Death-associated protein kinase 1 is associated with cognitive dysfunction in major depressive disorder

We previously showed that death-associated protein kinase 1(DAPK1)expression is increased in hippocampal tissue in a mouse model of major depressive disorde and is related to cognitive dysfunction in Alzheimer's disease.In addition,depression is a risk factor for developing Alzheimer's disease,as well as an early clinical manifestation of Alzheimer's disease.Meanwhile,cognitive dysfunction is a distinctive feature of major depressive disorder.Therefore,DAPK1 may be related to cognitive dysfunction in major depressive disorder.In this study,we established a mouse model of major depressive disorder by housing mice individually and exposing them to chronic,mild,unpredictable stressors.We found that DAPK1 and tau protein levels were increased in the hippocampal CA3 area,and tau was hyperphosphorylated at Thr231,Ser262,and Ser396 in these mice.Furthermore,DAPK1 shifted from axonal expression to overexpression on the cell membrane.Exercise and treatment with the antidepressant drug citalopram decreased DAPK1 expression and tau protein phosphorylation in hippocampal tissue and improved both depressive symptoms and cognitive dysfunction.These results indicate that DAPK1 may be a potential reason and therapeutic target of cognitive dysfunction in major depressive disorder.

Characterization and Expression Analysis of Protein Kinase C Gene from Dunaliella salina

Protein kinase C (PKC) has a crucial role in signal transduction for a variety of biologically active substances which activate cellular functions and proliferation. We previously isolated the full-length PKC gene from Dunaliella salina (DsPKC) using rapid amplification of cDNA ends (RACE) and RT-PCR methods. And we submitted the mRNA sequence of DsPKC gene to NCBI (Genbank No. JN625213). In the present paper, the DsPKC gene open reading frame obtained by PCR was cloned into pGS-21a vector and transformed into Escherichia coli to generate the fusion protein. Bioinformatics analysis revealed that DsPKC gene was a member of serine/threonine kinase with two conserved domains and highly conserved motifs. The DsPKC was highly expressed upon induction with isopropyl-β-d-thiogalactoside (IPTG) at a final concentration of 0.2 mmol L 1 at 37℃. Under salt stress, the fu- sion protein Green Fluorescent Protein (GFP)-DsPKC was transferred from the cytoplasm to the cell membrane. The expression pat- tern of DsPKC gene was analyzed using real-time quantitative PCR, and indicated that DsPKC gene was up-regulated by 3.0 mol L 1 NaCl at 12 h, which was significantly higher than in control values (P < 0.05). These results suggest that the DsPKC gene plays an important role in response to salt stress in D. salina.

Cloning and Expression Analysis of a Mitogen-Activated Protein Kinase Gene OsMPK14 in Rice

Mitogen activated-protein kinases （MAPKs） are important components in signal transduction pathways responding to various biotic and abiotic stresses. An MAPK gene, OsMPK14 （GenBank Accession No. GQ265780） from rice （Oryza sativa L.）, was cloned by RT-PCR. The full-length cDNA of OsMPK14 consists of 1660 bp in size, containing an open reading frame of 1629 bp, which encodes a 542-amino-acid polypeptide and has a typical protein kinase domain and a phosphorylation activation motif TDY. Sequence alignment and analysis revealed that OsMPK14 was located on rice chromosome 5, and composed of nine exons and eight introns in the coding region. Semi-quantitative RT-PCR was performed to detect the expression patterns of OsMPK14 in rice shoots and roots under darkness, drought, high salinity, low temperature and abscisic acid treatments. The OsMPK14 mRNA was induced by abscisic acid, low temperature and high salinity, but weakly inhibited by drought. In addition, the expression of OsMPK14 was up-regulated in roots, but down-regulated in shoots by light. The results indicate that OsMPK14 could be implicated in diverse rice stimuli-responsive signaling cascades, and its expression might be regulated by multiple factors.

Yinchenhao decoction attenuates obstructive jaundice-induced liver injury and hepatocyte apoptosis by suppressing protein kinase RNA-like endoplasmic reticulum kinase-induced pathway

BACKGROUND Chronic biliary obstruction results in ischemia and hypoxia of hepatocytes,and leads to apoptosis.Apoptosis is very important in regulating the homeostasis of the hepatobiliary system.Endoplasmic reticulum(ER)stress is one of the signaling pathways that induce apoptosis.Moreover,the protein kinase RNA-like endoplasmic reticulum kinase(PERK)-induced apoptotic pathway is the main way;but its role in liver injury remains unclear.Yinchenhao decoction(YCHD)is a traditional Chinese medicine formula that alleviates liver injury and apoptosis,yet its mechanism is unknown.We undertook this study to investigate the effects of YCHD on the expression of ER stress proteins and hepatocyte apoptosis in rats with obstructive jaundice(OJ).AIM To investigate whether YCHD can attenuate OJ-induced liver injury and hepatocyte apoptosis by inhibiting the PERK-CCAAT/enhancer-binding protein homologous protein(CHOP)-growth arrest and DNA damage-inducible protein 34(GADD34)pathway and B cell lymphoma/leukemia-2 related X protein(Bax)/B cell lymphoma/leukemia-2(Bcl-2)ratio.METHODS For in vivo experiments,30 rats were divided into three groups:control group,OJ model group,and YCHD-treated group.Blood was collected to detect the indicators of liver function,and liver tissues were used for histological analysis.For in vitro experiments,30 rats were divided into three groups:G1,G2,and G3.The rats in group G1 had their bile duct exposed without ligation,the rats in group G2 underwent total bile duct ligation,and the rats in group G3 were given a gavage of YCHD.According to the serum pharmacology,serum was extracted and centrifuged from the rat blood to cultivate the BRL-3A cells.Terminal deoxynucleotidyl transferase mediated dUTP nick end-labelling(TUNEL)assay was used to detect BRL-3A hepatocyte apoptosis.Alanine aminotransferase(ALT)and aspartate transaminase(AST)levels in the medium were detected.Western blot and quantitative real-time polymerase chain reaction(qRT-PCR)analyses were used to detect protein and gene expression levels of PERK,CHOP,GADD34,Bax,and Bcl-2 in the liver tissues and BRL-3A cells.RESULTS Biochemical assays and haematoxylin and eosin staining suggested severe liver function injury and liver tissue structure damage in the OJ model group.The TUNEL assay showed that massive BRL-3A rat hepatocyte apoptosis was induced by OJ.Elevated ALT and AST levels in the medium also demonstrated that hepatocytes could be destroyed by OJ.Western blot or qRT-PCR analyses showed that the protein and mRNA expression levels of PERK,CHOP,and GADD34 were significantly increased both in the rat liver tissue and BRL-3A rat hepatocytes by OJ.The Bax and Bcl-2 levels were increased,and the Bax/Bcl-2 ratio was also increased.When YCHD was used,the PERK,CHOP,GADD34,and Bax levels quickly decreased,while the Bcl-2 levels increased,and the Bax/Bcl-2 ratio decreased.CONCLUSION OJ-induced liver injury and hepatocyte apoptosis are associated with the activation of the PERK-CHOP-GADD34 pathway and increased Bax/Bcl-2 ratio.YCHD can attenuate these changes.

Bcl-2 over-expression and activation of protein kinase C suppress the Trail-induced apoptosis in Jurkat T cells

Trail, a tumor necrosis factor-related apoptosis-inducing ligand, is a novel potent endogenous activator of the cell death pathway through the activation of cell surface death receptors Trail-R1 and Trail-R2. Its role, like FasL in activation-induced cell death (AICD), has been demonstrated in immune system. However the mechanism of Trail induced apoptosis remains unclear. In this report, the recombinant Trail protein was expressed and purified. The apoptosis-inducing activity and the regulation mechanism of recombinant Trail on Jurkat T cells were explored in vitro. Trypan blue exclusion assay demonstrated that the recombinant Trail protein actively killed Jurkat T cells in a dose-dependent manner. Trail-induced apoptosis in Jurkat T cells were remarkably reduced by Bcl-2 over expression in Bcl-2 gene transfected cells. Treatment with PMA (phorbol 12-myristate 13-acetate), a PKC activator, suppressed Trail-induced apoptosis in Jurkat T cells. The inhibition of apoptosis by PMA was abolished by pretreatment with Bis, a PKC inhibitor. Taken together, it was suggested that Bcl-2 over-expression and PMA activated PKC actively down-regulated the Trail-mediated apoptosis in Jurkat T cell.

Wheat kinase TaSnRK2.4 forms a functional module with phosphatase TaPP2C01 and transcription factor TaABF2 to regulate drought response

SNF1-related protein kinase 2(SnRK2)family members are essential components of the plant abscisic acid(ABA)signaling pathway initiated by osmotic stress and triggering a drought stress response.This study characterized the molecular properties of TaSnRK2.4 and its function in mediating adaptation to drought in Triticum aestivum.Transcripts of TaSnRK2.4 were upregulated upon drought and ABA signaling and associated with drought-and ABA-responsive cis-elements ABRE and DRE,and MYB and MYC binding sites in the promoter as indicated by reporter GUS protein staining and activity driven by truncations of the promoter.Yeast two-hybrid,BiFC,and Co-IP assays indicated that TaSnRK2.4 protein interacts with TaPP2C01 and an ABF transcription factor(TF)TaABF2.The results suggested that TaSnRK2.4 forms a functional TaPP2C01-TaSnRK2.4-TaABF2 module with its upstream and downstream partners.Transgene analysis revealed that TaSnRK2.4 and TaABF2 positively regulate drought tolerance whereas TaPP2C01 acts negatively by modulating stomatal movement,osmotic adjustment,reactive oxygen species(ROS)homeostasis,and root morphology.Expression analysis,yeast one-hybrid,and transcriptional activation assays indicated that several osmotic stress-responsive genes,including TaSLAC1-4,TaP5CS3,TaSOD5,TaCAT1,and TaPIN4,are regulated by TaABF2.Transgene analysis verified their functions in positively regulating stomatal movement(TaSLAC1-4),proline accumulation(TaP5CS3),SOD activity(TaSOD5),CAT activity(TaCAT1),and root morphology(TaPIN4).There were high correlations between plant biomass and yield with module transcripts in a wheat variety panel cultivated under drought conditions in the field.Our findings provide insights into understanding plant drought response underlying the SnRK2 signaling pathway in common wheat.

MicroRNA-219 alleviates glutamate-induced neurotoxicity in cultured hippocampal neurons by targeting calmodulin-dependent protein kinase Ⅱ gamma

Septic encephalopathy is a frequent complication of sepsis,but there are few studies examining the role of micro RNAs（mi Rs） in its pathogenesis.In this study,a mi R-219 mimic was transfected into rat hippocampal neurons to model mi R-219 overexpression.A protective effect of mi R-219 was observed for glutamate-induced neurotoxicity of rat hippocampal neurons,and an underlying mechanism involving calmodulin-dependent protein kinase II γ（Ca MKIIγ） was demonstrated.mi R-219 and Ca MKIIγ m RNA expression induced by glutamate in hippocampal neurons was determined by quantitative real-time reverse transcription-polymerase chain reaction（q RT-PCR）.After neurons were transfected with mi R-219 mimic,effects on cell viability and apoptosis were measured by 3-（4,5-dimethylthiazolyl-2）-2,5-diphenyltetrazolium bromide（MTT） assay and flow cytometry.In addition,a luciferase reporter gene system was used to confirm Ca MKIIγ as a target gene of mi R-219.Western blot assay and rescue experiments were also utilized to detect Ca MKIIγ expression and further verify that mi R-219 in hippocampal neurons exerted its effect through regulation of Ca MKIIγ.MTT assay and q RT-PCR results revealed obvious decreases in cell viability and mi R-219 expression after glutamate stimulation,while Ca MKIIγ m RNA expression was increased.MTT,flow cytometry,and caspase-3 activity assays showed that mi R-219 overexpression could elevate glutamate-induced cell viability,and reduce cell apoptosis and caspase-3 activity.Moreover,luciferase Ca MKIIγ-reporter activity was remarkably decreased by co-transfection with mi R-219 mimic,and the results of a rescue experiment showed that Ca MKIIγ overexpression could reverse the biological effects of mi R-219.Collectively,these findings verify that mi R-219 expression was decreased in glutamate-induced neurons,Ca MKIIγ was a target gene of mi R-219,and mi R-219 alleviated glutamate-induced neuronal excitotoxicity by negatively controlling Ca MKIIγ expression.

Genome-wide identification of the mitogen-activated protein kinase kinases in pear and their functional analysis in response to black spot

The mitogen-activated protein kinase(MAPK)cascade is crucial to plant growth,development,and stress responses.MAPK kinases(MAPKK)play a vital role in linking upstream MAPKK kinases(MAPKKK)with the downstream MAPK.Black spot is one of the most serious fungal diseases of pear which is an important part of the fruit industry in China.The MAPKK genes have been identified in many plants,however,none has been reported in pear(Pyrus bretschneideri).In order to explore whether MAPK gene of pear is related to black spot disease,we designed this experiment.The present study investigated eight putative PbrMAPKK genes obtained from the Chinese white pear genome.The phylogenetic analysis revealed that PbrMAPKK genes were divided into A,B,C,and D groups.These PbrMAPKK genes are randomly distributed on 7 out of 17 chromosomes and mainly originated from the whole-genome duplication(WGD)event.The expression analysis of PbrMAPKK genes in seven pear tissues and the leaves of susceptible and resistant varieties after Alternaria alternata infection by quantitative real-time PCR(qRT-PCR)identified seven candidate genes associated with resistance.Furthermore,virus-induced gene silencing(VIGS)indicated that PbrMAPKK6 gene enhanced resistance to pear black spot disease in pear.

Investigation of the relationship between DNA-dependent protein kinase and lymphatic metastasis in colorectal cancer

Objective: To investigate DNA-dependent protein kinase (DNA-PK) expression,and its relationship with lymphat-ic metastasis in colorectal cancer. Methods: Tumor tissues from 60 patients,divided into two groups according to lymphatic metastasis,were immunohistochemically stained to detect the DNA-PK expression including Ku70,Ku80 and PKcs proteins. Results: Positivity of both Ku70 and Ku80 in colorectal cancer was negatively correlated with lymphatic metastasis with an r value of -0.57 and -0.38,respectively. Similar correlation was found between Ku expression,especially Ku70,and long-term survival. PKcs,however,displayed no significant correlation. Statistical analysis failed to detect any correlation between DNA-PK expression,and clinical characteristics,such as age,sex,tumor location,tumor thickness and distant metastasis (P>0.05). Conclusion: DNA-PK expression,especially Ku70 expression,is negatively correlated with lymphatic metastasis,and the survival of patients with colorectal cancer. Ku70 expression may be a potential indicator for the preoperative evaluation,and prognosis in colorectal cancer.

汉黄芩素干预糖尿病脑梗死模型大鼠的神经损伤

背景:汉黄芩素是黄芩根中提取的一种黄酮类化合物,既往研究表明汉黄芩素对脑缺血再灌注损伤有保护作用,还能降低糖尿病小鼠的血糖及其并发症,但其在糖尿病脑梗死中的作用及机制还不清楚。目的:探究汉黄芩素对糖尿病脑梗死大鼠神经损伤的影响,并探究其作用机制。方法:将SD大鼠随机分为6组:对照组、模型组、汉黄芩素低、中、高剂量组和汉黄芩素高剂量+RhoA激活剂组,每组10只。除对照组外,其余组通过腹腔注射链脲佐菌素和大脑中动脉闭塞法构建糖尿病脑梗死大鼠模型,汉黄芩素低、中、高剂量组分别灌胃10,20,40 mg/kg汉黄芩素,汉黄芩素高剂量+RhoA激活剂组灌胃40 mg/kg汉黄芩素并腹腔注射10 mg/kg溶血磷脂酸,对照组和模型组给予等量生理盐水,1次/d,连续7 d。末次给药结束后,各组大鼠进行神经功能缺损评分,检测血糖水平,TTC染色检测脑梗死体积,苏木精-伊红染色观察脑组织病理变化,ELISA试剂盒检测脑组织中肿瘤坏死因子α、白细胞介素6和丙二醛、超氧化物歧化酶水平,Western blot检测脑组织中RhoA、ROCK2蛋白表达。结果与结论:(1)与对照组相比,模型组大鼠神经元结构严重受损,细胞坏死、变性;神经功能缺损评分、血糖水平、脑梗死体积升高(P<0.05);脑组织中肿瘤坏死因子α、白细胞介素6、丙二醛水平升高(P<0.05),超氧化物歧化酶水平下降(P<0.05);脑组织中RhoA、ROCK2蛋白表达升高(P<0.05);(2)与模型组相比,汉黄芩素低、中、高剂量组神经元损伤改善,细胞变性和坏死减少;神经功能缺损评分、血糖水平、脑梗死体积下降(P<0.05);脑组织中肿瘤坏死因子α、白细胞介素6、丙二醛水平下降(P<0.05),超氧化物歧化酶水平升高(P<0.05);脑组织中RhoA、ROCK2蛋白表达下降(P<0.05);(3)与汉黄芩素高剂量组相比,汉黄芩素高剂量+RhoA激活剂组明显抑制糖尿病脑梗死大鼠上述指标的改善(P<0.05)。结果表明:汉黄芩素能够改善糖尿病脑梗死大鼠血糖水平,减轻脑梗死和神经损伤,其作用机制可能与抑制RhoA/ROCK信号通路有关。

Plant protein kinase genes induced by drought, high salt and cold stresses

Drought, high salt and cold are three different kinds of environment stresses that severely influence the growth, development and productivity of crops. They all decrease the water state of plant cells, and consequently result in the harm of plant from water deficit. Several genes encoding protein kinases and induced by drought, high salt and low temperature have been isolated from Arabidopsis. These protein kinases include receptor protein kinase (RPK), MAP kinases, ribosomal-protein kinases and transcription-regulation protein kinase. The expression features of these genes and the regulatory roles of these protein kinases in stress response and signal transduction are discussed.

Let-7a gene knockdown protects against cerebral ischemia/reperfusion injury

The micro RNA（mi RNA） let-7 was one of the first mi RNAs to be discovered, and is highly conserved and widely expressed among species. let-7 expression increases in brain tissue after cerebral ischemia/reperfusion injury; however, no studies have reported let-7 effects on nerve injury after cerebral ischemia/reperfusion injury. To investigate the effects of let-7 gene knockdown on cerebral ischemia/reperfusion injury, we established a rat model of cerebral ischemia/reperfusion injury. Quantitative reverse transcription-polymerase chain reaction demonstrated that 12 hours after cerebral ischemia/reperfusion injury, let-7 expression was up-regulated, peaked at 24 hours, and was still higher than that in control rats after 72 hours. Let-7 gene knockdown in rats suppressed microglial activation and inflammatory factor release, reduced neuronal apoptosis and infarct volume in brain tissue after cerebral ischemia/reperfusion injury. Western blot assays and luciferase assays revealed that mitogen-activated protein kinase phosphatase-1（MKP1） is a direct target of let-7. Let-7 enhanced phosphorylated p38 mitogen-activated protein kinase（MAPK） and c-Jun N-terminal kinase（JNK） expression by down-regulating MKP1. These findings suggest that knockdown of let-7 inhibited the activation of p38 MAPK and JNK signaling pathways by up-regulating MKP1 expression, reduced apoptosis and the inflammatory reaction, and exerted a neuroprotective effect following cerebral ischemia/reperfusion injury.

Electroacupuncture preconditioning protects against focal cerebral ischemia/reperfusion injury via suppression of dynamin-related protein 1

Electroacupuncture preconditioning at acupoint Baihui （GV20） can reduce focal cerebral ischemia/reperfusion injury. However, the precise protective mechanism remains unknown. Mitochondrial fission mediated by dynamin-related protein 1 （Drp1） can trigger neuronal apoptosis following cerebral ischemia/reperfusion injury. Herein, we examined the hypothesis that electroacupuncture pretreatment can regulate Drp1, and thus inhibit mitochondrial fission to provide cerebral protection. Rat models of focal cerebral ischemia/reperfusion injury were established by middle cerebral artery occlusion at 24 hours after 5 consecutive days of preconditioning with electroacupuncture at GV20 （depth 2 mm, intensity 1 mA, frequency 2/15 Hz, for 30 minutes, once a day）. Neurological function was assessed using the Longa neurological deficit score. Pathological changes in the ischemic penumbra on the injury side were assessed by hematoxylin-eosin staining. Cellular apoptosis in the ischemic penumbra on the injury side was assessed by terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling staining. Mitochondrial ultrastructure in the ischemic penumbra on the injury side was assessed by transmission electron microscopy. Drp1 and cytochrome c expression in the ischemic penumbra on the injury side were assessed by western blot assay. Results showed that electroacupuncture preconditioning decreased expression of total and mitochondrial Drp1, decreased expression of total and cytosolic cytochrome c, maintained mitochondrial morphology and reduced the proportion of apoptotic cells in the ischemic penumbra on the injury side, with associated improvements in neurological function. These data suggest that electroacupuncture preconditioning-induced neuronal protection involves inhibition of the expression and translocation of Drp1.

Gene polymorphisms of interleukin-28, p21-activated protein kinases 4, and response to interferon-α based therapy in Chinese patients with chronic hepatitis B

Background Peg-lnterferon-a treatment is expensive and associated with considerable adverse effects, selection of patients with the highest probability of response is essential for clinical practice. The objective of this study was to assess the relationship between the gene polymorphisms of interleukin-28 （IL-28）, p21-activated protein kinase 4 （PAK4） and the response to interferon treatment in chronic hepatitis B patients. Methods Two hundred and forty interferon-naive treatment HBeAg seropositive chronic hepatitis B patients were enrolled in the present prospective nested case-control study. Peripheral blood samples were collected, including 92 with favorable response and 148 without response to the interferon treatment. Rs8099917, rs12980602, and rs9676717 SNP was genotyped using matrix assisted laser desorption/ionization time of flight mass spectrometry （MALDI-TOF MS）. Results IL-28 genotype was not associated with response to interferon treatment （OR for GT/GG vs. TT, 0.881 （95% CI 0.388-2.002）; P=0.762; OR for CT/CC vs. TT, 0.902 （95% CI 0.458-1.778）; P=-0.766）. Rs9676717 in PAK4 genotype was independently associated with the response （OR for CT/CC vs. TT, 0.524 （95% CI 0.310-0.888）; P=0.016）. When adjusting for age, gender, smoking, drinking, levels of hepatitis B virus DNA, and alanine aminotransferase （ALT）, rs9676717 genotype TT appeared to be associated with a higher probability of response for interferon treatment （OR, 0.155 （95% CI 0.034-0.700）; P=0.015）. Conclusion Genotype TTfor rs9676717 in PAK4 gene and no drinking may be predictive of the interferon-a treatment success.

Preliminary Investigation on Regulating Effects of Different TCM Treatments on Transcription of the Correlated Genes of Liver Cancer in Rats

The regulating effects of TCM treatments including clearing away heat and toxic materials,promoting blood circulation and removing blood stasis,and strengthening the spleen and regulating qi on the oncogene transcription were observed in the liver cancer model rats.The preliminary results indicated that the mRNA levels of H-ras N-ras and K-ras,and signal molecules correlated with the ras/MAPK signal transduction pathway were down-regulated by the different TCM treatments in varying degrees.Also,the regulating effects of the treatments on differently-displayed genes were discrepant.It is suggested that the molecular mechanisms of the TCM treatments for liver cancer was complex with different target genes.

Identification of key genes involved in axon regeneration and Wallerian degeneration by weighted gene co-expression network analysis

Peripheral nerve injury repair requires a certain degree of cooperation between axon regeneration and Wallerian degeneration.Therefore,investigating how axon regeneration and degeneration work together to repair peripheral nerve injury may uncover the molecular mechanisms and signal cascades underlying peripheral nerve repair and provide potential strategies for improving the low axon regeneration capacity of the central nervous system.In this study,we applied weighted gene co-expression network analysis to identify differentially expressed genes in proximal and distal sciatic nerve segments from rats with sciatic nerve injury.We identified 31 and 15 co-expression modules from the proximal and distal sciatic nerve segments,respectively.Functional enrichment analysis revealed that the differentially expressed genes in proximal modules promoted regeneration,while the differentially expressed genes in distal modules promoted neurodegeneration.Next,we constructed hub gene networks for selected modules and identified a key hub gene,Kif22,which was up-regulated in both nerve segments.In vitro experiments confirmed that Kif22 knockdown inhibited proliferation and migration of Schwann cells by modulating the activity of the extracellular signal-regulated kinase signaling pathway.Collectively,our findings provide a comparative framework of gene modules that are co-expressed in injured proximal and distal sciatic nerve segments,and identify Kif22 as a potential therapeutic target for promoting peripheral nerve injury repair via Schwann cell proliferation and migration.All animal experiments were approved by the Institutional Animal Ethics Committee of Nantong University,China(approval No.S20210322-008)on March 22,2021.

EFFECT OF INTERLEUKIN-1β ON GROWTH HORMONE GENE EXPRESSION AND ITS POSSIBLE MOLECULAR MECHANISM IN RAT MtT/S SOMATOTROPH CELLS

Objective To elucidate the effect of interleukin-1β （IL- 1β） on human growth hormone （hGH） gene expression in a rat somatotropic pituitary cell line MtT/S. Methods Stably transfected MtT/S cells were firstly established by transfecting 484-Lucl plasmid which contained hGH gene promoter --484 to ＋30 bp and luciferase reporter gene. The effect of IL-1β on hGH gene expression was determined by assaying the luciferase activities. RT-PCR method was also used to determine whether IL-1 recepor mRNA was expressed in MtT/S cells. Results The 10^3 U/mL IL-1β stimulated secretion and synthesis of GH, and promoted the 5＇-promoter activity of GH gene in stably transfected MtT/SGL cells with the action of 1.38 times above the control. Among inhibitors of signaling transduction pathways, mitogen-activated protein kinase kinase （MAPKK/MEK） inhibitor PD98059 （40 μmol/L） and p38 mitogen-activated protein kinase （MAPK） inhibitor SB203580 （5 μmol/L） completely blocked the stimulatory effect of IL-1μ, and phosphatidylinositol-3-kinase （PI3-K） inhibitor LY294002 partly abolished the effect of IL-1μ. Western blot analysis further confirmed the activation of phosphorylated MEK and p38 MAPK in MtT/SGL cells. Neither over-expression of Pit- 1 nor inhibition of Pit- 1 expression affected induction of hGH promoter activity by IL-1μ. A series of deletion constructs of hGH promoter were created to identify the DNA sequence that mediated the effect of IL-1β, and results showed that the stimulatory effect of IL-1β was abolished following deletion of the --196 to -- 132 bp fragment. Conclusions IL-1β promotes GH secretion and synthesis in rat MtT/S somatotroph cells. The stimulatory effect of IL-1β on hGH gene promoter appears to require the activation of MEK, p38 MAPK, PI3-K, and a fragment of promoter sequence that spans the -196 to -132 bp of the gene, but it may be unlinked with Pit-1 protein.

MEK AND p38 MAPK-DEPENDANT PATHWAYS ARE INVOLOVED IN THE POSITIVE EFFECT OF INTERLEUKIN-6 ON HUMAN GROWTH HORMONE GENE EXPRESSION IN RAT MtT/S SOMATOTROPH CELLS

Objective To investigate the effect of interleukin-6(IL-6)on the human growth hormone(hGH)gene expression in a rat somatotropic pituitary cell line MtT/S.Methods The plasmids containing various lengths of hGH gene 5'-promoter fragments were constructed.Stably transfected MtT/S cells were created by cotransfecting the above plasmids and pcDNA3.1(+)with DMRIE-C transfection reagent.After the administration of these cells with IL-6 and/or various inhibitors of signaling transduction pathways,the luciferase activities in MtT/S cells lysis were assayed to demonstrate the effects of IL-6 on hGH gene promoter activity and possibly involved mechanism.Results The 103 U/mL IL-6 stimulated GH secretion and synthesis,and promoted the 5'-promoter activity of GH gene in stably transfected MtT/SGL cells with the action of 1.69 times above the control.Among inhibitors of signaling transduction pathways,mitogen-activated protein kinase kinase(MAPKK/MEK)inhibitor PD98059(40 μmol/L)and p38 mitogen-activated protein kinase(MAPK)inhibitor SB203580(5 μmol/L)completely blocked the stimulatory effect of IL-6.Western blot analysis further confirmed the activation of phosphorylated MEK and p38 MAPK in MtT/SGL cells.Neither over-expression of Pit-1 nor inhibition of Pit-1 expression affected IL-6 induction of hGH promoter activity.A series of deletion constructs of hGH promoter were created to identify the DNA sequence that mediated the effect of IL-6.The results showed that the stimulatory effect of IL-6 was abolished following deletion of the-196 to-132 bp fragment.Conclusions IL-6 promotes GH secretion and synthesis by rat MtT/S somatotroph cells.The stimulatory effect of IL-6 on hGH gene promoter appears to require the activation of MEK and p38 MAPK,and a fragment of promoter sequence that spans the-196 to-132 bp of the gene,but may be unlinked with Pit-1 protein.

Methylation of DAPK and THBS1 genes in esophageal gastric-type columnar metaplasia

AIM: To explore methylation of DAPK, THBS1, CDH-1, and p14 genes, and Helicobacter pylori(H. pylori) status in individuals harboring esophageal columnar metaplasia.METHODS: Distal esophageal mucosal samples obtained by endoscopy and histologically diagnosed as gastric-type(non-specialized) columnar metaplasia, were studied thoroughly. DNA was extracted from paraffin blocks, and methylation status of deathassociated protein kinase(DAPK), thrombospondin-1(THBS1), cadherin-1(CDH1), and p14 genes, was examined using a methyl-sensitive polymerase chain reaction(MS-PCR) and sodium bisulfite modification protocol. H. pylori cag A status was determined by PCR.RESULTS: In total, 68 subjects(33 females and 35 males), with a mean age of 52 years, were included. H. pylori cag A positive was present in the esophageal gastric-type metaplastic mucosa of 18 individuals. DAPK, THSB1, CDH1, and p14 gene promoters were methylated by MS-PCR in 40(58.8%), 33(48.5%), 46(67.6%), and 23(33.8%) cases of the 68 esophageal samples. H. pyloristatus was associated with methylation of DAPK(P = 0.003) and THBS1(P = 0.019).CONCLUSION: DNA methylation occurs in cases of gastric-type(non-specialized) columnar metaplasia of the esophagus, and this modification is associated with H. pylori cag A positive infection.

Cloning and Sequence Analysis of Disease Resistance Gene Analogues from Three Wild Rice Species in Yunnan

Two sets of degenerate oligonucleotide primers were designed according to amino acid conserved regions of reported plant disease resistance genes which encode proteins that contain nucleotide-binding site and leucine-rich repeats(NBS-LRR), and the plant disease resistance genes which encode serine/threonine protein kinase(STK). By polymerase chain reaction(PCR), disease resistance gene analogues have been amplified from three wild rice species in Yunnan Province, China. The DIN A fragments from amplification have been cloned into the pGEM-T vector respectively. Sequencing of the DNA fragments indicated that 7 classes, 2 classes and 6 classes NBS-LRR disease resistance gene analogues from Oryza rufipogon Griff. , Oryza officinalis Wall. , and Oryza meyeriana Baill. were obtained respectively. The two representative fragments of TO12 from Oryza officinalis Wall, and TR19 from Oryza rufipogon Griff, belong to the same class and homology of their sequences are 100%. The result shows that the sequences of the same class disease resistance gene analogues have no difference among different species of wild rice. 5 classes STK disease resistance gene analogues were also obtained among which 4 classes from Oryza rufipogon Griff. , 1 class from Oryza officinalis Wall. By comparison analysis of amino acid sequences. we found that the obtained disease resistance gene analogues have very low identity(low to 25%) with the reported disease resistance gene L6, N, Bs2, Prf, Pto, Lr10 and Xa21 etc. The finding suggests that the obtained disease resistance gene analogues are analogues of putative disease resistance genes that have not been isolated so far.