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Sample records for distinct subunit species

  1. Salamander rods and cones contain distinct transducin alpha subunits.

    PubMed

    Ryan, J C; Znoiko, S; Xu, L; Crouch, R K; Ma, J X

    2000-01-01

    The mammalian retina is known to contain two distinct transducins that interact with their respective rod and cone pigments. However, there are no reports of a nonmammalian species having two distinct transducins. In the present study, we report the cloning and cellular localization of two transducin a subunits (G alpha t) from the tiger salamander. Through degenerate polymerase chain reaction (PCR) and subsequent screening of a salamander retina cDNA library, we have identified two forms of G alpha t. When compared to existing sequences in GenBank, the cloned subunits showed high similarity to rod and cone transducins. The salamander G alpha t-1 has 91.2-93.7% amino acid sequence identity to mammalian rod G alpha t subunits and 79.7-80.9% to mammalian cone Gats. The salamander G alpha t-2 has 86.2-87.9% sequence identity to mammalian cone G alpha ts and 78.9-80.9% to mammalian rod G alpha ts at the amino acid level. The G alpha t-1 cDNA encodes 350 amino acids while the G alpha t-2 cDNA encodes 354 residues, which is typical for rod and cone G alpha ts, respectively, and we thus identified the G alpha t- 1 as rod and G alpha t-2 as cone G alpha t. Sequences identified as effector binding sites and GTPase activity regions are highly conserved between the two subunits. Genomic Southern blot analysis showed that rod and cone G alpha t subunits are both encoded by single-copy genes. Northern blot analysis identified retina-specific transcripts of 3.0 kb for rod G alpha t and 2.6 kb for cone G alpha t. Immunohistochemistry in the flat-mounted salamander retina demonstrated that rod G alpha t is localized to rods, predominantly in the outer segments; similarly, cone G alpha t is localized to cone outer segments. The results confirm that the two sequences encode rod and cone transducins and demonstrate that this lower vertebrate contains two distinct transducins that are localized specifically to rod and cone photoreceptors.

  2. The regulatory subunits of PI3Kγ control distinct neutrophil responses.

    PubMed

    Deladeriere, Arnaud; Gambardella, Laure; Pan, Dingxin; Anderson, Karen E; Hawkins, Phillip T; Stephens, Len R

    2015-01-20

    Neutrophils, which migrate toward inflamed sites and kill pathogens by producing reactive oxygen species (ROS), are important in the defense against bacterial and fungal pathogens, but their inappropriate regulation causes various chronic inflammatory diseases. Phosphoinositide 3-kinase γ (PI3Kγ) functions downstream of proinflammatory G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors (GPCRs) in neutrophils and is a therapeutic target. In neutrophils, PI3Kγ consists of a p110γ catalytic subunit, which is activated by the guanosine triphosphatase Ras, and either a p84 or p101 regulatory subunit. Loss or inhibition of p110γ or expression of a Ras-insensitive variant p110γ (p110γ(DASAA/DASAA)) impairs PIP3 production, Akt phosphorylation, migration, and ROS formation in response to GPCR activation. The p101 subunit binds to, and mediates PI3Kγ activation by, G protein βγ subunits, and p101(-/-) neutrophils have a similar phenotype to that of p110γ(-/-) neutrophils, except that ROS responses are normal. We found that p84(-/-) neutrophils displayed reduced GPCR-stimulated PIP3 and Akt signaling, which was indistinguishable from that of p101(-/-) neutrophils. However, p84(-/-) neutrophils produced less ROS and exhibited normal migration in response to GPCR stimulation. These data suggest that p84-containing PI3Kγ controls GPCR-dependent ROS production. Thus, the PI3Kγ regulatory subunits enable PI3Kγ to mediate distinct neutrophil responses, which may occur by targeting PIP3 signaling into spatially distinct domains. Copyright © 2015, American Association for the Advancement of Science.

  3. 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

  4. Distinct roles for ATP binding and hydrolysis at individual subunits of an archaeal clamp loader

    PubMed Central

    Seybert, Anja; Wigley, Dale B

    2004-01-01

    Circular clamps are utilised by replicative polymerases to enhance processivity. The topological problem of loading a toroidal clamp onto DNA is overcome by ATP-dependent clamp loader complexes. Different organisms use related protein machines to load clamps, but the mechanisms by which they utilise ATP are surprisingly different. Using mutant clamp loaders that are deficient in either ATP binding or hydrolysis in different subunits, we show how the different subunits of an archaeal clamp loader use ATP binding and hydrolysis in distinct ways at different steps in the loading process. Binding of nucleotide by the large subunit and three of the four small subunits is sufficient for clamp loading. However, ATP hydrolysis by the small subunits is required for release of PCNA to allow formation of the complex between PCNA and the polymerase, while hydrolysis by the large subunit is required for catalytic clamp loading. PMID:15014449

  5. Distinct forms of the. beta. subunit of GTP-binding regulatory proteins identified by molecular cloning

    SciTech Connect

    Fong, H.K.W.; Amatruda, T.T. III; Birren, B.W.; Simon, M.I.

    1987-06-01

    Two distinct ..beta.. subunits of guanine nucleotide-binding regulatory proteins have been identified by cDNA cloning and are referred to as ..beta../sub 1/ and ..beta../sub 1/ subunits. The bovine transducin ..beta.. subunit (..beta../sub 1/) has been cloned previously. The author now isolated and analyzed cDNA clones that encode the ..beta../sub 2/ subunit from bovine adrenal, bovine brain, and a human myeloid leukemia cell line, HL-60. The 340-residue M/sub r/ 37,329 BETA/sub 2/ protein is 90% identical with ..beta../sub 1/ in predicted amino acid sequence, and it is also organized as a series of repetitive homologous segments. The major mRNA that encodes the bovine ..beta../sub 2/ subunit is 1.7 kilobases in length. It is expressed at lower levels than ..beta../sub 1/ subunit mRNA in all tissues examined. The ..beta../sub 1/ and ..beta../sub 2/ messages are expressed in cloned human cell lines. Hybridization of cDNA probes to bovine DNA showed that ..beta../sub 1/ and ..beta../sub 2/ are encoded by separate genes. The amino acid sequences for the bovine and human ..beta../sub 2/ subunit are identical, as are the amino acid sequences for the bovine and human ..beta../sub 1/ subunit. This evolutionary conservation suggests that the two ..beta.. subunits have different roles in the signal transduction process.

  6. 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.

  7. Cardiolipin: characterization of distinct oxidized molecular species

    PubMed Central

    Kim, Junhwan; Minkler, Paul E.; Salomon, Robert G.; Anderson, Vernon E.; Hoppel, Charles L.

    2011-01-01

    Cardiolipin (CL) is a phospholipid predominantly found in the mitochondrial inner membrane and is associated structurally with individual complexes of the electron transport chain (ETC). Because the ETC is the major mitochondrial reactive oxygen species (ROS)-generating site, the proximity to the ETC and bisallylic methylenes of the PUFA chains of CL make it a likely target of ROS in the mitochondrial inner membrane. Oxidized cellular CL products, uniquely derived from ROS-induced autoxidation, could serve as biomarkers for the presence of the ROS and could help in the understanding of the mechanism of oxidative stress. Because major CL species have four unsaturated acyl chains, whereas other phospholipids usually have only one in the sn-2 position, characterization of oxidized CL is highly challenging. In the current study, we exposed CL, under aerobic conditions, to singlet oxygen (1O2), the radical initiator 2,2′-azobis(2-methylpropionamidine) dihydrochloride, or room air, and the oxidized CL species were characterized by HPLC-tandem mass spectrometry (MS/MS). Our reverse-phase ion-pair HPLC-MS/MS method can characterize the major and minor oxidized CL species by detecting distinctive fragment ions associated with specific oxidized species. The HPLC-MS/MS results show that monohydroperoxides and bis monohydroperoxides were generated under all three conditions. However, significant amounts of CL dihydroperoxides were produced only by 1O2-mediated oxidation. These products were barely detectable from radical oxidation either in a liposome bilayer or in thin film. These observations are only possible due to the chromatographic separation of the different oxidized species. PMID:20858593

  8. 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

  9. 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

  10. A single mutation in the acetylcholine receptor δ-subunit causes distinct effects in two types of neuromuscular synapses.

    PubMed

    Park, Jee-Young; Mott, Meghan; Williams, Tory; Ikeda, Hiromi; Wen, Hua; Linhoff, Michael; Ono, Fumihito

    2014-07-30

    Mutations in AChR subunits, expressed as pentamers in neuromuscular junctions (NMJs), cause various types of congenital myasthenic syndromes. In AChR pentamers, the adult ε subunit gradually replaces the embryonic γ subunit as the animal develops. Because of this switch in subunit composition, mutations in specific subunits result in synaptic phenotypes that change with developmental age. However, a mutation in any AChR subunit is considered to affect the NMJs of all muscle fibers equally. Here, we report a zebrafish mutant of the AChR δ subunit that exhibits two distinct NMJ phenotypes specific to two muscle fiber types: slow or fast. Homozygous fish harboring a point mutation in the δ subunit form functional AChRs in slow muscles, whereas receptors in fast muscles are nonfunctional. To test the hypothesis that different subunit compositions in slow and fast muscles underlie distinct phenotypes, we examined the presence of ε/γ subunits in NMJs using specific antibodies. Both wild-type and mutant larvae lacked ε/γ subunits in slow muscle synapses. These findings in zebrafish suggest that some mutations in human congenital myasthenic syndromes may affect slow and fast muscle fibers differently. Copyright © 2014 the authors 0270-6474/14/3410211-08$15.00/0.

  11. A functionally distinct member of the DP family of E2F subunits.

    PubMed

    Milton, A; Luoto, K; Ingram, L; Munro, S; Logan, N; Graham, A L; Brummelkamp, T R; Hijmans, E M; Bernards, R; La Thangue, N B

    2006-05-25

    E2F transcription factors regulate genes involved in cell-cycle progression. In mammalian cells, physiological E2F exists as an E2F/DP heterodimer. Currently, eight E2F and two DP subunits have been characterized. We report here the characterization of a new member of the DP family, DP-4. While DP-4 exhibits certain similarities with members of the DP family, it also possesses a number of significant differences. Thus, DP-4 forms a heterodimer with E2F subunits, binds to the E2F site and associates with pocket proteins including pRb. In contrast to DP-1, however, DP-4/E2F-1 complexes exhibit reduced DNA binding activity. Furthermore, DP-4 interferes with E2F-1-dependent transcription and delays cell-cycle progression. These results highlight an emerging complexity in the DP family of E2F subunits, and suggest that DP-4 may endow E2F heterodimers with distinct transcription properties.

  12. 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.

  13. 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.

  14. 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

  15. Analysis of Exocyst Subunit EXO70 Family Reveals Distinct Membrane Polar Domains in Tobacco Pollen Tubes.

    PubMed

    Sekereš, Juraj; Pejchar, Přemysl; Šantrůček, Jiří; Vukašinović, Nemanja; Žárský, Viktor; Potocký, Martin

    2017-03-01

    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. © 2017 American Society of Plant Biologists. All Rights Reserved.

  16. 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.

  17. Two distinct effects of PIP2 underlie auxiliary subunit-dependent modulation of Slo1 BK channels

    PubMed Central

    Ullrich, Florian; Xu, Rong; Heinemann, Stefan H.; Hou, Shangwei

    2015-01-01

    Phosphatidylinositol 4,5-bisphosphate (PIP2) plays a critical role in modulating the function of numerous ion channels, including large-conductance Ca2+- and voltage-dependent K+ (BK, Slo1) channels. Slo1 BK channel complexes include four pore-forming Slo1 (α) subunits as well as various regulatory auxiliary subunits (β and γ) that are expressed in different tissues. We examined the molecular and biophysical mechanisms underlying the effects of brain-derived PIP2 on human Slo1 BK channel complexes with different subunit compositions that were heterologously expressed in human embryonic kidney cells. PIP2 inhibited macroscopic currents through Slo1 channels without auxiliary subunits and through Slo1 + γ1 complexes. In contrast, PIP2 markedly increased macroscopic currents through Slo1 + β1 and Slo1 + β4 channel complexes and failed to alter macroscopic currents through Slo1 + β2 and Slo1 + β2 Δ2–19 channel complexes. Results obtained at various membrane potentials and divalent cation concentrations suggest that PIP2 promotes opening of the ion conduction gate in all channel types, regardless of the specific subunit composition. However, in the absence of β subunits positioned near the voltage-sensor domains (VSDs), as in Slo1 and probably Slo1 + γ1, PIP2 augments the negative surface charge on the cytoplasmic side of the membrane, thereby shifting the voltage dependence of VSD-mediated activation in the positive direction. When β1 or β4 subunits occupy the space surrounding the VSDs, only the stimulatory effect of PIP2 is evident. The subunit compositions of native Slo1 BK channels differ in various cell types; thus, PIP2 may exert distinct tissue- and divalent cation–dependent modulatory influences. PMID:25825171

  18. Two distinct effects of PIP2 underlie auxiliary subunit-dependent modulation of Slo1 BK channels.

    PubMed

    Tian, Yutao; Ullrich, Florian; Xu, Rong; Heinemann, Stefan H; Hou, Shangwei; Hoshi, Toshinori

    2015-04-01

    Phosphatidylinositol 4,5-bisphosphate (PIP2) plays a critical role in modulating the function of numerous ion channels, including large-conductance Ca(2+)- and voltage-dependent K(+) (BK, Slo1) channels. Slo1 BK channel complexes include four pore-forming Slo1 (α) subunits as well as various regulatory auxiliary subunits (β and γ) that are expressed in different tissues. We examined the molecular and biophysical mechanisms underlying the effects of brain-derived PIP2 on human Slo1 BK channel complexes with different subunit compositions that were heterologously expressed in human embryonic kidney cells. PIP2 inhibited macroscopic currents through Slo1 channels without auxiliary subunits and through Slo1 + γ1 complexes. In contrast, PIP2 markedly increased macroscopic currents through Slo1 + β1 and Slo1 + β4 channel complexes and failed to alter macroscopic currents through Slo1 + β2 and Slo1 + β2 Δ2-19 channel complexes. Results obtained at various membrane potentials and divalent cation concentrations suggest that PIP2 promotes opening of the ion conduction gate in all channel types, regardless of the specific subunit composition. However, in the absence of β subunits positioned near the voltage-sensor domains (VSDs), as in Slo1 and probably Slo1 + γ1, PIP2 augments the negative surface charge on the cytoplasmic side of the membrane, thereby shifting the voltage dependence of VSD-mediated activation in the positive direction. When β1 or β4 subunits occupy the space surrounding the VSDs, only the stimulatory effect of PIP2 is evident. The subunit compositions of native Slo1 BK channels differ in various cell types; thus, PIP2 may exert distinct tissue- and divalent cation-dependent modulatory influences. © 2015 Tian et al.

  19. 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.

  20. Distinct structural features of the. cap alpha. and. beta. subunits of nitrogenase molybdenum-iron protein of Clostridium pasteurianum: an analysis of amino acid sequences

    SciTech Connect

    Wang, S.Z.; Chen, J.S.; Johnson, J.L.

    1988-04-19

    Nitrogenase is composed of two separately purified proteins, a molybdenum-iron (MoFe) protein and an iron (Fe) protein. Structural genes (nifD and nifK) encoding ..cap alpha.. and ..beta.. subunits of the MoFe protein of Clostridium pasteurianum (Cp) have been cloned and sequenced. The deduced amino acid sequences were analyzed for structures that could be related to the unique properties of the Cp protein, particularly its low capacity to form an active enzyme with a heterologous Fe protein. Cp nifK is located immediately downstream from Cp nifD, with the start codon of nifK overlapping by one base with the stop codon of nifD. An open reading frame following nifK was identified as nifE. The amino acid sequence deduced from nifK encompasses the partial amino acid sequences previously reported from the isolated ..beta.. subunit. Cp nifK encodes a polypeptide of 458 amino acid residues (M/sub r/ 50,115) whose amino-terminal region is about 50 resides shorter than the otherwise conserved corresponding polypeptides from four other organisms. In contrast, Cp ..cap alpha.. subunit (nifD product) contains an additional stretch of 50 amino acid residues in the 380-430 region, which is unique to the Cp protein. It therefore appears that the combined size of the ..cap alpha.. and ..beta.. subunits could be important to nitrogenase function. An analysis of the predicted secondary structure from the amino acid sequence of each subunit from three species (C. pasteurianum, Azotobacter vinelandii, and Rhizobium japonicum) further revealed structural features, including regions adjacent to some of the conserved cysteine resides, differentiating the Cp MoFe protein from others. These different regions may be further tested for correlation with distinct properties of Cp nitrogenase.

  1. Distinctive biophysical and pharmacological properties of class A (BI) calcium channel alpha 1 subunits.

    PubMed

    Sather, W A; Tanabe, T; Zhang, J F; Mori, Y; Adams, M E; Tsien, R W

    1993-08-01

    Transcripts for the class A Ca2+ channel alpha 1 subunit (also known as BI) are present at high levels in many parts of the mammalian CNS and are widely assumed to encode the P-type Ca2+ channel. To characterize the biophysical and pharmacological properties of alpha 1A channels, macroscopic and single-channel recordings were made in Xenopus oocytes injected with alpha 1A cRNA. alpha 1-specific properties were identified by making systematic comparisons with the more familiar class C alpha 1 subunit under the condition of a standard ancillary subunit (alpha 2/delta + beta) makeup. alpha 1A currents activate and inactivate more rapidly and display steeper voltage dependence of gating than alpha 1C currents. Unlike alpha 1C, alpha 1A channels are largely insensitive to dihydropyridines and FPL 64176, but respond to the cone snail peptide omega-CTx-MVIIC(SNX-230), a potent and fairly selective inhibitor. In comparison with P-type Ca2+ channels in rat cerebellar Purkinje cells, alpha 1A channels in oocytes are approximately 10(2)-fold less sensitive to omega-Aga-IVA and approximately 10-fold more sensitive to omega-CTx-MVIIC. alpha 1A channels are not inhibited by Bay K 8644 and inactivate much more rapidly than P-type Ca2+ channels. Thus, alpha 1A is capable of generating a Ca2+ channel phenotype quite different from P-type current.

  2. 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.

  3. 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. Copyright © 2014 by the Genetics Society of America.

  4. Diverse Gene-Silencing Mechanisms with Distinct Requirements for RNA Polymerase Subunits in Zea mays

    PubMed Central

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

    2014-01-01

    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. PMID:25164883

  5. 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

  6. 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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  8. 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

  9. 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.

  10. 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).

  11. Molecular Evidence for Species-Level Distinctions in Clouded Leopards

    PubMed Central

    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.

    2017-01-01

    Summary 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 [1–5]. This secretive, mid-sized (16–23 kg) carnivore, now severely endangered, is traditionally subdivided into four southeast Asian subspecies (Figure 1A) [4–8]. 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 and 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). PMID:17141620

  12. 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

  13. 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

  14. 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.

  15. 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. Copyright © 2011 Wiley-Liss, Inc.

  16. 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

  17. Genetic variation in the mitochondrial cytochrome c oxidase subunit 1 within three species of Progamotaenia (Cestoda: Anoplocephalidae) from macropodid marsupials.

    PubMed

    Hu, M; Gasser, R B; Chilton, N B; Beveridge, I

    2005-01-01

    Sequence variation within 3 morphologically defined species of the anoplocephalid cestode genus Progamotaenia (P. ewersi, P. macropodis and P. zschokkei) was investigated using the cytochrome c oxidase subunit 1 gene. The magnitude of genetic variation detected within each morphospecies suggests that, in each instance, several cryptic species are present. Within P. ewersi, 5 genetically distict groups of cestodes were detected, 1 shared by Macropus robustus and M. parryi in Queensland, 1 in M. agilis from Queensland, 1 in Petrogale assimilis from Queensland, 1 in Macropus fuliginosus from South Australia and 1 in Wallabia bicolor from Victoria. In P. macropodis, cestodes from M. robustus from Queensland, Western Australia and the Northern Territory, M. parryi from Queensland and M. eugenii from South Australia were genetically distinct from those in Wallabia bicolor from Queensland and Victoria and from M. fuliginosus from South Australia. P. zschokkei consisted of a number of genetically distinct groups of cestodes, 1 in Lagorchestes conspicillatus and L. hirsutus from Queensland and the Northern Territory respectively, 1 in Petrogale herberti, P. assimilis and M. dorsalis from Queensland, 1 in Onychogalea fraenata from Queensland, 1 in M. agilis from Queensland and 1 in Thylogale stigmatica and T. thetis from Queensland. In general, genetic groups within each morphospecies were host specific and occurred predominantly in a particular macropodid host clade. Comparison of genetic relationships of cestodes with the phylogeny of their hosts revealed examples of colonization (P. zschokkei in M. agilis) and of host switching (P. zschokkei in M. dorsalis).

  18. The production effect in memory: multiple species of distinctiveness

    PubMed Central

    Icht, Michal; Mama, Yaniv; Algom, Daniel

    2014-01-01

    The production effect is the difference in memory favoring words read aloud relative to words read silently during study. According to a currently popular explanation, the distinctiveness of aloud words relative to silent words at the time of encoding underlies the better memory for the former. This distinctiveness is attributable to the additional dimension(s) of encoding for the aloud items that can be subsequently used during retrieval. In this study we argue that encoding distinctiveness is not the sole source of distinctiveness and that, in fact, there is an independent source of distinctiveness, statistical distinctiveness, which may or may not work in harmony with encoding distinctiveness in influencing memory. Statistical distinctiveness refers to the relative size of a subset of items marked by a(ny) unique property. Silently read words can carry statistical distinctiveness if they form a salient minority on the background of a majority of vocalized words. We show that, when the two sources are placed in opposition, statistical distinctiveness modifies the PE in a profound way. PMID:25157237

  19. Distinct contributions of T1R2 and T1R3 taste receptor subunits to the detection of sweet stimuli.

    PubMed

    Nie, Yiling; Vigues, Stephan; Hobbs, Jeanette R; Conn, Graeme L; Munger, Steven D

    2005-11-08

    Animals utilize hundreds of distinct G protein-coupled receptor (GPCR)-type chemosensory receptors to detect a diverse array of chemical signals in their environment, including odors, pheromones, and tastants. However, the molecular mechanisms by which these receptors 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. Here, we report that each of the two subunits in the 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 behaving mice. Thus, individual T1R subunits increase the receptive range of the sweet taste receptor, offering a functional mechanism for phenotypic variations in sweet taste.

  20. Specific residues within the alpha 2 integrin subunit cytoplasmic domain regulate migration and cell cycle progression via distinct MAPK pathways.

    PubMed

    Klekotka, P A; Santoro, S A; Wang, H; Zutter, M M

    2001-08-24

    The alpha(2) integrin subunit cytoplasmic domain is necessary for epidermal growth factor (EGF)-stimulated chemotactic migration and insulin-dependent entry into S-phase of mammary epithelial cells adherent to type I collagen. Truncation mutants revealed that the seven amino acids, KYEKMTK, in addition to the GFFKR motif were sufficient for these functions. Mutation of tyrosine 1134 to alanine inhibited the ability of the cells to phosphorylate p38 MAPK and to migrate in response to EGF but had only a modest effect on the ability of the cells to induce sustained phosphorylation of the ERK MAPK, to up-regulate cyclin E and cdk2 expression, and to enter S-phase when adherent to type I collagen. Conversely, mutation of the lysine 1136 inhibited the ability of the cells to increase cyclin E and cdk2 expression, to maintain long term phosphorylation of the ERK MAPK, and to enter S-phase but had no effect on the ability of the cells to phosphorylate the p38 MAPK or to migrate on type I collagen in response to EGF. Methionine 1137 was essential for both migration and entry into S-phase. Thus, distinctly different structural elements of the alpha(2) integrin cytoplasmic domain are required to engage the signaling pathways leading to cell migration or cell cycle progression.

  1. Cephalosporium maydis is a distinct species in the Gaeumannomyces-Harpophora species complex.

    PubMed

    Saleh, Amgad A; Leslie, John F

    2004-01-01

    Cephalosporium maydis is an important plant pathogen whose phylogenetic position relative to other fungi has not been established clearly. We compared strains of C. maydis, strains from several other plant-pathogenic Cephalosporium spp. and several possible relatives within the Gaeumannomyces-Harpophora species complex, to which C. maydis has been suggested to belong based on previous preliminary DNA sequence analyses. DNA sequences of the nuclear genes encoding the rDNA ITS region, β-tubulin, histone H3, and MAT-2 support the hypothesis that C. maydis is a distinct taxon within the Gaeumannomyces-Harpophora species complex. Based on amplified fragment length polymorphism (AFLP) profiles, C. maydis also is distinct from the other tested species of Cephalosporium, Phialophora sensu lato and members of Gaeumannomyces-Harpophora species complex, which supports its classification as Harpophora maydis. Oligonucleotide primers for H. maydis were developed that can be used in a PCR diagnostic protocol to rapidly and reliably detect and identify this pathogen. These diagnostic PCR primers will aid the detection of H. maydis in diseased maize because this fungus can be difficult to detect and isolate, and the movement of authentic cultures may be limited by quarantine restrictions.

  2. 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.

  3. 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.

  4. 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.

  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 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.

  7. 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 as...

  8. 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.

  9. 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.

  10. 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

  11. Distribution of Plasmids in Distinct Leptospira Pathogenic Species.

    PubMed

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

    2015-11-01

    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

  12. Three alpha-subunits of heterotrimeric G proteins and an adenylyl cyclase have distinct roles in fruiting body development in the homothallic fungus Sordaria macrospora.

    PubMed

    Kamerewerd, Jens; Jansson, Malin; Nowrousian, Minou; Pöggeler, Stefanie; Kück, Ulrich

    2008-09-01

    Sordaria macrospora, a self-fertile filamentous ascomycete, carries genes encoding three different alpha-subunits of heterotrimeric G proteins (gsa, G protein Sordaria alpha subunit). We generated knockout strains for all three gsa genes (Deltagsa1, Deltagsa2, and Deltagsa3) as well as all combinations of double mutants. Phenotypic analysis of single and double mutants showed that the genes for Galpha-subunits have distinct roles in the sexual life cycle. While single mutants show some reduction of fertility, double mutants Deltagsa1Deltagsa2 and Deltagsa1Deltagsa3 are completely sterile. To test whether the pheromone receptors PRE1 and PRE2 mediate signaling via distinct Galpha-subunits, two recently generated Deltapre strains were crossed with all Deltagsa strains. Analyses of the corresponding double mutants revealed that compared to GSA2, GSA1 is a more predominant regulator of a signal transduction cascade downstream of the pheromone receptors and that GSA3 is involved in another signaling pathway that also contributes to fruiting body development and fertility. We further isolated the gene encoding adenylyl cyclase (AC) (sac1) for construction of a knockout strain. Analyses of the three DeltagsaDeltasac1 double mutants and one Deltagsa2Deltagsa3Deltasac1 triple mutant indicate that SAC1 acts downstream of GSA3, parallel to a GSA1-GSA2-mediated signaling pathway. In addition, the function of STE12 and PRO41, two presumptive signaling components, was investigated in diverse double mutants lacking those developmental genes in combination with the gsa genes. This analysis was further completed by expression studies of the ste12 and pro41 transcripts in wild-type and mutant strains. From the sum of all our data, we propose a model for how different Galpha-subunits interact with pheromone receptors, adenylyl cyclase, and STE12 and thus cooperatively regulate sexual development in S. macrospora.

  13. Human leukocyte inhibitory factor (LIF): two distinct molecular species.

    PubMed

    Brown, A P; Rocklin, R E

    1979-03-01

    Human leukocyte inhibitory factor or LIF was generated in vitro by stimulating blood lymphocytes with concanavalin A (Con A). The control and Con A active supernatants were partially purified by gel filtration on Sephadex G-100. The fraction containing LIF (68,000 daltons) activity was then subjected to isoelectric focusing (pH 3 to 10 ampholines) in a sucrose gradient. Two LIF activities were reproducibly recovered by this procedure. One molecular form was found to have an isoelectric point of approximately pH 5.0 and the other approximately pH 8.5. Both molecular species were rechromatographed on Sephadex G-75 and found to have the same apparent m.w. (68 to 75,000). Furthermore, the biologic activity of both factors was destroyed after treatment with diisopropylphosphofluoridate, suggesting that they may be esterases.

  14. 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.

  15. 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

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

    PubMed

    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.

  17. Two Drosophila DEG/ENaC channel subunits have distinct functions in gustatory neurons that activate male courtship

    PubMed Central

    Liu, Tong; Starostina, Elena; Vijayan, Vinoy; Pikielny, Claudio W.

    2012-01-01

    Trimeric sodium channels of the DEG/ENaC family have important roles in neurons, but the specific functions of different subunits present in heteromeric channels are poorly understood. We previously reported that the Drosophila DEG/ENaC subunit Ppk25 is essential in a small subset of gustatory neurons for activation of male courtship behavior, likely through detection of female pheromones. Here we show that, like mutations in ppk25, mutations in another Drosophila DEG/ENaC subunit, nope, specifically impair male courtship of females. nope regulatory sequences drive reporter gene expression in gustatory neurons of the labellum, wings and legs, including all gustatory neurons in which ppk25 function is required for male courtship of females. In addition, gustatory-specific knockdown of nope impairs male courtship. Further, the impaired courtship response of nope mutant males to females is rescued by targeted expression of nope in the subset of gustatory neurons in which ppk25 functions. However, nope and ppk25 have non-redundant functions, as targeted expression of ppk25 does not compensate for the lack of nope and vice versa. Moreover, Nope and Ppk25 form specific complexes when co-expressed in cultured cells. Together, these data indicate that the Nope and Ppk25 subunits have specific, non-redundant functions in a subset of gustatory neurons required for activation of male courtship in response to females, and suggest the hypothesis that Nope and Ppk25 function as subunits of a heteromeric DEG/ENaC channel required for gustatory detection of female pheromones. PMID:22915128

  18. 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.

  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. 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

  1. 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.

  2. Three α-Subunits of Heterotrimeric G Proteins and an Adenylyl Cyclase Have Distinct Roles in Fruiting Body Development in the Homothallic Fungus Sordaria macrospora

    PubMed Central

    Kamerewerd, Jens; Jansson, Malin; Nowrousian, Minou; Pöggeler, Stefanie; Kück, Ulrich

    2008-01-01

    Sordaria macrospora, a self-fertile filamentous ascomycete, carries genes encoding three different α-subunits of heterotrimeric G proteins (gsa, G protein Sordaria alpha subunit). We generated knockout strains for all three gsa genes (Δgsa1, Δgsa2, and Δgsa3) as well as all combinations of double mutants. Phenotypic analysis of single and double mutants showed that the genes for Gα-subunits have distinct roles in the sexual life cycle. While single mutants show some reduction of fertility, double mutants Δgsa1Δgsa2 and Δgsa1Δgsa3 are completely sterile. To test whether the pheromone receptors PRE1 and PRE2 mediate signaling via distinct Gα-subunits, two recently generated Δpre strains were crossed with all Δgsa strains. Analyses of the corresponding double mutants revealed that compared to GSA2, GSA1 is a more predominant regulator of a signal transduction cascade downstream of the pheromone receptors and that GSA3 is involved in another signaling pathway that also contributes to fruiting body development and fertility. We further isolated the gene encoding adenylyl cyclase (AC) (sac1) for construction of a knockout strain. Analyses of the three ΔgsaΔsac1 double mutants and one Δgsa2Δgsa3Δsac1 triple mutant indicate that SAC1 acts downstream of GSA3, parallel to a GSA1–GSA2-mediated signaling pathway. In addition, the function of STE12 and PRO41, two presumptive signaling components, was investigated in diverse double mutants lacking those developmental genes in combination with the gsa genes. This analysis was further completed by expression studies of the ste12 and pro41 transcripts in wild-type and mutant strains. From the sum of all our data, we propose a model for how different Gα-subunits interact with pheromone receptors, adenylyl cyclase, and STE12 and thus cooperatively regulate sexual development in S. macrospora. PMID:18723884

  3. 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......

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

    USDA-ARS?s Scientific Manuscript database

    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. ...

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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

  10. The Physcomitrella patens exocyst subunit EXO70.3d has distinct roles in growth and development, and is essential for completion of the moss life cycle.

    PubMed

    Rawat, Anamika; Brejšková, Lucie; Hála, Michal; Cvrčková, Fatima; Žárský, Viktor

    2017-04-11

    The exocyst, an evolutionarily conserved secretory vesicle-tethering complex, spatially controls exocytosis and membrane turnover in fungi, metazoans and plants. The exocyst subunit EXO70 exists in multiple paralogs in land plants, forming three conserved clades with assumed distinct roles. Here we report functional analysis of the first moss exocyst subunit to be studied, Physcomitrella patens PpEXO70.3d (Pp1s97_91V6), from the, as yet, poorly characterized EXO70.3 clade. Following phylogenetic analysis to confirm the presence of three ancestral land plant EXO70 clades outside angiosperms, we prepared and phenotypically characterized loss-of-function Ppexo70.3d mutants and localized PpEXO70.3d in vivo using green fluorescent protein-tagged protein expression. Disruption of PpEXO70.3d caused pleiotropic cell elongation and differentiation defects in protonemata, altered response towards exogenous auxin, increased endogenous IAA concentrations, along with defects in bud and gametophore development. During mid-archegonia development, an abnormal egg cell is formed and subsequently collapses, resulting in mutant sterility. Mutants exhibited altered cell wall and cuticle deposition, as well as compromised cytokinesis, consistent with the protein localization to the cell plate. Despite some functional redundancy allowing survival of moss lacking PpEXO70.3d, this subunit has an essential role in the moss life cycle, indicating sub-functionalization within the moss EXO70 family. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  11. 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.

  12. 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

  13. Barcoding Tetrahymena: discriminating species and identifying unknowns using the cytochrome c oxidase subunit I (cox-1) barcode.

    PubMed

    Kher, Chandni P; Doerder, F Paul; Cooper, Jason; Ikonomi, Pranvera; Achilles-Day, Undine; Küpper, Frithjof C; Lynn, Denis H

    2011-01-01

    DNA barcoding using the mitochondrial cytochromecoxidase subunit I (cox-1) gene has recently gained popularity as a tool for species identification of a variety of taxa. The primary objective of our research was to explore the efficacy of using cox-1 barcoding for species identification within the genusTetrahymena. We first increased intraspecific sampling forTetrahymena canadensis, Tetrahymena hegewischi, Tetrahymena pyriformis, Tetrahymena rostrata, Tetrahymena thermophila, and Tetrahymena tropicalis. Increased sampling efforts show that intraspecific sequence divergence is typically less than 1%, though it may be more in some species. The barcoding also showed that some strains might be misidentified or mislabeled. We also used cox-1 barcodes to provide species identifications for 51 unidentified environmental isolates, with a success rate of 98%. Thus, cox-1 barcoding is an invaluable tool for protistologists, especially when used in conjunction with morphological studies. 2010 Elsevier GmbH. All rights reserved.

  14. A new distinctive species of pagurid hermit crab (Crustacea: Decapoda: Anomura) from Japan.

    PubMed

    Komai, T; Osawa, M

    2001-12-01

    A new species of pagurid hermit crab, Pagurus decimbranchiae, is described and illustrated based on 20 specimens collected from shallow waters of the Pacific coast of Japan ranging from Boso Peninsula to Tanegashima Island. It is quite distinctive in having the rudimentary arthrobranch on the third maxilliped represented by a single bud, however close morphological similarity is found between the new species and P. moluccensis Haig and Ball. Comparisons are also made among other species, including P. boriaustraliensis Morgan, P. sp. cf. boriaustraliensis sensu Rahayu and Komai (2000) and the members of the P. anachoretus group. The present generic assignment of the new species should be considered provisional, as more extensive study is needed to investigate phylogenetic relationships of the new species and the other species of Pagurus.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  16. 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: National...

  17. 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

  18. 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

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

    USDA-ARS?s Scientific Manuscript database

    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...

  20. Range shift and introgression of the rear and leading populations in two ecologically distinct Rubus species.

    PubMed

    Mimura, Makiko; Mishima, Misako; Lascoux, Martin; Yahara, Tetsukazu

    2014-10-25

    The margins of a species' range might be located at the margins of a species' niche, and in such cases, can be highly vulnerable to climate changes. They, however, may also undergo significant evolutionary changes due to drastic population dynamics in response to climate changes, which may increase the chances of isolation and contact among species. Such species interactions induced by climate changes could then regulate or facilitate further responses to climatic changes. We hypothesized that climate changes lead to species contacts and subsequent genetic exchanges due to differences in population dynamics at the species boundaries. We sampled two closely related Rubus species, one temperate (Rubus palmatus) and the other subtropical (R. grayanus) near their joint species boundaries in southern Japan. Coalescent analysis, based on molecular data and ecological niche modelling during the Last Glacial Maximum (LGM), were used to infer past population dynamics. At the contact zones on Yakushima (Yaku Island), where the two species are parapatrically distributed, we tested hybridization along altitudinal gradients. Coalescent analysis suggested that the southernmost populations of R. palmatus predated the LGM (~20,000 ya). Conversely, populations at the current northern limit of R. grayanus diverged relatively recently and likely represent young outposts of a northbound range shift. These population dynamics were partly supported by the ensemble forecasting of six different species distribution models. Both past and ongoing hybridizations were detected near and on Yakushima. Backcrosses and advanced-generation hybrids likely generated the clinal hybrid zones along altitudinal gradients on the island where the two species are currently parapatrically distributed. Climate oscillations during the Quaternary Period and the response of a species in range shifts likely led to repeated contacts with the gene pools of ecologically distinct relatives. Such species interactions

  1. 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. Copyright © 2015 the American Physiological Society.

  2. Transcriptome analysis of complex I-deficient patients reveals distinct expression programs for subunits and assembly factors of the oxidative phosphorylation system.

    PubMed

    van der Lee, Robin; Szklarczyk, Radek; Smeitink, Jan; Smeets, Hubert J M; Huynen, Martijn A; Vogel, Rutger

    2015-09-15

    Transcriptional control of mitochondrial metabolism is essential for cellular function. A better understanding of this process will aid the elucidation of mitochondrial disorders, in particular of the many genetically unsolved cases of oxidative phosphorylation (OXPHOS) deficiency. Yet, to date only few studies have investigated nuclear gene regulation in the context of OXPHOS deficiency. In this study we performed RNA sequencing of two control and two complex I-deficient patient cell lines cultured in the presence of compounds that perturb mitochondrial metabolism: chloramphenicol, AICAR, or resveratrol. We combined this with a comprehensive analysis of mitochondrial and nuclear gene expression patterns, co-expression calculations and transcription factor binding sites. Our analyses show that subsets of mitochondrial OXPHOS genes respond opposingly to chloramphenicol and AICAR, whereas the response of nuclear OXPHOS genes is less consistent between cell lines and treatments. Across all samples nuclear OXPHOS genes have a significantly higher co-expression with each other than with other genes, including those encoding mitochondrial proteins. We found no evidence for complex-specific mRNA expression regulation: subunits of different OXPHOS complexes are similarly (co-)expressed and regulated by a common set of transcription factors. However, we did observe significant differences between the expression of nuclear genes for OXPHOS subunits versus assembly factors, suggesting divergent transcription programs. Furthermore, complex I co-expression calculations identified 684 genes with a likely role in OXPHOS biogenesis and function. Analysis of evolutionarily conserved transcription factor binding sites in the promoters of these genes revealed almost all known OXPHOS regulators (including GABP, NRF1/2, SP1, YY1, E-box factors) and a set of novel candidates (ELK1, KLF7, SP4, EHF, ZNF143, and TEL2). OXPHOS genes share an expression program distinct from other genes

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. Multichromophoric hybrid species made of perylene bisimide derivatives and Ru(ii) and Os(ii) polypyridine subunits.

    PubMed

    Nastasi, Francesco; La Ganga, Giuseppina; Campagna, Sebastiano; Syrgiannis, Zois; Rigodanza, Francesco; Vitale, Stefania; Licciardello, Antonino; Prato, Maurizio

    2017-05-31

    Herein, the synthesis and the photophysical and redox properties of a new perylene bisimide (PBI) species (L), bearing two 1,10-phenanthroline (phen) ligands at the two imide positions of the PBI, and its dinuclear Ru(ii) and Os(ii) complexes, [(bpy)2Ru(μ-L)Ru(bpy)2](PF6)4 (Ru2; bpy = 2,2'-bipyridine) and [(Me2-bpy)2Os(μ-L)Os(Me2-bpy)2](PF6)4 (Os2; Me2-bpy = (4,4'-dimethyl)-2,2'-bipyridine), are reported. The absorption spectra of the compounds are dominated by the structured bands of the PBI subunit due to the lowest-energy spin-allowed π-π* transition. The spin-allowed MLCT transitions in Ru2 and Os2 are inferred by the absorption at 350-470 nm, where the PBI absorption is negligible. The absorption band extends towards the red region for Os2 due to the spin-forbidden MLCT transitions, intensified by the heavy osmium center. The reduction processes of the compounds are dominated by two successive mono-electronic PBI-based processes, which in the metal complexes are slightly shifted compared to the free ligand. On oxidation, both metal complexes undergo an apparent bi-electronic process (at 1.31 V vs. SCE for Ru2 and 0.77 V for Os2), attributed to the simultaneous one-electron oxidation of the two weakly-interacting metal centers. In Ru2 and Os2, the intense fluorescence of L subunit (λmax, 535 nm; τ, 4.3 ns; Φ, 0.91) is fully quenched, mainly by photoinduced electron transfer from the metal centers, on the ps timescale (time constant, 11 ps in Ru2 and 3 ps in Os2). Such photoinduced electron transfer leads to the formation of a charge-separated state, which directly decays to the ground state in about 70 ps in Os2, but produces the triplet π-π* state of the PBI subunit in 35 ps in Ru2. The results provide information on the excited-state processes of the hybrid species combining two dominant classes of chromophore/luminophore species, the PBI and the metal polypyridine complexes, and can be used for future design on new hybrid species with made

  8. 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.

  9. 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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. Testing the utility of mitochondrial cytochrome oxidase subunit 1 sequences for phylogenetic estimates of relationships between crane (Grus) species.

    PubMed

    Yu, D B; Chen, R; Kaleri, H A; Jiang, B C; Xu, H X; Du, W-X

    2011-12-21

    Morphology and biogeography are widely used in animal taxonomy. Recent study has suggested that a DNA-based identification system, using a 648-bp portion of the mitochondrial gene cytochrome oxidase subunit 1 (CO1), also known as the barcoding gene, can aid in the resolution of inferences concerning phylogenetic relationships and for identification of species. However, the effectiveness of DNA barcoding for identifying crane species is unknown. We amplified and sequenced 894-bp DNA fragments of CO1 from Grus japonensis (Japanese crane), G. grus (Eurasian crane), G. monacha (hooded crane), G. canadensis (sandhill crane), G. leucogeranus (Siberian crane), and Balearica pavonina (crowned crane), along with those of 15 species obtained from GenBank and DNA barcoding, to construct four algorithms using Tringa stagnatilis, Scolopax rusticola, and T. erythropus as outgroups. The four phylum profiles showed good resolution of the major taxonomic groups. We concluded that reconstruction of the molecular phylogenetic tree can be helpful for classification and that CO1 sequences are suitable for studying the molecular evolution of cranes. Although support for several deeper branches was limited, CO1 data gave remarkably good separations, especially considering that our analysis was based on just a fragment of the gene and that CO1 has generally been viewed as useful only for resolving shallow divergences.

  15. Moniezia benedeni and Moniezia expansa are distinct cestode species based on complete mitochondrial genomes.

    PubMed

    Guo, Aijiang

    2017-02-01

    Moniezia spp. parasitize the intestines of ruminants, causing monieziasis. In this study, the complete mitochondrial (mt) genomes of M. benedeni and M. expansa have been determined, characterized and employed to test the hypothesis that M. benedeni and M. expansa are distinct species by phylogenetic analysis based on the concatenated amino acid sequences derived from 12 protein-coding genes, inferred with Bayesian and Maximum-likelihood methods. The complete mt genomes of M. benedeni and M. expansa were 13,958bp and 13,934bp in size, respectively. Nucleotide sequence identity between the two mt genomes was 83.4%. Each of the two circular mt genomes encodes 36 genes including two ribosomal RNA genes, 22 transfer RNA genes and 12 protein-coding genes, which are transcribed from the same direction. The gene orders of the two mt genomes are identical to those of Anoplocephala spp. (Anoplocephalidae), Hymenolepis spp. (Hymenolepididae) and Dipylidium caninum (Dipylidiidae), but distinct from the species of the Taeniidae family. Phylogenetic analysis confirmed that M. benedeni and M. expansa are taxonomically valid species and have a sister relationship, regardless of the analytical method employed. Furthermore, comparing the cox1 gene sequences of Moniezia spp. from the NCBI deposited sequences and the ones obtained in the present study revealed that the nucleotide sequence differences were 12.5% for M. benedeni and 6.2% for M. expansa, respectively, suggesting the existence of cryptic species in these parasites. The complete mt genome sequences reported herein will be valuable in further studies of diagnoses, molecular ecology and population genetics of Moniezia spp. of socio-economic importance. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. 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.

  17. The minimum activation peptide from ilvH can activate the catalytic subunit of AHAS from different species.

    PubMed

    Zhao, Yuefang; Niu, Congwei; Wen, Xin; Xi, Zhen

    2013-04-15

    Acetohydroxyacid synthases (AHASs), which catalyze the first step in the biosynthesis of branched-chain amino acids, are composed of a catalytic subunit (CSU) and a regulatory subunit (RSU). The CSU harbors the catalytic site, and the RSU is responsible for the activation and feedback regulation of the CSU. Previous results from Chipman and co-workers and our lab have shown that heterologous activation can be achieved among isozymes of Escherichia coli AHAS. It would be interesting to find the minimum peptide of ilvH (the RSU of E. coli AHAS III) that could activate other E. coli CSUs, or even those of ## species. In this paper, C-terminal, N-terminal, and C- and N-terminal truncation mutants of ilvH were constructed. The minimum peptide to activate ilvI (the CSU of E. coli AHAS III) was found to be ΔN 14-ΔC 89. Moreover, this peptide could not only activate its homologous ilvI and heterologous ilvB (CSU of E. coli AHAS I), but also heterologously activate the CSUs of AHAS from Saccharomyces cerevisiae, Arabidopsis thaliana, and Nicotiana plumbaginifolia. However, this peptide totally lost its ability for feedback regulation by valine, thus suggesting different elements for enzymatic activation and feedback regulation. Additionally, the apparent dissociation constant (Kd ) of ΔN 14-ΔC 89 when binding CSUs of different species was found to be 9.3-66.5 μM by using microscale thermophoresis. The ability of this peptide to activate different CSUs does not correlate well with its binding ability (Kd ) to these CSUs, thus implying that key interactions by specific residues is more important than binding ability in promoting enzymatic reactions. The high sequence similarity of the peptide ΔN 14-ΔC 89 to RSUs across species hints that this peptide represents the minimum activation motif in RSU and that it regulates all AHASs. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Phenotypic convergence in genetically distinct lineages of a Rhinolophus species complex (Mammalia, Chiroptera).

    PubMed

    Jacobs, David S; Babiker, Hassan; Bastian, Anna; Kearney, Teresa; van Eeden, Rowen; Bishop, Jacqueline M

    2013-01-01

    Phenotypes of distantly related species may converge through adaptation to similar habitats and/or because they share biological constraints that limit the phenotypic variants produced. A common theme in bats is the sympatric occurrence of cryptic species that are convergent in morphology but divergent in echolocation frequency, suggesting that echolocation may facilitate niche partitioning, reducing competition. If so, allopatric populations freed from competition, could converge in both morphology and echolocation provided they occupy similar niches or share biological constraints. We investigated the evolutionary history of a widely distributed African horseshoe bat, Rhinolophus darlingi, in the context of phenotypic convergence. We used phylogenetic inference to identify and date lineage divergence together with phenotypic comparisons and ecological niche modelling to identify morphological and geographical correlates of those lineages. Our results indicate that R. darlingi is paraphyletic, the eastern and western parts of its distribution forming two distinct non-sister lineages that diverged ~9.7 Mya. We retain R. darlingi for the eastern lineage and argue that the western lineage, currently the sub-species R. d. damarensis, should be elevated to full species status. R. damarensis comprises two lineages that diverged ~5 Mya. Our findings concur with patterns of divergence of other co-distributed taxa which are associated with increased regional aridification between 7-5 Mya suggesting possible vicariant evolution. The morphology and echolocation calls of R. darlingi and R. damarensis are convergent despite occupying different biomes. This suggests that adaptation to similar habitats is not responsible for the convergence. Furthermore, R. darlingi forms part of a clade comprising species that are bigger and echolocate at lower frequencies than R. darlingi, suggesting that biological constraints are unlikely to have influenced the convergence. Instead, the

  19. Beyond the EDGE with EDAM: Prioritising British Plant Species According to Evolutionary Distinctiveness, and Accuracy and Magnitude of Decline

    PubMed Central

    Pearse, William D.; Chase, Mark W.; Crawley, Michael J.; Dolphin, Konrad; Fay, Michael F.; Joseph, Jeffrey A.; Powney, Gary; Preston, Chris D.; Rapacciuolo, Giovanni; Roy, David B.; Purvis, Andy

    2015-01-01

    Conservation biologists have only finite resources, and so must prioritise some species over others. The EDGE-listing approach ranks species according to their combined evolutionary distinctiveness and degree of threat, but ignores the uncertainty surrounding both threat and evolutionary distinctiveness. We develop a new family of measures for species, which we name EDAM, that incorporates evolutionary distinctiveness, the magnitude of decline, and the accuracy with which decline can be predicted. Further, we show how the method can be extended to explore phyogenetic uncertainty. Using the vascular plants of Britain as a case study, we find that the various EDAM measures emphasise different species and parts of Britain, and that phylogenetic uncertainty can strongly affect the prioritisation scores of some species. PMID:26018568

  20. 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

  1. 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.

  2. Molecular arguments for splitting of Schistosoma intercalatum, into two distinct species.

    PubMed

    Pagès, J R; Durand, P; Southgate, V R; Tchuem Tchuenté, L A; Jourdane, J

    2001-01-01

    The taxonomic status of the two known strains of Schistosoma intercalatum, the Lower Guinea strain (originating from Edea, Cameroon) and the Zaire strain (originating from Kinshasa, Democratic Republic of Congo, formerly Zaire) was examined using random amplified polymorphic DNA (RAPD) markers. Two additional species within the S. haematobium group, S. haematobium and S. mattheei, were included in the study. DNA was extracted from four male and four female worms of each species and strain under investigation. In all, 13 primers gave reproducible and informative marker patterns; the monomorphic bands in all the males and females of each sample were scored, and 138 bands were included in the final analysis. Overall, 14 RAPD fragments were shared by all the schistosomes studied, and 19 RAPD fragments were considered to be sex markers. Only 22% (20/91) of the RAPD fragments were shared between S. intercalatum Zaire and S. intercalatum Cameroon. The mean values recorded for the Nei and Li's genetic distances between S. haematobium and S. mattheei and between S. intercalatum Zaire and S. intercalatum Cameroon were 0.546 and 0.596, respectively. A principal component analysis and one-way analysis of variance (ANOVA/MANOVA) showed a significant separation between S. intercalatum Zaire and S. intercalatum Cameroon. The data support the hypothesis that S. intercalatum Zaire and S. intercalatum Cameroon are distinct species. Additional molecular-biology studies are in progress that involve the use of nuclear and mitochondrial markers to confirm the extent of the genetic divergence prior to the establishment of final decision on the taxonomic status of the two strains of S. intercalatum.

  3. Subunit composition and substrate specificity of a MOF-containing histone acetyltransferase distinct from the male-specific lethal (MSL) complex.

    PubMed

    Cai, Yong; Jin, Jingji; Swanson, Selene K; Cole, Michael D; Choi, Seung Hyuk; Florens, Laurence; Washburn, Michael P; Conaway, Joan W; Conaway, Ronald C

    2010-02-12

    Human MOF (MYST1), a member of the MYST (Moz-Ybf2/Sas3-Sas2-Tip60) family of histone acetyltransferases (HATs), is the human ortholog of the Drosophila males absent on the first (MOF) protein. MOF is the catalytic subunit of the male-specific lethal (MSL) HAT complex, which plays a key role in dosage compensation in the fly and is responsible for a large fraction of histone H4 lysine 16 (H4K16) acetylation in vivo. MOF was recently reported to be a component of a second HAT complex, designated the non-specific lethal (NSL) complex (Mendjan, S., Taipale, M., Kind, J., Holz, H., Gebhardt, P., Schelder, M., Vermeulen, M., Buscaino, A., Duncan, K., Mueller, J., Wilm, M., Stunnenberg, H. G., Saumweber, H., and Akhtar, A. (2006) Mol. Cell 21, 811-823). Here we report an analysis of the subunit composition and substrate specificity of the NSL complex. Proteomic analyses of complexes purified through multiple candidate subunits reveal that NSL is composed of nine subunits. Two of its subunits, WD repeat domain 5 (WDR5) and host cell factor 1 (HCF1), are shared with members of the MLL/SET family of histone H3 lysine 4 (H3K4) methyltransferase complexes, and a third subunit, MCRS1, is shared with the human INO80 chromatin-remodeling complex. In addition, we show that assembly of the MOF HAT into MSL or NSL complexes controls its substrate specificity. Although MSL-associated MOF acetylates nucleosomal histone H4 almost exclusively on lysine 16, NSL-associated MOF exhibits a relaxed specificity and also acetylates nucleosomal histone H4 on lysines 5 and 8.

  4. A distinctive and host-restricted gut microbiota in populations of a cactophilic Drosophila species.

    PubMed

    Martinson, Vincent G; Carpinteyro-Ponce, Javier; Moran, Nancy A; Markow, Therese A

    2017-09-22

    species possesses a distinctive microbiome, composed of bacterial types absent from their natural food but widespread in other wild-caught insects. This study highlights the importance of fieldwork-informed microbiota research. Copyright © 2017 American Society for Microbiology.

  5. Distinctive role of KV1.1 subunit in the biology and functions of low threshold K(+) channels with implications for neurological disease.

    PubMed

    Ovsepian, Saak V; LeBerre, Marie; Steuber, Volker; O'Leary, Valerie B; Leibold, Christian; Oliver Dolly, J

    2016-03-01

    The diversity of pore-forming subunits of KV1 channels (KV1.1-KV1.8) affords their physiological versatility and predicts a range of functional impairments resulting from genetic aberrations. Curiously, identified so far human neurological conditions associated with dysfunctions of KV1 channels have been linked exclusively to mutations in the KCNA1 gene encoding for the KV1.1 subunit. The absence of phenotypes related to irregularities in other subunits, including the prevalent KV1.2 subunit of neurons is highly perplexing given that deletion of the corresponding kcna2 gene in mouse models precipitates symptoms reminiscent to those of KV1.1 knockouts. Herein, we critically evaluate the molecular and biophysical characteristics of the KV1.1 protein in comparison with others and discuss their role in the greater penetrance of KCNA1 mutations in humans leading to the neurological signs of episodic ataxia type 1 (EA1). Future research and interpretation of emerging data should afford new insights towards a better understanding of the role of KV1.1 in integrative mechanisms of neurons and synaptic functions under normal and disease conditions. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Distinct Functions of Regions 1.1 and 1.2 of RNA Polymerase σ Subunits from Escherichia coli and Thermus aquaticus in Transcription Initiation*

    PubMed Central

    Miropolskaya, Nataliya; Ignatov, Artem; Bass, Irina; Zhilina, Ekaterina; Pupov, Danil; Kulbachinskiy, Andrey

    2012-01-01

    RNA polymerase (RNAP) from thermophilic Thermus aquaticus is characterized by higher temperature of promoter opening, lower promoter complex stability, and higher promoter escape efficiency than RNAP from mesophilic Escherichia coli. We demonstrate that these differences are in part explained by differences in the structures of the N-terminal regions 1.1 and 1.2 of the E. coli σ70 and T. aquaticus σA subunits. In particular, region 1.1 and, to a lesser extent, region 1.2 of the E. coli σ70 subunit determine higher promoter complex stability of E. coli RNAP. On the other hand, nonconserved amino acid substitutions in region 1.2, but not region 1.1, contribute to the differences in promoter opening between E. coli and T. aquaticus RNAPs, likely through affecting the σ subunit contacts with DNA nucleotides downstream of the −10 element. At the same time, substitutions in σ regions 1.1 and 1.2 do not affect promoter escape by E. coli and T. aquaticus RNAPs. Thus, evolutionary substitutions in various regions of the σ subunit modulate different steps of the open promoter complex formation pathway, with regions 1.1 and 1.2 affecting promoter complex stability and region 1.2 involved in DNA melting during initiation. PMID:22605342

  7. 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 Steelhead...

  8. 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.

  9. 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

  10. 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

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

    PubMed

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

    2016-09-14

    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.

  12. 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

  13. 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.

  14. 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

  15. Multilocus DNA sequence analysis shows that Penicillium biourgeianum is a distinct species closely related to P. brevicompactum and P. olsonii.

    PubMed

    Peterson, Stephen W

    2004-04-01

    Penicillium brevicompactum and other isolates with the compact, complex conidiogenous apparatus typical of the species were sequenced in one ribosomal and two protein coding regions. The aligned DNA sequences were analyzed by maximum parsimony and the data from different loci were tested for compatibility using the partition homogeneity test. Analysis of each of the three loci revealed three clades corresponding to P. brevicompactum, P. olsonii and P. biourgeianum. Using the phylogenetic species concept and the genetic isolation of the clades, P. hagemi, P. patrismei, P. stoloniferum, and P. griseobrunneum are all synonyms of P. brevicompactum. P. volgaense is a synonym of Penicillium olsonii, while P. biourgeianum is a distinct species closely related to P. brevicompactum and P. olsonii. Phenotypic distinctions between the species are mostly based on colony characteristics such as colour. P. bialowiezenze, often treated as a synonym of P. brevicompactum, is most closely related to P. polonicum.

  16. 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.

  17. 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

  18. 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

    ... definition of an endangered or threatened species under the ESA: It is not in danger of extinction or likely... ESA further defines an endangered species as ``any species which is in danger of extinction throughout... extinction. A ``threatened species,'' on the other hand, is not presently in danger of extinction, but is...

  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. 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... the Steller Sea Lion (Eumetopias jubatus) under the Endangered Species Act of 1973, as amended (ESA... subject line of the e-mail: ``Comments on the 5-year review for the eastern DPS of Steller sea lion.'' Fax...

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

    PubMed Central

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

    2000-01-01

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

  2. 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

  3. 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.

  4. Poles Apart: The “Bipolar” Pteropod Species Limacina helicina Is Genetically Distinct Between the Arctic and Antarctic Oceans

    PubMed Central

    Bednarsek, Nina; Linse, Katrin; Nelson, R. John; Pakhomov, Evgeny; Seibel, Brad; Steinke, Dirk; Würzberg, Laura

    2010-01-01

    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. PMID:20360985

  5. First comparative characterization of three distinct ferritin subunits from a teleost: Evidence for immune-responsive mRNA expression and iron depriving activity of seahorse (Hippocampus abdominalis) ferritins.

    PubMed

    Oh, Minyoung; Umasuthan, Navaneethaiyer; Elvitigala, Don Anushka Sandaruwan; Wan, Qiang; Jo, Eunyoung; Ko, Jiyeon; Noh, Gyeong Eon; Shin, Sangok; Rho, Sum; Lee, Jehee

    2016-02-01

    Ferritins play an indispensable role in iron homeostasis through their iron-withholding function in living beings. In the current study, cDNA sequences of three distinct ferritin subunits, including a ferritin H, a ferritin M, and a ferritin L, were identified from big belly seahorse, Hippocampus abdominalis, and molecularly characterized. Complete coding sequences (CDS) of seahorse ferritin H (HaFerH), ferritin M (HaFerM), and ferritin L (HaFerL) subunits were comprised of 531, 528, and 522 base pairs (bp), respectively, which encode polypeptides of 177, 176, and 174 amino acids, respectively, with molecular masses of ∼20-21 kDa. Our in silico analyses demonstrate that these three ferritin subunits exhibit the typical characteristics of ferritin superfamily members including iron regulatory elements, domain signatures, and reactive centers. The coding sequences of HaFerH, M, and L were cloned and the corresponding proteins were overexpressed in a bacterial system. Recombinantly expressed HaFer proteins demonstrated detectable in vivo iron sequestrating (ferroxidase) activity, consistent with their putative iron binding capability. Quantification of the basal expression of these three HaFer sequences in selected tissues demonstrated a gene-specific ubiquitous spatial distribution pattern, with abundance of mRNA in HaFerM in the liver and predominant expression of HaFerH and HaFerL in blood. Interestingly, the basal expression of all three ferritin genes was found to be significantly modulated against pathogenic stress mounted by lipopolysaccharides (LPS), poly I:C, Streptococcus iniae, and Edwardsiella tarda. Collectively, our findings suggest that the three HaFer subunits may be involved in iron (II) homeostasis in big belly seahorse and that they are important in its host defense mechanisms. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. 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.

  7. The use of large and small subunits of ribosomal DNA in evaluating phylogenetic relationships between species of Cornudiscoides Kulkarni, 1969 (Monogenoidea: Dactylogyridae) from India.

    PubMed

    Verma, J; Agrawal, N; Verma, A K

    2017-03-01

    Two partial regions of ribosomal DNA (28S and 18S) were used to evaluate genetic variations among the species of Cornudiscoides, viz. C. proximus, C. geminus and C. agarwali, all parasites of Mystus vittatus (Bagridae) from River Gomati, Ganges River basin, India. Our findings demonstrated that both the large and small ribosomal subunits are useful for species identification and genetic characterization of parasites, leading to resolution of inter/intra-relationships at generic and specific levels. The secondary structures of all three species for 28S and 18S rRNA genes contained exact pattern matches (EMPs) displaying the high degree of similarity among them. The phylogenetic analyses within the members of Dactylogyridae demonstrated that species of Cornudiscoides cluster together for 28S rRNA and 18S rRNA genes.

  8. Diversity of heterotrimeric G-protein γ subunits in plants.

    PubMed

    Trusov, Yuri; Chakravorty, David; Botella, José Ramón

    2012-10-31

    Heterotrimeric G-proteins, consisting of three subunits Gα, Gβ and Gγ are present in most eukaryotes and mediate signaling in numerous biological processes. In plants, Gγ subunits were shown to provide functional selectivity to G-proteins. Three unconventional Gγ subunits were recently reported in Arabidopsis, rice and soybean but no structural analysis has been reported so far. Their relationship with conventional Gγ subunits and taxonomical distribution has not been yet demonstrated. After an extensive similarity search through plant genomes, transcriptomes and proteomes we assembled over 200 non-redundant proteins related to the known Gγ subunits. Structural analysis of these sequences revealed that most of them lack the obligatory C-terminal prenylation motif (CaaX). According to their C-terminal structures we classified the plant Gγ subunits into three distinct types. Type A consists of Gγ subunits with a putative prenylation motif. Type B subunits lack a prenylation motif and do not have any cysteine residues in the C-terminal region, while type C subunits contain an extended C-terminal domain highly enriched with cysteines. Comparative analysis of C-terminal domains of the proteins, intron-exon arrangement of the corresponding genes and phylogenetic studies suggested a common origin of all plant Gγ subunits. Phylogenetic analyses suggest that types C and B most probably originated independently from type A ancestors. We speculate on a potential mechanism used by those Gγ subunits lacking isoprenylation motifs to anchor the Gβγ dimer to the plasma membrane and propose a new flexible nomenclature for plant Gγ subunits. Finally, in the light of our new classification, we give a word of caution about the interpretation of Gγ research in Arabidopsis and its generalization to other plant species.

  9. 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.

  10. 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.

  11. Distinct antimicrobial peptide expression determines host species-specific bacterial associations

    PubMed Central

    Franzenburg, Sören; Walter, Jonas; Künzel, Sven; Wang, Jun; Baines, John F.; Bosch, Thomas C. G.; Fraune, Sebastian

    2013-01-01

    Animals are colonized by coevolved bacterial communities, which contribute to the host’s health. This commensal microbiota is often highly specific to its host-species, inferring strong selective pressures on the associated microbes. Several factors, including diet, mucus composition, and the immune system have been proposed as putative determinants of host-associated bacterial communities. Here we report that species-specific antimicrobial peptides account for different bacterial communities associated with closely related species of the cnidarian Hydra. Gene family extensions for potent antimicrobial peptides, the arminins, were detected in four Hydra species, with each species possessing a unique composition and expression profile of arminins. For functional analysis, we inoculated arminin-deficient and control polyps with bacterial consortia characteristic for different Hydra species and compared their selective preferences by 454 pyrosequencing of the bacterial microbiota. In contrast to control polyps, arminin-deficient polyps displayed decreased potential to select for bacterial communities resembling their native microbiota. This finding indicates that species-specific antimicrobial peptides shape species-specific bacterial associations. PMID:24003149

  12. Differential transcriptome analysis supports Rhodnius montenegrensis and Rhodnius robustus (Hemiptera, Reduviidae, Triatominae) as distinct species.

    PubMed

    de Carvalho, Danila Blanco; Congrains, Carlos; Chahad-Ehlers, Samira; Pinotti, Heloisa; Brito, Reinaldo Alves de; da Rosa, João Aristeu

    2017-01-01

    Chagas disease is one of the main parasitic diseases found in Latin America and it is estimated that between six and seven million people are infected worldwide. Its etiologic agent, the protozoan Trypanosoma cruzi, is transmitted by triatomines, some of which from the genus Rhodnius. Twenty species are currently recognized in this genus, including some closely related species with low levels of morphological differentiation, such as Rhodnius montenegrensis and Rhodnius robustus. In order to investigate genetic differences between these two species, we generated large-scale RNA-sequencing data (consisting of four RNA-seq libraries) from the heads and salivary glands of males of R. montenegrensis and R. robustus. Transcriptome assemblies produced for each species resulted in 64,952 contigs for R. montenegrensis and 70,894 contigs for R. robustus, with N50 of approximately 2,100 for both species. SNP calling based on the more complete R. robustus assembly revealed 3,055 fixed interspecific differences and 216 transcripts with high levels of divergence which contained only fixed differences between the two species. A gene ontology enrichment analysis revealed that these highly differentiated transcripts were enriched for eight GO terms related to AP-2 adaptor complex, as well as other interesting genes that could be involved in their differentiation. The results show that R. montenegrensis and R. robustus have a substantial quantity of fixed interspecific polymorphisms, which suggests a high degree of genetic divergence between the two species and likely corroborates the species status of R. montenegrensis.

  13. 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

  14. The Goldfish SG2NA Gene Encodes Two α-Type Regulatory Subunits for PP-2A and Displays Distinct Developmental Expression Pattern

    PubMed Central

    Ma, Hai-Li; Peng, Yun-Lei; Gong, Lili; Liu, Wen-Bin; Sun, Shuming; Liu, Jiao; Zheng, Chun-Bing; Fu, Hu; Yuan, Dan; Zhao, Junqiong; Chen, Pei-Chao; Xie, Si-si; Zeng, Xiao-Ming; Xiao, Ya-Mei; Liu, Yun; Li, David Wan-Cheng

    2009-01-01

    SG2NA is a member of the striatin protein family. In human and mouse, the SG2NA gene encodes two major protein isoforms: SG2NAα and SG2NAβ. The functions of these proteins, except for acting as the regulatory subunits for PP-2A, remain largely unknown. To explore the possible functions of SG2NA in lower vertebrates, we have isolated two SG2NA cDNAs from goldfish, Carassius auratus. Our results reveal that the first cDNA contains an ORF of 2118 bp encoding a deduced protein with 705 amino acids, and the second one 2148 bp coding for a deduced protein of 715 amino acids. Comparative analysis reveals that both isoforms belong to the α-type, and are named SG2NAα and SG2NAα+. RT-PCR and western blot analysis reveal that the SG2NA gene is differentially expressed in 9 tissues examined. During goldfish development, while the SG2NA mRNAs remain relatively constant in the first 3 stages and then become decreased and fluctuated from gastrula to larval hatching, the SG2NA proteins are fluctuated, displaying a peak every 3 to 4 stages. Each later peak is higher than the earlier one and the protein expression level becomes maximal at hatching stage. Together, our results reveal that SG2NA may play an important role during goldfish development and also in homeostasis of most adult tissues. PMID:19838339

  15. 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

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. Protein phosphatase 2B (PP2B, calcineurin) in Paramecium: partial characterization reveals that two members of the unusually large catalytic subunit family have distinct roles in calcium-dependent processes.

    PubMed

    Fraga, D; Sehring, I M; Kissmehl, R; Reiss, M; Gaines, R; Hinrichsen, R; Plattner, H

    2010-07-01

    We characterized the calcineurin (CaN) gene family, including the subunits CaNA and CaNB, based upon sequence information obtained from the Paramecium genome project. Paramecium tetraurelia has seven subfamilies of the catalytic CaNA subunit and one subfamily of the regulatory CaNB subunit, with each subfamily having two members of considerable identity on the amino acid level (>or=55% between subfamilies, >or=94% within CaNA subfamilies, and full identity in the CaNB subfamily). Within CaNA subfamily members, the catalytic domain and the CaNB binding region are highly conserved and molecular modeling revealed a three-dimensional structure almost identical to a human ortholog. At 14 members, the size of the CaNA family is unprecedented, and we hypothesized that the different CaNA subfamily members were not strictly redundant and that at least some fulfill different roles in the cell. This was tested by selecting two phylogenetically distinct members of this large family for posttranscriptional silencing by RNA interference. The two targets resulted in differing effects in exocytosis, calcium dynamics, and backward swimming behavior that supported our hypothesis that the large, highly conserved CaNA family members are not strictly redundant and that at least two members have evolved diverse but overlapping functions. In sum, the occurrence of CaN in Paramecium spp., although disputed in the past, has been established on a molecular level. Its role in exocytosis and ciliary beat regulation in a protozoan, as well as in more complex organisms, suggests that these roles for CaN were acquired early in the evolution of this protein family.

  1. Protein Phosphatase 2B (PP2B, Calcineurin) in Paramecium: Partial Characterization Reveals That Two Members of the Unusually Large Catalytic Subunit Family Have Distinct Roles in Calcium-Dependent Processes▿‡

    PubMed Central

    Fraga, D.; Sehring, I. M.; Kissmehl, R.; Reiss, M.; Gaines, R.; Hinrichsen, R.; Plattner, H.

    2010-01-01

    We characterized the calcineurin (CaN) gene family, including the subunits CaNA and CaNB, based upon sequence information obtained from the Paramecium genome project. Paramecium tetraurelia has seven subfamilies of the catalytic CaNA subunit and one subfamily of the regulatory CaNB subunit, with each subfamily having two members of considerable identity on the amino acid level (≥55% between subfamilies, ≥94% within CaNA subfamilies, and full identity in the CaNB subfamily). Within CaNA subfamily members, the catalytic domain and the CaNB binding region are highly conserved and molecular modeling revealed a three-dimensional structure almost identical to a human ortholog. At 14 members, the size of the CaNA family is unprecedented, and we hypothesized that the different CaNA subfamily members were not strictly redundant and that at least some fulfill different roles in the cell. This was tested by selecting two phylogenetically distinct members of this large family for posttranscriptional silencing by RNA interference. The two targets resulted in differing effects in exocytosis, calcium dynamics, and backward swimming behavior that supported our hypothesis that the large, highly conserved CaNA family members are not strictly redundant and that at least two members have evolved diverse but overlapping functions. In sum, the occurrence of CaN in Paramecium spp., although disputed in the past, has been established on a molecular level. Its role in exocytosis and ciliary beat regulation in a protozoan, as well as in more complex organisms, suggests that these roles for CaN were acquired early in the evolution of this protein family. PMID:20435698

  2. 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.

  3. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. 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.

  5. 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.

  6. Radiizonates arcuatus, a distinctive new miospore species from the Lower Carboniferous of Western Gondwana.

    PubMed

    Loboziak; Playford; Melo

    2000-05-01

    A new species of trilete zonate miospores, Radiizonates arcuatus, is established for Lower Carboniferous Western Gondwanan forms hitherto ascribed misguidedly to Radiizonates genuinus (Jushko) Loboziak and Alpern (1978), a Russian Lower Carboniferous species. The latter binomen is, moreover, not a valid combination and is more correctly designated as Vallatisporites genuinus (Jushko) Byvsheva, 1980. R. arcuatus is, from records to date, confined to westerly parts of Gondwana (Brazil, North Africa and Middle East), in which it is characteristic of Early Carboniferous strata, albeit with some slightly older and slightly younger occurrences.

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. Characterization of the Uukuniemi Virus Group (Phlebovirus: Bunyaviridae): Evidence for Seven Distinct Species

    PubMed Central

    Savji, Nazir; Travassos da Rosa, Amelia; Guzman, Hilda; Yu, Xuejie; Desai, Aaloki; Rosen, Gail Emilia; Hutchison, Stephen; Lipkin, W. Ian; Tesh, Robert

    2013-01-01

    Evolutionary insights into the phleboviruses are limited because of an imprecise classification scheme based on partial nucleotide sequences and scattered antigenic relationships. In this report, the serologic and phylogenetic relationships of the Uukuniemi group viruses and their relationships with other recently characterized tick-borne phleboviruses are described using full-length genome sequences. We propose that the viruses currently included in the Uukuniemi virus group be assigned to five different species as follows: Uukuniemi virus, EgAn 1825-61 virus, Fin V707 virus, Chizé virus, and Zaliv Terpenia virus would be classified into the Uukuniemi species; Murre virus, RML-105-105355 virus, and Sunday Canyon virus would be classified into a Murre virus species; and Grand Arbaud virus, Precarious Point virus, and Manawa virus would each be given individual species status. Although limited sequence similarity was detected between current members of the Uukuniemi group and Severe fever with thrombocytopenia syndrome virus (SFTSV) and Heartland virus, a clear serological reaction was observed between some of them, indicating that SFTSV and Heartland virus should be considered part of the Uukuniemi virus group. Moreover, based on the genomic diversity of the phleboviruses and given the low correlation observed between complement fixation titers and genetic distance, we propose a system for classification of the Bunyaviridae based on genetic as well as serological data. Finally, the recent descriptions of SFTSV and Heartland virus also indicate that the public health importance of the Uukuniemi group viruses must be reevaluated. PMID:23283959

  12. 76 FR 65323 - Endangered and Threatened Species; Designation of Critical Habitat for the Southern Distinct...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-20

    ... from the ocean to spawn in freshwater creeks and rivers where their offspring hatch and migrate back to the ocean to forage until maturity. Although they spend 95 to 98 percent of their lives at sea (Hay... existence. The species is endemic to the northeastern Pacific Ocean, ranging from northern California to...

  13. 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).

  14. Newly discovered populations of salamanders from Siskiyou County California represent a species distinct from Plethodon Stormi.

    Treesearch

    Louise S. Mead; David R. Clayton; Richard S. Nauman; Deanna H. Olson; Michael E. Pfrender

    2005-01-01

    Plethodon stormi and Plethodon elongatus are two closely related species of plethodontid salamanders that are restricted to the Klamath Province of northwestern California and southwestern Oregon. Discovery of three localities south of the Klamath River, in the Scott River drainage, not assignable to either P....

  15. 77 FR 76740 - Endangered and Threatened Species; Threatened Status for the Beringia and Okhotsk Distinct...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-12-28

    .... Based on the best scientific and commercial data available on the bearded seals' taxonomy, the BRT... bearded seal under the ESA. Species Information A thorough review of the taxonomy, life history, and... of pollutants from lower latitudes to the Arctic, highlighting the importance of continued monitoring...

  16. 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.

  17. 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.

  18. Medium term water deficit elicits distinct transcriptome responses in Eucalyptus species of contrasting environmental origin.

    PubMed

    Spokevicius, Antanas V; Tibbits, Josquin; Rigault, Philippe; Nolin, Marc-Alexandre; Müller, Caroline; Merchant, Andrew

    2017-04-07

    Climatic and edaphic conditions over geological timescales have generated enormous diversity of adaptive traits and high speciation within the genus Eucalyptus (L. Hér.). Eucalypt species occur from high rainfall to semi-arid zones and from the tropics to latitudes as high as 43°S. Despite several morphological and metabolomic characterizations, little is known regarding gene expression differences that underpin differences in tolerance to environmental change. Using species of contrasting taxonomy, morphology and physiology (E. globulus and E. cladocalyx), this study combines physiological characterizations with 'second-generation' sequencing to identify key genes involved in eucalypt responses to medium-term water limitation. One hundred twenty Million high-quality HiSeq reads were created from 14 tissue samples in plants that had been successfully subjected to a water deficit treatment or a well-watered control. Alignment to the E. grandis genome saw 23,623 genes of which 468 exhibited differential expression (FDR < 0.01) in one or both ecotypes in response to the treatment. Further analysis identified 80 genes that demonstrated a significant species-specific response of which 74 were linked to the 'dry' species E. cladocalyx where 23 of these genes were uncharacterised. The majority (approximately 80%) of these differentially expressed genes, were expressed in stem tissue. Key genes that differentiated species responses were linked to photoprotection/redox balance, phytohormone/signalling, primary photosynthesis/cellular metabolism and secondary metabolism based on plant metabolic pathway network analysis. These results highlight a more definitive response to water deficit by a 'dry' climate eucalypt, particularly in stem tissue, identifying key pathways and associated genes that are responsible for the differences between 'wet' and 'dry' climate eucalypts. This knowledge provides the opportunity to further investigate and understand the mechanisms and

  19. 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.

  20. 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.

  1. 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

  2. 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].

  3. Nitrogen use strategies of seedlings from neotropical tree species of distinct successional groups.

    PubMed

    Oliveira, Halley Caixeta; da Silva, Ligia Maria Inocêncio; de Freitas, Letícia Dias; Debiasi, Tatiane Viegas; Marchiori, Nidia Mara; Aidar, Marcos Pereira Marinho; Bianchini, Edmilson; Pimenta, José Antonio; Stolf-Moreira, Renata

    2017-05-01

    Few studies have analyzed the strategies of neotropical tree seedlings for absorbing, translocating and assimilating the nitrogen. Here, we compared the nitrogen use strategies of seedlings from six tree species that are native to the Brazilian Atlantic Forest and that belong to different successional groups: Trema micrantha, Heliocarpus popayanensis and Cecropia pachystachya (pioneers), Cariniana estrellensis, Eugenia brasiliensis and Guarea kunthiana (non-pioneers). The effects of cultivating seedlings with nitrate or ammonium on the growth, physiology and nitrogen metabolism were analyzed. Nitrate-grown pioneer species had much higher leaf nitrate reductase activity than non-pioneer ones, but non-pioneer seedlings were also able to use nitrate as a nitrogen source. In addition to this remarkable difference between the groups in the capacity for leaf nitrate assimilation, substantial variations in the nitrogen use strategies were observed within the successional classes. Differently from the other non-pioneers, the canopy species C. estrellensis seemed to assimilate nitrate mainly in the leaves. Morphophysiological analyses showed a gradient of ammonium toxicity response, with E. brasiliensis as the most tolerant species, and T. micrantha and H. popayanensis as the most sensitive ones. Guarea kunthiana showed a relatively low tolerance to ammonium and an unusual high translocation of this cation in the xylem sap. In contrast to the other pioneers, C. pachystachya had a high plasticity in the use of nitrogen sources. Overall, these results suggest that nitrogen use strategies of neotropical tree seedlings were not determined solely by their successional position. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  4. Lignin extraction distinctively enhances biomass enzymatic saccharification in hemicelluloses-rich Miscanthus species under various alkali and acid pretreatments.

    PubMed

    Si, Shengli; Chen, Yan; Fan, Chunfen; Hu, Huizhen; Li, Ying; Huang, Jiangfeng; Liao, Haofeng; Hao, Bo; Li, Qing; Peng, Liangcai; Tu, Yuanyuan

    2015-05-01

    In this study, one- and two-step pretreatments with alkali and acid were performed in the three Miscanthus species that exhibit distinct hemicelluloses levels. As a result, one-step with 4% NaOH or two-step with 2% NaOH and 1% H2SO4 was examined to be optimal for high biomass saccharification, indicating that alkali was the main effecter of pretreatments. Notably, both one- and two-step pretreatments largely enhanced biomass digestibility distinctive in hemicelluloses-rich samples by effectively co-extracting hemicelluloses and lignin. However, correlation analysis further indicated that the effective lignin extraction, other than the hemicelluloses removals, predominately determined biomass saccharification under various alkali and acid pretreatments, leading to a significant alteration of cellulose crystallinity. Hence, this study has suggested the potential approaches in bioenergy crop breeding and biomass process technology. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. 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

  10. 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.

  11. Differential Costs of Two Distinct Resistance Mechanisms Induced by Different Herbivore Species in Arabidopsis1

    PubMed Central

    Reichelt, Michael; van Doorn, Arjen; Schuurink, Robert C.

    2016-01-01

    Plants respond to herbivory with the induction of resistance, mediated by distinct phytohormonal signaling pathways and their interactions. Phloem feeders are known to induce plant resistance via the salicylic acid pathway, whereas biting-chewing herbivores induce plant resistance mainly via the jasmonate pathway. Here, we show that a specialist caterpillar (biting-chewing herbivore) and a specialist aphid (phloem feeder) differentially induce resistance against Pieris brassicae caterpillars in Arabidopsis (Arabidopsis thaliana) plants. Caterpillar feeding induces resistance through the jasmonate signaling pathway that is associated with the induction of kaempferol 3,7-dirhamnoside, whereas aphid feeding induces resistance via a novel mechanism involving sinapoyl malate. The role of sinapoyl malate is confirmed through the use of a mutant compromised in the biosynthesis of this compound. Caterpillar-induced resistance is associated with a lower cost in terms of plant growth reduction than aphid-induced resistance. A strong constitutive resistance against P. brassicae caterpillars in combination with a strong growth attenuation in plants of a transfer DNA (T-DNA) insertion mutant of WRKY70 (wrky70) suggest that the WRKY70 transcription factor, a regulator of downstream responses mediated by jasmonate-salicylic acid signaling cross talk, is involved in the negative regulation of caterpillar resistance and in the tradeoff between growth and defense. In conclusion, different mechanisms of herbivore-induced resistance come with different costs, and a functional WRKY70 transcription factor is required for the induction of low-cost resistance. PMID:26603653

  12. 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.

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

    PubMed Central

    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.

    2005-01-01

    The structure of an AKAP docked to the dimerization/docking (D/D) domain of the type II (RIIα) 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 (RIα) 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 α-helical A-kinase binding (AKB) motif from D-AKAP2 docked to both RIα and RIIα 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 RIα compared to RIIα, 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 RIα indicated that there were more binding constraints for the AKB ligand when bound to RIα. This was in contrast to RIIα, which has a preformed, localized binding surface. These distinct modes of AKAP binding may contribute to the more discriminating nature of the RIα 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. PMID:16260760

  14. 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

  15. 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.

  16. Distinct sesquiterpene pyridine alkaloids from in Salvadoran and Peruvian Celastraceae species.

    PubMed

    Callies, Oliver; Núñez, Marvin J; Perestelo, Nayra R; Reyes, Carolina P; Torres-Romero, David; Jiménez, Ignacio A; Bazzocchi, Isabel L

    2017-10-01

    As part of a bioprospecting program aimed at the discovery of undescribed natural products from Salvadoran and Peruvian flora, the phytochemical investigations of four Celastraceae species, Celastrus vulcanicola, Maytenus segoviarum, Maytenus jeslkii, and Maytenus cuzcoina, were performed. The current study reports the isolation and structural characterization of five previously undescribed macrolide sesquiterpene pyridine alkaloids, named vulcanicoline-A, cuzcoinine, vulcanicoline-B, jelskiine, and vulcanicoline-C, along with sixteen known alkaloids. The structures of the alkaloids were established by spectrometric and extensive 1D and 2D NMR spectroscopic analysis, including COSY, HSQC, HMBC, and ROESY experiments. The absolute configurations of alkaloids were proposed based on optical rotation sign, and biogenetic considerations. This study represents the first phytochemical analysis of Maytenus segoviarum. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. 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

  18. 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...

  19. 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...

  20. A Cross-Species Analysis in Pancreatic Neuroendocrine Tumors Reveals Molecular Subtypes with Distinctive Clinical, Metastatic, Developmental, and Metabolic Characteristics

    PubMed Central

    Sadanandam, Anguraj; Wullschleger, Stephan; Lyssiotis, Costas A.; Grötzinger, Carsten; Barbi, Stefano; Bersani, Samantha; Körner, Jan; Wafy, Ismael; Mafficini, Andrea; Lawlor, Rita T.; Simbolo, Michele; Asara, John M.; Bläker, Hendrik; Cantley, Lewis C.; Wiedenmann, Bertram; Scarpa, Aldo; Hanahan, Douglas

    2016-01-01

    Seeking to assess the representative and instructive value of an engineered mouse model of pancreatic neuroendocrine tumors (PanNET) for its cognate human cancer, we profiled and compared mRNA and miRNA transcriptomes of tumors from both. Mouse PanNET tumors could be classified into two distinctive subtypes, well-differentiated islet/insulinoma tumors (IT) and poorly differentiated tumors associated with liver metastases, dubbed metastasis-like primary (MLP). Human PanNETs were independently classified into these same two subtypes, along with a third, specific gene mutation–enriched subtype. The MLP subtypes in human and mouse were similar to liver metastases in terms of miRNA and mRNA transcriptome profiles and signature genes. The human/mouse MLP subtypes also similarly expressed genes known to regulate early pancreas development, whereas the IT subtypes expressed genes characteristic of mature islet cells, suggesting different tumorigenesis pathways. In addition, these subtypes exhibit distinct metabolic profiles marked by differential pyruvate metabolism, substantiating the significance of their separate identities. SIGNIFICANCE This study involves a comprehensive cross-species integrated analysis of multi-omics profiles and histology to stratify PanNETs into subtypes with distinctive characteristics. We provide support for the RIP1-TAG2 mouse model as representative of its cognate human cancer with prospects to better understand PanNET heterogeneity and consider future applications of personalized cancer therapy. PMID:26446169

  1. 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.

  2. Geographically distinct patterns of reproductive isolation and hybridization in two sympatric species of the Jaera albifrons complex (marine isopods).

    PubMed

    Ribardière, Ambre; Daguin-Thiébaut, Claire; Houbin, Céline; Coudret, Jérôme; Broudin, Caroline; Timsit, Olivier; Broquet, Thomas

    2017-07-01

    Sympatric species that in some populations hybridize and in other populations remain reproductively isolated open interesting research possibilities for the study of hybridization and speciation. Here, we test for such a situation in two littoral isopods (Jaera albifrons and J. praehirsuta) that occur in mixed populations and where past morphological descriptions suggested that the two species are generally reproductively isolated except in rare populations where hybridization may be happening. Using field surveys and microsatellite genetic structure analyses in two regions from France (Normandy and Brittany), we confirmed that introgressive hybridization occurs in a subset of mixed J. albifrons/J. praehirsuta populations (region Normandy) where the two species are found in the same habitat (pebbles on the shore). Moreover, we found that introgression in these populations is differential, 21 of 23 microsatellite markers showing little genetic divergence between species (hierarchical analysis of molecular variance FCT = 0.017) while the remaining two loci were strongly differentiated (FCT = 0.428). By contrast, J. albifrons and J. praehirsuta in mixed populations from region Brittany occupied distinct habitats (pebbles and seaweeds, respectively) with little overlap and showed stronger genetic divergence (FCT = 0.132). In hybridizing populations, the majority of individuals show morphological traits that are characteristic of one or the other species. This raises the question of the forces that act to maintain this polymorphism, noting that hybridizing populations seem to be geographically isolated from potential source parental populations and show no detectable habitat divergence between species.

  3. Distinctive stable isotope ratios in important zooplankton species in relation to estuarine salinity gradients: Potential tracer of fish migration

    NASA Astrophysics Data System (ADS)

    Suzuki, Keita W.; Kasai, Akihide; Isoda, Takane; Nakayama, Kouji; Tanaka, Masaru

    2008-07-01

    To assess the potential of stable isotope ratios as an indicator of fish migration within estuaries, stable isotope ratios in important zooplankton species were analyzed in relation to estuarine salinity gradients. Gut contents from migratory juveniles of the euryhaline marine fish Lateolabrax japonicus were examined along the Chikugo River estuary of the Ariake Sea, which has the most developed estuarine turbidity maximum (ETM) in Japan. Early juveniles in March and April preyed primarily on two copepod species; Sinocalanus sinensis at lower salinities and Acartia omorii at higher salinities. Late juveniles (standard length > 40 mm) at lower salinities preyed exclusively on the mysid Acanthomysis longirostris until July and complementarily on the decapod Acetes japonicus in August. These prey species were collected along the estuary during the spring-summer seasons of 2003 and 2004, and their carbon and nitrogen stable isotope ratios ( δ13C and δ15N) were evaluated. The δ13C values of prey species were distinct from each other and were primarily depleted within and in close proximity to the ETM (salinity < 10); S. sinensis (-26.6‰) < Acanthomysis longirostris (-23.3‰) < Acartia omorii (-21.1‰) < Acetes japonicus (-18.5‰). The overall gradient of δ13C with salinity occurred for all prey species and showed minor temporal fluctuations, while it was not directly influenced by the δ13C values in particulate organic matter along the estuary. In contrast to δ13C, the δ15N values of prey species did not exhibit any clear relationship with salinity. The present study demonstrated that δ13C has the potential for application as a tracer of fish migration into lower salinity areas including the ETM.

  4. 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.

  5. 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.

  6. 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

  7. Induction of heme oxygenase in mammalian cells by mineral fibers: distinctive effect of reactive oxygen species.

    PubMed

    Suzuki, K; Hei, T K

    1996-04-01

    Exponentially growing human-hamster hybrid [AL] cells treated with a 40 micrograms/ml (8 micrograms/cm2) dose of UICC standard reference chrysotile fibers induced heme oxygenase (HO) protein with a maximum expression level at 8 h post-treatment. While the constitutive HO expression was detectable in non-treated AL cells, the protein level was increased approximately 4.5-fold in fiber-treated cells. The induction was dose-dependent at fiber concentration between 2.5 micrograms/ml (0.5 microgram/cm2) and 40 micrograms/ml (8 micrograms/cm2) with the induced HO concentrated mostly in the cytoplasm as shown by immunostaining. Several other types of mineral fibers examined including crocidolites, tremolites, and erionites also induced HO synthesis with varying degree of efficiency. In general, chrysotile and crocidolite were more efficient inducers of HO than tremolite and erionite when compared at fiber doses that resulted in approximately 50% survival (LD50) level. The effects of antioxidant enzymes on HO induction were examined by concurrent treatment of fiber-exposed cultures with SOD and catalase. Although addition of superoxide dismutase (SOD) and catalase inhibited HO induction in a dose-dependent manner, they offered no protection on fiber-mediated clonogenic toxicity in the same population of treated cells. These results suggest that reactive oxygen species (ROS) produced by asbestos fibers play an essential role in the induction of HO and that different mineral fibers, when applied at equitoxic doses, often result in different oxidative stress status as determined by the induction of HO proteins.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. Mitochondrial and nuclear ribosomal DNA dataset supports that Paramphistomum leydeni (Trematoda: Digenea) is a distinct rumen fluke species.

    PubMed

    Ma, Jun; He, Jun-Jun; Liu, Guo-Hua; Zhou, Dong-Hui; Liu, Jian-Zhi; Liu, Yi; Zhu, Xing-Quan

    2015-04-02

    Rumen flukes parasitize the rumen and reticulum of ruminants, causing paramphistomiasis. Over the years, there has been considerable debate as to whether Paramphistomum leydeni and Paramphistomum cervi are the same or distant species. In the present study, the complete mitochondrial (mt) genome of P. leydeni was amplified using PCR-based sequencing and compared with that of P. cervi. The second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA) of P. leydeni specimens (n = 6) and P. cervi specimens (n = 8) was amplified and then sequenced. Phylogenetic relationship of the concatenated amino acid sequence data for 12 protein-coding genes of the two rumen flukes and selected members of Trematoda was evaluated using Bayesian inference (BI). The complete mt genome of P. leydeni was 14,050 bp in size. Significant nucleotide difference between the P. leydeni mt genome and that of P. cervi (14.7%) was observed. For genetic divergence in ITS-2, sequence difference between P. leydeni and P. cervi was 3.1%, while no sequence variation was detected within each of them. Phylogenetic analysis indicated that P. leydeni and P. cervi are closely-related but distinct rumen flukes. Results of the present study support the proposal that P. leydeni and P. cervi represent two distinct valid species. The mt genome sequences of P. leydeni provide plentiful resources of mitochondrial markers, which can be combined with nuclear markers, for further comparative studies of the biology of P. leydeni and its congeners from China and other countries.

  13. Physicochemical characterization of the ribosomal RNA species of the Mollusca. Molecular weight, integrity and secondary-structure features of the RNA of the large and small ribosomal subunits.

    PubMed Central

    Cammarano, P; Londei, P; Mazzei, F; Felsani, A

    1980-01-01

    1. The rRNA species of the Cephalopoda Octopus vulgaris and Loligo vulgaris were found to have unexpectedly high sedimentation coefficients and molecular weights. In 0.1 M-NaCl the L-rRNA (RNA from large ribosomal subunit) has the same s20 value as the L-rRNA of the mammals (30.7S), whereas the S-rRNA (RNA from small ribosomal subunit) sediments at a faster rate (20.1S) than the S-rRNA of both the mammals and the fungi (Neurospora crassa) (17.5S). The molecular weights of the L-rRNA were determined by gel electrophoresis in formamide and found to be 1.66 X 10(6) (Octupus) and 1.89 X 10(6) (Loligo); the mol.wt. of the S-rRNA of both species is 0.96 X 10(6), i.e. much larger than that of the mammals (0.65 X 10(6)) and almost coincident with that of the '23S' RNA of the prokaryotes. 2. By contrast, the less evolved Gastropoda and Lamellibranchiata (Murex trunculus and Macrocallista chione) have S-rRNA and L-rRNA species with mol.wts. of 0.65 X 10(6) and approx. 1.40 X 10(6).3. All the mature L-rRNA molecules of the cephalopoda are composed of two unequal fragments held together by regions of hydrogen-bonding having a similar, low, thermal stability in the two species; the molecular weights of the two fragments composing the L-rRNA are estimated to be 0.96 X 10(6) and 0.88 X 10(6) (Loligo) and 0.96 X 10(6) and 0.65 X 10(6) (Octupus). THe S-rRNA of both species is a continuous chain with exactly the same molecular weight (0.96 X 10(6)) as the heavier of the two fragments of the L-rRNA. 4. The secondary-structure features of the L-rRNA and S-rRNA species of the Caphalopoda were investigated by thermal 'melting' analysis in 4.0 M-guanidinium chloride; 60-70% of the residues are estimated to form short, independently 'melting' bihelical segments not more than 10 base-pairs in length. 5. Bases are unevenly distributed between non-helical and bihelical portions of the rRNA molecules, G and C residues being preferentially concentrated in bihelical comains. 6. The secondary

  14. 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

  15. 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.

  16. 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

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. 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. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

  2. 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.

  3. 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.

  4. 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

    ... of a 5-Year Review of the Eastern Distinct Population Segment of the Steller Sea Lion AGENCY... of a 5-year review of the eastern Distinct Population Segment (DPS) of the Steller Sea Lion... for the eastern Distinct Population Segment of the Steller sea lion (75 FR 37385). NMFS inadvertently...

  5. 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.

  6. 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.

  7. Gene flow analysis demonstrates that Phytophthora fragariae var. rubi constitutes a distinct species, Phytophthora rubi comb. nov.

    PubMed

    Man in 't Veld, Willem A

    2007-01-01

    Isozyme analysis and cytochrome oxidase sequences were used to examine whether differentiation of P. fragariae var. fragariae and P. fragariae var. rubi at the variety level is justified. In isozyme studies six strains of both P. fragariae varieties were analyzed with malate dehydrogenase (MDH), glucose phosphate isomerase (GPI), aconitase (ACO), isocitrate dehydrogenase (IDH) and phosphogluconate dehydrogenase (PGD), comprising altogether seven putative loci. Five unique alleles (Mdh-1(A), Mdh-2(B), Gpi(A), Aco(B) and Idh-1(B)) were found in strains of P. fragariae var. fragariae, whereas five unique alleles (Mdh-1(B), Mdh-2(A), Gpi(B), Aco(A) and Idh-1(A)) were present in strains of P. fragariae var. rubi. It was inferred from these data that there is no gene flow between the two P. fragariae varieties. Cytochrome oxidase I (Cox I) sequences showed consistent differences at 15 positions between strains of Fragaria and Rubus respectively. Based on isozyme data, cytochrome oxidase I sequences, and previously published differences in restyriction enzyme patterns of mitochondrial DNA, sequences of nuclear and mitochondrial genes, AFLP patterns and pathogenicity, it was concluded that both specific pathogenic varieties of P. fragariae are reproductively isolated and constitute a distinct species. Consequently strains isolated from Rubus idaeus are assigned to Phytophthora rubi comb. nov.

  8. 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.

  9. Early signaling components in ultraviolet-B responses: distinct roles for different reactive oxygen species and nitric oxide.

    PubMed

    A -H -Mackerness, S; John, C F; Jordan, B; Thomas, B

    2001-02-02

    The nature and origin of the reactive oxygen species (ROS) involved in the early part of Ultraviolet-B (UV-B)-induced signaling pathways were investigated in Arabidopsis thaliana using a range of enzyme inhibitors and free radical scavengers. The increase in PR-1 transcript and decrease in Lhcb transcript in response to UV-B exposure was shown to be mediated through pathways involving hydrogen peroxide (H(2)O(2)) derived from superoxide (O(2)(&z.rad;-)). In contrast, the up-regulation of PDF1.2 transcript was mediated through a pathway involving O(2)(&z.rad;-) directly. The origins of the ROS were also shown to be distinct and to involve NADPH oxidase and peroxidase(s). The up-regulation of Chs by UV-B was not affected by ROS scavengers, but was reduced by inhibitors of nitric oxide synthase (NOS) or NO scavengers. Together these results suggest that UV-B exposure leads to the generation of ROS, from multiple sources, and NO, through increased NOS activity, giving rise to parallel signaling pathways mediating responses of specific genes to UV-B radiation.

  10. 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

  11. 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

  12. 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.

  13. 76 FR 76386 - Endangered and Threatened Species; 5-Year Reviews for 4 Distinct Population Segments of Steelhead...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-07

    ... Reviews for 4 Distinct Population Segments of Steelhead in California AGENCY: National Marine Fisheries... Viable Salmonid Population framework, which relies on evaluating four key population parameters...

  14. 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.

  15. 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

  16. 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

  17. Extralarge XL(alpha)s (XXL(alpha)s), a variant of stimulatory G protein alpha-subunit (Gs(alpha)), is a distinct, membrane-anchored GNAS product that can mimic Gs(alpha).

    PubMed

    Aydin, Cumhur; Aytan, Nurgul; Mahon, Mathew J; Tawfeek, Hesham A W; Kowall, Neil W; Dedeoglu, Alpaslan; Bastepe, Murat

    2009-08-01

    GNAS gives rise to multiple imprinted gene products, including the alpha-subunit of the stimulatory G protein (Gs(alpha)) and its variant XL(alpha)s. Based on genomic sequence, the translation of XL(alpha)s begins from the middle of a long open reading frame, suggesting the existence of an N-terminally extended variant termed extralarge XLalphas (XXL(alpha)s). Although XXL(alpha), like Gs(alpha) and XL(alpha)s, would be affected by most disease-causing GNAS mutations, its authenticity and biological significance remained unknown. Here we identified a mouse cDNA clone that comprises the entire open reading frame encoding XXL(alpha)s. Whereas XXL(alpha)s mRNA was readily detected in mouse heart by RT-PCR, it appeared virtually absent in insulinoma-derived INS-1 cells. By Northern blots and RT-PCR, XXL(alpha)s mRNA was detected primarily in the mouse brain, cerebellum, and spleen. Immunohistochemistry using a specific anti-XXL(alpha)s antibody demonstrated XXL(alpha)s protein in multiple brain areas, including dorsal hippocampus and cortex. In transfected cells, full-length human XXL(alpha)s was localized to the plasma membrane and mediated isoproterenol- and cholera toxin-stimulated cAMP accumulation. XXL(alpha)s-R844H, which bears a mutation analogous to that in the constitutively active Gs(alpha) mutant Gs(alpha)-R201H (gsp oncogene), displayed elevated basal signaling. However, unlike Gs(alpha)-R201H, which mostly remains in the cytoplasm, both XXL(alpha)s-R844H and a constitutively active XL(alpha)s mutant localized to the plasma membrane. Hence, XXL(alpha)s is a distinct GNAS product and can mimic Gs(alpha), but the constitutively active XXL(alpha)s and Gs(alpha) mutants differ from each other regarding subcellular targeting. Our findings suggest that XXL(alpha)s deficiency or hyperactivity may contribute to the pathogenesis of diseases caused by GNAS mutations.

  18. 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 be...

  19. 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

  20. Synthesis in vitro of precursors of the catalytic subunits of acetylcholinesterase from Torpedo marmorata and Electrophorus electricus.

    PubMed

    Sikorav, J L; Grassi, J; Bon, S

    1984-12-17

    We translated poly(A-rich messenger RNA prepared from the electric organs of Electrophorus electricus and Torpedo marmorata in a reticulocyte lysate system. In the case of Electrophorus, which appears to contain only one type of acetylcholinesterase catalytic subunit, an anti-(Electrophorus acetylcholinesterase) antiserum precipitated a single 65-kDa polypeptide from the products translation obtained in vitro. In the case of Torpedo, where a number of distinct catalytic subunits corresponding to different fractions of the enzyme have been described, an anti-(Torpedo acetylcholinesterase) antiserum precipitated two main polypeptides, 61 kDa and 65 kDa, both of which could be displaced by unlabelled purified Torpedo acetylcholinesterase. Synthesis in vitro thus appears to produce a single type of precursor of the acetylcholinesterase catalytic subunit for Electrophorus, and at least two distinct precursors for Torpedo, suggesting that several mRNAs code for the catalytic subunits in the latter species.

  1. Conservation prioritization in widespread species: the use of genetic and morphological data to assess population distinctiveness in rainbow trout (Oncorhynchus mykiss) from British Columbia, Canada

    PubMed Central

    Taylor, Eric B; Tamkee, Patrick; Keeley, Ernest R; Parkinson, Eric A

    2011-01-01

    Prioritization of efforts to maintain biodiversity is an important component of conservation, but is more often applied to ecosystems or species than within species. We assessed distinctiveness among 27 populations of rainbow trout (Salmonidae: Oncorhynchus mykiss) from British Columbia, Canada, using microsatellite DNA variation (representing historical or contemporary demography) and morphology (representing adaptive variation). Standardized genetic scores, that is, the average deviation across individuals within populations from the overall genetic score generated by factorial correspondence analysis, ranged from 1.05 to 4.90 among populations. Similar standardized morphological scores, generated by principal components analysis, ranged from 1.19 to 5.35. There was little correlation between genetic and morphological distinctiveness across populations, although one population was genetically and morphologically the most distinctive. There was, however, a significant correlation (r = 0.26, P = 0.008) between microsatellite (FST) and morphological (PST) divergence. We combined measures of allelic richness, genetic variation within, and divergence among, populations and morphological variation to provide a conservation ranking of populations. Our approach can be combined with other measures of biodiversity value (habitat, rarity, human uses, threat status) to rationalize the prioritization of populations, especially for widespread species where geographic isolation across distinct environments promotes intraspecific variability. PMID:25567956

  2. [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).

  3. Distinct genetic differentiation and species diversification within two marine nematodes with different habitat preference in Antarctic sediments.

    PubMed

    Hauquier, Freija; Leliaert, Frederik; Rigaux, Annelien; Derycke, Sofie; Vanreusel, Ann

    2017-05-30

    Dispersal ability, population genetic structure and species divergence in marine nematodes are still poorly understood, especially in remote areas such as the Southern Ocean. We investigated genetic differentiation of species and populations of the free-living endobenthic nematode genera Sabatieria and Desmodora using nuclear 18S rDNA, internal transcribed spacer (ITS) rDNA, and mitochondrial cytochrome oxidase I (COI) gene sequences. Specimens were collected at continental shelf depths (200-500 m) near the Antarctic Peninsula, Scotia Arc and eastern side of the Weddell Sea. The two nematode genera co-occurred at all sampled locations, but with different vertical distribution in the sediment. A combination of phylogenetic (GMYC, Bayesian Inference, Maximum Likelihood) and population genetic (AMOVA) analyses were used for species delimitation and assessment of gene flow between sampling locations. Sequence analyses resulted in the delimitation of four divergent species lineages in Sabatieria, two of which could not be discriminated morphologically and most likely constitute cryptic species. Two species were recognised in Desmodora, one of which showed large intraspecific morphological variation. Both genera comprised species that were restricted to one side of the Weddell Sea and species that were widely spread across it. Population genetic structuring was highly significant and more pronounced in the deeper sediment-dwelling Sabatieria species, which are generally less prone to resuspension and passive dispersal in the water column than surface Desmodora species. Our results indicate that gene flow is restricted at large geographic distance in the Southern Ocean, which casts doubt on the efficiency of the Weddell gyre and Antarctic Circumpolar Current in facilitating circum-Antarctic nematode species distributions. We also show that genetic structuring and cryptic speciation can be very different in nematode species isolated from the same geographic area, but with

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

    USDA-ARS?s Scientific Manuscript database

    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...

  5. 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...

  6. 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...

  7. 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

  8. 75 FR 77602 - Endangered and Threatened Species; 90-Day Finding on Petitions To Delist the Eastern Distinct...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-12-13

    ... Lion AGENCY: National Marine Fisheries Service (NMFS), National Oceanic and Atmospheric Administration... Steller Sea Lion (Eumetopias jubatus) under the Endangered Species Act of 1973, as amended (ESA). We find...)). Background The Steller sea lion (Eumetopias jubatus) was listed as a threatened species under the ESA on...

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. The first record of Orthocis casey (Coleoptera: Ciidae) from the Andean region, with the description of a distinctive new species.

    PubMed

    Lopes-Andrade, Cristiano

    2010-10-01

    Orthocis Casey is recorded for the first time from the Andean Region, with the description of O. elguetai sp. nov., for which diagnostic characters and details on the male genitalia and other external morphological structures are provided. One record of the new species, from Punta Espora (Chile, higher latitude than 52°S), constitutes the southernmost record of the family Ciidae in the world, and the first in the Subantarctic subregion. A brief discussion on morphological similarities to other species in the genus is also provided.

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    USDA-ARS?s Scientific Manuscript database

    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...

  17. 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

  18. Evolutionary Genetic Analysis Uncovers Multiple Species with Distinct Habitat Preferences and Antibiotic Resistance Phenotypes in the Stenotrophomonas maltophilia Complex.

    PubMed

    Ochoa-Sánchez, Luz E; Vinuesa, Pablo

    2017-01-01

    The genus Stenotrophomonas (Gammaproteobacteria) has a broad environmental distribution. Stenotrophomonas maltophilia is its best known species because it is a globally emerging, multidrug-resistant (MDR), opportunistic pathogen. Members of this species are known to display high genetic, ecological and phenotypic diversity, forming the so-called S. maltophilia complex (Smc). Heterogeneous resistance and virulence phenotypes have been reported for environmental Smc isolates of diverse ecological origin. We hypothesized that this heterogeneity could be in part due to the potential lumping of several cryptic species in the Smc. Here we used state-of-the-art phylogenetic and population genetics methods to test this hypothesis based on the multilocus dataset available for the genus at pubmlst.org. It was extended with sequences from complete and draft genome sequences to assemble a comprehensive set of reference sequences. This framework was used to analyze 108 environmental isolates obtained in this study from the sediment and water column of four rivers and streams in Central Mexico, affected by contrasting levels of anthropogenic pollution. The aim of the study was to identify species in this collection, defined as genetically cohesive sequence clusters, and to determine the extent of their genetic, ecological and phenotypic differentiation. The multispecies coalescent, coupled with Bayes factor analysis was used to delimit species borders, together with population genetic structure analyses, recombination and gene flow estimates between sequence clusters. These analyses consistently revealed that the Smc contains at least 5 significantly differentiated lineages: S. maltophilia and Smc1 to Smc4. Only S. maltophilia was found to be intrinsically MDR, all its members expressing metallo-β-lactamases (MBLs). The other Smc lineages were not MDR and did not express MBLs. We also obtained isolates related to S. acidaminiphila, S. humi and S. terrae. They were significantly

  19. 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.

  20. Photolysis of Hi-CO Nitrogenase - Observation of a Plethora of Distinct CO Species using Infrared Spectroscopy.

    PubMed

    Yan, Lifen; Dapper, Christie H; George, Simon J; Wang, Hongxin; Mitra, Devrani; Dong, Weibing; Newton, William E; Cramer, Stephen P

    2011-05-01

    Fourier transform infrared spectroscopy (FT-IR) was used to study the photochemistry of CO-inhibited Azotobacter vinelandii nitrogenase using visible light at cryogenic temperatures. The FT-IR difference spectrum of photolyzed hi-CO at 4 K comprises negative bands at 1973 cm(-1) and 1679 cm(-1) together with positive bands at 1711 cm(-1), 2135 and 2123 cm(-1). The negative bands are assigned to a hi-CO state that comprises 2 metal-bound CO ligands, one terminally bound, and one bridged and/or protonated species. The positive band at 1711 cm(-1) is assigned to a lo-CO product with a single bridged and/or protonated metal-CO group. We term these species 'Hi-1' and 'Lo-1' respectively. The high-energy bands are assigned to a liberated CO trapped in the protein pocket. Warming results in CO recombination, and the temperature dependence of the recombination rate yields an activation energy of 4 kJ mol(-1). Two α-H195 variant enzymes yielded additional signals. Asparagine substitution, α-H195N, gives a spectrum containing 2 negative 'Hi-2' bands at 1936 and 1858 cm(-1) with a positive 'Lo-2' band at 1780 cm(-1), while glutamine substitution, α-H195Q, produces a complex spectrum that includes a third CO species, with negative 'Hi-3' bands at 1938 and 1911 cm(-1) and a positive feature 'Lo-3' band at 1921 cm(-1). These species can be assigned to a combination of terminal, bridged, and possibly protonated CO groups bound to the FeMo-cofactor active site. The proposed structures are discussed in terms of both CO inhibition and the mechanism nitrogenase catalysis. Given the intractability of observing nitrogenase intermediates by crystallographic methods, IR-monitored photolysis appears to be a promising and information-rich probe of nitrogenase structure and chemistry.

  1. 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. © 2016 John Wiley & Sons Ltd.

  2. 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

  3. 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.

  4. Tawny owl (Strix aluco) and Hume's Tawny owl (Strix butleri) are distinct species: evidence from nucleotide sequences of the cytochrome b gene.

    PubMed

    Heidrich, P; Wink, M

    1994-01-01

    The cytochrome b gene of the Tawny Owl (Strix aluco), Hume's Tawny Owl (Strix butleri) and the African wood owl (Strix woodfordii) was amplified by polymerase chain reaction (PCR) and partially sequenced (300 base pairs). Sequences differ substantially (9 to 12% nucleotide substitutions) between these taxa indicating that they represent distinct species, which is also implicated from morphological and biogeographic differences. Using cytochrome b sequences of S. aluco, S. butleri, S. woodfordii, Athene noctua and Tyto alba phylogenetic relationship were reconstructed using the "maximum parsimony" principal (PAUP 3.1.1) and the neighbour-joining method (MEGA).

  5. 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.

  6. 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

  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. Copyright © 2016 Elsevier GmbH. All rights reserved.

  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. Involvement of proteasomal subunits zeta and iota in RNA degradation.

    PubMed Central

    Petit, F; Jarrousse, A S; Dahlmann, B; Sobek, A; Hendil, K B; Buri, J; Briand, Y; Schmid, H P

    1997-01-01

    We have identified two distinct subunits of 20 S proteasomes that are associated with RNase activity. Proteasome subunits zeta and iota, eluted from two-dimensional Western blots, hydrolysed tobacco mosaic virus RNA, whereas none of the other subunits degraded this substrate under the same conditions. Additionally, proteasomes were dissociated by 6 M urea, and subunit zeta, containing the highest RNase activity, was isolated by anion-exchange chromatography and gel filtration. Purified subunit zeta migrated as a single spot on two-dimensional PAGE with a molecular mass of approx. 28 kDa. Addition of anti-(subunit zeta) antibodies led to the co-precipitation of this proteasome subunit and nuclease activity. This is the first evidence that proteasomal alpha-type subunits are associated with an enzymic activity, and our results provide further evidence that proteasomes may be involved in cellular RNA metabolism. PMID:9337855

  10. 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

  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. 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.

  14. Distinct contributions of reactive oxygen species in amygdala to bee venom-induced spontaneous pain-related behaviors

    PubMed Central

    Lu, Yun-Fei; Neugebauer, Volker; Chen, Jun; Li, Zhen

    2016-01-01

    Reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, play essential roles in physiological plasticity and are also involved in the pathogenesis of persistent pain. Roles of peripheral and spinal ROS in pain have been well established, but much less is known about ROS in the amygdala, a brain region that plays an important role in pain modulation. The present study explored the contribution of ROS in the amygdala to bee venom (BV)-induced pain behaviors. Our data show that the amygdala is activated following subcutaneous BV injection into the left hindpaw, which is reflected in the increased number of c-Fos positive cells in the central and basolateral amygdala nuclei in the right hemisphere. Stereotaxic administration of a ROS scavenger (tempol, 10 mM), NADPH oxidase inhibitor (baicalein, 5 mM) or lipoxygenase inhibitor (apocynin, 10 mM) into the right amygdala attenuated the BV-induced spontaneous licking and lifting behaviors, but had no effect on BV-induced paw flinch reflexes. Our study provides further evidence for the involvement of the amygdala in nociceptive processing and pain behaviors, and that ROS in amygdala may be a potential target for treatment strategies to inhibit pain. PMID:26971700

  15. 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

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. Assessment of a quantitative 5' nuclease real-time polymerase chain reaction using the nicotinamide adenine dinucleotide dehydrogenase gamma subunit (nuoG) for Bartonella species in domiciled and stray cats in Brazil.

    PubMed

    André, Marcos Rogério; Dumler, John Stephen; Herrera, Heitor M; Gonçalves, Luiz R; de Sousa, Keyla Cm; Scorpio, Diana Gerardi; de Santis, Ana Cláudia Gabriela Alexandre; Domingos, Iara Helena; de Macedo, Gabriel Carvalho; Machado, Rosangela Zacarias

    2016-10-01

    The objective of this study was to develop a quantitative 5' nuclease real-time polymerase chain reaction (PCR) assay to diagnose infections caused by Bartonella species. Between January and April 2013 whole blood samples were collected by convenience from 151 cats (86 domiciled and 65 stray cats). The feline blood samples were subjected to a novel quantitative 5' nuclease real-time PCR (qPCR) for Bartonella species targeting the nictonamide adenine dinucleotide dehydrogenase gamma subunit (nuoG) and conventional PCR assays targeting intergenic transcribed spacer, ribC, gltA, pap31 and rpoB, followed by sequencing and basic local alignment search tool analysis. The qPCR assay detected as few as 10 copies of plasmid per reaction. Forty-six (54.4% domiciled and 45.6% stray cats) of 151 sampled cats showed positive results in nuoG qPCR for Bartonella species. The absolute quantification of nuoG Bartonella DNA in sampled cats ranged from 1.1 × 10(4) to 1.3 × 10(4). Eighteen (39.1%) of 46 positive samples in the qPCR were also positive in conventional PCR assays. The sequencing confirmed that Bartonella henselae and Bartonella clarridgeiae circulate in cats in midwestern Brazil. The present work provides details of a novel qPCR assay to diagnose infections caused by Bartonella species. © The Author(s) 2015.

  1. 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...

  2. 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...

  3. 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...

  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. 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.

  6. 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.

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

    PubMed Central

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

    2017-01-01

    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. PMID:28375172

  8. 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.

  9. 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

  10. Phylogenetic relationship and virulence inference of Streptococcus Anginosus Group: curated annotation and whole-genome comparative analysis support distinct species designation

    PubMed Central

    2013-01-01

    VNTR numbers that occurred over the course of one year. Conclusions The comparative genomic analysis of the SAG clarifies the phylogenetics of these bacteria and supports the distinct species classification. Numerous potential virulence determinants were identified and provide a foundation for further studies into SAG pathogenesis. Furthermore, the data may be used to enable the development of rapid diagnostic assays and therapeutics for these pathogens. PMID:24341328

  11. 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

  12. 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

  13. 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.

  14. 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.

  15. Impact of Detergent on Biophysical Properties and Immune Response of the IpaDB Fusion Protein, a Candidate Subunit Vaccine against Shigella Species

    PubMed Central

    Chen, Xiaotong; Choudhari, Shyamal P.; Martinez-Becerra, Francisco J.; Kim, Jae Hyun; Dickenson, Nicholas E.; Toth, Ronald T.; Joshi, Sangeeta B.; Greenwood, Jamie C.; Clements, John D.; Picking, William D.; Middaugh, C. Russell

    2014-01-01

    Shigella spp. are causative agents of bacillary dysentery, a human illness with high global morbidity levels, particularly among elderly and infant populations. Shigella infects via the fecal-oral route, and its virulence is dependent upon a type III secretion system (T3SS). Two components of the exposed needle tip complex of the Shigella T3SS, invasion plasmid antigen D (IpaD) and IpaB, have been identified as broadly protective antigens in the mouse lethal pneumonia model. A recombinant fusion protein (DB fusion) was created by joining the coding sequences of IpaD and IpaB. The DB fusion is coexpressed with IpaB's cognate chaperone, IpgC, for proper recombinant expression. The chaperone can then be removed by using the mild detergents octyl oligooxyethelene (OPOE) or N,N-dimethyldodecylamine N-oxide (LDAO). The DB fusion in OPOE or LDAO was used for biophysical characterization and subsequent construction of an empirical phase diagram (EPD). The EPD showed that the DB fusion in OPOE is most stable at neutral pH below 55°C. In contrast, the DB fusion in LDAO exhibited remarkable thermal plasticity, since this detergent prevents the loss of secondary and tertiary structures after thermal unfolding at 90°C, as well as preventing thermally induced aggregation. Moreover, the DB fusion in LDAO induced higher interleukin-17 secretion and provided a higher protective efficacy in a mouse challenge model than did the DB fusion in OPOE. These data indicate that LDAO might introduce plasticity to the protein, promoting thermal resilience and enhanced protective efficacy, which may be important in its use as a subunit vaccine. PMID:25368115

  16. 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 Central

    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.

    2011-01-01

    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•−) 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. PMID:21268708

  17. 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

  18. 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.

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

    PubMed

    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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. Quantification of Mycobacterium avium subsp. paratuberculosis strains representing distinct genotypes and isolated from domestic and wildlife animal species by use of an automatic liquid culture system.

    PubMed

    Abendaño, Naiara; Sevilla, Iker; Prieto, José M; Garrido, Joseba M; Juste, Ramon A; Alonso-Hearn, Marta

    2012-08-01

    Quantification of 11 clinical strains of Mycobacterium avium subsp. paratuberculosis isolated from domestic (cattle, sheep, and goat) and wildlife (fallow deer, deer, wild boar, and bison) animal species in an automatic liquid culture system (Bactec MGIT 960) was accomplished. The strains were previously isolated and typed using IS1311 PCR followed by restriction endonuclease analysis (PCR-REA) into type C, S, or B. A strain-specific quantification curve was generated for each M. avium subsp. paratuberculosis strain by relating the time to detection in the liquid culture system to the estimated log(10) CFU in each inoculum. According to their growth curves, the tested M. avium subsp. paratuberculosis strains were classified into two distinct groups. The first group included the S-type strain isolated from goat and all the sheep strains with C, S, and B genotypes. A second group contained the C- and B-type strains isolated from cattle, goat, and wildlife animals with the exception of the fallow deer strain. The strains isolated from cattle or sheep showed similar strain-specific standard curves irrespective of their genotype. In contrast, the strains isolated from goat or from wildlife animal species varied in their rates of growth in liquid culture. Universal-standard curves and algorithms for the quantification of each group of strains were generated. In addition, the liquid culture system was compared with a real-time quantitative PCR system for the quantification of the 11 M. avium subsp. paratuberculosis strains. Correlations between the estimated log(10) CFU and M. avium subsp. paratuberculosis DNA copy numbers were very high for all the tested strains (R ≥ 0.9).

  9. 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.

  10. Genetic diversity of Bemisia tabaci species colonizing cassava in Central African Republic characterized by analysis of cytochrome c oxidase subunit I

    PubMed Central

    Tocko-Marabena, Brice Kette; Silla, Semballa; Simiand, Christophe; Zinga, Innocent; Legg, James; Reynaud, Bernard

    2017-01-01

    After 2007, upsurges of whiteflies on cassava plants and high incidences of cassava diseases were observed in Central African Republic. This recent upsurge in the abundance of Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) was directly linked to serious damage to cassava crops resulting from spread of whitefly-borne cassava mosaic geminiviruses (CMGs). There is currently very little information describing whitefly populations on cassava and associated crops in Central African Republic. The current study aimed to address this gap, and to determine whether the increasing damage associated with B. tabaci whiteflies was the consequence of a new invasion, or an upsurge of a local population. The molecular genetic identification and phylogenetic relationships of 898 B. tabaci adult individuals collected from representative locations (54) throughout CAR were determined based on their mitochondrial cytochrome oxidase I sequences (mtCOI). Field and ecological data were also collected from each site, including whitefly abundance, CMD incidence, host plants colonized by B. tabaci and agro-ecological zone. Phylogenetic analysis of the whitefly mtCOI sequences indicated that SSA1 (-SG1, -SG2), SSA3, MED, MEAM1 and Indian Ocean (IO) putative species occur in CAR. One specific haplotype of SSA1-SG1 (SSA1-SG1-P18F5) predominated on most cassava plants and at the majority of sites. This haplotype was identical to the SSA1-SG1 Mukono8-4 (KM377961) haplotype that was recorded from Uganda but that also occurs widely in CMD pandemic-affected areas of East Africa. These results suggest that the SSA1-SG1-P18F5 haplotype occurring in CAR represents a recent invasive population, and that it is the likely cause of the increased spread and severity of CMD in CAR. Furthermore, the high mtDNA sequence diversity observed for SSA1 and its broad presence on all sites and host plants sampled suggest that this genetic group was the dominant resident species even before the arrival of this new

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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

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

    PubMed

    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; Lau, Yee Ling

    2016-08-01

    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.

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. The evolution of new lipoprotein subunits of the bacterial outer membrane BAM complex

    PubMed Central

    Anwari, Khatira; Webb, Chaille T; Poggio, Sebastian; Perry, Andrew J; Belousoff, Matthew; Celik, Nermin; Ramm, Georg; Lovering, Andrew; Sockett, R Elizabeth; Smit, John; Jacobs-Wagner, Christine; Lithgow, Trevor

    2012-01-01

    The β-barrel assembly machine (BAM) complex is an essential feature of all bacteria with an outer membrane. The core subunit of the BAM complex is BamA and, in Escherichia coli, four lipoprotein subunits: BamB, BamC, BamD and BamE, also function in the BAM complex. Hidden Markov model analysis was used to comprehensively assess the distribution of subunits of the BAM lipoproteins across all subclasses of proteobacteria. A patchwork distribution was detected which is readily reconciled with the evolution of the α-, β-, γ-, δ- and ε-proteobacteria. Our findings lead to a proposal that the ancestral BAM complex was composed of two subunits: BamA and BamD, and that BamB, BamC and BamE evolved later in a distinct sequence of events. Furthermore, in some lineages novel lipoproteins have evolved instead of the lipoproteins found in E. coli. As an example of this concept, we show that no known species of α-proteobacteria has a homologue of BamC. However, purification of the BAM complex from the model α-proteobacterium Caulobacter crescentus identified a novel subunit we refer to as BamF, which has a conserved sequence motif related to sequences found in BamC. BamF and BamD can be eluted from the BAM complex under similar conditions, mirroring the BamC:D module seen in the BAM complex of γ-proteobacteria such as E. coli. PMID:22524202

  8. High-resolution melting of 12S rRNA and cytochrome b DNA sequences for discrimination of species within distinct European animal families.

    PubMed

    Naue, Jana; Hansmann, Tobias; Schmidt, Ulrike

    2014-01-01

    The cheap and easy identification of species is necessary within multiple fields of molecular biology. The use of high-resolution melting (HRM) of DNA provides a fast closed-tube method for analysis of the sequence composition of the mitochondrial genes 12S rRNA and cytochrome b. We investigated the potential use of HRM for species identification within eleven different animal groups commonly found in Europe by animal-group-specific DNA amplification followed by DNA melting. Influence factors as DNA amount, additional single base alterations, and the existence of mixed samples were taken into consideration. Visual inspection combined with mathematical evaluation of the curve shapes did resolve nearly all species within an animal group. The assay can therefore not only be used for identification of animal groups and mixture analysis but also for species identification within the respective groups. The use of a universal 12S rRNA system additionally revealed a possible approach for species discrimination, mostly by exclusion. The use of the HRM assay showed to be a reliable, fast, and cheap method for species discrimination within a broad range of different animal species and can be used in a flexible "modular" manner depending on the question to be solved.

  9. Comparison of sequencing the D2 region of the large subunit ribosomal RNA gene (MicroSEQ®) versus the internal transcribed spacer (ITS) regions using two public databases for identification of common and uncommon clinically relevant fungal species.

    PubMed

    Arbefeville, S; Harris, A; Ferrieri, P

    2017-09-01

    Fungal infections cause considerable morbidity and mortality in immunocompromised patients. Rapid and accurate identification of fungi is essential to guide accurately targeted antifungal therapy. With the advent of molecular methods, clinical laboratories can use new technologies to supplement traditional phenotypic identification of fungi. The aims of the study were to evaluate the sole commercially available MicroSEQ® D2 LSU rDNA Fungal Identification Kit compared to the in-house developed internal transcribed spacer (ITS) regions assay in identifying moulds, using two well-known online public databases to analyze sequenced data. 85 common and uncommon clinically relevant fungi isolated from clinical specimens were sequenced for the D2 region of the large subunit (LSU) of ribosomal RNA (rRNA) gene with the MicroSEQ® Kit and the ITS regions with the in house developed assay. The generated sequenced data were analyzed with the online GenBank and MycoBank public databases. The D2 region of the LSU rRNA gene identified 89.4% or 92.9% of the 85 isolates to the genus level and the full ITS region (f-ITS) 96.5% or 100%, using GenBank or MycoBank, respectively, when compared to the consensus ID. When comparing species-level designations to the consensus ID, D2 region of the LSU rRNA gene aligned with 44.7% (38/85) or 52.9% (45/85) of these isolates in GenBank or MycoBank, respectively. By comparison, f-ITS possessed greater specificity, followed by ITS1, then ITS2 regions using GenBank or MycoBank. Using GenBank or MycoBank, D2 region of the LSU rRNA gene outperformed phenotypic based ID at the genus level. Comparing rates of ID between D2 region of the LSU rRNA gene and the ITS regions in GenBank or MycoBank at the species level against the consensus ID, f-ITS and ITS2 exceeded performance of the D2 region of the LSU rRNA gene, but ITS1 had similar performance to the D2 region of the LSU rRNA gene using MycoBank. Our results indicated that the MicroSEQ® D2 LSU r

  10. Localization of Saccharomyces cerevisiae Protein Phosphatase 2A Subunits throughout Mitotic Cell Cycle

    PubMed Central

    Gentry, Matthew S.; Hallberg, Richard L.

    2002-01-01

    Protein phosphatase 2A (PP2A) regulates a broad spectrum of cellular processes. This enzyme is a collection of varied heterotrimeric complexes, each composed of a catalytic (C) and regulatory (B) subunit bound together by a structural (A) subunit. To understand the cell cycle dynamics of this enzyme population, we carried out quantitative and qualitative analyses of the PP2A subunits of Saccharomyces cerevisiae. We found the following: the level of each subunit remained constant throughout the cell cycle; there is at least 10 times more of one of the regulatory subunits (Rts1p) than the other (Cdc55p); Tpd3p, the structural subunit, is limiting for both catalytic and regulatory subunit binding. Using green fluorescent protein-tagged forms of each subunit, we monitored the sites of significant accumulation of each protein throughout the cell cycle. The two regulatory subunits displayed distinctly different dynamic localization patterns that overlap with the A and C subunits at the bud tip, kinetochore, bud neck, and nucleus. Using strains null for single subunit genes, we confirmed the hypothesis that regulatory subunits determine sites of PP2A accumulation. Although Rts1p and Tpd3p required heterotrimer formation to achieve normal localization, Cdc55p achieved its normal localization in the absence of either an A or C subunit. PMID:12388751

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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

    USDA-ARS?s Scientific Manuscript database

    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...

  16. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. 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

  18. 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.

  19. 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

  20. Identification of Dermatophyte Species by 28S Ribosomal DNA Sequencing with a Commercial Kit

    PubMed Central

    Ninet, Béatrice; Jan, Isabelle; Bontems, Olympia; Léchenne, Barbara; Jousson, Olivier; Panizzon, Renato; Lew, Daniel; Monod, Michel

    2003-01-01

    We have shown that dermatophyte species can be easily identified on the basis of a DNA sequence encoding a part of the large-subunit (LSU) rRNA (28S rRNA) by using the MicroSeq D2 LSU rRNA Fungal Sequencing Kit. Two taxa causing distinct dermatophytoses were clearly distinguished among isolates of the Trichophyton mentagrophytes species complex. PMID:12574293

  1. 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.

  2. 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

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

    USDA-ARS?s Scientific Manuscript database

    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...

  4. 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

    USDA-ARS?s Scientific Manuscript database

    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...

  5. DNA barcoding and morphology reveal three cryptic species of Anania (Lepidoptera: Crambidae: Pyraustinae) in North America, all distinct from their European counterpart

    USDA-ARS?s Scientific Manuscript database

    Anania coronata (Hufnagel), a Holarctic species of pyraustine crambid moth, has long been treated as having two geographically separated subspecies, the nominotypical Anania coronata in the Palaearctic Region, and Anania coronata tertialis (Guenée) in the Nearctic Region. Maximum likelihood and Baye...

  6. 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

  7. Novel consortium of Klebsiella variicola and Lactobacillus species enhances the functional potential of fermented dairy products by increasing the availability of branched-chain amino acids and the amount of distinctive volatiles.

    PubMed

    Rosales-Bravo, H; Morales-Torres, H C; Vázquez-Martínez, J; Molina-Torres, J; Olalde-Portugal, V; Partida-Martínez, L P

    2017-08-17

    Identify novel bacterial taxa that could increase the availability of branched-chain amino acids and the amount of distinctive volatiles during skim milk fermentation. We recovered 344 bacterial isolates from stool samples of healthy and breastfed infants. Five were selected based on their ability to produce branched-chain amino acids. Three strains were identified as Escherichia coli, one as Klebsiella pneumoniae and other as Klebsiella variicola by molecular and biochemical methods. HPLC and solid-phase microextraction with GC-MS were used for the determination of free amino acids and volatile compounds respectively. The consortium formed by K. variicola and four Lactobacillus species showed the highest production of Leu and Ile in skim milk fermentation. In addition, the production of volatile compounds, such as acetoin, ethanol, 2-nonanone, and acetic, hexanoic and octanoic acids, increased in comparison to commercial yogurt, Emmental and Gouda cheese. Also, distinctive volatiles, such as 2,3-butanediol, 4-methyl-2- hexanone and octanol, were identified. The use of K. variicola in combination with probiotic Lactobacillus species enhances the availability of Leu and Ile and the amount of distinctive volatiles during skim milk fermentation. The identified consortium increases the functional potential of fermented dairy products. © 2017 The Society for Applied Microbiology.

  8. 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

  9. 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

  10. Genetic variation in the mitochondrial cytochrome c oxidase subunit 1 within Progamotaenia festiva (Cestoda: Anoplocephalidae) from macropodid marsupials.

    PubMed

    Beveridge, I; Shamsi, S; Hu, M; Chilton, N B; Gasser, R B

    2007-09-01

    Genetic variation was examined in the anoplocephalid cestode Progamotaenia festiva, from Australian marsupials, in order to test the hypothesis that P. festiva, is a complex of sibling species and to assess the extent of host switching reported previously based on multilocus enzyme electrophoresis (MEE). Polymerase chain reaction (PCR)-based single-strand conformational polymorphism (SSCP) was used for the analysis of sequence variation in the cytochrome c oxidase subunit 1 (cox1) gene among 179 specimens of P. festiva (identified based on morphology and predilection site in the host) from 13 different host species, followed by selective DNA sequencing. Fifty-three distinct sequence types (haplotypes) representing all specimens were defined. Phylogenetic analyses of these sequence data (utilizing maximum parsimony and neighbour-joining methods) revealed 12 distinct clades. Other heterologous species, P. ewersi and P. macropodis, were used as outgroups and the remaining bile-duct inhabiting species, P. diaphana and P. effigia, were included in the analysis for comparative purposes. The latter 2 species were nested within the clades representing P. festiva. Most clades of P. festiva identified were restricted to a single host species; one clade primarily in Macropus robustus was also found in the related host species M. antilopinus in an area of host sympatry; another clade occurring primarily in M. robustus occurred also in additional kangaroo species, M. rufus and M. dorsalis. High levels of genetic divergence, the existence of distinct clades and their occurrence in sympatry provide support for the hypothesis that P. festiva represents a complex of numerous species, most of which, but not all, are host specific. Three distinct clades of cestodes were found within a single host, M. robustus, but there was no evidence of within-host speciation.

  11. The distinctive character of shell morphology Rectidens sumatrensis (DUNKER, 1852) against Elongaria orientalis (LEA, 1840) (two local species, bivalvia: Unionidae) from the Brantas river, Indonesia

    NASA Astrophysics Data System (ADS)

    Affandi, Moch.; Hariyanto, Sucipto; Soegianto, Agoes

    2017-09-01

    This research is directed to recognize the main characters of shell morphology that can easily be used to distinguish the two local species of Bivalia Unionidae in the Brantas River in East Java (Indonesia) which are very similar, namely Rectidens sumatrensis and Elongaria orientalis. A total of 49 individuals sampled specimens of both species were characterized based on identification guide Jutting (1953). About of 30 of shell morphological characters were observed, there are three characters identified in specimens Rectidens sumatrensis that can be used to distinguish from the specimen Elongaria orientalis, namely the presence of wrinkles noticeable on the surface structure of the interior of the shell; anteriorly of the greatest diameter of the shell, there is generally a shallow concavity in the flanks; and the two cardinal teeth in each valve shells left and right are relatively undeveloped.

  12. 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.

  13. Distinct ribosome maturation defects in yeast models of Diamond-Blackfan anemia and Shwachman-Diamond syndrome.

    PubMed

    Moore, Joseph B; Farrar, Jason E; Arceci, Robert J; Liu, Johnson M; Ellis, Steven R

    2010-01-01

    Diamond-Blackfan anemia and Shwachman-Diamond syndrome are inherited bone marrow failure syndromes linked to defects in ribosome synthesis. The purpose of this study was to determine whether yeast models for Diamond-Blackfan anemia and Shwachman-Diamond syndrome differed in the mechanism by which ribosome synthesis was affected. Northern blotting, pulse-chase analysis, and polysome profiling were used to study ribosome synthesis in yeast models. Localization of 60S ribosomal subunits was assessed using RPL25eGFP. Relative to wild-type controls, each disease model showed defects in 60S subunit maturation, but with distinct underlying mechanisms. In the model of Diamond-Blackfan anemia, 60S subunit maturation was disrupted at a relatively early stage with abortive complexes subject to rapid degradation. 5S ribosomal RNA, unlike other large subunit ribosomal RNA in this model, accumulated as an extra-ribosomal species. In contrast, subunit maturation in the Shwachman-Diamond syndrome model was affected at a later step, giving rise to relatively stable pre-60S particles with associated 5S ribosomal RNA retained in the nucleus. Conclusions These differences between the yeast Diamond-Blackfan anemia and Shwachman-Diamond syndrome models have implications for signaling mechanisms linking abortive ribosome assembly to cell fate decisions and may contribute to the divergent clinical presentations of Diamond-Blackfan anemia and Shwachman-Diamond syndrome.

  14. Interactions among rice ORC subunits.

    PubMed

    Tan, Deyong; Lv, Qundan; Chen, Xinai; Shi, Jianghua; Ren, Meiyan; Wu, Ping; Mao, Chuanzao

    2013-08-01

    The origin recognition complex (ORC) is composed of six subunits and plays an important role in DNA replication in all eukaryotes. The ORC subunits OsORC6 as well as the other five ORC subunits in rice were experimentally isolated and sequenced. It indicated that there also exist six ORC subunits in rice. Results of RT-PCR indicated that expression of all the rice ORC genes are no significant difference under 26°C and 34°C. Yeast two hybridization indicated that OsORC2, -3, -5 interact with each other. OsORC5 can then bind OsORC4 to form the OsORC2, -3,-4,-5 core complex. It suggested that the basic interactions have been conserved through evolution. No binding of OsORC1 and OsORC6 with the other subunits were observed. A model of ORC complex in rice is proposed.

  15. 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.

  16. 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.

  17. Anaerobic utilization of Fe(III)-xenosiderophores among Bacteroides species and the distinct assimilation of Fe(III)-ferrichrome by Bacteroides fragilis within the genus.

    PubMed

    Rocha, Edson R; Krykunivsky, Anna S

    2017-04-11

    In this study, we show that Bacteroides species utilize Fe(III)-xenosiderophores as the only source of exogenous iron to support growth under iron-limiting conditions in vitro anaerobically. Bacteroides fragilis was the only species able to utilize Fe(III)-ferrichrome while Bacteroides vulgatus ATCC 8482 and Bacteroides thetaiotaomicron VPI 5482 were able to utilize both Fe(III)-enterobactin and Fe(III)-salmochelin S4 as the only source of iron in a dose-dependent manner. We have investigated the way B. fragilis assimilates Fe(III)-ferrichrome as initial model to understand the utilization of xenosiderophores in anaerobes. B. fragilis contains two outer membrane TonB-dependent transporters (TBDTs), FchA1 and FchA2, which are homologues to Escherichia coli ferrichrome transporter FhuA. The disruption of fchA1 gene had only partial growth defect on Fe(III)-ferrichrome while the fchA2 mutant had no growth defect compared to the parent strain. The genetic complementation of fchA1 gene restored growth to parent strain levels indicating that it plays a role in Fe(III)-ferrichrome assimilation though we cannot rule out some functional overlap in transport systems as B. fragilis contains abundant TBDTs whose functions are yet not understood. However, the growth of B. fragilis on Fe(III)-ferrichrome was abolished in a feoAB mutant indicating that Fe(III)-ferrichrome transported into the periplasmic space was reduced in the periplasm releasing ferrous iron prior to transport through the FeoAB transport system. Moreover, the release of iron from the ferrichrome may be linked to the thiol redox system as the trxB deletion mutant was also unable to grow in the presence of Fe(III)-ferrichrome. The genetic complementation of feoAB and trxB mutants completely restored growth on Fe(III)-ferrichrome. Taken together, these findings show that Bacteroides species have developed mechanisms to utilize ferric iron bound to xenosiderophores under anaerobic growth conditions though the

  18. 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.

  19. 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

  20. 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

  1. 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

  2. Characterization of the basic glutathione S-transferase B1 and B2 subunits from human liver.

    PubMed Central

    Stockman, P K; McLellan, L I; Hayes, J D

    1987-01-01

    The basic glutathione S-transferases in human liver are composed of at least two immunochemically distinct polypeptides, designated B1 and B2. These subunits exist as homodimers, but can hybridize to form the B1B2 heterodimer [Stockman, Beckett & Hayes (1985) Biochem. J. 227, 457-465]. Although these basic glutathione S-transferases possess similar catalytic properties, the B2 subunit exhibits significantly greater selenium-independent glutathione peroxidase activity than subunit B1. The use of the ligands haematin, tributyltin acetate and Bromosulphophthalein as inhibitors of 1-chloro-2,4-dinitrobenzene-GSH-conjugating activity clearly discriminate between the B1 and B2 subunits and should help facilitate their identification. Peptide mapping experiments showed that B1 and B2 are structurally distinct, but related, subunits; subunit B1 yielded 43 tryptic peptides, seven of which were unique, whereas subunit B2 yielded 40 tryptic peptides, four of which were unique. PMID:3663118

  3. Two species of Strobilomyces (Boletaceae, Boletales), S. seminudus and S. hongoi sp. nov. from Japan.

    PubMed

    Sato, Hirotoshi; Hattori, Tsutomu; Lee, Su-See; Murakami, Noriaki

    2011-01-01

    We describe and illustrate two Strobilomyces species, S. seminudus and S. hongoi sp. nov. These two species have been confused and treated as a single species (i.e. S. seminudus). However recent studies based on population genetics have implied that they are reproductively isolated. In the present study we found that they are phylogenetically and morphologically distinct. The molecular phylogenetic trees inferred from the partial sequences of the largest subunit of RNA polymerase II (RPB1) and the second-largest subunit of RNA polymerase II (RPB2) support the differentiation of these two species as well as their differentiation from other related species. Strobilomyces seminudus is characterized by a stipe with an annular zone, becoming distinctly thickened near the apex and mottled with appressed-tomentose scales near the base. In contrast S. hongoi is characterized by a stipe with a remarkable reticulum at the upper and middle part and with minutely warty scales downward. Stipe characteristics also are useful for distinguishing these two species from other related species. In addition the incompletely reticulated basidiospores of these two species are also distinct from those of related species (i.e. S. foveatus).

  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. 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

  6. The Subunit Structure of Potato Tuber ADPglucose Pyrophosphorylase 1

    PubMed Central

    Okita, Thomas W.; Nakata, Paul A.; Anderson, Joseph M.; Sowokinos, Joseph; Morell, Matthew; Preiss, Jack

    1990-01-01

    ADPglucose pyrophosphorylase has been extensively purified from potato (Solanum tuberosum L.) tuber tissue to study its structure. By employing a modified published procedure (JR Sowokinos, J Preiss [1982] Plant Physiol 69: 1459-1466) 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. Images Figure 1 Figure 2 Figure 3 PMID:16667537

  7. 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.

  8. Molecular evolution of pentatricopeptide repeat genes reveals truncation in species lacking an editing target and structural domains under distinct selective pressures.

    PubMed

    Hayes, Michael L; Giang, Karolyn; Mulligan, R Michael

    2012-05-14

    Pentatricopeptide repeat (PPR) proteins are required for numerous RNA processing events in plant organelles including C-to-U editing, splicing, stabilization, and cleavage. Fifteen PPR proteins are known to be required for RNA editing at 21 sites in Arabidopsis chloroplasts, and belong to the PLS class of PPR proteins. In this study, we investigate the co-evolution of four PPR genes (CRR4, CRR21, CLB19, and OTP82) and their six editing targets in Brassicaceae species. PPR genes are composed of approximately 10 to 20 tandem repeats and each repeat has two α-helical regions, helix A and helix B, that are separated by short coil regions. Each repeat and structural feature was examined to determine the selective pressures on these regions. All of the PPR genes examined are under strong negative selection. Multiple independent losses of editing site targets are observed for both CRR21 and OTP82. In several species lacking the known editing target for CRR21, PPR genes are truncated near the 17th PPR repeat. The coding sequences of the truncated CRR21 genes are maintained under strong negative selection; however, the 3' UTR sequences beyond the truncation site have substantially diverged. Phylogenetic analyses of four PPR genes show that sequences corresponding to helix A are high compared to helix B sequences. Differential evolutionary selection of helix A versus helix B is observed in both plant and mammalian PPR genes. PPR genes and their cognate editing sites are mutually constrained in evolution. Editing sites are frequently lost by replacement of an edited C with a genomic T. After the loss of an editing site, the PPR genes are observed with three outcomes: first, few changes are detected in some cases; second, the PPR gene is present as a pseudogene; and third, the PPR gene is present but truncated in the C-terminal region. The retention of truncated forms of CRR21 that are maintained under strong negative selection even in the absence of an editing site target

  9. Molecular evolution of pentatricopeptide repeat genes reveals truncation in species lacking an editing target and structural domains under distinct selective pressures

    PubMed Central

    2012-01-01

    Background Pentatricopeptide repeat (PPR) proteins are required for numerous RNA processing events in plant organelles including C-to-U editing, splicing, stabilization, and cleavage. Fifteen PPR proteins are known to be required for RNA editing at 21 sites in Arabidopsis chloroplasts, and belong to the PLS class of PPR proteins. In this study, we investigate the co-evolution of four PPR genes (CRR4, CRR21, CLB19, and OTP82) and their six editing targets in Brassicaceae species. PPR genes are composed of approximately 10 to 20 tandem repeats and each repeat has two α-helical regions, helix A and helix B, that are separated by short coil regions. Each repeat and structural feature was examined to determine the selective pressures on these regions. Results All of the PPR genes examined are under strong negative selection. Multiple independent losses of editing site targets are observed for both CRR21 and OTP82. In several species lacking the known editing target for CRR21, PPR genes are truncated near the 17th PPR repeat. The coding sequences of the truncated CRR21 genes are maintained under strong negative selection; however, the 3’ UTR sequences beyond the truncation site have substantially diverged. Phylogenetic analyses of four PPR genes show that sequences corresponding to helix A are high compared to helix B sequences. Differential evolutionary selection of helix A versus helix B is observed in both plant and mammalian PPR genes. Conclusion PPR genes and their cognate editing sites are mutually constrained in evolution. Editing sites are frequently lost by replacement of an edited C with a genomic T. After the loss of an editing site, the PPR genes are observed with three outcomes: first, few changes are detected in some cases; second, the PPR gene is present as a pseudogene; and third, the PPR gene is present but truncated in the C-terminal region. The retention of truncated forms of CRR21 that are maintained under strong negative selection even in the

  10. Distinct subpopulations of head and neck cancer cells with different levels of intracellular reactive oxygen species exhibit diverse stemness, proliferation, and chemosensitivity.

    PubMed

    Chang, Ching-Wen; Chen, Yu-Syuan; Chou, Shiu-Huey; Han, Chia-Li; Chen, Yu-Ju; Yang, Cheng-Chieh; Huang, Chih-Yang; Lo, Jeng-Fan

    2014-11-01

    Head and neck squamous cell carcinoma (HNSCC) is driven by cancer-initiating cells (CIC), but their maintenance mechanisms are obscure. For hematopoietic stem cells, low levels of intracellular reactive oxygen species (ROS(Low)) is known to help sustain stemness properties. In this report, we evaluated the hypothesis that ROS(Low) character conferred CIC properties in HNSCC. Sphere cultures define CIC in HNSCC cell populations (HN-CIC). We found that ROS(Low) cells in HN-CIC defined in this manner were more numerous than in parental HNSCC cells. Further, ROS(Low) cells frequently coexpressed CIC surface markers such as memGrp78 and Glut3. Exploiting flow cytometry to sort cells on the basis of their ROS level, we found that isolated ROS(Low) cells displayed relatively more CIC properties, including quiescence, chemoresistance, in vitro malignant properties, and tumorigenicity. Pharmacological depletion of ROS modulators in cisplatin-treated HN-CIC reduced CIC properties, enhancing cell differentiation and enhancing cisplatin-induced cell death. Overall, our work defined cell subpopulations in HNSCC on the basis of differential intracellular ROS levels, which associated with stemness and chemoresistance properties. On the basis of our findings, we suggest that strategies to promote intracellular ROS levels may heighten the efficacy of conventional chemotherapy used for HNSCC treatment. ©2014 American Association for Cancer Research.

  11. 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

  12. 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.

  13. Genome-wide transcriptomic and phylogenetic analyses reveal distinct aluminum-tolerance mechanisms in the aluminum-accumulating species buckwheat (Fagopyrum tataricum).

    PubMed

    Zhu, Haifeng; Wang, Hua; Zhu, Yifang; Zou, Jianwen; Zhao, Fang-Jie; Huang, Chao-Feng

    2015-01-21

    Similar to common buckwheat (Fagopyrum esculentum), tartary buckwheat (Fagopyrum tataricum) shows a high level of aluminum (Al) tolerance and accumulation. However, the molecular mechanisms for Al detoxification and accumulation are still poorly understood. To begin to elucidate the molecular basis of Al tolerance and accumulation, we used the Illumina high-throughput mRNA sequencing (RNA-seq) technology to conduct a genome-wide transcriptome analysis on both tip and basal segments of the roots exposed to Al. By using the Trinity method for the de novo assembly and cap3 software to reduce the redundancy and chimeras of the transcripts, we constructed 39,815 transcripts with an average length of 1184 bp, among which 20,605 transcripts were annotated by BLAST searches in the NCBI non-redundant protein database. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that expression of genes involved in the defense of cell wall toxicity and oxidative stress was preferentially induced by Al stress. Our RNA-seq data also revealed that organic acid metabolism was unlikely to be a rate-limiting step for the Al-induced secretion of organic acids in buckwheat. We identified two citrate transporter genes that were highly induced by Al and potentially involved in the release of citrate into the xylem. In addition, three of four conserved Al-tolerance genes were found to be duplicated in tartary buckwheat and display diverse expression patterns. Nearly 40,000 high quality transcript contigs were de novo assembled for tartary buckwheat, providing a reference platform for future research work in this plant species. Our differential expression and phylogenetic analysis revealed novel aspects of Al-tolerant mechanisms in buckwheat.

  14. 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.

  15. 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

  16. 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

  17. A new Sparassis species from Spain described using morphological and molecular data.

    PubMed

    Blanco-Dios, Jaime B; Wang, Zheng; Binder, Manfred; Hibbett, David S

    2006-10-01

    Sparassis miniensis, collected in Pinus pinaster forests in Galicia (northwest Iberian Peninsula) is described as a new species, based on morphological and molecular data. Sparassis miniensis is morphologically distinct from all other species in the genus Sparassis based on scattered flabellae, which are strongly laciniated, azonate, and arise from an orange to rose-purplish base. The sporadic presence of clamp connections is restricted to subhymenial hyphae. Molecular data from LSU-rDNA, ITS and partial gene coding RNA polymerase subunit II (rpb2) suggest a close relationship between the new species S. miniensis and S. brevipes, another European species producing large fruiting bodies but with entire flabellae and no clamp connections.

  18. Specific subunits of heterotrimeric G proteins play important roles during nodulation in soybean.

    PubMed

    Choudhury, Swarup Roy; Pandey, Sona

    2013-05-01

    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.

  19. 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.

  20. 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

  1. 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

  2. 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

  3. Subunit structure of the acetylcholine receptor from Electrophorus electricus.

    PubMed

    Conti-Tronconi, B M; Hunkapiller, M W; Lindstrom, J M; Raftery, M A

    1982-11-01

    The amino-terminal amino acid sequences of the four major peptides (Mr 41,000, 50,000, 55,000, and 62,000) present in purified preparations of Electrophorus electricus nicotinic acetylcholine receptor (AcChoR) have been determined for 24 cycles by automated sequence analysis procedures yielding four unique polypeptide sequences. The sequences showed a high degree of similarity, having identical residues in a number of positions ranging between 37% and 50% for specific pairs of subunits. Comparison of the sequences obtained with those of the subunits of similar molecular weight from Torpedo californica AcChoR revealed an even higher degree of homology (from 46% to 71%) for these two highly diverged species. Simultaneous sequence analysis of the amino termini present in native, purified Electrophorus AcChoR showed that these four related sequences were the only ones present and that they occur in a ratio of 2:1:1:1, with the smallest subunit ("alpha 1") being present in two copies. Genealogical analysis suggests that the subunits of both Torpedo and Electrophorus AcChoRs derive from a common ancestral gene, the divergence having occurred early in the evolution of the receptor. This shared ancestry and the very early divergence of the four subunits, as well as the highly conserved structure of the AcChoR complex along animal evolution, suggest that each of the subunits evolved to perform discrete crucial roles in the physiological function of the AcChoR.

  4. The land crabs of the Discoplax longipes A. Milne-Edwards, 1867 species group, with description of a new species from Guam (Crustacea: Decapoda: Brachyura: Gecarcinidae).

    PubMed

    Ng, Peter K L; Shih, Hsi-Te

    2015-06-30

    Specimens of the gecarcinid land crab Discoplax longipes A. Milne-Edwards, 1867, from the western Pacific, can be separated into two distinct groups on the basis of DNA (mitochondrial 16S rDNA and cytochrome oxidase subunit I) and structure of the male first gonopod. On the basis of this data, the material that occurs from the Loyalty Islands to French Polynesia is shown to be D. longipes s. str., whereas specimens from Guam are here referred to a new pseudocryptic species, D. michalis n. sp. The two species are described and figured; and a revised key to the long-legged Discoplax species is provided.

  5. 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

  6. Small subunit ribosomal DNA-based phylogenetic analysis of foliar nematodes (Aphelenchoides spp.) and their quantitative detection in complex DNA backgrounds.

    PubMed

    Rybarczyk-Mydłowska, Katarzyna; Mooyman, Paul; van Megen, Hanny; van den Elsen, Sven; Vervoort, Mariëtte; Veenhuizen, Peter; van Doorn, Joop; Dees, Robert; Karssen, Gerrit; Bakker, Jaap; Helder, Johannes

    2012-12-01

    Foliar nematodes, plant-parasitic representatives of the genus Aphelenchoides, constitute a minority in a group dominated by fungivorous species. Distinction between (mostly harmless) fungal feeding Aphelenchoides species and high impact plant parasites such as A. besseyi, A. fragariae, A. ritzemabosi, and A. subtenuis is severely hampered by the scarcity of informative morphological characters, some of which are only observable in specific developmental stages. Poor description of a number of non-plant-parasitic Aphelenchoides species further complicates identification. Based on (nearly) full-length small subunit ribosomal DNA (SSU rDNA) sequences (≈1,700 bp), a phylogenetic tree was generated, and the four target species appeared as distinct, well-supported groups. Notably, this genus does not constitute a monophyletic group: A. besseyi and A. ritzemabosi cluster together and they are phylogenetically isolated from A. fragariae, A. subtenuis, and most other fungivorous species. A phylum-wide SSU rDNA framework was used to identify species-specific DNA motifs. For the molecular detection of four plant-parasitic Aphelenchoides species, polymerase chain reaction primers were developed with high, identical annealing temperatures (63°C). Within the molecular framework presented here, these primers can be used for the rapid screening of plant material and soil for the presence of one or multiple foliar nematode species.

  7. 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

  8. 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).

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. Talaromyces rubrifaciens, a new species discovered from heating, ventilation and air conditioning systems in China.

    PubMed

    Luo, Yi; Lu, Xiaohong; Bi, Wu; Liu, Fan; Gao, Weiwei

    2016-01-01

    A new Talaromyces species, T. rubrifaciens, was isolated from supply air outlets of heating, ventilation and air conditioning (HVAC) systems in three kinds of public building in Beijing and Nanjing, China. Morphologically it exhibits many characters of section Trachyspermi but is distinguished from other species of this section by restricted growth and broad and strictly biverticillate conidiophores. Phylogenetic analyses based on the internal transcribed spacer rDNA (ITS), β-tubulin (BenA), calmodulin (CaM) and RNA polymerase second largest subunit (RPB2) genes reveal that T. rubrifaciens is a distinct species in section Trachyspermi. © 2016 by The Mycological Society of America.

  14. 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

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. Inactivation of the protein phosphatase 2A regulatory subunit A results in morphological and transcriptional defects in Saccharomyces cerevisiae.

    PubMed Central

    van Zyl, W; Huang, W; Sneddon, A A; Stark, M; Camier, S; Werner, M; Marck, C; Sentenac, A; Broach, J R

    1992-01-01

    We have determined that TPD3, a gene previously identified in a screen for mutants defective in tRNA biosynthesis, most likely encodes the A regulatory subunit of the major protein phosphatase 2A species in the yeast Saccharomyces cerevisiae. The predicted amino acid sequence of the product of TPD3 is highly homologous to the sequence of the mammalian A subunit of protein phosphatase 2A. In addition, antibodies raised against Tpd3p specifically precipitate a significant fraction of the protein phosphatase 2A activity in the cell, and extracts of tpd3 strains yield a different chromatographic profile of protein phosphatase 2A than do extracts of isogenic TPD3 strains. tpd3 deletion strains generally grow poorly and have at least two distinct phenotypes. At reduced temperatures, tpd3 strains appear to be defective in cytokinesis, since most cells become multibudded and multinucleate following a shift to 13 degrees C. This is similar to the phenotype obtained by overexpression of the protein phosphatase 2A catalytic subunit or by loss of CDC55, a gene that encodes a protein with homology to a second regulatory subunit of protein phosphatase 2A. At elevated temperatures, tpd3 strains are defective in transcription by RNA polymerase III. Consistent with this in vivo phenotype, extracts of tpd3 strains fail to support in vitro transcription of tRNA genes, a defect that can be reversed by addition of either purified RNA polymerase III or TFIIIB. These results reinforce the notion that protein phosphatase 2A affects a variety of biological processes in the cell and provide an initial identification of critical substrates for this phosphatase. Images PMID:1328868

  20. 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.

  1. 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

  2. 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

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

    PubMed

    Bicknell, Brendan A; Goodhill, Geoffrey J

    2016-09-06

    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 Ca(2+) 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.

  4. 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

  5. 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

  6. 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.

  7. Subunit asymmetry in the three-dimensional structure of a human CuZnSOD mutant found in familial amyotrophic lateral sclerosis.

    PubMed Central

    Hart, P. J.; Liu, H.; Pellegrini, M.; Nersissian, A. M.; Gralla, E. B.; Valentine, J. S.; Eisenberg, D.

    1998-01-01

    The X-ray crystal structure of a human copper/zinc superoxide dismutase mutant (G37R CuZnSOD) found in some patients with the inherited form of Lou Gehrig's disease (FALS) has been determined to 1.9 angstroms resolution. The two SOD subunits have distinct environments in the crystal and are different in structure at their copper binding sites. One subunit (subunit[intact]) shows a four-coordinate ligand geometry of the copper ion, whereas the other subunit (subunit[broken]) shows a three-coordinate geometry of the copper ion. Also, subunit(intact) displays higher atomic displacement parameters for backbone atoms ((B) = 30 +/- 10 angstroms2) than subunit(broken) ((B) = 24 +/- 11 angstroms2). This structure is the first CuZnSOD to show large differences between the two subunits. Factors that may contribute to these differences are discussed and a possible link of a looser structure to FALS is suggested. PMID:9541385

  8. 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.

  9. 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

  10. 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.

  11. Subunit structure of the acetylcholine receptor from Electrophorus electricus.

    PubMed Central

    Conti-Tronconi, B M; Hunkapiller, M W; Lindstrom, J M; Raftery, M A

    1982-01-01

    The amino-terminal amino acid sequences of the four major peptides (Mr 41,000, 50,000, 55,000, and 62,000) present in purified preparations of Electrophorus electricus nicotinic acetylcholine receptor (AcChoR) have been determined for 24 cycles by automated sequence analysis procedures yielding four unique polypeptide sequences. The sequences showed a high degree of similarity, having identical residues in a number of positions ranging between 37% and 50% for specific pairs of subunits. Comparison of the sequences obtained with those of the subunits of similar molecular weight from Torpedo californica AcChoR revealed an even higher degree of homology (from 46% to 71%) for these two highly diverged species. Simultaneous sequence analysis of the amino termini present in native, purified Electrophorus AcChoR showed that these four related sequences were the only ones present and that they occur in a ratio of 2:1:1:1, with the smallest subunit ("alpha 1") being present in two copies. Genealogical analysis suggests that the subunits of both Torpedo and Electrophorus AcChoRs derive from a common ancestral gene, the divergence having occurred early in the evolution of the receptor. This shared ancestry and the very early divergence of the four subunits, as well as the highly conserved structure of the AcChoR complex along animal evolution, suggest that each of the subunits evolved to perform discrete crucial roles in the physiological function of the AcChoR. Images PMID:6959131

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

    PubMed

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

    2017-05-15

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

  13. Stoichiometry of δ subunit containing GABAA receptors

    PubMed Central

    Patel, B; Mortensen, M; Smart, T G

    2014-01-01

    Background and Purpose Although the stoichiometry of the major synaptic αβγ subunit-containing GABAA receptors has consensus support for 2α:2β:1γ, a clear view of the stoichiometry of extrasynaptic receptors containing δ subunits has remained elusive. Here we examine the subunit stoichiometry of recombinant α4β3δ receptors using a reporter mutation and a functional electrophysiological approach. Experimental Approach Using site-directed mutagenesis, we inserted a highly characterized 9′ serine to leucine mutation into the second transmembrane (M2) region of α4, β3 and δ subunits that increases receptor sensitivity to GABA. Whole-cell, GABA-activated currents were recorded from HEK-293 cells co-expressing different combinations of wild-type (WT) and/or mutant α4(L297S), β3(L284S) and δ(L288S) subunits. Key Results Recombinant receptors containing one or more mutant subunits showed increased GABA sensitivity relative to WT receptors by approximately fourfold, independent of the subunit class (α, β or δ) carrying the mutation. GABA dose–response curves of cells co-expressing WT subunits with their respective L9′S mutants exhibited multiple components, with the number of discernible components enabling a subunit stoichiometry of 2α, 2β and 1δ to be deduced for α4β3δ receptors. Varying the cDNA transfection ratio by 10-fold had no significant effect on the number of incorporated δ subunits. Conclusions and Implications Subunit stoichiometry is an important determinant of GABAA receptor function and pharmacology, and δ subunit-containing receptors are important mediators of tonic inhibition in several brain regions. Here we demonstrate a preferred subunit stoichiometry for α4β3δ receptors of 2α, 2β and 1δ. PMID:24206220

  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. 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.

  17. 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

  18. 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.

  19. 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

  20. Phylogenetic position of Gromia oviformis Dujardin inferred from nuclear-encoded small subunit ribosomal DNA.

    PubMed

    Burki, Fabien; Berney, Cédric; Pawlowski, Jan

    2002-09-01

    Gromia oviformis Dujardin is a common marine protist characterised by a large, globular test and filose pseudopodia. First considered a foraminifer, Gromia was later placed within the Filosea and recently included among amoebae of uncertain affinities. In order to clarify the phylogenetic position of this genus, we sequenced the complete small-subunit ribosomal DNA gene of G. oviformis collected at five different geographic localities. The high divergence of obtained sequences suggests that G. oviformis is a species complex composed of several genetically distinct sibling species. Sequence analyses show Gromia to be a member of the Cercozoa, a heterogeneous assemblage which includes filose amoebae, the amoeboflagellate cercomonads, the chlorarachniophytes and the plasmodiophorid plant pathogens. Contrary to traditional classification, Gromia is not closely related to other testate filose amoebae (the Euglyphida), but seems to branch early among the Cercozoa. Our analyses also show a close relationship between the Cercozoa and the Acantharea. Because the Cercozoa are related to the Foraminifera based on other molecular data, we propose that most protists possessing filopodia, reticulopodia and axopodia have a common origin.

  1. 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

  2. Folding of active calcium channel beta(1b) -subunit by size-exclusion chromatography and its role on channel function.

    PubMed

    Neely, Alan; Garcia-Olivares, Jennie; Voswinkel, Stephan; Horstkott, Hannelore; Hidalgo, Patricia

    2004-05-21

    Voltage-gated calcium channels mediate the influx of Ca(2+) ions into eukaryotic cells in response to membrane depolarization. They are hetero-multimer membrane proteins formed by at least three subunits, the poreforming alpha(1)-subunit and the auxiliary beta- and alpha(2)delta-subunits. The beta-subunit is essential for channel performance because it regulates two distinct features of voltage-gated calcium channels, the surface expression and the channel activity. Four beta-subunit genes have been cloned, beta(1-4), with molecular masses ranging from 52 to 78 kDa, and several splice variants have been identified. The beta(1b)-subunit, expressed at high levels in mammalian brain, has been used extensively to study the interaction between the pore forming alpha(1)- and the regulatory beta-subunit. However, structural characterization has been impaired for its tendency to form aggregates when expressed in bacteria. We applied an on-column refolding procedure based on size exclusion chromatography to fold the beta(1b)-subunit of the voltage gated-calcium channels from Escherichia coli inclusion bodies. The beta(1b)-subunit refolds into monomers, as shown by sucrose gradient analysis, and binds to a glutathione S-transferase protein fused to the known target in the alpha(1)-subunit (the alpha-interaction domain). Using the cut-open oocyte voltage clamp technique, we measured gating and ionic currents in Xenopus oocytes expressing cardiac alpha(1)-subunit (alpha(1C)) co-injected with folded-beta(1b)-protein or beta(1b)-cRNA. We demonstrate that the co-expression of the alpha(1C)-subunit with either folded-beta(1b)-protein or beta(1b)-cRNA increases ionic currents to a similar extent and with no changes in charge movement, indicating that the beta(1b)-subunit primarily modulates channel activity, rather than expression.

  3. Subunit Compositions of the RNA-Silencing Enzymes Pol IV and Pol V Reveal Their Origins as Specialized Forms of RNA Polymerase II

    SciTech Connect

    Ream, Thomas S.; Haag, J. R.; Wierzbicki, A. T.; Nicora, Carrie D.; Norbeck, Angela D.; Zhu, Jian-Kang; Hagen, Gretchen; Guilfoyle, Thomas J.; Pasa-Tolic, Ljiljana; Pikaard, Craig S.

    2009-01-30

    In addition to RNA polymerases I, II, and III, the essential RNA polymerases present in all eukaryotes, plants have two additional nuclear RNA polymerases, abbreviated as Pol IV and Pol V, that play nonredundant roles in siRNA-directed DNA methylation and gene silencing. We show that Arabidopsis Pol IV and Pol V are composed of subunits that are paralogous or identical to the 12 subunits of Pol II. Four subunits of Pol IV are distinct from their Pol II paralogs, six subunits of Pol V are distinct from their Pol II paralogs, and four subunits differ between Pol IV and Pol V. Importantly, the subunit differences occur in key positions relative to the template entry and RNA exit paths. Our findings support the hypothesis that Pol IV and Pol V are Pol II-like enzymes that evolved specialized roles in the production of noncoding transcripts for RNA silencing and genome defense.

  4. Developmental expression of human hemoglobins mediated by maturation of their subunit interfaces

    PubMed Central

    Manning, Lois R; Popowicz, Anthony M; Padovan, Julio; Chait, Brian T; Russell, J Eric; Manning, James M

    2010-01-01

    Different types of human hemoglobins (Hbs) consisting of various combinations of the embryonic, fetal, and adult Hb subunits are present at certain times during development representing a major paradigm of developmental biology that is still not understood and one which we address here. We show that the subunit interfaces of these Hbs have increasing bonding strengths as demonstrated by their distinct distribution of tetramers, dimers, and monomers during gel filtration at very low-Hb concentration. This maturation is mediated by competition between subunits for more favorable partners with stronger subunit interactions. Thus, the protein products of gene expression can themselves have a role in the developmental process due to their intrinsic properties. PMID:20572018

  5. The subunit composition of hinokiresinol synthase controls geometrical selectivity in norlignan formation

    PubMed Central

    Suzuki, Shiro; Yamamura, Masaomi; Hattori, Takefumi; Nakatsubo, Tomoyuki; Umezawa, Toshiaki

    2007-01-01

    The selective formation of E- or Z-isomers is an important process in natural product metabolism. We show that the subunit composition of an enzyme can alter the geometrical composition of the enzymatic products. Hinokiresinol synthase, purified from Asparagus officinalis cell cultures, is responsible for the conversion of (7E,7′E)-4-coumaryl 4-coumarate to (Z)-hinokiresinol, the first step in norlignan formation. The protein is most likely a heterodimer composed of two distinct subunits, which share identity with members of the phloem protein 2 gene superfamily. Interestingly, each recombinant subunit of hinokiresinol synthase expressed in Escherichia coli solely converted (7E,7′E)-4-coumaryl 4-coumarate to the unnatural (E)-hinokiresinol, the E-isomer of (Z)-hinokiresinol. By contrast, a mixture of recombinant subunits catalyzed the formation of (Z)-hinokiresinol from the same substrate. PMID:18093914

  6. 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

  7. Mitochondrial and cytoplasmic ribosomes from mammalian tissues. Further characterization of ribosomal subunits and validity of buoyant-density methods for determination of the chemical composition and partial specific volume of ribonucleoprotein particles

    PubMed Central

    Sacchi, Ada; Ferrini, Ugo; Londei, Paola; Cammarano, Piero; Maraldi, Nadir

    1977-01-01

    1. At 0–4°C mitochondrial ribosomes (55S) dissociate into 39S and 29S subunits after exposure to 300mm-K+ in the presence of 3.0mm-Mg2+. When these subunits are placed in a medium containing a lower concentration of K+ ions (25mm), approx. 75% of the subparticles recombine giving 55S monomers. 2. After negative staining the large subunits (20.3nm width) usually show a roundish profile, whereas the small subunits (12nm width) show an elongated, often bipartite, profile. The dimensions of the 55S ribosomes are 25.5nm×20.0nm×21.0nm, indicating a volume ratio of mitochondrial to cytosol ribosomes of 1:1.5. 3. The 39S and 29S subunits obtained in high-salt media at 0–4°C have a buoyant density of 1.45g/cm3; from the rRNA content calculated from buoyant density and from the rRNA molecular weights it is confirmed that the two subparticles have weights of 2.0×106 daltons and 1.20×106 daltons; the weights of the two subunits of cytosol ribosomes are 2.67×106 and 1.30×106 daltons. 4. The validity of the isodensity-equilibrium-centrifugation methods used to calculate the chemical composition of ribosomes was reinvestigated; it is confirmed that (a) reaction of ribosomal subunits with 6.0% (v/v) formaldehyde at 0°C is sufficient to fix the particles, so that they remain essentially stable after exposure to dodecyl sulphate or centrifugation in CsCl, and (b) the partial specific volume of ribosomal subunits is a simple additive function of the partial specific volumes of RNA and protein. The RNA content is linearly related to buoyant density by the equation RNA (% by wt.)=349.5−(471.2×1/ρCsCl), where 1/ρCsCl=[unk]RNP (partial specific volume of ribonucleoprotein). 5. The nucleotide compositions of the two subunit rRNA species of mitochondrial ribosomes from rodents (42% and 43% G+C) are distinctly different from those of cytoplasmic ribosomes. ImagesPLATE 1PLATE 2 PMID:563718

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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.

  16. The taxonomic status and sister group relationship of the cardinalfish species Jaydia striatodes (Percomorphaceae: Apogonidae).

    PubMed

    Yu, Zhengsen; Song, Na; Han, Zhiqiang; Gao, Tianxiang; Shui, Bonian; Gon, Ofer

    2016-10-12

    Two similar cardinalfish species, Jaydia striatodes and J. striata, were compared morphologically and genetically, using the fragment of cytochrome oxidase subunit I (COI) gene of the mitochondrial DNA. The results confirmed the validity of both species and their sister group relationship. The species formed well-supported monophyletic clades that were distinctly separate with mean sequence divergence of 12.2%. Jaydia striatodes is distinct in having 4-5 + 12-13 gill rakers; 3 + 11-12 developed gill rakers; 9 gill rakers on the first ceratobranchial; 3-9 weak serration at the angle of preopercular edge; and a usually blackish distal half of anal fin. Jaydia striatodes was recorded for the first time from Beibu Gulf, China, and from Vietnam.

  17. Voltage-gated sodium channel β subunits: The power outside the pore in brain development and disease.

    PubMed

    Hull, Jacob M; Isom, Lori L

    2017-09-16

    Voltage gated sodium channels (VGSCs) were first identified in terms of their role in the upstroke of the action potential. The underlying proteins were later identified as saxitoxin and scorpion toxin receptors consisting of α and β subunits. We now know that VGSCs are heterotrimeric complexes consisting of a single pore forming α subunit joined by two β subunits; a noncovalently linked β1 or β3 and a covalently linked β2 or β4 subunit. VGSC α subunits contain all the machinery necessary for channel cell surface expression, ion conduction, voltage sensing, gating, and inactivation, in one central, polytopic, transmembrane protein. VGSC β subunits are more than simple accessories to α subunits. In the more than two decades since the original cloning of β1, our knowledge of their roles in physiology and pathophysiology has expanded immensely. VGSC β subunits are multifunctional. They confer unique gating mechanisms, regulate cellular excitability, affect brain development, confer distinct channel pharmacology, and have functions that are independent of the α subunits. The vast array of functions of these proteins stems from their special station in the channelome: being the only known constituents that are cell adhesion and intra/extracellular signaling molecules in addition to being part of channel complexes. This functional trifecta and how it goes awry demonstrates the power outside the pore in ion channel signaling complexes, broadening the term channelopathy beyond defects in ion conduction. Copyright © 2017. Published by Elsevier Ltd.

  18. 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

  19. Diversity of ribulose-1,5-bisphosphate carboxylase/oxygenase large-subunit genes from groundwater and aquifer microorganisms.

    PubMed

    Alfreider, A; Vogt, C; Hoffmann, D; Babel, W

    2003-05-01

    To test our hypothesis that microbial autotrophic CO2 fixation plays an important role in subsurface systems of two large groundwater remediation projects, several anaerobic/microaerobic aquifer and groundwater samples were taken and used to investigate the distribution and phylogenetic diversity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large-subunit genes. Two primer sets were designed for amplifying partial-subunit genes of RubisCO forms I and II from the DNA, directly extracted from the samples. PCR products were used to construct five clone libraries with putative RubisCO form I sequences, and two libraries of DNA amplified by form II primers. Selected clones were screened for variation by restriction fragment length polymorphism analysis, and a total of 28 clone inserts were sequenced and further analyzed. The phylogenies constructed from amino acid sequences derived from the partial RubisCO large-subunit sequences showed a distinct pattern. Diverse sequences affiliated to the cluster of green-like type IA RubisCO sequences were found, representing various obligate and facultative chemolithoautotrophic Proteobacteria, whereas type II RubisCO sequences detected were most closely related to those of thiobacilli species. An isolate obtained from aquifer enrichment culture, which has been provisionally named Halothiobacillus sp. RA13 on the basis of its 16S rDNA sequence, was found to contain both types of RubisCO genes, i.e., forms I and II. Physiological and ecological considerations are discussed in the context of additional microbial data and physicochemical properties.

  20. NADH dehydrogenase subunit genes in the mitochondrial DNA of yeasts.

    PubMed Central

    Nosek, J; Fukuhara, H

    1994-01-01

    The genes encoding the NADH dehydrogenase subunits of respiratory complex I have not been identified so far in the mitochondrial DNA (mtDNA) of yeasts. In the linear mtDNA of Candida parapsilosis, we found six new open reading frames whose sequences were unambiguously homologous to those of the genes known to code for NADH dehydrogenase subunit proteins of different organisms, i.e., ND1, ND2, ND3, ND4L, ND5, and ND6. The gene for ND4 also appears to be present, as judged from hybridization experiments with a Podospora gene probe. Specific transcripts from these open reading frames (ND genes) could be detected in the mitochondria. Hybridization experiments using C. parapsilosis genes as probes suggested that ND genes are present in the mtDNAs of a wide range of yeast species including Candida catenulata, Pichia guilliermondii, Clavispora lusitaniae, Debaryomyces hansenii, Hansenula polymorpha, and others. Images PMID:7521869

  1. 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

  2. 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

  3. Three New Soil-inhabiting Species of Trichoderma in the Stromaticum Clade with Test of Their Antagonism to Pathogens.

    PubMed

    Chen, Kai; Zhuang, Wen-Ying

    2017-09-01

    Trichoderma is a dominant component of the soil mycoflora. During the field investigations of northern, central, and southwestern China, three new species in the Stromaticum clade were encountered from soil, and named as T. hebeiense, T. sichuanense, and T. verticillatum. Their phylogenetic positions were determined by analyses of the combined two genes: partial sequences of translation elongation factor 1-alpha and the second largest RNA polymerase subunit-encoding genes. Distinctions between the new species and their close relatives were discussed. Trichoderma hebeiense appeared as a separate terminal branch. The species is distinctive by its oblong conidia and aggregated pustules in culture. Trichoderma sichuanense features in concentric colony and produces numerous clean exudates on aerial mycelium in culture. Trichoderma verticillatum is characterized by its verticillium-like synanamorph and production of abundant chlamydospores. In vitro antagonism towards the new species was tested by dual culture technique.

  4. 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

  5. 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...

  6. Spectroscopic properties of Callinectes sapidus hemocyanin subunits

    NASA Astrophysics Data System (ADS)

    Stoeva, Stanka; Dolashka, Pavlina; Bankov, Banko; Voelter, Wolfgang; Salvato, Benedeto; Genov, Nicolay

    1995-10-01

    The two major subunits of the Callinectes sapidus hemocyanin were isolated and characterized by spectroscopic techniques. They consist of 641 and 652 residues, respectively. Circular dichroism spectra showed that the structural integrity of the isolated polypeptide chains is preserved. Tryptophan fluorescence parameters were determined for the hemocyanin aggregates and for the subunits Cs1 and Cs2. The emitting tryptophyl fluorophores in the native hemocyanin are deeply buried in hydrophobic regions and are shielded from the solvent by the quaternary structure of the protein aggregates. In two subunits, obtained after dissociation of the aggregates, these residues become "exposed". It is concluded that the tryptophyl side chains in Cs1 and Cs2 are located in subunit interfaces (contact regions) in a negatively charged environment when the polypeptide chains are aggregated. Most probably they participate in hydrophobic protein-protein interactions. The environment of these fluorophores is more negatively charged after the dissociation of the aggregates to subunits.

  7. 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

  8. Malassezia nana sp. nov., a novel lipid-dependent yeast species isolated from animals.

    PubMed

    Hirai, Asuka; Kano, Rui; Makimura, Koichi; Duarte, Eduardo Robson; Hamdan, Júnia Soares; Lachance, Marc-André; Yamaguchi, Hideyo; Hasegawa, Atsuhiko

    2004-03-01

    Five isolates of a novel species of the yeast genus Malassezia were isolated from animals in Japan and Brazil. Phylogenetic trees based on the D1/D2 domains of the large-subunit (26S) rDNA sequences and nucleotide sequences of the internal transcribed spacer 1 region showed that the isolates were conspecific and belonged to the genus Malassezia. They were related closely to Malassezia dermatis and Malassezia sympodialis, but were clearly distinct from these two species and the other six species of Malassezia that have been reported, indicating that they should be classified as a novel species, Malassezia nana sp. nov. Morphologically and physiologically, M. nana resembles M. dermatis and M. sympodialis, but can be distinguished from these species by its inability to use Cremophor EL (Sigma) as the sole lipid source and to hydrolyse aesculin. The type strain of M. nana is NUSV 1003(T) (=CBS 9557(T)=JCM 12085(T)).

  9. 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.

  10. 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

  11. 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.

  12. The herpes zoster subunit vaccine.

    PubMed

    Cunningham, Anthony L

    2016-01-01

    Herpes zoster (HZ) causes severe pain and rash in older people and may be complicated by prolonged pain (postherpetic neuralgia; PHN). HZ results from reactivation of latent varicella-zoster virus (VZV) infection, often associated with age related or other causes of decreased T cell immunity. A concentrated live attenuated vaccine boosts this immunity and provides partial protection against HZ, but this decreases with age and declines over 5-8 years. The new HZ subunit (HZ/su or Shingrix) vaccine combines a key surface VZV glycoprotein (E) with T cell boosting adjuvant (AS01B). It is highly efficacious in protection (97%) against HZ in immunocompetent subjects, with no decline in advancing age and protection maintained for >3 years. Phase I-II trials showed safety and similar immunogenicity in severely immunocompromised patients. Local injection site pain and swelling can be severe in a minority (9.5%) but is transient (2 days). The HZ/su vaccine appears very promising in immunocompetent patients in the ZoE-50 controlled trial. The unblinding of the current ZoE-50 trial and publication of results from the accompanying ZoE-70 trial will reveal more about its mechanism of action and its efficacy against PHN, particularly in subjects >70 years. Phase III trial results in immunocompromised patients are eagerly awaited.

  13. 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.

  14. [Co-expression of beta-subunit with other subunits of Qbeta replicase].

    PubMed

    Wang, Dong

    2004-12-01

    In researches involving in vitro protein synthesis and self-replication system, Qbeta replicase is one of the key enzymes, which are demanded for the high availability. Qbeta replicase is a RNA-dependent RNA polymerase of Qbeta coliphage. It consists of four subunits (alpha, beta, gamma, and delta subunit), where the beta-subunit is encoded by the viral genome, while the other three subunits are host proteins normally involved in protein synthesis, namely, ribosomal protein S1 (alpha), elongation factors EF-Tu (gamma) and EF-Ts (delta). To increase the production of the Qbeta replicase holoenzyme, several types of expression vectors, including pKK, pET and others, were employed to produce Qbeta replicase. However, the beta-subunit was almost in the precipitate fraction. Considering that the four subunits of Qbeta rep