Biomedical subjects

T Niidome

Publications and source records attributed to T Niidome.

At least 19 recordsLinked to original sources

PubMed2001

Differential nociceptive responses in mice lacking the alpha(1B) subunit of N-type Ca(2+) channels.

The role of N-type Ca(2+) channels in nociceptive transmission was examined in genetically engineered mice lacking the alpha(1B) subunit of N-type channels and in their heterozygote and wild-type littermates. In alpha(1B)-deficient mice, N-type channel activities in dorsal root ganglion neurons and spinal synaptoneurosomes were eliminated without compensation by other types of voltage-dependent Ca(2+) channels. The alpha(1B)-deficient mice showed a diminution in the phase 2 nociceptive responses more extensively than in the phase 1 nociceptive responses of the formalin test. The alpha(1B)-deficient mice exhibited significantly increased thermal nociceptive thresholds in the hot plate test, but failed to increase mechanical nociceptive thresholds in the tail pinch test. These results suggest a crucial role of N-type channels in nociceptive transmission, especially for persistent pain like phase 2 of the formalin test and for nociception induced by thermal stimuli.

Animals↗

PubMed2001

Carbohydrate recognition of gramicidin S analogues in aqueous medium.

We have designed and synthesized of carbohydrate-binding peptides, gramicidin S analogues. Asn/Asp/Gln and Trp residues in the peptides were employed as the binding sites for carbohydrates by hydrogen-bonding interaction and the creation units for hydrophobic pocket to promote the interaction, respectively. The data of fluorescence spectroscopy and affinity column chromatography indicated that the peptides possessed the binding ability for some carbohydrates in aqueous medium. As a result of 1H NMR study, nuclear Overhauser effects between aromatic side chains of a peptide, [Gln(1,1'),Trp(3,3')]-gramisidin S and mannose were observed, indicating that the interaction of the peptide with the sugar occurred in the hydrophobic environment formed by Trp and Phe residues.

Bacillus↗

PubMed2001

Salmonella enteritidis FliC (flagella filament protein) induces human beta-defensin-2 mRNA production by Caco-2 cells.

Antimicrobial peptides are crucial for host defense at mucosal surfaces. Bacterial factors responsible for induction of human beta-defensin-2 (hBD-2) mRNA expression in Caco-2 human carcinoma cells were determined. Salmonella enteritidis, Salmonella typhimurium, Salmonella typhi, Salmonella dublin, and culture supernatants of these strains induced hBD-2 mRNA expression in Caco-2 human carcinoma cells. Using luciferase as a reporter gene for a approximately 2.1-kilobase pair hBD-2 promoter, the hBD-2-inducing factor in culture supernatant of S. enteritidis was isolated. The supernatant factor was heat-stable and proteinase-sensitive. After purification by anion exchange and gel filtration chromatography, the hBD-2-inducing factor was identified as a 53-kDa monomeric protein with the amino-terminal sequence AQVINTNSLSLLTQNNLNK, which is identical to that of the flagella filament structural protein (FliC) of S. enteritidis. Consistent with this finding, the 53-kDa protein reacted with anti-FliC antibody, which prevented its induction of hBD-2 mRNA in Caco-2 cells. In agreement, the hBD-2-inducing activity in culture supernatant was completely neutralized by anti-FliC antibody. In gel retardation analyses, FliC increased binding of NF-kappaB (p65 homodimer) to hBD-2 gene promoter sequences. We conclude that S. enteritidis FliC induces hBD-2 expression in Caco-2 cells via NF-kappaB activation and thus plays an important role in up-regulation of the innate immune response.

Amino Acid Sequence↗

PubMed2001

Functional disorders of the sympathetic nervous system in mice lacking the alpha 1B subunit (Cav 2.2) of N-type calcium channels.

N-type voltage-dependent Ca(2+) channels (VDCCs), predominantly localized in the nervous system, have been considered to play an essential role in a variety of neuronal functions, including neurotransmitter release at sympathetic nerve terminals. As a direct approach to elucidating the physiological significance of N-type VDCCs, we have generated mice genetically deficient in the alpha(1B) subunit (Ca(v) 2.2). The alpha(1B)-deficient null mice, surprisingly, have a normal life span and are free from apparent behavioral defects. A complete and selective elimination of N-type currents, sensitive to omega-conotoxin GVIA, was observed without significant changes in the activity of other VDCC types in neuronal preparations of mutant mice. The baroreflex response, mediated by the sympathetic nervous system, was markedly reduced after bilateral carotid occlusion. In isolated left atria prepared from N-type-deficient mice, the positive inotropic responses to electrical sympathetic neuronal stimulation were dramatically decreased compared with those of normal mice. In contrast, parasympathetic nervous activity in the mutant mice was nearly identical to that of wild-type mice. Interestingly, the mutant mice showed sustained elevation of heart rate and blood pressure. These results provide direct evidence that N-type VDCCs are indispensable for the function of the sympathetic nervous system in circulatory regulation and indicate that N-type VDCC-deficient mice will be a useful model for studying disorders attributable to sympathetic nerve dysfunction.

Animals↗

PubMed2001

Antibacterial activity of bactenecin 5 fragments and their interaction with phospholipid membranes.

Bactenecin 5 (Bac 5) is an antibacterial 43mer peptide isolated from bovine neutrophils. It consists of an Arg-rich N-terminal region and successive repeats of Arg-Pro-Pro-Ile (or Phe). We synthesized Bac 5(1-23) and several related peptides to clarify the roles these regions play in antibacterial activity. An assay of antibacterial activity revealed that such activity requires the presence of Arg residues at or near the N-terminus, as well as a chain length exceeding 15 residues. None of the peptides exhibited haemolytic activity. Polyproline II-like CD curves were observed for most of the peptides. Measurements of the membrane perturbation and fusion indicated that the perturbation and fusogenic activities of the peptides were, generally, parallel to their antibacterial activities. Amino acid substitution in the repeating region had some effect on antibacterial activity.

Amino Acid Sequence↗

PubMed2001

Impairment of glutathione metabolism in human gastric epithelial cells treated with vacuolating cytotoxin from Helicobacter pylori.

Helicobacter pylori vacuolating cytotoxin (VacA) is believed to be one of the factors that induces gastric disease. Our previous study indicated that VacA causes a decrease in the intracellular ATP level in human gastric epithelial cells, suggesting to impair mitochondrial membrane potential followed by a decrease in energy metabolism (Kimura et al., Microb. Pathog., 1999, 26: 45--52). In the present study, we investigated whether the decrease in ATP level affects glutathione metabolism, in which its synthesis and efflux are ATP-dependent. Treatment of AZ-521 human gastric epithelial cells with 120 nM VacA for 6 h suppressed the efflux of oxidized glutathione (GSSG) in a dose- and time-dependent manner. The efflux of GSSG from the cells and glutathione (GSH) synthesis of cells treated with VacA were approximately 50 and 70% of those of the control, respectively. The turnover rate of intracellular GSH was also suppressed by VacA. Viability of the cells pretreated with VacA, then further incubated with H(2)O(2), was decreased by 50% at 6 h and 70% at 12 h. These results suggested that VacA impairs GSH metabolism in the gastric epithelial cells, which weakens the resistance of the cells against oxidative stress or cellular redox regulation by GSH.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2001

Interaction of pleurocidin and its analogs with phospholipid membrane and their antibacterial activity.

A 25-mer cationic peptide pleurocidin, isolated from the winter flounder, has broad antibacterial activity. To clarify the structure-activity relationship, its properties and biological activity were examined. CD measurements showed that pleurocidin took an alpha-helical structure in the presence of DOPC/DOPG (3:1, anionic) vesicles. Very weak hemolytic activity of pleurocidin was observed and its antibacterial activity was moderate. Tryptophan fluorescence shift measurements showed that pleurocidin interacted weakly with a neutral phospholipid, but strongly with an acidic phospholipid. The peptide exhibited weak dye-leakage activity for DOPC (neutral) vesicles and moderate activity for acidic vesicles. From experiments on dye-leakage activity and membrane translocation of the peptide, it seemed likely that pleurocidin, like magainin 2, forms pores in the lipid membrane. A study of amino acid substitution in pleurocidin revealed that alpha-helicity, rather than hydrophobicity, affects the properties and activity of the peptide.

Animals↗

PubMed2001

Interaction of mastoparan with membranes studied by 1H-NMR spectroscopy in detergent micelles and by solid-state 2H-NMR and 15N-NMR spectroscopy in oriented lipid bilayers.

Several complementary NMR approaches were used to study the interaction of mastoparan, a 14-residue peptide toxin from wasp venom, with lipid membranes. First, the 3D structure of mastoparan was determined using 1H-NMR spectroscopy in perdeuterated (SDS-d25) micelles. NOESY experiments and distance geometry calculations yielded a straight amphiphilic alpha-helix with high-order parameters, and the chemical shifts of the amide protons showed a characteristic periodicity of 3-4 residues. Secondly, solid-state 2H-NMR spectoscopy was used to describe the binding of mastoparan to lipid bilayers, composed of headgroup-deuterated dimyristoylglycerophosphocholine (DMPC-d4) and dimyristoylphosphatidylglycerol (DMPG). By correlating the deuterium quadrupole splittings of the alpha-segments and beta-segments, it was possible to differentiate the electrostatically induced structural response of the choline headgroup from dynamic effects induced by the peptide. A partial phase separation was observed, leading to a DMPG-rich phase and a DMPG-depleted phase, each containing some mastoparan. Finally, the insertion and orientation of a specifically 15N-labeled mastoparan (at position Ala10) in the bilayer environment was investigated by solid-state 15N-NMR spectroscopy, using macroscopically oriented samples. Two distinct orientational states were observed for the mastoparan helix, namely an in-plane and a trans-membrane alignment. The two populations of 90% in-plane and 10% trans-membrane helices are characterized by a mosaic spread of +/- 30 degrees and +/- 10 degrees, respectively. The biological activity of mastoparan is discussed in terms of a pore-forming model, as the peptide is known to be able to induce nonlamellar phases and facilitate a flip-flop between the monolayers.

Deuterium↗

PubMed2000

Morphologic differentiation of HL-60 cells is associated with appearance of RPTPbeta and induction of Helicobacter pylori VacA sensitivity.

Phorbol 12-myristate 13-acetate (PMA) induces differentiation of human leukemic HL-60 cells into cells with macrophage-like characteristics and enhances the susceptibility of HL-60 cells to the Helicobacter pylori VacA toxin (de Bernard, M., Moschioni., M., Papini, E., Telford, J. L., Rappuoli, R., and Montecucco, C. (1998) FEBS Lett. 436, 218-222). We examined the mechanism by which HL-60 cells acquire sensitivity to VacA, in particular, looking for expression of RPTPbeta, a VacA-binding protein postulated to be the VacA receptor (Yahiro, K., Niidome, T., Kimura, M., Hatakeyama, T., Aoyagi, H., Kurazono, H., Imagawa, K., Wada, A., Moss, J., and Hirayama, T. (1999) J. Biol. Chem. 274, 36693-36699). PMA induced expression of RPTPbeta mRNA and protein as determined by RNase protection assay and indirect immunofluorescence studies, respectively. Vitamin D(3) and interferon-gamma, which stimulate differentiation of HL-60 cells into monocyte-like cells, also induced VacA sensitivity and expression of RPTPbeta mRNA, whereas 1. 2% Me(2)SO and retinoic acid, which stimulated the maturation of HL-60 into granulocyte-like cells, did not. RPTPbeta antisense oligonucleotide inhibited induction of VacA sensitivity and expression of RPTPbeta. Double immunostaining studies also indicated that newly expressed RPTPbeta colocalized with VacA in PMA-treated HL-60 cells. In agreement with these data, BHK-21 cells, which are insensitive to VacA, when transfected with the RPTPbeta cDNA, acquired VacA sensitivity. All data are consistent with the conclusion that acquisition of VacA sensitivity by PMA-treated HL-60 cells results from induction of RPTPbeta, a protein that functions as the VacA receptor.

Bacterial Proteins↗

PubMed2000

Required structure of cationic peptide for oligonucleotide-binding and -delivering into cells.

Improvement of the methods for oligonucleotide delivery into cells is necessary for the development of antisense therapy. In the present work, a new strategy for oligonucleotide delivery into cells was tested using cationic peptides as a vector. At first, to understand what structure of the peptide is required for binding with an oligonucleotide, several kinds of alpha-helical and non-alpha-helical peptides containing cationic amino acids were employed. As a result, the amphiphilic alpha-helix peptides were best for binding with the oligonucleotide, and the long chain length and large hydrophobic region in the amphiphilic structure of the peptide were necessary for the binding and forming of aggregates with the oligonucleotide. In the case of non-alpha-helical peptides, no significant binding ability was observed even if their chain lengths and number of cationic amino acid residues were equal to those of the alpha-helical peptides. The remarkable ability of oligonucleotide delivery into COS-7 cells was observed in the alpha-helical peptides with a long chain length and large hydrophobic region in the amphiphilic structure, but was not observed in the non-alpha-helical peptides. It is considered that such alpha-helical peptides could form optimum aggregates with the ODN for uptake into cells. Based on these results, the alpha-helical peptide with a long chain length and large hydrophobic region is applicable as a vector for the delivery of oligonucleotides into cells.

Amino Acids↗

PubMed2000

Gene transfer into hepatoma cells mediated by galactose-modified alpha-helical peptides.

To develop a receptor-mediated gene delivery system into hepatoma cells using the cationic alpha-helical peptide as the gene carrier molecule, we modified an alpha-helical peptide, which is known to have transfection abilities into cells, with a multi-antennary ligand containing several galactose residues that provide efficient binding to the asialoglycoprotein receptor. The galactose-modified peptides formed complexes with a plasmid DNA and showed gene transfer abilities into HuH-7 cells, a human hepatoma cell line. The transfection efficiency of the peptide was increased by increasing the number of modified galactose residues on the peptide. Furthermore, considerable inhibition of the transfection efficiency by the addition of asialofetuin, which is a ligand for the asialoglycoprotein receptor, was observed in all galactose-modified peptides. Based on this result, we could confirm that the internalization of the galactose-modified peptides occurred by the receptor-mediated endocytosis pathway. In addition, to understand the transport route of the peptide-DNA complex in the cell, the effects on the transfection efficiencies with several endocytosis inhibitors were examined. As a result, it was suggested that the translocation of the peptide-DNA complex from the endocytic compartments to the cytosol mainly occurred during an early endosome step.

Carcinoma, Hepatocellular↗

PubMed1999

Activation of Helicobacter pylori VacA toxin by alkaline or acid conditions increases its binding to a 250-kDa receptor protein-tyrosine phosphatase beta.

Helicobacter pylori, a Gram-negative gastric bacterium, secretes VacA, a cytotoxin that causes vacuolar degeneration of susceptible cells. Velocity sedimentation analysis showed that treatment of VacA at alkaline pH led to disassembly of VacA oligomers, an observation reported previously for acid-treated VacA. Exposure of VacA to acid or alkali increased its binding to AZ-521 cells, as shown by indirect immunofluorescence and flow cytometry. Moreover, immunoprecipitates with polyclonal antibodies against VacA from AZ-521 cells previously exposed to acid- or alkali-treated VacA had a 250-kDa glycoprotein containing galactose-beta(1-3)-N-acetylgalactosamine and galactose-beta(1-4)-N-acetylglucosamine. p250, purified by chromatography on peanut agglutinin affinity and Superose 6 columns, contained N-terminal and internal amino acid sequences of YRQQRKLVEEIGWSYT and LIIQDHILEATQDDY, respectively. These sequences are identical to those of a receptor protein-tyrosine phosphatase (RPTPbeta/PTPzeta); in agreement, p250 reacted with anti-human RPTPbeta monoclonal antibody. Immunoprecipitation with anti-human RPTPbeta antibody of solubilized membrane preparations previously incubated with VacA or heat-inactivated VacA demonstrated that RPTPbeta bound native, but not denatured, VacA. Acidic and alkaline treatments were associated with activation of VacA and increased binding to the cell surface RPTPbeta.

Amino Acid Sequence↗

PubMed1999

Neuron-specific expression of reporter gene in transgenic mice carrying the 5'-upstream region of mouse P/Q-type Ca2+ channel alpha 1A subunit gene fused to E. coli lacZ reporter gene.

To dissect the molecular mechanisms underlying the neuron-specific expression of the P/Q type calcium channel alpha 1A subunit gene, transgenic mice carrying a 0.5-kb, 1.5-kb, 3.0-kb or 6.3-kb 5'-upstream region of the gene fused to Escherichia coli lacZ reporter gene were produced. In transgenic mice carrying the 1.5-kb, 3.0-kb or 6.3-kb 5'-upstream region, the reporter gene was exclusively expressed in the nervous system, although those with the 0.5-kb 5'-upstream region failed to show reporter expression. Histological examinations showed that the three 5'-upstream regions induced distinct expression patterns of the reporter gene in the CNS and adrenal medulla. The 1.5-kb 5'-upstream region drove reporter gene expression in the olfactory bulb, dorsal cortex and hippocampus, while the regulatory element for the expression in the amygdaloid nucleus, septum, habenula medial nucleus, choroid plexus, substantia nigra, inferior colliculus, pontine nucleus and cerebellum was located in the 5'-upstream sequence between 1.5 kb and 6.3 kb. In the cerebellum, the expression of the reporter gene was induced by the 3.0-kb region in granule cells, whereas it was induced by the 6.3-kb region in Purkinje cells. The expression of the reporter gene in chromaffin cells in the adrenal medulla was induced only by the 6.3-kb 5'-upstream region. These results suggest that the expression of the mouse P/Q-type Ca2+ channel alpha 1A subunit gene is regulated in a complex fashion by both positive and negative cis-regulatory elements.

5' Untranslated Regions↗

PubMed1999

Induction of human beta-defensin-2 mRNA expression by Helicobacter pylori in human gastric cell line MKN45 cells on cag pathogenicity island.

Helicobacter pylori is an etiological agent of gastritis, peptic ulcer, and gastric cancer. Human beta-defensin-2 (hBD-2) is an antimicrobial peptide which belongs to one of the most important host defense systems against bacterial infection in several epithelial tissues. We studied the effect of H. pylori on the expression of hBD-2 mRNA in MKN45 gastric mucosal cells. H. pylori, but not culture filtrate, increased the hBD-2 mRNA level in MKN45 cells; the inductive effect of H. pylori was not detected with Intestine 407 cells. Among H. pylori strains, strain OHPC0002, which lacks a cag Pathogenicity Island (PAI), did not induce hBD-2 mRNA in MKN45 cells. These results suggested that H. pylori cag PAI is critical for the induction of hBD-2 mRNA in MKN45 cells. Exposure of MKN45 cells to Salmonella typhimurium, S. enteritidis, S. typhi, and S. dublin, but not Escherichia coli ML35, also resulted in induction of hBD-2 mRNA.

Blood Bactericidal Activity↗

PubMed1999

Orientational behavior of phospholipid membranes with mastoparan studied by 31P solid state NMR.

Solid state 31P NMR spectroscopy was used to study the perturbing effect of the wasp venom peptide mastoparan (MP) on lipid bilayers composed of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG). The 31P chemical shift anisotropy of multilamellar vesicles decreased with increasing peptide concentration, indicating that MP interacts strongly and selectively with the charged DMPG head group. Macroscopically oriented MP-lipid samples between glass plates were studied by 31P NMR as a function of tilt angle. These spectra showed the coexistence of orientation-dependent lamellar signals as well as an isotropic peak, suggesting that MP can induce non-lamellar phases in DMPC/DMPG membranes.

Amino Acid Sequence↗

PubMed1999

Isolation and functional characterization of the 5'-upstream region of mouse P/Q-type Ca2+ channel alpha1A subunit gene.

The omega-agatoxin-IVA-sensitive P/Q-type Ca2+ channel is predominantly expressed in the nervous system. To dissect the molecular mechanisms underlying the neuron-specific expression of the P/Q-type channel, we have isolated and characterized the 5'-upstream region of the mouse alpha1A subunit gene. A transcription start site appeared to exist at -269 bp upstream from the start codon as found by 5' RACE analysis. The proximal promoter of the alpha1A subunit gene lacks a typical TATA box, but contains several transcription factor binding sequences, including two Sp1 sites. When linked to a placental alkaline phosphatase (PLAP) reporter gene to examine the promoter activity, the 6.3-kb (-6,273 to +269) 5'-upstream region, but not a smaller 3.0-kb construct (-3, 021 to +269), was able to drive the reporter gene in neuron-like PC12 cells. In contrast, neither of these constructs enhanced the PLAP expression in fibroblast NIH3T3 cells. The sequence between 6.3 and 3.0 kb of the 5'-upstream region did not show promoter activity in either of the cell lines, but enhanced TK promoter activity in PC12 cells, though not in NIH3T3 cells. These results suggest that neuron-specific elements of the alpha1A subunit gene are likely to be located in the distal upstream regions (-6,273 to -3,021) of the 5'-upstream sequence.

3T3 Cells↗

PubMed1999

Effect of amino acid substitution in amphiphilic alpha-helical peptides on peptide-phospholipid membrane interaction.

It was previously found that a cationic amphiphilic peptide, Ac-(Leu-Ala-Arg-Leu)3-NHCH3 (4(3)), caused the destabilization of a phospholipid membrane and showed strong antibacterial activity [Lee et al. Biochim. Biophys. Acta 1986; 862: 211-219]. In order to investigate the effect of changing alpha-helix propensity, hydrophobicity and basicity in 4(3) on the peptide conformation and activity, the 4(3) analogs, [Gly (or Val)6]4(3), [Gly (or Val)2,6]4(3), [Gly (or Val)2,6,10]4(3), [Gln3]4(3), [Gln3,7]4(3) and [Gln3,7,11]4(3) were synthesized. Except for [Val2,6]4(3) and [Val2,6,10]4(3), which mainly formed a beta-structure, other peptides formed an alpha-helix and showed moderate membrane-perturbing activity toward neutral and acidic lipid vesicles. All the peptides other than [Val2,6,10]4(3) and [Gln3,7,10]4(3) had the antibacterial activity comparable with that of 4(3). The relationship between the membrane-perturbing activity and the antibacterial activity was not always parallel. Conclusively, the Ala-->Val substitution in 4(3) causes the change of peptide conformation and the presence of a cationic amino acid residue is necessary for the antibacterial activity.

Amino Acid Sequence↗

PubMed1999

Interaction of bundled Ser-rich amphiphilic peptides with phospholipid membranes.

To investigate properties of hydrophilic bundled peptides and their interactions with phospholipid membranes, bundled peptides named [Trp2]- and [Trp12]-4alpha-46S9, which are composed of four fragments of amphiphilic 24-mer peptide, were designed and synthesized. Tryptophan (Trp) was introduced at the 2nd position from the N-terminal or at the centre (12th) of the helix to monitor the peptide-lipid interaction. Circular dichroism measurements indicated that the peptides had low alpha-helicities in a buffer solution (pH 7.4) and also in the presence of dipalmitoyl-DL-3-phosphatidylcholine (DPPC) vesicles. In the presence of DPPC/dipalmitoyl-DL-3-phosphatidylglycerol (DPPG) (3:1) vesicles, the measurement could not be taken because of turbidity induced by vesicle aggregation. Both peptides had moderate perturbation activity for both the neutral and acidic vesicles at 25 degrees C. The perturbation patterns at 50 degrees C were much different from those at 25 degrees C and the maximum activity reached 100% at a low peptide concentration. The results of the measurement of membrane fusion activity of peptides showed a similar tendency to that found in the perturbation experiment. A quenching experiment indicated that the Trp2 and Trp12 residues in [Trp2]- and [Trp12]-4alpha-46S9 were scarcely embedded in neutral lipid membranes.

Amino Acid Sequence↗