scholarly journals Targeted Engineering of the Antibacterial Peptide Apidaecin, Based on an In Vivo Monitoring Assay System

2008 ◽  
Vol 75 (5) ◽  
pp. 1460-1464 ◽  
Author(s):  
Seiichi Taguchi ◽  
Kensuke Mita ◽  
Kenta Ichinohe ◽  
Shigeki Hashimoto
Keyword(s):  
Amino Acid ◽  
Variable Region ◽  
Antibacterial Peptide ◽  
Amino Acid Residues ◽  
Terminal Amino ◽  
Combinatorial Mutagenesis ◽  
Mutant Forms ◽  
Increased Activity

ABSTRACT Seven mutant forms of the antibacterial peptide apidaecin with increased activity were created by combinatorial mutagenesis targeted to the three N-terminal amino acid residues that had previously been identified as a nonessential region. An in vitro MIC assay revealed that the amino acid substitutions in the functionally variable region were effective in improving differential activity toward the four gram-negative bacteria tested, while a gram-positive bacterium was unaffected.

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.

Synthesis of Fragments of the Vasoactive Intestinal Peptide (VIP) and Analysis of Their Residual Biological Activities

1995 ◽  
Vol 60 (7) ◽  
pp. 1229-1235 ◽  
Author(s):  
Ivana Zoulíková ◽  
Ivan Svoboda ◽  
Jiří Velek ◽  
Václav Kašička ◽  
Jiřina Slaninová ◽  
...  
Keyword(s):  
Amino Acid ◽  
Biological Activities ◽  
Residual Activity ◽  
Detailed Examination ◽  
Amino Acid Residues ◽  
Linear Peptide ◽  
Zone Electrophoresis ◽  
Capillary Zone

The vasoactive intestinal (poly)peptide (VIP) is a linear peptide containing 28 amino acid residues, whose primary structure indicates a low metabolic stability. The following VIP fragments, as potential metabolites, and their analogues were prepared by synthesis on a solid: [His(Dnp)1]VIP(1-10), VIP(11-14), [D-Arg12]VIP(11-14), [Lys(Pac)15,21,Arg20]VIP(15-22), and VIP(23-28). After purification, the peptides were characterized by amino acid analysis, mass spectrometry, RP HPLC, and capillary zone electrophoresis. In some tests, detailed examination of the biological activity of the substances in vivo and in vitro gave evidence of a low, residual activity of some fragments, viz. a depressoric activity in vivo for [His(Dnp)1]VIP(1-10) and a stimulating activity for the release of α-amylase in vitro and in vivo for [Lys(Pac)15,21,Arg20]VIP(15-22) and VIP(23-28).

scholarly journals A synthetic glucagon-like peptide-1 analog with improved plasma stability

1998 ◽  
Vol 159 (1) ◽  
pp. 93-102 ◽  
Author(s):  
U Ritzel ◽  
U Leonhardt ◽  
M Ottleben ◽  
A Ruhmann ◽  
K Eckart ◽  
...  
Keyword(s):  
Amino Acid ◽  
Plasma Insulin ◽  
Rat Plasma ◽  
Plasma Stability ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1

Glucagon-like peptide-1 (GLP-1) is the most potent endogenous insulin-stimulating hormone. In the present study the plasma stability and biological activity of a GLP-1 analog, [Ser]GLP-1(7-36)amide, in which the second N-terminal amino acid alanine was replaced by serine, was evaluated in vitro and in vivo. Incubation of GLP-1 with human or rat plasma resulted in degradation of native GLP-1(7-36)amide to GLP-1(9-36)amide, while [Ser]GLP-1(7-36)amide was not significantly degraded by plasma enzymes. Using glucose-responsive HIT-T15 cells, [Ser]GLP-1(7-36)amide showed strong insulinotropic activity, which was inhibited by the specific GLP-1 receptor antagonist exendin-4(9-39)amide. Simultaneous i.v. injection of [Ser]GLP-1(7-36)amide and glucose in rats induced a twofold higher increase in plasma insulin levels than unmodified GLP-1(7-36)amide with glucose and a fivefold higher increase than glucose alone. [Ser]GLP-1(7-36)amide induced a 1.5-fold higher increase in plasma insulin than GLP-1(7-36)amide when given 1 h before i.v. application of glucose. The insulinotropic effect of [Ser]GLP-1(7-36)amide was suppressed by i.v. application of exendin-4(9-39)amide. The present data demonstrate that replacement of the second N-terminal amino acid alanine by serine improves the plasma stability of GLP-1(7-36)amide. The insulinotropic action in vitro and in vivo was not impaired significantly by this modification.

scholarly journals The functional dyad of scorpion toxin Pi1 is not itself a prerequisite for toxin binding to the voltage-gated Kv1.2 potassium channels

2004 ◽  
Vol 377 (1) ◽  
pp. 25-36 ◽  
Author(s):  
Stéphanie MOUHAT ◽  
Amor MOSBAH ◽  
Violeta VISAN ◽  
Heike WULFF ◽  
Muriel DELEPIERRE ◽  
...  
Keyword(s):  
Amino Acid ◽  
Scorpion Toxin ◽  
Xenopus Laevis Oocytes ◽  
Amino Acid Residues ◽  
Voltage Gated ◽  
Toxin Binding ◽  
Ic50 Values

Pi1 is a 35-residue scorpion toxin cross-linked by four disulphide bridges that acts potently on both small-conductance Ca2+-activated (SK) and voltage-gated (Kv) K+ channel subtypes. Two approaches were used to investigate the relative contribution of the Pi1 functional dyad (Tyr-33 and Lys-24) to the toxin action: (i) the chemical synthesis of a [A24,A33]-Pi1 analogue, lacking the functional dyad, and (ii) the production of a Pi1 analogue that is phosphorylated on Tyr-33 (P-Pi1). According to molecular modelling, this phosphorylation is expected to selectively impact the two amino acid residues belonging to the functional dyad without altering the nature and three-dimensional positioning of other residues. P-Pi1 was directly produced by peptide synthesis to rule out any possibility of trace contamination by the unphosphorylated product. Both Pi1 analogues were compared with synthetic Pi1 for bioactivity. In vivo, [A24,A33]-Pi1 and P-Pi1 are lethal by intracerebroventricular injection in mice (LD50 values of 100 and 40 µg/mouse, respectively). In vitro, [A24,A33]-Pi1 and P-Pi1 compete with 125I-apamin for binding to SK channels of rat brain synaptosomes (IC50 values of 30 and 10 nM, respectively) and block rat voltage-gated Kv1.2 channels expressed in Xenopus laevis oocytes (IC50 values of 22 µM and 75 nM, respectively), whereas they are inactive on Kv1.1 or Kv1.3 channels at micromolar concentrations. Therefore, although both analogues are less active than Pi1 both in vivo and in vitro, the integrity of the Pi1 functional dyad does not appear to be a prerequisite for the recognition and binding of the toxin to the Kv1.2 channels, thereby highlighting the crucial role of other toxin residues with regard to Pi1 action on these channels. The computed simulations detailing the docking of Pi1 peptides on to the Kv1.2 channels support an unexpected key role of specific basic amino acid residues, which form a basic ring (Arg-5, Arg-12, Arg-28 and Lys-31 residues), in toxin binding.

scholarly journals An Evaluation of 3-Rhamnosylquercetin, a Glycosylated Form of Quercetin, against the Myotoxic and Edematogenic Effects of sPLA2fromCrotalus durissus terrificus

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
Daniela de Oliveira Toyama ◽  
Henrique Hessel Gaeta ◽  
Marcus Vinícius Terashima de Pinho ◽  
Marcelo José Pena Ferreira ◽  
Paulete Romoff ◽  
...  
Keyword(s):  
Amino Acid ◽  
Docking Studies ◽  
Pharmacological Effects ◽  
Amino Acid Residues ◽  
Substrate Molecule ◽  
Pharmacological Activities ◽  
Potent Inhibition ◽  
Crotalus Durissus

This paper shows the results of quercitrin effects on the structure and biological activity of secretory phospholipase (sPLA2) fromCrotalus durissus terrificus, which is the main toxin involved in the pharmacological effects of this snake venom. According to our mass spectrometry and circular dichroism results, quercetin was able to promote a chemical modification of some amino acid residues and modify the secondary structure ofC. d. terrificussPLA2. Moreover, molecular docking studies showed that quercitrin can establish chemical interactions with some of the crucial amino acid residues involved in the enzymatic activity of the sPLA2, indicating that this flavonoid could also physically impair substrate molecule access to the catalytic site of the toxin. Additionally,in vitroandin vivoassays showed that the quercitrin strongly diminished the catalytic activity of the protein, altered its Vmax and Km values, and presented a more potent inhibition of essential pharmacological activities in theC. d. terrificussPLA2, such as its myotoxicity and edematogenic effect, in comparison to quercetin. Thus, we concluded that the rhamnose group found in quercitrin is most likely essential to the antivenom activities of this flavonoid againstC. d. terrificussPLA2.

scholarly journals Leishmania major parasites express cyclophilin isoforms with an unusual interaction with calcineurin

1998 ◽  
Vol 334 (3) ◽  
pp. 659-667 ◽  
Author(s):  
Christine RASCHER ◽  
Andreas PAHL ◽  
Anja PECHT ◽  
Kay BRUNE ◽  
Werner SOLBACH ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Leishmania Major ◽  
Protozoan Parasite ◽  
Isomerase Activity ◽  
Terminal Amino ◽  
Pathogenic Protozoan ◽  
Parasite Leishmania

The immunosuppressive effects of the fungal metabolite cyclosporin A (CsA) are mediated primarily by binding to cyclophilins (Cyps). The resulting CsA–Cyp complex inhibits the Ca2+-regulated protein phosphatase calcineurin and down-regulates signal transduction events. Previously we reported that CsA is a potent inhibitor of infections transmitted by the human pathogenic protozoan parasite Leishmania major in vitro and in vivo, but does not effect the extracellular growth of L. major itself. It is unknown how L. major exerts this resistance to CsA. Here we report that a major Cyp, besides additional isoforms with the same N-terminal amino acid sequence, was expressed in L. major. The cloned and sequenced gene encodes a putative 174-residue protein called L. major Cyp 19 (LmCyp19). The recombinant LmCyp19 exhibits peptidyl-prolyl cis/trans isomerase activity with a substrate specificity and an inhibition by CsA that are characteristic of other eukaryotic Cyps. To determine whether calcineurin is involved in the discrimination of the effects of CsA we also examined the presence of a parasitic calcineurin and tested the interaction with Cyps. Despite the expression of functionally active calcineurin by L. major, neither LmCyp19 nor other L. major Cyps bound to its own or mammalian calcineurin. The amino acid sequence of most Cyps includes an essential arginine residue around the calcineurin-docking side. In LmCyp19 this is replaced by an asparagine residue. This exchange and additional charged residues are apparently responsible for the lack of LmCyp19 interaction with calcineurin. These observations indicate that resistance of L. major to CsA in vitro is mediated by the lack of complex formation with calcineurin despite CsA binding by parasitic Cyp.

scholarly journals Highly Conserved Configuration of Catalytic Amino Acid Residues among Calicivirus-Encoded Proteases

2007 ◽  
Vol 81 (13) ◽  
pp. 6798-6806 ◽  
Author(s):  
Tomoichiro Oka ◽  
Mami Yamamoto ◽  
Masaru Yokoyama ◽  
Satoko Ogawa ◽  
Grant S. Hansman ◽  
...  
Keyword(s):  
Amino Acid ◽  
Proteolytic Processing ◽  
Site Directed Mutagenesis ◽  
Computer Assisted ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Binding Cleft ◽  
Viral Polyprotein ◽  
Mutant Forms

ABSTRACT A common feature of caliciviruses is the proteolytic processing of the viral polyprotein catalyzed by the viral 3C-like protease encoded in open reading frame 1 (ORF1). Here we report the identification and structural characterization of the protease domains and amino acid residues in sapovirus (SaV) and feline calicivirus (FCV). The in vitro expression and processing of a panel of truncated ORF1 polyproteins and corresponding mutant forms showed that the functional protease domain is 146 amino acids (aa) in SaV and 154 aa in FCV. Site-directed mutagenesis of the protease domains identified four amino acid residues essential to protease activities: H31, E52, C116, and H131 in SaV and H39, E60, C122, and H137 in FCV. A computer-assisted structural analysis showed that despite high levels of diversity in the primary structures of the protease domains in the family Caliciviridae, the configurations of the H, E, C, and H residues are highly conserved, with these residues positioned closely along the inner surface of the potential binding cleft for the substrate. These results strongly suggest that the H, E, C, and H residues are involved in the formation of a conserved catalytic surface of the SaV and FCV 3C-like proteases.

scholarly journals Different epitope structures select distinct mutant forms of an antibody variable region for expression during the immune response.

1991 ◽  
Vol 173 (3) ◽  
pp. 665-672 ◽  
Author(s):  
S Fish ◽  
M Fleming ◽  
J Sharon ◽  
T Manser
Keyword(s):  
Immune Response ◽  
Amino Acid ◽  
Somatic Mutation ◽  
Amino Acid Substitution ◽  
Variable Region ◽  
Single Amino Acid ◽  
Amino Acid Residues ◽  
V Region ◽  
Mutant Forms ◽  
Selection Of

Antibody variable (V) regions that initially differ from one another by only single amino acid residues at VH-D and D-JH segment junctions (termed canonical V regions) can be elicited in strain A/J mice by three different haptens. Among such V regions an amino acid substitution due to somatic mutation is recurrently observed at VH CDR2 position 58, regardless of which of these haptens is used for immunization. This substitution confers upon a canonical V region a generic increase in affinity for all the haptens. Conversely, the type of amino acid substitution at VH position 59 resulting from somatic mutation that is recurrently observed among such V regions changes with the eliciting hapten, in a manner that correlates directly with the cognate affinity increases (or decreases) for hapten conferred by the observed substitutions. This small subregion of VH CDR2 therefore plays a major role in determining both affinity and specificity for antigen. The data confirm that affinity for antigen is of pivotal importance in determining the degree of selection of different mutant forms of a V region. Moreover, during an immune response a sufficiently diverse mutant repertoire can be generated from a single canonical V region to allow adaptation to increase affinity for three different epitopes.

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