scholarly journals New renin inhibitors homologous with pepstatin

1981 ◽  
Vol 197 (2) ◽  
pp. 465-471 ◽  
Author(s):  
M Eid ◽  
G Evin ◽  
B Castro ◽  
J Menard ◽  
P Corvol
Keyword(s):  
Methyl Ester ◽  
Glutamic Acid ◽  
Water Soluble ◽  
Amino Acid Residues ◽  
Renin Inhibitors ◽  
C Terminus ◽  
Coupling Reagent ◽  
Order Of Magnitude ◽  
High Yields

Four homologues of pepstatin, the potent but poorly soluble inhibitor of aspartic proteinases, were synthesized by coupling to the C-terminus of the natural pentapeptide the following amino acid residues: L-arginine methyl ester, L-aspartic acid, L-glutamic acid and the dipeptide L-aspartyl-L-arginine. The peptide-coupling reagent we used, benzotriazolyloxytris(dimethylamino)phosphonium hexafluorophosphate, allowed us to obtain readily pure pepstatin homologues with high yields (60-83%). Pepstatylarginine methyl ester and pepstatylglutamic acid were about one order of magnitude more water-soluble than pepstatin. The four homologues and pepstatin were tested in vitro as inhibitors for highly purified pig and human renins acting on the N-acetyltetradecapeptide substrate. The 50% inhibitory concentrations (IC50) of the homologues were ranged from 0.01 to 1 microM against porcine renin at pH 6.0 (pepstatin IC50 approximately 0.32 microM) and from 5.8 to 41 microM against human renin at pH 6.5 (pepstatin IC 50 approximately 17 microM). By three different graphical methods we showed that pepstatin and the four homologues behaved as competitive inhibitors for porcine renin. The most potent inhibitors were pepstatylaspartic acid and pepstatylglutamic acid, with inhibitory constants respectively 2- and 10-fold smaller than that of pepstatin. By coupling glutamic acid to pepstatin, the ratio solubility/Ki was increased by two orders of magnitude.

Peroxisome targeting signal of rat liver acyl-coenzyme A oxidase resides at the carboxy terminus

1989 ◽  
Vol 9 (1) ◽  
pp. 83-91
Author(s):  
S Miyazawa ◽  
T Osumi ◽  
T Hashimoto ◽  
K Ohno ◽  
S Miura ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rat Liver ◽  
Coenzyme A ◽  
Terminal Region ◽  
Proteinase K ◽  
Amino Acid Residues ◽  
C Terminus ◽  
Targeting Signal ◽  
Acyl Coenzyme A

To identify the topogenic signal of peroxisomal acyl-coenzyme A oxidase (AOX) of rat liver, we carried out in vitro import experiments with mutant polypeptides of the enzyme. Full-length AOX and polypeptides that were truncated at the N-terminal region were efficiently imported into peroxisomes, as determined by resistance to externally added proteinase K. Polypeptides carrying internal deletions in the C-terminal region exhibited much lower import activities. Polypeptides that were truncated or mutated at the extreme C terminus were totally import negative. When the five amino acid residues at the extreme C terminus were attached to some of the import-negative polypeptides, the import activities were rescued. Moreover, the C-terminal 199 and 70 amino acid residues of AOX directed fusion proteins with two bacterial enzymes to peroxisomes. These results are interpreted to mean that the peroxisome targeting signal of AOX residues at the C terminus and the five or fewer residues at the extreme terminus have an obligatory function in targeting. The C-terminal internal region also has an important role for efficient import, possibly through a conformational effect.

scholarly journals Characterization of Chimpanzee/Human Monoclonal Antibodies to Vaccinia Virus A33 Glycoprotein and Its Variola Virus Homolog In Vitro and in a Vaccinia Virus Mouse Protection Model

2007 ◽  
Vol 81 (17) ◽  
pp. 8989-8995 ◽  
Author(s):  
Zhaochun Chen ◽  
Patricia Earl ◽  
Jeffrey Americo ◽  
Inger Damon ◽  
Scott K. Smith ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Vaccinia Virus ◽  
Protective Efficacy ◽  
Variola Virus ◽  
Binding Affinities ◽  
Amino Acid Residues ◽  
C Terminus ◽  
20 Nm

ABSTRACT Three distinct chimpanzee Fabs against the A33 envelope glycoprotein of vaccinia virus were isolated and converted into complete monoclonal antibodies (MAbs) with human γ1 heavy-chain constant regions. The three MAbs (6C, 12C, and 12F) displayed high binding affinities to A33 (Kd of 0.14 nM to 20 nM) and may recognize the same epitope, which was determined to be conformational and located within amino acid residues 99 to 185 at the C terminus of A33. One or more of the MAbs were shown to reduce the spread of vaccinia virus as well as variola virus (the causative agent of smallpox) in vitro and to more effectively protect mice when administered before or 2 days after intranasal challenge with virulent vaccinia virus than a previously isolated mouse anti-A33 MAb (1G10) or vaccinia virus immunoglobulin. The protective efficacy afforded by anti-A33 MAb was comparable to that of a previously isolated chimpanzee/human anti-B5 MAb. The combination of anti-A33 MAb and anti-B5 MAb did not synergize the protective efficacy. These chimpanzee/human anti-A33 MAbs may be useful in the prevention and treatment of vaccinia virus-induced complications of vaccination against smallpox and may also be effective in the immunoprophylaxis and immunotherapy of smallpox and other orthopoxvirus diseases.

scholarly journals Σύνθεση και χαρακτηρισμός πρωτότυπων ολιγομερών κυκλοδεξτρινών για μεταφορά φαρμάκων

2014 ◽  
Author(s):  
Μαλαματένια Μανουηλίδου
Keyword(s):  
Binding Capacity ◽  
Drug Carrier ◽  
Aqueous Solubility ◽  
Single Step ◽  
Drug Formulation ◽  
Water Soluble ◽  
Amide Bond ◽  
Mild Conditions ◽  
Order Of Magnitude ◽  
High Yields

Cyclodextrins (CDs) are cyclic hollow oligosaccharide molecules that form water solublehost-guest systems, with many applications in drug formulation and delivery. CDoligomers have been previously studied due to the interest towards smart hosts withenhanced molecular recognition and binding capacity as sensors, catalysts, enzymemimics, photoreactive systems, etc. The aim of this dissertation was to prepare αCDoligomers for drug inclusion and transport with criteria: (i) ease of preparation, inaqueous media, in short steps, under mild conditions and in good yields, (ii) to obtainoligomers with satisfactory aqueous solubility and full availability of the CD cavities (iv)to achieve multiple binding with strengths better or comparable to those of parent αCD.The copper catalyzed azide-alkyne cyclization (CuAAC) reaction was utilized to preparea new water soluble cyclodextrin trimer very efficiently. The trimer engulfed threemolecules of a model guest and satisfactorily solubilized the chemotherapeutictamoxifen citrate and its active metabolite, N-desmethyltamoxifen, increasing theirsolubility by >1 order of magnitude. Moreover, for the first time the bioorthogonalStaudinger Ligation was applied to prepare αCD-dimers. For this purpose, a doublyactive linker was specifically developed that enabled dimer preparation in a single step,in aqueous/organic media, under mild conditions and with high yields. The aboveprepared products were studied in detail by NMR spectroscopy and were found toadopt, by self-inclusion, a closed conformation in aqueous solution, which completelyopened up in the presence of a suitable guest, leaving the cavities fully available to formthe corresponding inclusion complexes. Titration and DOSY NMR experimentsconfirmed the above and showed that the dimeric species form slowly diffusingaggregates in water, that in the presence of the guest partially disperse. The StaudingerLigation could thus become the method of choice for preparing CD dimers.Solubilization of practically insoluble N-desmethyl-tamoxifen was also achieved to 0.3mM. Moreover, CD dimers prepared via amide bond formation were less efficient andrequired harsh conditions. Finally, SNO-αCD derivatives were prepared andcharacterized as bimodal NO and drug carrier systems.

Structure and transforming potential of the human cot oncogene encoding a putative protein kinase

1991 ◽  
Vol 11 (8) ◽  
pp. 4088-4096
Author(s):  
J Miyoshi ◽  
T Higashi ◽  
H Mukai ◽  
T Ohuchi ◽  
T Kakunaga
Keyword(s):  
Protein Kinase ◽  
Transcriptional Control ◽  
Thyroid Tumor ◽  
Kinase Assay ◽  
Human Thyroid ◽  
Protein Kinase Activity ◽  
Amino Acid Residues ◽  
Morphological Transformation ◽  
C Terminus

A new transforming gene has been molecularly cloned from hamster SHOK cells transformed with DNA extracted from a human thyroid carcinoma cell line and named the cot (cancer Osaka thyroid) oncogene. cDNA sequencing disclosed that this oncogene codes for a protein with 415 amino acid residues, and computer matching showed 42 to 48% similarity matches with serine protein kinases. Its gene product was identified as a 52-kDa protein by transcription and translation in vitro. Expression of cot cDNA under transcriptional control by a retroviral long terminal repeat induced morphological transformation of NIH 3T3 cells as well as SHOK cells. Protein kinase activity associated with constructed p60gag-cot was detected by immune complex kinase assay with anti-gag antiserum. The cot oncogene was overexpressed in transformed SHOK cells and found to have a rearranged 3' end in the last coding exon, which probably resulted in a deletion and an altered C' terminus in the transforming protein. This DNA rearrangement appeared to have occurred during transfection of the tumor DNA into hamster SHOK cells and not in the original thyroid tumor.

scholarly journals Improvement of Peptide Affinity and Stability by Complexing to Cyclodextrin-Grafted Ammonium Chitosan

Polymers ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 474 ◽  
Author(s):  
Andrea Cesari ◽  
Alessandra Recchimurzo ◽  
Angela Fabiano ◽  
Federica Balzano ◽  
Nicolò Rossi ◽  
...  
Keyword(s):  
In Vitro Models ◽  
Water Soluble ◽  
Complexing Agent ◽  
Grafted Polymers ◽  
Polymeric Derivative ◽  
Nmr Characterization ◽  
Order Of Magnitude ◽  
Covalently Linked ◽  
Peptide Affinity

Cyclodextrin-grafted polymers are attractive biomaterials that could bring together the host–guest complexing capability of pristine cyclodextrin and the pharmaceutical features of the polymeric backbone. The present paper is aimed at characterizing the potential application of ammonium–chitosan grafted with 2-methyl-β-cyclodextrin (N+-rCh-MCD) as the functional macromolecular complexing agent for the oral administration of the neuropeptide dalargin (DAL). Specific NMR characterization procedures, along with UV and fluorescence techniques, as well as biological in vitro assessments have been performed. The results indicate that N+-rCh-MCD forms water-soluble complexes with DAL, with a prevalent involvement of Tyr or Phe over Leu and Ala residues. The association constant of DAL with the polymeric derivative is one order of magnitude higher than that with the pristine cyclodextrin (Ka: 2600 M−1 and 120 M−1, respectively). Additionally, N+-rCh-MCD shields DAL from enzymatic degradation in gastrointestinal in vitro models with a three-fold time delay, suggesting a future pharmaceutical exploitation of the polymeric derivative. Therefore, the greater affinity of N+-rCh-MCD for DAL and its protective effect against enzymatic hydrolysis can be attributed to the synergistic cooperation between cyclodextrin and the polymer, which is realized only when the former is covalently linked to the latter.

Sequence Determination of a Protein with N-Terminal Glutamic Acid after Modification of Carboxyl Groups with Carbodiimide as the Coupling Agent

1974 ◽  
Vol 52 (6) ◽  
pp. 560-562
Author(s):  
C. Gilardeau ◽  
M. Chrétien
Keyword(s):  
Methyl Ester ◽  
Glutamic Acid ◽  
Water Soluble ◽  
Carboxyl Groups ◽  
Edman Degradation ◽  
Sequence Determination ◽  
Glutamic Acid Residue ◽  
N Terminus ◽  
Glycine Methyl Ester

Glutamine and asparagine residues in proteins can be differentiated from glutamic and aspartic residues, during the Edman degradation, after modification of the carboxyl groups by glycine methyl ester in presence of a water-soluble carbodiimide. When applied to ovine and porcine beta-lipotropic hormones, which have a glutamic acid residue at the N-terminus, the carbodiimide blocks the N-terminus. However, the Edman degradation proceeds normally, if the phenylthiocarbamyl derivative is formed prior to the modification reaction with glycine. In this communication, radioactive glycine was used to modify the carboxyl groups.

scholarly journals Construction and Application of Variants of the Pseudomonas fluorescens EBC191 Arylacetonitrilase for Increased Production of Acids or Amides

2010 ◽  
Vol 76 (11) ◽  
pp. 3668-3674 ◽  
Author(s):  
Olga Sosedov ◽  
Stefanie Baum ◽  
Sibylle B�rger ◽  
Kathrin Matzer ◽  
Christoph Kiziak ◽  
...  
Keyword(s):  
Pseudomonas Fluorescens ◽  
Mandelic Acid ◽  
Cysteine Residue ◽  
Amino Terminus ◽  
Amino Acid Residues ◽  
Amide Formation ◽  
C Terminus ◽  
Reaction Specificity ◽  
Homologous Position ◽  
High Yields

ABSTRACT The arylacetonitrilase from Pseudomonas fluorescens EBC191 differs from previously studied arylacetonitrilases by its low enantiospecificity during the turnover of mandelonitrile and by the large amounts of amides that are formed in the course of this reaction. In the sequence of the nitrilase from P. fluorescens, a cysteine residue (Cys163) is present in direct neighborhood (toward the amino terminus) to the catalytic active cysteine residue, which is rather unique among bacterial nitrilases. Therefore, this cysteine residue was exchanged in the nitrilase from P. fluorescens EBC191 for various amino acid residues which are present in other nitrilases at the homologous position. The influence of these mutations on the reaction specificity and enantiospecificity was analyzed with (R,S)-mandelonitrile and (R,S)-2-phenylpropionitrile as substrates. The mutants obtained demonstrated significant differences in their amide-forming capacities. The exchange of Cys163 for asparagine or glutamine residues resulted in significantly increased amounts of amides formed. In contrast, a substitution for alanine or serine residues decreased the amounts of amides formed. The newly discovered mutation was combined with previously identified mutations which also resulted in increased amide formation. Thus, variants which possessed in addition to the mutation Cys163Asn also a deletion at the C terminus of the enzyme and/or the modification Ala165Arg were constructed. These constructs demonstrated increased amide formation capacity in comparison to the mutants carrying only single mutations. The recombinant plasmids that encoded enzyme variants which formed large amounts of mandeloamide or that formed almost stoichiometric amounts of mandelic acid from mandelonitrile were used to transform Escherichia coli strains that expressed a plant-derived (S)-hydroxynitrile lyase. The whole-cell biocatalysts obtained in this way converted benzaldehyde plus cyanide either to (S)-mandeloamide or (S)-mandelic acid with high yields and enantiopurities.

scholarly journals Antileishmanial Activity, Uptake, and Biodistribution of an Amphotericin B and Poly(α-Glutamic Acid) Complex

2013 ◽  
Vol 57 (10) ◽  
pp. 4608-4614 ◽  
Author(s):  
Abeer H. A. Mohamed-Ahmed ◽  
Karin Seifert ◽  
Vanessa Yardley ◽  
Hollie Burrell-Saward ◽  
Stephen Brocchini ◽  
...  
Keyword(s):  
Glutamic Acid ◽  
Amphotericin B ◽  
Leishmania Donovani ◽  
Water Soluble ◽  
Molecular Weight Range ◽  
Acid Complex ◽  
Content Type ◽  
New Treatment

ABSTRACTA noncovalent, water-soluble complex of amphotericin B (AMB) and poly(α-glutamic acid) (PGA), with AMB loadings ranging from 25 to 55% (wt/wt) using PGA with a molecular weight range of 50,000 to 70,000, was prepared as a potential new treatment for visceral leishmaniasis (VL). The AMB-PGA complex was shown to be as active as Fungizone (AMB deoxycholate) against intracellularLeishmania donovaniamastigotes in differentiated THP-1 cells. Thein vitrouptake of the AMB-PGA complex by differentiated THP-1 cells was similar to that of Fungizone and higher than that of AmBisome (liposomal AMB). The AMB-PGA complex also displayed a dose-response profile similar to that of AmBisomein vivoin BALB/c mice againstL. donovani, with 50% effective doses (ED50s) of 0.24 ± 0.03 mg/kg of body weight for the AMB-PGA complex and 0.24 ± 0.06 mg/kg for AmBisome. A biodistribution study with mice indicated that the AMB-PGA complex cleared more rapidly from plasma than AmBisome, with a comparable low level of distribution to the kidneys.

scholarly journals Invading the Yeast Nucleus: a Nuclear Localization Signal at the C Terminus of Ty1 Integrase Is Required for Transposition In Vivo

1998 ◽  
Vol 18 (2) ◽  
pp. 1115-1124 ◽  
Author(s):  
Margaret A. Kenna ◽  
Carrie Baker Brachmann ◽  
Scott E. Devine ◽  
Jef D. Boeke
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Reverse Transcriptase Activity ◽  
Deletion Mutants ◽  
Amino Acid Residues ◽  
Wild Type ◽  
C Terminus ◽  
Localization Signal

ABSTRACT Retrotransposon Ty1 faces a formidable cell barrier during transposition—the yeast nuclear membrane which remains intact throughout the cell cycle. We investigated the mechanism by which transposition intermediates are transported from the cytoplasm (the presumed site of Ty1 DNA synthesis) to the nucleus, where they are integrated into the genome. Ty1 integrase has a nuclear localization signal (NLS) at its C terminus. Both full-length integrase and a C-terminal fragment localize to the nucleus. C-terminal deletion mutants in Ty1 integrase were used to map the putative NLS to the last 74 amino acid residues of integrase. Mutations in basic segments within this region decreased retrotransposition at least 50-fold in vivo. Furthermore, these mutant integrase proteins failed to localize to the nucleus. Production of virus-like particles, reverse transcriptase activity, and complete in vitro Ty1 integration resembled wild-type levels, consistent with failure of the mutant integrases to enter the nucleus.

scholarly journals Influence of N-terminal acetylation and C-terminal proteolysis on the analgesic activity of β-endorphin

1980 ◽  
Vol 189 (3) ◽  
pp. 501-506 ◽  
Author(s):  
J F W Deakin ◽  
J O Doströvsky ◽  
D G Smyth
Keyword(s):  
Amino Acid ◽  
Opiate Receptors ◽  
Acetyl Derivative ◽  
Amino Acid Residues ◽  
Duration Of Action ◽  
Beta Endorphin ◽  
C Terminus ◽  
N Terminus ◽  
Specific Affinity

Removal of one, two and four amino-acid residues from the C-terminus of beta-endorphin (‘lipotropin C-Fragment’, lipotropin residues 61–91) led to the formation of peptides with progressively decreased analgesic potency; there was no change in the persistence of the analgesic effects. The four C-terminal residues are thus important for the activity of beta-endorphin, but not for the duration of action. Removal of eight amino-acid residues from the N-terminus provided a peptide that had no specific affinity for brain opiate receptors in vitro and was devoid of analgesic properties. The N-terminal sequence of beta-endorphin is therefore necessary for the production of analgesia, whereas the C-terminal residues confer potency. The N alpha-acetyl form of beta-endorphin had no specific affinity for brain opiate receptors in vitro and possessed no significant analgesic properties. Since lipotropin C'-Fragment (lipotropin residues 61–87) and the N alpha-acetyl derivative of beta-endorphin occur naturally in brain and pituitary and are only weakly active or inactive as opiates, it is suggested that proteolysis at the C-terminus and acetylation of the N-terminus of beta-endorphin may constitute physiological mechanisms for inactivation of this potent analgesic peptide.

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