Virulence Factor NSs of Rift Valley Fever Virus Recruits the F-Box Protein FBXO3 To Degrade Subunit p62 of General Transcription Factor TFIIH

PubMed Central

Kainulainen, Markus; Habjan, Matthias; Hubel, Philipp; Busch, Laura; Lau, Simone; Colinge, Jacques; Superti-Furga, Giulio; Pichlmair, Andreas

2014-01-01

ABSTRACT The nonstructural protein NSs is the main virulence factor of Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus), a serious pathogen of livestock and humans in Africa. RVFV NSs blocks transcriptional upregulation of antiviral type I interferons (IFN) and destroys the general transcription factor TFIIH subunit p62 via the ubiquitin/proteasome pathway. Here, we identified a subunit of E3 ubiquitin ligases, F-box protein FBXO3, as a host cell interactor of NSs. Small interfering RNA (siRNA)-mediated depletion of FBXO3 rescued p62 protein levels in RVFV-infected cells and elevated IFN transcription by 1 order of magnitude. NSs interacts with the full-length FBXO3 protein as well as with a truncated isoform that lacks the C-terminal acidic and poly(R)-rich domains. These isoforms are present in both the nucleus and the cytoplasm. NSs exclusively removes the nuclear pool of full-length FBXO3, likely due to consumption during the degradation process. F-box proteins form the variable substrate recognition subunit of the so-called SCF ubiquitin ligases, which also contain the constant components Skp1, cullin 1 (or cullin 7), and Rbx1. siRNA knockdown of Skp1 also protected p62 from degradation, suggesting involvement in NSs action. However, knockdown of cullin 1, cullin 7, or Rbx1 could not rescue p62 degradation by NSs. Our data show that the enzymatic removal of p62 via the host cell factor FBXO3 is a major mechanism of IFN suppression by RVFV. IMPORTANCE Rift Valley fever virus is a serious emerging pathogen of animals and humans. Its main virulence factor, NSs, enables unhindered virus replication by suppressing the antiviral innate immune system. We identified the E3 ubiquitin ligase FBXO3 as a novel host cell interactor of NSs. NSs recruits FBXO3 to destroy the general host cell transcription factor TFIIH-p62, resulting in suppression of the transcriptional upregulation of innate immunity. PMID:24403578

Cloning and functional expression of the small subunit of acetolactate synthase from Nicotiana plumbaginifolia.

PubMed

Hershey, H P; Schwartz, L J; Gale, J P; Abell, L M

1999-07-01

Acetolactate synthase (ALS) is the first committed step of branched-chain amino acid biosynthesis in plants and bacteria. The bacterial holoenzyme has been well characterized and is a tetramer of two identical large subunits (LSUs) of 60 kDa and two identical small subunits (SSUs) ranging in molecular mass from 9 to 17 kDa depending on the isozyme. The enzyme from plants is much less well characterized. Attempts to purify the protein have yielded an enzyme which appears to be an oligomer of LSUs, with the potential existence of a SSU for the plant enzyme remaining a matter of considerable speculation. We report here the discovery of a cDNA clone that encodes a SSU of plant ALS based upon the homology of the encoded peptide with various bacterial ALS SSUs. The plant ALS SSU is more than twice as large as any of its prokaryotic homologues and contains two domains that each encode a full-length copy of the prokaryotic SSU polypeptide. The cDNA clone was used to express Nicotiana plumbaginifolia SSU in Escherichia coli. Mixing a partially purified preparation of this SSU with the LSU of ALS from either N. plumbaginifolia or Arabidopsis thaliana results in both increased specific activity and increased stability of the enzymic activity. These results are consistent with those observed for the bacterial enzyme in similar experiments and represent the first functional demonstration of the existence of a SSU for plant ALS.

Heterodimerization with the β1 subunit directs the α2 subunit of nitric oxide-sensitive guanylyl cyclase to calcium-insensitive cell-cell contacts in HEK293 cells: Interaction with Lin7a.

PubMed

Hochheiser, Julia; Haase, Tobias; Busker, Mareike; Sömmer, Anne; Kreienkamp, Hans-Jürgen; Behrends, Sönke

2016-12-15

Nitric oxide-sensitive guanylyl cyclase is a heterodimeric enzyme consisting of an α and a β subunit. Two different α subunits (α 1 and α 2 ) give rise to two heterodimeric enzymes α 1 /β 1 and α 2 /β 1 . Both coexist in a wide range of tissues including blood vessels and the lung, but expression of the α 2 /β 1 form is generally much lower and approaches levels similar to the α 1 /β 1 form in the brain only. In the present paper, we show that the α 2 /β 1 form interacts with Lin7a in mouse brain synaptosomes based on co-precipitation analysis. In HEK293 cells, we found that the overexpressed α 2 /β 1 form, but not the α 1 /β 1 form is directed to calcium-insensitive cell-cell contacts. The isolated PDZ binding motif of an amino-terminally truncated α 2 subunit was sufficient for cell-cell contact localization. For the full length α 2 subunit with the PDZ binding motif this was only the case in the heterodimer configuration with the β 1 subunit, but not as isolated α 2 subunit. We conclude that the PDZ binding motif of the α 2 subunit is only accessible in the heterodimer conformation of the mature nitric oxide-sensitive enzyme. Interaction with Lin7a, a small scaffold protein important for synaptic function and cell polarity, can direct this complex to nectin based cell-cell contacts via MPP3 in HEK293 cells. We conclude that heterodimerization is a prerequisite for further protein-protein interactions that direct the α 2 /β 1 form to strategic sites of the cell membrane with adjacent neighbouring cells. Drugs increasing the nitric oxide-sensitivity of this specific form may be particularly effective. Copyright © 2016 Elsevier Inc. All rights reserved.

Modeling the full length HIV-1 Gag polyprotein reveals the role of its p6 subunit in viral maturation and the effect of non-cleavage site mutations in protease drug resistance.

PubMed

Su, Chinh Tran-To; Kwoh, Chee-Keong; Verma, Chandra Shekhar; Gan, Samuel Ken-En

2017-12-27

HIV polyprotein Gag is increasingly found to contribute to protease inhibitor resistance. Despite its role in viral maturation and in developing drug resistance, there remain gaps in the knowledge of the role of certain Gag subunits (e.g. p6), and that of non-cleavage mutations in drug resistance. As p6 is flexible, it poses a problem for structural experiments, and is hence often omitted in experimental Gag structural studies. Nonetheless, as p6 is an indispensable component for viral assembly and maturation, we have modeled the full length Gag structure based on several experimentally determined constraints and studied its structural dynamics. Our findings suggest that p6 can mechanistically modulate Gag conformations. In addition, the full length Gag model reveals that allosteric communication between the non-cleavage site mutations and the first Gag cleavage site could possibly result in protease drug resistance, particularly in the absence of mutations in Gag cleavage sites. Our study provides a mechanistic understanding to the structural dynamics of HIV-1 Gag, and also proposes p6 as a possible drug target in anti-HIV therapy.

[Molecular cloning and characterization of cDNA of the rpc10+ gene encoding the smallest subunit of nuclear RNA polymerases of Schizosaccharomyces pombe].

PubMed

Shpakovskiĭ, G V; Lebedenko, E N

1997-05-01

The full-length cDNA of the rpc10+ gene encoding mini-subunit Rpc10, which is common for all three nuclear RNA polymerases of the fission yeast Schizosaccharomyces pombe, was cloned and sequenced. The Rpc10 subunit of Sz. pombe and its homologs from S. cerevisiae and H. sapiens are positively charged proteins with a highly conserved C-terminal region and an invariant zinc-binding domain (Zn-finger) of a typical amino acid composition: YxCx2Cx12RCx2CGxR. Functional tests of heterospecific complementation, using tetrad analysis or plasmid shuffling, showed that the Rpc10 subunit of Sz. pombe can successfully replace the homologous ABC10 alpha subunit in nuclear RNA polymerases I-III of S. cerevisiae.

Isolated spinach ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit .sup..epsilon. N-methyltransferase and method of inactivating ribulose-1,5-bisphosphatase carboxylase/oxygenase large subunit .sup..epsilon. N-methyltransferase activity

DOEpatents

Houtz, Robert L.

1999-01-01

The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .sup..epsilon. N-methyltransferase (protein methylase III or Rubisco LSMT) from a plant which has a des(methyl) lysyl residue in the LS is disclosed. In addition, the full-length cDNA clones for Rubisco LSMT are disclosed. Transgenic plants and methods of producing same which have the Rubisco LSMT gene inserted into the DNA are also provided. Further, methods of inactivating the enzymatic activity of Rubisco LSMT are also disclosed.

Crystal structure of calpain-3 penta-EF-hand (PEF) domain - a homodimerized PEF family member with calcium bound at the fifth EF-hand

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

Partha, Sarathy K.; Ravulapalli, Ravikiran; Allingham, John S.

2014-08-21

Calpains are Ca 2+dependent intracellular cysteine proteases that cleave a wide range of protein substrates to help implement Ca 2+ signaling in the cell. The major isoforms of this enzyme family, calpain-1 and calpain-2, are heterodimers of a large and a small subunit, with the main dimer interface being formed through their C-terminal penta-EF hand (PEF) domains. Calpain-3, or p94, is a skeletal muscle-specific isoform that is genetically linked to limb-girdle muscular dystrophy. Biophysical and modeling studies with the PEF domain of calpain-3 support the suggestion that full-length calpain-3 exists as a homodimer. Here, we report the crystallization of calpain-3'smore » PEF domain and its crystal structure in the presence of Ca 2+, which provides evidence for the homodimer architecture of calpain-3 and supports the molecular model that places a protease core at either end of the elongated dimer. Unlike other calpain PEF domain structures, the calpain-3 PEF domain contains a Ca 2+ bound at the EF5-hand used for homodimer association. Three of the four Ca 2+-binding EF-hands of the PEF domains are concentrated near the protease core, and have the potential to radically change the local charge within the dimer during Ca 2+ signaling. Examination of the homodimer interface shows that there would be steric clashes if the calpain-3 large subunit were to try to pair with a calpain small subunit.« less

Identification of Species and Sources of Cryptosporidium Oocysts in Storm Waters with a Small-Subunit rRNA-Based Diagnostic and Genotyping Tool

PubMed Central

Xiao, Lihua; Alderisio, Kerri; Limor, Josef; Royer, Michael; Lal, Altaf A.

2000-01-01

The identification of Cryptosporidium oocysts in environmental samples is largely made by the use of an immunofluorescent assay. In this study, we have used a small-subunit rRNA-based PCR-restriction fragment length polymorphism technique to identify species and sources of Cryptosporidium oocysts present in 29 storm water samples collected from a stream in New York. A total of 12 genotypes were found in 27 positive samples; for 4 the species and probable origins were identified by sequence analysis, whereas the rest represent new genotypes from wildlife. Thus, this technique provides an alternative method for the detection and differentiation of Cryptosporidium parasites in environmental samples. PMID:11097935

Identification of the Dimer Exchange Interface of the Bacterial DNA Damage Response Protein UmuD.

PubMed

Murison, David A; Timson, Rebecca C; Koleva, Bilyana N; Ordazzo, Michael; Beuning, Penny J

2017-09-12

The Escherichia coli SOS response, an induced DNA damage response pathway, confers survival on bacterial cells by providing accurate repair mechanisms as well as the potentially mutagenic pathway translesion synthesis (TLS). The umuD gene products are upregulated after DNA damage and play roles in both nonmutagenic and mutagenic aspects of the SOS response. Full-length UmuD is expressed as a homodimer of 139-amino-acid subunits, which eventually cleaves its N-terminal 24 amino acids to form UmuD'. The cleavage product UmuD' and UmuC form the Y-family polymerase DNA Pol V (UmuD' 2 C) capable of performing TLS. UmuD and UmuD' exist as homodimers, but their subunits can readily exchange to form UmuDD' heterodimers preferentially. Heterodimer formation is an essential step in the degradation pathway of UmuD'. The recognition sequence for ClpXP protease is located within the first 24 amino acids of full-length UmuD, and the partner of full-length UmuD, whether UmuD or UmuD', is degraded by ClpXP. To better understand the mechanism by which UmuD subunits exchange, we measured the kinetics of exchange of a number of fluorescently labeled single-cysteine UmuD variants as detected by Förster resonance energy transfer. Labeling sites near the dimer interface correlate with increased rates of exchange, indicating that weakening the dimer interface facilitates exchange, whereas labeling sites on the exterior decrease the rate of exchange. In most but not all cases, homodimer and heterodimer exchange exhibit similar rates, indicating that somewhat different molecular surfaces mediate homodimer exchange and heterodimer formation.

Kv channel subunits that contribute to voltage-gated K+ current in renal vascular smooth muscle.

PubMed

Fergus, Daniel J; Martens, Jeffrey R; England, Sarah K

2003-03-01

The rat renal arterial vasculature displays differences in K(+) channel current phenotypes along its length. Small arcuate to cortical radial arteries express a delayed rectifier phenotype, while the predominant Kv current in larger arcuate and interlobar arteries is composed of both transient and sustained components. We sought to determine whether Kvalpha subunits in the rat renal interlobar and arcuate arteries form heterotetramers, which may account for the unique currents, and whether modulatory Kvbeta subunits are present in renal vascular smooth muscle cells. RT-PCR indicated the presence of several different Kvalpha subunit isoform transcripts. Co-immunoprecipitation with immunoblotting and immunohistochemical evidence suggests that a portion of the K(+) current phenotype is a heteromultimer containing delayed-rectifier Kv1.2 and A-type Kv1.4 channel subunits. RT-PCR and immunoblot analyses also demonstrated the presence of both Kvbeta1.2 and Kvbeta1.3 in renal arteries. These results suggest that heteromultimeric formation of Kvalpha subunits and the presence of modulatory Kvbeta subunits are important factors in mediating Kv currents in the renal microvasculature and suggest a potentially critical role for these channel subunits in blood pressure regulation.

The Small Subunit of Snapdragon Geranyl Diphosphate Synthase Modifies the Chain Length Specificity of Tobacco Geranylgeranyl Diphosphate Synthase in Planta[W

PubMed Central

Orlova, Irina; Nagegowda, Dinesh A.; Kish, Christine M.; Gutensohn, Michael; Maeda, Hiroshi; Varbanova, Marina; Fridman, Eyal; Yamaguchi, Shinjiro; Hanada, Atsushi; Kamiya, Yuji; Krichevsky, Alexander; Citovsky, Vitaly; Pichersky, Eran; Dudareva, Natalia

2009-01-01

Geranyl diphosphate (GPP), the precursor of many monoterpene end products, is synthesized in plastids by a condensation of dimethylallyl diphosphate and isopentenyl diphosphate (IPP) in a reaction catalyzed by homodimeric or heterodimeric GPP synthase (GPPS). In the heterodimeric enzymes, a noncatalytic small subunit (GPPS.SSU) determines the product specificity of the catalytic large subunit, which may be either an active geranylgeranyl diphosphate synthase (GGPPS) or an inactive GGPPS-like protein. Here, we show that expression of snapdragon (Antirrhinum majus) GPPS.SSU in tobacco (Nicotiana tabacum) plants increased the total GPPS activity and monoterpene emission from leaves and flowers, indicating that the introduced catalytically inactive GPPS.SSU found endogenous large subunit partner(s) and formed an active snapdragon/tobacco GPPS in planta. Bimolecular fluorescence complementation and in vitro enzyme analysis of individual and hybrid proteins revealed that two of four GGPPS-like candidates from tobacco EST databases encode bona fide GGPPS that can interact with snapdragon GPPS.SSU and form a functional GPPS enzyme in plastids. The formation of chimeric GPPS in transgenic plants also resulted in leaf chlorosis, increased light sensitivity, and dwarfism due to decreased levels of chlorophylls, carotenoids, and gibberellins. In addition, these transgenic plants had reduced levels of sesquiterpene emission, suggesting that the export of isoprenoid intermediates from the plastids into the cytosol was decreased. These results provide genetic evidence that GPPS.SSU modifies the chain length specificity of phylogenetically distant GGPPS and can modulate IPP flux distribution between GPP and GGPP synthesis in planta. PMID:20028839

TFIID TAF6-TAF9 Complex Formation Involves the HEAT Repeat-containing C-terminal Domain of TAF6 and Is Modulated by TAF5 Protein*

PubMed Central

Scheer, Elisabeth; Delbac, Frédéric; Tora, Laszlo; Moras, Dino; Romier, Christophe

2012-01-01

The general transcription factor TFIID recognizes specifically the core promoter of genes transcribed by eukaryotic RNA polymerase II, nucleating the assembly of the preinitiation complex at the transcription start site. However, the understanding in molecular terms of TFIID assembly and function remains poorly understood. Histone fold motifs have been shown to be extremely important for the heterodimerization of many TFIID subunits. However, these subunits display several evolutionary conserved noncanonical features when compared with histones, including additional regions whose role is unknown. Here we show that the conserved additional C-terminal region of TFIID subunit TAF6 can be divided into two domains: a small middle domain (TAF6M) and a large C-terminal domain (TAF6C). Our crystal structure of the TAF6C domain from Antonospora locustae at 1.9 Å resolution reveals the presence of five conserved HEAT repeats. Based on these data, we designed several mutants that were introduced into full-length human TAF6. Surprisingly, the mutants affect the interaction between TAF6 and TAF9, suggesting that the formation of the complex between these two TFIID subunits do not only depend on their histone fold motifs. In addition, the same mutants affect even more strongly the interaction between TAF6 and TAF9 in the context of a TAF5-TAF6-TAF9 complex. Expression of these mutants in HeLa cells reveals that most of them are unstable, suggesting their poor incorporation within endogenous TFIID. Taken together, our results suggest that the conserved additional domains in histone fold-containing subunits of TFIID and of co-activator SAGA are important for the assembly of these complexes. PMID:22696218

Nature of KaiB-KaiC binding in the cyanobacterial circadian oscillator

PubMed Central

Pattanayek, Rekha; Yadagiri, Kirthi Kiran; Ohi, Melanie D.; Egli, Martin

2013-01-01

In the cyanobacteria Synechococcus elongatus and Thermosynechococcus elongatus, the KaiA, KaiB and KaiC proteins in the presence of ATP generate a post-translational oscillator (PTO) that can be reconstituted in vitro. KaiC is the result of a gene duplication and resembles a double doughnut with N-terminal CI and C-terminal CII hexameric rings. Six ATPs are bound between subunits in both the CI and CII ring. CI harbors ATPase activity, and CII catalyzes phosphorylation and dephosphorylation at T432 and S431 with a ca. 24-h period. KaiA stimulates KaiC phosphorylation, and KaiB promotes KaiC subunit exchange and sequesters KaiA on the KaiB-KaiC interface in the final stage of the clock cycle. Studies of the PTO protein-protein interactions are convergent in terms of KaiA binding to CII but have led to two opposing models of the KaiB-KaiC interaction. Electron microscopy (EM) and small angle X-ray scattering (SAXS), together with native PAGE using full-length proteins and separate CI and CII rings, are consistent with binding of KaiB to CII. Conversely, NMR together with gel filtration chromatography and denatured PAGE using monomeric CI and CII domains support KaiB binding to CI. To resolve the existing controversy, we studied complexes between KaiB and gold-labeled, full-length KaiC with negative stain EM. The EM data clearly demonstrate that KaiB contacts the CII ring. Together with the outcomes of previous analyses, our work establishes that only CII participates in interactions with KaiA and KaiB as well as with the His kinase SasA involved in the clock output pathway. PMID:23388462

Studies of Ribonucleotide Reductase in Crucian Carp—An Oxygen Dependent Enzyme in an Anoxia Tolerant Vertebrate

PubMed Central

Sandvik, Guro K.; Tomter, Ane B.; Bergan, Jonas; Zoppellaro, Giorgio; Barra, Anne-Laure; Røhr, Åsmund K.; Kolberg, Matthias; Ellefsen, Stian

2012-01-01

The enzyme ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to deoxyribonucleotides, the precursors for DNA. RNR requires a thiyl radical to activate the substrate. In RNR of eukaryotes (class Ia RNR), this radical originates from a tyrosyl radical formed in reaction with oxygen (O2) and a ferrous di-iron center in RNR. The crucian carp (Carassius carassius) is one of very few vertebrates that can tolerate several months completely without oxygen (anoxia), a trait that enables this fish to survive under the ice in small ponds that become anoxic during the winter. Previous studies have found indications of cell division in this fish after 7 days of anoxia. This appears nearly impossible, as DNA synthesis requires the production of new deoxyribonucleotides and therefore active RNR. We have here characterized RNR in crucian carp, to search for adaptations to anoxia. We report the full-length sequences of two paralogs of each of the RNR subunits (R1i, R1ii, R2i, R2ii, p53R2i and p53R2ii), obtained by cloning and sequencing. The mRNA levels of these subunits were measured with quantitative PCR and were generally well maintained in hypoxia and anoxia in heart and brain. We also report maintained or increased mRNA levels of the cell division markers proliferating cell nuclear antigen (PCNA), brain derived neurotrophic factor (BDNF) and Ki67 in anoxic hearts and brains. Electron paramagnetic resonance (EPR) measurements on in vitro expressed crucian carp R2 and p53R2 proteins gave spectra similar to mammalian RNRs, including previously unpublished human and mouse p53R2 EPR spectra. However, the radicals in crucian carp RNR small subunits, especially in the p53R2ii subunit, were very stable at 0°C. A long half-life of the tyrosyl radical during wintertime anoxia could allow for continued cell division in crucian carp. PMID:22916159

MgATP hydrolysis destabilizes the interaction between subunit H and yeast V1-ATPase, highlighting H's role in V-ATPase regulation by reversible disassembly.

PubMed

Sharma, Stuti; Oot, Rebecca A; Wilkens, Stephan

2018-05-12

Vacuolar H+-ATPases (V-ATPases; V1Vo-ATPases) are rotary motor proton pumps that acidify intracellular compartments and in some tissues, the extracellular space. V-ATPase is regulated by reversible disassembly into autoinhibited V1-ATPase and Vo proton channel sectors. An important player in V-ATPase regulation is subunit H, which binds at the interface of V1 and Vo. H is required for MgATPase activity in holo V-ATPase, but also for stabilizing the MgADP inhibited state in membrane detached V1. However, how H fulfills these two functions is poorly understood. To characterize the H-V1 interaction and its role in reversible disassembly, we determined binding affinities of full length H and its N-terminal domain (HNT) for an isolated heterodimer of subunits E and G (EG), the N-terminal domain of subunit a (aNT), and V1 lacking subunit H (V1ΔH). Using isothermal titration calorimetry (ITC) and biolayer interferometry (BLI), we show that HNT binds EG with moderate affinity, that full length H binds aNT weakly, and that both H and HNT bind V1ΔH with high affinity. We also found that only one molecule of HNT binds V1ΔH with high affinity, suggesting conformational asymmetry of the three EG heterodimers in V1ΔH. Moreover, MgATP hydrolysis-driven conformational changes in V1 destabilized the interaction of H, or HNT, with V1ΔH, suggesting an interplay between MgADP inhibition and subunit H. Our observation that H binding is affected by MgATP hydrolysis in V1 points to H's role in the mechanism of reversible disassembly. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Suppression of the heterotrimeric G protein causes abnormal morphology, including dwarfism, in rice

PubMed Central

Fujisawa, Yukiko; Kato, Teruhisa; Ohki, Shizuka; Ishikawa, Atsushi; Kitano, Hidemi; Sasaki, Takuji; Asahi, Tadashi; Iwasaki, Yukimoto

1999-01-01

Transgenic rice containing an antisense cDNA for the α subunit of rice heterotrimeric G protein produced little or no mRNA for the subunit and exhibited abnormal morphology, including dwarf traits and the setting of small seeds. In normal rice, the mRNA for the α subunit was abundant in the internodes and florets, the tissues closely related to abnormality in the dwarf transformants. The position of the α-subunit gene was mapped on rice chromosome 5 by mapping with the restriction fragment length polymorphism. The position was closely linked to the locus of a rice dwarf mutant, Daikoku dwarf (d-1), which is known to exhibit abnormal phenotypes similar to those of the transformants that suppressed the endogenous mRNA for the α subunit by antisense technology. Analysis of the cDNAs for the α subunits of five alleles of Daikoku dwarf (d-1), ID-1, DK22, DKT-1, DKT-2, and CM1361–1, showed that these dwarf mutants had mutated in the coding region of the α-subunit gene. These results show that the G protein functions in the formation of normal internodes and seeds in rice. PMID:10377457

Escherichia coli F1Fo-ATP synthase with a b/δ fusion protein allows analysis of the function of the individual b subunits.

PubMed

Gajadeera, Chathurada S; Weber, Joachim

2013-09-13

The "stator stalk" of F1Fo-ATP synthase is essential for rotational catalysis as it connects the nonrotating portions of the enzyme. In Escherichia coli, the stator stalk consists of two (identical) b subunits and the δ subunit. In mycobacteria, one of the b subunits and the δ subunit are replaced by a b/δ fusion protein; the remaining b subunit is of the shorter b' type. In the present study, it is shown that it is possible to generate a functional E. coli ATP synthase containing a b/δ fusion protein. This construct allowed the analysis of the roles of the individual b subunits. The full-length b subunit (which in this case is covalently linked to δ in the fusion protein) is responsible for connecting the stalk to the catalytic F1 subcomplex. It is not required for interaction with the membrane-embedded Fo subcomplex, as its transmembrane helix can be removed. Attachment to Fo is the function of the other b subunit which in turn has only a minor (if any at all) role in binding to δ. Also in E. coli the second b subunit can be shortened to a b' type.

Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit N-methyltransferase

DOEpatents

Houtz, Robert L.

1998-01-01

The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .epsilon.N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the .epsilon.-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed.

Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit epsilon N-methyltransferase

DOEpatents

Houtz, Robert L.

1999-01-01

The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .sup..epsilon. N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the .epsilon.-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed.

Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit N-methyltransferase

DOEpatents

Houtz, R.L.

1998-03-03

The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) {epsilon}N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the {epsilon}-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed. 5 figs.

Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit epsilon N-methyltransferase

DOEpatents

Houtz, R.L.

1999-02-02

The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS){sup {epsilon}}N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the {epsilon}-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed. 8 figs.

Conservation of the Human Integrin-Type Beta-Propeller Domain in Bacteria

PubMed Central

Chouhan, Bhanupratap; Denesyuk, Alexander; Heino, Jyrki; Johnson, Mark S.; Denessiouk, Konstantin

2011-01-01

Integrins are heterodimeric cell-surface receptors with key functions in cell-cell and cell-matrix adhesion. Integrin α and β subunits are present throughout the metazoans, but it is unclear whether the subunits predate the origin of multicellular organisms. Several component domains have been detected in bacteria, one of which, a specific 7-bladed β-propeller domain, is a unique feature of the integrin α subunits. Here, we describe a structure-derived motif, which incorporates key features of each blade from the X-ray structures of human αIIbβ3 and αVβ3, includes elements of the FG-GAP/Cage and Ca2+-binding motifs, and is specific only for the metazoan integrin domains. Separately, we searched for the metazoan integrin type β-propeller domains among all available sequences from bacteria and unicellular eukaryotic organisms, which must incorporate seven repeats, corresponding to the seven blades of the β-propeller domain, and so that the newly found structure-derived motif would exist in every repeat. As the result, among 47 available genomes of unicellular eukaryotes we could not find a single instance of seven repeats with the motif. Several sequences contained three repeats, a predicted transmembrane segment, and a short cytoplasmic motif associated with some integrins, but otherwise differ from the metazoan integrin α subunits. Among the available bacterial sequences, we found five examples containing seven sequential metazoan integrin-specific motifs within the seven repeats. The motifs differ in having one Ca2+-binding site per repeat, whereas metazoan integrins have three or four sites. The bacterial sequences are more conserved in terms of motif conservation and loop length, suggesting that the structure is more regular and compact than those example structures from human integrins. Although the bacterial examples are not full-length integrins, the full-length metazoan-type 7-bladed β-propeller domains are present, and sometimes two tandem copies are found. PMID:22022374

Use of a purified and functional recombinant calcium-channel beta4 subunit in surface-plasmon resonance studies.

PubMed Central

Geib, Sandrine; Sandoz, Guillaume; Mabrouk, Kamel; Matavel, Alessandra; Marchot, Pascale; Hoshi, Toshinori; Villaz, Michel; Ronjat, Michel; Miquelis, Raymond; Lévêque, Christian; de Waard, Michel

2002-01-01

Native high-voltage-gated calcium channels are multi-subunit complexes comprising a pore-forming subunit Ca(v) and at least two auxiliary subunits alpha(2)delta and beta. The beta subunit facilitates cell-surface expression of the channel and contributes significantly to its biophysical properties. In spite of its importance, detailed structural and functional studies are hampered by the limited availability of native beta subunit. Here, we report the purification of a recombinant calcium-channel beta(4) subunit from bacterial extracts by using a polyhistidine tag. The purified protein is fully functional since it binds on the alpha1 interaction domain, its main Ca(v)-binding site, and regulates the activity of P/Q calcium channel expressed in Xenopus oocytes in a similar way to the beta(4) subunit produced by cRNA injection. We took advantage of the functionality of the purified material to (i) develop an efficient surface-plasmon resonance assay of the interaction between two calcium channel subunits and (ii) measure, for the first time, the affinity of the recombinant His-beta(4) subunit for the full-length Ca(v)2.1 channel. The availability of this purified material and the development of a surface-plasmon resonance assay opens two immediate research perspectives: (i) drug screening programmes applied to the Ca(v)/beta interaction and (ii) crystallographic studies of the calcium-channel beta(4) subunit. PMID:11988102

Ligand-Binding Properties and Conformational Dynamics of Autolysin Repeat Domains in Staphylococcal Cell Wall Recognition

PubMed Central

Zoll, Sebastian; Schlag, Martin; Shkumatov, Alexander V.; Rautenberg, Maren; Svergun, Dmitri I.; Götz, Friedrich

2012-01-01

The bifunctional major autolysin Atl plays a key role in staphylococcal cell separation. Processing of Atl yields catalytically active amidase (AM) and glucosaminidase (GL) domains that are each fused to repeating units. The two repeats of AM (R1 and R2) target the enzyme to the septum, where it cleaves murein between dividing cells. We have determined the crystal structure of R2, which reveals that each repeat folds into two half-open β-barrel subunits. We further demonstrate that lipoteichoic acid serves as a receptor for the repeats and that this interaction depends on conserved surfaces in each subunit. Small-angle X-ray scattering of the mature amidase reveals the presence of flexible linkers separating the AM, R1, and R2 units. Different levels of flexibility for each linker provide mechanistic insights into the conformational dynamics of the full-length protein and the roles of its components in cell wall association and catalysis. Our analysis supports a model in which the repeats direct the catalytic AM domain to the septum, where it can optimally perform the final step of cell division. PMID:22609916

Isolated spinach ribulose-1,5-bisphosphate carboxylase/oxgenase large subunit .epsilon. n-methyltransferase and method of inactivating ribulose-1,5-bishosphatase .epsilon. n-methyltransferase activity

DOEpatents

Houtz, Robert L.

2001-01-01

The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .sup..epsilon. N-methyltansferase (protein methylase III or Rubisco LSMT) from a plant which has a des(methyl) lysyl residue in the LS is disclosed. In addition, the full-length cDNA clones for Rubisco LSMT are disclosed. Transgenic plants and methods of producing same which have the Rubisco LSMT gene inserted into the DNA are also provided. Further, methods of inactivating the enzymatic activity of Rubisco LSMT are also disclosed.

Neutralizing activity and protective immunity to ricin toxin conferred by B subunit (RTB)-specific Fab fragments.

PubMed

Yermakova, Anastasiya; Mantis, Nicholas J

2013-09-01

SylH3 and 24B11 are murine monoclonal antibodies directed against different epitopes on ricin toxin's binding (RTB) subunit that have been shown to passively protect mice against ricin challenge. Here we report that Fab fragments of SylH3 and 24B11 neutralize ricin in a cell based assay, and in a mouse challenge model as effectively as their respective full length parental IgGs. These data demonstrate that immunity to ricin can occur independent of Fc-mediated clearance. Copyright © 2013 Elsevier Ltd. All rights reserved.

The multifaceted subunit interfaces of ionotropic glutamate receptors.

PubMed

Green, Tim; Nayeem, Naushaba

2015-01-01

The past fifteen years has seen a revolution in our understanding of ionotropic glutamate receptor (iGluR) structure, starting with the first view of the ligand binding domain (LBD) published in 1998, and in many ways culminating in the publication of the full-length structure of GluA2 in 2009. These reports have revealed not only the central role played by subunit interfaces in iGluR function, but also myriad binding sites within interfaces for endogenous and exogenous factors. Changes in the conformation of inter-subunit interfaces are central to transmission of ligand gating into pore opening (itself a rearrangement of interfaces), and subsequent closure through desensitization. With the exception of the agonist binding site, which is located entirely within individual subunits, almost all modulatory factors affecting iGluRs appear to bind to sites in subunit interfaces. This review seeks to summarize what we currently understand about the diverse roles interfaces play in iGluR function, and to highlight questions for future research. © 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.

Occurrence of hemocyanin in ostracod crustaceans.

PubMed

Marxen, Julia C; Pick, Christian; Oakley, Todd H; Burmester, Thorsten

2014-08-01

Hemocyanin is a copper-containing protein that transports O2 in the hemolymph of many arthropod species. Within the crustaceans, hemocyanin appeared to be restricted to Malacostraca but has recently been identified in Remipedia. Here, we report the occurrence of hemocyanin in ostracods, indicating that this respiratory protein is more widespread within crustaceans than previously thought. By analyses of expressed sequence tags and by RT-PCR, we obtained four full length and nine partial hemocyanin sequences from six of ten investigated ostracod species. Hemocyanin was identified in Myodocopida (Actinoseta jonesi, Cypridininae sp., Euphilomedes morini, Skogsbergia lerneri, Vargula tsujii) and Platycopida (Cytherelloidea californica) but not in Podocopida. We found no evidence for the presence of hemoglobin in any of these ostracod species. Like in other arthropods, we identified multiple hemocyanin subunits (up to six) to occur in a single ostracod species. Bayesian phylogenetic analyses showed that ostracod hemocyanin subunit diversity evolved independently from that of other crustaceans. Ostracod hemocyanin subunits were found paraphyletic, with myodocopid and platycopid subunits forming distinct clades within those of the crustaceans. This pattern suggests that ostracod hemocyanins originated from distinct subunits in the pancrustacean stemline.

The complete mitochondrial genome sequence of Eimeria innocua (Eimeriidae, Coccidia, Apicomplexa).

PubMed

Hafeez, Mian Abdul; Vrba, Vladimir; Barta, John Robert

2016-07-01

The complete mitochondrial genome of Eimeria innocua KR strain (Eimeriidae, Coccidia, Apicomplexa) was sequenced. This coccidium infects turkeys (Meleagris gallopavo), Bobwhite quails (Colinus virginianus), and Grey partridges (Perdix perdix). Genome organization and gene contents were comparable with other Eimeria spp. infecting galliform birds. The circular-mapping mt genome of E. innocua is 6247 bp in length with three protein-coding genes (cox1, cox3, and cytb), 19 gene fragments encoding large subunit (LSU) rRNA and 14 gene fragments encoding small subunit (SSU) rRNA. Like other Apicomplexa, no tRNA was encoded. The mitochondrial genome of E. innocua confirms its close phylogenetic affinities to Eimeria dispersa.

Identification and transcription profiling of NDUFS8 in Aedes taeniorhynchus (Diptera:Culididae): developmental regulation and environmental response

USDA-ARS?s Scientific Manuscript database

The cDNA of a NADH dehydrogenase -ubiquinone Fe-S protein 8 subunit (NDUFS8) gene from Aedes (Ochlerotatus) taeniorhynchus Wiedemann has been cloned and sequenced. The full-length mRNA sequence (824 bp) of AetNDUFS8 encodes an open reading region of 651 bp (i.e., 217 amino acids). To detect whether ...

Toxic β-Amyloid (Aβ) Alzheimer's Ion Channels: From Structure to Function and Design

NASA Astrophysics Data System (ADS)

Nussinov, Ruth

2012-02-01

Full-length amyloid beta peptides (Aβ1-40/42) form neuritic amyloid plaques in Alzheimer's disease (AD) patients and are implicated in AD pathology. Recent biophysical and cell biological studies suggest a direct mechanism of amyloid beta toxicity -- ion channel mediated loss of calcium homeostasis. Truncated amyloid beta fragments (Aβ11-42 and Aβ17-42), commonly termed as non-amyloidogenic are also found in amyloid plaques of Alzheimer's disease (AD) and in the preamyloid lesions of Down's syndrome (DS), a model system for early onset AD study. Very little is known about the structure and activity of these smaller peptides although they could be key AD and DS pathological agents. Using complementary techniques of explicit solvent molecular dynamics (MD) simulations, atomic force microscopy (AFM), channel conductance measurements, cell calcium uptake assays, neurite degeneration and cell death assays, we have shown that non-amyloidogenic Aβ9-42 and Aβ17-42 peptides form ion channels with loosely attached subunits and elicit single channel conductances. The subunits appear mobile suggesting insertion of small oligomers, followed by dynamic channel assembly and dissociation. These channels allow calcium uptake in APP-deficient cells and cause neurite degeneration in human cortical neurons. Channel conductance, calcium uptake and neurite degeneration are selectively inhibited by zinc, a blocker of amyloid ion channel activity. Thus truncated Aβ fragments could account for undefined roles played by full length Aβs and provide a novel mechanism of AD and DS pathology. The emerging picture from our large-scale simulations is that toxic ion channels formed by β-sheets are highly polymorphic, and spontaneously break into loosely interacting dynamic units (though still maintaining ion channel structures as imaged with AFM), that associate and dissociate leading to toxic ion flux. This sharply contrasts intact conventional gated ion channels that consist of tightly interacting α-helices that robustly prevent ion leakage, rather than hydrogen-bonded β-strands. Moreover, in comparison with β-rich antimicrobial peptide (AMP) such as a protegrin-1 (PG-1), both Aβ and PG-1 are cytotoxic, and capable of forming fibrils and dynamic channels which consist of subunits with similar dimensions. These combined properties support a functional relationship between amyloidogenic peptides and β-sheet-rich cytolytic AMPs, suggesting that PG-1 is amyloidogenic and amyloids may have an antimicrobial function.

The complete mitochondrial genomes of five Eimeria species infecting domestic rabbits.

PubMed

Liu, Guo-Hua; Tian, Si-Qin; Cui, Ping; Fang, Su-Fang; Wang, Chun-Ren; Zhu, Xing-Quan

2015-12-01

Rabbit coccidiosis caused by members of the genus Eimeria can cause enormous economic impact worldwide, but the genetics, epidemiology and biology of these parasites remain poorly understood. In the present study, we sequenced and annotated the complete mitochondrial (mt) genomes of five Eimeria species that commonly infect the domestic rabbits. The complete mt genomes of Eimeria intestinalis, Eimeria flavescens, Eimeria media, Eimeria vejdovskyi and Eimeria irresidua were 6261bp, 6258bp, 6168bp, 6254bp, 6259bp in length, respectively. All of the mt genomes consist of 3 genes for proteins (cytb, cox1, and cox3), 14 gene fragments for the large subunit (LSU) rRNA and 11 gene fragments for the small subunit (SSU) rRNA, but no transfer RNA (tRNA) genes. The gene order of the mt genomes is similar to that of Plasmodium, but distinct from Haemosporida and Theileria. Phylogenetic analyses based on full nucleotide sequences using Bayesian analysis revealed that the monophyly of the Eimeria of rabbits was strongly statistically supported with a Bayesian posterior probabilities. These data provide novel mtDNA markers for studying the population genetics and molecular epidemiology of the Eimeria species, and should have implications for the molecular diagnosis, prevention and control of coccidiosis in rabbits. Copyright © 2015 Elsevier Inc. All rights reserved.

Subunit association of gamma-glutamyltranspeptidase of Escherichia coli K-12.

PubMed

Hashimoto, W; Suzuki, H; Nohara, S; Tachi, H; Yamamoto, K; Kumagai, H

1995-12-01

gamma-Glutamyltranspeptidase [EC 2.3.2.2] of Escherichia coli K-12 consists of one large subunit and one small subunit, which can be separated from each other by high-performance liquid chromatography. Using ion spray mass spectrometry, the masses of the large and the small subunit were determined to be 39,207 and 20,015, respectively. The large subunit exhibited no gamma-glutamyltranspeptidase activity and the small subunit had little enzymatic activity, but a mixture of the two subunits showed partial recovery of the enzymatic activity. The results of native-polyacrylamide gel electrophoresis suggested that they could partially recombine, and that the recombined dimer exhibited enzymatic activity. The gene of gamma-glutamyltranspeptidase encoded a signal peptide, and the large and small subunits in a single open reading frame in that order. Two kinds of plasmid were constructed encoding the signal peptide and either the large or the small subunit. A gamma-glutamyltranspeptidase-less mutant of E. coli K-12 was transformed with each plasmid or with both of them. The strain harboring the plasmid encoding each subunit produced a small amount of the corresponding subunit protein in the periplasmic space but exhibited no enzymatic activity. The strain transformed with both plasmids together exhibited the enzymatic activity, but its specific activity was approximately 3% of that of a strain harboring a plasmid encoding the intact structural gene. These results indicate that a portion of the separated large and small subunits can be reconstituted in vitro and exhibit the enzymatic activity, and that the expressed large and small subunits independently are able to associate in vivo and be folded into an active structure, though the specific activity of the associated subunits was much lower than that of native enzyme. This suggests that the synthesis of gamma-glutamyltranspeptidase in a single precursor polypeptide and subsequent processing are more effective to construct the intact structure of gamma-glutamyltranspeptidase than the association of the separated large and small subunits.

The chorionic gonadotropin alpha-subunit gene is on human chromosome 18 in JEG cells.

PubMed Central

Hardin, J W; Riser, M E; Trent, J M; Kohler, P O

1983-01-01

The gene for the alpha subunit of human chorionic gonadotropin (hCG) has been tentatively assigned to human chromosome 18. This localization was accomplished through the use of Southern blot analysis. A full-length cDNA probe for the hCG alpha subunit and DNA isolated from a series of somatic hybrids between mouse and human cells were utilized to make this assignment. In addition, in situ hybridization with normal human peripheral blood lymphocytes as a source of human chromosomes and with the same cDNA probe confirmed this result. The presence of human chromosome 18 was required for the detection of DNA fragments characteristic of the alpha-hCG gene. These results are consistent with our previous observation that human chromosomes 10 and 18 are required for the production of hCG in cultured cells. Images PMID:6578509

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

Spreitzer, Robert J.

CO{sub 2} and O{sub 2} are mutually competitive at the active site of ribulose-1,5-biphosphate (RuBP) carboxylase/oxygenase (Rubisco). Rubisco contains two subunits, each present in eight copies. The 15-kD small subunit is coded by a family of nuclear RbcS genes. Until now, the role of the small subunit in Rubisco structure or catalytic efficiency is not known. Because of other work in eliminating the two RbcS genes in the green algo Chlamydomonas reinhardtii, it is now possible to address questions about the structure-function relationships of the eukaryotic small subunit. There are three specific aims in this project: (1) Alanine scanning mutagenesismore » is being used to dissect the importance of the {beta}A/{beta}B loop, a feature unique to the eukaryotic small subunit. (2) Random mutagenesis is being used to identify additional residues or regions of the small subunit that are important for holoenzyme assembly and function. (3) Attempts are being made to express foreign small subunits in Chlamydomonas to examine the contribution of small subunits to holoenzyme assembly, catalytic efficiency, and CO{sub 2}/O{sub 2} specificity.« less

Differential toxicity of TDP-43 isoforms depends on their sub-mitochondrial localization in neuronal cells.

PubMed

Salvatori, Illari; Ferri, Alberto; Scaricamazza, Silvia; Giovannelli, Ilaria; Serrano, Alessia; Rossi, Simona; D'Ambrosi, Nadia; Cozzolino, Mauro; Di Giulio, Andrea; Moreno, Sandra; Valle, Cristiana; Carrì, Maria Teresa

2018-05-20

TAR DNA binding protein 43 (TDP-43) is an RNA binding protein and a major component of protein aggregates found in Amyotrophic Lateral Sclerosis and several other neurodegenerative diseases. TDP-43 exists as a full length protein and as two shorter forms of 25 and 35 kDa. Full length mutant TDP-43s found in ALS patients re-localize from the nucleus to the cytoplasm and in part to mitochondria, where they exert a toxic role associated with neurodegeneration. However, induction of mitochondrial damage by TDP-43 fragments is yet to be clarified. In this work, we show that the mitochondrial 35 kDa truncated form of TDP-43 is restricted to the intermembrane space while the full length forms also localise in the mitochondrial matrix in cultured neuronal NSC-34 cells. Interestingly, the full length forms clearly affect mitochondrial metabolism and morphology, possibly via their ability to inhibit the expression of Complex I subunits encoded by the mitochondrial-transcribed mRNAs, while the 35 kDa form does not. In the light of the known differential contribution of the full length and short isoforms to generate toxic aggregates, we propose that the presence of full length TDP-43s in the matrix is a primary cause of mitochondrial damage. This in turn may cause oxidative stress inducing toxic oligomers formation, in which short TDP-43 forms play a major role. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Small angle X-ray scattering and cross-linking for data assisted protein structure prediction in CASP 12 with prospects for improved accuracy.

PubMed

Ogorzalek, Tadeusz L; Hura, Greg L; Belsom, Adam; Burnett, Kathryn H; Kryshtafovych, Andriy; Tainer, John A; Rappsilber, Juri; Tsutakawa, Susan E; Fidelis, Krzysztof

2018-03-01

Experimental data offers empowering constraints for structure prediction. These constraints can be used to filter equivalently scored models or more powerfully within optimization functions toward prediction. In CASP12, Small Angle X-ray Scattering (SAXS) and Cross-Linking Mass Spectrometry (CLMS) data, measured on an exemplary set of novel fold targets, were provided to the CASP community of protein structure predictors. As solution-based techniques, SAXS and CLMS can efficiently measure states of the full-length sequence in its native solution conformation and assembly. However, this experimental data did not substantially improve prediction accuracy judged by fits to crystallographic models. One issue, beyond intrinsic limitations of the algorithms, was a disconnect between crystal structures and solution-based measurements. Our analyses show that many targets had substantial percentages of disordered regions (up to 40%) or were multimeric or both. Thus, solution measurements of flexibility and assembly support variations that may confound prediction algorithms trained on crystallographic data and expecting globular fully-folded monomeric proteins. Here, we consider the CLMS and SAXS data collected, the information in these solution measurements, and the challenges in incorporating them into computational prediction. As improvement opportunities were only partly realized in CASP12, we provide guidance on how data from the full-length biological unit and the solution state can better aid prediction of the folded monomer or subunit. We furthermore describe strategic integrations of solution measurements with computational prediction programs with the aim of substantially improving foundational knowledge and the accuracy of computational algorithms for biologically-relevant structure predictions for proteins in solution. © 2018 Wiley Periodicals, Inc.

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

Jack Preiss

Conversion of the Potato tuber ADP-glucose Pyrophopshorylase Regulatory Subunit into a Catalytic Subunit. ADP-glucose synthesis, a rate-limiting reaction in starch synthesis, is catalyzed by ADP-glucose pyrophosphorylase (ADPGlc PPase). The enzyme in plants is allosterically activated by 3-phosphoglycerate (3PGA) and inhibited by inorganic phosphate (Pi) and is composed of two subunits as a heterotetramer, a2b2. Subunit a is the catalytic subunit and subunit b is designated as the regulatory subunit.The b subunit increases the affinty of the activator for the catalytic subunit. Recent results have shown that the subunits are derived from the same ancestor subunit as the regulatory subunit canmore » be converted to a catalytically subunit via mutation of just two amino acids. Lys44 and Thr54 in the large subunit from potato tuber were converted to the homologous catalytic subunit residues, Arg33 and Lys43. The activity of the large subunit mutants cannot be readily tested with a co-expressed wild-type small (catalytic) subunit because of the intrinsic activity of the latter. We co-expressed the regulatory-subunit mutants with SmallD145N, an inactive S subunit in which the catalytic Asp145 was mutated. The activity of the small (catalytic) subunit was reduced more than three orders of magnitude. Coexpression of the L subunit double mutant LargeK44R/T54K with SmallD145N generated an enzyme with considerable activity, 10% and 18% of the wildtype enzyme, in the ADP-glucose synthetic and pyrophosphorolytic direction, respectively. Replacement of those two residues in the small subunit by the homologous amino acids in the L subunits (mutations R33K and K43T) decreased the activity one and two orders of magnitude. The wild-type enzyme and SmallD145NLargeK44R/T54K had very similar kinetic properties indicating that the substrate site has been conserved. The fact that only two mutations in the L subunit restored enzyme activity is very strong evidence that the large subunit is derived from the catalytic ancestor. Previous results showed that Asp145 in the small subunit of the wild-type is essential for catalysis, whereas the homologous Asp160 in the Large WT subunit is not. However, in this study, mutation D160N or D160E in the LK44R/T54K subunit abolished the activity, which shows the ancestral essential role of this residue and confirms that the catalysis of SmallD145NLarge K44R/T54K occurs in the L(b) subunit. A phylogenetic tree of the ADP-Glc PPases present in photosynthetic eukaryotes also sheds information about the origin of the subunits. The tree showed that plant Small and Large subunits can be divided into two and four distinct groups, respectively. The two main groups of S subunits are from dicot and monocot plants, whereas Large subunit groups correlate better with their documented tissue expression. The first Large-subunit group is generally expressed in photosynthetic tissues and comprises Large subunits from dicots and monocots. Group II displays a broader expression pattern, whereas groups III and IV are expressed in storage organs (roots, stems, tubers, seeds). Subunits from group III are only from dicot plants, whereas group IV are seed-specific subunits from monocots. These last two groups stem from the same branch of the phylogenetic tree and split before monocot and dicot separation. Thus few as two mutations turned the L subunit from Solanum tuberosum catalytic, showing that L and S subunits share a common catalytic ancestor, rather than a non-catalytic one. The L subunit evolved to have a regulatory role, lost catalytic residues more than 130 million years ago before monocots and dicots diverged, and preserved, possibly as a byproduct, the active site domain.« less

Exact Length Distribution of Filamentous Structures Assembled from a Finite Pool of Subunits.

PubMed

Harbage, David; Kondev, Jané

2016-07-07

Self-assembling filamentous structures made of protein subunits are ubiquitous in cell biology. These structures are often highly dynamic, with subunits in a continuous state of flux, binding to and falling off of filaments. In spite of this constant turnover of their molecular parts, many cellular structures seem to maintain a well-defined size over time, which is often required for their proper functioning. One widely discussed mechanism of size regulation involves the cell maintaining a finite pool of protein subunits available for assembly. This finite pool mechanism can control the length of a single filament by having assembly proceed until the pool of free subunits is depleted to the point when assembly and disassembly are balanced. Still, this leaves open the question of whether the same mechanism can provide size control for multiple filamentous structures that are assembled from a common pool of protein subunits, as is often the case in cells. We address this question by solving the steady-state master equation governing the stochastic assembly and disassembly of multifilament structures made from a shared finite pool of subunits. We find that, while the total number of subunits within a multifilament structure is well-defined, individual filaments within the structure have a wide, power-law distribution of lengths. We also compute the phase diagram for two multifilament structures competing for the same pool of subunits and identify conditions for coexistence when both have a well-defined size. These predictions can be tested in cell experiments in which the size of the subunit pool or the number of filament nucleators is tuned.

Morphology and Description of Bursaphelenchus platzeri n. sp. (Nematoda: Parasitaphelenchidae), an Associate of Nitidulid Beetles

PubMed Central

Giblin-Davis, Robin M.; Kanzaki, Natsumi; Ye, Weimin; Mundo-Ocampo, Manuel; Baldwin, James G.; Thomas, W. Kelley

2006-01-01

Bursaphelenchus platzeri n. sp., an associate of nitidulid beetles in southern California, is described and illustrated. Adult males and females of B. platzeri n. sp. were examined by scanning electron microscopy for ultrastructural comparisons with other members of the genus. Bursaphelenchus cocophilus (red ring nematode) appears to be the closest related taxon to B. platzeri n. sp. based upon shared morphological features of the fused spicules, female tail shape, phoresy with non-scolytid beetles, and molecular analysis of the near full-length small subunit (SSU) rDNA. Unfortunately, sequence data from the D2D3 expansion segments of the large subunit (LSU) rDNA and partial mitochondrial DNA COI did not help resolve the relationship of nearest relative. In addition to significant molecular sequence differences in SSU, LSU, and COI, B. platzeri n. sp., which is an obligate fungal feeder, can be differentiated from B. cocophilus because it is an obligate parasite of palms. Bursaphelenchus platzeri n. sp. can be differentiated from all other species of Bursaphelenchus by the length and shape of the female tail and spicule morphology. The spicules are fused along the ventral midline and possess unfused cucullae; the fused unit appears to function as a conduit for sperm. Population growth of B. platzeri n. sp. was measured in a time-course experiment at 25°C in the laboratory on cultures of the fungus Monilinia fructicola grown on 5% glycerol-supplemented potato dextrose agar (GPDA). Nematode population densities rapidly increased from 25 to approximately 200,000/culture within 14 d and then plateaued for up to 28 d. PMID:19259440

From genus to phylum: large-subunit and internal transcribed spacer rRNA operon regions show similar classification accuracies influenced by database composition.

PubMed

Porras-Alfaro, Andrea; Liu, Kuan-Liang; Kuske, Cheryl R; Xie, Gary

2014-02-01

We compared the classification accuracy of two sections of the fungal internal transcribed spacer (ITS) region, individually and combined, and the 5' section (about 600 bp) of the large-subunit rRNA (LSU), using a naive Bayesian classifier and BLASTN. A hand-curated ITS-LSU training set of 1,091 sequences and a larger training set of 8,967 ITS region sequences were used. Of the factors evaluated, database composition and quality had the largest effect on classification accuracy, followed by fragment size and use of a bootstrap cutoff to improve classification confidence. The naive Bayesian classifier and BLASTN gave similar results at higher taxonomic levels, but the classifier was faster and more accurate at the genus level when a bootstrap cutoff was used. All of the ITS and LSU sections performed well (>97.7% accuracy) at higher taxonomic ranks from kingdom to family, and differences between them were small at the genus level (within 0.66 to 1.23%). When full-length sequence sections were used, the LSU outperformed the ITS1 and ITS2 fragments at the genus level, but the ITS1 and ITS2 showed higher accuracy when smaller fragment sizes of the same length and a 50% bootstrap cutoff were used. In a comparison using the larger ITS training set, ITS1 and ITS2 had very similar accuracy classification for fragments between 100 and 200 bp. Collectively, the results show that any of the ITS or LSU sections we tested provided comparable classification accuracy to the genus level and underscore the need for larger and more diverse classification training sets.

From Genus to Phylum: Large-Subunit and Internal Transcribed Spacer rRNA Operon Regions Show Similar Classification Accuracies Influenced by Database Composition

PubMed Central

Liu, Kuan-Liang; Kuske, Cheryl R.

2014-01-01

We compared the classification accuracy of two sections of the fungal internal transcribed spacer (ITS) region, individually and combined, and the 5′ section (about 600 bp) of the large-subunit rRNA (LSU), using a naive Bayesian classifier and BLASTN. A hand-curated ITS-LSU training set of 1,091 sequences and a larger training set of 8,967 ITS region sequences were used. Of the factors evaluated, database composition and quality had the largest effect on classification accuracy, followed by fragment size and use of a bootstrap cutoff to improve classification confidence. The naive Bayesian classifier and BLASTN gave similar results at higher taxonomic levels, but the classifier was faster and more accurate at the genus level when a bootstrap cutoff was used. All of the ITS and LSU sections performed well (>97.7% accuracy) at higher taxonomic ranks from kingdom to family, and differences between them were small at the genus level (within 0.66 to 1.23%). When full-length sequence sections were used, the LSU outperformed the ITS1 and ITS2 fragments at the genus level, but the ITS1 and ITS2 showed higher accuracy when smaller fragment sizes of the same length and a 50% bootstrap cutoff were used. In a comparison using the larger ITS training set, ITS1 and ITS2 had very similar accuracy classification for fragments between 100 and 200 bp. Collectively, the results show that any of the ITS or LSU sections we tested provided comparable classification accuracy to the genus level and underscore the need for larger and more diverse classification training sets. PMID:24242255

Cloning and sequence analysis of a full-length cDNA of SmPP1cb encoding turbot protein phosphatase 1 beta catalytic subunit

NASA Astrophysics Data System (ADS)

Qi, Fei; Guo, Huarong; Wang, Jian

2008-02-01

Reversible protein phosphorylation, catalyzed by protein kinases and phosphatases, is an important and versatile mechanism by which eukaryotic cells regulate almost all the signaling processes. Protein phosphatase 1 (PP1) is the first and well-characterized member of the protein serine/threonine phosphatase family. In the present study, a full-length cDNA encoding the beta isoform of the catalytic subunit of protein phosphatase 1(PP1cb), was for the first time isolated and sequenced from the skin tissue of flatfish turbot Scophthalmus maximus, designated SmPP1cb, by the rapid amplification of cDNA ends (RACE) technique. The cDNA sequence of SmPP1cb we obtained contains a 984 bp open reading frame (ORF), flanked by a complete 39 bp 5' untranslated region and 462 bp 3' untranslated region. The ORF encodes a putative 327 amino acid protein, and the N-terminal section of this protein is highly acidic, Met-Ala-Glu-Gly-Glu-Leu-Asp-Val-Asp, a common feature for PP1 catalytic subunit but absent in protein phosphatase 2B (PP2B). And its calculated molecular mass is 37 193 Da and pI 5.8. Sequence analysis indicated that, SmPP1cb is extremely conserved in both amino acid and nucleotide acid levels compared with the PP1cb of other vertebrates and invertebrates, and its Kozak motif contained in the 5'UTR around ATG start codon is GXXAXXGXX ATGG, which is different from mammalian in two positions A-6 and G-3, indicating the possibility of different initiation of translation in turbot, and also the 3'UTR of SmPP1cb is highly diverse in the sequence similarity and length compared with other animals, especially zebrafish. The cloning and sequencing of SmPP1cb gene lays a good foundation for the future work on the biological functions of PP1 in the flatfish turbot.

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

Spreitzer, Robert Joseph

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of CO 2 fixation in photosynthesis. However, it is a slow enzyme, and O 2 competes with CO 2 at the active site. Oxygenation initiates the photorespiratory pathway, which also results in the loss of CO 2. If carboxylation could be increased or oxygenation decreased, an increase in net CO 2 fixation would be realized. Because Rubisco provides the primary means by which carbon enters all life on earth, there is much interest in engineering Rubisco to increase the production of food and renewable energy. Rubisco is located in the chloroplasts of plants,more » and it is comprised of two subunits. Much is known about the chloroplast-gene-encoded large subunit (rbcL gene), which contains the active site, but much less is known about the role of the nuclear-gene-encoded small subunit in Rubisco function (rbcS gene). Both subunits are coded by multiple genes in plants, which makes genetic engineering difficult. In the eukaryotic, green alga Chlamydomonas reinhardtii, it has been possible to eliminate all the Rubisco genes. These Rubisco-less mutants can be maintained by providing acetate as an alternative carbon source. In this project, focus has been placed on determining whether the small subunit might be a better genetic-engineering target for improving Rubisco. Analysis of a variable-loop structure (βA-βB loop) of the small subunit by genetic selection, directed mutagenesis, and construction of chimeras has shown that the small subunit can influence CO 2/O 2 specificity. X-ray crystal structures of engineered chimeric-loop enzymes have indicated that additional residues and regions of the small subunit may also contribute to Rubisco function. Structural dynamics of the small-subunit carboxyl terminus was also investigated. Alanine-scanning mutagenesis of the most-conserved small-subunit residues has identified a possible structural pathway between the small-subunit βA-βB loop and alpha-helix 8 of the large-subunit α/β-barrel active site. Hybrid enzymes were also created comprised of plant small subunits and Chlamydomonas large subunits, and these enzymes have increases in CO 2/O 2 specificity, further indicating that small subunits may be the key for ultimately engineering an improved Rubisco enzyme.« less

Creation of a data base for sequences of ribosomal nucleic acids and detection of conserved restriction endonucleases sites through computerized processing.

PubMed Central

Patarca, R; Dorta, B; Ramirez, J L

1982-01-01

As part of a project pertaining the organization of ribosomal genes in Kinetoplastidae, we have created a data base for published sequences of ribosomal nucleic acids, with information in Spanish. As a first step in their processing, we have written a computer program which introduces the new feature of determining the length of the fragments produced after single or multiple digestion with any of the known restriction enzymes. With this information we have detected conserved SAU 3A sites: (i) at the 5' end of the 5.8S rRNA and at the 3' end of the small subunit rRNA, both included in similar larger sequences; (ii) in the 5.8S rRNA of vertebrates (a second one), which is not present in lower eukaryotes, showing a clear evolutive divergence; and, (iii) at the 5' terminal of the small subunit rRNA, included in a larger conserved sequence. The possible biological importance of these sequences is discussed. PMID:6278402

Identification of a short sequence in the HCMV terminase pUL56 essential for interaction with pUL89 subunit.

PubMed

Ligat, G; Jacquet, C; Chou, S; Couvreux, A; Alain, S; Hantz, S

2017-08-18

The human cytomegalovirus (HCMV) terminase complex consists of several components acting together to cleave viral DNA into unit length genomes and translocate them into capsids, a critical process in the production of infectious virions subsequent to DNA replication. Previous studies suggest that the carboxyl-terminal portion of the pUL56 subunit interacts with the pUL89 subunit. However, the specific interacting residues of pUL56 remain unknown. We identified a conserved sequence in the C-terminal moiety of pUL56 ( 671 WMVVKYMGFF 680 ). Overrepresentation of conserved aromatic amino acids through 20 herpesviruses homologues of pUL56 suggests an involvement of this short peptide into the interaction between the larger pUL56 terminase subunit and the smaller pUL89 subunit. Use of Alpha technology highlighted an interaction between pUL56 and pUL89 driven through the peptide 671 WMVVKYMGFF 680 . A deletion of these residues blocks viral replication. We hypothesize that it is the consequence of the disruption of the pUL56-pUL89 interaction. These results show that this motif is essential for HCMV replication and could be a target for development of new small antiviral drugs or peptidomimetics.

A novel role for the condensin II complex in cellular senescence.

PubMed

Yokoyama, Yuhki; Zhu, Hengrui; Zhang, Rugang; Noma, Ken-ichi

2015-01-01

Although cellular senescence is accompanied by global alterations in genome architecture, how the genome is restructured during the senescent processes is not well understood. Here, we show that the hCAP-H2 subunit of the condensin II complex exists as either a full-length protein or an N-terminus truncated variant (ΔN). While the full-length hCAP-H2 associates with mitotic chromosomes, the ΔN variant exists as an insoluble nuclear structure. When overexpressed, both hCAP-H2 isoforms assemble this nuclear architecture and induce senescence-associated heterochromatic foci (SAHF). The hCAP-H2ΔN protein accumulates as cells approach senescence, and hCAP-H2 knockdown inhibits oncogene-induced senescence. This study identifies a novel mechanism whereby condensin drives senescence via nuclear/genomic reorganization.

Cloning and restriction enzyme mapping of ribosomal DNA of Giardia duodenalis, Giardia ardeae and Giardia muris.

PubMed

van Keulen, H; Campbell, S R; Erlandsen, S L; Jarroll, E L

1991-06-01

In an attempt to study Giardia at the DNA sequence level, the rRNA genes of three species, Giardia duodenalis, Giardia ardeae and Giardia muris were cloned and restriction enzyme maps were constructed. The rDNA repeats of these Giardia show completely different restriction enzyme recognition patterns. The size of the rDNA repeat ranges from approximately 5.6 kb in G. duodenalis to 7.6 kb in both G. muris and G. ardeae. These size differences are mainly attributable to the variation in length of the spacer. Minor differences exist among these Giardia in the sizes of their small subunit rRNA and the internal transcribed spacer between small and large subunit rRNA. The genetic maps were constructed by sequence analysis of the DNA around the 5' and 3' ends of the mature rRNA genes and between the rRNA covering the 5.8S rRNA gene and internal transcribed spacer. Comparison of the 5.8S rDNA and 3' end of large subunit rDNA from these three Giardia species showed considerable sequence variation, but the rDNA sequences of G. duodenalis and G. ardeae appear more closely related to each other than to G. muris.

Molecular Architecture of Full-length TRF1 Favors Its Interaction with DNA.

PubMed

Boskovic, Jasminka; Martinez-Gago, Jaime; Mendez-Pertuz, Marinela; Buscato, Alberto; Martinez-Torrecuadrada, Jorge Luis; Blasco, Maria A

2016-10-07

Telomeres are specific DNA-protein structures found at both ends of eukaryotic chromosomes that protect the genome from degradation and from being recognized as double-stranded breaks. In vertebrates, telomeres are composed of tandem repeats of the TTAGGG sequence that are bound by a six-subunit complex called shelterin. Molecular mechanisms of telomere functions remain unknown in large part due to lack of structural data on shelterins, shelterin complex, and its interaction with the telomeric DNA repeats. TRF1 is one of the best studied shelterin components; however, the molecular architecture of the full-length protein remains unknown. We have used single-particle electron microscopy to elucidate the structure of TRF1 and its interaction with telomeric DNA sequence. Our results demonstrate that full-length TRF1 presents a molecular architecture that assists its interaction with telometic DNA and at the same time makes TRFH domains accessible to other TRF1 binding partners. Furthermore, our studies suggest hypothetical models on how other proteins as TIN2 and tankyrase contribute to regulate TRF1 function. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Molecular Architecture of Full-length TRF1 Favors Its Interaction with DNA*

PubMed Central

Boskovic, Jasminka; Martinez-Gago, Jaime; Mendez-Pertuz, Marinela; Buscato, Alberto; Martinez-Torrecuadrada, Jorge Luis; Blasco, Maria A.

2016-01-01

Telomeres are specific DNA-protein structures found at both ends of eukaryotic chromosomes that protect the genome from degradation and from being recognized as double-stranded breaks. In vertebrates, telomeres are composed of tandem repeats of the TTAGGG sequence that are bound by a six-subunit complex called shelterin. Molecular mechanisms of telomere functions remain unknown in large part due to lack of structural data on shelterins, shelterin complex, and its interaction with the telomeric DNA repeats. TRF1 is one of the best studied shelterin components; however, the molecular architecture of the full-length protein remains unknown. We have used single-particle electron microscopy to elucidate the structure of TRF1 and its interaction with telomeric DNA sequence. Our results demonstrate that full-length TRF1 presents a molecular architecture that assists its interaction with telometic DNA and at the same time makes TRFH domains accessible to other TRF1 binding partners. Furthermore, our studies suggest hypothetical models on how other proteins as TIN2 and tankyrase contribute to regulate TRF1 function. PMID:27563064

The delta-subunit of murine guanine nucleotide exchange factor eIF-2B. Characterization of cDNAs predicts isoforms differing at the amino-terminal end.

PubMed

Henderson, R A; Krissansen, G W; Yong, R Y; Leung, E; Watson, J D; Dholakia, J N

1994-12-02

Protein synthesis in mammalian cells is regulated at the level of the guanine nucleotide exchange factor, eIF-2B, which catalyzes the exchange of eukaryotic initiation factor 2-bound GDP for GTP. We have isolated and sequenced cDNA clones encoding the delta-subunit of murine eIF-2B. The cDNA sequence encodes a polypeptide of 544 amino acids with molecular mass of 60 kDa. Antibodies against a synthetic polypeptide of 30 amino acids deduced from the cDNA sequence specifically react with the delta-subunit of mammalian eIF-2B. The cDNA-derived amino acid sequence shows significant homology with the yeast translational regulator Gcd2, supporting the hypothesis that Gcd2 may be the yeast homolog of the delta-subunit of mammalian eIF-2B. Primer extension studies and anchor polymerase chain reaction analysis were performed to determine the 5'-end of the transcript for the delta-subunit of eIF-2B. Results of these experiments demonstrate two different mRNAs for the delta-subunit of eIF-2B in murine cells. The isolation and characterization of two different full-length cDNAs also predicts the presence of two alternate forms of the delta-subunit of eIF-2B in murine cells. These differ at their amino-terminal end but have identical nucleotide sequences coding for amino acids 31-544.

Identification of a tetramerization domain in the C terminus of the vanilloid receptor.

PubMed

García-Sanz, Nuria; Fernández-Carvajal, Asia; Morenilla-Palao, Cruz; Planells-Cases, Rosa; Fajardo-Sánchez, Emmanuel; Fernández-Ballester, Gregorio; Ferrer-Montiel, Antonio

2004-06-09

TRPV1 (transient receptor potential vanilloid receptor subtype 1) is a member of the TRP channel family gated by vanilloids, protons, and heat. Structurally, TRPV1 appears to be a tetramer formed by the assembly of four identical subunits around a central aqueous pore. The molecular determinants that govern its subunit oligomerization remain elusive. Here, we report the identification of a segment comprising 684Glu-721Arg (referred to as the TRP-like domain) in the C terminus of TRPV1 as an association domain (AD) of the protein. Purified recombinant C terminus of TRPV1 (TRPV1-C) formed discrete and stable multimers in vitro. Yeast two-hybrid and pull-down assays showed that self-association of the TRPV1-C is blocked when segment 684Glu-721Arg is deleted. Biochemical and immunological analysis indicate that removal of the AD from full-length TRPV1 monomers blocks the formation of stable heteromeric assemblies with wild-type TRPV1 subunits. Deletion of the AD in a poreless TRPV1 subunit suppressed its robust dominant-negative phenotype. Together, these findings are consistent with the tenet that the TRP-like domain in TRPV1 is a molecular determinant of the tetramerization of receptor subunits into functional channels. Our observations suggest that the homologous TRP domain in the TRP protein family may function as a general, evolutionary conserved AD involved in subunit multimerization.

In vivo reconstitution of a homodimeric cytochrome b559 like structure: The role of the N-terminus α-subunit from Synechocystis sp. PCC 6803.

PubMed

Luján, María A; Martínez, Jesús I; Alonso, Pablo J; Torrado, Alejandro; Roncel, Mercedes; Ortega, José M; Sancho, Javier; Picorel, Rafael

2015-11-01

The cytochrome b559 is a heme-bridged heterodimeric protein with two subunits, α and β. Both subunits from Synechocystis sp. PCC 6803 have previously been cloned and overexpressed in Escherichia coli and in vivo reconstitution experiments have been carried out. The formation of homodimers in the bacterial membrane with endogenous heme was only observed in the case of the β-subunit (β/β) but not with the full length α-subunit. In the present work, reconstitution of a homodimer (α/α) cytochrome b559 like structure was possible using a chimeric N-terminus α-subunit truncated before the amino acid isoleucine 17, eliminating completely a short amphipathic α-helix that lays on the surface of the membrane. Overexpression and in vivo reconstitution in the bacteria was clearly demonstrated by the brownish color of the culture pellet and the use of a commercial monoclonal antibody against the fusion protein carrier, the maltoside binding protein, and polyclonal antibodies against a synthetic peptide of the α-subunit from Thermosynechococcus elongatus. Moreover, a simple partial purification after membrane solubilization with Triton X-100 confirmed that the overexpressed protein complex corresponded with the maltoside binding protein-chimeric α-subunit cytochrome b559 like structure. The features of the new structure were determined by UV-Vis, electron paramagnetic resonance and redox potentiometric techniques. Ribbon representations of all possible structures are also shown to better understand the mechanism of the cytochrome b559 maturation in the bacterial cytoplasmic membrane. Copyright © 2015 Elsevier B.V. All rights reserved.

Binding-dependent disorder-order transition in PKI alpha: a fluorescence anisotropy study.

PubMed

Hauer, J A; Taylor, S S; Johnson, D A

1999-05-25

The conformational flexibility of peptidyl ligands may be an essential element of many peptide-macromolecular interactions. Consequently, the alpha-carbonyl backbone flexibility of the 8 kDa protein kinase inhibitor (PKI alpha) peptide of cAMP-dependent protein kinase (cAPK) free in solution and bound to cAPK was assessed by time-resolved fluorescence anisotropy. Specifically, three full-length, single-site PKI alpha mutants (V3C, S28C, and S59C) were prepared, and fluorescein iodoacetamide (FI) was selectively conjugated to the side chains of each substituted cysteine. The time-resolved anisotropy decay profiles of the labeled mutants were well fit to a model-free nonassociative biexponential equation. Free in solution, the three labeled proteins had very similar anisotropy decays arising primarily from local alpha-carbonyl backbone movements. Only a small fraction of the anisotropy decay was associated with slower, whole-body tumbling, confirming that PKI alpha is highly disordered at all three locations. Complexation of the mutants with the catalytic (C) subunit of cAPK decreased the rate of whole-body tumbling for all three mutants. The effects on the rapid decay processes, however, were dependent upon the site of conjugation. The anisotropy decay profiles of both FI-V3C- and FI-S28C-PKI alpha were associated with significantly reduced contributions from the fast decay processes, while that of FI-S59C-PKI alpha was largely unaffected by binding to the C-subunit. The results suggest that the cAPK-binding domain of PKI alpha extends from the its N-terminus to residues beyond Ser28 but does not include the segment around Ser59, which is still part of a highly flexible domain when bound to the C-subunit.

Superstructure for high current applications in superconducting linear accelerators

DOEpatents

Sekutowicz, Jacek [Elbchaussee, DE; Kneisel, Peter [Williamsburg, VA

2008-03-18

A superstructure for accelerating charged particles at relativistic speeds. The superstructure consists of two weakly coupled multi-cell subunits equipped with HOM couplers. A beam pipe connects the subunits and an HOM damper is included at the entrance and the exit of each of the subunits. A coupling device feeds rf power into the subunits. The subunits are constructed of niobium and maintained at cryogenic temperatures. The length of the beam pipe between the subunits is selected to provide synchronism between particles and rf fields in both subunits.

Small subunit of a cold-resistant plant, Timothy, does not significantly alter the catalytic properties of Rubisco in transgenic rice.

PubMed

Fukayama, Hiroshi; Koga, Atsushi; Hatanaka, Tomoko; Misoo, Shuji

2015-04-01

Effects of overexpression of high activity-type Rubisco small subunit (RbcS) from a cold-resistant plant, timothy (Phleum pratense), on kinetic properties of Rubisco were studied in rice (Oryza sativa). The full-length mRNA sequence of timothy RbcS (PpRbcS1) was determined by 5'RACE and 3'RACE. The coding sequence of PpRbcS1 was fused to the chlorophyll a/b-binding protein promoter and introduced into rice. PpRbcS was highly expressed in leaf blade and accounted for approximately 30 % of total RbcS in homozygous transgenic lines. However, the catalytic turnover rate and K m for CO2 of Rubisco did not significantly change in these transgenic lines compared to non-transgenic rice, suggesting that PpRbcS1 is not effective for improvement of catalytic efficiency of rice Rubisco. The photosynthetic rate and growth were essentially unchanged, whereas the photosynthetic rate at low CO2 condition was marginally increased in transgenic lines. Rubisco content was significantly increased, whereas soluble protein, nitrogen, and chlorophyll contents were unchanged in transgenic lines compared to non-transgenic rice. Because the kinetic properties were similar, observed slight increase in photosynthetic rate at low CO2 is considered to be large due to increase in Rubisco content in transgenic lines. Introduction of foreign RbcS is an effective approach for the improvement of Rubisco kinetics and photosynthesis. However, in this study, it was suggested that RbcS of high activity-type Rubisco, even showing higher amino acid identity with rice RbcS, did not always enhance the catalytic turnover rate of Rubisco in rice. Thus, we should carefully select RbcS to be overexpressed before introduction.

Reconstitution of the Recombinant RanBP2 SUMO E3 Ligase Complex.

PubMed

Ritterhoff, Tobias; Das, Hrishikesh; Hao, Yuqing; Sakin, Volkan; Flotho, Annette; Werner, Andreas; Melchior, Frauke

2016-01-01

One of the few proteins that have SUMO E3 ligase activity is the 358 kDa nucleoporin RanBP2 (Nup358). While small fragments of RanBP2 can stimulate SUMOylation in vitro, the physiologically relevant E3 ligase is a stable multi-subunit complex comprised of RanBP2, SUMOylated RanGAP1, and Ubc9. Here, we provide a detailed protocol to in vitro reconstitute the RanBP2 SUMO E3 ligase complex. With the exception of RanBP2, reconstitution involves untagged full-length proteins. We describe the bacterial expression and purification of all complex components, namely an 86 kDa His-tagged RanBP2 fragment, the SUMO E2-conjugating enzyme Ubc9, RanGAP1, and SUMO1, and we provide a protocol for quantitative SUMOylation of RanGAP1. Finally, we present details for the assembly and final purification of the catalytically active RanBP2/RanGAP1*SUMO1/Ubc9 complex.

Roles of yeast eIF2α and eIF2β subunits in the binding of the initiator methionyl-tRNA

PubMed Central

Naveau, Marie; Lazennec-Schurdevin, Christine; Panvert, Michel; Dubiez, Etienne; Mechulam, Yves; Schmitt, Emmanuelle

2013-01-01

Heterotrimeric eukaryotic/archaeal translation initiation factor 2 (e/aIF2) binds initiator methionyl-tRNA and plays a key role in the selection of the start codon on messenger RNA. tRNA binding was extensively studied in the archaeal system. The γ subunit is able to bind tRNA, but the α subunit is required to reach high affinity whereas the β subunit has only a minor role. In Saccharomyces cerevisiae however, the available data suggest an opposite scenario with β having the most important contribution to tRNA-binding affinity. In order to overcome difficulties with purification of the yeast eIF2γ subunit, we designed chimeric eIF2 by assembling yeast α and β subunits to archaeal γ subunit. We show that the β subunit of yeast has indeed an important role, with the eukaryote-specific N- and C-terminal domains being necessary to obtain full tRNA-binding affinity. The α subunit apparently has a modest contribution. However, the positive effect of α on tRNA binding can be progressively increased upon shortening the acidic C-terminal extension. These results, together with small angle X-ray scattering experiments, support the idea that in yeast eIF2, the tRNA molecule is bound by the α subunit in a manner similar to that observed in the archaeal aIF2–GDPNP–tRNA complex. PMID:23193270

Highly conserved small subunit residues influence rubisco large subunit catalysis.

PubMed

Genkov, Todor; Spreitzer, Robert J

2009-10-30

The chloroplast enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of photosynthetic CO(2) fixation. With a deeper understanding of its structure-function relationships and competitive inhibition by O(2), it may be possible to engineer an increase in agricultural productivity and renewable energy. The chloroplast-encoded large subunits form the active site, but the nuclear-encoded small subunits can also influence catalytic efficiency and CO(2)/O(2) specificity. To further define the role of the small subunit in Rubisco function, the 10 most conserved residues in all small subunits were substituted with alanine by transformation of a Chlamydomonas reinhardtii mutant that lacks the small subunit gene family. All the mutant strains were able to grow photosynthetically, indicating that none of the residues is essential for function. Three of the substitutions have little or no effect (S16A, P19A, and E92A), one primarily affects holoenzyme stability (L18A), and the remainder affect catalysis with or without some level of associated structural instability (Y32A, E43A, W73A, L78A, P79A, and F81A). Y32A and E43A cause decreases in CO(2)/O(2) specificity. Based on the x-ray crystal structure of Chlamydomonas Rubisco, all but one (Glu-92) of the conserved residues are in contact with large subunits and cluster near the amino- or carboxyl-terminal ends of large subunit alpha-helix 8, which is a structural element of the alpha/beta-barrel active site. Small subunit residues Glu-43 and Trp-73 identify a possible structural connection between active site alpha-helix 8 and the highly variable small subunit loop between beta-strands A and B, which can also influence Rubisco CO(2)/O(2) specificity.

Analysis of colonization factor antigen I, an adhesin of enterotoxigenic Escherichia coli O78:H11: fimbrial morphology and location of the receptor-binding site.

PubMed Central

Bühler, T; Hoschützky, H; Jann, K

1991-01-01

Colonization factor antigen I (CFA/I) of enterotoxigenic Escherichia coli was dissociated into one type of subunit (15 kDa). The dissociation was achieved either by heating CFA/I in sodium dodecyl sulfate at 100 degrees C or by heating it for 20 min in water. Heating in water to 100 degrees C yielded only in the 15-kDa subunit, but heating to 85 degree C yielded small amounts of oligomers in addition. The monomeric subunits obtained after heating in water are stable, as demonstrated by gel permeation chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis without heating prior to the electrophoretic run. These subunits inhibited CFA/I-induced hemagglutination, indicating that they had maintained their receptor-binding properties. When the hybridoma technique was used, two types of monoclonal anti-CFA/I antibodies were obtained. Antibodies obtained by immunization with the purified subunits were more reactive with subunits than with fimbriae, as shown by enzyme-linked immunosorbent assay. These antibodies strongly inhibited CFA/I-induced hemagglutination. When examined by immunoelectron microscopy, these antibodies seemed to label the fimbrial tips. A similar labeling pattern was obtained with gold particles modified with the receptor ganglioside GM2. Antibodies obtained by immunization with fimbriae reacted in enzyme-linked immunosorbent assays equally well with fimbriae and subunits. They inhibited CFA/I-induced hemagglutination only slightly. Immunoelectron microscopy revealed that these antibodies labeled the fimbriae densely and regularly over their entire lengths. In a coagglutination experiment with Staphylococcus aureus and monoclonal antibodies, the subunits retained their receptor-binding properties. From these results, we conclude that CFA/I fimbriae consist entirely of one type of adhesive subunit, of which only the one at the tip is accessible to the receptor. Images PMID:1682253

Protein synthesis during acquisition of long-term facilitation is needed for the persistent loss of regulatory subunits of the Aplysia cAMP-dependent protein kinase.

PubMed Central

Bergold, P J; Sweatt, J D; Winicov, I; Weiss, K R; Kandel, E R; Schwartz, J H

1990-01-01

Depending on the number or the length of exposure, application of serotonin can produce either short-term or long-term presynaptic facilitation of Aplysia sensory-to-motor synapses. The cAMP-dependent protein kinase, a heterodimer of two regulatory and two catalytic subunits, has been shown to become stably activated only during long-term facilitation. Both acquisition of long-term facilitation and persistent activation of the kinase is blocked by anisomycin, an effective, reversible, and specific inhibitor of protein synthesis in Aplysia. We report here that 2-hr exposure of pleural sensory cells to serotonin lowers the concentration of regulatory subunits but does not change the concentration of catalytic subunits, as assayed 24 hr later; 5-min exposure to serotonin has no effect on either type of subunit. Increasing intracellular cAMP with a permeable analog of cAMP together with the phosphodiesterase inhibitor isobutyl methylxanthine also decreased regulatory subunits, suggesting that cAMP is the second messenger mediating serotonin action. Anisomycin blocked the loss of regulatory subunits only when applied with serotonin; application after the 2-hr treatment with serotonin had no effect. In the Aplysia accessory radula contractor muscle, prolonged exposure to serotonin or to the peptide transmitter small cardioactive peptide B, both of which produce large increases in intracellular cAMP, does not decrease regulatory subunits. This mechanism of regulating the cAMP-dependent protein kinase therefore may be specific to the nervous system. We conclude that during long-term facilitation, new protein is synthesized in response to the facilitatory stimulus, which changes the ratio of subunits of the cAMP-dependent protein kinase. This alteration in ratio could persistently activate the kinase and produce the persistent phosphorylation seen in long-term facilitated sensory cells. Images PMID:1692622

Subunits of ADA-two-A-containing (ATAC) or Spt-Ada-Gcn5-acetyltrasferase (SAGA) Coactivator Complexes Enhance the Acetyltransferase Activity of GCN5.

PubMed

Riss, Anne; Scheer, Elisabeth; Joint, Mathilde; Trowitzsch, Simon; Berger, Imre; Tora, László

2015-11-27

Histone acetyl transferases (HATs) play a crucial role in eukaryotes by regulating chromatin architecture and locus specific transcription. GCN5 (KAT2A) is a member of the GNAT (Gcn5-related N-acetyltransferase) family of HATs. In metazoans this enzyme is found in two functionally distinct coactivator complexes, SAGA (Spt Ada Gcn5 acetyltransferase) and ATAC (Ada Two A-containing). These two multiprotein complexes comprise complex-specific and shared subunits, which are organized in functional modules. The HAT module of ATAC is composed of GCN5, ADA2a, ADA3, and SGF29, whereas in the SAGA HAT module ADA2b is present instead of ADA2a. To better understand how the activity of human (h) hGCN5 is regulated in the two related, but different, HAT complexes we carried out in vitro HAT assays. We compared the activity of hGCN5 alone with its activity when it was part of purified recombinant hATAC or hSAGA HAT modules or endogenous hATAC or hSAGA complexes using histone tail peptides and full-length histones as substrates. We demonstrated that the subunit environment of the HAT complexes into which GCN5 incorporates determines the enhancement of GCN5 activity. On histone peptides we show that all the tested GCN5-containing complexes acetylate mainly histone H3K14. Our results suggest a stronger influence of ADA2b as compared with ADA2a on the activity of GCN5. However, the lysine acetylation specificity of GCN5 on histone tails or full-length histones was not changed when incorporated in the HAT modules of ATAC or SAGA complexes. Our results thus demonstrate that the catalytic activity of GCN5 is stimulated by subunits of the ADA2a- or ADA2b-containing HAT modules and is further increased by incorporation of the distinct HAT modules in the ATAC or SAGA holo-complexes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Allosteric mechanism of water channel gating by Ca2+–calmodulin

PubMed Central

Reichow, Steve L.; Clemens, Daniel M.; Freites, J. Alfredo; Németh-Cahalan, Karin L.; Heyden, Matthias; Tobias, Douglas J.; Hall, James E.; Gonen, Tamir

2013-01-01

Calmodulin (CaM) is a universal regulatory protein that communicates the presence of calcium to its molecular targets and correspondingly modulates their function. This key signaling protein is important for controlling the activity of hundreds of membrane channels and transporters. However, our understanding of the structural mechanisms driving CaM regulation of full-length membrane proteins has remained elusive. In this study, we determined the pseudo-atomic structure of full-length mammalian aquaporin-0 (AQP0, Bos Taurus) in complex with CaM using electron microscopy to understand how this signaling protein modulates water channel function. Molecular dynamics and functional mutation studies reveal how CaM binding inhibits AQP0 water permeability by allosterically closing the cytoplasmic gate of AQP0. Our mechanistic model provides new insight, only possible in the context of the fully assembled channel, into how CaM regulates multimeric channels by facilitating cooperativity between adjacent subunits. PMID:23893133

Delivery of Iron-Sulfur Clusters to the Hydrogen-Oxidizing [NiFe]-Hydrogenases in Escherichia coli Requires the A-Type Carrier Proteins ErpA and IscA

PubMed Central

Pinske, Constanze; Sawers, R. Gary

2012-01-01

During anaerobic growth Escherichia coli synthesizes two membrane-associated hydrogen-oxidizing [NiFe]-hydrogenases, termed hydrogenase 1 and hydrogenase 2. Each enzyme comprises a catalytic subunit containing the [NiFe] cofactor, an electron-transferring small subunit with a particular complement of [Fe-S] (iron-sulfur) clusters and a membrane-anchor subunit. How the [Fe-S] clusters are delivered to the small subunit of these enzymes is unclear. A-type carrier (ATC) proteins of the Isc (iron-sulfur-cluster) and Suf (sulfur mobilization) [Fe-S] cluster biogenesis pathways are proposed to traffic pre-formed [Fe-S] clusters to apoprotein targets. Mutants that could not synthesize SufA had active hydrogenase 1 and hydrogenase 2 enzymes, thus demonstrating that the Suf machinery is not required for hydrogenase maturation. In contrast, mutants devoid of the IscA, ErpA or IscU proteins of the Isc machinery had no detectable hydrogenase 1 or 2 activities. Lack of activity of both enzymes correlated with the absence of the respective [Fe-S]-cluster-containing small subunit, which was apparently rapidly degraded. During biosynthesis the hydrogenase large subunits receive their [NiFe] cofactor from the Hyp maturation machinery. Subsequent to cofactor insertion a specific C-terminal processing step occurs before association of the large subunit with the small subunit. This processing step is independent of small subunit maturation. Using western blotting experiments it could be shown that although the amount of each hydrogenase large subunit was strongly reduced in the iscA and erpA mutants, some maturation of the large subunit still occurred. Moreover, in contrast to the situation in Isc-proficient strains, these processed large subunits were not membrane-associated. Taken together, our findings demonstrate that both IscA and ErpA are required for [Fe-S] cluster delivery to the small subunits of the hydrogen-oxidizing hydrogenases; however, delivery of the Fe atom to the active site might have different requirements. PMID:22363723

Molecular Cloning of Pituitary Glycoprotein α-Subunit and Follicle Stimulating Hormone and Chorionic Gonadotropin β-Subunits from New World Squirrel Monkey and Owl Monkey

PubMed Central

Scammell, Jonathan G.; Funkhouser, Jane D.; Moyer, Felricia S.; Gibson, Susan V.; Willis, Donna L.

2008-01-01

The goal of this study was to characterize the gonadotropins expressed in pituitary glands of the New World squirrel monkey (Saimiri sp.) and owl monkey (Aotus sp.). The various subunits were amplified from total RNA from squirrel monkey and owl monkey pituitary glands by reverse transcription-polymerase chain reaction and the deduced amino acid sequences compared to those of other species. Mature squirrel monkey and owl monkey glycoprotein hormone α-polypeptides (96 amino acids in length) were determined to be 80% homologous to the human sequence. The sequences of mature β subunits of follicle stimulating hormone (FSHβ) from squirrel monkey and owl monkey (111 amino acids in length) are 92% homologous to human FSHβ. New World primate glycoprotein hormone α-polypeptides and FSHβ subunits showed conservation of all cysteine residues and consensus N-linked glycosylation sites. Attempts to amplify the β-subunit of luteinizing hormone from squirrel monkey and owl monkey pituitary glands were unsuccessful. Rather, the β-subunit of chorionic gonadotropin (CG) was amplified from pituitaries of both New World primates. Squirrel monkey and owl monkey CGβ are 143 and 144 amino acids in length and 77% homologous with human CGβ. The greatest divergence is in the C terminus, where all four sites for O-linked glycosylation in human CGβ, responsible for delayed metabolic clearance, are predicted to be absent in New World primate CGβs. It is likely that CG secreted from pituitary of New World primates exhibits a relatively short half-life compared to human CG. PMID:17897645

Molecular cloning of pituitary glycoprotein alpha-subunit and follicle stimulating hormone and chorionic gonadotropin beta-subunits from New World squirrel monkey and owl monkey.

PubMed

Scammell, Jonathan G; Funkhouser, Jane D; Moyer, Felricia S; Gibson, Susan V; Willis, Donna L

2008-02-01

The goal of this study was to characterize the gonadotropins expressed in pituitary glands of the New World squirrel monkey (Saimiri sp.) and owl monkey (Aotus sp.). The various subunits were amplified from total RNA from squirrel monkey and owl monkey pituitary glands by reverse transcription-polymerase chain reaction and the deduced amino acid sequences compared to those of other species. Mature squirrel monkey and owl monkey glycoprotein hormone alpha-polypeptides (96 amino acids in length) were determined to be 80% homologous to the human sequence. The sequences of mature beta subunits of follicle stimulating hormone (FSHbeta) from squirrel monkey and owl monkey (111 amino acids in length) are 92% homologous to human FSHbeta. New World primate glycoprotein hormone alpha-polypeptides and FSHbeta subunits showed conservation of all cysteine residues and consensus N-linked glycosylation sites. Attempts to amplify the beta-subunit of luteinizing hormone from squirrel monkey and owl monkey pituitary glands were unsuccessful. Rather, the beta-subunit of chorionic gonadotropin (CG) was amplified from pituitaries of both New World primates. Squirrel monkey and owl monkey CGbeta are 143 and 144 amino acids in length and 77% homologous with human CGbeta. The greatest divergence is in the C terminus, where all four sites for O-linked glycosylation in human CGbeta, responsible for delayed metabolic clearance, are predicted to be absent in New World primate CGbetas. It is likely that CG secreted from pituitary of New World primates exhibits a relatively short half-life compared to human CG.

Photoinduced reduction of the medial FeS center in the hydrogenase small subunit HupS from Nostoc punctiforme.

PubMed

Raleiras, Patrícia; Hammarström, Leif; Lindblad, Peter; Styring, Stenbjörn; Magnuson, Ann

2015-07-01

The small subunit from the NiFe uptake hydrogenase, HupSL, in the cyanobacterium Nostoc punctiforme ATCC 29133, has been isolated in the absence of the large subunit (P. Raleiras, P. Kellers, P. Lindblad, S. Styring, A. Magnuson, J. Biol. Chem. 288 (2013) 18,345-18,352). Here, we have used flash photolysis to reduce the iron-sulfur clusters in the isolated small subunit, HupS. We used ascorbate as electron donor to the photogenerated excited state of Ru(II)-trisbipyridine (Ru(bpy)3), to generate Ru(I)(bpy)3 as reducing agent. Our results show that the isolated small subunit can be reduced by the Ru(I)(bpy)3 generated through flash photolysis. Copyright © 2015 Elsevier Inc. All rights reserved.

Cloning, Purification, and Characterization of a Heterodimeric β-Galactosidase from Lactobacillus kefiranofaciens ZW3.

PubMed

He, Xi; Han, Ning; Wang, Yan-Ping

2016-01-01

Lactobacillus kefiranofaciens ZW3 was obtained from kefir grains, which have high lactose hydrolytic activity. In this study, a heterodimeric LacLM-type β-galactosidase gene (lacLM) from ZW3 was isolated, which was composed of two overlapping genes, lacL (1,884 bp) and lacM (960 bp) encoding large and small subunits with calculated molecular masses of 73,620 and 35,682 Da, respectively. LacLM, LacL, and LacM were expressed in Escherichia coli BL21(DE3) and these recombinant proteins were purified and characterized. The results showed that, compared with the recombinant holoenzyme, the recombinant large subunit exhibits obviously lower thermostability and hydrolytic activity. Moreover, the optimal temperature and pH of the holoenzyme and large subunit are 60°C and 7.0, and 50°C and 8.0, respectively. However, the recombinant small subunit alone has no activity. Interestingly, the activity and thermostability of the large subunit were greatly improved after mixing it with the recombinant small subunit. Therefore, the results suggest that the small subunit might play an important role in maintaining the stability of the structure of the catalytic center located in the large subunit.

A Possible Role of the Full-Length Nascent Protein in Post-Translational Ribosome Recycling.

PubMed

Das, Debasis; Samanta, Dibyendu; Bhattacharya, Arpita; Basu, Arunima; Das, Anindita; Ghosh, Jaydip; Chakrabarti, Abhijit; Das Gupta, Chanchal

2017-01-01

Each cycle of translation initiation in bacterial cell requires free 50S and 30S ribosomal subunits originating from the post-translational dissociation of 70S ribosome from the previous cycle. Literature shows stable dissociation of 70S from model post-termination complexes by the concerted action of Ribosome Recycling Factor (RRF) and Elongation Factor G (EF-G) that interact with the rRNA bridge B2a/B2b joining 50S to 30S. In such experimental models, the role of full-length nascent protein was never considered seriously. We observed relatively slow release of full-length nascent protein from 50Sof post translation ribosome, and in that process, its toe prints on the rRNA in vivo and in in vitro translation with E.coli S30 extract. We reported earlier that a number of chemically unfolded proteins like bovine carbonic anhydrase (BCA), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), lysozyme, ovalbumin etc., when added to free 70Sin lieu of the full length nascent proteins, also interact with identical RNA regions of the 23S rRNA. Interestingly the rRNA nucleotides that slow down release of the C-terminus of full-length unfolded protein were found in close proximity to the B2a/B2b bridge. It indicated a potentially important chemical reaction conserved throughout the evolution. Here we set out to probe that conserved role of unfolded protein conformation in splitting the free or post-termination 70S. How both the RRF-EFG dependent and the plausible nascent protein-EFG dependent ribosome recycling pathways might be relevant in bacteria is discussed here.

A Possible Role of the Full-Length Nascent Protein in Post-Translational Ribosome Recycling

PubMed Central

Das, Debasis; Samanta, Dibyendu; Bhattacharya, Arpita; Basu, Arunima; Das, Anindita; Ghosh, Jaydip; Chakrabarti, Abhijit; Das Gupta, Chanchal

2017-01-01

Each cycle of translation initiation in bacterial cell requires free 50S and 30S ribosomal subunits originating from the post-translational dissociation of 70S ribosome from the previous cycle. Literature shows stable dissociation of 70S from model post-termination complexes by the concerted action of Ribosome Recycling Factor (RRF) and Elongation Factor G (EF-G) that interact with the rRNA bridge B2a/B2b joining 50S to 30S. In such experimental models, the role of full-length nascent protein was never considered seriously. We observed relatively slow release of full-length nascent protein from 50Sof post translation ribosome, and in that process, its toe prints on the rRNA in vivo and in in vitro translation with E.coli S30 extract. We reported earlier that a number of chemically unfolded proteins like bovine carbonic anhydrase (BCA), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), lysozyme, ovalbumin etc., when added to free 70Sin lieu of the full length nascent proteins, also interact with identical RNA regions of the 23S rRNA. Interestingly the rRNA nucleotides that slow down release of the C-terminus of full-length unfolded protein were found in close proximity to the B2a/B2b bridge. It indicated a potentially important chemical reaction conserved throughout the evolution. Here we set out to probe that conserved role of unfolded protein conformation in splitting the free or post-termination 70S. How both the RRF-EFG dependent and the plausible nascent protein–EFG dependent ribosome recycling pathways might be relevant in bacteria is discussed here. PMID:28099529

Activation of interspecies-hybrid Rubisco enzymes to assess different models for the Rubisco-Rubisco activase interaction.

PubMed

Wachter, Rebekka M; Salvucci, Michael E; Carmo-Silva, A Elizabete; Barta, Csengele; Genkov, Todor; Spreitzer, Robert J

2013-11-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is prone to inactivation from non-productive binding of sugar-phosphates. Reactivation of Rubisco requires conformational remodeling by a specific chaperone, Rubisco activase. Rubisco activase from tobacco and other plants in the family Solanaceae is an inefficient activator of Rubisco from non-Solanaceae plants and from the green alga Chlamydomonas reinhardtii. To determine if the Rubisco small subunit plays a role in the interaction with Rubisco activase, a hybrid Rubisco (SSNT) composed of tobacco small subunits and Chlamydomonas large subunits was constructed. The SSNT hybrid, like other hybrid Rubiscos containing plant small subunits, supported photoautotrophic growth in Chlamydomonas, but growth in air was much slower than for cells containing wild-type Rubisco. The kinetic properties of the SSNT hybrid Rubisco were similar to the wild-type enzyme, indicating that the poor growth in air was probably caused by disruption of pyrenoid formation and the consequent impairment of the CO2concentrating mechanism. Recombinant Rubisco activase from Arabidopsis activated the SSNT hybrid Rubisco and hybrid Rubiscos containing spinach and Arabidopsis small subunits at rates similar to the rates with wild-type Rubisco. However, none of the hybrid Rubiscos was activated by tobacco Rubisco activase. That replacement of Chlamydomonas small subunits with plant small subunits does not affect the species-specific interaction between Rubisco and Rubisco activase suggests that the association is not dominated by the small subunits that surround the Rubisco central solvent channel. Therefore, the geometry of a side-on binding mode is more consistent with the data than a top-on or ring-stacking binding mode.

Characterization of the Genes Encoding the Cytosolic and Plastidial Forms of ADP-Glucose Pyrophosphorylase in Wheat Endosperm1

PubMed Central

Burton, Rachel A.; Johnson, Philip E.; Beckles, Diane M.; Fincher, Geoffrey B.; Jenner, Helen L.; Naldrett, Mike J.; Denyer, Kay

2002-01-01

In most species, the synthesis of ADP-glucose (Glc) by the enzyme ADP-Glc pyrophosphorylase (AGPase) occurs entirely within the plastids in all tissues so far examined. However, in the endosperm of many, if not all grasses, a second form of AGPase synthesizes ADP-Glc outside the plastid, presumably in the cytosol. In this paper, we show that in the endosperm of wheat (Triticum aestivum), the cytosolic form accounts for most of the AGPase activity. Using a combination of molecular and biochemical approaches to identify the cytosolic and plastidial protein components of wheat endosperm AGPase we show that the large and small subunits of the cytosolic enzyme are encoded by genes previously thought to encode plastidial subunits, and that a gene, Ta.AGP.S.1, which encodes the small subunit of the cytosolic form of AGPase, also gives rise to a second transcript by the use of an alternate first exon. This second transcript encodes an AGPase small subunit with a transit peptide. However, we could not find a plastidial small subunit protein corresponding to this transcript. The protein sequence of the purified plastidial small subunit does not match precisely to that encoded by Ta.AGP.S.1 or to the predicted sequences of any other known gene from wheat or barley (Hordeum vulgare). Instead, the protein sequence is most similar to those of the plastidial small subunits from chickpea (Cicer arietinum) and maize (Zea mays) and rice (Oryza sativa) seeds. These data suggest that the gene encoding the major plastidial small subunit of AGPase in wheat endosperm has yet to be identified. PMID:12428011

A human-specific, truncated α7 nicotinic receptor subunit assembles with full-length α7 and forms functional receptors with different stoichiometries.

PubMed

Lasala, Matías; Corradi, Jeremías; Bruzzone, Ariana; Esandi, María Del Carmen; Bouzat, Cecilia

2018-05-21

The cholinergic α7 nicotinic receptor gene, CHRNA7, encodes a subunit that forms the homopentameric α7 receptor, involved in learning and memory. In humans, exons 5-10 in CHRNA7 are duplicated and fused to the FAM7A genetic element, giving rise to the hybrid gene CHRFAM7A. Its product, dupα7, is a truncated subunit lacking part of the N-terminal extracellular ligand-binding domain and is associated with neurological disorders, including schizophrenia, and immunomodulation.We combined dupα7 expression on mammalian cells with patch clamp recordings to understand its functional role. Transfected cells expressed dupα7 protein, but they exhibited neither surface binding of the α7 antagonist α-bungarotoxin nor responses to acetylcholine (ACh) or to an allosteric agonist that binds to the conserved transmembrane region. To determine if dupα7 assembles with α7, we generated receptors comprising α7 and dupα7 subunits, one of which was tagged with conductance substitutions that report subunit stoichiometry and monitored ACh-elicited channel openings elicited by ACh in the presence of a positive allosteric α7 modulator. We found that α7 and dupα7 subunits co-assemble into functional heteromeric receptors, that at least two α7 subunits are required for channel opening, and that dupα7's presence in the pentameric arrangement does not affect the duration of the potentiated events compare with that of α7. Using an α7 subunit mutant, we found that activation of (α7)2(dupα7)3 receptors occurs through ACh binding at the α7/α7 interfacial binding site. Our study contributes to the understanding of the modulation of α7 function by the human specific, duplicated subunit, associated with human disorders. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Na+/K+-ATPase α-subunit in swimming crab Portunus trituberculatus: molecular cloning, characterization, and expression under low salinity stress

NASA Astrophysics Data System (ADS)

Han, Xiaolin; Liu, Ping; Gao, Baoquan; Wang, Haofeng; Duan, Yafei; Xu, Wenfei; Chen, Ping

2015-07-01

Na+/K+-ATPases are membrane-associated enzymes responsible for the active transport of Na+ and K+ ions across cell membranes, generating chemical and electrical gradients. These enzymes' α-subunit provides catalytic function, binding and hydrolyzing ATP, and itself becoming phosphorylated during the transport cycle. In this study, Na+/K+-ATPase α-subunit cDNA was cloned from gill tissue of the swimming crab Portunus trituberculatus by reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end methods. Analysis of the nucleotide sequence revealed that the cDNA had a full-length of 3 833 base pairs (bp), with an open reading frame of 3 120 bp, 5' untranslated region (UTR) of 317 bp, and 3' UTR of 396 bp. The sequence encoded a 1 039 amino acid protein with a predicted molecular weight of 115.57 kDa and with estimated pI of 5.21. It was predicted here to possess all expected features of Na+/K+-ATPase members, including eight transmembrane domains, putative ATP-binding site, and phosphorylation site. Comparison of amino acid sequences showed that the P. trituberculatus α-subunit possessed an overall identity of 75%-99% to that of other organisms. Phylogenetic analysis revealed that this α-subunit was in the same category as those of crustaceans. Quantitative real-time RT-PCR analysis indicated that this α-subunit's transcript were most highly expressed in gill and lowest in muscle. RT-PCR analysis also revealed that α-subunit expression in crab gill decreased after 2 and 6 h, but increased after 12, 24, 48, and 72 h. In addition, α-subunit expression in hepatopancreas of crab decreased after 2-72 h. These facts indicated that the crab's Na+/K+-ATPase α-subunit was potentially involved in the observed acute response to low salinity stress.

The NH2-terminal php domain of the alpha subunit of the Escherichia coli replicase binds the epsilon proofreading subunit.

PubMed

Wieczorek, Anna; McHenry, Charles S

2006-05-05

The alpha subunit of the replicase of all bacteria contains a php domain, initially identified by its similarity to histidinol phosphatase but of otherwise unknown function (Aravind, L., and Koonin, E. V. (1998) Nucleic Acids Res. 26, 3746-3752). Deletion of 60 residues from the NH2 terminus of the alpha php domain destroys epsilon binding. The minimal 255-residue php domain, estimated by sequence alignment with homolog YcdX, is insufficient for epsilon binding. However, a 320-residue segment including sequences that immediately precede the polymerase domain binds epsilon with the same affinity as the 1160-residue full-length alpha subunit. A subset of mutations of a conserved acidic residue (Asp43 in Escherichia coli alpha) present in the php domain of all bacterial replicases resulted in defects in epsilon binding. Using sequence alignments, we show that the prototypical gram+ Pol C, which contains the polymerase and proofreading activities within the same polypeptide chain, has an epsilon-like sequence inserted in a surface loop near the center of the homologous YcdX protein. These findings suggest that the php domain serves as a platform to enable coordination of proofreading and polymerase activities during chromosomal replication.

A definition of the domains Archaea, Bacteria and Eucarya in terms of small subunit ribosomal RNA characteristics

NASA Technical Reports Server (NTRS)

Winker, S.; Woese, C. R.

1991-01-01

The number of small subunit rRNA sequences is now great enough that the three domains Archaea, Bacteria and Eucarya (Woese et al., 1990) can be reliably defined in terms of their sequence "signatures". Approximately 50 homologous positions (or nucleotide pairs) in the small subunit rRNA characterize and distinguish among the three. In addition, the three can be recognized by a variety of nonhomologous rRNA characters, either individual positions and/or higher-order structural features. The Crenarchaeota and the Euryarchaeota, the two archaeal kingdoms, can also be defined and distinguished by their characteristic compositions at approximately fifteen positions in the small subunit rRNA molecule.

Small things considered: the small accessory subunits of RNA polymerase in Gram-positive bacteria

PubMed Central

Weiss, Andy; Shaw, Lindsey N.

2015-01-01

The DNA-dependent RNA polymerase core enzyme in Gram-positive bacteria consists of seven subunits. Whilst four of them (α2ββ′) are essential, three smaller subunits, δ, ε and ω (∼9–21.5 kDa), are considered accessory. Both δ and ω have been viewed as integral components of RNAP for several decades; however, ε has only recently been described. Functionally these three small subunits carry out a variety of tasks, imparting important, supportive effects on the transcriptional process of Gram-positive bacteria. While ω is thought to have a wide range of roles, reaching from maintaining structural integrity of RNAP to σ factor recruitment, the only suggested function for ε thus far is in protecting cells from phage infection. The third subunit, δ, has been shown to have distinct influences in maintaining transcriptional specificity, and thus has a key role in cellular fitness. Collectively, all three accessory subunits, although dispensable under laboratory conditions, are often thought to be crucial for proper RNAP function. Herein we provide an overview of the available literature on each subunit, summarizing landmark findings that have deepened our understanding of these proteins and their function, and outline future challenges in understanding the role of these small subunits in the transcriptional process. PMID:25878038

Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates

PubMed Central

Noy-Porat, Tal; Rosenfeld, Ronit; Ariel, Naomi; Epstein, Eyal; Alcalay, Ron; Zvi, Anat; Kronman, Chanoch; Ordentlich, Arie; Mazor, Ohad

2016-01-01

Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit) was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (koff < 1 × 10−7 s−1) that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication. PMID:26950154

Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates.

PubMed

Noy-Porat, Tal; Rosenfeld, Ronit; Ariel, Naomi; Epstein, Eyal; Alcalay, Ron; Zvi, Anat; Kronman, Chanoch; Ordentlich, Arie; Mazor, Ohad

2016-03-03

Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit) was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (k(off )< 1 × 10(-7) s(-1)) that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication.

HER2 isoforms co-expression differently tunes mammary tumor phenotypes affecting onset, vasculature and therapeutic response

PubMed Central

Balboni, Tania; Ianzano, Marianna L.; Laranga, Roberta; Landuzzi, Lorena; Giusti, Veronica; Ceccarelli, Claudio; Santini, Donatella; Taffurelli, Mario; Di Oto, Enrico; Asioli, Sofia; Amici, Augusto; Pupa, Serenella M.; De Giovanni, Carla; Tagliabue, Elda; Iezzi, Manuela; Nanni, Patrizia; Lollini, Pier-Luigi

2017-01-01

Full-length HER2 oncoprotein and splice variant Delta16 are co-expressed in human breast cancer. We studied their interaction in hybrid transgenic mice bearing human full-length HER2 and Delta16 (F1 HER2/Delta16) in comparison to parental HER2 and Delta16 transgenic mice. Mammary carcinomas onset was faster in F1 HER2/Delta16 and Delta16 than in HER2 mice, however tumor growth was slower, and metastatic spread was comparable in all transgenic mice. Full-length HER2 tumors contained few large vessels or vascular lacunae, whereas Delta16 tumors presented a more regular vascularization with numerous endothelium-lined small vessels. Delta16-expressing tumors showed a higher accumulation of i.v. injected doxorubicin than tumors expressing full-length HER2. F1 HER2/Delta16 tumors with high full-length HER2 expression made few large vessels, whereas tumors with low full-length HER2 and high Delta16 contained numerous small vessels and expressed higher levels of VEGF and VEGFR2. Trastuzumab strongly inhibited tumor onset in F1 HER2/Delta16 and Delta16 mice, but not in full-length HER2 mice. Addiction of F1 tumors to Delta16 was also shown by long-term stability of Delta16 levels during serial transplants, in contrast full-length HER2 levels underwent wide fluctuations. In conclusion, full-length HER2 leads to a faster tumor growth and to an irregular vascularization, whereas Delta16 leads to a faster tumor onset, with more regular vessels, which in turn could better transport cytotoxic drugs within the tumor, and to a higher sensitivity to targeted therapeutic agents. F1 HER2/Delta16 mice are a new immunocompetent mouse model, complementary to patient-derived xenografts, for studies of mammary carcinoma onset, prevention and therapy. PMID:28903354

Recombinant Production, Reconstruction in Lipid-Protein Nanodiscs, and Electron Microscopy of Full-Length α-Subunit of Human Potassium Channel Kv7.1.

PubMed

Shenkarev, Z O; Karlova, M G; Kulbatskii, D S; Kirpichnikov, M P; Lyukmanova, E N; Sokolova, O S

2018-05-01

Voltage-gated potassium channel Kv7.1 plays an important role in the excitability of cardiac muscle. The α-subunit of Kv7.1 (KCNQ1) is the main structural element of this channel. Tetramerization of KCNQ1 in the membrane results in formation of an ion channel, which comprises a pore and four voltage-sensing domains. Mutations in the human KCNQ1 gene are one of the major causes of inherited arrhythmias, long QT syndrome in particular. The construct encoding full-length human KCNQ1 protein was synthesized in this work, and an expression system in the Pichia pastoris yeast cells was developed. The membrane fraction of the yeast cells containing the recombinant protein (rKCNQ1) was solubilized with CHAPS detergent. To better mimic the lipid environment of the channel, lipid-protein nanodiscs were formed using solubilized membrane fraction and MSP2N2 protein. The rKCNQ1/nanodisc and rKCNQ1/CHAPS samples were purified using the Rho1D4 tag introduced at the C-terminus of the protein. Protein samples were examined using transmission electron microscopy with negative staining. In both cases, homogeneous rKCNQ1 samples were observed based on image analysis. Statistical analysis of the images of individual protein particles solubilized in the detergent revealed the presence of a tetrameric structure confirming intact subunit assembly. A three-dimensional channel structure reconstructed at 2.5-nm resolution represents a compact density with diameter of the membrane part of ~9 nm and height ~11 nm. Analysis of the images of rKCNQ1 in nanodiscs revealed additional electron density corresponding to the lipid bilayer fragment and the MSP2N2 protein. These results indicate that the nanodiscs facilitate protein isolation, purification, and stabilization in solution and can be used for further structural studies of human Kv7.1.

Cryo-EM structure of the large subunit of the spinach chloroplast ribosome

PubMed Central

Ahmed, Tofayel; Yin, Zhan; Bhushan, Shashi

2016-01-01

Protein synthesis in the chloroplast is mediated by the chloroplast ribosome (chloro-ribosome). Overall architecture of the chloro-ribosome is considerably similar to the Escherichia coli (E. coli) ribosome but certain differences are evident. The chloro-ribosome proteins are generally larger because of the presence of chloroplast-specific extensions in their N- and C-termini. The chloro-ribosome harbours six plastid-specific ribosomal proteins (PSRPs); four in the small subunit and two in the large subunit. Deletions and insertions occur throughout the rRNA sequence of the chloro-ribosome (except for the conserved peptidyl transferase center region) but the overall length of the rRNAs do not change significantly, compared to the E. coli. Although, recent advancements in cryo-electron microscopy (cryo-EM) have provided detailed high-resolution structures of ribosomes from many different sources, a high-resolution structure of the chloro-ribosome is still lacking. Here, we present a cryo-EM structure of the large subunit of the chloro-ribosome from spinach (Spinacia oleracea) at an average resolution of 3.5 Å. High-resolution map enabled us to localize and model chloro-ribosome proteins, chloroplast-specific protein extensions, two PSRPs (PSRP5 and 6) and three rRNA molecules present in the chloro-ribosome. Although comparable to E. coli, the polypeptide tunnel and the tunnel exit site show chloroplast-specific features. PMID:27762343

Molecular Characterization and Functional Analysis of a Ferritin Heavy Chain Subunit from the Eri-Silkworm, Samia cynthia ricini

PubMed Central

Yu, Hai-Zhong; Zhang, Shang-Zhi; Ma, Yan; Fei, Dong-Qiong; Li, Bing; Yang, Li-Ang; Wang, Jie; Li, Zhen; Muhammad, Azharuddin; Xu, Jia-Ping

2017-01-01

Ferritins are conserved iron-binding proteins that are primarily involved in iron storage, detoxification and the immune response. Despite the importance of ferritin in organisms, little is known about their roles in the eri-silkworm (Samia cynthia ricini). We previously identified a ferritin heavy chain subunit named ScFerHCH in the S. c. ricini transcriptome database. The full-length S. c. ricini ferritin heavy chain subunit (ScFerHCH) was 1863 bp and encoded a protein of 231 amino acids with a deduced molecular weight of 25.89 kDa. Phylogenetic analysis revealed that ScFerHCH shared a high amino acid identity with the Bombyx mori and Danaus plexippus heavy chain subunits. Higher ScFerHCH expression levels were found in the silk gland, fat body and midgut of S. c. ricini by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting. Injection of Staphylococcus aureus and Pseudomonas aeruginosa was associated with an upregulation of ScFerHCH in the midgut, fat body and hemolymph, indicating that ScFerHCH may contribute to the host’s defense against invading pathogens. In addition, the anti-oxidation activity and iron-binding capacity of recombinant ScFerHCH protein were examined. Taken together, our results suggest that the ferritin heavy chain subunit from eri-silkworm may play critical roles not only in innate immune defense, but also in organismic iron homeostasis. PMID:29036914

Foccα6, a truncated nAChR subunit, positively correlates with spinosad resistance in the western flower thrips, Frankliniella occidentalis (Pergande).

PubMed

Wan, Yanran; Yuan, Guangdi; He, Bingqing; Xu, Baoyun; Xie, Wen; Wang, Shaoli; Zhang, Youjun; Wu, Qingjun; Zhou, Xuguo

2018-08-01

Nicotinic acetylcholine receptors (nAChRs), a molecular target for spinosyns and neonicotinoids, mediate rapid cholinergic transmission in insect central nervous system by binding acetylcholine. Previous studies have shown that mutations in nAChRs contribute to the high level of resistance to these two classes of insecticides. In this study, we identified nine nAChR subunits from a transcriptome of the western flower thrips, Frankliniella occidentalis, including α1-7, β1, and β2. Exon 4 of α4 and exons 3 and 8 of α6 each have two splicing variants, respectively. In addition, altered or incorrect splicing leads to truncated forms of α3, α5, and α6 subunits. The abundance of every nAChRs in both spinosad susceptible and resistant strains was highest in the 1st instar nymph. Significantly more truncated forms of α6 subunit were detected in spinosad resistant strains, whereas, hardly any full-length form was found in the two highly resistant F. occidentalis strains (resistance ratio >10 4 -fold). Under laboratory conditions, spinosad resistance was positively correlated with truncated α6 transcripts. The correlation was later confirmed under the field conditions using five field strains. As the molecular target of spinosad, the percentage of truncated nAChR α6 subunits can be used as a diagnostic tool to detect and quantify spinosad resistance in the field. Copyright © 2018. Published by Elsevier Ltd.

Subunit Conformations and Assembly States of a DNA Translocating Motor: The Terminase of Bacteriophage P22

PubMed Central

Němeček, Daniel; Gilcrease, Eddie B.; Kang, Sebyung; Prevelige, Peter E.; Casjens, Sherwood; Thomas, George J.

2007-01-01

Bacteriophage P22, a podovirus infecting strains of Salmonella typhimurium, packages a 42 kbp genome using a headful mechanism. DNA translocation is accomplished by the phage terminase, a powerful molecular motor consisting of large and small subunits. Although many of the structural proteins of the P22 virion have been well characterized, little is known about the terminase subunits and their molecular mechanism of DNA translocation. We report here structural and assembly properties of ectopically expressed and highly purified terminase large and small subunits. The large subunit (gp2), which contains the nuclease and ATPase activities of terminase, exists as a stable monomer with an α/β fold. The small subunit (gp3), which recognizes DNA for packaging and may regulate gp2 activity, exhibits a highly α-helical secondary structure and self-associates to form a stable oligomeric ring in solution. For wildtype gp3, the ring contains nine subunits, as demonstrated by hydrodynamic measurements, electron microscopy and native mass spectrometry. We have also characterized a gp3 mutant (Ala 112 → Thr) that forms a ten subunit ring, despite a subunit fold indistinguishable from wildtype. Both the nonameric and decameric gp3 rings exhibit nonspecific DNA binding activity, and gp2 is able to bind strongly to the DNA/gp3 complex but not to DNA alone. We propose a scheme for the roles of P22 terminase large and small subunits in the recruitment and packaging of viral DNA and discuss the model in relation to proposals for terminase-driven DNA translocation in other phages. PMID:17945256

Crystal Structure of the Cul2-Rbx1-EloBC-VHL Ubiquitin Ligase Complex.

PubMed

Cardote, Teresa A F; Gadd, Morgan S; Ciulli, Alessio

2017-06-06

Cullin RING E3 ubiquitin ligases (CRLs) function in the ubiquitin proteasome system to catalyze the transfer of ubiquitin from E2 conjugating enzymes to specific substrate proteins. CRLs are large dynamic complexes and attractive drug targets for the development of small-molecule inhibitors and chemical inducers of protein degradation. The atomic details of whole CRL assembly and interactions that dictate subunit specificity remain elusive. Here we present the crystal structure of a pentameric CRL2 VHL complex, composed of Cul2, Rbx1, Elongin B, Elongin C, and pVHL. The structure traps a closed state of full-length Cul2 and a new pose of Rbx1 in a trajectory from closed to open conformation. We characterize hotspots and binding thermodynamics at the interface between Cul2 and pVHL-EloBC and identify mutations that contribute toward a selectivity switch for Cul2 versus Cul5 recognition. Our findings provide structural and biophysical insights into the whole Cul2 complex that could aid future drug targeting. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Feast/famine regulatory proteins (FFRPs): Escherichia coli Lrp, AsnC and related archaeal transcription factors.

PubMed

Yokoyama, Katsushi; Ishijima, Sanae A; Clowney, Lester; Koike, Hideaki; Aramaki, Hironori; Tanaka, Chikako; Makino, Kozo; Suzuki, Masashi

2006-01-01

Feast/famine regulatory proteins comprise a diverse family of transcription factors, which have been referred to in various individual identifications, including Escherichia coli leucine-responsive regulatory protein and asparagine synthase C gene product. A full length feast/famine regulatory protein consists of the N-terminal DNA-binding domain and the C-domain, which is involved in dimerization and further assembly, thereby producing, for example, a disc or a chromatin-like cylinder. Various ligands of the size of amino acids bind at the interface between feast/famine regulatory protein dimers, thereby altering their assembly forms. Also, the combination of feast/famine regulatory protein subunits forming the same assembly is altered. In this way, a small number of feast/famine regulatory proteins are able to regulate a large number of genes in response to various environmental changes. Because feast/famine regulatory proteins are shared by archaea and eubacteria, the genome-wide regulation by feast/famine regulatory proteins is traceable back to their common ancestor, being the prototype of highly differentiated transcription regulatory mechanisms found in organisms nowadays.

Biodiversity and Emerging Biogeography of the Neutrophilic Iron-Oxidizing Zetaproteobacteria ▿ †

PubMed Central

McAllister, Sean M.; Davis, Richard E.; McBeth, Joyce M.; Tebo, Bradley M.; Emerson, David; Moyer, Craig L.

2011-01-01

Members of the neutrophilic iron-oxidizing candidate class Zetaproteobacteria have predominantly been found at sites of microbially mediated iron oxidation in marine environments around the Pacific Ocean. Eighty-four full-length (>1,400-bp) and 48 partial-length Zetaproteobacteria small-subunit (SSU) rRNA gene sequences from five novel clone libraries, one novel Zetaproteobacteria isolate, and the GenBank database were analyzed to assess the biodiversity of this burgeoning class of the Proteobacteria and to investigate its biogeography between three major sampling regions in the Pacific Ocean: Loihi Seamount, the Southern Mariana Trough, and the Tonga Arc. Sequences were grouped into operational taxonomic units (OTUs) on the basis of a 97% minimum similarity. Of the 28 OTUs detected, 13 were found to be endemic to one of the three main sampling regions and 2 were ubiquitous throughout the Pacific Ocean. Additionally, two deeply rooted OTUs that potentially dominate communities of iron oxidizers originating in the deep subsurface were identified. Spatial autocorrelation analysis and analysis of molecular variance (AMOVA) showed that geographic distance played a significant role in the distribution of Zetaproteobacteria biodiversity, whereas environmental parameters, such as temperature, pH, or total Fe concentration, did not have a significant effect. These results, detected using the coarse resolution of the SSU rRNA gene, indicate that the Zetaproteobacteria have a strong biogeographic signal. PMID:21666021

Morphology and small-subunit rRNA gene sequences of two novel marine ciliates, Metanophrys orientalis spec. nov. and Uronemella sinensis spec. nov. (Protista, Ciliophora, Scuticociliatia), with an improved diagnosis of the genus Uronemella.

PubMed

Pan, Xuming; Zhu, Mingzhuang; Ma, Honggang; Al-Rasheid, Khaled A S; Hu, Xiaozhong

2013-09-01

The morphology and infraciliature of two novel marine scuticociliates, Metanophrys orientalis spec. nov. and Uronemella sinensis spec. nov., collected from sandy beaches at Qingdao, China, were investigated using live observation and protargol-staining methods. Metanophrys orientalis spec. nov. is distinguished by the following characteristics: marine habitat and a slender to elongate oval body with pointed anterior end and rounded caudal end, in vivo about 25-50 µm long; buccal field about a quarter to a third of body length; nine or ten somatic kineties with dikinetids approximately in anterior half of body, monokinetids in posterior half; membranelles 1 and 2 almost equal in length and composed of two and three longitudinal rows of kinetids respectively; paroral membrane with zigzag structure extending anteriorly to middle portion of membranelle 2; contractile vacuole pore located at posterior end of somatic kinety 1. The genus Uronemella is redefined as follows: marine form with an elongate-elliptical or inverted pear-shaped body; apical plate conspicuous; buccal field about two-thirds of body length, cytostome subequatorially located; oral apparatus Uronema-like; somatic kineties comprising a mixture of dikinetids and monokinetids. Uronemella sinensis spec. nov. is recognized by having an elongate-elliptical body with truncated apical frontal plate, size in vivo about 25-35 × 15-20 µm, nine or ten somatic kineties, membranelle 1 consisting of two or three basal bodies, contractile vacuole pore at posterior end of somatic kinety 1. This study also compared the small-subunit rRNA gene sequences of these two species with other closely related species to show the sequence divergence, which ranged from 3.53 to 9.60%. Phylogenetic analyses support the contention that the genus Uronemella is monophyletic, while Metanophrys is non-monophyletic.

Accumulation of the mitochondrial form of the sulphydryl oxidase Erv1p/Alrp during the early stages of spermatogenesis.

PubMed

Klissenbauer, Monika; Winters, Silke; Heinlein, Uwe A O; Lisowsky, Thomas

2002-07-01

In this study, we investigated the expression of the mammalian FAD-dependent sulphydryl oxidase Erv1p/Alrp in the rat and mouse and during mouse spermatogenesis. Up to three forms of Alrp were identified in protein extracts from different tissues and organs, but very little enzyme was present in blood samples. The three forms of Alrp represent the full-length protein of 23 kDa and fragments of 21 kDa and 15 kDa. All forms of Alrp were assembled into dimers in vivo. In contrast to samples from other organs, the protein analysis of mouse testis identified predominantly full-length 23 kDa Alrp. This finding prompted us to investigate in more detail the expression of Alrp during spermatogenesis. Testis samples of individual mice from postnatal days 13-29 were probed with an antibody specific for mammalian Alrp. In addition, cells from whole testis preparations were fractionated on a bovine serum albumin column gradient. Protein expression of mouse Alrp was compared with those of testis-specific cyritestin, the cytoskeleton marker actin and mitochondrial subunit Vb of cytochrome oxidase and cytochrome c. These studies demonstrated a specific accumulation of full-length mouse Alrp during the early stages of spermatogenesis. The highest levels of Alrp were found in spermatogonia and primary spermatocytes. Levels of expression of Alrp did not correlate with the synthesis of components of the respiratory chain, indicating that full-length Alrp in the mitochondria of spermatogonia and spermatocytes has another function in addition to its role in oxidative phosphorylation.

Isolation and in vitro cultivation of the aphid pathogenic fungus Entomophthora planchoniana.

PubMed

Freimoser, F M; Jensen, A B; Tuor, U; Aebi, M; Eilenberg, J

2001-12-01

Entomophthora planchoniana is an important fungal pathogen of aphids. Although Entomophthora chromaphidis has been considered a synonym for E. planchoniana, the two species are now separated, and E. planchoniana is reported not to grow in vitro. In this paper, we describe for the first time the isolation and cultivation of this species. Entomophthora planchoniana was isolated from a population of Ovatus crataegarius (Homoptera, Aphididae), which was infected by E. planchoniana only. The isolates did not sporulate, but the sequence of the small subunit rDNA and the restriction fragment length polymorphism patterns of the first part of the large subunit rDNA and the ITS II region confirm that the isolates were E. planchoniana. The isolated fungus grew in a medium consisting of Grace's insect cell culture medium supplemented with lactalbumin hydrolysate, yeastolate, and 10% fetal bovine serum or in GLEN medium with 10% fetal bovine serum. Vegetative cells of E. planchoniana were long and club-shaped and did not stain with Calcofluor, thus suggesting that they were protoplasts.

Regulation of human cardiac potassium channels by full-length KCNE3 and KCNE4.

PubMed

Abbott, Geoffrey W

2016-12-06

Voltage-gated potassium (Kv) channels comprise pore-forming α subunits and a multiplicity of regulatory proteins, including the cardiac-expressed and cardiac arrhythmia-linked transmembrane KCNE subunits. After recently uncovering novel, N-terminally extended (L) KCNE3 and KCNE4 isoforms and detecting their transcripts in human atrium, reported here are their functional effects on human cardiac Kv channel α subunits expressed in Xenopus laevis oocytes. As previously reported for short isoforms KCNE3S and KCNE4S, KCNE3L inhibited hERG; KCNE4L inhibited Kv1.1; neither form regulated the HCN1 pacemaker channel. Unlike KCNE4S, KCNE4L was a potent inhibitor of Kv4.2 and Kv4.3; co-expression of cytosolic β subunit KChIP2, which regulates Kv4 channels in cardiac myocytes, partially relieved Kv4.3 but not Kv4.2 inhibition. Inhibition of Kv4.2 and Kv4.3 by KCNE3L was weaker, and its inhibition of Kv4.2 abolished by KChIP2. KCNE3L and KCNE4L also exhibited subunit-specific effects on Kv4 channel complex inactivation kinetics, voltage dependence and recovery. Further supporting the potential physiological significance of the robust functional effects of KCNE4L on Kv4 channels, KCNE4L protein was detected in human atrium, where it co-localized with Kv4.3. The findings establish functional effects of novel human cardiac-expressed KCNE isoforms and further contribute to our understanding of the potential mechanisms influencing cardiomyocyte repolarization.

Toward a Whole-Cell Model of Ribosome Biogenesis: Kinetic Modeling of SSU Assembly

PubMed Central

Earnest, Tyler M.; Lai, Jonathan; Chen, Ke; Hallock, Michael J.; Williamson, James R.; Luthey-Schulten, Zaida

2015-01-01

Central to all life is the assembly of the ribosome: a coordinated process involving the hierarchical association of ribosomal proteins to the RNAs forming the small and large ribosomal subunits. The process is further complicated by effects arising from the intracellular heterogeneous environment and the location of ribosomal operons within the cell. We provide a simplified model of ribosome biogenesis in slow-growing Escherichia coli. Kinetic models of in vitro small-subunit reconstitution at the level of individual protein/ribosomal RNA interactions are developed for two temperature regimes. The model at low temperatures predicts the existence of a novel 5′→3′→central assembly pathway, which we investigate further using molecular dynamics. The high-temperature assembly network is incorporated into a model of in vivo ribosome biogenesis in slow-growing E. coli. The model, described in terms of reaction-diffusion master equations, contains 1336 reactions and 251 species that dynamically couple transcription and translation to ribosome assembly. We use the Lattice Microbes software package to simulate the stochastic production of mRNA, proteins, and ribosome intermediates over a full cell cycle of 120 min. The whole-cell model captures the correct growth rate of ribosomes, predicts the localization of early assembly intermediates to the nucleoid region, and reproduces the known assembly timescales for the small subunit with no modifications made to the embedded in vitro assembly network. PMID:26333594

Late-assembly of human ribosomal protein S20 in the cytoplasm is essential for the functioning of the small subunit ribosome

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

Tai, Lin-Ru; Chou, Chang-Wei; Wu, Jing-Ying

2013-11-15

Using immuno-fluorescent probing and Western blotting analysis, we reveal the exclusive cytoplasm nature of the small subunit ribosomal protein S20. To illustrate the importance of the cellular compartmentation of S20 to the function of small subunit 40S, we created a nuclear resident S20{sub NLS} mutant gene and examined polysome profile of cells that had been transfected with the S20{sub NLS} gene. As a result, we observed the formation of recombinant 40S carried S20{sub NLS} but this recombinant 40S was never found in the polysome, suggesting such a recombinant 40S was translation incompetent. Moreover, by the tactic of the energy depletionmore » and restoration, we were able to restrain the nuclear-resided S20{sub NLS} in the cytoplasm. Yet, along a progressive energy restoration, we observed the presence of recombinant 40S subunits carrying the S20{sub NLS} in the polysome. This proves that S20 needs to be cytoplasmic in order to make a functional 40S subunit. Furthermore, it also implies that the assembly order of ribosomal protein in eukaryote is orderly regulated. - Highlights: • The step of S20 assembled on 40S is happened in the cytoplasm. • A small subunit assembled with a nuclear S20{sub NLS} is translational incompetence. • Using energy depletion and recovery to manipulate the cellular compartment of S20{sub NLS}. • Cytoplasm-retained S20{sub NLS} is crucial for creating a functional small subunit.« less

Molecular characterization and phylogenetic relationships among microsporidian isolates infecting silkworm, Bombyx mori using small subunit rRNA (SSU-rRNA) gene sequence analysis.

PubMed

Nath, B Surendra; Gupta, S K; Bajpai, A K

2012-12-01

The life cycle, spore morphology, pathogenicity, tissue specificity, mode of transmission and small subunit rRNA (SSU-rRNA) gene sequence analysis of the five new microsporidian isolates viz., NIWB-11bp, NIWB-12n, NIWB-13md, NIWB-14b and NIWB-15mb identified from the silkworm, Bombyx mori have been studied along with type species, NIK-1s_mys. The life cycle of the microsporidians identified exhibited the sequential developmental cycles that are similar to the general developmental cycle of the genus, Nosema. The spores showed considerable variations in their shape, length and width. The pathogenicity observed was dose-dependent and differed from each of the microsporidian isolates; the NIWB-15mb was found to be more virulent than other isolates. All of the microsporidians were found to infect most of the tissues examined and showed gonadal infection and transovarial transmission in the infected silkworms. SSU-rRNA sequence based phylogenetic tree placed NIWB-14b, NIWB-12n and NIWB-11bp in a separate branch along with other Nosema species and Nosema bombycis; while NIWB-15mb and NIWB-13md together formed another cluster along with other Nosema species. NIK-1s_mys revealed a signature sequence similar to standard type species, N. bombycis, indicating that NIK-1s_mys is similar to N. bombycis. Based on phylogenetic relationships, branch length information based on genetic distance and nucleotide differences, we conclude that the microsporidian isolates identified are distinctly different from the other known species and belonging to the genus, Nosema. This SSU-rRNA gene sequence analysis method is found to be more useful approach in detecting different and closely related microsporidians of this economically important domestic insect.

Molecular cloning and expression of heteromeric ACCase subunit genes from Jatropha curcas.

PubMed

Gu, Keyu; Chiam, Huihui; Tian, Dongsheng; Yin, Zhongchao

2011-04-01

Acetyl-CoA carboxylase (ACCase) catalyzes the biotin-dependent carboxylation of acetyl-CoA to produce malonyl-CoA, which is the essential first step in the biosynthesis of long-chain fatty acids. ACCase exists as a multi-subunit enzyme in most prokaryotes and the chloroplasts of most plants and algae, while it is present as a multi-domain enzyme in the endoplasmic reticulum of most eukaryotes. The heteromeric ACCase of higher plants consists of four subunits: an α-subunit of carboxyltransferase (α-CT, encoded by accA gene), a biotin carboxyl carrier protein (BCCP, encoded by accB gene), a biotin carboxylase (BC, encoded by accC gene) and a β-subunit of carboxyltransferase (β-CT, encoded by accD gene). In this study, we cloned and characterized the genes accA, accB1, accC and accD that encode the subunits of heteromeric ACCase in Jatropha (Jatropha curcas), a potential biofuel plant. The full-length cDNAs of the four subunit genes were isolated from a Jatropha cDNA library and by using 5' RACE, whereas the genomic clones were obtained from a Jatropha BAC library. They encode a 771 amino acid (aa) α-CT, a 286-aa BCCP1, a 537-aa BC and a 494-aa β-CT, respectively. The single-copy accA, accB1 and accC genes are nuclear genes, while the accD gene is located in chloroplast genome. Jatropha α-CT, BCCP1, BC and β-CT show high identity to their homologues in other higher plants at amino acid level and contain all conserved domains for ACCase activity. The accA, accB1, accC and accD genes are temporally and spatially expressed in the leaves and endosperm of Jatropha plants, which are regulated by plant development and environmental factors. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Differential targeting of Gbetagamma-subunit signaling with small molecules.

PubMed

Bonacci, Tabetha M; Mathews, Jennifer L; Yuan, Chujun; Lehmann, David M; Malik, Sundeep; Wu, Dianqing; Font, Jose L; Bidlack, Jean M; Smrcka, Alan V

2006-04-21

G protein betagamma subunits have potential as a target for therapeutic treatment of a number of diseases. We performed virtual docking of a small-molecule library to a site on Gbetagamma subunits that mediates protein interactions. We hypothesized that differential targeting of this surface could allow for selective modulation of Gbetagamma subunit functions. Several compounds bound to Gbetagamma subunits with affinities from 0.1 to 60 muM and selectively modulated functional Gbetagamma-protein-protein interactions in vitro, chemotactic peptide signaling pathways in HL-60 leukocytes, and opioid receptor-dependent analgesia in vivo. These data demonstrate an approach for modulation of G protein-coupled receptor signaling that may represent an important therapeutic strategy.

Novel exon 1 protein-coding regions N-terminally extend human KCNE3 and KCNE4.

PubMed

Abbott, Geoffrey W

2016-08-01

The 5 human (h)KCNE β subunits each regulate various cation channels and are linked to inherited cardiac arrhythmias. Reported here are previously undiscovered protein-coding regions in exon 1 of hKCNE3 and hKCNE4 that extend their encoded extracellular domains by 44 and 51 residues, which yields full-length proteins of 147 and 221 residues, respectively. Full-length hKCNE3 and hKCNE4 transcript and protein are expressed in multiple human tissues; for hKCNE4, only the longer protein isoform is detectable. Two-electrode voltage-clamp electrophysiology revealed that, when coexpressed in Xenopus laevis oocytes with various potassium channels, the newly discovered segment preserved conversion of KCNQ1 by hKCNE3 to a constitutively open channel, but prevented its inhibition of Kv4.2 and KCNQ4. hKCNE4 slowing of Kv4.2 inactivation and positive-shifted steady-state inactivation were also preserved in the longer form. In contrast, full-length hKCNE4 inhibition of KCNQ1 was limited to 40% at +40 mV vs. 80% inhibition by the shorter form, and augmentation of KCNQ4 activity by hKCNE4 was entirely abolished by the additional segment. Among the genome databases analyzed, the longer KCNE3 is confined to primates; full-length KCNE4 is widespread in vertebrates but is notably absent from Mus musculus Findings highlight unexpected KCNE gene diversity, raise the possibility of dynamic regulation of KCNE partner modulation via splice variation, and suggest that the longer hKCNE3 and hKCNE4 proteins should be adopted in future mechanistic and genetic screening studies.-Abbott, G. W. Novel exon 1 protein-coding regions N-terminally extend human KCNE3 and KCNE4. © FASEB.

One-step affinity tag purification of full-length recombinant human AP-1 complexes from bacterial inclusion bodies using a polycistronic expression system

PubMed Central

Wang, Wei-Ming; Lee, A-Young; Chiang, Cheng-Ming

2008-01-01

The AP-1 transcription factor is a dimeric protein complex formed primarily between Jun (c-Jun, JunB, JunD) and Fos (c-Fos, FosB, Fra-1, Fra-2) family members. These distinct AP-1 complexes are expressed in many cell types and modulate target gene expression implicated in cell proliferation, differentiation, and stress responses. Although the importance of AP-1 has long been recognized, the biochemical characterization of AP-1 remains limited in part due to the difficulty in purifying full-length, reconstituted dimers with active DNA-binding and transcriptional activity. Using a combination of bacterial coexpression and epitope-tagging methods, we successfully purified all 12 heterodimers (3 Jun × 4 Fos) of full-length human AP-1 complexes as well as c-Jun/c-Jun, JunD/JunD, and c-Jun/JunD dimers from bacterial inclusion bodies using one-step nickel-NTA affinity tag purification following denaturation and renaturation of coexpressed AP-1 subunits. Coexpression of two constitutive components in a dimeric AP-1 complex helps stabilize the proteins when compared with individual protein expression in bacteria. Purified dimeric AP-1 complexes are functional in sequence-specific DNA binding, as illustrated by electrophoretic mobility shift assays and DNase I footprinting, and are also active in transcription with in vitro-reconstituted human papillomavirus (HPV) chromatin containing AP-1-binding sites in the native configuration of HPV nucleosomes. The availability of these recombinant full-length human AP-1 complexes has greatly facilitated mechanistic studies of AP-1-regulated gene transcription in many biological systems. PMID:18329890

Three Immunoproteasome-Associated Subunits Cooperatively Generate a Cytotoxic T-Lymphocyte Epitope of Epstein-Barr Virus LMP2A by Overcoming Specific Structures Resistant to Epitope Liberation

PubMed Central

Ito, Yoshinori; Kondo, Eisei; Demachi-Okamura, Ayako; Akatsuka, Yoshiki; Tsujimura, Kunio; Tanimoto, Mitsune; Morishima, Yasuo; Takahashi, Toshitada; Kuzushima, Kiyotaka

2006-01-01

The precise roles of gamma interferon-inducible immunoproteasome-associated molecules in generation of cytotoxic T-lymphocyte (CTL) epitopes have yet to be fully elucidated. We describe here a unique epitope derived from the Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) presented by HLA-A*2402 molecules. Generation of the epitope, designated LMP2A222-230, from the full-length protein requires the immunoproteasome subunit low-molecular-weight protein 7 (ip-LMP7) and the proteasome activator 28-α subunit and is accelerated by ip-LMP2, as revealed by gene expression experiments using an LMP2A222-230-specific CTL clone as a responder in enzyme-linked immunospot assays. The unequivocal involvement of all three components was confirmed by RNA interference gene silencing. Interestingly, the LMP2A222-230 epitope could be efficiently generated from incomplete EBV-LMP2A fragments that were produced by puromycin treatment or gene-engineered shortened EBV-LMP2A lacking some of its hydrophobic domains. In addition, epitope generation was increased by a single amino acid substitution from leucine to alanine immediately flanking the C terminus, this being predicted by a web-accessible program to increase the cleavage strength. Taken together, the data indicate that the generation of LMP2A222-230 is influenced not only by extrinsic factors such as immunoproteasomes but also by intrinsic factors such as the length of the EBV-LMP2A protein and proteasomal cleavage strength at specific positions in the source antigen. PMID:16378990

Differential Targeting of Gβγ-Subunit Signaling with Small Molecules

NASA Astrophysics Data System (ADS)

Bonacci, Tabetha M.; Mathews, Jennifer L.; Yuan, Chujun; Lehmann, David M.; Malik, Sundeep; Wu, Dianqing; Font, Jose L.; Bidlack, Jean M.; Smrcka, Alan V.

2006-04-01

G protein βγ subunits have potential as a target for therapeutic treatment of a number of diseases. We performed virtual docking of a small-molecule library to a site on Gβγ subunits that mediates protein interactions. We hypothesized that differential targeting of this surface could allow for selective modulation of Gβγ subunit functions. Several compounds bound to Gβγ subunits with affinities from 0.1 to 60 μM and selectively modulated functional Gβγ-protein-protein interactions in vitro, chemotactic peptide signaling pathways in HL-60 leukocytes, and opioid receptor-dependent analgesia in vivo. These data demonstrate an approach for modulation of G protein-coupled receptor signaling that may represent an important therapeutic strategy.

Identification and molecular characterization of five putative toxins from the venom gland of the snake Philodryas chamissonis (Serpentes: Dipsadidae).

PubMed

Urra, Félix A; Pulgar, Rodrigo; Gutiérrez, Ricardo; Hodar, Christian; Cambiazo, Verónica; Labra, Antonieta

2015-12-15

Philodryas chamissonis is a rear-fanged snake endemic to Chile. Its bite produces mild to moderate symptoms with proteolytic and anti-coagulant effects. Presently, the composition of the venom, as well as, the biochemical and structural characteristics of its toxins, remains unknown. In this study, we cloned and reported the first full-length sequences of five toxin-encoding genes from the venom gland of this species: Type III snake venom metalloprotease (SVMP), snake venom serine protease (SVSP), Cysteine-rich secretory protein (CRISP), α and β subunits of C-type lectin-like protein (CLP) and C-type natriuretic peptide (NP). These genes are highly expressed in the venom gland and their sequences exhibited a putative signal peptide, suggesting that these are components of the venom. These putative toxins had different evolutionary relationships with those reported for some front-fanged snakes, being SVMP, SVSP and CRISP of P. chamissonis closely related to the toxins present in Elapidae species, while NP was more related to those of Viperidae species. In addition, analyses suggest that the α and β subunits of CLP of P. chamissonis might have a α-subunit scaffold in common with Viperidae species, whose highly variable C-terminal region might have allowed the diversification in α and β subunits. Our results provide the first molecular description of the toxins possibly implicated in the envenomation of prey and humans by the bite of P. chamissonis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Cloning and functional characterization of inward-rectifying potassium (Kir) channels from Malpighian tubules of the mosquito Aedes aegypti

PubMed Central

Piermarini, Peter M.; Rouhier, Matthew F.; Schepel, Matthew; Kosse, Christin; Beyenbach, Klaus W.

2013-01-01

Inward-rectifying K+ (Kir) channels play critical physiological roles in a variety of vertebrate cells/tissues, including the regulation of membrane potential in nerve and muscle, and the transepithelial transport of ions in osmoregulatory epithelia, such as kidneys and gills. It remains to be determined whether Kir channels play similar physiological roles in insects. In the present study, we sought to 1) clone the cDNAs of Kir channel subunits expressed in the renal (Malpighian) tubules of the mosquito Aedes aegypti, and 2) characterize the electrophysiological properties of the cloned Kir subunits when expressed heterologously in oocytes of Xenopus laevis. Here, we reveal that three Kir subunits are expressed abundantly in Aedes Malpighian tubules (AeKir1, AeKir2B, and AeKir3); each of their full-length cDNAs was cloned. Heterologous expression of the AeKir1 or the AeKir2B subunits in Xenopus oocytes elicits inward-rectifying K+ currents that are blocked by barium. Relative to the AeKir2B-expressing oocytes, the AeKir1-expressing oocytes 1) produce larger macroscopic currents, and 2) exhibit a modulation of their conductive properties by extracellular Na+. Attempts to functionally characterize the AeKir3 subunit in Xenopus oocytes were unsuccessful. Lastly, we show that in isolated Aedes Malpighian tubules, the cation permeability sequence of the basolateral membrane of principal cells (Tl+ > K+ > Rb+ > NH4+) is consistent with the presence of functional Kir channels. We conclude that in Aedes Malpighian tubules, Kir channels contribute to the majority of the barium-sensitive transepithelial transport of K+. PMID:23085358

YC-1 BINDING TO THE BETA SUBUNIT OF SOLUBLE GUANYLYL CYCLASE OVERCOMES ALLOSTERIC INHIBITION BY THE ALPHA SUBUNIT

PubMed Central

Purohit, Rahul; Fritz, Bradley G.; The, Juliana; Issaian, Aaron; Weichsel, Andrzej; David, Cynthia L.; Campbell, Eric; Hausrath, Andrew C.; Rassouli-Taylor, Leida; Garcin, Elsa D.; Gage, Matthew J.; Montfort, William R.

2014-01-01

Soluble guanylate cyclase (sGC) is a heterodimeric heme protein and the primary nitric oxide receptor. NO binding stimulates cyclase activity, leading to regulation of cardiovascular physiology and making sGC an attractive target for drug discovery. YC-1 and related compounds stimulate sGC both independently and synergistically with NO and CO binding; however, where the compounds bind and how they work remains unknown. Using linked-equilibria binding measurements, surface plasmon resonance, and domain truncations in Manduca sexta and bovine sGC, we demonstrate that YC-1 binds near or directly to the heme-containing domain of the beta subunit. In the absence of CO, YC-1 binds with Kd = 9–21 μM, depending on construct. In the presence of CO, these values decrease to 0.6–1.1 μM. Pfizer compound 25 bound ~10-fold weaker than YC-1 in the absence of CO whereas compound BAY 41–2272 bound particularly tightly in the presence of CO (Kd = 30–90 nM). Additionally, we found that CO binding is much weaker to heterodimeric sGC proteins (Kd = 50–100 μM) than to the isolated heme domain (Kd = 0.2 μM for Manduca beta H-NOX/PAS). YC-1 greatly enhanced CO binding to heterodimeric sGC, as expected (Kd = ~1 μM). These data indicate the alpha subunit induces a heme pocket conformation with lower affinity for CO and NO. YC-1 family compounds bind near the heme domain, overcoming the alpha subunit effect and inducing a heme pocket conformation with high affinity. We propose this high-affinity conformation is required for the full-length protein to achieve high catalytic activity. PMID:24328155

Identification of a Kinase in Wheat Germ that Phosphorylates the Large Subunit of Initiation Factor 4F 1

PubMed Central

Humphreys, Jean; Browning, Karen S.; Ravel, Joanne M.

1988-01-01

A kinase has been isolated from wheat (Triticum aestivum) germ that phosphorylates the 220 kilodaltons (kD) subunit of wheat germ initiation factor (eIF) 4F, the 80 kD subunit of eIF-4B (an isozyme form of eIF-4F) and eIF-4G (the functional equivalent to mammalian eIF-4B). The kinase elutes from Sephacryl S-200 slightly in front of ovalbumin. The kinase phosphorylates casein and histone IIA to a small extent, but does not phosphorylate phosvitin. Of the wheat germ initiation factors, elongation factors, and small and large ribosomal subunits, only eIF-4F, eIF-4B, and eIF-4G are phosphorylated to a significant extent. The kinase phosphorylates eIF-4F to the extent of two phosphates per mole of the 220 kD subunit and phosphorylates eIF-4B to the extent of one phosphate per mole of the 80 kD subunit. The 26 kD subunit of eIF-4F and the 28 kD subunit of eIF-4B are not phosphorylated by the kinase. The kinase phosphorylates the 59 kD component of eIF-4G to the extent of 0.25 phosphate per mole of eIF-4G. Phosphorylation of eIF-4F and eIF-4B does not affect their ability to support the binding of mRNA to small ribosomal subunits in vitro. Images Fig. 2 Fig. 3 PMID:16666331

Identification of verotoxin type 2 variant B subunit genes in Escherichia coli by the polymerase chain reaction and restriction fragment length polymorphism analysis.

PubMed Central

Tyler, S D; Johnson, W M; Lior, H; Wang, G; Rozee, K R

1991-01-01

A set of synthetic oligonucleotide primers was designed for use in a polymerase chain reaction protocol to specifically detect the B subunit genes in vtx2ha and vtx2hb, which code for the production of the VT2 (Shiga-like toxin II) variant cytotoxins VT2v-a and VT2v-b, respectively. An additional set of primers amplified a fragment common to the B subunits of the VT2 and the VT2 variant genes. Subsequent restriction endonuclease digestion of this amplicon permitted prediction of specific VT2 and variant genotypes on the basis of predetermined restriction fragment length polymorphisms. Genotypes of 21 VT2-producing strains of Escherichia coli were determined using this polymerase chain reaction-restriction fragment length polymorphism procedure. Four strains contained B subunit target sequences only for VT2 genes, 9 strains contained sequences only for VT2v-a genes, and 3 strains contained sequences only for VT2v-b. For genes in combination, one strain contained B subunit genes for both VT2 and VT2v-a and two strains contained B subunit genes for VT2 and VT2v-b. Two strains of E. coli O91:H21 contained both VT2v-a and VT2v-b B subunit genes. The VT2 reference strain of E. coli, E32511, was found to contain the targeted sequences from both VT2 and VT2v-a genes, whereas the recombinant E. coli, pEB1, possessed only that of the VT2 gene. The specific activities of extracellular VT2 determined in HeLa cells ranged from 0.3 to 41.7 TCD50 per microgram of protein in strains carrying the VT2 gene target and from 0 to 50.0 TCD50 per microgram of protein in strains carrying only the VT2 variant target (TCD50 is the tissue culture dose by which 50% of the cells were affected), suggesting that phenotypic expression does not correlate with genotype. Images PMID:1679436

Multiple effects of S13 in modulating the strength of intersubunit interactions in the ribosome during translation.

PubMed

Cukras, Anthony R; Green, Rachel

2005-05-27

The ribosomal protein S13 is found in the head region of the small subunit, where it interacts with the central protuberance of the large ribosomal subunit and with the P site-bound tRNA through its extended C terminus. The bridging interactions between the large and small subunits are dynamic, and are thought to be critical in orchestrating the molecular motions of the translation cycle. S13 provides a direct link between the tRNA-binding site and the movements in the head of the small subunit seen during translocation, thereby providing a possible pathway of signal transduction. We have created and characterized an rpsM(S13)-deficient strain of Escherichia coli and have found significant defects in subunit association, initiation and translocation through in vitro assays of S13-deficient ribosomes. Targeted mutagenesis of specific bridge and tRNA contact elements in S13 provides evidence that these two interaction domains play critical roles in maintaining the fidelity of translation. This ribosomal protein thus appears to play a non-essential, yet important role by modulating subunit interactions in multiple steps of the translation cycle.

Functional Analysis of a Wheat AGPase Plastidial Small Subunit with a Truncated Transit Peptide.

PubMed

Yang, Yang; Gao, Tian; Xu, Mengjun; Dong, Jie; Li, Hanxiao; Wang, Pengfei; Li, Gezi; Guo, Tiancai; Kang, Guozhang; Wang, Yonghua

2017-03-01

ADP-glucose pyrophosphorylase (AGPase), the key enzyme in starch synthesis, consists of two small subunits and two large subunits with cytosolic and plastidial isoforms. In our previous study, a cDNA sequence encoding the plastidial small subunit (TaAGPS1b) of AGPase in grains of bread wheat ( Triticum aestivum L.) was isolated and the protein subunit encoded by this gene was characterized as a truncated transit peptide (about 50% shorter than those of other plant AGPS1bs). In the present study, TaAGPS1b was fused with green fluorescent protein (GFP) in rice protoplast cells, and confocal fluorescence microscopy observations revealed that like other AGPS1b containing the normal transit peptide, TaAGPS1b-GFP was localized in chloroplasts. TaAGPS1b was further overexpressed in a Chinese bread wheat cultivar, and the transgenic wheat lines exhibited a significant increase in endosperm AGPase activities, starch contents, and grain weights. These suggested that TaAGPS1b subunit was targeted into plastids by its truncated transit peptide and it could play an important role in starch synthesis in bread wheat grains.

Characterization of ribulose-1, 5-bisphosphate carboxylase/oxygenase and transcriptional analysis of its related genes in Saccharina japonica (Laminariales, Phaeophyta)

NASA Astrophysics Data System (ADS)

Shao, Zhanru; Liu, Fuli; Li, Qiuying; Yao, Jianting; Duan, Delin

2014-03-01

Saccharina japonica is a common macroalga in sublittoral communities of cold seawater environments, and consequently may have highly efficient ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) activity for carbon assimilation. In our study, we cloned the full-length Rubisco gene from S. japonica ( SJ-rbc). It contained an open reading frame for a large subunit gene ( SJ — rbcL) of 1 467 bp, a small subunit gene ( SJ-rbcS) of 420 bp, and a SJ-rbcL/S intergenic spacer of 269 bp. The deduced peptides of SJ-rbcL and SJ-rbcS were 488 and 139 amino acids with theoretical molecular weights and isoelectric points of 53.97 kDa, 5.81 and 15.84 kDa, 4.71, respectively. After induction with 1 mmol/L isopropyl- β-D-thiogalactopyranoside for 5 h and purification by Ni2+ affinity chromatography, electrophoresis and western blot detection demonstrated successful expression of the 55 kDa SJ-rbcL protein. Real-time quantitative PCR showed that the mRNA levels of SJ-rbcL in gametophytes increased when transferred into normal growth conditions and exhibited diurnal variations: increased expression during the day but suppressed expression at night. This observation implied that Rubisco played a role in normal gametophytic growth and development. In juvenile sporophytes, mRNA levels of SJ-rbcL, carbonic anhydrase, Calvin-Benson-Bassham cycle-related enzyme, and chloroplast light-harvesting protein were remarkably increased under continuous light irradiance. Similarly, expression of these genes was up-regulated under blue light irradiance at 350 μmol/(m2·s). Our results indicate that long-term white light and short-term blue light irradiance enhances juvenile sporophytic growth by synergistic effects of various photosynthetic elements.

Human alpha 7 acetylcholine receptor: cloning of the alpha 7 subunit from the SH-SY5Y cell line and determination of pharmacological properties of native receptors and functional alpha 7 homomers expressed in Xenopus oocytes.

PubMed

Peng, X; Katz, M; Gerzanich, V; Anand, R; Lindstrom, J

1994-03-01

The alpha-bungarotoxin-binding acetylcholine receptors from the human neuroblastoma cell line SH-SY5Y were found to cross-react with some monoclonal antibodies to alpha 7 subunits of nicotinic acetylcholine receptors from chicken brain. The human alpha 7 subunit cDNA from SH-SY5Y was cloned, revealing 94% amino acid sequence identity to rat alpha 7 subunits and 92% identity to chicken alpha 7 subunits. Native human alpha 7 receptors showed affinities for some ligands similar to those previously observed with native chicken alpha 7 receptors, but for other ligands there were large species-specific differences in binding affinity. These results paralleled properties of alpha 7 homomers expressed in Xenopus oocytes. Human alpha 7 homomers exhibited rapidly desensitizing, inwardly rectifying, agonist-induced, cation currents that triggered Ca(2+)-sensitive Cl- channels in the oocytes. A change in efficacy from partial agonist for chicken alpha 7 homomers to full agonist for human alpha 7 homomers was exhibited by 1,1-dimethyl-4-phenylpiperazinium. This result reveals a large species-specific pharmacological difference, despite small differences in alpha 7 sequences. This is important for understanding the effects of these drugs in humans and for identifying amino acids that may contribute to the acetylcholine binding site, for analysis by in vitro mutagenesis. These results also characterize properties of native alpha 7 receptors and alpha 7 homomers that will provide criteria for functional properties expected of structural subunits, when these can be identified, cloned, and coexpressed with alpha 7 subunits.

Stochastic Severing of Actin Filaments by Actin Depolymerizing Factor/Cofilin Controls the Emergence of a Steady Dynamical Regime

PubMed Central

Roland, Jeremy; Berro, Julien; Michelot, Alphée; Blanchoin, Laurent; Martiel, Jean-Louis

2008-01-01

Actin dynamics (i.e., polymerization/depolymerization) powers a large number of cellular processes. However, a great deal remains to be learned to explain the rapid actin filament turnover observed in vivo. Here, we developed a minimal kinetic model that describes key details of actin filament dynamics in the presence of actin depolymerizing factor (ADF)/cofilin. We limited the molecular mechanism to 1), the spontaneous growth of filaments by polymerization of actin monomers, 2), the ageing of actin subunits in filaments, 3), the cooperative binding of ADF/cofilin to actin filament subunits, and 4), filament severing by ADF/cofilin. First, from numerical simulations and mathematical analysis, we found that the average filament length, 〈L〉, is controlled by the concentration of actin monomers (power law: 5/6) and ADF/cofilin (power law: −2/3). We also showed that the average subunit residence time inside the filament, 〈T〉, depends on the actin monomer (power law: −1/6) and ADF/cofilin (power law: −2/3) concentrations. In addition, filament length fluctuations are ∼20% of the average filament length. Moreover, ADF/cofilin fragmentation while modulating filament length keeps filaments in a high molar ratio of ATP- or ADP-Pi versus ADP-bound subunits. This latter property has a protective effect against a too high severing activity of ADF/cofilin. We propose that the activity of ADF/cofilin in vivo is under the control of an affinity gradient that builds up dynamically along growing actin filaments. Our analysis shows that ADF/cofilin regulation maintains actin filaments in a highly dynamical state compatible with the cytoskeleton dynamics observed in vivo. PMID:18065447

Photoaffinity labeling of the Torpedo californica nicotinic acetylcholine receptor with an aryl azide derivative of phosphatidylserine

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

Blanton, M.P.; Wang, H.H.

1990-02-06

A photoactivatable analogue of phosphatidylserine, {sup 125}I-labeled 4-azidosalicylic acid-phosphatidylserine ({sup 125}I ASA-PS), was used to label both native acetylcholine receptor (AchR)-rich membranes from Torpedo californica and AchR membranes affinity purified from Torpedo reconstituted into asolectin vesicles. The radioiodinated arylazido group attaches directly to the phospholipid head group and thus probes for regions of the AchR structure in contact with the negatively charged head group of phosphatidylserine. All four subunits of the AchR incorporated the label, with the {alpha} subunit incorporating approximately twice as much as each of the other subunits on a per mole basis. The regions of the AchRmore » {alpha} subunit that incorporated {sup 125}I ASA-PS were mapped by Staphylococcus aureus V8 protease digestion. The majority of label incorporated into fragments representing a more complete digestion of the {alpha} subunit was localized to 11.7- and 10.1-kDa V8 cleavage fragments, both beginning at Asn-339 and of sufficient length to contain the hydrophobic region M4. An 18.7-kDa fragment beginning at Ser-173 and of sufficient length to contain the hydrophobic regions M1, M2, and M3 was also significantly labeled. In contrast, V8 cleavage fragments representing roughly a third of the amino-terminal portion of the {alpha} subunit incorporated little or no detectable amount of probe.« less

Proteolytic processing of endogenous and recombinant beta 4 integrin subunit

PubMed Central

1992-01-01

The alpha 6 beta 4 integrin is a receptor involved in the interaction of epithelial cells with basement membranes. This integrin is unique among the known integrins in that its beta 4 subunit has a large cytoplasmic domain. The function of this cytoplasmic domain is not known. In this paper we show that the beta 4 subunit undergoes proteolytic processing in cultured cells and provide evidence that this also happens in tissues. Immunoprecipitation experiments indicated that the cytoplasmic domain of beta 4 is susceptible to a calcium-dependent protease present in cellular extracts. In vitro assays with purified calpain showed that this enzyme can cleave beta 4 at two distinct sites in the cytoplasmic domain, generating truncated molecules of 165 and 130 kD. Immunoblotting experiments performed on cultured epithelial cells using an antibody to a peptide modeled after the COOH-terminus of the beta 4 subunit showed 70-kD fragments and several fragments of molecular masses between 185 and 115 kD. Similar fragments were detected in CHO cells transfected with the full-length beta 4 cDNA, but not in control transfected cells or in cells transfected with a mutant cDNA lacking the epitope of the cytoplasmic peptide antibody. The sizes of the fragments indicated that both the intracellular and extracellular domains of beta 4 are proteolytically processed. To examine the processing of the beta 4 subunit in epithelial tissues in vivo, human skin frozen sections were stained with antibodies to the ectodomain or the cytoplasmic domain of beta 4. The distinct staining patterns obtained with the two types of antibodies provided evidence that beta 4 is proteolytically processed in vivo in skin. Analogous experiments performed on sections of the cornea suggested that beta 4 is not proteolytically processed at a detectable level in this tissue. Thus, cleavage of the beta 4 subunit occurs in a tissue-specific fashion. These results suggest a potential mechanism of modulating the activities of the alpha 6 beta 4 integrin. PMID:1500432

Morphology and Small-Subunit Ribosomal DNA Sequence of Henneguya Adiposa (Myxosporea) From Ictalurus punctatus (Siluriformes)

USDA-ARS?s Scientific Manuscript database

The original description of Henneguya adiposa, a myxozoan parasitizing channel catfish Ictalurus punctatus, is supplemented with new data on spore morphology, including photomicrographs and line drawings, as well as 18S small-subunit (SSU) ribosomal DNA (rDNA) sequence. Elongate, translucent, linear...

Natural Endophytic Occurrence of Acetobacter diazotrophicus in Pineapple Plants.

PubMed

Tapia-Hernández; Bustillos-Cristales; Jiménez-Salgado; Caballero-Mellado; Fuentes-Ramírez

2000-01-01

The presence of endophytic Acetobacter diazotrophicus was tested for pineapple plants (Ananas comosus [L.] Merr.) grown in the field. Diazotrophic bacteria were isolated from the inner tissues of surface sterilized roots, stems, and leaves of pineapple plants. Phenotypic tests permitted the selection of presumptive nitrogen-fixing A. diazotrophicus isolates. Restriction fragment length polymorphisms (RFLPs) of small subunit (SSU) rDNA using total DNA digested with endonuclease SphI and with endonuclease NcoI, hybridizations of RNA with an A. diazotrophicus large subunit (LSU) rRNA specific probe, as well as patterns in denaturing protein electrophoresis (SDS-PAGE) and multilocus enzyme tests allowed the identification of A. diazotrophicus isolates. High frequencies of isolation were obtained from propagative buds that had not been nitrogen-fertilized, and lower frequencies from 3-month-old plants that had been nitrogen-fertilized. No isolates were recovered from 5- to 7-month-old nitrogen-fertilized plants. All the A. diazotrophicus isolates recovered from pineapple plants belonged to the multilocus genotype which shows the most extensive distribution among all host species previously analyzed.

Polyubiquitin-Photoactivatable Crosslinking Reagents for Mapping Ubiquitin Interactome Identify Rpn1 as a Proteasome Ubiquitin-Associating Subunit.

PubMed

Chojnacki, Michal; Mansour, Wissam; Hameed, Dharjath S; Singh, Rajesh K; El Oualid, Farid; Rosenzweig, Rina; Nakasone, Mark A; Yu, Zanlin; Glaser, Fabian; Kay, Lewis E; Fushman, David; Ovaa, Huib; Glickman, Michael H

2017-04-20

Ubiquitin (Ub) signaling is a diverse group of processes controlled by covalent attachment of small protein Ub and polyUb chains to a range of cellular protein targets. The best documented Ub signaling pathway is the one that delivers polyUb proteins to the 26S proteasome for degradation. However, studies of molecular interactions involved in this process have been hampered by the transient and hydrophobic nature of these interactions and the lack of tools to study them. Here, we develop Ub-phototrap (Ub PT ), a synthetic Ub variant containing a photoactivatable crosslinking side chain. Enzymatic polymerization into chains of defined lengths and linkage types provided a set of reagents that led to identification of Rpn1 as a third proteasome ubiquitin-associating subunit that coordinates docking of substrate shuttles, unloading of substrates, and anchoring of polyUb conjugates. Our work demonstrates the value of Ub PT , and we expect that its future uses will help define and investigate the ubiquitin interactome. Copyright © 2017 Elsevier Ltd. All rights reserved.

MUC1 and MUC4: Switching the Emphasis from Large to Small

PubMed Central

Carraway, Kermit L.

2011-01-01

Summation The MUC1 and MUC4 membrane mucins are each composed of a large alpha (α) and a small beta (β) subunit. The α subunits are fully exposed at the cell surface and contain variable numbers of repeated amino acid sequences that are heavily glycosylated. In contrast, the β subunits are much smaller and are anchored within the cell membrane, with their amino-terminal portions exposed at the cell surface and their carboxy-terminal tails facing the cytosol. Studies over the last several years are challenging the long-held belief that α subunits play the predominant role in cancer by conferring cellular properties that allow tumor cells to evade immune recognition and destruction. Indeed, the β subunits of MUC1 and MUC4 have emerged as oncogenes, as they engage signaling pathways responsible for tumor initiation and progression. Thus, a switch in the emphasis from the large α to the small β subunits offers attractive possibilities for successful clinical application. Such a focus shift is further supported by the absence of allelic polymorphism and variable glycosylation in the β subunit as well as by the presence of the β subunit in most MUC1 and MUC4 isoforms expressed by tumors. MUC1α, also known as CA15.3, is a Food and Drug Administration-approved serum biomarker for breast cancer, but its use is no longer recommended by the American Society of Clinical Oncology. However, comparison of β subunit expression in normal and malignant breast tissues may offer a novel approach to the exploitation of membrane mucins as biomarkers, as MUC1β-induced gene signatures with prognostic and predictive values in breast cancer have been reported. Preclinical studies with peptides that interfere with MUC1β oncogenic functions also look promising. PMID:21728842

Community Structure Comparisons of Hydrothermal Vent Microbial Mats Along the Mariana Arc and Back-arc

NASA Astrophysics Data System (ADS)

Hager, K. W.; Fullerton, H.; Moyer, C. L.

2015-12-01

Hydrothermal vents along the Mariana Arc and back-arc represent a hotspot of microbial diversity that has not yet been fully recognized. The Mariana Arc and back-arc contain hydrothermal vents with varied vent effluent chemistry and temperature, which translates to diverse community composition. We have focused on iron-rich sites where the dominant primary producers are iron oxidizing bacteria. Because microbes from these environments have proven elusive in culturing efforts, we performed culture independent analysis among different microbial communities found at these hydrothermal vents. Terminal-restriction fragment length polymorphism (T-RFLP) and Illumina sequencing of small subunit ribosomal gene amplicons were used to characterize community members and identify samples for shotgun metagenomics. Used in combination, these methods will better elucidate the composition and characteristics of the bacterial communities at these hydrothermal vent systems. The overarching goal of this study is to evaluate and compare taxonomic and metabolic diversity among different communities of microbial mats. We compared communities collected on a fine scale to analyze the bacterial community based on gross mat morphology, geography, and nearby vent effluent chemistry. Taxa richness and evenness are compared with rarefaction curves to visualize diversity. As well as providing a survey of diversity this study also presents a juxtaposition of three methods in which ribosomal small subunit diversity is compared with T-RFLP, next generation amplicon sequencing, and metagenomic shotgun sequencing.

Biogenesis of the bacterial cbb3 cytochrome c oxidase: Active subcomplexes support a sequential assembly model.

PubMed

Durand, Anne; Bourbon, Marie-Line; Steunou, Anne-Soisig; Khalfaoui-Hassani, Bahia; Legrand, Camille; Guitton, Audrey; Astier, Chantal; Ouchane, Soufian

2018-01-19

The cbb 3 oxidase has a high affinity for oxygen and is required for growth of bacteria, including pathogens, in oxygen-limited environments. However, the assembly of this oxidase is poorly understood. Most cbb 3 are composed of four subunits: the catalytic CcoN subunit, the two cytochrome c subunits (CcoO and CcoP) involved in electron transfer, and the small CcoQ subunit with an unclear function. Here, we address the role of these four subunits in cbb 3 biogenesis in the purple bacterium Rubrivivax gelatinosus Analyses of membrane proteins from different mutants revealed the presence of active CcoNQO and CcoNO subcomplexes and also showed that the CcoP subunit is not essential for their assembly. However, CcoP was required for the oxygen reduction activity in the absence of CcoQ. We also found that CcoQ is dispensable for forming an active CcoNOP subcomplex in membranes. CcoNOP exhibited oxygen reductase activity, indicating that the cofactors (hemes b and copper for CcoN and cytochromes c for CcoO and CcoP) were present within the subunits. Finally, we discovered the presence of a CcoNQ subcomplex and showed that CcoN is the required anchor for the assembly of the full CcoNQOP complex. On the basis of these findings, we propose a sequential assembly model in which the CcoQ subunit is required for the early maturation step: CcoQ first associates with CcoN before the CcoNQ-CcoO interaction. CcoP associates to CcoNQO subcomplex in the late maturation step, and once the CcoNQOP complex is fully formed, CcoQ is released for degradation by the FtsH protease. This model could be conserved in other bacteria, including the pathogenic bacteria lacking the assembly factor CcoH as in R. gelatinosus . © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Three Group-I introns in 18S rDNA of Endosymbiotic Algae of Paramecium bursaria from Japan

NASA Astrophysics Data System (ADS)

Hoshina, Ryo; Kamako, Shin-ichiro; Imamura, Nobutaka

2004-08-01

In the nuclear encoded small subunit ribosomal DNA (18S rDNA) of symbiotic alga of Paramecium bursaria (F36 collected in Japan) possesses three intron-like insertions (Hoshina et al., unpubl. data, 2003). The present study confirmed these exact lengths and insertion sites by reverse transcription-PCR. Two of them were inserted at Escherichia coli 16S rRNA genic position 943 and 1512 that are frequent intron insertion positions, but another insertion position (nearly 1370) was the first finding. Their secondary structures suggested they belong to Group-I intron; one belongs to subgroup IE, others belong to subgroup IC1. Similarity search indicated these introns are ancestral ones.

Small subunits of RNA polymerase: localization, levels and implications for core enzyme composition.

PubMed

Doherty, Geoff P; Fogg, Mark J; Wilkinson, Anthony J; Lewis, Peter J

2010-12-01

Bacterial RNA polymerases (RNAPs) contain several small auxiliary subunits known to co-purify with the core α, β and β' subunits. The ω subunit is conserved between Gram-positive and Gram-negative bacteria, while the δ subunit is conserved within, but restricted to, Gram-positive bacteria. Although various functions have been assigned to these subunits via in vitro assays, very little is known about their in vivo roles. In this work we constructed a pair of vectors to investigate the subcellular localization of the δ and ω subunits in Bacillus subtilis with respect to the core RNAP. We found these subunits to be closely associated with RNAP involved in transcribing both mRNA and rRNA operons. Quantification of these subunits revealed δ to be present at equimolar levels with RNAP and ω to be present at around half the level of core RNAP. For comparison, the localization and quantification of RNAP β' and ω subunits in Escherichia coli was also investigated. Similar to B. subtilis, β' and ω closely associated with the nucleoid and formed subnucleoid regions of high green fluorescent protein intensity, but, unlike ω in B. subtilis, ω levels in E. coli were close to parity with those of β'. These results indicate that δ is likely to be an integral RNAP subunit in Gram-positives, whereas ω levels differ substantially between Gram-positives and -negatives. The ω subunit may be required for RNAP assembly and subsequently be turned over at different rates or it may play roles in Gram-negative bacteria that are performed by other factors in Gram-positives.

Altered Distribution of RNA Polymerase Lacking the Omega Subunit within the Prophages along the Escherichia coli K-12 Genome.

PubMed

Yamamoto, Kaneyoshi; Yamanaka, Yuki; Shimada, Tomohiro; Sarkar, Paramita; Yoshida, Myu; Bhardwaj, Neerupma; Watanabe, Hiroki; Taira, Yuki; Chatterji, Dipankar; Ishihama, Akira

2018-01-01

The RNA polymerase (RNAP) of Escherichia coli K-12 is a complex enzyme consisting of the core enzyme with the subunit structure α 2 ββ'ω and one of the σ subunits with promoter recognition properties. The smallest subunit, omega (the rpoZ gene product), participates in subunit assembly by supporting the folding of the largest subunit, β', but its functional role remains unsolved except for its involvement in ppGpp binding and stringent response. As an initial approach for elucidation of its functional role, we performed in this study ChIP-chip (chromatin immunoprecipitation with microarray technology) analysis of wild-type and rpoZ -defective mutant strains. The altered distribution of RpoZ-defective RNAP was identified mostly within open reading frames, in particular, of the genes inside prophages. For the genes that exhibited increased or decreased distribution of RpoZ-defective RNAP, the level of transcripts increased or decreased, respectively, as detected by reverse transcription-quantitative PCR (qRT-PCR). In parallel, we analyzed, using genomic SELEX (systemic evolution of ligands by exponential enrichment), the distribution of constitutive promoters that are recognized by RNAP RpoD holoenzyme alone and of general silencer H-NS within prophages. Since all 10 prophages in E. coli K-12 carry only a small number of promoters, the altered occupancy of RpoZ-defective RNAP and of transcripts might represent transcription initiated from as-yet-unidentified host promoters. The genes that exhibited transcription enhanced by RpoZ-defective RNAP are located in the regions of low-level H-NS binding. By using phenotype microarray (PM) assay, alterations of some phenotypes were detected for the rpoZ -deleted mutant, indicating the involvement of RpoZ in regulation of some genes. Possible mechanisms of altered distribution of RNAP inside prophages are discussed. IMPORTANCE The 91-amino-acid-residue small-subunit omega (the rpoZ gene product) of Escherichia coli RNA polymerase plays a structural role in the formation of RNA polymerase (RNAP) as a chaperone in folding the largest subunit (β', of 1,407 residues in length), but except for binding of the stringent signal ppGpp, little is known of its role in the control of RNAP function. After analysis of genomewide distribution of wild-type and RpoZ-defective RNAP by the ChIP-chip method, we found alteration of the RpoZ-defective RNAP inside open reading frames, in particular, of the genes within prophages. For a set of the genes that exhibited altered occupancy of the RpoZ-defective RNAP, transcription was found to be altered as observed by qRT-PCR assay. All the observations here described indicate the involvement of RpoZ in recognition of some of the prophage genes. This study advances understanding of not only the regulatory role of omega subunit in the functions of RNAP but also the regulatory interplay between prophages and the host E. coli for adjustment of cellular physiology to a variety of environments in nature.

Altered Distribution of RNA Polymerase Lacking the Omega Subunit within the Prophages along the Escherichia coli K-12 Genome

PubMed Central

Yamamoto, Kaneyoshi; Yamanaka, Yuki; Shimada, Tomohiro; Sarkar, Paramita; Yoshida, Myu; Bhardwaj, Neerupma; Watanabe, Hiroki; Taira, Yuki

2018-01-01

ABSTRACT The RNA polymerase (RNAP) of Escherichia coli K-12 is a complex enzyme consisting of the core enzyme with the subunit structure α2ββ′ω and one of the σ subunits with promoter recognition properties. The smallest subunit, omega (the rpoZ gene product), participates in subunit assembly by supporting the folding of the largest subunit, β′, but its functional role remains unsolved except for its involvement in ppGpp binding and stringent response. As an initial approach for elucidation of its functional role, we performed in this study ChIP-chip (chromatin immunoprecipitation with microarray technology) analysis of wild-type and rpoZ-defective mutant strains. The altered distribution of RpoZ-defective RNAP was identified mostly within open reading frames, in particular, of the genes inside prophages. For the genes that exhibited increased or decreased distribution of RpoZ-defective RNAP, the level of transcripts increased or decreased, respectively, as detected by reverse transcription-quantitative PCR (qRT-PCR). In parallel, we analyzed, using genomic SELEX (systemic evolution of ligands by exponential enrichment), the distribution of constitutive promoters that are recognized by RNAP RpoD holoenzyme alone and of general silencer H-NS within prophages. Since all 10 prophages in E. coli K-12 carry only a small number of promoters, the altered occupancy of RpoZ-defective RNAP and of transcripts might represent transcription initiated from as-yet-unidentified host promoters. The genes that exhibited transcription enhanced by RpoZ-defective RNAP are located in the regions of low-level H-NS binding. By using phenotype microarray (PM) assay, alterations of some phenotypes were detected for the rpoZ-deleted mutant, indicating the involvement of RpoZ in regulation of some genes. Possible mechanisms of altered distribution of RNAP inside prophages are discussed. IMPORTANCE The 91-amino-acid-residue small-subunit omega (the rpoZ gene product) of Escherichia coli RNA polymerase plays a structural role in the formation of RNA polymerase (RNAP) as a chaperone in folding the largest subunit (β′, of 1,407 residues in length), but except for binding of the stringent signal ppGpp, little is known of its role in the control of RNAP function. After analysis of genomewide distribution of wild-type and RpoZ-defective RNAP by the ChIP-chip method, we found alteration of the RpoZ-defective RNAP inside open reading frames, in particular, of the genes within prophages. For a set of the genes that exhibited altered occupancy of the RpoZ-defective RNAP, transcription was found to be altered as observed by qRT-PCR assay. All the observations here described indicate the involvement of RpoZ in recognition of some of the prophage genes. This study advances understanding of not only the regulatory role of omega subunit in the functions of RNAP but also the regulatory interplay between prophages and the host E. coli for adjustment of cellular physiology to a variety of environments in nature. PMID:29468196

Chemotactic Signaling by Single-Chain Chemoreceptors

PubMed Central

Mowery, Patricia; Ames, Peter; Reiser, Rebecca H.; Parkinson, John S.

2015-01-01

Bacterial chemoreceptors of the methyl-accepting chemotaxis protein (MCP) family operate in commingled clusters that enable cells to detect and track environmental chemical gradients with high sensitivity and precision. MCP homodimers of different detection specificities form mixed trimers of dimers that facilitate inter-receptor communication in core signaling complexes, which in turn assemble into a large signaling network. The two subunits of each homodimeric receptor molecule occupy different locations in the core complexes. One subunit participates in trimer-stabilizing interactions at the trimer axis, the other lies on the periphery of the trimer, where it can interact with two cytoplasmic proteins: CheA, a signaling autokinase, and CheW, which couples CheA activity to receptor control. As a possible tool for independently manipulating receptor subunits in these two structural environments, we constructed and characterized fused genes for the E. coli serine chemoreceptor Tsr that encoded single-chain receptor molecules in which the C-terminus of the first Tsr subunit was covalently connected to the N-terminus of the second with a polypeptide linker. We showed with soft agar assays and with a FRET-based in vivo CheA kinase assay that single-chain Tsr~Tsr molecules could promote serine sensing and chemotaxis responses. The length of the connection between the joined subunits was critical. Linkers nine residues or shorter locked the receptor in a kinase-on state, most likely by distorting the native structure of the receptor HAMP domain. Linkers 22 or more residues in length permitted near-normal Tsr function. Few single-chain molecules were found as monomer-sized proteolytic fragments in cells, indicating that covalently joined receptor subunits were responsible for mediating the signaling responses we observed. However, cysteine-directed crosslinking, spoiling by dominant-negative Tsr subunits, and rearrangement of ligand-binding site lesions revealed subunit swapping interactions that will need to be taken into account in experimental applications of single-chain chemoreceptors. PMID:26709829

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

Januszyk, Kurt; Liu, Quansheng; Lima, Christopher D.

The eukaryotic RNA exosome is a highly conserved multi-subunit complex that catalyzes degradation and processing of coding and noncoding RNA. A noncatalytic nine-subunit exosome core interacts with Rrp44 and Rrp6, two subunits that possess processive and distributive 3'-to-5' exoribonuclease activity, respectively. While both Rrp6 and Rrp44 are responsible for RNA processing in budding yeast, Rrp6 may play a more prominent role in processing, as it has been demonstrated to be inhibited by stable RNA secondary structure in vitro and because the null allele in budding yeast leads to the buildup of specific structured RNA substrates. Human RRP6, otherwise known asmore » PM/SCL-100 or EXOSC10, shares sequence similarity to budding yeast Rrp6 and is proposed to catalyze 3'-to-5' exoribonuclease activity on a variety of nuclear transcripts including ribosomal RNA subunits, RNA that has been poly-adenylated by TRAMP, as well as other nuclear RNA transcripts destined for processing and/or destruction. To characterize human RRP6, we expressed the full-length enzyme as well as truncation mutants that retain catalytic activity, compared their activities to analogous constructs for Saccharomyces cerevisiae Rrp6, and determined the X-ray structure of a human construct containing the exoribonuclease and HRDC domains that retains catalytic activity. Structural data show that the human active site is more exposed when compared to the yeast structure, and biochemical data suggest that this feature may play a role in the ability of human RRP6 to productively engage and degrade structured RNA substrates more effectively than the analogous budding yeast enzyme.« less

Metataxonomics reveal vultures as a reservoir for Clostridium perfringens.

PubMed

Meng, Xiangli; Lu, Shan; Yang, Jing; Jin, Dong; Wang, Xiaohong; Bai, Xiangning; Wen, Yumeng; Wang, Yiting; Niu, Lina; Ye, Changyun; Rosselló-Móra, Ramon; Xu, Jianguo

2017-02-22

The Old World vulture may carry and spread pathogens for emerging infections since they feed on the carcasses of dead animals and participate in the sky burials of humans, some of whom have died from communicable diseases. Therefore, we studied the precise fecal microbiome of the Old World vulture with metataxonomics, integrating the high-throughput sequencing of almost full-length small subunit ribosomal RNA (16S rRNA) gene amplicons in tandem with the operational phylogenetic unit (OPU) analysis strategy. Nine vultures of three species were sampled using rectal swabs on the Qinghai-Tibet Plateau, China. Using the Pacific Biosciences sequencing platform, we obtained 54 135 high-quality reads of 16S rRNA amplicons with an average of 1442±6.9 bp in length and 6015±1058 reads per vulture. Those sequences were classified into 314 OPUs, including 102 known species, 50 yet to be described species and 161 unknown new lineages of uncultured representatives. Forty-five species have been reported to be responsible for human outbreaks or infections, and 23 yet to be described species belong to genera that include pathogenic species. Only six species were common to all vultures. Clostridium perfringens was the most abundant and present in all vultures, accounting for 30.8% of the total reads. Therefore, using the new technology, we found that vultures are an important reservoir for C. perfringens as evidenced by the isolation of 107 strains encoding for virulence genes, representing 45 sequence types. Our study suggests that the soil-related C. perfringens and other pathogens could have a reservoir in vultures and other animals.

Metataxonomics reveal vultures as a reservoir for Clostridium perfringens

PubMed Central

Meng, Xiangli; Lu, Shan; Yang, Jing; Jin, Dong; Wang, Xiaohong; Bai, Xiangning; Wen, Yumeng; Wang, Yiting; Niu, Lina; Ye, Changyun; Rosselló-Móra, Ramon; Xu, Jianguo

2017-01-01

The Old World vulture may carry and spread pathogens for emerging infections since they feed on the carcasses of dead animals and participate in the sky burials of humans, some of whom have died from communicable diseases. Therefore, we studied the precise fecal microbiome of the Old World vulture with metataxonomics, integrating the high-throughput sequencing of almost full-length small subunit ribosomal RNA (16S rRNA) gene amplicons in tandem with the operational phylogenetic unit (OPU) analysis strategy. Nine vultures of three species were sampled using rectal swabs on the Qinghai-Tibet Plateau, China. Using the Pacific Biosciences sequencing platform, we obtained 54 135 high-quality reads of 16S rRNA amplicons with an average of 1442±6.9 bp in length and 6015±1058 reads per vulture. Those sequences were classified into 314 OPUs, including 102 known species, 50 yet to be described species and 161 unknown new lineages of uncultured representatives. Forty-five species have been reported to be responsible for human outbreaks or infections, and 23 yet to be described species belong to genera that include pathogenic species. Only six species were common to all vultures. Clostridium perfringens was the most abundant and present in all vultures, accounting for 30.8% of the total reads. Therefore, using the new technology, we found that vultures are an important reservoir for C. perfringens as evidenced by the isolation of 107 strains encoding for virulence genes, representing 45 sequence types. Our study suggests that the soil-related C. perfringens and other pathogens could have a reservoir in vultures and other animals. PMID:28223683

Altered stoichiometry Escherichia coli Cascade complexes with shortened CRISPR RNA spacers are capable of interference and primed adaptation

DOE PAGES

Kuznedelov, Konstantin; Mekler, Vladimir; Lemak, Sofia; ...

2016-10-13

The Escherichia coli type I-E CRISPR-Cas system Cascade effector is a multisubunit complex that binds CRISPR RNA (crRNA). Through its 32-nucleotide spacer sequence, Cascade-bound crRNA recognizes protospacers in foreign DNA, causing its destruction during CRISPR interference or acquisition of additional spacers in CRISPR array during primed CRISPR adaptation. Within Cascade, the crRNA spacer interacts with a hexamer of Cas7 subunits. We show that crRNAs with a spacer length reduced to 14 nucleotides cause primed adaptation, while crRNAs with spacer lengths of more than 20 nucleotides cause both primed adaptation and target interference in vivo. Shortened crRNAs assemble into altered-stoichiometry Cascademore » effector complexes containing less than the normal amount of Cas7 subunits. The results show that Cascade assembly is driven by crRNA and suggest that multi-subunit type I CRISPR effectors may have evolved from much simpler ancestral complexes.« less

Altered stoichiometry Escherichia coli Cascade complexes with shortened CRISPR RNA spacers are capable of interference and primed adaptation

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

Kuznedelov, Konstantin; Mekler, Vladimir; Lemak, Sofia

The Escherichia coli type I-E CRISPR-Cas system Cascade effector is a multisubunit complex that binds CRISPR RNA (crRNA). Through its 32-nucleotide spacer sequence, Cascade-bound crRNA recognizes protospacers in foreign DNA, causing its destruction during CRISPR interference or acquisition of additional spacers in CRISPR array during primed CRISPR adaptation. Within Cascade, the crRNA spacer interacts with a hexamer of Cas7 subunits. We show that crRNAs with a spacer length reduced to 14 nucleotides cause primed adaptation, while crRNAs with spacer lengths of more than 20 nucleotides cause both primed adaptation and target interference in vivo. Shortened crRNAs assemble into altered-stoichiometry Cascademore » effector complexes containing less than the normal amount of Cas7 subunits. The results show that Cascade assembly is driven by crRNA and suggest that multi-subunit type I CRISPR effectors may have evolved from much simpler ancestral complexes.« less

Genetic diversity and stock identification of small abalone (Haliotis diversicolor) in Taiwan and Japan

PubMed Central

Hsu, Te-Hua; Gwo, Jin-Chywan

2017-01-01

Small abalone (Haliotis diversicolor) is a commercially valuable species for both fisheries and aquaculture. The production of annual farmed small abalone in Taiwan, once the highest in the world, has dramatically decreased in the past 15 years, and currently, the industry is close to collapse. Understanding the genetic diversity of small abalone and developing stock identification methods will be useful for genetic breeding, restoring collapsed stocks, managing stocks, and preventing illegal trade. We investigated 307 cultured and wild individuals from Taiwan, Japan, and Bali Island (Indonesia) by using the mitochondrial cytochrome c oxidase subunit I (COI) gene. Network analysis of mtDNA COI gene sequences revealed that the individuals collected from Taiwan, Japan, and Indonesia could be identified, and showed significant genetic divergence. In addition, the Indonesian population (Haliotis diversicolor squamata) was significantly different from the other populations and might need to be considered a separate species. We discovered a single nucleotide polymorphism marker in the mtDNA COI gene that can be used to distinguish the Taiwan population from the Japan population. We also developed a polymerase chain reaction-restriction fragment length polymorphism method for rapid detection. Furthermore, we could identify the cultured stocks, wild population, and hybrid stocks by using 6 microsatellites and amplified fragment length polymorphism. This study contributes useful tools for stock identification and the production of high-disease resistant small abalone strains (Japan × Taiwan or Taiwan × Japan). Efforts should be made to avoid unintentional random genetic mixing of the Taiwan population with the Japan population and subsequent breakdown of population differentiation, which impair local adaptation of the Taiwan wild population. Molecular markers revealed a split between the Taiwan and Japan populations, and the existence of a possible barrier to the free dispersal of small abalone is discussed. PMID:28662122

Genetic diversity and stock identification of small abalone (Haliotis diversicolor) in Taiwan and Japan.

PubMed

Hsu, Te-Hua; Gwo, Jin-Chywan

2017-01-01

Small abalone (Haliotis diversicolor) is a commercially valuable species for both fisheries and aquaculture. The production of annual farmed small abalone in Taiwan, once the highest in the world, has dramatically decreased in the past 15 years, and currently, the industry is close to collapse. Understanding the genetic diversity of small abalone and developing stock identification methods will be useful for genetic breeding, restoring collapsed stocks, managing stocks, and preventing illegal trade. We investigated 307 cultured and wild individuals from Taiwan, Japan, and Bali Island (Indonesia) by using the mitochondrial cytochrome c oxidase subunit I (COI) gene. Network analysis of mtDNA COI gene sequences revealed that the individuals collected from Taiwan, Japan, and Indonesia could be identified, and showed significant genetic divergence. In addition, the Indonesian population (Haliotis diversicolor squamata) was significantly different from the other populations and might need to be considered a separate species. We discovered a single nucleotide polymorphism marker in the mtDNA COI gene that can be used to distinguish the Taiwan population from the Japan population. We also developed a polymerase chain reaction-restriction fragment length polymorphism method for rapid detection. Furthermore, we could identify the cultured stocks, wild population, and hybrid stocks by using 6 microsatellites and amplified fragment length polymorphism. This study contributes useful tools for stock identification and the production of high-disease resistant small abalone strains (Japan × Taiwan or Taiwan × Japan). Efforts should be made to avoid unintentional random genetic mixing of the Taiwan population with the Japan population and subsequent breakdown of population differentiation, which impair local adaptation of the Taiwan wild population. Molecular markers revealed a split between the Taiwan and Japan populations, and the existence of a possible barrier to the free dispersal of small abalone is discussed.

Structural Variation of Type I-F CRISPR RNA Guided DNA Surveillance.

PubMed

Pausch, Patrick; Müller-Esparza, Hanna; Gleditzsch, Daniel; Altegoer, Florian; Randau, Lennart; Bange, Gert

2017-08-17

CRISPR-Cas systems are prokaryotic immune systems against invading nucleic acids. Type I CRISPR-Cas systems employ highly diverse, multi-subunit surveillance Cascade complexes that facilitate duplex formation between crRNA and complementary target DNA for R-loop formation, retention, and DNA degradation by the subsequently recruited nuclease Cas3. Typically, the large subunit recognizes bona fide targets through the PAM (protospacer adjacent motif), and the small subunit guides the non-target DNA strand. Here, we present the Apo- and target-DNA-bound structures of the I-Fv (type I-F variant) Cascade lacking the small and large subunits. Large and small subunits are functionally replaced by the 5' terminal crRNA cap Cas5fv and the backbone protein Cas7fv, respectively. Cas5fv facilitates PAM recognition from the DNA major groove site, in contrast to all other described type I systems. Comparison of the type I-Fv Cascade with an anti-CRISPR protein-bound I-F Cascade reveals that the type I-Fv structure differs substantially at known anti-CRISPR protein target sites and might therefore be resistant to viral Cascade interception. Copyright © 2017 Elsevier Inc. All rights reserved.

Crystallization and preliminary X-ray crystallographic analysis of the small subunit of the heterodimeric laccase POXA3b from Pleurotus ostreatus

PubMed Central

Ferraroni, Marta; Scozzafava, Andrea; Ullah, Sana; Tron, Thierry; Piscitelli, Alessandra; Sannia, Giovanni

2014-01-01

Laccases are multicopper oxidases of great biotechnological potential. While laccases are generally monomeric glycoproteins, the white-rot fungus Pleurotus ostreatus produces two closely related heterodimeric isoenzymes composed of a large subunit, homologous to the other fungal laccases, and a small subunit. The sequence of the small subunit does not show significant homology to any other protein or domain of known function and consequently its function is unknown. The highest similarity to proteins of known structure is to a putative enoyl-CoA hydratase/isomerase from Acinetobacter baumannii, which shows an identity of 27.8%. Diffraction-quality crystals of the small subunit of the heterodimeric laccase POXA3b (sPOXA3b) from P. ostreatus were obtained using the sitting-drop vapour-diffusion method at 294 K from a solution consisting of 1.8 M sodium formate, 0.1 M Tris–HCl pH 8.5. The crystals belonged to the tetragonal space group P41212 or P43212, with unit-cell parameters a = 126.6, c = 53.9 Å. The asymmetric unit contains two molecules related by a noncrystallographic twofold axis. A complete data set extending to a maximum resolution of 2.5 Å was collected at 100 K using a wavelength of 1.140 Å. PMID:24419623

Spontaneous quaternary and tertiary T-R transitions of human hemoglobin in molecular dynamics simulation.

PubMed

Hub, Jochen S; Kubitzki, Marcus B; de Groot, Bert L

2010-05-06

We present molecular dynamics simulations of unliganded human hemoglobin (Hb) A under physiological conditions, starting from the R, R2, and T state. The simulations were carried out with protonated and deprotonated HC3 histidines His(beta)146, and they sum up to a total length of 5.6 micros. We observe spontaneous and reproducible T-->R quaternary transitions of the Hb tetramer and tertiary transitions of the alpha and beta subunits, as detected from principal component projections, from an RMSD measure, and from rigid body rotation analysis. The simulations reveal a marked asymmetry between the alpha and beta subunits. Using the mutual information as correlation measure, we find that the beta subunits are substantially more strongly linked to the quaternary transition than the alpha subunits. In addition, the tertiary populations of the alpha and beta subunits differ substantially, with the beta subunits showing a tendency towards R, and the alpha subunits showing a tendency towards T. Based on the simulation results, we present a transition pathway for coupled quaternary and tertiary transitions between the R and T conformations of Hb.

Spontaneous Quaternary and Tertiary T-R Transitions of Human Hemoglobin in Molecular Dynamics Simulation

PubMed Central

de Groot, Bert L.

2010-01-01

We present molecular dynamics simulations of unliganded human hemoglobin (Hb) A under physiological conditions, starting from the R, R2, and T state. The simulations were carried out with protonated and deprotonated HC3 histidines His(β)146, and they sum up to a total length of 5.6µs. We observe spontaneous and reproducible T→R quaternary transitions of the Hb tetramer and tertiary transitions of the α and β subunits, as detected from principal component projections, from an RMSD measure, and from rigid body rotation analysis. The simulations reveal a marked asymmetry between the α and β subunits. Using the mutual information as correlation measure, we find that the β subunits are substantially more strongly linked to the quaternary transition than the α subunits. In addition, the tertiary populations of the α and β subunits differ substantially, with the β subunits showing a tendency towards R, and the α subunits showing a tendency towards T. Based on the simulation results, we present a transition pathway for coupled quaternary and tertiary transitions between the R and T conformations of Hb. PMID:20463873

Hepatitis C virus NS3 helicase forms oligomeric structures that exhibit optimal DNA unwinding activity in vitro.

PubMed

Sikora, Bartek; Chen, Yingfeng; Lichti, Cheryl F; Harrison, Melody K; Jennings, Thomas A; Tang, Yong; Tackett, Alan J; Jordan, John B; Sakon, Joshua; Cameron, Craig E; Raney, Kevin D

2008-04-25

HCV NS3 helicase exhibits activity toward DNA and RNA substrates. The DNA helicase activity of NS3 has been proposed to be optimal when multiple NS3 molecules are bound to the same substrate molecule. NS3 catalyzes little or no measurable DNA unwinding under single cycle conditions in which the concentration of substrate exceeds the concentration of enzyme by 5-fold. However, when NS3 (100 nm) is equimolar with the substrate, a small burst amplitude of approximately 8 nm is observed. The burst amplitude increases as the enzyme concentration increases, consistent with the idea that multiple molecules are needed for optimal unwinding. Protein-protein interactions may facilitate optimal activity, so the oligomeric properties of the enzyme were investigated. Chemical cross-linking indicates that full-length NS3 forms higher order oligomers much more readily than the NS3 helicase domain. Dynamic light scattering indicates that full-length NS3 exists as an oligomer, whereas NS3 helicase domain exists in a monomeric form in solution. Size exclusion chromatography also indicates that full-length NS3 behaves as an oligomer in solution, whereas the NS3 helicase domain behaves as a monomer. When NS3 was passed through a small pore filter capable of removing protein aggregates, greater than 95% of the protein and the DNA unwinding activity was removed from solution. In contrast, only approximately 10% of NS3 helicase domain and approximately 20% of the associated DNA unwinding activity was removed from solution after passage through the small pore filter. The results indicate that the optimally active form of full-length NS3 is part of an oligomeric species in vitro.

Cop9/signalosome subunits and Pcu4 regulate ribonucleotide reductase by both checkpoint-dependent and -independent mechanisms

PubMed Central

Liu, Cong; Powell, Kelly A.; Mundt, Kirsten; Wu, LeJung; Carr, Antony M.; Caspari, Thomas

2003-01-01

The signalosome is implicated in regulating cullin-dependent ubiquitin ligases. We find that two signalosome subunits, Csn1 and Csn2, are required to regulate ribonucleotide reductase (RNR) through the degradation of a small protein, Spd1, that acts to anchor the small RNR subunit in the nucleus. Spd1 destruction correlates with the nuclear export of the small RNR subunit, which, in turn, correlates with a requirement for RNR in replication and repair. Spd1 degradation is promoted by two separate CSN-dependent mechanisms. During unperturbed S phase, Spd1 degradation is independent of checkpoint proteins. In irradiated G2 cells, Spd1 degradation requires the DNA damage checkpoint. The signalosome copurifies with Pcu4 (cullin 4). Pcu4, Csn1, and Csn2 promote the degradation of Spd1, identifying a new function for the signalosome as a regulator of Pcu4-containing E3 ubiquitin ligase. PMID:12695334

Engineering behavior of small-scale foundation piers constructed from alternative materials

NASA Astrophysics Data System (ADS)

Prokudin, Maxim Mikhaylovich

Testing small-scale prototype pier foundations to evaluate engineering behavior is an alternative to full-scale testing that facilitates testing of several piers and pier groups at relatively low cost. In this study, various pier systems and pier groups at one tenth scale were subjected to static vertical loading under controlled conditions to evaluate stiffness, bearing capacity, and group efficiency. Pier length, material properties and methods of installation were evaluated. Pier length to diameter ratios varied between four and eight. A unique soil pit with dimensions of 2.1 m in width, 1.5 m in length and 2.0 m in depth was designed to carry out this research. The test pit was filled with moisture conditioned and compacted Western Iowa loess. A special load test frame was designed and fabricated to provide up to 25,000 kg vertical reaction force for load testing. A load cell and displacement instrumentation was setup to capture the load test data. Alternative materials to conventional cement concrete were studied. The pier materials evaluated in this study included compacted aggregate, cement stabilized silt, cementitious grouts, and fiber reinforced silt. Key findings from this study demonstrated that (1) the construction method influences the behavior of aggregate piers, (2) the composition of the pier has a significant impact on the stiffness, (3) group efficiencies were found to be a function of pier length and pier material, (4) in comparison to full-scale testing the scaled piers were found to produce a stiffer response with load-settlement and bearing capacities to be similar. Further, although full-scale test results were not available for all pier materials, the small-scale testing provided a means for comparing results between pier systems. Finally, duplicate pier tests for a given length and material were found to be repeatable.

Overexpression of human NR2B receptor subunit in LMAN causes stuttering and song sequence changes in adult zebra finches.

PubMed

Chakraborty, Mukta; Chen, Liang-Fu; Fridel, Emma E; Klein, Marguerita E; Senft, Rebecca A; Sarkar, Abhra; Jarvis, Erich D

2017-04-21

Zebra finches (Taeniopygia guttata) learn to produce songs in a manner reminiscent of spoken language development in humans. One candidate gene implicated in influencing learning is the N-methyl-D-aspartate (NMDA) subtype 2B glutamate receptor (NR2B). Consistent with this idea, NR2B levels are high in the song learning nucleus LMAN (lateral magnocellular nucleus of the anterior nidopallium) during juvenile vocal learning, and decreases to low levels in adults after learning is complete and the song becomes more stereotyped. To test for the role of NR2B in generating song plasticity, we manipulated NR2B expression in LMAN of adult male zebra finches by increasing its protein levels to those found in juvenile birds, using a lentivirus containing the full-length coding sequence of the human NR2B subunit. We found that increased NR2B expression in adult LMAN induced increases in song sequence diversity and slower song tempo more similar to juvenile songs, but also increased syllable repetitions similar to stuttering. We did not observe these effects in control birds with overexpression of NR2B outside of LMAN or with the green fluorescent protein (GFP) in LMAN. Our results suggest that low NR2B subunit expression in adult LMAN is important in conserving features of stereotyped adult courtship song.

Disulfide-mediated stabilization of the IκB kinase binding domain of NF-κB essential modulator (NEMO).

PubMed

Zhou, Li; Yeo, Alan T; Ballarano, Carmine; Weber, Urs; Allen, Karen N; Gilmore, Thomas D; Whitty, Adrian

2014-12-23

Human NEMO (NF-κB essential modulator) is a 419 residue scaffolding protein that, together with catalytic subunits IKKα and IKKβ, forms the IκB kinase (IKK) complex, a key regulator of NF-κB pathway signaling. NEMO is an elongated homodimer comprising mostly α-helix. It has been shown that a NEMO fragment spanning residues 44-111, which contains the IKKα/β binding site, is structurally disordered in the absence of bound IKKβ. Herein we show that enforcing dimerization of NEMO1-120 or NEMO44-111 constructs through introduction of one or two interchain disulfide bonds, through oxidation of the native Cys54 residue and/or at position 107 through a Leu107Cys mutation, induces a stable α-helical coiled-coil structure that is preorganized to bind IKKβ with high affinity. Chemical and thermal denaturation studies showed that, in the context of a covalent dimer, the ordered structure was stabilized relative to the denatured state by up to 3 kcal/mol. A full-length NEMO-L107C protein formed covalent dimers upon treatment of mammalian cells with H2O2. Furthermore, NEMO-L107C bound endogenous IKKβ in A293T cells, reconstituted TNF-induced NF-κB signaling in NEMO-deficient cells, and interacted with TRAF6. Our results indicate that the IKKβ binding domain of NEMO possesses an ordered structure in the unbound state, provided that it is constrained within a dimer as is the case in the constitutively dimeric full-length NEMO protein. The stability of the NEMO coiled coil is maintained by strong interhelix interactions in the region centered on residue 54. The disulfide-linked constructs we describe herein may be useful for crystallization of NEMO's IKKβ binding domain in the absence of bound IKKβ, thereby facilitating the structural characterization of small-molecule inhibitors.

Evaluation of candidate vaccine approaches for MERS-CoV

DOE PAGES

Wang, Lingshu; Shi, Wei; Joyce, M. Gordon; ...

2015-07-28

The emergence of Middle East respiratory syndrome coronavirus (MERS-CoV) as a cause of severe respiratory disease highlights the need for effective approaches to CoV vaccine development. Efforts focused solely on the receptor-binding domain (RBD) of the viral Spike (S) glycoprotein may not optimize neutralizing antibody (NAb) responses. Here we show that immunogens based on full-length S DNA and S1 subunit protein elicit robust serum-neutralizing activity against several MERS-CoV strains in mice and non-human primates. Serological analysis and isolation of murine monoclonal antibodies revealed that immunization elicits NAbs to RBD and, non-RBD portions of S1 and S2 subunit. Multiple neutralization mechanismsmore » were demonstrated by solving the atomic structure of a NAb-RBD complex, through sequencing of neutralization escape viruses and by constructing MERS-CoV S variants for serological assays. Immunization of rhesus macaques confers protection against MERS-CoV-induced radiographic pneumonia, as assessed using computerized tomography, supporting this strategy as a promising approach for MERS-CoV vaccine development.« less

Cloning of the chaperonin t-complex polypeptide 1 gene from Schistosoma mansoni and studies of its expression levels under heat shock and oxidative stress.

PubMed

Campos, E G; Hamdan, F F

2000-03-01

The protein TCP-1 (t-complex polypeptide 1) is a subunit of the hetero-oligomeric complex CCT (chaperonin containing TCP- 1) present in the eukaryotic cytosol. Chaperone function may be critical for the development and survival of the different life stages of Schistosoma mansoni, a parasite that is exposed to drastic environmental changes during its development. We isolated a full-length S. mansoni TCP-1 cDNA (SmTCP-1A) encoding a protein highly homologous with TCP-1. The deduced SmTCP-1A amino-acid sequence shows up to 65% identity with other eukaryotic CCT family members. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that the mRNA expression levels of SmTCP-1A in adult S. mansoni were down-regulated in worms subjected to heat shock and oxidative stress conditions. This down-regulation of SmTCP-1A mRNA may reflect a switch in CCT subunits as an adaptive response to heat shock and oxidative stress conditions.

Structural analysis and modeling reveals new mechanisms governing ESCRT-III spiral filament assembly

PubMed Central

Shen, Qing-Tao; Schuh, Amber L.; Zheng, Yuqing; Quinney, Kyle; Wang, Lei; Hanna, Michael; Mitchell, Julie C.; Otegui, Marisa S.; Ahlquist, Paul; Cui, Qiang

2014-01-01

The scission of biological membranes is facilitated by a variety of protein complexes that bind and manipulate lipid bilayers. ESCRT-III (endosomal sorting complex required for transport III) filaments mediate membrane scission during the ostensibly disparate processes of multivesicular endosome biogenesis, cytokinesis, and retroviral budding. However, mechanisms by which ESCRT-III subunits assemble into a polymer remain unknown. Using cryogenic electron microscopy (cryo-EM), we found that the full-length ESCRT-III subunit Vps32/CHMP4B spontaneously forms single-stranded spiral filaments. The resolution afforded by two-dimensional cryo-EM combined with molecular dynamics simulations revealed that individual Vps32/CHMP4B monomers within a filament are flexible and able to accommodate a range of bending angles. In contrast, the interface between monomers is stable and refractory to changes in conformation. We additionally found that the carboxyl terminus of Vps32/CHMP4B plays a key role in restricting the lateral association of filaments. Our findings highlight new mechanisms by which ESCRT-III filaments assemble to generate a unique polymer capable of membrane remodeling in multiple cellular contexts. PMID:25202029

Evaluation of candidate vaccine approaches for MERS-CoV

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

Wang, Lingshu; Shi, Wei; Joyce, M. Gordon

The emergence of Middle East respiratory syndrome coronavirus (MERS-CoV) as a cause of severe respiratory disease highlights the need for effective approaches to CoV vaccine development. Efforts focused solely on the receptor-binding domain (RBD) of the viral Spike (S) glycoprotein may not optimize neutralizing antibody (NAb) responses. Here we show that immunogens based on full-length S DNA and S1 subunit protein elicit robust serum-neutralizing activity against several MERS-CoV strains in mice and non-human primates. Serological analysis and isolation of murine monoclonal antibodies revealed that immunization elicits NAbs to RBD and, non-RBD portions of S1 and S2 subunit. Multiple neutralization mechanismsmore » were demonstrated by solving the atomic structure of a NAb-RBD complex, through sequencing of neutralization escape viruses and by constructing MERS-CoV S variants for serological assays. Immunization of rhesus macaques confers protection against MERS-CoV-induced radiographic pneumonia, as assessed using computerized tomography, supporting this strategy as a promising approach for MERS-CoV vaccine development.« less

The Fate of Major Royal Jelly Proteins during Proteolytic Digestion in the Human Gastrointestinal Tract.

PubMed

Mureşan, Carmen I; Schierhorn, Angelika; Buttstedt, Anja

2018-04-25

Royal jelly (RJ) is a beehive product with a complex composition, major royal jelly proteins (MRJPs) being the most abundant proteins. Cell culture and animal studies suggest various biological activities for the full-length/native MRJPs. In the field of apitherapy, it is assumed that MRJPs can positively affect human health. However, whenever RJ is administered orally, the availability for assimilation in the gastrointestinal tract is a prerequisite for MRJPs to have any effect on humans. We here show that MRJPs vary in resistance to pepsin digestion with MRJP2 being most stable and still present as full-length protein after 24 h of digestion. In the intestinal phase, using trypsin and chymotrypsin, MRJPs are rapidly digested with MRJP2 again showing longest stability (40 min), suggesting that MRJPs can reach the small intestine as full-length proteins but then have to be resorbed quickly if full-length proteins are to fulfill any biological activity.

Mechanism of Telomerase Activation by v-Rel and Its Contribution to Transformation

PubMed Central

Hrdličková, Radmila; Nehyba, Jiří; Liss, Andrew S.; Bose, Henry R.

2006-01-01

Telomerase is activated during the transformation of lymphoid cells and fibroblasts by v-Rel, the oncogenic member of the Rel/NF-κB family of transcription factors. v-Rel-transformed cell lines have longer telomeres than untransformed chicken lymphoid cells and have high levels of telomerase activity. v-Rel-mediated activation of telomerase is achieved by multiple mechanisms. The expression of the gene encoding the catalytic subunit of telomerase (TERT) was directly upregulated by v-Rel. Moreover, the expression of v-Rel altered the ratio of alternatively spliced and full-length TERT transcripts in favor of the full-length forms. The activation of telomerase by v-Rel in lymphocytes was also accompanied by inactivation of nuclear inhibitors. The inhibition of telomerase activity in v-Rel-transformed cell lines led to apoptosis within 24 h. The expression of v-Rel in a macrophage cell line resulted in elevated levels of reactive oxygen species (ROS), increased telomerase activity, and increased sensitivity to telomerase inhibitors. In contrast, the ectopic expression of TERT decreased the extent of apoptosis induced by ROS. The activation of telomerase by v-Rel may, therefore, partially protect the transformed cells from apoptosis induced by ROS. PMID:16352553

Structure-guided Design and Immunological Characterization of Immunogens Presenting the HIV-1 gp120 V3 Loop on a CTB Scaffold

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

M Totrov; X Jiang; X Kong

2011-12-31

V3 loop is a major neutralizing determinant of the HIV-1 gp120. Using 3D structures of cholera toxin B subunit (CTB), complete V3 in the gp120 context, and V3 bound to a monoclonal antibody (mAb), we designed two V3-scaffold immunogen constructs (V3-CTB). The full-length V3-CTB presenting the complete V3 in a structural context mimicking gp120 was recognized by the large majority of our panel of 24 mAbs. The short V3-CTB presenting a V3 fragment in the conformation observed in the complex with the 447-52D Fab, exhibited high-affinity binding to this mAb. The immunogens were evaluated in rabbits using DNA-prime/protein-boost protocol. Boostingmore » with the full-length V3-CTB induced high anti-V3 titers in sera that potently neutralize multiple HIV virus strains. The short V3-CTB was ineffective. The results suggest that very narrow antigenic profile of an immunogen is associated with poor Ab response. An immunogen with broader antigenic activity elicits robust Ab response.« less

Recognition of chimeric small-subunit ribosomal DNAs composed of genes from uncultivated microorganisms

NASA Technical Reports Server (NTRS)

Kopczynski, E. D.; Bateson, M. M.; Ward, D. M.

1994-01-01

When PCR was used to recover small-subunit (SSU) rRNA genes from a hot spring cyanobacterial mat community, chimeric SSU rRNA sequences which exhibited little or no secondary structural abnormality were recovered. They were revealed as chimeras of SSU rRNA genes of uncultivated species through separate phylogenetic analysis of short sequence domains.

Ribosomal small subunit domains radiate from a central core

NASA Astrophysics Data System (ADS)

Gulen, Burak; Petrov, Anton S.; Okafor, C. Denise; Vander Wood, Drew; O'Neill, Eric B.; Hud, Nicholas V.; Williams, Loren Dean

2016-02-01

The domain architecture of a large RNA can help explain and/or predict folding, function, biogenesis and evolution. We offer a formal and general definition of an RNA domain and use that definition to experimentally characterize the rRNA of the ribosomal small subunit. Here the rRNA comprising a domain is compact, with a self-contained system of molecular interactions. A given rRNA helix or stem-loop must be allocated uniquely to a single domain. Local changes such as mutations can give domain-wide effects. Helices within a domain have interdependent orientations, stabilities and interactions. With these criteria we identify a core domain (domain A) of small subunit rRNA. Domain A acts as a hub, linking the four peripheral domains and imposing orientational and positional restraints on the other domains. Experimental characterization of isolated domain A, and mutations and truncations of it, by methods including selective 2‧OH acylation analyzed by primer extension and circular dichroism spectroscopy are consistent with our architectural model. The results support the utility of the concept of an RNA domain. Domain A, which exhibits structural similarity to tRNA, appears to be an essential core of the small ribosomal subunit.

Post-translational generation of constitutively active cores from larger phosphatases in the malaria parasite, Plasmodium falciparum: implications for proteomics

PubMed Central

Kumar, Rajinder; Musiyenko, Alla; Oldenburg, Anja; Adams, Brian; Barik, Sailen

2004-01-01

Background Although the complete genome sequences of a large number of organisms have been determined, the exact proteomes need to be characterized. More specifically, the extent to which post-translational processes such as proteolysis affect the synthesized proteins has remained unappreciated. We examined this issue in selected protein phosphatases of the protease-rich malaria parasite, Plasmodium falciparum. Results P. falciparum encodes a number of Ser/Thr protein phosphatases (PP) whose catalytic subunits are composed of a catalytic core and accessory domains essential for regulation of the catalytic activity. Two examples of such regulatory domains are found in the Ca+2-regulated phosphatases, PP7 and PP2B (calcineurin). The EF-hand domains of PP7 and the calmodulin-binding domain of PP2B are essential for stimulation of the phosphatase activity by Ca+2. We present biochemical evidence that P. falciparum generates these full-length phosphatases as well as their catalytic cores, most likely as intermediates of a proteolytic degradation pathway. While the full-length phosphatases are activated by Ca+2, the processed cores are constitutively active and either less responsive or unresponsive to Ca+2. The processing is extremely rapid, specific, and occurs in vivo. Conclusions Post-translational cleavage efficiently degrades complex full-length phosphatases in P. falciparum. In the course of such degradation, enzymatically active catalytic cores are produced as relatively stable intermediates. The universality of such proteolysis in other phosphatases or other multi-domain proteins and its potential impact on the overall proteome of a cell merits further investigation. PMID:15230980

Direct observation of subunit exchange along mature vimentin intermediate filaments.

PubMed

Nöding, Bernd; Herrmann, Harald; Köster, Sarah

2014-12-16

Actin filaments, microtubules, and intermediate filaments (IFs) are central elements of the metazoan cytoskeleton. At the molecular level, the assembly mechanism for actin filaments and microtubules is fundamentally different from that of IFs. The former two types of filaments assemble from globular proteins. By contrast, IFs assemble from tetrameric complexes of extended, half-staggered, and antiparallel oriented coiled-coils. These tetramers laterally associate into unit-length filaments; subsequent longitudinal annealing of unit-length filaments yields mature IFs. In vitro, IFs form open structures without a fixed number of tetramers per cross-section along the filament. Therefore, a central question for the structural biology of IFs is whether individual subunits can dissociate from assembled filaments and rebind at other sites. Using the fluorescently labeled IF-protein vimentin for assembly, we directly observe and quantitatively determine subunit exchange events between filaments as well as with soluble vimentin pools. Thereby we demonstrate that the cross-sectional polymorphism of donor and acceptor filaments plays an important role. We propose that in segments of donor filaments with more than the standard 32 molecules per cross-section, subunits are not as tightly bound and are predisposed to be released from the filament. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

The cAMP-induced G protein subunits dissociation monitored in live Dictyostelium cells by BRET reveals two activation rates, a positive effect of caffeine and potential role of microtubules.

PubMed

Tariqul Islam, A F M; Yue, Haicen; Scavello, Margarethakay; Haldeman, Pearce; Rappel, Wouter-Jan; Charest, Pascale G

2018-08-01

To study the dynamics and mechanisms controlling activation of the heterotrimeric G protein Gα2βγ in Dictyostelium in response to stimulation by the chemoattractant cyclic AMP (cAMP), we monitored the G protein subunit interaction in live cells using bioluminescence resonance energy transfer (BRET). We found that cAMP induces the cAR1-mediated dissociation of the G protein subunits to a similar extent in both undifferentiated and differentiated cells, suggesting that only a small number of cAR1 (as expressed in undifferentiated cells) is necessary to induce the full activation of Gα2βγ. In addition, we found that treating cells with caffeine increases the potency of cAMP-induced Gα2βγ activation; and that disrupting the microtubule network but not F-actin inhibits the cAMP-induced dissociation of Gα2βγ. Thus, microtubules are necessary for efficient cAR1-mediated activation of the heterotrimeric G protein. Finally, kinetics analyses of Gα2βγ subunit dissociation induced by different cAMP concentrations indicate that there are two distinct rates at which the heterotrimeric G protein subunits dissociate when cells are stimulated with cAMP concentrations above 500 nM versus only one rate at lower cAMP concentrations. Quantitative modeling suggests that the kinetics profile of Gα2βγ subunit dissociation results from the presence of both uncoupled and G protein pre-coupled cAR1 that have differential affinities for cAMP and, consequently, induce G protein subunit dissociation through different rates. We suggest that these different signaling kinetic profiles may play an important role in initial chemoattractant gradient sensing. Copyright © 2018 Elsevier Inc. All rights reserved.

Improved purification of brine-shrimp (Artemia saline) (Na+ + K+)-activated adenosine triphosphatase and amino-acid and carbohydrate analyses of the isolated subunits.

PubMed Central

Peterson, G L; Hokin, L E

1980-01-01

Purification of the (Na+ + K+)-activated ATPase has been improved 2-fold the respect to both purity and yield over the previous method [Peterson, Ewing, Hootman & Conte (1978) J. Biol. Chem. 253, 4762-4770] by using Lubrol WX and non-denaturing concentrations of sodium dodecyl sulphate (SDS). The enzyme was purified 200-fold over the homogenate. The preparation had a specific activity of about 600 mumol of Pi/h per mg of protein, and was about 60% pure according to quantification of Coomassie Blue-stained SDS/polyacrylamide gels. The yield of purified enzyme was about 10 mg of protein per 100g of dry brine-shrimp (Artemia salina) cysts. The method is highly suitable for purification either on a small scale (10-25g of dry cysts) or on a large scale (900g of dry cysts) and methods are described for both. The large (Na+ + K+)-activated ATPase subunit (alpha-subunit) was isolated in pure form by SDS-gel filtration on Bio-Gel A 1.5m. The small subunit (beta-subunit) was eluted with other contaminating proteins on the Bio-Gel column, but was isolated in pure form by extraction from SDS/polyacrylamide gels. The amino acid and carbohydrate compositions of both subunits are reported. The alpha-subunit contained 5.2% carbohydrate by weight, and the beta-subunit 9.2%. Sialic acid was absent from both subunits. Images Fig. 3. Fig. 4. PMID:6272692

Comparison of the kinetic parameters of the truncated catalytic subunit and holoenzyme of human DNA polymerase ε

PubMed Central

Zahurancik, Walter J.; Baranovskiy, Andrey G.; Tahirov, Tahir H.; Suo, Zucai

2015-01-01

Numerous genetic studies have provided compelling evidence to establish DNA polymerase ε (Polε) as the primary DNA polymerase responsible for leading strand synthesis during eukaryotic nuclear genome replication. Polε is a heterotetramer consisting of a large catalytic subunit that contains the conserved polymerase core domain as well as a 3′ → 5′ exonuclease domain common to many replicative polymerases. In addition, Polε possesses three small subunits that lack a known catalytic activity but associate with components involved in a variety of DNA replication and maintenance processes. Previous enzymatic characterization of the Polε heterotetramer from budding yeast suggested that the small subunits slightly enhance DNA synthesis by Polε in vitro. However, similar studies of the human Polε heterote-tramer (hPolε) have been limited by the difficulty of obtaining hPolε in quantities suitable for thorough investigation of its catalytic activity. Utilization of a baculovirus expression system for overexpression and purification of hPolε from insect host cells has allowed for isolation of greater amounts of active hPolε, thus enabling a more detailed kinetic comparison between hPolε and an active N-terminal fragment of the hPolε catalytic subunit (p261N), which is readily overexpressed in Escherichia coli. Here, we report the first pre-steady-state studies of fully-assembled hPolε. We observe that the small subunits increase DNA binding by hPolε relative to p261N, but do not increase processivity during DNA synthesis on a single-stranded M13 template. Interestingly, the 3′ → 5′ exonuclease activity of hPolε is reduced relative to p261N on matched and mismatched DNA substrates, indicating that the presence of the small subunits may regulate the proofreading activity of hPolε and sway hPolε toward DNA synthesis rather than proofreading. PMID:25684708

C-terminal interactions mediate the quaternary dynamics of αB-crystallin

PubMed Central

Hilton, Gillian R.; Hochberg, Georg K. A.; Laganowsky, Arthur; McGinnigle, Scott I.; Baldwin, Andrew J.; Benesch, Justin L. P.

2013-01-01

αB-crystallin is a highly dynamic, polydisperse small heat-shock protein that can form oligomers ranging in mass from 200 to 800 kDa. Here we use a multifaceted mass spectrometry approach to assess the role of the C-terminal tail in the self-assembly of αB-crystallin. Titration experiments allow us to monitor the binding of peptides representing the C-terminus to the αB-crystallin core domain, and observe individual affinities to both monomeric and dimeric forms. Notably, we find that binding the second peptide equivalent to the core domain dimer is considerably more difficult than the first, suggesting a role of the C-terminus in regulating assembly. This finding motivates us to examine the effect of point mutations in the C-terminus in the full-length protein, by quantifying the changes in oligomeric distribution and corresponding subunit exchange rates. Our results combine to demonstrate that alterations in the C-terminal tail have a significant impact on the thermodynamics and kinetics of αB-crystallin. Remarkably, we find that there is energy compensation between the inter- and intra-dimer interfaces: when one interaction is weakened, the other is strengthened. This allosteric communication between binding sites on αB-crystallin is likely important for its role in binding target proteins. PMID:23530258

A New Model for Allosteric Regulation of Phenylalanine Hydroxylase: Implications for Disease and Therapeutics

PubMed Central

Stith, Linda; Lawrence, Sarah H.; Andrake, Mark; Dunbrack, Roland L.

2013-01-01

The structural basis for allosteric regulation of phenylalanine hydroxylase (PAH), whose dysfunction causes phenylketonuria (PKU), is poorly understood. A new morpheein model for PAH allostery is proposed to consist of a dissociative equilibrium between two architecturally different tetramers whose interconversion requires a ~90° rotation between the PAH catalytic and regulatory domains, the latter of which contains an ACT domain. This unprecedented model is supported by in vitro data on purified full length rat and human PAH. The conformational change is both predicted to and shown to render the tetramers chromatographically separable using ion exchange methods. One novel aspect of the activated tetramer model is an allosteric phenylalanine binding site at the inter-subunit interface of ACT domains. Amino acid ligand-stabilized ACT domain dimerization follows the multimerization and ligand binding behavior of ACT domains present in other proteins in the PDB. Spectroscopic, chromatographic, and electrophoretic methods demonstrate a PAH equilibrium consisting of two architecturally distinct tetramers as well as dimers. We postulate that PKU-associated mutations may shift the PAH quaternary structure equilibrium in favor of the low activity assemblies. Pharmacological chaperones that stabilize the ACT:ACT interface can potentially provide PKU patients with a novel small molecule therapeutic. PMID:23296088

Distinct cellular distributions of Kv4 pore-forming and auxiliary subunits in rat dorsal root ganglion neurons.

PubMed

Matsuyoshi, Hiroko; Takimoto, Koichi; Yunoki, Takakazu; Erickson, Vickie L; Tyagi, Pradeep; Hirao, Yoshihiko; Wanaka, Akio; Yoshimura, Naoki

2012-09-17

Dorsal root ganglia contain heterogeneous populations of primary afferent neurons that transmit various sensory stimuli. This functional diversity may be correlated with differential expression of voltage-gated K(+) (Kv) channels. Here, we examine cellular distributions of Kv4 pore-forming and ancillary subunits that are responsible for fast-inactivating A-type K(+) current. Expression pattern of Kv α-subunit, β-subunit and auxiliary subunit was investigated using immunohistochemistry, in situ hybridization and RT-PCR technique. The two pore-forming subunits Kv4.1 and Kv4.3 show distinct cellular distributions: Kv4.3 is predominantly in small-sized C-fiber neurons, whereas Kv4.1 is seen in DRG neurons in various sizes. Furthermore, the two classes of Kv4 channel auxiliary subunits are also distributed in different-sized cells. KChIP3 is the only significantly expressed Ca(2+)-binding cytosolic ancillary subunit in DRGs and present in medium to large-sized neurons. The membrane-spanning auxiliary subunit DPP6 is seen in a large number of DRG neurons in various sizes, whereas DPP10 is restricted in small-sized neurons. Distinct combinations of Kv4 pore-forming and auxiliary subunits may constitute A-type channels in DRG neurons with different physiological roles. Kv4.1 subunit, in combination with KChIP3 and/or DPP6, form A-type K(+) channels in medium to large-sized A-fiber DRG neurons. In contrast, Kv4.3 and DPP10 may contribute to A-type K(+) current in non-peptidergic, C-fiber somatic afferent neurons. Copyright © 2012 Elsevier Inc. All rights reserved.

Gonadotropin subunits of the characiform Astyanax altiparanae: Molecular characterization, spatiotemporal expression and their possible role on female reproductive dysfunction in captivity.

PubMed

de Jesus, Lázaro Wender O; Bogerd, Jan; Vieceli, Felipe M; Branco, Giovana S; Camargo, Marília P; Cassel, Mônica; Moreira, Renata G; Yan, Chao Y I; Borella, Maria I

2017-05-15

To better understand the endocrine control of reproduction in Characiformes and the reproductive dysfunctions that commonly occur in migratory fish of this order when kept in captivity, we chose Astyanax altiparanae, which has asynchronous ovarian development and multiple spawning events, as model species. From A. altiparanae pituitary total RNA, we cloned the full-length cDNAs coding for the follicle-stimulating hormone β subunit (fshb), the luteinizing hormone β subunit (lhb), and the common gonadotropin α subunit (gpha). All three sequences showed the highest degree of amino acid identity with other homologous sequences from Siluriformes and Cypriniformes. Real-time, quantitative PCR analysis showed that gpha, fshb and lhb mRNAs were restricted to the pituitary gland. In situ hybridization and immunofluorescence, using specific-developed and characterized polyclonal antibodies, revealed that both gonadotropin β subunits mRNAs/proteins are expressed by distinct populations of gonadotropic cells in the proximal pars distalis. No marked variations for lhb transcripts levels were detected during the reproductive cycle, and 17α,20β-dihydroxy-4-pregnen-3-one plasma levels were also constant, suggesting that the reproductive dysfunction seen in A. altiparanae females in captivity are probably due to a lack of increase of Lh synthesis during spawning season. In contrast, fshb transcripts changed significantly during the reproductive cycle, although estradiol-17β (E 2 ) levels remained constant during the experiment, possibly due to a differential regulation of E 2 synthesis. Taken together, these data demonstrate the putative involvement of gonadotropin signaling on the impairment of the reproductive function in a migratory species when kept in captivity. Future experimental studies must be carried to clarify this hypothesis. All these data open the possibility for further basic and applied studies related to reproduction in this fish model. Copyright © 2016 Elsevier Inc. All rights reserved.

Comparison of Voltage Gated K+ Currents in Arterial Myocytes with Heterologously Expressed K v Subunits.

PubMed

Cox, Robert H; Fromme, Samantha

2016-12-01

We have shown that three components contribute to functional voltage gated K + (K v ) currents in rat small mesenteric artery myocytes: (1) Kv1.2 plus Kv1.5 with Kvβ1.2 subunits, (2) Kv2.1 probably associated with Kv9.3 subunits, and (3) Kv7.4 subunits. To confirm and address subunit stoichiometry of the first two, we have compared the biophysical properties of K v currents in small mesenteric artery myocytes with those of K v subunits heterologously expressed in HEK293 cells using whole cell voltage clamp methods. Selective inhibitors of Kv1 (correolide, COR) and Kv2 (stromatoxin, ScTx) channels were used to separate these K v current components. Conductance-voltage and steady state inactivation data along with time constants of activation, inactivation, and deactivation of native K v components were generally well represented by those of Kv1.2-1.5-β1.2 and Kv2.1-9.3 channels. The slope of the steady state inactivation-voltage curve (availability slope) proved to be the most sensitive measure of accessory subunit presence. The availability slope curves exhibited a single peak for both native K v components. Availability slope curves for Kv1.2-1.5-β1.2 and Kv2.1-9.3 channels expressed in human embryonic kidney cells also exhibited a single peak that shifted to more depolarized voltages with increasing accessory to α subunit transfection ratio. Availability slope curves for SxTc-insensitive currents were similar to those of Kv1.2-1.5 expressed with Kvβ1.2 at a 1:5 molar ratio while curves for COR-insensitive currents closely resembled those of Kv2.1 expressed with Kv9.3 at a 1:1 molar ratio. These results support the suggested K v subunit combinations in small mesenteric artery, and further suggest that Kv1 α and Kvβ1.2 but not Kv2.1 and Kv9.3 subunits are present in a saturated (4:4) stoichiometry.

Reversal of a full-length mutant huntingtin neuronal cell phenotype by chemical inhibitors of polyglutamine-mediated aggregation

PubMed Central

Wang, Jin; Gines, Silvia; MacDonald, Marcy E; Gusella, James F

2005-01-01

Background Huntington's disease (HD) is an inherited neurodegenerative disorder triggered by an expanded polyglutamine tract in huntingtin that is thought to confer a new conformational property on this large protein. The propensity of small amino-terminal fragments with mutant, but not wild-type, glutamine tracts to self-aggregate is consistent with an altered conformation but such fragments occur relatively late in the disease process in human patients and mouse models expressing full-length mutant protein. This suggests that the altered conformational property may act within the full-length mutant huntingtin to initially trigger pathogenesis. Indeed, genotype-phenotype studies in HD have defined genetic criteria for the disease initiating mechanism, and these are all fulfilled by phenotypes associated with expression of full-length mutant huntingtin, but not amino-terminal fragment, in mouse models. As the in vitro aggregation of amino-terminal mutant huntingtin fragment offers a ready assay to identify small compounds that interfere with the conformation of the polyglutamine tract, we have identified a number of aggregation inhibitors, and tested whether these are also capable of reversing a phenotype caused by endogenous expression of mutant huntingtin in a striatal cell line from the HdhQ111/Q111 knock-in mouse. Results We screened the NINDS Custom Collection of 1,040 FDA approved drugs and bioactive compounds for their ability to prevent in vitro aggregation of Q58-htn 1–171 amino terminal fragment. Ten compounds were identified that inhibited aggregation with IC50 < 15 μM, including gossypol, gambogic acid, juglone, celastrol, sanguinarine and anthralin. Of these, both juglone and celastrol were effective in reversing the abnormal cellular localization of full-length mutant huntingtin observed in mutant HdhQ111/Q111 striatal cells. Conclusions At least some compounds identified as aggregation inhibitors also prevent a neuronal cellular phenotype caused by full-length mutant huntingtin, suggesting that in vitro fragment aggregation can act as a proxy for monitoring the disease-producing conformational property in HD. Thus, identification and testing of compounds that alter in vitro aggregation is a viable approach for defining potential therapeutic compounds that may act on the deleterious conformational property of full-length mutant huntingtin. PMID:15649316

Effects of the α subunit on imidacloprid sensitivity of recombinant nicotinic acetylcholine receptors

PubMed Central

Matsuda, K; Buckingham, S D; Freeman, J C; Squire, M D; Baylis, H A; Sattelle, D B

1998-01-01

Imidacloprid is a new insecticide with selective toxicity for insects over vertebrates. Recombinant (α4β2) chicken neuronal nicotinic acetylcholine receptors (AChRs) and a hybrid nicotinic AChR formed by co-expression of a Drosophila melanogaster neuronal α subunit (SAD) with the chicken β2 subunit were heterologously expressed in Xenopus oocytes by nuclear injection of cDNAs. The agonist actions of imidacloprid and other nicotinic AChR ligands ((+)-epibatidine, (−)-nicotine and acetylcholine) were compared on both recombinant nicotinic AChRs by use of two-electrode, voltage-clamp electrophysiology. Imidacloprid alone of the 4 agonists behaved as a partial agonist on the α4β2 receptor; (+)-epibatidine, (−)-nicotine and acetylcholine were all full, or near full, agonists. Imidacloprid was also a partial agonist of the hybrid Drosophila SAD chicken β2 receptor, as was (−)-nicotine, whereas (+)-epibatidine and acetylcholine were full agonists. The EC50 of imidacloprid was decreased by replacing the chicken α4 subunit with the Drosophila SAD α subunit. This α subunit substitution also resulted in an increase in the EC50 for (+)-epibatidine, (−)-nicotine and acetylcholine. Thus, the Drosophila (SAD) α subunit contributes to the greater apparent affinity of imidacloprid for recombinant insect/vertebrate nicotinic AChRs. Imidacloprid acted as a weak antagonist of ACh-mediated responses mediated by SADβ2 hybrid receptors and as a weak potentiator of ACh responses mediated by α4β2 receptors. This suggests that imidacloprid has complex effects upon these recombinant receptors, determined at least in part by the α subunit. PMID:9504393

Crystal Structure of the Human Pol α B Subunit in Complex with the C-terminal Domain of the Catalytic Subunit*

PubMed Central

Suwa, Yoshiaki; Gu, Jianyou; Baranovskiy, Andrey G.; Babayeva, Nigar D.; Pavlov, Youri I.; Tahirov, Tahir H.

2015-01-01

In eukaryotic DNA replication, short RNA-DNA hybrid primers synthesized by primase-DNA polymerase α (Prim-Pol α) are needed to start DNA replication by the replicative DNA polymerases, Pol δ and Pol ϵ. The C terminus of the Pol α catalytic subunit (p180C) in complex with the B subunit (p70) regulates the RNA priming and DNA polymerizing activities of Prim-Pol α. It tethers Pol α and primase, facilitating RNA primer handover from primase to Pol α. To understand these regulatory mechanisms and to reveal the details of human Pol α organization, we determined the crystal structure of p70 in complex with p180C. The structured portion of p70 includes a phosphodiesterase (PDE) domain and an oligonucleotide/oligosaccharide binding (OB) domain. The N-terminal domain and the linker connecting it to the PDE domain are disordered in the reported crystal structure. The p180C adopts an elongated asymmetric saddle shape, with a three-helix bundle in the middle and zinc-binding modules (Zn1 and Zn2) on each side. The extensive p180C-p70 interactions involve 20 hydrogen bonds and a number of hydrophobic interactions resulting in an extended buried surface of 4080 Å2. Importantly, in the structure of the p180C-p70 complex with full-length p70, the residues from the N-terminal to the OB domain contribute to interactions with p180C. The comparative structural analysis revealed both the conserved features and the differences between the human and yeast Pol α complexes. PMID:25847248

Ligand-Induced Conformational Change in the α7 Nicotinic Receptor Ligand Binding Domain

PubMed Central

Henchman, Richard H.; Wang, Hai-Long; Sine, Steven M.; Taylor, Palmer; McCammon, J. Andrew

2005-01-01

Molecular dynamics simulations of a homology model of the ligand binding domain of the α7 nicotinic receptor are conducted with a range of bound ligands to induce different conformational states. Four simulations of 15 ns each are run with no ligand, antagonist d-tubocurarine (dTC), agonist acetylcholine (ACh), and agonist ACh with potentiator Ca2+, to give insight into the conformations of the active and inactive states of the receptor and suggest the mechanism for conformational change. The main structural factor distinguishing the active and inactive states is that a more open, symmetric arrangement of the five subunits arises for the two agonist simulations, whereas a more closed and asymmetric arrangement results for the apo and dTC cases. Most of the difference arises in the lower portion of the ligand binding domain near its connection to the adjacent transmembrane domain. The transfer of the more open state to the transmembrane domain could then promote ion flow through the channel. Variation in how subunits pack together with no ligand bound appears to give rise to asymmetry in the apo case. The presence of dTC expands the receptor but induces rotations in alternate directions in adjacent subunits that lead to an asymmetric arrangement as in the apo case. Ca2+ appears to promote a slightly greater expansion in the subunits than ACh alone by stabilizing the C-loop and ACh positions. Although the simulations are unlikely to be long enough to view the full conformational changes between open and closed states, a collection of different motions at a range of length scales are observed that are likely to participate in the conformational change. PMID:15665135

The β1a Subunit of the Skeletal DHPR Binds to Skeletal RyR1 and Activates the Channel via Its 35-Residue C-Terminal Tail

PubMed Central

Rebbeck, Robyn T.; Karunasekara, Yamuna; Gallant, Esther M.; Board, Philip G.; Beard, Nicole A.; Casarotto, Marco G.; Dulhunty, Angela F.

2011-01-01

Although it has been suggested that the C-terminal tail of the β1a subunit of the skeletal dihyropyridine receptor (DHPR) may contribute to voltage-activated Ca2+ release in skeletal muscle by interacting with the skeletal ryanodine receptor (RyR1), a direct functional interaction between the two proteins has not been demonstrated previously. Such an interaction is reported here. A peptide with the sequence of the C-terminal 35 residues of β1a bound to RyR1 in affinity chromatography. The full-length β1a subunit and the C-terminal peptide increased [3H]ryanodine binding and RyR1 channel activity with an AC50 of 450–600 pM under optimal conditions. The effect of the peptide was dependent on cytoplasmic Ca2+, ATP, and Mg2+ concentrations. There was no effect of the peptide when channel activity was very low as a result of Mg2+ inhibition or addition of 100 nM Ca2+ (without ATP). Maximum increases were seen with 1–10 μM Ca2+, in the absence of Mg2+ inhibition. A control peptide with the C-terminal 35 residues in a scrambled sequence did not bind to RyR1 or alter [3H]ryanodine binding or channel activity. This high-affinity in vitro functional interaction between the C-terminal 35 residues of the DHPR β1a subunit and RyR1 may support an in vivo function of β1a during voltage-activated Ca2+ release. PMID:21320436

ATP-sensitive potassium currents from channels formed by Kir6 and a modified cardiac mitochondrial SUR2 variant

PubMed Central

Aggarwal, Nitin T; Shi, Nian-Qing; Makielski, Jonathan C

2013-01-01

Cardiac ATP-sensitive potassium channels (KATP) are found in both the sarcoplasmic reticulum (sarcKATP) and the inner membrane of mitochondria (mitoKATP). SarcKATP are composed of a pore containing subunit Kir6.2 and a regulatory sulfonylurea receptor subunit (SUR2), but the composition of mitoKATP remains unclear. An unusual intra-exonic splice variant of SUR2 (SUR2A-55) was previously identified in mitochondria of mammalian heart and brain, and by analogy with sarcKATP we proposed SUR2A-55 as a candidate regulatory subunit of mitoKATP. Although SUR2A-55 lacks the first nucleotide binding domain (NBD) and 2 transmembrane domains (TMD), it has a hybrid TMD and retains the second NBD. It resembles a hemi-ABC transporter suggesting it could multimerize to function as a regulatory subunit. A putative mitochondrial targeting signal in the N-terminal domain of SUR2A-55 was removed by truncation and when co-expressed with Kir6.1 and Kir6.2 it targeted to the plasma membrane and yielded KATP currents. Single channel conductance, mean open time, and burst open time of SUR2A-55 based KATP was similar to the full-length SUR2A based KATP. However, the SUR2A-55 KATP were 70-fold less sensitive to block by ATP, and twice as resistant to intracellular Ca2+ inhibition compared with the SUR2A KATP, and were markedly insensitive to KATP drugs, pinacidil, diazoxide, and glybenclamide. These results suggest that the SUR2A-55 based channels would tend to be open under physiological conditions and in ischemia, and could account for cardiac and mitochondrial phenotypes protective for ischemia. PMID:24037327

A potentially novel nicotinic receptor in Aplysia neuroendocrine cells.

PubMed

White, Sean H; Carter, Christopher J; Magoski, Neil S

2014-07-15

Nicotinic receptors form a diverse group of ligand-gated ionotropic receptors with roles in both synaptic transmission and the control of excitability. In the bag cell neurons of Aplysia, acetylcholine activates an ionotropic receptor, which passes inward current to produce a long-lasting afterdischarge and hormone release, leading to reproduction. While testing the agonist profile of the cholinergic response, we observed a second current that appeared to be gated only by nicotine and not acetylcholine. The peak nicotine-evoked current was markedly smaller in magnitude than the acetylcholine-induced current, cooperative (Hill value of 2.7), had an EC50 near 500 μM, readily recovered from desensitization, showed Ca(2+) permeability, and was blocked by mecamylamine, dihydro-β-erythroidine, or strychnine, but not by α-conotoxin ImI, methyllycaconitine, or hexamethonium. Aplysia transcriptome analysis followed by PCR yielded 20 full-length potential nicotinic receptor subunits. Sixteen of these were predicted to be cation selective, and real-time PCR suggested that 15 of the 16 subunits were expressed to varying degrees in the bag cell neurons. The acetylcholine-induced current, but not the nicotine current, was reduced by double-strand RNA treatment targeted to both subunits ApAChR-C and -E. Conversely, the nicotine-evoked current, but not the acetylcholine current, was lessened by targeting both subunits ApAChR-H and -P. To the best of our knowledge, this is the first report suggesting that a nicotinic receptor is not gated by acetylcholine. Separate receptors may serve as a means to differentially trigger plasticity or safeguard propagation by assuring that only acetylcholine, the endogenous agonist, initiates large enough responses to trigger reproduction. Copyright © 2014 the American Physiological Society.

An Interleukin 12 Adjuvanted Herpes Simplex Virus 2 DNA Vaccine Is More Protective Than a Glycoprotein D Subunit Vaccine in a High-Dose Murine Challenge Model.

PubMed

Bagley, Kenneth C; Schwartz, Jennifer A; Andersen, Hanne; Eldridge, John H; Xu, Rong; Ota-Setlik, Ayuko; Geltz, Joshua J; Halford, William P; Fouts, Timothy R

2017-04-01

Vaccination is a proven intervention against human viral diseases; however, success against Herpes Simplex Virus 2 (HSV-2) remains elusive. Most HSV-2 vaccines tested in humans to date contained just one or two immunogens, such as the virion attachment receptor glycoprotein D (gD) and/or the envelope fusion protein, glycoprotein B (gB). At least three factors may have contributed to the failures of subunit-based HSV-2 vaccines. First, immune responses directed against one or two viral antigens may lack sufficient antigenic breadth for efficacy. Second, the antibody responses elicited by these vaccines may have lacked necessary Fc-mediated effector functions. Third, these subunit vaccines may not have generated necessary protective cellular immune responses. We hypothesized that a polyvalent combination of HSV-2 antigens expressed from a DNA vaccine with an adjuvant that polarizes immune responses toward a T helper 1 (Th1) phenotype would compose a more effective vaccine. We demonstrate that delivery of DNA expressing full-length HSV-2 glycoprotein immunogens by electroporation with the adjuvant interleukin 12 (IL-12) generates substantially greater protection against a high-dose HSV-2 vaginal challenge than a recombinant gD subunit vaccine adjuvanted with alum and monophosphoryl lipid A (MPL). Our results further show that DNA vaccines targeting optimal combinations of surface glycoproteins provide better protection than gD alone and provide similar survival benefits and disease symptom reductions compared with a potent live attenuated HSV-2 0ΔNLS vaccine, but that mice vaccinated with HSV-2 0ΔNLS clear the virus much faster. Together, our data indicate that adjuvanted multivalent DNA vaccines hold promise for an effective HSV-2 vaccine, but that further improvements may be required.

A dominant mutation in mediator of paramutation2, one of three second-largest subunits of a plant-specific RNA polymerase, disrupts multiple siRNA silencing processes.

PubMed

Sidorenko, Lyudmila; Dorweiler, Jane E; Cigan, A Mark; Arteaga-Vazquez, Mario; Vyas, Meenal; Kermicle, Jerry; Jurcin, Diane; Brzeski, Jan; Cai, Yu; Chandler, Vicki L

2009-11-01

Paramutation involves homologous sequence communication that leads to meiotically heritable transcriptional silencing. We demonstrate that mop2 (mediator of paramutation2), which alters paramutation at multiple loci, encodes a gene similar to Arabidopsis NRPD2/E2, the second-largest subunit of plant-specific RNA polymerases IV and V. In Arabidopsis, Pol-IV and Pol-V play major roles in RNA-mediated silencing and a single second-largest subunit is shared between Pol-IV and Pol-V. Maize encodes three second-largest subunit genes: all three genes potentially encode full length proteins with highly conserved polymerase domains, and each are expressed in multiple overlapping tissues. The isolation of a recessive paramutation mutation in mop2 from a forward genetic screen suggests limited or no functional redundancy of these three genes. Potential alternative Pol-IV/Pol-V-like complexes could provide maize with a greater diversification of RNA-mediated transcriptional silencing machinery relative to Arabidopsis. Mop2-1 disrupts paramutation at multiple loci when heterozygous, whereas previously silenced alleles are only up-regulated when Mop2-1 is homozygous. The dramatic reduction in b1 tandem repeat siRNAs, but no disruption of silencing in Mop2-1 heterozygotes, suggests the major role for tandem repeat siRNAs is not to maintain silencing. Instead, we hypothesize the tandem repeat siRNAs mediate the establishment of the heritable silent state-a process fully disrupted in Mop2-1 heterozygotes. The dominant Mop2-1 mutation, which has a single nucleotide change in a domain highly conserved among all polymerases (E. coli to eukaryotes), disrupts both siRNA biogenesis (Pol-IV-like) and potentially processes downstream (Pol-V-like). These results suggest either the wild-type protein is a subunit in both complexes or the dominant mutant protein disrupts both complexes. Dominant mutations in the same domain in E. coli RNA polymerase suggest a model for Mop2-1 dominance: complexes containing Mop2-1 subunits are non-functional and compete with wild-type complexes.

Cryphodera sinensis n. sp. (Nematoda: Heteroderidae), a non-cyst-forming parasitic nematode from the root of ramie Boehmeria nivea in China.

PubMed

Zhuo, K; Wang, H H; Ye, W; Peng, D L; Liao, J L

2014-12-01

Cryphodera sinensis n. sp. is described from ramie (Boehmeria nivea) based on the morphology and molecular analyses of rRNA small subunit (SSU), D2D3 expansion domains of large subunit (LSU D2D3) and internal transcribed spacer (ITS). This new species is characterized by oval females with a distinct subcrystalline layer and pronounced and protruding vulval lip, distinctly concave vulva-anus profile and a vulva-anus distance of 29.5-35.8 μm. Males possess two annuli in the lip region, a stylet 27-32.5 μm in length with round knobs sloping slightly posteriorly, lateral fields with three lines, spicules 20-28 μm long and the presence of a short cloacal tube. Second-stage juveniles possess three lip annuli, a stylet 28-31 μm in length with well-developed knobs projected anteriorly and three lines along the lateral field. The pointed tail, 52-65 μm long, possesses a mucro-like tip and a hyaline region, 24.5-35 μm long. Large phasmids with a lens-like structure are located 2-6 annuli posterior to the anus. Phylogenetic analysis shows that the species has unique SSU, LSU D2D3 and ITS rRNA sequences. Phylogenetic relationships of the three rDNA sequences of C. sinensis n. sp. and other cystoid/cyst nematodes are analysed together with a comparison of other species within the genus Cryphodera.

The morphological and chemical characteristics of striatal neurons immunoreactive for the alpha1-subunit of the GABA(A) receptor in the rat.

PubMed

Waldvogel, H J; Kubota, Y; Trevallyan, S C; Kawaguchi, Y; Fritschy, J M; Mohler, H; Faull, R L

1997-10-01

The distribution, morphology and chemical characteristics of neurons immunoreactive for the alpha1-subunit of the GABA(A) receptor in the striatum of the basal ganglia in the rat brain were investigated at the light, confocal and electron microscope levels using single, double and triple immunohistochemical labelling techniques. The results showed that alpha1-subunit immunoreactive neurons were sparsely distributed throughout the rat striatum. Double and triple labelling results showed that all the alpha1-subunit-immunoreactive neurons were positive for glutamate decarboxylase and immunoreactive for the beta2,3 and gamma2 subunits of the GABA(A) receptor. Three types of alpha1-subunit-immunoreactive neurons were identified in the striatum on the basis of cellular morphology and chemical characteristics. The most numerous alpha1-subunit-immunoreactive neurons were medium-sized, aspiny neurons with a widely branching dendritic tree. They were parvalbumin-negative and were located mainly in the dorsolateral regions of the striatum. Electron microscopy showed that these neurons had an indented nuclear membrane, typical of striatal interneurons, and were surrounded by small numbers of axon terminals which established alpha1-subunit-immunoreactive synaptic contacts with the soma and dendrites. These cells were classified as type 1 alpha1-subunit-immunoreactive neurons and comprised 75% of the total population of alpha1-subunit-immunoreactive neurons in the striatum. The remaining alpha1-subunit-immunoreactive neurons comprised of a heterogeneous population of large-sized neurons localized in the ventral and medial regions of the striatum. The most numerous large-sized cells were parvalbumin-negative, had two to three relatively short branching dendrites and were designated type 2 alpha1-subunit-immunoreactive neurons. Electron microscopy showed that the type 2 neurons were characterized by a highly convoluted nuclear membrane and were sparsely covered with small axon terminals. The type 2 neurons comprised 20% of the total population of alpha1-subunit-immunoreactive neurons. The remaining large-sized alpha1-immunoreactive cells were designated type 3 cells; they were positive for parvalbumin and were distinguished by long branching dendrites extending dorsally for 600-800 microm into the striatum. These neurons comprised 5% of the total population of alpha1-subunit-immunoreactive neurons and were surrounded by enkephalin-immunoreactive terminals. Electron microscopy showed that the alpha1-subunit type 3 neurons had an indented nuclear membrane and were densely covered with small axon terminals which established alpha1-subunit-immunoreactive symmetrical synaptic contacts with the soma and dendrites. These results provide a detailed characterization of the distribution, morphology and chemical characteristics of the alpha1-subunit-immunoreactive neurons in the rat striatum and suggest that the type 1 and type 2 neurons comprise of separate populations of striatal interneurons while the type 3 neurons may represent the large striatonigral projection neurons described by Bolam et al. [Bolam J. P., Somogyi P., Totterdell S. and Smith A. D. (1981) Neuroscience 6, 2141-2157.].

The Second Subunit of DNA Polymerase Delta Is Required for Genomic Stability and Epigenetic Regulation1[OPEN

PubMed Central

Cheng, Jinkui; Lai, Jinsheng; Gong, Zhizhong

2016-01-01

DNA polymerase δ plays crucial roles in DNA repair and replication as well as maintaining genomic stability. However, the function of POLD2, the second small subunit of DNA polymerase δ, has not been characterized yet in Arabidopsis (Arabidopsis thaliana). During a genetic screen for release of transcriptional gene silencing, we identified a mutation in POLD2. Whole-genome bisulfite sequencing indicated that POLD2 is not involved in the regulation of DNA methylation. POLD2 genetically interacts with Ataxia Telangiectasia-mutated and Rad3-related and DNA polymerase α. The pold2-1 mutant exhibits genomic instability with a high frequency of homologous recombination. It also exhibits hypersensitivity to DNA-damaging reagents and short telomere length. Whole-genome chromatin immunoprecipitation sequencing and RNA sequencing analyses suggest that pold2-1 changes H3K27me3 and H3K4me3 modifications, and these changes are correlated with the gene expression levels. Our study suggests that POLD2 is required for maintaining genome integrity and properly establishing the epigenetic markers during DNA replication to modulate gene expression. PMID:27208288

COP9 signalosome subunit 7 from Arabidopsis interacts with and regulates the small subunit of ribonucleotide reductase (RNR2).

PubMed

Halimi, Yair; Dessau, Moshe; Pollak, Shaul; Ast, Tslil; Erez, Tamir; Livnat-Levanon, Nurit; Karniol, Baruch; Hirsch, Joel A; Chamovitz, Daniel A

2011-09-01

The COP9 Signalosome protein complex (CSN) is a pleiotropic regulator of plant development and contains eight-subunits. Six of these subunits contain the PCI motif which mediates specific protein interactions necessary for the integrity of the complex. COP9 complex subunit 7 (CSN7) contains an N-terminal PCI motif followed by a C-terminal extension which is also necessary for CSN function. A yeast-interaction trap assay identified the small subunit of ribonucelotide reductase (RNR2) from Arabidopsis as interacting with the C-terminal section of CSN7. This interaction was confirmed in planta by both bimolecular fluorescence complementation and immuoprecipitation assays with endogenous proteins. The subcellular localization of RNR2 was primarily nuclear in meristematic regions, and cytoplasmic in adult cells. RNR2 was constitutively nuclear in csn7 mutant seedlings, and was also primarily nuclear in wild type seedlings following exposure to UV-C. These two results correlate with constitutive expression of several DNA-damage response genes in csn7 mutants, and to increased tolerance of csn7 seedlings to UV-C treatment. We propose that the CSN is a negative regulator of RNR activity in Arabidopsis.

AFM study of Escherichia coli RNA polymerase σ⁷⁰ subunit aggregation.

PubMed

Dubrovin, Evgeniy V; Koroleva, Olga N; Khodak, Yulia A; Kuzmina, Natalia V; Yaminsky, Igor V; Drutsa, Valeriy L

2012-01-01

The self-assembly of Escherichia coli RNA polymerase σ⁷⁰ subunit was investigated using several experimental approaches. A novel rodlike shape was reported for σ⁷⁰ subunit aggregates. Atomic force microscopy reveals that these aggregates, or σ⁷⁰ polymers, have a straight rodlike shape 5.4 nm in diameter and up to 300 nm in length. Atomic force microscopy data, Congo red binding assay, and sodium dodecyl sulfate gel electrophoresis confirm the amyloid nature of observed aggregates. The process of formation of rodlike structures proceeds spontaneously under nearly physiological conditions. E. coli RNA polymerase σ⁷⁰ subunit may be an interesting object for investigation of amyloidosis as well as for biotechnological applications that exploit self-assembled bionanostructures. Polymerization of σ⁷⁰ subunit may be a competitive process with its three-dimensional crystallization and association with core RNA polymerase. In this basic science study, the self-assembly of Escherichia coli RNA polymerase σ⁷⁰( subunit was investigated using atomic force microscopy and other complementary approaches. 2012 Elsevier Inc. All rights reserved.

Structure-function of proteins interacting with the α1 pore-forming subunit of high-voltage-activated calcium channels

PubMed Central

Neely, Alan; Hidalgo, Patricia

2014-01-01

Openings of high-voltage-activated (HVA) calcium channels lead to a transient increase in calcium concentration that in turn activate a plethora of cellular functions, including muscle contraction, secretion and gene transcription. To coordinate all these responses calcium channels form supramolecular assemblies containing effectors and regulatory proteins that couple calcium influx to the downstream signal cascades and to feedback elements. According to the original biochemical characterization of skeletal muscle Dihydropyridine receptors, HVA calcium channels are multi-subunit protein complexes consisting of a pore-forming subunit (α1) associated with four additional polypeptide chains β, α2, δ, and γ, often referred to as accessory subunits. Twenty-five years after the first purification of a high-voltage calcium channel, the concept of a flexible stoichiometry to expand the repertoire of mechanisms that regulate calcium channel influx has emerged. Several other proteins have been identified that associate directly with the α1-subunit, including calmodulin and multiple members of the small and large GTPase family. Some of these proteins only interact with a subset of α1-subunits and during specific stages of biogenesis. More strikingly, most of the α1-subunit interacting proteins, such as the β-subunit and small GTPases, regulate both gating and trafficking through a variety of mechanisms. Modulation of channel activity covers almost all biophysical properties of the channel. Likewise, regulation of the number of channels in the plasma membrane is performed by altering the release of the α1-subunit from the endoplasmic reticulum, by reducing its degradation or enhancing its recycling back to the cell surface. In this review, we discuss the structural basis, interplay and functional role of selected proteins that interact with the central pore-forming subunit of HVA calcium channels. PMID:24917826

Hydroxyurea enhances SMN2 gene expression through nitric oxide release.

PubMed

Xu, Cheng; Chen, Xin; Grzeschik, Susanna M; Ganta, Madhuri; Wang, Ching H

2011-02-01

Small molecules that increase full-length survivor motor neuron (SMN) gene transcript are promising therapeutic candidates for spinal muscular atrophy (SMA). Hydroxyurea (HU) has recently been shown to increase full-length SMN transcript in cultured lymphocytes from patients with SMA. We investigate the mechanism by which HU enhances full-length SMN2 gene expression in SMA lymphocytes. Nitric oxide (NO) is a major intracellular metabolite of HU. We test whether NO donors can themselves enhance full-length SMN2 expression. Eighteen cell lines (five type I, five type II, six type III SMA, and two non-SMA controls) were treated with or without NO donors for 48 h. SMA cells treated with HU and three NO donors: two long-acting donors, Deta-NONOate and S-nitrosoglutathione, and one short-acting donor, 3-ethyl-3-(ethylaminoethyl)-1-hydroxy-2-oxo-1-triazene, resulted in significant increase in full-length SMN2 mRNA. These effects were abolished by co-treatment with an NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide. One short-acting NO donor, S-nitroso-N-acetyl-DL-penicillamine, failed to show significant effect on full-length SMN2 expression, possibly due to high degree of cytotoxicity. These results were observed using both densitometry and quantitative PCR methods. We conclude that HU enhances SMN2 expression through the release of NO. NO donors may themselves be considered as new therapeutic candidates for SMA.

Description of a new planktonic mixotrophic dinoflagellate Paragymnodinium shiwhaense n. gen., n. sp. from the coastal waters off Western Korea: morphology, pigments, and ribosomal DNA gene sequence.

PubMed

Kang, Nam Seon; Jeong, Hae Jin; Moestrup, Øjvind; Shin, Woongghi; Nam, Seung Won; Park, Jae Yeon; De Salas, Miguel F; Kim, Ki Woo; Noh, Jae Hoon

2010-01-01

The mixotrophic dinoflagellate Paragymnodinium shiwhaense n. gen., n. sp. is described from living cells and from cells prepared by light, scanning electron, and transmission electron microscopy. In addition, sequences of the small subunit (SSU) and large subunit (LSU) rDNA and photosynthetic pigments are reported. The episome is conical, while the hyposome is hemispherical. Cells are covered with polygonal amphiesmal vesicles arranged in 16 rows and containing a very thin plate-like component. There is neither an apical groove nor apical line of narrow plates. Instead, there is a sulcal extension-like furrow. The cingulum is as wide as 0.2-0.3 x cell length and displaced by 0.2-0.3 x cell length. Cell length and width of live cells fed Amphidinium carterae were 8.4-19.3 and 6.1-16.0 microm, respectively. Paragymnodinium shiwhaense does not have a nuclear envelope chamber nor a nuclear fibrous connective (NFC). Cells contain chloroplasts, nematocysts, trichocysts, and peduncle, though eyespots, pyrenoids, and pusules are absent. The main accessory pigment is peridinin. The sequence of the SSU rDNA of this dinoflagellate (GenBank AM408889) is 4% different from that of Gymnodinium aureolum, Lepidodinium viride, and Gymnodinium catenatum, the three closest species, while the LSU rDNA was 17-18% different from that of G. catenatum, Lepidodinium chlorophorum, and Gymnodinium nolleri. The phylogenetic trees show that this dinoflagellate belongs within the Gymnodinium sensu stricto clade. However, in contrast to Gymnodinium spp., cells lack nuclear envelope chambers, NFC, and an apical groove. Unlike Polykrikos spp., which have a taeniocyst-nematocyst complex, P. shiwhaense has nematocysts without taeniocysts. In addition, P. shiwhaense does not have ocelloids in contrast to Warnowia spp. and Nematodinium spp. Therefore, based on morphological and molecular analyses, we suggest that this taxon is a new species, also within a new genus.

Architecture and Assembly of HIV Integrase Multimers in the Absence of DNA Substrates*

PubMed Central

Bojja, Ravi Shankar; Andrake, Mark D.; Merkel, George; Weigand, Steven; Dunbrack, Roland L.; Skalka, Anna Marie

2013-01-01

We have applied small angle x-ray scattering and protein cross-linking coupled with mass spectrometry to determine the architectures of full-length HIV integrase (IN) dimers in solution. By blocking interactions that stabilize either a core-core domain interface or N-terminal domain intermolecular contacts, we show that full-length HIV IN can form two dimer types. One is an expected dimer, characterized by interactions between two catalytic core domains. The other dimer is stabilized by interactions of the N-terminal domain of one monomer with the C-terminal domain and catalytic core domain of the second monomer as well as direct interactions between the two C-terminal domains. This organization is similar to the “reaching dimer” previously described for wild type ASV apoIN and resembles the inner, substrate binding dimer in the crystal structure of the PFV intasome. Results from our small angle x-ray scattering and modeling studies indicate that in the absence of its DNA substrate, the HIV IN tetramer assembles as two stacked reaching dimers that are stabilized by core-core interactions. These models of full-length HIV IN provide new insight into multimer assembly and suggest additional approaches for enzyme inhibition. PMID:23322775

The Staphylococcus aureus extracellular adherence protein (Eap) adopts an elongated but structured conformation in solution.

PubMed

Hammel, Michal; Nemecek, Daniel; Keightley, J Andrew; Thomas, George J; Geisbrecht, Brian V

2007-12-01

The extracellular adherence protein (Eap) of Staphylococcus aureus participates in a wide range of protein-protein interactions that facilitate the initiation and dissemination of Staphylococcal disease. In this report, we describe the use of a multidisciplinary approach to characterize the solution structure of full-length Eap. In contrast to previous reports suggesting that a six-domain isoform of Eap undergoes multimerization, sedimentation equilibrium analytical ultracentrifugation data revealed that a four-domain isoform of Eap is a monomer in solution. In vitro proteolysis and solution small angle X-ray scattering studies both indicate that Eap adopts an extended conformation in solution, where the linkers connecting sequential EAP modules are solvent exposed. Construction of a low-resolution model of full-length Eap using a combination of ab initio deconvolution of the SAXS data and rigid body modeling of the EAP domain crystal structure suggests that full-length Eap may present several unique concave surfaces capable of participating in ligand binding. These results also raise the possibility that such surfaces may be held together by additional interactions between adjacent EAP modules. This hypothesis is supported by a comparative Raman spectroscopic analysis of full-length Eap and a stoichiometric solution of the individual EAP modules, which indicates the presence of additional secondary structure and a greater extent of hydrogen/deuterium exchange protection in full-length Eap. Our results provide the first insight into the solution structure of full-length Eap and an experimental basis for interpreting the EAP domain crystal structures within the context of the full-length molecule. They also lay a foundation for future studies into the structural and molecular bases of Eap-mediated protein-protein interactions with its many ligands.

The Staphylococcus aureus extracellular adherence protein (Eap) adopts an elongated but structured conformation in solution

PubMed Central

Hammel, Michal; Němeček, Daniel; Keightley, J. Andrew; Thomas, George J.; Geisbrecht, Brian V.

2007-01-01

The extracellular adherence protein (Eap) of Staphylococcus aureus participates in a wide range of protein–protein interactions that facilitate the initiation and dissemination of Staphylococcal disease. In this report, we describe the use of a multidisciplinary approach to characterize the solution structure of full-length Eap. In contrast to previous reports suggesting that a six-domain isoform of Eap undergoes multimerization, sedimentation equilibrium analytical ultracentrifugation data revealed that a four-domain isoform of Eap is a monomer in solution. In vitro proteolysis and solution small angle X-ray scattering studies both indicate that Eap adopts an extended conformation in solution, where the linkers connecting sequential EAP modules are solvent exposed. Construction of a low-resolution model of full-length Eap using a combination of ab initio deconvolution of the SAXS data and rigid body modeling of the EAP domain crystal structure suggests that full-length Eap may present several unique concave surfaces capable of participating in ligand binding. These results also raise the possibility that such surfaces may be held together by additional interactions between adjacent EAP modules. This hypothesis is supported by a comparative Raman spectroscopic analysis of full-length Eap and a stoichiometric solution of the individual EAP modules, which indicates the presence of additional secondary structure and a greater extent of hydrogen/deuterium exchange protection in full-length Eap. Our results provide the first insight into the solution structure of full-length Eap and an experimental basis for interpreting the EAP domain crystal structures within the context of the full-length molecule. They also lay a foundation for future studies into the structural and molecular bases of Eap-mediated protein–protein interactions with its many ligands. PMID:18029416

Oligothiophene bipyridine alternate copolymers and their ruthenium metalated analogues: in situ ESR and UV-vis investigations of metal-chain interactions.

PubMed

Lafolet, F; Genoud, F; Divisia-Blohorn, B; Aronica, C; Guillerez, S

2005-07-07

In situ electron spin resonance (ESR) and UV-vis spectro-electrochemical studies have been performed on two copolymers consisting of alternating subunits of regioregular head to tail (HT) coupled 3-octylthiophene tetramer and 2,2'-bipyridine subunits (P4) or 3-octylthiophene hexamer subunits of the same regioregularity and 2,2'-bipyridine subunits (P6). Both P4 and P6 have been investigated in their metal-free form as well as in the ruthenium(II) metalated form (P4-Ru and P6-Ru). P4 and P6 in the p-doped state exhibit a clear ESR signal characteristic of the presence of polarons in the oligothienylene subunits. In the case of P4, no recombination of polarons into bipolarons is observed, whereas the recombination process takes place in P6. The formation of bipolarons is well-rationalized in terms of the conjugation length, and it seems clear that the higher length of the oligothiophene subunit in P6( )()stabilizes bipolarons(.)() The same effect, is induced by the coordination of -Ru(bpy)(2)(2+) to the bipyridine unit in the metalated form of both polymers, which results in an increase of the conjugation length. Important information is gained from the analysis of the ESR spectra of both nonmetalated and metalated in the oxidized (p-doped) and reduced (n-doped) forms. In the p-doped state both nonmetalated and metalated polymers reveal the presence of a narrow ESR line characteristic of the mobile spin carriers in the polymer matrix. The oxidation of the metal center occurs at higher potentials and leads to an irreversible destruction of the system. To the contrary, in the reduced (n-doped) state the ESR lines of the nonmetalated and metalated polymers markedly differ. A significant line broadening with simultaneous change of the g-value is caused by spin-orbit coupling phenomenon induced by the presence of the coordinating metal. Finally, the observation of a clear polaronic band in the UV-vis spectrum of p-doped P4 and its strong dependence on the applied potential can be clearly correlated with the potential induced changes in the ESR spin density. The same applies to P4-Ru, where the changes in the polaronic and bipolaronic bands can also be correlated with the ESR spin density changes.

Morphology and 18S rDNA of Henneguya gurlei (Myxosporea) from Ameiurus nebulosus (Siluriformes) in North Carolina.

PubMed

Iwanowicz, Luke R; Iwanowicz, Deborah D; Pote, Linda M; Blazer, Vicki S; Schill, William B

2008-02-01

Henneguya gurlei was isolated from Ameiurus nebulosus captured in North Carolina and redescribed using critical morphological features and 18S small-subunit ribosomal RNA (SSU rDNA) gene sequence. Plasmodia are white, spherical, or subspherical, occur in clusters, measure up to 1.8 mm in length, and are located on the dorsal, pectoral, and anal fins. Histologically, plasmodia are located in the dermis and subdermally, and the larger cysts disrupt the melanocyte pigment layer. The spore body is lanceolate, 18.2 +/- 0.3 microm (range 15.7-20.3) in length, and 5.4 +/- 0.1 microm (range 3.8-6.1) in width in valvular view. The caudal appendages are 41.1 +/- 1.1 microm (range 34.0-49.7) in length. Polar capsules are pyriform and of unequal size. The longer polar capsule measures 6.2 +/- 0.1 microm (range 5.48-7.06), while the shorter is 5.7 +/- 0.1 microm (range 4.8-6.4) in length. Polar capsule width is 1.2 +/- 0.03 microm (range 1.0-1.54). The total length of the spore is 60.9 +/- 1.2 microm (range 48.7-68.5). Morphologically, this species is similar to other species of Henneguya that are known to infect ictalurids. Based on SSU rDNA sequences, this species is most closely related to H. exilis and H. ictaluri, which infect Ictalurus punctatus.

Morphology and 18S rDNA of Henneguya gurlei (Myxosporea) from Ameiurus nebulosus (Siluriformes) in North Carolina

USGS Publications Warehouse

Iwanowicz, L.R.; Iwanowicz, D.D.; Pote, L.M.; Blazer, V.S.; Schill, W.B.

2008-01-01

Henneguya gurlei was isolated from Ameiurus nebulosus captured in North Carolina and redescribed using critical morphological features and 18S small-subunit ribosomal RNA (SSU rDNA) gene sequence. Plasmodia are white, spherical, or subspherical, occur in clusters, measure up to 1.8 mm in length, and are located on the dorsal, pectoral, and anal fins. Histologically, plasmodia are located in the dermis and subdermally, and the larger cysts disrupt the melanocyte pigment layer. The spore body is lanceolate, 18.2 ?? 0.3 ??m (range 15.7-20.3) in length, and 5.4 ?? 0.1 ??m (range 3.8-6.1) in width in valvular view. The caudal appendages are 41.1 ?? 1.1 ??m (range 34.0-49.7) in length. Polar capsules are pyriform and of unequal size. The longer polar capsule measures 6.2 ?? 0.1 ??m (range 5.48-7.06), while the shorter is 5.7 ?? 0.1 ??m (range 4.8-6.4) in length. Polar capsule width is 1.2 ?? 0.03 ??m (range 1.0-1.54). The total length of the spore is 60.9 ?? 1.2 ??m (range 48.7-68.5). Morphologically, this species is similar to other species of Henneguya that are known to infect ictalurids. Based on SSU rDNA sequences, this species is most closely related to H. exilis and H. ictaluri, which infect Ictalurus punctatus. ?? American Society of Parasitologists 2008.

Geranyl diphosphate synthase large subunit, and methods of use

DOEpatents

Croteau, Rodney B.; Burke, Charles C.; Wildung, Mark R.

2001-10-16

A cDNA encoding geranyl diphosphate synthase large subunit from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase large subunit). In another aspect, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase large subunit. In yet another aspect, the present invention provides isolated, recombinant geranyl diphosphate synthase protein comprising an isolated, recombinant geranyl diphosphate synthase large subunit protein and an isolated, recombinant geranyl diphosphate synthase small subunit protein. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase.

Revisiting the phylogeny of Ocellularieae, the second largest tribe within Graphidaceae (lichenized Ascomycota: Ostropales)

Treesearch

Ekaphan Kraichak; Sittiporn Parnmen; Robert Lücking; Eimy Rivas Plata; Andre Aptroot; Marcela E.S. Caceres; Damien Ertz; Armin Mangold; Joel A. Mercado-Diaz; Khwanruan Papong; Dries Van der Broeck; Gothamie Weerakoon; H. Thorsten Lumbsch; NO-VALUE

2014-01-01

We present an updated 3-locus molecular phylogeny of tribe Ocellularieae, the second largest tribe within subfamily Graphidoideae in the Graphidaceae. Adding 165 newly generated sequences from the mitochondrial small subunit rDNA (mtSSU), the nuclear large subunit rDNA (nuLSU), and the second largest subunit of the DNA-directed RNA polymerase II (RPB2), we currently...

D1/D2 domain of large-subunit ribosomal DNA for differentiation of Orpinomyces spp.

PubMed

Dagar, Sumit S; Kumar, Sanjay; Mudgil, Priti; Singh, Rameshwar; Puniya, Anil K

2011-09-01

This study presents the suitability of D1/D2 domain of large-subunit (LSU) ribosomal DNA (rDNA) for differentiation of Orpinomyces joyonii and Orpinomyces intercalaris based on PCR-restriction fragment length polymorphism (RFLP). A variation of G/T in O. intercalaris created an additional restriction site for AluI, which was used as an RFLP marker. The results demonstrate adequate heterogeneity in the LSU rDNA for species-level differentiation.

Identification of Telomerase Components and Telomerase Regulating Factors in Yeast

DTIC Science & Technology

1998-07-01

subunit of telomerase in S. cerevisiae is encoded by TLCJ (7). Recently , through sequence comparison with the telomerase catalytic 6 subunit from Euplotes...length maintenance has been unclear, although very recent data has shown that Ku80p can be found specifically associated with telomeric DNA in vivo...chromatin structure. It has been recently observed that loss of either YKU80 or HDF1 results in altered telomere end structure, such that there appears to

Identification of a small molecule that inhibits herpes simplex virus DNA Polymerase subunit interactions and viral replication.

PubMed

Pilger, Beatrice D; Cui, Can; Coen, Donald M

2004-05-01

The interaction between the catalytic subunit Pol and the processivity subunit UL42 of herpes simplex virus DNA polymerase has been characterized structurally and mutationally and is a potential target for novel antiviral drugs. We developed and validated an assay for small molecules that could disrupt the interaction of UL42 and a Pol-derived peptide and used it to screen approximately 16,000 compounds. Of 37 "hits" identified, four inhibited UL42-stimulated long-chain DNA synthesis by Pol in vitro, of which two exhibited little inhibition of polymerase activity by Pol alone. One of these specifically inhibited the physical interaction of Pol and UL42 and also inhibited viral replication at concentrations below those that caused cytotoxic effects. Thus, a small molecule can inhibit this protein-protein interaction, which provides a starting point for the discovery of new antiviral drugs.

Photocontrol of the expression of genes encoding chlorophyll a/b binding proteins and small subunit of ribulose-1,5-bisphosphate carboxylase in etiolated seedlings of Lycopersicon esculentum (L. ) and Nicotiana tabacum (L. )

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

Wehmeyer, B.; Cashmore, A.R.; Schaefer, E.

Phytochrome and the blue ultraviolet-A photoreceptor control light-induced expression of genes encoding the chlorophyll a/b binding protein of photosystem II and photosystem I and the genes for the small subunit of the ribulose-1,5-bisphosphate carboxylase in etiolated seedlings of Lycopersicon esculentum (tomato) and Nicotiana tabacum (tobacco). A high irradiance response also controls the induction of these genes. Genes encoding photosystem II- and I-associated chlorophyll a/b binding proteins both exhibit a transient rapid increase in expression in response to light pulse or to continuous irradiation. In contrast, genes encoding the small subunit exhibit a continuous increase in expression in response to light.more » These distinct expression characteristics are shown to reflect differences at the level of transcription.« less

Transcript levels, alternative splicing and proteolytic cleavage of TFIIIA control 5S rRNA accumulation during Arabidopsis thaliana development.

PubMed

Layat, Elodie; Cotterell, Sylviane; Vaillant, Isabelle; Yukawa, Yasushi; Tutois, Sylvie; Tourmente, Sylvette

2012-07-01

Ribosome biogenesis is critical for eukaryotic cells and requires coordinated synthesis of the protein and rRNA moieties of the ribosome, which are therefore highly regulated. 5S ribosomal RNA, an essential component of the large ribosomal subunit, is transcribed by RNA polymerase III and specifically requires transcription factor IIIA (TFIIIA). To obtain insight into the regulation of 5S rRNA transcription, we have investigated the expression of 5S rRNA and the exon-skipped (ES) and exon-including (EI) TFIIIA transcripts, two transcript isoforms that result from alternative splicing of the TFIIIA gene, and TFIIIA protein amounts with respect to requirements for 5S rRNA during development. We show that 5S rRNA quantities are regulated through distinct but complementary mechanisms operating through transcriptional and post-transcriptional control of TFIIIA transcripts as well as at the post-translational level through proteolytic cleavage of the TFIIIA protein. During the reproductive phase, high expression of the TFIIIA gene together with low proteolytic cleavage contributes to accumulation of functional, full-length TFIIIA protein, and results in 5S rRNA accumulation in the seed. In contrast, just after germination, the levels of TFIIIA-encoding transcripts are low and stable. Full-length TFIIIA protein is undetectable, and the level of 5S rRNA stored in the embryo progressively decreases. After day 4, in correlation with the reorganization of 5S rDNA chromatin to a mature state, full-length TFIIIA protein with transcriptional activity accumulates and permits de novo transcription of 5S rRNA. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

Inhibition of Ebola virus glycoprotein-mediated cytotoxicity by targeting its transmembrane domain and cholesterol.

PubMed

Hacke, Moritz; Björkholm, Patrik; Hellwig, Andrea; Himmels, Patricia; Ruiz de Almodóvar, Carmen; Brügger, Britta; Wieland, Felix; Ernst, Andreas M

2015-07-09

The high pathogenicity of the Ebola virus reflects multiple concurrent processes on infection. Among other important determinants, Ebola fusogenic glycoprotein (GP) has been associated with the detachment of infected cells and eventually leads to vascular leakage and haemorrhagic fever. Here we report that the membrane-anchored GP is sufficient to induce the detachment of adherent cells. The results show that the detachment induced through either full-length GP1,2 or the subunit GP2 depends on cholesterol and the structure of the transmembrane domain. These data reveal a novel molecular mechanism in which GP regulates Ebola virus assembly and suggest that cholesterol-reducing agents could be useful as therapeutics to counteract GP-mediated cell detachment.

Structure and organization of heteromeric AMPA-type glutamate receptors.

PubMed

Herguedas, Beatriz; García-Nafría, Javier; Cais, Ondrej; Fernández-Leiro, Rafael; Krieger, James; Ho, Hinze; Greger, Ingo H

2016-04-29

AMPA-type glutamate receptors (AMPARs), which are central mediators of rapid neurotransmission and synaptic plasticity, predominantly exist as heteromers of the subunits GluA1 to GluA4. Here we report the first AMPAR heteromer structures, which deviate substantially from existing GluA2 homomer structures. Crystal structures of the GluA2/3 and GluA2/4 N-terminal domains reveal a novel compact conformation with an alternating arrangement of the four subunits around a central axis. This organization is confirmed by cysteine cross-linking in full-length receptors, and it permitted us to determine the structure of an intact GluA2/3 receptor by cryogenic electron microscopy. Two models in the ligand-free state, at resolutions of 8.25 and 10.3 angstroms, exhibit substantial vertical compression and close associations between domain layers, reminiscent of N-methyl-D-aspartate receptors. Model 1 resembles a resting state and model 2 a desensitized state, thus providing snapshots of gating transitions in the nominal absence of ligand. Our data reveal organizational features of heteromeric AMPARs and provide a framework to decipher AMPAR architecture and signaling. Copyright © 2016, American Association for the Advancement of Science.

Cooperative autoinhibition and multi-level activation mechanisms of calcineurin

PubMed Central

Li, Sheng-Jie; Wang, Jue; Ma, Lei; Lu, Chang; Wang, Jie; Wu, Jia-Wei; Wang, Zhi-Xin

2016-01-01

The Ca2+/calmodulin-dependent protein phosphatase calcineurin (CN), a heterodimer composed of a catalytic subunit A and an essential regulatory subunit B, plays critical functions in various cellular processes such as cardiac hypertrophy and T cell activation. It is the target of the most widely used immunosuppressants for transplantation, tacrolimus (FK506) and cyclosporin A. However, the structure of a large part of the CNA regulatory region remains to be determined, and there has been considerable debate concerning the regulation of CN activity. Here, we report the crystal structure of full-length CN (β isoform), which revealed a novel autoinhibitory segment (AIS) in addition to the well-known autoinhibitory domain (AID). The AIS nestles in a hydrophobic intersubunit groove, which overlaps the recognition site for substrates and immunosuppressant-immunophilin complexes. Indeed, disruption of this AIS interaction results in partial stimulation of CN activity. More importantly, our biochemical studies demonstrate that calmodulin does not remove AID from the active site, but only regulates the orientation of AID with respect to the catalytic core, causing incomplete activation of CN. Our findings challenge the current model for CN activation, and provide a better understanding of molecular mechanisms of CN activity regulation. PMID:26794871

Rat nicotinic ACh receptor α7 and β2 subunits co-assemble to form functional heteromeric nicotinic receptor channels

PubMed Central

Khiroug, Serguei S; Harkness, Patricia C; Lamb, Patricia W; Sudweeks, Sterling N; Khiroug, Leonard; Millar, Neil S; Yakel, Jerrel L

2002-01-01

Rat hippocampal interneurons express diverse subtypes of functional nicotinic acetylcholine receptors (nAChRs), including α7-containing receptors that have properties unlike those expected for homomeric α7 nAChRs. We previously reported a strong correlation between expression of the α7 and of the β2 subunits in individual neurons. To explore whether co-assembly of the α7 and β2 subunits might occur, these subunits were co-expressed in Xenopus oocytes and the functional properties of heterologously expressed nAChRs were characterized by two-electrode voltage clamp. Co-expression of the β2 subunit, both wild-type and mutant forms, with the α7 subunit significantly slowed the rate of nAChR desensitization and altered the pharmacological properties. Whereas ACh, carbachol and choline were full or near-full agonists for homomeric α7 receptor channels, both carbachol and choline were only partial agonists in oocytes expressing both α7 and β2 subunits. In addition the EC50 values for all three agonists significantly increased when the β2 subunit was co-expressed with the α7 subunit. Co-expression with the β2 subunit did not result in any significant change in the current-voltage curve. Biochemical evidence for the co-assembly of the α7 and β2 subunits was obtained by co-immunoprecipitation of these subunits from transiently transfected human embryonic kidney (TSA201) cells. These data provide direct biophysical and molecular evidence that the nAChR α7 and β2 subunits co-assemble to form a functional heteromeric nAChR with functional and pharmacological properties different from those of homomeric α7 channels. This co-assembly may help to explain nAChR channel diversity in rat hippocampal interneurons, and perhaps in other areas of the nervous system. PMID:11956333

Development and use of domain-specific antibodies in a characterization of the large subunits of soybean photosystem 1

NASA Technical Reports Server (NTRS)

Henry, R. L.; Takemoto, L. J.; Murphy, J.; Gallegos, G. L.; Guikema, J. A.; Spooner, B. S. (Principal Investigator)

1992-01-01

The molecular architecture of the soybean photosystem 1 reaction center complex was examined using a combination of surface labeling and immunological methodology on isolated thylakoid membranes. Synthetic peptides (12 to 14 amino acids in length) were prepared which correspond to the N-terminal regions of the 83 and 82.4 kDa subunits of photosystem 1 (the PsaA and PsaB proteins, respectively). Similarly, a synthetic peptide was prepared corresponding to the C-terminal region of the PsaB subunit. These peptides were conjugated to a carrier protein, and were used for the production of polyclonal antibodies in rabbits. The resulting sera could distinguish between the PsaA and PsaB photosystem 1 subunits by Western blot analysis, and could identify appropriate size classes of cyanogen bromide cleavage fragments as predicted from the primary sequences of these two subunits. When soybean thylakoid membranes were surface-labeled with N-hydroxysuccinimidobiotin, several subunits of the complete photosystem 1 lipid/protein complex incorporated label. These included the light harvesting chlorophyll proteins of photosystem 1, and peptides thought to aid in the docking of ferredoxin to the complex during photosynthetic electron transport. However, the PsaA and PsaB subunits showed very little biotinylation. When these subunits were examined for the domains to which biotin did attach, most of the observed label was associated with the N-terminal domain of the PsaA subunit, as identified using a domain-specific polyclonal antisera.

Nucleotide sequence of an exceptionally long 5.8S ribosomal RNA from Crithidia fasciculata.

PubMed Central

Schnare, M N; Gray, M W

1982-01-01

In Crithidia fasciculata, a trypanosomatid protozoan, the large ribosomal subunit contains five small RNA species (e, f, g, i, j) in addition to 5S rRNA [Gray, M.W. (1981) Mol. Cell. Biol. 1, 347-357]. The complete primary sequence of species i is shown here to be pAACGUGUmCGCGAUGGAUGACUUGGCUUCCUAUCUCGUUGA ... AGAmACGCAGUAAAGUGCGAUAAGUGGUApsiCAAUUGmCAGAAUCAUUCAAUUACCGAAUCUUUGAACGAAACGG ... CGCAUGGGAGAAGCUCUUUUGAGUCAUCCCCGUGCAUGCCAUAUUCUCCAmGUGUCGAA(C)OH. This sequence establishes that species i is a 5.8S rRNA, despite its exceptional length (171-172 nucleotides). The extra nucleotides in C. fasciculata 5.8S rRNA are located in a region whose primary sequence and length are highly variable among 5.8S rRNAs, but which is capable of forming a stable hairpin loop structure (the "G+C-rich hairpin"). The sequence of C. fasciculata 5.8S rRNA is no more closely related to that of another protozoan, Acanthamoeba castellanii, than it is to representative 5.8S rRNA sequences from the other eukaryotic kingdoms, emphasizing the deep phylogenetic divisions that seem to exist within the Kingdom Protista. Images PMID:7079176

Quantification of Transthyretin Kinetic Stability in Human Plasma Using Subunit Exchange

PubMed Central

2015-01-01

The transthyretin (TTR) amyloidoses are a group of degenerative diseases caused by TTR aggregation, requiring rate-limiting tetramer dissociation. Kinetic stabilization of TTR, by preferential binding of a drug to the native tetramer over the dissociative transition state, dramatically slows the progression of familial amyloid polyneuropathy. An established method for quantifying the kinetic stability of recombinant TTR tetramers in buffer is subunit exchange, in which tagged TTR homotetramers are added to untagged homotetramers at equal concentrations to measure the rate at which the subunits exchange. Herein, we report a subunit exchange method for quantifying the kinetic stability of endogenous TTR in human plasma. The subunit exchange reaction is initiated by the addition of a substoichiometric quantity of FLAG-tagged TTR homotetramers to endogenous TTR in plasma. Aliquots of the subunit exchange reaction, taken as a function of time, are then added to an excess of a fluorogenic small molecule, which immediately arrests further subunit exchange. After binding, the small molecule reacts with the TTR tetramers, rendering them fluorescent and detectable in human plasma after subsequent ion exchange chromatography. The ability to report on the extent of TTR kinetic stabilization resulting from treatment with oral tafamidis is important, especially for selection of the appropriate dose for patients carrying rare mutations. This method could also serve as a surrogate biomarker for the prediction of the clinical outcome. Subunit exchange was used to quantify the stabilization of WT TTR from senile systemic amyloidosis patients currently being treated with tafamidis (20 mg orally, once daily). TTR kinetic stability correlated with the tafamidis plasma concentration. PMID:24661308

Differential endothelial transcriptomics identifies semaphorin 3G as a vascular class 3 semaphorin.

PubMed

Kutschera, Simone; Weber, Holger; Weick, Anja; De Smet, Frederik; Genove, Guillem; Takemoto, Minoru; Prahst, Claudia; Riedel, Maria; Mikelis, Constantinos; Baulande, Sylvain; Champseix, Catherine; Kummerer, Petra; Conseiller, Emmanuel; Multon, Marie-Christine; Heroult, Melanie; Bicknell, Roy; Carmeliet, Peter; Betsholtz, Christer; Augustin, Hellmut G

2011-01-01

To characterize the role of a vascular-expressed class 3 semaphorin (semaphorin 3G [Sema3G]). Semaphorins have been identified as axon guidance molecules. Yet, they have more recently also been characterized as attractive and repulsive regulators of angiogenesis. Through a transcriptomic screen, we identified Sema3G as a molecule of angiogenic endothelial cells. Sema3G-deficient mice are viable and exhibit no overt vascular phenotype. Yet, LacZ expression in the Sema3G locus revealed intense arterial vascular staining in the angiogenic vasculature, starting at E9.5, which was detectable throughout adolescence and downregulated in adult vasculature. Sema3G is expressed as a full-length 100-kDa secreted molecule that is processed by furin proteases to yield 95- and a 65-kDa Sema domain-containing subunits. Full-length Sema3G binds to NP2, whereas processed Sema3G binds to NP1 and NP2. Expression profiling and cellular experiments identified autocrine effects of Sema3G on endothelial cells and paracrine effects on smooth muscle cells. Although the mouse knockout phenotype suggests compensatory mechanisms, the experiments identify Sema3G as a primarily endothelial cell-expressed class 3 semaphorin that controls endothelial and smooth muscle cell functions in autocrine and paracrine manners, respectively.

D1/D2 Domain of Large-Subunit Ribosomal DNA for Differentiation of Orpinomyces spp.▿

PubMed Central

Dagar, Sumit S.; Kumar, Sanjay; Mudgil, Priti; Singh, Rameshwar; Puniya, Anil K.

2011-01-01

This study presents the suitability of D1/D2 domain of large-subunit (LSU) ribosomal DNA (rDNA) for differentiation of Orpinomyces joyonii and Orpinomyces intercalaris based on PCR-restriction fragment length polymorphism (RFLP). A variation of G/T in O. intercalaris created an additional restriction site for AluI, which was used as an RFLP marker. The results demonstrate adequate heterogeneity in the LSU rDNA for species-level differentiation. PMID:21784906

Assessing self-care and social function using a computer adaptive testing version of the pediatric evaluation of disability inventory.

PubMed

Coster, Wendy J; Haley, Stephen M; Ni, Pengsheng; Dumas, Helene M; Fragala-Pinkham, Maria A

2008-04-01

To examine score agreement, validity, precision, and response burden of a prototype computer adaptive testing (CAT) version of the self-care and social function scales of the Pediatric Evaluation of Disability Inventory compared with the full-length version of these scales. Computer simulation analysis of cross-sectional and longitudinal retrospective data; cross-sectional prospective study. Pediatric rehabilitation hospital, including inpatient acute rehabilitation, day school program, outpatient clinics; community-based day care, preschool, and children's homes. Children with disabilities (n=469) and 412 children with no disabilities (analytic sample); 38 children with disabilities and 35 children without disabilities (cross-validation sample). Not applicable. Summary scores from prototype CAT applications of each scale using 15-, 10-, and 5-item stopping rules; scores from the full-length self-care and social function scales; time (in seconds) to complete assessments and respondent ratings of burden. Scores from both computer simulations and field administration of the prototype CATs were highly consistent with scores from full-length administration (r range, .94-.99). Using computer simulation of retrospective data, discriminant validity, and sensitivity to change of the CATs closely approximated that of the full-length scales, especially when the 15- and 10-item stopping rules were applied. In the cross-validation study the time to administer both CATs was 4 minutes, compared with over 16 minutes to complete the full-length scales. Self-care and social function score estimates from CAT administration are highly comparable with those obtained from full-length scale administration, with small losses in validity and precision and substantial decreases in administration time.

Nuclear export of the small ribosomal subunit requires the Ran–GTPase cycle and certain nucleoporins

PubMed Central

Moy, Terence I.; Silver, Pamela A.

1999-01-01

After their assembly in the nucleolus, ribosomal subunits are exported from the nucleus to the cytoplasm. After export, the 20S rRNA in the small ribosomal subunit is cleaved to yield 18S rRNA and the small 5′ ITS1 fragment. The 5′ ITS1 RNA is normally degraded by the cytoplasmic Xrn1 exonuclease, but in strains lacking XRN1, the 5′ ITS1 fragment accumulates in the cytoplasm. Using the cytoplasmic localization of the 5′ ITS1 fragment as an indicator for the export of the small ribosomal subunit, we have identified genes that are required for ribosome export. Ribosome export is dependent on the Ran–GTPase as mutations in Ran or its regulators caused 5′ ITS1 to accumulate in the nucleoplasm. Mutations in the genes encoding the nucleoporin Nup82 and in the NES exporter Xpo1/Crm1 also caused the nucleoplasmic accumulation of 5′ ITS1. Mutants in a subset of nucleoporins and in the nuclear transport factors Srp1, Kap95, Pse1, Cse1, and Mtr10 accumulate the 5′ ITS1 in the nucleolus and affect ribosome assembly. In contrast, we did not detect nuclear accumulation of 5′ ITS1 in 28 yeast strains that have mutations in other genes affecting nuclear trafficking. PMID:10465789

Feeding habits of the deep-snouted pipefish Syngnathus typhle in a temperate coastal lagoon

NASA Astrophysics Data System (ADS)

Oliveira, Frederico; Erzini, Karim; Gonçalves, Jorge M. S.

2007-03-01

Feeding habits of Syngnathus typhle were determined based on monthly sampling during experimental fishing in a coastal lagoon (Ria Formosa, South Portugal) from April 2001 to May 2002. The gut contents of 856 individuals were analysed and quantified with numerical and gravimetric methods, as well as with some complementary indices and measures (vacuity, fullness and relative importance). In this study, this species fed mainly on Copepoda, Mysidacea, small caridean grass shrimps (Hippolytidae and Palaemonidae) and small fishes (Gobiidae) and to a lesser degree on other small invertebrates. No significant differences were found between the diets of males and females. However, significant differences in the fullness indexes of both sexes were observed during the reproductive period. Significant differences in the diet among seasons were also verified but no seasonal trends were discovered. Snout length showed a linear increase with growth while mouth area and total length displayed an allometric relation. Mouth shape varied between an ellipse and an almost perfect circle. The increase in mouth area and snout length as S. typhle grows contributed to the observed ontogenic diet shift with specimens smaller than 10 cm feeding almost exclusively on Copepoda, medium sized specimens feeding mainly on Hippolytidae and Mysidacea, while larger specimens preyed on Hippolytidae, Palaemonidae and Gobiidae. Prey size generally increased with size of S. typhle.

Characterization of antigenic determinants in ApxIIA exotoxin capable of inducing protective immunity to Actinobacillus pleuropneumoniae challenge.

PubMed

Seo, Ki-Weon; Kim, Dong-Heon; Kim, Ah Hyun; Yoo, Han-Sang; Lee, Kyung-Yeol; Jang, Yong-Suk

2011-01-01

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia. Among the virulence factors of the pathogen, ApxIIA, a bacterial exotoxin, is expressed by many serotypes and presents a plausible target for vaccine development. We characterized the region within ApxIIA that induces a protective immune response against bacterial infection using mouse challenge model. Recombinant proteins spanning the length of ApxIIA were produced and antiserum to the full-length ApxIIA was induced in mice. This antiserum recognized fragments #2, #3 and #5 with high binding specificity, but showed poor recognition for fragments #1 and #4. Of the antisera induced in mice by injection of each fragments, only the antiserum to fragment #4 failed to efficiently recognize the full-length antigen, although the individual antisera recognized their cognate antigens with almost equal efficiency. The protective potency of the immunogenic proteins against a challenge injection of bacteria in vivo correlated well with the antibody titer. Fragment #5 induced the highest level of protective activity, comparable to that by the full-length protein. These results support the use of fragment #5 to produce a vaccine against A. pleuropneumoniae challenge, since the small antigen peptide is easier to handle than is the full-length protein and can be expressed efficiently in heterologous expression systems.

Construction of a small Mus musculus repetitive DNA library: identification of a new satellite sequence in Mus musculus.

PubMed Central

Pietras, D F; Bennett, K L; Siracusa, L D; Woodworth-Gutai, M; Chapman, V M; Gross, K W; Kane-Haas, C; Hastie, N D

1983-01-01

We report the construction of a small library of recombinant plasmids containing Mus musculus repetitive DNA inserts. The repetitive cloned fraction was derived from denatured genomic DNA by reassociation to a Cot value at which repetitive, but not unique, sequences have reannealed followed by exhaustive S1 nuclease treatment to degrade single stranded DNA. Initial characterizations of this library by colony filter hybridizations have led to the identification of a previously undetected M. musculus minor satellite as well as to clones containing M. musculus major satellite sequences. This new satellite is repeated 10-20 times less than the major satellite in the M. musculus genome. It has a repeat length of 130 nucleotides compared with the M. musculus major satellite with a repeat length of 234 nucleotides. Sequence analysis of the minor satellite has shown that it has a 29 base pair region with extensive homology to one of the major satellite repeating subunits. We also show by in situ hybridization that this minor satellite sequence is located at the centromeres and possibly the arms of at least half the M musculus chromosomes. Sequences related to the minor satellite have been found in the DNA of a related Mus species, Mus spretus, and may represent the major satellite of that species. Images PMID:6314268

A morphological and molecular study of two species of Raphidascaroides Yamaguti, 1941 (Nematoda: Anisakidae), parasites of doradid catfish (Siluriformes) in South America, with a description of R. moraveci n. sp.

PubMed

Pereira, Felipe B; Tavares, Luiz E R; Scholz, Tomáš; Luque, José L

2015-05-01

Nematodes of the genus Raphidascaroides Yamaguti, 1941 parasitising doradid catfishes (Siluriformes: Doradidae) in Brazil were studied based on morphological and molecular evaluation of newly collected material. A new species, Raphidascaroides moraveci n. sp., is described from the intestine of Platydoras armatulus (Valenciennes) from River Miranda, River Paraguay basin, Pantanal, Mato Grosso do Sul. The new species differs from all of the congeners in having short spicules (163-217 μm in length) representing less than 1% of the total body length and in the posterior region of cloacal opening covered by small rudimentary spines. In addition, it differs from the other congeneric species in the number and arrangement of the caudal papillae and the structure of lips and tail. Raphidascaroides moraveci n. sp. is the third species described from freshwater fishes and the second one in the Neotropical Region. New morphological data on R. brasiliensis Moravec & Thatcher, 1997 from Megalodoras uranoscopus (Eigenmann & Eigenmann) and Platydoras costatus (Linnaeus) (both new host records) from River Xingu, River Amazon basin, Pará, are provided including scanning electron micrographs of taxonomically important structures. The differentiation of the new species is supported by molecular data (partial sequences of the small and large subunits of the rRNA gene).

The roles of RIIbeta linker and N-terminal cyclic nucleotide-binding domain in determining the unique structures of Type IIbeta Protein Kinase A. A small angle X-ray and neutron scattering study

DOE PAGES

Blumenthal, Donald K.; Copps, Jeffrey; Smith-Nguyen, Eric V.; ...

2014-08-11

Protein kinase A (PKA) is ubiquitously expressed and is responsible for regulating many important cellular functions in response to changes in intracellular cAMP concentrations. Moreover, the PKA holoenzyme is a tetramer (R 2:C 2), with a regulatory subunit homodimer (R 2) that binds and inhibits two catalytic (C) subunits; binding of cAMP to the regulatory subunit homodimer causes activation of the catalytic subunits. Four different R subunit isoforms exist in mammalian cells, and these confer different structural features, subcellular localization, and biochemical properties upon the PKA holoenzymes they form. The holoenzyme containing RIIβ is structurally unique in that the typemore » IIβ holoenzyme is much more compact than the free RIIβ homodimer. We have used small angle x-ray scattering and small angle neutron scattering to study the solution structure and subunit organization of a holoenzyme containing an RIIβ C-terminal deletion mutant (RIIβ(1–280)), which is missing the C-terminal cAMP-binding domain to better understand the structural organization of the type IIβ holoenzyme and the RIIβ domains that contribute to stabilizing the holoenzyme conformation. These results demonstrate that compaction of the type IIβ holoenzyme does not require the C-terminal cAMP-binding domain but rather involves large structural rearrangements within the linker and N-terminal cyclic nucleotide-binding domain of the RIIβ homodimer. The structural rearrangements are significantly greater than seen previously with RIIα and are likely to be important in mediating short range and long range interdomain and intersubunit interactions that uniquely regulate the activity of the type IIβ isoform of PKA.« less

Isolation and characterization of the stage-specific cytochrome b small subunit (CybS) of Ascaris suum complex II from the aerobic respiratory chain of larval mitochondria.

PubMed

Amino, Hisako; Osanai, Arihiro; Miyadera, Hiroko; Shinjyo, Noriko; Tomitsuka, Eriko; Taka, Hikari; Mineki, Reiko; Murayama, Kimie; Takamiya, Shinzaburo; Aoki, Takashi; Miyoshi, Hideto; Sakamoto, Kimitoshi; Kojima, Somei; Kita, Kiyoshi

2003-05-01

We recently reported that Ascaris suum mitochondria express stage-specific isoforms of complex II: the flavoprotein subunit and the small subunit of cytochrome b (CybS) of the larval complex II differ from those of adult enzyme, while two complex IIs share a common iron-sulfur cluster subunit (Ip). In the present study, A. suum larval complex II was highly purified to characterize the larval cytochrome b subunits in more detail. Peptide mass fingerprinting and N-terminal amino acid sequencing showed that the larval and adult cytochrome b (CybL) proteins are identical. In contrast, cDNA sequences revealed that the small subunit of larval cytochrome b (CybS(L)) is distinct from the adult CybS (CybS(A)). Furthermore, Northern analysis and immunoblotting showed stage-specific expression of CybS(L) and CybS(A) in larval and adult mitochondria, respectively. Enzymatic assays revealed that the ratio of rhodoquinol-fumarate reductase (RQFR) to succinate-ubiquinone reductase (SQR) activities and the K(m) values for quinones are almost identical for the adult and larval complex IIs, but that the fumarate reductase (FRD) activity is higher for the adult form than for the larval form. These results indicate that the adult and larval A. suum complex IIs have different properties than the complex II of the mammalian host and that the larval complex II is able to function as a RQFR. Such RQFR activity of the larval complex II would be essential for rapid adaptation to the dramatic change of oxygen availability during infection of the host.

The roles of the RIIβ linker and N-terminal cyclic nucleotide-binding domain in determining the unique structures of the type IIβ protein kinase A: a small angle x-ray and neutron scattering study.

PubMed

Blumenthal, Donald K; Copps, Jeffrey; Smith-Nguyen, Eric V; Zhang, Ping; Heller, William T; Taylor, Susan S

2014-10-10

Protein kinase A (PKA) is ubiquitously expressed and is responsible for regulating many important cellular functions in response to changes in intracellular cAMP concentrations. The PKA holoenzyme is a tetramer (R2:C2), with a regulatory subunit homodimer (R2) that binds and inhibits two catalytic (C) subunits; binding of cAMP to the regulatory subunit homodimer causes activation of the catalytic subunits. Four different R subunit isoforms exist in mammalian cells, and these confer different structural features, subcellular localization, and biochemical properties upon the PKA holoenzymes they form. The holoenzyme containing RIIβ is structurally unique in that the type IIβ holoenzyme is much more compact than the free RIIβ homodimer. We have used small angle x-ray scattering and small angle neutron scattering to study the solution structure and subunit organization of a holoenzyme containing an RIIβ C-terminal deletion mutant (RIIβ(1-280)), which is missing the C-terminal cAMP-binding domain to better understand the structural organization of the type IIβ holoenzyme and the RIIβ domains that contribute to stabilizing the holoenzyme conformation. Our results demonstrate that compaction of the type IIβ holoenzyme does not require the C-terminal cAMP-binding domain but rather involves large structural rearrangements within the linker and N-terminal cyclic nucleotide-binding domain of the RIIβ homodimer. The structural rearrangements are significantly greater than seen previously with RIIα and are likely to be important in mediating short range and long range interdomain and intersubunit interactions that uniquely regulate the activity of the type IIβ isoform of PKA. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

The linkage between ribosomal crystallography, metal ions, heteropolytungstates and functional flexibility

PubMed Central

Bashan, Anat; Yonath, Ada

2009-01-01

Crystallography of ribosomes, the universal cell nucleoprotein assemblies facilitating the translation of the genetic-code into proteins, met with severe problems owing to their large size, complex structure, inherent flexibility and high conformational variability. For the case of the small ribosomal subunit, which caused extreme difficulties, post crystallization treatment by minute amounts of a heteropolytungstate cluster allowed structure determination at atomic resolution. This cluster played a dual role in ribosomal crystallography: providing anomalous phasing power and dramatically increased the resolution, by stabilization of a selected functional conformation. Thus, four out of the fourteen clusters that bind to each of the crystallized small subunits are attached to a specific ribosomal protein in a fashion that may control a significant component of the subunit internal flexibility, by “gluing” symmetrical related subunits. Here we highlight basic issues in the relationship between metal ions and macromolecules and present common traits controlling in the interactions between polymetalates and various macromolecules, which may be extended towards the exploitation of polymetalates for therapeutical treatment. PMID:19915655

Targeted discovery of glycoside hydrolases from a switchgrass-adapted compost community

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

Allgaier, M.; Reddy, A.; Park, J. I.

2009-11-15

Development of cellulosic biofuels from non-food crops is currently an area of intense research interest. Tailoring depolymerizing enzymes to particular feedstocks and pretreatment conditions is one promising avenue of research in this area. Here we added a green-waste compost inoculum to switchgrass (Panicum virgatum) and simulated thermophilic composting in a bioreactor to select for a switchgrass-adapted community and to facilitate targeted discovery of glycoside hydrolases. Small-subunit (SSU) rRNA-based community profiles revealed that the microbial community changed dramatically between the initial and switchgrass-adapted compost (SAC) with some bacterial populations being enriched over 20-fold. We obtained 225 Mbp of 454-titanium pyrosequence datamore » from the SAC community and conservatively identified 800 genes encoding glycoside hydrolase domains that were biased toward depolymerizing grass cell wall components. Of these, {approx}10% were putative cellulases mostly belonging to families GH5 and GH9. We synthesized two SAC GH9 genes with codon optimization for heterologous expression in Escherichia coli and observed activity for one on carboxymethyl cellulose. The active GH9 enzyme has a temperature optimum of 50 C and pH range of 5.5 to 8 consistent with the composting conditions applied. We demonstrate that microbial communities adapt to switchgrass decomposition using simulated composting condition and that full-length genes can be identified from complex metagenomic sequence data, synthesized and expressed resulting in active enzyme.« less

Dissecting substrate specificities of the mitochondrial AFG3L2 protease.

PubMed

Ding, Bojian; Martin, Dwight W; Rampello, Anthony J; Glynn, Steven E

2018-06-22

Human AFG3L2 is a compartmental AAA+ protease that performs ATP-fueled degradation at the matrix face of the inner mitochondrial membrane. Identifying how AFG3L2 selects substrates from the diverse complement of matrix-localized proteins is essential for understanding mitochondrial protein biogenesis and quality control. Here, we create solubilized forms of AFG3L2 to examine the enzyme's substrate specificity mechanisms. We show that conserved residues within the pre-sequence of the mitochondrial ribosomal protein, MrpL32, target the subunit to the protease for processing into a mature form. Moreover, these residues can act as a degron, delivering diverse model proteins to AFG3L2 for degradation. By determining the sequence of degra-dation products from multiple substrates using mass spectrometry, we construct a peptidase specificity pro-file that displays constrained product lengths and is dominated by the identity of the residue at the P1' posi-tion, with a strong preference for hydrophobic and small polar residues. This specificity profile is validated by examining the cleavage of both fluorogenic reporter peptides and full polypeptide substrates bearing different P1' residues. Together, these results demonstrate that AFG3L2 contains multiple modes of specificity, dis-criminating between potential substrates by recognizing accessible degron sequences, and performing peptide bond cleavage at preferred patterns of residues within the compartmental chamber.

Photosynthetic Trichomes Contain a Specific Rubisco with a Modified pH-Dependent Activity.

PubMed

Laterre, Raphaëlle; Pottier, Mathieu; Remacle, Claire; Boutry, Marc

2017-04-01

Ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) is the most abundant enzyme in plants and is responsible for CO 2 fixation during photosynthesis. This enzyme is assembled from eight large subunits (RbcL) encoded by a single chloroplast gene and eight small subunits (RbcS) encoded by a nuclear gene family. Rubisco is primarily found in the chloroplasts of mesophyll (C3 plants), bundle-sheath (C4 plants), and guard cells. In certain species, photosynthesis also takes place in the secretory cells of glandular trichomes, which are epidermal outgrowths (hairs) involved in the secretion of specialized metabolites. However, photosynthesis and, in particular, Rubisco have not been characterized in trichomes. Here, we show that tobacco ( Nicotiana tabacum ) trichomes contain a specific Rubisco small subunit, NtRbcS-T, which belongs to an uncharacterized phylogenetic cluster (T). This cluster contains RbcS from at least 33 species, including monocots, many of which are known to possess glandular trichomes. Cluster T is distinct from the cluster M, which includes the abundant, functionally characterized RbcS isoforms expressed in mesophyll or bundle-sheath cells. Expression of NtRbcS-T in Chlamydomonas reinhardtii and purification of the full Rubisco complex showed that this isoform conferred higher V max and K m values as well as higher acidic pH-dependent activity than NtRbcS-M, an isoform expressed in the mesophyll. This observation was confirmed with trichome extracts. These data show that an ancient divergence allowed for the emergence of a so-far-uncharacterized RbcS cluster. We propose that secretory trichomes have a particular Rubisco uniquely adapted to secretory cells where CO 2 is released by the active specialized metabolism. © 2017 American Society of Plant Biologists. All Rights Reserved.

Decreased surface expression of the δ subunit of the GABAA receptor contributes to reduced tonic inhibition in dentate granule cells in a mouse model of fragile X syndrome.

PubMed

Zhang, Nianhui; Peng, Zechun; Tong, Xiaoping; Lindemeyer, A Kerstin; Cetina, Yliana; Huang, Christine S; Olsen, Richard W; Otis, Thomas S; Houser, Carolyn R

2017-11-01

While numerous changes in the GABA system have been identified in models of Fragile X Syndrome (FXS), alterations in subunits of the GABA A receptors (GABA A Rs) that mediate tonic inhibition are particularly intriguing. Considering the key role of tonic inhibition in controlling neuronal excitability, reduced tonic inhibition could contribute to FXS-associated disorders such as hyperactivity, hypersensitivity, and increased seizure susceptibility. The current study has focused on the expression and function of the δ subunit of the GABA A R, a major subunit involved in tonic inhibition, in granule cells of the dentate gyrus in the Fmr1 knockout (KO) mouse model of FXS. Electrophysiological studies of dentate granule cells revealed a marked, nearly four-fold, decrease in tonic inhibition in the Fmr1 KO mice, as well as reduced effects of two δ subunit-preferring pharmacological agents, THIP and DS2, supporting the suggestion that δ subunit-containing GABA A Rs are compromised in the Fmr1 KO mice. Immunohistochemistry demonstrated a small but statistically significant decrease in δ subunit labeling in the molecular layer of the dentate gyrus in Fmr1 KO mice compared to wildtype (WT) littermates. The discrepancy between the large deficits in GABA-mediated tonic inhibition in granule cells in the Fmr1 KO mice and only modest reductions in immunolabeling of the δ subunit led to studies of surface expression of the δ subunit. Cross-linking experiments followed by Western blot analysis demonstrated a small, non-significant decrease in total δ subunit protein in the hippocampus of Fmr1 KO mice, but a four-fold decrease in surface expression of the δ subunit in these mice. No significant changes were observed in total or surface expression of the α4 subunit protein, a major partner of the δ subunit in the forebrain. Postembedding immunogold labeling for the δ subunit demonstrated a large, three-fold, decrease in the number of symmetric synapses with immunolabeling at perisynaptic locations in Fmr1 KO mice. While α4 immunogold particles were also reduced at perisynaptic locations in the Fmr1 KO mice, the labeling was increased at synaptic sites. Together these findings suggest that, in the dentate gyrus, altered surface expression of the δ subunit, rather than a decrease in δ subunit expression alone, could be limiting δ subunit-mediated tonic inhibition in this model of FXS. Finding ways to increase surface expression of the δ subunit of the GABA A R could be a novel approach to treatment of hyperexcitability-related alterations in FXS. Copyright © 2017 Elsevier Inc. All rights reserved.

Direct and Indirect Targeting of PP2A by Conserved Bacterial Type-III Effector Proteins

PubMed Central

Jin, Lin; Ham, Jong Hyun; Hage, Rosemary; Zhao, Wanying; Soto-Hernández, Jaricelis; Lee, Sang Yeol; Paek, Seung-Mann; Kim, Min Gab; Boone, Charles; Coplin, David L.; Mackey, David

2016-01-01

Bacterial AvrE-family Type-III effector proteins (T3Es) contribute significantly to the virulence of plant-pathogenic species of Pseudomonas, Pantoea, Ralstonia, Erwinia, Dickeya and Pectobacterium, with hosts ranging from monocots to dicots. However, the mode of action of AvrE-family T3Es remains enigmatic, due in large part to their toxicity when expressed in plant or yeast cells. To search for targets of WtsE, an AvrE-family T3E from the maize pathogen Pantoea stewartii subsp. stewartii, we employed a yeast-two-hybrid screen with non-lethal fragments of WtsE and a synthetic genetic array with full-length WtsE. Together these screens indicate that WtsE targets maize protein phosphatase 2A (PP2A) heterotrimeric enzyme complexes via direct interaction with B’ regulatory subunits. AvrE1, another AvrE-family T3E from Pseudomonas syringae pv. tomato strain DC3000 (Pto DC3000), associates with specific PP2A B’ subunit proteins from its susceptible host Arabidopsis that are homologous to the maize B’ subunits shown to interact with WtsE. Additionally, AvrE1 was observed to associate with the WtsE-interacting maize proteins, indicating that PP2A B’ subunits are likely conserved targets of AvrE-family T3Es. Notably, the ability of AvrE1 to promote bacterial growth and/or suppress callose deposition was compromised in Arabidopsis plants with mutations of PP2A genes. Also, chemical inhibition of PP2A activity blocked the virulence activity of both WtsE and AvrE1 in planta. The function of HopM1, a Pto DC3000 T3E that is functionally redundant to AvrE1, was also impaired in specific PP2A mutant lines, although no direct interaction with B’ subunits was observed. These results indicate that sub-component specific PP2A complexes are targeted by bacterial T3Es, including direct targeting by members of the widely conserved AvrE-family. PMID:27191168

The role of GluN2A and GluN2B NMDA receptor subunits in AgRP and POMC neurons on body weight and glucose homeostasis.

PubMed

Üner, Aykut; Gonçalves, Gabriel H M; Li, Wenjing; Porceban, Matheus; Caron, Nicole; Schönke, Milena; Delpire, Eric; Sakimura, Kenji; Bjørbæk, Christian

2015-10-01

Hypothalamic agouti-related peptide (AgRP) and pro-opiomelanocortin (POMC) expressing neurons play critical roles in control of energy balance. Glutamatergic input via n-methyl-d-aspartate receptors (NMDARs) is pivotal for regulation of neuronal activity and is required in AgRP neurons for normal body weight homeostasis. NMDARs typically consist of the obligatory GluN1 subunit and different GluN2 subunits, the latter exerting crucial differential effects on channel activity and neuronal function. Currently, the role of specific GluN2 subunits in AgRP and POMC neurons on whole body energy and glucose balance is unknown. We used the cre-lox system to genetically delete GluN2A or GluN2B only from AgRP or POMC neurons in mice. Mice were then subjected to metabolic analyses and assessment of AgRP and POMC neuronal function through morphological studies. We show that loss of GluN2B from AgRP neurons reduces body weight, fat mass, and food intake, whereas GluN2B in POMC neurons is not required for normal energy balance control. GluN2A subunits in either AgRP or POMC neurons are not required for regulation of body weight. Deletion of GluN2B reduces the number of AgRP neurons and decreases their dendritic length. In addition, loss of GluN2B in AgRP neurons of the morbidly obese and severely diabetic leptin-deficient Lep (ob/ob) mice does not affect body weight and food intake but, remarkably, leads to full correction of hyperglycemia. Lep (ob/ob) mice lacking GluN2B in AgRP neurons are also more sensitive to leptin's anti-obesity actions. GluN2B-containing NMDA receptors in AgRP neurons play a critical role in central control of body weight homeostasis and blood glucose balance via mechanisms that likely involve regulation of AgRP neuronal survival and structure, and modulation of hypothalamic leptin action.

Small-angle scattering study of Aspergillus awamori glycoprotein glucoamylase

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

Schmidt, A. E., E-mail: schmidt@omrb.pnpi.spb.ru; Shvetsov, A. V.; Kuklin, A. I.

2016-01-15

Glucoamylase from fungus Aspergillus awamori is glycoside hydrolase that catalyzes the hydrolysis of α-1,4- and α-1,6-glucosidic bonds in glucose polymers and oligomers. This glycoprotein consists of a catalytic domain and a starch-binding domain connected by an O-glycosylated polypeptide chain. The conformation of the linker, the relative arrangement of the domains, and the structure of the full-length enzyme are unknown. The structure of the recombinant glucoamylase GA1 was studied by molecular modelling and small-angle neutron scattering (SANS) methods. The experimental SANS data provide evidence that glucoamylase exists as a monomer in solution and contains a glycoside component, which makes a substantialmore » contribution to the scattering. The model of full-length glucoamylase, which was calculated without taking into account the effect of glycosylation, is consistent with the experimental data and has a radius of gyration of 33.4 ± 0.6 Å.« less

Cytochrome b in human complex II (succinate-ubiquinone oxidoreductase): cDNA cloning of the components in liver mitochondria and chromosome assignment of the genes for the large (SDHC) and small (SDHD) subunits to 1q21 and 11q23.

PubMed

Hirawake, H; Taniwaki, M; Tamura, A; Kojima, S; Kita, K

1997-01-01

Complex II (succinate-ubiquinone oxidoreductase) is an important enzyme complex in both the tricarboxylic acid cycle and the aerobic respiratory chains of mitochondria in eukaryotic cells and prokaryotic organisms. In this study, the amino acid sequences of the large (cybL) and small (cybS) subunits of cytochrome b in human liver complex II were deduced from cDNAs isolated by homology probing with mixed primers for the polymerase chain reaction. The mature cybL and cybS contain 140 and 103 amino acids, respectively, and show little similarity to the amino acid sequences of the subunits from other species in contrast to the highly conserved features of the flavoprotein (Fp) subunit and iron-sulfur protein (Ip) subunit. From hydrophobicity analysis, both cybL and cybS appear to have three transmembrane segments, indicating their role as membrane-anchors for the enzyme complex. Histidine residues, which are possible heme axial ligands in cytochrome b of complex II, were found in the second transmembrane segment of each subunit. The genes for cybL (SDHC) and cybS (SDHD) were mapped to chromosome 1q21 and 11q23, respectively by fluorescent in situ hybridization (FISH).

Assessing subunit dependency of the Plasmodium proteasome using small molecule inhibitors and active site probes.

PubMed

Li, Hao; van der Linden, Wouter A; Verdoes, Martijn; Florea, Bogdan I; McAllister, Fiona E; Govindaswamy, Kavitha; Elias, Joshua E; Bhanot, Purnima; Overkleeft, Herman S; Bogyo, Matthew

2014-08-15

The ubiquitin-proteasome system (UPS) is a potential pathway for therapeutic intervention for pathogens such as Plasmodium, the causative agent of malaria. However, due to the essential nature of this proteolytic pathway, proteasome inhibitors must avoid inhibition of the host enzyme complex to prevent toxic side effects. The Plasmodium proteasome is poorly characterized, making rational design of inhibitors that induce selective parasite killing difficult. In this study, we developed a chemical probe that labels all catalytic sites of the Plasmodium proteasome. Using this probe, we identified several subunit selective small molecule inhibitors of the parasite enzyme complex. Treatment with an inhibitor that is specific for the β5 subunit during blood stage schizogony led to a dramatic decrease in parasite replication while short-term inhibition of the β2 subunit did not affect viability. Interestingly, coinhibition of both the β2 and β5 catalytic subunits resulted in enhanced parasite killing at all stages of the blood stage life cycle and reduced parasite levels in vivo to barely detectable levels. Parasite killing was achieved with overall low host toxicity, something that has not been possible with existing proteasome inhibitors. Our results highlight differences in the subunit dependency of the parasite and human proteasome, thus providing a strategy for development of potent antimalarial drugs with overall low host toxicity.

Regulation of Cilium Length and Intraflagellar Transport by the RCK-Kinases ICK and MOK in Renal Epithelial Cells

PubMed Central

Broekhuis, Joost R.; Verhey, Kristen J.; Jansen, Gert

2014-01-01

Primary cilia are important sensory organelles. They exist in a wide variety of lengths, which could reflect different cell-specific functions. How cilium length is regulated is unclear, but it probably involves intraflagellar transport (IFT), which transports protein complexes along the ciliary axoneme. Studies in various organisms have identified the small, conserved family of ros-cross hybridizing kinases (RCK) as regulators of cilium length. Here we show that Intestinal Cell Kinase (ICK) and MAPK/MAK/MRK overlapping kinase (MOK), two members of this family, localize to cilia of mouse renal epithelial (IMCD-3) cells and negatively regulate cilium length. To analyze the effects of ICK and MOK on the IFT machinery, we set up live imaging of five fluorescently tagged IFT proteins: KIF3B, a subunit of kinesin-II, the main anterograde IFT motor, complex A protein IFT43, complex B protein IFT20, BBSome protein BBS8 and homodimeric kinesin KIF17, whose function in mammalian cilia is unclear. Interestingly, all five proteins moved at ∼0.45 µm/s in anterograde and retrograde direction, suggesting they are all transported by the same machinery. Moreover, GFP tagged ICK and MOK moved at similar velocities as the IFT proteins, suggesting they are part of, or transported by the IFT machinery. Indeed, loss- or gain-of-function of ICK affected IFT speeds: knockdown increased anterograde velocities, whereas overexpression reduced retrograde speed. In contrast, MOK knockdown or overexpression did not affect IFT speeds. Finally, we found that the effects of ICK or MOK knockdown on cilium length and IFT are suppressed by rapamycin treatment, suggesting that these effects require the mTORC1 pathway. Our results confirm the importance of RCK kinases as regulators of cilium length and IFT. However, whereas some of our results suggest a direct correlation between cilium length and IFT speed, other results indicate that cilium length can be modulated independent of IFT speed. PMID:25243405

Sequence and functional characterization of hypoxia-inducible factors, HIF1α, HIF2αa, and HIF3α, from the estuarine fish, Fundulus heteroclitus

PubMed Central

Townley, Ian K.; Karchner, Sibel I.; Skripnikova, Elena; Wiese, Thomas E.; Hahn, Mark E.

2017-01-01

The hypoxia-inducible factor (HIF) family of transcription factors plays central roles in the development, physiology, pathology, and environmental adaptation of animals. Because many aquatic habitats are characterized by episodes of low dissolved oxygen, fish represent ideal models to study the roles of HIF in the response to aquatic hypoxia. The estuarine fish Fundulus heteroclitus is found in habitats prone to hypoxia. It responds to low oxygen via behavioral, physiological, and molecular changes, and one member of the HIF family, HIF2α, has been previously described. Herein, cDNA sequencing, phylogenetic analyses, and genomic approaches were used to determine other members of the HIFα family from F. heteroclitus and their relationships to HIFα subunits from other vertebrates. In vitro and cellular approaches demonstrated that full-length forms of HIF1α, HIF2α, and HIF3α independently formed complexes with the β-subunit, aryl hydrocarbon receptor nuclear translocator, to bind to hypoxia response elements and activate reporter gene expression. Quantitative PCR showed that HIFα mRNA abundance varied among organs of normoxic fish in an isoform-specific fashion. Analysis of the F. heteroclitus genome revealed a locus encoding a second HIF2α—HIF2αb—a predicted protein lacking oxygen sensing and transactivation domains. Finally, sequence analyses demonstrated polymorphism in the coding sequence of each F. heteroclitus HIFα subunit, suggesting that genetic variation in these transcription factors may play a role in the variation in hypoxia responses among individuals or populations. PMID:28039194

Sequence and functional characterization of hypoxia-inducible factors, HIF1α, HIF2αa, and HIF3α, from the estuarine fish, Fundulus heteroclitus.

PubMed

Townley, Ian K; Karchner, Sibel I; Skripnikova, Elena; Wiese, Thomas E; Hahn, Mark E; Rees, Bernard B

2017-03-01

The hypoxia-inducible factor (HIF) family of transcription factors plays central roles in the development, physiology, pathology, and environmental adaptation of animals. Because many aquatic habitats are characterized by episodes of low dissolved oxygen, fish represent ideal models to study the roles of HIF in the response to aquatic hypoxia. The estuarine fish Fundulus heteroclitus is found in habitats prone to hypoxia. It responds to low oxygen via behavioral, physiological, and molecular changes, and one member of the HIF family, HIF2α, has been previously described. Herein, cDNA sequencing, phylogenetic analyses, and genomic approaches were used to determine other members of the HIFα family from F. heteroclitus and their relationships to HIFα subunits from other vertebrates. In vitro and cellular approaches demonstrated that full-length forms of HIF1α, HIF2α, and HIF3α independently formed complexes with the β-subunit, aryl hydrocarbon receptor nuclear translocator, to bind to hypoxia response elements and activate reporter gene expression. Quantitative PCR showed that HIFα mRNA abundance varied among organs of normoxic fish in an isoform-specific fashion. Analysis of the F. heteroclitus genome revealed a locus encoding a second HIF2α-HIF2αb-a predicted protein lacking oxygen sensing and transactivation domains. Finally, sequence analyses demonstrated polymorphism in the coding sequence of each F. heteroclitus HIFα subunit, suggesting that genetic variation in these transcription factors may play a role in the variation in hypoxia responses among individuals or populations. Copyright © 2017 the American Physiological Society.

Physical and functional interactions of Caenorhabditis elegans WRN-1 helicase with RPA-1.

PubMed

Hyun, Moonjung; Park, Sojin; Kim, Eunsun; Kim, Do-Hyung; Lee, Se-Jin; Koo, Hyeon-Sook; Seo, Yeon-Soo; Ahn, Byungchan

2012-02-21

The Caenorhabditis elegans Werner syndrome protein, WRN-1, a member of the RecQ helicase family, has a 3'-5' DNA helicase activity. Worms with defective wrn-1 exhibit premature aging phenotypes and an increased level of genome instability. In response to DNA damage, WRN-1 participates in the initial stages of checkpoint activation in concert with C. elegans replication protein A (RPA-1). WRN-1 helicase is stimulated by RPA-1 on long DNA duplex substrates. However, the mechanism by which RPA-1 stimulates DNA unwinding and the function of the WRN-1-RPA-1 interaction are not clearly understood. We have found that WRN-1 physically interacts with two RPA-1 subunits, CeRPA73 and CeRPA32; however, full-length WRN-1 helicase activity is stimulated by only the CeRPA73 subunit, while the WRN-1(162-1056) fragment that harbors the helicase activity requires both the CeRPA73 and CeRPA32 subunits for the stimulation. We also found that the CeRPA73(1-464) fragment can stimulate WRN-1 helicase activity and that residues 335-464 of CeRPA73 are important for physical interaction with WRN-1. Because CeRPA73 and the CeRPA73(1-464) fragment are able to bind single-stranded DNA (ssDNA), the stimulation of WRN-1 helicase by RPA-1 is most likely due to the ssDNA binding activity of CeRPA73 and the direct interaction of WRN-1 and CeRPA73.

Assessing self-care and social function using a computer adaptive testing version of the Pediatric Evaluation of Disability Inventory Accepted for Publication, Archives of Physical Medicine and Rehabilitation

PubMed Central

Coster, Wendy J.; Haley, Stephen M.; Ni, Pengsheng; Dumas, Helene M.; Fragala-Pinkham, Maria A.

2009-01-01

Objective To examine score agreement, validity, precision, and response burden of a prototype computer adaptive testing (CAT) version of the Self-Care and Social Function scales of the Pediatric Evaluation of Disability Inventory (PEDI) compared to the full-length version of these scales. Design Computer simulation analysis of cross-sectional and longitudinal retrospective data; cross-sectional prospective study. Settings Pediatric rehabilitation hospital, including inpatient acute rehabilitation, day school program, outpatient clinics; community-based day care, preschool, and children’s homes. Participants Four hundred sixty-nine children with disabilities and 412 children with no disabilities (analytic sample); 38 children with disabilities and 35 children without disabilities (cross-validation sample). Interventions Not applicable. Main Outcome Measures Summary scores from prototype CAT applications of each scale using 15-, 10-, and 5-item stopping rules; scores from the full-length Self-Care and Social Function scales; time (in seconds) to complete assessments and respondent ratings of burden. Results Scores from both computer simulations and field administration of the prototype CATs were highly consistent with scores from full-length administration (all r’s between .94 and .99). Using computer simulation of retrospective data, discriminant validity and sensitivity to change of the CATs closely approximated that of the full-length scales, especially when the 15- and 10-item stopping rules were applied. In the cross-validation study the time to administer both CATs was 4 minutes, compared to over 16 minutes to complete the full-length scales. Conclusions Self-care and Social Function score estimates from CAT administration are highly comparable to those obtained from full-length scale administration, with small losses in validity and precision and substantial decreases in administration time. PMID:18373991

Pharmacological efficacy of anti-IL-1β scFv, Fab and full-length antibodies in treatment of rheumatoid arthritis.

PubMed

Qi, Jianying; Ye, Xianlong; Ren, Guiping; Kan, Fangming; Zhang, Yu; Guo, Mo; Zhang, Zhiyi; Li, Deshan

2014-02-01

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease that mainly causes the synovial joint inflammation and cartilage destruction. Interleukin-1β (IL-1β) is an important proinflammatory cytokine involved in the pathogenesis of RA. In this study, we constructed and expressed anti-IL-1β-full-length antibody in CHO-K1-SV, anti-IL-1β-Fab and anti-IL-1β-scFv in Rosetta. We compared the therapeutic efficacy of three anti-IL-1β antibodies for CIA mice. Mice with CIA were subcutaneously injected with humanized anti-IL-1β-scFv, anti-IL-1β-Fab or anti-IL-1β-full-length antibody. The effects of treatment were determined by arthritis severity score, autoreactive humoral, cellular immune responses, histological lesion and cytokines production. Compared with anti-IL-1β-scFv treatments, anti-IL-1β-Fab and anti-IL-1β-full-length antibody therapy resulted in more significant effect in alleviating the severity of arthritis by preventing bone damage and cartilage destruction, reducing humoral and cellular immune responses, and down-regulating the expression of IL-1β, IL-6, IL-2, IFN-γ, TNF-α and MMP-3 in inflammatory tissue. The therapeutic effects of anti-IL-1β-Fab and anti-IL-1β-full-length antibodies on CIA mice had no significant difference. However, production of anti-IL-1β-full-length antibody in eukaryotic system is, in general, time-consuming and more expensive than that of anti-IL-1β-Fab in prokaryotic systems. In conclusion, as a small molecule antibody, anti-IL-1β-Fab is an ideal candidate for RA therapy. Copyright © 2013 Elsevier Ltd. All rights reserved.

Development of Small-molecule HIV Entry Inhibitors Specifically Targeting gp120 or gp41

PubMed Central

Lu, Lu; Yu, Fei; Cai, Lifeng; Debnath, Asim K.; Jiang, Shibo

2015-01-01

Human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein surface subunit gp120 and transmembrane subunit gp41 play important roles in HIV-1 entry, thus serving as key targets for the development of HIV-1 entry inhibitors. T20 peptide (enfuvirtide) is the first U.S. FDA-approved HIV entry inhibitor; however, its clinical application is limited by the lack of oral availability. Here, we have described the structure and function of the HIV-1 gp120 and gp41 subunits and reviewed advancements in the development of small-molecule HIV entry inhibitors specifically targeting these two Env glycoproteins. We then compared the advantages and disadvantages of different categories of HIV entry inhibitor candidates and further predicted the future trend of HIV entry inhibitor development. PMID:26324044

Implications of High Molecular Divergence of Nuclear rRNA and Phylogenetic Structure for the Dinoflagellate Prorocentrum (Dinophyceae, Prorocentrales).

PubMed

Boopathi, Thangavelu; Faria, Daphne Georgina; Cheon, Ju-Yong; Youn, Seok Hyun; Ki, Jang-Seu

2015-01-01

The small and large nuclear subunit molecular phylogeny of the genus Prorocentrum demonstrated that the species are dichotomized into two clades. These two clades were significantly different (one-factor ANOVA, p < 0.01) with patterns compatible for both small and large subunit Bayesian phylogenetic trees, and for a larger taxon sampled dinoflagellate phylogeny. Evaluation of the molecular divergence levels showed that intraspecies genetic variations were significantly low (t-test, p < 0.05), than those for interspecies variations (> 2.9% and > 26.8% dissimilarity in the small and large subunit [D1/D2], respectively). Based on the calculated molecular divergence, the genus comprises two genetically distinct groups that should be considered as two separate genera, thereby setting the pace for major systematic changes for the genus Prorocentrum sensu Dodge. Moreover, the information presented in this study would be useful for improving species identification, detection of novel clades from environmental samples. © 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists.

The Stability of the Small Nucleolar Ribonucleoprotein (snoRNP) Assembly Protein Pih1 in Saccharomyces cerevisiae Is Modulated by Its C Terminus*

PubMed Central

Paci, Alexandr; Liu, Xiao Hu; Huang, Hao; Lim, Abelyn; Houry, Walid A.; Zhao, Rongmin

2012-01-01

Pih1 is an unstable protein and a subunit of the R2TP complex that, in yeast Saccharomyces cerevisiae, also contains the helicases Rvb1, Rvb2, and the Hsp90 cofactor Tah1. Pih1 and the R2TP complex are required for the box C/D small nucleolar ribonucleoprotein (snoRNP) assembly and ribosomal RNA processing. Purified Pih1 tends to aggregate in vitro. Molecular chaperone Hsp90 and its cochaperone Tah1 are required for the stability of Pih1 in vivo. We had shown earlier that the C terminus of Pih1 destabilizes the protein and that the C terminus of Tah1 binds to the Pih1 C terminus to form a stable complex. Here, we analyzed the secondary structure of the Pih1 C terminus and identified two intrinsically disordered regions and five hydrophobic clusters. Site-directed mutagenesis indicated that one predicted intrinsically disordered region IDR2 is involved in Tah1 binding, and that the C terminus of Pih1 contains multiple destabilization or degron elements. Additionally, the Pih1 N-terminal domain, Pih11–230, was found to be able to complement the physiological role of full-length Pih1 at 37 °C. Pih11–230 as well as a shorter Pih1 N-terminal fragment Pih11–195 is able to bind Rvb1/Rvb2 heterocomplex. However, the sequence between the two disordered regions in Pih1 significantly enhances the Pih1 N-terminal domain binding to Rvb1/Rvb2. Based on these data, a model of protein-protein interactions within the R2TP complex is proposed. PMID:23139418

PCR amplification and sequences of cDNA clones for the small and large subunits of ADP-glucose pyrophosphorylase from barley tissues.

PubMed

Villand, P; Aalen, R; Olsen, O A; Lüthi, E; Lönneborg, A; Kleczkowski, L A

1992-06-01

Several cDNAs encoding the small and large subunit of ADP-glucose pyrophosphorylase (AGP) were isolated from total RNA of the starchy endosperm, roots and leaves of barley by polymerase chain reaction (PCR). Sets of degenerate oligonucleotide primers, based on previously published conserved amino acid sequences of plant AGP, were used for synthesis and amplification of the cDNAs. For either the endosperm, roots and leaves, the restriction analysis of PCR products (ca. 550 nucleotides each) has revealed heterogeneity, suggesting presence of three transcripts for AGP in the endosperm and roots, and up to two AGP transcripts in the leaf tissue. Based on the derived amino acid sequences, two clones from the endosperm, beps and bepl, were identified as coding for the small and large subunit of AGP, respectively, while a leaf transcript (blpl) encoded the putative large subunit of AGP. There was about 50% identity between the endosperm clones, and both of them were about 60% identical to the leaf cDNA. Northern blot analysis has indicated that beps and bepl are expressed in both the endosperm and roots, while blpl is detectable only in leaves. Application of the PCR technique in studies on gene structure and gene expression of plant AGP is discussed.

Functional conservation of RNA polymerase II in fission and budding yeasts.

PubMed

Shpakovski, G V; Gadal, O; Labarre-Mariotte, S; Lebedenko, E N; Miklos, I; Sakurai, H; Proshkin, S A; Van Mullem, V; Ishihama, A; Thuriaux, P

2000-02-04

The complementary DNAs of the 12 subunits of fission yeast (Schizosaccharomyces pombe) RNA polymerase II were expressed from strong promoters in Saccharomyces cerevisiae and tested for heterospecific complementation by monitoring their ability to replace in vivo the null mutants of the corresponding host genes. Rpb1 and Rpb2, the two largest subunits and Rpb8, a small subunit shared by all three polymerases, failed to support growth in S. cerevisiae. The remaining nine subunits were all proficient for heterospecific complementation and led in most cases to a wild-type level of growth. The two alpha-like subunits (Rpb3 and Rpb11), however, did not support growth at high (37 degrees C) or low (25 degrees C) temperatures. In the case of Rpb3, growth was restored by increasing the gene dosage of the host Rpb11 or Rpb10 subunits, confirming previous evidence of a close genetic interaction between these three subunits. Copyright 2000 Academic Press.

Inhibition of herpesvirus and influenza virus replication by blocking polymerase subunit interactions.

PubMed

Palù, Giorgio; Loregian, Arianna

2013-09-01

Protein-protein interactions (PPIs) play a key role in many biological processes, including virus replication in the host cell. Since most of the PPIs are functionally essential, a possible strategy to inhibit virus replication is based on the disruption of viral protein complexes by peptides or small molecules that interfere with subunit interactions. In particular, an attractive target for antiviral drugs is the binding between the subunits of essential viral enzymes. This review describes the development of new antiviral compounds that inhibit herpesvirus and influenza virus replication by blocking interactions between subunit proteins of their polymerase complexes. Copyright © 2013 Elsevier B.V. All rights reserved.

Role of Small Subunit in Mediating Assembly of Red-type Form I Rubisco

PubMed Central

Joshi, Jidnyasa; Mueller-Cajar, Oliver; Tsai, Yi-Chin C.; Hartl, F. Ulrich; Hayer-Hartl, Manajit

2015-01-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the key enzyme involved in photosynthetic carbon fixation, converting atmospheric CO2 to organic compounds. Form I Rubisco is a cylindrical complex composed of eight large (RbcL) subunits that are capped by four small subunits (RbcS) at the top and four at the bottom. Form I Rubiscos are phylogenetically divided into green- and red-type. Some red-type enzymes have catalytically superior properties. Thus, understanding their folding and assembly is of considerable biotechnological interest. Folding of the green-type RbcL subunits in cyanobacteria is mediated by the GroEL/ES chaperonin system, and assembly to holoenzyme requires specialized chaperones such as RbcX and RAF1. Here, we show that the red-type RbcL subunits in the proteobacterium Rhodobacter sphaeroides also fold with GroEL/ES. However, assembly proceeds in a chaperone-independent manner. We find that the C-terminal β-hairpin extension of red-type RbcS, which is absent in green-type RbcS, is critical for efficient assembly. The β-hairpins of four RbcS subunits form an eight-stranded β-barrel that protrudes into the central solvent channel of the RbcL core complex. The two β-barrels stabilize the complex through multiple interactions with the RbcL subunits. A chimeric green-type RbcS carrying the C-terminal β-hairpin renders the assembly of a cyanobacterial Rubisco independent of RbcX. Our results may facilitate the engineering of crop plants with improved growth properties expressing red-type Rubisco. PMID:25371207

Molecular phylogeny of Laetiporus and other brown rot polypore genera in North America

Treesearch

Daniel L. Lindner; Mark T. Banik

2008-01-01

Phylogenetic relationships were investigated among North American species of Laetiporus, Leptoporus, Phaeolus, Pycnoporellus, and Wolfiporia using ITS, nuclear large subunit and mitochondrial small subunit rDNA sequences. Members of these genera have poroid hymenophores, simple septate hyphae and cause brown rots in a variety of...

Development of a PCR-restriction fragment length polymorphism protocol for rapid detection and differentiation of four cockroach vectors (group I "Dirty 22" species) responsible for food contamination and spreading of foodborne pathogens: public health importance.

PubMed

Sulaiman, Irshad M; Anderson, Mickey; Khristova, Marina; Tang, Kevin; Sulaiman, Nikhat; Phifer, Edwin; Simpson, Steven; Kerdahi, Khalil

2011-11-01

Assessing the adulteration of food products and the presence of filth and extraneous materials is one of the measures that the U.S. Food and Drug Administration (FDA) utilizes in implementing regulatory actions of public health importance. To date, 22 common pest species (also known as the "Dirty 22" species) have been regarded by this agency as the spreaders of foodborne diseases. We have further categorized the Dirty 22 species into four groups: I has four cockroach species, II has two ant species, III has 12 fly species, and IV has four rodent species. The presence of any Dirty 22 species is also considered an indicator of unsanitary conditions in food processing and storage facilities. In this study, we describe the development of a two-step nested PCR protocol to amplify the small subunit ribosomal gene of group I Dirty 22 species that include four cockroach species: Blattella germanica, Blatta orientalis, Periplaneta americana, and Supella longipalpa, along with the development of a PCR-restriction fragment length polymorphism method for rapid detection and differentiation of these violative species. This method will be utilized when the specimen cannot be identified with conventional microscopic taxonomic methods, especially when only small body parts are separated and recovered from food samples for analysis or when these body parts are in a decomposed state. This new PCR-restriction fragment length polymorphism will provide correct identification of group I Dirty 22 species; this information can then be used in regulation and prevention of foodborne pathogens.

Sequence and properties of HMW subunit 1Bx20 from pasta wheat (Triticum durum) which is associated with poor end use properties.

PubMed

Shewry, P R; Gilbert, S M; Savage, A W J; Tatham, A S; Wan, Y-F; Belton, P S; Wellner, N; D'Ovidio, R; Békés, F; Halford, N G

2003-02-01

The gene encoding high-molecular-weight (HMW) subunit 1Bx20 was isolated from durum wheat cv. Lira. It encodes a mature protein of 774 amino acid residues with an M(r) of 83,913. Comparison with the sequence of subunit 1Bx7 showed over 96% identity, the main difference being the substitution of two cysteine residues in the N-terminal domain of subunit 1Bx7 with tyrosine residues in 1Bx20. Comparison of the structures and stabilities of the two subunits purified from wheat using Fourier-transform infra-red and circular dichroism spectroscopy showed no significant differences. However, incorporation of subunit 1Bx7 into a base flour gave increased dough strength and stability measured by Mixograph analysis, while incorporation of subunit 1Bx20 resulted in small positive or negative effects on the parameters measured. It is concluded that the different effects of the two subunits could relate to the differences in their cysteine contents, thereby affecting the cross-linking and hence properties of the glutenin polymers.

Molecular cloning of the alpha subunit of complement component C8 (CpC8α) of whitespotted bamboo shark (Chiloscyllium plagiosum).

PubMed

Wang, Ying; Zhang, Mengmeng; Wang, Conghui; Ye, Boping; Hua, Zichun

2013-12-01

Complement-mediated cytolysis is the important effect of immune response, which results from the assembly of terminal complement components (C5b-9). Among them, α subunit of C8 (C8α) is the first protein that traverses the lipid bilayer, and then initiates the recruitment of C9 molecules to form pore on target membranes. In this article, a full-length cDNA of C8α (CpC8α) is identified from the whitespotted bamboo shark (Chiloscyllium plagiosum) by RACE. The CpC8α cDNA is 2183 bp in length, encoding a protein of 591 amino acids. The deduced CpC8α exhibits 89%, 49% and 44% identity with nurse shark, frog and human orthologs, respectively. Sequence alignment indicates that the C8α is well conserved during the evolution process from sharks to mammals, with the same modular architecture as well as the identical cysteine composition in the mature protein. Phylogenetic analysis places CpC8α and nurse shark C8α in cartilaginous fish clade, in parallel with the teleost taxa, to form the C8α cluster with higher vertebrates. Hydrophobicity analysis also indicates a similar hydrophobicity of CpC8α to mammals. Finally, expression analysis revealed CpC8α transcripts were constitutively highly expressed in shark liver, with much less expression in other tissues. The well conserved structure and properties suggests an analogous function of CpC8α to mammalian C8α, though it remains to be confirmed by further study. Copyright © 2013 Elsevier Ltd. All rights reserved.

Function of Protein Phosphatase 2A in Control of Proliferation: Isolation and Analysis of Dominant-Defective Mutants

DTIC Science & Technology

1999-06-01

subunits are expressed ubiquitously and appear to be encoded by small and quite homogeneous gene families. In plants , however, A and C subunit gene...1996). In both plants and animals, different B subunit isoforms are encoded by two or more unrelated gene families, some of which are expressed in a...PP2A functions in whole plants and in mammalian tissue culture cells. This genetic system may also prove useful for analyzing interactions between

Structural analysis of the α subunit of Na(+)/K(+) ATPase genes in invertebrates.

PubMed

Thabet, Rahma; Rouault, J-D; Ayadi, Habib; Leignel, Vincent

2016-01-01

The Na(+)/K(+) ATPase is a ubiquitous pump coordinating the transport of Na(+) and K(+) across the membrane of cells and its role is fundamental to cellular functions. It is heteromer in eukaryotes including two or three subunits (α, β and γ which is specific to the vertebrates). The catalytic functions of the enzyme have been attributed to the α subunit. Several complete α protein sequences are available, but only few gene structures were characterized. We identified the genomic sequences coding the α-subunit of the Na(+)/K(+) ATPase, from the whole-genome shotgun contigs (WGS), NCBI Genomes (chromosome), Genomic Survey Sequences (GSS) and High Throughput Genomic Sequences (HTGS) databases across distinct phyla. One copy of the α subunit gene was found in Annelida, Arthropoda, Cnidaria, Echinodermata, Hemichordata, Mollusca, Placozoa, Porifera, Platyhelminthes, Urochordata, but the nematodes seem to possess 2 to 4 copies. The number of introns varied from 0 (Platyhelminthes) to 26 (Porifera); and their localization and length are also highly variable. Molecular phylogenies (Maximum Likelihood and Maximum Parsimony methods) showed some clusters constituted by (Chordata/(Echinodermata/Hemichordata)) or (Plathelminthes/(Annelida/Mollusca)) and a basal position for Porifera. These structural analyses increase our knowledge about the evolutionary events of the α subunit genes in the invertebrates. Copyright © 2016 Elsevier Inc. All rights reserved.

Subunit mass fingerprinting of mitochondrial complex I.

PubMed

Morgner, Nina; Zickermann, Volker; Kerscher, Stefan; Wittig, Ilka; Abdrakhmanova, Albina; Barth, Hans-Dieter; Brutschy, Bernhard; Brandt, Ulrich

2008-10-01

We have employed laser induced liquid bead ion desorption (LILBID) mass spectrometry to determine the total mass and to study the subunit composition of respiratory chain complex I from Yarrowia lipolytica. Using 5-10 pmol of purified complex I, we could assign all 40 known subunits of this membrane bound multiprotein complex to peaks in LILBID subunit fingerprint spectra by comparing predicted protein masses to observed ion masses. Notably, even the highly hydrophobic subunits encoded by the mitochondrial genome were easily detectable. Moreover, the LILBID approach allowed us to spot and correct several errors in the genome-derived protein sequences of complex I subunits. Typically, the masses of the individual subunits as determined by LILBID mass spectrometry were within 100 Da of the predicted values. For the first time, we demonstrate that LILBID spectrometry can be successfully applied to a complex I band eluted from a blue-native polyacrylamide gel, making small amounts of large multiprotein complexes accessible for subunit mass fingerprint analysis even if they are membrane bound. Thus, the LILBID subunit mass fingerprint method will be of great value for efficient proteomic analysis of complex I and its assembly intermediates, as well as of other water soluble and membrane bound multiprotein complexes.

Structural and functional aspects of hetero-oligomers formed by the small heat shock proteins αB-crystallin and HSP27.

PubMed

Aquilina, J Andrew; Shrestha, Sudichhya; Morris, Amie M; Ecroyd, Heath

2013-05-10

αB-crystallin and HSP27 are mammalian intracellular small heat shock proteins. These proteins exchange subunits in a rapid and temperature-dependent manner. This facile subunit exchange suggests that differential expression could be used by the cell to regulate the response to stress. A robust technique defines parameters for the dynamic interaction between the major mammalian small heat shock proteins. Small heat shock proteins (sHSPs) exist as large polydisperse species in which there is constant dynamic subunit exchange between oligomeric and dissociated forms. Their primary role in vivo is to bind destabilized proteins and prevent their misfolding and aggregation. αB-Crystallin (αB) and HSP27 are the two most widely distributed and most studied sHSPs in the human body. They are coexpressed in different tissues, where they are known to associate with each other to form hetero-oligomeric complexes. In this study, we aimed to determine how these two sHSPs interact to form hetero-oligomers in vitro and whether, by doing so, there is an increase in their chaperone activity and stability compared with their homo-oligomeric forms. Our results demonstrate that HSP27 and αB formed polydisperse hetero-oligomers in vitro, which had an average molecular mass that was intermediate of each of the homo-oligomers and which were more thermostable than αB, but less so than HSP27. The hetero-oligomer chaperone function was found to be equivalent to that of αB, with each being significantly better in preventing the amorphous aggregation of α-lactalbumin and the amyloid fibril formation of α-synuclein in comparison with HSP27. Using mass spectrometry to monitor subunit exchange over time, we found that HSP27 and αB exchanged subunits 23% faster than the reported rate for HSP27 and αA and almost twice that for αA and αB. This represents the first quantitative evaluation of αB/HSP27 subunit exchange, and the results are discussed in the broader context of regulation of function and cellular proteostasis.

Analysis and design of coaxial three-mirror anastigmat with long effective focal length and full two-dimensional field

NASA Astrophysics Data System (ADS)

Lin, Han; Baoqi, Mao; Wen, Sun; Weimin, Shen

2016-10-01

There is a race to develop spaceborne high-resolution video cameras since Skybox's success. For low manufacture cost and adaption to micro and small satellites, it is urgent to design and develop compact long focal length optical system with not only small volume, light weight and easy implementation, and also two dimensional field. Our focus is on the Coaxial Three-Mirror Anastigmat (CTMA) with intermediate real image for its no need outer hood and compactness and for its easy alignment, low-order aspheric surface and low cost. The means to deflect its image space beam for accessibility of focal plane array detector and to eliminate its inherent secondary obscuration from its primary mirror central hole and deflection flat mirror is discussed. The conditions to satisfy the above-mentioned requirements are presented with our derived relationship among its optical and structural parameters based on Gaussian optics and geometry. One flat mirror near its exit pupil can be used to deflect its image plane from its axis. And its total length can be decreased with other some flat mirrors. Method for determination of its initial structure with the derived formulae is described through one design example. Furthermore, optimized CTMA without secondary obscuration and with effective focal length (EFFL) of 10m is reported. Its full field, F-number and total length are respectively 1.1°×1°, F/14.3, and one eighth of its EFFL. And its imaging quality is near diffraction limit.

Evaluation of vector-primed cDNA library production from microgram quantities of total RNA.

PubMed

Kuo, Jonathan; Inman, Jason; Brownstein, Michael; Usdin, Ted B

2004-12-15

cDNA sequences are important for defining the coding region of genes, and full-length cDNA clones have proven to be useful for investigation of the function of gene products. We produced cDNA libraries containing 3.5-5 x 10(5) primary transformants, starting with 5 mug of total RNA prepared from mouse pituitary, adrenal, thymus, and pineal tissue, using a vector-primed cDNA synthesis method. Of approximately 1000 clones sequenced, approximately 20% contained the full open reading frames (ORFs) of known transcripts, based on the presence of the initiating methionine residue codon. The libraries were complex, with 94, 91, 83 and 55% of the clones from the thymus, adrenal, pineal and pituitary libraries, respectively, represented only once. Twenty-five full-length clones, not yet represented in the Mammalian Gene Collection, were identified. Thus, we have produced useful cDNA libraries for the isolation of full-length cDNA clones that are not yet available in the public domain, and demonstrated the utility of a simple method for making high-quality libraries from small amounts of starting material.

Casimir interaction of rodlike particles in a two-dimensional critical system.

PubMed

Eisenriegler, E; Burkhardt, T W

2016-09-01

We consider the fluctuation-induced interaction of two thin, rodlike particles, or "needles," immersed in a two-dimensional critical fluid of Ising symmetry right at the critical point. Conformally mapping the plane containing the needles onto a simpler geometry in which the stress tensor is known, we analyze the force and torque between needles of arbitrary length, separation, and orientation. For infinite and semi-infinite needles we utilize the mapping of the plane bounded by the needles onto the half plane, and for two needles of finite length we use the mapping onto an annulus. For semi-infinite and infinite needles the force is expressed in terms of elementary functions, and we also obtain analytical results for the force and torque between needles of finite length with separation much greater than their length. Evaluating formulas in our approach numerically for several needle geometries and surface universality classes, we study the full crossover from small to large values of the separation to length ratio. In these two limits the numerical results agree with results for infinitely long needles and with predictions of the small-particle operator expansion, respectively.

Both α and β Subunits of Human Choriogonadotropin Photoaffinity Label the Hormone Receptor

NASA Astrophysics Data System (ADS)

Ji, Inhae; Ji, Tae H.

1981-09-01

It has been shown that a photoactivable derivative of human choriogonadotropin (hCG) labels the lutropin receptor on porcine granulosa cells [Ji, I. & Ji, T. H. (1980) Proc. Natl. Acad. Sci. USA 77, 7167-7170]. In an attempt to identify which of the hCG subunits labeled the receptor, three sets of different hCG derivatives were prepared. In the first set, hCG was coupled to the N-hydroxysuccinimide ester of 4-azidobenzoylglycine and radioiodinated. In the second set, only one of the subunits was radioiodinated, but both subunits were allowed to react with the reagent. In the third set, both the reagent and [125I]iodine were coupled to only one of the subunits. The binding activity of each hormone derivative was comparable to that of 125I-labeled hCG. After binding of these hormone derivatives to the granulosa cell surface, they were photolyzed. After solubilization, autoradiographs of sodium dodecyl sulfate/polyacrylamide gels of each sample revealed a number of labeled bands; the hCG derivatives containing 125I-labeled alpha subunit produced four bands (molecular weights 120,000 +/- 6,000, 96,000 +/- 5,000, 76,000 +/- 4,000, and 73,000 +/- 4,000) and those containing 125I-labeled beta subunit produced three bands (molecular weights 106,000 +/- 6,000, 88,000 +/- 5,000, and 83,000 +/- 4,000). Results were the same when the hormone-receptor complexes were solubilized in 0.5% Triton X-100 and then photolyzed or when the hormone was derivatized with a family of reagents having arms of various lengths. We conclude that both the alpha subunit and the beta subunit of hCG photoaffinity labeled certain membrane polypeptides and that these polypeptides are related to the hormone receptor.

Modulation of Starch Digestion for Slow Glucose Release through “Toggling” of Activities of Mucosal α-Glucosidases*

PubMed Central

Lee, Byung-Hoo; Eskandari, Razieh; Jones, Kyra; Reddy, Kongara Ravinder; Quezada-Calvillo, Roberto; Nichols, Buford L.; Rose, David R.; Hamaker, Bruce R.; Pinto, B. Mario

2012-01-01

Starch digestion involves the breakdown by α-amylase to small linear and branched malto-oligosaccharides, which are in turn hydrolyzed to glucose by the mucosal α-glucosidases, maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI). MGAM and SI are anchored to the small intestinal brush-border epithelial cells, and each contains a catalytic N- and C-terminal subunit. All four subunits have α-1,4-exohydrolytic glucosidase activity, and the SI N-terminal subunit has an additional exo-debranching activity on the α-1,6-linkage. Inhibition of α-amylase and/or α-glucosidases is a strategy for treatment of type 2 diabetes. We illustrate here the concept of “toggling”: differential inhibition of subunits to examine more refined control of glucogenesis of the α-amylolyzed starch malto-oligosaccharides with the aim of slow glucose delivery. Recombinant MGAM and SI subunits were individually assayed with α-amylolyzed waxy corn starch, consisting mainly of maltose, maltotriose, and branched α-limit dextrins, as substrate in the presence of four different inhibitors: acarbose and three sulfonium ion compounds. The IC50 values show that the four α-glucosidase subunits could be differentially inhibited. The results support the prospect of controlling starch digestion rates to induce slow glucose release through the toggling of activities of the mucosal α-glucosidases by selective enzyme inhibition. This approach could also be used to probe associated metabolic diseases. PMID:22851177

Stress and transcriptional regulation of tick ferritin HC.

PubMed

Mulenga, A; Simser, J A; Macaluso, K R; Azad, A F

2004-08-01

We previously identified a partial Dermacentor variabilis cDNA encoding ferritin HC (HC) subunit homolog (DVFER) that was differentially upregulated in Rickettsia montanensis infected ticks (Mulenga et al., 2003a). We have used rapid amplification of cDNA ends to clone full-length DVFER cDNA and its apparent ortholog from the wood tick, D. andersoni (DAFER), both of which show high sequence similarity to vertebrate than insect ferritin. Both DVFER and DAFER contain the stem-loop structure of a putative iron responsive element in the 5' untranslated region (nucleotide positions, 16-42) and the feroxidase centre loop typical for vertebrate ferritin HC subunits. Quantitative Western and Northern blotting analyses of protein and RNA from unfed and partially fed whole tick as well as dissected tick tissues demonstrated that DVFER is constitutively and ubiquitously expressed. Based on densitometric analysis of detected protein and mRNA bands, DVFER is predominantly expressed in the midgut, and to a lesser extent in the salivary glands, ovary and fatbody. Sham treatment (mechanical injury) and Escherichia coli challenge of D. variabilis ticks stimulated statistically significant (approximately 1.5- and approximately 3.0-fold, respectively) increases in DVFER mRNA abundance over time point matched naive control ticks. These data suggest that DVFER mRNA is nonspecifically up regulated in response to mechanical injury or bacterial infection induced stress.

Isolation and Characterization of the PKAr Gene From a Plant Pathogen, Curvularia lunata.

PubMed

Liu, T; Ma, B C; Hou, J M; Zuo, Y H

2014-09-01

By using EST database from a full-length cDNA library of Curvularia lunata, we have isolated a 2.9 kb cDNA, termed PKAr. An ORF of 1,383 bp encoding a polypeptide of 460 amino acids with molecular weight 50.1 kDa, (GeneBank Acc. No. KF675744) was cloned. The deduced amino acid sequence of the PKAr shows 90 and 88 % identity with cAMP-dependent protein kinase A regulatory subunit from Alternaria alternate and Pyrenophora tritici-repentis Pt-1C-BFP, respectively. Database analysis revealed that the deduced amino acid sequence of PKAr shares considerable similarity with that of PKA regulatory subunits in other organisms, particularly in the conserved regions. No introns were identified within the 1,383 bp of ORF compared with PKAr genomic DNA sequence. Southern blot indicated that PKAr existed as a single copy per genome. The mRNA expression level of PKAr in different development stages were demonstrated using real-time quantitative PCR. The results showed that the level of PKAr expression was highest in vegetative growth mycelium, which indicated it might play an important role in the vegetative growth of C. lunata. These results provided a fundamental supporting research on the function of PKAr in plant pathogen, C. lunata.

A high-throughput immobilized bead screen for stable proteins and multi-protein complexes

PubMed Central

Lockard, Meghan A.; Listwan, Pawel; Pedelacq, Jean-Denis; Cabantous, Stéphanie; Nguyen, Hau B.; Terwilliger, Thomas C.; Waldo, Geoffrey S.

2011-01-01

We describe an in vitro colony screen to identify Escherichia coli expressing soluble proteins and stable, assembled multiprotein complexes. Proteins with an N-terminal 6His tag and C-terminal green fluorescent protein (GFP) S11 tag are fluorescently labeled in cells by complementation with a coexpressed GFP 1–10 fragment. After partial colony lysis, the fluorescent soluble proteins or complexes diffuse through a supporting filtration membrane and are captured on Talon® resin metal affinity beads immobilized in agarose. Images of the fluorescent colonies convey total expression and the level of fluorescence bound to the beads indicates how much protein is soluble. Both pieces of information can be used together when selecting clones. After the assay, colonies can be picked and propagated, eliminating the need to make replica plates. We used the method to screen a DNA fragment library of the human protein p85 and preferentially obtained clones expressing the full-length ‘breakpoint cluster region-homology' and NSH2 domains. The assay also distinguished clones expressing stable multi-protein complexes from those that are unstable due to missing subunits. Clones expressing stable, intact heterotrimeric E.coli YheNML complexes were readily identified in libraries dominated by complexes of YheML missing the N subunit. PMID:21642284

Extensive frameshift at all AGG and CCC codons in the mitochondrial cytochrome c oxidase subunit 1 gene of Perkinsus marinus (Alveolata; Dinoflagellata).

PubMed

Masuda, Isao; Matsuzaki, Motomichi; Kita, Kiyoshi

2010-10-01

Diverse mitochondrial (mt) genetic systems have evolved independently of the more uniform nuclear system and often employ modified genetic codes. The organization and genetic system of dinoflagellate mt genomes are particularly unusual and remain an evolutionary enigma. We determined the sequence of full-length cytochrome c oxidase subunit 1 (cox1) mRNA of the earliest diverging dinoflagellate Perkinsus and show that this gene resides in the mt genome. Apparently, this mRNA is not translated in a single reading frame with standard codon usage. Our examination of the nucleotide sequence and three-frame translation of the mRNA suggest that the reading frame must be shifted 10 times, at every AGG and CCC codon, to yield a consensus COX1 protein. We suggest two possible mechanisms for these translational frameshifts: a ribosomal frameshift in which stalled ribosomes skip the first bases of these codons or specialized tRNAs recognizing non-triplet codons, AGGY and CCCCU. Regardless of the mechanism, active and efficient machinery would be required to tolerate the frameshifts predicted in Perkinsus mitochondria. To our knowledge, this is the first evidence of translational frameshifts in protist mitochondria and, by far, is the most extensive case in mitochondria.

Unexpected high digestion rate of cooked starch by the Ct-Maltase-Glucoamylase small intestine mucosal alpha-glucosidase subunit

USDA-ARS?s Scientific Manuscript database

For starch digestion to glucose, two luminal alpha-amylases and four gut mucosal alpha-glucosidase subunits are employed. The aim of this research was to investigate, for the first time, direct digestion capability of individual mucosal alpha-glucosidases on cooked (gelatinized) starch. Gelatinized ...

Phylogeny, divergence time and historical biogeography of Laetiporus (Basidiomycota, Polyporales).

PubMed

Song, Jie; Cui, Bao-Kai

2017-04-20

The aim of this study was to characterize the molecular relationship, origin and historical biogeography of the species in important brown rot fungal genus Laetiporus from East Asia, Europe, Pan-America, Hawaii and South Africa. We used six genetic markers to estimate a genus-level phylogeny including (1) the internal transcribed spacer (ITS), (2) nuclear large subunit rDNA (nrLSU), (3) nuclear small subunit rDNA (nrSSU), (4) translation elongation factor 1-α (EF-1α), (5) DNA-directed RNA polymerase II subunit 2 (RPB2), and (6) mitochondrial small subunit rDNA (mtSSU). Results of multi-locus phylogenetic analyses show clade support for at least seventeen species-level lineages including two new Laetiporus in China. Molecular dating using BEAST estimated the present crown group diverged approximately 20.16 million years ago (Mya) in the early Miocene. Biogeographic analyses using RASP indicated that Laetiporus most likely originated in temperate zones with East Asia and North America having the highest probability (48%) of being the ancestral area. Four intercontinental dispersal routes and a possible concealed dispersal route were established for the first time.

Calmodulin overexpression does not alter Cav1.2 function or oligomerization state.

PubMed

Findeisen, Felix; Tolia, Alexandra; Arant, Ryan; Kim, Eun Young; Isacoff, Ehud; Minor, Daniel L

2011-01-01

Interactions between calmodulin (CaM) and voltage-gated calcium channels (Ca(v)s) are crucial for Ca(v) activity-dependent feedback modulation. We recently reported an X-ray structure that shows two Ca(2+)/CaM molecules bound to the Ca(v)1.2 C terminal tail, one at the PreIQ region and one at the IQ domain. Surprisingly, the asymmetric unit of the crystal showed a dimer in which Ca(2+)/CaM bridged two PreIQ helixes to form a 4:2 Ca(2+)/CaM:Ca(v) C-terminal tail assembly. Contrary to previous proposals based on a similar crystallographic dimer, extensive biochemical analysis together with subunit counting experiments of full-length channels in live cell membranes failed to find evidence for multimers that would be compatible with the 4:2 crossbridged complex. Here, we examine this possibility further. We find that CaM over-expression has no functional effect on Ca(v)1.2 inactivation or on the stoichiometry of full-length Ca(v)1.2. These data provide further support for the monomeric Ca(v)1.2 stoichiometry. Analysis of the electrostatic surfaces of the 2:1 Ca(2+)/CaM:Ca(V) C-terminal tail assembly reveals notable patches of electronegativity. These could influence various forms of channel modulation by interacting with positively charged elements from other intracellular channel domains.

The 5′ Untranslated Region of the Human T-Cell Lymphotropic Virus Type 1 mRNA Enables Cap-Independent Translation Initiation

PubMed Central

Olivares, Eduardo; Landry, Dori M.; Cáceres, C. Joaquín; Pino, Karla; Rossi, Federico; Navarrete, Camilo; Huidobro-Toro, Juan Pablo; Thompson, Sunnie R.

2014-01-01

ABSTRACT The human T-cell leukemia virus type 1 (HTLV-1) is a complex human retrovirus that causes adult T cell leukemia and of HTLV-associated myelopathy/tropical spastic paraparesis. The mRNA of some complex retroviruses, including the human and simian immunodeficiency viruses (HIV and SIV), can initiate translation using a canonical cap-dependent mechanism or through an internal ribosome entry site (IRES). In this study, we present strong evidence showing that like HIV-1 and SIV, the 5′-untranslated region (5′UTR) of the HTLV-1 full-length mRNA harbors an IRES. Cap-independent translational activity was evaluated and demonstrated using dual luciferase bicistronic mRNAs in rabbit reticulocyte lysate, in mammalian cell culture, and in Xenopus laevis oocytes. Characterization of the HTLV-1 IRES shows that its activity is dependent on the ribosomal protein S25 (RPS25) and that its function is highly sensitive to the drug edeine. Together, these findings suggest that the 5′UTR of the HTLV-1 full-length mRNA enables internal recruitment of the eukaryotic translation initiation complex. However, the recognition of the initiation codon requires ribosome scanning. These results suggest that, after internal recruitment by the HTLV-1 IRES, a scanning step takes place for the 40S ribosomal subunit to be positioned at the translation initiation codon. IMPORTANCE The mechanism by which retroviral mRNAs recruit the 40S ribosomal subunit internally is not understood. This study provides new insights into the mechanism of translation initiation used by the human T-cell lymphotropic virus type 1 (HTLV-1). The results show that the HTLV-1 mRNA can initiate translation via a noncanonical mechanism mediated by an internal ribosome entry site (IRES). This study also provides evidence showing the involvement of cellular proteins in HTLV-1 IRES-mediated translation initiation. Together, the data presented in this report significantly contribute to the understanding of HTLV-1 gene expression. PMID:24623421

The NMR-Rosetta capsid model of M13 bacteriophage reveals a quadrupled hydrophobic packing epitope.

PubMed

Morag, Omry; Sgourakis, Nikolaos G; Baker, David; Goldbourt, Amir

2015-01-27

Filamentous phage are elongated semiflexible ssDNA viruses that infect bacteria. The M13 phage, belonging to the family inoviridae, has a length of ∼1 μm and a diameter of ∼7 nm. Here we present a structural model for the capsid of intact M13 bacteriophage using Rosetta model building guided by structure restraints obtained from magic-angle spinning solid-state NMR experimental data. The C5 subunit symmetry observed in fiber diffraction studies was enforced during model building. The structure consists of stacked pentamers with largely alpha helical subunits containing an N-terminal type II β-turn; there is a rise of 16.6-16.7 Å and a tilt of 36.1-36.6° between consecutive pentamers. The packing of the subunits is stabilized by a repeating hydrophobic stacking pocket; each subunit participates in four pockets by contributing different hydrophobic residues, which are spread along the subunit sequence. Our study provides, to our knowledge, the first magic-angle spinning NMR structure of an intact filamentous virus capsid and further demonstrates the strength of this technique as a method of choice to study noncrystalline, high-molecular-weight molecular assemblies.

The NMR–Rosetta capsid model of M13 bacteriophage reveals a quadrupled hydrophobic packing epitope

PubMed Central

Morag, Omry; Sgourakis, Nikolaos G.; Baker, David; Goldbourt, Amir

2015-01-01

Filamentous phage are elongated semiflexible ssDNA viruses that infect bacteria. The M13 phage, belonging to the family inoviridae, has a length of ∼1 μm and a diameter of ∼7 nm. Here we present a structural model for the capsid of intact M13 bacteriophage using Rosetta model building guided by structure restraints obtained from magic-angle spinning solid-state NMR experimental data. The C5 subunit symmetry observed in fiber diffraction studies was enforced during model building. The structure consists of stacked pentamers with largely alpha helical subunits containing an N-terminal type II β-turn; there is a rise of 16.6–16.7 Å and a tilt of 36.1–36.6° between consecutive pentamers. The packing of the subunits is stabilized by a repeating hydrophobic stacking pocket; each subunit participates in four pockets by contributing different hydrophobic residues, which are spread along the subunit sequence. Our study provides, to our knowledge, the first magic-angle spinning NMR structure of an intact filamentous virus capsid and further demonstrates the strength of this technique as a method of choice to study noncrystalline, high-molecular-weight molecular assemblies. PMID:25587134

An Fe-S cluster in the conserved Cys-rich region in the catalytic subunit of FAD-dependent dehydrogenase complexes.

PubMed

Shiota, Masaki; Yamazaki, Tomohiko; Yoshimatsu, Keiichi; Kojima, Katsuhiro; Tsugawa, Wakako; Ferri, Stefano; Sode, Koji

2016-12-01

Several bacterial flavin adenine dinucleotide (FAD)-harboring dehydrogenase complexes comprise three distinct subunits: a catalytic subunit with FAD, a cytochrome c subunit containing three hemes, and a small subunit. Owing to the cytochrome c subunit, these dehydrogenase complexes have the potential to transfer electrons directly to an electrode. Despite various electrochemical applications and engineering studies of FAD-dependent dehydrogenase complexes, the intra/inter-molecular electron transfer pathway has not yet been revealed. In this study, we focused on the conserved Cys-rich region in the catalytic subunits using the catalytic subunit of FAD dependent glucose dehydrogenase complex (FADGDH) as a model, and site-directed mutagenesis and electron paramagnetic resonance (EPR) were performed. By co-expressing a hitch-hiker protein (γ-subunit) and a catalytic subunit (α-subunit), FADGDH γα complexes were prepared, and the properties of the catalytic subunit of both wild type and mutant FADGDHs were investigated. Substitution of the conserved Cys residues with Ser resulted in the loss of dye-mediated glucose dehydrogenase activity. ICP-AEM and EPR analyses of the wild-type FADGDH catalytic subunit revealed the presence of a 3Fe-4S-type iron-sulfur cluster, whereas none of the Ser-substituted mutants showed the EPR spectrum characteristic for this cluster. The results suggested that three Cys residues in the Cys-rich region constitute an iron-sulfur cluster that may play an important role in the electron transfer from FAD (intra-molecular) to the multi-heme cytochrome c subunit (inter-molecular) electron transfer pathway. These features appear to be conserved in the other three-subunit dehydrogenases having an FAD cofactor. Copyright © 2016 Elsevier B.V. All rights reserved.

Role of small subunit in mediating assembly of red-type form I Rubisco.

PubMed

Joshi, Jidnyasa; Mueller-Cajar, Oliver; Tsai, Yi-Chin C; Hartl, F Ulrich; Hayer-Hartl, Manajit

2015-01-09

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the key enzyme involved in photosynthetic carbon fixation, converting atmospheric CO2 to organic compounds. Form I Rubisco is a cylindrical complex composed of eight large (RbcL) subunits that are capped by four small subunits (RbcS) at the top and four at the bottom. Form I Rubiscos are phylogenetically divided into green- and red-type. Some red-type enzymes have catalytically superior properties. Thus, understanding their folding and assembly is of considerable biotechnological interest. Folding of the green-type RbcL subunits in cyanobacteria is mediated by the GroEL/ES chaperonin system, and assembly to holoenzyme requires specialized chaperones such as RbcX and RAF1. Here, we show that the red-type RbcL subunits in the proteobacterium Rhodobacter sphaeroides also fold with GroEL/ES. However, assembly proceeds in a chaperone-independent manner. We find that the C-terminal β-hairpin extension of red-type RbcS, which is absent in green-type RbcS, is critical for efficient assembly. The β-hairpins of four RbcS subunits form an eight-stranded β-barrel that protrudes into the central solvent channel of the RbcL core complex. The two β-barrels stabilize the complex through multiple interactions with the RbcL subunits. A chimeric green-type RbcS carrying the C-terminal β-hairpin renders the assembly of a cyanobacterial Rubisco independent of RbcX. Our results may facilitate the engineering of crop plants with improved growth properties expressing red-type Rubisco. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Small Angle Neutron Scattering Observation of Chain Retraction after a Large Step Deformation

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

Blanchard, A.; Heinrich, M.; Pyckhout-Hintzen, W.

The process of retraction in entangled linear chains after a fast nonlinear stretch was detected from time-resolved but quenched small angle neutron scattering (SANS) experiments on long, well-entangled polyisoprene chains. The statically obtained SANS data cover the relevant time regime for retraction, and they provide a direct, microscopic verification of this nonlinear process as predicted by the tube model. Clear, quantitative agreement is found with recent theories of contour length fluctuations and convective constraint release, using parameters obtained mainly from linear rheology. The theory captures the full range of scattering vectors once the crossover to fluctuations on length scales belowmore » the tube diameter is accounted for.« less

Cloning and characterization of two novel zebrafish P2X receptor subunits.

PubMed

Diaz-Hernandez, Miguel; Cox, Jane A; Migita, Keisuke; Haines, William; Egan, Terrance M; Voigt, Mark M

2002-07-26

In this report we describe the cloning and characterization of two P2X receptor subunits cloned from the zebrafish (Danio rerio). Primary sequence analysis suggests that one cDNA encodes an ortholog of the mammalian P2X(4) subunit and the second cDNA encodes the ortholog of the mammalian P2X(5) subunit. The zP2X(4) subunit forms a homo-oligomeric receptor that displays a low affinity for ATP (EC(50)=274+/-48 microM) and very low affinity (EC(50)>500 microM) for other purinergic ligands such as alphabetameATP, suramin, and PPADS. As seen with the mammalian orthologs, the zP2X(5) subunit forms a homo-oligomeric receptor that yields very small whole-cell currents (<20pA), making determination of an EC(50) problematic. Both subunit genes were physically mapped onto the zebrafish genome using radiation hybrid analysis of the T51 panel, with the zp2x4 localized to LG21 and zp2x5 to LG5.

Functional role of the MrpA- and MrpD-homologous protein subunits in enzyme complexes evolutionary related to respiratory chain complex I.

PubMed

Moparthi, Vamsi K; Kumar, Brijesh; Al-Eryani, Yusra; Sperling, Eva; Górecki, Kamil; Drakenberg, Torbjörn; Hägerhäll, Cecilia

2014-01-01

NADH:quinone oxidoreductase or complex I is a large membrane bound enzyme complex that has evolved from the combination of smaller functional building blocks. Intermediate size enzyme complexes exist in nature that comprise some, but not all of the protein subunits in full size 14-subunit complex I. The membrane spanning complex I subunits NuoL, NuoM and NuoN are homologous to each other and to two proteins from one particular class of Na(+)/H(+) antiporters, denoted MrpA and MrpD. In complex I, these ion transporter protein subunits are prime candidates for harboring important parts of the proton pumping machinery. Using a model system, consisting of Bacillus subtilis MrpA and MrpD deletion strains and a low copy expression plasmid, it was recently demonstrated that NuoN can rescue the strain deleted for MrpD but not that deleted for MrpA, whereas the opposite tendency was seen for NuoL. This demonstrated that the MrpA-type and MrpD-type proteins have unique functional specializations. In this work, the corresponding antiporter-like protein subunits from the smaller enzymes evolutionarily related to complex I were tested in the same model system. The subunits from 11-subunit complex I from Bacillus cereus behaved essentially as those from full size complex I, corroborating that this enzyme should be regarded as a bona fide complex I. The hydrogenase-3 and hydrogenase-4 antiporter-like proteins on the other hand, could substitute equally well for MrpA or MrpD at pH7.4, suggesting that these enzymes have intermediate forms of the antiporter-like proteins, which seemingly lack the functional specificity. © 2013. Published by Elsevier B.V. All rights reserved.

Liquid Lens module with wide field-of-view and variable focal length

NASA Astrophysics Data System (ADS)

Seo, Sang Won; Han, Seungoh; Seo, Jun Ho; Choi, Woo Bum; Sung, Man Young

2010-12-01

A novel wide angle and variable-focus imaging module based on a miniaturized liquid lens is presented for capsule endoscopy applications. For these applications, it is desirable to have features such as a wide field of view (FOV), variable focus, small size, and low power consumption, thereby taking full advantage of the miniaturized liquid lens. The proposed imaging module has three aspheric plastic lenses for a wide FOV, and one liquid lens that can change the focal length by as much as 24.5 cm with a bias voltage difference of 23 Vrms for variable focusing. The assembled lens module has an overall length of 8.4 mm and a FOV of 120.5°. The realized imaging module including the proposed lenses is small enough to be inserted into a capsule endoscope, and it is expected to improve the diagnostic capability of capsule endoscopes.

Two Small Molecules Restore Stability to a Subpopulation of the Cystic Fibrosis Transmembrane Conductance Regulator with the Predominant Disease-causing Mutation.

PubMed

Meng, Xin; Wang, Yiting; Wang, Xiaomeng; Wrennall, Joe A; Rimington, Tracy L; Li, Hongyu; Cai, Zhiwei; Ford, Robert C; Sheppard, David N

2017-03-03

Cystic fibrosis (CF) is caused by mutations that disrupt the plasma membrane expression, stability, and function of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl - channel. Two small molecules, the CFTR corrector lumacaftor and the potentiator ivacaftor, are now used clinically to treat CF, although some studies suggest that they have counteracting effects on CFTR stability. Here, we investigated the impact of these compounds on the instability of F508del-CFTR, the most common CF mutation. To study individual CFTR Cl - channels, we performed single-channel recording, whereas to assess entire CFTR populations, we used purified CFTR proteins and macroscopic CFTR Cl - currents. At 37 °C, low temperature-rescued F508del-CFTR more rapidly lost function in cell-free membrane patches and showed altered channel gating and current flow through open channels. Compared with purified wild-type CFTR, the full-length F508del-CFTR was about 10 °C less thermostable. Lumacaftor partially stabilized purified full-length F508del-CFTR and slightly delayed deactivation of individual F508del-CFTR Cl - channels. By contrast, ivacaftor further destabilized full-length F508del-CFTR and accelerated channel deactivation. Chronic (prolonged) co-incubation of F508del-CFTR-expressing cells with lumacaftor and ivacaftor deactivated macroscopic F508del-CFTR Cl - currents. However, at the single-channel level, chronic co-incubation greatly increased F508del-CFTR channel activity and temporal stability in most, but not all, cell-free membrane patches. We conclude that chronic lumacaftor and ivacaftor co-treatment restores stability in a small subpopulation of F508del-CFTR Cl - channels but that the majority remain destabilized. A fuller understanding of these effects and the characterization of the small F508del-CFTR subpopulation might be crucial for CF therapy development. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Similarities in transcription factor IIIC subunits that bind to the posterior regions of internal promoters for RNA polymerase III.

PubMed

Matsutani, Sachiko

2004-08-09

In eukaryotes, RNA polymerase III (RNAP III) transcribes the genes for small RNAs like tRNAs, 5S rRNA, and several viral RNAs, and short interspersed repetitive elements (SINEs). The genes for these RNAs and SINEs have internal promoters that consist of two regions. These two regions are called the A and B blocks. The multisubunit transcription factor TFIIIC is required for transcription initiation of RNAP III; in transcription of tRNAs, the B-block binding subunit of TFIIIC recognizes a promoter. Although internal promoter sequences are conserved in eukaryotes, no evidence of homology between the B-block binding subunits of vertebrates and yeasts has been reported previously. Here, I reported the results of PSI-BLAST searches using the B-block binding subunits of human and Shizosacchromyces pombe as queries, showing that the same Arabidopsis proteins were hit with low E-values in both searches. Comparison of the convergent iterative alignments obtained by these PSI-BLAST searches revealed that the vertebrate, yeast, and Arabidopsis proteins have similarities in their N-terminal one-third regions. In these regions, there were three domains with conserved sequence similarities, one located in the N-terminal end region. The N-terminal end region of the B-block binding subunit of Saccharomyces cerevisiae is tentatively identified as a HMG box, which is the DNA binding motif. Although I compared the alignment of the N-terminal end regions of the B-block binding subunits, and their homologs, with that of the HMG boxes, it is not clear whether they are related. Molecular phylogenetic analyses using the small subunit rRNA and ubiquitous proteins like actin and alpha-tubulin, show that fungi are more closely related to animals than either is to plants. Interestingly, the results obtained in this study show that, with respect to the B-block binding subunits of TFIIICs, animals appear to be evolutionarily closer to plants than to fungi.

A saposin-like domain influences the intracellular localization, stability, and catalytic activity of human acyloxyacyl hydrolase.

PubMed

Staab, J F; Ginkel, D L; Rosenberg, G B; Munford, R S

1994-09-23

Acyloxyacyl hydrolase, a leukocyte enzyme that acts on bacterial lipopolysaccharides (LPSs) and many glycerolipids, is synthesized as a precursor polypeptide that undergoes internal disulfide linkage before being proteolytically processed into two subunits. The larger subunit contains an amino acid sequence (Gly-X-Ser-X-Gly) that is found at the active sites of many lipases, while the smaller subunit has amino acid sequence similarity to saposins (sphingolipid activator proteins), cofactors for sphingolipid glycohydrolases. We show here that both acyloxyacyl hydrolase subunits are required for catalytic activity toward LPS and glycerophosphatidylcholine. In addition, mutations that truncate or delete the small subunit have profound effects on the intracellular localization, proteolytic processing, and stability of the enzyme in baby hamster kidney cells. Remarkably, proteolytic cleavage of the precursor protein increases the activity of the enzyme toward LPS by 10-20-fold without altering its activity toward glycerophosphatidylcholine. Proper orientation of the two subunits thus seems very important for the substrate specificity of this unusual enzyme.

Genome-Wide Identification and Comprehensive Expression Profiling of Ribosomal Protein Small Subunit (RPS) Genes and their Comparative Analysis with the Large Subunit (RPL) Genes in Rice

PubMed Central

Saha, Anusree; Das, Shubhajit; Moin, Mazahar; Dutta, Mouboni; Bakshi, Achala; Madhav, M. S.; Kirti, P. B.

2017-01-01

Ribosomal proteins (RPs) are indispensable in ribosome biogenesis and protein synthesis, and play a crucial role in diverse developmental processes. Our previous studies on Ribosomal Protein Large subunit (RPL) genes provided insights into their stress responsive roles in rice. In the present study, we have explored the developmental and stress regulated expression patterns of Ribosomal Protein Small (RPS) subunit genes for their differential expression in a spatiotemporal and stress dependent manner. We have also performed an in silico analysis of gene structure, cis-elements in upstream regulatory regions, protein properties and phylogeny. Expression studies of the 34 RPS genes in 13 different tissues of rice covering major growth and developmental stages revealed that their expression was substantially elevated, mostly in shoots and leaves indicating their possible involvement in the development of vegetative organs. The majority of the RPS genes have manifested significant expression under all abiotic stress treatments with ABA, PEG, NaCl, and H2O2. Infection with important rice pathogens, Xanthomonas oryzae pv. oryzae (Xoo) and Rhizoctonia solani also induced the up-regulation of several of the RPS genes. RPS4, 13a, 18a, and 4a have shown higher transcript levels under all the abiotic stresses, whereas, RPS4 is up-regulated in both the biotic stress treatments. The information obtained from the present investigation would be useful in appreciating the possible stress-regulatory attributes of the genes coding for rice ribosomal small subunit proteins apart from their functions as house-keeping proteins. A detailed functional analysis of independent genes is required to study their roles in stress tolerance and generating stress- tolerant crops. PMID:28966624

Dietary phenolic compounds selectively inhibit the individual subunits of maltase-glucoamylase and sucrase-isomaltase with the potential of modulating glucose release

USDA-ARS?s Scientific Manuscript database

In this study, it was hypothesized that dietary phenolic compounds selectively inhibit the individual C- and N-terminal (Ct, Nt) subunits of the two small intestinal alpha-glucosidases, maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI), for a modulated glycemic carbohydrate digestion. The inhi...

Three new anascosporic genera of the Saccharomycotina: Danielozyma gen. nov., Deakozyma gen. nov. and Middelhovenomyces gen. nov.

USDA-ARS?s Scientific Manuscript database

Three new non-ascosporic, ascomycetous yeast genera are proposed based on their isolation from currently described species and genera. Phylogenetic placement of the genera was determined from analysis of nuclear gene sequences for D1/D2 large subunit rRNA, small subunit rRNA, translation elongation...

Molecular cloning and characterization of ADP-glucose pyrophosphorylase cDNA clones isolated from pea cotyledons.

PubMed

Burgess, D; Penton, A; Dunsmuir, P; Dooner, H

1997-02-01

Three ADP-glucose pyrophosphorylase (ADPG-PPase) cDNA clones have been isolated and characterized from a pea cotyledon cDNA library. Two of these clones (Psagps1 and Psagps2) encode the small subunit of ADPG-PPase. The deduced amino acid sequences for these two clones are 95% identical. Expression of these two genes differs in that the Psagps2 gene shows comparatively higher expression in seeds relative to its expression in other tissues. Psagps2 expression also peaks midway through seed development at a time in which Psagps1 transcripts are still accumulating. The third cDNA isolated (Psagp11) encodes the large subunit of ADPG-PPase. It shows greater selectivity in expression than either of the small subunit clones. It is highly expressed in sink organs (seed, pod, and seed coat) and undetectable in leaves.

First report of Sapajus cay naturally infected by Trypanosoma cruzi in San Pedro Department, Paraguay.

PubMed

Acosta, Nidia; Miret, Jorge; López, Elsa; Schinini, Alicia

2016-08-29

To verify the occurrence of natural Trypanosoma cruzi infection in non-human primates from a rural endemic area of the east region of Paraguay, xenodiagnosis was performed in 35 animals belonging to two species. For genotyping and T. cruzi discrete typing unit (DTU) assignment, a combination of four markers was used, including amplification products of the small (18S) and large (24Sα) subunits of ribosomal ribonucleic acid gene, the intergenic region of mini-exon gene and the heat shock protein 60 Eco-RV polymerase chain reaction-restriction fragment length polymorphism (HSP60/EcoRV-PCR-RFLP). One specimen of Sapajus cay was found positive and infected by the DTU TcII. This result constitutes the first record of natural T. cruzi infection in a sylvatic monkey in Paraguay, harbouring a DTU associated with severe Chagas disease in humans.

The complete mitochondrial genome of Chinese green hydra, Hydra sinensis (Hydroida: Hydridae).

PubMed

Pan, Hong-Chun; Qian, Xiao-Cheng; Li, Ping; Li, Xiao-Fei; Wang, An-Tai

2014-02-01

The complete mitochondrial genome of Chinese green hydra, Hydra sinensis (Hydroida: Hydridae) is a linear molecule of 16,189 bp in length, containing 13 protein-coding genes, small and large subunit ribosomal RNAs, methionine and tryptophan transfer RNAs, a pseudogene consisting of a partial copy of COI and terminal sequences at two ends of the linear mitochondrial DNA. The A + T content of the overall base composition of H-strand is 77.2% (T: 41.7%; C: 10.9%; A: 35.5%; and G: 11.9%). COI and ND1 genes begin with GTG as start codon, while other 11 protein-coding genes start with a typical ATG initiation codon. COII, ATP8, ATP6, COIII, ND5, ND6, ND3, ND1, ND4 and COI genes are terminated with TAA as stop codon, ND4L ends with TAG, ND2 ends with TA and Cyt b ends with T.

Genetic Diversity of Cryptosporidium spp. in Captive Reptiles

PubMed Central

Xiao, Lihua; Ryan, Una M.; Graczyk, Thaddeus K.; Limor, Josef; Li, Lixia; Kombert, Mark; Junge, Randy; Sulaiman, Irshad M.; Zhou, Ling; Arrowood, Michael J.; Koudela, Břetislav; Modrý, David; Lal, Altaf A.

2004-01-01

The genetic diversity of Cryptosporidium in reptiles was analyzed by PCR-restriction fragment length polymorphism and sequence analysis of the small subunit rRNA gene. A total of 123 samples were analyzed, of which 48 snake samples, 24 lizard samples, and 3 tortoise samples were positive for Cryptosporidium. Nine different types of Cryptosporidium were found, including Cryptosporidium serpentis, Cryptosporidium desert monitor genotype, Cryptosporidium muris, Cryptosporidium parvum bovine and mouse genotypes, one C. serpentis-like parasite in a lizard, two new Cryptosporidium spp. in snakes, and one new Cryptosporidium sp. in tortoises. C. serpentis and the desert monitor genotype were the most common parasites and were found in both snakes and lizards, whereas the C. muris and C. parvum parasites detected were probably the result of ingestion of infected rodents. Sequence and biologic characterizations indicated that the desert monitor genotype was Cryptosporidium saurophilum. Two host-adapted C. serpentis genotypes were found in snakes and lizards. PMID:14766569

A Metabotropic-Like Flux-Independent NMDA Receptor Regulates Ca2+ Exit from Endoplasmic Reticulum and Mitochondrial Membrane Potential in Cultured Astrocytes.

PubMed

Montes de Oca Balderas, Pavel; Aguilera, Penélope

2015-01-01

Astrocytes were long thought to be only structural cells in the CNS; however, their functional properties support their role in information processing and cognition. The ionotropic glutamate N-methyl D-aspartate (NMDA) receptor (NMDAR) is critical for CNS functions, but its expression and function in astrocytes is still a matter of research and debate. Here, we report immunofluorescence (IF) labeling in rat cultured cortical astrocytes (rCCA) of all NMDAR subunits, with phenotypes suggesting their intracellular transport, and their mRNA were detected by qRT-PCR. IF and Western Blot revealed GluN1 full-length synthesis, subunit critical for NMDAR assembly and transport, and its plasma membrane localization. Functionally, we found an iCa2+ rise after NMDA treatment in Fluo-4-AM labeled rCCA, an effect blocked by the NMDAR competitive inhibitors D(-)-2-amino-5-phosphonopentanoic acid (APV) and Kynurenic acid (KYNA) and dependent upon GluN1 expression as evidenced by siRNA knock down. Surprisingly, the iCa2+ rise was not blocked by MK-801, an NMDAR channel blocker, or by extracellular Ca2+ depletion, indicating flux-independent NMDAR function. In contrast, the IP3 receptor (IP3R) inhibitor XestosponginC did block this response, whereas a Ryanodine Receptor inhibitor did so only partially. Furthermore, tyrosine kinase inhibition with genistein enhanced the NMDA elicited iCa2+ rise to levels comparable to those reached by the gliotransmitter ATP, but with different population dynamics. Finally, NMDA depleted the rCCA mitochondrial membrane potential (mΔψ) measured with JC-1. Our results demonstrate that rCCA express NMDAR subunits which assemble into functional receptors that mediate a metabotropic-like, non-canonical, flux-independent iCa2+ increase.

Concholepas concholepas Ferritin H-like subunit (CcFer): Molecular characterization and single nucleotide polymorphism associated to innate immune response.

PubMed

Chávez-Mardones, Jacqueline; Valenzuela-Muñoz, Valentina; Núñez-Acuña, Gustavo; Maldonado-Aguayo, Waleska; Gallardo-Escárate, Cristian

2013-09-01

Ferritin has been identified as the principal protein of iron storage and iron detoxification, playing a pivotal role for the cellular homeostasis in living organisms. However, recent studies in marine invertebrates have suggested its association with innate immune system. In the present study, one Ferritin subunit was identified from the gastropod Concholepas concholepas (CcFer), which was fully characterized by Rapid Amplification of cDNA Ends technique. Simultaneously, a challenge test was performed to evaluate the immune response against Vibrio anguillarum. The full length of cDNA Ccfer was 1030 bp, containing 513 bp of open reading frame that encodes to 170 amino acid peptide, which was similar to the Ferritin H subunit described in vertebrates. Untranslated Regions (UTRs) were identified with a 5'UTR of 244 bp that contains iron responsive element (IRE), and a 3'UTR of 273 bp. The predicted molecular mass of deduced amino acid of CcFer was 19.66 kDa and isoelectric point of 4.92. Gene transcription analysis revealed that CcFer increases against infections with V. anguillarum, showing a peak expression at 6 h post-infection. Moreover, a single nucleotide polymorphism was detected at -64 downstream 5'UTR sequence (SNP-64). Quantitative real time analysis showed that homozygous mutant allele (TT) was significantly associated with higher expression levels of the challenged group compared to wild (CC) and heterozygous (CT) variants. Our findings suggest that CcFer is associated to innate immune response in C. concholepas and that the presence of SNPs may involve differential transcriptional expression of CcFer. Copyright © 2013 Elsevier Ltd. All rights reserved.

Electron cryomicroscopy structure of a membrane-anchored mitochondrial AAA protease.

PubMed

Lee, Sukyeong; Augustin, Steffen; Tatsuta, Takashi; Gerdes, Florian; Langer, Thomas; Tsai, Francis T F

2011-02-11

FtsH-related AAA proteases are conserved membrane-anchored, ATP-dependent molecular machines, which mediate the processing and turnover of soluble and membrane-embedded proteins in eubacteria, mitochondria, and chloroplasts. Homo- and hetero-oligomeric proteolytic complexes exist, which are composed of homologous subunits harboring an ATPase domain of the AAA family and an H41 metallopeptidase domain. Mutations in subunits of mitochondrial m-AAA proteases have been associated with different neurodegenerative disorders in human, raising questions on the functional differences between homo- and hetero-oligomeric AAA proteases. Here, we have analyzed the hetero-oligomeric yeast m-AAA protease composed of homologous Yta10 and Yta12 subunits. We combined genetic and structural approaches to define the molecular determinants for oligomer assembly and to assess functional similarities between Yta10 and Yta12. We demonstrate that replacement of only two amino acid residues within the metallopeptidase domain of Yta12 allows its assembly into homo-oligomeric complexes. To provide a molecular explanation, we determined the 12 Å resolution structure of the intact yeast m-AAA protease with its transmembrane domains by electron cryomicroscopy (cryo-EM) and atomic structure fitting. The full-length m-AAA protease has a bipartite structure and is a hexamer in solution. We found that residues in Yta12, which facilitate homo-oligomerization when mutated, are located at the interface between neighboring protomers in the hexamer ring. Notably, the transmembrane and intermembrane space domains are separated from the main body, creating a passage on the matrix side, which is wide enough to accommodate unfolded but not folded polypeptides. These results suggest a mechanism regarding how proteins are recognized and degraded by m-AAA proteases.

Microbial community biofabrics in a geothermal mine adit.

PubMed

Spear, John R; Barton, Hazel A; Robertson, Charles E; Francis, Christopher A; Pace, Norman R

2007-10-01

Speleothems such as stalactites and stalagmites are usually considered to be mineralogical in composition and origin; however, microorganisms have been implicated in the development of some speleothems. We have identified and characterized the biological and mineralogical composition of mat-like biofabrics in two novel kinds of speleothems from a 50 degrees C geothermal mine adit near Glenwood Springs, CO. One type of structure consists of 2- to 3-cm-long, 3- to 4-mm-wide, leather-like, hollow, soda straw stalactites. Light and electron microscopy indicated that the stalactites are composed of a mineralized biofabric with several cell morphotypes in a laminated form, with gypsum and sulfur as the dominant mineral components. A small-subunit rRNA gene phylogenetic community analysis along the stalactite length yielded a diverse gradient of organisms, with a relatively simple suite of main constituents: Thermus spp., crenarchaeotes, Chloroflexi, and Gammaproteobacteria. PCR analysis also detected putative crenarchaeal ammonia monooxygenase subunit A (amoA) genes in this community, the majority related to sequences from other geothermal systems. The second type of speleothem, dumpling-like rafts floating on a 50 degrees C pool on the floor of the adit, showed a mat-like fabric of evidently living organisms on the outside of the dumpling, with a multimineral, amorphous, gypsum-based internal composition. These two novel types of biofabrics are examples of the complex roles that microbes can play in mineralization, weathering, and deposition processes in karst environments.

GABAA receptors: Various stoichiometrics of subunit arrangement in α1β3 and α1β3ε receptors.

PubMed

Has, Ahmad Tarmizi Che; Chebib, Mary

2018-05-15

GABAA receptors (GABAARs) are members of the Cys-loop ligand-gated ion channel (LGIC) superfamily, which includes nicotinic acetylcholine, glycine, and serotonin (5HT3) receptors [1,2,3,4]. LGICs typically mediate fast synaptic transmission via the movement of ions through channels gated by neurotransmitters, such as acetylcholine for nicotinic receptors and GABA for GABAARs [5]. The term Cys-loop receptors originates from the presence of a conserved disulphide bond (or bridge) which holds together two cysteine amino acids of the loop that forms from the structure of polypeptides in the extracellular domain of the receptor's subunit [6]. GABAARs are pentameric transmembrane protein complexes consisting of five subunits from a variety of polypeptide subunits [7,8]. All of these subunits are pseudo-symmetrically organized in the plane of the membrane, with a Cl--selective channel in the middle of the complex. To date, nineteen GABAAR subunits have been identified and categorized into eight classes, α1-6, β1-3, γ1-3, δ, ε, θ, π and ρ1-3, but their variety is further broadened by the existence of several splice forms for certain subunits (e.g., α6, β2 and γ2) [9,10,11,12]. The subunits within each class have an amino acid sequence homology of 70% or more, whereas those with a sequence homology of 30% or less are grouped into different classes [13,14]. A subunit consists of four transmembrane domains (TM1-TM4), each forming an α-helix; a large extracellular N-terminal domain that incorporates part of the orthosteric agonist/antagonist binding site; a large intracellular loop between the TM3 and TM4; a small intracellular loop between TM1 and TM2; a small extracellular loop between TM2 and TM3; and a C-terminal extracellular domain [15,16]. Each subunit is arranged in such a way as to create principal and complementary interfaces with the other subunits, and in a position such that the TM2 of each subunit forms the wall of the channel pore [17,18,19]. The major subunit combination found in the brain comprises α1, β2 and γ2 subunits with the stoichiometry 2α1:2β2:1γ2 [18,20]. For the GABAA α1β2γ2 receptors, the subunits form a specific arrangement in which α1 and β2 subunits alternate with each other and are connected by a γ2 subunit (Figure A) [16,20,21]. For minor subtypes, different α and β subunits have been detected to co-exist as proven by the existence of GABAARs containing α1α2, α1α3, α1α5, α2α3, α3α5, α4α1, α4α2 and α4α3 in the central nervous system [22,23]. Meanwhile, the same pattern has been detected with β and γ subunits, at least the co-occurrence of β in the same GABAAR as well as γ2 with γ3, indicating that these subunits have the capacity to co-exist with each other [24,25,26]. Since different subunits can actually occur in one receptor, GABAARs are considered to exist in a multi-subunit arrangement, leading to ambiguity in the determination of a receptor's stoichiometry. In this review, we first briefly discuss the different subunit arrangements of α1 and β3 subunits in the binary α1β3 receptors. Then we review the GABAA ε-containing receptors predominantly in terms of the ability of ε subunit to present in different position in the ternary α1β3ε receptors, which introduce distinct populations of receptor. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

How the structure of the large subunit controls function in an oxygen-tolerant [NiFe]-hydrogenase

PubMed Central

Bowman, Lisa; Flanagan, Lindsey; Fyfe, Paul K.; Parkin, Alison; Hunter, William N.; Sargent, Frank

2014-01-01

Salmonella enterica is an opportunistic pathogen that produces a [NiFe]-hydrogenase under aerobic conditions. In the present study, genetic engineering approaches were used to facilitate isolation of this enzyme, termed Hyd-5. The crystal structure was determined to a resolution of 3.2 Å and the hydro-genase was observed to comprise associated large and small subunits. The structure indicated that His229 from the large subunit was close to the proximal [4Fe–3S] cluster in the small subunit. In addition, His229 was observed to lie close to a buried glutamic acid (Glu73), which is conserved in oxygen-tolerant hydrogenases. His229 and Glu73 of the Hyd-5 large subunit were found to be important in both hydrogen oxidation activity and the oxygen-tolerance mechanism. Substitution of His229 or Glu73 with alanine led to a loss in the ability of Hyd-5 to oxidize hydrogen in air. Furthermore, the H229A variant was found to have lost the overpotential requirement for activity that is always observed with oxygen-tolerant [NiFe]-hydrogenases. It is possible that His229 has a role in stabilizing the super-oxidized form of the proximal cluster in the presence of oxygen, and it is proposed that Glu73could play a supporting role in fine-tuning the chemistry of His229 to enable this function. PMID:24428762

The Genetic Basis of Natural Variation in Caenorhabditis elegans Telomere Length

PubMed Central

Cook, Daniel E.; Zdraljevic, Stefan; Tanny, Robyn E.; Seo, Beomseok; Riccardi, David D.; Noble, Luke M.; Rockman, Matthew V.; Alkema, Mark J.; Braendle, Christian; Kammenga, Jan E.; Wang, John; Kruglyak, Leonid; Félix, Marie-Anne; Lee, Junho; Andersen, Erik C.

2016-01-01

Telomeres are involved in the maintenance of chromosomes and the prevention of genome instability. Despite this central importance, significant variation in telomere length has been observed in a variety of organisms. The genetic determinants of telomere-length variation and their effects on organismal fitness are largely unexplored. Here, we describe natural variation in telomere length across the Caenorhabditis elegans species. We identify a large-effect variant that contributes to differences in telomere length. The variant alters the conserved oligonucleotide/oligosaccharide-binding fold of protection of telomeres 2 (POT-2), a homolog of a human telomere-capping shelterin complex subunit. Mutations within this domain likely reduce the ability of POT-2 to bind telomeric DNA, thereby increasing telomere length. We find that telomere-length variation does not correlate with offspring production or longevity in C. elegans wild isolates, suggesting that naturally long telomeres play a limited role in modifying fitness phenotypes in C. elegans. PMID:27449056

Crystallographic structure of the turbine C-ring from spinach chloroplast F-ATP synthase

PubMed Central

Balakrishna, Asha Manikkoth; Seelert, Holger; Marx, Sven-Hendric; Dencher, Norbert A.; Grüber, Gerhard

2014-01-01

In eukaryotic and prokaryotic cells, F-ATP synthases provide energy through the synthesis of ATP. The chloroplast F-ATP synthase (CF1FO-ATP synthase) of plants is integrated into the thylakoid membrane via its FO-domain subunits a, b, b’ and c. Subunit c with a stoichiometry of 14 and subunit a form the gate for H+-pumping, enabling the coupling of electrochemical energy with ATP synthesis in the F1 sector. Here we report the crystallization and structure determination of the c14-ring of subunit c of the CF1FO-ATP synthase from spinach chloroplasts. The crystals belonged to space group C2, with unit-cell parameters a=144.420, b=99.295, c=123.51 Å, and β=104.34° and diffracted to 4.5 Å resolution. Each c-ring contains 14 monomers in the asymmetric unit. The length of the c-ring is 60.32 Å, with an outer ring diameter 52.30 Å and an inner ring width of 40 Å. PMID:27919036

Intestinal disease of scattered mirror carp Cyprinus carpio caused by Thelohanellus kitauei and notes on the morphology and phylogeny of the myxosporean from Sichuan Province, southwest China

NASA Astrophysics Data System (ADS)

Ye, Lingtong; Lu, Mingmiao; Quan, Keyan; Li, Wenxiang; Zou, Hong; Wu, Shangong; Wang, Jiangyong; Wang, Guitang

2017-05-01

The mass mortality of pond-reared scattered mirror carp, Cyprinus carpio, caused by Thelohanellus kitauei, occurred at fish farms in Sichuan Province, southwest China. Morphological and molecular analyses were supplemented with histological evaluation of infected tissues to better understand the route of infection and the pathological effects of T. kitauei on the fish host. The intestine of the diseased host was full of large cysts of the myxosporean. The cysts range from 2 cm to 3.6 cm in diameter. Histopathology indicated that T. kitaue i first invaded the submucosa of the host intestine and then moved into the mucosa layers with the development of their spores, finally entering into the enteric cavity of the hosts after the disruption of mucosa layers. The pyriform spores of T. kitauei were surrounded by the transparent spore sheath, measuring 25.98 μm±0.95 μm in body length, 8.72 μm±0.51 μm in body width, and 7.86 μm±0.26 μm in body thickness. The single polar capsule was pyriform, measuring 14.73 μm±0.92 μm in length and 6.82 μm±0.45 μm in width, with eight to 10 turns of filament coils winding inside. Phylogenetic analysis based on the 18S small-subunit ribosomal DNA sequences indicated that minimal genetic differences were present between T. kitauei samples from South Korea and from China. Close affinity was found between the genus Thelohanellus and Myxobolus. Additionally, two polar capsule nuclei were found at the anterior end of the single polar capsule in spores of T. kitauei stained with hematoxylin and eosin, which suggested the separation of the genus Thelohanellus from Myxobolus.

Voltage-gated calcium channel and antisense oligonucleotides thereto

NASA Technical Reports Server (NTRS)

Friedman, Peter A. (Inventor); Duncan, Randall L. (Inventor); Hruska, Keith A. (Inventor); Barry, Elizabeth L. R. (Inventor)

1998-01-01

An antisense oligonucleotide of 10 to 35 nucleotides in length that can hybridize with a region of the .alpha..sub.1 subunit of the SA-Cat channel gene DNA or mRNA is provided, together with pharmaceutical compositions containing and methods utilizing such antisense oligonucleotide.

Building a flagellum in biological outer space.

PubMed

Evans, Lewis D B; Hughes, Colin; Fraser, Gillian M

2014-02-01

Flagella, the rotary propellers on the surface of bacteria, present a paradigm for how cells build and operate complex molecular 'nanomachines'. Flagella grow at a constant rate to extend several times the length of the cell, and this is achieved by thousands of secreted structural subunits transiting through a central channel in the lengthening flagellum to incorporate into the nascent structure at the distant extending tip. A great mystery has been how flagella can assemble far outside the cell where there is no conventional energy supply to fuel their growth. Recent work published by Evans et al. [ Nature (2013) 504: 287-290], has gone some way towards solving this puzzle, presenting a simple and elegant transit mechanism in which growth is powered by the subunits them selves as they link head-to-tail in a chain that is pulled through the length of the growing structure to the tip. This new mechanism answers an old question and may have resonance in other assembly processes.

Deletion of Marek’s disease virus large subunit of ribonucleotide reductase (RR) impairs virus growth in vitro and in vivo

USDA-ARS?s Scientific Manuscript database

Marek’s disease virus (MDV), a highly cell-associated lymphotropic alphaherpesvirus, is the causative agent of a neoplastic disease in domestic chickens, called Marek’s disease (MD). In the unique long region of the MDV genome, open reading frames UL39 and UL40 encode the large and small subunits o...

Marek’s Disease Virus Encoded Ribonucleotide Reductase Large Subunit is not Essential for In Vitro Replication

USDA-ARS?s Scientific Manuscript database

Marek’s disease virus (MDV) infected cells express a viral ribonucleotide reductase (RR) that is distinguishable from that present in uninfected cells by monoclonal antibody T81. Open reading frames UL39 and UL40 of the MDV genome encode the large (RR1) and small (RR2) subunits of RR enzyme, respe...

Multiple forms of ADP-glucose pyrophosphorylase from tomato fruit

NASA Technical Reports Server (NTRS)

Chen, B. Y.; Janes, H. W.

1997-01-01

ADP-glucose pyrophosphorylase (AGP) was purified from tomato (Lycopersicon esculentum Mill.) fruit to apparent homogeneity. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis the enzyme migrated as two close bands with molecular weights of 50,000 and 51,000. Two-dimensional polyacrylamide gel electrophoresis analysis of the purified enzyme, however, revealed at least five major protein spots that could be distinguished by their slight differences in net charge and molecular weight. Whereas all of the spots were recognized by the antiserum raised against tomato fruit AGP holoenzyme, only three of them reacted strongly with antiserum raised against the potato tuber AGP large subunit, and the other two spots (with lower molecular weights) reacted specifically with antisera raised against spinach leaf AGP holoenzyme and the potato tuber AGP small subunit. The results suggest the existence of at least three isoforms of the AGP large subunit and two isoforms of the small subunit in tomato fruit in vivo. The native molecular mass of the enzyme determined by gel filtration was 220 +/- 10 kD, indicating a tetrameric structure for AGP from tomato fruit. The purified enzyme is very sensitive to 3-phosphoglycerate/inorganic phosphate regulation.

Protein Phosphatase 2A Isoforms Utilizing Aβ Scaffolds Regulate Differentiation through Control of Akt Protein*

PubMed Central

Hwang, Justin H.; Jiang, Tao; Kulkarni, Shreya; Faure, Nathalie; Schaffhausen, Brian S.

2013-01-01

Protein phosphatase 2A (PP2A) regulates almost all cell signaling pathways. It consists of a scaffolding A subunit to which a catalytic C subunit and one of many regulatory B subunits bind. Of the more than 80 PP2A isoforms, 10% use Aβ as a scaffold. This study demonstrates the isoform-specific function of the A scaffold subunits. Polyomaviruses have shown the importance of phosphotyrosine, PI3K, and p53 in transformation. Comparisons of polyoma and SV40 small T antigens implicate Aβ in the control of differentiation. Knockdown of Aβ enhanced differentiation. Akt signaling regulated differentiation; its activation or inhibition promoted or blocked it, respectively. Aβ bound Akt. Enhancement of PP2A Aβ/Akt interaction by polyoma small T antigen increased turnover of Akt Ser-473 phosphorylation. Conversely, knockdown of Aβ promoted Akt activity and reduced turnover of phosphate at Ser-473 of Akt. These data provide new insight into the regulation of Akt, a protein of extreme importance in cancer. Furthermore, our results suggest that the role for Aβ in differentiation and perhaps tumor suppression may lie partly in its ability to negatively regulate Akt. PMID:24052256

Expression of the ribulose-1,5-bisphosphate carboxylase large subunit gene and three small subunit genes in two cell types of maize leaves

PubMed Central

Sheen, Jenq-Yunn; Bogorad, Lawrence

1986-01-01

Transcripts of three distinct ribulose-1,5-bisphosphate carboxylase (RuBPC) small subunit (SS) genes account for ∼90% of the mRNA for this protein in maize leaves. Transcripts of two of them constitute >80% of the SS mRNA in 24-h greening maize leaves. The third gene contribute ∼10%. Transcripts of all three nuclear-encoded SS genes are detectable in bundle sheath (BSC) and mesophyll cells (MC) of etiolated maize leaves. The level of mRNA for each gene is different in etioplasts of MC but all drop during photoregulated development of chloroplasts in MC and follow a pattern of transitory rise and fall in BSC. The amounts of LS and SS proteins continue to increase steadily well after the mRNA levels reach their peaks in BSC. The molar ratio of mRNA for chloroplast-encoded RuBPC large subunit (LS) to the nuclear genome encoded SS is about 10:1 although LS and SS proteins are present in about equimolar amounts. ImagesFig. 1.Fig. 2.Fig. 3.Fig. 4.Fig. 5.Fig. 6. PMID:16453739

The localisation of the heparin binding sites of human and murine interleukin-12 within the carboxyterminal domain of the P40 subunit.

PubMed

Garnier, Pascale; Mummery, Rosemary; Forster, Mark J; Mulloy, Barbara; Gibbs, Roslyn V; Rider, Christopher C

2018-05-09

We have previously shown that the heterodimeric cytokine interleukin-12, and the homodimer of its larger subunit p40, both bind to heparin and heparan sulfate with relatively high affinity. In the present study we characterised these interactions using a series of chemically modified heparins as competitive inhibitors. Human interleukin-12 and p40 homodimer show indistinguishable binding profiles with a panel of heparin derivatives, but that of murine interleukin-12 is distinct. Heparin markedly protects the human and murine p40 subunits, but not the p35 subunits, from cleavage by the bacterial endoprotease LysC, further implicating the larger subunit as the location of the heparin binding site. Moreover the essential role of the carboxyterminal D3 domain in heparin binding is established by the failure of a truncated construct of the p40 subunit lacking this domain to bind. Predictive docking calculations indicate that a cluster of basic residues at the tip of the exposed C'D' loop within D3 is important in heparin binding. However since the human and murine C'D' loops differ considerably in length, the mode and three dimensional orientation of heparin binding are likely to differ substantially between the human and murine p40s. Thus overall the binding of IL-12 via its p40 subunit to heparin-related polysaccharides of the extracellular matrix appears to be functionally important since it has been conserved across mammalian species despite this structural divergence. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Vimentin filament precursors exchange subunits in an ATP-dependent manner

PubMed Central

Robert, Amélie; Rossow, Molly J.; Hookway, Caroline; Adam, Stephen A.; Gelfand, Vladimir I.

2015-01-01

Intermediate filaments (IFs) are a component of the cytoskeleton capable of profound reorganization in response to specific physiological situations, such as differentiation, cell division, and motility. Various mechanisms were proposed to be responsible for this plasticity depending on the type of IF polymer and the biological context. For example, recent studies suggest that mature vimentin IFs (VIFs) undergo rearrangement by severing and reannealing, but direct subunit exchange within the filament plays little role in filament dynamics at steady state. Here, we studied the dynamics of subunit exchange in VIF precursors, called unit-length filaments (ULFs), formed by the lateral association of eight vimentin tetramers. To block vimentin assembly at the ULF stage, we used the Y117L vimentin mutant (vimentinY117L). By tagging vimentinY117L with a photoconvertible protein mEos3.2 and photoconverting ULFs in a limited area of the cytoplasm, we found that ULFs, unlike mature filaments, were highly dynamic. Subunit exchange among ULFs occurred within seconds and was limited by the diffusion of soluble subunits in the cytoplasm rather than by the association and dissociation of subunits from ULFs. Our data demonstrate that cells expressing vimentinY117L contained a large pool of soluble vimentin tetramers that was in rapid equilibrium with ULFs. Furthermore, vimentin exchange in ULFs required ATP, and ATP depletion caused a dramatic reduction of the soluble tetramer pool. We believe that the dynamic exchange of subunits plays a role in the regulation of ULF assembly and the maintenance of a soluble vimentin pool during the reorganization of filament networks. PMID:26109569

Assembly of the stator in Escherichia coli ATP synthase. Complexation of alpha subunit with other F1 subunits is prerequisite for delta subunit binding to the N-terminal region of alpha

PubMed Central

Senior, Alan E.; Muharemagi, Alma; Wilke-Mounts, Susan

2008-01-01

Alpha subunit of Escherichia coli ATP synthase was expressed with a C-terminal 6-His tag and purified. Pure alpha was monomeric, competent in nucleotide binding, and had normal N-terminal sequence. In F1-subunit dissociation/reassociation experiments it supported full reconstitution of ATPase, and reassociated complexes were able to bind to F1-depleted membranes with restoration of ATP-driven proton pumping. Therefore interaction between the stator delta subunit and the N-terminal residue 1-22 region of alpha occurred normally when pure alpha was complexed with other F1 subunits. On the other hand, three different types of experiment showed that no interaction occurred between pure delta and isolated alpha subunit. Unlike in F1, the N-terminal region of isolated alpha was not susceptible to trypsin cleavage. Therefore, during assembly of ATP synthase, complexation of alpha subunit with other F1 subunits is prerequisite for delta subunit binding to the N-terminal region of alpha. We suggest that the N-terminal 1-22 residues of alpha are sequestered in isolated alpha until released by binding of beta to alpha subunit. This prevents 1/1 delta/alpha complexes from forming, and provides a satisfactory explanation of the stoichiometry of one delta per three alpha seen in the F1 sector of ATP synthase, assuming that steric hindrance prevents binding of more than one delta to the alpha3/beta3 hexagon. The cytoplasmic fragment of the b subunit (bsol) did not bind to isolated alpha. It might also be that complexation of alpha with beta subunits is prerequisite for direct binding of stator b subunit to the F1-sector. PMID:17176112

Substitutions at the opening of the Rubisco central solvent channel affect holoenzyme stability and CO2/O 2 specificity but not activation by Rubisco activase.

PubMed

Esquivel, M Gloria; Genkov, Todor; Nogueira, Ana S; Salvucci, Michael E; Spreitzer, Robert J

2013-12-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the initial step of carbon metabolism in photosynthesis. The holoenzyme comprises eight large subunits, arranged as a tetramer of dimers around a central solvent channel that defines a fourfold axis of symmetry, and eight small subunits, arranged as two tetramers at the poles of the axis. The phylogenetically divergent small-subunit loops between β-strands A and B form the entrance to the solvent channel. In the green alga Chlamydomonas reinhardtii, Ile-58 from each of the four small-subunit βA-βB loops defines the minimal diameter of the channel opening. To understand the role of the central solvent channel in Rubisco function, directed mutagenesis and transformation of Chlamydomonas were employed to replace Ile-58 with Ala, Lys, Glu, Trp, or three Trp residues (I58W3) to close the entrance to the channel. The I58E, I58K, and I58W substitutions caused only small decreases in photosynthetic growth at 25 and 35 °C, whereas I58W3 had a substantial effect at both temperatures. The mutant enzymes had decreased carboxylation rates, but the I58W3 enzyme had decreases in both carboxylation and CO2/O2 specificity. The I58E, I58W, and I58W3 enzymes were inactivated at lower temperatures than wild-type Rubisco, and were degraded at slower rates under oxidative stress. However, these mutant enzymes were activated by Rubisco activase at normal rates, indicating that the structural transition required for carboxylation is not affected by altering the solvent channel opening. Structural dynamics alone may not be responsible for these distant effects on the Rubisco active site.

Fragmentation of the CRISPR-Cas Type I-B signature protein Cas8b.

PubMed

Richter, Hagen; Rompf, Judith; Wiegel, Julia; Rau, Kristina; Randau, Lennart

2017-11-01

CRISPR arrays are transcribed into long precursor RNA species, which are further processed into mature CRISPR RNAs (crRNAs). Cas proteins utilize these crRNAs, which contain spacer sequences that can be derived from mobile genetic elements, to mediate immunity during a reoccurring virus infection. Type I CRISPR-Cas systems are defined by the presence of different Cascade interference complexes containing large and small subunits that play major roles during target DNA selection. Here, we produce the protein and crRNA components of the Type I-B CRISPR-Cas complex of Clostridium thermocellum and Methanococcus maripaludis. The C. thermocellum Cascade complexes were reconstituted and analyzed via size-exclusion chromatography. Activity of the heterologous M. maripaludis CRISPR-Cas system was followed using phage lambda plaques assays. The reconstituted Type-I-B Cascade complex contains Cas7, Cas5, Cas6b and the large subunit Cas8b. Cas6b can be omitted from the reconstitution protocol. The large subunit Cas8b was found to be represented by two tightly associated protein fragments and a small C-terminal Cas8b segment was identified in recombinant complexes and C. thermocellum cell lysate. Production of Cas8b generates a small C-terminal fragment, which is suggested to fulfill the role of the missing small subunit. A heterologous, synthetic M. maripaludis Type I-B system is active in E. coli against phage lambda, highlighting a potential for genome editing using endogenous Type-I-B CRISPR-Cas machineries. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2017 Elsevier B.V. All rights reserved.

The nucleotide binding dynamics of human MSH2-MSH3 are lesion dependent.

PubMed

Owen, Barbara A L; H Lang, Walter; McMurray, Cynthia T

2009-05-01

Here we report that the human DNA mismatch complex MSH2-MSH3 recognizes small loops by a mechanism different from that of MSH2-MSH6 for single-base mismatches. The subunits MSH2 and MSH3 can bind either ADP or ATP with similar affinities. Upon binding to a DNA loop, however, MSH2-MSH3 adopts a single 'nucleotide signature', in which the MSH2 subunit is occupied by an ADP molecule and the MSH3 subunit is empty. Subsequent ATP binding and hydrolysis in the MSH3 subunit promote ADP-ATP exchange in the MSH2 subunit to yield a hydrolysis-independent ATP-MSH2-MSH3-ADP intermediate. Human MSH2-MSH3 and yeast Msh2-Msh6 both undergo ADP-ATP exchange in the Msh2 subunit but, apparently, have opposite requirements for ATP hydrolysis: ADP release from DNA-bound Msh2-Msh6 requires ATP stabilization in the Msh6 subunit, whereas ADP release from DNA-bound MSH2-MSH3 requires ATP hydrolysis in the MSH3 subunit. We propose a model in which lesion binding converts MSH2-MSH3 into a distinct nucleotide-bound form that is poised to be a molecular sensor for lesion specificity.

Purification and properties of a third form of anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferase from the enterobacteriaceae

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

Largen, M.; Mills, S.E.; Rowe, J.

1978-01-25

Anthranilate-5-phosphoribosypyrophosphate phosphoribosyltransferase was purified from the bacterium Erwinia carotovora, a member of the Enterobacteriaceae. The enzyme was homogeneous according to the criteria of gel electrophoresis and NH/sub 2/-terminal amino acid sequence analysis. The molecular weight of the enzyme as determined on a calibrated Sephadex G-200 column was 67,000 +- 2,000. Sodium dodecyl sulfate-polyacrylamide gels gave a subunit molecular weight of 40,000 +- 1,000, suggesting that the enzyme was a dimer. A comparison of the NH/sub 2/-terminal sequence of the enzyme with the (previously determined) homologue from Serratia marcescens, a monomer with a molecular weight of 45,000, showed that the largermore » Serratia subunit came into register with amino acid 14 of the Erwinia subunit. The register for the length of the known overlap, 26 amino acids, was highly conserved.« less

Nmd3p Is a Crm1p-Dependent Adapter Protein for Nuclear Export of the Large Ribosomal Subunit

PubMed Central

Ho, Jennifer Hei-Ngam; Kallstrom, George; Johnson, Arlen W.

2000-01-01

In eukaryotic cells, nuclear export of nascent ribosomal subunits through the nuclear pore complex depends on the small GTPase Ran. However, neither the nuclear export signals (NESs) for the ribosomal subunits nor the receptor proteins, which recognize the NESs and mediate export of the subunits, have been identified. We showed previously that Nmd3p is an essential protein from yeast that is required for a late step in biogenesis of the large (60S) ribosomal subunit. Here, we show that Nmd3p shuttles and that deletion of the NES from Nmd3p leads to nuclear accumulation of the mutant protein, inhibition of the 60S subunit biogenesis, and inhibition of the nuclear export of 60S subunits. Moreover, the 60S subunits that accumulate in the nucleus can be coimmunoprecipitated with the NES-deficient Nmd3p. 60S subunit biogenesis and export of truncated Nmd3p were restored by the addition of an exogenous NES. To identify the export receptor for Nmd3p we show that Nmd3p shuttling and 60S export is blocked by the Crm1p-specific inhibitor leptomycin B. These results identify Crm1p as the receptor for Nmd3p export. Thus, export of the 60S subunit is mediated by the adapter protein Nmd3p in a Crm1p-dependent pathway. PMID:11086007

Nucleoplasmin-like domain of FKBP39 from Drosophila melanogaster forms a tetramer with partly disordered tentacle-like C-terminal segments

PubMed Central

Kozłowska, Małgorzata; Tarczewska, Aneta; Jakób, Michał; Bystranowska, Dominika; Taube, Michał; Kozak, Maciej; Czarnocki-Cieciura, Mariusz; Dziembowski, Andrzej; Orłowski, Marek; Tkocz, Katarzyna; Ożyhar, Andrzej

2017-01-01

Nucleoplasmins are a nuclear chaperone family defined by the presence of a highly conserved N-terminal core domain. X-ray crystallographic studies of isolated nucleoplasmin core domains revealed a β-propeller structure consisting of a set of five monomers that together form a stable pentamer. Recent studies on isolated N-terminal domains from Drosophila 39-kDa FK506-binding protein (FKBP39) and from other chromatin-associated proteins showed analogous, nucleoplasmin-like (NPL) pentameric structures. Here, we report that the NPL domain of the full-length FKBP39 does not form pentameric complexes. Multi-angle light scattering (MALS) and sedimentation equilibrium ultracentrifugation (SE AUC) analyses of the molecular mass of the full-length protein indicated that FKBP39 forms homotetrameric complexes. Molecular models reconstructed from small-angle X-ray scattering (SAXS) revealed that the NPL domain forms a stable, tetrameric core and that FK506-binding domains are linked to it by intrinsically disordered, flexible chains that form tentacle-like segments. Analyses of full-length FKBP39 and its isolated NPL domain suggested that the distal regions of the polypeptide chain influence and determine the quaternary conformation of the nucleoplasmin-like protein. These results provide new insights regarding the conserved structure of nucleoplasmin core domains and provide a potential explanation for the importance of the tetrameric structural organization of full-length nucleoplasmins. PMID:28074868

Structural and molecular characterization of the prefoldin beta subunit from Thermococcus strain KS-1.

PubMed

Kida, Hiroshi; Sugano, Yuri; Iizuka, Ryo; Fujihashi, Masahiro; Yohda, Masafumi; Miki, Kunio

2008-11-14

Prefoldin (PFD) is a heterohexameric molecular chaperone that is found in eukaryotic cytosol and archaea. PFD is composed of alpha and beta subunits and forms a "jellyfish-like" structure. PFD binds and stabilizes nascent polypeptide chains and transfers them to group II chaperonins for completion of their folding. Recently, the whole genome of Thermococcus kodakaraensis KOD1 was reported and shown to contain the genes of two alpha and two beta subunits of PFD. The genome of Thermococcus strain KS-1 also possesses two sets of alpha (alpha1 and alpha2) and beta subunits (beta1 and beta2) of PFD (TsPFD). However, the functions and roles of each of these PFD subunits have not been investigated in detail. Here, we report the crystal structure of the TsPFD beta1 subunit at 1.9 A resolution and its functional analysis. TsPFD beta1 subunits form a tetramer with four coiled-coil tentacles resembling the jellyfish-like structure of heterohexameric PFD. The beta hairpin linkers of beta1 subunits assemble to form a beta barrel "body" around a central fourfold axis. Size-exclusion chromatography and multi-angle light-scattering analyses show that the beta1 subunits form a tetramer at pH 8.0 and a dimer of tetramers at pH 6.8. The tetrameric beta1 subunits can protect against aggregation of relatively small proteins, insulin or lysozyme. The structural and biochemical analyses imply that PFD beta1 subunits act as molecular chaperones in living cells of some archaea.

Peering down the barrel of a bacteriophage portal: the genome packaging and release valve in p22.

PubMed

Tang, Jinghua; Lander, Gabriel C; Olia, Adam S; Olia, Adam; Li, Rui; Casjens, Sherwood; Prevelige, Peter; Cingolani, Gino; Baker, Timothy S; Johnson, John E

2011-04-13

The encapsidated genome in all double-strand DNA bacteriophages is packaged to liquid crystalline density through a unique vertex in the procapsid assembly intermediate, which has a portal protein dodecamer in place of five coat protein subunits. The portal orchestrates DNA packaging and exit, through a series of varying interactions with the scaffolding, terminase, and closure proteins. Here, we report an asymmetric cryoEM reconstruction of the entire P22 virion at 7.8 Å resolution. X-ray crystal structure models of the full-length portal and of the portal lacking 123 residues at the C terminus in complex with gene product 4 (Δ123portal-gp4) obtained by Olia et al. (2011) were fitted into this reconstruction. The interpreted density map revealed that the 150 Å, coiled-coil, barrel portion of the portal entraps the last DNA to be packaged and suggests a mechanism for head-full DNA signaling and transient stabilization of the genome during addition of closure proteins. Copyright © 2011 Elsevier Ltd. All rights reserved.

Cotesia vestalis parasitization suppresses expression of a Plutella xylostella thioredoxin

USDA-ARS?s Scientific Manuscript database

Thioredoxins (Trxs) are a family of small, highly conserved and ubiquitous proteins involved in protecting organisms against toxic reactive oxygen species (ROS). In this study, a typical thioredoxin gene, PxTrx, was isolated from Plutella xylostella. The full-length cDNA sequence is composed of 959 ...

California mild CTV strains that break resistance in Trifoliate Orange

USDA-ARS?s Scientific Manuscript database

This is the final report of a project to characterize California isolates of Citrus tristeza virus (CTV) that replicate in Poncirus trifoliata (trifoliate orange). Next Generation Sequencing (NGS) of viral small interfering RNAs (siRNAs) and assembly of full-length sequences of mild California CTV i...

Regulation of RNA polymerase III transcription during transformation of human IMR90 fibroblasts with defined genetic elements.

PubMed

Durrieu-Gaillard, Stéphanie; Dumay-Odelot, Hélène; Boldina, Galina; Tourasse, Nicolas J; Allard, Delphine; André, Fabrice; Macari, Françoise; Choquet, Armelle; Lagarde, Pauline; Drutel, Guillaume; Leste-Lasserre, Thierry; Petitet, Marion; Lesluyes, Tom; Lartigue-Faustin, Lydia; Dupuy, Jean-William; Chibon, Frédéric; Roeder, Robert G; Joubert, Dominique; Vagner, Stéphan; Teichmann, Martin

2018-01-01

RNA polymerase (Pol) III transcribes small untranslated RNAs that are essential for cellular homeostasis and growth. Its activity is regulated by inactivation of tumor suppressor proteins and overexpression of the oncogene c-MYC, but the concerted action of these tumor-promoting factors on Pol III transcription has not yet been assessed. In order to comprehensively analyse the regulation of Pol III transcription during tumorigenesis we employ a model system that relies on the expression of five genetic elements to achieve cellular transformation. Expression of these elements in six distinct transformation intermediate cell lines leads to the inactivation of TP53, RB1, and protein phosphatase 2A, as well as the activation of RAS and the protection of telomeres by TERT, thereby conducting to full tumoral transformation of IMR90 fibroblasts. Transformation is accompanied by moderately enhanced levels of a subset of Pol III-transcribed RNAs (7SK; MRP; H1). In addition, mRNA and/or protein levels of several Pol III subunits and transcription factors are upregulated, including increased protein levels of TFIIIB and TFIIIC subunits, of SNAPC1 and of Pol III subunits. Strikingly, the expression of POLR3G and of SNAPC1 is strongly enhanced during transformation in this cellular transformation model. Collectively, our data indicate that increased expression of several components of the Pol III transcription system accompanied by a 2-fold increase in steady state levels of a subset of Pol III RNAs is sufficient for sustaining tumor formation.

The electrophoretically 'slow' and 'fast' forms of the alpha 2-macroglobulin molecule.

PubMed Central

Barrett, A J; Brown, M A; Sayers, C A

1979-01-01

alpha 2-Macroglobulin (alpha 2M) was isolated from human plasma by a four-step procedure: poly(ethylene glyco) fractionation, gel chromatography, euglobulin precipitation and immunoadsorption. No contaminants were detected in the final preparations by electrophoresis or immunoprecipitation. The protein ran as a single slow band in gel electrophoresis, and was designated 'S-alpha 2M'. S-alpha 2M bound about 2 mol of trypsin/mol. Treatment of S-alpha 2M with a proteinase or ammonium salts produced a form of the molecule more mobile in electrophoresis, and lacking proteinase-binding activity (F-alpha 2M). The electrophoretic mobility of the F-alpha 2M resulting from reaction with NH4+ salts was identical with that of proteinase complexes. We attribute the change in electrophoretic mobility of the alpha 2M to a conformation change, but there was no evidence of a change in pI or Strokes radius. Electrophoresis of S-alpha 2M in the presence of sodium dodecylsulphate gave results consistent with the view that the alpha 2M molecule is a tetramer of identical subunits, assembled as a non-covalent pair of disulphide-linked dimers. Some of the subunits seemed to be 'nicked' into two-thires-length and one-third-length chains, however. This was not apparent with F-alpha 2M produced by ammonium salts. F-alpha 2M produced by trypsin showed two new bands attributable to cleavage of the subunit polypeptide chain near the middle. Immunoassays of F-alpha 2M gave 'rockets' 12-29% lower than those with S-alpha 2M. The nature of the interactions between subunits in S-alpha 2M and F-alpha 2M was investigated by treating each form with glutaraldehyde before electrophoresis in the presence of sodium dodecyl sulphate. A much greater degree of cross-linking was observed with the F-alpha 2M, indicating that the subunits interact most closely in this form of the molecule. Exposure of S-alpha 2M to 3 M-urea or pH3 resulted in dissociation to the disulphide-bonded half-molecules; these did not show the proteinase-binding activity characteristic of the intact alpha 2M. F-alpha 2M was less easily dissociated than was S-alpha 2M. S-alpha 2M was readily dissociated to the quarter-subunits by mild reduction, with the formation of 3-4 new thiol groups per subunit. Inact reactive alpha 2M could then be regenerated in high yield by reoxidation of the subunits. F-alpha 2M formed by reaction with a proteinase or ammonium salts was not dissociated under the same conditions, although the interchain disulphide bonds were reduced. If the thiol groups of the quarter-subunits of S-alpha 2M were blocked by carboxymethylation, oxidative reassociation did not occur. Nevertheless treatment of these subunits with methylammonium salts or a proteinase caused the reassembly of half-molecules and intact (F-) tetramers. It is emphasized that F-alpha 2M does not have the properties of a denatured form of the protein... Images Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:91367

Unexpected High Digestion Rate of Cooked Starch by the Ct-Maltase-Glucoamylase Small Intestine Mucosal α-Glucosidase Subunit

PubMed Central

Lin, Amy Hui-Mei; Nichols, Buford L.; Quezada-Calvillo, Roberto; Avery, Stephen E.; Sim, Lyann; Rose, David R.; Naim, Hassan Y.; Hamaker, Bruce R.

2012-01-01

For starch digestion to glucose, two luminal α-amylases and four gut mucosal α-glucosidase subunits are employed. The aim of this research was to investigate, for the first time, direct digestion capability of individual mucosal α-glucosidases on cooked (gelatinized) starch. Gelatinized normal maize starch was digested with N- and C-terminal subunits of recombinant mammalian maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) of varying amounts and digestion periods. Without the aid of α-amylase, Ct-MGAM demonstrated an unexpected rapid and high digestion degree near 80%, while other subunits showed 20 to 30% digestion. These findings suggest that Ct-MGAM assists α-amylase in digesting starch molecules and potentially may compensate for developmental or pathological amylase deficiencies. PMID:22563462

Unexpected high digestion rate of cooked starch by the Ct-maltase-glucoamylase small intestine mucosal α-glucosidase subunit.

PubMed

Lin, Amy Hui-Mei; Nichols, Buford L; Quezada-Calvillo, Roberto; Avery, Stephen E; Sim, Lyann; Rose, David R; Naim, Hassan Y; Hamaker, Bruce R

2012-01-01

For starch digestion to glucose, two luminal α-amylases and four gut mucosal α-glucosidase subunits are employed. The aim of this research was to investigate, for the first time, direct digestion capability of individual mucosal α-glucosidases on cooked (gelatinized) starch. Gelatinized normal maize starch was digested with N- and C-terminal subunits of recombinant mammalian maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) of varying amounts and digestion periods. Without the aid of α-amylase, Ct-MGAM demonstrated an unexpected rapid and high digestion degree near 80%, while other subunits showed 20 to 30% digestion. These findings suggest that Ct-MGAM assists α-amylase in digesting starch molecules and potentially may compensate for developmental or pathological amylase deficiencies.

The structure of subunit E of the Pyrococcus horikoshii OT3 A-ATP synthase gives insight into the elasticity of the peripheral stalk.

PubMed

Balakrishna, Asha Manikkoth; Hunke, Cornelia; Grüber, Gerhard

2012-07-13

A(1)A(O) ATP synthases are the major energy converters of archaea. They are composed of an A(1) region that synthesizes ATP and an integral part A(O) that conducts ions. Subunit E is a component of the peripheral stalk that links the A(1) with the A(O) part of the A-ATP synthase. We have determined the crystal structure of the entire subunit E (PhE) of the Pyrococcus horikoshii OT3 A-ATP synthase at 3.6 Å resolution. The structure reveals an extended S-shaped N-terminal α-helix with 112.29 Å in length, followed by a globular head group. The S-shaped feature, common in elastic connectors and spacers, would facilitate the storage of transient elastic energy during rotary motion in the enzyme. The structure has been superimposed into the asymmetric peripheral stalks of the three-dimensional reconstruction of the Pyrococcus furiosus enzyme, revealing that the S-shaped subunit PhE fits well into the bent peripheral stalk, whereas the previously solved E subunit structure (3.1 Å resolution) of Thermus thermophilus A-ATP synthase is well accommodated in the density of the straight stator domain. The different features of the two stalk subunits are discussed in light of a novel coupling mechanism in A-ATP synthases proposed to differ from the Wankel engine of F-ATP synthases. Copyright © 2012 Elsevier Ltd. All rights reserved.

Group I introns are inherited through common ancestry in the nuclear-encoded rRNA of Zygnematales (Charophyceae).

PubMed Central

Bhattacharya, D; Surek, B; Rüsing, M; Damberger, S; Melkonian, M

1994-01-01

Group I introns are found in organellar genomes, in the genomes of eubacteria and phages, and in nuclear-encoded rRNAs. The origin and distribution of nuclear-encoded rRNA group I introns are not understood. To elucidate their evolutionary relationships, we analyzed diverse nuclear-encoded small-subunit rRNA group I introns including nine sequences from the green-algal order Zygnematales (Charophyceae). Phylogenetic analyses of group I introns and rRNA coding regions suggest that lateral transfers have occurred in the evolutionary history of group I introns and that, after transfer, some of these elements may form stable components of the host-cell nuclear genomes. The Zygnematales introns, which share a common insertion site (position 1506 relative to the Escherichia coli small-subunit rRNA), form one subfamily of group I introns that has, after its origin, been inherited through common ancestry. Since the first Zygnematales appear in the middle Devonian within the fossil record, the "1506" group I intron presumably has been a stable component of the Zygnematales small-subunit rRNA coding region for 350-400 million years. PMID:7937917

Biology of Symbioses between Marine Invertebrates and Intracellular Bacteria

DTIC Science & Technology

1990-01-30

bisphosphate carboxylase We designed from published sequence information oligonucleotide primers which are complementary to conserved regions on RubisCO ...large and small subunit genes. These primers were used successfully to amplify using polymerase chain reaction (PCR) specific regions of RubisCO ...for the large subunit of ribulose bisphosphate carboxylase/oxygenase ( RubisCO ) to symbiont DNA shows that the symbionts from both deep-sea and shallow

Phylogenetic relationships of the Gomphales based on nuc-25S-rDNA, mit-12S-rDNA, and mit-atp6-DNA combined sequences

Treesearch

Admir J. Giachini; Kentaro Hosaka; Eduardo Nouhra; Joseph Spatafora; James M. Trappe

2010-01-01

Phylogenetic relationships among Geastrales, Gomphales, Hysterangiales, and Phallales were estimated via combined sequences: nuclear large subunit ribosomal DNA (nuc-25S-rDNA), mitochondrial small subunit ribosomal DNA (mit-12S-rDNA), and mitochondrial atp6 DNA (mit-atp6-DNA). Eighty-one taxa comprising 19 genera and 58 species...

Topography of succinate dehydrogenase in the mitochondrial inner membrane. A study using limited proteolysis and immunoblotting.

PubMed Central

Clarkson, G H; Neagle, J; Lindsay, J G

1991-01-01

The arrangement of the large (70,000-Mr) and small (30,000-Mr) subunits of succinate dehydrogenase in the mitochondrial inner membrane was investigated by immunoblot analysis of bovine heart mitochondria (right-side-out, outer membrane disrupted) or submitochondrial particles (inside-out) that had been subjected to surface-specific proteolysis. Both subunits were resistant to proteinase treatment provided that the integrity of the inner membrane was preserved, suggesting that neither subunit is exposed at the cytoplasmic surface of the membrane. The bulk of the small subunit appears to protrude into the matrix compartment, since the 30,000-Mr polypeptide is degraded extensively during limited proteolysis of submitochondrial particles without the appearance of an immunologically reactive membrane-associated fragment: moreover, a soluble 27,000-Mr peptide derived from this subunit is observed transiently on incubation with trypsin. Similar data obtained from the large subunit suggest that this polypeptide interacts with the matrix side of the inner membrane via two distinct domains; these are detected as stable membrane-associated fragments of 32,000 Mr and 27,000 Mr after treatment of submitochondrial particles with papain or proteinase K, although the 27,000-Mr fragment can be degraded further to low-Mr peptides with trypsin or alpha-chymotrypsin. A stable 32,000-34,000-Mr fragment is generated by a variety of specific and non-specific proteinases, indicating that it may be embedded largely within the lipid bilayer, or is inaccessible to proteolytic attack owing to its proximity to the surface of the intact membrane, possibly interacting with the hydrophobic membrane anchoring polypeptides of the succinate: ubiquinone reductase complex. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:1996968

Dual role of imidazole as activator/inhibitor of sweet almond (Prunus dulcis) β-glucosidase.

PubMed

Caramia, Sara; Gatius, Angela Gala Morena; Dal Piaz, Fabrizio; Gaja, Denis; Hochkoeppler, Alejandro

2017-07-01

The activity of Prunus dulcis (sweet almond) β-glucosidase at the expense of p -nitrophenyl-β-d-glucopyranoside at pH 6 was determined, both under steady-state and pre-steady-state conditions. Using crude enzyme preparations, competitive inhibition by 1-5 mM imidazole was observed under both kinetic conditions tested. However, when imidazole was added to reaction mixtures at 0.125-0.250 mM, we detected a significant enzyme activation. To further inspect this effect exerted by imidazole, β-glucosidase was purified to homogeneity. Two enzyme isoforms were isolated, i.e. a full-length monomer, and a dimer containing a full-length and a truncated subunit. Dimeric β-glucosidase was found to perform much better than the monomeric enzyme, independently of the kinetic conditions used to assay enzyme activity. In addition, the sensitivity towards imidazole was found to differ between the two isoforms. While monomeric enzyme was indeed found to be relatively insensitive to imidazole, dimeric β-glucosidase was observed to be significantly activated by 0.125-0.250 mM imidazole under pre-steady-state conditions. Further, steady-state assays revealed that the addition of 0.125 mM imidazole to reaction mixtures increases the K m of dimeric enzyme from 2.3 to 6.7 mM. The activation of β-glucosidase dimer by imidazole is proposed to be exerted via a conformational transition poising the enzyme towards proficient catalysis.

Studies on the regulation of the human E1 subunit of the 2-oxoglutarate dehydrogenase complex, including the identification of a novel calcium-binding site.

PubMed

Armstrong, Craig T; Anderson, J L Ross; Denton, Richard M

2014-04-15

The regulation of the 2-oxoglutarate dehydrogenase complex is central to intramitochondrial energy metabolism. In the present study, the active full-length E1 subunit of the human complex has been expressed and shown to be regulated by Ca2+, adenine nucleotides and NADH, with NADH exerting a major influence on the K0.5 value for Ca2+. We investigated two potential Ca2+-binding sites on E1, which we term site 1 (D114ADLD) and site 2 (E139SDLD). Comparison of sequences from vertebrates with those from Ca2+-insensitive non-vertebrate complexes suggest that site 1 may be the more important. Consistent with this view, a mutated form of E1, D114A, shows a 6-fold decrease in sensitivity for Ca2+, whereas variant ∆site1 (in which the sequence of site 1 is replaced by A114AALA) exhibits an almost complete loss of Ca2+ activation. Variant ∆site2 (in which the sequence is replaced with A139SALA) shows no measurable change in Ca2+ sensitivity. We conclude that site 1, but not site 2, forms part of a regulatory Ca2+-binding site, which is distinct from other previously described Ca2+-binding sites.

Molecular determinants of tetramerization in the KcsA cytoplasmic domain.

PubMed

Kamnesky, Guy; Hirschhorn, Orel; Shaked, Hadassa; Chen, Jingfei; Yao, Lishan; Chill, Jordan H

2014-10-01

The cytoplasmic C-terminal domain (CTD) of KcsA, a bacterial homotetrameric potassium channel, is an amphiphilic domain that forms a helical bundle with four-fold symmetry mediated by hydrophobic and electrostatic interactions. Previously we have established that a CTD-derived 34-residue peptide associates into a tetramer in a pH-dependent manner (Kamnesky et al., JMB 2012;418:237-247). Here we further investigate the molecular determinants of tetramer formation in the CTD by characterizing the kinetics of monomer-tetramer equilibrium for 10 alanine mutants using NMR, sedimentation equilibrium (SE) and molecular dynamics simulation. NMR and SE concur in finding single-residue contributions to tetramer stability to be in the 0.5 to 3.5 kcal/mol range. Hydrophobic interactions between residues lining the tetramer core generally contributed more to formation of tetramer than electrostatic interactions between residues R147, D149 and E152. In particular, alanine replacement of residue R147, a key contributor to inter-subunit salt bridges, resulted in only a minor effect on tetramer dissociation. Mutations outside of the inter-subunit interface also influenced tetramer stability by affecting the tetramerization on-rate, possibly by changing the inherent helical propensity of the peptide. These findings are interpreted in the context of established paradigms of protein-protein interactions and protein folding, and lay the groundwork for further studies of the CTD in full-length KcsA channels. © 2014 The Protein Society.

Molecular determinants of tetramerization in the KcsA cytoplasmic domain

PubMed Central

Kamnesky, Guy; Hirschhorn, Orel; Shaked, Hadassa; Chen, Jingfei; Yao, Lishan; Chill, Jordan H

2014-01-01

The cytoplasmic C-terminal domain (CTD) of KcsA, a bacterial homotetrameric potassium channel, is an amphiphilic domain that forms a helical bundle with four-fold symmetry mediated by hydrophobic and electrostatic interactions. Previously we have established that a CTD-derived 34-residue peptide associates into a tetramer in a pH-dependent manner (Kamnesky et al., JMB 2012;418:237-247). Here we further investigate the molecular determinants of tetramer formation in the CTD by characterizing the kinetics of monomer-tetramer equilibrium for 10 alanine mutants using NMR, sedimentation equilibrium (SE) and molecular dynamics simulation. NMR and SE concur in finding single-residue contributions to tetramer stability to be in the 0.5 to 3.5 kcal/mol range. Hydrophobic interactions between residues lining the tetramer core generally contributed more to formation of tetramer than electrostatic interactions between residues R147, D149 and E152. In particular, alanine replacement of residue R147, a key contributor to inter-subunit salt bridges, resulted in only a minor effect on tetramer dissociation. Mutations outside of the inter-subunit interface also influenced tetramer stability by affecting the tetramerization on-rate, possibly by changing the inherent helical propensity of the peptide. These findings are interpreted in the context of established paradigms of protein-protein interactions and protein folding, and lay the groundwork for further studies of the CTD in full-length KcsA channels. PMID:25042120

Molecular cloning, characterization and functional analysis of QRFP in orange-spotted grouper (Epinephelus coioides).

PubMed

Shu, Hu; Chen, Huapu; Liu, Yun; Yang, Lidong; Yang, Yuqing; Zhang, Haifa

2014-10-01

The peptide QRFP plays an important role in the regulation of vertebrate feeding behavior. In this study, we cloned the full length cDNA of a QRFP precursor in a teleost fish, the orange-spotted grouper (Epinephelus coioides). Sequence analysis has shown that the functional regions of QRFP in other vertebrates (QRFP-25 and QRFP-7) are conserved in orange-spotted grouper. RT-PCR demonstrated that the pre-processed mRNA of QRFP is widely expressed in orange-spotted grouper. Three days of food deprivation did not change the hypothalamic pre-processed QRFP expression. However, QRFP expression significantly increased when the fish were reefed after three days of fasting. Intraperitoneal injection of QRFP-25 peptide to orange-spotted grouper suppressed expression of orexin, but elevated expression of pro-opiomelanocortin (POMC) in the hypothalamus. We also investigated the effects of QRFP-25 on the expression of reproductive genes. The peptide suppressed the expression of seabream-type gonadotropin-releasing hormones (sbGnRH), luteinizing hormone beta subunit (LHβ) and follicle-stimulating hormone beta subunit (FSHβ) in vivo, as well as inhibited the expression of LHβ and FSHβ in pituitary cells in primary culture. Our results indicate that QRFP may play an inhibitory role in the regulation of feeding behavior and reproduction in orange-spotted grouper. Copyright © 2014 Elsevier Inc. All rights reserved.

Role of the Cytoplasmic N-terminal Cap and Per-Arnt-Sim (PAS) Domain in Trafficking and Stabilization of Kv11.1 Channels*

PubMed Central

Ke, Ying; Hunter, Mark J.; Ng, Chai Ann; Perry, Matthew D.; Vandenberg, Jamie I.

2014-01-01

The N-terminal cytoplasmic region of the Kv11.1a potassium channel contains a Per-Arnt-Sim (PAS) domain that is essential for the unique slow deactivation gating kinetics of the channel. The PAS domain has also been implicated in the assembly and stabilization of the assembled tetrameric channel, with many clinical mutants in the PAS domain resulting in reduced stability of the domain and reduced trafficking. Here, we use quantitative Western blotting to show that the PAS domain is not required for normal channel trafficking nor for subunit-subunit interactions, and it is not necessary for stabilizing assembled channels. However, when the PAS domain is present, the N-Cap amphipathic helix must also be present for channels to traffic to the cell membrane. Serine scan mutagenesis of the N-Cap amphipathic helix identified Leu-15, Ile-18, and Ile-19 as residues critical for the stabilization of full-length proteins when the PAS domain is present. Furthermore, mutant cycle analysis experiments support recent crystallography studies, indicating that the hydrophobic face of the N-Cap amphipathic helix interacts with a surface-exposed hydrophobic patch on the core of the PAS domain to stabilize the structure of this critical gating domain. Our data demonstrate that the N-Cap amphipathic helix is critical for channel stability and trafficking. PMID:24695734

Morphology and small subunit rDNA-based phylogeny of a new Henneguya species, infecting the ornamental fish Corydoras leucomelas from the Peruvian Amazon.

PubMed

Mathews, Patrick D; Naldoni, Juliana; Adriano, Edson A

2017-12-01

A new species of Myxosporea, Henneguya loreotoensis n. sp. is described parasitizing the gill filaments from 17 of 35 specimens (48.5%) of Corydoras leucomelas (Siluriformes: Callichthyidae) caught in the Nanay River, near village Ninarumi, in the Loreto state, Peru. Mature spores were ellipsoidal in shape from the frontal view, measuring 36.2±0.1μm (36.1-36.3) in total length, 14.3±0.1μm (14.2-14.4) in body length, 5.1±0.1μm (4.9-5.3) in width and 21.9±0.1μm (21.8-22.0) in the caudal process. The two polar capsules were symmetrical and elongated, measuring 5.1±0.1μm (4.9-5.3) in length and 2.4±0.2μm (2.1-2.7) in width, containing a polar filament with five coils arranged obliquely to the longitudinal axis. The sporoplasm was binucleate. Partial sequencing of the ssu-rDNA of H. loretoensis n. sp. resulted in a total of 1676 nucleotides, and this sequence did not match any of the myxozoan available in the GenBank. The phylogenetic analysis shows H. loretoensis n. sp. as a sister species of Henneguya paraensis, another amazonian myxozoan parasite of Cichla temensis (Perciformes: Cichlidae). Copyright © 2017 Elsevier B.V. All rights reserved.

The small GTPase Arl8b regulates assembly of the mammalian HOPS complex on lysosomes

PubMed Central

Khatter, Divya; Raina, Vivek B.; Dwivedi, Devashish; Sindhwani, Aastha; Bahl, Surbhi; Sharma, Mahak

2015-01-01

The homotypic fusion and protein sorting (HOPS) complex is a multi-subunit complex conserved from yeast to mammals that regulates late endosome and lysosome fusion. However, little is known about how the HOPS complex is recruited to lysosomes in mammalian cells. Here, we report that the small GTPase Arl8b, but not Rab7 (also known as RAB7A), is essential for membrane localization of the human (h)Vps41 subunit of the HOPS complex. Assembly of the core HOPS subunits to Arl8b- and hVps41-positive lysosomes is guided by their subunit–subunit interactions. RNA interference (RNAi)-mediated depletion of hVps41 resulted in the impaired degradation of EGFR that was rescued upon expression of wild-type but not an Arl8b-binding-defective mutant of hVps41, suggesting that Arl8b-dependent lysosomal localization of hVps41 is required for its endocytic function. Furthermore, we have also identified that the Arl8b effector SKIP (also known as PLEKHM2) interacts with and recruits HOPS subunits to Arl8b and kinesin-positive peripheral lysosomes. Accordingly, RNAi-mediated depletion of SKIP impaired lysosomal trafficking and degradation of EGFR. These findings reveal that Arl8b regulates the association of the human HOPS complex with lysosomal membranes, which is crucial for the function of this tethering complex in endocytic degradation. PMID:25908847

A protein with an inactive pterin-4a-carbinolamine dehydratase domain is required for Rubisco biogenesis in plants.

PubMed

Feiz, Leila; Williams-Carrier, Rosalind; Belcher, Susan; Montano, Monica; Barkan, Alice; Stern, David B

2014-12-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) plays a critical role in sustaining life by catalysis of carbon fixation in the Calvin-Benson pathway. Incomplete knowledge of the assembly pathway of chloroplast Rubisco has hampered efforts to fully delineate the enzyme's properties, or seek improved catalytic characteristics via directed evolution. Here we report that a Mu transposon insertion in the Zea mays (maize) gene encoding a chloroplast dimerization co-factor of hepatocyte nuclear factor 1 (DCoH)/pterin-4α-carbinolamine dehydratases (PCD)-like protein is the causative mutation in a seedling-lethal, Rubisco-deficient mutant named Rubisco accumulation factor 2 (raf2-1). In raf2 mutants newly synthesized Rubisco large subunit accumulates in a high-molecular weight complex, the formation of which requires a specific chaperonin 60-kDa isoform. Analogous observations had been made previously with maize mutants lacking the Rubisco biogenesis proteins RAF1 and BSD2. Chemical cross-linking of maize leaves followed by immunoprecipitation with antibodies to RAF2, RAF1 or BSD2 demonstrated co-immunoprecipitation of each with Rubisco small subunit, and to a lesser extent, co-immunoprecipitation with Rubisco large subunit. We propose that RAF2, RAF1 and BSD2 form transient complexes with the Rubisco small subunit, which in turn assembles with the large subunit as it is released from chaperonins. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

Fluorescent speckle microscopy of microtubules: how low can you go?

PubMed

Waterman-Storer, C M; Salmon, E D

1999-12-01

Fluorescent speckle microscopy (FSM) is a new technique for visualizing the movement, assembly, and turnover of macromolecular assemblies like the cytoskeleton in living cells. In this method, contrast is created by coassembly of a small fraction of fluorescent subunits in a pool of unlabeled subunits. Random variation in association creates a nonuniform "fluorescent speckle" pattern. Fluorescent speckle movements in time-lapse recordings stand out to the eye and can be measured. Because fluorescent speckles represent fiduciary marks on the polymer lattice, FSM provides the opportunity for the first time to see the 2- and 3-dimensional trajectories of lattice movements within large arrays of polymers as well as identifying sites of assembly and disassembly of individual polymers. The technique works with either microinjection of fluorescently labeled subunits or expression of subunits ligated to green fluorescent protein (GFP). We have found for microtubules assembled in vitro that speckles containing one fluorophore can be detected and recorded using a conventional wide-field epi-fluorescence light microscope and digital imaging with a low noise cooled CCD camera. In living cells, optimal speckle contrast occurs at fractions of labeled tubulin of approximately 0.1-0.5% where the fluorescence of each speckle corresponds to one to seven fluorophores per resolvable unit (approximately 0.27 microm) in the microscope. This small fraction of labeled subunits significantly reduces out-of-focus fluorescence and greatly improves visibility of fluorescently labeled structures and their dynamics in thick regions of living cells.

Perception of Age, Attractiveness, and Tiredness After Isolated and Combined Facial Subunit Aging.

PubMed

Forte, Antonio Jorge; Andrew, Tom W; Colasante, Cesar; Persing, John A

2015-12-01

Patients often seek help to redress aging that affects various regions of the face (subunits). The purpose of this study was to determine how aging of different facial subunits impacts perception of age, attractiveness, and tiredness. Frontal and lateral view facial photographs of a middle-aged woman were modified using imaging software to independently age different facial features. Sixty-six subjects were administered with a questionnaire, and presented with a baseline unmodified picture and others containing different individual or grouped aging of facial subunits. Test subjects were asked to estimate the age of the subject in the image and quantify (0-10 scale) how "tired" and "attractive" they appeared. Facial subunits were organized following rank assignment regarding impact on perception of age, attractiveness, and tiredness. The correlation coefficient between age and attractiveness had a strong inverse relationship of approximately -0.95 in both lateral and frontal views. From most to least impact in age, the rank assignment for frontal view facial subunits was full facial aging, middle third, lower third, upper third, vertical lip rhytides, horizontal forehead rhytides, jowls, upper eyelid ptosis, loss of malar volume, lower lid fat herniation, deepening glabellar furrows, and deepening nasolabial folds. From most to least impact in age, the rank assignment for lateral view facial subunits was severe neck ptosis, jowls, moderate neck ptosis, vertical lip rhytides, crow's feet, lower lid fat herniation, loss of malar volume, and elongated earlobe. This study provides a preliminary template for further research to determine which anatomical subunit will have the most substantial effect on an aged appearance, as well as on the perception of tiredness and attractiveness. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

MutSβ and histone deacetylase complexes promote expansions of trinucleotide repeats in human cells

PubMed Central

Gannon, Anne-Marie M.; Frizzell, Aisling; Healy, Evan; Lahue, Robert S.

2012-01-01

Trinucleotide repeat (TNR) expansions cause at least 17 heritable neurological diseases, including Huntington’s disease. Expansions are thought to arise from abnormal processing of TNR DNA by specific trans-acting proteins. For example, the DNA repair complex MutSβ (MSH2–MSH3 heterodimer) is required in mice for on-going expansions of long, disease-causing alleles. A distinctive feature of TNR expansions is a threshold effect, a narrow range of repeat units (∼30–40 in humans) at which mutation frequency rises dramatically and disease can initiate. The goal of this study was to identify factors that promote expansion of threshold-length CTG•CAG repeats in a human astrocytic cell line. siRNA knockdown of the MutSβ subunits MSH2 or MSH3 impeded expansions of threshold-length repeats, while knockdown of the MutSα subunit MSH6 had no effect. Chromatin immunoprecipitation experiments indicated that MutSβ, but not MutSα, was enriched at the TNR. These findings imply a direct role for MutSβ in promoting expansion of threshold-length CTG•CAG tracts. We identified the class II deacetylase HDAC5 as a novel promoting factor for expansions, joining the class I deacetylase HDAC3 that was previously identified. Double knockdowns were consistent with the possibility that MutSβ, HDAC3 and HDAC5 act through a common pathway to promote expansions of threshold-length TNRs. PMID:22941650

Rotation of Subunits During Catalysis by Escherichia coli F_1-ATPase

NASA Astrophysics Data System (ADS)

Duncan, Thomas M.; Bulygin, Vladimir V.; Zhou, Yuantai; Hutcheon, Marcus L.; Cross, Richard L.

1995-11-01

During oxidative and photo-phosphorylation, F_0F_1-ATP synthases couple the movement of protons down an electrochemical gradient to the synthesis of ATP. One proposed mechanistic feature that has remained speculative is that this coupling process requires the rotation of subunits within F_0F_1. Guided by a recent, high-resolution structure for bovine F_1 [Abrahams, J. P., Leslie, A. G., Lutter, R. & Walker, J. E. (1994) Nature (London) 370, 621-628], we have developed a critical test for rotation of the central γ subunit relative to the three catalytic β subunits in soluble F_1 from Escherichia coli. In the bovine F_1 structure, a specific point of contact between the γ subunit and one of the three catalytic β subunits includes positioning of the homolog of E. coli γ-subunit C87 (γC87) close to the β-subunit 380DELSEED386 sequence. A βD380C mutation allowed us to induce formation of a specific disulfide bond between β and γC87 in soluble E. coli F_1. Formation of the crosslink inactivated βD380C-F_1, and reduction restored full activity. Using a dissociation/reassembly approach with crosslinked βD380C-F_1, we incorporated radiolabeled β subunits into the two noncrosslinked β-subunit positions of F_1. After reduction of the initial nonradio-active β-γ crosslink, only exposure to conditions for catalytic turnover results in similar reactivities of unlabeled and radiolabeled β subunits with γC87 upon reoxidation. The results demonstrate that γ subunit rotates relative to the β subunits during catalysis.

Preferential cross-linking of the small subunit of the electron-transfer flavoprotein to general acyl-CoA dehydrogenase.

PubMed Central

Steenkamp, D J

1987-01-01

The interaction between pig liver mitochondrial electron-transfer flavoprotein (ETF) and general acyl-CoA dehydrogenase (GAD) was investigated by means of the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio)propionate. Neither ETF or GAD contained reactive thiol groups. The substitution of 9.4 lysine residues/FAD group in GAD with pyridyl disulphide structures did not affect the catalytic activity of the enzyme. Thiol groups were introduced into ETF by thiolation with methyl 4-mercaptobutyrimidate. ETF containing 10.5 reactive thiol groups/FAD group showed undiminished electron-acceptor activity with respect to GAD. The reaction of thiolated ETF and GAD containing pyridyl disulphide structures resulted in a decreased staining intensity of the small subunit of ETF on SDS/polyacrylamide-gel electrophoresis. Preferential cross-linking of the smaller subunit of ETF to GAD did not take place when ETF was first treated with SDS, but was unaffected by reduction of GAD by octanoyl-CoA. Images Fig. 2. Fig. 3. Fig. 5. PMID:3115254

Evolution of the α-Subunit of Na/K-ATPase from Paramecium to Homo sapiens: Invariance of Transmembrane Helix Topology.

PubMed

Morrill, Gene A; Kostellow, Adele B; Liu, Lijun; Gupta, Raj K; Askari, Amir

2016-05-01

Na/K-ATPase is a key plasma membrane enzyme involved in cell signaling, volume regulation, and maintenance of electrochemical gradients. The α-subunit, central to these functions, belongs to a large family of P-type ATPases. Differences in transmembrane (TM) helix topology, sequence homology, helix-helix contacts, cell signaling, and protein domains of Na/K-ATPase α-subunit were compared in fungi (Beauveria), unicellular organisms (Paramecia), primitive multicellular organisms (Hydra), and vertebrates (Xenopus, Homo sapiens), and correlated with evolution of physiological functions in the α-subunit. All α-subunits are of similar length, with groupings of four and six helices in the N- and C-terminal regions, respectively. Minimal homology was seen for protein domain patterns in Paramecium and Hydra, with high correlation between Hydra and vertebrates. Paramecium α-subunits display extensive disorder, with minimal helix contacts. Increases in helix contacts in Hydra approached vertebrates. Protein motifs known to be associated with membrane lipid rafts and cell signaling reveal significant positional shifts between Paramecium and Hydra vulgaris, indicating that regional membrane fluidity changes occur during evolution. Putative steroid binding sites overlapping TM-3 occurred in all species. Sites associated with G-protein-receptor stimulation occur both in vertebrates and amphibia but not in Hydra or Paramecia. The C-terminus moiety "KETYY," necessary for the Na(+) activation of pump phosphorylation, is not present in unicellular species indicating the absence of classical Na(+)/K(+)-pumps. The basic protein topology evolved earliest, followed by increases in protein domains and ordered helical arrays, correlated with appearance of α-subunit regions known to involve cell signaling, membrane recycling, and ion channel formation.

Architecture of human translation initiation factor 3

PubMed Central

Querol-Audi, Jordi; Sun, Chaomin; Vogan, Jacob M.; Smith, Duane; Gu, Yu; Cate, Jamie; Nogales, Eva

2013-01-01

SUMMARY Eukaryotic translation initiation factor 3 (eIF3) plays a central role in protein synthesis by organizing the formation of the 43S preinitiation complex. Using genetic tag visualization by electron microscopy, we reveal the molecular organization of ten human eIF3 subunits, including an octameric core. The structure of eIF3 bears a close resemblance to that of the proteasome lid, with a conserved spatial organization of eight core subunits containing PCI and MPN domains that coordinate functional interactions in both complexes. We further show that eIF3 subunits a and c interact with initiation factors eIF1 and eIF1A, which control the stringency of start codon selection. Finally, we find that subunit j, which modulates messenger RNA interactions with the small ribosomal subunit, makes multiple independent interactions with the eIF3 octameric core. These results highlight the conserved architecture of eIF3 and how it scaffolds key factors that control translation initiation in higher eukaryotes, including humans. PMID:23623729

Chemically related 4,5-linked aminoglycoside antibiotics drive subunit rotation in opposite directions

PubMed Central

Wasserman, Michael R.; Pulk, Arto; Zhou, Zhou; Altman, Roger B.; Zinder, John C.; Green, Keith D.; Garneau-Tsodikova, Sylvie; Doudna Cate, Jamie H.; Blanchard, Scott C.

2015-01-01

Dynamic remodelling of intersubunit bridge B2, a conserved RNA domain of the bacterial ribosome connecting helices 44 (h44) and 69 (H69) of the small and large subunit, respectively, impacts translation by controlling intersubunit rotation. Here we show that aminoglycosides chemically related to neomycin—paromomycin, ribostamycin and neamine—each bind to sites within h44 and H69 to perturb bridge B2 and affect subunit rotation. Neomycin and paromomycin, which only differ by their ring-I 6′-polar group, drive subunit rotation in opposite directions. This suggests that their distinct actions hinge on the 6′-substituent and the drug's net positive charge. By solving the crystal structure of the paromomycin–ribosome complex, we observe specific contacts between the apical tip of H69 and the 6′-hydroxyl on paromomycin from within the drug's canonical h44-binding site. These results indicate that aminoglycoside actions must be framed in the context of bridge B2 and their regulation of subunit rotation. PMID:26224058

Two hydrophobic subunits are essential for the heme b ligation and functional assembly of complex II (succinate-ubiquinone oxidoreductase) from Escherichia coli.

PubMed

Nakamura, K; Yamaki, M; Sarada, M; Nakayama, S; Vibat, C R; Gennis, R B; Nakayashiki, T; Inokuchi, H; Kojima, S; Kita, K

1996-01-05

Complex II (succinate-ubiquinone oxidoreductase) from Escherichia coli is composed of four nonidentical subunits encoded by the sdhCDAB operon. Gene products of sdhC and sdhD are small hydrophobic subunits that anchor the hydrophilic catalytic subunits (flavoprotein and iron-sulfur protein) to the cytoplasmic membrane and are believed to be the components of cytochrome b556 in E. coli complex II. In the present study, to elucidate the role of two hydrophobic subunits in the heme b ligation and functional assembly of complex II, plasmids carrying portions of the sdh gene were constructed and introduced into E. coli MK3, which lacks succinate dehydrogenase and fumarate reductase activities. The expression of polypeptides with molecular masses of about 19 and 17 kDa was observed when sdhC and sdhD were introduced into MK3, respectively, indicating that sdhC encodes the large subunit (cybL) and sdhD the small subunit (cybS) of cytochrome b556. An increase in cytochrome b content was found in the membrane when sdhD was introduced, while the cytochrome b content did not change when sdhC was introduced. However, the cytochrome b expressed by the plasmid carrying sdhD differed from cytochrome b556 in its CO reactivity and red shift of the alpha absorption peak to 557.5 nm at 77 K. Neither hydrophobic subunit was able to bind the catalytic portion to the membrane, and only succinate dehydrogenase activity, not succinate-ubiquinone oxidoreductase activity, was found in the cytoplasmic fractions of the cells. In contrast, significantly higher amounts of cytochrome b556 were expressed in the membrane when sdhC and sdhD genes were both present, and the catalytic portion was found to be localized in the membrane with succinate-ubiquitnone oxidoreductase and succinate oxidase activities. These results strongly suggest that both hydrophobic subunits are required for heme insertion into cytochrome b556 and are essential for the functional assembly of E. coli complex II in the membrane. Accumulation of the catalytic portion in the cytoplasm was found when sdhCDAB was introduced into a heme synthesis mutant, suggesting the importance of heme in the assembly of E. coli complex II.

A novel myxosporean parasite Myxobolus klamathellus n. sp. (Cnidaria: Myxosporea) from native blue chub (Gila coerulea) in Klamath Lake, Oregon.

PubMed

Atkinson, Stephen Douglas; Banner, Craig Randall

2017-01-01

Blue chub, Gila coerulea Girard, 1856 is a freshwater cyprinid fish native to inland drainages of western North America. It has not previously been recorded as a host of any myxosporean parasite (Cnidaria: Myxosporea), despite myxosporeans being cosmopolitan in freshwater and marine fishes worldwide and sympatric with this host. Herein, we describe a novel myxosporean from subcutaneous cysts in native blue chub from Klamath Lake, Oregon. Myxospores were consistent with genus Myxobolus, being obovoid but compressed in thickness, length 14.3 ± 0.4 (13-15) μm, width 9.7 ± 0.4 (9-10) μm, thickness 7.7 ± 0.3 (7-8) μm; two polar capsules ovoid slightly dissimilar in size, length 6.4 ± 0.4 (6-7) μm, width 3.8 ± 0.3 (3-4) μm, with four (3-5) turns of the polar filament (tubule); capsule openings apical, one in each valve cell. The small subunit ribosomal DNA sequence was up to 97 % similar to Myxobolus spp. from other cyprinids from North America and Europe. Given the novel host, unique myxospore morphometrics, and DNA sequence, we describe this as Myxobolus klamathellus n. sp.

Biallelic Mutations in MRPS34 Lead to Instability of the Small Mitoribosomal Subunit and Leigh Syndrome.

PubMed

Lake, Nicole J; Webb, Bryn D; Stroud, David A; Richman, Tara R; Ruzzenente, Benedetta; Compton, Alison G; Mountford, Hayley S; Pulman, Juliette; Zangarelli, Coralie; Rio, Marlene; Boddaert, Nathalie; Assouline, Zahra; Sherpa, Mingma D; Schadt, Eric E; Houten, Sander M; Byrnes, James; McCormick, Elizabeth M; Zolkipli-Cunningham, Zarazuela; Haude, Katrina; Zhang, Zhancheng; Retterer, Kyle; Bai, Renkui; Calvo, Sarah E; Mootha, Vamsi K; Christodoulou, John; Rötig, Agnes; Filipovska, Aleksandra; Cristian, Ingrid; Falk, Marni J; Metodiev, Metodi D; Thorburn, David R

2017-08-03

The synthesis of all 13 mitochondrial DNA (mtDNA)-encoded protein subunits of the human oxidative phosphorylation (OXPHOS) system is carried out by mitochondrial ribosomes (mitoribosomes). Defects in the stability of mitoribosomal proteins or mitoribosome assembly impair mitochondrial protein translation, causing combined OXPHOS enzyme deficiency and clinical disease. Here we report four autosomal-recessive pathogenic mutations in the gene encoding the small mitoribosomal subunit protein, MRPS34, in six subjects from four unrelated families with Leigh syndrome and combined OXPHOS defects. Whole-exome sequencing was used to independently identify all variants. Two splice-site mutations were identified, including homozygous c.321+1G>T in a subject of Italian ancestry and homozygous c.322-10G>A in affected sibling pairs from two unrelated families of Puerto Rican descent. In addition, compound heterozygous MRPS34 mutations were identified in a proband of French ancestry; a missense (c.37G>A [p.Glu13Lys]) and a nonsense (c.94C>T [p.Gln32 ∗ ]) variant. We demonstrated that these mutations reduce MRPS34 protein levels and the synthesis of OXPHOS subunits encoded by mtDNA. Examination of the mitoribosome profile and quantitative proteomics showed that the mitochondrial translation defect was caused by destabilization of the small mitoribosomal subunit and impaired monosome assembly. Lentiviral-mediated expression of wild-type MRPS34 rescued the defect in mitochondrial translation observed in skin fibroblasts from affected subjects, confirming the pathogenicity of MRPS34 mutations. Our data establish that MRPS34 is required for normal function of the mitoribosome in humans and furthermore demonstrate the power of quantitative proteomic analysis to identify signatures of defects in specific cellular pathways in fibroblasts from subjects with inherited disease. Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Imaging of subunit complexes of thermophilic bacterium H(+)-ATPase with scanning tunneling microscopy.

PubMed

Masai, J; Shibata, T; Kagawa, Y; Kondo, S

1992-07-01

Using a scanning tunneling microscope (STM), we observed reconstructed subunit complexes of H(+)-ATPase of a thermophilic bacterium. The measurement was carried out in air without conductive coating on the samples deposited on a highly oriented pyrolytic graphite (HOPG). The F1 subunit complex of the H(+)-ATPase, and an H(+)-ATPase whose F0 portion was embedded into liposomes prepared from soybean lecithin were imaged. Overall structural images of the subunit complex F1 were obtained: the structural dimensions of the STM images are in agreement with those deduced from conventional methods such as an transmission electron microscopy (TEM) and small-angle X-ray scattering (SAX) experimentation. Regarding the STM imaging of these samples, we discuss the advantages and disadvantages of the STM over those of conventional methods such as a TEM and SAX.

Polypeptide composition of fraction 1 protein of the somatic hybrid between Petunia parodii and Petunia parviflora.

PubMed

Kumar, A; Wilson, D; Cocking, E C

1981-04-01

The analysis of the subunit polypeptide composition of Fraction 1 protein provides information on the expression of both chloroplast and nuclear genomes. Fraction 1 protein, isolated from leaves of the somatic hybrid plants derived form the fusion of protoplasts of Petunia parodii and P. parviflora, was analyzed for its subunit polypeptide composition by isoelectric focusing in 8 M urea. The fraction 1 protein enzyme oligomer in the somatic hybrid plants contained small subunits resulting from the expression of both parental nuclear genomes, but probably only one of the parental large subunits, namely that of P. parodii. The relevance of such somatic hybrid material for the study of nucleocytoplasmic interrelationship is discussed, as well as the use of these fraction 1 protein isoelectric focusing patterns for the analysis of taxonomic relationships in Petunia.

Tunable color parallel tandem organic light emitting devices with carbon nanotube and metallic sheet interlayers

NASA Astrophysics Data System (ADS)

Oliva, Jorge; Papadimitratos, Alexios; Desirena, Haggeo; De la Rosa, Elder; Zakhidov, Anvar A.

2015-11-01

Parallel tandem organic light emitting devices (OLEDs) were fabricated with transparent multiwall carbon nanotube sheets (MWCNT) and thin metal films (Al, Ag) as interlayers. In parallel monolithic tandem architecture, the MWCNT (or metallic films) interlayers are an active electrode which injects similar charges into subunits. In the case of parallel tandems with common anode (C.A.) of this study, holes are injected into top and bottom subunits from the common interlayer electrode; whereas in the configuration of common cathode (C.C.), electrons are injected into the top and bottom subunits. Both subunits of the tandem can thus be monolithically connected functionally in an active structure in which each subunit can be electrically addressed separately. Our tandem OLEDs have a polymer as emitter in the bottom subunit and a small molecule emitter in the top subunit. We also compared the performance of the parallel tandem with that of in series and the additional advantages of the parallel architecture over the in-series were: tunable chromaticity, lower voltage operation, and higher brightness. Finally, we demonstrate that processing of the MWCNT sheets as a common anode in parallel tandems is an easy and low cost process, since their integration as electrodes in OLEDs is achieved by simple dry lamination process.

The Genetic Basis of Natural Variation in Caenorhabditis elegans Telomere Length.

PubMed

Cook, Daniel E; Zdraljevic, Stefan; Tanny, Robyn E; Seo, Beomseok; Riccardi, David D; Noble, Luke M; Rockman, Matthew V; Alkema, Mark J; Braendle, Christian; Kammenga, Jan E; Wang, John; Kruglyak, Leonid; Félix, Marie-Anne; Lee, Junho; Andersen, Erik C

2016-09-01

Telomeres are involved in the maintenance of chromosomes and the prevention of genome instability. Despite this central importance, significant variation in telomere length has been observed in a variety of organisms. The genetic determinants of telomere-length variation and their effects on organismal fitness are largely unexplored. Here, we describe natural variation in telomere length across the Caenorhabditis elegans species. We identify a large-effect variant that contributes to differences in telomere length. The variant alters the conserved oligonucleotide/oligosaccharide-binding fold of protection of telomeres 2 (POT-2), a homolog of a human telomere-capping shelterin complex subunit. Mutations within this domain likely reduce the ability of POT-2 to bind telomeric DNA, thereby increasing telomere length. We find that telomere-length variation does not correlate with offspring production or longevity in C. elegans wild isolates, suggesting that naturally long telomeres play a limited role in modifying fitness phenotypes in C. elegans. Copyright © 2016 by the Genetics Society of America.

M1.3 - a small scaffold for DNA origami

NASA Astrophysics Data System (ADS)

Said, Hassan; Schüller, Verena J.; Eber, Fabian J.; Wege, Christina; Liedl, Tim; Richert, Clemens

2012-12-01

The DNA origami method produces programmable nanoscale objects that form when one long scaffold strand hybridizes to numerous oligonucleotide staple strands. One scaffold strand is dominating the field: M13mp18, a bacteriophage-derived vector 7249 nucleotides in length. The full-length M13 is typically folded by using over 200 staple oligonucleotides. Here we report the convenient preparation of a 704 nt fragment dubbed ``M1.3'' as a linear or cyclic scaffold and the assembly of small origami structures with just 15-24 staple strands. A typical M1.3 origami is large enough to be visualized by TEM, but small enough to show a cooperativity in its assembly and thermal denaturation that is reminiscent of oligonucleotide duplexes. Due to its medium size, M1.3 origami with globally modified staples is affordable. As a proof of principle, two origami structures with globally 5'-capped staples were prepared and were shown to give higher UV-melting points than the corresponding assembly with unmodified DNA. M1.3 has the size of a gene, not a genome, and may function as a model for gene-based nanostructures. Small origami with M1.3 as a scaffold may serve as a workbench for chemical, physical, and biological experiments.The DNA origami method produces programmable nanoscale objects that form when one long scaffold strand hybridizes to numerous oligonucleotide staple strands. One scaffold strand is dominating the field: M13mp18, a bacteriophage-derived vector 7249 nucleotides in length. The full-length M13 is typically folded by using over 200 staple oligonucleotides. Here we report the convenient preparation of a 704 nt fragment dubbed ``M1.3'' as a linear or cyclic scaffold and the assembly of small origami structures with just 15-24 staple strands. A typical M1.3 origami is large enough to be visualized by TEM, but small enough to show a cooperativity in its assembly and thermal denaturation that is reminiscent of oligonucleotide duplexes. Due to its medium size, M1.3 origami with globally modified staples is affordable. As a proof of principle, two origami structures with globally 5'-capped staples were prepared and were shown to give higher UV-melting points than the corresponding assembly with unmodified DNA. M1.3 has the size of a gene, not a genome, and may function as a model for gene-based nanostructures. Small origami with M1.3 as a scaffold may serve as a workbench for chemical, physical, and biological experiments. Electronic supplementary information (ESI) available: Materials, full sequence of M1.3, alternative restriction reactions, sequences and origami designs, ALEX data, estimated cost of producing M13, additional melting data for origami, and MALDI spectra of individual capped oligonucleotides. See DOI: 10.1039/c2nr32393a

Microbial Community Biofabrics in a Geothermal Mine Adit▿ †

PubMed Central

Spear, John R.; Barton, Hazel A.; Robertson, Charles E.; Francis, Christopher A.; Pace, Norman R.

2007-01-01

Speleothems such as stalactites and stalagmites are usually considered to be mineralogical in composition and origin; however, microorganisms have been implicated in the development of some speleothems. We have identified and characterized the biological and mineralogical composition of mat-like biofabrics in two novel kinds of speleothems from a 50°C geothermal mine adit near Glenwood Springs, CO. One type of structure consists of 2- to 3-cm-long, 3- to 4-mm-wide, leather-like, hollow, soda straw stalactites. Light and electron microscopy indicated that the stalactites are composed of a mineralized biofabric with several cell morphotypes in a laminated form, with gypsum and sulfur as the dominant mineral components. A small-subunit rRNA gene phylogenetic community analysis along the stalactite length yielded a diverse gradient of organisms, with a relatively simple suite of main constituents: Thermus spp., crenarchaeotes, Chloroflexi, and Gammaproteobacteria. PCR analysis also detected putative crenarchaeal ammonia monooxygenase subunit A (amoA) genes in this community, the majority related to sequences from other geothermal systems. The second type of speleothem, dumpling-like rafts floating on a 50°C pool on the floor of the adit, showed a mat-like fabric of evidently living organisms on the outside of the dumpling, with a multimineral, amorphous, gypsum-based internal composition. These two novel types of biofabrics are examples of the complex roles that microbes can play in mineralization, weathering, and deposition processes in karst environments. PMID:17693567

Lateral Comparative Investigation of Stromatolites: Astrobiological Implications and Assessment of Scales of Control

NASA Astrophysics Data System (ADS)

Ibarra, Yadira; Corsetti, Frank A.

2016-04-01

The processes that govern the formation of stromatolites, structures that may represent macroscopic manifestation of microbial processes and a clear target for astrobiological investigation, occur at various scales (local versus regional), yet determining their relative importance remains a challenge, particularly for ancient deposits and/or if similar deposits are discovered elsewhere in the Solar System. We build upon the traditional multiscale level approach of investigation (micro-, meso-, macro-, mega-) by including a lateral comparative investigational component of fine- to large-scale features to determine the relative significance of local and/or nonlocal controls on stromatolite morphology, and in the process, help constrain the dominant influences on microbialite formation. In one example of lateral comparative investigation, lacustrine microbialites from the Miocene Barstow Formation (California) display two main mesofabrics: (1) micritic bands that drastically change in thickness and cannot directly be traced between adjacent decimeter-scale subunits and (2) sparry fibrous layers that are strikingly consistent across subunits, suggesting the formation of sparry fibrous layers was influenced by a process larger than the length scale between the subunits (likely lake chemistry). Microbialites from the uppermost Triassic Cotham Member, United Kingdom, occur as meter-scale mounds and contain a characteristic succession of laminated and dendrolitic mesofabrics. The same succession of laminated/dendrolitic couplets can be traced, not only from mound to mound, but over 100 km, indicating a regional-scale influence on very small structures (microns to centimeters) that would otherwise not be apparent without the lateral comparative approach, and demonstrating that the scale of the feature does not necessarily scale with the scope of the process. Thus, the combination of lateral comparative investigations and multiscale analyses can provide an effective approach for evaluating the dominant controls on stromatolite texture and morphology throughout the rock record and potentially on other planets via rover-scale analyses (e.g., Mars).

Comparative Analysis of AGPase Genes and Encoded Proteins in Eight Monocots and Three Dicots with Emphasis on Wheat

PubMed Central

Batra, Ritu; Saripalli, Gautam; Mohan, Amita; Gupta, Saurabh; Gill, Kulvinder S.; Varadwaj, Pritish K.; Balyan, Harindra S.; Gupta, Pushpendra K.

2017-01-01

ADP-glucose pyrophosphorylase (AGPase) is a heterotetrameric enzyme with two large subunits (LS) and two small subunits (SS). It plays a critical role in starch biosynthesis. We are reporting here detailed structure, function and evolution of the genes encoding the LS and the SS among monocots and dicots. “True” orthologs of maize Sh2 (AGPase LS) and Bt2 (AGPase SS) were identified in seven other monocots and three dicots; structure of the enzyme at protein level was also studied. Novel findings of the current study include the following: (i) at the DNA level, the genes controlling the SS are more conserved than those controlling the LS; the variation in both is mainly due to intron number, intron length and intron phase distribution; (ii) at protein level, the SS genes are more conserved relative to those for LS; (iii) “QTCL” motif present in SS showed evolutionary differences in AGPase belonging to wheat 7BS, T. urartu, rice and sorghum, while “LGGG” motif in LS was present in all species except T. urartu and chickpea; SS provides thermostability to AGPase, while LS is involved in regulation of AGPase activity; (iv) heterotetrameric structure of AGPase was predicted and analyzed in real time environment through molecular dynamics simulation for all the species; (v) several cis-acting regulatory elements were identified in the AGPase promoters with their possible role in regulating spatial and temporal expression (endosperm and leaf tissue) and also the expression, in response to abiotic stresses; and (vi) expression analysis revealed downregulation of both subunits under conditions of heat and drought stress. The results of the present study have allowed better understanding of structure and evolution of the genes and the encoded proteins and provided clues for exploitation of variability in these genes for engineering thermostable AGPase. PMID:28174576

Compilation of small ribosomal subunit RNA structures.

PubMed Central

Neefs, J M; Van de Peer, Y; De Rijk, P; Chapelle, S; De Wachter, R

1993-01-01

The database on small ribosomal subunit RNA structure contained 1804 nucleotide sequences on April 23, 1993. This number comprises 365 eukaryotic, 65 archaeal, 1260 bacterial, 30 plastidial, and 84 mitochondrial sequences. These are stored in the form of an alignment in order to facilitate the use of the database as input for comparative studies on higher-order structure and for reconstruction of phylogenetic trees. The elements of the postulated secondary structure for each molecule are indicated by special symbols. The database is available on-line directly from the authors by ftp and can also be obtained from the EMBL nucleotide sequence library by electronic mail, ftp, and on CD ROM disk. PMID:8332525

Detection of trypanosomatid Phytomonas parasitic in plants by polymerase chain reaction amplification of small subunit ribosomal DNA.

PubMed

Teixeira, M M; Campaner, M; Camargo, E P

1994-01-01

To improve the diagnosis of Phytomonas infections in plants, we developed a polymerase chain reaction (PCR) assay using synthetic oligonucleotides complementary to conserved sequences of the 18S small subunit ribosomal (SSU) gene. From 10 ng upward of DNA of cultures of Phytomonas isolated from plants, fruits, and insects, PCR amplified an 800-bp DNA band that, after restriction analysis and probe hybridization, proved to be of 18S rDNA Phytomonas origin. PCR was also done with sap samples of tomatoes experimentally infected with Phytomonas, yielding amplified 800-bp ribosomal DNA bands before any flagellate could be detected by microscopic examination of the fruit sap.

Theileria sp. Infections Associated with Bovine Fatalities in the United States Confirmed by Small-Subunit rRNA Gene Analyses of Blood and Tick Samples

PubMed Central

Chae, Joon-seok; Levy, Michael; Hunt, John; Schlater, Jack; Snider, Glen; Waghela, Suryakant D.; Holman, Patricia J.; Wagner, G. Gale

1999-01-01

Theileria sp.-specific small subunit (SSU) rRNA gene amplification confirmed the presence of the organism in cattle and in Amblyomma americanum and Dermacentor variabilis ticks collected from a cattle herd in Missouri. Blood from the index animal had type A and type D Theileria SSU rRNA genes. The type D gene was also found in blood from two cohort cattle and tick tissues. The type A SSU rRNA gene was previously reported from bovine Theileria isolates from Texas and North Carolina; the type D gene was reported from a Texas cow with theileriosis. PMID:10449501

Phylogenetic position of the genus Perkinsus (Protista, Apicomplexa) based on small subunit ribosomal RNA.

PubMed

Goggin, C L; Barker, S C

1993-07-01

Parasites of the genus Perkinsus destroy marine molluscs worldwide. Their phylogenetic position within the kingdom Protista is controversial. Nucleotide sequence data (1792 bp) from the small subunit rRNA gene of Perkinsus sp. from Anadara trapezia (Mollusca: Bivalvia) from Moreton Bay, Queensland, was used to examine the phylogenetic affinities of this enigmatic genus. These data were aligned with nucleotide sequences from 6 apicomplexans, 3 ciliates, 3 flagellates, a dinoflagellate, 3 fungi, maize and human. Phylogenetic trees were constructed after analysis with maximum parsimony and distance matrix methods. Our analyses indicate that Perkinsus is phylogenetically closer to dinoflagellates and to coccidean and piroplasm apicomplexans than to fungi or flagellates.

Moving Iron through ferritin protein nanocages depends on residues throughout each four α-helix bundle subunit.

PubMed

Haldar, Suranjana; Bevers, Loes E; Tosha, Takehiko; Theil, Elizabeth C

2011-07-22

Eukaryotic H ferritins move iron through protein cages to form biologically required, iron mineral concentrates. The biominerals are synthesized during protein-based Fe²⁺/O₂ oxidoreduction and formation of [Fe³⁺O](n) multimers within the protein cage, en route to the cavity, at sites distributed over ~50 Å. Recent NMR and Co²⁺-protein x-ray diffraction (XRD) studies identified the entire iron path and new metal-protein interactions: (i) lines of metal ions in 8 Fe²⁺ ion entry channels with three-way metal distribution points at channel exits and (ii) interior Fe³⁺O nucleation channels. To obtain functional information on the newly identified metal-protein interactions, we analyzed effects of amino acid substitution on formation of the earliest catalytic intermediate (diferric peroxo-A(650 nm)) and on mineral growth (Fe³⁺O-A(350 nm)), in A26S, V42G, D127A, E130A, and T149C. The results show that all of the residues influenced catalysis significantly (p < 0.01), with effects on four functions: (i) Fe²⁺ access/selectivity to the active sites (Glu¹³⁰), (ii) distribution of Fe²⁺ to each of the three active sites near each ion channel (Asp¹²⁷), (iii) product (diferric oxo) release into the Fe³⁺O nucleation channels (Ala²⁶), and (iv) [Fe³⁺O](n) transit through subunits (Val⁴², Thr¹⁴⁹). Synthesis of ferritin biominerals depends on residues along the entire length of H subunits from Fe²⁺ substrate entry at 3-fold cage axes at one subunit end through active sites and nucleation channels, at the other subunit end, inside the cage at 4-fold cage axes. Ferritin subunit-subunit geometry contributes to mineral order and explains the physiological impact of ferritin H and L subunits.

Posttranslational Amelogenin Processing and Changes in Matrix Assembly during Enamel Development

PubMed Central

Pandya, Mirali; Lin, Tiffani; Li, Leo; Allen, Michael J.; Jin, Tianquan; Luan, Xianghong; Diekwisch, Thomas G. H.

2017-01-01

The extracellular tooth enamel matrix is a unique, protein-rich environment that provides the structural basis for the growth of long and parallel oriented enamel crystals. Here we have conducted a series of in vivo and in vitro studies to characterize the changes in matrix shape and organization that take place during the transition from ameloblast intravesicular matrices to extracellular subunit compartments and pericrystalline sheath proteins, and correlated these changes with stages of amelogenin matrix protein posttranslational processing. Our transmission electron microscopic studies revealed a 2.5-fold difference in matrix subunit compartment dimensions between secretory vesicle and extracellular enamel protein matrix as well as conformational changes in matrix structure between vesicles, stippled materials, and pericrystalline matrix. Enamel crystal growth in organ culture demonstrated granular mineral deposits associated with the enamel matrix framework, dot-like mineral deposits along elongating initial enamel crystallites, and dramatic changes in enamel matrix configuration following the onset of enamel crystal formation. Atomic force micrographs provided evidence for the presence of both linear and hexagonal/ring-shaped full-length recombinant amelogenin protein assemblies on mica surfaces, while nickel-staining of the N-terminal amelogenin N92 His-tag revealed 20 nm diameter oval and globular amelogenin assemblies in N92 amelogenin matrices. Western blot analysis comparing loosely bound and mineral-associated protein fractions of developing porcine enamel organs, superficial and deep enamel layers demonstrated (i) a single, full-length amelogenin band in the enamel organ followed by 3 kDa cleavage upon entry into the enamel layer, (ii) a close association of 8–16 kDa C-terminal amelogenin cleavage products with the growing enamel apatite crystal surface, and (iii) a remaining pool of N-terminal amelogenin fragments loosely retained between the crystalline phases of the deep enamel layer. Together, our data establish a temporo-spatial correlation between amelogenin protein processing and the changes in enamel matrix configuration that take place during the transition from intracellular vesicle compartments to extracellular matrix assemblies and the formation of protein coats along elongating apatite crystal surfaces. In conclusion, our study suggests that enzymatic cleavage of the amelogenin enamel matrix protein plays a key role in the patterning of the organic matrix framework as it affects enamel apatite crystal growth and habit. PMID:29089900

Posttranslational Amelogenin Processing and Changes in Matrix Assembly during Enamel Development.

PubMed

Pandya, Mirali; Lin, Tiffani; Li, Leo; Allen, Michael J; Jin, Tianquan; Luan, Xianghong; Diekwisch, Thomas G H

2017-01-01

The extracellular tooth enamel matrix is a unique, protein-rich environment that provides the structural basis for the growth of long and parallel oriented enamel crystals. Here we have conducted a series of in vivo and in vitro studies to characterize the changes in matrix shape and organization that take place during the transition from ameloblast intravesicular matrices to extracellular subunit compartments and pericrystalline sheath proteins, and correlated these changes with stages of amelogenin matrix protein posttranslational processing. Our transmission electron microscopic studies revealed a 2.5-fold difference in matrix subunit compartment dimensions between secretory vesicle and extracellular enamel protein matrix as well as conformational changes in matrix structure between vesicles, stippled materials, and pericrystalline matrix. Enamel crystal growth in organ culture demonstrated granular mineral deposits associated with the enamel matrix framework, dot-like mineral deposits along elongating initial enamel crystallites, and dramatic changes in enamel matrix configuration following the onset of enamel crystal formation. Atomic force micrographs provided evidence for the presence of both linear and hexagonal/ring-shaped full-length recombinant amelogenin protein assemblies on mica surfaces, while nickel-staining of the N-terminal amelogenin N92 His-tag revealed 20 nm diameter oval and globular amelogenin assemblies in N92 amelogenin matrices. Western blot analysis comparing loosely bound and mineral-associated protein fractions of developing porcine enamel organs, superficial and deep enamel layers demonstrated (i) a single, full-length amelogenin band in the enamel organ followed by 3 kDa cleavage upon entry into the enamel layer, (ii) a close association of 8-16 kDa C-terminal amelogenin cleavage products with the growing enamel apatite crystal surface, and (iii) a remaining pool of N-terminal amelogenin fragments loosely retained between the crystalline phases of the deep enamel layer. Together, our data establish a temporo-spatial correlation between amelogenin protein processing and the changes in enamel matrix configuration that take place during the transition from intracellular vesicle compartments to extracellular matrix assemblies and the formation of protein coats along elongating apatite crystal surfaces. In conclusion, our study suggests that enzymatic cleavage of the amelogenin enamel matrix protein plays a key role in the patterning of the organic matrix framework as it affects enamel apatite crystal growth and habit.

Linkage of genes for laminin B1 and B2 subunits on chromosome 1 in mouse.

PubMed

Elliott, R W; Barlow, D; Hogan, B L

1985-08-01

We have used cDNA clones for the B1 and B2 subunits of laminin to find restriction fragment length DNA polymorphisms for the genes encoding these polypeptides in the mouse. Three alleles were found for LamB2 and two for LamB1 among the inbred mouse strains. The segregation of these polymorphisms among recombinant inbred strains showed that these genes are tightly linked in the central region of mouse Chromosome 1 between Sas-1 and Ly-m22, 7.4 +/- 3.2 cM distal to the Pep-3 locus. There is no evidence in the mouse for pseudogenes for these proteins.

The nucleotide binding properties of human MSH2/MSH3 are lesion-dependent and distinct from those of human MSH2/MSH6

PubMed Central

Owen, Barbara A. L.; Lang, Walter; McMurray, Cynthia T.

2010-01-01

Summary Here, we report that MSH2/MSH3 maintains lesion specificity for small loops by a distinctly different mechanism than does MHSH2/MSH6 for single base mismatches. ADP and ATP have no preference for the subunits of hMSH2/MSH3. Upon lesion binding, however, hMSH2/MSH3 adopts a single “nucleotide signature” in which one ADP binds within the hMSH2 subunit and the hMSH3 subunit is empty. On the lesion, ADP-hMSH2/MSH3-empty binds and hydrolyzes ATP in the empty hMSH3 subunit, which reduces ADP affinity and increases ATP affinity for the hMSH2 subunit. ADP/ATP exchange converts (CA)4-loop-bound ADP-MSH2/MSH3-ATP into an ATP-hMSH2/MSH3-ADP intermediate in which ATP hydrolysis is inhibited in the hMSH2 subunit. We propose a model in which lesion binding converts hMSH2/MSH3 into a distinct nucleotide-bound form, and poises it to be a molecular sensor for lesion specificity. PMID:19377479

Genetic characterization of human herpesvirus type 1: Full-length genome sequence of strain obtained from an encephalitis case from India.

PubMed

Bondre, Vijay P; Sankararaman, Vasudha; Andhare, Vijaysinh; Tupekar, Manisha; Sapkal, Gajanan N

2016-11-01

Human herpes simplex virus 1 (HSV-1) is the most common cause of sporadic encephalitis in humans that contributes to >10 per cent of the encephalitis cases occurring worldwide. Availability of limited full genome sequences from a small number of isolates resulted in poor understanding of host and viral factors responsible for variable clinical outcome. In this study genetic relationship, extent and source of recombination using full-length genome sequence derived from a newly isolated HSV-1 isolate was studied in comparison with those sampled from patients with varied clinical outcome. Full genome sequence of HSV-1 isolated from cerebrospinal fluid (CSF) of a patient with acute encephalitis syndrome (AES) by inoculation in baby hamster kidney-21 (BHK-21) cells was determined using next-generation sequencing (NGS) technology. Phylogenetic analysis of the newly generated sequence in comparison with 33 additional full-length genomes defined genetic relationship with worldwide distributed strains. The bootscan and similarity plot analysis defined recombination crossovers and similarities between newly isolated Indian HSV-1 with six Asian and a total of 34 worldwide isolated strains. Mapping of 376,332 reads amplified from HSV-1 DNA by NGS generated full-length genome of 151,024 bp from newly isolated Indian HSV-1. Phylogenetic analysis classified worldwide distributed strains into three major evolutionary lineages correlating to their geographic distribution. Lineage 1 containing strains were isolated from America and Europe; lineage 2 contained all the strains from Asian countries along with the North American KOS and RE strains whereas the South African isolates were distributed into two groups under lineage 3. Recombination analysis confirmed events of recombination in Indian HSV-1 genome resulting from mixing of different strains evolved in Asian countries. Our results showed that the full-length genome sequence generated from an Indian HSV-1 isolate shared close genetic relationship with the American KOS and Chinese CR38 strains which belonged to the Asian genetic lineage. Recombination analysis of Indian isolate demonstrated multiple recombination crossover points throughout the genome. This full-length genome sequence amplified from the Indian isolate would be helpful to study HSV evolution, genetic basis of differential pathogenesis, host-virus interactions and viral factors contributing towards differential clinical outcome in human infections.

Genetic characterization of human herpesvirus type 1: Full-length genome sequence of strain obtained from an encephalitis case from India

PubMed Central

Bondre, Vijay P.; Sankararaman, Vasudha; Andhare, Vijaysinh; Tupekar, Manisha; Sapkal, Gajanan N.

2016-01-01

Background & objectives: Human herpes simplex virus 1 (HSV-1) is the most common cause of sporadic encephalitis in humans that contributes to >10 per cent of the encephalitis cases occurring worldwide. Availability of limited full genome sequences from a small number of isolates resulted in poor understanding of host and viral factors responsible for variable clinical outcome. In this study genetic relationship, extent and source of recombination using full-length genome sequence derived from a newly isolated HSV-1 isolate was studied in comparison with those sampled from patients with varied clinical outcome. Methods: Full genome sequence of HSV-1 isolated from cerebrospinal fluid (CSF) of a patient with acute encephalitis syndrome (AES) by inoculation in baby hamster kidney-21 (BHK-21) cells was determined using next-generation sequencing (NGS) technology. Phylogenetic analysis of the newly generated sequence in comparison with 33 additional full-length genomes defined genetic relationship with worldwide distributed strains. The bootscan and similarity plot analysis defined recombination crossovers and similarities between newly isolated Indian HSV-1 with six Asian and a total of 34 worldwide isolated strains. Results: Mapping of 376,332 reads amplified from HSV-1 DNA by NGS generated full-length genome of 151,024 bp from newly isolated Indian HSV-1. Phylogenetic analysis classified worldwide distributed strains into three major evolutionary lineages correlating to their geographic distribution. Lineage 1 containing strains were isolated from America and Europe; lineage 2 contained all the strains from Asian countries along with the North American KOS and RE strains whereas the South African isolates were distributed into two groups under lineage 3. Recombination analysis confirmed events of recombination in Indian HSV-1 genome resulting from mixing of different strains evolved in Asian countries. Interpretation & conclusions: Our results showed that the full-length genome sequence generated from an Indian HSV-1 isolate shared close genetic relationship with the American KOS and Chinese CR38 strains which belonged to the Asian genetic lineage. Recombination analysis of Indian isolate demonstrated multiple recombination crossover points throughout the genome. This full-length genome sequence amplified from the Indian isolate would be helpful to study HSV evolution, genetic basis of differential pathogenesis, host-virus interactions and viral factors contributing towards differential clinical outcome in human infections. PMID:28361829

Escherichia coli gamma-glutamyltranspeptidase mutants deficient in processing to subunits.

PubMed

Hashimoto, W; Suzuki, H; Nohara, S; Kumagai, H

1992-11-30

Arginyl residues 513 and 571 of Escherichia coli K-12 gamma-glutamyl-transpeptidase (EC 2.3.2.2) were substituted with alanyl and glycyl residues, respectively, by oligonucleotide-directed in vitro mutagenesis. Both mutants were devoid of the enzymatic activity. On Western blot analysis, we found that both mutants accumulated a gamma-glutamyltranspeptidase precursor which was not processed into large and small subunits in the periplasmic space of Escherichia coli.

Influenza A virus PB1-F2 protein prolongs viral shedding in chickens lengthening the transmission window

PubMed Central

James, Joe; Howard, Wendy; Iqbal, Munir; Nair, Venugopal K.; Barclay, Wendy S.

2016-01-01

Avian influenza is a significant economic burden on the poultry industry in geographical regions where it is enzootic. It also poses a public health concern when avian influenza subtypes infect humans, often with high mortality. Understanding viral genetic factors which positively contribute to influenza A virus (IAV) fitness – infectivity, spread and pathogenesis – is of great importance both for human and livestock health. PB1-F2 is a small accessory protein encoded by IAV and in mammalian hosts has been implicated in a wide range of functions that contribute to increased pathogenesis. In the avian host, the protein has been understudied despite high-level full-length conservation in avian IAV isolates, which is in contrast to the truncations of the PB1-F2 length frequently found in mammalian host isolates. Here we report that the presence of a full-length PB1-F2 protein, from a low pathogenicity H9N2 avian influenza virus, prolongs infectious virus shedding from directly inoculated chickens, thereby enhancing transmission of the virus by lengthening the transmission window to contact birds. As well as extending transmission, the presence of a full-length PB1-F2 suppresses pathogenicity evidenced by an increased minimum lethal dose in embryonated chicken eggs and increasing survival in directly infected birds when compared to a virus lacking an ORF for PB1-F2. We propose that there is a positive pressure to maintain a full-length functional PB1-F2 protein upon infection of avian hosts as it contributes to the effective transmission of IAV in the field. PMID:27558742

Influenza A virus PB1-F2 protein prolongs viral shedding in chickens lengthening the transmission window.

PubMed

James, Joe; Howard, Wendy; Iqbal, Munir; Nair, Venugopal K; Barclay, Wendy S; Shelton, Holly

2016-10-01

Avian influenza is a significant economic burden on the poultry industry in geographical regions where it is enzootic. It also poses a public health concern when avian influenza subtypes infect humans, often with high mortality. Understanding viral genetic factors which positively contribute to influenza A virus (IAV) fitness - infectivity, spread and pathogenesis - is of great importance both for human and livestock health. PB1-F2 is a small accessory protein encoded by IAV and in mammalian hosts has been implicated in a wide range of functions that contribute to increased pathogenesis. In the avian host, the protein has been understudied despite high-level full-length conservation in avian IAV isolates, which is in contrast to the truncations of the PB1-F2 length frequently found in mammalian host isolates. Here we report that the presence of a full-length PB1-F2 protein, from a low pathogenicity H9N2 avian influenza virus, prolongs infectious virus shedding from directly inoculated chickens, thereby enhancing transmission of the virus by lengthening the transmission window to contact birds. As well as extending transmission, the presence of a full-length PB1-F2 suppresses pathogenicity evidenced by an increased minimum lethal dose in embryonated chicken eggs and increasing survival in directly infected birds when compared to a virus lacking an ORF for PB1-F2. We propose that there is a positive pressure to maintain a full-length functional PB1-F2 protein upon infection of avian hosts as it contributes to the effective transmission of IAV in the field.

Mutational analysis of S12 protein and implications for the accuracy of decoding by the ribosome.

PubMed

Sharma, Divya; Cukras, Anthony R; Rogers, Elizabeth J; Southworth, Daniel R; Green, Rachel

2007-12-07

The fidelity of aminoacyl-tRNA selection by the ribosome depends on a conformational switch in the decoding center of the small ribosomal subunit induced by cognate but not by near-cognate aminoacyl-tRNA. The aminoglycosides paromomycin and streptomycin bind to the decoding center and induce related structural rearrangements that explain their observed effects on miscoding. Structural and biochemical studies have identified ribosomal protein S12 (as well as specific nucleotides in 16S ribosomal RNA) as a critical molecular contributor in distinguishing between cognate and near-cognate tRNA species as well as in promoting more global rearrangements in the small subunit, referred to as "closure." Here we use a mutational approach to define contributions made by two highly conserved loops in S12 to the process of tRNA selection. Most S12 variant ribosomes tested display increased levels of fidelity (a "restrictive" phenotype). Interestingly, several variants, K42A and R53A, were substantially resistant to the miscoding effects of paromomycin. Further characterization of the compromised paromomycin response identified a probable second, fidelity-modulating binding site for paromomycin in the 16S ribosomal RNA that facilitates closure of the small subunit and compensates for defects associated with the S12 mutations.

Phylogeny of the Haplosporidia (Eukaryota: Alveolata) based on small subunit ribosomal RNA gene sequence.

PubMed

Flores, B S; Siddall, M E; Burreson, E M

1996-08-01

The phylogenetic position of the phylum Haplosporidia was investigated with the complete small subunit rRNA gene sequences from 5 species in the phylum: Haplosporidium nelsoni and Haplosporidium costale, parasites of the eastern oyster Crassostrea virginica; Haplosporidium louisiana, a parasite of the mudcrab Panopeus herbstii; Minchinia teredinis, a parasite of shipworms (Teredo spp.) and Urosporidium crescens, a hyperparasite found in metacercariae of the trematode Megalophallus sp. in the blue crab, Callinectes sapidus. Multiple alignments of small subunit rRNA gene sequences included the 5 haplosporidian taxa and 14 taxa in the alveolate phyla Ciliophora, Dinoflagellida, and Apicomplexa. Maximum parsimony analysis placed the phylum Haplosporidia as a monophyletic group within the alveolate clade, as a taxon of equal rank with the other 3 alveolate phyla, and as a sister taxon to the clade composed of the phyla Dinoflagellida and Apicomplexa. Transversionally weighted parsimony placed the haplosporidians as a sister taxon to the ciliates. A separate analysis focused on the relationships of species in the genus Haplosporidium. Analyses were conducted with the haplosporidians as a functional ingroup, using each of the alveolate phyla individually as functional outgroups. The results indicated that species in the genus Haplosporidium do not form a monophyletic assemblage. As such, the present morphological criteria for distinguishing the genera Haplosporidium and Minchinia are insufficient.

Immunization with neuronal nicotinic acetylcholine receptor induces neurological autoimmune disease

PubMed Central

Lennon, Vanda A.; Ermilov, Leonid G.; Szurszewski, Joseph H.; Vernino, Steven

2003-01-01

Neuronal nicotinic AChRs (nAChRs) are implicated in the pathogenesis of diverse neurological disorders and in the regulation of small-cell lung carcinoma growth. Twelve subunits have been identified in vertebrates, and mutations of one are recognized in a rare form of human epilepsy. Mice with genetically manipulated neuronal nAChR subunits exhibit behavioral or autonomic phenotypes. Here, we report the first model of an acquired neuronal nAChR disorder and evidence for its pertinence to paraneoplastic neurological autoimmunity. Rabbits immunized once with recombinant α3 subunit (residues 1–205) develop profound gastrointestinal hypomotility, dilated pupils with impaired light response, and grossly distended bladders. As in patients with idiopathic and paraneoplastic autoimmune autonomic neuropathy, the severity parallels serum levels of ganglionic nAChR autoantibody. Failure of neurotransmission through abdominal sympathetic ganglia, with retention of neuronal viability, confirms that the disorder is a postsynaptic channelopathy. In addition, we found ganglionic nAChR protein in small-cell carcinoma lines, identifying this cancer as a potential initiator of ganglionic nAChR autoimmunity. The data support our hypothesis that immune responses driven by distinct neuronal nAChR subtypes expressed in small-cell carcinomas account for several lung cancer–related paraneoplastic disorders affecting cholinergic systems, including autoimmune autonomic neuropathy, seizures, dementia, and movement disorders. PMID:12639997

Synthesis and photophysical properties of a single bond linked tetracene dimer

NASA Astrophysics Data System (ADS)

Sun, Tingting; Shen, Li; Liu, Heyuan; Sun, Xuan; Li, Xiyou

2016-07-01

A tetracene dimer linked directly by a single bond has been successfully prepared by using electron withdrawing groups to improve the stability. The molecular structure of this dimer is characterized by 1H NMR, MALDI-TOF mass spectroscopy, and elemental analysis. The minimized molecular structure and X-ray crystallography reveal that the tetracene subunits of this dimer adopt an orthogonal configuration. Its absorption spectrum differs significantly from that of its monomeric counterpart, suggesting the presence of strong interactions between the two tetracene subunits. The excited state of this dimer is delocalized on both two tetracene subunits, which is significantly different from that of orthogonal anthracene dimers, but similar with that observed for orthogonal pentacene dimer. Most of the excited states of this dimer decay by radioactive channels, which is different from the localized twisted charge transfer state (LTCT) channel of anthracene dimers and the singlet fission (SF) channel of pentacene dimers. The results of this research suggest that similar orthogonal configurations caused different propertied for acene dimers with different conjugation length.

A comparative molecular analysis of water-filled limestone sinkholes in north-eastern Mexico.

PubMed

Sahl, Jason W; Gary, Marcus O; Harris, J Kirk; Spear, John R

2011-01-01

Sistema Zacatón in north-eastern Mexico is host to several deep, water-filled, anoxic, karstic sinkholes (cenotes). These cenotes were explored, mapped, and geochemically and microbiologically sampled by the autonomous underwater vehicle deep phreatic thermal explorer (DEPTHX). The community structure of the filterable fraction of the water column and extensive microbial mats that coat the cenote walls was investigated by comparative analysis of small-subunit (SSU) 16S rRNA gene sequences. Full-length Sanger gene sequence analysis revealed novel microbial diversity that included three putative bacterial candidate phyla and three additional groups that showed high intra-clade distance with poorly characterized bacterial candidate phyla. Limited functional gene sequence analysis in these anoxic environments identified genes associated with methanogenesis, sulfate reduction and anaerobic ammonium oxidation. A directed, barcoded amplicon, multiplex pyrosequencing approach was employed to compare ∼100,000 bacterial SSU gene sequences from water column and wall microbial mat samples from five cenotes in Sistema Zacatón. A new, high-resolution sequence distribution profile (SDP) method identified changes in specific phylogenetic types (phylotypes) in microbial mats at varied depths; Mantel tests showed a correlation of the genetic distances between mat communities in two cenotes and the geographic location of each cenote. Community structure profiles from the water column of three neighbouring cenotes showed distinct variation; statistically significant differences in the concentration of geochemical constituents suggest that the variation observed in microbial communities between neighbouring cenotes are due to geochemical variation. © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.

Structure of L-A Virus: A Specialized Compartment for the Transcription and Replication of Double-stranded RNA

PubMed Central

Castón, José R.; Trus, Benes L.; Booy, Frank P.; Wickner, Reed B.; Wall, Joseph S.; Steven, Alasdair C.

1997-01-01

The genomes of double-stranded (ds)RNA viruses are never exposed to the cytoplasm but are confined to and replicated from a specialized protein-bound compartment—the viral capsid. We have used cryoelectron microscopy and three-dimensional image reconstruction to study this compartment in the case of L-A, a yeast virus whose capsid consists of 60 asymmetric dimers of Gag protein (76 kD). At 16-Å resolution, we distinguish multiple domains in the elongated Gag subunits, whose nonequivalent packing is reflected in subtly different morphologies of the two protomers. Small holes, 10–15 Å across, perforate the capsid wall, which functions as a molecular sieve, allowing the exit of transcripts and the influx of metabolites, while retaining dsRNA and excluding degradative enzymes. Scanning transmission electron microscope measurements of mass-per-unit length suggest that L-A RNA is an A-form duplex, and that RNA filaments emanating from disrupted virions often consist of two or more closely associated duplexes. Nuclease protection experiments confirm that the genome is entirely sequestered inside full capsids, but it is packed relatively loosely; in L-A, the center-to-center spacing between duplexes is 40–45 Å, compared with 25–30 Å in other double-stranded viruses. The looser packing of L-A RNA allows for maneuverability in the crowded capsid interior, in which the genome (in both replication and transcription) must be translocated sequentially past the polymerase immobilized on the inner capsid wall. PMID:9281577

Phylogenetic stratigraphy in the Guerrero Negro hypersaline microbial mat.

PubMed

Harris, J Kirk; Caporaso, J Gregory; Walker, Jeffrey J; Spear, John R; Gold, Nicholas J; Robertson, Charles E; Hugenholtz, Philip; Goodrich, Julia; McDonald, Daniel; Knights, Dan; Marshall, Paul; Tufo, Henry; Knight, Rob; Pace, Norman R

2013-01-01

The microbial mats of Guerrero Negro (GN), Baja California Sur, Mexico historically were considered a simple environment, dominated by cyanobacteria and sulfate-reducing bacteria. Culture-independent rRNA community profiling instead revealed these microbial mats as among the most phylogenetically diverse environments known. A preliminary molecular survey of the GN mat based on only ∼1500 small subunit rRNA gene sequences discovered several new phylum-level groups in the bacterial phylogenetic domain and many previously undetected lower-level taxa. We determined an additional ∼119,000 nearly full-length sequences and 28,000 >200 nucleotide 454 reads from a 10-layer depth profile of the GN mat. With this unprecedented coverage of long sequences from one environment, we confirm the mat is phylogenetically stratified, presumably corresponding to light and geochemical gradients throughout the depth of the mat. Previous shotgun metagenomic data from the same depth profile show the same stratified pattern and suggest that metagenome properties may be predictable from rRNA gene sequences. We verify previously identified novel lineages and identify new phylogenetic diversity at lower taxonomic levels, for example, thousands of operational taxonomic units at the family-genus levels differ considerably from known sequences. The new sequences populate parts of the bacterial phylogenetic tree that previously were poorly described, but indicate that any comprehensive survey of GN diversity has only begun. Finally, we show that taxonomic conclusions are generally congruent between Sanger and 454 sequencing technologies, with the taxonomic resolution achieved dependent on the abundance of reference sequences in the relevant region of the rRNA tree of life.

Qualification of a Quantitative Laryngeal Imaging System Using Videostroboscopy and Videokymography

PubMed Central

Popolo, Peter S.; Titze, Ingo R.

2008-01-01

Objectives: We sought to determine whether full-cycle glottal width measurements could be obtained with a quantitative laryngeal imaging system using videostroboscopy, and whether glottal width and vocal fold length measurements were repeatable and reliable. Methods: Synthetic vocal folds were phonated on a laboratory bench, and dynamic images were obtained in repeated trials by use of videostroboscopy and videokymography (VKG) with an imaging system equipped with a 2-point laser projection device for measuring absolute dimensions. Video images were also obtained with an industrial videoscope system with a built-in laser measurement capability. Maximum glottal width and vocal fold length were compared among these 3 methods. Results: The average variation in maximum glottal width measurements between stroboscopic data and VKG data was 3.10%. The average variations in width measurements between the clinical system and the industrial system were 1.93% (stroboscopy) and 3.49% (VKG). The variations in vocal fold length were similarly small. The standard deviations across trials were 0.29 mm for width and 0.48 mm for length (stroboscopy), 0.18 mm for width (VKG), and 0.25 mm for width and 0.84 mm for length (industrial). Conclusions: For stable, periodic vibration, the full extent of the glottal width can be reliably measured with the quantitative videostroboscopy system. PMID:18646436

Reassessment of MxiH subunit orientation and fold within native Shigella T3SS needles using surface labelling and solid-state NMR.

PubMed

Verasdonck, Joeri; Shen, Da-Kang; Treadgold, Alexander; Arthur, Christopher; Böckmann, Anja; Meier, Beat H; Blocker, Ariel J

2015-12-01

T3SSs are essential virulence determinants of many Gram-negative bacteria, used to inject bacterial effectors of virulence into eukaryotic host cells. Their major extracellular portion, a ∼50 nm hollow, needle-like structure, is essential to host cell sensing and the conduit for effector secretion. It is formed of a small, conserved subunit arranged as a helical polymer. The structure of the subunit has been studied by electron cryomicroscopy within native polymers and by solid-state NMR in recombinant polymers, yielding two incompatible atomic models. To resolve this controversy, we re-examined the native polymer used for electron cryomicroscopy via surface labelling and solid-state NMR. Our data show the orientation and overall fold of the subunit within this polymer is as established by solid-state NMR for recombinant polymers. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

CALCOM: a software for calculating the center of mass of proteins.

PubMed

Costantini, Susan; Paladino, Antonella; Facchiano, Angelo M

2008-02-09

The center of mass of a protein is an artificial point useful for detecting important and simple features of proteins structure, shape and association.CALCOM is a software which calculates the center of mass of a protein, starting from PDB protein structure files. In the case of protein complexes and of protein-small ligand complexes, the position of protein residues or of ligand atoms respect to each protein subunit can be evaluated, as well as the distance among the center of mass of the protein subunits, in order to compare different conformations and evaluate the relative motion of subunits. THE SERVICE IS AVAILABLE AT THE URL: http://bioinformatica.isa.cnr.it/CALCOM/.

A broad survey reveals substitution tolerance of residues ligating FeS clusters in [NiFe] hydrogenase

PubMed Central

2014-01-01

Background In order to understand the effects of FeS cluster attachment in [NiFe] hydrogenase, we undertook a study to substitute all 12 amino acid positions normally ligating the three FeS clusters in the hydrogenase small subunit. Using the hydrogenase from Alteromonas macleodii “deep ecotype” as a model, we substituted one of four amino acids (Asp, His, Asn, Gln) at each of the 12 ligating positions because these amino acids are alternative coordinating residues in otherwise conserved-cysteine positions found in a broad survey of NiFe hydrogenase sequences. We also hoped to discover an enzyme with elevated hydrogen evolution activity relative to a previously reported “G1” (H230C/P285C) improved enzyme in which the medial FeS cluster Pro and the distal FeS cluster His were each substituted for Cys. Results Among all the substitutions screened, aspartic acid substitutions were generally well-tolerated, and examination suggests that the observed deficiency in enzyme activity may be largely due to misprocessing of the small subunit of the enzyme. Alignment of hydrogenase sequences from sequence databases revealed many rare substitutions; the five substitutions present in databases that we tested all exhibited measurable hydrogen evolution activity. Select substitutions were purified and tested, supporting the results of the screening assay. Analysis of these results confirms the importance of small subunit processing. Normalizing activity to quantity of mature small subunit, indicative of total enzyme maturation, weakly suggests an improvement over the “G1” enzyme. Conclusions We have comprehensively screened 48 amino acid substitutions of the hydrogenase from A. macleodii “deep ecotype”, to understand non-canonical ligations of amino acids to FeS clusters and to improve hydrogen evolution activity of this class of hydrogenase. Our studies show that non-canonical ligations can be functional and also suggests a new limiting factor in the production of active enzyme. PMID:24934472

Early Depositional History of the Eocene Izu-Bonin Mariana Arc, Western Pacific Ocean

NASA Astrophysics Data System (ADS)

Waldman, R.; Marsaglia, K. M.; Tepley, F. J., III

2015-12-01

Expedition 351 of the International Ocean Discovery Program cored an Eocene section at Site U1438 in the Philippine Sea that provides insight into the early history of the Izu-Bonin arc. Subduction here is hypothesized to have initiated spontaneously, leaving a characteristic depositional sequence of post-subduction-initiation localized extension and volcanism. We conducted detailed macroscopic and microscopic study of the cores of the lowermost 100m of volcaniclastic and sedimentary rocks (Unit IV) directly overlying subduction initiation igneous basement, to identify depositional facies and trends. We subdivided Unit IV into three subunits based on lithologic characteristics. Transitions between the subunits are relatively abrupt, occurring within the length of a single core. The lowermost subunit (IVA) consists of 4 meters of laminated pelagic claystone with thin beds of graded volcaniclastic siltstone, and fine-grained tuff laminae composed of plagioclase feldspar and green-brown amphibole. The middle subunit (IVB) comprises 51 meters of texturally variable, thick-bedded, coarse-grained gravity flow deposits. These are composed of volcaniclastic sandstone and conglomerate containing glassy and tachylitic volcanic grains as well as sedimentary lithic fragments, along with traces of shallow-water carbonate bioclasts. Subunit IVB sediments are poorer in feldspar than IVA and contain only trace amphibole. They show variable grain rounding and an upsection increase in vitric components. Tachylite grains range from sub-angular to well rounded throughout, and other volcanic grain types show upward increases in angularity and vesicularity. The abrupt transition from pelagic sediments in subunit IVA to shallow-water-sourced gravity flows in subunit IVB suggests a rapid emergence of shallow-water to subaerial volcanic center early in the arc's development. The upper part of subunit IVB also contains igneous intrusions, providing possible evidence for more proximal volcanism at the site. Subunit IVC consists of 46 meters of fine-grained turbidites composed of tuffaceous siltstone, along with radiolarian mudstone and vitric tuff layers. Ongoing studies of the volcanic components will provide additional insight into the source(s) of volcanics throughout the arc's early development.

Information Literacy Development at a Distance: Embedded or Reality?

ERIC Educational Resources Information Center

Chisholm, Elizabeth; Lamond, Heather M.

2012-01-01

A small library using two full time equivalent (FTE) professional staff integrated into the Moodle environment of over 40 postgraduate distance courses with the potential to reach over 1,800 students and getting results. How? This is not embedding as many would think of it, with the librarian an active teacher throughout the entire length of the…

Identification of an Enterocytozoon bieneusi-like microsporidian parasite in simian-immunodeficiency-virus-inoculated macaques with hepatobiliary disease.

PubMed Central

Mansfield, K. G.; Carville, A.; Shvetz, D.; MacKey, J.; Tzipori, S.; Lackner, A. A.

1997-01-01

Enterocytozoon bieneusi is a common opportunistic pathogen of human patients with acquired immune deficiency syndrome (AIDS) causing significant morbidity and mortality. In a retrospective analysis utilizing conventional histochemical techniques, in situ hybridization, polymerase chain reaction, and ultrastructural examination, we identified 18 simian-immunodeficiency-virus-infected macaques (16 Macaca mulatta, 1 M. nemestrina, and 1 M. cyclopis) with Enterocytozoon infection of the hepatobiliary system and small intestine. The organisms were readily identified in the bile ducts and gall bladder by special stains and by in situ hybridization using a probe directed against the small subunit ribosomal RNA of human origin E. bieneusi. Infection of the biliary system was associated with a nonsuppurative and proliferative cholecystitis and choledochitis. Hepatic involvement was characterized by bridging portal fibrosis and nodular hepatocellular regeneration accompanied by marked bile ductular and septal duct hyperplasia. Ultrastructurally, all developmental stages of the organism were found in direct contact with the host cell cytoplasm; spores and sporoblasts contained a double layer of polar tubes. Sequencing of a 607-bp segment of the small subunit ribosomal RNA revealed 97 and 100% identity to two clones of small subunit ribosomal RNA derived from E. bieneusi of human origin. Extensive morphological and genetic similarities between the simian and human enterocytozoons suggest that experimentally infected macaques may serve as a useful model of microsporidial infection in AIDS. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:9094995

Characterization of a new myxozoan species (Myxozoa: Myxobolidae: Myxosporea) in largescale stonerollers (Campostoma oligolepis) from the Mobile River Basin (Alabama).

PubMed

Iwanowicz, D D; Iwanowicz, L R; Howerth, E W; Schill, W B; Blazer, V S; Johnson, R L

2013-02-01

Myxobolus stanlii sp. n. was described from largescale stonerollers ( Campostoma oligolepis ) from the Mobile River Basin in Alabama. The parasite was described using critical identifying morphological features, and the 18S small subunit ribosomal RNA (SSU rRNA) gene sequence. The spore body was ovoid, 10.03 ± 0.7 (7.5-11.0) μm long and 8.8 ± 1.5 (6.3-11.3) μm wide in frontal view. Spore thickness was 6.3 ± 2.7 (6.2-8.6) μm in sutural view. Polar capsules were pyriform, of equal size, and oriented in plane with the sutural ridge. Polar capsules were 2.45 ± 1.5 (range 2.1-4.3) μm in width and 4.6 ± 2.7 (range 4.5-6.9) μm in length. Based on the SSU rRNA gene sequence of Myxobolus stanlii sp. n. is most closely related to M. pseudodispar.

Morphological and Molecular Characterization of Paramecium (Viridoparamecium nov. subgen.) chlorelligerum Kahl, 1935 (Ciliophora)

PubMed Central

KREUTZ, MARTIN; STOECK, THORSTEN; FOISSNER, WILHELM

2013-01-01

We redescribe Paramecium chlorelligerum, a forgotten species, which Kahl (1935) briefly but precisely described in the addendum to his ciliate monographs as a Paramecium with symbiotic green algae. The redescription is based on classical morphological methods and the analysis of the small subunit (SSU) rDNA. Morphologically, P. chlorelligerum differs from P. (Chloroparamecium) bursaria, the second green species in the genus, by having a special swimming shape, the length of the caudal cilia, the size of the micronucleus, the size of the symbiotic algae, the contractile vacuoles (with collecting vesicles vs. collecting canals), and the number of excretory pores /contractile vacuole (1 vs. 2--3). The molecular investigations show that P. chlorelligerum forms a distinct branch distant from the P. (Chloroparamecium) bursaria clade. Thus, we classify P. chlorelligerum in a new subgenus: Paramecium (Viridoparamecium) chlorelligerum. The symbiotic alga belongs to the little-known genus Meyerella, as yet recorded only from the plankton of a North American lake. PMID:22827482

Organization of Circadian Behavior Relies on Glycinergic Transmission.

PubMed

Frenkel, Lia; Muraro, Nara I; Beltrán González, Andrea N; Marcora, María S; Bernabó, Guillermo; Hermann-Luibl, Christiane; Romero, Juan I; Helfrich-Förster, Charlotte; Castaño, Eduardo M; Marino-Busjle, Cristina; Calvo, Daniel J; Ceriani, M Fernanda

2017-04-04

The small ventral lateral neurons (sLNvs) constitute a central circadian pacemaker in the Drosophila brain. They organize daily locomotor activity, partly through the release of the neuropeptide pigment-dispersing factor (PDF), coordinating the action of the remaining clusters required for network synchronization. Despite extensive efforts, the basic principles underlying communication among circadian clusters remain obscure. We identified classical neurotransmitters released by sLNvs through disruption of specific transporters. Adult-specific RNAi-mediated downregulation of the glycine transporter or impairment of glycine synthesis in LNv neurons increased period length by nearly an hour without affecting rhythmicity of locomotor activity. Electrophysiological recordings showed that glycine reduces spiking frequency in circadian neurons. Interestingly, downregulation of glycine receptor subunits in specific sLNv targets impaired rhythmicity, revealing involvement of glycine in information processing within the network. These data identify glycinergic inhibition of specific targets as a cue that contributes to the synchronization of the circadian network. Copyright © 2017 Elsevier Inc. All rights reserved.

The dose response relation for rat spinal cord paralysis analyzed in terms of the effective size of the functional subunit

NASA Astrophysics Data System (ADS)

Adamus-Górka, Magdalena; Mavroidis, Panayiotis; Brahme, Anders; Lind, Bengt K.

2008-11-01

Radiobiological models for estimating normal tissue complication probability (NTCP) are increasingly used in order to quantify or optimize the clinical outcome of radiation therapy. A good NTCP model should fulfill at least the following two requirements: (a) it should predict the sigmoid shape of the corresponding dose-response curve and (b) it should accurately describe the probability of a specified response for arbitrary non-uniform dose delivery for a given endpoint as accurately as possible, i.e. predict the volume dependence. In recent studies of the volume effect of a rat spinal cord after irradiation with narrow and broad proton beams the authors claim that none of the existing NTCP models is able to describe their results. Published experimental data have been used here to try to quantify the change in the effective dose (D50) causing 50% response for different field sizes. The present study was initiated to describe the induction of white matter necrosis in a rat spinal cord after irradiation with narrow proton beams in terms of the mean dose to the effective volume of the functional subunit (FSU). The physically delivered dose distribution was convolved with a function describing the effective size or, more accurately, the sensitivity distribution of the FSU to obtain the effective mean dose deposited in it. This procedure allows the determination of the mean D50 value of the FSUs of a certain size which is of interest for example if the cell nucleus of the oligodendrocyte is the sensitive target. Using the least-squares method to compare the effective doses for different sizes of the functional subunits with the experimental data the best fit was obtained with a length of about 9 mm. For the non-uniform dose distributions an effective FSU length of 8 mm gave the optimal fit with the probit dose-response model. The method could also be used to interpret the so-called bath and shower experiments where the heterogeneous dose delivery was used in the convolution process. The assumption of an effective FSU size is consistent with most of the effects seen when different portions of the rat spinal cord are irradiated to different doses. The effective FSU length from these experiments is about 8.5 ± 0.5 mm. This length could be interpreted as an effective size of the functional subunits in a rat spinal cord, where multiple myelin sheaths are connected by a single oligodendrocyte and repair is limited by the range of oligodendrocyte progenitor cell diffusion. It was even possible to suggest a more likely than uniform effective FSU sensitivity distribution from the experimental data.

Altering the N-terminal arms of the polymerase manager protein UmuD modulates protein interactions.

PubMed

Murison, David A; Ollivierre, Jaylene N; Huang, Qiuying; Budil, David E; Beuning, Penny J

2017-01-01

Escherichia coli cells that are exposed to DNA damaging agents invoke the SOS response that involves expression of the umuD gene products, along with more than 50 other genes. Full-length UmuD is expressed as a 139-amino-acid protein, which eventually cleaves its N-terminal 24 amino acids to form UmuD'. The N-terminal arms of UmuD are dynamic and contain recognition sites for multiple partner proteins. Cleavage of UmuD to UmuD' dramatically affects the function of the protein and activates UmuC for translesion synthesis (TLS) by forming DNA Polymerase V. To probe the roles of the N-terminal arms in the cellular functions of the umuD gene products, we constructed additional N-terminal truncated versions of UmuD: UmuD 8 (UmuD Δ1-7) and UmuD 18 (UmuD Δ1-17). We found that the loss of just the N-terminal seven (7) amino acids of UmuD results in changes in conformation of the N-terminal arms, as determined by electron paramagnetic resonance spectroscopy with site-directed spin labeling. UmuD 8 is cleaved as efficiently as full-length UmuD in vitro and in vivo, but expression of a plasmid-borne non-cleavable variant of UmuD 8 causes hypersensitivity to UV irradiation, which we determined is the result of a copy-number effect. UmuD 18 does not cleave to form UmuD', but confers resistance to UV radiation. Moreover, removal of the N-terminal seven residues of UmuD maintained its interactions with the alpha polymerase subunit of DNA polymerase III as well as its ability to disrupt interactions between alpha and the beta processivity clamp, whereas deletion of the N-terminal 17 residues resulted in decreases in binding to alpha and in the ability to disrupt the alpha-beta interaction. We find that UmuD 8 mimics full-length UmuD in many respects, whereas UmuD 18 lacks a number of functions characteristic of UmuD.

Distinctive Photosystem II Photoinactivation and Protein Dynamics in Marine Diatoms1[W

PubMed Central

Wu, Hongyan; Cockshutt, Amanda M.; McCarthy, Avery; Campbell, Douglas A.

2011-01-01

Diatoms host chlorophyll a/c chloroplasts distinct from green chloroplasts. Diatoms now dominate the eukaryotic oceanic phytoplankton, in part through their exploitation of environments with variable light. We grew marine diatoms across a range of temperatures and then analyzed their PSII function and subunit turnover during an increase in light to mimic an upward mixing event. The small diatom Thalassiosira pseudonana initially responds to increased photoinactivation under blue or white light with rapid acceleration of the photosystem II (PSII) repair cycle. Increased red light provoked only modest PSII photoinactivation but triggered a rapid clearance of a subpool of PsbA. Furthermore, PsbD and PsbB content was greater than PsbA content, indicating a large pool of partly assembled PSII repair cycle intermediates lacking PsbA. The initial replacement rates for PsbD (D2) were, surprisingly, comparable to or higher than those for PsbA (D1), and even the supposedly stable PsbB (CP47) dropped rapidly upon the light shift, showing a novel aspect of rapid protein subunit turnover in the PSII repair cycle in small diatoms. Under sustained high light, T. pseudonana induces sustained nonphotochemical quenching, which correlates with stabilization of PSII function and the PsbA pool. The larger diatom Coscinodiscus radiatus showed generally similar responses but had a smaller allocation of PSII complexes relative to total protein content, with nearly equal stiochiometries of PsbA and PsbD subunits. Fast turnover of multiple PSII subunits, pools of PSII repair cycle intermediates, and photoprotective induction of nonphotochemical quenching are important interacting factors, particularly for small diatoms, to withstand and exploit high, fluctuating light. PMID:21617029

Plant RuBisCo assembly in E. coli with five chloroplast chaperones including BSD2.

PubMed

Aigner, H; Wilson, R H; Bracher, A; Calisse, L; Bhat, J Y; Hartl, F U; Hayer-Hartl, M

2017-12-08

Plant RuBisCo, a complex of eight large and eight small subunits, catalyzes the fixation of CO 2 in photosynthesis. The low catalytic efficiency of RuBisCo provides strong motivation to reengineer the enzyme with the goal of increasing crop yields. However, genetic manipulation has been hampered by the failure to express plant RuBisCo in a bacterial host. We achieved the functional expression of Arabidopsis thaliana RuBisCo in Escherichia coli by coexpressing multiple chloroplast chaperones. These include the chaperonins Cpn60/Cpn20, RuBisCo accumulation factors 1 and 2, RbcX, and bundle-sheath defective-2 (BSD2). Our structural and functional analysis revealed the role of BSD2 in stabilizing an end-state assembly intermediate of eight RuBisCo large subunits until the small subunits become available. The ability to produce plant RuBisCo recombinantly will facilitate efforts to improve the enzyme through mutagenesis. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Structural integration in hypoxia-inducible factors

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

Wu, Dalei; Potluri, Nalini; Lu, Jingping

The hypoxia-inducible factors (HIFs) coordinate cellular adaptations to low oxygen stress by regulating transcriptional programs in erythropoiesis, angiogenesis and metabolism. These programs promote the growth and progression of many tumours, making HIFs attractive anticancer targets. Transcriptionally active HIFs consist of HIF-alpha and ARNT (also called HIF-1 beta) subunits. Here we describe crystal structures for each of mouse HIF-2 alpha-ARNT and HIF-1 alpha-ARNT heterodimers in states that include bound small molecules and their hypoxia response element. A highly integrated quaternary architecture is shared by HIF-2 alpha-ARNT and HIF-1 alpha-ARNT, wherein ARNT spirals around the outside of each HIF-alpha subunit. Five distinctmore » pockets are observed that permit small-molecule binding, including PAS domain encapsulated sites and an interfacial cavity formed through subunit heterodimerization. The DNA-reading head rotates, extends and cooperates with a distal PAS domain to bind hypoxia response elements. HIF-alpha mutations linked to human cancers map to sensitive sites that establish DNA binding and the stability of PAS domains and pockets.« less

The nucleotide sequence of the entire ribosomal DNA operon and the structure of the large subunit rRNA of Giardia muris.

PubMed

van Keulen, H; Gutell, R R; Campbell, S R; Erlandsen, S L; Jarroll, E L

1992-10-01

The total nucleotide sequence of the rDNA of Giardia muris, an intestinal protozoan parasite of rodents, has been determined. The repeat unit is 7668 basepairs (bp) in size and consists of a spacer of 3314 bp, a small-subunit rRNA (SSU-rRNA) gene of 1429, and a large-subunit rRNA (LSU-rRNA) gene of 2698 bp. The spacer contains long direct repeats and is heterogeneous in size. The LSU-rRNA of G. muris was compared to that of the human intestinal parasite Giardia duodenalis, to the bird parasite Giardia ardeae, and to that of Escherichia coli. The LSU-rRNA has a size comparable to the 23S rRNA of E. coli but shows structural features typical for eukaryotes. Some variable regions are typically small and account for the overall smaller size of this rRNA. The structure of the G. muris LSU-rRNA is similar to that of the other Giardia rRNA, but each rRNA has characteristic features residing in a number of variable regions.

The Nanomechanical Properties of Lactococcus lactis Pili Are Conditioned by the Polymerized Backbone Pilin

PubMed Central

Castelain, Mickaël; Duviau, Marie-Pierre; Canette, Alexis; Schmitz, Philippe; Loubière, Pascal; Cocaign-Bousquet, Muriel; Piard, Jean-Christophe; Mercier-Bonin, Muriel

2016-01-01

Pili produced by Lactococcus lactis subsp. lactis are putative linear structures consisting of repetitive subunits of the major pilin PilB that forms the backbone, pilin PilA situated at the distal end of the pilus, and an anchoring pilin PilC that tethers the pilus to the peptidoglycan. We determined the nanomechanical properties of pili using optical-tweezers force spectroscopy. Single pili were exposed to optical forces that yielded force-versus-extension spectra fitted using the Worm-Like Chain model. Native pili subjected to a force of 0–200 pN exhibit an inextensible, but highly flexible ultrastructure, reflected by their short persistence length. We tested a panel of derived strains to understand the functional role of the different pilins. First, we found that both the major pilin PilB and sortase C organize the backbone into a full-length organelle and dictate the nanomechanical properties of the pili. Second, we found that both PilA tip pilin and PilC anchoring pilin were not essential for the nanomechanical properties of pili. However, PilC maintains the pilus on the bacterial surface and may play a crucial role in the adhesion- and biofilm-forming properties of L. lactis. PMID:27010408

Differences in cholinergic responses from outer hair cells of rat and guinea pig.

PubMed

Chen, C; LeBlanc, C; Bobbin, R P

1996-09-01

A cholinergic receptor on outer hair cells (OHC) in guinea pig cochlea induces a K+ current when it is activated by acetylcholine and suberyldicholine but not by nicotine or muscarine (Bobbin, 1995). This unusual receptor may contain an alpha 9-subunit. However, the pharmacology of the alpha 9-subunit cloned from rat and expressed in Xenopus oocytes does not completely match that obtained for the ACh receptor in guinea pig OHCs. The response to 1,1-dimethyl-4-phenylpiperazinium (DMPP) is large in guinea pig OHCs and small in oocytes containing receptors of the alpha 9-subunit. Therefore, we compared the effects of cholinergic receptor agonists in rat and guinea pig OHCs using the whole-cell variant of the patch-clamp technique. ACh caused the largest outward K+ current in OHCs from both rat and guinea pig. Carbachol- and suberyldicholine-induced responses were similar in magnitude in OHCs of rat and guinea pig. However, DMPP produced a small response in OHCs from rat and a large response in OHCs from guinea pig. At a concentration of 100 microM, muscarine, oxotremorine M, nicotine and cytisine induced little response in guinea pig OHCs and none in rat OHCs. Results suggest that the ACh receptor on rat OHCs is similar to the alpha 9-subunit-containing receptor expressed in oocytes but different from the ACh receptor on guinea pig OHCs.

Modulation of procaspase-7 self-activation by PEST amino acid residues of the N-terminal prodomain and intersubunit linker.

PubMed

Alves, Juliano; Garay-Malpartida, Miguel; Occhiucci, João M; Belizário, José E

2017-12-01

Procaspase-7 zymogen polypeptide is composed of a short prodomain, a large subunit (p20), and a small subunit (p10) connected to an intersubunit linker. Caspase-7 is activated by an initiator caspase-8 and -9, or by autocatalysis after specific cleavage at IQAD 198 ↓S located at the intersubunit linker. Previously, we identified that PEST regions made of amino acid residues Pro (P), Glu (E), Asp (D), Ser (S), Thr (T), Asn (N), and Gln (Q) are conserved flanking amino acid residues in the cleavage sites within a prodomain and intersubunit linker of all caspase family members. Here we tested the impact of alanine substitution of PEST amino acid residues on procaspase-7 proteolytic self-activation directly in Escherichia coli. The p20 and p10 subunit cleavage were significantly delayed in double caspase-7 mutants in the prodomain (N18A/P26A) and intersubunit linker (S199A/P201A), compared with the wild-type caspase-7. The S199A/P201A mutants effectively inhibited the p10 small subunit cleavage. However, the mutations did not change the kinetic parameters (k cat /K M ) and optimal tetrapeptide specificity (DEVD) of the purified mutant enzymes. The results suggest a role of PEST-amino acid residues in the molecular mechanism for prodomain and intersubunit cleavage and caspase-7 self-activation.

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

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

Sinophysis and Pseudophalacroma are distantly related to typical Dinophysoid dinoflagellates (Dinophysales, Dinophyceae).

PubMed

Gómez, Fernando; Moreira, David; López-García, Purificación

2012-01-01

Dinophysoid dinoflagellates are usually considered a large monophyletic group. Large subunit and small subunit (SSU) rDNA phylogenies suggest a basal position for Amphisoleniaceae (Amphisolenia,Triposolenia) with respect to two sister groups, one containing most Phalacroma species plus Oxyphysis and the other Dinophysis,Ornithocercus, Dinophysoid dinoflagellates are usually considered a large monophyletic group. Large subunit and small subunit (SSU) rDNA phylogenies suggest a basal position for Amphisoleniaceae (Amphisolenia,Triposolenia) with respect to two sister groups, one containing most Phalacroma species plus Oxyphysis and the other Dinophysis,Ornithocercus, Histioneis,Citharistes and some Phalacroma species. We provide here new SSU rDNA sequences of Pseudophalacroma (pelagic) and Sinophysis (the only benthic dinophysoid genus). Molecular phylogenies support that they are very divergent with respect to the main clade of Dinophysales. Additional molecular markers of these two key genera are needed to elucidate the evolutionary relations among the dinophysoid dinoflagellates. Histioneis,Citharistes and some Phalacroma species. We provide here new SSU rDNA sequences of Pseudophalacroma (pelagic) and Sinophysis (the only benthic dinophysoid genus). Molecular phylogenies support that they are very divergent with respect to the main clade of Dinophysales. Additional molecular markers of these two key genera are needed to elucidate the evolutionary relations among the dinophysoid dinoflagellates. © 2011 The Author(s) Journal of Eukaryotic Microbiology © 2011 International Society of Protistologists.

Structures of the Porphyromonas gingivalis OxyR regulatory domain explain differences in expression of the OxyR regulon in Escherichia coli and P. gingivalis

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

Svintradze, David V.; Virginia Commonwealth University, Richmond, VA 23219-1540; Peterson, Darrell L.

Differences in OxyR regulated expression of oxidative stress genes between Escherichia coli and Porphyromonas gingivalis are explained by very minor differences in structure and amino-acid sequence of the respective oxidized and reduced OxyR regulatory domains. These differences affect OxyR quaternary structures and are predicted from model building of full length OxyR–DNA complexes to confer distinct modes of DNA binding on this transcriptional regulator. OxyR transcriptionally regulates Escherichia coli oxidative stress response genes through a reversibly reducible cysteine disulfide biosensor of cellular redox status. Structural changes induced by redox changes in these cysteines are conformationally transmitted to the dimer subunit interfaces,more » which alters dimer and tetramer interactions with DNA. In contrast to E. coli OxyR regulatory-domain structures, crystal structures of Porphyromonas gingivalis OxyR regulatory domains show minimal differences in dimer configuration on changes in cysteine disulfide redox status. This locked configuration of the P. gingivalis OxyR regulatory-domain dimer closely resembles the oxidized (activating) form of the E. coli OxyR regulatory-domain dimer. It correlates with the observed constitutive activation of some oxidative stress genes in P. gingivalis and is attributable to a single amino-acid insertion in P. gingivalis OxyR relative to E. coli OxyR. Modelling of full-length P. gingivalis, E. coli and Neisseria meningitidis OxyR–DNA complexes predicts different modes of DNA binding for the reduced and oxidized forms of each.« less

Filling reference gaps via assembling DNA barcodes using high-throughput sequencing—moving toward barcoding the world

PubMed Central

Zhou, Chengran

2017-01-01

Abstract Over the past decade, biodiversity researchers have dedicated tremendous efforts to constructing DNA reference barcodes for rapid species registration and identification. Although analytical cost for standard DNA barcoding has been significantly reduced since early 2000, further dramatic reduction in barcoding costs is unlikely because Sanger sequencing is approaching its limits in throughput and chemistry cost. Constraints in barcoding cost not only led to unbalanced barcoding efforts around the globe, but also prevented high-throughput sequencing (HTS)–based taxonomic identification from applying binomial species names, which provide crucial linkages to biological knowledge. We developed an Illumina-based pipeline, HIFI-Barcode, to produce full-length Cytochrome c oxidase subunit I (COI) barcodes from pooled polymerase chain reaction amplicons generated by individual specimens. The new pipeline generated accurate barcode sequences that were comparable to Sanger standards, even for different haplotypes of the same species that were only a few nucleotides different from each other. Additionally, the new pipeline was much more sensitive in recovering amplicons at low quantity. The HIFI-Barcode pipeline successfully recovered barcodes from more than 78% of the polymerase chain reactions that didn’t show clear bands on the electrophoresis gel. Moreover, sequencing results based on the single molecular sequencing platform Pacbio confirmed the accuracy of the HIFI-Barcode results. Altogether, the new pipeline can provide an improved solution to produce full-length reference barcodes at about one-tenth of the current cost, enabling construction of comprehensive barcode libraries for local fauna, leading to a feasible direction for DNA barcoding global biomes. PMID:29077841

Full-length cellular β-secretase has a trimeric subunit stoichiometry, and its sulfur-rich transmembrane interaction site modulates cytosolic copper compartmentalization.

PubMed

Liebsch, Filip; Aurousseau, Mark R P; Bethge, Tobias; McGuire, Hugo; Scolari, Silvia; Herrmann, Andreas; Blunck, Rikard; Bowie, Derek; Multhaup, Gerd

2017-08-11

The β-secretase (BACE1) initiates processing of the amyloid precursor protein (APP) into Aβ peptides, which have been implicated as central players in the pathology of Alzheimer disease. BACE1 has been described as a copper-binding protein and its oligomeric state as being monomeric, dimeric, and/or multimeric, but the native cellular stoichiometry has remained elusive. Here, by using single-molecule fluorescence and in vitro cross-linking experiments with photo-activatable unnatural amino acids, we show that full-length BACE1, independently of its subcellular localization, exists as trimers in human cells. We found that trimerization requires the BACE1 transmembrane sequences (TMSs) and cytoplasmic domains, with residues Ala 463 and Cys 466 buried within the trimer interface of the sulfur-rich core of the TMSs. Our 3D model predicts that the sulfur-rich core of the trimeric BACE1 TMS is accessible to metal ions, but copper ions did not trigger trimerization. The results of functional assays of endogenous BACE1 suggest that it has a role in intracellular copper compartmentalization by transferring cytosolic copper to intracellular compartments, while leaving the overall cellular copper concentration unaltered. Adding to existing physiological models, our results provide novel insight into the atypical interactions between copper and BACE1 and into its non-enzymatic activities. In conclusion, therapeutic Alzheimer disease prevention strategies aimed at decreasing BACE1 protein levels should be regarded with caution, because adverse effects in copper homeostasis may occur. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The C-terminal SH2 domain of p85 accounts for the high affinity and specificity of the binding of phosphatidylinositol 3-kinase to phosphorylated platelet-derived growth factor beta receptor.

PubMed Central

Klippel, A; Escobedo, J A; Fantl, W J; Williams, L T

1992-01-01

Upon stimulation by its ligand, the platelet-derived growth factor (PDGF) receptor associates with the 85-kDa subunit of phosphatidylinositol (PI) 3-kinase. The 85-kDa protein (p85) contains two Src homology 2 (SH2) domains and one SH3 domain. To define the part of p85 that interacts with the PDGF receptor, a series of truncated p85 mutants was analyzed for association with immobilized PDGF receptor in vitro. We found that a fragment of p85 that contains a single Src homology domain, the C-terminal SH2 domain (SH2-C), was sufficient for directing the high-affinity interaction with the receptor. Half-maximal binding of SH2-C to the receptor was observed at an SH2-C concentration of 0.06 nM. SH2-C, like full-length p85, was able to distinguish between wild-type PDGF receptor and a mutant receptor lacking the PI 3-kinase binding site. An excess of SH2-C blocked binding of full-length p85 and PI 3-kinase to the receptor but did not interfere with the binding of two other SH2-containing proteins, phospholipase C-gamma and GTPase-activating protein. These results demonstrate that a region of p85 containing a single SH2 domain accounts both for the high affinity and specificity of binding of PI 3-kinase to the PDGF receptor. Images PMID:1312663

Filling reference gaps via assembling DNA barcodes using high-throughput sequencing-moving toward barcoding the world.

PubMed

Liu, Shanlin; Yang, Chentao; Zhou, Chengran; Zhou, Xin

2017-12-01

Over the past decade, biodiversity researchers have dedicated tremendous efforts to constructing DNA reference barcodes for rapid species registration and identification. Although analytical cost for standard DNA barcoding has been significantly reduced since early 2000, further dramatic reduction in barcoding costs is unlikely because Sanger sequencing is approaching its limits in throughput and chemistry cost. Constraints in barcoding cost not only led to unbalanced barcoding efforts around the globe, but also prevented high-throughput sequencing (HTS)-based taxonomic identification from applying binomial species names, which provide crucial linkages to biological knowledge. We developed an Illumina-based pipeline, HIFI-Barcode, to produce full-length Cytochrome c oxidase subunit I (COI) barcodes from pooled polymerase chain reaction amplicons generated by individual specimens. The new pipeline generated accurate barcode sequences that were comparable to Sanger standards, even for different haplotypes of the same species that were only a few nucleotides different from each other. Additionally, the new pipeline was much more sensitive in recovering amplicons at low quantity. The HIFI-Barcode pipeline successfully recovered barcodes from more than 78% of the polymerase chain reactions that didn't show clear bands on the electrophoresis gel. Moreover, sequencing results based on the single molecular sequencing platform Pacbio confirmed the accuracy of the HIFI-Barcode results. Altogether, the new pipeline can provide an improved solution to produce full-length reference barcodes at about one-tenth of the current cost, enabling construction of comprehensive barcode libraries for local fauna, leading to a feasible direction for DNA barcoding global biomes. © The Authors 2017. Published by Oxford University Press.

Characterization of two genes encoding the Mycobacterium tuberculosis ribonucleotide reductase small subunit.

PubMed Central

Yang, F; Curran, S C; Li, L S; Avarbock, D; Graf, J D; Chua, M M; Lu, G; Salem, J; Rubin, H

1997-01-01

Two nrdF genes, nrdF1 and nrdF2, encoding the small subunit (R2) of ribonucleotide reductase (RR) from Mycobacterium tuberculosis have 71% identity at the amino acid level and are both highly homologous with Salmonella typhimurium R2F. The calculated molecular masses of R2-1 and R2-2 are 36,588 (322 amino acids [aa]) and 36,957 (324 aa) Da, respectively. Western blot analysis of crude M. tuberculosis extracts indicates that both R2s are expressed in vivo. Recombinant R2-2 is enzymatically active when assayed with pure recombinant M. tuberculosis R1 subunit. Both ATP and dATP are activators for CDP reduction up to 2 and 1 mM, respectively. The gene encoding M. tuberculosis R2-1, nrdF1, is not linked to nrdF2, nor is either gene linked to the gene encoding the large subunit, M. tuberculosis nrdE. The gene encoding MTP64 was found downstream from nrdF1, and the gene encoding alcohol dehydrogenase was found downstream from nrdF2. A nrdA(Ts) strain of E. coli (E101) could be complemented by simultaneous transformation with M. tuberculosis nrdE and nrdF2. An M. tuberculosis nrdF2 variant in which the codon for the catalytically necessary tyrosine was replaced by the phenylalanine codon did not complement E101 when cotransformed with M. tuberculosis nrdE. Similarly, M. tuberculosis nrdF1 and nrdE did not complement E101. Activity of recombinant M. tuberculosis RR was inhibited by incubating the enzyme with a peptide corresponding to the 7 C-terminal amino acid residues of the R2-2 subunit. M. tuberculosis is a species in which a nrdEF system appears to encode the biologically active species of RR and also the only bacterial species identified so far in which class I RR subunits are not arranged on an operon. PMID:9335290

Beta3 subunits promote expression and nicotine-induced up-regulation of human nicotinic alpha6* nicotinic acetylcholine receptors expressed in transfected cell lines.

PubMed

Tumkosit, Prem; Kuryatov, Alexander; Luo, Jie; Lindstrom, Jon

2006-10-01

Nicotinic acetylcholine receptors (AChRs) containing alpha6 subunits are typically found at aminergic nerve endings where they play important roles in nicotine addiction and Parkinson's disease. alpha6* AChRs usually contain beta3 subunits. beta3 subunits are presumed to assemble only in the accessory subunit position within AChRs where they do not participate in forming acetylcholine binding sites. Assembly of subunits in the accessory position may be a critical final step in assembly of mature AChRs. Human alpha6 AChRs subtypes were permanently transfected into human tsA201 human embryonic kidney (HEK) cell lines. alpha6beta2beta3 and alpha6beta4beta3 cell lines were found to express much larger amounts of AChRs and were more sensitive to nicotine-induced increase in the amount of AChRs than were alpha6beta2 or alpha6beta4 cell lines. The increased sensitivity to nicotine-induced up-regulation was due not to a beta3-induced increase in affinity for nicotine but probably to a direct effect on assembly of AChR subunits. HEK cells express only a small amount of mature alpha6beta2 AChRs, but many of these subunits are on the cell surface. This contrasts with Xenopus laevis oocytes, which express a large amount of incorrectly assembled alpha6beta2 subunits that bind cholinergic ligands but form large amorphous intracellular aggregates. Monoclonal antibodies (mAbs) were made to the alpha6 and beta3 subunits to aid in the characterization of these AChRs. The alpha6 mAbs bind to epitopes C-terminal of the extracellular domain. These data demonstrate that both cell type and the accessory subunit beta3 can play important roles in alpha6* AChR expression, stability, and up-regulation by nicotine.

Detection of rearrangements and transcriptional up-regulation of ALK in FFPE lung cancer specimens using a novel, sensitive, quantitative reverse transcription polymerase chain reaction assay.

PubMed

Gruber, Kim; Horn, Heike; Kalla, Jörg; Fritz, Peter; Rosenwald, Andreas; Kohlhäufl, Martin; Friedel, Godehard; Schwab, Matthias; Ott, German; Kalla, Claudia

2014-03-01

The approved dual-color fluorescence in situ hybridization (FISH) test for the detection of anaplastic lymphoma receptor tyrosine kinase (ALK) gene rearrangements in non-small-cell lung cancer (NSCLC) is complex and represents a low-throughput assay difficult to use in daily diagnostic practice. We devised a sensitive and robust routine diagnostic test for the detection of rearrangements and transcriptional up-regulation of ALK. We developed a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay adapted to RNA isolated from routine formalin-fixed, paraffin-embedded material and applied it to 652 NSCLC specimens. The reliability of this technique to detect ALK dysregulation was shown by comparison with FISH and immunohistochemistry. qRT-PCR analysis detected unbalanced ALK expression indicative of a gene rearrangement in 24 (4.6%) and full-length ALK transcript expression in six (1.1%) of 523 interpretable tumors. Among 182 tumors simultaneously analyzed by FISH and qRT-PCR, the latter accurately typed 97% of 19 rearranged and 158 nonrearranged tumors and identified ALK deregulation in two cases with insufficient FISH. Six tumors expressing full-length ALK transcripts did not show rearrangements of the gene. Immunohistochemistry detected ALK protein overexpression in tumors with gene fusions and transcriptional up-regulation, but did not distinguish between the two. One case with full-length ALK expression carried a heterozygous point mutation (S1220Y) within the kinase domain potentially interfering with kinase activity and/or inhibitor binding. Our qRT-PCR assay reliably identifies and distinguishes ALK rearrangements and full-length transcript expression in formalin-fixed, paraffin-embedded material. It is an easy-to-perform, cost-effective, and high-throughput tool for the diagnosis of ALK activation. The expression of full-length ALK transcripts may be relevant for ALK inhibitor therapy in NSCLC.

Ebolavirus Glycoprotein Fc Fusion Protein Protects Guinea Pigs against Lethal Challenge.

PubMed

Konduru, Krishnamurthy; Shurtleff, Amy C; Bradfute, Steven B; Nakamura, Siham; Bavari, Sina; Kaplan, Gerardo

2016-01-01

Ebola virus (EBOV), a member of the Filoviridae that can cause severe hemorrhagic fever in humans and nonhuman primates, poses a significant threat to the public health. Currently, there are no licensed vaccines or therapeutics to prevent and treat EBOV infection. Several vaccines based on the EBOV glycoprotein (GP) are under development, including vectored, virus-like particles, and protein-based subunit vaccines. We previously demonstrated that a subunit vaccine containing the extracellular domain of the Ebola ebolavirus (EBOV) GP fused to the Fc fragment of human IgG1 (EBOVgp-Fc) protected mice against EBOV lethal challenge. Here, we show that the EBOVgp-Fc vaccine formulated with QS-21, alum, or polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC) adjuvants induced strong humoral immune responses in guinea pigs. The vaccinated animals developed anti-GP total antibody titers of approximately 105-106 and neutralizing antibody titers of approximately 103 as assessed by a BSL-2 neutralization assay based on vesicular stomatitis virus (VSV) pseudotypes. The poly-ICLC formulated EBOVgp-Fc vaccine protected all the guinea pigs against EBOV lethal challenge performed under BSL-4 conditions whereas the same vaccine formulated with QS-21 or alum only induced partial protection. Vaccination with a mucin-deleted EBOVgp-Fc construct formulated with QS-21 adjuvant did not have a significant effect in anti-GP antibody levels and protection against EBOV lethal challenge compared to the full-length GP construct. The bulk of the humoral response induced by the EBOVgp-Fc vaccine was directed against epitopes outside the EBOV mucin region. Our findings indicate that different adjuvants can eliciting varying levels of protection against lethal EBOV challenge in guinea pigs vaccinated with EBOVgp-Fc, and suggest that levels of total anti-GP antibodies elicit by protein-based GP subunit vaccines do not correlate with protection. Our data further support the development of Fc fusions of GP as a candidate vaccine for human use.

The Varicella-zoster virus DNA encapsidation genes: Identification and characterization of the putative terminase subunits

PubMed Central

Visalli, Robert J.; Nicolosi, Denise M.; Irven, Karen L.; Goshorn, Bradley; Khan, Tamseel; Visalli, Melissa A.

2007-01-01

The putative DNA encapsidation genes encoded by open reading frames (ORFs) 25, 26, 30, 34, 43, 45/42 and 54 were cloned from Varicella-zoster virus (VZV) strain Ellen. Sequencing revealed that the Ellen ORFs were highly conserved at the amino acid level when compared to those of nineteen previously published VZV isolates. Additionally, RT-PCR provided the first evidence that ORF45/42 was expressed as a spliced transcript in VZV-infected cells. All seven ORFs were expressed in vitro and full length products were identified using a C-terminal V5 epitope tag. The in vitro products of the putative VZV terminase subunits encoded by ORFs 30 and 45/42 proved useful in protein-protein interaction assays. Previous studies have reported the formation of a heterodimeric terminase complex involved in DNA encapsidation for both herpes simplex virus-type 1 (HSV-1) and human cytomegalovirus (HCMV). Here we report that the C-terminal portion of exon II of ORF45/42 (ORF42-C269) interacted in GST-pull down experiments with in vitro synthesized ORF30 and ORF45/42. The interactions were maintained in the presence of anionic detergents and in buffers of increasing ionic strength. Cells transiently transfected with epitope tagged ORF45/42 or ORF30 showed primarily cytoplasmic staining. In contrast, an antiserum directed to the N-terminal portion of ORF45 showed nearly exclusive nuclear localization of the ORF45/42 gene product in infected cells. An ORF30 specific antiserum detected an 87 kDa protein in both the cytoplasmic and nuclear fractions of VZV infected cells. The results were consistent with the localization and function of herpesviral terminase subunits. This is the first study aimed at the identification and characterization of the VZV DNA encapsidation gene products. PMID:17868947

Ebolavirus Glycoprotein Fc Fusion Protein Protects Guinea Pigs against Lethal Challenge

DOE PAGES

Konduru, Krishnamurthy; Shurtleff, Amy C.; Bradfute, Steven B.; ...

2016-09-13

Ebola virus (EBOV), a member of the Filoviridae that can cause severe hemorrhagic fever in humans and nonhuman primates, poses a significant threat to the public health. Currently, there are no licensed vaccines or therapeutics to prevent and treat EBOV infection. Several vaccines based on the EBOV glycoprotein (GP) are under development, including vectored, virus-like particles, and protein-based subunit vaccines. We previously demonstrated that a subunit vaccine containing the extracellular domain of the Ebola ebolavirus (EBOV) GP fused to the Fc fragment of human IgG1 (EBOVgp-Fc) protected mice against EBOV lethal challenge. Here, we show that the EBOVgp-Fc vaccine formulatedmore » with QS-21, alum, or polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC) adjuvants induced strong humoral immune responses in guinea pigs. The vaccinated animals developed anti-GP total antibody titers of approximately 10 5–10 6 and neutralizing antibody titers of approximately 10 3 as assessed by a BSL-2 neutralization assay based on vesicular stomatitis virus (VSV) pseudotypes. The poly-ICLC formulated EBOVgp-Fc vaccine protected all the guinea pigs against EBOV lethal challenge performed under BSL-4 conditions whereas the same vaccine formulated with QS-21 or alum only induced partial protection. Vaccination with a mucin-deletedEBOVgp-Fc construct formulated with QS-21 adjuvant did not have a significant effect in anti-GP antibody levels and protection against EBOV lethal challenge compared to the full-lengthGP construct. The bulk of the humoral response induced by the EBOVgp-Fc vaccine was directed against epitopes outside the EBOV mucin region. Our findings indicate that different adjuvants can eliciting varying levels of protection against lethal EBOV challenge in guinea pigs vaccinated with EBOVgp-Fc,and suggest that levels of total anti-GP antibodies elicit by protein-based GP subunit vaccines do not correlate with protection. In conclusion, our data further support the development of Fc fusions of GP as a candidate vaccine for human use.« less

ScMED7, a sugarcane mediator subunit gene, acts as a regulator of plant immunity and is responsive to diverse stress and hormone treatments.

PubMed

Zhang, Xu; Yang, Yuting; Zou, Jiake; Chen, Yun; Wu, Qibin; Guo, Jinlong; Que, Youxiong; Xu, Liping

2017-12-01

The Mediator complex, is an essential component of the RNA polymerase II general transcriptional machinery in eukaryotes. Mediator subunit 7 (MED7), a key subunit in the central module of this complex, plays an important role in gene transcriptional regulation. The present study isolated the full-length cDNA of the MED7 gene of sugarcane, hereby designated as ScMED7, which was characterized to harbor a 525-bp open reading frame that is predicted to encode a 174-amino acid protein with a molecular mass of 19.9 kDa and was localized to the nucleus and cytoplasm. ScMED7 contains one typical conserved domain of MED7 proteins and shares 98% homology with that from Sorghum bicolor (XP_002447862.1). ScMED7 was constitutively expressed, yet significantly higher in bud tissues. ScMED7 transcription was obviously induced by heavy metal (CdCl 2 ), low temperature (4 °C), and hormone (SA and MeJA) treatments, while inhibited by osmotic stresses of NaCl and PEG. The role of ScMED7 in plant immunity was demonstrated by transient overexpression in tobacco, which in turn induces the expression of six out of nine defense-related marker genes, including all the three hypersensitive response genes. The responses of defense-related marker genes in the mock and in the ScMED7 transiently overexpressed leaves challenged by pathogenic Pseudomonas solanacearum and Fusarium solani var. coeruleum suggest that ScMED7 acts as a negative regulator during pathogen infections, whereas only fungal infection was clearly phenotypically expressed. In sum, ScMED7 plays an important role in modulating sugarcane responses to biotic and abiotic stresses, and may have dual roles in hypersensitive responses and basal defense against pathogens.

Ebolavirus Glycoprotein Fc Fusion Protein Protects Guinea Pigs against Lethal Challenge

PubMed Central

Konduru, Krishnamurthy; Shurtleff, Amy C.; Bradfute, Steven B.; Nakamura, Siham; Bavari, Sina; Kaplan, Gerardo

2016-01-01

Ebola virus (EBOV), a member of the Filoviridae that can cause severe hemorrhagic fever in humans and nonhuman primates, poses a significant threat to the public health. Currently, there are no licensed vaccines or therapeutics to prevent and treat EBOV infection. Several vaccines based on the EBOV glycoprotein (GP) are under development, including vectored, virus-like particles, and protein-based subunit vaccines. We previously demonstrated that a subunit vaccine containing the extracellular domain of the Ebola ebolavirus (EBOV) GP fused to the Fc fragment of human IgG1 (EBOVgp-Fc) protected mice against EBOV lethal challenge. Here, we show that the EBOVgp-Fc vaccine formulated with QS-21, alum, or polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC) adjuvants induced strong humoral immune responses in guinea pigs. The vaccinated animals developed anti-GP total antibody titers of approximately 105−106 and neutralizing antibody titers of approximately 103 as assessed by a BSL-2 neutralization assay based on vesicular stomatitis virus (VSV) pseudotypes. The poly-ICLC formulated EBOVgp-Fc vaccine protected all the guinea pigs against EBOV lethal challenge performed under BSL-4 conditions whereas the same vaccine formulated with QS-21 or alum only induced partial protection. Vaccination with a mucin-deleted EBOVgp-Fc construct formulated with QS-21 adjuvant did not have a significant effect in anti-GP antibody levels and protection against EBOV lethal challenge compared to the full-length GP construct. The bulk of the humoral response induced by the EBOVgp-Fc vaccine was directed against epitopes outside the EBOV mucin region. Our findings indicate that different adjuvants can eliciting varying levels of protection against lethal EBOV challenge in guinea pigs vaccinated with EBOVgp-Fc, and suggest that levels of total anti-GP antibodies elicit by protein-based GP subunit vaccines do not correlate with protection. Our data further support the development of Fc fusions of GP as a candidate vaccine for human use. PMID:27622456

Ebolavirus Glycoprotein Fc Fusion Protein Protects Guinea Pigs against Lethal Challenge

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

Konduru, Krishnamurthy; Shurtleff, Amy C.; Bradfute, Steven B.

Ebola virus (EBOV), a member of the Filoviridae that can cause severe hemorrhagic fever in humans and nonhuman primates, poses a significant threat to the public health. Currently, there are no licensed vaccines or therapeutics to prevent and treat EBOV infection. Several vaccines based on the EBOV glycoprotein (GP) are under development, including vectored, virus-like particles, and protein-based subunit vaccines. We previously demonstrated that a subunit vaccine containing the extracellular domain of the Ebola ebolavirus (EBOV) GP fused to the Fc fragment of human IgG1 (EBOVgp-Fc) protected mice against EBOV lethal challenge. Here, we show that the EBOVgp-Fc vaccine formulatedmore » with QS-21, alum, or polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC) adjuvants induced strong humoral immune responses in guinea pigs. The vaccinated animals developed anti-GP total antibody titers of approximately 10 5–10 6 and neutralizing antibody titers of approximately 10 3 as assessed by a BSL-2 neutralization assay based on vesicular stomatitis virus (VSV) pseudotypes. The poly-ICLC formulated EBOVgp-Fc vaccine protected all the guinea pigs against EBOV lethal challenge performed under BSL-4 conditions whereas the same vaccine formulated with QS-21 or alum only induced partial protection. Vaccination with a mucin-deletedEBOVgp-Fc construct formulated with QS-21 adjuvant did not have a significant effect in anti-GP antibody levels and protection against EBOV lethal challenge compared to the full-lengthGP construct. The bulk of the humoral response induced by the EBOVgp-Fc vaccine was directed against epitopes outside the EBOV mucin region. Our findings indicate that different adjuvants can eliciting varying levels of protection against lethal EBOV challenge in guinea pigs vaccinated with EBOVgp-Fc,and suggest that levels of total anti-GP antibodies elicit by protein-based GP subunit vaccines do not correlate with protection. In conclusion, our data further support the development of Fc fusions of GP as a candidate vaccine for human use.« less

Fixed-focus camera objective for small remote sensing satellites

NASA Astrophysics Data System (ADS)

Topaz, Jeremy M.; Braun, Ofer; Freiman, Dov

1993-09-01

An athermalized objective has been designed for a compact, lightweight push-broom camera which is under development at El-Op Ltd. for use in small remote-sensing satellites. The high performance objective has a fixed focus setting, but maintains focus passively over the full range of temperatures encountered in small satellites. The lens is an F/5.0, 320 mm focal length Tessar type, operating over the range 0.5 - 0.9 micrometers . It has a 16 degree(s) field of view and accommodates various state-of-the-art silicon detector arrays. The design and performance of the objective is described in this paper.

Diaphragm disease: the limitation of laparoscopy and assessment of the small bowel for strictures using a ball bearing.

PubMed

Moffat, C E; Khyan, M K; Davies, C G; Ghauri, A S K; Ranaboldo, C J

2006-09-07

Diaphragm disease is a rare cause of intestinal obstruction that will be seen with increasing frequency with the widespread use of nonsteroidal anti-inflammatory drugs (NSAIDs). We present a case study of a patient with diaphragm disease where the diagnosis was not apparent at laparoscopy, and passage of a steel ball through the small intestine was required to identify all strictures present. A high index of suspicion, recognition of the limitations of conventional diagnostic aids, and the need to assess the full length of the small bowel are all important in the surgical management of this condition.

Adipose and muscle tissue expression of two genes (NCAPG and LCORL) located in a chromosomal region associated with cattle feed intake and gain

USDA-ARS?s Scientific Manuscript database

A region on bovine chromosome 6 has been implicated in cattle birth weight, growth, and length. Non-SMC conodensin I complex subunit G (NCAPG) and ligand dependent nuclear receptor corepressor-like protein (LCORL) are positional candidate genes within this region. We previously identified genetic ...

Expression of Wheat High Molecular Weight Glutenin Subunit 1Bx Is Affected by Large Insertions and Deletions Located in the Upstream Flanking Sequences

PubMed Central

Hao, Chenyang; Tang, Saijun; Zhang, Xueyong; Li, Tian

2014-01-01

To better understand the transcriptional regulation of high molecular weight glutenin subunit (HMW-GS) expression, we isolated four Glu-1Bx promoters from six wheat cultivars exhibiting diverse protein expression levels. The activities of the diverse Glu-1Bx promoters were tested and compared with β-glucuronidase (GUS) reporter fusions. Although all the full-length Glu-1Bx promoters showed endosperm-specific activities, the strongest GUS activity was observed with the 1Bx7OE promoter in both transient expression assays and stable transgenic rice lines. A 43 bp insertion in the 1Bx7OE promoter, which is absent in the 1Bx7 promoter, led to enhanced expression. Analysis of promoter deletion constructs confirmed that a 185 bp MITE (miniature inverted-repeat transposable element) in the 1Bx14 promoter had a weak positive effect on Glu-1Bx expression, and a 54 bp deletion in the 1Bx13 promoter reduced endosperm-specific activity. To investigate the effect of the 43 bp insertion in the 1Bx7OE promoter, a functional marker was developed to screen 505 Chinese varieties and 160 European varieties, and only 1Bx7-type varieties harboring the 43 bp insertion in their promoters showed similar overexpression patterns. Hence, the 1Bx7OE promoter should be important tool in crop genetic engineering as well as in molecular assisted breeding. PMID:25133580

Transcriptional regulation of the cytosolic chaperonin theta subunit gene, Cctq, by Ets domain transcription factors Elk-1, Sap-1a, and Net in the absence of serum response factor.

PubMed

Yamazaki, Yuji; Kubota, Hiroshi; Nozaki, Masami; Nagata, Kazuhiro

2003-08-15

The chaperonin-containing t-complex polypeptide 1 (CCT) is a molecular chaperone that facilitates protein folding in eukaryotic cytosol, and the expression of CCT is highly dependent on cell growth. We show here that transcription of the gene encoding the theta subunit of mouse CCT, Cctq, is regulated by the ternary complex factors (TCFs), Elk-1, Sap-1a, and Net (Sap-2). Reporter gene assay using HeLa cells indicated that the Cctq gene promoter contains a cis-acting element of the CCGGAAGT sequence (CQE1) at -36 bp. The major CQE1-binding proteins in HeLa cell nuclear extract was recognized by anti-Elk-1 or anti-Sap-1a antibodies in electrophoretic mobility shift assay, and recombinant Elk-1, Sap-1a, or Net specifically recognized CQE1. The CQE1-dependent transcriptional activity in HeLa cells was virtually abolished by overexpression of the DNA binding domains of TCFs. Overexpression of full-length TCFs with Ras indicated that exogenous TCFs can regulate the CQE1-dependent transcription in a Ras-dependent manner. PD98059, an inhibitor of MAPK, significantly repressed the CQE1-dependent transcription. However, no serum response factor was detected by electrophoretic mobility shift assay using the CQE1 element. These results indicate that transcription of the Cctq gene is regulated by TCFs under the control of the Ras/MAPK pathway, probably independently of serum response factor.

Synthesis of prolyl 4-hydroxylase alpha subunit and type IV collagen in hemocytic granular cells of silkworm, Bombyx mori: Involvement of type IV collagen in self-defense reaction and metamorphosis.

PubMed

Adachi, Takahiro; Tomita, Masahiro; Yoshizato, Katsutoshi

2005-04-01

The present study shows that hemocytic granular cells synthesize and secrete type IV collagen (ColIV) in the silkworm Bombyx mori (B. mori) and suggests that these cells play roles in the formation of basement membrane, the encapsulation of foreign bodies, and the metamorphic remodeling of the gut. The full- and partial-length cDNA of B. mori prolyl 4-hydroxylase alpha subunit (BmP4Halpha) and B. mori ColIV (BmColIV) were cloned, respectively. In situ hybridization and immunocytochemistry on larval tissues and cells identified hemocytic granular cells as the cells that express mRNAs and proteins of both BmP4Halpha and BmColIV. Immunohistochemistry and immunocytochemistry demonstrated that BmColIV was present in the basement membrane and in the secretory granules of granular cells, respectively. Granular cells in culture secreted BmColIV without accompanying the degranulation and discharged it from the granules when the cells were degranulated. Nylon threads were inserted into the hemocoel of larvae. Granular cells concentrated around the nylon threads and encapsulated them as a self-defense reaction. BmColIV was found to be a component of the capsules. Furthermore, the present study showed that actively BmColIV-expressing granular cells accumulated around the midgut epithelium and formed BmColIV-rich thick basal lamina-like structures there in larval to pupal metamorphosis.

Bacterial effector binds host cell adenylyl cyclase to potentiate Gαs-dependent cAMP production

PubMed Central

Pulliainen, Arto T.; Pieles, Kathrin; Brand, Cameron S.; Hauert, Barbara; Böhm, Alex; Quebatte, Maxime; Wepf, Alexander; Gstaiger, Matthias; Aebersold, Ruedi; Dessauer, Carmen W.; Dehio, Christoph

2012-01-01

Subversion of host organism cAMP signaling is an efficient and widespread mechanism of microbial pathogenesis. Bartonella effector protein A (BepA) of vasculotumorigenic Bartonella henselae protects the infected human endothelial cells against apoptotic stimuli by elevation of cellular cAMP levels by an as yet unknown mechanism. Here, adenylyl cyclase (AC) and the α-subunit of the AC-stimulating G protein (Gαs) were identified as potential cellular target proteins for BepA by gel-free proteomics. Results of the proteomics screen were evaluated for physical and functional interaction by: (i) a heterologous in vivo coexpression system, where human AC activity was reconstituted under the regulation of Gαs and BepA in Escherichia coli; (ii) in vitro AC assays with membrane-anchored full-length human AC and recombinant BepA and Gαs; (iii) surface plasmon resonance experiments; and (iv) an in vivo fluorescence bimolecular complementation-analysis. The data demonstrate that BepA directly binds host cell AC to potentiate the Gαs-dependent cAMP production. As opposed to the known microbial mechanisms, such as ADP ribosylation of G protein α-subunits by cholera and pertussis toxins, the fundamentally different BepA-mediated elevation of host cell cAMP concentration appears subtle and is dependent on the stimulus of a G protein-coupled receptor-released Gαs. We propose that this mechanism contributes to the persistence of Bartonella henselae in the chronically infected vascular endothelium. PMID:22635269

Generation and characterization of anti-MUC4 monoclonal antibodies reactive with normal and cancer cells in humans.

PubMed

Moniaux, Nicolas; Varshney, Grish Chandra; Chauhan, Subhash Chand; Copin, Marie Christine; Jain, Maneesh; Wittel, Uwe A; Andrianifahanana, Mahefatiana; Aubert, Jean-Pierre; Batra, Surinder Kumar

2004-02-01

We have previously cloned the full-length cDNA (approximately 28 Kb) and established the complete genomic organization (25 exons/introns over 100 kb) of the human MUC4 mucin. This large molecule is predicted to protrude over 2 microm above the cell surface, in which MUC4alpha is an extracellular mucin-type glycoprotein subunit and MUC4beta is the transmembrane subunit. Over two thirds of the encoded protein sequence consists of 16-amino-acid tandem repeats (TR), which are flanked by unique sequences. In this study we generated and characterized monoclonal antibodies (MAbs) directed against the TR region of MUC4. Mice were immunized with a KLH-conjugated MUC4 TR peptide, STGDTTPLPVTDTSSV. Several clones were purified by three rounds of limited dilutions and stable clones presenting a sustained antibody production were selected for subsequent characterization. Antibodies were tested for their reactivity and specificity to recognize the MUC4 peptide and further screened by enzyme-linked immunosorbent assay (ELISA) and Western blotting analyses. One of the MAbs (8G7) was strongly reactive against the MUC4 peptide and with native MUC4 from human tissues or pancreatic cancer cells in Western blotting, immunohistochemistry, and confocal analysis. Anti-MUC4 MAb may represent a powerful tool for the study of MUC4 function under normal and pathological conditions and for diagnosis of solid tumors including those in the breast, pancreas, lungs, and ovaries.

Symmetric scrolled packings of multilayered carbon nanoribbons

NASA Astrophysics Data System (ADS)

Savin, A. V.; Korznikova, E. A.; Lobzenko, I. P.; Baimova, Yu. A.; Dmitriev, S. V.

2016-06-01

Scrolled packings of single-layer and multilayer graphene can be used for the creation of supercapacitors, nanopumps, nanofilters, and other nanodevices. The full atomistic simulation of graphene scrolls is restricted to consideration of relatively small systems in small time intervals. To overcome this difficulty, a two-dimensional chain model making possible an efficient calculation of static and dynamic characteristics of nanoribbon scrolls with allowance for the longitudinal and bending stiffness of nanoribbons is proposed. The model is extended to the case of scrolls of multilayer graphene. Possible equilibrium states of symmetric scrolls of multilayer carbon nanotribbons rolled up so that all nanoribbons in the scroll are equivalent are found. Dependences of the number of coils, the inner and outer radii, lowest vibrational eigenfrequencies of rolled packages on the length L of nanoribbons are obtained. It is shown that the lowest vibrational eigenfrequency of a symmetric scroll decreases with a nanoribbon length proportionally to L -1. It is energetically unfavorable for too short nanoribbons to roll up, and their ground state is a stack of plane nanoribbons. With an increasing number k of layers, the nanoribbon length L necessary for creation of symmetric scrolls increases. For a sufficiently small number of layers k and a sufficiently large nanoribbon length L, the scrolled packing has the lowest energy as compared to that of stack of plane nanoribbons and folded structures. The results can be used for development of nanomaterials and nanodevices on the basis of graphene scrolled packings.

Coexpression of the KCNA3B gene product with Kv1.5 leads to a novel A-type potassium channel.

PubMed

Leicher, T; Bähring, R; Isbrandt, D; Pongs, O

1998-12-25

Shaker-related voltage-gated potassium (Kv) channels may be heterooligomers consisting of membrane-integral alpha-subunits associated with auxiliary cytoplasmic beta-subunits. In this study we have cloned the human Kvbeta3.1 subunit and the corresponding KCNA3B gene. Identification of sequence-tagged sites in the gene mapped KCNA3B to band p13.1 of human chromosome 17. Comparison of the KCNA1B, KCNA2B, and KCNA3B gene structures showed that the three Kvbeta genes have very disparate lengths varying from >/=350 kb (KCNA1B) to approximately 7 kb (KCNA3B). Yet, the exon patterns of the three genes, which code for the seven known mammalian Kvbeta subunits, are very similar. The Kvbeta1 and Kvbeta2 splice variants are generated by alternative use of 5'-exons. Mouse Kvbeta4, a potential splice variant of Kvbeta3, is a read-through product where the open reading frame starts within the sequence intervening between Kvbeta3 exons 7 and 8. The human KCNA3B sequence does not contain a mouse Kvbeta4-like open reading frame. Human Kvbeta3 mRNA is specifically expressed in the brain, where it is predominantly detected in the cerebellum. The heterologous coexpression of human Kv1.5 and Kvbeta3.1 subunits in Chinese hamster ovary cells yielded a novel Kv channel mediating very fast inactivating (A-type) outward currents upon depolarization. Thus, the expression of Kvbeta3.1 subunits potentially extends the possibilities to express diverse A-type Kv channels in the human brain.

Ternary nylon-3 copolymers as host-defense peptide mimics: beyond hydrophobic and cationic subunits.

PubMed

Chakraborty, Saswata; Liu, Runhui; Hayouka, Zvi; Chen, Xinyu; Ehrhardt, Jeffrey; Lu, Qin; Burke, Eileen; Yang, Yiqing; Weisblum, Bernard; Wong, Gerard C L; Masters, Kristyn S; Gellman, Samuel H

2014-10-15

Host-defense peptides (HDPs) are produced by eukaryotes to defend against bacterial infection, and diverse synthetic polymers have recently been explored as mimics of these natural peptides. HDPs are rich in both hydrophobic and cationic amino acid residues, and most HDP-mimetic polymers have therefore contained binary combinations of hydrophobic and cationic subunits. However, HDP-mimetic polymers rarely duplicate the hydrophobic surface and cationic charge density found among HDPs ( Hu , K. ; et al. Macromolecules 2013 , 46 , 1908 ); the charge and hydrophobicity are generally higher among the polymers. Statistical analysis of HDP sequences ( Wang , G. ; et al. Nucleic Acids Res. 2009 , 37 , D933 ) has revealed that serine (polar but uncharged) is a very common HDP constituent and that glycine is more prevalent among HDPs than among proteins in general. These observations prompted us to prepare and evaluate ternary nylon-3 copolymers that contain a modestly polar but uncharged subunit, either serine-like or glycine-like, along with a hydrophobic subunit and a cationic subunit. Starting from binary hydrophobic-cationic copolymers that were previously shown to be highly active against bacteria but also highly hemolytic, we found that replacing a small proportion of the hydrophobic subunit with either of the polar, uncharged subunits can diminish the hemolytic activity with minimal impact on the antibacterial activity. These results indicate that the incorporation of polar, uncharged subunits may be generally useful for optimizing the biological activity profiles of antimicrobial polymers. In the context of HDP evolution, our findings suggest that there is a selective advantage to retaining polar, uncharged residues in natural antimicrobial peptides.

Structure of Rv1848 (UreA), the Mycobacterium tuberculosis urease γ subunit

PubMed Central

Habel, Jeff E.; Bursey, Evan H.; Rho, Beom-Seop; Kim, Chang-Yub; Segelke, Brent W.; Rupp, Bernhard; Park, Min S.; Terwilliger, Thomas C.; Hung, Li-Wei

2010-01-01

The crystal structure of the urease γ subunit (UreA) from Mycobacterium tuberculosis, Rv1848, has been determined at 1.8 Å resolution. The asymmetric unit contains three copies of Rv1848 arranged into a homotrimer that is similar to the UreA trimer in the structure of urease from Klebsiella aerogenes. Small-angle X-ray scattering experiments indicate that the Rv1848 protein also forms trimers in solution. The observed homotrimer and the organization of urease genes within the M. tuberculosis genome suggest that M. tuberculosis urease has the (αβγ)3 composition observed for other bacterial ureases. The γ subunit may be of primary importance for the formation of the urease quaternary structure. PMID:20606272

Size, Shape, and Lateral Correlation of Highly Uniform, Mesoscopic, Self-Assembled Domains of Fluorocarbon-Hydrocarbon Diblocks at the Air/Water Interface: A GISAXS Study.

PubMed

Veschgini, Mariam; Abuillan, Wasim; Inoue, Shigeto; Yamamoto, Akihisa; Mielke, Salomé; Liu, Xianhe; Konovalov, Oleg; Krafft, Marie Pierre; Tanaka, Motomu

2017-10-06

The shape and size of self-assembled mesoscopic surface domains of fluorocarbon-hydrocarbon (FnHm) diblocks and the lateral correlation between these domains were quantitatively determined from grazing incidence small-angle X-ray scattering (GISAXS). The full calculation of structure and form factors unravels the influence of fluorocarbon and hydrocarbon block lengths on the diameter and height of the domains, and provides the inter-domain correlation length. The diameter of the domains, as determined from the form factor analysis, exhibits a monotonic increase in response to the systematic lengthening of each block, which can be attributed to the increase in van der Waals attraction between molecules. The pair correlation function in real space calculated from the structure factor implies that the inter-domain correlation can reach a distance that is over 25 times larger than the domain's size. The full calculation of the GISAXS signals introduced here opens a potential towards the hierarchical design of mesoscale domains of self-assembled small organic molecules, covering several orders of magnitude in space. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Conformational analysis of the Streptococcus pneumoniae hyaluronate lyase and characterization of its hyaluronan-specific carbohydrate-binding module.

PubMed

Suits, Michael D L; Pluvinage, Benjamin; Law, Adrienne; Liu, Yan; Palma, Angelina S; Chai, Wengang; Feizi, Ten; Boraston, Alisdair B

2014-09-26

For a subset of pathogenic microorganisms, including Streptococcus pneumoniae, the recognition and degradation of host hyaluronan contributes to bacterial spreading through the extracellular matrix and enhancing access to host cell surfaces. The hyaluronate lyase (Hyl) presented on the surface of S. pneumoniae performs this role. Using glycan microarray screening, affinity electrophoresis, and isothermal titration calorimetry we show that the N-terminal module of Hyl is a hyaluronan-specific carbohydrate-binding module (CBM) and the founding member of CBM family 70. The 1.2 Å resolution x-ray crystal structure of CBM70 revealed it to have a β-sandwich fold, similar to other CBMs. The electrostatic properties of the binding site, which was identified by site-directed mutagenesis, are distinct from other CBMs and complementary to its acidic ligand, hyaluronan. Dynamic light scattering and solution small angle x-ray scattering revealed the full-length Hyl protein to exist as a monomer/dimer mixture in solution. Through a detailed analysis of the small angle x-ray scattering data, we report the pseudoatomic solution structures of the monomer and dimer forms of the full-length multimodular Hyl. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

First Record of Raillietina celebensis (Cestoda: Cyclophyllidea) in South America: Redescription and Phylogeny.

PubMed

de Oliveira Simões, Raquel; Simões, Susana Balmant Enrique; Luque, José Luis; Iñiguez, Alena Mayo; Júnior, Arnaldo Maldonado

2017-08-01

Raillietina celebensis is a cestode that parasitizes the small intestine of rats and humans. Here, we detail the morphology and morphometry of R. celebensis based on specimens collected from Rattus norvegicus in the municipality of São Gonçalo, state of Rio de Janeiro, Brazil, by light and confocal scanning laser microscopies and also report the results of molecular phylogenetic analyses to determine its relationships within the family Davaineidae. Analysis of the number and size of testes, number and shape of rostellar hooks, cirrus sac length, capsules and eggs per capsule, and morphology of the mature proglottid allowed concluding that the present specimens constitute a new record of R. celebensis in South America. Our genetic and phylogenetic analyses, based on the partial small subunit 18S rRNA gene, revealed R. celebensis to be in the family Davaineidae within the genus Raillietina, in agreement with the morphological taxonomy. Phylogenetic trees obtained by neighbor-joining and maximum likelihood methods demonstrated R. celebensis as a unique taxonomic unit, and also demonstrated some taxonomic inconsistences. The incorporation of Brazilian R. celebensis sequences derived from mammals in the phylogeny of davaineids is consistent with the assertion that neither Raillietina nor Fuhrmannetta can be supported as distinct genera.

Accumulation of unstable promoter-associated transcripts upon loss of the nuclear exosome subunit Rrp6p in Saccharomyces cerevisiae

PubMed Central

Davis, Carrie Anne; Ares, Manuel

2006-01-01

Mutations in RRP6 result in the accumulation of aberrant polyadenylated transcripts from small nucleolar RNA genes. We exploited this observation to search for novel noncoding RNA genes in the yeast genome. When RNA from rrp6Δ yeast is compared with wild-type on whole-genome microarrays, numerous intergenic loci exhibit an increased mutant/wild type signal ratio. Among these loci, we found one encoding a new C/D box small nucleolar RNA, as well as a surprising number that gave rise to heterogeneous Trf4p-polyadenylated RNAs with lengths of ≈250–500 nt. This class of RNAs is not easily detected in wild-type cells and appears associated with promoters. Fine mapping of several such transcripts shows they originate near known promoter elements but do not usually extend far enough to act as mRNAs, and may regulate the transcription of downstream mRNAs. Rather than being uninformative transcriptional “noise,” we hypothesize that these transcripts reflect important features of RNA polymerase activity at the promoter. This activity is normally undetectable in wild-type cells because the transcripts are somehow distinguished from true mRNAs and are degraded in an Rrp6p-dependent fashion in the nucleus. PMID:16484372

Design and performance investigation of a highly accurate apodized fiber Bragg grating-based strain sensor in single and quasi-distributed systems.

PubMed

Ali, Taha A; Shehata, Mohamed I; Mohamed, Nazmi A

2015-06-01

In this work, fiber Bragg grating (FBG) strain sensors in single and quasi-distributed systems are investigated, seeking high-accuracy measurement. Since FBG-based strain sensors of small lengths are preferred in medical applications, and that causes the full width at half-maximum (FWHM) to be larger, a new apodization profile is introduced for the first time, to the best of our knowledge, with a remarkable FWHM at small sensor lengths compared to the Gaussian and Nuttall profiles, in addition to a higher mainlobe slope at these lengths. A careful selection of apodization profiles with detailed investigation is performed-using sidelobe analysis and the FWHM, which are primary judgment factors especially in a quasi-distributed configuration. A comparison between the elite selection of apodization profiles (extracted from related literature) and the proposed new profile is carried out covering the reflectivity peak, FWHM, and sidelobe analysis. The optimization process concludes that the proposed new profile with a chosen small length (L) of 10 mm and Δn_ac of 1.4×10^-4 is the optimum choice for single stage and quasi-distributed strain-sensor networks, even better than the Gaussian profile at small sensor lengths. The proposed profile achieves the smallest FWHM of 15 GHz (suitable for UDWDM), and the highest mainlobe slope of 130 dB/nm. For the quasi-distributed scenario, a noteworthy high isolation of 6.953 dB is achieved while applying a high strain value of 1500 μstrain (με) for a five-stage strain-sensing network. Further investigation was undertaken, proving that consistency in choosing the apodization profile in the quasi-distributed network is mandatory. A test was made of the inclusion of a uniform apodized sensor among other apodized sensors with the proposed profile in an FBG strain-sensor network.

Multiple regression based imputation for individualizing template human model from a small number of measured dimensions.

PubMed

Nohara, Ryuki; Endo, Yui; Murai, Akihiko; Takemura, Hiroshi; Kouchi, Makiko; Tada, Mitsunori

2016-08-01

Individual human models are usually created by direct 3D scanning or deforming a template model according to the measured dimensions. In this paper, we propose a method to estimate all the necessary dimensions (full set) for the human model individualization from a small number of measured dimensions (subset) and human dimension database. For this purpose, we solved multiple regression equation from the dimension database given full set dimensions as the objective variable and subset dimensions as the explanatory variables. Thus, the full set dimensions are obtained by simply multiplying the subset dimensions to the coefficient matrix of the regression equation. We verified the accuracy of our method by imputing hand, foot, and whole body dimensions from their dimension database. The leave-one-out cross validation is employed in this evaluation. The mean absolute errors (MAE) between the measured and the estimated dimensions computed from 4 dimensions (hand length, breadth, middle finger breadth at proximal, and middle finger depth at proximal) in the hand, 2 dimensions (foot length, breadth, and lateral malleolus height) in the foot, and 1 dimension (height) and weight in the whole body are computed. The average MAE of non-measured dimensions were 4.58% in the hand, 4.42% in the foot, and 3.54% in the whole body, while that of measured dimensions were 0.00%.

Miscoding-induced stalling of substrate translocation on the bacterial ribosome.

PubMed

Alejo, Jose L; Blanchard, Scott C

2017-10-10

Directional transit of the ribosome along the messenger RNA (mRNA) template is a key determinant of the rate and processivity of protein synthesis. Imaging of the multistep translocation mechanism using single-molecule FRET has led to the hypothesis that substrate movements relative to the ribosome resolve through relatively long-lived late intermediates wherein peptidyl-tRNA enters the P site of the small ribosomal subunit via reversible, swivel-like motions of the small subunit head domain within the elongation factor G (GDP)-bound ribosome complex. Consistent with translocation being rate-limited by recognition and productive engagement of peptidyl-tRNA within the P site, we now show that base-pairing mismatches between the peptidyl-tRNA anticodon and the mRNA codon dramatically delay this rate-limiting, intramolecular process. This unexpected relationship between aminoacyl-tRNA decoding and translocation suggests that miscoding antibiotics may impact protein synthesis by impairing the recognition of peptidyl-tRNA in the small subunit P site during EF-G-catalyzed translocation. Strikingly, we show that elongation factor P (EF-P), traditionally known to alleviate ribosome stalling at polyproline motifs, can efficiently rescue translocation defects arising from miscoding. These findings help reveal the nature and origin of the rate-limiting steps in substrate translocation on the bacterial ribosome and indicate that EF-P can aid in resuming translation elongation stalled by miscoding errors.

Miscoding-induced stalling of substrate translocation on the bacterial ribosome

PubMed Central

Alejo, Jose L.; Blanchard, Scott C.

2017-01-01

Directional transit of the ribosome along the messenger RNA (mRNA) template is a key determinant of the rate and processivity of protein synthesis. Imaging of the multistep translocation mechanism using single-molecule FRET has led to the hypothesis that substrate movements relative to the ribosome resolve through relatively long-lived late intermediates wherein peptidyl-tRNA enters the P site of the small ribosomal subunit via reversible, swivel-like motions of the small subunit head domain within the elongation factor G (GDP)-bound ribosome complex. Consistent with translocation being rate-limited by recognition and productive engagement of peptidyl-tRNA within the P site, we now show that base-pairing mismatches between the peptidyl-tRNA anticodon and the mRNA codon dramatically delay this rate-limiting, intramolecular process. This unexpected relationship between aminoacyl-tRNA decoding and translocation suggests that miscoding antibiotics may impact protein synthesis by impairing the recognition of peptidyl-tRNA in the small subunit P site during EF-G–catalyzed translocation. Strikingly, we show that elongation factor P (EF-P), traditionally known to alleviate ribosome stalling at polyproline motifs, can efficiently rescue translocation defects arising from miscoding. These findings help reveal the nature and origin of the rate-limiting steps in substrate translocation on the bacterial ribosome and indicate that EF-P can aid in resuming translation elongation stalled by miscoding errors. PMID:28973849

Superconducting magnets for traveling-wave maser application. Technical documentary report, Oct 1960--Mar 1962

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

Okwit, S.; Siegel, K.; Smith, J.G.

1962-09-01

Results of an investigation to determine the feasibility of incorporating superconducting magnet techniques in the design of traveling-wave maser systems are reported. Several different types of magnet configurations were investigated: isomagnets, Helmholtz coils, modified Helmholtz coils, air-core solenoids, and magnetic end-loaded air-core solenoids. The magnetic end-loaded air-core solenoid was found to be the best configuration for the S-band maser under consideration. This technique yielded relatively large regions of field homogeneity with relatively small aspect ratios (length of solenoid/diameter of solenoid). Several small-scale models of full-length superconducting magnets and foreshortened end-loaded superconducting magnets were constructed using un-annealed niobium wire. Measurements havemore » shown that these magnets were adequate for traveling-wave maser applications that require magnetic fields up to 2,200 G and marginal for magnetic fields up to 2,500 G.« less

The 28S–18S rDNA intergenic spacer from Crithidia fasciculata: repeated sequences, length heterogeneity, putative processing sites and potential interactions between U3 small nucleolar RNA and the ribosomal RNA precursor

PubMed Central

Schnare, Murray N.; Collings, James C.; Spencer, David F.; Gray, Michael W.

2000-01-01

In Crithidia fasciculata, the ribosomal RNA (rRNA) gene repeats range in size from ∼11 to 12 kb. This length heterogeneity is localized to a region of the intergenic spacer (IGS) that contains tandemly repeated copies of a 19mer sequence. The IGS also contains four copies of an ∼55 nt repeat that has an internal inverted repeat and is also present in the IGS of Leishmania species. We have mapped the C.fasciculata transcription initiation site as well as two other reverse transcriptase stop sites that may be analogous to the A0 and A′ pre-rRNA processing sites within the 5′ external transcribed spacer (ETS) of other eukaryotes. Features that could influence processing at these sites include two stretches of conserved primary sequence and three secondary structure elements present in the 5′ ETS. We also characterized the C.fasciculata U3 snoRNA, which has the potential for base-pairing with pre-rRNA sequences. Finally, we demonstrate that biosynthesis of large subunit rRNA in both C.fasciculata and Trypanosoma brucei involves 3′-terminal addition of three A residues that are not present in the corresponding DNA sequences. PMID:10982863

Metal-coupled folding as the driving force for the extreme stability of Rad50 zinc hook dimer assembly

NASA Astrophysics Data System (ADS)

Kochańczyk, Tomasz; Nowakowski, Michał; Wojewska, Dominika; Kocyła, Anna; Ejchart, Andrzej; Koźmiński, Wiktor; Krężel, Artur

2016-11-01

The binding of metal ions at the interface of protein complexes presents a unique and poorly understood mechanism of molecular assembly. A remarkable example is the Rad50 zinc hook domain, which is highly conserved and facilitates the Zn2+-mediated homodimerization of Rad50 proteins. Here, we present a detailed analysis of the structural and thermodynamic effects governing the formation and stability (logK12 = 20.74) of this evolutionarily conserved protein assembly. We have dissected the determinants of the stability contributed by the small β-hairpin of the domain surrounding the zinc binding motif and the coiled-coiled regions using peptides of various lengths from 4 to 45 amino acid residues, alanine substitutions and peptide bond-to-ester perturbations. In the studied series of peptides, an >650 000-fold increase of the formation constant of the dimeric complex arises from favorable enthalpy because of the increased acidity of the cysteine thiols in metal-free form and the structural properties of the dimer. The dependence of the enthalpy on the domain fragment length is partially compensated by the entropic penalty of domain folding, indicating enthalpy-entropy compensation. This study facilitates understanding of the metal-mediated protein-protein interactions in which the metal ion is critical for the tight association of protein subunits.

Metal-coupled folding as the driving force for the extreme stability of Rad50 zinc hook dimer assembly

PubMed Central

Kochańczyk, Tomasz; Nowakowski, Michał; Wojewska, Dominika; Kocyła, Anna; Ejchart, Andrzej; Koźmiński, Wiktor; Krężel, Artur

2016-01-01

The binding of metal ions at the interface of protein complexes presents a unique and poorly understood mechanism of molecular assembly. A remarkable example is the Rad50 zinc hook domain, which is highly conserved and facilitates the Zn2+-mediated homodimerization of Rad50 proteins. Here, we present a detailed analysis of the structural and thermodynamic effects governing the formation and stability (logK12 = 20.74) of this evolutionarily conserved protein assembly. We have dissected the determinants of the stability contributed by the small β-hairpin of the domain surrounding the zinc binding motif and the coiled-coiled regions using peptides of various lengths from 4 to 45 amino acid residues, alanine substitutions and peptide bond-to-ester perturbations. In the studied series of peptides, an >650 000-fold increase of the formation constant of the dimeric complex arises from favorable enthalpy because of the increased acidity of the cysteine thiols in metal-free form and the structural properties of the dimer. The dependence of the enthalpy on the domain fragment length is partially compensated by the entropic penalty of domain folding, indicating enthalpy-entropy compensation. This study facilitates understanding of the metal-mediated protein-protein interactions in which the metal ion is critical for the tight association of protein subunits. PMID:27808280

The tapeworm Ligula intestinalis (Cestoda: Pseudophyllidea) inhibits LH expression and puberty in its teleost host, Rutilus rutilus.

PubMed

Carter, V; Pierce, R; Dufour, S; Arme, C; Hoole, D

2005-12-01

The tapeworm Ligula intestinalis occurs in the body cavity of its cyprinid second intermediate host, in this study the roach Rutilus rutilus, and inhibits host gonadal development. The mechanism by which infected fish are prevented from reproducing is unknown. Comparison of parameters, such as body length and weight, and condition factor and age, between infected and uninfected individuals, indicated only minor effects of parasitism on growth and condition. In contrast, seasonal gonadal development, as observed in uninfected fish, did not occur in infected fish, and gonads remained small and blocked at the primary oocyte stage in female roach. As immature ovaries and testes are still present, the parasite is presumed to act upon the brain-pituitary-gonadal axis of the fish to inhibit further development of reproductive organs. We investigated the Ligula/fish interaction at the level of the pituitary gland by determination of gonadotrophin (LH) content using a heterologous RIA for carp (Cyprinus carpio) LHbeta subunit. The results indicated that the pituitary glands of infected roach contained approximately 50% less LH than non-infected fish. After the cloning and sequencing of roach LHbeta subunit, we measured roach LHbeta mRNA levels by real-time RT-PCR. A corresponding 50% reduction in LHbeta mRNA pituitary levels was determined. These results reflect a significant and measurable effect of parasitism on the pituitary gland, and lend support to the hypothesis that excretory/secretory products released from the parasite interact with the brain-pituitary-gonadal axis of the fish host and thus inhibit gonadal development.

P. berghei Telomerase Subunit TERT is Essential for Parasite Survival

PubMed Central

Religa, Agnieszka A.; Ramesar, Jai; Janse, Chris J.; Scherf, Artur; Waters, Andrew P.

2014-01-01

Telomeres define the ends of chromosomes protecting eukaryotic cells from chromosome instability and eventual cell death. The complex regulation of telomeres involves various proteins including telomerase, which is a specialized ribonucleoprotein responsible for telomere maintenance. Telomeres of chromosomes of malaria parasites are kept at a constant length during blood stage proliferation. The 7-bp telomere repeat sequence is universal across different Plasmodium species (GGGTTT/CA), though the average telomere length varies. The catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), is present in all sequenced Plasmodium species and is approximately three times larger than other eukaryotic TERTs. The Plasmodium RNA component of TERT has recently been identified in silico. A strategy to delete the gene encoding TERT via double cross-over (DXO) homologous recombination was undertaken to study the telomerase function in P. berghei. Expression of both TERT and the RNA component (TR) in P. berghei blood stages was analysed by Western blotting and Northern analysis. Average telomere length was measured in several Plasmodium species using Telomere Restriction Fragment (TRF) analysis. TERT and TR were detected in blood stages and an average telomere length of ∼950 bp established. Deletion of the tert gene was performed using standard transfection methodologies and we show the presence of tert − mutants in the transfected parasite populations. Cloning of tert- mutants has been attempted multiple times without success. Thorough analysis of the transfected parasite populations and the parasite obtained from extensive parasite cloning from these populations provide evidence for a so called delayed death phenotype as observed in different organisms lacking TERT. The findings indicate that TERT is essential for P. berghei cell survival. The study extends our current knowledge on telomere biology in malaria parasites and validates further investigations to identify telomerase inhibitors to induce parasite cell death. PMID:25275500

Characterization of the human SDHD gene encoding the small subunit of cytochrome b (cybS) in mitochondrial succinate-ubiquinone oxidoreductase.

PubMed

Hirawake, H; Taniwaki, M; Tamura, A; Amino, H; Tomitsuka, E; Kita, K

1999-08-04

We have mapped large (cybL) and small (cybS) subunits of cytochrome b in the succinate-ubiquinone oxidoreductase (complex II) of human mitochondria to chromosome 1q21 and 11q23, respectively (H. Hirawake et al., Cytogenet. Cell Genet. 79 (1997) 132-138). In the present study, the human SDHD gene encoding cybS was cloned and characterized. The gene comprises four exons and three introns extending over 19 kb. Sequence analysis of the 5' promoter region showed several motifs for the binding of transcription factors including nuclear respiratory factors NRF-1 and NRF-2 at positions -137 and -104, respectively. In addition to this gene, six pseudogenes of cybS were isolated and mapped on the chromosome.

Redescriptions of three trachelocercid ciliates (Protista, Ciliophora, Karyorelictea), with notes on their phylogeny based on small subunit rRNA gene sequences.

PubMed

Yan, Ying; Xu, Yuan; Yi, Zhenzhen; Warren, Alan

2013-09-01

Three trachelocercid ciliates, Kovalevaia sulcata (Kovaleva, 1966) Foissner, 1997, Trachelocerca sagitta (Müller, 1786) Ehrenberg, 1840 and Trachelocerca ditis (Wright, 1982) Foissner, 1996, isolated from two coastal habitats at Qingdao, China, were investigated using live observation and silver impregnation methods. Data on their infraciliature and morphology are supplied. The small subunit rRNA (SSU rRNA) genes of K. sulcata and Trachelocerca sagitta were sequenced for the first time. Phylogenetic analyses based on SSU rRNA gene sequence data indicate that both organisms, and the previously sequenced Trachelocerca ditis, are located within the trachelocercid assemblage and that K. sulcata is sister to an unidentified taxon forming a clade that is basal to the core trachelocercids.

Adipose and muscle tissue gene expression of two genes (NCAPG and LCORL) located in a chromosomal region associated with cattle feed intake and gain

USDA-ARS?s Scientific Manuscript database

A region on bovine chromosome 6 has been implicated in cattle birth weight, growth, and length. Non-SMC conodensin I complex subunit G (NCAPG) and ligand dependent nuclear receptor corepressor-like protein (LCORL) are positional candidate genes within this region. Previously identified genetic mark...

The electrochemical behavior of a FAD dependent glucose dehydrogenase with direct electron transfer subunit by immobilization on self-assembled monolayers.

PubMed

Lee, Inyoung; Loew, Noya; Tsugawa, Wakako; Lin, Chi-En; Probst, David; La Belle, Jeffrey T; Sode, Koji

2018-06-01

Continuous glucose monitoring (CGM) is a vital technology for diabetes patients by providing tight glycemic control. Currently, many commercially available CGM sensors use glucose oxidase (GOD) as sensor element, but this enzyme is not able to transfer electrons directly to the electrode without oxygen or an electronic mediator. We previously reported a mutated FAD dependent glucose dehydrogenase complex (FADGDH) capable of direct electron transfer (DET) via an electron transfer subunit without involving oxygen or a mediator. In this study, we investigated the electrochemical response of DET by controlling the immobilization of DET-FADGDH using 3 types of self-assembled monolayers (SAMs) with varying lengths. With the employment of DET-FADGDH and SAM, high current densities were achieved without being affected by interfering substances such as acetaminophen and ascorbic acid. Additionally, the current generated from DET-FADGDH electrodes decreased with increasing length of SAM, suggesting that the DET ability can be affected by the distance between the enzyme and the electrode. These results indicate the feasibility of controlling the immobilization state of the enzymes on the electrode surface. Copyright © 2017. Published by Elsevier B.V.

An ancestral host defence peptide within human β-defensin 3 recapitulates the antibacterial and antiviral activity of the full-length molecule

PubMed Central

Nigro, Ersilia; Colavita, Irene; Sarnataro, Daniela; Scudiero, Olga; Zambrano, Gerardo; Granata, Vincenzo; Daniele, Aurora; Carotenuto, Alfonso; Galdiero, Stefania; Folliero, Veronica; Galdiero, Massimiliano; Urbanowicz, Richard A.; Ball, Jonathan K.; Salvatore, Francesco; Pessi, Antonello

2015-01-01

Host defence peptides (HDPs) are critical components of innate immunity. Despite their diversity, they share common features including a structural signature, designated “γ-core motif”. We reasoned that for each HDPs evolved from an ancestral γ-core, the latter should be the evolutionary starting point of the molecule, i.e. it should represent a structural scaffold for the modular construction of the full-length molecule, and possess biological properties. We explored the γ-core of human β-defensin 3 (HBD3) and found that it: (a) is the folding nucleus of HBD3; (b) folds rapidly and is stable in human serum; (c) displays antibacterial activity; (d) binds to CD98, which mediates HBD3 internalization in eukaryotic cells; (e) exerts antiviral activity against human immunodeficiency virus and herpes simplex virus; and (f) is not toxic to human cells. These results demonstrate that the γ-core within HBD3 is the ancestral core of the full-length molecule and is a viable HDP per se, since it is endowed with the most important biological features of HBD3. Notably, the small, stable scaffold of the HBD3 γ-core can be exploited to design disease-specific antimicrobial agents. PMID:26688341

Dual functions of a small regulatory subunit in the mitochondrial calcium uniporter complex.

PubMed

Tsai, Ming-Feng; Phillips, Charles B; Ranaghan, Matthew; Tsai, Chen-Wei; Wu, Yujiao; Willliams, Carole; Miller, Christopher

2016-04-21

Mitochondrial Ca(2+) uptake, a process crucial for bioenergetics and Ca(2+) signaling, is catalyzed by the mitochondrial calcium uniporter. The uniporter is a multi-subunit Ca(2+)-activated Ca(2+) channel, with the Ca(2+) pore formed by the MCU protein and Ca(2+)-dependent activation mediated by MICU subunits. Recently, a mitochondrial inner membrane protein EMRE was identified as a uniporter subunit absolutely required for Ca(2+) permeation. However, the molecular mechanism and regulatory purpose of EMRE remain largely unexplored. Here, we determine the transmembrane orientation of EMRE, and show that its known MCU-activating function is mediated by the interaction of transmembrane helices from both proteins. We also reveal a second function of EMRE: to maintain tight MICU regulation of the MCU pore, a role that requires EMRE to bind MICU1 using its conserved C-terminal polyaspartate tail. This dual functionality of EMRE ensures that all transport-competent uniporters are tightly regulated, responding appropriately to a dynamic intracellular Ca(2+) landscape.

Elastic rotation of Escherichia coli F(O)F(1) having ε subunit fused with cytochrome b(562) or flavodoxin reductase.

PubMed

Oka, Hideyuki; Hosokawa, Hiroyuki; Nakanishi-Matsui, Mayumi; Dunn, Stanley D; Futai, Masamitsu; Iwamoto-Kihara, Atsuko

2014-04-18

Intra-molecular rotation of FOF1 ATP synthase enables cooperative synthesis and hydrolysis of ATP. In this study, using a small gold bead probe, we observed fast rotation close to the real rate that would be exhibited without probes. Using this experimental system, we tested the rotation of FOF1 with the ε subunit connected to a globular protein [cytochrome b562 (ε-Cyt) or flavodoxin reductase (ε-FlavR)], which is apparently larger than the space between the central and the peripheral stalks. The enzymes containing ε-Cyt and ε-FlavR showed continual rotations with average rates of 185 and 148 rps, respectively, similar to the wild type (172 rps). However, the enzymes with ε-Cyt or ε-FlavR showed a reduced proton transport. These results indicate that the intra-molecular rotation is elastic but proton transport requires more strict subunit/subunit interaction. Copyright © 2014 Elsevier Inc. All rights reserved.

Crystal Structure and Proteomics Analysis of Empty Virus-like Particles of Cowpea Mosaic Virus

PubMed Central

Huynh, Nhung T.; Hesketh, Emma L.; Saxena, Pooja; Meshcheriakova, Yulia; Ku, You-Chan; Hoang, Linh T.; Johnson, John E.; Ranson, Neil A.; Lomonossoff, George P.; Reddy, Vijay S.

2016-01-01

SUMMARY Empty virus-like particles (eVLPs) of Cowpea mosaic virus (CPMV) are currently being utilized as reagents in various biomedical and nanotechnology applications. Here, we report the crystal structure of CPMV eVLPs determined using X-ray crystallography at 2.3 Å resolution and compare it with previously reported cryo-electron microscopy (cryo-EM) of eVLPs and virion crystal structures. Although the X-ray and cryo-EM structures of eVLPs are mostly similar, there exist significant differences at the C terminus of the small (S) subunit. The intact C terminus of the S subunit plays a critical role in enabling the efficient assembly of CPMV virions and eVLPs, but undergoes proteolysis after particle formation. In addition, we report the results of mass spectrometry-based proteomics analysis of coat protein subunits from CPMV eVLPs and virions that identify the C termini of S subunits undergo proteolytic cleavages at multiple sites instead of a single cleavage site as previously observed. PMID:27021160

Targeting Cullin–RING E3 ubiquitin ligases for drug discovery: structure, assembly and small-molecule modulation

PubMed Central

Bulatov, Emil; Ciulli, Alessio

2015-01-01

In the last decade, the ubiquitin–proteasome system has emerged as a valid target for the development of novel therapeutics. E3 ubiquitin ligases are particularly attractive targets because they confer substrate specificity on the ubiquitin system. CRLs [Cullin–RING (really interesting new gene) E3 ubiquitin ligases] draw particular attention, being the largest family of E3s. The CRLs assemble into functional multisubunit complexes using a repertoire of substrate receptors, adaptors, Cullin scaffolds and RING-box proteins. Drug discovery targeting CRLs is growing in importance due to mounting evidence pointing to significant roles of these enzymes in diverse biological processes and human diseases, including cancer, where CRLs and their substrates often function as tumour suppressors or oncogenes. In the present review, we provide an account of the assembly and structure of CRL complexes, and outline the current state of the field in terms of available knowledge of small-molecule inhibitors and modulators of CRL activity. A comprehensive overview of the reported crystal structures of CRL subunits, components and full-size complexes, alone or with bound small molecules and substrate peptides, is included. This information is providing increasing opportunities to aid the rational structure-based design of chemical probes and potential small-molecule therapeutics targeting CRLs. PMID:25886174

DNA-launched live-attenuated vaccines for biodefense applications

PubMed Central

Pushko, Peter; Lukashevich, Igor S.; Weaver, Scott C.; Tretyakova, Irina

2016-01-01

Summary A novel vaccine platform uses DNA immunization to launch live-attenuated virus vaccines in vivo. This technology has been applied for vaccine development against positive-strand RNA viruses with global public health impact including alphaviruses and flaviviruses. The DNA-launched vaccine represents the recombinant plasmid that encodes the full-length genomic RNA of live-attenuated virus downstream from a eukaryotic promoter. When administered in vivo, the genomic RNA of live-attenuated virus is transcribed. The RNA initiates limited replication of a genetically defined, live-attenuated vaccine virus in the tissues of the vaccine recipient, thereby inducing a protective immune response. This platform combines the strengths of reverse genetics, DNA immunization and the advantages of live-attenuated vaccines, resulting in a reduced chance of genetic reversions, increased safety, and improved immunization. With this vaccine technology, the field of DNA vaccines is expanded from those that express subunit antigens to include a novel type of DNA vaccines that launch live-attenuated viruses. PMID:27055100

MDA5 cooperatively forms dimers and ATP-sensitive filaments upon binding double-stranded RNA

PubMed Central

Berke, Ian C; Modis, Yorgo

2012-01-01

Melanoma differentiation-associated gene-5 (MDA5) detects viral double-stranded RNA in the cytoplasm. RNA binding induces MDA5 to activate the signalling adaptor MAVS through interactions between the caspase recruitment domains (CARDs) of the two proteins. The molecular mechanism of MDA5 signalling is not well understood. Here, we show that MDA5 cooperatively binds short RNA ligands as a dimer with a 16–18-basepair footprint. A crystal structure of the MDA5 helicase-insert domain demonstrates an evolutionary relationship with the archaeal Hef helicases. In X-ray solution structures, the CARDs in unliganded MDA5 are flexible, and RNA binds on one side of an asymmetric MDA5 dimer, bridging the two subunits. On longer RNA, full-length and CARD-deleted MDA5 constructs assemble into ATP-sensitive filaments. We propose a signalling model in which the CARDs on MDA5–RNA filaments nucleate the assembly of MAVS filaments with the same polymeric geometry. PMID:22314235

Sequence analysis of the phs operon in Salmonella typhimurium and the contribution of thiosulfate reduction to anaerobic energy metabolism.

PubMed Central

Heinzinger, N K; Fujimoto, S Y; Clark, M A; Moreno, M S; Barrett, E L

1995-01-01

The phs chromosomal locus of Salmonella typhimurium is essential for the dissimilatory anaerobic reduction of thiosulfate to hydrogen sulfide. Sequence analysis of the phs region revealed a functional operon with three open reading frames, designated phsA, phsB, and phsC, which encode peptides of 82.7, 21.3, and 28.5 kDa, respectively. The predicted products of phsA and phsB exhibited significant homology with the catalytic and electron transfer subunits of several other anaerobic molybdoprotein oxidoreductases, including Escherichia coli dimethyl sulfoxide reductase, nitrate reductase, and formate dehydrogenase. Simultaneous comparison of PhsA to seven homologous molybdoproteins revealed numerous similarities among all eight throughout the entire frame, hence, significant amino acid conservation among molybdoprotein oxidoreductases. Comparison of PhsB to six other homologous sequences revealed four highly conserved iron-sulfur clusters. The predicted phsC product was highly hydrophobic and similar in size to the hydrophobic subunits of the molybdoprotein oxidoreductases containing subunits homologous to phsA and phsB. Thus, phsABC appears to encode thiosulfate reductase. Single-copy phs-lac translational fusions required both anaerobiosis and thiosulfate for full expression, whereas multicopy phs-lac translational fusions responded to either thiosulfate or anaerobiosis, suggesting that oxygen and thiosulfate control of phs involves negative regulation. A possible role for thiosulfate reduction in anaerobic respiration was examined. Thiosulfate did not significantly augment the final densities of anaerobic cultures grown on any of the 18 carbon sources tested. on the other hand, washed stationary-phase cells depleted of ATP were shown to synthesize small amounts of ATP on the addition of the formate and thiosulfate, suggesting that the thiosulfate reduction plays a unique role in anaerobic energy conservation by S typhimurium. PMID:7751291

Dynactin Function in Mitotic Spindle Positioning

PubMed Central

Moore, Jeffrey K.; Li, Jun; Cooper, John A.

2008-01-01

Dynactin is a multisubunit protein complex necessary for dynein function. Here, we investigated the function of dynactin in budding yeast. Loss of dynactin impaired movement and positioning of the mitotic spindle, similar to loss of dynein. Dynactin subunits required for function included p150Glued, dynamitin, actin-related protein (Arp) 1 and p24. Arp10 and capping protein were dispensable, even in combination. All dynactin subunits tested localized to dynamic plus ends of cytoplasmic microtubules, to stationary foci on the cell cortex and to spindle pole bodies. The number of molecules of dynactin in those locations was small, less than five. In the absence of dynactin, dynein accumulated at plus ends and did not appear at the cell cortex, consistent with a role for dynactin in offloading dynein from the plus end to the cortex. Dynein at the plus end was necessary for dynactin plus-end targeting. p150Glued was the only dynactin subunit sufficient for plus-end targeting. Interactions among the subunits support a molecular model that resembles the current model for brain dynactin in many respects; however, three subunits at the pointed end of brain dynactin appear to be absent from yeast. PMID:18221362

Binding of adenine to Stx2, the protein toxin from Escherichia coli O157:H7

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

Fraser, Marie E., E-mail: frasm@ucalgary.ca; Cherney, Maia M.; Marcato, Paola

2006-07-01

Crystals of Stx2 were grown in the presence of adenosine and adenine. In both cases, the resulting electron density showed only adenine bound at the active site of the A subunit, proving that the holotoxin is an active N-glycosidase. Stx2 is a protein toxin whose catalytic subunit acts as an N-glycosidase to depurinate a specific adenine base from 28S rRNA. In the holotoxin, the catalytic portion, A1, is linked to the rest of the A subunit, A2, and A2 interacts with the pentameric ring formed by the five B subunits. In order to test whether the holotoxin is active asmore » an N-glycosidase, Stx2 was crystallized in the presence of adenosine and adenine. The crystals diffracted to ∼1.8 Å and showed clear electron density for adenine in the active site. Adenosine had been cleaved, proving that Stx2 is an active N-glycosidase. While the holotoxin is active against small substrates, it would be expected that the B subunits would interfere with the binding of the 28S rRNA.« less

Direct interaction of the bacteriophage SPP1 packaging ATPase with the portal protein.

PubMed

Oliveira, Leonor; Cuervo, Ana; Tavares, Paulo

2010-03-05

DNA packaging in tailed bacteriophages and other viruses requires assembly of a complex molecular machine at a specific vertex of the procapsid. This machine is composed of the portal protein that provides a tunnel for DNA entry, an ATPase that fuels DNA translocation (large terminase subunit), and most frequently, a small terminase subunit. Here we characterized the interaction between the terminase ATPase subunit of bacteriophage SPP1 (gp2) and the procapsid portal vertex. We found, by affinity pulldown assays with purified proteins, that gp2 interacts with the portal protein, gp6, independently of the terminase small subunit gp1, DNA, or ATP. The gp2-procapsid interaction via the portal protein depends on gp2 concentration and requires the presence of divalent cations. Competition experiments showed that isolated gp6 can only inhibit gp2-procapsid interactions and DNA packaging at gp6:procapsid molar ratios above 10-fold. Assays with gp6 carrying mutations in distinct regions of its structure that affect the portal-induced stimulation of ATPase and DNA packaging revealed that none of these mutations impedes gp2-gp6 binding. Our results demonstrate that the SPP1 packaging ATPase binds directly to the portal and that the interaction is stronger with the portal embedded in procapsids. Identification of mutations in gp6 that allow for assembly of the ATPase-portal complex but impair DNA packaging support an intricate cross-talk between the two proteins for activity of the DNA translocation motor.

Translation Initiation Factor eIF4B Interacts with a Picornavirus Internal Ribosome Entry Site in both 48S and 80S Initiation Complexes Independently of Initiator AUG Location

PubMed Central

Ochs, Kerstin; Rust, René C.; Niepmann, Michael

1999-01-01

Most eukaryotic initiation factors (eIFs) are required for internal translation initiation at the internal ribosome entry site (IRES) of picornaviruses. eIF4B is incorporated into ribosomal 48S initiation complexes with the IRES RNA of foot-and-mouth disease virus (FMDV). In contrast to the weak interaction of eIF4B with capped cellular mRNAs and its release upon entry of the ribosomal 60S subunit, eIF4B remains tightly associated with the FMDV IRES during formation of complete 80S ribosomes. Binding of eIF4B to the IRES is energy dependent, and binding of the small ribosomal subunit to the IRES requires the previous energy-dependent association of initiation factors with the IRES. The interaction of eIF4B with the IRES in 48S and 80S complexes is independent of the location of the initiator AUG and thus independent of the mechanism by which the small ribosomal subunit is placed at the actual start codon, either by direct internal ribosomal entry or by scanning. eIF4B does not greatly rearrange its binding to the IRES upon entry of the ribosomal subunits, and the interaction of eIF4B with the IRES is independent of the polypyrimidine tract-binding protein, which enhances FMDV translation. PMID:10438840

Differential senescence capacities in meibomian gland carcinoma and basal cell carcinoma.

PubMed

Zhang, Leilei; Huang, Xiaolin; Zhu, Xiaowei; Ge, Shengfang; Gilson, Eric; Jia, Renbing; Ye, Jing; Fan, Xianqun

2016-03-15

Meibomian gland carcinoma (MGC) and basal cell carcinoma (BCC) are common eyelid carcinomas that exhibit highly dissimilar degrees of proliferation and prognoses. We address here the question of the differential mechanisms between these two eyelid cancers that explain their different outcome. A total of 102 confirmed MGC and 175 diagnosed BCC cases were analyzed. Twenty confirmed MGC and twenty diagnosed BCC cases were collected to determine the telomere length, the presence of senescent cells, and the expression levels of the telomere capping shelterin complex, P53, and the E3 ubiquitin ligase Siah1. Decreased protein levels of the shelterin subunits, shortened telomere length, over-expressed Ki-67, and Bcl2 as well as mutations in P53 were detected both in MGC and BCC. It suggests that the decreased protein levels of the shelterin complex and the shortened telomere length contribute to the tumorigenesis of MGC and BCC. However, several parameters distinguish MGC from BCC samples: (i) the mRNA level of the shelterin subunits decreased in MGC but it increased in BCC; (ii) P53 was more highly mutated in MGC; (iii) Siah1 mRNA was over-expressed in BCC; (iv) BCC samples contain a higher level of senescent cells; (v) Ki-67 and Bcl2 expression were lower in BCC. These results support a model where a preserved P53 checkpoint in BCC leads to cellular senescence and reduced tumor proliferation as compared to MGC. © 2015 UICC.

Polyglutamine length-dependent toxicity from α1ACT in Drosophila models of spinocerebellar ataxia type 6

PubMed Central

Tsou, Wei-Ling; Qiblawi, Sultan H.; Hosking, Ryan R.; Gomez, Christopher M.

2016-01-01

ABSTRACT Spinocerebellar ataxia type 6 (SCA6) is a neurodegenerative disease that results from abnormal expansion of a polyglutamine (polyQ) repeat. SCA6 is caused by CAG triplet repeat expansion in the gene CACNA1A, resulting in a polyQ tract of 19-33 in patients. CACNA1A, a bicistronic gene, encodes the α1A calcium channel subunit and the transcription factor, α1ACT. PolyQ expansion in α1ACT causes degeneration in mice. We recently described the first Drosophila models of SCA6 that express α1ACT with a normal (11Q) or hyper-expanded (70Q) polyQ. Here, we report additional α1ACT transgenic flies, which express full-length α1ACT with a 33Q repeat. We show that α1ACT33Q is toxic in Drosophila, but less so than the 70Q version. When expressed everywhere, α1ACT33Q-expressing adults die earlier than flies expressing the normal allele. α1ACT33Q causes retinal degeneration and leads to aggregated species in an age-dependent manner, but at a slower pace than the 70Q counterpart. According to western blots, α1ACT33Q localizes less readily in the nucleus than α1ACT70Q, providing clues into the importance of polyQ tract length on α1ACT localization and its site of toxicity. We expect that these new lines will be highly valuable for future work on SCA6. PMID:27979829

Experimental Study of Heat Transfer Enhancements from Array of Alternate Rectangular Dwarf Fins at Different Inclinations

NASA Astrophysics Data System (ADS)

Awasarmol, Umesh Vandeorao; Pise, Ashok T.

2018-02-01

The main objective of this experimental work is to investigate and compare heat transfer enhancement of alternate dwarf fin array at different angles of inclination. In this study, the steady state heat transfer from the full length fin arrays and alternate dwarf fin arrays are measured in natural convection and radiation environment. Largest increase in the Nusselt number was achieved with alternate dwarf fin at angle of orientation 90°, which shows about 28% enhanced heat transfer coefficient as opposed to the full-length fin array with 25% saving in material. In case of non-black FAB, contribution of radiation heat transfer is found to be very small nearly within 1% of the heater input. After coating lamp black contribution of radiation heat transfer is found to increase to about 3-4% of the heater input in the range of temperatures considered in this study.

Description of two new gill myxozoans from smallmouth (Micropterus dolomieu) and largemouth bass (Micropterus salmoides)

USGS Publications Warehouse

Walsh, Heather L.; Iwanowicz, Luke R.; Glenney, Gavin W.; Iwanowicz, Deborah D.; Blazer, Vicki

2012-01-01

Two previously undescribed species of myxozoan parasites were observed in the gills of bass inhabiting the Potomac and James River basins. They are described using morphological characteristics and small-subunit (SSU) rDNA gene sequences. Both were taxonomically identified as new species of Myxobolus; Myxobolus branchiarum n. sp. was found exclusively in smallmouth bass, and Myxobolus micropterii n. sp. was found in largemouth and smallmouth bass. Small, spherical, white plasmodia of M. branchiarum from smallmouth bass were observed grossly in the gills; these plasmodia had an average length of 320.3 µm and width of 246.1 µm. The development of the plasmodia is intralamellar in the secondary lamellae of the gills. Mature spores were pyriform in shape with a length of 12.8 ± 1.4 (8.1–15.1) µm and width of 6.9 ± 1.1 (4.0–9.0) µm. Analysis of SSU rDNA identified M. branchiarum in a sister-group to 3 species of Henneguya, although morphologically caudal appendages were absent. Myxobolus micropterii observed in the gills of largemouth and smallmouth bass had larger, ovoid, cream-colored plasmodia with an average length of 568.1 µm and width of 148.1 µm. The cysts developed at the distal end of the gill filament within the primary lamellae. The mature spores were ovoid in shape with a length of 10.8 ± 0.7 (9.2–12.2) µm and width of 10.6 ± 0.6 (9.0–11.8) µm. SSU rDNA analysis placed M. micropterii in a sister group with Henneguya lobosa and Myxobolus oliveirai. The highest prevalence of M. branchiarum was observed in the gills of bass collected from the Cowpasture River (50.9%). Prevalence was 44.6% in bass from the Potomac River and only 4.3% in bass collected from the Shenandoah River. A seasonal study of M. branchiarum, which included both infected and uninfected smallmouth bass, determined that a significantly higher intensity was observed in the spring than in the summer (P < 0.001) or fall (P  =  0.004). In an analysis excluding uninfected bass, a higher intensity was observed in the spring than in the summer (P  =  0.001) or fall (P  =  0.008). Prevalence and seasonal differences were not determined for M. micropterii.

Description of two new gill myxozoans from smallmouth (Micropterus dolomieu) and largemouth (Micropterus salmoides) bass.

PubMed

Walsh, Heather L; Iwanowicz, Luke R; Glenney, Gavin W; Iwanowicz, Deborah D; Blazer, Vicki S

2012-04-01

Two previously undescribed species of myxozoan parasites were observed in the gills of bass inhabiting the Potomac and James River basins. They are described using morphological characteristics and small-subunit (SSU) rDNA gene sequences. Both were taxonomically identified as new species of Myxobolus; Myxobolus branchiarum n. sp. was found exclusively in smallmouth bass, and Myxobolus micropterii n. sp. was found in largemouth and smallmouth bass. Small, spherical, white plasmodia of M. branchiarum from smallmouth bass were observed grossly in the gills; these plasmodia had an average length of 320.3 µm and width of 246.1 µm. The development of the plasmodia is intralamellar in the secondary lamellae of the gills. Mature spores were pyriform in shape with a length of 12.8 ± 1.4 (8.1-15.1) µm and width of 6.9 ± 1.1 (4.0-9.0) µm. Analysis of SSU rDNA identified M. branchiarum in a sister-group to 3 species of Henneguya , although morphologically caudal appendages were absent. Myxobolus micropterii observed in the gills of largemouth and smallmouth bass had larger, ovoid, cream-colored plasmodia with an average length of 568.1 µm and width of 148.1 µm. The cysts developed at the distal end of the gill filament within the primary lamellae. The mature spores were ovoid in shape with a length of 10.8 ± 0.7 (9.2-12.2) µm and width of 10.6 ± 0.6 (9.0-11.8) µm. SSU rDNA analysis placed M. micropterii in a sister group with Henneguya lobosa and Myxobolus oliveirai . The highest prevalence of M. branchiarum was observed in the gills of bass collected from the Cowpasture River (50.9%). Prevalence was 44.6% in bass from the Potomac River and only 4.3% in bass collected from the Shenandoah River. A seasonal study of M. branchiarum , which included both infected and uninfected smallmouth bass, determined that a significantly higher intensity was observed in the spring than in the summer (P < 0.001) or fall (P  =  0.004). In an analysis excluding uninfected bass, a higher intensity was observed in the spring than in the summer (P  =  0.001) or fall (P  =  0.008). Prevalence and seasonal differences were not determined for M. micropterii .

Inter-subunit electrostatic interactions in ferritin molecule: comparison with inter-molecular interactions in crystals

NASA Astrophysics Data System (ADS)

Takahashi, Takuya; Hogyoku, Michiru; Nagayama, Kuniaki

1996-10-01

We evaluated the contribution of electrostatic interactions to the stability of macromolecular assembly in a horse L ferritin molecule composed of 24 subunits and the three-dimensional crystal of the ferritin molecules with numerical calculation of Poisson-Boltzmann equation based on dielectric model. The calculation showed that the electrostatic energy both favors the assembly of the 24 subunits and the crystalline assembly of the ferritin molecules (i.e., 24-mers). Short-range interactions less than 5 Å such as salt bridges and hydrogen bonds were important for both the subunit assembly and the crystalline assembly. To elucidate the strong stabilization by electrostatic interactions in both the ferritin 24-mer and its crystal, we analyzed the contribution of individual atoms. It revealed that the stabilization was arising from buried salt bridges or hydrogen bonds, which yielded more than 5 kcal/mol in some interactions. These large electrostatic stabilization and also the unexpected small ionic strength dependence was different from those of bovine pancreatic trypsin inhibitor (BPTI) orthorhombic and pig-insulin cubic crystals previously calculated. We also evaluated changes of the accessible surface area (ASA) and hydration free energy in accordance with the process of the subunit assembly. The change of hydration free energy, which was very large (i.e. ˜ + 100 kcal/mol/subunit) and unfavorable for the assembly, was proportional to the electrostatic hydration energy (i.e. Born energy change in hydration process). Hydrophobic groups were likely to appear more frequently than hydrophilic groups at the subunit interfaces. These results suggest that the molecular structure of the ferritin 24-mer and the crystal structure of the 24-mers were both stabilized by local electrostatic interactions, in particular. We view protein crystals as an extension of the protein oligomer to an infinite number of subunits association.

PDB-NMA of a protein homodimer reproduces distinct experimental motility asymmetry.

PubMed

Tirion, Monique M; Ben-Avraham, Daniel

2018-01-16

We have extended our analytically derived PDB-NMA formulation, Atomic Torsional Modal Analysis or ATMAN (Tirion and ben-Avraham 2015 Phys. Rev. E 91 032712), to include protein dimers using mixed internal and Cartesian coordinates. A test case on a 1.3 [Formula: see text] resolution model of a small homodimer, ActVA-ORF6, consisting of two 112-residue subunits identically folded in a compact 50 [Formula: see text] sphere, reproduces the distinct experimental Debye-Waller motility asymmetry for the two chains, demonstrating that structure sensitively selects vibrational signatures. The vibrational analysis of this PDB entry, together with biochemical and crystallographic data, demonstrates the cooperative nature of the dimeric interaction of the two subunits and suggests a mechanical model for subunit interconversion during the catalytic cycle.

PDB-NMA of a protein homodimer reproduces distinct experimental motility asymmetry

NASA Astrophysics Data System (ADS)

Tirion, Monique M.; ben-Avraham, Daniel

2018-03-01

We have extended our analytically derived PDB-NMA formulation, Atomic Torsional Modal Analysis or ATMAN (Tirion and ben-Avraham 2015 Phys. Rev. E 91 032712), to include protein dimers using mixed internal and Cartesian coordinates. A test case on a 1.3 {\\mathringA} resolution model of a small homodimer, ActVA-ORF6, consisting of two 112-residue subunits identically folded in a compact 50 {\\mathringA} sphere, reproduces the distinct experimental Debye-Waller motility asymmetry for the two chains, demonstrating that structure sensitively selects vibrational signatures. The vibrational analysis of this PDB entry, together with biochemical and crystallographic data, demonstrates the cooperative nature of the dimeric interaction of the two subunits and suggests a mechanical model for subunit interconversion during the catalytic cycle.

The ω Subunit Governs RNA Polymerase Stability and Transcriptional Specificity in Staphylococcus aureus.

PubMed

Weiss, Andy; Moore, Brittney D; Tremblay, Miguel H J; Chaput, Dale; Kremer, Astrid; Shaw, Lindsey N

2017-01-15

Staphylococcus aureus is a major human pathogen that causes infection in a wide variety of sites within the human body. Its ability to adapt to the human host and to produce a successful infection requires precise orchestration of gene expression. While DNA-dependent RNA polymerase (RNAP) is generally well characterized, the roles of several small accessory subunits within the complex have yet to be fully explored. This is particularly true for the omega (ω or RpoZ) subunit, which has been extensively studied in Gram-negative bacteria but largely neglected in Gram-positive counterparts. In Escherichia coli, it has been shown that ppGpp binding, and thus control of the stringent response, is facilitated by ω. Interestingly, key residues that facilitate ppGpp binding by ω are not conserved in S. aureus, and consequently, survival under starvation conditions is unaffected by rpoZ deletion. Further to this, ω-lacking strains of S. aureus display structural changes in the RNAP complex, which result from increased degradation and misfolding of the β' subunit, alterations in δ and σ factor abundance, and a general dissociation of RNAP in the absence of ω. Through RNA sequencing analysis we detected a variety of transcriptional changes in the rpoZ-deficient strain, presumably as a response to the negative effects of ω depletion on the transcription machinery. These transcriptional changes translated to an impaired ability of the rpoZ mutant to resist stress and to fully form a biofilm. Collectively, our data underline, for the first time, the importance of ω for RNAP stability, function, and cellular physiology in S. aureus IMPORTANCE: In order for bacteria to adjust to changing environments, such as within the host, the transcriptional process must be tightly controlled. Transcription is carried out by DNA-dependent RNA polymerase (RNAP). In addition to its major subunits (α 2 ββ') a fifth, smaller subunit, ω, is present in all forms of life. Although this small subunit is well studied in eukaryotes and Gram-negative bacteria, only limited information is available for Gram-positive and pathogenic species. In this study, we investigated the structural and functional importance of ω, revealing key roles in subunit folding/stability, complex assembly, and maintenance of transcriptional integrity. Collectively, our data underline, for the first time, the importance of ω for RNAP function and cellular harmony in S. aureus. Copyright © 2016 American Society for Microbiology.

How the morphology of dusts influences packing density in small solar system bodies

NASA Astrophysics Data System (ADS)

Zangmeister, C.; Radney, J. G.; Zachariah, M. R.

2014-12-01

Large planetary seedlings, comets, and nanoscale soot particles are made from rigid, aggregated subunits that are compacted under low compression into larger structures spanning over 10 orders of magnitude in dimensional space. Here, we demonstrate that the packing density (Φf) of compacted rigid aggregates is independent of spatial scale for systems under weak compaction, a regime that includes small solar system bodies. The Φf of rigid aggregated structures across 6 orders of magnitude were measured using nanoscale spherical soot aerosol composed of aggregates with ≈ 17 nm monomeric subunits and aggregates made from uniform monomeric 6 mm spherical subunits at the macroscale. We find Φf = 0.36 ± 0.02 at both the nano- and macroscale. These values are remarkably similar to qf observed for comet nuclei and measured values of other rigid aggregated systems across a wide variety of spatial and formative conditions. We present a packing model that incorporates the aggregate morphology and show that Φf is independent of both monomer and aggregate size. These observations suggest thatqf of rigid aggregates is independent of spatial dimension across varied formative conditions ranging from interstellar space to pharmaceutical manufacturing.

Live-cell imaging of budding yeast telomerase RNA and TERRA.

PubMed

Laprade, Hadrien; Lalonde, Maxime; Guérit, David; Chartrand, Pascal

2017-02-01

In most eukaryotes, the ribonucleoprotein complex telomerase is responsible for maintaining telomere length. In recent years, single-cell microscopy techniques such as fluorescent in situ hybridization and live-cell imaging have been developed to image the RNA subunit of the telomerase holoenzyme. These techniques are now becoming important tools for the study of telomerase biogenesis, its association with telomeres and its regulation. Here, we present detailed protocols for live-cell imaging of the Saccharomyces cerevisiae telomerase RNA subunit, called TLC1, and also of the non-coding telomeric repeat-containing RNA TERRA. We describe the approach used for genomic integration of MS2 stem-loops in these transcripts, and provide information for optimal live-cell imaging of these non-coding RNAs. Copyright © 2016 Elsevier Inc. All rights reserved.

Subunit interface dynamics in hexadecameric rubisco.

PubMed

van Lun, Michiel; van der Spoel, David; Andersson, Inger

2011-09-02

Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) plays an important role in the global carbon cycle as a hub for biomass. Rubisco catalyzes not only the carboxylation of RuBP with carbon dioxide but also a competing oxygenation reaction of RuBP with a negative impact on photosynthetic yield. The functional active site is built from two large (L) subunits that form a dimer. The octameric core of four L(2) dimers is held at each end by a cluster of four small (S) subunits, forming a hexadecamer. Each large subunit contacts more than one S subunit. These interactions exploit the dynamic flexibility of Rubisco, which we address in this study. Here, we describe seven different types of interfaces of hexadecameric Rubisco. We have analyzed these interfaces with respect to the size of the interface area and the number of polar interactions, including salt bridges and hydrogen bonds in a variety of Rubisco enzymes from different organisms and different kingdoms of life, including the Rubisco-like proteins. We have also performed molecular dynamics simulations of Rubisco from Chlamydomonas reinhardtii and mutants thereof. From our computational analyses, we propose structural checkpoints of the S subunit to ensure the functionality and/or assembly of the Rubisco holoenzyme. These checkpoints appear to fine-tune the dynamics of the enzyme in a way that could influence enzyme performance. Copyright © 2011 Elsevier Ltd. All rights reserved.

The Carboxyl Tail of Connexin32 Regulates Gap Junction Assembly in Human Prostate and Pancreatic Cancer Cells*

PubMed Central

Katoch, Parul; Mitra, Shalini; Ray, Anuttoma; Kelsey, Linda; Roberts, Brett J.; Wahl, James K.; Johnson, Keith R.; Mehta, Parmender P.

2015-01-01

Connexins, the constituent proteins of gap junctions, are transmembrane proteins. A connexin (Cx) traverses the membrane four times and has one intracellular and two extracellular loops with the amino and carboxyl termini facing the cytoplasm. The transmembrane and the extracellular loop domains are highly conserved among different Cxs, whereas the carboxyl termini, often called the cytoplasmic tails, are highly divergent. We have explored the role of the cytoplasmic tail of Cx32, a Cx expressed in polarized and differentiated cells, in regulating gap junction assembly. Our results demonstrate that compared with the full-length Cx32, the cytoplasmic tail-deleted Cx32 is assembled into small gap junctions in human pancreatic and prostatic cancer cells. Our results further document that the expression of the full-length Cx32 in cells, which express the tail-deleted Cx32, increases the size of gap junctions, whereas the expression of the tail-deleted Cx32 in cells, which express the full-length Cx32, has the opposite effect. Moreover, we show that the tail is required for the clustering of cell-cell channels and that in cells expressing the tail-deleted Cx32, the expression of cell surface-targeted cytoplasmic tail alone is sufficient to enhance the size of gap junctions. Our live-cell imaging data further demonstrate that gap junctions formed of the tail-deleted Cx32 are highly mobile compared with those formed of full-length Cx32. Our results suggest that the cytoplasmic tail of Cx32 is not required to initiate the assembly of gap junctions but for their subsequent growth and stability. Our findings suggest that the cytoplasmic tail of Cx32 may be involved in regulating the permeability of gap junctions by regulating their size. PMID:25548281

Comparing K-mer based methods for improved classification of 16S sequences.

PubMed

Vinje, Hilde; Liland, Kristian Hovde; Almøy, Trygve; Snipen, Lars

2015-07-01

The need for precise and stable taxonomic classification is highly relevant in modern microbiology. Parallel to the explosion in the amount of sequence data accessible, there has also been a shift in focus for classification methods. Previously, alignment-based methods were the most applicable tools. Now, methods based on counting K-mers by sliding windows are the most interesting classification approach with respect to both speed and accuracy. Here, we present a systematic comparison on five different K-mer based classification methods for the 16S rRNA gene. The methods differ from each other both in data usage and modelling strategies. We have based our study on the commonly known and well-used naïve Bayes classifier from the RDP project, and four other methods were implemented and tested on two different data sets, on full-length sequences as well as fragments of typical read-length. The difference in classification error obtained by the methods seemed to be small, but they were stable and for both data sets tested. The Preprocessed nearest-neighbour (PLSNN) method performed best for full-length 16S rRNA sequences, significantly better than the naïve Bayes RDP method. On fragmented sequences the naïve Bayes Multinomial method performed best, significantly better than all other methods. For both data sets explored, and on both full-length and fragmented sequences, all the five methods reached an error-plateau. We conclude that no K-mer based method is universally best for classifying both full-length sequences and fragments (reads). All methods approach an error plateau indicating improved training data is needed to improve classification from here. Classification errors occur most frequent for genera with few sequences present. For improving the taxonomy and testing new classification methods, the need for a better and more universal and robust training data set is crucial.

Intrinsic Pleckstrin Homology (PH) Domain Motion in Phospholipase C-β Exposes a Gβγ Protein Binding Site.

PubMed

Kadamur, Ganesh; Ross, Elliott M

2016-05-20

Mammalian phospholipase C-β (PLC-β) isoforms are stimulated by heterotrimeric G protein subunits and members of the Rho GTPase family of small G proteins. Although recent structural studies showed how Gαq and Rac1 bind PLC-β, there is a lack of consensus regarding the Gβγ binding site in PLC-β. Using FRET between cerulean fluorescent protein-labeled Gβγ and the Alexa Fluor 594-labeled PLC-β pleckstrin homology (PH) domain, we demonstrate that the PH domain is the minimal Gβγ binding region in PLC-β3. We show that the isolated PH domain can compete with full-length PLC-β3 for binding Gβγ but not Gαq, Using sequence conservation, structural analyses, and mutagenesis, we identify a hydrophobic face of the PLC-β PH domain as the Gβγ binding interface. This PH domain surface is not solvent-exposed in crystal structures of PLC-β, necessitating conformational rearrangement to allow Gβγ binding. Blocking PH domain motion in PLC-β by cross-linking it to the EF hand domain inhibits stimulation by Gβγ without altering basal activity or Gαq response. The fraction of PLC-β cross-linked is proportional to the fractional loss of Gβγ response. Cross-linked PLC-β does not bind Gβγ in a FRET-based Gβγ-PLC-β binding assay. We propose that unliganded PLC-β exists in equilibrium between a closed conformation observed in crystal structures and an open conformation where the PH domain moves away from the EF hands. Therefore, intrinsic movement of the PH domain in PLC-β modulates Gβγ access to its binding site. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Intrinsic Pleckstrin Homology (PH) Domain Motion in Phospholipase C-β Exposes a Gβγ Protein Binding Site*

PubMed Central

Kadamur, Ganesh

2016-01-01

Mammalian phospholipase C-β (PLC-β) isoforms are stimulated by heterotrimeric G protein subunits and members of the Rho GTPase family of small G proteins. Although recent structural studies showed how Gαq and Rac1 bind PLC-β, there is a lack of consensus regarding the Gβγ binding site in PLC-β. Using FRET between cerulean fluorescent protein-labeled Gβγ and the Alexa Fluor 594-labeled PLC-β pleckstrin homology (PH) domain, we demonstrate that the PH domain is the minimal Gβγ binding region in PLC-β3. We show that the isolated PH domain can compete with full-length PLC-β3 for binding Gβγ but not Gαq, Using sequence conservation, structural analyses, and mutagenesis, we identify a hydrophobic face of the PLC-β PH domain as the Gβγ binding interface. This PH domain surface is not solvent-exposed in crystal structures of PLC-β, necessitating conformational rearrangement to allow Gβγ binding. Blocking PH domain motion in PLC-β by cross-linking it to the EF hand domain inhibits stimulation by Gβγ without altering basal activity or Gαq response. The fraction of PLC-β cross-linked is proportional to the fractional loss of Gβγ response. Cross-linked PLC-β does not bind Gβγ in a FRET-based Gβγ-PLC-β binding assay. We propose that unliganded PLC-β exists in equilibrium between a closed conformation observed in crystal structures and an open conformation where the PH domain moves away from the EF hands. Therefore, intrinsic movement of the PH domain in PLC-β modulates Gβγ access to its binding site. PMID:27002154

Broad genomic and transcriptional analysis reveals a highly derived genome in dinoflagellate mitochondria

PubMed Central

Jackson, Christopher J; Norman, John E; Schnare, Murray N; Gray, Michael W; Keeling, Patrick J; Waller, Ross F

2007-01-01

Background Dinoflagellates comprise an ecologically significant and diverse eukaryotic phylum that is sister to the phylum containing apicomplexan endoparasites. The mitochondrial genome of apicomplexans is uniquely reduced in gene content and size, encoding only three proteins and two ribosomal RNAs (rRNAs) within a highly compacted 6 kb DNA. Dinoflagellate mitochondrial genomes have been comparatively poorly studied: limited available data suggest some similarities with apicomplexan mitochondrial genomes but an even more radical type of genomic organization. Here, we investigate structure, content and expression of dinoflagellate mitochondrial genomes. Results From two dinoflagellates, Crypthecodinium cohnii and Karlodinium micrum, we generated over 42 kb of mitochondrial genomic data that indicate a reduced gene content paralleling that of mitochondrial genomes in apicomplexans, i.e., only three protein-encoding genes and at least eight conserved components of the highly fragmented large and small subunit rRNAs. Unlike in apicomplexans, dinoflagellate mitochondrial genes occur in multiple copies, often as gene fragments, and in numerous genomic contexts. Analysis of cDNAs suggests several novel aspects of dinoflagellate mitochondrial gene expression. Polycistronic transcripts were found, standard start codons are absent, and oligoadenylation occurs upstream of stop codons, resulting in the absence of termination codons. Transcripts of at least one gene, cox3, are apparently trans-spliced to generate full-length mRNAs. RNA substitutional editing, a process previously identified for mRNAs in dinoflagellate mitochondria, is also implicated in rRNA expression. Conclusion The dinoflagellate mitochondrial genome shares the same gene complement and fragmentation of rRNA genes with its apicomplexan counterpart. However, it also exhibits several unique characteristics. Most notable are the expansion of gene copy numbers and their arrangements within the genome, RNA editing, loss of stop codons, and use of trans-splicing. PMID:17897476

Murine recessive hereditary spherocytosis, sph/sph, is caused by a mutation in the erythroid alpha-spectrin gene.

PubMed

Wandersee, N J; Birkenmeier, C S; Gifford, E J; Mohandas, N; Barker, J E

2000-01-01

Spectrin, a heterodimer of alpha- and beta-subunits, is the major protein component of the red blood cell membrane skeleton. The mouse mutation, sph, causes an alpha-spectrin-deficient hereditary spherocytosis with the severe phenotype typical of recessive hereditary spherocytosis in humans. The sph mutation maps to the erythroid alpha-spectrin locus, Spna1, on Chromosome 1. Scanning electron microscopy, osmotic gradient ektacytometry, cDNA cloning, RT-PCR, nucleic acid sequencing, and Northern blot analyses were used to characterize the wild type and sph alleles of the Spna1 locus. Our results confirm the spherocytic nature of sph/sph red blood cells and document a mild spherocytic transition in the +/sph heterozygotes. Sequencing of the full length coding region of the Spna1 wild type allele from the C57BL/6J strain of mice reveals a 2414 residue deduced amino acid sequence that shows the typical 106-amino-acid repeat structure previously described for other members of the spectrin protein family. Sequence analysis of RT-PCR clones from sph/sph alpha-spectrin mRNA identified a single base deletion in repeat 5 that would cause a frame shift and premature termination of the protein. This deletion was confirmed in sph/sph genomic DNA. Northern blot analyses of the distribution of Spna1 mRNA in non-erythroid tissues detects the expression of 8, 2.5 and 2.0 kb transcripts in adult heart. These results predict the heart as an additional site where alpha-spectrin mutations may produce a phenotype and raise the possibility that a novel functional class of small alpha-spectrin isoforms may exist.

Estimating the effective radiation dose imparted to patients by intraoperative cone-beam computed tomography in thoracolumbar spinal surgery.

PubMed

Lange, Jeffrey; Karellas, Andrew; Street, John; Eck, Jason C; Lapinsky, Anthony; Connolly, Patrick J; Dipaola, Christian P

2013-03-01

Observational. To estimate the radiation dose imparted to patients during typical thoracolumbar spinal surgical scenarios. Minimally invasive techniques continue to become more common in spine surgery. Computer-assisted navigation systems coupled with intraoperative cone-beam computed tomography (CT) represent one such method used to aid in instrumented spinal procedures. Some studies indicate that cone-beam CT technology delivers a relatively low dose of radiation to patients compared with other x-ray-based imaging modalities. The goal of this study was to estimate the radiation exposure to the patient imparted during typical posterior thoracolumbar instrumented spinal procedures, using intraoperative cone-beam CT and to place these values in the context of standard CT doses. Cone-beam CT scans were obtained using Medtronic O-arm (Medtronic, Minneapolis, MN). Thermoluminescence dosimeters were placed in a linear array on a foam-plastic thoracolumbar spine model centered above the radiation source for O-arm presets of lumbar scans for small or large patients. In-air dosimeter measurements were converted to skin surface measurements, using published conversion factors. Dose-length product was calculated from these values. Effective dose was estimated using published effective dose to dose-length product conversion factors. Calculated dosages for many full-length procedures using the small-patient setting fell within the range of published effective doses of abdominal CT scans (1-31 mSv). Calculated dosages for many full-length procedures using the large-patient setting fell within the range of published effective doses of abdominal CT scans when the number of scans did not exceed 3. We have demonstrated that single cone-beam CT scans and most full-length posterior instrumented spinal procedures using O-arm in standard mode would likely impart a radiation dose within the range of those imparted by a single standard CT scan of the abdomen. Radiation dose increases with patient size, and the radiation dose received by larger patients as a result of more than 3 O-arm scans in standard mode may exceed the dose received during standard CT of the abdomen. Understanding radiation imparted to patients by cone-beam CT is important for assessing risks and benefits of this technology, especially when spinal surgical procedures require multiple intraoperative scans.

The first genetically confirmed case of Dioctophyme renale (Nematoda: Dioctophymatida) in a patient with a subcutaneous nodule.

PubMed

Tokiwa, Toshihiro; Ueda, Wataru; Takatsuka, Satoshi; Okawa, Kiyotaka; Onodera, Masayuki; Ohta, Nobuo; Akao, Nobuaki

2014-02-01

We describe a nematode larva in a subcutaneous nodule excised from a 44-year-old Chinese male who had been living in Japan for 15 years. Morphological features suggested that the worm was a dioctophimatid nematode. PCR amplification and sequencing of small subunit ribosomal DNA and mitochondrial cytochrome subunit c oxidase genes allowed us to identify the larva as the giant kidney worm, Dioctophyme renale (Goeze, 1972). This is the first molecularly confirmed human case of a dermal D. renale infection. © 2013.

Potent and Selective Peptide-based Inhibition of the G Protein Gαq*

PubMed Central

Charpentier, Thomas H.; Waldo, Gary L.; Lowery-Gionta, Emily G.; Krajewski, Krzysztof; Strahl, Brian D.; Kash, Thomas L.; Harden, T. Kendall; Sondek, John

2016-01-01

In contrast to G protein-coupled receptors, for which chemical and peptidic inhibitors have been extensively explored, few compounds are available that directly modulate heterotrimeric G proteins. Active Gαq binds its two major classes of effectors, the phospholipase C (PLC)-β isozymes and Rho guanine nucleotide exchange factors (RhoGEFs) related to Trio, in a strikingly similar fashion: a continuous helix-turn-helix of the effectors engages Gαq within its canonical binding site consisting of a groove formed between switch II and helix α3. This information was exploited to synthesize peptides that bound active Gαq in vitro with affinities similar to full-length effectors and directly competed with effectors for engagement of Gαq. A representative peptide was specific for active Gαq because it did not bind inactive Gαq or other classes of active Gα subunits and did not inhibit the activation of PLC-β3 by Gβ1γ2. In contrast, the peptide robustly prevented activation of PLC-β3 or p63RhoGEF by Gαq; it also prevented G protein-coupled receptor-promoted neuronal depolarization downstream of Gαq in the mouse prefrontal cortex. Moreover, a genetically encoded form of this peptide flanked by fluorescent proteins inhibited Gαq-dependent activation of PLC-β3 at least as effectively as a dominant-negative form of full-length PLC-β3. These attributes suggest that related, cell-penetrating peptides should effectively inhibit active Gαq in cells and that these and genetically encoded sequences may find application as molecular probes, drug leads, and biosensors to monitor the spatiotemporal activation of Gαq in cells. PMID:27742837

Potent and Selective Peptide-based Inhibition of the G Protein Gαq.

PubMed

Charpentier, Thomas H; Waldo, Gary L; Lowery-Gionta, Emily G; Krajewski, Krzysztof; Strahl, Brian D; Kash, Thomas L; Harden, T Kendall; Sondek, John

2016-12-02

In contrast to G protein-coupled receptors, for which chemical and peptidic inhibitors have been extensively explored, few compounds are available that directly modulate heterotrimeric G proteins. Active Gα q binds its two major classes of effectors, the phospholipase C (PLC)-β isozymes and Rho guanine nucleotide exchange factors (RhoGEFs) related to Trio, in a strikingly similar fashion: a continuous helix-turn-helix of the effectors engages Gα q within its canonical binding site consisting of a groove formed between switch II and helix α3. This information was exploited to synthesize peptides that bound active Gα q in vitro with affinities similar to full-length effectors and directly competed with effectors for engagement of Gα q A representative peptide was specific for active Gα q because it did not bind inactive Gα q or other classes of active Gα subunits and did not inhibit the activation of PLC-β3 by Gβ 1 γ 2 In contrast, the peptide robustly prevented activation of PLC-β3 or p63RhoGEF by Gα q ; it also prevented G protein-coupled receptor-promoted neuronal depolarization downstream of Gα q in the mouse prefrontal cortex. Moreover, a genetically encoded form of this peptide flanked by fluorescent proteins inhibited Gα q -dependent activation of PLC-β3 at least as effectively as a dominant-negative form of full-length PLC-β3. These attributes suggest that related, cell-penetrating peptides should effectively inhibit active Gα q in cells and that these and genetically encoded sequences may find application as molecular probes, drug leads, and biosensors to monitor the spatiotemporal activation of Gα q in cells. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Human Antibodies that Recognize Novel Immunodominant Quaternary Epitopes on the HIV-1 Env Protein

PubMed Central

Hicar, Mark D.; Chen, Xuemin; Sulli, Chidananda; Barnes, Trevor; Goodman, Jason; Sojar, Hakimuddin; Briney, Bryan; Willis, Jordan; Chukwuma, Valentine U.; Kalams, Spyros A.; Doranz, Benjamin J.; Spearman, Paul; Crowe, James E.

2016-01-01

Numerous broadly neutralizing antibodies (Abs) target epitopes that are formed or enhanced during mature HIV envelope formation (i.e. quaternary epitopes). Generally, it is thought that Env epitopes that induce broadly neutralizing Abs are difficult to access and poorly immunogenic because of the characteristic oligomerization, conformational flexibility, sequence diversity and extensive glycosylation of Env protein. To enhance for isolation of quaternary epitope-targeting Abs (QtAbs), we previously used HIV virus-like particles (VLPs) to bind B cells from long-term non-progressor subjects to identify a panel of monoclonal Abs. When expressed as recombinant full-length Abs, a subset of these novel Abs exhibited the binding profiles of QtAbs, as they either failed to bind to monomeric Env protein or showed much higher affinity for Env trimers and VLPs. These QtAbs represented a significant proportion of the B-cell response identified with VLPs. The Ab genes of these clones were highly mutated, but they did not neutralize common HIV strains. We sought to further define the epitopes targeted by these QtAbs. Competition-binding and mapping studies revealed these Abs targeted four separate epitopes; they also failed to compete for binding by Abs to known major neutralizing epitopes. Detailed epitope mapping studies revealed that two of the four epitopes were located in the gp41 subunit of Env. These QtAbs bound pre-fusion forms of antigen and showed differential binding kinetics depending on whether oligomers were produced as recombinant gp140 trimers or as full-length Env incorporated into VLPs. Antigenic regions within gp41 present unexpectedly diverse structural epitopes, including these QtAb epitopes, which may be targeted by the naturally occurring Ab response to HIV infection. PMID:27411063

An inducible amphipathic helix within the intrinsically disordered C terminus can participate in membrane curvature generation by peripherin-2/rds.

PubMed

Milstein, Michelle L; Kimler, Victoria A; Ghatak, Chiranjib; Ladokhin, Alexey S; Goldberg, Andrew F X

2017-05-12

Peripherin-2/rds is required for biogenesis of vertebrate photoreceptor outer segment organelles. Its localization at the high-curvature rim domains of outer segment disk membranes suggests that it may act to shape these structures; however, the molecular function of this protein is not yet resolved. Here, we apply biochemical, biophysical, and imaging techniques to elucidate the role(s) played by the protein's intrinsically disordered C-terminal domain and an incipient amphipathic α-helix contained within it. We investigated a deletion mutant lacking only this α-helix in stable cell lines and Xenopus laevis photoreceptors. We also studied a soluble form of the full-length ∼7-kDa cytoplasmic C terminus in cultured cells and purified from Escherichia coli The α-helical motif was not required for protein biosynthesis, tetrameric subunit assembly, tetramer polymerization, localization at disk rims, interaction with GARP2, or the generation of membrane curvature. Interestingly, however, loss of the helical motif up-regulated membrane curvature generation in cellulo , introducing the possibility that it may regulate this activity in photoreceptors. Furthermore, the incipient α-helix (within the purified soluble C terminus) partitioned into membranes only when its acidic residues were neutralized by protonation. This suggests that within the context of full-length peripherin-2/rds, partitioning would most likely occur at a bilayer interfacial region, potentially adjacent to the protein's transmembrane domains. In sum, this study significantly strengthens the evidence that peripherin-2/rds functions directly to shape the high-curvature rim domains of the outer segment disk and suggests that the protein's C terminus may modulate membrane curvature-generating activity present in other protein domains. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

A simple strategy for the purification of native recombinant full-length human RPL10 protein from inclusion bodies.

PubMed

Pereira, Larissa M; Silva, Luana R; Alves, Joseane F; Marin, Nélida; Silva, Flavio Sousa; Morganti, Ligia; Silva, Ismael D C G; Affonso, Regina

2014-09-01

The L10 ribosomal protein (RPL10) plays a role in the binding of the 60 S and 40 S ribosomal subunits and in mRNA translation. The evidence indicates that RPL10 also has multiple extra-ribosomal functions, including tumor suppression. Recently, the presence of RPL10 in prostate and ovarian cancers was evaluated, and it was demonstrated to be associated with autistic disorders and premature ovarian failure. In the present work, we successfully cloned and expressed full-length human RPL10 (hRPL10) protein and isolated inclusion bodies containing this protein that had formed under mild growth conditions. The culture produced 376mg of hRPL10 protein per liter of induced bacterial culture, of which 102.4mg was present in the soluble fraction, and 25.6mg was recovered at approximately 94% purity. These results were obtained using a two-step process of non-denaturing protein extraction from pelleted inclusion bodies. We studied the characteristics of this protein using circular dichroism spectroscopy and by monitoring the changes induced by the presence or absence of zinc ions using fluorescence spectrometry. The results demonstrated that the protein obtained using these non-conventional methods retained its secondary and tertiary structure. The conformational changes induced by the incorporation of zinc suggested that this protein could interact with Jun or the SH3 domain of c-yes. The results suggested that the strategy used to obtain hRPL10 is simple and could be applied to obtaining other proteins that are susceptible to degradation. Copyright © 2014 Elsevier Inc. All rights reserved.

Mediator Tail Module Is Required for Tac1-Activated CDR1 Expression and Azole Resistance in Candida albicans.

PubMed

Liu, Zhongle; Myers, Lawrence C

2017-11-01

The human fungal pathogen Candida albicans develops drug resistance after long-term exposure to azole drugs in the treatment of chronic candidiasis. Gain-of-function (GOF) mutations in the transcription factor Tac1 and the consequent expression of its targets, drug efflux pumps Cdr1 and Cdr2, are a common mechanism by which C. albicans acquires fluconazole resistance. The mechanism by which GOF mutations hyperactivate Tac1 is currently unknown. Here, we define a transcriptional activation domain (TAD) at the C terminus of Tac1. GOF mutations within the Tac1 TAD, outside the context of full-length Tac1, generally do not enhance its absolute potential as a transcriptional activator. Negative regulation of the Tac1 TAD by the Tac1 middle region is necessary for the activating effect of GOF mutations or fluphenazine to be realized. We have found that full-length Tac1, when hyperactivated by xenobiotics or GOF mutations, facilitates the recruitment of the Mediator coactivator complex to the CDR1 promoter. Azole resistance and the activation of Tac1 target genes, such as CDR1 , are dependent on the Tac1 TAD and subunits of the Mediator tail module. The dependence of different Tac1 target promoters on the Mediator tail module, however, varies widely. Lastly, we show that hyperactivation of Tac1 is correlated with its Mediator-dependent phosphorylation, a potentially useful biomarker for Tac1 hyperactivation. The role of Mediator in events downstream of Tac1 hyperactivation in fluconazole-resistant clinical isolates is complex and provides opportunities and challenges for therapeutic intervention. Copyright © 2017 American Society for Microbiology.

Disease-associated extracellular loop mutations in the adhesion G protein-coupled receptor G1 (ADGRG1; GPR56) differentially regulate downstream signaling.

PubMed

Kishore, Ayush; Hall, Randy A

2017-06-09

Mutations to the adhesion G protein-coupled receptor ADGRG1 (G1; also known as GPR56) underlie the neurological disorder bilateral frontoparietal polymicrogyria. Disease-associated mutations in G1 studied to date are believed to induce complete loss of receptor function through disruption of either receptor trafficking or signaling activity. Given that N-terminal truncation of G1 and other adhesion G protein-coupled receptors has been shown to significantly increase the receptors' constitutive signaling, we examined two different bilateral frontoparietal polymicrogyria-inducing extracellular loop mutations (R565W and L640R) in the context of both full-length and N-terminally truncated (ΔNT) G1. Interestingly, we found that these mutations reduced surface expression of full-length G1 but not G1-ΔNT in HEK-293 cells. Moreover, the mutations ablated receptor-mediated activation of serum response factor luciferase, a classic measure of Gα 12/13 -mediated signaling, but had no effect on G1-mediated signaling to nuclear factor of activated T cells (NFAT) luciferase. Given these differential signaling results, we sought to further elucidate the pathway by which G1 can activate NFAT luciferase. We found no evidence that ΔNT activation of NFAT is dependent on Gα q/11 -mediated or β-arrestin-mediated signaling but rather involves liberation of Gβγ subunits and activation of calcium channels. These findings reveal that disease-associated mutations to the extracellular loops of G1 differentially alter receptor trafficking, depending on the presence of the N terminus, and differentially alter signaling to distinct downstream pathways. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Stiffened yeast telomerase RNA supports RNP function in vitro and in vivo

PubMed Central

Lebo, Kevin J.; Zappulla, David C.

2012-01-01

The 1157-nt Saccharomyces cerevisiae telomerase RNA, TLC1, in addition to providing a 16-nt template region for reverse transcription, has been proposed to act as a scaffold for protein subunits. Although accessory subunits of the telomerase ribonucleoprotein (RNP) complex function even when their binding sites are relocated on the yeast telomerase RNA, the physical nature of the RNA scaffold has not been directly analyzed. Here we explore the structure–function organization of the yeast telomerase RNP by extensively stiffening the three long arms of TLC1, which connect essential and important accessory protein subunits Ku, Est1, and Sm7, to its central catalytic hub. This 956-nt triple-stiff-arm TLC1 (TSA-T) reconstitutes active telomerase with TERT (Est2) in vitro. Furthermore, TSA-T functions in vivo, even maintaining longer telomeres than TLC1 on a per RNA basis. We also tested functional contributions of each stiffened arm within TSA-T and found that the stiffened Est1 and Ku arms contribute to telomere lengthening, while stiffening the terminal arm reduces telomere length and telomerase RNA abundance. The fact that yeast telomerase tolerates significant stiffening of its RNA subunit in vivo advances our understanding of the architectural and functional organization of this RNP and, more broadly, our conception of the world of lncRNPs. PMID:22850424

Principal component and normal mode analysis of proteins; a quantitative comparison using the GroEL subunit.

PubMed

Skjaerven, Lars; Martinez, Aurora; Reuter, Nathalie

2011-01-01

Principal component analysis (PCA) and normal mode analysis (NMA) have emerged as two invaluable tools for studying conformational changes in proteins. To compare these approaches for studying protein dynamics, we have used a subunit of the GroEL chaperone, whose dynamics is well characterized. We first show that both PCA on trajectories from molecular dynamics (MD) simulations and NMA reveal a general dynamical behavior in agreement with what has previously been described for GroEL. We thus compare the reproducibility of PCA on independent MD runs and subsequently investigate the influence of the length of the MD simulations. We show that there is a relatively poor one-to-one correspondence between eigenvectors obtained from two independent runs and conclude that caution should be taken when analyzing principal components individually. We also observe that increasing the simulation length does not improve the agreement with the experimental structural difference. In fact, relatively short MD simulations are sufficient for this purpose. We observe a rapid convergence of the eigenvectors (after ca. 6 ns). Although there is not always a clear one-to-one correspondence, there is a qualitatively good agreement between the movements described by the first five modes obtained with the three different approaches; PCA, all-atoms NMA, and coarse-grained NMA. It is particularly interesting to relate this to the computational cost of the three methods. The results we obtain on the GroEL subunit contribute to the generalization of robust and reproducible strategies for the study of protein dynamics, using either NMA or PCA of trajectories from MD simulations. © 2010 Wiley-Liss, Inc.

Structural changes of homodimers in the PDB.

PubMed

Koike, Ryotaro; Amemiya, Takayuki; Horii, Tatsuya; Ota, Motonori

2018-04-01

Protein complexes are involved in various biological phenomena. These complexes are intrinsically flexible, and structural changes are essential to their functions. To perform a large-scale automated analysis of the structural changes of complexes, we combined two original methods. An application, SCPC, compares two structures of protein complexes and decides the match of binding mode. Another application, Motion Tree, identifies rigid-body motions in various sizes and magnitude from the two structural complexes with the same binding mode. This approach was applied to all available homodimers in the Protein Data Bank (PDB). We defined two complex-specific motions: interface motion and subunit-spanning motion. In the former, each subunit of a complex constitutes a rigid body, and the relative movement between subunits occurs at the interface. In the latter, structural parts from distinct subunits constitute a rigid body, providing the relative movement spanning subunits. All structural changes were classified and examined. It was revealed that the complex-specific motions were common in the homodimers, detected in around 40% of families. The dimeric interfaces were likely to be small and flat for interface motion, while large and rugged for subunit-spanning motion. Interface motion was accompanied by a drastic change in contacts at the interface, while the change in the subunit-spanning motion was moderate. These results indicate that the interface properties of homodimers correlated with the type of complex-specific motion. The study demonstrates that the pipeline of SCPC and Motion Tree is useful for the massive analysis of structural change of protein complexes. Copyright © 2017 Elsevier Inc. All rights reserved.

Ultrastructural morphology and phylogeny of Henneguya gilbert n. sp. (Myxozoa) infecting the teleostean Cyphocharax gilbert (Characiformes: Curimatidae) from Brazil.

PubMed

Casal, Graça; São Clemente, Sérgio C; Lopes, Leila; Rocha, Sónia; Felizardo, Nilza; Oliveira, Elsa; Al-Quraishy, Saleh; Azevedo, Carlos

2017-10-01

This paper describes light and ultrastructural observations and molecular analysis of a fish-infecting myxosporean, Henneguya gilbert n. sp., which was found infecting the gill epithelium of the commercially important freshwater teleost fish Cyphocharax gilbert (Curimatidae) collected in the estuarine region of Guandu River, Rio de Janeiro State, Brazil. The parasite occurs in the gills, forming whitish spherical to ellipsoidal polysporic cysts measuring up to ~ 750 μm, and displaying asynchronous development. Mature myxospores are ellipsoidal with a bifurcated caudal process. The length, width and thickness of the body of the myxospore are 12.0 × 5.3 × 3.6 μm, respectively; two equal caudal processes are 16.8 μm long, and the total length of the myxospore is 27.2 μm. There are two unequal polar capsules: the larger measures 5.5 μm length × 1.3 μm width and has a polar filament with 9-10 coils; the smaller is 4.0 μm long × 1.3 μm wide and has a polar filament with 7-8 coils. The sporoplasm is binucleated and presents a spherical vacuole surrounded by numerous globular sporoplasmosomes. Phylogenetic analysis, based on the small subunit rRNA sequencing, using maximum likelihood method reveals the parasite clustering together with other myxobolids that are histozoic and parasitize freshwater fish of the order Characiformes, thereby strengthening the contention that the host phylogenetic relationships and aquatic environment are the strongest evolutionary signals for myxosporeans of the family Myxobolidae.

DPN-Generated Combinatorial Libraries

DTIC Science & Technology

2012-02-29

µM final concentration) is added (Fig. 6). Control over deposition parameters was examined for two model proteins, cholera toxin β subunit (CTβ...conjugated anti- cholera toxin beta (anti-CTb). The wells in the mould are inverted pyramids with an average depth of 86 µm, edge length of 120 µm, and...Therapeutics,” (2011). 91. University of New Mexico , Chemistry Department Colloquium, Albuquerque, NM, “The Polyvalent Gold Nanoparticle Conjugate

Characterization of North American Armillaria species: Genetic relationships determined by ribosomal DNA sequences and AFLP markers

Treesearch

M. -S. Kim; N. B. Klopfenstein; J. W. Hanna; G. I. McDonald

2006-01-01

Phylogenetic and genetic relationships among 10 North American Armillaria species were analysed using sequence data from ribosomal DNA (rDNA), including intergenic spacer (IGS-1), internal transcribed spacers with associated 5.8S (ITS + 5.8S), and nuclear large subunit rDNA (nLSU), and amplified fragment length polymorphism (AFLP) markers. Based on rDNA sequence data,...

Phylogenetic position of parabasalid symbionts from the termite Calotermes flavicollis based on small subunit rRNA sequences.

PubMed

Gerbod, D; Edgcomb, V P; Noël, C; Delgado-Viscogliosi, P; Viscogliosi, E

2000-09-01

Small subunit rDNA genes were amplified by polymerase chain reaction using specific primers from mixed-population DNA obtained from the whole hindgut of the termite Calotermes flavicollis. Comparative sequence analysis of the clones revealed two kinds of sequences that were both from parabasalid symbionts. In a molecular tree inferred by distance, parsimony and likelihood methods, and including 27 parabasalid sequences retrieved from the data bases, the sequences of the group II (clones Cf5 and Cf6) were closely related to the Devescovinidae/Calonymphidae species and thus were assigned to the Devescovinidae Foaina. The sequence of the group I (clone Cf1) emerged within the Trichomonadinae and strongly clustered with Tetratrichomonas gallinarum. On the basis of morphological data, the Monocercomonadidae Hexamastix termitis might be the most likely origin of this sequence.

Isolation and characterization of cbbL and cbbS genes encoding form I ribulose-1,5-bisphosphate carboxylase/oxygenase large and small subunits in Nitrosomonas sp. strain ENI-11.

PubMed

Hirota, Ryuichi; Kato, Junichi; Morita, Hiromu; Kuroda, Akio; Ikeda, Tsukasa; Takiguchi, Noboru; Ohtake, Hisao

2002-03-01

The cbbL and cbbS genes encoding form I ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large and small subunits in the ammonia-oxidizing bacterium Nitrosomonas sp. strain ENI-11 were cloned and sequenced. The deduced gene products, CbbL and CbbS, had 93 and 87% identity with Thiobacillus intermedius CbbL and Nitrobacter winogradskyi CbbS, respectively. Expression of cbbL and cbbS in Escherichia coli led to the detection of RubisCO activity in the presence of 0.1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG). To our knowledge, this is the first paper to report the genes involved in the carbon fixation reaction in chemolithotrophic ammonia-oxidizing bacteria.

Development of an oligonucleotide probe for Aureobasidium pullulans based on the small-subunit rRNA gene.

PubMed Central

Li, S; Cullen, D; Hjort, M; Spear, R; Andrews, J H

1996-01-01

Aureobasidium pullulans, a cosmopolitan yeast-like fungus, colonizes leaf surfaces and has potential as a biocontrol agent of pathogens. To assess the feasibility of rRNA as a target for A. pullulans-specific oligonucleotide probes, we compared the nucleotide sequences of the small-subunit rRNA (18S) genes of 12 geographically diverse A. pullulans strains. Extreme sequence conservation was observed. The consensus A. pullulans sequence was compared with other fungal sequences to identify potential probes. A 21-mer probe which hybridized to the 12 A. pullulans strains but not to 98 other fungi, including 82 isolates from the phylloplane, was identified. A 17-mer highly specific for Cladosporium herbarum was also identified. These probes have potential in monitoring and quantifying fungi in leaf surface and other microbial communities. PMID:8633850

Amazonian waters harbour an ancient freshwater Ceratomyxa lineage (Cnidaria: Myxosporea).

PubMed

Zatti, Suellen A; Atkinson, Stephen D; Bartholomew, Jerri L; Maia, Antônio A M; Adriano, Edson A

2017-05-01

A new species of Ceratomyxa parasitizing the gall bladder of Cichla monoculus, an endemic cichlid fish from the Amazon basin in Brazil, is described using morphological and molecular data. In the bile, both immature and mature myxospores were found floating freely or inside elongated plasmodia: length 304 (196-402) μm and width 35.7 (18.3-55.1) μm. Mature spores were elongated and only slightly crescent-shaped in frontal view with a prominent sutural line between two valve cells, which had rounded ends. Measurements of formalin-fixed myxospores: length 6.3±0.6 (5.1-7.5) μm, thickness 41.2±2.9 (37.1-47.6) μm, posterior angle 147°. Lateral projections slightly asymmetric, with lengths 19.3±1.4μm and 20.5±1.3μm. Two ovoid, equal size polar capsules, length 2.6±0.3 (2-3.3) μm, width 2.5±0.4 (1.8-3.7) μm, located adjacent to the suture and containing polar filaments with 3-4 turns. The small subunit ribosomal DNA sequence of 1605 nt was no more than 97% similar to any other sequence in GenBank, and together with the host, locality and morphometric data, supports diagnosis of the parasite as a new species, Ceratomyxa brasiliensis n. sp. Maximum parsimony and maximum likelihood analyses showed that C. brasiliensis n. sp. clustered within the marine Ceratomyxa clade, but was in a basally divergent lineage with two other freshwater species from the Amazon basin. Our results are consistent with previous studies that show Ceratomyxa species can cluster according to both geography and host ecotype, and that the few known freshwater species diverged from marine cousins relatively early in evolution of the genus, possibly driven by marine incursions into riverine environments. Copyright © 2017 Elsevier B.V. All rights reserved.

Characterization of a novel organic solute transporter homologue from Clonorchis sinensis

PubMed Central

Dai, Fuhong; Lee, Ji-Yun; Pak, Jhang Ho; Sohn, Woon-Mok

2018-01-01

Clonorchis sinensis is a liver fluke that can dwell in the bile ducts of mammals. Bile acid transporters function to maintain the homeostasis of bile acids in C. sinensis, as they induce physiological changes or have harmful effects on C. sinensis survival. The organic solute transporter (OST) transports mainly bile acid and belongs to the SLC51 subfamily of solute carrier transporters. OST plays a critical role in the recirculation of bile acids in higher animals. In this study, we cloned full-length cDNA of the 480-amino acid OST from C. sinensis (CsOST). Genomic analysis revealed 11 exons and nine introns. The CsOST protein had a ‘Solute_trans_a’ domain with 67% homology to Schistosoma japonicum OST. For further analysis, the CsOST protein sequence was split into the ordered domain (CsOST-N) at the N-terminus and disordered domain (CsOST-C) at the C-terminus. The tertiary structure of each domain was built using a threading-based method and determined by manual comparison. In a phylogenetic tree, the CsOST-N domain belonged to the OSTα and CsOST-C to the OSTβ clade. These two domains were more highly conserved with the OST α- and β-subunits at the structure level than at sequence level. These findings suggested that CsOST comprised the OST α- and β-subunits. CsOST was localized in the oral and ventral suckers and in the mesenchymal tissues abundant around the intestine, vitelline glands, uterus, and testes. This study provides fundamental data for the further understanding of homologues in other flukes. PMID:29702646

The Regulatory Subunit of PKA-I Remains Partially Structured and Undergoes β-Aggregation upon Thermal Denaturation

PubMed Central

Dao, Khanh K.; Pey, Angel L.; Gjerde, Anja Underhaug; Teigen, Knut; Byeon, In-Ja L.; Døskeland, Stein O.; Gronenborn, Angela M.; Martinez, Aurora

2011-01-01

Background The regulatory subunit (R) of cAMP-dependent protein kinase (PKA) is a modular flexible protein that responds with large conformational changes to the binding of the effector cAMP. Considering its highly dynamic nature, the protein is rather stable. We studied the thermal denaturation of full-length RIα and a truncated RIα(92-381) that contains the tandem cyclic nucleotide binding (CNB) domains A and B. Methodology/Principal Findings As revealed by circular dichroism (CD) and differential scanning calorimetry, both RIα proteins contain significant residual structure in the heat-denatured state. As evidenced by CD, the predominantly α-helical spectrum at 25°C with double negative peaks at 209 and 222 nm changes to a spectrum with a single negative peak at 212–216 nm, characteristic of β-structure. A similar α→β transition occurs at higher temperature in the presence of cAMP. Thioflavin T fluorescence and atomic force microscopy studies support the notion that the structural transition is associated with cross-β-intermolecular aggregation and formation of non-fibrillar oligomers. Conclusions/Significance Thermal denaturation of RIα leads to partial loss of native packing with exposure of aggregation-prone motifs, such as the B' helices in the phosphate-binding cassettes of both CNB domains. The topology of the β-sandwiches in these domains favors inter-molecular β-aggregation, which is suppressed in the ligand-bound states of RIα under physiological conditions. Moreover, our results reveal that the CNB domains persist as structural cores through heat-denaturation. PMID:21394209

Matrix metalloproteinase-9 and -2 enhance the ligand sensitivity of photoreceptor cyclic nucleotide-gated channels.

PubMed

Meighan, Peter C; Meighan, Starla E; Rich, Elizabeth D; Brown, R Lane; Varnum, Michael D

2012-01-01

Photoreceptor cyclic nucleotide-gated (CNG) channels are the principal ion channels responsible for transduction of the light-induced change in cGMP concentration into an electrical signal. The ligand sensitivity of photoreceptor CNG channels is subject to regulation by intracellular signaling effectors, including calcium-calmodulin, tyrosine kinases and phosphoinositides. Little is known, however, about regulation of channel activity by modification to extracellular regions of CNG channel subunits. Extracellular proteases MMP9 and -2 are present in the interphotoreceptor matrix adjacent to photoreceptor outer segments. Given that MMPs have been implicated in retinal dysfunction and degeneration, we hypothesized that MMP activity may alter the functional properties of photoreceptor CNG channels. For heterologously expressed rod and cone CNG channels, extracellular exposure to MMPs dramatically increased the apparent affinity for cGMP and the efficacy of cAMP. These changes to ligand sensitivity were not prevented by destabilization of the actin cytoskeleton or by disruption of integrin mediated cell adhesion, but could be attenuated by inhibition of MMP catalytic activity. MMP-mediated gating changes exhibited saturable kinetic properties consistent with enzymatic processing of the CNG channels. In addition, exposure to MMPs decreased the abundance of full-length expressed CNGA3 subunits, with a concomitant increase in putative degradation products. Similar gating effects and apparent proteolysis were observed also for native rod photoreceptor CNG channels. Furthermore, constitutive apparent proteolysis of retinal CNGA1 and retinal MMP9 levels were both elevated in aged mice compared with young mice. Together, these results provide evidence that MMP-mediated proteolysis can regulate the ligand sensitivity of CNG channels.

Matrix metalloproteinase-9 and -2 enhance the ligand sensitivity of photoreceptor cyclic nucleotide-gated channels

PubMed Central

Meighan, Peter C.; Meighan, Starla E.; Rich, Elizabeth D.; Brown, R. Lane; Varnum, Michael D.

2012-01-01

Photoreceptor cyclic nucleotide-gated (CNG) channels are the principal ion channels responsible for transduction of the light-induced change in cGMP concentration into an electrical signal. The ligand sensitivity of photoreceptor CNG channels is subject to regulation by intracellular signaling effectors, including calcium-calmodulin, tyrosine kinases and phosphoinositides. Little is known, however, about regulation of channel activity by modification to extracellular regions of CNG channel subunits. Extracellular proteases MMP9 and -2 are present in the interphotoreceptor matrix adjacent to photoreceptor outer segments. Given that MMPs have been implicated in retinal dysfunction and degeneration, we hypothesized that MMP activity may alter the functional properties of photoreceptor CNG channels. For heterologously expressed rod and cone CNG channels, extracellular exposure to MMPs dramatically increased the apparent affinity for cGMP and the efficacy of cAMP. These changes to ligand sensitivity were not prevented by destabilization of the actin cytoskeleton or by disruption of integrin mediated cell adhesion, but could be attenuated by inhibition of MMP catalytic activity. MMP-mediated gating changes exhibited saturable kinetic properties consistent with enzymatic processing of the CNG channels. In addition, exposure to MMPs decreased the abundance of full-length expressed CNGA3 subunits, with a concomitant increase in putative degradation products. Similar gating effects and apparent proteolysis were observed also for native rod photoreceptor CNG channels. Furthermore, constitutive apparent proteolysis of retinal CNGA1 and retinal MMP9 levels were both elevated in aged mice compared with young mice. Together, these results provide evidence that MMP-mediated proteolysis can regulate the ligand sensitivity of CNG channels. PMID:22699690

Intact Arabidopsis RPB1 functions in stem cell niches maintenance and cell cycling control.

PubMed

Zhang, Qian-Qian; Li, Ying; Fu, Zhao-Ying; Liu, Xun-Biao; Yuan, Kai; Fang, Ying; Liu, Yan; Li, Gang; Zhang, Xian-Sheng; Chong, Kang; Ge, Lei

2018-05-12

Plant meristem activity depends on accurate execution of transcriptional networks required for establishing optimum functioning of stem cell niches. An Arabidopsis mutant card1-1 (constitutive auxin response with DR5:GFP) that encodes a truncated RPB1 (RNA Polymerase II's largest subunit) with shortened C-terminal domain (CTD) was identified. Phosphorylation of the CTD repeats of RPB1 is coupled to transcription in eukaryotes. Here we uncover that the truncated CTD of RPB1 disturbed cell cycling and enlarged the size of shoot and root meristem. The defects in patterning of root stem cell niche in card1-1 indicates that intact CTD of RPB1 is necessary for fine-tuning the specific expression of genes responsible for cell-fate determination. The gene-edited plants with different CTD length of RPB1, created by CRISPR-CAS9 technology, confirmed that both the full length and the DK-rich tail of RPB1's CTD play roles in the accurate transcription of CYCB1;1 encoding a cell-cycle marker protein in root meristem and hence participate in maintaining root meristem size. Our experiment proves that the intact RPB1 CTD is necessary for stem cell niche maintenance, which is mediated by transcriptional regulation of cell cycling genes. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

Every three-hour versus every six-hour oral feeding in preterm infants: a randomised clinical trial.

PubMed

Gray, Megan M; Medoff-Cooper, Barbara; Enlow, Elizabeth M; Mukhopadhyay, Sagori; DeMauro, Sara B

2017-02-01

This trial compares two oral feeding schedules, every three-hour and every six-hour oral feeding attempts, to determine which schedule allows for more rapid attainment of full oral feeding in preterm infants. Infants born at ≤33-week gestation were randomly assigned to receive oral feeding every three hours or every six hours if feeding cues were present. The primary outcome was time to full oral feeding; secondary outcomes include respiratory and apnoea rates, growth and length of stay. A total of 55 infants were recruited. There was no difference between the groups in the primary or secondary outcomes. For preterm infants fed when oral feeding cues are present, an every six-hour schedule did not alter the time to full oral feeding and had no effect on rates of tachypnoea, apnoea or length of hospital stay compared to every three-hour feeding schedule. An every six-hour oral feeding schedule led to only small reductions in number of oral feeding attempts per day. ©2016 Foundation Acta Paediatrica. Published by John Wiley & Sons Ltd.

78 FR 13071 - Guidance for Industry: Implementation of an Acceptable Full-Length and Abbreviated Donor History...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-02-26

...] Guidance for Industry: Implementation of an Acceptable Full- Length and Abbreviated Donor History... Full-Length and Abbreviated Donor History Questionnaires and Accompanying Materials for Use in... full-length and abbreviated donor history questionnaires and accompanying materials, version 1.2 dated...

Principles of 60S ribosomal subunit assembly emerging from recent studies in yeast

PubMed Central

Konikkat, Salini; Woolford, John L.

2017-01-01

Ribosome biogenesis requires the intertwined processes of folding, modification, and processing of ribosomal RNA, together with binding of ribosomal proteins. In eukaryotic cells, ribosome assembly begins in the nucleolus, continues in the nucleoplasm, and is not completed until after nascent particles are exported to the cytoplasm. The efficiency and fidelity of ribosome biogenesis are facilitated by >200 assembly factors and ~76 different small nucleolar RNAs. The pathway is driven forward by numerous remodeling events to rearrange the ribonucleoprotein architecture of pre-ribosomes. Here, we describe principles of ribosome assembly that have emerged from recent studies of biogenesis of the large ribosomal subunit in the yeast Saccharomyces cerevisiae. We describe tools that have empowered investigations of ribosome biogenesis, and then summarize recent discoveries about each of the consecutive steps of subunit assembly. PMID:28062837

Coastal bacterioplankton community diversity along a latitudinal gradient in Latin America by means of V6 tag pyrosequencing.

PubMed

Thompson, Fabiano L; Bruce, Thiago; Gonzalez, Alessandra; Cardoso, Alexander; Clementino, Maysa; Costagliola, Marcela; Hozbor, Constanza; Otero, Ernesto; Piccini, Claudia; Peressutti, Silvia; Schmieder, Robert; Edwards, Robert; Smith, Mathew; Takiyama, Luis Roberto; Vieira, Ricardo; Paranhos, Rodolfo; Artigas, Luis Felipe

2011-02-01

The bacterioplankton diversity of coastal waters along a latitudinal gradient between Puerto Rico and Argentina was analyzed using a total of 134,197 high-quality sequences from the V6 hypervariable region of the small-subunit ribosomal RNA gene (16S rRNA) (mean length of 60 nt). Most of the OTUs were identified into Proteobacteria, Bacteriodetes, Cyanobacteria, and Actinobacteria, corresponding to approx. 80% of the total number of sequences. The number of OTUs corresponding to species varied between 937 and 1946 in the seven locations. Proteobacteria appeared at high frequency in the seven locations. An enrichment of Cyanobacteria was observed in Puerto Rico, whereas an enrichment of Bacteroidetes was detected in the Argentinian shelf and Uruguayan coastal lagoons. The highest number of sequences of Actinobacteria and Acidobacteria were obtained in the Amazon estuary mouth. The rarefaction curves and Good coverage estimator for species diversity suggested a significant coverage, with values ranging between 92 and 97% for Good coverage. Conserved taxa corresponded to aprox. 52% of all sequences. This study suggests that human-contaminated environments may influence bacterioplankton diversity.