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Biomedical subjects

Yoichi Hayakawa

Publications and source records attributed to Yoichi Hayakawa.

At least 19 recordsLinked to original sources
PubMed2005

Peptide mimics of epidermal growth factor (EGF) with antagonistic activity.

Epidermal growth factor is a potent growth-promoting factor for a variety of tissue cells in vivo and in vitro. Epidermal growth factor binds, phosphorylates, and activates epidermal growth factor receptors on the cell surface. In this study, we attempted to design functional peptide mimics by panning a phage display library on the anti-epidermal growth factor monoclonal antibody. By using anti-epidermal growth factor monoclonal antibody as a mold of the structure of the binding site of epidermal growth factor, high-efficiency probing was expected. From a random peptide phage display library, phage clones that bind to the anti-epidermal growth factor monoclonal antibody were isolated. One of the phage clones also exhibited binding activity to the epidermal growth factor receptor. The amino acid sequence of this phage clone showed slight similarity to the primary sequence of epidermal growth factor. We synthesized this motif to a 9-amino-acid intramolecularly disulfide-linked peptide. This synthetic peptide inhibited mitogenesis as well as epidermal growth factor receptor tyrosine phosphorylation, which is induced by epidermal growth factor. The present results suggest that the peptide synthesized in this study may mimic the epidermal growth factor receptor-binding region in epidermal growth factor.

Animals↗
PubMed2005

A cytokine secreted from the suboesophageal body is essential for morphogenesis of the insect head.

The suboesophageal body of insects was identified over a century ago in the silkworm embryo, but its biological function is still unknown. We discovered that this tissue is differentiated in the earliest embryonic stages of the cabbage armyworm and secretes the insect cytokine, growth-blocking peptide (GBP), transiently from 24 to 60 h after oviposition when gastrulation is in progress. Over-expression of GBP, achieved by microinjection of the GBP gene driven by a cytomegalovirus (CMV) constitutive promoter, resulted in complex deformities of the procephalon (embryonic head). Severe abnormal phenotypes of the head structure were produced by silencing the GBP expression in the embryo by treating with GBP double-stranded RNA: the procephalon-containing optic lobes diminished and completely separated into bilateral halves. This indicates that GBP secreted from the suboesophageal body plays an essential role in the formation of the procephalic domain during early embryogenesis. The cytokine-induced fusion of bilateral procephalic lobes is thought to be evolutionarily conserved at least in insects, because of the widespread occurrence of the suboesophageal body in insect embryos.

Animals↗
PubMed2004

Legioliulin, a new isocoumarin compound responsible for blue-white autofluorescence in Legionella (Fluoribacter) dumoffii under long-wavelength UV light.

Legionella dumoffii is one of the causative agents of Legionnaires' disease. There are 50 species in the genus Legionella, of which 10 species including L. dumoffii are known to exhibit uncharacterized blue-white autofluorescence. We constructed an L. dumoffii strain that exhibited a high intensity blue-white fluorescence, isolated a fluorescent pigment from the strain, and determined its molecular formula to be C(19)H(14)O(3) by high-resolution mass spectrometry. An NMR analysis revealed that this was a new isocoumarin compound, which was named legioliulin.

Coumarins↗
PubMed2004

The Gly-Gly linker region of the insect cytokine growth-blocking peptide is essential for activity.

Growth-blocking peptide (GBP) is a 25-amino acid cytokine isolated from the lepidopteran insect Pseudaletia separata. GBP exhibits various biological activities such as regulation of larval growth of insects, proliferation of a few kinds of cultured cells, and stimulation of a class of insect immune cells called plasmatocytes. The tertiary structure of GBP consists of a well structured core domain and disordered N and C termini. Our previous studies revealed that, in addition to the structured core, specific residues in the unstructured N-terminal region (Glu1 and Phe3) are also essential for the plasmatocyte-stimulating activity. In this study, a number of deletion, insertion, and site-directed mutants targeting the unstructured N-terminal residues of GBP were constructed to gain more detailed insight into the mode of interaction between the N-terminal region and GBP receptor. Alteration of the backbone length of the linker region between the core structure and N-terminal domain reduced plasmatocyte-stimulating activity. The substitutions of Gly5 or Gly6 in this linker region with more bulky residues, such as Phe and Pro, also remarkably reduced this activity. We conclude that the interaction of GBP with its receptor depends on the relative position of the N-terminal domain to the core structure, and therefore the backbone flexibility of Gly residues in the linker region is necessary for adoption of a proper conformation suited to receptor binding. Additionally, antagonistic experiments using deletion mutants confirmed that not only the core domain but also the N-terminal region of GBP are required for "receptor-binding," and furthermore Phe3 is a binding determinant of the N-terminal domain.

Amino Acid Motifs↗
PubMed2004

Effect on tumor cells of blocking survival response to glucose deprivation.

BACKGROUND: Glucose deprivation, a feature of poorly vascularized solid tumors, activates the unfolded protein response (UPR), a stress-signaling pathway, in tumor cells. We recently isolated a novel macrocyclic compound, versipelostatin (VST), that inhibits transcription from the promoter of GRP78, a gene that is activated as part of the UPR. We examined the effect of VST on the UPR induced by glucose deprivation or other stressors and on tumor growth in vivo. METHODS: Human colon cancer HT-29, fibrosarcoma HT1080, and stomach cancer MKN74 cells were cultured in the absence of glucose or in the presence of glucose and a UPR-inducing chemical stressor (the N-glycosylation inhibitor tunicamycin, the calcium ionophore A23187, or the hypoglycemia-mimicking agent 2-deoxyglucose [2DG]). The effect of VST on UPR induction was determined by reverse transcription-polymerase chain reaction and immunoblot analysis of the UPR target genes GRP78 and GRP94; by immunoblot analysis of the UPR transcriptional activators ATF6, XBP1, and ATF4; and by analyzing reporter gene expression in cells transiently transfected with a GRP78 promoter-reporter gene. Cell sensitivity to VST was examined with a colony formation assay and flow cytometry. In vivo antitumor activity of VST was assessed with an MKN74 xenograft model. RESULTS: VST inhibited expression of UPR target genes in glucose-deprived or 2DG-treated cells but not in cells treated with tunicamycin or A23187. VST also inhibited the production of the UPR transcriptional activators XBP1 and ATF4 during glucose deprivation. The UPR-inhibitory action of VST was seen only in conditions of glucose deprivation and caused selective and massive killing of the glucose-deprived cells. VST alone and in combination with cisplatin statistically significantly (P =.004 and P<.001 for comparisons with untreated control, respectively) inhibited tumor growth of MKN74 xenografts. CONCLUSION: Disruption of the UPR may provide a novel therapeutic approach to targeting glucose-deprived solid tumors.

Activating Transcription Factor 4↗
PubMed2004

Bacillus subtilis ypgA gene is fni, a nonessential gene encoding type 2 isopentenyl diphosphate isomerase.

We previously identified the fni gene of Streptomyces sp. strain CL190 as type 2 isopentenyl diphosphate (IPP) isomerase, which needs both FMN and NADPH for enzyme activity. An fni gene homolog, ypgA, was detected in the database of the Bacillus subtilis genome. However, the ypgA product was about 140 amino acids shorter in the N-terminal than the Streptomyces fni gene product. A database search found three new putative start codons in 129, 225, and 411 bases upstream of the original start codon of the ypgA gene. The longest gene product, which was named ypgA3, showed the most significant homology to the Streptomyces fni gene product. The ypgA3 gene was expressed with an N-terminal His-tag in Escherichia coli and the purified soluble protein was characterized in detail. The ypgA3 protein converted IPP to its isomer dimethylallyl diphosphate in the presence of both FMN and NADPH. The enzyme also catalyzed the reverse reaction in the presence of both the cofactors. Disruption of the ypgA3 gene was not lethal to B. subtilis. These results indicate that Bacillus ypgA3 gene is fni, a nonessential gene encoding type 2 IPP isomerase.

Amino Acid Sequence↗
PubMed2004

Structure of tyroscherin, an antitumor antibiotic against IGF-1-dependent cells from Pseudallescheria sp.

An antitumor antibiotic, tyroscherin, was isolated from the culture of a fungus identified as Pseudallescheria sp. The structure of tyroscherin including the absolute stereochemistry was determined as shown in Fig. 1 by NMR and degradation studies. Tyroscherin selectively inhibited IGF-1-dependent growth of MCF-7 human breast cancer cells with an IC50 of 9.7 ng/ml.

Antibiotics, Antineoplastic↗
PubMed2004

[Structure-activity relationship analysis].

All samples for anticancer drug screening were classified according to their structural features and their structure-activity relationships were analyzed. Synthetic gymnastatin analogs including JCI: 11788 and JCI: 11786 altered their selectivity for protein kinase inhibition with the length of a fatty acid chain. Although a new inhibitor of tubulin depolymerization, JCI: 11578, displayed a high correlation to known tubulin binders, novel inhibitors of tubulin polymerization, JCI: 11534, JCI: 11675 and JCI: 11676, exhibited poor correlations to tubulin binders. JCI: 11403 and JCI: 11407 inhibited topoisomerase I selectively and appear to belong to a new family of topoisomerase inhibitors. They are expected to be important key compounds for structure-activity relationship analysis as well as new lead compounds for anticancer drugs.

Antineoplastic Agents↗
PubMed2003

Insect cytokine growth-blocking peptide triggers a termination system of cellular immunity by inducing its binding protein.

Growth-blocking peptide (GBP) is a 25-amino acid cytokine found in lepidopteran insects that possesses diverse biological activities such as stimulation of immune cells (plasmatocytes), cell proliferation, and larval growth regulation. We found another novel function of GBP that induces a hemolysis of another class of blood cells (oenocytoids). In the lysate of oenocytoids we identified a GBP-binding protein that shows a specific affinity for GBP. The characterization of purified GBP-binding protein and its cDNA demonstrated it as a 49.5-kDa novel protein with a C-terminal region displaying limited homology to several insect lipoproteins. Results of Northern and Western blotting indicated that the GBP-binding protein should be synthesized only in blood cells. Immunoelectron microscopic analyses confirmed that indirect immunoreactive signals were mostly localized in oenocytoids. Kinetic and biological analyses of interaction between GBP and the binding protein showed their strong binding was followed by clearance of GBP from hemolymph, thus indicating that this protein might function as an inhibitory factor against GBP. Based on these results, we propose that insect cytokine GBP shows multifunctions even in cellular immunity: it serves to stimulate immune cells and afterward silences its own action by inducing the binding protein through specific hemolysis.

Amino Acid Sequence↗
PubMed2003

Cause of mortality in insects under severe stress.

Mortality in the host armyworm larvae Pseudaletia separata parasitized by the parasitic wasp Cotesia kariyai was dramatically increased when they were simultaneously infected by the entomopathogen Serratia marcescens. Previous studies have shown that this strong insecticidal effect is due to a metalloprotease-like insecticide (MPLI) released from S. marcescens enterobacter. This study was conducted to elucidate the exact cause of the mortality resulting from MPLI. Injection of MPLI caused a sharp increase in hemolymph dopamine concentration followed by elevated levels of brain dopamine in armyworm larvae. [3H]Dopamine injected into the hemocoel, was incorporated into the brains of MPLI-injected larvae to a level eight times greater than in BSA-injected control larvae. Transmission electron microscopy showed an obvious decrease in thickness and density of the brain sheath in insects injected with MPLI. This was probably due to the MPLI-induced elevation of hemocyte metalloprotease activities. Further, electron microscopic and TUNEL staining analyses showed a significant increase in apoptotic cells in the brain 12 h after the injection. Injection of 3-iodotyrosine (a tyrosine hydroxylase inhibitor) before MPLI completely prevented the increase in hemolymph dopamine in test larvae and their following death. From these observations, we conclude that MPLI-injected larvae may have suffered mortal damage through increased apoptosis of brain cells caused by an influx of dopamine from the hemolymph.

Animals↗
PubMed2003

Byssochlamysol, a new antitumor steroid against IGF-1-dependent cells from Byssochlamys nivea. I. Taxonomy, fermentation, isolation and biological activity.

A new antitumor steroid, byssochlamysol, was isolated from the mycelium of Byssochlamys nivea M#5187. Byssochlamysol inhibited IGF-1-dependent growth of MCF-7 human breast cancer cells with an IC50 of 20 ng/ml, whereas serum-dependent cell growth was not inhibited by less than 10 microg/ml of byssochlamysol. This substance induced apoptosis in IGF-1-dependent Colo320DM human colon cancer cells.

Animals↗
PubMed2003

Oximidine III, a new antitumor antibiotic against transformed cells from Pseudomonas sp. I. Taxonomy, fermentation, isolation, physico-chemical properties and biological activity.

Our screening for antitumor antibiotics against transformed cells resulted in the isolation of a new active metabolite, oximidine III, from Pseudomonas sp. QN05727. This substance selectively inhibited the growth of rat 3Y1 fibroblasts transformed with various oncogenes. In ras- or src-transformed cells, oximidine III arrested the cell cycle at G1 phase and increased the expression of p21WAF1.

Animals↗
PubMed2002

Roles of aromatic residues in the structure and biological activity of the small cytokine, growth-blocking peptide (GBP).

Growth-blocking peptide (GBP) is a small (25 amino acids) insect cytokine with a variety of functions: controlling the larval development of lepidopteran insects, acting as a mitogen for various types of cultured cells, and stimulating insect blood cells. The aromatic residues of GBP (Phe-3, Tyr-11, and Phe-23) are highly conserved in the ENF peptide family found in lepidopteran insects. We investigated the relationship between the biological activities and structural properties of a series of GBP mutants, in which each of the three aromatic residues is replaced by a different residue. The results of the hemocytes-stimulating assays of GBP mutants indicated that Phe-3 is the key residue in this activity: Ala or Tyr replacement resulted in significant loss of the activity, but Leu replacement did not. The replacements of other aromatic residues hardly affected the activity. On the other hand, NMR analysis of the mutants suggested that Tyr-11 is a key residue for maintaining the core structure of GBP. Surprisingly, the Y11A mutant maintained its biological activity, although its native-like secondary structure was disordered. Detailed analyses of the (15)N-labeled Y11A mutant by heteronuclear NMR spectroscopy showed that the native-like beta-sheet structure of Y11A was induced by the addition of 2,2,2-trifluoroethanol. The results suggest that Y11A has a tendency to form a native-like structure, and this property may give the Y11A mutant native-like activity.

Amino Acid Sequence↗
PubMed2002

Hygrolidin induces p21 expression and abrogates cell cycle progression at G1 and S phases.

Hygrolidin family antibiotics showed selective cytotoxicity against both cyclin E- and cyclin A-overexpressing cells. Among them, hygrolidin was the most potent and inhibited growth of solid tumor-derived cell lines such as DLD-1 human colon cancer cells efficiently more than that of hematopoietic tumor cells and normal fibroblasts. FACS analysis revealed that hygrolidin increased cells in G1 and S phases in DLD-1 cells. While hygrolidin decreased amounts of cyclin-dependent kinase (cdk) 4, cyclin D, and cyclin B, it increased cyclin E and p21 levels. Hygrolidin-induced p21 bound to and inhibit cyclin A-cdk2 complex more strongly than cyclin E-cdk2 complex. Furthermore, hygrolidin was found to increase p21 mRNA in DLD-1 cells, but not in normal fibroblasts. Thus, hygrolidin inhibited tumor cell growth through induction of p21. In respect to p21 induction, inhibition of vacuolar-type (H+)-ATPase by hygrolidin was suggested to be involved.

Animals↗
PubMed2002

Structure and activity of insect cytokine GBP which stimulates the EGF receptor.

Growth-blocking peptide (GBP) is an insect cytokine that possesses diverse biological activities such as larval growth regulation, cell proliferation, and stimulation of immune cells. GBP is a 25-amino acid peptide with one disulfide bond. It has been revealed that the tertiary structure of GBP consists of an N- and C-terminal disordered region and a well-structured core. Although there is only a slight similarity between the primary structures of GBP and EGF and the molecular weight of GBP is about half that of EGF, GBP directly binds and activates the EGF receptor of human keratinocyte cells. Furthermore, the tertiary structure of the well-defined region of GBP is similar to that of the C-terminal domain of EGF. This review will focus on the tertiary structure of GBP and its activities, as compared with those of EGF.

Amino Acid Sequence↗