VPS 15 protein is a component of the phosphatidylinositol 3-kinase complex which plays a pivotal role in the development of yeast and mammalian cells. The knowledge about the function of its homologue in plants remain...VPS 15 protein is a component of the phosphatidylinositol 3-kinase complex which plays a pivotal role in the development of yeast and mammalian cells. The knowledge about the function of its homologue in plants remains limited. Here we report that AtVPS15, a homologue of yeast VPS15p in Arabidopsis, plays an essential role in pollen germination. Homozygous T-DNA insertion mutants of AtVPS15 could not be obtained from the progenies of self-pollinated heterozygous mutants. Reciprocal crosses between atvps15 mutants and wild-type Arabidopsis revealed that the T-DNA insertion was not able to be transmitted by male gametophytes. DAPI staining, Alexander＇s stain and scanning electron microscopic analysis showed that atvps15 heterozygous plants produced pollen grains that were morphologically indistinguishable from wild-type pollen, whereas in vitro germination experiments revealed that germination of the pollen grains was defective. GUS staining analysis of transgenic plants expressing the GUS reporter gene driven by the AtVPS15 promoter showed that AtVPS15 was mainly expressed in pollen grains. Finally, DUALmembrane yeast two-hybrid analysis demonstrated that AtVPS15 might interact directly with AtVPS34. These results suggest that AtVPS15 is very important for pollen germination, possibly through modulation of the activity of PI3-kinase.展开更多
We describe a pre-clinical spinal cord motor neuron injury model that is minimal invasive, reproducible, focal and easily applied to small rodents.Retrograde axonal transport of a pro-apoptotic phosphatidylinosotol 3&...We describe a pre-clinical spinal cord motor neuron injury model that is minimal invasive, reproducible, focal and easily applied to small rodents.Retrograde axonal transport of a pro-apoptotic phosphatidylinosotol 3'-kinase inhibitor, wortmannin, via the sciatic nerve results in loss of ipsilateral lumbar motor neurons proportional to the level of drug administered.Motor neuron loss was detected by choline acetyltransferase(ChAT) immunostaining and with a transgenic thy1-eGFP marker.The short half-life of wortmannin generates minimal wound spread, and wortmannin does not affect axon transport, as determined by co-injection of a pseudorabies virus tracer.Using quantitative transcript analysis, we found that ChAT transcripts significantly decreased at 14 days post-delivery of 1 μg wortmannin, relative to sham controls, and remained low after 90 days.Smaller effects were observed with 200 ng and 100 ng wortmannin.Wortmannin also generated a transient and significant increase in astrocyte Gfap transcripts after 14 days with a return to control levels at 90 days.Treated mice had hind limb spasticity and a forced motor function defect that was quantified using a water exit test.Controls rapidly exit a shallow water tray, and wortmannin treated animals were up to 12-fold slower, a phenotype that persisted for at least 3 months.Thus the focal delivery of wortmannin to motor neurons generates a reproducible and scalable injury that can facilitate quantitative studies on neural regeneration and repair.The efficacy of sciatic nerve suicide transport can also explain neurotoxin-mediated selective loss of motor neurons in diseases such as amyotrophic lateral sclerosis.All procedures were performed at Rutgers under established Institutional Animal Care and Use protocols(eIACUC_TR201800022, approved on March 20, 2018).展开更多
Much research has focused on the PI3-kinase and PTEN signaling pathway with the aim to stimulate repair of the injured central nervous system.Axons in the central nervous system fail to regenerate,meaning that injurie...Much research has focused on the PI3-kinase and PTEN signaling pathway with the aim to stimulate repair of the injured central nervous system.Axons in the central nervous system fail to regenerate,meaning that injuries or diseases that cause loss of axonal connectivity have life-changing consequences.In 2008,genetic deletion of PTEN was identified as a means of stimulating robust regeneration in the optic nerve.PTEN is a phosphatase that opposes the actions of PI3-kinase,a family of enzymes that function to generate the membrane phospholipid PIP_(3) from PIP_(2)(phosphatidylinositol(3,4,5)-trisphosphate from phosphatidylinositol(4,5)-bisphosphate).Deletion of PTEN therefore allows elevated signaling downstream of PI3-kinase,and was initially demonstrated to promote axon regeneration by signaling through mTOR.More recently,additional mechanisms have been identified that contribute to the neuron-intrinsic control of regenerative ability.This review describes neuronal signaling pathways downstream of PI3-kinase and PIP3,and considers them in relation to both developmental and regenerative axon growth.We briefly discuss the key neuron-intrinsic mechanisms that govern regenerative ability,and describe how these are affected by signaling through PI3-kinase.We highlight the recent finding of a developmental decline in the generation of PIP_(3) as a key reason for regenerative failure,and summarize the studies that target an increase in signaling downstream of PI3-kinase to facilitate regeneration in the adult central nervous system.Finally,we discuss obstacles that remain to be overcome in order to generate a robust strategy for repairing the injured central nervous system through manipulation of PI3-kinase signaling.展开更多
Objective: It is well known that systemic insulin resistance (IR) is closely associated with the metabolic syndrome including type 2 diabetes and hypertension. However, it remains
Berberine is identified to lower the serum cholesterol level in human and hamster through the induction of low density lipoproteins （LDL） receptor in hepatic cells. To evaluate its potential in preventing atheroscle...Berberine is identified to lower the serum cholesterol level in human and hamster through the induction of low density lipoproteins （LDL） receptor in hepatic cells. To evaluate its potential in preventing atherosclerosis, the effect of berberine on atherosclerosis development in apolipoprotcin E-deficient （apoE^-/-） mice was investigated. In apoE^-/- mice, berberine induced in rivo foam cell formation and promoted atheroselerosis development. The foam cell formation induced by berberinc was also observed in mouse RAW264.7 cells, as well as in mouse and human primary macrophages. By inducing scavenger receptor A （SR-A） expression in macrophages, berberine increased the uptake of modified LDL （DiO-Ac-LDL）. Bcrberine-induced SR-A expression was also observed in macrophage foam cells in vivo and in the cells at atherosclerotic lesion. Analysis in RAW264.7 cells indicated that berberine induced SR-A expression by suppressing PTEN expression, which led to sustained Akt activation. Our results suggest that to evaluate the potential of a cholesterol-reducing compound in alleviating atherosclerosis, its effect on the ceils involved in atherosclerosis development, such as macrophages, should also be considered. Promotion of foam cell formation could counter-balance the beneficial effect of lowering serum cholesterol.展开更多
Endothelial nitric oxide synthase (eNOS) is a key enzyme responsible for the regu-lation of vascular homeostasis. Many humor factors and mechanical forces can affect eNOS ac-tivity via phosphorylation modification but...Endothelial nitric oxide synthase (eNOS) is a key enzyme responsible for the regu-lation of vascular homeostasis. Many humor factors and mechanical forces can affect eNOS ac-tivity via phosphorylation modification but the mechanisms involved vary with stimuli applied. We have demonstrated that cytochrome P450 (CYP) epoxygenase-dependent metabolites of ara-chidonic acid, epoxyeicosatrienoic acids (EETs), can robustly up-regulate eNOS expression and its activity, however the relevant signaling pathways responsible for activity regulation are not well known. In this study, we explored the role of PI3 kinase (PI3K)/protein kinase B (Akt) sig-naling pathway in eNOS expression and its phosphorylation in response to EETs via direct addi-tion of EETs into cultured bovine aorta endothelial cells (BAECs) and recombinant adeno- asso-ciated virus-mediated transfection of CYP epoxygenase genes CYPF87V and CYP2C11 to pro-duce endogenous EETs followed by co-treatment with PI3K or Akt inhibitor. Results show that both exogenous and endogenous EETs could remarkably enhance eNOS expression and its phosphorylation at Ser1179 and Thr497 residues; PI3K inhibitor LY294002 could inhibit EETs-induced increase in eNOS-Ser(P)1179 but had no effect on the change of eNOS-Thr(P)497, while Akt inhibitor could attenuate the increase in phosphor-eNOS at both residues; both of the two inhibitors could block EETs-enhanced eNOS expression. These results lead to conclusions: (i) EETs-mediated regulation of eNOS activity may be related with the changes of phosphoryla-tion level at eNOS-Ser1179 via PI3K/Akt and eNOS-Thr497 via Akt; (ii) PI3K/Akt signaling pathway is involved in the up-regulation of eNOS expression by EETs.展开更多
A small nonpeptidyl compoud extracted from Pseudomassaria sp. was found to induce the activity of human insulin receptor tyrosine kinase in vitro. The compound was identified as demethylasterriquinone B-1 (DMAQ-B1). D...A small nonpeptidyl compoud extracted from Pseudomassaria sp. was found to induce the activity of human insulin receptor tyrosine kinase in vitro. The compound was identified as demethylasterriquinone B-1 (DMAQ-B1). DMAQ- B1 also induced an increase in [Ca2+]i and insulin secretion in mice pancreatic beta-cells at low glucose (3 mM) concentration via insulin receptor substrate-1/phosphatidylinositol-3-kinase (PI3 kinase) pathway. By using rat pancreatic perfusion technique, we found that 10 μM DMAQ-B1 directly stimulated insulin secretion up to 240% in normal rat pancreas. In the dosage from 1 to 20 μM, DMAQ-B1 stimulated insulin secretion in a dose dependent manner. Furthermore, DMAQ-B1 enhanced glucose-induced insulin secretion by 17.6% (first stage) and 19.0% (second stage), respectively. The PI3 kinase inhibitors, LY 294002 (3.9 μM) or wortmannin (100 nM), inhibited DMAQ-B1-induced insulin secretion by 46.3% and 57.4%, respectively. LY 294002 or wortmannin also inhibited DMAQ-B1 with10 mMglucose-induced insulin secretion by 70.3% and 79.0%, respectively. All the results suggested that DMAQ-B1 directly stimulated insulin secretion and enhanced glucose-induced insulin secretion. The effect of DMAQ-B1 may mediate through the activation of PI3 kinase pathway to stimulate insulin secretion in normal rat pancreas.展开更多
We aim to investigate the effect of transforming growth factor （TGF）-β1 on the expression of enhancer of split- and hairy-related protein-2 （SHARP-2） messenger RNA （mRNA） and its signaling pathway. In this stud...We aim to investigate the effect of transforming growth factor （TGF）-β1 on the expression of enhancer of split- and hairy-related protein-2 （SHARP-2） messenger RNA （mRNA） and its signaling pathway. In this study, several cell lines including LLC-PK1 （a porcine kidney tubular epithelial cell line）, MDCK （Madin-Darby canine kidney） and CTLL-2 （cytotoxic T-lymphocyte line） were treated with recombinant human TGF-131, and a series of experiments were carried out, involving Northern blot analysis of total RNA from these cells. Further, several specific chemical inhibitors were applied before TGF-β1 treatment to probe the signaling pathway. The results showed that TGF-β1 can significadtly up-regulate SHARP-2 mRNA expression in the LLC-PK1 cell line. The peak level of induction was found 2 h after TGF-β1 stimulation. While one phospho- inositide 3-kinases （PI-3） kinase inhibitor, LY294002, completely blocked the effect of TGF-131 on SHARP-2 mRNA expression in LLC-PK1 cells at a low concentration, other inhibitors, including PD98059, staurosporine, AG490, wortmannin, okadaic acid and rapamycin, had no effect. The effect of LY294002 was dose-dependent. We conclude that, in LLC-PK1 cells at least, TGF-β1 can effectively induce the SHARP-2 mRNA expression and that the PI-3 kinase pathway can mediate this effect.展开更多
基金supported by the National Basic Research Program of China(No.2009CB941502)
文摘VPS 15 protein is a component of the phosphatidylinositol 3-kinase complex which plays a pivotal role in the development of yeast and mammalian cells. The knowledge about the function of its homologue in plants remains limited. Here we report that AtVPS15, a homologue of yeast VPS15p in Arabidopsis, plays an essential role in pollen germination. Homozygous T-DNA insertion mutants of AtVPS15 could not be obtained from the progenies of self-pollinated heterozygous mutants. Reciprocal crosses between atvps15 mutants and wild-type Arabidopsis revealed that the T-DNA insertion was not able to be transmitted by male gametophytes. DAPI staining, Alexander＇s stain and scanning electron microscopic analysis showed that atvps15 heterozygous plants produced pollen grains that were morphologically indistinguishable from wild-type pollen, whereas in vitro germination experiments revealed that germination of the pollen grains was defective. GUS staining analysis of transgenic plants expressing the GUS reporter gene driven by the AtVPS15 promoter showed that AtVPS15 was mainly expressed in pollen grains. Finally, DUALmembrane yeast two-hybrid analysis demonstrated that AtVPS15 might interact directly with AtVPS34. These results suggest that AtVPS15 is very important for pollen germination, possibly through modulation of the activity of PI3-kinase.
基金Supported by grants to Dr McKinnon (PI) from the New Jersey Commission on Spinal Cord Research (05-304711-015)。
文摘We describe a pre-clinical spinal cord motor neuron injury model that is minimal invasive, reproducible, focal and easily applied to small rodents.Retrograde axonal transport of a pro-apoptotic phosphatidylinosotol 3'-kinase inhibitor, wortmannin, via the sciatic nerve results in loss of ipsilateral lumbar motor neurons proportional to the level of drug administered.Motor neuron loss was detected by choline acetyltransferase(ChAT) immunostaining and with a transgenic thy1-eGFP marker.The short half-life of wortmannin generates minimal wound spread, and wortmannin does not affect axon transport, as determined by co-injection of a pseudorabies virus tracer.Using quantitative transcript analysis, we found that ChAT transcripts significantly decreased at 14 days post-delivery of 1 μg wortmannin, relative to sham controls, and remained low after 90 days.Smaller effects were observed with 200 ng and 100 ng wortmannin.Wortmannin also generated a transient and significant increase in astrocyte Gfap transcripts after 14 days with a return to control levels at 90 days.Treated mice had hind limb spasticity and a forced motor function defect that was quantified using a water exit test.Controls rapidly exit a shallow water tray, and wortmannin treated animals were up to 12-fold slower, a phenotype that persisted for at least 3 months.Thus the focal delivery of wortmannin to motor neurons generates a reproducible and scalable injury that can facilitate quantitative studies on neural regeneration and repair.The efficacy of sciatic nerve suicide transport can also explain neurotoxin-mediated selective loss of motor neurons in diseases such as amyotrophic lateral sclerosis.All procedures were performed at Rutgers under established Institutional Animal Care and Use protocols(eIACUC_TR201800022, approved on March 20, 2018).
基金the Medical Research Council(MR/R004544/1,MR/R004463/1,to RE)EU ERA-NET NEURON(AxonRepair grant,to BN)+1 种基金Fight for Sight(5119/5120,and 5065-5066,to RE)National Eye Research Centre(to RE).
文摘Much research has focused on the PI3-kinase and PTEN signaling pathway with the aim to stimulate repair of the injured central nervous system.Axons in the central nervous system fail to regenerate,meaning that injuries or diseases that cause loss of axonal connectivity have life-changing consequences.In 2008,genetic deletion of PTEN was identified as a means of stimulating robust regeneration in the optic nerve.PTEN is a phosphatase that opposes the actions of PI3-kinase,a family of enzymes that function to generate the membrane phospholipid PIP_(3) from PIP_(2)(phosphatidylinositol(3,4,5)-trisphosphate from phosphatidylinositol(4,5)-bisphosphate).Deletion of PTEN therefore allows elevated signaling downstream of PI3-kinase,and was initially demonstrated to promote axon regeneration by signaling through mTOR.More recently,additional mechanisms have been identified that contribute to the neuron-intrinsic control of regenerative ability.This review describes neuronal signaling pathways downstream of PI3-kinase and PIP3,and considers them in relation to both developmental and regenerative axon growth.We briefly discuss the key neuron-intrinsic mechanisms that govern regenerative ability,and describe how these are affected by signaling through PI3-kinase.We highlight the recent finding of a developmental decline in the generation of PIP_(3) as a key reason for regenerative failure,and summarize the studies that target an increase in signaling downstream of PI3-kinase to facilitate regeneration in the adult central nervous system.Finally,we discuss obstacles that remain to be overcome in order to generate a robust strategy for repairing the injured central nervous system through manipulation of PI3-kinase signaling.
文摘Objective: It is well known that systemic insulin resistance (IR) is closely associated with the metabolic syndrome including type 2 diabetes and hypertension. However, it remains
文摘Berberine is identified to lower the serum cholesterol level in human and hamster through the induction of low density lipoproteins （LDL） receptor in hepatic cells. To evaluate its potential in preventing atherosclerosis, the effect of berberine on atherosclerosis development in apolipoprotcin E-deficient （apoE^-/-） mice was investigated. In apoE^-/- mice, berberine induced in rivo foam cell formation and promoted atheroselerosis development. The foam cell formation induced by berberinc was also observed in mouse RAW264.7 cells, as well as in mouse and human primary macrophages. By inducing scavenger receptor A （SR-A） expression in macrophages, berberine increased the uptake of modified LDL （DiO-Ac-LDL）. Bcrberine-induced SR-A expression was also observed in macrophage foam cells in vivo and in the cells at atherosclerotic lesion. Analysis in RAW264.7 cells indicated that berberine induced SR-A expression by suppressing PTEN expression, which led to sustained Akt activation. Our results suggest that to evaluate the potential of a cholesterol-reducing compound in alleviating atherosclerosis, its effect on the ceils involved in atherosclerosis development, such as macrophages, should also be considered. Promotion of foam cell formation could counter-balance the beneficial effect of lowering serum cholesterol.
基金This work was supported by the National Natural Science Foundation(Grant Nos.39870307,30270561&30430320).
文摘Endothelial nitric oxide synthase (eNOS) is a key enzyme responsible for the regu-lation of vascular homeostasis. Many humor factors and mechanical forces can affect eNOS ac-tivity via phosphorylation modification but the mechanisms involved vary with stimuli applied. We have demonstrated that cytochrome P450 (CYP) epoxygenase-dependent metabolites of ara-chidonic acid, epoxyeicosatrienoic acids (EETs), can robustly up-regulate eNOS expression and its activity, however the relevant signaling pathways responsible for activity regulation are not well known. In this study, we explored the role of PI3 kinase (PI3K)/protein kinase B (Akt) sig-naling pathway in eNOS expression and its phosphorylation in response to EETs via direct addi-tion of EETs into cultured bovine aorta endothelial cells (BAECs) and recombinant adeno- asso-ciated virus-mediated transfection of CYP epoxygenase genes CYPF87V and CYP2C11 to pro-duce endogenous EETs followed by co-treatment with PI3K or Akt inhibitor. Results show that both exogenous and endogenous EETs could remarkably enhance eNOS expression and its phosphorylation at Ser1179 and Thr497 residues; PI3K inhibitor LY294002 could inhibit EETs-induced increase in eNOS-Ser(P)1179 but had no effect on the change of eNOS-Thr(P)497, while Akt inhibitor could attenuate the increase in phosphor-eNOS at both residues; both of the two inhibitors could block EETs-enhanced eNOS expression. These results lead to conclusions: (i) EETs-mediated regulation of eNOS activity may be related with the changes of phosphoryla-tion level at eNOS-Ser1179 via PI3K/Akt and eNOS-Thr497 via Akt; (ii) PI3K/Akt signaling pathway is involved in the up-regulation of eNOS expression by EETs.
文摘A small nonpeptidyl compoud extracted from Pseudomassaria sp. was found to induce the activity of human insulin receptor tyrosine kinase in vitro. The compound was identified as demethylasterriquinone B-1 (DMAQ-B1). DMAQ- B1 also induced an increase in [Ca2+]i and insulin secretion in mice pancreatic beta-cells at low glucose (3 mM) concentration via insulin receptor substrate-1/phosphatidylinositol-3-kinase (PI3 kinase) pathway. By using rat pancreatic perfusion technique, we found that 10 μM DMAQ-B1 directly stimulated insulin secretion up to 240% in normal rat pancreas. In the dosage from 1 to 20 μM, DMAQ-B1 stimulated insulin secretion in a dose dependent manner. Furthermore, DMAQ-B1 enhanced glucose-induced insulin secretion by 17.6% (first stage) and 19.0% (second stage), respectively. The PI3 kinase inhibitors, LY 294002 (3.9 μM) or wortmannin (100 nM), inhibited DMAQ-B1-induced insulin secretion by 46.3% and 57.4%, respectively. LY 294002 or wortmannin also inhibited DMAQ-B1 with10 mMglucose-induced insulin secretion by 70.3% and 79.0%, respectively. All the results suggested that DMAQ-B1 directly stimulated insulin secretion and enhanced glucose-induced insulin secretion. The effect of DMAQ-B1 may mediate through the activation of PI3 kinase pathway to stimulate insulin secretion in normal rat pancreas.
基金supported by the National Natural Science Foundation of China (Nos. 30471641 and 30872389)the Natural Science Foundation of Zhejiang Province, China (No. Y207088)
文摘We aim to investigate the effect of transforming growth factor （TGF）-β1 on the expression of enhancer of split- and hairy-related protein-2 （SHARP-2） messenger RNA （mRNA） and its signaling pathway. In this study, several cell lines including LLC-PK1 （a porcine kidney tubular epithelial cell line）, MDCK （Madin-Darby canine kidney） and CTLL-2 （cytotoxic T-lymphocyte line） were treated with recombinant human TGF-131, and a series of experiments were carried out, involving Northern blot analysis of total RNA from these cells. Further, several specific chemical inhibitors were applied before TGF-β1 treatment to probe the signaling pathway. The results showed that TGF-β1 can significadtly up-regulate SHARP-2 mRNA expression in the LLC-PK1 cell line. The peak level of induction was found 2 h after TGF-β1 stimulation. While one phospho- inositide 3-kinases （PI-3） kinase inhibitor, LY294002, completely blocked the effect of TGF-131 on SHARP-2 mRNA expression in LLC-PK1 cells at a low concentration, other inhibitors, including PD98059, staurosporine, AG490, wortmannin, okadaic acid and rapamycin, had no effect. The effect of LY294002 was dose-dependent. We conclude that, in LLC-PK1 cells at least, TGF-β1 can effectively induce the SHARP-2 mRNA expression and that the PI-3 kinase pathway can mediate this effect.
