Vav1 transduces T cell receptor signals to the activation of phospholipase C-gamma1 via phosphoinositide 3-kinase-dependent and -independent pathways

Vav1 is a signal transducing protein required for T cell receptor (TCR) signals that drive positive and negative selection in the thymus. Furthermore, Vav1-deficient thymocytes show greatly reduced TCR-induced intracellular calcium flux. Using a novel genetic system which allows the study of signaling in highly enriched populations of CD4(+)CD8(+) double positive thymocytes, we have studied the mechanism by which Vav1 regulates TCR-induced calcium flux. We show that in Vav1-deficient double positive thymocytes, phosphorylation, and activation of phospholipase C-gamma1 (PLCgamma1) is defective. Furthermore, we demonstrate that Vav1 regulates PLCgamma1 phosphorylation by at least two distinct pathways. First, in the absence of Vav1 the Tec-family kinases Itk and Tec are no longer activated, most likely as a result of a defect in phosphoinositide 3-kinase (PI3K) activation. Second, Vav1-deficient thymocytes show defective assembly of a signaling complex containing PLCgamma1 and the adaptor molecule Src homology 2 domain-containing leukocyte phosphoprotein 76. We show that this latter function is independent of PI3K.     

Normal insulin-dependent activation of Akt/protein kinase B, with diminished activation of phosphoinositide 3-kinase, in muscle in type 2 diabetes

To determine whether the serine/threonine kinase Akt (also known as protein kinase B) is activated in vivo by insulin administration in humans, and whether impaired activation of Akt could play a role in insulin resistance, we measured the activity and phosphorylation of Akt isoforms in skeletal muscle from 3 groups of subjects: lean, obese nondiabetic, and obese type 2 diabetic. Vastus lateralis biopsies were taken in the basal (overnight fast) and insulin-stimulated (euglycemic clamp) states. Insulin-stimulated glucose disposal was reduced 31% in obese subjects and 63% in diabetic subjects, compared with lean subjects. Glycogen synthase (GS) activity in the basal state was reduced 28% in obese subjects and 49% in diabetic subjects, compared with lean subjects. Insulin-stimulated GS activity was reduced 30% in diabetic subjects. Insulin treatment activated the insulin receptor substrate-1-associated (IRS-1-associated) phosphoinositide 3-kinase (PI 3-kinase) 6.1-fold in lean, 3.7-fold in obese, and 2.4-fold in diabetic subjects. Insulin also stimulated IRS-2-associated PI 3-kinase activity 2.2-fold in lean subjects, but only 1.4-fold in diabetic subjects. Basal activity of Akt1/Akt2 (Akt1/2) and Akt3 was similar in all groups. Insulin increased Akt1/2 activity 1.7- to 2. 0-fold, and tended to activate Akt3, in all groups. Insulin-stimulated phosphorylation of Akt1/2 was normal in obese and diabetic subjects. In lean subjects only, insulin-stimulated Akt1/2 activity correlated with glucose disposal rate. Thus, insulin activation of Akt isoforms is normal in muscle of obese nondiabetic and obese diabetic subjects, despite decreases of approximately 50% and 39% in IRS-1- and IRS-2-associated PI 3-kinase activity, respectively, in obese diabetic subjects. It is therefore unlikely that Akt plays a major role in the resistance to insulin action on glucose disposal or GS activation that is observed in muscle of obese type 2 diabetic subjects.     

B cell receptor signaling strength modulates cancer immunity

Tumor-infiltrating B cells exert antitumor effects by producing antibodies against tumor-associated antigens. Conversely, B cells may promote tumors through the production of factors that dampen antitumor immunity. In this issue of the JCI, Bing Yang, Zhen Zhang, et al. investigated the roles of B cell receptor (BCR) signaling in antitumor immunity, focusing on the role of an Asia-specific variant of human immunoglobulin G1 (IgG1) containing a Gly396 to Arg396 substitution (hIgG1-G396R) in colorectal cancer (CRC). Epidemiological analysis revealed an association between hIgG1-G396R and progression-free survival in CRC. Human samples and mouse models of CRC showed plasma cells, as opposed to B cells, infiltrating the tumor microenvironment. Notably, patients with the hIgG1-G396R variant had increased CD8⁺ T cells, dendritic cells, and tertiary lymphoid structure density. These findings indicate that the hIgG1-G396R variant represses tumorigenesis by enhancing B cell responses, and suggest that modulating BCR signaling could improve the efficacy of immunotherapy in cancer.     

T cell regulation of B cell activation. T cells independently regulate the responses mediated by distinct B cell subpopulations

The present studies have been carried out to characterize the regulatory influences acting upon defined pathways of T cell-dependent B cell activation. In these studies, it was demonstrated that high concentrations of free carrier strongly inhibited the MHC-restricted in vitro T cell-dependent antibody responses of primed Lyb-5- B cells to the corresponding carrier-hapten conjugate. In contrast, these same concentrations of free carrier failed to inhibit the T cell dependent responses of Lyb-5+ B cells to the same antigen. The inhibition of Lyb-5- B cell responses by free carrier was shown to result from active suppression mediated by carrier-specific primed Lyt-1+2- T cells and to require the additional participation of unprimed Lyt-1-2+ T cells. The activation of this suppression was antigen-specific, but suppression once activated was antigen nonspecific in its effect. These findings thus demonstrate that distinct pathways of B cell activation can be independently regulated by T suppressor network influences, and that these pathways therefore constitute potentially independent components of the immune response to a given antigenic stimulus.     

Phosphoinositide 3-kinase gamma participates in T cell receptor-induced T cell activation

Class I phosphoinositide 3-kinases (PI3Ks) constitute a family of enzymes that generates 3-phosphorylated polyphosphoinositides at the cell membrane after stimulation of protein tyrosine (Tyr) kinase-associated receptors or G protein-coupled receptors (GPCRs). The class I PI3Ks are divided into two types: class I(A) p85/p110 heterodimers, which are activated by Tyr kinases, and the class I(B) p110gamma isoform, which is activated by GPCR. Although the T cell receptor (TCR) is a protein Tyr kinase-associated receptor, p110gamma deletion affects TCR-induced T cell stimulation. We examined whether the TCR activates p110gamma, as well as the consequences of interfering with p110gamma expression or function for T cell activation. We found that after TCR ligation, p110gamma interacts with G alpha(q/11), lymphocyte-specific Tyr kinase, and zeta-associated protein. TCR stimulation activates p110gamma, which affects 3-phosphorylated polyphosphoinositide levels at the immunological synapse. We show that TCR-stimulated p110gamma controls RAS-related C3 botulinum substrate 1 activity, F-actin polarization, and the interaction between T cells and antigen-presenting cells, illustrating a crucial role for p110gamma in TCR-induced T cell activation.     

FcγRIIB regulates autoantibody responses by limiting marginal zone B cell activation

FcγRIIB is an inhibitory receptor expressed throughout B cell development. Diminished expression or function is associated with lupus in mice and humans, in particular through an effect on autoantibody production and plasma cell (PC) differentiation. Here, we analyzed the effect of B cell–intrinsic FcγRIIB expression on B cell activation and PC differentiation. Loss of FcγRIIB on B cells in Fcgr2b–conditional KO (Fcgr2b-cKO) mice led to a spontaneous increase in autoantibody titers. This increase was most striking for IgG3, suggestive of increased extrafollicular responses. Marginal zone (MZ) B cells had the highest expression of FcγRIIB in both mice and humans. This high expression of FcγRIIB was linked to increased MZ B cell activation, Erk phosphorylation, and calcium flux in the absence of FcγRIIB triggering. We observed a marked increase in IgG3⁺ PCs and B cells during extrafollicular PC responses in Fcgr2b-cKO mice. The increased IgG3 response following immunization of Fcgr2b-cKO mice was lost in MZ-deficient Notch2 Fcgr2b–double KO mice. Importantly, patients with systemic lupus erythematosus (SLE) had a decrease in FcγRIIB expression that was strongest in MZ B cells. Thus, we present a model in which high FcγRIIB expression in MZ B cells prevented their hyperactivation and ensuing autoimmunity.     

Isoginkgetin inhibits tumor cell invasion by regulating phosphatidylinositol 3-kinase/Akt-dependent matrix metalloproteinase-9 expression

Matrix metalloproteinase (MMP)-9 plays a key role in tumor invasion. Inhibitors of MMP-9 were screened from Metasequoia glyptostroboides (Dawn redwood) and one potent inhibitor, isoginkgetin, a biflavonoid, was identified. Noncytotoxic levels of isoginkgetin decreased MMP-9 production profoundly, but up-regulated the level of tissue inhibitor of metalloproteinase (TIMP)-1, an inhibitor of MMP-9, in HT1080 human fibrosarcoma cells. The major mechanism of Ras-dependent MMP-9 production in HT1080 cells was phosphatidylinositol 3-kinase (PI3K)/Akt/nuclear factor-kappaB (NF-kappaB) activation. Expression of dominant-active H-Ras and p85 (a subunit of PI3K) increased MMP-9 activity, whereas dominant-negative forms of these molecules decreased the level of MMP-9. H-Ras did not increase MMP-9 in the presence of a PI3K inhibitor, LY294002, and a NF-kappaB inhibitor, SN50. Further studies showed that isoginkgetin regulated MMP-9 production via PI3K/Akt/NF-kappaB pathway, as evidenced by the findings that isoginkgetin inhibited activities of both Akt and NF-kappaB. PI3K/Akt is a well-known key pathway for cell invasion, and isoginkgetin inhibited HT1080 tumor cell invasion substantially. Isoginkgetin was also quite effective in inhibiting the activities of Akt and MMP-9 in MDA-MB-231 breast carcinomas and B16F10 melanoma. Moreover, isoginkgetin treatment resulted in marked decrease in invasion of these cells. In summary, PI3K/Akt is a major pathway for MMP-9 expression and isoginkgetin markedly decreased MMP-9 expression and invasion through inhibition of this pathway. This suggests that isoginkgetin could be a potential candidate as a therapeutic agent against tumor invasion.     

Impaired activation of phosphoinositide 3-kinase by insulin in fibroblasts from patients with severe insulin resistance and pseudoacromegaly. A disorder characterized by selective postreceptor insulin resistance.

Some patients with severe insulin resistance develop pathological tissue growth reminiscent of acromegaly. Previous studies of such patients have suggested the presence of a selective postreceptor defect of insulin signaling, resulting in the impairment of metabolic but preservation of mitogenic signaling. As the activation of phosphoinositide 3-kinase (PI 3-kinase) is considered essential for insulin's metabolic signaling, we have examined insulin-stimulated PI 3-kinase activity in anti-insulin receptor substrate (IRS)-1 immunoprecipitates from cultured dermal fibroblasts obtained from pseudoacromegalic (PA) patients and controls. At a concentration of insulin (1 nM) similar to that seen in vivo in PA patients, the activation of IRS-1-associated PI 3-kinase was reduced markedly in fibroblasts from the PA patients (32+/-7% of the activity of normal controls, P < 0.01). Genetic and biochemical studies indicated that this impairment was not secondary to a defect in the structure, expression, or activation of the insulin receptor, IRS-1, or p85alpha. Insulin stimulation of mitogenesis in PA fibroblasts, as determined by thymidine incorporation, was indistinguishable from controls, as was mitogen-activated protein kinase phosphorylation, confirming the integrity of insulin's mitogenic signaling pathways in this condition. These findings support the existence of an intrinsic defect of postreceptor insulin signaling in the PA subtype of insulin resistance, which involves impairment of the activation of PI 3-kinase. The PA tissue growth seen in such patients is likely to result from severe in vivo hyperinsulinemia activating intact mitogenic signaling pathways emanating from the insulin receptor.     

Pentoxifylline modulates activation of human neutrophils by amphotericin B in vitro.

The antifungal agent amphotericin B (AmB) alters neutrophil (polymorphonuclear leukocyte [PMN]) function, and this may be the mechanism for some of the adverse effects caused by AmB. AmB is a potent inhibitor of PMN migration, increases PMN adherence and aggregation, and primes PMN for increased oxidative activity in response to a second stimulus. AmB also stimulates mononuclear leukocytes (MNLs) to release inflammatory mediators which augment the effects of AmB on PMN function. In the present study, we observed that the methylxanthine derivative pentoxifylline decreased the effects of AmB on PMN function. AmB (2 micrograms/ml) priming doubled PMN chemiluminescence stimulated by fMet-Leu-Phe. In the presence of MNLs, AmB priming increased fMet-Leu-Phe-stimulated PMN chemiluminescence to 622% of unprimed PMN activity. Pentoxifylline (100 microM) blunted the rise in AmB-augmented PMN chemiluminescence in the presence of MNLs to 282% of unprimed PMN activity, and pentoxifylline metabolites were active at 10 microM. Pentoxifylline (100 microM) also blocked AmB-augmented PMN oxidative activity in whole blood, as measured by nitroblue tetrazolium reduction. In the presence of MNL, AmB (2 micrograms/ml) doubled the expression of the important PMN adherence factor Mac-1. Pentoxifylline (1 mM) decreased AmB-stimulated PMN Mac-1 expression back to unstimulated amounts. In the presence of MNLs, AmB (2 micrograms/ml) decreased PMN nondirected and directed migration to fMet-Leu-Phe to 40 and 38% of control PMN migration, respectively. Pentoxifylline (300 microM) counteracted AmB inhibition of nondirected and directed migration to fMet-Leu-Phe, resulting in migration that was 71 and 87% of control PMN migration, respectively. In contrast, the methylxanthine caffeine (100 muM) increased AmB-enhanced chemiluminescence but did not affect AmB-inhibited PMN migration. Pentoxifylline should be evaluated as adjunctive therapy to lessen the inflammatory damage caused by AmB.     

B cell activation by cytomegalovirus

Human cytomegalovirus is shown to be a nonspecific polyclonal B cell activator. The B cell response is independent of virus replication and requires little, if any, T cell help.     

Ig beta tyrosine residues contribute to the control of B cell receptor signaling by regulating receptor internalization

Immunoglobulin (Ig)alpha and Igbeta initiate B cell receptor (BCR) signaling through immune receptor tyrosine activation motifs (ITAMs) that are targets of SH2 domain-containing kinases. To examine the function of Igbeta ITAM tyrosine resides in mature B cells in vivo, we exchanged these residues for alanine by gene targeting (Igbeta(AA)). Mutant mice showed normal development of all B cell subtypes with the exception of B1 cells that were reduced by fivefold. However, primary B cells purified from Igbeta(AA) mice showed significantly decreased steady-state and ligand-mediated BCR internalization and higher levels of cell surface IgM and IgD. BCR cross-linking resulted in decreased Src and Syk activation but paradoxically enhanced and prolonged BCR signaling, as measured by cellular tyrosine phosphorylation, Ca(++) flux, AKT, and ERK activation. In addition, B cells with the ITAM mutant receptor showed an enhanced response to a T-independent antigen. Thus, Igbeta ITAM tyrosines help set BCR signaling threshold by regulating receptor internalization.     

B cell activation. III. B cell plasma membrane depolarization and hyper- Ia antigen expression induced by receptor immunoglobulin cross-linking are coupled

We report investigation of the relationship between ligand-induced B cell plasma membrane depolarization and increased expression of membrane-associated, I-A subregion encoded (mI-A) antigens. Results demonstrate that equal frequencies of B cells are stimulated to undergo membrane depolarization and to increase mI-A expression in response to mitogen, anti-Ig, and thymus-independent (TI) or thymus-dependent (TD) antigens. Further, a cause-and-effect relationship between these two events is suggested by results that demonstrate that inhibition of anti- Fab--induced depolarization by valinomycin also inhibits the subsequent increase in mI-A antigen expression and "passive" (non-ligand-mediated) depolarization of murine B cells by K+ results in hyper-mI-A antigen expression. Based upon these results we hypothesize that antigen- mediated receptor cross-linking results in signal transduction via membrane depolarization, which is resultant in increased mI-A antigen synthesis and cell surface expression. This increase in mI-A antigen density may render the B cell more receptive to subsequent interaction with I-region-restricted helper T cells.     

Cbl suppresses B cell receptor-mediated phospholipase C (PLC)-gamma2 activation by regulating B cell linker protein-PLC-gamma2 binding

Accumulating evidence indicates that the Cbl protein plays a negative role in immune receptor signaling; however, the mode of Cbl action in B cell receptor (BCR) signaling still remains unclear. DT40 B cells deficient in Cbl showed enhanced BCR-mediated phospholipase C (PLC)-gamma2 activation, thereby leading to increased apoptosis. A possible explanation for the involvement of Cbl in PLC-gamma2 activation was provided by findings that Cbl interacts via its Src homology 2 (SH2) domain with B cell linker protein (BLNK) after BCR ligation. BLNK is a critical adaptor molecule for PLC-gamma2 tyrosine phosphorylation through its binding to the PLC-gamma2 SH2 domains. As a consequence of the interaction between Cbl and BLNK, the BCR-induced recruitment of PLC-gamma2 to BLNK and the subsequent PLC-gamma2 tyrosine phosphorylation were inhibited. Thus, our data suggest that Cbl negatively regulates the PLC-gamma2 pathway by inhibiting the association of PLC-gamma2 with BLNK.     

Signals of oxidant-induced cardiomyocyte hypertrophy: key activation of p70 S6 kinase-1 and phosphoinositide 3-kinase

Cardiomyocytes in culture can survive low or mild doses of oxidants but later increase cell volume and protein content. To understand the mechanism, we determined the early signaling events of oxidative stress. With 200 microM H2O2, the activity of p70 S6 kinase-1 (p70S6K1) increased at 30 min and reached a plateau at 90 min. Dose-response studies at the 60 min time point show that p70S6K1 activity reached its highest level with 150 microM H2O2. Increased p70S6K1 activity correlated with phosphorylation of Thr389 and Thr421/Ser424 residues, suggesting the involvement of an upstream kinase. Phosphoinositide 3-kinase (PI3K) activity was elevated by 5 min, reached a plateau at 10 min, and remained more than 6-fold induced for at least 60 min after 200 microM H2O2 exposure. The dose-response studies at 10 min found that 150 microM H2O2 induced the highest PI3K activity. Increased PI3K activity correlated with tyrosine phosphorylation of the 85-kDa regulatory subunit. Inactivating PI3K with wortmannin prevented H2O2 from inducing Thr389 phosphorylation and p70S6K1 activation. Wortmannin and rapamycin prevented H2O2 from inducing increases in cell volume and protein content. The antineoplastic drugs doxorubicin and daunorubicin also induced significant enlargement of cardiomyocytes at 10 to 100 nM dose range. Although the glutathione synthesis inhibitor buthionine sulfoximine potentiated the effect of doxorubicin and H2O2, the antioxidant N-acetylcysteine prevented induction of cell enlargement. Our data suggest that oxidative stress induces activation of PI3K, which leads to p70S6K1 activation and enlargement of cell size.     

A crucial role for the p110delta subunit of phosphatidylinositol 3-kinase in B cell development and activation

Mice lacking the p110delta catalytic subunit of phosphatidylinositol 3-kinase have reduced numbers of B1 and marginal zone B cells, reduced levels of serum immunoglobulins, respond poorly to immunization with type II thymus-independent antigen, and are defective in their primary and secondary responses to thymus-dependent antigen. p110delta(-/-) B cells proliferate poorly in response to B cell receptor (BCR) or CD40 signals in vitro, fail to activate protein kinase B, and are prone to apoptosis. p110delta function is required for BCR-mediated calcium flux, activation of phosphlipaseCgamma2, and Bruton's tyrosine kinase. Thus, p110delta plays a critical role in B cell homeostasis and function.     

A platelet biomarker for assessing phosphoinositide 3-kinase inhibition during cancer chemotherapy

Thrombin cleavages of selective proteinase-activated receptors (PAR) as well as PAR-activating peptide ligands can initiate the phosphoinositide 3-kinase (PI3K) signaling cascade in platelets. Downstream to this event, fibrinogen receptors on platelets undergo conformational changes that enhance fibrinogen binding. In our study, we used this phenomenon as a surrogate biomarker for assessing effects on PI3K activity. Our method, using flow cytometric measurement of fluorescent ligand and antibody binding, uncovered a 16- to 45-fold signal window after PAR-induced platelet activation. Pretreatment (in vitro) with the PI3K inhibitors wortmannin and LY294002 resulted in concentration-dependent inhibition at predicted potencies. In addition, platelets taken from mice treated with wortmannin were blocked from PAR-induced ex vivo activation concomitantly with a decrease in phosphorylation of AKT from excised tumor xenografts. This surrogate biomarker assay was successfully tested (in vitro) on blood specimens received from volunteer cancer patients. Our results indicate that measurement of platelet activation could serve as an effective drug activity biomarker during clinical evaluation of putative PI3K inhibitors.     

Role of the major histocompatibility complex in T cell activation of B cell subpopulations. Major histocompatibility complex-restricted and -unrestricted B cell responses are mediated by distinct B cell subpopulations

The present study has evaluated the identity of the B cell subpopulations participating in T dependent antibody responses that differ in their requirements for major histocompatibility complex-restricted T cell recognition. In vitro responses of keyhole limpet hemocyanin (KLH)-primed T cells and trinitrophenyl (TNP)-primed B cells were studied to both low and high concentrations of the antigen TNP-KLH. It was first demonstrated that for responses to low concentrations of TNP-KLH, (A × B)F(1) {arrow} parent(A) chimeric helper T cells were restricted in their ability to recognize parent(A) but not parent(B) H-2 determinants expressed by both B cells and antigen-presenting cells (APC). In contrast, at higher antigen concentrations, helper T cells were not restricted in their interaction with B cells. It was then determined whether these observed differences in T cell recognition resulted from the activation of distinct B cell subpopulations with different activation requirements. At low concentrations of TNP-KLH it was demonstrated that Lyb-5(-) B cells were activated, and that it was thus the activation of the Lyb-5(-) subpopulation that required T cell recognition of B cell H-2 under these conditions. In contrast, responses to high concentration of antigen required the participation of Lyb-5(+) B cells, and these Lyb-5(+) B cells were activated by a pathway that required H-2- restricted T cell interaction with APC, but not with B cells. The findings presented here have demonstrated that Lyb-5(-) and Lyb-5(+) B cells constitute B cell subpopulations that differ significantly in their activation requirements for T cell-dependent antibody responses to TNP-KLH. In so doing, these findings have established that the function of genetic restrictions in immune response regulation is critically dependent upon the activation pathways employed by functionally distinct subpopulations of B, as well as T, lymphocytes.     

依布硒啉对大鼠心肌缺血再灌注损伤后磷酸肌醇-3激酶/蛋白激酶B信号通路的影响

目的探讨依布硒啉预处理对大鼠心肌缺血再灌注后磷酸肌醇-3激酶/蛋白激酶B（P13K/Akt）信号及其内质网应激的影响。 方法将30只大鼠分成对照组,缺血再灌注组及依布硒啉组,每组10只。对照组大鼠冠状动脉左室支左心耳下缘约0.5 cm处只穿线,不结扎,常规腹腔注射生理盐水2 ml;缺血再灌注组大鼠缺血再灌注前30 min腹腔注射生理盐水2 ml;依布硒啉组大鼠缺血再灌注前30 min腹腔注射依布硒啉溶液5 mg/kg。每组各取8只大鼠,采用酶联免疫吸附试验（ELISA）检测各组血清炎症因子高迁移率族蛋白1（HMGB1）、丙二醛、超氧化物歧化酶（SOD）、天冬氨酸转氨酶（AST）及乳酸脱氢酶（LDH）的表达,Tunel法检测缺血心肌细胞凋亡指数,Western-blotting检测各组心肌心肌葡萄糖调节蛋白78（GRP78）蛋白表达及P13K/Akt通路磷酸化水平。 结果三组大鼠间HMGB1、丙二醛、SOD、AST及LDH水平比较,差异均有统计学意义（F = 63.755、73.023、99.220、110.439、120.557,P均< 0.05）,进一步比较发现,缺血再灌注组及依布硒啉组的HMGB1[（9.2 ± 2.7）、（5.5 ± 1.1）、（2.2 ± 0.3）U/L]、丙二醛[（7.2 ± 0.4）、（5.6 ± 0.7）、（4.1 ± 0.9）μmol/L]、AST [（1 011 ± 226）、（813 ± 82）、（671 ± 60）U/L]及LDH [（2 783 ± 674）、（2 043 ± 489）、（1 528 ± 524）U/L]水平较对照组均明显升高,SOD水平[（249 ± 28）、（149 ± 10）、（172 ± 17）kU/L]较对照组明显降低（P均< 0.05）。三组大鼠间的凋亡指数（F = 139.942,P < 0.001）、GRP78蛋白表达（F = 177.846,P < 0.001）及P13K/Akt磷酸化水平（F = 86.286,P < 0.001）比较差异均存在统计学意义,进一步比较发现,缺血再灌注组和依布硒啉组凋亡指数[（38.1 ± 4.6）、（25.4 ± 3.9）、（8.2 ± 1.5）%]明显高于对照组,且I/R组更高（P均< 0.05）。 结论依布硒啉预处理可以抑制大鼠内质网应激,可能与调节P13K/Akt信号通路磷酸化水平有关。     

Water-soluble HPMA copolymer-wortmannin conjugate retains phosphoinositide 3-kinase inhibitory activity in vitro and in vivo.

Phosphoinositide kinases and ATM-related genes play a central role in many physiological processes. Activation of phosphoinositide 3-kinase (PI 3-kinase) is essential for signal transduction by many growth factors and oncogenes and may contribute to tumor progression. In the nanomolar range, Wortmannin (WM), a fungal metabolite, is a potent inhibitor of type I PI 3-kinase; it covalently modifies its catalytic subunit. Because WM is soluble only in organic solvents and unstable in water, there are difficulties in its use in vivo. To generate a water-soluble WM derivative, we used a conjugate of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer and 11-O-desacetylwortmannin (DAWM), which has a slightly lower inhibitory activity than WM. We covalently attached DAWM to HPMA copolymer containing oligopeptide (GFLG) side-chains. The final product had an estimated molecular mass of 20 kDa and contained 2 wt.% of DAWM. The HPMA copolymer (PHPMA)-DAWM conjugate inhibited type I PI 3-kinase activity in vitro and growth factor-stimulated activation of Akt in vivo; it possessed approximately 50% of the inhibitory activity of DMSO solubilized WM. The specificity and stability of the PHPMA-DAWM conjugate is currently under investigation. The new water-soluble form of WM may be useful in investigations of the role of PI 3-kinase in tumor progression and other cellular biological functions in vivo.