Poststro ke cognitive impairment is a major secondary effect of ischemic stroke in many patients;however,few options are available for the early diagnosis and treatment of this condition.The aims of this study were to...Poststro ke cognitive impairment is a major secondary effect of ischemic stroke in many patients;however,few options are available for the early diagnosis and treatment of this condition.The aims of this study were to(1)determine the specific relationship between hypoxic andα-synuclein during the occur of poststroke cognitive impairment and(2)assess whether the serum phosphorylatedα-synuclein level can be used as a biomarker for poststro ke cognitive impairment.We found that the phosphorylatedα-synuclein level was significantly increased and showed pathological aggregation around the cerebral infa rct area in a mouse model of ischemic stroke.In addition,neuronalα-synuclein phosphorylation and aggregation were observed in the brain tissue of mice subjected to chronic hypoxia,suggesting that hypoxia is the underlying cause ofα-synuclein-mediated pathology in the brains of mice with ischemic stroke.Serum phosphorylatedα-synuclein levels in patients with ischemic stroke were significantly lower than those in healt hy subjects,and were positively correlated with cognition levels in patients with ischemic stroke.Furthermore,a decrease in serum high-density lipoprotein levels in stroke patie nts was significantly correlated with a decrease in phosphorylatedα-synuclein levels.Although ischemic stroke mice did not show significant cognitive impairment or disrupted lipid metabolism 14 days after injury,some of them exhibited decreased cognitive function and reduced phosphorylatedα-synuclein levels.Taken together,our results suggest that serum phosphorylatedα-synuclein is a potential biomarker for poststroke cognitive impairment.展开更多
Meiotic resumption in mammalian oocytes involves nuclear and organelle structural changes,notably the chromatin configuration transition from a non-surrounding nucleolus(NSN)to surrounding nucleolus(SN)in germinal ves...Meiotic resumption in mammalian oocytes involves nuclear and organelle structural changes,notably the chromatin configuration transition from a non-surrounding nucleolus(NSN)to surrounding nucleolus(SN)in germinal vesicle oocytes.In the current study,we found that nuclear speckles(NSs),a subnuclear structure mainly composed of serine-arginine(SR)proteins,changed from a diffuse spotted distribution in mouse NSN oocytes to an aggregated pattern in SN oocytes.We also found that the SR protein-specific kinase 1(SRPK1),an enzyme that phosphorylates SR proteins,co-localized with NSs at the SN stage,and that NSN oocytes failed to transition to SN oocytes after the inhibition of SRPK1 activity.Furthermore,the typical structure of the chromatin ring around the nucleolus in SN oocytes collapsed after treatment with an SRPK1 inhibitor.Mechanistically,phosphorylated SR proteins were found to be related to chromatin as shown by a salt extraction experiment,and in situ DNaseⅠassay showed that the accessibility of chromatin was enhanced in SN oocytes when SRPK1 was inhibited,accompanied by a decreased repressive modification on histone and the abnormal recurrence of a transcriptional signal.In conclusion,our results indicated that SRPK1-regulated phosphorylation of SR proteins was involved in the NSN-SN transition and played an important role in maintaining the condensed nucleus of SN oocytes via interacting with chromatin.展开更多
Objectives:Weaning induces oxidative stress in pigs,increasing the risk of diarrhea and death.Intestinal damage is associated with obstructed intestinal cell cycles.To stop damage caused by reactive oxygen species(ROS...Objectives:Weaning induces oxidative stress in pigs,increasing the risk of diarrhea and death.Intestinal damage is associated with obstructed intestinal cell cycles.To stop damage caused by reactive oxygen species(ROS),N-acetyl cysteine(NAC)has been widely employed.In this study,we examined changes in the intestinal cyclin of weaning piglets and assessed the impact of NAC on intestinal cell cycle arrest and intracellular signaling pathways.Methods:We conducted two animal experiments.In the first,we divided 12 litters of 120 newborn piglets into two groups:a control group and a weaning group.The control piglets were allowed to suckle normally.The weaning group was weaned after 3 weeks and fed a normal diet for piglets.We slaughtered six piglets from the control group and six from the weaning group.We observed cyclin changes and intestinal development at days 0,1,4,and 7 after weaning.In the second experiment,we divided 15 litters of 150 piglets that were 2 weeks old into three groups:the control group,the weaning group,and the NAC group.Control piglets were allowed to suckle normally.Piglets in the weaning and NAC groups were weaned when they were 21 days old.The NAC group was fed a basal diet supplemented with 500 mg/kg NAC,and the weaning group was fed the basal diet alone.The experimental period was 14–25 days of age.Four days after weaning,we slaughtered one piglet from each litter.We then analyzed intestinal cell cycle indexes,intestinal oxidative stress,c-Jun N-terminal kinase(JNK),extracellular signal-regulated kinase(ERK),and p38 phosphorylation.Results:Weaning decreased the piglets’feed intake and daily gain,reduced the serum antioxidant capacity,and increased the intestinal ROS level.Furthermore,the jejunum histology and barrier development of the jejunum exhibited damage after weaning,the microvilli displayed hypoplasia,and the p21 and p27 protein expression levels of the jejunum were significantly elevated.We did not observe any significant differences in cyclin D and E after days 1,4,and 7 post-weaning compared with the control group.We observed,however,significantly increased cyclin D and E expression,lower ERK,JNK,and p38 kinase phosphorylation;villus atrophy alleviation;decreased p21 and p27 expression;and increased average daily intake of feed and weight gain.Conclusion:This research demonstrates that weaning stress inhibits piglet intestinal proliferation by reducing cyclin D and cyclin E expression.NAC downregulates p21 and p27 through modulating mitogen-activated protein kinases(MAPKase)phosphorylation,thereby promoting cell proliferation.The results indicate that NAC promotes intestinal function and the integrity of enterocytes and holds promise as a new feed additive for animal health.展开更多
Objective Machado-Joseph disease （MJD）/Spinocerebellar ataxia type 3 （SCA3） is an autosomal dominant neurodegenerative disorder caused by an expansion of polyglutamine tract near the C-terminus of the MJD1 gene pr...Objective Machado-Joseph disease （MJD）/Spinocerebellar ataxia type 3 （SCA3） is an autosomal dominant neurodegenerative disorder caused by an expansion of polyglutamine tract near the C-terminus of the MJD1 gene product, ataxin-3. The precise mechanism of the MJD/SCA3 pathogenesis remains unclear. A growing body of evidence demonstrates that phosphorylation plays an important role in the pathogenesis of many neurodegenerative diseases. However, few kinases are known to phosphorylate ataxin-3. The present study is to explore whether ataxin-3 is a substrate of casein kinase 2 （CK2）. Methods The interaction between ataxin-3 and CK2 was identified by glutathione S-transferase （GST） pull-down assay and co-immunoprecipition assay. The phosphorylation of ataxin-3 by CK2 was measured by in vitro phosphorylation assays. Results （1） Both wild type and expanded ataxin-3 interacted with CK2α and CK2β in vitro. （2） In 293 cells, both wild type and expanded ataxin-3 interacted with CK2β, but not CK2α. （3） CK2 phosphorylated wild type and expanded ataxin-3. Conclusion Ataxin-3 is a substrate of protein kinase CK2.展开更多
Parathyroid hormone(PTH) regulates bone remodeling by activating PTH type 1 receptor(PTH1R) in osteoblasts/osteocytes. Insulinlike growth factor type 1(IGF-1) stimulates mesenchymal stem cell differentiation to osteob...Parathyroid hormone(PTH) regulates bone remodeling by activating PTH type 1 receptor(PTH1R) in osteoblasts/osteocytes. Insulinlike growth factor type 1(IGF-1) stimulates mesenchymal stem cell differentiation to osteoblasts. However, little is known about the signaling mechanisms that regulates the osteoblast-to-osteocyte transition. Here we report that PTH and IGF-I synergistically enhance osteoblast-to-osteocyte differentiation. We identified that a specific tyrosine residue, Y494, on the cytoplasmic domain of PTH1R can be phosphorylated by insulin-like growth factor type I receptor(IGF1R) in vitro. Phosphorylated PTH1R localized to the barbed ends of actin filaments and increased actin polymerization during morphological change of osteoblasts into osteocytes.Disruption of the phosphorylation site reduced actin polymerization and dendrite length. Mouse models with conditional ablation of PTH1R in osteoblasts demonstrated a reduction in the number of osteoctyes and dendrites per osteocyte, with complete overlap of PTH1R with phosphorylated-PTH1R positioning in osteocyte dendrites in wild-type mice. Thus, our findings reveal a novel signaling mechanism that enhances osteoblast-to-osteocyte transition by direct phosphorylation of PTH1R by IGF1R.展开更多
Different hydroxy substituted coumarins were successfully phosphorylated with diisopropylphophite （DIPPH） by the Atherton-Todd reaction in 76-89% yields. Moreover, the reaction activities of different hydroxys of th...Different hydroxy substituted coumarins were successfully phosphorylated with diisopropylphophite （DIPPH） by the Atherton-Todd reaction in 76-89% yields. Moreover, the reaction activities of different hydroxys of the coumarins in the Atherton-Todd reaction were studied.展开更多
three kinds of N-(diisopropyloxyphosphoryl) amino acids containing hydroxyl group were prepared in high yield by using diisopropyl phosphite as the phosphorylating agent, sodium hypochlorite as the chlorinating agent ...three kinds of N-(diisopropyloxyphosphoryl) amino acids containing hydroxyl group were prepared in high yield by using diisopropyl phosphite as the phosphorylating agent, sodium hypochlorite as the chlorinating agent and tetrabutyl ammonium bromide as the phase transfer catalyst in basic aqueous media.展开更多
O-Phosphoryl serine derivative can perform self-catalytic esterification reaction in the mixture of CH3OH and CHCl3 at the room temperature. The phosphoryl group participation was the key step of the esterification. T...O-Phosphoryl serine derivative can perform self-catalytic esterification reaction in the mixture of CH3OH and CHCl3 at the room temperature. The phosphoryl group participation was the key step of the esterification. This type of reactions were proposed through an intermediate of mixed phosphoric-carboxylic anhydride that might provide a clue to the function of the phosphoryl group in the phosphorylated enzymes and in the prebiotic synthesis of protein.展开更多
One of the prominent cell cycle related modifications of histone proteins, whose function is correlated with chromosome condensation, is the phosphorylation of histone H3. Wheat (Triticum aestivum L.) mitotic and meio...One of the prominent cell cycle related modifications of histone proteins, whose function is correlated with chromosome condensation, is the phosphorylation of histone H3. Wheat (Triticum aestivum L.) mitotic and meiotic cells were analyzed with indirect immunoflurorescence labeling with an antibody recognizing histone H3 phosphorylated at Serine 10 to study the localization of phosphorylated histone H3 at mitosis and meiosis. Our results showed that, during mitotic division, the phosphoryiation of H3 started from early prophase and vanished at telophase, remaining mainly in the pericentromeric regions at metaphase and anaphase. During meiotic division, phosphorylation of H3 initiated at the transition from leptotene to zygotene and remained uniform, along the chromosomes from prophase I until telophase whereas it showed slightly stronger in the pericentromeric regions than along the chromosome arms from metaphase II until Lelophase II The different patterns of H3 phophorylation at mitosis and meiosis in wheat suggested that this evolutionarily conserved post-translational chromatin modification might be involved in more roles besides chromosome condensation.展开更多
Spermatozoa are highly specialized cells. Adenosine triphosphate （ATP）, which provides the energy for supporting the key functions of the spermatozoa, is formed by 2 metabolic pathways, namely glycolysis and oxidati...Spermatozoa are highly specialized cells. Adenosine triphosphate （ATP）, which provides the energy for supporting the key functions of the spermatozoa, is formed by 2 metabolic pathways, namely glycolysis and oxidative phosphorylation （OXPHOS）. It is produced in the mitochondria through OXPHOS as well as in the head and principal piece of the flagellum through glycolysis. However, there is a great discrepancy as to which method of ATP production is primarily utilized by the spermatozoa for successful fertilization. Mitochondrial respiration is considered to be a more efficient metabolic process for ATP synthesis in comparison to glycolysis. However, studies have shown that the diffusion potential of ATP from the mitochondria to the distal end of the flagellum is not sufficient to support sperm motility, suggesting that glycolysis in the tail region is the preferred pathway for energy production. It is suggested by many investigators that although glycolysis forms the major source of ATP along the flagellum, energy required for sperm motility is mainly produced during mitochondrial respiration. Nevertheless, some studies have shown that when glycolysis is inhibited, proper functioning and motility of spermatozoa remains intact although it is unclear whether such motility can be sustained for prolonged periods of time, or is sufficiently vigorous to achieve optimal fertilization. The purpose of this article is to provide an overview of mammalian sperm energy metabolism and identify the preferred metabolic pathway for ATP generation which forms the basis of energy Droduction in human spermatozoa during fertilization.展开更多
Sexually reproducing animals require an orchestrated communication between spermatozoa and the egg to generate a new individual. Capacitation, a maturational complex phenomenon that occurs in the female reproductive t...Sexually reproducing animals require an orchestrated communication between spermatozoa and the egg to generate a new individual. Capacitation, a maturational complex phenomenon that occurs in the female reproductive tract, renders spermatozoa capable of binding and fusing with the oocyte, and it is a requirement for mammalian fertilization. Capacitation encompasses plasma membrane reorganization, ion permeability regulation, cholesterol loss and changes in the phosphorylation state of many proteins. Novel tools to study sperm ion channels, image intracellular ionic changes and proteins with better spatial and temporal resolution, are unraveling how modifications in sperm ion transport and phosphorylation states lead to capacitation. Recent evidence indicates that two parallel pathways regulate phosphorylation events leading to capacitation, one of them requiring activation of protein kinase A and the second one involving inactivation of ser/thr phosphatases. This review examines the involvement of ion transporters and phosphorylation signaling processes needed for spermatozoa to achieve capacitation. Understanding the molecular mechanisms leading to fertilization is central for societies to deal with rising male infertility rates, to develop safe male gamete-based contraceptives and to preserve biodiversity through better assisted fertilization strategies.展开更多
NPR1 （Nonexpressor of Pathogenesis-Related gene 1） is a major co-activator of plant defense. Phosphorylations of NPR1 play important roles in fine-tuning its activity, however a kinase corresponding to such modifica...NPR1 （Nonexpressor of Pathogenesis-Related gene 1） is a major co-activator of plant defense. Phosphorylations of NPR1 play important roles in fine-tuning its activity, however a kinase corresponding to such modification remains uncharacterized. Here, we report that NPR1 interacts with PKS5 （SOS2-1ike Protein Kinase 5）. The AKR （AnKyrin Repeats） motif of NPR1 is required for this interaction. PKS5 phosphorylates NPR1 at the C-terminal region. Expression of PKS5 is induced quickly by Pseudomonas syringae pv. tomato DC3000. Expression level of two NPR1 target genes, WRKY38 and WRKY62, is reduced and/or delayed in pks5 mutants. Moreover, the expression of WRKY38 and WRKY62 displays a similar pattern in nprl-lpks5-1 double mutant comparing to that in nprl-1. Our results suggest that PKS5 functions at the upstream of NPR1 and might mediate expression of WRKY38 and WRKY62 possibly by interacting with and phosphorylating NPR 1.展开更多
AIM: To investigate the molecular signaling mechanism by which the plant-derived, pentacyclic triterpene maslinic acid(MA) exerts anti-diabetic effects. METHOD: HepG2 cells were stimulated with various concentrations ...AIM: To investigate the molecular signaling mechanism by which the plant-derived, pentacyclic triterpene maslinic acid(MA) exerts anti-diabetic effects. METHOD: HepG2 cells were stimulated with various concentrations of MA. The effects of MA on glycogen phosphorylase a(GPa) activity and the cellular glycogen content were measured. Western blot analyses were performed with anti-insulin receptor β(IRβ), protein kinase B(also known as Akt), and glycogen synthase kinase-3β(GSK3β) antibodies. Activation status of the insulin pathway was investigated using phospho-IRβ, as well as phospho-Akt, and phospho-GSK3β antibodies. The specific PI3-kinase inhibitor wortmannin was added to the cells to analyze the Akt expression. Enzyme-linked immunosorbent assay(ELISA) was used to measure the effect of MA on IRβ auto-phosphorylation. Furthermore, the effect of MA on glycogen metabolism was investigated in C57BL/6J mice fed with a high-fat diet(HFD). RESULTS: The results showed that MA exerts anti-diabetic effects by increasing glycogen content and inhibiting glycogen phosphorylase activity in HepG2 cells. Furthermore, MA was shown to induce the phosphorylation level of IRβ-subunit, Akt, and GSK3β. The MA-induced activation of Akt appeared to be specific, since it could be blocked by wortmannin. Finally, MA treatment of mice fed with a high-fat diet reduced the model-associated adiposity and insulin resistance, and increased the accumulated hepatic glycogen content. CONCLUSION: The results suggested that maslinic acid modulates glycogen metabolism by enhancing the insulin signaling pathway and inhibiting glycogen phosphorylase.展开更多
Dendritic cells (DCs) are the most potent antigen-presen ting cells that play crucial roles in the regulation of immune response. Triptol ide, an active component purified from the medicinal plant Tripterygium wilfor ...Dendritic cells (DCs) are the most potent antigen-presen ting cells that play crucial roles in the regulation of immune response. Triptol ide, an active component purified from the medicinal plant Tripterygium wilfor dii Hook F., has been demonstrated to act as a potent immunosuppressive drug c apab le of inhibiting T cell activation and proliferation. However, little is known a bout the effects of triptolide on DCs. The present study shows that triptolide d oes not affect phenotypic differentiation and LPS-induced maturation of murine DCs. But triptolide can dramatically reduce cell recovery by inducing apoptosis of DCs at concentration as low as 10 ng/ml, as demonstrated by phosphatidylserin e exposure, mitochondria potential decrease, and nuclear DNA condensation. Tript olide induces activation of p38 in DCs, which precedes the activation of caspase 3. SB203580, a specific kinase inhibitor for p38, can block the activation of caspase 3 and inhibit the resultant apoptosis of DCs. Our results suggest that t he anti-inflammatory and immunosuppressive activities of triptolide may be due, in part, to its apoptosis-inducing effects on DCs.展开更多
Hyperphosphorylated tau is the major protein component of neurofibrillary tangles in the brains of patients with Alzheimer’s disease(AD). However, the mechanism underlying tau hyperphosphorylation is not fully unders...Hyperphosphorylated tau is the major protein component of neurofibrillary tangles in the brains of patients with Alzheimer’s disease(AD). However, the mechanism underlying tau hyperphosphorylation is not fully understood. Here, we demonstrated that exogenously expressed wild-type human tau40 was detectable in the phosphorylated form at multiple AD-associated sites in cytoplasmic and nuclear fractions from HEK293 cells.Among these sites, tau phosphorylated at Thr205 and Ser214 was almost exclusively found in the nuclear fraction at the conditions used in the present study. With the intracellular tau accumulation, the Ca2+concentration was significantly increased in both cytoplasmic and nuclear fractions. Further studies using site-specific mutagenesis and pharmacological treatment demonstrated that phosphorylation of tau at Thr205 increased nuclear Ca2+concentration with a simultaneous increase in the phosphorylation of Ca2+/calmodulin-dependent protein kinase IV(Ca MKIV) at Ser196. On the other hand, phosphorylation of tau at Ser214 did not significantly change the nuclear Ca2+/Ca MKIV signaling. Finally, expressing calmodulin-binding protein-4 that disrupts formation ofthe Ca2+/calmodulin complex abolished the okadaic acidinduced tau hyperphosphorylation in the nuclear fraction.We conclude that the intracellular accumulation of phosphorylated tau, as detected in the brains of AD patients, can trigger nuclear Ca2+/Ca MKIV signaling, which in turn aggravates tau hyperphosphorylation. Our findings provide new insights for tauopathies: hyperphosphorylation of intracellular tau and an increased Ca2+concentration may induce a self-perpetuating harmful loop to promote neurodegeneration.展开更多
Nowadays,the cumulative intake of glucocorticoids has become the most common pathogenic factor for non-traumatic osteonecrosis of the femoral head(ONFH).Apoptosis of osteoblasts is considered as the main reason of ONF...Nowadays,the cumulative intake of glucocorticoids has become the most common pathogenic factor for non-traumatic osteonecrosis of the femoral head(ONFH).Apoptosis of osteoblasts is considered as the main reason of ONFH at the molecular level.Glycogen synthase kinase 3β(GSK3β)is an important regulator of cellular differentiation and apoptosis pathway,which can modulate the balance between osteoblasts and osteoclasts.Several studies have reported about its function in osteoporosis,but little is known about it in osteonecrosis.In our study,lipopolysaccharide and methylprednisolone were utilized to establish a rat ONFH model.The phosphorylation of GSK3βSer-9 was decreased in the model.Western blotting examination ofβ-catenin,Bcl-2,Bax and caspase-3 revealed that the osteoblasts were apoptotic.In dexamethasone(Dex)-incubated primary osteoblasts,the expression profile of GSK3βphosphorylation and apoptotic factors were consistent with those in the rat ONFH model.To further investigate the regulation of osteonecrosis caused by GSK3β,the expression and function of GSK3βwere inhibited in Dex-incubated primary osteoblasts.The knockdown of GSK3βby siRNA decreased the expression of Bax and cleaved caspase-3,but increased Bcl-2 andβ-catenin.On the other hand,selective inhibition of GSK3βfunction by LiCl counteracted the activation of caspase-3 induced by Dex.Our work is the first study about the GSK3P phosphorylation in ONFH,and provides evidence for further therapeutic methods.展开更多
Objective: Tumor cells rely heavily on glycolysis regardless of oxygen tension, a phenomenon called the Warburg effect. Hexokinase II(HKII) catalyzes the first irreversible step of glycolysis and is often overexpresse...Objective: Tumor cells rely heavily on glycolysis regardless of oxygen tension, a phenomenon called the Warburg effect. Hexokinase II(HKII) catalyzes the first irreversible step of glycolysis and is often overexpressed in tumor cells. Mitochondrial HKII couples glycolysis and oxidative phosphorylation while maintaining mitochondrial membrane integrity. In this study, we investigated the role of HKII in promoting the Warburg effect in cancer cells.Methods: HKII-mediated phosphorylation of the alpha subunit of pyruvate dehydrogenase(PDHA1) was tested in HEK293 T cells and clear cell renal cell carcinoma(cc RCC) specimens using gene knockdown, western blotting,immunohistochemistry, and immunofluorescence.Results: It was determined that HKII could not only transform glucose into glucose-6-phosphate, but also transfer the phosphate group of ATP onto PDHA1. In addition, it was found that HKII increased the phosphorylation of Ser293 on PDHA1, decreasing pyruvate dehydrogenase(PDH) complex activity and thus rerouting the metabolic pathway and promoting the Warburg effect. The overexpression of HKII correlated with the phosphorylation of PDHA1 and disease progression in cc RCC.Conclusions: The data presented here suggest that HKII is an important biomarker in the evaluation and treatment of cancer.展开更多
The reversing effect of wild-type PTEN gene on resistance of C 13K cells to cisplatin and its inhibitory effect on the phosphorylation of protein kinase B （AKT） were studied. The expression of PTEN mRNA and protein ...The reversing effect of wild-type PTEN gene on resistance of C 13K cells to cisplatin and its inhibitory effect on the phosphorylation of protein kinase B （AKT） were studied. The expression of PTEN mRNA and protein in OV2008 cells and C13K cells were semi-quantitatively detected by using RT-PCR and Western blotting. Recombinant eukaryotic expression plasmid containing human wild-type PTEN gene was transfected into C13K cells by lipofectamine2000. The expression of PTEN mRNA was monitored by RT-PCR and the expression of PTEN, Akt, p-Akt protein were ana- lyzed by Western blotting in PTEN-transfected and non-transfected C13K cells. Proliferation and chemosensitivity of cells to DDP were measured by MTT, and cell apoptosis was detected by flow cytometry after treatment with cisplatin. The expression of PTEN mRNA and protein in OV2008 cells were significantly higher than those in C13K cells. After transfection with PTEN gene for 48 h, the expression of PTEN mRNA and protein in C 13K cells were 2.04 ± 0.10, 0.94± 0.04 respectively and the expression of p-Akt protein （ 0.94± 0.07） was lower than those in control groups （1.68 ±0.14, 1.66± 0.10） （P〈 0.05）. The IC50 of DDP to C 13 K cells transfected with PTEN （7.2± 0.3 la mol/L） was obviously lower than those of empty-vector transfected cells and non-transfected cells （12.7±0.4 lamol/1, 13.0±0.3 lamol/L） （P〈0.05）. The apopototis ratio of wild-type PTEN-transfected, empty vector transfected and non-transfected C13K cells were （41.65___0.87）%, （18.61 ±0.70）% and （15.28±0.80）% respectively, and the difference was statistically significant （P〈0.05）. PTEN gene plays an important role in ovarian cancer multidrug resistance. Transfection of PTEN could increase the expression of PTEN and restore drug sensitivity to cisplatin in human ovarian cancer cell line C 13K with multidrug-resistance by decreasing the expression of p-Akt.展开更多
Astrocytes and microglia play an orchestrated role following spinal cord injury;however,the molecular mechanisms through which microglia regulate astrocytes after spinal cord injury are not yet fully understood.Herein...Astrocytes and microglia play an orchestrated role following spinal cord injury;however,the molecular mechanisms through which microglia regulate astrocytes after spinal cord injury are not yet fully understood.Herein,microglia were pharmacologically depleted and the effects on the astrocytic response were examined.We further explored the potential mechanisms involving the signal transducers and activators of transcription 3(STAT3)pathway.For in vivo experiments,we constructed a contusion spinal cord injury model in C57BL/6 mice.To deplete microglia,all mice were treated with colony-stimulating factor 1 receptor inhibitor PLX3397,starting 2 weeks prior to surgery until they were sacrificed.Cell proliferation was examined by 5-ethynyl-2-deoxyuridine(EdU)and three pivotal inflammatory cytokines were detected by a specific Bio-Plex Pro^(TM) Reagent Kit.Locomotor function,neuroinflammation,astrocyte activation and phosphorylated STAT3(pSTAT3,a maker of activation of STAT3 signaling)levels were determined.For in vitro experiments,a microglia and astrocyte coculture system was established,and the small molecule STA21,which blocks STAT3 activation,was applied to investigate whether STAT3 signaling is involved in mediating astrocyte proliferation induced by microglia.PLX3397 administration disrupted glial scar formation,increased inflammatory spillover,induced diffuse tissue damage and impaired functional recovery after spinal cord injury.Microglial depletion markedly reduced EdU+proliferating cells,especially proliferating astrocytes at 7 days after spinal cord injury.RNA sequencing analysis showed that the JAK/STAT3 pathway was downregulated in mice treated with PLX3397.Double immunofluorescence staining confirmed that PLX3397 significantly decreased STAT3 expression in astrocytes.Importantly,in vitro coculture of astrocytes and microglia showed that microglia-induced astrocyte proliferation was abolished by STA21 administration.These findings suggest that microglial depletion impaired astrocyte proliferation and astrocytic scar formation,and induced inflammatory diffusion partly by inhibiting STAT3 phosphorylation in astrocytes following spinal cord injury.展开更多
3-hydroxy-3-methylgulutaryl-coenzyme A （HMG-CoA） reductase inhibitors or statins are a kind of lipid-lowering agents and have been used for the prevention and treatment of Cardiovascular diseases. Recent studies sug...3-hydroxy-3-methylgulutaryl-coenzyme A （HMG-CoA） reductase inhibitors or statins are a kind of lipid-lowering agents and have been used for the prevention and treatment of Cardiovascular diseases. Recent studies suggested that statins, besides lowering cholesterol, may protect vessels by enhancing the activity of endothelial nitric oxide synthase （eNOS）. In the present study, we investigated if simvastatin increases eNOS activity through its phosphorylation in 293 cells （293-eNOS） with stable expression of eNOS. The results showed that incubation of 293-eNOS cells with simvastatin （10 μm/L） for 2 h significantly increased in the activity of eNOS as shown by the conversion of L-arginine to L-citrulline （2889.70±201.51 versus 5630.18＋218.75 pmol/min . mg proteins） （P〈0.01）. Western blotting revealed that simvastatin increased phosphorylation of eNOS at 1177 （ser） and also 495 （thr） but did not affect the overall expression of eNOS or inducible NOS. Further study found that simvastatin raised phosphorylation levels of Akt and AMPK, and such effect could be antagonized by Akt inhibitor or AMPK inhibitor. These results suggest that simvastatin could stimulate,the activity of eNOS via its phosphorylation by Akt and AMPK, which provides a new mechanism, other than lipid-lowering effect, for the cardiovascular protection of statins.展开更多
基金supported by the Scientific Research Project of China Rehabilitation Research Center,No.2021zx-23the National Natural Science Foundation of China,No.32100925the Beijing Nova Program,No.Z211100002121038。
文摘Poststro ke cognitive impairment is a major secondary effect of ischemic stroke in many patients;however,few options are available for the early diagnosis and treatment of this condition.The aims of this study were to(1)determine the specific relationship between hypoxic andα-synuclein during the occur of poststroke cognitive impairment and(2)assess whether the serum phosphorylatedα-synuclein level can be used as a biomarker for poststro ke cognitive impairment.We found that the phosphorylatedα-synuclein level was significantly increased and showed pathological aggregation around the cerebral infa rct area in a mouse model of ischemic stroke.In addition,neuronalα-synuclein phosphorylation and aggregation were observed in the brain tissue of mice subjected to chronic hypoxia,suggesting that hypoxia is the underlying cause ofα-synuclein-mediated pathology in the brains of mice with ischemic stroke.Serum phosphorylatedα-synuclein levels in patients with ischemic stroke were significantly lower than those in healt hy subjects,and were positively correlated with cognition levels in patients with ischemic stroke.Furthermore,a decrease in serum high-density lipoprotein levels in stroke patie nts was significantly correlated with a decrease in phosphorylatedα-synuclein levels.Although ischemic stroke mice did not show significant cognitive impairment or disrupted lipid metabolism 14 days after injury,some of them exhibited decreased cognitive function and reduced phosphorylatedα-synuclein levels.Taken together,our results suggest that serum phosphorylatedα-synuclein is a potential biomarker for poststroke cognitive impairment.
基金National Natural Science Foundation of China(Grant Nos.32070838 and 82301874)Open Fund of State Key Laboratory of Reproductive Medicine,Nanjing Medical University(Grant No.SKLRM K202102)。
文摘Meiotic resumption in mammalian oocytes involves nuclear and organelle structural changes,notably the chromatin configuration transition from a non-surrounding nucleolus(NSN)to surrounding nucleolus(SN)in germinal vesicle oocytes.In the current study,we found that nuclear speckles(NSs),a subnuclear structure mainly composed of serine-arginine(SR)proteins,changed from a diffuse spotted distribution in mouse NSN oocytes to an aggregated pattern in SN oocytes.We also found that the SR protein-specific kinase 1(SRPK1),an enzyme that phosphorylates SR proteins,co-localized with NSs at the SN stage,and that NSN oocytes failed to transition to SN oocytes after the inhibition of SRPK1 activity.Furthermore,the typical structure of the chromatin ring around the nucleolus in SN oocytes collapsed after treatment with an SRPK1 inhibitor.Mechanistically,phosphorylated SR proteins were found to be related to chromatin as shown by a salt extraction experiment,and in situ DNaseⅠassay showed that the accessibility of chromatin was enhanced in SN oocytes when SRPK1 was inhibited,accompanied by a decreased repressive modification on histone and the abnormal recurrence of a transcriptional signal.In conclusion,our results indicated that SRPK1-regulated phosphorylation of SR proteins was involved in the NSN-SN transition and played an important role in maintaining the condensed nucleus of SN oocytes via interacting with chromatin.
基金supported by the Jilin Agricultural Science and Technology University under the Scientific Startup Foundation for Doctors((2022)733)Shanghai Jiao Tong University under the National Natural Science Foundation of China(30972103).
文摘Objectives:Weaning induces oxidative stress in pigs,increasing the risk of diarrhea and death.Intestinal damage is associated with obstructed intestinal cell cycles.To stop damage caused by reactive oxygen species(ROS),N-acetyl cysteine(NAC)has been widely employed.In this study,we examined changes in the intestinal cyclin of weaning piglets and assessed the impact of NAC on intestinal cell cycle arrest and intracellular signaling pathways.Methods:We conducted two animal experiments.In the first,we divided 12 litters of 120 newborn piglets into two groups:a control group and a weaning group.The control piglets were allowed to suckle normally.The weaning group was weaned after 3 weeks and fed a normal diet for piglets.We slaughtered six piglets from the control group and six from the weaning group.We observed cyclin changes and intestinal development at days 0,1,4,and 7 after weaning.In the second experiment,we divided 15 litters of 150 piglets that were 2 weeks old into three groups:the control group,the weaning group,and the NAC group.Control piglets were allowed to suckle normally.Piglets in the weaning and NAC groups were weaned when they were 21 days old.The NAC group was fed a basal diet supplemented with 500 mg/kg NAC,and the weaning group was fed the basal diet alone.The experimental period was 14–25 days of age.Four days after weaning,we slaughtered one piglet from each litter.We then analyzed intestinal cell cycle indexes,intestinal oxidative stress,c-Jun N-terminal kinase(JNK),extracellular signal-regulated kinase(ERK),and p38 phosphorylation.Results:Weaning decreased the piglets’feed intake and daily gain,reduced the serum antioxidant capacity,and increased the intestinal ROS level.Furthermore,the jejunum histology and barrier development of the jejunum exhibited damage after weaning,the microvilli displayed hypoplasia,and the p21 and p27 protein expression levels of the jejunum were significantly elevated.We did not observe any significant differences in cyclin D and E after days 1,4,and 7 post-weaning compared with the control group.We observed,however,significantly increased cyclin D and E expression,lower ERK,JNK,and p38 kinase phosphorylation;villus atrophy alleviation;decreased p21 and p27 expression;and increased average daily intake of feed and weight gain.Conclusion:This research demonstrates that weaning stress inhibits piglet intestinal proliferation by reducing cyclin D and cyclin E expression.NAC downregulates p21 and p27 through modulating mitogen-activated protein kinases(MAPKase)phosphorylation,thereby promoting cell proliferation.The results indicate that NAC promotes intestinal function and the integrity of enterocytes and holds promise as a new feed additive for animal health.
基金the National Natural Sciences Foundation of China (No. 30770664)a grant from Educational Committee of Anhui Province, China (No. ZD2008008-2).
文摘Objective Machado-Joseph disease （MJD）/Spinocerebellar ataxia type 3 （SCA3） is an autosomal dominant neurodegenerative disorder caused by an expansion of polyglutamine tract near the C-terminus of the MJD1 gene product, ataxin-3. The precise mechanism of the MJD/SCA3 pathogenesis remains unclear. A growing body of evidence demonstrates that phosphorylation plays an important role in the pathogenesis of many neurodegenerative diseases. However, few kinases are known to phosphorylate ataxin-3. The present study is to explore whether ataxin-3 is a substrate of casein kinase 2 （CK2）. Methods The interaction between ataxin-3 and CK2 was identified by glutathione S-transferase （GST） pull-down assay and co-immunoprecipition assay. The phosphorylation of ataxin-3 by CK2 was measured by in vitro phosphorylation assays. Results （1） Both wild type and expanded ataxin-3 interacted with CK2α and CK2β in vitro. （2） In 293 cells, both wild type and expanded ataxin-3 interacted with CK2β, but not CK2α. （3） CK2 phosphorylated wild type and expanded ataxin-3. Conclusion Ataxin-3 is a substrate of protein kinase CK2.
基金provided by K01-AR060433 (T.Q.)K08-AR064833 (J.C)R01-AR063943 (X.C)
文摘Parathyroid hormone(PTH) regulates bone remodeling by activating PTH type 1 receptor(PTH1R) in osteoblasts/osteocytes. Insulinlike growth factor type 1(IGF-1) stimulates mesenchymal stem cell differentiation to osteoblasts. However, little is known about the signaling mechanisms that regulates the osteoblast-to-osteocyte transition. Here we report that PTH and IGF-I synergistically enhance osteoblast-to-osteocyte differentiation. We identified that a specific tyrosine residue, Y494, on the cytoplasmic domain of PTH1R can be phosphorylated by insulin-like growth factor type I receptor(IGF1R) in vitro. Phosphorylated PTH1R localized to the barbed ends of actin filaments and increased actin polymerization during morphological change of osteoblasts into osteocytes.Disruption of the phosphorylation site reduced actin polymerization and dendrite length. Mouse models with conditional ablation of PTH1R in osteoblasts demonstrated a reduction in the number of osteoctyes and dendrites per osteocyte, with complete overlap of PTH1R with phosphorylated-PTH1R positioning in osteocyte dendrites in wild-type mice. Thus, our findings reveal a novel signaling mechanism that enhances osteoblast-to-osteocyte transition by direct phosphorylation of PTH1R by IGF1R.
基金The authors thank the financial supports from the National Natural Science Foundation of China(No.20132020)the Ministry of Science and Technology,the Chinese Ministry of Education and Zhengzhou University.
文摘Different hydroxy substituted coumarins were successfully phosphorylated with diisopropylphophite （DIPPH） by the Atherton-Todd reaction in 76-89% yields. Moreover, the reaction activities of different hydroxys of the coumarins in the Atherton-Todd reaction were studied.
文摘three kinds of N-(diisopropyloxyphosphoryl) amino acids containing hydroxyl group were prepared in high yield by using diisopropyl phosphite as the phosphorylating agent, sodium hypochlorite as the chlorinating agent and tetrabutyl ammonium bromide as the phase transfer catalyst in basic aqueous media.
基金The authors would like to thank the financial supports from the National Natural Science Foundation of China(No.20272032 and No.20320130046)Ministry of Education of China and Tsinghua University.
文摘O-Phosphoryl serine derivative can perform self-catalytic esterification reaction in the mixture of CH3OH and CHCl3 at the room temperature. The phosphoryl group participation was the key step of the esterification. This type of reactions were proposed through an intermediate of mixed phosphoric-carboxylic anhydride that might provide a clue to the function of the phosphoryl group in the phosphorylated enzymes and in the prebiotic synthesis of protein.
文摘One of the prominent cell cycle related modifications of histone proteins, whose function is correlated with chromosome condensation, is the phosphorylation of histone H3. Wheat (Triticum aestivum L.) mitotic and meiotic cells were analyzed with indirect immunoflurorescence labeling with an antibody recognizing histone H3 phosphorylated at Serine 10 to study the localization of phosphorylated histone H3 at mitosis and meiosis. Our results showed that, during mitotic division, the phosphoryiation of H3 started from early prophase and vanished at telophase, remaining mainly in the pericentromeric regions at metaphase and anaphase. During meiotic division, phosphorylation of H3 initiated at the transition from leptotene to zygotene and remained uniform, along the chromosomes from prophase I until telophase whereas it showed slightly stronger in the pericentromeric regions than along the chromosome arms from metaphase II until Lelophase II The different patterns of H3 phophorylation at mitosis and meiosis in wheat suggested that this evolutionarily conserved post-translational chromatin modification might be involved in more roles besides chromosome condensation.
文摘Spermatozoa are highly specialized cells. Adenosine triphosphate （ATP）, which provides the energy for supporting the key functions of the spermatozoa, is formed by 2 metabolic pathways, namely glycolysis and oxidative phosphorylation （OXPHOS）. It is produced in the mitochondria through OXPHOS as well as in the head and principal piece of the flagellum through glycolysis. However, there is a great discrepancy as to which method of ATP production is primarily utilized by the spermatozoa for successful fertilization. Mitochondrial respiration is considered to be a more efficient metabolic process for ATP synthesis in comparison to glycolysis. However, studies have shown that the diffusion potential of ATP from the mitochondria to the distal end of the flagellum is not sufficient to support sperm motility, suggesting that glycolysis in the tail region is the preferred pathway for energy production. It is suggested by many investigators that although glycolysis forms the major source of ATP along the flagellum, energy required for sperm motility is mainly produced during mitochondrial respiration. Nevertheless, some studies have shown that when glycolysis is inhibited, proper functioning and motility of spermatozoa remains intact although it is unclear whether such motility can be sustained for prolonged periods of time, or is sufficiently vigorous to achieve optimal fertilization. The purpose of this article is to provide an overview of mammalian sperm energy metabolism and identify the preferred metabolic pathway for ATP generation which forms the basis of energy Droduction in human spermatozoa during fertilization.
文摘Sexually reproducing animals require an orchestrated communication between spermatozoa and the egg to generate a new individual. Capacitation, a maturational complex phenomenon that occurs in the female reproductive tract, renders spermatozoa capable of binding and fusing with the oocyte, and it is a requirement for mammalian fertilization. Capacitation encompasses plasma membrane reorganization, ion permeability regulation, cholesterol loss and changes in the phosphorylation state of many proteins. Novel tools to study sperm ion channels, image intracellular ionic changes and proteins with better spatial and temporal resolution, are unraveling how modifications in sperm ion transport and phosphorylation states lead to capacitation. Recent evidence indicates that two parallel pathways regulate phosphorylation events leading to capacitation, one of them requiring activation of protein kinase A and the second one involving inactivation of ser/thr phosphatases. This review examines the involvement of ion transporters and phosphorylation signaling processes needed for spermatozoa to achieve capacitation. Understanding the molecular mechanisms leading to fertilization is central for societies to deal with rising male infertility rates, to develop safe male gamete-based contraceptives and to preserve biodiversity through better assisted fertilization strategies.
基金supported by the National Basic Re-search Program of China (No. 2006CB100100)the National High Technology Research and Development Program of China (863 Programme) (No. 2003AA210100)
文摘NPR1 （Nonexpressor of Pathogenesis-Related gene 1） is a major co-activator of plant defense. Phosphorylations of NPR1 play important roles in fine-tuning its activity, however a kinase corresponding to such modification remains uncharacterized. Here, we report that NPR1 interacts with PKS5 （SOS2-1ike Protein Kinase 5）. The AKR （AnKyrin Repeats） motif of NPR1 is required for this interaction. PKS5 phosphorylates NPR1 at the C-terminal region. Expression of PKS5 is induced quickly by Pseudomonas syringae pv. tomato DC3000. Expression level of two NPR1 target genes, WRKY38 and WRKY62, is reduced and/or delayed in pks5 mutants. Moreover, the expression of WRKY38 and WRKY62 displays a similar pattern in nprl-lpks5-1 double mutant comparing to that in nprl-1. Our results suggest that PKS5 functions at the upstream of NPR1 and might mediate expression of WRKY38 and WRKY62 possibly by interacting with and phosphorylating NPR 1.
基金supported by the FundamentalResearch Funds for the Central Universities(No.JKP2011004)
文摘AIM: To investigate the molecular signaling mechanism by which the plant-derived, pentacyclic triterpene maslinic acid(MA) exerts anti-diabetic effects. METHOD: HepG2 cells were stimulated with various concentrations of MA. The effects of MA on glycogen phosphorylase a(GPa) activity and the cellular glycogen content were measured. Western blot analyses were performed with anti-insulin receptor β(IRβ), protein kinase B(also known as Akt), and glycogen synthase kinase-3β(GSK3β) antibodies. Activation status of the insulin pathway was investigated using phospho-IRβ, as well as phospho-Akt, and phospho-GSK3β antibodies. The specific PI3-kinase inhibitor wortmannin was added to the cells to analyze the Akt expression. Enzyme-linked immunosorbent assay(ELISA) was used to measure the effect of MA on IRβ auto-phosphorylation. Furthermore, the effect of MA on glycogen metabolism was investigated in C57BL/6J mice fed with a high-fat diet(HFD). RESULTS: The results showed that MA exerts anti-diabetic effects by increasing glycogen content and inhibiting glycogen phosphorylase activity in HepG2 cells. Furthermore, MA was shown to induce the phosphorylation level of IRβ-subunit, Akt, and GSK3β. The MA-induced activation of Akt appeared to be specific, since it could be blocked by wortmannin. Finally, MA treatment of mice fed with a high-fat diet reduced the model-associated adiposity and insulin resistance, and increased the accumulated hepatic glycogen content. CONCLUSION: The results suggested that maslinic acid modulates glycogen metabolism by enhancing the insulin signaling pathway and inhibiting glycogen phosphorylase.
文摘Dendritic cells (DCs) are the most potent antigen-presen ting cells that play crucial roles in the regulation of immune response. Triptol ide, an active component purified from the medicinal plant Tripterygium wilfor dii Hook F., has been demonstrated to act as a potent immunosuppressive drug c apab le of inhibiting T cell activation and proliferation. However, little is known a bout the effects of triptolide on DCs. The present study shows that triptolide d oes not affect phenotypic differentiation and LPS-induced maturation of murine DCs. But triptolide can dramatically reduce cell recovery by inducing apoptosis of DCs at concentration as low as 10 ng/ml, as demonstrated by phosphatidylserin e exposure, mitochondria potential decrease, and nuclear DNA condensation. Tript olide induces activation of p38 in DCs, which precedes the activation of caspase 3. SB203580, a specific kinase inhibitor for p38, can block the activation of caspase 3 and inhibit the resultant apoptosis of DCs. Our results suggest that t he anti-inflammatory and immunosuppressive activities of triptolide may be due, in part, to its apoptosis-inducing effects on DCs.
基金supported by the National Natural Science Foundation of China(91632305)the National Key Research and Development Program of China(2016YFC13058001)
文摘Hyperphosphorylated tau is the major protein component of neurofibrillary tangles in the brains of patients with Alzheimer’s disease(AD). However, the mechanism underlying tau hyperphosphorylation is not fully understood. Here, we demonstrated that exogenously expressed wild-type human tau40 was detectable in the phosphorylated form at multiple AD-associated sites in cytoplasmic and nuclear fractions from HEK293 cells.Among these sites, tau phosphorylated at Thr205 and Ser214 was almost exclusively found in the nuclear fraction at the conditions used in the present study. With the intracellular tau accumulation, the Ca2+concentration was significantly increased in both cytoplasmic and nuclear fractions. Further studies using site-specific mutagenesis and pharmacological treatment demonstrated that phosphorylation of tau at Thr205 increased nuclear Ca2+concentration with a simultaneous increase in the phosphorylation of Ca2+/calmodulin-dependent protein kinase IV(Ca MKIV) at Ser196. On the other hand, phosphorylation of tau at Ser214 did not significantly change the nuclear Ca2+/Ca MKIV signaling. Finally, expressing calmodulin-binding protein-4 that disrupts formation ofthe Ca2+/calmodulin complex abolished the okadaic acidinduced tau hyperphosphorylation in the nuclear fraction.We conclude that the intracellular accumulation of phosphorylated tau, as detected in the brains of AD patients, can trigger nuclear Ca2+/Ca MKIV signaling, which in turn aggravates tau hyperphosphorylation. Our findings provide new insights for tauopathies: hyperphosphorylation of intracellular tau and an increased Ca2+concentration may induce a self-perpetuating harmful loop to promote neurodegeneration.
文摘Nowadays,the cumulative intake of glucocorticoids has become the most common pathogenic factor for non-traumatic osteonecrosis of the femoral head(ONFH).Apoptosis of osteoblasts is considered as the main reason of ONFH at the molecular level.Glycogen synthase kinase 3β(GSK3β)is an important regulator of cellular differentiation and apoptosis pathway,which can modulate the balance between osteoblasts and osteoclasts.Several studies have reported about its function in osteoporosis,but little is known about it in osteonecrosis.In our study,lipopolysaccharide and methylprednisolone were utilized to establish a rat ONFH model.The phosphorylation of GSK3βSer-9 was decreased in the model.Western blotting examination ofβ-catenin,Bcl-2,Bax and caspase-3 revealed that the osteoblasts were apoptotic.In dexamethasone(Dex)-incubated primary osteoblasts,the expression profile of GSK3βphosphorylation and apoptotic factors were consistent with those in the rat ONFH model.To further investigate the regulation of osteonecrosis caused by GSK3β,the expression and function of GSK3βwere inhibited in Dex-incubated primary osteoblasts.The knockdown of GSK3βby siRNA decreased the expression of Bax and cleaved caspase-3,but increased Bcl-2 andβ-catenin.On the other hand,selective inhibition of GSK3βfunction by LiCl counteracted the activation of caspase-3 induced by Dex.Our work is the first study about the GSK3P phosphorylation in ONFH,and provides evidence for further therapeutic methods.
文摘Objective: Tumor cells rely heavily on glycolysis regardless of oxygen tension, a phenomenon called the Warburg effect. Hexokinase II(HKII) catalyzes the first irreversible step of glycolysis and is often overexpressed in tumor cells. Mitochondrial HKII couples glycolysis and oxidative phosphorylation while maintaining mitochondrial membrane integrity. In this study, we investigated the role of HKII in promoting the Warburg effect in cancer cells.Methods: HKII-mediated phosphorylation of the alpha subunit of pyruvate dehydrogenase(PDHA1) was tested in HEK293 T cells and clear cell renal cell carcinoma(cc RCC) specimens using gene knockdown, western blotting,immunohistochemistry, and immunofluorescence.Results: It was determined that HKII could not only transform glucose into glucose-6-phosphate, but also transfer the phosphate group of ATP onto PDHA1. In addition, it was found that HKII increased the phosphorylation of Ser293 on PDHA1, decreasing pyruvate dehydrogenase(PDH) complex activity and thus rerouting the metabolic pathway and promoting the Warburg effect. The overexpression of HKII correlated with the phosphorylation of PDHA1 and disease progression in cc RCC.Conclusions: The data presented here suggest that HKII is an important biomarker in the evaluation and treatment of cancer.
基金a grant from the National Natural Sciences Foundation of China (No. 30571950)National Key Basic Research Program Foundation (N0.2002CB513107).
文摘The reversing effect of wild-type PTEN gene on resistance of C 13K cells to cisplatin and its inhibitory effect on the phosphorylation of protein kinase B （AKT） were studied. The expression of PTEN mRNA and protein in OV2008 cells and C13K cells were semi-quantitatively detected by using RT-PCR and Western blotting. Recombinant eukaryotic expression plasmid containing human wild-type PTEN gene was transfected into C13K cells by lipofectamine2000. The expression of PTEN mRNA was monitored by RT-PCR and the expression of PTEN, Akt, p-Akt protein were ana- lyzed by Western blotting in PTEN-transfected and non-transfected C13K cells. Proliferation and chemosensitivity of cells to DDP were measured by MTT, and cell apoptosis was detected by flow cytometry after treatment with cisplatin. The expression of PTEN mRNA and protein in OV2008 cells were significantly higher than those in C13K cells. After transfection with PTEN gene for 48 h, the expression of PTEN mRNA and protein in C 13K cells were 2.04 ± 0.10, 0.94± 0.04 respectively and the expression of p-Akt protein （ 0.94± 0.07） was lower than those in control groups （1.68 ±0.14, 1.66± 0.10） （P〈 0.05）. The IC50 of DDP to C 13 K cells transfected with PTEN （7.2± 0.3 la mol/L） was obviously lower than those of empty-vector transfected cells and non-transfected cells （12.7±0.4 lamol/1, 13.0±0.3 lamol/L） （P〈0.05）. The apopototis ratio of wild-type PTEN-transfected, empty vector transfected and non-transfected C13K cells were （41.65___0.87）%, （18.61 ±0.70）% and （15.28±0.80）% respectively, and the difference was statistically significant （P〈0.05）. PTEN gene plays an important role in ovarian cancer multidrug resistance. Transfection of PTEN could increase the expression of PTEN and restore drug sensitivity to cisplatin in human ovarian cancer cell line C 13K with multidrug-resistance by decreasing the expression of p-Akt.
基金supported by the Natural Science Foundation of Guangdong Province,No.2020A1515010090(to ZLZ)the Science and Technology Project Foundation of Guangzhou City,No.202002030004(to HZ).
文摘Astrocytes and microglia play an orchestrated role following spinal cord injury;however,the molecular mechanisms through which microglia regulate astrocytes after spinal cord injury are not yet fully understood.Herein,microglia were pharmacologically depleted and the effects on the astrocytic response were examined.We further explored the potential mechanisms involving the signal transducers and activators of transcription 3(STAT3)pathway.For in vivo experiments,we constructed a contusion spinal cord injury model in C57BL/6 mice.To deplete microglia,all mice were treated with colony-stimulating factor 1 receptor inhibitor PLX3397,starting 2 weeks prior to surgery until they were sacrificed.Cell proliferation was examined by 5-ethynyl-2-deoxyuridine(EdU)and three pivotal inflammatory cytokines were detected by a specific Bio-Plex Pro^(TM) Reagent Kit.Locomotor function,neuroinflammation,astrocyte activation and phosphorylated STAT3(pSTAT3,a maker of activation of STAT3 signaling)levels were determined.For in vitro experiments,a microglia and astrocyte coculture system was established,and the small molecule STA21,which blocks STAT3 activation,was applied to investigate whether STAT3 signaling is involved in mediating astrocyte proliferation induced by microglia.PLX3397 administration disrupted glial scar formation,increased inflammatory spillover,induced diffuse tissue damage and impaired functional recovery after spinal cord injury.Microglial depletion markedly reduced EdU+proliferating cells,especially proliferating astrocytes at 7 days after spinal cord injury.RNA sequencing analysis showed that the JAK/STAT3 pathway was downregulated in mice treated with PLX3397.Double immunofluorescence staining confirmed that PLX3397 significantly decreased STAT3 expression in astrocytes.Importantly,in vitro coculture of astrocytes and microglia showed that microglia-induced astrocyte proliferation was abolished by STA21 administration.These findings suggest that microglial depletion impaired astrocyte proliferation and astrocytic scar formation,and induced inflammatory diffusion partly by inhibiting STAT3 phosphorylation in astrocytes following spinal cord injury.
基金supported by grants from National Natural Sciences Foundation of China (No. 30430320 and 30770882)National 973 Project (No. 2007CB512004)
文摘3-hydroxy-3-methylgulutaryl-coenzyme A （HMG-CoA） reductase inhibitors or statins are a kind of lipid-lowering agents and have been used for the prevention and treatment of Cardiovascular diseases. Recent studies suggested that statins, besides lowering cholesterol, may protect vessels by enhancing the activity of endothelial nitric oxide synthase （eNOS）. In the present study, we investigated if simvastatin increases eNOS activity through its phosphorylation in 293 cells （293-eNOS） with stable expression of eNOS. The results showed that incubation of 293-eNOS cells with simvastatin （10 μm/L） for 2 h significantly increased in the activity of eNOS as shown by the conversion of L-arginine to L-citrulline （2889.70±201.51 versus 5630.18＋218.75 pmol/min . mg proteins） （P〈0.01）. Western blotting revealed that simvastatin increased phosphorylation of eNOS at 1177 （ser） and also 495 （thr） but did not affect the overall expression of eNOS or inducible NOS. Further study found that simvastatin raised phosphorylation levels of Akt and AMPK, and such effect could be antagonized by Akt inhibitor or AMPK inhibitor. These results suggest that simvastatin could stimulate,the activity of eNOS via its phosphorylation by Akt and AMPK, which provides a new mechanism, other than lipid-lowering effect, for the cardiovascular protection of statins.
