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Biomedical subjects

H Maruta

Publications and source records attributed to H Maruta.

At least 163 records · Page 9Linked to original sources
PubMedDate not supplied

Solution synthesis of human midkine, a novel heparin-binding neurotrophic factor consisting of 121 amino acid residues with five disulphide bonds.

Human midkine (hMK), a novel heparin-binding neurotrophic factor consisting of 121 amino acid residues with five intramolecular disulphide bonds, was synthesized by solution procedure in order to demonstrate the usefulness of our newly developed solvent system, a mixture of dichloromethane or chloroform and trifluoroethanol. The final protected 121-residue peptide was assembled from two large fully protected intermediates, Boc-(1-59)-OH and H-(60-121)-OBzl, in CHL/TFE(3:1, v/v) using water-soluble carbodiimide in the presence of HOOBt as coupling reagents. After removal of the protecting groups by HF followed by treatment with Hg(OAc)2 in 50% acetic acid, the fully deprotected peptide was subjected to the oxidative folding reaction. The final product was confirmed to have the correct disulphide structure from its tryptic peptide mapping and to possess the same biological activities as those of the natural product. In order to clarify the active region of the hMK molecule, the N-terminal half domains [(1-59) and (60-121)] were also synthesized by the same procedure used for the hMK synthesis. The C-half domain was confirmed to show the full pattern of bioactivities except for the neuronal cell survival activity, while the N-half one showed much less activity in general.

Amino Acid Sequence↗
PubMedDate not supplied

Disruption of microfilament organization after injection of F-actin capping proteins into living tissue culture cells.

Capping proteins are F-actin binding proteins which interfere with the in vitro growth of an actin filament by blocking one of its ends (for recent reviews see refs 1-3). The majority of such proteins described so far "cap' the fast-growing (positive) end of the polar filament, thus reducing the velocity of filament growth while increasing the number of filaments being formed de novo from a monomer pool. We have studied the effects of capping proteins on the organization of actin filaments in living tissue culture cells by microinjection in conjunction with fluorescence, reflection contrast and electron microscopy. Our results, reported here, indicate that capping proteins from different sources disrupt microfilament bundles in a variety of cell types causing their disintegration from the distal end towards the centre of the cell.

Animals↗
PubMedDate not supplied

Regulation of actin polymerization by non-polymerizable actin-like proteins.

Three functionally distinct actin-capping proteins from the slime mould Physarum are structurally closely related to actin itself. In Physarum, actin polymerization is regulated by a set of non-polymerizable actin-like proteins. It remains to be established whether these proteins and actin are each encoded by separate genes.

Actins↗
PubMedDate not supplied

Role of the hypothalamic opioidergic system in the control of gonadotropin secretion in elderly men.

The role of opioidergic system in controlling gonadotropin secretion in elderly men still remains uncertain. In the present study, we attempted to examine the opioidergic inhibitory tone imposed on GnRH release mechanism in elderly men by monitoring the plasma gonadotropin response to the opiate receptor antagonist naloxone hydrochloride. Six normal young men and 10 elderly men with no endocrinological diseases volunteered for the present study. In young men, plasma LH showed a biphasic increase in response to naloxone and plasma LH level during naloxone treatment was significantly higher than the mean of prenaloxone control levels. In contrast, plasma LH in elderly men was not affected by naloxone. The ratio of the peak plasma LH response during naloxone treatment to the mean pre treatment LH level (plasma peak/basal LH ratio) declined with the advance of age (r = -0.74, P < 0.005) and also correlated significantly with plasma free testosterone level (r = 0.45, P < 0.05). These data suggest (1) that the hypothalamic opioidergic tone is reduced in elderly men and (2) that the primary testicular insufficiency with the advance of age may play a major role in the decline of the hypothalamic opioidergic tone in elderly men.

Adolescent↗
PubMedDate not supplied

Treatment of Ras-induced cancers by the F-actin cappers tensin and chaetoglobosin K, in combination with the caspase-1 inhibitor N1445.

UNLABELLED: For transforming normal fibroblasts to malignant cells, oncogenic Ras mutants such as v-Ha-ras require Rho family GTPases (Rho, Rac, and CDC42) that are responsible for controlling actin-cytoskeleton organization. Ras activates Rac through a PI-3 kinase-mediated pathway. Rac causes uncapping of actin filaments (F-actin) at the plus-ends, through phosphatidylinositol 4,5 bisphosphate (PIP2), and eventually induces membrane ruffling. Several distinct F-actin/PIP2-binding proteins, such as gelsolin, which severs and caps the plus-ends of actin filaments, or HS1, which cross-links actin filaments, have been shown to suppress v-Ha-Ras-induced malignant transformation when they are overexpressed. Interestingly, an F-actin cross-linking drug (photosensitizer) called MKT-077 suppresses Ras transformation. Thus, an F-actin capping/severing drug might also have an anticancer potential. PURPOSE: This study was conducted to determine first whether Ras-induced malignant phenotype (anchorage-independent growth) is suppressed by overexpression of the gene encoding a large plus-end F-actin capping protein called tensin and second to test the anti-Ras potential of a unique fungal antibiotic (small compound) called chaetoglobosin K (CK) that also caps the plus-ends of actin filaments. METHODS AND RESULTS: DNA transfection with a retroviral vector carrying the tensin cDNA was used to overexpress tensin in v-Ha-Ras-transformed NIH 3T3 cells. All stable tensin transfectants rarely formed colonies in soft agar, indicating that tensin suppresses the anchorage-independent growth. The anti-Ras action of CK was determined by incubating the Ras-transformants in the presence of CK in soft agar. Two microM CK almost completely inhibited their colony formation, indicating that CK also suppresses the malignant phenotype. However, unlike tensin, CK causes an apoptosis of Ras-transformed NIH 3T3 cells and, less effectively, of normal NIH 3T3 cells, indicating that CK has an F-actin capping-independent side effect(s). CK-induced apoptosis is at least in part caused by CK-induced inhibition of the kinase PKB/AKT. However, a specific ICE/caspase-1 inhibitor called N1445 completely abolished the CK-induced apoptosis by reactivating PKB, but without affecting the CK-induced suppression of Ras transformation. CONCLUSIONS: Like the F-actin cross-linking drug MKT-077, the F-actin capping drug CK may be useful for the treatment of Ras-associated cancers if it is combined with the ICE inhibitor N1445, which abolishes the side effect of CK. Our observations that two distinct F-actin capping molecules (i.e., tensin and CK) suppress Ras-induced malignant phenotype strongly suggest, if not prove, that capping of actin filaments at the plus-ends alone is sufficient to block one of the Ras signaling pathways essential for its oncogenicity. This notion is compatible with the fact that Ras induces the uncapping of actin filaments at the plus-ends through the Rac/PIP2 pathway.

3T3 Cells↗
PubMedDate not supplied

Treatment of ras-induced cancers by the F-actin-bundling drug MKT-077.

A rhodacyanine dye called MKT-077 has shown a highly selective toxicity toward several distinct human malignant cell lines, including bladder carcinoma EJ, and has been subjected to clinical trials for cancer therapy. In the pancreatic carcinoma cell line CRL-1420, but not in normal African green monkey kidney cell line CV-1, it is selectively accumulated in mitochondria. However, both the specific oncogenes responsible for its selective toxicity toward cancer cells, and its target proteins in these cancer cells, still remain to be determined. This study was conducted using normal and ras-transformed NIH 3T3 fibroblasts to determine whether oncogenic ras mutants such as v-Ha-ras are responsible for the selective toxicity of MKT-077 and also to identify its targets, using its derivative called "compound 1" as a specific ligand. We have found that v-Ha-ras is responsible for the selective toxicity of MKT-077 in both in vitro and in vivo. Furthermore, we have identified and affinity purified at least two distinct proteins of 45 kD (p45) and 75 kD (p75), which bind MKT-077 in v-Ha-ras-transformed cells but not in parental normal cells. Microsequencing analysis has revealed that the p45 is a mixture of beta- and gamma-actin, whereas the p75 is HSC70, a constitutive member of the Hsp70 heat shock adenosine triphosphatase family, which inactivates the tumor suppressor p53. MKT-077 binds actin directly, bundles actin filaments by cross-linking, and blocks membrane ruffling. Like a few F-actin-bundling proteins such as HS1, alpha-actinin, and vinculin as well as F-actin cappers such as tensin and chaetoglobosin K (CK), the F-actin-bundling drug MKT-077 suppresses ras transformation by blocking membrane ruffling. These findings suggest that other selective F-actin-bundling/capping compounds are also potentially useful for the chemotherapy of ras-associated cancers.

3T3 Cells↗
PubMedDate not supplied

An anti-Ras cancer potential of PP1, an inhibitor specific for Src family kinases: in vitro and in vivo studies.

BACKGROUND: We previously found that both PAK, a Rac/CDC42-activated Ser/Thr kinase, and its binding partner PIX are required for malignant transformation caused by oncogenic Ras mutants, such as v-Ha-Ras. Furthermore, oncogenic Ras requires an autocrine pathway to activate PAK. This pathway involves at least two distinct receptor kinases: EGF receptor (ErbB1) and ErbB2. Interestingly, both of these kinases are known to activate Src family kinases that phosphorylate CAT, another binding partner of PIX. PURPOSE: The major aim of this study was to determine whether Src family kinases are required for both Ras-induced PAK activation and malignant transformation. For this purpose, we used PP1, an inhibitor specific for Src family kinases, which does not inhibit either EGF receptor or ErbB2. METHODS AND RESULTS: We studied the effect of PP1 on the anchorage-dependent growth of normal and v-Ha-Ras transformed NIH 3T3 fibroblasts, PAK activation and anchorage-independent growth of Ras transformants, and development of Ras-induced sarcomas in nude mice. We found that PP1 (10 nM) strongly inhibits PAK activity in Ras transformants. PP1 at this concentration is known to inhibit c-Fyn kinase, but not c-Src kinase, and none of the three known Src family kinases (c-Src, c-Fyn, and c-Yes) expressed in fibroblasts is activated by v-Ha-Ras. Thus, it is most likely that the primary target of this drug is an as yet unidentified Ras-activated Tyr (Y) kinase or kinases, which we call "Ray." Although PP1 has no effect on their anchorage-dependent growth, it significantly inhibits their anchorage-independent growth in soft agar, as well as a rapid growth of Ras-induced sarcomas in mice. CONCLUSION: Like EGF receptor and ErbB2, a member of Src family kinases (most likely a new Src-related kinase called "Ray") is essential for the Ras-induced activation of PAK and the malignant transformation both in vitro and in vivo. These findings suggest that PP1 and other inhibitors specific for Src family kinases are potentially useful for the treatment of Ras-associated cancers.

3T3 Cells↗
PubMedDate not supplied

Signal therapy for RAS-induced cancers in combination of AG 879 and PP1, specific inhibitors for ErbB2 and Src family kinases, that block PAK activation.

BACKGROUND: Both EGF family ligands and ErbB family receptor kinases act upstream of RAS to induce mitogenesis of normal cells, such as NIH 3T3 fibroblasts. However, oncogenically mutated RAS, such as v-Ha-RAS is constitutively activated and therefore no longer requires these ligands or receptors for its activation. Nevertheless, it up-regulates the expression of these EGF family ligands. To understand the biologic significance of RAS-induced up-regulation of these ligands in both RAS-induced PAK activation and malignant transformation, we have conducted the following studies, based on the previous observations that (1) the N-terminal SH3 domain of PIX selectively binds a Pro-rich domain of 18 amino acids of PAKs, CDC42/Rac-dependent Ser/Thr kinase family, and (2) this specific interaction is essential for both PAK activation and membrane ruffling RESULTS: Using four distinct, cell-permeable, and highly specific inhibitors, namely WR-PAK18, which blocks the PAK-PIX interaction; AG 1478, which inhibits ErbB1 kinase activity; and AG 825 or AG 879, which inhibits ErbB2 kinase activity, we demonstrate that (1) the PAK-PIX interaction is essential for v-Ha-RAS-induced malignant transformation; (2) v-Ha-RAS requires not only ErbB1 but also ErbB2, which are activated through two independent autocrine pathways to induce both the PIX/Rac/CDC42-dependent PAK activation and malignant transformation in vitro; and (3) a combination of AG 879 and the Src family kinase-specific inhibitor PP1 suppresses almost completely the growth of RAS-induced sarcomas in nude mice. CONCLUSION: These findings not only change our conventional view on the role of these RAS-inducible ligands and ErbB family receptors (serving as RAS activators) but also suggest a new avenue for the treatment of RAS-associated cancers by a combination of inhibitors specific for ERbB, Src, or PAK family kinases.

3T3 Cells↗