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1
Akada, R., K. Minomi, J. Kai, I. Yamashita, T. Miyakawa, and S. Fukui. "Multiple genes coding for precursors of rhodotorucine A, a farnesyl peptide mating pheromone of the basidiomycetous yeast Rhodosporidium toruloides." Molecular and Cellular Biology 9, no. 8 (August 1989): 3491–98. http://dx.doi.org/10.1128/mcb.9.8.3491-3498.1989.
Повний текст джерелаАнотація:
Haploid cells of mating type A of the basidiomycetous yeast Rhodosporidium toruloides secrete a mating pheromone, rhodotorucine A, which is an undecapeptide containing S-farnesyl cysteine at its carboxy terminus. To analyze the processing and secretion pathway of rhodotorucine A, we isolated both genomic and complementary DNAs encoding the peptide moiety. We identified three distinct genes, RHA1, RHA2, and RHA3, encoding four, five, and three copies of the pheromone peptide, respectively. Complementary DNA clones were classified into two types. One type was homologous to RHA1, and the other type was homologous to RHA2. Transcription start sites were identified by primer extension and S1 nuclease protection, from which the site of the initiator methionine was verified. A primary precursor of rhodotorucine A was detected as a 7-kilodalton protein by immunoprecipitation of in vitro translation products. On the basis of these results, we propose similar three-precursor structures of rhodotorucine A, each containing the amino-terminal peptide sequence Met-Val-Ala. The precursors contain three, four, or five tandem repeats of the pheromone peptide, each separated by a spacer peptide, Thr-Val-Ser(Ala)-Lys, and each precursor has the carboxy-terminal sequence Thr-Val-Ala. This structure suggests that primary precursors of rhodotorucine A do not contain canonical signal sequences.
2
Akada, R., K. Minomi, J. Kai, I. Yamashita, T. Miyakawa, and S. Fukui. "Multiple genes coding for precursors of rhodotorucine A, a farnesyl peptide mating pheromone of the basidiomycetous yeast Rhodosporidium toruloides." Molecular and Cellular Biology 9, no. 8 (August 1989): 3491–98. http://dx.doi.org/10.1128/mcb.9.8.3491.
Повний текст джерелаАнотація:
Haploid cells of mating type A of the basidiomycetous yeast Rhodosporidium toruloides secrete a mating pheromone, rhodotorucine A, which is an undecapeptide containing S-farnesyl cysteine at its carboxy terminus. To analyze the processing and secretion pathway of rhodotorucine A, we isolated both genomic and complementary DNAs encoding the peptide moiety. We identified three distinct genes, RHA1, RHA2, and RHA3, encoding four, five, and three copies of the pheromone peptide, respectively. Complementary DNA clones were classified into two types. One type was homologous to RHA1, and the other type was homologous to RHA2. Transcription start sites were identified by primer extension and S1 nuclease protection, from which the site of the initiator methionine was verified. A primary precursor of rhodotorucine A was detected as a 7-kilodalton protein by immunoprecipitation of in vitro translation products. On the basis of these results, we propose similar three-precursor structures of rhodotorucine A, each containing the amino-terminal peptide sequence Met-Val-Ala. The precursors contain three, four, or five tandem repeats of the pheromone peptide, each separated by a spacer peptide, Thr-Val-Ser(Ala)-Lys, and each precursor has the carboxy-terminal sequence Thr-Val-Ala. This structure suggests that primary precursors of rhodotorucine A do not contain canonical signal sequences.
3
Coelho, Marco A., André Rosa, Nádia Rodrigues, Álvaro Fonseca, and Paula Gonçalves. "Identification of Mating Type Genes in the Bipolar Basidiomycetous Yeast Rhodosporidium toruloides: First Insight into the MAT Locus Structure of the Sporidiobolales." Eukaryotic Cell 7, no. 6 (April 11, 2008): 1053–61. http://dx.doi.org/10.1128/ec.00025-08.
Повний текст джерелаАнотація:
ABSTRACT Rhodosporidium toruloides is a heterothallic, bipolar, red yeast that belongs to the Sporidiobolales, an order within a major lineage of basidiomycetes, the Pucciniomycotina. In contrast to other basidiomycetes, considerably less is known about the nature of the mating type (MAT) loci that control sexual reproduction in this lineage. Three genes (RHA1, RHA2, and RHA3) encoding precursors of the MAT A1 pheromone (rhodotorucine A) were previously identified and formed the basis for a genome walking approach that led to the identification of additional MAT genes in complementary mating strains of R. toruloides. Two mating type-specific alleles encoding a p21-activated kinase (PAK; Ste20 homolog) were found between the RHA2 and RHA3 genes, and identification in MAT A2 strains of a gene encoding a presumptive pheromone precursor enabled prediction of the structure of rhodotorucine a. In addition, a putative pheromone receptor gene (STE3 homolog) was identified upstream of RHA1. Analyses of genomic data from two closely related species, Sporobolomyces roseus and Sporidiobolus salmonicolor, identified syntenic regions that contain homologs of all the above-mentioned genes. Notably, six novel pheromone precursor genes were uncovered, which encoded, similarly to the RHA genes, multiple tandem copies of the peptide moiety. This suggests that this structure, which is unique among fungal lipopeptide pheromones, seems to be prevalent in red yeasts. Species comparisons provided evidence for a large, multigenic MAT locus structure in the Sporidiobolales, but no putative homeodomain transcription factor genes (which are present in all basidiomycetous MAT loci characterized thus far) could be found in any of the three species in the vicinity of the MAT genes identified.
4
Nguyen, Duong. "EFFECT OF DIFFERENT TYPES OF RICE HUSK ASH ON SOME GEOTECHNICAL PROPERTIES OF CEMENT-ADMIXED SOIL." Iraqi Geological Journal 53, no. 2C (September 30, 2020): 1–12. http://dx.doi.org/10.46717/igj.53.2c.1rs-2020-09-01.
Повний текст джерелаАнотація:
Rice husk ash (RHA) is an agricultural residue and has shown great potential for soil stabilization. However, the research on the utilization of RHA for soft soil improvement using cement deep mixing method is still limited and the efficiency of using different RHA types for soil improvement needs to be clarified. In this study, the effect of different RHA types on Atterberg limits, unconfined compressive strength (UCS), and elastic modulus (E50) of soil-cement mixtures will be investigated. Two types of RHA which obtained from open fire burning (RHA1) and burning in a furnace under controlled conditions of temperature and duration of burning (RHA2), were used for this study. The RHA contents from 0 to 15% and 10% cement of the dry weight of the soil were used to treat the soft soil. The research results show that the types of RHA insignificantly affect the change in Atterberg limits of cement-admixed soil. Regarding the soil strength, the RHA2 shows a higher efficiency in the enhancement of treated soil strength at 28 days of curing than the RHA1. The addition of 12% RHA2 to the cement-admixed soil can increase the UCS and E50 values of treated soil by more than 50%.
5
Bhende, Prasanna M., and Susan M. Egan. "Amino Acid-DNA Contacts by RhaS: an AraC Family Transcription Activator." Journal of Bacteriology 181, no. 17 (September 1, 1999): 5185–92. http://dx.doi.org/10.1128/jb.181.17.5185-5192.1999.
Повний текст джерелаАнотація:
ABSTRACT RhaS, an AraC family protein, activates rhaBADtranscription by binding to rhaI, a site consisting of two 17-bp inverted repeat half-sites. In this work, amino acids in RhaS that make base-specific contacts with rhaI were identified. Sequence similarity with AraC suggested that the first contacting motif of RhaS was a helix-turn-helix. Assays of rhaB-lacZactivation by alanine mutants within this potential motif indicated that residues 201, 202, 205, and 206 might contact rhaI. The second motif was identified based on the hypothesis that a region of especially high amino acid similarity between RhaS and RhaR (another AraC family member) might contact the nearly identical DNA sequences in one major groove of their half-sites. We first made targeted, random mutations and then made alanine substitutions within this region of RhaS. Our analysis identified residues 247, 248, 250, 252, 253, and 254 as potentially important for DNA binding. A genetic loss-of-contact approach was used to identify whether any of the RhaS amino acids in the first or second contacting motif make base-specific DNA contacts. In motif 1, we found that Arg202 and Arg206 both make specific contacts with bp −65 and −67 in rhaI 1, and that Arg202 contacts −46 and Arg206 contacts −48 inrhaI 2. In motif 2, we found that Asp250 and Asn252 both contact the bp −79 in rhaI 1. Alignment with the recently crystallized MarA protein suggest that both RhaS motifs are likely helix-turn-helix DNA-binding motifs.
6
Christova, Nelly, Boryana Tuleva, Rashel Cohen, Galya Ivanova, Georgy Stoev, Margarita Stoilova-Disheva, and Ivanka Stoineva. "Chemical Characterization and Physical and Biological Activities of Rhamnolipids Produced by Pseudomonas aeruginosa BN10." Zeitschrift für Naturforschung C 66, no. 7-8 (August 1, 2011): 394–402. http://dx.doi.org/10.1515/znc-2011-7-811.
Повний текст джерелаАнотація:
Pseudomonas aeruginosa BN10 isolated from hydrocarbon-polluted soil was found to produce rhamnolipids when cultivated on 2% glycerol, glucose, n-hexadecane, and n-alkanes. The rhamnolipids were partially purified on silica gel columns and their chemical structures elucidated by combination of one- and two-dimensional 1H and 13C NMR techniques and ESI-MS analysis. Eight structural rhamnolipid homologues were identified: Rha-C10- C8, Rha-C10-C10, Rha-C10-C12:1, Rha-C10-C12, Rha2-C10-C8, Rha2-C10-C10, Rha2-C10-C12:1, and Rha2-C10-C12. The chemical composition of the rhamnolipid mixtures produced on different carbon sources did not vary with the type of carbon source used. The rhamnolipid mixture produced by Pseudomonas aeruginosa BN10 on glycerol reduced the surface tension of pure water from 72 to 29 mN m-1 at a critical micellar concentration of 40 mg l-1, and the interfacial tension was 0.9 mN m-1. The new surfactant product formed stable emulsions with hydrocarbons and showed high antimicrobial activity against Gram-positive bacteria. The present study shows that the new strain Pseudomonas aeruginosa BN10 demonstrates enhanced production of the di-rhamnolipid Rha2-C10-C10 on all carbon sources used. Due to its excellent surface and good antimicrobial activities the rhamnolipid homologue mixture from Pseudomonas aeruginosa BN10 can be exploited for use in bioremediation, petroleum and pharmaceutical industries.
7
Wickstrum, Jason R., Jeff M. Skredenske, Vinitha Balasubramaniam, Kyle Jones, and Susan M. Egan. "The AraC/XylS Family Activator RhaS Negatively Autoregulates rhaSR Expression by Preventing Cyclic AMP Receptor Protein Activation." Journal of Bacteriology 192, no. 1 (October 23, 2009): 225–32. http://dx.doi.org/10.1128/jb.00829-08.
Повний текст джерелаАнотація:
ABSTRACT The Escherichia coli RhaR protein activates expression of the rhaSR operon in the presence of its effector, l-rhamnose. The resulting RhaS protein (plus l-rhamnose) activates expression of the l-rhamnose catabolic and transport operons, rhaBAD and rhaT, respectively. Here, we further investigated our previous finding that rhaS deletion resulted in a threefold increase in rhaSR promoter activity, suggesting RhaS negative autoregulation of rhaSR. We found that RhaS autoregulation required the cyclic AMP receptor protein (CRP) binding site at rhaSR and that RhaS was able to bind to the RhaR binding site at rhaSR. In contrast to the expected repression, we found that in the absence of both RhaR and the CRP binding site at the rhaSR promoter, RhaS activated expression to a level comparable with RhaR activation of the same promoter. However, when the promoter included the RhaR and CRP binding sites, the level of activation by RhaS and CRP was much lower than that by RhaR and CRP, suggesting that CRP could not fully coactivate with RhaS. Taken together, our results indicate that RhaS negative autoregulation involves RhaS competition with RhaR for binding to the RhaR binding site at rhaSR. Although RhaS and RhaR activate rhaSR transcription to similar levels, CRP cannot effectively coactivate with RhaS. Therefore, once RhaS reaches a relatively high protein concentration, presumably sufficient to saturate the RhaS-activated promoters, there will be a decrease in rhaSR transcription. We propose a model in which differential DNA bending by RhaS and RhaR may be the basis for the difference in CRP coactivation.
8
Cristache, Corina Marilena, Eugenia Eftimie Totu, Daniel Petre, Roxana Buga, Gheorghe Cristache, and Tiberiu Totu. "Melatonin and Hyaluronic Acid Mixture as a Possible Therapeutic Agent in Dental Medicine." Revista de Chimie 69, no. 8 (September 15, 2018): 1996–99. http://dx.doi.org/10.37358/rc.18.8.6461.
Повний текст джерелаАнотація:
The present work reports an efficient procedure for a simultaneous determination of melatonin (MEL) and hyaluronic acid (HA) mixture applying first derivative UV-Vis spectrophotometry. At selected wavelength (205 nm), the linearity range for each component of the mixture was established. The linear regression correlation coefficients for the direct UV-Vis spectra (RHA2 = 0.9627) and their first derivate (RHA2 = 0.9986) put in evidence a better fit for the derivative procedure. Through such simple analysis method, it was possible to emphasize that each component of MEL-HA mixture preserves its identity and characteristic properties. Consequently, the association of MEL and HA biomolecules could be used for obtaining a complex therapeutic agent with extensive applications in dental medicine.
9
Wickstrum, Jason R., and Susan M. Egan. "Amino Acid Contacts between Sigma 70 Domain 4 and the Transcription Activators RhaS and RhaR." Journal of Bacteriology 186, no. 18 (September 15, 2004): 6277–85. http://dx.doi.org/10.1128/jb.186.18.6277-6285.2004.
Повний текст джерелаАнотація:
ABSTRACT The RhaS and RhaR proteins are transcription activators that respond to the availability of l-rhamnose and activate transcription of the operons in the Escherichia coli l-rhamnose catabolic regulon. RhaR activates transcription of rhaSR, and RhaS activates transcription of the operon that encodes the l-rhamnose catabolic enzymes, rhaBAD, as well as the operon that encodes the l-rhamnose transport protein, rhaT. RhaS is 30% identical to RhaR at the amino acid level, and both are members of the AraC/XylS family of transcription activators. The RhaS and RhaR binding sites overlap the −35 hexamers of the promoters they regulate, suggesting they may contact the σ70 subunit of RNA polymerase as part of their mechanisms of transcription activation. In support of this hypothesis, our lab previously identified an interaction between RhaS residue D241 and σ70 residue R599. In the present study, we first identified two positively charged amino acids in σ70, K593 and R599, and three negatively charged amino acids in RhaR, D276, E284, and D285, that were important for RhaR-mediated transcription activation of the rhaSR operon. Using a genetic loss-of-contact approach we have obtained evidence for a specific contact between RhaR D276 and σ70 R599. Finally, previous results from our lab separately showed that RhaS D250A and σ70 K593A were defective at the rhaBAD promoter. Our genetic loss-of-contact analysis of these residues indicates that they identify a second site of contact between RhaS and σ70.
10
Wickstrum, Jason R., Jeff M. Skredenske, Ana Kolin, Ding J. Jin, Jianwen Fang, and Susan M. Egan. "Transcription Activation by the DNA-Binding Domain of the AraC Family Protein RhaS in the Absence of Its Effector-Binding Domain." Journal of Bacteriology 189, no. 14 (May 18, 2007): 4984–93. http://dx.doi.org/10.1128/jb.00530-07.
Повний текст джерелаАнотація:
ABSTRACT The Escherichia coli l-rhamnose-responsive transcription activators RhaS and RhaR both consist of two domains, a C-terminal DNA-binding domain and an N-terminal dimerization domain. Both function as dimers and only activate transcription in the presence of l-rhamnose. Here, we examined the ability of the DNA-binding domains of RhaS (RhaS-CTD) and RhaR (RhaR-CTD) to bind to DNA and activate transcription. RhaS-CTD and RhaR-CTD were both shown by DNase I footprinting to be capable of binding specifically to the appropriate DNA sites. In vivo as well as in vitro transcription assays showed that RhaS-CTD could activate transcription to high levels, whereas RhaR-CTD was capable of only very low levels of transcription activation. As expected, RhaS-CTD did not require the presence of l-rhamnose to activate transcription. The upstream half-site at rhaBAD and the downstream half-site at rhaT were found to be the strongest of the known RhaS half-sites, and a new putative RhaS half-site with comparable strength to known sites was identified. Given that cyclic AMP receptor protein (CRP), the second activator required for full rhaBAD expression, cannot activate rhaBAD expression in a ΔrhaS strain, it was of interest to test whether CRP could activate transcription in combination with RhaS-CTD. We found that RhaS-CTD allowed significant activation by CRP, both in vivo and in vitro, although full-length RhaS allowed somewhat greater CRP activation. We conclude that RhaS-CTD contains all of the determinants necessary for transcription activation by RhaS.
11
DeCarlo, Arthur A., Mayuri Paramaesvaran, Peter L. W. Yun, Charles Collyer, and Neil Hunter. "Porphyrin-Mediated Binding to Hemoglobin by the HA2 Domain of Cysteine Proteinases (Gingipains) and Hemagglutinins from the Periodontal Pathogen Porphyromonas gingivalis." Journal of Bacteriology 181, no. 12 (June 15, 1999): 3784–91. http://dx.doi.org/10.1128/jb.181.12.3784-3791.1999.
Повний текст джерелаАнотація:
ABSTRACT Heme binding and uptake are considered fundamental to the growth and virulence of the gram-negative periodontal pathogenPorphyromonas gingivalis. We therefore examined the potential role of the dominant P. gingivalis cysteine proteinases (gingipains) in the acquisition of heme from the environment. A recombinant hemoglobin-binding domain that is conserved between two predominant gingipains (domain HA2) demonstrated tight binding to hemin (Kd = 16 nM), and binding was inhibited by iron-free protoporphyrin IX (Ki = 2.5 μM). Hemoglobin binding to the gingipains and the recombinant HA2 (rHA2) domain (Kd = 2.1 nM) was also inhibited by protoporphyrin IX (Ki = 10 μM), demonstrating an essential interaction between the HA2 domain and the heme moiety in hemoglobin binding. Binding of rHA2 with either hemin, protoporphyrin IX, or hematoporphyrin was abolished by establishing covalent linkage of the protoporphyrin propionic acid side chains to fixed amines, demonstrating specific and directed binding of rHA2 to these protoporphyrins. A monoclonal antibody which recognizes a peptide epitope within the HA2 domain was employed to demonstrate that HA2-associated hemoglobin-binding activity was expressed and released by P. gingivalis cells in a batch culture, in parallel with proteinase activity. Cysteine proteinases from P. gingivalis appear to be multidomain proteins with functions for hemagglutination, erythrocyte lysis, proteolysis, and heme binding, as demonstrated here. Detailed understanding of the biochemical pathways for heme acquisition in P. gingivalis may allow precise targeting of this critical metabolic aspect for periodontal disease prevention.
12
DeCarlo, A. A., Y. Huang, C. A. Collyer, D. B. Langley, and J. Katz. "Feasibility of an HA2 Domain-Based Periodontitis Vaccine." Infection and Immunity 71, no. 1 (January 2003): 562–66. http://dx.doi.org/10.1128/iai.71.1.562-566.2003.
Повний текст джерелаАнотація:
ABSTRACT In a rat periodontitis model, preinoculation with the Porphyromonas gingivalis HA2 binding domain for hemoglobin provided protection from disease. Protection was associated with induced anti-HA2 immunoglobulin G (IgG) humoral antibodies. The IgG subclass ratios suggested that relatively lower Th2/Th1-driven responses were directly associated with protection when rHA2 was administered in saline.
13
Manzanares, Paloma, Hetty C. van den Broeck, Leo H. de Graaff та Jaap Visser. "Purification and Characterization of Two Different α-l-Rhamnosidases, RhaA and RhaB, fromAspergillus aculeatus". Applied and Environmental Microbiology 67, № 5 (1 травня 2001): 2230–34. http://dx.doi.org/10.1128/aem.67.5.2230-2234.2001.
Повний текст джерелаАнотація:
ABSTRACT Two proteins exhibiting α-l-rhamnosidase activity, RhaA and RhaB, were identified upon fractionation and purification of a culture filtrate from Aspergillus aculeatus grown on hesperidin. Both proteins were shown to be N glycosylated and had molecular masses of 92 and 85 kDa, of which approximately 24 and 15%, respectively, were contributed by carbohydrate. RhaA and RhaB, optimally active at pH 4.5 to 5, showedK m and V maxvalues of 2.8 mM and 24 U/mg (RhaA) and 0.30 mM and 14 U/mg (RhaB) when tested forp-nitrophenyl-α-l-rhamnopyranoside. Both enzymes were able to hydrolyze α-1,2 and α-1,6 linkages to β-d-glucosides. Using polyclonal antibodies, the corresponding cDNA of both α-l-rhamnosidases,rhaA and rhaB, was cloned. On the basis of the amino acid sequences derived from the cDNA clones, both proteins are highly homologous (60% identity).
14
Smiley, Richard W., Guiping Yan, and John N. Pinkerton. "Resistance of wheat, barley and oat to Heterodera avenae in the Pacific Northwest, USA." Nematology 13, no. 5 (2011): 539–52. http://dx.doi.org/10.1163/138855410x531862.
Повний текст джерелаАнотація:
Abstract The cereal cyst nematode, Heterodera avenae, occurs in at least seven western states of the USA and reduces grain yield in localised regions and in selected crop management systems. Virulence phenotypes for H. avenae populations in North America have not been reported. Nine individual assays in six experiments were conducted to determine the reactions of barley, oat and wheat cultivars to five H. avenae populations in the Pacific Northwest (PNW) states of Idaho, Oregon and Washington. Three populations were evaluated for virulence to 23 entries of the 'International Test Assortment for Defining Cereal Cyst Nematode Pathotypes', plus selected local cultivars and entries representing a greater diversity of resistance genes. The virulence phenotype(s) for populations of H. avenae in the PNW did not correspond to any of the 11 pathotypes defined by the Test Assortment. Five PNW populations exhibited affinities with Group 2 but were not defined by pathotypes Ha12 and Ha22. Reproduction was prevented or greatly inhibited by barley carrying the Rha3 resistance gene and by most carriers of Rha2 resistance, and by selected oat cultivars with multigenic resistance. Wheat cultivars carrying the Cre1 resistance gene were highly effective in suppressing H. avenae reproduction. Current PNW wheat cultivars do not carry the Cre1 resistance gene. Crosses between Ouyen, an Australian bread wheat with Cre1 resistance, and several PNW wheat cultivars were resistant. The CreR gene also prevented H. avenae reproduction in the trial where it was tested. Intermediate levels of reproduction occurred on wheat cultivars carrying the Cre5, Cre7 and Cre8 resistance genes, each of which was considered useful for pyramiding into cultivars with Cre1 resistance. This research identified genetic resources of value in PNW cereal crop breeding programmes.
15
Bhende, Prasanna M., and Susan M. Egan. "Genetic Evidence that Transcription Activation by RhaS Involves Specific Amino Acid Contacts with Sigma 70." Journal of Bacteriology 182, no. 17 (September 1, 2000): 4959–69. http://dx.doi.org/10.1128/jb.182.17.4959-4969.2000.
Повний текст джерелаАнотація:
ABSTRACT RhaS activates transcription of the Escherichia coli rhaBAD and rhaT operons in response tol-rhamnose and is a member of the AraC/XylS family of transcription activators. We wished to determine whether ς70 might be an activation target for RhaS. We found that ς70 K593 and R599 appear to be important for RhaS activation at both rhaBAD and rhaT, but only at truncated promoters lacking the binding site for the second activator, CRP. To determine whether these positively charged ς70residues might contact RhaS, we constructed alanine substitutions at negatively charged residues in the C-terminal domain of RhaS. Substitutions at four RhaS residues, E181A, D182A, D186A, and D241A, were defective at both truncated promoters. Finally, we assayed combinations of the RhaS and ς70 substitutions and found that RhaS D241 and ς70 R599 met the criteria for interacting residues at both promoters. Molecular modeling suggests that ς70 R599 is located in very close proximity to RhaS D241; hence, this work provides the first evidence for a specific residue within an AraC/XylS family protein that may contact ς70. More than 50% of AraC/XylS family members have Asp or Glu at the position of RhaS D241, suggesting that this interaction with ς70 may be conserved.
16
Van Gansbeke, Bart, Kelvin H. P. Khoo, John G. Lewis, Kenneth J. Chalmers, and Diane E. Mather. "Fine mapping of Rha2 in barley reveals candidate genes for resistance against cereal cyst nematode." Theoretical and Applied Genetics 132, no. 5 (January 18, 2019): 1309–20. http://dx.doi.org/10.1007/s00122-019-03279-3.
Повний текст джерела
17
Rivers, Damien M. R., and Ivan J. Oresnik. "The Sugar Kinase That Is Necessary for the Catabolism of Rhamnose in Rhizobium leguminosarum Directly Interacts with the ABC Transporter Necessary for Rhamnose Transport." Journal of Bacteriology 197, no. 24 (September 28, 2015): 3812–21. http://dx.doi.org/10.1128/jb.00510-15.
Повний текст джерелаАнотація:
ABSTRACTRhamnose catabolism inRhizobium leguminosarumwas found to be necessary for the ability of the organism to compete for nodule occupancy. Characterization of the locus necessary for the catabolism of rhamnose showed that the transport of rhamnose was dependent upon a carbohydrate uptake transporter 2 (CUT2) ABC transporter encoded byrhaSTPQand on the presence of RhaK, a protein known to have sugar kinase activity. A linker-scanning mutagenesis analysis ofrhaKshowed that the kinase and transport activities of RhaK could be separated genetically. More specifically, two pentapeptide insertions defined by the allelesrhaK72andrhaK73were able to uncouple the transport and kinase activities of RhaK, such that the kinase activity was retained, but cells carrying these alleles did not have measurable rhamnose transport rates. These linker-scanning alleles were localized to the C terminus and N terminus of RhaK, respectively. Taken together, the data led to the hypothesis that RhaK might interact either directly or indirectly with the ABC transporter defined byrhaSTPQ. In this work, we show that both N- and C-terminal fragments of RhaK are capable of interacting with the N-terminal fragment of the ABC protein RhaT using a 2-hybrid system. Moreover, if RhaK fragments carrying either therhaK72orrhaK73allele were used, this interaction was abolished. Phylogenetic and bioinformatic analysis of the RhaK fragments suggested that a conserved region in the N terminus of RhaK may represent a putative binding domain. Alanine-scanning mutagenesis of this region followed by 2-hybrid analysis revealed that a substitution of any of the conserved residues greatly affected the interaction between RhaT and RhaK fragments, suggesting that the sugar kinase RhaK and the ABC protein RhaT interact directly.IMPORTANCEABC transporters involved in the transport of carbohydrates help define the overall physiological fitness of bacteria. The two largest groups of transporters are thecarbohydrateuptaketransporter classes 1 and 2 (CUT1 and CUT2, respectively). This work provides the first evidence that a kinase that is necessary for the catabolism of a sugar can directly interact with a domain from the ABC protein that is necessary for its transport.
18
Hirooka, Kazutake, Yusuke Kodoi, Takenori Satomura, and Yasutaro Fujita. "Regulation of therhaEWRBMAOperon Involved in l-Rhamnose Catabolism through Two Transcriptional Factors, RhaR and CcpA, in Bacillus subtilis." Journal of Bacteriology 198, no. 5 (December 28, 2015): 830–45. http://dx.doi.org/10.1128/jb.00856-15.
Повний текст джерелаАнотація:
ABSTRACTTheBacillus subtilisrhaEWRBMA(formerlyyuxG-yulBCDE) operon consists of four genes encoding enzymes forl-rhamnose catabolism and therhaRgene encoding a DeoR-type transcriptional regulator. DNase I footprinting analysis showed that the RhaR protein specifically binds to the regulatory region upstream of therhaEWgene, in which two imperfect direct repeats are included. Gel retardation analysis revealed that the direct repeat farther upstream is essential for the high-affinity binding of RhaR and that the DNA binding of RhaR was effectively inhibited byl-rhamnulose-1-phosphate, an intermediate ofl-rhamnose catabolism. Moreover, it was demonstrated that the CcpA/P-Ser-HPr complex, primarily governing the carbon catabolite control inB. subtilis, binds to the catabolite-responsive element, which overlaps the RhaR binding site.In vivoanalysis of therhaEWpromoter-lacZfusion in the background ofccpAdeletion showed that thel-rhamnose-responsive induction of therhaEWpromoter was negated by the disruption ofrhaAorrhaBbut notrhaEWorrhaM, whereasrhaRdisruption resulted in constitutiverhaEWpromoter activity. Thesein vitroandin vivoresults clearly indicate that RhaR represses the operon by binding to the operator site, which is detached byl-rhamnulose-1-phosphate formed froml-rhamnose through a sequence of isomerization by RhaA and phosphorylation by RhaB, leading to the derepression of the operon. In addition, thelacZreporter analysis using the strains with or without theccpAdeletion under the background ofrhaRdisruption supported the involvement of CcpA in the carbon catabolite repression of the operon.IMPORTANCESincel-rhamnose is a component of various plant-derived compounds, it is a potential carbon source for plant-associating bacteria. Moreover, it is suggested thatl-rhamnose catabolism plays a significant role in some bacteria-plant interactions, e.g., invasion of plant pathogens and nodulation of rhizobia. Despite the physiological importance ofl-rhamnose catabolism for various bacterial species, the transcriptional regulation of the relevant genes has been poorly understood, except for the regulatory system ofEscherichia coli. In this study, we show that, inBacillus subtilis, one of the plant growth-promoting rhizobacteria, therhaEWRBMAoperon forl-rhamnose catabolism is controlled by RhaR and CcpA. This regulatory system can be another standard model for better understanding the regulatory mechanisms ofl-rhamnose catabolism in other bacterial species.
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Li, Dongmei, Weiyi Tao, Dinghua Yu, and Shuang Li. "Emulsifying Properties of Rhamnolipids and Their In Vitro Antifungal Activity against Plant Pathogenic Fungi." Molecules 27, no. 22 (November 10, 2022): 7746. http://dx.doi.org/10.3390/molecules27227746.
Повний текст джерелаАнотація:
Rhamnolipids have significant emulsifying activity and the potential to become a component of pesticide emulsifier. Rhamnolipids are usually composed of two main components: mono-rhamnolipids (Rha-C10-C10) and di-rhamnolipids (Rha2-C10-C10). The proportion of di-rhamnolipids in the products ranged between 15% and 90%, affected by the production strains and fermentation process. In this paper, three kinds of rhamnolipid products containing di-rhamnolipids proportions, of 25.45, 46.46 and 89.52%, were used to test their emulsifying ability toward three conventional solvents used in pesticide (S-200, xylene, cyclohexanone) and antifungal activities against five strains of plant pathogenic fungi (Phytophthora capsici, Phytophthora parasitica var.nicotianae, Colletotrichum destructivum, Colletotrichum sublineolum, Fusarium oxysporum). The results indicated that although the CMC of the three rhamnolipids were significantly different, their emulsification properties had no remarkable differences, at a concentration of 10 g/L. However, their antifungal activities were significantly different: the more di-rhamnolipids, the stronger the antifungal activity. This work helps to promote the application of rhamnolipids as pesticides adjuvants.
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Badía, J. "Identification of the rhaA, rhaB and rhaD gene products from Escherichia coli K-12." FEMS Microbiology Letters 65, no. 3 (December 1989): 253–57. http://dx.doi.org/10.1016/0378-1097(89)90226-7.
Повний текст джерела
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Skredenske, Jeff M., Veerendra Koppolu, Ana Kolin, James Deng, Bria Kettle, Byron Taylor, and Susan M. Egan. "Identification of a Small-Molecule Inhibitor of Bacterial AraC Family Activators." Journal of Biomolecular Screening 18, no. 5 (January 30, 2013): 588–98. http://dx.doi.org/10.1177/1087057112474690.
Повний текст джерелаАнотація:
Protein members of the AraC family of bacterial transcriptional activators have great promise as targets for the development of novel antibacterial agents. Here, we describe an in vivo high-throughput screen to identify inhibitors of the AraC family activator protein RhaS. The screen used two Escherichia coli reporter fusions: one to identify potential RhaS inhibitors and a second to eliminate nonspecific inhibitors from consideration. One compound with excellent selectivity, OSSL_051168, was chosen for further study. OSSL_051168 inhibited in vivo transcription activation by the RhaS DNA-binding domain to the same extent as the full-length protein, indicating that this domain was the target of its inhibition. Growth curves showed that OSSL_051168 did not affect bacterial cell growth at the concentrations used in this study. In vitro DNA-binding assays with purified protein suggest that OSSL_051168 inhibits DNA binding by RhaS. In addition, we found that it inhibits DNA binding by a second AraC family protein, RhaR, which shares 30% amino acid identity with RhaS. OSSL_051168 did not have a significant impact on DNA binding by the non-AraC family proteins CRP and LacI, suggesting that the inhibition is likely specific for RhaS, RhaR, and possibly additional AraC family activator proteins.
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Holcroft, Carolyn C., and Susan M. Egan. "Interdependence of Activation at rhaSR by Cyclic AMP Receptor Protein, the RNA Polymerase Alpha Subunit C-Terminal Domain, and RhaR." Journal of Bacteriology 182, no. 23 (December 1, 2000): 6774–82. http://dx.doi.org/10.1128/jb.182.23.6774-6782.2000.
Повний текст джерелаАнотація:
ABSTRACT The Escherichia coli rhaSR operon encodes two AraC family transcription activators, RhaS and RhaR, and is activated by RhaR in the presence of l-rhamnose. β-Galactosidase assays of various rhaS-lacZ promoter fusions combined with mobility shift assays indicated that a cyclic AMP receptor protein (CRP) site located at −111.5 is also required for full activation of rhaSR expression. To address the mechanisms of activation by CRP and the RNA polymerase α-subunit C-terminal domain (α-CTD) at rhaSR, we tested the effects of alanine substitutions in CRP activating regions 1 and 2, overexpression of a truncated version of α (α-Δ235), and alanine substitutions throughout α-CTD. We found that DNA-contacting residues in α-CTD are required for full activation, and for simplicity, we discuss α-CTD as a third activator of rhaSR. CRP and RhaR could each partially activate transcription in the absence of the other two activators, and α-CTD was not capable of activation alone. In the case of CRP, this suggests that this activation involves neither an α-CTD interaction nor cooperative binding with RhaR, while in the case of RhaR, this suggests the likelihood of direct interactions with core RNA polymerase. We also found that CRP, RhaR, and α-CTD each have synergistic effects on activation by the others, suggesting direct or indirect interactions among all three. We have some evidence that the α-CTD–CRP and α-CTD–RhaR interactions might be direct. The magnitude of the synergistic effects was usually greater with just two activators than with all three, suggesting possible redundancies in the mechanisms of activation by CRP, α-CTD, and RhaR.
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Santos, Marcus Vinícius Alves dos, Juliana Cardoso de Morais, Shyrlane Torres Soares Veras, Wanderli Rogério Moreira Leite, Savia Gavazza, Lourdinha Florencio, and Mario Takayuki Kato. "Reatores híbridos anaeróbio e aeróbio para remoção de matéria orgânica e nitrogênio em esgoto doméstico diluído." Engenharia Sanitaria e Ambiental 26, no. 3 (June 2021): 591–600. http://dx.doi.org/10.1590/s1413-41522020284.
Повний текст джерелаАнотація:
RESUMO A remoção de matéria orgânica e de nitrogênio em esgoto doméstico diluído foi avaliada em dois reatores híbridos, um anaeróbio (RHAN) e outro aeróbio (RHAE). O RHAN era formado por uma câmara tipo upflow anaerobic sludge blanket sobreposta por outra de filtro anaeróbio, enquanto o RHAE tinha uma câmara de lodo ativado sobreposta por outra de biofilme aerado submerso. A operação do sistema foi dividida em duas fases, FI e FII, com razões de recirculação de 50 e 75% e duração de 94 e 110 dias, respectivamente. Para a remoção de nitrogênio, o RHAE foi operado com oxigênio dissolvido de 3,0 mg.L-1. A técnica da reação em cadeia da polimerase foi empregada tanto para o lodo suspenso das câmaras inferiores, como para o biofilme aderido nas câmaras superiores, para identificar a presença de micro-organismos desnitrificantes e nitrificantes. As maiores eficiências de remoção em termos de demanda química de oxigênio e nitrogênio total foram obtidas em FII, sendo 91% e ~50%, respectivamente; as concentrações no efluente foram ~40 mg O2.L-1 e ~15 mg N-NT.L-1. A presença de três grupos de bactérias, as desnitrificantes, as oxidantes de amônia e as oxidantes de nitrito, foi confirmada no biofilme aderido do RHAE, indicando uma biomassa mixotrófica e sugerindo a possibilidade do processo de nitrificação e desnitrificação simultânea.
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Kolin, Ana, Visnja Jevtic, Liskin Swint-Kruse, and Susan M. Egan. "Linker Regions of the RhaS and RhaR Proteins." Journal of Bacteriology 189, no. 1 (October 27, 2006): 269–71. http://dx.doi.org/10.1128/jb.01456-06.
Повний текст джерелаАнотація:
ABSTRACT Substitutions within the interdomain linkers of the AraC/XylS family proteins RhaS and RhaR were tested to determine whether side chain identity or linker structure was required for function. Neither was found crucial, suggesting that the linkers do not play a direct role in activation, but rather simply connect the two domains.
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Kook, MooChang, Heung-Min Son, Hien T. T. Ngo, and Tae-Hoo Yi. "Chryseobacterium camelliae sp. nov., isolated from green tea." International Journal of Systematic and Evolutionary Microbiology 64, Pt_3 (March 1, 2014): 851–57. http://dx.doi.org/10.1099/ijs.0.057398-0.
Повний текст джерелаАнотація:
A Gram-staining-negative, strictly aerobic, non-motile, rod-shaped and flexirubin-type-pigmented strain, THG C4-1T, was isolated from green tea leaves in Jangheung-gun, Republic of Korea. Strain THG C4-1T grew well at 20–30 °C, at pH 7.0–7.5 and in the absence of NaCl on nutrient agar. Based on 16S rRNA gene sequence comparisons, strain THG C4-1T was most closely related to Chryseobacterium taiwanense Soil-3-27T (97.7 %), C. hagamense RHA2-9T (97.2 %), C. gregarium P 461/12T (97.2 %), C. ginsenosidimutans THG 15T (97.1 %), C. taeanense PHA3-4T (97.0 %) and C. daeguense K105T (97.0 %), but DNA–DNA relatedness between strain THG C4-1T and its closest phylogenetic neighbours was below 21 %. The DNA G+C content was 41.7 mol%. The only isoprenoid quinone detected in strain THG C4-1T was menaquinone 6 (MK-6). The major component of the polyamine pattern was sym-homospermidine. The major polar lipids were phosphatidylethanolamine and unidentified aminolipids. The major fatty acids were iso-C15 : 0, iso-C17 : 0 3-OH and iso-C17 : 1ω9c. These data supported the affiliation of strain THG C4-1T to the genus Chryseobacterium . The results of physiological and biochemical tests enabled strain THG C4-1T to be differentiated genotypically and phenotypically from recognized species of the genus Chryseobacterium . Therefore, the novel isolate represents a novel species, for which the name Chryseobacterium camelliae sp. nov. is proposed, with THG C4-1T ( = KACC 16985T = JCM 18745T) as the type strain.
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Wickstrum, Jason R., Thomas J. Santangelo, and Susan M. Egan. "Cyclic AMP Receptor Protein and RhaR Synergistically Activate Transcription from the l-Rhamnose-Responsive rhaSR Promoter in Escherichia coli." Journal of Bacteriology 187, no. 19 (October 1, 2005): 6708–18. http://dx.doi.org/10.1128/jb.187.19.6708-6718.2005.
Повний текст джерелаАнотація:
ABSTRACT The Escherichia coli rhaSR operon encodes two AraC family transcription activator proteins, RhaS and RhaR, which regulate expression of the l-rhamnose catabolic regulon in response to l-rhamnose availability. RhaR positively regulates rhaSR in response to l-rhamnose, and RhaR activation can be enhanced by the cyclic AMP (cAMP) receptor protein (CRP) protein. CRP is a well-studied global transcription regulator that binds to DNA as a dimer and activates transcription in the presence of cAMP. We investigated the mechanism of CRP activation at rhaSR both alone and in combination with RhaR in vivo and in vitro. Base pair substitutions at potential CRP binding sites in the rhaSR-rhaBAD intergenic region demonstrate that CRP site 3, centered at position −111.5 relative to the rhaSR transcription start site, is required for the majority of the CRP-dependent activation of rhaSR. DNase I footprinting confirms that CRP binds to site 3; CRP binding to the other potential CRP sites at rhaSR was not detected. We show that, at least in vitro, CRP is capable of both RhaR-dependent and RhaR-independent activation of rhaSR from a total of three transcription start sites. In vitro transcription assays indicate that the carboxy-terminal domain of the alpha subunit (α-CTD) of RNA polymerase is at least partially dispensable for RhaR-dependent activation but that the α-CTD is required for CRP activation of rhaSR. Although CRP requires the presence of RhaR for efficient in vivo activation of rhaSR, DNase I footprinting assays indicated that cooperative binding between RhaR and CRP does not make a significant contribution to the mechanism of CRP activation at rhaSR. It therefore appears that CRP activates transcription from rhaSR as it would at simple class I promoters, albeit from a relatively distant position.
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Johnes, Geraint. "Effeithlonrwydd Canolig: Cymharu Mewnbynnau ac Allbynnau yn ôl Iaith y Cyfarwyddyd mewn Ysgolion Uwchradd yng Nghymru." Cylchgrawn Addysg Cymru / Wales Journal of Education 22, no. 2 (September 1, 2020): 53–68. http://dx.doi.org/10.16922/wje.22.2.3.
Повний текст джерелаАнотація:
Defnyddir dull meta-derfyn i werthuso effeithlonrwydd y modd y caiff mewnbynnau i'r broses addysg eu trosi'n allbynnau, gan ddefnyddio data ar gyfer ysgolion uwchradd yng Nghymru a gafwyd o astudiaeth PISA 2015. Mae effeithlonrwydd ysgolion lle mae'r addysgu'n cael ei wneud drwy gyfrwng y Saesneg yn cael ei gymharu ag effeithlonrwydd ysgolion cyfrwng Cymraeg. Ar y rhan fwyaf o fesurau, mae mewnbynnau i'r olaf yn gymharol uchel, gan effeithio ar y lefelau effeithlonrwydd a welwyd. Er mwyn gwella sgorau PISA yng Nghymru, mae'n debygol y bydd angen gwella perfformiad ysgolion cyfrwng Cymraeg, ond mae angen rhoi cydnabyddiaeth bendant i unrhyw gyfaddawdu rhwng gwelliant o'r fath a gwireddu nodau polisi sy'n ehangach na'r rhai a werthusir yn PISA.
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Garcia-Martin, C., L. Baldoma, J. Badia, and J. Aguilar. "Nucleotide sequence of the rhaR-sodA interval specifying rhaT in Escherichia coli." Journal of General Microbiology 138, no. 6 (June 1, 1992): 1109–16. http://dx.doi.org/10.1099/00221287-138-6-1109.
Повний текст джерела
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Harris, Alma, Michelle Jones, Helen Lewis, Neil Lucas, and Joanna Thomas. "Dylunio Rhaglen Integredig o Addysg Gychwynnol i Athrawon Cynnydd, Ystyriaethau a Myfyrdodau." Cylchgrawn Addysg Cymru / Wales Journal of Education 22, no. 1 (March 1, 2020): 142–64. http://dx.doi.org/10.16922/wje.22.1.7.
Повний текст джерелаАнотація:
Mae'r erthygl hon yn edrych ar ddatblygu rhaglen integredig o addysg gychwynnol i athrawon wedi'i seilio ar ddull ymarfer clinigol sy'n ceisio datblygu athrawon dan hyfforddiant fel ymarferwyr adfyfyriol sy'n gweithio ar sail ymchwil. Mae'r erthygl yn amlinellu penderfyniadau allweddol wrth ddatblygu ymagwedd integredig at raglen newydd o addysg gychwynnol i athrawon sy'n llawn ymchwil ac yn seiliedig ar ymchwil. Mae'n tynnu sylw at ba mor ganolog yw dull partneriaeth wrth ddylunio'r rhaglen ac yn amlinellu'r broses ddatblygu ar y cyd wrth wraidd y rhaglen. Mae'r erthygl hefyd yn egluro sut mae arbenigwyr pwnc Prifysgol yn rhan annatod o'r rhaglen AGA, gan ddarparu arbenigedd pwnc ac ymchwil. Amlinellir prif nodweddion y rhaglen integredig hon o AGA, ac mae'r erthygl yn gorffen drwy gynnig rhai myfyrdodau ac ystyriaethau ynghylch dull o gyflwyno addysg gychwynnol i athrawon sy'n wirioneddol seiliedig ar ymchwil ac wedi'i seilio ar waith ymchwilio gan athrawon.
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Dupont, Magali, Severine Cornaton, Esma Kahoul, Nolwen Prie, and Claudine Giroux-Lathuile. "Profil d’anti-RH1+anti-RH2 à la RAE : attention à l’anti-RH12 !" Transfusion Clinique et Biologique 26, no. 3 (September 2019): S70. http://dx.doi.org/10.1016/j.tracli.2019.06.122.
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31
Ávila, Marta, Muriel Jaquet, Deborah Moine, Teresa Requena, Carmen Peláez, Fabrizio Arigoni та Ivana Jankovic. "Physiological and biochemical characterization of the two α-l-rhamnosidases of Lactobacillus plantarum NCC245". Microbiology 155, № 8 (1 серпня 2009): 2739–49. http://dx.doi.org/10.1099/mic.0.027789-0.
Повний текст джерелаАнотація:
This work is believed to be the first report on the physiological and biochemical characterization of α-l-rhamnosidases in lactic acid bacteria. A total of 216 strains representing 37 species and eight genera of food-grade bacteria were screened for α-l-rhamnosidase activity. The majority of positive bacteria (25 out of 35) were Lactobacillus plantarum strains, and activity of the L. plantarum strain NCC245 was examined in more detail. The analysis of α-l-rhamnosidase activity under different growth conditions revealed dual regulation of the enzyme activity, involving carbon catabolite repression and induction: the enzyme activity was downregulated by glucose and upregulated by l-rhamnose. The expression of the two α-l-rhamnosidase genes rhaB1 and rhaB2 and two predicted permease genes rhaP1 and rhaP2, identified in a probable operon rhaP2B2P1B1, was repressed by glucose and induced by l-rhamnose, showing regulation at the transcriptional level. The two α-l-rhamnosidase genes were overexpressed and purified from Escherichia coli. RhaB1 activity was maximal at 50 °C and at neutral pH and RhaB2 maximal activity was detected at 60 °C and at pH 5, with high residual activity at 70 °C. Both enzymes showed a preference for the α-1,6 linkage of l-rhamnose to β-d-glucose, hesperidin and rutin being their best substrates, but, surprisingly, no activity was detected towards the α-1,2 linkage in naringin under the tested conditions. In conclusion, we identified and characterized the strain L. plantarum NCC245 and its two α-l-rhamnosidase enzymes, which might be applied for improvement of bioavailability of health-beneficial polyphenols, such as hesperidin, in humans.
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Stewart, Andrew K., Boris E. Shmukler, David H. Vandorpe, Alicia Rivera, John F. Heneghan, Xiaojin Li, Ann Hsu, et al. "Loss-of-function and gain-of-function phenotypes of stomatocytosis mutant RhAG F65S." American Journal of Physiology-Cell Physiology 301, no. 6 (December 2011): C1325—C1343. http://dx.doi.org/10.1152/ajpcell.00054.2011.
Повний текст джерелаАнотація:
Four patients with overhydrated cation leak stomatocytosis (OHSt) exhibited the heterozygous RhAG missense mutation F65S. OHSt erythrocytes were osmotically fragile, with elevated Na and decreased K contents and increased cation channel-like activity. Xenopus oocytes expressing wild-type RhAG and RhAG F65S exhibited increased ouabain and bumetanide-resistant uptake of Li+and86Rb+, with secondarily increased86Rb+influx sensitive to ouabain and to bumetanide. Increased RhAG-associated14C-methylammonium (MA) influx was severely reduced in RhAG F65S-expressing oocytes. RhAG-associated influxes of Li+,86Rb+, and14C-MA were pharmacologically distinct, and Li+uptakes associated with RhAG and RhAG F65S were differentially inhibited by NH4+and Gd3+. RhAG-expressing oocytes were acidified and depolarized by 5 mM bath NH3/NH4+, but alkalinized and depolarized by subsequent bath exposure to 5 mM methylammonium chloride (MA/MA+). RhAG F65S-expressing oocytes exhibited near-wild-type responses to NH4Cl, but MA/MA+elicited attenuated alkalinization and strong hyperpolarization. Expression of RhAG or RhAG F65S increased steady-state cation currents unaltered by bath Li+substitution or bath addition of 5 mM NH4Cl or MA/MA+. These oocyte studies suggest that 1) RhAG expression increases oocyte transport of NH3/NH4+and MA/MA+; 2) RhAG F65S exhibits gain-of-function phenotypes of increased cation conductance/permeability, and loss-of-function phenotypes of decreased and modified MA/MA+transport, and decreased NH3/NH4+-associated depolarization; and 3) RhAG transports NH3/NH4+and MA/MA+by distinct mechanisms, and/or the substrates elicit distinct cellular responses. Thus, RhAG F65S is a loss-of-function mutation for amine transport. The altered oocyte intracellular pH, membrane potential, and currents associated with RhAG or RhAG F65S expression may reflect distinct transport mechanisms.
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Mouro-Chanteloup, Isabelle, Anne Marie D'Ambrosio, Pierre Gane, Caroline Le Van Kim, Virginie Raynal, Didier Dhermy, Jean-Pierre Cartron, and Yves Colin. "Cell-surface expression of RhD blood group polypeptide is posttranscriptionally regulated by the RhAG glycoprotein." Blood 100, no. 3 (August 1, 2002): 1038–47. http://dx.doi.org/10.1182/blood.v100.3.1038.
Повний текст джерелаАнотація:
Abstract In most cases, the lack of Rh in Rhnull red cells is associated with RHAG gene mutations. We explored the role of RhAG in the surface expression of Rh. Nonerythroid HEK293 cells, which lack Rh and RhAG, or erythroid K562 cells, which endogenously express RhAG but not Rh, were transfected with RhD and/or RhAG cDNAs using cytomegalovirus (CMV) promoter–based expression vectors. In HEK293 cells, a low but significant expression of RhD was obtained only when RhAG was expressed at a high level. In K562 cells, as expected from the opposite effects of the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) on erythroid and CMV promoters, the levels of endogenous RhAG and recombinant RhD transcripts were substantially decreased and enhanced upon TPA treatment of RhD-transfected cells (K562/RhD), respectively. However, flow cytometry and fluorescence microscopy analysis revealed a decreased cell-surface expression of both RhAG and RhD proteins. Conversely, TPA treatment of RhAG-transfected cells increased both the transcript and surface expression levels of RhAG. When K562/RhD cells were cotransfected by the RhAG cDNA, the TPA-mediated induction of recombinant RhAG and RhD transcription was associated with an increased membrane expression of both RhAG and RhD proteins. These results demonstrate the role of RhAG as a strictly required posttranscriptional factor regulating Rh membrane expression. In addition, because the postulated 2:2 stoichiometry between Rh and RhAG observed in the native red cell membrane could not be obtained in cotransfected K562 cells, our study also suggests that as yet unidentified protein(s) might be involved for optimal membrane expression of Rh.
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Mouro-Chanteloup, Isabelle, Anne Marie D'Ambrosio, Pierre Gane, Caroline Le Van Kim, Virginie Raynal, Didier Dhermy, Jean-Pierre Cartron, and Yves Colin. "Cell-surface expression of RhD blood group polypeptide is posttranscriptionally regulated by the RhAG glycoprotein." Blood 100, no. 3 (August 1, 2002): 1038–47. http://dx.doi.org/10.1182/blood.v100.3.1038.h81502001038_1038_1047.
Повний текст джерелаАнотація:
In most cases, the lack of Rh in Rhnull red cells is associated with RHAG gene mutations. We explored the role of RhAG in the surface expression of Rh. Nonerythroid HEK293 cells, which lack Rh and RhAG, or erythroid K562 cells, which endogenously express RhAG but not Rh, were transfected with RhD and/or RhAG cDNAs using cytomegalovirus (CMV) promoter–based expression vectors. In HEK293 cells, a low but significant expression of RhD was obtained only when RhAG was expressed at a high level. In K562 cells, as expected from the opposite effects of the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) on erythroid and CMV promoters, the levels of endogenous RhAG and recombinant RhD transcripts were substantially decreased and enhanced upon TPA treatment of RhD-transfected cells (K562/RhD), respectively. However, flow cytometry and fluorescence microscopy analysis revealed a decreased cell-surface expression of both RhAG and RhD proteins. Conversely, TPA treatment of RhAG-transfected cells increased both the transcript and surface expression levels of RhAG. When K562/RhD cells were cotransfected by the RhAG cDNA, the TPA-mediated induction of recombinant RhAG and RhD transcription was associated with an increased membrane expression of both RhAG and RhD proteins. These results demonstrate the role of RhAG as a strictly required posttranscriptional factor regulating Rh membrane expression. In addition, because the postulated 2:2 stoichiometry between Rh and RhAG observed in the native red cell membrane could not be obtained in cotransfected K562 cells, our study also suggests that as yet unidentified protein(s) might be involved for optimal membrane expression of Rh.
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Westhoff, Connie M., and Seth Alper. "Hereditary Stomatocytosis Associated with a Loss of Function Mutation In Rh-Associated Glycoprotein (RhAG)." Blood 116, no. 21 (November 19, 2010): 2040. http://dx.doi.org/10.1182/blood.v116.21.2040.2040.
Повний текст джерелаАнотація:
Abstract Abstract 2040 The erythroid Rh family of proteins includes RhCE and RhD which carry the common Rh antigens, and the related Rh-associated glycoprotein, RhAG. RhAG is required for trafficking of the blood group proteins to the membrane and forms the core of a macro-complex in the membrane which includes glycophorin B, Band 3, CD47, and LW. The Rh proteins are structurally and functionally related to the Amt superfamily of NH3/NH4+ transport proteins, and RhAG and its nonerythroid paralogs, RhCG and RhBG, have been shown to mediate NH3/NH4+ transport. RhCG is responsible for part of renal collecting duct epithelial cell NH3/NH4+ secretion, and Rhcg-/- mice exhibit incomplete distal renal tubular acidosis due to impaired urinary NH4+ excretion. The Rhag-/- mouse is grossly normal, and the significance of RhAG-mediated NH3/NH4+ transport in human erythrocytes remains unclear. Over-hydrated hereditary stomatocytosis (OHSt) is a rare dominant disorder characterized by moderate hemolytic anemia, increased mean red cell volumes, stomatocytes and echinocytes, and increased red cell permeability to the monovalent cations, Na+ and K+. Six of the seven OHSt kindred studied by Bruce et al. (Blood. 2009;113:1350) displayed a heterozygous Phe65Ser mutation in RhAG. Expression studies of the mutant 65Ser-RhAG in Xenopus oocytes induced a monovalent cation flux compatible with the cation leak seen in RBCs. The increased Na+ and decreased K+ contents of mutant RhAG-expressing oocytes suggested that F65S is a gain-of-function mutation that opens a cation leak, likely within the RhAG polypeptide. In this study the ammonia transport properties of the OHSt mutant 65Ser-RhAG were investigated. Xenopus oocytes were injected with cRNA encoding wild-type RhAG, the OHSt mutant 65Ser-RhAG, and 65Val-RhAG, an engineered mutation with a smaller hydrophobic side chain at position 65. Wild-type and mutant RhAG polypeptides were well-expressed in the oocyte membrane as measured by quantitative immunoblotting. Uptake of the NH3/NH4+ substrate analog 14C-methylammonium (MA), was assayed in oocytes previously injected with water (control) or with cRNA. Expression of wild-type RhAG mediated MA uptake at rates 6-fold greater than that of water-injected controls. Uptake of MA by oocytes expressing 65Val-RhAG was equivalent to that of wild type RhAG. However, MA uptake by oocytes expressing OHSt mutant 65Ser-RhAG was greatly reduced to less than 20% that of oocytes expressing wild-type RHAG or 65Val-RhAG, and was only 1.5-fold greater than that of water-injected control oocytes. Co-expression with other, individual Rh complex members glycophorin B, RhD, RhCE, or Band 3 did not alter MA-mediated uptake by RhAG-expressing oocytes. Importantly, this study reveals that the RhAG mutation Phe65Ser found in patients with type 1 over-hydrated stomatocytosis is a loss of function mutation. Further study is required to define the relationship between loss of NH3/NH4+ transport and erythrocyte Na+ and K+ cation content. Disclosures: Westhoff: Immucor: Scientific Advisor.
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Berry, Leslie, Bruce Thong, and Anthony Chan. "Comparison of recombinant and plasma-derived antithrombin biodistribution in a rabbit model." Thrombosis and Haemostasis 102, no. 08 (2009): 302–8. http://dx.doi.org/10.1160/th09-01-0062.
Повний текст джерелаАнотація:
SummaryAntithrombin (AT) is a native plasma protein that acts as the main inhibitor of enzymes generated by the coagulation cascade. In extreme thrombotic conditions, consumption of plasma AT can make treatment with AT-associated heparin therapies less effective. Supplementation with recombinant human AT (rhAT) has shown promise but altered pharmacokinetics were observed when comparing stable heparin complexes of the plasma-derived AT (pAT) and rhAT proteins. To understand the differential clearance mechanisms,biodistribution of rhAT and pAT was determined. 125I-labelled ATryn (rhAT) or Kybernin P (pAT) was intravenously injected into rabbits. At various time points, animals were sacrificed and organs analysed for bound radioactivity. 131I-albumin, injected shortly before termination, was used as a marker for trapped blood. Levels of circulating protein + metabolites were significantly less for rhAT than pAT (p < 0.001) and removal of acid soluble fragments confirmed that differences were due to more rapid rhAT clearance. More rhAT (28% dose) than pAT (3% dose) was liver-associated by the earliest time points, corresponding to elevated rhAT degradation products in urine/feces. However, at intermediate times (4 hours), rhAT showed significantly increased arterial and venous uptake over pAT (p ≤0.001).These vessel wall interactions accounted for the primary differences between clearance of rhAT and pAT during these time periods. Overall, circulating rhAT is more rapidly lost to the liver and vessels than pAT. Increased vessel wall binding may facilitate rhAT treatment of vascular thrombosis.
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Bruce, Lesley J., Hélène Guizouarn, Nicholas M. Burton, Nicole Gabillat, Joyce Poole, Joanna F. Flatt, R. Leo Brady, Franck Borgese, Jean Delaunay, and Gordon W. Stewart. "The monovalent cation leak in overhydrated stomatocytic red blood cells results from amino acid substitutions in the Rh-associated glycoprotein." Blood 113, no. 6 (February 5, 2009): 1350–57. http://dx.doi.org/10.1182/blood-2008-07-171140.
Повний текст джерелаАнотація:
Abstract Overhydrated hereditary stomatocytosis (OHSt) is a rare dominantly inherited hemolytic anemia characterized by a profuse membrane leak to monovalent cations. Here, we show that OHSt red cell membranes contain slightly reduced amounts of Rh-associated glycoprotein (RhAG), a putative gas channel protein. DNA analysis revealed that the OHSt patients have 1 of 2 heterozygous mutations (t182g, t194c) in RHAG that lead to substitutions of 2 highly conserved amino acids (Ile61Arg, Phe65Ser). Unexpectedly, expression of wild-type RhAG in Xenopus laevis oocytes induced a monovalent cation leak; expression of the mutant RhAG proteins induced a leak about 6 times greater than that in wild type. RhAG belongs to the ammonium transporter family of proteins that form pore-like structures. We have modeled RhAG on the homologous Nitrosomonas europaea Rh50 protein and shown that these mutations are likely to lead to an opening of the pore. Although the function of RhAG remains controversial, this first report of functional RhAG mutations supports a role for RhAG as a cation pore.
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M, Ajay, Dinesh M. N, Gaurav Misha, Siddanna R, Shivayogi B. N, and Rajendra V. M. "Performance Studies of Biomass Derived RHAC and MgO Nanocomposite Electrode Materials for Supercapacitor Applications." ECS Transactions 107, no. 1 (April 24, 2022): 7607–18. http://dx.doi.org/10.1149/10701.7607ecst.
Повний текст джерелаАнотація:
The electrode materials for supercapacitors based on biomass-produced Rice Husk Activated Carbon (RHAC) and RHAC+50% MgO nanocomposite are compared in this article. The crystallinity, structural morphology, and crystalline size of the particles were determined using microstructural and electrochemical characterization. Scanning Electron Microscope and X-Ray Diffraction techniques were used to determine the surface morphology and crystallinity of the materials. The XRD results reveal that RHAC which has broad peaks indicates amorphous nature whereas RHAC+50% MgO nanocomposite which has sharp peaks exhibits crystalline nature. The RHAC+50% MgO nanocomposite electrode exhibits a specific capacitance of 220.52 F/g however RHAC electrode exhibited only 162.91 F/g at a scan rate of 2mV/s by Cyclic Voltammetry in 1 M KOH electrolyte. After 2500 cycles of Galvanic charge-discharge, it was discovered that RHAC+50% MgO nanocomposite has capacitance retention of 76.71% and RHAC has capacitance retention of 80.49%.
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Michon, Francis, Samuel L. Moore, John Kim, Milan S. Blake, France-Isabelle Auzanneau, Blair D. Johnston, Margaret A. Johnson, and B. Mario Pinto. "Doubly Branched Hexasaccharide Epitope on the Cell Wall Polysaccharide of Group A Streptococci Recognized by Human and Rabbit Antisera." Infection and Immunity 73, no. 10 (October 2005): 6383–89. http://dx.doi.org/10.1128/iai.73.10.6383-6389.2005.
Повний текст джерелаАнотація:
ABSTRACT A number of epitope specificities associated with the cell wall polysaccharide antigen of group A streptococci were identified in a polyclonal rabbit antiserum induced in rabbits by whole group A streptococci and in polyclonal convalescent human antisera from children that had recovered from streptococcal A infections. The identification was achieved by using a series of synthetic oligosaccharides, glycoconjugates, and bacterial polysaccharide inhibitors to inhibit the binding of the group A helical polysaccharide to the polyclonal antisera. The exclusively dominant epitope expressed in the convalescent human antisera was the doubly branched extended helical hexasaccharide with the structure α-l-Rhap(1→2)[β-d-GlcpNAc(1→3)]α-l-Rhap(1→3)α-l-Rhap(1→2)[β-d-GlcpNAc(1→3)]α-l-Rhap. The hexasaccharide epitope also bound with the highest immunoreactivity to the rabbit antiserum. In contrast, the human antisera did not show significant binding to the singly branched pentasaccharide with the structure α-l-Rhap(1→2)α-l-Rhap(1→3)α-l-Rhap(1→2)[β-d-GlcpNAc(1→3)]α-l-Rhap or the branched trisaccharide α-l-Rhap(1→2)[β-d-GlcpNAc(1→3)]α-l-Rhap, although both these haptens bound significantly to the same rabbit antiserum, albeit with less immunoreactivity than the hexasaccharide. Inhibition studies using streptococcal group A and B rabbit antisera and the inhibitors indicated above also suggested that the group A carbohydrate, unlike the group B streptococcal polysaccharide, does not contain the disaccharide α-l-Rhap(1→2)α-l-Rhap motif at its nonreducing chain terminus, stressing the importance of mapping the determinant specificities of these two important streptococcal subcapsular group polysaccharides to fully understand the serological relationships between group A and group B streptococci.
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Genetet, Sandrine, Pierre Ripoche, Julien Picot, Sylvain Bigot, Jean Delaunay, Corinne Armari-Alla, Yves Colin, and Isabelle Mouro-Chanteloup. "Human RhAG ammonia channel is impaired by the Phe65Ser mutation in overhydrated stomatocytic red cells." American Journal of Physiology-Cell Physiology 302, no. 2 (January 15, 2012): C419—C428. http://dx.doi.org/10.1152/ajpcell.00092.2011.
Повний текст джерелаАнотація:
In red cells, Rh-associated glycoprotein (RhAG) acts as an ammonia channel, as demonstrated by stopped-flow analysis of ghost intracellular pH (pHi) changes. Recently, overhydrated hereditary stomatocytosis (OHSt), a rare dominantly inherited hemolytic anemia, was found to be associated with a mutation (Phe65Ser or Ile61Arg) in RHAG. Ghosts from the erythrocytes of four of the OHSt patients with a Phe65Ser mutation were resealed with a pH-sensitive probe and submitted to ammonium gradients. Alkalinization rate constants, reflecting NH3transport through the channel and NH3diffusion unmediated by RhAG, were deduced from time courses of fluorescence changes. After subtraction of the constant value found for Rhnulllacking RhAG, we observed that alkalinization rate constant values decreased ∼50% in OHSt compared with those of controls. Similar RhAG expression levels were found in control and OHSt. Since half of the expressed RhAG in OHSt most probably corresponds to the mutated form of RhAG, as expected from the OHSt heterozygous status, this dramatic decrease can be therefore related to the loss of function of the Phe65Ser-mutated RhAG monomer.
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Lai, Janice Ching, Svetlana Ponti, Dejing Pan, Hubertus Kohler, Radek C. Skoda, Patrick Matthias, and Yoshikuni Nagamine. "The DEAH-box helicase RHAU is an essential gene and critical for mouse hematopoiesis." Blood 119, no. 18 (May 3, 2012): 4291–300. http://dx.doi.org/10.1182/blood-2011-08-362954.
Повний текст джерелаАнотація:
Abstract The DEAH helicase RHAU (alias DHX36, G4R1) is the only helicase shown to have G-quadruplex (G4)–RNA resolvase activity and the major source of G4-DNA resolvase activity. Previous report showed RHAU mRNA expression to be elevated in human lymphoid and CD34+ BM cells, suggesting a potential role in hematopoiesis. Here, we generated a conditional knockout of the RHAU gene in mice. Germ line deletion of RHAU led to embryonic lethality. We then targeted the RHAU gene specifically in the hematopoiesis system, using a Cre-inducible system in which an optimized variant of Cre recombinase was expressed under the control of the Vav1 promoter. RHAU deletion in hematopoietic system caused hemolytic anemia and differentiation defect at the proerythroblast stage. The partial differentiation block of proerythroblasts was because of a proliferation defect. Transcriptome analysis of RHAU knockout proerythroblasts showed that a statistically significant portion of the deregulated genes contain G4 motifs in their promoters. This suggests that RHAU may play a role in the regulation of gene expression that relies on its G4 resolvase activity.
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Brown, Carlos V. R., Peter Rhee, Kelly Evans, Demetrios Demetriades, and George Velmahos. "Rhabdomyolysis after Penetrating Trauma." American Surgeon 70, no. 10 (October 2004): 890–92. http://dx.doi.org/10.1177/000313480407001013.
Повний текст джерелаАнотація:
Rhabdomyolysis (RHAB) is a known complication following blunt trauma. RHAB after penetrating trauma has not been studied. The objective of this study was to evaluate the incidence, risk factors, and complications of RHAB following penetrating trauma. Over a 5-year period, penetrating trauma patients admitted to our intensive care unit (ICU) were studied. Significant RHAB was defined as a CK level of 5000 U/L or higher. There were 873 patients (29 ± 12 years old, 92% male), of whom 767 (88%) had abnormal CK levels (range 520–165,943 U/L), and 111 patients (13%) developed significant RHAB. Victims of penetrating trauma who sustain vascular and severe extremity injury are at a sixfold increased risk to develop significant RHAB. Patients with significant RHAB had a higher rate of renal failure (23% vs 7%, P < 0.0001) and longer ICU stay (15 ± 26 days vs 8 ± 12 days, P < 0.0001). CK elevations and significant RHAB are common after penetrating trauma. Patients who sustain vascular and severe extremity injury as a result of their penetrating wounds are at high risk to develop significant RHAB, resulting in renal failure and prolonged ICU stay. Therefore, critically injured penetrating trauma patients should be routinely screened with CK levels.
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Kim, K. S., and H. C. Choi. "Characteristics of adsorption of rice-hull activated carbon." Water Science and Technology 38, no. 4-5 (August 1, 1998): 95–101. http://dx.doi.org/10.2166/wst.1998.0591.
Повний текст джерелаАнотація:
An experiment was conducted to compare the adsorption capacity and characteristics between activated carbon made of rice-hull(RHAC) and F-400 by Calgon to remove phenol, heavy metal and ammonia-nitrogen. While F-400 could not remove ammonia-nitrogen, RHAC was able to adsorb it. This is considered to be due to the ionic sorption capability of SiO2 remaining on the surface of RHAC. From the sorption equilibrium test, it was found that RHAC has higher adsorption capacity than F-400. In column tests, the slope of breakthrough curves of RHAC which represent the affinity of an adsorbent, was observed to be more gradual than F-400. This may be attributed to the inner diffusion of adsorbent once attached on macropores, into micropores well developed, with higher specific surface area of RHAC than F-400. For heavy metals, F-400 would remove chromium and lead but not cadmium, whereas RHAC was able to remove cadmium, lead but not chromium. This phenomenon is considered to have something to do with the distinct surface functional group of RHAC and the various surface charge densities of heavy metals tested.
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Edmunds, Tim, Scott M. Van Patten, Julie Pollock, Eric Hanson, Richard Bernasconi, Elizabeth Higgins, Partha Manavalan, et al. "Transgenically Produced Human Antithrombin: Structural and Functional Comparison to Human Plasma–Derived Antithrombin." Blood 91, no. 12 (June 15, 1998): 4561–71. http://dx.doi.org/10.1182/blood.v91.12.4561.
Повний текст джерелаАнотація:
Abstract Recombinant human antithrombin (rhAT) produced in transgenic goat milk was purified to greater than 99%. The specific activity of the rhAT was identical to human plasma–derived AT (phAT) in an in vitro thrombin inhibition assay. However, rhAT had a fourfold higher affinity for heparin than phAT. The rhAT was analyzed and compared with phAT by reverse phase high-performance liquid chromatography, circular dichroism, fluorophore-assisted carbohydrate electrophoresis (FACE), amino acid sequence, and liquid chromatography/mass spectrography peptide mapping. Based on these analyses, rhAT was determined to be structurally identical to phAT except for differences in glycosylation. Oligomannose structures were found on the Asn 155 site of the transgenic protein, whereas only complex structures were observed on the plasma protein. RhAT contained a GalNAc for galactose substitution on some N-linked oligosaccharides, as well as a high degree of fucosylation. RhAT was less sialylated than phAT and contained both N-acetylneuraminic and N-glycolylneuraminic acid. We postulate that the increase in affinity for heparin found with rhAT resulted from the presence of oligomannose-type structures on the Asn 155 glycosylation site and differences in sialylation.
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Edmunds, Tim, Scott M. Van Patten, Julie Pollock, Eric Hanson, Richard Bernasconi, Elizabeth Higgins, Partha Manavalan, et al. "Transgenically Produced Human Antithrombin: Structural and Functional Comparison to Human Plasma–Derived Antithrombin." Blood 91, no. 12 (June 15, 1998): 4561–71. http://dx.doi.org/10.1182/blood.v91.12.4561.412k21_4561_4571.
Повний текст джерелаАнотація:
Recombinant human antithrombin (rhAT) produced in transgenic goat milk was purified to greater than 99%. The specific activity of the rhAT was identical to human plasma–derived AT (phAT) in an in vitro thrombin inhibition assay. However, rhAT had a fourfold higher affinity for heparin than phAT. The rhAT was analyzed and compared with phAT by reverse phase high-performance liquid chromatography, circular dichroism, fluorophore-assisted carbohydrate electrophoresis (FACE), amino acid sequence, and liquid chromatography/mass spectrography peptide mapping. Based on these analyses, rhAT was determined to be structurally identical to phAT except for differences in glycosylation. Oligomannose structures were found on the Asn 155 site of the transgenic protein, whereas only complex structures were observed on the plasma protein. RhAT contained a GalNAc for galactose substitution on some N-linked oligosaccharides, as well as a high degree of fucosylation. RhAT was less sialylated than phAT and contained both N-acetylneuraminic and N-glycolylneuraminic acid. We postulate that the increase in affinity for heparin found with rhAT resulted from the presence of oligomannose-type structures on the Asn 155 glycosylation site and differences in sialylation.
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Booy, Evan P., Ryan Howard, Oksana Marushchak, Emmanuel O. Ariyo, Markus Meier, Stefanie K. Novakowski, Soumya R. Deo, Edis Dzananovic, Jörg Stetefeld, and Sean A. McKenna. "The RNA helicase RHAU (DHX36) suppresses expression of the transcription factor PITX1." Nucleic Acids Research 42, no. 5 (December 24, 2013): 3346–61. http://dx.doi.org/10.1093/nar/gkt1340.
Повний текст джерелаАнотація:
Abstract RNA Helicase associated with AU-rich element (RHAU) (DHX36) is a DEAH (Aspartic acid, Glumatic Acid, Alanine, Histidine)-box RNA helicase that can bind and unwind G4-quadruplexes in DNA and RNA. To detect novel RNA targets of RHAU, we performed an RNA co-immunoprecipitation screen and identified the PITX1 messenger RNA (mRNA) as specifically and highly enriched. PITX1 is a homeobox transcription factor with roles in both development and cancer. Primary sequence analysis identified three probable quadruplexes within the 3′-untranslated region of the PITX1 mRNA. Each of these sequences, when isolated, forms stable quadruplex structures that interact with RHAU. We provide evidence that these quadruplexes exist in the endogenous mRNA; however, we discovered that RHAU is tethered to the mRNA via an alternative non–quadruplex-forming region. RHAU knockdown by small interfering RNA results in significant increases in PITX1 protein levels with only marginal changes in mRNA, suggesting a role for RHAU in translational regulation. Involvement of components of the microRNA machinery is supported by similar and non-additive increases in PITX1 protein expression on Dicer and combined RHAU/Dicer knockdown. We also demonstrate a requirement of argonaute-2, a key RNA-induced silencing complex component, to mediate RHAU-dependent changes in PITX1 protein levels. These results demonstrate a novel role for RHAU in microRNA-mediated translational regulation at a quadruplex-containing 3′-untranslated region.
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Suyama, Kimita, Hua Li, and Alex Zhu. "Surface expression of Rh-associated glycoprotein (RhAG) in nonerythroid COS-1 cells." Blood 95, no. 1 (January 1, 2000): 336–41. http://dx.doi.org/10.1182/blood.v95.1.336.
Повний текст джерелаАнотація:
Abstract In the Rh blood system, RhAG (Rh-associated glycoprotein, or Rh50) is thought to be involved in Rh30 (D, CE) expression by forming a protein complex on the red cell surface. To obtain further insight into the Rh complex, we chose nonerythroid COS-1 cells instead of proerythroblast-like K562 cells, which produce endogenous Rh proteins as cell host, for the expression of both RhAG and RhD. The RhAG cDNA was subcloned into a retroviral vector, and a stable COS-1 cell line was then established via retroviral transduction. Surface expression of RhAG on the COS-1 cells was monitored by flow cytometry using mouse monoclonal anti-RhAG(2D10). Under these conditions, we detected significant expression of RhAG on the cell surface, compared to stable COS-1 cells transduced with the vector alone. To confirm the results, we isolated RhAG by immunoprecipitation from the lysate of the COS-1 cells, which were metabolically labeled with [35S]-methionine. A strong band of the 32 kd on SDS-PAGE was obtained, corresponding to the results obtained from other cultured cells (K562 cell and others), which always produce partially glycosylated RhAG with a molecular weight of 32 kd. Thus, RhAG was expressed without Rh30 and other Rh-related glycoproteins (LW, glycophorin B) in nonerythroid cells. Using the same strategy, however, we could not express RhD epitopes on COS-1 cells even in the presence of RhAG cDNA, suggesting that other factors might be required for the surface expression of RhD antigen. (Blood. 2000;95:336-341)
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Suyama, Kimita, Hua Li, and Alex Zhu. "Surface expression of Rh-associated glycoprotein (RhAG) in nonerythroid COS-1 cells." Blood 95, no. 1 (January 1, 2000): 336–41. http://dx.doi.org/10.1182/blood.v95.1.336.001k46_336_341.
Повний текст джерелаАнотація:
In the Rh blood system, RhAG (Rh-associated glycoprotein, or Rh50) is thought to be involved in Rh30 (D, CE) expression by forming a protein complex on the red cell surface. To obtain further insight into the Rh complex, we chose nonerythroid COS-1 cells instead of proerythroblast-like K562 cells, which produce endogenous Rh proteins as cell host, for the expression of both RhAG and RhD. The RhAG cDNA was subcloned into a retroviral vector, and a stable COS-1 cell line was then established via retroviral transduction. Surface expression of RhAG on the COS-1 cells was monitored by flow cytometry using mouse monoclonal anti-RhAG(2D10). Under these conditions, we detected significant expression of RhAG on the cell surface, compared to stable COS-1 cells transduced with the vector alone. To confirm the results, we isolated RhAG by immunoprecipitation from the lysate of the COS-1 cells, which were metabolically labeled with [35S]-methionine. A strong band of the 32 kd on SDS-PAGE was obtained, corresponding to the results obtained from other cultured cells (K562 cell and others), which always produce partially glycosylated RhAG with a molecular weight of 32 kd. Thus, RhAG was expressed without Rh30 and other Rh-related glycoproteins (LW, glycophorin B) in nonerythroid cells. Using the same strategy, however, we could not express RhD epitopes on COS-1 cells even in the presence of RhAG cDNA, suggesting that other factors might be required for the surface expression of RhD antigen. (Blood. 2000;95:336-341)
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Kuběnová, Lenka, Michaela Tichá, Jozef Šamaj, and Miroslav Ovečka. "ROOT HAIR DEFECTIVE 2 vesicular delivery to the apical plasma membrane domain during Arabidopsis root hair development." Plant Physiology 188, no. 3 (January 5, 2022): 1563–85. http://dx.doi.org/10.1093/plphys/kiab595.
Повний текст джерелаАнотація:
Abstract Arabidopsis (Arabidopsis thaliana) root hairs develop as long tubular extensions from the rootward pole of trichoblasts and exert polarized tip growth. The establishment and maintenance of root hair polarity is a complex process involving the local apical production of reactive oxygen species generated by A. thaliana nicotinamide adenine dinucleotide phosphate (NADPH) oxidase respiratory burst oxidase homolog protein C/ROOT HAIR-DEFECTIVE 2 (AtRBOHC/RHD2). Loss-of-function root hair defective 2 (rhd2) mutants have short root hairs that are unable to elongate by tip growth, and this phenotype is fully complemented by GREEN FLUORESCENT PROTEIN (GFP)-RHD2 expressed under the RHD2 promoter. However, the spatiotemporal mechanism of AtRBOHC/RHD2 subcellular redistribution and delivery to the plasma membrane (PM) during root hair initiation and tip growth are still unclear. Here, we used advanced microscopy for detailed qualitative and quantitative analysis of vesicular compartments containing GFP-RHD2 and characterization of their movements in developing bulges and growing root hairs. These compartments, identified by an independent molecular marker mCherry-VTI12 as the trans-Golgi network (TGN), deliver GFP-RHD2 to the apical PM domain, the extent of which corresponds with the stage of root hair formation. Movements of TGN/early endosomes, but not late endosomes, were affected in the bulging domains of the rhd2-1 mutant. Finally, we revealed that structural sterols might be involved in the accumulation, docking, and incorporation of TGN compartments containing GFP-RHD2 to the apical PM of root hairs. These results help in clarifying the mechanism of polarized AtRBOHC/RHD2 targeting, maintenance, and recycling at the apical PM domain, coordinated with different developmental stages of root hair initiation and growth.
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Jilma, Bernd, Judith M. Leitner, Florian B. Mayr, Christa Firbas, Rosemarie A. Reiter, and Peter Kalhs. "Anticoagulant and Anti-Inflammatory Effects of Recombinant Human Antithrombin in Man after LPS Challenge." Blood 106, no. 11 (November 16, 2005): 1880. http://dx.doi.org/10.1182/blood.v106.11.1880.1880.
Повний текст джерелаАнотація:
Abstract Background: Antithrombin (AT) had beneficial effects on mortality of septic patients in an a priori defined subpopulation of the Kypersept trial, which received no concomitant administration of heparin. Objectives: We hypothesized that recombinant human (rh)AT (without concomitant heparin) has anticoagulant properties and may decrease cytokine production in a well standardized model of human endotoxemia. Methods: This study was randomized, double-blind, placebo-controlled in parallel groups in 30 healthy male volunteers. The active treatment groups received bolus primed continuous infusion of rhAT (recombinant human Antithrombin) to increase AT-levels to 200% and 500% iv. before infusion of 2ng/kg endotoxin Results: Infusion of rhAT rapidly decreased neutrophil (p<0.01) and monocyte counts (p<0.05) before LPS-challenge, demonstrating that rhAT directly interacts with these leukocyte subsets. rhAT exhibited intrinsic anticoagulant efficacy ex vivo as shown by thrombelastography ((p<0.01). More importantly, in vivo thrombin formation decreased in a dose dependent fashion as measured by prothrombin fragment (F1+2) and thrombin antithrombin complexes (TAT). rhAT significantly decreased interleukin-6 (IL-6) release by 40%. Conclusion: rhAT exhibited intrinsic anticoagulant effects, which are stronger than the minor effects of drotrecogin alfa (rhAPC; Blood2003;102:2093ff) in this model of tissue factor triggered coagulation. Effects of rhAT on leukocytes and inhibition of IL-6 release were not previously observed with various different synthetic or natural anticoagulants in this model and therefore represent specific pharmacodynamic properties of rhAT. The demonstrated anticoagulant and anti-inflammatory properties of rhAT may have beneficial effects in critically ill patients, and may explain the efficacy of rhAT in septic patients without concomitant heparin administration.