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Background: Dried blood spot (DBS) testing is a well-known method of bio-sampling by which blood samples are blotted and dried on filter paper. The dried samples can then be analyzed by several techniques such as DNA amplification and HPLC. We have developed a homemade DBS method followed by an alternative protocol for genomic DNA extraction from a drop of blood adsorbed on paper support. This protocol consists of two separate steps: (1) organic DNA extraction from the DBS, followed by (2) DNA amplification by polymerase chain reaction (PCR). The PCR-restriction fragment length polymorphism (PCR-RFLP) is an advantageous and simple approach to detect single nucleotide polymorphisms (SNPs). Results: We have evaluated the efficiency of our method for the extraction of genomic DNA from DBS by testing its performance in genotyping mouse models of obesity and herein discuss the sensitivity, specificity and feasibility of this novel procedure. Conclusions: Our protocol is easy to perform, fast and inexpensive and allows the isolation of pure DNA from a miniscule amount of sample.

Ras signaling is involved in the development of autoimmunity in general. Multiple sclerosis (MS) is a T cell-mediated autoimmune disease of the central nervous system. It is widely recognized that a reduction of Foxp3+ regulatory T (Treg) cells is an immunological hallmark of MS, but the underlying mechanisms are unclear. In experimental autoimmune models, N-Ras and K-Ras inhibition triggers an anti-inflammatory effect up-regulating, via foxp3 elevation, the numbers and the functional suppressive properties of Tregs. Similarly, an increase in natural Tregs number during Experimental Autoimmune Encephalomyelitis (EAE) in R-RAS -/- mice results in attenuated disease. In humans, only KRAS GTPase isoform is involved in mechanism causing tolerance defects in rheumatoid arthritis (RA). T cells from these patients have increased transcription of KRAS (but not NRAS). RAS genes are major drivers in human cancers. Consequently, there has been considerable interest in developing anti-RAS inhibitors for cancer treatment. Despite efforts, no anti-RAS therapy has succeeded in the clinic. The major strategy that has so far reached the clinic aimed to inhibit activated Ras indirectly through blocking its post-translational modification and inducing its mis-localization. The disappointing clinical outcome of Farnesyl Transferase Inhibitors (FTIs) in cancers has decreased interest in these drugs. However, FTIs suppress EAE by downregulation of myelin-reactive activated T-lymphocytes and statins are currently studied in clinical trials for MS. However, no pharmacologic approaches to targeting Ras proteins directly have yet succeeded. The therapeutic strategy to recover immune function through the restoration of impaired Tregs function with the mounting evidences regarding KRAS in autoimmune mediated disorder (MS, SLE, RA, T1D) suggest as working hypothesis the direct targeting KRAS activation using cancer-derived small molecules may be clinically relevant. Abbreviations FTIs: Farnesyl Transferase Inhibitors; MS: Multiple Sclerosis; RRMS: Relapsing Remitting Multiple Sclerosis; PPMS: Primary Progressive Multiple Sclerosis; Tregs: regulatory T-cells; Foxp3: Forkhead box P3; EAE: Experimental Autoimmune Encephalomyelitis; T1D: Type 1 Diabete; SLE: Systemic Lupus Erythematosus; RA: Rheumatoid Arthritis; CNS: Central Nervous System; TMEV: Theiler's murine encephalomyelitis virus; FTS: farnesyl thiosalicylic acid; TCR: T-Cell Receptor; AIA: Adjuvant-induced Arthritis; EAN: experimental autoimmune neuritis; HVR: hypervariable region; HMG-CoA: 3-hydroxy-3-methylglutaryl coenzyme A reductase; PBMC: Peripheral Blood Mononuclear Cells.

[This corrects the article DOI: 10.1371/journal.pgen.0030110.].

Astrocytes, one of the predominant types of glial cells, function as both supportive and metabolic cells for the brain. Among mammalian tissues, the highest levels of p21Ras protein are detected in the brain. Here, we investigated the expression of KRAS and HRAS proto-oncogenes in primary astrocytes following acute oxidative stimulation. Reactive oxygen species (ROS) changed the expression of proto-oncogenes at both transcriptional and translational levels. De novo protein synthesis analysis measured approximate values of proteins half-life, ranging from 1–4 h, of the different H- and K- isoforms by western blot analysis. Quantitative gene expression analysis of KRAS and HRAS revealed an unexpected short-term induction of KRAS mRNA in primary astrocytes in response to acute stimulation. Indeed, cultured astrocytes responded to proteasomal inhibition by preventing the reduction of c-K-Ras. A fraction of K-Ras protein accumulated in the presence of ROS and cycloheximide, while a substantial proportion was continuously synthesized. These data indicate that ROS regulate in a complementary fashion p21Ras isoforms in primary astrocytes: K-Ras is rapidly and transiently induced by post-translational and post-transcriptional mechanisms, while H-Ras is stably induced by mRNA accumulation. We suggest that K-Ras and H-Ras are ROS sensors that adapt cells to metabolic needs and oxidative stress.

Addition of hydrogen peroxide to cultured astrocytes induced a rapid and transient increase in the expression of Ha-Ras and Ki-Ras. Pull-down experiments with the GTP-Ras-binding domain of Raf-1 showed that oxidative stress substantially increased the activation of Ha-Ras, whereas a putative farnesylated activated form of Ki-Ras was only slightly increased. The increase in both Ha-Ras and Ki-Ras was insensitive to the protein synthesis inhibitor, cycloheximide, and was occluded by the proteasomal inhibitor, MG-132. In addition, exposure to hydrogen peroxide reduced the levels of ubiquitinated Ras protein, indicating that oxidative stress leads to a reduced degradation of both isoforms through the ubiquitin/proteasome pathway. Indeed, the late reduction in Ha-Ras and Ki-Ras was due to a recovery of proteasomal degradation because it was sensitive to MG-132. The late reduction of Ha-Ras levels was abrogated by compound PD98059, which inhibits the MAP kinase pathway, whereas the late reduction of Ki-Ras was unaffected by PD98059. We conclude that oxidative stress differentially regulates the expression of Ha-Ras and Ki-Ras in cultured astrocytes, and that activation of the MAP kinase pathway by oxidative stress itself or by additional factors may act as a fail-safe mechanism limiting a sustained expression of the potentially detrimental Ha-Ras.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that activates Src family kinases via SH2- and SH3-mediated interactions. Specific FAK isoforms (FAK+), responsive to depolarization and neurotransmitters, are enriched in neurons. We analyzed the interactions of endogenous FAK+ and recombinant FAK+ isoforms containing amino acid insertions (boxes 6,7,28) with an array of SH3 domains and the c-Src SH2/SH3 domain tandem. Endogenous FAK+ bound specifically to the SH3 domains of c-Src (but not n-Src), Fyn, Yes, phosphtidylinositol-3 kinase, amphiphysin II, amphiphysin I, phospholipase Cγ and NH2-terminal Grb2. The inclusion of boxes 6,7 was associated with a significant decrease in the binding of FAK+ to the c-Src and Fyn SH3 domains, and a significant increase in the binding to the Src SH2 domain, as a consequence of the higher phosphorylation of Tyr-397. The novel interaction with the amphiphysin SH3 domain, involving the COOH-terminal proline-rich region of FAK, was confirmed by coimmunoprecipitation of the two proteins and a closely similar response to stimuli affecting the actin cytoskeleton. Moreover, an impairment of endocytosis was observed in synaptosomes after internalization of a proline-rich peptide corresponding to the site of interaction. The data account for the different subcellular distribution of FAK and Src kinases and the specific regulation of the transduction pathways linked to FAK activation in the brain and implicate FAK in the regulation of membrane trafficking in nerve terminals.

We describe the use of phage libraries to derive new antibodies against p21Ras to be used for intracellular expression in mammalian cells. A panel of single-chain antibody fragments, binding to Ras, were analyzed and characterized for their capacity to interfere in vitro with (a) the intrinsic GTPase activity of Ras and (b) the binding of Ras to its effector Raf, and were found not to neutralize its function, according to these biochemical criteria. When expressed intracellularly in mouse 3T3 K-Ras transformed cells all the anti-Ras single-chain variable fragments (scFv) tested inhibited cell proliferation, as assessed by bromodeoxyuridine incorporation. Double immunofluorescence analysis of transfected cells using confocal microscopy confirmed that anti-Ras antibody fragments colocalize with endogenous Ras, at subcellular locations where the protein Ras is not normally found. These data suggest that the ability of phage-derived anti-Ras scFv fragments to inhibit the function of Ras in vivo is a rather general and frequent property and that the range of antibodies that can be successfully used for intracellular inhibition studies is much greater than anticipated, exploiting the mode of action of diverting protein traffic.

Dual-specificity phosphatase 6 (DUSP6, mitogen-activated protein kinase (MAPK) phosphatase 3 or PYST1) dephosphorylates phosphotyrosine and phosphothreonine residues on extracellular signal-regulated kinase (ERK1/2; MAPK1/2) to inactivate the ERK1/2 kinase. DUSP6 is a critical regulator of the ERK signaling cascade and has been implicated as a tumor suppressor. We report here experimental evidences that DUSP6 is transcriptionally upregulated in primary and long-term cultures of human glioblastoma, as assayed by northern hybridization and real-time quantitative PCR, producing constitutive high level of protein expression. Functional assays were performed with adenovirus-mediated expression of DUSP6 in glioblastoma cultures. Protein overexpression inhibits growth by inducing G1-phase delay and increased mitogenic/anchorage dependence and clonogenic potential in vitro. Changes in cell morphology were associated with an increased tumor growth in vivo. Chemoresistance is a major cause of treatment failure and poor outcome in human glioblastomas. Importantly, DUSP6 overexpression increased resistance to cisplatin-mediated cell death in vitro and in vivo. Antisense-mediated depletion of DUSP6 acted in lowering the threshold to anticancer DNA-damaging drugs. We conclude that upregulation of DUSP6 exerts a tumor-promoting role in human glioblastomas exacerbating the malignant phenotype.

Trophic deprivation contributes to astrocyte damage that oc- curs in acute and chronic neurodegenerative disorders. Unraveling the underlying mechanisms may pave way to no- vel cytoprotective strategies. Cultured mouse astrocytes re- sponded to trophic deprivation with a large and transient increase in the expression of p21ras, which was secondary to an enhanced formation of reactive oxygen species (ROS) detected by cytofluorimetric analysis after preloading with 2¢,7¢-dichlorofluorescein diacetate. The increase in p21ras levels was largely attenuated by the reducing agent, N-ace- tylcysteine, which was proven to reduce ROS formation in astrocytes subjected to serum deprivation. We extended the analysis to the Ha-Ras isoform, which has been implicated in mechanisms of cytotoxicity. We found that serum deprivation enhanced the expression and activity of Ha-Ras without changing Ha-Ras mRNA levels. The increase in Ha-Ras lev- els was sensitive to the protein synthesis inhibitor, cyclohex- The mechanisms underlying astrocyte damage are largely unexplored, although this process is relevant to the patho- physiology of acute and chronic neurodegenerative disorders (Bridges et al. 1992; Le Prince et al. 1995; Bruijn et al. 1997; Rafałowska and Podlecka 1998; Lee et al. 1999; Mouser et al. 2006). Unraveling these mechanisms might offer new targets for therapeutic intervention. Activation of P2Y1 receptors, for example, has been shown to protect astrocytes against oxidative damage (Shinozaki et al. 2005). We focused on the small monomeric G protein, p21ras, which is a key regulator of the signaling cascade triggered by oxidative stress (Cuda et al. 2002) and is also a target for reactive oxygen species (ROS) and cellular redox stress (Cheng and Meinkoth 2001). Ras-expressing cells produce imide, suggesting that serum deprivation increases translation of preformed Ha-Ras mRNA. The late decline in Ha-Ras levels observed after 60 min was prevented by the protea- some inhibitor, MG132, as well as by the selective mitogen- activated protein kinase (MAPK) inhibitor, PD98059. Serum deprivation led to the activation of the MAPK pathway in cul- tured astrocytes, as shown by an increase in phosphorylated extracellular signal-regulated kinase 1/2 levels after 5 and 30 min. Finally, using the siRNA technology, we found that an acute knock-down of Ha-Ras was protective against astrocyte damage induced by serum deprivation. We conclude that cultured astrocytes respond to trophic deprivation with an in- creased expression in Ha-Ras, which is limited by the con- comitant activation of the MAPK pathway, but is nevertheless involved in the pathophysiology of cell damage.

Phosphatidylinositol 3-kinase (PI3K) is necessary for thyroid stimulating hormone (TSH)-induced cell cycle progression. To determine the molecular mechanism linking PI3K to TSH, we have identified a serine residue in p85alpha(PI3K) phosphorylated by protein kinase A (PKA) in vitro and in vivo. Expression of an alanine mutant (p85A) abolished cyclic AMP/TSH-induced cell cycle progression and was lethal in thyroid cells (FRTL-5). The aspartic version of the p85alpha(PI3K) (p85D) inhibited apoptosis following TSH withdrawal. The p85alpha(PI3K) wild type not the p85A bound PKA regulatory subunit RIIbeta in cells stimulated with cAMP or TSH. The binding of the aspartic version of p85alpha(PI3K) to RIIbeta was independent of cAMP or TSH stimulation. Similarly, binding of PI3K to p21Ras and activation of AKT, a downstream PI3K target, were severely impaired in cells expressing the p85A mutant. Finally, we found that the catalytic activity of PI3K was stimulated by TSH in cells expressing the wild-type p85alpha(PI3K) but not in cells expressing p85A. This latter mutant did not affect the epidermal growth factor-stimulated PI3K activity. We suggest that (1) TSH-cAMP-induced PKA phosphorylates p85alpha(PI3K) at serine 83, (2) phosphorylated p85alpha(PI3K) binds RIIbeta-PKA and targets PKAII to the membrane, and (3) PI3K activity and p21Ras binding to PI3K increase and activate PI3K downstream targets. This pathway is essential for the transmission of TSH-cAMP growth signals.

Resistance to chemotherapy is a common feature of malignant gliomas. This resistance is mediated by receptor tyrosine kinase (RTK)-regulated signaling. p21-Ras protein is pivotal in the propagation of the signal originated from many RTKs. Our aim was to investigate whether inhibition of Ras pathway affects the response to cisplatin in malignant gliomas. We found an enhanced sensitivity to cisplatin of two glioblastoma cell lines expressing dominant negative Ras. Moreover, DN-Ras expressing cells, implanted in nude mice, resulted in being extremely sensitive to cisplatin. The growth of all the tumors was significantly inhibited by combining DN-Ras adenovirus infection with cisplatin treatment. The majority of glioma cells expressing DN-Ras underwent apoptosis in response to cisplatin. In vivo, DN-Ras alone did not influence the growth of tumors, suggesting that the effects of Ras-inhibition observed in vitro could not be extrapolated in vivo. The survival signal pathway transduced by Ras was essentially mediated by inhibition of caspase-9 cleavage via PI3K/Akt.

TSH activates its specific receptor in thyroid cells and induces cAMP, a robust stimulator of thyroid cell proliferation. Conversely, cAMP is a potent inhibitor of growth in mouse fibroblasts. To dissect the signals mediating cAMP-dependent growth, we have expressed in mouse fibroblasts the human thyrotropin receptor (TSHR) or a constitutively active mutant, under the control of the tetracyclin promoter. Both TSHR and cAMP levels were modulated by tetracyclin. In the presence of serum, activation of TSHR by TSH induced growth arrest. In the absence of serum, cells expressing TSHR stimulated with TSH, replicated their DNA, but underwent apoptosis. Co-expression of cAMP-dependent protein kinase (PKA) regulatory subunit type II (RIIbeta) inhibited apoptosis and stimulated the growth of cells only in the presence of TSH. Expression of RIIbeta-PKA, in the absence of TSHR, induced apoptosis, which was reversed by cAMP. Growth, stimulated by TSHR-RIIbeta-PKA in mouse fibroblasts, was also dependent on Rap1 activity, indicating cAMP-dependent growth in thyroid cells. As for the molecular mechanism underlying these effects, we found that in normal fibroblasts, TSH induced AKT and ERK1/2 only in cells expressing TSHR and RII. Similarly, activation of TSHR increased cAMP levels greatly, but was unable to stimulate CREB phosphorylation and transcription of cAMP-induced genes in the absence of RII. These data provide a simple explanation for the anti-proliferative and proliferative effects of cAMP in different cell types and indicate that RII-PKAII complements TSHR action by stably propagating robust cAMP signals in cell compartments.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that activates Src family kinases via SH2- and SH3-mediated interactions. Specific FAK isoforms (FAK+), responsive to depolarization and neurotransmitters, are enriched in neurons. We analyzed the interactions of endogenous FAK+ and recombinant FAK+ isoforms containing amino acid insertions (boxes 6,7,28) with an array of SH3 domains and the c-Src SH2/SH3 domain tandem. Endogenous FAK+ bound specifically to the SH3 domains of c-Src (but not n-Src), Fyn, Yes, phosphtidylinositol-3 kinase, amphiphysin II, amphiphysin I, phospholipase Cgamma and NH2-terminal Grb2. The inclusion of boxes 6,7 was associated with a significant decrease in the binding of FAK+ to the c-Src and Fyn SH3 domains, and a significant increase in the binding to the Src SH2 domain, as a consequence of the higher phosphorylation of Tyr-397. The novel interaction with the amphiphysin SH3 domain, involving the COOH-terminal proline-rich region of FAK, was confirmed by coimmunoprecipitation of the two proteins and a closely similar response to stimuli affecting the actin cytoskeleton. Moreover, an impairment of endocytosis was observed in synaptosomes after internalization of a proline-rich peptide corresponding to the site of interaction. The data account for the different subcellular distribution of FAK and Src kinases and the specific regulation of the transduction pathways linked to FAK activation in the brain and implicate FAK in the regulation of membrane trafficking in nerve terminals.

The anti-p21ras Y13-259 single-chain Fv fragment (scFv) neutralizes the activity of p21-ras when intracellularly expressed in different systems. We have studied the mode of action of this inhibition in 3T3 K-ras fibroblasts and demonstrated that (i) this antibody fragment is highly aggregating when cytoplasmically expressed and (ii) the p21-ras antigen is sequestered in these aggregates in an antibody-dependent manner. This co-segregation leads to an efficient inhibition of DNA synthesis. These results suggest that an antigen can be diverted from its normal location inside the cells in an antibody mediated way, prospecting a new mode of action for intracellular antibodies in vivo.

In the search for potential nucleoside/non-nucleoside mixed type inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase, we synthesized a new set of rifamycin S derivatives, containing AZT connected via its hydroxyl at 5′ C, through a spacer, to the third C of rifamycin S. The length of the spacer was eight, nine or 14 atoms. Rifamycin S was also used in its 21, 23-O, O-isopropylidene derivative form, and in one case thymidine replaced AZT. These nucleosidyl rifamycins were weak inhibitors of isolated HIV-1 reverse transcriptase. The inhibitory power was weak most probably because their large molecular volume hindered the inhibition process. With the exception of the thymidine derivative, the AZT derivatives, at concentrations in the range 0.04–0.07 μM, proved non-toxic and inhibited the replication of HIV-1 in C8166 T lymphocytes. This activity appears to be owing to AZT released by the derivatives upon hydrolysis in solution. The present compounds require further development as mixed type reverse transcriptase inhibitors and can be considered non-toxic lipophilic prodrugs of AZT.