Biomedical subjects

Y Nishimura

Publications and source records attributed to Y Nishimura.

At least 289 records · Page 16Linked to original sources

PubMed1998

Molecular cloning and expression of feline CD3epsilon.

The cDNA of feline CD3epsilon, one of the T-cell receptor components, was cloned from a feline T-lymphoblastoid cell line (MYA-1 cells) and peripheral blood mononuclear cells and thymocytes of cats by polymerase chain reaction. Sequencing analysis revealed that the open reading frame of feline CD3epsilon consists of 606 base pairs encoding a predicted molecular mass of 25 kDa transmembrane protein which lacks N-glycosylation site. Comparison of the predicted amino acid sequence of feline CD3epsilon with those of other mammalians' homologues revealed that a relatively low homology was present in the extracellular domain. However, the cytoplasmic domain contained several characteristic motifs highly conserved across the species. These motifs were known to be important for signal transduction upon T-cell activation or endoplasmic reticulum retention. In addition, the feline CD3epsilon protein was expressed in an insect cell line (Sf9) by a baculovirus expression system. The expression was confirmed by indirect immunofluorescence assay and immunoblotting analysis using an anti-human CD3epsilon polyclonal antibody. These results will provide additional information for understanding the feline immune system.

Amino Acid Sequence↗

PubMed1998

Metabolic changes in hibernating myocardium after percutaneous transluminal coronary angioplasty and the relation between recovery in left ventricular function and free fatty acid metabolism.

To elucidate the changes in oxidative metabolism in hibernating myocardium after coronary revascularization, we performed myocardial single-photon emission computed tomography with a free fatty acid analog, I-123 beta-methyliodophenylpentadecanoic acid (BMIPP), and thallium-201 before and 1 month after percutaneous transluminal coronary angioplasty (PTCA) in 11 patients with angina pectoris caused by single artery stenosis. All patients had improvement in wall motion after PTCA at the region with coronary stenosis; the wall motion abnormality score evaluated by left ventriculography decreased from 5.5+/-0.8 (mean +/- SE) to 2.1+/-0.9, p <0.01) after PTCA. The defect score of I-123 BMIPP images was significantly larger than that of thallium-201 images either before (14+/-1.3 vs 8.9+/-1.1, p <0.01) or 1 month after (7.4+/-1.5 vs 3.7+/-0.8, p <0.01) PTCA. The decrease in the defect score of both images was significant (p <0.01). Changes in the wall motion abnormality score showed a significant correlation with both the change in the defect score of thallium-201 images (r = 0.58, p < 0.01) and that of I-123 BMIPP images (r = 0.75, p <0.01). These results indicate that the metabolism of free fatty acid is impaired in hibernating myocardium, and that improvement in left ventricular function after successful PTCA is strongly associated with the recovery of oxidative metabolism.

Adult↗

PubMed1998

Solution structure of the DNA-binding domain of human telomeric protein, hTRF1.

BACKGROUND: Mammalian telomeres consist of long tandem arrays of the double-stranded TTAGGG sequence motif packaged by a telomere repeat binding factor, TRF1. The DNA-binding domain of TRF1 shows sequence homology to each of three tandem repeats of the DNA-binding domain of the transcriptional activator c-Myb. The isolated c-Myb-like domain of human TRF1 (hTRF1) binds specifically to telomeric DNA as a monomer, in a similar manner to that of homeodomains. So far, the only three-dimensional structure of a telomeric protein to be determined is that of a yeast telomeric protein, Rap 1p. The DNA-binding domain of Rap 1p contains two subdomains that are structurally closely related to c-Myb repeats. We set out to determine the solution structure of the DNA-binding domain of hTRF1 in order to establish its mode of DNA binding. RESULTS: The solution structure of the DNA-binding domain of hTRF1 has been determined and shown to comprise three helices. The architecture of the three helices is very similar to that of each Rap 1p subdomain and also to that of each c-Myb repeat. The second and third helix form a helix-turn-helix (HTH) variant. The length of the third helix of hTRF1 is similar to that of the second subdomain of Rap 1p. CONCLUSIONS: The hTRF1 DNA-binding domain is likely to bind to DNA in a similar manner to that of the second subdomain of Rap 1p. On the basis of the Rap 1p-DNA complex, a model of the hTRF1 DNA-binding domain in complex with human telomeric DNA was constructed. In addition to DNA recognition by the HTH variant, a flexible N-terminal arm of hTRF1 is likely to interact with DNA.

Amino Acid Sequence↗

PubMed1998

The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy.

Using confocal microscopy, onset of the mitochondrial permeability transition (MPT) in individual mitochondria within living cells can be visualized by the redistribution of the cytosolic fluorophore, calcein, into mitochondria. Simultaneously, mitochondria release membrane potential-indicating fluorophores like tetramethylrhodamine methylester. The MPT occurs in several forms of necrotic cell death, including oxidative stress, pH-dependent ischemia/reperfusion injury and Ca2+ ionophore toxicity. Cyclosporin A (CsA) and trifluoperazine block the MPT in these models and prevent cell killing, showing that the MPT is a causative factor in necrotic cell death. During oxidative injury induced by t-butylhydroperoxide, onset of the MPT is preceded by pyridine nucleotide oxidation, mitochondrial generation of reactive oxygen species, and an increase of mitochondrial free Ca2+, all changes that promote the MPT. During tissue ischemia, acidosis develops. Because of acidotic pH, anoxic cell death is substantially delayed. However, when pH is restored to normal after reperfusion (reoxygenation at pH 7.4), cell death occurs rapidly (pH paradox). This killing is caused by pH-dependent onset of the MPT, which is blocked by reperfusion at acidotic pH or with CsA. In isolated mitochondria, toxicants causing Reye's syndrome, such as salicylate and valproate, induce the MPT. Similarly, salicylate induces a CsA-sensitive MPT and killing of cultured hepatocytes. These in vitro findings suggest that the MPT is the pathophysiological mechanism underlying Reye's syndrome in vivo. Kroemer and coworkers proposed that the MPT is a critical event in the progression of apoptotic cell death. Using confocal microscopy, the MPT can be directly documented during tumor necrosis factor-alpha induced apoptosis in hepatocytes. CsA blocks this MPT and prevents apoptosis. The MPT does not occur uniformly during apoptosis. Initially, a small proportion of mitochondria undergo the MPT, which increases to nearly 100% over 1-3 h. A technique based on fluorescence resonance energy transfer can selectively reveal mitochondrial depolarization. After nutrient deprivation, a small fraction of mitochondria spontaneously depolarize and enter an acidic lysosomal compartment, suggesting that the MPT precedes the normal process of mitochondrial autophagy. A model is proposed in which onset of the MPT to increasing numbers of mitochondria within a cell leads progressively to autophagy, apoptosis and necrotic cell death.

Animals↗

PubMed1998

Two metal-binding sites in a lead ribozyme bound to competitively by Pb2+ and Mg2+--induced structural changes as revealed by NMR.

We reported recently that a lead ribozyme with modified bases cleaved at an additional site at high Pb2+ concentrations (>0.1 mM), and that the cleavage at a canonical site was enhanced nearly fourfold at the optimum combination of Pb2+ and Mg2+ concentrations [Kim, M. H., Katahira, M., Sugiyama, T. & Uesugi, S. (1997) J. Biochem. (Tokyo) 122, 1062-1067]. Here we have identified two metal-binding sites (sites 1 and 2) of the lead ribozyme at the residue level by NMR. Both sites are located in an asymmetric internal loop of the lead ribozyme. Site 1 is composed of residues of an enzyme strand and site 2 of residues of a substrate strand. The two sites are bound to competitively by Pb2+ and Mg2+. It was revealed that at certain Pb2+ and Mg2+ concentrations, site 1 is occupied by Pb2+ and site 2 is occupied by Mg2+. The dependency of the cleavage at the canonical and other sites on the Pb2+ and Mg2+ concentrations is interpreted by considering the species of metal ions bound to the two sites. It is suggested that the addition of the two metal ions produces similar and different effects on the structure of the lead ribozyme, and the two metal ions have a synergistic effect on the structure.

Binding Sites↗

PubMed1998

Silent nucleotide substitution in the sterol 27-hydroxylase gene (CYP 27) leads to alternative pre-mRNA splicing by activating a cryptic 5' splice site at the mutant codon in cerebrotendinous xanthomatosis patients.

A functionally silent nucleotide substitution of the sterol 27-hydroxylase gene (CYP 27), identified in two families with cerebrotendinous xanthomatosis (CTX), was confirmed to cause alternative pre-mRNA splicing of the gene. Full-length RT-PCR analysis of the CYP 27 gene in a patient from one of the CTX families revealed one major and an additional faint band. Sequence analysis of the cloned RT-PCR product showed three species of cDNA: 3' terminal 13 bp of exon 2 deleted cDNA, exon 2 skipped cDNA, and full-length cDNA with a functionally silent G to T mutation at codon 112 (GGG 112Gly to GGT 112Gly). Only a single base change was identified by genomic DNA sequence analysis of the CYP 27 gene in the patient: T replaced G at the third position of codon 112, 13 bp upstream from the 3' terminus of exon 2. Transfection of constructed minigenes, with or without the mutation, confirmed that this silent mutation resulted in alternative pre-mRNA splicing by activating a cryptic 5' splice site around the mutant codon. The mutation was also identified in two patients from another CTX family, with a compound heterozygous pattern of A for G substitution at codon 372, a mutation reported previously by our group. The results elucidate a novel molecular basis for the CTX and suggest the significance of a silent nucleotide substitution with regard to pre-RNA splicing.

Alternative Splicing↗

PubMed1998

Ceramide formation leads to caspase-3 activation during hypoxic PC12 cell death. Inhibitory effects of Bcl-2 on ceramide formation and caspase-3 activation.

PC12 cells undergo apoptosis as well as necrosis following exposure to hypoxia. Following a 6-h hypoxic treatment, a time-dependent increase in intracellular ceramide level was observed with a concurrent decrease in sphingomyelin. It was also shown that the hypoxia-induced ceramide accumulation resulted from activation of neutral magnesium-dependent sphingomyelinase. Comparative kinetic analyses of the neutral sphingomyelinase in the cells under normoxia and hypoxia showed that hypoxia increased Vmax but did not affect Km of the enzyme. In PC12 cells overexpressing Bcl-2 which show strong resistance to hypoxia, sphingomyelin hydrolysis was decreased and activation of neutral sphingomyelinase was reduced. Addition of exogenous C2-ceramide induced cell death and activated caspase-3 as markedly as the hypoxia treatment. On the other hand, in PC12 cells overexpressing Bcl-2, significant decreases in cell death and inhibition of caspase-3 activation were observed after exogenous addition of C2-ceramide. The inhibitors of caspase-3 prevented cell death by either hypoxia or C2-ceramide. These results suggest that ceramide generated by activation of neutral magnesium-dependent sphingomyelinase mediates hypoxic cell death and that Bcl-2 has inhibitory effects on ceramide formation and caspase activation.

Animals↗

PubMed1998

Molecular cloning of a cDNA encoding a serine protease homologous to complement C1s precursor from rat C6 glial cells and its expression during glial differentiation.

A cDNA of rat C6 cells was cloned, which was considered to be involved in glial cell differentiation induced by dibutyryl cyclic AMP and theophylline. The cDNA fragment of the gene, termed r-gsp, was originally isolated by mRNA fingerprinting using arbitrarily primed polymerase chain reaction, and was homologous to complement C1s precursors of hamster and human. It encodes a protein of 694 amino acids containing a potential signal peptide, an epidermal growth factor-like domain surrounded by two complement C1r/C1s-related repeats, and a putative trypsin-type serine protease domain. Since the hamster and human C1s, and a protein encoded by r-gsp shared high similarity in primary structure, the r-gsp gene could encode a C1s counterpart of the rat. Messenger RNA expression of this gene was markedly increased during cyclic AMP-induced glial cell differentiation. Its expression profile was well correlated with those of glial fibrillary acidic protein (GFAP) and S100B, which are known as glial differentiation markers. It was, moreover, observed that the r-gsp expression in brain increased considerably after birth, like those of S100B and GFAP. The results presented here suggest that the rat C1s gene would be also implicated in glial differentiation besides the complement cascade.

Amino Acid Sequence↗

PubMed1998

Substitutions of conserved aromatic amino acid residues in subunit I perturb the metal centers of the Escherichia coli bo-type ubiquinol oxidase.

Cytochrome bo is a four-subunit quinol oxidase in the aerobic respiratory chain of Escherichia coli and functions as a redox-coupled proton pump. Subunit I binds all the redox metal centers, low-spin heme b, high-spin heme o, and CuB, whose axial ligands have been identified to be six invariant histidines. This work explored the possible roles of the aromatic amino acid residues conserved in the putative transmembrane helices (or at the boundary of the membrane) of subunit I. Sixteen aromatic amino acid residues were individually substituted by Leu, except for Tyr61 and Trp282 by Phe and Phe415 by Trp. Leu substitutions of Trp280 and Tyr288 in helix VI, Trp331 in loop VII-VIII, and Phe348 in helix VIII reduced the catalytic activity, whereas all other mutations did not affect the in vivo activity. Spectroscopic analyses of the purified mutant enzymes revealed that the defects were attributable to perturbations of the binuclear center. On the basis of these findings and recent crystallographic studies on cytochrome c oxidases, we discuss the possible roles of the conserved aromatic amino acid residues in subunit I of the heme-copper terminal oxidases.

Amino Acid Sequence↗

PubMed1998

Induction of expression of MHC-class-II antigen on human thyroid carcinoma by wild-type p53.

Mutation of the tumor-suppressor gene p53 is involved in carcinogenetics. We investigated the role of p53 in the induction of anti-tumor immune responses by establishing a thyroid carcinoma cell line (1F3) prepared by transfection of wild-type human p53 gene into a p53-deficient cell line (FRO). Our results showed for the first time the involvement of p53 in the induction of anti-tumor immune responses, as demonstrated by: (i) expression of the major-histocompatibility-complex(MHC)-class-II antigen on 1F3, but not FRO; (ii) mRNA of class-II gene was expressed both in 1F3 and in FRO, but was stable at post-transcriptional level in FRO, which restrained protein synthesis; (iii) 1F3 induced MHC-class-II-specific CD4+ cytotoxic-T-cell activity through allo-antigen presentation and co-stimulation. Although our novel results are limited to the wild-type-p53-expressing clone from a p53-deficient cell line, we suggest that the absence of p53 in carcinoma cells may reduce the induction of CD4+ cytotoxic-T-cell activity against carcinoma cells by diminishing the expression of class-II antigen.

Blotting, Western↗

PubMed1998

Hyperglycaemia due to insulin resistance caused by interferon-gamma.

Marked hyperglycaemia (30.9 mmol l(-1)) during interferon-gamma (IFN-gamma) therapy for asymptomatic recurrent renal cancer as multiple lung metastases in a 52-year-old man is described. Although the involvement of IFN-gamma has been reported in the development of autoimmune diabetes, in this case, antibodies against pancreatic beta-cells including anti-islet cell antibody (ICA) and anti-glutamic acid decarboxylase (GAD) antibody were negative. Moreover, serum level of immunoreactive insulin (IRI) (11 microU ml(-1) at fasting) and urinary excretion of C-peptide (108 microg day(-1), reference range: 20-130) suggested insulin resistance, supported by results of insulin tolerance tests. With insulin therapy and cessation of IFN-gamma, fasting blood glucose concentration returned to 6.2 mmol l(-1), and insulin therapy was discontinued. The injection of IFN-gamma may cause hyperglycaemia because of insulin resistance, rather than beta-cell injury.

Adenocarcinoma, Clear Cell↗

PubMed1998

Evaluation of a novel alginate gel dressing: cytotoxicity to fibroblasts in vitro and foreign-body reaction in pig skin in vivo.

Calcium alginate dressings have beneficial effects on wound healing by providing a moist wound environment. However, cytotoxicity and the nonbiodegradable nature of calcium alginate dressings induce unresolved chronic foreign-body reaction. In this study, a novel freeze-dried alginate gel dressing (AGA-100) low in calcium ions was evaluated for cytotoxicity to L929 cells in vitro and in full-thickness pig wounds in vivo. Cytotoxicity testing on L929 cells showed the cytocompatibility of AGA-100 extracts, while extracts from Kaltostat, a well-established alginate dressing, induced cytopathic effects. In an in vivo study using pigskin, AGA-100, Kaltostat, and gauze were applied on 1-in-diameter circular full-thickness wounds on the back of pigs and the time course of wound closure was evaluated. Kaltostat and gauze dressings were used as controls. For histologic evaluation, wound tissue was harvested on day 18. AGA-100-treated wounds showed rapid wound closure compared to control wounds on day 15. Foreign-body reaction was marked in Kaltostat- and gauze-treated wounds, and differed significantly from AGA-100-treated wounds. Based on these data, AGA-100 could reduce the cytotoxicity to fibroblasts and foreign-body reaction that have been observed with currently available calcium alginate dressings; it was also found to be useful as an alginate dressing.

Alginates↗

PubMed1998

A new drug delivery system with controlled release of antibiotic only in the presence of infection.

An ideal drug delivery system (DDS) releases an appropriate drug at specific locations and times. We tried to create a new antibiotic delivery system that releases gentamicin only when wounds are infected by Pseudomonas aeruginosa (P.A.). Exudate from the dorsal pouch of rats infected with P.A. showed significantly higher hydrolytic activity-thrombin-like activity-toward Boc-Val-Pro-Arg-MCA than exudate from noninfected wounds. We therefore constructed a device for controlled release of an antimicrobial drug triggered by thrombin-like activity. Briefly, gentamicin was bound to a polyvinyl alcohol derivative (PVA) hydrogel through a newly developed peptide linker cleavable by the proteinase, PVA-(linker)-gentamicin. In vitro experiments showed that proteinases from wounds infected with P.A. cleaved the linker and gentamicin was released while the exudate from noninfected wounds had no hydrolytic activity toward the linker. This device shows potential as an occlusive dressing with an effective antibiotic delivery system for treating infected wounds.

Amino Acid Sequence↗

PubMed1998

Evidence that HLA class II-restricted human CD4+ T cells specific to p53 self peptides respond to p53 proteins of both wild and mutant forms.

By stimulating peripheral blood mononuclear cells of four healthy donors with a mixture of overlapping peptides representing the core domain of p53, we established two CD4+ alphabeta T cell clones and four lines that recognized wild-type and mutant p53 proteins as well as p53 self peptides in an HLA class II-restricted fashion. Two T cell lines established from two unrelated donors reacted to the p53 peptide (p)153-166 and p108-122, respectively, in the context of DP5 molecules. Two T cell clones established from two other unrelated donors were specific for p193-204 in the context of DRB1*1401 and for p153-165 in the context of DP5, respectively. These two T cell clones responded almost equally to both wild-type and four mutant recombinant p53 proteins. The proliferative responses of these T cell clones to p53 recombinant proteins were augmented by heat denaturing, thereby suggesting that altered conformation of the protein facilitates proteolytic processing to produce antigenic peptides. The DRB1*1401-restricted T cell clone specific for p193-204 killed a B lymphoblastoid cell line homozygous for HLA-DRB1*1401 when the cell line was pre-pulsed with p53 protein as well as peptide. These results indicate that CD4+ T cells reactive to p53 do exist in healthy individuals and the epitopes are probably ignored by the immune system under physiological conditions. It is suggested that such epitopes stimulate T cells to induce anti-p53 antibody production in cancer patients as previously reported by others. The possible involvement of p53-reactive T cells in anti-tumor immunity is discussed.

Adult↗

PubMed1998

Modulation of calcium responses by altered peptide ligands in a human T cell clone.

To determine whether altered peptide ligands (APL) affect calcium signaling events, we investigated changes in intracellular calcium concentration ([Ca2+]i) in human T cell clone stimulated with either the fully agonistic peptide M12p54-68, the partially agonistic analogue E63V or the simple antagonistic analogue E58M. Both E63V and E58M stimulated a Ca2+ response in approximately 40% of T cells, whereas M12p54-68 did so in approximately 70% of T cells. The most predominant pattern of a Ca2+ increase induced by M12p54-68 was a small sinusoidal peak followed by a sustained high response. The most frequent pattern of calcium response induced by E63V was a continuous high response without a preceding sinusoidal peak, whereas that induced by E58M was large with frequent oscillations. Genistein, an inhibitor of the protein tyrosine kinases (PTK), markedly inhibited the wild-type peptide-induced increase in [Ca2+]i, whereas it marginally inhibited the response induced by E63V or E58M. In contrast, GF109203X, a protein kinase C (PKC)-specific inhibitor, markedly inhibited the E63V- or E58M-induced Ca2+ response, whereas it marginally affected the wild peptide-induced Ca2+ response. Furthermore, in nominal Ca2+-free medium, the E58M-induced Ca2+ response was almost completely blocked, while the M12p54-68- or E63V-induced responses were only partially inhibited. Our results suggest that the Ca2+ response induced by the fully agonistic peptide depends on activation of the genistein-sensitive signaling pathway, including PTK, whereas the Ca2+ response to a simple antagonistic APL completely depends on extracellular Ca2+ and activation of the GF109203X-sensitive signaling pathway, including PKC. These differences in the CA2+i response in recognition of different APL may parallel the unique T cell activation patterns induced by APL in human T cells.

Amino Acid Sequence↗

PubMed1998

Confocal microscopy of the mitochondrial permeability transition in necrotic cell killing, apoptosis and autophagy.

Onset of the cyclosporin-A-sensitive mitochondrial permeability transition (MPT) in individual mitochondria within living cells can be visualized by laser scanning confocal microscopy. The MPT is a causative event in many types of necrotic and apoptotic cell death, including oxidative stress, ischemia/reperfusion injury, Ca2+ ionophore toxicity and tumor necrosis factor alpha (TNF alpha) induced apoptosis, and may contribute to Reye's-related drug toxicity. Pyridine nucleotide oxidation, mitochondrial generation of reactive oxygen species, and increased mitochondrial Ca2+ and pH can each promote onset of the MPT in situ. The MPT can also be directly visualized during TNF alpha-induced apoptosis to hepatocytes. Mitochondria spontaneously depolarize in situ after nutrient deprivation before entering an acidic lysosomal compartment, suggesting that the MPT precedes the normal process of mitochondrial autophagy. We propose a model in which onset of the MPT to increasing numbers of mitochondria leads progressively to autophagy, apoptosis and necrotic cell death.

Animals↗

PubMed1998

Mitochondrial dysfunction and cytoskeletal disruption during chemical hypoxia to cultured rat hepatic sinusoidal endothelial cells: the pH paradox and cytoprotection by glucose, acidotic pH, and glycine.

We investigated mechanisms underlying death of cultured rat liver sinusoidal endothelial cells exposed to chemical hypoxia with KCN (2.5 mmol/L) to simulate the adenosine triphosphate (ATP) depletion and reductive stress of anoxia. During chemical hypoxia, acidotic pH prevented cell death. Glucose (0.3-10 mmol/L) also prevented cell killing. Cytoprotection by glucose but not acidosis was associated with prevention of ATP depletion. After 4 hours of chemical hypoxia at pH 6.2 (simulated ischemia), rapid cell death occurred when pH was restored to pH 7.4 with or without washout of KCN (simulated reperfusion). This pH-dependent reperfusion injury (pH paradox) was prevented after KCN washout at pH 6.2. Glycine (0.3-3 mmol/L) also prevented the pH paradox, but glucose did not. The initial protection by acidotic pH and glycine during simulated reperfusion was lost when pH was later restored to 7.4 or glycine was subsequently removed. Mitochondria depolarized during chemical hypoxia. After washout of cyanide, mitochondrial membrane potential (delta psi) did not recover in cells that subsequently lost viability. Conversely, those cells that repolarized after cyanide washout did not subsequently lose viability. The actin cytoskeleton and focal adhesions became severely disrupted during chemical hypoxia at both pH 6.2 and 7.4 and did not recover after cyanide washout under any condition. Glucose during chemical hypoxia prevented cytoskeletal disruption. In conclusion, endothelial cell damage during simulated ischemia/reperfusion involves mitochondrial dysfunction, ATP depletion, and ATP-dependent cytoskeletal disruption. Glycine and acidotic pH prevented cell killing after reperfusion but did not reverse mitochondrial injury or the profound disruption to the cytoskeleton.

Adenosine Triphosphate↗

PubMed1998

Malignant transformation alters intracellular trafficking of lysosomal cathepsin D in human breast epithelial cells.

Increased expression and alteration of intracellular trafficking of lysosomal cathepsins have been reported in malignant tumors, or in cells transformed by the transfection with the ras oncogene. In the present study, immortal MCF-10A human breast epithelial cells were transformed with the mutated ras oncogene. Both cell lines were investigated for changes in the intracellular localization of lysosomal cathepsin D and lamp-1 (lysosome-associated membrane protein) employing specific antibodies and confocal immunofluorescence microscopy. The results revealed that staining for cathepsin D along with for lamp-1 was mostly localized in the perinuclear region of MCF-10A cells. In contrast, the staining for these proteins was found to be widely distributed throughout the cytoplasm and at the cell periphery in MCF-10AneoT cells. The organization of microtubules, but not actin, appeared to differ between MCF-10A cells and their oncogenic ras transfectants. When the microtubules were depolymerized by treatment of MCF-10A cells with nocodazole, vesicles containing the lysosomal cathepsin D were dispersed in the cytoplasm and translocation of these vesicles to the cell periphery was observed. The intracellular localization of cathepsin D in the nocodazole-treated MCF-10A cells seemed to be similar to that observed in the oncogenic ras transfectants of these cells. When taxol, which inhibits microtubule depolymerization, was added to the culture medium of neoT cells, a polymerized microtubule network was observed, and the reclustering of cathepsin D and lamp-1 occurred in an unidirectional manner towards the perinuclear region. These findings support a model in which cytoskeletal microtubule organization is closely related to the trafficking of lysosomes/endosomes, and in which oncogenic ras interferes with such organization in human breast epithelial cells.

Animals↗