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H Miki

Publications and source records attributed to H Miki.

At least 55 records · Page 3Linked to original sources
PubMed2001

N-WASP, WAVE and Mena play different roles in the organization of actin cytoskeleton in lamellipodia.

WASP- and Ena/VASP-family proteins have been reported to regulate the cortical actin cytoskeleton as downstream effectors of the Rho-family small G-proteins Rac and Cdc42, but their functions are little understood. We observed the localization of the WASP family proteins, N-WASP and WAVE, and the Ena/VASP family protein, Mena, in protruding lamellipodia. Rat fibroblast cell line 3Y1 protruded lamellipodia on poly-L-lysine-coated substrate without any trophic factor. N-WASP and Cdc42 were concentrated along the actin filament bundles of microspikes but not at the tips. By immunofluorescence and immunoelectron microscopy, both WAVE and Mena were observed to localize at the lamellipodium edge. Interestingly, Mena tended to concentrate at the microspike tips but WAVE did not. At the edge of the lamellipodium, the correlation between the fluorescence from Mena and actin filaments stained with the specific antibody and rhodamine-phalloidin, respectively, was much higher than that between WAVE and actin filament. The Ena/VASP homology 2 (EVH2) domain of avian Ena, an avian homolog of Mena, was localized to the lamellipodium edge and concentrated at the tip of microspikes. The SCAR homology domain (SHD) of human WAVE was distributed along the lamellipodium edge. These results indicate that N-WASP, WAVE and Mena have different roles in the regulation of the cortical actin cytoskeleton in the protruding lamellipodium. WAVE and Mena should be recruited to the lamellipodium edge through SHD and the EVH2 domain, respectively, to regulate the actin polymerization near the cell membrane. N-WASP should regulate the formation of the actin filament bundle in addition to activating Arp2/3 complex in lamellipodium under the control of Cdc42.

Actins↗
PubMed2001

Phase transformation of a zirconia ceramic head after total hip arthroplasty.

We report two cases of surface deterioration of a zirconia ceramic femoral head associated with phase transformation after total hip arthroplasty. One head was retrieved at revision due to recurrent dislocation after six years and the other because of failure of the locking mechanism of the polyethylene liner after three years. The monoclinic content of the zirconia ceramics rose from 1% to about 30% on the surface of the heads. SEM revealed numerous craters indicating extraction of the zirconia ceramics at the surface. Surface roughness increased from an initial value of 0.006 microm up to 0.12 microm. This is the first report to show that phase transformation of zirconia ceramics causes deterioration of the surface roughness of the head in vivo after total hip arthroplasty.

Female↗
PubMed2001

Discrimination of brain abscess and cystic tumor by in vivo proton magnetic resonance spectroscopy.

Proton magnetic resonance (MR) spectroscopy was evaluated for the differentiation of brain abscesses and cystic brain tumors. Proton MR spectroscopy was performed in vivo in two patients with brain abscess and eight patients with various cystic brain tumors (anaplastic astrocytoma, glioblastoma, and metastatic brain tumor). MR imaging with contrast medium demonstrated ring-like enhanced mass lesions in all patients. The various resonance peaks in proton MR spectra were assigned to metabolites according to chemical shifts. Treatment of the cystic brain lesions was based on the information from proton MR spectroscopy. Aspirated pus from one patient with brain abscess was examined using ex vivo proton MR spectroscopy. The in vivo spectra of brain abscess contained resonance peaks attributed to acetate, lactate, alanine, amino acids, and lipids in both cases, and an additional peak of succinate in one case. In vivo spectra of the neoplasms contained resonance peaks corresponding to lactate, lipids, choline, creatine, and N-acetyl aspartate. Proton MR spectroscopy is useful for discriminating brain abscess from cystic tumors with similar neuroimaging appearance, which is very important for determining the treatment strategy.

Acetates↗
PubMed2001

Measurement of cerebral hemodynamics with perfusion-weighted MR imaging: comparison with pre- and post-acetazolamide 133Xe-SPECT in occlusive carotid disease.

BACKGROUND AND PURPOSE: We generated regional cerebral blood volume (rCBV) and regional cerebral blood flow (rCBF) studies from dynamic susceptibility contrast-enhanced MR images after an intravenous bolus injection of contrast agent (perfusion-weighted imaging [PWI]) by applying indicator dilution theory. We used a multishot echo-planar imaging (EPI) sequence to obtain adequate arterial input function (AIF). Our purpose was to compare the cerebral hemodynamics measured by PWI with the rCBF values and cerebral perfusion reserve obtained by xenon-133 single-photon emission CT (133Xe-SPECT). METHODS: Eight patients with chronic internal carotid artery occlusion or stenosis were examined. PWI data were acquired using a multishot EPI sequence, and the AIF was determined automatically. Our procedure was based on indicator dilution theory and deconvolution analysis. To eliminate the effect of superficial vessels, the automatic threshold selection method was used. RESULTS: AIF was adequate to generate rCBF and rCBV images. The rCBF and rCBV images by PWI were superior to 133Xe-SPECT scans in spatial resolution, and the rCBF values obtained by PWI correlated well with those obtained by 133Xe-SPECT. The regions with severely decreased perfusion reserve, which were determined by pre- and post-acetazolamide 133Xe-SPECT, showed significantly lower rCBF and higher rCBV by PWI than did regions with normal and moderately decreased perfusion reserve. CONCLUSION: The rCBF and rCBV images generated by our procedure using PWI data appear to provide important clinical information for evaluating the degree of cerebral perfusion reserve impairment in patients with chronic ischemia.

Acetazolamide↗
PubMed2001

Fractal analysis of cerebral blood flow distribution in Alzheimer's disease.

UNLABELLED: This study describes a method for quantifying cerebral blood flow (CBF) distribution in Alzheimer's disease (AD) from SPECT images obtained with (99m)Tc-hexamethylpropyleneamine oxime (HMPAO) by 3-dimensional fractal analysis (3D-FA). METHODS: HMPAO SPECT was performed on 68 patients with probable AD and 14 healthy control subjects. We delineated the CBF images using 4 cutoff levels (35%, 40%, 45%, and 50% of the maximal voxel radioactivity) and measured the total number of voxels in the areas surrounded by the contours obtained with each cutoff level. We calculated fractal dimensions from the relationship between the total number of voxels and the cutoff levels transformed into natural logarithms. RESULTS: The fractal dimensions (mean +/- SD) for patients with probable AD and healthy subjects were 0.74 +/- 0.33 and 0.52 +/- 0.09, respectively. A significant difference in the fractal dimension was found between groups (P = 0.001). Statistically significant correlations were obtained between the fractal dimension and the Mini-Mental State Examination score (r = -0.598; P < 0.0001) and between the fractal dimension and the AD Assessment Scale (r = 0.670; P < 0.0001). The fractal dimensions for subjects with clinical dementia rates (CDRs) of 0, 1, 2, and 3 were 0.52 +/- 0.09, 0.63 +/- 0.21, 0.77 +/- 0.23 (P < 0.05 vs. the group with a CDR of 1), and 1.43 +/- 0.49 (P < 0.0001 vs. the group with a CDR of 2), respectively. CONCLUSION: Quantification of CBF distribution on SPECT images in AD was possible using 3D-FA. The fractal dimension was well correlated with the cognitive impairment, as assessed in neuropsychologic tests. 3D-FA may be a useful method for objectively evaluating the progression of AD.

Aged↗
PubMed2001

Rectus sheath hematoma in an elderly woman under anti-coagulant therapy.

Rectal sheath hematoma has been a well-known clinical entity from the ruin of the ancient Greece. It is relatively rare, however, to encounter this abdominal disorder in the clinical setting. Furthermore, the initial symptoms of rectus sheath hematoma are often similar to those of acute abdominal disorders. Therefore, the majority of the patients with rectus sheath hematoma have been treated with operative procedures because of the difficulty of a differential diagnosis from other abdominal disorders. We recently treated a 74-year female diagnosed with rectus sheath hematoma with the anticoagulants after an episode of cerebral infarction. From the findings of the physical examinations, ultrasound, and computed tomography, we could correctly diagnose, and could treat her with completely conservative methods without any invasive techniques. It is stressed that it is important to recognize this entity of rectus sheath hematoma when patients are examined, after complaining of acute abdominal pain and with evidence abdominal masses in the clinical setting.

Abdomen, Acute↗
PubMed2000

IRSp53 is an essential intermediate between Rac and WAVE in the regulation of membrane ruffling.

Neural Wiskott-Aldrich syndrome protein (N-WASP) functions in several intracellular events including filopodium formation, vesicle transport and movement of Shigella frexneri and vaccinia virus, by stimulating rapid actin polymerization through the Arp2/3 complex. N-WASP is regulated by the direct binding of Cdc42 (refs 7, 8), which exposes the domain in N-WASP that activates the Arp2/3 complex. A WASP-related protein, WAVE/Scar, functions in Rac-induced membrane ruffling; however, Rac does not bind directly to WAVE, raising the question of how WAVE is regulated by Rac. Here we demonstrate that IRSp53, a substrate for insulin receptor with unknown function, is the 'missing link' between Rac and WAVE. Activated Rac binds to the amino terminus of IRSp53, and carboxy-terminal Src-homology-3 domain of IRSp53 binds to WAVE to form a trimolecular complex. From studies of ectopic expression, we found that IRSp53 is essential for Rac to induce membrane ruffling, probably because it recruits WAVE, which stimulates actin polymerization mediated by the Arp2/3 complex.

3T3 Cells↗
PubMed2000

Two tandem verprolin homology domains are necessary for a strong activation of Arp2/3 complex-induced actin polymerization and induction of microspike formation by N-WASP.

All WASP family proteins share a common C terminus that consists of the verprolin homology domain (V), cofilin homology domain (C), and acidic region (A), through which they activate Arp2/3 complex-induced actin polymerization. In this study, we characterized the Arp2/3 complex-mediated actin polymerization activity of VCA fragments of all of the WASP family proteins: WASP, N-WASP, WAVE1, WAVE2, and WAVE3. All of the VCA fragments stimulated the nucleating activity of Arp2/3 complex. Among them, N-WASP VCA, which possesses two tandem V motifs, had a more potent activity than other VCA proteins. The chimeric protein experiments revealed that the V motif was more important to the activation potency than the CA region; two V motifs were required for full activity of N-WASP. COS7 cells overexpressing N-WASP form microspikes in response to epidermal growth factor. However, when a chimeric protein in which the VCA region of N-WASP is replaced with WAVE1 VCA was overexpressed, microspike formation was suppressed. Interestingly, when the N-WASP VCA region was replaced with WAVE1 VCA, having two V motifs, this chimeric protein could induce microspike formation. These results indicate that strong activation of Arp2/3 complex by N-WASP is mainly caused by its two tandem V motifs, which are essential for actin microspike formation.

Actin-Related Protein 2↗
PubMed2000

Constitutive tyrosine phosphorylation of ErbB-2 via Jak2 by autocrine secretion of prolactin in human breast cancer.

Overexpression of the oncogene for ErbB-2 is an unfavorable prognostic marker in human breast cancer. Its oncogenic potential appears to depend on the state of tyrosine phosphorylation. However, the mechanisms by which ErbB-2 is constitutively tyrosine-phosphorylated in human breast cancer are poorly understood. We now show that human breast carcinoma samples with ErbB-2 overexpression have higher proliferative and metastatic activity in the presence of autocrine secretion of prolactin (PRL). By using a neutralizing antibody or dominant negative (DN) strategies or specific inhibitors, we also show that activation of Janus kinase Jak2 by autocrine secretion of PRL is one of the significant components of constitutive tyrosine phosphorylation of ErbB-2, its association with Grb2 and activation of mitogen-activated protein (MAP) kinase in human breast cancer cell lines that overexpress ErbB-2. Furthermore, the neutralizing anti-PRL antibody or erbB-2 antisense oligonucleotide or DN Jak2 or Jak2 inhibitor or DNRas or MAP kinase kinase inhibitor inhibits the proliferation of both untreated and PRL-treated cells. Our results indicate that autocrine secretion of PRL stimulates tyrosine phosphorylation of ErbB-2 by Jak2, provides docking sites for Grb2 and stimulates Ras-MAP kinase cascade, thereby causing unrestricted cellular proliferation. The identification of this novel cross-talk between ErbB-2 and the autocrine growth stimulatory loop for PRL may provide new targets for therapeutic and preventive intervention of human breast cancer.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Profilin is required for sustaining efficient intra- and intercellular spreading of Shigella flexneri.

The ability of Shigella to mediate actin-based motility within the host cell is a prominent pathogenic feature of bacillary dysentery. The ability is dependent on the interaction of VirG with neural Wiskott-Aldrich syndrome protein (N-WASP), which in turn mediates recruitment of Arp2/3 complex and several actin-related proteins. In the present study, we show that profilin I is essential to the rapid movement of Shigella in epithelial cells, for which the capacity of profilin to interact with G-actin and N-WASP is critical. In COS-7 cells overexpressing either mutated profilin H119E, which failed to bind G-actin, or H133S, which is unable to interact with poly-l-proline, Shigella motility was significantly inhibited. Similarly, depletion of profilin from Xenopus egg extracts resulted in a decrease in bacterial motility that was completely rescued by adding back profilin I but not H119E or H133S. In COS-7 cells overexpressing a N-WASP mutant lacking the proline-rich domain (Deltap) unable to interact with profilin, the actin tail formation of intracellular Shigella was inhibited. In N-WASP-depleted extracts, addition of Deltap but not full-length N-WASP was unable to restore the bacterial motility. Furthermore, in a plaque formation assay with Madin-Darby canine kidney cell monolayers infected by Shigella, Madin-Darby canine kidney cells stably expressing H119E, H133S, or Deltap reduced the bacterial cell-to-cell spreading. These results indicate that profilin I associated with N-WASP is an essential host factor for sustaining efficient intra- and intercellular spreading of Shigella.

Actins↗
PubMed2000

Profilin enhances Cdc42-induced nucleation of actin polymerization.

We find that profilin contributes in several ways to Cdc42-induced nucleation of actin filaments in high speed supernatant of lysed neutrophils. Depletion of profilin inhibited Cdc42-induced nucleation; re-addition of profilin restored much of the activity. Mutant profilins with a decreased affinity for either actin or poly-l-proline were less effective at restoring activity. Whereas Cdc42 must activate Wiskott-Aldrich Syndrome protein (WASP) to stimulate nucleation by the Arp2/3 complex, VCA (verpolin homology, cofilin, and acidic domain contained in the COOH-terminal fragment of N-WASP) constitutively activates the Arp2/3 complex. Nucleation by VCA was not inhibited by profilin depletion. With purified N-WASP and Arp2/3 complex, Cdc42-induced nucleation did not require profilin but was enhanced by profilin, wild-type profilin being more effective than mutant profilin with reduced affinity for poly-l-proline. Nucleation by the Arp2/3 complex is a function of the free G-actin concentration. Thus, when profilin addition decreased the free G-actin concentration, it inhibited Cdc42- and VCA-induced nucleation. However, when profilin was added with G-actin in a ratio that maintained the initial free G-actin concentration, it increased the rate of both Cdc42- and VCA-induced nucleation. This enhancement, also seen with purified proteins, was greatest when the free G-actin concentration was low. These data suggest that under conditions present in intact cells, profilin enhances nucleation by activated Arp2/3 complex.

Actins↗
PubMed2000

Arp2/3 complex-independent actin regulatory function of WAVE.

We report that WAVE1/Scar1, a WASP-family protein that functions downstream of Rac in membrane ruffling, can induce part of the reorganization of the actin cytoskeleton without Arp2/3 complex. WAVE1 has been reported to associate and activate Arp2/3 complex at its C-terminal region that is rich in acidic residues. The deletion of the acidic residues abolished the interaction with and the activation ability of Arp2/3 complex. The expression of the mutant WAVE1 lacking the acidic residues (DeltaA), however, induced actin-clustering in cells as the wild-type WAVE1 did. In addition, this actin-clustering could not be suppressed by the coexpression of the Arp2/3 complex-sequestering fragment (CA-region) derived from N-WASP, which clearly inhibits Rac-induced membrane ruffling. This study therefore demonstrates that WAVE1 reorganizes the actin cytoskeleton not only through Arp2/3 complex but also through another unidentified mechanism that may be important but has been neglected thus far.

3T3 Cells↗
PubMed2000

Rho family GTPase Cdc42 is essential for the actin-based motility of Shigella in mammalian cells.

Shigella, the causative agent of bacillary dysentery, is capable of directing its movement within host cells by exploiting actin dynamics. The VirG protein expressed at one pole of the bacterium can recruit neural Wiskott-Aldrich syndrome protein (N-WASP), a downstream effector of Cdc42. Here, we show that Cdc42 is required for the actin-based motility of Shigella. Microinjection of a dominant active mutant Cdc42, but not Rac1 or RhoA, into Swiss 3T3 cells accelerated Shigella motility. In add-back experiments in Xenopus egg extracts, addition of a guanine nucleotide dissociation inhibitor for the Rho family, RhoGDI, greatly diminished the bacterial motility or actin assembly, which was restored by adding activated Cdc42. In N-WASP-depleted extracts, the bacterial movement almost arrested was restored by adding exogenous N-WASP but not H208D, an N-WASP mutant defective in binding to Cdc42. In pyrene actin assay, Cdc42 enhanced VirG-stimulating actin polymerization by N-WASP-actin-related protein (Arp)2/3 complex. Actually, Cdc42 stimulated actin cloud formation on the surface of bacteria expressing VirG in a solution containing N-WASP, Arp2/3 complex, and G-actin. Immunohistological study of Shigella-infected cells expressing green fluorescent protein-tagged Cdc42 revealed that Cdc42 accumulated by being colocalized with actin cloud at one pole of intracellular bacterium. Furthermore, overexpression of H208D mutant in cells interfered with the actin assembly of infected Shigella and diminished the intra- and intercellular spreading. These results suggest that Cdc42 activity is involved in initiating actin nucleation mediated by VirG-N-WASP-Arp2/3 complex formed on intracellular Shigella.

3T3 Cells↗
PubMed2000

Essential role of neural Wiskott-Aldrich syndrome protein in neurite extension in PC12 cells and rat hippocampal primary culture cells.

Neural Wiskott-Aldrich syndrome protein (N-WASP) is an actin-regulating protein that induces filopodium formation downstream of Cdc42. It has been shown that filopodia actively extend from the growth cone, a guidance apparatus located at the tip of neurites, suggesting their role in neurite extension. Here we examined the possible involvement of N-WASP in the neurite extension process. Since verprolin, cofilin homology and acidic region (VCA) of N-WASP is known to be required for the activation of Arp2/3 complex that induces actin polymerization, we prepared a mutant (Deltacof) lacking four amino acid residues in the cofilin homology region. The corresponding residues in WASP had been reported to be mutated in some Wiskott-Aldrich syndrome patients. Expression of Deltacof N-WASP suppressed neurite extension of PC12 cells. In support of this, the VCA region of Deltacof cannot activate Arp2/3 complex enough compared with wild-type VCA. Furthermore, H208D mutant, which has been shown unable to bind to Cdc42, also works as a dominant negative mutant in neurite extension assay. Interestingly, the expression of H208D-Deltacof double mutant has no significant dominant negative effect. Finally, the expression of the Deltacof mutant also severely inhibited the neurite extension of primary neurons from rat hippocampus. Thus, N-WASP is thought to be a general regulator of the actin cytoskeleton indispensable for neurite extension, which is probably caused through Cdc42 signaling and Arp2/3 complex-induced actin polymerization.

Actin Depolymerizing Factors↗
PubMed2000

Caspase-3 inhibitor rescues N -methyl- N -nitrosourea-induced retinal degeneration in Sprague-Dawley rats.

The effect of a caspase-3 inhibitor on N -methyl- N -nitrosourea (MNU)-induced retinal degeneration was investigated. Sixty mg kg(-1)MNU was given intraperitoneally to 50 day old female Sprague-Dawley rats, and 4000 ng Ac-DEVD-CHO, a caspase-3 inhibitor, was injected intravitreally twice at 0 and 10 hr after MNU. In both peripheral and central retina, an apoptotic index of the photoreceptor cells 24 hr after MNU treatment was calculated by TUNEL labeling, and retinal damage 7 days after MNU treatment was evaluated from retinal thickness and a retinal damage ratio (length of damaged retina : whole retinal length). In MNU-treated rats, the TUNEL index 24 hr post-MNU was 79.5% in the peripheral and 83.7% in the central retina, while the Ac-DEVD-CHO injection significantly reduced it to 59.7 and 71.8%, respectively. Total retinal thickness 7 days after MNU was 38 microm in the peripheral and 75 microm in the central retina. Ac-DEVD-CHO injection increased these values to 72 and 77 microm, respectively. The retinal damage ratio 7 days after MNU was 98.5%. Ac-DEVD-CHO injection significantly reduced this value to 54.4%. The use of a caspase-3 inhibitor was effective in the suppression of MNU-induced retinal apoptosis and may be a therapeutic intervention in human retinitis pigmentosa.

Animals↗
PubMed2000

Arthrographic examination of the pseudocapsule of the hip after posterior dislocation of total hip arthroplasty.

We examined the pseudocapsule in ten patients with posterior dislocation after total hip arthroplasty (THA) using arthrography to investigate the relationship between dislocation and the state of the pseudocapsule. The pattern of contrast medium pooling demonstrated on tangential views was evaluated. In five patients with early dislocation arthrography showed leakage indicating lack of pseudocapsule whereas there was no leakage in five patients with late dislocation. Repeat arthrography revealed healing of the pseudocapsule could progress by 6-8 weeks after dislocation. These findings suggest that the difference in the pseudocapsule between those who dislocate early and late may explain different clinical outcomes in the two groups.

Arthrography↗
PubMed2000

Intrathyroidal branchial cleft-like cyst in chronic thyroiditis.

An extremely rare case of intrathyroidal branchial cleft-like cyst is reported. A 71-year-old man complained of a growing mass in the right lateral neck. A cystic mass in the upper lobe of the right thyroid was demonstrated by ultrasonography and computed tomography. The surgical specimen revealed a cystic mass with dense fibrous capsule, 22 x 20 x 10 mm in size. Microscopically, the cyst walls and the surrounding thyroid tissue contained severe lymphoid cell infiltration with lymphoid follicle. Squamous epithelium lined the cyst wall. Immunohistochemically, squamous epithelium was positive for keratin, cytokeratin 19, carcinoembryonic antigen, and epithelial membrane antigen, but negative for calcitonin and chromogranin A. The patient is currently well with no evidence or recurrence for 43 months.

Aged↗