Classification of stevia sweeteners in soft drinks using liquid chromatography and time-of-flight mass spectrometry.

The aim of this study was to develop a comprehensive analytical method for the characterisation of stevia sweeteners in soft drinks. By using LC and time-of-flight MS, we detected 30 steviol glycosides from nine stevia sweeteners. The mass spectral data of these compounds were applied to the analysis to determine steviol glycosides in nine soft drinks. On the basis of chromatographic data and principal-component analysis, these soft drinks were classified into three groups, and the soft drinks of each group, respectively, contained high-rebaudioside A extract, normal stevia extract or alfa-glucosyltransferase-treated stevia extract.

Involvement of EphA2-mediated tyrosine phosphorylation of Shp2 in Shp2-regulated activation of extracellular signal-regulated kinase.

Shp2 is a positive regulator for Erk activation downstream of receptor tyrosine kinases for growth factors. It has been controversial how Shp2 induces Erk activation. We here demonstrate that EphA2 is responsible for Shp2-mediated Erk activation by phosphorylating Tyr542 and Tyr580 of Shp2 in the cells stimulated with growth factors. In NMuMG mammary epithelial cells stimulated with hepatocyte growth factor (HGF), HGF-dependent Erk phosphorylation was prolonged only in the presence of EphA2. This Erk activation paralleled the phosphorylation of Tyr542/580 of Shp2 and the association of Grb2 with Shp2, suggesting the positive signal involving Grb2 signal to activate Ras-Erk pathway. Immunohistochemical studies of mammary cancer specimens revealed that the cancer progression was associated with both Tyr580 phosphorylation of Shp2 and increased expression of EphA2, which were also correlated with increased Erk phosphorylation. Overexpression of either Shp2Thr468Met (a phosphatase-defective mutant found in Lentigines, Electrocardiographic abnormalities, Ocular hypertelorism, Pulmonary stenosis, Abnormal genitalia, Retardation of growth and sensorineural Deafness (LEOPARD) syndrome) or Shp2Asn308Asp (a phosphatase-active mutant found in Noonan syndrome) with EphA2 exhibited comparable activation of Erk and stronger activation than wild-type Shp2, suggesting the phosphatase-independent Erk activation. Expression of Shp2Thr468Met with Tyr542/580Phe mutations resulted in the suppression of Erk activation. Phosphatase-active and -inactive, and wild-type Shp2s bound equally to Grb2, suggesting that phosphorylation of Tyr542/580 of Shp2 was essential but not sufficient for Shp2-mediated Erk activation. We found that Gab1 (Grb2-associated binder 1) was involved in the mutant Shp2-mediated Erk activation. Zebrafish injected with Shp2Thr468Met mRNA showed cardiac edema, whereas those depleted of EphA2b showed less phenotype, suggesting that EphA2 might partly account for the phenotype of LEOPARD syndrome. Collectively, tyrosine phosphorylation of Shp2 by EphA2 contributes to the phosphatase-independent Shp2-mediated activation of Erk and might be involved in Shp2-associated diseases.

Detecting young Japanese adults with undetected low skeletal bone density using panoramic radiographs.

OBJECTIVE: The cortical width below the mental foramen of the mandible determined from panoramic radiographs is a useful screening tool for identifying elderly individuals with a low skeletal bone mineral density (BMD). However, whether the mandible cortical width (MCW) is useful for identifying a low skeletal BMD in men and women of 40 years or younger is not known. METHODS: The BMD of the calcaneus was measured by ultrasonography bone densitometry in 158 men and 76 women aged 18-36 years. A logistic regression analysis adjusted for age was used to calculate the odds ratios and 95% confidence interval (CI) of having a low calcaneal BMD, according to the quartiles of the MCW. The areas under the receiver operator characteristic curve (AUC) for identifying participants with a low calcaneal BMD using the MCW were assessed to evaluate the diagnostic efficacy of the MCW. RESULTS: In men, the adjusted odds ratios of a low calcaneal BMD associated with the second, third and lowest quartiles of MCW were 5.66 (95% CI, 0.61-52.23), 5.43 (95% CI, 0.59-50.18) and 33.22 (95% CI, 3.97-276.94), respectively, compared with the highest quartile, while no significant trend in the adjusted odds ratios was observed in women. The AUC for identifying participants with a low calcaneal BMD based on the MCW was 0.796 (95% CI, 0.702-0.890) in men and 0.593 (95% CI, 0.398-0.788) in women. CONCLUSION: MCW determined from panoramic radiographs can be used to identify undetected low calcaneus BMD in young adult men, but not in young adult women.

Caracterização genética e distribuição geográfica do vírus rábico isolado de bovinos no estado de Mato Grosso, Brasil / Genetic characterization and geographic distribution of rabies virus isolates from cattle in Mato Grosso, Brazil

Foram caracterizados, geneticamente e geograficamente, o sequenciamento parcial da nucleoproteína (gene N) de 53 isolados do vírus da raiva (VR) originários do Estado de Mato Grosso, Brasil. Os isolados de bovinos, que se encontravam no grupo do VR relacionado a morcegos hematófagos, foram posteriormente subdivididos em sete subgrupos genéticos. Estes subgrupos foram distribuídos em regiões de terras planas, com alguns subgrupos separados por formações de pequenas montanhas e hidrografia. Estes resultados indicam que a raiva em bovinos é derivada de diversas variantes regionalmente definidas, o que sugere que sua distribuição geográfica está relacionada as populações de morcegos hematófagos.

A total of 53 rabies virus (RV) isolates originating from cattle in the state of Mato Grosso, Brazil, were genetically characterized. Partial nucleoprotein gene sequences of these isolates were phylogenetically and geographically analyzed. Cattle isolates, which clustered with the vampire bat related RV group, were further subdivided into 7 subgroups. These subgroups were distributed widely in lowland regions, with some subgroups separated from each other by small mountains and hydrographical features. These results indicate that cattle rabies is derived from several regionally-defined variants, which suggests that its geographical distribution is related to that of the vampire bat population.

The role of dynamin 3 in the testis.

We report here that dynamin 3 in the testis is associated with structures termed tubulobulbar complexes that internalize intact intercellular junctions during sperm release and turnover of the blood-testis barrier. The protein lies adjacent to an actin-Arp2/3 network that cuffs the double plasma membrane tubular invagination at the core of each complex. To explore the possible relationship between dynamin 3 and nectin-based adhesion junctions, we transiently transfected DsRed-tagged dynamin 3 into MDCK cells stably transfected with eGFP-tagged nectin 2, one of the adhesion molecules known to be expressed in Sertoli cells at adhesion junctions. Cells transfected with the dynamin 3 construct had less uniformly distributed nectin 2 at intercellular contacts when compared to control cells expressing only nectin 2 or transfected with the DsRed plasmid alone. Significantly, tubular extensions positive for nectin 2 were visible projecting into the cells from regions of intercellular contact. Our findings are consistent with the conclusion that dynamin 3 is involved with tubulobulbar morphogenesis. Dynamin 3 also occurs in concentrated deposits around the capitulum and striated columns in the connecting piece of sperm tails suggesting that the protein in these cells may function to stabilize the base of the tail or serve as a reservoir for use during or after fertilization.

Effects of hepatic CYP3A4 activity on disposition of micafungin in liver transplant recipients with markedly small-for-size grafts.

Micafungin, the first candin antifungal drug developed in Japan, has a significant therapeutic effect against deep-seated mycoses caused by Candida or Aspergillus. Little is known, however, about the optimal dosage or disposition of micafungin in patients with severe hepatic impairment. Nine liver transplant recipients (5 males and 4 females) were enrolled in this study. In 1 recipient with a markedly small-for-size graft (ratio of graft volume to standard liver volume at the time of transplantation: 25.9%), the areas under the plasma concentration-time curves up to 12 hours postdose (AUC(0-12 h)) at doses of 50 and 100 mg/d were 79.38 and 601.17 mug.h/mL, respectively. The corresponding elimination half-life (T(1/2)) values were 16.01 and 75.75 hours, and saturated elimination was observed only at the dose of 100 mg/d. The mean urinary ratio of 6beta-hydroxycortisol to cortisol (6beta-OHF/F) in the small-for-size graft recipient was significantly (P < .05) lower than that in the other recipients. In conclusion, graft size was an important factor affecting disposition of micafungin. For liver transplant recipients with markedly small-for-size grafts, the optimal dosage of micafungin to reach and maintain therapeutic plasma levels is estimated to be 50 mg/d.

Inhibitor of vascular endothelial growth factor receptor tyrosine kinase attenuates cellular proliferation and differentiation to mature neurons in the hippocampal dentate gyrus after transient forebrain ischemia in the adult rat.

Neurogenesis in the adult hippocampal dentate gyrus is promoted by transient forebrain ischemia. The mechanism responsible for this ischemia-induced neurogenesis, however, remains to be determined. It has been suggested that there may be a close relationship between neurogenesis and the expression of vascular endothelial growth factor, an angiogenic factor. The purpose of the present study was to examine the relationship between vascular endothelial growth factor and cell proliferation in the dentate gyrus after transient forebrain ischemia. The mRNA expression of vascular endothelial growth factor was increased in the dentate gyrus on day 1 after ischemia. Immunohistochemical analysis on day 9 after ischemia, when a significant increase in cell proliferation was seen, showed that the cerebral vessel space in the subgranular zone of the dentate gyrus had not been affected by the ischemia. Neither were the vascular densities on days 1 and 3 after ischemia altered compared with those of non-operated naïve control rats. Furthermore, the distance from the center of the proliferative cells to the nearest cerebral vessel of ischemic rats was comparable to that of the sham-operated rats. We demonstrated that transient forebrain ischemia-induced cell proliferation and differentiation to mature neurons in the hippocampal dentate gyrus was attenuated by the i.c.v. administration of a vascular endothelial growth factor receptor tyrosine kinase inhibitor. These results suggest that vascular endothelial growth factor receptor at the early period of reperfusion may contribute to neurogenesis rather than to angiogenesis in the hippocampal dentate gyrus.

Involvement of adaptor protein Crk in malignant feature of human ovarian cancer cell line MCAS.

Signaling adaptor protein Crk regulates cell motility and growth through its targets Dock180 and C3G, those are the guanine-nucleotide exchange factors (GEFs) for small GTPases Rac and Rap, respectively. Recently, overexpression of Crk has been reported in various human cancers. To define the role for Crk in human cancer cells, Crk expression was targeted in the human ovarian cancer cell line MCAS through RNA interference, resulting in the establishment of three Crk knockdown cell lines. These cell lines exhibited disorganized actin fibers, reduced number of focal adhesions, and abolishment of lamellipodia formation. Decreased Rac activity was demonstrated by pull-down assay and FRET-based time-lapse microscopy, in association with suppression of both motility and invasion by phagokinetic track assay and transwell assay in these cells. Furthermore, Crk knockdown cells exhibited slow growth rates in culture and suppressed anchorage-dependent growth in soft agar. Tumor forming potential in nude mice was attenuated, and intraperitoneal dissemination was not observed when Crk knockdown cells were injected into the peritoneal cavity. These results suggest that the Crk is a key component of focal adhesion and involved in cell growth, invasion, and dissemination of human ovarian cancer cell line MCAS.

Sore throat and hoarseness after total intravenous anaesthesia.

BACKGROUND: Sore throat and hoarseness are common complications, but these have not been studied after total i.v. anaesthesia. METHODS: We prospectively studied 418 surgical patients, aged 15-92 yr, after total i.v. anaesthesia with propofol, fentanyl and ketamine to assess possible factors associated with sore throat and hoarseness. RESULT: We found sore throat in 50% and hoarseness in 55% of patients immediately after surgery. This decreased to 25% for sore throat and 24% for hoarseness on the day after surgery. Both sore throat and hoarseness were more common in females and when lidocaine spray had been used. Cricoid pressure during laryngoscopy was inversely associated with the risk of sore throat. CONCLUSION: Knowledge of these factors may reduce postoperative throat complications, and improve patient satisfaction.

Requirement of nef for HIV-1 infectivity is biased by the expression levels of Env in the virus-producing cells and CD4 in the target cells.

The nef gene of human immunodeficiency virus type 1 (HIV-1) encodes a 27 to 34 kDa myristoylated protein, which enhances viral infectivity in a single-round infection assay. The level of Nef enhancement of HIV-1 infectivity depends on the viral strains, on the target cells, and on the cells used for propagating the viruses. In this study, we aimed at clarifying the molecular basis of these differences in the requirement for Nef. We found that the requirement for Nef was increased when we decreased the quantity of Env protein in the virus-producing cells or the quantity of CD4 in the target cells. Both the wild-type and Nef-defective HIV-1 viruses were propagated in 293T cells, which did not express any CD4; therefore, Nef-induced CD4 down-regulation did not explain this phenomenon. Moreover, we did not observe any increase in the viral entry or fusion activity of gp120env in the wild-type HIV-1 compared to that in the Nef-defective HIV-1. Thus, we propose that Env on the virion and CD4 on the target cells have inhibitory effects on the post-entry step of the HIV-1 replication cycle, and that Nef functions to counteract this negative effect.

Requirement for C3G-dependent Rap1 activation for cell adhesion and embryogenesis.

C3G is a guanine nucleotide exchange factor (GEF) for Rap1, and is activated via Crk adaptor protein. To understand the physiological role of C3G, we generated C3G knockout mice. C3G(-/-) homozygous mice died before embryonic day 7.5. The lethality was rescued by the expression of the human C3G transgene, which could be excised upon the expression of Cre recombinase. From the embryo of this mouse, we prepared fibroblast cell lines, MEF-hC3G. Expression of Cre abolished the expression of C3G in MEF-hC3G and inhibited cell adhesion-induced activation of Rap1. The Cre-expressing MEF-hC3G showed impaired cell adhesion, delayed cell spreading and accelerated cell migration. The accelerated cell migration was suppressed by the expression of active Rap1, Rap2 and R-Ras. Expression of Epac and CalDAG-GEFI, GEFs for Rap1, also suppressed the accelerated migration of the C3G-deficient cells. This observation indicated that Rap1 activation was sufficient to complement the C3G deficiency. In conclusion, C3G-dependent activation of Rap1 is required for adhesion and spreading of embryonic fibroblasts and for the early embryogenesis of the mouse.

A pair of fluorescent resonance energy transfer-based probes for tyrosine phosphorylation of the CrkII adaptor protein in vivo.

An adaptor protein, CrkII, which is involved in a variety of signaling cascades such as cell growth, migration, and apoptosis, becomes phosphorylated on Tyr(221) upon stimulation. Here, we report on a fluorescent resonance energy transfer-based sensor, which consists of CrkII sandwiched with cyan- and yellow-emitting variants of green fluorescent protein. This protein enabled us to monitor rapid and transient phosphorylation of CrkII upon epidermal growth factor stimulation in a living cell. However, rapid diffusion of the probes prevented us from specifying where the phosphorylation started within the cell. To overcome this problem, we fused the CAAX box of Ki-Ras to the carboxyl terminus of this probe and restricted its localization mostly to the plasma membrane. With this modified probe, we found that epidermal growth factor-induced phosphorylation of CrkII was initiated at the peripheral plasma membrane, moving toward the center of the cell. Moreover, this CAAX box-fused probe showed improvement in sensitivity and time resolution of the monitoring of CrkII phosphorylation. Thus, this pair of CrkII probes visualizes dynamic changes in the total and local levels of the tyrosine phosphorylation of CrkII in a living cell.

Spatio-temporal images of growth-factor-induced activation of Ras and Rap1.

G proteins of the Ras family function as molecular switches in many signalling cascades; however, little is known about where they become activated in living cells. Here we use FRET (fluorescent resonance energy transfer)-based sensors to report on the spatio-temporal images of growth-factor-induced activation of Ras and Rap1. Epidermal growth factor activated Ras at the peripheral plasma membrane and Rap1 at the intracellular perinuclear region of COS-1 cells. In PC12 cells, nerve growth factor-induced activation of Ras was initiated at the plasma membrane and transmitted to the whole cell body. After three hours, high Ras activity was observed at the extending neurites. By using the FRAP (fluorescence recovery after photobleaching) technique, we found that Ras at the neurites turned over rapidly; therefore, the sustained Ras activity at neurites was due to high GTP/GDP exchange rate and/or low GTPase activity, but not to the retention of the active Ras. These observations may resolve long-standing questions as to how Ras and Rap1 induce different cellular responses and how the signals for differentiation and survival are distinguished by neuronal cells.

Membrane recruitment of DOCK180 by binding to PtdIns(3,4,5)P3.

DOCK180 was originally identified as one of two major proteins bound to the Crk oncogene product and became an archetype of the CDM family of proteins, including Ced-5 of Caenorhabditis elegans and Mbc of Drosophila melanogaster. Further study has suggested that DOCK180 is involved in the activation of Rac by the CrkII-p130(Cas) complex. With the use of deletion mutants of DOCK180, we found that the C-terminal region containing a cluster of basic amino acids was required for binding to and activation of Rac. This region showed high amino-acid sequence similarity to the consensus sequence of the phosphoinositide-binding site; this led us to examine whether this basic region binds to phosphoinositides. For this purpose we used PtdIns(3,4,5)P(3)-APB beads, as reported previously [Shirai, Tanaka, Terada, Sawada, Shirai, Hashimoto, Nagata, Iwamatsu, Okawa, Li et al. (1998) Biochim. Biophys. Acta 1402, 292-302]. By using various competitors, we demonstrated the specific binding of DOCK180 to PtdIns(3,4,5)P(3). The expression of active phosphoinositide 3-kinase (PI-3K) did not enhance a DOCK180-induced increase in GTP-Rac; however, the expression of PI-3K translocated DOCK180 to the plasma membrane. Thus DOCK180 contained a phosphoinositide-binding domain, as did the other guanine nucleotide exchange factors with a Dbl homology domain, and was translocated to the plasma membrane on the activation of PI-3K.

Arabidopsis genomes uncoupled 5 (GUN5) mutant reveals the involvement of Mg-chelatase H subunit in plastid-to-nucleus signal transduction.

A plastid-derived signal plays an important role in the coordinated expression of both nuclear- and chloroplast-localized genes that encode photosynthesis-related proteins. Arabidopsis GUN (genomes uncoupled) loci have been identified as components of plastid-to-nucleus signal transduction. Unlike wild-type plants, gun mutants have nuclear Lhcb1 expression in the absence of chloroplast development. We observed a synergistic phenotype in some gun double-mutant combinations, suggesting there are at least two independent pathways in plastid-to-nucleus signal transduction. There is a reduction of chlorophyll accumulation in gun4 and gun5 mutant plants, and a gun4gun5 double mutant shows an albino phenotype. We cloned the GUN5 gene, which encodes the ChlH subunit of Mg-chelatase. We also show that gun2 and gun3 are alleles of the known photomorphogenic mutants, hy1 and hy2, which are required for phytochromobilin synthesis from heme. These findings suggest that certain perturbations of the tetrapyrrole biosynthetic pathway generate a signal from chloroplasts that causes transcriptional repression of nuclear genes encoding plastid-localized proteins. The comparison of mutant phenotypes of gun5 and another Mg-chelatase subunit (ChlI) mutant suggests a specific function for ChlH protein in the plastid-signaling pathway.

A novel gene, MEL1, mapped to 1p36.3 is highly homologous to the MDS1/EVI1 gene and is transcriptionally activated in t(1;3)(p36;q21)-positive leukemia cells.

The reciprocal translocation t(1;3)(p36;q21) occurs in a subset of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), which is frequently characterized by trilineage dysplasia, in particular dysmegakaryocytopoiesis, and poor prognosis. Previously, the breakpoint cluster region (BCR) at 3q21 was identified within a 60-kilobase (kb) region centromeric to the BCR of 3q21q26 syndrome and that at 1p36.3 within a 90-kb region. In this study, genes were searched near the breakpoints at 1p36.3, and a novel gene was isolated that encoded a zinc finger protein with a PR domain, which is highly homologous to the MDS1/EVI1 gene. The novel gene, designated as MEL1 (MDS1/EVI1-like gene 1), with 1257 amino acid residues is 64% similar in nucleotide and 63% similar in amino acid sequences to MDS1/EVI1 with the same domain structure. The MEL1 gene is expressed in leukemia cells with t(1;3) but not in other cell lines or bone marrow, spleen, and fetal liver, suggesting that MEL1 is specifically in the t(1;3)(p36;q21)-positive MDS/AML. On the basis of the positional relationship between the EVI1 and MEL1 genes in each translocation, it was suggested that both genes are transcriptionally activated by the translocation of the 3q21 region with the Ribophorin I gene. Because of the transcriptional activation of the EVI1 family genes in both t(1;3)(p36;q21)-positive MDS/AML and 3q21q26 syndrome, it is suggested that they share a common molecular mechanism for the leukemogenic transformation of the cells.

Antiplatelet and antithrombotic effects of a novel selective phosphodiesterase 3 inhibitor, NSP-513, in mice and rats.

We investigated the effects of NSP-513, (R)-4,5-dihydro-5-methyl-6-[4-(2-propyl-3-oxo-1-cyclohexenyl)amino] phenyl-3(2H)-pyridazinone, on phosphodiesterase (PDE) isozyme activities, in vitro platelet aggregation and in vivo thrombus formation. NSP-513 selectively inhibited human platelet PDE 3 isozyme with an IC50 value of 0.039 microM. In an in vitro human platelet aggregation assay, the IC50 values (microM) of NSP-513 for platelet aggregation induced by collagen, U-46619, arachidonic acid, adenosine diphosphate (ADP), epinephrine and thrombin were 0.31, 0.25, 0.082, 0.66, 0.23 and 0.73, respectively. In a mouse pulmonary thromboembolism model, orally administered NSP-513 showed in vivo antithrombotic effects that were 320 to 470 times more potent than those of cilostazol. In a rat carotid arterial thrombosis model, intraduodenally administered NSP-513 (0.1 mg/kg), cilostazol (30 mg/kg) and aspirin (30 mg/kg) reduced thrombus formation by 75%, 66% and 48%, respectively. However, intravenously administered dipyridamole (10 mg/kg) did not significantly prevent thrombus formation. These results demonstrate that NSP-513 has the potential to prevent not only in vitro platelet aggregation but also in vivo thrombus formation and indicate that the highly selective PDE 3 inhibitory effect of NSP-513 may make this compound useful for assessing the physiological role of PDE 3.

Rap2 as a slowly responding molecular switch in the Rap1 signaling cascade.

Rap2 is a member of the Ras family of GTPases and exhibits 60% identity to Rap1, but the function and regulation of Rap2 remain obscure. We found that, unlike the other Ras family proteins, the GTP-bound active form exceeded 50% of total Rap2 protein in adherent cells. Guanine nucleotide exchange factors (GEFs) for Rap1, C3G, Epac (or cyclic AMP [cAMP]-GEF), CalDAG-GEFI, PDZ-GEF1, and GFR efficiently increased the level of GTP-Rap2 both in 293T cells and in vitro. GTPase-activating proteins (GAPs) for Rap1, rap1GAPII and SPA-1, stimulated Rap2 GTPase, but with low efficiency. The half-life of GTP-Rap2 was significantly longer than that of GTP-Rap1 in 293T cells, indicating that low sensitivity to GAPs caused a high GTP/GDP ratio on Rap2. Rap2 bound to the Ras-binding domain of Raf and inhibited Ras-dependent activation of Elk1 transcription factor, as did Rap1. The level of GTP-Rap2 in rat 3Y1 fibroblasts was decreased by the expression of v-Src, and expression of a GTPase-deficient Rap2 mutant inhibited v-Src-dependent transformation of 3Y1 cells. Altogether, Rap2 is regulated by a similar set of GEFs and GAPs as Rap1 and functions as a slowly responding molecular switch in the Rap1 signaling cascade.

CalDAG-GEFIII activation of Ras, R-ras, and Rap1.

We characterized a novel guanine nucleotide exchange factor (GEF) for Ras family G proteins that is highly homologous to CalDAG-GEFI, a GEF for Rap1 and R-Ras, and to RasGRP/CalDAG-GEFII, a GEF for Ras and R-Ras. This novel GEF, referred to as CalDAG-GEFIII, increased the GTP/GDP ratio of Ha-Ras, R-Ras, and Rap1 in 293T cells. CalDAG-GEFIII promoted the guanine nucleotide exchange of Ha-Ras, R-Ras, and Rap1 in vitro also, indicating that CalDAG-GEFIII exhibited the widest substrate specificity among the known GEFs for Ras family G proteins. Expression of CalDAG-GEFIII was detected in the glial cells of the brain and the glomerular mesangial cells of the kidney by in situ hybridization. CalDAG-GEFIII activated ERK/MAPK most efficiently, followed by CalDAG-GEFII and CalDAG-GEFI in 293T cells. JNK activation was most prominent in cells expressing CalDAG-GEFII, followed by CalDAG-GEFIII and CalDAG-GEFI. Expression of CalDAG-GEFIII induced neuronal differentiation of PC12 cells and anchorage-independent growth of Rat1A cells less efficiently than did CalDAG-GEFII. Thus, co-activation of Rap1 by CalDAG-GEFIII apparently attenuated Ras-MAPK-dependent neuronal differentiation and cellular transformation. Altogether, CalDAG-GEFIII activated a broad range of Ras family G proteins and exhibited a biological activity different from that of either CalDAG-GEFI or CalDAG-GEFII.

Antithrombotic activity of NSP-513, a novel selective phosphodiesterase 3 inhibitor, on femoral arterial thrombosis induced by physical stenosis and electrical current: comparison of antithrombotic and hemodynamic effects.

NSP-513, a novel potent and selective phosphodiesterase 3 (PDE 3) inhibitor, and cilostazol, a previously developed PDE 3 inhibitor, were compared with respect to antiplatelet, antithrombotic, and hemodynamic effects. In the in vitro antiplatelet aggregation studies, NSP-513 and cilostazol inhibited collagen-induced canine platelet aggregation with median inhibitory concentration (IC50) values of 0.093 and 3.1 miccroM, respectively, and inhibited adenosine diphosphate (ADP)-induced canine platelet aggregation with IC50 values of 0.15 and 12 microM, respectively. For ADP-induced platelet aggregation, the presence of prostaglandin E1 (PGE1; 3 and 10 nM) further decreased the IC50 values for NSP-513 to 0.11 and 0.032 microM, respectively. In ex vivo antiplatelet aggregation studies, orally administered NSP-513 (0.03-1 mg/kg) and cilostazol (50 mg/kg) inhibited collagen-induced canine platelet aggregation. In an in vivo canine femoral arterial thrombosis model, intraduodenally administered NSP-513 (0.01-0.03 mg/ kg) dose-dependently prevented thrombus formation without any changes in blood pressure, heart rate, or bleeding time. In conscious dogs, NSP-513 at oral doses of > or =0.3 mg/kg produced hemodynamic changes such as decreased blood pressure and increased heart rate and LVdP/dt(max). Thus the minimal hemodynamically effective dose of NSP-513 was 0.3 mg/kg, and the hemodynamic effects of this dose were comparable to those of 50 mg/kg of cilostazol. In conclusion, these data suggest that NSP-513 has in vivo selectivity for antiplatelet and antithrombotic activities over hemodynamic activity, and that the selectivity of NSP-513 is higher than that of cilostazol in dogs.