Biomedical subjects

K Kato

Publications and source records attributed to K Kato.

At least 127 records · Page 7Linked to original sources

PubMed2001

Galpha11 induces caspase-mediated proteolytic activation of Rho-associated kinase, ROCK-I, in HeLa cells.

Expression of the constitutively active mutant of Galpha(11) (Galpha(11)QL) induces the formation of vinculin-containing focal adhesion-like structures in HeLa cells. This was found to be inhibited by Y-27632, a specific inhibitor of Rho-associated kinases (ROCK), but not by co-expression with a dominant negative mutant of RhoA, suggesting Rho-independent activation of ROCK by Galpha(11)QL. Investigation of trypan blue exclusion and immunocytochemistry with an antibody against cleaved caspase revealed the cellular phenotype of Galpha(11)QL-expressing cells to be identical to that displayed by cells undergoing apoptosis, and the caspase inhibitor zVAD-fmk blocked all morphological changes induced by Galpha(11)QL. Transfection of Galpha(11)QL induced cleavage of ROCK-I, and this proteolysis was also prevented by zVAD-fmk. ROCK-I C-terminally truncated at its authentic caspase sites also induced the formation of vinculin-containing focal adhesion-like structures. In addition, cleavage of ROCK-I was observed when cells overexpressing m1 muscarinic acetylcholine receptors were stimulated with carbachol. These results suggest that Galpha(11) induces proteolytic activation of ROCK-I by caspase and thereby regulates the actin cytoskeleton during apoptosis.

Calcium↗

PubMed2001

Replacing factor-dependency with that for lysozyme: affordable culture of IL-6-dependent hybridoma by transfecting artificial cell surface receptor.

Cytokines and growth factors are indispensable for the propagation and maintenance of factor-dependent mammalian cells. However, cytokines are often so expensive that the use of factor-dependent cells for industrial applications such as protein production is often not practical. Based on our previous design of a binary hen egg lysozyme (HEL)-specific receptor composed of portions of the anti-HEL antibody and the erythropoietin receptor, a new pair of chimeric receptors having the intracellular domain of gp130 were made and transfected to an interleukin-6 (IL-6)-dependent hybridoma, 7TD1. The clone expressing the two new receptors showed clear HEL dose-dependent cell growth and monoclonal antibody production in both serum-based and serum-free media without IL-6. These results establish the feasibility of applying receptor design to tailor cells for the inexpensive induction of cell growth for the purpose of producing therapeutic products.

Animals↗

PubMed2001

Photoalkylation of 10-alkylacridinium ion via a charge-shift type of photoinduced electron transfer controlled by solvent polarity.

A charge-shift type of photoinduced electron-transfer reactions from various electron donors to the singlet excited state of 10-decylacridinium cation (DeAcrH+) in a nonpolar solvent (benzene) is found to be as efficient as those of 10-methylacridinium cation (MeAcrH+) and DeAcrH+ in a polar solvent (acetonitrile). Irradiation of the absorption bands of MeAcrH+ in acetonitrile solution containing tetraalkyltin compounds (R(4)Sn) results in the efficient and selective reduction of MeAcrH+ to yield the 10-methyl-9-alkyl-9,10-dihydroacridine (AcrHR). The same type of reaction proceeds in benzene when MeAcrH+ is replaced by DeAcrH+ which is soluble in benzene. The photoalkylation of R'AcrH+ (R' = Me and De) also proceeds in acetonitrile and benzene using 4-tert-butyl-1-benzyl-1,4-dihydronicotinamide (Bu(t)BNAH) instead of R(4)Sn, yielding MeAcrHBu(t). The quantum yield determinations, the fluorescence quenching of R'AcrH+ by electron donors, and direct detection of the reaction intermediates by means of laser flash photolysis experiments indicate that the photoalkylation of R'AcrH+ in benzene as well as in acetonitrile proceeds via photoinduced electron transfer from the alkylating agents (R(4)Sn and Bu(t)BNAH) to the singlet excited states of R'AcrH+. The limiting quantum yields are determined by the competition between the back electron-transfer process and the bond-cleavage process in the radical pair produced by the photoinduced electron transfer. The rates of back electron transfer have been shown to be controlled by the solvent polarity which affects the solvent reorganization energy of the back electron transfer. When the free energy change of the back electron transfer (DeltaG(0)(bet)) in a polar solvent is in the Marcus inverted region, the rate of back electron transfer decreases with decreasing the solvent polarity, leading to the larger limiting quantum yield for the photoalkylation reaction. In contrast, the opposite trend is obtained when the DeltaG(0)(bet) value is in the normal region: the limiting quantum yield decreases with decreasing the solvent polarity.

Journal Article↗

PubMed2001

Systematic search for single nucleotide polymorphisms in the insulin gene: evidence for a high frequency of -23T-->A in Japanese subjects.

It has recently been shown that the A/A genotype at g.-23 of the insulin gene correlates with impaired insulin secretion in response to body weight gain in subjects of European descent. To examine whether there are single nucleotide polymorphisms (SNPs) in the insulin gene associated with type 2 diabetes, all exons with their flanking sequences for 113 Japanese type 2 diabetic patients and 99 nondiabetic control subjects were analyzed using PCR direct sequencing. We have only found g.-23T --> A, 806G --> C, 1128T --> C, and 1141A --> C, which have previously been reported in alpha (A-C-C-C) and beta (T-G-T-A) alleles. The allele frequency of -23T --> A in control Japanese subjects was 97.4%, whereas that in Europeans is about 30%. The A/A genotype was found in 94 of 99 Japanese subjects (94.9%) and the allele frequencies of 806G --> C, 1128T --> C, and 1141A --> C were all 96.5%. The estimated haplotype frequencies were (A-C-C-C) (96.0%), (T-G-T-A) (2.0%), (A-G-T-A) (1.5%), and (T-C-C-C) (0.5%). No association of these SNPs or haplotypes with type 2 diabetes was evident. Thus, the A/A genotype at the g.-23 of insulin gene was generally high in Japanese subjects, which could account for the fact that they typically secrete lower levels of insulin.

Adult↗

PubMed2001

A comprehensive collection of mouse zinc finger motifs compiled by molecular indexing.

The C(2)H(2) zinc finger motif found in many transcription factors is thought to be for nucleic acid binding and/or dimerization. Nearly 1% of eukaryote genes are estimated to encode this motif. We investigated the gene family encoding this motif in the Mus musculus mRNA by molecular indexing, a technique used to select a subpopulation of cDNA by ligation of adapters to cDNA fragments digested by a class IIS restriction enzyme(s). In place of oligo-dT primers in the original method, a polymerase chain reaction primer designed based on the conserved sequence of the C(2)H(2) zinc finger protein stranded cDNA was made from various mouse tissue mRNAs, digested with FokI and BsmAI, and joined with adapters. Amplification of the cDNA with an adapter primer and zinc finger-specific primer yielded products enriched in zinc finger protein genes. Fragments were separated by subsequent denaturing polyacrylamide gel electrophoresis, and characterized by DNA sequencing. Consequently, 259 C(2)H(2) zinc finger motif sequences were obtained, among which 166 were novel. Combined with the reported sequences, these mouse motif sequences were compared with those of other species such as Saccharomyces cerevisiae and Caenorhabditis elegans. Some of the amino acids in the motif sequence showed strong bias among species. Most of the novel sequences were supposed to be DNA-binding according to the surface potential of predicted tertiary structures.

Amino Acid Motifs↗

PubMed2001

Polyglutamine expansions cause decreased CRE-mediated transcription and early gene expression changes prior to cell death in an inducible cell model of Huntington's disease.

Huntington's disease (HD) is one of 10 known diseases caused by a (CAG)(n) trinucleotide repeat expansion that is translated into an abnormally long polyglutamine tract. We have developed stable inducible neuronal (PC12) cell lines that express huntingtin exon 1 with varying CAG repeat lengths under doxycycline (dox) control. The expression of expanded repeats is associated with aggregate formation, caspase-dependent cell death and decreased neurite outgrowth. Post-mitotic cells expressing mutant alleles were more prone to cell death compared with identical cycling cells. To determine early metabolic changes induced by this mutation in cell models, we studied changes in gene expression after 18 h dox induction, using Affymetrix arrays, cDNA filters and adapter-tagged competitive PCR (ATAC-PCR). At this time point there were low rates of inclusion formation, no evidence of mitochondrial compromise and no excess cell death in the lines expressing expanded compared with wild-type repeats. The expression profiles suggest novel targets for the HD mutation and were compatible with impaired cAMP response element (CRE)-mediated transcription, which we confirmed using CRE-luciferase reporter assays. Reduced CRE-mediated transcription may contribute to the loss of neurite outgrowth and cell death in polyglutamine diseases, as these phenotypes were partially rescued by treating cells with cAMP or forskolin.

Animals↗

PubMed2001

Two-domain hemoglobin gene of the water flea Moina macrocopa: duplication in the ancestral Cladocera, diversification, and loss of a bridge intron.

Two cDNAs encoding the two-domain hemoglobin (Hb) chains of a crustacean Cladocera, Moina macrocopa, were cloned and their nucleotide (nt) sequences were determined. The amino acid (aa) sequences of both the gene products deduced from the nt sequences consisted of 348 residues and showed 98% identity with each other. These sequences together with the NH(2)-terminal aa sequences of the Hb chains determined after separation by two-dimensional gel electrophoresis showed that the Hb chains are synthesized as a secretory precursor with a signal peptide of 17 aa residues. The aa sequences of M. macrocopa Hb chains shared the following features with those of Daphnia Hb chains. Firstly, the signal peptide is followed by an NH(2)-terminal extension containing a threonine-rich sequence that might play a role in the multimerization of subunit chains. Secondly, the identity between the aa sequences of the first and second domains is exceptionally low. These facts suggest that duplication of the cladoceran Hb gene occurred before the divergence of families Moinidae and Daphniidae. Analysis of genomic DNA showed that the M. macrocopa Hb genes consist of two large repeated regions, encoding the first and second domains of Hb chains, respectively. The intron-exon organization of the first region of the M. macrocopa Hb genes was similar to that found in the Daphnia Hb genes, having the three-exon, two-intron structure characteristic of animal Hb genes. However, the intron bridging the two regions and the most downstream intron in the second region were missing in the Moina genes, providing a new example of intron loss. The following elements in the 5' flanking region were conserved in the Moina and Daphnia genes: (1) TATAAA, a typical TATA box sequence accompanied by a downstream sequence, GAAXAGCATCAGTT (the fourth residue X was G or A in Daphnia and absent in Moina); (2) CCAAT boxes, located upstream of the TATA box; (3) the binding sites for HIF-1 and GATA-1, also located upstream of the TATA box, that may be responsible for up-regulation of the cladoceran Hb genes under hypoxia.

Amino Acid Sequence↗

PubMed2001

Cluster algorithms for general-S quantum spin systems.

We present a general strategy to extend quantum cluster algorithms for S = 1 / 2 spin systems, such as the loop algorithm, to those with an arbitrary size of spins. The partition function of a high- S spin system is generally represented by the path integral of a S = 1 / 2 model with special boundary conditions in the imaginary-time direction. We introduce additional graphs for the boundary part and give the labeling probability explicitly, which completes the algorithm together with an existing S = 1 / 2 algorithm. As a demonstration, we simulate the integer-spin antiferromagnetic Heisenberg chains. The magnitude of the first excitation gap is estimated to be 0.41048(6), 0.08917(4), and 0.01002(3) for S = 1, 2, and 3, respectively.

Journal Article↗

PubMed2001

Electroretinogram analysis of relative spectral sensitivity in genetically identified dichromatic macaques.

The retinas of macaque monkeys usually contain three types of photopigment, providing them with trichromatic color vision homologous to that of humans. However, we recently used molecular genetic analysis to identify several macaques with a dichromatic genotype. The affected X chromosome of these animals contains a hybrid gene of long-wavelength-sensitive (L) and middle-wavelength-sensitive (M) photopigments instead of separate genes encoding L and M photopigments. The product of the hybrid gene exhibits a spectral sensitivity close to that of M photopigment; consequently, male monkeys carrying the hybrid gene are genetic protanopes, effectively lacking L photopigment. In the present study, we assessed retinal expression of L photopigment in monkeys carrying the hybrid gene. The relative sensitivities to middle-wavelength (green) and long-wavelength (red) light were measured by electroretinogram flicker photometry. We found the sensitivity to red light to be extremely low in protanopic male monkeys compared with monkeys with the normal genotype. In female heterozygotes, sensitivity to red light was intermediate between the genetic protanopes and normal monkeys. Decreased sensitivity to long wavelengths was thus consistent with genetic loss of L photopigment.

Animals↗

PubMed2001

Structural features of arabinogalactan-proteins from the fruit of Lycium chinense Mill.

In addition to the other arabinogalactan-proteins (AGPs) (Cp-1-C and -D) already reported, two kinds of AGP (Cp-2-B and Hp-2-C) were obtained from the fruit of Lycium chinense Mill. The ratio of arabinose to galactose was approximately 1:1 in both samples, and the carbohydrate was linked O-glycosidically to serine in Cp-2-B, and to both serine and threonine residues of the protein in Hp-2-C. The weight-average molecular weight was 71,000 for Cp-2-B and 120,000 for Hp-2-C. Both samples also contained non-reducing terminal 3-O- and 4-O-substituted galacturonic acids. The ratio of 6-O-substituted galactose (linear part) and 3,6-di-O-substituted galactose (branching point) was almost unity in both samples, being obviously different from the case of Cp-1-C (predominant in the branching domain) and Cp-1-D (predominant in the linear domain). These results offer fresh insight into the grouping of the AGPs, based on the ratio of 6-O- and 3,6-di-O-substituted galactosyl residues.

Amino Acids↗

PubMed2001

Regulation of the levels of small heat-shock proteins during differentiation of C2C12 cells.

Levels of the small heat-shock proteins (sHSPs) HSP27 and alphaB-crystallin during differentiation of mouse C2C12 cells were determined using specific immunoassays. Increases of these proteins were about 3-fold and 10-fold, respectively. Under the same conditions, however, the level of HSP70 in C2C12 cells barely increased, indicating selective accumulation of HSP27 and alphaB-crystallin with differentiation. While expression of mRNA for alphaB-crystallin was also markedly increased and that for HSP27 was but to a lesser extent, mRNA for HSP70 could barely be detected during differentiation. Activation of the heat-shock factor was not observed, in contrast to the case with heat-stressed undifferentiated cells. Various inhibitors of protein kinases affected the differentiation and the associated increase of sHSPs. Rapamycin, an inhibitor of p70 S6 kinase, completely inhibited the differentiation and suppressed the accumulation of HSP27 and alphaB-crystallin. SB203580, an inhibitor of p38 MAP kinase, also inhibited differentiation, but the accumulation of alphaB-crystallin was rather enhanced. PD98059, an inhibitor of MAP kinase kinase, significantly increased expression of a differentiation marker for muscle cells, creatine kinase M isozyme, as well as accumulation of alphaB-crystallin. These results suggest that accumulation of sHSPs during differentiation of C2C12 cells is regulated in a complex manner.

Animals↗

PubMed2001

A unique sequence of the laminin alpha 3 G domain binds to heparin and promotes cell adhesion through syndecan-2 and -4.

Laminin-5, consisting of the alpha 3, beta 3, and gamma 2 chains, is localized in the skin basement membrane and supports the structural stability of the epidermo-dermal linkage and regulates various cellular functions. The alpha chains of laminins have been shown to have various biological activities. In this study, we identified a sequence of the alpha 3 chain C-terminal globular domain (LG1-LG5 modules) required for both heparin binding and cell adhesion using recombinant proteins and synthetic peptides. We found that the LG3 and LG4 modules have activity for heparin binding and that LG4 has activity for cell adhesion. Studies with synthetic peptides delineated the A3G75aR sequence (NSFMALYLSKGR, residues 1412--1423) within LG4 as a major site for both heparin and cell binding. Substitution mutations in LG4 and A3G75aR identified the Lys and Arg of the A3G75aR sequence as critical for these activities. Cell adhesion to LG4 and A3G75aR was inhibited by heparitinase I treatment of cells, suggesting that cell binding to the A3G75aR site was mediated by cell surface heparan sulfate proteoglycans. We showed by affinity chromatography that syndecan-2 from fibroblasts bound to LG4. Solid-phase assays confirmed that syndecan-2 interacted with the A3G75aR peptide sequence. Stably transfected 293T cells with expression vectors for syndecan-2 and -4, but not glypican-1, specifically adhered to LG4 and A3G75aR. These results indicate that the A3G75aR sequence within the laminin alpha 3 LG4 module is responsible for cell adhesion and suggest that syndecan-2 and -4 mediate this activity.

Antibodies↗

PubMed2001

Dendritic aberrations in the hippocampal granular layer and the amygdalohippocampal area following kindled-seizures.

Amygdaloid kindling is a model of human temporal lobe epilepsy, in which excitability in limbic structures is permanently enhanced by repeated stimulations. We report here dendritic aberrations occurring in mice following kindled-seizures. Adult mice received a biphasic square wave pulse [495+/-25.5 (S.E.M.) microA 60 Hz, 200 micros duration, for 2 s] unilaterally in the basolateral amygdaloid complex once a day and mice with electrophysiologically and behaviorally verified seizures were used in the experiments. The hippocampus and amygdaloid complex contralateral to the lesions were observed by immunofluorescence histochemistry with a somatodendritic marker, microtubule-associated protein 2 (MAP2), showing that kindled-seizures caused hypertrophy of proximal dendrites in the granule cells of the dentate gyrus and in neurons of the amygdalohippocampal area. To further characterize the morphological changes of the dendrites, electron micrographic analysis was performed on the contralateral side. (1) In the granular layer of the dentate gyrus and the amygdalohippocampal area, kindled-seizures generated an increase in the number of dendrites containing polymerized microtubules and width of dendritic profiles showing the increase was in the range 0.2-3.0 and 0.2-1.4 microm, respectively. (2) In the granular layer, bundles between dendrites separated by the puncta adhaerentia increased. (3) In the granular layer, the seizure-induced dendritic aberration was more severe in the rostral than the caudal region. These results suggested that growth of dendrites with enriched-stable microtubules is part of the structural plasticity in response to seizure activity in specific areas of the adult brain.

Amygdala↗

PubMed2001

Neuronal plasticity in hippocampal mossy fiber-CA3 synapses of mice lacking the inositol-1,4,5-trisphosphate type 1 receptor.

In the present study, we used inositol-1,4,5-trisphosphate (IP3) type 1 receptor (IP3R1) knockout mice to examine the role of this receptor in the induction of LTP, LTD, and DP at mossy fiber-CA3 synapses. No difference in synaptically induced field-EPSPs was seen between the wild-type (IP3R1(+/+)) and IP3R1 knockout mice (IP3R1(-/-)), showing that basic synaptic transmission does not involve IP3R1 activation. Tetanus induced LTP in both wild-type and IP(3)R1(-/-) mice, but the magnitude of LTP was significantly greater in IP3R1(-/-) mice (149.8+/-3.5%, mean+/-S.E.M., n=15) than in wild-type mice (132.4+/-1.5%, n=17), suggesting that the IP3R1 has a suppressive effect on LTP induction. To determine whether this effect involved N-methyl-D-aspartate receptor (NMDAR)-dependent LTP, the effect of tetanus was tested in the present of the NMDAR antagonist, D,L-AP5 (50 microM); under these conditions, the LTP in both IP3R1(-/-) and IP3R1(+/+) mice was not significantly reduced. In addition, group I mGluR activation was shown to be necessary for LTP induction, as the LTP was almost blocked by the group I mGluR antagonist, RS-4CPG (500 microM) in both IP3R1(-/-) (117.6+/-1.7%, n=8) and IP3R1(+/+) (116.9+/-1.8%, n=5) mice. The IP3R1 also plays an essential role in LTD induction, as low-frequency stimulation (LFS) failed to induce LTD in the mutant mice (104.5+/-2.1%, n=10). DP was induced in both IP3R1(-/-) and wild-type mice.

Animals↗

PubMed2001

A narrow window for rescuing cells by the inhibition of calcium influx and the importance of influx route in rat cortical neuronal cell death induced by glutamate.

A rescue window for glutamate-insulted cells with regard to Ca(2+) influx was first investigated. The addition of EGTA, an impermeable calcium chelator to the culture within 5 min after the beginning of glutamate stimulation potently suppressed the neuronal cell death examined at 22 h. The effect of EGTA on rescuing cells decreased with the time delay of its addition to the system. MK-801, an antagonist of N-methyl-D-asparate (NMDA) receptor channels also inhibited the neuronal cell death in a similar manner to EGTA, suggesting Ca(2+) influx up to 5 min after the insult determined the fate of cells. But we also demonstrated that the elevated intracellular Ca(2+) did not always induce neurotoxicity. High concentration of potassium chloride plus FPL64176, an agonist of L-type Ca(2+) channels did not induce neuronal cell death, even though their combination elicited equal elevation of intracellular Ca(2+) to that by toxic concentration of glutamate, demonstrating that locally elevated intracellular Ca(2+) around NMDA receptors is important in the induction of neuronal cell death.

Animals↗

PubMed2001

Controlled expansion of a molecular cavity in a steroid host compound.

Expansion of a molecular cavity is described by using elongation of the side chain of a bile acid host compound. Bishomocholic acid (2), which has a side chain that is longer by two methylene unit than cholic acid (1), includes many organic substances at 1:1 host:guest ratios. X-ray crystallographic studies revealed that 2 has two types of open host frameworks: a bilayer type and a crossing type. Both of them are isostructural to those of 1, indicating that they are robust against the elongation of the side chain. In the former type, the increment of the width of the host channel corresponds to that of the length of the molecular structures. Larger aromatic guest components such as 1-methylnaphthalene and 1-tetralone, are included in 2, but not in 1.

Bile Acids and Salts↗

PubMed2001

Dorsal and ventral positional cues required for the onset of planarian regeneration may reside in differentiated cells.

We previously showed by grafting experiments that the dorsoventral (DV) interaction evokes morphogenetic events similar to those that occur in regeneration. However, it is not yet understood whether the stem cells themselves or differentiated cells have the ability to induce regeneration. Here we demonstrated by a combination of X-ray irradiation and grafting experiments that the dorsal and ventral positional cues inducing morphogenetic events are retained in X-ray-irradiated tissues, suggesting that the differentiated cells may be responsible for the positional cues. We grafted a small piece of irradiated worm, in which the stem cells were certainly eliminated, to an intact one in DV-reversed orientation. We observed that projections were developed from the host-donor boundary, as in the previous experiments. Whole-mount in situ hybridization with several markers demonstrated that the projections had a newly established DV axis and also had anterior or posterior characteristics. Furthermore, chimeric analysis with a strain-specific marker showed that the projections consisted of nonirradiated cells and that IFb-expressing cells, which normally belonged to the ventral tissue, could be generated even from the stem cells located on the dorsal side. Taken together, the findings suggest that the stem cells may simply differentiate depending on the surroundings and that differentiated cells may present positional cues that induce morphogenesis.

Animals↗

PubMed2001

Stimulatory effect of basic fibroblast growth factor on induction of heat shock protein 27 in osteoblasts: role of protein kinase C.

In a previous study we showed that basic fibroblast growth factor (bFGF) stimulates activation of protein kinase C through phosphoinositide hydrolysis by phospholipase C and phosphatidylcholine hydrolysis by phospholipase D in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether bFGF stimulates the induction of heat shock protein (HSP) 27, a low-molecular-weight HSP, and HSP70, a high-molecular-weight HSP, in MC3T3-E1 cells and the mechanism behind the induction. bFGF increased the level of HSP27 while having little effect on HSP70 level. bFGF stimulated the accumulation of HSP27 dose-dependently in the range between 1 and 30 ng/ml. bFGF induced an increase in the level of the mRNA for HSP27. The bFGF-stimulated accumulation of HSP27 was reduced by inhibitors of protein kinase C. The bFGF-induced HSP27 accumulation was reduced in protein kinase C-downregulated MC3T3-E1 cells. U-73122, an inhibitor of phospholipase C, and propranolol, a phosphatidic acid phosphohydrolase inhibitor, suppressed the bFGF-stimulated HSP27 accumulation. These results strongly suggest that bFGF stimulates HSP27 induction through protein kinase C activation in osteoblasts.

3T3 Cells↗