Biomedical subjects

E Munekata

Publications and source records attributed to E Munekata.

At least 19 recordsLinked to original sources

PubMed1996

Multiple intracellular signal transduction pathways mediating inward current produced by the neuropeptide, achatin-I.

The effects of intracellular signal transduction system inhibitors on the inward current (Iin) caused by achatin-I (Gly-D-Phe-Ala-Asp), an Achatina endogenous tetrapeptide having a D-phenylalanine residue, applied locally onto the neurone tested, were examined under voltage clamp using two identifiable Achatina giant neurone types, v-RCDN (ventral-right cerebral distinct neurone) and PON (periodically oscillating neurone). H-89 (N-[2-(p-bromocinnamylamino)-ethyl]-5-isoquinolinesulfonamide) (adenosine-3',5'-cyclic monophosphate (cyclic AMP)-dependent protein kinase inhibitor) markedly suppressed the achatin-I-induced Iin on PON, whereas this drug was ineffective on the Iin of v-RCDN. Dose (pressure duration)-response study of achatin-I on PON in a physiological solution and in the presence of H-89, and Lineweaver-Burk plot of these data, indicated that H-89 inhibited the Iin in a noncompetitive manner. KT5823 (N-methyl-(8R*,9S*,11S*)-(-)-9-methoxy-9-methoxycarbonyl-8-methyl-2,3,9, 10-tetrahydro-8,11-epoxy-1H,8H,11H-2, 7b,11a-triazadibenzo[a,g]cycloocta[c,d,e]-trinden-1-on e) (guanosine-3',5'-cyclic monophosphate (cyclic GMP)-dependent protein kinase inhibitor) suppressed the achatin-I-induced Iin of v-RCDN in mainly noncompetitive and partly uncompetitive manners, but this drug had no effect on the Iin of PON. W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide) (calmodulin inhibitor) suppressed noncompetitively the Iin of PON, but this drug had no effect on the Iin of v-RCDN. IBMX (3-isobutyl-1-methylxanthine) (cyclic nucleotide phosphodiesterase inhibitor) enhanced the achatin-I-induced Iin of v-RCDN, but this drug was ineffective on the Iin of PON. However, IBMX might have effects on the achatin-I receptor sites on v-RCDN. These findings suggest multiple intracellular signal transduction pathways mediating the achatin-I-induced Iin: the Iin of PON is via cyclic AMP-dependent and probably Ca2+/calmodulin-dependent protein kinases, and that of v-RCDN via cyclic GMP-dependent protein kinase. Other signal transduction system inhibitors including calphostin C (2-[12-[2-(benzyloxy)-propyl]-3, 10-dihydro-4,9-dihydroxy-2,6,7,11-tetramethoxy-3,10-dioxo-1-per yleny]-1 -methylethyl carbonic acid 4-hydroxyphenyl ester) (protein kinase C inhibitor) did not significantly affect the Iin of both v-RCDN and PON.

1-Methyl-3-isobutylxanthine↗

PubMed1996

Further identification of neurokinin receptor types and mechanisms of calcium signaling evoked by neurokinins in the murine neuroblastoma C1300 cell line.

It has been suggested that murine neuroblastoma C1300 cells express endogenous neurokinin NK2 receptors with features that differ from those of NK2 receptors characterized in other systems. In this study, we have further characterized the neurokinin receptor types present in this cell line. RNA blots showed that mRNAs of NK2 and NK3 receptors, but not of NK1 receptors, were expressed in C1300 cells. The increase in the cytosolic calcium concentration ([Ca2+]i) induced by 0.33 microM neurokinin A was completely inhibited by SR 48968, an NK2 receptor antagonist, whereas the partial response to 0.33 microM neurokinin B was unaffected, and the response was completely inhibited by SR 142801, and NK3 receptor antagonist. In addition, the [Ca2+]i increase by 0.33 microM senktide, an NK3 receptor agonist, was inhibited by SR 142801 but not by SR 48968. These findings indicated that C1300 cells endogenously express functional NK2 and NK3 receptors. It was also demonstrated that NK2 and NK3 receptors can be activated independently by 3.3 microM neurokinin A in the presence of 1.0 microM SR 142801 or 1.0 microM senktide, respectively. Therefore, the mechanisms of Ca2+ signaling mediated by endogenous NK2 and NK3 receptors were investigated. The independent activation of NK2 or NK3 receptors induced not only the [Ca2+]i increase, but also stimulated the formation of inositol trisphosphates; both these responses were inhibited by U73122, a phospholipase C (PLC) inhibitor. In addition, NK2 and NK3 receptor-mediated [Ca2+]i increase was partially attenuated in the absence of extracellular Ca2+ or in the presence of nickel, an inorganic Ca2+ influx blocker, but was unaffected by nifedipine and omega-conotoxin, L- and N-type voltage-dependent Ca2+ channel blockers, respectively. Furthermore, the depolarization by 60 mM K+ did not affect the [Ca2+]i. These findings suggested that the NK2 and NK3 receptor-mediated [Ca2+]i increase was due to the activation of PLC and was dependent on the mobilization of internal Ca2+ and the entry of extracellular Ca2+ through voltage-independent channels. This study showed that the C1300 cell line is a useful system with which to investigate pharmacological functions and signaling pathways of endogenous NK2 and NK3 receptors.

Animals↗

PubMed1995

Comparative study on the conformation of phalloidin, viroisin, and related derivatives in aqueous solution.

We investigated the conformations of toxic phalloidin and viroisin in aqueous solution using 500-MHz 1H-NMR spectroscopy in conjunction with molecular modeling. The conformations of two non-toxic phalloidin derivatives, secophalloidin and dethiophalloidin, were also correspondingly studied for comparison purposes. Results indicate that the non-toxic peptides have a multiple conformation, whereas the toxic peptides are comprised of a rigid molecule. It was found that the conformation of phalloidin partially resembles that of viroisin in the region of Cys3-Pro4-Ala5-Trp6, being different from that of the non-toxic peptides; thereby suggesting this region plays an important role leading to their toxicity.

Amino Acid Sequence↗

PubMed1995

Complement assembly of two fragments of the streptococcal protein G B1 domain in aqueous solution.

We examined the complementation of various pairs of fragments derived from the streptococcal protein G B1 domain by NMR and CD. Most were not associated; however, one pair of fragments (1-40) and (41-56) interacted sufficiently enough to regenerated a stable 1:1 complex, Kd = 9 x 10(-6) M. A 2D-NMR analysis showed that the structure of the complex resembled that of native domain. Here we discuss the complementation from the viewpoint of the folding pathway of the protein.

Bacterial Proteins↗

PubMed1995

Trophic effects of substance P and beta-amyloid peptide on dibutyryl cyclic AMP-differentiated human leukemic (HL-60) cells.

The neuropeptide substance P (SP) is a mediator of neurogenic inflammation. Also, beta-amyloid protein (beta AP) can directly activate the cell types involved in inflammatory processes. The relationship between SP and beta AP on their biological actions has attracted much interest. SP is trophic in neuronal cells and protected them against the death induced by beta AP. In this study, we examined the effects of SP and beta-amyloid peptide on cell viability in neutrophil-like HL-60 cells, by means of the WST-1 tetrazolium and lactate dehydrogenase (LDH) release assays. The results showed that SP promoted the cell survival on neutrophil-like HL-60 cells in serum-free conditions. Also, beta-amyloid peptide showed trophic effects rather than toxic in these cells in WST-1 assay, though it is reportedly toxic in neuronal cells.

Amino Acid Sequence↗

PubMed1995

Pharmacological evidence for neurokinin receptors in murine neuroblastoma C1300 cells.

We found that neurokinin A (NKA) and neurokinin B (NKB) induce an increase in the concentration of intracellular free Ca2+ ([Ca2+]i) in murine neuroblastoma C1300 cells (EC50: NKA 87 +/- 13 nM, NKB 97 +/- 15 nM). Substance P (SP) also caused a transient Ca2+ increase, although the potency of SP was much less than that of NKA and NKB. The increase in [Ca2+]i induced by NKA and NKB was inhibited by SR 48,968, a selective antagonist for NK2, and [beta Ala8]NKA(4-10), a selective agonist for NK2, did not stimulate the increase in [Ca2+]i. NKA- and NKB-induced Ca2+ mobilization was not inhibited by CP-96,345 and [Trp7, beta Ala8]NKA(4-10), selective antagonists for NK1 and NK3, respectively. These results suggested that C1300 cells express endogenous NK2 neurokinin receptors that have different features from known NK2 receptors.

Animals↗

PubMed1995

Synthesis and biological activity of N-terminal-truncated derivatives of human epidermal growth factor (h-EGF).

To investigate the contribution of the N-terminal sequence of h-EGF to its biological activity and the formation of three intramolecular disulfide bonds by oxidative refolding via air oxidation, five derivatives of h-EGF with a single N-terminal amino acid deletion were synthesized by solid-phase synthesis. The homogeneity of the synthetic peptides was confirmed by analytical reversed-phase HPLC, amino acid analysis, and FAB-MS. The pairing of the three disulfide bridges in synthetic peptides was determined by thermolytic digestion. All N-truncated derivatives of h-FGF formed the correct intramolecular three disulfide linkages during oxidative refolding and had equipotent activity in both EGF receptor binding on A-431 epidermoid carcinoma cells and mitogenesis on NIH-3T3 fibroblast cells, compared with authentic h-EGF. The results suggested that the five residues from N-terminal sequence of h-EGF have no effect on the formation of the correct disulfide linkages in h-EGF and do not exert a significant influence on its biological activity.

3T3 Cells↗

PubMed1995

beta-Amyloid peptide, substance P, and SEC receptor ligand activate cytoplasmic Ca2+ in neutrophil-like HL-60 cells: effect of chemotactic peptide antagonist BocMLF.

It has been reported that a discrete peptide fragment of beta-amyloid protein, beta A(25-35), and neuropeptide substance P (SP) possessed sequence homology and could bind to the serine protease inhibitor (serpin) enzyme complex (SEC) receptor. Thus, it has been thought that these peptides and SEC receptor ligand might have similar biological activities. In the present study, we found that C-terminal amidated beta A(25-35)-NH2, SP, and the SEC receptor ligand, Phe-Val-Phe-Leu-Met(FVFLM), could induce an increase in the intracellular free Ca2+ concentration ([Ca2+]i) in neutrophil-like human leukemic (HL-60) cells. Pretreatment with pertussis toxin (PTX) potently inhibited the increase in [Ca2+]i stimulated by these peptides, suggesting that these responses might be mediated by PTX-sensitive G-proteins. Furthermore, we examined the effect on these responses of t-butyloxycarbonyl-methionyl-leucyl-phenylalanine (BocMLF), which is a competitive antagonist of chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLF) at its receptor. BocMLF scarcely inhibited the [Ca2+]i increase stimulated by beta A(25-35)-NH2. However, the increase in FVFLM-induced [Ca2+]i was potently inhibited by BocMLF. The results suggest that the [Ca2+]i activation of beta A(25-35)-NH2 may have a different mechanism from that of FVFLM in neutrophil-like HL-60 cells, which is not mediated by the SEC-receptor.

Amino Acid Sequence↗

PubMed1995

Further mapping of the Achatina giant neurone types sensitive to the neuroactive peptides isolated from invertebrates.

1. The effects of the 10 synthetic neuroactive peptides originally isolated from invertebrates, applied locally to the neurone tested by the brief pneumatic pressure ejection on the identifiable neurone types of Achatina fulica Ferussac were examined. 2. Achatin-1 (Gly-D-Phe-Ala-Asp), an Achatina endogenous tetrapeptide having a D-phenylalanine residue, ejected locally, showed the depolarizing effects on nearly half of the number of neurone types tested. 3. ACEP-1 (Ser-Gly-Gln-Ser-Trp-Arg-Pro-Gln-Gly-Arg-Phe-NH2), isolated originally from Achatina atria, and pedal peptide (Pro-Leu-Asp-Ser-Val-Tyr-Gly-Thr-His-Gly-Met-Ser-Gly-Phe-Ala) and buccalin (Gly-Met-Asp-Ser-Leu-Ala-Phe-Ser-Gly-Gly-Leu-NH2), found in Aplysia neurones, showed excitatory effects on some Achatina neurone types. 4. Myomodulin (Pro-Met-Ser-Met-Leu-Arg-Leu-NH2), found in Aplysia neurones, produced a hyperpolarization on nearly half of the number of Achatina neurone types tested. The two FMRFamide-like peptides, <EDPFLRFamide (<Glu-Asp-Pro-Phe-Leu-Arg-Phe-NH2), isolated from Helix heart, and AF1 (Lys-Asn-Glu-Phe-Ile-Arg-Phe-NH2), from Ascaris head, also showed hyperpolarizing effects on more than half of the number of Achatina neurone types. 5. SALMFamide 1 (Gly-Phe-Asn-Ser-Ala-Leu-Met-Phe-NH2), isolated from Asterias nervous system, CCAP (Pro-Phe-Cys-Asn-Ala-Phe-Thr-Gly-Cys-NH2), from Carcinus pericardial organ, and corazonin (<Glu-Thr-Phe-Gln-Tyr-Ser-Arg-Gly-Trp-Thr-Asn-NH2), from Periplaneta cardiac corpus, had no effect on Achatina neurones.

Amino Acid Sequence↗

PubMed1995

Chemical synthesis and biological activities of the EGF-like domain of mouse schwannoma-derived growth factor (SDGF).

Schwannoma-derived growth factor (SDGF), an epidermal growth factor (EGF) family peptide recently discovered, has an EGF-like domain in the carboxyl terminal portion. In this study, we synthesized mSDGF(38-80) corresponding to the EGF-like domain of mouse SDGF by means of stepwise solid-phase method using Fmoc chemistry in order to evaluate the biological function of the EGF-like domain in SDGF. The linear peptide of mSDGF(38-80) was folded by direct oxidation with reduced and oxidized glutathione to form intramolecular disulfide bridges in synthetic peptide. On the biological activity, we examined mitogenic activity induced by mSDGF(38-80) in NIH/3T3 fibroblast cells and interaction with EGF receptor in A431 cells. In the results, mSDGF(38-80) was confirmed to form three disulfide linkages that were similar in pattern to EGF by amino acid and sequence analysis of fragments obtained after thermolytic digestion. However, mSDGF(38-80) possessed weak mitogenic activity in NIH/3T3 cells and weak binding affinity for the EGF receptor in A431 cells compared with those of human EGF. These results suggest that the EGF-like domain of SDGF may have little effect upon mitogenic activity and the EGF receptor binding of SDGF.

3T3 Cells↗

PubMed1994

Determination of electrostatic potential around specific locations on the surface of actin by diffusion-enhanced fluorescence resonance energy transfer.

In this study we have established systematic procedures for the measurement of electrostatic potentials around specific and localized portions of protein surfaces. Diffusion-enhanced fluorescence energy transfer was used for these determinations. Energy transfer from donor molecules to freely diffusing acceptors is sensitive to the electrostatic potential around the donor when the acceptors possess electric charges. To quantify this sensitivity phenomenon in a controllable environment, we observed energy transfer from excited terbium chelate donors of known electric charge to a series of acceptors of different charges but bearing the same chromophore group. The rate of energy transfer was calculated theoretically (considering the structural arrangement of charged groups around the chromophore center), as well as determined experimentally (by time-resolved detection of terbium luminescence), with the two values obtained by the different means in close agreement. Having established the validity of this procedure using a relatively simple system, we studied the electrostatic conditions around two specific sites on the surface of actin molecules. A negative potential was found at both sites; the potential at one location (around the myosin binding site) was nearly neutralized by the addition of myosin subfragment-1, while the potential at the other site (around the phalloidin binding site) was not significantly affected.

Actins↗

PubMed1994

Inhibitory effects of beta-amyloid peptides on nicotine-induced Ca2+ influx in PC12h cells in culture.

Synthetic beta-amyloid peptides and the neuropeptide substance P (SP) were examined for their ability to modulate nicotinic response in PC12h cells, a subclone of PC12 cells, SP, beta A1-40 and its peptide fragment beta A25-35-NH2 significantly inhibited an increase in cytoplasmic calcium concentrations ([Ca2+]i) induced by nicotine in a dose-dependent manner. Furthermore, beta A1-40 was found to inhibit the [Ca2+]i increase induced by depolarization with a high concentration of potassium. These findings show that both beta A1-40 and beta A25-35-NH2 may mimic the function of SP on inhibition of nicotinic response through different mechanisms.

Amino Acid Sequence↗

PubMed1994

Structure-activity relationships of human epidermal growth factor(h-EGF).

The 53 amino acid regulatory peptide, human epidermal growth factor (h-EGF), is a potent mitogen that stimulates cellular proliferation and differentiation in a wide variety of cells. To identify the critical residues that elicit the biological activity of h-EGF, peptides were constructed by stepwise solid-phase synthesis using the Boc-HF strategy. These synthetic peptides were characterized by HPLC, FAB-MS, amino acid analysis and thermolytic digestion. The mitogenic activity of these h-EGF analogues was determined by the stimulation of [3H]-thymidine uptake into DNA in NIH-3T3 fibroblast cell lines. Substituting Tyr with Phe at position's 37 and 13 had little effect on the mitogenic activity of h-EGF. In contrast, Ala at these positions resulted in a severe loss of activity (20 and 10(3)-fold). These results indicate that the hydrophobicity of the side chain at positions 13 and 37 of h-EGF is essential for its biological activity. A semiconservative substitution of Leu with Ala at position 15 and a conservative change of Lys at position 41 also drastically reduced mitogenic activity (10(4) and 10(5)-fold). Thus, the bulky hydrophobic side chain at position 15 and the guanidino group at position 41 are indispensable in determining the biological activity of h-EGF.

3T3 Cells↗

PubMed1994

Chemical synthesis and biological activity of the EGF-like domain of heparin-binding epidermal growth factor-like growth factor (HB-EGF).

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a recently discovered member of the epidermal growth factor (EGF) family. This novel growth factor possesses the EGF-like domain in the carboxyl portion. In order to evaluate the biological function of the EGF-like domain in HB-EGF, human HB-EGF(44-86) corresponding to the EGF-like domain was synthesized by the solid-phase procedure using the Fmoc strategy. It was confirmed by amino acid microsequencing of cysteine-containing fragments derived from thermolytic digestion that the pattern of three disulfide bond pairings in synthetic HB-EGF(44-86) was consistent with that of EGF and transforming growth factor-alpha (TGF-alpha). The homogeneity of the synthetic peptide was confirmed by analytical RP-HPLC, amino acid analysis and fast atom bombardment mass spectrometer (FAB-MS). Compared with h-EGF, the EGF-like domain of human HB-EGF showed a comparable mitogenic activity in the proliferation of NIH/3T3 fibroblast cells. These results suggest that the EGF-like domain of human HB-EGF may play an important role in mitogenic activity.

3T3 Cells↗

PubMed1993

A structurally flexible and antigenically variable N-terminal domain of the hepatitis C virus E2/NS1 protein: implication for an escape from antibody.

Hepatitis C virus persists in most infected hosts, and causes chronic hepatitis, liver cirrhosis, and/or hepatocellular carcinoma in humans. During the infection the RNA genome of hepatitis C virus undergoes frequent missense mutations at one or two "hypervariable" regions within a presumptive envelope gene (Okamoto et al., 1992, Virology 190, 894-899; Ogata et al., 1991, Proc. Natl. Acad. Sci. USA 88, 3392-3396). In the present study, we analyzed three cases of hepatitis C virus infection, two in chimpanzees and one in humans, for the antigenicity of peptides predicted from the hypervariable region of viral RNA obtained during the follow-up. Our results showed a successive appearance of hepatitis C virus mutants with antigenically distinct amino acid sequence within the domain; and the amino acid replacement was associated with an alteration of predicted local secondary structure of the epitope region. Hence, the hypervariable domain of the hepatitis C virus envelope appeared to be structurally flexible and antigenically variable, providing the virus a way to escape from host immunity.

Amino Acid Sequence↗

PubMed1993

Electrostatic potential around actin.

We presume that tension of contracting muscle originates from electrostatic force experienced by actin and myosin. We suppose that a high-energy state of myosin-ADP-Pi interacts with actin, transferring the stored energy to actin, and that the actin excited in this way develops around itself electric field which exerts sliding force against charged myosin heads. To explore the idea, first we conjectured how electric charges on actin produce electric field in the axial direction, and second we experimentally examined electrostatic circumstances around actin in solution and in muscle fibers by optimizing diffusion-enhanced fluorescence energy transfer. In the experiments, Tb ion, which has a long excited-state lifetime, was used as donor. To introduce Tb to actin, Tb-DTPA-phalloin and Tb-DTPA-maleimide were synthesized. As acceptors with electric charges (Za = -3 to +2), rhodamine B that was conjugated with various amino acids or their derivatives was used. The fluorescence energy transfer efficiency (ET) was estimated from the shortening in the lifetime of Tb. The electrostatic circumstances around actin were inferred from the ET-Za relation. When Tb was introduced at Cys-374 of actin, the Tb-site was found in negative electric potential. S-1 binding to the labeled actin neutralized the electric potential almost completely. Tb-DTPA-phalloin bound to actin seemed to reside in the vicinity of tryptophan residue(s). Electric potential around the phalloin site was negative. S-1 binding to the actin slightly reduced the negativity. In glycerinated fibers in the rigor state, the phalloin site was again found in negative potential. When fibers were transferred from an ADP-rigor solution to an active solution, the negative electric potential was neutralized to some extent. The direction of this change could not be explained by detachment of crossbridges from actin, since the detachment should have given an opposite direction of changes in the electric potential. Thus, this observation may indicate that electric potential characteristic of the active state occurs at actin surfaces.

Actins↗

PubMed1992

G protein antagonists. A novel hydrophobic peptide competes with receptor for G protein binding.

A substance P (SP) analog, [D-Pro4,D-Trp7,9,10] SP4-11, is known to inhibit the actions of various structurally unrelated messenger molecules as well as SP. Our studies on the effects of this peptide on the regulation of purified G proteins by receptor showed that at least some of the biological effects of the peptide can be explained by the ability of the peptide to block the activation of G proteins by receptors. Here we report that a novel truncated SP-related peptide, pGlu-Gln-D-Trp-Phe-D-Trp-D-Trp-Met-NH2, inhibited the activation of G(i) or G(o) by M2 muscarinic cholinergic receptor (M2 mAChR) or of Gs by beta-adrenergic receptor in the reconstituted phospholipid vesicles, assayed by receptor-promoted GTP hydrolysis. The inhibition by the peptide was apparently reversible and competitive with respect to receptor binding to G proteins; the inhibition could be overcome by increasing the concentration of receptor in the vesicles and was not altered by changes in the concentration of G protein. The competing effects of the peptide were used to analyze the effect of agonist on receptor-G protein interaction. The concentration change of muscarinic agonist did not alter the inhibitory effects of the peptide on M2 mAChR-promoted GTPase by G(o), which is consistent with the idea that agonist increases the regulatory efficiency of the receptor but does not alter its affinity for G proteins. This new group of compounds (G protein antagonists) is a promising tool to study receptor-G protein interaction quantitatively.

Amino Acid Sequence↗

PubMed1992

1H NMR study on the conformation of bacitracin A in aqueous solution.

The conformation of bacitracin A, a widely used cyclic dodecapeptide antibiotic in aqueous solution, has been investigated using 500 MHz 1H NMR and molecular modeling. Findings revealed that a region (residues 1-6) is folded over the cyclic ring, resulting in metal coordination sites, a thiazoline ring, and Glu4 and His10 being proximate to each other.

Amino Acid Sequence↗