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Journal articles on the topic 'Molecular cloning; Mutagenesis; 4-HPPD'
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Stirdivant, Steven M., Janet Ahern, Robert R. Conroy, Stanley F. Barnett, Lynette M. Ledder, Allen Oliff, and David C. Heimbrook. "Cloning and mutagenesis of the p110α subunit of human phosphoinositide 3′-hydroxykinase." Bioorganic & Medicinal Chemistry 5, no. 1 (January 1997): 65–74. http://dx.doi.org/10.1016/s0968-0896(96)00196-4.
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FAVALORO, Bartolo, Antonio TAMBURRO, Stefania ANGELUCCI, Antonella DE LUCA, Sonia MELINO, Carmine DI ILIO, and Domenico ROTILIO. "Molecular cloning, expression and site-directed mutagenesis of glutathione S-transferase from Ochrobactrum anthropi." Biochemical Journal 335, no. 3 (November 1, 1998): 573–79. http://dx.doi.org/10.1042/bj3350573.
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The gene coding for a novel glutathione S-transferase (GST) has been isolated from the bacterium Ochrobactrum anthropi. A PCR fragment of 230 bp was obtained using oligonucleotide primers deduced from N-terminal and ‘internal ’ sequences of the purified enzyme. The gene was obtained by screening of a genomic DNA partial library from O. anthropi constructed in pBluescript with a PCR fragment probe. The gene encodes a protein (OaGST) of 201 amino acids with a calculated molecular mass of 21738 Da. The product of the gene was expressed and characterized; it showed GST activity with substrates 1-chloro-2,4-dinitrobenzene (CDNB), p-nitrobenzyl chloride and 4-nitroquinoline 1-oxide, and glutathione-dependent peroxidase activity towards cumene hydroperoxide. The overexpressed product of the gene was also confirmed to have in vivo GST activity towards CDNB. The interaction of the recombinant GST with several antibiotics indicated that the enzyme is involved in the binding of rifamycin and tetracycline. The OaGST amino acid sequence showed the greatest identity (45%) with a GST from Pseudomonas sp. strain LB400. A serine residue in the N-terminal region is conserved in almost all known bacterial GSTs, and it appears to be the counterpart of the catalytic serine residue present in Theta-class GSTs. Substitution of the Ser-11 residue resulted in a mutant OaGST protein lacking CDNB-conjugating activity; moreover the mutant enzyme was not able to bind Sepharose–GSH affinity matrices. The amino acid and nucleotide sequences reported in this paper have been submitted to the EMBL Data Bank with the accession number Y17279.
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Garfinkel, D. J., M. F. Mastrangelo, N. J. Sanders, B. K. Shafer, and J. N. Strathern. "Transposon tagging using Ty elements in yeast." Genetics 120, no. 1 (September 1, 1988): 95–108. http://dx.doi.org/10.1093/genetics/120.1.95.
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Abstract We have used the ability to induce high levels of Ty transposition to develop a method for transposon mutagenesis in Saccharomyces cerevisiae. To facilitate genetic and molecular analysis, we have constructed GAL1-promoted TyH3 or Ty917 elements that contain unique cloning sites, and marked these elements with selectable genes. These genes include the yeast HIS3 gene, and the plasmid PiAN7 containing the Tn903 NEO gene. The marked Ty elements retain their ability to transpose, to mutate the LYS2, LYS5, or STE2 genes, and to activate the promoterless his3 delta 4 target gene. Ty elements containing selectable genes are also useful in strain construction, in chromosomal mapping, and in gene cloning strategies.
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Park, I. K., P. Roach, J. Bondor, S. P. Fox, and A. A. DePaoli-Roach. "Molecular mechanism of the synergistic phosphorylation of phosphatase inhibitor-2. Cloning, expression, and site-directed mutagenesis of inhibitor-2." Journal of Biological Chemistry 269, no. 2 (January 1994): 944–54. http://dx.doi.org/10.1016/s0021-9258(17)42203-4.
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Popham, D. L., and P. Setlow. "Cloning, nucleotide sequence, mutagenesis, and mapping of the Bacillus subtilis pbpD gene, which codes for penicillin-binding protein 4." Journal of Bacteriology 176, no. 23 (1994): 7197–205. http://dx.doi.org/10.1128/jb.176.23.7197-7205.1994.
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Richard, Nathalie, Horacio Salomon, Robert Rando, Tarek Mansour, Terry L. Bowlin, and Mark A. Wainberg. "Selection and Characterization of Human Immunodeficiency Virus Type 1 Variants Resistant to the (+) and (−) Enantiomers of 2′-Deoxy-3′-Oxa-4′-Thio-5-Fluorocytidine." Antimicrobial Agents and Chemotherapy 44, no. 5 (May 1, 2000): 1127–31. http://dx.doi.org/10.1128/aac.44.5.1127-1131.2000.
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ABSTRACT Human immunodeficiency virus (HIV) type 1 (HIV-1) variants were selected for resistance to the (+) and (−) enantiomers of a novel nucleoside analogue, 2′-deoxy-3′-oxa-4′-thio-5-fluorocytidine (dOTFC), by use of the infectious molecular clone HIV HXB2D and the human T-cell line MT-4. The dOTFC-resistant variants that were selected were 10-fold less sensitive than wild-type virus, and cloning and sequencing of the complete reverse transcriptase (RT)-coding region identified the mutation M184V. Studies with mutated recombinant HXB2D virus confirmed the importance of the M184V mutation in conferring resistance to (−)dOTFC in MT-4 cells, although no difference in sensitivity was observed in primary cells. The M184V substitution also displayed decreased susceptibility to (+)dOTFC. Selection with (+)dOTFC also produced variants which were 10-fold more resistant than the wild type, and a novel mutation, D67G, was identified following cloning and sequencing of the RT genes. The D67G mutation was introduced into HXB2D by site-directed mutagenesis, and the data obtained confirmed the importance of this mutation in conferring resistance to both (+)dOTFC and (−)dOTFC. Mutated recombinant molecular clone HXB2D-D67G was further selected with (+)dOTFC, and three of six clones sequenced contained both the D67G and M184V mutations, while the other three of the six clones contained only the D67G mutation. Clinical isolates of HIV-1 which are (−) 2′-deoxy-3′-thiacytidine-resistant also displayed resistance to both (+)dOTFC and (−)dOTFC.
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Betz, Andrea, Sandra J. Facey, Bernhard Hauer, Barbara Tshisuaka, and Franz Lingens. "Molecular cloning, sequencing, expression, and site-directed mutagenesis of the 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase gene fromArthrobacter spec. R?61a." Journal of Basic Microbiology 40, no. 1 (February 2000): 7–23. http://dx.doi.org/10.1002/(sici)1521-4028(200002)40:1<7::aid-jobm7>3.0.co;2-5.
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Chitnis, P. R., D. Purvis, and N. Nelson. "Molecular cloning and targeted mutagenesis of the gene psaF encoding subunit III of photosystem I from the cyanobacterium Synechocystis sp. PCC 6803." Journal of Biological Chemistry 266, no. 30 (October 1991): 20146–51. http://dx.doi.org/10.1016/s0021-9258(18)54902-4.
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Jia, Airong, and Xiao-Hua Zhang. "Molecular Cloning, Characterization, and Expression Analysis of the CXCR4 Gene from Turbot:Scophthalmus maximus." Journal of Biomedicine and Biotechnology 2009 (2009): 1–8. http://dx.doi.org/10.1155/2009/767893.
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Chemokine receptor 4 (CXCR4) belongs to the large superfamily of G protein-coupled receptors. The EST sequence of CXCR4 from turbot (Scophthalmus maximusL.) was obtained from a subtractive cDNA library. In the present study, the full-length cDNA sequence of turbot CXCR4 was obtained, and sequence analysis indicated that its primary structure was highly similar to CXCR4 from other vertebrates. Quantitative real-time PCR demonstrated that the highest expression level of turbot CXCR4 was in the spleen following injection with physiological saline (PS). After turbot were challenged withVibrio harveyi, the lowest expression level of CXCR4 was detected at 8 hours in the spleen and 12 hours in the head kidney, and then increased gradually to 36 hours. These findings suggested that CXCR4 may play a significant role in the immune response of turbot.
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Dai, Ding, Re Bai, Ernest Hodgson, and Randy L. Rose. "Cloning, sequencing, heterologous expression, and characterization of murine cytochrome P450 3a25*(Cyp3a25), a testosterone 6?-hydroxylase." Journal of Biochemical and Molecular Toxicology 15, no. 2 (2001): 90–99. http://dx.doi.org/10.1002/jbt.4.
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Fleer, R., C. M. Nicolet, G. A. Pure, and E. C. Friedberg. "RAD4 gene of Saccharomyces cerevisiae: molecular cloning and partial characterization of a gene that is inactivated in Escherichia coli." Molecular and Cellular Biology 7, no. 3 (March 1987): 1180–92. http://dx.doi.org/10.1128/mcb.7.3.1180.
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In contrast to other Saccharomyces cerevisiae RAD genes involved in nucleotide excision repair of DNA, the RAD4 gene could not be isolated by screening a yeast genomic library for recombinant plasmids which complement the UV sensitivity of rad4 mutants (Pure et al., J. Mol. Biol. 183:31-42, 1985). We therefore attempted to walk to RAD4 from the neighboring SPT2 gene and obtained an integrating derivative of a plasmid isolated by Roeder et al. (Mol. Cell. Biol. 5:1543-1553, 1985) which contains a 4-kilobase fragment of yeast DNA including a mutant allele of SPT2. When integrated into several different rad4 mutant strains, this plasmid (pR169) complements UV sensitivity at a frequency of approximately 10%. However, a centromeric plasmid containing rescued sequences which include flanking yeast DNA no longer complements the phenotype of rad4 mutants. Complementing activity was restored by in vivo repair of a defined gap in the centromeric plasmid. The repaired plasmid fully complements the UV sensitivity of all rad4 mutants tested when isolated directly from yeast cells, but when this plasmid is propagated in Escherichia coli complementing activity is lost. We have mapped the physical location of the RAD4 gene by insertional mutagenesis and by transcript mapping. The gene is approximately 2.3 kilobases in size and is located immediately upstream of the SPT2 gene. Both genes are transcribed in the same direction. RAD4 is not an essential gene, and no increased transcription of this gene is observed in cells exposed to the DNA-damaging agent 4-nitroquinoline-1-oxide. The site of inactivation of RAD4 in a particular plasmid propagated in E. coli was localized to a 100-base-pair region by gene disruption and gap repair experiments. In addition, we have identified the approximate locations of the chromosomal rad4-2, rad4-3, and rad4-4 mutations.
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Fleer, R., C. M. Nicolet, G. A. Pure, and E. C. Friedberg. "RAD4 gene of Saccharomyces cerevisiae: molecular cloning and partial characterization of a gene that is inactivated in Escherichia coli." Molecular and Cellular Biology 7, no. 3 (March 1987): 1180–92. http://dx.doi.org/10.1128/mcb.7.3.1180-1192.1987.
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In contrast to other Saccharomyces cerevisiae RAD genes involved in nucleotide excision repair of DNA, the RAD4 gene could not be isolated by screening a yeast genomic library for recombinant plasmids which complement the UV sensitivity of rad4 mutants (Pure et al., J. Mol. Biol. 183:31-42, 1985). We therefore attempted to walk to RAD4 from the neighboring SPT2 gene and obtained an integrating derivative of a plasmid isolated by Roeder et al. (Mol. Cell. Biol. 5:1543-1553, 1985) which contains a 4-kilobase fragment of yeast DNA including a mutant allele of SPT2. When integrated into several different rad4 mutant strains, this plasmid (pR169) complements UV sensitivity at a frequency of approximately 10%. However, a centromeric plasmid containing rescued sequences which include flanking yeast DNA no longer complements the phenotype of rad4 mutants. Complementing activity was restored by in vivo repair of a defined gap in the centromeric plasmid. The repaired plasmid fully complements the UV sensitivity of all rad4 mutants tested when isolated directly from yeast cells, but when this plasmid is propagated in Escherichia coli complementing activity is lost. We have mapped the physical location of the RAD4 gene by insertional mutagenesis and by transcript mapping. The gene is approximately 2.3 kilobases in size and is located immediately upstream of the SPT2 gene. Both genes are transcribed in the same direction. RAD4 is not an essential gene, and no increased transcription of this gene is observed in cells exposed to the DNA-damaging agent 4-nitroquinoline-1-oxide. The site of inactivation of RAD4 in a particular plasmid propagated in E. coli was localized to a 100-base-pair region by gene disruption and gap repair experiments. In addition, we have identified the approximate locations of the chromosomal rad4-2, rad4-3, and rad4-4 mutations.
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Chuang, Duen-yau, Yung-chei Chien, and Huang-Pin Wu. "Cloning and Expression of the Erwinia carotovora subsp. carotovora Gene Encoding the Low-Molecular-Weight Bacteriocin Carocin S1." Journal of Bacteriology 189, no. 2 (October 27, 2006): 620–26. http://dx.doi.org/10.1128/jb.01090-06.
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ABSTRACT The purpose of this study was to clone the carocin S1 gene and express it in a non-carocin-producing strain of Erwinia carotovora. A mutant, TH22-10, which produced a high-molecular-weight bacteriocin but not a low-molecular-weight bacteriocin, was obtained by Tn5 insertional mutagenesis using H-rif-8-2 (a spontaneous rifampin-resistant mutant of Erwinia carotovora subsp. carotovora 89-H-4). Using thermal asymmetric interlaced PCR, the DNA sequence from the Tn5 insertion site and the DNA sequence of the contiguous 2,280-bp region were determined. Two complete open reading frames (ORF), designated ORF2 and ORF3, were identified within the sequence fragment. ORF2 and ORF3 were identified with the carocin S1 genes, caroS1K (ORF2) and caroS1I (ORF3), which, respectively, encode a killing protein (CaroS1K) and an immunity protein (CaroS1I). These genes were homologous to the pyocin S3 gene and the pyocin AP41 gene. Carocin S1 was expressed in E. carotovora subsp. carotovora Ea1068 and replicated in TH22-10 but could not be expressed in Escherichia coli (JM101) because a consensus sequence resembling an SOS box was absent. A putative sequence similar to the consensus sequence for the E. coli cyclic AMP receptor protein binding site (−312 bp) was found upstream of the start codon. Production of this bacteriocin was also induced by glucose and lactose. The homology search results indicated that the carocin S1 gene (between bp 1078 and bp 1704) was homologous to the pyocin S3 and pyocin AP41 genes in Pseudomonas aeruginosa. These genes encode proteins with nuclease activity (domain 4). This study found that carocin S1 also has nuclease activity.
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Schromm, Andra B., Egil Lien, Philipp Henneke, Jesse C. Chow, Atsutoshi Yoshimura, Holger Heine, Eicke Latz, et al. "Molecular Genetic Analysis of an Endotoxin Nonresponder Mutant Cell Line." Journal of Experimental Medicine 194, no. 1 (July 2, 2001): 79–88. http://dx.doi.org/10.1084/jem.194.1.79.
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Somatic cell mutagenesis is a powerful tool for characterizing receptor systems. We reported previously two complementation groups of mutant cell lines derived from CD14-transfected Chinese hamster ovary–K1 fibroblasts defective in responses to bacterial endotoxin. Both classes of mutants expressed a normal gene product for Toll-like receptor (TLR)4, and fully responded to stimulation by tumor necrosis factor (TNF)-α or interleukin (IL)-1β. We identified the lesion in one of the complementation groups in the gene for MD-2, a putative TLR4 coreceptor. The nonresponder phenotype of this mutant was reversed by transfection with MD-2. Cloning of MD-2 from the nonresponder cell line revealed a point mutation in a highly conserved region resulting in a C95Y amino acid exchange. Both forms of MD-2 colocalized with TLR4 on the cell surface after transfection, but only the wild-type cDNA reverted the lipopolysaccharide (LPS) nonresponder phenotype. Furthermore, soluble MD-2, but not soluble MD-2C95Y, functioned to enable LPS responses in cells that expressed TLR4. Thus, MD-2 is a required component of the LPS signaling complex and can function as a soluble receptor for cells that do not otherwise express it. We hypothesize that MD-2 conformationally affects the extracellular domain of TLR4, perhaps resulting in a change in affinity for LPS or functioning as a portion of the true ligand for TLR4.
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Wu, Ting-Ting, Hsiang-I. Liao, Leming Tong, Ronika Sitapara Leang, Greg Smith, and Ren Sun. "Construction and Characterization of an Infectious Murine Gammaherpesivrus-68 Bacterial Artificial Chromosome." Journal of Biomedicine and Biotechnology 2011 (2011): 1–11. http://dx.doi.org/10.1155/2011/926258.
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Here we describe the cloning of a sequenced WUMS isolate of murine gammaherpesvirus-68 (MHV-68, γHV-68, also known as MuHV-4) as a bacterial artificial chromosome (BAC). We engineered the insertion of the BAC sequence flanked by loxP sites into the left end of the viral genome before the M1 open reading frame. The infectious viruses were reconstituted following transfection of the MHV-68 BAC DNA into cells. The MHV-68 BAC-derived virus replicated indistinguishably from the wild-type virus in cultured cells. Excision of the BAC insert was efficiently achieved by coexpressing the Cre recombinase. Although the BAC insertion did not significantly affect acute productive infection in the lung, it severely compromised the ability of MHV-68 to establish splenic latency. Removal of the BAC sequence restored the wild-type level of latency. Site-specific mutagenesis was carried out by RecA-mediated recombination to demonstrate that this infectious BAC clone can be used for genetic studies of MHV-68.
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Tremblay, Gilles B., André Tremblay, Neal G. Copeland, Debra J. Gilbert, Nancy A. Jenkins, Fernand Labrie, and Vincent Giguère. "Cloning, Chromosomal Localization, and Functional Analysis of the Murine Estrogen Receptor β." Molecular Endocrinology 11, no. 3 (March 1, 1997): 353–65. http://dx.doi.org/10.1210/mend.11.3.9902.
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Abstract Estrogen receptor β (ERβ) is a novel steroid receptor that is expressed in rat prostate and ovary. We have cloned the mouse homolog of ERβ and mapped the gene, designated Estrb, to the central region of chromosome 12. The cDNA encodes a protein of 485 amino acids that shares, respectively, 97% and 60% identity with the DNA- and ligand-binding domains of mouse (m) ERα. Mouse ERβ binds to an inverted repeat spaced by three nucleotides in a gel mobility shift assay and transactivates promoters containing synthetic or natural estrogen response elements in an estradiol (E2)-dependent manner. Scatchard analysis indicates that mERβ has slightly lower affinity for E2 [dissociation constant (Kd) = 0.5 nm] when compared with mERα (Kd = 0.2 nm). Antiestrogens, including 4-hydroxytamoxifen (OHT), ICI 182,780, and a novel compound, EM-800, inhibit E2-dependent transactivation efficiently. However, while OHT displays partial agonistic activity with ERα on a basal promoter linked to estrogen response elements in Cos-1 cells, this effect is not observed with mERβ. Cotransfection of mERβ and H-RasV12 causes enhanced activation in the presence of E2. Mutagenesis of a serine residue (position 60), located within a mitogen-activated protein kinase consensus phosphorylation site abolishes the stimulatory effect of Ras, suggesting that the activity of mERβ is also regulated by the mitogen-activated protein kinase pathway. Surprisingly, the coactivator SRC-1 up-regulates mERβ transactivation both in the absence and presence of E2, and in vitro interaction between SRC-1 and the ERβ ligand-binding domain is enhanced by E2. Moreover, the ligand-independent stimulatory effect of SRC-1 on ERβ transcriptional activity is abolished by ICI 182,780, but not by OHT. Our results demonstrate that while ERβ shares many of the functional characteristics of ERα, the molecular mechanisms regulating the transcriptional activity of mERβ may be distinct from those of ERα.
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Takeda, Kayoko, Homare Itoh, Issei Yoshioka, Megumi Yamamoto, Hideo Misaki, Sachiko Kajita, Kengo Shirai, et al. "Cloning of a thermostable ascorbate oxidase gene from Acremonium sp. HI-25 and modification of the azide sensitivity of the enzyme by site-directed mutagenesis." Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology 1388, no. 2 (November 1998): 444–56. http://dx.doi.org/10.1016/s0167-4838(98)00206-4.
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Zhang, Xiao-Mei, Hua-Jian Zhang, Min Liu, Bin Liu, Xia-Fang Zhang, Cheng-Jun Ma, Ting-Ting Fu, You-Ming Hou, and Bao-Zhen Tang. "Cloning and Immunosuppressive Properties of an Acyl-Activating Enzyme from the Venom Apparatus of Tetrastichus brontispae (Hymenoptera: Eulophidae)." Toxins 11, no. 11 (November 18, 2019): 672. http://dx.doi.org/10.3390/toxins11110672.
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Venom injected into the host plays vital roles in facilitating successful parasitization and development for parasitoid wasps, especially those devoid of polydnavirus, and the abundant venom proteins appear to be most likely involved in parasitization success. Previously, we found the four most abundant venom proteins, including 4-coumarate:CoA ligase-like 4 (4CL4-like), in the Tetrastichus brontispae (Hymenoptera: Eulophidae) venom apparatus. In this study, we cloned, expressed T. brontispae 4CL4-like (Tb4CL4-like) in Escherichia coli, and investigated its immunosuppressive properties. The deduced amino acid sequence for Tb4CL4-like shares high identity at conserved amino acids associated with the acyl-activating enzyme (AAE) consensus motif but shows only <40% identity with the members in the AAE superfamily. mRNA abundance analysis indicated that Tb4CL4-like was transcribed mainly in the venom apparatus. Recombinant Tb4CL4-like inhibited Octodonta nipae (Coleoptera: Chrysomelidae) pupal cellular encapsulation and spreading by targeting the hemocyte cytoskeleton and reduced the hemocyte-mediated phagocytosis of E. coli in vivo. Moreover, Tb4CL4-like exhibited greater affinity to palmitic acid and linolenic acid based on the molecular docking assay and is hypothesized to be involved in fatty acid metabolism. In conclusion, our results suggest that Tb4CL4-like may be an immunity-related AAE protein that is involved in the regulation of host immunity through fatty acid metabolism-derived signaling pathways.
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Rapala-Kozik, Maria, Mariusz Olczak, Katarzyna Ostrowska, Agata Starosta, and Andrzej Kozik. "Molecular characterization of the thi3 gene involved in thiamine biosynthesis in Zea mays: cDNA sequence and enzymatic and structural properties of the recombinant bifunctional protein with 4-amino-5-hydroxymethyl-2-methylpyrimidine (phosphate) kinase and thiamine monophosphate synthase activities." Biochemical Journal 408, no. 2 (November 14, 2007): 149–59. http://dx.doi.org/10.1042/bj20070677.
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A thiamine biosynthesis gene, thi3, from maize Zea mays has been identified through cloning and sequencing of cDNA and heterologous overexpression of the encoded protein, THI3, in Escherichia coli. The recombinant THI3 protein was purified to homogeneity and shown to possess two essentially different enzymatic activities of HMP(-P) [4-amino-5-hydroxymethyl-2-methylpyrimidine (phosphate)] kinase and TMP (thiamine monophosphate) synthase. Both activities were characterized in terms of basic kinetic constants, with interesting findings that TMP synthase is uncompetitively inhibited by excess of one of the substrates [HMP-PP (HMP diphosphate)] and ATP. A bioinformatic analysis of the THI3 sequence suggested that these activities were located in two distinct, N-terminal kinase and C-terminal synthase, domains. Models of the overall folds of THI3 domains and the arrangements of active centre residues were obtained with the SWISS-MODEL protein modelling server, on the basis of the known three-dimensional structures of Salmonella enterica serotype Typhimurium HMP(-P) kinase and Bacillus subtilis TMP synthase. The essential roles of Gln98 and Met134 residues for HMP kinase activity and of Ser444 for TMP synthase activity were experimentally confirmed by site-directed mutagenesis.
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Cherrington, Nathan J., J. Greg Falls, Randy L. Rose, Kieran M. Clements, Richard M. Philpot, Patricia E. Levi, and Ernest Hodgson. "Molecular Cloning, Sequence, and Expression of Mouse Flavin-Containing Monooxygenases 1 and 5 (FMO1 and FMO5)." Journal of Biochemical and Molecular Toxicology 12, no. 4 (1998): 205–12. http://dx.doi.org/10.1002/(sici)1099-0461(1998)12:4<205::aid-jbt2>3.0.co;2-p.
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Hakoda, Masayuki, Mitoshi Akiyama, Seishi Kyoizumi, Kyoko Kobuke, Akio A. Awa, and Michio Yamakido. "Measurement of in vivo HGPRT-deficient mutant cell frequency using a modified method for cloning human peripheral blood T-lymphocytes." Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 197, no. 1 (January 1988): 161–69. http://dx.doi.org/10.1016/0027-5107(88)90153-4.
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Cerioli, Sergio, Stefania Ballarini, Helmut Uhrig, Eugenio Scalzotto, and Adriano Marocco. "Induction of unstable alleles at the temperature-sensitive Virescent-1 gene of maize using the transposable element Dissociation." Genetical Research 66, no. 3 (December 1995): 203–12. http://dx.doi.org/10.1017/s0016672300034650.
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SummaryTranspositive mutagenesis was employed to prepare genetic strains useful in cloning the Virescent-1 locus (V1) of maize. A stepwise approach was used based on: (1) the isolation of putative insertion phenotypes (62 cases); (2) the verification of the genetic nature of the selected events (36 v1-m mutant alleles induced); (3) the accurate genetic study of 11 alleles; (4) the genetic assessment that the alleles v1-m1 and v1-m4 are due to the insertion of a Ds element into the locus V1; (5) the proof that a Ds-like DNA element induces the inactivation of the wild type function in the allele v1-m1. The phenotype of the unstable alleles, studied by germinating and keeping maize seedlings at the temperature of 18 °C, are the following: alleles v1-m1, v1-m9, v1-m11, v1-m17 and v1-m18 showing a few revertant green sectors on their leaves; v1-m4 exhibiting a reverse type of variegation; alleles v1-m2 and v1-m13 with a coarse pattern of variegation; alleles v1-m12, v1-m21 and v1-m23 frequently showing leaves part green with white stripes and part white with green stripes. For the alleles studied, in addition to somatic instability, germinal reversions also occurred. In some cases, these reversions resulted in stable derivatives with a different colour from that of the wild-type (‘near green’ or pale phenotypes). The results presented not only allow the v1-m1 allele to be chosen as a starting material for cloning the V1 locus, but also define the molecular strategy to be followed.
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Gardella, J. E., G. A. Gorgone, L. Candela, J. Ghiso, E. M. Castaño, B. Frangione, and P. D. Gorevic. "High-level expression and in vitro mutagenesis of a fibrillogenic 109-amino-acid C-terminal fragment of Alzheimer's-disease amyloid precursor protein." Biochemical Journal 294, no. 3 (September 15, 1993): 667–74. http://dx.doi.org/10.1042/bj2940667.
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We amplified DNA encoding the 3′ 109 codons of Alzheimer's-disease amyloid precursor protein (APP) inclusive of the beta protein (A beta) and cytoplasmic domains from cDNA using oligonucleotide primers designed to facilitate cloning into the T7 expression vector pT7Ad23K13. We also modified this construct to generate recombinant molecules incorporating two recently described APP mutants by site-directed mutagenesis. Both native C109 (deletion construct inclusive of the C-terminal 109 residues of APP) and constructs with a single mutation at codon 642 (T-->G, resulting in a substitution of glycine for valine) or a double mutation at codons 595 (G-->T, substituting asparagine for lysine) and 596 (A-->C, substituting leucine for methionine) were expressed in Escherichia coli to levels of 5-20% of total bacterial protein after induction. The major constituent of expressed C109 protein had an apparent molecular mass of 16-18 kDa by SDS/PAGE and appeared to be the full-length construct by size and N-terminal microsequencing. Also present was a 4-5 kDa species that co-purified with C109, constituting only approximately 1% of expressed protein, which was revealed by Western-blot analysis with antibodies specific for A beta epitopes and after biotinylation of purified recombinant C109. This fragment shared N-terminal sequence with, and appeared to arise by proteolysis of, full-length C109 in biosynthetic labelling experiments. C109 spontaneously precipitated after dialysis against NaCl or water, and with prolonged (> 20 weeks) standing was found by electron microscopy to contain a minor (< 5%) fibrillar component that was reactive with antibodies to a C-terminal epitope of APP. Recombinant C109 appears to duplicate some of the biochemical and physicochemical properties of C-terminal A beta-inclusive fragments of APP that have been found in transfected cells, brain cortex and cerebral microvessels.
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Burgess, Shawn, Gerlinde Reim, Wenbiao Chen, Nancy Hopkins, and Michael Brand. "The zebrafishspiel-ohne-grenzen(spg) gene encodes the POU domain protein Pou2 related to mammalianOct4and is essential for formation of the midbrain and hindbrain, and for pre-gastrula morphogenesis." Development 129, no. 4 (February 15, 2002): 905–16. http://dx.doi.org/10.1242/dev.129.4.905.
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In early embryonic development, the brain is divided into three main regions along the anteroposterior axis: the forebrain, midbrain and hindbrain. Through retroviral insertional mutagenesis and chemical mutagenesis experiments in zebrafish, we have isolated mutations that cause abnormal hindbrain organization and a failure of the midbrain-hindbrain boundary (MHB) to form, a region that acts as an organizer for the adjacent brain regions. The mutations fail to complement the spiel-ohne-grenzen (spg) mutation, which causes a similar phenotype, but for which the affected gene is unknown. We show through genetic mapping, cloning of the proviral insertion site and allele sequencing that spg mutations disrupt pou2, a gene encoding the Pou2 transcription factor. Based on chromosomal synteny, phylogenetic sequence comparison, and expression and functional data, we suggest that pou2 is the zebrafish ortholog of mouse Oct3/Oct4 and human POU5F1. For the mammalian genes, a function in brain development has so far not been described. In the absence of functional pou2, expression of markers for the midbrain, MHB and the hindbrain primordium (pax2.1, wnt1, krox20) are severely reduced, correlating with the neuroectoderm-specific expression phase of pou2. Injection of pou2 mRNA restores these defects in spg mutant embryos, but does not activate these markers ectopically, demonstrating a permissive role for pou2. Injections of pou2-morpholinos phenocopy the spg phenotype at low concentration, further proving that spg encodes pou2. Two observations suggest that pou2 has an additional earlier function: higher pou2-morpholino concentrations specifically cause a pre-gastrula arrest of cell division and morphogenesis, and expression of pou2 mRNA itself is reduced in spg-homozygous embryos at this stage. These experiments suggest two roles for pou2. Initially, Pou2 functions during early proliferation and morphogenesis of the blastomeres, similar to Oct3/4 in mammals during formation of the inner cell mass. During zebrafish brain formation, Pou2 then functions a second time to activate gene expression in the midbrain and hindbrain primordium, which is reflected at later stages in the specific lack in spg embryos of the MHB and associated defects in the mid- and hindbrain.
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Wang, N., Y. Z. Xie, Y. Z. Li, S. N. Wu, H. S. Wei, and C. S. Wang. "Molecular mapping of a novel early leaf-senescence gene Els2 in common wheat by SNP genotyping arrays." Crop and Pasture Science 71, no. 4 (2020): 356. http://dx.doi.org/10.1071/cp19435.
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Early leaf senescence in wheat (Triticum aestivum L.) is one of the limiting factors for developing high yield potential. In this study, a stably inherited, early leaf-senescence mutant LF2099 was initially identified in an M2 population of the common wheat accession H261 after ethyl methanesulfonate (EMS) mutagenesis. Early leaf senescence was observed in the LF2099 mutant during the three-leaf-stage, and then the etiolated area of the wheat leaf increased gradually from the bottom to the top throughout development. Compared with H261, the chlorophyll (Chl a, Chl b) and carotenoid contents and photosynthetic capacity of the mutant were significantly decreased. All of its yield-related traits except for spike length were also significantly reduced. Dissolved cytoplasm, abnormal chloroplast structure, dissolved chloroplast membrane, abnormal thylakoid development, and more plastoglobules were observed in the senescent leaf region of the mutant by transmission electronic microscope. Genetic analysis indicated that the early leaf-senescence phenotype is controlled by an incomplete-dominance nuclear gene, here designated Els2. Using single nucleotide polymorphisms and bulked segregant analysis, the els2 gene was anchored in a region on chromosome 2BL between simple sequence repeat (SSR) markers gpw4043 and wmc149. Six new polymorphic SSR markers were developed from the Chinese Spring 2BL shotgun survey sequence contigs. By means of comparative genomics analyses, the collinearity genomic regions of the els2 locus on wheat 2BL were identified in Brachypodium distachyon chromosome 5, rice (Oryza sativa) chromosome 4 and sorghum (Sorghum bicolor) chromosome 6. Five intron polymorphism (IP) markers were further developed from this collinearity genomic region. Ultimately, Els2 was mapped in a genetic interval of 0.95 cM flanked by IP markers 2BIP09 and 2BIP14. The co-segregating IP markers 2BIP12 and 2BIP17 provide a starting point for the fine mapping and map-based cloning of Els2.
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de Bruijn, I., M. J. D. de Kock, P. de Waard, T. A. van Beek, and J. M. Raaijmakers. "Massetolide A Biosynthesis in Pseudomonas fluorescens." Journal of Bacteriology 190, no. 8 (November 9, 2007): 2777–89. http://dx.doi.org/10.1128/jb.01563-07.
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ABSTRACT Massetolide A is a cyclic lipopeptide (CLP) antibiotic produced by various Pseudomonas strains from diverse environments. Cloning, sequencing, site-directed mutagenesis, and complementation showed that massetolide A biosynthesis in P. fluorescens SS101 is governed by three nonribosomal peptide synthetase (NRPS) genes, designated massA, massB, and massC, spanning approximately 30 kb. Prediction of the nature and configuration of the amino acids by in silico analysis of adenylation and condensation domains of the NRPSs was consistent with the chemically determined structure of the peptide moiety of massetolide A. Structural analysis of massetolide A derivatives produced by SS101 indicated that most of the variations in the peptide moiety occur at amino acid positions 4 and 9. Regions flanking the mass genes contained several genes found in other Pseudomonas CLP biosynthesis clusters, which encode LuxR-type transcriptional regulators, ABC transporters, and an RND-like outer membrane protein. In contrast to most Pseudomonas CLP gene clusters known to date, the mass genes are not physically linked but are organized in two separate clusters, with massA disconnected from massB and massC. Quantitative real-time PCR analysis indicated that transcription of massC is strongly reduced when massB is mutated, suggesting that these two genes function in an operon, whereas transcription of massA is independent of massBC and vice versa. Massetolide A is produced in the early exponential growth phase, and biosynthesis appears not to be regulated by N-acylhomoserine lactone-based quorum sensing. Massetolide A production is essential in swarming motility of P. fluorescens SS101 and plays an important role in biofilm formation.
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Basu, Soumik, Piyali Pal Chowdhury, Satamita Deb, and Tapan K. Dutta. "Degradation Pathways of 2- and 4-Nitrobenzoates in Cupriavidus sp. Strain ST-14 and Construction of a Recombinant Strain, ST-14::3NBA, Capable of Degrading 3-Nitrobenzoate." Applied and Environmental Microbiology 82, no. 14 (May 6, 2016): 4253–63. http://dx.doi.org/10.1128/aem.00739-16.
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ABSTRACTStrain ST-14, characterized as a member of the genusCupriavidus, was capable of utilizing 2- and 4-nitrobenzoates individually as sole sources of carbon and energy. Biochemical studies revealed the assimilation of 2- and 4-nitrobenzoates via 3-hydroxyanthranilate and protocatechuate, respectively. Screening of a genomic fosmid library of strain ST-14 constructed inEscherichia coliidentified two gene clusters,onbandpob-pca, to be responsible for the complete degradation of 2-nitrobenzoate and protocatechuate, respectively. Additionally, a gene segment (pnb) harboring the genes for the conversion of 4-nitrobenzoate to protocatechuate was unveiled by transposome mutagenesis. Reverse transcription-PCR analysis showed the polycistronic nature of the gene clusters, and their importance in the degradation of 2- and 4-nitrobenzoates was ascertained by gene knockout analysis. Cloning and expression of the relevant pathway genes revealed the transformation of 2-nitrobenzoate to 3-hydroxyanthranilate and of 4-nitrobenzoate to protocatechuate. Finally, incorporation of functional 3-nitrobenzoate dioxygenase into strain ST-14 allowed the recombinant strain to utilize 3-nitrobenzoate via the existing protocatechuate metabolic pathway, thereby allowing the degradation of all three isomers of mononitrobenzoate by a single bacterial strain.IMPORTANCEMononitrobenzoates are toxic chemicals largely used for the production of various value-added products and enter the ecosystem through industrial wastes. Bacteria capable of degrading mononitrobenzoates are relatively limited. Unlike other contaminants, these man-made chemicals have entered the environment since the last century, and it is believed that bacteria in nature evolved not quite efficiently to assimilate these compounds; as a consequence, to date, there are only a few reports on the bacterial degradation of one or more isomers of mononitrobenzoate. In the present study, fortunately, we have been able to isolate aCupriavidussp. strain capable of assimilating both 2- and 4-nitrobenzoates as the sole carbon source. Results of the biochemical and molecular characterization of catabolic genes responsible for the degradation of mononitrobenzoates led us to manipulate a single enzymatic step, allowing the recombinant host organism to expand its catabolic potential to assimilate 3-nitrobenzoate.
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Segura, Ana, Estrella Duque, Ana Hurtado, and Juan L. Ramos. "Mutations in Genes Involved in the Flagellar Export Apparatus of the Solvent-Tolerant Pseudomonas putida DOT-T1E Strain Impair Motility and Lead to Hypersensitivity to Toluene Shocks." Journal of Bacteriology 183, no. 14 (July 15, 2001): 4127–33. http://dx.doi.org/10.1128/jb.183.14.4127-4133.2001.
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ABSTRACT Pseudomonas putida DOT-T1E is a solvent-tolerant strain able to grow in the presence of 1% (vol/vol) toluene in the culture medium. Random mutagenesis with mini-Tn5-′phoA-Km allowed us to isolate a mutant strain (DOT-T1E-42) that formed blue colonies on Luria-Bertani medium supplemented with 5-bromo-4-chloro-3-indolylphosphate and that, in contrast to the wild-type strain, was unable to tolerate toluene shocks (0.3%, vol/vol). The mutant strain exhibited patterns of tolerance or sensitivity to a number of antibiotics, detergents, and chelating agents similar to those of the wild-type strain. The mutation in this strain therefore seemed to specifically affect toluene tolerance. Cloning and sequencing of the mutation revealed that the mini-Tn5-′phoA-Km was inserted within the fliPgene, which is part of the fliLMNOPQRflhBA cluster, a set of genes that encode flagellar structure components. FliP is involved in the export of flagellar proteins, and in fact, theP. putida fliP mutant was nonmotile. The finding that, after replacing the mutant allele with the wild-type one, the strain recovered the wild-type pattern of toluene tolerance and motility unequivocally assigned FliP a function in solvent resistance. An flhB knockout mutant, another gene component of the flagellar export apparatus, was also nonmotile and hypersensitive to toluene. In contrast, a nonpolar mutation at the fliLgene, which encodes a cytoplasmic membrane protein associated with the flagellar basal body, yielded a nonmotile yet toluene-resistant strain. The results are discussed regarding a possible role of the flagellar export apparatus in the transport of one or more proteins necessary for toluene tolerance in P. putida DOT-T1E to the periplasm.
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Lin, Hong-Yan, Xi Chen, Jia-Nan Chen, Da-Wei Wang, Feng-Xu Wu, Song-Yun Lin, Chang-Guo Zhan, Jia-Wei Wu, Wen-Chao Yang, and Guang-Fu Yang. "Crystal Structure of 4-Hydroxyphenylpyruvate Dioxygenase in Complex with Substrate Reveals a New Starting Point for Herbicide Discovery." Research 2019 (July 8, 2019): 1–11. http://dx.doi.org/10.34133/2019/2602414.
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4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a promising target for drug and pesticide discovery. The unknown binding mode of substrate is still a big challenge for the understanding of enzymatic reaction mechanism and novel HPPD inhibitor design. Herein, we determined the first crystal structure of Arabidopsis thaliana HPPD (AtHPPD) in complex with its natural substrate (HPPA) at a resolution of 2.80 Å. Then, combination of hybrid quantum mechanics/molecular mechanics (QM/MM) calculations confirmed that HPPA takes keto rather than enol form inside the HPPD active pocket. Subsequent site-directed mutagenesis and kinetic analysis further showed that residues (Phe424, Asn423, Glu394, Gln307, Asn282, and Ser267) played important roles in substrate binding and catalytic cycle. Structural comparison between HPPA-AtHPPD and holo-AtHPPD revealed that Gln293 underwent a remarkable rotation upon the HPPA binding and formed H-bond network of Ser267-Asn282-Gln307-Gln293, resulting in the transformation of HPPD from an inactive state to active state. Finally, taking the conformation change of Gln293 as a target, we proposed a new strategy of blocking the transformation of HPPD from inactive state to active state to design a novel inhibitor with Ki value of 24.10 nM towards AtHPPD. The inhibitor has entered into industry development as the first selective herbicide used for the weed control in sorghum field. The crystal structure of AtHPPD in complex with the inhibitor (2.40 Å) confirmed the rationality of the design strategy. We believe that the present work provides a new starting point for the understanding of enzymatic reaction mechanism and the design of next generation HPPD inhibitors.
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Gillet, L., V. Daix, G. Donofrio, M. Wagner, U. H. Koszinowski, B. China, M. Ackermann, N. Markine-Goriaynoff, and A. Vanderplasschen. "Development of bovine herpesvirus 4 as an expression vector using bacterial artificial chromosome cloning." Journal of General Virology 86, no. 4 (April 1, 2005): 907–17. http://dx.doi.org/10.1099/vir.0.80718-0.
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Several features make bovine herpesvirus 4 (BoHV-4) attractive as a backbone for use as a viral expression vector and/or as a model to study gammaherpesvirus biology. However, these developments have been impeded by the difficulty in manipulating its large genome using classical homologous recombination in eukaryotic cells. In the present study, the feasibility of exploiting bacterial artificial chromosome (BAC) cloning and prokaryotic recombination technology for production of BoHV-4 recombinants was explored. Firstly, the BoHV-4 genome was BAC cloned using two potential insertion sites. Both sites of insertion gave rise to BoHV-4 BAC clones stably maintained in bacteria and able to regenerate virions when transfected into permissive cells. Reconstituted virus replicated comparably to wild-type parental virus and the loxP-flanked BAC cassette was excised by growing them on permissive cells stably expressing Cre recombinase. Secondly, BoHV-4 recombinants expressing Ixodes ricinus anti-complement protein I or II (IRAC I/II) were produced using a two-step mutagenesis procedure in Escherichia coli. Both recombinants induced expression of high levels of functional IRAC molecules in the supernatant of infected cells. This study demonstrates that BAC cloning and prokaryotic recombination technology are powerful tools for the development of BoHV-4 as an expression vector and for further fundamental studies of this gammaherpesvirus.
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Soverini, Simona, Angela Poerio, Caterina Debenedittis, Ilaria Iacobucci, Sabrina Colarossi, Alessandra Gnani, Fausto Castagnetti, et al. "Low-Level Bcr-Abl Kinase Domain Mutations Are Very Rare In Chronic Myeloid Leukemia Patients Who Are In Major Molecular Response After 12 Months of First-Line Nilotinib Therapy." Blood 116, no. 21 (November 19, 2010): 1666. http://dx.doi.org/10.1182/blood.v116.21.1666.1666.
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Abstract Abstract 1666 Bcr-Abl kinase domain (KD) mutations have been documented in a variable proportion of residual Ph+ cells in some patients (pts) with stable response to imatinib. In general, the higher the sensitivity of the screening method, the greater the number of pts found to harbour mutations – although the clinical significance of low-level mutations is still controversial. Nilotinib is an imatinib derivative more selective and more potent in Bcr-Abl inhibition – with reported major molecular response (MMR) rates at 12 months ranging between 44 and 85%. We wondered whether low-level Bcr-Abl KD mutations are detectable in pts in MMR on nilotinib. To address this issue, we retrospectively analyzed the samples collected after 12 months of therapy from 12 pts enrolled on the GIMEMA CML working party study of nilotinib 400 mg BID as frontline treatment of CML. These pts had all achieved MMR (>3-log reduction in Bcr-Abl transcript according to the IS) between 3 and 6 months from nilotinib start and had a Bcr-Abl/Abl ratio ranging from 0.009%IS and 0.02%IS at the time of analysis. In all 12 pts, MMR was maintained at last follow up (24 months after nilotinib start), with four pts achieving complete molecular response (CMR; >4.5-log reduction) at 18 months and one at 24 months. Pts were equally distributed across Sokal risk categories (low Sokal risk, n=4; intermediate Sokal risk, n=4; high Sokal risk, n=4). Screening for low level mutations was performed by cloning the Bcr-Abl KD (a.a.240-502) in a bacterial vector and sequencing 150 independent clones for each patient. To rule out false positive results, a mutation was considered to be present in a sample if it was detected on both strands of two or more independent clones. The KD of Abl in 3 healthy individuals was analyzed in parallel. Our approach showed evidence of Bcr-Abl KD mutations in only 1 out of 12 pts analyzed. In this high Sokal risk patient, a Q346L mutation was detected in 3/150 independent clones, and an additional T315I mutation was present in 2 out of these 3 clones. The Q346L has never been reported in imatinib-resistant pts, neither is it among the mutants emerged in the in vitro random mutagenesis screenings for nilotinib-resistant mutations – hence it should be devoid of any clinical relevance. The T315I, in contrast, is known to be highly insensitive to nilotinib both in vitro and in vivo. Nevertheless, in this patient the Bcr-Abl transcript level continued to decline down to CMR (achieved after 24 months from nilotinib start). It may also be hypothesized that the Q346L-T315I double mutant may have decreased degree of resistance with respect to the T315I mutant. The remaining 11 pts scored negative for mutations – showing only evidence of some single, mutated clones as also the three healthy individuals did. To increase the number of pts analyzed and assess whether high Sokal risk pts are more prone to develop low level mutations, we have now set up a massively parallel amplicon sequencing approach of the Abl KD on the Roche 454 GS FLX instrument, allowing to increase both throughput and sensitivity (a 100.000x coverage will allow to detect mutations with a lower detection limit of 0.01%). Our current results suggest that a) low level Bcr-Abl KD mutations seem to be very rare in pts in MMR after 12 months of nilotinib therapy, a milestone achieved by the majority of pts. This may be due to the higher efficacy of nilotinib resulting in a more rapid clearance of the reservoir of Ph+ cells where mutations arise; b) as hypothesized by some authors, tyrosine kinase inhibitor-resistant mutations at low levels do not always predict for subsequent relapse and should not trigger changes in therapy. Supported by: European LeukemiaNet, AIL, AIRC, PRIN 2008, Fondazione del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2008, Ateneo RFO grants, Project of integrated program (PIO), Programma di Ricerca Regione – Università 2007 – 2009. Disclosures: Rosti: Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Baccarani:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Martinelli:Novartis: Consultancy, Honoraria; BMS: Honoraria; Pfizer: Consultancy.
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Rao, D. N., and D. L. Gupta. "Immunology of COVID-19 and its clinical implications for therapy." Journal of Environmental Biology 41, no. 6 (November 15, 2020): XIII—XV. http://dx.doi.org/10.22438/jeb/41/6/editorial.
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The world has already experienced several viral attacks in the 21st Century (Christou, 2011) have affected millions of people globally and caused substantial mortality and morbidity (Sutton, 2018). One that caused global pandemic and fear is newly emerged novel coronavirus (COVID-19) (Wilder-Smith et al., 2020). It is highly contagious, large enveloped, single-stranded, non-segmented, positive-sense RNA viruse that belong to coronaviridae family and genus Beta coronavirus (Yan et al., 2020). The viruses of coronaviridae family are further divided into two subfamilies; Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV) (Kindler et al., 2016). COVID-19 show close similarities to SARS-CoV due to phylogenetic relationship and genome structure. Word Health Organisation (WHO) has designated the current pandemic of SARS-CoV-2 infection as COVID-19 (Sohrabi et al., 2020). The virus shows high infectivity and mainly transmitted in the form of droplets with an incubation period of about 4-5 days. The onset of symptoms in patients is usually within 11-12 days (Linton et al., 2020). The most prominent symptoms seen in almost every patient includes dry cough, fever, fatigue, sore throat, lack of smell and taste, and severe patients suffer from breathing difficulties (Guan et al., 2019). We do not have any effective therapeutics or drugs approved by the U.S. Food and Drug Administration (FDA) to prevent or treat COVID-19 infections presently. The only treatment available for patients with COVID-19 is supportive care, including supplement oxygen and mechanical ventilator support. Therefore, there is an urgent need to develop an effective therapy to treat patients infected with COVID-19. The present situation calls much attention on prophylactics and therapeutics approach. This virus contain four structural proteins viz. the spike (S), membrane(M),envelop (E) and nucleocapsid (N).The S protein is considered to be highly immunogenic and contribute protective immunity in contrast to N, M and E proteins(Saha et al., 2020). Many laboratories have confirmed the entry of virus into human cells after its fusion with cell membrane of target tissues. After fusion, the S protein undergoes proteolysis by host proteases and give rise to S1 and S2 fragments. Thus, S1 protein is ready to interact with angiotensin converting 2 (ACE2) receptors present on respiratory and intestine epithelia besides alveolar macrophages present in the lungs(Cheng et al., 2020). Few studies have confirmed that ACE2 receptors are present in other cells too. S1 protein has a domain that interacts specifically with a domain on the ACE2 receptor. This interaction is must to produce the symptoms of disease (Nadeem et al., 2020).One must understand the role of chloroquine analogues works here by inhibiting virus fusion into the cell membrane. Some laboratories have generated both polyclonal and monoclonal antibodies against S1 domain and inhibited the interaction (Ravichandran et al., 2020; Zhao et al., 2020). This is one of the areas of immunotherapy. Some key amino acids present on the S1 domain initiate this interaction with high affinity (Saha et al., 2020). If these amino acids are blocked by monoclonal antibodies or lacked by chance due to mutation, the rate of infection would be reduced and patients may show minimal symptoms. This protective effect can be seen by reduced affinity of viruses to ACE2 receptor that would result in decreased infection. As the virus is getting mutated, some infected persons shown critical symptoms and other escape the disease severity. If the virus continues to mutate to lower its pathogenicity, there is a high possibility that it might coexist with humans. Using computational and bioinformatics, few laboratories have predicted B cells, T cells and cytotoxic T cells epitopes on S protein as well as other structural proteins (Baruah et al., 2020). This will help us to develop virus specific neutralizing antibodies by gene cloning or peptide synthesis-based approach. We personally feel if we have antisera from recovered patients of COVID-19 infection, using panel of peptides from different regions of viral proteins will help us to develop diagnosis kits and peptide-based vaccine. Since immune response is based on individual Human Leukocytes Antigens (HLA), the above approach will help to identify immunodominant regions on viral proteins. In addition, we can identify the polymorphic loci on HLA genes among infected persons showing clinical symptoms and others escape the disease severity to confirm the susceptibility towards infection, immunity and outcomes. COVID-19 infection has led to inflammatory storm due to presence of hyper activated immune cells and over-production of pro-inflammatory cytokines that may determine the outcome (Ye et al., 2020). Several studies have shown the presence of elevated levels of serum pro-inflammatory cytokines (IL-6, IL-1β and TNF-α) and increased immune inflammatory cells leading to cytokine storm, which could be partially responsible for immune mediated problems in the lungs of the patients(Ye et al., 2020; Felsenstein et al., 2020). Immune mediated, acute respiratory distress syndrome (ARDS) are commonly seen in ICU that are associated with poor outcome in patients with COVID-19 (Zhang et al., 2020).Therefore, specific blockade of inflammatory storm associated with hyper-inflammation during COVID-19 is an important therapeutic approach when compared to the systemic immunosuppression. To our knowledge, few clinical trials are ongoing to test the safety and efficacy of targeted therapies associated with IL-6, IL-1 receptor and Janus kinases (JAK) blockade in severe COVID-19 patients (Neurath, 2020; Russell et al., 2020). Like targeted therapies, several clinical trials of anti-viral drugs therapies such as Remdesivir, Lopinavin and Favipiravin are ongoing study (Şimşek and Ünal, 2020; Cao et al., 2020). The safety and efficacy of these trials are still unclear which need furthermore investigations in patients across the globe. Recently, few studies have confirmed blood clots in lungs and heart of patients with COVI-19 (Connors and Levy, 2020). We feel that treatment with blood thinner or low molecular weight heparin can reduce the problems of blood clots. Development of animal models of coronavirus infection are useful for testing vaccines and antiviral drugs. However, currently available animal models do not reproduce the inflammatory storm or pulmonary disease observed in individuals infected with SARS-CoV (Cleary et al., 2020). SARS-CoV viruses has been found to replicate in the lungs of domestic cats, hamsters, and mice. These animal models remain symptomatic (Tiwari et al., 2020). Therefore, we believe that development of animal models that establishes COVID-19 infections can show symptoms require a transgenic animal approach. The knowledge obtained from the study of animal models will help us in developing specific therapies, and vaccine would minimize pulmonary disease and optimise the inflammatory storm. Finally, we conclude that vaccine is the best for protection against COVID-19 infections, but development of antiviral drugs or targeted therapies are urgently required to cure COVID-19 patients.
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Tao, Zhang, Hu Chun-Yan, Peng Hua, Yang Bin-Bin, and Tang Xiaoping. "Phyllathin From Phyllanthus Amarus Ameliorates Epileptic Convulsion and Kindling Associated Post-Ictal Depression in Mice via Inhibition of NF-κB/TLR-4 Pathway." Dose-Response 18, no. 3 (July 1, 2020): 155932582094691. http://dx.doi.org/10.1177/1559325820946914.
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Background: Epilepsy is a chronic, complex, unprovoked, and recurrent disorder of the nervous system that affected several people worldwide. Phyllanthus amarus (PA) has been documented to have neuroprotective potential. Aim: To evaluate the potential of standardized extract of PA and its possible mechanism of action against the Pentylenetetrazol (PTZ)-induced convulsion and kindling associated post-ictal depression in experimental mice. Materials and Methods: Phyllathin was isolated from methanolic extract of PA and well-characterized using HPTLC, ESI-MS/MS, and LC/MS. Phyllathin containing a standardized extract of PA (50, 100, and 200 mg/kg) was administered in convulsed and kindled mice, followed by an assessment of various parameters. Results: The spectral analysis confirmed the molecular formula and weight of phyllanthin as C24H34O6 and 418.2342 Da. PA (100 and 200 mg/kg) significantly ameliorated PTZ-induced ( p < 0.05) duration, onset of tonic-clonic convulsion, and mortality in mice. It also significantly attenuated ( p < 0.05) PTZ-induced kindling in mice. Alteration in brain GABA, dopamine, and glutamate, Na+K+ATPase, Ca+2-ATPase activities, and oxido-nitrosative stress in kindled mice was significantly restored ( p < 0.05) by PA treatment. It also significantly ( p < 0.05) down-regulated brain mRNA expressions of NF-κB, TNF-α, IL-1β, COX-2, and TLR-4. Histological aberrations induced by PTZ in the brain of a kindled rat was significantly ( p < 0.05) ameliorated by PA. Conclusion: Phyllanthin containing a standardized extract of PA exerts its antiepileptic potential via balancing excitatory (glutamate) and inhibitory (GABA) brain monoamines, voltage-gated ion channels (Na+K+/Ca+2-ATPase) and inhibition of NF-κB/TLR-4 pathway to ameliorate neuroinflammation (TNF-α, IL-1β, and COX-2) in experimental mice.
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Mintmier, Breeanna, Jennifer M. McGarry, Courtney E. Sparacino-Watkins, Joseph Sallmen, Katrin Fischer-Schrader, Axel Magalon, Joseph R. McCormick, et al. "Molecular cloning, expression and biochemical characterization of periplasmic nitrate reductase from Campylobacter jejuni." FEMS Microbiology Letters 365, no. 16 (June 19, 2018). http://dx.doi.org/10.1093/femsle/fny151.
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AbstractCampylobacter jejuni, a human gastrointestinal pathogen, uses nitrate for growth under microaerophilic conditions using periplasmic nitrate reductase (Nap). The catalytic subunit, NapA, contains two prosthetic groups, an iron sulfur cluster and a molybdenum cofactor. Here we describe the cloning, expression, purification, and Michaelis-Menten kinetics (kcat of 5.91 ± 0.18 s−1 and a KM (nitrate) of 3.40 ± 0.44 μM) in solution using methyl viologen as an electron donor. The data suggest that the high affinity of NapA for nitrate could support growth of C. jejuni on nitrate in the gastrointestinal tract. Site-directed mutagenesis was used and the codon for the molybdenum coordinating cysteine residue has been exchanged for serine. The resulting variant NapA is 4-fold less active than the native enzyme confirming the importance of this residue. The properties of the C. jejuni enzyme reported here represent the first isolation and characterization of an epsilonproteobacterial NapA. Therefore, the fundamental knowledge of Nap has been expanded.
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Bhattarai, Krishna, Ana Conesa, Shunyuan Xiao, Natalia A. Peres, David G. Clark, Saroj Parajuli, and Zhanao Deng. "Sequencing and analysis of gerbera daisy leaf transcriptomes reveal disease resistance and susceptibility genes differentially expressed and associated with powdery mildew resistance." BMC Plant Biology 20, no. 1 (November 30, 2020). http://dx.doi.org/10.1186/s12870-020-02742-4.
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Abstract Background RNA sequencing has been widely used to profile genome-wide gene expression and identify candidate genes controlling disease resistance and other important traits in plants. Gerbera daisy is one of the most important flowers in the global floricultural trade, and powdery mildew (PM) is the most important disease of gerbera. Genetic improvement of gerbera PM resistance has become a crucial goal in gerbera breeding. A better understanding of the genetic control of gerbera resistance to PM can expedite the development of PM-resistant cultivars. Results The objectives of this study were to identify gerbera genotypes with contrasting phenotypes in PM resistance and sequence and analyze their leaf transcriptomes to identify disease resistance and susceptibility genes differentially expressed and associated with PM resistance. An additional objective was to identify SNPs and SSRs for use in future genetic studies. We identified two gerbera genotypes, UFGE 4033 and 06–245-03, that were resistant and susceptible to PM, respectively. De novo assembly of their leaf transcriptomes using four complementary pipelines resulted in 145,348 transcripts with a N50 of 1124 bp, of which 67,312 transcripts contained open reading frames and 48,268 were expressed in both genotypes. A total of 494 transcripts were likely involved in disease resistance, and 17 and 24 transcripts were up- and down-regulated, respectively, in UFGE 4033 compared to 06–245-03. These gerbera disease resistance transcripts were most similar to the NBS-LRR class of plant resistance genes conferring resistance to various pathogens in plants. Four disease susceptibility transcripts (MLO-like) were expressed only or highly expressed in 06–245-03, offering excellent candidate targets for gene editing for PM resistance in gerbera. A total of 449,897 SNPs and 19,393 SSRs were revealed in the gerbera transcriptomes, which can be a valuable resource for developing new molecular markers. Conclusion This study represents the first transcriptomic analysis of gerbera PM resistance, a highly important yet complex trait in a globally important floral crop. The differentially expressed disease resistance and susceptibility transcripts identified provide excellent targets for development of molecular markers and genetic maps, cloning of disease resistance genes, or targeted mutagenesis of disease susceptibility genes for PM resistance in gerbera.
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Kargar, Farzane, Mojtaba Mortazavi, Mahmood Maleki, Masoud Torkzadeh Mahani, Younes Ghasemi, and Amir Savardashtaki. "Isolation, identification and in silico study of native cellulase producing bacteria." Current Proteomics 17 (November 27, 2019). http://dx.doi.org/10.2174/1570164617666191127142035.
Full textAbstract:
Aims: The purpose of this study was to screen the bacteria producing cellulase enzymes and their bioinformatics studies. Background: Cellulose is a long-chain polymer of glucose that hydrolyzes by cellulases to glucose molecules. In order to design the new biotechnological applications, some strategies have been used as increasing the efficiency of enzyme production, generating cost-effective enzymes, producing stable enzymes and identification of new strains. Objective: On the other hand, some bacteria special features have made them suitable candidates for the identification of the new source of enzymes. In this regard, some native strains of bacteria were screened. Method: These bacteria were grown on a culture containing the liquid M9 media containing CMC to ensure the synthesis of cellulase. The formation of a clear area in the culture medium indicated decomposition of cellulose. In the following, the DNA of these bacteria were extracted and their 16S rDNA genes were amplified. Result: The results show that nine samples were able to synthesize cellulase. In following, these strains were identified using 16S rDNA. The results show that these screened bacteria belonged to the Bacillus sp., Alcaligenes sp., Alcaligenes sp., and Enterobacter sp.conclusionThe enzyme activity analysis shows that the Bacillus toyonensis, Bacillus sp. strain XA15-411 Bacillus cereus have produced the maximum yield of cellulases. However, these amounts of enzyme production in these samples are not proportional to their growth rate. As the bacterial growth chart within 4 consecutive days shows that the Alcaligenes sp. Bacillus cereus, Bacillus toyonensis, Bacillus sp. strain XA15-411 have a maximum growth rate. The study of the phylogenetic tree also shows that Bacillus species are more abundant in the production of cellulase enzyme. These bioinformatics analyses show that the Bacillus species have different evolutionary relationships and evolved in different evolutionary time. Other: However, for maximum cellulase production by this bacteria, some information as optimum temperature, optimum pH, carbon and nitrogen sources are needed for the ideal formulation of media composition. The cellulase production is closely controlled in microorganisms and the cellulase yields appear to depend on a variety of factors. However, the further studies are needed for cloning, purification and application of these new microbial cellulases in the different commercial fields as in food, detergent, and pharmaceutical, paper, textile industries and also various chemical industries. However, these novel enzymes can be further engineered through rational design or using random mutagenesis techniques.