Biomedical subjects

E Kominami

Publications and source records attributed to E Kominami.

At least 127 records · Page 7Linked to original sources

PubMed1993

Lysosomal cysteine and aspartic proteinases, acid phosphatase, and an endogenous cysteine proteinase inhibitor, cystatin-beta, in rat osteoclasts.

To understand the bone resorption and lysosomal proteinases in osteoclasts, we examined by immunohistochemistry the localization of lysosomal cysteine and aspartic proteinases, acid phosphatase, and cystatin-beta in the rat tibial bone. Immunoreactivity for cathepsins B, C, H, and L, cathepsin D, acid phosphatase, and cystatin-beta was demonstrated in various cells of the bone tissue; in particular, large multinucleated osteoclasts attached to the bone surface and chondroclasts in the proximal growth plate. These cells showed intense immunoreactivity for these lysosomal enzymes and cystatin-beta. Bone surface-lining osteoblasts displayed distinct immunoreactivity for cathepsins B, C, D, H, and acid phosphatase, while osteocytes often exhibited that for cathepsins D, H and acid phosphatase. Chondrocytes in the growth plate demonstrated intense immunoreactivity for cathepsins B, D, and acid phosphatase. Immunoreactivity for cystatin-beta was detected in osteoclasts and chondroclasts only. Large, round multinucleated cells free from the bone surface exhibited weak, faint, or no immunoreactivity for the lysosomal enzymes and cystatin-beta. These results suggest that lysosomal cysteine and aspartic proteinases may play a role in the degradation of organic constituents of the bone matrix. Moreover, cystatin-beta can serve as an excellent marker protein for osteoclasts.

Acid Phosphatase↗

PubMed1993

Partial characterization of a rabbit liver Ca(2+)-calmodulin-dependent kinase with myosin light chain phosphorylating activity.

A Ca(2+)-calmodulin-dependent protein kinase that phosphorylates the regulatory light chain of hepatocyte myosin was purified from rabbit liver. The kinase catalyzed the incorporation of phosphate into the 22-k light chains of hepatocyte myosin, resulting in a 7-fold activation of the Mg(2+)-ATPase activity by F-actin. The kinase did not show any glycogen synthase kinase activity which has previously been shown to phosphorylate isolated chicken gizzard myosin light chain. ML-7, an inhibitor specific for smooth muscle myosin light chain kinase, inhibited the liver kinase with a Ki value of 13.2 microM.

Actins↗

PubMed1992

Structural organization of the gene encoding rat cystatin beta.

A genomic DNA clone encompassing the gene (cy beta) encoding rat cystatin beta (Cy beta) was isolated by screening with a rat cy beta cDNA as a probe. The gene spans about 2.6 kb and comprises three exons. The first intron is located between Lys22 and Val23 and the second between Lys56 and Val57 in the deduced amino acid sequence of Cy beta. The second exon contains the highly conserved QVVAG sequence which, unlike the sequence of other cystatin family members, is not split by an intron. In the 5'-upstream region, three SP-1-binding sites exist, but no typical TATA-box or CAAT-box sequences are found. The difference in the organization of the rat cy beta gene, encoding a family-1 cystatin, from that encoding members of the other cystatin families, suggests that cy beta diverged from a common ancestral gene earlier than the separation of genes encoding family-2 and family-3 cystatins.

Amino Acid Sequence↗

PubMed1992

Immunocytochemical localization of a cysteine protease in adult worms of the liver fluke Fasciola sp.

The intracellular localization of a cysteine protease from Fasciola sp. that hydrolyzes host hemoglobin as a nutritional source was examined by light and electron microscopic immunocytochemistry using a monoclonal antibody specific to the enzyme. Immunoperoxidase staining was predominantly restricted to large numbers of granules in the parasite intestinal epithelial cells and to host erythrocytes present in the intestinal lumens. In immunogold electron microscopy, the gold particles were consistently deposited on the electron-dense secretory granules in intestinal epithelial cells and on the intestinal contents. These findings suggest that the Fasciola cysteine protease in the secretory granules is secreted as a digestive enzyme into the intestinal lumen, where it may play an important role in the extracellular degradation of host proteins, including hemoglobin.

Animals↗

PubMed1992

Specific storage of subunit c of mitochondrial ATP synthase in lysosomes of neuronal ceroid lipofuscinosis (Batten's disease).

Immunochemical studies demonstrated the specific accumulation of subunit c of mitochondrial ATP synthase in the brain homogenates of late infantile and juvenile forms of Batten's disease. It is not stored in the infantile form. Storage of subunit alpha of mitochondrial ATP synthase and cytochrome c oxidase subunit IV, an inner membrane protein of mitochondria was not detected in the brains. There was also no difference in the levels of cathepsin B between the two forms of Batten's disease and controls. In cultured skin fibroblasts subunit c accumulates in the late infantile form, whereas it does not in other lysosomal storage diseases. Crude mitochondrial lysosomal preparations of control fibroblasts were separated into high-density fractions rich in a lysosomal marker and low-density fractions rich in a mitochondrial marker on Percoll density gradients. Subunit c was mostly recovered in low-density mitochondrial fractions, but in cells from the late infantile disease a part of subunit c was recovered in the high-density lysosomal fractions. Immunolocalization studies demonstrated a dot-like staining of storage materials for subunit c in the cells from late infantile patients and the staining pattern of subunit c is similar to that of a lysosomal membrane marker, lgp120. Immunostaining failed to detect subunit c in control cells. These results indicate a specific accumulation of subunit c in lysosomes, and suggest that the two forms of Batten's disease are caused by a specific failure in the degradation of subunit c.

Blotting, Western↗

PubMed1992

The primary structure and tissue distribution of cathepsin C.

A cDNA for rat cathepsin C (dipeptidylaminopeptidase I) was isolated. The encoded protein is composed of the signal peptide of 28 residues, the propeptide of 201 residues and the mature enzyme region of 233 residues. The amino acid sequence of the mature enzyme region has 39.5 to 30.5% identity to other papain family proteinases. Cathepsin C is, therefore, belongs to papain family, although its propeptide region is much longer than those of other cysteine proteinases and show no significant sequence similarity to any other cysteine proteinase. The mRNA and protein for cathepsin C are broadly distributed in rat tissues, but the relative proportions of cathepsin C and other cysteine proteinases are found to vary from tissue to tissue.

Amino Acid Sequence↗

PubMed1992

Changes in subcellular structures of parietal cells in the rat gastric gland after omeprazole.

Omeprazole, an inhibitor of gastric acid secretion, was administered to rats at a dosage of 20 mg/kg/day for 14 and 35 days, and subsequent changes in subcellular structures of parietal cells were analyzed using morphometry and immunocytochemistry. Plasma gastrin levels were also examined, showing two times higher levels in the experimental groups than in the non-treated control. The volume and surface densities significantly decreased in tubulovesicles of the cells in the experimental rats. In the long term treatment of omeprazole (35 days), the volume density of microvilli on the membranes of secretory canaliculi in the cells also decreased significantly, whereas that of lysosomes clearly increased. By electron microscopy, many dense bodies of various shapes often appeared in the cytoplasm of parietal cells after the omeprazole treatment. Immunocytochemistry revealed that large granular immunodeposits for cathepsin B increase in the epithelial cells of the gastric glands after omeprazole treatment. These results suggest that omeprazole induces quantitatively significant decreases in both tubulovesicles and canalicular microvilli. The decreases in these membrane structures may possibly be ascribed to the degradation of the membrane in lysosomes; the proton pump on the membranes bound irreversibly with omeprazole is believed destined to be degraded in lysosomes.

Animals↗

PubMed1991

Lysosomal hydrolases of different classes are abnormally distributed in brains of patients with Alzheimer disease.

beta-Amyloid formation requires multiple abnormal proteolytic cleavages of amyloid precursor protein (APP), including one within its intramembrane domain. Lysosomes, which contain a wide variety of proteases (cathepsins) and other acid hydrolases, are major sites for the turnover of membrane proteins and other cell constituents. Using immunocytochemistry, immunoelectron microscopy, and enzyme histochemistry, we studied the expression and cellular distributions of 10 lysosomal hydrolases, including 4 cathepsins, in neocortex from patients with Alzheimer disease and control (non-Alzheimer-disease) individuals. In control brains, acid hydrolases were localized exclusively to intracellular lysosome-related compartments, and 8 of the 10 enzymes predominated in neurons. In Alzheimer disease brains, strongly immunoreactive lysosomes and lipofuscin granules accumulated markedly in the perikarya and proximal dendrites of many cortical neurons, some of which were undergoing degeneration. More strikingly, these same hydrolases were present in equally high or higher levels in senile plaques in Alzheimer disease, but they were not found extracellularly in control brains, including those from Parkinson or Huntington disease patients. At the ultrastructural level, hydrolase immunoreactivity in senile plaques was localized to extracellular lipofuscin granules similar in morphology to those within degenerating neurons. Two cathepsins that were undetectable in neurons were absent from senile plaques. These results show that lysosome function is altered in cortical neurons in Alzheimer disease. The presence of a broad spectrum of acid hydrolases in senile plaques indicates that lysosomes and their contents may be liberated from cells, principally neurons and their processes, as they degenerate. Because cathepsins can cleave polypeptide sites on APP relevant for beta-amyloid formation, their abnormal extracellular localization and dysregulation in Alzheimer disease can account for the multiple hydrolytic events in beta-amyloid formation. The actions of membrane-degrading acid hydrolases could also explain how the intramembrane portion of APP containing the C terminus of beta-amyloid becomes accessible to proteases.

Aged↗

PubMed1991

Membrane markers of endoplasmic reticulum preserved in autophagic vacuolar membranes isolated from leupeptin-administered rat liver.

We isolated membranes from leupeptin-induced autophagic vacuoles and compared them with lysosomal membranes purified from dextran-administered rats. In protein composition, autophagic vacuole membranes prepared from long term-starved (36 h) rats bear marked resemblance to lysosomal membranes, whereas vacuole membranes prepared from short term-starved (12 h) animals differ significantly from lysosomal membranes. Immunoblotting analyses showed that only autophagic vacuole membranes from short term-starved rats possess endoplasmic reticulum markers such as cytochrome P450 and NADPH-cytochrome c reductase. None of the membranes contain sialyltransferase, a Golgi membrane marker. In experiments in which rats were starved after feeding to induce autophagy, the appearance of the endoplasmic reticulum markers occurred during 6-12 h of starvation, concomitantly with increases in vacuolar proteins and sequestered cytosolic aldolase. The endoplasmic reticulum membrane markers and sequestered aldolase declined gradually after 20-36 h of starvation, suggesting that prolonged starvation causes no further increase in the formation of autophagic vacuoles but an increase in the population of matured autophagic vacuoles. Thus, the prominent markers of endoplasmic reticulum from which autophagosomes originate are well preserved in autophagic vacuole membranes, and retention of these markers is highly dependent on the formation and subsequent maturation process of autophagic vacuoles.

Animals↗

PubMed1991

Changes of lysosomal proteinase activities and their expression in rat cultured keratinocytes during differentiation.

The cathepsins B, H and L, lysosomal cysteine proteinases, play a major role in intracellular protein degradation. These proteinase activities and expressions were examined in a Ca2+ regulated epidermal culture system which consists of two morphological cell types: undifferentiated cells grown in low Ca2+ (0.1 mM concentration) and differentiated cells grown in high Ca2+ (1.8 mM concentration), respectively. Cathepsin B and L activities of the differentiated cells showed a several-fold increase compared to that of the undifferentiated cells. In addition, by using CM-cellulose column chromatography, cathepsin B and L were separated and the level of cathepsin L activity increased significantly. Cathepsin B, L and H were also detected by using an immunoblotting procedure in which their bands were expressed after differentiation was induced by the increasing calcium concentration. Cathepsin L activity and immunostaining intensity reached a maximum at 1 or 2 days of differentiation. In contrast, cystatin alpha (an endogenous inhibitor of cysteine-dependent cathepsins) appeared in the final stage of differentiation. These results indicate that the expression of epidermal cathepsins and their endogenous inhibitor are involved in part of the program of cell differentiation and the terminal differentiation process in cultured rat keratinocytes.

Animals↗

PubMed1991

Molecular cloning of cDNA for rat cathepsin C. Cathepsin C, a cysteine proteinase with an extremely long propeptide.

A cDNA for rat cathepsin C (dipeptidylaminopeptidase I) was isolated. The deduced amino acid sequence of cathepsin C comprises 462 amino acid residues: 28 NH2-terminal residues corresponding to the signal peptide, 201 residues corresponding to the propeptide, and 233 COOH-terminal residues corresponding to the mature enzyme region. Four potential glycosylation sites were found, three located in the propeptide region, and one in the mature enzyme region. The amino acid sequence of mature cathepsin C has 39.5% identity to that of cathepsin H, 35.1% to that of cathepsin L, 30.1% to that of cathepsin B, and 33.3% to that of papain. Cathepsin C, therefore, is a member of the papain family, although its propeptide region is much longer than those of other cysteine proteinases and shows no significant amino acid sequence similarity to any other cysteine proteinase.

Amino Acid Sequence↗

PubMed1991

The selective role of cathepsins B and D in the lysosomal degradation of endogenous and exogenous proteins.

A selective inhibitor of cathepsin B, a derivative of E-64 (compound CA-074), and pepstatin-asialofetuin, a potent inhibitor of cathepsin D, were used for an in vivo study of the selective role of these proteinases in lysosomal proteolysis. Administration of compound CA-074 or pepstatinasialofetuin to rats caused only a slight shift of the lysosomal density and no increase in sequestered enzymes in the autolysosomal fraction, although cathepsin B or D activity in the liver was markedly inhibited. These treatments also had little effect on the inhibition of the degradation of endocytosed FITC-labeled asialofetuin. In contrast, leupeptin treatment caused marked inhibition of lysosomal degradation of endogenous and exogenous proteins. These results suggest a small contribution of cathepsins B and D to the initiation of lysosomal proteolysis.

Animals↗

PubMed1991

Alzheimer's disease amyloid beta-clipping enzyme (APP secretase): identification, purification, and characterization of the enzyme.

Alzheimer's disease (AD) is the most frequent cause of dementia, although no genetic abnormality has been identified. Recent studies have elucidated the molecular defect in AD, including the abnormal deposition of amyloid beta peptide (beta/A4) in senile plaques of affected individuals. Normal brain contains the enzyme, APP secretase, which cleaves inside the beta/A4 portion of the precursor protein (APP); abnormal processing of APP occurs in AD brain. Until now, no evidence has been provided that APP secretase is an intracellular proteinase. We have now prepared two synthetic substrates of APP secretase, both of which contain the cleavage point and are much more sensitive than substrates previously available to identify APP secretase. Using these substrates, we found an intracellular proteinase that has APP secretase activity. This proteinase has been identified as cathepsin B.

Alzheimer Disease↗

PubMed1991

Changes in immunoreactivity for cathepsin H in rat type II alveolar epithelial cells and its proteolytic activity in bronchoalveolar lavage fluid over 24 hours.

Variations in immunoreactivity for cathepsin H in rat type II alveolar epithelial (type II) cells and in its proteolytic activity in bronchoalveolar lavage fluid (BALF) were examined at six evenly spaced times over 24 hr (light period: 0600-1800 hr). Ring-shaped immunodeposits for cathepsin H were detected in type II cells (lamellar bodies), their sizes varying over 24 hr. The large ring-shaped immunodeposits increased during the light period, decreasing rapidly from 2000 to 0000 hr. Small intense immunodeposits abundantly appeared in the cells at 0000 hr. The area densities of immunodeposits in type II cells and their optical densities also varied with the time of day; both densities were high during the dark period, peaking at the mid dark period, whereas they were decreased during the light period. Proteolytic activities of cathepsin H in BALF (6 ml/rat) examined at each time revealed a distinct variation over 24 hr, corresponding to the variation in the immunoreactivity in type II cells. The activities in BALF were high from 1600 to 0400 hr and low at 0800 hr. These results suggest that the variation in immunoreactivity for cathepsin H in type II cells over 24 hr reflects the intracellular growth of lamellar bodies and secretory activity of the cells. Similar variations in the immunoreactivity and proteolytic activity of cathepsin H in the cells and BALF indicate its cosecretion with surfactants.

Animals↗

PubMed1991

Immunocytochemical localization of cathepsins B, H, and L in the rat gastro-duodenal mucosa.

Cathepsins B, H, and L are representative cysteine proteinases in lysosomes of a large variety of cells. Previous immunochemical studies indicated the presence of these enzymes also in the gastrointestinal wall. Using specific antisera, the cellular and subcellular distribution of cathepsins B, H, and L in rat gastric (oxyntic and pyloric part) and duodenal mucosa was investigated by light and electron microscopical immunocytochemistry. The subtypes of cathepsins were distributed differently in the cellular constituents of the epithelia: Cathepsin B was localized to lysosomes of all cells except goblet cells. Cathepsin H was found predominantly in gastric parietal cells (lysosomes) and in secretion granules of pyloric gastrin and duodenal cholecystokinin cells. Cathepsin L immunoreactivities were weak and restricted to a minority of cells (gastric mucous cells, enterocytes). Interstitial cells of the lamina propria immunoreactive for cathepsins H and L were identified as macrophages. The present findings suggest a dual function of cathepsins in the gastro-duodenal mucosa. They (1) cleave enzymatically proteins and peptides ingested in lysosomes, and (2) they may be involved in the processing of biologically active peptides (enteric hormones) from their precursor proteins.

Animals↗

PubMed1991

Myosin in hepatocytes is essential for bile canalicular contraction.

Active dynamic contraction of bile canaliculi has been observed in cultured doublet hepatocytes using time-lapse cinephotomicrography. This contractile movement plays an important role in normal bile formation. The mechanism of bile canalicular contraction has been proved to involve the Ca(2+)-calmodulin system and pericanalicular actin filaments. However, the role of myosin in this system is still unknown. In this study, using the newly synthesized myosin light-chain kinase inhibitor ML-9, we found that the treatment of cultured doublet hepatocytes with ML-9 inhibited canalicular contraction. This inhibitory effect suggests that myosin is involved in this complex cellular function and that the integrity of the actin-myosin system, as well as the Ca(2+)-calmodulin system is essential for normal bile canalicular contraction.

Actins↗

PubMed1991

Cysteine proteinases in bronchoalveolar epithelial cells and lavage fluid of rat lung.

We examined the presence of cathepsins B, H, and L in bronchoalveolar epithelial cells, including alveolar macrophages, and in bronchoalveolar lavage fluid (BALF), using immunocytochemistry and immunoblotting. By light and electron microscopy, immunoreactivity for cathepsins B, H, and L was detected in lysosomes of ciliated and non-ciliated epithelial cells of bronchi and bronchioles, and in macrophages. Immunodeposits for cathepsin H only were demonstrated in lamellar bodies of Type II alveolar epithelial cells, suggesting the cosecretion of surfactants and cathepsin H from the cells into the alveolar space. By immunoblotting, cathepsins B and H were found to be present in BALF. To further investigate the origin of these enzymes in BALF, alveolar macrophages obtained from BALF were cultured for 6 hr in a serum-free medium. Immunoblotting revealed that protein bands corresponding to the pro-form and mature form of cathepsin B and the mature form of cathepsin H were present in the culture medium. From these results, the presence of cathepsins B and H in BALF can be explained by the fact that cathepsin B is secreted from alveolar macrophages and cathepsin H is secreted mainly with surfactants from Type II cells and also from macrophages.

Animals↗

PubMed1991

Immunocytochemical localization of cathepsin B in rat anterior pituitary endocrine cells, with special reference to its co-localization with renin and prorenin in gonadotrophs.

We examined by immunocytochemistry the localization of cathepsin B in endocrine cells of rat anterior pituitary lobe, using a monospecific antibody to cathepsin B. By light microscopy, granular immunodeposits for cathepsin B were detected in most endocrine cells of anterior pituitary lobe. Cells immunoreactive for luteinizing hormone (LH) were diffusely immunostained by anti-cathepsin B. By electron microscopy, immunogold particles for cathepsin B were localized in lysosomes of thyrotrophs, somatotrophs, and mammotrophs. In mammotrophs, immunogold particles for cathepsin B were also detected in crinophagic bodies. Double immunostaining co-localized immunogold particles for LH and cathepsin B in secretory granules of gonadotrophs. Immunocytochemistry was also applied to demonstrate localization of renin and prorenin in LH-producing gonadotrophs; immunogold particles for renin were co-localized with those for LH, cathepsin B, or prorenin in their secretory granules. Immunogold particles for prorenin were also co-localized with those for LH or cathepsin B in secretory granules, but prorenin-positive granules appeared less frequently than renin-positive granules. These results suggest that cathepsin B not only plays a role in the protein degradation in lysosomes of anterior pituitary endocrine cells but also participates in the activation of renin in gonadotrophs, as has been demonstrated in secretory granules of juxtaglomerular cells.

Animals↗