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Biomedical subjects

E Kominami

Publications and source records attributed to E Kominami.

At least 73 records · Page 4Linked to original sources
PubMed1997

Cytoplasmic processing of human profilaggrin by active mu-calpain.

The differentiation of keratinocytes involves numerous steps including the formation of the cornified envelope and the aggregation of keratin filaments by filaggrin monomer molecules. In this study, we investigated whether mu-calpain is involved in the processing of profilaggrin to filaggrin monomers by using both an active mu-calpain specific antibody and a 27-mer synthetic calpastatin peptide, a cell-permeable calpain-specific inhibitor. Upon incubation of cultured keratinocytes with Ca2+ for 96 hours, active mu-calpain with a molecular mass of 76 kDa appeared preceded by an increase in mu-calpain mRNA. In synchrony with the appearance of active mu-calpain, the processing of profilaggrin occurred. Furthermore, the Ca2+-induced activation of mu-calpain and the processing of profilaggrin were affected by the addition of the synthetic calpastatin inhibitor. These results indicate that the activation of mu-calpain plays a major role in the profilaggrin processing.

Amino Acid Sequence↗
PubMed1997

Accumulation of mitochondrial ATP synthase subunit c in muscle in a patient with neuronal ceroid lipofuscinosis (late infantile form).

We report a case late infantile neuronal ceroid lipofuscinosis (NCL). Abnormal granules were found in the skeletal muscle fibers, Schwann cells, perineurial cells, endothelial cells, fibroblasts, and perivascular smooth muscle cells in the sural nerve. Electron microscopy revealed that these granules showed fingerprint profiles, curvilinear profiles or membrane-bound membranous structures. Acid phosphatase reaction was increased in these cells. Immunohistochemical studies for mitochondrial ATP synthase subunit c showed a strong reaction in these cells, suggesting abnormal accumulation of subunit c. Immunohistochemistry for subunit c in muscle may be useful in the diagnosis of late infantile NCL.

Adult↗
PubMed1997

Processing of cathepsins L, B and D in psoriatic epidermis.

Proteinase activity is increased in psoriatic epidermis. To elucidate the involvement of enzymes in psoriatic epidermis, the expression of cathepsins, L, B and D was investigated by Western blotting and immunohistological studies. Normal epidermis contained abundant inactive precursors (39 kDa) of cathepsins L and B and an inactive intermediate form (45 kDa) of cathepsin D. Cathepsin L in psoriasis was processed to a variable extent from the precursor to a single-chain form (30 kDa) and a mixture of single- and heavy-chain (25 kDa) forms of the active mature enzyme, corresponding to the immunohistological staining patterns 'diffuse dense', 'small granular', and unevenly distributed 'condensed granular'. Cathepsin B showed a mixture of precursor form (39 kDa) and single-chain (30 kDa) forms and was expressed as a 'diffuse dense' staining pattern in the mid-spinous layer and as a 'condensed' pattern in the upper spinous and granular layers. Cathepsin D was processed to the heavy-chain (31 kDa) form of activated mature enzyme with small granular staining and a mixture of heavy-chain and degraded protein (28 kDa) with larger and more condensed granular staining. The distribution patterns of 'small granular' cathepsin L, and of cathepsins B and D expression in suprabasal keratinocytes were very similar to that of involucrin. After complete clinical resolution of psoriasis by 8-methoxypsoralen plus UVA treatment, the expression of the three cathepsins was normalized. These results suggest that cathepsins L, B and D in forms activated to a variable extent may be involved in the pathology of psoriasis.

Adolescent↗
PubMed1997

Decreased lysosomal subunit c-degrading activity in fibroblasts from patients with late infantile neuronal ceroid lipofuscinosis.

We investigated in in-vitro cell-free incubation experiments which factor, lysosomal proteolytic dysfunction or structural alteration of subunit c, is responsible for the specific delay in the degradation of subunit c in patient cells with the late infantile form of neuronal ceroid lipofuscinosis. Experiments using substrates and soluble lysosomal fractions isolated separately from control and patient cells indicated that lysosomes from control cells are able to degrade mitochondrial subunit c either from control or patient cells at much faster rate than lysosomes from patient cells. Subunit c stored in patient cell lysosomes showed much more resistance to proteolytic attack than mitochondrial subunit c, suggesting that conformation of subunit c as well as lysosomal proteolytic dysfunction both participate in the specific lysosomal accumulation of subunit c in the late infantile disease.

ATP Synthetase Complexes↗
PubMed1997

Involvement of interleukin-6 in activation of lysosomal cathepsin and atrophy of muscle fibers induced by intramuscular injection of turpentine oil in mice.

Serum IL-6 level increased after the injection of turpentine oil into the right gastrocnemius muscle in mice. The mRNA level of IL-6 was highest in the injected muscle at 12 h after injection, but was not identified in the opposite muscle. The activities of cathepsins B and B+L started to elevate after 12 h in the injected muscle and markedly increased after day 3. Likewise, the mRNA levels of cathepsins B and L markedly increased from day 1 to day 5 in the injected muscle. However, a very mild increase was also observed in the opposite muscle. Immunohistochemical staining of cathepsins B and L exhibited positive reactions as fine granules in myofibers at 12 h and strong positive reactions in the infiltrating macrophages at 3 days. Atrophy of myofibers type 1 and 2 was evident in a time-dependent manner in the injected muscle. Treatment with rat anti-mouse IL-6 receptor monoclonal antibody inhibited the increase in cathepsin activities in the injected muscle. We conclude that IL-6 produced in the inflamed muscle is involved in the process of muscle degeneration, especially through the activation of lysosomal cathepsins.

Animals↗
PubMed1997

MHC class I presentation of an exogenous polypeptide antigen encoded by the murine AIDS defective virus.

Peptides derived from endogenous proteins are presented by MHC class I molecules, whereas those derived from exogenous proteins are presented by MHC class II molecules. This strict segregation has been reconsidered in recent reports in which exogenous antigens are shown to be presented by MHC class I molecules in the phagocytic pathway. In this report, the presentation pathway of an exogenously added highly antigenic polypeptide encoded by the murine AIDS (MAIDS) defective virus gag p12 gene is investigated. A 25-mer polypeptide (P12-25) encoded within the gag p12 region of the MAIDS defective virus was found to be effective in stimulating unprimed B6 (H-2b) CD8+ T cells in vitro. The presentation of P12-25 is sensitive to cytochalasin B and D, brefeldin A and gelonin, a ribosome-inactivating protein synthesis inhibitor, but less sensitive or resistant to lactacystin, a highly specific inhibitor of the proteasome. Interestingly, CA-074, a selective inhibitor of cathepsin B, inhibited presentation of the polypeptide, indicating its involvement in the degradation of the P12-25 polypeptide. In fact, when P12-25 was digested with purified cathepsin B in vitro, a highly antigenic 11-mer peptide containing the class I (H-2Db)-binding motif was obtained. Our results favor the phagosome/macropinosome-to-cytosol-to-endoplasmic reticulum (ER)-to-cell surface pathway for exogenous antigens presented by MHC class I molecules. These findings may be relevant to exploiting peptide vaccines that specifically elicit CD8+ T cell immunity in vivo.

Acetylcysteine↗
PubMed1997

Calpain and cathepsins in the skeletal muscle of inflammatory myopathies.

To clarify the significance of intracellular lysosomal (cathepsins B, L and H) and extralysosomal (calpain) proteolytic systems in the process of muscle fiber degradation in inflammatory myopathies, biopsied muscle specimens were examined from patients with polymyositis (PM) and dermatomyositis (DM). Generally, in specimens from patients with PM and DM, but not in those from normal controls, muscle fibers surrounding inflammatory infiltrates or in the perifascicular regions, and occasionally mononuclear cell infiltrates demonstrated positive immunostaining for calpain and cathepsins B, L, and H. In addition, enzyme activities of cathepsins B and L increased in specimens with inflammatory myopathy. These results suggest that calpain and cathepsins play a significant role in the process of muscle fiber destruction in inflammatory myopathy.

Adult↗
PubMed1997

Immunocytochemical localization of lysosomal cysteine and aspartic proteinases, and ubiquitin in rat epidermis.

To analyze the degradation system in epidermal cells during their generation, differentiation, and cell death, immunocytochemical localization of lysosomal cysteine and aspartic proteinases, an endogenous cysteine proteinase inhibitor, cystatin beta, and ubiquitin were examined using rat sole skin. By confocal laser microscopy, granular immunodeposits for lysosomal proteinases were well demonstrated in epidermal cells; immunoreactivity for cathepsins B and C was prominent in the lower spinous and basal layers, while that for cathepsins L and D was intense in the upper spinous and granular layers, although immunoreactivity for cathepsin D was also detected in the lower epidermal layers. Immunoreactivity for cathepsin H was weakly detected only in the spinous layer, where there were some intensely immunopositive cells with processes which were also immunopositive for S-100 alpha, indicating that these cells were Langerhans cells. Diffuse immunoreactivity for cystatin beta was intense in the spinous and granular layers and weak in the basal layer. In addition, we also examined the localization of ubiquitin, which is a signal peptide for cytosolic proteolysis; clear-cut granular immunodeposits for ubiquitin were detected in spinous and granular cells, and some were co-localized with cathepsin B immunoreactivity. In the basal layer, mitotic cells were strongly immunopositive for ubiquitin. These results suggest that cysteine and aspartic proteinases are involved in the lysosomal system of the epidermis, showing different distributions in the epidermal layers depending on the enzymes examined. Moreover, ubiquitin may be associated with the cell cycle-dependent degradation in basal cells while it also participates in the non-lysosomal proteolysis and probably, lysosomal proteolysis in the spinous and granular cells.

Animals↗
PubMed1997

Genomic structure of human cystatin A.

Cystatin A is a cysteine proteinase inhibitor with a molecular mass of 11 kDa, and is located mainly in the keratohyaline granules of the stratum granulosum and the cornified envelope of the stratum corneum in the epidermis. In this study, we demonstrated the genomic structure of this proteinase inhibitor in which there were three exons of 111 bp, 102 bp and 226 bp in length, while the lengths of the 1st and 2nd intron were approximately 14 Kbp and 4 Kbp, respectively. The conserved sequence of QVVAG was encoded in the 2nd exon and was not inserted by any introns. There were binding sites for SP-1 and AP-2 in the promoter region and an AP-1 binding site in the 1st intron. The successful amplification of each exon of cystatin A may possibly contribute to the detection of the genomic abnormality of some skin disorders e.g. keratinization disorder, chronic bacterial infection or photophobia.

Amino Acid Sequence↗
PubMed1997

Expression of cathepsin D and B in invasion and metastasis of squamous cell carcinoma.

To determine the involvement of proteinases with hydrolytic activity towards extracellular matrix and basement membrane, in invasion and metastasis of tumour cells, the expression of cathepsin D, an aspartic proteinase, and cathepsin B, a cysteine proteinase, was studied. Formalin-fixed paraffin-embedded specimens from 13 patients who had squamous cell carcinomas (SCC) with local recurrence, skin and/or lymph node metastasis were examined. Cathepsin D stained intensely as a granular pattern (mature enzyme) in tumour cells of 69% of primary lesions and all the secondary lesions of the patients with SCC. Cathepsin B stained more intensely in SCC cells of all of the primary and secondary lesions than in normal epidermis; staining patterns were almost diffuse (procathepsin B). Granular and diffuse patterns (mature enzyme of cathepsin D and procathepsin B, respectively) appeared in the outer and inner parts of tumour islands, respectively. The presence of the active mature form of cathepsin D and procathepsin B in metastatic skin lesions of SCC was confirmed by Western blotting analysis. The presence and localization of the active mature form of cathepsin D suggests that activated cathepsin D may be involved in the invasion and metastasis of SCC.

Blotting, Western↗
PubMed1996

Assembly of hair keratins in transfected epithelial cells.

To define the interactions required for the filament assembly of differentiation-specific keratins, active copies of mouse hair keratin mHa1 and mHb4 genes were introduced into a rat kangaroo kidney epithelial cell line (PtK2) and a rat stratified squamous epithelial cell line (rat epidermal keratinocyte). In PtK2 transient transfectants, when introduced individually or in combination, mHa1 and mHb4 formed aggregates of ring-like structures of various sizes at the perinuclear region with no evidence of organization into a keratin network. These aggregates altered the distribution of the endogenous keratins and vimentin. In most of the cells carrying the ring-like structures of mHa1 and mHb4 around the nucleus, the endogenous keratin network collapsed and localized around the nucleus. Furthermore, the densely accumulated endogenous keratin surrounded the ring-like aggregates with partial co-localization. However, when transfected into the rat epidermal keratinocytes, mHa1 and mHb4 were able to co-localize with the well-developed cytoskeleton of endogenous keratins. These results showed that, in contrast to keratin pairs K5/K14 and K8/K18, the mHa1/mHb4 pair is unable to develop an extensive keratin network on its own and that there are possible differential abilities among these hair keratins and other keratins to form well-developed cytoplasmic networks.

Animals↗
PubMed1996

Dynamic changes of cathepsins B and L expression in the monkey hippocampus after transient ischemia.

Enzymatic activities, expressions, and the immunohistochemical localization of lysosomal cystein proteases, cathepsins B and L, were analyzed in the monkey hippocampus after transient ischemia to clarify the mechanism of delayed cornu Ammonis (CA)-1 neuronal death. By enzymatic assay, the activity of cathepsin B increased in CA-1, 24 h after the ischemic insult, while that of cathepsin L decreased. On Western blotting, the protein contents of both cathepsins B and L increased immediately after ischemia. By immunohistochemistry, cathepsins B and L were stained as coarse granules in the perikarya of control CA-1 neurons, but in postischemic CA-1 neurons they were released from lysosome granules. In contrast, in CA-2 and the remaining sectors, enzymatic activities increased after ischemia, and immunoreactivities of cathepsins B and L increased only within lysosome granules. These results suggest that cathepsins B and L may play an important role in the breakdown of certain cell proteins in the postischemic CA-1 neurons.

Animals↗
PubMed1996

Interleukin 6 receptor antibody inhibits muscle atrophy and modulates proteolytic systems in interleukin 6 transgenic mice.

The muscles of IL-6 transgenic mice suffer from atrophy. Experiments were carried out on these transgenic mice to elucidate activation of proteolytic systems in the gastrocnemius muscles and blockage of this activation by treatment with the anti-mouse IL-6 receptor (mIL-6R) antibody. Muscle atrophy observed in 16-wk-old transgenic mice was completely blocked by treatment with the mIL-6R antibody. In association with muscle atrophy, enzymatic activities and mRNA levels of cathepsins (B and L) and mRNA levels of ubiquitins (poly- and mono-ubiquitins) increased, whereas the mRNA level of muscle-specific calpain (calpain 3) decreased. All these changes were completely eliminated by treatment with the mIL-6R antibody. This IL-6 receptor antibody could, therefore, be effective against muscle wasting in sepsis and cancer cachexia, where IL-6 plays an important role.

Animals↗
PubMed1996

Genomic structure and PCR-SSCP analysis of the human CD40 ligand gene: its application to prenatal screening for X-linked hyper-IgM syndrome.

To develop a general method for analysis of the mutation and prenatal diagnosis of X-linked hyper-IgM syndrome (XHM), the human CD40 ligand (hCD40L) gene was cloned and sequenced with special reference to the 5' and 3' flanking regions and exon/intron boundaries. The hCD40L gene consists of five exons and four introns, as already reported by others. Two major transcription initiation sites were identified at 67 bp and 64 bp upstream from the ATG initiation codon. The hCD40L mRNA transcripts terminated at 321 bp, 327 bp and 987 bp downstream from the TGA stop codon. Based on the intronic sequences, oligonucleotide primers were designed for amplifying the coding region of each exon separately. Polymerase chain reaction--single-strand conformational polymorphism (PCR-SSCP) analysis was successfully applied to screening for the defective hCD40L gene in a family with XHM. The nonsense mutation, Trp140 (TGG)-->stop (TAG) in exon 5, was found in the mother and an affected child. We also performed prenatal diagnosis by PCR-SSCP during the first trimester of pregnancy in this family.

Amino Acid Sequence↗