Biological, antigenic and phylogenetic characterization of the flavivirus Alfuy

Alfuy virus (ALFV) is classified as a subtype of the flavivirus Murray Valley encephalitis virus (MVEV); however, despite preliminary reports of antigenic and ecological similarities with MVEV, ALFV has not been associated with human disease. Here, it was shown that ALFV is at least 104-fold less neuroinvasive than MVEV after peripheral inoculation of 3-week-old Swiss outbred mice, but ALFV demonstrates similar neurovirulence. In addition, it was shown that ALFV is partially attenuated in mice that are deficient in α/β interferon responses, in contrast to MVEV which is uniformly lethal in these mice. To assess the antigenic relationship between these viruses, a panel of monoclonal antibodies was tested for the ability to bind to ALFV and MVEV in ELISA. Although the majority of monoclonal antibodies recognized both viruses, confirming their antigenic similarity, several discriminating antibodies were identified. Finally, the entire genome of the prototype strain of ALFV (MRM3929) was sequenced and phylogenetically analysed. Nucleotide (73 %) and amino acid sequence (83 %) identity between ALFV and MVEV confirmed previous reports of their close relationship. Several nucleotide and amino acid deletions and/or substitutions with putative functional significance were identified in ALFV, including the abolition of a conserved glycosylation site in the envelope protein and the deletion of the terminal dinucleotide 5′-CUOH-3′ found in all other members of the genus. These findings confirm previous reports that ALFV is closely related to MVEV, but also highlights significant antigenic, genetic and phenotypic divergence from MVEV. Accordingly, the data suggest that ALFV is a distinct species within the serogroup Japanese encephalitis virus.

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Characterization of Mouse Monoclonal Antibodies Against the HA of A(H7N9) Influenza Virus

Viruses ◽

10.3390/v11020149 ◽

2019 ◽

Vol 11 (2) ◽

pp. 149 ◽

Cited By ~ 4

Author(s):

Mutsumi Ito ◽

Seiya Yamayoshi ◽

Kazushi Murakami ◽

Kenji Saito ◽

Atsuo Motojima ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Amino Acid ◽

Antigenic Site ◽

Prototype Strain ◽

Human Infection ◽

Antigenic Drift ◽

Amino Acid Substitutions ◽

H7n9 Virus ◽

Lethal Infection

Many cases of human infection with the H7N9 virus have been detected in China since 2013. H7N9 viruses are maintained in chickens and are transmitted to humans at live bird markets. During circulation in birds, H7N9 viruses have accumulated amino acid substitutions in their hemagglutinin (HA), which resulted in an antigenically change in the recent H7N9 viruses. Here, we characterized 46 mouse monoclonal antibodies against the HA of the prototype strain. 16 H7-HA-specific monoclonal antibodies (mAbs) possessed hemagglutination inhibition (HI) and neutralization activities by recognizing the major antigenic site A; four other H7-HA-specific clones also showed HI and neutralizing activities via recognition of the major antigenic sites A and D; seven mAbs that reacted with several HA subtypes and possibly recognized the HA stem partially protected mice from lethal infection with prototype H7N9 virus; and the remaining 19 mAbs had neither HI nor neutralization activity. All human H7N9 viruses tested showed a similar neutralization sensitivity to the first group of 16 mAbs, whereas human H7N9 viruses isolated in 2016‒2017 were not neutralized by a second group of 4 mAbs. These results suggest that amino acid substitutions at the epitope of the second mAb group appear to be involved in the antigenic drift of the H7N9 viruses. Further analysis is required to fully understand the antigenic change in H7N9 viruses.

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The Influence of Carbohydrate Structure on the Clearance of Recombinant Tissue-Type Plasminogen Activator

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647041 ◽

1988 ◽

Vol 60 (02) ◽

pp. 255-261 ◽

Cited By ~ 81

Author(s):

A Hotchkiss ◽

C J Refino ◽

C K Leonard ◽

J V O'Connor ◽

C Crowley ◽

...

Keyword(s):

Amino Acid ◽

Plasminogen Activator ◽

Sodium Periodate ◽

Glycosylation Site ◽

Tissue Type ◽

Carbohydrate Structure ◽

Tissue Type Plasminogen Activator ◽

Type Plasminogen Activator ◽

High Mannose ◽

Oligosaccharide Structures

SummaryModification of the carbohydrate structures of recombinant tissue-type plasminogen activator (rt-PA) can increase or decrease its rate of clearance in rabbits. When rt-PA was treated with sodium periodate to oxidize carbohydrate residues, the rate of clearance was decreased from 9.6 ± 1.9 ml min−1 kg−1 to 3.5 ± 0.6 ml min−1 kg−1 (mean ± SD, n = 5). A similar change in the clearance of rt-PA was introduced by the use of endo-β-N-acetyl- glucosaminidase H (Endo-H), which selectively removes high mannose asparagine-linked oligosaccharides; the clearance of Endo-H-treated rt-PA was 5.0 ± 0.5 ml min−1 kg−1. A mutant of rt-PA was produced with an amino acid substitution at position 117 (Asn replaced with Gin) to remove a potential glycosylation site that normally contains a high mannose structure. The clearance of this material was also decreased, similar to the periodate and Endo-H-treated rt-PA. Conversely, when rt-PA was produced in the CHO 15B cell line, which can produce only high mannose oligosaccharide structures on glycoproteins, the clearance was increased by a factor of 1.8. These results demonstrate that the removal of rt-PA from the blood depends significantly upon the nature of its oligosaccharide structures.

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Sequence Comparison of Vaginolysin from Different Gardnerella Species

Pathogens ◽

10.3390/pathogens10020086 ◽

2021 ◽

Vol 10 (2) ◽

pp. 86

Author(s):

Erin M. Garcia ◽

Myrna G. Serrano ◽

Laahirie Edupuganti ◽

David J. Edwards ◽

Gregory A. Buck ◽

...

Keyword(s):

Amino Acid ◽

Human Microbiome ◽

Human Microbiome Project ◽

Distinct Species ◽

Vaginal Swab ◽

Metagenomic Data ◽

Gardnerella Vaginalis ◽

Vaginal Epithelial Cells ◽

Species Specific

Gardnerella vaginalis has recently been split into 13 distinct species. In this study, we tested the hypotheses that species-specific variations in the vaginolysin (VLY) amino acid sequence could influence the interaction between the toxin and vaginal epithelial cells and that VLY variation may be one factor that distinguishes less virulent or commensal strains from more virulent strains. This was assessed by bioinformatic analyses of publicly available Gardnerella spp. sequences and quantification of cytotoxicity and cytokine production from purified, recombinantly produced versions of VLY. After identifying conserved differences that could distinguish distinct VLY types, we analyzed metagenomic data from a cohort of female subjects from the Vaginal Human Microbiome Project to investigate whether these different VLY types exhibited any significant associations with symptoms or Gardnerella spp.-relative abundance in vaginal swab samples. While Type 1 VLY was most prevalent among the subjects and may be associated with increased reports of symptoms, subjects with Type 2 VLY dominant profiles exhibited increased relative Gardnerella spp. abundance. Our findings suggest that amino acid differences alter the interaction of VLY with vaginal keratinocytes, which may potentiate differences in bacterial vaginosis (BV) immunopathology in vivo.

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Investigation of major amino acid residues of anti-norfloxacin monoclonal antibodies responsible for binding with fluoroquinolones

Scientific Reports ◽

10.1038/s41598-021-96466-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Patamalai Boonserm ◽

Songchan Puthong ◽

Thanaporn Wichai ◽

Sajee Noitang ◽

Pongsak Khunrae ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Amino Acid ◽

3D Structure ◽

Cross Reactivity ◽

Enzyme Linked Immunosorbent Assay ◽

Amino Acid Residues ◽

Variable Regions ◽

Complementarity Determining Regions ◽

Crucial Part ◽

Major Amino Acid

AbstractIt is important to understand the amino acid residues that govern the properties of the binding between antibodies and ligands. We studied the binding of two anti-norfloxacins, anti-nor 132 and anti-nor 155, and the fluoroquinolones norfloxacin, enrofloxacin, ciprofloxacin, and ofloxacin. Binding cross-reactivities tested by an indirect competitive enzyme-linked immunosorbent assay indicated that anti-nor 132 (22–100%) had a broader range of cross-reactivity than anti-nor 155 (62–100%). These cross-reactivities correlated with variations in the numbers of interacting amino acid residues and their positions. Molecular docking was employed to investigate the molecular interactions between the fluoroquinolones and the monoclonal antibodies. Homology models of the heavy chain and light chain variable regions of each mAb 3D structure were docked with the fluoroquinolones targeting the crucial part of the complementarity-determining regions. The fluoroquinolone binding site of anti-nor 155 was a region of the HCDR3 and LCDR3 loops in which hydrogen bonds were formed with TYR (H:35), ASN (H:101), LYS (H:106), ASN (L:92), and ASN (L:93). These regions were further away in anti-nor 132 and could not contact the fluoroquinolones. Another binding region consisting of HIS (L:38) and ASP (H:100) was found for norfloxacin, enrofloxacin, and ciprofloxacin, whereas only ASP (H:100) was found for ofloxacin.

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DNA barcodes provide evidence for the independent origin of the cultivated silkworms Samia ricini and Samia cynthia

Journal of Insects as Food and Feed ◽

10.3920/jiff2021.0077 ◽

2021 ◽

pp. 1-8

Author(s):

J.-C. Huang ◽

X.-Y. Li ◽

Y.-P. Li ◽

R.-S. Zhang ◽

D.-B. Chen ◽

...

Keyword(s):

Genetic Distance ◽

Phylogenetic Relationship ◽

Dna Barcode ◽

Distinct Species ◽

Coi Gene ◽

Molecular Evidence ◽

Dna Barcodes ◽

Close Relationship ◽

Molecular Phylogenetic ◽

Phylogenetic Framework

Samia ricini (Wm. Jones) and Samia cynthia (Drury) (Lepidoptera: Saturniidae) have been used as traditional sources of food as well as silk-producing insects. However, the phylogenetic relationship between the two silkworms remains to be addressed. In this study, the mitochondrial cytochrome c oxidase subunit 1 (COI) gene sequences corresponding to DNA barcodes from 13 Samia species were analysed, and a DNA barcode-based phylogenetic framework for these Samia species was provided. Phylogenetic analysis showed that multiple individuals of a species could be clustered together. Our analysis revealed a close relationship among Samia yayukae Paukstadt, Peigler and Paukstadt, Samia abrerai Naumann and Peigler, Samia kohlli Naumann and Peigler, Samia naessigi Naumann and Peigler, Samia naumanni Paukstadt, Peigler and Paukstadt, and Samia kalimantanensis Paukstadt and Paukstadt. The mixed clustering relationship and low Kimura-2-parameter (K2P) genetic distance (0.006) between individuals of S. ricini and Samia canningi (Hutton) indicated that the cultivated silkworm S. ricini was derived from the non-cultivated silkworm S. canningi. The remote phylogenetic relationship and high K2P genetic distance (0.039) indicated that S. ricini and S. cynthia are distinct species, thus providing solid molecular evidence that they had entirely independent origins. The relationships between S. kalimantanensis and S. naumanni and between S. cynthia and Samia wangi Naumann and Peigler, as well as the potential cryptic species within S. abrerai were also discussed. This is the first study to assess the DNA barcodes of the genus Samia, which supplements the knowledge of species identification and provides the first molecular phylogenetic framework for Samia species.

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Detection of fos protein during osteogenesis by monoclonal antibodies

Molecular and Cellular Biology ◽

10.1128/mcb.8.5.2251-2256.1988 ◽

1988 ◽

Vol 8 (5) ◽

pp. 2251-2256

Author(s):

P De Togni ◽

H Niman ◽

V Raymond ◽

P Sawchenko ◽

I M Verma

Keyword(s):

Monoclonal Antibodies ◽

Amino Acid ◽

Indirect Immunofluorescence ◽

Protein Complex ◽

Synthetic Peptide ◽

Immunohistochemical Staining ◽

Nuclear Staining ◽

Rat Embryos ◽

Fos Protein ◽

Modified Forms

We have generated monoclonal antibodies by using a synthetic peptide corresponding to amino acid positions 4 to 17 of the human fos protein. The antibodies detected both v- and c-fos proteins by immunoprecipitation, immunoblotting, and indirect immunofluorescence. The monoclonal antibodies not only identified the fos protein complex with the cellular 39-kilodalton protein, but also recognized the modified forms of the mouse, rat, and human fos proteins. In day-17 rat embryos, nuclear-staining fos protein could be identified in the cartilage by immunohistochemical staining.

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Comparison of 5 monoclonal antibodies for immunopurification of human butyrylcholinesterase on Dynabeads: KD values, binding pairs, and amino acid sequences

Chemico-Biological Interactions ◽

10.1016/j.cbi.2015.08.024 ◽

2015 ◽

Vol 240 ◽

pp. 336-345 ◽

Cited By ~ 14

Author(s):

Hong Peng ◽

Stephen Brimijoin ◽

Anna Hrabovska ◽

Katarina Targosova ◽

Eric Krejci ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Amino Acid ◽

Amino Acid Sequences ◽

Kd Values ◽

Human Butyrylcholinesterase

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Selective Cytotoxicity of Carboxypeptidase-activated Methotrexate ex-Peptides

Pteridines ◽

10.1515/pteridines.1989.1.1.65 ◽

1989 ◽

Vol 1 (1) ◽

pp. 65-69 ◽

Cited By ~ 6

Author(s):

Karin S. Vitols ◽

Yolanda D. Montejano ◽

Ulrike Kuefner ◽

F. M. Huennekens

Keyword(s):

Monoclonal Antibodies ◽

Amino Acid ◽

Suspension Culture ◽

Parent Drug ◽

Model System ◽

The Other ◽

Carboxypeptidase A ◽

Selective Cytotoxicity ◽

Cell Kill ◽

Semi Solid

Summary Methotrexate ex-pep tides (derivatives in which an amino acid is linked covalently to the ex-carboxyl of the glutamate residue on the parent drug) can be hydrolyzed by specific carboxypeptidases to yield free Methotrexate (MTX) and the corresponding amino acid. Studies with L12l0 cells in suspension culture have shown that the MTX pep tides can serve as "pro-drugs": Because of their inability to be taken up by cells, they are relatively non-toxic. When co-administered with appropriate carboxypeptidases, however, they become equitoxic with MTX. In the present investigation, the potential of the MTX-ala/carboxypeptidase A combination for providing regional cytotoxicity was demonstrated in a model system involving L12l0 cells propagated in semi-solid agarose. When cells and one of the components (MTX peptide or carboxypeptidase) were distributed uniformly throughout the agarose, and the other component was immobilized at the center, a discrete zone of cell kill radiated from the fixed component. These results suggest the possibility of developing a new mode of cancer chemotherapy involving circulating MTX peptides in conjunction with carboxypeptidases linked covalently to tumor-targeted monoclonal antibodies.

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Crotalase, a Fibrinogen-Clotting Snake Venom Enzyme: Primary Structure and Evidence for a Fibrinogen Recognition Exosite Different from Thrombin

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614423 ◽

1999 ◽

Vol 81 (01) ◽

pp. 81-86 ◽

Cited By ~ 19

Author(s):

Agnes Henschen-Edman ◽

Ida Theodor ◽

Brian Edwards ◽

Hubert Pirkle

Keyword(s):

Molecular Weight ◽

Amino Acid ◽

Amino Acid Sequence ◽

Spatial Modeling ◽

Glycosylation Site ◽

Amino Sugars ◽

Edman Degradation ◽

Heparin Binding ◽

Heparin Binding Site ◽

Total Molecular Weight

SummaryCrotalase, a fibrinogen-clotting enzyme isolated from the venom of Crotalus adamanteus, and its overlapping fragments were subjected to Edman degradation. The resulting amino acid sequence, VIGGDEC NINEHRFLVALYDYWSQLFLCGGTLINNEWVLTAAHCDRTHI LIYVGVHDRSVQFDKEQRRFPKEKYFFDCSNNFTKWDKDIM LIRLNKPVSYSEHIAPLSLPSSPPIVGSVCRAMGWGQTTSPQET LPDVPHCANINLLDYEVCRTAHPQFRLPATSRTLCAGVLEG GIDTCNRDSGGPLICNGQFQGIVFWGPDPCAQPDKPGLYTK VFDHLDWIQSIIAGEKTVNCP, is characteristic of a serine protein-ase. Comparison with thrombin, the physiological fibrinogen-clotting enzyme, showed that thrombin’s fibrinogen-recognition exosite (FRE) is poorly represented in crotalase. Hirudin, a FRE-dependent inhibitor, had no effect on crotalase. Spatial modeling of crotalase yielded a possible alternative fibrinogen-recognition site comprised of Arg 60F, Lys 85, Lys 87, and Arg 107 (underlined in the sequence above). Crotalase also lacks thrombin’s YPPW loop, as well as its functionally important ETW 146-148, and its heparin-binding site. The enzyme contains a single asparagine-linked glycosylation site, NFT, bearing neutral and amino sugars that account for 8.3% of the enzyme’s total molecular weight of 29,027. The calculated absorbance of crotalase at 280 nm, 1%, cm-1is 15.2.

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