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Biomedical subjects

K Ito

Publications and source records attributed to K Ito.

At least 163 records · Page 9Linked to original sources
PubMed2001

Deficiency of triad junction and contraction in mutant skeletal muscle lacking junctophilin type 1.

In skeletal muscle excitation-contraction (E-C) coupling, the depolarization signal is converted from the intracellular Ca2+ store into Ca2+ release by functional coupling between the cell surface voltage sensor and the Ca2+ release channel on the sarcoplasmic reticulum (SR). The signal conversion occurs in the junctional membrane complex known as the triad junction, where the invaginated plasma membrane called the transverse-tubule (T-tubule) is pinched from both sides by SR membranes. Previous studies have suggested that junctophilins (JPs) contribute to the formation of the junctional membrane complexes by spanning the intracellular store membrane and interacting with the plasma membrane (PM) in excitable cells. Of the three JP subtypes, both type 1 (JP-1) and type 2 (JP-2) are abundantly expressed in skeletal muscle. To examine the physiological role of JP-1 in skeletal muscle, we generated mutant mice lacking JP-1. The JP-1 knockout mice showed no milk suckling and died shortly after birth. Ultrastructural analysis demonstrated that triad junctions were reduced in number, and that the SR was often structurally abnormal in the skeletal muscles of the mutant mice. The mutant muscle developed less contractile force (evoked by low-frequency electrical stimuli) and showed abnormal sensitivities to extracellular Ca2+. Our results indicate that JP-1 contributes to the construction of triad junctions and that it is essential for the efficiency of signal conversion during E-C coupling in skeletal muscle.

Animals↗
PubMed2001

Transgenic expression of sarcoplasmic reticulum Ca(2+) atpase modifies the transition from hypertrophy to early heart failure.

To examine the contribution of sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a) to early heart failure, we subjected transgenic (TG) mice expressing SERCA2a gene and wild-type (WT) mice to aortic stenosis (AS) for 7 weeks. At an early stage of hypertrophy (4-week AS), in vivo hemodynamic and echocardiographic indices were similar in TG and WT mice. By 7 weeks of AS, which is the stage of early failure in this model, TG mice with AS had lower mortality than WT mice with AS (6.7% versus 29%). The magnitude of left ventricular (LV) hypertrophy was similar in WT and TG 7-week AS mice. In vivo LV systolic function was higher in TG than in WT 7-week AS mice. In LV myocytes loaded with fluo-3, fractional cell shortening and the amplitude of the [Ca(2+)](i) transients were higher in TG than in WT 7-week AS mice under baseline conditions (0.5 Hz, 1.5 mmol/L [Ca(2+)](o), 25 degrees C). The rates of relengthening and decay in [Ca(2+)](i) were faster in TG than in WT 7-week AS myocytes. In myocytes from WT 7-week AS compared with sham-operated WT mice, contractile reserve in response to rapid pacing was depressed with impaired augmentation of both peak-systolic [Ca(2+)](i) and the SR Ca(2+) load. In contrast, contractile reserve and the capacity to augment SR Ca(2+) load were maintained in TG 7-week AS mice. SERCA2a protein levels were depressed in WT 7-week AS mice, but were preserved in TG 7-week AS mice. These data suggest that defective SR Ca(2+) loading contributes to the onset of contractile failure in animals with chronic pressure overload.

Animals↗
PubMed2001

Functional expression of a chimeric myosin-containing motor domain of Chara myosin and neck and tail domains of Dictyostelium myosin II.

We succeeded in expressing a chimeric myosin that comprises the motor domain of characean algal myosin, (the fastest known motor protein), and the neck and tail domains of Dictyostelium myosin II. Although the chimeric myosin showed an ATPase activity comparable to that of muscle myosin (15 times higher than that of the wild-type Dictyostelium myosin II), the motile activity of the chimera was only 1.3 times higher than that of the wild-type. However, this is the first chimeric myosin that showed motile activity faster than at least one of the parent myosins. It was suggested, therefore, that the motor domain of Chara myosin has the potential for performing fast sliding movement.

Actins↗
PubMed2001

Mutation of Trp1254 in the multispecific organic anion transporter, multidrug resistance protein 2 (MRP2) (ABCC2), alters substrate specificity and results in loss of methotrexate transport activity.

The ATP-binding cassette (ABC) proteins comprise a large superfamily of transmembrane transporters that utilize the energy of ATP hydrolysis to translocate their substrates across biological membranes. Multidrug resistance protein (MRP) 2 (ABCC2) belongs to subfamily C of the ABC superfamily and, when overexpressed in tumor cells, confers resistance to a wide variety of anticancer chemotherapeutic agents. MRP2 is also an active transporter of organic anions such as methotrexate (MTX), estradiol glucuronide (E217betaG), and leukotriene C4 and is located on the apical membrane of polarized cells including hepatocytes where it acts as a biliary transporter. We recently identified a highly conserved tryptophan residue in the related MRP1 that is critical for the substrate specificity of this protein. In the present study, we have examined the effect of replacing the analogous tryptophan residue at position 1254 of MRP2. We found that only nonconservative substitutions (Ala and Cys) of Trp1254 eliminated [3H]E217betaG transport by MRP2, whereas more conservative substitutions (Phe and Tyr) had no effect. In addition, only the most conservatively substituted mutant (W1254Y) transported [3H]leukotriene C4, whereas all other substitutions eliminated transport of this substrate. On the other hand, all substitutions of Trp1254 eliminated transport of [3H]MTX. Finally, we found that sulfinpyrazone stimulated [3H]E217betaG transport by wild-type MRP2 4-fold, whereas transport by the Trp1254 substituted mutants was enhanced 6-10-fold. In contrast, sulfinpyrazone failed to stimulate [3H]MTX transport by either wild-type MRP2 or the MRP2-Trp1254 mutants. Taken together, our results demonstrate that Trp1254 plays an important role in the ability of MRP2 to transport conjugated organic anions and identify this amino acid in the putative last transmembrane segment (TM17) of this ABC protein as being critical for transport of MTX.

Amino Acid Sequence↗
PubMed2001

"Devil's staircase"-type phase transition in NaV2O5 under high pressure.

The "devil's staircase"-type phase transition in the quarter-filled spin-ladder compound NaV2O5 has been discovered at low temperature and high pressure by synchrotron radiation x-ray diffraction. A large number of transitions are found to successively take place among higher-order commensurate phases with 2a x 2b x zc type superstructures. The observed temperature and pressure dependence of modulation wave number q(c), defined by 1/z, is well reproduced by the axial next nearest neighbor Ising model. The q(c) is suggested to reflect atomic displacements presumably coupled with charge ordering in this system.

Journal Article↗
PubMed2001

The mechanism of tryptophan induction of tryptophanase operon expression: tryptophan inhibits release factor-mediated cleavage of TnaC-peptidyl-tRNA(Pro).

Expression of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and tryptophan-induced transcription antitermination. In a previous study, we reproduced the regulatory features of this operon observed in vivo by using an in vitro S-30 system. We also found that, under inducing conditions, the leader peptidyl-tRNA (TnaC-peptidyl-tRNA(Pro)) is not cleaved; it accumulates in the S-30 reaction mixture. In this paper, we examine the requirements for TnaC-peptidyl-tRNA(Pro) accumulation and cleavage, in vitro. We show that this peptidyl-tRNA remains bound to the translating ribosome. Removal of free tryptophan and addition of release factor 1 or 2 leads to hydrolysis of TnaC-peptidyl-tRNA(Pro) and release of TnaC from the ribosome-mRNA complex. Release factor-mediated cleavage is prevented by the addition of tryptophan. TnaC of the ribosome-bound TnaC-peptidyl-tRNA(Pro) was transferable to puromycin. This transfer was also blocked by tryptophan. Tests with various tryptophan analogs as substitutes for tryptophan revealed the existence of strict structural requirements for tryptophan action. Our findings demonstrate that the addition of tryptophan to ribosomes bearing nascent TnaC-peptidyl-tRNA(Pro) inhibits both TnaC peptidyl-tRNA(Pro) hydrolysis and TnaC peptidyl transfer. The associated translating ribosome therefore remains attached to the leader transcript where it blocks Rho factor binding and subsequent transcription termination.

Bacterial Proteins↗
PubMed2001

Real-time observation of micrometastasis formation in the living mouse liver using a green fluorescent protein gene-tagged rat tongue carcinoma cell line.

Initial arrest, attachment, extravasation and subsequent extravascular growth of tumor cells in the secondary organs are believed to be crucial events for hematogenous metastasis, but the actual processes in living animals remain unclear. For the present study, we established green fluorescent protein (GFP)-expressing rat tongue carcinoma cell lines (RSC3) that permit real-time analysis of micrometastasis formation in combination with intravital video microscopy (IVVM). With this system, GFP-expressing metastatic (LM-EGFP) and non-metastatic (E2-EGFP) cell lines could be visualized at the cellular level in live mice for more than 1 month. Real-time IVVM analysis of liver metastases after intraportal injection of cells via a mesenteric vein revealed that both LM-EGFP and E2-EGFP tumor cells arrest similarly in sinusoidal vessels near terminal portal venules within 0.4 sec, during which time no evidence of a "rolling"-like movement along endothelial cell surfaces was observed. Quantitative analysis of GFP-positive foci showed that E2-EGFP cells were completely sheared from the liver sinusoid within 3 days, with no solitary dormant cells, whereas a substantial number of LM-EGFP cells remained in the liver, probably due to stable attachment to the sinusoidal wall. Confocal laser scanning microscopic study in combination with laminin immunohistochemistry revealed that only LM-EGFP cells started growth at 3 to 4 days after inoculation and that most of the growing foci were surrounded by subsinusoidal basement membrane. Our results suggest that micrometastasis formation by LM-EGFP cells consists of initial tumor cell arrest due to size constraints of the vessel, stable attachment to subsinusoidal basement membrane and subsequent intravascular growth before extravasation. The difference in metastatic potential between the 2 lines may reside in their capacity to attach stably to the vessel wall rather than their potential for initial cell arrest or subsequent growth. The system used in the present study may be a powerful tool for analyzing targets for various anti-metastatic agents in the sequential process of metastasis.

Animals↗
PubMed2001

Dynamics and post-collision interaction effects in two electron decay from the Xenon 4d hole.

Two Auger electrons, one very slow, one fast, have been detected in coincidence following near threshold 4d photoionization of the Xe atom. The distribution in the energy the two electrons share has been measured for the first time revealing the presence of post-collision interaction effects that provide unique information on the decay dynamics of the 4d hole. Analysis of the distorted line shapes indicates that the dominant process is decay of Xe+(4d(-1)) to Xe3+ through cascade emission of a zero kinetic energy Auger electron followed by a fast Auger electron. The widths of the intermediate Xe2+* states are estimated to be about 60 meV.

Journal Article↗
PubMed2001

Roles of homooligomerization and membrane association in ATPase and proteolytic activities of FtsH in vitro.

Escherichia coli FtsH is a membrane-bound and ATP-dependent protease which degrades some soluble and integral membrane proteins. The N-terminal region of FtsH mediates membrane association as well as homooligomeric interaction of this enzyme. Previously, we studied in vivo functionality of FtsH derivatives, in which the N-terminal membrane region was either deleted (FtsH(DeltaTM)), replaced by a leucine zipper (Zip-FtsH(DeltaTM)), or replaced by a lactose permease transmembrane segment (LacY-FtsH). It was indicated that homooligomerization is required for the minimum proteolytic activity, whereas a transmembrane sequence is required for membrane protein degradation. Here we characterized these proteins in vitro. Although these mutant enzymes were very low in their activities, they were significantly stimulated by dimethyl sulfoxide, which enabled us to characterize their activities. LacY-FtsH degraded both soluble and membrane proteins, but Zip-FtsH(DeltaTM) only degraded soluble proteins. These proteins also exhibited significant ATPase activities. However, FtsH(DeltaTM) remained inactive both in ATPase and in protease activities even in the presence of dimethyl sulfoxide. The monomeric FtsH(DeltaTM) was able to bind ATP and a denatured protein. These results indicate that subunit association is important for the enzymatic catalysis by FtsH and that the additional presence of the transmembrane sequence is required for this enzyme to degrade a membrane protein even under detergent-solubilized conditions.

ATP-Dependent Proteases↗
PubMed2001

Spontaneously active GABAergic interneurons in the subfornical organ of rat slice preparations.

Inhibitory postsynaptic currents (IPSCs) were recorded from subfornical organ (SFO) neurons in slice preparations of rats, using whole-cell voltage clamp techniques. Some SFO neurons showed bimodal distributions in amplitude with the large and small IPSCs. The large IPSCs vanished in the tetrodotoxin perfusion medium, but the small did not. Both sizes of the IPSCs were completely abolished by application of bicuculline and picrotoxin. Further subpopulation of SFO neurons with the bimodal distributions showed intermittent bursts of the large IPSCs. Immunohistochemical approach revealed existence of gamma-aminobutyric acid (GABA)-immunoreactive neurons and axons in the SFO. These suggest that spontaneously-active and intermittently-burst-firing GABA interneurons affect other SFO neurons in slice preparations of rats.

Animals↗
PubMed2001

Preliminary crystallographic study of Thermus aquaticus glycerol kinase.

Glycerol kinase (GlpK) is an important enzyme which catalyzes the rate-limiting step in a central biochemical pathway involving glycerol metabolism. GlpK from the thermophile Thermus aquaticus has been overexpressed in glpK-deficient Escherichia coli and crystallized by the hanging-drop method. The crystal belongs to the cubic space group I23, with unit-cell parameters a = b = c = 163.94 (3) A. Native data were collected to 2.87 A resolution on a Cu Kalpha rotating-anode X-ray source.

Crystallization↗
PubMed2001

Isotachophoretic separation behavior of rare-earth EDTA chelates and analysis of minor rare-earth elements in an iron ore by bidirectional isotachophoresis-particle-induced X-ray emission.

Mobilities of 16 anions of rare-earth-EDTA 1:1 chelate (RE-EDTAs) were isotachophoretically measured by using two leading electrolytes (pH 3.6 and 6.0) in order to assess their separation behavior. The leading electrolyte was 20 mM hydrochloric acid. The pH of the solution was adjusted to 3.6 by adding beta-alanine and to 6.0 by adding histidine. The obtained mobilities were very close to each other in the range 20.1x10(-5)-21.9x10(-5) cm2 V(-1) s(-1) with the minimum mobilities for Pr-EDTA and Nd-EDTA for pH 3.6 and 6.0, respectively, and pH dependence was hardly observed. On the basis of the above knowledge. minor rare-earth elements in a standard iron ore sample were determined as RE-EDTAs by bidirectional isotachophoresis-particle-induced X-ray emission (PIXE), where the Fe(II) matrix digested by alkali fusion was separated as Fe(II)Phen3(2+) (phen = 1,10-phenanthroline). Since 5% of the total iron was still detected as Fe(III)EDTA- and might disturb PIXE analysis of RE-EDTA-, itaconic acid was used as the spacer for Fe(III)EDTA- and RE-EDTA-. The fractions of RE-EDTA- were successfully analyzed off-line by a multielemental analytical method, PIXE [analytical result (3.62% (w/w) as RE2O3]; the nominal value was 3.37% (w/w) as RExOy.

Chelating Agents↗
PubMed2001

2,4,5-Trichlorophenoxyacetic acid inhibits apoptosis in PC12 cells.

2,4,5-Trichlorophenoxyacetic acid (2,4,5-T) is an endocrine disrupter that exerts cytotoxic effects on organisms. In this study, the influence of 2,4,5-T at low concentrations on apoptosis in PC 12 cells was investigated. Although no apoptotic features were observed in PC12 cells treated with 2,4,5-T, it inhibited the DNA fragmentation induced by serum deprivation. In addition, the cell viability of PC12 cells increased after treatment with 2,4,5-T. In conclusion, 2,4,5-T suppressed the apoptosis of the cultured cells. Since apoptosis is a morphological and biochemical description of a physiological mechanism of cell death that is commonly associated with programmed events necessary for development of individuals and organs, the inhibitory effect of 2,4,5-T on apoptosis might cause serious damage to cell homeostasis and differentiation.

2,4,5-Trichlorophenoxyacetic Acid↗
PubMed2001

Identification of susceptibility loci for autoimmune thyroid disease to 5q31-q33 and Hashimoto's thyroiditis to 8q23-q24 by multipoint affected sib-pair linkage analysis in Japanese.

Autoimmune thyroid disease (AITD), including Graves' disease (GD) and Hashimoto's thyroiditis (HT), is caused by multiple genetic and environmental factors. The clinical and immunological features of GD and HT are distinct; however, there are multiplex families with both GD and HT, and cases in which GD evolves into HT. Thus, there may be specific susceptibility loci for GD or HT, and common loci controlling the susceptibility to both GD and HT may exist. A genome-wide analysis of data on 123 Japanese sib-pairs affected with AITD was made in which GD- or HT-affected sib-pairs (ASPs) were studied to detect GD- or HT-specific susceptibility loci, and all AITD-ASPs were used to detect AITD-common susceptibility loci. Our study revealed 19 regions on 14 chromosomes (1, 2, 3, 5, 6, 8, 9, 10, 11, 12, 13, 15, 18 and 22) where the multipoint maximum LOD score (MLS) was >1. Especially, chromosome 5q31-q33 yielded suggestive evidence for linkage to AITD as a whole, with an MLS of 3.14 at D5S436, and chromosome 8q23-q24 yielded suggestive evidence for linkage to HT, with an MLS of 3.77 at D8S272. These observations suggest the presence of an AITD susceptibility locus at 5q31-q33 and a HT susceptibility locus at 8q23-q24.

Abatacept↗
PubMed2001

An enhanced mutant of red fluorescent protein DsRed for double labeling and developmental timer of neural fiber bundle formation.

We developed a new variant of coral-derived red fluorescent protein, DsRed S197Y, which is brighter and essentially free from secondary fluorescence peak. This makes it an ideal reporter for double labeling with green fluorescent protein (GFP). Though purified protein shows only 20% stronger fluorescence emission, culture cells that express DsRed S197Y exhibit a 3-3.5 times higher level of fluorescence than the cells that express wild-type DsRed. The much slower fluorescence maturation of DsRed than that of GFP is a beneficial feature for a fluorescent developmental timer application. When GFP and DsRed S197Y are expressed simultaneously, emissions start at different latency. This provides information about the time after the onset of expression. It reflects the order of cell differentiation if the expression is activated upon differentiation of certain types of cells. We applied this system to the developing brain of Drosophila and visualized, for the first time, the formation order of neural fibers within a large bundle. Our results showed that newly extending fibers of the mushroom body neurons mainly run into the core rather than to the periphery of the existing bundle. DsRed-based timer thus presents an indispensable tool for developmental biology and genetics of model organisms.

Animals↗
PubMed2001

Enhancement of the thermal stability of pyroglutamyl peptidase I by introduction of an intersubunit disulfide bond.

From the comparison of the three-dimensional structure of mesophilic pyroglutamyl peptidase from Bacillus amyloliquefaciens and the thermophilic enzyme from Thermococcus litoralis, the intersubunit disulfide bond was estimated to be one of the factors for thermal stability. Since Ser185 was corresponded to Cys190 of the thermophilic enzyme by sequence alignment, the Ser185 residue was replaced with cysteine by site-directed mutagenesis. The S185C mutant enzyme appeared to form a disulfide bond, which was confirmed by SDS-PAGE with and without 2-mercaptoethanol. The mutant enzyme showed a catalytic efficiency equivalent to that of the wild-type enzyme for hydrolysis of a synthetic peptide substrate. However, the thermal stability of the S185C mutant was found to be 30 degrees C higher than that of wild-type. Thus the introduction of a disulfide bond enhanced thermal stability without changing the catalytic efficiency of the enzyme.

Amino Acid Sequence↗
PubMed2001

p65-activated histone acetyltransferase activity is repressed by glucocorticoids: mifepristone fails to recruit HDAC2 to the p65-HAT complex.

Glucocorticoids acting through their specific receptor can either enhance or repress gene transcription. Dexamethasone represses interleukin-1beta-stimulated histone acetylation and granulocyte-macrophage colony-stimulating factor expression through a combination of direct inhibition of p65-associated histone acetyltransferase (HAT) activity and by recruiting histone deacetylase 2 (HDAC2) to the p65-HAT complex. Here we show that mifepristone, a glucocorticoid receptor partial agonist, has no ability to induce gene expression but represses interleukin-1beta-stimulated histone acetylation and granulocyte-macrophage colony-stimulating factor release by 50% maximally. Mifepristone was able to inhibit p65-associated HAT activity to the same extent as dexamethasone but failed to inhibit the natural promoter to an equal extent due to an inability to recruit HDAC2 to the p65-associated HAT complex. These data suggest that the maximal repressive actions of glucocorticoids require recruitment of HDAC2 to a p65-HAT complex. These data also suggest that pharmacological manipulation of specific histone acetylation status is a potentially useful approach for the treatment of inflammatory diseases.

Acetylation↗