Effects of rapamycin on cerebral oxygen supply and consumption during reperfusion after cerebral ischemia.

Activation of the mammalian target of rapamycin (mTOR) leads to cell growth and survival. We tested the hypothesis that inhibition of mTOR would increase infarct size and decrease microregional O2 supply/consumption balance after cerebral ischemia-reperfusion. This was tested in isoflurane-anesthetized rats with middle cerebral artery blockade for 1h and reperfusion for 2h with and without rapamycin (20mg/kg once daily for two days prior to ischemia). Regional cerebral blood flow was determined using a C(14)-iodoantipyrine autoradiographic technique. Regional small-vessel arterial and venous oxygen saturations were determined microspectrophotometrically. The control ischemic-reperfused cortex had a similar blood flow and O2 consumption to the contralateral cortex. However, microregional O2 supply/consumption balance was significantly reduced in the ischemic-reperfused cortex. Rapamycin significantly increased cerebral O2 consumption and further reduced O2 supply/consumption balance in the reperfused area. This was associated with an increased cortical infarct size (13.5±0.8% control vs. 21.5±0.9% rapamycin). We also found that ischemia-reperfusion increased AKT and S6K1 phosphorylation, while rapamycin decreased this phosphorylation in both the control and ischemic-reperfused cortex. This suggests that mTOR is important for not only cell survival, but also for the control of oxygen balance after cerebral ischemia-reperfusion.

Situación socio-económica del trabajo infantil en la minería artesanal (caso Huanuni)

La informalidad y la marginalidad caracterizan el trabajo infantil en la minería, donde no existen condiciones técnicas mínimas de seguridad. Los impactos sobre la salud de los niños trabajadores mineros son severos y en muchos casos irreversibles.

TIP41 interacts with TAP42 and negatively regulates the TOR signaling pathway.

In Saccharomyces cerevisiae, the rapamycin-sensitive TOR kinases negatively regulate the type 2A-related phosphatase SIT4 by promoting the association of this phosphatase with the inhibitor TAP42. Here, we describe TIP41, a conserved TAP42-interacting protein involved in the regulation of SIT4. Deletion of the TIP41 gene confers rapamycin resistance, suppresses a tap42 mutation, and prevents dissociation of SIT4 from TAP42. Furthermore, a TIP41 deletion prevents SIT4-dependent events such as dephosphorylation of the kinase NPR1 and nuclear translocation of the transcription factor GLN3. Thus, TIP41 negatively regulates the TOR pathway by binding and inhibiting TAP42. The binding of TIP41 to TAP42 is stimulated upon rapamycin treatment via SIT4-dependent dephosphorylation of TIP41, suggesting that TIP41 is part of a feedback loop that rapidly amplifies SIT4 phosphatase activity under TOR-inactivating conditions.

Calcineurin preferentially synergizes with PKC-theta to activate JNK and IL-2 promoter in T lymphocytes.

Costimulation of the T cell receptor (TCR) and CD28 is required for optimal interleukin-2 (IL-2) induction. These signals, which can be replaced by the pharmacological agents phorbol ester (PMA) and Ca2+ ionophore, synergistically activate the mitogen-activated protein kinase (MAPK) JNK. Cyclosporin A, an inhibitor of the Ca2+-dependent phosphatase calcineurin which blocks IL-2 induction, abrogates Ca2+-triggered synergistic JNK activation. As protein kinase C (PKC) downregulation inhibits PMA+ionophore-induced JNK activation, we examined whether a particular PKC isoform is preferentially involved in this response. We found that PKC-theta but neither PKC-alpha nor PKC-epsilon participates in JNK activation, whereas all three PKCs lead to ERK MAPK activation. PKC-theta specifically cooperates with calcineurin, and together their signals converge on (or upstream of) Rac leading to potent JNK activation. Similarly, calcineurin and PKC-theta specifically synergize to induce transcription of reporters driven by the c-jun and IL-2 promoters. PKC-theta and calcineurin are also partially responsible for the synergistic activation of JNK following TCR and CD28 ligation. Preferential cooperation between PKC-theta and calcineurin is observed in Jurkat T cells but not in HeLa cells. These results indicate that PKC isozymes have distinct biological functions and suggest that synergistic JNK activation is an important function for PKC-theta in T-cell activation.

Cooperation between Syk and Rac1 leads to synergistic JNK activation in T lymphocytes.

The MAP kinase (MAPK) JNK but not ERK is synergistically activated during costimulation of T cells. We examined how protein tyrosine kinases (PTKs) and GTPases differentially regulate JNK and ERK in T cells. While PTKs are not selective, small GTPases display distinct MAPK-activating functions. Whereas Ras activates ERK, Rac activates JNK. Rac cooperates with a Syk-generated signal to enhance JNK activation and appears to be at a nodal point for pathways emanating from CD28, calcineurin, and protein kinase C. AP-1- and NF-AT-dependent reporters are stimulated by Rac and Syk and are dependent on JNK. Unlike Syk, the PTK Lck activates JNK but does not cooperate with Rac, resulting in weak AP-1 and NF-AT activation. Therefore, signals generated by PTKs are functionally distinct and need to be integrated to induce transcriptional responses.

Molecular cloning and characterization of human JNKK2, a novel Jun NH2-terminal kinase-specific kinase.

At least three mitogen-activated protein kinase (MAPK) cascades were identified in mammals, each consisting of a well-defined three-kinase module composed of a MAPK, a MAPK kinase (MAPKK), and a MAPKK kinase (MAPKKK). These cascades play key roles in relaying various physiological, environmental, or pathological signals from the environment to the transcriptional machinery in the nucleus. One of these MAPKs, c-Jun N-terminal kinase (JNK), stimulates the transcriptional activity of c-Jun in response to growth factors, proinflammatory cytokines, and certain environmental stresses, such as short wavelength UV light or osmotic shock. The JNKs are directly activated by the MAPKK JNKK1/SEK1/MKK4. However, inactivation of the gene encoding this MAPKK by homologous recombination suggested the existence of at least one more JNK-activating kinase. Recently, the JNK cascade was found to be structurally and functionally conserved in Drosophila, where DJNK is activated by the MAPKK DJNKK (hep). By a database search, we identified an expressed sequence tag (EST) encoding a portion of human MAPKK that is highly related to DJNKK (hep). We used this EST to isolate a full-length cDNA clone encoding a human JNKK2. We show that JNKK2 is a highly specific JNK kinase. Unlike JNKK1, it does not activate the related MAPK, p38. Although the regulation of JNKK1 activities and that of JNKK2 activities could be very similar, the two kinases may play somewhat different regulatory roles in a cell-type-dependent manner.

JNK is involved in signal integration during costimulation of T lymphocytes.

T lymphocyte activation and interleukin-2 (IL-2) production require at least two signals, generated by phorbol ester (TPA) and Ca2+ ionophore or costimulation of the T cell receptor (TCR) and the CD28 auxiliary receptor. We investigated how these stimuli affect mitogen activated protein (MAP) kinases. Full activation of the MAP kinases that phosphorylate the Jun activation domain, JNK1 and JNK2, required costimulation of T cells with either TPA and Ca2+ ionophore or antibodies to TCR and CD28. Alone, each stimulus resulted in little or no activation. Similar to its effect on IL-2 induction, cyclosporin A (CsA) inhibited the synergistic activation of JNK, and a competitive inhibitor of Jun phosphorylation by JNK inhibited IL-2 promoter activation. By contrast, the MAP kinases ERK1 and ERK2 were fully activated by TPA or TCR stimulation and were not affected by Ca2+, CD28, or CsA. Hence, integration of signals that lead to T cell activation occurs at the level of JNK activation.