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S Kamada

Publications and source records attributed to S Kamada.

At least 19 recordsLinked to original sources
PubMed1998

A cloning method for caspase substrates that uses the yeast two-hybrid system: cloning of the antiapoptotic gene gelsolin.

Caspase-mediated proteolysis is a critical and central element of the apoptotic process; therefore, it is important to identify the downstream molecular targets of caspases. We established a method for cloning the genes of caspase substrates by two major modifications of the yeast two-hybrid system: (i) both large and small subunits of active caspases were expressed in yeast under ADH1 promoters and the small subunit was fused to the LexA DNA-binding domain; and (ii) a point mutation was introduced that substituted serine for the active site cysteine and thereby prevented proteolytic cleavage of the substrates, possibly stabilizing the enzyme-substrate complexes in yeast. After screening a mouse embryo cDNA expression library by using the bait plasmid for caspase-3, we obtained 13 clones that encoded proteins binding to caspase-3, and showed that 10 clones including gelsolin, an actin-regulatory protein implicated in apoptosis, were cleaved by recombinant caspase-3 in vitro. Using the same bait, we also isolated human gelsolin cDNA from a human thymus cDNA expression library. We showed that human gelsolin was cleaved during Fas-mediated apoptosis in vivo and that the caspase-3 cleavage site of human gelsolin was at D352 of DQTD352G, findings consistent with previous observations on murine gelsolin. In addition, we ascribed the antiapoptotic activity of gelsolin (which we previously reported) to prevention of a step leading to cytochrome c release from the mitochondria into the cytosol. Our results indicate that this cloning method is useful for identification of the substrates of caspases and possibly also of other enzymes.

Apoptosis↗
PubMed1997

Evidence against a functional site for Bcl-2 downstream of caspase cascade in preventing apoptosis.

Apoptotic cell death is driven by ICE family proteases (caspases) and negatively regulated by Bcl-2 family proteins. Although it has been shown that Bcl-2 exerts anti-apoptotic activity by blocking a step(s) leading to the activation of caspases, a role for Bcl-2 and Bcl-xL downstream of the caspase cascade has remained unclear. Here, we show that purified active caspase-3 (CPP32/Yama/apopain) and caspase-1 (ICE) induces apoptosis when microinjected into the cytoplasm of cells, confirming our recent observations, and that the apoptosis is not at all prevented by Bcl-2 and Bcl-xL, which are overexpressed more than sufficiently to prevent Fas-mediated and overexpressed procaspase-1-mediated apoptosis. Thus, Bcl-2 and Bcl-xL do not act downstream of the caspase cascade.

Apoptosis↗
PubMed1997

Involvement of caspase-4(-like) protease in Fas-mediated apoptotic pathway.

Proteases of the caspase family, especially caspase-1 (ICE)(-like), caspase-3 (CPP32/Yama/apopain)(-like) and caspase-8 (MACH/FLICE/Mch5) proteases, are implicated in Fas (APO-1/CD95)-mediated apoptosis. Here, we show that the caspase-4 (TX/ICH-2/ICE(rel)II)(-like) protease, another member of the caspase family, is also involved in Fas-mediated apoptosis, based upon the observations: (i) caspase-4 is processed in response to an agonistic anti-Fas antibody treatment, (ii) overexpression of a mutant caspase-4 with active site mutations in both p20 and p10 subunits delays Fas-mediated apoptosis, (iii) microinjected anti-caspase-4 antibodies inhibit Fas-mediated apoptosis. Together with our observations that the mutant caspase-4 inhibits the Fas-mediated activation of caspase-3(-like) proteases and purified caspase-4 cleaves pro-caspase-3 to generate a subunit of active form, these results suggest that Fas-mediated apoptosis is driven by a caspase cascade in which the caspase-4(-like) protease transmits a death signal from caspase-8 to caspase-3(-like) proteases probably through directly cleaving pro-caspase-3(-like) proteases.

Antibodies↗
PubMed1997

Susceptibility of cerebellar granule neurons derived from Bcl-2-deficient and transgenic mice to cell death.

Overproduced Bcl-2 oncoprotein has been shown to suppress cell death induced by a variety of stimuli in many cell types, including neuronal cells. Because bcl-2 is expressed in the nervous system where massive cell death is observed during development, endogenous Bcl-2 is likely to be involved in regulating neuronal cell death. Here we examined the possible role of endogenous Bcl-2 in the regulation of neuronal cell survival in the central nervous system using primary cultured cerebellar granule neurons from bcl-2-deficient, wild-type and NSE-bcl-2-transgenic mice. Cerebellar granule neurons from bcl-2-deficient mice were more susceptible than those from normal littermates to death induced by reducing the K+ concentration of the medium from high (25 mM) to low (5 mM), and neurons from bcl-2-transgenic mice were least susceptible. Similar results were obtained when cell death was induced by serum withdrawal under high K+ conditions or by the presence of etoposide, A23187 or nimodipine. Consistently, bcl-2 deficiency reduced the number of cerebellar granule neurons per mouse. These results indicate that Bcl-2 impedes neuronal cell death induced by various stimuli in a dose-dependent manner, and that endogenous levels of Bcl-2 are able to regulate neuronal cell survival in the central nervous system.

Animals↗
PubMed1996

Accelerated disappearance of melanocytes in bcl-2-deficient mice.

Follicular melanocytes in bcl-2(-/-) mice have been reported to turn gray during the second hair cycle. Light microscopic analysis revealed that about half of bcl-2(-/-) mouse hair shafts had no detectable melanin granules after the second hair follicle cycle, but the remaining hair appeared to be pigmented normally. After depilation to induce new anagen hair, more than 97% of the hair shafts did not have visible melanin granules in bcl-2(-/-) mice, whereas 100% of the hair shafts in bcl-2(+/+) mice were pigmented. In bcl-2(+/+) mice, dopa-positive melanocytes appeared on day 4 after depilation, whereas bcl-2(-/-) mice developed few dopa-positive melanocytes after depilation, as assessed by light and electron microscopic observation. bcl-2(-/-) mouse hair in the second hair cycle contained about 60-70% less melanin than normal mouse hair, and newly generated bcl-2(-/-) mouse hair after depilation contained a level of melanin as low as that of albino mouse hair. These observations suggest that the expression of bcl-2 might be essential for melanocyte maintenance after the second hair cycle.

Animals↗
PubMed1996

Involvement of CPP32/Yama(-like) proteases in Fas-mediated apoptosis.

Fas (Apo-1/CD95) belongs to the tumor necrosis factor/nerve growth factor receptor family and transmits apoptotic signals by binding to its ligand. Interleukin-1beta-converting enzyme (ICE), which shows substantial homology to the product of the cell death gene, ced-3, of Caenorhabditis elegans, is reported to be involved in Fas-mediated apoptosis. Using two human carcinoma-derived cell lines with undetectable levels of ICE, we found that an agonistic antihuman Fas antibody induces the activation of CPP32/Yama(-like) proteases that are ICE(-like) protease family members, and that a tetrapeptide inhibitor of CPP32/Yama protease, DEVD-CHO, inhibits the Fas-mediated activation of the proteases, Fas-mediated apoptosis, and CPP32/Yama(-like) proteolytic activities in vitro. Fas-mediated apoptosis is inhibited by the CPP32/Yama inhibitor DEVD-CHO, but not by the ICE inhibitor YVAD-CHO, suggesting a dominant role for the CPP32/Yama(-like) proteases and not ICE itself in Fas-mediated apoptosis of the human carcinoma cell lines.

Amino Acid Sequence↗
PubMed1996

[Inhibition of apoptosis by a baculovirus p35 gene].

The baculovirus gene p35 inhibits virus-induced apoptosis in insect cells. p35 can also inhibit developmentally programmed cell death in Caenorhabditis elegans and Drosophila, mammalian neuronal cell death induced by serum or NGF deprivation, and Fas- and tumor necrosis factor (TNF)-induced apoptosis in mammalian cells, indicating that p35 may interrupt an evolutionally conserved component of the death machinery. Recently it has been shown that p35 protein functions as an inhibitor of ICE/CED-3 cysteine protease family that seem to play an important role in an apoptotic pathway. This observation indicates that p35 may inhibit apoptosis by directly blocking the activities of these cysteine proteases in diverse animals.

Amino Acid Sequence↗
PubMed1995

bcl-2 deficiency in mice leads to pleiotropic abnormalities: accelerated lymphoid cell death in thymus and spleen, polycystic kidney, hair hypopigmentation, and distorted small intestine.

Transgenic mice homozygously lacking in the bcl-2 gene were generated using homologous recombination in embryonal stem cells. The complete absence of Bcl-2 alpha and -beta proteins did not interfere with normal embryonic development. Abnormalities became evident after birth, although the severity varied among homozygous null mice, bcl-2-/- mice displayed pleiotropic abnormalities similar to those in the previously described bcl-2-/- mice, including growth retardation, smaller ears, short lives, polycystic kidney, atrophic thymus and spleen with accelerated apoptotic cell death of lymphocytes, and hair hypopigmentation in the second hair follicle cycle. Our bcl-2-/- mice also revealed novel defects in the small intestine, characterized by retarded development, accelerated exfoliation of epithelial cells, and very few mitotic progenitor cells.

Abnormalities, Multiple↗
PubMed1995

Endothelin synthesis and receptors in human endometrium throughout the normal menstrual cycle.

This study was undertaken to investigate the presence of messenger RNA (mRNA) for prepro-endothelin-I (ET-1) and the known receptor subtypes (ETA and ETB) in human endometrium at different stages of the menstrual cycle obtained at hysterectomy. Northern blot analysis revealed expression of ET-1 mRNA in human endometrium during the normal menstrual cycle. The concentration of ET-1 mRNA in endometrial tissue was greater during the menstrual and proliferative phases than during the ovulatory and secretory phases. Immunoreactive ET-1 was secreted into the medium of isolated endometrial stromal cells. Oestradiol and progesterone significantly attenuated ET-1 release in endometrial stromal cells cultured for 6 days. ETA and ETB mRNA were also present in endometrial tissue of the normal cycle. The concentration of ETA receptor mRNA was greater in the proliferative phase than in the secretory phase, whereas expression of ETB mRNA increased in menstrual phase. ET-1 significantly increased extracellular accumulation of cyclic AMP (cAMP), intracellular generation of inositol phosphates and significantly enhanced DNA synthesis in cultured endometrial stromal cells from the proliferative phase. Our results showed that human endometrial cells synthesized and released ET-1, and contained ETA and ETB receptors which were functionally coupled to phosphoinositide breakdown and to adenylate cyclase with the increase of cAMP by ET-1 stimulation. Our findings suggest that ET-1 may have a potential autocrine and/or paracrine function in human endometrial stromal cells.

Cells, Cultured↗
PubMed1995

The role of endothelin-1 in regulating human granulosa cell proliferation and steroidogenesis in vitro.

The effects of endothelin-1 (ET-1) on luteinized human granulosa cells (L-HGCs) have not been examined. It is well known that there are differences of actions of several autocrine/paracrine regulators between L-HGCs and GCs of other species, and therefore the present study was designed to examine the effects of ET-1 1) on intracellular Ca2+ concentrations ([Ca2+]i) using the Ca(2+)-responsive fluorescent indicator Fura-2, 2) on cell proliferation by the nonradioactive method using bromodeoxyuridine, and 3) on basal and gonadotropin-stimulated steroidogenesis, and to examine the expression of ET receptor messenger RNA (mRNA) using freshly isolated and cultured L-HGCs obtained from patients undergoing in vitro fertilization. ET-1 increased [Ca2+]i in L-HGCs in a dose-dependent manner between 1 and 1000 nmol/L. High concentrations (100-1000 nmol/L) of ET-1 produced a more rapid and transient increase in [Ca2+]i than that observed with low concentrations (1-10 nmol/L) of ET-1. The increase in [Ca2+]i elicited by ET-3 (1000 nmol/L) and IRL-1620 (1000 nmol/L), a selective ETB receptor agonist, was 16% and 3% (vs. ET-1, 100%), respectively. BQ-123 (1000 nmol/L), an ETA receptor antagonist, inhibited the increase in [Ca2+]i elicited by ET-1 (by 50% at 1000 nmol/L ET-1 and by > 90% at < 500 nmol/L ET-1). mRNAs for the two known receptor subtypes (ETA and ETB) were also present in L-HGCs; however, the expression of ETA receptor mRNA was much greater than that of ETB receptors. ET-1 stimulated cell proliferation in L-HGCs in a dose-dependent manner (1000 nmol/L, 210.5 +/- 13.1%; 100 nmol/L, 198 +/- 11%; 10 nmol/L, 146 +/- 18%; and 1 nmol/L, 103 +/- 9%; vs. control, 100%). These stimulatory effects were completely blocked by BQ-123 (1000 nmol/L). ET-3 and IRL-1620 had no effects on cell proliferation in L-HGCs. Significant stimulatory effects on cell proliferation by the calcium ionophore, ionomycin (10-1000 nmol/L), were observed. ET-1, ET-3, and IRL-1620 attenuated basal progesterone secretion in L-HGCs. These results suggest that ETA receptor predominantly exist in L-HGCs and that ET-1 may stimulate cell proliferation of L-HGCs by increasing [Ca2+]i via ETA receptors.

Calcium↗
PubMed1994

Serum-independent phosphorylation of c-Jun and alterations in AP-1 components by transformation with various oncogenes.

To understand the mechanisms regulating the transactivating activity of Jun/AP-1, we analyzed alterations in c-Jun induced by growth stimulation and cell transformation. Serum stimulation of quiescent NIH3T3 cells induced a marked increase in phosphorylation of c-Jun in its amino-terminal activation domain. On the other hand, this domain was highly phosphorylated, in a serum-independent manner, in cells transformed with various oncogenes, including active c-raf-1, v-src, active Ha-ras, and active erbB-2. There were no obvious differences in the phosphorylation states of c-Jun in exponentially growing normal and transformed cells. However, in the exponentially growing state, the TRECAT activity in transformed cells was markedly higher than that in normal cells. Gel retardation analysis indicated that the AP-1 components in transformed cells were significantly different from those in normal cells. These results suggest that some other alterations besides phosphorylation of c-Jun are involved in enhancement of AP-1 activity in exponentially growing transformed cells.

3T3 Cells↗
PubMed1994

Three-month oral repeated administration toxicity study of seed saponins of Thea sinensis L. (Ryokucha saponin) in rats.

Ryokucha saponin (RSP), which is one of the ingredients of green tea, was administered orally to rats for 3 months at dose levels of 50, 150 and 500 mg/kg/day to assess any toxic effects. As positive control, 1200 mg/kg/day of quillaia saponin (QSP), which contains saponins equivalent in amount to those in 500 mg RSP/kg/day, was administered. The no-effect level of RSP was 50 mg/kg/day and the lowest-observed-effect level of RSP was 150 mg/kg/day for both sexes, so that the true no-effect level of RSP was estimated to be between these dose levels. In addition, it was confirmed that the toxicity of RSP at 500 mg/kg/day was less than that of QSP at 1200 mg/kg/day and RSP was demonstrated to be safer than QSP, which is a permitted food additive.

Administration, Oral↗
PubMed1994

Autocrine/paracrine function of transforming growth factor-beta 1 in porcine granulosa cells.

The present study was undertaken to investigate the effects of transforming growth factor (TGF)-beta 1 on ovarian steroidogenesis in immature or moderately mature porcine granulosa cells in vitro. TGF-beta 1 (0.01-10 ng/ml) significantly attenuated progesterone release from the basal and follicle stimulating hormone-stimulated porcine granulosa cells, and significantly increased DNA synthesis. TGF-beta 1 also significantly increased the extracellular accumulation of cyclic AMP, but did not change the production of inositol phosphate or the intracellular calcium concentration in these cells. Thus, TGF-beta 1 appears to have a direct effect on porcine granulosa cell function, regulating the differential synthesis of progesterone mainly via the intracellular signal transduction of the cyclic AMP-protein kinase A pathway. This growth factor may play a physiologically significant role in controlling differentiation of immature and moderately mature porcine granulosa cells via an autocrine/paracrine mechanism.

Animals↗
PubMed1994

Endothelin-1 as a local ovarian regulator in porcine granulosa cells.

This study was conducted to investigate whether endothelin-1 (ET-1) has direct effects on the functions of porcine granulosa cells harvested from small or medium follicles. Positive ET-1-like immunoreactivity (ET-1-LI) was observed in the cultured porcine granulosa cells, and Northern blot analysis demonstrated the expression of mRNA for prepro-ET-1 in these cells. The binding study showed the presence of a nonselective, single class of binding sites which predominantly included the subtype ETB. Increased ET-1-LI was detected in human follicular fluid obtained from the patients with stimulated ovarian cycles. ET-1 stimulated basal and follicle-stimulating hormone (FSH)-stimulated secretion of progesterone during short-term incubation (2 h), while it inhibited FSH- and human-chorionic-gonadotropin-stimulated accumulation of progesterone during long-term incubation (48 h) of immature or moderately mature granulosa cells. ET-1 significantly increased DNA synthesis in the cells. These biological actions were induced by ET-1, probably via a cAMP-protein kinase A pathway and intracellular calcium mobilization-protein kinase C pathway. The results suggest that ET-1 exerts autocrine/paracrine effects on porcine granulosa cell functions.

Animals↗
PubMed1994

Existence of P2-purinoceptors on human and porcine granulosa cells.

This study examines the possible roles of extracellular purine nucleotides in regulating ovarian granulosa cell function. Using luteinized human (L-hGC) and porcine granulosa cells (PGC), we examined the effects of purine nucleotides on intracellular free Ca2+ concentrations ([Ca2+]i), and whether they have any effect on steroidogenesis and cell proliferation. L-hGC and PGC were responsive to ATP and ADP at concentrations ranging from 0.1-100 mumol/L in a dose-dependent manner. There was a difference between L-hGC and PGC in the rank orders of agonist potencies for stimulating [Ca2+]i; for L-hGC, UTP > ATP > ATP gamma s > ADP > AMPPNP >> AMP and adenosine; for PGC, 2-methylthio ADP (2-meS ADP) > ADP > ATP = ATP gamma s = UTP > AMPPNP >> AMP and adenosine. No apparent additivity between the responses elicited by UTP and the purine nucleotides (ATP and ADP) was shown in L-hGC. On the other hand, an apparent additive effect between the responses to ADP and UTP was shown on PGC. Furthermore, ATP was a competitive antagonist of the action of ADP on [Ca2+]i levels in PGC. ATP, ADP, and AMP as well as adenosine stimulated basal progesterone and estradiol secretion from L-hGC, however, UTP had no effect on steroidogenesis in L-hGC. On the other hand, purine nucleotides and UTP as well as adenosine inhibited progesterone and estradiol secretion on PGC. However, 2-meS ADP, the most potent agonist for P2T-purinoceptors, did not affect steroidogenesis in PGC. Purine nucleotides had no influence on cell proliferation of L-hGC and PGC. These results indicate the existence of P2U-purinoceptors on L-hGC and P2U- plus P2T-purinoceptors on PGC, and that the inhibitory effect of purine nucleotides on steroidogenesis in PGC is most likely due to A1-adenosine receptors and also P2U-purinoceptors.

Animals↗
PubMed1994

[A case of ventricular septal defect and patent ductus arteriosus associated with absent right pulmonary artery, scimitar syndrome and severe pulmonary hypertension].

A 8 month-old female was diagnosed with ventricular septal defect and patent ductus arteriosus associated with absent right pulmonary artery and scimitar syndrome. Cardiac catheterization revealed severe pulmonary hypertension as follows; 72 mmHg of mean pulmonary artery pressure, 0.56 Qp/Qs and 1.73 Pp/Ps. Temporary closure of the ductus reduced the mean pulmonary artery pressure from 72 to 40 mmHg. PDA was ligated and VSD was closed successfully. However, 4 months after initial operation she was readmitted due to infection of the hypoplastic right lung. Removal of the hypoplastic right lung was performed and postoperative course was uneventful.

Ductus Arteriosus, Patent↗
PubMed1993

Endothelin-1 is an autocrine/paracrine regulator of porcine granulosa cells.

We studied whether a novel vasoconstrictor, endothelin-1 (ET-1), is synthesized by and released from porcine granulosa cells, and whether ET-1 acts directly on granulosa cells in an autocrine/paracrine fashion. The dilution curve of the conditioned medium from cultured porcine granulosa cells was parallel to a standard curve of ET-1 in RIA. Reverse-phase HPLC of the conditioned media from the granulosa cells revealed a major peak of ET-1-like immunoreactivity (ET-1-LI) coeluting with standard ET-1. ET-1-LI was released from cultured porcine granulosa cells as a function of time. Northern blot analysis demonstrated the expression of mRNA for prepro-ET-1 in the granulosa cells. We demonstrated the presence of ET-1 in the follicular fluid of porcine ovaries, and that the concentration of ET-1 in large follicles was higher than that in small-medium follicles. ET-1 dose-dependently stimulated cell growth and DNA synthesis in porcine granulosa cells. ET-1 inhibited the FSH- and hCG stimulated accumulation of progesterone in porcine granulosa cells in long-term incubation. These findings suggest that ET-1 produced by porcine granulosa cells may function as an autocrine/paracrine growth factor and modulator of steroidogenesis in ovarian granulosa cells.

Animals↗