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K Kusano

Publications and source records attributed to K Kusano.

At least 37 records · Page 2Linked to original sources
PubMed1999

Detection of a sequence involved in actin-binding and phosphoinositide-binding in the N-terminal side of cofilin.

Cofilin is an actin-binding protein of low molecular weight which is widely distributed in eukaryotes and is deeply involved in the dynamics of actin assembly in the cytoplasm. The actin-binding ability of cofilin is inhibited by inositol phosphates (PIP2), and the PIP2- and actin-binding site(s) has been localized in residues W104-M115 of the cofilin primary sequence (Yonezawa et al. 1991 ). In the present study, in order to further clarify the functional domains in cofilin molecule, we constructed expression vectors containing cDNAs of different size with deletion at the 3'-region of the open reading frame. The truncated cofilin molecules produced in E. coli were purified and examined for their actin-binding and PIP2-binding ability. We found that the truncated cofilin molecule without C-terminal residues #100-#166 including the previously-described actin-binding site could be cross-linked with actin by EDC, a zero-length cross-linker. In addition, these truncated peptides as well as synthetic peptides corresponding to the N-terminal sequence of cofilin suppressed the inhibitory action of PIP2 on actin-cofilin interaction. These results strongly suggest that additional actin- and PIP2-binding sites exist in the N-terminal region of cofilin.

Actin Depolymerizing Factors↗
PubMed1998

Stationary organotypic cultures of oxytocin and vasopressin magnocellular neurones from rat and mouse hypothalamus.

Rat and mouse hypothalami from postnatal animals containing highly differentiated neurones survive very well in long-term (>15 days in vitro, DIV) stationary organotypic cultures. Magnocellular oxytocin (OT) and vasopressin (VP) neurones are present in identifiable paraventricular (PVN), supraoptic (SON) and accessory (ACC) nuclei in these cultures. After 15 DIV in standard medium immunocytochemistry revealed 427 +/- 63 OT cells and 217 +/- 27 VP cells per cultured rat hypothalamus, and 380 +/- 72 OT cells and 622 +/- 91 VP cells per cultured mouse hypothalamus. Following a 7-day adaptation period in standard culture medium containing serum, the rat slice-explants survived very well after subsequent transfer to defined, serum- free media (SFM) for an additional 8 days. The number of OT cells surviving in SFM was 612 +/- 147 OT cells per cultured rat hypothalamus. Only 0.5% of the magnocellular OT and VP neurones in the cultures appeared to express both peptides. Experiments on c-fos gene expression in these cultures showed that while only 12% of the magnocellular OT and VP neurones contained barely detectable Fos protein in their nuclei under control conditions, potassium depolarization of these cultures for 3 h produced intense c-fos expression in 87-91% of these cells. Thus, magnocellular neurones in these cultures are sufficiently stable and responsive to permit long-term physiological and gene expression studies to be done under defined media conditions.

Animals↗
PubMed1998

Regulation of matrix metalloproteinases (MMP-2, -3, -9, and -13) by interleukin-1 and interleukin-6 in mouse calvaria: association of MMP induction with bone resorption.

Interleukin-1 (IL-1) greatly induces osteoclast formation and stimulates bone resorption of mouse calvaria in culture. In the presence of soluble IL-6 receptor (sIL-6R), IL-6 similarly induces osteoclast formation, but the potency of IL-6 in inducing bone resorption in organ culture is weaker than that of IL-1. To study the differences in bone-resorbing activity between IL-1 and IL-6, we examined the effects of the two cytokines on the induction of matrix metalloproteinases (MMPs). In mouse calvarial cultures, IL-1 markedly enhanced the messenger RNA (mRNA) expression of MMP-13 (collagenase 3), MMP-2 (gelatinase A), MMP-9 (gelatinase B), and MMP-3 (stromelysin 1), which associated with increases in bone matrix degradation. A hydroxamate inhibitor of MMPs significantly suppressed bone-resorbing activity induced by IL-1. Gelatin zymography showed that both pro- and active-forms of MMP-2 and MMP-9 were detected in the conditioned medium collected from calvarial cultures, and IL-1 markedly stimulated both pro- and active-forms of the two gelatinases. IL-6 with sIL-6R also stimulated mRNA expression and biological activities of these MMPs, but the potency was much weaker than that of IL-1. Conditioned medium collected from IL-1-treated calvariae degraded native type I collagen, but 3/4- and 1/4-length collagen fragments were not detected, suggesting that both collagenases and gelatinases synergistically degraded type I collagen into smaller fragments. In mouse osteoblastic cells, the expression ofMMP-2, MMP-3, and MMP-13 mRNAs could be detected, and they were markedly enhanced by IL-1alpha on days 2 and 5. IL-6 with sIL-6R also induced expression of MMP-13 and MMP-2 mRNAs on day 2, but the expression was rather transient. These results demonstrate that the potency of induction of MMPs by IL-1 and IL-6 is closely linked to the respective bone-resorbing activity, suggesting that MMP-dependent degradation of bone matrix plays a key role in bone resorption induced by these cytokines.

Animals↗
PubMed1998

Anthelmintic efficacy of milbemycin oxime against Trichuris vulpis in dogs.

The anthelmintic efficacy of milbemycin oxime against dog whipworm, Trichuris vulpis, was evaluated. A total of 21 T. vulpis positive dogs were divided into 3 groups, one (5 dogs) for control and the other two (8 dogs each) for anthelmintic treatment with oral administration of milbemycin oxime.

Administration, Oral↗
PubMed1997

Chi-star, a chi-related 11-mer sequence partially active in an E. coli recC1004 strain.

BACKGROUND: chi sequence (5'GCTGGTGG) of Escherichia coli was first identified as a site that increased the plaque size of bacteriophage lambda. Subsequent studies showed that this site is responsible for both the attenuation ofRecBCD exonuclease activity and the promotion of RecA, RecBCD-mediated recombination. It is known that bacteriophage lambda containing the chi site makes very small plaques on a recC* (recC1004) mutant because chi is not recognized by the RecBC*D mutant enzyme. RESULTS: We cloned E. coli chromosomal fragments in lambda which allowed lambda to form larger plaques on this recC1004 mutant. The fragments were found to share a chi-like 11-mer sequence, 5'GCTGGTGCTCG. Substitution of these fragments with a synthetic 11-mer of this sequence and single-base-pair substitution analysis of its last four nucleotides demonstrated that this sequence is both necessary and sufficient for the observed activity. The sequence, designated X* (chi-star), protected rolling-circle DNA replication in the recC1004 mutant and in the recBCD+ strain, most likely because it attenuated the exonuclease activity of the RecBC*D and RecBCD+ enzyme. chi-star, did not significantly stimulate lambda recombination in two assays. CONCLUSION: We have discovered that a mutant RecBCD enzyme responds, in vivo, to a longer chi variant.

Bacteriophage lambda↗
PubMed1997

Genetic recombination through double-strand break repair: shift from two-progeny mode to one-progeny mode by heterologous inserts.

Double-strand break repair models of genetic recombination propose that a double-strand break is introduced into an otherwise intact DNA and that the break is then repaired by copying a homologous DNA segment. Evidence for these models has been found among lambdoid phages and during yeast meiosis. In an earlier report, we demonstrated such repair of a preformed double-strand break by the Escherichia coli RecE pathway. Here, our experiments with plasmids demonstrate that such reciprocal or conservative recombination (two parental DNAs resulting in two progeny DNAs) is frequent at a double-strand break even when there exists the alternative route of nonreciprocal or nonconservative recombination (two parental DNAs resulting in only one progeny DNA). The presence of a long heterologous DNA at the double-strand break, however, resulted in a shift from the conservative (two-progeny) mode to the nonconservative (one-progeny) mode. The product is a DNA free from the heterologous insert containing recombinant flanking sequences. The potential ability of the homology-dependent double-strand break repair reaction to detect and eliminate heterologous inserts may have contributed to the evolution of homologous recombination, meiosis and sexual reproduction.

DNA Damage↗
PubMed1997

Propionibacterium cyclohexanicum sp. nov., a new acid-tolerant omega-cyclohexyl fatty acid-containing propionibacterium isolated from spoiled orange juice.

A non-spore-forming, coryneform bacterium, strain TA-12T, was isolated from spoiled off-flavor orange juice. Growth of this organism occurs at pH 3.2 to 7.5, and optimum growth occurs at pH values between 5.5 and 6.5. This organism produces lactic acid, propionic acid, and acetic acid from glucose. It is catalase negative. The cells are heat resistant and can withstand a temperature of 90 degrees C for 10 min. The DNA G + C content is 66.8 mol%. This strain has as MK-9(H4) respiratory quinone system and contains meso-diaminopimelic acid in its cell wall, and omega-cyclohexyl undecanoic acid is the major cellular fatty acid. The results of a phylogenetic analysis of the 168 rRNA gene of this organism indicated that its highest level of homology is its level of homology with the representative of the classical propionibacteria, Propionibacterium freudenreichii (97.1%). Strain TA-12T is phenotypically similar to P. freudenreichii, but it produces a large amount of lactic acid and has a distinct fatty acid composition, acid tolerance, and heat resistance, which differentiate it from P. freudenreichii and other propionic acid-producing bacteria. On the basis of these findings we propose the name Propionibacterium cyclohexanicum sp. nov. for this organism. The type strain is TA-12 (= IAM 14535 = NRIC 0247).

Acids↗
PubMed1997

A new type of illegitimate recombination is dependent on restriction and homologous interaction.

Illegitimate (nonhomologous) recombination requires little or no sequence homology between recombining DNAs and has been regarded as being a process distinct from homologous recombination, which requires a long stretch of homology between recombining DNAs. Under special conditions in Escherichia coli, we have found a new type of illegitimate recombination that requires an interaction between homologous DNA sequences. It was detected when a plasmid that carried 2-kb-long inverted repeats was subjected to type II restriction in vitro and type I (EcoKI) restriction in vivo within a delta rac recBC recG ruvC strain. Removal of one of the repeats or its replacement with heterologous DNA resulted in a reduction in the level of recombination. The recombining sites themselves shared, at most, a few base pairs of homology. Many of the recombination events joined a site in one of the repeats with a site in another repeat. In two of the products, one of the recombining sites was at the end of one of the repeats. Removal of one of the EcoKI sites resulted in decreased recombination. We discuss the possibility that some structure made by homologous interaction between the long repeats is used by the EcoKI restriction enzyme to promote illegitimate recombination. The possible roles and consequences of this type of homologous interaction are discussed.

Base Sequence↗
PubMed1997

Low molecular-weight G-actin binding proteins involved in the regulation of actin assembly during myofibrillogenesis.

We previously demonstrated that small G-actin binding proteins, cofilin, ADF and profilin, are involved in the actin dynamics during myofibrillogenesis (OBINATA, T. (1993). Int. Rev. Cytol., 143: 153-189.). To better understand how they are responsible for the regulation of actin assembly, the amounts of the actin-binding proteins were quantified by means of quantitative immunoblotting and compared with that of G-actin pool. The sum of the amounts of cofilin, ADF and profilin was insufficient at early developmental stages but sufficient at later stages to account for the pool of G-actin in muscle cells. We detected expression of thymosin beta 4 at a considerable level in young embryonic but not in adult skeletal muscles. We, therefore, conclude that the G-actin pool in young embryonic skeletal muscle is mainly due to cofilin, ADF, profilin and thymosin beta 4. Switching from a non-muscle-type (NM-) cofilin to a muscle-type (M-) cofilin was observed during muscle development of mammals. In order to clarify cofilin-dependent regulation of actin assembly in muscle cells, cofilin tagged with fluorescence dyes was introduced into C2 myoblasts by a micro injection method. The exogeneous cofilin, but not ADF, caused quick disassembly of actin filaments and accumulated in furrow region of dividing cells. The analogs of the unphosphorylated form (A3-cofilin) and the phosphorylated form (D3-cofilin) were prepared by converting Ser3, a regulatory phosphorylation site, to Ala or Asp. When A3-cofilin and D3-cofilin were injected into living cells, the former was concentrated at the membrane ruffles and cleavage furrow, while the latter showed only diffuse distribution in the cytoplasm. These results suggest that the subcellular distribution of cofilin as well as its interaction with actin in vivo is regulated by its phosphorylation and dephosphorylation.

Actin Depolymerizing Factors↗
PubMed1997

Primary structure of mouse actin-related protein 1 (Arp1) and its tissue expression.

Different types of actin-related proteins which constitute an actin-superfamily together with conventional actin have recently been described (Mullins et al., 1996). Among them, Arp1 exhibits the highest homology with conventional actin. With the aim of clarifying the cellular function of Arp1 in mammalian cells, we cloned the cDNA encoding mouse alpha-Arp1, one of the variants of Arp1, from a mouse diaphragm cDNA library; two types of alpha-Arp1 cDNAs, which are probably generated by alternative RNA splicing from a single gene, were obtained and the entire sequences were determined. They differed only in the presence or absence of an insertion of 1.3 kb in the 3'-non-cooling region but shared a common open reading frame. The deduced amino acid sequence was identical with that of human alpha-Arp1. Northern blot analysis showed that the alpha-Arp1 mRNA corresponding to the longer cDNA is transcribed not only in various non-muscle tissues but also in muscle tissues, while the transcript corresponding to the shorter one becomes expressed only in skeletal muscle as development progresses. It is suggested that alpha-Arp1 may play some role in muscle, as judged by the significant level of its expression.

Actins↗
PubMed1996

GABAergic neurons in the embryonic olfactory pit/vomeronasal organ: maintenance of functional GABAergic synapses in olfactory explants.

In previous work, we showed a robust gamma-aminobutyric acid (GABAergic) synaptic input onto embryonic luteinizing hormone-releasing hormone (LHRH) neurons maintained in olfactory explants. In this study, we identify GABAergic neurons in olfactory pit (OP) of embryonic mice in vivo and study, using patch-pipet whole-cell current and voltage clamp techniques, synaptic interactions of these neurons in explant cultures. In vivo, glutamate decarboxylase (GAD, the enzyme which synthesizes GABA) mRNA was first detected in nasal regions on Embryonic Day (E) 11.5. From E12.5 to E13.5, robust GAD expression was localized to cells primarily in the ventral aspect of the OP. GAD mRNA was not detected over dorsally located cells in olfactory sensory or respiratory epithelium. In addition, GAD mRNA was not observed in cells along olfactory axons. GAD mRNA was dramatically reduced in the OP/vomeronasal organ by E16.5. Using antibodies against both GABA and GAD, immunopositive axonal-like tracts were detected in the nasal septum on E12.5. GABAergic staining decreased by E13.5. To examine synaptic interactions of these GABAergic cells, embryonic olfactory explants were generated and maintained in serum-free media. As explants spread, neuron-like cells migrated into the periphery, sometimes forming ganglion-like clusters. Cells were recorded, marked intracellularly with Lucifer Yellow and post-fixation, immunocytochemically examined. Forty-six cells, typically multipolar, were GABAergic, had resting potentials around -50 mV, and exhibited spontaneous action potentials which were generated by spontaneous depolarizing GABAergic (GABAA) synaptic activity. OP neurons depolarized in response to GABA by increasing Cl- conductance. The biophysical properties of OP-derived GABAergic neurons were distinct from those reported for olfactory receptor neurons but similar to embryonic LHRH neurons. However, unlike LHRH neurons, GABAergic neurons did not migrate large distances in olfactory explants or appear to leave the olfactory pit in vivo.

Acetylcholine↗
PubMed1996

GABAA receptors modulate early spontaneous excitatory activity in differentiating P19 neurons.

P19 embryonic carcinoma (EC) stem cells are pluripotent and are efficiently induced to differentiate into neurons and glia with retinoic acid (RA) treatment. Within 5 days, a substantial number of differentiating P19 cells express gene products that are characteristic of a neuronal phenotype. P19 neurons were used as a model to explore the relationship between neuronal "differentiation" in vitro and the acquisition of gamma-aminobutyric acid (GABAA) receptors and functional GABA responses. Pulse-labeling experiments using bromodeoxyuridine indicated that all neurons had become postmitotic within 3-4 days after treatment with RA. This was confirmed by a reduction in the immunocytochemical detection of the undifferentiated stem cell antigen SSEA-1. Subsequently, a transient expression of nestin was observed during the first 5 days in vitro (DIV) after exposure to RA. By 5-10 DIV after RA, a significant number of neurons (approximately 80-90%) expressed immunocytochemically detectable glutamate decarboxylase and GABA coincident with the acquisition of membrane binding sites for tetanus toxin. These phenotypic markers were maintained for > 30 DIV after RA. Under current-clamp conditions, random, low-amplitude, spontaneous electrical activity appeared in neurons within the first few days after RA treatment and this was blocked by the specific GABAA receptor antagonist bicuculline. Thereafter, the appearance and progressive increases in the frequency of spontaneous action potentials in P19 neurons were observed that were similarly attenuated by bicuculline. In neurons > 5 DIV after RA, exogenous application of GABA elicited similar action potentials. The onset of excitatory responses to GABA or muscimol in voltage-clamped neurons appeared immediately after the cessation of neuronal mitosis and before the previously reported acquisition of responses to glutamate. In fura-2 imaging studies, the exogenous application of GABA resulted in neuron-specific increases in intracellular Ca2+. Thus, P19 neurons provide an in vitro model for the study of the early acquisition and properties of electrical excitability to GABA and the expression of functional GABAA receptors.

Action Potentials↗
PubMed1996

Purification and characterization of rhesus monkey liver amido hydrolases and their roles in the metabolic polymorphism for E6123, a platelet-activating factor receptor antagonist.

We previously showed that a polymorphism for E6123 [(S)-(+)-6- (2-chlorophenyl)-3-cyclopropanecarbonyl-8,11-dimethyl-2,3,4,5- tetrahydro-8H-pyrido[4',3':4,5]thieno[3,2-f][1,2,4]triazolo[4,3-a] [1,4]diazepine] metabolism exists only in rhesus monkeys. In the present study, we purified, from rhesus monkey hepatic microsomes, three amido hydrolases that are involved in the metabolic polymorphism. Two forms of amido hydrolase from an extensive metabolizer and one from a poor metabolizer were purified by Q-Sepharose Fast Flow, Red A-agarose, octylamino-Sepharose 4B, and hydroxyapatite-Ultrogel chromatography, after solubilization with Lubrol. The three purified enzymes had the same molecular mass (47 kDa), and their amino-terminal amino acid sequences were identical. The enzymes were different from various known carboxylesterases in terms of substrate specificity, molecular mass, and amino-terminal amino acid sequence. They resembled arylacetamide deacetylase from human hepatic microsomes with respect to molecular mass and amino-terminal amino acid sequence. The KM values of the high and low affinity enzymes in the extensive metabolizer and the sole enzyme in the poor metabolizer were 37.6, 73.0, and 76.5 microM, respectively. The Vmax values were 3312.4, 504.8, and 427.9 pmol/min/mg of protein, respectively. The high affinity enzyme in extensive metabolizer appears to be quite distinct, whereas the low affinity enzyme in extensive metabolizer in similar or identical to the sole enzyme in poor metabolizer. Thus, the metabolic polymorphism in rhesus monkey may depend upon the existence of the high affinity enzyme in extensive metabolizer.

Amidohydrolases↗
PubMed1995

Restriction-modification systems as genomic parasites in competition for specific sequences.

Restriction-modification (RM) systems are believed to have evolved to protect cells from foreign DNA. However, this hypothesis may not be sufficient to explain the diversity and specificity in sequence recognition, as well as other properties, of these systems. We report that the EcoRI restriction endonuclease-modification methylase (rm) gene pair stabilizes plasmids that carry it and that this stabilization is blocked by an RM of the same sequence specificity (EcoRI or its isoschizomer, Rsr I) but not by an RM of a different specificity (PaeR7I) on another plasmid. The PaeR7I rm likewise stabilizes plasmids, unless an rm gene pair with identical sequence specificity is present. Our analysis supports the following model for stabilization and incompatibility: the descendants of cells that have lost an rm gene pair expose the recognition sites in their chromosomes to lethal attack by any remaining restriction enzymes unless modification by another RM system of the same specificity protects these sites. Competition for specific sequences among these selfish genes may have generated the great diversity and specificity in sequence recognition among RM systems. Such altruistic suicide strategies, similar to those found in virus-infected cells, may have allowed selfish RM systems to spread by effectively competing with other selfish genes.

Binding, Competitive↗
PubMed1995

Electrical and synaptic properties of embryonic luteinizing hormone-releasing hormone neurons in explant cultures.

Voltage- and ligand-activated channels in embryonic neurons containing luteinizing hormone-releasing hormone (LHRH) were studied by patch-pipette, whole-cell current and voltage clamp techniques. LHRH neurons were maintained in explant cultures derived from olfactory pit regions of embryonic mice. Cells were marked intracellularly with Lucifer yellow following recording. Sixty-two cells were unequivocally identified as LHRH neurons by Lucifer yellow and LHRH immunocytochemistry. The cultured LHRH neurons had resting potentials around -50 mV, exhibited spontaneous discharges generated by intrinsic and/or synaptic activities and contained a time-dependent inward rectifier (Iir). Voltage clamp analysis of ionic currents in the LHRH neuron soma revealed a tetrodotoxin-sensitive Na+ current (INa) and two major types of K+ currents, a transient current (IA), a delayed rectifier current (IK) and low- and high-voltage-activated Ca2+ currents. Spontaneous depolarizing synaptic potentials and depolarizations induced by direct application of gamma-aminobutyrate were both inhibited by picrotoxin or bicuculline, demonstrating the presence of functional gamma-aminobutyrate type A synapses on these neurons. Responses to glutamate were found in LHRH neurons in older cultures. Thus, embryonic LHRH neurons not yet positioned in their postnatal environment in the forebrain contained a highly differentiated repertoire of voltage- and ligand-gated channels.

Action Potentials↗
PubMed1995

Selfish behavior of restriction-modification systems.

Plasmids carrying gene pairs encoding type II DNA restriction endonucleases and their cognate modification enzymes were shown to have increased stability in Escherichia coli. The descendants of cells that had lost these genes appeared unable to modify a sufficient number of recognition sites in their chromosomes to protect them from lethal attack by the remaining restriction enzyme molecules. The capacity of these genes to act as a selfish symbiont is likely to have contributed to the evolution of restriction-modification gene pairs.

Apoptosis↗
PubMed1995

Concentration of cofilin, a small actin-binding protein, at the cleavage furrow during cytokinesis.

Cofilin is a small actin-binding protein which regulates actin polymerization in a pH-dependent manner. Immunofluorescence microscopy with a monoclonal antibody for cofilin revealed that this protein is temporarily concentrated at the contractile ring during cytokinesis. Cofilin appeared to accumulate rapidly at the contractile ring during late stages of furrowing, and was finally enriched at the midbody. The concentration of cofilin at the contractile ring was observed in several kinds of cultured cells. Furthermore, cofilin introduced into living cells by a microinjection method was also concentrated at the contractile ring. These results suggest that cofilin is involved in actin reorganization during cytokinesis.

Actin Cytoskeleton↗