Nucleotide sequence of the M genome segment of hemorrhagic fever with renal syndrome virus strain B-1.
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Amino Acid Sequence↗
Biomedical subjects
R Fukunaga
Publications and source records attributed to R Fukunaga.
Purification and characterization of the receptor for murine granulocyte colony-stimulating factor.
A receptor for mouse granulocyte colony-stimulating factor (G-CSF) has been found on the cell surface of mouse myeloid leukemia cell line NFS-60. Chemical cross-linking of the receptor with radioiodinated G-CSF, followed by gel electrophoresis in the presence of sodium dodecyl sulfate, has revealed that the G-CSF receptor in the NFS-60 cells is a single polypeptide of Mr approximately 100,000-130,000. The receptor in the membrane fraction of NFS-60 cells were solubilized in an active form with 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic acid. The solubilized receptor was purified approximately 100,000-fold to near homogeneity using a G-CSF affinity gel and gel filtration on a Superose 12 column, as measured by the selective precipitation of the 125I-G-CSF-receptor complex by polyethylene glycol. The purified G-CSF receptor has two classes of binding characteristics, one with an equilibrium dissociation constant (Kd) of 120-360 pM which is comparable with the Kd value for the cell-surface receptor, and the other with a higher Kd value of 2.6-4.2 nM. Analyses of the purified receptor by ligand blotting and sucrose density gradient centrifugation indicated that the low-affinity receptor is the monomer of the Mr 100,000-130,000 protein, whereas the high-affinity receptor consists of oligomers of the protein.
Animals↗
Expression cloning of a receptor for murine granulocyte colony-stimulating factor.
Two cDNAs encoding the receptor for murine granulocyte colony-stimulating factor (G-CSF) were isolated from a CDM8 expression library of mouse myeloid leukemia NFS-60 cells, and their nucleotide sequences were determined. Murine G-CSF receptor expressed in COS cells could bind G-CSF with an affinity and specificity similar to that of the native receptor expressed by mouse NFS-60 cells. The amino acid sequence encoded by the cDNAs has demonstrated that murine G-CSF receptor is an 812 amino acid polypeptide (Mr, 90,814) with a single transmembrane domain. The extracellular domain consists of 601 amino acids with a region of 220 amino acids that shows a remarkable similarity to rat prolactin receptor. The cytoplasmic domain of the G-CSF receptor shows a significant similarity with parts of the cytoplasmic domain of murine interleukin-4 receptor. A 3.7 kb mRNA coding for the G-CSF receptor could be detected in mouse myeloid leukemia NFS-60 and WEHI-3B D+ cells as well as in bone marrow cells.
Amino Acid Sequence↗
Changes in signal transduction in Alzheimer's disease.
We studied the signal transduction system including the receptor and protein kinase C (PKC) in Alzheimer's disease (AD) brains. We used 3H-TCP as a ligand for the NMDA receptor-ion channel complex. The total concentrations of 3H-TCP binding sites were significantly reduced in AD frontal cortex. 3H-TCP binding sites spared in AD brains retained the affinity for the ligand and the reactivity to NMDA, L-glutamate, and glycine. We utilized antibodies to assess the degree of involvement of different PKC isoforms in AD. The concentration of PKC (beta II) was lower in AD particulate fractions and higher in AD cytosol fractions. Immunocytochemical studies revealed reduced numbers of anti-PKC (beta II)-immunopositive neurons. Anti-PKC (alpha) faintly stained entire plaques and surrounding glial cells. Anti-PKC (beta I) stained dystrophic plaque neurites. Anti-PKC (beta II) stained the amyloid-containing portions of plaques. These results suggest an involvement of second messenger cascades in the pathogenesis of AD in addition to neurotransmitters and their receptors.
Aged↗
A validation study on the reproducibility of transcranial Doppler velocimetry.
The transcranial Doppler method for the measurement of intracranial arterial blood flow velocity is a useful noninvasive technique with a number of applications. The present study validated the reproducibility of this method for repeated measurements of flow velocity in the middle cerebral and basilar arteries. Fifteen healthy volunteers were studied. Measurements were made twice by one examiner and once by another in a single day and again by the first examiner on another day. The reproducibility was evaluated by calculating the correlation coefficient (r) and the coefficient of variation (CV) of the difference between the values obtained from each pair of measurements. Although depending on the examiner, the time interval between the examinations and the vessels studied some differences were noted in the reproducibility, both the r (0.69-0.95) and CV (6.7%-19.5%) values in the whole study were good enough to warrant the applicability of this method for the repeated measurements of the intracranial arterial blood flow velocity in future studies.
Cerebral Arteries↗
Free radical generation during brief period of cerebral ischemia may trigger delayed neuronal death.
We investigated the pathogenic role of free radical formation in ischemic neuronal death using radical scavenger, superoxide dismutase. Cerebral ischemia was produced in the gerbil by bilateral common carotid occlusion for 5 min, which consistently resulted in delayed neuronal death in the CA1 region of the hippocampus. The effects of free superoxide dismutase and a derivatized superoxide dismutase, pyran copolymer conjugated superoxide dismutase, on early ischemic damages, detected sensitively by the immunohistochemical reaction for microtubule associated protein 2, and a subsequent delayed neuronal death after restoration of blood flow were investigated. Preischemic treatment by pyran conjugated superoxide dismutase showed clear protective effects against both the neuronal damages detected by immunohistochemistry after 5 min ischemia and the delayed neuronal necrosis after one week of recovery, although no clear beneficial effects were observed when this drug was administered just before the recirculation or free superoxide dismutase was used. These results strongly suggest that free radical generation during brief period of ischemia plays a pivotal role in triggering the ischemic neuronal damages causing delayed neuronal death at the selectively vulnerable areas of the brain.
Animals↗
Three different mRNAs encoding human granulocyte colony-stimulating factor receptor.
Three cDNAs for the human granulocyte colony-stimulating factor (G-CSF) receptor were isolated from the cDNA libraries of human U937 leukemia cells and placenta by using a murine G-CSF receptor cDNA as the probe. The human G-CSF receptor containing 813 amino acids had a marked homology (62.5%) with its murine counterpart and consisted of extracellular, transmembrane, and cytoplasmic domains. The WSXWS motif found in members of the newly identified growth factor receptor family was also present in the extracellular domain of the human G-CSF receptor. Expression of the cloned cDNA in monkey COS cells gave rise to a protein that could specifically bind G-CSF with a high affinity (Kd, 550 pM). Two other classes of the human G-CSF receptor were also identified, one of which had a deletion of the transmembrane domain and seemed to encode a secreted, soluble receptor. The third class of the G-CSF receptor contained a 27-amino acid insertion in the cytoplasmic domain and was highly expressed in placenta.
Amino Acid Sequence↗
[3H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine ([3H]TCP) binding in human frontal cortex: decreases in Alzheimer-type dementia.
We studied [3H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine [( 3H]TCP) binding to human frontal cortex obtained at autopsy from 10 histologically normal controls and eight histopathologically verified cases with Alzheimer-type dementia (ATD). Extensively washed membrane preparations were used to minimize the effects of endogenous substances. In ATD frontal cortex, the total concentration (Bmax) of [3H]TCP binding sites was significantly reduced by 40-50%. The apparent dissociation constant (KD) values showed no significant change. The reduction in binding capacity was also apparent in Triton X-100-treated membrane preparations, and there was a linear correlation between the number of [3H]TCP binding sites and that of N-methyl-D-aspartate (NMDA)-sensitive [3H]glutamate binding sites. [3H]TCP binding sites spared in ATD brains retained the affinity for the ligand and the reactivity to NMDA, L-glutamate, and glycine. These results suggest that the primary change in NMDA receptor-ion channel complex in ATD brains is the reduction of its number, possibly reflecting the loss of neurons bearing these receptor complexes, and that the functional linkage within the receptor complexes spared in ATD brains remains normal.
Alzheimer Disease↗
Ultrasonic evaluation of early carotid atherosclerosis.
We investigated the prevalence of carotid atherosclerosis, including mild early lesions, and its association with cervical bruits and various risk factors (age, male sex, hypertension, hyperlipidemia, diabetes mellitus, obesity, and cigarette smoking) in 232 consecutive Japanese patients. High-resolution real-time B-mode ultrasonography was performed to determine the extent of atherosclerosis, and it was quantified by using a scoring system. The prevalence of carotid atherosclerosis was 49%, 59%, and 41% in all 232 patients, the 100 symptomatic patients, and the 132 asymptomatic patients, respectively. Although carotid lesions were detected frequently (87%) in the 30 patients with cervical bruits, bruits were noted in only 30% of the 88 examined patients with carotid atherosclerosis. Independent risk factors for carotid atherosclerosis in these patients were found to be age, male sex, and hyperlipidemia; diabetes mellitus was a possible risk factor for carotid atherosclerosis. Our study did not show a close association between hypertension and carotid atherosclerosis, and this might be caused by the high prevalence of hypertension in our patients. Our findings suggest an increasing prevalence of carotid atherosclerosis in the Japanese, though this should be confirmed in a population-based study. Our study demonstrates the clinical usefulness of high-resolution B-mode ultrasonography for the evaluation of early carotid atherosclerosis.
Adult↗
An ATP-driven Cl- pump in the brain.
EDTA-treated microsomes prepared from rat brain mainly consisted of sealed membrane vesicles 200-500 nm in diameter and were rich in both Cl- -ATPase and Na+,K+-ATPase activities. Such Cl- -ATPase-rich membrane vesicles accumulated Cl- in an ATP-dependent and osmotically reactive manner in the presence of 1 nM ouabain. The Cl- uptake was maximally stimulated by ATP with a Km value of 1.5 mM; GTP, ITP, and UTP partially stimulated Cl- uptake, but CTP, beta, gamma-methylene ATP, ADP, and AMP did not. The ATP-dependent Cl- uptake was accelerated by an increase in the medium Cl- concentration with a Km value of 7.4 mM. Such stimulation of Cl- uptake by ATP was dependent on the pH of the medium, with an optimal pH of 7.4, and also on the temperature of the medium, with an optimal range of 37-42 degrees C. Ethacrynic acid dose dependently inhibited the ATP-dependent Cl- uptake with a concentration for half-maximal inhibition at 57 microM. N-ethylmaleimide (0.1 mM) completely inhibited and sodium vanadate (1 mM) partially inhibited the ATP-dependent Cl- uptake. The membrane vesicles did not accumulate H+ in the Cl- uptake assay medium. The ATP-dependent Cl- uptake profile agreed with that of Cl- -ATPase activity reported previously (Inagaki, C., Tanaka, T., Hara, M., and Ishiko, J. (1985) Biochem. Pharmacol. 34, 1705-1712), and this strongly supports the idea that Cl- -ATPase in the brain actively transports Cl-.
Adenosine Triphosphatases↗
ATP-dependent H+ transport in synaptosomal membrane vesicles of the rat brain.
H+ transport into synaptosomal membrane vesicles of the rat brain was stimulated by ATP and to a lesser extent by GTP, but not by ITP, CTP, UTP, ADP, AMP or beta, gamma-methylene ATP. ATP at concentrations up to 200 mM concentration-dependently stimulated the rate of H+ transport with a Km value of 0.6 mM, but at higher concentrations of this nucleotide the rate decreased. Other nucleotides such as CTP, UTP, GTP and AMP, or products of ATP hydrolysis i.e. ADP and Pi also reduced the ATP-stimulated H+ transport. The inhibition by GTP and ADP was not affected by the ATP concentration. These findings suggest that plasma membranes of nerve endings transport H+ from inside to outside of the cells utilizing energy from ATP hydrolysis, and that this transport is regulated by the intracellular concentration of nucleotides and Pi on sites other than those involved in substrate binding.
Adenosine Triphosphate↗
Prediction of stroke-prone gerbils and their cerebral circulation.
We attempted to predict the severity of cerebral ischemia, prior to permanent occlusion of a common carotid artery (CCA), in the gerbil by measuring the diameter of the distal CCA before and during temporary ligation and correlated the severity of cerebral ischemia and the pattern of cerebral circulation. All gerbils with reduction of the diameter over 44% after temporary occlusion developed severe neurological signs following permanent ligation. These gerbils lacked any connection between the left and right anterior cerebral arteries (ACA) and the pericallosal arteries originated from the ACA of the occluded side. No gerbils with reduction of less than 30% developed neurological signs and those gerbils possessed a definite anastomosis between the ACAs from both sides. Six of 7 gerbils without connection between the ACAs whose pericallosal arteries originated from the ACA of the non-occluded side proved to be moderately symptomatic. It was concluded that we could reliably predict severely symptomatic and moderately symptomatic gerbils with specific cerebral circulation patterns prior to permanent occlusion of the CCA. The preselected 'stroke-prone' gerbils should be useful for the investigation of cerebral ischemia and the evaluation of pharmacological agents.
Animals↗
Microtubule-associated protein 2 as a sensitive marker for cerebral ischemic damage--immunohistochemical investigation of dendritic damage.
We investigated the neuronal distribution of microtubule-associated protein 2 in gerbil brain and monitored the progression of ischemic damage immunohistochemically by using this protein as a dendritic marker. The reaction for microtubule-associated protein 2 in normal gerbil brain clearly visualized neuronal soma and dendrites but other structures such as axonal bundles, glia and endothelial cells exhibited little immunoreactivity. In a reproducible gerbil model of unilateral cerebral ischemia, we could detect the ischemic lesions as early as 3 min after right common carotid occlusion at the subiculum-CA1 region of the ipsilateral hippocampus as faint loss of the reaction in the dendrites. After ischemia for 30 min, the ischemic lesions were clearly detected as loss of the reaction in the nerve cell bodies, dendrites and the neuropil in the hippocampus, cerebral cortex, thalamus and the caudoputamen. Although the mechanism for prompt disappearance of the immunohistochemical reaction for microtubule-associated protein 2 is not clear, the present investigation suggests that dendrites in the vulnerable regions may be quite susceptible to ischemic stress and that the immunohistochemical procedure for microtubule-associated protein 2 may be very useful for demonstration of dendritic damage in various pathophysiological states of the central nervous system.
Animals↗
[Effect of ethanol on synaptosomal membrane potential in the rat brain].
The membrane potential of rat brain synaptosomes was depolarized by ethanol at concentrations of over 0.8-1.6%. This ethanol-induced depolarization was augmented by 4-aminopyridine (4-AP) or the gluconate- -medium, but not affected by tetrodotoxin, choline-Cl, NH4Cl, picrotoxin, ethacrynic acid, furosemide or SCN- -medium. The ethanol-induced depolarization was augmented by 4-AP in Cl- - or gluconate- -medium, but not in SCN- -medium. These findings suggest that ethanol depolarizes the synaptosomal membrane potential through potassium and anion-related mechanisms, probably through potassium channels.
4-Aminopyridine↗
Efficacy of echo-Doppler examination for the evaluation of renovascular disease.
The accuracy of the diagnosis of renal artery stenosis using noninvasive echo-Doppler velocimetry was compared with that of angiography in 40 renal arteries. The duplex-Doppler signals were detected through the muscle of the back (the translumbar approach). Renal artery stenosis was diagnosed by three objectively defined Doppler parameters, the acceleration index (AI), the acceleration time (AT), and the acceleration time ratio (ATR). The normal range obtained on 11 control subjects was defined as AI greater than or equal to 3.78, AT less than or equal to 0.07 s, and ATR less than or equal to 1.35. High technological success (98%) was obtained using the translumbar approach. In comparison with angiography (cases of significant stenosis), the accuracy of the echo-Doppler method using the criteria of the AI was 95%, the sensitivity was 100%, and the specificity was 93%. This noninvasive method may be one of the most accurate screening methods for diagnosing significant renal artery stenosis.
Blood Flow Velocity↗
[Effect of NH4+ on synaptosomal membrane potential in the rat brain].
The effect of NH4+ on synaptosomal membrane potential was examined using rat brains. The membrane potential was measured by the rhodamine 6G fluorescence method. Both NH4+ diffusion potential (NH4+-potential) and K+ diffusion potential (K+-potential) were observed in the synaptosomes. Upon replacement of medium Cl- with SCN-, both K+- and NH4+-potentials depolarized. On the other hand, replacement of medium Cl- with gluconate resulted in the hyperpolarization of K+-potential, but not of NH4+-potential. Ethacrynic acid (0.3 mM), a Cl- -ATPase inhibitor, depolarized both K+(Cl-)- and NH4+ (Cl-)-potentials. In the presence of ethacrynic acid, both of the potentials were further depolarized by replacement of medium Cl- with SCN-, but not with gluconate-. Picrotoxin (5 mM), a Cl- channel inhibitor, did not significantly affect either K+- or NH4+-potential. In the presence of picrotoxin, replacement of medium Cl- with SCN- depolarized both K+- and NH4+-potentials with or without ethacrynic acid. Gluconate depolarized the K+-potential with ethacrynic acid and the NH4+-potential with or without ethacrynic acid. These findings suggest that NH4+ forms a diffusion potential in nerve endings, and inhibits the anion-mediated hyperpolarization through mechanisms other than anion channels.
Ammonia↗
Mouse 2-5A synthetase cDNA: nucleotide sequence and comparison to human 2-5A synthetase.
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2',5'-Oligoadenylate Synthetase↗
Undermethylation of interferon-gamma gene in human T cell lines and normal T lymphocytes.
The relative levels of DNA methylation at CCGG sequences within and around the interferon-gamma (IFN-gamma) gene in normal human tissues and cell lines were examined by Southern blot analysis using isoschizomeric restriction enzymes, HpaII and MspI. On the test of normal tissues, the IFN-gamma gene was undermethylated only in a small population of T lymphocyte, whereas the gene was fully methylated in T cell-depleted lymphocytes and uterus cells. In TCL-Fuj cell line which is a T cell line producing a high level of IFN-gamma spontaneously, the IFN-gamma gene was undermethylated. Moreover, the extent of DNA methylation was inversely correlated to the level of expression of the IFN-gamma gene in several T cell lines including sublines derived from TCL-Fuj cells. However, partial or complete unmethylation at the CCGG sites of IFN-gamma gene was observed in a promyelocytic leukemia cell line and two epithelial cell lines that fail to produce IFN-gamma irrespective of induction. These results suggest that undermethylation of IFN-gamma gene is necessary but not sufficient for its efficient expression.
Cell Line↗