A 90-day oral (dietary) toxicity study of arruva, an R, R-monatin salt isomer, in Crl:CD-1(ICR) mice.

R,R-Monatin [2R,4R- isomer of 2-hydroxy-2-(indol-3-ylmethyl)-4-aminoglutaric acid] is one of four natural constituent isomers in the root bark of Sclerochitin ilicifolius; and "arruva" is the common/usual name that is proposed to represent R,R-monatin salt forms, which have potential use as high potency sweetener food ingredients. In the present study, groups of male and female Crl:CD-1(ICR) mice were exposed to 0 (control), 5000, 10,000, 20,000, or 35,000ppm of arruva in the diet for 90days. There were no toxicologically relevant clinical or histopathological findings in any of the test article-treated groups. Significantly lower mean body weights and cumulative body weight gains were noted in the 35,000ppm group when compared to the control group. Mean body weights in the 35,000ppm group males and females were 9% and 7% less than the control group, respectively, at week 13. In the absence of observations associated with systemic toxicity and in consideration of the magnitude of body weight difference, these effects were not considered toxicologically significant. Based on the results of this study, the dietary no-observed-adverse-effect level (NOAEL) of arruva for 90days in male and female mice was 35,000ppm (equivalent to an exposure level of 5764 and 8013mg/kg bw/day, respectively).

A 90-day oral (dietary) toxicity study of the 2R,4R-isomer of monatin salt in Sprague-Dawley rats.

The root bark of Sclerochitin ilicifolius contains an intensely sweet substance analytically identified as isomers of 2-hydroxy-2-(indol-3-ylmethyl)-4-aminoglutaric acid and generically coined "monatin." Groups of male and female Crl:CD(SD) rats were fed 0 (control), 5000, 10,000, 20,000 or 35,000 ppm R,R-monatin salt in the diet for 90 days. There were no toxicologically relevant clinical or histopathological findings in any of the test article-treated groups. Significantly lower cumulative body weight gains were noted in the 35,000 ppm group. Mean body weights in the 35,000 ppm group males and females were 7% and 12% lower, respectively, than the control group at study week 13. In the absence of other observations associated with systemic toxicity and lower food consumption, the magnitude of the body weight difference in the 35,000 ppm group females relative to the control group exceeded 10%, which indicated attainment of a maximum tolerated dose (MTD) level. Based on the results of this study, and conservatively assuming the body weight observations at the MTD to be indicative of an adverse effect, the dietary no-observed-adverse-effect level (NOAEL) of R,R-monatin salt for 90 days was 20,000 ppm in female rats (approximately 1544 mg/kg bw/day) and 35,000 ppm in male rats (approximately 2368 mg/kg bw/day).

Dna damage-induced G(1) arrest in hematopoietic cells is overridden following phosphatidylinositol 3-kinase-dependent activation of cyclin-dependent kinase 2.

Exposure of hematopoietic cells to DNA-damaging agents induces p53-independent cell cycle arrest at a G(1) checkpoint. Previously, we have shown that this growth arrest can be overridden by cytokine growth factors, such as erythropoietin or interleukin-3, through activation of a phosphatidylinositol 3-kinase (PI 3-kinase)/Akt-dependent signaling pathway. Here, we show that gamma-irradiated murine myeloid 32D cells arrest in G(1) with active cyclin D-cyclin-dependent kinase 4 (Cdk4) but with inactive cyclin E-Cdk2 kinases. The arrest was associated with elevated levels of the Cdk inhibitors p21(Cip1) and p27(Kip1), yet neither was associated with Cdk2. Instead, irradiation-induced inhibition of cyclin E-Cdk2 correlated with absence of the activating threonine-160 phosphorylation on Cdk2. Cytokine treatment of irradiated cells induced Cdk2 phosphorylation and activation, and cells entered into S phase despite sustained high-level expression of p21 and p27. Notably, the PI 3-kinase inhibitor, LY294002, completely blocked cytokine-induced Cdk2 activation and cell growth in irradiated 32D cells but not in nonirradiated cells. Together, these findings demonstrate a novel mechanism underlying the DNA damage-induced G(1) arrest of hematopoietic cells, that is, inhibition of Cdk2 phosphorylation and activation. These observations link PI 3-kinase signaling pathways with the regulation of Cdk2 activity.

DNA damage-induced cell-cycle arrest of hematopoietic cells is overridden by activation of the PI-3 kinase/Akt signaling pathway.

Exposure of hematopoietic cells to DNA-damaging agents induces cell-cycle arrest at G1 and G2/M checkpoints. Previously, it was shown that DNA damage-induced growth arrest of hematopoietic cells can be overridden by treatment with cytokine growth factors, such as erythropoietin (EPO) or interleukin-3 (IL-3). Here, the cytokine-activated signaling pathways required to override G1 and G2/M checkpoints induced by gamma-irradiation (gamma-IR) are characterized. Using factor-dependent myeloid cells stably expressing EPO receptor (EPO-R) mutants, it is shown that removal of a minimal domain required for PI-3K signaling abrogated the ability of EPO to override gamma-IR-induced cell-cycle arrest. Similarly, the ability of cytokines to override gamma-IR-induced arrest was abolished by an inhibitor of PI-3K (LY294002) or by overexpression of dominant-negative Akt. Moreover, the ability of EPO to override these checkpoints in cells expressing defective EPO-R mutants could be restored by overexpression of a constitutively active Akt. Thus, activation of a PI-3K/Akt signaling pathway is required for cytokine-dependent suppression of DNA-damage induced checkpoints. Together, these findings suggest a novel role for PI-3K/Akt pathways in the modulation of growth arrest responses to DNA damage in hematopoietic cells. (Blood. 2001;98:834-841)

A truncated form of RGS3 negatively regulates G protein-coupled receptor stimulation of adenylyl cyclase and phosphoinositide phospholipase C.

Identification of a new family of proteins (RGS proteins) that function as negative regulators of G protein signaling has sparked new understanding of desensitization of this signaling process. Recent studies with several mammalian RGS proteins has delineated their ability to interact with and function as GTPase-activating proteins specifically for G proteins in the Gi family. Here, we investigated the functional activity of RGS3 and a truncated form of RGS3 on G protein-coupled receptor-mediated activation of adenylyl cyclase, phosphoinositide phospholipase C, and mitogen-activated protein kinase in intact cells. Polymerase chain reaction and 5'-rapid amplification of cDNA ends analyses revealed the tissue-specific expression of a short form of the RGS3 transcript that encodes the approximate carboxyl-terminal half of RGS3. This truncated form of RGS3 (RGS3T) was shown recently to function as a negative regulator of pheromone signaling in yeast (Druey, K. M., Blumer, K. J., Kang, V. R., and Kehrl, J. H. (1996) Nature 379, 742-746). Baby hamster kidney cells transiently transfected with RGS3T cDNA exhibited a pronounced impairment in platelet-activating factor receptor-stimulated inositol phosphate production, a pertussis toxin-insensitive response. Similarly, calcitonin gene-related peptide receptor-stimulated increases in intracellular cAMP and pituitary adenylate-cyclase activating polypeptide receptor-stimulated increases in both cAMP and inositol phosphates were reduced significantly in RGS3T transfectants compared with vector-transfected control cells. In contrast, baby hamster kidney cells transfected with the full-length RGS3 cDNA showed no impairment in cAMP and inositol phosphate production mediated by these G protein-coupled receptors. However, lysophosphatidic acid receptor-stimulated phosphorylation of endogenous ERK1 and ERK2 was impaired markedly in both RGS3 and RGS3T transfectants, demonstrating the functional ability of both RGS forms to modulate Gi-mediated signaling. These results provide the first evidence for regulatory effects of an RGS protein on Gs- and Gq-mediated signaling in intact cells and document that the carboxyl-terminal region of RGS3 comprises the structural domain for this activity.