Biomedical subjects

H Itoh

Publications and source records attributed to H Itoh.

At least 73 records · Page 4Linked to original sources

PubMed2001

Resolution of distinct rotational substeps by submillisecond kinetic analysis of F1-ATPase.

The enzyme F1-ATPase has been shown to be a rotary motor in which the central gamma-subunit rotates inside the cylinder made of alpha3beta3 subunits. At low ATP concentrations, the motor rotates in discrete 120 degrees steps, consistent with sequential ATP hydrolysis on the three beta-subunits. The mechanism of stepping is unknown. Here we show by high-speed imaging that the 120 degrees step consists of roughly 90 degrees and 30 degrees substeps, each taking only a fraction of a millisecond. ATP binding drives the 90 degrees substep, and the 30 degrees substep is probably driven by release of a hydrolysis product. The two substeps are separated by two reactions of about 1 ms, which together occupy most of the ATP hydrolysis cycle. This scheme probably applies to rotation at full speed ( approximately 130 revolutions per second at saturating ATP) down to occasional stepping at nanomolar ATP concentrations, and supports the binding-change model for ATP synthesis by reverse rotation of F1-ATPase.

Adenosine Triphosphate↗

PubMed2001

Parallel regulation of mitogen-activated protein kinase kinase 3 (MKK3) and MKK6 in Gq-signaling cascade.

Heterotrimeric G protein G(q) stimulates the activity of p38 mitogen-activated protein kinase (MAPK) in mammalian cells. To investigate the signaling mechanism whereby alpha and betagamma subunits of G(q) activate p38 MAPK, we introduced kinase-deficient mutants of mitogen-activated protein kinase kinase 3 (MKK3), MKK4, and MKK6 into human embryonal kidney 293 cells. The activation of p38 MAPK by Galpha(q) and Gbetagamma was blocked by kinase-deficient MKK3 and MKK6 but not by kinase-deficient MKK4. In addition, Galpha(q) and Gbetagamma stimulated MKK3 and MKK6 activities. The MKK3 and MKK6 activations by Galpha(q), but not by Gbetagamma, were dependent on phospholipase C and c-Src. Galpha(q) stimulated MKK3 in a Rac- and Cdc42-dependent manner and MKK6 in a Rho-dependent manner. On the other hand, Gbetagamma activated MKK3 in a Rac- and Cdc42-dependent manner and MKK6 in a Rho-, Rac-, and Cdc42-dependent manner. Gbetagamma-induced MKK3 and MKK6 activations were dependent on a tyrosine kinase other than c-Src. These results suggest that Galpha(q) and Gbetagamma stimulate the activity of p38 MAPK by regulating MKK3 and MKK6 through parallel signaling pathways.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed2001

Amyloid beta protein precursor is involved in the growth of human colon carcinoma cell in vitro and in vivo.

Amyloid beta protein precursor (APP) is a membrane-bound protein ubiquitously expressed in a variety of types of cells. However, its biological functions remain largely uncertain, particularly in non-neural cells and tumors. Our previous studies revealed that a secreted form of APP having a Kunitz-type inhibitor domain is a major serine proteinase inhibitor secreted by human colon carcinoma cells. In our study, we used an antisense RNA strategy to selectively inhibit the expression of APP in the human colon carcinoma cell line SW837. A vector capable of expressing an antisense mRNA complementary to 911 bases of the 5' end of APP mRNA was transfected into SW837 cells. After selection, 2 stably transfected antisense clones were obtained in which both the APP protein and mRNA were significantly suppressed. The proliferative potential and colony-forming efficiency of the antisense clones in vitro were markedly suppressed compared with the parent and mock-transfected clones. The addition of the conditioned medium of parent cells or purified secretory APP enabled these antisense effects to be overcome in vitro. The suppressed growth was also observed in vivo when the cells were injected subcutaneously into nude mice. Histologically, formation of tubular structures appeared to be suppressed in the antisense clones in vivo. These observations suggest potentially important roles of APP in cellular proliferation and differentiation of colon carcinoma cells.

Amyloid beta-Protein Precursor↗

PubMed2001

Induction of mitochondrial heat shock protein 60 and 10 mRNAs following transient focal cerebral ischemia in the rat.

Heat shock proteins (HSPs) 60 and 10 are stress-inducible mitochondrial matrix proteins that form a chaperonin complex that is important for mitochondrial protein folding and function. The effect of cerebral ischemia on mitochondrial HSPs is unclear. The topographical and chronological patterns of HSP60 and HSP10 messenger ribonucleic acid (mRNA) expression and induction were investigated in the rat focal cerebral ischemia model. Focal cerebral ischemia was produced by transient middle cerebral artery occlusion for 30 or 90 min. Expression of mRNAs was analyzed using reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. RT-PCR analysis showed that both HSP60 and HSP10 mRNA levels increased significantly in the ischemic cortex from 4 to 24 h of reperfusion after 30 min of occlusion. In situ hybridization analysis demonstrated significant induction of both mRNAs in the whole ischemic cortex after 30 min of occlusion and in the dorsomedial border (penumbra) of the ischemic cortex and ipsilateral hippocampus after 90 min of occlusion. Expression patterns and the timing of the induction of both HSP60 and HSP10 mRNAs were identical throughout the experiments. Simultaneous induction of the mRNAs for the mitochondrial chaperonins, HSP60 and HSP10, in various regions in focal cerebral ischemia demonstrates that mitochondrial stress conditions persist concomitantly with cytosolic stress conditions in focal cerebral ischemia.

Animals↗

PubMed2001

Purine but not pyrimidine nucleotides support rotation of F(1)-ATPase.

The binding change model for the F(1)-ATPase predicts that its rotation is intimately correlated with the changes in the affinities of the three catalytic sites for nucleotides. If so, subtle differences in the nucleotide structure may have pronounced effects on rotation. Here we show by single-molecule imaging that purine nucleotides ATP, GTP, and ITP support rotation but pyrimidine nucleotides UTP and CTP do not, suggesting that the extra ring in purine is indispensable for proper operation of this molecular motor. Although the three purine nucleotides were bound to the enzyme at different rates, all showed similar rotational characteristics: counterclockwise rotation, 120 degrees steps each driven by hydrolysis of one nucleotide molecule, occasional back steps, rotary torque of approximately 40 piconewtons (pN).nm, and mechanical work done in a step of approximately 80 pN.nm. These latter characteristics are likely to be determined by the rotational mechanism built in the protein structure, which purine nucleotides can energize. With ATP and GTP, rotation was observed even when the free energy of hydrolysis was -80 pN.nm/molecule, indicating approximately 100% efficiency. Reconstituted F(o)F(1)-ATPase actively translocated protons by hydrolyzing ATP, GTP, and ITP, but CTP and UTP were not even hydrolyzed. Isolated F(1) very slowly hydrolyzed UTP (but not CTP), suggesting possible uncoupling from rotation.

Adenosine Triphosphate↗

PubMed2001

Peripheral PAR-2 triggers thermal hyperalgesia and nociceptive responses in rats.

Protease-activated receptor-2 (PAR-2), a member of the G protein-coupled, seven trans-membrane domain receptor family, is activated by trypsin/tryptase and present in various tissues including the primary sensory neurons, playing a role in development of neurogenic inflammation. The present study examined if activation of peripheral PAR-2 could modulate nociception in the rat. Expression of mRNA for PAR-2 was confirmed in the L4-6 dorsal root ganglia, but not spinal cord. The PAR-2-activating peptide SLIGRL-NH2 administered by the intraplantar (i.pl.) route, produced thermal, but not mechanical, hyperalgesia in the rat, although the PAR-2-inactive control peptide LSIGRL-NH2 had no effect. Not only the PAR-2-activating but also inactive peptides elicited nociceptive behavior (licking/biting) in the intact rats, whereas only the former peptide produced such behavior in the rats that had received repeated administration of compound 48/80 for mast cell depletion. These data provide novel evidence that activation of peripheral PAR-2 is pro-nociceptive, producing thermal hyperalgesia and also triggering pain sensation, by itself, independently of mast cell degranulation.

Animals↗

PubMed2001

Dwarfism and early death in mice lacking C-type natriuretic peptide.

Longitudinal bone growth is determined by endochondral ossification that occurs as chondrocytes in the cartilaginous growth plate undergo proliferation, hypertrophy, cell death, and osteoblastic replacement. The natriuretic peptide family consists of three structurally related endogenous ligands, atrial, brain, and C-type natriuretic peptides (ANP, BNP, and CNP), and is thought to be involved in a variety of homeostatic processes. To investigate the physiological significance of CNP in vivo, we generated mice with targeted disruption of CNP (Nppc(-/-) mice). The Nppc(-/-) mice show severe dwarfism as a result of impaired endochondral ossification. They are all viable perinatally, but less than half can survive during postnatal development. The skeletal phenotypes are histologically similar to those seen in patients with achondroplasia, the most common genetic form of human dwarfism. Targeted expression of CNP in the growth plate chondrocytes can rescue the skeletal defect of Nppc(-/-) mice and allow their prolonged survival. This study demonstrates that CNP acts locally as a positive regulator of endochondral ossification in vivo and suggests its pathophysiological and therapeutic implication in some forms of skeletal dysplasia.

Animals↗

PubMed2001

Involvement of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase in alpha1B-adrenergic receptor/Galphaq-induced inhibition of cell proliferation.

Certain G protein-coupled receptors (GPCRs) stimulate the activities of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), members of the MAPK family. We investigated the role of JNK and p38 MAPK activation induced by the alpha1B-adrenergic receptor in the proliferation of human embryonic kidney 293T cells. Activation of the alpha1B-adrenergic receptor resulted in inhibition of cell proliferation. This receptor-induced inhibition of proliferation was blocked by a kinase-deficient MKK4 and by the p38 MAPK inhibitor SB203580. Additionally, transfection of constitutively activated Galphaq into cells also led to inhibition of proliferation in a JNK- and p38 MAPK-dependent manner. These results demonstrate that the alpha1B-adrenergic receptor/Galphaq signaling inhibits cell proliferation through pathways involving JNK and p38 MAPK.

Adrenergic alpha-Agonists↗

PubMed2001

cGMP-dependent protein kinase phosphorylates and inactivates RhoA.

Small GTPase Rho and cGMP/cGMP-dependent protein kinase (cGK) pathways exert opposing effects in specific systems such as vascular contraction and growth. However, the direct interaction between these pathways has remained elusive. We demonstrate that cGK phosphorylates RhoA in vitro at Ser188, the same residue phosphorylated by cAMP-dependent protein kinase. In HeLa cells transfected with constitutively active cGK (C-cGK), stress fiber formation induced by lysophosphatidic acid or V14RhoA was blocked. By contrast, C-cGK failed to inhibit stress fiber formation in cells transfected with mutant RhoA with substitution of Ser188 to Ala. C-cGK did not affect actin reorganization induced by Rac1 or Rho-associated kinase, one of the effectors for RhoA. Furthermore, C-cGK expression inhibited the membrane translocation of RhoA. Collectively, our findings suggest that cGK phosphorylates RhoA at Ser188 and inactivates RhoA signaling. The physiological relevance of the direct interaction between RhoA and cGK awaits further investigation.

Actins↗

PubMed2001

Direct observation of DNA rotation during transcription by Escherichia coli RNA polymerase.

Helical filaments driven by linear molecular motors are anticipated to rotate around their axis, but rotation consistent with the helical pitch has not been observed. 14S dynein and non-claret disjunctional protein (ncd) rotated a microtubule more efficiently than expected for its helical pitch, and myosin rotated an actin filament only poorly. For DNA-based motors such as RNA polymerase, transcription-induced supercoiling of DNA supports the general picture of tracking along the DNA helix. Here we report direct and real-time optical microscopy measurements of rotation rate that are consistent with high-fidelity tracking. Single RNA polymerase molecules attached to a glass surface rotated DNA for >100 revolutions around the right-handed screw axis of the double helix with a rotary torque of >5 pN nm. This real-time observation of rotation opens the possibility of resolving individual transcription steps.

DNA↗

PubMed2001

MRI white matter hyperintensities, (1)H-MR spectroscopy and cognitive function in geriatric depression: a comparison of early- and late-onset cases.

UNLABELLED: BACKGROUND AND OBJECTIVES Geriatric depression is often thought to differ from that at other times of adulthood. Recently, several studies have shown that the incidence of white matter hyperintense lesions identified by brain MRI is higher in patients with geriatric depression than in healthy elderly subjects, but a consensus has not yet been reached on the relationship between the severity of white matter lesions and either cognitive impairment or depressive symptoms. METHOD: Forty-seven patients aged 50 to 75 years with major depression were divided into two groups based on age at onset of depression: early-onset (< 50 years) group (20 patients; mean age, 62.7 +/- 6.7) and late-onset (> or =50 years) group (27 patients; mean age, 65.6 +/- 5.4). The severity of hyperintense white matter lesions on MRI was classified by region, then a proton magnetic resonance spectroscopy ((1)H-MRS) focusing on the white matter of the frontal lobes, multidimensional neuropsychological tests and evaluation of depressive symptoms were conducted. RESULTS: The severity of the deep white matter lesions, the deterioration of cognitive function related to subcortical/frontal brain system and clinician-rated depressive symptoms were all more pronounced in the late-onset group compared with those in the early-onset group. It was further observed that the more severe the deep white matter lesions, the lower the levels of N-acetylaspartate/creatine. With the age of onset as the covariate, the patients with moderate deep white matter lesions had more pronounced cognitive impairment and clinician-rated depressive symptoms than those with none and/or mild lesions. CONCLUSION: These results suggest that subcortical/frontal type cognitive impairment and the persistence of depressive symptoms in geriatric depression is related to moderate deep white matter lesions more often complicated in the late-onset group. The (1)H-MRS findings were suggested to be a useful indicator of neuronal/axonal loss in the white matter of the frontal lobes which precedes cognitive impairment.

Aged↗

PubMed2001

Time course of arterial hyperintensity with fast fluid-attenuated inversion-recovery imaging in acute and subacute middle cerebral arterial infarction.

The purpose of this study was to evaluate the time course of arterial hyperintensity (AH) in acute and subacute cerebral infarctions of the middle cerebral artery (MCA) distribution by using fast fluid-attenuated inversion-recovery (FLAIR) imaging. A total of 40 FLAIR MR examinations were performed in 27 patients with MCA infarction within 13 days after ictus. Thirteen patients underwent two MR examinations during this period. Thrombotic or embolic infarctions were included in this study, but lacunar infarctions were excluded. The presence or absence of AH and the location of AH were analyzed. Overall, AH was found in 24 (60%) of 40 FLAIR examinations within 13 days after onset. AH was seen in 17 (100%) examinations less than 24 hours old, four (40%) of 10 examinations 1-4 days old, two (18%) of 11 examinations 5-9 days old, and one (50%) of two examinations 10-13 days old. AH was most frequently found at the sylvian fissure (87%), followed by the sulci (54%), and the horizontal segment of MCA (29%) in the affected MCA distribution. Although AH could be seen even at 13 days after ictus, the presence of AH declined over time. AH represented an early sign of acute embolic or thrombotic infarction. J. Magn. Reson. Imaging 2001;13:987-990.

Aged↗

PubMed2001

Movement-related magnetic fields to tongue protrusion.

Movement-related magnetic fields (MRFs) associated with tongue protrusion were measured in five normal subjects using a helmet-shaped magnetoencephalography system. Bihemispherical two-dipolar patterns appeared from approximately -2000 or -1000 to 0 ms to the trigger signal indicating when protrusion of the tongue tip reached the frontal part of the palate. Equivalent current dipoles (ECDs) for the MRFs were localized on the central sulcus, 14.4 +/- 6.1 mm inferior (P < 0.0001) and 7.6 +/- 6.9 mm anterior (P < 0.01) to the ECD for the N20m in the somatosensory evoked fields for median nerve stimuli. The ECD orientations of MRFs were anterior and perpendicular to the central sulcus. These results correspond to the movement-related potentials for tongue protrusion previously recorded from subdural electrodes in patients with epilepsy. Magnetoencephalography can be applied to analyze cortical functions related to tongue movement with high resolution in time and space in normal subjects.

Adult↗

PubMed2001

Copper-62 ATSM as a hypoxic tissue tracer in myocardial ischemia.

Copper-62 labeled diacetyl-bis(N4-methylthiosemicarbazone) (62Cu-ATSM) has been proposed as a generator produced positron-emitting tracer for hypoxic tissue imaging. To clarify the usefulness of 62Cu-ATSM for myocardial ischemia, 62Cu-ATSM PET was performed in 7 patients with coronary artery disease. Increased myocardial uptake of 62Cu-ATSM was observed (myocardium/blood ratio: 3.09) in one patient with unstable angina, who had increased 18F-fluorodeoxyglucose (18F-FDG) uptake under the fasting condition. The other 6 patients, who were clinically stable, did not have increased 62Cu-ATSM uptake, although abnormal 18F-FDG uptake was seen in 4 patients. This preliminary study suggests that 62Cu-ATSM is a promising PET tracer for hypoxic imaging in acute ischemia.

Aged↗

PubMed2001

Myocardial FDG-PET examination during fasting and glucose loading states by means of a one-day protocol.

We propose a new method to measure the myocardial FDG uptake during fasting and glucose loading in one day, a myocardial FDG-PET one-day protocol, with both 2- and 3-dimensional data acquisition (2D and 3D) without background activity subtraction. To confirm it, we evaluated the effect of scatter correction in the 2D and 3D modes of a PET scanner both in phantom and patient studies. In the phantom study, we used a cardiac phantom with six divided chambers and two cylindrical phantoms placed as the activity outside the field of view. Each chamber was filled with a different concentration of F- 18 solution. Regions of interest (ROI) were placed on a polar map generated from reconstructed images and were compared to the concentration of the solution in each chamber in both 2D and 3D. In the patient study, 10 non-diabetic patients with coronary artery disease were studied. Each patient received a myocardial FDG study during fasting (F) and glucose loading (L). L images with background subtraction (Lsub(+)) and without background subtraction (Lsub(-)) were compared by polar map analysis. The ROI counts for the true activity in 2D and 3D demonstrated a linear relationship, and quite similar slopes were observed (0.72 in 2D, 0.69 in 3D). The background fraction in Lsub(-) was 3.59+/-1.83%. There were significant differences between Lsub(-) or Lsub(+) and F in both normal and ischemic myocardium. Scatter correction was successfully performed in both 2D and 3D modes. Background activity is thought to be negligible and this proposed method is simple touse in measuring the myocardial FDG uptake in one day.

Aged↗

PubMed2001

Acute effects of stereotactic radiosurgery on the kinetics of glucose metabolism in metastatic brain tumors: FDG PET study.

UNLABELLED: Hyperacute changes in the expression of glycolysis-associate gene products as well as FDG uptake in tumor cells after high-dose irradiation reflect response of the cells to noxious intervention and may be a potential indicator of the outcome of treatment. To understand acute effects on the kinetics of glucose metabolism of tumors in vivo after high-dose irradiation, we analyzed dynamic FDG PET data in patients with metastatic brain tumors receiving stereotactic radiosurgery. MATERIALS AND METHODS: We studied 5 patients with metastatic brain tumors by means of dynamic FDG PET before and 4 hours after stereotactic radiosurgery. Rate constants of glucose metabolism (K1*- k3*) were determined in a total of 13 tumors by a non-linear least squares fitting method for dynamic PET and arterial blood sampling data. Rate constants after radiosurgery were compared with those before radiosurgery. Changes in the rate constants induced by the therapy were also correlated with changes in tumor size evaluated by CT and/or MRI 6 months later. RESULTS: Four hours after radiosurgery, the phosphorylation rate indicated by k3* was significantly higher (0.080 +/- 0.058) than that before radiosurgery (0.049 +/- 0.023) (p < 0.05, paired t test), but there was no significant change in the membrane transport rates indicated by K1* and k2*. Although increases in the net influx rate constant K* (= K1*k3*/(k2* + k3*)) were correlated with increases in k3*, K* after radiosurgery (0.027 +/- 0.011) was not significantly different from that before the therapy (0.024 +/- 0.012). The reduction in the tumor size was correlated with k3* after radiosurgery. CONCLUSION: Acceleration of the phosphorylation process was demonstrated in vivo in metastatic brain tumors as early as 4 hours after stereotactic radiosurgery, as shown experimentally in vitro in a previous report. The phenomenon may be a sensitive indicator of cell damage.

Aged↗

PubMed2001

Brain perfusion SPECT study with 99mTc-bicisate: clinical pitfalls and improved diagnostic accuracy with a combination of linearization and scatter-attenuation correction.

To evaluate the usefulness of a combination of linearization and scatter-attenuation correction on 99mTc-bicisate (ECD)-single photon emission tomographic (SPECT) images, both cerebral blood flow (CBF)-positron emission tomographic (PET) images and ECD-SPECT images from fifteen patients with chronic cerebral infarction were acquired. We measured radioactivity counts in regions of interest (ROIs) on all sets of both images and obtained a 2D scattered graph between ECD-SPECT and CBF-PET data. To evaluate diagnostic accuracy, the sensitivity, specificity and accuracy of ECD-SPECT images were calculated by means of discriminant analysis. The same analysis was also performed on the ECD-SPECT images corrected by a combination of linearization and scatter-attenuation correction. An overall nonlinear relationship was observed between ECD-SPECT and CBF-PET. The sensitivity, specificity, and accuracy of ECD-SPECT images were 69.6%, 91.4% and 73.0%, and those of ECD images corrected by the combination of linearization and scatter-attenuation correction were 79.5%, 95.7% and 82.0% respectively. The clinically diagnostic accuracy of ECD-SPECT images corrected by the combined method apparently increased. So that the linearization with the scatter-attenuation method is useful for improving the diagnostic accuracy of ECD-SPECT images.

Aged↗

PubMed2001

Apoptosis in the basal ganglia of the developing human nervous system.

Programmed cell death (PCD) plays a crucial role in the development of the central nervous system through controlling neuronal numbers and adequate synaptic connections. PCD has been considered to occur in the form of apoptosis. To examine how apoptosis occurs in the developing human brain, we performed a morphometric TUNEL study, using a commercially available kit (ApopTag Kit, Oncor Inc.). We examined apoptotic cells in the basal ganglia of 47 fetuses and newborns without macroscopical and microscopical evident congenital anomalies. Gestational age ranged from 12 to 40 weeks. The numerical density as well as the labeling index of TUNEL-positively labeled nuclei were evaluated. In the caudate nucleus and putamen, TUNEL-labeled cells were observed around the 12th week of gestation. The numerical density of total cells was significantly decreased, whereas the labeling index of apoptotic cells was significantly increased with advanced gestational age. In the globus pallidus, the numerical density of total cells decreased with advancing gestational age, while the labeling index of apoptotic cells increased between the 20th and 28th week, followed by a decrease until the 40th week. The analysis of TUNEL-positive cells revealed a different reaction pattern for the various basal ganglia with regard to the timing and degree of the apoptotic process in regulating cell numbers.

Age Factors↗