Biomedical subjects
Publications and source records attributed to M Yamada.
A new approach to the generation of biologically active peptides from non-peptide lead was demonstrated. C,N-termini protected Phe-Phe-His derivatives possessing macrophage migration-enhancing activity were designed on the basis of the structure of imidazoline derivative 1, and these structure-activity relationships were also described.
It has been suggested that the structure at the head-rod junction of smooth muscle myosin is important for the phosphorylation-mediated regulation of myosin motor activity. To investigate whether a specific amino acid sequence at the head-rod junction is critical for the regulation, three smooth muscle myosin mutants in which the sequence at the N-terminal end of S2 is deleted to various extents were expressed in Sf9 cells; 28, 56, and 84 amino acid residues, respectively, at the position immediately C-terminal to the invariant proline (Pro849) were deleted, and the S1 domain was directly linked to the downstream sequence of the rod. The mutant myosins were expressed, purified, and biochemically characterized. All three myosin mutants showed a stable double-headed structure based upon electron microscopic observation. Both the actin-activated ATPase activity and the actin translocating activity of the mutants were completely regulated by the phosphorylation of the regulatory light chain. The actin sliding velocity of the three mutant myosins was the same as the wild-type recombinant myosin. These results indicate that a specific amino acid sequence at the head-rod junction is not required for the regulation of smooth muscle myosin. The results also suggest that there is no functionally important interaction between the regulatory light chain and the heavy chain at the head-rod junction.
1. We have examined the properties of intracellular nucleotide-mediated gating of K+ channel constructs composed of the sulphonylurea receptor 2B and the inwardly rectifying K+ channel subunits Kir6.1 and Kir6.2 (SUR2B/Kir6.1 and SUR2B/Kir6.2 complex K+ channels) heterologously expressed in human embryonic kidney (HEK) 293T cells. In the cell-attached form, both types of K+ channel were activated by pinacidil. 2. In inside-out (IO) patches, the SUR2B/Kir6.2 channels opened spontaneously and were inhibited by intracellular ATP (ATPi). Pinacidil attenuated the ATPi-mediated channel inhibition in a concentration-dependent manner. In contrast, the SUR2B/Kir6.1 channels required intracellular nucleoside di- or tri-, but not mono-, phosphates for opening. The potency of adenine, guanine or uracil nucleotides to activate SUR2B/Kir6.1 channels was enhanced by pinacidil. 3. In the presence of pinacidil, adenine and guanine, but not uracil, nucleotides exhibited bell-shaped concentration-dependent activating effects on SUR2B/Kir6.1 channels. This was due to channel inhibition caused by adenine and guanine nucleotides, which was unaffected by pinacidil. 4. From power density spectrum analysis of SUR2B/Kir6.1 currents, channel activation could be described by the product of two gates, a nucleotide-independent fast channel gate and a nucleotide-dependent slow gate, which controlled the number of functional channels. Pinacidil specifically increased the potency of nucleotide action on the slow gate. 5. We conclude that Kir6.0 subunits play a crucial role in the nucleotide-mediated gating of SUR/Kir6.0 complex K+ channels and may determine the molecular mode of pinacidil action.
Saccharomyces cerevisiae CDC7 encodes a serine/threonine kinase required for G1/S transition of the yeast cells. We previously reported human and Xenopus cDNAs encoding CDC7-related kinases and suggested the possibility that chromosomal replication of higher eukaryotes may be regulated through conserved mechanisms involving Cdc7-related kinases. Here we report a murine cDNA and gene (muCdc7) encoding a serine/threonine kinase related to CDC7. The predicted coding frame for the longest cDNA for muCdc7 consists of 564 amino acids, which shares 46, 77, and 93% identity, respectively, with those of budding yeast, Xenopus, and human in kinase conserved domains. The chromosomal gene for muCdc7, located at the band 5E5 on the mouse chromosome 5, consists of 12 exons, and its exon/intron organization shares some similarity with that of other protein kinases including Cdk and cAMP-dependent kinase. Transcription of muCdc7, initiated at multiple sites over the 370-base pair promoter region, is repressed in the resting state and is induced at the G1/S boundary after growth factor stimulation in a growth factor-dependent cell line. Transient transfection assays indicated that a 231-base pair segment of the muCdc7 promoter containing three putative E2F binding sites and one Sp1 site but lacking TATA sequence is sufficient for response to growth stimulation.
Several mutants of quinoprotein glucose dehydrogenase (GDH) in Escherichia coli were obtained and characterized. Of these, significant mutants were further characterized by kinetic analysis after purification or by site-directed mutagenesis to introduce different amino acid substitutions. H775R and H775A showed a pronounced reduction of affinity for a prosthetic group, pyrroloquinoline quinone (PQQ), suggesting that His-775 may directly interact with PQQ. D730N and D730A showed low glucose oxidase activity without influence on the affinity for PQQ, Mg2+, or substrate, but D730R showed reduced affinity for PQQ. The spectrum of tryptophan fluorescence revealed that the local structure surrounding PQQ was not changed by D730N mutation. Based on these data, we assume that Asp-730 may occur close to PQQ and function as a proton (and also electron) donor to PQQ or acceptor from PQQH2. Substitutions of Gly-689, that are located at the end of a unique segment of GDH among homologous quinoprotein dehydrogenases, directed reduction of the affinity for PQQ or GDH activity. Therefore, the unique segment and Asp-730 may play a specific role for GDH, which might be related to the intramolecular electron transfer from PQQ to ubiquinone.
2'-Deoxyoxanosine (dOxo) is a novel DNA lesion produced by the reaction of 2'-deoxyguanosine (dGuo) with nitrous acid and nitric oxide [Suzuki, T., Yamaoka, R., Nishi, M., Ide, H., and Makino, K. (1996) J. Am. Chem. Soc. 118, 2515-2516]. In this work, 2'-deoxyoxanosine 5'-triphosphate (dOTP) was prepared by nitrous acid treatment of 2'-deoxyguanosine 5'-triphosphate (dGTP), and its incorporation into DNA by DNA polymerases was investigated to elucidate the substrate and mutagenic properties of dOTP. Primed M13mp18 DNA was replicated by Escherichia coli DNA polymerase I Klenow fragment (Pol I Kf) in the presence of three normal dNTPs and dOTP or 2'-deoxyxanthosine 5'-triphosphate (dXTP), another major product of reaction of dGTP with nitrous acid and nitric oxide. dOTP substituted for dGTP and to a lesser extent for dATP, while dXTP substituted slightly for dGTP but not for dATP. Neither dOTP nor dXTP substituted for dCTP and dTTP. The similar results were obtained for the incorporation by T7 DNA polymerase deficient in 3'-5' exonuclease [T7(exo-)]. To quantify the substitution efficiency, kinetic parameters for incorporation of dOTP and dXTP opposite template C or T by Pol I Kf (exo-) were determined and compared with those for dGTP using oligodeoxynucleotide templates. Incorporation efficiencies (f = Vmax/Km) of dOTP (f = 0.28% min-1 microM-1) and dXTP (f = 0.10% min-1 microM-1) opposite template C were much lower than that of dGTP (f = 1506% min-1 microM-1). Frequencies of mutagenic incorporation of dOTP opposite template T were dependent on the nearest neighbor base pairs, and 1.6-3.9-fold higher than those for dGTP with the nearest neighbors containing G.C pairs. dXTP was not incorporated opposite template T with all four nearest neighbors. These data suggest that formation of dOTP, but not dXTP, from dGTP with nitrous acid or nitric oxide in the intracellular nucleotide pool would result in the elevation of the mutation frequency.
Peptidylarginine deiminases (PADs) are a group of enzymes which convert protein arginine residues to citrulline residues. Using rat muscle PAD cDNA as a probe, we obtained two novel cDNAs, PAD-R11 and PAD-R4, from immortalized rat keratinocytes treated with all-trans retinoic acid. Comparison of the deduced amino acid sequences with those of muscle and hair follicle enzymes showed high conservation in the C-terminal region. Recombinant proteins encoded by both PAD-R11 and PAD-R4 showed the enzyme activities. That of PAD-R11 showed a characteristic feature of the enzyme found in the epidermis.
Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disorder caused by the abnormal CAG triplet-repeat expansion resulting in an elongated polyglutamine (polyQ) stretch. We have recently showed that the DRPLA protein is cleaved during apoptosis by caspase-3, one of the cysteine protease family members known to be activated during apoptosis. We report here the subcellular localization of the DRPLA protein by fusing the green fluorescent protein as a tag. The full length DRPLA protein is localized predominantly but not exclusively in the nucleus regardless of the length of the polyQ stretch. In contrast, an N-terminal-deleted fragment containing polyQ produced by the proteolytic cleavage with caspase-3 is found both in the nucleus and the cytoplasm. Moreover, the same fragment with the elongated polyQ showed aggregation when overexpressed. Some cells with aggregate formation showed apoptotic phenotype. These findings raise the possibility that the DRPLA protein processed by caspase-3 may lead to aggregation of the protein resulting in the development of neurodegeneration.
We cloned and characterized the mouse uncoupling protein 2 (UCP2) gene and its promoter region. The gene spans approximately 6.3 kb and contains eight exons and seven introns. Two short exons are located in the 5' untranslated region, and each of the remaining exons encodes one of the transmembrane domains. 3'-RACE analysis showed that a polyadenylation signal 257 bp downstream from the stop codon was functional. Primer extension analysis indicated a single transcriptional start site 369 bp upstream from the translational start site. The promoter region lacks both TATA and CAAT boxes but is GC-rich. A construct containing 1250 bp of the promoter region showed significant activity in all 6 cell lines examined, and the region between -160 and -678 bp exhibited strong positive regulatory activity. These features of the UCP2 gene are different from those of the UCP1 gene and may contribute to its ubiquitous expression.
A series of benzoxazoles with a nitrogen-containing heterocyclic substituent at the 2-position was prepared and evaluated for 5-HT3 partial agonist activity on isolated guinea pig ileum. The nature of the substituent at the 5-position of the benzoxazole ring affected the potency for the 5-HT3 receptor, and the 5-chloro derivatives showed increased potency and lowered intrinsic activity. 5-Chloro-7-methyl-2-(4-methyl-1-homopiperazinyl)benzoxazole (6v) exhibited a high binding affinity in the same range as that of the 5-HT3 antagonist granisetron, and its intrinsic activity was 12% of that of 5-HT. Compound 6v inhibited 5-HT-evoked diarrhea but did not prolong the transition time of glass beads in the normal distal colon even at a dose of 100 times the ED50 for diarrhea inhibition in mice. Compounds of this type are expected to be effective for the treatment of irritable bowel syndrome without the side effect of constipation.
GTP:AMP phosphotransferase (adenylate kinase isozyme 3, AK3) mutants were obtained by using the ability of AK3 to complement a temperature-sensitive mutation of Escherichia coli adenylate kinase (AKe). Five mutants, P16L, G19S, G91D, G91S, and P93L, had mutation sites located at two loops that are involved in substrate binding of the enzyme. P16L and G19S bearing changes at the first loop showed reduced affinity for both GTP and AMP, the extent of reduction being slightly higher for GTP than AMP. In contrast, G91S and P93L having alterations at the second loop had lower affinities for AMP. Only the alterations at the second loop strongly influenced the Vmax value of the enzyme. Another mutant, D163N, had a substitution at the site forming a salt bridge in adenylate kinase isozyme 1 (AK1), which influenced the Vmax as well as the Km values for both substrates. The kinetic characteristics of these mutants were comparable to those of the corresponding AK1 or AKe mutants. Furthermore, from the results of mutations T201P and T201A that had alterations in all the kinetic parameters of AK3 and from a comparison with the structure and the kinetic parameters of AKe, we expect that a residue(s) around Thr201 is involved in recognition of the base of nucleoside triphosphate.
Explore the source record for details and available documents.
BAG-1 is a multifunctional protein that blocks apoptosis and interacts with several types of proteins, including Bcl-2 family proteins, the kinase Raf-1, certain tyrosine kinase growth factor receptors, and steroid hormone receptors, possibly by virtue of its ability to regulate the Hsp70/Hsc70 family of molecular chaperones. Two major forms of the human and mouse BAG-1 proteins were detected by immunoblotting. The longer human and mouse BAG-1 proteins (BAG-1L) appear to arise through translation initiation at noncanonical CTG codons located upstream of and in-frame with the usual ATG codon used for production of the originally described BAG-1 protein. Immunoblotting experiments using normal tissues revealed that BAG-1L is far more restricted in its expression and is present at lower levels than the more prevalent BAG-1 protein. Human but not mouse tissues also produce small amounts of an additional isoform of BAG-1 of intermediate size (BAG-1M) that probably arises through translation initiation at yet another site involving an ATG codon. All three isoforms of human BAG-1 (BAG-1, BAG-1M, and BAG-1L) retained the ability to bind Hsc70. Subcellular fractionation and immunofluorescence confocal microscopy studies indicated that BAG-1L often resides in the nucleus, consistent with the presence of a nuclear localization sequence in the NH2-terminal unique domain of this protein. In immunohistochemical assays, BAG-1 immunoreactivity was detected in a wide variety of types of cells in normal adult tissues and was localized to either cytosol, nucleus, or both, depending on the particular type of cell. In some cases, cytosolic BAG-1 immunostaining was clearly associated with organelles resembling mitochondria, consistent with the reported interaction of BAG-1 with Bcl-2 and related proteins. Furthermore, experiments using a green fluorescence protein (GFP)-BAG-1 fusion protein demonstrated that overexpression of Bcl-2 in cultured cells can cause intracellular redistribution of GFP-BAG-1, producing a membranous pattern typical of Bcl-2 family proteins. The BAG-1 protein was found at high levels in several types of human tumor cell lines among the 67 tested, particularly leukemias, breast, prostate, and colon cancers. In contrast to normal tissues, which only rarely expressed BAG-1L, tumor cell lines commonly contained BAG-1L protein, including most prostate, breast, and leukemia cell lines, suggesting that a change in BAG-1 mRNA translation frequently accompanies malignant transformation.
Monocyte chemoattractant protein-1 (MCP-1) is a member of the beta chemokine family which acts through specific seven transmembrane receptors to recruit monocytes, basophils, and T lymphocytes to sites of inflammation. To identify regions of the human MCP-1 protein which are important for its biological activity, we have synthesized domain-specific peptides and tested their ability to antagonize MCP-1 binding and chemotaxis in THP-1 cells. We have found that an intercysteine first loop peptide encompassing amino acids 13-35 inhibits MCP-1 binding and chemotactic activity, while peptides representing the amino-terminus (amino acids 1-10), second loop (amino acids 37-51), and carboxy-terminus (amino acids 56-71) of MCP-1 have no effect. In addition, we have found that cyclization of the first loop peptide by disulfide linkage and blocking the C-terminus of the peptide by amidation increases the activity of this peptide to block MCP-1 binding and chemotaxis. In order to specifically identify amino acid residues within the first loop that are crucial for MCP-1 functional activity, we have substituted alanine for tyrosine (Y13A) or arginine (R18A) in MCP-1 recombinant proteins. While baculovirus produced wild type and R18A MCP-1 proteins are indistinguishable in their ability to induce THP-1 chemotaxis and show modest effects in binding activity compared to commercially available recombinant MCP-1 protein, the Y13A point mutation causes a dramatic loss in function. The identification of functional domains of MCP-1 will assist in the design of MCP-1 receptor antagonists which may be clinically beneficial in a number of inflammatory diseases.
TGF beta family members are implicated in cardiac organogenesis, growth control, and positional information, including the direction of cardiac looping. However, genetic analysis of TGF beta signaling in mice has been confounded, in some cases, by noncardiac and generalized defects. Hence, deciphering TGF beta function in myocardium would benefit from cardiac-restricted mutations. We developed a constitutively activated type I receptor, ALK5L193A,P194A,T204D, and directed it to embryonic myocardium in transgenic mice. Expression of the activated ALK5 gene arrests looping morphogenesis and causes a linear, dilated, hypoplastic heart tube, despite normal expression of Nkx2.5 and dHAND, cardiogenic transcription factors whose absence provokes a similar phenotype. Ventricular hypoplasia was associated with precocious induction of the cyclin-dependent kinase inhibitor, p21. Thus, an ALK5-sensitive pathway mediates looping, perhaps through control of cardiac myocyte proliferation.
The present study was undertaken to examine the role of the benzodiazepine/GABA and N-methyl-d-aspartate (NMDA) systems in the convulsive effect of cocaine in mice. When cocaine (3.5 mg/ml) solution was infused into the tail vein at a rate of 0.3 ml/min, mice showed clonic and tonic convulsions. These seizures were not affected by low doses of bicuculline or picrotoxin, a GABAA receptor antagonist and a Cl ion channel blocker, respectively. Aminooxyacetic acid (AOAA), a GABA deaminase inhibitor, and phenobarbital, a Cl ion channel activator, and baclofen, a GABAB receptor agonist, also had no effect on these convulsions. Benzodiazepine inverse agonist beta-DMCM, at a dose which by itself had no convulsive effect lowered the convulsive threshold of cocaine. This lowered convulsive threshold was reversed by flumazenil, a benzodiazepine inverse antagonist, and diazepam, a benzodiazepine full agonist, which by themselves did not inhibit cocaine seizure. It is likely that cocaine seizure involves a benzodiazepine (beta-carboline) recognition site other than the benzodiazepine/GABAA receptor-Cl ionophore complex system. CPP and MK-801, competitive and noncompetitive NMDA receptor antagonists, respectively, inhibited cocaine seizures. The inhibitory effects of CPP on cocaine convulsion were reversed by a low dose of NMDA, which by itself did not induce seizure. A dopamine D1 receptor agonist SKF38393 enhanced both clonic and tonic convulsions, while a dopamine D2 receptor agonist bromocriptine inhibited these convulsions. These stimulatory and inhibitory effects were reversed by the D1 and D2 receptor antagonists, SCH23390 and haloperidol, respectively. These results suggest that the cocaine-induced convulsion may involve an activation of the NMDA-Ca ionophore complex system, which is mediated by the dopaminergic system, and a beta-carboline recognition site other than the benzodiazepine/GABAA receptor-Cl ionophore complex system.
A series of novel potent inhibitors of dipeptidyl peptidase IV (DPP-IV) has been developed. A brief structure-activity relationship of the inhibitors was investigated. The dipeptide TSL-225, tryptophyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, was identified with the critical structure for the inhibitory activity.
The neuroepithelium (NE) of the cerebrum of the developing brain has been reported to 'regenerate' within a certain period after being injured. To clarify the process of repair of the NE during neurogenesis, we chronologically examined the number of cells in the neocortex, and the rate of DNA-replicating cells and the mitotic figure ratio in the NE of rats after injury. The injury was induced by transplacental administration of ethylnitrosourea (ENU), which is cytotoxic immediately after administration, to the pregnant rats on embryonic day 16. The number of living and pyknotic cells in a 220-micron width of the neocortex was evaluated in each of three groups, such as NE, subventricular zone (SVZ) + intermediate zone (IMZ) + subplate (SP), and cortical plate (CP). The 5-bromo-2-deoxyuridine (BrdU)-labeling index and the mitotic index were examined 4, 8, 16, 24, 36, and 48 h after ENU administration. Up to 16 h after ENU administration, the number of living cells per 220 microns width in the NE and SVZ + IMZ + SP decreased, but increased in CP. From 16 to 24 h, the living cell number per 220 microns width in the NE and CP was unchanged, but increased in the SVZ + IMZ + SP. From 24 to 36 h, the living cell number per 220 microns width increased in all the groups, NE, SVZ + IMZ + SP and CP. From 36 to 48 h, the living cell number per 220 microns width in the NE was unchanged, but increased in the SVZ + IMZ + SP and CP. The BrdU-labeling index reached its nadir at 8 h, but markedly increased by 16 h, and then decreased to the control level by 36 h. No mitotic figures were observed at 16 h after administration, but a significant increase in mitotic index was noted at 24 h, and after which it decreased to almost the control value by 36 h. These findings indicate: (i) that temporary arrest of neuroepithelial cell cycle occurs in the G1-phase from 4 to 8 h after ENU administration, (ii) that the cell synchronizes in the S-phase at 16 h, (iii) that a proportion of neuroepithelial cells of rat fetal neocortex at the neurogenesis stage return to neuroepithelial cell proliferation stage, to repair the NE, and (iv) that regulation of the cell kinetics of neuroepithelial cells depends on the number of cells in a certain width of NE during regeneration.