Showing papers by "Kumamoto University published in 1995"

Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice

Abstract: The intracellular protein tyrosine kinase FAK (focal adhesion kinase) was originally identified gy its high level of tyrosine phosphorylation in v-src-transformed cells. FAK is also highly phosphorylated during early development. In cultured cells it is localized to focal adhesion contacts and becomes phosphorylated and activated in response to integrin-mediated binding of cells to the extracellular matrix, suggesting an important role in cell adhesion and/or migration. We have generated FAK-deficient mice by gene targeting to examine the role of FAK during development. Mutant embryos displayed a general defect of mesoderm development, and cells from these embryos had reduced mobility in vitro. Surprisingly, the number of focal adhesions was increased in FAK-deficient cells, suggesting that FAK may be involved in the turnover of focal adhesion contacts during cell migration.

Minamata disease: methylmercury poisoning in Japan caused by environmental pollution

Abstract: Minamata disease (M. d.) is methylmercury (MeHg) poisoning that occurred in humans who ingested fish and shellfish contaminated by MeHg discharged in waste water from a chemical plant (Chisso Co. L...

Reduced hippocampal LTP and spatial learning in mice lacking NMDA receptor ε1 subunit

Abstract: THE NMDA (TV-methyl-D-aspartate) receptor channel is important for synaptic plasticity, which is thought to underlie learning, memory and development1, 2. The NMDA receptor channel is formed by at least two members of the glutamate receptor (GluR) channel subunit families, the GluRe (NR2) and GiuRζ (NR1) sub-unit families3–8. The four e subunits are distinct in distribution, properties and regulation5–14. On the basis of the Mg2+ sensitivity and expression patterns, we have proposed that the ei (NR2A) and e2 (NR2B) subunits play a role in synaptic plasticity6, 14. Here we show that targeted disruption of the mouse el subunit gene resulted in significant reduction of the NMDA receptor channel current and long-term potentiation at the hippocampal CA1 synapses. The mutant mice also showed a moderate deficiency in spatial learning. These results support the notion that the NMDA receptor channel-dependent synaptic plasticity is the cellular basis of certain forms of learning.

Mouse Otx2 functions in the formation and patterning of rostral head.

Abstract: The anterior part of the vertebrate head expresses a group of homeo box genes in segmentally restricted patterns during embryogenesis. Among these, Otx2 expression covers the entire fore- and midbrains and takes place earliest. To examine its role in development of the rostral head, a mutation was introduced into this locus. The homozygous mutants did not develop structures anterior to rhombomere 3, indicating an essential role of Otx2 in the formation of the rostral head. In contrast, heterozygous mutants displayed craniofacial malformations designated as otocephaly; affected structures appeared to correspond to the most posterior and most anterior domains of Otx expression where Otxl is not expressed. The homo- and heterozygous mutant phenotypes suggest Otx2 functions as a gap-like gene in the rostral head where Hox code is not present. The evolutionary significance of Otx2 mutant phenotypes was discussed for the innovation of the neurocranium and the jaw.

Direct measurement of high-density lipoprotein cholesterol in serum with polyethylene glycol-modified enzymes and sulfated alpha-cyclodextrin.

Abstract: We have developed an automated method for measuring high-density lipoprotein (HDL)-cholesterol in serum without prior separation, using polyethylene glycol (PEG)-modified enzymes and sulfated alpha-cyclodextrin. When cholesterol esterase and cholesterol oxidase enzymes were modified with PEG, they showed selective catalytic activities towards lipoprotein fractions, with the reactivity increasing in the order: low-density lipoprotein < very-low-density lipoprotein approximately chylomicron < HDL. In the presence of magnesium ions, alpha-cyclodextrin sulfate reduced the reactivity of cholesterol, especially in chylomicrons and very-low-density lipoprotein, without the need for precipitation of those lipoprotein fractions. The combination of PEG-modified enzymes with alpha-cyclodextrin sulfate provided selectivity for the determination of HDL-cholesterol in serum in the presence of a small amount of dextran sulfate without the need for precipitation of lipoprotein aggregates. The results of the HDL-cholesterol assayed in serum by this direct method correlated well with those obtained by precipitation-based methods and also that by an ultracentrifugation method.

Escherichia coli FtsH is a membrane-bound, ATP-dependent protease which degrades the heat-shock transcription factor sigma 32.

Abstract: Escherichia coli FtsH is an essential integral membrane protein that has an AAA-type ATPase domain at its C-terminal cytoplasmic part, which is homologous to at least three ATPase subunits of the eukaryotic 26S proteasome. We report here that FtsH is involved in degradation of the heat-shock transcription factor sigma 32, a key element in the regulation of the E. coli heat-shock response. In the temperature-sensitive ftsH1 mutant, the amount of sigma 32 at a non-permissive temperature was higher than in the wild-type under certain conditions due to a reduced rate of degradation. In an in vitro system with purified components, FtsH catalyzed ATP-dependent degradation of biologically active histidine-tagged sigma 32. FtsH has a zinc-binding motif similar to the active site of zinc-metalloproteases. Protease activity of FtsH for histidine-tagged sigma 32 was stimulated by Zn2+ and strongly inhibited by the heavy metal chelating agent o-phenanthroline. We conclude that FtsH is a novel membrane-bound, ATP-dependent metalloprotease with activity for sigma 32. These findings indicate a new mechanism of gene regulation in E. coli.

An IRF-1-dependent pathway of DNA damage-induced apoptosis in mitogen-activated T lymphocytes

Abstract: Lymphocytes are particularly susceptible to DNA damage-induced apoptosis, a response which may serve as a form of 'altruistic suicide' to counter their intrinsic high potential for mutation and clonal expansion. The tumour suppressor p53 has been shown to regulate this type of apoptosis in thymocytes, but an as yet unknown, p53-independent pathway(s) appears to mediate the same event in mitogen-activated mature T lymphocytes. Here we show DNA damage-induced apoptosis in these T lymphocytes is dependent on the antioncogenic transcription factor interferon regulatory factor (IRF)-1. Thus two different anti-onco-genic transcription factors, p53 and IRF-1, are required for distinct apoptotic pathways in T lymphocytes. We also show that mitogen induction of the interleukin-1 beta converting enzyme (ICE) gene, a mammalian homologue of the Caenorhabditis elegans cell death gene ced-3, is IRF-1-dependent. Ectopic overexpression of IRF-1 results in the activation of the endogenous gene for ICE and enhances the sensitivity of cells to radiation-induced apoptosis.

Rapid ventricular induction of brain natriuretic peptide gene expression in experimental acute myocardial infarction

Abstract: Background We have demonstrated that brain natriuretic peptide (BNP) is a cardiac hormone predominantly synthesized in and secreted from the ventricle. We have also reported that, compared with atrial natriuretic peptide (ANP), the plasma concentration of BNP is increased to a greater degree in patients with congestive heart failure and more rapidly in patients with acute myocardial infarction (AMI). Methods and Results To investigate ventricular gene expression of BNP in AMI, we analyzed plasma and ventricular BNP concentrations along with ventricular BNP mRNA in rats with AMI produced by coronary artery ligation. The BNP concentration in the left ventricle increased about 2-fold as early as 12 hours postinfarction and 5-fold 1 day postinfarction compared with sham-operated rats, whereas left ventricular ANP concentration remained unchanged within 1 day. The tissue concentration of BNP increased in the noninfarcted region as well as in the infarcted region. The surviving myocytes in and around the necrot...

c-kit-dependent development of interstitial cells and electrical activity in the murine gastrointestinal tract.

Abstract: In vivo injection of a neutralizing, monoclonal antibody (ACK2) to the receptor tyrosine kinase (c-kit) disrupts the normal motility patterns of the mouse small intestine. Immunohistochemical studies showed that cells expressing c-kit-like immunoreactivity (c-kit-LI) decreased in numbers in response to ACK2, but the identity of these cells is unknown. We investigated the identity and development of the cells that express c-kit-LI in the mouse small intestine and colon. Cells in the region of the myenteric plexus and deep muscular plexus of the small intestine and in the subserosa, in the myenteric plexus region, within the circular and longitudinal muscle layers, and along the submucosal surface of the circular muscle in the colon were labeled with ACK2. The distribution of cells that express c-kit-LI was the same as that of interstitial cells (ICs). In whole-mount preparations cells with c-kit-LI were interconnected, forming a network similar to the network formed by cells that stained with methylene blue, which has been used as a marker for ICs in the mouse gastrointestinal tract. Immunocytochemistry verified that ICs were labeled with ACK2. Multiple injections of animals with ACK2 between days 0 and 8 post partum (pp) caused a dramatic reduction in the number of ICs compared to control animals. From an ultrastructural point of view, the proliferation and development appeared to be suppressed in some classes of ICs, while others displayed an altered course of development. Functional studies showed that the decrease in ICs was accompanied by a loss of electrical rhythmicity in the small intestine and reduced neural responses in the small bowel and colon. Morphological experiments showed that c-kit-positive cells are ICs, and physiological evidence reinforced the concept that ICs are involved in generation of rhythmicity and translation of neural inputs in gastrointestinal smooth muscles. Controlling the development of ICs provides a powerful new tool for the investigation of the physiological role of these cells.

Identification of optimal operating point of PV modules using neural network for real time maximum power tracking control

Abstract: This paper presents an application of a neural network for the identification of the optimal operating point of PV modules for the real time maximum power tracking control. The output power from the modules depends on the environmental factors such as insolation, cell temperature, and so on. Therefore, accurate identification of optimal operating point and real time continuous control are required to achieve the maximum output efficiency. The proposed neural network has a quite simple structure and provides a highly accurate identification of the optimal operating point and also a highly accurate estimation of the maximum power from the PV modules. >

Immunohistochemical and ultrastructural detection of advanced glycation end products in atherosclerotic lesions of human aorta with a novel specific monoclonal antibody.

Abstract: To elucidate the deposition of advanced glycation end products (AGEs) in aortic atherosclerosis, aortic walls were obtained from 25 autopsy cases and examined immunohistochemically and immunoelectron microscopically with a monoclonal antibody specific for AGEs, 6D12. Among the autopsy cases, atherosclerotic lesions were found in the aortas of 22 cases and were composed of diffuse intimal thickening, fatty streaks, atherosclerotic plaques, and/or complicated lesions. In these cases, intracellular AGE accumulation was demonstrated in the intimal lesions of aortic atherosclerosis in 12 cases. Compared with the diffuse intimal thickening, intracellular AGE accumulation was marked in the fatty streaks and atherosclerotic plaques. Immunohistochemical double staining with 6D12 and monoclonal antibodies for macrophages or muscle actin or a polyclonal antibody for scavenger receptors demonstrated that the AGE accumulation in macrophages or their related foam cells was marked in the diffuse intimal thickening and fatty streak lesions and that almost all macrophages and macrophage-derived foam cells possessed scavenger receptors. Immunoelectron microscopic observation revealed the localization of 6D12-positive reaction in lysosomal lipid vacuoles or electron-dense granules of the foam cells. These results indicate that AGE accumulation occurs in macrophages, smooth muscle cells, and their related foam cells.

Macrophage Scavenger Receptor Mediates the Endocytic Uptake and Degradation of Advanced Glycation End Products of the Maillard Reaction

Abstract: Modification of proteins by long-term incubation with glucose leads to the formation of advanced glycation end products (AGE). Recent immunological demonstration of the presence of AGE proteins in several human tissues suggests that they may be involved in aging, diabetic complications and atherosclerosis. AGE proteins are taken up by macrophages via the AGE receptor, which is similar to the macro-phage scavenger receptor (MSR). In the present study, we examined whether MSR could mediate the endocytic uptake of AGE proteins by using Chinese hamster ovary (CHO) cells overexpressing bovine type II MSR (CHO-SRII). 125I-labelled AGE bovine serum albumin (125I-AGE-BSA) as well as 125I-acetylated low-density lipoprotein (125I-acetyl-LDL) underwent endocytic degradation by CHO-SRII cells, but not by control CHO cells. Endocytic degradation of 125I-acetyl-LDL and 125I-AGE-BSA by CHO-SRII cells was significantly inhibited by unlabeled AGE-BSA, as well as by acetyl-LDL. Immunoelectron microscopic studies using both AGE-BSA conjugated with gold particles and anti-(bovine MSR) antibody (D2) revealed co-localization of gold particles and the reactive sites for the antibody at coated pits of plasma membranes as well as in endosomes. These results clearly show that MSR mediates the endocytic uptake and degradation of AGE proteins, suggesting a new role of MSR in biological recognition of AGE in vivo.

Mesodermal defect in late phase of gastrulation by a targeted mutation of focal adhesion kinase, FAK.

Abstract: FAK is a unique non-receptor protein tyrosine kinase that was found in cellular focal adhesions. An increasing number of in vitro observations has suggested that FAK mediates signaling through integrins brought about by interactions with extracellular matrix (ECM). It is highly tyrosine-phosphorylated in v-src-transformed cells and during embryogenesis. To clarify the function of FAK in cell-ECM interactions, embryonic phenotype of its mutant was analysed. FAK-deficient embryos could implant and initiate gastrulation normally, but showed abnormalities in subsequent development. The abnormalities were characterized as a general deficiency in mesoderm, and the phenotype was quite similar to that caused by fibronectin-deficiency. The results suggest that FAK mediates fibronectin-integrin interactions uniquely at this stage of development, thereby playing an essential role in development of mesodermal cell lineages.

Intravenous Injection of Rabbit Apolipoprotein A-I Inhibits the Progression of Atherosclerosis in Cholesterol-Fed Rabbits

Abstract: The effects of intravenous injection of purified rabbit apoA-I on the progression of aortic atherosclerosis in cholesterol-fed rabbits were examined. In experiment 1, 28 rabbits were equally divided into groups A and B and fed a 0.5% cholesterol diet for 90 days. For the last 30 days, group B received 40 mg apoA-I every week. The fatty streak lesions in group B (23.9±15.6%) were significantly suppressed compared with those in group A (46.0±24.9%) ( P <.05). In experiment 2, 33 rabbits were divided into four groups (8 or 9 rabbits per group) and fed a 0.5% cholesterol diet. Group A was killed on day 105, while groups B, C, and D were maintained for an additional 60 days on a normal diet, during which time groups C and D received 1 mg apoA-I every other day or 40 mg apoA-I every week, respectively. The lesions in group C (70.2±15.4%) and group D (65.7±20.0%) were significantly suppressed compared with those in group B (86.2±13.7%) ( P <.05) but were not reduced to the level of group A (50.0±22.9%). Although apparent regression was not observed under these conditions, the present study provided the first evidence for the antiatherogenic effect of homologous apoA-I on the progression of atherosclerosis in cholesterol-fed rabbits.

Prevalence of dry eye in Japanese eye centers

Abstract: • Background: The purpose of the investigation was to ascertain the prevalence of dry eye in new outpatients. • Methods: A total of 2127 consecutive new outpatients seen in eight Japanese centers from April 1992 to January 1993 underwent comprehensive examinations, including double vital staining and measurement of tear film break-up time, basal tear secretion, and tear clearance. Dry eye was diagnosed if patients had abnormalities of both the tear film and the ocular surface. • Results: Three hundred fifty-nine patients (17%) had dry eye. There was no seasonal pattern for dry eye. The condition was significantly more common in Tokyo than in suburban areas (P < 0.01). The prevalence of dry eye in visual display terminal (VDT) users and contact lens (CL) wearers was significantly higher than in non-VDT users and non-CL wearers (P < 0.05 and P < 0.02, respectively). • Conclusion: Our findings suggest that dry eye is one of the most common ocular disorders encountered by physicians. Furthermore, if patients use VDTs or wear CLs, the likelihood of dry eye occurring is higher.

Evaluation of neural network based real time maximum power tracking controller for PV system

Abstract: This paper presents a neural network based maximum power tracking controller for interconnected PV power systems The neural network is utilized to identify the optimal operating voltage of the PV power system The controller generates the control signal in real-time, and the control signal is fed back to the voltage control loop of the inverter to shift the terminal voltage of the PV power system to its identified optimum, which yields maximum power generation The controller is of the PI type The proportional and the integral gains are set to their optimal values to achieve fast response and also to prevent overshoot and also undershoot Continuous measurement is required for the open circuit voltage on the monitoring cell, and also for the terminal voltage of the PV power system Because of the accurate identification of the optimal operating voltage of the PV power system, more than 99% power is drawn from the actual maximum power >

Development of a ferrocene-mediated needle-type glucose sensor covered with newly designed biocompatible membrane, 2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate.

Abstract: To prepare the long-life and stable glucose sensor, we developed the ferrocene-mediated needle-type glucose sensor covered with newly designed biocompatible membrane, 2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate (MPC-co-BMA) membrane. In this membrane, the hydrophilic phosphorylcholine chains were grafted on the hydrophobic polymer surface. 1. The poly(MPC-co-BMA) membrane inhibited platelet activation and protein adhesion on the surface, showing excellent biocompatibility. These results suggested that the hydrophilic phospholipids chains might have the potential for suppressing activation and adsorption of biochemical molecules. 2. The ferrocene-mediated needle-type glucose sensor covered with poly(MPC-co-BMA) membrane achieved excellent results in vitro. Subcutaneous tissue glucose concentrations were measured in a wide range from 1.7 to more than 16.7 mmol/l. The correlation between subcutaneous tissue (Y) and blood (X) glucose concentrations was Y = 1.04X + 0.12 (r = 0.98). The subcutaneous tissue glucose concentrations could be monitored precisely for 7 days without any in vivo calibrations, and for 14 days by introducing in vivo calibrations. We therefore conclude that this sensor is stable and reliable, as compared to any other glucose sensors we developed.

Adnexal masses: accuracy of characterization with transvaginal US and precontrast and postcontrast MR imaging.

Abstract: PURPOSE: To determine the accuracy of transvaginal ultrasound (TVUS) and of precontrast and contrast material-enhanced magnetic resonance (MR) imaging in the differentiation of adnexal masses. MATERIALS AND METHODS: Five blinded readers analyzed images of 80 masses in 72 patients. MR and TVUS images were interpreted in separate sessions. Findings were confirmed at surgery or laparoscopy. RESULTS: Higher diagnostic accuracy was attained with MR imaging in mature cystic teratomas and endometriomas. However, better accuracy was achieved with contrast-enhanced MR imaging and TVUS in simple cysts, cystadenomas, and malignant tumors because internal details could be visualized. Receiver operating characteristic study indicated that observer confidence was significantly higher with contrast-enhanced MR imaging than with precontrast MR imaging (P = .011) or TVUS (P = .002) in the differentiation of benign and malignant masses. CONCLUSION: Contrast-enhanced MR imaging is superior to precontrast MR and TVUS imaging...

Dentatorubral‐pallidoluysian atrophy: Clinical features are closely related to unstable expansions of trinucleotide (CAG) repeat

Abstract: Dentatorubral-pallidoluysian atrophy is an autosomal dominant neurodegenerative disease characterized by various combinations of ataxia, choreoathetosis, myoclonus, epilepsy, and dementia as well as a wide range of ages at onset. A specific unstable trinucleotide repeat expansion in a gene on the short arm of chromosome 12 was recently identified as the pathogenic mutation for this disease. We investigated how the degree of expansion of the CAG repeat effects the clinical manifestations of dentatorubral-pallidoluysian atrophy. The size of the expanded alleles was well correlated with the age at onset (r = -0.696, p < 0.001). Patients with the progressive myoclonus epilepsy phenotype had larger expansions (62-79 repeats) and an earlier age at onset (onset before age 21). Furthermore, most of the patients with the progressive myoclonus epilepsy phenotype inherited their expanded alleles from their affected fathers. On the other hand, patients with the non-progressive myoclonus epilepsy phenotype showed smaller expansions (54-67 repeats) and a later age at onset (onset at or after age 21). Detailed analyses of clinical features demonstrated that ataxia, involuntary movement of either myoclonus or choreoathetosis, and intellectual decline are cardinal features of dentatorubral-pallidoluysian atrophy, with myoclonus and epilepsy being observed more frequently in patients with an earlier age at onset. Thus the wide variation in clinical manifestations of dentatorubral-pallidoluysian atrophy can now be clearly explained based on the degree of CAG repeat expansion, which strongly indicates that the expanded alleles are intimately involved in the neuronal degeneration in dentatofugal and pallidofugal systems.