Eisuke Munekata

Mammalian Copper Chaperone Cox17p Has an Essential Role in Activation of Cytochrome c Oxidase and Embryonic Development

ABSTRACT Cox17p is essential for the assembly of functional cytochrome c oxidase (CCO) and for delivery of copper ions to the mitochondrion for insertion into the enzyme in yeast. Although this small protein has already been cloned or purified from humans, mice, and pigs, the function of Cox17p in the mammalian system has not yet been elucidated. In vitro biochemical data for mammalian Cox17p indicate that the copper binds to the sequence -KPCCAC-. Although mouse embryos homozygous for COX17 disruption die between embryonic days E8.5 and E10, they develop normally until E6.5. This phenotype is strikingly similar to embryos of Ctr1(−/−), a cell surface copper transporter, in its lethality around the time of gastrulation. COX17-deficient embryos exhibit severe reductions in CCO activity at E6.5. Succinate dehydrogenase activity and immunoreactivities for anti-COX subunit antibodies were normal in the COX17(−/−) embryos, indicating that this defect was not caused by the deficiency of other complexes and/or subunits but was caused by impaired CCO activation by Cox17p. Since other copper chaperone (Atox1 and CCS)-deficient mice show a more moderate defect, the disruption of the COX17 locus causes the expression of only the phenotype of Ctr1(−/−). We found that the activity of lactate dehydrogenase was also normal in E6.5 embryos, implying that the activation of CCO by Cox17p may not be essential to the progress of embryogenesis before gastrulation.

Characterization of a Novel Kinetochore Protein, CENP-H

Macromolecular centromere-kinetochore complex plays a critical role in sister chromatid separation, but its complete protein composition as well as its precise dynamic function during mitosis has not yet been clearly determined. Here we report the isolation of a novel mouse kinetochore protein, CENP-H. The CENP-H, with an apparent molecular mass of 33 kDa, was found to contain a coiled-coil structure and a nuclear localization signal. The CENP-H transcripts were relatively scarce but were detectable in most tissues and embryos at various stages of development. Immunofluorescence stainings of mouse fibroblast cells with anti-CENP-H-specific antibody demonstrated that the CENP-H is specifically and constitutively localized in kinetochores throughout the cell cycle; this was also confirmed by stainings with anti-centromere-specific antibody. Thus the newly isolated CENP-H may play a role in kinetochore organization and function throughout the cell cycle.

Intermolecular and Interdomain Interactions of a Dynamin-related GTP-binding Protein, Dnm1p/Vps1p-like Protein

Dnm1p/Vps1p-like protein (DVLP) is a mammalian member of the dynamin GTPase family, which is classified into subfamilies on the basis of the structural similarity. Mammalian dynamins constitute the dynamin subfamily. DVLP belongs to the Vps1 subfamily, which also includes yeast Vps1p and Dnm1p. Typical structural features that discriminate between members of the Vps1 and dynamin subfamilies are that the former lacks the pleckstrin homology and Pro-rich domains. Dynamin exists as tetramers under physiological salt conditions, whereas under low salt conditions, it can polymerize into spirals that resemble the collar structures seen at the necks of constricted coated pits. In this study, we found that DVLP is also oligomeric, probably tetrameric, under physiological salt conditions and forms sedimentable large aggregates under low salt conditions. The data indicate that neither the pleckstrin homology nor Pro-rich domain is required for the self-assembly. Analyses using the two-hybrid system and co-immunoprecipitation show that the N-terminal region containing the GTPase domain and a domain (DVH1) conserved across members of the dynamin and Vps1 subfamilies, can interact with the C-terminal region containing another conserved domain (DVH2). The data on the interdomain interaction of DVLP is compatible with the previous reports on the interdomain interaction of dynamin. Thus, the self-assembly mechanism of DVLP appears to resemble that of dynamin, suggesting that DVLP may also be involved in the formation of transport vesicles.

The contractile activities of neurokinin A, B and related peptides on smooth muscles.

The contractile activities of neurokinin A (NKA), neurokinin B (NKB) and related peptides on the guinea-pig ileum, rat vas deferens and rat duodenum were compared to the activity of substance P (SP). The potencies of NKA and NKB on the guinea-pig ileum (SP-P tissue) were nearly the same as that of SP. NKA was approximately 250-400 times more potent than SP on the rat vas deferens (EC50 = 59.5 nM; SP, EC50 = 1500 nM) and rat duodenum (EC50 = 1.8 nM; SP, EC50 = 674 nM) (SP-E tissues). NKB also showed high contracting activity on the rat duodenum (EC50 = 3.1 nM) but was 10 fold less active than NKA on the rat vas deferens. These results suggest that neurokinin peptides are possible endogenous agonists for the SP-E tissues. The contractile potency of NKA and NKB remained nearly complete after removal of N-terminal tripeptide portions, i.e., His-Lys-Thr and Asp-Met-His from the native peptides, respectively. However, the removal of the Asp residue from both NKA7 and NKB7 decreased activity until it was similar to that of SP.

Demonstration of a hepatitis C virus-specific antigen predicted from the putative core gene in the circulation of infected hosts

An ELISA was used to detect a protein derived from the core gene of the hepatitis C virus (HCV) in human plasma. The solid phase antibody in the assay was a murine monoclonal antibody against a synthetic peptide deduced from the putative core gene of HCV (residues 39 to 74). An enzyme-labelled affinity-purified human antibody directed at another region within the HCV core (residues 5 to 23) was the second antibody tracer. The ELISA had a sensitivity capable of detecting a few ng/ml of the HCV core polypeptide expressed in Escherichia coli. Core antigen activity in plasma of infected hosts was detected after treatment of HCV RNA-rich fractions from buoyant density centrifugation with the detergent Tween 80. There was a direct correlation between core antigen ELISA values of a plasma fraction and intensities of polymerase chain reaction signals for HCV RNA. These observations are consistent with the proposal that the N-terminal sequence of the predicted polyprotein of HCV is a nucleocapsid protein, and that improved core antigen assays may correlate with viraemia.

Trophic effects of substance P and β-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.

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 enoughto regenerate a stable 1:1 complex, K d = 9 × 10−6M. 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.

The excitatory action of the newly-discovered mammalian tachykinins, neurokinin α and neurokinin β, on neurons of the isolated spinal cord of the newborn rat

The actions of two new mammalian tachykinins, neurokinin alpha and neurokinin beta, were examined using the isolated spinal cords of newborn rats. Depolarizing responses of spinal motoneurons were recorded extracellularly from the lumbar ventral root during application of neurokinin alpha or neurokinin beta at concentrations ranging from 3 X 10(-8) M to 10(-6) M. The potencies of various tachykinins in depolarizing the motoneurons showed the following order: physalaemin greater than neurokinin beta divided by kassinin divided by substance P greater than neurokinin alpha. When the synaptic transmission in the spinal cord was blocked by tetrodotoxin, the depolarizing action of neurokinin alpha and neurokinin beta was markedly reduced but not completely abolished. The depolarizing action of neurokinin alpha and neurokinin beta was depressed by a substance P antagonist, [D-Arg1, D-Pro2, D-Trp7,9, Leu11]SP. The possibility that neurokinin alpha and neurokinin beta act as neurotransmitters in the mammalian spinal cord is discussed.

Chemo-enzymatic synthesis of a bioactive peptide containing a glutamine-linked oligosaccharide and its characterization

A bioactive peptide containing a glutamine-linked oligosaccharide was chemo-enzymatically synthesized by use of the solid-phase method of peptide synthesis and the transglycosylation activity of endo-beta-N-acetylglucosaminidase. Substance P, a neuropeptide, is an undecapeptide containing two L-glutamine residues. A substance P derivative with an N-acetyl-D-glucosamine residue attached to the fifth or sixth L-glutamine residue from the N-terminal region was chemically synthesized. A sialo complex-type oligosaccharide derived from a glycopeptide of hen egg yolk was added to the N-acetyl-D-glucosamine moiety of the substance P derivative using the transglycosylation activity of endo-beta-N-acetylglucosaminidase from Mucor hiemalis, and a substance P derivative with a sialo complex-type oligosaccharide attached to the L-glutamine residue was synthesized. This glycosylated substance P was biologically active, although the activity was rather low, and stable against peptidase digestion. The oligosaccharide moiety attached to the L-glutamine residue of the peptide was not liberated by peptide-N(4)-(N-acetyl-beta-D-glucosaminyl) asparagine amidase F.

Amyloid β-Peptide-induced Inhibition of MTT Reduction in PC12h and C1300 Neuroblastoma Cells: Effect of Nitroprusside

We have investigated the effect of amyloid beta-peptide (Abeta) in rat pheochromocytoma PC 12h and murine C 1300 neuroblastoma cells by using MTT ¿3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assay. Exposure of the cells to Abeta peptides, Abeta1-40 and its fragment Abeta25-35, induced a concentration-dependent inhibition of MTT reduction in both cell lines, and MTT-dependent LDH release due to cell lysis in PC12h cells. We also found that sodium nitroprusside (SNP), a spontaneous nitric oxide (NO) generator, significantly prevented the inhibition of MTT reduction and MTT-dependent LDH release caused by Abeta peptides at 10-100 microM, although a high concentration of SNP (> or = 333 microM) was remarkably toxic by itself. Since the inhibition of MTT reduction caused by Abeta is known as one of the first indicators of its toxicity, these findings suggest that Abeta peptides have a toxic effect in these cell lines, and SNP may attenuate the Abeta peptide-induced toxicity. In regard the mechanisms of the actions of SNP, hydroxylamine which also generates NO and 8-Br-cGMP, a membrane-permeable analogue of cyclic GMP (cGMP), failed to prevent the inhibition of MTT reduction caused by Abeta25-35 in PC12h cells, implying that the effect of SNP may be mediated by the NO-independent pathway. Since potassium ferrocyanide showed a significant prevention at 333 microM although it had toxic effect at this concentration, it is considered that the ferrocyanide portion of the SNP metabolite may be partially involved. The cell death induced by other oxidative insults, such as glutamate and hydrogen peroxide (H2O2), could not be attenuated by SNP in both cell lines. Thus, the observed effect of SNP might not be due to its direct antioxidative action.