Potential Hypocholesterolemic Activity of Flour from Leaves of Moringa (Moringa oleifera L.)

Introduction: Moringa (Moringa oleifera L.) leaves contain phytosterols and dietary fibres which may be beneficial in controlling blood cholesterol levels. This study was aimed at assessing the hypocholesterolemic effect of flour from leaves of M. oleifera L. (MLF) with white and red stalk in rats. Methods: Thirty male rats were divided into 6 groups, comprising a normal group (negative control), a hypercholestrolemic group (positive control) both of which were without MLF feeding, and 4 hypercholesterolemic groups fed MLF for 4 weeks in the following manner: (i) 0.822 mg/g bw/d white stalk (WM); (ii) 0.822 mg/g bw/d red stalk (RM); (iii) 0.02 ml/g bw/d commercial plant stanol ester (FS); and (iv) 0.001 mg/g bw/d ezetimibe (ET). At the end, serum total cholesterol (TC) and low density lipoprotein cholesterol LDL-c), viscosity and pH of digesta, faecal cholesterol, and short chain fatty acids (SCFAs) were analysed. Results: TC levels in the WM, RM, FS and ET groups decreased by 42.0, 48.8, 48.4 and 52.8% respectively, compared to initial levels. The four groups also showed decreases in serum LDL-c levels by 30.3, 39.2, 37.9 and 46.7% respectively, over the feeding period. Faecal cholesterol levels of WM and RM were higher (63.93?±1.87 and 90.11?±1.77 mg/100 g faeces, respectively) than that of the positive control (51.30?±4.03 mg/100 g) after 4 weeks. Conclusion: Flour from moringa leaves of white and red stalk trees showed potential hypocholesterolemic activity in ra

Plasma CRH Level Difference Between Wistar Rats Exposed To Acute Stress Due To Predator And To The Psychological Stress Device

Objective: Stress triggers and causes psychiatric disorders. This study compared stress generated by different stressors: a cat as the predator of rats and a Psychological Stress Device (PSD) which was developed and modified by the researchers based on the model by Xu and Rocher. Methods: Twenty-eight Wistar rats were simple randomly divided into one control group and six treatment groups, each consisting of 4 rats. Each treatment group was individually exposed to stressor for 30, 60, and 90 minutes. The first three treatment groups were treated using the PSD while the other three treatment groups were treated exposed to the cat. Plasma CRH level was measured using the ELISA (Cusabio) method. Result: Plasma CRH levels in the rat exposed to stressor using the PSD ranged from 9.89 to 50.22 ng/mL, higher than plasma CRH level in the groups exposed to cat ranged from 0.22 to 23.44 ng/mL with significance level (p0.05). Conclusion: Plasma CRH level of the rats exposed to stressor using the PSD was higher and positively correlate with the length of exposure compared to those exposed to cat.

Different roles for the TOS and RAIP motifs of the translational regulator protein 4E-BP1 in the association with raptor and phosphorylation by mTOR in the regulation of cell size.

The translational regulator protein 4E-BP1, that binds to eukaryotic initiation factor-4E (eIF4E) to prevent the formation of the active translation complex, dissociates from eIF4E by phosphorylation through the mammalian target of rapamycin (mTOR) in the cells stimulated by amino acids. 4E-BP1 has been shown to associate with the scaffold protein raptor through its TOS and RAIP motifs to be recognized by mTOR. We revealed that the TOS motif mutant was phosphorylated by mTOR only at the priming sites of Thr37/46 but the RAIP motif mutant was phosphorylated not only at the priming sites but also at the subsequent site of Thr70 in vitro and in response to amino acid treatment in HEK293 cells. Analysis using the phosphorylation site mutants indicated that phosphorylation of the priming and subsequent sites of 4E-BP1 was required for dissociation from raptor as well as for the release of eIF4E. The expression of the 4E-BP1 mutants replacing the TOS motif and phosphorylation sites, that are poor substrates for mTOR and have no or little dissociation ability from raptor and eIF4E, respectively, significantly reduced the size of K562 cells. These results indicate that the the TOS motif has an essential function whereas the RAIP motif has an accessory role in the association with raptor and mTOR-mediated phosphorylation of 4E-BP1 to dissociate it from raptor and release eIF4E in response to amino acid stimulation leading to the control of cell size.

Suppression of the mTOR-raptor signaling pathway by the inhibitor of heat shock protein 90 geldanamycin.

Heat shock protein 90 (Hsp90) was co-immunoprecipitated with raptor, the binding partner of the mammalian target of rapamycin (mTOR) from HEK293 cells. Hsp90 was detected in the anti-raptor antibody immunoprecipitates prepared from the cell extract by immunoblot analysis using the anti-Hsp90 antibody, and the association of these two proteins was confirmed by immunoprecipitation from the cells co-expressing Hsp90 and raptor as epitope-tagged molecules. Geldanamycin, a potent inhibitor of Hsp90, disrupted the in vivo binding of Hsp90 to raptor without affecting the association of raptor and mTOR, and suppressed the phosphorylation by mTOR of the downstream translational regulators p70 S6 kinase (S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). The protein kinase activity of S6K as well as the phosphorylation of the substrate, 40S ribosomal protein S6, were lowered in the geldanamycin-treated cells. These results indicate that Hsp90 is involved in the regulation of protein translation by facilitating the phosphorylation reaction of 4E-BP1 and S6K catalyzed by the mTOR/raptor complex through the association with raptor, and that the mTOR signaling pathway is a novel target of geldanamycin.

Dissociation of raptor from mTOR is a mechanism of rapamycin-induced inhibition of mTOR function.

The mammalian target of rapamycin (mTOR) is a Ser/Thr protein kinase that plays a crucial role in a nutrient-sensitive signalling pathway that regulates cell growth. TOR signalling is potently inhibited by rapamycin, through the direct binding of a FK506-binding protein 12 (FKBP12)/rapamycin complex to the TOR FRB domain, a segment amino terminal to the kinase catalytic domain. The molecular basis for the inhibitory action of FKBP12/rapamycin remains uncertain. Raptor (regulatory associated protein of mTOR) is a recently identified mTOR binding partner that is essential for mTOR signalling in vivo, and whose binding to mTOR is critical for mTOR-catalysed substrate phosphorylation in vitro. Here we investigated the stability of endogenous mTOR/raptor complex in response to rapamycin in vivo, and to the direct addition of a FKBP12/rapamycin complex in vitro. Rapamycin diminished the recovery of endogenous raptor with endogenous or recombinant mTOR in vivo; this inhibition required the ability of mTOR to bind the FKBP12/rapamycin complex, but was independent of mTOR kinase activity. Rapamycin, in the presence of FKBP12, inhibited the association of raptor with mTOR directly in vitro, and concomitantly reduced the mTOR-catalysed phosphorylation of raptor-dependent, but not raptor-independent substrates; mTOR autophosphorylation was unaltered. These observations indicate that rapamycin inhibits mTOR function, at least in part, by inhibiting the interaction of raptor with mTOR; this action uncouples mTOR from its substrates, and inhibits mTOR signalling without altering mTOR's intrinsic catalytic activity.

Inhibition of amino acid-mTOR signaling by a leucine derivative induces G1 arrest in Jurkat cells.

We have previously demonstrated that N-acetylleucine amide, a derivative of L-leucine, inhibits leucine-induced p70(S6k) activation in a rat hepatoma cell line. In the present study, we investigated whether N-acetylleucine amide is capable of inhibiting amino acid-mTOR signaling. N-Acetylleucine amide caused cell cycle arrest at G1 stage in Jurkat cells, a human leukemia T cell line, concomitant with the inhibition of serum-induced p70(S6k) activation and p27 degradation. Treatment of Jurkat cells with this compound also exhibited dephosphorylation of retinoblastoma protein. These effects are similar to the inhibitory effects of rapamycin on amino acid-mTOR signaling pathway and suggest that N-acetylleucine amide acts as a rapamycin-like reagent to inhibit cell cycle progression in Jurkat cells.

The mammalian target of rapamycin (mTOR) partner, raptor, binds the mTOR substrates p70 S6 kinase and 4E-BP1 through their TOR signaling (TOS) motif.

The mammalian target of rapamycin (mTOR) controls multiple cellular functions in response to amino acids and growth factors, in part by regulating the phosphorylation of p70 S6 kinase (p70S6k) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). Raptor (regulatory associated protein of mTOR) is a recently identified mTOR binding partner that also binds p70S6k and 4E-BP1 and is essential for TOR signaling in vivo. Herein we demonstrate that raptor binds to p70S6k and 4E-BP1 through their respective TOS (conserved TOR signaling) motifs to be required for amino acid- and mTOR-dependent regulation of these mTOR substrates in vivo. A point mutation of the TOS motif also eliminates all in vitro mTOR-catalyzed 4E-BP1 phosphorylation and abolishes the raptor-dependent component of mTOR-catalyzed p70S6k phosphorylation in vitro. Raptor appears to serve as an mTOR scaffold protein, the binding of which to the TOS motif of mTOR substrates is necessary for effective mTOR-catalyzed phosphorylation in vivo and perhaps for conferring their sensitivity to rapamycin and amino acid sufficiency.

Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action.

mTOR controls cell growth, in part by regulating p70 S6 kinase alpha (p70alpha) and eukaryotic initiation factor 4E binding protein 1 (4EBP1). Raptor is a 150 kDa mTOR binding protein that also binds 4EBP1 and p70alpha. The binding of raptor to mTOR is necessary for the mTOR-catalyzed phosphorylation of 4EBP1 in vitro, and it strongly enhances the mTOR kinase activity toward p70alpha. Rapamycin or amino acid withdrawal increases, whereas insulin strongly inhibits, the recovery of 4EBP1 and raptor on 7-methyl-GTP Sepharose. Partial inhibition of raptor expression by RNA interference (RNAi) reduces mTOR-catalyzed 4EBP1 phosphorylation in vitro. RNAi of C. elegans raptor yields an array of phenotypes that closely resemble those produced by inactivation of Ce-TOR. Thus, raptor is an essential scaffold for the mTOR-catalyzed phosphorylation of 4EBP1 and mediates TOR action in vivo.