Biomedical subjects

T Ebina

Publications and source records attributed to T Ebina.

At least 19 recordsLinked to original sources

PubMed1999

A high-Mr glycoprotein fraction from cow's milk potent in inhibiting replication of human rotavirus in vitro.

Rotavirus is the major cause of infectious diarrhea in infants and young children all over the world. We have found that a high-M(r) glycoprotein fraction from cow's milk is potent in inhibiting replication of human rotaviruses in vitro. Since the activity seems to be unique and specific, this fraction may be useful as a novel agent for treatment or prevention of rotavirus diarrhea.

Animals↗

PubMed1998

Selective tumoricidal effect of soluble proteoglucan extracted from the basidiomycete, Agaricus blazei Murill, mediated via natural killer cell activation and apoptosis.

We have isolated a novel type of natural tumoricidal product from the basidiomycete strain, Agaricus blazei Murill. Using the double-grafted tumor system in Balb/c mice, treatment of the primary tumor with an acid-treated fraction (ATF) obtained from the fruit bodies resulted in infiltration of the distant tumor by natural killer (NK) cells with marked tumoricidal activity. As shown by electrophoresis and DNA fragmentation assay, the ATF also directly inhibited tumor cell growth in vitro by inducing apoptotic processing; this apoptotic effect was also demonstrated by increased expression of the Apo2.7 antigen on the mitochondrial membranes of tumor cells, as shown by flow-cytometric analysis. The ATF had no effect on normal mouse splenic or interleukin-2-treated splenic mononuclear cells, indicating that it is selectively cytotoxic for the tumor cells. Cell-cycle analysis demonstrated that ATF induced the loss of S phase in MethA tumor cells, but did not affect normal splenic mononuclear cells, which were mainly in the G0G1 phase. Various chromatofocussing purification steps and NMR analysis showed the tumoricidal activity to be chiefly present in fractions containing (1-->4)-alpha-D-glucan and (1-->6)-beta-D-glucan, present in a ratio of approximately 1:2 in the ATF (molecular mass 170 kDa), while the final purified fraction, HM3-G (molecular mass 380 kDa), with the highest tumoricidal activity, consisted of more than 90% glucose, the main component being (1-->4)-alpha-D-glucan with (1-->6)-beta branching, in the ratio of approximately 4:1.

Agaricus↗

PubMed1998

Interleukin-15 effectively potentiates the in vitro tumor-specific activity and proliferation of peripheral blood gammadeltaT cells isolated from glioblastoma patients.

GammadeltaT cells play a regulatory role in both primary and metastatic tumor growth in humans. The mechanisms responsible for the activation and proliferation of circulating gammadeltaT cells should be fully understood prior to their adoptive transfer to cancer patients. We have examined in vitro functional effects of interleukin-15 (IL-15) on highly purified gammadeltaT cells isolated from glioblastoma patients. GammadeltaT cells constitutively express the heterotrimeric IL-2 receptor (IL-2R) alpha betagamma, but the levels of IL-2Rbeta or gamma expression were not increased by incubation with saturating amounts of IL-15. IL-15 was shown to induce a maximal gammadeltaT cell proliferation, although at much higher concentrations (at least 2000 U/ml) than IL-2 (100 U/ml). Submaximal concentrations of IL-15 plus low concentrations of IL-2 produced an additive proliferative response. In contrast to the IL-2-induced response, this activity was completely or partially abrogated by anti-IL-2Rbeta, or anti-IL-2Rgamma antibodies, but not by anti-IL-2R alpha antibodies. Incubation of gammadeltaT cells in the presence of IL-15 resulted not only in the appearance of NK and LAK activity, but also in specific autologous tumor cell killing activity, an additive effect being seen with IL-15 and IL-2. This IL-15-induced tumor-specific activity could be significantly blocked by anti-IL-2Rgamma and anti-IL-2R-beta mAb, but not by anti-IL-2R alpha mAb. Thus, in contrast to IL-2, IL-15 activates tumor-specific gammadeltaT cells through the components of IL-2Rbeta and IL-2Rgamma, but not IL-2R alpha. These enhanced in vitro tumor-specific and proliferative responses of gammadeltaT cells seen with IL-15 suggest a rational adjuvant imunotherapeutic use of gammadeltaT cells in cancer patients.

Antibodies, Monoclonal↗

PubMed1998

The use of BRM-activated killer cells in adoptive immunotherapy: a pilot study with nine advanced cancer patients.

Adoptive immunotherapy using MHC-nonrestricted-lymphocytes, peripheral blood gammadelta T cells and NK cells was devised. Peripheral blood mononuclear cells (3 x 10(7)) were selected by immobilization to anti-CD3 monoclonal antibody for 4 days and cultured for 2 weeks in the presence of IL-2. Thereafter they were reactivated by 500 U/ml of IFN-alpha and 1000 U/ml of IL-2 for 1 hour. Enhancement of NK and LAK activities was confirmed. Peripheral blood gammadelta T cells proliferated in response to immobilized anti-CD3 antibody (3% to 30%). Approximately 6 x 10(9) BRM-activated killer (BAK) cells composed of CD56+ gammadelta T cells and CD56+ NK cells, were dispensed to cancer patients via intravenous drip infusion. Nine patients were treated with BAK cells every 2 weeks or every month on an outpatient basis. During the course of adoptive immunotherapy, the crossed affinity immunoelectrophoresis (CAIE) pattern of serum immunosuppressive acidic protein (IAP) was analysed. Both the production and glycosylation pattern of IAP is changed in response to tumor enlargement and may therefore act as a marker of the disease progression. During the course of BAK therapy, the glycosylation IAP pattern of 6 patients changed from tumor (T) to normal (N). In addition, the performance status of all patients was maintained at 90-100% of the Karnofsky scale and any side effects including fever were not observed during treatments with BAK cells. Moreover, the overall quality of life (QOL) of the patients, scored at the Face scale was favorable. In addition, blood levels of activated gammadelta T cells producing IFN-gamma were assayed as an indication marker of BAK therapy. The normal range of IFN-gamma producing gammadelta T cells comprised 6.9 +/- 0.9% of peripheral blood mononuclear cells (PBMC), according to a single cell FACScan analyses of PBMCs derived from normal individuals. IFN-gamma producing gammadelta T cells of Patients No. 8 and 9, who received extensive chemotherapy before initiation of BAK therapy, comprised only 0.2% and 2% of PBMC, respectively. These patients died 3 and 6 months after beginning BAK therapy. Peripheral blood gammadelta T cells of Patients Nos. 1-7 proliferated in response to immobilized anti-CD3 antibody and the frequency of IFN-gamma producing gammadelta T cells in PBMC preparation of these patients were over 3% before initiation of BAK therapy. Since our data show a positive correlation between survival time and initial gammadelta T cell counts, a low frequency of these cells may contraindicate BAK therapy.

Adult↗

PubMed1998

Antitumor effect of a peptide-glucan preparation extracted from Agaricus blazei in a double-grafted tumor system in mice.

The antitumor effect of extracts obtained from the fruit body of Agaricus blazei Murill was examined in a double-grafted tumor system, in which BALB/c mice received simultaneous intradermal injections of Meth-A tumor cells in both the right (10(6) cells) and left flank (2 x 10(5) cells), and were then injected with 5 mg of extracts of A. blazei in the right tumor on days 3, 4 and 5. Intratumoral administration of ethanol-soluble (Fraction 1), water-ethanol-soluble (Fraction 2), ammonium oxalate-soluble (Fraction 3) and ammonium oxalate-insoluble (Fraction 4) fractions resulted in inhibition of tumor growth, with Fraction 3 showing the most tumoricidal activity, producing regression of the right tumor and inhibition of growth of the left, non-injected tumor. The maximum effect was obtained using 0.5 mg of Fraction 3 and this amount was used in subsequent experiments. The antitumor effect of intratumorally administered Fraction 3 was enhanced by oral ad lib administration of feed containing 0.083% of Fraction 3. When immunized spleen cells from mice that had been cured by intratumoral administration of 0.5 mg of Fraction 3 were directly injected (2 x 10(7) cells/mouse) into the Meth-A tumor, tumor growth was inhibited. The tumor cells on day 7 from the Fraction 3-treated right tumor and from the left tumor were cultured for 24 h and their culture supernatants were assayed for neutrophil or macrophage chemotactic activity. Significant macrophage chemotactic factor activity was detected in the culture media from the left tumor tissue. Serum levels of immunosuppressive acidic protein (IAP), produced by activated macrophages and neutrophils, increased transiently soon after intradermal injection of 0.5 mg of Fraction 3. These results suggest that regression of the left non-injected tumor was due to an immune reaction, involving induction of cytotoxic cells in the spleen, and the release of chemotactic factors in the distant tumor.

Adoptive Transfer↗

PubMed1998

[Pharmacokinetics of propofol and ketamine during and after total intravenous anesthesia with propofol, fentanyl and ketamine for pediatric patients].

Pharmacokinetics of propofol and ketamine during propofol-fentanyl-ketamine (PFK) anesthesia for pediatric surgery was studied. Plasma levels of propofol (Pp) were maintained approximately at 2.5 micrograms.ml-1 during surgery. Fifteen minutes after the cessation of propofol infusion, Pp decreased to 1.5 micrograms.ml-1. Plasma levels of ketamine (Pk) were maintained at 150-200 ng.ml-1 during the surgery. After the cessation of ketamine infusion, Pk decreased as quickly as Pp. Pk values at 15 minutes and 120 minutes after the cessation of the infusion were 93 ng.ml-1, 24 ng.ml-1, respectively. On the other hand, plasma norketamine (Pn) levels increased gradually during surgery and stayed at 100-150 ng.ml-1 after the end of ketamine infusion to play an important role in post-operative sedation and pain relief. In conclusion, pharmacokinetics of propofol and ketamine in pediatric patients was similar to that in adult patients. PFK anesthesia can be used safely for pediatric as well as for adult patients.

Anesthesia, Intravenous↗

PubMed1997

Caveolin interaction with protein kinase C. Isoenzyme-dependent regulation of kinase activity by the caveolin scaffolding domain peptide.

Caveolar localization of protein kinase C and the regulation of caveolar function by protein kinase C are well known. This study was undertaken to examine whether caveolin subtypes interact with various protein kinase C isoenzymes using the caveolin scaffolding domain peptide. When protein kinase C-alpha, -epsilon, and -zeta were overexpressed in COS cells followed by subcellular fractionation using the sucrose gradient method, all the isoenzymes (alpha, epsilon, and zeta) were detected in the same fraction as caveolin. The scaffolding domain peptide of caveolin-1 and -3, but not -2, inhibited the kinase activity and autophosphorylation of protein kinase C-alpha and -zeta, but not of protein kinase C-epsilon, overexpressed in insect cells. Truncation mutation studies of the caveolin-1 and -3 peptides demonstrated that a minimum of 16 or 14 amino acid residues of the peptide were required for the inhibition or direct binding of protein kinase C. Thus, the caveolin peptide physically interacted with protein kinase C and regulated its function. Further, this regulation occurred in a protein kinase C isoenzyme-dependent manner. Our results may provide a new mechanism regarding the regulation of protein kinase C isoenzyme activity and the molecular interaction of protein kinase C with its putative binding proteins.

Amino Acid Sequence↗

PubMed1997

Regulation of adenylyl cyclase isoforms by N-alkanols.

We examined the effect of n-alkanols on adenylyl cyclase isoforms (types II and V) overexpressed in insect cells. Ethanol stimulated the type II isoform but not the type V isoform. Ethanol stimulated type II adenylyl cyclase greater than GTP gamma S, and the treatment of the membrane with GDP beta S or cholera toxin did not affect this stimulation. Other n-alkanols inhibited type V adenylyl cyclase activity in proportion to their lipophilic potency. In contrast, type II adenylyl cyclase was stimulated by weakly lipophilic n-alkanols and inhibited by strongly lipophilic n-alkanols. When solubilized membranes and purified preparations were used, all the n-alkanols inhibited type II adenylyl cyclase. Our data suggest that n-alkanols regulated adenylyl cyclase isoform-dependently. Stimulation of the type II isoform was independent from the interaction with Gs alpha but required the presence of an intact membrane structure. Our study may provide another step to understanding how membrane protein subtypes are differentially regulated by n-alkanols.

Adenylyl Cyclase Inhibitors↗

PubMed1997

A simple method for the propagation and purification of gamma delta T cells from the peripheral blood of glioblastoma patients using solid-phase anti-CD3 antibody and soluble IL-2.

Although gamma delta T cells make up no more than 10% of the peripheral blood mononuclear (PBM) cells, they appear to play an important role in host defense against tumor growth. In order to evaluate their functional activity against tumors and their response to various cytokines, large numbers of cells are required. Here, we describe a newly-devised method for the isolation and expansion of gamma delta T cells from the peripheral blood of cancer patients, in particular those with glioblastoma. Using this approach, a 1000-1500-fold increase in total cell numbers was achieved in two weeks, the proportion of gamma delta T cells in the expanded population being, on average, approximately 30% after 14 days of culture. The method therefore gives a yield of approximately 10-15 x 10(8) gamma delta T cells from only 5 ml of peripheral blood from glioblastoma patients and normal controls. The highly purified gamma delta T cells of glioblastoma patients were shown to bear both a high-affinity interleukin-2 receptor (IL-2R) and a low-affinity IL-12 receptor (IL-12R). They also displayed significant cytotoxicity against autologous tumor cells, but not against autologous fresh or IL-2-treated lymphocytes, and proliferated in response to IL-2, both effects being dependent on the dose of IL-2 used for activation. In addition, overnight incubation with 700 U/ml of IL-2 or 50 ng/ml of IL-12 resulted in significant cytotoxic activity of patients' gamma delta T cells against K562 target cells, the level of activity being almost the same as with similarly-treated gamma delta T cells from normal controls (P > 0.05). These results demonstrate that the patients' gamma delta T cells obtained using this method are intact in terms of cytotoxic function. Thus, this method not only makes it possible to produce large numbers of purified gamma delta T cells but also to produce populations containing both gamma delta T cells and NK cells, both active against tumor targets which might be suitable for clinical trials of adoptive-immunotherapy, especially in cancer patients for whom no effective therapy is available.

Adolescent↗

PubMed1997

Conformation-dependent activation of type II adenylyl cyclase by protein kinase C.

Phorbol ester treatment enhanced the catalytic activity of type II adenylyl cyclase overexpressed in insect cells. In cells coexpressing type II adenylyl cyclase and protein kinase C-alpha, type II adenylyl cyclase catalytic activity was higher even in the absence of phorbol ester treatment; phorbol ester treatment further and markedly enhanced type II adenylyl cyclase catalytic activity. However, this enhancement, either by phorbol ester treatment or by coexpression of protein kinase C-alpha, was lost following membrane solubilization with detergents. This attenuation was unaffected by phosphatase inhibitor or salts. In contrast, membrane solubilization did not affect forskolin-stimulated type II adenylyl cyclase catalytic activity. Purified type II adenylyl cyclase was stimulated by forskolin and Gs alpha, but not by protein kinase C-alpha. Therefore, a specific mammalian protein kinase C isoenzyme can activate type II adenylyl cyclase, but the mechanism clearly differs from that underlying either Gs alpha- or forskolin-mediated stimulation.

Adenylyl Cyclases↗

PubMed1997

Isoform-dependent activation of adenylyl cyclase by proteolysis.

Recent findings have suggested that the cellular proteolytic system plays a major role in the regulation of various intra- and extra-cellular signaling. It was previously shown that proteolytic treatment of adenylyl cyclase leads to the activation of this enzyme. We demonstrate that this activation occurs in an adenylyl cyclase isoform-dependent manner. The type II isoform was strongly activated (approximately 500%), the type III isoform was modestly activated (approximately 30%),and the type V isoform was inhibited by trypsin. Activation of type II adenylyl cyclase occurred in trypsin dose- and time-dependent manners and was blocked by a trypsin inhibitor in a dose-dependent manner. Other proteases, such as thrombin and plasminogen, similarly activated the type II isoform, but not the others. Our data suggest that proteolytic activation is an isoform- and thus cell type-dependent mechanism of altering adenylyl cyclase catalytic activity.

Adenylyl Cyclases↗

PubMed1997

Isoform-specific regulation of adenylyl cyclase by oxidized catecholamines.

Both epinephrine and manganese are known to stimulate cAMP production in cardiac homogenates. When added together, however, they inhibited adenylyl cyclase catalytic activity. Type V adenylyl cyclase, the major isoform in the heart, was also inhibited when an increasing concentration of epinephrine was added in the presence of manganese. Inhibition was not dependent on the condition of stimulation or preparation of the enzyme. However, this inhibition was abolished in the presence of anti-oxidant. Other catecholamines, including dopamine and isoproterenol, as well as adrenochrome, an oxidized product of epinephrine, similarly inhibited the activity of this enzyme. Kinetic analyses revealed that the K(m) for the substrate ATP was unchanged, but the V(max) was significantly decreased. In contrast, type II adenylyl cyclase, a non-cardiac isoform, was resistant to such inhibition by adrenochrome and was somewhat stimulated by it. Thus, catecholamines, when oxidized, directly interacted with adenylyl cyclase in an isoform-specific manner in the absence of G proteins. Our findings suggest that adenylyl cyclase isoforms have different sensitivity to various stresses, including oxidative stress.

Adenylyl Cyclases↗

PubMed1997

Crohn's disease associated with renal amyloidosis successfully treated with an elemental diet.

We report a case of Crohn's disease associated with nephrotic syndrome due to renal amyloidosis in a 21-year-old man in whom remission of both Crohn's disease and the nephrotic syndrome has been maintained with an elemental diet. The patient developed toxic megacolon and nephrotic syndrome due to renal amyloidosis. Intensive intravenous prednisolone therapy with total parenteral nutrition was dramatically effective in treating the toxic megacolon and inducing remission in Crohn's disease and afterward, remission of the nephrotic syndrome. Remission of both conditions has been maintained for more than 2 years with the elemental diet. To our knowledge, this is the first confirmed case of Crohn's disease complicated with renal amyloidosis in which only slight proteinuria (below 0.3 g/day) was shown with an elemental diet used for a long period.

Adult↗

PubMed1997

Computer simulation study on the conversion of spirolactone to enones over H-Y zeolite.

We report here the results of computer modeling studies and quantum chemical calculations on the spirolactone-to-enone conversion reaction over the zeolite catalysts, especially H-Y zeolite. We studied the adsorption mode of the molecules inside the supercage of H-Y and the mechanism of electron transfer between organic molecules and the framework of zeolite H-Y by density functional theory (DFT) calculations. Because the organic molecules considered in the present study are less symmetrical we docked the molecule inside the supercage of H-Y and energy minimization was then applied to these docked structures to yield representative low-energy binding sites for the molecules within the host structure. The interaction energy results show that the major interaction is between the methylene hydrogen of the molecule and the oxygen of the framework. The molecular electrostatic potential maps show that the ketonic oxygen of the reactant molecules abstract proton from Brönsted acid site. Thus the mechanism proposed by DFT calculation matches well with the experimental observations.

Computer Simulation↗

PubMed1997

[Catecholamine and dopamine].

Almost all the genes of the enzymes which synthesize and metabolize the catecholamines (dopamine, norepinephrine, epinephrine) have been cloned and the gene targeting technology have been applied to introduce the gene knockout mouse such as thyrosine hydroxylase and dopamine beta hydroxylase. At least nine adrenergic receptors and five dopamine receptors have been cloned, which include alpha 1A-, alpha 1 B-, alpha 1 D-, alpha 2 A-, alpha 2B-, alpha 2C-, beta 1-, beta 2-, beta 3-adrenergic receptors and D1-, D2-, D3-, D4-, D5-dopamine receptors. Transgenic mouse as well as gene knockout mouse of these genes have been also produced. Furthermore, intracellular signal transduction systems of the catecholamines have been clarified using molecular techniques, including nine subtypes of adenylyl cyclase. Using these cloned genes and transgenic and gene knockout mouse, more detailed features of the catecholamine systems and those receptors and intracellular signal transduction systems will be clarified in near future.

Adenylyl Cyclases↗

PubMed1997

In vitro interleukin 12 activation of peripheral blood CD3(+)CD56(+) and CD3(+)CD56(-) gammadelta T cells from glioblastoma patients.

Interleukin (IL)-12 has recently been shown to be directly involved in the activation of natural killer and alphabeta T cells via an IL-2-independent pathway. We show here that another type of human cytotoxic cell, gammadelta T cells activated by solid-phase anti-CD3 antibody and expanded using IL-2, obtained, in this case, from the peripheral blood of glioblastoma patients, displays significant tumoricidal activity. In addition, its cytotoxic activity against K562 or Daudi cells or against autologous glioblastoma targets (but not lymphocytes) is significantly enhanced when costimulated with IL-2 and IL-12. To study this synergistic activation by the two interleukins of the patients' gammadelta T cells, we screened the cells for the presence of the IL-2 receptor (IL-2R) and IL-12 receptor (IL-12R) using both flow cytometric analysis and PCR. The patients' gammadelta T cells constitutively expressed the high-affinity IL-2R; when stimulated with IL-12 plus IL-2, the levels of IL-2Ralpha and IL-2Rbeta increased, whereas that of IL-2gamma did not. They also expressed marginal levels of low-affinity IL-12R both immediately after IL-2 expansion and after 24-h incubation, and significantly higher levels after 72-h incubation, consistent with the level of gammadelta T-cell activation. IL-12 alone induced little proliferation of patients' gammadelta T cells in a 24-h assay and none in a 72-h assay; however, it caused a marked inhibition of the IL-2-induced proliferative response in the 72-h assay. The synergistic action of IL-2 and IL-12 was completely abolished by combined pretreatment with anti-IL-2alpha, beta, and gamma mAbs. IL-12-mediated enhancement of gammadelta T cell cytotoxic activity was inhibited by anti-IL-2Rbeta mAb in a dose-dependent manner but not by anti-IL-2Ralpha or anti-IL-2Rgamma mAbs. Thus, the increased expression of the IL-2Rbeta is critical for the synergistic activation of gammadelta T cells by IL-12 plus IL-2; it is also probable that at least the low-affinity IL-12R contributes to the activation of gammadelta T cells mediated by either IL-12 alone or IL-12 plus IL-2. We have, therefore, demonstrated that IL-12 can stimulate the cytotoxic activity of gammadelta T cells from glioblastoma patients, acting via the IL-2Rbeta component of the IL-2R and low-affinity IL-12R. IL-12 activation of patients' gammadelta T cells could possibly be of potential use in the treatment of glioblastoma patients.

Antigens, CD↗

PubMed1996

Soluble adenylyl cyclase from Spodoptera frugiperda (Sf9) cells. Purification and biochemical characterization.

An insect ovarian cell, Spodoptera frugiperda (Sf9), has been widely used to express recombinant proteins, including adenylyl cyclase, as a host cell in the baculovirus expression system. We report the presence and characterization of a soluble adenylyl cyclase (sAC) distinct from a membrane-bound form of adenylyl cyclase (mAC) that is also present in Sf9 cells. sAC was purified 3,500-fold to near homogeneity; a single band at 25 kDa on SDS-polyacrylamide gel electrophoresis correlated well with adenylyl cyclase catalytic activity. The purified enzyme had a catalytic activity of 0.1 micromol/min.mg and the Km of 0.55 mM for the substrate ATP. In contrast to mAC, sAC was heat-stable. Enzymatic activity of sAC was not stimulated by forskolin and was inhibited by salts at high concentrations. sAC utilized both manganese- and magnesium-ATP as substrate. Di- or triphosphate-containing nucleotides, such as GTP and GDP, as well as pyrophosphate, noncompetitively inhibited sAC. Our data suggest that the physical and biochemical characteristics of sAC are different from those of mAC in Sf9 cells as well as from those of other known forms of adenylyl cyclase in animal cells; sAC in Sf9 cells may constitute a new member of adenylyl cyclase found in animals.

Adenylyl Cyclase Inhibitors↗

PubMed1996

Regulation of type V adenylyl cyclase by PMA-sensitive and -insensitive protein kinase C isoenzymes in intact cells.

Abstract Type V adenylyl cyclase (AC) was stably over-expressed in HEK293 cells (293AC-V). Forskolin-stimulated cAMP accumulation in 293AC-V was 5 times as great as that in control cells. PMA, a protein kinase C (PKC) activator, enhanced cAMP accumulation in 293AC-V cells dose-and time-dependently and this enhancement was abolished by staurosporine. Insulin also enhanced cAMP accumulation in 293AC-V cells. Co-transfection of PKC-zeta, but not PKC-alpha, potentiated the effects of insulin. These data suggest that type V AC activity is regulated in cells by PKC isoenzymes through different extracellular stimuli.

Adenylyl Cyclases↗