Knockdown of lncRNA PVT1 attenuated macrophage M1 polarization and relieved sepsis induced myocardial injury via miR-29a/HMGB1 axis.

BACKGROUND: LncRNA PVT1 was reported to be elevated in septic myocardial tissue. The underlying mechanism by which PVT1 aggravated sepsis induced myocardial injury needs further investigation. METHODS: Mice was subjected to LPS injection to mimic in vivo sepsis model. HE staining was applied to observe tissue injury. Cardiac function of mice was determined by echocardiography. Bone marrow derived macrophage (BMDM) was used to confirm the regulatory effect of PVT1 in macrophage polarization. Western blotting or qRT-PCR were performed to evaluate protein or mRNA levels, respectively. ELISA was conducted to determine cytokine levels. Interaction between PVT1 and miR-29a, miR-29a and HMGB1 were accessed by dual luciferase assay. RESULTS: Expression of PVT1 was elevated in myocardial tissue and heart infiltrating macrophages of sepsis mice. PVT1 knockdown alleviated LPS induced myocardial injury and attenuated M1 macrophage polarization. The mechanic study suggested that PVT1 targeted miR-29a, thus elevated expression of HMGB1, which was repressed by miR-29a targeting. The effect of PVT1 on M1 macrophage polarization was dependent on targeting miR-29a. CONCLUSION: PVT1 promoted M1 polarization and aggravated LPS induced myocardial injury via miR-29a/HMGB1 axis.

Studies on anti-angiogenesis of ginsenoside structure modification HRG in vitro.

This study investigates the anti-angiogenic effect of 3ß, 12ß, 20(S)-trihydroxy dammarane-3-O-ß-d-glucopyranosyl(1-2)-ß-d-glucopyranoside(HRG), a new chemical compound obtained by structure modification on Ginseng saponins Rg3, associated with the regulation of matrix metalloproteinases(MMPs) and its upstream signal-regulated molecule of vascular endothelial growth factor(VEGF) and basic fibroblast growth factor(b-FGF) in vitro, which plays an critical role in angiogenesis during the process of carcinoma. In our study, to investigate the anti-angiogenesis effect of HRG in HUVECs, we utilized cell proliferation assay, tube formation assay, wound-healing assay, Semi-quantitative reverse transcription PCR, and Western blot assay. Our results demonstrated that HRG plays a major role in the regulation of proliferation, migration and tube formation of HUVECs by suppressing the expression of VEGF and b-FGF in both transcriptional and post-transcriptional levels. In addition, the expression of MMP-2 and MMP-9, which were related to the ECM degradation, were down-regulated after administration of HRG as well. Overall, our results revealed that HRG strongly inhibited the process of angiogenesis and shows better effectiveness than Rg3.

Hydrogen Sulfide Selectively Inhibits γ-Secretase Activity and Decreases Mitochondrial Aß Production in Neurons from APP/PS1 Transgenic Mice.

Hydrogen sulfide (H2S) is now considered to be a gasotransmitter and may be involved in the pathological process of Alzheimer's disease (AD). A majority of APP is associated with mitochondria and is a substrate for the mitochondrial γ-secretase. The mitochondria-associated APP metabolism where APP intracellular domains (AICD) and Aß are generated locally and may contribute to mitochondrial dysfunction in AD. Here, we aimed to investigate the ability of H2S to mediate APP processing in mitochondria and assessed the possible mechanisms underlying H2S-mediated AD development. We treated neurons from APP/PS1 transgenic mice with a range of sodium hydrosulfide (NaHS) concentrations. NaHS attenuated APP processing and decreased Aß production in mitochondria. Meanwhile, NaHS did not changed BACE-1 and ADAM10 (a disintegrin and metalloprotease 10) protein levels, but NaHS (30 µM) significantly increased the levels of presenilin 1(PS1), PEN-2, and NCT, as well as improved the γ-secretase activity, while NaHS (50 µM) exhibits the opposing effects. Furthermore, the intracellular ATP and the COX IV activity of APP/PS1 neurons were increased after 30 µM NaHS treatment, while the ROS level was decreased and the MMP was stabilized. The effect of NaHS differs from DAPT (a non-selective γ-secretase inhibitor), and it selectively inhibited γ-secretase in vitro, without interacting with Notch and modulating its cleavage. The results indicated that NaHS decreases Aß accumulation in mitochondria by selectively inhibiting γ-secretase. Thus, we provide a mechanistic view of NaHS is a potential anti-AD drug candidate and it may decrease Aß deposition in mitochondria by selectively inhibiting γ-secretase activity and therefore protecting the mitochondrial function during AD conditions.

Genome-wide Identification and Expression Analysis of the CDPK Gene Family in Grape, Vitis spp.

BACKGROUND: Calcium-dependent protein kinases (CDPKs) play vital roles in plant growth and development, biotic and abiotic stress responses, and hormone signaling. Little is known about the CDPK gene family in grapevine. RESULTS: In this study, we performed a genome-wide analysis of the 12X grape genome (Vitis vinifera) and identified nineteen CDPK genes. Comparison of the structures of grape CDPK genes allowed us to examine their functional conservation and differentiation. Segmentally duplicated grape CDPK genes showed high structural conservation and contributed to gene family expansion. Additional comparisons between grape and Arabidopsis thaliana demonstrated that several grape CDPK genes occured in the corresponding syntenic blocks of Arabidopsis, suggesting that these genes arose before the divergence of grapevine and Arabidopsis. Phylogenetic analysis divided the grape CDPK genes into four groups. Furthermore, we examined the expression of the corresponding nineteen homologous CDPK genes in the Chinese wild grape (Vitis pseudoreticulata) under various conditions, including biotic stress, abiotic stress, and hormone treatments. The expression profiles derived from reverse transcription and quantitative PCR suggested that a large number of VpCDPKs responded to various stimuli on the transcriptional level, indicating their versatile roles in the responses to biotic and abiotic stresses. Moreover, we examined the subcellular localization of VpCDPKs by transiently expressing six VpCDPK-GFP fusion proteins in Arabidopsis mesophyll protoplasts; this revealed high variability consistent with potential functional differences. CONCLUSIONS: Taken as a whole, our data provide significant insights into the evolution and function of grape CDPKs and a framework for future investigation of grape CDPK genes.

Hydrogen sulfide-induced processing of the amyloid precursor protein in SH-SY5Y human neuroblastoma cells involves the PI3-K/Akt signaling pathway.

Hydrogen sulfide (H2S) has been recently categorized as a gasotransmitter, and it may be involved in the pathology of Alzheimer's disease. However, whether H2S induces amyloid precursor protein (APP) processing remains unknown. In the present study, we tested the ability of H2S to mediate APP processing in SH-SY5Y human neuroblastoma cells. We treated SH-SY5Y human neuroblastoma cells with a range of sodium hydrosulfide (H2S donor) concentrations. Western blot analysis showed that H2S increased the generation of C83 and decreased the production of C99. Meanwhile, H2S increased the levels of a disintegrin and metalloprotease 10 (ADAM10) mRNA and protein, but had no effect on TNF-α-converting enzyme (TACE, also known as ADAM17) mRNA and protein levels. H2S also induced a significant decrease of extracellular amyloid-ß42 (Aß42). Furthermore, SH-SY5Y human neuroblastoma cells were assayed for activation of the phosphoinositide 3-kinase (PI3-K) pathway. H2S activated the PI3-K pathway. Using specific inhibitor of PI3-K, we determined that the effects of H2S on APP processing and Aß42 were blocked by LY 294002 (PI3-K inhibitor). These data indicate that H2S can induce APP processing, and this effect is dependent on activation of the PI3-K signaling pathway.

PPAR-α agonist regulates amyloid-ß generation via inhibiting BACE-1 activity in human neuroblastoma SH-SY5Y cells transfected with APPswe gene.

Alzheimer's disease is a neuroinflammatory disease and is the most common cause of dementia in the elderly. Studies have shown the beneficial effects of the peroxisome proliferator-activated receptor alpha (PPAR-α) agonists on the treatment of neuroinflammatory diseases. The aim of the present study is to examine the ability of GW7647 (a PPAR-α agonist) to regulate amyloid precursor protein (APP) amyloidogenic processing in human neuroblastoma SH-SY5Y cells transfected with APPswe gene. After administration of GW7647 for 24 h, the levels of APP, soluble APPß (sAPPß), and presenilin 1 (PS-1) were assessed by Western blot. Cellular culture medium levels of amyloid-ß 42 (Aß42) were analyzed by ELISA, and the activity of beta-site APP cleaving enzyme 1 (BACE-1) was measured by fluorometric assay. We found that GW7647 decreased the expression of sAPPß and the activity of BACE-1, and also reduced Aß42 release. However, GW7647 did not modify the levels of APP and PS-1. Furthermore, LY294002, the phosphoinositide 3-kinase (PI3-K) inhibitor, reversed the effects of GW7647 on the BACE-1 activity and the levels of sAPPß and Aß42. Our data demonstrate that GW7647 may reduce Aß production via inhibiting BACE-1 activity, and this may involve in PI3-K pathway.

[Clinical effect of electroacupuncture at Baihui and Shuigou points in treatment of brain injury in patients with sepsis-associated encephalopathy].

OBJECTIVE: To investigate the clinical effect of electroacupuncture at Baihui (GV20) and Shuigou (GV26) points in the treatment of brain injury in patients with sepsis-associated encephalopathy(SAE). METHODS: A total of 70 patients with SAE were randomly divided into control group and treatment group, with 35 patients in each group. The patients in the control group were given routine western medicine treatment, including anti-infective therapy, nerve nutrition, and mechanical ventilation, and those in the treatment group were given electroacupuncture at GV20 and GV26 in addition to the treatment in the control group. The course of treatment was 1 week for both groups. Serum levels of C-reactive protein (CRP), interleukin-6 (IL-6), and neuron-specific enolase (NSE) were measured for both groups, the Montreal Cognitive Assessment (MoCA) scale was used to assess the change in cognitive function, and Glasgow Coma Scale (GCS) score was determined before and after treatment and was used to evaluate treatment outcome after treatment. RESULTS: Both groups had significant reductions in the serum levels of CRP, IL-6, and NSE after 24 h and one week of treatment (P<0.05), and compared with the control group, the treatment group had significant reductions in the levels of CRP, IL-6 and NSE after treatment (P<0.05). The treatment group had significant increases in the total score of MoCA and the scores of all dimensions except attention after one week of treatment (P<0.05), and the treatment group had significantly higher scores than the control group after treatment (P<0.05). Both groups had a significant increase in GCS score after one week of treatment (P<0.05), and the treatment group had a significantly higher GCS score than the control group after treatment (P<0.05). The treatment group had a significantly higher total effective rate than the control group ï¼»88.6% (31/35) vs 57.1% (20/35), P<0.05ï¼½. CONCLUSION: Electroacupuncture at GV20 and GV26 can effectively improve brain injury and effective rate in SAE patients.

A cohort study of isolated brainstem infarction based on head MR imaging and clinical findings.

OBJECTIVE: The prognosis of patients with isolated brainstem infarction (BSI) differs on an individual patient basis. This study was undertaken to analyze the influences of different imaging and clinical features with the prognosis of patients with BSI. METHODS: The study population was derived from a multicenter study of intracranial atherosclerosis in China. In the present study, 300 patients were selected who had experienced non-cardiogenic brain stem infarction within the prior 7 days. Evaluations included clinical characteristics, location and size of the brainstem infarction, and whether the infarction was located in multiple perforating branches of the brainstem. Poor prognosis was defined as the presence of disability within 1 year from the onset of disease. RESULTS: In total, 281 patients were followed up at 1 year post-infarction. Of these 281 patients, 84 (29.9%) exhibited disability at 1 year; these patients showed a median National Institutes of Health Stroke Scale score of 6 on admission. Multiple logistic regression analysis showed that patients with BSI located in the territory of multiple perforating arteries, who were discharged without administration of statins, showed a poor 1-year prognosis. CONCLUSION: Isolated BSI involving multiple perforating arteries, without statin medication at discharge, indicated poor prognosis for patients with BSI.

AP39, a Mitochondria-Targeted Hydrogen Sulfide Donor, Supports Cellular Bioenergetics and Protects against Alzheimer's Disease by Preserving Mitochondrial Function in APP/PS1 Mice and Neurons.

Increasing evidence suggests that mitochondrial functions are altered in AD and play an important role in AD pathogenesis. It has been established that H2S homeostasis is balanced in AD. The emerging mitochondrial roles of H2S include antioxidation, antiapoptosis, and the modulation of cellular bioenergetics. Here, using primary neurons from the well-characterized APP/PS1 transgenic mouse model, we studied the effects of AP39 (a newly synthesized mitochondrially targeted H2S donor) on mitochondrial function. AP39 increased intracellular H2S levels, mainly in mitochondrial regions. AP39 exerted dose-dependent effects on mitochondrial activity in APP/PS1 neurons, including increased cellular bioenergy metabolism and cell viability at low concentrations (25-100 nM) and decreased energy production and cell viability at a high concentration (250 nM). Furthermore, AP39 (100 nM) increased ATP levels, protected mitochondrial DNA, and decreased ROS generation. AP39 regulated mitochondrial dynamics, shifting from fission toward fusion. After 6 weeks, AP39 administration to APP/PS1 mice significantly ameliorated their spatial memory deficits in the Morris water maze and NORT and reduced Aß deposition in their brains. Additionally, AP39 inhibited brain atrophy in APP/PS1 mice. Based on these results, AP39 was proposed as a promising drug candidate for AD treatment, and its anti-AD mechanism may involve protection against mitochondrial damage.

The WRKY transcription factors in the diploid woodland strawberry Fragaria vesca: Identification and expression analysis under biotic and abiotic stresses.

WRKY proteins comprise a large family of transcription factors that play important roles in response to biotic and abiotic stresses and in plant growth and development. To date, little is known about the WRKY gene family in strawberry. In this study, we identified 62 WRKY genes (FvWRKYs) in the wild diploid woodland strawberry (Fragaria vesca, 2n = 2x = 14) accession Heilongjiang-3. According to the phylogenetic analysis and structural features, these identified strawberry FvWRKY genes were classified into three main groups. In addition, eight FvWRKY-GFP fusion proteins showed distinct subcellular localizations in Arabidopsis mesophyll protoplasts. Furthermore, we examined the expression of the 62 FvWRKY genes in 'Heilongjiang-3' under various conditions, including biotic stress (Podosphaera aphanis), abiotic stresses (drought, salt, cold, and heat), and hormone treatments (abscisic acid, ethephon, methyl jasmonate, and salicylic acid). The expression levels of 33 FvWRKY genes were upregulated, while 12 FvWRKY genes were downregulated during powdery mildew infection. FvWRKY genes responded to drought and salt treatment to a greater extent than to temperature stress. Expression profiles derived from quantitative real-time PCR suggested that 11 FvWRKY genes responded dramatically to various stimuli at the transcriptional level, indicating versatile roles in responses to biotic and abiotic stresses. Interaction networks revealed that the crucial pathways controlled by WRKY proteins may be involved in the differential response to biotic stress. Taken together, the present work may provide the basis for future studies of the genetic modification of WRKY genes for pathogen resistance and stress tolerance in strawberry.

Identification and expression analysis of heat shock transcription factors in the wild Chinese grapevine (Vitis pseudoreticulata).

Heat shock transcription factors (Hsfs) are known to play pivotal roles in the adaptation of plants to heat stress and other stress stimuli. While grapevine (Vitis vinifera L.) is one of the most important fruit crops worldwide, little is known about the Hsf family in Vitis spp. Here, we identified nineteen putative Hsf genes (VviHsfs) in Vitis spp based on the 12 × grape genome (V. vinifera L.). Phylogenetic analysis revealed three classes of grape Hsf genes (classes A, B, and C). Additional comparisons between grape and Arabidopsis thaliana demonstrated that several VviHsfs genes occurred in corresponding syntenic blocks of Arabidopsis. Moreover, we examined the expression profiles of the homologs of the VviHsfs genes (VpHsfs) in the wild Chinese Vitis pseudoreticulata accession Baihe-35-1, which is tolerant to various environmental stresses. Among the nineteen VpHsfs, ten VpHsfs displayed lower transcript levels under non-stress conditions and marked up-regulation during heat stress treatment; several VpHsfs also displayed altered expression levels in response to cold, salt, and hormone treatments, suggesting their versatile roles in response to stress stimuli. In addition, eight VpHsf-GFP fusion proteins showed differential subcellular localization in V. pseudoreticulata mesophyll protoplasts. Taken together, our data may provide an important reference for further studies of Hsf genes in Vitis spp.

Identification and utilization of a new Erysiphe necator isolate NAFU1 to quickly evaluate powdery mildew resistance in wild Chinese grapevine species using detached leaves.

The most economically important disease of cultivated grapevines worldwide is powdery mildew caused by the biotrophic fungal pathogen Erysiphe necator. To integrate effective genetic resistance into cultivated grapevines, numerous disease resistance screens of diverse Vitis germplasm, including wild species, have been conducted to identify powdery mildew resistance, but the results have been inconsistent. Here, a new powdery mildew isolate that is infectious on grapevines, designated Erysiphe necator NAFU1 (En. NAFU1), was identified and characterized by phylogeny inferred from the internal transcribed spacer (ITS) of pathogen ribosomal DNA sequences. Three classical methods were compared for the maintenance of En. NAFU1, and the most convenient method was maintenance on detached leaves and propagation by contact with infected leaves. Furthermore, controlled inoculations of En. NAFU1 were performed using detached leaves from 57 wild Chinese grapevine accessions to quickly evaluate powdery mildew resistance based on trypan blue staining of leaf sections. The results were compared with previous natural epidemics in the field. Among the screened accessions inoculated with En. NAFU1, 22.8% were resistant, 33.3% were moderately resistant, and 43.9% were susceptible. None of the accessions assessed herein were immune from infection. These results support previous findings documenting the presence of race-specific resistance to E. necator in wild Chinese grapevine. The resistance of wild Chinese grapevine to En. NAFU1 could be due to programmed cell death. The present results suggest that En. NAFU1 isolate could be used for future large-scale screens of resistance to powdery mildew in diverse Vitis germplasms and investigations of the interaction between grapevines and pathogens.

Identification, isolation, and expression analysis of heat shock transcription factors in the diploid woodland strawberry Fragaria vesca.

Heat shock transcription factors (Hsfs) are known to play dominant roles in plant responses to heat, as well as other abiotic or biotic stress stimuli. While the strawberry is an economically important fruit plant, little is known about the Hsf family in the strawberry. To explore the functions of strawberry Hsfs in abiotic and biotic stress responses, this study identified 17 Hsf genes (FvHsfs) in a wild diploid woodland strawberry (Fragaria vesca, 2n = 2x = 14) and isolated 14 of these genes. Phylogenetic analysis divided the strawberry FvHsfs genes into three main groups. The evolutionary and structural analyses revealed that the FvHsf family is conserved. The promoter sequences of the FvHsf genes contain upstream regulatory elements corresponding to different stress stimuli. In addition, 14 FvHsf-GFP fusion proteins showed differential subcellular localization in Arabidopsis mesophyll protoplasts. Furthermore, we examined the expression of the 17 FvHsf genes in wild diploid woodland strawberries under various conditions, including abiotic stresses (heat, cold, drought, and salt), biotic stress (powdery mildew infection), and hormone treatments (abscisic acid, ethephon, methyl jasmonate, and salicylic acid). Fifteen of the seventeen FvHsf genes exhibited distinct changes on the transcriptional level during heat treatment. Of these 15 FvHsfs, 8 FvHsfs also exhibited distinct responses to other stimuli on the transcriptional level, indicating versatile roles in the response to abiotic and biotic stresses. Taken together, the present work may provide the basis for further studies to dissect FvHsf function in response to stress stimuli.

Fenofibrate reduces amyloidogenic processing of APP in APP/PS1 transgenic mice via PPAR-α/PI3-K pathway.

The peroxisome proliferator-activated receptor alpha (PPAR-α), a member of the family of ligand-activated nuclear hormone receptors, plays a relevant role in the development of Alzheimer's disease (AD). To better understand the role of PPAR-α in AD, we examined the ability of fenofibrate (a PPAR-α agonist) to regulate amyloid precursor protein (APP) processing in APP/PS1 transgenic mice. After intragastric administration of fenofibrate into 3-month-old APP/PS1 transgenic mice for 6 months, and the levels of relative proteins were quantified by quantitative reverse transcription-PCR, Western blotting and ELISA. We found that fenofibrate increased the expression of PPAR-α, and decreased beta-site amyloid precursor protein cleaving enzyme 1 (BACE-1) mRNA and protein levels, and also reduced soluble APPß (sAPPß) and amyloid-ß 42 (Aß42) releases. However. fenofibrate did not modify the levels of APP and presenilin 1 (PS1). Furthermore, LY294002, the phosphoinositide 3-kinase (PI3-K) inhibitor, suppressed the effects of fenofibrate on BACE-1, sAPPß, and Aß42, but not PPAR-α. Our data suggest that fenofibrate may reduce the amyloidogenic processing of APP in APP/PS1 transgenic mice via PPAR-α/PI3-K pathway.

Augmenting the antitumor effect of TRAIL by SOCS3 with double-regulated replicating oncolytic adenovirus in hepatocellular carcinoma.

Aberrant JAK/STAT3 pathway has been reported to be related to hepatocellular carcinoma (HCC) in many cell lines. In this study, a double-regulated oncolytic adenovirus vector that can replicate and induce a cytopathic effect in alpha-fetoprotein (AFP)-positive HCC cell lines with p53 dysfunction was successfully constructed. Two therapeutic genes, suppressor of cytokine signaling 3 (SOCS3) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), were chosen and incorporated into this vector system, respectively. The combined treatment of AFP-D55-SOCS3 and AFP-D55-TRAIL (2:3 ratio) exhibited potent antitumor activity in AFP-positive HCC cell lines compared with any other treatment both in vitro and in vivo. Specific replication and low progeny yield in AFP-positive HCC cell lines rendered these double-regulated oncolytic adenoviruses remarkably safe. Our data demonstrated that restoration of SOCS3, which inhibits the JAK/STAT3 pathway, by AFP-D55-SOCS3 not only could antagonize HCC therapeutic resistance to TRAIL and adenoviruses, but could also induce cell cycle arrest in HCC cell lines. SOCS3 could down-regulate Cyclin D1 and anti-apoptotic proteins such as XIAP, Survivin, Bcl-xL, and Mcl-1, which are responsible for the synergistic inhibitory effects of AFP-D55-SOCS3 and AFP-D55-TRAIL. Dual gene and double-regulated oncolytic adenoviruses may provide safety and excellent antitumor effects for liver cancer, which is the advantage of a cancer-targeting gene virotherapy strategy.

Circulating ghrelin was negatively correlated with pulmonary arterial pressure in atrial septal defect patients / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Ghrelin was found to attenuate the magnitude of pulmonary arterial hypertension and pulmonary vascular remodeling in rats. The objective of this study was to explore the fasting plasma ghrelin level and the relationships between ghrelin and pulmonary arterial pressure (PAP) in atrial septal defect (ASD) patients with pulmonary arterial hypertension (PAH).</p><p><b>METHODS</b>Fasting plasma ghrelin, obestatin, and insulin levels were measured by enzyme linked immunosorbent assay (ELISA) method in ASD patients with or without PAH according to the manufacturer's instructions. Insulin resistance was calculated by the homeostasis model of assessment for insulin resistance (HOMA-IR) approach, calculated as fasting insulin (microunits/ml)× fasting blood glucose (mmol/L)/22.5. Comparisons between the parameters of patients with PAH and those of patients with normal PAP were performed with an unpaired Student's t test. The relationships between ghrelin and various clinical parameters were examined by bivariate correlations and multiple regression analysis.</p><p><b>RESULTS</b>We found that the fasting plasma ghrelin level and the ratio of ghrelin to obestatin were significantly lower in the PAH group compared with the control group ((582.4±12.8) pg/ml vs. (1045.2±95.5) pg/ml, P < 0.05 and 30.5±4.9 vs. 70.0±9.7, P < 0.01). The fasting plasma obestatin level was higher in the PAH group compared with the control group, but the difference between them was not significant ((23.2±3.1) pg/ml vs. (16.3±1.6) pg/ml, P > 0.05). In a multiple regression model analysis, only mean PAP was an independent predictor of ghrelin and the ratio of ghrelin to obestatin (standardized coefficient = -0.737, P < 0.001 and standardized coefficient = -0.588, P = 0.006, respectively).</p><p><b>CONCLUSION</b>Ghrelin is negatively correlated with mean PAP and this suggests that circulating ghrelin might predict the severity of pulmonary hypertension in ASD patients with PAH.</p>

Preparation of anti-P21-activated kinase 5 polyclonal antibody and its application in dental germ cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To clone PAK5-N terminal sequence for expression in E. coli to prepare its polyclonal antibody, and examine the role of PAK5 in dental germ cells.</p><p><b>METHODS</b>Based on human PAK5 cDNA sequence, PCR primers were designed to amplify PAK5-N terminal sequence. The PCR product was cloned into the expression vector pGEX-4T-1 EcoRI/XhoI sites, and the recombinant plasmids were identified by agarose gel electrophoresis followed by DNA sequence analysis. The recombinant plasmids were transformed into E. coli BL21 and the expression of GST-fusion protein was induced by IPTG. Glutathione-Sepharose beads were used to purify GST-fusion PAK5-N-terminal fragment. Anti-PAK5 polyclonal antibody was obtained in immunizing rabbits with purified GST-PAK5 N-terminal fusion protein, and the antibodies were purified by protein A beads and used for detection of PAK5 expression in dental germ cells.</p><p><b>RESULTS AND CONCLUSIONS</b>We successfully cloned PAK5-N terminal gene fragment, and achieved protein expression, purification and production of PAK5-NT polyclonal antibody. The results of Western blotting indicated that PAK5 can be highly expressed in the dental germ cells, suggesting that PAK5 may play an important role in biological function of dental germ cells.</p>

Colocalization of 11beta-hydroxysteroid dehydrogenase type I and glucocorticoid receptor and its significance in rat hippocampus / 生理学报

This paper was designed to observe the colocalization of 11beta-HSD1 and GR, and its significance in the rat hippocampus. Immunocytochemical dual-staining showed that not only 11beta-HSD1 but also GR immunoreactive substances were present in the cultured rat hippocampal neurons. Moreover, they were colocalized in the same hippocampal neuron. Synthetic glucocorticoid dexamethasone (DEX) up-regulated the protein expression and activity of 11beta-HSD1 in the cultured hippocampal neurons, as determined by Western blot and thin layer chromatography (TLC) respectively. The transfection of PC12 cells with the plasmid containing promoter sequence of 11beta-HSD1 gene and the reporter gene of CAT enzyme was conducted. DEX up-regulated the reporter gene expression in the system described above. The up-regulation of 11beta-HSD1 and reporter gene expression induced by DEX were both blocked by GR antagonist RU38486. Our study suggests that the colocalization of 11beta-HSD1 and GR in the hippocampus may be implicated in the up-regulation of 11beta-HSD1 expression by glucocorticoids combining to its promoter region, which in turn produces more biologically active glucocorticoids necessary for the binding of low affinity of GR.