scholarly journals Involvement of Sialoadhesin in Entry of Porcine Reproductive and Respiratory Syndrome Virus into Porcine Alveolar Macrophages

2003 ◽  
Vol 77 (15) ◽  
pp. 8207-8215 ◽  
Author(s):  
Nathalie Vanderheijden ◽  
Peter L. Delputte ◽  
Herman W. Favoreel ◽  
Joël Vandekerckhove ◽  
Jozef Van Damme ◽  
...  
Keyword(s):  
Amino Acid ◽  
Viral Entry ◽  
Alveolar Macrophages ◽  
Amino Acid Identity ◽  
Peptide Sequencing ◽  
Respiratory Syndrome Virus ◽  
Vesicle Membrane ◽  
Virus Particles ◽  
Internal Peptide ◽  
Peptide Data

ABSTRACT Porcine reproductive and respiratory syndrome virus (PRRSV) shows a very restricted tropism for cells of the monocyte/macrophage lineage. It enters cells via receptor-mediated endocytosis. A monoclonal antibody (MAb) that is able to block PRRSV infection of porcine alveolar macrophages (PAM) and that recognizes a 210-kDa protein (p210) was described previously (MAb41D3) (X. Duan, H. Nauwynck, H. Favoreel, and M. Pensaert, J. Virol. 72:4520-4523, 1998). In the present study, the p210 protein was purified from PAM by immunoaffinity using MAb41D3 and was subjected to internal peptide sequencing after tryptic digestion. Amino acid sequence identities ranging from 56 to 91% with mouse sialoadhesin, a macrophage-restricted receptor, were obtained with four p210 peptides. Using these peptide data, the full p210 cDNA sequence (5,193 bp) was subsequently determined. It shared 69 and 78% amino acid identity, respectively, with mouse and human sialoadhesins. Swine (PK-15) cells resistant to viral entry were transfected with the cloned p210 cDNA and inoculated with European or American PRRSV strains. Internalized virus particles were detected only in PK-15 cells expressing the recombinant sialoadhesin, demonstrating that this glycoprotein mediated uptake of both types of strains. However, nucleocapsid disintegration, like that observed in infected Marc-145 cells as a result of virus uncoating after fusion of the virus with the endocytic vesicle membrane, was not observed, suggesting a block in the fusion process. The ability of porcine sialoadhesin to mediate endocytosis was demonstrated by specific internalization of MAb41D3 into PAM. Altogether, these results show that sialoadhesin is involved in the entry process of PRRSV in PAM.

scholarly journals Cloning and Biochemical Characterization of a Novel Carbendazim (Methyl-1H-Benzimidazol-2-ylcarbamate)-Hydrolyzing Esterase from the Newly Isolated Nocardioides sp. Strain SG-4G and Its Potential for Use in Enzymatic Bioremediation

2010 ◽  
Vol 76 (9) ◽  
pp. 2940-2945 ◽  
Author(s):  
Gunjan Pandey ◽  
Susan J. Dorrian ◽  
Robyn J. Russell ◽  
Clint Brearley ◽  
Steven Kotsonis ◽  
...  
Keyword(s):  
Amino Acid ◽  
Reverse Genetics ◽  
Biochemical Characterization ◽  
Hypothetical Protein ◽  
Amino Acid Identity ◽  
Peptide Sequencing ◽  
Amino Acid Residues ◽  
Serine Hydrolase ◽  
Freeze Dried ◽  
Cognate Gene

ABSTRACT A highly efficient carbendazim (methyl-1H-benzimidazol-2-ylcarbamate, or MBC)-mineralizing bacterium was isolated from enrichment cultures originating from MBC-contaminated soil samples. This bacterium, Nocardioides sp. strain SG-4G, hydrolyzed MBC to 2-aminobenzimidazole, which in turn was converted to the previously unknown metabolite 2-hydroxybenzimidazole. The initial steps of this novel metabolic pathway were confirmed by growth and enzyme assays and liquid chromatography-mass spectrometry (LC-MS) studies. The enzyme responsible for carrying out the first step was purified and subjected to N-terminal and internal peptide sequencing. The cognate gene, named mheI (for MBC-hydrolyzing enzyme), was cloned using a reverse genetics approach. The MheI enzyme was found to be a serine hydrolase of 242 amino acid residues. Its nearest known relative is an uncharacterized hypothetical protein with only 40% amino acid identity to it. Codon optimized mheI was heterologously expressed in Escherichia coli, and the His-tagged enzyme was purified and biochemically characterized. The enzyme has a Km and k cat of 6.1 μM and 170 min−1, respectively, for MBC. Radiation-killed, freeze-dried SG-4G cells showed strong and stable MBC detoxification activity suitable for use in enzymatic bioremediation applications.

scholarly journals Characterization and cloning of avian-hepatic glutathione S-transferases

1999 ◽  
Vol 343 (1) ◽  
pp. 87-93 ◽  
Author(s):  
Cheng-Hsilin HSIEH ◽  
Li-Fan LIU ◽  
Shu-Ping TSAI ◽  
Ming F. TAM
Keyword(s):  
Amino Acid ◽  
Peptide Sequencing ◽  
Cdna Sequences ◽  
Coding Regions ◽  
Liver Cdna Library ◽  
Cytosolic Glutathione ◽  
Glutathione S Transferases ◽  
Molecular Masses ◽  
Internal Peptide ◽  
Terminal Amino

Cytosolic glutathione S-transferases (GSTs) were isolated from 1-day-old Leghorn chick livers by glutathione (GSH)-affinity chromatography. After sample loading and extensive washing with 0.2 M NaCl, the column was sequentially eluted with 5 mM GSH and 1 mM S-hexylglutathione. The isolated GSTs were subjected to reverse-phase HPLC, electrospray ionization-MS, N-terminal and internal peptide sequencing analyses. The proteins recovered from the 5 mM GSH eluant were predominantly cGSTM1. A protein (cGSTM1′) with an N-terminal amino acid sequence identical to that of cGSTM1 but with the initiator methionine retained and a novel class-mu isozyme (cGSTM2*) were also recovered from this fraction. Nine class-alpha isozymes with distinctive molecular masses were identified from the 1 mM S-hexylglutathione eluant. Three of these proteins are probably variants with minor amino acid substitutions of other isozymes. Of the six remaining class-alpha isozymes, three of them have had their complete (cGSTA1 and cGSTA2) or partial (cGSTA3) cDNA sequences reported previously in the literature. A chicken liver cDNA library was screened with oligonucleotides generated from the cGSTA2 sequence as probes. Clones that encompass the complete coding regions of cGSTA3 and cGSTA4 were obtained. A clone encoding the C-terminal 187 residues of cGSTA5 was also isolated.

scholarly journals A theoretical analysis of single molecule protein sequencing via weak binding spectra

2018 ◽  
Author(s):  
Samuel Rodriques ◽  
Adam Marblestone ◽  
Ed Boyden
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Single Molecule ◽  
Amino Acid Identity ◽  
Peptide Sequencing ◽  
Protein Sequencing ◽  
Single Amino Acid ◽  
Weak Binding ◽  
Binding Spectra ◽  
Accuracy Determination

AbstractWe propose and theoretically study an approach to massively parallel single molecule peptide sequencing, based on single molecule measurement of the kinetics of probe binding [1] to the N-termini of immobilized peptides. Unlike previous proposals, this method is robust to both weak and non-specific probe-target affinities, which we demonstrate by applying the method to a range of randomized affinity matrices consisting of relatively low-quality binders. This suggests a novel principle for proteomic measurement whereby highly non-optimized sets of low-affinity binders could be applicable for protein sequencing, thus shifting the burden of amino acid identification from biomolecular design to readout. Measurement of probe occupancy times, or of time-averaged fluorescence, should allow high-accuracy determination of N-terminal amino acid identity for realistic probe sets. The time-averaged fluorescence method scales well to extremely weak-binding probes. We argue that this method could lead to an approach with single amino acid resolution and the ability to distinguish many canonical and modified amino acids, even using highly non-optimized probe sets. This readout method should expand the design space for single molecule peptide sequencing by removing constraints on the properties of the fluorescent binding probes.Author summaryWe simplify the problem of single molecule protein sequencing by proposing and analyzing an approach that makes use of low-affinity, low-specificity binding reagents. This decouples the problem of protein sequencing from the problem of generating a high-quality library of binding reagents against each of the amino acids.

Variation analysis of the ORF5 gene of Porcine reproductive and respiratory syndrome virus

2004 ◽  
Vol 1 (3) ◽  
pp. 173-179
Author(s):  
Gao Zhi-Qiang ◽  
Guo Xin ◽  
Cha Zhen-Lin ◽  
Chen Yan-Hong ◽  
Yang Han-Chun
Keyword(s):  
Amino Acids ◽  
Phylogenetic Analysis ◽  
Amino Acid ◽  
Amino Acid Identity ◽  
Respiratory Syndrome Virus ◽  
Variation Analysis ◽  
Rt Pcr ◽  
Acid Identity ◽  
Pig Farms ◽  
Orf5 Gene

AbstractThree Porcine reproductive and respiratory syndrome virus (PRRSV) isolates (HB-1(sh)/2002, HB-2(sh)/2002 and JX-1/2002) were obtained from pig farms in Hebei and Jiangxi provinces, China. The complete ORF5 gene of the isolates was amplified using RT-PCR and sequenced. It was shown that ORF5 genes of all isolates encoded 200 amino acids. Comparing ORF5 genes of the three isolates and published sequences for five other PRRSV isolates in China, variation analysis showed that all of the isolates were of the American genotype, with 88.2–99.0% amino acid identity. ORF5 genes among BJ-4, S1 and J1 had higher similarity, sharing 98–99% identity of the deduced amino acids. HB-1(sh)/2002, HB-2(sh)/2002 and JX-1/2002 and CH-1a presented 92–96% identity among their ORF5 genes. Phylogenetic analysis revealed that these isolates could be divided into two subgroups based on the genetic distance of their ORF5 gene: the first subgroup comprised BJ-4, S1 and J1 and was closer to VR2332 and vaccine strains; the second included HB-1(sh)/2002, HB-2(sh)/2002, JX-1/2002 and CH-1a.

scholarly journals First Report of Zantedeschia mosaic virus Infecting a Zantedeschia sp. in New Zealand

Plant Disease ◽  
2008 ◽  
Vol 92 (8) ◽  
pp. 1253-1253 ◽  
Author(s):  
T. Wei ◽  
M. N. Pearson ◽  
D. Cohen ◽  
J. Z. Tang ◽  
G. R. G. Clover
Keyword(s):  
New Zealand ◽  
Amino Acid ◽  
Mosaic Virus ◽  
Amino Acid Identity ◽  
Cut Flowers ◽  
Rt Pcr ◽  
Acid Identity ◽  
Virus Particles ◽  
Blast Search ◽  
Cp Gene

In February 2004, leaf yellowing, mottling, and mosaics were observed on a few plants of a Zantedeschia sp. (calla lily) growing in Rangiora, Canterbury, New Zealand. Zantedeschia spp. are known to be susceptible to at least 13 virus species (1). No symptoms were observed on Chenopodium amaranticolor, C. quinoa, Cucumis sativus, Gomphrena globosa, Nicotiana benthamiana, N. clevelandii, N. occidentalis, or N. tabacum when inoculated with sap from symptomatic plants. However, electron microscopy of crude sap preparations from a symptomatic Zantedeschia sp. and inoculated N. clevelandii plants revealed the presence of flexuous, filamentous virus particles approximately 700 nm long and 12 nm wide. No virus particles were seen in the other inoculated indicator species. Nucleic acid was extracted from leaves of the infected Zantedeschia sp. and N. clevelandii plants and tested in reverse transcription (RT)-PCR using published potyvirus-specific primers (4). PCR amplicons of the expected size (327 bp) were obtained from both plant species and sequenced directly. The products were identical, and a BLAST search in GenBank showed 99% nucleotide identity with a Taiwanese isolate of the species Zantedeschia mosaic virus (ZaMV) (GenBank Accession No. AY026463). A product of 1,531 bp (GenBank Accession No. EU544542) was amplified from symptomatic Zantedeschia by RT-PCR using novel forward (5′-GCACGGCAGATAAACACGAC-3′) and reverse (5′-GTGGGCAACCTTCAACTGTG-3′) primers designed to amplify the 3′ untranslated region (3′UTR), coat protein (CP), and partial nuclear inclusion b protein (NIb) genes. The product was sequenced and had 94% nucleotide identity with a South Korean ZaMV isolate (GenBank Accession No. AB081519), with 95% nucleotide (97% amino acid) identity in the CP gene. A second crop of Zantedeschia spp. in Tauranga, New Zealand (approximately 700 km north of Rangiora) was observed to have similar disease symptoms. Symptomatic plants tested positive in ELISA using a potyvirus-specific monoclonal antibody (Agdia Inc., Elkhart, IN). Nucleic acid was extracted from leaves of symptomatic plants and tested in RT-PCR using potyvirus-specific primer pairs, PV2I/T7 and D335 and U335 and PV1/SP6, which amplify overlapping regions within the 3′UTR, CP, and NIb genes (2,3). The products were sequenced and a consensus sequence of 1,793 bp was generated (GenBank Accession No. EU532065). A BLAST search showed that the sequence had 78% nucleotide (88% amino acid) identity with Zantedeschia mild mosaic virus (ZaMMV) (GenBank Accession No. AY626825). However, the sequences had only 73% nucleotide (79% amino acid) identity in the CP gene, and therefore, this second virus may be a distinct species. To our knowledge, this is the first report of ZaMV in New Zealand. Cut flowers are an increasingly important commodity in New Zealand and Zantedeschia is one of the most important crops; in 2005, exports of rhizomes and cut flowers of the genus were worth NZ$10.9 million. These viral diseases may require management to ensure that the quality of production is maintained. References: (1) C. H. Huang et al. Plant Pathol. 56:183, 2007. (2) S. A. Langeveld et al. J. Gen. Virol. 72:1531, 1991. (3) A. M. Mackenzie et al. Arch. Virol. 143:903, 1998. (4) V. Marie-Jeanne et al. J. Phytopathol. 148:141, 2000.

scholarly journals Recent Advances in PRRS Virus Receptors and the Targeting of Receptor–Ligand for Control

Vaccines ◽  
2021 ◽  
Vol 9 (4) ◽  
pp. 354
Author(s):  
Chia-Ming Su ◽  
Raymond Robert Richard Rowland ◽  
Dongwan Yoo
Keyword(s):  
Viral Entry ◽  
Critical Role ◽  
Scavenger Receptor ◽  
Viral Pathogenesis ◽  
Respiratory Syndrome Virus ◽  
Receptor Ligand ◽  
Cellular Receptors ◽  
Prrs Virus ◽  
Recent Advances ◽  
Virus Receptors

Cellular receptors play a critical role in viral infection. At least seven cellular molecules have been identified as putative viral entry mediators for porcine reproductive and respiratory syndrome virus (PRRSV). Accumulating data indicate that among these candidates, CD163, a cysteine-rich scavenger receptor on macrophages, is the major receptor for PRRSV. This review discusses the recent advances and understanding of the entry of PRRSV into cells, viral pathogenesis in CD163 gene-edited swine, and CD163 as a potential target of receptor–ligand for the control of PRRS.

scholarly journals ampG Gene of Pseudomonas aeruginosa and Its Role in β-Lactamase Expression

2010 ◽  
Vol 54 (11) ◽  
pp. 4772-4779 ◽  
Author(s):  
Ying Zhang ◽  
Qiyu Bao ◽  
Luc A. Gagnon ◽  
Ann Huletsky ◽  
Antonio Oliver ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Transmembrane Protein ◽  
Amino Acid Identity ◽  
Clinical Isolates ◽  
Signal Molecules ◽  
Carbonyl Cyanide ◽  
Increased Sensitivity ◽  
Potential Strategy ◽  
High Level

ABSTRACT In enterobacteria, the ampG gene encodes a transmembrane protein (permease) that transports 1,6-GlcNAc-anhydro-MurNAc and the 1,6-GlcNAc-anhydro-MurNAc peptide from the periplasm to the cytoplasm, which serve as signal molecules for the induction of ampC β-lactamase. The role of AmpG as a transporter is also essential for cell wall recycling. Pseudomonas aeruginosa carries two AmpG homologues, AmpG (PA4393) and AmpGh1 (PA4218), with 45 and 41% amino acid sequence identity, respectively, to Escherichia coli AmpG, while the two homologues share only 19% amino acid identity. In P. aeruginosa strains PAO1 and PAK, inactivation of ampG drastically repressed the intrinsic β-lactam resistance while ampGh1 deletion had little effect on the resistance. Further, deletion of ampG in an ampD-null mutant abolished the high-level β-lactam resistance that is associated with the loss of AmpD activity. The cloned ampG gene is able to complement both the P. aeruginosa and the E. coli ampG mutants, while that of ampGh1 failed to do so, suggesting that PA4393 encodes the only functional AmpG protein in P. aeruginosa. We also demonstrate that the function of AmpG in laboratory strains of P. aeruginosa can effectively be inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP), causing an increased sensitivity to β-lactams among laboratory as well as clinical isolates of P. aeruginosa. Our results suggest that inhibition of the AmpG activity is a potential strategy for enhancing the efficacy of β-lactams against P. aeruginosa, which carries inducible chromosomal ampC, especially in AmpC-hyperproducing clinical isolates.

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