Biomedical subjects

Y Tsujimoto

Publications and source records attributed to Y Tsujimoto.

At least 37 records · Page 2Linked to original sources

PubMed2000

Gelsolin inhibits apoptosis by blocking mitochondrial membrane potential loss and cytochrome c release.

Apoptotic cell death, characterized by chromatin condensation, nuclear fragmentation, cell membrane blebbing, and apoptotic body formation, is also accompanied by typical mitochondrial changes. The latter includes enhanced membrane permeability, fall in mitochondrial membrane potential (Deltapsi(m)) and release of cytochrome c into the cytosol. Gelsolin, an actin regulatory protein, has been shown to inhibit apoptosis, but when cleaved by caspase-3, a fragment that is implicated as an effector of apoptosis is generated. The mechanism by which the full-length form of gelsolin inhibits apoptosis is unclear. Here we show that the overexpression of gelsolin inhibits the loss of Deltapsi(m) and cytochrome c release from mitochondria resulting in the lack of activation of caspase-3, -8, and -9 in Jurkat cells treated with staurosporine, thapsigargin, and protoporphyrin IX. These effects were corroborated in vitro using recombinant gelsolin protein on isolated rat mitochondria stimulated with Ca(2+), atractyloside, or Bax. This protective function of gelsolin, which was not due to simple Ca(2+) sequestration, was inhibited by polyphosphoinositide binding. In addition we confirmed that gelsolin, besides its localization in the cytosol, is also present in the mitochondrial fraction of cells. Gelsolin thus acts on an early step in the apoptotic signaling at the level of mitochondria.

Animals↗

PubMed2000

Electrophysiological study of a novel large pore formed by Bax and the voltage-dependent anion channel that is permeable to cytochrome c.

The Bcl-2 family of proteins, consisting of anti-apoptotic and pro-apoptotic members, regulates cell death by controlling mitochondrial membrane permeability that is crucial for apoptotic signal transduction. We have recently shown that some of these proteins, such as Bcl-x(L), Bax, and Bak, directly modulate the mitochondrial voltage-dependent anion channel (VDAC) and thus regulate apoptogenic cytochrome c release and potential loss. To elucidate the molecular mechanisms of VDAC regulation by Bcl-2 family proteins, an electrophysiological study was carried out. It was found that VDAC and pro-apoptotic Bax created a large pore, with conductance levels 4- and 10-fold greater than those of the VDAC and Bax channels, respectively. Although the VDAC and Bax channels both show ion selectivity and voltage-dependent modulation of their activity, the VDAC-Bax channel had neither of their properties. Anti-apoptotic Bcl-x(L) and its BH4 oligopeptide completely closed the VDAC, in contrast to the Bax. Cytochrome c passed through a single VDAC-Bax channel but not through the VDAC or Bax channel in a planar lipid bilayer. These data provide direct evidence that VDAC forms a novel large pore together with Bax.

Animals↗

PubMed2000

BH4 domain of antiapoptotic Bcl-2 family members closes voltage-dependent anion channel and inhibits apoptotic mitochondrial changes and cell death.

A change of mitochondrial membrane permeability is essential for apoptosis, leading to translocation of apoptogenic cytochrome c and apoptosis-inducing factor into the cytoplasm. We recently showed that the Bcl-2 family of proteins regulate cytochrome c release and the mitochondrial membrane potential (Deltapsi) by directly modulating the activity of the voltage-dependent anion channel (VDAC) through binding. Here we investigated the biochemical role of the conserved N-terminal homology domain (BH4) of Bcl-x(L), which has been shown to be essential for inhibition of apoptosis, with respect to the regulation of mitochondrial membrane permeability and found that BH4 was required for Bcl-x(L) to prevent cytochrome c release and Deltapsi loss. A study using VDAC liposomes revealed that Bcl-x(L), but not Bcl-x(L) lacking the BH4 domain, inhibited VDAC activity. Furthermore, BH4 oligopeptides of Bcl-2 and Bcl-x(L), but not mutant peptides, were able to inhibit both VDAC activity on liposomes even in the presence of Bax and apoptotic Deltapsi loss in isolated mitochondria. It was also shown that the BH4 domain, fused to the protein transduction domain of HIV TAT protein (TAT-BH4), efficiently prevented apoptotic cell death. These results indicate that the BH4 of Bcl-2/Bcl-x(L) is essential and sufficient for inhibiting VDAC activity, which in turn prevents apoptotic mitochondrial changes, and for preventing apoptotic cell death. Finally, the data suggest that the TAT-BH4 peptide is potentially useful as a therapeutic agent for diseases caused by accelerated apoptosis.

Apoptosis↗

PubMed2000

Bcl-2 family: life-or-death switch.

The Bcl-2 family of proteins that consists of anti-apoptotic and pro-apoptotic members determines life-or-death of a cell by controlling the release of mitochondrial apoptogenic factors, cytochrome c and apoptosis-inducing factor (AIF), that activate downstream executional phases, including the activation of death proteases called caspases. Cytochrome c release is, thus, central to apoptotic signal transduction in mammals, making study of the mechanism for cytochrome c release a major issue. Several models for cytochrome c release have been proposed, including rupture of mitochondrial outer membrane and involvement of a specific channel. Here, we provide an overview of recent findings on the role of Bcl-2 family members in the life-or-death decision of a cell.

Animals↗

PubMed2000

Proapoptotic BH3-only Bcl-2 family members induce cytochrome c release, but not mitochondrial membrane potential loss, and do not directly modulate voltage-dependent anion channel activity.

Through direct interaction with the voltage-dependent anion channel (VDAC), proapoptotic Bcl-2 family members such as Bax and Bak induce apoptogenic mitochondrial cytochrome c release and membrane potential (Deltapsi) loss in isolated mitochondria. Using isolated mitochondria, we showed that Bid and Bik, BH3-only proteins from the Bcl-2 family, induced cytochrome c release but not Deltapsi loss. Unlike Bax/Bak, the cytochrome c release induced by Bid/Bik was Ca(2+)-independent, cyclosporin A-insensitive, and respiration-independent. Furthermore, in contrast to Bax/Bak, Bid/Bik neither interacted with VDAC nor directly affected the VDAC activity in liposomes. Consistently, Bid/Bik induced apoptosis without Deltapsi loss, whereas Bax induced apoptosis with Deltapsi loss. These findings indicated the involvement of a different mechanism in BH3-only, protein-induced apoptogenic cytochrome c release.

Animals↗

PubMed2000

Role of anti-apoptotic Bcl-2 protein in spinal muscular atrophy.

Apoptosis is an essential physiological cell death for selective elimination of cells, involved in a variety of biological events including morphogenesis, cell turn over and removal of harmful cells. Disruption of the regulation of apoptosis may result in various diseases, including cancer and autoimmune diseases both associated with inhibition of apoptosis and various degenerative disorders associated with enhancement of apoptosis, and therefore, apoptosis is an important theme in the medical field. Apoptosis is driven by a family of cysteine proteases, called caspases and regulated by a Bcl-2 family of proteins, which is the best characterized apoptosis regulators. The Bcl-2 family consists of anti-apoptotic and pro-apoptotic members, and some members are implicated in cancer and nuronal diseases. Here, I overview the mechanism of how Bcl-2 family proteins regulate cell death, and how they are implicated in human diseases, particularly focusing on role of Bcl-2 in spinal muscular atropy.

Apoptosis↗

PubMed2000

Identification of the dimethylbenzyl mercapturic acid in urine of rats administered with 1,2,4-trimethylbenzene.

A study was undertaken of the mercapturic acid metabolism of 1,2,4-trimethylbenzene in the rat. Of three regioisomeric dimethylbenzyl mercapturic acids, i.e. 2,4-, 2,5- and 3,4-dimethylbenzyl isomers, the third isomer was not found in the urinary mercapturic acid isolated by preparative HPLC, from the comparison of NMR spectrum of the isolate with those of authentic specimens. The urinary mercapturate was then assigned to 2,4- and/or 2,5-dimethylbenzyl isomers. The excretion rate of the mercapturic acid was 14-20% of dose as 2,4-dimethylbenzyl isomer.

Acetylcysteine↗

PubMed2000

Regions essential for the interaction between Bcl-2 and SMN, the spinal muscular atrophy disease gene product.

The SMN gene is implicated in spinal muscular atrophy (SMA), and its product has been shown to interact with Bcl-2 protein to enhance its anti-apoptotic activity. In this study, we determined the regions that were essential for the interaction of Bcl-2 and SMN by co-immunoprecipitation of deletion mutants. Bcl-2 lacking its amino-terminal 20 amino acid residues or its carboxyl-terminal membrane-anchoring domain showed no or greatly reduced binding with SMN, respectively. However, Bcl-2 lacking other regions could still bind to SMN. Because Bcl-2 lacking the membrane-anchoring domain could bind to SMN in a yeast two-hybrid system, the amino-terminal region of Bcl-2 seems to be the most important domain for binding with SMN. A fragment of SMN encoded by exon 6 could bind to Bcl-2, but SMN lacking this region could not. From these results, we concluded that Bcl-2 and SMN proteins bound with each other at the amino-terminal region near the BH4 domain of Bcl-2 and the region encoded by exon 6 of SMN, both regions known to be important for their function.

Amino Acid Sequence↗

PubMed2000

VDAC regulation by the Bcl-2 family of proteins.

The Bcl-2 family of proteins consists of anti-apoptotic and pro-apoptotic members, which determine the life or death of cells by altering mitochondrial membrane permeability. Pro-apoptotic Bcl-2 family members increase mitochondrial membrane permeability, resulting in the release of mitochondrial apoptogenic factors such as cytochrome c that activates death proteases called caspases, whereas anti-apoptotic family members prevent this increase of mitochondrial membrane permeability. The release of cytochrome c is central to apoptotic signal transduction in mammals, and has been studied extensively, leading to the development of several models for cytochrome c release including rupture of the mitochondrial outer membrane and involvement of specific channels. This article describes the important role of a mitochondrial outer membrane channel, the voltage-dependent anion channel (VDAC), in apoptogenic cytochrome c release and its regulation by Bcl-2 family members, and also discusses the molecular architecture of the life - death switch in mammalian cells. Cell Death and Differentiation (2000) 7, 1174 - 1181

Animals↗

PubMed2000

Determination of mercapturic acids in urine by solid-phase extraction followed by liquid chromatography-electrospray ionization mass spectrometry.

A novel method for the determination of five kinds of mercapturic acids, found in urine as metabolites of alkylbenzenes, based on liquid chromatography-electrospray ionization mass spectrometry is described. A solid-phase extraction procedure was used for the extraction of the mercapturic acids from urine and the separation was performed on a reversed-phase C30 column. The detection limits were in the range 2.4-3.2 ng ml-1.

Acetylcysteine↗

PubMed2000

Calmodulin and cyclic ADP-ribose interaction in Ca2+ signaling related to cardiac sarcoplasmic reticulum: superoxide anion radical-triggered Ca2+ release.

Reactive oxygen species (ROS) are often shown to damage cellular functions. The targets of oxidative damage depend on the nature of ROS produced and the site of generation. In contrast, ROS can also regulate signal transduction. In this case, ROS may either induce or enhance events, which lead to forward directions of cellular signaling. The consequences of regulation of signal transduction can be observed in physiological processes such as muscle contraction. Here, we discuss the concentration-dependent effects of superoxide anion radical (*O2-) on Ca2+ release from the cardiac sarcoplasmic reticulum (SR). Recent studies suggest that the ADP-ribosyl cyclase pathway, through its production of cyclic adenosine 5'-diphosphoribose (cADPR), may control Ca2+ mobilization in cardiac muscle cells. *O2- has dual effects that are concentration dependent. At low concentrations (nearly nanomolar levels), *O2- induces Ca2+ release by stimulating synthesis of cADPR, which requires calmodulin for sensitization of ryanodine-sensitive Ca2+-release channels (RyRC). At these low concentrations, *O2- is responsible for regulation of cellular signal transduction. At higher concentrations (micromolar levels), *O2- produces a loss in the function of calmodulin that is to inhibit RyRC. This results in an increase in Ca2+ release, which is linked to cell injury. The difference in the functions of low and high concentrations of *O2- may result in two distinct physiological roles in cardiac muscle Ca2+ signaling.

ADP-ribosyl Cyclase↗

PubMed2000

Association of insulin receptor substrate proteins with Bcl-2 and their effects on its phosphorylation and antiapoptotic function.

Insulin receptor substrate (IRS) proteins are docking proteins that couple growth factor receptors to various effector molecules, including phosphoinositide-3 kinase, Grb-2, Syp, and Nck. Here we show that IRS-1 associates with the loop domain of Bcl-2 and synergistically up-regulates antiapoptotic function of Bcl-2. IRS-2 but not IRS-3 binds to Bcl-2, and IRS-1 associates with Bcl-XL but not with Bax or Bik. Overexpression of IRS-1 suppresses phosphorylation of Bcl-2 induced by stimulation with insulin, and the hypophosphorylation may lead to its enhanced antiapoptotic activity. The binding site for Bcl-2 is located on the carboxyl half-domain of IRS-1. IRS-3, which lacks the corresponding region, dominant-negatively abrogates the survival effects of IRS-1 and Bcl-2. For the antiapoptotic activity of IRS-1, binding to Bcl-2 is more critical than activating phosphoinositide-3 kinase. Our results indicate that IRS proteins transmit signals from the insulin receptor to Bcl-2, thus regulating cell survival probably through regulating phosphorylation of Bcl-2.

Animals↗

PubMed2000

Cooperative regulation of DOG2, encoding 2-deoxyglucose-6-phosphate phosphatase, by Snf1 kinase and the high-osmolarity glycerol-mitogen-activated protein kinase cascade in stress responses of Saccharomyces cerevisiae.

We screened the genome of Saccharomyces cerevisiae for the genes responsive to oxidative stress by using the lacZ transposon-insertion library. As a result, we found that expression of the DOG2 gene coding for 2-deoxyglucose-6-phosphate phosphatase was induced by oxidative stress. The expression of DOG2 was also induced by osmotic stress. We found a putative cis element (STRE, a stress response element) in the DOG2 promoter adjacent to a consensus sequence to which the Mig1p repressor is known to bind. The basal levels of DOG2 gene expression were increased in a mig1Delta mutant, while the derepression of DOG2 was not observed in a snf1Delta mutant under glucose-deprived conditions. Induction of the DOG2 gene expression by osmotic stress was observed in any of the three disruptants pbs2Delta, hog1Delta, and snf1Delta. However, the osmotic induction was completely abolished in both the snf1Delta pbs2Delta mutant and the snf1Delta hog1Delta mutant. Additionally, these single mutants as well as double mutants failed to induce DOG2 expression by oxidative stress. These results suggest that Snf1p kinase and the high-osmolarity glycerol-mitogen-activated protein kinase cascade are likely to be involved in the signaling pathway of oxidative stress and osmotic stress in regulation of DOG2.

Base Sequence↗

PubMed2000

Rare somatic inactivation of the multiple endocrine neoplasia type 1 gene in secondary hyperparathyroidism of uremia.

The molecular pathway of autonomous growth of the parathyroid glands in uremic patients is poorly understood. Loss of heterozygosity at the recently identified multiple endocrine neoplasia type 1 (MEN1) gene locus on chromosome 11q13 has been found in a subset of parathyroid glands from patients with refractory hyperparathyroidism. To clarify the role of the MEN1 gene in parathyroid tumorigenesis, we analyzed 81 parathyroid glands from 22 Japanese uremic patients for allelic loss on chromosomal arm 11q13 DNA using 3 flanking markers (PYGM, D11S4946, and D11S449) and for mutations of the MEN1-coding exons by PCR-based single strand conformation polymorphism analysis and sequencing. Allelic loss on 11q13 was observed in 6 glands (7%), and 1 of 6 demonstrated a previously unrecognized somatic frameshift deletion (331delG) of the MEN1 gene. This mutation would probably result in a nonfunctional menin protein, consistent with a tumor suppressor mechanism. Clinical and pathological characteristics of hyperparathyroidism were unrelated to the presence or absence of loss of heterozygosity on 11q13 and MEN1 gene mutations. These observations indicate that somatic inactivation of the MEN1 gene contributes to the pathogenesis of uremia-associated parathyroid tumors, but its role in this disease appears to be very limited.

Chromosome Mapping↗

PubMed2000

Characterization of the activity of L-ascorbic acid 2-[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-1-be nzopyran-6-yl-hydrogen phosphate] potassium salt in hydroxyl radical elimination.

The effect of L-ascorbic acid 2-[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H -1-benzopyran-6-yl-hydrogen phosphate] potassium salt (EPC-K1) on hydroxyl radical (*OH) elimination was studied using electron spin resonance (ESR) and spectrophotometric experiments. The addition of EPC-K, and *OH scavengers eliminated the *OH generated from Cu2+/H2O2, Fe2+/H2O2 and H2O2/UV-irradiation reaction systems. However, in competitive reactions using different concentrations of a spin-trap agent, the addition of the *OH scavenger altered the IC50 values, whereas the addition of EPC-K1 and a metal chelater did not change the value in the Cu2+/H2O2 and Fe2+/H2O2 reaction systems. The addition of EPC-K1 and metal chelater changed the ESR signal for free Cu2+. The spectrophotometric experiments confirmed that the addition of EPC-K1 and metal chelater altered the absorption spectra due to CuCl2 and FeSO4, whereas the *OH scavenger did not alter the spectra. Therefore, it was demonstrated that EPC-K, has the ability both to scavenge *OH directly and to inhibit the generation of *OH by the chelation of Cu2+ and Fe2+.

Antioxidants↗

PubMed2000

[Renocolic fistula: a case report].

A 78-year-old woman had disregarded pneumaturia since April 1998. In March 1999, computed tomography and barium enema were done to examine anemia and positive fecal occult blood, revealing a left renocolic fistula and bilateral renal stones. Intravenous pyelography revealed a left staghorn calculus, non-functioning kidney, and right partial staghorn calculus. Urinalysis showed pyuria and the culture grew Proteus vulgaris and Klebsiella oxytoca. Smear and culture of the urine were negative for acid-fast bacilli. In consideration of the patient's age and conservation of right renal function, right pyelolithotomy was performed first. Three weeks later, left nephrectomy and partial colectomy were done. The postoperative course was uneventful. A renocolic fistula is relatively rare and to our knowledge there have been 37 cases reported in Japan, including our case. Surgery is the main therapy and was performed in 31 patients. Among them, surgery was not curative in 1 and 5 died of postoperative complications. Thus, surgery is not safe in all cases. However, despite her age and bilateral renal dysfunction, our patient was successfully operated on.

Aged↗

PubMed2000

Cloning and secretive expression of the gene encoding the proteinaceous alpha-amylase inhibitor paim from Streptomyces corchorusii.

A gene encoding the proteinaceous alpha-amylase inhibitor Paim was cloned and sequenced. Southern analysis and the amino acid sequence deduced from the cloned gene indicated that Paim isoforms were encoded in the same gene. When the gene was expressed in Escherichia coli and Streptomyces lividans, recombinant Paim inhibitors were produced in the periplasmic space and in the culture supernatant, respectively. The purified inhibitors had different N-terminal sequences from those of the authentic inhibitors.

Journal Article↗

PubMed1999

Bis, a Bcl-2-binding protein that synergizes with Bcl-2 in preventing cell death.

Bcl-2 is the best characterized inhibitor of apoptosis, although the molecular basis of this action is not fully understood. Using a protein interaction cloning procedure, we identified a human gene designated as bis (mapped to chromosome 10q25) that encoded a novel Bcl-2-interacting protein. Bis protein showed no significant homology with Bcl-2 family proteins and had no prominent functional motif. Co-immunoprecipitation analysis confirmed that Bis interacted with Bcl-2 in vivo. DNA transfection experiments indicated that Bis itself exerted only weak anti-apoptotic activity, but was synergistic with Bcl-2 in preventing Bax-induced and Fas-mediated apoptosis. These results suggest that Bis is a novel modulator of cellular anti-apoptotic activity that functions through its interaction with Bcl-2.

Adaptor Proteins, Signal Transducing↗