In vitro Characterization of Fitness and Convalescent Antibody Neutralization of SARS-CoV-2 Cluster 5 Variant Emerging in Mink at Danish Farms

In addition to humans, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transmit to animals that include hamsters, cats, dogs, mink, ferrets, tigers, lions, cynomolgus macaques, rhesus macaques, and treeshrew. Among these, mink are particularly susceptible. Indeed, 10 countries in Europe and North America reported SARS-CoV-2 infection among mink on fur farms. In Denmark, SARS-CoV-2 spread rapidly among mink farms and spilled-over back into humans, acquiring mutations/deletions with unknown consequences for virulence and antigenicity. Here we describe a mink-associated SARS-CoV-2 variant (Cluster 5) characterized by 11 amino acid substitutions and four amino acid deletions relative to Wuhan-Hu-1. Temporal virus titration, together with genomic and subgenomic viral RNA quantitation, demonstrated a modest in vitro fitness attenuation of the Cluster 5 virus in the Vero-E6 cell line. Potential alterations in antigenicity conferred by amino acid changes in the spike protein that include three substitutions (Y453F, I692V, and M1229I) and a loss of two amino acid residues 69 and 70 (ΔH69/V70), were evaluated in a virus microneutralization assay. Compared to a reference strain, the Cluster 5 variant showed reduced neutralization in a proportion of convalescent human COVID-19 samples. The findings underscore the need for active surveillance SARS-CoV-2 infection and virus evolution in susceptible animal hosts.

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Structural Modelling and in-silico characterization of a Novel Thermophilic β-amylase from Clostridium thermosulfuregenes

Biotechnology Journal International ◽

10.9734/bji/2021/v25i430144 ◽

2021 ◽

pp. 1-22

Author(s):

Shafiqa Nayel ◽

Mohd Shahir Shamsir ◽

Farid Ahmad Danishfar

Keyword(s):

Amino Acid ◽

Binding Sites ◽

Hydrolytic Enzyme ◽

Beta Sheets ◽

Amino Acid Residues ◽

Structural Domains ◽

Structural Conformation ◽

In Silico Characterization

β-amylase is a hydrolytic enzyme that is involved in breaking down starch and producing energy. Since the discovery of β-amylase, it has been applied in various applications especially in the food industry. In this study, a novel β-amylase from Clostridium thermosuluregen, a thermophilic anaerobic bacterium that ferments its extracellular emulsion to ethanol at 62 ℃ was modelled and studied using bioinformatics tools and compared with B. cereus β-amylases that functions at mesophilic conditions. The results showed that the overall structural conformations, secondary structures, and important residues involved in active and binding sites were identified in both proteins. The results revealed that the modelled β-amylase of C. thermosulfuregen is very similar with respect to the global conformation, location of active and binding sites. Both proteins showed identical structural domains with the thermophilic variant possessing a high percentage of hydrophobic amino acid residues, polar amino acid residues, and differences in secondary composition such as loops and beta sheets as the potential evolutionary thermal adaptations that make it stable enzyme that functions up to 70 ℃. The results suggest that the thermal stability are not dependent on one single unique mechanism and may use one or a combination of the mechanisms to sustain its structural conformation at a higher operating temperature. Overall, considering the common properties of this modelled protein with the β-amylase of B. cereus, it can be assumed that if the β-amylase of C. thermosulfuregen were expressed in-vitro, it would produce a stable protein that possesses the hydrolysis function for C. thermosulfuregen to break down the starch and sugar formation.

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The isolation and characterization of corticotropin from the pituitary gland of the ostrich Struthio camelus

Biochemical Journal ◽

10.1042/bj1650519 ◽

1977 ◽

Vol 165 (3) ◽

pp. 519-523 ◽

Cited By ~ 17

Author(s):

R J Naudé ◽

W Oelofsen

Keyword(s):

Amino Acid ◽

Polyacrylamide Gel Electrophoresis ◽

Amino Acid Residues ◽

Struthio Camelus ◽

Isolation And Characterization ◽

Isoelectric Points ◽

Pituitary Glands ◽

Amide Content

1. Avian corticotropin (ACTH) was purified from both fresh and aged pituitary glands of the ostrich Struthio camelus. 2. The isolation of corticotropin in pure form involved acid/acetone extraction, NaCl fractionation, CM-cellulose chromatography and Sephadex G-50 chromatography. 3. The hormone preparations from fresh and aged glands behaved as single substances on polyacrylamide-gel electrophoresis, and both preparations were found to consist of 39 amino acid residues, in identical molar proportions for the different amino acids. 4. The isoelectric points of the two hormone preparations were estimated to be in the range pH 8.3-8.7, indicating possible differences in amide content, and the N-terminal amino acid of both preparations appeared to be serine. 5. The hormone preparations from fresh and aged glands exhibited similar biological potencies (73 and 77 i.u./mg respectively), as measured by steroidogenesis in vitro. 6. Apart from possible differences in amide content, the corticotropin preparations obtained from fresh and aged glands appear to be indistinguishable.

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Characterization of a minus end-directed kinesin-like motor protein from cultured mammalian cells.

The Journal of Cell Biology ◽

10.1083/jcb.129.4.1049 ◽

1995 ◽

Vol 129 (4) ◽

pp. 1049-1059 ◽

Cited By ~ 50

Author(s):

R Kuriyama ◽

M Kofron ◽

R Essner ◽

T Kato ◽

S Dragas-Granoic ◽

...

Keyword(s):

Monoclonal Antibody ◽

Amino Acid ◽

Mammalian Cells ◽

Amino Acid Residues ◽

Central Stalk ◽

Vitro Analysis ◽

Kinesin Superfamily ◽

In Vitro Analysis

Using the CHO2 monoclonal antibody raised against CHO spindles (Sellitto, C., M. Kimble, and R. Kuriyama. 1992. Cell Motil. Cytoskeleton. 22:7-24) we identified a 66-kD protein located at the interphase centrosome and mitotic spindle. Isolated cDNAs for the antigen encode a 622-amino acid polypeptide. Sequence analysis revealed the presence of 340-amino acid residues in the COOH terminus, which is homologous to the motor domain conserved among other members of the kinesin superfamily. The protein is composed of a central alpha-helical portion with globular domains at both NH2 and COOH termini, and the epitope to the monoclonal antibody resides in the central alpha-helical stalk. A series of deletion constructs were created for in vitro analysis of microtubule interactions. While the microtubule binding and bundling activities require both the presence of the COOH terminus and the alpha-helical domain, the NH2-terminal half of the antigen lacked the ability to interact with microtubules. The full-length as well as deleted proteins consisting of the COOH-terminal motor and the central alpha-helical stalk supported microtubule gliding, with velocity ranging from 1.0 to 8.4 microns/minute. The speed of microtubule movement decreased with decreasing lengths of the central stalk attached to the COOH-terminal motor. The microtubules moved with their plus end leading, indicating that the antigen is a minus end-directed motor. The CHO2 sequence shows 86% identify to HSET, a gene located at the centromeric end of the human MHC region in chromosome 6 (Ando, A., Y. Y. Kikuti, H. Kawata, N. Okamoto, T. Imai, T. Eki, K. Yokoyama, E. Soeda, T. Ikemura, K. Abe, and H. Inoko. 1994. Immunogenetics. 39:194-200), indicating that HSET might represent a human homologue of the CHO2 antigen.

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Purification of a high-molecular-weight somatoliberin (growth-hormone-releasing factor) from pig hypothalami

Biochemical Journal ◽

10.1042/bj2090643 ◽

1983 ◽

Vol 209 (3) ◽

pp. 643-651 ◽

Cited By ~ 6

Author(s):

J E C Sykes ◽

P J Lowry

Keyword(s):

Molecular Weight ◽

Growth Hormone ◽

Amino Acid ◽

Response Curve ◽

Gel Filtration ◽

Apparent Molecular Weight ◽

Amino Acid Residues ◽

Growth Hormone Releasing Factor

Preliminary observations [Sykes & Lowry (1980) J. Endocrinol. 85, 42P-43P] had suggested that the major hypothalamic somatoliberin (growth-hormone-releasing factor) was a larger peptide than the other characterized hypothalamic factors, with an elution position on Sephadex G-50 between those of neurophysin and corticotropin. The present paper reports the isolation and preliminary characterization of pig hypothalamic somatoliberin. Acid extracts of pig stalk median eminence were purified by gel filtration and preparative and analytical high-pressure liquid chromatography to yield a preparation that was specific in the release of somatotropin (growth hormone) in vitro, giving a steep dose-response curve at doses in the range 0.20-3.0 ng. Amino acid analysis revealed a non-cysteine-containing peptide with a high number of glutamate (or glutamine) and aspartate (or asparagine) residues. The peptide had about 56-57 amino acid residues and an apparent molecular weight of 6400, in keeping with its elution position on a column of Sephadex G-50.

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In vitro characterization of multidrug-resistant influenza A(H1N1)pdm09 viruses carrying a dual amino acid substitution associated with reduced susceptibility to neuraminidase inhibitors

10.1101/334185 ◽

2018 ◽

Author(s):

Emi Takashita ◽

Seiichiro Fujisaki ◽

Masaru Yokoyama ◽

Masayuki Shirakura ◽

Kazuya Nakamura ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Substitution ◽

Influenza A ◽

Multidrug Resistant ◽

Cross Resistance ◽

Neuraminidase Inhibitors ◽

In Vitro Characterization ◽

Influenza A H1n1

AbstractWe detected influenza A(H1N1)pdm09 viruses carrying dual H275Y/I223R, H275Y/I223K, or H275Y/G147R substitutions in their neuraminidase protein, respectively. These viruses showed cross-resistance to oseltamivir and peramivir and reduced susceptibility to zanamivir. The H275Y/G147R virus retained its replication capability at least in vitro, but the H275Y/I223R and H275Y/I223K viruses did not.

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