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Sample records for distinct subunit species

  1. A distinct holoenzyme organization for two-subunit pyruvate carboxylase

    PubMed Central

    Choi, Philip H.; Jo, Jeanyoung; Lin, Yu-Cheng; Lin, Min-Han; Chou, Chi-Yuan; Dietrich, Lars E. P.; Tong, Liang

    2016-01-01

    Pyruvate carboxylase (PC) has important roles in metabolism and is crucial for virulence for some pathogenic bacteria. PC contains biotin carboxylase (BC), carboxyltransferase (CT) and biotin carboxyl carrier protein (BCCP) components. It is a single-chain enzyme in eukaryotes and most bacteria, and functions as a 500 kD homo-tetramer. In contrast, PC is a two-subunit enzyme in a collection of Gram-negative bacteria, with the α subunit containing the BC and the β subunit the CT and BCCP domains, and it is believed that the holoenzyme has α4β4 stoichiometry. We report here the crystal structures of a two-subunit PC from Methylobacillus flagellatus. Surprisingly, our structures reveal an α2β4 stoichiometry, and the overall architecture of the holoenzyme is strikingly different from that of the homo-tetrameric PCs. Biochemical and mutagenesis studies confirm the stoichiometry and other structural observations. Our functional studies in Pseudomonas aeruginosa show that its two-subunit PC is important for colony morphogenesis. PMID:27708276

  2. Identification of Four Distinct Subunit Types in the Unique 6×6 Hemocyanin of the Centipede Scutigera coleoptrata

    NASA Astrophysics Data System (ADS)

    Gebauer, W.; Markl, J.

    We isolated 6×6 hemocyanin, dissociated it into subunits, and examined it by electron microscopy. The subunits were separated by native polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate PAGE, and crossed immunoelectrophoresis. Single subunits were isolated by gel cutting from native PAGE and identified as hemocyanin by measuring their ultraviolet spectrum. A total of four distinct hemocyanin subunits were identified, and the subunit pattern of the three electrophoresis systems assigned to each other. The relative proportion of subunits a:b:c:d were 2 : 2 :>: 1 as determined by densitometry. Presumably, c and d act as linkers between hexamers.

  3. Distinct Structural Pathways Coordinate the Activation of AMPA Receptor-Auxiliary Subunit Complexes

    PubMed Central

    Dawe, G. Brent; Musgaard, Maria; Aurousseau, Mark R.P.; Nayeem, Naushaba; Green, Tim; Biggin, Philip C.; Bowie, Derek

    2016-01-01

    Summary Neurotransmitter-gated ion channels adopt different gating modes to fine-tune signaling at central synapses. At glutamatergic synapses, high and low activity of AMPA receptors (AMPARs) is observed when pore-forming subunits coassemble with or without auxiliary subunits, respectively. Whether a common structural pathway accounts for these different gating modes is unclear. Here, we identify two structural motifs that determine the time course of AMPAR channel activation. A network of electrostatic interactions at the apex of the AMPAR ligand-binding domain (LBD) is essential for gating by pore-forming subunits, whereas a conserved motif on the lower, D2 lobe of the LBD prolongs channel activity when auxiliary subunits are present. Accordingly, channel activity is almost entirely abolished by elimination of the electrostatic network but restored via auxiliary protein interactions at the D2 lobe. In summary, we propose that activation of native AMPAR complexes is coordinated by distinct structural pathways, favored by the association/dissociation of auxiliary subunits. PMID:26924438

  4. Species-specific Differences among KCNMB3 BK β3 Auxiliary Subunits: Some β3 N-terminal Variants May Be Primate-specific Subunits

    PubMed Central

    Zeng, Xuhui; Xia, Xiao-Ming; Lingle, Christopher J.

    2008-01-01

    The KCNMB3 gene encodes one of a family of four auxiliary β subunits found in the mammalian genome that associate with Slo1 α subunits and regulate BK channel function. In humans, the KCNMB3 gene contains four N-terminal alternative exons that produce four functionally distinct β3 subunits, β3a–d. Three variants, β3a–c, exhibit kinetically distinct inactivation behaviors. Since investigation of the physiological roles of BK auxiliary subunits will depend on studies in rodents, here we have determined the identity and functional properties of mouse β3 variants. Whereas β1, β2, and β4 subunits exhibit 83.2%, 95.3%, and 93.8% identity between mouse and human, the mouse β3 subunit, excluding N-terminal splice variants, shares only 62.8% amino acid identity with its human counterpart. Based on an examination of the mouse genome and screening of mouse cDNA libraries, here we have identified only two N-terminal candidates, β3a and β3b, of the four found in humans. Both human and mouse β3a subunits produce a characteristic use-dependent inactivation. Surprisingly, whereas the hβ3b exhibits rapid inactivation, the putative mβ3b does not inactivate. Furthermore, unlike hβ3, the mβ3 subunit, irrespective of the N terminus, mediates a shift in gating to more negative potentials at a given Ca2+ concentration. The shift in gating gradually is lost following patch excision, suggesting that the gating shift involves some regulatory process dependent on the cytosolic milieu. Examination of additional genomes to assess conservation among splice variants suggests that the putative mβ3b N terminus may not be a true orthologue of the hβ3b N terminus and that both β3c and β3d appear likely to be primate-specific N-terminal variants. These results have three key implications: first, functional properties of homologous β3 subunits may differ among mammalian species; second, the specific physiological roles of homologous β3 subunits may differ among mammalian

  5. Thermostable cross-protective subunit vaccine against Brucella species.

    PubMed

    Cherwonogrodzky, John W; Barabé, Nicole D; Grigat, Michelle L; Lee, William E; Poirier, Robert T; Jager, Scott J; Berger, Bradley J

    2014-12-01

    A subunit vaccine candidate was produced from Brucella suis 145 (biovar 4; expressing both the A antigen of Brucella abortus and the M antigen of Brucella melitensis). The preparation consisted mostly of polysaccharide (PS; >90% [wt/wt]; both cell-associated PS and exo-PS were combined) and a small amount of protein (1 to 3%) with no apparent nucleic acids. Vaccinated mice were protected (these had a statistically significant reduction in bacterial colonization compared to that of unvaccinated controls) when challenged with representative strains of three Brucella species most pathogenic for humans, i.e., B. abortus, B. melitensis, and B. suis. As little as 1 ng of the vaccine, without added adjuvant, protected mice against B. suis 145 infection (5 × 10(5) CFU), and a single injection of 1 μg of this subunit vaccine protected mice from B. suis 145 challenge for at least 14 months. A single immunization induced a serum IgG response to Brucella antigens that remained elevated for up to 9 weeks. The use of heat (i.e., boiling-water bath, autoclaving) in the vaccine preparation showed that it was thermostable. This method also ensured safety and security. The vaccine produced was immunogenic and highly protective against multiple strains of Brucella and represents a promising candidate for further evaluation.

  6. Subunit NDUFV3 is present in two distinct isoforms in mammalian complex I.

    PubMed

    Bridges, Hannah R; Mohammed, Khairunnisa; Harbour, Michael E; Hirst, Judy

    2017-03-01

    Complex I (NADH:ubiquinone oxidoreductase) is the first enzyme of the electron transport chain in mammalian mitochondria. Extensive proteomic and structural analyses of complex I from Bos taurus heart mitochondria have shown it comprises 45 subunits encoded on both the nuclear and mitochondrial genomes; 44 of them are different and one is present in two copies. The bovine heart enzyme has provided a model for studying the composition of complex I in other mammalian species, including humans, but the possibility of additional subunits or isoforms in other species or tissues has not been explored. Here, we describe characterization of the complexes I purified from five rat tissues and from a rat hepatoma cell line. We identify a~50kDa isoform of subunit NDUFV3, for which the canonical isoform is only ~10kDa in size. We combine LC-MS and MALDI-TOF mass spectrometry data from two different purification methods (chromatography and immuno-purification) with information from blue native PAGE analyses to show the long isoform is present in the mature complex, but at substoichiometric levels. It is also present in complex I in cultured human cells. We describe evidence that the long isoform is more abundant in both the mitochondria and purified complexes from brain (relative to in heart, liver, kidney and skeletal muscle) and more abundant still in complex I in cultured cells. We propose that the long 50kDa isoform competes with its canonical 10kDa counterpart for a common binding site on the flavoprotein domain of complex I.

  7. Dictyostelium discoideum protein phosphatase-1 catalytic subunit exhibits distinct biochemical properties.

    PubMed Central

    Andrioli, Luiz P M; Zaini, Paulo A; Viviani, Wladia; Da Silva, Aline M

    2003-01-01

    Protein phosphatase-1 (PP1) is expressed ubiquitously and is involved in many eukaryotic cellular functions, although PP1 enzyme activity could not be detected in the social amoeba Dictyostelium discoideum cell extracts. In the present paper, we show that D. discoideum has a single copy gene that codes for the catalytic subunit of PP1 (DdPP1c). DdPP1c is expressed throughout the D. discoideum life cycle with constant levels of mRNA, and its protein and amino acid sequence show a mean identity of 80% with other PP1c enzymes. However, it has a distinctive difference: the substitution of a phenylalanine residue (Phe(269) in the DdPP1c) for a highly conserved cysteine residue (Cys(273) in rabbit PP1c) in a region that was shown to have a critical role in the interaction of rabbit PP1c with toxin inhibitors. Wild-type DdPP1c and an engineered mutant form in which Phe(269) was replaced by a cysteine residue were expressed in Escherichia coli. Both recombinant activities were similarly inhibited by okadaic acid, tautomycin and microcystin. However, the Phe(269)-->Cys mutation resulted in a large increase in enzyme activity towards phosphorylase a and a higher sensitivity to calyculin A. These results, together with the molecular modelling of DdPP1c structure, indicate that the Phe(269) residue, which occurs naturally in D. discoideum, confers distinct biochemical properties on this enzyme. PMID:12737629

  8. Two subunits of the 55 K porcine zona pellucida glycoprotein family are immunologically distinct

    SciTech Connect

    Subramanian, M.G.; Yurewicz, E.C.; Sacco, A.G.

    1986-03-01

    The 55K glycoprotein family (ZP3) of the porcine zona pellucida is comprised of two subunits of 46 K and 45 K which can be resolved by endo-..beta..-galactosidase digestion of ZP3 followed by reversed phase HPLC on Vydac C4 resin. Gel electrophoresis revealed that the 46 K component (EBDG..cap alpha..) is approx. 95% pure and the 45 K component (EBGD..beta..) is 100% pure. In the present study, these two subunits were evaluated immunologically by RIA. Under similar reaction protocols (chloramine-T iodination procedure) comparable specific activities were obtained for EBGD..cap alpha.. (33.06 +/- 7.5 ..mu..ci/..mu..gm), EBGD..beta.. (30.45 +/- 1.6) and ZP3 (26.3 +/- 1.3). Antibody (Ab) titration studies revealed that EBGD..cap alpha.. and ..beta.. are potent immunogens and /sup 125/I-EBGD..cap alpha.. showed minimal cross reactivity to EBGD..beta..-Ab (8% bound at 1:500 dilution), whereas, /sup 125/I-EBGD..beta.. showed a greater degree of cross reactivity to EBGD..cap alpha..-Ab (23% bound at 1:500 dilution). Maximum binding for the two labeled antigens against homologous Abs (1:500) was > 60%. Dose response studies revealed that in the /sup 125/I-EBGD..cap alpha.. vs EBGD..cap alpha.. -Ab system, the 50% intercept was 3.25 +/- 0.32 ng for EBGD..cap alpha.. and 472.43 +/- 30.26 ng for EBGD..beta.. (p < 0.01), whereas, in the /sup 125/I-EBGD..beta.. vs EBGD..beta..-Ab system the 50% intercept was 3.51 +/- 0.58 for EBGD..beta.. and 166.77 +/- 49.20 for EBGD..cap alpha.. (p < 0.01). No significant differences were observed in the slopes of the dose response curves. It is concluded that the two subunits of ZP3 possess distinct immunologic characteristics as evaluated by RIA.

  9. Distribution of Kv3.3 potassium channel subunits in distinct neuronal populations of mouse brain.

    PubMed

    Chang, Su Ying; Zagha, Edward; Kwon, Elaine S; Ozaita, Andres; Bobik, Marketta; Martone, Maryann E; Ellisman, Mark H; Heintz, Nathaniel; Rudy, Bernardo

    2007-06-20

    Kv3.3 proteins are pore-forming subunits of voltage-dependent potassium channels, and mutations in the gene encoding for Kv3.3 have recently been linked to human disease, spinocerebellar ataxia 13, with cerebellar and extracerebellar symptoms. To understand better the functions of Kv3.3 subunits in brain, we developed highly specific antibodies to Kv3.3 and analyzed immunoreactivity throughout mouse brain. We found that Kv3.3 subunits are widely expressed, present in important forebrain structures but particularly prominent in brainstem and cerebellum. In forebrain and midbrain, Kv3.3 expression was often found colocalized with parvalbumin and other Kv3 subunits in inhibitory neurons. In brainstem, Kv3.3 was strongly expressed in auditory and other sensory nuclei. In cerebellar cortex, Kv3.3 expression was found in Purkinje and granule cells. Kv3.3 proteins were observed in axons, terminals, somas, and, unlike other Kv3 proteins, also in distal dendrites, although precise subcellular localization depended on cell type. For example, hippocampal dentate granule cells expressed Kv3.3 subunits specifically in their mossy fiber axons, whereas Purkinje cells of the cerebellar cortex strongly expressed Kv3.3 subunits in axons, somas, and proximal and distal, but not second- and third-order, dendrites. Expression in Purkinje cell dendrites was confirmed by immunoelectron microscopy. Kv3 channels have been demonstrated to rapidly repolarize action potentials and support high-frequency firing in various neuronal populations. In this study, we identified additional populations and subcellular compartments that are likely to sustain high-frequency firing because of the expression of Kv3.3 and other Kv3 subunits.

  10. PAFAH Ib Phospholipase A2 Subunits Have Distinct Roles in Maintaining Golgi Structure and Function

    PubMed Central

    Bechler, Marie E.; Brown, William J.

    2013-01-01

    Recent studies showed that the phospholipase subunits of Platelet Activating Factor Acetylhydrolase (PAFAH) Ib, α1 and α2, partially localize to the Golgi complex and regulate its structure and function. Using siRNA knockdown of individual subunits, we find that α1 and α2 perform overlapping and unique roles in regulating Golgi morphology, assembly, and secretory cargo trafficking. Knockdown of either α1 or α2 reduced secretion of soluble proteins, but neither single knockdown reduced secretion to the same degree as knockdown of both. Knockdown of α1 or α2 inhibited reassembly of an intact Golgi complex to the same extent as knockdown of both. Transport of VSV-G was slowed but at different steps in the secretory pathway: reduction of α1 slowed trans Golgi network to plasma membrane transport, whereas α2 loss reduced endoplasmic reticulum to Golgi trafficking. Similarly, knockdown of either subunit alone disrupted the Golgi complex but with markedly different morphologies. Finally, knockdown of α1, or double knockdown of α1 and α2, resulted in a significant redistribution of kinase dead protein kinase D from the Golgi to the plasma membrane, whereas loss of α2 alone had no such effect. These studies reveal an unexpected complexity in the regulation of Golgi structure and function by PAFAH Ib. PMID:23262398

  11. Distinct contributions of MSL complex subunits to the transcriptional enhancement responsible for dosage compensation in Drosophila.

    PubMed

    Dunlap, David; Yokoyama, Ruth; Ling, Huiping; Sun, He-Ying; McGill, Kerry; Cugusi, Simona; Lucchesi, John C

    2012-12-01

    The regulatory mechanism of dosage compensation is the paramount example of epigenetic regulation at the chromosomal level. In Drosophila, this mechanism, designed to compensate for the difference in the dosage of X-linked genes between the sexes, depends on the MSL complex that enhances the transcription of the single dose of these genes in males. We have investigated the function of various subunits of the complex in mediating dosage compensation. Our results confirm that the highly enriched specific acetylation of histone H4 at lysine 16 of compensated genes by the histone acetyl transferase subunit MOF induces a more disorganized state of their chromatin. We have determined that the association of the MSL complex reduces the level of negative supercoiling of the deoxyribonucleic acid of compensated genes, and we have defined the role that the other subunits of the complex play in this topological modification. Lastly, we have analyzed the potential contribution of ISWI-containing remodeling complexes to the architecture of compensated chromatin, and we suggest a role for this remodeling factor in dosage compensation.

  12. Distinct Elements in the Proteasomal β5 Subunit Propeptide Required for Autocatalytic Processing and Proteasome Assembly*

    PubMed Central

    Li, Xia; Li, Yanjie; Arendt, Cassandra S.; Hochstrasser, Mark

    2016-01-01

    Eukaryotic 20S proteasome assembly remains poorly understood. The subunits stack into four heteroheptameric rings; three inner-ring subunits (β1, β2, and β5) bear the protease catalytic residues and are synthesized with N-terminal propeptides. These propeptides are removed autocatalytically late in assembly. In Saccharomyces cerevisiae, β5 (Doa3/Pre2) has a 75-residue propeptide, β5pro, that is essential for proteasome assembly and can work in trans. We show that deletion of the poorly conserved N-terminal half of the β5 propeptide nonetheless causes substantial defects in proteasome maturation. Sequences closer to the cleavage site have critical but redundant roles in both assembly and self-cleavage. A conserved histidine two residues upstream of the autocleavage site strongly promotes processing. Surprisingly, although β5pro is functionally linked to the Ump1 assembly factor, trans-expressed β5pro associates only weakly with Ump1-containing precursors. Several genes were identified as dosage suppressors of trans-expressed β5pro mutants; the strongest encoded the β7 proteasome subunit. Previous data suggested that β7 and β5pro have overlapping roles in bringing together two half-proteasomes, but the timing of β7 addition relative to half-mer joining was unclear. Here we report conditions where dimerization lags behind β7 incorporation into the half-mer. Our results suggest that β7 insertion precedes half-mer dimerization, and the β7 tail and β5 propeptide have unequal roles in half-mer joining. PMID:26627836

  13. Distinct Elements in the Proteasomal β5 Subunit Propeptide Required for Autocatalytic Processing and Proteasome Assembly.

    PubMed

    Li, Xia; Li, Yanjie; Arendt, Cassandra S; Hochstrasser, Mark

    2016-01-22

    Eukaryotic 20S proteasome assembly remains poorly understood. The subunits stack into four heteroheptameric rings; three inner-ring subunits (β1, β2, and β5) bear the protease catalytic residues and are synthesized with N-terminal propeptides. These propeptides are removed autocatalytically late in assembly. In Saccharomyces cerevisiae, β5 (Doa3/Pre2) has a 75-residue propeptide, β5pro, that is essential for proteasome assembly and can work in trans. We show that deletion of the poorly conserved N-terminal half of the β5 propeptide nonetheless causes substantial defects in proteasome maturation. Sequences closer to the cleavage site have critical but redundant roles in both assembly and self-cleavage. A conserved histidine two residues upstream of the autocleavage site strongly promotes processing. Surprisingly, although β5pro is functionally linked to the Ump1 assembly factor, trans-expressed β5pro associates only weakly with Ump1-containing precursors. Several genes were identified as dosage suppressors of trans-expressed β5pro mutants; the strongest encoded the β7 proteasome subunit. Previous data suggested that β7 and β5pro have overlapping roles in bringing together two half-proteasomes, but the timing of β7 addition relative to half-mer joining was unclear. Here we report conditions where dimerization lags behind β7 incorporation into the half-mer. Our results suggest that β7 insertion precedes half-mer dimerization, and the β7 tail and β5 propeptide have unequal roles in half-mer joining.

  14. Diverse gene-silencing mechanisms with distinct requirements for RNA polymerase subunits in Zea mays.

    PubMed

    Sloan, Amy E; Sidorenko, Lyudmila; McGinnis, Karen M

    2014-11-01

    In Zea mays, transcriptional regulation of the b1 (booster1) gene requires a distal enhancer and MEDIATOR OF PARAMUTATION1 (MOP1), MOP2, and MOP3 proteins orthologous to Arabidopsis components of the RNA-dependent DNA methylation pathway. We compared the genetic requirements for MOP1, MOP2, and MOP3 for endogenous gene silencing by two hairpin transgenes with inverted repeats of the a1 (anthocyaninless1) gene promoter (a1pIR) and the b1 gene enhancer (b1IR), respectively. The a1pIR transgene induced silencing of endogenous A1 in mop1-1 and mop3-1, but not in Mop2-1 homozygous plants. This finding suggests that transgene-derived small interfering RNAs (siRNAs) circumvented the requirement for MOP1, a predicted RNA-dependent RNA polymerase, and MOP3, the predicted largest subunit of RNA polymerase IV (Pol IV). Because the Arabidopsis protein orthologous to MOP2 is the second largest subunit of Pol IV and V, our results may indicate that hairpin-induced siRNAs cannot bypass the requirement for the predicted scaffolding activity of Pol V. In contrast to a1pIR, the b1IR transgene silenced endogenous B1 in all three homozygous mutant genotypes--mop1-1, Mop2-1, and mop3-1--suggesting that transgene mediated b1 silencing did not involve MOP2-containing Pol V complexes. Based on the combined results for a1, b1, and three previously described loci, we propose a speculative hypothesis of locus-specific deployment of Pol II, MOP2-containing Pol V, or alternative versions of Pol V with second largest subunits other than MOP2 to explain the mechanistic differences in silencing at specific loci, including one example associated with paramutation.

  15. A distinct three-helix centipede toxin SSD609 inhibits Iks channels by interacting with the KCNE1 auxiliary subunit

    PubMed Central

    Sun, Peibei; Wu, Fangming; Wen, Ming; Yang, Xingwang; Wang, Chenyang; Li, Yiming; He, Shufang; Zhang, Longhua; Zhang, Yun; Tian, Changlin

    2015-01-01

    KCNE1 is a single-span transmembrane auxiliary protein that modulates the voltage-gated potassium channel KCNQ1. The KCNQ1/KCNE1 complex in cardiomyocytes exhibited slow activated potassium (Iks) currents. Recently, a novel 47-residue polypeptide toxin SSD609 was purified from Scolopendra subspinipes dehaani venom and showed Iks current inhibition. Here, chemically synthesized SSD609 was shown to exert Iks inhibition in extracted guinea pig cardiomyocytes and KCNQ1/KCNE1 current attenuation in CHO cells. The K+ current attenuation of SSD609 showed decent selectivity among different auxiliary subunits. Solution nuclear magnetic resonance analysis of SSD609 revealed a distinctive three-helix conformation that was stabilized by a new disulfide bonding pattern as well as segregated surface charge distribution. Structure-activity studies demonstrated that negatively charged Glu19 in the amphipathic extracellular helix of KCNE1 was the key residue that interacted with SSD609. The distinctive three-helix centipede toxin SSD609 is known to be the first polypeptide toxin acting on channel auxiliary subunit KCNE1, which suggests a new type of pharmacological regulation for ion channels in cardiomyocytes. PMID:26307551

  16. Group I introns in small subunit ribosomal DNA of several Phaeosphaeria species

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In a study of small subunit ribosomal RNA (SSU-rRNA) gene sequences in Phaeosphaeria species, group I introns were found in 9 of 10 P. avenaria f.sp. avenaria (Paa) isolates, 1 of 2 Phaeosphaeria sp. (P-rye) isolates from Polish rye (Sn48-1), 1 Phaeosphaeria sp. from dallis grass (P-dg) (S-93-48) an...

  17. Separation of distinct photoexcitation species in femtosecond transient absorption microscopy

    SciTech Connect

    Xiao, Kai; Ma, Ying -Zhong; Simpson, Mary Jane; Doughty, Benjamin; Yang, Bing

    2016-02-03

    Femtosecond transient absorption microscopy is a novel chemical imaging capability with simultaneous high spatial and temporal resolution. Although several powerful data analysis approaches have been developed and successfully applied to separate distinct chemical species in such images, the application of such analysis to distinguish different photoexcited species is rare. In this paper, we demonstrate a combined approach based on phasor and linear decomposition analysis on a microscopic level that allows us to separate the contributions of both the excitons and free charge carriers in the observed transient absorption response of a composite organometallic lead halide perovskite film. We found spatial regions where the transient absorption response was predominately a result of excitons and others where it was predominately due to charge carriers, and regions consisting of signals from both contributors. Lastly, quantitative decomposition of the transient absorption response curves further enabled us to reveal the relative contribution of each photoexcitation to the measured response at spatially resolved locations in the film.

  18. Molecular evidence for species-level distinctions in clouded leopards.

    PubMed

    Buckley-Beason, Valerie A; Johnson, Warren E; Nash, Willliam G; Stanyon, Roscoe; Menninger, Joan C; Driscoll, Carlos A; Howard, JoGayle; Bush, Mitch; Page, John E; Roelke, Melody E; Stone, Gary; Martelli, Paolo P; Wen, Ci; Ling, Lin; Duraisingam, Ratna K; Lam, Phan V; O'Brien, Stephen J

    2006-12-05

    Among the 37 living species of Felidae, the clouded leopard (Neofelis nebulosa) is generally classified as a monotypic genus basal to the Panthera lineage of great cats. This secretive, mid-sized (16-23 kg) carnivore, now severely endangered, is traditionally subdivided into four southeast Asian subspecies (Figure 1A). We used molecular genetic methods to re-evaluate subspecies partitions and to quantify patterns of population genetic variation among 109 clouded leopards of known geographic origin (Figure 1A, Tables S1 ans S2 in the Supplemental Data available online). We found strong phylogeographic monophyly and large genetic distances between N. n. nebulosa (mainland) and N. n. diardi (Borneo; n = 3 individuals) with mtDNA (771 bp), nuclear DNA (3100 bp), and 51 microsatellite loci. Thirty-six fixed mitochondrial and nuclear nucleotide differences and 20 microsatellite loci with nonoverlapping allele-size ranges distinguished N. n. nebulosa from N. n. diardi. Along with fixed subspecies-specific chromosomal differences, this degree of differentiation is equivalent to, or greater than, comparable measures among five recognized Panthera species (lion, tiger, leopard, jaguar, and snow leopard). These distinctions increase the urgency of clouded leopard conservation efforts, and if affirmed by morphological analysis and wider sampling of N. n. diardi in Borneo and Sumatra, would support reclassification of N. n. diardi as a new species (Neofelis diardi).

  19. Distinct functions of the RNA polymerase σ subunit region 3.2 in RNA priming and promoter escape

    PubMed Central

    Pupov, Danil; Kuzin, Ivan; Bass, Irina; Kulbachinskiy, Andrey

    2014-01-01

    The σ subunit of bacterial RNA polymerase (RNAP) has been implicated in all steps of transcription initiation, including promoter recognition and opening, priming of RNA synthesis, abortive initiation and promoter escape. The post-promoter-recognition σ functions were proposed to depend on its conserved region σ3.2 that directly contacts promoter DNA immediately upstream of the RNAP active centre and occupies the RNA exit path. Analysis of the transcription effects of substitutions and deletions in this region in Escherichia coli σ70 subunit, performed in this work, suggests that (i) individual residues in the σ3.2 finger collectively contribute to RNA priming by RNAP, likely by the positioning of the template DNA strand in the active centre, but are not critical to promoter escape; (ii) the physical presence of σ3.2 in the RNA exit channel is important for promoter escape; (iii) σ3.2 promotes σ dissociation during initiation and suppresses σ-dependent promoter-proximal pausing; (iv) σ3.2 contributes to allosteric inhibition of the initiating NTP binding by rifamycins. Thus, region σ3.2 performs distinct functions in transcription initiation and its inhibition by antibiotics. The B-reader element of eukaryotic factor TFIIB likely plays similar roles in RNAPII transcription, revealing common principles in transcription initiation in various domains of life. PMID:24452800

  20. Separation of distinct photoexcitation species in femtosecond transient absorption microscopy

    DOE PAGES

    Xiao, Kai; Ma, Ying -Zhong; Simpson, Mary Jane; ...

    2016-02-03

    Femtosecond transient absorption microscopy is a novel chemical imaging capability with simultaneous high spatial and temporal resolution. Although several powerful data analysis approaches have been developed and successfully applied to separate distinct chemical species in such images, the application of such analysis to distinguish different photoexcited species is rare. In this paper, we demonstrate a combined approach based on phasor and linear decomposition analysis on a microscopic level that allows us to separate the contributions of both the excitons and free charge carriers in the observed transient absorption response of a composite organometallic lead halide perovskite film. We found spatialmore » regions where the transient absorption response was predominately a result of excitons and others where it was predominately due to charge carriers, and regions consisting of signals from both contributors. Lastly, quantitative decomposition of the transient absorption response curves further enabled us to reveal the relative contribution of each photoexcitation to the measured response at spatially resolved locations in the film.« less

  1. Lungfish aestivating activities are locked in distinct encephalic γ-aminobutyric acid type A receptor α subunits.

    PubMed

    Giusi, Giuseppina; Crudo, Michele; Di Vito, Anna; Facciolo, Rosa Maria; Garofalo, Filippo; Chew, Shit Fun; Ip, Yuen Kwong; Canonaco, Marcello

    2011-03-01

    Ammonia in dipnoans plays a crucial role on neuronal homeostasis, especially for those brain areas that maintain torpor and awakening states in equilibrium. In the present study, specific α subunits of the major neuroreceptor inhibitory complex (GABA(A) R), which predominated during some phases of aestivation of the lungfish Protopterus annectens, turned out to be key adaptive factors of this species. From the isolation, for the first time, of the encoding sequence for GABA(A) R α₁, α₄ , and α₅ subunits in Protopterus annectens, qPCR and in situ hybridization levels of α₄ transcript in thalamic (P < 0.001) and mesencephalic (P < 0.01) areas proved to be significantly higher during long aestivating maintenance states. Very evident α₅ mRNA levels were detected in diencephalon during short inductive aestivating states, whereas an α₄ /α₁ turnover characterized the arousal state. Contextually, the recovery of physiological activities appeared to be tightly related to an evident up-regulation of α₁ transcripts in telencephalic and cerebellar sites. Surprisingly, TUNEL and amino cupric silver methods corroborated apoptotic and neurodegenerative cellular events, respectively, above all in telencephalon and cerebellum of lungfish exposed to long maintenance aestivating conditions. Overall, these results tend to underlie a novel GABAergic-related ON/OFF molecular switch operating during aestivation of the lungfish, which might have a bearing on sleeping disorders.

  2. Phylogenetic relationships between Vorticella convallaria and other species inferred from small subunit rRNA gene sequences.

    PubMed

    Itabashi, Takeshi; Mikami, Kazuyuki; Fang, Jie; Asai, Hiroshi

    2002-08-01

    Vorticellid ciliates generally dwell in freshwater. In nature, the species have up until now been identified by comparison with previous descriptions. It is difficult to identify between species of the genus Vorticella, because the morphological markers of vorticellid ciliates described in reports are limited and variable. Unfortunately, culturing them has only succeeded with certain species such as Vorticella convallaria, but many others have been impossible to culture. To find out whether the sequence of a small subunit rRNA gene was an appropriate marker to identify vorticellid ciliates, the gene was aligned and compared. Finding a new convenient method will contribute to research on vorticellid ciliates. In strains of V. convallaria, classified morphologically, some varieties of the SSrRNA gene sequences were recognized, but there were large variations within the same species. According to the phylogenetic tree, these strains are closely related. However, the difference was not as big as between Vorticella and Carchesium. In addition, Carchesium constructed a distinct clade from the genus Vorticella and Epistylis. These results show the possibility that the SSrRNA gene is one of the important markers to identify species of Vorticella. This study is first to approach and clarify the complicated taxa in the genus Vorticella.

  3. Analysis of Exocyst Subunit EXO70 Family Reveals Distinct Membrane Polar Domains in Tobacco Pollen Tubes1[OPEN

    PubMed Central

    Šantrůček, Jiří; Vukašinović, Nemanja

    2017-01-01

    The vesicle-tethering complex exocyst is one of the crucial cell polarity regulators. The EXO70 subunit is required for the targeting of the complex and is represented by many isoforms in angiosperm plant cells. This diversity could be partly responsible for the establishment and maintenance of membrane domains with different composition. To address this hypothesis, we employed the growing pollen tube, a well-established cell polarity model system, and performed large-scale expression, localization, and functional analysis of tobacco (Nicotiana tabacum) EXO70 isoforms. Various isoforms localized to different regions of the pollen tube plasma membrane, apical vesicle-rich inverted cone region, nucleus, and cytoplasm. The overexpression of major pollen-expressed EXO70 isoforms resulted in growth arrest and characteristic phenotypic deviations of tip swelling and apical invaginations. NtEXO70A1a and NtEXO70B1 occupied two distinct and mutually exclusive plasma membrane domains. Both isoforms partly colocalized with the exocyst subunit NtSEC3a at the plasma membrane, possibly forming different exocyst complex subpopulations. NtEXO70A1a localized to the small area previously characterized as the site of exocytosis in the tobacco pollen tube, while NtEXO70B1 surprisingly colocalized with the zone of clathrin-mediated endocytosis. Both NtEXO70A1a and NtEXO70B1 colocalized to different degrees with markers for the anionic signaling phospholipids phosphatidylinositol 4,5-bisphosphate and phosphatidic acid. In contrast, members of the EXO70 C class, which are specifically expressed in tip-growing cells, exhibited exocytosis-related functional effects in pollen tubes despite the absence of apparent plasma membrane localization. Taken together, our data support the existence of multiple membrane-trafficking domains regulated by different EXO70-containing exocyst complexes within a single cell. PMID:28082718

  4. γ-aminobutyric acid type A α4, β2, and δ subunits assemble to produce more than one functionally distinct receptor type.

    PubMed

    Eaton, Megan M; Bracamontes, John; Shu, Hong-Jin; Li, Ping; Mennerick, Steven; Steinbach, Joe Henry; Akk, Gustav

    2014-12-01

    Native γ-aminobutyric acid (GABA)A receptors consisting of α4, β1-3, and δ subunits mediate responses to the low, tonic concentration of GABA present in the extracellular milieu. Previous studies on heterologously expressed α4βδ receptors have shown a large degree of variability in functional properties, including sensitivity to the transmitter. We studied properties of α4β2δ receptors employing free subunits and concatemeric constructs, expressed in Xenopus oocytes, HEK 293 cells, and cultured hippocampal neurons. The expression system had a strong effect on the properties of receptors containing free subunits. The midpoint of GABA activation curve was 10 nM for receptors in oocytes versus 2300 nM in HEK cells. Receptors activated by the steroid alfaxalone had an estimated maximal open probability of 0.6 in oocytes and 0.01 in HEK cells. Irrespective of the expression system, receptors resulting from combining the tandem construct β2-δ and a free α4 subunit exhibited large steroid responses. We propose that free α4, β2, and δ subunits assemble in different configurations with distinct properties in oocytes and HEK cells, and that subunit linkage can overcome the expression system-dependent preferential assembly of free subunits. Hippocampal neurons transfected with α4 and the picrotoxin-resistant δ(T269Y) subunit showed large responses to alfaxalone in the presence of picrotoxin, suggesting that α4βδ receptors may assemble in a similar configuration in neurons and oocytes.

  5. Distinct Expression Patterns of Glycoprotein Hormone Subunits in the Lophotrochozoan Aplysia: Implications for the Evolution of Neuroendocrine Systems in Animals

    PubMed Central

    Plachetzki, David; Donelly, Evonne; Gunaratne, Dinuka; Bobkova, Yelena; Jacobson, John; Kohn, Andrea B.; Moroz, Leonid L.

    2012-01-01

    Glycoprotein hormones (GPHs) comprise a group of signaling molecules critical for major metabolic and reproductive functions. In vertebrates they include chorionic gonadotropin, LH, FSH, and TSH. The active hormones are characterized by heterodimerization between a common α and hormone-specific β subunit, which activate leucine-rich repeat-containing G protein coupled receptors. To date, genes referred to as GPHα2 and GPHβ5 have been the only glycoprotein hormone subunits identified in invertebrates, suggesting that other GPHα and GPHβ subunits diversified during vertebrate evolution. Still the functions of GPHα2 and GPHβ5 remain largely unknown for both vertebrates and invertebrates. To further understand the evolution and putative function of these subunits, we cloned and analyzed phylogenetically two glycoprotein subunits, AcaGPHα and AcaGPHβ, from the sea hare Aplysia californica. Model based three-dimensional predictions of AcaGPHβ confirm the presence of a complete cysteine knot, two hairpin loops, and a long loop. As in the human GPHβ5 subunit the seatbelt structure is absent in AcaGPHβ. We also found that AcaGPHα and AcaGPHβ subunits are expressed in larval stages of Aplysia, and we present a detailed expression map of the subunits in the adult central nervous system using in situ hybridizations. Both subunits are expressed in subpopulations of pleural and buccal mechanosensory neurons, suggesting a neuronal modulatory function of these subunits in Aplysia. Furthermore it supports the model of a relatively diffuse neuroendocrine-like system in molluscs, where specific primary sensory neurons release peptides extrasynaptically (paracrine secretion). This is in contrast to vertebrates and insects, in which releasing and stimulating factor from centralized sensory regions of the central nervous system ultimately regulate hormone release in peripheral glands. PMID:22977258

  6. Distinct expression patterns of glycoprotein hormone subunits in the lophotrochozoan Aplysia: implications for the evolution of neuroendocrine systems in animals.

    PubMed

    Heyland, Andreas; Plachetzki, David; Donelly, Evonne; Gunaratne, Dinuka; Bobkova, Yelena; Jacobson, John; Kohn, Andrea B; Moroz, Leonid L

    2012-11-01

    Glycoprotein hormones (GPHs) comprise a group of signaling molecules critical for major metabolic and reproductive functions. In vertebrates they include chorionic gonadotropin, LH, FSH, and TSH. The active hormones are characterized by heterodimerization between a common α and hormone-specific β subunit, which activate leucine-rich repeat-containing G protein coupled receptors. To date, genes referred to as GPHα2 and GPHβ5 have been the only glycoprotein hormone subunits identified in invertebrates, suggesting that other GPHα and GPHβ subunits diversified during vertebrate evolution. Still the functions of GPHα2 and GPHβ5 remain largely unknown for both vertebrates and invertebrates. To further understand the evolution and putative function of these subunits, we cloned and analyzed phylogenetically two glycoprotein subunits, AcaGPHα and AcaGPHβ, from the sea hare Aplysia californica. Model based three-dimensional predictions of AcaGPHβ confirm the presence of a complete cysteine knot, two hairpin loops, and a long loop. As in the human GPHβ5 subunit the seatbelt structure is absent in AcaGPHβ. We also found that AcaGPHα and AcaGPHβ subunits are expressed in larval stages of Aplysia, and we present a detailed expression map of the subunits in the adult central nervous system using in situ hybridizations. Both subunits are expressed in subpopulations of pleural and buccal mechanosensory neurons, suggesting a neuronal modulatory function of these subunits in Aplysia. Furthermore it supports the model of a relatively diffuse neuroendocrine-like system in molluscs, where specific primary sensory neurons release peptides extrasynaptically (paracrine secretion). This is in contrast to vertebrates and insects, in which releasing and stimulating factor from centralized sensory regions of the central nervous system ultimately regulate hormone release in peripheral glands.

  7. Reassortment and mutation of the avian influenza virus polymerase PA subunit overcome species barriers.

    PubMed

    Mehle, Andrew; Dugan, Vivien G; Taubenberger, Jeffery K; Doudna, Jennifer A

    2012-02-01

    The emergence of new pandemic influenza A viruses requires overcoming barriers to cross-species transmission as viruses move from animal reservoirs into humans. This complicated process is driven by both individual gene mutations and genome reassortments. The viral polymerase complex, composed of the proteins PB1, PB2, and PA, is a major factor controlling host adaptation, and reassortment events involving polymerase gene segments occurred with past pandemic viruses. Here we investigate the ability of polymerase reassortment to restore the activity of an avian influenza virus polymerase that is normally impaired in human cells. Our data show that the substitution of human-origin PA subunits into an avian influenza virus polymerase alleviates restriction in human cells and increases polymerase activity in vitro. Reassortants with 2009 pandemic H1N1 PA proteins were the most active. Mutational analyses demonstrated that the majority of the enhancing activity in human PA results from a threonine-to-serine change at residue 552. Reassortant viruses with avian polymerases and human PA subunits, or simply the T552S mutation, displayed faster replication kinetics in culture and increased pathogenicity in mice compared to those containing a wholly avian polymerase complex. Thus, the acquisition of a human PA subunit, or the signature T552S mutation, is a potential mechanism to overcome the species-specific restriction of avian polymerases and increase virus replication. Our data suggest that the human, avian, swine, and 2009 H1N1-like viruses that are currently cocirculating in pig populations set the stage for PA reassortments with the potential to generate novel viruses that could possess expanded tropism and enhanced pathogenicity.

  8. Distinct Contributions of T1R2 and T1R3 Taste Receptor Subunits to the Detection of Sweet Stimuli

    SciTech Connect

    Nie,Y.; Vigues, S.; Hobbs, J.; Conn, G.; Munger, S.

    2005-01-01

    The molecular mechanisms by which G protein-coupled receptor (GPCR)-type chemosensory receptors of animals selectively interact with their cognate ligands remain poorly understood. There is growing evidence that many chemosensory receptors exist in multimeric complexes, though little is known about the relative contributions of individual subunits to receptor functions. This study showed that each of the two subunits in the mammalian heteromeric T1R2:T1R3 sweet taste receptor binds sweet stimuli, though with distinct affinities and conformational changes. Furthermore, ligand affinities for T1R3 are drastically reduced by the introduction of a single amino acid change associated with decreased sweet taste sensitivity in mice. Thus, individual T1R subunits increase the receptive range of the sweet taste receptor, offering a functional mechanism for phenotypic variations in sweet taste.

  9. 76 FR 14883 - Endangered and Threatened Species; Proposed Threatened Status for Distinct Population Segments of...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-18

    ... Species; Proposed Threatened Status for Distinct Population Segments of the Bearded Seal AGENCY: National... Beringia and Okhotsk Distinct Population Segments (DPSs) of the bearded seal (Erignathus barbatus) as... proposed rule to list the Beringia and Okhotsk Distinct Population Segments (DPSs) of the bearded seal...

  10. Co-existing grass species have distinctive arbuscular mycorrhizal communities.

    PubMed

    Vandenkoornhuyse, P; Ridgway, K P; Watson, I J; Fitter, A H; Young, J P W

    2003-11-01

    Arbuscular mycorrhizal (AM) fungi are biotrophic symbionts colonizing the majority of land plants, and are of major importance in plant nutrient supply. Their diversity is suggested to be an important determinant of plant community structure, but the influence of host-plant and environmental factors on AM fungal community in plant roots is poorly documented. Using the terminal restriction fragment length polymorphism (T-RFLP) strategy, the diversity of AM fungi was assessed in 89 roots of three grass species (Agrostis capillaris, Festuca rubra, Poa pratensis) that co-occurred in the same plots of a field experiment. The impact of different soil amendments (nitrogen, lime, nitrogen and lime) and insecticide application on AM fungal community was also studied. The level of diversity found in AM fungal communities using the T-RFLP strategy was consistent with previous studies based on clone libraries. Our results clearly confirm that an AM fungal host-plant preference exists, even between different grass species. AM communities colonizing A. capillaris were statistically different from the others (P < 0.05). Although grass species evenness changed in amended soils, AM fungal community composition in roots of a given grass species remained stable. Conversely, in plots where insecticide was applied, we found higher AM fungal diversity and, in F. rubra roots, a statistically different AM fungal community.

  11. Retention of duplicated ITAM-containing transmembrane signaling subunits in the tetraploid amphibian species Xenopus laevis.

    PubMed

    Guselnikov, S V; Grayfer, L; De Jesús Andino, F; Rogozin, I B; Robert, J; Taranin, A V

    2015-11-01

    The ITAM-bearing transmembrane signaling subunits (TSS) are indispensable components of activating leukocyte receptor complexes. The TSS-encoding genes map to paralogous chromosomal regions, which are thought to arise from ancient genome tetraploidization(s). To assess a possible role of tetraploidization in the TSS evolution, we studied TSS and other functionally linked genes in the amphibian species Xenopus laevis whose genome was duplicated about 40 MYR ago. We found that X. laevis has retained a duplicated set of sixteen TSS genes, all except one being transcribed. Furthermore, duplicated TCRα loci and genes encoding TSS-coupling protein kinases have also been retained. No clear evidence for functional divergence of the TSS paralogs was obtained from gene expression and sequence analyses. We suggest that the main factor of maintenance of duplicated TSS genes in X. laevis was a protein dosage effect and that this effect might have facilitated the TSS set expansion in early vertebrates.

  12. Distribution of Plasmids in Distinct Leptospira Pathogenic Species

    PubMed Central

    Wang, Yanzhuo; Zhuang, Xuran; Zhong, Yi; Zhang, Cuicai; Zhang, Yan; Zeng, Lingbing; Zhu, Yongzhang; He, Ping; Dong, Ke; Pal, Utpal; Guo, Xiaokui; Qin, Jinhong

    2015-01-01

    Leptospirosis, caused by pathogenic Leptospira, is a worldwide zoonotic infection. The genus Leptospira includes at least 21 species clustered into three groups—pathogens, non-pathogens, and intermediates—based on 16S rRNA phylogeny. Research on Leptospira is difficult due to slow growth and poor transformability of the pathogens. Recent identification of extrachromosomal elements besides the two chromosomes in L. interrogans has provided new insight into genome complexity of the genus Leptospira. The large size, low copy number, and high similarity of the sequence of these extrachromosomal elements with the chromosomes present challenges in isolating and detecting them without careful genome assembly. In this study, two extrachromosomal elements were identified in L. borgpetersenii serovar Ballum strain 56604 through whole genome assembly combined with S1 nuclease digestion following pulsed-field gel electrophoresis (S1-PFGE) analysis. Further, extrachromosomal elements in additional 15 Chinese epidemic strains of Leptospira, comprising L. borgpetersenii, L. weilii, and L. interrogans, were successfully separated and identified, independent of genome sequence data. Southern blot hybridization with extrachromosomal element-specific probes, designated as lcp1, lcp2 and lcp3-rep, further confirmed their occurrences as extrachromosomal elements. In total, 24 plasmids were detected in 13 out of 15 tested strains, among which 11 can hybridize with the lcp1-rep probe and 11 with the lcp2-rep probe, whereas two can hybridize with the lcp3-rep probe. None of them are likely to be species-specific. Blastp search of the lcp1, lcp2, and lcp3-rep genes with a nonredundant protein database of Leptospira species genomes showed that their homologous sequences are widely distributed among clades of pathogens but not non-pathogens or intermediates. These results suggest that the plasmids are widely distributed in Leptospira species, and further elucidation of their biological

  13. Conformational Changes in the Endosomal Sorting Complex Required for the Transport III Subunit Ist1 Lead to Distinct Modes of ATPase Vps4 Regulation.

    PubMed

    Tan, Jason; Davies, Brian A; Payne, Johanna A; Benson, Linda M; Katzmann, David J

    2015-12-11

    Intralumenal vesicle formation of the multivesicular body is a critical step in the delivery of endocytic cargoes to the lysosome for degradation. Endosomal sorting complex required for transport III (ESCRT-III) subunits polymerize on endosomal membranes to facilitate membrane budding away from the cytoplasm to generate these intralumenal vesicles. The ATPase Vps4 remodels and disassembles ESCRT-III, but the manner in which Vps4 activity is coordinated with ESCRT-III function remains unclear. Ist1 is structurally homologous to ESCRT-III subunits and has been reported to inhibit Vps4 function despite the presence of a microtubule-interacting and trafficking domain-interacting motif (MIM) capable of stimulating Vps4 in the context of other ESCRT-III subunits. Here we report that Ist1 inhibition of Vps4 ATPase activity involves two elements in Ist1: the MIM itself and a surface containing a conserved ELYC sequence. In contrast, the MIM interaction, in concert with a more open conformation of the Ist1 core, resulted in stimulation of Vps4. Addition of the ESCRT-III subunit binding partner of Ist1, Did2, also converted Ist1 from an inhibitor to a stimulator of Vps4 ATPase activity. Finally, distinct regulation of Vps4 by Ist1 corresponded with altered ESCRT-III disassembly in vitro. Together, these data support a model in which Ist1-Did2 interactions during ESCRT-III polymerization coordinate Vps4 activity with the timing of ESCRT-III disassembly.

  14. Effects of Salt Stress on Three Ecologically Distinct Plantago Species

    PubMed Central

    Al Hassan, Mohamad; Pacurar, Andrea; López-Gresa, María P.; Donat-Torres, María P.; Llinares, Josep V.; Boscaiu, Monica; Vicente, Oscar

    2016-01-01

    Comparative studies on the responses to salt stress of taxonomically related taxa should help to elucidate relevant mechanisms of stress tolerance in plants. We have applied this strategy to three Plantago species adapted to different natural habitats, P. crassifolia and P. coronopus–both halophytes–and P. major, considered as salt-sensitive since it is never found in natural saline habitats. Growth inhibition measurements in controlled salt treatments indicated, however, that P. major is quite resistant to salt stress, although less than its halophytic congeners. The contents of monovalent ions and specific osmolytes were determined in plant leaves after four-week salt treatments. Salt-treated plants of the three taxa accumulated Na+ and Cl- in response to increasing external NaCl concentrations, to a lesser extent in P. major than in the halophytes; the latter species also showed higher ion contents in the non-stressed plants. In the halophytes, K+ concentration decreased at moderate salinity levels, to increase again under high salt conditions, whereas in P. major K+ contents were reduced only above 400 mM NaCl. Sorbitol contents augmented in all plants, roughly in parallel with increasing salinity, but the relative increments and the absolute values reached did not differ much in the three taxa. On the contrary, a strong (relative) accumulation of proline in response to high salt concentrations (600–800 mM NaCl) was observed in the halophytes, but not in P. major. These results indicate that the responses to salt stress triggered specifically in the halophytes, and therefore the most relevant for tolerance in the genus Plantago are: a higher efficiency in the transport of toxic ions to the leaves, the capacity to use inorganic ions as osmotica, even under low salinity conditions, and the activation, in response to very high salt concentrations, of proline accumulation and K+ transport to the leaves of the plants. PMID:27490924

  15. Dizocilpine (MK-801) induces distinct changes of N-methyl-D-aspartic acid receptor subunits in parvalbumin-containing interneurons in young adult rat prefrontal cortex.

    PubMed

    Xi, Dong; Zhang, Wentong; Wang, Huai-Xing; Stradtman, George G; Gao, Wen-Jun

    2009-11-01

    N-methyl-D-aspartic acid receptor (NMDAR) hypofunction has long been implicated in schizophrenia and NMDARs on gamma-aminobutyric acid (GABA)ergic interneurons are proposed to play an essential role in the pathogenesis. However, controversial results have been reported regarding the regulation of NMDAR expression, and direct evidence of how NMDAR antagonists act on specific subpopulations of prefrontal interneurons is missing. We investigated the effects of the NMDAR antagonist dizocilpine (MK-801) on the expression of NMDAR subtypes in the identified interneurons in young adult rat prefrontal cortex (PFC) by using laser microdissection and real-time polymerase chain reaction, combined with Western blotting and immunofluorescent staining. We found that MK-801 induced distinct changes of NMDAR subunits in the parvalbumin-immunoreactive (PV-ir) interneurons vs. pyramidal neurons in the PFC circuitry. The messenger RNA (mRNA) expression of all NMDAR subtypes, including NR1 and NR2A to 2D, exhibited inverted-U dose-dependent changes in response to MK-801 treatment in the PFC. In contrast, subunit mRNAs of NMDARs in PV-ir interneurons were significantly down-regulated at low doses, unaltered at medium doses, and significantly decreased again at high doses, suggesting a biphasic dose response to MK-801. The differential effects of MK-801 in mRNA expression of NMDAR subunits were consistent with the protein expression of NR2A and NR2B subunits revealed with Western blotting and double immunofluorescent staining. These results suggest that PV-containing interneurons in the PFC exhibit a distinct responsiveness to NMDAR antagonism and that NMDA antagonist can differentially and dose-dependently regulate the functions of pyramidal neurons and GABAergic interneurons in the prefrontal cortical circuitry.

  16. Rice G-protein subunits qPE9-1 and RGB1 play distinct roles in abscisic acid responses and drought adaptation.

    PubMed

    Zhang, Dong-Ping; Zhou, Yong; Yin, Jian-Feng; Yan, Xue-Jiao; Lin, Sheng; Xu, Wei-Feng; Baluška, František; Wang, Yi-Ping; Xia, Yi-Ji; Liang, Guo-hua; Liang, Jian-Sheng

    2015-10-01

    Heterotrimeric GTP-binding protein (G-protein)-mediated abscisic acid (ABA) and drought-stress responses have been documented in numerous plant species. However, our understanding of the function of rice G-protein subunits in ABA signalling and drought tolerance is limited. In this study, the function of G-protein subunits in ABA response and drought resistance in rice plants was explored. It was found that the transcription level of qPE9-1 (rice Gγ subunit) gradually decreased with increasing ABA concentration and the lack of qPE9-1 showed an enhanced drought tolerance in rice plants. In contrast, mRNA levels of RGB1 (rice Gβ subunit) were significantly upregulated by ABA treatment and the lack of RGB1 led to reduced drought tolerance. Furthermore, the results suggested that qPE9-1 negatively regulates the ABA response by suppressing the expression of key transcription factors involved in ABA and stress responses, while RGB1 positively regulates ABA biosynthesis by upregulating NCED gene expression under both normal and drought stress conditions. Taken together, it is proposed that RGB1 is a positive regulator of the ABA response and drought adaption in rice plants, whereas qPE9-1 is modulated by RGB1 and functions as a negative regulator in the ABA-dependent drought-stress responses.

  17. 77 FR 64959 - Endangered and Threatened Species; Initiation of 5-Year Review for the Southern Distinct...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-10-24

    ...; Initiation of 5-Year Review for the Southern Distinct Population Segment of North American Green Sturgeon... 5-year review of the Southern Distinct Population Segment (DPS) of North American green sturgeon... of requested information include: (1) Species biology including, but not limited to,...

  18. A Streamlined System for Species Diagnosis in Caenorhabditis (Nematoda: Rhabditidae) with Name Designations for 15 Distinct Biological Species

    PubMed Central

    Félix, Marie-Anne; Braendle, Christian; Cutter, Asher D.

    2014-01-01

    The rapid pace of species discovery outstrips the rate of species description in many taxa. This problem is especially acute for Caenorhabditis nematodes, where the naming of distinct species would greatly improve their visibility and usage for biological research, given the thousands of scientists studying Caenorhabditis. Species description and naming has been hampered in Caenorhabditis, in part due to the presence of morphologically cryptic species despite complete biological reproductive isolation and often enormous molecular divergence. With the aim of expediting species designations, here we propose and apply a revised framework for species diagnosis and description in this group. Our solution prioritizes reproductive isolation over traditional morphological characters as the key feature in delineating and diagnosing new species, reflecting both practical considerations and conceptual justifications. DNA sequence divergence criteria help prioritize crosses for establishing patterns of reproductive isolation among the many species of Caenorhabditis known to science, such as with the ribosomal internal transcribed spacer-2 (ITS2) DNA barcode. By adopting this approach, we provide new species name designations for 15 distinct biological species, thus increasing the number of named Caenorhabditis species in laboratory culture by nearly 3-fold. We anticipate that the improved accessibility of these species to the research community will expand the opportunities for study and accelerate our understanding of diverse biological phenomena. PMID:24727800

  19. Assembly of in vitro synthesized large subunits into ribulose-bisphosphate carboxylase/oxygenase. Formation and discharge of an L8-like species.

    PubMed

    Hubbs, A E; Roy, H

    1993-06-25

    Ribulose-bisphosphate carboxylase/oxygenase (Rubisco) from higher plants consists of eight approximately 53-kDa large subunits and eight approximately 14-kDa small subunits. Cytosolic ribosomes synthesize the small subunits as precursors, which enter the chloroplast, undergo proteolytic processing, and assemble with large subunits. Large subunits, synthesized in the chloroplast, first form a complex with the chloroplast chaperonin 60 (Cpn60(14)). In the presence of ATP, large subunits dissociate from Cpn60(14) and assemble into Rubisco. We now describe partial characterization of a new species, Z, containing radiotracer-labeled, newly synthesized pea Rubisco large subunits. Rubisco assembly occurs in low salt in the presence of small subunits and ATP. As with Rubisco assembly, the formation of Z is ATP-dependent and is inhibited by high chloride. Once formed, Z is stable except in high chloride. Z does not appear to interact directly with small subunits. However, after Z formation, Rubisco assembly occurs in an ATP-independent reaction that requires KCl and small subunits. These results are consistent with the hypothesis that Z is a large subunit containing structure that can contribute large subunits to Rubisco under appropriate conditions. Z shares some physical characteristics with reported cyanobacterial L8 core particles. However, formation of Rubisco from Z in the absence of ATP and the presence of small subunits appears to require conditions that otherwise destabilize Z.

  20. Culturing-based Temperature Calibration of a Genetically Distinct, Alkenone-producing Haptophyte Species isolated from Lake George, ND

    NASA Astrophysics Data System (ADS)

    Zheng, Y.; Andersen, R. A.; Huang, Y.; Amaral-Zettler, L. A.

    2014-12-01

    Lacustrine alkenones are rapidly becoming an important tool for continental paleoclimate reconstructions. However, DNA sequencing of 18S ribosomal RNA marker genes has uncovered multiple species of haptophytes in different lakes. To date, there are only two isolated lacustrine species Chrysotila lamellosa and Isochrysis galbana available for culture studies. In our study, we report the isolation of a new haptophyte species from Lake George (LG) that, based on analyses of partial large subunit rRNA gene sequences, is genetically distinct from both Chrysotila lamellosa and Isochrysis galbana. We examined alkenone unsaturation index UK37 values for the LG species at 4°C, 10°C, 15°C, 20°C and 25°C as a function of temperature in a culture experiment. The temperature sensitivity of the new species was significantly higher than previously cultured Isochrysis galbana and Chrysotila lamellosa strains, with a slope that was 25 to 100 % higher. We found that the best linear relationship was obtained when two double-bond alkenones were excluded from the calibration (we developed an index termed UK''37 = [C37:4] / [C37:3+C37:4]). In particular, UK''37 is more linear to the growth temperature than UK37 at low (4-10°C) and high (20-25°C) temperature ranges. Our experiments show that both UK37 and UK''37 of this new alkenone-produced species is strongly controlled by culture temperature and can be used for paleoclimate reconstruction. However, we recommend the use of UK''37 index to reconstruct temperature if the haptophyte's growing environment falls within temperature extremes (4-10°C and 20-25°C). This newly cultivated species broadens our ability of applying lacustrine haptophyte calibrations to continental paleothermometry.

  1. 76 FR 58867 - Endangered and Threatened Species; Determination of Nine Distinct Population Segments of...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-09-22

    ...We (NMFS and USFWS; also collectively referred to as the Services) have determined that the loggerhead sea turtle (Caretta caretta) is composed of nine distinct population segments (DPSs) that constitute ``species'' that may be listed as threatened or endangered under the Endangered Species Act (ESA). In this final rule, we are listing four DPSs as threatened and five as endangered under the......

  2. Hidden species complexes within distinctive taxa: the case of Epanthidium bicoloratum (Smith) (Hymenoptera: Megachilidae)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Epanthidium bicoloratum (Smith) differs from all other Neotropical Anthidiini in the distinctive tegula, which is narrowed posteriorly forming a slender wedge. Examination of material standing under this name revealed that it represents a species complex that includes three new cryptic species, E. ...

  3. A distinct three-helix centipede toxin SSD609 inhibits I(ks) channels by interacting with the KCNE1 auxiliary subunit.

    PubMed

    Sun, Peibei; Wu, Fangming; Wen, Ming; Yang, Xingwang; Wang, Chenyang; Li, Yiming; He, Shufang; Zhang, Longhua; Zhang, Yun; Tian, Changlin

    2015-08-26

    KCNE1 is a single-span transmembrane auxiliary protein that modulates the voltage-gated potassium channel KCNQ1. The KCNQ1/KCNE1 complex in cardiomyocytes exhibited slow activated potassium (I(ks)) currents. Recently, a novel 47-residue polypeptide toxin SSD609 was purified from Scolopendra subspinipes dehaani venom and showed I(ks) current inhibition. Here, chemically synthesized SSD609 was shown to exert I(ks) inhibition in extracted guinea pig cardiomyocytes and KCNQ1/KCNE1 current attenuation in CHO cells. The K(+) current attenuation of SSD609 showed decent selectivity among different auxiliary subunits. Solution nuclear magnetic resonance analysis of SSD609 revealed a distinctive three-helix conformation that was stabilized by a new disulfide bonding pattern as well as segregated surface charge distribution. Structure-activity studies demonstrated that negatively charged Glu19 in the amphipathic extracellular helix of KCNE1 was the key residue that interacted with SSD609. The distinctive three-helix centipede toxin SSD609 is known to be the first polypeptide toxin acting on channel auxiliary subunit KCNE1, which suggests a new type of pharmacological regulation for ion channels in cardiomyocytes.

  4. Identification of Egyptian Fasciola species by PCR and restriction endonucleases digestion of the nuclear small subunit ribosomal RNA gene.

    PubMed

    El-Gozamy, Bothina R; Shoukry, Nahla M

    2009-08-01

    Fascioliasis is one of the familiar zoonotic health problems of worldwide distribution including Egypt. In this study, a simple and rapid polymerase chain reaction/restriction fragment length polymorphisms (PCR/RFLPs) assay, using the common restriction endonucleases Aval, EcoRI, Eael, Sac11 and Avail was applied to differentiate between both Fasciola gigantica and F. hepatica. The five restriction endonucleases were used to differentiate between the two species of Fasciola based on -1950 bp long sequence of the 18S nuclear small subunit ribosomal RNA gene. Aval and EcoRI restriction endonucleases failed to differentiate between the two Fasciola species when each restriction enzyme gave the same restriction patterns in both of them. However, F. gigantica and F. hepatica were well-differentiated when their small subunit ribosomal DNA were digested with Eael and Sac 11 restriction endonucleases.

  5. Signal transduction by the high-affinity GM-CSF receptor: two distinct cytoplasmic regions of the common beta subunit responsible for different signaling.

    PubMed Central

    Sato, N; Sakamaki, K; Terada, N; Arai, K; Miyajima, A

    1993-01-01

    The high-affinity receptors for granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 3 (IL-3) and IL-5 consist of two subunits, alpha and beta. The alpha subunits are specific to each cytokine and the same beta subunit (beta c) is shared by these three receptors. Although none of these receptor subunits has intrinsic kinase activity, these cytokines induce protein tyrosine phosphorylation, activation of Ras, Raf-1 and MAP kinase, and transcriptional activation of nuclear proto-oncogenes such as c-myc, c-fos and c-jun. In this paper, we describe a detailed analysis of the signaling potential of the beta c subunit by using a series of cytoplasmic deletion mutants. The human beta c consists of 881 amino acid residues. A C-terminal deletion mutant of beta c at amino acid 763 (beta 763) induced phosphorylation of Shc and activation of Ras, Raf-1, MAP kinase and p70 S6 kinase, whereas a deletion at amino acid 626 (beta 626) induced none of these effects. The beta 763 mutant, as well as the full-length beta c, induced transcription of c-myc, c-fos and c-jun. Deletions at amino acid 517 (beta 517) and 626 (beta 626) induced c-myc and pim-1, but no induction of c-fos and c-jun was observed. GM-CSF increased phosphatidylinositol 3 kinase (PI3-K) activity in anti-phosphotyrosine immunoprecipitates from cells expressing beta 763 as well as beta c, whereas it was only marginally increased from cells expressing beta 517 or beta 626. Thus, there are at least two distinct regions within the cytoplasmic domain of beta c that are responsible for different signals, i.e. a membrane proximal region of approximately 60 amino acid residues upstream of Glu517 is essential for induction of c-myc and pim-1, and a distal region of approximately 140 amino acid residues (between Leu626 and Ser763) is required for activation of Ras, Raf-1, MAP kinase and p70 S6 kinase, as well as induction of c-fos and c-jun. Images PMID:8223433

  6. The nicotinic acetylcholine receptors of the parasitic nematode Ascaris suum: formation of two distinct drug targets by varying the relative expression levels of two subunits.

    PubMed

    Williamson, Sally M; Robertson, Alan P; Brown, Laurence; Williams, Tracey; Woods, Debra J; Martin, Richard J; Sattelle, David B; Wolstenholme, Adrian J

    2009-07-01

    Parasitic nematodes are of medical and veterinary importance, adversely affecting human health and animal welfare. Ascaris suum is a gastrointestinal parasite of pigs; in addition to its veterinary significance it is a good model of the human parasite Ascaris lumbricoides, estimated to infect approximately 1.4 billion people globally. Anthelmintic drugs are essential to control nematode parasites, and nicotinic acetylcholine receptors (nAChRs) on nerve and muscle are the targets of cholinergic anthelmintics such as levamisole and pyrantel. Previous genetic analyses of nematode nAChRs have been confined to Caenorhabditis elegans, which is phylogenetically distinct from Ascaris spp. and many other important parasites. Here we report the cloning and expression of two nAChR subunit cDNAs from A. suum. The subunits are very similar in sequence to C. elegans UNC-29 and UNC-38, are expressed on muscle cells and can be expressed robustly in Xenopus oocytes to form acetylcholine-, nicotine-, levamisole- and pyrantel-sensitive channels. We also demonstrate that changing the stoichiometry of the receptor by injecting different ratios of the subunit cRNAs can reproduce two of the three pharmacological subtypes of nAChR present in A. suum muscle cells. When the ratio was 5:1 (Asu-unc-38ratioAsu-unc-29), nicotine was a full agonist and levamisole was a partial agonist, and oocytes responded to oxantel, but not pyrantel. At the reverse ratio (1:5 Asu-unc-38ratioAsu-unc-29), levamisole was a full agonist and nicotine was a partial agonist, and the oocytes responded to pyrantel, but not oxantel. These results represent the first in vitro expression of any parasitic nicotinic receptor and show that their properties are substantially different from those of C. elegans. The results also show that changing the expression level of a single receptor subunit dramatically altered the efficacy of some anthelmintic drugs. In vitro expression of these subunits may permit the development of

  7. Distinct Developmental Function of Two Caenorhabditis elegans Homologs of the Cohesin Subunit Scc1/Rad21V⃞

    PubMed Central

    Mito, Yoshiko; Sugimoto, Asako; Yamamoto, Masayuki

    2003-01-01

    Cohesin, which mediates sister chromatid cohesion, is composed of four subunits, named Scc1/Rad21, Scc3, Smc1, and Smc3 in yeast. Caenorhabditis elegans has a single homolog for each of Scc3, Smc1, and Smc3, but as many as four for Scc1/Rad21 (COH-1, SCC-1/COH-2, COH-3, and REC-8). Except for REC-8 required for meiosis, function of these C. elegans proteins remains largely unknown. Herein, we examined their possible involvement in mitosis and development. Embryos depleted of the homolog of either Scc3, or Smc1, or Smc3 by RNA interference revealed a defect in mitotic chromosome segregation but not in chromosome condensation and cytokinesis. Depletion of SCC-1/COH-2 caused similar phenotypes. SCC-1/COH-2 was present in cells destined to divide. It localized to chromosomes in a cell cycle-dependent manner. Worms depleted of COH-1 arrested at either the late embryonic or the larval stage, with no indication of mitotic dysfunction. COH-1 associated chromosomes throughout the cell cycle in all somatic cells undergoing late embryogenesis or larval development. Thus, SCC-1/COH-2 and the homologs of Scc3, Smc1, and Smc3 facilitate mitotic chromosome segregation during the development, presumably by forming a cohesin complex, whereas COH-1 seems to play a role important for development but unrelated to mitosis. PMID:12808038

  8. Janus Model of The Na,K-ATPase β-subunit Transmembrane Domain: Distinct Faces Mediate α /β Assembly and β-β Homo-Oligomerization

    PubMed Central

    Barwe, Sonali P.; Kim, Sanguk; Rajasekaran, Sigrid A.; Bowie, James U.; Rajasekaran, Ayyappan K.

    2007-01-01

    Summary Na,K-ATPase is a hetero-oligomer of α- and β-subunits. The Na,K-ATPase β-subunit (Na,K-β ) is involved in both the regulation of ion transport activity, and in cell-cell adhesion. By structure prediction and evolutionary analysis, we identified two distinct faces on the Na,K-β transmembrane domain (TMD) that could mediate protein-protein interactions: a glycine zipper motif and a conserved heptad repeat. Here, we show that the heptad repeat face is involved in the hetero-oligomeric interaction of Na,K-β with Na,K-α , and the glycine zipper face is involved in the homo-oligomerization of Na,K-β . Point mutations in the heptad repeat motif reduced Na,K-β binding to Na,K-α , and Na,K-ATPase activity. Na,K-β TMD homo-oligomerized in biological membranes, and mutation of the glycine zipper motif affected oligomerization and cell-cell adhesion. These results provide a structural basis for understanding how Na,K-β links ion transport and cell-cell adhesion. PMID:17078968

  9. Efficient distinction of invasive aquatic plant species from non-invasive related species using DNA barcoding.

    PubMed

    Ghahramanzadeh, R; Esselink, G; Kodde, L P; Duistermaat, H; van Valkenburg, J L C H; Marashi, S H; Smulders, M J M; van de Wiel, C C M

    2013-01-01

    Biological invasions are regarded as threats to global biodiversity. Among invasive aliens, a number of plant species belonging to the genera Myriophyllum, Ludwigia and Cabomba, and to the Hydrocharitaceae family pose a particular ecological threat to water bodies. Therefore, one would try to prevent them from entering a country. However, many related species are commercially traded, and distinguishing invasive from non-invasive species based on morphology alone is often difficult for plants in a vegetative stage. In this regard, DNA barcoding could become a good alternative. In this study, 242 samples belonging to 26 species from 10 genera of aquatic plants were assessed using the chloroplast loci trnH-psbA, matK and rbcL. Despite testing a large number of primer sets and several PCR protocols, the matK locus could not be amplified or sequenced reliably and therefore was left out of the analysis. Using the other two loci, eight invasive species could be distinguished from their respective related species, a ninth one failed to produce sequences of sufficient quality. Based on the criteria of universal application, high sequence divergence and level of species discrimination, the trnH-psbA noncoding spacer was the best performing barcode in the aquatic plant species studied. Thus, DNA barcoding may be helpful with enforcing a ban on trade of such invasive species, such as is already in place in the Netherlands. This will become even more so once DNA barcoding would be turned into machinery routinely operable by a nonspecialist in botany and molecular genetics.

  10. Functional Characterization of Rpn3 Uncovers a Distinct 19S Proteasomal Subunit Requirement for Ubiquitin-Dependent Proteolysis of Cell Cycle Regulatory Proteins in Budding Yeast

    PubMed Central

    Bailly, Eric; Reed, Steven I.

    1999-01-01

    By selectively eliminating ubiquitin-conjugated proteins, the 26S proteasome plays a pivotal role in a large variety of cellular regulatory processes, particularly in the control of cell cycle transitions. Access of ubiquitinated substrates to the inner catalytic chamber within the 20S core particle is mediated by the 19S regulatory particle (RP), whose subunit composition in budding yeast has been recently elucidated. In this study, we have investigated the cell cycle defects resulting from conditional inactivation of one of these RP components, the essential non-ATPase Rpn3/Sun2 subunit. Using temperature-sensitive mutant alleles, we show that rpn3 mutations do not prevent the G1/S transition but cause a metaphase arrest, indicating that the essential Rpn3 function is limiting for mitosis. rpn3 mutants appear severely compromised in the ubiquitin-dependent proteolysis of several physiologically important proteasome substrates. Thus, RPN3 function is required for the degradation of the G1-phase cyclin Cln2 targeted by SCF; the S-phase cyclin Clb5, whose ubiquitination is likely to involve a combination of E3 (ubiquitin protein ligase) enzymes; and anaphase-promoting complex targets, such as the B-type cyclin Clb2 and the anaphase inhibitor Pds1. Our results indicate that the Pds1 degradation defect of the rpn3 mutants most likely accounts for the metaphase arrest phenotype observed. Surprisingly, but consistent with the lack of a G1 arrest phenotype in thermosensitive rpn3 strains, the Cdk inhibitor Sic1 exhibits a short half-life regardless of the RPN3 genotype. In striking contrast, Sic1 turnover is severely impaired by a temperature-sensitive mutation in RPN12/NIN1, encoding another essential RP subunit. While other interpretations are possible, these data strongly argue for the requirement of distinct RP subunits for efficient proteolysis of specific cell cycle regulators. The potential implications of these data are discussed in the context of possible Rpn3

  11. Distinctive interactions of the Arabidopsis homolog of the 30 kD subunit of the cleavage and polyadenylation specificity factor (AtCPSF30) with other polyadenylation factor subunits

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: The Arabidopsis ortholog of the 30 kD subunit of the mammalian Cleavage and Polyadenylation Specificity Factor (AtCPSF30) is an RNA-binding endonuclease that is associated with other Arabidopsis CPSF subunits (orthologs of the 160, 100, and 73 kD subunits of CPSF). In order to better u...

  12. A Comparative In Silico Study of the Antioxidant Defense Gene Repertoire of Distinct Lifestyle Trypanosomatid Species

    PubMed Central

    Beltrame-Botelho, Ingrid Thaís; Talavera-López, Carlos; Andersson, Björn; Grisard, Edmundo Carlos; Stoco, Patricia Hermes

    2016-01-01

    Kinetoplastids are an ancestral group of protists that contains free-living species and parasites with distinct mechanisms in response to stress. Here, we compared genes involved in antioxidant defense (AD), proposing an evolution model among trypanosomatids. All genes were identified in Bodo saltans, suggesting that AD mechanisms have evolved prior to adaptation for parasitic lifestyles. While most of the monoxenous and dixenous parasites revealed minor differences from B. saltans, the endosymbiont-bearing species have an increased number of genes. The absence of these genes was mainly observed in the extracellular parasites of the genera Phytomonas and Trypanosoma. In trypanosomes, a distinction was observed between stercorarian and salivarian parasites, except for Trypanosoma rangeli. Our analyses indicate that the variability of AD among trypanosomatids at the genomic level is not solely due to the geographical isolation, being mainly related to specific adaptations of their distinct biological cycles within insect vectors and to a parasitism of a wide range of hosts. PMID:27840574

  13. A natural M RNA reassortant arising from two distinct tospovirus species

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The complete nucleotide sequence of a tospovirus isolate from south Florida tomatoes was determined. Phylogenetic reconstructions of each genomic RNA segment showed that this isolate was produced by reassortment of segments from two distinct tospovirus species. The S and L segments are most closel...

  14. 77 FR 20774 - Endangered and Threatened Species; Proposed Threatened Status for Distinct Population Segments of...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-04-06

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF COMMERCE National Oceanic and Atmospheric Administration 50 CFR Part 223 RIN 0648-XZ58 Endangered and Threatened Species; Proposed Threatened Status for Distinct Population Segments of the Bearded Seal AGENCY:...

  15. Differential localization of ion transporters suggests distinct cellular mechanisms for calcification and photosynthesis between two coral species.

    PubMed

    Barott, Katie L; Perez, Sidney O; Linsmayer, Lauren B; Tresguerres, Martin

    2015-08-01

    Ion transport is fundamental for multiple physiological processes, including but not limited to pH regulation, calcification, and photosynthesis. Here, we investigated ion-transporting processes in tissues from the corals Acropora yongei and Stylophora pistillata, representatives of the complex and robust clades that diverged over 250 million years ago. Antibodies against complex IV revealed that mitochondria, an essential source of ATP for energetically costly ion transporters, were abundant throughout the tissues of A. yongei. Additionally, transmission electron microscopy revealed septate junctions in all cell layers of A. yongei, as previously reported for S. pistillata, as well as evidence for transcellular vesicular transport in calicoblastic cells. Antibodies against the alpha subunit of Na(+)/K(+)-ATPase (NKA) and plasma membrane Ca(2+)-ATPase (PMCA) immunolabeled cells in the calicoblastic epithelium of both species, suggesting conserved roles in calcification. However, NKA was abundant in the apical membrane of the oral epithelium in A. yongei but not S. pistillata, while PMCA was abundant in the gastroderm of S. pistillata but not A. yongei. These differences indicate that these two coral species utilize distinct pathways to deliver ions to the sites of calcification and photosynthesis. Finally, antibodies against mammalian sodium bicarbonate cotransporters (NBC; SLC4 family) resulted in strong immunostaining in the apical membrane of oral epithelial cells and in calicoblastic cells in A. yongei, a pattern identical to NKA. Characterization of ion transport mechanisms is an essential step toward understanding the cellular mechanisms of coral physiology and will help predict how different coral species respond to environmental stress.

  16. Genome-wide Evidence Reveals that African and Eurasian Golden Jackals Are Distinct Species.

    PubMed

    Koepfli, Klaus-Peter; Pollinger, John; Godinho, Raquel; Robinson, Jacqueline; Lea, Amanda; Hendricks, Sarah; Schweizer, Rena M; Thalmann, Olaf; Silva, Pedro; Fan, Zhenxin; Yurchenko, Andrey A; Dobrynin, Pavel; Makunin, Alexey; Cahill, James A; Shapiro, Beth; Álvares, Francisco; Brito, José C; Geffen, Eli; Leonard, Jennifer A; Helgen, Kristofer M; Johnson, Warren E; O'Brien, Stephen J; Van Valkenburgh, Blaire; Wayne, Robert K

    2015-08-17

    The golden jackal of Africa (Canis aureus) has long been considered a conspecific of jackals distributed throughout Eurasia, with the nearest source populations in the Middle East. However, two recent reports found that mitochondrial haplotypes of some African golden jackals aligned more closely to gray wolves (Canis lupus), which is surprising given the absence of gray wolves in Africa and the phenotypic divergence between the two species. Moreover, these results imply the existence of a previously unrecognized phylogenetically distinct species despite a long history of taxonomic work on African canids. To test the distinct-species hypothesis and understand the evolutionary history that would account for this puzzling result, we analyzed extensive genomic data including mitochondrial genome sequences, sequences from 20 autosomal loci (17 introns and 3 exon segments), microsatellite loci, X- and Y-linked zinc-finger protein gene (ZFX and ZFY) sequences, and whole-genome nuclear sequences in African and Eurasian golden jackals and gray wolves. Our results provide consistent and robust evidence that populations of golden jackals from Africa and Eurasia represent distinct monophyletic lineages separated for more than one million years, sufficient to merit formal recognition as different species: C. anthus (African golden wolf) and C. aureus (Eurasian golden jackal). Using morphologic data, we demonstrate a striking morphologic similarity between East African and Eurasian golden jackals, suggesting parallelism, which may have misled taxonomists and likely reflects uniquely intense interspecific competition in the East African carnivore guild. Our study shows how ecology can confound taxonomy if interspecific competition constrains size diversification.

  17. Distinct symbiont lineages in three thyasirid species (Bivalvia: Thyasiridae) from the eastern Atlantic and Mediterranean Sea

    NASA Astrophysics Data System (ADS)

    Rodrigues, Clara F.; Duperron, Sébastien

    2011-04-01

    Thyasiridae are one of the less studied groups of chemosymbiotic bivalves. Here, we investigated symbioses in three different thyasirid species collected at three cold seeps from the Atlantic and Mediterranean. Phylogenetic analysis of bacterial 16S ribosomal RNA gene sequences demonstrated that each thyasirid species harbours a single phylotype of symbiont that belongs to a distinct lineage of putative sulphur-oxidizing Gammaproteobacteria. This result is confirmed by other marker genes (encoding 23S rRNA and APS reductase) and fluorescence in situ hybridization. This work highlights the diversity of bacteria involved in symbiosis with thyasirids and underlines the relevance of this group as a target for future symbiosis studies.

  18. Identification of distinct physiochemical properties of toxic prefibrillar species formed by A{beta} peptide variants

    SciTech Connect

    Goeransson, Anna-Lena; Nilsson, K. Peter R.; Kagedal, Katarina; Brorsson, Ann-Christin

    2012-04-20

    Highlights: Black-Right-Pointing-Pointer Identification of toxic prefibrillar A{beta} species. Black-Right-Pointing-Pointer Fluorescence measurements using a combined set of fluorophores. Black-Right-Pointing-Pointer Morphology studies using transmission electron microscopy. -- Abstract: The formation of amyloid-{beta} peptide (A{beta}) aggregates at an early stage during the self-assembly process is an important factor in the development of Alzheimer's disease. The toxic effect is believed to be exerted by prefibrillar species of A{beta}. It is therefore important to identify which prefibrillar species are toxic and characterize their distinct properties. In the present study, we investigated the in vitro aggregation behavior of A{beta}-derived peptides possessing different levels of neurotoxic activity, using fluorescence spectroscopy in combination with transmission electron microscopy. The toxicity of various A{beta} aggregates was assessed by using cultures of human neuroblastoma cells. Through combined use of the fluorescence probe 8-anilino-1-napthalenesulfonate (ANS) and the novel luminescent probe pentamer formyl thiophene acetic acid (p-FTAA), we were able to identify those A{beta} peptide-derived prefibrillar species which exhibited cellular toxicity. In particular, species, which formed early during the aggregation process and showed strong p-FTAA and ANS fluorescence, were the species that possessed toxic activities. Moreover, by manipulating the aggregation conditions, it was possible to change the capacity of the A{beta} peptide to form nontoxic versus toxic species.

  19. Morphological Differences between Larvae of the Ciona intestinalis Species Complex: Hints for a Valid Taxonomic Definition of Distinct Species.

    PubMed

    Pennati, Roberta; Ficetola, Gentile Francesco; Brunetti, Riccardo; Caicci, Federico; Gasparini, Fabio; Griggio, Francesca; Sato, Atsuko; Stach, Thomas; Kaul-Strehlow, Sabrina; Gissi, Carmela; Manni, Lucia

    2015-01-01

    The cosmopolitan ascidian Ciona intestinalis is the most common model species of Tunicata, the sister-group of Vertebrata, and widely used in developmental biology, genomics and evolutionary studies. Recently, molecular studies suggested the presence of cryptic species hidden within the C. intestinalis species, namely C. intestinalis type A and type B. So far, no substantial morphological differences have been identified between individuals belonging to the two types. Here we present morphometric, immunohistochemical, and histological analyses, as well as 3-D reconstructions, of late larvae obtained by cross-fertilization experiments of molecularly determined type A and type B adults, sampled in different seasons and in four different localities. Our data point to quantitative and qualitative differences in the trunk shape of larvae belonging to the two types. In particular, type B larvae exhibit a longer pre-oral lobe, longer and relatively narrower total body length, and a shorter ocellus-tail distance than type A larvae. All these differences were found to be statistically significant in a Discriminant Analysis. Depending on the number of analyzed parameters, the obtained discriminant function was able to correctly classify > 93% of the larvae, with the remaining misclassified larvae attributable to the existence of intra-type seasonal variability. No larval differences were observed at the level of histology and immunohistochemical localization of peripheral sensory neurons. We conclude that type A and type B are two distinct species that can be distinguished on the basis of larval morphology and molecular data. Since the identified larval differences appear to be valid diagnostic characters, we suggest to raise both types to the rank of species and to assign them distinct names.

  20. Morphological Differences between Larvae of the Ciona intestinalis Species Complex: Hints for a Valid Taxonomic Definition of Distinct Species

    PubMed Central

    Pennati, Roberta; Ficetola, Gentile Francesco; Brunetti, Riccardo; Caicci, Federico; Gasparini, Fabio; Griggio, Francesca; Sato, Atsuko; Stach, Thomas; Kaul-Strehlow, Sabrina; Gissi, Carmela; Manni, Lucia

    2015-01-01

    The cosmopolitan ascidian Ciona intestinalis is the most common model species of Tunicata, the sister-group of Vertebrata, and widely used in developmental biology, genomics and evolutionary studies. Recently, molecular studies suggested the presence of cryptic species hidden within the C. intestinalis species, namely C. intestinalis type A and type B. So far, no substantial morphological differences have been identified between individuals belonging to the two types. Here we present morphometric, immunohistochemical, and histological analyses, as well as 3-D reconstructions, of late larvae obtained by cross-fertilization experiments of molecularly determined type A and type B adults, sampled in different seasons and in four different localities. Our data point to quantitative and qualitative differences in the trunk shape of larvae belonging to the two types. In particular, type B larvae exhibit a longer pre-oral lobe, longer and relatively narrower total body length, and a shorter ocellus-tail distance than type A larvae. All these differences were found to be statistically significant in a Discriminant Analysis. Depending on the number of analyzed parameters, the obtained discriminant function was able to correctly classify > 93% of the larvae, with the remaining misclassified larvae attributable to the existence of intra-type seasonal variability. No larval differences were observed at the level of histology and immunohistochemical localization of peripheral sensory neurons. We conclude that type A and type B are two distinct species that can be distinguished on the basis of larval morphology and molecular data. Since the identified larval differences appear to be valid diagnostic characters, we suggest to raise both types to the rank of species and to assign them distinct names. PMID:25955391

  1. Use of an α3β4 nicotinic acetylcholine receptor subunit concatamer to characterize ganglionic receptor subtypes with specific subunit composition reveals species-specific pharmacologic properties.

    PubMed

    Stokes, Clare; Papke, Roger L

    2012-09-01

    Drug development for nicotinic acetylcholine receptors (nAChR) is challenged by subtype diversity arising from variations in subunit composition. On-target activity for neuronal heteromeric receptors is typically associated with CNS receptors that contain α4 and other subunits, while off-target activity could be associated with ganglionic-type receptors containing α3β4 binding sites and other subunits, including β4, β2, α5, or α3 as a structural subunit in the pentamer. Additional interest in α3 β4 α5-containing receptors arises from genome-wide association studies linking these genes, and a single nucleotide polymorphism (SNP) in α5 in particular, to lung cancer and heavy smoking. While α3 and β4 readily form receptors in expression system such as the Xenopus oocyte, since α5 is not required for function, simple co-expression approaches may under-represent α5-containing receptors. We used a concatamer of human α3 and β4 subunits to form ligand-binding domains, and show that we can force the insertions of alternative structural subunits into the functional pentamers. These α3β4 variants differ in sensitivity to ACh, nicotine, varenicline, and cytisine. Our data indicated lower efficacy for varenicline and cytisine than expected for β4-containing receptors, based on previous studies of rodent receptors. We confirm that these therapeutically important α4 receptor partial agonists may present different autonomic-based side-effect profiles in humans than will be seen in rodent models, with varenicline being more potent for human than rat receptors and cytisine less potent. Our initial characterizations failed to find functional effects of the α5 SNP. However, our data validate this approach for further investigations.

  2. The cane or marine toad, Rhinella marina (Anura, Bufonidae): two genetically and morphologically distinct species.

    PubMed

    Acevedo, Aldemar A; Lampo, Margarita; Cipriani, Roberto

    2016-04-18

    Rhinella marina is a Neotropical toad that has been introduced widely worldwide. Its toxic effects to frog-eating predators threaten the native and domestic fauna of some regions where it has been introduced. Despite previous studies suggesting two genetically distinct cryptic species within R. marina, one east and one west of the Andes, its taxonomic status remained unresolved due to the absence of morphological complementary evidence. For the first time, data from two mitochondrial genes (ND3 and CR) and 23 morphometric landmarks are combined to evaluate the taxonomic status of this species. Our results support the hypothesis of two separate evolutionary lineages within R. marina and demonstrate that these lineages have significantly diverged in skull shape. We identified two distinct morphotypes, one eastern and one Andean western, with no overlapping morphospaces. The geographic pattern of genetic variation was consistent with a stable structured population with no evidence of recent demographic or geographic expansions. The concordance between the observed geographic patterns in morphometric and genic traits calls for the recognition of two species under R. marina name.

  3. Two Rumex species from contrasting hydrological niches regulate flooding tolerance through distinct mechanisms.

    PubMed

    van Veen, Hans; Mustroph, Angelika; Barding, Gregory A; Vergeer-van Eijk, Marleen; Welschen-Evertman, Rob A M; Pedersen, Ole; Visser, Eric J W; Larive, Cynthia K; Pierik, Ronald; Bailey-Serres, Julia; Voesenek, Laurentius A C J; Sasidharan, Rashmi

    2013-11-01

    Global climate change has increased flooding events, which affect both natural vegetation dynamics and crop productivity. The flooded environment is lethal for most plant species because it restricts gas exchange and induces an energy and carbon crisis. Flooding survival strategies have been studied in Oryza sativa, a cultivated monocot. However, our understanding of plant adaptation to natural flood-prone environments remains scant, even though wild plants represent a valuable resource of tolerance mechanisms that could be used to generate stress-tolerant crops. Here we identify mechanisms that mediate the distinct flooding survival strategies of two related wild dicot species: Rumex palustris and Rumex acetosa. Whole transcriptome sequencing and metabolite profiling reveal flooding-induced metabolic reprogramming specific to R. acetosa. By contrast, R. palustris uses the early flooding signal ethylene to increase survival by regulating shade avoidance and photomorphogenesis genes to outgrow submergence and by priming submerged plants for future low oxygen stress. These results provide molecular resolution of flooding survival strategies of two species occupying distinct hydrological niches. Learning how these contrasting flood adaptive strategies evolved in nature will be instrumental for the development of stress-tolerant crop varieties that deliver enhanced yields in a changing climate.

  4. Phylogenetic analysis of nuclear small subunit rDNA sequences suggests that the endangered African Pencil Cedar, Juniperus procera, is associated with distinct members of Glomeraceae.

    PubMed

    Wubet, Tesfaye; Weiss, Michael; Kottke, Ingrid; Teketay, Demel; Oberwinkler, Franz

    2006-09-01

    The endangered indigenous tree species Juniperus procera, commonly known as African Pencil Cedar, is an important component of the dry Afromontane vegetation of Ethiopia and was shown to be AM in earlier studies. Here we describe the composition of AM fungi in colonized roots of J. procera from two dry Afromontane forests of Ethiopia. The nuSSU rDNA gene was amplified from colonized roots, cloned and sequenced using AM fungal specific primers that were partly developed for this study. Molecular phylogenetic analysis revealed that all the glomeralean sequences obtained belonged exclusively to the genus Glomus (Glomeraceae). Seven distinct Glomus sequence types were identified that all are new to science. The composition of the AM fungal communities between the sampled trees, and between the two study sites in general, differed significantly. Isolation and utilization of the indigenous AM fungal taxa from the respective sites might be required for successful enrichment plantation of this threatened Juniperus species.

  5. Coexisting orchid species have distinct mycorrhizal communities and display strong spatial segregation.

    PubMed

    Jacquemyn, Hans; Brys, Rein; Merckx, Vincent S F T; Waud, Michael; Lievens, Bart; Wiegand, Thorsten

    2014-04-01

    Because orchids are dependent on mycorrhizal fungi for germination and establishment of seedlings, differences in the mycorrhizal communities associating with orchids can be expected to mediate the abundance, spatial distribution and coexistence of terrestrial orchids in natural communities. We assessed the small-scale spatial distribution of seven orchid species co-occurring in 25 × 25 m plots in two Mediterranean grasslands. In order to characterize the mycorrhizal community associating with each orchid species, 454 pyrosequencing was used. The extent of spatial clustering was assessed using techniques of spatial point pattern analysis. The community of mycorrhizal fungi consisted mainly of members of the Tulasnellaceae, Thelephoraceae and Ceratobasidiaceae, although sporadically members of the Sebacinaceae, Russulaceae and Cortinariaceae were observed. Pronounced differences in mycorrhizal communities were observed between species, whereas strong clustering and significant segregation characterized the spatial distribution of orchid species. However, spatial segregation was not significantly related to phylogenetic dissimilarity of fungal communities. Our results indicate that co-occurring orchid species have distinctive mycorrhizal communities and show strong spatial segregation, suggesting that mycorrhizal fungi are important factors driving niche partitioning in terrestrial orchids and may therefore contribute to orchid coexistence.

  6. Re-consideration of Peronospora farinosa infecting Spinacia oleracea as distinct species, Peronospora effusa.

    PubMed

    Choi, Young-Joon; Hong, Seung-Beom; Shin, Hyeon-Dong

    2007-04-01

    Downy mildew is probably the most widespread and potentially destructive global disease of spinach (Spinacia oleracea). The causal agent of downy mildew disease on various plants of Chenopodiaceae, including spinach, is regarded as a single species, Peronospora farinosa. In the present study, the ITS rDNA sequence and morphological data demonstrated that P. farinosa from S. oleracea is distinct from downy mildew of other chenopodiaceous hosts. Fifty-eight spinach specimens were collected or loaned from 17 countries of Asia, Europe, Oceania, North and South America, which all formed a distinct monophyletic group. No intercontinental genetic variation of the ITS rDNA within Peronospora accessions causing spinach downy mildew disease was found. Phylogenetic trees supported recognition of Peronospora from spinach as a separate species. Microscopic examination also revealed morphological differences between Peronospora specimens from Spinacia and P. farinosa s. lat. specimens from Atriplex, Bassia, Beta, and Chenopodium. Consequently, the name Peronospora effusa should be reinstated for the downy mildew fungus found on spinach. Here, a specimen of the original collections of Peronospora effusa is designated as lectotype.

  7. The β and γ subunits play distinct functional roles in the α2βγ heterotetramer of human NAD-dependent isocitrate dehydrogenase

    NASA Astrophysics Data System (ADS)

    Ma, Tengfei; Peng, Yingjie; Huang, Wei; Liu, Yabing; Ding, Jianping

    2017-01-01

    Human NAD-dependent isocitrate dehydrogenase existing as the α2βγ heterotetramer, catalyzes the decarboxylation of isocitrate into α-ketoglutarate in the Krebs cycle, and is allosterically regulated by citrate, ADP and ATP. To explore the functional roles of the regulatory β and γ subunits, we systematically characterized the enzymatic properties of the holoenzyme and the composing αβ and αγ heterodimers in the absence and presence of regulators. The biochemical and mutagenesis data show that αβ and αγ alone have considerable basal activity but the full activity of α2βγ requires the assembly and cooperative function of both heterodimers. α2βγ and αγ can be activated by citrate or/and ADP, whereas αβ cannot. The binding of citrate or/and ADP decreases the S0.5,isocitrate and thus enhances the catalytic efficiencies of the enzymes, and the two activators can act independently or synergistically. Moreover, ATP can activate α2βγ and αγ at low concentration and inhibit the enzymes at high concentration, but has only inhibitory effect on αβ. Furthermore, the allosteric activation of α2βγ is through the γ subunit not the β subunit. These results demonstrate that the γ subunit plays regulatory role to activate the holoenzyme, and the β subunit the structural role to facilitate the assembly of the holoenzyme.

  8. The β and γ subunits play distinct functional roles in the α2βγ heterotetramer of human NAD-dependent isocitrate dehydrogenase

    PubMed Central

    Ma, Tengfei; Peng, Yingjie; Huang, Wei; Liu, Yabing; Ding, Jianping

    2017-01-01

    Human NAD-dependent isocitrate dehydrogenase existing as the α2βγ heterotetramer, catalyzes the decarboxylation of isocitrate into α-ketoglutarate in the Krebs cycle, and is allosterically regulated by citrate, ADP and ATP. To explore the functional roles of the regulatory β and γ subunits, we systematically characterized the enzymatic properties of the holoenzyme and the composing αβ and αγ heterodimers in the absence and presence of regulators. The biochemical and mutagenesis data show that αβ and αγ alone have considerable basal activity but the full activity of α2βγ requires the assembly and cooperative function of both heterodimers. α2βγ and αγ can be activated by citrate or/and ADP, whereas αβ cannot. The binding of citrate or/and ADP decreases the S0.5,isocitrate and thus enhances the catalytic efficiencies of the enzymes, and the two activators can act independently or synergistically. Moreover, ATP can activate α2βγ and αγ at low concentration and inhibit the enzymes at high concentration, but has only inhibitory effect on αβ. Furthermore, the allosteric activation of α2βγ is through the γ subunit not the β subunit. These results demonstrate that the γ subunit plays regulatory role to activate the holoenzyme, and the β subunit the structural role to facilitate the assembly of the holoenzyme. PMID:28139779

  9. 77 FR 1669 - Endangered and Threatened Species; Recovery Plan for the Southern California Steelhead Distinct...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-01-11

    ... Plan for the Southern California Steelhead Distinct Population Segment AGENCY: National Marine... (ESA) recovery plan for the Southern California Steelhead (Oncorhynchus mykiss) Distinct Population.... mykiss were most closely related to the more northern populations of the Southern California...

  10. Cryptococcus gattii induces a cytokine pattern that is distinct from other cryptococcal species.

    PubMed

    Schoffelen, Teske; Illnait-Zaragozi, Maria-Teresa; Joosten, Leo A B; Netea, Mihai G; Boekhout, Teun; Meis, Jacques F; Sprong, Tom

    2013-01-01

    Understanding more about the host's immune response to different Cryptococcus spp. will provide additional insight into the pathogenesis of cryptocococcis. We hypothesized that the ability of C. gattii to cause disease in immunocompetent humans depends on a distinct innate cytokine response of the host to this emerging pathogen. In the current study we assessed the cytokine profile of human peripheral blood mononuclear cells (PBMCs) of healthy individuals, after in vitro stimulation with 40 different well-defined heat-killed isolates of C. gattii, C. neoformans and several hybrid strains. In addition, we investigated the involvement of TLR2, TLR4 and TLR9 in the pro-inflammatory cytokine response to C. gattii. Isolates of C. gattii induced higher concentrations of the pro-inflammatory cytokines IL-1β, TNF-α and IL-6 and the Th17/22 cytokine IL-17 and IL-22 compared to C. neoformans var neoformans and C. neoformans var grubii. In addition, clinical C. gattii isolates induced higher amounts of cytokines than environmental isolates. This difference was not observed in C. neoformans var. grubii isolates. Furthermore, we demonstrated a likely contribution of TLR4 and TLR9, but no role for TLR2, in the host's cytokine response to C. gattii. In conclusion, clinical heat-killed C. gattii isolates induced a more pronounced inflammatory response compared to other Cryptococcus species and non-clinical C. gattii. This is dependent on TLR4 and TLR9 as cellular receptors.

  11. Differential production of reactive oxygen species in distinct brain regions of hypoglycemic mice.

    PubMed

    Amador-Alvarado, Leticia; Montiel, Teresa; Massieu, Lourdes

    2014-09-01

    Hypoglycemia is a serious complication of insulin therapy in patients suffering from type 1 Diabetes Mellitus. Severe hypoglycemia leading to coma (isoelectricity) induces massive neuronal death in vulnerable brain regions such as the hippocampus, the striatum and the cerebral cortex. It has been suggested that the production of reactive oxygen species (ROS) and oxidative stress is involved in hypoglycemic brain damage, and that ROS generation is stimulated by glucose reintroduction (GR) after the hypoglycemic coma. However, the distribution of ROS in discrete brain regions has not been studied in detail. Using the oxidation sensitive marker dihydroethidium (DHE) we have investigated the distribution of ROS in different regions of the mouse brain during prolonged severe hypoglycemia without isoelectricity, as well as the effect of GR on ROS levels. Results show that ROS generation increases in the hippocampus, the cerebral cortex and the striatum after prolonged severe hypoglycemia before the coma. The hippocampus showed the largest increases in ROS levels. GR further stimulated ROS production in the hippocampus and the striatum while in the cerebral cortex, only the somatosensory and parietal areas were significantly affected by GR. Results suggest that ROS are differentially produced during the hypoglycemic insult and that a different response to GR is present among distinct brain regions.

  12. Genome Sequencing and Analysis of Catopsilia pomona nucleopolyhedrovirus: A Distinct Species in Group I Alphabaculovirus

    PubMed Central

    Wang, Jun; Zhu, Zheng; Zhang, Lei; Hou, Dianhai; Wang, Manli; Arif, Basil; Kou, Zheng; Wang, Hualin; Deng, Fei; Hu, Zhihong

    2016-01-01

    The genome sequence of Catopsilia pomona nucleopolyhedrovirus (CapoNPV) was determined by the Roche 454 sequencing system. The genome consisted of 128,058 bp and had an overall G+C content of 40%. There were 130 hypothetical open reading frames (ORFs) potentially encoding proteins of more than 50 amino acids and covering 92% of the genome. Among all the hypothetical ORFs, 37 baculovirus core genes, 23 lepidopteran baculovirus conserved genes and 10 genes conserved in Group I alphabaculoviruses were identified. In addition, the genome included regions of 8 typical baculoviral homologous repeat sequences (hrs). Phylogenic analysis showed that CapoNPV was in a distinct branch of clade “a” in Group I alphabaculoviruses. Gene parity plot analysis and overall similarity of ORFs indicated that CapoNPV is more closely related to the Group I alphabaculoviruses than to other baculoviruses. Interesting, CapoNPV lacks the genes encoding the fibroblast growth factor (fgf) and ac30, which are conserved in most lepidopteran and Group I baculoviruses, respectively. Sequence analysis of the F-like protein of CapoNPV showed that some amino acids were inserted into the fusion peptide region and the pre-transmembrane region of the protein. All these unique features imply that CapoNPV represents a member of a new baculovirus species. PMID:27166956

  13. Achromobacter Species Isolated from Cystic Fibrosis Patients Reveal Distinctly Different Biofilm Morphotypes

    PubMed Central

    Nielsen, Signe M.; Nørskov-Lauritsen, Niels; Bjarnsholt, Thomas; Meyer, Rikke L.

    2016-01-01

    Achromobacter species have attracted attention as emerging pathogens in cystic fibrosis. The clinical significance of Achromobacter infection is not yet fully elucidated; however, their intrinsic resistance to antimicrobials and ability to form biofilms renders them capable of establishing long-term chronic infections. Still, many aspects of Achromobacter biofilm formation remain uncharacterized. In this study, we characterized biofilm formation in clinical isolates of Achromobacter and investigated the effect of challenging the biofilm with antimicrobials and/or enzymes targeting the extracellular matrix. In vitro biofilm growth and subsequent visualization by confocal microscopy revealed distinctly different biofilm morphotypes: a surface-attached biofilm morphotype of small aggregates and an unattached biofilm morphotype of large suspended aggregates. Aggregates consistent with our in vitro findings were visualized in sputum samples from cystic fibrosis patients using an Achromobacter specific peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) probe, confirming the presence of Achromobacter biofilms in the CF lung. High antibiotic tolerance was associated with the biofilm phenotype, and biocidal antibiotic concentrations were up to 1000 fold higher than for planktonic cultures. Treatment with DNase or subtilisin partially dispersed the biofilm and reduced the tolerance to specific antimicrobials, paving the way for further research into using dispersal mechanisms to improve treatment strategies. PMID:27681927

  14. Evidence for a Common Origin of Homomorphic and Heteromorphic Sex Chromosomes in Distinct Spinacia Species

    PubMed Central

    Fujito, Satoshi; Takahata, Satoshi; Suzuki, Reimi; Hoshino, Yoichiro; Ohmido, Nobuko; Onodera, Yasuyuki

    2015-01-01

    The dioecious genus Spinacia is thought to include two wild relatives (S. turkestanica Ilj. and S. tetrandra Stev.) of cultivated spinach (S. oleracea L.). In this study, nuclear and chloroplast sequences from 21 accessions of Spinacia germplasm and six spinach cultivars or lines were subjected to phylogenetic analysis to define the relationships among the three species. Maximum-likelihood sequence analysis suggested that the Spinacia plant samples could be classified into two monophyletic groups (Group 1 and Group 2): Group 1 consisted of all accessions, cultivars, and lines of S. oleracea L. and S. turkestanica Ilj. and two of five S. tetrandra Stev. accessions, whereas Group 2 was composed of the three remaining S. tetrandra Stev. accessions. By using flow cytometry, we detected a distinct difference in nuclear genome size between the groups. Group 2 also was characterized by a sexual dimorphism in inflorescence structure, which was not observed in Group 1. Interspecific crosses between the groups produced hybrids with drastically reduced pollen fertility and showed that the male is the heterogametic sex (XY) in Group 2, as is the case in S. oleracea L. (Group 1). Cytogenetic and DNA marker analyses suggested that Group 1 and Group 2 have homomorphic and heteromorphic sex chromosome pairs (XY), respectively, and that the sex chromosome pairs of the two groups evolved from a common ancestral pair. Our data suggest that the Spinacia genus may serve as a good model for investigation of evolutionary mechanisms underlying the emergence of heteromorphic sex chromosome pairs from ancestral homomorphic pairs. PMID:26048564

  15. α7 and β2 Nicotinic Acetylcholine Receptor Subunits Form Heteromeric Receptor Complexes that Are Expressed in the Human Cortex and Display Distinct Pharmacological Properties

    PubMed Central

    Thomsen, Morten Skøtt; Zwart, Ruud; Ursu, Daniel; Jensen, Majbrit Myrup; Pinborg, Lars Hageman; Gilmour, Gary; Wu, Jie; Sher, Emanuele; Mikkelsen, Jens Damsgaard

    2015-01-01

    The existence of α7β2 nicotinic acetylcholine receptors (nAChRs) has recently been demonstrated in both the rodent and human brain. Since α7-containing nAChRs are promising drug targets for schizophrenia and Alzheimer’s disease, it is critical to determine whether α7β2 nAChRs are present in the human brain, in which brain areas, and whether they differ functionally from α7 nAChR homomers. We used α-bungarotoxin to affinity purify α7-containing nAChRs from surgically excised human temporal cortex, and found that α7 subunits co-purify with β2 subunits, indicating the presence of α7β2 nAChRs in the human brain. We validated these results by demonstrating co-purification of β2 from wild-type, but not α7 or β2 knock-out mice. The pharmacology and kinetics of human α7β2 nAChRs differed significantly from that of α7 homomers in response to nAChR agonists when expressed in Xenopus oocytes and HEK293 cells. Notably, α7β2 heteromers expressed in HEK293 cells display markedly slower rise and decay phases. These results demonstrate that α7 subunits in the human brain form heteromeric complexes with β2 subunits, and that human α7β2 nAChR heteromers respond to nAChR agonists with a unique pharmacology and kinetic profile. α7β2 nAChRs thus represent an alternative mechanism for the reported clinical efficacy of α7 nAChR ligands. PMID:26086615

  16. Simultaneous Single Molecule Fluorescence and Conductivity Studies Reveal Distinct Classes of Aβ Species on Lipid Bilayers

    PubMed Central

    Schauerte, Joseph A.; Wong, Pamela T.; Wisser, Kathleen C.; Ding, Hao; Steel, Duncan G.; Gafni, Ari

    2010-01-01

    The extracellular senile plaques prevalent in brain tissue in Alzheimer's disease (AD) are composed of amyloid fibrils formed by the Aβ peptide. These fibrils have been traditionally believed to feature in neurotoxicity; however, numerous recent studies provide evidence that cytotoxicity in AD may be associated with low molecular weight oligomers of Aβ that associate with neuronal membranes and may lead to membrane permeabilization and disruption of the ion balance in the cell. The underlying mechanism leading to disruption of the membrane is the subject of many recent studies. Here we report the application of single molecule optical detection, using fluorescently labeled human Aβ40, combined with membrane conductivity measurements, to monitor the interaction of single oligomeric peptide structures with model planar black lipid membranes (BLM). In a qualitative study, we show that the binding of Aβ to the membrane can be described by three distinctly different behaviors, depending on the Aβ monomer concentration. For concentrations much below 10 nM, there is uniform binding of monomers over the surface of the membrane with no evidence of oligomer formation or membrane permeabilization. Between 10 nM and a few 100 nM, the uniform monomer binding is accompanied by the presence of peptide species ranging from dimers to small oligomers. The dimers are not found to permeabilize the membrane but the larger oligomers lead to permeabilization with individual oligomers producing ion conductances of less than 10 pS/pore. At higher concentration, perhaps beyond physiologically relevant concentrations, larger extended and dynamic structures are found with large conductance (100's of pS) suggesting major disruption of the membrane. PMID:20201586

  17. DNA barcoding, MALDI-TOF, and AFLP data support Fusarium ficicrescens as a distinct species within the Fusarium fujikuroi species complex.

    PubMed

    Al-Hatmi, Abdullah M S; Mirabolfathy, Mansoureh; Hagen, Ferry; Normand, Anne-Cécile; Stielow, J Benjamin; Karami-Osbo, Rouhollah; van Diepeningen, Anne D; Meis, Jacques F; de Hoog, G Sybren

    2016-02-01

    The Fusarium fujikuroi species complex (FFSC) is one of the most common groups of fusaria associated with plant diseases, mycotoxin production and traumatic and disseminated human infections. Here we present the description and taxonomy of a new taxon, Fusarium ficicrescens sp. nov., collected from contaminated fig fruits in Iran. Initially this species was identified as Fusarium andiyazi by morphology. In the present study the species was studied by multilocus sequence analysis, amplified fragment length polymorphism (AFLP), matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and phenotypic characters. Multilocus analyses were based on translation elongation factor 1α (TEF1), RNA polymerase subunit (RPB2) and beta-tubulin (BT2) and proved F. ficicrescens as a member of the FFSC. Phylogenetic analysis showed that the fungus is closely related to Fusarium lactis, Fusarium ramigenum, and Fusarium napiforme; known plant pathogens, mycotoxin producers, and occasionally occurring multidrug resistant opportunists. The new species differed by being able to grow at 37 °C and by the absence of mycotoxin production. TEF1 was confirmed as an essential barcode for identifying Fusarium species. In addition to TEF1, we evaluated BT2 and RPB2 in order to provide sufficient genetic and species boundaries information for recognition of the novel species.

  18. Phosphatidylinositol 3-kinase activation is mediated by high-affinity interactions between distinct domains within the p110 and p85 subunits.

    PubMed Central

    Holt, K H; Olson, L; Moye-Rowley, W S; Pessin, J E

    1994-01-01

    Domains of interaction between the p85 and p110 subunits of phosphatidylinositol 3-kinase (PI 3-kinase) were studied with the yeast two-hybrid expression system. A gene fusion between the GAL4 transactivation domain and p85 activated transcription from a GAL1-lacZ reporter gene when complemented with a gene fusion between the GAL4 DNA binding domain and p110. To define subdomains responsible for this interaction, a series of p85 deletion mutants were analyzed. A 192-amino-acid inter-SH2 (IS) fragment (residues 429 to 621) was the smallest determinant identified that specifically associated with p110. In analogous experiments, the subdomain within p110 responsible for interaction with p85 was localized to an EcoRI fragment encoding the amino-terminal 127 residues. Expression of these two subdomains [p85(IS) with p110RI] resulted in 100-fold greater reporter activity than that obtained with full-length p85 and p110. Although the p85(IS) domain conferred a strong interaction with the p110 catalytic subunit, this region was not sufficient to impart phosphotyrosine peptide stimulation of PI 3-kinase activity. In contrast, coexpression of the p110 subunit with full-length p85 or with constructs containing the IS sequences flanked by both SH2 domains of p85 [p85(n/cSH2)] or either of the individual SH2 domains [p85(nSH2+IS) or p85(IS+cSH2)] resulted in PI 3-kinase activity that was activated by a phosphotyrosine peptide. These data suggest that phosphotyrosine peptide binding to either SH2 domain generates an intramolecular signal propagated through the IS region to allosterically activate p110. Images PMID:8264609

  19. 75 FR 30769 - Endangered and Threatened Species; Proposed Listing of Nine Distinct Population Segments of...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-02

    ... Distinct Population Segments of Loggerhead Sea Turtles as Endangered or Threatened; Extension of Comment... proposed listing of nine distinct population segments of loggerhead sea turtles as endangered or threatened... . Mail: NMFS National Sea Turtle Coordinator, Attn: Loggerhead Proposed Listing Rule, Office of...

  20. 76 FR 15932 - Endangered and Threatened Species; Proposed Listing of Nine Distinct Population Segments of...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-22

    ... Population Segments of Loggerhead Sea Turtles as Endangered or Threatened AGENCIES: National Marine Fisheries... to list nine Distinct Population Segments (DPS) of loggerhead sea turtles, Caretta caretta, as... Ocean DPS of the loggerhead turtle. Additionally, considerable disagreement exists regarding...

  1. Poles apart: the "bipolar" pteropod species Limacina helicina is genetically distinct between the Arctic and Antarctic oceans.

    PubMed

    Hunt, Brian; Strugnell, Jan; Bednarsek, Nina; Linse, Katrin; Nelson, R John; Pakhomov, Evgeny; Seibel, Brad; Steinke, Dirk; Würzberg, Laura

    2010-03-23

    The shelled pteropod (sea butterfly) Limacina helicina is currently recognised as a species complex comprising two sub-species and at least five "forma". However, at the species level it is considered to be bipolar, occurring in both the Arctic and Antarctic oceans. Due to its aragonite shell and polar distribution L. helicina is particularly vulnerable to ocean acidification. As a key indicator of the acidification process, and a major component of polar ecosystems, L. helicina has become a focus for acidification research. New observations that taxonomic groups may respond quite differently to acidification prompted us to reassess the taxonomic status of this important species. We found a 33.56% (+/-0.09) difference in cytochrome c oxidase subunit I (COI) gene sequences between L. helicina collected from the Arctic and Antarctic oceans. This degree of separation is sufficient for ordinal level taxonomic separation in other organisms and provides strong evidence for the Arctic and Antarctic populations of L. helicina differing at least at the species level. Recent research has highlighted substantial physiological differences between the poles for another supposedly bipolar pteropod species, Clione limacina. Given the large genetic divergence between Arctic and Antarctic L. helicina populations shown here, similarly large physiological differences may exist between the poles for the L. helicina species group. Therefore, in addition to indicating that L. helicina is in fact not bipolar, our study demonstrates the need for acidification research to take into account the possibility that the L. helicina species group may not respond in the same way to ocean acidification in Arctic and Antarctic ecosystems.

  2. Sequences of the cytochrome C oxidase subunit I (COI) gene are suitable for species identification of Korean Calliphorinae flies of forensic importance (Diptera: Calliphoridae).

    PubMed

    Park, Seong Hwan; Zhang, Yong; Piao, Huguo; Yu, Dong Ha; Jeong, Hyun Ju; Yoo, Ga Young; Jo, Tae-Ho; Hwang, Juck-Joon

    2009-09-01

    Calliphorinae fly species are important indicators of the postmortem interval especially during early spring and late fall in Korea. Although nucleotide sequences of various Calliphorinae fly species are available, there has been no research on the cytochrome c oxidase subunit I (COI) nucleotide sequences of Korean Calliphorinae flies. Here, we report the full-length sequences of the COI gene of four Calliphorinae fly species collected in Korea (five individuals of Calliphora vicina, five Calliphora lata, four Triceratopyga calliphoroides and three Aldrichina grahami). Each COI gene was amplified by polymerase chain reaction and directly sequenced and the resulting nucleotide sequences were aligned and analyzed by MEGA4 software. The results indicate that COI nucleotide sequences can be used to distinguish between these four species. Our phylogenetic result coincides with recent taxonomic views on the subfamily Calliphorinae in that the genera Aldrichina and Triceratopyga are nested within the genus Calliphora.

  3. Multilocus evidence for globally distributed cryptic species and distinct populations across ocean gyres in a mesopelagic copepod.

    PubMed

    Andrews, Kimberly R; Norton, Emily L; Fernandez-Silva, Iria; Portner, Elan; Goetze, Erica

    2014-11-01

    Zooplanktonic taxa have a greater number of distinct populations and species than might be predicted based on their large population sizes and open-ocean habitat, which lacks obvious physical barriers to dispersal and gene flow. To gain insight into the evolutionary mechanisms driving genetic diversification in zooplankton, we developed eight microsatellite markers to examine the population structure of an abundant, globally distributed mesopelagic copepod, Haloptilus longicornis, at 18 sample sites across the Atlantic and Pacific Oceans (n = 761). When comparing our microsatellite results with those of a prior study that used a mtDNA marker (mtCOII, n = 1059, 43 sample sites), we unexpectedly found evidence for the presence of a cryptic species pair. These species were globally distributed and apparently sympatric, and were separated by relatively weak genetic divergence (reciprocally monophyletic mtCOII lineages 1.6% divergent; microsatellite FST ranging from 0.28 to 0.88 across loci, P < 0.00001). Using both mtDNA and microsatellite data for the most common of the two species (n = 669 for microsatellites, n = 572 for mtDNA), we also found evidence for allopatric barriers to gene flow within species, with distinct populations separated by continental landmasses and equatorial waters in both the Atlantic and Pacific Ocean basins. Our study shows that oceanic barriers to gene flow can act as a mechanism promoting allopatric diversification in holoplanktonic taxa, despite the high potential dispersal abilities and pelagic habitat for these species.

  4. Distinctive life traits and distribution along environmental gradients of dominant and subordinate Mediterranean ant species.

    PubMed

    Arnan, Xavier; Cerdá, Xim; Retana, Javier

    2012-10-01

    For most animal and plant species, life traits strongly affect their species-specific role, function or position within ecological communities. Previous studies on ant communities have mostly focused on the role of dominant species and the outcome of interspecific interactions. However, life traits of ant species have seldom been considered within a community framework. This study (1) analyses life traits related to ecological and behavioural characteristics of dominant and subordinate ant species from 13 sites distributed throughout the Iberian Peninsula, (2) determines how similar the ant species are within each of the two levels of the dominance hierarchy, and (3) establishes the distribution patterns of these different groups of species along environmental gradients. Our results showed that the differences between dominants and subordinates fall into two main categories: resource exploitation and thermal tolerance. Dominant species have more populated colonies and defend food resources more fiercely than subordinates, but they display low tolerance to high temperatures. We have identified different assemblages of species included within each of these two levels in the dominance hierarchy. The distribution of these assemblages varied along the environmental gradient, shifting from dominant Dolichoderinae and cryptic species in moist areas, to dominant Myrmicinae and hot climate specialists mainly in open and hot sites. We have been able to identify a set of life traits of the most common Iberian ant species that has enabled us to characterise groups of dominant and subordinate species. Although certain common features within the groups of both dominants and subordinates always emerge, other different features allow for differentiating subgroups within each of these groups. These different traits allow the different subgroups coping with particular conditions across environmental gradients.

  5. 78 FR 69310 - Endangered and Threatened Species; Protective Regulations for the Gulf of Maine Distinct...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-19

    ... oxyrinchus). On February 6, 2012, we listed the Gulf of Maine DPS of Atlantic sturgeon as threatened under the Endangered Species Act (ESA). When a species is listed as threatened under the ESA, we are required to issue protective regulations under section 4(d) of the ESA. Such protective regulations...

  6. Meiotic cohesin subunits RAD21L and REC8 are positioned at distinct regions between lateral elements and transverse filaments in the synaptonemal complex of mouse spermatocytes

    PubMed Central

    RONG, Mei; MATSUDA, Atsushi; HIRAOKA, Yasushi; LEE, Jibak

    2016-01-01

    Cohesins containing a meiosis-specific α-kleisin subunit, RAD21L or REC8, play roles in diverse aspects of meiotic chromosome dynamics including formation of axial elements (AEs), assembly of the synaptonemal complex (SC), recombination of homologous chromosomes (homologs), and cohesion of sister chromatids. However, the exact functions of individual α-kleisins remain to be elucidated. Here, we examined the localization of RAD21L and REC8 within the SC by super-resolution microscopy, 3D-SIM. We found that both RAD21L and REC8 were localized at the connection sites between lateral elements (LEs) and transverse filaments (TFs) of pachynema with RAD21L locating interior to REC8 sites. RAD21L and REC8 were not symmetrical in terms of synaptic homologs, suggesting that the arrangement of different cohesins is not strictly fixed along all chromosome axes. Intriguingly, some RAD21L signals, but not REC8 signals, were observed between unsynapsed regions of AEs of zygonema as if they formed a bridge between homologs. Furthermore, the signals of recombination intermediates overlapped with those of RAD21L to a greater degree than with those of REC8. These results highlight the different properties of two meiotic α-kleisins, and strongly support the previous proposition that RAD21L is an atypical cohesin that establishes the association between homologs rather than sister chromatids. PMID:27665783

  7. Identification and Typing of Malassezia Species by Amplified Fragment Length Polymorphism and Sequence Analyses of the Internal Transcribed Spacer and Large-Subunit Regions of Ribosomal DNA

    PubMed Central

    Gupta, Aditya K.; Boekhout, Teun; Theelen, Bart; Summerbell, Richard; Batra, Roma

    2004-01-01

    Malassezia yeasts are associated with several dermatological disorders. The conventional identification of Malassezia species by phenotypic methods is complicated and time-consuming, and the results based on culture methods are difficult to interpret. A comparative molecular approach based on the use of three molecular techniques, namely, amplified fragment length polymorphism (AFLP) analysis, sequencing of the internal transcribed spacer, and sequencing of the D1 and D2 domains of the large-subunit ribosomal DNA region, was applied for the identification of Malassezia species. All species could be correctly identified by means of these methods. The results of AFLP analysis and sequencing were in complete agreement with each other. However, some discrepancies were noted when the molecular methods were compared with the phenotypic method of identification. Specific genotypes were distinguished within a collection of Malassezia furfur isolates from Canadian sources. AFLP analysis revealed significant geographical differences between the North American and European M. furfur strains. PMID:15365020

  8. Distinct Leishmania Species Infecting Wild Caviomorph Rodents (Rodentia: Hystricognathi) from Brazil

    PubMed Central

    Cássia-Pires, Renata; Boité, Mariana C.; D'Andrea, Paulo S.; Herrera, Heitor M.; Cupolillo, Elisa; Jansen, Ana Maria; Roque, André Luiz R.

    2014-01-01

    Background Caviomorph rodents, some of the oldest Leishmania spp. hosts, are widely dispersed in Brazil. Despite both experimental and field studies having suggested that these rodents are potential reservoirs of Leishmania parasites, not more than 88 specimens were analyzed in the few studies of natural infection. Our hypothesis was that caviomorph rodents are inserted in the transmission cycles of Leishmania in different regions, more so than is currently recognized. Methodology We investigated the Leishmania infection in spleen fragments of 373 caviomorph rodents from 20 different species collected in five Brazilian biomes in a period of 13 years. PCR reactions targeting kDNA of Leishmania sp. were used to diagnose infection, while Leishmania species identification was performed by DNA sequencing of the amplified products obtained in the HSP70 (234) targeting. Serology by IFAT was performed on the available serum of these rodents. Principal findings In 13 caviomorph rodents, DNA sequencing analyses allowed the identification of 4 species of the subgenus L. (Viannia): L. shawi, L. guyanensis, L. naiffi, and L. braziliensis; and 1 species of the subgenus L. (Leishmania): L. infantum. These include the description of parasite species in areas not previously included in their known distribution: L. shawi in Thrichomys inermis from Northeastern Brazil and L. naiffi in T. fosteri from Western Brazil. From the four other positive rodents, two were positive for HSP70 (234) targeting but did not generate sequences that enabled the species identification, and another two were positive only in kDNA targeting. Conclusions/Significance The infection rate demonstrated by the serology (51.3%) points out that the natural Leishmania infection in caviomorph rodents is much higher than that observed in the molecular diagnosis (4.6%), highlighting that, in terms of the host species responsible for maintaining Leishmania species in the wild, our current knowledge represents only the

  9. Fingerprinting species and strains of Bacilli spores by distinctive coat surface morphology.

    PubMed

    Wang, Rong; Krishnamurthy, Soumya N; Jeong, Jae-Sun; Driks, Adam; Mehta, Manav; Gingras, Bruce A

    2007-09-25

    In this work, we applied high-resolution atomic force microscopy (AFM) to identify and characterize similarities and differences in the spore surface morphology of strains from four species of Bacilli: B. anthracis, B. cereus, B. pumilis, and B. subtilis. Common features of the examined spores in the dry state included ridges that spanned the long axis of each spore, and nanometer-scale fine rodlets that covered the entire spore surface. However, important differences in these features between species permitted them to be distinguished by AFM. Specifically, each species possessed significant variation in ridge architecture, and the rodlet width in B. anthracis was significantly less than that of the other species. To characterize similarities and differences within a species, we examined three B. subtilis strains. The ridge patterns among the three strains were largely the same; however, we detected significant differences in the ridge dimensions. Taken together, these experiments provide important information about natural variation in spore surface morphology, define structural features that can serve as species- and strain-specific signatures, and give insight into the dynamics of spore coat flexibility and its role during spore dormancy and germination.

  10. Analyses of mitochondrial amino acid sequence datasets support the proposal that specimens of Hypodontus macropi from three species of macropodid hosts represent distinct species

    PubMed Central

    2013-01-01

    Background Hypodontus macropi is a common intestinal nematode of a range of kangaroos and wallabies (macropodid marsupials). Based on previous multilocus enzyme electrophoresis (MEE) and nuclear ribosomal DNA sequence data sets, H. macropi has been proposed to be complex of species. To test this proposal using independent molecular data, we sequenced the whole mitochondrial (mt) genomes of individuals of H. macropi from three different species of hosts (Macropus robustus robustus, Thylogale billardierii and Macropus [Wallabia] bicolor) as well as that of Macropicola ocydromi (a related nematode), and undertook a comparative analysis of the amino acid sequence datasets derived from these genomes. Results The mt genomes sequenced by next-generation (454) technology from H. macropi from the three host species varied from 13,634 bp to 13,699 bp in size. Pairwise comparisons of the amino acid sequences predicted from these three mt genomes revealed differences of 5.8% to 18%. Phylogenetic analysis of the amino acid sequence data sets using Bayesian Inference (BI) showed that H. macropi from the three different host species formed distinct, well-supported clades. In addition, sliding window analysis of the mt genomes defined variable regions for future population genetic studies of H. macropi in different macropodid hosts and geographical regions around Australia. Conclusions The present analyses of inferred mt protein sequence datasets clearly supported the hypothesis that H. macropi from M. robustus robustus, M. bicolor and T. billardierii represent distinct species. PMID:24261823

  11. Four tropical, closely related fern species belonging to the genus Adiantum L. are genetically distinct as revealed by ISSR fingerprinting.

    PubMed

    Korpelainen, Helena; de Britto, John; Doublet, Jérémy; Pravin, Sahaya

    2005-11-01

    The level and pattern of genetic variation was analyzed in four species of the fern genus Adiantum L., A. hispidulum Sw., A. incisum Forrsk., A. raddianum C.Presl, and A. zollingeri Mett. ex Kuhn, originating from South India, using the ISSR fingerprinting method. The populations of Adiantum possessed a considerable level of genetic variation, the diversity indices ranging from 0.284 to 0.464. Only 12% of the ISSR markers found were restricted to one species only, and 54% were detected in all four species. The analysis of molecular variance revealed that 71.1% of variation was present within populations. The proportion of variation detected among species was only 18.5% while the proportion of variation among populations within species equalled 10.4%. Despite the low level of intrageneric differentiation, the discriminant analysis and clustering of genetic distances indicated that the four Adiantum species are genetically distinct. The F(ST) values calculated for the species were low, varying from 0.089 to 0.179. No linkage disequilibrium was detected between the loci. Such low level of differentiation among populations and the presence of linkage equilibrium reflect that the life history of Adiantum ferns apparently involves common or relatively common sexuality, effective wind-dispersal of spores and outcrossing.

  12. Development of a multiplex RT-PCR-ELISA to identify four distinct species of tospovirus.

    PubMed

    Charoenvilaisiri, Saengsoon; Seepiban, Channarong; Bhunchoth, Anjana; Warin, Nuchnard; Luxananil, Plearnpis; Gajanandana, Oraprapai

    2014-06-01

    In this study, a multiplex RT-PCR-ELISA was developed to detect and differentiate four tospovirus species found in Thailand, namely Capsicum chlorosis virus (CaCV), Melon yellow spot virus (MYSV), Tomato necrotic ringspot virus (TNRV), and Watermelon silver mottle virus (WSMoV). In this system, nucleocapsid (N) gene fragments of four tospoviruses were simultaneously amplified and labeled with digoxigenin (DIG) in a single RT-PCR reaction using a pair of degenerate primers binding to the same conserved regions in all four tospovirus N genes. The DIG-labeled amplicons were distinguished into species by four parallel hybridizations to species-specific biotinylated probes in streptavidin-coated microtiter wells followed by ELISA detection using a peroxidase-conjugated anti-DIG antibody. Results indicated that the multiplex RT-PCR-ELISA assay could specifically identify each of these four tospoviruses without cross-reactivity between species or reactivity to healthy plant negative controls. Assay sensitivity was 10- to 1000-fold higher than conventional RT-PCR. When applied to naturally infected plants, all samples yielded concordant results between RT-PCR-ELISA and the reference RT-PCR. In conclusion, the multiplex RT-PCR-ELISA developed in this study has superior specificity, sensitivity, and high-throughput capacity compared to conventional RT-PCR and is an attractive alternative for the identification of different tospovirus species.

  13. A distinct alleles and genetic recombination of pmrCAB operon in species of Acinetobacter baumannii complex isolates.

    PubMed

    Kim, Dae Hun; Ko, Kwan Soo

    2015-07-01

    To investigate pmrCAB sequence divergence in 5 species of Acinetobacter baumannii complex, a total of 80 isolates from a Korean hospital were explored. We evaluated nucleotide and amino acid polymorphisms of pmrCAB operon, and phylogenetic trees were constructed for each gene of prmCAB operon. Colistin and polymyxin B susceptibility was determined for all isolates, and multilocus sequence typing was also performed for A. baumannii isolates. Our results showed that each species of A. baumannii complex has divergent pmrCAB operon sequences. We identified a distinct pmrCAB allele allied with Acinetobacter nosocomialis in gene trees. Different grouping in each gene tree suggests sporadic recombination or emergence of pmrCAB genes among Acinetobacter species. Sequence polymorphisms among Acinetobacter species might not be associated with colistin resistance. We revealed that a distinct pmrCAB allele may be widespread across the continents such as North America and Asia and that sporadic genetic recombination or emergence of pmrCAB genes might occur.

  14. Are Phenacoccus solani Ferris and P. defectus Ferris (Hemiptera: Pseudococcidae) distinct species?

    PubMed

    Chatzidimitriou, Evangelia; Simonato, Mauro; Watson, Gillian W; Martinez-Sañudo, Isabel; Tanaka, Hirotaka; Zhao, Jing; Pellizzari, Giuseppina

    2016-03-24

    Among the Nearctic species of Phenacoccus (Hemiptera: Pseudococcidae), Phenacoccus solani Ferris and P. defectus Ferris are morphologically similar and it can be difficult to separate them on the basis of microscopic morphological characters of the adult female alone. In order to resolve their identity, a canonical variates morphological analysis of 199 specimens from different geographical origins and host plants and a molecular analysis of the COI and 28S genes were performed. The morphological analysis supported synonymy of the two species, as although the type specimens of the "species" are widely separated from each other in the canonical variates plot, they are all part of a continuous range of variation. The molecular analysis showed that P. solani and P. defectus are grouped in the same clade. On the basis of the morphological and molecular analyses, P. defectus is synonymized under the senior name P. solani, syn. n.

  15. Mercury's Exosphere During MESSENGER's Second Flyby: Detection of Magnesium and Distinct Distributions of Neutral Species

    NASA Technical Reports Server (NTRS)

    McClintock, William E.; Vervack, Ronald J., Jr.; Bradley, E. Todd; Killen, Rosemary M.; Mouawad, Nelly; Sprague, Ann L.; Burger, Matthew H.; Solomon, Sean C.; Izenberg, Noam R.

    2009-01-01

    During MESSENGER's second Mercury flyby, the Mercury Atmospheric and Surface Composition Spectrometer observed emission from Mercury's neutral exosphere. These observations include the first detection of emission from magnesium. Differing spatial distributions for sodium, calcium, and magnesium were revealed by observations beginning in Mercury's tail region, approximately 8 Mercury radii anti-sunward of the planet, continuing past the nightside, and ending near the dawn terminator. Analysis of these observations, supplemented by observations during the first Mercury flyby as well as those by other MESSENGER instruments, suggests that the distinct spatial distributions arise from a combination of differences in source, transfer, and loss processes.

  16. The discovery of the two types of small subunit ribosomal RNA gene in Eimeria mitis contests the existence of E. mivati as an independent species.

    PubMed

    Vrba, Vladimir; Poplstein, Martin; Pakandl, Michal

    2011-12-29

    Although the validity of the coccidian species, Eimeria mivati, has been questioned by many researchers for a long time there has not been any molecular analysis that would help resolve this issue. Here we report on the discovery of the two types of small ribosomal subunit (18S) gene within the Eimeria mitis genome that correspond to the known 18S sequences of E. mitis and E. mivati, and this is in conflict with the existence of E. mivati as an independent species. We have carried out five single oocyst isolations to obtain five single-oocyst-derived strains of E. mitis and these were analyzed by the sequencing of 18S and mitochondrial cytochrome c oxidase subunit I genes. The two types of 18S gene were found to be present in each strain in roughly equal ratios. This indicates that if the strains carrying only one or the other 18S type exist, they will likely cross-breed and still represent a single species. However, the more probable explanation is that all strains of E. mitis contain two types of 18S gene and that the occasional detection of only one or the other type by sequencing might be caused by insufficient sampling. This is also the first report of the two types of 18S gene in Eimeria, which has already been described in some other apicomplexan species, most notably Plasmodium. We also found that these two types of ribosomal RNA differ significantly in their secondary structure. The biological significance of the two 18S gene variants in E. mitis is not known, however, we hypothesize that these variants might be used in different stages of the parasite's life-cycle as it is in other apicomplexan species investigated so far.

  17. 78 FR 66139 - Endangered and Threatened Species; Delisting of the Eastern Distinct Population Segment of...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-04

    ..., management of habitat, conservation status, or regulatory mechanisms exist that are significant in light of... species and to evaluate the adequacy of ongoing research and management. The Team recommended that NMFS..., particularly in regard to prey populations, must be maintained through appropriate fisheries management...

  18. Occurrence of putative pathogenicity islands in enterococci from distinct species and of differing origins.

    PubMed

    Semedo-Lemsaddek, Teresa; Barreto-Crespo, Maria Teresa; Tenreiro, Rogério

    2009-11-01

    Enterococci isolated from ewe's milk and cheese, clinical isolates of human and veterinary origins, and reference strains obtained from culture collections were screened for the occurrence of putative pathogenicity island (PAIs). Results obtained after PCR amplification and hybridization point toward PAI dissemination among enterococci of diverse origins (food/clinical) and species (Enterococcus faecalis/non-E. faecalis).

  19. The wild tomato species Solanum chilense shows variation in pathogen resistance between geographically distinct populations

    PubMed Central

    Scheikl, Daniela; Tellier, Aurélien

    2017-01-01

    Wild tomatoes are a valuable source of disease resistance germplasm for tomato (Solanum lycopersicum) breeders. Many species are known to possess a certain degree of resistance against certain pathogens; however, evolution of resistance traits is yet poorly understood. For some species, like Solanum chilense, both differences in habitat and within species genetic diversity are very large. Here we aim to investigate the occurrence of spatially heterogeneous coevolutionary pressures between populations of S. chilense. We investigate the phenotypic differences in disease resistance within S. chilense against three common tomato pathogens (Alternaria solani, Phytophthora infestans and a Fusarium sp.) and confirm high degrees of variability in resistance properties between selected populations. Using generalised linear mixed models, we show that disease resistance does not follow the known demographic patterns of the species. Models with up to five available climatic and geographic variables are required to best describe resistance differences, confirming the complexity of factors involved in local resistance variation. We confirm that within S. chilense, resistance properties against various pathogens show a mosaic pattern and do not follow environmental patterns, indicating the strength of local pathogen pressures. Our study can form the basis for further investigations of the genetic traits involved. PMID:28133579

  20. A Hepatozoon species genetically distinct from H. canis infecting spotted hyenas in the Serengeti ecosystem, Tanzania.

    PubMed

    East, Marion L; Wibbelt, Gudrun; Lieckfeldt, Dietmar; Ludwig, Arne; Goller, Katja; Wilhelm, Kerstin; Schares, Gereon; Thierer, Dagmar; Hofer, Heribert

    2008-01-01

    Health monitoring of spotted hyenas (Crocuta crocuta) in the Serengeti ecosystem, Tanzania, revealed Hepatozoon infection in all of 11 immature individuals examined following death from natural causes. Hepatozoon infection was probably an important factor contributing to mortality in two cases that exhibited clinical signs of ataxia, lethargy, ocular discharge, retching, and labored breathing before death. Whether Hepatozoon infection contributed to six deaths from fire, probable lion predation and unknown causes could not be determined. Four deaths from infanticide and starvation were unlikely to be associated with Hepatozoon infection. Histologic examination revealed lung tissue infected with cyst-like structures containing protozoan stages in all eight cases examined and interstitial pneumonia in most cases. Systemic spread of infection to several organs was found in three cases. Alignment of a 426 bp sequence from the parasite's 18s rRNA gene revealed a Hepatozoon species identical to that recently described from two domestic cats in Spain and only 7 bp substitutions when a 853 bp sequence was aligned to this cat Hepatozoon species. Previous reports of infection of wild carnivores in eastern and southern Africa with an unspecified Hepatozoon species similar in appearance to H. canis may have involved the species described in this study.

  1. Subunit sequences of the 4 x 6-mer hemocyanin from the golden orb-web spider, Nephila inaurata.

    PubMed

    Averdam, Anne; Markl, Jürgen; Burmester, Thorsten

    2003-08-01

    The transport of oxygen in the hemolymph of many arthropod and mollusc species is mediated by large copper-proteins that are referred to as hemocyanins. Arthropod hemocyanins are composed of hexamers and oligomers of hexamers. Arachnid hemocyanins usually form 4 x 6-mers consisting of seven distinct subunit types (termed a-g), although in some spider taxa deviations from this standard scheme have been observed. Applying immunological and electrophoretic methods, six distinct hemocyanin subunits were identified in the red-legged golden orb-web spider Nephila inaurata madagascariensis (Araneae: Tetragnathidae). The complete cDNA sequences of six subunits were obtained that corresponded to a-, b-, d-, e-, f- and g-type subunits. No evidence for a c-type subunit was found in this species. The inclusion of the N. inaurata hemocyanins in a multiple alignment of the arthropod hemocyanins and the application of the Bayesian method of phylogenetic inference allow, for the first time, a solid reconstruction of the intramolecular evolution of the chelicerate hemocyanin subunits. The branch leading to subunit a diverged first, followed by the common branch of the dimer-forming b and c subunits, while subunits d and f, as well as subunits e and g form common branches. Assuming a clock-like evolution of the chelicerate hemocyanins, a timescale for the evolution of the Chelicerata was obtained that agrees with the fossil record.

  2. Sequences of conserved region in the A subunit of DNA gyrase from nine species of the genus Mycobacterium: phylogenetic analysis and implication for intrinsic susceptibility to quinolones.

    PubMed

    Guillemin, I; Cambau, E; Jarlier, V

    1995-09-01

    The sequences of a conserved region in the A subunit of DNA gyrase corresponding to the quinolone resistance-determining region were determined for nine mycobacterial species and were compared. Although the nucleotide sequences were highly conserved, they clearly differentiated one species from another. The results of the phylogenetic analysis based on the sequences of the quinolone resistance-determining regions were compared with those provided by the 16S rRNA sequences. Deduced amino acid sequences were identical within the nine species except for amino acid 83, which was frequently involved in acquired resistance to quinolones in many genera, including mycobacteria. The presence at position 83 of an alanine for seven mycobacterial species (M. tuberculosis, M. bovis BCG, M. leprae, M. avium, M. kansasii, M. chelonae, and M. smegmatis) and of a serine for the two remaining mycobacterial species (M. fortuitum and M. aurum) correlated well with the MICs of ofloxacin for both groups of species, suggesting the role of this residue in intrinsic susceptibility to quinolones in mycobacteria.

  3. Use of cytochrome c oxidase subunit i (COI) nucleotide sequences for identification of the Korean Luciliinae fly species (Diptera: Calliphoridae) in forensic investigations.

    PubMed

    Park, Seong Hwan; Zhang, Yong; Piao, Huguo; Yu, Dong Ha; Jeong, Hyun Ju; Yoo, Ga Young; Chung, Ukhee; Jo, Tae-Ho; Hwang, Juck-Joon

    2009-12-01

    Blowflies, especially species belonging to the subfamily Luciliinae, are the first insects to lay eggs on corpses in Korea. Fast and accurate species identification has been a key task for forensic entomologists. Because conventional morphologic identification methods have many limitations with respect to forensic practice, molecular methods have been proposed to identify fly species of forensic importance. To this end, the authors amplified and sequenced the full length of the cytochrome c oxidase subunit I (COI) gene of the Luciliinae fly species collected in Korea. The results showed the COI sequences are instrumental in identifying Luciliinae fly species. However, when compared with previously reported data, considerable inconsistencies were noted. Hemipyrellia ligurriens data in this study differed significantly from two of the five pre-existing data. Two closely related species, Lucilia illustris and Lucilia caesar, showed an overlap of COI haplotypes due to four European sequences. The results suggest that more individuals from various geographic regions and additive nuclear DNA markers should be analyzed, and morphologic identification keys must be reconfirmed to overcome these inconsistencies.

  4. Sink-source characteristics of two distinctly different forest species as affected by elevated carbon dioxide

    SciTech Connect

    Pushnik, J.C.; Florv, W.B.; Demaree, R.S. ); Anderson, P.D.; Houpis J.L.J. )

    1993-05-01

    The basic physiology and biochemistry of photosynthesis is being correlated with the leaf level processes and morphology of the Sierra Nevada varieties of Taxus brevifolia and Pinus ponderosa in an attempt to identify control mechanisms of carbohydrate partitioning. We are evaluating sink/source relationships in terms of carbon assimilation (gas-exchange (A[ci] curves and temperature effects); RuBPCase activity, chloroplast structure, integrity, and distributions, stomatal densities, internal leaf organization); transport functions (sucrose-phosphate synthetase (SPS) activity); long-term sink (immunoelectron microscopic detection of taxol). The results of these investigations suggest carbon acquisition characteristics are similar among the conifers, but with distinct differences in carboxylation efficiencies, SPS activity, needle starch content/chloroplast, and vascular tissue areas. These baseline characteristics are currently being evaluated in response to elevated CO[sub 2].

  5. Contributions of distinct gold species to catalytic reactivity for carbon monoxide oxidation

    NASA Astrophysics Data System (ADS)

    Guo, Li-Wen; Du, Pei-Pei; Fu, Xin-Pu; Ma, Chao; Zeng, Jie; Si, Rui; Huang, Yu-Ying; Jia, Chun-Jiang; Zhang, Ya-Wen; Yan, Chun-Hua

    2016-11-01

    Small-size (<5 nm) gold nanostructures supported on reducible metal oxides have been widely investigated because of the unique catalytic properties they exhibit in diverse redox reactions. However, arguments about the nature of the gold active site have continued for two decades, due to the lack of comparable catalyst systems with specific gold species, as well as the scarcity of direct experimental evidence for the reaction mechanism under realistic working conditions. Here we report the determination of the contribution of single atoms, clusters and particles to the oxidation of carbon monoxide at room temperature, by the aid of in situ X-ray absorption fine structure analysis and in situ diffuse reflectance infrared Fourier transform spectroscopy. We find that the metallic gold component in clusters or particles plays a much more critical role as the active site than the cationic single-atom gold species for the room-temperature carbon monoxide oxidation reaction.

  6. Contributions of distinct gold species to catalytic reactivity for carbon monoxide oxidation

    PubMed Central

    Guo, Li-Wen; Du, Pei-Pei; Fu, Xin-Pu; Ma, Chao; Zeng, Jie; Si, Rui; Huang, Yu-Ying; Jia, Chun-Jiang; Zhang, Ya-Wen; Yan, Chun-Hua

    2016-01-01

    Small-size (<5 nm) gold nanostructures supported on reducible metal oxides have been widely investigated because of the unique catalytic properties they exhibit in diverse redox reactions. However, arguments about the nature of the gold active site have continued for two decades, due to the lack of comparable catalyst systems with specific gold species, as well as the scarcity of direct experimental evidence for the reaction mechanism under realistic working conditions. Here we report the determination of the contribution of single atoms, clusters and particles to the oxidation of carbon monoxide at room temperature, by the aid of in situ X-ray absorption fine structure analysis and in situ diffuse reflectance infrared Fourier transform spectroscopy. We find that the metallic gold component in clusters or particles plays a much more critical role as the active site than the cationic single-atom gold species for the room-temperature carbon monoxide oxidation reaction. PMID:27848964

  7. Two Rumex Species from Contrasting Hydrological Niches Regulate Flooding Tolerance through Distinct Mechanisms[C][W][OPEN

    PubMed Central

    van Veen, Hans; Mustroph, Angelika; Barding, Gregory A.; Vergeer-van Eijk, Marleen; Welschen-Evertman, Rob A.M.; Pedersen, Ole; Visser, Eric J.W.; Larive, Cynthia K.; Pierik, Ronald; Bailey-Serres, Julia; Voesenek, Laurentius A.C.J.; Sasidharan, Rashmi

    2013-01-01

    Global climate change has increased flooding events, which affect both natural vegetation dynamics and crop productivity. The flooded environment is lethal for most plant species because it restricts gas exchange and induces an energy and carbon crisis. Flooding survival strategies have been studied in Oryza sativa, a cultivated monocot. However, our understanding of plant adaptation to natural flood-prone environments remains scant, even though wild plants represent a valuable resource of tolerance mechanisms that could be used to generate stress-tolerant crops. Here we identify mechanisms that mediate the distinct flooding survival strategies of two related wild dicot species: Rumex palustris and Rumex acetosa. Whole transcriptome sequencing and metabolite profiling reveal flooding-induced metabolic reprogramming specific to R. acetosa. By contrast, R. palustris uses the early flooding signal ethylene to increase survival by regulating shade avoidance and photomorphogenesis genes to outgrow submergence and by priming submerged plants for future low oxygen stress. These results provide molecular resolution of flooding survival strategies of two species occupying distinct hydrological niches. Learning how these contrasting flood adaptive strategies evolved in nature will be instrumental for the development of stress-tolerant crop varieties that deliver enhanced yields in a changing climate. PMID:24285788

  8. Simultaneous identification and DNA barcoding of six Eimeria species infecting turkeys using PCR primers targeting the mitochondrial cytochrome c oxidase subunit I (mtCOI) locus.

    PubMed

    Hafeez, Mian A; Shivaramaiah, Srichaitanya; Dorsey, Kristi Moore; Ogedengbe, Mosun E; El-Sherry, Shiem; Whale, Julia; Cobean, Julie; Barta, John R

    2015-05-01

    Species-specific PCR primers targeting the mitochondrial cytochrome c oxidase subunit I (mtCOI) locus were generated that allow for the specific identification of the most common Eimeria species infecting turkeys (i.e., Eimeria adenoeides, Eimeria meleagrimitis, Eimeria gallopavonis, Eimeria meleagridis, Eimeria dispersa, and Eimeria innocua). PCR reaction chemistries were optimized with respect to divalent cation (MgCl2) and dNTP concentrations, as well as PCR cycling conditions (particularly anneal temperature for primers). Genomic DNA samples from single oocyst-derived lines of six Eimeria species were tested to establish specificity and sensitivity of these newly designed primer pairs. A mixed 60-ng total DNA sample containing 10 ng of each of the six Eimeria species was used as DNA template to demonstrate specific amplification of the correct product using each of the species-specific primer pairs. Ten nanograms of each of the five non-target Eimeria species was pooled to provide a non-target, control DNA sample suitable to test the specificity of each primer pair. The amplifications of the COI region with species-specific primer pairs from pooled samples yielded products of expected sizes (209 to 1,012 bp) and no amplification of non-target Eimeria sp. DNA was detected using the non-target, control DNA samples. These primer pairs specific for Eimeria spp. of turkeys did not amplify any of the seven Eimeria species infecting chickens. The newly developed PCR primers can be used as a diagnostic tool capable of specifically identifying six turkey Eimeria species; additionally, sequencing of the PCR amplification products yields sequence-based genotyping data suitable for identification and molecular phylogenetics.

  9. Distinct interaction modes of an AKAP bound to two regulatory subunit isoforms of protein kinase A revealed by amide hydrogen/deuterium exchange.

    PubMed

    Burns-Hamuro, Lora L; Hamuro, Yoshitomo; Kim, Jack S; Sigala, Paul; Fayos, Rosa; Stranz, David D; Jennings, Patricia A; Taylor, Susan S; Woods, Virgil L

    2005-12-01

    The structure of an AKAP docked to the dimerization/docking (D/D) domain of the type II (RIIalpha) isoform of protein kinase A (PKA) has been well characterized, but there currently is no detailed structural information of an AKAP docked to the type I (RIalpha) isoform. Dual-specific AKAP2 (D-AKAP2) binds in the nanomolar range to both isoforms and provided us with an opportunity to characterize the isoform-selective nature of AKAP binding using a common docked ligand. Hydrogen/deuterium (H/D) exchange combined with mass spectrometry (DXMS) was used to probe backbone structural changes of an alpha-helical A-kinase binding (AKB) motif from D-AKAP2 docked to both RIalpha and RIIalpha D/D domains. The region of protection upon complex formation and the magnitude of protection from H/D exchange were determined for both interacting partners in each complex. The backbone of the AKB ligand was more protected when bound to RIalpha compared to RIIalpha, suggesting an increased helical stabilization of the docked AKB ligand. This combined with a broader region of backbone protection induced by the AKAP on the docking surface of RIalpha indicated that there were more binding constraints for the AKB ligand when bound to RIalpha. This was in contrast to RIIalpha, which has a preformed, localized binding surface. These distinct modes of AKAP binding may contribute to the more discriminating nature of the RIalpha AKAP-docking surface. DXMS provides valuable structural information for understanding binding specificity in the absence of a high-resolution structure, and can readily be applied to other protein-ligand and protein-protein interactions.

  10. Genetic panmixia within a narrow contact zone between chromosomally and ecologically distinct black fly sibling species (Diptera: Simuliidae).

    PubMed

    Conflitti, I M; Shields, G F; Murphy, R W; Currie, D C

    2015-09-01

    Hybrid zones are windows into the speciation process, and their study can give clues into the maintenance and breakdown of species boundaries. Using both genetic and ecological tools, we investigate lineage diversification across a contact zone characterized by chromosome rearrangements. We show that black fly sibling species, Simulium arcticum sensu stricto (s.s.) and Simulium saxosum, lack genetic differentiation at both microsatellite and mtDNA loci in allopatry and sympatry, as well as exhibit high levels of gene flow and continuous chromosome variation in sympatry. Furthermore, hybrid frequencies at the contact zone are similar to those seen between races, rather than species. In contrast, S. arcticum s.s. and S. saxosum maintain ecological differences and distinct habitat associations - the contact zone situated at the margin of suitable habitat for each sibling species. Moreover, gene flow occurs only in a narrow band along an ecological transition. Except for the contact zone, S. arcticum s.s. and S. saxosum hybrids do not occur elsewhere within the sibling species' ranges. Although S. arcticum s.s. and S. saxosum maintain the potential to interbreed freely, we conclude that habitat associations and, perhaps, chromosome systems prevent expansion of ranges and assimilation of lineages.

  11. Closely related intertidal and deep-sea Halomonhystera species have distinct fatty acid compositions

    NASA Astrophysics Data System (ADS)

    Van Campenhout, Jelle; Vanreusel, Ann

    2017-01-01

    The deep-sea free-living nematode Halomonhystera hermesi, dominant in the sulphidic sediments of the Håkon Mosby mud volcano (1280 m, Barent sea slope), is part of the mainly estuarine Halomonhystera disjuncta species complex consisting of five cryptic species (GD1-GD5). Cryptic species have a very similar morphology raising questions on their specific environmental differences. This study analyzed total fatty acid (FA) compositions of H. hermesi and GD1, one of H. hermesi's closest relatives. Additionally, we experimentally investigated the effect of a temperature reduction, salinity increase and sulphide concentrations on GD1's FA composition. Because nematodes are expected to have low amounts of storage FA, total FA compositions most likely reflect FA contents of cellular membranes. The deep-sea nematode H. hermesi had significantly lower saturation levels and increased highly unsaturated fatty acid (HUFAs) proportions due to the presence of docosahexanoic acid (DHA—22:6ω3) and higher eicosapentaenoic acid (EPA—20:5ω3) proportions. HUFAs were absent in H. hermesi's food source indicating the ability and need for this nematode to synthesize HUFAs in a deep-sea environment. Our experimental data revealed that only a decrease in temperature resulted in lower saturated fatty acids proportions, indicating that the FA content of H. hermesi is most likely a response to temperature but not to sulphide concentrations or salinity differences. In experimental nematodes, EPA proportions were low and DHA was absent indicating that other factors than temperature, salinity and sulphides mediate the presence of these HUFAs in H. hermesi.

  12. Two distinct types of the inhibition of vasculogenesis by different species of charged particles

    PubMed Central

    2013-01-01

    Background Charged particle radiation is known to be more biologically effective than photon radiation. One example of this is the inhibition of the formation of human blood vessels. This effect is an important factor influencing human health and is relevant to space travel as well as to cancer radiotherapy. We have previously shown that ion particles with a high energy deposition, or linear energy transfer (LET) are more than four times more effective at disrupting mature vessel tissue models than particles with a lower LET. For vasculogenesis however, the relative biological effectiveness between particles is the same. This unexpected result prompted us to investigate whether the inhibition of vasculogenesis was occurring by distinct mechanisms. Methods Using 3-Dimensional human vessel models, we developed assays that determine at what stage angiogenesis is inhibited. Vessel morphology, the presence of motile tip structures, and changes in the matrix architecture were assessed. To confirm that the mechanisms are distinct, stimulation of Protein Kinase C (PKC) with phorbol ester (PMA) was employed to selectively restore vessel formation in cultures where early motile tip activity was inhibited. Results Endothelial cells in 3-D culture exposed to low LET protons failed to make connections with other cells but eventually developed a central lumen. Conversely, cells exposed to high LET Fe charged particles extended cellular processes and made connections to other cells but did not develop a central lumen. The microtubule and actin cytoskeletons indicated that motility at the extending tips of endothelial cells is inhibited by low LET but not high LET particles. Actin-rich protrusive structures that contain bundled microtubules showed a 65% decrease when exposed to low LET particles but not high LET particles, with commensurate changes in the matrix architecture. Stimulation of PKC with PMA restored tip motility and capillary formation in low but not high LET particle

  13. Is Homo heidelbergensis a distinct species? New insight on the Mauer mandible.

    PubMed

    Mounier, Aurélien; Marchal, François; Condemi, Silvana

    2009-03-01

    The discovery of new fossils in Africa, Asia, and Europe, and the recognition of a greater diversity in the middle Pleistocene fossil record, has led to a reconsideration of the species Homo heidelbergensis. This nomen, formulated by Schoetensack in 1908 to describe the Mauer jaw (Germany), was almost forgotten during most of the past century. Numerous fossils have been attributed to it but no consensus has arisen concerning their classification. The holotype anatomical traits are still poorly understood, and numerous fossils with no mandibular remains have been placed in the taxon. Some researchers propose H. heidelbergensis as an Afro-European taxon that is ancestral to both modern humans and Neandertals whereas others think it is a strictly European species that is part of the Neandertal lineage. We focus on the validity of H. heidelbergensis, using the traditional basis of species recognition: anatomical description. We provide a comparative morphological analysis using 47 anatomical traits of 36 Pleistocene fossils from Africa, Asia, and Europe and 35 extant human mandibles. We re-examine the mandibular features of Mauer and discuss the specimen's inclusion in H. heidelbergensis, as well as alternative evolutionary theories. To lend objectivity to specimen grouping, we use multiple correspondence analysis associated with hierarchical classification that creates clusters corresponding to phenetic similarities between jaws. Our phenetic and comparative morphological analyses support the validity of H. heidelbergensis as a taxon. A set of morphological features can be statistically identified for the definition of the species. Some traits can be used to delimit H. heidelbergensis in an evolutionary framework (e.g., foramina mentale posteriorly positioned, horizontal retromolar surface). Those traits are also present on African (e.g., Tighenif) and European (e.g., Sima de los Huesos) specimens that show a close relationship with the Mauer mandible. Therefore, the

  14. Antigenic diversity is generated by distinct evolutionary mechanisms in African trypanosome species.

    PubMed

    Jackson, Andrew P; Berry, Andrew; Aslett, Martin; Allison, Harriet C; Burton, Peter; Vavrova-Anderson, Jana; Brown, Robert; Browne, Hilary; Corton, Nicola; Hauser, Heidi; Gamble, John; Gilderthorp, Ruth; Marcello, Lucio; McQuillan, Jacqueline; Otto, Thomas D; Quail, Michael A; Sanders, Mandy J; van Tonder, Andries; Ginger, Michael L; Field, Mark C; Barry, J David; Hertz-Fowler, Christiane; Berriman, Matthew

    2012-02-28

    Antigenic variation enables pathogens to avoid the host immune response by continual switching of surface proteins. The protozoan blood parasite Trypanosoma brucei causes human African trypanosomiasis ("sleeping sickness") across sub-Saharan Africa and is a model system for antigenic variation, surviving by periodically replacing a monolayer of variant surface glycoproteins (VSG) that covers its cell surface. We compared the genome of Trypanosoma brucei with two closely related parasites Trypanosoma congolense and Trypanosoma vivax, to reveal how the variant antigen repertoire has evolved and how it might affect contemporary antigenic diversity. We reconstruct VSG diversification showing that Trypanosoma congolense uses variant antigens derived from multiple ancestral VSG lineages, whereas in Trypanosoma brucei VSG have recent origins, and ancestral gene lineages have been repeatedly co-opted to novel functions. These historical differences are reflected in fundamental differences between species in the scale and mechanism of recombination. Using phylogenetic incompatibility as a metric for genetic exchange, we show that the frequency of recombination is comparable between Trypanosoma congolense and Trypanosoma brucei but is much lower in Trypanosoma vivax. Furthermore, in showing that the C-terminal domain of Trypanosoma brucei VSG plays a crucial role in facilitating exchange, we reveal substantial species differences in the mechanism of VSG diversification. Our results demonstrate how past VSG evolution indirectly determines the ability of contemporary parasites to generate novel variant antigens through recombination and suggest that the current model for antigenic variation in Trypanosoma brucei is only one means by which these parasites maintain chronic infections.

  15. Phylogenetic Reassessment of Antarctic Tetillidae (Demospongiae, Tetractinellida) Reveals New Genera and Genetic Similarity among Morphologically Distinct Species

    PubMed Central

    Carella, Mirco; Agell, Gemma; Cárdenas, Paco; Uriz, Maria J.

    2016-01-01

    Species of Tetillidae are distributed worldwide. However, some genera are unresolved and only a few genera and species of this family have been described from the Antarctic. The incorporation of 25 new COI and 18S sequences of Antarctic Tetillidae to those used recently for assessing the genera phylogeny, has allowed us to improve the resolution of some poorly resolved nodes and to confirm the monophyly of previously identified clades. Classical genera such as Craniella recovered their traditional diagnosis by moving the Antarctic Tetilla from Craniella, where they were placed in the previous family phylogeny, to Antarctotetilla gen. nov. The morphological re-examination of specimens used in the previous phylogeny and their comparison to the type material revealed misidentifications. The proposed monotypic new genus Levantinella had uncertain phylogenetic relationships depending on the gene partition used. Two more clades would require the inclusion of additional species to be formally established as new genera. The parsimony tree based on morphological characters and the secondary structure of the 18S (V4 region) almost completely matched the COI M1-M6 and the COI+18S concatenated phylogenies. Morphological synapomorphies have been identified for the genera proposed. New 15 28S (D3-D5) and 11 COI I3-M11 partitions were exclusively sequenced for the Antarctic species subset. Remarkably, species within the Antarctic genera Cinachyra (C. barbata and C. antarctica) and Antarctotetilla (A. leptoderma, A. grandis, and A. sagitta), which are clearly distinguishable morphologically, were not genetically differentiated with any of the markers assayed. Thus, as it has been reported for other Antarctic sponges, both the mitochondrial and nuclear partitions used did not differentiate species that were well characterized morphologically. Antarctic Tetillidae offers a rare example of genetically cryptic (with the traditional markers used for sponges), morphologically distinct

  16. The genome of African yam (Dioscorea cayenensis-rotundata complex) hosts endogenous sequences from four distinct Badnavirus species.

    PubMed

    Umber, Marie; Filloux, Denis; Muller, Emmanuelle; Laboureau, Nathalie; Galzi, Serge; Roumagnac, Philippe; Iskra-Caruana, Marie-Line; Pavis, Claudie; Teycheney, Pierre-Yves; Seal, Susan E

    2014-10-01

    Several endogenous viral elements (EVEs) have been identified in plant genomes, including endogenous pararetroviruses (EPRVs). Here, we report the first characterization of EPRV sequences in the genome of African yam of the Dioscorea cayenensis-rotundata complex. We propose that these sequences should be termed 'endogenous Dioscorea bacilliform viruses' (eDBVs). Molecular characterization of eDBVs shows that they constitute sequences originating from various parts of badnavirus genomes, resulting in a mosaic structure that is typical of most EPRVs characterized to date. Using complementary molecular approaches, we show that eDBVs belong to at least four distinct Badnavirus species, indicating multiple, independent, endogenization events. Phylogenetic analyses of eDBVs support and enrich the current taxonomy of yam badnaviruses and lead to the characterization of a new Badnavirus species in yam. The impact of eDBVs on diagnosis, yam germplasm conservation and movement, and breeding is discussed.

  17. Dicrocoelium chinensis and Dicrocoelium dendriticum (Trematoda: Digenea) are distinct lancet fluke species based on mitochondrial and nuclear ribosomal DNA sequences.

    PubMed

    Liu, Guo-Hua; Yan, Hong-Bin; Otranto, Domenico; Wang, Xing-Ye; Zhao, Guang-Hui; Jia, Wan-Zhong; Zhu, Xing-Quan

    2014-10-01

    Lancet flukes parasitize the bile ducts and gall bladder of a range of mammals, including humans, causing dicrocoeliosis. In the present study, we sequenced and characterized the complete mitochondrial (mt) genomes as well as the first and second internal transcribed spacers (ITS-1 and ITS-2=ITS) of nuclear ribosomal DNA (rDNA) of two lancet flukes, Dicrocoelium chinensis and D. dendriticum. Sequence comparison of a conserved mt gene and nuclear rDNA sequences among multiple individual lancet flukes revealed substantial nucleotide differences between the species but limited sequence variation within each of them. Phylogenetic analysis of the concatenated amino acid and multiple mt rrnS sequences using Bayesian inference supported the separation of D. chinensis and D. dendriticum into two distinct species-specific clades. Results of the present study support the proposal that D. dendriticum and D. chinensis represent two distinct lancet flukes. While providing the first mt genomes from members of the superfamily Plagiorchioidea, the novel mt markers described herein will be useful for further studies of the diagnosis, epidemiology and systematics of the lancet flukes and other trematodes of human and animal health significance.

  18. Distinctive green recovery of silver species from modified cellulose: mechanism and spectroscopic studies.

    PubMed

    Dwivedi, Amarendra Dhar; Dubey, Shashi Prabha; Sillanpää, Mika; Liimatainen, Henrikki; Suopajärvi, Terhi; Niinimäki, Jouko; Kwon, Young-Nam; Lee, Changha

    2015-05-01

    The present study aimed to recover precious silver in order to identify the adsorption coupled reduction pathways that determine this process. A combination technique of adsorption and nanocrystallization was used to investigate the recovery of silver species from taurine-cellulose (T-DAC) samples. The non-synthetic route of nanocrystallization yielded spherical zero-valent silver sized ∼ 18 nm. Rate-controlling steps were modeled by adsorption parameters by the best fit of Langmuir capacity (55 mg/g), pseudo-second order curves, and exothermic chemical reactions. The T-DAC was an excellent sorbing phase for the treatment of silver-polluted waters over a broad range of pH (2.1-10.1) and varying ionic strengths (8.5-850 mM, as NaCl), which are the conditions often encountered in industrial and mining effluents. A good recovery of silver (40-65%) was also obtained in the presence of Cd(II), Co(II), Cr(VI), Ni(II), and As(V) at lower or equivalent concentrations with Ag(I), either from individually added metals or from all metal ions mixed together. Desorption was compared with a series of five eluents including complexing agents. In these experiments acidified thiourea yielded 86% desorption of Ag(I). Aqueous silver reduced to metallic silver on the surface of the T-DAC samples, which was confirmed by X-ray photo electron spectroscopy.

  19. Cryptosporidiosis caused by two distinct species in Russian tortoises and a pancake tortoise.

    PubMed

    Griffin, Chris; Reavill, Drury R; Stacy, Brian A; Childress, April L; Wellehan, James F X

    2010-05-28

    Cryptosporidiosis in squamates is well documented, but there is very limited information available on cryptosporidiosis in testudines. We describe three cases of cryptosporidiosis in tortoises with associated pathology. Two Russian tortoises (Agrionemys [Testudo] horsfieldii) and a pancake tortoise (Malacochersus tornieri), all from separate collections, were found dead. At necropsy, two had histological evidence of intestinal cryptosporidiosis and one had gastric cryptosporidiosis. Consensus Cryptosporidium sp. PCR and sequencing was used to identify the Cryptosporidium sp. present in these three tortoises. In the juvenile Russian tortoise with gastric cryptosporidiosis, the organism had 98% homology with a previously reported sequence from an Indian star tortoise isolate. A second chelonian Cryptosporidium sp. was identified in the pancake tortoise and the second Russian tortoise. This sequence was 100% identical to a shorter gene sequence previously reported in a marginated tortoise. This is the first report coordinating pathology with Cryptosporidium characterization in chelonians. The two Cryptosporidium sp. found in tortoises segregate according to site of infection, and there may be further differences in pathology, host range, and transmission. These Cryptosporidium sp. appear to be able to infect diverse tortoise host species. This may be an under-recognized problem in tortoises.

  20. The Mitochondrial Cytochrome Oxidase Subunit I Gene Occurs on a Minichromosome with Extensive Heteroplasmy in Two Species of Chewing Lice, Geomydoecus aurei and Thomomydoecus minor

    PubMed Central

    Pietan, Lucas L.; Spradling, Theresa A.

    2016-01-01

    In animals, mitochondrial DNA (mtDNA) typically occurs as a single circular chromosome with 13 protein-coding genes and 22 tRNA genes. The various species of lice examined previously, however, have shown mitochondrial genome rearrangements with a range of chromosome sizes and numbers. Our research demonstrates that the mitochondrial genomes of two species of chewing lice found on pocket gophers, Geomydoecus aurei and Thomomydoecus minor, are fragmented with the 1,536 base-pair (bp) cytochrome-oxidase subunit I (cox1) gene occurring as the only protein-coding gene on a 1,916–1,964 bp minicircular chromosome in the two species, respectively. The cox1 gene of T. minor begins with an atypical start codon, while that of G. aurei does not. Components of the non-protein coding sequence of G. aurei and T. minor include a tRNA (isoleucine) gene, inverted repeat sequences consistent with origins of replication, and an additional non-coding region that is smaller than the non-coding sequence of other lice with such fragmented mitochondrial genomes. Sequences of cox1 minichromosome clones for each species reveal extensive length and sequence heteroplasmy in both coding and noncoding regions. The highly variable non-gene regions of G. aurei and T. minor have little sequence similarity with one another except for a 19-bp region of phylogenetically conserved sequence with unknown function. PMID:27589589

  1. IDENTIFICATION OF CRYPTOSPORIDIUM SPECIES AND SOURCES IN RAW WASTEWATER USING A SMALL SUBUNIT RRNA-BASED PCR-RFLP TOOL

    EPA Science Inventory

    The species composition and source of Cryptosporidium oocysts in wastewater have never been determined, even though it is widely assumed that these oocysts are from human sewage. Recent molecular characterizations of Cryptosporidium parasites make it possible to differentiate hum...

  2. Revision of the western Palaearctic species of Aleiodes Wesmael (Hymenoptera, Braconidae, Rogadinae). Part 1: Introduction, key to species groups, outlying distinctive species, and revisionary notes on some further species

    PubMed Central

    van Achterberg, Cornelis; Shaw, Mark R.

    2016-01-01

    Abstract Seven new species of the genus Aleiodes Wesmael, 1838 (Braconidae: Rogadinae) are described and illustrated: Aleiodes abraxanae sp. n., Aleiodes angustipterus sp. n., Aleiodes artesiariae sp. n., Aleiodes carminatus sp. n., Aleiodes diarsianae sp. n., Aleiodes leptofemur sp. n., and Aleiodes ryrholmi sp. n. A neotype is designated for each of Aleiodes circumscriptus (Nees, 1834) and Aleiodes pictus (Herrich-Schäffer, 1838), and both species are redescribed and illustrated. Aleiodes ochraceus Hellén, 1927 (not Aleiodes ochraceus (Curtis, 1834)) is renamed as Aleiodes curticornis nom. n. & stat. rev., and redescribed and illustrated. Aleiodes bistrigatus Roman, 1917, Aleiodes nigriceps Wesmael, 1838, and Aleiodes reticulatus (Noskiewicz, 1956), are re-instated as valid species. A lectotype is designated for Aleiodes bistrigatus Roman. An illustrated key is given to some distinctive species and the residual species groups along which further parts of an entire revision of western Palaearctic species of Aleiodes and Heterogamus will be organised. Biology, host associations and phenology are discussed for the keyed species (in addition to the above, Aleiodes albitibia (Herrich-Schäffer, 1838), Aleiodes apiculatus (Fahringer, 1932), Aleiodes arcticus (Thomson, 1892), Aleiodes cantherius (Lyle, 1919), Aleiodes esenbeckii (Hartig, 1834), Aleiodes jakowlewi (Kokujev, 1898), Aleiodes modestus (Reinhard, 1863), Aleiodes nigricornis Wesmael, 1838, Aleiodes pallidator (Thunberg, 1822), Aleiodes praetor (Reinhard, 1863), Aleiodes seriatus (Herrich- Schäffer, 1838) sensu lato, Aleiodes testaceus (Telenga, 1941), Aleiodes ungularis (Thomson, 1892), and Aleiodes varius (Herrich-Schäffer, 1838)) which are dealt with in full here (with the exception of Aleiodes seriatus s.l. which is, however, included in the key). The experimental methodology covering the revision as a whole, which involves some behavioural investigation, is outlined. PMID:28138281

  3. On the specificity of antibiotics targeting the large ribosomal subunit.

    PubMed

    Wilson, Daniel N

    2011-12-01

    The peptidyltransferase center of the large ribosomal subunit is responsible for catalyzing peptide bonds. This active site is the target of a variety of diverse antibiotics, many of which are used clinically. The past decade has seen a plethora of structures of antibiotics in complex with the large ribosomal subunit, providing unprecedented insight into the mechanism of action of these inhibitors. Ten distinct antibiotics (chloramphenicol, clindamycin, linezolid, tiamulin, sparsomycin, and five macrolides) have been crystallized in complex with four distinct ribosomal species, three bacterial, and one archaeal. This review aims to compare these structures in order to provide insight into the conserved and species-specific modes of interaction for particular members of each class of antibiotics. Coupled with the wealth of biochemical data, a picture is emerging defining the specific functional states of the ribosome that antibiotics preferentially target. Such mechanistic insight into antibiotic inhibition will be important for the development of the next generation of antimicrobial agents.

  4. Experimental Crossing of Two Distinct Species of Leopard Geckos, Eublepharis angramainyu and E. macularius: Viability, Fertility and Phenotypic Variation of the Hybrids.

    PubMed

    Jančúchová-Lásková, Jitka; Landová, Eva; Frynta, Daniel

    2015-01-01

    Hybridization between distinct species of animals and subsequent genetic introgression plays a considerable role in the speciation process and the emergence of adaptive characters. Fitness of between-species hybrids usually sharply decreases with the divergence time of the concerned species and the divergence depth, which still allows for a successful crossing differs among principal clades of vertebrates. Recently, a review of hybridization events among distinct lizard species revealed that lizards belong to vertebrates with a highly developed ability to hybridize. In spite of this, reliable reports of experimental hybridizations between genetically fairly divergent species are only exceptional. Here, we show the results of the crossing of two distinct allopatric species of eyelid geckos possessing temperature sex determination and lacking sex chromosomes: Eublepharis macularius distributed in Pakistan/Afghanistan area and E. angramainyu, which inhabits Mesopotamia and adjacent areas. We demonstrated that F1 hybrids were viable and fertile, and the introgression of E. angramainyu genes into the E. macularius genome can be enabled via a backcrossing. The examined hybrids (except those of the F2 generation) displayed neither malformations nor a reduced survival. Analyses of morphometric and coloration traits confirmed phenotypic distinctness of both parental species and their F1 hybrids. These findings contrast with long-term geographic and an evolutionary separation of the studied species. Thus, the occurrence of fertile hybrids of comparably divergent species, such as E. angramainyu and E. macularius, may also be expected in other taxa of squamates. This would violate the current estimates of species diversity in lizards.

  5. Molecular characterization of a distinct bipartite Begomovirus species infecting ivy gourd (Coccinia grandis L.) in Tamil Nadu, India.

    PubMed

    Nagendran, K; Satya, V K; Mohankumar, S; Karthikeyan, G

    2016-02-01

    A distinct bipartite begomovirus was found to be associated with the mosaic disease on ivy gourd (Coccinia grandis L.) in Tamil Nadu, India. The complete DNA A and DNA B components were cloned by rolling circle amplification. Genome organization of this virus is found to be typical of Old World bipartite begomovirus. The association of betasatellite component with this virus is absent. The closest nucleotide identity of 73.4 % was seen with the Loofa yellow mosaic virus (LYMV-[VN]-AF509739) suggesting that it is a new virus species Coccinia mosaic virus (CoMoV-Ivy gourd [TN TDV Coc1]) and distantly related to the other known begomoviruses. The DNA B component shared a maximum identity of 55 % with that of Tomato leaf curl New Delhi virus (ToLCNDV). In the phylogenetic analysis, CoMoV-Ivy gourd form cluster separate from other begomoviruses. Recombination analysis showed that there was no recombination event in the genome. This is the distinct begomovirus infecting ivy gourd.

  6. Evolutionary relationships between the former species Saccharomyces uvarum and the hybrids Saccharomyces bayanus and Saccharomyces pastorianus; reinstatement of Saccharomyces uvarum (Beijerinck) as a distinct species.

    PubMed

    Nguyen, Huu-Vang; Gaillardin, Claude

    2005-02-01

    Analysis of the nucleotide sequence of the GDH1 homologues from Saccharomyces bayanus strain CBS 380T and S. pastorianus strains showed that they share an almost identical sequence, SuGDH1*, which is a diverged form of the SuGDH1 from the type strain of the former species S. uvarum, considered as synonym of S. bayanus. SuGDH1* is close to but differs from SuGDH1 by the accumulation of a high number of neutral substitutions designated as Multiple Neutral Mutations Accumulation (MNMA). Further analysis carried out with three other markers, BAP2, HO and MET2 showed that they have also diverged from their S. uvarum counterparts by MNMA. S. bayanus CBS 380T is placed between S. uvarum and S. pastorianus sharing MET2, CDC91 sequences with the former and BAP2, GDH1, HO sequences with the latter. S. bayanus CBS 380T has been proposed to be a S. uvarum/S. cerevisiae hybrid and this proposal is confirmed by the presence in its genome a S. cerevisiae SUC4 gene. Strain S. bayanus CBS 380T, with a composite genome, is genetically isolated from strains of the former S. uvarum species, thus justifying the reinstatement of S. uvarum as a distinct species.

  7. RT-PCR for detecting five distinct Tospovirus species using degenerate primers and dsRNA template.

    PubMed

    Okuda, M; Hanada, K

    2001-08-01

    RT-PCR procedures for detection of multiple species of tospovirus from plant tissues were investigated. Downstream primers were designated from the 3' untranslated sequences of the S RNA. An upstream primer was designated from the degenerated sequences of the nucleocapsid protein. Approximately 450 bp DNA fragments were detected when Tomato spotted wilt virus (TSWV)- or Impatiens necrotic spot virus (INSV)- infected tissues were examined. Approximately 350 bp DNA fragments were detected when Watermelon silver mottle virus (WSMoV)- or Melon yellow spot virus (MYSV)-infected tissues were examined. Template RNA was extracted using CF 11 cellulose powder, and nonspecific amplification became unnoticeable when double-stranded RNA was used. The amplified fragments of WSMoV were differentiated from those of MYSV by AluI or TaqI digestion. The amplified fragments of TSWV were differentiated from those of INSV by DraI or HindIII digestion. An alstroemeria plant that was infected with an unidentified tospovirus was also examined, and a 350 bp fragment that was 97% identical to Iris yellow spot virus was detected. This method, therefore, detected at least five distinct Tospovirus species.

  8. Sequential Utilization of Hosts from Different Fly Families by Genetically Distinct, Sympatric Populations within the Entomophthora muscae Species Complex

    PubMed Central

    Gryganskyi, Andrii P.; Humber, Richard A.; Stajich, Jason E.; Mullens, Bradley; Anishchenko, Iryna M.; Vilgalys, Rytas

    2013-01-01

    The fungus Entomophthora muscae (Entomophthoromycota, Entomophthorales, Entomophthoraceae) is a widespread insect pathogen responsible for fatal epizootic events in many dipteran fly hosts. During epizootics in 2011 and 2012 in Durham, North Carolina, we observed a transition of fungal infections from one host, the plant-feeding fly Delia radicum, to a second host, the predatory fly Coenosia tigrina. Infections first appeared on Delia in the middle of March, but by the end of May, Coenosia comprised 100% of infected hosts. Multilocus sequence typing revealed that E. muscae in Durham comprises two distinct subpopulations (clades) with several haplotypes in each. Fungi from either clade are able to infect both fly species, but vary in their infection phenologies and host-specificities. Individuals of the more phylogenetically diverse clade I predominated during the beginning of the spring epizootic, infecting mostly phytophagous Delia flies. Clade II dominated in late April and May and affected mostly predatory Coenosia flies. Analysis of population structure revealed two subpopulations within E. muscae with limited gene exchange. This study provides the first evidence of recombination and population structure within the E. muscae species complex, and illustrates the complexity of insect-fungus relationships that should be considered for development of biological control methods. PMID:23951101

  9. Adaptive sequence evolution is driven by biotic stress in a pair of orchid species (Dactylorhiza) with distinct ecological optima.

    PubMed

    Balao, Francisco; Trucchi, Emiliano; Wolfe, Thomas; Hao, Bao-Hai; Lorenzo, Maria Teresa; Baar, Juliane; Sedman, Laura; Kosiol, Carolin; Amman, Fabian; Chase, Mark W; Hedrén, Mikael; Paun, Ovidiu

    2017-03-30

    The orchid family is the largest in the angiosperms, but little is known about the molecular basis of the significant variation they exhibit. We investigate here the transcriptomic divergence between two European terrestrial orchids, Dactylorhiza incarnata and D. fuchsii, and integrate these results in the context of their distinct ecologies that we also document. Clear signals of lineage-specific adaptive evolution of protein-coding sequences are identified, notably targeting elements of biotic defence, including both physical and chemical adaptations in the context of divergent pools of pathogens and herbivores. In turn, a substantial regulatory divergence between the two species appears linked to adaptation/acclimation to abiotic conditions. Several of the pathways affected by differential expression are also targeted by deviating post-transcriptional regulation via sRNAs. Finally, Dactylorhiza incarnata appears to suffer from insufficient sRNA control over the activity of RNA-dependent DNA polymerase, resulting in increased activity of class I transposable elements and, over time, in larger genome size than that of D. fuchsii. The extensive molecular divergence between the two species suggests significant genomic and transcriptomic shock in their hybrids and offers insights into the difficulty of coexistence at the homoploid level. Altogether, biological response to selection, accumulated during the history of these orchids, appears governed by their microenvironmental context, in which biotic and abiotic pressures act synergistically to shape transcriptome structure, expression and regulation. This article is protected by copyright. All rights reserved.

  10. Molecular evidence for genetic distinctions between Chlamydiaceae detected in Siamese crocodiles (Crocodylus siamensis) and known Chlamydiaceae species.

    PubMed

    Sariya, Ladawan; Kladmanee, Kan; Bhusri, Benjaporn; Thaijongrak, Prawporn; Tonchiangsai, Kanittha; Chaichoun, Kridsada; Ratanakorn, Parntep

    2015-02-01

    Chlamydiosis, caused by Chlamydiaceae, is a zoonotic disease found in humans and several species of animals, including reptiles and amphibians. Although chlamydiosis in saltwater crocodiles has been previously reported in South Africa and Papua New Guinea, the reported strains have not been identified or confirmed. Therefore, the main aim of this study was to sequence and characterize Chamydiaceae isolated from Siamese crocodiles. Results showed the 16S ribosomal (r) RNA and the 16S/23S rRNA gene of the crocodile isolates were closely related to the genus Chlamydophila with matched identity greater than 98%. The phylogenetic tree constructed from the 16S/23S rRNA gene showed the crocodile cluster diverges far from Cp. caviae with a 100% bootstrap value. The tree based on the ompA gene loci distinguished the crocodile strains into genotypes I, II, and III. The present study is the first report on Chlamydophila detected in Siamese crocodiles that is genetically distinct from the known species of Chlamydiaceae.

  11. Subterranean, Herbivore-Induced Plant Volatile Increases Biological Control Activity of Multiple Beneficial Nematode Species in Distinct Habitats

    PubMed Central

    Ali, Jared G.; Alborn, Hans T.; Campos-Herrera, Raquel; Kaplan, Fatma; Duncan, Larry W.; Rodriguez-Saona, Cesar; Koppenhöfer, Albrecht M.; Stelinski, Lukasz L.

    2012-01-01

    While the role of herbivore-induced volatiles in plant-herbivore-natural enemy interactions is well documented aboveground, new evidence suggests that belowground volatile emissions can protect plants by attracting entomopathogenic nematodes (EPNs). However, due to methodological limitations, no study has previously detected belowground herbivore-induced volatiles in the field or quantified their impact on attraction of diverse EPN species. Here we show how a belowground herbivore-induced volatile can enhance mortality of agriculturally significant root pests. First, in real time, we identified pregeijerene (1,5-dimethylcyclodeca-1,5,7-triene) from citrus roots 9–12 hours after initiation of larval Diaprepes abbreviatus feeding. This compound was also detected in the root zone of mature citrus trees in the field. Application of collected volatiles from weevil-damaged citrus roots attracted native EPNs and increased mortality of beetle larvae (D. abbreviatus) compared to controls in a citrus orchard. In addition, field applications of isolated pregeijerene caused similar results. Quantitative real-time PCR revealed that pregeijerene increased pest mortality by attracting four species of naturally occurring EPNs in the field. Finally, we tested the generality of this root-zone signal by application of pregeijerene in blueberry fields; mortality of larvae (Galleria mellonella and Anomala orientalis) again increased by attracting naturally occurring populations of an EPN. Thus, this specific belowground signal attracts natural enemies of widespread root pests in distinct agricultural systems and may have broad potential in biological control of root pests. PMID:22761668

  12. Development and Application of Small-Subunit rRNA Probes for Assessment of Selected Thiobacillus Species and Members of the Genus Acidiphilium

    PubMed Central

    Peccia, Jordan; Marchand, Eric A.; Silverstein, Joann; Hernandez, Mark

    2000-01-01

    Culture-dependent studies have implicated sulfur-oxidizing bacteria as the causative agents of acid mine drainage and concrete corrosion in sewers. Thiobacillus species are considered the major representatives of the acid-producing bacteria in these environments. Small-subunit rRNA genes from all of the Thiobacillus and Acidiphilium species catalogued by the Ribosomal Database Project were identified and used to design oligonucleotide DNA probes. Two oligonucleotide probes were synthesized to complement variable regions of 16S rRNA in the following acidophilic bacteria: Thiobacillus ferrooxidans and T. thiooxidans (probe Thio820) and members of the genus Acidiphilium (probe Acdp821). Using 32P radiolabels, probe specificity was characterized by hybridization dissociation temperature (Td) with membrane-immobilized RNA extracted from a suite of 21 strains representing three groups of bacteria. Fluorochrome-conjugated probes were evaluated for use with fluorescent in situ hybridization (FISH) at the experimentally determined Tds. FISH was used to identify and enumerate bacteria in laboratory reactors and environmental samples. Probing of laboratory reactors inoculated with a mixed culture of acidophilic bacteria validated the ability of the oligonucleotide probes to track specific cell numbers with time. Additionally, probing of sediments from an active acid mine drainage site in Colorado demonstrated the ability to identify numbers of active bacteria in natural environments that contain high concentrations of metals, associated precipitates, and other mineral debris. PMID:10877807

  13. Development and application of small-subunit rRNA probes for assessment of selected Thiobacillus species and members of the genus Acidiphilium.

    PubMed

    Peccia, J; Marchand, E A; Silverstein, J; Hernandez, M

    2000-07-01

    Culture-dependent studies have implicated sulfur-oxidizing bacteria as the causative agents of acid mine drainage and concrete corrosion in sewers. Thiobacillus species are considered the major representatives of the acid-producing bacteria in these environments. Small-subunit rRNA genes from all of the Thiobacillus and Acidiphilium species catalogued by the Ribosomal Database Project were identified and used to design oligonucleotide DNA probes. Two oligonucleotide probes were synthesized to complement variable regions of 16S rRNA in the following acidophilic bacteria: Thiobacillus ferrooxidans and T. thiooxidans (probe Thio820) and members of the genus Acidiphilium (probe Acdp821). Using (32)P radiolabels, probe specificity was characterized by hybridization dissociation temperature (T(d)) with membrane-immobilized RNA extracted from a suite of 21 strains representing three groups of bacteria. Fluorochrome-conjugated probes were evaluated for use with fluorescent in situ hybridization (FISH) at the experimentally determined T(d)s. FISH was used to identify and enumerate bacteria in laboratory reactors and environmental samples. Probing of laboratory reactors inoculated with a mixed culture of acidophilic bacteria validated the ability of the oligonucleotide probes to track specific cell numbers with time. Additionally, probing of sediments from an active acid mine drainage site in Colorado demonstrated the ability to identify numbers of active bacteria in natural environments that contain high concentrations of metals, associated precipitates, and other mineral debris.

  14. Nine novel DNA components associated with the foorkey disease of large cardamom: evidence of a distinct babuvirus species in Nanoviridae.

    PubMed

    Mandal, Bikash; Shilpi, S; Barman, Ashis Roy; Mandal, Seema; Varma, Anupam

    2013-12-26

    Foorkey disease is a serious constraint to the production of large cardamom (Amomum subulatum, family Zingiberaceae). The disease is characterized by profuse proliferation of excessive stunted shoots, which makes the clump totally unproductive. The disease has been known in India since 1936 but the complete genome of the virus had not yet been characterized. In a preliminary study, an associated virus tentatively named as Cardamom bushy dwarf virus (CBDV) was identified based on the partial sequence of a single DNA component (DNA-R). In the present study, a high incidence (37.2-39.3%) of foorkey was recorded in certain plantations in the Darjeeling hills located at lower altitudes (300-1380 m) and CBDV was detected in several field samples by PCR. Nine novel DNA components were isolated and characterized from foorkey affected plants. CBDV contained six major DNA components (DNA-R, -S, -M, -C, -N and -U3) similar to the integral genome components known for the members of the genus Babuvirus in the family Nanoviridae. Additional components, satellite Rep (DNA-sRep1) and unknown components (DNA-Uf1 and -Uf2) were also identified. The size of the genome components ranged from 1028 to 1127. The sequence identity and phylogeny based on the individual components as well as overall genome (59.8-62% identity) distinguished CBDV from the two existing babuvirus species, Banana bunchy top virus and Abaca bunchy top virus. CBDV is the first distinct babuvirus species that affects plant species outside family Musaceae. This study shows further diversity in the genus Babuvirus.

  15. 75 FR 53272 - Endangered and Threatened Species; Initiation of 5-Year Review of the Eastern Distinct Population...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-31

    ... of 5-Year Review of the Eastern Distinct Population Segment of the Steller Sea Lion AGENCY: National..., 2010, announcing the initiation of a 5-year review of the eastern Distinct Population Segment (DPS) of..., announcing the initiation of a 5-year review of the eastern Distinct Population Segment (DPS) of the...

  16. Comparison of tick-borne microorganism communities in Ixodes spp. of the Ixodes ricinus species complex at distinct geographical regions.

    PubMed

    Movila, Alexandru; Dubinina, Helen V; Sitnicova, Natalia; Bespyatova, Liubov; Uspenskaia, Inga; Efremova, Galina; Toderas, Ion; Alekseev, Andrey N

    2014-05-01

    Characterizing the tick-borne microorganism communities of Ixodes ricinus (sheep tick) and Ixodes persulcatus (taiga tick) from the I. ricinus species complex in distinct geographical regions of Eastern Europe and European Russia, we demonstrated differences between the two ticks. Taiga ticks were more frequently mono- and co-infected than sheep ticks: 24.4 % (45/184 tested ticks) versus 17.5 % (52/297) and 4.3 % (8/184) versus 3.4 % (10/297), respectively. Ginsberg co-infection index values were significant at the various sites. Diversity of the tick-borne microorganism communities was estimated by the Shannon index, reaching values of 1.71 ± 0.46 and 1.20 ± 0.15 at the sheep-tick and the taiga-tick harbored sites, respectively. Richness of the tick-borne microorganism community in the sheep tick collection sites was about twice the value of the taiga tick collection sites. Future investigations are warranted to further characterize the peculiarities of the tick-borne microorganism communities among the ticks of the Ixodes ricinus complex.

  17. Simultaneous Observation of an Intraband Transition and Distinct Transient Species in the Infrared Region for Perovskite Solar Cells.

    PubMed

    Narra, Sudhakar; Chung, Chih-Chun; Diau, Eric Wei-Guang; Shigeto, Shinsuke

    2016-07-07

    Solar cells based on organometal-halide perovskites such as CH3NH3PbI3 have emerged as a promising next-generation photovoltaic system, but the underlying photophysics and photochemistry remain to be established because of the limited availability of methods to implement the simultaneous and direct measurement of various charge carriers and ions that play a crucial role in the operating device. We used nanosecond time-resolved infrared (IR) spectroscopy to investigate, with high molecular specificity, distinct transient species that are formed in perovskite solar cells after photoexcitation. In CH3NH3PbI3 planar-heterojuction solar cells, we simultaneously observed infrared spectral signatures that are associated with an intraband transition of conduction-band electrons, Fano resonance, and the spiro-OMeTAD cation having an exceptionally short lifetime of 1.0 μs (at ∼1485 cm(-1)). The present results show that the time-resolved IR method offers a unique capability to elucidate these important transients in perovskite solar cells and their dynamic interplay in a comprehensive manner.

  18. Different reactive oxygen species lead to distinct changes of cellular metal ions in the eukaryotic model organism Saccharomyces cerevisiae.

    PubMed

    Wu, Ming J; O'Doherty, Patrick J; Murphy, Patricia A; Lyons, Victoria; Christophersen, Melinda; Rogers, Peter J; Bailey, Trevor D; Higgins, Vincent J

    2011-01-01

    Elemental uptake and export of the cell are tightly regulated thereby maintaining the ionomic homeostasis. This equilibrium can be disrupted upon exposure to exogenous reactive oxygen species (ROS), leading to reduction or elevation of the intracellular metal ions. In this study, the ionomic composition in the eukaryotic model organism Saccharomyces cerevisiae was profiled using the inductively-coupled plasma optical emission spectrometer (ICP-OES) following the treatment with individual ROS, including hydrogen peroxide, cumen hydroperoxide, linoleic acid hydroperoxide (LAH), the superoxide-generating agent menadione, the thiol-oxidising agent diamide [diazine-dicarboxylic acid-bis(dimethylamide)], dimedone and peroxynitrite. The findings demonstrated that different ROS resulted in distinct changes in cellular metal ions. Aluminium (Al(3+)) level rose up to 50-fold after the diamide treatment. Cellular potassium (K(+)) in LAH-treated cells was 26-fold less compared to the non-treated controls. The diamide-induced Al(3+) accumulation was further validated by the enhanced Al(3+) uptake along the time course and diamide doses. Pre-incubation of yeast with individual elements including iron, copper, manganese and magnesium failed to block diamide-induced Al(3+) uptake, suggesting Al(3+)-specific transporters could be involved in Al(3+) uptake. Furthermore, LAH-induced potassium depletion was validated by a rescue experiment in which addition of potassium increased yeast growth in LAH-containing media by 26% compared to LAH alone. Taken together, the data, for the first time, demonstrated the linkage between ionomic profiles and individual oxidative conditions.

  19. Reactive oxygen species signaling facilitates FOXO-3a/FBXO-dependent vascular BK channel β1 subunit degradation in diabetic mice.

    PubMed

    Lu, Tong; Chai, Qiang; Yu, Ling; d'Uscio, Livius V; Katusic, Zvonimir S; He, Tongrong; Lee, Hon-Chi

    2012-07-01

    Activity of the vascular large conductance Ca(2+)-activated K(+) (BK) channel is tightly regulated by its accessory β(1) subunit (BK-β(1)). Downregulation of BK-β(1) expression in diabetic vessels is associated with upregulation of the forkhead box O subfamily transcription factor-3a (FOXO-3a)-dependent F-box-only protein (FBXO) expression. However, the upstream signaling regulating this process is unclear. Overproduction of reactive oxygen species (ROS) is a common finding in diabetic vasculopathy. We hypothesized that ROS signaling cascade facilitates the FOXO-3a/FBXO-mediated BK-β(1) degradation and leads to diabetic BK channel dysfunction. Using cellular biology, patch clamp, and videomicroscopy techniques, we found that reduced BK-β(1) expression in streptozotocin (STZ)-induced diabetic mouse arteries and in human coronary smooth muscle cells (SMCs) cultured with high glucose was attributable to an increase in protein kinase C (PKC)-β and NADPH oxidase expressions and accompanied by attenuation of Akt phosphorylation and augmentation of atrogin-1 expression. Treatment with ruboxistaurin (a PKCβ inhibitor) or with GW501516 (a peroxisome proliferator-activated receptor δ activator) reduced atrogin-1 expression and restored BK channel-mediated coronary vasodilation in diabetic mice. Our results suggested that oxidative stress inhibited Akt signaling and facilitated the FOXO-3a/FBXO-dependent BK-β(1) degradation in diabetic vessels. Suppression of the FOXO-3a/FBXO pathway prevented vascular BK-β(1) degradation and protected coronary function in diabetes.

  20. GNG11 (G-protein γ subunit 11) suppresses cell growth with induction of reactive oxygen species and abnormal nuclear morphology in human SUSM-1 cells.

    PubMed

    Takauji, Yuki; Kudo, Ikuru; En, Atsuki; Matsuo, Ryo; Hossain, Mohammad; Nakabayashi, Kazuhiko; Miki, Kensuke; Fujii, Michihiko; Ayusawa, Dai

    2017-04-05

    Enforced expression of GNG11, G-protein γ subunit 11, induces cellular senescence in normal human diploid fibroblasts. We here examined the effect of the expression of GNG11 on the growth of immortalized human cell lines, and found that it suppressed the growth of SUSM-1 cells, but not of HeLa cells. We then compared these two cell lines to understand the molecular basis for the action of GNG11. We found that expression of GNG11 induced the generation of reactive oxygen species (ROS) and abnormal nuclear morphology in SUSM-1 cells but not in HeLa cells. Increased ROS generation by GNG11 would likely be caused by the down-regulation of the antioxidant enzymes in SUSM-1 cells. We also found that SUSM-1 cells, even under normal culture conditions, showed higher levels of ROS and higher incidence of abnormal nuclear morphology than HeLa cells, and that abnormal nuclear morphology was relevant to the increased ROS generation in SUSM-1 cells. Thus, SUSM-1 and HeLa cells showed differences in the regulation of ROS and nuclear morphology, which might account for their different responses to the expression of GNG11. Then, SUSM-1 cells may provide a unique system to study the regulatory relationship between ROS generation, nuclear morphology, and G-protein signaling.

  1. Discovery of a Novel Bottlenose Dolphin Coronavirus Reveals a Distinct Species of Marine Mammal Coronavirus in Gammacoronavirus

    PubMed Central

    Woo, Patrick C. Y.; Lau, Susanna K. P.; Lam, Carol S. F.; Tsang, Alan K. L.; Hui, Suk-Wai; Fan, Rachel Y. Y.; Martelli, Paolo

    2014-01-01

    While gammacoronaviruses mainly comprise infectious bronchitis virus (IBV) and its closely related bird coronaviruses (CoVs), the only mammalian gammacoronavirus was discovered from a white beluga whale (beluga whale CoV [BWCoV] SW1) in 2008. In this study, we discovered a novel gammacoronavirus from fecal samples from three Indo-Pacific bottlenose dolphins (Tursiops aduncus), which we named bottlenose dolphin CoV (BdCoV) HKU22. All the three BdCoV HKU22-positive samples were collected on the same date, suggesting a cluster of infection, with viral loads of 1 × 103 to 1 × 105 copies per ml. Clearance of virus was associated with a specific antibody response against the nucleocapsid of BdCoV HKU22. Complete genome sequencing and comparative genome analysis showed that BdCoV HKU22 and BWCoV SW1 have similar genome characteristics and structures. Their genome size is about 32,000 nucleotides, the largest among all CoVs, as a result of multiple unique open reading frames (NS5a, NS5b, NS5c, NS6, NS7, NS8, NS9, and NS10) between their membrane (M) and nucleocapsid (N) protein genes. Although comparative genome analysis showed that BdCoV HKU22 and BWCoV SW1 should belong to the same species, a major difference was observed in the proteins encoded by their spike (S) genes, which showed only 74.3 to 74.7% amino acid identities. The high ratios of the number of synonymous substitutions per synonymous site (Ks) to the number of nonsynonymous substitutions per nonsynonymous site (Ka) in multiple regions of the genome, especially the S gene (Ka/Ks ratio, 2.5), indicated that BdCoV HKU22 may be evolving rapidly, supporting a recent transmission event to the bottlenose dolphins. We propose a distinct species, Cetacean coronavirus, in Gammacoronavirus, to include BdCoV HKU22 and BWCoV SW1, whereas IBV and its closely related bird CoVs represent another species, Avian coronavirus, in Gammacoronavirus. PMID:24227844

  2. Experimental Crossing of Two Distinct Species of Leopard Geckos, Eublepharis angramainyu and E. macularius: Viability, Fertility and Phenotypic Variation of the Hybrids

    PubMed Central

    Jančúchová-Lásková, Jitka; Landová, Eva; Frynta, Daniel

    2015-01-01

    Hybridization between distinct species of animals and subsequent genetic introgression plays a considerable role in the speciation process and the emergence of adaptive characters. Fitness of between-species hybrids usually sharply decreases with the divergence time of the concerned species and the divergence depth, which still allows for a successful crossing differs among principal clades of vertebrates. Recently, a review of hybridization events among distinct lizard species revealed that lizards belong to vertebrates with a highly developed ability to hybridize. In spite of this, reliable reports of experimental hybridizations between genetically fairly divergent species are only exceptional. Here, we show the results of the crossing of two distinct allopatric species of eyelid geckos possessing temperature sex determination and lacking sex chromosomes: Eublepharis macularius distributed in Pakistan/Afghanistan area and E. angramainyu, which inhabits Mesopotamia and adjacent areas. We demonstrated that F1 hybrids were viable and fertile, and the introgression of E. angramainyu genes into the E. macularius genome can be enabled via a backcrossing. The examined hybrids (except those of the F2 generation) displayed neither malformations nor a reduced survival. Analyses of morphometric and coloration traits confirmed phenotypic distinctness of both parental species and their F1 hybrids. These findings contrast with long-term geographic and an evolutionary separation of the studied species. Thus, the occurrence of fertile hybrids of comparably divergent species, such as E. angramainyu and E. macularius, may also be expected in other taxa of squamates. This would violate the current estimates of species diversity in lizards. PMID:26633648

  3. Induction of reactive oxygen species-stimulated distinctive autophagy by chelerythrine in non-small cell lung cancer cells.

    PubMed

    Tang, Zheng-Hai; Cao, Wen-Xiang; Wang, Zhao-Yu; Lu, Jia-Hong; Liu, Bo; Chen, Xiuping; Lu, Jin-Jian

    2017-03-09

    Chelerythrine (CHE), a natural benzo[c]phenanthridine alkaloid, shows anti-cancer effect through a number of mechanisms. Herein, the effect and mechanism of the CHE-induced autophagy, a type II programmed cell death, in non-small cell lung cancer (NSCLC) cells were studied for the first time. CHE induced cell viability decrease, colony formation inhibition, and apoptosis in a concentration-dependent manner in NSCLC A549 and NCI-H1299 cells. In addition, CHE triggered the expression of phosphatidylethanolamine-modified microtubule-associated protein light-chain 3 (LC3-II). The CHE-induced expression of LC3-II was further increased in the combination treatment with chloroquine (CQ), an autophagy inhibitor, and large amounts of red-puncta were observed in the CHE-treated A549 cells with stable expression of mRFP-EGFP-LC3, indicating that CHE induces autophagy flux. Silence of beclin 1 reversed the CHE-induced expression of LC3-II. Inhibition of autophagy remarkably reversed the CHE-induced cell viability decrease and apoptosis in NCI-H1299 cells but not in A549 cells. Furthermore, CHE triggered reactive oxygen species (ROS) generation in both cell lines. A decreased level of ROS through pretreatment with N-acetyl-L-cysteine reversed the CHE-induced cell viability decrease, apoptosis, and autophagy. Taken together, CHE induced distinctive autophagy in A549 (accompanied autophagy) and NCI-H1299 (pro-death autophagy) cells and a decreased level of ROS reversed the effect of CHE in NSCLC cells in terms of cell viability, apoptosis, and autophagy.

  4. The identity of Cintractia carpophila var. kenaica: reclassification of a North American smut on Carex micropoda as a distinct species of Anthracoidea.

    PubMed

    Piątek, Marcin

    2013-07-01

    Cintractia carpophila var. kenaica, a neglected taxon described from Alaska more than half a century ago, is re-described and illustrated. Its nomenclature and taxonomic status are discussed. This smut species is characterised by small spores with a very finely verruculose surface rarely enclosed by a thin, hyaline, mucilaginous sheath, a wall with 2-5 distinct internal swellings, and parasitism on Carex micropoda (Carex sect. Dornera). It is reallocated to the genus Anthracoidea as a distinct species, Anthracoidea kenaica comb. nov., and assigned to Anthracoidea section Leiosporae which includes species having smooth or very finely verruculose spores. Morphological and biological characteristics of the five most similar Anthracoidea species are contrasted and discussed.

  5. 75 FR 37385 - Endangered and Threatened Species; Initiation of a 5-Year Review of the Eastern Distinct...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-29

    ... Sea Lion is listed as a Distinct Population Segment of a vertebrate taxon. We will also be considering application of the DPS policy for vertebrate taxa. DPS is defined in the February 7, 1996, Policy Regarding the Recognition of Distinct Vertebrate in Population Segments (61 FR 4722). For a population to...

  6. Genes encoding the biotin carboxylase subunit of acetyl-CoA carboxylase from Brassica napus and parental species: cloning, expression patterns, and evolution.

    PubMed

    Li, Zhi-Guo; Yin, Wei-Bo; Song, Li-Ying; Chen, Yu-Hong; Guan, Rong-Zhan; Wang, Jing-Qiao; Wang, Richard R-C; Hu, Zan-Min

    2011-03-01

    Comparative genomics is a useful tool to investigate gene and genome evolution. Biotin carboxylase (BC), an important subunit of heteromeric acetyl-CoA carboxylase (ACCase) that is a rate-limiting enzyme in fatty acid biosynthesis in dicots, catalyzes ATP, biotin carboxyl carrier protein, and CO2 to form carboxybiotin carboxyl carrier protein. In this study, we cloned four genes encoding BC from Brassica napus L. (namely BnaC.BC.a, BnaC.BC.b, BnaA.BC.a, and BnaA.BC.b), and two were cloned from each of the two parental species Brassica rapa L. (BraA.BC.a and BraA.BC.b) and Brassica oleracea L. (BolC.BC.a and BolC.BC.b). Sequence analyses revealed that in B. napus the genes BnaC.BC.a and BnaC.BC.b were from the C genome of B. oleracea, whereas BnaA.BC.a and BnaA.BC.b were from the A genome of B. rapa. Comparative and cluster analysis indicated that these genes were divided into two major groups, BnaC.BC.a, BnaA.BC.a, BraA.BC.a, and BolC.BC.a in group-1 and BnaC.BC.b, BnaA.BC.b, BraA.BC.b, and BolC.BC.b in group-2. The divergence of group-1 and group-2 genes occurred in their common ancestor 13-17 million years ago (MYA), soon after the divergence of Arabidopsis and Brassica (15-20 MYA). This time of divergence is identical to the previously reported triplicated time of paralogous subgenomes of diploid Brassica species and the divergence date of group-1 and group-2 genes of α-carboxyltransferase, another subunit of heteromeric ACCase, in Brassica. Reverse transcription PCR revealed that the expression level of group-1 and group-2 genes varied in different organs, and the expression patterns of the two groups of genes were similar in different organs, except in flower. However, two paralogs of group-2 BC genes from B. napus could express differently in mature plants tested by generating BnaA.BC.b and BnaC.BC.b promoter-β-glucuronidase (GUS) fusions. The amino acid sequences of proteins encoded by these genes were highly conserved, except the sequence encoding

  7. Isotopic 'fingerprinting' of distinct water reservoirs in the critical zone and their exploitation by different tree species

    NASA Astrophysics Data System (ADS)

    Oshun, J.; Dietrich, W. E.; Dawson, T. E.; Rempe, D. M.; Fung, I. Y.

    2013-12-01

    Channel incision, surface erosion, and drainage of landscapes leads to emergent hillslopes underlain by varying degrees of soil development and weathered bedrock. Vertical and lateral differentiation by weathering leads to a structured heterogeneity of soil and rock properties that affect water pathways, moisture retention, and moisture availability to plants. Near surface evaporative losses cause seasonal isotopic enrichment of soil water, but we asked, what is the isotopic pattern of the deeper moisture and how important is it as a water source to the vegetation? Here, we report on the use of stable isotopic data to 'fingerprint' and then characterize the structure of subsurface water reservoirs that lie beneath an old-growth forested hillslope in the Eel River basin of northern California. The site, Rivendell, a 4000 m2 heavily instrumented, unchannled basin draining to Elder Creek 0.5 km upstream from the confluence with the South Fork Eel River, is composed of turbidite sequences of vertically dipping mudstone and minor sandstone. Soils are thin (0-60 cm), and are underlain by a 1-4 m thick saprolite layer, and a 3-20 m thick weathered rock zone. Annual precipitation of 1900 mm, predominantly from October to May, raises the water table from summer lows of 5-28 m below the ground to winter highs of 2.5 m below the ground, with depth to the water table increasing with upslope distance from Elder Creek. Whereas the north-facing slope has a dominant forest canopy composed of Douglas-fir (Pseudotsuga menziesii), and a mid-story of hardwood species (Quercus agrifolia, Umbellularia californica, Notholithocarpus densiflorus, Arbutus menziesii, and the riparian tree, Acer macrophyllum), the south-facing slope, which drains directly to the South Fork Eel River, is primarily hardwood. Intensive sampling over 20 months reveals subsurface moisture reservoirs in the mobile and bulk water of the soil, saprolite, and weathered rock, with persistent and distinct isotopic

  8. Binding of EBP50 to Nox organizing subunit p47phox is pivotal to cellular reactive species generation and altered vascular phenotype

    PubMed Central

    Al Ghouleh, Imad; Meijles, Daniel N.; Zhang, Qiangmin; Sahoo, Sanghamitra; Gorelova, Anastasia; Henrich Amaral, Jefferson; Rodríguez, Andrés I.; Mamonova, Tatyana; Song, Gyun Jee; Bisello, Alessandro; Friedman, Peter A.; Cifuentes-Pagano, M. Eugenia; Pagano, Patrick J.

    2016-01-01

    Despite numerous reports implicating NADPH oxidases (Nox) in the pathogenesis of many diseases, precise regulation of this family of professional reactive oxygen species (ROS) producers remains unclear. A unique member of this family, Nox1 oxidase, functions as either a canonical or hybrid system using Nox organizing subunit 1 (NoxO1) or p47phox, respectively, the latter of which is functional in vascular smooth muscle cells (VSMC). In this manuscript, we identify critical requirement of ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50; aka NHERF1) for Nox1 activation and downstream responses. Superoxide (O2•−) production induced by angiotensin II (AngII) was absent in mouse EBP50 KO VSMC vs. WT. Moreover, ex vivo incubation of aortas with AngII showed a significant increase in O2•− in WT but not EBP50 or Nox1 nulls. Similarly, lipopolysaccharide (LPS)-induced oxidative stress was attenuated in femoral arteries from EBP50 KO vs. WT. In silico analyses confirmed by confocal microscopy, immunoprecipitation, proximity ligation assay, FRET, and gain-/loss-of-function mutagenesis revealed binding of EBP50, via its PDZ domains, to a specific motif in p47phox. Functional studies revealed AngII-induced hypertrophy was absent in EBP50 KOs, and in VSMC overexpressing EBP50, Nox1 gene silencing abolished VSMC hypertrophy. Finally, ex vivo measurement of lumen diameter in mouse resistance arteries exhibited attenuated AngII-induced vasoconstriction in EBP50 KO vs. WT. Taken together, our data identify EBP50 as a previously unidentified regulator of Nox1 and support that it promotes Nox1 activity by binding p47phox. This interaction is pivotal for agonist-induced smooth muscle ROS, hypertrophy, and vasoconstriction and has implications for ROS-mediated physiological and pathophysiological processes. PMID:27540115

  9. Secondary structure features of ribosomal RNA species within intact ribosomal subunits and efficiency of RNA-protein interactions in thermoacidophilic (Caldariella acidophila, Bacillus acidocaldarius) and mesophilic (Escherichia coli) bacteria.

    PubMed

    Cammarano, P; Mazzei, F; Londei, P; Teichner, A; de Rosa, M; Gambacorta, A

    1983-08-02

    Ribosomal subunits of Caldariella acidophila (max.growth temp., 90 degrees C) have been compared to subunits of Bacillus acidocaldarius (max. growth temp., 70 degrees C) and Escherichia coli (max. growth temp., 47 degrees C) with respect to (a) bihelical content of rRNA; (b) G . C content of bihelical domains and (c) tightness of rRNA-protein interactions. The principal results are as follows. Subunits of C. acidophilia ribosomes (Tm = 90-93 degrees C) exhibit considerable thermal tolerance over their B. acidocaldarius (Tm = 77 degrees C) and E. coli counterparts (Tm = 72 degrees C). Based on the "melting' hyperchromicities of the intact ribosomal subunits a 51-55% fraction of the nucleotides appears to participate in hydrogen-bonded base pairing regardless of ribosome source, whereas a larger fraction, 67-70%, appears to be involved in hydrogen bonding in the naked rRNA species. The G . C content of bihelical domains of both free and ribosome-bound rRNA increases with increasing thermophily; based on hyperchromicity dispersion spectra of intact subunits and free rRNA, the bihelical parts of C. acidophila rRNA are estimated to contain 63-64% G . C, compared to 58.5% G . C for B. acidocaldarius and 55% G . C for E. coli. The increment of ribosome Tm values with increasing thermophily is greater than the increase in Tm for the free rRNA, indicating that within ribosomes bihelical domains of the thermophile rRNA species are stabilized more efficiently than their mesophile counterparts by proteins or/ and other component(s). The efficiency of the rRNA-protein interactions in the mesophile and thermophile ribosomes has been probed by comparing the releases, with LiCl-urea, of the rRNA species from the corresponding ribosomal subunits stuck to a Celite column through their protein moiety; it has been established that the release of C. acidophila rRNA from the Celite-bound ribosomes occurs at salt-urea concentrations about 4-fold higher than those required to release r

  10. [Molecular cloning of the DNA sequence of activin beta A subunit gene mature peptides from panda and related species and its application in the research of phylogeny and taxonomy].

    PubMed

    Wang, Xiao-Jing; Wang, Xiao-Xing; Wang, Ya-Jun; Wang, Xi-Zhong; He, Guang-Xin; Chen, Hong-Wei; Fei, Li-Song

    2002-09-01

    Activin, which is included in the transforming growth factor-beta (TGF beta) superfamily of proteins and receptors, is known to have broad-ranging effects in the creatures. The mature peptide of beta A subunit of this gene, one of the most highly conserved sequence, can elevate the basal secretion of follicle-stimulating hormone (FSH) in the pituitary and FSH is pivotal to organism's reproduction. Reproduction block is one of the main reasons which cause giant panda to extinct. The sequence of Activin beta A subunit gene mature peptides has been successfully amplified from giant panda, red panda and malayan sun bear's genomic DNA by using polymerase chain reaction (PCR) with a pair of degenerate primers. The PCR products were cloned into the vector pBlueScript+ of Esherichia coli. Sequence analysis of Activin beta A subunit gene mature peptides shows that the length of this gene segment is the same (359 bp) and there is no intron in all three species. The sequence encodes a peptide of 119 amino acid residues. The homology comparison demonstrates 93.9% DNA homology and 99% homology in amino acid among these three species. Both GenBank blast search result and restriction enzyme map reveal that the sequences of Activin beta A subunit gene mature peptides of different species are highly conserved during the evolution process. Phylogeny analysis is performed with PHYLIP software package. A consistent phylogeny tree has been drawn with three different methods. The software analysis outcome accords with the academic view that giant panda has a closer relationship to the malayan sun bear than the red panda. Giant panda should be grouped into the bear family (Uersidae) with the malayan sun bear. As to the red panda, it would be better that this animal be grouped into the unique family (red panda family) because of great difference between the red panda and the bears (Uersidae).

  11. Rare failures of DNA barcodes [corrected] to separate morphologically distinct species in a biodiversity survey of Iberian leaf beetles.

    PubMed

    Baselga, Andrés; Gómez-Rodríguez, Carola; Novoa, Francisco; Vogler, Alfried P

    2013-01-01

    During a survey of genetic and species diversity patterns of leaf beetle (Coleoptera: Chrysomelidae) assemblages across the Iberian Peninsula we found a broad congruence between morphologically delimited species and variation in the cytochrome oxidase (cox1) gene. However, one species pair each in the genera Longitarsus Berthold and Pachybrachis Chevrolat was inseparable using molecular methods, whereas diagnostic morphological characters (including male or female genitalia) unequivocally separated the named species. Parsimony haplotype networks and maximum likelihood trees built from cox1 showed high genetic structure within each species pair, but no correlation with the morphological types and neither with geographic distributions. This contrasted with all analysed congeneric species, which were recovered as monophyletic. A limited number of specimens were sequenced for the nuclear 18S rRNA gene, which showed no or very limited variation within the species pair and no separation of morphological types. These results suggest that processes of lineage sorting for either group are lagging behind the clear morphological and presumably reproductive separation. In the Iberian chrysomelids, incongruence between DNA-based and morphological delimitations is a rare exception, but the discovery of these species pairs may be useful as an evolutionary model for studying the process of speciation in this ecological and geographical setting. In addition, the study of biodiversity patterns based on DNA requires an evolutionary understanding of these incongruences and their potential causes.

  12. 75 FR 38979 - Endangered and Threatened Species; Initiation of a 5-Year Review of the Eastern Distinct...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-07-07

    ... (Eumetopias jubatus) under the Endangered Species Act of 1973, as amended (ESA) and requesting information... National Oceanic and Atmospheric Administration RIN 0648-XU50 Endangered and Threatened Species; Initiation... number to which comments and information should be sent. DATES: This correction is effective July 7,...

  13. Rare Failures of DNA Bar Codes to Separate Morphologically Distinct Species in a Biodiversity Survey of Iberian Leaf Beetles

    PubMed Central

    Baselga, Andrés; Gómez-Rodríguez, Carola; Novoa, Francisco; Vogler, Alfried P.

    2013-01-01

    During a survey of genetic and species diversity patterns of leaf beetle (Coleoptera: Chrysomelidae) assemblages across the Iberian Peninsula we found a broad congruence between morphologically delimited species and variation in the cytochrome oxidase (cox1) gene. However, one species pair each in the genera Longitarsus Berthold and Pachybrachis Chevrolat was inseparable using molecular methods, whereas diagnostic morphological characters (including male or female genitalia) unequivocally separated the named species. Parsimony haplotype networks and maximum likelihood trees built from cox1 showed high genetic structure within each species pair, but no correlation with the morphological types and neither with geographic distributions. This contrasted with all analysed congeneric species, which were recovered as monophyletic. A limited number of specimens were sequenced for the nuclear 18S rRNA gene, which showed no or very limited variation within the species pair and no separation of morphological types. These results suggest that processes of lineage sorting for either group are lagging behind the clear morphological and presumably reproductive separation. In the Iberian chrysomelids, incongruence between DNA-based and morphological delimitations is a rare exception, but the discovery of these species pairs may be useful as an evolutionary model for studying the process of speciation in this ecological and geographical setting. In addition, the study of biodiversity patterns based on DNA requires an evolutionary understanding of these incongruences and their potential causes. PMID:24040352

  14. Genes encoding biotin carboxylase subunit of acetyl-CoA carboxylase from Brassica napus and parental species: cloning, expression patterns, and evolution

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Comparative genomics is a useful tool to investigate gene and genome evolution. Biotin carboxylase (BC), an important subunit of heteromeric ACCase that is a rate-limiting enzyme in fatty acid biosynthesis in dicots, catalyzes ATP, biotin-carboxyl-carrier protein and CO2 to form carboxybiotin-carbo...

  15. Oogonial biometry and phylogenetic analyses of the Pythium vexans species group from woody agricultural hosts in South Africa reveal distinct groups within this taxon.

    PubMed

    Spies, Christoffel F J; Mazzola, Mark; Botha, Wilhelm J; Van Der Rijst, Marieta; Mostert, Lizel; Mcleod, Adéle

    2011-02-01

    Pythium vexans fits into the internal transcribed spacer (ITS) clade K sensu Lévesque & De Cock (2004). Within clade K, P. vexans forms a distinct clade containing two enigmatic species, Pythium indigoferae and Pythium cucurbitacearum of which no ex-type strains are available. In South Africa, as well as in other regions of the world, P. vexans isolates are known to be heterogeneous in their ITS sequences and may consist of more than one species. This study aimed to investigate the diversity of South African P. vexans isolates, mainly from grapevines, but also citrus and apple using (i) phylogenetic analyses of the ITS, cytochrome c oxidase (cox) I, cox II, and β-tubulin regions and (ii) seven biometric oogonial parameters. Each of the phylogenies clustered P. vexans isolates into a single well-supported clade, distinct from other clade K species. The β-tubulin region was phylogenetically uninformative regarding the P. vexans group. The ITS phylogeny and combined cox I and II phylogenies, although each revealing several P. vexans subclades, were incongruent. One of the most striking incongruences was the presence of one cox subclade that contained two distinct ITS subclades (Ib and IV). Three groups (A-C) were subjectively identified among South African P. vexans isolates using (i) phylogenetic clades (ITS and cox), (ii) univariate analysis of oogonial diameters, and (iii) multivariate analyses of biometric oogonial parameters. Group A is considered to be P. vexans s. str. since it contained the P. vexans CBS reference strain from Van der Plaats-Niterink (1981). This group had significantly smaller oogonial diameters than group B and C isolates. Group B contained the isolates from ITS subclades Ib and IV, which formed a single cox subclade. The ITS subclade IV isolates were all sexually sterile or produced mainly abortive oospores, as opposed to the sexually fertile subclade Ib isolates, and may thus represent a distinct assemblage within group B. Although ITS

  16. Comparison of four species-delimitation methods applied to a DNA barcode data set of insect larvae for use in routine bioassessment for use in routine bioassessment

    EPA Science Inventory

    Species delimitation (grouping individuals into distinct taxonomic groups) is an essential part of evolutionary, conservation, and molecular ecology. Deoxyribonucleic acid (DNA) barcodes, short fragments of the cytochrome c oxidase subunit I (COI) gene, are being used in environm...

  17. Bird and mammal species composition in distinct geographic regions and their relationships with environmental factors across multiple spatial scales.

    PubMed

    Kent, Rafi; Bar-Massada, Avi; Carmel, Yohay

    2014-05-01

    Global patters of species distributions and their underlying mechanisms are a major question in ecology, and the need for multi-scale analyses has been recognized. Previous studies recognized climate, topography, habitat heterogeneity and disturbance as important variables affecting such patterns. Here we report on analyses of species composition - environment relationships among different taxonomic groups in two continents, and the components of such relationships, in the contiguous USA and Australia. We used partial Canonical Correspondence Analysis of occurrence records of mammals and breeding birds from the Global Biodiversity Information Facility, to quantify relationships between species composition and environmental variables in remote geographic regions at multiple spatial scales, with extents ranging from 10(5) to 10(7) km(2) and sampling grids from 10 to 10,000 km(2). We evaluated the concept that two elements contribute to the impact of environmental variables on composition: the strength of species' affinity to an environmental variable, and the amount of variance in the variable. To disentangle these two elements, we analyzed correlations between resulting trends and the amount of variance contained in different environmental variables to isolate the mechanisms behind the observed relationships. We found that climate and land use-land cover are responsible for most explained variance in species composition, regardless of scale, taxonomic group and geographic region. However, the amount of variance in species composition attributed to land use / land cover (LULC) was closely related to the amount of intrinsic variability in LULC in the USA, but not in Australia, while the effect of climate on species composition was negatively correlated to the variability found in the climatic variables. The low variance in climate, compared to LULC, suggests that species in both taxonomic groups have strong affinity to climate, thus it has a strong effect on species

  18. Trypanosoma cruzi: distinct patterns of infection in the sibling caviomorph rodent species Thrichomys apereoides laurentius and Thrichomys pachyurus (Rodentia, Echimyidae).

    PubMed

    Roque, André Luiz Rodrigues; D'Andrea, Paulo Sérgio; de Andrade, Gisele Braziliano; Jansen, Ana Maria

    2005-09-01

    Thrichomys apereoides, a caviomorph rodent species common in a highly endemic area for Chagas disease in Brazil, may act as reservoir of the parasite. However, no information is available concerning its sibling species Thrichomys pachyurus, found in the Pantanal region, where Trypanosoma cruzi is found only in the enzootic cycle. We followed up the cross infection of these cryptic species with two isolates derived from naturally infected T. pachyurus and Thrichomys apereoides laurentius. No regional co-adaptation between Thrichomys species and the regional isolates were noticed. However, significant differences in the outcome of the infection were observed. T. a. laurentius was more resistant than T. pachyurus, as expressed by lower parasitemia and less histopathological damage. The routine biochemical markers used for laboratory rodents were unsuitable for follow up of infection in Thrichomys spp, since they did not correlate with the histopathological findings or allowed the kinetic follow-up of tissue colonization by the parasite.

  19. Super-resolution fluorescence of huntingtin reveals growth of globular species into short fibers and coexistence of distinct aggregates.

    PubMed

    Duim, Whitney C; Jiang, Yan; Shen, Koning; Frydman, Judith; Moerner, W E

    2014-12-19

    Polyglutamine-expanded huntingtin, the protein encoded by HTT mutations associated with Huntington's disease, forms aggregate species in vitro and in vivo. Elucidation of the mechanism of growth of fibrillar aggregates from soluble monomeric protein is critical to understanding the progression of Huntington's disease and to designing therapeutics for the disease, as well as for aggregates implicated in Alzheimer's and Parkinson's diseases. We used the technique of multicolor single-molecule, super-resolution fluorescence imaging to characterize the growth of huntingtin exon 1 aggregates. The huntingtin exon 1 aggregation followed a pathway from exclusively spherical or globular species of ∼80 nm to fibers ∼1 μm in length that increased in width, but not length, over time with the addition of more huntingtin monomers. The fibers further aggregated with one another into aggregate assemblies of increasing size. Seeds created by sonication, which were comparable in shape and size to the globular species in the pathway, were observed to grow through multidirectional elongation into fibers, suggesting a mechanism for growth of globular species into fibers. The single-molecule sensitivity of our approach made it possible to characterize the aggregation pathway across a large range of size scales, from monomers to fiber assemblies, and revealed the coexistence of different aggregate species (globular species, fibers, fiber assemblies) even at late time points.

  20. Single and mixed-species trypanosome and microsporidia infections elicit distinct, ephemeral cellular and humoral immune responses in honey bees

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Frequently encountered parasite species impart strong selective pressures on host immune system evolution and are more apt to concurrently infect the same host, yet molecular impacts in light of this are often overlooked. We have contrasted immune responses in honey bees to two common eukaryotic en...

  1. Taxonomic utility of environmental niche models for species distinction: A case study in Anthophora (Heliophila) (Hymenoptera: Apidae)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Taxonomy has far-reaching effects throughout biology, and incorrect taxonomy can be detrimental in many ways. Polymorphic species complexes, many of which exist in the bee genus Anthophora Latreille, lend themselves to such difficulties. This study employs environmental niche mapping (ENM) and tradi...

  2. Distinct migratory and non-migratory ecotypes of an endemic New Zealand eleotrid (Gobiomorphus cotidianus) – implications for incipient speciation in island freshwater fish species

    PubMed Central

    2008-01-01

    Background Many postglacial lakes contain fish species with distinct ecomorphs. Similar evolutionary scenarios might be acting on evolutionarily young fish communities in lakes of remote islands. One process that drives diversification in island freshwater fish species is the colonization of depauperate freshwater environments by diadromous (migratory) taxa, which secondarily lose their migratory behaviour. The loss of migration limits dispersal and gene flow between distant populations, and, therefore, is expected to facilitate local morphological and genetic differentiation. To date, most studies have focused on interspecific relationships among migratory species and their non-migratory sister taxa. We hypothesize that the loss of migration facilitates intraspecific morphological, behavioural, and genetic differentiation between migratory and non-migratory populations of facultatively diadromous taxa, and, hence, incipient speciation of island freshwater fish species. Results Microchemical analyses of otolith isotopes (88Sr, 137Ba and 43Ca) differentiated migratory and non-migratory stocks of the New Zealand endemic Gobiomorphus cotidianus McDowall (Eleotridae). Samples were taken from two rivers, one lake and two geographically-separated outgroup locations. Meristic analyses of oculoscapular lateral line canals documented a gradual reduction of these structures in the non-migratory populations. Amplified fragment length polymorphism (AFLP) fingerprints revealed considerable genetic isolation between migratory and non-migratory populations. Temporal differences in reproductive timing (migratory = winter spawners, non-migratory = summer spawners; as inferred from gonadosomatic indices) provide a prezygotic reproductive isolation mechanism between the two ecotypes. Conclusion This study provides a holistic look at the role of diadromy in incipient speciation of island freshwater fish species. All four analytical approaches (otolith microchemistry, morphology

  3. Possible natural hybridization of two morphologically distinct species of Acropora (Cnidaria, Scleractinia) in the Pacific: fertilization and larval survival rates.

    PubMed

    Isomura, Naoko; Iwao, Kenji; Fukami, Hironobu

    2013-01-01

    Natural hybridization of corals in the Indo-Pacific has been considered rather rare. However, field studies have observed many corals with intermediate interspecific or unusual morphologies. Given that the existence of F1 hybrids with intermediate interspecific morphologies has been proven in the Caribbean, hybrids may also inhabit the Indo-Pacific and occur more frequently than expected. In this study, we focused on two morphologically different species, Acropora florida and A. intermedia, and performed crossing experiments at Akajima Island, Japan. Results showed that these species could hybridize in both directions via eggs and sperm, but that fertilization rates significantly differed according to which species provided eggs. These results are similar to those reported from the Caribbean. Although all embryos developed normally to the planular larval stage, the developmental processes of some hybrid embryos were delayed by approximately 1 h compared with conspecific embryos, suggesting that fertilization occurred 1 h later in interspecific crosses than in intraspecific crosses. More successful hybridization could occur under conditions with low numbers of conspecific colonies. Additionally, a comparison of survival rates between hybrid and intraspecific larvae revealed that intra- and interspecific larvae produced from eggs of A. florida survived for significantly longer than those produced from eggs of A. intermedia. Considering these data, under specific conditions, hybrids can be expected to be produced and survive in nature in the Pacific. Furthermore, we identified one colony with intermediate morphology between A. florida and A. intermedia in the field. This colony was fertilized only by eggs of A. florida, with high fertilization rates, suggesting that this colony would be a hybrid of these two species and might be backcrossed.

  4. Coexisting cryptic species of the Litoditis marina complex (Nematoda) show differential resource use and have distinct microbiomes with high intraspecific variability.

    PubMed

    Derycke, S; De Meester, N; Rigaux, A; Creer, S; Bik, H; Thomas, W K; Moens, T

    2016-05-01

    Differences in resource use or in tolerances to abiotic conditions are often invoked as potential mechanisms underlying the sympatric distribution of cryptic species. Additionally, the microbiome can provide physiological adaptations of the host to environmental conditions. We determined the intra- and interspecific variability of the microbiomes of three cryptic nematode species of the Litoditis marina species complex that co-occur, but show differences in abiotic tolerances. Roche 454 pyrosequencing of the microbial 16S rRNA gene revealed distinct bacterial communities characterized by a substantial diversity (85-513 OTUs) and many rare OTUs. The core microbiome of each species contained only very few OTUs (2-6), and four OTUs were identified as potentially generating tolerance to abiotic conditions. A controlled experiment in which nematodes from two cryptic species (Pm1 and Pm3) were fed with either an E. coli suspension or a bacterial mix was performed, and the 16S rRNA gene was sequenced using the MiSeq technology. OTU richness was 10-fold higher compared to the 454 data set and ranged between 1118 and 7864. This experiment confirmed the existence of species-specific microbiomes, a core microbiome with few OTUs, and high interindividual variability. The offered food source affected the bacterial community and illustrated different feeding behaviour between the cryptic species, with Pm3 exhibiting a higher degree of selective feeding than Pm1. Morphologically similar species belonging to the same feeding guild (bacterivores) can thus have substantial differences in their associated microbiomes and feeding strategy, which in turn may have important ramifications for biodiversity-ecosystem functioning relationships.

  5. The molecular dimension of microbial species: 1. Ecological distinctions among, and homogeneity within, putative ecotypes of Synechococcus inhabiting the cyanobacterial mat of Mushroom Spring, Yellowstone National Park.

    PubMed

    Becraft, Eric D; Wood, Jason M; Rusch, Douglas B; Kühl, Michael; Jensen, Sheila I; Bryant, Donald A; Roberts, David W; Cohan, Frederick M; Ward, David M

    2015-01-01

    Based on the Stable Ecotype Model, evolution leads to the divergence of ecologically distinct populations (e.g., with different niches and/or behaviors) of ecologically interchangeable membership. In this study, pyrosequencing was used to provide deep sequence coverage of Synechococcus psaA genes and transcripts over a large number of habitat types in the Mushroom Spring microbial mat. Putative ecological species [putative ecotypes (PEs)], which were predicted by an evolutionary simulation based on the Stable Ecotype Model (Ecotype Simulation), exhibited distinct distributions relative to temperature-defined positions in the effluent channel and vertical position in the upper 1 mm-thick mat layer. Importantly, in most cases variants predicted to belong to the same PE formed unique clusters relative to temperature and depth in the mat in canonical correspondence analysis, supporting the hypothesis that while the PEs are ecologically distinct, the members of each ecotype are ecologically homogeneous. PEs responded differently to experimental perturbations of temperature and light, but the genetic variation within each PE was maintained as the relative abundances of PEs changed, further indicating that each population responded as a set of ecologically interchangeable individuals. Compared to PEs that predominate deeper within the mat photic zone, the timing of transcript abundances for selected genes differed for PEs that predominate in microenvironments closer to upper surface of the mat with spatiotemporal differences in light and O2 concentration. All of these findings are consistent with the hypotheses that Synechococcus species in hot spring mats are sets of ecologically interchangeable individuals that are differently adapted, that these adaptations control their distributions, and that the resulting distributions constrain the activities of the species in space and time.

  6. The molecular dimension of microbial species: 1. Ecological distinctions among, and homogeneity within, putative ecotypes of Synechococcus inhabiting the cyanobacterial mat of Mushroom Spring, Yellowstone National Park

    PubMed Central

    Becraft, Eric D.; Wood, Jason M.; Rusch, Douglas B.; Kühl, Michael; Jensen, Sheila I.; Bryant, Donald A.; Roberts, David W.; Cohan, Frederick M.; Ward, David M.

    2015-01-01

    Based on the Stable Ecotype Model, evolution leads to the divergence of ecologically distinct populations (e.g., with different niches and/or behaviors) of ecologically interchangeable membership. In this study, pyrosequencing was used to provide deep sequence coverage of Synechococcus psaA genes and transcripts over a large number of habitat types in the Mushroom Spring microbial mat. Putative ecological species [putative ecotypes (PEs)], which were predicted by an evolutionary simulation based on the Stable Ecotype Model (Ecotype Simulation), exhibited distinct distributions relative to temperature-defined positions in the effluent channel and vertical position in the upper 1 mm-thick mat layer. Importantly, in most cases variants predicted to belong to the same PE formed unique clusters relative to temperature and depth in the mat in canonical correspondence analysis, supporting the hypothesis that while the PEs are ecologically distinct, the members of each ecotype are ecologically homogeneous. PEs responded differently to experimental perturbations of temperature and light, but the genetic variation within each PE was maintained as the relative abundances of PEs changed, further indicating that each population responded as a set of ecologically interchangeable individuals. Compared to PEs that predominate deeper within the mat photic zone, the timing of transcript abundances for selected genes differed for PEs that predominate in microenvironments closer to upper surface of the mat with spatiotemporal differences in light and O2 concentration. All of these findings are consistent with the hypotheses that Synechococcus species in hot spring mats are sets of ecologically interchangeable individuals that are differently adapted, that these adaptations control their distributions, and that the resulting distributions constrain the activities of the species in space and time. PMID:26157420

  7. Distinct galactofuranose antigens in the cell wall and culture supernatants as a means to differentiate Fusarium from Aspergillus species.

    PubMed

    Wiedemann, Annegret; Kakoschke, Tamara Katharina; Speth, Cornelia; Rambach, Günter; Ensinger, Christian; Jensen, Henrik Elvang; Ebel, Frank

    2016-09-01

    Detection of carbohydrate antigens is an important means for diagnosis of invasive fungal infections. For diagnosis of systemic Aspergillus infections, galactomannan is commonly used, the core antigenic structure of which consists of chains of several galactofuranose moieties. In this study, we provide evidence that Fusarium produces at least two distinct galactofuranose antigens: Smaller amounts of galactomannan and larger quantities of a novel antigen recognized by the monoclonal antibody AB135-8. In A. fumigatus, only minor amounts of the AB135-8 antigen are found in supernatants and in the apical regions of hyphae. A galactofuranose-deficient A. fumigatus mutant lacks the AB135-8 antigen, which strongly suggests that galactofuranose is an essential constituent of this antigen. Using a combination of AB135-8 and a galactomannan-specific antibody, we were able to unambiguously differentiate A. fumigatus and Fusarium hyphae in immunohistology. Moreover, since Fusarium releases the AB135-8 antigen, it appears to be a promising target antigen for a serological detection of Fusarium infections.

  8. Combinations of distinct long-chain polyunsaturated fatty acid species for improved dietary treatment against allergic bronchial asthma.

    PubMed

    Beermann, Christopher; Neumann, Sandy; Fußbroich, Daniela; Zielen, Stefan; Schubert, Ralf

    2016-01-01

    Allergic bronchial asthma is a chronic inflammatory disease of the airways with an increasing incidence in Western societies. Exposure to allergens provokes recurrent attacks of breathlessness, airway hyperreactivity, wheezing, and coughing. For the early phase and milder forms of allergic asthma, dietary supplementation with long-chain polyunsaturated fatty acids (LCPUFA), predominantly fish oil-associated eicosapentaenoic (C20:5 ω-3) and docosahexaenoic acid (C22:6 ω-3), and distinct crop oil-derived fatty acids might provide a sustainable treatment strategy, as discussed in several studies. In addition to immune-controlling prostaglandins, leukotrienes, and thromboxanes, specialized proresolving mediators, such as lipoxins, resolvins, protectins, and maresins, are metabolized from different LCPUFA, which actively resolve inflammation. The aim of this review was to discuss the possible synergistic effects of ω-3 and ω-6 LCPUFA combinations concerning rebuilding fatty acid homeostasis in cellular membranes, modifying eicosanoid metabolic pathways, controlling inflammatory processes by focusing on resolving inflammation in the bronchoalveolar system on the cellular level, and helping to control clinical symptoms in bronchial asthma.

  9. Subunit compositions of Arabidopsis RNA polymerases I and III reveal Pol I- and Pol III-specific forms of the AC40 subunit and alternative forms of the C53 subunit

    SciTech Connect

    Ream, Thomas S.; Haag, Jeremy R.; Pontvianne, Frederic; Nicora, Carrie D.; Norbeck, Angela D.; Pasa-Tolic, Ljiljana; Pikaard, Craig S.

    2015-05-02

    Using affinity purification and mass spectrometry, we identified the subunits of Arabidopsis thaliana multisubunit RNA Polymerases I and III (abbreviated as Pol I and Pol III), providing the first description of their physical compositions in plants. AC40 and AC19 subunits are typically common to Pol I (a.k.a. Pol A) and Pol III (a.k.a. Pol C) and are encoded by single genes whose mutation, in humans, is a cause of the craniofacial disorder, Treacher-Collins Syndrome. Surprisingly, A. thaliana, and related species, express two distinct AC40 paralogs, one of which assembles into Pol I and the other of which assembles into Pol III. Changes at eight amino acid positions correlate with this functional divergence of Pol I and Pol III-specific AC40 paralogs. Two genes encode homologs of the yeast C53 subunit, and either variant can assemble into Pol III. By contrast, only one of two potential C17 variants, and one of two potential C31 variants were detected in Pol III. We introduce a new nomenclature system for plant Pol I and Pol III subunits in which the twelve subunits that are structurally and functionally homologous among Pols I through V are assigned equivalent numbers.

  10. Subunit compositions of Arabidopsis RNA polymerases I and III reveal Pol I- and Pol III-specific forms of the AC40 subunit and alternative forms of the C53 subunit

    DOE PAGES

    Ream, Thomas S.; Haag, Jeremy R.; Pontvianne, Frederic; ...

    2015-05-02

    Using affinity purification and mass spectrometry, we identified the subunits of Arabidopsis thaliana multisubunit RNA Polymerases I and III (abbreviated as Pol I and Pol III), providing the first description of their physical compositions in plants. AC40 and AC19 subunits are typically common to Pol I (a.k.a. Pol A) and Pol III (a.k.a. Pol C) and are encoded by single genes whose mutation, in humans, is a cause of the craniofacial disorder, Treacher-Collins Syndrome. Surprisingly, A. thaliana, and related species, express two distinct AC40 paralogs, one of which assembles into Pol I and the other of which assembles into Polmore » III. Changes at eight amino acid positions correlate with this functional divergence of Pol I and Pol III-specific AC40 paralogs. Two genes encode homologs of the yeast C53 subunit, and either variant can assemble into Pol III. By contrast, only one of two potential C17 variants, and one of two potential C31 variants were detected in Pol III. We introduce a new nomenclature system for plant Pol I and Pol III subunits in which the twelve subunits that are structurally and functionally homologous among Pols I through V are assigned equivalent numbers.« less

  11. Adaptation of the Mitochondrial Genome in Cephalopods: Enhancing Proton Translocation Channels and the Subunit Interactions

    PubMed Central

    Almeida, Daniela; Maldonado, Emanuel; Vasconcelos, Vitor; Antunes, Agostinho

    2015-01-01

    Mitochondrial protein-coding genes (mt genes) encode subunits forming complexes of crucial cellular pathways, including those involved in the vital process of oxidative phosphorylation (OXPHOS). Despite the vital role of the mitochondrial genome (mt genome) in the survival of organisms, little is known with respect to its adaptive implications within marine invertebrates. The molluscan Class Cephalopoda is represented by a marine group of species known to occupy contrasting environments ranging from the intertidal to the deep sea, having distinct metabolic requirements, varied body shapes and highly advanced visual and nervous systems that make them highly competitive and successful worldwide predators. Thus, cephalopods are valuable models for testing natural selection acting on their mitochondrial subunits (mt subunits). Here, we used concatenated mt genes from 17 fully sequenced mt genomes of diverse cephalopod species to generate a robust mitochondrial phylogeny for the Class Cephalopoda. We followed an integrative approach considering several branches of interest–covering cephalopods with distinct morphologies, metabolic rates and habitats–to identify sites under positive selection and localize them in the respective protein alignment and/or tridimensional structure of the mt subunits. Our results revealed significant adaptive variation in several mt subunits involved in the energy production pathway of cephalopods: ND5 and ND6 from Complex I, CYTB from Complex III, COX2 and COX3 from Complex IV, and in ATP8 from Complex V. Furthermore, we identified relevant sites involved in protein-interactions, lining proton translocation channels, as well as disease/deficiencies related sites in the aforementioned complexes. A particular case, revealed by this study, is the involvement of some positively selected sites, found in Octopoda lineage in lining proton translocation channels (site 74 from ND5) and in interactions between subunits (site 507 from ND5) of

  12. Adaptation of the Mitochondrial Genome in Cephalopods: Enhancing Proton Translocation Channels and the Subunit Interactions.

    PubMed

    Almeida, Daniela; Maldonado, Emanuel; Vasconcelos, Vitor; Antunes, Agostinho

    2015-01-01

    Mitochondrial protein-coding genes (mt genes) encode subunits forming complexes of crucial cellular pathways, including those involved in the vital process of oxidative phosphorylation (OXPHOS). Despite the vital role of the mitochondrial genome (mt genome) in the survival of organisms, little is known with respect to its adaptive implications within marine invertebrates. The molluscan Class Cephalopoda is represented by a marine group of species known to occupy contrasting environments ranging from the intertidal to the deep sea, having distinct metabolic requirements, varied body shapes and highly advanced visual and nervous systems that make them highly competitive and successful worldwide predators. Thus, cephalopods are valuable models for testing natural selection acting on their mitochondrial subunits (mt subunits). Here, we used concatenated mt genes from 17 fully sequenced mt genomes of diverse cephalopod species to generate a robust mitochondrial phylogeny for the Class Cephalopoda. We followed an integrative approach considering several branches of interest-covering cephalopods with distinct morphologies, metabolic rates and habitats-to identify sites under positive selection and localize them in the respective protein alignment and/or tridimensional structure of the mt subunits. Our results revealed significant adaptive variation in several mt subunits involved in the energy production pathway of cephalopods: ND5 and ND6 from Complex I, CYTB from Complex III, COX2 and COX3 from Complex IV, and in ATP8 from Complex V. Furthermore, we identified relevant sites involved in protein-interactions, lining proton translocation channels, as well as disease/deficiencies related sites in the aforementioned complexes. A particular case, revealed by this study, is the involvement of some positively selected sites, found in Octopoda lineage in lining proton translocation channels (site 74 from ND5) and in interactions between subunits (site 507 from ND5) of Complex I.

  13. Molecular Identification of Sibling Species of Sclerodermus (Hymenoptera: Bethylidae) That Parasitize Buprestid and Cerambycid Beetles by Using Partial Sequences of Mitochondrial DNA Cytochrome Oxidase Subunit 1 and 28S Ribosomal RNA Gene

    PubMed Central

    Jiang, Yuan; Yang, Zhongqi; Wang, Xiaoyi; Hou, Yuxia

    2015-01-01

    The species belonging to Sclerodermus (Hymenoptera: Bethylidae) are currently the most important insect natural enemies of wood borer pests, mainly buprestid and cerambycid beetles, in China. However, some sibling species of this genus are very difficult to distinguish because of their similar morphological features. To address this issue, we conducted phylogenetic and genetic analyses of cytochrome oxidase subunit I (COI) and 28S RNA gene sequences from eight species of Sclerodermus reared from different wood borer pests. The eight sibling species were as follows: S. guani Xiao et Wu, S. sichuanensis Xiao, S. pupariae Yang et Yao, and Sclerodermus spp. (Nos. 1–5). A 594-bp fragment of COI and 750-bp fragment of 28S were subsequently sequenced. For COI, the G-C content was found to be low in all the species, averaging to about 30.0%. Sequence divergences (Kimura-2-parameter distances) between congeneric species averaged to 4.5%, and intraspecific divergences averaged to about 0.09%. Further, the maximum sequence divergences between congeneric species and Sclerodermus sp. (No. 5) averaged to about 16.5%. All 136 samples analyzed were included in six reciprocally monophyletic clades in the COI neighbor-joining (NJ) tree. The NJ tree inferred from the 28S rRNA sequence yielded almost identical results, but the samples from S. guani, S. sichuanensis, S. pupariae, and Sclerodermus spp. (Nos. 1–4) clustered together and only Sclerodermus sp. (No. 5) clustered separately. Our findings indicate that the standard barcode region of COI can be efficiently used to distinguish morphologically similar Sclerodermus species. Further, we speculate that Sclerodermus sp. (No. 5) might be a new species of Sclerodermus. PMID:25782000

  14. Regulatory Genes Controlling Anthocyanin Pigmentation Are Functionally Conserved among Plant Species and Have Distinct Sets of Target Genes.

    PubMed Central

    Quattrocchio, F; Wing, JF; Leppen, H; Mol, J; Koes, RE

    1993-01-01

    In this study, we demonstrate that in petunia at least four regulatory genes (anthocyanin-1 [an1], an2, an4, and an11) control transcription of a subset of structural genes from the anthocyanin pathway by using a combination of RNA gel blot analysis, transcription run-on assays, and transient expression assays. an2- and an11- mutants could be transiently complemented by the maize regulatory genes Leaf color (Lc) or Colorless-1 (C1), respectively, whereas an1- mutants only by Lc and C1 together. In addition, the combination of Lc and C1 induces pigment accumulation in young leaves. This indicates that Lc and C1 are both necessary and sufficient to produce pigmentation in leaf cells. Regulatory pigmentation genes in maize and petunia control different sets of structural genes. The maize Lc and C1 genes expressed in petunia differentially activate the promoters of the chalcone synthase genes chsA and chsJ in the same way that the homologous petunia genes do. This suggests that the regulatory proteins in both species are functionally similar and that the choice of target genes is determined by their promoter sequences. We present an evolutionary model that explains the differences in regulation of pigmentation pathways of maize, petunia, and snapdragon. PMID:12271045

  15. Two distinct plant respiratory physiotypes might exist which correspond to fast-growing and slow-growing species.

    PubMed

    Nogués, Salvador; Aljazairi, Salvador; Arias, Claudia; Sánchez, Elena; Aranjuelo, Iker

    2014-08-15

    The origin of the carbon atoms in CO2 respired by leaves in the dark of several plant species has been studied using 13C/12C stable isotopes. This study was conducted using an open gas exchange system for isotope labeling that was coupled to an elemental analyzer and further linked to an isotope ratio mass spectrometer (EA-IRMS) or coupled to a gas chromatography-combustion-isotope ratio mass spectrometer (GC-C-IRMS). We demonstrate here that the carbon, which is recently assimilated during photosynthesis, accounts for nearly ca. 50% of the carbon in the CO2 lost through dark respiration (Rd) after illumination in fast-growing and cultivated plants and trees and, accounts for only ca. 10% in slow-growing plants. Moreover, our study shows that fast-growing plants, which had the largest percentages of newly fixed carbon of leaf-respired CO2, were also those with the largest shoot/root ratios, whereas slow-growing plants showed the lowest shoot/root values.

  16. Distinction of three wood species by Fourier transform infrared spectroscopy and two-dimensional correlation IR spectroscopy

    NASA Astrophysics Data System (ADS)

    Huang, Anmin; Zhou, Qun; Liu, Junliang; Fei, Benhua; Sun, Suqin

    2008-07-01

    Dalbergia odorifera T. Chen, Pterocarpus santalinus L.F. and Pterocarpus soyauxii are three kinds of the most valuable wood species, which are hard to distinguish. In this paper, differentiation of D. odorifera, P. santalinus and P. soyauxii was carried out by using Fourier transform infrared spectroscopy (FT-IR), second derivative IR spectra and two-dimensional correlation infrared (2D-IR) spectroscopy. The three woods have their characteristic peaks in conventional IR spectra. For example, D. odorifera has obvious absorption peaks at 1640 and 1612 cm -1; P. santalinus has only one peak at 1614 cm -1; and P. soyauxii has one peak at 1619 cm -1 and one shoulder peak at 1597 cm -1. To enhance spectrum resolution and amplify the differences between the IR spectra of different woods, the second derivative technology was adopted to examine the three wood samples. More differences could be observed in the region of 800-1700 cm -1. Then, the thermal perturbation is applied to distinguish different wood samples in an easier way, because of the spectral resolution being enhanced by the 2D correlation spectroscopy. In the region of 1300-1800 cm -1, D. odorifera has five auto-peaks at 1518, 1575, 1594, 1620 and 1667 cm -1; P. santalinus has four auto-peaks at 1469, 1518, 1627 and 1639 cm -1 and P. soyauxii has only two auto-peaks at 1627 and 1639 cm -1. It is proved that the 2D correlation IR spectroscopy can be a new method to distinguish D. odorifera, P. santalinus and P. soyauxii.

  17. Local vasotocin modulation of the pacemaker nucleus resembles distinct electric behaviors in two species of weakly electric fish.

    PubMed

    Perrone, Rossana; Migliaro, Adriana; Comas, Virginia; Quintana, Laura; Borde, Michel; Silva, Ana

    2014-01-01

    The neural bases of social behavior diversity in vertebrates have evolved in close association with hypothalamic neuropeptides. In particular, arginine-vasotocin (AVT) is a key integrator underlying differences in behavior across vertebrate taxa. Behavioral displays in weakly electric fish are channeled through specific patterns in their electric organ discharges (EODs), whose rate is ultimately controlled by a medullary pacemaker nucleus (PN). We first explored interspecific differences in the role of AVT as modulator of electric behavior in terms of EOD rate between the solitary Gymnotus omarorum and the gregarious Brachyhypopomus gauderio. In both species, AVT IP injection (10μg/gbw) caused a progressive increase of EOD rate of about 30%, which was persistent in B. gauderio, and attenuated after 30min in G. omarorum. Secondly, we demonstrated by in vitro electrophysiological experiments that these behavioral differences can be accounted by dissimilar effects of AVT upon the PN in itself. AVT administration (1μM) to the perfusion bath of brainstem slices containing the PN produced a small and transient increase of PN activity rate in G. omarorum vs the larger and persistent increase previously reported in B. gauderio. We also identified AVT neurons, for the first time in electric fish, using immunohistochemistry techniques and confirmed the presence of hindbrain AVT projections close to the PN that might constitute the anatomical substrate for AVT influences on PN activity. Taken together, our data reinforce the view of the PN as an extremely plastic medullary central pattern generator that not only responds to higher influences to adapt its function to diverse contexts, but also is able to intrinsically shape its response to neuropeptide actions, thus adding a hindbrain target level to the complexity of the global integration of central neuromodulation of electric behavior.

  18. Two distinct abnormalities in patients with C8 alpha-gamma deficiency. Low level of C8 beta chain and presence of dysfunctional C8 alpha-gamma subunit.

    PubMed Central

    Tedesco, F; Roncelli, L; Petersen, B H; Agnello, V; Sodetz, J M

    1990-01-01

    The sera from three C8 alpha-gamma deficient patients previously reported to have a selective C8 alpha-gamma defect were analyzed by SDS-PAGE and Western blot using two polyclonal antisera to C8 alpha-gamma and a monoclonal antibody to C8 alpha. All three sera exhibited C8 alpha-gamma bands that dissociated into alpha and gamma chains under reducing conditions. Quantitation of the alpha-gamma subunit in these sera by a sensitive ELISA revealed an amount approximately 1% of that found in normal human serum. A similar assay performed with a specific antiserum to C8 beta showed unexpectedly low levels of C8 beta in these sera, which were confirmed by hemolytic titration of C8 beta. The remarkable differences between C8 alpha-gamma and C8 beta in the C8 alpha-gamma deficient sera was that in spite of their comparable immunochemical levels, C8 beta still exhibited functional activity whereas C8 alpha-gamma was totally inactive. That the residual C8 alpha-gamma was inactive was also proved by its inability to show lytic bands in an overlay system after SDS-PAGE and subsequent removal of SDS. The implications of these findings for a novel concept of C8 deficiency are discussed. Images PMID:2394837

  19. Electrospray Ionization Tandem Mass Spectrometry (Esi-Ms/Ms) Analysis of the Lipid Molecular Species Composition of Yeast Subcellular Membranes Reveals Acyl Chain-Based Sorting/Remodeling of Distinct Molecular Species En Route to the Plasma Membrane

    PubMed Central

    Schneiter, Roger; Brügger, Britta; Sandhoff, Roger; Zellnig, Günther; Leber, Andrea; Lampl, Manfred; Athenstaedt, Karin; Hrastnik, Claudia; Eder, Sandra; Daum, Günther; Paltauf, Fritz; Wieland, Felix T.; Kohlwein, Sepp D.

    1999-01-01

    Nano-electrospray ionization tandem mass spectrometry (nano-ESI-MS/MS) was employed to determine qualitative differences in the lipid molecular species composition of a comprehensive set of organellar membranes, isolated from a single culture of Saccharomyces cerevisiae cells. Remarkable differences in the acyl chain composition of biosynthetically related phospholipid classes were observed. Acyl chain saturation was lowest in phosphatidylcholine (15.4%) and phosphatidylethanolamine (PE; 16.2%), followed by phosphatidylserine (PS; 29.4%), and highest in phosphatidylinositol (53.1%). The lipid molecular species profiles of the various membranes were generally similar, with a deviation from a calculated average profile of ∼± 20%. Nevertheless, clear distinctions between the molecular species profiles of different membranes were observed, suggesting that lipid sorting mechanisms are operating at the level of individual molecular species to maintain the specific lipid composition of a given membrane. Most notably, the plasma membrane is enriched in saturated species of PS and PE. The nature of the sorting mechanism that determines the lipid composition of the plasma membrane was investigated further. The accumulation of monounsaturated species of PS at the expense of diunsaturated species in the plasma membrane of wild-type cells was reversed in elo3Δ mutant cells, which synthesize C24 fatty acid-substituted sphingolipids instead of the normal C26 fatty acid-substituted species. This observation suggests that acyl chain-based sorting and/or remodeling mechanisms are operating to maintain the specific lipid molecular species composition of the yeast plasma membrane. PMID:10459010

  20. The functional analysis of distinct tospovirus movement proteins (NSM) reveals different capabilities in tubule formation, cell-to-cell and systemic virus movement among the tospovirus species.

    PubMed

    Leastro, Mikhail O; Pallás, Vicente; Resende, Renato O; Sánchez-Navarro, Jesús A

    2017-01-02

    The lack of infectious tospovirus clones to address reverse genetic experiments has compromised the functional analysis of viral proteins. In the present study we have performed a functional analysis of the movement proteins (NSM) of four tospovirus species Bean necrotic mosaic virus (BeNMV), Chrysanthemum stem necrosis virus (CSNV), Tomato chlorotic spot virus (TCSV) and Tomato spotted wilt virus (TSWV), which differ biologically and molecularly, by using the Alfalfa mosaic virus (AMV) model system. All NSM proteins were competent to: i) support the cell-to-cell and systemic transport of AMV, ii) generate tubular structures on infected protoplast and iii) transport only virus particles. However, the NSM of BeNMV (one of the most phylogenetically distant species) was very inefficient to support the systemic transport. Deletion assays revealed that the C-terminal region of the BeNMV NSM, but not that of the CSNV, TCSV and TSWV NSM proteins, was dispensable for cell-to-cell transport, and that all the non-functional C-terminal NSM mutants were unable to generate tubular structures. Bimolecular fluorescence complementation analysis revealed that the C-terminus of the BeNMV NSM was not required for the interaction with the cognate nucleocapsid protein, showing a different protein organization when compared with other movement proteins of the '30K family'. Overall, our results revealed clearly differences in functional aspects among movement proteins from divergent tospovirus species that have a distinct biological behavior.

  1. Post-Colonization Interval Estimates Using Multi-Species Calliphoridae Larval Masses and Spatially Distinct Temperature Data Sets: A Case Study.

    PubMed

    Weatherbee, Courtney R; Pechal, Jennifer L; Stamper, Trevor; Benbow, M Eric

    2017-04-04

    Common forensic entomology practice has been to collect the largest Diptera larvae from a scene and use published developmental data, with temperature data from the nearest weather station, to estimate larval development time and post-colonization intervals (PCIs). To evaluate the accuracy of PCI estimates among Calliphoridae species and spatially distinct temperature sources, larval communities and ambient air temperature were collected at replicate swine carcasses (N = 6) throughout decomposition. Expected accumulated degree hours (ADH) associated with Cochliomyia macellaria and Phormia regina third instars (presence and length) were calculated using published developmental data sets. Actual ADH ranges were calculated using temperatures recorded from multiple sources at varying distances (0.90 m-7.61 km) from the study carcasses: individual temperature loggers at each carcass, a local weather station, and a regional weather station. Third instars greatly varied in length and abundance. The expected ADH range for each species successfully encompassed the average actual ADH for each temperature source, but overall under-represented the range. For both calliphorid species, weather station data were associated with more accurate PCI estimates than temperature loggers associated with each carcass. These results provide an important step towards improving entomological evidence collection and analysis techniques, and developing forensic error rates.

  2. Sodium channel auxiliary subunits.

    PubMed

    Tseng, Tsai-Tien; McMahon, Allison M; Johnson, Victoria T; Mangubat, Erwin Z; Zahm, Robert J; Pacold, Mary E; Jakobsson, Eric

    2007-01-01

    Voltage-gated ion channels are well known for their functional roles in excitable tissues. Excitable tissues rely on voltage-gated ion channels and their auxiliary subunits to achieve concerted electrical activity in living cells. Auxiliary subunits are also known to provide functional diversity towards the transport and biogenesis properties of the principal subunits. Recent interests in pharmacological properties of these auxiliary subunits have prompted significant amounts of efforts in understanding their physiological roles. Some auxiliary subunits can potentially serve as drug targets for novel analgesics. Three families of sodium channel auxiliary subunits are described here: beta1 and beta3, beta2 and beta4, and temperature-induced paralytic E (TipE). While sodium channel beta-subunits are encoded in many animal genomes, TipE has only been found exclusively in insects. In this review, we present phylogenetic analyses, discuss potential evolutionary origins and functional data available for each of these subunits. For each family, we also correlate the functional specificity with the history of evolution for the individual auxiliary subunits.

  3. IDENTIFICATION OF SPECIES AND SOURCES OF CRYPTOSPORIDIUM OOCYSTS IN STORM WATERS BY A SMALL SUBUNIT RRNA-BASED DIAGNOSTIC AND GENOTYPING TOOL

    EPA Science Inventory

    The identification of Cryptosporidium oocysts in environmental samples is largely made by the use of immunofluorescent assay (IFA). because IFA detects oocysts from all Cryptosporidium parasites, the species distribution and source of Cryptosporidium parasites in environmental sa...

  4. IDENTIFICATION OF CRYPTOSPORIDIUM SPECIES AND THE SOURCES IN RAW WASTEWATER USING A SMALL SUBUNIT RRNA-BASED PCR-RFLP TOOL

    EPA Science Inventory

    The species composition and source of Cryptosporidium oocysts in wastewater have never been determined, even though it is widely assumed that these oocysts are from human sewage. Recent molecular characterizations of Cryptosporidium parasites make it possible to differentiate hum...

  5. Steady state or non-steady state? Identifying driving mechanisms of oxygen isotope signatures of leaf transpiration in functionally distinct plant species

    NASA Astrophysics Data System (ADS)

    Dubbert, Maren; Kübert, Angelika; Cuntz, Matthias; Werner, Christiane

    2015-04-01

    Isotope techniques are widely applied in ecosystem studies. For example, isoflux models are used to separate soil evaporation from transpiration in ecosystems. These models often assume that plant transpiration occurs at isotopic steady state, i.e. that the transpired water shows the same isotopic signature as the source water. Yet, several studies found that transpiration did not occur at isotopic steady state, under both controlled and field conditions. Here we focused on identifying the internal and external factors which drive the isotopic signature of leaf transpiration. Using cavity ring-down spectroscopy (CRDS), the effect of both environmental variables and leaf physiological traits on δ18OT was investigated under controlled conditions. Six plant species with distinct leaf physiological traits were exposed to step changes in relative air humidity (RH), their response in δ18OT and gas exchange parameters and their leaf physiological traits were assessed. Moreover, two functionally distinct plant types (tree, i.e. Quercus suber, and grassland) of a semi-arid Mediterranean oak-woodland where observed under natural conditions throughout an entire growth period in the field. The species differed substantially in their leaf physiological traits and their turn-over times of leaf water. They could be grouped in species with fast (<60 min.), intermediate (ca. 120 min.) and slow (>240 min.) turn-over times, mostly due to differences in stomatal conductance, leaf water content or a combination of both. Changes in RH caused an immediate response in δ18OT, which were similarly strong in all species, while leaf physiological traits affected the subsequent response in δ18OT. The turn-over time of leaf water determined the speed of return to the isotopic steady or a stable δ18OT value (Dubbert & Kübert et al., in prep.). Under natural conditions, changes in environmental conditions over the diurnal cycle had a huge impact on the diurnal development of δ18OT in both

  6. Extremely low microsatellite diversity but distinct population structure in a long-lived threatened species, the Australian lungfish Neoceratodus forsteri (Dipnoi).

    PubMed

    Hughes, Jane M; Schmidt, Daniel J; Huey, Joel A; Real, Kathryn M; Espinoza, Thomas; McDougall, Andrew; Kind, Peter K; Brooks, Steven; Roberts, David T

    2015-01-01

    The Australian lungfish is a unique living representative of an ancient dipnoan lineage, listed as 'vulnerable' to extinction under Australia's Environment Protection and Biodiversity Conservation Act 1999. Historical accounts indicate this species occurred naturally in two adjacent river systems in Australia, the Burnett and Mary. Current day populations in other rivers are thought to have arisen by translocation from these source populations. Early genetic work detected very little variation and so had limited power to answer questions relevant for management including how genetic variation is partitioned within and among sub-populations. In this study, we use newly developed microsatellite markers to examine samples from the Burnett and Mary Rivers, as well as from two populations thought to be of translocated origin, Brisbane and North Pine. We test whether there is significant genetic structure among and within river drainages; assign putatively translocated populations to potential source populations; and estimate effective population sizes. Eleven polymorphic microsatellite loci genotyped in 218 individuals gave an average within-population heterozygosity of 0.39 which is low relative to other threatened taxa and for freshwater fishes in general. Based on FST values (average over loci = 0.11) and STRUCTURE analyses, we identify three distinct populations in the natural range, one in the Burnett and two distinct populations in the Mary. These analyses also support the hypothesis that the Mary River is the likely source of translocated populations in the Brisbane and North Pine rivers, which agrees with historical published records of a translocation event giving rise to these populations. We were unable to obtain bounded estimates of effective population size, as we have too few genotype combinations, although point estimates were low, ranging from 29 - 129. We recommend that, in order to preserve any local adaptation in the three distinct populations that

  7. Extremely Low Microsatellite Diversity but Distinct Population Structure in a Long-Lived Threatened Species, the Australian Lungfish Neoceratodus forsteri (Dipnoi)

    PubMed Central

    Hughes, Jane M.; Schmidt, Daniel J.; Huey, Joel A.; Real, Kathryn M.; Espinoza, Thomas; McDougall, Andrew; Kind, Peter K.; Brooks, Steven; Roberts, David T.

    2015-01-01

    The Australian lungfish is a unique living representative of an ancient dipnoan lineage, listed as ‘vulnerable’ to extinction under Australia’s Environment Protection and Biodiversity Conservation Act 1999. Historical accounts indicate this species occurred naturally in two adjacent river systems in Australia, the Burnett and Mary. Current day populations in other rivers are thought to have arisen by translocation from these source populations. Early genetic work detected very little variation and so had limited power to answer questions relevant for management including how genetic variation is partitioned within and among sub-populations. In this study, we use newly developed microsatellite markers to examine samples from the Burnett and Mary Rivers, as well as from two populations thought to be of translocated origin, Brisbane and North Pine. We test whether there is significant genetic structure among and within river drainages; assign putatively translocated populations to potential source populations; and estimate effective population sizes. Eleven polymorphic microsatellite loci genotyped in 218 individuals gave an average within-population heterozygosity of 0.39 which is low relative to other threatened taxa and for freshwater fishes in general. Based on FST values (average over loci = 0.11) and STRUCTURE analyses, we identify three distinct populations in the natural range, one in the Burnett and two distinct populations in the Mary. These analyses also support the hypothesis that the Mary River is the likely source of translocated populations in the Brisbane and North Pine rivers, which agrees with historical published records of a translocation event giving rise to these populations. We were unable to obtain bounded estimates of effective population size, as we have too few genotype combinations, although point estimates were low, ranging from 29 - 129. We recommend that, in order to preserve any local adaptation in the three distinct populations

  8. RNA sequencing supports distinct reactive oxygen species-mediated pathways of apoptosis by high and low size mass fractions of Bay leaf (Lauris nobilis) in HT-29 cells.

    PubMed

    Rodd, Annabelle L; Ververis, Katherine; Sayakkarage, Dheeshana; Khan, Abdul W; Rafehi, Haloom; Ziemann, Mark; Loveridge, Shanon J; Lazarus, Ross; Kerr, Caroline; Lockett, Trevor; El-Osta, Assam; Karagiannis, Tom C; Bennett, Louise E

    2015-08-01

    Anti-proliferative and pro-apoptotic effects of Bay leaf (Laurus nobilis) in mammalian cancer and HT-29 adenocarcinoma cells have been previously attributed to effects of polyphenolic and essential oil chemical species. Recently, we demonstrated differentiated growth-regulating effects of high (HFBL) versus low molecular mass (LFBL) aqueous fractions of bay leaf and now confirm by comparative effects on gene expression, that HFBL and LFBL suppress HT-29 growth by distinct mechanisms. Induction of intra-cellular lesions including DNA strand breakage by extra-cellular HFBL, invoked the hypothesis that iron-mediated reactive oxygen species with capacity to penetrate cell membrane, were responsible for HFBL-mediated effects, supported by equivalent effects of HFBL in combination with γ radiation. Activities of HFBL and LFBL were interpreted to reflect differentiated responses to iron-mediated reactive oxygen species (ROS), occurring either outside or inside cells. In the presence of LFBL, apoptotic death was relatively delayed compared with HFBL. ROS production by LFBL mediated p53-dependent apoptosis and recovery was suppressed by promoting G1/S phase arrest and failure of cellular tight junctions. In comparison, intra-cellular anti-oxidant protection exerted by LFBL was absent for extra-cellular HFBL (likely polysaccharide-rich), which potentiated more rapid apoptosis by producing DNA double strand breaks. Differentiated effects on expression of genes regulating ROS defense and chromatic condensation by LFBL versus HFBL, were observed. The results support ferrous iron in cell culture systems and potentially in vivo, can invoke different extra-cellular versus intra-cellular ROS-mediated chemistries, that may be regulated by exogenous, including dietary species.

  9. Sensitivity to low-dose/low-LET ionizing radiation in mammalian cells harboring mutations in succinate dehydrogenase subunit C is governed by mitochondria-derived reactive oxygen species.

    PubMed

    Aykin-Burns, Nukhet; Slane, Benjamin G; Liu, Annie T Y; Owens, Kjerstin M; O'Malley, Malinda S; Smith, Brian J; Domann, Frederick E; Spitz, Douglas R

    2011-02-01

    It has been hypothesized that ionizing radiation-induced disruptions in mitochondrial O₂ metabolism lead to persistent heritable increases in steady-state levels of intracellular superoxide (O₂(•U+2212)) and hydrogen peroxide (H₂O₂) that contribute to the biological effects of radiation. Hamster fibroblasts (B9 cells) expressing a mutation in the gene coding for the mitochondrial electron transport chain protein succinate dehydrogenase subunit C (SDHC) demonstrate increases in steady-state levels of O₂•- and H₂O₂. When B9 cells were exposed to low-dose/low-LET radiation (5-50 cGy), they displayed significantly increased clonogenic cell killing compared with parental cells. Clones derived from B9 cells overexpressing a wild-type human SDHC (T4, T8) demonstrated significantly increased surviving fractions after exposure to 5-50 cGy relative to B9 vector controls. In addition, pretreatment with polyethylene glycol-conjugated CuZn superoxide dismutase and catalase as well as adenoviral-mediated overexpression of MnSOD and/or mitochondria-targeted catalase resulted in significantly increased survival of B9 cells exposed to 10 cGy ionizing radiation relative to vector controls. Adenoviral-mediated overexpression of either MnSOD or mitochondria-targeted catalase alone was equally as effective as when both were combined. These results show that mammalian cells over expressing mutations in SDHC demonstrate low-dose/low-LET radiation sensitization that is mediated by increased levels of O₂•- and H₂O₂. These results also support the hypothesis that mitochondrial O₂•- and H₂O₂ originating from SDH are capable of playing a role in low-dose ionizing radiation-induced biological responses.

  10. A comparative study on Ca content and distribution in two Gesneriaceae species reveals distinctive mechanisms to cope with high rhizospheric soluble calcium

    PubMed Central

    Li, Wenlong; Xu, Falun; Chen, Shixuan; Zhang, Zhennan; Zhao, Yan; Jin, Yukuan; Li, Meijing; Zhu, Yan; Liu, Yongxiu; Yang, Yi; Deng, Xin

    2014-01-01

    Excessive Ca is toxic to plants thus significantly affects plant growth and species distribution in Ca-rich karst areas. To understand how plants survive high Ca soil, laboratory experiments were established to compare the physiological responses and internal Ca distribution in organ, tissue, cell, and intracellular levels under different Ca levels for Lysionotus pauciflorus and Boea hygrometrica, two karst habitant Gesneriaceae species in Southwest China. In the controlled condition, L. pauciflorus could survive as high as 200 mM rhizospheric soluble Ca, attributed to a series of physiological responses and preferential storage that limited Ca accumulation in chloroplasts of palisade cells. In contrast, B. hygrometrica could survive only 20 mM rhizospheric soluble Ca, but accumulated a high level of internal Ca in both palisade and spongy cells without disturbance on photosynthetic activity. By phenotype screening of transgenic plants expressing high Ca-inducible genes from B. hygrometrica, the expression of BhDNAJC2 in A. thaliana was found to enhance plant growth and photosynthesis under high soluble Ca stress. BhDNAJC2 encodes a recently reported heat shock protein (HSP) 40 family DnaJ-domain protein. The Ca-resistant phenotype of BhDNAJC2 highlights the important role of chaperone-mediated protein quality control in Ca tolerance in B. hygrometrica. Taken together, our results revealed that distinctive mechanisms were employed in the two Gesneriaceae karst habitants to cope with a high Ca environment. PMID:25477893

  11. Comprehensive genetic analyses reveal evolutionary distinction of a mouse (Zapus hudsonius preblei) proposed for delisting from the US Endangered Species Act

    USGS Publications Warehouse

    King, Timothy L.; Switzer, John F.; Morrison, Cheryl L.; Eackles, Michael S.; Young, Colleen C.; Lubinski, Barbara A.; Cryan, Paul M.

    2006-01-01

    Zapus hudsonius preblei, listed as threatened under the US Endangered Species Act (ESA), is one of 12 recognized subspecies of meadow jumping mice found in North America. Recent morphometric and phylogenetic comparisons among Z. h. preblei and neighbouring conspecifics questioned the taxonomic status of selected subspecies, resulting in a proposal to delist the Z. h. preblei from the ESA. We present additional analyses of the phylogeographic structure within Z. hudsonius that calls into question previously published data (and conclusions) and confirms the original taxonomic designations. A survey of 21 microsatellite DNA loci and 1380 base pairs from two mitochondrial DNA (mtDNA) regions (control region and cytochrome b) revealed that each Z. hudsonius subspecies is genetically distinct. These data do not support the null hypothesis of a homogeneous gene pool among the five subspecies found within the southwestern portion of the species' range. The magnitude of the observed differentiation was considerable and supported by significant findings for nearly every statistical comparison made, regardless of the genome or the taxa under consideration. Structuring of nuclear multilocus genotypes and subspecies-specific mtDNA haplotypes corresponded directly with the disjunct distributions of the subspecies investigated. Given the level of correspondence between the observed genetic population structure and previously proposed taxonomic classification of subspecies (based on the geographic separation and surveys of morphological variation), we conclude that the nominal subspecies surveyed in this study do not warrant synonymy, as has been proposed for Z. h. preblei, Z. h. campestris, and Z. h. intermedius. ?? 2006 The Authors.

  12. Alpine bistort (Bistorta vivipara) in edge habitat associates with fewer but distinct ectomycorrhizal fungal species: a comparative study of three contrasting soil environments in Svalbard.

    PubMed

    Mundra, Sunil; Bahram, Mohammad; Eidesen, Pernille Bronken

    2016-11-01

    Bistorta vivipara is a widespread arctic-alpine ectomycorrhizal (ECM) plant species. Recent findings suggest that fungal communities associated with B. vivipara roots appear random over short distances, but at larger scales, environmental filtering structure fungal communities. Habitats in highly stressful environments where specialist species with narrower niches may have an advantage represent unique opportunity to test the effect of environmental filtering. We utilised high-throughput amplicon sequencing to identify ECM communities associated with B. vivipara in Svalbard. We compared ECM communities in a core habitat where B. vivipara is frequent (Dryas-heath) with edge habitats representing extremes in terms of nutrient availability where B. vivipara is less frequent (bird-manured meadow and a nutrient-depleted mine tilling). Our analysis revealed that soil conditions in edge habitats favour less diverse but more distinct ECM fungal communities with functional traits adapted to local conditions. ECM richness was overall lower in both edge habitats, and the taxonomic compositions of ECM fungi were in line with our functional expectations. Stress-tolerant genera such as Laccaria and Hebeloma were abundant in nutrient-poor mine site whereas functional competitors genera such as Lactarius and Russula were dominant in the nutrient-rich bird-cliff site. Our results suggest that ECM communities in rare edge habitats are most likely not subsets of the larger pool of ECM fungi found in natural tundra, and they may represent a significant contribution to the overall diversity of ECM fungi in the Arctic.

  13. Genetic differentiation among distinct karyomorphs of the wolf fish Hoplias malabaricus species complex (Characiformes, Erythrinidae) and report of unusual hybridization with natural triploidy.

    PubMed

    Utsunomia, R; Alves, J C Pansonato; Paiva, L R S; Silva, G J Costa; Oliveira, C; Bertollo, L A C; Foresti, F

    2014-11-01

    In this study, genetic differentiation between karyomorphs A (2n = 42) and D (2n = 39/40) of the wolf fish Hoplias malabaricus, which is comprised of several cryptic species that present a wide variety of diploid chromosome numbers and sex chromosome systems, resulting in the identification of seven distinct karyomorphs (A-G), was investigated using a combination of molecular and cytogenetic tools. Deep sequence divergences for both karyomorphs were observed and indicate a long period of reproductive isolation between karyomorphs A and D. Additionally, one individual with 61 chromosomes was identified, which, as far as is known, is the first case of natural triploidy resulting from the hybridization between these highly differentiated karyomorphs of H. malabaricus. Molecular and cytogenetic analyses revealed that this allotriploid specimen carries two sets of maternal chromosomes from karyomorph D (2n = 40) and one set of chromosomes from karyomorph A (n = 21). Moreover, ribosomal sites and active nucleolus organizer regions from both parental contributors were found in the triploid hybrid. Considering the significant genetic distances between karyomorphs A and D, one of the primary reasons for the lack of recurrent reports of hybridization in the H. malabaricus species complex may be due to post-zygotic barriers, such as hybrid sterility or unviability.

  14. Comprehensive genetic analyses reveal evolutionary distinction of a mouse (Zapus hudsonius preblei) proposed for delisting from the US Endangered Species Act.

    PubMed

    King, Tim L; Switzer, John F; Morrison, Cheryl L; Eackles, Michael S; Young, Colleen C; Lubinski, Barbara A; Cryan, Paul

    2006-12-01

    Zapus hudsonius preblei, listed as threatened under the US Endangered Species Act (ESA), is one of 12 recognized subspecies of meadow jumping mice found in North America. Recent morphometric and phylogenetic comparisons among Z. h. preblei and neighbouring conspecifics questioned the taxonomic status of selected subspecies, resulting in a proposal to delist the Z. h. preblei from the ESA. We present additional analyses of the phylogeographic structure within Z. hudsonius that calls into question previously published data (and conclusions) and confirms the original taxonomic designations. A survey of 21 microsatellite DNA loci and 1380 base pairs from two mitochondrial DNA (mtDNA) regions (control region and cytochrome b) revealed that each Z. hudsonius subspecies is genetically distinct. These data do not support the null hypothesis of a homogeneous gene pool among the five subspecies found within the southwestern portion of the species' range. The magnitude of the observed differentiation was considerable and supported by significant findings for nearly every statistical comparison made, regardless of the genome or the taxa under consideration. Structuring of nuclear multilocus genotypes and subspecies-specific mtDNA haplotypes corresponded directly with the disjunct distributions of the subspecies investigated. Given the level of correspondence between the observed genetic population structure and previously proposed taxonomic classification of subspecies (based on the geographic separation and surveys of morphological variation), we conclude that the nominal subspecies surveyed in this study do not warrant synonymy, as has been proposed for Z. h. preblei, Z. h. campestris, and Z. h. intermedius.

  15. Phenolic antioxidants in some Vigna species of legumes and their distinct inhibitory effects on α-glucosidase and pancreatic lipase activities.

    PubMed

    Sreerama, Yadahally N; Takahashi, Yoko; Yamaki, Kohji

    2012-09-01

    Phenolic extracts of 4 Vigna species of legumes (mung bean, moth bean, and black and red varieties of adzuki beans) were evaluated for phenolic contents, antioxidant activities, and inhibitory properties against α-glucosidase and pancreatic lipase. Results showed that adzuki bean varieties contain higher phenolic indexes than mung bean and moth beans. Adzuki bean (black) variety was found to be the most active 2,2'-diphenyl-1-picrylhydrazyl and superoxide anion scavenger. However, the hydrogen peroxide scavenging and metal chelating abilities were significantly higher in adzuki bean (red) variety. Mung bean exhibited least antioxidant activities in all the methods tested. Phenolic extracts from these legumes also showed distinct variations in the inhibition of enzymes associated with hyperglycemia and hyperlipidemia. Inhibitory activities of all the extracts against lipase were found to be more potent than α-glucosidase. Although, α-glucosidase inhibitory activity was superior in the black variety of adzuki bean (IC(50,) 26.28 mg/mL), both adzuki bean varieties (black and red) along with moth bean showed strong inhibitory activities on lipase with no significant difference in their IC(50) values (7.32 to 9.85 mg/mL). These results suggest that Vigna species of legumes are potential source of antioxidant phenolics and also great sources of strong natural inhibitors for α-glucosidase and lipase activities. This information may help for effective utilization of these legumes as functional food ingredients for promoting health. Practical Application:  Vigna species of legumes are good sources of phenolic antioxidants and strong natural inhibitors of enzymes associated with diabetes and obesity. Therefore, utilization of these legumes in the development of functional foods with increased therapeutic value would be a significant step toward health promotion and wellness.

  16. Distinct Macrophage Fates after in vitro Infection with Different Species of Leishmania: Induction of Apoptosis by Leishmania (Leishmania) amazonensis, but Not by Leishmania (Viannia) guyanensis

    PubMed Central

    DaMata, Jarina Pena; Mendes, Bárbara Pinheiro; Maciel-Lima, Kátia; Menezes, Cristiane Alves Silva; Dutra, Walderez Ornelas; Sousa, Lirlândia Pires; Horta, Maria Fátima

    2015-01-01

    Leishmania is an intracellular parasite in vertebrate hosts, including man. During infection, amastigotes replicate inside macrophages and are transmitted to healthy cells, leading to amplification of the infection. Although transfer of amastigotes from infected to healthy cells is a crucial step that may shape the outcome of the infection, it is not fully understood. Here we compare L. amazonensis and L. guyanensis infection in C57BL/6 and BALB/c mice and investigate the fate of macrophages when infected with these species of Leishmania in vitro. As previously shown, infection of mice results in distinct outcomes: L. amazonensis causes a chronic infection in both strains of mice (although milder in C57BL/6), whereas L. guyanensis does not cause them disease. In vitro, infection is persistent in L. amazonensis-infected macrophages whereas L. guyanensis growth is controlled by host cells from both strains of mice. We demonstrate that, in vitro, L. amazonensis induces apoptosis of both C57BL/6 and BALB/c macrophages, characterized by PS exposure, DNA cleavage into nucleosomal size fragments, and consequent hypodiploidy. None of these signs were seen in macrophages infected with L. guyanensis, which seem to die through necrosis, as indicated by increased PI-, but not Annexin V-, positive cells. L. amazonensis-induced macrophage apoptosis was associated to activation of caspases-3, -8 and -9 in both strains of mice. Considering these two species of Leishmania and strains of mice, macrophage apoptosis, induced at the initial moments of infection, correlates with chronic infection, regardless of its severity. We present evidence suggestive that macrophages phagocytize L. amazonensis-infected cells, which has not been verified so far. The ingestion of apoptotic infected macrophages by healthy macrophages could be a way of amastigote spreading, leading to the establishment of infection. PMID:26513474

  17. Distinct Macrophage Fates after in vitro Infection with Different Species of Leishmania: Induction of Apoptosis by Leishmania (Leishmania) amazonensis, but Not by Leishmania (Viannia) guyanensis.

    PubMed

    DaMata, Jarina Pena; Mendes, Bárbara Pinheiro; Maciel-Lima, Kátia; Menezes, Cristiane Alves Silva; Dutra, Walderez Ornelas; Sousa, Lirlândia Pires; Horta, Maria Fátima

    2015-01-01

    Leishmania is an intracellular parasite in vertebrate hosts, including man. During infection, amastigotes replicate inside macrophages and are transmitted to healthy cells, leading to amplification of the infection. Although transfer of amastigotes from infected to healthy cells is a crucial step that may shape the outcome of the infection, it is not fully understood. Here we compare L. amazonensis and L. guyanensis infection in C57BL/6 and BALB/c mice and investigate the fate of macrophages when infected with these species of Leishmania in vitro. As previously shown, infection of mice results in distinct outcomes: L. amazonensis causes a chronic infection in both strains of mice (although milder in C57BL/6), whereas L. guyanensis does not cause them disease. In vitro, infection is persistent in L. amazonensis-infected macrophages whereas L. guyanensis growth is controlled by host cells from both strains of mice. We demonstrate that, in vitro, L. amazonensis induces apoptosis of both C57BL/6 and BALB/c macrophages, characterized by PS exposure, DNA cleavage into nucleosomal size fragments, and consequent hypodiploidy. None of these signs were seen in macrophages infected with L. guyanensis, which seem to die through necrosis, as indicated by increased PI-, but not Annexin V-, positive cells. L. amazonensis-induced macrophage apoptosis was associated to activation of caspases-3, -8 and -9 in both strains of mice. Considering these two species of Leishmania and strains of mice, macrophage apoptosis, induced at the initial moments of infection, correlates with chronic infection, regardless of its severity. We present evidence suggestive that macrophages phagocytize L. amazonensis-infected cells, which has not been verified so far. The ingestion of apoptotic infected macrophages by healthy macrophages could be a way of amastigote spreading, leading to the establishment of infection.

  18. Characterization and charge distribution of the asparagine-linked oligosaccharides on secreted mouse thyrotropin and free alpha-subunits

    SciTech Connect

    Gesundheit, N.; Gyves, P.W.; DeCherney, G.S.; Stannard, B.S.; Winston, R.L.; Weintraub, B.D.

    1989-06-01

    Mouse hemipituitaries in vitro secrete TSH, composed of an alpha-beta heterodimer, as well as excess (free) alpha-subunits. By dual metabolic labeling with (35S)sulfate and (3H)mannose, we have characterized oligosaccharides from secreted TSH alpha, TSH beta, and free alpha-subunits released from the apoprotein by enzymatic deglycosylation. Oligosaccharides from each subunit displayed a distinct anion exchange HPLC profile due to a specific pattern of sialylation and sulfation. Six species were obtained from TSH alpha (with two glycosylation sites), including neutral oligosaccharides as well as those with one or two negative charges. For TSH beta (with one glycosylation site) at least eight oligosaccharide species were noted, representing nearly every permutation of sialylation and sulfation; approximately 30% contained three or more negative charges. Analysis of (3H)mannose-labeled oligosaccharides on Concanavalin-A-agarose showed 85% binding for those from TSH alpha, 70% for free alpha, and 50% for those from TSH beta. These data demonstrate that oligosaccharides from secreted TSH beta were more sialylated and sulfated, consistent with a more complex branching pattern, than those from TSH alpha. Oligosaccharides from free alpha-subunit were more sialylated than those from TSH alpha, and the net negative charge was intermediate between those of TSH alpha and TSH beta. Although great microheterogeneity is present even at the single glycosylation site on the beta-subunit of secreted TSH, a pattern of sialylation and sulfation could be discerned.

  19. Genes encoding the alpha-carboxyltransferase subunit of acetyl-CoA carboxylase from Brassica napus and parental species: cloning, expression patterns, and evolution.

    PubMed

    Li, Zhi-Guo; Yin, Wei-Bo; Guo, Huan; Song, Li-Ying; Chen, Yu-Hong; Guan, Rong-Zhan; Wang, Jing-Qiao; Wang, Richard R-C; Hu, Zan-Min

    2010-05-01

    Heteromeric acetyl coenzyme A carboxylase (ACCase), a rate-limiting enzyme in fatty acid biosynthesis in dicots, is a multi-enzyme complex consisting of biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase (alpha-CT and beta-CT). In the present study, four genes encoding alpha-CT were cloned from Brassica napus, and two were cloned from each of the two parental species, B. rapa and B. oleracea. Comparative and cluster analyses indicated that these genes were divided into two major groups. The major divergence between group-1 and group-2 occurred in the second intron. Group-2 alpha-CT genes represented the ancestral form in the genus Brassica. The divergence of group-1 and group-2 genes occurred in their common ancestor 12.96-17.78 million years ago (MYA), soon after the divergence of Arabidopsis thaliana and Brassica (15-20 MYA). This time of divergence is identical to that reported for the paralogous subgenomes of diploid Brassica species (13-17 MYA). Real-time reverse transcription PCR revealed that the expression patterns of the two groups of genes were similar in different organs, except in leaves. To better understand the regulation and evolution of alpha-CT genes, promoter regions from two sets of orthologous gene copies from B. napus, B. rapa, and B. oleracea were cloned and compared. The function of the promoter of gene Bnalpha-CT-1-1 in group-1 and gene Bnalpha-CT-2-1 in group-2 was examined by assaying beta-glucuronidase activity in transgenic A. thaliana. Our results will be helpful in elucidating the evolution and regulation of ACCase in oilseed rape.

  20. Diversity of insect nicotinic acetylcholine receptor subunits.

    PubMed

    Jones, Andrew K; Sattelle, David B

    2010-01-01

    Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that mediate fast synaptic transmission in the insect nervous system and are targets of a major group of insecticides, the neonicotinoids. They consist of five subunits arranged around a central ion channeL Since the subunit composition determines the functional and pharmacological properties of the receptor the presence of nAChR families comprising several subunit-encodinggenes provides a molecular basis for broad functional diversity. Analyses of genome sequences have shown that nAChR gene families remain compact in diverse insect species, when compared to their nematode andvertebrate counterparts. Thus, the fruit fly (Drosophila melanogaster), malaria mosquito (Anopheles gambiae), honey bee (Apis mellifera), silk worm (Bombyx mon) and the red flour beetle (Tribolium castaneum) possess 10-12 nAChR genes while human and the nematode Caenorhabditis elegans have 16 and 29 respectively. Although insect nAChRgene families are amongst the smallest known, receptor diversity can be considerably increased by the posttranscriptional processes alternative splicing and mRNA A-to-I editingwhich can potentially generate protein products which far outnumber the nAChR genes. These two processes can also generate species-specific subunit isoforms. In addition, each insect possesses at least one highly divergent nAChR subunit which may perform species-specific functions. Species-specific subunit diversification may offer promising targets for future rational design of insecticides that target specific pest insects while sparing beneficial species.

  1. The pathogen-inducible promoter of defense-related LsGRP1 gene from Lilium functioning in phylogenetically distinct species of plants.

    PubMed

    Lin, Chia-Hua; Chen, Chao-Ying

    2017-01-01

    A suitable promoter greatly enhances the efficiency of target gene expression of plant molecular breeding and farming; however, only very few promoters are available for economically important non-graminaceous ornamental monocots. In this study, an 868-bp upstream region of defense-related LsGRP1 of Lilium, named PLsGRP1, was cloned by genome walking and proven to exhibit promoter activity in Nicotiana benthamiana and Lilium 'Stargazer' as assayed by agroinfiltration-based β-glucuronidase (GUS) expression system. Many putative biotic stress-, abiotic stress- and physiological regulation-related cis-acting elements were found in PLsGRP1. Serial deletion analysis of PLsGRP1 performed in Nicotiana tabacum var. Wisconsin 38 accompanied with types of treatments indicated that 868-bp PLsGRP1 was highly induced upon pathogen challenges and cold stress while the 131-bp 3'-end region of PLsGRP1 could be dramatically induced by many kinds of abiotic stresses, biotic stresses and phytohormone treatments. Besides, transient GUS expression in a fern, gymnosperms, monocots and dicots revealed good promotor activity of PLsGRP1 in many phylogenetically distinct plant species. Thus, pathogen-inducible PLsGRP1 and its 131-bp 3'-end region are presumed potential as tools for plant molecular breeding and farming.

  2. A class I KNOX gene from the palm species Elaeis guineensis (Arecaceae) is associated with meristem function and a distinct mode of leaf dissection.

    PubMed

    Jouannic, Stefan; Collin, Myriam; Vidal, Benjamin; Verdeil, Jean-Luc; Tregear, James W

    2007-01-01

    Class I Knotted-like homeobox (KNOX) transcription factors are important regulators of shoot apical meristem function and leaf morphology by their contribution to dissected leaf development. Palms are of particular interest as they produce dissected leaves generated by a distinct mechanism compared with eudicots. The question addressed here was whether class I KNOX genes might be involved in meristem function and leaf dissection in palms. Here, we characterized the EgKNOX1 gene from oil palm (Elaeis guineensis, Arecaceae) and compared it with available sequences from other plant species using phylogenetic analysis. Gene expression pattern was investigated using reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. Functional analysis was carried out by ectopic expression in Arabidopsis and rice. EgKNOX1 was orthologous to STM from Arabidopsis and to OSH1 from rice. It was expressed in the central zone of both vegetative and reproductive meristems. During leaf development, its expression was associated with plications from which the leaflets originate. Different modes of leaf dissection are seen to involve a similar class of genes to control meristematic activities, which govern the production of dissected morphologies.

  3. Covalent dimerization of ribulose bisphosphate carboxylase subunits by UV radiation.

    PubMed

    Ferreira, R M; Franco, E; Teixeira, A R

    1996-08-15

    The effect of UV radiation (UV-A, UV-B and UV-C) on ribulose bisphosphate carboxylase from a variety of plant species was examined. The exposition of plant leaves or the pure enzyme to UV radiation produced a UV-dependent accumulation of a +5 kDa polypeptide (P65). Different approaches were utilized to elucidate the origin and structure of P65: electrophoretic and fluorographic analyses of 35S-labelled ribulose bisphosphate carboxylase exposed to UV radiation and immunological experiments using antibodies specific for P65, for the large and small subunits of ribulose bisphosphate carboxylase and for high-molecular-mass aggregates of the enzyme. These studies revealed that P65 is a dimer, formed by the covalent, non-disulphide linkage of one small subunit with one large subunit of ribulose bisphosphate carboxylase. For short periods of time (< 1 h), the amount of P65 formed increased with the duration of the exposure to the UV radiation and with the energy of the radiation applied. Prolonged exposure to UV radiation (1-6 h) resulted in the formation of high-molecular-mass aggregates of ribulose bisphosphate carboxylase. Formation of P65 was shown to depend on the native state of the protein, was stimulated by inhibitors of enzyme activity, and was inhibited by activators of enzyme activity. A UV-independent accumulation of P65 was also achieved by the in vitro incubation of plant crude extracts. However, the UV-dependent and the UV-independent formation of P65 seemed to occur by distinct molecular mechanisms. The UV-dependent accumulation of P65 was immunologically detected in all species examined, including Lemna minor, Arum italicum, Brassica oleracea, Triticum aestivum, Zea mays, Pisum sativum and Phaseolus vulgaris, suggesting that it may constitute a universal response to UV radiation, common to all photo-synthetic tissues.

  4. Formation of active bacterial luciferase between interspecific subunits in vivo.

    PubMed

    Almashanu, S; Tuby, A; Hadar, R; Einy, R; Kuhn, J

    1995-01-01

    Interspecific complementation between luxAs and luxBs from Vibrio harveyi, Vibrio fischeri, Photobacterium leiognathi and Xenorhabdus luminescens was examined in vivo. The individual genes from these species were cloned on different compatible plasmids or amplified by PCR and brought together to yield cis combinations without extraneous DNA. The beta subunits from V. harveyi and X. luminescens form active enzyme only with alpha subunits from one of these species. All other combinations yield active enzymes. The lack of activity of the V. harveyi and X. luminescens beta subunits with the alpha subunits from V. fischeri and P. leiognathi results from a lack of association. This was shown by in vivo competition in which these beta subunits were overproduced in comparison with the beta and alpha of V. fischeri. No reduction in light was found. Overall, the in vivo results parallel those found in vitro using isolated denatured subunits and renaturation by removal of the denaturant.

  5. Genetic relatedness among Filobasidiella species.

    PubMed

    Sivakumaran, Swarna; Bridge, Paul; Roberts, Peter

    2002-01-01

    The three accepted species of Filobasidiella, F. neoformans, F. depauperata, and F. lutea, are compared morphologically and by molecular analysis. Sequences of the internally transcribed spacer (ITS) and the small subunit (SSU) gene of the ribosomal RNA (rRNA) gene cluster were obtained, and analysed by Neighbor-joining and Maximum parsimony methods. The three species of Filobsidiella are shown to form a single monophyletic clade, rooted by Tremella mesenterica. F. lutea was recovered as a distinct, but closely related taxon with the Filobasidiella clade. This is the first report of DNA sequences from herbarium specimens of F. lutea.

  6. The impact of copper, nitrate and carbon status on the emission of nitrous oxide by two species of bacteria with biochemically distinct denitrification pathways.

    PubMed

    Felgate, Heather; Giannopoulos, Georgios; Sullivan, Matthew J; Gates, Andrew J; Clarke, Thomas A; Baggs, Elizabeth; Rowley, Gary; Richardson, David J

    2012-07-01

    Denitrifying bacteria convert nitrate (NO(3) (-) ) to dinitrogen (N(2) ) gas through an anaerobic respiratory process in which the potent greenhouse gas nitrous oxide (N(2) O) is a free intermediate. These bacteria can be grouped into classes that synthesize a nitrite (NO(2) (-) ) reductase (Nir) that is solely dependent on haem-iron as a cofactor (e.g. Paracoccus denitrificans) or a Nir that is solely dependent on copper (Cu) as a cofactor (e.g. Achromobacter xylosoxidans). Regardless of which form of Nir these groups synthesize, they are both dependent on a Cu-containing nitrous oxide reductase (NosZ) for the conversion of N(2) O to N(2) . Agriculture makes a major contribution to N(2) O release and it is recognized that a number of agricultural lands are becoming Cu-limited but are N-rich because of fertilizer addition. Here we utilize continuous cultures to explore the denitrification phenotypes of P. denitrificans and A. xylosoxidans at a range of extracellular NO(3) (-) , organic carbon and Cu concentrations. Quite distinct phenotypes are observed between the two species. Notably, P. denitrificans emits approximately 40% of NO(3) (-) consumed as N(2) O under NO(3) (-) -rich Cu-deficient conditions, while under the same conditions A. xylosoxidans releases approximately 40% of the NO(3) (-) consumed as NO(2) (-) . However, the denitrification phenotypes are very similar under NO(3) (-) -limited conditions where denitrification intermediates do not accumulate significantly. The results have potential implications for understanding denitrification flux in a range of agricultural environments.

  7. Complementation of subunits from different bacterial luciferases. Evidence for the role of the. beta. subunit in the bioluminescent mechanism

    SciTech Connect

    Meighen, E.A.; Bartlet, I.

    1980-12-10

    Complementation of the nonidentical subunits (..cap alpha.. and ..beta..) of luciferases isolated from two different bioluminescent strains, Beneckea harveyi and Photobacterium phosphoreum, has resulted in the formation of a functional hybrid luciferase (..cap alpha../sub h/..beta../sub p/) containing the ..cap alpha.. subunit from B. harveyi luciferase (..cap alpha../sub h/) and the ..beta.. subunit from P. phosphoreum luciferase (..beta../sub p/). The complementation was unidirectional; activity could not be restored by complementing the ..cap alpha.. subunit of P. phosphoreum luciferase with the ..beta.. subunit of B. harveyi luciferase, showing that the subunits from these luciferases were not identical. Kinetic parameters of the hybrid luciferase reflecting the intermediate and later steps of the bioluminescent reaction as well as the overall activity and specificity were essentially identical to the same kinetic parameters for B. harveyi luciferase, the source of the ..cap alpha.. subunit, and quite distinct from those of P. phosphoreum luciferase. However, kinetic parameters that reflected the initial step in the reaction involving interaction of FMNH/sub 2/ and luciferase were altered in the hybrid luciferase compared to both the parental luciferases, the K/sub d/ for FMNH/sub 2/ actually being closer to that observed for the P. phosphoreum luciferase (the source of the ..beta.. subunit). These results provide direct evidence that modification or alteration of the ..beta.. subunit in a dimeric luciferase molecule can affect the kinetic properties and indicates that the ..beta.. subunit plays a functional role in the bioluminescent mechanism. It is proposed that both the ..cap alpha.. and ..beta.. subunits are involved with the initial interaction with FMNH/sub 2/, whereas subsequent steps in the mechanism are dictated exclusively by the ..cap alpha.. subunit and are unaffected by alterations in the ..beta.. subunit.

  8. Phylogeographic Evidence for 2 Genetically Distinct Zoonotic Plasmodium knowlesi Parasites, Malaysia

    PubMed Central

    Yusof, Ruhani; Ahmed, Md Atique; Jelip, Jenarun; Ngian, Hie Ung; Mustakim, Sahlawati; Hussin, Hani Mat; Fong, Mun Yik; Mahmud, Rohela; Sitam, Frankie Anak Thomas; Japning, J. Rovie-Ryan; Snounou, Georges; Escalante, Ananias A.

    2016-01-01

    Infections of humans with the zoonotic simian malaria parasite Plasmodium knowlesi occur throughout Southeast Asia, although most cases have occurred in Malaysia, where P. knowlesi is now the dominant malaria species. This apparently skewed distribution prompted an investigation of the phylogeography of this parasite in 2 geographically separated regions of Malaysia, Peninsular Malaysia and Malaysian Borneo. We investigated samples collected from humans and macaques in these regions. Haplotype network analyses of sequences from 2 P. knowlesi genes, type A small subunit ribosomal 18S RNA and cytochrome c oxidase subunit I, showed 2 genetically distinct divergent clusters, 1 from each of the 2 regions of Malaysia. We propose that these parasites represent 2 distinct P. knowlesi types that independently became zoonotic. These types would have evolved after the sea-level rise at the end of the last ice age, which separated Malaysian Borneo from Peninsular Malaysia. PMID:27433965

  9. Subunit organization in cytoplasmic dynein subcomplexes

    PubMed Central

    King, Stephen J.; Bonilla, Myriam; Rodgers, Michael E.; Schroer, Trina A.

    2002-01-01

    Because cytoplasmic dynein plays numerous critical roles in eukaryotic cells, determining the subunit composition and the organization and functions of the subunits within dynein are important goals. This has been difficult partly because of accessory polypeptide heterogeneity of dynein populations. The motor domain containing heavy chains of cytoplasmic dynein are associated with multiple intermediate, light intermediate, and light chain accessory polypeptides. We examined the organization of these subunits within cytoplasmic dynein by separating the molecule into two distinct subcomplexes. These subcomplexes were competent to reassemble into a molecule with dynein-like properties. One subcomplex was composed of the dynein heavy and light intermediate chains whereas the other subcomplex was composed of the intermediate and light chains. The intermediate and light chain subcomplex could be further separated into two pools, only one of which contained dynein light chains. The two pools had distinct intermediate chain compositions, suggesting that intermediate chain isoforms have different light chain–binding properties. When the two intermediate chain pools were characterized by analytical velocity sedimentation, at least four molecular components were seen: intermediate chain monomers, intermediate chain dimers, intermediate chain monomers with bound light chains, and a mixture of intermediate chain dimers with assorted bound light chains. These data provide new insights into the compositional heterogeneity and assembly of the cytoplasmic dynein complex and suggest that individual dynein molecules have distinct molecular compositions in vivo. PMID:11967380

  10. Carbon and Oxygen Isotope Measurements of Ordinary Chondrite (OC) Meteorites from Antarctica Indicate Distinct Terrestrial Carbonate Species using a Stepped Acid Extraction Procedure Impacting Mars Carbonate Research

    NASA Astrophysics Data System (ADS)

    Evans, M. E.; Niles, P. B.; Locke, D.

    2015-12-01

    The purpose of this study is to characterize the stable isotope values of terrestrial, secondary carbonate minerals from five OC meteorites collected in Antarctica. These samples were selected for analysis based upon their size and collection proximity to known Martian meteorites. They were also selected based on petrologic type (3+) such that they were likely to be carbonate-free before falling to Earth. This study has two main tasks: 1) characterize the isotopic composition of terrestrial, secondary carbonate minerals formed on meteorites in Antarctica, and 2) study the mechanisms of carbonate formation in cold and arid environments with Antarctica as an analog for Mars. Two samples from each meteorite, each ~0.5g, was crushed and dissolved in pure phosphoric acid for 3 sequential reactions: a) Rx0 for 1 hour at 30°C, b) Rx1 for 18 hours at 30°C, and c) Rx2 for 3 hours at 150°C. CO2 was distilled by freezing with liquid nitrogen from each sample tube, then separated from organics and sulfides with a TRACE GC using a Restek HayeSep Q 80/100 6' 2mm stainless column, and then analyzed on a Thermo MAT 253 IRMS in Dual Inlet mode. This system was built at NASA/JSC over the past 3 years and proof tested with known carbonate standards to develop procedures, assess yield, and quantify expected uncertainties. Two distinct species of carbonates are found based on the stepped extraction technique: 1) Ca-rich carbonate released at low temperatures, and 2) Mg, or Fe-rich carbonate released at high temperatures. Preliminary results indicate that most of the carbonates present in the ordinary chondrites analyzed have δ13C=+5‰, which is consistent with formation from atmospheric CO2 δ13C=-7‰ at -20°C. The oxygen isotopic compositions of the carbonates vary between +4‰ and +34‰ with the Mg-rich and/or Fe-rich carbonates possessing the lowest δ18O values. This suggests that the carbonates formed under a wide range of temperatures. However, the carbonate oxygen

  11. Normal Hematopoietic Progenitor Subsets Have Distinct Reactive Oxygen Species, BCL2 and Cell-Cycle Profiles That Are Decoupled from Maturation in Acute Myeloid Leukemia

    PubMed Central

    Hills, Robert K.; Knapper, Steve; Steadman, Lora; Qureshi, Ushna; Rector, Jerrald L.; Bradbury, Charlotte; Russell, Nigel H.; Vyas, Paresh; Burnett, Alan K.; Grimwade, David; Hole, Paul S.; Freeman, Sylvie D.

    2016-01-01

    In acute myeloid leukemia (AML) quiescence and low oxidative state, linked to BCL2 mitochondrial regulation, endow leukemic stem cells (LSC) with treatment-resistance. LSC in CD34+ and more mature CD34− AML have heterogeneous immunophenotypes overlapping with normal stem/progenitor cells (SPC) but may be differentiated by functional markers. We therefore investigated the oxidative/reactive oxygen species (ROS) profile, its relationship with cell-cycle/BCL2 for normal SPC, and whether altered in AML and myelodysplasia (MDS). In control BM (n = 24), ROS levels were highest in granulocyte-macrophage progenitors (GMP) and CD34− myeloid precursors but megakaryocyte-erythroid progenitors had equivalent levels to CD34+CD38low immature-SPC although they were ki67high. BCL2 upregulation was specific to GMPs. This profile was also observed for CD34+SPC in MDS-without-excess-blasts (MDS-noEB, n = 12). Erythroid CD34− precursors were, however, abnormally ROS-high in MDS-noEB, potentially linking oxidative stress to cell loss. In pre-treatment AML (n = 93) and MDS-with-excess-blasts (MDS-RAEB) (n = 14), immunophenotypic mature-SPC had similar ROS levels to co-existing immature-SPC. However ROS levels varied between AMLs; Flt3ITD+/NPM1wild-type CD34+SPC had higher ROS than NPM1mutated CD34+ or CD34− SPC. An aberrant ki67lowBCL2high immunophenotype was observed in CD34+AML (most prominent in Flt3ITD AMLs) but also in CD34− AMLs and MDS-RAEB, suggesting a shared redox/pro-survival adaptation. Some patients had BCL2 overexpression in CD34+ ROS-high as well as ROS-low fractions which may be indicative of poor early response to standard chemotherapy. Thus normal SPC subsets have distinct ROS, cell-cycle, BCL2 profiles that in AML /MDS-RAEB are decoupled from maturation. The combined profile of these functional properties in AML subpopulations may be relevant to differential treatment resistance. PMID:27669008

  12. Carbon and Oxygen Isotope Measurements of Ordinary Chondrite (OC) Meteorites from Antarctica Indicate Distinct Carbonate Species Using a Stepped Acid Extraction Procedure

    NASA Technical Reports Server (NTRS)

    Evans, Michael E.

    2015-01-01

    The purpose of this study is to characterize the stable isotope values of terrestrial, secondary carbonate minerals from five Ordinary Chondrite (OC) meteorites collected in Antarctica. These samples were identified and requested from NASA based upon their size, alteration history, and collection proximity to known Martian meteorites. They are also assumed to be carbonate-free before falling to Earth. This research addresses two questions involving Mars carbonates: 1) characterize terrestrial, secondary carbonate isotope values to apply to Martian meteorites for isolating in-situ carbonates, and 2) increase understanding of carbonates formed in cold and arid environments with Antarctica as an analog for Mars. Two samples from each meteorite, each approximately 0.5 grams, were crushed and dissolved in pure phosphoric acid for 3 sequential reactions: a) R times 0 for 1 hour at 30 degrees Centigrade (fine calcite extraction), b) R times 1 for 18 hours at 30 degrees Centigrade (course calcite extraction), and c) R times 2 for 3 hours at 150 degrees Centigrade (siderite and/or magnesite extraction). CO (sub 2) was distilled by freezing with liquid nitrogen from each sample tube, then separated from organics and sulfides with a TRACE GC using a Restek HayeSep Q 80/100 6 foot 2 millimeter stainless column, and then analyzed on a Thermo MAT 253 Isotope Ratio Mass Spectrometer (IRMS) in Dual Inlet mode. This system was built at NASA/JSC over the past 3 years and proof-tested with known carbonate standards to develop procedures, assess yield, and quantify expected error bands. Two distinct species of carbonates are found: 1) calcite, and 2) non-calcite carbonate (future testing will attempt to differentiate siderite from magnesite). Preliminary results indicate the terrestrial carbonates are formed at approximately sigma (sup 13) C equal to plus 5 per mille, which is consistent with atmospheric CO (sub 2) sigma (sup 13) C equal to minus 7 per mille and fractionation of plus

  13. The Arabidopsis KINβγ Subunit of the SnRK1 Complex Regulates Pollen Hydration on the Stigma by Mediating the Level of Reactive Oxygen Species in Pollen.

    PubMed

    Gao, Xin-Qi; Liu, Chang Zhen; Li, Dan Dan; Zhao, Ting Ting; Li, Fei; Jia, Xiao Na; Zhao, Xin-Ying; Zhang, Xian Sheng

    2016-07-01

    Pollen-stigma interactions are essential for pollen germination. The highly regulated process of pollen germination includes pollen adhesion, hydration, and germination on the stigma. However, the internal signaling of pollen that regulates pollen-stigma interactions is poorly understood. KINβγ is a plant-specific subunit of the SNF1-related protein kinase 1 complex which plays important roles in the regulation of plant development. Here, we showed that KINβγ was a cytoplasm- and nucleus-localized protein in the vegetative cells of pollen grains in Arabidopsis. The pollen of the Arabidopsis kinβγ mutant could not germinate on stigma, although it germinated normally in vitro. Further analysis revealed the hydration of kinβγ mutant pollen on the stigma was compromised. However, adding water to the stigma promoted the germination of the mutant pollen in vivo, suggesting that the compromised hydration of the mutant pollen led to its defective germination. In kinβγ mutant pollen, the structure of the mitochondria and peroxisomes was destroyed, and their numbers were significantly reduced compared with those in the wild type. Furthermore, we found that the kinβγ mutant exhibited reduced levels of reactive oxygen species (ROS) in pollen. The addition of H2O2 in vitro partially compensated for the reduced water absorption of the mutant pollen, and reducing ROS levels in pollen by overexpressing Arabidopsis CATALASE 3 resulted in compromised hydration of pollen on the stigma. These results indicate that Arabidopsis KINβγ is critical for the regulation of ROS levels by mediating the biogenesis of mitochondria and peroxisomes in pollen, which is required for pollen-stigma interactions during pollination.

  14. The Arabidopsis KINβγ Subunit of the SnRK1 Complex Regulates Pollen Hydration on the Stigma by Mediating the Level of Reactive Oxygen Species in Pollen

    PubMed Central

    Zhao, Ting Ting; Li, Fei; Jia, Xiao Na; Zhao, Xin-Ying; Zhang, Xian Sheng

    2016-01-01

    Pollen–stigma interactions are essential for pollen germination. The highly regulated process of pollen germination includes pollen adhesion, hydration, and germination on the stigma. However, the internal signaling of pollen that regulates pollen–stigma interactions is poorly understood. KINβγ is a plant-specific subunit of the SNF1-related protein kinase 1 complex which plays important roles in the regulation of plant development. Here, we showed that KINβγ was a cytoplasm- and nucleus-localized protein in the vegetative cells of pollen grains in Arabidopsis. The pollen of the Arabidopsis kinβγ mutant could not germinate on stigma, although it germinated normally in vitro. Further analysis revealed the hydration of kinβγ mutant pollen on the stigma was compromised. However, adding water to the stigma promoted the germination of the mutant pollen in vivo, suggesting that the compromised hydration of the mutant pollen led to its defective germination. In kinβγ mutant pollen, the structure of the mitochondria and peroxisomes was destroyed, and their numbers were significantly reduced compared with those in the wild type. Furthermore, we found that the kinβγ mutant exhibited reduced levels of reactive oxygen species (ROS) in pollen. The addition of H2O2 in vitro partially compensated for the reduced water absorption of the mutant pollen, and reducing ROS levels in pollen by overexpressing Arabidopsis CATALASE 3 resulted in compromised hydration of pollen on the stigma. These results indicate that Arabidopsis KINβγ is critical for the regulation of ROS levels by mediating the biogenesis of mitochondria and peroxisomes in pollen, which is required for pollen–stigma interactions during pollination. PMID:27472382

  15. Subunit-selective proteasome activity profiling uncovers uncoupled proteasome subunit activities during bacterial infections.

    PubMed

    Misas-Villamil, Johana C; van der Burgh, Aranka M; Grosse-Holz, Friederike; Bach-Pages, Marcel; Kovács, Judit; Kaschani, Farnusch; Schilasky, Sören; Emon, Asif Emran Khan; Ruben, Mark; Kaiser, Markus; Overkleeft, Hermen S; van der Hoorn, Renier A L

    2017-01-24

    The proteasome is a nuclear - cytoplasmic proteolytic complex involved in nearly all regulatory pathways in plant cells. The three different catalytic activities of the proteasome can have different functions but tools to monitor and control these subunits selectively are not yet available in plant science. Here, we introduce subunit-selective inhibitors and dual-color fluorescent activity-based probes for studying two of the three active catalytic subunits of the plant proteasome. We validate these tools in two model plants and use this to study the proteasome during plant-microbe interactions. Our data reveals that Nicotiana benthamiana incorporates two different paralogs of each catalytic subunit into active proteasomes. Interestingly, both β1 and β5 activities are significantly increased upon infection with pathogenic Pseudomonas syringae pv. tomato DC3000 lacking hopQ1-1 (PtoDC3000(ΔhQ)) whilst the activity profile of the β1 subunit changes. Infection with wild-type PtoDC3000 causes proteasome activities that range from strongly induced β1 and β5 activities to strongly suppressed β5 activities, revealing that β1 and β5 activities can be uncoupled during bacterial infection. These selective probes and inhibitors are now available to the plant science community and can be widely and easily applied to study the activity and role of the different catalytic subunits of the proteasome in different plant species. This article is protected by copyright. All rights reserved.

  16. Molecular and expression characterization of three gonadotropin subunits common alpha, FSHbeta and LHbeta in groupers.

    PubMed

    Li, Chuang-Ju; Zhou, Li; Wang, Yang; Hong, Yun-Han; Gui, Jian-Fang

    2005-04-15

    A SMART cDNA plasmid library was constructed from protogyous greasy grouper (Epinephelus coioides) pituitary, and the full-length cDNAs of three gonadotropin (GTH) subunits common alpha, FSHbeta and LHbeta were cloned and sequenced from the library. The nucleotide sequences of common alpha, FSHbeta and LHbeta subunit cDNAs are 647, 594 and 574 bp in length, and encode for mature peptides of 94, 99 and 115 aa, respectively. High homology was observed by amino acid sequence alignment and identity comparison of the grouper mature peptides of common alpha, FSHbeta and LHbeta with that of other fishes. Phylogenetic tree analyses of the three GTH mature subunits revealed similar phylogeny relationships among the studied fish species. Three polyclonal antibodies were prepared from the in vitro expressed common alpha, FSHbeta and LHbeta mature proteins, respectively. Western blot analysis and immunofluoresence localization were performed on two typical stages of ovarian development stages in red-spotted grouper. Significant differences in protein expression levels of three gonadotropin subunits were revealed between the two ovarian development stages. In the individuals with resting ovary, common alpha was almost not detected in pituitaries, and FSHbeta and LHbeta expression levels were very low. While in the individuals with developing ovary, the expression of all three gonadotropin subunits reached to a high level. Immunofluoresence localization indicated that the grouper FSHbeta cells mainly distributed in the middle area of PPD, while the LHbeta cells distributed more widely, including in the area similar to the FSHbeta cells and at the external periphery of pituitary near to the PI side. The common alpha might be expressed in both FSHbeta and LHbeta cells. Double immunofluoresence localization further demonstrated FSHbeta and LHbeta expression in distinct cells in the PPD area, although the FSHbeta and LHbeta cells were detected in the identical area of PPD.

  17. The Lesser of Two Weevils: Molecular-Genetics of Pest Palm Weevil Populations Confirm Rhynchophorus vulneratus (Panzer 1798) as a Valid Species Distinct from R. ferrugineus (Olivier 1790), and Reveal the Global Extent of Both

    PubMed Central

    Rugman-Jones, Paul F.; Hoddle, Christina D.; Hoddle, Mark S.; Stouthamer, Richard

    2013-01-01

    The red palm weevil (RPW) is a major pest of palms. It is native to southeast Asia and Melanesia, but in recent decades has vastly expanded its range as the result of multiple accidental anthropogenic introductions into the Middle East, Mediterranean Basin, Caribbean, and U.S.A. Currently regarded as a single species, Rhynchophorus ferrugineus (Olivier), RPW displays remarkable color variation across its range, and consequently has a taxonomic history littered with new species descriptions and synonymization. We compared DNA sequences of the mitochondrial cytochrome oxidase subunit I (COI) gene from RPW populations throughout the native and invaded ranges, to investigate the specific status and invasion history of this serious economic pest, and to identify possible common routes of entry. Analyses of COI haplotype data provide conclusive support, corroborated by sequences of additional nuclear gene regions, for the existence of at least two predominantly allopatric species. The true R. ferrugineus is native only to the northern and western parts of continental southeast Asia, Sri Lanka and the Philippines, and is responsible for almost all invasive populations worldwide. In contrast, the second species, which is currently synonymized under R. ferrugineus and should be resurrected under the name R. vulneratus (Panzer), has a more southern distribution across Indonesia, and is responsible for only one invasive population; that in California, U.S.A. The distribution of COI haplotypes is used to discuss the possible existence of further cryptic species, sources and routes of entry of different invasive populations, and the implications of our findings for current control methods. PMID:24143263

  18. The lesser of two weevils: molecular-genetics of pest palm weevil populations confirm Rhynchophorus vulneratus (Panzer 1798) as a valid species distinct from R. ferrugineus (Olivier 1790), and reveal the global extent of both.

    PubMed

    Rugman-Jones, Paul F; Hoddle, Christina D; Hoddle, Mark S; Stouthamer, Richard

    2013-01-01

    The red palm weevil (RPW) is a major pest of palms. It is native to southeast Asia and Melanesia, but in recent decades has vastly expanded its range as the result of multiple accidental anthropogenic introductions into the Middle East, Mediterranean Basin, Caribbean, and U.S.A. Currently regarded as a single species, Rhynchophorus ferrugineus (Olivier), RPW displays remarkable color variation across its range, and consequently has a taxonomic history littered with new species descriptions and synonymization. We compared DNA sequences of the mitochondrial cytochrome oxidase subunit I (COI) gene from RPW populations throughout the native and invaded ranges, to investigate the specific status and invasion history of this serious economic pest, and to identify possible common routes of entry. Analyses of COI haplotype data provide conclusive support, corroborated by sequences of additional nuclear gene regions, for the existence of at least two predominantly allopatric species. The true R. ferrugineus is native only to the northern and western parts of continental southeast Asia, Sri Lanka and the Philippines, and is responsible for almost all invasive populations worldwide. In contrast, the second species, which is currently synonymized under R. ferrugineus and should be resurrected under the name R. vulneratus (Panzer), has a more southern distribution across Indonesia, and is responsible for only one invasive population; that in California, U.S.A. The distribution of COI haplotypes is used to discuss the possible existence of further cryptic species, sources and routes of entry of different invasive populations, and the implications of our findings for current control methods.

  19. Multigene Phylogeography of Bactrocera caudata (Insecta: Tephritidae): Distinct Genetic Lineages in Northern and Southern Hemispheres

    PubMed Central

    Yong, Hoi-Sen; Lim, Phaik-Eem; Tan, Ji; Song, Sze-Looi; Suana, I Wayan; Eamsobhana, Praphathip

    2015-01-01

    Bactrocera caudata is a pest of pumpkin flower. Specimens of B. caudata from the northern hemisphere (mainland Asia) and southern hemisphere (Indonesia) were analysed using the partial DNA sequences of the nuclear 28S rRNA and internal transcribed spacer region 2 (ITS-2) genes, and the mitochondrial cytochrome c oxidase subunit I (COI), cytochrome c oxidase subunit II (COII) and 16S rRNA genes. The COI, COII, 16S rDNA and concatenated COI+COII+16S and COI+COII+16S+28S+ITS-2 nucleotide sequences revealed that B. caudata from the northern hemisphere (Peninsular Malaysia, East Malaysia, Thailand) was distinctly different from the southern hemisphere (Indonesia: Java, Bali and Lombok), without common haplotype between them. Phylogenetic analysis revealed two distinct clades (northern and southern hemispheres), indicating distinct genetic lineage. The uncorrected ‘p’ distance for the concatenated COI+COII+16S nucleotide sequences between the taxa from the northern and southern hemispheres (‘p’ = 4.46-4.94%) was several folds higher than the ‘p’ distance for the taxa in the northern hemisphere (‘p’ = 0.00-0.77%) and the southern hemisphere (‘p’ = 0.00%). This distinct difference was also reflected by concatenated COI+COII+16S+28S+ITS-2 nucleotide sequences with an uncorrected 'p' distance of 2.34-2.69% between the taxa of northern and southern hemispheres. In accordance with the type locality the Indonesian taxa belong to the nominal species. Thus the taxa from the northern hemisphere, if they were to constitute a cryptic species of the B. caudata species complex based on molecular data, need to be formally described as a new species. The Thailand and Malaysian B. caudata populations in the northern hemisphere showed distinct genetic structure and phylogeographic pattern. PMID:26090853

  20. Multigene Phylogeography of Bactrocera caudata (Insecta: Tephritidae): Distinct Genetic Lineages in Northern and Southern Hemispheres.

    PubMed

    Yong, Hoi-Sen; Lim, Phaik-Eem; Tan, Ji; Song, Sze-Looi; Suana, I Wayan; Eamsobhana, Praphathip

    2015-01-01

    Bactrocera caudata is a pest of pumpkin flower. Specimens of B. caudata from the northern hemisphere (mainland Asia) and southern hemisphere (Indonesia) were analysed using the partial DNA sequences of the nuclear 28S rRNA and internal transcribed spacer region 2 (ITS-2) genes, and the mitochondrial cytochrome c oxidase subunit I (COI), cytochrome c oxidase subunit II (COII) and 16S rRNA genes. The COI, COII, 16S rDNA and concatenated COI+COII+16S and COI+COII+16S+28S+ITS-2 nucleotide sequences revealed that B. caudata from the northern hemisphere (Peninsular Malaysia, East Malaysia, Thailand) was distinctly different from the southern hemisphere (Indonesia: Java, Bali and Lombok), without common haplotype between them. Phylogenetic analysis revealed two distinct clades (northern and southern hemispheres), indicating distinct genetic lineage. The uncorrected 'p' distance for the concatenated COI+COII+16S nucleotide sequences between the taxa from the northern and southern hemispheres ('p' = 4.46-4.94%) was several folds higher than the 'p' distance for the taxa in the northern hemisphere ('p' = 0.00-0.77%) and the southern hemisphere ('p' = 0.00%). This distinct difference was also reflected by concatenated COI+COII+16S+28S+ITS-2 nucleotide sequences with an uncorrected 'p' distance of 2.34-2.69% between the taxa of northern and southern hemispheres. In accordance with the type locality the Indonesian taxa belong to the nominal species. Thus the taxa from the northern hemisphere, if they were to constitute a cryptic species of the B. caudata species complex based on molecular data, need to be formally described as a new species. The Thailand and Malaysian B. caudata populations in the northern hemisphere showed distinct genetic structure and phylogeographic pattern.

  1. The subunit composition and function of mammalian cytochrome c oxidase.

    PubMed

    Kadenbach, Bernhard; Hüttemann, Maik

    2015-09-01

    Cytochrome c oxidase (COX) from mammals and birds is composed of 13 subunits. The three catalytic subunits I-III are encoded by mitochondrial DNA, the ten nuclear-coded subunits (IV, Va, Vb, VIa, VIb, VIc, VIIa, VIIb, VIIc, VIII) by nuclear DNA. The nuclear-coded subunits are essentially involved in the regulation of oxygen consumption and proton translocation by COX, since their removal or modification changes the activity and their mutation causes mitochondrial diseases. Respiration, the basis for ATP synthesis in mitochondria, is differently regulated in organs and species by expression of tissue-, developmental-, and species-specific isoforms for COX subunits IV, VIa, VIb, VIIa, VIIb, and VIII, but the holoenzyme in mammals is always composed of 13 subunits. Various proteins and enzymes were shown, e.g., by co-immunoprecipitation, to bind to specific COX subunits and modify its activity, but these interactions are reversible, in contrast to the tightly bound 13 subunits. In addition, the formation of supercomplexes with other oxidative phosphorylation complexes has been shown to be largely variable. The regulatory complexity of COX is increased by protein phosphorylation. Up to now 18 phosphorylation sites have been identified under in vivo conditions in mammals. However, only for a few phosphorylation sites and four nuclear-coded subunits could a specific function be identified. Research on the signaling pathways leading to specific COX phosphorylations remains a great challenge for understanding the regulation of respiration and ATP synthesis in mammalian organisms. This article reviews the function of the individual COX subunits and their isoforms, as well as proteins and small molecules interacting and regulating the enzyme.

  2. Within-island speciation with an exceptional case of distinct separation between two sibling lizard species divided by a narrow stream.

    PubMed

    Tseng, Shu-Ping; Wang, Chao-Jun; Li, Shou-Hsien; Lin, Si-Min

    2015-09-01

    Delimitation of genetic and geographic boundaries between species is a focus of evolutionary biology. In this study, we demonstrated fine-scale differentiation of Takydromus formosanus species complex comprising four insular endemics on Taiwan Island. Phylogeny and ancestral range reconstruction based on mitochondrial DNA sequences of 430 Takydromus lizards (405 lizards of this complex throughout their distribution range, and 25 lizards from 11 other species) indicated that the major branching process occurred within Taiwan, which represented a solid evidence of within-island speciation on this small island. We further demonstrated an exceptional case of a pair of sister species, T. viridipunctatus and T. luyeanus, that were separated by a narrow stream with a width of only 15m. This pattern might be one of the narrowest contact zones ever documented in terrestrial vertebrates. To evaluate the level of genetic introgression between these sister species, a fine-scale collection of another 382 lizards was conducted along a transect line across the stream. A total of 13 microsatellite markers and mtDNA genotyping was used to detect a low proportion of hybrids (5.7-9.9% from STRUCTURE, and 2.3% from DAPC). Our results indicated that the two clades are highly differentiated across this extremely short distance.

  3. Structural determinants of alpha-bungarotoxin binding to the sequence segment 181-200 of the muscle nicotinic acetylcholine receptor. alpha. subunit: Effects of cysteine/cystine modification and species-specific amino acid substitution

    SciTech Connect

    McLane, K.E.; Wu, Xiadong; Diethelm, B.; Conti-Tronconi, B.M. )

    1991-05-21

    The sequence segment 181-200 of the Torpedo nicotinic acetylcholine receptor (nAChR) {alpha}subunit forms a binding site for {alpha}-bungarotoxin ({alpha}-BTX). Synthetic peptides corresponding to the homologous sequences of human, calf, mouse, chicken, frog, and cobra muscle nAChR {alpha}1 subunits were tested for their ability to bind {sup 125}I-{alpha}-BTX, and differences in {alpha}-BTX affinity were determined by using solution (IC{sub 50}s) and solid-phase (K{sub d}s) assays. Panels of overlapping peptides corresponding to the complete {alpha}1 subunit of mouse and human were also tested for {alpha}-BTX binding, but other sequence segments forming the {alpha}-BTX site were not consistently detectable. The role of a putative vicinal disulfide bound between Cys-192 and -193, relative to the Torpedo sequence, was determined by modifying the peptides with sulfhydryl reagents. Reduction and alkylation of the peptides decreased {alpha}-BTX binding, whereas oxidation of the peptides had little effect. These results indicate that while the adjacent cysteines are likely to be involved in forming the toxin/{alpha}1-subunit interface a vicinal disulfide bound was not required for {alpha}-BTX binding.

  4. Megraft: A software package to graft ribosomal small subunit (16S/18S) fragments onto full-length sequences for accurate species richness and sequencing depth analysis in pyrosequencing-length metagenomes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Metagenomic libraries represent subsamples of the total DNA found at a study site and offer unprecedented opportunities to study ecological and functional aspects of microbial communities. To examine the depth of the sequencing effort, rarefaction analysis of the ribosomal small sub-unit (SSU/16S/18...

  5. Transcription, Signaling Receptor Activity, Oxidative Phosphorylation, and Fatty Acid Metabolism Mediate the Presence of Closely Related Species in Distinct Intertidal and Cold-Seep Habitats.

    PubMed

    Van Campenhout, Jelle; Vanreusel, Ann; Van Belleghem, Steven; Derycke, Sofie

    2015-12-03

    Bathyal cold seeps are isolated extreme deep-sea environments characterized by low species diversity while biomass can be high. The Håkon Mosby mud volcano (Barents Sea, 1,280 m) is a rather stable chemosynthetic driven habitat characterized by prominent surface bacterial mats with high sulfide concentrations and low oxygen levels. Here, the nematode Halomonhystera hermesi thrives in high abundances (11,000 individuals 10 cm(-2)). Halomonhystera hermesi is a member of the intertidal Halomonhystera disjuncta species complex that includes five cryptic species (GD1-5). GD1-5's common habitat is characterized by strong environmental fluctuations. Here, we compared the transcriptomes of H. hermesi and GD1, H. hermesi's closest relative. Genes encoding proteins involved in oxidative phosphorylation are more strongly expressed in H. hermesi than in GD1, and many genes were only observed in H. hermesi while being completely absent in GD1. Both observations could in part be attributed to high sulfide concentrations and low oxygen levels. Additionally, fatty acid elongation was also prominent in H. hermesi confirming the importance of highly unsaturated fatty acids in this species. Significant higher amounts of transcription factors and genes involved in signaling receptor activity were observed in GD1 (many of which were completely absent in H. hermesi), allowing fast signaling and transcriptional reprogramming which can mediate survival in dynamic intertidal environments. GC content was approximately 8% higher in H. hermesi coding unigenes resulting in differential codon usage between both species and a higher proportion of amino acids with GC-rich codons in H. hermesi. In general our results showed that most pathways were active in both environments and that only three genes are under natural selection. This indicates that also plasticity should be taken in consideration in the evolutionary history of Halomonhystera species. Such plasticity, as well as possible

  6. Altered 40 S ribosomal subunits in omnipotent suppressors of yeast.

    PubMed

    Eustice, D C; Wakem, L P; Wilhelm, J M; Sherman, F

    1986-03-20

    The five suppressors SUP35, SUP43, SUP44, SUP45 and SUP46, each mapping at a different chromosomal locus in the yeast Saccharomyces cerevisiae, suppress a wide range of mutations, including representatives of all three types of nonsense mutations, UAA, UAG and UGA. We have demonstrated that ribosomes from the four suppressors SUP35, SUP44, SUP45 and SUP46 translate polyuridylate templates in vitro with higher errors than ribosomes from the normal stain, and that this misreading is substantially enhanced by the antibiotic paromomycin. Furthermore, ribosomal subunit mixing experiments established that the 40 S ribosomal subunit, and this subunit only, is responsible for the higher levels of misreading. Thus, the gene products of SUP35, SUP44, SUP45 and SUP46 are components of the 40 S subunit or are enzymes that modify the subunit. In addition, a protein from the 40 S subunit of the SUP35 suppressor has an altered electrophoretic mobility; this protein is distinct from the altered protein previously uncovered in the 40 S subunit of the SUP46 suppressor. In contrast to the ribosomes from the four suppressors SUP35, SUP44, SUP45 and SUP46, the ribosomes from the SUP43 suppressor do not significantly misread polyuridylate templates in vitro, suggesting that this locus may not encode a ribosomal component or that the misreading is highly specific.

  7. Genetic Analysis of the Cytoplasmic Dynein Subunit Families

    PubMed Central

    Pfister, K. Kevin; Shah, Paresh R; Hummerich, Holger; Russ, Andreas; Cotton, James; Annuar, Azlina Ahmad; King, Stephen M; Fisher, Elizabeth M. C

    2006-01-01

    Cytoplasmic dyneins, the principal microtubule minus-end-directed motor proteins of the cell, are involved in many essential cellular processes. The major form of this enzyme is a complex of at least six protein subunits, and in mammals all but one of the subunits are encoded by at least two genes. Here we review current knowledge concerning the subunits, their interactions, and their functional roles as derived from biochemical and genetic analyses. We also carried out extensive database searches to look for new genes and to clarify anomalies in the databases. Our analysis documents evolutionary relationships among the dynein subunits of mammals and other model organisms, and sheds new light on the role of this diverse group of proteins, highlighting the existence of two cytoplasmic dynein complexes with distinct cellular roles. PMID:16440056

  8. Echinococcus granulosus Antigen B Structure: Subunit Composition and Oligomeric States

    PubMed Central

    Monteiro, Karina M.; Cardoso, Mateus B.; Follmer, Cristian; da Silveira, Nádya P.; Vargas, Daiani M.; Kitajima, Elliot W.; Zaha, Arnaldo; Ferreira, Henrique B.

    2012-01-01

    Background Antigen B (AgB) is the major protein secreted by the Echinococcus granulosus metacestode and is involved in key host-parasite interactions during infection. The full comprehension of AgB functions depends on the elucidation of several structural aspects that remain unknown, such as its subunit composition and oligomeric states. Methodology/Principal Findings The subunit composition of E. granulosus AgB oligomers from individual bovine and human cysts was assessed by mass spectrometry associated with electrophoretic analysis. AgB8/1, AgB8/2, AgB8/3 and AgB8/4 subunits were identified in all samples analyzed, and an AgB8/2 variant (AgB8/2v8) was found in one bovine sample. The exponentially modified protein abundance index (emPAI) was used to estimate the relative abundance of the AgB subunits, revealing that AgB8/1 subunit was relatively overrepresented in all samples. The abundance of AgB8/3 subunit varied between bovine and human cysts. The oligomeric states formed by E. granulosus AgB and recombinant subunits available, rAgB8/1, rAgB8/2 and rAgB8/3, were characterized by native PAGE, light scattering and microscopy. Recombinant subunits showed markedly distinct oligomerization behaviors, forming oligomers with a maximum size relation of rAgB8/3>rAgB8/2>rAgB8/1. Moreover, the oligomeric states formed by rAgB8/3 subunit were more similar to those observed for AgB purified from hydatid fluid. Pressure-induced dissociation experiments demonstrated that the molecular assemblies formed by the more aggregative subunits, rAgB8/2 and rAgB8/3, also display higher structural stability. Conclusions/Significance For the first time, AgB subunit composition was analyzed in samples from single hydatid cysts, revealing qualitative and quantitative differences between samples. We showed that AgB oligomers are formed by different subunits, which have distinct abundances and oligomerization properties. Overall, our findings have significantly contributed to increase the

  9. Comparative analysis of Edwardsiella tarda isolates from fish in the eastern United States suggests the existence of two genetically distinct species, Edwardsiella tarda and Edwardsiella pseudotarda sp. nov

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Edwardsiella tarda, a Gram-negative member of the family Enterobacteriaceae, is often implicated in significant losses in aquaculture facilities worldwide. Here, we assessed the intra-specific variability of a collection of E. tarda isolates from 4 different fish species in the eastern United State...

  10. Oogonal biometry and phylogenetic analysis of the Pythium vexans species group from woody agricultural hosts in South Africa reveal distinct groups within this taxon

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pythium vexans fits into the internal transcribed spacer (ITS) clade K. In South Africa, as well as in other regions of the world, P. vexans isolates are known to be heterogeneous in their ITS sequences and may consist of more than one species. Therefore, this study examined the diversity of South...

  11. Modulation of BK Channel Function by Auxiliary Beta and Gamma Subunits

    PubMed Central

    Li, Q.; Yan, J.

    2016-01-01

    The large-conductance, Ca2+- and voltage-activated K+ (BK) channel is ubiquitously expressed in mammalian tissues and displays diverse biophysical or pharmacological characteristics. This diversity is in part conferred by channel modulation with different regulatory auxiliary subunits. To date, two distinct classes of BK channel auxiliary subunits have been identified: β subunits and γ subunits. Modulation of BK channels by the four auxiliary β (β1–β4) subunits has been well established and intensively investigated over the past two decades. The auxiliary γ subunits, however, were identified only very recently, which adds a new dimension to BK channel regulation and improves our understanding of the physiological functions of BK channels in various tissues and cell types. This chapter will review the current understanding of BK channel modulation by auxiliary β and γ subunits, especially the latest findings. PMID:27238261

  12. Transcription, Signaling Receptor Activity, Oxidative Phosphorylation, and Fatty Acid Metabolism Mediate the Presence of Closely Related Species in Distinct Intertidal and Cold-Seep Habitats

    PubMed Central

    Van Campenhout, Jelle; Vanreusel, Ann; Van Belleghem, Steven; Derycke, Sofie

    2016-01-01

    Bathyal cold seeps are isolated extreme deep-sea environments characterized by low species diversity while biomass can be high. The Håkon Mosby mud volcano (Barents Sea, 1,280 m) is a rather stable chemosynthetic driven habitat characterized by prominent surface bacterial mats with high sulfide concentrations and low oxygen levels. Here, the nematode Halomonhystera hermesi thrives in high abundances (11,000 individuals 10 cm−2). Halomonhystera hermesi is a member of the intertidal Halomonhystera disjuncta species complex that includes five cryptic species (GD1-5). GD1-5’s common habitat is characterized by strong environmental fluctuations. Here, we compared the transcriptomes of H. hermesi and GD1, H. hermesi’s closest relative. Genes encoding proteins involved in oxidative phosphorylation are more strongly expressed in H. hermesi than in GD1, and many genes were only observed in H. hermesi while being completely absent in GD1. Both observations could in part be attributed to high sulfide concentrations and low oxygen levels. Additionally, fatty acid elongation was also prominent in H. hermesi confirming the importance of highly unsaturated fatty acids in this species. Significant higher amounts of transcription factors and genes involved in signaling receptor activity were observed in GD1 (many of which were completely absent in H. hermesi), allowing fast signaling and transcriptional reprogramming which can mediate survival in dynamic intertidal environments. GC content was approximately 8% higher in H. hermesi coding unigenes resulting in differential codon usage between both species and a higher proportion of amino acids with GC-rich codons in H. hermesi. In general our results showed that most pathways were active in both environments and that only three genes are under natural selection. This indicates that also plasticity should be taken in consideration in the evolutionary history of Halomonhystera species. Such plasticity, as well as possible

  13. Distinct retinohypothalamic innervation patterns predict the developmental emergence of species-typical circadian phase preference in nocturnal Norway rats and diurnal nile grass rats.

    PubMed

    Todd, William D; Gall, Andrew J; Weiner, Joshua A; Blumberg, Mark S

    2012-10-01

    How does the brain develop differently to support nocturnality in some mammals, but diurnality in others? To answer this question, one might look to the suprachiasmatic nucleus (SCN), which is entrained by light via the retinohypothalamic tract (RHT). However, because the SCN is more active during the day in all mammals studied thus far, it alone cannot determine circadian phase preference. In adult Norway rats (Rattus norvegicus), which are nocturnal, the RHT also projects to the ventral subparaventricular zone (vSPVZ), an adjacent region that expresses an in-phase pattern of SCN-vSPVZ neuronal activity. In contrast, in adult Nile grass rats (Arvicanthis niloticus), which are diurnal, an anti-phase pattern of SCN-vSPVZ neuronal activity is expressed. We hypothesized that these species differences result in part from a weak or absent RHT-to-vSPVZ projection in grass rats. Here, using a developmental comparative approach, we assessed species differences in behavior, hypothalamic activity, and RHT anatomy. We report that a robust retina-to-vSPVZ projection develops in Norway rats around the end of the second postnatal week when nocturnal wakefulness and the in-phase pattern of neuronal activity emerge. In grass rats, however, such a projection does not develop and the emergence of the anti-phase pattern during the second postnatal week is accompanied by increased diurnal wakefulness. When considered within the context of previously published reports on RHT projections in a variety of species, the current findings suggest that how and when the retina connects to the hypothalamus differentially shapes brain and behavior to produce animals that occupy opposing temporal niches.

  14. Microheliella maris (Microhelida ord. n.), an ultrastructurally highly distinctive new axopodial protist species and genus, and the unity of phylum Heliozoa.

    PubMed

    Yabuki, Akinori; Chao, Ema E; Ishida, Ken-Ichiro; Cavalier-Smith, Thomas

    2012-05-01

    A new heliozoan, Microheliella maris, has sufficiently distinctive ultrastructure to merit a new order, Microhelida. Its 18S and 28S rRNA genes were sequenced earlier under the informal name 'marine microheliozoan'; we here sequenced its Hsp90 gene. A three-gene tree suggests that it is distantly related to centrohelids and others in chromist subkingdom Hacrobia; but it is too divergent to be placed accurately by few genes. Unlike centrohelids, its central spherical centrosome has two concentric granular shells and a dense core devoid of a trilaminar central disc. Microtubules radiate from the centrosomal shells. Unlike centrohelids, axopodia have only three microtubules, fixed basally by dense plasma membrane anchors, and bear terminal and lateral haptosome-like extrusomes. As in the heliomonad Heliomorpha, the centrosome is embedded in a nuclear cavity, and centrosomal microtubules traverse the nucleus inside cytoplasmic channels. A novel filogranular network interconnects mitochondria, ER, and plasma membrane. The microbody is attached to the nucleus and mitochondrion, which has vermicular tubular cristae. We group Microhelida and Heliomonadida, purged of dissimilar flagellates, as a new tubulicristate class Endohelea within phylum Heliozoa. Previously misassigned GenBank 18S rDNA sequences reveal Microhelida as diverse and ancient. We discuss principles underlying the biogenesis and diversity of axopodial patterns.

  15. 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.

  16. Distinct Cell-Specific Expression of Homospermidine Synthase Involved in Pyrrolizidine Alkaloid Biosynthesis in Three Species of the Boraginales1[C][W][OA

    PubMed Central

    Niemüller, Daniel; Reimann, Andreas; Ober, Dietrich

    2012-01-01

    Homospermidine synthase (HSS) is the first specific enzyme in pyrrolizidine alkaloid (PA) biosynthesis, a pathway involved in the plant’s chemical defense. HSS has been shown to be recruited repeatedly by duplication of a gene involved in primary metabolism. Within the lineage of the Boraginales, only one gene duplication event gave rise to HSS. Here, we demonstrate that the tissue-specific expression of HSS in three boraginaceous species, Heliotropium indicum, Symphytum officinale, and Cynoglossum officinale, is unique with respect to plant organ, tissue, and cell type. Within H. indicum, HSS is expressed exclusively in nonspecialized cells of the lower epidermis of young leaves and shoots. In S. officinale, HSS expression has been detected in the cells of the root endodermis and in leaves directly underneath developing inflorescences. In young roots of C. officinale, HSS is detected only in cells of the endodermis, but in a later developmental stage, additionally in the pericycle. The individual expression patterns are compared with those within the Senecioneae lineage (Asteraceae), where HSS expression is reproducibly found in specific cells of the endodermis and the adjacent cortex parenchyma of the roots. The individual expression patterns within the Boraginales species are discussed as being a requirement for the successful recruitment of HSS after gene duplication. The diversity of HSS expression within this lineage adds a further facet to the already diverse patterns of expression that have been observed for HSS in other PA-producing plant lineages, making this PA-specific enzyme one of the most diverse expressed proteins described in the literature. PMID:22566491

  17. Comparative Analyses of Three Chlorella Species in Response to Light and Sugar Reveal Distinctive Lipid Accumulation Patterns in the Microalga C. sorokiniana

    PubMed Central

    Barnes, Austin; Noel, Eric A.; Betenbaugh, Michael J.; Oyler, George A.

    2014-01-01

    While photosynthetic microalgae, such as Chlorella, serve as feedstocks for nutritional oils and biofuels, heterotrophic cultivation can augment growth rates, support high cell densities, and increase triacylglycerol (TAG) lipid content. However, these species differ significantly in their photoautotrophic and heterotrophic characteristics. In this study, the phylogeny of thirty Chlorella strains was determined in order to inform bioprospecting efforts and detailed physiological assessment of three species. The growth kinetics and lipid biochemistry of C. protothecoides UTEX 411, C. vulgaris UTEX 265, and C. sorokiniana UTEX 1230 were quantified during photoautotrophy in Bold's basal medium (BBM) and heterotrophy in BBM supplemented with glucose (10 g L−1). Heterotrophic growth rates of UTEX 411, 265, and 1230 were found to be 1.5-, 3.7-, and 5-fold higher than their respective autotrophic rates. With a rapid nine-hour heterotrophic doubling time, Chlorella sorokiniana UTEX 1230 maximally accumulated 39% total lipids by dry weight during heterotrophy compared to 18% autotrophically. Furthermore, the discrete fatty acid composition of each strain was examined in order to elucidate lipid accumulation patterns under the two trophic conditions. In both modes of growth, UTEX 411 and 265 produced 18∶1 as the principal fatty acid while UTEX 1230 exhibited a 2.5-fold enrichment in 18∶2 relative to 18∶1. Although the total lipid content was highest in UTEX 411 during heterotrophy, UTEX 1230 demonstrated a two-fold increase in its heterotrophic TAG fraction at a rate of 28.9 mg L−1 d−1 to reach 22% of the biomass, corresponding to as much as 90% of its total lipids. Interestingly, UTEX 1230 growth was restricted during mixotrophy and its TAG production rate was suppressed to 18.2 mg L−1 d−1. This constraint on carbon flow raises intriguing questions about the impact of sugar and light on the metabolic regulation of microalgal lipid biosynthesis. PMID:24699196

  18. Molecular distinction between populations of Gonatocerus morrilli, egg parasitoids of the glassy-winged sharpshooter from Texas and California: Do cryptic species exist?

    PubMed Central

    de León, Jesse H.; Jones, Walker A.; Morgan, David J. W.

    2004-01-01

    Two molecular methods were utilized to distinguish geographic populations of Gonatocerus morrilli (Howard) from Texas and California and to test the possibility that this species could exist as a species-complex. Inter-Simple Sequence Repeat–Polymerase Chain Reactions (ISSR–PCR) were performed with a 5′-anchored ISSR primer. Twenty-five markers were generated with four populations (40 individuals) of G. morrilli. Twenty-three were polymorphic and the percentage of polymorphic loci was 92%. Most markers could be considered diagnostic since there was no band sharing between the Texas and California populations. Such differences typically are not found unless the populations are reproductively isolated. Exact tests for population differentiation indicated significant differences in marker frequencies among the populations. Comparison of other genetic differentiation estimates, which evaluate the degree of genetic subdivision, demonstrated excellent agreement between GST and θ values, 0.92 and 0.94, respectively, indicating that about 92 to 94% of the variance was distributed among populations. The average genetic divergence (D), as measured by genetic distance, was extremely high (Nei = 0.82 and Reynolds = 2.79). A dendrogram based on Nei's genetic distance separated the Texas and California populations into two clusters, respectively. Amplification of the Internal Transcribed Spacer-1 (ITS-1) region showed no size differences, whereas the ITS-2 DNA fragment varied in size between the two geographic populations. The ITS-2 fragment sizes were about 865 and 1099 base pairs for the California and Texas populations, respectively. The present study using the two molecular methods provides novel data critical to the glassy-winged sharpshooter/Pierce's disease biological control program in California. Abbreviations: ISSR–PCR Inter-Simple Sequence Repeat–Polymerase Chain Reaction ITS Internal Transcribed Spacer PMID:15861254

  19. Comparative analyses of three Chlorella species in response to light and sugar reveal distinctive lipid accumulation patterns in the Microalga C. sorokiniana.

    PubMed

    Rosenberg, Julian N; Kobayashi, Naoko; Barnes, Austin; Noel, Eric A; Betenbaugh, Michael J; Oyler, George A

    2014-01-01

    While photosynthetic microalgae, such as Chlorella, serve as feedstocks for nutritional oils and biofuels, heterotrophic cultivation can augment growth rates, support high cell densities, and increase triacylglycerol (TAG) lipid content. However, these species differ significantly in their photoautotrophic and heterotrophic characteristics. In this study, the phylogeny of thirty Chlorella strains was determined in order to inform bioprospecting efforts and detailed physiological assessment of three species. The growth kinetics and lipid biochemistry of C. protothecoides UTEX 411, C. vulgaris UTEX 265, and C. sorokiniana UTEX 1230 were quantified during photoautotrophy in Bold's basal medium (BBM) and heterotrophy in BBM supplemented with glucose (10 g L-1). Heterotrophic growth rates of UTEX 411, 265, and 1230 were found to be 1.5-, 3.7-, and 5-fold higher than their respective autotrophic rates. With a rapid nine-hour heterotrophic doubling time, Chlorella sorokiniana UTEX 1230 maximally accumulated 39% total lipids by dry weight during heterotrophy compared to 18% autotrophically. Furthermore, the discrete fatty acid composition of each strain was examined in order to elucidate lipid accumulation patterns under the two trophic conditions. In both modes of growth, UTEX 411 and 265 produced 18:1 as the principal fatty acid while UTEX 1230 exhibited a 2.5-fold enrichment in 18:2 relative to 18:1. Although the total lipid content was highest in UTEX 411 during heterotrophy, UTEX 1230 demonstrated a two-fold increase in its heterotrophic TAG fraction at a rate of 28.9 mg L(-1) d(-1) to reach 22% of the biomass, corresponding to as much as 90% of its total lipids. Interestingly, UTEX 1230 growth was restricted during mixotrophy and its TAG production rate was suppressed to 18.2 mg L-1 d-1. This constraint on carbon flow raises intriguing questions about the impact of sugar and light on the metabolic regulation of microalgal lipid biosynthesis.

  20. Transcription Activator Interactions with Multiple SWI/SNF Subunits

    PubMed Central

    Neely, Kristen E.; Hassan, Ahmed H.; Brown, Christine E.; Howe, LeAnn; Workman, Jerry L.

    2002-01-01

    We have previously shown that the yeast SWI/SNF complex stimulates in vitro transcription from chromatin templates in an ATP-dependent manner. SWI/SNF function in this regard requires the presence of an activator with which it can interact directly, linking activator recruitment of SWI/SNF to transcriptional stimulation. In this study, we determine the SWI/SNF subunits that mediate its interaction with activators. Using a photo-cross-linking label transfer strategy, we show that the Snf5, Swi1, and Swi2/Snf2 subunits are contacted by the yeast acidic activators, Gcn4 and Hap4, in the context of the intact native SWI/SNF complex. In addition, we show that the same three subunits can interact individually with acidic activation domains, indicating that each subunit contributes to binding activators. Furthermore, mutations that reduce the activation potential of these activators also diminish its interaction with each of these SWI/SNF subunits. Thus, three distinct subunits of the SWI/SNF complex contribute to its interactions with activation domains. PMID:11865042

  1. Evolutionary relatedness of mackerels of the genus Scomber based on complete mitochondrial genomes: strong support to the recognition of Atlantic Scomber colias and Pacific Scomber japonicus as distinct species.

    PubMed

    Catanese, Gaetano; Manchado, Manuel; Infante, Carlos

    2010-02-15

    Mackerels of the genus Scomber are commercially important species, but their taxonomic status is still controversial. Although previous phylogenetic data support the recognition of Atlantic Scomber colias and Pacific Scomber japonicus as separate species, it is only based on the analysis of partial mitochondrial and nuclear DNA sequences. In an attempt to shed light on this relevant issue, we have determined the complete mitochondrial DNA sequence of S. colias, S. japonicus, and Scomber australasicus. The total length of the mitogenomes was 16,568 bp for S. colias and 16,570 bp for both S. japonicus and S. australasicus. All mitogenomes had a gene content (13 protein-coding, 2 rRNAs, and 22 tRNAs) and organization similar to that observed in Scomber scombrus and most other vertebrates. The major noncoding region (control region) ranged between 865 and 866 bp in length and showed the typical conserved blocks. Phylogenetic analyses revealed a monophyletic origin of Scomber species with regard to other scombrid fish. The major finding of this study is that S. colias and S. japonicus were significantly grouped in distinct lineages within Scomber cluster, which phylogenetically constitutes evidence that they may be considered as separate species. Additionally, molecular data here presented provide a useful tool for evolutionary as well as population genetic studies.

  2. Distinct transcriptome profiles reveal gene expression patterns during fruit development and maturation in five main cultivated species of pear (Pyrus L.)

    PubMed Central

    Zhang, Ming-Yue; Xue, Cheng; Xu, Linlin; Sun, Honghe; Qin, Meng-Fan; Zhang, Shaoling; Wu, Jun

    2016-01-01

    The transcriptomes of five pear cultivars, ‘Hosui’ (P. pyrifolia), ‘Yali’ (P. bretschneideri), ‘Kuerlexiangli’ (P. sinkiangensis), ‘Nanguoli’ (P. ussuriensis), and ‘Starkrimson’ (P. communis) were sequenced at seven key fruit developmental stages, from fruit setting to maturation and fruit senescence after harvesting. In total, 33,136 genes that could be mapped by reads, were analyzed. Most gene expression cluster models showed a steadily decreasing trend. Gene expression patterns had obvious differences according to maturity type, that is, post-ripening cultivars were still vigorous at maturity, and showed a higher proportion of up-regulated genes; non post-ripening cultivars had a gradually decreasing tendency during fruit maturation. Meanwhile, differentially expressed genes related to fruit quality and development, such as stone cells, sugar, acid and hormones, were identified. Co-expression analysis revealed that several ethylene synthesis genes and polyphenoloxidase-related genes interacted with each other directly, and an indirect relationship was reflected between ethylene synthesis genes and ethylene response genes. In addition, the highly diverse SNPs represented the great differences between oriental and occidental pears. Understanding how RNA-seq based gene-expression patterns and differential gene expression contribute to fruit quality allows us to build models for gene-expression for fruit development of Pyrus species. PMID:27305967

  3. Morphological and ITS1, 5.8S, and partial ITS2 ribosomal DNA sequence distinctions between two species Platygyra (Cnidaria: Scleractinia) from Hong Kong [corrected].

    PubMed

    Lam, Katherine; Morton, Brian

    2003-01-01

    Two sympatric species of Platygyra have been identified from Hong Kong waters: i.e., P. sinensis and P. pini. The former has been further subdivided into 4 morphotypes based on colony growth form as follows: classic, encrusting, hillocky, and long-valley. Taxonomic confusion raised by overlapping morphological variations and frequent sympatric occurrences, however, has posed problems in relation to Platygyra ecology and population dynamics. This study attempted to differentiate Platygyra pini and morphotypes of P. sinensis by both morphological and ITS1, 5.8S, and partial ITS2 ribosomal DNA sequence analysis. Morphological data based on 9 skeletal characters were subjected to multivariate analysis. No clear groupings were obtained using a multidimensional scaling plot. Most parsimony analysis was conducted using either the rDNA data set including ITS1, 5.8S, and partial ITS2 or the ITS1 region only. Maximum parsimony (MP) and neighbor-joining (NJ) trees obtained from both data sets, clustered samples of P. sinensis and P. pini into 2 clades. The interspecific Kimura 2-parameter sequence divergence value (k2) obtained by the former rDNA data set was 14.275 +/- 0.507%, which is greater than the intraspecific values (1.239 +/- 1.147% for P. sinensis and 0.469 +/- 0.364% for P. pini), indicating that this marker of ITS1, 5.8S, and ITS2 contains substantially high levels of inherent diversity and is useful in resolving the problematic taxonomy of Platygyra.

  4. The gene sml0013 of Synechocystis species strain PCC 6803 encodes for a novel subunit of the NAD(P)H oxidoreductase or complex I that is ubiquitously distributed among Cyanobacteria.

    PubMed

    Schwarz, Doreen; Schubert, Hendrik; Georg, Jens; Hess, Wolfgang R; Hagemann, Martin

    2013-11-01

    The NAD(P)H oxidoreductase or complex I (NDH1) complex participates in many processes such as respiration, cyclic electron flow, and inorganic carbon concentration in the cyanobacterial cell. Despite immense progress in our understanding of the structure-function relation of the cyanobacterial NDH1 complex, the subunits catalyzing NAD(P)H docking and oxidation are still missing. The gene sml0013 of Synechocystis 6803 encodes for a small protein of unknown function for which homologs exist in all completely known cyanobacterial genomes. The protein exhibits weak similarities to the NDH-dependent flow6 (NDF6) protein, which was reported from Arabidopsis (Arabidopsis thaliana) chloroplasts as a NDH subunit. An sml0013 inactivation mutant of Synechocystis 6803 was generated and characterized. It showed only weak differences regarding growth and pigmentation in various culture conditions; most remarkably, it exhibited a glucose-sensitive phenotype in the light. The genome-wide expression pattern of the Δsml0013::Km mutant was almost identical to the wild type when grown under high CO2 conditions as well as after shifts to low CO2 conditions. However, measurements of the photosystem I redox kinetic in cells of the Δsml0013::Km mutant revealed differences, such as a decreased capability of cyclic electron flow as well as electron flow into respiration in comparison with the wild type. These results suggest that the Sml0013 protein (named NdhP) represents a novel subunit of the cyanobacterial NDH1 complex, mediating its coupling either to the respiratory or the photosynthetic electron flow.

  5. Species differences in Cl- affinity and in electrogenicity of SLC26A6-mediated oxalate/Cl- exchange correlate with the distinct human and mouse susceptibilities to nephrolithiasis.

    PubMed

    Clark, Jeffrey S; Vandorpe, David H; Chernova, Marina N; Heneghan, John F; Stewart, Andrew K; Alper, Seth L

    2008-03-01

    The mouse is refractory to lithogenic agents active in rats and humans, and so has been traditionally considered a poor experimental model for nephrolithiasis. However, recent studies have identified slc26a6 as an oxalate nephrolithiasis gene in the mouse. Here we extend our earlier demonstration of different anion selectivities of the orthologous mouse and human SLC26A6 polypeptides to investigate the correlation between species-specific differences in SLC26A6 oxalate/anion exchange properties as expressed in Xenopus oocytes and in reported nephrolithiasis susceptibility. We find that human SLC26A6 mediates minimal rates of Cl(-) exchange for Cl(-), sulphate or formate, but rates of oxalate/Cl(-) exchange roughly equivalent to those of mouse slc2a6. Both transporters exhibit highly cooperative dependence of oxalate efflux rate on extracellular [Cl(-)], but whereas the K(1/2) for extracellular [Cl(-)] is only 8 mM for mouse slc26a6, that for human SLC26A6 is 62 mM. This latter value approximates the reported mean luminal [Cl(-)] of postprandial human jejunal chyme, and reflects contributions from both transmembrane and C-terminal cytoplasmic domains of human SLC26A6. Human SLC26A6 variant V185M exhibits altered [Cl(-)] dependence and reduced rates of oxalate/Cl(-) exchange. Whereas mouse slc26a6 mediates bidirectional electrogenic oxalate/Cl(-) exchange, human SLC26A6-mediated oxalate transport appears to be electroneutral. We hypothesize that the low extracellular Cl(-) affinity and apparent electroneutrality of oxalate efflux characterizing human SLC26A6 may partially explain the high human susceptibility to nephrolithiasis relative to that of mouse. SLC26A6 sequence variant(s) are candidate risk modifiers for nephrolithiasis.

  6. High ploidy diversity and distinct patterns of cytotype distribution in a widespread species of Oxalis in the Greater Cape Floristic Region

    PubMed Central

    Krejčíková, Jana; Sudová, Radka; Lučanová, Magdalena; Trávníček, Pavel; Urfus, Tomáš; Vít, Petr; Weiss-Schneeweiss, Hanna; Kolano, Bożena; Oberlander, Kenneth; Dreyer, Leanne L.; Suda, Jan

    2013-01-01

    Background and Aims Genome duplication is widely acknowledged as a major force in the evolution of angiosperms, although the incidence of polyploidy in different floras may differ dramatically. The Greater Cape Floristic Region of southern Africa is one of the world's biodiversity hotspots and is considered depauperate in polyploids. To test this assumption, ploidy variation was assessed in a widespread member of the largest geophytic genus in the Cape flora: Oxalis obtusa. Methods DNA flow cytometry complemented by confirmatory chromosome counts was used to determine ploidy levels in 355 populations of O. obtusa (1014 individuals) across its entire distribution range. Ecological differentiation among cytotypes was tested by comparing sets of vegetation and climatic variables extracted for each locality. Key Results Three majority (2x, 4x, 6x) and three minority (3x, 5x, 8x) cytotypes were detected in situ, in addition to a heptaploid individual originating from a botanical garden. While single-cytotype populations predominate, 12 mixed-ploidy populations were also found. The overall pattern of ploidy level distribution is quite complex, but some ecological segregation was observed. Hexaploids are the most common cytotype and prevail in the Fynbos biome. In contrast, tetraploids dominate in the Succulent Karoo biome. Precipitation parameters were identified as the most important climatic variables associated with cytotype distribution. Conclusions Although it would be premature to make generalizations regarding the role of genome duplication in the genesis of hyperdiversity of the Cape flora, the substantial and unexpected ploidy diversity in Oxalis obtusa is unparalleled in comparison with any other cytologically known native Cape plant species. The results suggest that ploidy variation in the Greater Cape Floristic Region may be much greater than currently assumed, which, given the documented role of polyploidy in speciation, has direct implications for radiation

  7. Similar GABAA receptor subunit composition in somatic and axon initial segment synapses of hippocampal pyramidal cells

    PubMed Central

    Kerti-Szigeti, Katalin; Nusser, Zoltan

    2016-01-01

    Hippocampal pyramidal cells (PCs) express many GABAAR subunit types and receive GABAergic inputs from distinct interneurons. Previous experiments revealed input-specific differences in α1 and α2 subunit densities in perisomatic synapses, suggesting distinct IPSC decay kinetics. However, IPSC decays evoked by axo-axonic, parvalbumin- or cholecystokinin-expressing basket cells were found to be similar. Using replica immunogold labeling, here we show that all CA1 PC somatic and AIS synapses contain the α1, α2, β1, β2, β3 and γ2 subunits. In CA3 PCs, 90% of the perisomatic synapses are immunopositive for the α1 subunit and all synapses are positive for the remaining five subunits. Somatic synapses form unimodal distributions based on their immunoreactivity for these subunits. The α2 subunit densities in somatic synapses facing Cav2.1 (i.e. parvalbumin) or Cav2.2 (cholecystokinin) positive presynaptic active zones are comparable. We conclude that perisomatic synapses made by three distinct interneuron types have similar GABAA receptor subunit content. DOI: http://dx.doi.org/10.7554/eLife.18426.001 PMID:27537197

  8. Zinc induces distinct changes in the metabolism of reactive oxygen and nitrogen species (ROS and RNS) in the roots of two Brassica species with different sensitivity to zinc stress

    PubMed Central

    Feigl, Gábor; Lehotai, Nóra; Molnár, Árpád; Ördög, Attila; Rodríguez-Ruiz, Marta; Palma, José M.; Corpas, Francisco J.; Erdei, László; Kolbert, Zsuzsanna

    2015-01-01

    Background and Aims Zinc (Zn) is an essential micronutrient naturally present in soils, but anthropogenic activities can lead to accumulation in the environment and resulting damage to plants. Heavy metals such as Zn can induce oxidative stress and the generation of reactive oxygen and nitrogen species (ROS and RNS), which can reduce growth and yield in crop plants. This study assesses the interplay of these two families of molecules in order to evaluate the responses in roots of two Brassica species under high concentrations of Zn. Methods Nine-day-old hydroponically grown Brassica juncea (Indian mustard) and B. napus (oilseed rape) seedlings were treated with ZnSO4 (0, 50, 150 and 300 µm) for 7 d. Stress intensity was assessed through analyses of cell wall damage and cell viability. Biochemical and cellular techniques were used to measure key components of the metabolism of ROS and RNS including lipid peroxidation, enzymatic antioxidants, protein nitration and content of superoxide radical (O2·−), nitric oxide (NO) and peroxynitrite (ONOO−). Key Results Analysis of morphological root damage and alterations of microelement homeostasis indicate that B. juncea is more tolerant to Zn stress than B. napus. ROS and RNS parameters suggest that the oxidative components are predominant compared with the nitrosative components in the root system of both species. Conclusions The results indicate a clear relationship between ROS and RNS metabolism as a mechanism of response against stress caused by an excess of Zn. The oxidative stress components seem to be more dominant than the elements of the nitrosative stress in the root system of these two Brassica species. PMID:25538112

  9. Molecular and morphological evidence supports the species status of the Mahachai fighter Betta sp. Mahachai and reveals new species of Betta from Thailand.

    PubMed

    Sriwattanarothai, N; Steinke, D; Ruenwongsa, P; Hanner, R; Panijpan, B

    2010-08-01

    Two regions of mitochondrial (mt) DNA, cytochrome c oxidase subunit 1 (COI) and 16S rRNA, were sequenced in nine species of Betta from Thailand and Indonesia. Most species showed little intraspecific COI variation (adjusted mean = 0.48%) including the putative species Betta sp. Mahachai, but one species (Betta smaragdina) included three lineages showing much greater divergence (7.03-13.48%) that probably represent overlooked species. These findings were confirmed by maximum likelihood analysis and Bayesian inference, which revealed well-supported corresponding monophyletic clades. Based on these results and morphological differences, the putative species Betta sp. Mahachai from central Thailand is a species distinct from other members of the B. splendens group and represents a new and hitherto undescribed species. Furthermore, this study also demonstrated the probable existence of two overlooked Betta species found in the Khorat plateau basin, illustrating the utility of mitochondrial genetic markers in the revelation of overlooked diversity.

  10. Phylogenetic Analysis of Cryptosporidium Parasites Based on the Small-Subunit rRNA Gene Locus

    PubMed Central

    Xiao, Lihua; Escalante, Lillian; Yang, Chunfu; Sulaiman, Irshad; Escalante, Anannias A.; Montali, Richard J.; Fayer, Ronald; Lal, Altaf A.

    1999-01-01

    Biological data support the hypothesis that there are multiple species in the genus Cryptosporidium, but a recent analysis of the available genetic data suggested that there is insufficient evidence for species differentiation. In order to resolve the controversy in the taxonomy of this parasite genus, we characterized the small-subunit rRNA genes of Cryptosporidium parvum, Cryptosporidium baileyi, Cryptosporidium muris, and Cryptosporidium serpentis and performed a phylogenetic analysis of the genus Cryptosporidium. Our study revealed that the genus Cryptosporidium contains the phylogenetically distinct species C. parvum, C. muris, C. baileyi, and C. serpentis, which is consistent with the biological characteristics and host specificity data. The Cryptosporidium species formed two clades, with C. parvum and C. baileyi belonging to one clade and C. muris and C. serpentis belonging to the other clade. Within C. parvum, human genotype isolates and guinea pig isolates (known as Cryptosporidium wrairi) each differed from bovine genotype isolates by the nucleotide sequence in four regions. A C. muris isolate from cattle was also different from parasites isolated from a rock hyrax and a Bactrian camel. Minor differences were also detected between C. serpentis isolates from snakes and lizards. Based on the genetic information, a species- and strain-specific PCR-restriction fragment length polymorphism diagnostic tool was developed. PMID:10103253

  11. RNA polymerase beta subunit (rpoB) gene and the 16S-23S rRNA intergenic transcribed spacer region (ITS) as complementary molecular markers in addition to the 16S rRNA gene for phylogenetic analysis and identification of the species of the family Mycoplasmataceae.

    PubMed

    Volokhov, Dmitriy V; Simonyan, Vahan; Davidson, Maureen K; Chizhikov, Vladimir E

    2012-01-01

    Conventional classification of the species in the family Mycoplasmataceae is mainly based on phenotypic criteria, which are complicated, can be difficult to measure, and have the potential to be hampered by phenotypic deviations among the isolates. The number of biochemical reactions suitable for phenotypic characterization of the Mycoplasmataceae is also very limited and therefore the strategy for the final identification of the Mycoplasmataceae species is based on comparative serological results. However, serological testing of the Mycoplasmataceae species requires a performance panel of hyperimmune sera which contains anti-serum to each known species of the family, a high level of technical expertise, and can only be properly performed by mycoplasma-reference laboratories. In addition, the existence of uncultivated and fastidious Mycoplasmataceae species/isolates in clinical materials significantly complicates, or even makes impossible, the application of conventional bacteriological tests. The analysis of available genetic markers is an additional approach for the primary identification and phylogenetic classification of cultivable species and uncultivable or fastidious organisms in standard microbiological laboratories. The partial nucleotide sequences of the RNA polymerase β-subunit gene (rpoB) and the 16S-23S rRNA intergenic transcribed spacer (ITS) were determined for all known type strains and the available non-type strains of the Mycoplasmataceae species. In addition to the available 16S rRNA gene data, the ITS and rpoB sequences were used to infer phylogenetic relationships among these species and to enable identification of the Mycoplasmataceae isolates to the species level. The comparison of the ITS and rpoB phylogenetic trees with the 16S rRNA reference phylogenetic tree revealed a similar clustering patterns for the Mycoplasmataceae species, with minor discrepancies for a few species that demonstrated higher divergence of their ITS and rpoB in

  12. Characterization and mutagenesis of the gene encoding the A49 subunit of RNA polymerase A in Saccharomyces cerevisiae.

    PubMed Central

    Liljelund, P; Mariotte, S; Buhler, J M; Sentenac, A

    1992-01-01

    The gene encoding the 49-kDa subunit of RNA polymerase A in Saccharomyces cerevisiae has been identified by formation of a hybrid enzyme between the S. cerevisiae A49 subunit and Saccharomyces douglasii subunits based on a polymorphism existing between the subunits of RNA polymerase A in these two species. The sequence of the gene reveals a basic protein with an unusually high lysine content, which may account for the affinity for DNA shown by the subunit. No appreciable homology with any polymerase subunits, enzymes, or transcription factors is found. Complete deletion of the single-copy RPA49 gene leads to viable but slowly growing colonies. Insertion of the HIS3 gene halfway into the RPA49 coding region results in synthesis of a truncated A49 subunit that is incorporated into the polymerase. The truncated and wild-type subunits compete equally for assembly in the heterozygous diploid, although the wild type is phenotypically dominant. Images PMID:1409638

  13. Determination of kainate receptor subunit ratios in mouse brain using novel chimeric protein standards.

    PubMed

    Watanabe-Iida, Izumi; Konno, Kohtarou; Akashi, Kaori; Abe, Manabu; Natsume, Rie; Watanabe, Masahiko; Sakimura, Kenji

    2016-01-01

    Kainate-type glutamate receptors (KARs) are tetrameric channels assembled from GluK1-5. GluK1-3 are low-affinity subunits that form homomeric and heteromeric KARs, while GluK4 and GluK5 are high-affinity subunits that require co-assembly with GluK1-3 for functional expression. Although the subunit composition is thought to be highly heterogeneous in the brain, the distribution of KAR subunits at the protein level and their relative abundance in given regions of the brain remain largely unknown. In the present study, we titrated C-terminal antibodies to each KAR subunit using chimeric GluA2-GluK fusion proteins, and measured their relative abundance in the P2 and post-synaptic density (PSD) fractions of the adult mouse hippocampus and cerebellum. Analytical western blots showed that GluK2 and GluK3 were the major KAR subunits, with additional expression of GluK5 in the hippocampus and cerebellum. In both regions, GluK4 was very low and GluK1 was below the detection threshold. The relative amount of low-affinity subunits (GluK2 plus GluK3) was several times higher than that of high-affinity subunits (GluK4 plus GluK5) in both regions. Of note, the highest ratio of high-affinity subunits to low-affinity subunits was found in the hippocampal PSD fraction (0.32), suggesting that heteromeric receptors consisting of high- and low-affinity subunits highly accumulate at hippocampal synapses. In comparison, this ratio was decreased to 0.15 in the cerebellar PSD fraction, suggesting that KARs consisting of low-affinity subunits are more prevalent in the cerebellum. Therefore, low-affinity KAR subunits are predominant in the brain, with distinct subunit combinations between the hippocampus and cerebellum. Kainate receptors, an unconventional member of the iGluR receptor family, have a tetrameric structure assembled from low-affinity (GluK1-3) and high-affinity (GluK4 and GluK5) subunits. We used a simple but novel procedure to measure the relative abundance of both low- and

  14. Distinct serum proteome profiles associated with collagen-induced arthritis and complete Freund's adjuvant-induced inflammation in CD38⁻/⁻ mice: The discriminative power of protein species or proteoforms.

    PubMed

    Rosal-Vela, Antonio; García-Rodríguez, Sonia; Postigo, Jorge; Iglesias, Marcos; Longobardo, Victoria; Lario, Antonio; Merino, Jesús; Merino, Ramón; Zubiaur, Mercedes; Sancho, Jaime

    2015-10-01

    Collagen-type-II-induced arthritis (CIA) is an autoimmune disease, which involves a complex host systemic response including inflammatory and autoimmune reactions. CIA is milder in CD38(-/-) than in wild-type (WT) mice. ProteoMiner-equalized serum samples were subjected to 2D-DiGE and MS-MALDI-TOF/TOF analyses to identify proteins that changed in their relative abundances in CD38(-/-) versus WT mice either with arthritis (CIA(+) ), with no arthritis (CIA(-) ), or with inflammation (complete Freund's adjuvant (CFA)-treated mice). Multivariate analyses revealed that a multiprotein signature (n = 28) was able to discriminate CIA(+) from CIA(-) mice, and WT from CD38(-/-) mice within each condition. Likewise, a distinct multiprotein signature (n = 16) was identified which differentiated CIA(+) CD38(-/-) mice from CIA(+) WT mice, and lastly, a third multiprotein signature (n = 18) indicated that CD38(-/-) and WT mice could be segregated in response to CFA treatment. Further analyses showed that the discriminative power to distinguish these groups was reached at protein species level and not at the protein level. Hence, the need to identify and quantify proteins at protein species level to better correlate proteome changes with disease processes. It is crucial for plasma proteomics at the low-abundance protein species level to apply the ProteoMiner enrichment. All MS data have been deposited in the ProteomeXchange with identifiers PXD001788, PXD001799 and PXD002071 (http://proteomecentral.proteomexchange.org/dataset/PXD001788, http://proteomecentral.proteomexchange.org/dataset/PXD001799 and http://proteomecentral.proteomexchange.org/dataset/PXD002071).

  15. Na(+), K(+)-ATPase β1 subunit associates with α1 subunit modulating a "higher-NKA-in-hyposmotic media" response in gills of euryhaline milkfish, Chanos chanos.

    PubMed

    Hu, Yau-Chung; Chu, Keng-Fu; Yang, Wen-Kai; Lee, Tsung-Han

    2017-03-10

    The euryhaline milkfish (Chanos chanos) is a popular aquaculture species that can be cultured in fresh water, brackish water, or seawater in Southeast Asia. In gills of the milkfish, Na(+), K(+)-ATPase (i.e., NKA; sodium pump) responds to salinity challenges including changes in mRNA abundance, protein amount, and activity. The functional pump is composed of a heterodimeric protein complex composed of α- and β-subunits. Among the NKA genes, α1-β1 isozyme comprises the major form of NKA subunits in mammalian osmoregulatory organs; however, most studies on fish gills have focused on the α1 subunit and did not verify the α1-β1 isozyme. Based on the sequenced milkfish transcriptome, an NKA β1 subunit gene was identified that had the highest amino acid homology to β233, a NKA β1 subunit paralog originally identified in the eel. Despite this high level of homology to β233, phylogenetic analysis and the fact that only a single NKA β1 subunit gene exists in the milkfish suggest that the milkfish gene should be referred to as the NKA β1 subunit gene. The results of accurate domain prediction of the β1 subunit, co-localization of α1 and β1 subunits in epithelial ionocytes, and co-immunoprecipitation of α1 and β1 subunits, indicated the formation of a α1-β1 complex in milkfish gills. Moreover, when transferred to hyposmotic media (fresh water) from seawater, parallel increases in branchial mRNA and protein expression of NKA α1 and β1 subunits suggested their roles in hypo-osmoregulation of euryhaline milkfish. This study molecularly characterized the NKA β1 subunit and provided the first evidence for an NKA α1-β1 association in gill ionocytes of euryhaline teleosts.

  16. A CAPS test allowing a rapid distinction of Penicillium expansum among fungal species collected on grape berries, inferred from the sequence and secondary structure of the mitochondrial SSU-rRNA.

    PubMed

    Garcia, Carole; La Guerche, Stéphane; Mouhamadou, Bello; Férandon, Cyril; Labarère, Jacques; Blancard, Dominique; Darriet, Philippe; Barroso, Gérard

    2006-10-01

    Penicillium expansum is a fungal species highly damageable for the postharvest conservation of numerous fruits. In vineyards, this fungus is sometimes isolated from grape berries where its presence may lead to the production of geosmin, a powerful earthy odorant, which can impair grapes and wines aromas. However, the discrimination of P. expansum from related fungi is difficult because it is based on ambiguous phenotypic characters and/or expensive and time-consuming molecular tests. In this context, the complete sequences and secondary structures of Penicillium expansum and Penicillium thomii mitochondrial SSU-rRNAs were achieved and compared with those of two other phylogenetically related Ascomycota: Penicillium chrysogenum and Emericella nidulans. The comparison has shown a high conservation in size and sequence of the core and of the variable domains (more than 80% of nt identity) of the four SSU-rRNAs, arguing for a close phylogenetic relationship between these four species of the Trichocomaceae family. Large (from 10 to 18 nt) inserted/deleted (indel) sequences were evidenced in the V1, V5 and V6 variable domains. The size variations (10 to 18 nt) of the V1 indel sequence allowed the distinction of the four species; the V5 indel (15 nt) was specifically recovered in E. nidulans; the V6 indel (16 nt), shared by the three Penicillium species, was lacking in E. nidulans. A couple of conserved primers (UI/R2) were defined to generate a PCR product containing the V1 to V5 variable domains. This product contained the two regions of the four SSU-rRNAs showing the highest rates of nt substitutions, namely the V2 variable domain and, surprisingly, a helix (H17) of the core. The H17 sequence was shown to specifically possess in P. expansum a recognition site for the ClaI restriction endonuclease. Hence, this enzyme generates a digestion pattern of the PCR product with two bands (350 bp+500 bp), specific to P. expansum and easily separable by agarose gel

  17. beta-subunits of Snf1 kinase are required for kinase function and substrate definition.

    PubMed

    Schmidt, M C; McCartney, R R

    2000-09-15

    The Snf1 kinase and its mammalian homolog, the AMP-activated protein kinase, are heterotrimeric enzymes composed of a catalytic alpha-subunit, a regulatory gamma-subunit and a beta-subunit that mediates heterotrimer formation. Saccharomyces cerevisiae encodes three beta-subunit genes, SIP1, SIP2 and GAL83. Earlier studies suggested that these subunits may not be required for Snf1 kinase function. We show here that complete and precise deletion of all three beta-subunit genes inactivates the Snf1 kinase. The sip1Delta sip2Delta gal83Delta strain is unable to derepress invertase, grows poorly on alternative carbon sources and fails to direct the phosphorylation of the Mig1 and Sip4 proteins in vivo. The SIP1 sip2Delta gal83Delta strain manifests a subset of Snf phenotypes (Raf(+), Gly(-)) observed in the snf1Delta 10 strain (Raf(-), Gly(-)), suggesting that individual beta-subunits direct the Snf1 kinase to a subset of its targets in vivo. Indeed, deletion of individual beta-subunit genes causes distinct differences in the induction and phosphorylation of Sip4, strongly suggesting that the beta-subunits play an important role in substrate definition.

  18. The mongoose acetylcholine receptor alpha-subunit: analysis of glycosylation and alpha-bungarotoxin binding.

    PubMed

    Asher, O; Jensen, B S; Lupu-Meiri, M; Oron, Y; Fuchs, S

    1998-04-17

    The mongoose AChR alpha-subunit has been cloned and shown to be highly homologous to other AChR alpha-subunits, with only six differences in amino acid residues at positions that are conserved in animal species that bind alpha-bungarotoxin (alpha-BTX). Four of these six substitutions cluster in the ligand binding site, and one of them, Asn-187, forms a consensus N-glycosylation site. The mongoose glycosylated alpha-subunit has a higher apparent molecular mass than that of the rat glycosylated alpha-subunit, probably resulting from the additional glycosylation at Asn-187 of the mongoose subunit. The in vitro translated mongoose alpha-subunit, in a glycosylated or non-glycosylated form, does not bind alpha-BTX, indicating that lack of alpha-BTX binding can be achieved also in the absence of glycosylation.

  19. The ribosomal subunit assembly line

    PubMed Central

    Dlakić, Mensur

    2005-01-01

    Recent proteomic studies in Saccharomyces cerevisiae have identified nearly 200 proteins, other than the structural ribosomal proteins, that participate in the assembly of ribosomal subunits and their transport from the nucleus. In a separate line of research, proteomic studies of mature plant ribosomes have revealed considerable variability in the protein composition of individual ribosomes. PMID:16207363

  20. Subunits of the Snf1 kinase heterotrimer show interdependence for association and activity.

    PubMed

    Elbing, Karin; Rubenstein, Eric M; McCartney, Rhonda R; Schmidt, Martin C

    2006-09-08

    The Snf1 kinase and its mammalian orthologue, the AMP-activated protein kinase (AMPK), function as heterotrimers composed of a catalytic alpha-subunit and two non-catalytic subunits, beta and gamma. The beta-subunit is thought to hold the complex together and control subcellular localization whereas the gamma-subunit plays a regulatory role by binding to and blocking the function of an auto-inhibitory domain (AID) present in the alpha-subunit. In addition, catalytic activity requires phosphorylation by a distinct upstream kinase. In yeast, any one of three Snf1-activating kinases, Sak1, Tos3, or Elm1, can fulfill this role. We have previously shown that Sak1 is the only Snf1-activating kinase that forms a stable complex with Snf1. Here we show that the formation of the Sak1.Snf1 complex requires the beta- and gamma-subunits in vivo. However, formation of the Sak1.Snf1 complex is not necessary for glucose-regulated phosphorylation of the Snf1 activation loop. Snf1 kinase purified from cells lacking the beta-subunits do not contain any gamma-subunit, indicating that the Snf1 kinase does not form a stable alphagamma dimer in vivo. In vitro kinase assays using purified full-length and truncated Snf1 proteins demonstrate that the kinase domain, which lacks the AID, is significantly more active than the full-length Snf1 protein. Addition of purified beta- and gamma-subunits could stimulate the kinase activity of the full-length alpha-subunit but only when all three subunits were present, suggesting an interdependence of all three subunits for assembly of a functional complex.

  1. NOX Activation by Subunit Interaction and Underlying Mechanisms in Disease

    PubMed Central

    Rastogi, Radhika; Geng, Xiaokun; Li, Fengwu; Ding, Yuchuan

    2017-01-01

    Nicotinamide adenine dinucleotide phosphate (NAPDH) oxidase (NOX) is an enzyme complex with the sole function of producing superoxide anion and reactive oxygen species (ROS) at the expense of NADPH. Vital to the immune system as well as cellular signaling, NOX is also involved in the pathologies of a wide variety of disease states. Particularly, it is an integral player in many neurological diseases, including stroke, TBI, and neurodegenerative diseases. Pathologically, NOX produces an excessive amount of ROS that exceed the body’s antioxidant ability to neutralize them, leading to oxidative stress and aberrant signaling. This prevalence makes it an attractive therapeutic target and as such, NOX inhibitors have been studied and developed to counter NOX’s deleterious effects. However, recent studies of NOX have created a better understanding of the NOX complex. Comprised of independent cytosolic subunits, p47-phox, p67-phox, p40-phox and Rac, and membrane subunits, gp91-phox and p22-phox, the NOX complex requires a unique activation process through subunit interaction. Of these subunits, p47-phox plays the most important role in activation, binding and translocating the cytosolic subunits to the membrane and anchoring to p22-phox to organize the complex for NOX activation and function. Moreover, these interactions, particularly that between p47-phox and p22-phox, are dependent on phosphorylation initiated by upstream processes involving protein kinase C (PKC). This review will look at these interactions between subunits and with PKC. It will focus on the interaction involving p47-phox with p22-phox, key in bringing the cytosolic subunits to the membrane. Furthermore, the implication of these interactions as a target for NOX inhibitors such as apocynin will be discussed as a potential avenue for further investigation, in order to develop more specific NOX inhibitors based on the inhibition of NOX assembly and activation. PMID:28119569

  2. Balancing acts of two HEAT subunits of condensin I support dynamic assembly of chromosome axes.

    PubMed

    Kinoshita, Kazuhisa; Kobayashi, Tetsuya J; Hirano, Tatsuya

    2015-04-06

    Condensin I is a five-subunit protein complex that plays a central role in mitotic chromosome assembly and segregation in eukaryotes. To dissect its mechanism of action, we reconstituted wild-type and mutant complexes from recombinant subunits and tested their abilities to assemble chromosomes in Xenopus egg cell-free extracts depleted of endogenous condensins. We find that ATP binding and hydrolysis by SMC subunits have distinct contributions to the action of condensin I and that continuous ATP hydrolysis is required for structural maintenance of chromosomes. Mutant complexes lacking either one of two HEAT subunits produce abnormal chromosomes with highly characteristic defects and have contrasting structural effects on chromosome axes preassembled with the wild-type complex. We propose that balancing acts of the two HEAT subunits support dynamic assembly of chromosome axes under the control of the SMC ATPase cycle, thereby governing construction of rod-shaped chromosomes in eukaryotic cells.

  3. Structure of the archaeal Cascade subunit Csa5: relating the small subunits of CRISPR effector complexes.

    PubMed

    Reeks, Judith; Graham, Shirley; Anderson, Linzi; Liu, Huanting; White, Malcolm F; Naismith, James H

    2013-05-01

    The Cascade complex for CRISPR-mediated antiviral immunity uses CRISPR RNA (crRNA) to target invading DNA species from mobile elements such as viruses, leading to their destruction. The core of the Cascade effector complex consists of the Cas5 and Cas7 subunits, which are widely conserved in prokaryotes. Cas7 binds crRNA and forms the helical backbone of Cascade. Many archaea encode a version of the Cascade complex (denoted Type I-A) that includes a Csa5 (or small) subunit, which interacts weakly with the core proteins. Here, we report the crystal structure of the Csa5 protein from Sulfolobus solfataricus. Csa5 comprises a conserved α-helical domain with a small insertion consisting of a weakly conserved β-strand domain. In the crystal, the Csa5 monomers have multimerized into infinite helical threads. At each interface is a strictly conserved intersubunit salt bridge, deletion of which disrupts multimerization. Structural analysis indicates a shared evolutionary history among the small subunits of the CRISPR effector complexes. The same α-helical domain is found in the C-terminal domain of Cse2 (from Type I-E Cascade), while the N-terminal domain of Cse2 is found in Cmr5 of the CMR (Type III-B) effector complex. As Cmr5 shares no match with Csa5, two possibilities present themselves: selective domain loss from an ancestral Cse2 to create two new subfamilies or domain fusion of two separate families to create a new Cse2 family. A definitive answer awaits structural studies of further small subunits from other CRISPR effector complexes.

  4. Rotating proton pumping ATPases: subunit/subunit interactions and thermodynamics.

    PubMed

    Nakanishi-Matsui, Mayumi; Sekiya, Mizuki; Futai, Masamitsu

    2013-03-01

    In this article, we discuss single molecule observation of rotational catalysis by E. coli ATP synthase (F-ATPase) using small gold beads. Studies involving a low viscous drag probe showed the stochastic properties of the enzyme in alternating catalytically active and inhibited states. The importance of subunit interaction between the rotor and the stator, and thermodynamics of the catalysis are also discussed. "Single Molecule Enzymology" is a new trend for understanding enzyme mechanisms in biochemistry and physiology.

  5. The Evolution of the Four Subunits of Voltage-Gated Calcium Channels: Ancient Roots, Increasing Complexity, and Multiple Losses

    PubMed Central

    Moran, Yehu; Zakon, Harold H.

    2014-01-01

    The alpha subunits of voltage-gated calcium channels (Cavs) are large transmembrane proteins responsible for crucial physiological processes in excitable cells. They are assisted by three auxiliary subunits that can modulate their electrical behavior. Little is known about the evolution and roles of the various subunits of Cavs in nonbilaterian animals and in nonanimal lineages. For this reason, we mapped the phyletic distribution of the four channel subunits and reconstructed their phylogeny. Although alpha subunits have deep evolutionary roots as ancient as the split between plants and opistokonths, beta subunits appeared in the last common ancestor of animals and their close-relatives choanoflagellates, gamma subunits are a bilaterian novelty and alpha2/delta subunits appeared in the lineage of Placozoa, Cnidaria, and Bilateria. We note that gene losses were extremely common in the evolution of Cavs, with noticeable losses in multiple clades of subfamilies and also of whole Cav families. As in vertebrates, but not protostomes, Cav channel genes duplicated in Cnidaria. We characterized by in situ hybridization the tissue distribution of alpha subunits in the sea anemone Nematostella vectensis, a nonbilaterian animal possessing all three Cav subfamilies common to Bilateria. We find that some of the alpha subunit subtypes exhibit distinct spatiotemporal expression patterns. Further, all six sea anemone alpha subunit subtypes are conserved in stony corals, which separated from anemones 500 MA. This unexpected conservation together with the expression patterns strongly supports the notion that these subtypes carry unique functional roles. PMID:25146647

  6. Distinct Genetic Lineages of Bactrocera caudata (Insecta: Tephritidae) Revealed by COI and 16S DNA Sequences

    PubMed Central

    Lim, Phaik-Eem; Tan, Ji; Suana, I. Wayan; Eamsobhana, Praphathip; Yong, Hoi Sen

    2012-01-01

    The fruit fly Bactrocera caudata is a pest species of economic importance in Asia. Its larvae feed on the flowers of Cucurbitaceae such as Cucurbita moschata. To-date it is distinguished from related species based on morphological characters. Specimens of B. caudata from Peninsular Malaysia and Indonesia (Bali and Lombok) were analysed using the partial DNA sequences of cytochrome c oxidase subunit I (COI) and 16S rRNA genes. Both gene sequences revealed that B. caudata from Peninsular Malaysia was distinctly different from B. caudata of Bali and Lombok, without common haplotype between them. Phylogenetic analysis revealed two distinct clades, indicating distinct genetic lineage. The uncorrected ‘p’ distance for COI sequences between B. caudata of Malaysia-Thailand-China and B. caudata of Bali-Lombok was 5.65%, for 16S sequences from 2.76 to 2.99%, and for combined COI and 16S sequences 4.45 to 4.46%. The ‘p’ values are distinctly different from intraspecific ‘p’ distance (0–0.23%). Both the B. caudata lineages are distinctly separated from related species in the subgenus Zeugodacus – B. ascita, B. scutellata, B. ishigakiensis, B. diaphora, B. tau, B. cucurbitae, and B. depressa. Molecular phylogenetic analysis indicates that the B. caudata lineages are closely related to B. ascita sp. B, and form a clade with B. scutellata, B. ishigakiensis, B. diaphora and B. ascita sp. A. This study provides additional baseline for the phylogenetic relationships of Bactrocera fruit flies of the subgenus Zeugodacus. Both the COI and 16S genes could be useful markers for the molecular differentiation and phylogenetic analysis of tephritid fruit flies. PMID:22615962

  7. Distinct genetic lineages of Bactrocera caudata (Insecta: Tephritidae) revealed by COI and 16S DNA sequences.

    PubMed

    Lim, Phaik-Eem; Tan, Ji; Suana, I Wayan; Eamsobhana, Praphathip; Yong, Hoi Sen

    2012-01-01

    The fruit fly Bactrocera caudata is a pest species of economic importance in Asia. Its larvae feed on the flowers of Cucurbitaceae such as Cucurbita moschata. To-date it is distinguished from related species based on morphological characters. Specimens of B. caudata from Peninsular Malaysia and Indonesia (Bali and Lombok) were analysed using the partial DNA sequences of cytochrome c oxidase subunit I (COI) and 16S rRNA genes. Both gene sequences revealed that B. caudata from Peninsular Malaysia was distinctly different from B. caudata of Bali and Lombok, without common haplotype between them. Phylogenetic analysis revealed two distinct clades, indicating distinct genetic lineage. The uncorrected 'p' distance for COI sequences between B. caudata of Malaysia-Thailand-China and B. caudata of Bali-Lombok was 5.65%, for 16S sequences from 2.76 to 2.99%, and for combined COI and 16S sequences 4.45 to 4.46%. The 'p' values are distinctly different from intraspecific 'p' distance (0-0.23%). Both the B. caudata lineages are distinctly separated from related species in the subgenus Zeugodacus - B. ascita, B. scutellata, B. ishigakiensis, B. diaphora, B. tau, B. cucurbitae, and B. depressa. Molecular phylogenetic analysis indicates that the B. caudata lineages are closely related to B. ascita sp. B, and form a clade with B. scutellata, B. ishigakiensis, B. diaphora and B. ascita sp. A. This study provides additional baseline for the phylogenetic relationships of Bactrocera fruit flies of the subgenus Zeugodacus. Both the COI and 16S genes could be useful markers for the molecular differentiation and phylogenetic analysis of tephritid fruit flies.

  8. Enrichment of GABAA Receptor α-Subunits on the Axonal Initial Segment Shows Regional Differences

    PubMed Central

    Gao, Yudong; Heldt, Scott A.

    2016-01-01

    Although it is generally recognized that certain α-subunits of γ-aminobutyric acid type A receptors (GABAARs) form enriched clusters on the axonal initial segment (AIS), the degree to which these clusters vary in different brain areas is not well known. In the current study, we quantified the density, size, and enrichment ratio of fluorescently labeled α1-, α2-, or α3-subunits aggregates co-localized with the AIS-marker ankyrin G and compared them to aggregates in non-AIS locations among different brain areas including hippocampal subfields, basal lateral amygdala (BLA), prefrontal cortex (PFC), and sensory cortex (CTX). We found regional differences in the enrichment of GABAAR α-subunits on the AIS. Significant enrichment was identified in the CA3 of hippocampus for α1-subunits, in the CA1, CA3, and BLA for α2-subunits, and in the BLA for α3-subunits. Using α-subunit knock-out (KO) mice, we found that BLA enrichment of α2- and α3-subunits were physiologically independent of each other, as the enrichment of one subunit was unaffected by the genomic deletion of the other. To further investigate the unique pattern of α-subunit enrichment in the BLA, we examined the association of α2- and α3-subunits with the presynaptic vesicular GABA transporter (vGAT) and the anchoring protein gephyrin (Geph). As expected, both α2- and α3-subunits on the AIS within the BLA received prominent GABAergic innervation from vGAT-positive terminals. Further, we found that the association of α2- and α3-subunits with Geph was weaker in AIS versus non-AIS locations, suggesting that Geph might be playing a lesser role in the enrichment of α2- and α3-subunits on the AIS. Overall, these observations suggest that GABAARs on the AIS differ in subunit composition across brain regions. As with somatodendritic GABAARs, the distinctive expression pattern of AIS-located GABAAR α-subunits in the BLA, and other brain areas, likely contribute to unique forms of GABAergic inhibitory

  9. Emergence of ion channel modal gating from independent subunit kinetics

    PubMed Central

    Bicknell, Brendan A.

    2016-01-01

    Many ion channels exhibit a slow stochastic switching between distinct modes of gating activity. This feature of channel behavior has pronounced implications for the dynamics of ionic currents and the signaling pathways that they regulate. A canonical example is the inositol 1,4,5-trisphosphate receptor (IP3R) channel, whose regulation of intracellular Ca2+ concentration is essential for numerous cellular processes. However, the underlying biophysical mechanisms that give rise to modal gating in this and most other channels remain unknown. Although ion channels are composed of protein subunits, previous mathematical models of modal gating are coarse grained at the level of whole-channel states, limiting further dialogue between theory and experiment. Here we propose an origin for modal gating, by modeling the kinetics of ligand binding and conformational change in the IP3R at the subunit level. We find good agreement with experimental data over a wide range of ligand concentrations, accounting for equilibrium channel properties, transient responses to changing ligand conditions, and modal gating statistics. We show how this can be understood within a simple analytical framework and confirm our results with stochastic simulations. The model assumes that channel subunits are independent, demonstrating that cooperative binding or concerted conformational changes are not required for modal gating. Moreover, the model embodies a generally applicable principle: If a timescale separation exists in the kinetics of individual subunits, then modal gating can arise as an emergent property of channel behavior. PMID:27551100

  10. Accessory subunits are integral for assembly and function of human mitochondrial complex I.

    PubMed

    Stroud, David A; Surgenor, Elliot E; Formosa, Luke E; Reljic, Boris; Frazier, Ann E; Dibley, Marris G; Osellame, Laura D; Stait, Tegan; Beilharz, Traude H; Thorburn, David R; Salim, Agus; Ryan, Michael T

    2016-10-06

    Complex I (NADH:ubiquinone oxidoreductase) is the first enzyme of the mitochondrial respiratory chain and is composed of 45 subunits in humans, making it one of the largest known multi-subunit membrane protein complexes. Complex I exists in supercomplex forms with respiratory chain complexes III and IV, which are together required for the generation of a transmembrane proton gradient used for the synthesis of ATP. Complex I is also a major source of damaging reactive oxygen species and its dysfunction is associated with mitochondrial disease, Parkinson's disease and ageing. Bacterial and human complex I share 14 core subunits that are essential for enzymatic function; however, the role and necessity of the remaining 31 human accessory subunits is unclear. The incorporation of accessory subunits into the complex increases the cellular energetic cost and has necessitated the involvement of numerous assembly factors for complex I biogenesis. Here we use gene editing to generate human knockout cell lines for each accessory subunit. We show that 25 subunits are strictly required for assembly of a functional complex and 1 subunit is essential for cell viability. Quantitative proteomic analysis of cell lines revealed that loss of each subunit affects the stability of other subunits residing in the same structural module. Analysis of proteomic changes after the loss of specific modules revealed that ATP5SL and DMAC1 are required for assembly of the distal portion of the complex I membrane arm. Our results demonstrate the broad importance of accessory subunits in the structure and function of human complex I. Coupling gene-editing technology with proteomics represents a powerful tool for dissecting large multi-subunit complexes and enables the study of complex dysfunction at a cellular level.

  11. Specific Subunits of Heterotrimeric G Proteins Play Important Roles during Nodulation in Soybean1[W][OA

    PubMed Central

    Choudhury, Swarup Roy; Pandey, Sona

    2013-01-01

    Heterotrimeric G proteins comprising Gα, Gβ, and Gγ subunits regulate many fundamental growth and development processes in all eukaryotes. Plants possess a relatively limited number of G-protein components compared with mammalian systems, and their detailed functional characterization has been performed mostly in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa). However, the presence of single Gα and Gβ proteins in both these species has significantly undermined the complexity and specificity of response regulation in plant G-protein signaling. There is ample pharmacological evidence for the role of G proteins in regulation of legume-specific processes such as nodulation, but the lack of genetic data from a leguminous species has restricted its direct assessment. Our recent identification and characterization of an elaborate G-protein family in soybean (Glycine max) and the availability of appropriate molecular-genetic resources have allowed us to directly evaluate the role of G-protein subunits during nodulation. We demonstrate that all G-protein genes are expressed in nodules and exhibit significant changes in their expression in response to Bradyrhizobium japonicum infection and in representative supernodulating and nonnodulating soybean mutants. RNA interference suppression and overexpression of specific G-protein components results in lower and higher nodule numbers, respectively, validating their roles as positive regulators of nodule formation. Our data further show preferential usage of distinct G-protein subunits in the presence of an additional signal during nodulation. Interestingly, the Gα proteins directly interact with the soybean nodulation factor receptors NFR1α and NFR1β, suggesting that the plant G proteins may couple with receptors other than the canonical heptahelical receptors common in metazoans to modulate signaling. PMID:23569109

  12. Description of a new species of Niphargus (Crustacea: Amphipoda: Niphargidae): the first record of a lake ecomorph in the Carpathian Mountains.

    PubMed

    Petković, Matija; Delić, Teo; Lučić, Luka; Fišer, Cene

    2015-10-01

    We describe and phylogenetically characterize a new species Niphargus mirocensis from Mt. Miroč, eastern Serbia. This species shows distinct morphology typical for a lake ecomorph of niphargid amphipod, i.e. large and stout body, elongated appendages and raptorial gnathopods and presents the first record of this ecomorph in Carpathian Mountains. Phylogenetic analyses based on Cytochrome Oxidase Subunit 1 gene (COI), Histone (H3) and 28S rRNA (28S) suggests that species is nested within a clade of lake ecomorphs spread in Italy and Central Dinaric Region. The new finding is geographic extension of clade's range, the species of which are generally narrow endemics.

  13. 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

    2016-12-08

    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 (E2) levels remained constant during the experiment, possibly due to a differential regulation of E2 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.

  14. Translation initiation factor 3 regulates switching between different modes of ribosomal subunit joining.

    PubMed

    MacDougall, Daniel D; Gonzalez, Ruben L

    2015-05-08

    Ribosomal subunit joining is a key checkpoint in the bacterial translation initiation pathway during which initiation factors (IFs) regulate association of the 30S initiation complex (IC) with the 50S subunit to control formation of a 70S IC that can enter into the elongation stage of protein synthesis. The GTP-bound form of IF2 accelerates subunit joining, whereas IF3 antagonizes subunit joining and plays a prominent role in maintaining translation initiation fidelity. The molecular mechanisms through which IF2 and IF3 collaborate to regulate the efficiency of 70S IC formation, including how they affect the dynamics of subunit joining, remain poorly defined. Here, we use single-molecule fluorescence resonance energy transfer to monitor the interactions between IF2 and the GTPase-associated center (GAC) of the 50S subunit during real-time subunit joining reactions in the absence and presence of IF3. In the presence of IF3, IF2-mediated subunit joining becomes reversible, and subunit joining events cluster into two distinct classes corresponding to formation of shorter- and longer-lifetime 70S ICs. Inclusion of IF3 within the 30S IC was also found to alter the conformation of IF2 relative to the GAC, suggesting that IF3's regulatory effects may stem in part from allosteric modulation of IF2-GAC interactions. The results are consistent with a model in which IF3 can exert control over the efficiency of subunit joining by modulating the conformation of the 30S IC, which in turn influences the formation of stabilizing intersubunit contacts and thus the reaction's degree of reversibility.

  15. Phylogenetic relationships within Taenia taeniaeformis variants and other taeniid cestodes inferred from the nucleotide sequence of the cytochrome c oxidase subunit I gene.

    PubMed

    Okamoto, M; Bessho, Y; Kamiya, M; Kurosawa, T; Horii, T

    1995-01-01

    Nucleotide sequence variations in a region of the mitochondrial cytochrome c oxidase subunit I (COI) gene (391 bp) were examined within seven species of the genus Taenia and two species of the genus Echinococcus, including ten isolates of T. taeniaeformis and six isolates of E. multilocularis. More than a 12% rate of nucleotide differences between taeniid species was found, allowing the species to be distinguished. In E. multilocularis, no sequence variation was observed among isolates, regardless of the host (gray red-backed vole, tundra vole, pig, Norway rat) or area (Japan, Alaska) from which each metacestode had been isolated. In contrast, six distinct sequences were detected among the ten T. taeniaeformis isolates examined. The level of nucleotide variation in the COI gene within T. taeniaeformis isolates except for one isolate from the gray red-backed vole (TtACR), which has been proposed as a distinct strain or a different species, was about 0.3%-4.1%, whereas the COI gene sequence for TtACR differed from those of the other isolates, with levels being 9.0%-9.5%. Phylogenetic trees were then inferred from these sequence data using two different algorithms.

  16. Human alcohol dehydrogenase: structural differences between the beta and gamma subunits suggest parallel duplications in isoenzyme evolution and predominant expression of separate gene descendants in livers of different mammals.

    PubMed Central

    Bühler, R; Hempel, J; Kaiser, R; von Wartburg, J P; Vallee, B L; Jörnvall, H

    1984-01-01

    Human alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) occurs in multiple forms, which exhibit distinct electrophoretic mobilities and enzymatic properties. The homogeneous isoenzymes beta 1 beta 1 and gamma 1 gamma 1 were isolated from livers of Caucasians with "typical" ADH phenotype by double ternary complex affinity chromatography and ion exchange chromatography. The differences between the beta 1 and gamma 1 subunits were determined by structural analysis of all tryptic peptides from the carboxymethylated proteins. The human beta 1 and gamma 1 chains differ at 21 of the 373 positions (5.6%). Ten tryptic peptides account for the differences. All residue substitutions are compatible with one-base mutations and result in largely unaltered properties, but five lead to charge differences. Sixteen substitutions are at positions corresponding to the catalytic domain of the well-known horse enzyme; five correspond to the coenzyme-binding domain. Substitutions adjacent to important regions may correlate with differences in coenzyme binding, substrate specificities, and active-site relationships. The residue replacements between the beta 1 and gamma 1 subunits of human ADH are not identical to the known substitutions between ethanol-active (E) and steroid-active (S) subunits of horse ADH. Thus, the duplication leading to human beta 1 and gamma 1 subunits is separate and different from that leading to equine E and S subunits. Both duplications are likely to have occurred after the ancestral separation of human and equine ADH. Of the 21 residues that are different between beta 1/gamma 1, 13 in gamma 1 but only 6 in beta 1 are identical to those of the horse E chain. This suggests a closer relationship between gamma 1 and E, although beta 1 in man and E in the horse are the subunits recovered in highest yield from liver ADH preparations. Consequently, in these two mammalian species, relative activities of genes for an isoenzyme family appear to be

  17. Glycine activated ion channel subunits encoded by ctenophore glutamate receptor genes

    PubMed Central

    Alberstein, Robert; Grey, Richard; Zimmet, Austin; Simmons, David K.; Mayer, Mark L.

    2015-01-01

    Recent genome projects for ctenophores have revealed the presence of numerous ionotropic glutamate receptors (iGluRs) in Mnemiopsis leidyi and Pleurobrachia bachei, among our earliest metazoan ancestors. Sequence alignments and phylogenetic analysis show that these form a distinct clade from the well-characterized AMPA, kainate, and NMDA iGluR subtypes found in vertebrates. Although annotated as glutamate and kainate receptors, crystal structures of the ML032222a and PbiGluR3 ligand-binding domains (LBDs) reveal endogenous glycine in the binding pocket, whereas ligand-binding assays show that glycine binds with nanomolar affinity; biochemical assays and structural analysis establish that glutamate is occluded from the binding cavity. Further analysis reveals ctenophore-specific features, such as an interdomain Arg-Glu salt bridge, present only in subunits that bind glycine, but also a conserved disulfide in loop 1 of the LBD that is found in all vertebrate NMDA but not AMPA or kainate receptors. We hypothesize that ctenophore iGluRs are related to an early ancestor of NMDA receptors, suggesting a common evolutionary path for ctenophores and bilaterian species, and suggest that future work should consider both glycine and glutamate as candidate neurotransmitters in ctenophore species. PMID:26460032

  18. Glycine activated ion channel subunits encoded by ctenophore glutamate receptor genes

    DOE PAGES

    Alberstein, Robert; Grey, Richard; Zimmet, Austin; ...

    2015-10-12

    Recent genome projects for ctenophores have revealed the presence of numerous ionotropic glutamate receptors (iGluRs) in Mnemiopsis leidyi and Pleurobrachia bachei, among our earliest metazoan ancestors. Sequence alignments and phylogenetic analysis show that these form a distinct clade from the well-characterized AMPA, kainate, and NMDA iGluR subtypes found in vertebrates. Although annotated as glutamate and kainate receptors, crystal structures of the ML032222a and PbiGluR3 ligand-binding domains (LBDs) reveal endogenous glycine in the binding pocket, whereas ligand-binding assays show that glycine binds with nanomolar affinity; biochemical assays and structural analysis establish that glutamate is occluded from the binding cavity. Further analysismore » reveals ctenophore-specific features, such as an interdomain Arg-Glu salt bridge, present only in subunits that bind glycine, but also a conserved disulfide in loop 1 of the LBD that is found in all vertebrate NMDA but not AMPA or kainate receptors. In this paper, we hypothesize that ctenophore iGluRs are related to an early ancestor of NMDA receptors, suggesting a common evolutionary path for ctenophores and bilaterian species, and finally suggest that future work should consider both glycine and glutamate as candidate neurotransmitters in ctenophore species.« less

  19. Glycine activated ion channel subunits encoded by ctenophore glutamate receptor genes

    SciTech Connect

    Alberstein, Robert; Grey, Richard; Zimmet, Austin; Simmons, David K.; Mayer, Mark L.

    2015-10-12

    Recent genome projects for ctenophores have revealed the presence of numerous ionotropic glutamate receptors (iGluRs) in Mnemiopsis leidyi and Pleurobrachia bachei, among our earliest metazoan ancestors. Sequence alignments and phylogenetic analysis show that these form a distinct clade from the well-characterized AMPA, kainate, and NMDA iGluR subtypes found in vertebrates. Although annotated as glutamate and kainate receptors, crystal structures of the ML032222a and PbiGluR3 ligand-binding domains (LBDs) reveal endogenous glycine in the binding pocket, whereas ligand-binding assays show that glycine binds with nanomolar affinity; biochemical assays and structural analysis establish that glutamate is occluded from the binding cavity. Further analysis reveals ctenophore-specific features, such as an interdomain Arg-Glu salt bridge, present only in subunits that bind glycine, but also a conserved disulfide in loop 1 of the LBD that is found in all vertebrate NMDA but not AMPA or kainate receptors. In this paper, we hypothesize that ctenophore iGluRs are related to an early ancestor of NMDA receptors, suggesting a common evolutionary path for ctenophores and bilaterian species, and finally suggest that future work should consider both glycine and glutamate as candidate neurotransmitters in ctenophore species.

  20. Glycine activated ion channel subunits encoded by ctenophore glutamate receptor genes.

    PubMed

    Alberstein, Robert; Grey, Richard; Zimmet, Austin; Simmons, David K; Mayer, Mark L

    2015-11-03

    Recent genome projects for ctenophores have revealed the presence of numerous ionotropic glutamate receptors (iGluRs) in Mnemiopsis leidyi and Pleurobrachia bachei, among our earliest metazoan ancestors. Sequence alignments and phylogenetic analysis show that these form a distinct clade from the well-characterized AMPA, kainate, and NMDA iGluR subtypes found in vertebrates. Although annotated as glutamate and kainate receptors, crystal structures of the ML032222a and PbiGluR3 ligand-binding domains (LBDs) reveal endogenous glycine in the binding pocket, whereas ligand-binding assays show that glycine binds with nanomolar affinity; biochemical assays and structural analysis establish that glutamate is occluded from the binding cavity. Further analysis reveals ctenophore-specific features, such as an interdomain Arg-Glu salt bridge, present only in subunits that bind glycine, but also a conserved disulfide in loop 1 of the LBD that is found in all vertebrate NMDA but not AMPA or kainate receptors. We hypothesize that ctenophore iGluRs are related to an early ancestor of NMDA receptors, suggesting a common evolutionary path for ctenophores and bilaterian species, and suggest that future work should consider both glycine and glutamate as candidate neurotransmitters in ctenophore species.

  1. The Subunit Structure of Benzylsuccinate Synthase†

    PubMed Central

    Li, Lei; Patterson, Dustin P.; Fox, Christel C.; Lin, Brian; Coschigano, Peter W.; Marsh, E. Neil G.

    2010-01-01

    Benzylsuccinate synthase is a member of the glycyl radical family of enzymes. It catalyzes the addition of toluene to fumarate to form benzylsuccinate as the first step in the anaerobic pathway of toluene fermentation. The enzyme comprises three subunits α, β and γ that in Thauera Aromatica T1 strain are encoded by the tutD, tutG and tutF genes respectively. The large α-subunit contains the essential glycine and cysteine residues that are conserved in all glycyl radical enzymes. However, the function of the small β- and γ-subunits has remained unclear. We have over-expressed all three subunits of benzylsuccinate synthase in E. coli, both individually and in combination. Co-expression of the γ-subunit (but not the β-subunit) is essential for efficient expression of the α-subunit. The benzylsuccinate synthase complex lacking the glycyl radical could be purified as an α2β2γ2 hexamer by nickel-affinity chromatography through a ‘His6’ affinity tag engineered onto the C-terminus of the α-subunit. Unexpectedly, BSS was found to contain two iron-sulfur clusters, one associated with the β-subunit and the other with the γ-subunit that appear to be necessary for the structural integrity of the complex. The spectroscopic properties of these clusters suggest that they are most likely [4Fe-4S] clusters. Removal of iron with chelating agents results in dissociation of the complex; similarly a mutant γ-subunit lacking the [4Fe-4S] cluster is unable to stabilize the α-subunit when the proteins are co-expressed. PMID:19159265

  2. Evolution, expression differentiation and interaction specificity of heterotrimeric G-protein subunit gene family in the mesohexaploid Brassica rapa.

    PubMed

    Arya, Gulab C; Kumar, Roshan; Bisht, Naveen C

    2014-01-01

    Heterotrimeric G-proteins, comprising of Gα, Gβ, and Gγ subunits, are important signal transducers which regulate many aspects of fundamental growth and developmental processes in all eukaryotes. Initial studies in model plants Arabidopsis and rice suggest that the repertoire of plant G-protein is much simpler than that observed in metazoans. In order to assess the consequence of whole genome triplication events within Brassicaceae family, we investigated the multiplicity of G-protein subunit genes in mesohexaploid Brassica rapa, a globally important vegetable and oilseed crop. We identified one Gα (BraA.Gα1), three Gβ (BraA.Gβ1, BraA.Gβ2, and BraA.Gβ3), and five Gγ (BraA.Gγ1, BraA.Gγ2, BraA.Gγ3, BraA.Gγ4, and BraA.Gγ5) genes from B. rapa, with a possibility of 15 Gαβγ heterotrimer combinations. Our analysis suggested that the process of genome triplication coupled with gene-loss (gene-fractionation) phenomenon have shaped the quantitative and sequence diversity of G-protein subunit genes in the extant B. rapa genome. Detailed expression analysis using qRT-PCR assays revealed that the G-protein genes have retained ubiquitous but distinct expression profiles across plant development. The expression of multiple G-protein genes was differentially regulated during seed-maturation and germination stages, and in response to various phytohormone treatments and stress conditions. Yeast-based interaction analysis showed that G-protein subunits interacted in most of the possible combinations, with some degree of subunit-specific interaction specificity, to control the functional selectivity of G-protein heterotrimer in different cell and tissue-types or in response to different environmental conditions. Taken together, this research identifies a highly diverse G-protein signaling network known to date from B. rapa, and provides a clue about the possible complexity of G-protein signaling networks present across globally important Brassica species.

  3. Evolution, Expression Differentiation and Interaction Specificity of Heterotrimeric G-Protein Subunit Gene Family in the Mesohexaploid Brassica rapa

    PubMed Central

    Arya, Gulab C.; Kumar, Roshan; Bisht, Naveen C.

    2014-01-01

    Heterotrimeric G-proteins, comprising of Gα, Gβ, and Gγ subunits, are important signal transducers which regulate many aspects of fundamental growth and developmental processes in all eukaryotes. Initial studies in model plants Arabidopsis and rice suggest that the repertoire of plant G-protein is much simpler than that observed in metazoans. In order to assess the consequence of whole genome triplication events within Brassicaceae family, we investigated the multiplicity of G-protein subunit genes in mesohexaploid Brassica rapa, a globally important vegetable and oilseed crop. We identified one Gα (BraA.Gα1), three Gβ (BraA.Gβ1, BraA.Gβ2, and BraA.Gβ3), and five Gγ (BraA.Gγ1, BraA.Gγ2, BraA.Gγ3, BraA.Gγ4, and BraA.Gγ5) genes from B. rapa, with a possibility of 15 Gαβγ heterotrimer combinations. Our analysis suggested that the process of genome triplication coupled with gene-loss (gene-fractionation) phenomenon have shaped the quantitative and sequence diversity of G-protein subunit genes in the extant B. rapa genome. Detailed expression analysis using qRT-PCR assays revealed that the G-protein genes have retained ubiquitous but distinct expression profiles across plant development. The expression of multiple G-protein genes was differentially regulated during seed-maturation and germination stages, and in response to various phytohormone treatments and stress conditions. Yeast-based interaction analysis showed that G-protein subunits interacted in most of the possible combinations, with some degree of subunit-specific interaction specificity, to control the functional selectivity of G-protein heterotrimer in different cell and tissue-types or in response to different environmental conditions. Taken together, this research identifies a highly diverse G-protein signaling network known to date from B. rapa, and provides a clue about the possible complexity of G-protein signaling networks present across globally important Brassica species. PMID

  4. Identification of binding sites on the regulatory A subunit of protein phosphatase 2A for the catalytic C subunit and for tumor antigens of simian virus 40 and polyomavirus.

    PubMed Central

    Ruediger, R; Roeckel, D; Fait, J; Bergqvist, A; Magnusson, G; Walter, G

    1992-01-01

    Protein phosphatase 2A is composed of three subunits: the catalytic subunit C and two regulatory subunits, A and B. The A subunit consists of 15 nonidentical repeats and has a rodlike shape. It is associated with the B and C subunits as well as with the simian virus 40 small T, polyomavirus small T, and polyomavirus medium T tumor antigens. We determined the binding sites on subunit A for subunit C and tumor antigens by site-directed mutagenesis of A. Twenty-four N- and C-terminal truncations and internal deletions of A were assayed by coimmunoprecipitation for their ability to bind C and tumor antigens. It was found that C binds to repeats 11 to 15 at the C terminus of A, whereas T antigens bind to overlapping but distinct regions of the N terminus. Simian virus 40 small T binds to repeats 3 to 6, and polyomavirus small T and medium T bind to repeats 2 to 8. The data suggest cooperativity between C and T antigens in binding to A. This is most apparent for medium T antigen, which can only bind to those A subunit molecules that provide the entire binding region for the C subunit. We infer from our results that B also binds to N-terminal repeats. A model of the small T/medium T/B-A-C complexes is presented. Images PMID:1328865

  5. The centromere-kinetochore complex: a repeat subunit model

    PubMed Central

    1991-01-01

    The three-dimensional structure of the kinetochore and the DNA/protein composition of the centromere-kinetochore region was investigated using two novel techniques, caffeine-induced detachment of unreplicated kinetochores and stretching of kinetochores by hypotonic and/or shear forces generated in a cytocentrifuge. Kinetochore detachment was confirmed by EM and immunostaining with CREST autoantibodies. Electron microscopic analyses of serial sections demonstrated that detached kinetochores represented fragments derived from whole kinetochores. This was especially evident for the seven large kinetochores in the male Indian muntjac that gave rise to 80-100 fragments upon detachment. The kinetochore fragments, all of which interacted with spindle microtubules and progressed through the entire repertoire of mitotic movements, provide evidence for a subunit organization within the kinetochore. Further support for a repeat subunit model was obtained by stretching or uncoiling the metaphase centromere-kinetochore complex by hypotonic treatments. When immunostained with CREST autoantibodies and subsequently processed for in situ hybridization using synthetic centromere probes, stretched kinetochores displayed a linear array of fluorescent subunits arranged in a repetitive pattern along a centromeric DNA fiber. In addition to CREST antigens, each repetitive subunit was found to bind tubulin and contain cytoplasmic dynein, a microtubule motor localized in the zone of the corona. Collectively, the data suggest that the kinetochore, a plate-like structure seen by EM on many eukaryotic chromosomes is formed by the folding of a linear DNA fiber consisting of tandemly repeated subunits interspersed by DNA linkers. This model, unlike any previously proposed, can account for the structural and evolutional diversity of the kinetochore and its relationship to the centromere of eukaryotic chromosomes of many species. PMID:1828250

  6. Differential Localization of G Protein βγ Subunits

    PubMed Central

    2015-01-01

    G protein βγ subunits play essential roles in regulating cellular signaling cascades, yet little is known about their distribution in tissues or their subcellular localization. While previous studies have suggested specific isoforms may exhibit a wide range of distributions throughout the central nervous system, a thorough investigation of the expression patterns of both Gβ and Gγ isoforms within subcellular fractions has not been conducted. To address this, we applied a targeted proteomics approach known as multiple-reaction monitoring to analyze localization patterns of Gβ and Gγ isoforms in pre- and postsynaptic fractions isolated from cortex, cerebellum, hippocampus, and striatum. Particular Gβ and Gγ subunits were found to exhibit distinct regional and subcellular localization patterns throughout the brain. Significant differences in subcellular localization between pre- and postsynaptic fractions were observed within the striatum for most Gβ and Gγ isoforms, while others exhibited completely unique expression patterns in all four brain regions examined. Such differences are a prerequisite for understanding roles of individual subunits in regulating specific signaling pathways throughout the central nervous system. PMID:24568373

  7. Role of the Rubisco Small Subunit

    SciTech Connect

    Spreitzer, Robert Joseph

    2016-11-05

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of CO2 fixation in photosynthesis. However, it is a slow enzyme, and O2 competes with CO2 at the active site. Oxygenation initiates the photorespiratory pathway, which also results in the loss of CO2. If carboxylation could be increased or oxygenation decreased, an increase in net CO2 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, 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 CO2/O2 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

  8. Validity of a blue stripe snapper, Lutjanus octolineatus (Cuvier 1828) and a related species, L. bengalensis (Bloch 1790) with a new species (Pisces; Lutjanidae) from the Arabian Sea.

    PubMed

    Iwatsuki, Yukio; Al-Mamry, Juma M; Heemstra, Phillip C

    2016-04-07

    Lutjanus octolineatus (Cuvier 1828), previously considered a junior synonym of Lutjanus bengalensis (Bloch 1790), is shown to be a valid species and lectotypes are designated. Both species are redescribed. The two species have overlapping distributions in the Indian Ocean, but are clearly separable by different dorsal-fin spine counts, blue-striped pattern on the body and the presence or absence of a subocular extension of cheek scales. Lutjanus octovittata (Valenciennes 1830), formerly assigned to synonymy of L. bengalensis, is considered a junior synonym of L. octolineatus based on examination of the holotype. Lutjanus sapphirolineatus n. sp., a species formerly misidentified as L. bengalensis, is described based on 10 specimens from Oman and Somalia. The new species differs from the three species above by a combination of different characters. Analysis of the mitochondrial cytochrome c oxidase subunit 1 (CO1, 603 bp) genetic marker, also strongly supports the validity of each species of the blue-striped snapper complex as distinct.

  9. Sequence analysis of dolphin ferritin H and L subunits and possible iron-dependent translational control of dolphin ferritin gene

    PubMed Central

    Takaesu, Azusa; Watanabe, Kiyotaka; Takai, Shinji; Sasaki, Yukako; Orino, Koichi

    2008-01-01

    Background Iron-storage protein, ferritin plays a central role in iron metabolism. Ferritin has dual function to store iron and segregate iron for protection of iron-catalyzed reactive oxygen species. Tissue ferritin is composed of two kinds of subunits (H: heavy chain or heart-type subunit; L: light chain or liver-type subunit). Ferritin gene expression is controlled at translational level in iron-dependent manner or at transcriptional level in iron-independent manner. However, sequencing analysis of marine mammalian ferritin subunits has not yet been performed fully. The purpose of this study is to reveal cDNA-derived amino acid sequences of cetacean ferritin H and L subunits, and demonstrate the possibility of expression of these subunits, especially H subunit, by iron. Methods Sequence analyses of cetacean ferritin H and L subunits were performed by direct sequencing of polymerase chain reaction (PCR) fragments from cDNAs generated via reverse transcription-PCR of leukocyte total RNA prepared from blood samples of six different dolphin species (Pseudorca crassidens, Lagenorhynchus obliquidens, Grampus griseus, Globicephala macrorhynchus, Tursiops truncatus, and Delphinapterus leucas). The putative iron-responsive element sequence in the 5'-untranslated region of the six different dolphin species was revealed by direct sequencing of PCR fragments obtained using leukocyte genomic DNA. Results Dolphin H and L subunits consist of 182 and 174 amino acids, respectively, and amino acid sequence identities of ferritin subunits among these dolphins are highly conserved (H: 99–100%, (99→98) ; L: 98–100%). The conserved 28 bp IRE sequence was located -144 bp upstream from the initiation codon in the six different dolphin species. Conclusion These results indicate that six different dolphin species have conserved ferritin sequences, and suggest that these genes are iron-dependently expressed. PMID:18954429

  10. Mechanism of β4 Subunit Modulation of BK Channels

    PubMed Central

    Wang, Bin; Rothberg, Brad S.; Brenner, Robert

    2006-01-01

    Large-conductance (BK-type) Ca2+-activated potassium channels are activated by membrane depolarization and cytoplasmic Ca2+. BK channels are expressed in a broad variety of cells and have a corresponding diversity in properties. Underlying much of the functional diversity is a family of four tissue-specific accessory subunits (β1–β4). Biophysical characterization has shown that the β4 subunit confers properties of the so-called “type II” BK channel isotypes seen in brain. These properties include slow gating kinetics and resistance to iberiotoxin and charybdotoxin blockade. In addition, the β4 subunit reduces the apparent voltage sensitivity of channel activation and has complex effects on apparent Ca2+ sensitivity. Specifically, channel activity at low Ca2+ is inhibited, while at high Ca2+, activity is enhanced. The goal of this study is to understand the mechanism underlying β4 subunit action in the context of a dual allosteric model for BK channel gating. We observed that β4's most profound effect is a decrease in Po (at least 11-fold) in the absence of calcium binding and voltage sensor activation. However, β4 promotes channel opening by increasing voltage dependence of Po-V relations at negative membrane potentials. In the context of the dual allosteric model for BK channels, we find these properties are explained by distinct and opposing actions of β4 on BK channels. β4 reduces channel opening by decreasing the intrinsic gating equilibrium (L0), and decreasing the allosteric coupling between calcium binding and voltage sensor activation (E). However, β4 has a compensatory effect on channel opening following depolarization by shifting open channel voltage sensor activation (Vho) to more negative membrane potentials. The consequence is that β4 causes a net positive shift of the G-V relationship (relative to α subunit alone) at low calcium. At higher calcium, the contribution by Vho and an increase in allosteric coupling to Ca2+ binding (C

  11. 28 CFR 51.6 - Political subunits.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Political subunits. 51.6 Section 51.6 Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) PROCEDURES FOR THE ADMINISTRATION OF SECTION 5 OF THE VOTING RIGHTS ACT OF 1965, AS AMENDED General Provisions § 51.6 Political subunits. All...

  12. Sarcocysts of an unidentified species of Sarcocystis in the sea otter (Enhydra lutris)

    USGS Publications Warehouse

    Dubey, J.P.; Lindsay, D.S.; Rosenthal, B.M.; Thomas, N.J.

    2003-01-01

    The number of Sarcocystis species that infect sea otters (Enhydra lutris) is unknown. Sea otter tissues were recently shown to harbor sarcocysts of S. neurona and of unidentified species of Sarcocystis. Whereas sarcocysts of S. neurona have walls 1a??3 I?m thick with type 9 villar protrusions, ultrastructure of a distinct thin-walled sarcocyst (0.5a??0.7 I?m thick) lacking villar protrusions, but instead exhibiting minute type 1 undulations on the sarcocyst wall, is described in this report. Parasites characterized from a sea otter infection were inferred to be related to, but distinct from, other species belonging to Sarcocystis, based on sequencing and phylogenetic analysis of a portion of the beta subunit of the plastid-encoded RNA polymerase gene.

  13. Distinct Cytoplasmic and Nuclear Fractions of Drosophila Heterochromatin Protein 1: Their Phosphorylation Levels and Associations with Origin Recognition Complex Proteins

    PubMed Central

    Huang, Da Wei; Fanti, Laura; Pak, Daniel T.S.; Botchan, Michael R.; Pimpinelli, Sergio; Kellum, Rebecca

    1998-01-01

    The distinct structural properties of heterochromatin accommodate a diverse group of vital chromosome functions, yet we have only rudimentary molecular details of its structure. A powerful tool in the analyses of its structure in Drosophila has been a group of mutations that reverse the repressive effect of heterochromatin on the expression of a gene placed next to it ectopically. Several genes from this group are known to encode proteins enriched in heterochromatin. The best characterized of these is the heterochromatin-associated protein, HP1. HP1 has no known DNA-binding activity, hence its incorporation into heterochromatin is likely to be dependent upon other proteins. To examine HP1 interacting proteins, we isolated three distinct oligomeric species of HP1 from the cytoplasm of early Drosophila embryos and analyzed their compositions. The two larger oligomers share two properties with the fraction of HP1 that is most tightly associated with the chromatin of interphase nuclei: an underphosphorylated HP1 isoform profile and an association with subunits of the origin recognition complex (ORC). We also found that HP1 localization into heterochromatin is disrupted in mutants for the ORC2 subunit. These findings support a role for the ORC-containing oligomers in localizing HP1 into Drosophila heterochromatin that is strikingly similar to the role of ORC in recruiting the Sir1 protein to silencing nucleation sites in Saccharomyces cerevisiae. PMID:9679132

  14. Phylogeny of Fomitopsis pinicola: a species complex.

    PubMed

    Haight, John-Erich; Laursen, Gary A; Glaeser, Jessie A; Taylor, D Lee

    2016-09-01

    Fungal species with a broad distribution may exhibit considerable genetic variation over their geographic ranges. Variation may develop among populations based on geographic isolation, lack of migration, and genetic drift, though this genetic variation may not always be evident when examining phenotypic characters. Fomitopsis pinicola is an abundant saprotrophic fungus found on decaying logs throughout temperate regions of the Northern Hemisphere. Phylogenetic studies have addressed the relationship of F. pinicola to other wood-rotting fungi, but pan-continental variation within F. pinicola has not been addressed using molecular data. While forms found growing on hardwood and softwood hosts exhibit variation in habit and appearance, it is unknown if these forms are genetically distinct. In this study, we generated DNA sequences of the nuc rDNA internal transcribed spacers (ITS), the TEF1 gene encoding translation elongation factor 1-α, and the RPB2 gene encoding the second largest subunit of RNA polymerase II for collections across all major geographic regions where this fungus occurs, with a primary focus on North America. We used Bayesian and maximum likelihood analyses and evaluated the gene trees within the species tree using coalescent methods to elucidate evolutionarily independent lineages. We find that F. pinicola sensu lato encompasses four well-supported, congruent clades: a European clade, southwestern US clade, and two sympatric northern North American clades. Each clade represents distinct species according to phylogenetic and population-genetic species concepts. Morphological data currently available for F. pinicola do not delimit these species, and three of the species are not specific to either hardwood or softwood trees. Originally described from Europe, F. pinicola appears to be restricted to Eurasia. Based on DNA data obtained from an isotype, one well-defined and widespread clade found only in North America represents the recently described

  15. Phylogeny of Fomitopsis pinicola: a species complex.

    PubMed

    Haight, John-Erich; Laursen, Gary A; Glaeser, Jessie; Taylor, Lee

    2016-08-22

    Fungal species with a broad distribution may exhibit considerable genetic variation over their geographic ranges. Variation may develop among populations based on geographic isolation, lack of migration, and genetic drift, though this genetic variation may not always be evident when examining phenotypic characters. Fomitopsis pinicola is an abundant saprotrophic fungus found on decaying logs throughout temperate regions of the Northern Hemisphere. Phylogenetic studies have addressed the relationship of F. pinicola to other wood-rotting fungi, but pan-continental variation within F. pinicola has not been addressed using molecular data. While forms found growing on hardwood and softwood hosts exhibit variation in habit and appearance, it is unknown if these forms are genetically distinct. In this study, we generated DNA sequences of the nuc rDNA internal transcribed spacers (ITS), the TEF1 gene encoding translation elongation factor 1-α, and the RPB2 gene encoding the second largest subunit of RNA polymerase II for collections across all major geographic regions where this fungus occurs, with a primary focus on North America. We used Bayesian and maximum likelihood analyses and evaluated the gene trees within the species tree using coalescent methods to elucidate evolutionarily independent lineages. We find that F. pinicola sensu lato encompasses four well-supported, congruent clades: a European clade, southwestern US clade, and two sympatric northern North American clades. Each clade represents distinct species according to phylogenetic and population-genetic species concepts. Morphological data currently available for F. pinicola do not delimit these species, and three of the species are not specific to either hardwood or softwood trees. Originally described from Europe, F. pinicola appears to be restricted to Eurasia. Based on DNA data obtained from an isotype, one well-defined and widespread clade found only in North America represents the recently described

  16. Functional Consequences of Subunit Diversity in RNA Polymerases II and V

    SciTech Connect

    Tan, Ek Han; Blevins, Todd; Ream, Thomas S.; Pikaard, Craig S.

    2012-03-01

    Multisubunit RNA polymerases IV and V (Pol IV and Pol V) evolved as specialized forms of Pol II that mediate RNA-directed DNA methylation (RdDM) and transcriptional silencing of transposons, viruses, and endogenous repeats in plants. Among the subunits common to Arabidopsis thaliana Pols II, IV, and V are 93% identical alternative ninth subunits, NRP(B/D/E)9a and NRP(B/D/E)9b. The 9a and 9b subunit variants are incompletely redundant with respect to Pol II; whereas double mutants are embryo lethal, single mutants are viable, yet phenotypically distinct. Likewise, 9a or 9b can associate with Pols IV or V but RNA-directed DNA methylation is impaired only in 9b mutants. Based on genetic and molecular tests, we attribute the defect in RdDM to impaired Pol V function. Collectively, our results reveal a role for the ninth subunit in RNA silencing and demonstrate that subunit diversity generates functionally distinct subtypes of RNA polymerases II and V.

  17. Recent radiation in a marine and freshwater dinoflagellate species flock

    PubMed Central

    Annenkova, Nataliia V; Hansen, Gert; Moestrup, Øjvind; Rengefors, Karin

    2015-01-01

    Processes of rapid radiation among unicellular eukaryotes are much less studied than among multicellular organisms. We have investigated a lineage of cold-water microeukaryotes (protists) that appear to have diverged recently. This lineage stands in stark contrast to known examples of phylogenetically closely related protists, in which genetic difference is typically larger than morphological differences. We found that the group not only consists of the marine-brackish dinoflagellate species Scrippsiella hangoei and the freshwater species Peridinium aciculiferum as discovered previously but also of a whole species flock. The additional species include Peridinium euryceps and Peridinium baicalense, which are restricted to a few lakes, in particular to the ancient Lake Baikal, Russia, and freshwater S. hangoei from Lake Baikal. These species are characterized by relatively large conspicuous morphological differences, which have given rise to the different species descriptions. However, our scanning electron microscopic studies indicate that they belong to a single genus according to traditional morphological characterization of dinoflagellates (thecal plate patterns). Moreover, we found that they have identical SSU (small subunit) rDNA fragments and distinct but very small differences in the DNA markers LSU (large subunit) rDNA, ITS2 (internal transcribed spacer 2) and COB (cytochrome b) gene, which are used to delineate dinoflagellates species. As some of the species co-occur, and all four have small but species–specific sequence differences, we suggest that these taxa are not a case of phenotypic plasticity but originated via recent adaptive radiation. We propose that this is the first clear example among free-living microeukaryotes of recent rapid diversification into several species followed by dispersion to environments with different ecological conditions. PMID:25603395

  18. A new identification method for five species of oysters in genus Crassostrea from China based on high-resolution melting analysis

    NASA Astrophysics Data System (ADS)

    Wang, Jiafeng; Xu, Fei; Li, Li; Zhang, Guofan

    2014-03-01

    The high phenotypic plasticity in the shell of oysters presents a challenge during taxonomic and phylogenetic studies of these economically important bivalves. However, because DNA can exhibit marked differences among morphologically similar species, DNA barcoding offers a potential means for oyster identification. We analyzed the complete sequences of the cytochrome oxidase subunit I (COI) of five common Crassostrea species in China (including Hong Kong oyster C. hongkongensis, Jinjiang oyster C. ariakensis, Portuguese oyster C. angulata, Kumamoto oyster C. sikamea, and Pacific oyster C. gigas) and screened for distinct fragments. Using these distinct fragments on a high-resolution melting analysis platform, we developed an identification method that does not rely on species-specific PCR or fragment length polymorphism and is efficient, reliable, and easy to visualize. Using a single pair of primers (Oyster-COI-1), we were able to successfully distinguish among the five oyster species. This new method provides a simple and powerful tool for the identification of oyster species.

  19. Revisiting phylogenetic diversity and cryptic species of Cenococcum geophilum sensu lato.

    PubMed

    Obase, Keisuke; Douhan, Greg W; Matsuda, Yosuke; Smith, Matthew E

    2016-08-01

    The fungus Cenococcum geophilum Fr. (Dothideomycetes, Ascomycota) is one of the most common ectomycorrhizal fungi in boreal to temperate regions. A series of molecular studies has demonstrated that C. geophilum is monophyletic but a heterogeneous species or a species complex. Here, we revisit the phylogenetic diversity of C. geophilum sensu lato from a regional to intercontinental scale by using new data from Florida (USA) along with existing data in GenBank from Japan, Europe, and North America. The combination of internal transcribed spacer (ITS) ribosomal DNA and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene resolved six well-supported lineages (87-100 % bootstrap values) that are closely related to each other and a seventh lineage that is phylogenetically distinct. A multi-locus analysis (small subunit (SSU), large subunit (LSU), translational elongation factor (TEF), and the largest and second-largest subunits of RNA polymerase II (RPB1 and RPB2)) revealed that the divergent lineage is the sister group to all other known Cenococcum isolates. Isolates of the divergent lineage grow fast on nutrient media and do not form ectomycorrhizas on seedlings of several pine and oak species. Our results indicate that C. geophilum sensu lato includes more phylogenetically distinct cryptic species than have previously been reported. Furthermore, the divergent lineage appears to be a non-mycorrhizal sister group. We discuss the phylogenetic diversity of C. geophilum sensu lato and argue in favor of species recognition based on phylogenetic and ecological information in addition to morphological characteristics. A new genus and species (Pseudocenococcum floridanum gen. et sp. nov.) is proposed to accommodate a divergent and putatively non-mycorrhizal lineage.

  20. Two classes of regulatory subunits coassemble in the same BK channel and independently regulate gating

    NASA Astrophysics Data System (ADS)

    Gonzalez-Perez, Vivian; Xia, Xiao-Ming; Lingle, Christopher J.

    2015-09-01

    High resolution proteomics increasingly reveals that most native ion channels are assembled in macromolecular complexes. However, whether different partners have additive or cooperative functional effects, or whether some combinations of proteins may preclude assembly of others are largely unexplored topics. The large conductance Ca2+-and-voltage activated potassium channel (BK) is well-suited to discern nuanced differences in regulation arising from combinations of subunits. Here we examine whether assembly of two different classes of regulatory proteins, β and γ, in BK channels is exclusive or independent. Our results show that both γ1 and up to four β2-subunits can coexist in the same functional BK complex, with the gating shift caused by β2-subunits largely additive with that produced by the γ1-subunit(s). The multiplicity of β:γ combinations that can participate in a BK complex therefore allow a range of BK channels with distinct functional properties tuned by the specific stoichiometry of the contributing subunits.

  1. The TCP1γ subunit of Leishmania donovani forms a biologically active homo-oligomeric complex.

    PubMed

    Bhaskar; Mitra, Kalyan; Kuldeep, Jitendra; Siddiqi, Mohammad Imran; Goyal, Neena

    2015-12-01

    Chaperonins are a class of molecular chaperons that encapsulate nascent or stress-denatured proteins and assist their intracellular assembly and folding in an ATP-dependent manner. The ubiquitous eukaryotic chaperonin, TCP1 ring complex is a hetero-oligomeric complex comprising two rings, each formed of eight subunits that may have distinct substrate recognition and ATP hydrolysis properties. In Leishmania, only the TCP1γ subunit has been cloned and characterized. It exhibited differential expression at various growth stages of promastigotes. In the present study, we expressed the TCP1γ subunit in Escherichia coli to investigate whether it forms chaperonin-like complexes and plays a role in protein folding. LdTCP1γ formed high-molecular-weight complexes within E. coli cells as well as in Leishmania cell lysates. The recombinant protein is arranged into two back-to-back rings of seven subunits each, as predicted by homology modelling and observed by negative staining electron microscopy. This morphology is consistent with that of the oligomeric double-ring group I chaperonins found in mitochondria. The LdTCP1γ homo-oligomeric complex hydrolysed ATP, and was active as assayed by luciferase refolding. Thus, the homo-oligomer performs chaperonin reactions without partner subunit(s). Further, co-immunoprecipitation studies revealed that LdTCP1γ interacts with actin and tubulin proteins, suggesting that the complex may have a role in maintaining the structural dynamics of the cytoskeleton of parasites.

  2. Archaeal Mo-Containing Glyceraldehyde Oxidoreductase Isozymes Exhibit Diverse Substrate Specificities through Unique Subunit Assemblies

    PubMed Central

    Miyake, Masayuki; Fushinobu, Shinya

    2016-01-01

    Archaea use glycolytic pathways distinct from those found in bacteria and eukaryotes, where unique enzymes catalyze each reaction step. In this study, we isolated three isozymes of glyceraldehyde oxidoreductase (GAOR1, GAOR2 and GAOR3) from the thermoacidophilic archaeon Sulfolobus tokodaii. GAOR1–3 belong to the xanthine oxidoreductase superfamily, and are composed of a molybdo-pyranopterin subunit (L), a flavin subunit (M), and an iron-sulfur subunit (S), forming an LMS hetero-trimer unit. We found that GAOR1 is a tetramer of the STK17810/STK17830/STK17820 hetero-trimer, GAOR2 is a dimer of the STK23390/STK05620/STK05610 hetero-trimer, and GAOR3 is the STK24840/STK05620/STK05610 hetero-trimer. GAOR1–3 exhibited diverse substrate specificities for their electron donors and acceptors, due to their different L-subunits, and probably participate in the non-phosphorylative Entner-Doudoroff glycolytic pathway. We determined the crystal structure of GAOR2, as the first three-dimensional structure of an archaeal molybdenum-containing hydroxylase, to obtain structural insights into their substrate specificities and subunit assemblies. The gene arrangement and the crystal structure suggested that the M/S-complex serves as a structural scaffold for the binding of the L-subunit, to construct the three enzymes with different specificities. Collectively, our findings illustrate a novel principle of a prokaryotic multicomponent isozyme system. PMID:26808202

  3. The subunit structure of potato tuber ADPglucose pyrophosphorylase. [Solanum tuberosum L

    SciTech Connect

    Okita, T.W.; Nakata, P.A.; Anderson, J.M. ); Sowokinos, J. ); Morell, M.; Preiss, J. )

    1990-06-01

    ADPglucose pyrophosphorylase has been extensively purified from potato (Solanum tuberosum L.) tuber tissue to study its structure. By employing a modified published procedure together with Mono Q chromatography, a near homogeneous enzyme preparation was obtained with substantial improvement in enzyme yield and specific activity. In single dimensional sodium dodecyl sulfate polyacrylamide gels, the enzyme migrated as a single polypeptide band with a mobility of about 50,000 daltons. Analysis by two-dimensional polyacrylamide gel electrophoresis, however, revealed the presence of two types of subunits which could be distinguished by their slight differences in net charge and molecular weight. The smaller potato tuber subunit was recognized by antiserum prepared against the smaller spinach leaf 51 kilodalton ADPglucose pyrophosphorylase subunit. In contrast, the anti-54 kilodalton raised against the spinach leaf subunit did not significantly react to the tuber enzyme subunits. The results are consistent with the hypothesis that the potato tuber ADPglucose pyrophosphorylase is not composed of a simple homotetramer as previously suggested, but is a product of two separate and distinct subunits as observed for the spinach leaf and maize enzymes.

  4. Molecular architecture of the yeast Elongator complex reveals an unexpected asymmetric subunit arrangement.

    PubMed

    Setiaputra, Dheva T; Cheng, Derrick Th; Lu, Shan; Hansen, Jesse M; Dalwadi, Udit; Lam, Cindy Hy; To, Jeffrey L; Dong, Meng-Qiu; Yip, Calvin K

    2017-02-01

    Elongator is a ~850 kDa protein complex involved in multiple processes from transcription to tRNA modification. Conserved from yeast to humans, Elongator is assembled from two copies of six unique subunits (Elp1 to Elp6). Despite the wealth of structural data on the individual subunits, the overall architecture and subunit organization of the full Elongator and the molecular mechanisms of how it exerts its multiple activities remain unclear. Using single-particle electron microscopy (EM), we revealed that yeast Elongator adopts a bilobal architecture and an unexpected asymmetric subunit arrangement resulting from the hexameric Elp456 subassembly anchored to one of the two Elp123 lobes that form the structural scaffold. By integrating the EM data with available subunit crystal structures and restraints generated from cross-linking coupled to mass spectrometry, we constructed a multiscale molecular model that showed the two Elp3, the main catalytic subunit, are located in two distinct environments. This work provides the first structural insights into Elongator and a framework to understand the molecular basis of its multifunctionality.

  5. Cell-free synthesis and assembly of prolyl 4-hydroxylase: the role of the beta-subunit (PDI) in preventing misfolding and aggregation of the alpha-subunit.

    PubMed Central

    John, D C; Grant, M E; Bulleid, N J

    1993-01-01

    Prolyl 4-hydroxylase (P4-H) catalyses a vital post-translational modification in the biosynthesis of collagen. The enzyme consists of two distinct polypeptides forming an alpha 2 beta 2 tetramer (alpha = 64 kDa, beta = 60 kDa), the beta-subunit being identical to the multifunctional enzyme protein disulfide isomerase (PDI). By studying the cell-free synthesis of the rat alpha-subunit of P4-H we have shown that the alpha-subunit can be translocated, glycosylated and the signal peptide cleaved by dog pancreatic microsomal membranes to yield both singly and doubly glycosylated forms. When translations were carried out under conditions which prevent disulfide bond formation, the product synthesized formed aggregates which were associated with the immunoglobulin heavy chain binding protein (BiP). Translations carried out under conditions that promote disulfide bond formation yielded a product that was not associated with BiP but formed a complex with the endogenous beta-subunit (PDI). Complex formation was detected by co-precipitation of the newly synthesized alpha-subunit with antibodies raised against PDI, by sucrose gradient centrifugation and by chemical cross-linking. When microsomal vesicles were depleted of PDI, BiP and other soluble endoplasmic reticulum proteins, no complex formation was observed and the alpha-subunit aggregated even under conditions that promote disulfide bond formation. We have therefore demonstrated that the enzyme P4-H can be assembled at synthesis in a cell-free system and that the solubility of the alpha-subunit is dependent upon its association with PDI. Images PMID:8385607

  6. Subunit mass analysis for monitoring antibody oxidation

    PubMed Central

    Sokolowska, Izabela; Mo, Jingjie; Dong, Jia; Lewis, Michael J.; Hu, Ping

    2017-01-01

    ABSTRACT Methionine oxidation is a common posttranslational modification (PTM) of monoclonal antibodies (mAbs). Oxidation can reduce the in-vivo half-life, efficacy and stability of the product. Peptide mapping is commonly used to monitor the levels of oxidation, but this is a relatively time-consuming method. A high-throughput, automated subunit mass analysis method was developed to monitor antibody methionine oxidation. In this method, samples were treated with IdeS, EndoS and dithiothreitol to generate three individual IgG subunits (light chain, Fd’ and single chain Fc). These subunits were analyzed by reversed phase-ultra performance liquid chromatography coupled with an online quadrupole time-of-flight mass spectrometer and the levels of oxidation on each subunit were quantitated based on the deconvoluted mass spectra using the UNIFI software. The oxidation results obtained by subunit mass analysis correlated well with the results obtained by peptide mapping. Method qualification demonstrated that this subunit method had excellent repeatability and intermediate precision. In addition, UNIFI software used in this application allows automated data acquisition and processing, which makes this method suitable for high-throughput process monitoring and product characterization. Finally, subunit mass analysis revealed the different patterns of Fc methionine oxidation induced by chemical and photo stress, which makes it attractive for investigating the root cause of oxidation. PMID:28106519

  7. Subunit mass analysis for monitoring antibody oxidation.

    PubMed

    Sokolowska, Izabela; Mo, Jingjie; Dong, Jia; Lewis, Michael J; Hu, Ping

    2017-04-01

    Methionine oxidation is a common posttranslational modification (PTM) of monoclonal antibodies (mAbs). Oxidation can reduce the in-vivo half-life, efficacy and stability of the product. Peptide mapping is commonly used to monitor the levels of oxidation, but this is a relatively time-consuming method. A high-throughput, automated subunit mass analysis method was developed to monitor antibody methionine oxidation. In this method, samples were treated with IdeS, EndoS and dithiothreitol to generate three individual IgG subunits (light chain, Fd' and single chain Fc). These subunits were analyzed by reversed phase-ultra performance liquid chromatography coupled with an online quadrupole time-of-flight mass spectrometer and the levels of oxidation on each subunit were quantitated based on the deconvoluted mass spectra using the UNIFI software. The oxidation results obtained by subunit mass analysis correlated well with the results obtained by peptide mapping. Method qualification demonstrated that this subunit method had excellent repeatability and intermediate precision. In addition, UNIFI software used in this application allows automated data acquisition and processing, which makes this method suitable for high-throughput process monitoring and product characterization. Finally, subunit mass analysis revealed the different patterns of Fc methionine oxidation induced by chemical and photo stress, which makes it attractive for investigating the root cause of oxidation.

  8. The tip of the iceberg: a distinctive new spotted-wing Megaselia species (Diptera: Phoridae) from a tropical cloud forest survey and a new, streamlined method for Megaselia descriptions

    PubMed Central

    Brown, Brian V.

    2014-01-01

    Abstract A new Megaselia species, M. shadeae, with a large, central, pigmented and bubble-like wing spot and a greatly enlarged radial wing vein fork, is described from Zurquí de Moravia, Costa Rica. As part of the Zurquí All Diptera Biodiversity Inventory (ZADBI) project, it represents the first of an incredible number of new phorid species to be described from this one Costa Rican cloud forest site. A new, streamlined method of description for species of this enormous genus of phorid flies is presented. PMID:25425935

  9. The tip of the iceberg: a distinctive new spotted-wing Megaselia species (Diptera: Phoridae) from a tropical cloud forest survey and a new, streamlined method for Megaselia descriptions.

    PubMed

    Hartop, Emily A; Brown, Brian V

    2014-01-01

    A new Megaselia species, M.shadeae, with a large, central, pigmented and bubble-like wing spot and a greatly enlarged radial wing vein fork, is described from Zurquí de Moravia, Costa Rica. As part of the Zurquí All Diptera Biodiversity Inventory (ZADBI) project, it represents the first of an incredible number of new phorid species to be described from this one Costa Rican cloud forest site. A new, streamlined method of description for species of this enormous genus of phorid flies is presented.

  10. The complete genome sequences of a Peruvian and a Colombian isolate of Andean potato latent virus and partial sequences of further isolates suggest the existence of two distinct potato-infecting tymovirus species.

    PubMed

    Kreuze, Jan; Koenig, Renate; De Souza, Joao; Vetten, Heinrich Josef; Muller, Giovanna; Flores, Betty; Ziebell, Heiko; Cuellar, Wilmer

    2013-05-01

    The complete genomic RNA sequences of the tymovirus isolates Hu and Col from potato which originally had been considered to be strains of the same virus species, i.e. Andean potato latent virus (APLV), were determined by siRNA sequencing and assembly, and found to share only c. 65% nt sequence identity. This result together with those of serological tests and comparisons of the coat protein gene sequences of additional tymovirus isolates from potato suggest that the species Andean potato latent virus should be subdivided into two species, i.e. APLV and Andean potato mild mosaic virus (APMMV). Primers were designed for the broad specificity detection of both viruses.

  11. Candida bromeliacearum sp. nov. and Candida ubatubensis sp. nov., two yeast species isolated from the water tanks of Canistropsis seidelii (Bromeliaceae).

    PubMed

    Ruivo, Carla C C; Lachance, Marc-André; Rosa, Carlos A; Bacci, Maurício; Pagnocca, Fernando C

    2005-09-01

    Strains belonging to two novel yeast species, Candida bromeliacearum and Candida ubatubensis, were isolated from the bromeliad tank of Canistropsis seidelii (Bromeliaceae) in a sandy coastal plain (restinga) ecosystem site in an Atlantic rainforest of south-eastern Brazil. These species were genetically distinct from all other currently accepted ascomycetous yeasts, based on sequence divergence in the D1/D2 domains of the large-subunit rDNA and in the small-subunit rDNA. The species occupy basal positions in the Metschnikowiaceae clade. The type strains are Candida bromeliacearum UNESP 00-103(T) (=CBS 10002(T)=NRRL Y-27811(T)) and Candida ubatubensis UNESP 01-247R(T) (=CBS 10003(T)=NRRL Y-27812(T)).

  12. Description of Scheffersomyces henanensis sp. nov., a New D-Xylose-Fermenting Yeast Species Isolated from Rotten Wood

    PubMed Central

    Ren, Yongcheng; Chen, Liang; Niu, Qiuhong; Hui, Fengli

    2014-01-01

    Two strains of a D-xylose-fermenting yeast species were isolated from rotten wood samples collected from the Baotianman Nature Reserve in Henan Province, central China. These strains formed hat-shaped ascospores in conjugated and deliquescent asci. Multilocus phylogenetic analysis that included the nearly complete small subunit (SSU), the internal transcribed spacer (ITS) region and the D1/D2 domain of the large subunit (LSU) rRNA genes, as well as RNA polymerase II largest subunit (RPB1) gene demonstrated that the two strains represent a novel yeast species closely related to Scheffersomyces segobiensis. A sequence comparison of xylose reductase (XYL1) gene, which was recently recommended for rapid identification of cryptic species in the Scheffersomyces clade, revealed a significant sequence divergence of 25 nucleotides between the novel strains and their closest relative S. segobiensis, supporting their classification as a distinct species. Furthermore, these new strains can be clearly distinguished from S. segobiensis by a number of morphological and physiological characteristics. Therefore, a novel yeast species, Scheffersomyces henanensis sp. nov., is proposed to accommodate these strains. The type strain is BY-41T ( =  CICC 1974T  =  CBS 12475T). PMID:24647466

  13. Description of Scheffersomyces henanensis sp. nov., a new D-xylose-fermenting yeast species isolated from rotten wood.

    PubMed

    Ren, Yongcheng; Chen, Liang; Niu, Qiuhong; Hui, Fengli

    2014-01-01

    Two strains of a D-xylose-fermenting yeast species were isolated from rotten wood samples collected from the Baotianman Nature Reserve in Henan Province, central China. These strains formed hat-shaped ascospores in conjugated and deliquescent asci. Multilocus phylogenetic analysis that included the nearly complete small subunit (SSU), the internal transcribed spacer (ITS) region and the D1/D2 domain of the large subunit (LSU) rRNA genes, as well as RNA polymerase II largest subunit (RPB1) gene demonstrated that the two strains represent a novel yeast species closely related to Scheffersomyces segobiensis. A sequence comparison of xylose reductase (XYL1) gene, which was recently recommended for rapid identification of cryptic species in the Scheffersomyces clade, revealed a significant sequence divergence of 25 nucleotides between the novel strains and their closest relative S. segobiensis, supporting their classification as a distinct species. Furthermore, these new strains can be clearly distinguished from S. segobiensis by a number of morphological and physiological characteristics. Therefore, a novel yeast species, Scheffersomyces henanensis sp. nov., is proposed to accommodate these strains. The type strain is BY-41T ( =  CICC 1974T  =  CBS 12475T).

  14. Lack of immunological analogy between the beta-subunits of cholera toxin and human choriogonadotropin.

    PubMed

    Acevedo, H F; Kellen, J A

    1986-01-01

    A chemical relatedness has been described between the beta-subunit of cholera toxin and that of the four dimeric glycoprotein hormones (hCG, hLH, hFSH and hTSH). However, antibodies induced by cholera toxin did not crossreact, when tested by labeled hCG binding and immunocytochemistry, with the beta-subunit of hCG. It appears that differences in the tertiary structures, as shown in this study, account for distinct epitopes. Similarities in biological activity between these two compounds, such as induction of adenyl cyclase or a protective effect against some tumors, are not based on immunological mechanisms.

  15. Developmental Expression Patterns of GABAA Receptor Subunits in Layer 3 and 5 Pyramidal Cells of Monkey Prefrontal Cortex.

    PubMed

    Datta, Dibyadeep; Arion, Dominique; Lewis, David A

    2015-08-01

    Cortical pyramidal neuron activity is regulated in part through inhibitory inputs mediated by GABAA receptors. The subunit composition of these receptors confers distinct functional properties. Thus, developmental shifts in subunit expression will likely influence the characteristics of pyramidal cell firing and the functional maturation of processes that depend on these neurons. We used laser microdissection and PCR to quantify postnatal developmental changes in the expression of GABAA receptor subunits (α1, α2, α5, β2, γ2, and δ) in layer 3 pyramidal cells of monkey prefrontal cortex, which are critical for working memory. To determine the specificity of these changes, we examined glutamate receptor subunits (AMPA Glur1 and NMDA Grin1) and conducted the same analyses in layer 5 pyramidal cells. Expression of GABAA receptor subunit mRNAs changed substantially, whereas glutamate receptor subunit changes were modest over postnatal development. Some transcripts (e.g., GABAA α1) progressively increased from birth until adulthood, whereas others (e.g., GABAA α2) declined with age. Changes in some transcripts were present in only one layer (e.g., GABAA δ). The development of GABAA receptor subunit expression in primate prefrontal pyramidal neurons is protracted and subunit- and layer-specific. These trajectories might contribute to the molecular basis for the maturation of working memory.

  16. Diverse roles for auxiliary subunits in phosphorylation-dependent regulation of mammalian brain voltage-gated potassium channels.

    PubMed

    Vacher, Helene; Trimmer, James S

    2011-11-01

    Voltage-gated ion channels are a diverse family of signaling proteins that mediate rapid electrical signaling events. Among these, voltage-gated potassium or Kv channels are the most diverse partly due to the large number of principal (or α) subunits and auxiliary subunits that can assemble in different combinations to generate Kv channel complexes with distinct structures and functions. The diversity of Kv channels underlies much of the variability in the active properties between different mammalian central neurons and the dynamic changes that lead to experience-dependent plasticity in intrinsic excitability. Recent studies have revealed that Kv channel α subunits and auxiliary subunits are extensively phosphorylated, contributing to additional structural and functional diversity. Here, we highlight recent studies that show that auxiliary subunits exert some of their profound effects on dendritic Kv4 and axonal Kv1 channels through phosphorylation-dependent mechanisms, either due to phosphorylation on the auxiliary subunit itself or by influencing the extent and/or impact of α subunit phosphorylation. The complex effects of auxiliary subunits and phosphorylation provide a potent mechanism to generate additional diversity in the structure and function of Kv4 and Kv1 channels, as well as allowing for dynamic reversible regulation of these important ion channels.

  17. Distribution of NMDA and AMPA receptor subunits at thalamo-amygdaloid dendritic spines.

    PubMed

    Radley, Jason J; Farb, Claudia R; He, Yong; Janssen, William G M; Rodrigues, Sarina M; Johnson, Luke R; Hof, Patrick R; LeDoux, Joseph E; Morrison, John H

    2007-02-23

    Synapses onto dendritic spines in the lateral amygdala formed by afferents from the auditory thalamus represent a site of plasticity in Pavlovian fear conditioning. Previous work has demonstrated that thalamic afferents synapse onto LA spines expressing glutamate receptor (GluR) subunits, but the GluR subunit distribution at the synapse and within the cytoplasm has not been characterized. Therefore, we performed a quantitative analysis for alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunits GluR2 and GluR3 and N-methyl-D-aspartate (NMDA) receptor subunits NR1 and NR2B by combining anterograde labeling of thalamo-amygdaloid afferents with postembedding immunoelectron microscopy for the GluRs in adult rats. A high percentage of thalamo-amygdaloid spines was immunoreactive for GluR2 (80%), GluR3 (83%), and NR1 (83%), while a smaller proportion of spines expressed NR2B (59%). To compare across the various subunits, the cytoplasmic to synaptic ratios of GluRs were measured within thalamo-amygdaloid spines. Analyses revealed that the cytoplasmic pool of GluR2 receptors was twice as large compared to the GluR3, NR1, and NR2B subunits. Our data also show that in the adult brain, the NR2B subunit is expressed in the majority of in thalamo-amygdaloid spines and that within these spines, the various GluRs are differentially distributed between synaptic and non-synaptic sites. The prevalence of the NR2B subunit in thalamo-amygdaloid spines provides morphological evidence supporting its role in the fear conditioning circuit while the differential distribution of the GluR subtypes may reflect distinct roles for their involvement in this circuitry and synaptic plasticity.

  18. Type B Heterotrimeric G Protein γ-Subunit Regulates Auxin and ABA Signaling in Tomato.

    PubMed

    Subramaniam, Gayathery; Trusov, Yuri; Lopez-Encina, Carlos; Hayashi, Satomi; Batley, Jacqueline; Botella, José Ramón

    2016-02-01

    Heterotrimeric G proteins composed of α, β, and γ subunits are central signal transducers mediating the cellular response to multiple stimuli in most eukaryotes. Gγ subunits provide proper cellular localization and functional specificity to the heterotrimer complex. Plant Gγ subunits, divided into three structurally distinct types, are more diverse than their animal counterparts. Type B Gγ subunits, lacking a carboxyl-terminal isoprenylation motif, are found only in flowering plants. We present the functional characterization of type B Gγ subunit (SlGGB1) in tomato (Solanum lycopersicum). We show that SlGGB1 is the most abundant Gγ subunit in tomato and strongly interacts with the Gβ subunit. Importantly, the green fluorescent protein-SlGGB1 fusion protein as well as the carboxyl-terminal yellow fluorescent protein-SlGGB1/amino-terminal yellow fluorescent protein-Gβ heterodimer were localized in the plasma membrane, nucleus, and cytoplasm. RNA interference-mediated silencing of SlGGB1 resulted in smaller seeds, higher number of lateral roots, and pointy fruits. The silenced lines were hypersensitive to exogenous auxin, while levels of endogenous auxins were lower or similar to those of the wild type. SlGGB1-silenced plants also showed strong hyposensitivity to abscisic acid (ABA) during seed germination but not in other related assays. Transcriptome analysis of the transgenic seeds revealed abnormal expression of genes involved in ABA sensing, signaling, and response. We conclude that the type B Gγ subunit SlGGB1 mediates auxin and ABA signaling in tomato.

  19. Differential proteolysis of the subunits of pyrophosphate-dependent 6-phosphofructo-1-phosphotransferase.

    PubMed

    Cheng, H F; Tao, M

    1990-02-05

    Antibodies against the alpha (Mr 67,000) and beta (Mr 60,000) subunits of wheat seedling Fru-2,6-P2-stimulated pyrophosphate-dependent 6-phosphofructo-1-phosphotransferase (PFP) were used to probe the subunit structures of several partially purified plant PFPs after tryptic digestion. Antisera to the alpha and beta subunits of wheat seedling PFP cross-reacted with the corresponding alpha and beta subunits of PFP preparations from wheat germ, potato tubers, and lettuce leaves. With the mung bean PFP, both antisera reacted with a protein band of Mr 60,000. A protein band corresponding to the Mr 67,000 alpha subunit was not detected in the mung bean PFP preparation. Tryptic digestion of wheat seedling and potato tuber PFPs resulted in the preferential cleavage of the alpha subunit. The trypsinized PFP retained most of its Fru-2,6-P2-stimulated activity but not its basal activity. The proteolyzed enzyme also exhibited a 2-fold increase in Ka for Fru-2,6-P2. Studies with the mung bean enzyme revealed that the anti-alpha immunoreactive component was more sensitive to trypsinization than the anti-beta immunoreactive component of the Mr 60,000 protein band. Thus, the Mr 60,000 protein band of the mung bean PFP appears to be heterogeneous and contains both alpha and beta-like proteins. The above observations indicate that the alpha and beta subunits of PFP are two distinct polypeptides and that alpha acts as a regulatory protein in regulating both the catalytic activity and the Fru-2,6-P2-binding affinity of the beta subunit.

  20. Gene targeting of CK2 catalytic subunits

    PubMed Central

    Lou, David Y.; Toselli, Paul; Landesman-Bollag, Esther; Dominguez, Isabel

    2013-01-01

    Protein kinase CK2 is a highly conserved and ubiquitous serine–threonine kinase. It is a tetrameric enzyme that is made up of two regulatory CK2β subunits and two catalytic subunits, either CK2α/CK2α, CK2α/ CK2α′, or CK2α′/CK2α′. Although the two catalytic subunits diverge in their C termini, their enzymatic activities are similar. To identify the specific function of the two catalytic subunits in development, we have deleted them individually from the mouse genome by homologous recombination. We have previously reported that CK2α′is essential for male germ cell development, and we now demonstrate that CK2α has an essential role in embryogenesis, as mice lacking CK2α die in mid-embryogenesis, with cardiac and neural tube defects. PMID:18594950

  1. The human thyrotropin beta-subunit gene differs in 5' structure from murine TSH-beta genes.

    PubMed

    Guidon, P T; Whitfield, G K; Porti, D; Kourides, I A

    1988-12-01

    The gene encoding the beta-subunit of human thyrotropin (hTSH-beta) was isolated, and its nucleotide sequence was determined. The gene is 4.3 kb in length, consists of three exons and two introns, and is present as a single copy as determined by Southern blot analysis of total genomic DNA. The protein coding portion of the gene, which includes exons 2 and 3, was isolated from a human genomic phage library, while exon 1, which encodes only 5' untranslated mRNA sequence, was isolated from a plasmid library of size-selected genomic DNA fragments. Here we describe the isolation of the 5' untranslated exon of the hTSH-beta subunit and 5'-flanking region. The structure of the hTSH-beta gene is very similar to the previously characterized TSH-beta genes from mouse and rat. The genes from all three species have two distinct promoter regions, but while both promoters are utilized by the murine TSH-beta genes, the human TSH-beta gene apparently utilizes only the proximal promoter for transcription initiation. A striking difference in hTSH-beta gene structure compared to the murine genes is that exon 1 of the human gene is 36 nucleotides. An analysis of the mouse, rat, and human exon 1 and 5'-flanking region shows a high percentage of sequence homology, with the exception of a 9-nucleotide insertion 13 bases 3' from the proximal TATA box found in the human gene but not found in the other two species. We propose that this insertion results in the additional length of human exon 1 compared to the mouse and rat genes. By isolating the promoter region of the hTSH-beta gene, we can begin to identify specific sequences involved in the regulation of hTSH gene expression.

  2. Identification of New Agonists and Antagonists of the Insect Odorant Receptor Co-Receptor Subunit

    PubMed Central

    Chen, Sisi; Luetje, Charles W.

    2012-01-01

    Background Insects detect attractive and aversive chemicals using several families of chemosensory receptors, including the OR family of olfactory receptors, making these receptors appealing targets for the control of insects. Insect ORs are odorant-gated ion channels, comprised of at least one common subunit (the odorant receptor co-receptor subunit, Orco) and at least one variable odorant specificity subunit. Each of the many ORs of an insect species is activated or inhibited by an unique set of odorants that interact with the variable odorant specificity subunits, making the development of OR directed insect control agents complex and laborious. However, several N-,2-substituted triazolothioacetamide compounds (VUAA1, VU0450667 and VU0183254) were recently shown to act directly on the highly conserved Orco subunit, suggesting that broadly active compounds can be developed. We have explored the chemical space around the VUAA1 structure in order to identify new Orco ligands. Principal Findings We screened ORs from several insect species, using heterologous expression in Xenopus oocytes and an electrophysiological assay, with a panel of 22 compounds structurally related to VUAA1. By varying the nitrogen position in the pyridine ring and altering the moieties decorating the phenyl ring, we identified two new agonists and a series of competitive antagonists. Screening smaller compounds, similar to portions of the VUAA1 structure, also yielded competitive antagonists. Importantly, we show that Orco antagonists inhibit odorant activation of ORs from several insect species. Detailed examination of one antagonist demonstrated inhibition to be through a non-competitive mechanism. Conclusions A similar pattern of agonist and antagonist sensitivity displayed by Orco subunits from different species suggests a highly conserved binding site structure. The susceptibility to inhibition of odorant activation by Orco antagonism is conserved across disparate insect species

  3. Molecular and functional characterization of seven Na+/K+-ATPase β subunit paralogs in Senegalese sole (Solea senegalensis Kaup, 1858).

    PubMed

    Armesto, Paula; Infante, Carlos; Cousin, Xavier; Ponce, Marian; Manchado, Manuel

    2015-04-01

    In the present work, seven genes encoding Na(+),K(+)-ATPase (NKA) β-subunits in the teleost Solea senegalensis are described for the first time. Sequence analysis of the predicted polypeptides revealed a high degree of conservation with those of other vertebrate species and maintenance of important motifs involved in structure and function. Phylogenetic analysis clustered the seven genes into four main clades: β1 (atp1b1a and atp1b1b), β2 (atp1b2a and atp1b2b), β3 (atp1b3a and atp1b3b) and β4 (atp1b4). In juveniles, all paralogous transcripts were detected in the nine tissues examined albeit with different expression patterns. The most ubiquitous expressed gene was atp1b1a whereas atp1b1b was mainly detected in osmoregulatory organs (gill, kidney and intestine), and atp1b2a, atp1b2b, atp1b3a, atp1b3b and atp1b4 in brain. An expression analysis in three brain regions and pituitary revealed that β1-type transcripts were more abundant in pituitary than the other β paralogs with slight differences between brain regions. Quantification of mRNA abundance in gills after a salinity challenge showed an activation of atp1b1a and atp1b1b at high salinity water (60 ppt) and atp1b3a and atp1b3b in response to low salinity (5 ppt). Transcriptional analysis during larval development showed specific expression patterns for each paralog. Moreover, no differences in the expression profiles between larvae cultivated at 10 and 35 ppt were observed except for atp1b4 with higher mRNA levels at 10 than 35 ppt at 18 days post hatch. Whole-mount in situ hybridization analysis revealed that atp1b1b was mainly localized in gut, pronephric tubule, gill, otic vesicle, and chordacentrum of newly hatched larvae. All these data suggest distinct roles of NKA β subunits in tissues, during development and osmoregulation with β1 subunits involved in the adaptation to hyperosmotic conditions and β3 subunits to hypoosmotic environments.

  4. Anthranilate synthase subunit organization in Chromobacterium violaceum.

    PubMed

    Carminatti, C A; Oliveira, I L; Recouvreux, D O S; Antônio, R V; Porto, L M

    2008-09-16

    Tryptophan is an aromatic amino acid used for protein synthesis and cellular growth. Chromobacterium violaceum ATCC 12472 uses two tryptophan molecules to synthesize violacein, a secondary metabolite of pharmacological interest. The genome analysis of this bacterium revealed that the genes trpA-F and pabA-B encode the enzymes of the tryptophan pathway in which the first reaction is the conversion of chorismate to anthranilate by anthranilate synthase (AS), an enzyme complex. In the present study, the organization and structure of AS protein subunits from C. violaceum were analyzed using bioinformatics tools available on the Web. We showed by calculating molecular masses that AS in C. violaceum is composed of alpha (TrpE) and beta (PabA) subunits. This is in agreement with values determined experimentally. Catalytic and regulatory sites of the AS subunits were identified. The TrpE and PabA subunits contribute to the catalytic site while the TrpE subunit is involved in the allosteric site. Protein models for the TrpE and PabA subunits were built by restraint-based homology modeling using AS enzyme, chains A and B, from Salmonella typhimurium (PDB ID 1I1Q).

  5. A case of orthologous sequences of hemocyanin subunits for an evolutionary study of horseshoe crabs: amino acid sequence comparison of immunologically identical subunits of Carcinoscorpius rotundicauda and Tachypleus tridentatus.

    PubMed

    Sugita, H; Shishikura, F

    1995-10-01

    About 83% of the amino acid sequence of hemocyanin subunit HR6 from the Southeast Asian horseshoe crab, Carcinoscorpius rotundicauda, has been determined. There is a difference of about 43% between HR6 and complete sequences of chelicerate hemocyanin subunits from the American horseshoe crab, Limulus polyphemus, and a tarantula, Eurypelma californicum. However, the immunologically identical subunits HR6 and HT6 from Tachypleus tridentatus (Japanese horseshoe crab) show 2.7% sequence difference. Based on the amino acid sequences of HR6 and HT6, the divergence between C. rotundicauda and T. tridentatus occurred about 9.6 million years ago. In the case of horseshoe crab hemocyanin subunits, it seems that the orthologous homologues in many homologous subunits between species are immunologically detectable.

  6. Suppression of 19S proteasome subunits marks emergence of an altered cell state in diverse cancers.

    PubMed

    Tsvetkov, Peter; Sokol, Ethan; Jin, Dexter; Brune, Zarina; Thiru, Prathapan; Ghandi, Mahmoud; Garraway, Levi A; Gupta, Piyush B; Santagata, Sandro; Whitesell, Luke; Lindquist, Susan

    2017-01-10

    The use of proteasome inhibitors to target cancer's dependence on altered protein homeostasis has been greatly limited by intrinsic and acquired resistance. Analyzing data from thousands of cancer lines and tumors, we find that those with suppressed expression of one or more 19S proteasome subunits show intrinsic proteasome inhibitor resistance. Moreover, such proteasome subunit suppression is associated with poor outcome in myeloma patients, where proteasome inhibitors are a mainstay of treatment. Beyond conferring resistance to proteasome inhibitors, proteasome subunit suppression also serves as a sentinel of a more global remodeling of the transcriptome. This remodeling produces a distinct gene signature and new vulnerabilities to the proapoptotic drug, ABT-263. This frequent, naturally arising imbalance in 19S regulatory complex composition is achieved through a variety of mechanisms, including DNA methylation, and marks the emergence of a heritably altered and therapeutically relevant state in diverse cancers.

  7. Seven new species within western Atlantic Starksia atlantica, S. lepicoelia, and S. sluiteri (Teleostei, Labrisomidae), with comments on congruence of DNA barcodes and species

    PubMed Central

    Baldwin, Carole C.; Castillo, Cristina I.; Weigt, Lee A.; Benjamin C., Victor

    2011-01-01

    Abstract Specimens of Starksia were collected throughout the western Atlantic, and a 650-bp portion of the mitochondrial gene cytochrome oxidase-c subunit I (COl) was sequenced as part of a re-analysis of species diversity of western Central Atlantic shorefishes. A neighbor-joining tree constructed from the sequence data suggests the existence of several cryptic species. Voucher specimens from each genetically distinct lineage and color photographs of vouchers taken prior to dissection and preservation were examined for diagnostic morphological characters. The results suggest that Starksia atlantica, Starksia lepicoelia, and Starksia sluiteri are species complexes, and each comprises three or more species. Seven new species are described. DNA data usually support morphological features, but some incongruence between genetic and morphological data exists. Genetic lineages are only recognized as species if supported by morphology. Genetic lineages within western Atlantic Starksia generally correspond to geography, such that members of each species complex have a very restricted geographical distribution. Increasing geographical coverage of sampling locations will almost certainly increase the number of Starksia species and species complexes recognized in the western Atlantic. Combining molecular and morphological investigations is bringing clarity to the taxonomy of many genera of morphologically similar fishes and increasing the number of currently recognized species. Future phylogenetic studies should help resolve species relationships and shed light on patterns of speciation in western Atlantic Starksia. PMID:21594143

  8. The resurrection of a species: Sarcocystis bovifelis Heydorn et al., 1975 is distinct from the current Sarcocystis hirsuta in cattle and morphologically indistinguishable from Sarcocystis sinensis in water buffaloes.

    PubMed

    Gjerde, Bjørn

    2016-01-01

    In the mid-1970s, it was established through transmission experiments and ultrastructural studies of sarcocysts by transmission electron microscopy (TEM) that cattle was the intermediate host of three Sarcocystis spp. using dogs, cats and humans, respectively, as definitive hosts. The cat-transmitted species with microscopic sarcocysts was initially named Sarcocystis bovifelis, but it was soon renamed Sarcocystis hirsuta, since it was considered to be identical with a previously named species. In recent years, an apparently new species has been detected in cattle in several countries by molecular methods and TEM and found by both methods to be indistinguishable from Sarcocystis sinensis in water buffaloes. This species was recently named Sarcocystis rommeli. Beginning in August 2014, a thorough review of papers comprising TEM micrographs of thick-walled sarcocysts in cattle was made in order to determine whether S. sinensis-like sarcocysts had been reported previously under other designations. Surprisingly, the review showed that the species S. bovifelis Heydorn et al., 1975 as described from cattle in Germany was S. sinensis-like and that indistinguishable sarcocysts had also been found in cattle in New Zealand and Canada in the 1980s. However, in the New Zealand study, these small sarcocysts were erroneously thought to represent developmental stages of a species with ultrastructurally similar but macroscopic sarcocysts, since the macroscopic cysts were found to be infective for cats. Thus, in the late 1980s, the cat-transmitted S. bovifelis, after having been renamed S. hirsuta, was erroneously synonymised with a second cat-transmitted species in cattle and then slid into obscurity until recently being rediscovered as a S. sinensis-like species in cattle and then named S. rommeli. Following the erroneous synonymisation, the name S. hirsuta has consistently been used for a taxon with macroscopic sarcocysts, and this usage should be continued. The name S. bovifelis

  9. Ecology of cryptic invasions: latitudinal segregation among Watersipora (Bryozoa) species.

    PubMed

    Mackie, Joshua A; Darling, John A; Geller, Jonathan B

    2012-01-01

    Watersipora is an invasive genus of bryozoans, easily dispersed by fouled vessels. We examined Cytochrome c oxidase subunit I haplotypes from introduced populations on the US Pacific coastline to investigate geographic segregation of species and/or haplotypes. In California, the W. subtorquata group fell into three major sub-groups: W. subtorquata clades A and B, and W. "new sp.". W. subtorquata clades A and B were common in southern California south of Point Conception, a recognized biogeographic boundary, whereas further north, W. subtorquata clade A and W. n. sp. were frequent. The southern California region also had colonies of a morphologically distinct species, W. arcuata, also found in southern Australia and Hawaii; COI variation indicates a common ancestral source(s) in these introductions. The distribution of Watersipora-complex lineages on different coastlines is shown to be temperature correlated. Accordingly, pre-exisitng temperature-based adaptations may play a key role in determining invasion patterns.

  10. Taxonomy and phylogenetic relationships of nine species of Hypocrea with anamorphs assignable to Trichoderma section Hypocreanum

    PubMed Central

    Overton, Barrie E.; Stewart, Elwin L.; Geiser, David M.

    2006-01-01

    Morphological studies and phylogenetic analyses of DNA sequences from the internal transcribed spacer (ITS) regions of the nuclear ribosomal gene repeat, a partial sequence of RNA polymerase II subunit (rpb2), and a partial sequence of the large exon of tef1 (LEtef1) were used to investigate the taxonomy and systematics of nine Hypocrea species with anamorphs assignable to Trichoderma sect. Hypocreanum. Hypocrea corticioides and H. sulphurea are reevaluated. Their Trichoderma anamorphs are described and the phylogenetic positions of these species are determined. Hypocrea sulphurea and H. subcitrina are distinct species based on studies of the type specimens. Hypocrea egmontensis is a facultative synonym of the older name H. subcitrina. Hypocrea with anamorphs assignable to Trichoderma sect. Hypocreanum formed a well-supported clade. Five species with anamorphs morphologically similar to sect. Hypocreanum, H. avellanea, H. parmastoi, H. megalocitrina, H. alcalifuscescens, and H. pezizoides, are not located in this clade. Protocrea farinosa belongs to Hypocrea s.s. PMID:18490989

  11. Two new species of Lactarius associated with Alnus acuminata subsp. arguta in Mexico.

    PubMed

    Montoya, Leticia; Bandala, Victor M; Garay, Edith

    2014-01-01

    In pure stands of Alnus acuminata subsp. arguta trees from Sierra Norte de Puebla (central Mexico) two undescribed ectomycorrhizal species of Lactarius were discovered. Distinction of the two new species is based on morphological characters and supported with phylogenetic analyses of the nuclear ribosomal DNA ITS region and part of the gene that encodes for the second largest subunit of RNA polymerase II (rpb2). The phylogenies inferred recovered the two species in different clades strongly supported by posterior probabilities and bootstrap values. The new Lactarius species are recognized as part of the assemblage of ectomycorrhizal fungi associated with Alnus acuminata. Information about these taxa includes the morphological variation achieved along 16 monitories 2010-2013. Descriptions are provided. They are accompanied by photos including SEM photomicrographs of basidiospores and information on differences between them and other related taxa from Europe and the United States.

  12. Mosquitoes of Anopheles hyrcanus (Diptera, Culicidae) Group: Species Diagnostic and Phylogenetic Relationships

    PubMed Central

    Khrabrova, Natalia V.; Andreeva, Yulia V.; Sibataev, Anuarbek K.; Alekseeva, Svetlana S.; Esenbekova, Perizat A.

    2015-01-01

    Herein, we report the results of study of Anopheles species in Primorsk and Khabarovsk regions of Russia. Three species of the Anopheles hyrcanus group: An. kleini, An. pullus, and An. lesteri were identified by molecular taxonomic diagnostics for the first time in Russia. Surprisingly, An. sinensis, which earlier was considered the only species of Anopheles in Russian Far East, was not observed. We analyzed nucleotide variation in the 610-bp fragment of the 5′ end of the cytochrome c oxidase subunit I (COI) region. All species possessed a distinctive set of COI sequences. A maximum likelihood phylogenetic tree was constructed for members of the hyrcanus group. The examined Anopheles hyrcanus group members could be divided into two major subgroups: subgroup 1 (An. hyrcanus and An. pullus) and subgroup 2 (An. sinensis, An. kleini, and An. lesteri), which were found to be monophyletic. PMID:26149867

  13. Protein synthesis by ribosomes with tethered subunits.

    PubMed

    Orelle, Cédric; Carlson, Erik D; Szal, Teresa; Florin, Tanja; Jewett, Michael C; Mankin, Alexander S

    2015-08-06

    The ribosome is a ribonucleoprotein machine responsible for protein synthesis. In all kingdoms of life it is composed of two subunits, each built on its own ribosomal RNA (rRNA) scaffold. The independent but coordinated functions of the subunits, including their ability to associate at initiation, rotate during elongation, and dissociate after protein release, are an established model of protein synthesis. Furthermore, the bipartite nature of the ribosome is presumed to be essential for biogenesis, since dedicated assembly factors keep immature ribosomal subunits apart and prevent them from translation initiation. Free exchange of the subunits limits the development of specialized orthogonal genetic systems that could be evolved for novel functions without interfering with native translation. Here we show that ribosomes with tethered and thus inseparable subunits (termed Ribo-T) are capable of successfully carrying out protein synthesis. By engineering a hybrid rRNA composed of both small and large subunit rRNA sequences, we produced a functional ribosome in which the subunits are covalently linked into a single entity by short RNA linkers. Notably, Ribo-T was not only functional in vitro, but was also able to support the growth of Escherichia coli cells even in the absence of wild-type ribosomes. We used Ribo-T to create the first fully orthogonal ribosome-messenger RNA system, and demonstrate its evolvability by selecting otherwise dominantly lethal rRNA mutations in the peptidyl transferase centre that facilitate the translation of a problematic protein sequence. Ribo-T can be used for exploring poorly understood functions of the ribosome, enabling orthogonal genetic systems, and engineering ribosomes with new functions.

  14. Kainate receptor subunit diversity underlying response diversity in retinal Off bipolar cells

    PubMed Central

    Lindstrom, Sarah H; Ryan, David G; Shi, Jun; DeVries, Steven H

    2014-01-01

    Postsynaptic kainate receptors mediate excitatory synaptic transmission over a broad range of temporal frequencies. In heterologous systems, the temporal responses of kainate receptors vary when different channel-forming and auxiliary subunits are co-expressed but how this variability relates to the temporal differences at central synapses is incompletely understood. The mammalian cone photoreceptor synapse provides advantages for comparing the different temporal signalling roles of kainate receptors, as cones release glutamate over a range of temporal frequencies, and three functionally distinct Off bipolar cell types receive cone signals at synapses that contain either AMPA or kainate receptors, all with different temporal properties. A disadvantage is that the different receptor subunits are not identified. We used in situ hybridization, immunocytochemistry, and pharmacology to identify the kainate receptor and auxiliary subunits in ground squirrel (Ictidomys tridecimlineatus) cb1a/b, cb2, and cb3a/b Off bipolar cell types. As expected, the types showed distinct subunit expression patterns. Kainate receptors mediated ∼80% of the synaptic response in cb3a/b cells and were heteromers of GluK1 and GluK5. Cb3a/b cells contained message for GluK1 and GluK5, and also GluK3 and the auxiliary subunit Neto1. The synaptic responses in cb1a/b cells were mediated by GluK1-containing kainate receptors that behaved differently from the receptors expressed by cb3a/b cells. AMPA receptors mediated the entire synaptic response in cb2 cells and the remaining synaptic response in cb3a/b cells. We conclude that GluK1 is the predominant kainate receptor subunit in cb1 and cb3 Off bipolar cells. Different temporal response properties may result from selective association with GluK3, GluK5, or Neto1. PMID:24396054

  15. Ricin and Ricinus communis agglutinin subunits are all derived from a single-size polypeptide precursor.

    PubMed

    Butterworth, A G; Lord, J M

    1983-12-01

    Antibodies have been raised in rabbits against the individually purified A and B subunits of the toxic castor bean lectin, ricin, and against the A' and B' subunits of Ricinus communis agglutinin type I. Each of the antisera recognised a single polypeptide species of Mr 60 500 when maturing castor bean endosperm mRNA was translated in vitro in a rabbit-reticulocyte-derived system. When dog pancreatic microsomal vesicles were included in the translational system, each subunit antiserum precipitated a group of 66 000-68 000-Mr core-glycosylated polypeptides which had been translocated into the lumen of the vesicles. The 60 500-Mr polypeptide appeared to be a common precursor to all four individual lectin subunits since (a) its glycosylated (66 000-68 000-Mr) forms were readily detected in the endoplasmic reticulum fraction isolated from maturing castor bean endosperm and (b) pulse-chase studies showed that the glycosylated precursors disappeared from the endoplasmic reticulum fraction with the concomittant appearance of authentic lectin subunits in a soluble protein fraction which included protein body matrix components. Antiserum prepared against whole R. communis agglutinin, type I, also precipitated the 65 000-Mr precursor in vitro and in vivo, but in addition precipitated a non-glycosylated 34 000-Mr polypeptide. This smaller protein is not a lectin subunit precursor, contradicting an earlier suggestion. It is most probably a precursor to the 2-S albumin storage proteins found in castor bean endosperm protein bodies.

  16. 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.

  17. Binding of ATP by pertussis toxin and isolated toxin subunits

    SciTech Connect

    Hausman, S.Z.; Manclark, C.R.; Burns, D.L. )

    1990-07-03

    The binding of ATP to pertussis toxin and its components, the A subunit and B oligomer, was investigated. Whereas, radiolabeled ATP bound to the B oligomer and pertussis toxin, no binding to the A subunit was observed. The binding of ({sup 3}H)ATP to pertussis toxin and the B oligomer was inhibited by nucleotides. The relative effectiveness of the nucleotides was shown to be ATP > GTP > CTP > TTP for pertussis toxin and ATP > GTP > TTP > CTP for the B oligomer. Phosphate ions inhibited the binding of ({sup 3}H)ATP to pertussis toxin in a competitive manner; however, the presence of phosphate ions was essential for binding of ATP to the B oligomer. The toxin substrate, NAD, did not affect the binding of ({sup 3}H)ATP to pertussis toxin, although the glycoprotein fetuin significantly decreased binding. These results suggest that the binding site for ATP is located on the B oligomer and is distinct from the enzymatically active site but may be located near the eukaryotic receptor binding site.

  18. An assembly landscape for the 30S ribosomal subunit

    PubMed Central

    Talkington, Megan W. T.; Siuzdak, Gary

    2005-01-01

    Self-assembling macromolecular machines drive fundamental cellular processes, including transcription, mRNA processing, translation, DNA replication, and cellular transport. The ribosome, which carries out protein synthesis, is one such machine, and the 30S subunit of the bacterial ribosome is the preeminent model system for biophysical analysis of large RNA-protein complexes. Our understanding of 30S assembly is incomplete, due to the challenges of monitoring the association of many components simultaneously. We have developed a new method involving pulse-chase monitored by quantitative mass spectrometry (PC/QMS) to follow the assembly of the 20 ribosomal proteins with 16S rRNA during formation of the functional particle. These data represent the first detailed and quantitative kinetic characterization of the assembly of a large multicomponent macromolecular complex. By measuring the protein binding rates at a range of temperatures, we have found that local transformations throughout the assembling subunit have similar but distinct activation energies. This observation shows that the prevailing view of 30S assembly as a pathway proceeding through a global rate-limiting conformational change must give way to a view in which the assembly of the complex traverses a landscape dotted with a variety of local conformational transitions. PMID:16319883

  19. Dissecting structural basis of the unique substrate selectivity of human enteropeptidase catalytic subunit.

    PubMed

    Ostapchenko, Valeriy G; Gasparian, Marine E; Kosinsky, Yurij A; Efremov, Roman G; Dolgikh, Dmitry A; Kirpichnikov, Mikhail P

    2012-01-01

    Enteropeptidase is a key enzyme in the digestion system of higher animals. It initiates enzymatic cascade cleaving trypsinogen activation peptide after a unique sequence DDDDK. Recently, we have found specific activity of human enteropeptidase catalytic subunit (L-HEP) being significantly higher than that of its bovine ortholog (L-BEP). Moreover, we have discovered that L-HEP hydrolyzed several nonspecific peptidic substrates. In this work, we aimed to further characterize species-specific enteropeptidase activities and to reveal their structural basis. First, we compared hydrolysis of peptides and proteins lacking DDDDK sequence by L-HEP and L-BEP. In each case human enzyme was more efficient, with the highest hydrolysis rate observed for substrates with a large hydrophobic residue in P2-position. Computer modeling suggested enzyme exosite residues 96 (Arg in L-HEP, Lys in L-BEP) and 219 (Lys in L-HEP, Gln in L-BEP) to be responsible for these differences in enteropeptidase catalytic activity. Indeed, human-to-bovine mutations Arg96Lys, Lys219Gln shifted catalytic properties of L-HEP toward those of L-BEP. This effect was amplified in case of the double mutation Arg96Lys/Lys219Gln, but still did not cover the full difference in catalytic activities of human and bovine enzymes. To find a missing link, we studied monopeptide benzyl-arginine-β-naphthylamide hydrolysis. L-HEP catalyzed it with an order lower K (m) than L-BEP, suggesting the monopeptide-binding S1 site input into catalytic distinction between two enteropeptidase species. Together, our findings suggest structural basis of the unique catalytic properties of human enteropeptidase and instigate further studies of its tentative physiological and pathological roles.

  20. Mixed Infections of Four Viruses, the Incidence and Phylogenetic Relationships of Sweet Potato Chlorotic Fleck Virus (Betaflexiviridae) Isolates in Wild Species and Sweetpotatoes in Uganda and Evidence of Distinct Isolates in East Africa

    PubMed Central

    Tugume, Arthur K.; Mukasa, Settumba B.; Valkonen, Jari P. T.

    2016-01-01

    Viruses infecting wild flora may have a significant negative impact on nearby crops, and vice-versa. Only limited information is available on wild species able to host economically important viruses that infect sweetpotatoes (Ipomoea batatas). In this study, Sweet potato chlorotic fleck virus (SPCFV; Carlavirus, Betaflexiviridae) and Sweet potato chlorotic stunt virus (SPCSV; Crinivirus, Closteroviridae) were surveyed in wild plants of family Convolvulaceae (genera Astripomoea, Ipomoea, Hewittia and Lepistemon) in Uganda. Plants belonging to 26 wild species, including annuals, biannuals and perennials from four agro-ecological zones, were observed for virus-like symptoms in 2004 and 2007 and sampled for virus testing. SPCFV was detected in 84 (2.9%) of 2864 plants tested from 17 species. SPCSV was detected in 66 (5.4%) of the 1224 plants from 12 species sampled in 2007. Some SPCSV-infected plants were also infected with Sweet potato feathery mottle virus (SPFMV; Potyvirus, Potyviridae; 1.3%), Sweet potato mild mottle virus (SPMMV; Ipomovirus, Potyviridae; 0.5%) or both (0.4%), but none of these three viruses were detected in SPCFV-infected plants. Co-infection of SPFMV with SPMMV was detected in 1.2% of plants sampled. Virus-like symptoms were observed in 367 wild plants (12.8%), of which 42 plants (11.4%) were negative for the viruses tested. Almost all (92.4%) the 419 sweetpotato plants sampled from fields close to the tested wild plants displayed virus-like symptoms, and 87.1% were infected with one or more of the four viruses. Phylogenetic and evolutionary analyses of the 3′-proximal genomic region of SPCFV, including the silencing suppressor (NaBP)- and coat protein (CP)-coding regions implicated strong purifying selection on the CP and NaBP, and that the SPCFV strains from East Africa are distinguishable from those from other continents. However, the strains from wild species and sweetpotato were indistinguishable, suggesting reciprocal movement of SPCFV

  1. Mixed Infections of Four Viruses, the Incidence and Phylogenetic Relationships of Sweet Potato Chlorotic Fleck Virus (Betaflexiviridae) Isolates in Wild Species and Sweetpotatoes in Uganda and Evidence of Distinct Isolates in East Africa.

    PubMed

    Tugume, Arthur K; Mukasa, Settumba B; Valkonen, Jari P T

    2016-01-01

    Viruses infecting wild flora may have a significant negative impact on nearby crops, and vice-versa. Only limited information is available on wild species able to host economically important viruses that infect sweetpotatoes (Ipomoea batatas). In this study, Sweet potato chlorotic fleck virus (SPCFV; Carlavirus, Betaflexiviridae) and Sweet potato chlorotic stunt virus (SPCSV; Crinivirus, Closteroviridae) were surveyed in wild plants of family Convolvulaceae (genera Astripomoea, Ipomoea, Hewittia and Lepistemon) in Uganda. Plants belonging to 26 wild species, including annuals, biannuals and perennials from four agro-ecological zones, were observed for virus-like symptoms in 2004 and 2007 and sampled for virus testing. SPCFV was detected in 84 (2.9%) of 2864 plants tested from 17 species. SPCSV was detected in 66 (5.4%) of the 1224 plants from 12 species sampled in 2007. Some SPCSV-infected plants were also infected with Sweet potato feathery mottle virus (SPFMV; Potyvirus, Potyviridae; 1.3%), Sweet potato mild mottle virus (SPMMV; Ipomovirus, Potyviridae; 0.5%) or both (0.4%), but none of these three viruses were detected in SPCFV-infected plants. Co-infection of SPFMV with SPMMV was detected in 1.2% of plants sampled. Virus-like symptoms were observed in 367 wild plants (12.8%), of which 42 plants (11.4%) were negative for the viruses tested. Almost all (92.4%) the 419 sweetpotato plants sampled from fields close to the tested wild plants displayed virus-like symptoms, and 87.1% were infected with one or more of the four viruses. Phylogenetic and evolutionary analyses of the 3'-proximal genomic region of SPCFV, including the silencing suppressor (NaBP)- and coat protein (CP)-coding regions implicated strong purifying selection on the CP and NaBP, and that the SPCFV strains from East Africa are distinguishable from those from other continents. However, the strains from wild species and sweetpotato were indistinguishable, suggesting reciprocal movement of SPCFV

  2. Identification and characterization of high-molecular-weight glutenin subunits from Agropyron intermedium.

    PubMed

    Cao, Shuanghe; Li, Zhixin; Gong, Caiyan; Xu, Hong; Yang, Ran; Hao, Shanting; Wang, Xianping; Wang, Daowen; Zhang, Xiangqi

    2014-01-01

    High-molecular-weight glutenin subunit (HMW-GS) is a primary determinant of processing quality of wheat. Considerable progress has been made in understanding the structure, function and genetic regulation of HMW-GS in wheat and some of its related species, but less is known about their orthologs in Agropyron intermedium, a useful related species for wheat improvement. Here seven HMW-GSs in Ag. intermedium were identified using SDS-PAGE and Western blotting experiments. Subsequently, the seven genes (Glu-1Aix1 ∼ 4 and Glu-1Aiy1 ∼ 3) encoding the seven HMW-GSs were isolated using PCR technique with degenerate primers, and confirmed by bacterial expression and Western blotting. Sequence analysis indicated that the seven Ag. intermedium HMW-GSs shared high similarity in primary structure to those of wheat, but four of the seven subunits were unusually small compared to the representatives of HMW-GS from wheat and two of them possessed extra cysteine residues. The alignment and clustering analysis of deduced amino acid sequences revealed that 1Aix1 and 1Aiy1 subunits had special molecular structure, belonging to the hybrid type compounding between typical x- and y-type subunit. The xy-type subunit 1Aix1 is composed of the N-terminal of x-type and C-terminal of y-type, whereas yx-type subunit 1Aiy1 comprises the N-terminal of y-type and C-terminal of x-type. This result strongly supported the hypothesis of unequal crossover mechanism that might generate the novel coding sequence for the hybrid type of HMW-GSs. In addition to the aforementioned, the other novel characteristics of the seven subunits were also discussed. Finally, phylogenetic analysis based on HMW-GS genes was carried out and provided new insights into the evolutionary biology of Ag. intermedium.

  3. Carrier subunit of plasma membrane transporter is required for oxidative folding of its helper subunit.

    PubMed

    Rius, Mònica; Chillarón, Josep

    2012-05-25

    We study the amino acid transport system b(0,+) as a model for folding, assembly, and early traffic of membrane protein complexes. System b(0,+) is made of two disulfide-linked membrane subunits: the carrier, b(0,+) amino acid transporter (b(0,+)AT), a polytopic protein, and the helper, related to b(0,+) amino acid transporter (rBAT), a type II glycoprotein. rBAT ectodomain mutants display folding/trafficking defects that lead to type I cystinuria. Here we show that, in the presence of b(0,+)AT, three disulfides were formed in the rBAT ectodomain. Disulfides Cys-242-Cys-273 and Cys-571-Cys-666 were essential for biogenesis. Cys-673-Cys-685 was dispensable, but the single mutants C673S, and C685S showed compromised stability and trafficking. Cys-242-Cys-273 likely was the first disulfide to form, and unpaired Cys-242 or Cys-273 disrupted oxidative folding. Strikingly, unassembled rBAT was found as an ensemble of different redox species, mainly monomeric. The ensemble did not change upon inhibition of rBAT degradation. Overall, these results indicated a b(0,+)AT-dependent oxidative folding of the rBAT ectodomain, with the initial and probably cotranslational formation of Cys-242-Cys-273, followed by the oxidation of Cys-571-Cys-666 and Cys-673-Cys-685, that was completed posttranslationally.

  4. 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.

  5. 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.

  6. Molecular phylogeny of Stentor (Ciliophora: Heterotrichea) based on small subunit ribosomal RNA sequences.

    PubMed

    Gong, Ying-Chun; Yu, Yu-He; Zhu, Fei-Yun; Feng, Wei-Song

    2007-01-01

    To determine the phylogenetic position of Stentor within the Class Heterotrichea, the complete small subunit rRNA genes of three Stentor species, namely Stentor polymorphus, Stentor coeruleus, and Stentor roeseli, were sequenced and used to construct phylogenetic trees using the maximum parsimony, neighbor joining, and Bayesian analysis. With all phylogenetic methods, the genus Stentor was monophyletic, with S. roeseli branching basally.

  7. Electrophoretic analysis of the high-molecular-weight glutenin subunits of Triticum monococcum, T. urartu, and the A genome of bread wheat (T. aestivum).

    PubMed

    Waines, J G; Payne, P I

    1987-05-01

    The high molecular weight (HMW) subunit composition of glutenin was analysed by sodium dodecyl sulphate, polyacrylamide gel electrophoresis (SDS-PAGE) in the A genome of 497 diploid wheats and in 851 landraces of bread wheat. The material comprised 209 accessions of wild Triticum monococcum ssp. boeoticum from Greece, Turkey, Lebanon, Armenia, Iraq, and Iran; 132 accessions of the primitive domesticate T. monococcum ssp. monococcum from many different germplasm collections; one accession of free-threshing T. monococcum ssp. sinskajae; 155 accessions of wild T. urartu from Lebanon, Turkey, Armenia, Iraq, and Iran; and landraces of T. aestivum, mainly from the Mediterranean area and countries bordering on the Himalayan Mountains. Four novel HMW glutenin sub-units were discovered in the landraces of bread wheat, and the alleles that control them were designated Glu-Ald through Glu-Alg, respectively. The HMW subunits of T. monococcum ssp. boeoticum have a major, "x" subunit of slow mobility and several, less prominent, "y" subunits of greater mobility, all of which fall within the mobility range of HMW subunits reported for bread wheat. In T. monococcum ssp. monococcum the range of the banding patterns for HMW subunits was similar to that of ssp. boeoticum. However, two accessions, while containing "y" subunits were null for "x" subunits. The single accession of Triticum monococcum ssp. sinskajae had a banding pattern similar to that of most ssp. boeoticum and ssp. monococcum accessions. The HMW subunit banding patterns of T. urartu accessions were distinct from those of T. monococcum. All of them contained one major "x" and most contained one major "y" subunit. In the other accessions a "y" subunit was not expressed. The active genes for "y" subunits, if transferred to bread wheat, may be useful in improving bread-making quality.

  8. Drosophila laminin: sequence of B2 subunit and expression of all three subunits during embryogenesis

    PubMed Central

    1989-01-01

    In a previous study, we described the cloning of the genes encoding the three subunits of Drosophila laminin, a substrate adhesion molecule, and the cDNA sequence of the B1 subunit (Montell and Goodman, 1988). This analysis revealed the similarity of Drosophila laminin with the mouse and human complexes in subunit composition, domain structure, and amino acid sequence. In this paper, we report the deduced amino acid sequence of the B2 subunit. We then describe the expression and tissue distribution of the three subunits of laminin during Drosophila embryogenesis using both in situ hybridization and immunolocalization techniques, with particular emphasis on its expression in and around the developing nervous system. PMID:2808533

  9. Gonococcal pilus subunit size heterogeneity correlates with transitions in colony piliation phenotype, not with changes in colony opacity

    PubMed Central

    1983-01-01

    The apparent subunit sizes for pili of gonococci (Gc) have been visualized by using either Iodogen 125I-labeled whole Gc or immunoblotting with antipilus antiserum. These methods permitted definition of pilus subunit sizes for Gc of a given strain that had undergone changes either in piliation phenotype or in colonial opacity/protein II phenotype. The results indicate that pilus subunit size does not change coincident with changes in colony opacity/protein II phenotypes; but change in pilus subunit size is seen after a change in piliation phenotype (P+ leads to P++, and vice versa). Marked diversity in pilus subunit sizes is found for Gc of individual strains when P+ derivatives of P- colonies are compared. This diversity extends to pilus subunits of Gc found in single colonies; two distinct pilus forms were demonstrated for Gc residing in several single colonies. These findings show that Gc of a given strain are able to express any of a number of different pilus subunit size forms. PMID:6138388

  10. Functional characterisation of the regulatory subunit of cyclic AMP-dependent protein kinase A homologue of Giardia lamblia: Differential expression of the regulatory and catalytic subunits during encystation.

    PubMed

    Gibson, Candace; Schanen, Brian; Chakrabarti, Debopam; Chakrabarti, Ratna

    2006-06-01

    To understand the functional roles of protein kinase A (PKA) during vegetative and differentiating states of Giardia parasites, we studied the structural and functional characteristics of the regulatory subunit of PKA (gPKAr) and its involvement in the giardial encystment process. Molecular cloning and characterisation showed that gPKAr contains two tandem 3'5'-cyclic adenosine monphosphate (cyclic AMP) binding domains at the C-terminal end and the interaction domain for the catalytic subunit. A number of consensus residues including in vivo phosphorylation site for PKAc and dimerisation/docking domain are present in gPKAr. The regulatory subunit physically interacts with the catalytic subunit and inhibits its kinase activity in the absence of cyclic AMP, which could be partially restored upon addition of cyclic AMP. Western blot analysis showed a marked reduction in the endogenous gPKAr concentration during differentiation of Giardia into cysts. An increased activity of gPKAc was also detected during encystation without any significant change in the protein concentration. Distinct localisations of gPKAc to the anterior flagella, basal bodies and caudal flagella as noted in trophozoites were absent in encysting cells at later stages. Instead, PKAc staining was punctate and located mostly to the cell periphery. Our study indicates possible enrichment of the active gPKAc during late stages of encystation, which may have implications in completion of the encystment process or priming of cysts for efficient excystation.

  11. Characterization of three homologs of the large subunit of the magnesium chelatase from Chlorobaculum tepidum and interaction with the magnesium protoporphyrin IX methyltransferase.

    PubMed

    Johnson, Ethan T; Schmidt-Dannert, Claudia

    2008-10-10

    Green bacteria synthesize several types of (bacterio)chlorophylls for the assembly of functional photosynthetic reaction centers and antenna complexes. A distinctive feature of green bacteria compared with other photosynthetic microbes is that their genomes contain multiple homologs of the large subunit (BchH) of the magnesium chelatase which is a three-subunit enzyme complex (BchH, BchD, and BchI) that inserts magnesium into protoporphyrin IX as the first committed step of (bacterio)chlorophyll biosynthesis. There is speculation that the additional BchH homologs may regulate the biosynthesis of each type of chlorophyll, although the biochemical properties of the different magnesium chelatase complexes from a single species of green bacteria have not yet been compared. In this study, we investigated the activities of all three chelatase complexes from the green sulfur bacterium Chlorobaculum tepidum and interactions with the next enzyme in the pathway, magnesium protoporphyrin IX methyltransferase (BchM). Although all three chelatase complexes insert magnesium into protoporphyrin IX, the activities range by a factor of 10(5). Further, there are differences in the interactions between the BchH homologs and BchM; two of the subunits increase the methyltransferase activity by 30-60%, and the third decreases it by 30%. Expression of the chelatase complexes alone and together with BchM in Escherichia coli overproducing protoporphyrin IX suggests that the chelatase is the rate-limiting enzyme. We observed that BchM uses protoporphyrin IX without bound metal as a substrate. Our results conflict with expectations generated by previous gene inactivation studies and suggest a complex regulation of chlorophyll biosynthesis in green bacteria.

  12. Subunit Recombinant Vaccine Protects Against Monkeypox

    DTIC Science & Technology

    2006-05-27

    Subunit Recombinant Vaccine Protects against Monkeypox1 Jean-Michel Heraud,* Yvette Edghill-Smith,*† Victor Ayala,‡ Irene Kalisz,‡ Janie Parrino ...GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Heraud, JM Edghill-Smith, Y Ayala, V Kalisz, I Parrino , J Kalyanaraman, VS Manischewitz, J King

  13. Foundations of Distinctive Feature Theory.

    ERIC Educational Resources Information Center

    Baltaxe, Christiane A. M.

    This treatise on the theoretical and historical foundations of distinctive feature theory traces the evolution of the distinctive features concept in the context of related notions current in linguistic theory, discusses the evolution of individual distinctive features, and criticizes certain acoustic and perceptual correlates attributed to these…

  14. Flexible subunit stoichiometry of functional human P2X2/3 heteromeric receptors.

    PubMed

    Kowalski, Maria; Hausmann, Ralf; Schmid, Julia; Dopychai, Anke; Stephan, Gabriele; Tang, Yong; Schmalzing, Günther; Illes, Peter; Rubini, Patrizia

    2015-12-01

    The aim of the present work was to clarify whether heterotrimeric P2X2/3 receptors have a fixed subunit stoichiometry consisting of one P2X2 and two P2X3 subunits as previously suggested, or a flexible stoichiometry containing also the inverse subunit composition. For this purpose we transfected HEK293 cells with P2X2 and P2X3 encoding cDNA at the ratios of 1:2 and 4:1, and analysed the biophysical and pharmacological properties of the generated receptors by means of the whole-cell patch-clamp technique. The concentration-response curves for the selective agonist α,β-meATP did not differ from each other under the two transfection ratios. However, co-expression of an inactive P2X2 mutant and the wild type P2X3 subunit and vice versa resulted in characteristic distortions of the α,β-meATP concentration-response relationships, depending on which subunit was expressed in excess, suggesting that HEK293 cells express mixtures of (P2X2)1/(P2X3)2 and (P2X2)2/(P2X3)1 receptors. Whereas the allosteric modulators H+ and Zn2+ failed to discriminate between the two possible heterotrimeric receptor variants, the α,β-meATP-induced responses were blocked more potently by the competitive antagonist A317491, when the P2X2 subunit was expressed in deficit of the P2X3 subunit. Furthermore, blue-native PAGE analysis of P2X2 and P2X3 subunits co-expressed in Xenopus laevis oocytes and HEK293 cells revealed that plasma membrane-bound P2X2/3 receptors appeared in two clearly distinct heterotrimeric complexes: a (P2X2-GFP)2/(P2X3)1 complex and a (P2X2-GFP)1/(P2X3)2 complex. These data strongly indicate that the stoichiometry of the heteromeric P2X2/3 receptor is not fixed, but determined in a permutational manner by the relative availability of P2X2 and P2X3 subunits.

  15. Progress in the development of subunit vaccines for gastrointestinal nematodes of ruminants.

    PubMed

    Matthews, J B; Geldhof, P; Tzelos, T; Claerebout, E

    2016-12-01

    The global increase in anthelmintic resistant nematodes of ruminants, together with consumer concerns about chemicals in food, necessitates the development of alternative methods of control for these pathogens. Subunit recombinant vaccines are ideally placed to fill this gap. Indeed, they are probably the only valid option for the long-term control of ruminant parasitic nematodes given the increasing ubiquity of multidrug resistance in a range of worm species across the world. The development of a subunit multicellular parasite vaccine to the point of practical application would be a groundbreaking step in the control of these important endemic infections of livestock. This review summarizes the current status of subunit vaccine development for a number of important gastrointestinal nematodes of cattle and sheep, with a focus on the limitations and problems encountered thus far, and suggestions as to how these hurdles might be overcome.

  16. Heterogeneity of the alpha subunit of tubulin and the variability of tubulin within a single organism.

    PubMed

    Bibring, T; Baxandall, J; Denslow, S; Walker, B

    1976-05-01

    When tubulins obtained from particular microtubules of the sea urchin (ciliary doublet A tubules, flagellar doublet microtubules, and mitotic microtubules) are analyzed by electrophoresis in a polyacrylamide gel system containing sodium dodecyl sulfate and urea, heterogeneity of the alpha subunit, and differences between the tubulins are revealed. The alpha subunit of tubulin from mitotic apparatus and from A microtubules of ciliary doublets is resolved into two bands, while the alpha subunit of flagellar doublet tubulin gives a single band. The mitotic and ciliary tubulins differ in the mobilities of their two alpha species, or in the relative amounts present, or both. The existence of differences between the tubulins has been confirmed by a preliminary analysis of their cyanogen bromide peptides.

  17. Heterogeneity of the alpha subunit of tubulin and the variability of tubulin within a single organism

    PubMed Central

    1976-01-01

    When tubulins obtained from particular microtubules of the sea urchin (ciliary doublet A tubules, flagellar doublet microtubules, and mitotic microtubules) are analyzed by electrophoresis in a polyacrylamide gel system containing sodium dodecyl sulfate and urea, heterogeneity of the alpha subunit, and differences between the tubulins are revealed. The alpha subunit of tubulin from mitotic apparatus and from A microtubules of ciliary doublets is resolved into two bands, while the alpha subunit of flagellar doublet tubulin gives a single band. The mitotic and ciliary tubulins differ in the mobilities of their two alpha species, or in the relative amounts present, or both. The existence of differences between the tubulins has been confirmed by a preliminary analysis of their cyanogen bromide peptides. PMID:1262392

  18. Characteristic views of E. coli and B. stearothermophilus 30S ribosomal subunits in the electron microscope.

    PubMed Central

    van Heel, M; Stöffler-Meilicke, M

    1985-01-01

    Large sets of electron microscopic images of the 30S ribosomal subunits of Bacillus stearothermophilus (914 molecules) and Escherichia coli (422 molecules) were analysed with image processing techniques. Using computer alignment and a new multivariate statistical classification scheme, three predominant views of the subunit were found for both species. These views, which together account for approximately 90% of the population of images, were determined to a reproducible resolution of up to 1.7 nm, thus elucidating many new structural details. The angular spread of the molecular orientations around the three main stable positions is remarkably small (less than 8 degrees). Some of the current models for the small ribosomal subunit are incompatible with our new results. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:3908096

  19. Biological properties of two distinct pilus types produced by isogenic variants of Neisseria gonorrhoeae P9.

    PubMed Central

    Lambden, P R; Robertson, J N; Watt, P J

    1980-01-01

    Isogenic variants from a single strain of Neisseria gonorrhoeae were shown to produce two distinct types of pili. These pili, designated alpha and beta, differed in both subunit molecular weight and in ability to attach to buccal epithelial cells. Images PMID:6101593

  20. Catalase and superoxide dismutase conjugated with platelet-endothelial cell adhesion molecule antibody distinctly alleviate abnormal endothelial permeability caused by exogenous reactive oxygen species and vascular endothelial growth factor.

    PubMed

    Han, Jingyan; Shuvaev, Vladimir V; Muzykantov, Vladimir R

    2011-07-01

    Reactive oxygen species (ROS) superoxide anion (O(2)()) and hydrogen peroxide (H(2)O(2)) produced by activated leukocytes and endothelial cells in sites of inflammation or ischemia cause endothelial barrier dysfunction that may lead to tissue edema. Antioxidant enzymes (AOEs) catalase and superoxide dismutase (SOD) conjugated with antibodies to platelet-endothelial cell adhesion molecule-1 (PECAM-1) specifically bind to endothelium, quench the corresponding ROS, and alleviate vascular oxidative stress and inflammation. In the present work, we studied the effects of anti-PECAM/catalase and anti-PECAM/SOD conjugates on the abnormal permeability manifested by transendothelial electrical resistance decline, increased fluorescein isothiocyanate-dextran influx, and redistribution of vascular endothelial-cadherin in human umbilical vein endothelial cell (HUVEC) monolayers. Anti-PECAM/catalase protected HUVEC monolayers against H(2)O(2)-induced endothelial barrier dysfunction. Polyethylene glycol-conjugated catalase exerted orders of magnitude lower endothelial uptake and no protective effect, similarly to IgG/catalase. Anti-PECAM/catalase, but not anti-PECAM/SOD, alleviated endothelial hyperpermeability caused by exposure to hypoxanthine/xanthine oxidase, implicating primarily H(2)O(2) in the disruption of the endothelial barrier in this model. Thrombin-induced endothelial permeability was not affected by treatment with anti-PECAM/AOEs or the NADPH oxidase inhibitor apocynin or overexpression of AOEs, indicating that the endogenous ROS play no key role in thrombin-mediated endothelial barrier dysfunction. In contrast, anti-PECAM/SOD, but not anti-PECAM/catalase, inhibited a vascular endothelial growth factor (VEGF)-induced increase in endothelial permeability, identifying a key role of endogenous O(2)() in the VEGF-mediated regulation of endothelial barrier function. Therefore, AOEs targeted to endothelial cells provide versatile molecular tools for testing the roles of

  1. Characterization of the Testis-specific Proteasome Subunit α4s in Mammals

    PubMed Central

    Uechi, Hiroyuki; Hamazaki, Jun; Murata, Shigeo

    2014-01-01

    The 26 S proteasome is responsible for regulated proteolysis in eukaryotic cells. It is composed of one 20 S core particle (CP) flanked by one or two 19 S regulatory particles. The CP is composed of seven different α-type subunits (α1-α7) and seven different β-type subunits, three of which are catalytic. Vertebrates encode four additional catalytic β subunits that are expressed predominantly in immune tissues and produce distinct subtypes of CPs particularly well suited for the acquired immune system. In contrast, the diversity of α subunits remains poorly understood. Recently, another α subunit, referred to as α4s, was reported. However, little is known about α4s. Here we provide a detailed characterization of α4s and the α4s-containing CP. α4s is exclusively expressed in germ cells that enter the meiotic prophase and is incorporated into the CP in place of α4. A comparison of structural models revealed that the differences in the primary sequences between α4 and α4s are located on the outer surface of the CP, suggesting that α4s interacts with specific molecules via these unique regions. α4s-containing CPs account for the majority of the CPs in mouse sperm. The catalytic β subunits in the α4s-containing CP are β1, β2, and β5, and immunosubunits are not included in the α4s-containing CP. α4s-containing CPs have a set of peptidase activities almost identical to those of α4-containing CPs. Our results provide a basis for understanding the role of α4s and male germ cell-specific proteasomes in mammals. PMID:24668818

  2. Post-translational modifications in the large subunit of ribulose bisphosphate carboxylase/oxygenase.

    PubMed

    Houtz, R L; Stults, J T; Mulligan, R M; Tolbert, N E

    1989-03-01

    Two adjacent N-terminal tryptic peptides of the large subunit of ribulose bisphosphate carboxylase/oxygenase [3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39] from spinach, wheat, tobacco, and muskmelon were removed by limited tryptic proteolysis. Characterization by peptide sequencing, amino acid composition, and tandem mass spectrometry revealed that the N-terminal residue from the large subunit of the enzyme from each plant species was acetylated proline. The sequence of the penultimate N-terminal tryptic peptide from the large subunit of the spinach and wheat enzyme was consistent with previous primary structure determinations. However, the penultimate N-terminal peptide from the large subunit of both the tobacco and muskmelon enzymes, while identical, differed from the corresponding peptide from spinach and wheat by containing a trimethyllysyl residue at position 14. Thus, tryptic proteolysis occurred at lysine-18 rather than lysine-14 as with the spinach and wheat enzymes. A comparison of the DNA sequences for the large subunit of ribulose bisphosphate carboxylase/oxygenase indicates that the N terminus has been post-translationally processed by removal of methionine-1 and serine-2 followed by acetylation of proline-3. In addition, for the enzyme from tobacco and muskmelon a third post-translational modification occurs at lysine-14 in the form of N epsilon-trimethylation.

  3. Fusarium foetens, a new species pathogenic to begonia elatior hybrids (Begonia x hiemalis) and the sister taxon of the Fusarium oxysporum species complex.

    PubMed

    Schroers, H-J; Baayen, R P; Meffert, J P; de Gruyter, J; Hooftman, M; O'Donnell, K

    2004-01-01

    A new disease recently was discovered in begonia elatior hybrid (Begonia × hiemalis) nurseries in The Netherlands. Diseased plants showed a combination of basal rot, vein yellowing and wilting and the base of collapsing plants was covered by unusually large masses of Fusarium macroconidia. A species of Fusarium was isolated consistently from the discolored veins of leaves and stems. It differed morphologically from F. begoniae, a known agent of begonia flower, leaf and stem blight. The Fusarium species resembled members of the F. oxysporum species complex in producing short monophialides on the aerial mycelium and abundant chlamydospores. Other phenotypic characters such as polyphialides formed occasionally in at least some strains, relatively long monophialides intermingled with the short monophialides formed on the aerial mycelium, distinct sporodochial conidiomata, and distinct pungent colony odor distinguished it from the F. oxysporum species complex. Phylogenetic analyses of partial sequences of the mitochondrial small subunit of the ribosomal DNA (mtSSU rDNA), nuclear translation elongation factor 1α (EF-1α) and β-tubulin gene exons and introns indicate that the Fusarium species represents a sister group of the F. oxysporum species complex. Begonia × hiemalis cultivars Bazan, Bellona and Netja Dark proved to be highly susceptible to the new species. Inoculated plants developed tracheomycosis within 4 wk, and most died within 8 wk. The new taxon was not pathogenic to Euphorbia pulcherrima, Impatiens walleriana and Saintpaulia ionantha that commonly are grown in nurseries along with B. × hiemalis. Inoculated plants of Cyclamen persicum did not develop the disease but had discolored vessels from which the inoculated fungus was isolated. Given that the newly discovered begonia pathogen is distinct in pathogenicity, morphology and phylogeny from other fusaria, it is described here as a new species, Fusarium foetens.

  4. Studies of double-labeled mouse thyrotropin and free alpha-subunits to estimate relative fucose content

    SciTech Connect

    Magner, J.; Papagiannes, E.

    1986-11-01

    The composition and structure of the complex oligosaccharides of thyrotropin (TSH) and free alpha-subunits are not well established, but are believed to be important determinants of the biological properties of these glycoproteins. We employed a simple double-label technique to learn the relative fucose content of mouse thyrotropin and free alpha-subunits. Thyrotropic tumor minces were incubated simultaneously with (/sup 35/S)methionine and (/sup 3/H)fucose. Thyrotropin and free alpha-subunits were labeled with both isotopes, and the ratio of /sup 3/H//sup 35/S was higher in free alpha-subunits than in thyrotropin; free alpha-subunits were approximately fivefold richer in fucose than was thyrotropin. The /sup 3/H//sup 35/S ratio was not substantially altered in TSH or free alpha-subunits secreted after a brief incubation with 10(-7) M thyrotropin-releasing hormone. Species which incorporated (/sup 3/H)fucose were resistant to endoglycosidase H. Thus, mouse free alpha-subunits secreted by thyrotropic tumor are relatively rich in fucose. Double-isotope labeling using an amino acid and a sugar appears to be a useful technique for studies of the glycoprotein hormones.

  5. The conserved Mediator subunit MDT-15 is required for oxidative stress responses in Caenorhabditis elegans

    PubMed Central

    Goh, Grace Y S; Martelli, Katherine L; Parhar, Kulveer S; Kwong, Ada W L; Wong, Marcus A; Mah, Allan; Hou, Nicole S; Taubert, Stefan

    2014-01-01

    Reactive oxygen species (ROS) play important signaling roles in metazoans, but also cause significant molecular damage. Animals tightly control ROS levels using sophisticated defense mechanisms, yet the transcriptional pathways that induce ROS defense remain incompletely understood. In the nematode Caenorhabditis elegans, the transcription factor SKN-1 is considered a master regulator for detoxification and oxidative stress responses. Here, we show that MDT-15, a subunit of the conserved Mediator complex, is also required for oxidative stress responses in nematodes. Specifically, mdt-15 is required to express SKN-1 targets upon chemical and genetic increase in SKN-1 activity. mdt-15 is also required to express genes in SKN-1-dependent and SKN-1-independent fashions downstream of insulin/IGF-1 signaling and for the longevity of daf-2/insulin receptor mutants. At the molecular level, MDT-15 binds SKN-1 through a region distinct from the classical transcription-factor-binding KIX-domain. Moreover, mdt-15 is essential for the transcriptional response to and survival on the organic peroxide tert-butyl-hydroperoxide (tBOOH), a largely SKN-1-independent response. The MDT-15 interacting nuclear hormone receptor, NHR-64, is specifically required for tBOOH but not arsenite resistance, but NHR-64 is dispensable for the transcriptional response to tBOOH. Hence, NHR-64 and MDT-15’s mode of action remain elusive. Lastly, the role of MDT-15 in oxidative stress defense is functionally separable from its function in fatty acid metabolism, as exogenous polyunsaturated fatty acid complementation rescues developmental, but not stress sensitivity phenotypes of mdt-15 worms. Our findings reveal novel conserved players in the oxidative stress response and suggest a broad cytoprotective role for MDT-15. PMID:23957350

  6. Serological reactivity of patients with Echinococcus infections (E. granulosus, E. vogeli, and E. multilocularis) against three antigen B subunits.

    PubMed

    de la Rue, Mário L; Yamano, Kimiaki; Almeida, Cybele E; Iesbich, Margarete P; Fernandes, Cloé D; Goto, Akiko; Kouguchi, Hirokazu; Takahashi, Kenichi

    2010-02-01

    In serodiagnosis of cystic echinococcosis (CE) by Echinococcus granulosus infection, antigen B (AgB) has been utilized worldwide. However, it is known that about 40% of sera with alveolar echinococcosis (AE) by Echinococcus multilocularis infection recognize AgB. Furthermore, cross-reaction against AgB was also reported in sera from polycystic echinococcosis (PE) patients with Echinococcus vogeli infection. These findings indicate that AgB is widely common to the genus Echinococcus. On the other hand, AgB has several subunits, which are composed of the smallest 8-kDa subunit. In this study, reactivities of patient sera with three kinds of Echinococcus infections (CE, PE, and AE) were compared simultaneously under the same condition against three subunits of AgB (8, 16, and 24 kDa). Many articles have referred the fundamental 8- kDa subunit as a diagnostic antigen for CE. However, the reactivity for the 8-kDa subunit of the CE patient was not so high (47.7%) in this study. Furthermore, there are many cases in which serum of patients with PE or AE also recognizes this subunit (66.7% in PE; 45.9% in AE). AgB is effective for the detection of the genus Echinococcus infections, but it does not have high species specificity. Therefore, we need to pay attention to cross-reaction in serodiagnosis of CE in areas where plural species coexist.

  7. Subunit interface mutants of rabbit muscle aldolase form active dimers.

    PubMed Central

    Beernink, P. T.; Tolan, D. R.

    1994-01-01

    We report the construction of subunit interface mutants of rabbit muscle aldolase A with altered quaternary structure. A mutation has been described that causes nonspherocytic hemolytic anemia and produces a thermolabile aldolase (Kishi H et al., 1987, Proc Natl Acad Sci USA 84:8623-8627). The disease arises from substitution of Gly for Asp-128, a residue at the subunit interface of human aldolase A. To elucidate the role of this residue in the highly homologous rabbit aldolase A, site-directed mutagenesis is used to replace Asp-128 with Gly, Ala, Asn, Gln, or Val. Rabbit aldolase D128G purified from Escherichia coli is found to be similar to human D128G by kinetic analysis, CD, and thermal inactivation assays. All of the mutant rabbit aldolases are similar to the wild-type rabbit enzyme in secondary structure and kinetic properties. In contrast, whereas the wild-type enzyme is a tetramer, chemical crosslinking and gel filtration indicate that a new dimeric species exists for the mutants. In sedimentation velocity experiments, the mutant enzymes as mixtures of dimer and tetramer at 4 degrees C. Sedimentation at 20 degrees C shows that the mutant enzymes are > 99.5% dimeric and, in the presence of substrate, that the dimeric species is active. Differential scanning calorimetry demonstrates that Tm values of the mutant enzymes are decreased by 12 degrees C compared to wild-type enzyme. The results indicate that Asp-128 is important for interface stability and suggest that 1 role of the quaternary structure of aldolase is to provide thermostability. PMID:7833800

  8. Recent Advances in Subunit Vaccine Carriers

    PubMed Central

    Vartak, Abhishek; Sucheck, Steven J.

    2016-01-01

    The lower immunogenicity of synthetic subunit antigens, compared to live attenuated vaccines, is being addressed with improved vaccine carriers. Recent reports indicate that the physio-chemical properties of these carriers can be altered to achieve optimal antigen presentation, endosomal escape, particle bio-distribution, and cellular trafficking. The carriers can be modified with various antigens and ligands for dendritic cells targeting. They can also be modified with adjuvants, either covalently or entrapped in the matrix, to improve cellular and humoral immune responses against the antigen. As a result, these multi-functional carrier systems are being explored for use in active immunotherapy against cancer and infectious diseases. Advancing technology, improved analytical methods, and use of computational methodology have also contributed to the development of subunit vaccine carriers. This review details recent breakthroughs in the design of nano-particulate vaccine carriers, including liposomes, polymeric nanoparticles, and inorganic nanoparticles. PMID:27104575

  9. Expression of GABA(A) receptor alpha3-, theta-, and epsilon-subunit mRNAs during rat CNS development and immunolocalization of the epsilon subunit in developing postnatal spinal cord.

    PubMed

    Pape, J-R; Bertrand, S S; Lafon, P; Odessa, M-F; Chaigniau, M; Stiles, J K; Garret, M

    2009-04-21

    Ionotropic GABA(A) receptors are heteromeric structures composed of a combination of five from at least 16 different subunits. Subunit genes are expressed in distinct cell types at specific times during development. The most abundant native GABA(A) receptors consist of alpha1-, beta2-, and gamma2-subunits that are co-expressed in numerous brain areas. alpha3-, theta-, And epsilon-subunits are clustered on the X chromosome and show striking overlapping expression patterns throughout the adult rat brain. To establish whether these subunits are temporally and spatially co-expressed, we used in situ hybridization to analyze their expression throughout rat development from embryonic stage E14 to postnatal stage P12. Each transcript exhibited a unique or a shared regional and temporal developmental expression profile. The thalamic expression pattern evolved from a restricted expression of epsilon and theta transcripts before birth, to a theta and alpha3 expression at birth, and finally to a grouped epsilon, theta and alpha3 expression postpartum. However, strong similarities occurred, such as a grouped expression of the three subunits within the hypothalamus, tegmentum and pontine nuclei throughout the developmental process. At early stages of development (E17), epsilon and theta appeared to have a greater spatial distribution before the dominance of the alpha3 subunit transcript around birth. We also revealed expression of alpha3, theta, and epsilon in the developing spinal cord and identified neurons that express epsilon in the postnatal dorsal horn, intermediolateral column and motoneurons. Our findings suggest that various combinations of alpha3-, theta- and epsilon-subunits may be assembled at a regional and developmental level in the brain.

  10. EXPRESSION OF GABAA RECEPTOR α3-, θ-, AND ε-SUBUNIT mRNAs DURING RAT CNS DEVELOPMENT AND IMMUNOLOCALIZATION OF THE ε SUBUNIT IN DEVELOPING POSTNATAL SPINAL CORD

    PubMed Central

    PAPE, J.-R.; BERTRAND, S. S.; LAFON, P.; ODESSA, M.-F.; CHAIGNIAU, M.; STILES, J. K.; GARRET, M.

    2010-01-01

    Ionotropic GABAA receptors are heteromeric structures composed of a combination of five from at least 16 different subunits. Subunit genes are expressed in distinct cell types at specific times during development. The most abundant native GABAA receptors consist of α1-, β2-, and γ2-subunits that are co-expressed in numerous brain areas. α3-, θ-, And ε-subunits are clustered on the X chromosome and show striking overlapping expression patterns throughout the adult rat brain. To establish whether these subunits are temporally and spatially co-expressed, we used in situ hybridization to analyze their expression throughout rat development from embryonic stage E14 to postnatal stage P12. Each transcript exhibited a unique or a shared regional and temporal developmental expression profile. The thalamic expression pattern evolved from a restricted expression of ε and θ transcripts before birth, to a θ and α3 expression at birth, and finally to a grouped ε, θ and α3 expression post-partum. However, strong similarities occurred, such as a grouped expression of the three subunits within the hypothalamus, tegmentum and pontine nuclei throughout the developmental process. At early stages of development (E17), ε and θ appeared to have a greater spatial distribution before the dominance of the α3 subunit transcript around birth. We also revealed expression of α3, θ, and ε in the developing spinal cord and identified neurons that express ε in the post-natal dorsal horn, intermediolateral column and motoneurons. Our findings suggest that various combinations of α3-, θ- and ε-subunits may be assembled at a regional and developmental level in the brain. PMID:19249336

  11. Structure–Function Relationships in Fungal Large-Subunit Catalases

    SciTech Connect

    Diaz, A.; Valdez, V; Rudino-Pinera, E; Horjales, E; Hansberg, W

    2009-01-01

    Neurospora crassa has two large-subunit catalases, CAT-1 and CAT-3. CAT-1 is associated with non-growing cells and accumulates particularly in asexual spores; CAT-3 is associated with growing cells and is induced under different stress conditions. It is our interest to elucidate the structure-function relationships in large-subunit catalases. Here we have determined the CAT-3 crystal structure and compared it with the previously determined CAT-1 structure. Similar to CAT-1, CAT-3 hydrogen peroxide (H{sub 2}O{sub 2}) saturation kinetics exhibited two components, consistent with the existence of two active sites: one saturated in the millimolar range and the other in the molar range. In the CAT-1 structure, we found three interesting features related to its unusual kinetics: (a) a constriction in the channel that conveys H{sub 2}O{sub 2} to the active site; (b) a covalent bond between the tyrosine, which forms the fifth coordination bound to the iron of the heme, and a vicinal cysteine; (c) oxidation of the pyrrole ring III to form a cis-hydroxyl group in C5 and a cis-{gamma}-spirolactone in C6. The site of heme oxidation marks the starts of the central channel that communicates to the central cavity and the shortest way products can exit the active site. CAT-3 has a similar constriction in its major channel, which could function as a gating system regulated by the H{sub 2}O{sub 2} concentration before the gate. CAT-3 functional tyrosine is not covalently bonded, but has instead the electron relay mechanism described for the human catalase to divert electrons from it. Pyrrole ring III in CAT-3 is not oxidized as it is in other large-subunit catalases whose structure has been determined. Different in CAT-3 from these enzymes is an occupied central cavity. Results presented here indicate that CAT-3 and CAT-1 enzymes represent a functional group of catalases with distinctive structural characteristics that determine similar kinetics.

  12. Multi-site Phosphorylation of Voltage-Gated Sodium Channel α Subunits from Rat Brain

    PubMed Central

    Berendt, Frank J.; Park, Kang-Sik; Trimmer, James S.

    2010-01-01

    Reversible phosphorylation of ion channels underlies cellular plasticity in mammalian neurons. Voltage-gated sodium or Nav channels underlie action potential initiation and propagation, dendritic excitability, and many other aspects of neuronal excitability. Various protein kinases have been suggested to phosphorylate the primary α subunit of Nav channels, affecting diverse aspects of channel function. Previous studies of Nav α subunit phosphorylation have led to the identification of a small set of phosphorylation sites important in meditating aspects of Nav channel function. Here we use nanoflow liquid chromatography tandem mass spectrometry (nano-LC MS/MS) on Nav α subunits affinity-purified from rat brain with two distinct monoclonal antibodies to identify 15 phosphorylation sites on Nav1.2, 12 of which have not been previously reported. We also found 3 novel phosphorylation sites on Nav1.1. In general, commonly used phosphorylation site prediction algorithms did not accurately predict these novel in vivo phosphorylation sites. Our results demonstrate that specific Nav α subunits isolated from rat brain are highly phosphorylated, and suggest extensive modulation of Nav channel activity in mammalian brain. Identification of phosphorylation sites using monoclonal antibody-based immunopurification and mass spectrometry is an effective approach to define the phosphorylation status of Nav channels and important membrane proteins in mammalian brain. PMID:20131913

  13. Mutation of valine residue unique to alpha subunit of Gs abolishes activation.

    PubMed

    Devic, E; Journot, L; Pantaloni, C; Bockaert, J; Audigier, Y

    1994-08-19

    We recently characterized a decapeptide sequence (residues 367-376) that is important for the membrane association of the activated alpha subunit of Gs. We report here that when this sequence is replaced by the cognate sequence of Gi1 alpha subunit, the chimeric protein (Gsis alpha) still interacts with the membrane but cannot be activated, regardless of the mode of activation. Construction of various chimeras demonstrates that the single replacement of valine 367 by threonine, the cognate residue of Gi1 alpha subunit, fully reproduces the loss of activation. Analysis of nucleotide interaction reveals that the mutant V367T Gs alpha protein poorly binds GDP or GTP. On the other hand, the conservative change of valine to isoleucine does not alter activation. Interestingly, members of the Gs and G12 classes have a valine and an isoleucine, respectively, at this position, whereas members of the Gi or Gq class contain a threonine residue. The evolutionary relationship between the different classes suggests that the presence of a hydrophobic or a hydrophilic residue is not fortuitous in these alpha subunits and might provide distinctive structural and/or functional properties.

  14. Crystal structure of a flavoprotein related to the subunits of bacterial luciferase.

    PubMed Central

    Moore, S A; James, M N; O'Kane, D J; Lee, J

    1993-01-01

    The molecular structure of the luxF protein from the bioluminescent bacterium Photobacterium leiognathi has been determined by X-ray diffraction techniques and refined to a conventional R-factor of 17.8% at 2.3 A resolution. The 228 amino acid polypeptide exists as a symmetrical homodimer and 33% of the monomer's solvent-accessible surface area is buried upon dimerization. The monomer displays a novel fold that contains a central seven-stranded beta-barrel. The solvent-exposed surface of the monomer is covered by seven alpha-helices, whereas the dimer interface is primarily a flat surface composed of beta-strands. The protein monomer binds two molecules of flavin mononucleotide, each of which has C6 of the flavin isoalloxazine moiety covalently attached to the C3' carbon atom of myristic acid. Both myristyl groups of these adducts are buried within the hydrophobic core of the protein. One of the cofactors contributes to interactions at the dimer interface. The luxF protein displays considerable amino acid sequence homology with both alpha- and beta-subunits of bacterial luciferase, especially the beta-subunit. Conserved amino acid residues shared between luxF and the luciferase subunits cluster predominantly in two distinct regions of the luxF protein molecule. These homologous regions in the luciferase subunits probably share a three-dimensional fold similar to that of the luxF protein. Images PMID:8491169

  15. 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

  16. Toxocara canis and Toxocara vitulorum: molecular characterization, discrimination, and phylogenetic analysis based on mitochondrial (ATP synthase subunit 6 and 12S) and nuclear ribosomal (ITS-2 and 28S) genes.

    PubMed

    Wickramasinghe, Susiji; Yatawara, Lalani; Rajapakse, R P V J; Agatsuma, Takeshi

    2009-06-01

    Toxocara canis and Toxocara vitulorum are two important parasites of dogs and buffaloes with public health concern. The objectives of the present study are to identify molecular markers to discriminate these closely related parasites and to determine their phylogenetic position and genetic diversity within the genus Toxocara. Thus, two mitochondrial genes (complete ATPase 6 and partial small subunit ribosomal RNA (12S rDNA)), two nuclear ribosomal genes (second internal transcribed spacer region (ITS-2)), and part of the large subunit 28S region were analyzed. Nucleotide sequence (597 bp) and predicted amino acid sequences of the complete ATPase 6 gene (199 amino acids) of both species (T. canis and T. vitulorum) are similar in size with the Toxocara cati and Toxocara malaysiensis. There was 88% nucleotide similarity between T. canis and T. vitulorum and many transversions present in the 12S gene. Analyses of the ITS-2 and 28S regions revealed that the 28S region was more conserved (95% nucleotide similarity between T. canis and T. vitulorum) than the ITS-2 region (85%). This study has provided useful molecular markers for the molecular epidemiological investigation of Toxocara species. Further, phylogenetic analyses of the ITS-2 and 28S genes have indicated that the members of the genus Toxocara form a distinct group with reference to their definitive hosts.

  17. Vanderwaltozyma verrucispora sp. nov., a new ascomycetous yeast species.

    PubMed

    Lee, Ching-Fu; Liu, Chun-Hao; Ninomiya, Shinya; Kawasaki, Hiroko; Nakase, Takashi

    2009-02-01

    A new yeast species, Vanderwaltozyma verrucispora, is proposed in this study based on two strains isolated from partially decayed leaves in Japan and one strain from soil in Taiwan. The species is characterized by the fermentation of glucose and galactose, formation of one to four spheroidal to ellipsoidal ascospores with warty surfaces in each ascus, and assimilation of a few carbon and nitrogen compounds. Genus assignment and distinction of the species from the other two recognized species of Vanderwaltozyma is based on the morphological and physiological characteristics, and phylogenetic analysis of nucleotide sequences of the D1/D2 domains of the large subunit (LSU) rRNA gene. From these comparisons, the name V. verrucispora sp. nov. is proposed. Sequence analysis of the D1/D2 domains of the LSU rRNA gene reveals that the phylogenetically closest relative of V. verrucispora is Vanderwaltozyma yarrowii. The type strain of the new species, which was isolated from a partially decayed leaf in Kagoshima Prefecture, Japan, is NBRC 1884(T) (=CBS 10887(T)=BCRC 23141(T)).

  18. Species boundaries and nomenclature of Rhizopus arrhizus (syn. R. oryzae).

    PubMed

    Dolatabadi, Somayeh; de Hoog, G Sybren; Meis, Jacques F; Walther, Grit

    2014-12-01

    Rhizopus arrhizus (Mucorales, Mucoromycotina) is the prevalent opportunist worldwide among the mucoralean species causing human infections. On the other hand the species has been used since ancient times to ferment African and Asian traditional foods and condiments based on ground soybeans. As producer of organic acids and hydrolytic enzymes it is widely applied in food industry and biotechnology. Using a set of 82 strains we studied phylogenetic and biological species boundaries within Rhizopus arrhizus s.l. to test the taxonomic status of R. delemar that was recently separated from R. arrhizus. Sequence analyses based on the internal transcribed spacer region, the gene of the largest subunit of the RNA polymerase II, a part of the actin gene, and the translation elongation factor 1-α as well as amplified fragment length polymorphism analysis were performed. Phenotypic characters such as enzyme profiles and growth kinetics were examined and the mating behavior was tested. Molecular analyses supported the existence of two phylogenetic species. However, the results of the mating test suggest that the mating barrier is still not complete. No physiological, ecological or epidemiological distinction could be found beside the difference in the production of organic acids. Consequently the status of varieties is proposed for the two phylogenetic species. Because the description of the first described R. arrhizus is considered to be conclusive we recommend the use of Rhizopus arrhizus var. arrhizus and var. delemar.

  19. Subunit diversity and tissue distribution of human glutathione S-transferases: interpretations based on electrospray ionization-MS and peptide sequence-specific antisera.

    PubMed Central

    Rowe, J D; Nieves, E; Listowsky, I

    1997-01-01

    Uncertainties about the composition and identities of glutathione S-transferases (GSTs) in human tissue have impeded studies on their biological functions. A rigorous protocol has therefore been developed to characterize the human proteins. Cytosolic GST subunits were resolved by reverse-phase HPLC methods, individual components were assigned to Alpha, Mu and Pi classes on the basis of their immunoreactivities, and peptide-sequence-specific antisera were used to distinguish among five different Mu-class subunits (GSTM1-GSTM5). Each subunit type was characterized and identified unambiguously by electrospray ionization-MS. Acetylation of N-terminal residues in the GSTA1, GSTA2, GSTM3 and GSTM4 subunits were the only natural post-translational modifications detected. The unique structure of GSTM3, with N- and C-terminal peptide extensions predicted from cDNA sequences, was confirmed. Only testis and brain were rich sources of GSTM3 subunits. Subunit profiles were distinct and characteristic of the particular tissue type, and this tissue specificity in GST expression was evident even in organs from different individuals. For instance, livers had relatively simple GST compositions, consisting of a preponderance of Alpha-class subunits and GSTM1 (when present). By contrast, representation of most subunit types was a characteristic feature of testis, which had the highest levels of GSTs. GSTM4 and GSTM5 subunits, here identified for the first time in human tissue extracts, were minor components, with GSTM5 found only in brain, lung and testis. Specimens devoid of GSTM1 subunits, particularly those from null-genotype individuals, were readily discerned at the protein level. Liver was the only rich source of the GSTM1 subunit (although it also constituted a major fraction of adrenal GSTs), and so the functional consequences of the GSTM1 gene deletion are likely to vary in extrahepatic tissues. PMID:9230131

  20. 3D-localization of the a-subunit in F 0F I-ATP synthase by time resolved single-molecule FRET

    NASA Astrophysics Data System (ADS)

    Düser, Monika G.; Zarrabi, Nawid; Bi, Yumin; Zimmermann, Boris; Dunn, Stanley D.; Börsch, Michael

    2006-02-01

    F °F I-ATP synthases catalyze the ATP formation from ADP and phosphate in the membranes of mitochondria, chloroplasts and bacteria. Internal rotation of subunits couples the chemical reaction at the F I part to the proton translocation through the F ° part. In these enzymes, the membrane-embedded a-subunit is part of the non-rotating 'stator' subunits and provides the proton channel of the F ° motor. At present, the relative position of the a-subunit is not known. We examined the rotary movements of the ɛ-subunit with respect to the non-rotating a-subunit by time resolved singlemolecule fluorescence resonance energy transfer (FRET) using a novel pulsed laser diode. Rotation of the ɛ-subunit during ATP hydrolysis was divided into three major steps. The stopping positions of ɛ resulted in three distinct FRET efficiency levels and FRET donor lifetimes. From these FRET efficiencies the position of the FRET donor at the asubunit was calculated. Different populations of the three resting positions of ɛ, which were observed previously, enabled us to scrutinize the models for the position of the a-subunit in the F ° part.

  1. Type B Heterotrimeric G Protein γ-Subunit Regulates Auxin and ABA Signaling in Tomato[OPEN

    PubMed Central

    Subramaniam, Gayathery; Trusov, Yuri; Hayashi, Satomi; Batley, Jacqueline

    2016-01-01

    Heterotrimeric G proteins composed of α, β, and γ subunits are central signal transducers mediating the cellular response to multiple stimuli in most eukaryotes. Gγ subunits provide proper cellular localization and functional specificity to the heterotrimer complex. Plant Gγ subunits, divided into three structurally distinct types, are more diverse than their animal counterparts. Type B Gγ subunits, lacking a carboxyl-terminal isoprenylation motif, are found only in flowering plants. We present the functional characterization of type B Gγ subunit (SlGGB1) in tomato (Solanum lycopersicum). We show that SlGGB1 is the most abundant Gγ subunit in tomato and strongly interacts with the Gβ subunit. Importantly, the green fluorescent protein-SlGGB1 fusion protein as well as the carboxyl-terminal yellow fluorescent protein-SlGGB1/amino-terminal yellow fluorescent protein-Gβ heterodimer were localized in the plasma membrane, nucleus, and cytoplasm. RNA interference-mediated silencing of SlGGB1 resulted in smaller seeds, higher number of lateral roots, and pointy fruits. The silenced lines were hypersensitive to exogenous auxin, while levels of endogenous auxins were lower or similar to those of the wild type. SlGGB1-silenced plants also showed strong hyposensitivity to abscisic acid (ABA) during seed germination but not in other related assays. Transcriptome analysis of the transgenic seeds revealed abnormal expression of genes involved in ABA sensing, signaling, and response. We conclude that the type B Gγ subunit SlGGB1 mediates auxin and ABA signaling in tomato. PMID:26668332

  2. Unequal synthesis and differential degradation of propionyl CoA carboxylase subunits in cells from normal and propionic acidemia patients.

    PubMed Central

    Ohura, T; Kraus, J P; Rosenberg, L E

    1989-01-01

    We have characterized further the molecular basis of human inherited propionyl CoA carboxylase deficiency by measuring steady state levels of the mRNAs coding for the enzyme's two protein subunits (alpha and beta) and by estimating initial synthesis and steady state levels of the protein subunits in skin fibroblasts from controls and affected patients. We studied cell lines from both major complementation groups (pccA and pccBC) corresponding, respectively, to defects in the carboxylase's alpha and beta subunits. Analysis of pccA lines revealed the absence of alpha chain mRNA in three and an abnormally small alpha-mRNA in a fourth. Despite the presence of normal beta-mRNA in each of these pccA lines, there was complete absence of both alpha and beta protein subunits under steady state conditions, even though new synthesis and mitochondrial import of beta precursors was normal. Results in nine pccBC lines revealed normal alpha mRNA in each, while the amounts of beta-mRNA were distinctly reduced in every case. Correspondingly, alpha protein subunits were present in normal amounts at steady-state, but beta subunits were uniformly decreased. In addition, in six of the nine beta deficient cell lines, partially degraded beta-subunits were observed. To help interpret these results, synthesis and stability of carboxylase subunits were studied in intact HeLa cells using a pulse-chase protocol. Whereas alpha chains were stable over the four hour interval studied, beta chains--initially synthesized in large excess over alpha chains--were degraded rapidly reaching equivalence with alpha chains after two hours.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:2741949

  3. α4β2 Nicotinic Acetylcholine Receptors: RELATIONSHIPS BETWEEN SUBUNIT STOICHIOMETRY AND FUNCTION AT THE SINGLE CHANNEL LEVEL.

    PubMed

    Mazzaferro, Simone; Bermudez, Isabel; Sine, Steven M

    2017-02-17

    Acetylcholine receptors comprising α4 and β2 subunits are the most abundant class of nicotinic acetylcholine receptor in the brain. They contribute to cognition, reward, mood, and nociception and are implicated in a range of neurological disorders. Previous measurements of whole-cell macroscopic currents showed that α4 and β2 subunits assemble in two predominant pentameric stoichiometries, which differ in their sensitivity to agonists, antagonists, and allosteric modulators. Here we compare agonist-elicited single channel currents from receptors assembled with an excess of either the α4 or β2 subunit, forming receptor populations biased toward one or the other stoichiometry, with currents from receptors composed of five concatemeric subunits in which the subunit stoichiometry is predetermined. Our results associate each subunit stoichiometry with a unique single channel conductance, mean open channel lifetime, and sensitivity to the allosteric potentiator 3-[3-(3-pyridinyl)-1,2,4-oxadiazol-5-yl]benzonitrile (NS-9283). Receptors with the composition (α4β2)2α4 exhibit high single channel conductance, brief mean open lifetime, and strong potentiation by NS-9283, whereas receptors with the composition (α4β2)2β2 exhibit low single channel conductance and long mean open lifetime and are not potentiated by NS-9283. Thus single channel current measurements reveal bases for the distinct functional and pharmacological properties endowed by different stoichiometries of α4 and β2 subunits and establish pentameric concatemers as a means to delineate interactions between subunits that confer these properties.

  4. Three Members of the Bemisia tabaci (Hemiptera: Aleyrodidae) Cryptic Species Complex Occur Sympatrically in Argentine Horticultural Crops.

    PubMed

    Alemandri, V; Vaghi Medina, C G; Dumón, A D; Argüello Caro, E B; Mattio, M F; García Medina, S; López Lambertini, P M; Truol, G

    2015-04-01

    The whitefly, Bemisia tabaci (Gennadius), is a cryptic species complex that attacks >600 different species of plants and transmits several plant viruses causing severe economic losses. Until 2010, the B. tabaci complex comprised 24 distinct putative species. Recently, at least 15 new species have been reported. The objective of this study was to identify B. tabaci species present in bean, melon, and tomato crops in Argentina by applying phylogenetic analyses and pairwise comparison of genetic distances of mitochondrial cytochrome c oxidase subunit I (mtCOI) sequences. The 39 proposed whitefly species were identified with both analyses, and the presence in Argentina of one indigenous species, New World 2 (NW2), and two introduced species, Middle East-Asia Minor one (MEAM1) and Mediterranean, was confirmed. Common bean crop presented the three whitefly species detected, with NW2, MEAM1, and Mediterranean being present all together under field conditions. Also, Mediterranean was the only species identified in tomato, whereas MEAM1 was found in melon. To the best of our knowledge, Mediterranean is a recent invasive species in open-field agriculture in the American continent and in greenhouse tomato in Argentina. Additionally, we provide the first report of MEAM1 in common bean and melon. These findings raise several questions on the future scenario of B. tabaci and the viruses it transmits in Argentina.

  5. DNA sequences, recombinant DNA molecules and processes for producing the A and B subunits of cholera toxin and preparations containing so-obtained subunit or subunits

    SciTech Connect

    Harford, N.; De Wilde, M.

    1987-05-19

    A recombinant DNA molecule is described comprising at least a portion coding for subunits A and B of cholera toxin, or a fragment or derivative of the portion wherein the fragment or derivative codes for a polypeptide have an activity which can induce an immune response to subunit A; can induce an immune response to subunit A and cause epithelial cell penetration and the enzymatic effect leading to net loss of fluid into the gut lumen; can bind to the membrane receptor for the B subunit of cholera toxin; can induce an immune response to subunit B; can induce an immune response to subunit B and bind to the membrane receptor; or has a combination of the activities.

  6. A molecular phylogeny of the marine red algae (Rhodophyta) based on the nuclear small-subunit rRNA gene.

    PubMed Central

    Ragan, M A; Bird, C J; Rice, E L; Gutell, R R; Murphy, C A; Singh, R K

    1994-01-01

    A phylogeny of marine Rhodophyta has been inferred by a number of methods from nucleotide sequences of nuclear genes encoding small subunit rRNA from 39 species in 15 orders. Sequence divergences are relatively large, especially among bangiophytes and even among congeners in this group. Subclass Bangiophycidae appears polyphyletic, encompassing at least three lineages, with Porphyridiales distributed between two of these. Subclass Florideophycidae is monophyletic, with Hildenbrandiales, Corallinales, Ahnfeltiales, and a close association of Nemaliales, Acrochaetiales, and Palmariales forming the four deepest branches. Cermiales may represent a convergence of vegetative and reproductive morphologies, as family Ceramiaceae is at best weakly related to the rest of the order, and one of its members appears to be allied to Gelidiales. Except for Gigartinales, for which more data are required, the other florideophyte orders appear distinct and taxonomically justified. A good correlation was observed with taxonomy based on pit-plug ultrastructure. Tests under maximum-likelihood and parsimony of alternative phylogenies based on structure and chemistry refuted suggestions that Acrochaetiales is the most primitive florideophyte order and that Gelidiales and Hildenbrandiales are sister groups. PMID:8041780

  7. Unique subunit packing in mycobacterial nanoRNase leads to alternate substrate recognitions in DHH phosphodiesterases

    PubMed Central

    Srivastav, Rajpal; Kumar, Dilip; Grover, Amit; Singh, Ajit; Manjasetty, Babu A.; Sharma, Rakesh; Taneja, Bhupesh

    2014-01-01

    DHH superfamily includes RecJ, nanoRNases (NrnA), cyclic nucleotide phosphodiesterases and pyrophosphatases. In this study, we have carried out in vitro and in vivo investigations on the bifunctional NrnA-homolog from Mycobacterium smegmatis, MSMEG_2630. The crystal structure of MSMEG_2630 was determined to 2.2-Å resolution and reveals a dimer consisting of two identical subunits with each subunit folding into an N-terminal DHH domain and a C-terminal DHHA1 domain. The overall structure and fold of the individual domains is similar to other members of DHH superfamily. However, MSMEG_2630 exhibits a distinct quaternary structure in contrast to other DHH phosphodiesterases. This novel mode of subunit packing and variations in the linker region that enlarge the domain interface are responsible for alternate recognitions of substrates in the bifunctional nanoRNases. MSMEG_2630 exhibits bifunctional 3′-5′ exonuclease [on both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) substrates] as well as CysQ-like phosphatase activity (on pAp) in vitro with a preference for nanoRNA substrates over single-stranded DNA of equivalent lengths. A transposon disruption of MSMEG_2630 in M. smegmatis causes growth impairment in the presence of various DNA-damaging agents. Further phylogenetic analysis and genome organization reveals clustering of bacterial nanoRNases into two distinct subfamilies with possible role in transcriptional and translational events during stress. PMID:24878921

  8. Botrytis californica, a new cryptic species in the B. cinerea species complex causing gray mold in blueberries and table grapes.

    PubMed

    Saito, S; Margosan, D; Michailides, T J; Xiao, C L

    2016-01-01

    The Botrytis cinerea species complex comprises two cryptic species, originally referred to Group I and Group II based on Bc-hch gene RFLP haplotyping. Group I was described as a new cryptic species B. pseudocinerea During a survey of Botrytis spp. causing gray mold in blueberries and table grapes in the Central Valley of California, six isolates, three from blueberries and three from table grapes, were placed in Group I but had a distinct morphological character with conidiophores significantly longer than those of B. cinerea and B. pseudocinerea We compared these with B. cinerea and B. pseudocinerea by examining morphological and physiological characters, sensitivity to fenhexamid and phylogenetic analysis inferred from sequences of three nuclear genes. Phylogenetic analysis with the three partial gene sequences encoding glyceraldehyde-3-phosate dehydrogenase (G3PDH), heat-shock protein 60 (HSP60) and DNA-dependent RNA polymerase subunit II (RPB2) supported the proposal of a new Botrytis species, B. californica, which is closely related genetically to B. cinerea, B. pseudocinerea and B. sinoviticola, all known as causal agents of gray mold of grapes. Botrytis californica caused decay on blueberry and table grape fruit inoculated with the fungus. This study suggests that B. californica is a cryptic species sympatric with B. cinerea on blueberries and table grapes in California.

  9. Bacteriophage HK97 structure: wholesale covalent cross-linking between the major head shell subunits.

    PubMed Central

    Popa, M P; McKelvey, T A; Hempel, J; Hendrix, R W

    1991-01-01

    We describe initial genetic and structural characterizations of HK97, a temperate bacteriophage of Escherichia coli. We isolated 28 amber mutants, characterized them with respect to what phage-related structures they make, and mapped many of them to restriction fragments of genomic DNA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of HK97 virions revealed nine different protein species plus a substantial amount of material that failed to enter the gel, apparently because it is too large. Five proteins are tail components and are assigned functions as tail fiber subunit, tail length template, and major shaft subunit (two and possibly three species). The four remaining proteins and the material that did not enter the gel are head components. One of these proteins is assigned as the portal subunit, and the remaining three head proteins in the gel and the material that did not enter the gel are components of the head shell. All of the head shell protein species have apparent molecular masses well in excess of 100 kDa; they share amino acid sequence with each other and also with a 42-kDa protein that is found in infected lysates and as the major component of prohead structures that accumulate in infections by one of the amber mutants. We propose that all of the head shell species found in mature heads are covalently cross-linked oligomers derived from the 42-kDa precursor during head shell maturation. Images PMID:1709700

  10. Tilletia vankyi, a new species of reticulate-spored bunt fungus with non-conjugating basidiospores infecting species of Festuca and Lolium.

    PubMed

    Carris, Lori M; Castlebury, Lisa A; Huang, Guoming; Alderman, Steve C; Luo, Jiafeng; Bao, Xiaodong

    2007-12-01

    A bunt fungus, exhibiting a spore germination pattern unique to known reticulate-spored species of Tilletia was found infecting plants in seed production fields of Festuca rubra ssp. rubra (red fescue) and F. rubra ssp. fallax (Chewing's fescue) in Oregon, and in seed lots of Lolium perenne (perennial ryegrass) from Australia and Germany. Teliospores germinated to form 20-40 uninucleate, non-conjugating basidiospores, and colonies derived from single basidiospores produced teliospores in culture. In inoculation studies using single basidiospore colonies, perennial ryegrass and L. perenne ssp. multiflorum (Italian or annual ryegrass) were infected. A phylogenetic analysis, based on ITS region rDNA, eukaryotic translation elongation factor 1 alpha, and the second largest subunit of RNA polymerase II demonstrated that the fescue and ryegrass bunts are conspecific, and distinct from known species of Tilletia.

  11. Lactobacillus herbarum sp. nov., a species related to Lactobacillus plantarum.

    PubMed

    Mao, Yuejian; Chen, Meng; Horvath, Philippe

    2015-12-01

    Strain TCF032-E4 was isolated from a traditional Chinese fermented radish. It shares >99% 16S rRNA sequence identity with L. plantarum, L. pentosus and L. paraplantarum. This strain can ferment ribose, galactose, glucose, fructose, mannose, mannitol, N-acetylglucosamine, amygdalin, arbutin, salicin, cellobiose, maltose, lactose, melibiose, trehalose and gentiobiose. It cannot ferment sucrose, which can be used by L. pentosus, L. paraplantarum, L. fabifermentans, L. xiangfangensis and L. mudanjiangensis, as well as most of the L. plantarum strains (88.7%). TCF032-E4 cannot grow at temperature above 32 °C. This strain shares 78.2-83.6% pheS (phenylalanyl-tRNA synthetase alpha subunit) and 89.5-94.9% rpoA (RNA polymerase alpha subunit) sequence identity with L. plantarum, L. pentosus, L. paraplantarum, L. fabifermentans, L. xiangfangensis and L. mudanjiangensis. These results indicate that TCF032-E4 represents a distinct species. This hypothesis was further confirmed by whole-genome sequencing and comparison with available genomes of related species. The draft genome size of TCF032-E4 is approximately 2.9 Mb, with a DNA G+C content of 43.5 mol%. The average nucleotide identity (ANI) between TCF032-E4 and related species ranges from 79.0 to 81.1%, the highest ANI value being observed with L. plantarum subsp. plantarum ATCC 14917T. A novel species, Lactobacillus herbarum sp. nov., is proposed with TCF032-E4T ( = CCTCC AB2015090T = DSM 100358T) as the type strain.

  12. Differential accumulation of ribonucleotide reductase subunits in clam oocytes: the large subunit is stored as a polypeptide, the small subunit as untranslated mRNA

    PubMed Central

    1986-01-01

    Within minutes of fertilization of clam oocytes, translation of a set of maternal mRNAs is activated. One of the most abundant of these stored mRNAs encodes the small subunit of ribonucleotide reductase (Standart, N. M., S. J. Bray, E. L. George, T. Hunt, and J. V. Ruderman, 1985, J. Cell Biol., 100:1968-1976). Unfertilized oocytes do not contain any ribonucleotide reductase activity; such activity begins to appear shortly after fertilization. In virtually all organisms, this enzyme is composed of two dissimilar subunits with molecular masses of approximately 44 and 88 kD, both of which are required for activity. This paper reports the identification of the large subunit of clam ribonucleotide reductase isolated by dATP-Sepharose chromatography as a relatively abundant 86-kD polypeptide which is already present in oocytes, and whose level remains constant during early development. The enzyme activity of this large subunit was established in reconstitution assays using the small subunit isolated from embryos by virtue of its binding to the anti-tubulin antibody YL 1/2. Thus the two components of clam ribonucleotide reductase are differentially stored in the oocyte: the small subunit in the form of untranslated mRNA and the large subunit as protein. When fertilization triggers the activation of translation of the maternal mRNA, the newly synthesized small subunit combines with the preformed large subunit to generate active ribonucleotide reductase. PMID:3536960

  13. Phylogenetic relationships in European Ceriporiopsis species inferred from nuclear and mitochondrial ribosomal DNA sequences.

    PubMed

    Tomšovský, Michal; Menkis, Audrius; Vasaitis, Rimvydas

    2010-04-01

    The aim of this work was to clarify taxonomy and examine evolutionary relationships within European Ceriporiopsis species using a combined analysis of the large subunit (nLSU) nuclear rRNA and small subunit (mtSSU) mitochondrial rRNA gene sequences. Data from the ITS region were applied to enhance the view of the phylogenetic relationships among different species. The studied samples grouped into four complex clades, suggesting that the genus Ceriporiopsis is polyphyletic. The generic type Ceriporiopsis gilvescens formed a separate group together with Ceriporiopsis guidella and Phlebia spp. in the phlebioid clade. In this clade, the closely related species Ceriporiopsis resinascens and Ceriporiopsis pseudogilvescens grouped together with Ceriporiopsis aneirina. C. resinascens and C. pseudogilvescens have identical LSU and SSU sequences but differ in ITS. Ceriporiopsis pannocincta also fell in the phlebioid clade, but showed closer proximity to Gloeoporus dichrous than to C. gilvescens or C. aneirina-C. pseudogilvescens-C. resinascens group. Another clade was composed of a Ceriporiopsis balaenae-Ceriporiopsis consobrina group and was found to be closely related to Antrodiella and Frantisekia, with the overall clade highly reminiscent of the residual polyporoid clade. The monotypic genus Pouzaroporia, erected in the past for Ceriporiopsis subrufa due to its remarkable morphological differences, also fell within the residual polyporoid clade. Ceriporiopsis subvermispora held an isolated position from the other species of the genus. Therefore, the previously proposed name Gelatoporia subvermispora has been adopted for this species. Physisporinus rivulosus appeared unrelated to two other European Physisporinus species. Moreover, Ceriporiopsis (=Skeletocutis) jelicii grouped in a separate clade, distinct from Ceriporiopsis species. Finally, the ITS data demonstrated the proximity of some Ceriporiopsis species (Ceriporiopsis portcrosensis and Ceriporiopsis

  14. Is Face Distinctiveness Gender Based?

    ERIC Educational Resources Information Center

    Baudouin, Jean-Yves; Gallay, Mathieu

    2006-01-01

    Two experiments were carried out to study the role of gender category in evaluations of face distinctiveness. In Experiment 1, participants had to evaluate the distinctiveness and the femininity-masculinity of real or artificial composite faces. The composite faces were created by blending either faces of the same gender (sexed composite faces,…

  15. Counselor Identity: Conformity or Distinction?

    ERIC Educational Resources Information Center

    McLaughlin, Jerry E.; Boettcher, Kathryn

    2009-01-01

    The authors explore 3 debates in other disciplines similar to counseling's identity debate in order to learn about common themes and outcomes. Conformity, distinction, and cohesion emerged as common themes. They conclude that counselors should retain their distinctive, humanistic approach rather than conforming to the dominant, medical approach.

  16. Development of Distinctive Feature Theory.

    ERIC Educational Resources Information Center

    Meyer, Peggy L.

    Since the beginning of man's awareness of his language capabilities and language structure, he has assumed that speech is composed of discrete entities. The linguist attempts to establish a model of the workings of these distinctive sounds in a language. Utilizing an historical basis for discussion, this general survey of the distinctive feature…

  17. Prefoldin Subunits Are Protected from Ubiquitin-Proteasome System-mediated Degradation by Forming Complex with Other Constituent Subunits*

    PubMed Central

    Miyazawa, Makoto; Tashiro, Erika; Kitaura, Hirotake; Maita, Hiroshi; Suto, Hiroo; Iguchi-Ariga, Sanae M. M.; Ariga, Hiroyoshi

    2011-01-01

    The molecular chaperone prefoldin (PFD) is a complex comprised of six different subunits, PFD1-PFD6, and delivers newly synthesized unfolded proteins to cytosolic chaperonin TRiC/CCT to facilitate the folding of proteins. PFD subunits also have functions different from the function of the PFD complex. We previously identified MM-1α/PFD5 as a novel c-Myc-binding protein and found that MM-1α suppresses transformation activity of c-Myc. However, it remains unclear how cells regulate protein levels of individual subunits and what mechanisms alter the ratio of their activities between subunits and their complex. In this study, we found that knockdown of one subunit decreased protein levels of other subunits and that transfection of five subunits other than MM-1α into cells increased the level of endogenous MM-1α. We also found that treatment of cells with MG132, a proteasome inhibitor, increased the level of transfected/overexpressed MM-1α but not that of endogenous MM-1α, indicating that overexpressed MM-1α, but not endogenous MM-1α, was degraded by the ubiquitin proteasome system (UPS). Experiments using other PFD subunits showed that the UPS degraded a monomer of PFD subunits, though extents of degradation varied among subunits. Furthermore, the level of one subunit was increased after co-transfection with the respective subunit, indicating that there are specific combinations between subunits to be stabilized. These results suggest mutual regulation of protein levels among PFD subunits and show how individual subunits form the PFD complex without degradation. PMID:21478150

  18. Prefoldin subunits are protected from ubiquitin-proteasome system-mediated degradation by forming complex with other constituent subunits.

    PubMed

    Miyazawa, Makoto; Tashiro, Erika; Kitaura, Hirotake; Maita, Hiroshi; Suto, Hiroo; Iguchi-Ariga, Sanae M M; Ariga, Hiroyoshi

    2011-06-03

    The molecular chaperone prefoldin (PFD) is a complex comprised of six different subunits, PFD1-PFD6, and delivers newly synthesized unfolded proteins to cytosolic chaperonin TRiC/CCT to facilitate the folding of proteins. PFD subunits also have functions different from the function of the PFD complex. We previously identified MM-1α/PFD5 as a novel c-Myc-binding protein and found that MM-1α suppresses transformation activity of c-Myc. However, it remains unclear how cells regulate protein levels of individual subunits and what mechanisms alter the ratio of their activities between subunits and their complex. In this study, we found that knockdown of one subunit decreased protein levels of other subunits and that transfection of five subunits other than MM-1α into cells increased the level of endogenous MM-1α. We also found that treatment of cells with MG132, a proteasome inhibitor, increased the level of transfected/overexpressed MM-1α but not that of endogenous MM-1α, indicating that overexpressed MM-1α, but not endogenous MM-1α, was degraded by the ubiquitin proteasome system (UPS). Experiments using other PFD subunits showed that the UPS degraded a monomer of PFD subunits, though extents of degradation varied among subunits. Furthermore, the level of one subunit was increased after co-transfection with the respective subunit, indicating that there are specific combinations between subunits to be stabilized. These results suggest mutual regulation of protein levels among PFD subunits and show how individual subunits form the PFD complex without degradation.

  19. Activation of a Chimeric Rpb5/RpoH Subunit Using Library Selection

    PubMed Central

    Sommer, Bettina; Waege, Ingrid; Pöllmann, David; Seitz, Tobias; Thomm, Michael; Sterner, Reinhard; Hausner, Winfried

    2014-01-01

    Rpb5 is a general subunit of all eukaryotic RNA polymerases which consists of a N-terminal and a C-terminal domain. The corresponding archaeal subunit RpoH contains only the conserved C-terminal domain without any N-terminal extensions. A chimeric construct, termed rp5H, which encodes the N-terminal yeast domain and the C-terminal domain from Pyrococcus furiosus is unable to complement the lethal phenotype of a yeast rpb5 deletion strain (Δrpb5). By applying a random mutagenesis approach we found that the amino acid exchange E197K in the C-terminal domain of the chimeric Rp5H, either alone or with additional exchanges in the N-terminal domain, leads to heterospecific complementation of the growth deficiency of Δrpb5. Moreover, using a recently described genetic system for Pyrococcus we could demonstrate that the corresponding exchange E62K in the archaeal RpoH subunit alone without the eukaryotic N-terminal extension was stable, and growth experiments indicated no obvious impairment in vivo. In vitro transcription experiments with purified RNA polymerases showed an identical activity of the wild type and the mutant Pyrococcus RNA polymerase. A multiple alignment of RpoH sequences demonstrated that E62 is present in only a few archaeal species, whereas the great majority of sequences within archaea and eukarya contain a positively charged amino acid at this position. The crystal structures of the Sulfolobus and yeast RNA polymerases show that the positively charged arginine residues in subunits RpoH and Rpb5 most likely form salt bridges with negatively charged residues from subunit RpoK and Rpb1, respectively. A similar salt bridge might stabilize the interaction of Rp5H-E197K with a neighboring subunit of yeast RNA polymerase and thus lead to complementation of Δrpb5. PMID:24489922

  20. Molecular cloning and expression of a GABA receptor subunit from the crayfish Procambarus clarkii.

    PubMed

    Jiménez-Vázquez, Eric N; Díaz-Velásquez, Clara E; Uribe, R M; Arias, Juan M; García, Ubaldo

    2016-02-01

    Molecular cloning has introduced an unexpected, large diversity of neurotransmitter hetero- oligomeric receptors. Extensive research on the molecular structure of the γ-aminobutyric acid receptor (GABAR) has been of great significance for understanding how the nervous system works in both vertebrates and invertebrates. However, only two examples of functional homo-oligomeric GABA-activated Cl(-) channels have been reported. In the vertebrate retina, the GABAρ1 subunit of various species forms homo-oligomeric receptors; in invertebrates, a cDNA encoding a functional GABA-activated Cl(-) channel has been isolated from a Drosophila melanogaster head cDNA library. When expressed in Xenopus laevis oocytes, these subunits function efficiently as a homo-oligomeric complex. To investigate the structure-function of GABA channels from the crayfish Procambarus clarkii, we cloned a subunit and expressed it in human embryonic kidney cells. Electrophysiological recordings show that this subunit forms a homo-oligomeric ionotropic GABAR that gates a bicuculline-insensitive Cl(-) current. The order of potency of the agonists was GABA > trans-4-amino-crotonic acid = cis-4-aminocrotonic acid > muscimol. These data support the notion that X-organ sinus gland neurons express at least two GABA subunits responsible for the formation of hetero-oligomeric and homo-oligomeric receptors. In addition, by in situ hybridization studies we demonstrate that most X-organ neurons from crayfish eyestalk express the isolated pcGABAA β subunit. This study increases the knowledge of the genetics of the crayfish, furthers the understanding of this important neurotransmitter receptor family, and provides insight into the evolution of these genes among vertebrates and invertebrates.

  1. Optimal Distinctiveness Signals Membership Trust.

    PubMed

    Leonardelli, Geoffrey J; Loyd, Denise Lewin

    2016-07-01

    According to optimal distinctiveness theory, sufficiently small minority groups are associated with greater membership trust, even among members otherwise unknown, because the groups are seen as optimally distinctive. This article elaborates on the prediction's motivational and cognitive processes and tests whether sufficiently small minorities (defined by relative size; for example, 20%) are associated with greater membership trust relative to mere minorities (45%), and whether such trust is a function of optimal distinctiveness. Two experiments, examining observers' perceptions of minority and majority groups and using minimal groups and (in Experiment 2) a trust game, revealed greater membership trust in minorities than majorities. In Experiment 2, participants also preferred joining minorities over more powerful majorities. Both effects occurred only when minorities were 20% rather than 45%. In both studies, perceptions of optimal distinctiveness mediated effects. Discussion focuses on the value of relative size and optimal distinctiveness, and when membership trust manifests.

  2. Inherent conformational flexibility of F1-ATPase α-subunit.

    PubMed

    Hahn-Herrera, Otto; Salcedo, Guillermo; Barril, Xavier; García-Hernández, Enrique

    2016-09-01

    The core of F1-ATPase consists of three catalytic (β) and three noncatalytic (α) subunits, forming a hexameric ring in alternating positions. A wealth of experimental and theoretical data has provided a detailed picture of the complex role played by catalytic subunits. Although major conformational changes have only been seen in β-subunits, it is clear that α-subunits have to respond to these changes in order to be able to transmit information during the rotary mechanism. However, the conformational behavior of α-subunits has not been explored in detail. Here, we have combined unbiased molecular dynamics (MD) simulations and calorimetrically measured thermodynamic signatures to investigate the conformational flexibility of isolated α-subunits, as a step toward deepening our understanding of its function inside the α3β3 ring. The simulations indicate that the open-to-closed conformational transition of the α-subunit is essentially barrierless, which is ideal to accompany and transmit the movement of the catalytic subunits. Calorimetric measurements of the recombinant α-subunit from Geobacillus kaustophilus indicate that the isolated subunit undergoes no significant conformational changes upon nucleotide binding. Simulations confirm that the nucleotide-free and nucleotide-bound subunits show average conformations similar to that observed in the F1 crystal structure, but they reveal an increased conformational flexibility of the isolated α-subunit upon MgATP binding, which might explain the evolutionary conserved capacity of α-subunits to recognize nucleotides with considerable strength. Furthermore, we elucidate the different dependencies that α- and β-subunits show on Mg(II) for recognizing ATP.

  3. Microbial community composition and in silico predicted metabolic potential reflect biogeochemical gradients between distinct peatland types.

    PubMed

    Urbanová, Zuzana; Bárta, Jiří

    2014-12-01

    It is not well understood how the ecological status and microbial community composition of spruce swamp forests (SSF) relate to those found in bogs and fens. To clarify this, we investigated biogeochemical parameters and microbial community composition in a bog, a fen and two SSF using high throughput barcoded sequencing of the small ribosomal subunit (SSU) variable region V4. The results demonstrated that the microbial community of SSF is positioned between those of bogs and fens, and this was confirmed by in silico predicted metabolic potentials. This corresponds well with the position of SSF on the trophic gradient and reflects distinct responses of microbial communities to environmental variables. Species richness and microbial diversity increased significantly from bog to fen, with SSF in between, reflecting the variation in pH, nutrient availability and peat decomposability. The archaeal community, dominated by hydrogenotrophic methanogens, was more similar in SSF and the bog compared with the fen. The composition of the bacterial community of SSF was intermediate between those of bog and fen. However, the production of CO2 (an indicator of peat decomposability) did not differ between SSF and bog, suggesting the limiting effect of low pH and poor litter quality on the functioning of the bacterial community in SSF. These results help to clarify the transitional position of SSF between bogs and fens and showed the strong effect of environmental conditions on microbial community composition and functioning.

  4. The functionally distinct hemoglobins of the Arctic spotted wolffish Anarhichas minor.

    PubMed

    Verde, Cinzia; Carratore, Vito; Riccio, Antonio; Tamburrini, Maurizio; Parisi, Elio; Di Prisco, Guido

    2002-09-27

    The Arctic fish Anarhichas minor, a benthic sedentary species, displays high hemoglobin multiplicity. The three major hemoglobins (Hb 1, Hb 2, and Hb 3) show important functional differences in pH and organophosphate regulation, subunit cooperativity, and response of oxygen binding to temperature. Hb 1 and Hb 2 display a low, effector-enhanced Bohr effect and no Root effect. In contrast, Hb 3 displays pronounced Bohr and Root effects, accompanied by strong organophosphate regulation. Hb 1 has the beta (beta(1)) chain in common with Hb 2; Hb 3 and Hb 2 share the alpha (alpha(2)) chain. The amino acid sequences have been established. Several substitutions in crucial positions were observed, such as Cys in place of C-terminal His in the beta(1) chain of Hb 1 and Hb 2. In Hb 3, Val E11 of the beta(2) chain is replaced by Ile. Homology modeling revealed an unusual structure of the Hb 3 binding site of inositol hexakisphoshate. Phylogenetic analysis indicated that only Hb 2 displays higher overall similarity with the major Antarctic hemoglobins. The oxygen transport system of A. minor differs remarkably from those of Antarctic Notothenioidei, indicating distinct evolutionary pathways in the regulatory mechanisms of the fish respiratory system in the two polar environments.

  5. Motor neurons with differential vulnerability to degeneration show distinct protein signatures in health and ALS.

    PubMed

    Comley, L; Allodi, I; Nichterwitz, S; Nizzardo, M; Simone, C; Corti, S; Hedlund, E

    2015-04-16

    The lethal disease amyotrophic lateral sclerosis (ALS) is characterized by the loss of somatic motor neurons. However, not all motor neurons are equally vulnerable to disease; certain groups are spared, including those in the oculomotor nucleus controlling eye movement. The reasons for this differential vulnerability remain unknown. Here we have identified a protein signature for resistant oculomotor motor neurons and vulnerable hypoglossal and spinal motor neurons in mouse and man and in health and ALS with the aim of understanding motor neuron resistance. Several proteins with implications for motor neuron resistance, including GABAA receptor α1, guanylate cyclase soluble subunit alpha-3 and parvalbumin were persistently expressed in oculomotor neurons in man and mouse. Vulnerable motor neurons displayed higher protein levels of dynein, peripherin and GABAA receptor α2, which play roles in retrograde transport and excitability, respectively. These were dynamically regulated during disease and thus could place motor neurons at an increased risk. From our analysis is it evident that oculomotor motor neurons have a distinct protein signature compared to vulnerable motor neurons in brain stem and spinal cord, which could in part explain their resistance to degeneration in ALS. Our comparison of human and mouse shows the relative conservation of signals across species and infers that transgenic SOD1G93A mice could be used to predict mechanisms of neuronal vulnerability in man.

  6. DNA Barcodes of Rosy Tetras and Allied Species (Characiformes: Characidae: Hyphessobrycon) from the Brazilian Amazon Basin

    PubMed Central

    Castro Paz, Francis Paola; Batista, Jacqueline da Silva; Porto, Jorge Ivan Rebelo

    2014-01-01

    DNA barcoding can be an effective tool for fast and accurate species-level identification based on sequencing of the mitochondrial cytochrome c oxidase subunit (COI) gene. The diversity of this fragment can be used to estimate the richness of the respective species. In this study, we explored the use of DNA barcoding in a group of ornamental freshwater fish of the genus Hyphessobrycon. We sequenced the COI from 10 species of Hyphessobrycon belonging to the “Rosy Tetra Clade” collected from the Amazon and Negro River basins and combined our results with published data. The average conspecific and congeneric Kimura 2-parameter distances were 2.3% and 19.3%, respectively. Six of the 10 species were easily distinguishable by DNA barcoding (H. bentosi, H. copelandi, H. eques, H. epicharis, H. pulchrippinis, and H. sweglesi), whereas the remaining species (H. erythrostigma, H. pyrrhonotus, H. rosaceus and H. socolofi) lacked reciprocal monophyly. Although the COI gene was not fully diagnostic, the discovery of distinct evolutionary units in certain Hyphessobrycon species under the same specific epithet as well as haplotype sharing between different species suggest that DNA barcoding is useful for species identification in this speciose genus. PMID:24878569

  7. Nuclear stability and transcriptional directionality separate functionally distinct RNA species.

    PubMed

    Andersson, Robin; Refsing Andersen, Peter; Valen, Eivind; Core, Leighton J; Bornholdt, Jette; Boyd, Mette; Heick Jensen, Torben; Sandelin, Albin

    2014-11-12

    Mammalian genomes are pervasively transcribed, yielding a complex transcriptome with high variability in composition and cellular abundance. Although recent efforts have identified thousands of new long non-coding (lnc) RNAs and demonstrated a complex transcriptional repertoire produced by protein-coding (pc) genes, limited progress has been made in distinguishing functional RNA from spurious transcription events. This is partly due to present RNA classification, which is typically based on technical rather than biochemical criteria. Here we devise a strategy to systematically categorize human RNAs by their sensitivity to the ribonucleolytic RNA exosome complex and by the nature of their transcription initiation. These measures are surprisingly effective at correctly classifying annotated transcripts, including lncRNAs of known function. The approach also identifies uncharacterized stable lncRNAs, hidden among a vast majority of unstable transcripts. The predictive power of the approach promises to streamline the functional analysis of known and novel RNAs.

  8. Ecological distinctions between sympatric species of Saguinus and Sciurus.

    PubMed

    Garber, P A; Sussman, R W

    1984-10-01

    Tamarins are small New World monkeys that have been described as "squirrellike." Squirrels, along with bats and birds, are the taxa most likely to utilize resources similar to those used by primates in the tropical forest canopy. In this paper we compare differences in ecology, diet, locomotion, and habitat utilization between sympatric populations of tamarins (Saguinus oedipus) and tree squirrels (Sciurus granatensis) in Panama. Data presented indicate that although there is some degree of resource overlap, patterns of habitat utilization differ significantly. Rather than being "squirrellike," the Panamanian tamarin exhibits a pattern of locomotor and feeding behavior consistent with that found in other arboreal primates.

  9. The RCN1-encoded A subunit of protein phosphatase 2A increases phosphatase activity in vivo

    NASA Technical Reports Server (NTRS)

    Deruere, J.; Jackson, K.; Garbers, C.; Soll, D.; Delong, A.; Evans, M. L. (Principal Investigator)

    1999-01-01

    Protein phosphatase 2A (PP2A), a heterotrimeric serine/threonine-specific protein phosphatase, comprises a catalytic C subunit and two distinct regulatory subunits, A and B. The RCN1 gene encodes one of three A regulatory subunits in Arabidopsis thaliana. A T-DNA insertion mutation at this locus impairs root curling, seedling organ elongation and apical hypocotyl hook formation. We have used in vivo and in vitro assays to gauge the impact of the rcn1 mutation on PP2A activity in seedlings. PP2A activity is decreased in extracts from rcn1 mutant seedlings, and this decrease is not due to a reduction in catalytic subunit expression. Roots of mutant seedlings exhibit increased sensitivity to the phosphatase inhibitors okadaic acid and cantharidin in organ elongation assays. Shoots of dark-grown, but not light-grown seedlings also show increased inhibitor sensitivity. Furthermore, cantharidin treatment of wild-type seedlings mimics the rcn1 defect in root curling, root waving and hypocotyl hook formation assays. In roots of wild-type seedlings, RCN1 mRNA is expressed at high levels in root tips, and accumulates to lower levels in the pericycle and lateral root primordia. In shoots, RCN1 is expressed in the apical hook and the basal, rapidly elongating cells in etiolated hypocotyls, and in the shoot meristem and leaf primordia of light-grown seedlings. Our results show that the wild-type RCN1-encoded A subunit functions as a positive regulator of the PP2A holoenzyme, increasing activity towards substrates involved in organ elongation and differential cell elongation responses such as root curling.

  10. Apical endosomes isolated from kidney collecting duct principal cells lack subunits of the proton pumping ATPase

    PubMed Central

    1992-01-01

    Endocytic vesicles that are involved in the vasopressin-stimulated recycling of water channels to and from the apical membrane of kidney collecting duct principal cells were isolated from rat renal papilla by differential and Percoll density gradient centrifugation. Fluorescence quenching measurements showed that the isolated vesicles maintained a high, HgCl2-sensitive water permeability, consistent with the presence of vasopressin-sensitive water channels. They did not, however, exhibit ATP-dependent luminal acidification, nor any N-ethylmaleimide-sensitive ATPase activity, properties that are characteristic of most acidic endosomal compartments. Western blotting with specific antibodies showed that the 31- and 70-kD cytoplasmically oriented subunits of the vacuolar proton pump were not detectable in these apical endosomes from the papilla, whereas they were present in endosomes prepared in parallel from the cortex. In contrast, the 56-kD subunit of the proton pump was abundant in papillary endosomes, and was localized at the apical pole of principal cells by immunocytochemistry. Finally, an antibody that recognizes the 16-kD transmembrane subunit of oat tonoplast ATPase cross-reacted with a distinct 16-kD band in cortical endosomes, but no 16-kD band was detectable in endosomes from the papilla. This antibody also recognized a 16-kD band in affinity- purified H+ ATPase preparations from bovine kidney medulla. Therefore, early endosomes derived from the apical plasma membrane of collecting duct principal cells fail to acidify because they lack functionally important subunits of a vacuolar-type proton pumping ATPase, including the 16-kD transmembrane domain that serves as the proton-conducting channel, and the 70-kD cytoplasmic subunit that contains the ATPase catalytic site. This specialized, non-acidic early endosomal compartment appears to be involved primarily in the hormonally induced recycling of water channels to and from the apical plasma membrane of

  11. Initiation factor 2, tRNA, and 50S subunits cooperatively stabilize mRNAs on the ribosome during initiation

    PubMed Central

    Masuda, Tomoaki; Petrov, Alexey N.; Iizuka, Ryo; Funatsu, Takashi; Puglisi, Joseph D.; Uemura, Sotaro

    2012-01-01

    Initiation factor 2 (IF2) is a key factor in initiation of bacterial protein synthesis. It recruits initiator tRNA to the small ribosomal subunit and facilitates joining of the large ribosomal subunit. Using reconstituted translation system of Escherichia coli and optical tweezers, we directly measure the rupture force between single ribosomal complexes and mRNAs for initiation complexes in the presence and the absence of IF2. We demonstrate that IF2 together with codon recognition by initiator tRNA increases the force required to dislocate mRNA from the ribosome complexes; mRNA stabilization by IF2 required the presence of a joined 50S subunit, and was independent of bound guanine nucleotide. IF2 thus helps lock the 70S ribosome over the start codon during initiation, thus maintaining reading frame. Our results show how mRNA is progressively stabilized on the ribosome through distinct steps of initiation. PMID:22411833

  12. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates.

    PubMed

    Folmer, O; Black, M; Hoeh, W; Lutz, R; Vrijenhoek, R

    1994-10-01

    We describe "universal" DNA primers for polymerase chain reaction (PCR) amplification of a 710-bp fragment of the mitochondrial cytochrome c oxidase subunit I gene (COI) from 11 invertebrate phyla: Echinodermata, Mollusca, Annelida, Pogonophora, Arthropoda, Nemertinea, Echiura, Sipuncula, Platyhelminthes, Tardigrada, and Coelenterata, as well as the putative phylum Vestimentifera. Preliminary comparisons revealed that these COI primers generate informative sequences for phylogenetic analyses at the species and higher taxonomic levels.

  13. [Nose surgical anatomy in six aesthetic subunits].

    PubMed

    Chaput, B; Lauwers, F; Lopez, R; Saboye, J; André, A; Grolleau, J-L; Chavoin, J-P

    2013-04-01

    The nose is a complex entity, combining aesthetic and functional roles. Descriptive anatomy is a fundamental science that it can be difficult to relate directly to our daily surgical activity. Reasoning in terms of aesthetic subunits to decide on his actions appeared to us so obvious. The aim of this paper is to resume the anatomical bases relevant to our daily practice in order to fully apprehend the restorative or cosmetic procedures. We discuss the limits of the systematization of these principles in nasal oncology.

  14. Amino-terminal truncations of the ribulose-bisphosphate carboxylase small subunit influence catalysis and subunit interactions.

    PubMed Central

    Paul, K; Morell, M K; Andrews, T J

    1993-01-01

    The first 20 residues at the amino terminus of the small subunit of spinach ribulose-1,5-bisphosphate carboxylase form an irregular arm that makes extensive contacts with the large subunit and also with another small subunit (S. Knight, I. Andersson, and C.-I. Brändén [1990] J Mol Biol 215: 113-160). The influence of these contacts on subunit binding and, indirectly, on catalysis was investigated by constructing truncations from the amino terminus of the small subunit of the highly homologous enzyme from Synechococcus PCC 6301 expressed in Escherichia coli. Removal of the first six residues (and thus the region of contact with a neighboring small subunit) affected neither the affinity with which the small subunits bound to the large subunits nor the catalytic properties of the assembled holoenzyme. Extending the truncation to include the first 12 residues (which encroaches into a highly conserved region that interacts with the large subunit) also did not weaken intersubunit binding appreciably, but it reduced the catalytic activity of the holoenzyme nearly 5-fold. Removal of an additional single residue (i.e. removal of a total of 13 residues) weakened intersubunit binding approximately 80-fold. Paradoxically, this partially restored catalytic activity to approximately 40% of that of the wild-type holoenzyme. None of these truncations materially affected the Km values for ribulose-1,5-bisphosphate or CO2. Removal of all 20 residues of the irregular arm (thereby deleting the conserved region of contact with large subunits) totally abolished the small subunit's ability to bind to large subunits to form a stable holoenzyme. However, this truncated small subunit was still synthesized by the E. coli cells. These data are interpreted in terms of the role of the amino-terminal arm of the small subunit in maintaining the structure of the holoenzyme. PMID:8278544

  15. Reservoir floodplains support distinct fish assemblages

    USGS Publications Warehouse

    Miranda, Leandro E.; Wigen, S. L.; Dagel, Jonah D.

    2014-01-01

    Reservoirs constructed on floodplain rivers are unique because the upper reaches of the impoundment may include extensive floodplain environments. Moreover, reservoirs that experience large periodic water level fluctuations as part of their operational objectives seasonally inundate and dewater floodplains in their upper reaches, partly mimicking natural inundations of river floodplains. In four flood control reservoirs in Mississippi, USA, we explored the dynamics of connectivity between reservoirs and adjacent floodplains and the characteristics of fish assemblages that develop in reservoir floodplains relative to those that develop in reservoir bays. Although fish species richness in floodplains and bays were similar, species composition differed. Floodplains emphasized fish species largely associated with backwater shallow environments, often resistant to harsh environmental conditions. Conversely, dominant species in bays represented mainly generalists that benefit from the continuous connectivity between the bay and the main reservoir. Floodplains in the study reservoirs provided desirable vegetated habitats at lower water level elevations, earlier in the year, and more frequently than in bays. Inundating dense vegetation in bays requires raising reservoir water levels above the levels required to reach floodplains. Therefore, aside from promoting distinct fish assemblages within reservoirs and helping promote diversity in regulated rivers, reservoir floodplains are valued because they can provide suitable vegetated habitats for fish species at elevations below the normal pool, precluding the need to annually flood upland vegetation that would inevitably be impaired by regular flooding. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

  16. New Species of Boletellus Section Boletellus (Boletaceae, Boletales) from Japan, B. aurocontextus sp. nov. and B. areolatus sp. nov.

    PubMed Central

    Sato, Hirotoshi; Hattori, Tsutomu

    2015-01-01

    We describe and illustrate two new species of Boletellus section Boletellus, B. aurocontextus sp. nov. and B. areolatus sp. nov., which are generally assumed to be B. emodensis. In this study, we reconstructed separate molecular phylogenetic trees of section Boletellus using the nucleotide sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA, the largest subunit (RPB1) and the second-largest subunit (RPB2) of nuclear RNA polymerase II gene and mitochondrial cytochrome oxidase subunit 3 (cox3) gene. We also examined the morphologies of B. emodensis sensu lato (s.l.) and other related species for comparison. The molecular phylogenetic tree inferred from the sequences of nuclear DNA (ITS, and combined dataset of RPB1 and RPB2) indicated that three genetically and phylogenetically well-separated lineages were present within B. emodensis s.l. These three lineages were also distinguished on the basis of the molecular phylogenetic tree constructed using the sequences of mitochondrial DNA (cox3), suggesting distinct cytonuclear disequilibria (i.e., evidence of reproductive isolation) among these lineages. Therefore, these three lineages can be treated as independent species: B. aurocontextus, B. areolatus, and B. emodensis. Boletellus aurocontextus and B. areolatus are also distinct from B. emodensis by the macro- and microscopic morphologies. Boletellus aurocontextus is characterized by a pileus with bright yellow to lemon yellow context, which can be observed through a gap in the scales, and basidiospores with relatively large length (mean spore length, 21.4 μm; quotient of spore length and width, 2.51). In contrast, B. areolatus is characterized by a pileus with floccose to appressed thin scaly patches, a stipe with pallid or pale cream color at the upper half, and basidiospores with relatively small length (mean spore length, 16.5 μm; quotient of spore length and width, 1.80). PMID:26083243

  17. A revision of Cyanonectria and Geejayessia gen. nov., and related species with Fusarium-like anamorphs

    PubMed Central

    Schroers, H.-J.; Gräfenhan, T.; Nirenberg, H.I.; Seifert, K.A.

    2011-01-01

    A revision of Fusarium-like species associated with the plant genus Buxus led to a reconsideration of generic concepts in the Fusarium clade of the Nectriaceae. Phylogenetic analyses of the partial second largest subunit of the RNA polymerase II (rpb2) and the larger subunit of the ATP citrate lyase (acl1) gene exons confirm the existence of a clade, here called the terminal Fusarium clade, that includes genera such as Fusarium sensu stricto (including its Gibberella teleomorphs), Albonectria, Cyanonectria, “Haematonectria”, the newly described genus Geejayessia, and “Nectria” albida. Geejayessia accommodates five species. Four were previously classified in Nectria sensu lato, namely the black perithecial, KOH–species G. atrofusca and the orange or reddish, KOH+ G. cicatricum, G. desmazieri and G. zealandica. Geejayessia celtidicola is newly described. Following our phylogenetic analyses showing its close relationship with Cyanonectria cyanostoma, the former Gibbera buxi is recombined as the second species of Cyanonectria. A three gene phylogenetic analysis of multiple strains of each morphological species using translation elongation factor 1 α (tef-1), rpb2 and acl1 gene exons and introns confirms their status as distinct phylogenetic species. Internal transcribed spacer of the ribosomal RNA gene cluster and nuclear large ribosomal subunit sequences were generated as additional DNA barcodes for selected strains. The connection of Fusarium buxicola, often erroneously reported as the anamorph of G. desmazieri, with the bluish black and KOH+ perithecial species C. buxi is reinstated. Most Cyanonectria and Geejayessia species exhibit restricted host ranges on branches or twigs of Buxus species, Celtis occidentalis, or Staphylea trifolia. Their perithecia form caespitose clusters on well-developed, mostly erumpent stromata on the bark or outer cortex of the host and are relatively thin-walled, mostly smooth, and therefore reminiscent of the more or less

  18. Phylogenetic Analyses of Meloidogyne Small Subunit rDNA

    PubMed Central

    De Ley, Irma Tandingan; De Ley, Paul; Vierstraete, Andy; Karssen, Gerrit; Moens, Maurice; Vanfleteren, Jacques

    2002-01-01

    Phylogenies were inferred from nearly complete small subunit (SSU) 18S rDNA sequences of 12 species of Meloidogyne and 4 outgroup taxa (Globodera pallida, Nacobbus abberans, Subanguina radicicola, and Zygotylenchus guevarai). Alignments were generated manually from a secondary structure model, and computationally using ClustalX and Treealign. Trees were constructed using distance, parsimony, and likelihood algorithms in PAUP* 4.0b4a. Obtained tree topologies were stable across algorithms and alignments, supporting 3 clades: clade I = [M. incognita (M. javanica, M. arenaria)]; clade II = M. duytsi and M. maritima in an unresolved trichotomy with (M. hapla, M. microtyla); and clade III = (M. exigua (M. graminicola, M. chitwoodi)). Monophyly of [(clade I, clade II) clade III] was given maximal bootstrap support (mbs). M. artiellia was always a sister taxon to this joint clade, while M. ichinohei was consistently placed with mbs as a basal taxon within the genus. Affinities with the outgroup taxa remain unclear, although G. pallida and S. radicicola were never placed as closest relatives of Meloidogyne. Our results show that SSU sequence data are useful in addressing deeper phylogeny within Meloidogyne, and that both M. ichinohei and M. artiellia are credible outgroups for phylogenetic analysis of speciations among the major species. PMID:19265950

  19. Two new Neuratelia Rondani (Diptera, Mycetophilidae) species from Western Palaearctic: a case of limited congruence between morphology and DNA sequence data

    PubMed Central

    Kurina, Olavi; Õunap, Erki; Põldmaa, Kadri

    2015-01-01

    Abstract Two new Mycetophilidae species, Neuratelia jabalmoussae sp. n. and Neuratelia salmelai sp. n. are described on the basis of material collected from Lebanon, Estonia and Finland. Detailed figures of male terminalia and photographs of general facies are provided along with discussions of their morphological distinction from sibling species. For the first time molecular characters are used to distinguish new fungus gnat species. Molecular analysis relies on cytochrome oxidase subunit one (COI) but has additionally been corroborated by information from the 28S and ITS2 regions of nuclear ribosomal DNA. Situations where morphological and molecular data provide conflicting evidence for species delimitation are discussed. A new country record from Georgia is provided for Neuratelia caucasica. PMID:25931957

  20. Expression of α and β subunits of the integrin superfamily in articular cartilage from macroscopically normal and osteoarthritic human femoral heads

    PubMed Central

    Ostergaard, K.; Salter, D.; Petersen, J.; Bendtzen, K.; Hvolris, J.; Andersen, C.

    1998-01-01

    OBJECTIVE—The objective of this study was to detail the topographical and zonal distribution of α and β subunits of the integrin superfamily in normal and osteoarthritic cartilage.
METHODS—Immunohistochemistry utilising antibodies towards α and β subunits was performed on cryostat sections of human articular cartilage from macroscopically normal (n = 6) and osteoarthritic (n = 6) femoral heads. Samples of articular cartilage were obtained from 12 topographically distinct sites from each femoral head. Each section was divided into zones (superficial, middle, deep) and staining scores were recorded.
RESULTS—Normal cartilage stained for integrin subunits α1, α5, αV, β1, β4, and β5, but not for α2, α3, α4, α6, β2, β3, and β6. Intact and non-intact residual cartilage from osteoarthritic femoral heads stained for α1, α2, α5, αV, β1, β4, and β5. Staining was occasionally seen for α4 and β2, but not for α3, α6, β3, and β6. There was no topographical variation in the staining for any of the subunits in either normal or osteoarthritic cartilage. The only subunit that displayed a zonal variation was αV; staining for this subunit was most pronounced in the superficial zone compared with the middle and deep zones.
CONCLUSION—Chondrocytes in normal and osteoarthritic cartilage express the integrin subunits α1, α5, αV, β1, β4, and β5. Chondrocytes in osteoarthritic cartilage, in addition, express the α2, α4, and β2 subunits. The αv subunit is expressed by more chondrocytes in the superficial zone in comparison with cells in the deeper zones. None of the subunits display topographical variation in expression.

 Keywords: cartilage; integrins; immunohistochemistry; osteoarthritis PMID:9741315

  1. Alternative splicing in nicotinic acetylcholine receptor subunits from Locusta migratoria and its influence on acetylcholine potencies.

    PubMed

    Zhang, Yixi; Liu, Yang; Bao, Haibo; Sun, Huahua; Liu, Zewen

    2017-01-18

    Due to the great abundance within insect central nervous system (CNS), nicotinic acetylcholine receptors (nAChRs) play key roles in insect CNS, which makes it to be the targets of several classes of insecticides, such as neonicotinoids. Insect nAChRs are pentameric complexes consisting of five subunits, and a dozen subunits in one insect species can theoretically comprise diverse nAChRs. The alternative splicing in insect nAChR subunits may increase the diversity of insect nAChRs. In the oriental migratory locust (Locusta migratoria manilensis Meyen), a model insect species with agricultural importance, the alternative splicing was found in six α subunits among nine α and two β subunits, such as missing conserved residues in Loop D from Locα1, Locα6 and Locα9, a 34-residue insertion in Locα8 cytoplasmic loop, and truncated transcripts for Locα4, Locα7 and Locα9. Hybrid nAChRs were successfully constructed in Xenopus oocytes through co-expression with rat β2 and one α subunit from L. migratoria, which included Locα1, Locα2, Locα3, Locα4, Locα5, Locα8 and Locα9. Influences of alternative splicing in Locα1, Locα8 and Locα9 on acetylcholine potency were tested on hybrid nAChRs. The alternative splicing in Locα1 and Locα9 could increase acetylcholine sensitivities on recombinant receptors, while the splicing in Locα8 showed significant influences on the current amplitudes of oocytes. The results revealed that the alternative splicing at or close to the ligand-binding sites, as well as at cytoplasmic regions away from the ligand-binding sites, in insect nAChR subunits would change the agonist potencies on the receptors, which consequently increased nAChR diversity in functional and pharmacological properties.

  2. DNA barcoding of oomycetes with cytochrome c oxidase subunit I and internal transcribed spacer.

    PubMed

    Robideau, Gregg P; De Cock, Arthur W A M; Coffey, Michael D; Voglmayr, Hermann; Brouwer, Henk; Bala, Kanak; Chitty, David W; Désaulniers, Nicole; Eggertson, Quinn A; Gachon, Claire M M; Hu, Chia-Hui; Küpper, Frithjof C; Rintoul, Tara L; Sarhan, Ehab; Verstappen, Els C P; Zhang, Yonghong; Bonants, Peter J M; Ristaino, Jean B; Lévesque, C André

    2011-11-01

    Oomycete species occupy many different environments and many ecological niches. The genera Phytophthora and Pythium for example, contain many plant pathogens which cause enormous damage to a wide range of plant species. Proper identification to the species level is a critical first step in any investigation of oomycetes, whether it is research driven or compelled by the need for rapid and accurate diagnostics during a pathogen outbreak. The use of DNA for oomycete species identification is well established, but DNA barcoding with cytochrome c oxidase subunit I (COI) is a relatively new approach that has yet to be assessed over a significant sample of oomycete genera. In this study we have sequenced COI, from 1205 isolates representing 23 genera. A comparison to internal transcribed spacer (ITS) sequences from the same isolates showed that COI identification is a practical option; complementary because it uses the mitochondrial genome instead of nuclear DNA. In some cases COI was more discriminative than ITS at the species level. This is in contrast to the large ribosomal subunit, which showed poor species resolution when sequenced from a subset of the isolates used in this study. The results described in this paper indicate that COI sequencing and the dataset generated are a valuable addition to the currently available oomycete taxonomy resources, and that both COI, the default DNA barcode supported by GenBank, and ITS, the de facto barcode accepted by the oomycete and mycology community, are acceptable and complementary DNA barcodes to be used for identification of oomycetes.

  3. Binary Toxin Subunits of Lysinibacillus sphaericus Are Monomeric and Form Heterodimers after In Vitro Activation

    PubMed Central

    Choong, Yeu Khai; Torres, Jaume; Boonserm, Panadda

    2016-01-01

    The binary toxin from Lysinibacillus sphaericus has been successfully used for controlling mosquito-transmitted diseases. An activation step shortens both subunits BinA and BinB before their interaction with membranes and internalization in midgut cells, but the precise role of this activation step is unknown. Herein, we show conclusively using three orthogonal biophysical techniques that protoxin subunits form only monomers in aqueous solution. However, in vitro activated toxins readily form heterodimers. This oligomeric state did not change after incubation of these heterodimers with detergent. These results are consistent with the evidence that maximal toxicity in mosquito larvae is achieved when the two subunits, BinA and BinB, are in a 1:1 molar ratio, and directly link proteolytic activation to heterodimerization. Formation of a heterodimer must thus be necessary for subsequent steps, e.g., interaction with membranes, or with a suitable receptor in susceptible mosquito species. Lastly, despite existing similarities between BinB C-terminal domain with domains 3 and 4 of pore-forming aerolysin, no aerolysin-like SDS-resistant heptameric oligomers were observed when the activated Bin subunits were incubated in the presence of detergents or lipidic membranes. PMID:27341696

  4. Metal content and subunit composition of bean and bovine cytochrome C oxidases

    SciTech Connect

    Sowa, S.; Yewey, G.L.; Roos, E.E.; Caughey, W.S.

    1986-05-01

    Cytochrome c oxidase (CcO) isolated from imbibed whole beans (Phaseolus vulgaris) and bovine hearts have been compared in terms of metal content, subunit composition, and dioxygen reduction site. Both preparations contain Zn and Mg as well as Cu and Fe, with similar Fe/Zn ratios (2.5). Both enzymes also contain multiple subunits of comparable molecular weights and exhibit sharp CO-IR specta for heme iron bound carbonyl species. Alkaline detergent treatment of the bovine enzyme (i.e. incubation with 1% Triton X-100, pH 9.5, followed by ion exchange chromatography) has been reported by others to remove subunit III with loss of H+ pumping activity in reconstituted proteoliposomes without loss of oxidase activity. However, the authors find the treatment does not alter subunit distribution as visualized by SDS-Urea PAGE or HPLC analysis and markedly reduces activity (90%). Metal atom ratios of the treated enzyme indicate a loss of Fe and Mg (Cu/Fe 2.33 +/- 0.24, Fe/Zn 1.64 +/- 0.34, Mg/Zn 0.76 +/- 0.22) relative to native CcO (Cu/Fe 1.23 +/-0.06, Fe/Zn 2.47 +/- 0.14, Mg/Zn 0.94 +/- 0.05). These data provide evidence for similarity between plant and mammalian oxidases and lend further support for vital roles of Zn, Mg, Fe, and Cu in CcO.

  5. Differential roles of the glycogen-binding domains of beta subunits in regulation of the Snf1 kinase complex.

    PubMed

    Mangat, Simmanjeet; Chandrashekarappa, Dakshayini; McCartney, Rhonda R; Elbing, Karin; Schmidt, Martin C

    2010-01-01

    Members of the AMP-activated protein kinase family, including the Snf1 kinase of Saccharomyces cerevisiae, are activated under conditions of nutrient stress. AMP-activated protein kinases are heterotrimeric complexes composed of a catalytic alpha subunit and regulatory beta and gamma subunits. In this study, the role of the beta subunits in the regulation of Snf1 activity was examined. Yeasts express three isoforms of the AMP-activated protein kinase consisting of Snf1 (alpha), Snf4 (gamma), and one of three alternative beta subunits, either Sip1, Sip2, or Gal83. The Gal83 isoform of the Snf1 complex is the most abundant and was analyzed in the greatest detail. All three beta subunits contain a conserved domain referred to as the glycogen-binding domain. The deletion of this domain from Gal83 results in a deregulation of the Snf1 kinase, as judged by a constitutive activity independent of glucose availability. In contrast, the deletion of this homologous domain from the Sip1 and Sip2 subunits had little effect on Snf1 kinase regulation. Therefore, the different Snf1 kinase isoforms are regulated through distinct mechanisms, which may contribute to their specialized roles in different stress response pathways. In addition, the beta subunits are subjected to phosphorylation. The responsible kinases were identified as being Snf1 and casein kinase II. The significance of the phosphorylation is unclear since the deletion of the region containing the phosphorylation sites in Gal83 had little effect on the regulation of Snf1 in response to glucose limitation.

  6. Differential Roles of the Glycogen-Binding Domains of β Subunits in Regulation of the Snf1 Kinase Complex▿

    PubMed Central

    Mangat, Simmanjeet; Chandrashekarappa, Dakshayini; McCartney, Rhonda R.; Elbing, Karin; Schmidt, Martin C.

    2010-01-01

    Members of the AMP-activated protein kinase family, including the Snf1 kinase of Saccharomyces cerevisiae, are activated under conditions of nutrient stress. AMP-activated protein kinases are heterotrimeric complexes composed of a catalytic α subunit and regulatory β and γ subunits. In this study, the role of the β subunits in the regulation of Snf1 activity was examined. Yeasts express three isoforms of the AMP-activated protein kinase consisting of Snf1 (α), Snf4 (γ), and one of three alternative β subunits, either Sip1, Sip2, or Gal83. The Gal83 isoform of the Snf1 complex is the most abundant and was analyzed in the greatest detail. All three β subunits contain a conserved domain referred to as the glycogen-binding domain. The deletion of this domain from Gal83 results in a deregulation of the Snf1 kinase, as judged by a constitutive activity independent of glucose availability. In contrast, the deletion of this homologous domain from the Sip1 and Sip2 subunits had little effect on Snf1 kinase regulation. Therefore, the different Snf1 kinase isoforms are regulated through distinct mechanisms, which may contribute to their specialized roles in different stress response pathways. In addition, the β subunits are subjected to phosphorylation. The responsible kinases were identified as being Snf1 and casein kinase II. The significance of the phosphorylation is unclear since the deletion of the region containing the phosphorylation sites in Gal83 had little effect on the regulation of Snf1 in response to glucose limitation. PMID:19897735

  7. Spatial arrangement and functional role of α subunits of proteasome activator PA28 in hetero-oligomeric form

    SciTech Connect

    Sugiyama, Masaaki; Sahashi, Hiroki; Kurimoto, Eiji; Takata, Shin-ichi; Yagi, Hirokazu; Kanai, Keita; Sakata, Eri; Minami, Yasufumi; Tanaka, Keiji; Kato, Koichi

    2013-03-01

    Highlights: ► Homologous α and β subunits are alternatively arranged in the PA28 heptameric ring. ► The flexible loops of the three α subunits surround the site of substrate entry. ► The loops serve as gatekeepers that selectively hinder passage of longer peptides. - Abstract: A major form of proteasome activator PA28 is a heteroheptamer composed of interferon-γ-inducible α and β subunits, which share approximately 50% amino acid identity and possess distinct insert loops. This activator forms a complex with the 20S proteasome and thereby stimulates proteasomal degradation of peptides in an ATP-independent manner, giving rise to smaller antigenic peptides presented by major histocompatibility complex class I molecules. In this study, we performed biophysical and biochemical characterization of the structure and function of the PA28 hetero-oligomer. Deuteration-assisted small-angle neutron scattering demonstrated three α and four β subunits are alternately arranged in the heptameric ring. In this arrangement, PA28 loops surround the central pore of the heptameric ring (site for peptide entry). Activating the 20S proteasome with a PA28 mutant that lacked the α subunit loops cleaved model substrates longer than a nonapeptide with better efficiency when compared to wild-type PA28. Based on these data, we hypothesize that the flexible PA28 loops act as gatekeepers, which function to select the length of peptide substrates to be transported between the proteolytic chamber and the extra-proteasomal medium.

  8. Structure and function of an archaeal topoisomerase VI subunit with homology to the meiotic recombination factor Spo11.

    PubMed

    Nichols, M D; DeAngelis, K; Keck, J L; Berger, J M

    1999-11-01

    In all organisms, type II DNA topoisomerases are essential for untangling chromosomal DNA. We have determined the structure of the DNA-binding core of the Methanococcus jannaschii DNA topoisomerase VI A subunit at 2.0 A resolution. The overall structure of this subunit is unique, demonstrating that archaeal type II enzymes are distinct from other type II topoisomerases. However, the core structure contains a pair of domains that are also found in type IA and classic type II topoisomerases. Together, these regions may form the basis of a DNA cleavage mechanism shared among these enzymes. The core A subunit is a dimer that contains a deep groove that spans both protomers. The dimer architecture suggests that DNA is bound in the groove, across the A subunit interface, and that the two monomers separate during DNA transport. The A subunit of topoisomerase VI is homologous to the meiotic recombination factor, Spo11, and this structure can serve as a template for probing Spo11 function in eukaryotes.

  9. Re-examination of a proposed case of stasipatric speciation: phylogeography of the Australian morabine grasshoppers (Vandiemenella viatica species group).

    PubMed

    Kawakami, Takeshi; Butlin, Roger K; Adams, Mark; Saint, Kathleen M; Paull, David J; Cooper, Steven J B

    2009-08-01

    Karyotypic differences have been used for delimiting populations or species, although whether these mutations provide strong barriers to gene flow between populations and promote speciation remains contentious. In this study, we assessed whether 11 chromosomal races of Australian morabine grasshoppers (Vandiemenella viatica species group) represent genetically distinct populations by analyses of cytological and allozyme (35 loci) data and DNA sequences of the elongation factor-1 alpha (EF-1alpha), anonymous Mvia11, and mitochondrial cytochrome c oxidase subunit I (COI) loci. While the Vandiemenella chromosomal taxa generally represent genetically distinct units, a substantial portion of the total genetic variation in our samples was not explained by the chromosomal variation. Mantel tests indicated that Vandiemenella populations were spatially structured and have maintained gene flow at a local scale within each of the taxa. The group was subdivided into 13 genetic clusters; four chromosomal taxa comprised single exclusive clusters, while others comprised more than one cluster or clusters shared with other taxa. Boundaries of these cryptic population subdivisions correspond with several biogeographical barriers, such as straits, gulfs, the Murray River, and an ancient mega-lake, Lake Bungunnia. The viatica species group was previously proposed to have diversified without major geographical separation based on the stasipatric speciation model; however, the present study suggests the involvement of allopatric fragmentation. Given extensive nonmonophyly of chromosomal taxa and incomplete barriers to gene flow among taxa, all Vandiemenella chromosomal taxa and genetically distinct populations within chromosomal taxa, except Vandiemenella pichirichi, should be regarded as populations of one species: Vandiemenella viatica.

  10. An uncoupling channel within the c-subunit ring of the F1FO ATP synthase is the mitochondrial permeability transition pore.

    PubMed

    Alavian, Kambiz N; Beutner, Gisela; Lazrove, Emma; Sacchetti, Silvio; Park, Han-A; Licznerski, Pawel; Li, Hongmei; Nabili, Panah; Hockensmith, Kathryn; Graham, Morven; Porter, George A; Jonas, Elizabeth A

    2014-07-22

    Mitochondria maintain tight regulation of inner mitochondrial membrane (IMM) permeability to sustain ATP production. Stressful events cause cellular calcium (Ca(2+)) dysregulation followed by rapid loss of IMM potential known as permeability transition (PT), which produces osmotic shifts, metabolic dysfunction, and cell death. The molecular identity of the mitochondrial PT pore (mPTP) was previously unknown. We show that the purified reconstituted c-subunit ring of the FO of the F1FO ATP synthase forms a voltage-sensitive channel, the persistent opening of which leads to rapid and uncontrolled depolarization of the IMM in cells. Prolonged high matrix Ca(2+) enlarges the c-subunit ring and unhooks it from cyclophilin D/cyclosporine A binding sites in the ATP synthase F1, providing a mechanism for mPTP opening. In contrast, recombinant F1 beta-subunit applied exogenously to the purified c-subunit enhances the probability of pore closure. Depletion of the c-subunit attenuates Ca(2+)-induced IMM depolarization and inhibits Ca(2+) and reactive oxygen species-induced cell death whereas increasing the expression or single-channel conductance of the c-subunit sensitizes to death. We conclude that a highly regulated c-subunit leak channel is a candidate for the mPTP. Beyond cell death, these findings also imply that increasing the probability of c-subunit channel closure in a healthy cell will enhance IMM coupling and increase cellular metabolic efficiency.

  11. Sodium channel β subunits: emerging targets in channelopathies

    PubMed Central

    O’Malley, Heather A.; Isom, Lori L.

    2016-01-01

    Voltage-gated sodium channels (VGSCs) are responsible for initiation and propagation of action potentials in excitable cells. VGSCs in mammalian brain are heterotrimeric complexes of α and β subunits. Originally called “auxiliary,” we now know that β subunit proteins are multifunctional signaling molecules that play roles in both excitable and non-excitable cell types, and with or without the pore-forming α subunit present. β subunits function in VGSC and potassium channel modulation, cell adhesion, and gene regulation, with particularly important roles in brain development. Mutations in the genes encoding β subunits are linked to a number of diseases, including epilepsy, sudden death syndromes like SUDEP and SIDS, and cardiac arrhythmia. While VGSC β subunit-specific drugs have not yet been developed, this protein family is an emerging therapeutic target. PMID:25668026

  12. Nicotine normalizes intracellular subunit stoichiometry of nicotinic receptors carrying mutations linked to autosomal dominant nocturnal frontal lobe epilepsy.

    PubMed

    Son, Cagdas D; Moss, Fraser J; Cohen, Bruce N; Lester, Henry A

    2009-05-01

    Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is linked with high penetrance to several distinct nicotinic receptor (nAChR) mutations. We studied (alpha4)(3)(beta2)(2) versus (alpha4)(2)(beta2)(3) subunit stoichiometry for five channel-lining M2 domain mutations: S247F, S252L, 776ins3 in alpha4, V287L, and V287M in beta2. alpha4 and beta2 subunits were constructed with all possible combinations of mutant and wild-type (WT) M2 regions, of cyan and yellow fluorescent protein, and of fluorescent and nonfluorescent M3-M4 loops. Sixteen fluorescent subunit combinations were expressed in N2a cells. Förster resonance energy transfer (FRET) was analyzed by donor recovery after acceptor photobleaching and by pixel-by-pixel sensitized emission, with confirmation by fluorescence intensity ratios. Because FRET efficiency is much greater for adjacent than for nonadjacent subunits and the alpha4 and beta2 subunits occupy specific positions in nAChR pentamers, observed FRET efficiencies from (alpha4)(3)(beta2)(2) carrying fluorescent alpha4 subunits were significantly higher than for (alpha4)(2)(beta2)(3); the converse was found for fluorescent beta2 subunits. All tested ADNFLE mutants produced 10 to 20% increments in the percentage of intracellular (alpha4)(3)(beta2)(2) receptors compared with WT subunits. In contrast, 24- to 48-h nicotine (1 muM) exposure increased the proportion of (alpha4)(2)(beta2)(3) in WT receptors and also returned subunit stoichiometry to WT levels for alpha4S248F and beta2V287L nAChRs. These observations may be relevant to the decreased seizure frequency in patients with ADNFLE who use tobacco products or nicotine patches. Fluorescence-based investigations of nAChR subunit stoichiometry may provide efficient drug discovery methods for nicotine addiction or for other disorders that result from dysregulated nAChRs.

  13. Unexpected occurrence of Hemifridericia bivesiculata Christensen & Dózsa-Farkas, 2006 in Hungary, a species presumed to be endemic to Devon Island, Canada, and its comparative analysis with H. parva Nielsen & Christensen, 1959 (Enchytraeidae, Oligochaeta).

    PubMed

    Dózsa-Farkas, Klára; Felföldi, Tamás

    2015-01-27

    During the exploration of the enchytraeid fauna in forests and grasslands of Hungarian flat areas, a small enchytraeid species, Hemifridericia bivesiculata Christensen & Dózsa-Farkas, 2006, was found unexpectedly. This species was known previously only from one single location, Bank Island in the Canadian archipelago, and was presumed to be endemic. Here, the distinctive features of the two Hemifridericia species are described based on detailed morphological analyses, which were supported with molecular methods. The results demonstrated that the two Hemifridericia species are very similar morphologically, but can be clearly distinguished from each other by the presence or absence of the oesophageal vesicles and the shape of the brain. Some additional differences were observed between the Hungarian material and the type specimens of H. bivesiculata. The two species were unequivocally separated based on the studied molecular markers, the mitochondrial cytochrome c oxidase subunit I (CO1) gene, the nuclear histon 3 (H3) gene and the nuclear ribosomal ITS region. 

  14. An atypical heterotrimeric G-protein γ-subunit is involved in guard cell K⁺-channel regulation and morphological development in Arabidopsis thaliana.

    PubMed

    Chakravorty, David; Trusov, Yuri; Zhang, Wei; Acharya, Biswa R; Sheahan, Michael B; McCurdy, David W; Assmann, Sarah M; Botella, José Ramón

    2011-09-01

    Currently, there are strong inconsistencies in our knowledge of plant heterotrimeric G-proteins that suggest the existence of additional members of the family. We have identified a new Arabidopsis G-protein γ-subunit (AGG3) that modulates morphological development and ABA-regulation of stomatal aperture. AGG3 strongly interacts with the Arabidopsis G-protein β-subunit in vivo and in vitro. Most importantly, AGG3-deficient mutants account for all but one of the 'orphan' phenotypes previously unexplained by the two known γ-subunits in Arabidopsis. AGG3 has unique characteristics never before observed in plant or animal systems, such as its size (more than twice that of canonical γ-subunits) and the presence of a C-terminal Cys-rich domain. AGG3 thus represent a novel class of G-protein γ-subunits, widely spread throughout the plant kingdom but not present in animals. Homologues of AGG3 in rice have been identified as important quantitative trait loci for grain size and yield, but due to the atypical nature of the proteins their identity as G-protein subunits was thus far unknown. Our work demonstrates a similar trend in seeds of Arabidopsis agg3 mutants, and implicates G-proteins in such a crucial agronomic trait. The discovery of this highly atypical subunit reinforces the emerging notion that plant and animal G-proteins have distinct as well as shared evolutionary pathways.

  15. Descriptions of two azooxanthellate Palythoa species (Subclass Hexacorallia, Order Zoantharia) from the Ryukyu Archipelago, southern Japan

    PubMed Central

    Irei, Yuka; Sinniger, Frederic; Reimer, James Davis

    2015-01-01

    Abstract Two new species of zoantharians (Hexacorallia, Zoantharia, Sphenopidae), Palythoa mizigama sp. n. and Palythoa umbrosa sp. n., are described from the Ryukyu Archipelago, southern Japan. Unlike almost all other known Palythoa spp., both species