Biomedical subjects

M Enari

Publications and source records attributed to M Enari.

30 records · Page 2Linked to original sources

PubMed1996

Different apoptotic pathways mediated by Fas and the tumor-necrosis-factor receptor. Cytosolic phospholipase A2 is not involved in Fas-mediated apoptosis.

Fas is a cell-surface receptor that belongs to the tumor-necrosis factor (TNF)/nerve growth factor receptor family. Fas can transduce an apoptotic signal through the death domain in the cytoplasmic region, which has similarity with the corresponding region of the TNF type-I receptor. Here, we expressed human Fas in mouse L929 cells or its subline (C12), which express extremely low levels of cytosolic phospholipase A2 (cPLA2). L929 cells were sensitive to the cytotoxic activity of TNF, while C12 cells were resistant. Cross-linking of human Fas with anti-(human Fas antibody) Ig killed both L929 transformants and C12 transformants expressing human Fas. Various inhibitors of the arachidonate metabolism significantly inhibited the TNF-induced cytotoxicity in L929 cells, but they did not have any effect on Fas-mediated apoptosis. These results indicated that cPLA2 is required for TNF-induced apoptosis, whereas it is dispensable for Fas-mediated apoptosis, and suggested that the TNF receptor and Fas use different signaling pathways for apoptosis.

Animals↗

PubMed1996

Involvement of Fas in regression of vaginal epithelia after ovariectomy and during an estrous cycle.

Fas, also called APO-1, belongs to the tumor necrosis factor/nerve growth factor receptor family and transmits an apoptotic signal within the cell by binding to the Fas ligand. Fas has been implicated in the activation-induced suicide of T cells and cytotoxic T cell activity in the immune system. Non-immune cells such as those in liver, lung and ovary also express Fas, but its role in these cells remains unclear. Ovariectomy has been used to study homeostasis of female reproductive organs, which is regulated by sex hormones. Here we analyzed Fas function in the ovariectomy-induced regression of mouse vaginal epithelial cells. Fas expression was detected in vagina and was elevated after ovariectomy. Fas-deficient lpr and lpr(cg) mice did not exhibit ovariectomy-induced regression of vaginal epithelia, whereas uterine regression induced by ovariectomy was not affected in these mice. The vaginas of lpr and lpr(cg) mice were in a persistent estrous stage with cornification of vaginal epithelia, as judged from the cell types in the vaginal fluid. Thus, Fas appears to be involved directly in the regression of vaginal epithelia induced by ovariectomy and during the estrous cycle, suggesting that the physiological role of this receptor extends beyond that exerted on immune cells. This is the first evidence of a role for Fas inducing physiological apoptosis in non-immune cells.

Animals↗

PubMed1996

The cavity in the hydrophobic core of Myb DNA-binding domain is reserved for DNA recognition and trans-activation.

The DNA-binding domain of Myb consists of three imperfect repeats, R1, R2 and R3, each containing a helix-turn-helix motif variation. Among these repeats, R2 has distinct characteristics with high thermal instability. The NMR structure analysis found a cavity inside the hydrophobic core of R2 but not in R1 or R3. Here, we show that R2 has slow conformational fluctuations, and that a cavity-filling mutation which stabilizes the R2 structure significantly reduces specific Myb DNA-binding activity and trans-activation. Structural observations of the free and DNA-complexed stages suggest that the implied inherent conformational flexibility of R2, associated with the presence of the cavity, could be important for DNA recognition by Myb.

Amino Acid Sequence↗

PubMed1995

Structural comparison of the free and DNA-bound forms of the purine repressor DNA-binding domain.

BACKGROUND: The purine repressor (PurR) regulates genes that encode enzymes for purine biosynthesis. PurR has a two domain structure with an N-terminal DNA-binding domain (DBD) and a C-terminal corepressor-binding domain (CBD). The three dimensional structure of a ternary complex of PurR bound to both corepressor and a specific DNA sequence has recently been determined by X-ray crystallography. RESULTS: We have determined the solution structure of the PurR DBD by NMR. It contains three helices, with the first and second helices forming a helix-turn-helix motif. The tertiary structure of the three helices is very similar to that of the corresponding region in the ternary complex. The structure of the hinge helical region, however, which makes specific base contacts in the minor groove of DNA, is disordered in the DNA-free form. CONCLUSION: The stable formation of PurR hinge helices requires PurR dimerization, which brings the hinge regions proximal to each other. The dimerization of the hinge helices is likely to be controlled by the CBD dimerization interface, but is induced by specific-DNA binding.

Amino Acid Sequence↗

PubMed1995

Apoptosis by a cytosolic extract from Fas-activated cells.

Fas is a type I membrane protein and its activation by binding of the Fas ligand or an agonistic anti-Fas antibody induces apoptosis in Fas-bearing cells. In this report we prepared lysates from cells treated with anti-Fas antibody. The lysates induced apoptotic morphological changes in nuclei from normal mouse liver, accompanied by DNA degradation. The apoptosis-inducing activity was quickly generated in cells by anti-Fas antibody and was found in the soluble cytosolic fraction. Induction of the activity in cells was inhibited by a tetrapeptide, acetyl-Tyr-Val-Ala-Asp-chloromethylketone, a specific inhibitor of interleukin-1 beta converting enzyme. Addition of COS cell lysates containing Bcl-2 to the assay significantly inhibited the apoptotic process, indicating that the in vitro process reflected apoptosis that occurs in intact cells.

Animals↗

PubMed1995

Involvement of an ICE-like protease in Fas-mediated apoptosis.

Fas is a type-I membrane protein that transduces an apoptotic signal. Binding of Fas ligand or agonistic anti-Fas antibody to Fas kills the cells by apoptosis. Studies in the nematode Caenorhabditis elegans have suggested that proteases such as interleukin-1 beta-converting enzyme (ICE) or the product of the C. elegans cell-death gene ced-3 are involved in apoptotic signal transduction. The activity of ICE can be inhibited by the product of crmA, a cytokine-response modifier gene encoded by cowpox virus. We report here that expression of crmA inhibits cytotoxicity induced by anti-Fas antibody or tumour necrosis factor (TNF). We have found a specific ICE inhibitor tetrapeptide (acetyl-Tyr-Val-Ala-Asp-chloromethylketone) that also prevents apoptosis induced by anti-Fas antibody. These results suggest an involvement of an ICE-like protease in Fas-mediated apoptosis and TNF-induced cytotoxicity.

Amino Acid Sequence↗

PubMed1994

No requirement of reactive oxygen intermediates in Fas-mediated apoptosis.

Fas is a cell surface molecule that mediates apoptosis, but the intracellular mechanisms leading to apoptosis are not well understood. It is known that diethylmaleate (DEM)-induced cell death can be blocked by substances with antioxidant activity. Here we have studied whether antioxidants have any effect on Fas-mediated apoptosis and show that they are not able to block Fas-mediated apoptosis. Therefore, it seems that reactive oxygen intermediate (ROI)-dependent and -independent mechanisms which lead to apoptosis do exist. Fas-mediated apoptosis probably proceeds via a ROI-independent pathway.

Animals↗

PubMed1993

Recognition of specific DNA sequences by the c-myb protooncogene product: role of three repeat units in the DNA-binding domain.

The DNA-binding domain of c-Myb consists of three homologous tandem repeats of 52 amino acids. The structure of the third (C-terminal) repeat obtained by NMR analysis has a conformation related to the helix-turn-helix motif. To identify the role of each repeat in the sequence recognition of DNA, we analyzed specific interactions between c-Myb and DNA by measuring binding affinities for systematic mutants of Myb-binding DNA sites and various truncated c-Myb mutants. We found that specific interactions are localized unevenly in the AACTGAC region in the consensus binding site of c-Myb: The first adenine, third cytosine, and fifth guanine are involved in very specific interactions, in which any base substitutions reduce the binding affinity by > 500-fold. On the other hand, the interaction at the second adenine is less specific, with the affinity reduction in the range of 6- to 15-fold. The seventh cytosine involves a rather peculiar interaction, in which only guanine substitution abolishes the specific binding. The binding analyses, together with the chemical protection analyses, showed that the c-Myb fragment containing the second and third repeats covers the AACTGAC region from the major groove of DNA in such an orientation that the third repeat covers the core AAC sequence. These results suggest that the third repeat recognizes the core AAC sequence very specifically, whereas the second repeat recognizes the GAC sequence in a more redundant manner. The first (N-terminal) repeat, which covers the major groove of DNA only partially, is not significant in the sequence recognition, but it contributes to increase the stability of the Myb-DNA complex. The presence of an N-terminal acidic region upstream of the first repeat, which is important for the activation of c-myb protooncogene, was found to reduce the binding affinity by interfering with the first repeat in binding to DNA.

Base Sequence↗

PubMed1992

Interaction of a protooncogene product, Myb with DNAs.

The DNA-binding domain of Myb consists of three imperfect tandem repeats and the third one which is essential for sequence-specific binding was established to have a helix-turn-helix-related motif. DNA sequences recognized by Myb have been reported to contain TAACPy sequence. Here we have examined the details of Myb-binding sequence. Using DNAs with a single mutation on the various sites of two specific DNAs and some fragments of the DNA-binding domain of Myb, we have found that (i) in a specific DNA which contains only one AAC sequence, each AAC nucleotide is found to be essential for the specific binding of Myb, while any other mutations cause no serious binding loss, (ii) in a specific DNA which contains two AAC sequences separately, one AAC is not so important in the binding, and (iii) for the specific binding with DNA, at least both repeats 2 and 3 of Myb are required. These findings suggest that repeat 3 containing a helix-turn-helix-related structure recognizes the core AAC sequence and repeat 2 supports this recognition by interactions with phosphate groups of DNA.

Base Sequence↗

PubMedDate not supplied

Characterization and role of proteinases induced by estrogen-deprivation in female mouse reproductive tracts.

Normal mouse vaginae and uteri regress following ovariectomy, whereas the vagina of mice given five daily injections of 3 micrograms diethylstilbestrol (DES) from the day of birth exhibit ovary (estrogen)-independent persistent stratification and cornification of the epithelium. Zymography indicated expression of four proteinases in both vaginae and uteri of normal mice after ovariectomy. Two proteinases detected in gelatin-containing gel and two others in casein-containing gel proved to be metalloproteinases and serineproteinases, respectively. The two metalloproteinases were identified as gelatinases A and B. Only gelatinase B was intensified 1 d after ovariectomy; however, all four proteinases showed an increase in expression 3 d after ovariectomy. In the uterus, the two gelatinases showed increased expression after ovariectomy. Progelatinase B and serineproteinase II were expressed in the vagina of normal mice at estrus; ovariectomy intensified expression and activation of gelatinases and serineproteinases II in the vagina. Vaginae of mice treated neonatally with DES exhibited a weak expression of proteinases. Ovariectomy changed neither the histology nor the expression of proteinases in these DES-exposed vaginae. Expression of gelatinases was inhibited by estrogen; progesterone stimulated expression and activation of gelatinase B. Serineproteinases found in the vagina and uterus of ovariectomized mice were also inhibited by estrogen but neither was affected by progesterone. These results suggest that gelatinase B and both gelatinases participate in vaginal and uterine regression, respectively, following ovariectomy. Estrogen negatively regulates expression of gelatinases and serineproteinases in the vagina, and of gelatinase A and serineproteinase II in the uterus.

Animals↗

PubMedDate not supplied

Effect of neonatal exposure to DES in Fas and Bcl-2 expression in the adult mouse vagina and approach to the DES syndrome.

Neonatal exposure to a synthetic estrogen, diethylstilbestrol (DES), induces the ovary-independent persistent proliferation of vaginal epithelium in mice. Mouse vagina at the estrous stage in the normal estrous cycle shows 10 to 15 layers of epithelium with superficial keratinized layers, and ovariectomy induces a decrease of these epithelial cell layers and lymphocyte infiltration. Thus, cell proliferation and regression of vaginal epithelium are ovary dependent. Neonatally DES-treated mouse vagina showed the same phenotype as normal mouse vagina at the estrous stage, but ovariectomy did not induce a decrease of epithelial cell layers or a lymphocyte infiltration, and there was persistent proliferation of vaginal epithelium even after ovariectomy. In addition, apoptotic cell death characterized by oligonucleosomal DNA fragmentation, Fas expression, and Bcl-2 downregulation were induced after ovariectomy in normal mouse vagina, but not in DES-treated mouse vagina. These results suggest that neonatal DES-exposure in mice prevents vaginal Fas-mediated apoptosis following the downregulation of Bcl-2, and these abnormalities in expression are involved in persistent proliferation of the vaginal epithelium.

Animals↗

PubMedDate not supplied

Effect of ovariectomy on histological change and protein expression in female mouse reproductive tracts.

Proliferation of the vagina and uterus in rodents is stimulated by ovarian estrogen, and ovariectomy induces regression of these tracts. In mouse, reproductive tracts, organ weights, vaginal epithelial thickness, the number of epithelial cell layers, and uterine epithelial cell height were significantly reduced 1 to 20 days after ovariectomy when compared with those of intact, mice taken at the estrous stage. No significant differences in those parameters were found between ovariectomized mice implanted with estradiol and intact estrous mice. Vaginal epithelium at estrus was composed of 10-15 layers of epithelial cells with superficial keratinized layers, however, the vaginal epithelium began to regress one day after ovariectomy. Infiltration of leukocytes was encountered in the vaginal epithelium 2-3 days after ovariectomy. Protein expression in the regressing vagina and uterus after ovariectomy was examined by two-dimensional polyacrylamide gel electrophoresis. After ovariectomy, new proteins appeared in the regressing vagina, but not in the regressing uterus. Expressions of 7 and 6 proteins in the vagina and uterus, respectively, reached peak levels 2-3 days after ovariectomy. The expression of ovariectomy-specific proteins and infiltration of leukocytes in vaginal epithelium was inhibited by the injection of cycloheximide, an inhibitor of protein synthesis. These results suggest that some proteins participate in the regression of mouse reproductive tracts after estrogen withdrawal induced by ovariectomy.

Animals↗