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Biomedical subjects

E Kominami

Publications and source records attributed to E Kominami.

At least 91 records · Page 5Linked to original sources
PubMed1996

Cathepsin B and D expression in squamous cell carcinoma.

To elucidate involvement of proteinases in malignancy of keratinocytes, expression of cathepsin B, a cysteine proteinase, and cathepsin D, an aspartic proteinase, was ascertained in formalin-fixed paraffin-embedded specimens of normal skin, squamous cell carcinoma (SCC). Bowen's disease, seborrhoeic keratosis and basal cell carcinoma (BCC). Presence of procathepsin B and an intermediate form of cathepsin D was confirmed by Western blotting and enzyme activity analysis. Cathepsin B stained more intensely in SCC tumour cells than in normal epidermis; staining patterns were diffuse, granular or both. Diffuse and granular patterns (procathepsin B and mature enzyme, respectively) appeared in inner and outer parts of tumour islands, respectively. Five of 20 cases of Bowen's disease showed diffuse enhanced cathepsin B expression; 20 cases of seborrhoeic keratosis or BCC did not. Cathepsin D stained intensely in tumour cells of half the SCC cases. The staining manner and distribution of cathepsins B and D was similar in the cytoplasm of cancer cells. No enhanced staining of cathepsin D was seen in any cases of Bowen's disease, seborrhoeic keratosis, or BCC. Coexistence and localization of active mature forms of cathepsins B and D suggests that cooperation between the two enzymes may play an important part in invasion of SCC.

Blotting, Western↗
PubMed1996

Specific delay in the degradation of mitochondrial ATP synthase subunit c in late infantile neuronal ceroid lipofuscinosis is derived from cellular proteolytic dysfunction rather than structural alteration of subunit c.

Previously we indicated that a specific delay in subunit c degradation causes the accumulation of mitochondrial ATP synthase subunit c in lysosomes from the cells of patients with the late infantile form of neuronal ceroid lipofuscinosis (NCL). To explore the mechanism of lysosomal storage of subunit c in patient cells, we investigated the mechanism of the lysosomal accumulation of subunit c both in cultured normal fibroblasts and in in vitro cell-free incubation experiments. Addition of pepstatin to normal fibroblasts causes the marked lysosomal accumulation of subunit c and less accumulation of Mn(2+)-superoxide dismutase (SOD). In contrast, E-64-d stimulates greater lysosomal storage of Mn(2+)-SOD than of subunit c. Incubation of mitochondrial-lysosomal fractions from control and diseased cells at acidic pH leads to a much more rapid degradation of subunit c in control cells than in diseased cells, whereas other mitochondrial proteins, including Mn(2+)-SOD, beta subunit of ATP synthase, and subunit i.v. of cytochrome oxidase, are degraded at similar rates in both control and patient cells. The proteolysis of subunit c in normal cell extracts is inhibited markedly by pepstatin and weakly by E-64-c, as in the cultured cell experiments. However, there are no differences in the lysosomal protease levels, including the levels of the pepstatin-sensitive aspartic protease cathepsin D between control and patient cells. The stable subunit c in mitochondrial-lysosomal fractions from patient cells is degraded on incubation with mitochondrial-lysosomal fractions from control cells. Exchange experiments using radiolabeled substrates and nonlabeled proteolytic sources from control and patient cells showed that proteolytic dysfunction, rather than structural alterations such as the posttranslational modification of subunit c, is responsible for the specific delay in the degradation of subunit c in the late infantile form of NCL.

Cathepsin B↗
PubMed1996

Monoclonal antibodies to rabbit hepatocyte myosin that cross-react with human liver myosin.

We prepared polyclonal and monoclonal antibodies against myosin purified from rabbit hepatocytes. Immunoblotting analyses revealed that the polyclonal antibody and four (HM1, HM2, HM3 and HM4) of five monoclonal antibodies reacted with myosin heavy chain. Chymotryptic cleavage of myosin yielded a 130 kDa fragment comprising the tail portion of the myosin heavy chain and a 67 kDa fragment comprising the ATP-binding active site of the myosin head. All active antibodies reacted with epitopes localized in the 130 kDa fragment. Monoclonal antibodies HM3 and HM4 and the polyclonal antibody reacted strongly with myosin heavy chains from a human liver homogenate prepared from a surgically resected liver specimen. Immunocytochemical analyses showed that myosin is localized along the plasma membrane as well as around the bile canaliculi in both rabbit and human hepatocytes. Immunocytochemical analyses on liver blocks obtained from those patients who suffered various types of diseases accompanying cholestasis clearly indicated a marked increase in pericanalicular myosin. This altered myosin localization is analogous to that observed in phalloidin-treated liver. Thus, myosin localization, determined using these antibodies, can provide a valid morphological basis for diagnosing the pathological state of the patient liver.

Adult↗
PubMed1996

Neuronal ceroid-lipofuscinosis--late-infantile or Jansky-Bielschowsky type--revisited.

The tissues from three patients with late-infantile NCL originally described by Max Bielschowsky became available to apply modern techniques such as fluorescence microscopy, electron microscopy and immunohistochemistry. While regular tinctorial preparations of the tissues documented a neuronal storage disorder in all three patients' tissues, the accumulated material proved to be autofluorescent, showed the ultrastructure of curvilinear lipopigments, and reacted strongly with an antibody against the subunit-C of mitochondrial ATP synthase, a major component of lipopigments in NCL and also with an antibody against sphingolipid activator proteins. Thus, these modern morphological techniques demonstrated that the originally described three siblings with late-infantile "amaurotic familial idiocy" really had neuronal ceroid-lipofuscinosis of the late-infantile or Jansky-Bielschowsky type, according to current diagnostic criteria. This type of archival study may also contribute to the mosaic of medical history.

Brain↗
PubMed1996

Lysosomal cysteine and aspartic proteinases and ubiquitin in rat and human urinary bladder epithelium.

To examine localization of cysteine and aspartic proteinases, and ubiquitin in rat and human urinary bladders, immunocytochemistry was applied to the tissues. In semi-thin sections, immunoreactivity for cathepsins B and D was densely localized throughout epithelial layers of rats and humans, while that for cathepsins H and L was mainly localized in rat superficial and human intermediate cells. Immunoreactivity for cathepsin C was relatively high in rat and human epithelia, especially in humans. Immunoreactivity for ubiquitin was detected in rat and human epithelial cells. By electron microscopy, vesicular or heterogeneously dense lysosomes labeled with immunogold particles indicating cathepsin B were seen in rat and human epithelial cells; particularly, they often appeared near fusiform vesicles in rat superficial cells and in human intermediate and superficial cells. By double immunostaining, lysosomes with or without vesicular structures were co-labeled with immunogold particles showing both cathepsin B and ubiquitin. The results suggest that cathepsins B, C, H, and L, and cathepsin D are involved in the lysosomal system of rat and human bladder epithelia. Moreover, considering that ubiquitin is a cofactor in the soluble ATP-dependent proteolysis, the results may also indicate that epithelial cells actively form autophagolysosomes.

Animals↗
PubMed1995

Procathepsin L degrades extracellular matrix proteins in the presence of glycosaminoglycans in vitro.

The processing of procathepsin L on the surfaces of physiological glycosaminoglycans, heparan sulfate, chondroitin sulfate, etc., as well as dextran derivatives were studied. All glycosaminoglycans and dextran derivatives including dextran T-500 and DEAE dextran examined in this study accelerated the conversion of procathepsin L to processed cathepsin L in vitro with different time courses. Further, we examined whether procathepsin L digests protein substrates in the presence or absence of the surface materials. Laminin was degraded by both procathepsin L itself and the 31-kDa processed form in the presence of surface materials with the same profiles. In contrast, fibronectin was digested by procathepsin L without processing in the presence of surface materials. The proteolytic profiles of fibronectin by the processed form differed from those by procathepsin L. This is the first evidence that the proform of a cysteine proteinase proteolyzes protein substrates.

3T3 Cells↗
PubMed1995

Physical association of Ras and cathepsins B and L in the conditioned medium of v-Ha-ras-transformed NIH3T3 cells.

Immunoblot analysis of the conditioned medium of v-Ha-ras-transformed NIH3T3 cells showed that Ras protein is secreted in the culture fluid. Because cathepsins L and B whose activities are inhibited by Ras are also secreted, possible association between Ras and cathepsins was investigated by immunoprecipitation and following immunoblot analysis. The results showed that Ras is physically associated with cathepsins B and L in the culture fluid.

3T3 Cells↗
PubMed1995

Abnormal degradative pathway of mitochondrial ATP synthase subunit c in late infantile neuronal ceroid-lipofuscinosis (Batten disease).

Subunit c is normally present as an inner mitochondrial membrane component of the F0 sector of the ATP synthase complex, but in the late infantile form of neuronal ceroid-lipofuscinosis (NCL) it was also found in lysosomes in high concentrations. The rate of degradation of subunit c as measured by pulse-chase and immunoprecipitation showed a marked delay of degradation in patients' fibroblasts with late infantile form of NCL. There were no significant differences between control cells and cells with disease in the degradation of cytochrome oxidase subunit IV, an inner membrane protein of mitochondria. Measurement of labeled subunit c in mitochondrial and lysosomal fractions showed that the accumulation of labeled subunit c in the mitochondrial fraction can be detected before lysosomal appearance of radioactive subunit c, suggesting that subunit c accumulated as a consequence of abnormal catabolism in the mitochondrion and is transferred to lysosomes through an autophagic process. The biosynthetic rate of subunit c and mRNA levels for P1 and P2 genes that code for it were almost the same in both control and patient cells. These findings suggest that a specific failure in the degradation of subunit c after its normal inclusion in mitochondria and its consequent accumulation in lysosomes.

Electron Transport Complex IV↗
PubMed1995

Autocatalytic inactivation of lysosomal cathepsins is associated with inhibition of protein breakdown by insulin-like growth factor-1 (IGF-1) in myotubes.

Protein breakdown was monitored in C2C12 myotubes as the rate of release of radioactivity after prelabeling cell protein with [3H] tyrosine. IGF-1 (13 nM) and insulin (100 nM) prolonged the half-life of long-lived proteins. Enzymatic activities of cathepsins B and B+L were inhibited by the addition of IGF-1 or insulin. Immunoblotting of cathepsins B and L revealed extensive degradation of heavy chain forms by IGF-1. However, neither expression of cathepsins B and L genes nor expression of cystatin beta, an intrinsic inhibitor for cathepsins, was influenced. The addition of E-64, L-3-carboxy-trans-2,3-epoxypropionyl-leucylamide-(4-guanidin o) butane, a inhibitor of cathepsins B and L, increased protein contents of heavy chains of cathepsins B and L in the IGF-1 treated cells. Inhibition of protein breakdown by IGF-1 is mediated by autocatalytic inactivation of lysosomal cathepsins B and L.

Analysis of Variance↗
PubMed1995

Muscle undergoes atrophy in association with increase of lysosomal cathepsin activity in interleukin-6 transgenic mouse.

Interleukin(IL)-6 transgenic mice were produced by microinjection of human IL-6 cDNA fused with H-2Ld promoter into the pronucleus of fertilized eggs from C57BL/6J mice. At 16 weeks old, the gastrocnemius muscles of the IL-6 transgenic mice became atrophic as compared to those of the normal mice, while the body weights increased significantly. The activities and mRNA levels of lysosomal cathepsins B and L were increased in the muscles of the transgenic mice. Immunohistochemical study on the muscles showed increased staining of both cathepsins B and L in the transgenic mice. IL-6 is responsible for enhanced muscle catabolism by activating the lysosomal cathepsin (B and L) system.

Animals↗
PubMed1995

New insight into lysosomal protein storage disease: delayed catabolism of ATP synthase subunit c in Batten disease.

Subunit c is normally present as an inner mitochondrial membrane component of the Fo sector of the ATP synthase complex, but in the late infantile form of neuronal ceroid lipofuscinosis (NCL) it was also found in lysosomes in high concentrations. Mechanism for specific accumulation of subunit c in lysosomes is not known. The rate of degradation of subunit c as measured by pulsechase and immunoprecipitation showed a marked delay of degradation in patients fibroblasts with late infantile form of NCL. There were no significant differences between control cells and cells with disease in the degradation of cytochrome oxidase subunit IV, an inner membrane protein of mitochondria. Measurement of labeled subunit c in mitochondrial and lysosomal fractions showed that the accumulation of labeled subunit c in the mitochondrial fraction can be detected before lysosomal appearance of radioactive subunit c, suggesting that subunit c accumulated as a consequence of abnormal catabolism in the mitochondrion and is transferred to lysosomes, through an autophagic process. There were no large differences of various lysosomal protease activities between control and patient cells. In patient cells sucrose loading caused a marked shift of lysosomal density, but did not a shift of subunit c containing storage body. The biosynthetic rate of subunit c and mRNA levels for P1 and P2 genes that code for it were almost the same in both control and patient cells. These findings suggest that a specific failure in the degradation of subunit c after its normal inclusion in mitochondria and its consequent accumulation in lysosomes.

Humans↗
PubMed1995

Interleukin-6 induces proteolysis by activating intracellular proteases (cathepsins B and L, proteasome) in C2C12 myotubes.

1. A cell culture system of C2C12 myotubes was established as a model of the muscle. With the aid of this model, the half-lives of intracellular proteins as well as the activities and mRNA levels of proteasomes (26S and 20S) and cathepsins (B, L, and H) were examined in the presence of various amounts of cytokines. 2. It was found that 100 units/ml recombinant human interleukin-6 somewhat shortened the half-life of long-lived proteins to 23.79 +/- 1.55 h (control: 25.60 +/- 1.87 h). When 1% fetal bovine serum contained in the culture medium was replaced by 0.5 mg/ml bovine serum albumin, interleukin-6 was more effective since 10 units/ml of interleukin-6 shortened the half-life to 19.09 +/- 2.87 h (control: 22.26 +/- 321 h). Interleukin-6 (100 units/ml) increased the activity of 26S proteasome by 31.5%, of cathepsin B by 53.5% and of cathepsin B+L by 21.3%. These increases occurred in association with an increase in their transcription. 3. On the other hand, 1000 units/ml of recombinant human tumour necrosis factor alpha prolonged the half-life of long-lived proteins while reducing the protease activities of 20S proteasome (-27.1%), cathepsins B (-64.6%) and B+L (-54.9%). 4. These results suggest that interleukin-6 induces degradation of long-lived intracellular proteins by activating both the non-lysosomal (proteasomes) and lysosomal (cathepsins) proteolytic pathways. It is therefore concluded that interleukin-6 is a candidate for a proteolysis-inducing factor in myotubes and may play an important role in the progression of muscle degradation in systemic inflammatory responses induced by sepsis or severe injury.

Animals↗
PubMed1995

Specific delay of degradation of mitochondrial ATP synthase subunit c in late infantile neuronal ceroid lipofuscinosis (Batten disease).

Subunit c is normally present as an inner mitochondrial membrane component of the F0 section of the ATP synthase complex, but in the late infantile form of neuronal ceroid lipofuscinosis (NCL) it was also found in lysosomes in high concentrations. To explore the mechanism of storage of subunit c, the rates of degradation and synthesis of subunit c were measured in fibroblast cell types from controls and patients with the late infantile form of NCL. The radiolabel from subunit c decreased with time in control cells, whereas no apparent loss of radioactivity of subunit c was found in patients' cells. There were no significant differences between control cells and cells with disease in the degradation of cytochrome oxidase subunit IV, an inner membrane protein of mitochondria. A combination of pulse-chase and subcellular fractionation analysis showed that a delay of intramitochondrial loss from prelabeled subunit c was seen in all diseased cells tested. Lysosomal appearance of labeled subunit c could be detected after chase for more than 1 week and its radioactivities were variable among diseased cell types. The biosynthetic rate of subunit c was almost the same in both control and patient cells. Northern blotting analyses showed that mRNAs for P1 and P2 genes had no significant difference in lengths and amounts between control and patient cells. Results suggest a specific failure in the degradation of subunit c after its normal inclusion in mitochondria and its consequent accumulation in lysosomes. This is the first direct evidence to show a delay of subunit c degradation in the cells from the late infantile form of NCL.

Fibroblasts↗
PubMed1995

Changes of proteasomes and cathepsins activities and their expression during differentiation of C2C12 myoblasts.

C2C12 myoblasts fuse to form multinucleated myotubes and express muscle specific proteins during differentiation. To elucidate developmental regulation of intracellular proteolytic systems, enzymatic activities, protein and mRNA levels of proteasomes (20S and 26S) and lysosomal cathepsins (B, L, and H) were examined. Myoblasts were differentiated fully to myotubes 6 days after starting differentiation. In this developmental process, the 26S proteasome activity decreased, while the 20S proteasome activity increased. Expression of proteasome subunits of 20S (RC2, RC8) and regulatory components of 26S (S4, S7) was down-regulated, though total protein levels of proteasomes showed no remarkable changes. On the other hand, enzymatic activities of cathepsins B and B + L increased in association with an increase of their transcriptional and translational levels. Expression of their specific endogenous inhibitor, cystatin beta, also increased. Maturation of the lysosomal proteolytic system was tightly linked to the differentiation process. These results suggested that signals for differentiation of myoblasts mediate a change of intracellular proteolytic systems, involving up-regulation of lysosomal cathepsins and down-regulation of proteasomes.

Animals↗
PubMed1995

Defect of proteolysis of mitochondrial ATP synthase subunit C in neuronal ceroid lipofuscinosis.

Mechanism of lysosomal storage of mitochondrial ATP synthase subunit c in late infantile form of NCL was studied. Morphological and biochemical examinations with patient fibroblasts showed that subunit c, not other mitochondrial proteins was specifically localized in lysosomes. The biosynthetic rate of subunit c and mRNA levels for P1 and P2 genes that code for it were almost the same in both control and patient cells. Measurement of labeled subunit c in mitochondrial and lysosomal fractions showed a specific delay of degradation of subunit c in patient cells with late infantile form of NCL and lysosomal transfer of radioactive mitochondrial subunit c after chase for 1-2 weeks, suggesting that subunit c is transfered to lysosomes through an autophagic process and accumulated as a consequence of abnormal catabolism in lysosomes.

Humans↗
PubMed1995

Role of cathepsin B as prorenin processing enzyme in human kidney.

Renin is synthesized from an inactive precursor, prorenin, through cleavage at a pair of basic amino acids catalyzed by prorenin processing enzyme (PPE). A lysosomal protease, cathepsin B, has been suggested to be a strong candidate for PPE. However, there still remains a possibility that other protease(s) can also catalyze prorenin processing. We studied the subcellular distribution of PPE in human renal cortex using pure recombinant prorenin as a PPE assay substrate. PPE and renin activities, and cathepsin B activity and protein were colocalized in the lysosomal fraction. The PPE activity was completely inhibited by a cathepsin B specific inhibitor, CA074. Taken together with the immunohistochemical data showing that cathepsin B and PPE are colocalized in dense secretory granules of juxtaglomerular cells of kidney, we conclude that cathepsin B is the authentic PPE in human kidney.

Catalysis↗
PubMed1995

Cytochemical localization of hyaluronic acid in human synovium with special reference to its possible process of degradation.

Fibroblast-like type B cells are known to produce hyaluronic acid (HA), but the process of its degradation remains unknown. In order to examine the possible route for the degradation of HA in normal human synovium, histochemical and immunohistochemical techniques were applied to the synovial tissue, using biotinylated HA binding region (HABR) and antibodies against CD44 and cathepsin B. Reaction products for HA and CD44 were detected on the cell surface of all synovial lining cells, while half of these lining cells contained intracellular stainings of HA and CD44. Electron microscopically, the lining cells containing intracellularly stained HA and CD44 extended cytoplasmic processes (type A cells), while the other lining cells possessed a smooth cell surface (type B cells). By light microscopic double staining, the intracellular stainings of HA and CD44 appeared co-localized in the cells immunopositive for cystatin beta, an endogenous cysteine proteinase inhibitor which has been shown to be localized in alveolar macrophages and osteoclasts. Moreover, these intracellular stainings of HA and CD44 were co-localized with immunodeposits for cathepsin B, a representative cysteine proteinase in lysosomes. In the extracellular staining of HA, dot-like reaction products appeared on fibrous structures with a periodicity of 41.7 nm. These results suggest that Type A cells in the normal human synovium participate in the degradation of HA by its CD44 mediated intake. Furthermore, HA may be closely associated with fibrous structures, probably type III collagen molecules.

Adult↗