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T Maehama

Publications and source records attributed to T Maehama.

At least 37 records · Page 2Linked to original sources
PubMed1998

Selective down-regulation of human papillomavirus transcription by 2-deoxyglucose.

The glycolytic pathway inhibitor 2-deoxyglucose (2-DG) is capable of suppressing the transcription of the human pathogenic papillomavirus type 18 (HPV 18) in cervical carcinoma cells and derived non-tumorigenic somatic cell hybrids at the level of transcription initiation. HPV down-regulation is selective, since other reference genes are not affected or even up-regulated under the same experimental conditions. Moreover, 2-DG appears to restore the normal half-life of the tumor suppressor gene product p53, because the protein is strongly up-regulated after HPV 18 E6/E7 suppression. The observed 2-DG-effect is not cytotoxic and is reversible after refeeding with fresh medium. HPV 18 suppression by 2-DG can be completely abrogated by simultaneous treatment with the intracellular Ca2+ antagonist TMB-8, indicating that Ca2+, a known intracellular "second messenger", is involved in this process. Elevated c-myc and p53 expression appears to be responsible for the time-dependent accumulation of apoptotic cells after prolonged 2-DG treatment. The finding that 2-DG acts selectively against the expression of a human pathogenic papillomavirus strongly suggests that an appropriate level of glycolysis is not only a peculiarity of growing tumors, but even may be an essential prerequisite for the maintenance of virus-specific E6/E7 gene expression. Our results may have substantial implications for the potential therapeutic application of 2-DG or other glucose derivatives in the treatment of precancerous and malignant HPV-associated lesions.

Adenosine Triphosphate↗
PubMed1998

The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate.

Phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) is a key molecule involved in cell growth signaling. We demonstrated that overexpression of PTEN, a putative tumor suppressor, reduced insulin-induced PtdIns(3,4,5)P3 production in human 293 cells without effecting insulin-induced phosphoinositide 3-kinase activation. Further, transfection of the catalytically inactive mutant of PTEN (C124S) caused PtdIns(3,4,5)P3 accumulation in the absence of insulin stimulation. Purified recombinant PTEN catalyzed dephosphorylation of PtdIns(3,4,5)P3, specifically at position 3 on the inositol ring. PTEN also exhibited 3-phosphatase activity toward inositol 1,3,4,5-tetrakisphosphate. Our results raise the possibility that PTEN acts in vivo as a phosphoinositide 3-phosphatase by regulating PtdIns(3,4,5)P3 levels. As expected, the C124S mutant of PTEN was incapable of catalyzing dephosphorylation of PtdIns(3,4,5)P3 consistent with the mechanism observed in protein-tyrosine phosphatase-catalyzed reactions.

Cell Line↗
PubMed1998

Activation of c-Jun N-terminal kinase (JNK) by lysophosphatidic acid in Swiss 3T3 fibroblasts.

Lysophosphatidic acid (LPA) induced activation of c-Jun N-terminal kinase (JNK) in Swiss 3T3 fibroblasts. This activation reached the maximum at 20 min and required a high concentration of LPA with an EC50 value of approximately 3 microg/ml. LPA-induced activation of JNK was not suppressed by prior treatment of the cells with pertussis toxin, whereas it was completely blocked by suramin, a non-selective inhibitor of ligand-receptor interactions. The kinetics and concentration-dependency of LPA-induced JNK activation were in sharp contrast with those of LPA-induced extracellular signal-regulated kinase (ERK) activation, which reached the maximum within 3 min and occurred with an EC50 of 0.1 microg/ml. The ERK activation was susceptible to pertussis toxin, whereas it was not inhibited by suramin. These results indicate that the signal transduction pathways of LPA-induced JNK and ERK activations are distinct. Thus, this is the first report showing that LPA induces not only ERK activation but also JNK activation, which may be responsible for the induction of DNA synthesis in LPA-stimulated Swiss 3T3 fibroblasts.

3T3 Cells↗
PubMed1997

Heterodimeric phosphoinositide 3-kinase consisting of p85 and p110beta is synergistically activated by the betagamma subunits of G proteins and phosphotyrosyl peptide.

Phosphoinositide 3-kinase (PI 3-kinase) is a key signaling enzyme implicated in variety of receptor-stimulated cell responses. Receptors with intrinsic or associated tyrosine kinase activity recruit heterodimeric PI 3-kinases consisting of a 110-kDa catalytic subunit (p110) and an 85-kDa regulatory subunit (p85). We separated a PI 3-kinase that could be stimulated by the betagamma subunits of G protein (Gbetagamma) from rat liver. The Gbetagamma-sensitive PI 3-kinase appeared to be a heterodimer consisting of p110beta and p85 (or their related subunits). The stimulation by Gbetagamma was inhibited by the GDP-bound alpha subunit of the inhibitory GTP-binding protein. Moreover, the stimulatory action of Gbetagamma was markedly enhanced by the simultaneous addition of a phosphotyrosyl peptide synthesized according to the amino acid sequence of the insulin receptor substrate-1. Such enzymic properties could be observed with a recombinant p110beta/p85alpha expressed in COS-7 cells with their cDNAs. In contrast, another heterodimeric PI 3-kinase consisting of p110alpha and p85 in the same rat liver, together with a recombinant p110alpha/p85alpha, was not activated by Gbetagamma, although their activities were stimulated by the phosphotyrosyl peptide. These results indicate that p110beta/p85 PI 3-kinase may be regulated in a cooperative manner by two different types of membrane receptors, one possessing tyrosine kinase activity and the other activating GTP-binding proteins.

Amino Acid Sequence↗
PubMed1997

Molecular characterization of rat T lymphocyte alloantigen RT6.1 as an ADP-ribosyltransferase.

A family of glycosylphosphatidylinositol-linked ADP-ribosyltransferases, of which cDNAs were cloned from various mammalian cells, possess a common Glu-rich motif (EEEVLIP) near their carboxyl termini. Although the first Glu in the common motif is replaced by Gln (Q207EEVLIP) in rat T lymphocyte alloantigens RT6.1 and RT6.2, the two RT6s appear to have ADP-ribosyltransferase activity. To investigate the significance of the Glu-rich motif in the enzyme activity, we produced a mutant RT6.1, in which Gln207 was replaced by Glu (Q207E), together with wild-type RT6s, in Escherichia coli. The recombinant RT6.1 and RT6.2 displayed extremely low auto-ADP-ribosylation, though the latter modification was somewhat higher than the former one. In contrast, much higher the auto-modification was observed for Q207E mutant. Moreover, the mutant could effectively ADP-ribosylate agmatine as a substrate. Thus, the single amino acid mutation of RT6.1 caused remarkable increase in its ADP-ribosyltransferase activity, indicating that the Glu-rich motif near the carboxy terminus plays an important role in the enzyme activity.

ADP Ribose Transferases↗
PubMed1996

Increase in ADP-ribosyltransferase activity of rat T lymphocyte alloantigen RT6.1 by a single amino acid mutation.

A family of glycosylphosphatidylinositol-linked ADP-ribosyltransferases, of which cDNAs were cloned from various mammalian cells, possess a common Glu-rich motif (EEEVLIP) near their carboxyl termini. Although the first Glu in the common motif is replaced by Gln (Q207EEVLIP) in rat T lymphocyte alloantigens RT6.1 and RT6.2, the two RT6s appear to have both activities of NAD+ glycohydrolase and ADP-ribosyltransferase to a lesser extent. To investigate the significance of the Glu-rich motif in the two enzyme activities, we produced a mutant RT6.1 (Q207E), in which Gln207 was replaced by Glu, together with wild-type RT6s, in Escherichia coli. Kinetic analysis revealed that there were no marked differences in the Vmax and Km values of NAD+ glycohydrolases among the three recombinant proteins. The recombinant RT6.1 and RT6.2 displayed extremely low auto-ADP-ribosylation, although the latter modification was somewhat higher than the former. In contrast, much greater auto-modification was observed for the Q207E mutant. Moreover, the mutant could effectively ADP-ribosylate agmatine as a substrate. Thus, the single amino acid mutation of RT6.1 caused a marked increase in its ADP-ribosyltransferase activity, indicating that the Glu-rich motif near the carboxy terminus plays an important role in the enzyme activity.

ADP Ribose Transferases↗
PubMed1995

NAD(+)-dependent ADP-ribosylation of T lymphocyte alloantigen RT6.1 reversibly proceeding in intact rat lymphocytes.

Rat T lymphocyte alloantigen 6.1 (RT6.1), which was synthesized as the fusion protein with a maltose-binding protein in Escherichia coli, displayed NAD(+)-dependent auto-ADP-ribosylation in addition to an enzyme activity of NAD+ glycohydrolase. Such ADP-ribosylation of RT6.1 was also observed in lymphocytes isolated from rat tissues as follows. When intact rat lymphocytes expressing RT6.1 mRNA were incubated with [alpha-32P]NAD+, its radioactivity was incorporated into a cell surface protein with the M(r) of 31,000. The radiolabeled 31-kDa protein was released from the cell surface by treatment of the cells with phosphatidylinositol-specific phospholipase C and immunoprecipitated with anti-RT6.1 antiserum. The radioactivity incorporated into the 31-kDa protein was recovered as 5'-[32P]AMP upon incubation with snake venom phosphodiesterase and also removed by NH2OH treatment. These results suggested that the NAD(+)-dependent modification of the 31-kDa protein was due to ADP-ribosylation of glycosylphosphatidylinositol-anchored RT6.1 at an arginine residue. When intact lymphocytes, in which RT6.1 had been once modified by [32P]ADP-ribosylation, were further incubated in the absence of NAD+, there was reduction of the radioactivity in the [32P]ADP-ribosylated RT6.1. The reduced radioactivity was recovered from the incubation medium as [32P]ADP-ribose. This reduction was effectively inhibited by the addition of ADP-ribose to the reaction mixture. Moreover, readdition of NAD+ caused the ADP-ribosylation of RT6.1 again. Thus, the ADP-ribosylation of RT6.1 appeared to proceed reversibly in intact rat lymphocytes.

ADP Ribose Transferases↗
PubMed1995

Significance of Thr182 in the nucleotide-exchange and GTP-hydrolysis reactions of the alpha subunit of GTP-binding protein Gi2.

The crystal structures of the GTP- and GDP-bound alpha subunits of heterotrimeric GTP-binding proteins were recently determined, and a conserved Thr residue in the G2 (linker 2) region of the alpha subunits, which corresponds to Thr182 in Gi2 alpha, was deduced to interact with the gamma-phosphate of GTP and Mg2+. To investigate biochemically the significance of the Thr residue, we produced a mutant Gi2 alpha, in which Thr182 was substituted for Ala (T182A), in Escherichia coli. The rate of guanosine 5'-(gamma-thio)tri-phosphate (GTP gamma S) binding to T182A was higher than that to the wild-type Gi2 alpha, especially with a high concentration (10 mM) of Mg2+. The rate of dissociation of bound GDP from T182A was also much faster than that from the wild-type with the high Mg2+ concentration. Moreover, T182A had much lower GTPase activity than the wild-type, like the gip mutant (R179C) of Gi2 alpha found in human endocrine tumors. The ability of T182A to interact with beta gamma subunits and membrane-bound receptors was the same as that of the wild-type alpha subunit. T182A could take on a GTP-bound active conformation, as judged from its sensitivity to tryptic digestion. These results indicated that Thr182 plays an important role not only in the Mg(2+)-sensitive GDP-GTP exchange reaction but also in the GTPase activity of Gi2 alpha. The T182A mutant of Gi2 alpha, characterized by the faster GDP release and the slower GTP hydrolysis, would be a novel mutant that retains the ability to interact with receptors and beta gamma subunits.

Alanine↗
PubMed1994

Characterization of botulinum C3-catalyzed ADP-ribosylation of rho proteins and identification of mammalian C3-like ADP-ribosyltransferase.

The exoenzyme C3 produced by Clostridium botulinum catalyzes ADP-ribosylation of rho gene products which belong to a family of small molecular-weight GTP-binding proteins. The C3 enzyme-catalyzed ADP-ribosylation of rho proteins partially purified from bovine brain was markedly activated by certain types of detergents or phospholipids and by endogenous factors present in the brain cytosol. Rho A protein that had been expressed in E. coli and subsequential purified was readily ADP-ribosylated by the C3 enzyme even in the absence of the activating factors. These results suggest that partially purified rho proteins contain an inhibitor, probably rho GDI (GDP-dissociation inhibitor for rho p21), of C3-catalyzed ADP-ribosylation. The activity of an endogenous enzyme, having the same substrate as botulinum C3 enzyme, was also found in brain cytosol. The enzyme activity was partially purified and characterized. The enzyme appeared to have a molecular mass of approximately 20,000 on a gel filtration and displayed unique properties similar to those observed with the botulinum C3 enzyme. The alpha-subunits of alpha beta gamma-trimeric G proteins which served as the substrates of cholera or pertussis toxin were not ADP-ribosylated by the brain enzyme.

ADP Ribose Transferases↗
PubMed1994

ADP-ribosylarginine glycohydrolase catalyzing the release of ADP-ribose from the cholera toxin-modified alpha-subunits of GTP-binding proteins.

A rat glycohydrolase which catalyzes the hydrolysis of ADP-ribosylarginine was expressed in Escherichia coli and purified to homogeneity for characterization of its enzymatic properties. The purified glycohydrolase catalyzed the hydrolysis of N-glycoside linked ADP-ribosylarginine on the alpha-subunits of stimulatory GTP-binding proteins (Gs) and cholera toxin A1-subunit that had been modified by cholera toxin and NAD. Nonmuscle actin of which an arginine residue was ADP-ribosylated by botulinum C2 toxin also served as a substrate of the glycohydrolase. On the other hand, the glycohydrolase did not hydrolyze ADP-ribosylated cysteine on the alpha-subunits of pertussis toxin-substrate GTP-binding proteins, ADP-ribosylated diphthamide on elongation factor 2, or ADP-ribosylated asparagine on rho GTP-binding proteins. The rate of the reaction catalyzed by the glycohydrolase was affected by nucleotide-binding form of the ADP-ribosylated substrate proteins; the GDP-bound form of the modified Gs-alpha was more rapidly hydrolyzed than the guanosine 5'-(3-O-thio)triphosphate-bound form. Interestingly, the glycohydrolase activity was markedly inhibited by mM order concentration of ATP in addition to ADP-ribose, the product of the enzyme reaction, though ADP had no inhibitory effect on the activity. Moreover, alpha NAD, but not beta NAD, inhibited the enzyme activity, suggesting that the glycohydrolase reaction was stereospecific for the alpha-anomer.

Adenine Nucleotides↗
PubMed1992

[Studies on factor affecting mother-to-child HTLV-I transmission].

Epidemiological data indicate that breast feeding is the primary route of mother-to-child transmission of HTLV-I. However, serological studies show that 60 to 90% of infants who are fed breast milk from HTLV-I carriers avoid HTLV-I infection. To understand the mechanism of the virus transmission, the titer of HTLV-I antibodies and p40tax antibody and the copy number of the HTLV-I proviral DNA in peripheral lymphocytes in the HTLV-I carrier-mothers were surveyed. The seroprevalence of HTLV-I in the children born to carrier mothers was 6.1% in a normal breast feeding group, 7.7% in a group fed with freeze-thawed breast milk and none in a bottle-fed group. The difference between the seroconversion rate for the normal breast feeding group and others was statistically insignificant. Our studies also indicated that neither the titer of HTLV-I antibodies in carriers nor the duration of breast feeding was relevant to seroconversion in the children. The seroconversion rate in children was higher in p40tax antibody-positive mothers than in a seronegative group, but the difference was statistically insignificant. In conclusion, the copy number of the HTLV-I provirus in the lymphocytes of the carrier mothers was the most relevant factor in the seroconversion of infants who received breast milk from HTLV-I carriers.

Base Sequence↗
PubMed1991

Identification of a botulinum C3-like enzyme in bovine brain that catalyzes ADP-ribosylation of GTP-binding proteins.

A novel enzyme activity was found in bovine brain cytosol that transfers the ADP-ribosyl moiety of NAD to proteins with Mr values of 22,000 and 25,000. The substrates were the same GTP-binding proteins serving as the substrate of an ADP-ribosyltransferase C3 which was produced by a type C strain of Clostridium botulinum. The brain enzyme was partially purified from the cytosol and had a molecular mass of approximately 20,000 on a gel filtration column. The brain endogenous enzyme displayed unique properties similar to those observed with botulinum C3 enzyme. The enzyme activity was markedly stimulated by a protein factor that had been initially found in the cytosol as an activator for botulinum C3-catalyzed ADP-ribosylation (Ohtsuka, T., Nagata, K., Iiri, T., Nozawa, Y., Ueno, K., Ui, M., and Katada, T. (1989) J. Biol. Chem. 264, 15000-15005). The activity of the brain enzyme was also affected by certain types of detergents or phospholipids. The substrate of the brain enzyme was specific for GTP-binding proteins serving as the substrate of botulinum C3 enzyme; the alpha-subunits of trimeric GTP-binding proteins which served as the substrate of cholera or pertussis toxin were not ADP-ribosylated by the endogenous enzyme. Thus, this is the first report showing an endogenous enzyme in mammalian cells that catalyzes ADP-ribosylation of small molecular weight GTP-binding proteins.

ADP Ribose Transferases↗
PubMed1991

Purification and characterization of a new GTP-binding protein of Mr 24,000 in bovine brain membranes.

A GTP-binding protein with an Mr of 24,000 was purified from a cholate extract of bovine brain membranes in addition to the previously reported alpha beta gamma-trimeric GTP-binding proteins (G proteins). Partial amino acid sequence analysis of the purified 24-kDa protein revealed that it was not identical to any of the low Mr GTP-binding proteins already reported, but similar to the rac-gene products serving as the substrate of an ADP-ribosyltransferase (C3) purified from the culture medium of Clostridium botulinum type C. However, the 24-kDa protein was not ADP-ribosylated by the botulinum C3 enzyme. The 24-kDa protein was purified as a nucleotide-free form and characterized by the following unique properties distinct from those of alpha beta gamma-trimeric G proteins. (1) Mg2+ was essentially required for nucleotide binding to the 24-kDa protein; there was a progressive increase in its binding affinity for nucleotides as the concentration of the divalent cation was increased. (2) Nucleotides previously bound to the 24-kDa protein were rapidly dissociated from the protein in Mg(2+)-free medium, in accord with the fact that the protein was indeed purified as a nucleotide-free form with Mg(2+)-free solutions. (3) The 24-kDa protein apparently exhibited much lower GTPase activity than do alpha beta gamma-trimeric G proteins because the product GDP was released from the 24-kDa protein in exchange for the substrate GTP only at a very low rate. Based on these findings, a possible role of the 24-kDa protein in cellular signalling is discussed in comparison with well characterized alpha beta gamma-trimeric G proteins.

ADP Ribose Transferases↗
PubMed1990

Transmission of HTLV-I to rabbits via semen and breast milk from seropositive healthy persons.

Four rabbits inoculated intravenously with milk cells from 4 post-partum women seropositive for human T-cell leukemia virus type I (HTLV-I) and one rabbit inoculated with semen cells from a seropositive healthy man seroconverted for HTLV-I after 3-5 weeks but no seroconversion occurred in 2 rabbits inoculated with milk cells from a seronegative mother or with heated (56 degrees C, 30 min) milk cells from a seropositive mother. Attempts were made to isolate HTLV-I from peripheral blood lymphocytes harvested 5-15 weeks after cell inoculation and cultured in the presence of interleukin-2. An HTLV-I-carrying lymphoid cell line of rabbit origin was established from a rabbit inoculated with milk cells. Another long-term culture, derived from a rabbit inoculated with semen cells, also expressed HTLV-I antigens and harbored virus particles. Furthermore, transfusion of 20 ml of blood from all 5 seroconverted rabbits, but not from the 2 seronegative ones, caused seroconversion in normal recipient rabbits after 4-6 weeks.

Animals↗