The Bcr-Abl kinase inhibitor INNO-406 induces autophagy and different modes of cell death execution in Bcr-Abl-positive leukemias.

Bcr-Abl tyrosine kinase (TK) inhibitors are promising therapeutic agents for Bcr-Abl-positive (Bcr-Abl(+)) leukemias. Although they are known to promote caspase-mediated apoptosis, it remains unclear whether caspase-independent cell death-inducing mechanisms are also triggered. Here we demonstrated that INNO-406, a second-generation Bcr-Abl TK inhibitor, induces programmed cell death (PCD) in chronic myelogenous leukemia (CML) cell lines through both caspase-mediated and caspase-independent pathways. The latter pathways include caspase-independent apoptosis (CIA) and necrosis-like cell death (CIND), and the cell lines varied regarding which mechanism was elicited upon INNO-406 treatment. We also observed that the propensity toward CIA or CIND in cells was strongly associated with cellular dependency on apoptosome-mediated caspase activity. Cells that undergo CIND have a high apoptosome activity potential whereas cells that undergo CIA tend to have a lower potential. Moreover, we found that INNO-406 promotes autophagy. When autophagy was inhibited with chloroquine or gene knockdown of beclin1 by shRNA, INNO-406-induced cell death was enhanced, which indicates that the autophagic response of the tumor cells is protective. These findings suggest new insights into the biology and therapy of Bcr-Abl(+) leukemias.

The pharmacokinetics and effects of a long-acting preparation of superoxide dismutase (PC-SOD) in man.

AIM: To study the pharmacokinetics (PK), safety and tolerability of single rising doses up to 80 mg of superoxide dismutase covalently linked to lecithin (PC-SOD) in healthy White volunteers. METHODS: This double-blind, placebo-controlled, four-period cross-over study was performed in eight healthy volunteers (four male/four female). Three doses of PC-SOD (20, 40 and 80 mg) and placebo were administered intravenously in randomized order. Serum and urinary PC-SOD concentrations were measured predose and up to 96 h after dosing. In addition to standard safety measurements, the urinary excretion of N-acetyl-beta-glucosaminidase, alpha-glutathione S-transferase (alpha-GST) and pi-GST was measured to evaluate renal function. The PK of PC-SOD was analysed using noncompartmental and compartmental methods. RESULTS: All treatments were well tolerated, and no obvious relationship between adverse events and treatment was observed. No effects of PC-SOD on renal function could be detected. Dose normalized C(max) and AUC were not different between the different dosages, indicating linearity of plasma concentrations with dose. Estimated PC-SOD clearance was 2.54 ml min(-1)[95% confidence interval (CI) 2.07, 2.83]. The terminal half-life was estimated to be 1.54 days (95% CI 0.93, 2.15). SOD activity was elevated above baseline for 19 +/- 6 h after the 80-mg dose. CONCLUSIONS: Single intravenous administrations of PC-SOD in doses up to 80 mg were well tolerated in healthy White male and female volunteers. With the doses used, SOD activity was linearly related to the dose; after the 80-mg dose it was present for an appreciable period. These findings suggest that it is worthwhile to investigate PC-SOD in clinical conditions characterized by a high radical overload.

DDS for anti-aging and regenerative medicine (review).

In this paper we summarized, first the present status and history of the development of research in anti-aging and regenerative medicine in Japan, and secondly some of our research using DDS in the field of both medicine. The regenerative medicine has been developed in Japan by using the fund from the Government, particularly as the Millennium Project. While anti-aging medicine developed following the social interest on it in Japan and it was influenced by American Society (A4M). Next, we summarized our research on DDS for anti-aging and regenerative medicine. In most cases we used oily or solid nanoparticles as carriers of drug. Those particles have a property of both of targeting and slow release in the DDS technology. The two properties are important for anti-aging and regenerative medicine, since drugs have to be administered safely and for long time. We applied prostaglandin E1, granulocyte-colony stimulate factor (G-CSF), and retinoid into the systems.

Augmentation of specific cell-mediated immune responses to tumor cells in tumor-bearing rats pretreated wih the antileukemia drug busulfan.

Lethal growth of a syngeneic transplanted tumor (KMT-17) was inhibited in inbred WKA rats pretreated with the antileukemia drug busulfan (BU). However, the lethal growth of KMT-17 was not inhibited by pretreatment with cyclophosphamide, adriamycin, or ftorafur. With the Winn assay, spleen cells from BU-pretreated KMT-17-bearing rats (TBR) inhibited the growth of admixed KMT-17 cells more strongly than did spleen cells from BU-untreated TBR. The augmented tumor inhibitory activity of spleen cells was KMT-17-specific, and this activity was abrogated by in vitro treatment of spleen cells with anti-T serum and guinea pig complement. Augmentation of the immune response to KMT-17-associated antigen(s) in BU-pretreated TBR was also demonstrated in lymphocyte-mediated cytotoxicity, as detected by a 51Cr release assay and by a delayed-type hypersensitivity with a radioisotope footpad assay. Tumor regression in BU-pretreated rats was demonstrated to be mediated by the augmentation of T-cell-mediated immune responses to tumor-associated antigens. The tumor inhibitory effect of BU was abrogated by adoptive transfer with thymus cells from normal rats but not with those from BU-pretreated rats 1 day before tumor inoculation. The augmentation oif the antitumor immune responses by pretreatment with BU was suggested to be due to the fact that BU selectively inhibited the suppressor cells of their precursors.

Increased sensitivity of murine leukemia virus-infected tumor cells to lymphocyte-mediated cytotoxicity.

The cytotoxic sensitivity of murine leukemia virus (MuLV)-infected and noninfected fibrosarcoma cells in syngeneic inbred WKA/Hok rats was compared by in vitro cell-mediated 51Cr release cytotoxicity assay. A highly significant increase in cytotoxic sensitivity of target cells was observe in MuLV-infected tumor cells as compared with noninfected cells when spleen cells from syngeneic tumor-bearing hosts (TBH) were used as a source of effector lymphocytes. The cytotoxicity of spleen cells against MuLV-infected tumor cells was specifically directed to the tumor-associated antigen (TAA), but not to the virus-associated antigen. However, there was no quantitative difference in the amount of TAA on the cell membranes between virus-infected and noninfected tumor cells as measured by a quantitative absorption test of anti-TAA serum. The cytotoxic activity of spleen cells from TBH against MuLV-infected tumor cells was abrogated by the treatment of anti-T-serum plus complement and significantly decreased after trypsin treatment. Spleen cells from normal rats given injections of immune sera from TBH acquired the cytotoxic activity against MuLV-infected tumor cells.

Augmentation of antitumor immune response by trinitrophenyl (TNP)-reactive helper T-cells: enhanced induction of tumor-specific Lyt-1+2- T-cell-mediated delayed-type hypersensitivity from spleen cells of tumor-bearing mice by TNP helpers.

Spleen cells from X5563 tumor-bearing syngeneic C3H/HeN mice were stimulated in vitro with trinitrophenyl (TNP)-modified or unmodified X5563 tumor cells. TNP-reactive helper T-cells obtained from TNP-primed C3H/HeN mice were added to the above cultures in an attempt to augment the induction of anti-X5563 delayed-type hypersensitivity (DTH) responses. The DTH responses were measured by adoptive transfer of cultured cells together with unmodified X5563 cells into footpads of syngeneic mice. Cultures of spleen cells from tumor-bearing mice plus X5563 or TNP-modified X5563 cells failed to generate anti-X5563 DTH responses. In contrast, addition of TNP-helper T-cells to the cultures resulted in appreciable DTH as well as in cytotoxic responses to X5563 tumor cells. Demonstration of this immunity was dependent on the presence of TNP-X5563 tumor cells as stimulators during the culture period. The anti-X5563 DTH effector cells augmented by TNP helpers were found to be of the Lyt-1+2- phenotype and were tumor specific, since DTH responses were observed when the cultured cells were injected with X5563 but not when injected with another syngeneic tumor. These results demonstrate that TNP-helper T-cells are capable of augmenting the induction of tumor-specific Lyt-1+2- T-cell-mediated DTH responses from lymphoid cells of tumor-bearing mice upon the stimulation of TNP-reactive tumor cells. The results are discussed in relation to: a previously described tumor-specific immunotherapy model in which a growing tumor regressed by virtue of TNP helpers and the implications of augmenting induction of tumor-specific DTH responses in antitumor resistance.

Genetic control of hapten-reactive helper T-cell responses and its implications for the generation of augmented antitumor cytotoxic responses.

The genetic control of hapten-reactive helper T-cell activity involved in cytotoxic T-lymphocyte (CTL) responses and its implications for augmenting tumor-specific immunity were studied. C57BL/6N mice were immunized to trinitrophenyl (TNP) or N-iodoacetyl-N'-(5-sulfonic l-naphthyl)ethylenediamine (AED) hapten by inoculation of hapten-modified syngeneic spleen cells. Spleen cells from these hapten-immunized mice were tested for hapten-reactive helper T-cell activity for generation of CTL. TNP-primed spleen cells resulted in only marginal help for the generation of anti-TNP-modified syngeneic spleen cell (TNP-self) CTL response when cocultured with normal C57BL/6N spleen cells (responding cells) in the presence of TNP-self. In contrast, AED-primed spleen cells exhibited appreciable help for AED-induced CTL responses. Furthermore, AED-helper, but not TNP-helper, T-cell activity was demonstrated to augment the generation of antitumor (RBL-5 leukemia) CTL responses from normal syngeneic spleen cells when stimulated with the corresponding hapten-self plus RBL-5 tumor cells. These results indicate that the successful augmentation of syngeneic tumor immunity through T-T-cell interaction with the use of hapten-reactive helper T-cells can depend on selection of the appropriate haptenic reagent in an individual expressing a given major histocompatibility haplotype.

Modification of in vitro and in vivo BCG cell wall-induced immunosuppression by treatment with chemotherapeutic agents or indomethacin.

The in vitro inhibition of spleen cell blastogenesis response and the in vivo enhancement of tumor growth are phenomena associated with BCG cell wall (BCGcw) immunization. What effect treatment with chemotherapeutic agents and the prostaglandin inhibitor indomethacin would have on the in vitro and in vivo responses to BCGcw immunization was evaluated. In vitro blastogenesis studies showed that chemotherapy pretreatment prior to immunization with BCGcw resulted in a restoration of the spleen cell blastogenesis response. In blastogenesis addback studies, where BCGcw-induced irradiated splenic suppressor cells were admixed with normal cells, less inhibition of blastogenesis occurred when spleen cells were obtained from rats that had received the combined treatment of chemotherapy and BCGcw immunization versus only BCGcw immunization. The cocultivation of spleen cells from BCGcw-immunized rats with indomethacin resulted in a 30-40% restoration of the blastogenesis response. In vivo studies showed that BCGcw-mediated enhancement of intramuscular tumor growth of the 3924a ACI rat tumor could be abrogated by either pretreatment with busulfan or mitomycin or by the feeding of indomethacin.

Administration of optimum sustained-insulin release PLGA microcapsules to spontaneous diabetes-prone BB/Wor//Tky rats.

To show the possibility of sustained-release insulin formulation composed of PLGA, the optimum one was administered to BioBreeding rat, a model of spontaneous type I diabetes mellitus (IDDM). Every 2 weeks subcutaneous administration made their blood glucose level depend on the insulin release and food intake. However, all of them kept alive with little change or rather a little gain in body weight. Furthermore, some of pregnant rats with intermittent treatment bore fetuses, although additional insulin therapy seemed necessary. Therefore, the formulation could become a new tool as a provider of basal insulin for IDDM patients.

Differences in in vitro proliferative responsiveness to granulocyte colony-stimulating factor and interleukin 2 of bone marrow cells from mice treated with chemotherapeutic drugs.

The toxicity effects of several anticancer drugs on normal mouse bone marrow (BM) were estimated using the in vitro proliferative responsiveness [( 3H]thymidine incorporation) of the treated BM cells to recombinant human granulocyte colony-stimulating factor (G-CSF) and recombinant human interleukin 2 (IL-2). From the response pattern of the treated BM cells to G-CSF and IL-2, the anticancer drugs were classified into three groups: (a) BM cells from cyclophosphamide- or nimustine hydrochloride-treated mice showed an increased responsiveness to G-CSF but a decreased responsiveness to IL-2; (b) BM cells from vindesine- or peplomycin-treated mice showed an increased responsiveness to both G-CSF and IL-2; and (c) BM cells from mitomycin C-treated mice showed a decreased responsiveness to both G-CSF and IL-2. These different response patterns may reflect qualitative differences in the myelotoxicity effects of these anticancer drugs.

Diminution of cyclophosphamide-induced suppression of antitumor immunity by an immunomodulator PS-K and combined therapeutic effects of PS-K and cyclophosphamide on transplanted tumor in rats.

An immunomodulator PS-K was shown to diminish the cyclophosphamide (CY)-induced suppression of specific antitumor transplantation resistance in WKA rats immunized with X-irradiated KMT-17 tumor cells when PS-K was given before treatment of CY. In the immunochemotherapy of transplanted KMT-17 tumor in WKA rats by a combination of CY and PS-K, an enhanced therapeutic effect was also observed when PS-K was given before treatment of CY, with different doses of CY and at different days of CY treatment. However, when PS-K was given just after treatment of CY, the therapeutic effect was rather diminished in comparison with the group having CY treatment alone. By means of the Winn assay, spleen cells obtained from KMT-17-bearing rats (TBR) treated with PS-K followed by CY inhibited the admixed tumor cells more strongly than did spleen cells obtained from TBR treated with CY alone. Recovery of thymus weight from the damage caused by CY was accelerated in TBR treated with PS-K followed for CY and was delayed in TBR treated with CY followed by PS-K. These observations suggest that diminution of CY-induced immunosuppression by PS-K possibly results in an enhanced therapeutic effect in WKA rats treated with PS-K followed by CY.

Most effective route of administration and utilization of high-dose chemotherapy with bone marrow transplantation in rats.

In order to find out which anticancer drugs could utilize to the best advantage a syngeneic bone marrow transplantation in high-dose chemotherapy for cancer, we tested six drugs [nimustine hydrochloride (ACNU), Adriamycin, cyclophosphamide (CY), mitomycin c, vindesin, etoposide] in Sprague-Dawley rats from a standpoint of the beneficial effect of bone marrow transplantation (BMT). Two or three varying doses of each drug were administered i.v. on Day 0, followed by the injection of syngeneic bone marrow (BM) cells (5 x 10(7), i.v.) on Day 2, and the animals were observed for over 60 days. Adriamycin caused high rates of peripheral neuropathy, and was therefore judged to be inappropriate for high-dose chemotherapy-BMT in this animal model. Among the other five drugs, a beneficial effect of BMT was observed only with CY (300-400 mg/kg) and ACNU (40 mg/kg). In order to enhance the beneficial effect of BMT observed with CY and ACNU, a way of drug administration was designed and carried out. Consequently a higher survival rate was obtained in the following experimental groups: (a) (CY 200 mg/kg, Days 0 and 1) + BMT greater than (CY 400 mg/kg, Day 0) + BMT, (ACNU 20 mg/kg, Days 0 and 1) + BMT greater than (ACNU 40 mg/kg, Day 0) + BMT. (b) (CY 200 mg/kg + ACNU 20 mg/kg, Day 0) + BMT greater than (CY 400 mg/kg or ACNU 40 mg/kg, Day 0) + BMT. (c) (CY 200 mg/kg, Day 0) + (ACNU 20 mg/kg, Day 1) + BMT greater than (ACNU 20 mg/kg, Day 0) + (CY 200 mg/kg, Day 1) + BMT. Among the six anticancer drugs tested in this study, CY and ACNU were suggested to be more appropriate drugs for high-dose chemotherapy-BMT, but methods for reducing drug toxicity (dose, combination, sequence) were necessary so as to enhance the beneficial effect of the BMT.

Prevention of fatal infections by recombinant human interleukin 1 alpha in normal and anticancer drug-treated mice.

The preventive capability of interleukin 1 alpha (IL-1) against bacterial infections was estimated in normal and anticancer drug-treated BALB/c mice in comparison with OK432, granulocyte colony-stimulating factor, interferons alpha and gamma, and interleukin 2. Pretreatment with IL-1 (days -4 and -2) resulted in a significantly higher survival rate in normal mice inoculated i.p. with Klebsiella pneumoniae, Pseudomonas aeruginosa or Listeria monocytogenes (day 0). The i.p. and s.c. administrations of IL-1 were equally effective for the induction of antibacterial resistance. Pretreatment with OK432 showed an equal degree of resistance to i.p. infection but was effective only by i.p. administration. Enhanced antibacterial resistance by IL-1 and OK432 was also observed in cyclophosphamide- and aminomethylpyrimidinylmethylchloroethylnitrosourea hydrochloride-pretreated (day -5) normal hosts and in cyclophosphamide-treated tumor-bearing hosts. In the case of granulocyte colony-stimulating factor (i.p. or s.c.) (days -4 to -1), a statistical difference in survival rate between granulocyte colony-stimulating factor and its vehicle-treated groups was observed in cyclophosphamide-pretreated hosts, but not in normal hosts or aminomethylpyrimidinylmethylchloroethylnitrosourea hydrochloride-pretreated hosts. Viable bacteria in the peritoneal cavity and blood at 12 h after i.p. infection of K. pneumoniae correlated well with the survival rate. In IL-1-pretreated hosts, the earlier and increased accumulation of neutrophils into peritoneal cavity after the infection was observed and the number of inflammatory cells in peritoneal cavity correlated well with the survival rate. The enhanced resistance to bacterial infection by IL-1 was suggested to be in part due to the enhanced cellular defense mechanisms. The prophylactic administration of IL-1 would be beneficial for the management of serious infections in cancer patients.

Enhancement of antitumor transplantation resistance in rats by appropriately timed administration of busulfan.

Enhancement of specific transplantation resistance to a syngeneic tumor (KMT-17) was observed in WKA rats by treatment with the antileukemia drug busulfan (BU) (15 mg/kg) 5 days before and 5 days after immunization with X-irradiated KMT-17 tumor cells. Rats immunized with X-irradiated KMT-17 cells and then treated with BU showed specific transplantation resistance only against KMT-17 tumor. Carrageenan administration after BU treatment had no effect on enhancement by BU, which indicated that macrophages were not playing a major role in the observed enhancement. With the Winn assay, it was found that spleen cells from rats immunized with X-irradiated tumor cells followed by BU inhibited the growth of admixed tumor cells more strongly than did spleen cells from rats only immunized or only BU treated and that the tumor-neutralizing activity of spleen cells from rats treated by immunization followed by BU was abrogated by treatment with anti-T-serum and complement. It was suggested that the enhanced antitumor transplantation resistance caused by BU was due to enhanced T-cell immune responses to tumor cells. Enhancement of anti-tumor transplantation resistance by BU was significantly abrogated by adoptive transfer with thymus cells and was slightly abrogated with spleen cells from rats immunized with X-irradiated KMT-17 cells 1 day before tumor challenge but receiving no other treatment. Transfer of sera from the immunized rats had no effect on enhancement by BU. These results, taken together, suggest that the mechanism of the enhancement by BU involved a selective elimination of the immunosuppressor cells from the immunized hosts.

Characterization of immunosuppressor cells in rats immunized with solubilized tumor-associated antigens prepared from a methylcholanthrene-induced fibrosarcoma.

Antiimmune responses in rats previously immunized with soluble tumor antigens prepared by sodium deoxycholate (DOC-STA) from chemically induced fibrosarcoma KMT-17 were measured by the Winn assay. Enhancement of tumor growth was demonstrated at a tumor:effector ratio of 1:500 with DOC-STA-immune spleen cells, although inhibition of tumor growth was demonstrated at a tumor:effector ratio of 1:100. The tumor-neutralizing ability of KMT-17-immune spleen cells was abrogated when DOC-STA-immune spleen cells were added to a mixture of KMT-17 cells and KMT-17-immune spleen cells. This suppressor activity of the spleen cells was diminished by the treatment with rabbit anti-rat T-cell serum and immune complement. The suppressor activity of DOC-STA-immune spleen cells was also shown in 51Cr release assay and was specific for the tumor line used. After fractionation of spleen cells from DOC-STA-immune rats by the Ficoll density gradient, the cells in the light layer showed an enhancing effect on tumor growth detected by the Winn assay, whereas the cells in the heavier region of the gradient had an inhibiting effect.

Negative and positive immunobiological responses in mice pretreated with Bacillus Calmette-Guérin cell wall.

Spleen cells from C57BL/6 mice injected i.p. with Bacillus Calmette-Guérin cell walls (BCGcw) showed strongly depressed response to the T-cell mitogen phytohemagglutinin-P in vitro. Mitogen reactivity of normal spleen cells could be suppressed by the addition of spleen cells from BCGcw-treated mice. The suppressor cells mediating this effect appeared to belong to the plastic-adherent, radioresistant, and non-T-cell populations, maybe macrophages. Spleen cells from mice which had been passively transferred i.p. with the adherent cells from BCGcw-treated mice also showed the depressed mitogen response in vitro. Depressed T-cell reactivity of spleen cells obtained from animals immunized with BCGcw on a per-cell basis was also demonstrated in vivo: graft versus host reactivity of spleen cells obtained from animals immunized with BCGcw was depressed as compared to normal spleen cells. At times when strong suppressor cell activity could be detected in BCGcw-treated mice, activity of alloimmune cytotoxic lymphocytes generated in vivo by immunizing with X-irradiated allogeneic MH-134 tumor cells was weaker in BCGcw-pretreated mice than in untreated control groups (detected by means of 51Cr release assay). Furthermore, accelerated development of s.c. inoculated syngeneic B-16 melanoma cells was observed in BCGcw-pretreated mice. On the other hand, stronger resistance to i.v. inoculated B-16 tumor cells was observed in BCGcw-pretreated mice. BCGcw-treated mice responded normally to i.p. immunization with 2 X 10(8) sheep erythrocytes. Negative and positive immunobiological responses were observed in C57BL/6 mice pretreated with BCGcw.

Identification of a ubiquitin- and ATP-dependent protein degradation pathway in rat cerebral cortex.

To investigate the existence of a ubiquitin-dependent protein degradation system in the brain, the proteolytic activity of the cerebral cortex was examined. The soluble extract of rat cerebral cortex degraded 125I-radiolabeled lysozyme in an ATP-dependent manner. The ATP-dependent proteolysis was suppressed with iodoacetamide, which inhibits ubiquitin conjugation, and was abolished by blocking of the amino residues of lysozyme. These results suggest the participation of ubiquitination in the proteolytic activity. An ATP-dependent 125I-ubiquitin-conjugating activity was detected in fraction II from the cerebral cortex. The presence of ATP-dependent proteolytic activity which acted preferentially on ubiquitinated lysozyme was demonstrated, using ubiquitin-125I-lysozyme conjugates as a substrate. The proteinase had a molecular mass of 1500 kDa and displayed nucleotide dependence and sensitivity to various proteinase inhibitors similar to those of the 26S proteinase complex found in reticulocytes. Dialysis of the soluble fraction caused a decrease in the proteolytic activity of ATP-dependent and preferential for ubiquitin-lysozyme conjugates and a reciprocal increase in the ATP-independent free 125I-lysozyme-degrading activity which was scarcely detected before dialysis. The former ATP-dependent proteolytic activity may play a physiological role in the brain.

Absence of bicarbonate abolishes the glycogenic effect of cortisol in cultured fetal rat hepatocytes.

The glycogenic action of cortisol in cultured fetal rat hepatocytes was completely abolished by the absence of NaHCO3 from the medium, while its presence stimulated the action in relation to its concentration. The absence of NaHCO3 slightly reduced glycogen storage by insulin but did not affect glucose-dependent glycogen deposition in the basal state. Also, the cortisol-induced increase in glycogen synthase a activity was reduced but that in total synthase activity was not affected. The absence of NaHCO3 did not reduce the cortisol-induced increase in tyrosine aminotransferase activity and the incorporation of [3H]dexamethasone into the nuclei. These results show that the absence of NaHCO3 specifically inhibits the glycogenic action of glucocorticoids in cultured fetal rat hepatocytes and indicate the need for further investigation into the role of HCO3- in universally used bicarbonate-buffered media.

Alterations in the stimulatory G protein of the rat liver after partial hepatectomy.

In adult male rat livers, cAMP generation in response to beta-adrenergic agonists was dramatically stimulated after partial hepatectomy. Quantitation of the alpha subunits of the stimulatory G protein (Gs alpha) using ADP-ribosylation catalyzed by cholera toxin revealed the increment in the amounts of two forms of Gs alpha, Gs alpha-S and Gs alpha-L, during liver regeneration. These increases in the amounts of both Gs alpha proteins were associated with the stimulation in their mRNA levels. In addition, partial hepatectomy gave rise to a shift in the proportion of beta-adrenergic receptor (beta-AR) in the high affinity state produced by beta-AR-Gs complex. The susceptibility of Gs alpha to trypsin was used as a probe for beta-AR-Gs coupling. The GTP-bound forms of both Gs alpha-S and Gs alpha-L were more trypsin-sensitive than their GDP-bound forms. Preincubation of liver plasma membranes prepared from partially hepatectomized rats with the agonist isoproterenol resulted in an enhancement of trypsin-sensitivity of Gs alpha-L, but not Gs alpha-S. This effect was retarded by the addition of the antagonist propranolol. We conclude that the increase in the amount of Gs alpha can be contributed to the rise in beta-response after partial hepatectomy, and suggest that beta-AR is preferentially coupled with Gs alpha-L rather than Gs alpha-S.

Drug-incorporating calcium carbonate nanoparticles for a new delivery system.

We devised a simple method for incorporating drugs into solid calcium carbonate nanoparticles (nano-CaCO3). The size of nano-CaCO3 was controlled by mixing speed. Washing the nanoparticles released little incorporated drug but much drug that was adsorbed on the surface. In an in vitro releasing test, granulocyte colony-stimulating factor incorporated in nano-CaCO3 was chemically stable and released very slowly. Subcutaneous injection of nano-CaCO3 incorporating betamethasone phosphate (BP) resulted in a smaller initial increase in plasma concentration and a subsequent sustained release in compared with betamethasone phosphate solution. Nano-CaCO3 may be useful to deliver hydrophilic drugs and bioactive proteins.