scholarly journals Selective Cytotoxicity of Carboxypeptidase-activated Methotrexate ex-Peptides

Pteridines ◽  
1989 ◽  
Vol 1 (1) ◽  
pp. 65-69 ◽  
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.

scholarly journals The amino acid sequence of cytochrome c from Nigella damascena L. (love-in-a-mist)

1973 ◽  
Vol 133 (2) ◽  
pp. 251-254 ◽  
Author(s):  
R. H. Brown ◽  
D. Boulter
Keyword(s):  
Amino Acid ◽  
Cytochrome C ◽  
Amino Acid Sequence ◽  
Science And Technology ◽  
The Other ◽  
Carboxypeptidase A ◽  
Cytochromes C ◽  
Lending Library ◽  
Nigella Damascena

The amino acid sequence of cytochrome c from Nigella damascena L. was determined on 0.2μmol of protein. Peptides from a single chymotryptic digest were analysed by the dansyl–Edman procedure. These peptides were ordered by reference to the sequences of other plant cytochromes c, assuming that the Nigella cytochrome is homologous with the other cytochromes. Many of the Nigella peptides were one or two residues short when compared with the corresponding chymotryptic peptides from other plant cytochromes c. These residues are assumed to have been removed by an endogenous carboxypeptidase, and the identification and placing of these residues is entirely based on homology. These residues are numbered 3, 18, 42, 43, 44, 54, 67, 72, 73, 82 and 105. A number of other positions are almost entirely placed by homology. These are positions which could not be placed definitely by dansyl–Edman analysis or by dansylation after digestion with carboxypeptidase A, and are numbered 14, 15, 16, 39, 40, 85, 86, 87 and 88. Except for residue 15, all residues based entirely, or nearly so, on homology have been previously found invariant in sequences of plant cytochromes c. Experimental details are given in a supplementary paper that has been deposited as Supplementary Publication SUP 50017, at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973) 131, 5.

The mechanism of antigenic drift in influenza virus: sequence changes in the haemagglutinin of variants selected with monoclonal hybridoma antibodies

1980 ◽  
Vol 288 (1029) ◽  
pp. 313-326 ◽  
Keyword(s):  
Monoclonal Antibody ◽  
Influenza Virus ◽  
Monoclonal Antibodies ◽  
Hong Kong ◽  
Amino Acid ◽  
The Other ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Sequence Change

Antigenic variants of the A/PR8 (H0N1) and A/Hong Kong/68 (H3N2) strains of influenza virus were isolated after a single passage of these viruses in the presence of monoclonal hybridoma antibodies to the haemagglutinin. Hyperimmune rabbit antisera reacted (in haemagglutination-inhibition tests) to high titre with both wild-type and variant viruses, but the monoclonal antibodies, which reacted with the wild-type virus to titres of the order of 1/10 5 did not react at all (or to very low titre) with the variants that they selected. This suggests that the changes occurring in the monoclonal variants are restricted to a single antigenic site out of many on the haemagglutinin molecule. Amino acid analysis of the soluble tryptic peptides from the haemagglutinin ‘spikes’ of wild-type and variant viruses suggest that the dramatic loss in the ability of the variants to bind the monoclonal antibody used in their selection is associated with a single change in the amino acid sequence of the large haemagglutinin polypeptide, HA 1 . For PR8 virus, eight out of ten variants selected with one monoclonal antibody showed the same sequence change of serine to leucine in the HA 1 polypeptide. The change in the other two variants was not determined. No sequence data on PR8 haemagglutinin are available, so the experiments were continued with a Hong Kong (H3N2) strain where much of the sequence of HA 1 and HA 2 is known. Three different monoclonal hybridoma antibodies to A/Mem/1/71 (H3N2) haemagglutinin were used to select a total of ten variants of this virus. Variants selected with one monoclonal antibody were not recognized by the other two monoclonal antibodies as being different from wild-type virus, suggesting that the three antibodies bound to different sites on the surface of the haemagglutinin molecules. Each of the variants occurred with a frequency of about 1 in 105 in the wild-type virus. One group of our variants selected with H14/A2 monoclonal antibody showed the same antigenic properties and the same sequence change (asparagine to lysine) in the N-terminal half of HA 1 . Of three variants selected with H14/A20, two showed a different change at a locus also in the N-terminal region of HA 1 (a proline was replaced by serine in one variant and by leucine in the other). Of the other three variants (selected with H14/A21 monoclonal antibody) one showed a change in HAX of serine to tyrosine. This change occurred in residue number 37 of cyanogen bromide fragment 2 (CN2). In the other two variants the change in HAX has not been determined, but in these a tryptic peptide comprising residues 49-56 of CN2 was missing. The tryptic peptides of the HA 1 polypeptide, showing changes in the variants selected with monoclonal antibodies, were also found to undergo sequence changes in naturally occurring Hong Kong variants isolated from man. In each case, however, the sequence changes in the monoclonal variants were different from those in the field strains. No changes were found in the HA 2 polypeptide from any of the variants.

scholarly journals Complete mapping of viral escape from neutralizing antibodies

2016 ◽  
Author(s):  
Michael B. Doud ◽  
Scott E. Hensley ◽  
Jesse D. Bloom
Keyword(s):  
Monoclonal Antibodies ◽  
Amino Acid ◽  
Neutralizing Antibodies ◽  
Viral Evolution ◽  
The Other ◽  
Viral Escape ◽  
Amino Acid Mutation ◽  
Influenza Hemagglutinin ◽  
Complete Mapping ◽  
Protein Variants

AbstractIdentifying viral mutations that confer escape from antibodies is crucial for understanding the interplay between immunity and viral evolution. Here we quantify how every amino-acid mutation to influenza hemagglutinin affects neutralization by monoclonal antibodies targeting several antigenic regions. Our approach involves creating all replication-competent protein variants of the virus, selecting these variants with antibody, and using deep sequencing to identify enriched mutations. These high-throughput measurements are predictive of the effects of individual mutations in traditional neutralization assays. At many residues, only some of the possible mutations escape from an antibody. For instance, at a single residue targeted by two different antibodies, we identify some mutations that escape both antibodies and other mutations that escape only one or the other. Therefore, our approach maps how viruses can escape antibodies with mutation-level sensitivity, and shows that only some mutations at antigenic residues actually alter antigenicity.

scholarly journals Characterization of monoclonal antibodies against a type SAT 2 foot-and-mouth disease virus

1993 ◽  
Vol 111 (2) ◽  
pp. 391-406 ◽  
Author(s):  
J. R. Crowther ◽  
C. A. Rowe ◽  
R. Butcher
Keyword(s):  
Monoclonal Antibodies ◽  
Amino Acid ◽  
Sandwich Elisa ◽  
Foot And Mouth Disease ◽  
The Other ◽  
Linear Epitope ◽  
Field Isolates ◽  
Mouth Disease Virus ◽  
Escape Mutants

SummaryThis paper is the first to describe characterization of monoclonal antibodies (MAbs) against a South African Territories 2 (SAT 2) foot-and-mouth disease virus (isolate Rho 1/48). Twelve MAbs which neutralized homologous virus were characterized in indirect and sandwich ELISA using purified Rho 1/48 virus particles, subunits, trypsin-treated, and chemically denatured virus. All the Mabs inhibited haemagglutination by parental virus. Binding of the MAbs to 73 SAT 2 field isolates was measured in a sandwich ELISA and defined four distinct antigenic regions. Preliminary characterization of escape mutants selected with some of the MAbs using virus neutralization tests, ELISA, and amino acid sequencing is included. MAbs 2, 25, 40, 48 and 64, reacted with a linear epitope on the VP1 loop region. An amino acid change at position 149 (valine to glutamic acid) was detected in mutants selected by MAb 2 and 40 and this eliminated binding and neutralization by all the other MAb. This epitope was conformation-dependent and was conserved in all 73 isolates of SAT 2 examined. Escape mutants isolated with MAb 41 and 44, had changes at positions 156 (glycine to aspartic acid), or 158 (serine to leucine) respectively. These MAbs bound with Rho 1/48 only out of 73 field strain viruses studies and the reactions of MAbs from the other groups was unaltered. MAb 27, 28 and 37 reacted with a conformation-dependent epitope on VP1 which was not conserved in field isolates. All mutants selected by these MAbs had a single amino acid substitution at position 149 (valine to alanine). The same change was always found in field isolates which did not bind MAbs from this group. MAb 11 reacted with a linear epitope associated with amino acids 147 or 148 on VP1 and showed similar binding characteristics to a conformation dependent MAb 7, no amino acid residue changes were found within VP1 for monoclonal antibody 7 mutants.

Oxytocin analogues with non-coded amino acid residues in position 8: [8-Neopentylglycine]oxytocin and [8-cycloleucine]oxytocin

1987 ◽  
Vol 52 (9) ◽  
pp. 2317-2325 ◽  
Author(s):  
Jan Hlaváček ◽  
Jan Pospíšek ◽  
Jiřina Slaninová ◽  
Walter Y. Chan ◽  
Victor J. Hruby
Keyword(s):  
Amino Acid ◽  
Solid Phase Synthesis ◽  
Solid Phase ◽  
The Other ◽  
Amino Acid Residues ◽  
Phase Synthesis ◽  
Other Hand ◽  
Fragment Condensation

[8-Neopentylglycine]oxytocin (II) and [8-cycloleucine]oxytocin (III) were prepared by a combination of solid-phase synthesis and fragment condensation. Both analogues exhibited decreased uterotonic potency in vitro, each being about 15-30% that of oxytocin. Analogue II also displayed similarly decreased uterotonic potency in vivo and galactogogic potency. On the other hand, analogue III exhibited almost the same potency as oxytocin in the uterotonic assay in vivo and in the galactogogic assay.

C-Terminal amino acid sequence of chicken pepsinogen and its homology with sequences of other acid proteases

1980 ◽  
Vol 45 (4) ◽  
pp. 1144-1154 ◽  
Author(s):  
Miroslav Baudyš ◽  
Helena Keilová ◽  
Vladimír Kostka
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
The Other ◽  
Terminal Sequence ◽  
Terminal Part ◽  
Terminal Amino Acid ◽  
Tryptic Peptides ◽  
Terminal Amino ◽  
High Degree ◽  
Terminal Amino Acid Sequence

To determine the primary structure of the C-terminal part of the molecule of chicken pepsinogen the tryptic, chymotryptic and thermolytic digest of the protein were investigated and peptides derived from this region were sought. These peptides permitted the following 21-residue C-terminal sequence to be determined: ...Ile-Arg-Glu-Tyr-Tyr-Val-Ile-Phe-Asp-Arg-Ala-Asn-Asn-Lys-Val-Gly-Leu-Ser-Pro-Leu-Ser.COOH. A comparison of this structure with the C-terminal sequential regions of the other acid proteases shows a high degree of homology between chicken pepsinogen and these proteases (e.g., the degree of homology with respect to hog pepsinogen and calf prochymosin is about 66%). Additional tryptic peptides, derived from the N-terminal part of the zymogen molecule whose amino acid sequence has been reported before, were also obtained in this study. This sequence was extended by two residues using an overlapping peptide. An ancillary result of this study was the isolation of tryptic peptides derived from other regions of the zymogen molecule.

An ESR study of the peroxidase reaction catalyzed by human methaemoglobin and methaemoglobin-haptoglobin complex

1991 ◽  
Vol 56 (4) ◽  
pp. 923-932
Author(s):  
Jana Stejskalová ◽  
Pavel Stopka ◽  
Zdeněk Pavlíček
Keyword(s):  
Hydrogen Peroxide ◽  
Ascorbic Acid ◽  
Amino Acid ◽  
Peroxidase Activity ◽  
Amino Acid Analysis ◽  
Superoxide Radical ◽  
Esr Spectra ◽  
The Other ◽  
Delocalized Electron

The ESR spectra of peroxidase systems of methaemoglobin-ascorbic acid-hydrogen peroxide and methaemoglobin-haptoglobin complex-ascorbic acid-hydrogen peroxide have been measured in the acetate buffer of pH 4.5. For the system with methaemoglobin an asymmetrical signal with g ~ 2 has been observed which is interpreted as the perpendicular region of anisotropic spectrum of superoxide radical. On the other hand, for the system with methaemoglobin-haptoglobin complex the observed signal with g ~ 2 is symmetrical and is interpreted as a signal of delocalized electron. After realization of three repeatedly induced peroxidase processes the ESR signal of the perpendicular part of anisotropic spectrum of superoxide radical is distinctly diminished, whereas the signal of delocalized electron remains practically unchanged. An amino acid analysis of methaemoglobin along with results of the ESR measurements make it possible to derive a hypothesis about the role of haptoglobin in increasing of the peroxidase activity of methaemoglobin.

Comparison of the Potency of 8-L-Arginine, 8-D-Arginine and 8-D-Homoarginine Vasopressin Analogs with Substituted Phenylalanine in Position 2

1994 ◽  
Vol 59 (6) ◽  
pp. 1439-1450 ◽  
Author(s):  
Miroslava Žertová ◽  
Jiřina Slaninová ◽  
Zdenko Procházka
Keyword(s):  
Amino Acid ◽  
Solid Phase Synthesis ◽  
Solid Phase ◽  
Basic Amino Acid ◽  
The Other ◽  
Side Chain ◽  
Inhibitory Potency ◽  
Phase Synthesis ◽  
Other Hand ◽  
Order Of Magnitude

An analysis of the uterotonic potencies of all analogs having substituted L- or D-tyrosine or -phenylalanine in position 2 and L-arginine, D-arginine or D-homoarginine in position 8 was made. The series of analogs already published was completed by the solid phase synthesis of ten new analogs having L- or D-Phe, L- or D-Phe(2-Et), L- or D-Phe(2,4,6-triMe) or D-Tyr(Me) in position 2 and either L- or D-arginine in position 8. All newly synthesized analogs were found to be uterotonic inhibitors. Deamination increases both the agonistic and antagonistic potency. In the case of phenylalanine analogs the change of configuration from L to D in position 2 enhances the uterotonic inhibition for more than 1 order of magnitude. The L to D change in position 8 enhances the inhibitory potency negligibly. Prolongation of the side chain of the D-basic amino acid in position 8 seems to decrease slightly the inhibitory potency if there is L-substituted amino acid in position 2. On the other hand there is a tendency to the increase of the inhibitory potency if there is D-substituted amino acid in position 2.

Use of monoclonal and polyclonal antibodies to detect prostatic acid phosphatase by immunoblotting.

1986 ◽  
Vol 32 (10) ◽  
pp. 1832-1835 ◽  
Author(s):  
P C Patel ◽  
L Aubin ◽  
J Côte
Keyword(s):  
Monoclonal Antibodies ◽  
Acid Phosphatase ◽  
Western Blot ◽  
Polyclonal Antibodies ◽  
Prostatic Acid Phosphatase ◽  
The Other ◽  
Dot Blot ◽  
Western Blots ◽  
Dot Blot Assay ◽  
Polyclonal Antisera

Abstract We investigated two techniques of immunoblotting--the Western blot and the dot blot--for use in detecting prostatic acid phosphatase (PAP, EC 3.1.3.2). We used polyclonal antisera to human PAP, produced in rabbits by hyperimmunization with purified PAP, and PAP-specific monoclonal antibodies in the immunoenzymatic protocols. We conclude that PAP can be readily detected by Western blots with use of polyclonal antisera, but not with monoclonal antibodies. On the other hand, using a dot blot assay, we could easily detect PAP with both polyclonal and monoclonal antibodies.

scholarly journals Charged Residues Flanking the Transmembrane Domain of Two Related Toxin–Antitoxin System Toxins Affect Host Response

Toxins ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 329
Author(s):  
Andrew Holmes ◽  
Jessie Sadlon ◽  
Keith Weaver
Keyword(s):  
Amino Acid ◽  
Enterococcus Faecalis ◽  
Host Response ◽  
Cytoplasmic Membrane ◽  
Transmembrane Domain ◽  
The Other ◽  
Type I ◽  
Transcriptomic Response ◽  
Specific Amino Acid ◽  
Transporter Protein

A majority of toxins produced by type I toxin–antitoxin (TA-1) systems are small membrane-localized proteins that were initially proposed to kill cells by forming non-specific pores in the cytoplasmic membrane. The examination of the effects of numerous TA-1 systems indicates that this is not the mechanism of action of many of these proteins. Enterococcus faecalis produces two toxins of the Fst/Ldr family, one encoded on pheromone-responsive conjugative plasmids (FstpAD1) and the other on the chromosome, FstEF0409. Previous results demonstrated that overexpression of the toxins produced a differential transcriptomic response in E. faecalis cells. In this report, we identify the specific amino acid differences between the two toxins responsible for the differential response of a gene highly induced by FstpAD1 but not FstEF0409. In addition, we demonstrate that a transporter protein that is genetically linked to the chromosomal version of the TA-1 system functions to limit the toxicity of the protein.

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