Biomedical subjects

A Otaka

Publications and source records attributed to A Otaka.

At least 55 records · Page 3Linked to original sources

PubMed1995

Synthesis of protegrin-related peptides and their antibacterial and anti-human immunodeficiency virus activity.

All disulfide analogs (types I, II and III) of protegrin (PG)-1, an 18-residue antimicrobial peptide having two intramolecular disulfide bonds, were synthesized using regioselective disulfide bond formation. Random air-oxidation of the fully reduced PG-1 formed the type III PG-1. In addition, a type III analog containing an amidated carboxy-terminal residue was also prepared. Each analog showed significant and different antibacterial and anti-human immunodeficiency virus (HIV) activity. Deletion of two disulfide bridges caused a significant decrease in activity.

Amino Acid Sequence↗

PubMed1994

Structure-activity relationships of an anti-HIV peptide, T22.

T22 ([Tyr5,12,Lys7]-polyphemusin II) has been shown to have a strong anti-human immunodeficiency virus (HIV) activity comparable to that of 3'-azido-2',3'-dideoxythymidine (AZT). We studied the structure-anti-HIV activity relationships of T22 and determined the following information. The number of Arg residues in the N-terminal and C-terminal regions of T22 is closely related with anti-HIV activity. Disulfide rings, especially the major disulfide ring, are indispensable for anti-HIV activity and maintenance of the secondary structure. Between two repeats of Tyr-Arg-Lys, which are a characteristic structure contained in T22, Tyr-Arg-Lys in the N-terminal portion is more closely related with anti-HIV activity. We found some compounds having a higher selectivity index (50% cytotoxic concentration/50% effective concentration) than that of T22.

Amino Acid Sequence↗

PubMed1994

Assembly of synthetic laminin peptides into a triple-stranded coiled-coil structure.

Laminin, a large multidomain glycoprotein specific to basement membranes, is a heterotrimer with alpha, beta, and gamma chains held together in an alpha-helical coiled-coil structure. Synthetic peptides comprising two 51-mers (B1 and B2) from the beta 1 and gamma 1 subunits and a 55-mer (M) from alpha 2 were used to study the molecular mechanisms in laminin chain assembly. Using the synthetic peptides in various mixing experiments, the heterotrimer (B1-B2-M) was preferentially produced. The thermal stability of the heterotrimer increased dramatically (by approximately 20 degrees C) over that of the B1-B2 heterodimer as measured by circular dichroism (CD) spectroscopy. The B1-B1 homodimer (Tm = 60 degrees C) showed higher thermal stability when compared to B1-B2 and B2-B2 dimers. However, the B1 + B2 mixture produced principally the B1-B2 heterodimer. These results suggested that the preferential formations of heterodimer was regulated by kinetic interactions between each chain. The B2 and M peptides have many hydrophobic isoleucine residues which were replaced by leucines. These substitutions were predicted to favor an alpha-helical conformation and a higher propensity for zipper formation. B2L and ML, in which all isoleucine residues were replaced by leucine, showed significantly increased alpha-helicities. While B2L was able to form heterodimers and heterotrimers similar to B2, ML was not able to participate in heterotrimer formation as efficiently as the M peptide. The thermal stability of B1-B2L was comparable to that of B1-B2, but B2L and/or ML containing trimers showed lower thermal stability than B1-B2-M. These results suggest that the isoleucine residues in the alpha 2 and gamma 1 chains are critical for stabilizing the heteromeric triple-stranded coiled-coil structure.

Amino Acid Sequence↗

PubMed1994

Lymphocytes and promonocytes attach to the synthetic [Tyr5,12, Lys7]- polyphemusin II peptide.

The [Tyr5,12,Lys7]-polyphemusin II peptide (T22) has been shown to inhibit HIV-1 replication in lymphocytes. The mechanism of T22 inhibition of HIV-1 replication is not known but may involve T22 competition with HIV-1 for attachment sites on the plasma membrane of targeted cells. Here we find that three human immunocyte cell lines (H9, Jurkat, and U-937) attach to T22. The phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), has been shown to activate intracellular protein kinase C and to stimulate lymphocyte attachment to various substrates through specific cell surface receptors. Here we find that TPA treatment enhances attachment of the immunocytes to T22 by three- to four-fold. These data demonstrate that T22 binds to immunocyte cell surfaces and support the hypothesis that T22 may inhibit HIV-1 replication by competing with the virus for a common cell surface receptor(s).

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1994

Cyclic peptide inhibitors of phosphatidylinositol 3-kinase p85 SH2 domain binding.

Cyclic hexameric peptides based on the amino acid sequence "Gly-Xxx-Val-Pro-Met-Leu", where Xxx is either phosphotyrosyl (pTyr) residue or a hydrolytically stable pTyr mimetic, were examined for their ability to bind to the C-terminal SH2 domain of the p85 phosphoinositol 3-kinase (PI 3-kinase). The cyclic peptides retained significant binding affinity relative to their linear counterparts. Potency varied depending on Xxx in the order: phosphonomethyl phenylalanine (Pmp, ID50 = 5.2 microM) < phosphonodifluoromethyl phenylalanine (F2Pmp, ID50 = 2.2 microM) < pTyr (ID50 = 1.0 microM), with Xxx = Tyr being inactive (ID50 > 500 M). Greatly reduced potency was observed when Xxx was of the unnatural D-configuration. The cyclic peptides represent conformationally constrained ligands which should be useful in the development of p85 SH2 domain-directed inhibitors.

Amino Acid Sequence↗

PubMed1994

Nonhydrolyzable phosphotyrosyl mimetics for the preparation of phosphatase-resistant SH2 domain inhibitors.

Src homology 2 (SH2) domains participate in protein tyrosine kinase (PTK)-mediated cellular signal transduction through their ability to bind with high affinity to phosphotyrosyl (pTyr)-bearing protein sequences. Although peptides containing pTyr competitively inhibit the binding between phosphoproteins and cognate SH2 proteins in a sequence-specific manner, such peptides are rapidly dephosphorylated by cellular phosphatases. We now describe our efforts to develop SH2 inhibitory peptides containing phosphatase-resistant pTyr surrogates. The parent compound, (phosphonomethyl)phenylalanine (Pmp), is a phosphonate-based mimetic of pTyr in which the phosphate ester oxygen (> COPO3H2) has been replaced by a methylene unit (> CCX2PO3H2, X2 = H2). Pmp analogues bearing fluorine (X2 = H, F or X2 = F2) or hydroxyl (X2 = H, OH) substituents on the phosphonate alpha-methylene carbon have been prepared and incorporated into peptides for use as SH2 domain inhibitors. In an assay using the C-terminal SH2 domain of phosphatidylinositol (PI) 3-kinase, peptides having a GXVPML sequence [where X = pTyr, Pmp, hydroxy-Pmp (HPmp), monofluoro-Pmp (FPmp), and difluoro-Pmp (F2Pmp)] exhibited binding potency in the order HPmp < Pmp < FPmp < F2Pmp = pTyr. Distinct peptide sequences which bind selectively with Src and Grb2 SH2 domains were also prepared with pTyr and F2Pmp. The F2Pmp peptides bound with high (0.2- to 5-fold) relative affinity, compared to analogous pTyr peptides. We conclude that peptides containing F2Pmp bind to SH2 domains with high affinity and specificity and, being resistant to cellular phosphatases, should provide a generally useful tool for disrupting SH2 domain-mediated signaling pathways in intact cells.

Amino Acid Sequence↗

PubMed1994

Molecular parameters for the anti-human immunodeficiency virus activity of T22 ([Tyr5,12, Lys7]-polyphemusin II).

T22 ([Tyr5,12, Lys7]-polyphemusin II) was found to exhibit strong anti-human immunodeficiency virus (HIV) activity and exert its effects on a virus-cell fusion process. In the present study, the all-D enantiomer of T22 and its related compounds were synthesized to examine the molecular parameters required for the interaction of T22 with membrane components of cells or viruses in order to exert this anti-HIV activity. The anti-HIV activity of these analogs was investigated in comparison with their membrane permeability with aspect to large unilamellar vesicles (LUVs). The all-D enantiomer of T22 exhibited a 20-fold lower anti-HIV activity compared with T22, whereas they both showed the same membrane permeability. No positive correlation between anti-HIV activity and membrane permeability was observed. These results suggest that the anti-HIV activity of T22 is mediated through the interaction with chiral component(s) of the cell or virus.

Amino Acid Sequence↗

PubMed1993

Role of disulfide linkages in tachyplesin-lipid interactions.

In order to elucidate the role of the two disulfide linkages of tachyplesin I (T-SS), a membrane-acting cyclic antimicrobial peptide from Tachypleus tridentatus, we synthesized the acyclic analog (T-Acm) with the four SH groups protected by acetamidomethyl groups and also investigated the interactions of these peptides with lipid bilayers. T-SS induced leakage of calcein from egg yolk L-alpha-phosphatidylglycerol large unilamellar vesicles (PG LUVs) at peptide concentrations 1 order of magnitude smaller than those at which leakage was induced by T-Acm, which coincides with the stronger antimicrobial activities of T-SS. The micellization of PG LUVs was also more efficient for the cyclic peptide. Fluorescence titration studies revealed that binding affinities of both peptides to the PG membranes were similar. Fourier transform infrared polarized attenuated total reflection spectroscopy and fluorescence quenching experiments demonstrated that T-SS and T-Acm both form amphiphilic antiparallel beta-sheet structures in the membranes. They are formed in such a way that the sheet planes lie parallel to the membrane surface with the sheet hydrophobic surfaces penetrating slightly into the hydrophobic region of the bilayers. Furthermore, the observation that the linear T-Acm, the weaker membrane permeabilizer, caused a far more serious membrane disruption suggests the possibility that the mechanisms of membrane permeabilization by the cyclic peptide are different from those by the linear peptide, the latter being the disruption of the lipid organization.

Amino Acid Sequence↗

PubMed1993

A comparative study of the solution structures of tachyplesin I and a novel anti-HIV synthetic peptide, T22 ([Tyr5,12, Lys7]-polyphemusin II), determined by nuclear magnetic resonance.

The solution structure of tachyplesin I, which was isolated from membrane acid extracts of the hemocytes from the Japanese horseshoe crab (Tachypleus tridentatus), was determined by nuclear magnetic resonance (NMR) and distance geometry calculation. Tachyplesin I takes an antiparallel beta-sheet structure with a type-II beta-turn. Recently, among more than 20 synthetic peptides associated with tachyplesin and its isopeptide (polyphemusin), we found that a novel compound, which we designated as T22 ([Tyr5,12, Lys7]-polyphemusin II), strongly inhibited the human immunodeficiency virus (HIV)-1-induced cytopathic effect and viral antigen expression. The solution structure of T22 was investigated using NMR, and its secondary structure was confirmed to be similar to that of tachyplesin I. The anti-parallel beta-sheet structure and the several amino-acid side chains on the plane of the beta-sheet of T22 are thought to be associated with the expression of anti-HIV activity.

Amino Acid Sequence↗

PubMed1993

Multiplicity of virus-encoded helper T-cell epitopes expressed on FBL-3 tumor cells.

To identify retroviral antigenic determinants recognized by CD4+ T helper cells during tumor rejection, we established four noncytolytic, helper-type, CD4+ T-cell clones by limiting dilution cultures of mixed lymphocyte-tumor cultures from mice immune to a Friend virus-induced tumor, FBL-3. Among these, three T helper cell clones were isolated from C57BL/6 mice and the fourth was isolated from a (BALB/c x C57BL/6)F1 mouse. All these clones proliferated in response to the immunizing FBL-3 tumor cells in a major histocompatibility complex class II-restricted manner. Each clone expressed a distinct T-cell receptor with a characteristic combination of alpha and beta chains. The localization of helper T-cell determinants on viral proteins was analyzed with recombinant vaccinia viruses expressing Friend murine leukemia virus (F-MuLV) gag or env genes or shorter fragments of the env gene. Epitopes recognized by these T-cell clones were mapped to at least two distinct portions in the env region of the F-MuLV genome. These epitopes were identified more precisely with synthetic peptides derived from the F-MuLV envelope protein sequence. One of these epitopes was common to Friend and Moloney MuLVs and was located in the N-terminal region of the gp70 glycoprotein at amino acids 122 to 141. The second epitope, which was recognized in the context of hybrid I-Eb/d major histocompatibility complex class II molecule, was located close to the C-terminal end of gp70 at amino acids 462 to 479. In addition, a possible third epitope was located in the N-terminal half of the gp70 sequence and differed from the first epitope in that it was not cross-reactive with the Moloney MuLV envelope protein.

Amino Acid Sequence↗

PubMed1993

Preparation of peptide-carrying microspheres with bioactivity on platelets.

A tetrapeptide RGDS (Arg-Gly-Asp-Ser), which is one of the active sites of cell adhesive proteins, was synthesized according to the solution method and covalently coupled onto monodisperse microspheres. The biospecific activity of the RGDS-carrying microspheres was investigated by measuring the stimulatory effect of the microspheres on platelets. Large and tightly packed platelet aggregation accompanied with drastic ATP release was induced by the RGDS-carrying microspheres. It was confirmed that the specific platelet aggregation was attributed to the activity of the immobilized RGDS by comparison with those induced by other microspheres.

Acrylamides↗

PubMed1993

Investigation of the dimethylsulfoxide-trifluoroacetic acid oxidation system for the synthesis of cystine-containing peptides.

Disulfide bonds of peptides were effectively established between S-protected cysteine residues as well as free cysteine residues by the action of dimethylsulfoxide in trifluoroacetic acid. Oxytocin and alpha-human calcitonin gene-related peptide were synthesized using this oxidation system. The feasibility of this method for the formation of two disulfide bridges of apamin was also examined.

Amino Acid Sequence↗

PubMed1993

Syntheses and biological activities of selenium analogs of alpha-rat atrial natriuretic peptide.

alpha-Rat atrial natriuretic peptide (7--28) (rANP (7--28)) and a series of its analogs in which half cystine residue(s) were substituted with half selenocystine residue(s) were synthesized by using the Fmoc-based solid-phase method followed by cyclization by means of dimethylsulfoxide (DMSO)-trifluoroacetic acid (TFA) oxidation. These analogs possess comparable activities in both receptor binding and cGMP accumulation in rat vascular smooth muscle cells to those of rAMP (7--28).

Amino Acid Sequence↗

PubMed1993

Synthetic study on selenocystine-containing peptides.

N-9-Fluorenylmethoxycarbonyl-Se-4-methoxybenzylselenocysteine++ + [Fmoc-Sec(MBzl)-OH] was synthesized from selenocystine and successfully applied to Fmoc-based solid-phase peptide synthesis. The stability and the deprotection conditions of the Se-MBzl group were examined. The diselenide bond of a peptide was directly and effectively established between Sec(MBzl) residues by treatment with iodine or the dimethyl sulfoxide-trifluoroacetic acid system. Reduction kinetics of diselenide and disulfide in model peptides by reduced glutathione were also studied comparatively.

Amino Acid Sequence↗

PubMed1992

A novel anti-HIV synthetic peptide, T-22 ([Tyr5,12,Lys7]-polyphemusin II).

Tachyplesin and polyphemusin are antimicrobial peptides recently isolated from the hemocytes of horseshoe crabs (Tachypleus tridentatus and Limulus polyphemus). We synthesized them and their analogs and examined their antiviral activity against human immunodeficiency virus (HIV) type 1 in vitro. The infection of human T cells with the virus was markedly inhibited by some of them at low concentrations. In this structure-activity study, we found that [Tyr5,12, Lys7]-polyphemusin II, which was designated as T22, had extremely high anti-HIV activity. Its 50% inhibitory concentration (EC50) was 0.008 micrograms/ml, while its 50% cytotoxic concentration (CC50) was 54 micrograms/ml and these values were comparable to those of AZT. This result indicates that T22 would be a potential candidate for the therapy of HIV infection.

Amino Acid Sequence↗

PubMed1992

Enzyme immunoassay of thyrotropin releasing hormone (TRH).

A sensitive and specific double-antibody enzyme immunoassay (EIA) for a thyrotropin releasing hormone (TRH)-like immunoreactive substance has been developed. In order to synthesize TRH-labeled beta-D-galactosidase (beta-gal), a newly devised TRH derivative, pGlu-His-Pro-NH-(CH2)6-NH2 (TRH-Hex), was employed. TRH-Hex was linked to beta-gal by the N-(epsilon-maleimidocaproyloxy) succinimide coupling procedure. For competitive reactions, the TRH antibody was incubated with standard TRH and TRH-Hex-beta-gal (delayed addition). Free and antibody-bound enzyme hapten were separated by using an anti-rabbit immunoglobulin G coated immunoplate. Activity of the enzyme on the plate was fluorometrically determined. The present immunoassay allows detection of 0.8 to 100 pmol/well of TRH.

Amino Acid Sequence↗

PubMed1991

Gastrin releasing peptide-like immunoreactive substance in rat mammary glands during pregnancy.

The concentration of gastrin releasing peptide-like immunoreactive substance (GRP-IS) was measured by enzyme immunoassay (EIA) in rat mammary glands during pregnancy and after delivery. The GRP-IS concentration was high in the middle stage of pregnancy (10-14 days of gestation) and decreased in late pregnancy to reach a plateau range. By using HPLC, it was shown that GRP(20-29) and GRP(16-29) were mainly present in rat mammary glands. Immunohistochemical study revealed that epithelial cells of rat mammary glands were stained intensely with antiserum GP-6201. These results suggest that GRP-IS is produced and secreted in epithelial cells of mammary glands and takes part in the proliferation, differentiation and hypertrophy of mammary glands.

Animals↗

PubMed1991

Two-step hard acid deprotection/cleavage procedure for solid phase peptide synthesis.

A new two-step deprotection/cleavage procedure for t-butoxycarbonyl (Boc) based solid phase peptide synthesis is reported. First the protective groups are removed from 4-(oxymethyl)-phenylacetamidomethyl (PAM) resin attached peptide with the weak hard acid, trimethylsilyl bromide-thioanisole/trifluoroacetic acid (TFA). In the second step, the peptide is cleaved from the resin with a stronger hard acid such as trimethylsilyl trifluoromethanesulfonate in TFA or with HF. The method is also shown to deformylate Nin-formyltryptophan moiety efficiently. The usefulness of this procedure for practical solid phase peptide synthesis is demonstrated by comparison with other deprotection methods in the synthesis of urotensin II and human endothelin.

Acetamides↗