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Biomedical subjects

Ken Sato

Publications and source records attributed to Ken Sato.

At least 55 records · Page 3Linked to original sources
PubMed2005

Reconstitution of cargo-dependent COPII coat assembly on proteoliposomes.

The coat protein complex II (COPII) coat is responsible for direct capture of membrane cargo proteins and for the physical deformation of the endoplasmic reticulum (ER) membrane that drives the transport vesicle formation. The use of an in vitro reconstitution system comprising purified components is desirable for studies aimed at elucidating the role(s) of individual proteins in a specific biochemical reaction. To investigate the assembly-disassembly of COPII coats in a completely reconstituted reaction, we have developed a synthetic budding reaction involving purified coat proteins and cargo-reconstituted proteoliposomes. We describe here a fluorescence resonance energy transfer (FRET)-based method for monitoring the kinetics of COPII coat complex assembly and disassembly in cargo-reconstituted proteoliposomes. This assay allows comparison of the time course of the coat disassembly from the cargo as monitored by FRET signal with the time course of accompanying Sar1p GTP hydrolysis by tryptophan fluorescence.

COP-Coated Vesicles↗
PubMed2005

Elevated plasma adiponectin concentrations in patients with liver cirrhosis correlate with plasma insulin levels.

BACKGROUND: Adiponectin is a hormone secreted by adipocytes and has anti-diabetic and anti-atherogenic properties. Hypoadiponectinemia is associated with insulin-resistant diabetes and liver dysfunction. The aim of this study was to determine plasma adiponectin and insulin levels in patients with liver cirrhosis. METHODS: Adiponectin and insulin levels were determined in 38 patients with cirrhosis and 30 healthy controls, and were correlated with various clinical and biochemical parameters. Patients included 21 with Child A, eight Child B, and nine with Child C liver cirrhosis. RESULTS: Log adiponectin and insulin levels were significantly elevated in patients with cirrhosis compared with the control. In liver cirrhosis, the level of adiponectin increased proportionately with the Child's classification score. In control subjects, plasma adiponectin correlated inversely with insulin levels. In contrast, plasma adiponectin correlated positively with insulin levels in patients with liver cirrhosis. Plasma adiponectin levels did not correlate with age, sex, body mass index, total bilirubin, aspartate aminotransferase, and fasting blood sugar levels in both groups, while alanine aminotransferase correlated negatively with adiponectin in control subjects as reported previously. CONCLUSION: Our results of high plasma adiponectin in patients with liver cirrhosis could reflect an imbalance between its production by adipocytes and metabolism in the liver.

Adiponectin↗
PubMed2005

Type 2 diabetes mellitus complicated with smoldering myeloma and non-alcoholic steatohepatitis.

We report a 59-year-old woman with type 2 diabetes mellitus (DM) complicated with smoldering myeloma and non-alcoholic steatohepatitis. A diagnosis of smoldering myeloma was made on the basis of elevation of IgA, M-protein (type:IgA-lambda) and histological findings of bone marrow without bone lesion. As to liver dysfunction, anti-HBV, anti-HCV and a series of auto-immune antibody were negative. She had no alcohol drinking habit and histological findings showed pericellular fibrosis without fatty degeneration, suggesting liver cirrhosis due to non-alcoholic steatohepatitis (NASH). To date, there are no reports of cases with DM, NASH and myeloma. Particular attention may be necessary for these complications.

Diabetes Mellitus, Type 2↗
PubMed2005

[Idiopathic testicular infarction: a case report].

We herein report a case of idiopathic testicular infarction. The patient was a 20-year-old man with the chief complaint of right testicular swelling and pain. Ultrasonography and color Doppler sonography demonstrated a hypoechoic lesion without any blood supply. Torsion of the right spermatic cord was most suspected, but we could not exclude the possibility of testicular cancer judging from the findings obtained by computed tomography and magnetic resonance imaging. Surgical exploration did not reveal any torsion of the spermatic cord. Subsequently, right high orchiectomy was performed because of the risk of a testicular cancer. Histopathological examination revealed a hemorrhage and congestion of the testis and epididymis.

Adult↗
PubMed2005

Cisplatin represses transcriptional activity from the minimal promoter of the O6-methylguanine methyltransferase gene and increases sensitivity of human gallbladder cancer cells to 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-2-chloroethyl)-3-nitrosourea.

O(6)-methylguanine methyltransferase (MGMT) repairs O(6)-alkylguanine in cellular DNA introduced by the clinically used alkylating drug 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU). Thus, cancer cells with MGMT expression are resistant to ACNU treatment. Cisplatin has been reported to suppress MGMT expression; however, the molecular mechanism by which cisplatin reduces MGMT expression remains to be elucidated. Using gallbladder cancer cells (KMG-C) expressing MGMT, we analyzed whether a low dose of cisplatin suppresses MGMT expression, followed by an enhanced drug effect of ACNU in vitro and in vivo. We also investigated the promoter region critical for the transcriptional repression of MGMT gene by cisplatin using 5 deletion mutants in reporter promoter assays. In RT-PCR analysis, the expression of MGMT mRNA in KMG-C cells was dose- and time-dependently repressed. Drug sensitivity to ACNU was increased 2-fold by pretreatment with cisplatin, compared with only ACNU treatment, in MTT assays as well as tumor-bearing nude mice. Although the 5'-flanking region is deleted as far as -69 bp upstream of the transcription start site, cisplatin dose dependently inhibited luciferase activity. However, cisplatin did not cause such repression when 59 bp region from -69 to -10 bp was deleted. We confirmed that cisplatin enhanced sensitivity to ACNU in KMG-C cells expressing MGMT both in vitro and in vivo. Furthermore, a low dose of cisplatin repressed the transcription of the MGMT promoter. The 59 bp region in the MGMT promoter was crucial for repression by cisplatin. These results might form the basis of a chemotherapeutic strategy involving alkylating agents via prior cisplatin-induced biochemical modulation.

Antineoplastic Agents↗
PubMed2005

Deficient MGMT and proficient hMLH1 expression renders gallbladder carcinoma cells sensitive to alkylating agents through G2-M cell cycle arrest.

The aim of this study was to assess whether combined evaluation of O6-methylguanine methyltransferase (MGMT) and hMLH1 status determines sensitivity to monofuntional alkylating agents such as N-methyl-N-nitrosourea (MNU) and dacarbazine (DTIC) against gallbladder carcinoma cells. The molecular mechanism behind MGMT and hMLH1 status affecting the cell cycle was also addressed. Using 5 gallbladder cancer carcinoma lines and 1 colon carcinoma cell line (SW48), MGMT and hMLH1 expression was analyzed using RT-PCR and Western blotting. MGMT and hMLH1 status in the 6 cell lines was compared with drug sensitivity to MNU. As a result, cell lines that were MGMT-/hMLH1+ had the highest sensitivity to MNU, compared with MGMT+/hMLH1+ and MGMT-/hMLH1- cells. In flow cytometric analysis, G2-M cell cycle arrest was specifically observed in GB-d1 cells with MGMT-/hMLH1+ and expression of cyclin A and Cdc2 in GB-d1 cells was significantly reduced by MNU treatment, but not observed in KMG-C cells with MGMT+/hMLH1+. Finally, we assessed the in vitro and in vivo effect of the clinically used alkylating agent DTIC in these cells. The highest sensitivity to DTIC was also observed in MGMT-/hMLH1+. In conclusion, MNU suppressed cell proliferation of MGMT-/hMLH1+ gallbladder carcinoma cells by arresting the cell cycle at the G2-M phase, accompanied by down-regulation of cyclin A and Cdc2. These results indicated that expression of MGMT and hMLH1 could be used to select candidates for alkylating agent chemotherapy against gallbladder carcinoma.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Guanidine alkaloid analogs as inhibitors of HIV-1 Nef interactions with p53, actin, and p56lck.

With current anti-HIV treatments targeting only 4 of the 15 HIV proteins, many potential viral vulnerabilities remain unexploited. We report small-molecule inhibitors of the HIV-1 protein Nef. In addition to expanding the anti-HIV arsenal, small-molecule inhibitors against untargeted HIV proteins could be used to dissect key events in the HIV lifecycle. Numerous incompletely characterized interactions between Nef and cellular ligands, for example, present a challenge to understanding molecular events during HIV progression to AIDS. Assays with phage-displayed Nef from HIV(NL4-3) were used to identify a series of guanidine alkaloid-based inhibitors of Nef interactions with p53, actin, and p56(lck). The guanidines, synthetic analogs of batzellidine and crambescidin natural products, inhibit the Nef-ligand interactions with IC(50) values in the low micromolar range. In addition, sensitive in vivo assays for Nef inhibition are reported. Although compounds that are effective in vitro proved to be too cytotoxic for cellular assays, the reported Nef inhibitors provide proof-of-concept for disrupting a new HIV target and offer useful leads for drug development.

Actins↗
PubMed2004

A phage display selection of engrailed homeodomain mutants and the importance of residue Q50.

Mutants of engrailed homeodomain (HD) that retain DNA-binding activity were isolated using a phage display selection. This selection was used to enrich for active DNA-binding clones from a complex library consisting of over a billion members. A more focused library of mutant homeodomains consisting of all possible amino acid combinations at two DNA-contacting residues (I47 and Q50) was constructed and screened for members capable of binding tightly and specifically to the engrailed consensus sequence, TAATTA. The isolated mutants largely recapitulated the distribution of amino acids found at these positions in natural homeodomains thus validating the in vitro selection conditions. In particular, the unequivocal advantage enjoyed by glutamine at residue 50 is surprising in light of reports that minimize the importance of this residue. Here, the subtle contributions of residue Q50 are demonstrated to play a functionally important role in specific recognition of DNA. These results highlight the complex subtlety of protein-DNA interactions, underscoring the value of the first reported in vitro selection of a homeodomain.

Binding Sites↗
PubMed2004

Dissecting the Engrailed homeodomain-DNA interaction by phage-displayed shotgun scanning.

Phage-displayed alanine shotgun scanning was used to dissect contributions by engrailed homedomain (En-HD) residues 17 through 46, which indirectly influence recognition of DNA. The relative contributions of such indirect contacts, quantified by shotgun scanning, highlight previously unexplored En-HD residues. Two motifs dominate En-HD function in this region. First, two surface-exposed aromatic residues (F20 and Y25) bracket the hydrophobic core. Second, two sets of turn-forming residues are highlighted, including carboxamide-requiring residues E22/N23 and a leucine/isoleucine splint. The En-HD hydrophobic core exhibits a surprising degree of malleability, as demonstrated by homolog shotgun scanning. Most selectants from in vitro shotgun scanning mirror the consensus human homeodomain sequence. Thus, natural evolution and in vitro selection use similar selection criteria: affinity, specificity, and stability. However, homolog shotgun scanning identified mutations capable of improving the affinity and specificity of En-HD.

Bacteriophages↗
PubMed2004

COPII coat assembly and selective export from the endoplasmic reticulum.

The coat protein complex II (COPII) generates transport vesicles that mediate protein transport from the endoplasmic reticulum (ER). Recent structural and biochemical studies have suggested that the COPII coat is responsible for direct capture of membrane cargo proteins and for the physical deformation of the ER membrane that drives the transport vesicle formation. The COPII-coated vesicle formation at the ER membrane is triggered by the activation of the Ras-like small GTPase Sar1 by GDP/GTP exchange, and activated Sar1 in turn promotes COPII coat assembly. Subsequent GTP hydrolysis by Sar1 leads to disassembly of the coat proteins, which are then recycled for additional rounds of vesicle formation. Thus, the Sar1 GTPase cycle is thought to regulate COPII coat assembly and disassembly. Emerging evidence suggests that the cargo proteins modulate the Sar1 GTP hydrolysis to coordinate coat assembly with cargo selection. Here, I discuss the possible roles of the GTP hydrolysis by Sar1 in COPII coat assembly and selective uptake of cargo proteins into transport vesicles.

Animals↗
PubMed2004

Induction of redox imbalance and apoptosis in multiple myeloma cells by the novel triterpenoid 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid.

The synthetic oleanane triterpenoid 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO) and its chemical derivatives induce differentiation and apoptosis of human leukemia cells. The precise mechanisms responsible for the effects of CDDO, however, remain unclear. In the present study, we examined the effects of CDDO and its C-28 imidazolide ester (CDDO-Im) on apoptosis of multiple myeloma (MM) cells. The results show that both CDDO and CDDO-Im are potent inducers of MM cell apoptosis and that CDDO-Im is more active than CDDO. CDDO-Im treatment was associated with (a) depletion of glutathione, (b) increases in reactive oxygen species, (c) a reduction of the Fas-associated death domain (FADD)-like interleukin-1-converting enzyme (FLICE) inhibitory protein, (d) activation of caspase-8, and (e) a decrease of the mitochondrial transmembrane potential. The reducing agents, N-acetyl-L-cysteine, DTT, and catalase inhibited each of these CDDO-Im-induced proapoptotic signals. Inhibition of caspase-8 with z-IETD-fmk also abrogated CDDO-Im-induced decreases of the mitochondrial transmembrane potential and inhibited apoptosis. These results demonstrate that CDDO-Im disrupts intracellular redox balance and thereby activates the extrinsic caspase-8-dependent apoptotic pathway. We further show that CDDO-Im induces apoptosis of primary MM cells at submicromolar concentrations and that MM cells are more sensitive to this agent than normal bone marrow mononuclear cells. These results suggest that CDDO compounds have potential as new agents for the treatment of MM.

Antioxidants↗
PubMed2004

[Emergence and decrement of Ph-negative clone with trisomy 8 in CML during imatinib therapy].

An increase in the presence of the Ph-negative, trisomy 8 clone has been reported in chronic myelogenous leukemia (CML) under imatinib therapy, but any impact of the clone on patient prognosis remains uncertain. We report here on a 42-year-old male with CML who received imatinib after failure of interferon-alpha therapy. A chromosomal analysis revealed 18/20 trisomy 8 in bone marrow at 10 months of imatinib administration. Continuing imatinib at 300 mg daily resulted in a decrease in the number of trisomy 8 clones as well as the disappearance of Ph clone. Furthermore, the patient's pancytopenia was gradually improved. Imatinib therapy could be continued even with the emergence of trisomy 8.

Adult↗
PubMed2003

Endoplasmic reticulum quality control of unassembled iron transporter depends on Rer1p-mediated retrieval from the golgi.

Endoplasmic reticulum (ER) quality control is a conserved process by which misfolded or unassembled proteins are selectively retained in the endoplasmic reticulum (ER). Failure in oligomerization of multisubunit membrane proteins is one of the events that triggers ER quality control. The transmembrane domains (TMDs) of unassembled subunits are determinants of ER retention in many cases, although the mechanism of the TMD-mediated sorting of unassembled subunits remains elusive. We studied a yeast iron transporter complex on the cell surface as a new model system for ER quality control. When Fet3p, a transmembrane subunit, is not assembled with the other membrane subunit, Ftr1p, unassembled Fet3p is exclusively localized to the ER at steady state. The TMD of Fet3p contains a determinant for this process. However, pulse-chase analysis and in vitro budding assays indicate that unassembled Fet3p rapidly escapes from the ER. Furthermore, Rer1p, a retrieval receptor for ER-resident membrane proteins in the Golgi, is responsible for the TMD-dependent ER retrieval of unassembled Fet3p. These findings provide clear evidence that the ER quality control of unassembled membrane proteins can be achieved by retrieval from the Golgi and that Rer1p serves as a specific sorting receptor in this process.

Ceruloplasmin↗
PubMed2003

Reconstitution of coat protein complex II (COPII) vesicle formation from cargo-reconstituted proteoliposomes reveals the potential role of GTP hydrolysis by Sar1p in protein sorting.

Secretory proteins are transported from the endoplasmic reticulum (ER) in vesicles coated with coat protein complex II (COPII). To investigate the molecular mechanism of protein sorting into COPII vesicles, we have developed an in vitro budding reaction comprising purified coat proteins and cargo reconstituted proteolipsomes. Emp47p, a type-I membrane protein, is specifically required for the transport of an integral membrane protein, Emp46p, from the ER. Recombinant Emp46/47p proteins and the ER resident protein Ufe1p were reconstituted into liposomes whose composition resembles yeast ER membranes. When the proteoliposomes were mixed with COPII proteins and GMP-PNP, Emp46/47p, but not Ufe1p, were concentrated into COPII vesicles. We also show here that reconstituted Emp47p accelerates the GTP hydrolysis by Sar1p as stimulated by its GTPase-activating protein, Sec23/24p, both of which are components of the COPII coat. Furthermore, this GTP hydrolysis decreases the error of cargo sorting. We suggest that GTP hydrolysis by Sar1p promotes exclusion of improper proteins from COPII vesicles.

COP-Coated Vesicles↗
PubMed2003

Rer1p, a retrieval receptor for ER membrane proteins, recognizes transmembrane domains in multiple modes.

The yeast Golgi membrane protein Rer1p is required for the retrieval of various endoplasmic reticulum (ER) membrane proteins such as Sec12p and Sec71p to the ER. We demonstrate here that the transmembrane domain (TMD) of Sec71p, a type-III membrane protein, contains an ER localization signal, which is required for physical recognition by Rer1p. The Sec71TMD-GFP fusion protein is efficiently retrieved to the ER by Rer1p. The structural feature of this TMD signal turns out to be the spatial location of polar residues flanking the highly hydrophobic core sequence but not the whole length of the TMD. On the Rer1p side, Tyr152 residue in the 4th TMD is important for the recognition of Sec12p but not Sec71p, suggesting that Rer1p interacts with its ligands at least in two modes. Sec71TMD-GFP expressed in the Deltarer1 mutant cells is mislocalized from the ER to the lumen of vacuoles via the multivesicular body (MVB) sorting pathway. In this case, not only the presence of polar residues in the Sec71TMD but also the length of the TMD is critical for the MVB sorting. Thus, the Rer1p-dependent ER retrieval and the MVB sorting in late endosomes both watch polar residues in the TMD but in a different manner.

Amino Acid Sequence↗
PubMed2003

Oligomerization of a cargo receptor directs protein sorting into COPII-coated transport vesicles.

Secretory proteins are transported from the endoplasmic reticulum (ER) to the Golgi complex in vesicles coated with coat protein complex II (COPII). The incorporation of certain transport molecules (cargo) into the COPII vesicles is thought to be mediated by cargo receptors. Here we show that Emp47p, a type-I membrane protein, is specifically required for the transport of an integral membrane protein, Emp46p, from the ER. Exit of Emp46p from the ER was saturable and dependent on the expression level of Emp47p. Emp46p binding to Emp47p occurs in the ER through the coiled-coil region in the luminal domains of both Emp47p and Emp46p, and dissociation occurs in the Golgi. Further, this coiled-coil region is also required for Emp47p to form an oligomeric complex of itself in the ER, which is essential for exit of Emp47p from the ER. Our results suggest that Emp47p is a receptor protein for Emp46p that allows for the selective transport of this protein, and this event involves receptor oligomerization.

COP-Coated Vesicles↗