[Analysis of ovarian tumors treated at Nippon Medical School Hospital in a 5-year period. Problems in a frozen section diagnosis].
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Adult↗
Biomedical subjects
M Matsubara
Publications and source records attributed to M Matsubara.
Relationships among blood pressures obtained using different measurement methods in the general population of Ohasama, Japan.
To examine the relationships between casual, ambulatory and home blood pressure measurements in the general population, these measurements were obtained in 1,695 of 3,744 subjects aged 20 yr or older in Ohasama, Japan. Of these 1,695 subjects, 1,207 measured their home blood pressure more than 14 times in each of the morning and evening (881 untreated subjects including normotensives and untreated hypertensives, 56.4 +/- 11.5 yr of age; 326 treated subjects, 66.0 +/- 9.2 yr of age). We analyzed data in these 1,207 subjects, examining the distribution of each measurement, the relationships among measurements, and the factors affecting the blood pressure differences among the measurements. For systolic pressure, the casual measurement was the highest among the methods examined. The daytime ambulatory measurement was significantly higher than morning and evening home measurements. Morning home measurements were significantly higher than those in the evening. For diastolic pressure, however, the morning home measurement was the highest among the methods examined. Short-term pressure variability (standard deviation and variation coefficient of ambulatory measurements) was greater than long-term pressure variability (standard deviation and variation coefficient of home measurements). The pressure variability in treated subjects was greater than that in untreated subjects. The correlation between casual pressure and the other pressures was not as strong (r<0.567). Among the relationships between ambulatory and home measurements, the strongest correlation was observed between the 24-h ambulatory measurement and the morning home measurement (r=0.738) in untreated subjects. The morning home measurement was highly correlated with the evening home measurement (r>0.814). The differences among the methods examined were affected by blood pressure level and age. It should be noted that in elderly and treated subjects, blood pressure measurement using one method does not necessarily correlate with that obtained using the other methods. This information is useful for the estimation of the value of one type of blood pressure measurement from values obtained with other methods.
Adult↗
Non-invasive assessment of bone quality.
A morphological filter and pipeline analysis was applied to computed radiographs (CR) to abstract the geometrical characteristics of the trabecular pattern and to analyze bone mechanical properties. By applying a morphological filter to the CR images, trabecular patterns were divided into 8 subset groups (n=O through 7) by the trabecular thickness. Sumset images were also derived from the thicker trabeculae group (n=2-5, 3-6, and 4-7) to examine the criteria relating the percentage of thicker trabeculae to the strength of the bone. A method to analyze the bone mechanical properties was developed, based on the hypothesis that data of connective line segments were loaded mechanically. By this method, trabeculae were analyzed in the fifth lumbar vertebrae from etidronic acid-treated and non-treated ovariectomized osteoporosis rat models, and in human third lumbar vertebrae. Consequently, it was shown that total loss of bone mass accompanied a reduction of consecutive trabecular number in the rat osteoporosis model, especially in parts of the bone having thick trabeculae. The reduction in number of connective trabeculae, from one cortical side to the other, occurred at a similar rate, along both the craniocaudal and anteroposterior axes in the vertebral bodies of the experimental osteoporosis rats. In the dynamic study with human third vertebral blocks, no significant correlation was observed between the bone mineral density of the bone specimens and their ultimate strength (R2=0.093). In contrast, the abstracted percentage of thicker trabeculae, derived from the sumset (n=2-5) images, showed a strong correlation with bony mechanical properties (Young's modulus: R2=0.915, maximum point load: R2=0.815). The result calculated by this methodology can serve as an excellent index to indicate bone strength under non-destructive, living conditions.
Aged↗
MARCKS, a major protein kinase C substrate, assumes non-helical conformations both in solution and in complex with Ca2+-calmodulin.
MARCKS, a major cellular substrate for protein kinase C, plays important roles in various cellular functions and its functions are regulated by calmodulin. We have studied the conformational properties of recombinant human MARCKS in solution and in complex with calmodulin. Circular dichroism (CD) spectra showed a high content of random coil in physiological solution. When MARCKS or MARCKS-derived calmodulin-binding peptide was complexed with Ca2+-calmodulin, little change was observed in the CD spectra, suggesting that MARCKS binds with calmodulin in a non-helical conformation, which is unique among the calmodulin-binding proteins.
Amino Acid Sequence↗
An expression system of rat calmodulin using T7 phage promoter in Escherichia coli.
An efficient expression system of rat calmodulin in Escherichia coli is presented. To express rat calmodulin cDNA, we employed a pET expression vector which contains the T7 phage promoter and terminator. After transformation of E. coli BL21(DE3) strain which carries T7 phage RNA polymerase inducible with isopropyl-beta-D-thiogalactopyranoside, induction of the expression, and chromatography of soluble proteins on a phenyl-Sepharose column, about 250 mg of recombinant rat calmodulin was obtained from 1 liter of E. coli culture. The recombinant calmodulin lacked the N-terminal methionine, and posttranslational modifications such as Nalpha-acetylation and methylation. This system facilitates the large amount preparation of calmodulin and the mutant proteins required for the structural analysis by NMR spectrometry and/or X-ray crystallography.
Animals↗
An Escherichia coli protein that exhibits phosphohistidine phosphatase activity towards the HPt domain of the ArcB sensor involved in the multistep His-Asp phosphorelay.
The Escherichia coli sensory kinase, ArcB, possesses a histidine-containing phosphotransfer (HPt) domain, which is implicated in the His-Asp multistep phosphorelay. We searched for a presumed phosphohistidine phosphatase, if present, which affects the function of the HPt domain through its dephosphorylation activity. Using in vivo screening, we first identified a gene that appeared to interfere with the His-Asp phosphorelay between the HPt domain and the receiver domain of OmpR, provided that the gene product was expressed through a multicopy plasmid. The cloned gene (named sixA) was found to encode a protein consisting of 161 amino acids, which has a noticeable sequence motif, an arginine-histidine-glycine (RHG) signature, at its N-terminus. Such an RHG signature, which presumably functions as a nucleophilic phosphoacceptor, was previously found in a set of divergent enzymes, including eukaryotic fructose-2,6-bisphosphatase, E. coli periplasmic phosphatase and E. coli glucose-1-phosphate phosphatase, and ubiquitous phosphoglycerate mutase. Otherwise, the entire amino acid sequences of none of these enzymes resembles that of SixA. It was demonstrated in vitro that the purified SixA protein exhibited the ability to release the phosphoryl group from the HPt domain of ArcB, but the mutant protein lacking the crucial histidine residue in the RHG signature did not. Evidence was also provided that a deletion mutation of sixA on the chromosome affected the in vivo phosphotransfer signalling. These results support the view that SixA is capable of functioning as a phosphohistidine phosphatase that may be implicated in the His-Asp phosphorelay through regulating the phosphorylation state of the HPt domain.
Amino Acid Sequence↗
Differential upregulation of rat Na-K-Cl cotransporter, rBSC1, mRNA in the thick ascending limb of Henle in different pathological conditions.
BACKGROUND: Na-Cl cotransport across the apical membrane of epithelial cells in the thick ascending limb of the loop of Henle (TAL) plays a major role in salt accumulation for hypertonic medullary interstitium. The electroneutral, rat bumetanide-sensitive sodium transporter, rBSC1, is involved in this process. We studied the level of rBSC1 mRNA in dehydration and cardiac failure, since sodium transport in TAL may be enhanced in both conditions in spite of the difference in extracellular fluid accumulation. METHODS: Male Sprague-Dawley rats were deprived of water for 24 hours and myocardial infarction of about 40% of left ventricular circumference was induced in another group of rats that later developed congestive heart failure (CHF). Digoxigenin-labeled cRNA probe for rBSC1 was constructed using polymerase chain reaction (PCR), and Northern blot analysis was performed using RNAs from renal outer medulla. By inducing a point mutation at the middle of PCR product, we compared the amount of rBSC1 transcripts in the renal cortex using competitive PCR, since TAL represents a small fraction of the total cortical tissue. RESULTS: Northern analysis showed a significant increase in rBSC1 mRNA in the renal outer medulla of both dehydrated and CHF rats. In the renal cortex, however, the increase was noted only in CHF by competitive PCR. In situ hybridization using the riboprobe for northern analysis demonstrated that the transcript signal in dehydrated rats was intensified segmentally in TAL located in the inner stripe of outer medulla. Western analysis and immunohistochemistry using a specific antibody against rBSC1 confirmed the distinct segmental enhancement of apical protein expression in dehydration and diffuse enhanced expression in CHF. CONCLUSIONS: rBSC1 is differentially upregulated in different pathological conditions.
Animals↗
Familial lobular glomerulopathy: first case report in Asia.
A 23-year-old male Japanese student presented a unique lobular glomerulopathy characterized by mesangial and subendothelial expansion with numerous periodic acid-Schiff-positive deposits. Electron microscopy showed massive fine granular deposits with a homogeneous distribution. Fibrillar or microtubular structures were not demonstrated. Fibronectin was positive on immunostaining, as was immunoglobulin G and fibrinogen. Familial study revealed that the patient's grandfather, two aunts, and one cousin on his father's side had developed end-stage renal failure. Clinicopathologic features of this patient are identical with those of familial lobular glomerulopathy, which has been previously described by several investigators. Seven of the previously reported families were white and resided in the United States or in European countries. This is the first report of an Asian case, and indicates that this disease universally occurs independently of racial specificity.
Adult↗
Bone mineral density in periodontally healthy and edentulous postmenopausal women.
(Osteoporosis is the most common metabolic disease among postmenopausal women. Reduced masticatory function caused by tooth loss may be a contributing risk factor of osteoporosis. The present study examined the effect of dentate state on skeletal bone mineral density (BMD) in postmenopausal women. Fourteen periodontally healthy dentate subjects (group H; mean age: 64.0 + 5.5 years) and 12 edentulous subjects (group E; mean age: 67.1 + 2.9 years) were randomly selected from the clinics of the departments of Periodontology and Gerodontology, respectively. Informed consent was obtained from all participants. BMD of the lumbar spine (L2-L4) was measured by dual energy x-ray absorptiometry. In addition, occlusal force was measured in 11 group H subjects and 8 group E subjects by using an occlusal diagnostic system. Risk factors associated with osteoporosis including age, calcium intake, physical activity, and cigarette smoking and causes of tooth loss were assessed by interview and questionnaire sent to all participants. The BMD of group H was 1.07 t 0.21 g/cm2 and that of group E was 0.89 + 0.17 g/cm2, which was significantly different(P< 0.05). The occlusal force of group H and E patients was 312.4 + 148 Nand 56.3 + 36 N, respectively, which was significantly different (P< 0.05). Risk factors such as calcium intake, physical activity, and smoking did not differ significantly between the 2 groups. Thus, the periodontally healthy dentate women, who showed about 6 times higher occlusal force than edentulous women, maintained significantly higher BMD of the lumbar spine than edentulous women. Our results suggest that sufficient masticatory function with periodontally healthy dentition may inhibit or delay the progress of osteoporotic change in skeletal bone or that edentulous women may be more susceptible to osteoporosis.
Absorptiometry, Photon↗
[The effect of carbon dioxide on intraorbital hemodynamics in glaucoma determined by color Doppler imaging].
We developed a new system to safely supply carbon dioxide (CO2) to man to investigate the effect of the gas vasodilator on orbital blood flow in open angle glaucoma (OAG) patients. Using the system, we determined orbital hemodynamics in OAG by color Doppler imaging (CDI) at baseline conditions and during CO2 supplementation sufficient to increase end-tidal CO2% by 10%. Seven OAG patients (mean age, 60.9 +/- 16.4 years; normal-tension glaucoma/primary open-angle glaucoma = 5/2) were included in the study. CDI was performed to measure resistance index (RI), and peak-systolic and end-diastolic blood flow velocities (PSV & EDV) of the ophthalmic artery (OA) and the central retinal artery (CRA). Systemic conditions including oxygen saturation and blood pressure were monitored throughout the period of the CO2 inhalation. CO2 significantly increased PSV and EDV in the CRA (p = 0.0273, p = 0.0094, respectively; Wilcoxon signed-rank test), but not in the OA. Other parameters were not altered. The results suggest that CO2 inhalation increases blood flow velocities in distal arteries in OAG patients without affecting proximal vessels. The new system enables us to supply CO2 in a safe and controlled manner in glaucoma patients and to modify orbital hemodynamics.
Administration, Inhalation↗
High production of nondisjunction mutants in the offspring of Drosophila melanogaster females exposed to carbon dioxide at meiosis I.
Drosophila melanogaster females homozygous for X-linked recessive markers, y and wi, were exposed to CO2 and mated with y+ w+/Y males. The progeny were sampled and inspected for y wi/y wi/Y (XXY) and y+ w+/O (XO) mutants. The frequency of nondisjunction XXY mutants after a 90-min exposure to CO2 increased 100-fold above the control level in the first-day brood but did not increase above the control level in the second to sixth broods, showing that CO2 is an extremely potent inducer of nondisjunction in mature oocytes during meiotic metaphase I but is not harmful to immature oocytes. Nondisjunction-causing damage induced by CO2 in mature oocytes disappeared completely within one day after CO2 treatment, as evidenced by a reduction of the number of XXY mutants to the control level when the mating of CO2-treated females was delayed by one day. CO2-induced nondisjunction is probably due to damage to spindle microtubules in mature oocytes at metaphase I. N2 is a less potent inducer of nondisjunction than CO2. Maternal X-irradiation with 4 Gy did not induce XXY mutants, showing that medium-level radiation does not induce nondisjunction. The results support Gaulden's hypothesis that oxygen deficits and CO2 increases in the microenvironment of mature oocytes can be potent inducers of nondisjunction. The possible relationship to the cluster of Down syndrome seen in Berlin shortly after the Chernobyl accident is discussed.
Aneuploidy↗
A frequency-doubling perimetric study in normal-tension glaucoma with hemifield defect.
PURPOSE: The authors prospectively compare perimetric results obtained by conventional differential light sensitivity (DLS) perimetry and frequency-doubling perimetry (FDP) in normal-tension glaucoma (NTG) with a hemifield visual field defect, assuming that some of these eyes already have early glaucomatous changes in the "intact" hemifield. METHODS: Eleven eyes of 11 patients with NTG who met the enrollment criteria were studied. Seven eyes had an upper hemifield defect and four had a lower defect, determined by DLS with a Humphrey Field Analyzer (Zeiss-Humphrey, Inc., San Leandro, CA, U.S.A.). Frequency-doubling perimetry and DLS perimetry were conducted less than 7 days apart. To quantitatively detect nerve fiber layer defect (NFLD), scanning laser ophthalmoscopy was also performed. RESULTS: Of the 11 intact hemifields, seven showed NFLD. Of the seven, six also showed visual field defects by FDP. Of the four eyes without NFLD, two showed no defects in the intact hemifield by FDP. The threshold values of FDP and DLS perimetry did not significantly correlate in either the intact or the defective hemifields, or in the total field. The average test time of FDP was approximately 6 minutes. CONCLUSIONS: Frequency-doubling perimetry may detect early glaucomatous visual field defects.
Adult↗
Circular dichroism and 1H NMR studies on the structures of peptides derived from the calmodulin-binding domains of inducible and endothelial nitric-oxide synthase in solution and in complex with calmodulin. Nascent alpha-helical structures are stabilized by calmodulin both in the presence and absence of Ca2+.
There exist two types of nitric-oxide synthase (NOS); constitutive isozymes that are activated by binding calmodulin in response to elevated Ca2+ and an inducible isozyme that binds calmodulin regardless of Ca2+. To study the structural basis of the difference in Ca2+ sensitivity, we have designed synthetic peptides of minimal lengths derived from the calmodulin-binding domain of endothelial NOS (eNOS) and that of macrophage NOS (iNOS). A peptide, KRREIPLKVLVKAVLFACMLMRK, derived from human iNOS sequence, retained the ability to bind to calmodulin both in the presence and absence of Ca2+, while a peptide derived from human eNOS sequence, RKKTFKEVANAVKISASLMG, bound to calmodulin only in the presence of Ca2+. Circular dichroism and two-dimensional 1H nuclear magnetic resonance studies suggested that both peptides assume nascent alpha-helical structures in aqueous solution. When mixed with calmodulin, both peptides showed circular dichroism spectra characteristic for alpha-helix. In contrast to other target proteins, the addition of iNOS peptide to calmodulin did not affect the Ca2+ binding of calmodulin appreciably. The peptide derived from the calmodulin-binding domain of iNOS, therefore, binds in alpha-helical structures both to Ca2+-calmodulin and apo-calmodulin, which is unique among various target proteins of calmodulin.
Amino Acid Sequence↗
Circular dichroism and 1H nuclear magnetic resonance studies on the solution and membrane structures of GAP-43 calmodulin-binding domain.
Growth-associated protein-43 (GAP-43) is believed to be palmitoylated near the N terminus and the modification is assumed to be involved in the membrane anchoring of the protein. However, GAP-43 isolated from bovine brain is not palmitoylated as shown by mass spectrometric analysis, but still retains the ability to bind phospholipids, suggesting that other parts of the molecule are involved in the interaction. Upon addition of acidic phospholipids, purified GAP-43 showed a conformational change from random coil to alpha-helix as indicated by a change in CD spectra. A synthetic peptide corresponding to the calmodulin-binding domain showed a similar conformational change from random coil to alpha-helix in the presence of various acidic phospholipids. These results suggest that the calmodulin-binding domain of GAP-43 is directly involved in the GAP-43-membrane interaction and undergoes a conformational change upon binding to phospholipid membranes. After phosphorylation by protein kinase C, the phospholipid-induced conformational changes were no longer observed. Structural characteristics of the calmodulin-binding domain peptide in aqueous and hydrophobic solvents were further studied in detail by two-dimensional 1H nuclear magnetic resonance. The results obtained suggest that the domain assumes a nascent alpha-helical structure in aqueous solution, which is stabilized under hydrophobic environments.
Animals↗
[A study of the effects of antidepressants on the GABAA receptor and its complex based on the drug actions on the power-spectral changes of rat hippocampal EEG induced by GABA antagonists and inverse agonists].
To analyze the effects of antidepressants on the GABAA receptor, we investigated how the chronic administration of antidepressants (10 mg/kg twice a day for three or seven days, ip) influenced the power-spectral changes induced by pentylenetetrazol (PTZ; a GABA antagonist; 27.5 mg/kg) or beta-carboline-3-carboxylic-acid-methylester (beta-CCM; an inverse agonist; 1 mg/kg) on rat hippocampal EEGs. PTZ and beta-CCM are known to inhibit the chloride ionophore and benzodiazepine receptor (GABAA receptor complex), respectively. After the ip injection of both compounds, the EEG power under 12 Hz increased to about five times that before injection. Between the rats that did not receive any antidepressants and all those injected with the drugs for 3 days or treated with desipramine (DMI) for 7 days, there were no apparent changes in the effect of PTZ or beta-CCM. However, in the rats treated with imipramine, fluoxetine or trazodone for 7 days, the increase in power after the injection of PTZ or beta-CCM was apparently suppressed. In these rats, the power values were less than three times those before the dosing of PTZ or beta-CCM. DMI is known to inhibit the re-uptake of norepinephrine (NE), while the other three antidepressants inhibit that of serotonin (5-HT). Trazodone is also reported to block the 5-HT2 sites. These observations might indicate that the chronic administration of antidepressants prompted the function of the GABAA receptor complex. Moreover, it is also suggested that, to that action, the effect of antidepressants on the 5-HT system or interaction between the 5-HT system and GABA receptors might play some role.
Animals↗
Interaction of calmodulin-binding domain peptides of nitric oxide synthase with membrane phospholipids: regulation by protein phosphorylation and Ca(2+)-calmodulin.
Endothelial nitric oxide synthase (eNOS) is unique among the NO synthase isozymes in being modified with myristoyl group, which appears to be necessary for its membrane association. However, the presence of myristoylated eNOS in cytosolic fraction after the stimulation-dependent translocation of the enzyme from membrane to cytosol suggests that other regions may be involved in the eNOS-membrane interaction and its regulation. In this study, we have synthesized a 20-amino acid peptide corresponding to the putative calmodulin-binding domain of human eNOS and studied the interaction of the peptide with calmodulin and with various membrane phospholipids. The peptide formed a stoichiometric complex with calmodulin. Upon addition of various acidic phospholipids, the peptide showed a drastic conformational change from random coil to alpha-helix, as was evidenced by circular dichroism spectroscopy. These results suggest that the same domain of eNOS binds both calmodulin and membrane phospholipids. Furthermore, we found that the synthetic peptide was phosphorylated in vitro by protein kinase C. Phosphorylation of the peptide decreased its interaction with membrane phospholipids. Thus, our results raise the possibility that the calmodulin-binding domain is directly involved in the membrane association of eNOS and that phosphorylation of the domain and Ca(2+)-calmodulin may regulate the interaction. Synthetic peptides corresponding to the calmodulin-binding domains of macrophage and neuronal isozymes showed similar abilities to bind phospholipids, suggesting that the calmodulin-binding domains of NO synthase serve as the phospholipid-binding domains as well.
Amino Acid Sequence↗
Participation of endodermal epithelial cells on the synthesis of plasma LDL and HDL in the chick yolk sac.
We ultrastructually examined the chick yolk sac endodermal epithelium and evaluated our findings in combination with the biochemical analysis of serum and yolk lipoproteins. Twenty-five to 30 nm-sized particles were demonstrated to be a principal element of the extracellular yolk mass and these were determined to be yolk very low density lipoprotein (VLDL). The particles were shown to be taken up by the epithelial cells via coated pits and engulfed by plasma membrane invaginations together with yolk subdroplets, another element of the yolk mass. Through apical vacuoles, the two yolk elements were incorporated into yolk drops, which were identified to be one of the lysosomal structures by a cytochemical procedure using acid phosphatase (AcP)ase activity. During the last week of incubation, which is the final third of the incubation period, the digestion seemed to progress rapidly in the yolk drops, which came to resemble lipolysosomes; lipoprotein production became active as expressed by an enlarged Golgi apparatus. The newly produced lipoprotein particles were electron-lucent and irregular in size (50-120 nm). They were sequestered in secretory vacuoles and secreted from the vascular surface of the epithelial cells. Finally, the particles were thought to be taken into the vitelline circulation as plasma lipoproteins. The major component of lipoprotein in serum was determined to be low density lipoprotein (LDL) and high density lipoprotein (HDL), while cholesterol content was found to increase during incubation. We concluded that endodermal epithelial cells participate the synthesis of plasma LDL and HDL. For this synthesis the cells probably apply lipids and apo-protein generated from yolk VLDL degradation.
Acid Phosphatase↗
Site-specific phosphorylation of synapsin I by mitogen-activated protein kinase and Cdk5 and its effects on physiological functions.
Posttranslational modifications of synapsin I, a major phosphoprotein in synaptic terminals, were studied by mass spectrometry. In addition to a well known phosphorylation site by calmodulin-dependent protein kinase II (CaM kinase II), a hitherto unrecognized site (Ser553) was found phosphorylated in vivo. The phosphorylation site is immediately followed by a proline, suggesting that the protein is an in vivo substrate of so-called proline-directed protein kinase(s). To identify the kinase involved, three proline-directed protein kinases expressed highly in the brain, i.e. mitogen-activated protein (MAP) kinase, Cdk5-p23, and glycogen synthase kinase 3beta, were tested for the in vitro phosphorylation of synapsin I. Only MAP kinase and Cdk5-p23 phosphorylated synapsin I stoichiometrically. The phosphorylation sites were determined to be Ser551 and Ser553 with Cdk5-p23, and Ser62, Ser67, and Ser551 with MAP kinase. Upon phosphorylation with MAP kinase, synapsin I showed reduced F-actin bundling activity, while no significant effect on the interaction was observed with the protein phosphorylated with Cdk5-p23. These results raise the possibility that the so-called proline-directed protein kinases together with CaM kinase II and cAMP-dependent protein kinase play an important role in the regulation of synapsin I function.
Actins↗