A WXW Motif Is Required for the Anticancer Activity of the TAT-RasGAP317–326 Peptide

TAT-RasGAP_317–326, a cell-permeable 10-amino acid-long peptide derived from the N2 fragment of p120 Ras GTPase-activating protein (RasGAP), sensitizes tumor cells to apoptosis induced by various anticancer therapies. This RasGAP-derived peptide, by targeting the deleted in liver cancer-1 (DLC1) tumor suppressor, also hampers cell migration and invasion by promoting cell adherence and by inhibiting cell movement. Here, we systematically investigated the role of each amino acid within the RasGAP_317–326 sequence for the anticancer activities of TAT-RasGAP_317–326. We report here that the first three amino acids of this sequence, tryptophan, methionine, and tryptophan (WMW), are necessary and sufficient to sensitize cancer cells to cisplatin-induced apoptosis and to reduce cell migration. The WMW motif was found to be critical for the binding of fragment N2 to DLC1. These results define the interaction mode between the active anticancer sequence of RasGAP and DLC1. This knowledge will facilitate the design of small molecules bearing the tumor-sensitizing and antimetastatic activities of TAT-RasGAP_317–326.     

Mapacalcine Protects Mouse Neurons against Hypoxia by Blocking Cell Calcium Overload

Stroke is one of a major cause of death and adult disability. Despite intense researches, treatment for stroke remains reduced to fibrinolysis, a technique useful for less than 10% of patients. Finding molecules able to treat or at least to decrease the deleterious consequences of stroke is an urgent need. Here, we showed that mapacalcine, a homodimeric peptide purified from the marine sponge Cliona vastifica, is able to protect mouse cortical neurons against hypoxia. We have also identified a subtype of L-type calcium channel as a target for mapacalcine and we showed that the channel has to be open for mapacalcine binding. The two main L-type subunits at the brain level are CaV1.3 and CaV1.2 subunits but mapacalcine was unable to block these calcium channels.Mapacalcine did not interfere with N-, P/Q- and R-type calcium channels. The protective effect was studied by measuring internal calcium level variation triggered by Oxygen Glucose Deprivation protocol, which mimics stroke, or glutamate stimulation. We showed that NMDA/AMPA receptors are not involved in the mapacalcine protection. The protective effect was confirmed by measuring the cell survival rate after Oxygen Glucose Deprivation condition. Our data indicate that mapacalcine is a promising molecule for stroke treatment.     

Diffusion-Weighted Imaging Using a Readout-Segmented,Multishot EPI Sequence at 3 T Distinguishes between Morphologically Differentiated and Undifferentiated Subtypes of Thyroid Carcinoma—A Preliminary Study

BACKGROUND: Thyroid carcinomas represent the most frequent endocrine malignancies. Recent studies were able to distinguish malignant from benign nodules of the thyroid gland with diffusion-weighted imaging (DWI). Although this differentiation is undoubtedly helpful, presurgical discrimination between well-differentiated and undifferentiated carcinomas would be crucial to define the optimal treatment algorithm. Therefore, the aim of this study was to investigate if readout-segmented multishot echo planar DWI is able to differentiate between differentiated and undifferentiated subtypes of thyroid carcinomas. PATIENTS AND METHODS: Fourteen patients with different types of thyroid carcinomas who received preoperative DWI were included in our study. In all lesions, apparent diffusion coefficient (ADC)min, ADCmean, ADCmax, and D were estimated on the basis of region of interest measurements after coregistration with T1-weighted, postcontrast images. All tumors were resected and analyzed histopathologically. Ki-67 index, p53 synthesis, cellularity, and total and average nucleic areas were estimated using ImageJ version 1.48. RESULTS: Analysis of variance revealed a statistically significant difference in ADCmean values between differentiated and undifferentiated thyroid carcinomas (P = .022). Spearman Rho calculation identified significant correlations between ADCmax and cell count (r = 0.541, P = .046) as well as between ADCmax and total nuclei area (r = 0.605, P = .022). CONCLUSION: DWI can distinguish between differentiated and undifferentiated thyroid carcinomas.     

Role of mTOR,Bad, and Survivin in RasGAP Fragment N-Mediated Cell Protection

Partial cleavage of p120 RasGAP by caspase-3 in stressed cells generates an N-terminal fragment, called fragment N, which activates an anti-apoptotic Akt-dependent survival response. Akt regulates several effectors but which of these mediate fragment N-dependent cell protection has not been defined yet. Here we have investigated the role of mTORC1, Bad, and survivin in the capacity of fragment N to protect cells from apoptosis. Neither rapamycin, an inhibitor of mTORC1, nor silencing of raptor, a subunit of the mTORC1 complex, altered the ability of fragment N from inhibiting cisplatin- and Fas ligand-induced death. Cells lacking Bad, despite displaying a stronger resistance to apoptosis, were still protected by fragment N against cisplatin-induced death. Fragment N was also able to protect cells from Fas ligand-induced death in conditions where Bad plays no role in apoptosis regulation. Fragment N expression in cells did neither modulate survivin mRNA nor its protein expression. Moreover, the expression of cytoplasmic survivin, known to exert anti-apoptotic actions in cells, still occurred in UV-B-irradiated epidermis of mouse expressing a caspase-3-resistant RasGAP mutant that cannot produce fragment N. Additionally, survivin function in cell cycle progression was not affected by fragment N. These results indicate that, taken individually, mTOR, Bad, or Survivin are not required for fragment N to protect cells from cell death. We conclude that downstream targets of Akt other than mTORC1, Bad, or survivin mediate fragment N-induced protection or that several Akt effectors can compensate for each other to induce the pro-survival fragment N-dependent response.     

PyCogent: a toolkit for making sense from sequence

We have implemented in Python the COmparative GENomic Toolkit, a fully integrated and thoroughly tested framework for novel probabilistic analyses of biological sequences, devising workflows, and generating publication quality graphics. PyCogent includes connectors to remote databases, built-in generalized probabilistic techniques for working with biological sequences, and controllers for third-party applications. The toolkit takes advantage of parallel architectures and runs on a range of hardware and operating systems, and is available under the general public license from http://sourceforge.net/projects/pycogent.     

Estimation of an early meaningful time point of bone parameter changes in application to an osteoporotic rat model with in vivo microcomputed tomography measurements

The commonly used preclinical animal model of postmenopausal osteoporosis is the mature ovariectomized rat, whereby cessation of ovarian oestrogen production consequently results in bone volume reduction. The study aim was to precisely define the time course of structural changes resulting from ovariectomy and thereby reduce the time animals have to be treated to judge the effects of osteoporosis treatment. For this purpose, we assessed architectural changes by microcomputed tomography (μCT) during 10 weeks following ovariectomy or sham surgery at two-week intervals. Moreover, the trabecular microarchitecture of the lumbar vertebrae was assessed after necropsy. Besides this, serum biomarkers of bone turnover were determined. These data were in a new approach additionally correlated to femur mRNA expression profiles. We selected the osteoblast marker genes osteocalcin and type I collagen as well as the two osteoclast marker genes cathepsin k and tartrate-resistant acid phosphatase 5. The gene expression analysis suggested an activation of osteoblasts as well as octeoclasts. The significantly induced serum levels of osteocalcin and collagen degradation fragments also revealed this higher rate of bone turnover. Our results indicate that as soon as four weeks after ovariectomy the bone volume fraction exhibited a decline of 30% and 50% of the connectivity density. In addition, significant decreases of trabecular number and thickness as well as of the bone volume fraction were only observed in vertebrae of ovariectomized animals. Interestingly, changes of trabecular morphology were also found in the sham animals as a consequence of senescence.     

Caspase-3 Protects Stressed Organs against Cell Death

The ability to generate appropriate defense responses is crucial for the survival of an organism exposed to pathogenesis-inducing insults. However, the mechanisms that allow tissues and organs to cope with such stresses are poorly understood. Here we show that caspase-3-knockout mice or caspase inhibitor-treated mice were defective in activating the antiapoptotic Akt kinase in response to various chemical and environmental stresses causing sunburns, cardiomyopathy, or colitis. Defective Akt activation in caspase-3-knockout mice was accompanied by increased cell death and impaired survival in some cases. Mice homozygous for a mutation in RasGAP that prevents its cleavage by caspase-3 exhibited a similar defect in Akt activation, leading to increased apoptosis in stressed organs, marked deterioration of their physiological functions, and stronger disease development. Our results provide evidence for the relevance of caspase-3 as a stress intensity sensor that controls cell fate by either initiating a RasGAP cleavage-dependent cell resistance program or a cell suicide response.     

LDLs stimulate p38 MAPKs and wound healing through SR-BI independently of Ras and PI3 kinase

Intracellular signals elicited by LDLs are likely to play a role in the pathogenesis associated with increased LDL blood levels. We have previously determined that LDL stimulation of human skin fibroblasts, used as a model system for adventitial fibroblasts, activates p38 mitogen-activated protein kinases (MAPKs), followed by IL-8 production and increased wound-healing capacity of the cells. The proximal events triggering these responses had not been characterized, however. Here we show that MAPK kinases MKK3 and MKK6, but not MKK4, are the upstream kinases responsible for the activation of the p38 MAPKs and stimulation of wound closure in response to LDLs. Phosphoinositide 3 kinases (PI3Ks) and Ras have been suggested to participate in lipoprotein-induced MAPK activation. However, specific PI3K inhibitors or expression of a dominant-negative form of Ras failed to blunt LDL-induced p38 MAPK activation. The classical LDL receptor does not participate in LDL signaling, but the contribution of other candidate lipoprotein receptors has not been investigated. Using cells derived from scavenger receptor class B type I (SR-BI) knockout mice or the BLT-1 SR-BI inhibitor, we now show that this receptor is required for LDLs to stimulate p38 MAPKs and to promote wound healing. Identification of MKK3/6 and SR-BI as cellular relays in LDL-mediated p38 activation further defines the signaling events that could participate in LDL-mediated pathophysiological responses.