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Biomedical subjects

Y Tashiro

Publications and source records attributed to Y Tashiro.

At least 181 records · Page 10Linked to original sources
PubMed1985

Two chymotrypsin-susceptible sites of myosin rod from chicken gizzard.

The rod prepared from chicken gizzard myosin has been found to have two sites sensitive to limited digestion with chymotrypsin; these sites were located at a subfragment 2/light meromyosin junction (site 1), and at a site 10 kDa remote from either C-terminal or N-terminal of light meromyosin (site 2). The site 1 was more sensitive to the digestion than the site 2. The cleavage at site 2 of the light meromyosin yielded a 74-kDa fragment that was soluble in a low ionic strength solution, contrary to the insolubility of the parent light meromyosin in the same solution. Studies on the effects of MgCl2, ATP and pH on the susceptibilities of these sites to chymotrypsin have given following results. (a) Millimolar concentrations of MgCl2 protected site 1 and site 2 from the chymotryptic cleavage. (b) The cleavage at site 1 of myosin rod in the low salt solution free of Mg2+ at pH 7.0 and pH 8.5, was not affected by the presence of 5 mM ATP. However, MgCl2-induced protection of site 1 was relieved by addition of ATP. On the other hand, the cleavage at site 2 was stimulated by addition of ATP, irrespective of the presence or absence of MgCl2. (c) The alkaline condition of pH 8.5 was more favorable for the chymotryptic cleavages at both site 1 and site 2 than the neutral condition of pH 7.0. These results suggest that myosin rod contains two flexible regions, the structures of which are influenced by such an ambient factor as MgCl2, ATP or pH.

Adenosine Triphosphate↗
PubMed1985

Phosphorylation of the rat hepatocyte asialoglycoprotein receptor.

Phosphorylation of asialoglycoprotein receptor was investigated by using rat hepatocytes. Analysis of the purified receptor by SDS-PAGE and autoradiogram revealed that the 64 and 54 Kd polypeptides of the receptor were phosphorylated but the 43 Kd one was not and that phosphorylation took place at the cell surface. These results are compatible with the fact that the 64 and 54 Kd species exist predominantly at the cell surface. The sites of phosphorylation were identified as Ser and Thr with no detectable radioactivity in phosphotyrosine.

Amino Acids↗
PubMed1985

Is cytochrome P-450 transported from the endoplasmic reticulum to the Golgi apparatus in rat hepatocytes?

The Golgi apparatus mediates intracellular transport of not only secretory and lysosomal proteins but also membrane proteins. As a typical marker membrane protein for endoplasmic reticulum (ER) of rat hepatocytes, we have selected phenobarbital (PB)-inducible cytochrome P-450 (P-450[PB]) and investigated whether P-450(PB) is transported to the Golgi apparatus or not by combining biochemical and quantitative ferritin immunoelectron microscopic techniques. We found that P-450(PB) was not detectable on the membrane of Golgi cisternae either when P-450 was maximally induced by phenobarbital treatment or when P-450 content in the microsomes rapidly decreased after cessation of the treatment. The P-450 detected biochemically in the Golgi subcellular fraction can be explained by the contamination of the microsomal vesicles derived from fragmented ER membranes to the Golgi fraction. We conclude that when the transfer vesicles are formed by budding on the transitional elements of ER, P-450 is completely excluded from such regions and is not transported to the Golgi apparatus, and only the membrane proteins destined for the Golgi apparatus, plasma membranes, or lysosomes are selectively collected and transported.

Animals↗
PubMed1985

Intracellular transport of a newly synthesized asialoglycoprotein receptor in rat liver.

Intracellular transport of a newly synthesized asialoglycoprotein receptor was studied biochemically using a monospecific antibody for the receptor. Pulse-labeling by intravenous injection of [3H]leucine and pulse-chasing after 10 min by cycloheximide injection resulted in the maximal labeling of the receptor in the rough microsomes at 15 min, in the smooth microsomes and the heavy Golgi subfraction (GF3) at 25 min and in the intermediate plus light Golgi subfraction (GF1+2) at 30 min. By 60 min, the labeling in GF1+2 had decreased and leveled off. In the plasma membrane fraction, the labeled receptor first appeared at 20 min, increased rapidly and also reached a constant level at 40-60 min. Intracellular movement of the newly synthesized receptor in the GF1+2 and plasma membrane fractions was also investigated by purifying the receptor protein from the GF1+2 and plasma membrane fractions by affinity chromatography. It was revealed that the specific radioactivities of the receptor in the two fractions become equilibrated after 60-120 min. The receptor of the various membrane fractions was also pulse-labeled in vivo for 20 min simultaneously with [3H]glucosamine and [14C]leucine, and pulse-chased for the following 40 min. After pulse-labeling for 20 min, the ratio of the radioactivity of [3H]glucosamine or [3H]sialic acid to [14C]leucine of the receptor from the rough and smooth microsomes, and GF3, GF2, and GF1 increased in that order. That of the receptor from the plasma membrane fraction was infinitely higher, because, while a significant amount of 3H-radioactivity was incorporated into the receptor in the Golgi apparatus, only a negligible amount of 14C-radioactivity was incorporated into the same receptor in the plasma membrane due to the delay in the arrival of [14C]leucine labeled receptor to the plasma membrane. After chasing for 40 min, however, the same radioactivity ratios of the GF1 and plasma membrane fractions approached each other. All these results strongly suggest that the distribution of the newly synthesized receptor becomes rapidly equilibrated between the trans-Golgi components and plasma membranes probably by repeated recycling of the receptor protein between the two membranes.

Animals↗
PubMed1985

[Analysis of 53 ruptured middle cerebral artery aneurysms].

In spite of a recent remarkable progress in operative results of ruptured middle cerebral artery aneurysms, a mortality rate of 2-8% appears to be unavoidable. In the present study, 53 ruptured MCA aneurysms were retrospectively analyzed to determine causative factors of unfortunate outcome (fair, poor and dead). Surgical results of 53 ruptured MCA aneurysms are shown in Table 1, where the outcome was unfortunate in 7 cases (17%). Intracerebral hematoma was responsible for 4 cases, two of which were fatal and postoperative vasospasm for 3 cases. There were 13 cases with intracerebral hematomas (25%) ranging from 21 mm to 68 mm in diameter. Although hematomas less than 40 mm in diameter localized in temporal or frontal subcortical areas and yielded no neurological deficits, those more than 60mm extended to the caudate nucleus or thalamus through the internal capsule and led to deep coma (Table 2, Fig. 1). Intracerebral hematoma with the diameter between 50 to 60 mm seems to be critical in regard to postoperative outcome. Repeated rupture caused intracerebral hematoma (50%) more frequently than single rupture (21%) and aneurysm with intracerebral hematoma was liable to bleed (27%), resulting in acute deterioration of neurological conditions by marked enlargement of the hematoma (Fig. 2). Accordingly it is essential for the cases with intracerebral hematoma to prevent rerupture. Subarachnoid hemorrhage and symptomatic vasospasm were observed less frequently in hematoma group than in non-hematoma group. However, prophylactic treatment of vasospasm is important even in the cases with intracerebral hematoma since more than half of them suffer from relatively thicker subarachnoid clot.

Adult↗
PubMed1984

Requirement of the 20-kDa light chain for the papain-resistant conformation of gizzard myosin.

The limited chymotryptic digestion of unphosphorylated gizzard myosin in 0.15 M NaCl converted a papain-insensitive myosin in ATP to a papain-sensitive one. This conversion without phosphorylation of its 20-kDa light chain was accompanied with truncation of a 200-kDa heavy chain to a 195-kDa fragment and with the degradation of a 20-kDa light chain. Papain also yielded the 195-kDa fragment from the heavy chain, irrespective of the presence or absence of ATP. However, the ATP-induced protection of unphosphorylated myosin from the papain-digestion disappeared concurrently with degradation of the 20-kDa light chain by papain rather than the truncation of heavy chain. Papers from two laboratories [Onishi, H. & Watanabe, S. (1984) J. Biochem. (Tokyo) 95, 903-905; Kumon, A., Yasuda, S., Murakami, N., and Matsumura, S. (1984) Eur. J. Biochem. 140, 265-271] have reported that the ATP-protection of unphosphorylated myosin against papain is not observed after the 20-kDa light chain has been phosphorylated. The present results might indicate that the ATP-induced protection is also abolished through the chymotryptic degradation of the 20-kDa light chain.

Adenosine Triphosphate↗
PubMed1984

The phosphorylation site for casein kinase II on 20,000-Da light chain of gizzard myosin.

The 20-kDa light chain isolated from gizzard myosin has recently been reported to be phosphorylated by casein kinase II at a site distinct from that phosphorylated by Ca2+- and calmodulin-dependent myosin light-chain kinase. In the present study, the site phosphorylated by casein kinase II has been analyzed through procedures including tryptic digestion of the radioactively phosphorylated light chain and CNBr cleavage of the purified tryptic phosphopeptide, followed by amino acid analysis of these phosphopeptides. Comparison of the amino acid compositions of these peptides with the previously reported sequence has indicated that the phosphorylation site is threonine-134 of the light chain. The significance of the phosphorylation of the light chain by casein kinase II, as well as the substrate specificity of the protein kinase, is discussed on the basis of the result.

Amino Acids↗
PubMed1984

Morphological changes in myofibrils and glycerinated muscle fibers on treatment with cathepsins D and L.

Morphological changes in myofibrils and glycerinated muscle fibers on treatment with cathepsins D and L obtained from rabbit skeletal muscle were studied by phase contrast and electron microscopy. Cathepsin D degraded the Z-line and filaments adjacent to the H-zone. Cathepsin L caused the fragmentation of myofibrils, and degraded the Z-line and M-line. Both cathepsins induced disturbance of the lateral arrangement of myofibrils.

Animals↗
PubMed1984

Ferritin immunoelectron microscopic localization of 5'-nucleotidase on rat liver cell surface.

Rat livers were prefixed by perfusion with 0.6% glutaraldehyde and briefly homogenized with a Teflon-glass homogenizer. The prefixed cells isolated by low-speed centrifugation in high yield effectively preserved the original polygonal shape and polarity. These cells were incubated with ferritin-antibody conjugates monospecific for rat liver 5'-nucleotidase, and the localization of the enzymes on the surface of hepatocytes and endothelial cells was quantitatively investigated. It was revealed that the surface density of 5'-nucleotidase is much higher on the bile canalicular surface than on the sinusoidal surface and only a few ferritin particles were detected on the lateral surface. On the bile canalicular surface ferritin particles were almost exclusively found on the microvilli in larger clusters. Similar distribution was also observed on the sinusoidal surface but the size of cluster was much smaller. On both surfaces many fewer ferritin particles were found on the intermicrovillar region, including the coated pits region, than on the microvillar region. Ferritin particles were also found on the endothelial cell surface.

5'-Nucleotidase↗
PubMed1984

Quantitative immunoferritin localization of [Na+,K+]ATPase on canine hepatocyte cell surface.

Distribution of [Na+,K+]ATPase on the cell surface of canine hepatocytes was investigated quantitatively by incubating prefixed and dissociated liver cells with ferritin antibody conjugates against canine kidney holo[Na+,K+]ATPase. We found that [Na+,K+]-ATPase exists bilaterally both on the bile canalicular and sinusoid-lateral surfaces. The particle density on the bile canalicular surface was much higher (approximately 2.5 times) than that on the sinusoid-lateral surface. In the latter region, the enzyme was detected almost equally both on the sinusoidal and lateral surfaces. On all the surfaces, the distribution of the enzyme was homogeneous and no clustering of the enzyme was detected. Total number of the enzyme on the sinusoid-lateral surface was, however, approximately three times higher than that on the bile canalicular region, because the sinusoid-lateral surface represents approximately 87% of the total cell surface of a hepatocyte. We suggest that the [Na+, K+]ATPase on the bile canalicular surface is responsible for the bile acid-independent bile flow and the other transport processes on the bile canalicular cell surface, while that on the sinusoid-lateral surface is responsible not only for the active transport of Na+ but also for the secondary active transport of various substances in this region.

Animals↗
PubMed1984

Glycosylation of a receptor specific for asialoglycoproteins in rat liver.

Glycosylation of a rat receptor specific for asialoglycoproteins was investigated in vivo by using monospecific antibody. After intravenous injection of [3H]mannose, the receptor protein was immunoprecipitated from various subcellular fractions and the glycopeptide and oligosaccharide chains of the protein were prepared by treatment with pronase and endo-beta-N-acetylglucosaminidase H. The glycopeptides thus prepared from the rough and smooth microsomes and Golgi heavy fraction (GF3) were all sensitive to endo H and most of the endo H-sensitive oligosaccharides were eluted at the position considered to correspond to Man8GlcNAc on high-resolution Bio-Gel chromatography. Endo H-resistant forms were first detected in the Golgi intermediate fraction (GF2) and significantly in the light Golgi fraction (GF1), suggesting the formation of the complex-type oligosaccharide chains in the latter fraction. This view was also supported by the almost exclusive addition of the terminal sugars such as N-acetylglucosamine, galactose, and sialic acid in GF1. These results suggest that the major form of the oligosaccharide chains from the endoplasmic reticulum to the Golgi apparatus is Man8GlcNAc2 and that the processing of these large mannosyloligosaccharide chains and subsequent addition of terminal sugars to them are performed successively in the trans-Golgi region.

Animals↗
PubMed1984

Topology of asialoglycoprotein receptor in rat liver subcellular fractions: a ferritin immunoelectron microscopic study.

Direct ferritin immunoelectron microscopy was used to visualize the asialoglycoprotein receptor in various rat liver subcellular fractions. The cytoplasmic surfaces of cytoplasmic organelles such as the rough and smooth microsomes, Golgi cisternae and lysosomes showed hardly any ferritin label exception for the slight labeling of secretory granules found mainly in the light Golgi fraction (GF1). Occasionally, however, open membrane sheet structures, smooth vesicular or tubular structures heavily labeled with ferritin, were present in all these subcellular fractions. These structures probably correspond to fragmented sinusoidal or lateral hepatocyte plasma membranes recovered to these subcellular fractions. When the limiting membranes of the secretion granules were partially broken by mechanical force, a number of ferritin particles frequently were seen attached in large clusters to the luminal surface of the membrane, the cytoplasmic surface of the corresponding domain being slightly labeled. These observations are strong evidence that the receptor protein is never translocated vertically throughout the intracellular transport from ER to plasma membrane via Golgi apparatus and from plasma membrane back to trans-Golgi elements and also in lysosomes, always exposing the major antigenic sites to the luminal or extracellular surface and the minor counterparts to the cytoplasmic surface of the membranes. The receptor protein also is suggested to be concentrated in clusters on the luminal surface of secretion granules when they form on the trans-side of the Golgi apparatus.

Animals↗
PubMed1984

A biochemical and electron microscopic study of changes in the content of cytochrome P-450 in rat livers after cessation of treatment with phenobarbital, beta-naphtoflavone or 3-methylcholanthrene.

Changes in rat livers after treatment with phenobarbital (PB), beta- naphtoflavone (beta NF) or 3-methylcholanthrene (MC) was stopped were investigated by biochemical, immunochemical and electron microscopic methods. Repeated injections of each drug led to increases in the relative liver weights, in the microsomal proteins per gram of liver, in the microsomal contents of the total cytochrome P-450, and in the corresponding molecular species of P-450; P-450(PB), when treated with PB and P-450(MC) when treated with either beta NF or MC. After cessation of treatment with PB or beta NF, decreases in all these contents to what were essentially the control values took place approximately in parallel within 7 days. The apparent half lives of cytochrome P-450(PB) and P-450(MC) were calculated from the regression lines of cytochrome P-450 as approximately 15 hours. After treatment with MC, however, all the increases were maintained at approximately the maximal values even after 9 days. Electron microscopy revealed that the smooth ER, which markedly proliferated on PB or beta NF treatment, decreased rapidly after the drug treatment stopped, and was accompanied by an increase in the number of autophagosomes. After treatment with MC, however, the proliferated smooth ER remained even after 9 days.

Animals↗
PubMed1984

Hyperasialoglycoproteinemia in patients with chronic liver diseases and/or liver cell carcinoma. Asialoglycoprotein receptor in cirrhosis and liver cell carcinoma.

The pathogenesis of hyperasialoglycoproteinemia in cirrhosis and liver cell carcinoma was investigated by measuring the amount of asialoglycoprotein receptor in hepatic tissues from 30 patients with either cirrhosis or a malignancy. The asialoglycoprotein receptor activity in the cirrhotic liver was 770 +/- 423 U/g liver, i.e., 28% of the control value (2770 +/- 731 U/g liver). Receptor activity was negligible in tissues from patients with either liver cell carcinoma or metastatic tumor. The receptor levels in noncirrhotic and cirrhotic tissues in close proximity to liver cell carcinoma were 2281 +/- 659 and 1134 +/- 473 U/g liver, respectively. There was a close correlation between serum asialoglycoprotein levels and the size of the tumor mass in patients with liver cell carcinoma. Our results suggest that decreases in the asialoglycoprotein receptor levels may lead to an accumulation of serum asialoglycoproteins in patients with cirrhosis or liver cell carcinoma, or both.

Asialoglycoprotein Receptor↗