought after goal in this indication. Here, we report the results of a double blind, placebo controlled, phase I clinical trial of a novel antigen-specific therapeutic in 12 subjects with recently diagnosed T1DM. Our primary objective was to test its safety. The study drug, human insulin B-chain in incomplete Freund’s adjuvant (IFA) was administered as a single intramuscular injection, with subjects followed for 2 years. All subjects completed therapy and all follow-up visits. The therapy was generally safe and well-tolerated. Mixed meal stimulated C-peptide responses, measured every 6 months, showed no statistical differences between arms. All patients vaccinated with the autoantigen, but none who received placebo, developed robust insulin-specific humoral and T cell responses. Up to two years following the single injection, in peripheral blood from subjects in the experimental arm, but not the control arm, insulin B-chain-specific CD4+ T cells could be isolated and cloned that showed phenotypic and functional characteristics of regulatory T cells. The induction of a lasting, robust immune response generating autoantigen-specific regulatory T cells provides strong justification for further testing of this therapy in type 1 diabetes. (clinicaltrials.gov identifier NCT00057499). PMID:19931408

Trimming of two major type 1 diabetes driving antigens, GAD65 and IA-2, allows for successful expression in Lactococcus lactis.

PubMed

Robert, S; Van Huynegem, K; Gysemans, C; Mathieu, C; Rottiers, P; Steidler, L

2015-01-01

Type 1 diabetes (T1D) is a chronic autoimmune disease characterised by excessive immune reactions against auto-antigens of pancreatic β-cells. Restoring auto-antigen tolerance remains the superior therapeutic strategy. Oral auto-antigen administration uses the tolerogenic nature of the gut-associated immune system to induce antigen-specific tolerance. However, due to gastric degradation, proper mucosal product delivery often imposes a challenge. Recombinant Lactococcus lactis have proven to be effective and safe carriers for gastrointestinal delivery of therapeutic products: L. lactis secreting diabetes-associated auto-antigens in combination with interleukin (IL)-10 have demonstrated therapeutic efficacy in a well-defined mouse model for T1D. Here, we describe the construction of recombinant L. lactis secreting the 65 kDa isoform of glutamic acid decarboxylase (GAD65) and tyrosine phosphatase-like protein ICA512 (IA-2), two major T1D-related auto-antigens. Attempts to secrete full size human GAD65 and IA-2 protein by L. lactis were unsuccessful. Trimming of GAD65 and IA-2 was investigated to optimise antigen secretion while maintaining sufficient bacterial growth. GAD65370-575 and IA-2635-979 showed to be efficiently secreted by recombinant L. lactis. Antigen secretion was verified by immunoblotting. Plasmid-derived GAD65 and IA-2 expression was combined in single strains with human IL-10 expression, a desired combination to allow tolerance induction. This study reports the generation of recombinant L. lactis secreting two major diabetes-related auto-antigens: human GAD65 and IA-2, by themselves or combined with the anti-inflammatory cytokine human IL-10. Prohibitive sequence obstacles hampering antigen secretion were resolved by trimming the full size proteins.

Alternative exon definition events control the choice between nuclear retention and cytoplasmic export of U11/U12-65K mRNA

PubMed Central

Verbeeren, Jens; Verma, Bhupendra

2017-01-01

Cellular homeostasis of the minor spliceosome is regulated by a negative feed-back loop that targets U11-48K and U11/U12-65K mRNAs encoding essential components of the U12-type intron-specific U11/U12 di-snRNP. This involves interaction of the U11 snRNP with an evolutionarily conserved splicing enhancer giving rise to unproductive mRNA isoforms. In the case of U11/U12-65K, this mechanism controls the length of the 3′ untranslated region (3′UTR). We show that this process is dynamically regulated in developing neurons and some other cell types, and involves a binary switch between translation-competent mRNAs with a short 3′UTR to non-productive isoforms with a long 3′UTR that are retained in the nucleus or/and spliced to the downstream amylase locus. Importantly, the choice between these alternatives is determined by alternative terminal exon definition events regulated by conserved U12- and U2-type 5′ splice sites as well as sequence signals used for pre-mRNA cleavage and polyadenylation. We additionally show that U11 snRNP binding to the U11/U12-65K mRNA species with a long 3′UTR is required for their nuclear retention. Together, our studies uncover an intricate molecular circuitry regulating the abundance of a key spliceosomal protein and shed new light on the mechanisms limiting the export of non-productively spliced mRNAs from the nucleus to the cytoplasm. PMID:28549066

Alternative exon definition events control the choice between nuclear retention and cytoplasmic export of U11/U12-65K mRNA.

PubMed

Verbeeren, Jens; Verma, Bhupendra; Niemelä, Elina H; Yap, Karen; Makeyev, Eugene V; Frilander, Mikko J

2017-05-01

Cellular homeostasis of the minor spliceosome is regulated by a negative feed-back loop that targets U11-48K and U11/U12-65K mRNAs encoding essential components of the U12-type intron-specific U11/U12 di-snRNP. This involves interaction of the U11 snRNP with an evolutionarily conserved splicing enhancer giving rise to unproductive mRNA isoforms. In the case of U11/U12-65K, this mechanism controls the length of the 3' untranslated region (3'UTR). We show that this process is dynamically regulated in developing neurons and some other cell types, and involves a binary switch between translation-competent mRNAs with a short 3'UTR to non-productive isoforms with a long 3'UTR that are retained in the nucleus or/and spliced to the downstream amylase locus. Importantly, the choice between these alternatives is determined by alternative terminal exon definition events regulated by conserved U12- and U2-type 5' splice sites as well as sequence signals used for pre-mRNA cleavage and polyadenylation. We additionally show that U11 snRNP binding to the U11/U12-65K mRNA species with a long 3'UTR is required for their nuclear retention. Together, our studies uncover an intricate molecular circuitry regulating the abundance of a key spliceosomal protein and shed new light on the mechanisms limiting the export of non-productively spliced mRNAs from the nucleus to the cytoplasm.

Autoimmunity to the alpha 3 chain of type IV collagen in glomerulonephritis is triggered by ‘autoantigen complementarity’

PubMed Central

Reynolds, John; Preston, Gloria A.; Pressler, Barrak M.; Hewins, Peter; Brown, Michael; Roth, Aleeza; Alderman, Elizabeth; Bunch, Donna; Jennette, J. Charles; Cook, H. Terence; Falk, Ronald J.; Pusey, Charles D.

2015-01-01

‘Autoantigen complementarity’ is a theory proposing that the initiator of an autoimmune response is not necessarily the autoantigen or its molecular mimic, but may instead be a peptide that is ‘antisense/complementary’ to the autoantigen. We investigated whether such complementary proteins play a role in the immunopathogenesis of autoimmune glomerulonephritis. Experimental autoimmune glomerulonephritis, a model of anti-glomerular basement membrane (GBM) disease, can be induced in Wistar Kyoto (WKY) rats by immunization with the α3 chain of type IV collagen. In this study, WKY rats were immunized with a complementary α3 peptide (c-α3-Gly) comprised of amino acids that ‘complement’ the well characterized epitope on α3(IV)NC1, pCol(24–38). Within 8 weeks post-immunization, these animals developed cresentic glomerulonephritis, similar to pCol(24–38)-immunized rats, while animals immunized with scrambled peptide were normal. Anti-idiotypic antibodies to epitopes from c-α3-Gly-immunized animals were shown to be specific for α3 protein, binding in a region containing sense pCol(24–38) sequence. Interestingly, anticomplementary α3 antibodies were identified in sera from patients with anti-GBM disease, suggesting a role for ‘autoantigen complementarity’ in immunopathogenesis of the human disease. This work supports the idea that autoimmune glomerulonephritis can be initiated through an immune response against a peptide that is anti-sense or complementary to the autoantigen. The implications of this discovery may be far reaching, and other autoimmune diseases could be due to responses to these once unsuspected ‘complementary’ antigens. PMID:25841937

Autoantibodies against 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase (HMGCR) in Patients with Statin-Associated Autoimmune Myopathy

PubMed Central

Mammen, Andrew L.; Chung, Tae; Christopher-Stine, Lisa; Rosen, Paul; Rosen, Antony; Casciola-Rosen, Livia A.

2010-01-01

Objective In addition to inducing a self-limited myopathy, statin use is associated with an immune-mediated necrotizing (IMNM) myopathy with autoantibodies recognizing ~ 200 and ~100 kDa autoantigens. Identifying these molecules will clarify disease mechanism and facilitate diagnosis. Methods The effect of statin treatment on autoantigen expression was addressed by immunoprecipitation using patient sera. The identity of the ~100 kDa autoantigen was confirmed by immunoprecipitating in vitro-translated HMGCR protein. HMGCR expression in muscle was analyzed by immunofluorescence. A cohort of myopathy patients was screened for anti-HMGCR autoantibodies by ELISA and genotyped for the rs4149056 C allele, a predictor of self-limited statin myopathy. Results Statin exposure induced expression of the ~200/~100 kDa autoantigens in cultured cells. HMGCR was identified as the ~100 kDa autoantigen. Competition experiments demonstrated no distinct autoantibodies recognizing the ~200 kDa protein. In muscle biopsies from anti-HMGCR positive patients, HMGCR expression was up-regulated in cells expressing NCAM, a marker of muscle regeneration. Anti-HMGCR autoantibodies were found in 45 of 750 patients presenting to the Johns Hopkins Myositis Center (6%). Among patients age 50 or older, 92% were exposed to statins. The prevalence of the rs4149056 C allele was not increased in anti-HMGCR subjects. Conclusion Statins up-regulate expression of HMGCR, the major target of autoantibodies in statin-associated IMNM. Regenerating muscle cells express high levels of HMGCR, which may sustain the immune response even after statins are discontinued. These studies demonstrate a mechanistic link between an environmental trigger and the development of sustained autoimmunity. Detection of anti-HMGCR autoantibodies may facilitate diagnosis and direct therapy. PMID:21360500

Anti-inflammatory effect of garlic 14-kDa protein on LPS-stimulated-J774A.1 macrophages.

PubMed

Rabe, Shahrzad Zamani Taghizadeh; Ghazanfari, Tooba; Siadat, Zahra; Rastin, Maryam; Rabe, Shahin Zamani Taghizadeh; Mahmoudi, Mahmoud

2015-04-01

Garlic 14-kDa protein is purified from garlic (Allium sativum L.) which is used in traditional medicine and exerts various immunomodulatory activities. The present study investigated the suppressive effect of garlic 14-kDa protein on LPS-induced expression of pro-inflammatory mediators and underlying mechanism in inflammatory macrophages. J774A.1 macrophages were treated with 14-kDa protein (5-30 μg/ml) with/without LPS (1 μg/ml) and the production of inflammatory mediators such as prostaglandin E2 (PGE2), TNF-α, and IL-1β released were measured using ELISA. Nitric oxide (NO) production was determined using the Griess method. The anti-inflammatory activity of 14-kDa protein was examined by measuring inducible nitric oxide synthase and cyclooxygenase-2 proteins using western blot. The expression of nuclear NF-κB p65 subunit was assessed by western blot. Garlic 14-kDa protein significantly inhibited the excessive production of NO, PGE, TNF-α, and IL-1β in lipopolysaccharide (LPS)-activated J774A.1 macrophages in a concentration-related manner without cytotoxic effect. Western blot analysis demonstrated that garlic 14-kDa protein suppressed corresponding inducible NO synthase expression and activated cyclooxygenase-2 protein expression. The inhibitory effect was mediated partly by a reduction in the activity and expression of transcription factor NF-κB protein. Our results suggested, for the first time, garlic 14-kDa protein exhibits anti-inflammatory properties in macrophages possibly by suppressing the inflammatory mediators via the inhibition of transcription factor NF-κB signaling pathway. The traditional use of garlic as anti-inflammatory remedy could be ascribed partly to 14-kDa protein content. This protein might be a useful candidate for controlling inflammatory diseases and further investigations in vivo.

The splicing factor U2AF65 stabilizes TRF1 protein by inhibiting its ubiquitin-dependent proteolysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Jeonghee; Chung, In Kwon, E-mail: topoviro@yonsei.ac.kr

Highlights: •Identification of U2AF65 as a novel TRF1-interacting protein. •U2AF65 stabilizes TRF1 protein by inhibiting its ubiquitin-dependent proteolysis. •U2AF65 interferes with the interaction between TRF1 and Fbx4. •U2AF65 represents a new route for modulating TRF1 function at telomeres. -- Abstract: The human telomeric protein TRF1 is a component of the six-subunit protein complex shelterin, which provides telomere protection by organizing the telomere into a high-order structure. TRF1 functions as a negative regulator of telomere length by controlling the access of telomerase to telomeres. Thus, the cellular abundance of TRF1 at telomeres should be maintained and tightly regulated to ensure propermore » telomere function. Here, we identify U2 small nuclear ribonucleoprotein (snRNP) auxiliary factor 65 (U2AF65), an essential pre-mRNA splicing factor, as a novel TRF1-interacting protein. U2AF65 interacts with TRF1 in vitro and in vivo and is capable of stabilizing TRF1 protein by inhibiting its ubiquitin-dependent proteolysis. We also found that U2AF65 interferes with the interaction between TRF1 and Fbx4, an E3 ubiquitin ligase for TRF1. Depletion of endogenous U2AF65 expression by short interfering RNA (siRNA) reduced the stability of endogenous TRF1 whereas overexpression of U2AF65 significantly extended the half-life of TRF1. These findings demonstrate that U2AF65 plays a critical role in regulating the level of TRF1 through physical interaction and ubiquitin-mediated proteolysis. Hence, U2AF65 represents a new route for modulating TRF1 function at telomeres.« less

A conserved intronic U1 snRNP-binding sequence promotes trans-splicing in Drosophila

PubMed Central

Gao, Jun-Li; Fan, Yu-Jie; Wang, Xiu-Ye; Zhang, Yu; Pu, Jia; Li, Liang; Shao, Wei; Zhan, Shuai; Hao, Jianjiang

2015-01-01

Unlike typical cis-splicing, trans-splicing joins exons from two separate transcripts to produce chimeric mRNA and has been detected in most eukaryotes. Trans-splicing in trypanosomes and nematodes has been characterized as a spliced leader RNA-facilitated reaction; in contrast, its mechanism in higher eukaryotes remains unclear. Here we investigate mod(mdg4), a classic trans-spliced gene in Drosophila, and report that two critical RNA sequences in the middle of the last 5′ intron, TSA and TSB, promote trans-splicing of mod(mdg4). In TSA, a 13-nucleotide (nt) core motif is conserved across Drosophila species and is essential and sufficient for trans-splicing, which binds U1 small nuclear RNP (snRNP) through strong base-pairing with U1 snRNA. In TSB, a conserved secondary structure acts as an enhancer. Deletions of TSA and TSB using the CRISPR/Cas9 system result in developmental defects in flies. Although it is not clear how the 5′ intron finds the 3′ introns, compensatory changes in U1 snRNA rescue trans-splicing of TSA mutants, demonstrating that U1 recruitment is critical to promote trans-splicing in vivo. Furthermore, TSA core-like motifs are found in many other trans-spliced Drosophila genes, including lola. These findings represent a novel mechanism of trans-splicing, in which RNA motifs in the 5′ intron are sufficient to bring separate transcripts into close proximity to promote trans-splicing. PMID:25838544

U bodies respond to nutrient stress in Drosophila

DOE Office of Scientific and Technical Information (OSTI.GOV)

Buckingham, Mickey; Liu, Ji-Long, E-mail: jilong.liu@dpag.ox.ac.uk

The neurodegenerative disease spinal muscular atrophy (SMA) is caused by mutation of the survival motor neuron 1 (SMN1) gene. Cytoplasmic SMN protein-containing granules, known as U snRNP bodies (U bodies), are thought to be responsible for the assembly and storage of small nuclear ribonucleoproteins (snRNPs) which are essential for pre-mRNA splicing. U bodies exhibit close association with cytoplasmic processing bodies (P bodies), which are involved in mRNA decay and translational repression. The close association of the U body and P body in Drosophila resemble that of the stress granule and P body in yeast and mammalian cells. However, it ismore » unknown whether the U body is responsive to any stress. Using Drosophila oogenesis as a model, here we show that U bodies increase in size following nutritional deprivation. Despite nutritional stress, U bodies maintain their close association with P bodies. Our results show that U bodies are responsive to nutrition changes, presumably through the U body-P body pathway.« less

Expression of the autoantigen TRIM33/TIF1γ in skin and muscle of patients with dermatomyositis is upregulated, together with markers of cellular stress.

PubMed

Scholtissek, B; Ferring-Schmitt, S; Maier, J; Wenzel, J

2017-08-01

Dermatomyositis (DM) is an autoimmune disorder associated with a dysregulation of immune homeostasis of both the innate and adaptive immune system. Earlier data suggested that these two arms of the immune system interconnect in DM. In the current study, we analysed the association of autoantigen expression [adaptive system components: Mi2, transcriptional intermediary factor (TIF)1γ, small ubiquitin-like modifier 1 activating enzyme subunit (SAE)1, melanoma differentiation-associated protein (MDA)5] with markers of cellular stress (innate system components: MxA, p53) in skin and muscle (immunohistology and gene expression data, respectively). We found that distinctive self-antigens of DM were elevated in both skin and muscle tissue. In particular, TIF1γ expression was seen in autoimmune diseases including DM, but not in other inflammatory skin disorders. This upregulation was closely associated with p53 expression and type I interferon-regulated inflammation, suggesting that upregulation of autoantigens in the skin and muscle of patients with DM might be driven by cellular stress. Better understanding of these mechanisms could pave the way for new therapeutic concepts focusing on stress reduction. © 2017 British Association of Dermatologists.

Nuclear 82-kDa choline acetyltransferase decreases amyloidogenic APP metabolism in neurons from APP/PS1 transgenic mice.

PubMed

Albers, Shawn; Inthathirath, Fatima; Gill, Sandeep K; Winick-Ng, Warren; Jaworski, Ewa; Wong, Daisy Y L; Gros, Robert; Rylett, R Jane

2014-09-01

Alzheimer disease (AD) is associated with increased amyloidogenic processing of amyloid precursor protein (APP) to β-amyloid peptides (Aβ), cholinergic neuron loss with decreased choline acetyltransferase (ChAT) activity, and cognitive dysfunction. Both 69-kDa ChAT and 82-kDa ChAT are expressed in cholinergic neurons in human brain and spinal cord with 82-kDa ChAT localized predominantly to neuronal nuclei, suggesting potential alternative functional roles for the enzyme. By gene microarray analysis, we found that 82-kDa ChAT-expressing IMR32 neural cells have altered expression of genes involved in diverse cellular functions. Importantly, genes for several proteins that regulate APP processing along amyloidogenic and non-amyloidogenic pathways are differentially expressed in 82-kDa ChAT-containing cells. The predicted net effect based on observed changes in expression patterns of these genes would be decreased amyloidogenic APP processing with decreased Aβ production. This functional outcome was verified experimentally as a significant decrease in BACE1 protein levels and activity and a concomitant reduction in the release of endogenous Aβ1-42 from neurons cultured from brains of AD-model APP/PS1 transgenic mice. The expression of 82-kDa ChAT in neurons increased levels of GGA3, which is involved in trafficking BACE1 to lysosomes for degradation. shRNA-induced decreases in GGA3 protein levels attenuated the 82-kDa ChAT-mediated decreases in BACE1 protein and activity and Aβ1-42 release. Evidence that 82-kDa ChAT can enhance GGA3 gene expression is shown by enhanced GGA3 gene promoter activity in SN56 neural cells expressing this ChAT protein. These studies indicate a novel relationship between cholinergic neurons and APP processing, with 82-kDa ChAT acting as a negative regulator of Aβ production. This decreased formation of Aβ could result in protection for cholinergic neurons, as well as protection of other cells in the vicinity that are sensitive to

Drosophila SMN complex proteins Gemin2, Gemin3, and Gemin5 are components of U bodies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cauchi, Ruben J.; Sanchez-Pulido, Luis; Liu, Ji-Long, E-mail: jilong.liu@dpag.ox.ac.uk

2010-08-15

Uridine-rich small nuclear ribonucleoproteins (U snRNPs) play key roles in pre-mRNA processing in the nucleus. The assembly of most U snRNPs takes place in the cytoplasm and is facilitated by the survival motor neuron (SMN) complex. Discrete cytoplasmic RNA granules called U bodies have been proposed to be specific sites for snRNP assembly because they contain U snRNPs and SMN. U bodies invariably associate with P bodies, which are involved in mRNA decay and translational control. However, it remains unknown whether other SMN complex proteins also localise to U bodies. In Drosophila there are four SMN complex proteins, namely SMN,more » Gemin2/CG10419, Gemin3 and Gemin5/Rigor mortis. Drosophila Gemin3 was originally identified as the Drosophila orthologue of human and yeast Dhh1, a component of P bodies. Through an in silico analysis of the DEAD-box RNA helicases we confirmed that Gemin3 is the bona fide Drosophila orthologue of vertebrate Gemin3 whereas the Drosophila orthologue of Dhh1 is Me31B. We then made use of the Drosophila egg chamber as a model system to study the subcellular distribution of the Gemin proteins as well as Me31B. Our cytological investigations show that Gemin2, Gemin3 and Gemin5 colocalise with SMN in U bodies. Although they are excluded from P bodies, as components of U bodies, Gemin2, Gemin3 and Gemin5 are consistently found associated with P bodies, wherein Me31B resides. In addition to a role in snRNP biogenesis, SMN complexes residing in U bodies may also be involved in mRNP assembly and/or transport.« less

Anti-Saccharomyces cerevisiae autoantibodies in autoimmune diseases: from bread baking to autoimmunity.

PubMed

Rinaldi, Maurizio; Perricone, Roberto; Blank, Miri; Perricone, Carlo; Shoenfeld, Yehuda

2013-10-01

Saccharomyces cerevisiae is best known as the baker's and brewer's yeast, but its residual traces are also frequent excipients in some vaccines. Although anti-S. cerevisiae autoantibodies (ASCAs) are considered specific for Crohn's disease, a growing number of studies have detected high levels of ASCAs in patients affected with autoimmune diseases as compared with healthy controls, including antiphospholipid syndrome, systemic lupus erythematosus, type 1 diabetes mellitus, and rheumatoid arthritis. Commensal microorganisms such as Saccharomyces are required for nutrition, proper development of Peyer's aggregated lymphoid tissue, and tissue healing. However, even the commensal nonclassically pathogenic microbiota can trigger autoimmunity when fine regulation of immune tolerance does not work properly. For our purposes, the protein database of the National Center for Biotechnology Information (NCBI) was consulted, comparing Saccharomyces mannan to several molecules with a pathogenetic role in autoimmune diseases. Thanks to the NCBI bioinformation technology tool, several overlaps in molecular structures (50-100 %) were identified when yeast mannan, and the most common autoantigens were compared. The autoantigen U2 snRNP B″ was found to conserve a superfamily protein domain that shares 83 % of the S. cerevisiae mannan sequence. Furthermore, ASCAs may be present years before the diagnosis of some associated autoimmune diseases as they were retrospectively found in the preserved blood samples of soldiers who became affected by Crohn's disease years later. Our results strongly suggest that ASCAs' role in clinical practice should be better addressed in order to evaluate their predictive or prognostic relevance.

A common structural motif in immunopotentiating peptides with sequences present in human autoantigens. Elicitation of a response mediated by monocytes and Th1 cells.

PubMed

López-Moratalla, N; Ruíz, E; López-Zabalza, M J; Santiago, E

1996-12-16

We have found a common structural motif in human autoantigens, heat shock proteins and viral proteins. Peptides modelled after sequences present in those molecules were synthesized and immunomodulating properties tested. They share a core of 15 amino acid residues and a common pattern ('2-6-11' motif) characterized by requirements at fixed positions with respect to a Pro (position 6); an apolar residue or a Lys at position 2; and a Glu, Asp or Lys at position 11. Any of these peptides, when added to cultures of lymphomononuclear cells, caused the activation of monocytes manifested by a release of IL-1 alpha, IL-1 beta and TNF alpha. A release of INF gamma and IL-2 took also place; this release was abolished by anti-DR antibodies. Neither IL-4 nor IL-5 could be detected. This suggests a presentation by APCs and the appearance of cells with a Th1 phenotype. Monocytes and Th1 cells freshly obtained from 12 patients of Graves' disease, 8 of Hashimoto's disease and 8 of primary biliary cirrhosis exhibited activation features similar to those found in cells from healthy subjects incubated in the presence of peptides with a "2-6-11' motif and representing fragments of autoantigens. Their immunopotentiating properties suggest their involvement in the initiation or progression of the autoimmune response mediated by activated monocytes and Th1 cells.

The VPH1 gene encodes a 95-kDa integral membrane polypeptide required for in vivo assembly and activity of the yeast vacuolar H(+)-ATPase.

PubMed

Manolson, M F; Proteau, D; Preston, R A; Stenbit, A; Roberts, B T; Hoyt, M A; Preuss, D; Mulholland, J; Botstein, D; Jones, E W

1992-07-15

Yeast vacuolar acidification-defective (vph) mutants were identified using the pH-sensitive fluorescence of 6-carboxyfluorescein diacetate (Preston, R. A., Murphy, R. F., and Jones, E. W. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7027-7031). Vacuoles purified from yeast bearing the vph1-1 mutation had no detectable bafilomycin-sensitive ATPase activity or ATP-dependent proton pumping. The peripherally bound nucleotide-binding subunits of the vacuolar H(+)-ATPase (60 and 69 kDa) were no longer associated with vacuolar membranes yet were present in wild type levels in yeast whole cell extracts. The VPH1 gene was cloned by complementation of the vph1-1 mutation and independently cloned by screening a lambda gt11 expression library with antibodies directed against a 95-kDa vacuolar integral membrane protein. Deletion disruption of the VPH1 gene revealed that the VPH1 gene is not essential for viability but is required for vacuolar H(+)-ATPase assembly and vacuolar acidification. VPH1 encodes a predicted polypeptide of 840 amino acid residues (molecular mass 95.6 kDa) and contains six putative membrane-spanning regions. Cell fractionation and immunodetection demonstrate that Vph1p is a vacuolar integral membrane protein that co-purifies with vacuolar H(+)-ATPase activity. Multiple sequence alignments show extensive homology over the entire lengths of the following four polypeptides: Vph1p, the 116-kDa polypeptide of the rat clathrin-coated vesicles/synaptic vesicle proton pump, the predicted polypeptide encoded by the yeast gene STV1 (Similar To VPH1, identified as an open reading frame next to the BUB2 gene), and the TJ6 mouse immune suppressor factor.

A Drosophila model of spinal muscular atrophy uncouples snRNP biogenesis functions of survival motor neuron from locomotion and viability defects.

PubMed

Praveen, Kavita; Wen, Ying; Matera, A Gregory

2012-06-28

The spinal muscular atrophy (SMA) protein, survival motor neuron (SMN), functions in the biogenesis of small nuclear ribonucleoproteins (snRNPs). SMN has also been implicated in tissue-specific functions; however, it remains unclear which of these is important for the etiology of SMA. Smn null mutants display larval lethality and show significant locomotion defects as well as reductions in minor-class spliceosomal snRNAs. Despite these reductions, we found no appreciable defects in the splicing of mRNAs containing minor-class introns. Transgenic expression of low levels of either wild-type or an SMA patient-derived form of SMN rescued the larval lethality and locomotor defects; however, snRNA levels were not restored. Thus, the snRNP biogenesis function of SMN is not a major contributor to the phenotype of Smn null mutants. These findings have major implications for SMA etiology because they show that SMN's role in snRNP biogenesis can be uncoupled from the organismal viability and locomotor defects. Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.

Variability in clinical phenotypes of PRPF8-linked autosomal dominant retinitis pigmentosa correlates with differential PRPF8/SNRNP200 interactions.

PubMed

Escher, Pascal; Passarin, Olga; Munier, Francis L; Tran, Viet H; Vaclavik, Veronika

2018-01-01

To expand the genotype/phenotype correlations in patients with autosomal dominant retinitis pigmentosa (adRP) harboring PRPF8 variants. Two patients, a father and his daughter, harboring a novel p.PRPF8-Glu2331* variant, underwent ophthalmic examination at 3-year-interval, including fundus photography, fundus autofluorescence, optical coherence tomography, and ISCEV standard full field ERGs. All reported disease-causing PRPF8 variants were collected and localized in the PRPF8 and PRPF8/SNRNP200 protein structures. The p.PRPF8-Glu2331* variant results in a truncated PRPF8 protein lacking the last five C-terminal amino acids and caused in the two patients a severe clinical phenotype, with the macula being affected from the second decade on. All but two adRP-linked variants are located in the last exon 43 encoding the C-terminal tail of the C-terminal PRPF8 Jab1 domain. The p.PRPF8-Ser2118Phe and -Asn2280Lys variants encoded by exons 39 and 42, respectively, are located at the basis of the C-terminal tail. Frame-shift mutations and nonconservative amino acid changes in PRPF8 typically cause severe clinical phenotypes. The conservative missense variant p.PRPF8-Arg2310Lys that is not altering the global charge of the C-terminal tail, and variants located at the basis of the C-terminal tail show milder clinical phenotypes, in accordance with functional data on PRPF8/SNRNP200 interactions in yeast.

Autoantigen microarrays reveal autoantibodies associated with proliferative nephritis and active disease in pediatric systemic lupus erythematosus.

PubMed

Haddon, D James; Diep, Vivian K; Price, Jordan V; Limb, Cindy; Utz, Paul J; Balboni, Imelda

2015-06-17

Pediatric systemic lupus erythematosus (pSLE) patients often initially present with more active and severe disease than adults, including a higher frequency of lupus nephritis. Specific autoantibodies, including anti-C1q, anti-DNA and anti-alpha-actinin, have been associated with kidney involvement in SLE, and DNA antibodies are capable of initiating early-stage lupus nephritis in severe combined immunodeficiency (SCID) mice. Over 100 different autoantibodies have been described in SLE patients, highlighting the need for comprehensive autoantibody profiling. Knowledge of the antibodies associated with pSLE and proliferative nephritis will increase the understanding of SLE pathogenesis, and may aid in monitoring patients for renal flare. We used autoantigen microarrays composed of 140 recombinant or purified antigens to compare the serum autoantibody profiles of new-onset pSLE patients (n = 45) to healthy controls (n = 17). We also compared pSLE patients with biopsy-confirmed class III or IV proliferative nephritis (n = 23) and without significant renal involvement (n = 18). We performed ELISA with selected autoantigens to validate the microarray findings. We created a multiple logistic regression model, based on the ELISA and clinical information, to predict whether a patient had proliferative nephritis, and used a validation cohort (n = 23) and longitudinal samples (88 patient visits) to test its accuracy. Fifty autoantibodies were at significantly higher levels in the sera of pSLE patients compared to healthy controls, including anti-B cell-activating factor (BAFF). High levels of anti-BAFF were associated with active disease. Thirteen serum autoantibodies were present at significantly higher levels in pSLE patients with proliferative nephritis than those without, and we confirmed five autoantigens (dsDNA, C1q, collagens IV and X and aggrecan) by ELISA. Our model, based on ELISA measurements and clinical variables, correctly identified patients with proliferative

Autoantibodies of IgM and IgG classes show differences in recognition of multiple autoantigens in chronic obstructive pulmonary disease.

PubMed

Shindi, Reham; Almehairi, Amna; Negm, Ola H; Kalsheker, Noor; Gale, Nichola S; Shale, Dennis J; Harrison, Timothy W; Bolton, Charlotte E; John, Michelle; Todd, Ian; Tighe, Patrick J; Fairclough, Lucy C

2017-10-01

Autoimmunity occurs in chronic obstructive pulmonary disease (COPD). We describe an antigen microarray for detecting serum autoantibodies (AAbs) to determine how IgM, as well as IgG, AAbs distinguish patients with COPD from controls with a history of smoking without COPD. All COPD patients' sera contained elevated levels of AAbs to some of 30 autoantigens. There were significant differences in the autoantigenic specificities of IgM AAbs compared to IgG AAbs in the COPD sera: for example, AAbs to histone and scl-70 were mainly IgG, whereas AAbs to CENP-B and La/ssB were mainly IgM; by contrast, IgM and IgG AAbs to collagen-V were equally prevalent. Thus, a combination of IgM and IgG AAbs specific for multiple autoantigens are detected in all cases of COPD at a level at which all non-COPD controls are negative for AAbs. This highlights the importance of different classes of AAbs to a range of autoantigens in COPD. Copyright © 2017 Elsevier Inc. All rights reserved.

Large-scale remodeling of a repressed exon ribonucleoprotein to an exon definition complex active for splicing

PubMed Central

Wongpalee, Somsakul Pop; Vashisht, Ajay; Sharma, Shalini; Chui, Darryl; Wohlschlegel, James A; Black, Douglas L

2016-01-01

Polypyrimidine-tract binding protein PTBP1 can repress splicing during the exon definition phase of spliceosome assembly, but the assembly steps leading to an exon definition complex (EDC) and how PTBP1 might modulate them are not clear. We found that PTBP1 binding in the flanking introns allowed normal U2AF and U1 snRNP binding to the target exon splice sites but blocked U2 snRNP assembly in HeLa nuclear extract. Characterizing a purified PTBP1-repressed complex, as well as an active early complex and the final EDC by SILAC-MS, we identified extensive PTBP1-modulated changes in exon RNP composition. The active early complex formed in the absence of PTBP1 proceeded to assemble an EDC with the eviction of hnRNP proteins, the late recruitment of SR proteins, and binding of the U2 snRNP. These results demonstrate that during early stages of splicing, exon RNP complexes are highly dynamic with many proteins failing to bind during PTBP1 arrest. DOI: http://dx.doi.org/10.7554/eLife.19743.001 PMID:27882870

Detection of anticentromere antibodies using cloned autoantigen CENP-B.

PubMed

Rothfield, N; Whitaker, D; Bordwell, B; Weiner, E; Senecal, J L; Earnshaw, W

1987-12-01

A solid-phase enzyme-linked immunosorbent assay has been established using a cloned fusion protein, CtermCENP-B [beta-gal], as antigen. The fusion protein carries the major epitope of CENP-B, the major centromeric autoantigen. The enzyme-linked immunosorbent assay was more sensitive than immunofluorescence techniques in detecting anticentromere antibodies in patients with scleroderma or Raynaud's disease, and was weakly positive in 3% of normal controls and in 3% of 70 patients with other connective tissue diseases.

All Small Nuclear RNAs (snRNAs) of the [U4/U6.U5] Tri-snRNP Localize to Nucleoli; Identification of the Nucleolar Localization Element of U6 snRNA

PubMed Central

Gerbi, Susan A.; Lange, Thilo Sascha

2002-01-01

Previously, we showed that spliceosomal U6 small nuclear RNA (snRNA) transiently passes through the nucleolus. Herein, we report that all individual snRNAs of the [U4/U6.U5] tri-snRNP localize to nucleoli, demonstrated by fluorescence microscopy of nucleolar preparations after injection of fluorescein-labeled snRNA into Xenopus oocyte nuclei. Nucleolar localization of U6 is independent from [U4/U6] snRNP formation since sites of direct interaction of U6 snRNA with U4 snRNA are not nucleolar localization elements. Among all regions in U6, the only one required for nucleolar localization is its 3′ end, which associates with the La protein and subsequently during maturation of U6 is bound by Lsm proteins. This 3′-nucleolar localization element of U6 is both essential and sufficient for nucleolar localization and also required for localization to Cajal bodies. Conversion of the 3′ hydroxyl of U6 snRNA to a 3′ phosphate prevents association with the La protein but does not affect U6 localization to nucleoli or Cajal bodies. PMID:12221120

Tir8/Sigirr prevents murine lupus by suppressing the immunostimulatory effects of lupus autoantigens

PubMed Central

Lech, Maciej; Kulkarni, Onkar P.; Pfeiffer, Stephanie; Savarese, Emina; Krug, Anne; Garlanda, Cecilia; Mantovani, Alberto; Anders, Hans-Joachim

2008-01-01

The Sigirr gene (also known as Tir8) encodes for an orphan receptor of the Toll-like receptor (TLR)/interleukin 1 receptor family that inhibits TLR-mediated pathogen recognition in dendritic cells. Here, we show that Sigirr also inhibits the activation of dendritic cells and B cells upon exposure to RNA and DNA lupus autoantigens. To evaluate the functional role of Sigirr in the pathogenesis of systemic lupus erythematosus (SLE), we generated Sigirr-deficient C57BL/6-lpr/lpr mice. These mice developed a progressive lymphoproliferative syndrome followed by severe autoimmune lung disease and lupus nephritis within 6 mo of age as compared with the minor abnormalities observed in C57BL/6-lpr/lpr mice. Lack of Sigirr was associated with enhanced activation of dendritic cells and increased expression of multiple proinflammatory and antiapoptotic mediators. In the absence of Sigirr, CD4 T cell numbers were increased and CD4+CD25+ T cell numbers were reduced. Furthermore, lack of Sigirr enhanced the activation and proliferation of B cells, including the production of autoantibodies against multiple nuclear lupus autoantigens. These data identify Sigirr as a novel SLE susceptibility gene in mice. PMID:18644972

Relationships between major epitopes of the IA-2 autoantigen in Type 1 diabetes: Implications for determinant spreading.

PubMed

McLaughlin, Kerry A; Richardson, Carolyn C; Williams, Stefan; Bonifacio, Ezio; Morgan, Diana; Feltbower, Richard G; Powell, Michael; Rees Smith, Bernard; Furmaniak, Jadwiga; Christie, Michael R

2015-10-01

Diversification of autoimmunity to islet autoantigens is critical for progression to Type 1 diabetes. B-cells participate in diversification by modifying antigen processing, thereby influencing which peptides are presented to T-cells. In Type 1 diabetes, JM antibodies are associated with T-cell responses to PTP domain peptides. We investigated whether this is the consequence of close structural alignment of JM and PTP domain determinants on IA-2. Fab fragments of IA-2 antibodies with epitopes mapped to the JM domain blocked IA-2 binding of antibodies that recognise epitopes in the IA-2 PTP domain. Peptides from both the JM and PTP domains were protected from degradation during proteolysis of JM antibody:IA-2 complexes and included those representing major T-cell determinants in Type 1 diabetes. The results demonstrate close structural relationships between JM and PTP domain epitopes on IA-2. Stabilisation of PTP domain peptides during proteolysis in JM-specific B-cells may explain determinant spreading in IA-2 autoimmunity. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

CYCLIN-DEPENDENT KINASE G1 Is Associated with the Spliceosome to Regulate CALLOSE SYNTHASE5 Splicing and Pollen Wall Formation in Arabidopsis[C][W][OA

PubMed Central

Huang, Xue-Yong; Niu, Jin; Sun, Ming-Xi; Zhu, Jun; Gao, Ju-Fang; Yang, Jun; Zhou, Que; Yang, Zhong-Nan

2013-01-01

Arabidopsis thaliana CYCLIN-DEPEDENT KINASE G1 (CDKG1) belongs to the family of cyclin-dependent protein kinases that were originally characterized as cell cycle regulators in eukaryotes. Here, we report that CDKG1 regulates pre-mRNA splicing of CALLOSE SYNTHASE5 (CalS5) and, therefore, pollen wall formation. The knockout mutant cdkg1 exhibits reduced male fertility with impaired callose synthesis and abnormal pollen wall formation. The sixth intron in CalS5 pre-mRNA, a rare type of intron with a GC 5′ splice site, is abnormally spliced in cdkg1. RNA immunoprecipitation analysis suggests that CDKG1 is associated with this intron. CDKG1 contains N-terminal Ser/Arg (RS) motifs and interacts with splicing factor Arginine/Serine-Rich Zinc Knuckle-Containing Protein33 (RSZ33) through its RS region to regulate proper splicing. CDKG1 and RS-containing Zinc Finger Protein22 (SRZ22), a splicing factor interacting with RSZ33 and U1 small nuclear ribonucleoprotein particle (snRNP) component U1-70k, colocalize in nuclear speckles and reside in the same complex. We propose that CDKG1 is recruited to U1 snRNP through RSZ33 to facilitate the splicing of the sixth intron of CalS5. PMID:23404887

Trichohyalin is a potential major autoantigen in human alopecia areata.

PubMed

Leung, Man Ching; Sutton, Chris W; Fenton, David A; Tobin, Desmond J

2010-10-01

Several lines of evidence support an autoimmune basis for alopecia areata (AA), a common putative autoimmune hair loss disorder. However, definitive support is lacking largely because the identity of hair follicle (HF) autoantigen(s) involved in its pathogenesis remains unknown. Here, we isolated AA-reactive HF-specific antigens from normal human scalp anagen HF extracts by immunoprecipitation using serum antibodies from 10 AA patients. Samples were analyzed by LC-MALDI-TOF/TOF mass spectrometry, which indicated strong reactivity to the hair growth phase-specific structural protein trichohyalin in all AA sera. Keratin 16 (K16) was also identified as another potential AA-relevant target HF antigen. Double immunofluorescence studies using AA (and control sera) together with a monoclonal antibody to trichohyalin revealed that AA sera contained immunoreactivity that colocalized with trichohyalin in the growth phase-specific inner root sheath of HF. Furthermore, a partial colocalization of AA serum reactivity with anti-K16 antibody was observed in the outer root sheath of the HF. In summary, this study supports the involvement of an immune response to anagen-specific HFs antigens in AA and specifically suggests that an immune response to trichohyalin and K16 may have a role in the pathogenesis of the enigmatic disorder.

Identification of new autoantigens for primary biliary cirrhosis using human proteome microarrays.

PubMed

Hu, Chao-Jun; Song, Guang; Huang, Wei; Liu, Guo-Zhen; Deng, Chui-Wen; Zeng, Hai-Pan; Wang, Li; Zhang, Feng-Chun; Zhang, Xuan; Jeong, Jun Seop; Blackshaw, Seth; Jiang, Li-Zhi; Zhu, Heng; Wu, Lin; Li, Yong-Zhe

2012-09-01

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease of unknown etiology and is considered to be an autoimmune disease. Autoantibodies are important tools for accurate diagnosis of PBC. Here, we employed serum profiling analysis using a human proteome microarray composed of about 17,000 full-length unique proteins and identified 23 proteins that correlated with PBC. To validate these results, we fabricated a PBC-focused microarray with 21 of these newly identified candidates and nine additional known PBC antigens. By screening the PBC microarrays with additional cohorts of 191 PBC patients and 321 controls (43 autoimmune hepatitis, 55 hepatitis B virus, 31 hepatitis C virus, 48 rheumatoid arthritis, 45 systematic lupus erythematosus, 49 systemic sclerosis, and 50 healthy), six proteins were confirmed as novel PBC autoantigens with high sensitivities and specificities, including hexokinase-1 (isoforms I and II), Kelch-like protein 7, Kelch-like protein 12, zinc finger and BTB domain-containing protein 2, and eukaryotic translation initiation factor 2C, subunit 1. To facilitate clinical diagnosis, we developed ELISA for Kelch-like protein 12 and zinc finger and BTB domain-containing protein 2 and tested large cohorts (297 PBC and 637 control sera) to confirm the sensitivities and specificities observed in the microarray-based assays. In conclusion, our research showed that a strategy using high content protein microarray combined with a smaller but more focused protein microarray can effectively identify and validate novel PBC-specific autoantigens and has the capacity to be translated to clinical diagnosis by means of an ELISA-based method.

Investigations of Caspr2, an autoantigen of encephalitis and neuromyotonia

PubMed Central

Lancaster, Eric; Huijbers, Maartje GM; Bar, Vered; Boronat, Anna; Wong, Andrew; Martinez-Hernandez, Eugenia; Wilson, Christina; Jacobs, Dina; Lai, Meizan; Walker, Russell W; Graus, Francesc; Bataller, Luis; Illa, Isabel; Markx, Sander; Strauss, Kevin A.; Peles, Elior; Scherer, Steven S; Dalmau, Josep

2010-01-01

Objective To report clinical and immunological investigations of contactin-associated protein-like 2 (Caspr2), an autoantigen of encephalitis and peripheral nerve hyperexcitability (PNH) previously attributed to voltage-gated potassium channels (VGKC). Methods Clinical analysis of patients with encephalitis, PNH, or both. Immunoprecipitation and mass spectrometry were used to identify the antigen and to develop an assay with Caspr2-expressing cells. Immunoabsorption with Caspr2 and comparative immunostaining of brain and peripheral nerve of wild-type and Caspr2-null mice were used to assess antibody specificity. Results Using Caspr2-expressing cells, antibodies were identified in 8 patients but not in 140 patients with several types of autoimmune or viral encephalitis, PNH, or mutations of the Caspr2-encoding gene. Patients’ antibodies reacted with brain and peripheral nerve in a pattern that co-localized with Caspr2. This reactivity was abrogated after immunoabsorption with Caspr2 and was absent in tissues from Caspr2-null mice. Of the 8 patients with Caspr2 antibodies, 7 had encephalopathy or seizures, 5 neuropathy or PNH, and 1 isolated PNH. Three patients had also myasthenia gravis, bulbar weakness, or symptoms that initially suggested motor neuron disease. None of the patients had active cancer; 7 responded to immunotherapy and were healthy or only mildly disabled at last follow-up (median 8 months, range 6–84). Interpretation Caspr2 is an autoantigen of encephalitis and PNH previously attributed to VGKC antibodies. The occurrence of other autoantibodies may result in a complex syndrome that at presentation could be mistaken for a motor neuron disorder. Recognition of this disorder is important because it responds to immunotherapy. PMID:21387375

Ultratight crystal packing of a 10 kDa protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Trillo-Muyo, Sergio; Jasilionis, Andrius; Domagalski, Marcin J.

2013-03-01

The crystal structure of the C-terminal domain of a putative U32 peptidase from G. thermoleovorans is reported; it is one of the most tightly packed protein structures reported to date. While small organic molecules generally crystallize forming tightly packed lattices with little solvent content, proteins form air-sensitive high-solvent-content crystals. Here, the crystallization and full structure analysis of a novel recombinant 10 kDa protein corresponding to the C-terminal domain of a putative U32 peptidase are reported. The orthorhombic crystal contained only 24.5% solvent and is therefore among the most tightly packed protein lattices ever reported.

NETs are a source of citrullinated autoantigens and stimulate inflammatory responses in rheumatoid arthritis

PubMed Central

Khandpur, Ritika; Carmona-Rivera, Carmelo; Vivekanandan-Giri, Anuradha; Gizinski, Alison; Yalavarthi, Srilakshmi; Knight, Jason S.; Friday, Sean; Li, Sam; Patel, Rajiv M.; Subramanian, Venkataraman; Thompson, Paul; Chen, Pojen; Fox, David A.; Pennathur, Subramaniam; Kaplan, Mariana J.

2013-01-01

The early events leading to the development of rheumatoid arthritis (RA) remain unclear but formation of autoantibodies to citrullinated antigens (ACPA) is considered a key pathogenic phenomenon. Neutrophils isolated from patients with various autoimmune diseases display enhanced extracellular trap formation (NETs), a phenomenon that externalizes autoantigens and immunostimulatory molecules. We investigated whether aberrant NETosis occurs in RA, determined its triggers and examined its deleterious inflammatory consequences. Enhanced NETosis was observed in circulating and synovial fluid RA neutrophils, compared to neutrophils from healthy controls and from patients with osteoarthritis. Further, netting neutrophils infiltrated RA synovial tissue, rheumatoid nodules and skin. NETosis correlated with ACPA presence and levels and with systemic inflammatory markers. RA sera and immunoglobulin fractions from RA patients with high levels of ACPA and/or rheumatoid factor significantly enhanced NETosis, and the NETs induced by these autoantibodies displayed distinct protein content. During NETosis, neutrophils externalized citrullinated autoantigens implicated in RA pathogenesis, whereas anti-citrullinated vimentin antibodies potently induced NET formation. The inflammatory cytokines IL-17A and TNF-α induced NETosis in RA neutrophils. In turn, NETs significantly augmented inflammatory responses in RA and OA synovial fibroblasts, including induction of IL-6, IL-8, chemokines and adhesion molecules. These observations implicate accelerated NETosis in RA pathogenesis, through externalization of citrullinated autoantigens and immunostimulatory molecules that may promote aberrant adaptive and innate immune responses in the joint and in the periphery, and perpetuate pathogenic mechanisms in this disease. PMID:23536012

Osteopontin is a tumor autoantigen in prostate cancer patients

PubMed Central

TILLI, TATIANA M.; SILVA, ELOÍSIO A.; MATOS, LÍVIA C.; FAGET, DOUGLAS V.; DIAS, BIANCA F.P.; VASCONCELOS, JULIANA S.P.; YOKOSAKI, YASUYUKI; GIMBA, ETEL R.P.

2011-01-01

Anti-tumor antibodies act as biomarkers for the early diagnosis of prostate cancer (PCa). Osteopontin (OPN) is overexpressed in PCa cells and contributes to the progression of the disease. This study aimed to evaluate whether OPN evokes a humoral immune response in PCa patients and whether the reactivity levels of anti-OPN antibodies may be used to better differentiate PCa from benign and healthy donor plasma samples. Plasma samples from biopsy-proven PCa patients (29), benign prostate hyperplasia (BPH) (18) and control healthy donors (HD) (30) were tested by immunoblots using the recombinant human OPN. The frequency of anti-OPN antibodies was significantly higher in PCa (66%) plasma samples as compared to BPH (33%) and HD controls (10%). Anti-OPN antibodies were detected in a high proportion of plasma samples from patients with a Gleason score of less than 6 (57%), prostate-specific antigen levels lower than 10 ng/ml (67%) and pT2 organ-confined disease (70%), suggesting that anti-OPN antibodies may be used as an early serum marker for PCa. To the best of our knowledge, this is the first description of OPN as a tumor autoantigen and one of the most reactive individual autoantigens described thus far. These data support the inclusion of OPN in a multiplex of tumor antigens in order to perform antibody profiling in PCa as well as in other malignancies overexpressing OPN. PMID:22870138

Effect of 14-kDa and 47-kDa protein molecules of age garlic extract on peritoneal macrophages.

PubMed

Daneshmandi, Saeed; Hajimoradi, Monire; Ahmadabad, Hasan Namdar; Hassan, Zuhair Mohammad; Roudbary, Maryam; Ghazanfari, Tooba

2011-03-01

Garlic (Allium sativum), traditionally being used as a spice worldwide, has different applications and is claimed to possess beneficial effects in several health ailments such as tumor and atherosclerosis. Garlic is also an immunomodulator and its different components are responsible for different properties. The present work aimed to assess the effect of protein fractions of garlic on peritoneal macrophages. 14-kDa and 47-kDa protein fractions of garlic were purified. Mice peritoneal macrophages were lavaged and cultured in a microtiter plate and exposed to different concentrations of garlic proteins. MTT assay was performed to evaluate the viability of macrophage. The amount of nitric oxide (NO) was detected in culture supernatants of macrophages by Griess reagent and furthermore, the cytotoxicity study of culture supernatants was carried out on WEHI-164 fibrosarcoma cell line as tumor necrosis factor-α bioassay. MTT assay results for both 14-kDa and 47-kDa protein fractions of stimulated macrophages were not significant (P > 0.05). Both 14-kDa and 47-kDa fractions significantly suppressed production of NO from macrophages (P = 0.007 and P = 0.003, respectively). Cytotoxicity of macrophages' supernatant on WEHI-164 fibrosarcoma cells was not affected by garlic protein fractions (P = 0.066 for 14-kDa and P = 0.085 for 47-kDa fractions). according to our finding, 14-kDa and 47-kDa fractions of aged garlic extract are able to suppress NO production from macrophages, which can be used as a biological advantage. These molecules had no cytotoxic effect on macrophages and do not increase tumoricidal property of macrophages.

Progression through the spliceosome cycle requires Prp38p function for U4/U6 snRNA dissociation.

PubMed Central

Xie, J; Beickman, K; Otte, E; Rymond, B C

1998-01-01

The elaborate and energy-intensive spliceosome assembly pathway belies the seemingly simple chemistry of pre-mRNA splicing. Prp38p was previously identified as a protein required in vivo and in vitro for the first pre-mRNA cleavage reaction catalyzed by the spliceosome. Here we show that Prp38p is a unique component of the U4/U6.U5 tri-small nuclear ribonucleoprotein (snRNP) particle and is necessary for an essential step late in spliceosome maturation. Without Prp38p activity spliceosomes form, but arrest in a catalytically impaired state. Functional spliceosomes shed U4 snRNA before 5' splice-site cleavage. In contrast, Prp38p-defective spliceosomes retain U4 snRNA bound to its U6 snRNA base-pairing partner. Prp38p is the first tri-snRNP-specific protein shown to be dispensable for assembly, but required for conformational changes which lead to catalytic activation of the spliceosome. PMID:9582287

Liver/kidney microsomal antibody type 1 and liver cytosol antibody type 1 concentrations in type 2 autoimmune hepatitis.

PubMed

Muratori, L; Cataleta, M; Muratori, P; Lenzi, M; Bianchi, F B

1998-05-01

Liver/kidney microsomal antibody type 1 (LKM1) and liver cytosol antibody type 1 (LC1) are the serological markers of type 2 autoimmune hepatitis (AIH). Since LKM1 and LC1 react against two distinct liver specific autoantigens (cytochrome P450IID6 (CYP2D6) and a 58 kDa cytosolic polypeptide respectively), the aim was to see whether LKM1 and LC1 concentrations correlate with liver disease activity. Twenty one patients with type 2 AIH were studied. All sera were tested by indirect immunofluorescence, counterimmunoelectrophoresis, and immunoblotting visualised by enhanced chemiluminescence. To evaluate LKM1 and LC1 levels, the 50 kDa microsomal reactivity (corresponding to CYP2D6) and the 58 kDa cytosolic reactivity were quantified by densitometric analysis. Seven patients were positive for LKM1, nine for LC1, and five for both. Serial serum samples at onset and during immunosuppressive treatment were analysed in 13 patients (four positive for LKM1, six positive for LC1 and three positive for both). During remission, LKM1 concentration remained essentially unchanged in six of seven patients, and decreased in only one. Conversely, in two of nine patients, LC1 was completely lost, and, in the remaining seven, LC1 concentration was reduced by more than 50%. After immunosuppression tapering or withdrawal, flare ups of liver necrosis ensued with increasing LC1 concentration, but not LKM1. LC1 concentration, at variance with that of LKM1, parallels liver disease activity, and its participation in the pathogenic mechanisms of liver injury can be hypothesised.

NADH:ubiquinone oxidoreductase from bovine heart mitochondria. cDNA sequences of the import precursors of the nuclear-encoded 39 kDa and 42 kDa subunits.

PubMed Central

Fearnley, I M; Finel, M; Skehel, J M; Walker, J E

1991-01-01

The 39 kDa and 42 kDa subunits of NADH:ubiquinone oxidoreductase from bovine heart mitochondria are nuclear-coded components of the hydrophobic protein fraction of the enzyme. Their amino acid sequences have been deduced from the sequences of overlapping cDNA clones. These clones were amplified from total bovine heart cDNA by means of the polymerase chain reaction, with the use of complex mixtures of oligonucleotide primers based upon fragments of protein sequence determined at the N-terminals of the proteins and at internal sites. The protein sequences of the 39 kDa and 42 kDa subunits are 345 and 320 amino acid residues long respectively, and their calculated molecular masses are 39,115 Da and 36,693 Da. Both proteins are predominantly hydrophilic, but each contains one or two hydrophobic segments that could possibly be folded into transmembrane alpha-helices. The bovine 39 kDa protein sequence is related to that of a 40 kDa subunit from complex I from Neurospora crassa mitochondria; otherwise, it is not related significantly to any known sequence, including redox proteins and two polypeptides involved in import of proteins into mitochondria, known as the mitochondrial processing peptidase and the processing-enhancing protein. Therefore the functions of the 39 kDa and 42 kDa subunits of complex I are unknown. The mitochondrial gene product, ND4, a hydrophobic component of complex I with an apparent molecular mass of about 39 kDa, has been identified in preparations of the enzyme. This subunit stains faintly with Coomassie Blue dye, and in many gel systems it is not resolved from the nuclearcoded 36 kDa subunit. Images Fig. 1. PMID:1832859

Generation of novel covalent RNA-protein complexes in cells by ultraviolet B irradiation: implications for autoimmunity.

PubMed

Andrade, Felipe; Casciola-Rosen, Livia A; Rosen, Antony

2005-04-01

To determine whether ultraviolet B (UVB) irradiation induces novel modifications in autoantigens targeted during experimental photoinduced epidermal damage. To search for novel UVB-induced autoantigen modifications, lysates made from UVB-irradiated human keratinocytes or HeLa cells were immunoblotted using human autoantibodies that recognize ribonucleoprotein autoantigens. Novel autoantigen structures identified were further characterized using nucleases and RNA hybridization. Human sera that recognize U1-70 kd (U1-70K) and La by immunoblotting also recognized multiple novel species when they were used to immunoblot lysates of UVB-irradiated keratinocytes or HeLa cells. These species were not present in control cells and were not observed when apoptosis was induced by Fas ligation or cytotoxic lymphocyte granule contents. Biochemical analysis using multiple assays revealed that these novel UVB-induced molecular species result from the covalent crosslinking between the U1 RNA and the hYRNA molecules with their associated proteins, including U1-70K, La, and likely components of the Sm particle. These data demonstrate that UVB irradiation of live cells can directly induce covalent RNA-protein complexes, which are recognized by human autoantibodies. As previously described for other autoantigens, these covalent complexes of RNA and proteins may have important consequences in terms of antigen capture and processing.

Anti-liver-kidney microsome antibody type 1 recognizes human cytochrome P450 db1.

PubMed

Gueguen, M; Yamamoto, A M; Bernard, O; Alvarez, F

1989-03-15

Anti-liver-kidney microsome antibody type 1 (LKM1), present in the sera of a group of children with autoimmune hepatitis, was recently shown to recognize a 50 kDa protein identified as rat liver cytochromes P450 db1 and db2. High homology between these two members of the rat P450 IID subfamily and human P450 db1 suggested that anti-LKM1 antibody is directed against this human protein. To test this hypothesis, a human liver cDNA expression library in phage lambda GT-11 was screened using rat P450 db1 cDNA as a probe. Two human cDNA clones were found to be identical to human P450 db1 by restriction mapping. Immunoblot analysis using as antigen, the purified fusion protein from one of the human cDNA clones showed that only anti-LKM1 with anti-50 kDa reactivity recognized the fusion protein. This fusion protein was further used to develop an ELISA test that was shown to be specific for sera of children with this disease. These results: 1) identify the human liver antigen recognized by anti-LKM1 auto-antibodies as cytochrome P450 db1, 2) allow to speculate that mutation on the human P450 db1 gene could alter its expression in the hepatocyte and make it auto-antigenic, 3) provide a simple and specific diagnostic test for this disease.

Sequential recognition of the pre-mRNA branch point by U2AF65 and a novel spliceosome-associated 28-kDa protein.

PubMed Central

Gaur, R K; Valcárcel, J; Green, M R

1995-01-01

Splicing of pre-mRNAs occurs via a lariat intermediate in which an intronic adenosine, embedded within a branch point sequence, forms a 2',5'-phosphodiester bond (RNA branch) with the 5' end of the intron. How the branch point is recognized and activated remains largely unknown. Using site-specific photochemical cross-linking, we have identified two proteins that specifically interact with the branch point during the splicing reaction. U2AF65, an essential splicing factor that binds to the adjacent polypyrimidine tract, crosslinks to the branch point at the earliest stage of spliceosome formation in an ATP-independent manner. A novel 28-kDa protein, which is a constituent of the mature spliceosome, contacts the branch point after the first catalytic step. Our results indicate that the branch point is sequentially recognized by distinct splicing factors in the course of the splicing reaction. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 5 FIGURE 6 FIGURE 7 FIGURE 8 FIGURE 9 PMID:7493318

Liver/kidney microsomal antibody type 1 and liver cytosol antibody type 1 concentrations in type 2 autoimmune hepatitis

PubMed Central

Muratori, L; Cataleta, M; Muratori, P; Lenzi, M; Bianchi, F

1998-01-01

Background—Liver/kidney microsomal antibody type 1 (LKM1) and liver cytosol antibody type 1 (LC1) are the serological markers of type 2 autoimmune hepatitis (AIH). 
Aims—Since LKM1 and LC1 react against two distinct liver specific autoantigens (cytochrome P450IID6 (CYP2D6) and a 58 kDa cytosolic polypeptide respectively), the aim was to see whether LKM1 and LC1 concentrations correlate with liver disease activity. 
Patients—Twenty one patients with type 2 AIH were studied. 
Methods—All sera were tested by indirect immunofluorescence, counterimmunoelectrophoresis, and immunoblotting visualised by enhanced chemiluminescence. To evaluate LKM1 and LC1 levels, the 50 kDa microsomal reactivity (corresponding to CYP2D6) and the 58 kDa cytosolic reactivity were quantified by densitometric analysis. 
Results—Seven patients were positive for LKM1, nine for LC1, and five for both. Serial serum samples at onset and during immunosuppressive treatment were analysed in 13 patients (four positive for LKM1, six positive for LC1 and three positive for both). During remission, LKM1 concentration remained essentially unchanged in six of seven patients, and decreased in only one. Conversely, in two of nine patients, LC1 was completely lost, and, in the remaining seven, LC1 concentration was reduced by more than 50%. After immunosuppression tapering or withdrawal, flare ups of liver necrosis ensued with increasing LC1 concentration, but not LKM1. 
Conclusions—LC1 concentration, at variance with that of LKM1, parallels liver disease activity, and its participation in the pathogenic mechanisms of liver injury can be hypothesised. 

 Keywords: autoantibodies; immunoblotting; LKM1; LC1; immunosuppression PMID:9659171

Review of autoantigens in Sjögren's syndrome: an update.

PubMed

Tong, Louis; Koh, Vanessa; Thong, Bernard Yu-Hor

2017-01-01

Primary Sjögren's syndrome (pSS) is an autoimmune disease characterized by inflammation in exocrine glands, resulting in reduced secretion of tears and saliva, manifesting as xerophthalmia and xerostomia, respectively. It is commonly associated with Sjögren's syndrome type A (Ro) and Sjögren's syndrome type B (La) antigens. However, in most patients, the identity of the triggering antigen is not known. Factors such as genetics of histocompatibility, dysregulation of T-cells, B-cells and viral infections have been implicated. Several important studies on autoantigens in pSS have been published since a review in 2012, and the aim of this review is to provide an update on further peer-reviewed original articles in this field. Oxidative damage of Ro60 antigen may explain the epitope spreading during the immune activation in pSS. Immune-mediated destruction of the muscarinic receptor-3-expressing cells has been associated with a reduction in parasympathetic function, which could cause reduced secretory function of exocrine glands. Such a process also activates reactive oxidative species and antioxidants, which are linked to the triggering of inflammatory responses. Elevated levels of kallikrein, yet another antigen present in the lacrimal gland and other tissues, are similarly involved in triggering an autoimmune T-cell response against target glands. Studying additional antigens, the platelet-selectin and vasoactive intestinal peptides, in patients with pSS can help to elucidate the origin and process of autoimmunity, or even lead to potential biomarkers. In conclusion, the understanding of autoantigens has led to exciting major advances in the biology of pSS and may influence diagnosis and management of pSS in future.

On the mode of sperm autoantigen presentation to the regional lymph node of the testis after vasectomy in rats.

PubMed Central

McDonald, S W; Scothorne, R J

1987-01-01

The regional testicular lymph nodes of vasectomised rats, and of sham-operated controls, have been examined, in stained smears of cell suspensions, for the presence of spermatozoa, at intervals of 1-10 weeks after operation. Very few, if any, spermatozoa were found in either group. These findings differ from those reported in rams and boars by Ball & Setchell (1983), and indicate species differences in the mode of presentation of sperm autoantigens to the regional nodes after vasectomy. PMID:3429321

Possibility of the transformation of eEF-2 (100 kDa) to eEF-2 (65 kDa) in the peptide elongation process in vitro.

PubMed

Gajko, A; Sredzińska, K; Galasiński, W; Gindzieński, A

1999-02-16

Two active eEF-2 polypeptides of approximately 100 and 65 kDa were copurified from rat liver cells and separated. The fate of eEF-2 (100 kDa) during its binding to ribosomes and in the translocation step of the peptide elongation process was investigated. It was shown that eEF-2 (100 kDa) did not change its form during the process of binding to the ribosomes. In the postribosomal supernatant, obtained from the postincubation mixture of the elongation process, only eEF-2 (65 kDa) was found. These results suggest that the form of eEF-2 (100 kDa), when bound to the ribosome during the elongation process, is transformed to eEF-2 (65 kDa). Copyright 1999 Academic Press.

The 29-kDa proteins phosphorylated ion thrombin-activated human platelets are forms of the estrogen receptor-related 27-kDa heat shock protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mendelsohn, M.E.; Yan Zhu; O'Neill, S.

Thrombin plays a critical role in platelet activation, hemostasis, and thrombosis. Cellular activation by thrombin leads to the phosphorylation of multiple proteins, most of which are unidentified. The authors have characterized several 29-kDa proteins that are rapidly phosphorylated following exposure of intact human platelets to thrombin. A murine monoclonal antibody raised to an unidentified estrogen receptor-related 29-kDa protein selectively recognized these proteins as well as a more basic, unphosphorylated 27-kDa protein. Cellular activation by thrombin led to a marked shift in the proportion of protein from the 27-kDa unphosphorylated form to the 29-kDa phosphoprotein species. Using this antibody, they isolatedmore » and sequenced a human cDNA clone encoding a protein that was identical to the mammalian 27-kDa heat shock protein (HSP27), a protein of uncertain function that is known to be phosphorylated to several forms and to be transcriptionally induced by estrogen. The 29-kDa proteins were confirmed to be phosphorylated forms of HSP27 by immunoprecipitation studies. Thus, the estrogen receptor-related protein is HSP27, and the three major 20-kDa proteins phosphorylated in thrombin-activated platelets are forms of HSP27. These data suggest a role for HSP27 in the signal transduction events of platelet activation.« less

Two distinct arginine methyltransferases are required for biogenesis of Sm-class ribonucleoproteins.

PubMed

Gonsalvez, Graydon B; Tian, Liping; Ospina, Jason K; Boisvert, François-Michel; Lamond, Angus I; Matera, A Gregory

2007-08-27

Small nuclear ribonucleoproteins (snRNPs) are core components of the spliceosome. The U1, U2, U4, and U5 snRNPs each contain a common set of seven Sm proteins. Three of these Sm proteins are posttranslationally modified to contain symmetric dimethylarginine (sDMA) residues within their C-terminal tails. However, the precise function of this modification in the snRNP biogenesis pathway is unclear. Several lines of evidence suggest that the methyltransferase protein arginine methyltransferase 5 (PRMT5) is responsible for sDMA modification of Sm proteins. We found that in human cells, PRMT5 and a newly discovered type II methyltransferase, PRMT7, are each required for Sm protein sDMA modification. Furthermore, we show that the two enzymes function nonredundantly in Sm protein methylation. Lastly, we provide in vivo evidence demonstrating that Sm protein sDMA modification is required for snRNP biogenesis in human cells.

Multiple discrete encephalitogenic epitopes of the autoantigen myelin basic protein include a determinant for I-E class II-restricted T cells

PubMed Central

1988-01-01

Immunization with the autoantigen myelin basic protein (MBP) causes experimental allergic encephalomyelitis (EAE). Initial investigations indicated that encephalitogenic murine determinants of MBP were located only within MBP 1-37 and MBP 89-169. Encephalitogenic T cell epitopes within these fragments have been identified. Each epitope is recognized by T cells in association with separate allelic I-A molecules. A hybrid I-E-restricted T cell clone that recognizes intact mouse (self) MBP has been examined. The epitope recognized by this clone includes MBP residues 35-47. When tested in vivo, p35-47 causes EAE. T cell recognition of p35-47 occurs only in association with I-E molecules. These results provide the first clear example that antigen-specific T cells restricted by I-E class II molecules participate in murine autoimmune disease. Furthermore, it is clear that there are multiple (at least three) discrete encephalitogenic T cell epitopes of this autoantigen, each recognized in association with separate allelic class II molecules. These results may be relevant to human autoimmune diseases whose susceptibility is associated with more than one HLA-D molecule. PMID:2459291

Complementation of a mutant cell line: central role of the 91 kDa polypeptide of ISGF3 in the interferon-alpha and -gamma signal transduction pathways.

PubMed Central

Müller, M; Laxton, C; Briscoe, J; Schindler, C; Improta, T; Darnell, J E; Stark, G R; Kerr, I M

1993-01-01

Mutants in complementation group U3, completely defective in the response of all genes tested to interferons (IFNs) alpha and gamma, do not express the 91 and 84 kDa polypeptide components of interferon-stimulated gene factor 3 (ISGF3), a transcription factor known to play a primary role in the IFN-alpha response pathway. The 91 and 84 kDa polypeptides are products of a single gene. They result from differential splicing and differ only in a 38 amino acid extension at the C-terminus of the 91 kDa polypeptide. Complementation of U3 mutants with cDNA constructs expressing the 91 kDa product at levels comparable to those observed in induced wild-type cells completely restored the response to both IFN-alpha and -gamma and the ability to form ISGF3. Complementation with the 84 kDa component similarly restored the ability to form ISGF3 and, albeit to a lower level, the IFN-alpha response of all genes tested so far. It failed, however, to restore the IFN-gamma response of any gene analysed. The precise nature of the DNA motifs and combination of factors required for the transcriptional response of all genes inducible by IFN-alpha and -gamma remains to be established. The results presented here, however, emphasize the apparent general requirement of the 91 kDa polypeptide in the primary transcriptional response to both types of IFN. Images PMID:7693454

Immuno-proteomic discovery of tumor tissue autoantigens identifies olfactomedin 4, CD11b, and integrin alpha-2 as markers of colorectal cancer with liver metastases.

PubMed

Yang, Qian; Bavi, Prashant; Wang, Julia Y; Roehrl, Michael H

2017-09-25

Late-stage colorectal cancer with liver metastasis is common and affords poor prognosis, yet there is a dearth of reliable biomarkers. Cancer is often characterized by an increase in serologic autoantibodies. Hence, we embarked on an immuno-proteomic strategy by using autoantibodies to discover antigens in tumor tissue as potential cancer markers. Matched sets of tissues from primary colon cancer, liver metastases, and adjacent benign tissues were obtained from colon cancer patients. Tissue proteins were extracted, and autoantigens were uncovered by immunoblotting with autoantibodies and sequenced by mass spectrometry. Informatics analyses identified 48 proteins that were found in tumor only but were absent in normal tissue. Five of these were reproducibly found in two independent experiments, including olfactomedin 4 (OLFM4), CD11b, integrin α2 (ITGA2), periostin, and thrombospondin-2. Further confirmation with tissue from 43 patients by Western blotting, immunohistochemistry, and tissue microarray deemed OLFM4, CD11b, and ITGA2 to be significantly overexpressed in both primary colon tumors and liver metastases. These tumor tissue autoantigens may serve as promising markers for developing differential diagnostics and immunotherapies for colorectal cancers, in particular, those with tendency to progress to liver metastases. Late-stage colorectal cancer with liver metastasis is common and affords poor prognosis, yet there is a dearth of reliable biomarkers. Cancer is often characterized by an increase in serologic autoantibodies. Cancer tissue immunogens - antigens capable of inducing specific antibody production in patients - are promising targets for development of precision diagnostics and immunotherapies. In our manuscript, we describe on an immuno-proteomic strategy by using autoantibodies to discover antigens in tumor tissue as potential cancer markers. Matched sets of tissues from primary colon cancer, liver metastases, and adjacent benign tissues were

An endogenous 55 kDa TNF receptor mediates cell death in a neural cell line.

PubMed

Sipe, K J; Srisawasdi, D; Dantzer, R; Kelley, K W; Weyhenmeyer, J A

1996-06-01

Tumor necrosis factor-alpha (TNF) is associated with developmental and injury-related events in the central nervous system (CNS). In the present study, we have examined the role of TNF on neurons using the clonal murine neuroblastoma line, N1E-115 (N1E). N1E cells represent a well-defined model for studying neuronal development since they can be maintained as either undifferentiated, mitotically active neuroblasts or as differentiated, mature neurons. Northern and reverse transcription-polymerase chain reaction (RT-PCR) analyses revealed that both undifferentiated and differentiated N1Es express transcripts for the 55 kDa TNF receptor (TNFR), but not the 75 kDa TNFR. The biological activity of the expressed TNF receptor was demonstrated by a dose dependent cytotoxicity to either recombinant murine or human TNF when the cells were incubated with the transcriptional inhibitor actinomycin D. The lack of the 75 kDa receptor mRNA expression and the dose dependent response to rHuTNF, an agonist specific for the murine 55 kDa receptor, suggest that the TNF induced cytotoxicity is mediated through the 55 kDa receptor in both the undifferentiated and differentiated N1Es. Light microscopic observations, flow cytometric analysis of hypodiploid DNA, and electrophoretic analysis of nucleosomal DNA fragmentation of N1Es treated with actinomycin D and TNF revealed features characteristic of both necrotic and apoptotic cell death. These findings demonstrate that blast and mature N1E cells express the 55 kDa TNF receptor which is responsible for inducing both necrotic and apoptotic death in these cells. The observation that actinomycin D renders N1E cells susceptible to the cytotoxic effects of TNF indicates that a sensitization step, such as removal of an endogenous protective factor or viral-mediated inhibition of transcription, may be necessary for TNF cytotoxicity in neurons.

Cloning, expression and activation of a truncated 92-kDa gelatinase minienzyme.

PubMed

Kröger, M; Tschesche, H

1997-09-01

The matrix metalloproteinases (MMPs) are a family of highly homologous zinc-endopeptidases that degrade extracellular matrix components. Human 92-kDa gelatinase (MMP-9) represents one of the MMPs that cleaves native collagen type IV. As a basis for structural investigations, the short form (catalytic domain, amino acid residues 113-450) of the 92-kDa gelatinase cDNA was cloned and expressed in E. coli as a minienzyme. By combination of reverse transcription (RT) and polymerase chain reaction (PCR), the truncated 92-kDa gelatinase-cDNA was amplified from the corresponding mRNA derived from ovarian carcinoma cells. The cDNA fragment obtained was cloned in E. coli and sequenced. With the exception of one nucleotide inversion at position 745 (gt-->tg) the cDNA sequence was identical to the nucleotide sequence of the 92-kDa gelatinase as has been previously reported. The protein was expressed in E. coli using the vector pET-12b. The recombinant protein was stored in inclusion bodies and extracted as a 38 kDa species from the inclusion bodies by solubilization in 8 M urea. The product was purified by affinity chromatography and gel filtration. Amino-terminal sequence analysis confirmed the identity with the catalytic domain of 92-kDa gelatinase. The recombinant protein was refolded in the presence of Ca2+ and Zn2+ and yielded an active minienzyme with gelatinolytic activity. It degrades the native substrate collagen type IV and the synthetic substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 x AcOH like the full-length 92-kDa gelatinase. The catalytic activity could be inhibited by the specific MMP inhibitors TIMP-1 and TIMP-2.

Recombinant expression, isolation, and proteolysis of extracellular matrix-secreted phosphoprotein-24 kDa.

PubMed

Murray, Elsa J Brochmann; Murray, Samuel S; Simon, Robert; Behnam, Keyvan

2007-01-01

Secreted phosphoprotein-24 kDa (spp24) is an extracellular matrix protein first cloned from bone. Bovine spp24 is transcribed as a 203 amino acid residue protein that undergoes cleavage of a secretory peptide to form the mature protein (spp24, residues 24 to 203). While not osteogenic itself, spp24 is degraded to a pro-osteogenic protein, spp18.5, in bone. Both spp18.5 and spp24 contain a cyclic TRH1 (TGF-beta receptor II homology-1) domain similar to that found in the receptor itself and in fetuin. A synthetic peptide corresponding to the TRH1 domain of spp18.5 and spp24 specifically binds BMP-2 and enhances the rate and magnitude of BMP-2-induced ectopic bone formation in vivo. The parental protein, spp24, exhibits a high affinity for bone and mineral complexes, but its abundance there is low, suggesting that it is rapidly degraded. The availability of recombinant spp24 and its degradation products would facilitate the elucidation of their structure:function relationships. We describe here the expression of His(6)-tagged bovine spp24 (residues 24 to 203) in E. coli, its purification by high-resolution IMAC (immobilized metal affinity chromatography), and the characterization of the full-length recombinant 21.5 kDa protein and its two major 16 kDa and 14.5 kDa degradation products (spp24, residues 24 to 157, and spp24, residues 24 to 143) by mass spectroscopy. The recombinant spp24 protein was resistant to proteolysis by MC3T3-E1 osteoblastic cell extracts in the absence of calcium; however, in the presence of 4 mM Ca, it can undergo essentially complete proteolysis to small peptides, bypassing the 16 kDa and 14.5 kDa intermediates. This confirms the proteolytic susceptibility of spp24. It also suggests that the levels of spp24 in bone may be regulated, in part, by calcium-dependent proteolysis mediated by osteoblastic cells.

Two distinct arginine methyltransferases are required for biogenesis of Sm-class ribonucleoproteins

PubMed Central

Gonsalvez, Graydon B.; Tian, Liping; Ospina, Jason K.; Boisvert, François-Michel; Lamond, Angus I.; Matera, A. Gregory

2007-01-01

Small nuclear ribonucleoproteins (snRNPs) are core components of the spliceosome. The U1, U2, U4, and U5 snRNPs each contain a common set of seven Sm proteins. Three of these Sm proteins are posttranslationally modified to contain symmetric dimethylarginine (sDMA) residues within their C-terminal tails. However, the precise function of this modification in the snRNP biogenesis pathway is unclear. Several lines of evidence suggest that the methyltransferase protein arginine methyltransferase 5 (PRMT5) is responsible for sDMA modification of Sm proteins. We found that in human cells, PRMT5 and a newly discovered type II methyltransferase, PRMT7, are each required for Sm protein sDMA modification. Furthermore, we show that the two enzymes function nonredundantly in Sm protein methylation. Lastly, we provide in vivo evidence demonstrating that Sm protein sDMA modification is required for snRNP biogenesis in human cells. PMID:17709427

Modeling and Docking Studies on Novel Mutants (K71L and T204V) of the ATPase Domain of Human Heat Shock 70 kDa Protein 1

PubMed Central

Elengoe, Asita; Naser, Mohammed Abu; Hamdan, Salehhuddin

2014-01-01

The purpose of exploring protein interactions between human adenovirus and heat shock protein 70 is to exploit a potentially synergistic interaction to enhance anti-tumoral efficacy and decrease toxicity in cancer treatment. However, the protein interaction of Hsp70 with E1A32 kDa of human adenovirus serotype 5 remains to be elucidated. In this study, two residues of ATPase domain of human heat shock 70 kDa protein 1 (PDB: 1 HJO) were mutated. 3D mutant models (K71L and T204V) using PyMol software were then constructed. The structures were evaluated by PROCHECK, ProQ, ERRAT, Verify 3D and ProSA modules. All evidence suggests that all protein models are acceptable and of good quality. The E1A32 kDa motif was retrieved from UniProt (P03255), as well as subjected to docking interaction with NBD, K71L and T204V, using the Autodock 4.2 program. The best lowest binding energy value of −9.09 kcal/mol was selected for novel T204V. Moreover, the protein-ligand complex structures were validated by RMSD, RMSF, hydrogen bonds and salt bridge analysis. This revealed that the T204V-E1A32 kDa motif complex was the most stable among all three complex structures. This study provides information about the interaction between Hsp70 and the E1A32 kDa motif, which emphasizes future perspectives to design rational drugs and vaccines in cancer therapy. PMID:24758925

Gene targeting of Gemin2 in mice reveals a correlation between defects in the biogenesis of U snRNPs and motoneuron cell death

PubMed Central

Jablonka, Sibylle; Holtmann, Bettina; Meister, Gunter; Bandilla, Michael; Rossoll, Wilfried; Fischer, Utz; Sendtner, Michael

2002-01-01

Neuronal degeneration in spinal muscular atrophy is caused by reduced expression of the survival motor neuron (SMN) protein. SMN and the tightly interacting Gemin2 form part of a macromolecular complex (SMN complex) that mediates assembly of spliceosomal small nuclear ribonucleoproteins (U snRNPs). We used mouse genetics to investigate the function of this complex in motoneuron maintenance. Reduced Smn/Gemin2 protein levels lead to disturbed U snRNP assembly as indicated by reduced nuclear accumulation of Sm proteins. This finding correlates with enhanced motoneuron degeneration in Gemin2+/−/Smn+/− mice. Our data provide in vivo evidence that impaired production of U snRNPs contributes to motoneuron degeneration. PMID:12091709

Molecular cloning of metaphase chromosome protein 1 (MCP1), a novel human autoantigen that associates with condensed chromosomes during mitosis.

PubMed

Bronze-da-Rocha, E; Catita, J A; Sunkel, C E

1998-02-01

Systemic lupus erythematosus autoantibodies were used to identify and to characterize new human chromosome-associated proteins. Previous immunolocalization studies in human and murine tissue culture cells showed that some of these monoclonal antibodies recognize nuclear antigens that associate with condensed chromosomes during mitosis. One antibody was selected for screening a human HeLa S3 cDNA expression library, and cDNAs that code for an antigen of 31-33 kDa were isolated. Immunological, biochemical and cell fractionation data indicate that the 31- to 33-kDa antigen corresponds to the chromosome-associated protein recognized by the original monoclonal antibody. Sequence analysis shows that we isolated a novel human gene. Immunolocalization to human tissue culture cells shows that during interphase the antigen is dispersed in the nucleus and that during mitosis it associates exclusively with condensed chromosomes. A similar pattern of localization was also observed in mouse fibroblasts, suggesting that the antigen is conserved among different species. Finally, we show that part of the antigen remains bound to the scaffold/matrix component, even after high salt extraction.

Re-exposure to beta cell autoantigens in pancreatic allograft recipients with preexisting beta cell autoantibodies.

PubMed

Mujtaba, Muhammad Ahmad; Fridell, Jonathan; Book, Benita; Faiz, Sara; Sharfuddin, Asif; Wiebke, Eric; Rigby, Mark; Taber, Tim

2015-11-01

Re-exposure to beta cell autoantigens and its relevance in the presence of donor-specific antibodies (DSA) in pancreatic allograft recipients is not well known. Thirty-three patients requiring a pancreas transplant were enrolled in an IRB approved study. They underwent prospective monitoring for DSA and beta cell autoantibody (BCAA) levels to GAD65, insulinoma-associated antigen 2 (IA-2), insulin (micro-IAA [mIAA]), and islet-specific zinc transporter isoform-8 (ZnT8). Twenty-five (75.7%) had pre-transplant BCAA. Twenty had a single antibody (mIAA n = 15, GAD65 n = 5); five had two or more BCAA (GAD65 + mIAA n = 2, GAD65 + mIAA+IA-2 n = 2, GA65 + mIAA+IA-2 + ZnT8 = 1). No changes in GAD65 (p > 0.29), IA-2 (>0.16), and ZnT8 (p > 0.07) were observed between pre-transplant and post-transplant at 6 or 12 months. A decrease in mIAA from pre- to post-6 months (p < 0.0001), 12 months (p < 0.0001), and from post-6 to post-12 months (p = 0.0002) was seen. No new BCAA was observed at one yr. Seven (21.0%) developed de novo DSA. The incidence of DSA was 24% in patients with BCAA vs. 25% in patients without BCAA (p = 0.69). Pancreatic allograft function of patients with vs. without BCAA, and with and without BCAA + DSA was comparable until last follow-up (three yr). Re-exposure to beta cell autoantigens by pancreas transplant may not lead to increased levels or development of new BCAA or pancreatic allograft dysfunction. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Isoform composition and stoichiometry of the approx. 90-kDa heat shock protein associated with glucocorticoid receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mendel, D.B.; Orti, E.

1988-05-15

The authors observed that the approx. 90-kDa non-steroid-binding component of nonactivated glucocorticoid receptors purified from WEHI-7 mouse thymoma cells (which has been identified as the approx. 90-kDa heat shock protein) consistently migrates as a doublet during polyacrylamide gel electrophoresis under denaturing and reducing conditions. It has recently been reported that murine Meth A cells contain a tumor-specific transplantation antigen (TSTA) which is related or identical to the approx. 90-kDa heat shock protein. The observation that TSTA and the approx. 90-kDa heat shock protein isolated from these cells exists as two isoforms of similar molecular mass and charge has suggested thatmore » the doublet observed is also due to the existence of two isoforms. They have therefore conducted this study to determine whether TSTA and the approx. 90-kDa component of glucocorticoid receptors are indeed related, to establish whether the receptor preferentially binds one isoform of the approx. 90-kDa heat shock protein, and to investigate the stoichiometry of the nonactivated receptor complex. They used the BuGr1 and AC88 monoclonal antibodies to purify, respectively, receptor-associated and free approx. 90-kDa heat shock protein from WEHI-7 cells grown for 48 h with (/sup 35/S)methionine to metabolically label proteins to steady state. The long-term metabolic labeling approach has also enabled them to directly determine that the purified non-activated glucocorticoid receptor contains a single steroid-binding protein and two approx. 90-kDa non-steroid-binding subunits. The consistency with which a approx. 1:2 stoichiometric ratio of steroid binding to approx. 90-kDa protein is observed supports the view that the approx. 90-kDa heat shock protein is a true component of nonactivated glucocorticoid-receptor complexes.« less

Commensal orthologs of the human autoantigen Ro60 as triggers of autoimmunity in lupus | Center for Cancer Research

Cancer.gov

Lupus: No Longer a Lone Wolf. Lupus (represented by the wolf, Canis lupus) is a chronic autoimmune disorder that progresses over decades. Greiling et al. analyzed commensal bacteria in lupus patients and identified species with Ro60 proteins similar to human Ro60, an early autoantigen in lupus. Bacterial Ro60 could activate patient lymphocytes, and colonization of mice with

Oral delivery of human biopharmaceuticals, autoantigens and vaccine antigens bioencapsulated in plant cells

PubMed Central

Kwon, Kwang-Chul; Verma, Dheeraj; Singh, Nameirakpam D.; Herzog, Roland; Daniell, Henry

2012-01-01

Among 12 billion injections administered annually, unsafe delivery leads to >20 million infections and >100 million reactions. In an emerging new concept, freeze-dried plant cells (lettuce) expressing vaccine antigens/biopharmaceuticals are protected in the stomach from acids/enzymes but are released to the immune or blood circulatory system when plant cell walls are digested by microbes that colonize the gut. Vaccine antigens bioencapsulated in plant cells upon oral delivery after priming, conferred both mucosal and systemic immunity and protection against bacterial, viral or protozoan pathogens or toxin challenge. Oral delivery of autoantigens was effective against complications of type 1diabetes and hemophilia, by developing tolerance. Oral delivery of proinsulin or exendin-4 expressed in plant cells regulated blood glucose levels similar to injections. Therefore, this new platform offers a low cost alternative to deliver different therapeutic proteins to combat infectious or inherited diseases by eliminating inactivated pathogens, expensive purification, cold storage/transportation and sterile injections. PMID:23099275

A novel Amoeba proteus 120 kDa actin-binding protein with only 1 filamin repeat and a coiled-coil region.

PubMed

Sobczak, Magdalena; Kocik, Elzbieta; Redowicz, Maria Jolanta

2007-02-01

A novel 120 kDa actin-binding protein (ApABP-F1) was found in Amoeba proteus. It was distributed throughout the cytoplasm, mainly in the subplasma membrane and perinuclear-nuclear areas, enriched in actin. The full-length cDNA of ApABP consisted of 2672 nucleotides with an open reading frame of 878 amino acids, giving a ~95 kDa protein with a theoretical pI value of 5.11. It had a novel domain organization pattern: the N terminus (residues 1-104) contained 1 calponin-homology (CH) domain, followed by only 1 region that was homologous to the filamin repeat (FR, residues 209-324), and a central region (residues 344-577) exhibiting a very high probability of coiled-coil formation, probably engaged in the observed protein dimerization. A phylogenetic tree constructed for CH domains from 25 various proteins revealed that the CH domain of ApABP was most related to that of the hypothetical mouse KIAA0903-like protein, whereas not much relationship to either filamins or the gelation factor (ABP-120) of Dictyostelium discoideum and Entamoeba histolytica was found.

A mechanism underlying position-specific regulation of alternative splicing

PubMed Central

Hamid, Fursham M.

2017-01-01

Abstract Many RNA-binding proteins including a master regulator of splicing in developing brain and muscle, polypyrimidine tract-binding protein 1 (PTBP1), can either activate or repress alternative exons depending on the pre-mRNA recruitment position. When bound upstream or within regulated exons PTBP1 tends to promote their skipping, whereas binding to downstream sites often stimulates inclusion. How this switch is orchestrated at the molecular level is poorly understood. Using bioinformatics and biochemical approaches we show that interaction of PTBP1 with downstream intronic sequences can activate natural cassette exons by promoting productive docking of the spliceosomal U1 snRNP to a suboptimal 5′ splice site. Strikingly, introducing upstream PTBP1 sites to this circuitry leads to a potent splicing repression accompanied by the assembly of an exonic ribonucleoprotein complex with a tightly bound U1 but not U2 snRNP. Our data suggest a molecular mechanism underlying the transition between a better-known repressive function of PTBP1 and its role as a bona fide splicing activator. More generally, we argue that the functional outcome of individual RNA contacts made by an RNA-binding protein is subject to extensive context-specific modulation.

Negative and Translation Termination-Dependent Positive Control of FLI-1 Protein Synthesis by Conserved Overlapping 5′ Upstream Open Reading Frames in Fli-1 mRNA

PubMed Central

Sarrazin, Sandrine; Starck, Joëlle; Gonnet, Colette; Doubeikovski, Alexandre; Melet, Fabrice; Morle, François

2000-01-01

The proto-oncogene Fli-1 encodes a transcription factor of the ets family whose overexpression is associated with multiple virally induced leukemias in mouse, inhibits murine and avian erythroid cell differentiation, and induces drastic perturbations of early development in Xenopus. This study demonstrates the surprisingly sophisticated regulation of Fli-1 mRNA translation. We establish that two FLI-1 protein isoforms (of 51 and 48 kDa) detected by Western blotting in vivo are synthesized by alternative translation initiation through the use of two highly conserved in-frame initiation codons, AUG +1 and AUG +100. Furthermore, we show that the synthesis of these two FLI-1 isoforms is regulated by two short overlapping 5′ upstream open reading frames (uORF) beginning at two highly conserved upstream initiation codons, AUG −41 and GUG −37, and terminating at two highly conserved stop codons, UGA +35 and UAA +15. The mutational analysis of these two 5′ uORF revealed that each of them negatively regulates FLI-1 protein synthesis by precluding cap-dependent scanning to the 48- and 51-kDa AUG codons. Simultaneously, the translation termination of the two 5′ uORF appears to enhance 48-kDa protein synthesis, by allowing downstream reinitiation at the 48-kDa AUG codon, and 51-kDa protein synthesis, by allowing scanning ribosomes to pile up and consequently allowing upstream initiation at the 51-kDa AUG codon. To our knowledge, this is the first example of a cellular mRNA displaying overlapping 5′ uORF whose translation termination appears to be involved in the positive control of translation initiation at both downstream and upstream initiation codons. PMID:10757781

Biochemical characterization of the 49 kDa penicillin-binding protein of Mycobacterium smegmatis.

PubMed Central

Mukherjee, T; Basu, D; Mahapatra, S; Goffin, C; van Beeumen, J; Basu, J

1996-01-01

The 49 kDa penicillin-binding protein (PBP) of Mycobacterium smegmatis catalyses the hydrolysis of the peptide or S-ester bond of carbonyl donors R1-CONH-CHR2-COX-CHR2-COO- (where X is NH or S). In the presence of a suitable amino acceptor, the reaction partitions between the transpeptidation and hydrolysis pathways, with the amino acceptor, behaving as a simple alternative nucleophile at the level of the acyl-enzyme. By virtue of its N-terminal sequence similarity, the 49 kDa PBP represents one of the class of monofunctional low-molecular-mass PBPs. An immunologically related protein of M(r) 52,000 is present in M. tuberculosis. The 49 kDa PBP is sensitive towards amoxycillin, imipenem, flomoxef and cefoxitin. PMID:8947487

The location of a disease-associated polymorphism and genomic structure of the human 52-kDa Ro/SSA locus (SSA1)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsugu, H.; Horowitz, R.; Gibson, N.

1994-12-01

Sera from approximately 30% of patients with systemic lupus erythematosus (SLE) contain high titers of autoantibodies that bind to the 52-kDa Ro/SSA protein. We previously detected polymorphisms in the 52-kDa Ro/SSA gene (SSA1) with restriction enzymes, one of which is strongly associated with the presence of SLE (P < 0.0005) in African Americans. A higher disease frequency and more severe forms of the disease are commonly noted among these female patients. To determine the location and nature of this polymorphism, we obtained two clones that span 8.5 kb of the 52-kDa Ro/SSA locus including its upstream regulatory region. Six exonsmore » were identified, and their nucleotide sequences plus adjacent noncoding regions were determined. No differences were found between these exons and the coding region of one of the reported cDNAs. The disease-associated polymorphic site suggested by a restriction enzyme map and confirmed by DNA amplification and nucleotide sequencing was present upstream of exon 1. This polymorphism may be a genetic marker for a disease-related variation in the coding region for the protein or in the upstream regulatory region of this gene. Although this RFLP is present in Japanese, it is not associated with lupus in this race. 41 refs., 4 figs., 2 tabs.« less

Release of Active Peptidyl Arginine Deiminases by Neutrophils Can Explain Production of Extracellular Citrullinated Autoantigens in Rheumatoid Arthritis Synovial Fluid

PubMed Central

Spengler, Julia; Lugonja, Božo; Jimmy Ytterberg, A.; Zubarev, Roman A.; Creese, Andrew J.; Pearson, Mark J.; Grant, Melissa M.; Milward, Michael; Lundberg, Karin; Buckley, Christopher D.; Filer, Andrew; Raza, Karim; Cooper, Paul R.; Chapple, Iain L.

2015-01-01

Objective In the majority of patients with rheumatoid arthritis (RA), antibodies specifically recognize citrullinated autoantigens that are generated by peptidylarginine deiminases (PADs). Neutrophils express high levels of PAD and accumulate in the synovial fluid (SF) of RA patients during disease flares. This study was undertaken to test the hypothesis that neutrophil cell death, induced by either NETosis (extrusion of genomic DNA–protein complexes known as neutrophil extracellular traps [NETs]) or necrosis, can contribute to production of autoantigens in the inflamed joint. Methods Extracellular DNA was quantified in the SF of patients with RA, patients with osteoarthritis (OA), and patients with psoriatic arthritis (PsA). Release of PAD from neutrophils was investigated by Western blotting, mass spectrometry, immunofluorescence staining, and PAD activity assays. PAD2 and PAD4 protein expression, as well as PAD enzymatic activity, were assessed in the SF of patients with RA and those with OA. Results Extracellular DNA was detected at significantly higher levels in RA SF than in OA SF (P < 0.001) or PsA SF (P < 0.05), and its expression levels correlated with neutrophil concentrations and PAD activity in RA SF. Necrotic neutrophils released less soluble extracellular DNA compared to NETotic cells in vitro (P < 0.05). Higher PAD activity was detected in RA SF than in OA SF (P < 0.05). The citrullinated proteins PAD2 and PAD4 were found attached to NETs and also freely diffused in the supernatant. PAD enzymatic activity was detected in supernatants of neutrophils undergoing either NETosis or necrosis. Conclusion Release of active PAD isoforms into the SF by neutrophil cell death is a plausible explanation for the generation of extracellular autoantigens in RA. PMID:26245941

The aqueous phase of Alzheimer's disease brain contains assemblies built from ∼4 and ∼7 kDa Aβ species.

PubMed

Mc Donald, Jessica M; O'Malley, Tiernan T; Liu, Wen; Mably, Alexandra J; Brinkmalm, Gunnar; Portelius, Erik; Wittbold, William M; Frosch, Matthew P; Walsh, Dominic M

2015-11-01

Much knowledge about amyloid β (Aβ) aggregation and toxicity has been acquired using synthetic peptides and mouse models, whereas less is known about soluble Aβ in human brain. We analyzed aqueous extracts from multiple AD brains using an array of techniques. Brains can contain at least four different Aβ assembly forms including: (i) monomers, (ii) a ∼7 kDa Aβ species, and larger species (iii) from ∼30-150 kDa, and (iv) >160 kDa. High molecular weight species are by far the most prevalent and appear to be built from ∼7 kDa Aβ species. The ∼7 kDa Aβ species resist denaturation by chaotropic agents and have a higher Aβ42/Aβ40 ratio than monomers, and are unreactive with antibodies to Asp1 of Ab or APP residues N-terminal of Asp1. Further analysis of brain-derived ∼7 kDa Aβ species, the mechanism by which they assemble and the structures they form should reveal therapeutic and diagnostic opportunities. Copyright © 2015 The Alzheimer's Association. Published by Elsevier Inc. All rights reserved.

Identification of bovine sperm acrosomal proteins that interact with a 32-kDa acrosomal matrix protein.

PubMed

Nagdas, Subir K; Smith, Linda; Medina-Ortiz, Ilza; Hernandez-Encarnacion, Luisa; Raychoudhury, Samir

2016-03-01

Mammalian fertilization is accomplished by the interaction between sperm and egg. Previous studies from this laboratory have identified a stable acrosomal matrix assembly from the bovine sperm acrosome termed the outer acrosomal membrane-matrix complex (OMC). This stable matrix assembly exhibits precise binding activity for acrosin and N-acetylglucosaminidase. A highly purified OMC fraction comprises three major (54, 50, and 45 kDa) and several minor (38-19 kDa) polypeptides. The set of minor polypeptides (38-19 kDa) termed "OMCrpf polypeptides" is selectively solubilized by high-pH extraction (pH 10.5), while the three major polypeptides (55, 50, and 45 kDa) remain insoluble. Proteomic identification of the OMC32 polypeptide (32 kDa polypeptide isolated from high-pH soluble fraction of OMC) yielded two peptides that matched the NCBI database sequence of acrosin-binding protein. Anti-OMC32 recognized an antigenically related family of polypeptides (OMCrpf polypeptides) in the 38-19-kDa range with isoelectric points ranging between 4.0 and 5.1. Other than glycohydrolases, OMC32 may also be complexed to other acrosomal proteins. The present study was undertaken to identify and localize the OMC32 binding polypeptides and to elucidate the potential role of the acrosomal protein complex in sperm function. OMC32 affinity chromatography of a detergent-soluble fraction of bovine cauda sperm acrosome followed by mass spectrometry-based identification of bound proteins identified acrosin, lactadherin, SPACA3, and IZUMO1. Co-immunoprecipitation analysis also demonstrated the interaction of OMC32 with acrosin, lactadherin, SPACA3, and IZUMO1. Our immunofluorescence studies revealed the presence of SPACA3 and lactadherin over the apical segment, whereas IZUMO1 is localized over the equatorial segment of Triton X-100 permeabilized cauda sperm. Immunoblot analysis showed that a significant portion of SPACA3 was released after the lysophosphatidylcholine (LPC)-induced acrosome

Effects of porcine 25 kDa amelogenin and its proteolytic derivatives on bone sialoprotein expression.

PubMed

Nakayama, Y; Yang, L; Mezawa, M; Araki, S; Li, Z; Wang, Z; Sasaki, Y; Takai, H; Nakao, S; Fukae, M; Ogata, Y

2010-10-01

Amelogenins are hydrophobic proteins that are the major component of developing enamel. Enamel matrix derivative has been used for periodontal regeneration. Bone sialoprotein is an early phenotypic marker of osteoblast differentiation. In this study, we examined the ability of porcine amelogenins to regulate bone sialoprotein transcription. To determine the molecular basis of the transcriptional regulation of the bone sialoprotein gene by amelogenins, we conducted northern hybridization, transient transfection analyses and gel mobility shift assays using the osteoblast-like ROS 17/2.8 cells. Amelogenins (100 ng/mL) up-regulated bone sialoprotein mRNA at 3 h, with maximal mRNA expression occurring at 12 h (25 and 20 kDa) and 6 h (13 and 6 kDa). Amelogenins (100 ng/mL, 12 h) increased luciferase activities in pLUC3 (nucleotides -116 to +60), and 6 kDa amelogenin up-regulated pLUC4 (nucleotides -425 to +60) activity. The tyrosine kinase inhibitor inhibited amelogenin-induced luciferase activities, whereas the protein kinase A inhibitor abolished 25 kDa amelogenin-induced bone sialoprotein transcription. The effects of amelogenins were abrogated by 2-bp mutations in the fibroblast growth factor 2 response element (FRE). Gel-shift assays with radiolabeled FRE, homeodomain-protein binding site (HOX) and transforming growth factor-beta1 activation element (TAE) double-strand oligonucleotides revealed increased binding of nuclear proteins from amelogenin-stimulated ROS 17/2.8 cells at 3 h (25 and 13 kDa) and 6 h (20 and 6 kDa). These results demonstrate that porcine 25 kDa amelogenin and its proteolytic derivatives stimulate bone sialoprotein transcription by targeting FRE, HOX and TAE in the bone sialoprotein gene promoter, and that full-length amelogenin and amelogenin cleavage products are able to regulate bone sialoprotein transcription via different signaling pathways. (c) 2010 John Wiley & Sons A/S.

Trichinella spiralis: strong antibody response to a 49 kDa newborn larva antigen in infected rats.

PubMed

Salinas-Tobon, Maria Del Rosario; Navarrete-Leon, Anaid; Mendez-Loredo, Blanca Esther; Esquivel-Aguirre, Dalia; Martínez-Abrajan, Dulce Maria; Hernandez-Sanchez, Javier

2007-02-01

In this work, we analyzed the kinetics of anti-Trichinella spiralis newborn larva (NBL) antibodies (Ab) and the antigenic recognition pattern of NBL proteins and its dose effects. Wistar rats were infected with 0, 700, 2000, 4000 and 8000 muscle larvae (ML) and bled at different time intervals up to day 31 post infection (p.i.). Ab production was higher with 2000 ML dose and decreased with 8000, 4000 and 700 ML. Abs were not detected until day 10, peaked on day 14 for the 2000 ML dose and on day 19 for the other doses and thereafter declined slowly from 19 to 31 days p.i. In contrast, Abs to ML increased from day 10, peaked on day 19 and remained high until the end of the study. Abs bound strongly at least to three NBL components of 188, 205 and 49 kDa. NBL antigen of 188 and 205 kDa were recognized 10-26 days p.i. and that of 49 kDa from day 10 to day 31 p.i. A weak recognition towards antigens of 52, 54, 62 and 83 kDa was also observed during the infection. An early recognition of 31, 43, 45, 55, 68 and 85 kDa ML antigens was observed whereas the response to those of 43, 45, 48, 60, 64 and 97 kDa (described previously as TSL-1 antigens) occurred late in the infection. A follow-up of antigen recognition up to day 61 with the optimal immunization dose (2000 ML) evidenced a decline of Ab production to the 49 kDa NBL antigen 42 days p.i., which suggested antigenic differences with the previously reported 43 kDa ML antigen strongly recognized late in the infection. To analyze the stage-specificity of the 49 kDa NBL antigen, polyclonal antibodies (PoAb) were obtained in rats immunized with 49 kDa NBL antigen. PoAb reacted strongly with the 49 kDa NBL component in NBL total soluble extract but no reactivity was observed with soluble antigen of the other T. spiralis stages. Albeit with less intensity, the 49 kDa component was also recognized by PoAb together with other antigens of 53, 97 and 107 kDa, in NBL excretory-secretory products (NBL-ESP). Thus, our results reveal

B-Lymphocytes Expressing an Ig Specificity Recognizing the Pancreatic β-Cell Autoantigen Peripherin Are Potent Contributors to Type 1 Diabetes Development in NOD Mice

PubMed Central

Leeth, Caroline M.; Racine, Jeremy; Chapman, Harold D.; Arpa, Berta; Carrillo, Jorge; Carrascal, Jorge; Wang, Qiming; Ratiu, Jeremy; Egia-Mendikute, Leire; Rosell-Mases, Estela; Stratmann, Thomas

2016-01-01

Although the autoimmune destruction of pancreatic β-cells underlying type 1 diabetes (T1D) development is ultimately mediated by T cells in NOD mice and also likely in humans, B cells play an additional key pathogenic role. It appears that the expression of plasma membrane–bound Ig molecules that efficiently capture β-cell antigens allows autoreactive B cells that bypass normal tolerance induction processes to be the subset of antigen-presenting cells most efficiently activating diabetogenic T cells. NOD mice transgenically expressing Ig molecules recognizing antigens that are (insulin) or are not (hen egg lysozyme [HEL]) expressed by β-cells have proven useful in dissecting the developmental basis of diabetogenic B cells. However, these transgenic Ig specificities were originally selected for their ability to recognize insulin or HEL as foreign, rather than autoantigens. Thus, we generated and characterized NOD mice transgenically expressing an Ig molecule representative of a large proportion of naturally occurring islet-infiltrating B cells in NOD mice recognizing the neuronal antigen peripherin. Transgenic peripherin-autoreactive B cells infiltrate NOD pancreatic islets, acquire an activated proliferative phenotype, and potently support accelerated T1D development. These results support the concept of neuronal autoimmunity as a pathogenic feature of T1D, and targeting such responses could ultimately provide an effective disease intervention approach. PMID:26961115

Carboxyl methylation of 21-23 kDa membrane proteins in intact neuroblastoma cells is increased with differentiation.

PubMed

Haklai, R; Kloog, Y

1990-01-01

Evidence is presented for specific enzymatic methylation of 21-23 kDa membrane proteins in intact neuroblastoma N1E 115 cells, which is increased in dimethylsulfoxide-induced differentiated cells. Methylation of these proteins has characteristics typical of enzymatic reactions in which base labile volatile methyl groups are incorporated into proteins, consistent with the formation of protein carboxyl methylesters. However, these methylesters of the 21-23 kDa proteins are relatively stable compared to other protein carboxyl methylesters. The 3-fold increase in methylated 21-23 kDa proteins in the differentiated cells suggest biological significance in differentiation of the cell membranes.

Biofortification of soybean meal: immunological properties of the 27 kDa γ-zein.

PubMed

Krishnan, Hari B; Jang, Sungchan; Kim, Won-Seok; Kerley, Monty S; Oliver, Melvin J; Trick, Harold N

2011-02-23

Legumes, including soybeans ( Glycine max ), are deficient in sulfur-containing amino acids, which are required for the optimal growth of monogastric animals. This deficiency can be overcome by expressing heterologous proteins rich in sulfur-containing amino acids in soybean seeds. A maize 27 kDa γ-zein, a cysteine-rich protein, has been successfully expressed in several crops including soybean, barley, and alfalfa with the intent to biofortify these crops for animal feed. Previous work has shown that the maize 27 kDa zein can withstand digestion by pepsin and elicit an immunogenic response in young pigs. By use of sera from patients who tested positive by ImmunoCAP assay for elevated IgE to maize proteins, specific IgE binding to the 27 kDa γ-zein is demonstrated. Bioinformatic analysis using the full-length and 80 amino acid sliding window FASTA searches identified significant sequence homology of the 27 kDa γ-zein with several known allergens. Immunoblot analysis using human serum that cross-reacts with maize seed proteins also revealed specific IgE-binding to the 27 kDa γ-zein in soybean seed protein extracts containing the 27 kDa zein. This study demonstrates for the first time the allergenicity potential of the 27 kDa γ-zein and the potential that this protein has to limit livestock performance when used in soybeans that serve as a biofortified feed supplement.

A Bayesian framework based on a Gaussian mixture model and radial-basis-function Fisher discriminant analysis (BayGmmKda V1.1) for spatial prediction of floods

NASA Astrophysics Data System (ADS)

Tien Bui, Dieu; Hoang, Nhat-Duc

2017-09-01

In this study, a probabilistic model, named as BayGmmKda, is proposed for flood susceptibility assessment in a study area in central Vietnam. The new model is a Bayesian framework constructed by a combination of a Gaussian mixture model (GMM), radial-basis-function Fisher discriminant analysis (RBFDA), and a geographic information system (GIS) database. In the Bayesian framework, GMM is used for modeling the data distribution of flood-influencing factors in the GIS database, whereas RBFDA is utilized to construct a latent variable that aims at enhancing the model performance. As a result, the posterior probabilistic output of the BayGmmKda model is used as flood susceptibility index. Experiment results showed that the proposed hybrid framework is superior to other benchmark models, including the adaptive neuro-fuzzy inference system and the support vector machine. To facilitate the model implementation, a software program of BayGmmKda has been developed in MATLAB. The BayGmmKda program can accurately establish a flood susceptibility map for the study region. Accordingly, local authorities can overlay this susceptibility map onto various land-use maps for the purpose of land-use planning or management.

Cardioprotective effects of 70-kDa heat shock protein in transgenic mice.

PubMed Central

Radford, N B; Fina, M; Benjamin, I J; Moreadith, R W; Graves, K H; Zhao, P; Gavva, S; Wiethoff, A; Sherry, A D; Malloy, C R; Williams, R S

1996-01-01

Heat shock proteins are proposed to limit injury resulting from diverse environmental stresses, but direct metabolic evidence for such a cytoprotective function in vertebrates has been largely limited to studies of cultured cells. We generated lines of transgenic mice to express human 70-kDa heat shock protein constitutively in the myocardium. Hearts isolated from these animals demonstrated enhanced recovery of high energy phosphate stores and correction of metabolic acidosis following brief periods of global ischemia sufficient to induce sustained abnormalities of these variables in hearts from nontransgenic littermates. These data demonstrate a direct cardioprotective effect of 70-kDa heat shock protein to enhance postischemic recovery of the intact heart. Images Fig. 1 Fig. 3 PMID:8637874

Characterization of a novel isoform of alpha-nascent polypeptide-associated complex as IgE-defined autoantigen.

PubMed

Mossabeb, Roschanak; Seiberler, Susanne; Mittermann, Irene; Reininger, Renate; Spitzauer, Susanne; Natter, Susanne; Verdino, Petra; Keller, Walter; Kraft, Dietrich; Valenta, Rudolf

2002-10-01

The nascent polypeptide-associated complex is required for intracellular translocation of newly synthesized polypeptides in eukaryotic cells. It may also act as a transcriptional coactivator in humans and various eukaryotic organisms and binds to nucleic acids. Recently, we provided evidence that a component of nascent polypeptide-associated complex, alpha-nascent polypeptide-associated complex, represents an IgE-reactive autoantigen for atopic dermatitis patients. By oligonucleotide screening we isolated a complete cDNA coding for a so far unknown alpha-nascent polypeptide-associated complex isoform from a human epithelial cDNA library. Southern blot hybridization experiments provided further evidence that alpha-nascent polypeptide-associated complex is encoded by a gene family. Recombinant alpha-nascent polypeptide-associated complex was expressed in Escherichia coli as a soluble, His-tagged protein, and purified via nickel affinity chromatography. By circular dichroism analysis it is demonstrated that purified recombinant alpha-nascent polypeptide-associated complex represents a folded protein of mixed alpha-helical and beta-sheet conformation with unusual high thermal stability and remarkable refolding capacity. Complete recombinant alpha-nascent polypeptide-associated complex (215 amino acids) and its 86 amino acid C-terminal fragment specifically bound IgE autoantibodies. Recombinant alpha-nascent polypeptide-associated complex also inhibited IgE binding to natural alpha-nascent polypeptide-associated complex, demonstrating the presence of common IgE epitopes between the recombinant and natural protein. Furthermore, recombinant alpha-nascent polypeptide-associated complex induced specific lymphoproliferative responses in peripheral blood mononuclear cells of a sensitized atopic dermatitis patient. As has been proposed for environmental allergens it is possible that T cell responses to IgE-defined autoantigens may contribute to the chronic skin manifestations

Influence of 120 kDa Pyruvate:Ferredoxin Oxidoreductase on Pathogenicity of Trichomonas vaginalis.

PubMed

Song, Hyun-Ouk

2016-02-01

Trichomonas vaginalis is a flagellate protozoan parasite and commonly infected the lower genital tract in women and men. Iron is a known nutrient for growth of various pathogens, and also reported to be involved in establishment of trichomoniasis. However, the exact mechanism was not clarified. In this study, the author investigated whether the 120 kDa protein of T. vaginalis may be involved in pathogenicity of trichomonads. Antibodies against 120 kDa protein of T. vaginalis, which was identified as pyruvate:ferredoxin oxidoreductase (PFOR) by peptide analysis of MALDI-TOF-MS, were prepared in rabbits. Pretreatment of T. vaginalis with anti-120 kDa Ab decreased the proliferation and adherence to vaginal epithelial cells (MS74) of T. vaginalis. Subcutaneous tissue abscess in anti-120 kDa Ab-treated T. vaginalis-injected mice was smaller in size than that of untreated T. vaginalis-infected mice. Collectively, the 120 kDa protein expressed by iron may be involved in proliferation, adhesion to host cells, and abscess formation, thereby may influence on the pathogenicity of T. vaginalis.

Studying the highly bent spectra of FR II-type radio galaxies with the KDA EXT model

NASA Astrophysics Data System (ADS)

Kuligowska, Elżbieta

2018-04-01

Context. The Kaiser, Dennett-Thorpe & Alexander (KDA, 1997, MNRAS, 292, 723) EXT model, that is, the extension of the KDA model of Fanaroff & Riley (FR) II-type source evolution, is applied and confronted with the observational data for selected FR II-type radio sources with significantly aged radio spectra. Aim. A sample of FR II-type radio galaxies with radio spectra strongly bent at their highest frequencies is used for testing the usefulness of the KDA EXT model. Methods: The dynamical evolution of FR II-type sources predicted with the KDA EXT model is briefly presented and discussed. The results are then compared to the ones obtained with the classical KDA approach, assuming the source's continuous injection and self-similarity. Results: The results and corresponding diagrams obtained for the eight sample sources indicate that the KDA EXT model predicts the observed radio spectra significantly better than the best spectral fit provided by the original KDA model.

Th1-stimulatory polyproteins of soluble Leishmania donovani promastigotes ranging from 89.9 to 97.1 kDa offers long-lasting protection against experimental visceral leishmaniasis.

PubMed

Kumari, Shraddha; Samant, Mukesh; Misra, Pragya; Khare, Prashant; Sisodia, Brijesh; Shasany, Ajit K; Dube, Anuradha

2008-10-23

Our earlier studies identified a fraction (F2) of Leishmania donovani soluble promastigote antigen belonging to 97.4-68 kDa for its ability to stimulate Th1-type cellular responses in cured visceral leishmaniasis (VL) patients as well as in cured hamsters. A further fractionation of F2-fraction into seven subfractions (F2.1-F2.7) and re-assessment for their immunostimulatory responses revealed that out of these, only four (F2.4-F2.7) belonging to 89.9-97.1 kDa, stimulated remarkable Th1-type cellular responses either individually or in a pooled form (P4-7). In this study these potential subfractions were further assessed for their prophylactic potential in combination with BCG against L. donovani challenge in hamsters. Optimum parasite inhibition ( approximately 99%) was obtained in hamsters vaccinated with pooled subfractions and they survived for 1 year. The protection was further supported by remarkable lymphoproliferative, IFN-gamma and IL-12 responses along with profound delayed type hypersensitivity and increased levels of Leishmania-specific IgG2 antibody as observed on days 45, 90 and 120 post-challenge suggesting that a successful subunit vaccine against VL may require multiple Th1-immunostimulatory proteins. MALDI-TOF-MS/MS analysis of these subfractions further revealed that of the 19 identified immunostimulatory proteins, Elongation factor-2, p45, Heat shock protein-70/83, Aldolase, Enolase, Triosephosphate isomerase, Disulfideisomerase and Calreticulin were the major ones in these subfractions.

Purification and partial sequencing of the nuclear autoantigen RA33 shows that it is indistinguishable from the A2 protein of the heterogeneous nuclear ribonucleoprotein complex.

PubMed Central

Steiner, G; Hartmuth, K; Skriner, K; Maurer-Fogy, I; Sinski, A; Thalmann, E; Hassfeld, W; Barta, A; Smolen, J S

1992-01-01

RA33 is a nuclear autoantigen with an apparent molecular mass of 33 kD. Autoantibodies against RA33 are found in about 30% of sera from RA patients, but only occasionally in sera from patients with other connective tissue diseases. To characterize RA33, the antigen was purified from HeLa cell nuclear extracts to more than 90% homogeneity by affinity chromatography on heparin-Sepharose and by chromatofocusing. Sequence analysis of five tryptic peptides revealed that their sequences matched corresponding sequences of the A2 protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex. Furthermore, RA33 was shown to be present in the 40S hnRNP complex and to behave indistinguishably from A2 in binding to single stranded DNA. In summary, these data strongly indicate that RA33 and A2 are the same protein, and thus identify on a molecular level a new autoantigen. Images PMID:1522214

Identification of an abundant 56 kDa protein implicated in food allergy as granule-bound starch synthase

USDA-ARS?s Scientific Manuscript database

Rice, the staple food of South and East Asian counties, is considered to be hypoallergenic. However, several clinical studies have documented rice-induced allergy in sensitive patients. Rice proteins with molecular weights of 14-16 kDa, 26 kDa, 33 kDa and 56 kDa have been identified as allergens. Re...

Maize 27 kDa gamma-zein is a potential allergen for early weaned pigs.

PubMed

Krishnan, Hari B; Kerley, Monty S; Allee, Gary L; Jang, Sungchan; Kim, Won-Seok; Fu, Chunjiang J

2010-06-23

Soybean and maize are extensively used in animal feed, primarily in poultry, swine, and cattle diets. Soybean meal can affect pig performance in the first few weeks following weaning and elicit specific antibodies in weaned piglets. Though maize is a major component of pig feed, it is not known if any of the maize proteins can elicit immunological response in young pigs. In this study, we have identified a prominent 27 kDa protein from maize as an immunodominant protein in young pigs. This protein, like some known allergens, exhibited resistance to pepsin digestion in vitro. Several lines of evidence identify the immunodominant 27 kDa protein as a gamma-zein, a maize seed storage protein. First, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of different solubility classes of maize seed proteins revealed the presence of an abundant 27 kDa protein in the prolamin (zein) fraction. Antibodies raised against the purified maize 27 kDa gamma-zein also reacted against the same protein recognized by the young pig serum. Additionally, matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of the peptides generated by trypsin digestion of the immunodominant 27 kDa protein showed significant homology to the maize 27 kDa gamma-zein. Since eliminating the allergenic protein will have a great impact on the nutritive value of the maize meal and expand its use in the livestock industry, it will be highly desirable to develop maize cultivars completely lacking the 27 kDa allergenic protein.

Expression, purification, and characterization of a bifunctional 99-kDa peptidoglycan hydrolase from Pediococcus acidilactici ATCC 8042.

PubMed

García-Cano, Israel; Campos-Gómez, Manuel; Contreras-Cruz, Mariana; Serrano-Maldonado, Carlos Eduardo; González-Canto, Augusto; Peña-Montes, Carolina; Rodríguez-Sanoja, Romina; Sánchez, Sergio; Farrés, Amelia

2015-10-01

Pediococcus acidilactici ATCC 8042 is a lactic acid bacteria that inhibits pathogenic microorganisms such as Staphylococcus aureus through the production of two proteins with lytic activity, one of 110 kDa and the other of 99 kDa. The 99-kDa one has high homology to a putative peptidoglycan hydrolase (PGH) enzyme reported in the genome of P. acidilactici 7_4, where two different lytic domains have been identified but not characterized. The aim of this work was the biochemical characterization of the recombinant enzyme of 99 kDa. The enzyme was cloned and expressed successfully and retains its activity against Micrococcus lysodeikticus. It has a higher N-acetylglucosaminidase activity, but the N-acetylmuramoyl-L-alanine amidase can also be detected spectrophotometrically. The protein was then purified using gel filtration chromatography. Antibacterial activity showed an optimal pH of 6.0 and was stable between 5.0 and 7.0. The optimal temperature for activity was 60 °C, and all activity was lost after 1 h of incubation at 70 °C. The number of strains susceptible to the recombinant 99-kDa enzyme was lower than that susceptible to the mixture of the 110- and 99-kDa PGHs of P. acidilactici, a result that suggests synergy between these two enzymes. This is the first PGH from LAB that has been shown to possess two lytic sites. The results of this study will aid in the design of new antibacterial agents from natural origin that can combat foodborne disease and improve hygienic practices in the industrial sector.

A relevant IgE-reactive 28kDa protein identified from Salsola kali pollen extract by proteomics is a natural degradation product of an integral 47kDa polygalaturonase.

PubMed

Mas, Salvador; Oeo-Santos, Carmen; Cuesta-Herranz, Javier; Díaz-Perales, Araceli; Colás, Carlos; Fernández, Javier; Barber, Domingo; Rodríguez, Rosalía; de Los Ríos, Vivian; Barderas, Rodrigo; Villalba, Mayte

2017-08-01

A highly prevalent IgE-binding protein band of 28kDa is observed when Salsola kali pollen extract is incubated with individual sera from Amaranthaceae pollen sensitized patients. By an immunoproteomic analysis of S. kali pollen extract, we identified this protein band as an allergenic polygalacturonase enzyme. The allergen, named Sal k 6, exhibits a pI of 7.14 and a molecular mass of 39,554.2Da. It presents similarities to Platanaceae, Poaceae, and Cupressaceae allergenic polygalacturonases. cDNA-encoding sequence was subcloned into the pET41b vector and produced in bacteria as a His-tag fusion recombinant protein. The far-UV CD spectrum determined that rSal k 6 was folded. Immunostaining of the S. kali pollen protein extract with a rSal k 6-specific pAb and LC-MS/MS proteomic analyses confirmed the co-existence of the 28kDa band together with an allergenic band of about 47kDa in the pollen extract. Therefore, the 28kDa was assigned as a natural degradation product of the 47kDa integral polygalacturonase. The IgE-binding inhibition to S. kali pollen extract using rSal k 6 as inhibitor showed that signals directed to both protein bands of 28 and 47kDa were completely abrogated. The average prevalence of rSal k 6 among the three populations analyzed was 30%, with values correlating well with the levels of grains/m 3 of Amaranthaceae pollen. Sal k 6 shares IgE epitopes with Oleaceae members (Fraxinus excelsior, Olea europaea and Syringa vulgaris), with IgE-inhibition values ranging from 20% to 60%, respectively. No IgE-inhibition was observed with plant-derived food extracts. Copyright © 2017 Elsevier B.V. All rights reserved.

A 115 kDa calmodulin-binding protein is located in rat liver endosome fractions.

PubMed Central

Enrich, C; Bachs, O; Evans, W H

1988-01-01

The distribution of calmodulin-binding polypeptides in various rat liver subcellular fractions was investigated. Plasma-membrane, endosome, Golgi and lysosome fractions were prepared by established procedures. The calmodulin-binding polypeptides present in the subcellular fractions were identified by using an overlay technique after transfer from gels to nitrocellulose sheets. Distinctive populations of calmodulin-binding polypeptides were present in all the fractions examined except lysosomes. A major 115 kDa calmodulin-binding polypeptide of pI 4.3 was located to the endosome subfractions, and it emerges as a candidate endosome-specific protein. Partitioning of endosome fractions between aqueous and Triton X-114 phases indicated that the calmodulin-binding polypeptide was hydrophobic. Major calmodulin-binding polypeptides of 140 and 240 kDa and minor polypeptides of 40-60 kDa were present in plasma membranes. The distribution of calmodulin in the various endosome and plasma-membrane fractions was also analysed, and the results indicated that the amounts were high compared with those in the cytosol. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:3214436

Photoaffinity labelling of the cardiac calcium channel. (-)-[3H]azidopine labels a 165 kDa polypeptide, and evidence against a [3H]-1,4-dihydropyridine-isothiocyanate being a calcium-channel-specific affinity ligand.

PubMed

Ferry, D R; Goll, A; Glossmann, H

1987-04-01

The arylazide 1,4-dihydropyridine (-)-[3H]azidopine binds to a saturable population of sites in guinea-pig heart membranes with a dissociation constant (KD) of 30 +/- 7 pM and a density (Bmax.) of 670 +/- 97 fmol/mg of protein. This high-affinity binding site is assumed to reside on voltage-operated calcium channels because reversible binding is blocked stereoselectively by 1,4-dihydropyridine channel blockers and by the enantiomers of Bay K 8644. A low-affinity (KD 25 +/- 7 nM) high-capacity (Bmax. 21.6 +/- 9 pmol/mg of protein) site does not bind (-)- or (+)-Bay K 8644, but is blocked by high concentrations (greater than 500 nM) of dihydro-2,6-dimethyl-4-(2-isothiocyanatophenyl)-3,5-pyridinedicarboxy lic acid dimethyl ester (1,4-DHP-isothiocyanate) or, e.g., (+/-)-nicardipine. (-)-[3H]Azidopine was photoincorporated covalently into bands of 165 +/- 8, 39 +/- 2 and 35 +/- 3 kDa, as determined by SDS/polyacrylamide-gel electrophoresis. Labelling of the 165 kDa band is protected stereoselectively by 1,4-dihydropyridine enantiomers at low (nM) concentrations and by (-)- and (+)-Bay K 8644, whereas the lower-Mr bands are not. Thus, only the 165 kDa band is the calcium-channel-linked 1,4-dihydropyridine receptor. Photolabelling of the 39 or 35 kDa bands was only blocked by 10 microM-1,4-DHP-isothiocyanate or 50 microM-(+/-)-nicardipine but not by 10 microM-(-)-Bay K 8644. [3H]-1,4-DHP-isothiocyanate binds to guinea-pig heart membranes with a KD of 0.35 nM and dissociates with a k-1 of 0.2 min-1 at 30 degrees C. [3H]-1,4 DHP-isothiocyanate irreversibly labels bands of 39 and 35 kDa which are protected by greater than 10 microM-(+/-)-nicardipine or unlabelled ligand but not by 10 microM-(-)-Bay K 8644. Thus, [3H]-1,4-DHP-isothiocyanate is not an affinity probe for the calcium channel.

The Effects of Threonine Phosphorylation on the Stability and Dynamics of the Central Molecular Switch Region of 18.5-kDa Myelin Basic Protein

PubMed Central

De Avila, Miguel; Polverini, Eugenia; Harauz, George

2013-01-01

The classic isoforms of myelin basic protein (MBP) are essential for the formation and maintenance of myelin in the central nervous system of higher vertebrates. The protein is involved in all facets of the development, compaction, and stabilization of the multilamellar myelin sheath, and also interacts with cytoskeletal and signaling proteins. The predominant 18.5-kDa isoform of MBP is an intrinsically-disordered protein that is a candidate auto-antigen in the human demyelinating disease multiple sclerosis. A highly-conserved central segment within classic MBP consists of a proline-rich region (murine 18.5-kDa sequence –T92-P93-R94-T95-P96-P97-P98-S99–) containing a putative SH3-ligand, adjacent to a region that forms an amphipathic α-helix (P82-I90) upon interaction with membranes, or under membrane-mimetic conditions. The T92 and T95 residues within the proline-rich region can be post-translationally modified through phosphorylation by mitogen-activated protein (MAP) kinases. Here, we have investigated the structure of the α-helical and proline-rich regions in dilute aqueous buffer, and have evaluated the effects of phosphorylation at T92 and T95 on the stability and dynamics of the α-helical region, by utilizing four 36-residue peptides (S72–S107) with differing phosphorylation status. Nuclear magnetic resonance spectroscopy reveals that both the α-helical as well as the proline-rich regions are disordered in aqueous buffer, whereas they are both structured in a lipid environment (cf., Ahmed et al., Biochemistry 51, 7475-9487, 2012). Thermodynamic analysis of trifluoroethanol-titration curves monitored by circular dichroism spectroscopy reveals that phosphorylation, especially at residue T92, impedes formation of the amphipathic α-helix. This conclusion is supported by molecular dynamics simulations, which further illustrate that phosphorylation reduces the folding reversibility of the α-helix upon temperature perturbation and affect the global

The effects of threonine phosphorylation on the stability and dynamics of the central molecular switch region of 18.5-kDa myelin basic protein.

PubMed

Vassall, Kenrick A; Bessonov, Kyrylo; De Avila, Miguel; Polverini, Eugenia; Harauz, George

2013-01-01

The classic isoforms of myelin basic protein (MBP) are essential for the formation and maintenance of myelin in the central nervous system of higher vertebrates. The protein is involved in all facets of the development, compaction, and stabilization of the multilamellar myelin sheath, and also interacts with cytoskeletal and signaling proteins. The predominant 18.5-kDa isoform of MBP is an intrinsically-disordered protein that is a candidate auto-antigen in the human demyelinating disease multiple sclerosis. A highly-conserved central segment within classic MBP consists of a proline-rich region (murine 18.5-kDa sequence -T92-P93-R94-T95-P96-P97-P98-S99-) containing a putative SH3-ligand, adjacent to a region that forms an amphipathic α-helix (P82-I90) upon interaction with membranes, or under membrane-mimetic conditions. The T92 and T95 residues within the proline-rich region can be post-translationally modified through phosphorylation by mitogen-activated protein (MAP) kinases. Here, we have investigated the structure of the α-helical and proline-rich regions in dilute aqueous buffer, and have evaluated the effects of phosphorylation at T92 and T95 on the stability and dynamics of the α-helical region, by utilizing four 36-residue peptides (S72-S107) with differing phosphorylation status. Nuclear magnetic resonance spectroscopy reveals that both the α-helical as well as the proline-rich regions are disordered in aqueous buffer, whereas they are both structured in a lipid environment (cf., Ahmed et al., Biochemistry 51, 7475-9487, 2012). Thermodynamic analysis of trifluoroethanol-titration curves monitored by circular dichroism spectroscopy reveals that phosphorylation, especially at residue T92, impedes formation of the amphipathic α-helix. This conclusion is supported by molecular dynamics simulations, which further illustrate that phosphorylation reduces the folding reversibility of the α-helix upon temperature perturbation and affect the global structure

Agarose and Polyacrylamide Gel Electrophoresis Methods for Molecular Mass Analysis of 5–500 kDa Hyaluronan

PubMed Central

Bhilocha, Shardul; Amin, Ripal; Pandya, Monika; Yuan, Han; Tank, Mihir; LoBello, Jaclyn; Shytuhina, Anastasia; Wang, Wenlan; Wisniewski, Hans-Georg; de la Motte, Carol; Cowman, Mary K.

2011-01-01

Agarose and polyacrylamide gel electrophoresis systems for the molecular mass-dependent separation of hyaluronan (HA) in the size range of approximately 5–500 kDa have been investigated. For agarose-based systems, the suitability of different agarose types, agarose concentrations, and buffers systems were determined. Using chemoenzymatically synthesized HA standards of low polydispersity, the molecular mass range was determined for each gel composition, over which the relationship between HA mobility and logarithm of the molecular mass was linear. Excellent linear calibration was obtained for HA molecular mass as low as approximately 9 kDa in agarose gels. For higher resolution separation, and for extension to molecular masses as low as approximately 5 kDa, gradient polyacrylamide gels were superior. Densitometric scanning of stained gels allowed analysis of the range of molecular masses present in a sample, and calculation of weight-average and number-average values. The methods were validated for polydisperse HA samples with viscosity-average molecular masses of 112, 59, 37, and 22 kDa, at sample loads of 0.5 µg (for polyacrylamide) to 2.5 µg (for agarose). Use of the methods for electrophoretic mobility shift assays was demonstrated for binding of the HA-binding region of aggrecan (recombinant human aggrecan G1-IGD-G2 domains) to a 150 kDa HA standard. PMID:21684248

Desflurane Hepatitis Associated with Hapten and Autoantigen-Specific IgG4 Antibodies

PubMed Central

Anderson, James S.; Rose, Noel R.; Martin, Jackie L.; Eger, Edmond I.; Njoku, Dolores B.

2013-01-01

BACKGROUND Three cases of drug-induced liver injury (DILI) have been reported after desflurane anesthesia. However, no previous reports have detected serum autoantibodies such as that reported with DILI from halothane or isoflurane. METHODS AND RESULTS We describe the first documentation of cytochrome P450 2E1 IgG4 autoantibodies, as well as 58 kDa endoplasmic reticulum protein and trifluoroacetyl chloride hapten-specific IgG4 antibodies, in a patient who developed DILI after desflurane anesthesia. CONCLUSIONS These findings suggest that allergic and autoimmune mechanisms have critical roles in the development of desflurane DILI. PMID:17513640

Transcriptional activation of a 37 kDa ethylene responsive cysteine protease gene, RbCP1, is associated with protein degradation during petal abscission in rose

PubMed Central

Tripathi, Siddharth Kaushal; Singh, Amar Pal; Sane, Aniruddha P.; Nath, Pravendra

2009-01-01

Cysteine proteases play an important role in several developmental processes in plants, particularly those related to senescence and cell death. A cysteine protease gene, RbCP1, has been identified that encodes a putative protein of 357 amino acids and is expressed in the abscission zone (AZ) of petals in rose. The gene was responsive to ethylene in petals, petal abscission zones, leaves, and thalamus. The expression of RbCP1 increased during both ethylene-induced as well as natural abscission and was inhibited by 1-MCP. Transcript accumulation of RbCP1 was accompanied by the appearance of a 37 kDa cysteine protease, a concomitant increase in protease activity and a substantial decrease in total protein content in the AZ of petals. Agro-injection of rose petals with a 2.0 kb region upstream of the RbCP1 gene could drive GUS expression in an abscission zone-specific manner and was blocked by 1-MCP. It is concluded that petal abscission is associated with a decrease in total protein content resulting from rapid transcription of RbCP1 and the expression of a 37 kDa protease. PMID:19346241

7 CFR 51.2541 - U.S. Fancy, U.S. Extra No. 1, U.S. No. 1 And U.S. Select Grades.

Code of Federal Regulations, 2010 CFR

2010-01-01

... PRODUCTS 1,2 (INSPECTION, CERTIFICATION, AND STANDARDS) United States Standards for Grades of Pistachio.... Fancy,” “U.S. Extra No. 1,” “U.S. No. 1,” and “U.S. Select” consists of pistachio nuts in the shell...

ATP can be dispensable for prespliceosome formation in yeast

PubMed Central

Perriman, Rhonda; Ares, Manuel

2000-01-01

The first ATP-dependent step in pre-mRNA splicing involves the stable binding of U2 snRNP to form the prespliceosome. We show that a prespliceosome-like complex forms in the absence of ATP in yeast extracts lacking the U2 suppressor protein CUS2. These complexes display the same pre-mRNA and U snRNA requirements as authentic prespliceosomes and can be chased through the splicing pathway, indicating that they are a functional intermediate in the spliceosome assembly pathway. ATP-independent prespliceosome-like complexes are also observed in extracts containing a mutant U2 snRNA. Loss of CUS2 does not bypass the role of PRP5, an RNA helicase family member required for ATP-dependent prespliceosome formation. Genetic interactions between CUS2 and a heat-sensitive prp5 allele parallel those observed between CUS2 and U2, and suggest that CUS2 mediates functional interactions between U2 RNA and PRP5. We propose that CUS2 enforces ATP dependence during formation of the prespliceosome by brokering an interaction between PRP5 and the U2 snRNP that depends on correct U2 RNA structure. PMID:10640279

USP15 regulates dynamic protein–protein interactions of the spliceosome through deubiquitination of PRP31

PubMed Central

Das, Tanuza; Park, Joon Kyu; Park, Jinyoung; Kim, Eunji; Rape, Michael

2017-01-01

Abstract Post-translational modifications contribute to the spliceosome dynamics by facilitating the physical rearrangements of the spliceosome. Here, we report USP15, a deubiquitinating enzyme, as a regulator of protein–protein interactions for the spliceosome dynamics. We show that PRP31, a component of U4 snRNP, is modified with K63-linked ubiquitin chains by the PRP19 complex and deubiquitinated by USP15 and its substrate targeting factor SART3. USP15SART3 makes a complex with USP4 and this ternary complex serves as a platform to deubiquitinate PRP31 and PRP3. The ubiquitination and deubiquitination status of PRP31 regulates its interaction with the U5 snRNP component PRP8, which is required for the efficient splicing of chromosome segregation related genes, probably by stabilizing the U4/U6.U5 tri-snRNP complex. Collectively, our data suggest that USP15 plays a key role in the regulation of dynamic protein–protein interactions of the spliceosome. PMID:28088760

Usefulness of 8 kDa protein of Fasciola hepatica in diagnosis of fascioliasis

PubMed Central

Kim, Kwangsig; Yang, Hyun Jong

2003-01-01

This study was designed to detect and evaluate an antigenicity of low molecular weight proteins of Fasciola hepatica in fascioliasis. Low molecular weight protein of F. hepatica was purified by ammonium sulfate precipitation and Sephacryl S-100 HR gel filtration. The protein obtained was estimated to be 8 kDa on 7.5-15% gradient sodium dodecyl sulfate gel electrophoresis. Immunoblotting studies showed that the 8 kDa protein reacted with human fascioliasis sera, but not other trematodiasis sera. This result suggests that the 8 kDa protein of F. hepatica is one of diagnostic antigens in human fascioliasis without cross-reaction with other human trematodiasis. PMID:12815325

Phosphorylation of Tat-interactive protein 60 kDa by protein kinase C epsilon is important for its subcellular localisation.

PubMed

Sapountzi, Vasileia; Logan, Ian R; Nelson, Glyn; Cook, Susan; Robson, Craig N

2008-01-01

Tat-interactive protein 60 kDa is a nuclear acetyltransferase that both coactivates and corepresses transcription factors and has a definitive function in the DNA damage response. Here, we provide evidence that Tat-interactive protein 60 kDa is phosphorylated by protein kinase C epsilon. In vitro, protein kinase C epsilon phosphorylates Tat-interactive protein 60 kDa on at least two sites within the acetyltransferase domain. In whole cells, activation of protein kinase C increases the levels of phosphorylated Tat-interactive protein 60 kDa and the interaction of Tat-interactive protein 60 kDa with protein kinase C epsilon. A phosphomimetic mutant Tat-interactive protein 60 kDa has distinct subcellular localisation compared to the wild-type protein in whole cells. Taken together, these findings suggest that the protein kinase C epsilon phosphorylation sites on Tat-interactive protein 60 kDa are important for its subcellular localisation. Regulation of the subcellular localisation of Tat-interactive protein 60 kDa via phosphorylation provides a novel means of controlling Tat-interactive protein 60 kDa function.

The analysis Arabidopsis thaliana overexpressing a 14kDa self-folding protein [abstract

USDA-ARS?s Scientific Manuscript database

A recent study in banana identified a 14kDa protein that has been hypothesized to function in regulating the nucleation and growth of the needle-shaped crystals of calcium oxalate that accumulate within the tissues of this plant. To gain further insight in to the functional role of this 14 kDa prote...

Oleosins (24 and 18 kDa) are hydrolyzed not only in extracted soybean oil bodies but also in soybean germination.

PubMed

Chen, Yeming; Zhao, Luping; Cao, Yanyun; Kong, Xiangzhen; Hua, Yufei

2014-01-29

After oil bodies (OBs) were extracted from ungerminated soybean by pH 6.8 extraction, it was found that 24 and 18 kDa oleosins were hydrolyzed in the extracted OBs, which contained many OB extrinsic proteins (i.e., lipoxygenase, β-conglycinin, γ-conglycinin, β-amylase, glycinin, Gly m Bd 30K (Bd 30K), and P34 probable thiol protease (P34)) as well as OB intrinsic proteins. In this study, some properties (specificity, optimal pH and temperature) of the proteases of 24 and 18 kDa oleosins and the oleosin hydrolysis in soybean germination were examined, and the high relationship between Bd 30K/P34 and the proteases was also discussed. The results showed (1) the proteases were OB extrinsic proteins, which had high specificity to hydrolyze 24 and 18 kDa oleosins, and cleaved the specific peptide bonds to form limited hydrolyzed products; (2) 24 and 18 kDa oleosins were not hydrolyzed in the absence of Bd 30K and P34 (or some Tricine-SDS-PAGE undetectable proteins); (3) the protease of 24 kDa oleosin had strong resistance to alkaline pH while that of 18 kDa oleosin had weak resistance to alkaline pH, and Bd 30K and P34, resolved into two spots on two-dimensional electrophoresis gel, also showed the same trend; (4) 16 kDa oleosin as well as 24 and 18 kDa oleosins were hydrolyzed in soybean germination, and Bd 30K and P34 were always contained in the extracted OBs from germinated soybean even when all oleosins were hydrolyzed; (5) the optimal temperature and pH of the proteases were respectively determined as in the ranges of 35-50 °C and pH 6.0-6.5, while 60 °C or pH 11.0 could denature them.

Formation of the 67-kDa laminin receptor by acylation of the precursor.

PubMed

Butò, S; Tagliabue, E; Ardini, E; Magnifico, A; Ghirelli, C; van den Brûle, F; Castronovo, V; Colnaghi, M I; Sobel, M E; Ménard, S

1998-06-01

Even though the involvement of the 67-kDa laminin receptor (67LR) in tumor invasiveness has been clearly demonstrated, its molecular structure remains an open problem, since only a full-length gene encoding a 37-kDa precursor protein (37LRP) has been isolated so far. A pool of recently obtained monoclonal antibodies directed against the recombinant 37LRP molecule was used to investigate the processing that leads to the formation of the 67-kDa molecule. In soluble extracts of A431 human carcinoma cells, these reagents recognize the precursor molecule as well as the mature 67LR and a 120-kDa molecule. The recovery of these proteins was found to be strikingly dependent upon the cell solubilization conditions: the 67LR is soluble in NP-40-lysis buffer whereas the 37LRP is NP-40-insoluble. Inhibition of 67LR formation by cerulenin indicates that acylation is involved in the processing of the receptor. It is likely a palmitoylation process, as indicated by sensitivity of NP-40-soluble extracts to hydroxylamine treatment. Immunoblotting assays performed with a polyclonal serum directed against galectin3 showed that both the 67- and the 120-kDa proteins carry galectin3 epitopes whereas the 37LRP does not. These data suggest that the 67LR is a heterodimer stabilized by strong intramolecular hydrophobic interactions, carried by fatty acids bound to the 37LRP and to a galectin3 cross-reacting molecule.

Detection of brain-directed autoantibodies in the serum of non-small cell lung cancer patients.

PubMed

Banjara, Manoj; Ghosh, Chaitali; Dadas, Aaron; Mazzone, Peter; Janigro, Damir

2017-01-01

Antibodies against brain proteins were identified in the plasma of cancer patients and are defined to cause paraneoplastic neurological syndromes. The profiles of brain-directed antibodies in non-small cell lung cancer (NSCLC) are largely unknown. Here, for the first time, we compared autoantibodies against brain proteins in NSCLC (n = 18) against those present in age-matched non-cancer control subjects (n = 18) with a similar life-style, habit, and medical history. Self-recognizing immunoglobulin (IgG) are primarily directed against cells in the cortex (P = 0.008), hippocampus (P = 0.003-0.05), and cerebellum (P = 0.02). More specifically, IgG targets were prominent in the pyramidal, Purkinje, and granule cell layers. Furthermore, autoimmune IgG signals were localized to neurons (81%), astrocytes (48%), and endothelial (29%) cells. While cancer sera yielded overall higher intensity signals, autoantigens of 100, 65, 45, 37, and 30 kDa molecular weights were the most represented. Additionally, a group of 100 kDa proteins seem more prevalent in female adenocarcinoma patients (4/5, 80%). In conclusion, our results revealed autoantigen specificity in NSCLC, which implicitly depends on patient's demographics and disease history. Patients at risk for lung cancer but with no active disease revealed that the immune profile in NSCLC is disease-dependent.

Structure–function analysis and genetic interactions of the SmG, SmE, and SmF subunits of the yeast Sm protein ring

PubMed Central

Schwer, Beate; Kruchten, Joshua; Shuman, Stewart

2016-01-01

A seven-subunit Sm protein ring forms a core scaffold of the U1, U2, U4, and U5 snRNPs that direct pre-mRNA splicing. Using human snRNP structures to guide mutagenesis in Saccharomyces cerevisiae, we gained new insights into structure–function relationships of the SmG, SmE, and SmF subunits. An alanine scan of 19 conserved amino acids of these three proteins, comprising the Sm RNA binding sites or inter-subunit interfaces, revealed that, with the exception of Arg74 in SmF, none are essential for yeast growth. Yet, for SmG, SmE, and SmF, as for many components of the yeast spliceosome, the effects of perturbing protein–RNA and protein–protein interactions are masked by built-in functional redundancies of the splicing machine. For example, tests for genetic interactions with non-Sm splicing factors showed that many benign mutations of SmG, SmE, and SmF (and of SmB and SmD3) were synthetically lethal with null alleles of U2 snRNP subunits Lea1 and Msl1. Tests of pairwise combinations of SmG, SmE, SmF, SmB, and SmD3 alleles highlighted the inherent redundancies within the Sm ring, whereby simultaneous mutations of the RNA binding sites of any two of the Sm subunits are lethal. Our results suggest that six intact RNA binding sites in the Sm ring suffice for function but five sites may not. PMID:27417296

Definition of the HLA-A29 peptide ligand motif allows prediction of potential T-cell epitopes from the retinal soluble antigen, a candidate autoantigen in birdshot retinopathy.

PubMed Central

Boisgerault, F; Khalil, I; Tieng, V; Connan, F; Tabary, T; Cohen, J H; Choppin, J; Charron, D; Toubert, A

1996-01-01

The peptide-binding motif of HLA-A29, the predisposing allele for birdshot retinopathy, was determined after acid-elution of endogenous peptides from purified HLA-A29 molecules. Individual and pooled HPLC fractions were sequenced by Edman degradation. Major anchor residues could be defined as glutamate at the second position of the peptide and as tyrosine at the carboxyl terminus. In vitro binding of polyglycine synthetic peptides to purified HLA-A29 molecules also revealed the need for an auxiliary anchor residue at the third position, preferably phenylalanine. By using this motif, we synthesized six peptides from the retinal soluble antigen, a candidate autoantigen in autoimmune uveoretinitis. Their in vitro binding was tested on HLA-A29 and also on HLA-B44 and HLA-B61, two alleles sharing close peptide-binding motifs. Two peptides derived from the carboxyl-terminal sequence of the human retinal soluble antigen bound efficiently to HLA-A29. This study could contribute to the prediction of T-cell epitopes from retinal autoantigens implicated in birdshot retinopathy. PMID:8622959

Atomic features of an autoantigen in heparin-induced thrombocytopenia (HIT).

PubMed

Cai, Zheng; Zhu, Zhiqiang; Greene, Mark I; Cines, Douglas B

2016-07-01

Autoantigen development is poorly understood at the atomic level. Heparin-induced thrombocytopenia (HIT) is an autoimmune thrombotic disorder caused by antibodies to an antigen composed of platelet factor 4 (PF4) and heparin or cellular glycosaminoglycans (GAGs). In solution, PF4 exists as an equilibrium among monomers, dimers and tetramers. Structural studies of these interacting components helped delineate a multi-step process involved in the pathogenesis of HIT. First, heparin binds to the 'closed' end of the PF4 tetramer and stabilizes its conformation; exposing the 'open' end. Second, PF4 arrays along heparin/GAG chains, which approximate tetramers, form large antigenic complexes that enhance antibody avidity. Third, pathogenic HIT antibodies bind to the 'open' end of stabilized PF4 tetramers to form an IgG/PF4/heparin ternary immune complex and also to propagate the formation of 'ultralarge immune complexes' (ULCs) that contain multiple IgG antibodies. Fourth, ULCs signal through FcγRIIA receptors, activating platelets and monocytes directly and generating thrombin, which transactivates hematopoietic and endothelial cells. A non-pathogenic anti-PF4 antibody prevents tetramer formation, binding of pathogenic antibody, platelet activation and thrombosis, providing a new approach to manage HIT. An improved understanding of the pathogenesis of HIT may lead to novel diagnostics and therapeutics for this autoimmune disease. Copyright © 2016 Elsevier B.V. All rights reserved.

Translocation of an 89-kDa periplasmic protein is associated with Holospora infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Iwatani, Koichi; Dohra, Hideo; Lang, B. Franz

2005-12-02

The symbiotic bacterium Holospora obtusa infects the macronucleus of the ciliate Paramecium caudatum. After ingestion by its host, an infectious form of Holospora with an electron-translucent tip passes through the host digestive vacuole and penetrates the macronuclear envelope with this tip. To investigate the underlying molecular mechanism of this process, we raised a monoclonal antibody against the tip-specific 89-kDa protein, sequenced this partially, and identified the corresponding complete gene. The deduced protein sequence carries two actin-binding motifs. Indirect immunofluorescence microscopy shows that during escape from the host digestive vacuole, the 89-kDa proteins translocates from the inside to the outside ofmore » the tip. When the bacterium invades the macronucleus, the 89-kDa protein is left behind at the entry point of the nuclear envelope. Transmission electron microscopy shows the formation of fine fibrous structures that co-localize with the antibody-labeled regions of the bacterium. Our findings suggest that the 89-kDa protein plays a role in Holospora's escape from the host digestive vacuole, the migration through the host cytoplasm, and the invasion into the macronucleus.« less

The 33.1 kDa Excretory/secretory Protein Produced by Toxocara canis Larvae Serves as a Potential Common Biomarker for Serodiagnosis of Toxocariasis in Paratenic Animals and Human.

PubMed

Nguyen, Huu-Hung; Vo, Doan-Trung; Thai, Thi-Tuyet-Trinh; LE, Thi-Thanh-Thao; LE, Thanh-Dong; Hoang, Nghia-Son

2017-01-01

Toxocariasis is a prevalent zoonosis disease caused by the closely related nematode species Toxocara canis and Toxocara cati which parasitise Canidae and Felidae respectively. In paratenic hosts, larvae of these worms cause multiple organ damage. However, how these paratenic hosts response to these worms and whether any common biomarker can be applied for diagnosis are still unclear. Excreted/secreted (E/S) antigens were prepared by culture of T. canis larvae in vitro. Using a western blot (WB) assay the humoral IgG responses, induced by Toxocara spp. larvae to the worm's E/S antigens in different infected hosts including mice, rabbits and human, were examined. In a mouse model of toxocariasis, intraperitoneal injection of T. canis larvae induces inflammatory leukocyte accumulation in the liver and the lungs but not in the brain, although a remarkable number of larvae were detected in this organ. Mice and rabbits responded differently to Toxocara spp. resulting in distinct heterogenous WB band patterns. Mice and rabbits both responded to a 33.1 kDa E/S constituent that turned out to be the most sensitive protein for serodiagnosis. Sera from human toxocariasis patients showed heterogenous WB band patterns similar to those observed in rabbits and all responded to the 33.1 kDa band. 33.1 kDa E/S protein can be considered as a critical common biomarker for toxocariasis immuno-diagnosis in both paratenic animals and human and its specificity requires further investigation.

The 33.1 kDa Excretory/secretory Protein Produced by Toxocara canis Larvae Serves as a Potential Common Biomarker for Serodiagnosis of Toxocariasis in Paratenic Animals and Human

PubMed Central

NGUYEN, Huu-Hung; VO, Doan-Trung; THAI, Thi-Tuyet-Trinh; LE, Thi-Thanh-Thao; LE, Thanh-Dong; HOANG, Nghia-Son

2017-01-01

Background: Toxocariasis is a prevalent zoonosis disease caused by the closely related nematode species Toxocara canis and Toxocara cati which parasitise Canidae and Felidae respectively. In paratenic hosts, larvae of these worms cause multiple organ damage. However, how these paratenic hosts response to these worms and whether any common biomarker can be applied for diagnosis are still unclear. Methods: Excreted/secreted (E/S) antigens were prepared by culture of T. canis larvae in vitro. Using a western blot (WB) assay the humoral IgG responses, induced by Toxocara spp. larvae to the worm’s E/S antigens in different infected hosts including mice, rabbits and human, were examined. Results: In a mouse model of toxocariasis, intraperitoneal injection of T. canis larvae induces inflammatory leukocyte accumulation in the liver and the lungs but not in the brain, although a remarkable number of larvae were detected in this organ. Mice and rabbits responded differently to Toxocara spp. resulting in distinct heterogenous WB band patterns. Mice and rabbits both responded to a 33.1 kDa E/S constituent that turned out to be the most sensitive protein for serodiagnosis. Sera from human toxocariasis patients showed heterogenous WB band patterns similar to those observed in rabbits and all responded to the 33.1 kDa band. Conclusion: 33.1 kDa E/S protein can be considered as a critical common biomarker for toxocariasis immuno-diagnosis in both paratenic animals and human and its specificity requires further investigation. PMID:28761463

Functional Genomic Screening Reveals Splicing of the EWS-FLI1 Fusion Transcript as a Vulnerability in Ewing Sarcoma.

PubMed

Grohar, Patrick J; Kim, Suntae; Rangel Rivera, Guillermo O; Sen, Nirmalya; Haddock, Sara; Harlow, Matt L; Maloney, Nichole K; Zhu, Jack; O'Neill, Maura; Jones, Tamara L; Huppi, Konrad; Grandin, Magdalena; Gehlhaus, Kristen; Klumpp-Thomas, Carleen A; Buehler, Eugen; Helman, Lee J; Martin, Scott E; Caplen, Natasha J

2016-01-26

Ewing sarcoma cells depend on the EWS-FLI1 fusion transcription factor for cell survival. Using an assay of EWS-FLI1 activity and genome-wide RNAi screening, we have identified proteins required for the processing of the EWS-FLI1 pre-mRNA. We show that Ewing sarcoma cells harboring a genomic breakpoint that retains exon 8 of EWSR1 require the RNA-binding protein HNRNPH1 to express in-frame EWS-FLI1. We also demonstrate the sensitivity of EWS-FLI1 fusion transcripts to the loss of function of the U2 snRNP component, SF3B1. Disrupted splicing of the EWS-FLI1 transcript alters EWS-FLI1 protein expression and EWS-FLI1-driven expression. Our results show that the processing of the EWS-FLI1 fusion RNA is a potentially targetable vulnerability in Ewing sarcoma cells. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Identification of a major 50-kDa molecular weight human B-cell growth factor with Tac antigen-inducing activity on B cells.

PubMed

Kawano, M; Matsushima, K; Oppenheim, J J

1987-08-01

A bioassay was developed using human small B cells adherent to anti-human IgM (anti-mu)-coated wells. These B cells were stimulated to proliferate by culture supernatants of concanavalin A (Con A)-activated human peripheral blood lymphocytes (Con A Sup) even in the presence of high concentrations of anti-mu coated on assay wells. Human B-cell growth factor (BCGF) activities were partially purified from Con A Sup. Preparative chromatography (Sephacryl S-200 and isoelectrofocusing) yielded a major peak of BCGF activity for B cells adherent to anti-mu-coated wells with a molecular weight of 50,000 (50 kDa) and a pI 7.6. The 50-kDa BCGF was further purified by sequential chromatography using DEAE-Sephacel, CM-Sepharose, Sephacryl S-200, CM-high performance liquid chromatography (HPLC), and hydroxyapatite (HA)-HPLC. The HA-HPLC-purified 50-kDa BCGF was free of interleukin-1 (IL-1), interleukin-2 (IL-2), and interferon activities, but could support growth of BCL1 cells, similar to BCGF-II. Neither IL-1 nor interferon-gamma had any growth-stimulating effect in our B-cell proliferation assay with or without BCGF in Iscove's synthetic assay medium. BCGF-induced proliferation of B cells adherent to anti-mu-coated wells could be markedly augmented by the simultaneous or sequential addition of recombinant human IL-2 (rIL-2). When cultured for 3 days with 50-kDa BCGF, about 40% of B cells adherent to anti-mu-coated wells expressed Tac antigen, and monoclonal anti-Tac antibody inhibited rIL-2 enhancement of proliferation of 50-kDa BCGF-preactivated B cells. In addition, 50-kDa BCGF could induce Tac antigen on an Epstein-Barr virus-transformed B-cell line (ORSON) in the presence of a suboptimal dose of phorbol myristate acetate (PMA) and also on a natural killer-like cell line (YT cells). We have therefore identified a major 50-kDa BCGF activity with Tac antigen-inducing activity that also has a synergistic effect with IL-2 on normal B-cell proliferation.

Plasmodium falciparum spliceosomal RNAs: 3' and 5' end processing.

PubMed

Eliana, Calvo; Javier, Escobar; Moisés, Wasserman

2011-02-01

The major spliceosomal small nuclear ribonucleoproteins (snRNPs) consist of snRNA (U1, U2, U4 or U5) and several proteins which can be unique or common to each snRNP particle. The common proteins are known as Sm proteins; they are crucial for RNP assembly and nuclear import of spliceosomal RNPs. This paper reports detecting the interaction between Plasmodium falciparum snRNAs and Sm proteins, and the usual 5' trimethylated caps on the snRNAs, by immunoprecipitation with specific antibodies. Furthermore, an unusual poly(A) tail was detected on these non-coding RNAs. 2010 Elsevier B.V. All rights reserved.

Functioning of the Drosophila Wilms'-Tumor-1-Associated Protein Homolog, Fl(2)d, in Sex-Lethal-Dependent Alternative Splicing

PubMed Central

Penn, Jill K. M.; Graham, Patricia; Deshpande, Girish; Calhoun, Gretchen; Chaouki, Ahmad Sami; Salz, Helen K.; Schedl, Paul

2008-01-01

fl(2)d, the Drosophila homolog of Wilms'-tumor-1-associated protein (WTAP), regulates the alternative splicing of Sex-lethal (Sxl), transformer (tra), and Ultrabithorax (Ubx). Although WTAP has been found in functional human spliceosomes, exactly how it contributes to the splicing process remains unknown. Here we attempt to identify factors that interact genetically and physically with fl(2)d. We begin by analyzing the Sxl-Fl(2)d protein–protein interaction in detail and present evidence suggesting that the female-specific fl(2)d1 allele is antimorphic with respect to the process of sex determination. Next we show that fl(2)d interacts genetically with early acting general splicing regulators and that Fl(2)d is present in immunoprecipitable complexes with Snf, U2AF50, U2AF38, and U1-70K. By contrast, we could not detect Fl(2)d complexes containing the U5 snRNP protein U5-40K or with a protein that associates with the activated B spliceosomal complex SKIP. Significantly, the genetic and molecular interactions observed for Sxl are quite similar to those detected for fl(2)d. Taken together, our findings suggest that Sxl and fl(2)d function to alter splice-site selection at an early step in spliceosome assembly. PMID:18245840

The U(1)-Kepler Problems

NASA Astrophysics Data System (ADS)

Meng, Guowu

2010-12-01

Let n ⩾ 2 be a positive integer. To each irreducible representation σ of U(1), a U(1)-Kepler problem in dimension (2n - 1) is constructed and analyzed. This system is superintegrable and when n = 2 it is equivalent to a MICZ-Kepler problem. The dynamical symmetry group of this system is widetildeU(n, n), and the Hilbert space of bound states {{H}}(σ ) is the unitary highest weight representation of widetildeU(n, n) with the minimal positive Gelfand-Kirillov dimension. Furthermore, it is shown that the correspondence between σ ^* (the dual of σ) and {H}(σ ) is the theta-correspondence for dual pair (U(1), U(n,n))subseteq Sp_{4n}({R}).

Clinical and serologic parallels to APS-I in patients with thymomas, and autoantigen transcripts in their tumors1

PubMed Central

Wolff, Anette S. B.; Kärner, Jaanika; Owe, Jone F.; Oftedal, Bergithe E.V.; Gilhus, Nils Erik; Erichsen, Martina M.; Kämpe, Olle; Meager, Anthony; Peterson, Pärt; Kisand, Kai; Willcox, Nick; Husebye, Eystein S.

2014-01-01

Patients with the autoimmune polyendocrine syndrome type I (APS-I), caused by mutations in the autoimmune regulator (AIRE) gene, and myasthenia gravis (MG) with thymoma, show intriguing but unexplained parallels. They include uncommon manifestations like autoimmune adrenal insufficiency (AI), hypoparathyroidism (HP), and chronic mucocutaneous candidiasis (CMC) plus autoantibodies neutralizing IL-17, IL-22 and type I interferons. Thymopoiesis in the absence of AIRE is implicated in both syndromes. To test whether these parallels extend further, we screened 247 patients with MG and/or thymoma for clinical features and organ-specific autoantibodies characteristic of APS-I patients, and assayed 26 thymoma samples for transcripts for AIRE and 16 peripheral tissue-specific autoantigens (TSAgs) by quantitative PCR. We found APS-I-typical autoantibodies and clinical manifestations, including CMC, AI and asplenia, respectively in 49/121 (40%) and 10/121 (8%) thymoma patients, but clinical features seldom co-occurred with the corresponding autoantibodies. Both were rare in other MG subgroups (N=126). In 38 APS-I patients, by contrast, we observed neither autoantibodies against muscle antigens nor any neuromuscular disorders. Whereas relative transcript levels for AIRE and 7 of 16 TSAgs showed the expected under-expression in thymomas, levels were increased for 4 of the 5 TSAgs most frequently targeted by these patients’ autoAbs. Hence the clinical and serologic parallels to APS-I in patients with thymomas are not explained purely by deficient TSAg transcription in these aberrant AIRE-deficient tumors. We therefore propose additional explanations for the unusual autoimmune biases they provoke. Thymoma patients should be monitored for potentially life-threatening APS-I manifestations such as AI and HP. PMID:25230752

A conserved 19-kDa Eimeria tenella antigen is a profilin-like protein.

PubMed

Fetterer, R H; Miska, K B; Jenkins, M C; Barfield, R C

2004-12-01

A wide range of recombinant proteins from Eimeria species have been reported to offer some degree of protection against infection and disease, but the specific biological function of these proteins is largely unknown. Previous studies have demonstrated a 19-kDa protein of unknown function designated SZ-1 in sporozoites and merozoites of Eimeria acervulina that can be used to confer partial protection against coccidiosis. Reverse transcriptase-polymerase chain reaction indicated that the gene for SZ-1 is expressed by all the asexual stages of Eimeria tenella. Rabbit antisera to recombinant SZ-1 recognized an approximately 19-kDa protein from extracts of E. tenella sporozoites, merozoites, sporulated oocysts, and oocysts in various stages of sporulation. Immunofluorescence antibody staining indicated specific staining of E. tenella sporozoites and merozoites. Staining was most intense in the cytoplasm of the posterior end of the parasite. The primary amino acid sequence of the gene for E. tenella SZ-1 deduced from the E. tenella genome indicated a conserved domain for the actin-regulatory protein profilin. A conserved binding site for poly-L-proline (PLP), characteristic of profilin was also observed. SZ-1 was separated from soluble extract of E. tenella proteins by affinity chromatography using a PLP ligand, confirming the ability of SZ-1 to bind PLP. SZ-1 also partially inhibited the polymerization of actin. The current results are consistent with the classification of SZ-1 as a profilin-related protein.

High expression of 23 kDa protein of augmenter of liver regeneration (ALR) in human hepatocellular carcinoma

PubMed Central

Yu, Hai-Ying; Zhu, Man-Hua; Xiang, Dai-Rong; Li, Jun; Sheng, Ji-Fang

2014-01-01

Background Augmenter of liver regeneration (ALR) is an important polypeptide that participates in the process of liver regeneration. Two forms of ALR proteins are expressed in hepatocytes. Previous data have shown that ALR is essential for cell survival and has potential antimetastatic properties in hepatocellular carcinoma (HCC). Aims The study aimed to evaluate the expression levels of two forms of ALR proteins in HCC and their possible significance in HCC development. Methods Balb/c mouse monoclonal antibody against ALR protein was prepared in order to detect the ALR protein in HCC by Western blotting and immunohistochemistry. ALR mRNA expression levels were measured by real-time polymerase chain reaction in HCC tissues and compared to paracancerous liver tissues in 22 HCC patients. Results ALR mRNA expression in HCC liver tissues (1.51×106 copies/μL) was higher than in paracancerous tissues (1.04×104 copies/μL). ALR protein expression was also enhanced in HCC liver tissues. The enhanced ALR protein was shown to be 23 kDa by Western blotting. Immunohistochemical analysis showed that the 23 kDa ALR protein mainly existed in the hepatocyte cytosol. Conclusion The 23 kDa ALR protein was highly expressed in HCC and may play an important role in hepatocarcinogenesis. PMID:24940072

Structure-function analysis and genetic interactions of the SmG, SmE, and SmF subunits of the yeast Sm protein ring.

PubMed

Schwer, Beate; Kruchten, Joshua; Shuman, Stewart

2016-09-01

A seven-subunit Sm protein ring forms a core scaffold of the U1, U2, U4, and U5 snRNPs that direct pre-mRNA splicing. Using human snRNP structures to guide mutagenesis in Saccharomyces cerevisiae, we gained new insights into structure-function relationships of the SmG, SmE, and SmF subunits. An alanine scan of 19 conserved amino acids of these three proteins, comprising the Sm RNA binding sites or inter-subunit interfaces, revealed that, with the exception of Arg74 in SmF, none are essential for yeast growth. Yet, for SmG, SmE, and SmF, as for many components of the yeast spliceosome, the effects of perturbing protein-RNA and protein-protein interactions are masked by built-in functional redundancies of the splicing machine. For example, tests for genetic interactions with non-Sm splicing factors showed that many benign mutations of SmG, SmE, and SmF (and of SmB and SmD3) were synthetically lethal with null alleles of U2 snRNP subunits Lea1 and Msl1. Tests of pairwise combinations of SmG, SmE, SmF, SmB, and SmD3 alleles highlighted the inherent redundancies within the Sm ring, whereby simultaneous mutations of the RNA binding sites of any two of the Sm subunits are lethal. Our results suggest that six intact RNA binding sites in the Sm ring suffice for function but five sites may not. © 2016 Schwer et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Mixed Connective Tissue Disease and Epitope Spreading: An Historical Cohort Study.

PubMed

Escolà-Vergé, Laura; Pinal-Fernandez, Iago; Fernandez-Codina, Andreu; Callejas-Moraga, Eduardo L; Espinosa, Juan; Marin, Ana; Labrador-Horrillo, Moises; Selva-O'Callaghan, Albert

2017-04-01

Mixed connective tissue disease (MCTD) is characterized by the presence of anti-U1-snRNP autoantibodies and a variable set of associated clinical features. Some MCTD patients test positive over time to autoantibodies against Sm, proteins spatially related with U1-snRNP. This situation has been attributed to expanding of the autoimmune response by a phenomenon known as epitope spreading. Our aim was to study the frequency of this phenomenon in MCTD patients and the specific clinical features of those with epitope spreading. All anti-U1-RNP-positive patients (2010-2015) were retrospectively reviewed, and those meeting the MCTD criteria were included in the study. Patients showing epitope spreading were compared with the remainder of the MCTD cohort. In addition, the clinical features of patients with epitope spreading were compared before and after the phenomenon occurred. Among 72 anti-U1-RNP-positive patients, 40 (37 women) were diagnosed with MCTD. Thirteen MCTD patients (43%) presented epitope spreading, mainly during the first 2 years after the diagnosis of the disease (median, 1.4 years). Patients with epitope spreading had a significantly lower prevalence of skin sclerosis (0% vs. 44%, P = 0.004) and a greater prevalence of interstitial lung disease (46% vs. 15%, P = 0.05) than those without. Arthritis (92% vs. 25%, P = 0.02) and muscle involvement (67% vs. 17%, P = 0.02) were less frequent after epitope spreading had occurred. Epitope spreading is common in MCTD, occurring early after the diagnosis. The clinical manifestations in patients with this phenomenon differ from those without, and their clinical features change after the immunological phenomenon has occurred.

Effects of Liposomal Compositions with Oxidized Dextrans on Functional Activity of U937 Macrophage-Like Cells In Vitro.

PubMed

Kozhin, P M; Chechushkov, A V; Zaitseva, N S; Lemza, A E; Men'shchikova, E B; Troitskii, A V; Shkurupy, V A

2015-11-01

We studied the effects of liposomal pharmaceutical compositions with oxidized dextrans on functional activity of U937 monocyte/macrophage-like cells. Liposomes in the emulsion contained oxidized dextran with a molecular weights of 40 kDa or 70 kDa or isonicotinic acid hydrazide (INAH) conjugated with oxidized dextran (40 kDa). Cell viability was evaluated by MTT test; mitochondrial transmembrane potential and production of superoxide anion and H2O2 were studied by fluorescent methods. The studied compositions exhibited no cytotoxic effect and even improved cell viability and mitochondrial respiration. Liposomes with oxidized 40 kDa dextran, including those with INAH-conjugated dextran, inhibited production of superoxide anion, but increased H2O2 generation.

Targeting Anti-Insulin B Cell Receptors Improves Receptor Editing in Type 1 Diabetes-Prone Mice1, 2, 3

PubMed Central

Bonami, Rachel H.; Thomas, James W.

2015-01-01

Autoreactive B lymphocytes that commonly arise in the developing repertoire can be salvaged by receptor editing, a central tolerance mechanism that alters BCR specificity through continued L chain rearrangement. It is unknown whether autoantigens with weak cross-linking potential, such as insulin, elicit receptor editing, or if this process is dysregulated in related autoimmunity. To resolve these issues, an editing-competent model was developed in which anti-insulin Vκ125 was targeted to the Igκ locus and paired with anti-insulin VH125Tg. Physiologic, circulating insulin increased RAG-2 expression and was associated with BCR replacement that eliminated autoantigen recognition in a proportion of developing anti-insulin B lymphocytes. The proportion of anti-insulin B cells that underwent receptor editing was reduced in the type 1 diabetes-prone NOD strain relative to a non-autoimmune strain. Resistance to editing was associated with increased surface IgM expression on immature (but not transitional or mature) anti-insulin B cells in the NOD strain. The actions of mAb123 on central tolerance were also investigated, as selective targeting of insulin-occupied BCR by mAb123 eliminates anti-insulin B lymphocytes and prevents type 1 diabetes. Autoantigen-targeting by mAb123 increased RAG-2 expression and dramatically enhanced BCR replacement in newly developed B lymphocytes. Administering F(ab’)2123 induced IgM downregulation and reduced the frequency of anti-insulin B lymphocytes within the polyclonal repertoire of VH125Tg/NOD mice, suggesting enhanced central tolerance by direct BCR interaction. These findings indicate that weak or faulty checkpoints for central tolerance can be overcome by autoantigen-specific immunomodulatory therapy. PMID:26432895

Extremely High Peak Power Pulsed RF and UWB EMR Effects on Genomic Transcription - Microarray Assessment

DTIC Science & Technology

2008-06-26

Homo sapiens decorin variant C mRNA, complete cds. 2.117 PKNOX2 HUM408A08B Human fetal brain (TFujiwara) Homo sapiens cDNA clone GEN -408A08 5’, mRNA...mRNA, complete cds. 2.117 PKNOX2 HUM408A08B Human fetal brain (TFujiwara) Homo sapiens cDNA clone GEN -408A08 5’, mRNA sequence. 2.076 SEC23B...RAS oncogene family ; RAB33B, member RAS oncogene family 205300_s_at 0.37 U1SNRNPBP U11/U12 snRNP 35K 220728_at 0.349 218689_at 0.342 FANCF Fanconi

Molecular characterization of a 40 kDa OmpC-like porin from Serratia marcescens.

PubMed

Hutsul, J A; Worobec, E

1994-02-01

An oligonucleotide that encodes the N-terminal portion of a 41 kDa porin of Serratia marcescens was used to probe S. marcescens UOC-51 genomic DNA. An 11 kb EcoRI fragment which hybridized with the oligonucleotide was subcloned into Escherichia coli, examined for expression, and sequenced. The product expressed by the cloned gene was 40 kDa. The nucleotide sequence has an ORF of 1.13 kb. When the deduced amino acid sequence was aligned and compared to other enterobacterial porins the cloned S. marcescens porin most closely resembled E. coli OmpC. Although we did not detect osmoregulation or thermoregulation of any porins in S. marcescens UOC-51, sequences analogous to the E. coli osmoregulator OmpR-binding regions are seen upstream to the cloned gene. We examined the regulation of the S. marcescens porin in E. coli and found that its expression increased in a high salt environment. A micF gene, whose transcriptional product functions to inhibit synthesis of OmpF by hybridizing with the ompF transcript, was also seen upstream of the S. marcescens ompC. An alignment with the E. coli micF gene revealed that the functional region of the S. marcescens micF gene is conserved. Based on the results obtained we have determined that S. marcescens UOC-51 produces a 40 kDa porin similar to the E. coli OmpC porin.

Cardioprotective effects of 70-kDa heat shock protein in transgenic mice.

PubMed

Radford, N B; Fina, M; Benjamin, I J; Moreadith, R W; Graves, K H; Zhao, P; Gavva, S; Wiethoff, A; Sherry, A D; Malloy, C R; Williams, R S

1996-03-19

Heat shock proteins are proposed to limit injury resulting from diverse environmental stresses, but direct metabolic evidence for such a cytoprotective function in vertebrates has been largely limited to studies of cultured cells. We generated lines of transgenic mice to express human 70-kDa heat shock protein constitutively in the myocardium. Hearts isolated from these animals demonstrated enhanced recovery of high energy phosphate stores and correction of metabolic acidosis following brief periods of global ischemia sufficient to induce sustained abnormalities of these variables in hearts from nontransgenic littermates. These data demonstrate a direct cardioprotective effect of 70-kDa heat shock protein to enhance postischemic recovery of the intact heart.

Keratin sponge/hydrogel part 1. fabrication and characterization

USDA-ARS?s Scientific Manuscript database

Keratin sponge/hydrogel products formed by either the oxidation or reduction of U.S. domestic fine- or coarse-grade wool exhibited distinctively different topologies and molecular weights of 6- 8 kDa and 40-60 kDa, each with unique macro-porous structure and microstructural behaviors. The sponge/ ...

Dot-Blot Immunoassay of Fasciola gigantica Infection using 27 kDa and Adult Worm Regurge Antigens in Egyptian Patients

PubMed Central

Kamel, Hanan H.; Saad, Ghada A.

2013-01-01

The purpose of the present study was to evaluate the potential role of the 27-Kilodalton (KDa) antigen versus Fasciola gigantica adult worm regurge antigens in a DOT-Blot assay and to assess this assay as a practical tool for diagnosis fascioliasis in Egyptian patients. Fasciola gigantica antigen of an approximate molecular mass 27-(KDa) was obtained from adult worms by a simple elution SDS-PAGE. A Dot-Blot was developed comparatively to adult worm regurge antigens for the detection of specific antibodies from patients infected with F. gigantica in Egypt. Control sera were obtained from patients with other parasitic infections and healthy volunteers to assess the test and compare between the antigens. The sensitivity, specificity, positive and negative predictive values of Dot-Blot using the adult worm regurge were 80%, 90%, 94.1%, and 69.2% respectively, while those using 27-KDa were 100% which confirms the diagnostic potential of this antigen. All patients infected with Fasciola were positive, with cross reactivity reported with Schistosoma mansoni serum samples. This 27-KDa Dot-Blot assay showed to be a promising test which can be used for serodiagnosis of fascioliasis in Egyptian patients especially, those presenting with hepatic disease. It is specific, sensitive and easy to perform method for the rapid diagnosis particularly when more complex laboratory tests are unavailable. PMID:23710084

Dot-blot immunoassay of Fasciola gigantica infection using 27 kDa and adult worm regurge antigens in Egyptian patients.

PubMed

Kamel, Hanan H; Saad, Ghada A; Sarhan, Rania M

2013-04-01

The purpose of the present study was to evaluate the potential role of the 27-Kilodalton (KDa) antigen versus Fasciola gigantica adult worm regurge antigens in a DOT-Blot assay and to assess this assay as a practical tool for diagnosis fascioliasis in Egyptian patients. Fasciola gigantica antigen of an approximate molecular mass 27-(KDa) was obtained from adult worms by a simple elution SDS-PAGE. A Dot-Blot was developed comparatively to adult worm regurge antigens for the detection of specific antibodies from patients infected with F. gigantica in Egypt. Control sera were obtained from patients with other parasitic infections and healthy volunteers to assess the test and compare between the antigens. The sensitivity, specificity, positive and negative predictive values of Dot-Blot using the adult worm regurge were 80%, 90%, 94.1%, and 69.2% respectively, while those using 27-KDa were 100% which confirms the diagnostic potential of this antigen. All patients infected with Fasciola were positive, with cross reactivity reported with Schistosoma mansoni serum samples. This 27-KDa Dot-Blot assay showed to be a promising test which can be used for serodiagnosis of fascioliasis in Egyptian patients especially, those presenting with hepatic disease. It is specific, sensitive and easy to perform method for the rapid diagnosis particularly when more complex laboratory tests are unavailable.

Heat shock 70-kDa protein 8 isoform 1 is expressed on the surface of human embryonic stem cells and downregulated upon differentiation.

PubMed

Son, Yeon Sung; Park, Jae Hyun; Kang, Young Kook; Park, Jin-Sung; Choi, Hong Seo; Lim, Ji Young; Lee, Jeoung Eun; Lee, Jung Bok; Ko, Myoung Seok; Kim, Yong-Sam; Ko, Jeong-Heon; Yoon, Hyun Soo; Lee, Kwang-Woong; Seong, Rho Hyun; Moon, Shin Yong; Ryu, Chun Jeih; Hong, Hyo Jeong

2005-01-01

The cell-surface markers used routinely to define the undifferentiated state and pluripotency of human embryonic stem cells (hESCs) are those used in mouse embryonic stem cells (mESCs) because of a lack of markers directly originated from hESC itself. To identify more hESC-specific cell-surface markers, we generated a panel of monoclonal antibodies (MAbs) by immunizing the irradiated cell clumps of hESC line Miz-hES1, and selected 26 MAbs that were able to bind to Miz-hES1 cells but not to mESCs, mouse embryonic fibroblast cells, and STO cells. Most antibodies did not bind to human neural progenitor cells derived from the Miz-hES1 cells, either. Of these, MAb 20-202S (IgG1, kappa) immunoprecipitated a cell-surface protein of 72-kDa from the lysate of biotin-labeled Miz-hES1 cells, which was identified to be heat shock 70-kDa protein 8 isoform 1 (HSPA8) by quadrupole time-of-flight tandem mass spectrometry. Immunocytochemical analyses proved that the HSPA8 protein was also present on the surface of hESC lines Miz-hES4, Miz-hES6, and HSF6. Two-color flow cytometric analysis of Miz-hES1 and HSF6 showed the coexpression of the HSPA8 protein with other hESC markers such as stage-specific embryonic antigen 3 (SSEA3), SSEA4, TRA-1-60, and TRA-1-81. Flow cytometric and Western blot analyses using various cells showed that MAb 20-202S specifically bound to the HSPA8 protein on the surface of Miz-hES1, contrary to other anti-HSP70 antibodies examined. Furthermore, the surface expression of the HSPA8 protein on Miz-hES1 was markedly downregulated upon differentiation. These data indicate that a novel MAb 20-202S recognizes the HSPA8 protein on the surface of hESCs and suggest that the HSPA8 protein is a putative cell-surface marker for undifferentiated hESCs.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Montemayor, Eric J.; Didychuk, Allison L.; Liao, Honghong

U6 small nuclear RNA (snRNA) is a key component of the active site of the spliceosome, a large ribonucleoprotein complex that catalyzes the splicing of precursor messenger RNA. Prior to its incorporation into the spliceosome, U6 is bound by the protein Prp24, which facilitates unwinding of the U6 internal stem-loop (ISL) so that it can pair with U4 snRNA. A previously reported crystal structure of the `core' of the U6 small nuclear ribonucleoprotein (snRNP) contained an ISL-stabilized A62G mutant of U6 bound to all four RNA-recognition motif (RRM) domains of Prp24 [Montemayoret al.(2014),Nature Struct. Mol. Biol.21, 544–551]. The structure revealedmore » a novel topology containing interlocked rings of protein and RNA that was not predicted by prior biochemical and genetic data. Here, the crystal structure of the U6 snRNP core with a wild-type ISL is reported. This complex crystallized in a new space group, apparently owing in part to the presence of an intramolecular cross-link in RRM1 that was not observed in the previously reported U6-A62G structure. The structure exhibits the same protein–RNA interface and maintains the unique interlocked topology. However, the orientation of the wild-type ISL is altered relative to the A62G mutant structure, suggesting inherent structural dynamics that may facilitate its pairing with U4. Consistent with their similar architectures in the crystalline state, the wild-type and A62G variants of U6 exhibit similar Prp24-binding affinities and electrophoretic mobilities when analyzed by gel-shift assay.« less

Interaction of human platelets with laminin and identification of the 67 kDa laminin receptor on platelets.

PubMed Central

Tandon, N N; Holland, E A; Kralisz, U; Kleinman, H K; Robey, F A; Jamieson, G A

1991-01-01

A microtitre adhesion assay has been developed to define parameters affecting the adherence of washed platelets to laminin. Adherence was optimally supported by Mg2+ and was inhibited by Ca2+ and by anti-laminin Fab fragments, but significant adhesion (75-90% of control) was found both in heparinized plasma containing physiological levels of bivalent cations and in plasma anti-coagulated with EGTA. Adherence was unaffected by platelet activation with ADP but was decreased by 50% by treatment with alpha-thrombin (1 unit/ml, 5 min). Adherence was unaffected by monospecific polyclonal antibodies to glycoprotein (GP) Ib and GPIV, and was normal with platelets from two patients with Glanzmann's thrombasthaenia, indicating that GPIb, the GPIIb/IIIa complex and GPIV are not involved in platelet-laminin interaction. Affinity chromatography of Triton-solubilized membranes on laminin-Sepharose followed by elution with 0.2 M-glycine/HCl (pH 2.85) identified a major band with a molecular mass of 67 kDa in the reduced and of 53 kDa in the unreduced form. This protein gave a positive reaction on Western blotting with a monospecific polyclonal antibody raised against the high-affinity laminin receptor isolated from human breast carcinoma tissue. The adhesion of platelets to laminin was inhibited by two monoclonal IgM antibodies specific to the LR-1 domain of the 67 kDa receptor. The binding protein was surface-oriented, as shown by flow cytofluorimetry and by the fact that it could be iodinated in intact platelets, but it was not labelled by the periodate-borotritide procedure, suggesting that it did not contain terminal sialic acid. The laminin-derived peptides Tyr-Ile-Gly-Ser-Arg and Cys-Asp-Pro-Gly-Tyr-Ile-Gly-Ser-Arg-NH2, which constitute a complementary binding domain in laminin for the 67 kDa receptor, themselves supported platelet adhesion, bound to the receptor and inhibited the adhesion of platelets to laminin. In addition, Fab fragments of anti

Melanoma cultures show different susceptibility towards E1A-, E1B-19 kDa- and fiber-modified replication-competent adenoviruses.

PubMed

Schmitz, M; Graf, C; Gut, T; Sirena, D; Peter, I; Dummer, R; Greber, U F; Hemmi, S

2006-06-01

Replicating adenovirus (Ad) vectors with tumour tissue specificity hold great promise for treatment of cancer. We have recently constructed a conditionally replicating Ad5 AdDeltaEP-TETP inducing tumour regression in a xenograft mouse model. For further improvement of this vector, we introduced four genetic modifications and analysed the viral cytotoxicity in a large panel of melanoma cell lines and patient-derived melanoma cells. (1) The antiapoptotic gene E1B-19 kDa (Delta19 mutant) was deleted increasing the cytolytic activity in 18 of 21 melanoma cells. (2) Introduction of the E1A 122-129 deletion (Delta24 mutant), suggested to attenuate viral replication in cell cycle-arrested cells, did not abrogate this activity and increased the cytolytic activity in two of 21 melanoma cells. (3) We inserted an RGD sequence into the fiber to extend viral tropism to alphav integrin-expressing cells, and (4) swapped the fiber with the Ad35 fiber (F35) enhancing the tropism to malignant melanoma cells expressing CD46. The RGD-fiber modification strongly increased cytolysis in all of the 11 CAR-low melanoma cells. The F35 fiber-chimeric vector boosted the cytotoxicity in nine of 11 cells. Our results show that rational engineering additively enhances the cytolytic potential of Ad vectors, a prerequisite for the development of patient-customized viral therapies.

Microparticles prepared with 50-190kDa chitosan as promising non-toxic carriers for pulmonary delivery of isoniazid.

PubMed

Oliveira, Paula M; Matos, Breno N; Pereira, Priscilla A T; Gratieri, Taís; Faccioli, Lucia H; Cunha-Filho, Marcílio S S; Gelfuso, Guilherme M

2017-10-15

Chitosan biocompatibility and mucoadhesiveness make it an ideal polymer for antituberculotic drugs microcapsulation for pulmonary delivery. Yet, previous study indicated toxicity problems to J-774.1-cells treated with some medium molecular weight (190-310kDa) chitosan microparticles. As polymer molecular weight is a crucial factor to be considered, this paper describes the preparation and characterization of chitosan (50-190kDa) microparticles containing isoniazid (INH). Cytotoxicity assays were also performed on murine peritoneal (J-774.1) and alveolar (AMJ2-C11) macrophages cell lines, followed by cytokines detection from AMJ2-C11 cells. Spray-drying process produced mucoadhesive microparticles from 3.2μm to 3.9μm, entrapping more than 89% of the drug and preserving their chemical stability. Drug release behavior could be controlled by the use of cross-linked or uncross-linked chitosan, the latter leading to a rapid drug release. Mucoadhesive potential of the microparticles was characterized following in vitro and ex vivo assays. Finally, a significant reduction on toxicity against peritoneal macrophages and no toxic effect on alveolar macrophages with use of such microparticles were observed. In conclusion, 50-190kDa chitosan microparticles may act as promising non-cytotoxic carriers for pulmonary delivery of INH showing marked alveoli macrophage activation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Tryptic digestion of human GPIIIa. Isolation and biochemical characterization of the 23 kDa N-terminal glycopeptide carrying the antigenic determinant for a monoclonal antibody (P37) which inhibits platelet aggregation.

PubMed Central

Calvete, J J; Rivas, G; Maruri, M; Alvarez, M V; McGregor, J L; Hew, C L; Gonzalez-Rodriguez, J

1988-01-01

Early digestion of pure human platelet glycoprotein IIIa (GPIIIa) leads to a single cleavage of the molecule at 23 kDa far from one of the terminal amino acids. Automated Edman degradation demonstrates that GPIIIa and the smaller (23 kDa) tryptic fragment share the same N-terminal amino acid sequence. A further cleavage occurs in the larger fragment (80 kDa), reducing its apparent molecular mass by 10 kDa. The 23 kDa fragment remains attached to the larger ones in unreduced samples. Stepwise reduction of early digested GPIIIa with dithioerythritol selectively reduces the single disulphide bond joining the smaller (23 kDa) to the larger (80/70 kDa) fragments. Two fractions were obtained by size-exclusion chromatography of early digested GPIIIa after partial or full reduction and alkylation. The larger-size fraction contains the 80/70 kDa fragments, while the 23 kDa fragment is isolated in the smaller. The amino acid compositions of these fractions do not differ very significantly from the composition of GPIIIa; however the 23 kDa fragment contains only 10.2% by weight of sugars and is richer in neuraminic acid. Disulphide bonds are distributed four in the 23 kDa glycopeptide and 20-21 in the 80/70 kDa glycopeptide. The epitope for P37, a monoclonal antibody which inhibits platelet aggregation [Melero & González-Rodríguez (1984) Eur. J. Biochem. 141, 421-427] is situated within the first 17 kDa of the N-terminal region of GPIIIa, which gives a special functional interest to this extracellular region of GPIIIa. On the other hand, the epitopes for GPIIIa-specific monoclonal antibodies, P6, P35, P40 and P97, which do not interfere with platelet aggregation, are located within the larger tryptic fragment (80/70 kDa). Thus, the antigenic areas available in the extracellular surface of GPIIIa for these five monoclonal antibodies are now more precisely delineated. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:2455507

In vivo exposure to ozone produces an increase in a 72-kDa heat shock protein in guinea pigs.

PubMed

Su, W Y; Gordon, T

1997-09-01

Although several lines of evidence have suggested that oxidizing agents can induce heat shock proteins (HSPs) in vitro, little is known about the induction of HSPs during in vivo exposure to oxidants. Guinea pigs were exposed to ozone for 6 h and euthanized up to 72 h later. Proteins from lavage cells and lung tissue were characterized by immunoblotting with 72- and 73/72-kDa HSP monoclonal antibodies. Although 73-kDa HSP was expressed constituitively in lung tissue, it was not affected by ozone. In contrast, 72-kDa HSP was significantly increased in lavage cells and lung tissue of animals exposed to 0.4 and 0.66 parts/million of ozone. Both heat treatment and arsenite induced 72-kDa HSP in cultured alveolar macrophages. The increase in 72-kDa HSP in the lavage cell pellet peaked at 24 h after ozone, whereas the influx of polymorphonuclear leukocytes peaked at 4 h. Examination of the induction of HSPs by ozone may provide clues to the development of ozone tolerance in humans and animals.

Cloning and sequencing of a gene encoding the 69-kDa extracellular chitinase of Janthinobacterium lividum.

PubMed

Gleave, A P; Taylor, R K; Morris, B A; Greenwood, D R

1995-09-15

Janthinobacterium lividum secretes a major 56-kDa chitinase and a minor 69-kDa chitinase. A chitinase gene was defined on a 3-kb fragment of clone pRKT10, by virtue of fluorescent colonies in the presence of 4-methylumbelliferyl-beta-D-N,N',N"-chitotrioside. Nucleotide sequencing revealed an 1998-bp open reading frame with the potential to encode a 69,716-Da protein with amino acid sequences similar to those in other chitinases, suggesting it encodes the minor chitinase (Chi69). Chitinase activity of Escherichia coli (pRKT10) lysates was detected mainly in the periplasmic fraction and immunoblotting detected a 70-kDa protein in this fraction. Chi69 has an N-terminal secretory leader peptide preceding two probable chitin-binding domains and a catalytic domain. These functional domains are separated by linker regions of proline-threonine repeats. Amino acid sequencing of cyanogen bromide cleavage-derived peptides from the major 56-kDa chitinase suggested that Chi69 may be a precursor of Chi56. In addition, an N-terminally truncated version of Chi69 retained chitinase activity as expected if in vivo processing of Chi69 generates Chi56.

Isolation and initial structural characterization of a 27 kDa protein from Zingiber officinale

NASA Astrophysics Data System (ADS)

Rasheed, Saima; Malik, Shoaib Ahmad; Falke, Sven; Arslan, Ali; Fazel, Ramin; Schlüter, Hartmut; Betzel, Christian; Choudhary, M. Iqbal

2018-03-01

Zingiber officinale Roscoe (Ginger) is a widely used traditional medicinal plant (for different ailments such as arthritis, constipation, and hypertension). This article describes the isolation and characterization of a so far unknown protein from ginger rhizomes applying ion exchange, affinity, size-exclusion chromatography, small angle X-ray scattering (SAXS), and mass spectrometry techniques. One-dimensional Coomassie-stained SDS-PAGE was performed under non-reducing conditions, showing one band corresponding to approx. 27 kDa. Dynamic light scattering (DLS) analysis of the protein solution revealed monodispersity and a monomeric state of the purified protein. Circular dichroism (CD) spectroscopy strongly indicated a β-sheet-rich protein, and disordered regions. MALDI-TOF-MS, and LC-MS/MS analysis resulted in the identification of 27.29 kDa protein, having 32.13% and 25.34% sequence coverage with Zingipain-1 and 2, respectively. The monomeric state and molecular weight were verified by small angle X-ray scattering (SAXS) studies. An elongated ab-initio model was calculated based on the scattering intensity distribution.

Crystal structure of the 25 kDa subunit of human cleavage factor Im

PubMed Central

Coseno, Molly; Martin, Georges; Berger, Christopher; Gilmartin, Gregory; Keller, Walter; Doublié, Sylvie

2008-01-01

Cleavage factor Im is an essential component of the pre-messenger RNA 3′-end processing machinery in higher eukaryotes, participating in both the polyadenylation and cleavage steps. Cleavage factor Im is an oligomer composed of a small 25 kDa subunit (CF Im25) and a variable larger subunit of either 59, 68 or 72 kDa. The small subunit also interacts with RNA, poly(A) polymerase, and the nuclear poly(A)-binding protein. These protein–protein interactions are thought to be facilitated by the Nudix domain of CF Im25, a hydrolase motif with a characteristic α/β/α fold and a conserved catalytic sequence or Nudix box. We present here the crystal structures of human CF Im25 in its free and diadenosine tetraphosphate (Ap4A) bound forms at 1.85 and 1.80 Å, respectively. CF Im25 crystallizes as a dimer and presents the classical Nudix fold. Results from crystallographic and biochemical experiments suggest that CF Im25 makes use of its Nudix fold to bind but not hydrolyze ATP and Ap4A. The complex and apo protein structures provide insight into the active oligomeric state of CF Im and suggest a possible role of nucleotide binding in either the polyadenylation and/or cleavage steps of pre-messenger RNA 3′-end processing. PMID:18445629

Identification of a 27.8 kDa protein from flounder gill cells involved in lymphocystis disease virus binding and infection.

PubMed

Wang, Mu; Sheng, Xiu-Zhen; Xing, Jing; Tang, Xiao-Qian; Zhan, Wen-Bin

2011-03-16

In vitro, lymphocystis disease virus (LCDV) infection of flounder gill (FG) cell cultures causes obvious cytopathic effect (CPE). We describe attempts to isolate and characterize the LCDV-binding molecule(s) on the plasma membrane of FG cells that were responsible for virus entry. The results showed that the co-immunoprecipitation assay detected a 27.8 kDa molecule from FG cells that bound to LCDV. In a blocking ELISA, pre-incubation of FG cell membrane proteins with the specific antiserum developed against the 27.8 kDa protein could block LCDV binding. Similarly, antiserum against 27.8 kDa protein could also inhibit LCDV infection of FG cells in vitro. Mass spectrometric analysis established that the 27.8 kDa protein and beta-actin had a strong association. These results strongly supported the possibility that the 27.8 kDa protein was the putative receptor specific for LCDV infection of FG cells.

39 CFR 1.1 - Establishment of the U.S. Postal Service.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 39 Postal Service 1 2011-07-01 2011-07-01 false Establishment of the U.S. Postal Service. 1.1 Section 1.1 Postal Service UNITED STATES POSTAL SERVICE THE BOARD OF GOVERNORS OF THE U.S. POSTAL SERVICE POSTAL POLICY (ARTICLE I) § 1.1 Establishment of the U.S. Postal Service. The U.S. Postal Service is...

39 CFR 1.1 - Establishment of the U.S. Postal Service.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 39 Postal Service 1 2010-07-01 2010-07-01 false Establishment of the U.S. Postal Service. 1.1 Section 1.1 Postal Service UNITED STATES POSTAL SERVICE THE BOARD OF GOVERNORS OF THE U.S. POSTAL SERVICE POSTAL POLICY (ARTICLE I) § 1.1 Establishment of the U.S. Postal Service. The U.S. Postal Service is...

39 CFR 1.1 - Establishment of the U.S. Postal Service.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 39 Postal Service 1 2012-07-01 2012-07-01 false Establishment of the U.S. Postal Service. 1.1 Section 1.1 Postal Service UNITED STATES POSTAL SERVICE THE BOARD OF GOVERNORS OF THE U.S. POSTAL SERVICE POSTAL POLICY (ARTICLE I) § 1.1 Establishment of the U.S. Postal Service. The U.S. Postal Service is...

39 CFR 1.1 - Establishment of the U.S. Postal Service.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 39 Postal Service 1 2014-07-01 2014-07-01 false Establishment of the U.S. Postal Service. 1.1 Section 1.1 Postal Service UNITED STATES POSTAL SERVICE THE BOARD OF GOVERNORS OF THE U.S. POSTAL SERVICE POSTAL POLICY (ARTICLE I) § 1.1 Establishment of the U.S. Postal Service. The U.S. Postal Service is...

39 CFR 1.1 - Establishment of the U.S. Postal Service.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 39 Postal Service 1 2013-07-01 2013-07-01 false Establishment of the U.S. Postal Service. 1.1 Section 1.1 Postal Service UNITED STATES POSTAL SERVICE THE BOARD OF GOVERNORS OF THE U.S. POSTAL SERVICE POSTAL POLICY (ARTICLE I) § 1.1 Establishment of the U.S. Postal Service. The U.S. Postal Service is...

Structure, dynamics and RNA binding of the multi-domain splicing factor TIA-1

PubMed Central

Wang, Iren; Hennig, Janosch; Jagtap, Pravin Kumar Ankush; Sonntag, Miriam; Valcárcel, Juan; Sattler, Michael

2014-01-01

Alternative pre-messenger ribonucleic acid (pre-mRNA) splicing is an essential process in eukaryotic gene regulation. The T-cell intracellular antigen-1 (TIA-1) is an apoptosis-promoting factor that modulates alternative splicing of transcripts, including the pre-mRNA encoding the membrane receptor Fas. TIA-1 is a multi-domain ribonucleic acid (RNA) binding protein that recognizes poly-uridine tract RNA sequences to facilitate 5′ splice site recognition by the U1 small nuclear ribonucleoprotein (snRNP). Here, we characterize the RNA interaction and conformational dynamics of TIA-1 by nuclear magnetic resonance (NMR), isothermal titration calorimetry (ITC) and small angle X-ray scattering (SAXS). Our NMR-derived solution structure of TIA-1 RRM2–RRM3 (RRM2,3) reveals that RRM2 adopts a canonical RNA recognition motif (RRM) fold, while RRM3 is preceded by an non-canonical helix α0. NMR and SAXS data show that all three RRMs are largely independent structural modules in the absence of RNA, while RNA binding induces a compact arrangement. RRM2,3 binds to pyrimidine-rich FAS pre-mRNA or poly-uridine (U9) RNA with nanomolar affinities. RRM1 has little intrinsic RNA binding affinity and does not strongly contribute to RNA binding in the context of RRM1,2,3. Our data unravel the role of binding avidity and the contributions of the TIA-1 RRMs for recognition of pyrimidine-rich RNAs. PMID:24682828

Purification and characterization of a thermostable beta-1,3-1,4-glucanase from Laetiporus sulphureus var. miniatus.

PubMed

Hong, Mi-Ri; Kim, Yeong-Su; Joo, Ah-Reum; Lee, Jung-Kul; Kim, Yeong-Suk; Oh, Deok-Kun

2009-08-01

A beta-1,3-1,4-glucanase from the fungus Laetiporus sulphureus var. miniatus was purified as a single 26 kDa band by ammonium sulfate precipitation, HiTrap Q HP, and UNO Q ion-exchange chromatography, with a specific activity of 29 U/mg. The molecular mass of the native enzyme was 52 kDa as a dimer by gel filtration. beta-1,3-1,4-Glucanase showed optimum activity at pH 4.0 and 75 degrees . The half-lives of the enzyme at 70 degrees and 75 degrees were 152 h and 22 h, respectively. The enzyme showed the highest activity for barley beta- glucan as beta-1,3-1,4-glucan among the tested polysaccharides and p-nitrophenyl-beta-D-glycosides with a K(m) of 0.67 mg/ml, a k(cat) of 13.5 s(-1) and a k(cat)/K(m) of 20 mg/ml/s.

Evaluation of the immunomodulatory effect of the 14 kDa protein isolated from aged garlic extract on dendritic cells.

PubMed

Ahmadabad, Hasan Namdar; Hassan, Zuhair Mohammad; Safari, Elahe; Bozorgmehr, Mahmood; Ghazanfari, Tooba; Moazzeni, Seyed Mohammad

2011-01-01

Garlic is used all over the world for treatment of different diseases. A wide range of biological activities of garlic has been verified in vitro and in vivo. One of major proteins of garlic which has been isolated and purified is the 14 kDa protein. This protein has been shown to have immunomodulatory effects. In this study, the effect of the 14 kDa protein isolated from aged garlic extract (AGE) was investigated on maturation and immunomodulatory activity of dendritic cells (DC). Proteins were purified from AGE by biochemical method; the semi-purified 14 kDa protein was run on gel filtration Sephadex G50 and its purity was checked by SDS-PAGE. DC were isolated from spleen of BALB/c mice by Nycodenz centrifugation and their adhesiveness to plastic dish. 14 kDa protein from AGE was added to overnight culture of DC medium and the expression percentage of CD40, CD86, and MHC-II was evaluated by flowcytometric analysis. Also, proliferation of T-cells was measured by allogenic mixed lymphocyte reaction (MLR) test. The purified 14 kDa protein isolated from AGE increased the expression of CD40 molecule on DC, but it did not influence CD86 and MHCII molecules. Furthermore, no significant differences were noticed in the pulsed-DC with 14 kDa protein and non-pulsed DC on the MLR. Copyright © 2011 Elsevier Inc. All rights reserved.

Characterization of the transacylase activity of rat liver 60-kDa lysophospholipase-transacylase. Acyl transfer from the sn-2 to the sn-1 position.

PubMed

Sugimoto, H; Yamashita, S

1999-05-18

Rat liver 60-kDa lysophospholipase-transacylase catalyzes not only the hydrolysis of 1-acyl-sn-glycero-3-phosphocholine, but also the transfer of its acyl chain to a second molecule of 1-acyl-sn-glycero-3-phosphocholine to form phosphatidylcholine (H. Sugimoto, S. Yamashita, J. Biol. Chem. 269 (1994) 6252-6258). Here we report the detailed characterization of the transacylase activity of the enzyme. The enzyme mediated three types of acyl transfer between donor and acceptor lipids, transferring acyl residues from: (1) the sn-1 to -1(3); (2) sn-1 to -2; and (3) sn-2 to -1 positions. In the sn-1 to -1(3) transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1(3) positions of glycerol and 2-acyl-sn-glycerol, producing 1(3)-acyl-sn-glycerol and 1,2-diacyl-sn-glycerol, respectively. In the sn-1 to -2 transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to not only the sn-2 positions of 1-acyl-sn-glycero-3-phosphocholine, but also 1-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. 1-Acyl-sn-glycero-3-phospho-myo-inositol and 1-acyl-sn-glycero-3-phosphoserine were much less effectively transacylated by the enzyme. In the sn-2 to -1 transfer, the sn-2 acyl residue of 2-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1 position of 2-acyl-sn-glycero-3-phosphocholine and 2-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. Consistently, the enzyme hydrolyzed the sn-2 acyl residue from 2-acyl-sn-glycero-3-phosphocholine. By the sn-2 to -1 transfer activity, arachidonic acid was transferred from the sn-2 position of donor lipids to the sn-1 position of acceptor lipids, thus producing 1-arachidonoyl phosphatidylcholine. When 2-arachidonoyl-sn-glycero-3-phosphocholine was used as the sole substrate, diarachidonoyl phosphatidylcholine was synthesized at a rate of 0

Autoantigen La promotes efficient RNAi, antiviral response, and transposon silencing by facilitating multiple-turnover RISC catalysis

PubMed Central

Liu, Ying; Tan, Huiling; Tian, Hui; Liang, Chunyang; Chen, She; Liu, Qinghua

2011-01-01

SUMMARY The effector of RNA interference (RNAi) is the RNA-induced silencing complex (RISC). C3PO promotes the activation of RISC by degrading Argonaute2 (Ago2)-nicked passenger strand of duplex siRNA. Active RISC is a multiple-turnover enzyme that uses the guide strand of siRNA to direct Ago2-mediated sequence-specific cleavage of complementary mRNA. How this effector step of RNAi is regulated is currently unknown. Here, we used human Ago2 minimal RISC system to purify Sjögren’s syndrome antigen B (SSB)/autoantigen La as an activator of the RISC-mediated mRNA cleavage activity. Our reconstitution studies showed that La could promote multiple-turnover RISC catalysis by facilitating the release of cleaved mRNA from RISC. Moreover, we demonstrated that La was required for efficient RNAi, antiviral defense, and transposon silencing in vivo. Taken together, the findings of C3PO and La reveal a general concept that regulatory factors are required to remove Ago2-cleaved products to assemble or restore active RISC. PMID:22055194

The role of RNA structure in the interaction of U1A protein with U1 hairpin II RNA

PubMed Central

Law, Michael J.; Rice, Andrew J.; Lin, Patti; Laird-Offringa, Ite A.

2006-01-01

The N-terminal RNA Recognition Motif (RRM1) of the spliceosomal protein U1A interacting with its target U1 hairpin II (U1hpII) has been used as a paradigm for RRM-containing proteins interacting with their RNA targets. U1A binds to U1hpII via direct interactions with a 7-nucleotide (nt) consensus binding sequence at the 5′ end of a 10-nt loop, and via hydrogen bonds with the closing C–G base pair at the top of the RNA stem. Using surface plasmon resonance (Biacore), we have examined the role of structural features of U1hpII in binding to U1A RRM1. Mutational analysis of the closing base pair suggests it plays a minor role in binding and mainly prevents “breathing” of the loop. Lengthening the stem and nontarget part of the loop suggests that the increased negative charge of the RNA might slightly aid association. However, this is offset by an increase in dissociation, which may be caused by attraction of the RRM to nontarget parts of the RNA. Studies of a single stranded target and RNAs with untethered loops indicate that structure is not very relevant for association but is important for complex stability. In particular, breaking the link between the stem and the 5′ side of the loop greatly increases complex dissociation, presumably by hindering simultaneous contacts between the RRM and stem and loop nucleotides. While binding of U1A to a single stranded target is much weaker than to U1hpII, it occurs with nanomolar affinity, supporting recent evidence that binding of unstructured RNA by U1A has physiological significance. PMID:16738410

The role of RNA structure in the interaction of U1A protein with U1 hairpin II RNA.

PubMed

Law, Michael J; Rice, Andrew J; Lin, Patti; Laird-Offringa, Ite A

2006-07-01

The N-terminal RNA Recognition Motif (RRM1) of the spliceosomal protein U1A interacting with its target U1 hairpin II (U1hpII) has been used as a paradigm for RRM-containing proteins interacting with their RNA targets. U1A binds to U1hpII via direct interactions with a 7-nucleotide (nt) consensus binding sequence at the 5' end of a 10-nt loop, and via hydrogen bonds with the closing C-G base pair at the top of the RNA stem. Using surface plasmon resonance (Biacore), we have examined the role of structural features of U1hpII in binding to U1A RRM1. Mutational analysis of the closing base pair suggests it plays a minor role in binding and mainly prevents "breathing" of the loop. Lengthening the stem and nontarget part of the loop suggests that the increased negative charge of the RNA might slightly aid association. However, this is offset by an increase in dissociation, which may be caused by attraction of the RRM to nontarget parts of the RNA. Studies of a single stranded target and RNAs with untethered loops indicate that structure is not very relevant for association but is important for complex stability. In particular, breaking the link between the stem and the 5' side of the loop greatly increases complex dissociation, presumably by hindering simultaneous contacts between the RRM and stem and loop nucleotides. While binding of U1A to a single stranded target is much weaker than to U1hpII, it occurs with nanomolar affinity, supporting recent evidence that binding of unstructured RNA by U1A has physiological significance.

Anti-calponin-3 autoantibodies: a new specificity in patients with Sjögren's syndrome.

PubMed

Birnbaum, Julius; Hoke, Ahmet; Lalji, Aliya; Calabresi, Peter; Bhargava, Pavan; Casciola-Rosen, Livia

2018-05-11

Autoantibodies are clinically useful for phenotyping patients across the spectrum of autoimmune rheumatic diseases. Using serum from a patient with Sjögren's Syndrome (SS), we detected a new specificity by immunoblotting. The purpose of this study was to identify this autoantibody and to evaluate its disease specificity. A prominent 40 kDa band was detected when immunoblots were performed using a SS patient serum and lysate from rat dorsal root ganglia (DRG). Using two-dimensional gel electrophoresis and liquid chromatography tandem mass spectrometry peptide sequencing, the autoantigen was identified as calponin-3. Anti-calponin-3 antibodies were assayed by ELISA using sera from patients with primary SS (n=209), systemic lupus erythematosus (SLE, n=138), myositis (n=138), multiple sclerosis (MS, n=44) and healthy controls (n=46). Expression of calponin-3 was assessed by immunohistochemistry. These studies identified calponin-3 as a new autoantigen. Anti-calponin-3 antibodies were detected in 23/209 (11.0%) SS patients, 12/138 (8.7%) SLE patients, 7/138 (5.1%) myositis patients, 3/44 (6.8%) MS patients, and 1/46 (2.2%) healthy controls. Amongst SS patients, the frequency of anti-calponin-3 antibodies was highest in those with neuropathies (7/39, 17.9%). In this subset, the frequency of anti-calponin-3 antibodies differed significantly from the controls (p=0.02). Calponin-3 was expressed primarily in perineuronal satellite cells but not in DRG neurons. Calponin-3 is a novel autoantigen. Antibodies against this protein are found in SS, and associate with the subset of patients experiencing neuropathies. Intriguingly, we found that calponin-3 is expressed in DRG perineuronal satellite cells, suggesting that these may be one of the targets in SS. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Integral cross sections for the direct excitation of the A 3 (sigma) u +, B 3 (pi) g, W 3 (delta) u, B' 3 (sigma) u -, a' 1 (sigma) u -, a 1 (pi) g, w 1 (delta) u, and C 3 (pi) u electronic states in

NASA Technical Reports Server (NTRS)

Johnson, P. V.; Malone, C. P.; Kanik, I.

2005-01-01

Integral cross sections for electron impact excitation out of the ground state (X 1(sigma)g +) to the A 3(sigma)u +, B 3(pi)g, W 3(delta)u, B' 3(sigma)u -, a' 1(sigma)u -, a 1(pi)g, w 1(delta)u, and states in N2 are reported at incident energies ranging between 10 and 100 eV. These data have been derived by integrating differential cross sections previously reported by this group. New differential cross section measurements for the a 1(pi)g state at 200 eV are also presented to extend the range of the reported integral cross sections for this state, which is responsible for the emissions of the Lyman-Birge-Hopfield band system (a 1(pi)g (rightwards arrow) X 1(sigma)g +). The present results are compared and critically evaluated against existing cross sec In general, the present cross sections are smaller than previous results at low impact energies from threshold through the excitation function peak regions. These lower cross sections have potentially significant implications on our understanding of UV emissions in the atmospheres of Earth and Titan.

A 92-kDa human immunostimulatory protein.

PubMed Central

Fontan, E; Briend, E; Saklani-Jusforgues, H; d'Alayer, J; Vandekerckhove, J; Fauve, R M

1994-01-01

We purified to apparent homogeneity a human urinary glycoprotein of 92 kDa (HGP.92) that, administered intravenously at 250 micrograms/kg, fully protected mice against a lethal inoculum of Listeria monocytogenes. Since HGP.92 protected scid mice, which lack B and T lymphocytes, this increased resistance to Listeria did not appear to be lymphocyte mediated. Furthermore, inflammatory macrophages incubated with 6 nM HGP.92 inhibited the growth of Lewis carcinoma cells in vitro. These two activities appeared to depend on an oligosaccharide moiety, as they were lost after N-Glycanase treatment of HGP.92. Thus, the biological activity of HGP.92 was in some way related to a glycan moiety. Images PMID:8078887

Chaperonin-containing T-complex Protein 1 Subunit ζ Serves as an Autoantigen Recognized by Human Vδ2 γδ T Cells in Autoimmune Diseases.

PubMed

Chen, Hui; You, Hongqin; Wang, Lifang; Zhang, Xuan; Zhang, Jianmin; He, Wei

2016-09-16

Human γδ T cells recognize conserved endogenous and stress-induced antigens typically associated with autoimmune diseases. However, the role of γδ T cells in autoimmune diseases is not clear. Few autoimmune disease-related antigens recognized by T cell receptor (TCR) γδ have been defined. In this study, we compared Vδ2 TCR complementarity-determining region 3 (CDR3) between systemic lupus erythematosus (SLE) patients and healthy donors. Results show that CDR3 length distribution differed significantly and displayed oligoclonal characteristics in SLE patients when compared with healthy donors. We found no difference in the frequency of Jδ gene fragment usage between these two groups. According to the dominant CDR3δ sequences in SLE patients, synthesized SL2 peptides specifically bound to human renal proximal tubular epithelial cell line HK-2; SL2-Vm, a mutant V sequence of SL2, did not bind. We identified the putative protein ligand chaperonin-containing T-complex protein 1 subunit ζ (CCT6A) using SL2 as a probe in HK-2 cell protein extracts by affinity chromatography and liquid chromatography-electrospray ionization-tandem mass spectrometry analysis. We found CCT6A expression on the surface of HK-2 cells. Cytotoxicity of only Vδ2 γδ T cells to HK-2 cells was blocked by anti-CCT6A antibody. Finally, we note that CCT6A concentration was significantly increased in plasma of SLE and rheumatoid arthritis patients. These data suggest that CCT6A is a novel autoantigen recognized by Vδ2 γδ T cells, which deepens our understanding of mechanisms in autoimmune diseases. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Molecular differentiation and phylogenetic relationships of three Angiostrongylus species and Angiostrongylus cantonensis geographical isolates based on a 66-kDa protein gene of A. cantonensis (Nematoda: Angiostrongylidae).

PubMed

Eamsobhana, Praphathip; Lim, Phaik Eem; Zhang, Hongman; Gan, Xiaoxian; Yong, Hoi Sen

2010-12-01

The phylogenetic relationships and molecular differentiation of three species of angiostrongylid nematodes (Angiostrongylus cantonensis, Angiostrongylus costaricensis and Angiostrongylus malaysiensis) were studied using the AC primers for a 66-kDa protein gene of A. cantonensis. The AC primers successfully amplified the genomic DNA of these angiostrongylid nematodes. No amplification was detected for the DNA of Ascaris lumbricoides, Ascaris suum, Anisakis simplex, Gnathostoma spinigerum, Toxocara canis, and Trichinella spiralis. The maximum-parsimony (MP) consensus tree and the maximum-likelihood (ML) tree both showed that the Angiostrongylus taxa could be divided into two major clades - Clade 1 (A. costaricensis) and Clade 2 (A. cantonensis and A. malaysiensis) with a full support bootstrap value. A. costaricensis is the most distant taxon. A. cantonensis is a sister group to A. malaysiensis; these two taxa (species) are clearly separated. There is no clear distinction between the A. cantonensis samples from four different geographical localities (Thailand, China, Japan and Hawaii); only some of the samples are grouped ranging from no support or low support to moderate support of bootstrap values. The published nucleotide sequences of A. cantonensis adult-specific native 66kDa protein mRNA, clone L5-400 from Taiwan (U17585) appear to be very distant from the A. cantonensis samples from Thailand, China, Japan and Hawaii, with the uncorrected p-distance values ranging from 26.87% to 29.92%.

Crystal Structure of the 25 kDa Subunit of Human Cleavage Factor I{m}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Coseno,M.; Martin, G.; Berger, C.

Cleavage factor Im is an essential component of the pre-messenger RNA 3'-end processing machinery in higher eukaryotes, participating in both the polyadenylation and cleavage steps. Cleavage factor Im is an oligomer composed of a small 25 kDa subunit (CF Im25) and a variable larger subunit of either 59, 68 or 72 kDa. The small subunit also interacts with RNA, poly(A) polymerase, and the nuclear poly(A)-binding protein. These protein-protein interactions are thought to be facilitated by the Nudix domain of CF Im25, a hydrolase motif with a characteristic {alpha}/{beta}/{alpha} fold and a conserved catalytic sequence or Nudix box. We present heremore » the crystal structures of human CF Im25 in its free and diadenosine tetraphosphate (Ap4A) bound forms at 1.85 and 1.80 Angstroms, respectively. CF Im25 crystallizes as a dimer and presents the classical Nudix fold. Results from crystallographic and biochemical experiments suggest that CF Im25 makes use of its Nudix fold to bind but not hydrolyze ATP and Ap4A. The complex and apo protein structures provide insight into the active oligomeric state of CF Im and suggest a possible role of nucleotide binding in either the polyadenylation and/or cleavage steps of pre-messenger RNA 3'-end processing.« less

Draft Genome Sequences of Two Bacillus thuringiensis Strains and Characterization of a Putative 41.9-kDa Insecticidal Toxin

PubMed Central

Palma, Leopoldo; Muñoz, Delia; Berry, Colin; Murillo, Jesús; Caballero, Primitivo

2014-01-01

In this work, we report the genome sequencing of two Bacillus thuringiensis strains using Illumina next-generation sequencing technology (NGS). Strain Hu4-2, toxic to many lepidopteran pest species and to some mosquitoes, encoded genes for two insecticidal crystal (Cry) proteins, cry1Ia and cry9Ea, and a vegetative insecticidal protein (Vip) gene, vip3Ca2. Strain Leapi01 contained genes coding for seven Cry proteins (cry1Aa, cry1Ca, cry1Da, cry2Ab, cry9Ea and two cry1Ia gene variants) and a vip3 gene (vip3Aa10). A putative novel insecticidal protein gene 1143 bp long was found in both strains, whose sequences exhibited 100% nucleotide identity. The predicted protein showed 57 and 100% pairwise identity to protein sequence 72 from a patented Bt strain (US8318900) and to a putative 41.9-kDa insecticidal toxin from Bacillus cereus, respectively. The 41.9-kDa protein, containing a C-terminal 6× HisTag fusion, was expressed in Escherichia coli and tested for the first time against four lepidopteran species (Mamestra brassicae, Ostrinia nubilalis, Spodoptera frugiperda and S. littoralis) and the green-peach aphid Myzus persicae at doses as high as 4.8 µg/cm2 and 1.5 mg/mL, respectively. At these protein concentrations, the recombinant 41.9-kDa protein caused no mortality or symptoms of impaired growth against any of the insects tested, suggesting that these species are outside the protein’s target range or that the protein may not, in fact, be toxic. While the use of the polymerase chain reaction has allowed a significant increase in the number of Bt insecticidal genes characterized to date, novel NGS technologies promise a much faster, cheaper and efficient screening of Bt pesticidal proteins. PMID:24784323

The histone variant H2A.Z promotes efficient cotranscriptional splicing in S. cerevisiae

PubMed Central

Neves, Lauren T.; Douglass, Stephen; Spreafico, Roberto; Venkataramanan, Srivats; Kress, Tracy L.; Johnson, Tracy L.

2017-01-01

In eukaryotes, a dynamic ribonucleic protein machine known as the spliceosome catalyzes the removal of introns from premessenger RNA (pre-mRNA). Recent studies show the processes of RNA synthesis and RNA processing to be spatio–temporally coordinated, indicating that RNA splicing takes place in the context of chromatin. H2A.Z is a highly conserved histone variant of the canonical histone H2A. In Saccharomyces cerevisiae, H2A.Z is deposited into chromatin by the SWR-C complex, is found near the 5′ ends of protein-coding genes, and has been implicated in transcription regulation. Here we show that splicing of intron-containing genes in cells lacking H2A.Z is impaired, particularly under suboptimal splicing conditions. Cells lacking H2A.Z are especially dependent on a functional U2 snRNP (small nuclear RNA [snRNA] plus associated proteins), as H2A.Z shows extensive genetic interactions with U2 snRNP-associated proteins, and RNA sequencing (RNA-seq) reveals that introns with nonconsensus branch points are particularly sensitive to H2A.Z loss. Consistently, H2A.Z promotes efficient spliceosomal rearrangements involving the U2 snRNP, as H2A.Z loss results in persistent U2 snRNP association and decreased recruitment of downstream snRNPs to nascent RNA. H2A.Z impairs transcription elongation, suggesting that spliceosome rearrangements are tied to H2A.Z's role in elongation. Depletion of disassembly factor Prp43 suppresses H2A.Z-mediated splice defects, indicating that, in the absence of H2A.Z, stalled spliceosomes are disassembled, and unspliced RNAs are released. Together, these data demonstrate that H2A.Z is required for efficient pre-mRNA splicing and indicate a role for H2A.Z in coordinating the kinetics of transcription elongation and splicing. PMID:28446598

Association of HLA-DRB1 alleles with susceptibility to mixed connective tissue disease in Polish patients.

PubMed

Paradowska-Gorycka, A; Stypińska, B; Olesińska, M; Felis-Giemza, A; Mańczak, M; Czuszynska, Z; Zdrojewski, Z; Wojciechowicz, J; Jurkowska, M

2016-01-01

Mixed connective tissue disease (MCTD) is a systemic autoimmune disease, originally defined as a connective tissue inflammatory syndrome with overlapping features of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), polymyositis/dermatomyositis (PM/DM) and systemic sclerosis (SSc), characterized by the presence of antibodies against components of the U1 small nuclear ribonucleoprotein (U1snRNP). The aim of the study was to assess the frequency of (high-resolution-typed) DRB1 alleles in a cohort of Polish patients with MCTD (n = 103). Identification of the variants potentially associated with risk and protection was carried out by comparison with the DKMS Polish Bone Marrow Donor Registry (41306 alleles). DRB1*15:01 (odds ratio (OR): 6.06; 95% confidence interval (CI) 4.55-8.06), DRB1*04 (OR: 3.69; 95% CI 2.69-5.01) and *09:01 (OR: 8.12; 95% CI 2.15-21.75) were identified as risk alleles for MCTD, while HLA-DRB1*07:01 allele was found to be protective (OR: 0.50; 95% CI 0.28-0.83). The carrier frequency of the DRB1*01 was higher in MCTD patients compared with controls, although the differences were not statistically significant. Our results confirm the modulating influence of HLA-DRB1 genotypes on development of connective tissue diseases such as MCTD. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The 21.5-kDa isoform of myelin basic protein has a non-traditional PY-nuclear-localization signal

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, Graham S.T.; Seymour, Lauren V.; Boggs, Joan M.

2012-06-15

Highlights: Black-Right-Pointing-Pointer Full-length 21.5-kDa MBP isoform is translocated to the nucleus. Black-Right-Pointing-Pointer We hypothesized that the exon-II-encoded sequence contained the NLS. Black-Right-Pointing-Pointer We mutated this sequence in RFP-tagged constructs and transfected N19-cells. Black-Right-Pointing-Pointer Abolition of two key positively-charged residues resulted in loss of nuclear-trafficking. Black-Right-Pointing-Pointer The 21.5-kDa isoform of classic MBP contains a non-traditional PY-NLS. -- Abstract: The predominant 18.5-kDa classic myelin basic protein (MBP) is mainly responsible for compaction of the myelin sheath in the central nervous system, but is multifunctional, having numerous interactions with Ca{sup 2+}-calmodulin, actin, tubulin, and SH3-domains, and can tether these proteins to a lipidmore » membrane in vitro. The full-length 21.5-kDa MBP isoform has an additional 26 residues encoded by exon-II of the classic gene, which causes it to be trafficked to the nucleus of oligodendrocytes (OLGs). We have performed site-directed mutagenesis of selected residues within this segment in red fluorescent protein (RFP)-tagged constructs, which were then transfected into the immortalized N19-OLG cell line to view protein localization using epifluorescence microscopy. We found that 21.5-kDa MBP contains two non-traditional PY-nuclear-localization signals, and that arginine and lysine residues within these motifs were involved in subcellular trafficking of this protein to the nucleus, where it may have functional roles during myelinogenesis.« less

The purification and characterization of an 88-kDa Porphyromonas endodontalis 35406 protease.

PubMed

Rosen, G; Shoshani, M; Naor, R; Sela, M N

2001-12-01

A Porphyromonas endodontalis ATCC 35406 protease was purified from Triton X-114 cell extracts by preparative SDS-PAGE followed by electroelution. The purified enzyme exhibits a molecular size of 88 kDa and was dissociated into two polypeptides of 43 and 41 kDa upon heating in the presence of sodium dodecyl sulfate with or without a reducing agent. The protease (pH optimum 7.5-8.0) degraded the extracellular matrix proteins fibrinogen and fibronectin. Collagen IV was also degraded at 37 degrees C but not at 28 degrees C. The protease also cleaved the bioactive peptide angiotensin at amino acid residue phenylalanine-8 and tyrosine-4 but failed to hydrolyze bradykinin, vasopressin and synthetic chromogenic substrates with phenylalanine or tyrosine at the P1 position. In addition, two peptidases were detected in P. endodontalis cells: a proline aminopeptidase that remained associated with the cell pellet after detergent extraction and peptidase/s that partitioned into the Triton X-114 phase after phase separation and degraded the bioactive peptides bradykinin and vasopressin. These P. endodontalis peptidases and proteases may play an important role in both the nutrition and pathogenicity of these assacharolytic microorganisms. The inactivation of bioactive peptides and degradation of extracellular matrix proteins by bacterial enzymes may contribute to the damage of host tissues accompanied with endodontic infections.

Expression of Clara cell 10-kDa protein and trefoil factor family 1 in patients with chronic rhinosinusitis and nasal polyps

PubMed Central

Wang, Yuanyuan; Wang, Zong-Feng; Zhang, Zhili; Su, Yi

2018-01-01

The current study measured the expression of Clara cell 10-kDa protein (CC10) and trefoil factor family 1 (TFF1) in the sinus mucosa of patients exhibiting chronic rhinosinusitis (CRS) and nasal polyps (NP). CC10 and TFF1 expression in the sinus mucosa of the control group and patients with CRS and NP was determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blotting and immunohistochemistry. The correlation between CC10 and TFF1 expression was further analyzed using Spearman's correlation analysis. The expression of TFF1 was significantly increased in the sinus mucosa of patients with CRS and NP, whereas CC10 expression was significantly decreased compared with controls. Spearman's correlation analysis identified a negative correlation between CC10 and TFF1 expression in the sinus mucosa of patients with CRS and NP. The results of immunohistochemistry and RT-qPCR were consistent with each other. Hematoxylin and eosin staining revealed notable lesions in the mucous membranes, goblet cells and cilia of sinus mucosa samples from patients with CRS and NP. The negative correlation between CC10 and TFF1 expression during the progression of CRS and NP suggest that CC10 and TFF1 may serve important roles in its pathogenesis. PMID:29456658

Infrared radiative decay dynamics from the γ 1u (3P2), H 1u (3P1), and 1u (1D2) ion-pair states of I2 observed by a perturbation facilitated optical-optical double resonance technique

NASA Astrophysics Data System (ADS)

Hoshino, Shoma; Araki, Mitsunori; Nakano, Yukio; Ishiwata, Takashi; Tsukiyama, Koichi

2016-01-01

We report the spectroscopic and temporal analyses on the amplified spontaneous emission (ASE) from the single rovibrational levels of the Ω = 1u ion-pair series, γ 1u (3P2), H 1u (3P1), and 1u (1D2), of I2 by using a perturbation facilitated optical-optical double resonance technique through the c 1 Π g ˜ B 3 Π ( 0u + ) hyperfine mixed valence state as the intermediate state. The ASE detected in the infrared region was assigned to the parallel transitions from the Ω = 1u ion-pair states down to the nearby Ω = 1g ion-pair states. The subsequent ultraviolet (UV) fluorescence from the Ω = 1g states was also observed and the relative vibrational populations in the Ω = 1g states were derived through the Franck-Condon simulation of the intensity pattern of the vibrational progression. In the temporal profiles of the UV fluorescence, an obvious delay in the onset of the fluorescence was recognized after the excitation laser pulse. These results revealed that ASE is a dominant energy relaxation process between the Ω = 1u and 1g ion-pair states of I2. Finally, the lifetimes of the relevant ion-pair states were evaluated by temporal analyses of the UV fluorescence. The propensity was found which was the longer lifetime in the upper level of the ASE transitions tends to give intense ASE.

Autoantigen La promotes efficient RNAi, antiviral response, and transposon silencing by facilitating multiple-turnover RISC catalysis.

PubMed

Liu, Ying; Tan, Huiling; Tian, Hui; Liang, Chunyang; Chen, She; Liu, Qinghua

2011-11-04

The effector of RNA interference (RNAi) is the RNA-induced silencing complex (RISC). C3PO promotes the activation of RISC by degrading the Argonaute2 (Ago2)-nicked passenger strand of duplex siRNA. Active RISC is a multiple-turnover enzyme that uses the guide strand of siRNA to direct the Ago2-mediated sequence-specific cleavage of complementary mRNA. How this effector step of RNAi is regulated is currently unknown. Here, we used the human Ago2 minimal RISC system to purify Sjögren's syndrome antigen B (SSB)/autoantigen La as an activator of the RISC-mediated mRNA cleavage activity. Our reconstitution studies showed that La could promote multiple-turnover RISC catalysis by facilitating the release of cleaved mRNA from RISC. Moreover, we demonstrated that La was required for efficient RNAi, antiviral defense, and transposon silencing in vivo. Taken together, the findings of C3PO and La reveal a general concept that regulatory factors are required to remove Ago2-cleaved products to assemble or restore active RISC. Copyright © 2011 Elsevier Inc. All rights reserved.

Bcl-2-interacting mediator of cell death influences autoantigen-driven deletion and TCR revision

PubMed Central

Hale, J. Scott; Nelson, Lisa T.; Simmons, Kalynn B.; Fink, Pamela J.

2010-01-01

Peripheral CD4+Vβ5+ T cells are tolerized to an endogenous mouse mammary tumor virus superantigen either by deletion or TCR revision. Through TCR revision, RAG reexpression mediates extrathymic TCRβ rearrangement and results in a population of post-revision CD4+Vβ5− T cells expressing revised TCRβ chains. We have hypothesized that cell death pathways regulate the selection of cells undergoing TCR revision to ensure the safety and utility of the post-revision population. Here, we investigate the role of Bim-mediated cell death in autoantigen-driven deletion and TCR revision. Bim deficiency and Bcl-2 overexpression in Vβ5 transgenic (Tg) mice both impair peripheral deletion. Vβ5 Tg Bim deficient and Bcl-2 Tg mice exhibit an elevated frequency of CD4+ T cells expressing both the transgene-encoded Vβ5 chain and a revised TCRβ chain. We now show that these dual-TCR expressing cells are TCR revision intermediates, and that the population of RAG-expressing, revising CD4+ T cells is increased in Bim deficient Vβ5 Tg mice. These findings support a role for Bim and Bcl-2 in regulating the balance of survival versus apoptosis in peripheral T cells undergoing RAG-dependent TCR rearrangements during TCR revision, thereby ensuring the utility of the post-revision repertoire. PMID:21148799

Bcl-2-interacting mediator of cell death influences autoantigen-driven deletion and TCR revision.

PubMed

Hale, J Scott; Nelson, Lisa T; Simmons, Kalynn B; Fink, Pamela J

2011-01-15

Peripheral CD4(+)Vβ5(+) T cells are tolerized to an endogenous mouse mammary tumor virus superantigen either by deletion or TCR revision. Through TCR revision, RAG reexpression mediates extrathymic TCRβ rearrangement and results in a population of postrevision CD4(+)Vβ5(-) T cells expressing revised TCRβ chains. We have hypothesized that cell death pathways regulate the selection of cells undergoing TCR revision to ensure the safety and utility of the postrevision population. In this study, we investigate the role of Bcl-2-interacting mediator of cell death (Bim)-mediated cell death in autoantigen-driven deletion and TCR revision. Bim deficiency and Bcl-2 overexpression in Vβ5 transgenic (Tg) mice both impair peripheral deletion. Vβ5 Tg Bim-deficient and Bcl-2 Tg mice exhibit an elevated frequency of CD4(+) T cells expressing both the transgene-encoded Vβ5 chain and a revised TCRβ chain. We now show that these dual-TCR-expressing cells are TCR revision intermediates and that the population of RAG-expressing, revising CD4(+) T cells is increased in Bim-deficient Vβ5 Tg mice. These findings support a role for Bim and Bcl-2 in regulating the balance of survival versus apoptosis in peripheral T cells undergoing RAG-dependent TCR rearrangements during TCR revision, thereby ensuring the utility of the postrevision repertoire.

Mammalian Peptidylglycine α-Amidating Monooxygenase mRNA Expression Can Be Modulated by the La Autoantigen

PubMed Central

Brenet, Fabienne; Dussault, Nadège; Borch, Jonas; Ferracci, Géraldine; Delfino, Christine; Roepstorff, Peter; Miquelis, Raymond; Ouafik, L'Houcine

2005-01-01

Peptidylglycine α-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the COOH-terminal α-amidation of peptidylglycine substrates, yielding amidated products. We have previously reported a putative regulatory RNA binding protein (PAM mRNA-BP) that binds specifically to the 3′ untranslated region (UTR) of PAM-mRNA. Here, the PAM mRNA-BP was isolated and revealed to be La protein using affinity purification onto a 3′ UTR PAM RNA, followed by tandem mass spectrometry identification. We determined that the core binding sequence is approximately 15-nucleotides (nt) long and is located 471 nt downstream of the stop codon. Moreover, we identified the La autoantigen as a protein that specifically binds the 3′ UTR of PAM mRNA in vivo and in vitro. Furthermore, La protein overexpression caused a nuclear retention of PAM mRNAs and resulted in the down-regulation of endogenous PAM activity. Most interestingly, the nuclear retention of PAM mRNA is lost upon expressing the La proteins that lack a conserved nuclear retention element, suggesting a direct association between PAM mRNA and La protein in vivo. Reporter assays using a chimeric mRNA that combined luciferase and the 3′ UTR of PAM mRNA demonstrated a decrease of the reporter activity due to an increase in the nuclear localization of reporter mRNAs, while the deletion of the 15-nt La binding site led to their clear-cut cytoplasmic relocalization. The results suggest an important role for the La protein in the modulation of PAM expression, possibly by mechanisms that involve a nuclear retention and perhaps a processing of pre-PAM mRNA molecules. PMID:16107699

Accumulation of 19-kDa plasma membrane polypeptide during induction of freezing tolerance in wheat suspension-cultured cells by abscisic acid.

PubMed

Koike, M; Takezawa, D; Arakawa, K; Yoshida, S

1997-06-01

Suspension-cultured cells derived from immature embryos of winter wheat (Triticum aestivum L. cv. Chihoku) were used in experiments designed to obtain clues to the mechanism of the ABA-induced development of freezing tolerance. Cultured cells treated with 50 microM ABA for 5 d at 23 degrees C acquired the maximum level of freezing tolerance (LT50; -21.6 degrees C). The increased freezing tolerance of ABA-treated cells was closely associated with the remarkable accumulation of 19-kDa polypeptides in the plasma membrane. The 19-kDa polypeptide components were isolated by preparative gel electrophoresis and were further separated into one major (AWPM-19) and other minor polypeptide components by Tricine-SDS-PAGE. N-terminal amino acid sequence of AWPM-19 was determined, and a cDNA clone encoding AWPM-19 was isolated by PCR from the library prepared from the ABA-treated cultured cells. The cDNA clone (WPM-1) encoded a 18.9 kDa hydrophobic polypeptide with four putative membrane spanning domains and with a high pI value (10.2). Expression of WPM-1 mRNA was dramatically induced by 50 microM ABA within a few hours. These results suggest that the AWPM-19 might be closely associated with the ABA-induced increase in freezing tolerance in wheat cultured cells.

MALDI-TOF mass spectrometry analysis of small molecular weight compounds (under 10 KDa) as biomarkers of rat hearts undergoing arecoline challenge.

PubMed

Chen, Tung-Sheng; Chang, Mu-Hsin; Kuo, Wei-Wen; Lin, Yueh-Min; Yeh, Yu-Lan; Day, Cecilia Hsuan; Lin, Chien-Chung; Tsai, Fuu-Jen; Tsai, Chang-Hai; Huang, Chih-Yang

2013-04-01

Statistical and clinical reports indicate that betel nut chewing is strongly associated with progression of oral cancer because some ingredients in betel nuts are potential cancer promoters, especially arecoline. Early diagnosis for cancer biomarkers is the best strategy for prevention of cancer progression. Several methods are suggested for investigating cancer biomarkers. Among these methods, gel-based proteomics approach is the most powerful and recommended tool for investigating biomarkers due to its high-throughput. However, this proteomics approach is not suitable for screening biomarkers with molecular weight under 10 KDa because of the characteristics of gel electrophoresis. This study investigated biomarkers with molecular weight under 10 KDa in rats with arecoline challenge. The centrifuging vials with membrane (10 KDa molecular weight cut-off) played a crucial role in this study. After centrifuging, the filtrate (containing compounds with molecular weight under 10 KDa) was collected and spotted on a sample plate for MALDI-TOF mass spectrometry analysis. Compared to control, three extra peaks (m/z values were 1553.1611, 1668.2097 and 1740.1832, respectively) were found in sera and two extra peaks were found in heart tissue samples (408.9719 and 524.9961, respectively). These small compounds should play important roles and may be potential biomarker candidates in rats with arecoline. This study successfully reports a mass-based method for investigating biomarker candidates with small molecular weight in different types of sample (including serum and tissue). In addition, this reported method is more time-efficient (1 working day) than gel-based proteomics approach (5~7 working days).

Transcription boundaries of U1 small nuclear RNA.

PubMed Central

Kunkel, G R; Pederson, T

1985-01-01

Transcription-proximal stages of U1 small nuclear RNA biosynthesis were studied by 32P labeling of nascent chains in isolated HeLa cell nuclei. Labeled RNA was hybridized to nitrocellulose-immobilized, single-stranded M13 DNA clones corresponding to regions within or flanking a human U1 RNA gene. Transcription of U1 RNA was inhibited by greater than 95% by alpha-amanitin at 1 microgram/ml, consistent with previous evidence that it is synthesized by RNA polymerase II. No hybridization to DNA immediately adjacent to the 5' end of mature U1 RNA (-6 to -105 nucleotides) was detected, indicating that, like all studied polymerase II initiation, transcription of U1 RNA starts at or very near the cap site. However, in contrast to previously described transcription units for mRNA, in which equimolar transcription occurs for hundreds or thousands of nucleotides beyond the mature 3' end of the mRNA, labeled U1 RNA hybridization dropped off sharply within a very short region (approximately 60 nucleotides) immediately downstream from the 3' end of mature U1 RNA. Also in contrast to pre-mRNA, which is assembled into ribonucleoprotein (RNP) particles while still nascent RNA chains, the U1 RNA transcribed in isolated nuclei did not form RNP complexes by the criterion of reaction with a monoclonal antibody for the small nuclear RNP Sm proteins. This suggests that, unlike pre-mRNA-RNP particle formation, U1 small nuclear RNP assembly does not occur until after the completion of transcription. These results show that, despite their common synthesis by RNA polymerase II, mRNA and U1 small nuclear RNA differ markedly both in their extents of 3' processing and their temporal patterns of RNP assembly. Images PMID:2942763

Isolation and characterization of cDNA clones for carrot extensin and a proline-rich 33-kDa protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, J.; Varner, J.E.

1985-07-01

Extensins are hydroxyproline-rich glycoproteins associated with most dicotyledonous plant cell walls. To isolate cDNA clones encoding extensin, the authors started by isolating poly(A) RNA from carrot root tissue, and then translating the RNA in vitro, in the presence of tritiated leucine or proline. A 33-kDa peptide was identified in the translation products as a putative extensin precursor. From a cDNA library constructed with poly(A) RNA from wounded carrots, one cDNA clone (pDC5) was identified that specifically hybridized to poly(A) RNA encoding this 33-kDa peptide. They isolated three cDNA clones (pDC11, pDC12, and pDC16) from another cDNA library using pCD5 asmore » a probe. DNA sequence data, RNA hybridization analysis, and hybrid released in vitro translation indicate that the cDNA clones pDC11 encodes extensin and that cDNA clones pDC12 and pDC16 encode the 33-kDa peptide, which as yet has an unknown identity and function. The assumption that the 33-kDa peptide was an extensin precursor was invalid. RNA hybridization analysis showed that RNA encoded by both clone types is accumulated upon wounding.« less

Supersymmetric U(1)Y‧⊗ U(1)B-L extension of the Standard Model

NASA Astrophysics Data System (ADS)

Montero, J. C.; Pleitez, V.; Sánchez-Vega, B. L.; Rodriguez, M. C.

2017-06-01

We build a supersymmetric version with SU(3)C ⊗ SU(2)L ⊗ U(1)Y‧⊗ U(1)B-L gauge symmetry, where Y‧ is a new charge and B and L are the usual baryonic and leptonic numbers. The model has three right-handed neutrinos with identical B - L charges, and can accommodate all fermion masses at the tree level. In particular, the type I seesaw mechanism is implemented for the generation of the active neutrino masses. We obtain the mass spectra of all sectors and for the scalar one we also give the flat directions allowed by the model.

Modelling the thermal conductivity of (U xTh 1-x)O 2 and (U xPu 1-x)O 2

DOE PAGES

Cooper, M. W. D.; Middleburgh, S. C.; Grimes, R. W.

2015-07-15

The degradation of thermal conductivity due to the non-uniform cation lattice of (U xTh 1-x)O 2 and (U xPu 1-x)O 2 solid solutions has been investigated by molecular dynamics, using the non-equilibrium method, from 300 to 2000 K. Degradation of thermal conductivity is predicted in (U xTh 1-x)O 2 and (U xPu 1-x)O 2 as compositions deviate from the pure end members: UO 2, PuO 2 and ThO 2. The reduction in thermal conductivity is most apparent at low temperatures where phonon-defect scattering dominates over phonon-phonon interactions. The effect is greater for (U xTh 1-x)O 2 than U xPu 1-x)Omore » 2 due to the greater mismatch in cation size. Parameters for an analytical expressions have been developed that describe the predicted thermal conductivities over the full temperature and compositional ranges. Finally, these expressions may be used in higher level fuel performance codes.« less

The glyoxysomal and plastid molecular chaperones (70-kDa heat shock protein) of watermelon cotyledons are encoded by a single gene

PubMed Central

Wimmer, Bernhard; Lottspeich, Friedrich; van der Klei, Ida; Veenhuis, Marten; Gietl, Christine

1997-01-01

The monoclonal a-70-kDa heat shock protein (hsp70) antibody recognizes in crude extracts from watermelon (Citrullus vulgaris) cotyledons two hsps with molecular masses of 70 and 72 kDa. Immunocytochemistry on watermelon cotyledon tissue and on isolated glyoxysomes identified hsp70s in the matrix of glyoxysomes and plastids. Affinity purification and partial amino acid determination revealed the 70-kDa protein to share high sequence identity with cytosolic hsp70s from a number of plant species, while the 72 kDa protein was very similar to plastid hsp70s from pea and cucumber. A full-length cDNA clone encoding the 72-kDa hsp70 was isolated and identified two start methionines in frame within the N-terminal presequence leading either to an N-terminal extension of 67 amino acids or to a shorter one of 47 amino acids. The longer presequence was necessary and sufficient to target a reporter protein into watermelon proplastids in vitro. The shorter extension starting from the second methionine within the long version harbored a consensus peroxisomal targeting signal (RT-X5-KL) that directed in vivo a reporter protein into peroxisomes of the yeast Hansenula polymorpha. Peroxisomal targeting was however prevented, when the 67-residue presequence was fused to the reporter protein, indicating that the peroxisomal targeting signal 2 information is hidden in this context. We propose that the 72-kDa hsp70 is encoded by a single gene, but targeted alternatively into two organelles by the modulated use of its presequence. PMID:9391076

[Stimulation of cell cultures recovery after cryopreservation by the cattle cord blood FRACTION (below 5 kDa) or Actovegin].

PubMed

Gulevskiĭ, A K; Trifonova, A V; Lavrik, A A

2013-01-01

The capacities of the cattle cord blood low-molecular fraction (below 5 kDa) and Actovegin (the vealer blood fraction (below 5 kDa)) for recovering functions of cell cultures after cryopreservation compared. Their influence proliferation of the flozen-thawed cell cultures, certain stages of their growth, cell attachment, rate of cell spreading, and mitotic regiment has been studied. Both the cord blood low-molecular fraction and Actovegin were shown to stimulate growth of the cell cultures after cryopreservation more efficiently at the concentration of 224 μg/ml. However, despite the stimulating effect discovered, their application did not bring proliferative indices on the 1st passage after cryopreservation to the values of the native culture. The effects of the cord blood low-molecular fraction and Actovegin on the human fibroblast culture were identical by the following parameters: cell attachment, rates of cell spreading and proliferation. In culture BHK-21 clone 13/04 the efficiency of Actovegin was low, while the cord blood low-molecular fraction has a conspicuous stimulating effect on its adhesion and proliferation. The investigations carried out can serve as a basis for the development of regenerative media containing the cattle cord blood low-molecular fraction (below 5 kDa) or Actovegin as active components at the concentration of 224 μg/ml with the purpose of fast recovery of culture prolifetative properties after cryopreservation.

Allergenic Characterization of 27-kDa Glycoprotein, a Novel Heat Stable Allergen, from the Pupa of Silkworm, Bombyx mori.

PubMed

Jeong, Kyoung Yong; Son, Mina; Lee, June Yong; Park, Kyung Hee; Lee, Jae-Hyun; Park, Jung-Won

2016-01-01

Boiled silkworm pupa is a traditional food in Asia, and patients with silkworm pupa food allergy are common in these regions. Still now only one allergen from silkworm, arginine kinase, has been identified. The purpose of this study was to identify novel food allergens in silkworm pupa by analyzing a protein extract after heat treatment. Heat treated extracts were examined by proteomic analysis. A 27-kDa glycoprotein was identified, expressed in Escherichia coli, and purified. IgE reactivity of the recombinant protein was investigated by ELISA. High molecular weight proteins (above 100 kDa) elicited increased IgE binding after heat treatment compared to that before heat treatment. The molecular identities of these proteins, however, could not be determined. IgE reactivity toward a 27-kDa glycoprotein was also increased after heating the protein extract. The recombinant protein was recognized by IgE antibodies from allergic subjects (33.3%). Glycation or aggregation of protein by heating may create new IgE binding epitopes. Heat stable allergens are shown to be important in silkworm allergy. Sensitization to the 27-kDa glycoprotein from silkworm may contribute to elevation of IgE to silkworm.

Allergenic Characterization of 27-kDa Glycoprotein, a Novel Heat Stable Allergen, from the Pupa of Silkworm, Bombyx mori

PubMed Central

Son, Mina; Lee, June Yong

2016-01-01

Boiled silkworm pupa is a traditional food in Asia, and patients with silkworm pupa food allergy are common in these regions. Still now only one allergen from silkworm, arginine kinase, has been identified. The purpose of this study was to identify novel food allergens in silkworm pupa by analyzing a protein extract after heat treatment. Heat treated extracts were examined by proteomic analysis. A 27-kDa glycoprotein was identified, expressed in Escherichia coli, and purified. IgE reactivity of the recombinant protein was investigated by ELISA. High molecular weight proteins (above 100 kDa) elicited increased IgE binding after heat treatment compared to that before heat treatment. The molecular identities of these proteins, however, could not be determined. IgE reactivity toward a 27-kDa glycoprotein was also increased after heating the protein extract. The recombinant protein was recognized by IgE antibodies from allergic subjects (33.3%). Glycation or aggregation of protein by heating may create new IgE binding epitopes. Heat stable allergens are shown to be important in silkworm allergy. Sensitization to the 27-kDa glycoprotein from silkworm may contribute to elevation of IgE to silkworm. PMID:26770033

Monoclonal antibodies against 27.8 kDa protein receptor efficiently block lymphocystis disease virus infection in flounder Paralichthys olivaceus gill cells.

PubMed

Sheng, Xiu-Zhen; Wang, Mu; Xing, Jing; Zhan, Wen-Bin

2012-08-13

In previous research using co-immunoprecipitation, a 27.8 kDa protein in flounder Paralichthys olivaceus gill (FG) cells was found to bind lymphocystis disease virus (LCDV). In this paper, 13 hybridomas secreting monoclonal antibodies (MAbs) against the 27.8 kDa protein were obtained, and 2 MAbs designated as 2G11 and 3D9 were cloned by limiting dilution. Analyzed by indirect enzyme-linked immunosorbent assay (ELISA) and western blotting, the MAbs specifically reacted with the 27.8 kDa protein of FG cells. Confocal fluorescence microscopy and immunogold electron microscopy (IEM) provided evidence that the epitopes recognized by these MAbs were located primarily on the cell membrane and occasionally in the cytoplasm near the cell membrane of FG cells. The MAbs could block LCDV binding after MAbs were pre-incubated with isolated membrane proteins of FG cells in a blocking ELISA, and MAbs also could inhibit LCDV infection of FG cells in culture. Moreover, several target tissues of LCDV in flounder, including gill, stomach, intestine and liver, displayed the presence of the LCDV receptor-27.8 kDa. These results strongly supported the possibility that the 27.8 kDa protein is the putative receptor specific for LCDV infection of FG cells in flounder.

Diffusion of U(VI) in Opalinus Clay: Influence of temperature and humic acid

NASA Astrophysics Data System (ADS)

Joseph, C.; Van Loon, L. R.; Jakob, A.; Steudtner, R.; Schmeide, K.; Sachs, S.; Bernhard, G.

2013-05-01

The diffusion of U(VI) (c0 = 1 × 10-6 mol/L) in compacted Opalinus Clay from the Mont Terri underground laboratory, Switzerland, was studied in the absence and presence of humic acid (10 mg/L) at two different temperatures (25 °C, 60 °C) under anaerobic conditions. As background electrolyte synthetic Opalinus Clay pore water (pH 7.6, I = 0.36 mol/L) was used. The diffusion-accessible porosity, ɛ, was determined for each Opalinus Clay bore core sample by through-diffusion experiments with tritiated water (HTO) before the U(VI) diffusion experiments were carried out. The values for the effective diffusion and distribution coefficients De and Kd obtained for U(VI) and humic acid at 25 °C as well as at 60 °C showed that humic acid has no significant influence on the U(VI) diffusion. The diffusion profiles of humic acid in Opalinus Clay at 25 and 60 °C indicate the contributions of two different humic acid particle size fractions (<1 kDa and 10-100 kDa). The small-sized humic acid fraction diffused through the whole Opalinus Clay samples at both temperatures within the 3 month duration of the U(VI) diffusion experiments. At 60 °C, diffusion profiles of two different U(VI) species were observed. In a separate experiment the U(VI) speciation in the source reservoir solution at 60 °C was analyzed by laser-induced fluorescence spectroscopy, photon correlation spectroscopy and scanning electron microscopy with an energy dispersive X-ray detector. The two diffusion profiles could be attributed to an unknown colloidal and a known aquatic U(VI) species (Ca2UO2(CO3)3(aq)). The diffusion results showed that the interaction of U(VI) and of the large-sized humic acid colloid fraction with the clay is stronger at 60 °C. An increase of Kd from 0.025 ± 0.003 m3/kg at 25 °C to 0.25 ± 0.05 m3/kg for U(VI)colloidal at 60 °C was determined. In addition, the value for De of U(VI) increased with increasing temperature. Using the De values at 25 and 60 °C, a preliminary

Symmetry enriched U(1) quantum spin liquids

NASA Astrophysics Data System (ADS)

Zou, Liujun; Wang, Chong; Senthil, T.

2018-05-01

We classify and characterize three-dimensional U (1 ) quantum spin liquids [deconfined U (1 ) gauge theories] with global symmetries. These spin liquids have an emergent gapless photon and emergent electric/magnetic excitations (which we assume are gapped). We first discuss in great detail the case with time-reversal and SO(3 ) spin rotational symmetries. We find there are 15 distinct such quantum spin liquids based on the properties of bulk excitations. We show how to interpret them as gauged symmetry-protected topological states (SPTs). Some of these states possess fractional response to an external SO (3 ) gauge field, due to which we dub them "fractional topological paramagnets." We identify 11 other anomalous states that can be grouped into three anomaly classes. The classification is further refined by weakly coupling these quantum spin liquids to bosonic symmetry protected topological (SPT) phases with the same symmetry. This refinement does not modify the bulk excitation structure but modifies universal surface properties. Taking this refinement into account, we find there are 168 distinct such U (1 ) quantum spin liquids. After this warm-up, we provide a general framework to classify symmetry enriched U (1 ) quantum spin liquids for a large class of symmetries. As a more complex example, we discuss U (1 ) quantum spin liquids with time-reversal and Z2 symmetries in detail. Based on the properties of the bulk excitations, we find there are 38 distinct such spin liquids that are anomaly-free. There are also 37 anomalous U (1 ) quantum spin liquids with this symmetry. Finally, we briefly discuss the classification of U (1 ) quantum spin liquids enriched by some other symmetries.

Autoproteolysis and intramolecular dissociation of Yersinia YscU precedes secretion of its C-terminal polypeptide YscU(CC).

PubMed

Frost, Stefan; Ho, Oanh; Login, Frédéric H; Weise, Christoph F; Wolf-Watz, Hans; Wolf-Watz, Magnus

2012-01-01

Type III secretion system mediated secretion and translocation of Yop-effector proteins across the eukaryotic target cell membrane by pathogenic Yersinia is highly organized and is dependent on a switching event from secretion of early structural substrates to late effector substrates (Yops). Substrate switching can be mimicked in vitro by modulating the calcium levels in the growth medium. YscU that is essential for regulation of this switch undergoes autoproteolysis at a conserved N↑PTH motif, resulting in a 10 kDa C-terminal polypeptide fragment denoted YscU(CC). Here we show that depletion of calcium induces intramolecular dissociation of YscU(CC) from YscU followed by secretion of the YscU(CC) polypeptide. Thus, YscU(CC) behaved in vivo as a Yop protein with respect to secretion properties. Further, destabilized yscU mutants displayed increased rates of dissociation of YscU(CC)in vitro resulting in enhanced Yop secretion in vivo at 30°C relative to the wild-type strain.These findings provide strong support to the relevance of YscU(CC) dissociation for Yop secretion. We propose that YscU(CC) orchestrates a block in the secretion channel that is eliminated by calcium depletion. Further, the striking homology between different members of the YscU/FlhB family suggests that this protein family possess regulatory functions also in other bacteria using comparable mechanisms.

A novel PGC-1α isoform in brain localizes to mitochondria and associates with PINK1 and VDAC

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, Joungil, E-mail: jochoi@som.umaryland.edu; Veterans Affairs Medical Center, Baltimore, MD 21201; Batchu, Vera Venkatanaresh Kumar

2013-06-14

Highlights: •Novel 35 kDa PGC-1α localizes to mitochondrial inner membrane and matrix in brain. •Mitochondrial localization of 35 kDa PGC-1α depends on VDAC protein. •Mitochondrial localization of 35 kDa PGC-1α depends on membrane potential. •The 35 kDa PGC-1α associates and colocalizes with PINK in brain mitochondria. -- Abstract: Peroxisome proliferator-activated receptor-gamma co-activator 1α (PGC-1α) and PTEN-induced putative kinase 1 (PINK1) are powerful regulators of mitochondrial function. Here, we report that a previously unrecognized, novel 35 kDa PGC-1α isoform localizes to the mitochondrial inner membrane and matrix in brain as determined by protease protection and carbonate extraction assays, as well asmore » by immunoelectron microscopy. Immunoelectron microscopy and import experiments in vitro revealed that 35 kDa PGC-1α colocalizes and interacts with the voltage-dependent anion channel (VDAC), and that its import depends on VDAC. Valinomycin treatment which depolarizes the membrane potential, abolished mitochondrial localization of the 35 kDa PGC-1α. Using blue native-PAGE, co-immunoprecipitation, and immunoelectron microscopy analyses, we found that the 35 kDa PGC-1α binds and colocalizes with PINK1 in brain mitochondria. This is the first report regarding mitochondrial localization of a novel 35 kDa PGC-1α isoform and its association with PINK1, suggesting possible regulatory roles for mitochondrial function in the brain.« less

7SK snRNP/P-TEFb couples transcription elongation with alternative splicing and is essential for vertebrate development

PubMed Central

Barboric, Matjaz; Lenasi, Tina; Chen, Hui; Johansen, Eric B.; Guo, Su; Peterlin, B. Matija

2009-01-01

Eukaryotic gene expression is commonly controlled at the level of RNA polymerase II (RNAPII) pausing subsequent to transcription initiation. Transcription elongation is stimulated by the positive transcription elongation factor b (P-TEFb) kinase, which is suppressed within the 7SK small nuclear ribonucleoprotein (7SK snRNP). However, the biogenesis and functional significance of 7SK snRNP remain poorly understood. Here, we report that LARP7, BCDIN3, and the noncoding 7SK small nuclear RNA (7SK) are vital for the formation and stability of a cell stress-resistant core 7SK snRNP. Our functional studies demonstrate that 7SK snRNP is not only critical for controlling transcription elongation, but also for regulating alternative splicing of pre-mRNAs. Using a transient expression splicing assay, we find that 7SK snRNP disintegration promotes inclusion of an alternative exon via the increased occupancy of P-TEFb, Ser2-phosphorylated (Ser2-P) RNAPII, and the splicing factor SF2/ASF at the minigene. Importantly, knockdown of larp7 or bcdin3 orthologues in zebrafish embryos destabilizes 7SK and causes severe developmental defects and aberrant splicing of analyzed transcripts. These findings reveal a key role for P-TEFb in coupling transcription elongation with alternative splicing, and suggest that maintaining core 7SK snRNP is essential for vertebrate development. PMID:19416841

Gauged U(1) clockwork theory

NASA Astrophysics Data System (ADS)

Lee, Hyun Min

2018-03-01

We consider the gauged U (1) clockwork theory with a product of multiple gauge groups and discuss the continuum limit of the theory to a massless gauged U (1) with linear dilaton background in five dimensions. The localization of the lightest state of gauge fields on a site in the theory space naturally leads to exponentially small effective couplings of external matter fields localized away from the site. We discuss the implications of our general discussion with some examples, such as mediators of dark matter interactions, flavor-changing B-meson decays as well as D-term SUSY breaking.

Test-firing ammunition for spliceosome inhibition in cancer.

PubMed

Dehm, Scott M

2013-11-15

E7107 is a derivative of the pladienolide family of natural product spliceosome inhibitors, which targets the U2 small nuclear ribonucleoprotein (snRNP) subunit SF3b. The results of a first-in-human trial with E7107 have been reported, representing an important translational step toward the goal of modulating RNA splicing for cancer therapy. Clin Cancer Res; 19(22); 6064-6. ©2013 AACR.

General U(1)×U(1) F-theory compactifications and beyond: geometry of unHiggsings and novel matter structure

DOE PAGES

Cvetic, Mirjam; Klevers, Denis; Piragua, Hernan; ...

2015-11-30

We construct the general form of an F-theory compactification with two U(1) factors based on a general elliptically fibered Calabi-Yau manifold with Mordell-Weil group of rank two. This construction produces broad classes of models with diverse matter spectra, including many that are not realized in earlier F-theory constructions with U(1)×U(1) gauge symmetry. Generic U(1)×U(1) models can be related to a Higgsed non-Abelian model with gauge group SU(2)×SU(2)×SU(3), SU(2) 3×SU(3), or a subgroup thereof. The nonlocal horizontal divisors of the Mordell-Weil group are replaced with local vertical divisors associated with the Cartan generators of non-Abelian gauge groups from Kodaira singularities. Wemore » give a global resolution of codimension two singularities of the Abelian model; we identify the full anomaly free matter content, and match it to the unHiggsed non-Abelian model. The non-Abelian Weierstrass model exhibits a new algebraic description of the singularities in the fibration that results in the first explicit construction of matter in the symmetric representation of SU(3). This matter is realized on double point singularities of the discriminant locus. In conclusion, the construction suggests a generalization to U(1) k factors with k > 2, which can be studied by Higgsing theories with larger non-Abelian gauge groups.« less

The M2 autoantigen of central nervous system myelin, a glycoprotein present in oligodendrocyte membrane.

PubMed Central

Lebar, R; Lubetzki, C; Vincent, C; Lombrail, P; Boutry, J M

1986-01-01

Autoantibodies with in-vitro demyelinating capacity induced in Hartley and strain 13 guinea pigs with homologous central nervous system (CNS) tissue were used to characterize the target autoantigen M2. Using the Dot Immunobinding technique, M2 was found to be a component of CNS myelin different from basic protein (BP) and from cerebroside. The expression of M2 on oligodendrocytes, cells known to produce CNS myelin, also confirmed that M2 was a component of CNS myelin. Furthermore, the autoradiography of immunoprecipitates formed with radiolabelled guinea pig myelin and analysed in sodium dodecyl sulphate gels showed that M2 was specific to CNS myelin and absent in peripheral nervous system (PNS) myelin. On electrophoresis M2 appeared as two CNS myelin protein bands at the 27 and 54 KD molecular weight levels, distinct from the major protein bands of proteolipid and BP. M2 bands were of glycoprotein nature, as was demonstrated by affinity chromatography of CNS myelin on wheat germ agglutinin (WGA)-Sepharose. A monoclonal antibody induced by BP-free CNS glycoproteins recognized the same bands as anti-M2 serum in guinea pig CNS myelin. This would imply that both M2 bands share common determinants. M2 bands similar to the above in guinea pig were also shown in rat, rabbit and bovine CNS myelin with guinea pig antibodies. The same type of anti-M2 antibodies were induced in rabbit immunized with homologous CNS tissue. Although only a minor component of myelin, M2 is strongly immunogenic compared to BP. M2 antigen could thus be the target of chronic demyelinating processes such as experimental allergic encephalomyelitis. Images Fig. 1 Figure 2 Fig. 3 Fig. 4 PMID:2434274

Nano-LC FTICR tandem mass spectrometry for top-down proteomics: routine baseline unit mass resolution of whole cell lysate proteins up to 72 kDa.

PubMed

Tipton, Jeremiah D; Tran, John C; Catherman, Adam D; Ahlf, Dorothy R; Durbin, Kenneth R; Lee, Ji Eun; Kellie, John F; Kelleher, Neil L; Hendrickson, Christopher L; Marshall, Alan G

2012-03-06

Current high-throughput top-down proteomic platforms provide routine identification of proteins less than 25 kDa with 4-D separations. This short communication reports the application of technological developments over the past few years that improve protein identification and characterization for masses greater than 25 kDa. Advances in separation science have allowed increased numbers of proteins to be identified, especially by nanoliquid chromatography (nLC) prior to mass spectrometry (MS) analysis. Further, a goal of high-throughput top-down proteomics is to extend the mass range for routine nLC MS analysis up to 80 kDa because gene sequence analysis predicts that ~70% of the human proteome is transcribed to be less than 80 kDa. Normally, large proteins greater than 50 kDa are identified and characterized by top-down proteomics through fraction collection and direct infusion at relatively low throughput. Further, other MS-based techniques provide top-down protein characterization, however at low resolution for intact mass measurement. Here, we present analysis of standard (up to 78 kDa) and whole cell lysate proteins by Fourier transform ion cyclotron resonance mass spectrometry (nLC electrospray ionization (ESI) FTICR MS). The separation platform reduced the complexity of the protein matrix so that, at 14.5 T, proteins from whole cell lysate up to 72 kDa are baseline mass resolved on a nano-LC chromatographic time scale. Further, the results document routine identification of proteins at improved throughput based on accurate mass measurement (less than 10 ppm mass error) of precursor and fragment ions for proteins up to 50 kDa.

Spin Tests of 1/20-Scale Models of the Chance Vought Revised XF6U-1 and F6U-1 Airplanes, TED No. NACA 2390

NASA Technical Reports Server (NTRS)

Klinar, Walter J.; Berman, Theodore

1948-01-01

An investigation has been conducted in the Langley 20-foot free-spinning tunnel on the 1/20-scale model of the Chance Vought XF6U-1 airplane altered to represent the XF6U-1 airplane as it will be spin-tested in flight, and also altered to represent the F6U-1 airplane as it will be produced for service use. Spin tests were made to determine the effects of control settings and movements at the normal loading. The results show that the spins obtained on the revised XF6U-1 airplane will be oscillatory in roll and yaw and that recoveries by rudder reversal will be rapid. Model test results indicate that the F6U-1 airplane will probably not spin. Inasmuch as the results of this investigation show that the new designs are as good as or better than the original XF6U-1 design in regard to spin recovery, it is felt that the conclusions and recommendations reached for the original design can be applied to the new designs for all loading conditions.

Design of Chemical Conjugate for Targeted Therapy of Multiple Sclerosis Based of Constant Fragment of Human Antibody Heavy Chain and Peptoid Analog of Autoantigen MOG35-55.

PubMed

Lomakin, Y A; Stepanov, A V; Balabashin, D S; Ponomarenko, N A; Smirnov, I V; Belogurov, A A

2017-04-01

Elimination of B cells producing autoantibodies to neuroantigens is considered as beneficial in the treatment of multiple sclerosis. Myelin oligodendrocyte glycoprotein (MOG) is a significant autoantigen in multiple sclerosis. It was shown that MOG-like peptoid AMogP3 can bind autoantibodies produced by pathological lymphocytes. We propose a structure of an innovative drug for targeted elimination of the pool of autoreactive B cells responsible for multiple sclerosis pathogenesis; this compound is a complex of peptoid AMogP3 with Fc fragment of human immunoglobulin. The obtained Fc-PEG-AMogP3 conjugate effectively interact with autoreactive antibodies, which attests to their high therapeutic potential.

7 CFR 51.302 - U.S. No. 1.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 2 2011-01-01 2011-01-01 false U.S. No. 1. 51.302 Section 51.302 Agriculture... Standards for Grades of Apples Grades § 51.302 U.S. No. 1. “U.S. No. 1” consists of apples which meet the... color is required for all varieties listed in table I of § 51.305. Apples of this grade are free from...

7 CFR 51.302 - U.S. No. 1.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 2 2012-01-01 2012-01-01 false U.S. No. 1. 51.302 Section 51.302 Agriculture... Standards for Grades of Apples Grades § 51.302 U.S. No. 1. “U.S. No. 1” consists of apples which meet the... color is required for all varieties listed in table I of § 51.305. Apples of this grade are free from...

7 CFR 51.302 - U.S. No. 1.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 2 2010-01-01 2010-01-01 false U.S. No. 1. 51.302 Section 51.302 Agriculture... Standards for Grades of Apples Grades § 51.302 U.S. No. 1. “U.S. No. 1” consists of apples which meet the... color is required for all varieties listed in table I of § 51.305. Apples of this grade are free from...

Molluscan mega-hemocyanin: an ancient oxygen carrier tuned by a ~550 kDa polypeptide

PubMed Central

2010-01-01

Background The allosteric respiratory protein hemocyanin occurs in gastropods as tubular di-, tri- and multimers of a 35 × 18 nm, ring-like decamer with a collar complex at one opening. The decamer comprises five subunit dimers. The subunit, a 400 kDa polypeptide, is a concatenation of eight paralogous functional units. Their exact topology within the quaternary structure has recently been solved by 3D electron microscopy, providing a molecular model of an entire didecamer (two conjoined decamers). Here we study keyhole limpet hemocyanin (KLH2) tridecamers to unravel the exact association mode of the third decamer. Moreover, we introduce and describe a more complex type of hemocyanin tridecamer discovered in fresh/brackish-water cerithioid snails (Leptoxis, Melanoides, Terebralia). Results The "typical" KLH2 tridecamer is partially hollow, whereas the cerithioid tridecamer is almost completely filled with material; it was therefore termed "mega-hemocyanin". In both types, the staggering angle between adjoining decamers is 36°. The cerithioid tridecamer comprises two typical decamers based on the canonical 400 kDa subunit, flanking a central "mega-decamer" composed of ten unique ~550 kDa subunits. The additional ~150 kDa per subunit substantially enlarge the internal collar complex. Preliminary oxygen binding measurements indicate a moderate hemocyanin oxygen affinity in Leptoxis (p50 ~9 mmHg), and a very high affinity in Melanoides (~3 mmHg) and Terebralia (~2 mmHg). Species-specific and individual variation in the proportions of the two subunit types was also observed, leading to differences in the oligomeric states found in the hemolymph. Conclusions In cerithioid hemocyanin tridecamers ("mega-hemocyanin") the collar complex of the central decamer is substantially enlarged and modified. The preliminary O2 binding curves indicate that there are species-specific functional differences in the cerithioid mega-hemocyanins which might reflect different physiological

(234)U/(238)U signatures associated with uranium ore bodies: part 1 Ranger 3.

PubMed

Lowson, Richard T; McIntyre, Mark G

2013-04-01

The Ranger 3 ore body is an early Proterozoic U ore body in the Alligator Rivers U province, Northern Territory, Australia. It has surface expression with a redox front located between 30 and 50 m below the surface. The ground water U concentration and (234)U/(238)U AR signature in the top 10 m of the weathered zone are reported for 357 samples collected over 4 wet seasons, at 5 depths, along a transect in-line with the hydraulic gradient and along the centre line of the ore body and its associated dispersion halo. The results show that the weathered zone displays a general U isotope feature for this type of ore body with the (234)U/(238)U AR for the ground water and amorphous phase of the solid matrix being less than 1. The ground water (234)U/(238)U AR is independent of the annual monsoonal climate and depth within the range surface to 10 m. In the vicinity of the U ore body the ground water (234)U/(238)U AR is 0.75 and is very similar to the (234)U/(238)U AR of the amorphous phase of the solid (0.76). The (234)U/(238)U ARs of the amorphous phase and ground water rise and separate to values of 0.88 and 1.02 at the end of the transect. The rise and separation in (234)U/(238)U AR are interpreted as evidence that the source of the U in the ground water is from the water-soluble sub-phase of the amorphous phase and that the ground water flow is too fast to allow the processes occurring across the solid-water interface to reach chemical equilibrium. The data set is a robust characterisation of the coarse and fine detail of the (234)U/(238)U AR signature in the weathered zone of U ore bodies. Copyright © 2012 Elsevier Ltd. All rights reserved.

Genetic Diversity and Natural Selection in 42 kDa Region of Plasmodium vivax Merozoite Surface Protein-1 from China-Myanmar Endemic Border.

PubMed

Zhou, Xia; Tambo, Ernest; Su, Jing; Fang, Qiang; Ruan, Wei; Chen, Jun-Hu; Yin, Ming-Bo; Zhou, Xiao-Nong

2017-10-01

Plasmodium vivax merozoite surface protein-1 (PvMSP1) gene codes for a major malaria vaccine candidate antigen. However, its polymorphic nature represents an obstacle to the design of a protective vaccine. In this study, we analyzed the genetic polymorphism and natural selection of the C-terminal 42 kDa fragment within PvMSP1 gene (Pv MSP142) from 77 P. vivax isolates, collected from imported cases of China-Myanmar border (CMB) areas in Yunnan province and the inland cases from Anhui, Yunnan, and Zhejiang province in China during 2009-2012. Totally, 41 haplotypes were identified and 30 of them were new haplotypes. The differences between the rates of non-synonymous and synonymous mutations suggest that PvMSP142 has evolved under natural selection, and a high selective pressure preferentially acted on regions identified of PvMSP133. Our results also demonstrated that PvMSP142 of P. vivax isolates collected on China-Myanmar border areas display higher genetic polymorphisms than those collected from inland of China. Such results have significant implications for understanding the dynamic of the P. vivax population and may be useful information towards China malaria elimination campaign strategies.

U1A Complex

ScienceCinema

None

2018-01-16

Some of the most sophisticated experiments in the stockpile stewardship program are conducted in an environmentally safe manner, nearly 1000 feet below the ground at the site. The U1a complex a sprawling underground laboratory and tunnel complex is home to a number of unique capabilities.

Identification of a 170-kDa protein associated with the vacuolar Na+/H+ antiport of Beta vulgaris.

PubMed Central

Barkla, B J; Blumwald, E

1991-01-01

The effect of the addition of amiloride to the growth medium was tested on the Na+/H+ antiport activity of tonoplast vesicles isolated from sugar beet (beta vulgaris L.) cell suspensions. Cells grown in the presence of NaCl and amiloride displayed an increased antiport activity. Analysis of the kinetic data showed that while the affinity of the antiport for Na+ ions did not change, the maximal velocity of the Na+/H+ exchange increased markedly. These results suggest the addition of more antiport molecules to the tonoplast and/or an increase in the turnover rate of the Na+/H+ exchange. The increase in activity of the antiport by the presence of amiloride was correlated with the enhanced synthesis of a tonoplast 170-kDa polypeptide. The increased synthesis of this polypeptide was detected not only upon exposure of the cells to amiloride but also when the cells were exposed to high NaCl concentrations. Polyclonal antibodies against the 170-kDa polypeptide almost completely inhibited the antiport activity. These results suggest the association of the 170-kDa polypeptide with the vacuolar Na+/H+ antiport. Images PMID:1662387

Identification of a 170-kDa protein associated with the vacuolar Na+/H+ antiport of Beta vulgaris.

PubMed

Barkla, B J; Blumwald, E

1991-12-15

The effect of the addition of amiloride to the growth medium was tested on the Na+/H+ antiport activity of tonoplast vesicles isolated from sugar beet (beta vulgaris L.) cell suspensions. Cells grown in the presence of NaCl and amiloride displayed an increased antiport activity. Analysis of the kinetic data showed that while the affinity of the antiport for Na+ ions did not change, the maximal velocity of the Na+/H+ exchange increased markedly. These results suggest the addition of more antiport molecules to the tonoplast and/or an increase in the turnover rate of the Na+/H+ exchange. The increase in activity of the antiport by the presence of amiloride was correlated with the enhanced synthesis of a tonoplast 170-kDa polypeptide. The increased synthesis of this polypeptide was detected not only upon exposure of the cells to amiloride but also when the cells were exposed to high NaCl concentrations. Polyclonal antibodies against the 170-kDa polypeptide almost completely inhibited the antiport activity. These results suggest the association of the 170-kDa polypeptide with the vacuolar Na+/H+ antiport.

A DOUBLE KNOCKOUT; A NOVEL APPROACH TO UNDERSTANDING STRESS-INDUCIBLE 70 KDA HEAT SHOCK PROTEINS (HSP70S) ON DEVELOPMENT AND REPRODUCTION

EPA Science Inventory

Heat and chemical toxicants which disrupt spermatogenesis and cause male infertility are thought to induce the expression of Hsp70-1 and 70-3, the major inducible heat shock proteins of the 70kDa family. Previous studies from several laboratories including our own have characteri...

Wild-Type U2AF1 Antagonizes the Splicing Program Characteristic of U2AF1-Mutant Tumors and Is Required for Cell Survival

PubMed Central

Fei, Dennis Liang; Motowski, Hayley; Chatrikhi, Rakesh; Gao, Shaojian; Kielkopf, Clara L.; Varmus, Harold

2016-01-01

We have asked how the common S34F mutation in the splicing factor U2AF1 regulates alternative splicing in lung cancer, and why wild-type U2AF1 is retained in cancers with this mutation. A human lung epithelial cell line was genetically modified so that U2AF1S34F is expressed from one of the two endogenous U2AF1 loci. By altering levels of mutant or wild-type U2AF1 in this cell line and by analyzing published data on human lung adenocarcinomas, we show that S34F-associated changes in alternative splicing are proportional to the ratio of S34F:wild-type gene products and not to absolute levels of either the mutant or wild-type factor. Preferential recognition of specific 3′ splice sites in S34F-expressing cells is largely explained by differential in vitro RNA-binding affinities of mutant versus wild-type U2AF1 for those same 3′ splice sites. Finally, we show that lung adenocarcinoma cell lines bearing U2AF1 mutations do not require the mutant protein for growth in vitro or in vivo. In contrast, wild-type U2AF1 is required for survival, regardless of whether cells carry the U2AF1S34F allele. Our results provide mechanistic explanations of the magnitude of splicing changes observed in U2AF1-mutant cells and why tumors harboring U2AF1 mutations always retain an expressed copy of the wild-type allele. PMID:27776121

7 CFR 51.302 - U.S. No. 1.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 2 2013-01-01 2013-01-01 false U.S. No. 1. 51.302 Section 51.302 Agriculture..., CERTIFICATION, AND STANDARDS) United States Standards for Grades of Apples Grades § 51.302 U.S. No. 1. “U.S. No... § 51.305. Apples of this grade are free from excessive damage caused by russeting which means that...

7 CFR 51.302 - U.S. No. 1.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 2 2014-01-01 2014-01-01 false U.S. No. 1. 51.302 Section 51.302 Agriculture..., CERTIFICATION, AND STANDARDS) United States Standards for Grades of Apples Grades § 51.302 U.S. No. 1. “U.S. No... § 51.305. Apples of this grade are free from excessive damage caused by russeting which means that...

Structure of the putative 32 kDa myrosinase-binding protein from Arabidopsis (At3g16450.1) determined by SAIL-NMR.

PubMed

Takeda, Mitsuhiro; Sugimori, Nozomi; Torizawa, Takuya; Terauchi, Tsutomu; Ono, Akira M; Yagi, Hirokazu; Yamaguchi, Yoshiki; Kato, Koichi; Ikeya, Teppei; Jee, Jungoo; Güntert, Peter; Aceti, David J; Markley, John L; Kainosho, Masatsune

2008-12-01

The product of gene At3g16450.1 from Arabidopsis thaliana is a 32 kDa, 299-residue protein classified as resembling a myrosinase-binding protein (MyroBP). MyroBPs are found in plants as part of a complex with the glucosinolate-degrading enzyme myrosinase, and are suspected to play a role in myrosinase-dependent defense against pathogens. Many MyroBPs and MyroBP-related proteins are composed of repeated homologous sequences with unknown structure. We report here the three-dimensional structure of the At3g16450.1 protein from Arabidopsis, which consists of two tandem repeats. Because the size of the protein is larger than that amenable to high-throughput analysis by uniform (13)C/(15)N labeling methods, we used stereo-array isotope labeling (SAIL) technology to prepare an optimally (2)H/(13)C/(15)N-labeled sample. NMR data sets collected using the SAIL protein enabled us to assign (1)H, (13)C and (15)N chemical shifts to 95.5% of all atoms, even at a low concentration (0.2 mm) of protein product. We collected additional NOESY data and determined the three-dimensional structure using the cyana software package. The structure, the first for a MyroBP family member, revealed that the At3g16450.1 protein consists of two independent but similar lectin-fold domains, each composed of three beta-sheets.

7 CFR 51.2076 - U.S. No. 1 Mixed.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 2 2013-01-01 2013-01-01 false U.S. No. 1 Mixed. 51.2076 Section 51.2076 Agriculture.... No. 1 Mixed. “U.S. No. 1 Mixed” consists of almonds in the shell which meet the requirements of U.S. No. 1 grade, except that two or more varieties of sweet almonds are mixed. ...

7 CFR 51.2076 - U.S. No. 1 Mixed.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 2 2014-01-01 2014-01-01 false U.S. No. 1 Mixed. 51.2076 Section 51.2076 Agriculture.... No. 1 Mixed. “U.S. No. 1 Mixed” consists of almonds in the shell which meet the requirements of U.S. No. 1 grade, except that two or more varieties of sweet almonds are mixed. ...

Precipitation of the thyrotropin receptor and identification of thyroid autoantigens using Graves' disease immunoglobulins.

PubMed Central

Heyma, P; Harrison, L C

1984-01-01

The thyrotropin (TSH) receptor is a putative target for autoantibodies in Graves' hyperthyroidism and therefore, should be capable of being identified, isolated, and structurally characterized by immunological means. To this end, four sera from patients with hyperthyroidism, three of which inhibited the binding of 125I-TSH to Triton-solubilized human thyroid membranes, were used to isolate TSH receptors by immunoprecipitation. To account for an effect of TSH binding or receptor occupancy on the ability of Graves' immunoglobulins to precipitate TSH receptors, two approaches were taken: (a) specific 125I-TSH binding activity was measured after solubilized thyroid membranes had been incubated with Graves' sera followed by precipitation with Staphylococcus protein A ("receptor depletion"); (b) TSH binding sites were labeled with 125I-TSH and the complexes were precipitated using Graves' sera and Staphylococcus protein A ("receptor precipitation"). The three sera which inhibited 125I-TSH binding depleted 125I-TSH binding activity between 30-80%. Preformed complexes between Staphylococcus protein A and immunoglobulins in these sera were also able to deplete 125I-TSH binding activity. However, after receptor depletion, the one serum that did not inhibit 125I-TSH binding was associated with a significant increase in 125I-TSH binding. All four sera specifically precipitated 80-100% of receptors identified by prelabeling with 125I-TSH. The dilutions of sera that precipitated 50% of 125I-TSH-receptor complexes ranged from 1:150-1:20. Complexes were partially precipitated by high concentrations of control sera (1:20), but the relative potency of control sera was at least fourfold less than Graves' sera. Immunoprecipitates of 125I-labeled thyroid membranes were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography to reveal Graves'-specific bands of reduced molecular weights of 100-110,000, 80-90,000, and 70-75,000. These bands were similar

Plants transformed with a tobacco mosaic virus nonstructural gene sequence are resistant to the virus.

PubMed Central

Golemboski, D B; Lomonossoff, G P; Zaitlin, M

1990-01-01

Nicotiana tabacum cv. Xanthi nn plants were transformed with nucleotides 3472-4916 of tobacco mosaic virus (TMV) strain U1. This sequence contains all but the three 3 terminal nucleotides of the TMV 54-kDa gene, which encodes a putative component of the replicase complex. These plants were resistant to infection when challenged with either TMV U1 virions or TMV U1 RNA at concentrations of up to 500 micrograms/ml or 300 micrograms/ml, respectively, the highest concentrations tested. Resistance was also exhibited when plants were inoculated at 100 micrograms/ml with the closely related TMV mutant YSI/1 but was not shown in plants challenged at the same concentrations with the more distantly related TMV strains U2 or L or cucumber mosaic virus. Although the copy number of the 54-kDa gene sequence varied in individual transformants from 1 to approximately 5, the level of resistance in plants was not dependent on the number of copies of the 54-kDa gene sequence integrated. The transformed plants accumulated a 54-kDa gene sequence-specific RNA transcript of the expected size, but no protein product was detected. Images PMID:2385595

7 CFR 51.2076 - U.S. No. 1 Mixed.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 2 2012-01-01 2012-01-01 false U.S. No. 1 Mixed. 51.2076 Section 51.2076 Agriculture... Standards for Grades of Almonds in the Shell Grades § 51.2076 U.S. No. 1 Mixed. “U.S. No. 1 Mixed” consists... varieties of sweet almonds are mixed. ...

7 CFR 51.340 - U.S. No. 1.

Code of Federal Regulations, 2012 CFR

2012-01-01

... FRESH FRUITS, VEGETABLES AND OTHER PRODUCTS 1,2 (INSPECTION, CERTIFICATION, AND STANDARDS) United States Standards for Grades of Apples for Processing Grades § 51.340 U.S. No. 1. “U.S. No. 1” consists of apples of... percent, by weight, of the apple. ...

7 CFR 51.2833 - U.S. No. 1 Boilers.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 2 2010-01-01 2010-01-01 false U.S. No. 1 Boilers. 51.2833 Section 51.2833 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards...) Grades § 51.2833 U.S. No. 1 Boilers. U.S. No. 1 Boilers consists of onions which meet all the...

Sequencing Larger Intact Proteins (30-70 kDa) with Activated Ion Electron Transfer Dissociation

NASA Astrophysics Data System (ADS)

Riley, Nicholas M.; Westphall, Michael S.; Coon, Joshua J.

2018-01-01

The analysis of intact proteins via mass spectrometry can offer several benefits to proteome characterization, although the majority of top-down experiments focus on proteoforms in a relatively low mass range (<30 kDa). Recent studies have focused on improving the analysis of larger intact proteins (up to 75 kDa), but they have also highlighted several challenges to be addressed. One major hurdle is the efficient dissociation of larger protein ions, which often to do not yield extensive fragmentation via conventional tandem MS methods. Here we describe the first application of activated ion electron transfer dissociation (AI-ETD) to proteins in the 30-70 kDa range. AI-ETD leverages infrared photo-activation concurrent to ETD reactions to improve sequence-informative product ion generation. This method generates more product ions and greater sequence coverage than conventional ETD, higher-energy collisional dissociation (HCD), and ETD combined with supplemental HCD activation (EThcD). Importantly, AI-ETD provides the most thorough protein characterization for every precursor ion charge state investigated in this study, making it suitable as a universal fragmentation method in top-down experiments. Additionally, we highlight several acquisition strategies that can benefit characterization of larger proteins with AI-ETD, including combination of spectra from multiple ETD reaction times for a given precursor ion, multiple spectral acquisitions of the same precursor ion, and combination of spectra from two different dissociation methods (e.g., AI-ETD and HCD). In all, AI-ETD shows great promise as a method for dissociating larger intact protein ions as top-down proteomics continues to advance into larger mass ranges. [Figure not available: see fulltext.

Uranus moon - 1985U1

NASA Technical Reports Server (NTRS)

1986-01-01

Several craters are seen on the surface of 1985U1, one of several small moons of Uranus discovered by Voyager 2. The spacecraft acquired this single image -- the only close-up it obtained of any of the new moons -- on Jan. 24, 1986. At the time, Voyager was at a distance of about 500,000 kilometers (300,000 miles) from 1985U1, yielding a resolution of about 10 km (6 mi) in this clear-filter, narrow-angle image. The moon was found Dec. 3O, 1985; it was the first and largest of nearly a dozen satellites discovered by the spacecraft cameras. This image shows 1985U1 to be a dark, nearly spherical object, with a diameter of about 150 km (90 mi); the dark surface reflects only 7 percent of the incident light. The picture was inserted into the Voyager encounter sequence late in its development. This image has had a complex history, having been recorded on the spacecraft tape recorder and first played back during the late afternoon of Jan. 24. An antenna-pointing problem at one of the Australian tracking stations led to loss of the data, so the image had to be transmitted a second time. It was successfully received shortly before 6 p.m. PST Jan. 26. The Voyager project is managed for NASA by the Jet Propulsion Laboratory.

7 CFR 51.2730 - U.S. No. 1 Spanish.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 2 2010-01-01 2010-01-01 false U.S. No. 1 Spanish. 51.2730 Section 51.2730... STANDARDS) United States Standards for Grades of Shelled Spanish Type Peanuts Grades § 51.2730 U.S. No. 1 Spanish. “U.S. No. 1 Spanish” consists of shelled Spanish type peanut kernels which are whole and free...

The 170-kDa glucose-regulated stress protein is an endoplasmic reticulum protein that binds immunoglobulin.

PubMed Central

Lin, H Y; Masso-Welch, P; Di, Y P; Cai, J W; Shen, J W; Subjeck, J R

1993-01-01

Anoxia, glucose starvation, calcium ionophore A23187, EDTA, glucosamine, and several other conditions that adversely affect the function of the endoplasmic reticulum (ER) induce the synthesis of the glucose-regulated class of stress proteins (GRPs). The primary GRPs induced by these stresses migrate at 78 and 94 kDa (GRP78 and GRP94). In addition, another protein of approximately 150-170 kDa (GRP170) has been previously observed and is coordinately induced with GRP78 and GRP94. To characterize this novel stress protein, we have prepared an antisera against purified GRP170. Immunofluorescence, Endoglycosidase H sensitivity, and protease resistance of this protein in microsomes indicates that GRP170 is an ER lumenal glycoprotein retained in a pre-Golgi compartment. Immunoprecipitation of GRP170 with our antibody coprecipitates the GRP78 (also referred to as the B cell immunoglobulin-binding protein) and GRP94 members of this stress protein family in Chinese hamster ovary cells under stress conditions. ATP depletion, by immunoprecipitation in the presence of apyrase, does not affect the interaction between GRP78 and GRP170 but results in the coprecipitation of an unidentified 60-kDa protein. In addition, GRP170 is found to be coprecipitated with immunoglobulin (Ig) in four different B cell hybridomas expressing surface IgM, cytoplasmic Ig light chain only, cytoplasmic Ig heavy chain only, or an antigen specific secreted IgG. In addition, in IgM surface expressing WEHI-231 B cells, anti-IgM coprecipitates GRP78, GRP94, as well as GRP170; antibodies against GRP170 and GRP94 reciprocally coprecipitate GRP94/GRP170 as well as GRP78. Results suggest that this 170-kDa GRP is a retained ER lumenal glycoprotein that is constitutively present and that may play a role in immunoglobulin folding and assembly in conjunction or consecutively with GRP78 and GRP94. Images PMID:8305733

Calmyonemin: a 23 kDa analogue of algal centrin occurring in contractile myonemes of Eudiplodinium maggii (ciliate).

PubMed

David, C; Viguès, B

1994-01-01

Myonemes are bundles of thin filaments (3-6 nm in diameter) which mediate calcium-induced contraction of the whole or only parts of the cell body in a number of protists. In Eudiplodinium maggii, a rumen ciliate which lacks a uniform ciliation of the cell body, myonemes converge toward the bases of apical ciliary zones that can be retracted under stress conditions, entailing immobilization of the cell. An mAB (A69) has been produced that identifies a calcium-binding protein by immunoblot, immunoprecipitation experiments and specifically labels the myonemes in immunoelectron microscopy. Solubility properties, apparent molecular weight (23 kDa) and isoelectric point (4.9) of the myonemal protein, are similar to the values reported for the calcium-modulated contractile protein centrin. Western-blot analysis indicates that the 23 kDa protein cross-reacts antigenically with anti-centrin antibodies. In addition, the 23 kDa protein displays calcium-induced changes in both electrophoretic and chromatographic behaviour, and contains calcium-binding domains that conform to the EF-hand structure, as known for centrin. Based on these observations, we conclude that a calcium-binding protein with major similarities to centrin occurs in the myonemes of E. maggii. We postulate that this protein plays an essential role in myoneme-mediated retraction of the ciliature.

Hyaluronan 35kDa treatment protects mice from Citrobacter rodentium infection and induces epithelial tight junction protein ZO-1 in vivo.

PubMed

Kim, Yeojung; Kessler, Sean P; Obery, Dana R; Homer, Craig R; McDonald, Christine; de la Motte, Carol A

2017-10-01

Maintaining a healthy intestinal barrier, the primary physical barrier between intestinal microbiota and the underlying lamina propria, is critical for optimal health. Epithelial integrity is essential for the prevention of the entrance of luminal contents, such as bacteria and their products, through the large intestinal barrier. In this study, we investigated the protective functions of biosynthetic, specific sized, hyaluronan around 35kDa (HA35) on intestinal epithelium in healthy mice, as well as mice infected Citrobacter rodentium, an established model that mimics infection with a serious human pathogen, enteropathogenic E. coli (EPEC). Our results reveal that treatment with HA35 protects mice from Citrobacter infection and enhances the epithelial barrier function. In particular, we have found that HA35 induces the expression of tight junction protein zonula occludens (ZO)-1 in both healthy and Citrobacter infected mice, as demonstrated by immunoflurorescence and Western blot analyses. Furthermore, we determined that HA35 treatment enhances ZO-1 expression and reduces intestinal permeability at the early stages of dextran sulfate sodium (DSS)-induced colitis in mice. Together, our data demonstrate that the expression and functionality of tight junctions, are increased by HA35 treatment, suggesting a novel mechanism for the protection from Citrobacter infection. Copyright © 2016 Elsevier B.V. All rights reserved.

Left-handed and right-handed U(1) gauge symmetry

NASA Astrophysics Data System (ADS)

Nomura, Takaaki; Okada, Hiroshi

2018-01-01

We propose a model with the left-handed and right-handed continuous Abelian gauge symmetry; U(1) L × U(1) R . Then three right-handed neutrinos are naturally required to achieve U(1) R anomaly cancellations, while several mirror fermions are also needed to do U(1) L anomaly cancellations. Then we formulate the model, and discuss its testability of the new gauge interactions at collider physics such as the large hadron collider (LHC) and the international linear collider (ILC). In particular, we can investigate chiral structure of the interactions by the analysis of forward-backward asymmetry based on polarized beam at the ILC.

7 CFR 51.3740 - U.S. No. 1.

Code of Federal Regulations, 2011 CFR

2011-01-01

... Standards for Grades of Honey Dew and Honey Ball Type Melons Grades § 51.3740 U.S. No. 1. “U.S. No. 1” consists of honey dew or honey ball type melons which are mature, firm, well formed, which are free from...

7 CFR 51.3740 - U.S. No. 1.

Code of Federal Regulations, 2010 CFR

2010-01-01

... Standards for Grades of Honey Dew and Honey Ball Type Melons Grades § 51.3740 U.S. No. 1. “U.S. No. 1” consists of honey dew or honey ball type melons which are mature, firm, well formed, which are free from...

Structure of the Putative 32 kDa Myrosinase Binding Protein from Arabidopsis (At3g16450.1) Determined by SAIL-NMR

PubMed Central

Takeda, Mitsuhiro; Sugimori, Nozomi; Torizawa, Takuya; Terauchi, Tsutomu; Ono, Akira Mei; Yagi, Hirokazu; Yamaguchi, Yoshiki; Kato, Koichi; Ikeya, Teppei; Jee, JunGoo; Güntert, Peter; Aceti, David J.; Markley, John L.; Kainosho, Masatsune

2009-01-01

The product of gene At3g16450.1 from Arabidopsis thaliana is a 32 kDa, 299-residue protein classified as resembling a myrosinase-binding protein (MyroBP). MyroBPs are found in plants as part of a complex with the glucosinolate-degrading enzyme, myrosinase, and are suspected to play a role in myrosinase-dependent defense against pathogens. Many MyroBPs and MyroBP-related proteins are composed of repeated homologous sequences with unknown structure. We report here the three-dimensional structure of the At3g16450.1 protein from Arabidopsis, which consists of two tandem repeats. Because the size of the protein is larger than that amenable to high-throughput analysis by uniformly 13C/15N labeling methods, we used our stereo-array isotope labeling (SAIL) technology to prepare an optimally 2H/13C/15N-labeled sample. NMR data sets collected with the SAIL-protein enabled us to assign 1H, 13C and 15N chemical shifts to 95.5% of all atoms, even at the low concentration (0.2 mM) of the protein product. We collected additional NOESY data and solved the three-dimensional structure with the CYANA software package. The structure, the first for a MyroBP family member, revealed that the At3g16450.1 protein consists of two independent, but similar, lectin-fold domains composed of three β-sheets. PMID:19021763

Potential involvement of F0F1-ATP(synth)ase and reactive oxygen species in apoptosis induction by the antineoplastic agent erucylphosphohomocholine in glioblastoma cell lines : a mechanism for induction of apoptosis via the 18 kDa mitochondrial translocator protein.

PubMed

Veenman, Leo; Alten, Julia; Linnemannstöns, Karen; Shandalov, Yulia; Zeno, Sivan; Lakomek, Max; Gavish, Moshe; Kugler, Wilfried

2010-07-01

Erucylphosphohomocholine (ErPC3, Erufosine) was reported previously to induce apoptosis in otherwise highly apoptosis-resistant malignant glioma cell lines while sparing their non-tumorigenic counterparts. We also previously found that the mitochondrial 18 kDa Translocator Protein (TSPO) is required for apoptosis induction by ErPC3. These previous studies also suggested involvement of reactive oxygen species (ROS). In the present study we further investigated the potential involvement of ROS generation, the participation of the mitochondrial respiration chain, and the role of the mitochondrial F(O)F(1)-ATP(synth)ase in the pro-apoptotic effects of ErPC3 on U87MG and U118MG human glioblastoma cell lines. For this purpose, cells were treated with the ROS chelator butylated hydroxyanisole (BHA), the mitochondrial respiration chain inhibitors rotenone, antimycin A, myxothiazol, and the uncoupler CCCP. Also oligomycin and piceatannol were studied as inhibitors of the F(O) and F(1) subunits of the mitochondrial F(O)F(1)-ATP(synth)ase, respectively. BHA was able to attenuate apoptosis induction by ErPC3, including mitochondrial ROS generation as determined with cardiolipin oxidation, as well as collapse of the mitochondrial membrane potential (Deltapsi(m)). Similarly, we found that oligomycin attenuated apoptosis and collapse of the Deltapsi(m), normally induced by ErPC3, including the accompanying reductions in cellular ATP levels. Other inhibitors of the mitochondrial respiration chain, as well as piceatannol, did not show such effects. Consequently, our findings strongly point to a role for the F(O) subunit of the mitochondrial F(O)F(1)-ATP(synth)ase in ErPC3-induced apoptosis and dissipation of Deltapsi(m) as well as ROS generation by ErPC3 and TSPO.

Mature parasite-infected erythrocyte surface antigen (MESA) of Plasmodium falciparum binds to the 30-kDa domain of protein 4.1 in malaria-infected red blood cells.

PubMed

Waller, Karena L; Nunomura, Wataru; An, Xiuli; Cooke, Brian M; Mohandas, Narla; Coppel, Ross L

2003-09-01

The Plasmodium falciparum mature parasite-infected erythrocyte surface antigen (MESA) is exported from the parasite to the infected red blood cell (IRBC) membrane skeleton, where it binds to protein 4.1 (4.1R) via a 19-residue MESA sequence. Using purified RBC 4.1R and recombinant 4.1R fragments, we show MESA binds the 30-kDa region of RBC 4.1R, specifically to a 51-residue region encoded by exon 10 of the 4.1R gene. The 3D structure of this region reveals that the MESA binding site overlaps the region of 4.1R involved in the p55, glycophorin C, and 4.1R ternary complex. Further binding studies using p55, 4.1R, and MESA showed competition between p55 and MESA for 4.1R, implying that MESA bound at the IRBC membrane skeleton may modulate normal 4.1R and p55 interactions in vivo. Definition of minimal binding domains involved in critical protein interactions in IRBCs may aid the development of novel therapies for falciparum malaria.

Proteome-wide survey of the autoimmune target repertoire in autoimmune polyendocrine syndrome type 1

PubMed Central

Landegren, Nils; Sharon, Donald; Freyhult, Eva; Hallgren, Åsa; Eriksson, Daniel; Edqvist, Per-Henrik; Bensing, Sophie; Wahlberg, Jeanette; Nelson, Lawrence M.; Gustafsson, Jan; Husebye, Eystein S.; Anderson, Mark S.; Snyder, Michael; Kämpe, Olle

2016-01-01

Autoimmune polyendocrine syndrome type 1 (APS1) is a monogenic disorder that features multiple autoimmune disease manifestations. It is caused by mutations in the Autoimmune regulator (AIRE) gene, which promote thymic display of thousands of peripheral tissue antigens in a process critical for establishing central immune tolerance. We here used proteome arrays to perform a comprehensive study of autoimmune targets in APS1. Interrogation of established autoantigens revealed highly reliable detection of autoantibodies, and by exploring the full panel of more than 9000 proteins we further identified MAGEB2 and PDILT as novel major autoantigens in APS1. Our proteome-wide assessment revealed a marked enrichment for tissue-specific immune targets, mirroring AIRE’s selectiveness for this category of genes. Our findings also suggest that only a very limited portion of the proteome becomes targeted by the immune system in APS1, which contrasts the broad defect of thymic presentation associated with AIRE-deficiency and raises novel questions what other factors are needed for break of tolerance. PMID:26830021

Study of Infrared Emission Spectroscopy for the B1Δg-A1Πu and B'1Σg+-A1Πu Systems of C2

NASA Astrophysics Data System (ADS)

Tang, Jian; Chen, Wang; Kawaguchi, Kentarou; Bernath, Peter F.

2016-06-01

Recently, we carried out the perturbation analysis of C_2 spectra and identified forbidden singlet-triplet intersystem transitions, which aroused further interest in other C_2 spectra for the many low-lying electronic states of this fundamental molecule. In 1988, the B1Δg-A1Πu and B'1Σg+-A1Πu band systems were discovered by Douay et al., who observed eight bands of the B1Δg-A1Πu system with v up to 5 for the B1Δg state and six bands of the B'1Σg+-A1Πu system with v up to 3 for the B'1Σg+ state in the Fourier transform infrared emission spectra of hydrocarbon discharges. In the work presented here, we identified twenty-four bands of the two systems, among which the B'1Σg+ v = 4 and the B1Δg v = 6, 7 and 8 vibrational levels involved in nine bands were studied for the first time. A direct global analysis with Dunham parameters was carried out satisfactorily for the B1Δg-A1Πu system except for a small perturbation in the B1Δg v = 6 level. The calculated rovibrational term energies up to B1Δg v = 12 showed that the level crossing between the B1Δg and d3Πg states is responsible for many of the prominent perturbations in the Swan system observed previously. Nineteen lines of the B1Δg-a3Πu forbidden transitions were identified and the off-diagonal spin-orbit interaction constant AdB between d3Πg and B1Δg was derived as 8.3(1) wn. For the B'1Σg+-A1Πu system, only individual band analyses for each vibrational level in the B'1Σg+ state could be done satisfactorily and Dunham parameters obtained from these effective parameters showed that the anharmonic vibrational constant ω_e x_e is anomalously small (nearly zero). Inspection of the RKR potential curves for the B'1Σg+ and X1Σg+ states revealed that an avoided crossing may occur around 30000 wn, which is responsible for the anomalous molecular constants in these two states. W. Chen, K. Kawaguchi, P. F. Bernath, and J. Tang, J. Chem. Phys., 141, 064317 (2015) M. Douay, R. Nietmann and P. F. Bernath

Crystallization and X-ray data analysis of the 10 kDa C-terminal lid subdomain from Caenorhabditis elegans Hsp70

DOE Office of Scientific and Technical Information (OSTI.GOV)

Worrall, Liam; Walkinshaw, Malcolm D., E-mail: m.walkinshaw@ed.ac.uk

Crystals of the C-terminal 10 kDa lid subdomain from the C. elegans chaperone Hsp70 have been obtained that diffract X-rays to ∼3.5 Å and belong to space group I2{sub 1}2{sub 1}2{sub 1}. Analysis of X-ray data and initial heavy-atom phasing reveals 24 monomers in the asymmetric unit related by 432 non-crystallographic symmetry. Hsp70 is an important molecular chaperone involved in the regulation of protein folding. Crystals of the C-terminal 10 kDa helical lid domain (residues 542–640) from a Caenorhabditis elegans Hsp70 homologue have been produced that diffract X-rays to ∼3.4 Å. Crystals belong to space group I2{sub 1}2{sub 1}2{sub 1},more » with unit-cell parameters a = b = 197, c = 200 Å. The Matthews coefficient, self-rotation function and Patterson map indicate 24 monomers in the asymmetric unit, showing non-crystallographic 432 symmetry. Molecular-replacement studies using the corresponding domain from rat, the only eukaryotic homologue with a known structure, failed and a mercury derivative was obtained. Preliminary MAD phasing using SHELXD and SHARP for location and refinement of the heavy-atom substructure and SOLOMON for density modification produced interpretable maps with a clear protein–solvent boundary. Further density-modification, model-building and refinement are currently under way.« less

U(1) mediation of flux supersymmetry breaking

NASA Astrophysics Data System (ADS)

Grimm, Thomas W.; Klemm, Albrecht

2008-10-01

We study the mediation of supersymmetry breaking triggered by background fluxes in Type II string compactifications with Script N = 1 supersymmetry. The mediation arises due to an U(1) vector multiplet coupling to both a hidden supersymmetry breaking flux sector and a visible D-brane sector. The required internal manifolds can be constructed by non-Kähler resolutions of singular Calabi-Yau manifolds. The effective action encoding the U(1) coupling is then determined in terms of the global topological properties of the internal space. We investigate suitable local geometries for the hidden and visible sector in detail. This includes a systematic study of orientifold symmetries of del Pezzo surfaces realized in compact geometries after geometric transition. We construct compact examples admitting the key properties to realize flux supersymmetry breaking and U(1) mediation. Their toric realization allows us to analyze the geometry of curve classes and confirm the topological connection between the hidden and visible sector.

Modelling of Thyroid Peroxidase Reveals Insights into Its Enzyme Function and Autoantigenicity

PubMed Central

Fodor, James; Riley, Blake; Godlewska, Marlena; Góra, Monika; Czarnocka, Barbara; Banga, J Paul; Hoke, David E.; Kass, Itamar; Buckle, Ashley M.

2015-01-01

Thyroid peroxidase (TPO) catalyses the biosynthesis of thyroid hormones and is a major autoantigen in Hashimoto’s disease—the most common organ-specific autoimmune disease. Epitope mapping studies have shown that the autoimmune response to TPO is directed mainly at two surface regions on the molecule: immunodominant regions A and B (IDR-A, and IDR-B). TPO has been a major target for structural studies for over 20 years; however, to date, the structure of TPO remains to be determined. We have used a molecular modelling approach to investigate plausible modes of TPO structure and dimer organisation. Sequence features of the C-terminus are consistent with a coiled-coil dimerization motif that most likely anchors the TPO dimer in the apical membrane of thyroid follicular cells. Two contrasting models of TPO were produced, differing in the orientation and exposure of their active sites relative to the membrane. Both models are equally plausible based upon the known enzymatic function of TPO. The “trans” model places IDR-B on the membrane-facing side of the myeloperoxidase (MPO)-like domain, potentially hindering access of autoantibodies, necessitating considerable conformational change, and perhaps even dissociation of the dimer into monomers. IDR-A spans MPO- and CCP-like domains and is relatively fragmented compared to IDR-B, therefore most likely requiring domain rearrangements in order to coalesce into one compact epitope. Less epitope fragmentation and higher solvent accessibility of the “cis” model favours it slightly over the “trans” model. Here, IDR-B clusters towards the surface of the MPO-like domain facing the thyroid follicular lumen preventing steric hindrance of autoantibodies. However, conformational rearrangements may still be necessary to allow full engagement with autoantibodies, with IDR-B on both models being close to the dimer interface. Taken together, the modelling highlights the need to consider the oligomeric state of TPO, its

Modelling of Thyroid Peroxidase Reveals Insights into Its Enzyme Function and Autoantigenicity.

PubMed

Le, Sarah N; Porebski, Benjamin T; McCoey, Julia; Fodor, James; Riley, Blake; Godlewska, Marlena; Góra, Monika; Czarnocka, Barbara; Banga, J Paul; Hoke, David E; Kass, Itamar; Buckle, Ashley M

2015-01-01

Thyroid peroxidase (TPO) catalyses the biosynthesis of thyroid hormones and is a major autoantigen in Hashimoto's disease--the most common organ-specific autoimmune disease. Epitope mapping studies have shown that the autoimmune response to TPO is directed mainly at two surface regions on the molecule: immunodominant regions A and B (IDR-A, and IDR-B). TPO has been a major target for structural studies for over 20 years; however, to date, the structure of TPO remains to be determined. We have used a molecular modelling approach to investigate plausible modes of TPO structure and dimer organisation. Sequence features of the C-terminus are consistent with a coiled-coil dimerization motif that most likely anchors the TPO dimer in the apical membrane of thyroid follicular cells. Two contrasting models of TPO were produced, differing in the orientation and exposure of their active sites relative to the membrane. Both models are equally plausible based upon the known enzymatic function of TPO. The "trans" model places IDR-B on the membrane-facing side of the myeloperoxidase (MPO)-like domain, potentially hindering access of autoantibodies, necessitating considerable conformational change, and perhaps even dissociation of the dimer into monomers. IDR-A spans MPO- and CCP-like domains and is relatively fragmented compared to IDR-B, therefore most likely requiring domain rearrangements in order to coalesce into one compact epitope. Less epitope fragmentation and higher solvent accessibility of the "cis" model favours it slightly over the "trans" model. Here, IDR-B clusters towards the surface of the MPO-like domain facing the thyroid follicular lumen preventing steric hindrance of autoantibodies. However, conformational rearrangements may still be necessary to allow full engagement with autoantibodies, with IDR-B on both models being close to the dimer interface. Taken together, the modelling highlights the need to consider the oligomeric state of TPO, its conformational

7 CFR 51.3050 - U.S. No. 1.

Code of Federal Regulations, 2010 CFR

2010-01-01

... Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing Practices), DEPARTMENT OF AGRICULTURE REGULATIONS AND STANDARDS UNDER THE AGRICULTURAL MARKETING ACT OF 1946... Standards for Florida Avocados Grades § 51.3050 U.S. No. 1. “U.S. No. 1” consists of avocados of similar...

The 53-kDa proteolytic product of precursor starch-hydrolyzing enzyme of Aspergillus niger has Taka-amylase-like activity.

PubMed

Ravi-Kumar, K; Venkatesh, K S; Umesh-Kumar, S

2007-04-01

The 53-kDa amylase secreted by Aspergillus niger due to proteolytic processing of the precursor starch-hydrolyzing enzyme was resistant to acarbose, a potent alpha-glucosidase inhibitor. The enzyme production was induced when A. niger was grown in starch medium containing the inhibitor. Antibodies against the precursor enzyme cross-reacted with the 54-kDa Taka-amylase protein of A. oryzae. It resembled Taka-amylase in most of its properties and also hydrolyzed starch to maltose of alpha-anomeric configuration. However, it did not degrade maltotriose formed during the reaction and was not inhibited by zinc ions.

Subinhibitory Concentrations of Antimicrobial Agents Reduce the Uptake of Legionella pneumophila into Acanthamoeba castellanii and U937 Cells by Altering the Expression of Virulence-Associated Antigens

PubMed Central

Lück, P. Christian; Schmitt, Jürgen W.; Hengerer, Arne; Helbig, Jürgen H.

1998-01-01

We determined the MICs of ampicillin, ciprofloxacin, erythromycin, imipenem, and rifampin for two clinical isolates of Legionella pneumophila serogroup 1 by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay and by quantitative culture. To test the influence of subinhibitory concentrations (sub-MICs) of antimicrobial agents on Legionella uptake into Acanthamoeba castellanii and U937 macrophage-like cells, both strains were pretreated with 0.25 MICs of the antibiotics for 24 h. In comparison to that for the untreated control, subinhibitory concentrations of antibiotics significantly reduced Legionella uptake into the host cells. Measurement of the binding of monoclonal antibodies against several Legionella antigens by enzyme-linked immunoassays indicated that sub-MIC antibiotic treatment reduced the expression of the macrophage infectivity potentiator protein (Mip), the Hsp 60 protein, the outer membrane protein (OmpM), an as-yet-uncharacterized protein of 55 kDa, and a few lipopolysaccharide (LPS) epitopes. In contrast, the expression of some LPS epitopes recognized by monoclonal antibodies 8/5 and 30/4 as well as a 45-kDa protein, a 58-kDa protein, and the major outer membrane protein (OmpS) remained unaffected. PMID:9797218

7 CFR 51.1430 - U.S. No. 1 Halves.

Code of Federal Regulations, 2010 CFR

2010-01-01

... Standards for Grades of Shelled Pecans Grades § 51.1430 U.S. No. 1 Halves. “U.S. No. 1 Halves” consists of pecan half-kernels which meet the following requirements: (a) For quality: (1) Well dried; (2) Fairly...

Diagnostic potential of Fasciola gigantica-derived 14.5 kDa fatty acid binding protein in the immunodiagnosis of bubaline fascioliasis.

PubMed

Allam, G; Bauomy, I R; Hemyeda, Z M; Diab, T M; Sakran, T F

2013-06-01

The 14.5 kDa fatty acid binding protein (FABP) was isolated from the crude extract of adult Fasciola gigantica worms. Polyclonal anti-FABP IgG was generated in rabbits immunized with prepared FABP antigen. Sandwich enzyme-linked immunosorbent assay (ELISA) was applied to detect coproantigen in stools and circulating Fasciola antigen (CA) in sera of 126 water buffaloes by using purified and horseradish peroxidase (HRP)-conjugated anti-FABP IgG. Sandwich ELISA sensitivity was 96.97% and 94.95%; while specificity was 94.12% and 82.35% for coproantigen and CA detection, respectively. However, sensitivity and specificity of the Kato-Katz technique was 73.74% and 100%, respectively. The diagnostic efficacy of sandwich ELISA was 96.55% and 93.1% for coproantigen and CA detection, respectively. In contrast, the diagnostic efficacy of the Kato-Katz technique was 77.59%. In conclusion, these results demonstrate that the purified 14.5 kDa FABP provides a more suitable antigen for immunodiagnosis of early and current bubaline fascioliasis by using sandwich ELISA.

7 CFR 51.340 - U.S. No. 1.

Code of Federal Regulations, 2014 CFR

2014-01-01

..., CERTIFICATION, AND STANDARDS) United States Standards for Grades of Apples for Processing Grades § 51.340 U.S. No. 1. “U.S. No. 1” consists of apples of one variety, unless designated as mixed varieties, which... preparation for use will cause a loss of more than 5 percent, by weight, of the apple. ...

7 CFR 51.340 - U.S. No. 1.

Code of Federal Regulations, 2013 CFR

2013-01-01

..., CERTIFICATION, AND STANDARDS) United States Standards for Grades of Apples for Processing Grades § 51.340 U.S. No. 1. “U.S. No. 1” consists of apples of one variety, unless designated as mixed varieties, which... preparation for use will cause a loss of more than 5 percent, by weight, of the apple. ...

Sidechain biology and the immunogenicity of PDC-E2, the major autoantigen of primary biliary cirrhosis.

PubMed

Mao, Tin K; Davis, Paul A; Odin, Joseph A; Coppel, Ross L; Gershwin, M Eric

2004-12-01

The E2 component of mitochondrial pyruvate dehydrogenase complex (PDC-E2) is the immunodominant autoantigen of primary biliary cirrhosis. Whereas lipoylation of PDC-E2 is essential for enzymatic activity and predominates under normal conditions, other biochemical systems exist that also target the lysine residue, including acylation of fatty acids or xenobiotics and ubiquitinylation. More importantly, the immunogenicity can be affected by derivatization of the lysine residue, as the recognition of lipoylated PDC-E2 by patient autoantibodies is enhanced compared with octanoylated PDC-E2. Furthermore, our laboratory has shown that various xenobiotic modifications of a peptide representing the immunodominant region of PDC-E2 are immunoreactive against patient sera. The only purported regulatory system that prevents the accumulation of potentially autoreactive PDC-E2 is glutathionylation, in which the lysine-lipoic acid moiety is further modified with glutathione during apoptosis. Interestingly, this system is found in several cell lines, including HeLa, Jurkat, and Caco-2 cells, but not in cholangiocytes and salivary gland epithelial cells, both of which are targets for destruction in primary biliary cirrhosis. Hence, the failure of this or other regulatory system(s) may overwhelm the immune system with immunogenic PDC-E2 that can initiate the breakdown of tolerance in a genetically susceptible individual. In this review the authors survey the data available on the biochemical life of PDC-E2, with particular emphasis on the lysine residue and its known interactions with machinery involved in various posttranslational modifications.

Exploring the supersymmetric U(1 ) B -L×U(1 ) R model with dark matter, muon g - 2 , and Z' mass limits

NASA Astrophysics Data System (ADS)

Frank, Mariana; Özdal, Özer

2018-01-01

We study the low scale predictions of the supersymmetric standard model extended by U (1 )B -L×U (1 )R symmetry, obtained from S O (10 ) breaking via a left-right supersymmetric model, imposing universal boundary conditions. Two singlet Higgs fields are responsible for the radiative U (1 )B -L×U (1 )R symmetry breaking, and a singlet fermion S is introduced to generate neutrino masses through an inverse seesaw mechanism. The lightest neutralino or sneutrino emerge as dark matter candidates, with different low scale implications. We find that the composition of the neutralino lightest supersymmetric particle (LSP) changes considerably depending on the neutralino LSP mass, from roughly half U (1 )R bino, half minimal supersymmetric model (MSSM) bino, to a singlet higgsino, or completely dominated by the MSSM higgsino. The sneutrino LSP is statistically much less likely, and when it occurs it is a 50-50 mixture of right-handed sneutrino and the scalar S ˜. Most of the solutions consistent with the relic density constraint survive the XENON 1T exclusion curve for both LSP cases. We compare the two scenarios and investigate parameter space points and find consistency with the muon anomalous magnetic moment only at the edge of a 2 σ deviation from the measured value. However, we find that the sneutrino LSP solutions could be ruled out completely by the strict reinforcement of the recent Z' mass bounds. We finally discuss collider prospects for testing the model.

Accumulation of 52 kDa glycine rich protein in auxin-deprived strawberry fruits and its role in fruit growth. [Fragaria ananassa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reddy, A.S.N.; Poovaiah, B.W.

1987-04-01

Growth of strawberry (Fragaria ananassa Duch) receptacles can be stopped at any stage by deachening the fruits and can be resumed by exogenous application of auxin. In their earlier studies they demonstrated auxin regulated polypeptide changes at different stages of strawberry fruit development. Removal of achenes from fruits to deprive auxin resulted in the accumulation of 52 KDa polypeptide. This polypeptide is associated with cell wall and its concentration is increased in a time-dependent manner in auxin deprived receptacles. Incorporation studies with (/sup 35/S) methionine showed the promotion of labelling of 52 kDa polypeptide in the auxin-deprived receptacles within 12more » h after removal of the achenes. Amino acid analysis revealed that the 52 KDa polypeptide is rich in glycine. Their studies, with normal and mutant strawberry receptacles, indicate that the synthesis and accumulation of this glycine rich protein correlates with cessation of receptacle growth. These results suggest a role for the glycine rich protein in growth.« less

Faradaurate nanomolecules: a superstable plasmonic 76.3 kDa cluster.

PubMed

Dass, Amala

2011-12-07

Information on the emergence of the characteristic plasmonic optical properties of nanoscale noble-metal particles has been limited, due in part to the problem of preparing homogeneous material for ensemble measurements. Here, we report the identification, isolation, and mass spectrometric and optical characterization of a 76.3 kDa thiolate-protected gold nanoparticle. This giant molecule is far larger than any metal-cluster compound, those with direct metal-to-metal bonding, previously known as homogeneous molecular substances, and is the first to exhibit clear plasmonic properties. The observed plasmon emergence phenomena in nanomolecules are of great interest, and the availability of absolutely homogeneous and characterized samples is thus critical to establishing their origin. © 2011 American Chemical Society

Bombyx mori nucleopolyhedrovirus orf25 encodes a 30kDa late protein in the infection cycle.

PubMed

Wang, Haiyan; Chen, Keping; Guo, Zhongjian; Yao, Qin

2008-02-01

Bombyx mori nucleopolyhedrovirus (BmNPV) orf25 gene was characterized for the first time. The coding sequence of Bm25 was amplified and subcloned into the prokaryotic expression vector pGEX-4T-2 to produce glutathione S-transferase-tagged fusion protein in the BL21 (DE3) cells. The GST-Bm25 fusion protein was expressed efficiently after induction with IPTG. The purified fusion protein was used to immunize New Zealand white rabbits to prepare polyclonal antibody. Temporal expression analysis revealed a 30-kDa protein, which was detected beginning 24 hours post-infection using a polyclonal antibody against GST-Bm25 fusion protein. The transcript of Bm25 was detected by RT-PCR at 18-72 h p.i. In conclusion, the available data suggest that Bm25 encodes a 30kDa protein expressed in the late stage of infection cycle.

Characterization, efficacy, pharmacokinetics, and biodistribution of 5kDa mPEG modified tetrameric canine uricase variant.

PubMed

Zhang, Chun; Fan, Kai; Luo, Hua; Ma, Xuefeng; Liu, Riyong; Yang, Li; Hu, Chunlan; Chen, Zhenmin; Min, Zhiqiang; Wei, Dongzhi

2012-07-01

PEGylated uricase is a promising anti-gout drug, but the only commercially marketed 10kDa mPEG modified porcine-like uricase (Pegloticase) can only be used for intravenous infusion. In this study, tetrameric canine uricase variant was modified by covalent conjugation of all accessible ɛ amino sites of lysine residues with a smaller 5kDa mPEG (mPEG-UHC). The average modification degree and PEGylation homogeneity were evaluated. Approximately 9.4 5 kDa mPEG chains were coupled to each monomeric uricase and the main conjugates contained 7-11 mPEG chains per subunit. mPEG-UHC showed significantly therapeutic or preventive effect on uric acid nephropathy and acute urate arthritis based on three different animal models. The clearance rate from an intravenous injection of mPEG-UHC varied significantly between species, at 2.61 mL/h/kg for rats and 0.21 mL/h/kg for monkeys. The long elimination half-life of mPEG-UHC in non-human primate (191.48 h, intravenous injection) indicated the long-term effects in humans. Moreover, the acceptable bioavailability of mPEG-UHC after subcutaneous administration in monkeys (94.21%) suggested that subcutaneous injection may be regarded as a candidate administration route in clinical trails. Non-specific tissue distribution was observed after administration of (125)I-labeled mPEG-UHC in rats, and elimination by the kidneys into the urine is the primary excretion route. Copyright © 2012 Elsevier B.V. All rights reserved.

Production of the Main Celiac Disease Autoantigen by Transient Expression in Nicotiana benthamiana

PubMed Central

Marín Viegas, Vanesa S.; Acevedo, Gonzalo R.; Bayardo, Mariela P.; Chirdo, Fernando G.; Petruccelli, Silvana

2015-01-01

Celiac Disease (CD) is a gluten sensitive enteropathy that remains widely undiagnosed and implementation of massive screening tests is needed to reduce the long term complications associated to untreated CD. The main CD autoantigen, human tissue transglutaminase (TG2), is a challenge for the different expression systems available since its cross-linking activity affects cellular processes. Plant-based transient expression systems can be an alternative for the production of this protein. In this work, a transient expression system for the production of human TG2 in Nicotiana benthamiana leaves was optimized and reactivity of plant-produced TG2 in CD screening test was evaluated. First, a subcellular targeting strategy was tested. Cytosolic, secretory, endoplasmic reticulum (C-terminal SEKDEL fusion) and vacuolar (C-terminal KISIA fusion) TG2 versions were transiently expressed in leaves and recombinant protein yields were measured. ER-TG2 and vac-TG2 levels were 9- to 16-fold higher than their cytosolic and secretory counterparts. As second strategy, TG2 variants were co-expressed with a hydrophobic elastin-like polymer (ELP) construct encoding for 36 repeats of the pentapeptide VPGXG in which the guest residue X were V and F in ratio 8:1. Protein bodies (PB) were induced by the ELP, with a consequent two-fold-increase in accumulation of both ER-TG2 and vac-TG2. Subsequently, ER-TG2 and vac-TG2 were produced and purified using immobilized metal ion affinity chromatography. Plant purified ER-TG2 and vac-TG2 were recognized by three anti-TG2 monoclonal antibodies that bind different epitopes proving that plant-produced antigen has immunochemical characteristics similar to those of human TG2. Lastly, an ELISA was performed with sera of CD patients and healthy controls. Both vac-TG2 and ER-TG2 were positively recognized by IgA of CD patients while they were not recognized by serum from non-celiac controls. These results confirmed the usefulness of plant-produced TG2 to

26 CFR 1.679-1 - U.S. transferor treated as owner of foreign trust.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 26 Internal Revenue 8 2010-04-01 2010-04-01 false U.S. transferor treated as owner of foreign... (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES Grantors and Others Treated As Substantial Owners § 1.679-1 U.S. transferor treated as owner of foreign trust. (a) In general. A U.S. transferor who transfers...

7 CFR 51.1346 - U.S. No. 1.

Code of Federal Regulations, 2014 CFR

2014-01-01

... scald, hard end, black end, internal breakdown, decay, worms and worm holes, and from damage caused by..., CERTIFICATION, AND STANDARDS) United States Standards for Pears for Canning Grades § 51.1346 U.S. No. 1. “U.S...

7 CFR 51.1346 - U.S. No. 1.

Code of Federal Regulations, 2013 CFR

2013-01-01

... scald, hard end, black end, internal breakdown, decay, worms and worm holes, and from damage caused by..., CERTIFICATION, AND STANDARDS) United States Standards for Pears for Canning Grades § 51.1346 U.S. No. 1. “U.S...

MAP30 promotes apoptosis of U251 and U87 cells by suppressing the LGR5 and Wnt/β-catenin signaling pathway, and enhancing Smac expression

PubMed Central

Jiang, Yilin; Miao, Junjie; Wang, Dongliang; Zhou, Jingru; Liu, Bo; Jiao, Feng; Liang, Jiangfeng; Wang, Yangshuo; Fan, Cungang; Zhang, Qingjun

2018-01-01

Significant antitumor activity of Momordica anti-human immunodeficiency virus protein of 30 kDa (MAP30) purified from Momordica charantia has been the subject of previous research. However, the effective mechanism of MAP30 on malignant glioma cells has not yet been clarified. The aim of the present study was to investigate the effects and mechanism of MAP30 on U87 and U251 cell lines. A Cell Counting Kit-8 assay, wound healing assay and Transwell assay were used to detect the effects on U87 and U251 cells treated with different concentrations of MAP30 (0.5, 1, 2, 4, 8 and 16 µM) over different periods of time. Proliferation, migration and invasion of each cell line were markedly inhibited by MAP30 in a dose- and time-dependent manner. Flow cytometry and fluorescence staining demonstrated that apoptosis increased and the cell cycle was arrested in S-phase in the two investigated cell lines following MAP30 treatment. Western blot analysis demonstrated that leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5) expression and key proteins in the Wnt/β-catenin signaling pathway were apparently decreased, whereas second mitochondria-derived activator of caspase (Smac) protein expression significantly increased with MAP30 treatment in the same manner. These results suggest that MAP30 markedly induces apoptosis in U87 and U251 cell lines by suppressing LGR5 and the Wnt/β-catenin signaling pathway, and enhancing Smac expression in a dose- and time-dependent manner. PMID:29556310

Src homology 2-domain containing leukocyte-specific phosphoprotein of 76 kDa is mandatory for TCR-mediated inside-out signaling, but dispensable for CXCR4-mediated LFA-1 activation, adhesion, and migration of T cells.

PubMed

Horn, Jessica; Wang, Xiaoqian; Reichardt, Peter; Stradal, Theresia E; Warnecke, Nicole; Simeoni, Luca; Gunzer, Matthias; Yablonski, Deborah; Schraven, Burkhart; Kliche, Stefanie

2009-11-01

Engagement of the TCR or of chemokine receptors such as CXCR4 induces adhesion and migration of T cells via so-called inside-out signaling pathways. The molecular processes underlying inside-out signaling events are as yet not completely understood. In this study, we show that TCR- and CXCR4-mediated activation of integrins critically depends on the membrane recruitment of the adhesion- and degranulation-promoting adapter protein (ADAP)/Src kinase-associated phosphoprotein of 55 kDa (SKAP55)/Rap1-interacting adapter protein (RIAM)/Rap1 module. We further demonstrate that the Src homology 2 domain containing leukocyte-specific phosphoprotein of 76 kDa (SLP76) is crucial for TCR-mediated inside-out signaling and T cell/APC interaction. Besides facilitating membrane recruitment of ADAP, SKAP55, and RIAM, SLP76 regulates TCR-mediated inside-out signaling by controlling the activation of Rap1 as well as Rac-mediated actin polymerization. Surprisingly, however, SLP76 is not mandatory for CXCR4-mediated inside-out signaling. Indeed, both CXCR4-induced T cell adhesion and migration are not affected by loss of SLP76. Moreover, after CXCR4 stimulation, the ADAP/SKAP55/RIAM/Rap1 module is recruited to the plasma membrane independently of SLP76. Collectively, our data indicate a differential requirement for SLP76 in TCR- vs CXCR4-mediated inside-out signaling pathways regulating T cell adhesion and migration.

Tuned and non-Higgsable U(1)s in F-theory

DOE PAGES

Wang, Yi-Nan

2017-03-01

We study the tuning of U(1) gauge fields in F-theory models on a base of general dimension. We construct a formula that computes the change in Weierstrass moduli when such a U(1) is tuned, based on the Morrison-Park form of a Weierstrass model with an additional rational section. Using this formula, we propose the form of “minimal tuning” on any base, which corresponds to the case where the decrease in the number of Weierstrass moduli is minimal. Applying this result, we discover some universal features of bases with non-Higgsable U(1)s. Mathematically, a generic elliptic fibration over such a base hasmore » additional rational sections. Physically, this condition implies the existence of U(1) gauge group in the low-energy supergravity theory after compactification that cannot be Higgsed away. In particular, we show that the elliptic Calabi-Yau manifold over such a base has a small number of complex structure moduli. We also suggest that non-Higgsable U(1)s can never appear on any toric bases. Finally, we construct the first example of a threefold base with non-Higgsable U(1)s.« less

7 CFR 51.884 - U.S. No. 1 Table.

Code of Federal Regulations, 2013 CFR

2013-01-01

... Grades § 51.884 U.S. No. 1 Table. “U.S. No. 1 Table” consists of bunches of well developed grapes of one...) Mold; (2) Decay. (f) Berries not damaged by: (1) Any other cause. (g) Bunches not damaged by: (1) Shot...: Exclusive of shot berries and dried berries, 75 percent, by count, of the berries on each bunch shall have...

7 CFR 51.884 - U.S. No. 1 Table.

Code of Federal Regulations, 2014 CFR

2014-01-01

... Grades § 51.884 U.S. No. 1 Table. “U.S. No. 1 Table” consists of bunches of well developed grapes of one...) Mold; (2) Decay. (f) Berries not damaged by: (1) Any other cause. (g) Bunches not damaged by: (1) Shot...: Exclusive of shot berries and dried berries, 75 percent, by count, of the berries on each bunch shall have...

Properties of a U1 RNA enhancer-like sequence.

PubMed Central

Ciliberto, G; Palla, F; Tebb, G; Mattaj, I W; Philipson, L

1987-01-01

The properties of a X.laevis U1B snRNA gene enhancer have been studied by microinjection in Xenopus oocytes. The enhancer-like sequence, defined as a short DNA stretch that is able to activate transcription in an orientation independent manner, is interchangeable between different U snRNA genes. The enhancer sequence alone does not, however, efficiently activate transcription from an SV40 pol II promoter but regains its activity when combined with the U-gene specific proximal sequence element. DNase I protection experiments show that the X.laevis U1B enhancer can interact specifically with a nuclear factor present in mammalian cells. Images PMID:3031597

A novel 35 kDa frog liver acid metallophosphatase.

PubMed

Szalewicz, A; Radomska, B; Strzelczyk, B; Kubicz, A

1999-04-12

The lower molecular weight (35 kDa) acid phosphatase from the frog (Rana esculenta) liver is a glycometalloenzyme susceptible to activation by reducing agents and displaying tartrate and fluoride resistance. Metal chelators (EDTA, 1,10-phenanthroline) inactivate the enzyme reversibly in a time- and temperature-dependent manner. The apoenzyme is reactivated by divalent transition metal cations, i. e. cobalt, zinc, ferrous, manganese, cadmium and nickel to 130%, 75%, 63%, 62%, 55% and 34% of the original activity, respectively. Magnesium, calcium, cupric and ferric ions were shown to be ineffective in this process. Metal analysis by the emission spectrometry method (inductively coupled plasma-atomic emission spectrometry) revealed the presence of zinc, iron and magnesium. The time course of the apoenzyme reactivation, the stabilization effect and the relatively high resistance to oxidizing conditions indicate that the zinc ion is crucial for the enzyme activity. The presence of iron was additionally confirmed by the visible absorption spectrum of the enzyme with a shoulder at 417 nm and by the electron paramagnetic resonance line of high spin iron(III) with geff of 2.4. The active center containing only zinc or both zinc and iron ions is proposed. The frog liver lower molecular weight acid phosphatase is a novel metallophosphatase of lower vertebrate origin, distinct from the mammalian tartrate-resistant, purple acid phosphatases.

SU(3)_C× SU(2)_L× U(1)_Y( × U(1)_X ) as a symmetry of division algebraic ladder operators

NASA Astrophysics Data System (ADS)

Furey, C.

2018-05-01

We demonstrate a model which captures certain attractive features of SU(5) theory, while providing a possible escape from proton decay. In this paper we show how ladder operators arise from the division algebras R, C, H, and O. From the SU( n) symmetry of these ladder operators, we then demonstrate a model which has much structural similarity to Georgi and Glashow's SU(5) grand unified theory. However, in this case, the transitions leading to proton decay are expected to be blocked, given that they coincide with presumably forbidden transformations which would incorrectly mix distinct algebraic actions. As a result, we find that we are left with G_{sm} = SU(3)_C× SU(2)_L× U(1)_Y / Z_6. Finally, we point out that if U( n) ladder symmetries are used in place of SU( n), it may then be possible to find this same G_{sm}=SU(3)_C× SU(2)_L× U(1)_Y / Z_6, together with an extra U(1)_X symmetry, related to B-L.

A rationally designed peptide IA-2-P2 against type 1 diabetes in streptozotocin-induced diabetic mice.

PubMed

Shen, Lili; Lu, Shiping; Huang, Dongcheng; Li, Guoliang; Liu, Kunfeng; Cao, Rongyue; Zong, Li; Jin, Liang; Wu, Jie

2017-05-01

Recent studies have investigated the potential of type 1 diabetes mellitus-related autoantigens, such as heat shock protein 60, to induce immunological tolerance or to suppress the immune response. A functional 24-residue peptide derived from heat shock protein 60 (P277) has shown anti-type 1 diabetes mellitus potential in experimental animals and in clinical studies, but it also carries a potential atherogenic effect. In this study, we have modified P277 to retain an anti-type 1 diabetes mellitus effect and minimize the atherogenic potential by replacing the P277 B epitope with another diabetes-associated autoantigen, insulinoma antigen-2 (IA-2), to create the fusion peptide IA-2-P2. In streptozotocin-induced diabetic C57BL/6J mice, the IA-2-P2 peptide displayed similar anti-diabetic effects to the control P277 peptide. Also, the IA-2-P2 peptide did not show atherogenic activity in a rabbit model. Our findings indicate the potential of IA-2-P2 as a promising vaccine against type 1 diabetes mellitus.

CD4 T-cell autoreactivity to the mitochondrial autoantigen PDC-E2 in AMA-negative primary biliary cirrhosis.

PubMed

Shimoda, Shinji; Miyakawa, Hiroshi; Nakamura, Minoru; Ishibashi, Hiromi; Kikuchi, Kentaro; Kita, Hiroto; Niiro, Hiroaki; Arinobu, Youjirou; Ono, Nobuyuki; Mackay, Ian R; Gershwin, M Eric; Akashi, Koichi

2008-09-01

Approximately 5% of patients with primary biliary cirrhosis (PBC) lack characteristic anti-mitochondrial antibodies (AMA). Yet clinically AMA+ and AMA- patients are similar. Using both AMA+ and AMA- patients, we quantitated the frequency of autoreactive T cells that respond to the major CD4 T-cell epitope, PDC-E2 163-176, using limiting dilution assays and quantitation of IFN-gamma, IL-10 and IL-4. Further, based on data that both PBC patients and healthy subjects have CD4+ T cells that recognize PDC-E2 163-176 but with differing costimulation requirements, assays were performed using two different antigen-presenting cell (APC) systems: either autologous peripheral blood mononuclear cells (PBMC) or HLA DR53 transfected mouse fibroblast cell lines (L-DR53). When costimulation-incompetent L-DR53 were used as APCs, the PDC-E2 CD4 T-cell frequency and capacity for IFN-gamma production were equivalent in both AMA+ and AMA- patients but the frequencies of such cells were significantly lower in normals, with IL-10 production similar in all three groups. Thus, in PBC there is 'universal' autoreactive CD4+ T-cell immune responsiveness to the critical autoantigen, PDC-E2. These observations emphasize that the mitochondrial autoreactivity in PBC is a multi-lineage response and hence, AMA-negative PBC may be an anachronism that refers only to sera autoantibodies.

Role of Bruton's tyrosine kinase inhibitors in HIV-1-infected cells.

PubMed

Guendel, Irene; Iordanskiy, Sergey; Sampey, Gavin C; Van Duyne, Rachel; Calvert, Valerie; Petricoin, Emanuel; Saifuddin, Mohammed; Kehn-Hall, Kylene; Kashanchi, Fatah

2015-06-01

Many cellular cofactors have been documented to be critical for various stages of viral replication. Using high-throughput proteomic assays, we have previously identified Bruton's tyrosine kinase (BTK) as a host protein that was uniquely upregulated in the plasma membrane of human immunodeficiency virus (HIV-1)-infected T cells. Here, we have further characterized the BTK expression in HIV-1 infection and show that this cellular factor is specifically expressed in infected myeloid cells. Significant upregulation of the phosphorylated form of BTK was observed in infected cells. Using size exclusion chromatography, we found BTK to be virtually absent in the uninfected U937 cells; however, new BTK protein complexes were identified and distributed in both high molecular weight (∼600 kDa) and a small molecular weight complex (∼60-120 kDa) in the infected U1 cells. BTK levels were highest in cells either chronically expressing virus or induced/infected myeloid cells and that BTK translocated to the membrane following induction of the infected cells. BTK knockdown in HIV-1-infected cells using small interfering RNA (siRNA) resulted in selective death of infected, but not uninfected, cells. Using BTK-specific antibody and small-molecule inhibitors including LFM-A13 and a FDA-approved compound, ibrutinib (PCI-32765), we have found that HIV-1-infected cells are sensitive to apoptotic cell death and result in a decrease in virus production. Overall, our data suggests that HIV-1-infected cells are sensitive to treatments targeting BTK expressed in infected cells.

7 CFR 51.1145 - U.S. No. 1 Bronze.

Code of Federal Regulations, 2010 CFR

2010-01-01

...) Internal quality: Lots meeting the internal requirements for “U.S. Grade AA Juice (Double A)” or “U.S... 7 Agriculture 2 2010-01-01 2010-01-01 false U.S. No. 1 Bronze. 51.1145 Section 51.1145 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing...

7 CFR 51.1142 - U.S. No. 1 Bright.

Code of Federal Regulations, 2010 CFR

2010-01-01

...) Internal quality: Lots meeting the internal requirements for “U.S. Grade AA Juice (Double A)” or “U.S... 7 Agriculture 2 2010-01-01 2010-01-01 false U.S. No. 1 Bright. 51.1142 Section 51.1142 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing...

Multiplex giant magnetoresistive biosensor microarrays identify interferon-associated autoantibodies in systemic lupus erythematosus

NASA Astrophysics Data System (ADS)

Lee, Jung-Rok; Haddon, D. James; Wand, Hannah E.; Price, Jordan V.; Diep, Vivian K.; Hall, Drew A.; Petri, Michelle; Baechler, Emily C.; Balboni, Imelda M.; Utz, Paul J.; Wang, Shan X.

2016-06-01

High titer, class-switched autoantibodies are a hallmark of systemic lupus erythematosus (SLE). Dysregulation of the interferon (IFN) pathway is observed in individuals with active SLE, although the association of specific autoantibodies with chemokine score, a combined measurement of three IFN-regulated chemokines, is not known. To identify autoantibodies associated with chemokine score, we developed giant magnetoresistive (GMR) biosensor microarrays, which allow the parallel measurement of multiple serum antibodies to autoantigens and peptides. We used the microarrays to analyze serum samples from SLE patients and found individuals with high chemokine scores had significantly greater reactivity to 13 autoantigens than individuals with low chemokine scores. Our findings demonstrate that multiple autoantibodies, including antibodies to U1-70K and modified histone H2B tails, are associated with IFN dysregulation in SLE. Further, they show the microarrays are capable of identifying autoantibodies associated with relevant clinical manifestations of SLE, with potential for use as biomarkers in clinical practice.

The fusion of Toxoplasma gondii SAG1 vaccine candidate to Leishmania infantum heat shock protein 83-kDa improves expression levels in tobacco chloroplasts.

PubMed

Albarracín, Romina M; Becher, Melina Laguía; Farran, Inmaculada; Sander, Valeria A; Corigliano, Mariana G; Yácono, María L; Pariani, Sebastián; López, Edwin Sánchez; Veramendi, Jon; Clemente, Marina

2015-05-01

Chloroplast transformation technology has emerged as an alternative platform offering many advantages over nuclear transformation. SAG1 is the main surface antigen of the intracellular parasite Toxoplasma gondii and a promising candidate to produce an anti-T. gondii vaccine. The aim of this study was to investigate the expression of SAG1 using chloroplast transformation technology in tobacco plants. In order to improve expression in transplastomic plants, we also expressed the 90-kDa heat shock protein of Leishmania infantum (LiHsp83) as a carrier for the SAG1 antigen. SAG1 protein accumulation in transplastomic plants was approximately 0.1-0.2 μg per gram of fresh weight (FW). Fusion of SAG1 to LiHsp83 significantly increased the level of SAG1 accumulation in tobacco chloroplasts (by up to 500-fold). We also evaluated the functionality of the chLiHsp83-SAG1. Three human seropositive samples reacted with SAG1 expressed in transplastomic chLiHsp83-SAG1 plants. Oral immunization with chLiHsp83-SAG1 elicited a significant reduction of the cyst burden that correlated with an increase of SAG1-specific antibodies. We propose the fusion of foreign proteins to LiHsp83 as a novel strategy to increase the expression level of the recombinant proteins using chloroplast transformation technology, thus addressing one of the current challenges for this approach in antigen protein production. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A novel biomarker associated with distress in humans: calcium-binding protein, spermatid-specific 1 (CABS1)

PubMed Central

Ritz, Thomas; Rosenfield, David; St. Laurent, Chris D.; Trueba, Ana F.; Werchan, Chelsey A.; Vogel, Pia D.; Auchus, Richard J.; Reyes-Serratos, Eduardo

2017-01-01

Calcium-binding protein spermatid-specific 1 (CABS1) is expressed in the human submandibular gland and has an anti-inflammatory motif similar to that in submandibular rat 1 in rats. Here, we investigate CABS1 in human saliva and its association with psychological and physiological distress and inflammation in humans. Volunteers participated across three studies: 1) weekly baseline measures; 2) a psychosocial speech and mental arithmetic stressor under evaluative threat; and 3) during academic exam stress. Salivary samples were analyzed for CABS1 and cortisol. Additional measures included questionnaires of perceived stress and negative affect; exhaled nitric oxide; respiration and cardiac activity; lung function; and salivary and nasal inflammatory markers. We identified a CABS1 immunoreactive band at 27 kDa in all participants and additional molecular mass forms in some participants. One week temporal stability of the 27-kDa band was satisfactory (test–retest reliability estimate = 0.62–0.86). Acute stress increased intensity of 18, 27, and 55 kDa bands; 27-kDa increases were associated with more negative affect and lower heart rate, sympathetic activity, respiration rate, and minute ventilation. In both acute and academic stress, changes in 27 kDa were positively associated with salivary cortisol. The 27-kDa band was also positively associated with VEGF and salivary leukotriene B4 levels. Participants with low molecular weight CABS1 bands showed reduced habitual stress and negative affect in response to acute stress. CABS1 is readily detected in human saliva and is associated with psychological and physiological indicators of stress. The role of CABS1 in inflammatory processes, stress, and stress resilience requires careful study. PMID:28381457

Origin of the U(1) field mass in superconductors

NASA Astrophysics Data System (ADS)

Koizumi, Hiroyasu

2017-05-01

Recently, a new theory for superconductivity has been put forward, in which the persistent current generation is attributed to the emergent singularities of the electronic wave function that are created by the spin-twisting itinerant circular motion of electrons. The persistent current generated by this mechanism behaves in every respect like supercurrent in superconductors, yielding the flux quantum h/2e and the Josephson frequency 2eV/h, where h is Planck’s constant, -e is the electron charge, and V is the voltage across the Josephson junction. The mass generation of the U(1) gauge field (or the Meissner effect) in the new theory is due to the emergence of topological objects, ‘instantons’ generated by the single-valued requirement of the wave function in the presence of the emergent singularities. The current standard theory of superconductivity is based on the BCS theory, and explains the emergence of superconductivity as due to the global U(1) gauge symmetry breaking realized by the Cooper pair formation. The U(1) field mass generation is believed to be due to this global U(1) gauge symmetry breaking. However, the feasibility of this mechanism has been questioned since no known interaction can prepare the global U(1) symmetry broken state from the normal state. We argue here that the U(1) mass generation in the BCS superconductor can be attributed to the one by the instanton mentioned above if the Rashba spin-orbit interaction is added. Then, the occurrence of persistent current generation becomes due to the instanton formation, and the role of the Cooper pair formation is to stabilize the instanton by providing an energy gap for perturbative excitations. Upon forming the Cooper pair, the instanton is stabilized and persistent current generation becomes possible. Thus, the superconducting transition temperature coincides with the Cooper pair formation temperature.

Electron-impact excitation of the BΣ1u+ and CΠ1u electronic states of H2

NASA Astrophysics Data System (ADS)

Kato, H.; Kawahara, H.; Hoshino, M.; Tanaka, H.; Campbell, L.; Brunger, M. J.

2008-06-01

Differential and integral cross sections for electron-impact excitation of the dipole-allowed BΣ1u+ and CΠ1u electronic states of molecular hydrogen have been measured. The differential cross sections were determined by analysis of normalized energy-loss spectra obtained using a crossed-beam apparatus at the electron-impact energies of 40, 100, and 200 eV. Integral cross sections were subsequently derived from these data. The present work was undertaken in order to investigate some ambiguities between earlier experimental data and recent BEf-scaled cross sections as defined and calculated by Kim [J. Chem. Phys. 126, 064305 (2007)] and also to extend the energy range of the available data. Optical oscillator strengths, also determined as a part of the present investigation, were found to be in fair accordance with previous measurements and some calculations.

Cooperative Interactions between 480 kDa Ankyrin-G and EB Proteins Assemble the Axon Initial Segment.

PubMed

Fréal, Amélie; Fassier, Coralie; Le Bras, Barbara; Bullier, Erika; De Gois, Stéphanie; Hazan, Jamilé; Hoogenraad, Casper C; Couraud, François

2016-04-20

The axon initial segment (AIS) is required for generating action potentials and maintaining neuronal polarity. Significant progress has been made in deciphering the basic building blocks composing the AIS, but the underlying mechanisms required for AIS formation remains unclear. The scaffolding protein ankyrin-G is the master-organizer of the AIS. Microtubules and their interactors, particularly end-binding proteins (EBs), have emerged as potential key players in AIS formation. Here, we show that the longest isoform of ankyrin-G (480AnkG) selectively associates with EBs via its specific tail domain and that this interaction is crucial for AIS formation and neuronal polarity in cultured rodent hippocampal neurons. EBs are essential for 480AnkG localization and stabilization at the AIS, whereas 480AnkG is required for the specific accumulation of EBs in the proximal axon. Our findings thus provide a conceptual framework for understanding how the cooperative relationship between 480AnkG and EBs induces the assembly of microtubule-AIS structures in the proximal axon. Neuronal polarity is crucial for the proper function of neurons. The assembly of the axon initial segment (AIS), which is the hallmark of early neuronal polarization, relies on the longest 480 kDa ankyrin-G isoform. The microtubule cytoskeleton and its interacting proteins were suggested to be early key players in the process of AIS formation. In this study, we show that the crosstalk between 480 kDa ankyrin-G and the microtubule plus-end tracking proteins, EBs, at the proximal axon is decisive for AIS assembly and neuronal polarity. Our work thus provides insight into the functional mechanisms used by 480 kDa ankyrin-G to drive the AIS formation and thereby to establish neuronal polarity. Copyright © 2016 the authors 0270-6474/16/364421-13$15.00/0.

Role of Bruton’s Tyrosine Kinase inhibitors in HIV-1 infected cells

PubMed Central

Guendel, Irene; Iordanskiy, Sergey; Sampey, Gavin C; Van Duyne, Rachel; Calvert, Valerie; Petricoin, Emanuel; Saifuddin, Mohammed; Kehn-Hall, Kylene; Kashanchi, Fatah

2015-01-01

Many cellular cofactors have been documented to be critical for various stages of viral replication. Using high throughput proteomic assays, we have previously identified Bruton’s tyrosine kinase (BTK) as a host protein that was uniquely up-regulated in the plasma membrane of HIV-1 infected T-cells. Here, we have further characterized the BTK expression in HIV-1 infection and show that this cellular factor is specifically expressed in infected myeloid cells. Significant up-regulation of the phosphorylated form of BTK was observed in infected cells. Using size exclusion chromatography, we found BTK to be virtually absent in the uninfected U937 cells, however new BTK protein complexes were identified and distributed in both high molecular weight (~600 kDa) and a small molecular weight complex (~60–120 kDa) in the infected U1 cells. BTK levels were highest in cells either chronically expressing virus or induced/infected myeloid cells and that BTK translocated to the membrane following induction of the infected cells. BTK knockdown in HIV-1 infected cells using siRNA resulted in selective death of infected, but not uninfected, cells. Using BTK specific antibody and small molecule inhibitors including LFM-A13 and a FDA approved compound, Ibrutinib (PCI – 32765), we have found that HIV-1 infected cells are sensitive to apoptotic cell death and result in a decrease in virus production. Overall, our data suggests that HIV-1 infected cells are sensitive to treatments targeting BTK expressed in infected cells. PMID:25672887

Cloning and characterization of mouse extracellular-signal-regulated protein kinase 3 as a unique gene product of 100 kDa.

PubMed

Turgeon, B; Saba-El-Leil, M K; Meloche, S

2000-02-15

MAP (mitogen-activated protein) kinases are a family of serine/threonine kinases that have a pivotal role in signal transduction. Here we report the cloning and characterization of a mouse homologue of extracellular-signal-regulated protein kinase (ERK)3. The mouse Erk3 cDNA encodes a predicted protein of 720 residues, which displays 94% identity with human ERK3. Transcription and translation of this cDNA in vitro generates a 100 kDa protein similar to the human gene product ERK3. Immunoblot analysis with an antibody raised against a unique sequence of ERK3 also recognizes a 100 kDa protein in mouse tissues. A single transcript of Erk3 was detected in every adult mouse tissue examined, with the highest expression being found in the brain. Interestingly, expression of Erk3 mRNA is acutely regulated during mouse development, with a peak of expression observed at embryonic day 11. The mouse Erk3 gene was mapped to a single locus on central mouse chromosome 9, adjacent to the dilute mutation locus and in a region syntenic to human chromosome 15q21. Finally, we provide several lines of evidence to support the existence of a unique Erk3 gene product of 100 kDa in mammalian cells.

Platelet cytosolic 44-kDa protein is a substrate of cholera toxin-induced ADP-ribosylation and is not recognized by antisera against the. alpha. subunit of the stimulatory guanine nucleotide-binding regulatory protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Molina Y Vedia, L.M.; Reep, B.R.; Lapetina, E.G.

1988-08-01

ADP-ribosylation induced by cholera toxin and pertussis toxin was studied in particulate and cytosolic fractions of human platelets. Platelets were disrupted by a cycle of freezing and thawing in the presence of a hyposmotic buffer containing protease inhibitors. In both fractions, the A subunit of cholera toxin ADP-ribosylates two proteins with molecular masses of 42 and 44 kDa, whereas pertussis toxin ADP-ribosylates a 41-kDa polypeptide. Two antisera against the {alpha} subunit of the stimulatory guanine nucleotide-binding regulatory protein recognize only the 42-kDa polypeptide. Cholera toxin-induced ADP-ribosylation of the 42- and 44-kDa proteins is reduced by pretreatment of platelets with iloprost,more » a prostacyclin analog. The 44-kDa protein, which is substrate of cholera toxin, could be extracted completely from the membrane and recovered in the cytosolic fraction when the cells were disrupted by Dounce homogenization and the pellet was extensively washed. A 44-kDa protein can also be labeled with 8-azidoguanosine 5{prime}-({alpha}-{sup 32}P)triphosphate in the cytosol and membranes. These finding indicate that cholera and pertussis toxins produced covalent modifications of proteins present in particulate and cytosolic platelet fractions. Moreover, the 44-kDa protein might be an {alpha} subunit of a guanine nucleotide-binding regulatory protein that is not recognized by available antisera.« less

Cloning, expression, and functional analysis of molecular motor pilT and pilU genes of type IV pili in Acidithiobacillus ferrooxidans.

PubMed

Li, Yongquan; Huang, Shuangsheng; Zhang, Xiaosu; Huang, Tao; Li, Hongyu

2013-02-01

PilT is a hexameric ATPase required for type IV pili (Tfp) retraction in gram-negative bacterium. Retraction of Tfp mediates intimate attachment and motility on inorganic solid surfaces. We investigated the cloning and expression of pilT and pilU genes of Acidithiobacillus ferrooxidans strains ATCC 23270, and the results indicate that PilT and PilU contain the canonical conserved AIRNLIRE and GMQTXXXXLXXL motifs that are the characteristic motifs of the PilT protein family; PilT and PilU also contain the canonical nucleotide-binding motifs, named with Walker A box (GxxGxGKT/S) and Walker B box (hhhhDE), respectively. The pilT and pilU genes were expressed to produce 37.1- and 42.0-kDa proteins, respectively, and co-transcribed induced by 10 % mineral powder. However, ATPase activity of PilT was distinctly higher than those of PilU. These results indicated that the PilT protein was the real molecular motor of Tfp, while PilU could play a key role in the assembly, modification, and twitching motility of Tfp in A. ferrooxidans. However, PilT and PilU were nonetheless interrelated in the forming and function of the molecular motor of Tfp.

Annexin 1 Modulates Monocyte-Endothelial Cell Interaction In Vitro and Cell Migration In Vivo in the Human SCID Mouse Transplantation Model1

PubMed Central

Perretti, Mauro; Ingegnoli, Francesca; Wheller, Samantha K.; Blades, Mark C.; Solito, Egle; Pitzalis, Costantino

2015-01-01

The effect of the glucocorticoid inducible protein annexin 1 (ANXA1) on the process of monocytic cell migration was studied using transfected U937 cells expressing variable protein levels. An antisense (AS) (36.4AS; ~50% less ANXA1) and a sense (S) clone (15S; overexpressing the bioactive 24-kDa fragment) together with the empty plasmid CMV clone were obtained and compared with wild-type U937 cells in various models of cell migration in vitro and in vivo. 15S-transfected U937 cells displayed a reduced (50%) degree of trans-endothelial migration in response to stromal cell-derived factor-1α (CXC chemokine ligand 12 (CXCL12)). In addition, the inhibitory role of endogenous ANXA1 on U937 cell migration in vitro was confirmed by the potentiating effect of a neutralizing anti-ANXA1 serum. Importantly, overexpression of ANXA1 in clone 15S inhibited the extent of cell migration into rheumatoid synovial grafts transplanted into SCID mice. ANXA1 inhibitory effects were not due to modifications in adhesion molecule or CXCL12 receptor (CXCR4) expression as shown by the similar amounts of surface molecules found in transfected and wild-type U937 cells. Likewise, an equal chemotactic response to CXCL12 in vitro excluded an intrinsic defect in cell motility in clones 15S and 36.4AS. These data strongly support the notion that ANXA1 critically interferes with a leukocyte endothelial step essential for U937 cell, and possibly monocyte, transmigration both in vitro and in vivo. PMID:12165536

Inflection-point inflation in a hyper-charge oriented U ( 1 ) X model

DOE PAGES

Okada, Nobuchika; Okada, Satomi; Raut, Digesh

2017-03-31

Inflection-point inflation is an interesting possibility to realize a successful slow-roll inflation when inflation is driven by a single scalar field with its value during inflation below the Planck mass (ΦI≲M Pl). In order for a renormalization group (RG) improved effective λΦ 4 potential to develop an inflection-point, the running quartic coupling λ(Φ) must exhibit a minimum with an almost vanishing value in its RG evolution, namely λ(Φ I)≃0 and β λ(ΦI)≃0, where β λ is the beta-function of the quartic coupling. Here in this paper, we consider the inflection-point inflation in the context of the minimal gauged U(1) Xmore » extended Standard Model (SM), which is a generalization of the minimal U(1) B$-$L model, and is constructed as a linear combination of the SM U(1) Y and U(1) B$-$L gauge symmetries. We identify the U(1) X Higgs field with the inflaton field. For a successful inflection-point inflation to be consistent with the current cosmological observations, the mass ratios among the U(1) X gauge boson, the right-handed neutrinos and the U(1) X Higgs boson are fixed. Focusing on the case that the extra U(1) X gauge symmetry is mostly aligned along the SM U(1) Y direction, we investigate a consistency between the inflationary predictions and the latest LHC Run-2 results on the search for a narrow resonance with the di-lepton final state.« less

Inflection-point inflation in a hyper-charge oriented U ( 1 ) X model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Okada, Nobuchika; Okada, Satomi; Raut, Digesh

Inflection-point inflation is an interesting possibility to realize a successful slow-roll inflation when inflation is driven by a single scalar field with its value during inflation below the Planck mass (ΦI≲M Pl). In order for a renormalization group (RG) improved effective λΦ 4 potential to develop an inflection-point, the running quartic coupling λ(Φ) must exhibit a minimum with an almost vanishing value in its RG evolution, namely λ(Φ I)≃0 and β λ(ΦI)≃0, where β λ is the beta-function of the quartic coupling. Here in this paper, we consider the inflection-point inflation in the context of the minimal gauged U(1) Xmore » extended Standard Model (SM), which is a generalization of the minimal U(1) B$-$L model, and is constructed as a linear combination of the SM U(1) Y and U(1) B$-$L gauge symmetries. We identify the U(1) X Higgs field with the inflaton field. For a successful inflection-point inflation to be consistent with the current cosmological observations, the mass ratios among the U(1) X gauge boson, the right-handed neutrinos and the U(1) X Higgs boson are fixed. Focusing on the case that the extra U(1) X gauge symmetry is mostly aligned along the SM U(1) Y direction, we investigate a consistency between the inflationary predictions and the latest LHC Run-2 results on the search for a narrow resonance with the di-lepton final state.« less

Modified properties of a glycated and cross-linked soy protein isolate by transglutaminase and an oligochitosan of 5 kDa.

PubMed

Fu, Miao; Zhao, Xin-Huai

2017-01-01

Soy protein is an important protein ingredient for the food industry; however, its properties can be improved by enzymatic and chemical modifications. This study applied a new enzymatic glycation and cross-linking to modify soy protein isolate (SPI), using an oligochitosan of 5 kDa and transglutaminase. Properties of the obtained glycated and cross-linked SPI (GC-SPI) were unknown and thus assessed. GC-SPI contained glucosamine of 13.6 g kg -1 protein, but less reactable &bond;NH 2 than SPI (0.42 vs. 0.50 mol kg -1 protein). Infrared spectra and circular dichroism results showed that GC-SPI other than SPI and cross-linked SPI had more &bond;OH in molecules, and was more disordered in secondary structure. In comparison with SPI, GC-SPI showed enhanced water-binding capacity, could form aggregates with enlarged hydrodynamic radius (180.2 vs. 82.9 nm) and negative zeta-potential (-31.2 vs. -27.7 mV) in dispersion, but exhibited lower thermal stability (e.g. greater mass loss) upon heating at a temperature above 288 °C. GC-SPI also had lower in vitro proteolytic digestibility than SPI due to the protein cross-linking. Oligochitosan of 5 kDa and transglutaminase can be used to glycate and cross-link SPI. This approach is applicable to generate potential protein ingredient with good hydration and dispersive stabilisation. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

The Role of U2AF1 Mutations in the Pathogenesis of Myelodysplastic Syndromes

DTIC Science & Technology

2015-10-01

mutation, U2AF1(S34F), on hematopoiesis and pre-mRNA splicing in vivo, we created doxycycline-inducible U2AF1(WT) and U2AF1(S34F) transgenic mice...U2AF1(S34F) versus U2AF1(WT). Together, these results suggest that mutant U2AF1 expression contributes to the altered hematopoiesis and pre-mRNA...Spliceosome, Mouse Model, Hematopoiesis , RNA-seq, U2AF1 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME

Gauge U (1) dark symmetry and radiative light fermion masses

DOE PAGES

Kownacki, Corey; Ma, Ernest

2016-06-22

A gauge U (1) family symmetry is proposed, spanning the quarks and leptons as well as particles of the dark sector. The breaking of U (1) to Z(2) divides the two sectors and generates one-loop radiative masses for the first two families of quarks and leptons, as well as all three neutrinos. We study the phenomenological implications of this new connection between family symmetry and dark matter. In particular, a scalar or pseudoscalar particle associated with this U (1) breaking may be identified with the 750 GeV diphoton resonance recently observed at the Large Hadron Collider (LHC).

Release of carrot plasma membrane-associated phosphatidylinositol kinase by phospholipase A2 and activation by a 70 kDa protein.

PubMed

Gross, W; Yang, W; Boss, W F

1992-02-19

Plasma membranes were isolated from carrot (Daucus carota L.) cells grown in suspension culture and treated with phospholipase A2 from snake or bee venom for 10 min. As a result of this treatment, phosphatidylinositol kinase activity was recovered in the soluble fraction. There was no detectable diacylglycerol kinase or phosphatidylinositol monophosphate kinase activity released from the membranes after the phospholipase A2 treatment. Treating the plasma membranes with phospholipase C or D did not release PI kinase activity. The phospholipase A2-released PI kinase was activated over 2-fold by a heat stable, soluble 70 kDa protein. The partially purified 70 kDa activator increases the Vmax but does not affect the Km of the phospholipase A2-released PI kinase.

Over-expression of the gene (bglBC1) from Bacillus circulans encoding an endo-beta-(1-->3),(1-->4)-glucanase useful for the preparation of oligosaccharides from barley beta-glucan.

PubMed

Kim, Ki-Hoon; Kim, Yea-Oon; Ko, Bong-Sun; Youn, Hyun-Joo; Lee, Dong-Seok

2004-11-01

An endo-beta-(1-->3),(1-->4)-glucanase gene (bglBC1) from Bacillus circulans ATCC21367 was modified by substituting its native promoter with a strong promoter, BJ27X, to increase expression of the gene when cloned into B. subtilis RM125 and B. megaterium ATCC14945. A 771-bp endo-beta-(1-->3),(1-->4)-glucanase open reading frame was inserted into a new shuttle plasmid, pBLC771, by ligating the ORF and pBE1, the latter of which contained the strong promoter, BJ27X. B. subtilis , transformed with the recombinant plasmid pBLC771, produced an extracellular endo-beta-(1-->3),(1-->4)-glucanase that was 130 times (7176 mU ml(-1)) more active than that of the gene donor cells (55 mU ml(-1)), while the enzyme from the transformed B. megaterium was 7 times (378 mU ml(-1)) more active than that of the gene donor cells. M(r) of the enzyme was 28 kDa, with proteolytic processing of the enzyme being observed only in B. subtilis cells. The major products of water-soluble beta-glucan hydrolyzed by over-produced endo-beta-(1-->3),(1-->4)-glucanase were tri- and tetra-oligosaccharides which can be developed as useful products such as anti-hypercholesterolemic, anti-hypertriglyceridemic, and anti-hyperglycemic agents.

IgG and IgM anti-snRNP reactivity in sequentially obtained serum samples from patients with connective tissue diseases.

PubMed Central

Nyman, U; Lundberg, I; Hedfors, E; Wahren, M; Pettersson, I

1992-01-01

Sequentially obtained serum samples from 30 patients with connective tissue disease positive for antibody to ribonucleoprotein (RNP) were examined to determine the specificities of IgG and IgM antibodies to snRNP during the disease course using immunoblotting of nuclear extracts. The antibody patterns were correlated with disease activity. The patterns of antibody to snRNP of individual patients were mainly stable during the study but changes in levels of antibody to snRNP were seen corresponding to changes in clinical activity. These results indicate that increased reactivity of serum IgM antibodies against the B/B' proteins seems to precede a clinically evident exacerbation of disease whereas IgG antibody reactivity to the 70 K protein peaks at the time of a disease flare. Images PMID:1485812

Purification of a 6.5 kDa protease inhibitor from Amazon Inga umbratica seeds effective against serine proteases of the boll weevil Anthonomus grandis.

PubMed

Calderon, L A; Teles, R C L; Leite, J R S A; Franco, O L; Grossi-de-Sá, M F; Medrano, F J; Bloch, C; Freitas, S M

2005-08-01

A 6.5 kDa serine protease inhibitor was purified by anion-exchange chromatography from the crude extract of the Inga umbratica seeds, containing inhibitor isoforms ranging from 6.3 to 6.7 kDa and protease inhibitors of approximately 19 kDa. The purified protein was characterized as a potent inhibitor against trypsin and chymotrypsin and it was named I. umbratica trypsin and chymotrypsin inhibitor (IUTCI). MALDI-TOF spectra of the IUTCI, in the presence of DTT, showed six disulfide bonds content, suggesting that this inhibitor belongs to Bowman-Birk family. The circular dichroism spectroscopy indicates that IUTCI is predominantly formed by unordered and beta-sheet secondary structure. It was also characterized, by fluorescence spectroscopy, as a stable protein at range of pH from 5.0 to 7.0. Moreover, this inhibitor at concentration of 75 microM presented a remarkable inhibitory activity (60%) against digestive serine proteases from boll weevil Anthonomus grandis, an important economical cotton pest.

A 170kDa multi-domain cystatin of Fasciola gigantica is active in the male reproductive system.

PubMed

Geadkaew, Amornrat; Kosa, Nanthawat; Siricoon, Sinee; Grams, Suksiri Vichasri; Grams, Rudi

2014-09-01

Cystatins are functional as intra- and extracellular inhibitors of cysteine proteases and are expressed as single or multi-domain proteins. We have previously described two single domain type 1 cystatins in the trematode Fasciola gigantica that are released into the parasite's intestinal tract and exhibit inhibitory activity against endogenous and host cathepsin L and B proteases. In contrast, the here presented 170kDa multi-domain cystatin (FgMDC) comprises signal peptide and 12 tandem repeated cystatin-like domains with similarity to type 2 single domain cystatins. The domains show high sequence divergence with identity values often <20% and at only 26.8% between the highest matching domains 6 and 10. Several domains contain degenerated QVVAG core motifs and/or lack other important residues of active type 2 cystatins. Domain-specific antisera detected multiple forms of FgMDC ranging from <10 to >120kDa molecular mass in immunoblots of parasite crude extracts and ES product with different banding patterns for each antiserum demonstrating complex processing of the proprotein. The four domains with the highest conserved QVVAG motifs were expressed in Escherichia coli and the refolded recombinant proteins blocked cysteine protease activity in the parasite's ES product. Strikingly, immunohistochemical analysis using seven domain-specific antisera localized FgMDC in testis lobes and sperm. It is speculated that the processed cystatin-like domains have function analogous to the mammalian group of male reproductive tissue-specific type 2 cystatins and are functional in spermiogenesis and fertilization. Copyright © 2014 Elsevier B.V. All rights reserved.

Vibrational wave packets in the B 1Πu and D 1Σu+ states of Cs2: Determination of improved Cs2+(X) and Cs2(B) spectroscopic constants

NASA Astrophysics Data System (ADS)

Oldenburg, A. L.; John, P. C.; Eden, J. G.

2000-12-01

Vibrational wave packets in the B 1Πu and D 1Σu+ excited states of Cs2 have been studied on the ˜100 fs time scale by pump-probe laser spectroscopy. The temporal behavior of the wave packets was monitored by photoionizing the electronically excited molecule with a time-delayed probe pulse and recording the time and energy-integrated photoelectron signal as a function of time delay between the pump and probe pulses. For the B 1Σu+ experiments, wave packets were produced by exciting the B 1Σu+←X 1Σg+ transition in the ˜740-790 nm region and subsequently detected by photoionizing the molecule at wavelengths between 565 nm and 600 nm. By simulating the experimentally observed transients with the density matrix formalism (and explicitly accounting for laser chirp and |Δv|>1 coherences), improved values for the equilibrium internuclear separation for the Cs2(B1Πu) state and Te for the Cs2+(X) state were determined to be Re(B 1Πu)=4.93±0.03 Å and Te[Cs2+(X)]=29 930±100 cm-1, respectively. Similar experiments were conducted for the D 1Σu+ state. Wave packets composed of vibrational levels (v'≈40-50) perturbed by the bound 2 3Πou state were produced on the D 1Σu+ potential surface by driving the D 1Σu+←X 1Σg+ transition in the 575-610 nm spectral interval.

A first detection of singlet to triplet conversion from the 1 1B u- to the 1 3A g state and triplet internal conversion from the 1 3A g to the 1 3B u state in carotenoids: dependence on the conjugation length

NASA Astrophysics Data System (ADS)

Rondonuwu, Ferdy S.; Watanabe, Yasutaka; Fujii, Ritsuko; Koyama, Yasushi

2003-07-01

Subpicosecond time-resolved absorption spectra were recorded in the visible region for a set of photosynthetic carotenoids having different numbers of conjugated double bonds ( n), which include neurosporene ( n=9), spheroidene ( n=10), lycopene ( n=11), anhydrorhodovibrin ( n=12) and spirilloxanthin ( n=13). Singular-value decomposition and global fitting of the spectral-data matrices lead us to a branched relaxation scheme including both (1) the singlet internal conversion in the sequence of 1 1B u+ → 1 1B u- → 2 1A g- → 1 1A g-(ground), and (2) the singlet-to-triplet conversion of 1 1B u- → 1 3A g followed by triplet internal conversion of 1 3A g → 1 3B u.

In Vitro Assembly of Human H/ACA Small Nucleolar RNPs Reveals Unique Features of U17 and Telomerase RNAs

PubMed Central

Dragon, François; Pogačić, Vanda; Filipowicz, Witold

2000-01-01

The H/ACA small nucleolar RNAs (snoRNAs) are involved in pseudouridylation of pre-rRNAs. They usually fold into a two-domain hairpin-hinge-hairpin-tail structure, with the conserved motifs H and ACA located in the hinge and tail, respectively. Synthetic RNA transcripts and extracts from HeLa cells were used to reconstitute human U17 and other H/ACA ribonucleoproteins (RNPs) in vitro. Competition and UV cross-linking experiments showed that proteins of about 60, 29, 23, and 14 kDa interact specifically with U17 RNA. Except for U17, RNPs could be reconstituted only with full-length H/ACA snoRNAs. For U17, the 3′-terminal stem-loop followed by box ACA (U17/3′st) was sufficient to form an RNP, and U17/3′st could compete other full-length H/ACA snoRNAs for assembly. The H/ACA-like domain that constitutes the 3′ moiety of human telomerase RNA (hTR), and its 3′-terminal stem-loop (hTR/3′st), also could form an RNP by binding H/ACA proteins. Hence, the 3′-terminal stem-loops of U17 and hTR have some specific features that distinguish them from other H/ACA RNAs. Antibodies that specifically recognize the human GAR1 (hGAR1) protein could immunoprecipitate H/ACA snoRNAs and hTR from HeLa cell extracts, which demonstrates that hGAR1 is a component of H/ACA snoRNPs and telomerase in vivo. Moreover, we show that in vitro-reconstituted RNPs contain hGAR1 and that binding of hGAR1 does not appear to be a prerequisite for the assembly of the other H/ACA proteins. PMID:10757788

Purification and Immunobiochemical Characterization of a 31 kDa Cross-Reactive Allergen from Phaseolus vulgaris (Kidney Bean)

PubMed Central

Kasera, Ramkrashan; Singh, Anand Bahadur; Lavasa, Shakuntala; Nagendra, Komarla; Arora, Naveen

2013-01-01

Background Legumes are a rich source of proteins but are also potential elicitors of IgE-mediated food allergy. This study aimed to isolate and characterize a major allergen of Phaseolus vulgaris (kidney bean) and determine its allergenicity. Methodology Kidney bean allergen was purified using Q Sepharose column (anion exchanger) and eluates with high intensity were pooled to purify protein using Superdex 75 (gel filtration) and C18 column (RP-HPLC). Patients with history of kidney bean allergy were skin prick tested (SPT) with crude kidney bean extract and the purified protein. Specific IgE was estimated in sera by enzyme-linked immunosorbent assay (ELISA). Characterization of purified protein and its cross-reactivity was investigated by immunobiochemical methods. Identification of purified protein was carried out by tandem mass spectrometry. Principal Findings Purified protein appeared as a single band at 31 kDa on SDS-PAGE and showed IgE binding to 88% patients’ sera by ELISA and immunoblotting. SPT with purified protein identified 78% hypersensitive patients of kidney bean. Significant release of histamine from sensitized basophils was observed after challenge with purified protein. PAS staining suggested it to be a glycoprotein, but no change in IgE binding was observed after periodate oxidation. The 31 kDa protein remained stable for 60 min on incubation with pepsin. The purified protein had high allergenic potential since it required only 102 ng of self protein for 50% IgE inhibition. Mass spectrometric analysis identified it as Phytohemagglutinin. It also showed hemagglutination with human RBCs. Cross-reactivity was observed with peanut and black gram with IC50 of 185 and 228 ng respectively. Conclusion/Significance A 31 kDa major allergen of kidney bean was purified and identified as phytohemagglutinin with cross-reactivity to peanut and black gram. PMID:23671655

Conjugation of 10 kDa Linear PEG onto Trastuzumab Fab' Is Sufficient to Significantly Enhance Lymphatic Exposure while Preserving in Vitro Biological Activity.

PubMed

Chan, Linda J; Ascher, David B; Yadav, Rajbharan; Bulitta, Jürgen B; Williams, Charlotte C; Porter, Christopher J H; Landersdorfer, Cornelia B; Kaminskas, Lisa M

2016-04-04

The lymphatic system is a major conduit by which many diseases spread and proliferate. There is therefore increasing interest in promoting better lymphatic drug targeting. Further, antibody fragments such as Fabs have several advantages over full length monoclonal antibodies but are subject to rapid plasma clearance, which can limit the lymphatic exposure and activity of Fabs against lymph-resident diseases. This study therefore explored ideal PEGylation strategies to maximize biological activity and lymphatic exposure using trastuzumab Fab' as a model. Specifically, the Fab' was conjugated with single linear 10 or 40 kDa PEG chains at the hinge region. PEGylation led to a 3-4-fold reduction in binding affinity to HER2, but antiproliferative activity against HER2-expressing BT474 cells was preserved. Lymphatic pharmacokinetics were then examined in thoracic lymph duct cannulated rats after intravenous and subcutaneous dosing at 2 mg/kg, and the data were evaluated via population pharmacokinetic modeling. The Fab' displayed limited lymphatic exposure, but conjugation of 10 kDa PEG improved exposure by approximately 11- and 5-fold after intravenous (15% dose collected in thoracic lymph over 30 h) and subcutaneous (9%) administration, respectively. Increasing the molecular weight of the PEG to 40 kDa, however, had no significant impact on lymphatic exposure after intravenous (14%) administration and only doubled lymphatic exposure after subcutaneous administration (18%) when compared to 10 kDa PEG-Fab'. The data therefore suggests that minimal PEGylation has the potential to enhance the exposure and activity of Fab's against lymph-resident diseases, while no significant benefit is achieved with very large PEGs.

Type 1 diabetes vaccine candidates promote human Foxp3+Treg induction in humanized mice

PubMed Central

Serr, Isabelle; Fürst, Rainer W.; Achenbach, Peter; Scherm, Martin G.; Gökmen, Füsun; Haupt, Florian; Sedlmeier, Eva-Maria; Knopff, Annette; Shultz, Leonard; Willis, Richard A.; Ziegler, Anette-Gabriele; Daniel, Carolin

2016-01-01

Immune tolerance is executed partly by Foxp3+regulatory T (Treg) cells, which suppress autoreactive T cells. In autoimmune type 1 diabetes (T1D) impaired tolerance promotes destruction of insulin-producing β-cells. The development of autoantigen-specific vaccination strategies for Foxp3+Treg-induction and prevention of islet autoimmunity in patients is still in its infancy. Here, using human haematopoietic stem cell-engrafted NSG-HLA-DQ8 transgenic mice, we provide direct evidence for human autoantigen-specific Foxp3+Treg-induction in vivo. We identify HLA-DQ8-restricted insulin-specific CD4+T cells and demonstrate efficient human insulin-specific Foxp3+Treg-induction upon subimmunogenic vaccination with strong agonistic insulin mimetopes in vivo. Induced human Tregs are stable, show increased expression of Treg signature genes such as Foxp3, CTLA4, IL-2Rα and TIGIT and can efficiently suppress effector T cells. Such Foxp3+Treg-induction does not trigger any effector T cells. These T1D vaccine candidates could therefore represent an expedient improvement in the challenge to induce human Foxp3+Tregs and to develop novel precision medicines for prevention of islet autoimmunity in children at risk of T1D. PMID:26975663

Hidden gauged U (1 ) model: Unifying scotogenic neutrino and flavor dark matter

NASA Astrophysics Data System (ADS)

Yu, Jiang-Hao

2016-06-01

In both scotogenic neutrino and flavor dark matter models, the dark sector communicates with the standard model fermions via Yukawa portal couplings. We propose an economic scenario where the scotogenic neutrino and a flavored mediator share the same inert Higgs doublet and all are charged under a hidden gauged U (1 ) symmetry. The dark Z2 symmetry in the dark sector is regarded as the remnant of this hidden U (1 ) symmetry breaking. In particular, we investigate a dark U (1 )D [and also U (1 )B-L] model which unifies the scotogenic neutrino and top-flavored mediator. Thus dark tops and dark neutrinos are the standard model fermion partners, and the dark matter could be the inert Higgs or the lightest dark neutrino. We note that this model has rich collider signatures on dark tops, the inert Higgs and the Z' gauge boson. Moreover, the scalar associated to the U (1 )D [and also U (1 )B -L ] symmetry breaking could explain the 750 GeV diphoton excess reported by ATLAS and CMS recently.

Experimental measurements of U60 nanocluster stability in aqueous solution

NASA Astrophysics Data System (ADS)

Flynn, Shannon L.; Szymanowski, Jennifer E. S.; Gao, Yunyi; Liu, Tianbo; Burns, Peter C.; Fein, Jeremy B.

2015-05-01

In this study, the aqueous behavior of isolated U60 nanoclusters (K16Li25[UO2(O2)OH]60)-19 was studied under several pH conditions and nanocluster concentrations to determine if the nanoclusters exhibit solid phase buffering behavior or if they exhibit behavior more like aqueous complexes. U60 is a cage cluster consisting of 60 (UO2)(O2)2(OH)2 uranyl polyhedral which share OH and O2 groups with their neighboring uranyl polyhedral, resulting in negatively charged cage clusters whose charge is at least partially offset by K+ and Li+ in the aqueous phase. Batch experiments to monitor nanocluster stability were conducted for 16 days at pH 7.5, 8.0 and 8.5 at nanocluster suspension concentrations of 1.4, 2.8 and 6.0 g/L. The aqueous concentrations of U, Li, and K, determined after 10 kDa molecular weight filtration, achieved steady-state with the nanoclusters within 24 h. The steady-state aqueous U, Li, and K concentrations were independent of solution pH, however they increased with increasing nanocluster concentration, indicating that the nanoclusters do not buffer the aqueous activities as a bulk solid phase would, but exhibit behavior that is more characteristic of dissolved aqueous complexes. The ion activity product (I.A.P.) value was calculated using two approaches: (1) treating the nanoclusters as a solid phase with an activity of one, and (2) treating the nanoclusters as aqueous complexes with a non-unit activity equal to their concentration in solution. The I.A.P. values that were calculated with non-unit activity for the nanoclusters exhibited significantly less variation as a function of nanocluster concentration compared to the I.A.P. values calculated with a nanocluster activity of one. The results yield a calculated log dissociation constant for the U60 nanoclusters of 9.2 + 0.2/-0.3 (1σ). Our findings provide a better understanding of the thermodynamic stability and behavior of U60 nanoclusters in aqueous systems, and can be used to estimate the

7 CFR 51.1146 - U.S. No. 1 Russet.

Code of Federal Regulations, 2010 CFR

2010-01-01

.... (a) For tolerances see § 51.1151. (b) Internal quality: Lots meeting the internal requirements for “U... 7 Agriculture 2 2010-01-01 2010-01-01 false U.S. No. 1 Russet. 51.1146 Section 51.1146 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing...

7 CFR 51.1144 - U.S. No. 1 Golden.

Code of Federal Regulations, 2010 CFR

2010-01-01

...) For tolerances see § 51.1151. (b) Internal quality: Lots meeting the internal requirements for “U.S... 7 Agriculture 2 2010-01-01 2010-01-01 false U.S. No. 1 Golden. 51.1144 Section 51.1144 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing...

Differential Cross Sections for the Electron Impact Excitation of the A(sup 3)(Sigma)(sub u)(sup +), B(sup 3)Pi(sub g), W(sup 3)(Delta)(sub u), B'(sup 3)(Sigma)(sub u)(sup -), a'(sup 1)Sigma(sub u)(sup -), a(sup 1)Pi(sub g), w(sup 1)Delta(sub u), and C(sup 3)Pi(sub u) States of N(sub 2)

NASA Technical Reports Server (NTRS)

Khakoo, M. A.; Johnson, P. V.; Ozkay, I.; Yan, P.; Trajmar, S.; Kanik, I.

2005-01-01

Measurements of differential cross sections for the electron-impact excitation of molecular nitrogen from the ground X(sup 1)(Sigma)(sub g)(sup +)(v''=0)level to the A(sup 3)(Sigma)(sub u)(sup +)(v'), B(sup 3)Pi(sub g)(v'), W(sup 3)(Delta)(sub u)(v'),B'(sup 3)(Sigma)(sub u)(sup -)(v'), a(sup 1)(Pi)(sub g)(v'), w(sup 1)(Delta)(sub u)(v'), and C(sup 3)(Pi)(sub u)(v') levels are presented. The data are obtained at the incident energies of 10, 12.5, 15, 17.5, 20, 30, 50, and 100 eV over the angular range of 5(deg)-130(deg) in 5(deg) intervals. The individual electronic state excitation differential cross sections are obtained by unfolding electron energy-loss spectra of molecular nitrogen using available semiempirical Frank-Condon factors. The data are compared to previous measurements and to available theory. We also make several important suggestions regarding future work that, like the present, relies on the unfolding of electron energy-loss spectra for obtaining differential cross sections.

Oral delivery of Brucella spp. recombinant protein U-Omp16 abrogates the IgE-mediated milk allergy.

PubMed

Smaldini, Paola Lorena; Ibañez, Andrés Esteban; Fossati, Carlos Alberto; Cassataro, Juliana; Docena, Guillermo Horacio

2014-01-01

Food allergies are increasingly common disorders and no therapeutic strategies are yet approved. The unlipidated Omp16 (U-Omp16) is the outer membrane protein of 16 kDa from B. abortus and possesses a mucosal adjuvant property. In this study, we aimed to examine the U-Omp16 capacity to abrogate an allergen-specific Th2 immune response when it is administered as an oral adjuvant in a mouse model of food allergy.   Balb/c mice were sensitized with cholera toxin and cow's milk proteins (CMP) by gavage and simultaneously treated with U-Omp16 and CMP. Oral challenge with CMP was performed to evaluate the allergic status of mice. Symptoms, local (small bowel cytokine and transcription factor gene expression) and systemic (specific isotypes and spleen cell-secreted cytokines) parameters, and skin tests were done to evaluate the immune response. We found that the oral administration of U-Omp16 with CMP during sensitization dampened the allergic symptoms, with negativization of immediate skin test and increased skin DTH response. Serum specific IgE and IL-5 were inhibited and a Th1 response was promoted (specific IgG2a antibodies and CMP-induced IFN-γ secretion). We found at the mucosal site an inhibition of the gene expression corresponding to IL-13 and Gata-3, with an induction of IFN-γ and T-bet. These results indicated that the oral administration of U-Omp16 significantly controlled the allergic response in sensitized mice with a shift of the balance of Th1- and Th2-T cells toward Th1 predominance. These findings suggest that U-Omp16 may be useful as a Th1-directing adjuvant in an oral vaccine.

Oral delivery of Brucella spp. recombinant protein U-Omp16 abrogates the IgE-mediated milk allergy

PubMed Central

Smaldini, Paola Lorena; Ibañez, Andrés Esteban; Fossati, Carlos Alberto; Cassataro, Juliana; Docena, Guillermo Horacio

2014-01-01

Food allergies are increasingly common disorders and no therapeutic strategies are yet approved. The unlipidated Omp16 (U-Omp16) is the outer membrane protein of 16 kDa from B. abortus and possesses a mucosal adjuvant property. In this study, we aimed to examine the U-Omp16 capacity to abrogate an allergen-specific Th2 immune response when it is administered as an oral adjuvant in a mouse model of food allergy.   Balb/c mice were sensitized with cholera toxin and cow’s milk proteins (CMP) by gavage and simultaneously treated with U-Omp16 and CMP. Oral challenge with CMP was performed to evaluate the allergic status of mice. Symptoms, local (small bowel cytokine and transcription factor gene expression) and systemic (specific isotypes and spleen cell-secreted cytokines) parameters, and skin tests were done to evaluate the immune response. We found that the oral administration of U-Omp16 with CMP during sensitization dampened the allergic symptoms, with negativization of immediate skin test and increased skin DTH response. Serum specific IgE and IL-5 were inhibited and a Th1 response was promoted (specific IgG2a antibodies and CMP-induced IFN-γ secretion). We found at the mucosal site an inhibition of the gene expression corresponding to IL-13 and Gata-3, with an induction of IFN-γ and T-bet. These results indicated that the oral administration of U-Omp16 significantly controlled the allergic response in sensitized mice with a shift of the balance of Th1- and Th2-T cells toward Th1 predominance. These findings suggest that U-Omp16 may be useful as a Th1-directing adjuvant in an oral vaccine. PMID:25424811

A complete backbone spectral assignment of human apolipoprotein AI on a 38 kDa preβHDL (Lp1-AI) particle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ren, Xuefeng; Yang, Yunhuang; Neville, T.

2007-06-12

Apolipoprotein A-I (apoAI, 243-residues) is the major protein component of the high-density lipoprotein (HDL) that has been a hot subject of interests because of its anti-atherogenic properties. This important property of apoAI is related to its roles in reverse cholesterol transport pathway. Upon lipid-binding, apoAI undergoes conformational changes from lipid-free to several different HDL-associated states (1). These different conformational states regulate HDL formation, maturation and transportation. Two initial conformational states of apoAI are lipid-free apoAI and apoAI/preβHDL that recruit phospholipids and cholesterol to form HDL particles. In particular, lipid-free apoAI specifically binds to phospholipids to form lipid-poor apoAI, including apoAI/preβ-HDLmore » (~37 kDa). As a unique class of lipid poor HDL, both in vitro and in vivo evidence demonstrates that apoAI/preβ-HDLs are the most effective acceptors specifically for free cholesterol in human plasma and serves as the precursor of HDL particles (2). Here we report a complete backbone spectral assignment of human apoAI/preβHDL. Secondary structure prediction using backbone NMR parameters indicates that apoAI/preβHDL displays a two-domain structure: the N-terminal four helix-bundle domain (residues 1-186) and the C-terminal flexible domain (residues 187-243). A structure of apoAI/preβ-HDL is the first lipid-associated structure of apoAI and is critical for us to understand how apoAI recruits cholesterol to initialize HDL formation. BMRB deposit with accession number: 15093.« less

The major autoantibody epitope on factor H in atypical hemolytic uremic syndrome is structurally different from its homologous site in factor H-related protein 1, supporting a novel model for induction of autoimmunity in this disease.

PubMed

Bhattacharjee, Arnab; Reuter, Stefanie; Trojnár, Eszter; Kolodziejczyk, Robert; Seeberger, Harald; Hyvärinen, Satu; Uzonyi, Barbara; Szilágyi, Ágnes; Prohászka, Zoltán; Goldman, Adrian; Józsi, Mihály; Jokiranta, T Sakari

2015-04-10

Atypical hemolytic uremic syndrome (aHUS) is characterized by complement attack against host cells due to mutations in complement proteins or autoantibodies against complement factor H (CFH). It is unknown why nearly all patients with autoimmune aHUS lack CFHR1 (CFH-related protein-1). These patients have autoantibodies against CFH domains 19 and 20 (CFH19-20), which are nearly identical to CFHR1 domains 4 and 5 (CFHR14-5). Here, binding site mapping of autoantibodies from 17 patients using mutant CFH19-20 constructs revealed an autoantibody epitope cluster within a loop on domain 20, next to the two buried residues that are different in CFH19-20 and CFHR14-5. The crystal structure of CFHR14-5 revealed a difference in conformation of the autoantigenic loop in the C-terminal domains of CFH and CFHR1, explaining the variation in binding of autoantibodies from some aHUS patients to CFH19-20 and CFHR14-5. The autoantigenic loop on CFH seems to be generally flexible, as its conformation in previously published structures of CFH19-20 bound to the microbial protein OspE and a sialic acid glycan is somewhat altered. Cumulatively, our data suggest that association of CFHR1 deficiency with autoimmune aHUS could be due to the structural difference between CFHR1 and the autoantigenic CFH epitope, suggesting a novel explanation for CFHR1 deficiency in the pathogenesis of autoimmune aHUS. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

U2AF1 mutations alter splice site recognition in hematological malignancies.

PubMed

Ilagan, Janine O; Ramakrishnan, Aravind; Hayes, Brian; Murphy, Michele E; Zebari, Ahmad S; Bradley, Philip; Bradley, Robert K

2015-01-01

Whole-exome sequencing studies have identified common mutations affecting genes encoding components of the RNA splicing machinery in hematological malignancies. Here, we sought to determine how mutations affecting the 3' splice site recognition factor U2AF1 alter its normal role in RNA splicing. We find that U2AF1 mutations influence the similarity of splicing programs in leukemias, but do not give rise to widespread splicing failure. U2AF1 mutations cause differential splicing of hundreds of genes, affecting biological pathways such as DNA methylation (DNMT3B), X chromosome inactivation (H2AFY), the DNA damage response (ATR, FANCA), and apoptosis (CASP8). We show that U2AF1 mutations alter the preferred 3' splice site motif in patients, in cell culture, and in vitro. Mutations affecting the first and second zinc fingers give rise to different alterations in splice site preference and largely distinct downstream splicing programs. These allele-specific effects are consistent with a computationally predicted model of U2AF1 in complex with RNA. Our findings suggest that U2AF1 mutations contribute to pathogenesis by causing quantitative changes in splicing that affect diverse cellular pathways, and give insight into the normal function of U2AF1's zinc finger domains. © 2015 Ilagan et al.; Published by Cold Spring Harbor Laboratory Press.

Cloning and expression of the Aspergillus oryzae glucan 1,3-beta-glucosidase A (exgA) in Pichia pastoris.

PubMed

Boonvitthya, Nassapat; Tanapong, Phatrapan; Kanngan, Patcharaporn; Burapatana, Vorakan; Chulalaksananukul, Warawut

2012-10-01

The glucan 1,3-beta-glucosidase A gene (exgA) from Aspergillus oryzae and fused to the Saccharomyces cerevisiae signal peptide (α-factor) was expressed under the control of either a constitutive (GAP) or an inducible (AOX1) promoter in Pichia pastoris. A 1.4-fold higher extracellular enzyme activity (2 U/ml) was obtained using the AOX1 inducible expression system than with the GAP constitutive promoter (1.4 U/ml). The purified recombinant ExgA enzyme, with a yield of 10 mg protein/l culture supernatant, was about 40 kDa by SDS-PAGE analysis with a specific activity of 289 U/mg protein. The enzyme was optimally active at 35 °C and pH 5.0 and displayed a K(M) and V(max) of 0.56 mM and 10,042 μmol/(min mg protein), respectively, with p-nitrophenyl-β-D-glucopyranoside as the substrate. Moreover, it was tolerant to glucose inhibition with a K(i) of 365 mM.

Antimicrobial activity of a 48-kDa protease (AMP48) from Artocarpus heterophyllus latex.

PubMed

Siritapetawee, J; Thammasirirak, S; Samosornsuk, W

2012-01-01

Artocarpus heterophyllus (jackfruit) is a latex producing plant. Plant latex is produced from secretory cells and contains many intergradients. It also has been used in folk medicine. This study aimed to purify and characterize the biological activities of a protease from jackfruit latex. A protease was isolated and purified from crude latex of a jackfruit tree by acid precipitation and ion exchange chromatography. The proteolytic activities of protein were tested using gelatin- and casein-zymography. The molecular weight and isoelectric point (pl) of protein were analysed by SDS/12.5% PAGE and 2D-PAGE, respectively. Antimicrobial activity of protein was analysed by broth microdilution method. In addition, the antibacterial activity of protein against Pseudomonas aeruginosa ATCC 27853 was observed and measured using atomic force microscopy (AFM) technique. The purified protein contained protease activity by digesting gelatin- and casein-substrates. The protease was designated as antimicrobial protease-48 kDa or AMP48 due to its molecular mass on SDS-PAGE was approximately 48 kDa. The isoelectric point (pl) of AMP48 was approximately 4.2. In addition, AMP48 contained antimicrobial activities by it could inhibit the growths of Pseudomonas aeruginosa ATCC 27853 and clinical isolated Candida albicans at minimum inhibitory concentration (MIC) 2.2 mg/ml and Minimum microbicidal concentration (MMC) 8.8 mg/ml. AFM image also supported the antimicrobial activities of AMP48 by the treated bacterial morphology and size were altered from normal.

Nonminimal quartic inflation in classically conformal U(1 ) X extended standard model

NASA Astrophysics Data System (ADS)

Oda, Satsuki; Okada, Nobuchika; Raut, Digesh; Takahashi, Dai-suke

2018-03-01

We propose quartic inflation with nonminimal gravitational coupling in the context of the classically conformal U(1 ) X extension of the standard model (SM). In this model, the U(1 ) X gauge symmetry is radiatively broken through the Coleman-Weinberg mechanism, by which the U(1 ) X gauge boson (Z' boson) and the right-handed Majorana neutrinos acquire their masses. We consider their masses in the range of O (10 GeV )-O (10 TeV ) , which are accessible to high-energy collider experiments. The radiative U(1 ) X gauge symmetry breaking also generates a negative mass squared for the SM Higgs doublet, and the electroweak symmetry breaking occurs subsequently. We identify the U(1 ) X Higgs field with inflaton and calculate the inflationary predictions. Because of the Coleman-Weinberg mechanism, the inflaton quartic coupling during inflation, which determines the inflationary predictions, is correlated to the U(1 ) X gauge coupling. With this correlation, we investigate complementarities between the inflationary predictions and the current constraint from the Z' boson resonance search at the LHC Run 2 as well as the prospect of the search for the Z' boson and the right-handed neutrinos at the future collider experiments.

A 21-35 kDa Mixed Protein Component from Helicobacter pylori Activates Mast Cells Effectively in Chronic Spontaneous Urticaria.

PubMed

Tan, Ran-Jing; Sun, He-Qiang; Zhang, Wei; Yuan, Han-Mei; Li, Bin; Yan, Hong-Tao; Lan, Chun-Hui; Yang, Jun; Zhao, Zhuo; Wu, Jin-Jin; Wu, Chao

2016-12-01

Helicobacter pylori (H. pylori) seem to involve in the etiology of chronic spontaneous urticaria (CSU). But studies of the pathogenic mechanism are very little. In this study, we detected the serum-specific anti-H. pylori IgG and IgE antibodies in 211 CSU and 137 normal subjects by enzyme-linked immunosorbent assay (ELISA), evaluated the direct activation effects of H. pylori preparations and its protein components on human LAD 2 mast cell line in vitro, and analyzed the specific protein ingredients and functions of the most effective H. pylori mixed protein component using liquid chromatography-mass spectrometry and ELISA assay. In CSU patients, the positive rate of anti-H. pylori IgG positive rate was significantly higher than that in normal controls, and the anti-H. pylori IgE levels had no statistical difference between H. pylori-infected patients with and without CSU. Further studies suggested that H. pylori preparations can directly activate human LAD 2 mast cell line in a dose-dependent manner and its most powerful protein component was a mixture of 21-35 kDa proteins. Moreover, the 21-35 kDa mixed protein component mainly contained 23 kinds of proteins, which can stimulate the release of histamine, TNF-a, IL-3, IFN-γ, and LTB4 by LAD 2 cells in a dose-dependent or time-dependent manner. A 21-35 kDa mixed protein component should be regarded as the most promising pathogenic factor contributing to the CSU associated with H. pylori infection. © 2016 John Wiley & Sons Ltd.

Genome-Wide Protein Interaction Screens Reveal Functional Networks Involving Sm-Like Proteins

PubMed Central

Fromont-Racine, Micheline; Mayes, Andrew E.; Brunet-Simon, Adeline; Rain, Jean-Christophe; Colley, Alan; Dix, Ian; Decourty, Laurence; Joly, Nicolas; Ricard, Florence; Beggs, Jean D.

2000-01-01

A set of seven structurally related Sm proteins forms the core of the snRNP particles containing the spliceosomal U1, U2, U4 and U5 snRNAs. A search of the genomic sequence of Saccharomyces cerevisiae has identified a number of open reading frames that potentially encode structurally similar proteins termed Lsm (Like Sm) proteins. With the aim of analysing all possible interactions between the Lsm proteins and any protein encoded in the yeast genome, we performed exhaustive and iterative genomic two-hybrid screens, starting with the Lsm proteins as baits. Indeed, extensive interactions amongst eight Lsm proteins were found that suggest the existence of a Lsm complex or complexes. These Lsm interactions apparently involve the conserved Sm domain that also mediates interactions between the Sm proteins. The screens also reveal functionally significant interactions with splicing factors, in particular with Prp4 and Prp24, compatible with genetic studies and with the reported association of Lsm proteins with spliceosomal U6 and U4/U6 particles. In addition, interactions with proteins involved in mRNA turnover, such as Mrt1, Dcp1, Dcp2 and Xrn1, point to roles for Lsm complexes in distinct RNA metabolic processes, that are confirmed in independent functional studies. These results provide compelling evidence that two-hybrid screens yield functionally meaningful information about protein–protein interactions and can suggest functions for uncharacterized proteins, especially when they are performed on a genome-wide scale. PMID:10900456

Defective minor spliceosome mRNA processing results in isolated familial growth hormone deficiency

PubMed Central

Argente, Jesús; Flores, Raquel; Gutiérrez-Arumí, Armand; Verma, Bhupendra; Martos-Moreno, Gabriel Á; Cuscó, Ivon; Oghabian, Ali; Chowen, Julie A; Frilander, Mikko J; Pérez-Jurado, Luis A

2014-01-01

The molecular basis of a significant number of cases of isolated growth hormone deficiency remains unknown. We describe three sisters affected with severe isolated growth hormone deficiency and pituitary hypoplasia caused by biallelic mutations in the RNPC3 gene, which codes for a minor spliceosome protein required for U11/U12 small nuclear ribonucleoprotein (snRNP) formation and splicing of U12-type introns. We found anomalies in U11/U12 di-snRNP formation and in splicing of multiple U12-type introns in patient cells. Defective transcripts include preprohormone convertases SPCS2 and SPCS3 and actin-related ARPC5L genes, which are candidates for the somatotroph-restricted dysfunction. The reported novel mechanism for familial growth hormone deficiency demonstrates that general mRNA processing defects of the minor spliceosome can lead to very narrow tissue-specific consequences. Subject Categories Genetics, Gene Therapy ' Genetic Disease; Metabolism PMID:24480542

A model with isospin doublet U(1)D gauge symmetry

NASA Astrophysics Data System (ADS)

Nomura, Takaaki; Okada, Hiroshi

2018-05-01

We propose a model with an extra isospin doublet U(1)D gauge symmetry, in which we introduce several extra fermions with odd parity under a discrete Z2 symmetry in order to cancel the gauge anomalies out. A remarkable issue is that we impose nonzero U(1)D charge to the Standard Model Higgs, and it gives the most stringent constraint to the vacuum expectation value of a scalar field breaking the U(1)D symmetry that is severer than the LEP bound. We then explore relic density of a Majorana dark matter candidate without conflict of constraints from lepton flavor violating processes. A global analysis is carried out to search for parameters which can accommodate with the observed data.

Differential cross sections for the electron impact excitation of the A {sup 3}{sigma}{sub u}{sup +}, B {sup 3}{pi}{sub g}, W {sup 3}{delta}{sub u}, B{sup '} {sup 3}{sigma}{sub u}{sup -}, a{sup '} {sup 1}{sigma}{sub u}{sup -}, a {sup 1}{pi}{sub g}, w {sup 1}{delta}{sub u}, and C {sup 3}{pi}{sub u} states of N{sub 2}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khakoo, M.A.; Ozkay, I.; Johnson, P.V.

2005-06-15

Measurements of differential cross sections for the electron-impact excitation of molecular nitrogen from the ground X {sup 1}{sigma}{sub g}{sup +}(v{sup ''}=0) level to the A {sup 3}{sigma}{sub u}{sup +}(v{sup '}), B {sup 3}{pi}{sub g}(v{sup '}), W {sup 3}{delta}{sub u}(v{sup '}), B{sup '} {sup 3}{sigma}{sub u}{sup -}(v{sup '}), a{sup '} {sup 1}{sigma}{sub u}{sup -}(v{sup '}), a {sup 1}{pi}{sub g}(v{sup '}), w {sup 1}{delta}{sub u}(v{sup '}), and C {sup 3}{pi}{sub u}(v{sup '}) levels are presented. The data are obtained at the incident energies of 10, 12.5, 15, 17.5, 20, 30, 50, and 100 eV over the angular range of 5 deg. -130more » deg. in 5 deg. intervals. The individual electronic state excitation differential cross sections are obtained by unfolding electron energy-loss spectra of molecular nitrogen using available semiempirical Frank-Condon factors. The data are compared to previous measurements and to available theory. We also make several important suggestions regarding future work that, like the present, relies on the unfolding of electron energy-loss spectra for obtaining differential cross sections.« less

Radioligand binding and functional characterization of recombinant human NmU1 and NmU2 receptors stably expressed in clonal human embryonic kidney-293 cells.

PubMed

Aiyar, Nambi; Disa, Jyoti; Foley, James J; Buckley, Peter T; Wixted, William E; Pullen, Mark; Shabon, Usman; Dul, Edward; Szekeres, Philip G; Elshourbagy, Nabil A; Sarau, Henry M; Appelbaum, Edward; Bolaky, Jane

2004-09-01

Neuromedin U (NmU) is a smooth muscle contracting peptide. Recently, two G-protein-coupled receptors for NmU (NmU1R and NmU2R) have been cloned having approximately 50% homology. They have distinct patterns of expression suggesting they may have different biological functions. This study provides a comprehensive characterization of both NmU receptors expressed in human embryonic kidney 293 cells. [125I]hNmU binding to the recombinant NmU receptors was rapid, saturable, of high affinity and to a single population of binding sites. Exposure of these cells to NmU isopeptides resulted in an increase in intracellular [Ca2+]i release (EC50 value of 0.50 +/- 0.10 nmol/l) and inositol phosphate formation (EC50 1.6 +/- 0.2 and 1.50 +/- 0.4 nmol/l for NmU1R and NmU2R respectively). Furthermore, hNmU inhibited forskolin (3 micromol/l)-stimulated accumulation of cAMP in intact HEK-293 cells expressing either NmU1R or NmU2R. The inhibitory effect was significant for the cells expressing NmU2R with IC50 value of 0.80 +/- 0.21 nmol/l. In summary, both NmU1R and NmU2R in HEK-293 cells have similar signaling capability. Copyright 2004 S. Karger AG, Basel

Correlation between phosphorylation level of a hippocampal 86kDa protein and extinction of a behaviour in a model of Wernicke-Korsakoff syndrome.

PubMed

Pires, Rita G W; Pereira, Sílvia R C; Carvalho, Fabiana M; Oliveira-Silva, Ieda F; Ferraz, Vany P; Ribeiro, Angela M

2007-06-04

The effects of chronic ethanol and thiamine deficiency, alone or associated, on hippocampal protein phosphorylation profiles ranging in molecular weight from 30 to 250kDa molecular weight, in stimulated (high K(+) concentration) and unstimulated (basal) conditions were investigated. These treatments significantly changed the phosphorylation level of an 86kDa phosphoprotein. Thiamine deficiency, but not chronic ethanol, induced a decrease in a behavioural extinction index, which is significantly correlated to the phosphorylation level of the p86 protein. These data add to and extend previous findings by our laboratory implicating the involvement of hippocampal neurotransmission components in extinction of a behaviour which involves learning of environmental spatial cues.

Two forms of Vibrio cholerae O1 El Tor hemolysin derived from identical precursor protein.

PubMed

Ikigai, H; Ono, T; Nakae, T; Otsuru, H; Shimamura, T

1999-01-08

Vibrio cholerae O1 grown in heart infusion broth produces two forms of El Tor hemolysin (ETH) monomers of 65 and 50 kDa. These monomers form several different sizes of mixed oligomers ranging from 180 to 280 kDa in the liposomal membranes. We found that the N-terminal amino acid sequences, NH2-Trp-Pro-Ala-Pro-Ala-Asn-Ser-Glu, of both the 65- and 50-kDa toxins were identical. We assumed, therefore, that the 65- and 50-kDa toxins were derivatives of the identical precursor protein and the 50-kDa protein was a truncated derivative of 65-kDa ETH. To substantiate this assumption, we treated the 260-kDa oligomer with trypsin and obtained a 190-kDa oligomer. This 190-kDa oligomer consisted of only the 50-kDa subunits. Both 260- and 190-kDa oligomers formed ion channels indistinguishable from each other in planar lipid bilayers. These results suggest that the essential part of the ETH in forming the membrane-damaging aggregate is a 50-kDa protein.

7 CFR 51.1431 - U.S. No. 1 Halves and Pieces.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 2 2010-01-01 2010-01-01 false U.S. No. 1 Halves and Pieces. 51.1431 Section 51.1431... MARKETING ACT OF 1946 FRESH FRUITS, VEGETABLES AND OTHER PRODUCTS 1,2 (INSPECTION, CERTIFICATION, AND STANDARDS) United States Standards for Grades of Shelled Pecans Grades § 51.1431 U.S. No. 1 Halves and...

7 CFR 51.1431 - U.S. No. 1 Halves and Pieces.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 2 2011-01-01 2011-01-01 false U.S. No. 1 Halves and Pieces. 51.1431 Section 51.1431... MARKETING ACT OF 1946 FRESH FRUITS, VEGETABLES AND OTHER PRODUCTS 1,2 (INSPECTION, CERTIFICATION, AND STANDARDS) United States Standards for Grades of Shelled Pecans Grades § 51.1431 U.S. No. 1 Halves and...

Size-Sorting Combined with Improved Nanocapillary-LC-MS for Identification of Intact Proteins up to 80 kDa

PubMed Central

Vellaichamy, Adaikkalam; Tran, John C.; Catherman, Adam D.; Lee, Ji Eun; Kellie, John F.; Sweet, Steve M.M.; Zamdborg, Leonid; Thomas, Paul M.; Ahlf, Dorothy R.; Durbin, Kenneth R.; Valaskovic, Gary A.; Kelleher, Neil L.

2010-01-01

Despite the availability of ultra-high resolution mass spectrometers, methods for separation and detection of intact proteins for proteome-scale analyses are still in a developmental phase. Here we report robust protocols for on-line LC-MS to drive high-throughput top-down proteomics in a fashion similar to bottom-up. Comparative work on protein standards showed that a polymeric stationary phase led to superior sensitivity over a silica-based medium in reversed-phase nanocapillary-LC, with detection of proteins >50 kDa routinely accomplished in the linear ion trap of a hybrid Fourier-Transform mass spectrometer. Protein identification was enabled by nozzle-skimmer dissociation (NSD) and detection of fragment ions with <5 ppm mass accuracy for highly-specific database searching using custom software. This overall approach led to identification of proteins up to 80 kDa, with 10-60 proteins identified in single LC-MS runs of samples from yeast and human cell lines pre-fractionated by their molecular weight using a gel-based sieving system. PMID:20073486

Reversibly constraining spliceosome-substrate complexes by engineering disulfide crosslinks.

PubMed

McCarthy, Patrick; Garside, Erin; Meschede-Krasa, Yonatan; MacMillan, Andrew; Pomeranz Krummel, Daniel

2017-08-01

The spliceosome is a highly dynamic mega-Dalton enzyme, formed in part by assembly of U snRNPs onto its pre-mRNA substrate transcripts. Early steps in spliceosome assembly are challenging to study biochemically and structurally due to compositional and conformational dynamics. We detail an approach to covalently and reversibly constrain or trap non-covalent pre-mRNA/protein spliceosome complexes. This approach involves engineering a single disulfide bond between a thiol-bearing cysteine sidechain and a proximal backbone phosphate of the pre-mRNA, site-specifically modified with an N-thioalkyl moiety. When distance and angle between reactants is optimal, the sidechain will react with the single N-thioalkyl to form a crosslink upon oxidation. We provide protocols detailing how this has been applied successfully to trap an 11-subunit RNA-protein assembly, the human U1 snRNP, in complex with a pre-mRNA. Copyright © 2017 Elsevier Inc. All rights reserved.

A Targeted Oligonucleotide Enhancer of SMN2 Exon 7 Splicing Forms Competing Quadruplex and Protein Complexes in Functional Conditions

PubMed Central

Smith, Lindsay D.; Dickinson, Rachel L.; Lucas, Christian M.; Cousins, Alex; Malygin, Alexey A.; Weldon, Carika; Perrett, Andrew J.; Bottrill, Andrew R.; Searle, Mark S.; Burley, Glenn A.; Eperon, Ian C.

2014-01-01

Summary The use of oligonucleotides to activate the splicing of selected exons is limited by a poor understanding of the mechanisms affected. A targeted bifunctional oligonucleotide enhancer of splicing (TOES) anneals to SMN2 exon 7 and carries an exonic splicing enhancer (ESE) sequence. We show that it stimulates splicing specifically of intron 6 in the presence of repressing sequences in intron 7. Complementarity to the 5′ end of exon 7 increases U2AF65 binding, but the ESE sequence is required for efficient recruitment of U2 snRNP. The ESE forms at least three coexisting discrete states: a quadruplex, a complex containing only hnRNP F/H, and a complex enriched in the activator SRSF1. Neither hnRNP H nor quadruplex formation contributes to ESE activity. The results suggest that splicing limited by weak signals can be rescued by rapid exchange of TOES oligonucleotides in various complexes and raise the possibility that SR proteins associate transiently with ESEs. PMID:25263560

45. Master plan of 4th floor, building 1, U.S. Naval ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

45. Master plan of 4th floor, building 1, U.S. Naval supply activities, New York, Brooklyn, New York, public works department, March 2, 1953. Drawing #BK-S1-4. Scale 1/16=1. - U.S. Navy Fleet Supply Base, Storehouse No. 1, 830 Third Avenue, Brooklyn, Kings County, NY

Targeting of tolerogenic dendritic cells towards heat-shock proteins: a novel therapeutic strategy for autoimmune diseases?

PubMed

Jansen, Manon A A; Spiering, Rachel; Broere, Femke; van Laar, Jacob M; Isaacs, John D; van Eden, Willem; Hilkens, Catharien M U

2018-01-01

Tolerogenic dendritic cells (tolDCs) are a promising therapeutic tool to restore immune tolerance in autoimmune diseases. The rationale of using tolDCs is that they can specifically target the pathogenic T-cell response while leaving other, protective, T-cell responses intact. Several ways of generating therapeutic tolDCs have been described, but whether these tolDCs should be loaded with autoantigen(s), and if so, with which autoantigen(s), remains unclear. Autoimmune diseases, such as rheumatoid arthritis, are not commonly defined by a single, universal, autoantigen. A possible solution is to use surrogate autoantigens for loading of tolDCs. We propose that heat-shock proteins may be a relevant surrogate antigen, as they are evolutionarily conserved between species, ubiquitously expressed in inflamed tissues and have been shown to induce regulatory T cells, ameliorating disease in various arthritis mouse models. In this review, we provide an overview on how immune tolerance may be restored by tolDCs, the problem of selecting relevant autoantigens for loading of tolDCs, and why heat-shock proteins could be used as surrogate autoantigens. © 2017 John Wiley & Sons Ltd.

U1 Adaptor Oligonucleotides Targeting BCL2 and GRM1 Suppress Growth of Human Melanoma Xenografts In Vivo

PubMed Central

Goraczniak, Rafal; Wall, Brian A; Behlke, Mark A; Lennox, Kim A; Ho, Eric S; Zaphiros, Nikolas H; Jakubowski, Christopher; Patel, Neil R; Zhao, Steven; Magaway, Carlo; Subbie, Stacey A; Jenny Yu, Lumeng; LaCava, Stephanie; Reuhl, Kenneth R; Chen, Suzie; Gunderson, Samuel I

2013-01-01

U1 Adaptor is a recently discovered oligonucleotide-based gene-silencing technology with a unique mechanism of action that targets nuclear pre-mRNA processing. U1 Adaptors have two distinct functional domains, both of which must be present on the same oligonucleotide to exert their gene-silencing function. Here, we present the first in vivo use of U1 Adaptors by targeting two different human genes implicated in melanomagenesis, B-cell lymphoma 2 (BCL2) and metabotropic glutamate receptor 1 (GRM1), in a human melanoma cell xenograft mouse model system. Using a newly developed dendrimer delivery system, anti-BCL2 U1 Adaptors were very potent and suppressed tumor growth at doses as low as 34 µg/kg with twice weekly intravenous (iv) administration. Anti-GRM1 U1 Adaptors suppressed tumor xenograft growth with similar potency. Mechanism of action was demonstrated by showing target gene suppression in tumors and by observing that negative control U1 Adaptors with just one functional domain show no tumor suppression activity. The anti-BCL2 and anti-GRM1 treatments were equally effective against cell lines harboring either wild-type or a mutant V600E B-RAF allele, the most common mutation in melanoma. Treatment of normal immune-competent mice (C57BL6) indicated no organ toxicity or immune stimulation. These proof-of-concept studies represent an in-depth (over 800 mice in ~108 treatment groups) validation that U1 Adaptors are a highly potent gene-silencing therapeutic and open the way for their further development to treat other human diseases. PMID:23673539

37 CFR 201.1 - Communication with the U.S. Copyright Office.

Code of Federal Regulations, 2014 CFR

2014-07-01

.... Copyright Office. 201.1 Section 201.1 Patents, Trademarks, and Copyrights U.S. COPYRIGHT OFFICE, LIBRARY OF... below in paragraph (c) of this section. Please note that the Library of Congress no longer accepts on... the Library of Congress, U.S. Copyright Office, 101 Independence Avenue SE., Washington, DC 20559-6000...

Differential responses of Mcl-1 in photosensitized epithelial vs lymphoid-derived human cancer cells.

PubMed

Xue, Liang-yan; Chiu, Song-mao; Oleinick, Nancy L

2005-10-20

The antiapoptotic Bcl-2-family proteins, Bcl-2 and Bcl-xL, are recognized phototargets of photodynamic therapy (PDT) with the mitochondrion-targeting phthalocyanine photosensitizer Pc 4. In the present study, we found that myeloid cell leukemia 1 (Mcl-1), another antiapoptotic member of the Bcl-2 family, was not photodamaged in Pc 4-PDT-treated human carcinoma cells MCF-7c3, MDA-MB468, DU145, and A431, although Mcl-1 turnover was observed after exposure of HeLa or MCF-7c3 cells to a supralethal dose of UVC. In contrast, when human lymphoma U937 and Jurkat cells were treated with Pc 4-PDT, staurosporine (STS) or UVC, Mcl-1 was cleaved to generate a 28-kDa fragment over a 2-4 h period. The cleavage of Mcl-1 was accompanied by the activation of caspases-3, -9, and -8. The broad-specificity caspase inhibitor z-VAD-fmk completely blocked Mcl-1 cleavage induced by PDT, STS or UVC, providing evidence for Mcl-1 as a substrate for caspases. Western blot analysis localized Mcl-1 to mitochondria, ER, and cytosol of both MCF-7c3 and U937 cells, suggesting that Mcl-1 protein, unlike Bcl-2 and Bcl-xL, is not a target for Pc 4-PDT, probably due to its localization to sites removed from those of Pc 4 binding. The 28-kDa cleaved fragment of Mcl-1, which has proapoptotic activity, was produced in PDT-treated lymphoid-derived cells, but not in cells of epithelial origin, suggesting that PDT-induced rapid and extensive apoptosis in lymphoma cells may result in part from the sensitivity of their Mcl-1 to caspase cleavage, removing an important negative control on apoptosis.

Solution Structure of the 128 kDa Enzyme I Dimer from Escherichia coli and Its 146 kDa Complex with HPr Using Residual Dipolar Couplings and Small- and Wide-Angle X-ray Scattering

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schwieters, Charles D.; Suh, Jeong-Yong; Grishaev, Alexander

2010-09-17

The solution structures of free Enzyme I (EI, {approx}128 kDa, 575 x 2 residues), the first enzyme in the bacterial phosphotransferase system, and its complex with HPr ({approx}146 kDa) have been solved using novel methodology that makes use of prior structural knowledge (namely, the structures of the dimeric EIC domain and the isolated EIN domain both free and complexed to HPr), combined with residual dipolar coupling (RDC), small- (SAXS) and wide- (WAXS) angle X-ray scattering and small-angle neutron scattering (SANS) data. The calculational strategy employs conjoined rigid body/torsion/Cartesian simulated annealing, and incorporates improvements in calculating and refining against SAXS/WAXS datamore » that take into account complex molecular shapes in the description of the solvent layer resulting in a better representation of the SAXS/WAXS data. The RDC data orient the symmetrically related EIN domains relative to the C{sub 2} symmetry axis of the EIC dimer, while translational, shape, and size information is provided by SAXS/WAXS. The resulting structures are independently validated by SANS. Comparison of the structures of the free EI and the EI-HPr complex with that of the crystal structure of a trapped phosphorylated EI intermediate reveals large ({approx}70-90{sup o}) hinge body rotations of the two subdomains comprising the EIN domain, as well as of the EIN domain relative to the dimeric EIC domain. These large-scale interdomain motions shed light on the structural transitions that accompany the catalytic cycle of EI.« less

Electroweak theory based on S U (4 )L⊗U (1 )X gauge group

NASA Astrophysics Data System (ADS)

Long, H. N.; Hue, L. T.; Loi, D. V.

2016-07-01

This paper includes two main parts. In the first part, we present generalized gauge models based on the S U (3 )C⊗S U (4 )L⊗U (1 )X (3-4-1) gauge group with arbitrary electric charges of exotic leptons. The mixing matrix of neutral gauge bosons is analyzed, and the eigenmasses and eigenstates are obtained. The anomaly-free as well as matching conditions are discussed precisely. In the second part, we present a new development of the original 3-4-1 model [R. Foot, H. N. Long, and T. A. Tran, Phys. Rev. D 50, R34 (1994), F. Pisano and V. Pleitez, Phys. Rev. D 51, 3865 (1995).]. Different from previous works, in this paper the neutrinos, with the help of the scalar decuplet H , get the Dirac masses at the tree level. The vacuum expectation value (VEV) of the Higgs boson field in the decuplet H acquiring the VEV responsible for neutrino Dirac mass leads to mixing in separated pairs of singly charged gauge bosons, namely the Standard Model (SM) W boson and K , the new gauge boson acting in the right-handed lepton sector, as well as the singly charged bileptons X and Y . Due to the mixing, there occurs a right-handed current carried by the W boson. From the expression of the electromagnetic coupling constant, ones get the limit of the sine-squared of the Weinberg angle, sin2θW<0.25 , and a constraint on electric charges of extra leptons. In the limit of lepton number conservation, the Higgs sector contains all massless Goldstone bosons for massive gauge bosons and the SM-like Higgs boson. Some phenomenology is discussed.

Ca2+-induced changes in the secondary structure of a 60 kDa phosphoinositide-specific phospholipase C from bovine brain cytosol.

PubMed Central

Herrero, C; Cornet, M E; Lopez, C; Barreno, P G; Municio, A M; Moscat, J

1988-01-01

The purification to homogeneity of a 60 kDa phosphoinositide-specific phospholipase C from bovine brain cytosol is reported here. This enzyme exhibits the same properties, in terms of response to Ca2+, as does the cytosolic activity in a variety of cell types. We show here that Ca2+ does not appear to modulate the binding of the enzyme to the substrate, but induces dramatic changes in its secondary structure. Therefore we suggest that a decrease in the alpha-helix content of this enzyme correlates with its ability to be activated by Ca2+. Images Fig. 1. PMID:2850798

11 CFR 101.1 - Candidate designations (2 U.S.C. 432(e)(1)).

Code of Federal Regulations, 2010 CFR

2010-01-01

... 11 Federal Elections 1 2010-01-01 2010-01-01 false Candidate designations (2 U.S.C. 432(e)(1)). 101.1 Section 101.1 Federal Elections FEDERAL ELECTION COMMISSION GENERAL CANDIDATE STATUS AND... and address, party affiliation, and office sought, the District and State in which Federal office is...

Solution structure of the core SMN–Gemin2 complex