Enzymatic activity of bone markers on Lithobates catesbeianus (Shaw, 1802) growth during the ossification process / Atividade enzimática de marcadores ósseos no crescimento de Lithobates catesbeianus (Shaw, 1802) durante o processo de ossificação

Abstract In order to better understand the ossification processes in anurans our study was carried out on tadpoles and adults of Lithobates catesbeianus. In this sense, we characterized the kinetic properties of alkaline phosphatase with p-nitrophenylphosphatase (pNPP) and pyrophosphate (PPi) and evaluated the activities of tartrate-resistant acid phosphatase and acid phosphatase. The enzyme extracts were obtained from tadpoles and adult femurs, which were divided into epiphysis and diaphysis. After homogenization, the samples were submitted to differential centrifugation to obtain cell membranes and, further, to phospholipase C (PIPLC) treatment, to remove membrane-bound proteins anchored by phosphatidylinositol. The average of specific activity for pNPP hydrolysis (at pH 10.5) by alkaline phosphatase released by phosphatidylinositol-specific phospholipase C (PIPLC) from Bacillus cereus among different bone regions at different animal ages was 1,142.57 U.mg-1, while for PPi hydrolysis (at pH 8.0), it was 1,433.82 U.mg-1. Among the compounds tested for enzymatic activity, the one that influenced the most was EDTA, with approximately 67% of inhibition for pNPPase activity and 77% for PPase activity. In the case of kinetic parameters, the enzyme showed a "Michaelian" behavior for pNPP and PPi hydrolysis. The Km value was around 0.6mM for pNPPase activity and ranged from 0.01 to 0.11mM for PPase activity, indicating that the enzyme has a higher affinity for this substrate. The study of pNPP and PPi hydrolysis by the enzyme revealed that the optimum pH of actuation for pNPP was 10.5, while for PPi, which is considered the true substrate of alkaline phosphatase, was 8.0, close to the physiological value. The results show that regardless of the ossification type that occurs, the same enzyme or isoenzymes act on the different bone regions and different life stages of anurans. The similarity of the results of studies with other vertebrates shows that anurans can be considered excellent animal models for the study of biological calcification.

Resumo Para melhor compreender o processo de ossificação em anuros, nosso estudo foi conduzido em girinos e adultos de Lithobates catesbeianus. Nesse sentido, as propriedades cinéticas da fosfatase alcalina com p-nitrofenilfosfato (pNPP) e pirofosfato (PPi) foram caracterizadas, e as atividades enzimáticas das fosfatases ácida e ácida tartarato resistente foram avaliadas. Os extratos enzimáticos foram obtidos de fêmur de girinos e adultos, divididos em epífise e diáfise. Após a homogeneização as amostras foram submetidas à centrifugação diferencial para obter membrana celular e, em seguida, ao tratamento com fosfolipase C (PIPLC), para remover as proteínas de membrana ancoradas por fosfatidilinositol. A média da atividade específica da fosfatase alcalina, liberada pela PIPLC de Bacillus cereus, para a hidrólise de pNPP (pH 10,5) nas diferentes regiões do fêmur e idades dos animais foi de 1.142,57 U.mg-1, enquanto para a hidrólise do PPi (pH 8,0) foi de 1.433,82 U.mg-1. Entre os compostos testados para a atividade enzimática, o de maior influência foi o EDTA, inibindo aproximadamente 67% e 77% das atividades de pNPPase e PPase, respectivamente. Quanto aos parâmetros cinéticos, a enzima apresentou comportamento Michaeliano para a hidrólise dos dois substratos. O valor de Km foi de 0,6 mM para a atividade de pNPPase e variou de 0,01 a 0,11 para a atividade de PPase, indicando uma maior afinidade por esse substrato. O estudo da hidrólise de pNPP e PPi revelou que o pH ótimo aparente de atuação foi de 10,5 para o pNPP e 8,0 para o PPi, próximo ao fisiológico, sendo que esse é considerado o substrato natural da fosfatase alcalina. Os resultados demonstram que, apesar do tipo de ossificação que ocorre, a mesma enzima ou isoenzimas, atuam nos diferentes locais do osso e estágios de vida dos anuros. A similaridade dos estudos com os realizados com outros vertebrados apontam que os anuros podem ser considerados excelentes modelos animais para o estudo da calcificação biológica.

Enzymatic activity of bone markers on Lithobates catesbeianus (Shaw, 1802) growth during the ossification process

Abstract In order to better understand the ossification processes in anurans our study was carried out on tadpoles and adults of Lithobates catesbeianus. In this sense, we characterized the kinetic properties of alkaline phosphatase with p-nitrophenylphosphatase (pNPP) and pyrophosphate (PPi) and evaluated the activities of tartrate-resistant acid phosphatase and acid phosphatase. The enzyme extracts were obtained from tadpoles and adult femurs, which were divided into epiphysis and diaphysis. After homogenization, the samples were submitted to differential centrifugation to obtain cell membranes and, further, to phospholipase C (PIPLC) treatment, to remove membrane-bound proteins anchored by phosphatidylinositol. The average of specific activity for pNPP hydrolysis (at pH 10.5) by alkaline phosphatase released by phosphatidylinositol-specific phospholipase C (PIPLC) from Bacillus cereus among different bone regions at different animal ages was 1,142.57 U.mg-1, while for PPi hydrolysis (at pH 8.0), it was 1,433.82 U.mg-1. Among the compounds tested for enzymatic activity, the one that influenced the most was EDTA, with approximately 67% of inhibition for pNPPase activity and 77% for PPase activity. In the case of kinetic parameters, the enzyme showed a Michaelian behavior for pNPP and PPi hydrolysis. The Km value was around 0.6mM for pNPPase activity and ranged from 0.01 to 0.11mM for PPase activity, indicating that the enzyme has a higher affinity for this substrate. The study of pNPP and PPi hydrolysis by the enzyme revealed that the optimum pH of actuation for pNPP was 10.5, while for PPi, which is considered the true substrate of alkaline phosphatase, was 8.0, close to the physiological value. The results show that regardless of the ossification type that occurs, the same enzyme or isoenzymes act on the different bone regions and different life stages of anurans. The similarity of the results of studies with other vertebrates shows that anurans can be considered excellent animal models for the study of biological calcification.

Resumo Para melhor compreender o processo de ossificação em anuros, nosso estudo foi conduzido em girinos e adultos de Lithobates catesbeianus. Nesse sentido, as propriedades cinéticas da fosfatase alcalina com p-nitrofenilfosfato (pNPP) e pirofosfato (PPi) foram caracterizadas, e as atividades enzimáticas das fosfatases ácida e ácida tartarato resistente foram avaliadas. Os extratos enzimáticos foram obtidos de fêmur de girinos e adultos, divididos em epífise e diáfise. Após a homogeneização as amostras foram submetidas à centrifugação diferencial para obter membrana celular e, em seguida, ao tratamento com fosfolipase C (PIPLC), para remover as proteínas de membrana ancoradas por fosfatidilinositol. A média da atividade específica da fosfatase alcalina, liberada pela PIPLC de Bacillus cereus, para a hidrólise de pNPP (pH 10,5) nas diferentes regiões do fêmur e idades dos animais foi de 1.142,57 U.mg-1, enquanto para a hidrólise do PPi (pH 8,0) foi de 1.433,82 U.mg-1. Entre os compostos testados para a atividade enzimática, o de maior influência foi o EDTA, inibindo aproximadamente 67% e 77% das atividades de pNPPase e PPase, respectivamente. Quanto aos parâmetros cinéticos, a enzima apresentou comportamento Michaeliano para a hidrólise dos dois substratos. O valor de Km foi de 0,6 mM para a atividade de pNPPase e variou de 0,01 a 0,11 para a atividade de PPase, indicando uma maior afinidade por esse substrato. O estudo da hidrólise de pNPP e PPi revelou que o pH ótimo aparente de atuação foi de 10,5 para o pNPP e 8,0 para o PPi, próximo ao fisiológico, sendo que esse é considerado o substrato natural da fosfatase alcalina. Os resultados demonstram que, apesar do tipo de ossificação que ocorre, a mesma enzima ou isoenzimas, atuam nos diferentes locais do osso e estágios de vida dos anuros. A similaridade dos estudos com os realizados com outros vertebrados apontam que os anuros podem ser considerados excelentes modelos animais para o estudo da calcificação biológica.

Dual specificity phosphatases 8 directly interacts with mitogen-activated protein kinase 1 to regulate the proliferation and apoptosis of rheumatoid arthritis fibroblast-like synovial cells / 中华风湿病学杂志

Objective:To explore the interaction between dual specificity phosphatases 8 (DUSP8) and mitogen-activated protein kinase 1 (MAPK1) in rheumatoid arthritis fibroblast-like synovial (RA-FLS), and its effect on the proliferation and apoptosis of RA-FLSs.Methods:RA-FLS and normal fibroblast-like synovial cells (FLS) were cultured. Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) and Western blotting were used to detect the expression levels of DUSP8 mRNA and protein in the two groups of cells. DUSP8 overexpression cell lines and DUSP8 silencing cell lines were constructed using cell transfection technology and RNA interference technology, respectively. Cell counting kit 8 (CCK-8) method was used to detect cell proliferation in each group, and flow cytometry was used to detect cell apoptosis in each group. Western blotting was used to detect the expression levels of DUSP8, MAPK1, p-MAPK1, ki-67 and Bax protein in each group. The indirect immunofluorescence experiment was used to analyze the spatial co-localization of DUSP8 and MAPK1, and the co-immunoprecipitation experiment was used to analyze whether there was interaction between DUSP8 and MAPK1 protein. The t-test was used to compare the means of the two groups. One-way analysis of variance was used to compare the mean values of the three groups of samples, and then the LSD- t tests were used to compare the two groups. Results:In RA-FLS, both mRNA [(2.4±0.6) vs (11.2±0.8), t=21.63, P<0.001] and protein levels [(0.24±0.04) vs (0.74±0.08), t=9.45, P<0.001] of DUSP8 were significantly lower than FLS. Compared with the blank control group and the overexpression control group, RA-FLS cells transfected with pcDNA3.1-Myc-DUSP8 could inhibit the proliferation of RA-FLS cells [(90.5±5.6) vs (92.5±1.8) vs (56.4±4.4), F=138.60, P<0.001], increase the rate of apoptosis significantly [(12.7±1.4)% vs (12.6±1.3)% vs (27.5±3.0)%, F=16.98, P<0.001], increase the expression levels of DUSP8 [(0.49±0.05) vs (0.45±0.04) vs (0.73±0.07), Bax (0.39±0.06) vs (0.36±0.05) vs (0.89±0.10)] and down-regulate the expression levels of ki-67 [(1.07±0.12) vs (1.11±0.16) vs (0.70±0.08), and p-MAPK1/MAPK1 [(0.59±0.06) vs (0.65±0.07) vs (0.39±0.03) (all P<0.001). Compared with the blank control group and the silent control group, RA-FLS cells transfected with siRNA-DUSP8 could promote the proliferation of RA-FLS cells [(90.5±5.6) vs (91.1±2.9) vs (128.3±4.6), F=137.50, P<0.001) and decrease apoptosis rate [(12.7±1.4) vs (13.2±1.2) vs (5.4±0.7), F=16.98, P<0.001], down-regulate the expression levels of DUSP8 [(0.492±0.048) vs (0.432±0.051) vs (0.102±0.024)], Bax [(0.391±0.062) vs (0.411±0.058) vs (0.090±0.011)], and up-regulate the expression levels of ki-67 [(1.07±0.12) vs (1.11±0.15) vs (1.93±0.22)], p-MAPK1/MAPK1 [(0.59±0.06) vs (0.68±0.06) vs (0.93±0.11)] (all P<0.001). The results of indirect immunofluorescence tests showed that both DUSP8 and MAPK1 were ex-pressed in the cytoplasm and nucleus of RA-FLS. The co-immunoprecipitation study verified that DUSP8 and MAPK1 protein could interact with each other. Conclusion:DUSP8 can bind to MAPK1 and regulate the abundance of active phospho-MAPK1 through its phosphatase activity and by inhibiting the proliferation of RA-FLS and promoting apoptosis.

Identification and Expression Analysis of PP2C Gene Family Members in Cannabis sativa / 中国实验方剂学杂志

ObjectiveThe type 2C protein phosphatases （PP2C） are involved in numerous plant signal transduction pathways. They mainly participate in plant stress response and regulate second metabolites biosynthesis via negatively regulating MAPK signaling pathway. Herein，we were to identify and analyze PP2C （CsPP2C） gene family from hemp genome，in hope of providing comprehensive insights for studying CsPP2C function during the development of hemp. MethodMolecular Evolutionary Genetics Analysis （MAGA）-X was used to construct phylogenetic tree. Expert Protein Analysis System （ExPASy），WoLF PSORT，Multiple EM for Motif Elicitation （MEME），Batch Conserved Domain Search （Batch-CD-Search），PlantCare，and TBtools were used，respectively，to predict CsPP2C physicochemical properties，subcellular localization，conserved motifs，protein structure，cis-element in promoter and collinearity with Arabidopsis PP2C. Cannabis sativa transcriptome and Real-time polymerase chain reaction（Real-time PCR） were used to analyze and verify gene expressions，respectively. ResultFifty-two CsPP2C with conserved domains were identified from the entire genome of hemp，encoding proteins ranging from 244 to 1 089 aa in length and with molecular weights ranging from 26.76 to 122.53 kDa. Those genes were mainly distributed in the nucleus，cytoplasm and chloroplast. The 47 CsPP2C were divided into 10 subfamilies，and the remaining 5 were not clustered. Seven pairs of homologous genes between hemp and Arabidopsis thaliana were identified according to collinear analysis. The light-responsive elements and abscisic acid elements are most abundant in the prediction. The gene expression heat map showed varied expression pattern of CsPP2C in different tissues. Real-time PCR results of three CsPP2C were consistent with transcriptome data. Moreover，alternative splicing analysis showed that some CsPP2C had alternative-splicing genes during evolution. ConclusionWe predicted and analyzed CsPP2C gene family in genomic scale and showed that CsPP2C are involved in many biological processes，whereby provides foundation for CsPP2C functional study.

Identification and Expression Analysis of PP2C Gene Family Members in Cannabis sativa / 中国实验方剂学杂志

ObjectiveThe type 2C protein phosphatases （PP2C） are involved in numerous plant signal transduction pathways. They mainly participate in plant stress response and regulate second metabolites biosynthesis via negatively regulating MAPK signaling pathway. Herein，we were to identify and analyze PP2C （CsPP2C） gene family from hemp genome，in hope of providing comprehensive insights for studying CsPP2C function during the development of hemp. MethodMolecular Evolutionary Genetics Analysis （MAGA）-X was used to construct phylogenetic tree. Expert Protein Analysis System （ExPASy），WoLF PSORT，Multiple EM for Motif Elicitation （MEME），Batch Conserved Domain Search （Batch-CD-Search），PlantCare，and TBtools were used，respectively，to predict CsPP2C physicochemical properties，subcellular localization，conserved motifs，protein structure，cis-element in promoter and collinearity with Arabidopsis PP2C. Cannabis sativa transcriptome and Real-time polymerase chain reaction（Real-time PCR） were used to analyze and verify gene expressions，respectively. ResultFifty-two CsPP2C with conserved domains were identified from the entire genome of hemp，encoding proteins ranging from 244 to 1 089 aa in length and with molecular weights ranging from 26.76 to 122.53 kDa. Those genes were mainly distributed in the nucleus，cytoplasm and chloroplast. The 47 CsPP2C were divided into 10 subfamilies，and the remaining 5 were not clustered. Seven pairs of homologous genes between hemp and Arabidopsis thaliana were identified according to collinear analysis. The light-responsive elements and abscisic acid elements are most abundant in the prediction. The gene expression heat map showed varied expression pattern of CsPP2C in different tissues. Real-time PCR results of three CsPP2C were consistent with transcriptome data. Moreover，alternative splicing analysis showed that some CsPP2C had alternative-splicing genes during evolution. ConclusionWe predicted and analyzed CsPP2C gene family in genomic scale and showed that CsPP2C are involved in many biological processes，whereby provides foundation for CsPP2C functional study.

Kinetic and thermodynamic parameters of four acid phosphatases from edible seeds of oleaginous cucurbit Lagenaria siceraria (Molina) Standl.

In Sub-Saharan Africa, Lagenaria siceraria is an oleaginous cucurbit widely consumed for its edibleseeds. These seeds were previously categorized as good enzyme sources, including four non-specific acidphosphatases isolated and designated as BLsAP1, BLsAP2, RLsAP1, and RLsAP2. In this study, we investigateon the kinetic and thermodynamic characteristics of these biocatalysts in order to evaluate their thermostability.Thermal inactivation was carried out in the temperature range of 55°C to 80°C from 5 to 60 minutes. The resultsrevealed that thermal inactivation of studied acid phosphatases follows first order kinetics. At their optimumtemperatures, these enzymes showed high half-lives ranging from 169.06 to 495.10 minutes and D valuesfrom 561.71 to 1,645.03 minutes. Moreover, they exhibited high activation energies (from 155.08 to 200.55kJ/mol) and average enthalpy values (from 152.23 to 197.74 kJ.mol−1) suggesting their good thermostability.The comparison of all these values revealed that the two acid phosphatases (RLsAP1 and RLsAP2) from theround-fruited cultivar of L. siceraria showed better thermal stability than those (BLsAP1 and BLsAP2) fromthe blocky-fruited cultivar.

Justicia secunda Vahl leaf fraction protects against acetaminophen-induced liver damage in rats by alleviating oxidative stress and enhancing membrane-bound phosphatase activities

To investigate the effect of Justicia secunda Vahl leaf fraction against acetaminophen-induced oxidative damage in the liver of rats. Methods: In vitro antioxidant assays were performed on Justicia secunda leaf fractions. Gas chromatography-mass spectrometry analytical method was done. Experimental animals were orally administered with 2 g/kg b.wt. acetaminophen, 100-500 mg/kg b.wt. Justicia secunda ethyl acetate leaf fraction (JSELF), and 100 mg/kg b.wt. silymarin. Blood and liver were collected to measure hepatic, oxidative stress, and membrane-bound phosphatase markers. Results: JSELF had significantly (P0.05) high total antioxidant capacity and inhibition of lipid peroxidation. JSELF-treated animals had reduced plasma hepatic enzymes, serum C-reactive protein, and oxidized low-density lipoprotein while hepatic superoxide dismutase, catalase, and reduced glutathione levels were elevated compared with untreated control. Membrane-bound phosphatase activities were improved in JSELF-treated animals. GC-MS detected tentatively 7 antioxidants and 4 hepatoprotective compounds. Conclusions: JSELF could protect against oxidative stress and improve membrane-bound phosphatase activity in rats with acetaminophen-induced hepatic damage.

Justicia secunda Vahl leaf fraction protects against acetaminophen-induced liver damage in rats by alleviating oxidative stress and enhancing membrane-bound phosphatase activities

Objective: To investigate the effect of Justicia secunda Vahl leaf fraction against acetaminophen-induced oxidative damage in the liver of rats. Methods: In vitro antioxidant assays were performed on Justicia secunda leaf fractions. Gas chromatography-mass spectrometry analytical method was done. Experimental animals were orally administered with 2 g/kg b.wt. acetaminophen, 100-500 mg/kg b.wt. Justicia secunda ethyl acetate leaf fraction (JSELF), and 100 mg/kg b.wt. silymarin. Blood and liver were collected to measure hepatic, oxidative stress, and membrane-bound phosphatase markers. Results: JSELF had significantly (P<0.05) high total antioxidant capacity and inhibition of lipid peroxidation. JSELF-treated animals had reduced plasma hepatic enzymes, serum C-reactive protein, and oxidized low-density lipoprotein while hepatic superoxide dismutase, catalase, and reduced glutathione levels were elevated compared with untreated control. Membrane-bound phosphatase activities were improved in JSELF-treated animals. GC-MS detected tentatively 7 antioxidants and 4 hepatoprotective compounds. Conclusions: JSELF could protect against oxidative stress and improve membrane-bound phosphatase activity in rats with acetaminophen-induced hepatic damage.

Cloning and expression analysis on a protein phosphatase encoding gene DoPP2C1 in Dendrobium officinale / 中草药

Objective: To isolate and characterize a protein phosphatase (PP) encoding gene DoPP2C1 in a rare endangered medicinal orchid species Dendrobium officinale, followed by bioinformatics analysis and expression pattern detection. Methods: qRT-PCR and RACE technologies were used to isolate the full length cDNA of DoPP2C1. Characteristics of physiochemical properties, conserved domains, and subcellular localization of DoPP2C1 protein were determined using a series of bioinformatics tools. The analyses of multiple alignment and phylogenetic tree were performed using DNASTAR 7.0 and MEGA 6.0 softwares, respectively. Quantitative PCR was used for gene expression analysis. Results: The full length cDNA of DoPP2C1 (GenBank accession KJ995533) was 1 221 bp in length, and encoded a 285-aa protein with a molecular weight of 31 080 and an isoelectric point of 6.18; The deduced DoPP2C1 protein had one PP2C domain (27-285), which are all conserved among the PP2C proteins. DoPP2C1 protein did not contain a signal peptide or a transmembrane region, and was predicted to locate in cytoplasm; DoPP2C1 had high identities (56.3%-73.7%) with various PP2C proteins in plants; DoPP2C1 was closely related to Oryza sativa OsPP2C62, Sorghum bicolor XP_002462907, Hordeum vulgare BAK00362 and Triticum urartu EMS47641 proteins, and belonged to the Group F1 of the PP2C evolutionary tree; DoPP2C1 gene was differentially expressed in the three included organs. The transcripts were more abundant in the roots and stems, with 7.57 and 1.79 fold, respectively, over that in the leaves. Conclusion: Molecular characterization of DoPP2C1 gene was obtained, which will be useful for further functional determination of the gene involving in the growth and development, physiological stress adaptations, and secondary metabolic regulations of D. officinale.

The effect of acetated ringer's solution on inflammatory mediators on lung tissue and their signaling pathways in rats with shock / 中华急诊医学杂志

Objective To study the effects of acetated ringer's solution resuscitation in hemorrhagic shock rats on inflammatory mediators on lung tissue and their JNK (c-Jun N-terminal kinase) signaling pathways. Methods Thirty-two SD rats were randomly(random number) divided into four groups: shock without resuscitation group (CR, n=8), saline group (NR, n=8), lactated ringer's solution group (LR, n=8) and acetated ringer's solution group (AR,n=8). The rats of NR group, LR group and AR group were prepared into shock models (mean arterial blood pressure maintained at 40-45 mmHg),The rats of NR group, LR group and AR group were in the shock for 60 min and then the corresponding kinds of liquid were administered for 30 min and observation was carried out for 4 hours. The rats of CR group without liquid resuscitation were observed for 4 hours after shock. After that, the lung tissues of rats were taken from NR group, LR group and AR group as well as from CR group 4 hours after shock (if the rats died, the lung tissues were immediately taken). The levels of TNF-α, IL-4 and IL-10 mRNA in lung were measured by real-time polymerase chain reaction (RT-PCR),and Western blot was used to measure the levels of JNK phosphorylation and MKP-1 acetylation. The one-way ANOVA was used for comparison among groups. Between the two groups, the comparison was analyzed by using LSD-t test. Results The IL-4 mRNA expression of lung tissue in AR group was higher than that in CR group, NR group and LR group (CR group:0.42±0.34; NR group:2.60±0.66; LR group:6.24±2.95; AR group: 11.08±4.24; P＜0.05).The IL-10 mRNA expression of lung tissue in AR group was significantly higher than that in CR group, NR group and LR group (CR group:0.25±0.25; NR group:2.79±1.62; LR group:3.51±1.66; AR group:9.35±2.86;P＜0.01).The TNF-a mRNA expression in AR group was significantly lower than that in CR group, NR group and LR group (CR group:4.98±1.26; NR group:2.50±0.76; LR group:3.87±3.00; AR group:0.19±0.09; P＜0.01). The level of JNK phosphorylation in lung tissue of rats in AR group was significantly lower than that in CR group, NR group and LR group (CR group:0.52±0.12; NR group:0.42±0.08; LR group:0.30±0.08; AR group:0.17±0.06;P＜0.01). The level of MKP-1 acetylation in lung tissue of rats in AR group was significantly higher than that in CR group, NR group and LR group (CR group:0.14±0.07; NR group:0.30±0.07; LR group:0.37±0.02; AR group:0.48±0.06;P＜0.01). Compared with normal saline and lactated ringer's solution, acetated ringer's solution used in hemorrhagic shock rats could promote MKP-1 acetylation, inhibit the phosphorylation of JNK, significantly inhibit the lung tissue TNF-a released, promote the release of anti-inflammatory factors, IL-4 and IL-10. Conclusions The acetated ringer's solution for resuscitation of hemorrhagic shock in rats could reduce inflammation of lung tissue in a certain extent, probably by enhanced the acetylation of MKP-1 to inhibited JNK signaling pathway and reduced lung tissue inflammation.

Construction and characterization of the stk and stp1 knockout mutant in streptococcus suis type 2 / 医学研究生学报

Objective Serine /threonine kinases (STK) and phosphatases (STP) regulate various physiological activities of prokaryotes by reversible phosphorylation of proteins .This paper aimed to study the effects of simultaneous deletion of the stk and stp1 genes on the biological characteristics and pathogenicity of streptococcus suis type 2, the Chinese virulent strain 05ZYH33. Methods The double mutant of the stk and stp1 genes of 05ZYH33 was constructed by homologous recombination .The biological characteristics of the wild strain 05ZYH33 and the mutant strain Δstk/stp1 were compared.The effects of the stk and stp1 deletion on bacterial virulence was analyzed using cell adhesion assay , anti-phagocytosis assay and the mouse model of infection . Results RT-PCR showed that the stk and stp1 genes were replaced by the spectinomycin resistance gene Spc r and the mutant strain was successfully constructed .Experi-ments of biological characterization revealed gradually increased value of 05ZYH33 and Δstk/stp1 at 2 hours after inoculation and a plateau period at 7 hours.The logarithmic phase of the mutant strain (A600≈0.4) was 1 hour later than that of the wild one , and the bacterial den-sity of the former was lower than that of the latter after the plateau pe -riod (0.8 vs 1.0).On the blood plates of 05ZYH33 and Δstk/stp1 were observed greyish, round, semitransparent, wet and smooth-sur-faced tiny bacterial colonies , around which there were hemolysis rings with no significant differences in colony morphology and hemolytic ac -tivity.In the experiment on pathogenicity , the mice of the 05ZYH33 group all died within 12 hours while 9 of the 30 mice in the Δstk/stp1 group died within 12 hours and all died within 24 hours. Conclusion The simultaneous deletion of the stk and stp1 genes may mainly affect the regulation of the proteins associated with bacte -rial proliferation and division.

Association of the protein tyrosine phosphatase non-receptor 22 polymorphism (PTPN22) with endometriosis: a meta-analysis / Associação do polimorfismo da proteína tirosina fosfatase não receptora 22 (PTPN22) com endometriose: uma metanálise

ABSTRACT Objective To evaluate PTPN22 C1858T polymorphism and the risk of endometriosis. Methods A meta-analysis of 10 published case-control studies (from four articles), with a total sample of 971 cases and 1,181 controls, was performed. We estimated risk (odds ratio and 95% confidence intervals) of endometriosis associations with the C1858T polymorphism. Results A significant increased risk in all genetic models of the variant T allele with endometriosis (odds ratio: 3.14-5.55; p<0.00001-0.002) was found. The analysis without the study whose controls deviated from the Hardy-Weinberg equilibrium exacerbated these effects in the homozygous and recessive models (odds ratio: 7.19-9.45; p<0.00001-0.0002). In the Italian subgroup, a significant risk association was found in the homozygous and recessive models (odds ratio: 8.72-11.12; p=0.002). Conclusion The associations observed between PTPN22 (C1858T) and the risk of endometriosis suggest this polymorphism might be a useful susceptibility marker for this disease.

RESUMO Objetivo Avaliar o polimorfismo PTPN22 C1858T e o risco de endometriose. Métodos Foi realizada uma metanálise de 10 estudos caso-controle publicados (a partir de quatro artigos), com uma amostra total de 971 casos e 1.181 controles. O risco da associação da endometriose com o polimorfismo C1858T foi estimado em razão de chance e intervalo de confiança de 95%. Resultados Observou-se um aumento de risco significativo em todos os modelos genéticos com o alelo variante T e a endometriose (razão de chance: 3,14-5,55; p<0,00001-0,002). A análise sem incluir o estudo, em que os controles não estavam em equilíbrio de Hardy-Weinberg, mostrou aumento significativo nos modelos homozigotos e recessivos (razão de chance: 7,19-9,45; p<0,00001-0,0002). No subgrupo italiano, uma associação significativa foi encontrada considerando os modelos homozigoto e recessivo (razão de chance: 8,72-11,12; p=0,002). Conclusão As associações observadas entre PTPN22 (C1858T) e o risco de endometriose sugerem que este polimorfismo pode ser um marcador de suscetibilidade para a endometriose.

The effect of estradiol on renal tubular exression of ATP-binding cassette superfamily member 2 via PI3K/Akt pathway / 中华风湿病学杂志

Objective To compare the serum estradiol level of gouty patients with healthy controls and to investigate whether estradiol upregulatesthe expression of the uric acid transporter ATP-binding cassette superfamily member 2 (ABCG2) of human renal tubular epithelial cells (HK-2)..Methods Serum of 16 male gout patients and 16 male healthy controls and their estradiol level were assessed with ELISA.The HK-2 cells wer cultured with different concentrations of estradiol for 24 hours or 48 hours.mRNA expression of ABCG2 was assessed by quantitative polymerase chain reaction (qPCR).HK-2 cells were cultured with estradiol or estradiol and inhibitor of PI3K/Akt pathway LY294002.mRNA expression of ABCG2 was assessed by qPCR,while the Akt,p-Ser473-Akt,p-Thr308-Akt and ABCG2 expression were investigated by Western blot.Data was analyzed using either the one-way analysis of variance or the t test.Results The level of serum uric acid in gout patients was [(547±18) μmol/L],which was significantly higher than that of healthy controls [(344±12) μmol/L),t=-5.395,P＜0.01].The level of estradiol in gout patients was (45±6) μmol/L,which was significantly lower than that of healthy controls [(100±8) μmol/L,t=9.375,P＜0.01].The mRNA expression of ABCG2 of 10-4 mol/L estradiol group was elevated after 24 hours or 48 hours (t=3.168,t=3.990;P＜0.01).In the group of co-stimulation withestradiol and LY294002,the ABCG2,p-Ser473-Akt and p-Thr308-Akt expression were down regulated compared to the estradiol group.Conclusion There is a significant correlation between serum estradiol and uric acid.Estradiol inducesthe expression of ABCG2 of HK-2 cells by activating PI3K/Akt pathway.

Expression of PTPN13 in human gastric cancer and gastric cancer SGC-7901 cell line and its effect on cell proliferation and invasion / 基础医学与临床

Objective To investigate the expression of PTPN13 in human gastric cancer and gastric cancer SGC-7901celllineanditsassociationwithproliferationandinvasion.Methods 106casesgastriccancertissuesamples and matched normal peritumorial tissues were collected .SGC-7901 cells were cultured and divided into two groups including pcDNA3.1-PTPN13 transfection group and without transfection group .Immunohistochemical technique was used to detecte protein expression of PTPN 13.The association of PTPN13 expression with tumor location , tumor size , depth of invasion and tumor metastasis were analyzed .The survival rate of patients with different PT-PN13 expression was calculated by Kaplan-Meier curves.CCK-8 assay was used to estimate the proliferation chan-ges.Using Trans-well assay analyzed the invasion of SGC-7901 cells.Moreover, Western blot was performed to de-tect the markers of EMT including E-cadherin, Snail and MMP9.Results Immunohistochemistry showed the expression rate of PTPN13 in gastric cancer tissues was lower than normal tissue (31 %vs 83%, P<0.05).The expression of PTPN13 in patients was related to with different tumor size , depth of invasion and tumor metastasis (P<0.05).The 2-year survival rate of patients with negative PTPN13 expression were lower.Overexpression reduced both proliferation and invasion in SGC-7901 cells.Up-regulated PTPN13 may increase E-cadherin level but decreases the level of Snail and MMP9 .Conclusions PTPN13 plays a role in gastric cancer tissue and cells as a tumor suppressor.Lower PTPN13 may predict a poor prognosis.PTPN13 may be used as therapy target in gastric cancer.

Scutellaria barbata flavonoids inhibits NFT aggregation and regulatory mechanism in rats induced by composited Aβ / 中国病理生理杂志

AIM: To investigate the effects of Scutellaria barbata flavonoids (SBF) on neurofibrillary tangle (NFT) aggregation, tau protein phosphorylation and the regulated mechanism of glycogen synthase kinase (GSK) 3βand protein phosphatase (PP) 2A in the rats induced by amyloid βprotein 25-35 (Aβ25-35) in combination with AlCl3 and re-combinant human transforming growth factor ( RHTGF)-β1( composited Aβ) .METHODS:The male SD rats were used to establish the simulated Alzheimer disease ( AD) model by intracerebroventricular injection of composited Aβ.The Morris water maze was applied for screening the successful model rats with learning and memory deficits .The successful model rats were daily and orally administrated with SBF at doses of 35, 70 and 140 mg/kg or positive control drug Ginkgo biloba leaves flavonoids ( GLF) at 140 mg/kg for 37 d.The silver nitrate staining was used to determine the cortical NFT .The protein levels of total tau, phosphorylated protein of tau at Ser199 and Ser214 sites, GSK3βand PP2A in hippocampus and cortex were determined by Western blot .The mRNA expression of GSK3βand PP2A in the hippocampus and cortex was detected by RT-PCR.RESULTS:Compared with sham group , the cell number of positive NFT with silver nitrate staining in model rat cerebral cortex was significantly increased .The protein levels of phosphorylated tau protein at Ser 199 and Ser214 sites, GSK3βin the hippocampus and cerebral cortex in the model rats dramatically elevated , and PP2A was marked decreased as compared with the sham group rats.Meanwhile, the mRNA expression of GSK-3βsignificantly increased but PP2A was de-creased.However, these above abnormalities were differently attenuated by treating with SBF at different doses or GLF at 140 mg/kg for 37 d.CONCLUSION: SBF suppresses the NFT aggregation by inhibition of the regulatory functions of GSK-3βand PP2A, thus reducing the phosphorylation of tau protein .

Análise imunocitoquímica e de expressão gênica de efeitos do bevacizumabe em explantes de retina de ratos lister e em linhagem celular de glia de Müller humana

INTRODUÇÃO: As doenças retinianas associadas à neovascularização, tais como a degeneração macular relacionada à idade e as retinopatias diabética e da prematuridade são as principais e mais importantes causas da cegueira em todo o mundo. Nos últimos anos, injeções intravítreas de fármacos com ação antiangiogênica, como o bevacizumabe (BVZ), têm sido de grande valia tanto em pacientes na fase adulta quanto nos recém-natos. Todavia, estudos experimentais in vitro e in vivo sugerem que essas drogas promovam efeitos adversos sobre alguns processos celulares, interferindo diretamente em mecanismos fisiológicos que mantém a homeostase do tecido retiniano, incluindo os mecanismos de proliferação, diferenciação e morte celular. OBJETIVO: investigar o efeito do BVZ nos processos de transcrição e tradução de marcadores da gliose: GFAP e vimentina, de morte celular, caspase-3 e beclina-1, e dos proteoglicanos relacionados à manutenção e desenvolvimento de tecido retiniano: neurocam, fosfacam e sindecam-3. MÉTODOS: Dois modelos experimentais foram usados nesse estudo: 1) linhagem celular de Müller de Glia humana adulta (MIO-M1), cultivada em meio de cultura D-MEM na presença e ausência de BVZ por 12 e 24 horas nas concentrações de 0,25 mg/mL e 0,50 mg/mL e 2) explantes de retinas de ratos 2 dias pós-nascidos submetidos à 0,50 mg/mL da droga por 48 horas. Durante este período foram mantidos a 5% de dióxido de carbono à temperatura de 37°C. A análise de proteínas foi realizada por imunocitohistoquímica e Western Blotting e a expressão de RNAm, pela reação em cadeia da polimerase em tempo real (PCR Real Time). Foi utilizado o Teste de ANOVA - fator único para a comparação entre os grupos controle e tratados com BVZ de um mesmo período (12h ou 24h) e o teste t de Student para a comparação entre as mesmas concentrações de 12h e 24h, e para a comparação entre os grupos controle e tratado com BVZ dos explantes (p < 0,05). RESULTADOS...

Backgraound: Vasoproliferative retinal disorders such as age-related macular, degeneration, diabetic retinopathy and retinopathy of prematurity are major causes of blindness in the world. In recent years, intravitreal injections of drugs with antiangiogenic action, as bevacizumab, have been very useful for both patients in adulthood and in newborns. However, experimental studies, in vivo and in vitro, suggest that antiangiogenic drugs may promote side effects in cellular proceedings, interfering directly in physiological mechanisms of cellular proliferation, differentiation and death. POURPOSE: Investigate the bevacizumab effects in transcription and translation processes of gliosis, GFAP and vimentin, cellular death markers, caspase-3 and beclin-1, and proteoglycans involved in retinal tissue maintenance and development, neurocan, phosphacan and syndecan-3. METHODS: Two experimental models were used on this research: cellular lineage of adult and human Müller glial cell(MIO-M1) were cultivated on D-MEM medium with 0,25 and 0,50 mg/mL bevacizumab for 12 and 24 hours, and two days old rat retinal explants submitted to 0,50 mg/mL for 48 hours. During this period were stored in laboratory ovens at 5% carbon dioxide pressure and 37 °C average temperature. Molecular techniques were used to evaluate gene expression and protein content. Protein assessments were performed by immunocytochemistry and western blotting analysis, while Real Time PCR was used to measure mRNA content. ANOVA tests one factor were applied to compare the control and BVZ groups of the same period (12h or 24h) and t test from Student to compare the same conditions of 12h and 24h, and to compare the control and BVZ retinal explants groups (p<0.05). RESULTS...

Advance research on protein tyrosine phosphatase SHP-2 and the regulating role of it in osteoclastogenesis / 医学研究生学报

SHP-2 is one of the protein tyrosine phosphatases which plays a role in the progress of dephosphorylation in organ -isms, participating in many kinds of signal transduction pathways .The mutation of SHP-2 is associated with many kinds of malignant diseases .In recent years , scholars have found that SHP-2 is the key factor in osteoclastogenesis , playing a positive role in the progress of reversible phosphorylation of osteoclasts .The objective of this article is to review the molecular biological characteristics of SHP-2, th diseases associated with the mutation of SHP-2 and the effects of SHP-2 in regulation of osteoclastogenesis .

Research progress of cell division cycle 25 homolog C and tumor radiosensitivity / 国际肿瘤学杂志

AsoneoftheCdc25phosphatasefamilymembers,Cdc25Cplaysanimportantroleinregu-lating mitosis of eukaryotic cells.In eukaryotic cells,CDK1-cell cycle protein B (CyclinB)compound mainly control the process of G2-M.The activity of Cdc25 C is the key in cell cycle into M phase.It activates CDK1-cyclinB complexes to promote cells from G2 to M phase .Improving Cdc25 C activity can promote the G2-M phase transition,and remove the G2-M phase retardation induced by ionizing radiation,preventing the damaged DNA from repaired into the phase of cell division,resulting in cell death caused by excessive cell proliferation, thus enhance radiosensitivity.

Effect of the FKBP51 · PHLPP · Akt signal module on the phosphorylation of Akt and hippocampal neuronal injury after the cerebral ischemia/reperfusion / 中华行为医学与脑科学杂志

Objective To investigate the effects of the FKBP51 · PHLPP · AKT signal module on the phosphorylation of Akt and hippocampal neuronal injury after the cerebral ischemia / reperfusion induced neuronal death in rat hippocampus.Methods Transient(15 min)brain ischemia was induced by the four-vessel occlusion in Sprague-Dawley rats.6 rats were used in each group.The antisense oligodeoxynucletides(AS ODN)of PHLPP2 (PH domain and leucine rich repeat protein phosphatases) was used to suppress the assembly of FKBP51 · PHLPP · Akt signal module by intracerebroventricular infusion once per day for 3 days before ischemia.After 6 hours reperfusion,interactions of PHLPP2 and FKBP51 (FK506 binding protein 5) with Akt were detected by immunoprecipitation (IP) and the phosphorylation of Akt was detected by western blot (IB).After 5 days reperfusion,rats were perfusion-fixed with paraformaldehyde and Hematoxylin-Eosin staining was used to examine the survival number of CA1 pyramidal cells of hippocampus.Results Compared to PHLPP2 MS ODN group(1.24±0.24,1.68±0.11,0.58±0.01),PHLPP2 AS ODN suppressed the assembly of the FKBP51 · PHLPP · Akt signaling module(1.06±0.01,1.04±0.13),and increased the phosphorylation of Akt(0.76±0.02) (P＜0.05).Furthermore,compared to PHLPP2 MS ODN group (20.1±2.5),the number of surviving neurons significantly increased in PHLPP2 AS ODN group(88.3±2.7)(P＜0.05).Conclusion The increasing assembly of FKBP51 · PHLPP · Akt signal module can damage CA1 pyramidal cells of hippocampus by inhibiting the phosphorylation level of Akt.

Biochemical and cellularchanges in Oreochromis niloticus related to the water pollution of a degraded river / Alterações bioquímicas e celulares em Oreochromis niloticus relacionadas com a poluição da água de um rio degradado

The effects of polluted water at three sites in the Marinho River, Brazil, on Oreochromis niloticus (Nile tilápia) were investigated using histological, hematological and biochemical approaches. Fish exposed to the impacted water demonstrated that histological changes in gills were accompanied by nuclear and micronuclei abnormalities in cells. The activity of liver and plasma biomarkers (alkaline phosphatase (ALP), acid phosphatase (ACP), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and liver glutathione S-transferase (GST)) showed an expressive change due to the. The results were also correlated with the highest levels of Cu+2, Zn+2 and Mn+2 in the water. The data of this study evidenced the importance of using a set of biomarkers to quantify pollution in lentic ecosystems. Additionally, histological analyses of gills and erythrocytes have proven to be an important instrument for signaling the impact of pollutants in rivers.

Os efeitos da poluição da água de três locais do Rio Marinho, Brasil, em Oreochromis niloticus foram investigados usando técnicas histológicas, hematológicas e bioquímicas. Peixes expostos à água impactada demonstraram que alterações histológicas nas brânquias foram acompanhadas de anomalias nucleares e micronúcleo nas células. A determinação da atividade de biomarcadores em fígado e plasma de tilápia (fosfatase alcalina (ALP), fosfatase ácida (ACP), alanina aminotransferase (ALT), aspartato aminotransferase (AST) e glutationa S-transferase (GST)) mostrou uma substancial alteração em função da poluição. Os resultados são correlacionados com os níveis mais elevados de Cu+2, Zn+2 e Mn+2 na água. Os dados deste estudo demonstram a importância de utilizar um conjunto de biomarcadores para quantificar a poluição em ecossistemas lênticos. Adicionalmente, as análises histológicas das brânquias e de eritrocitos têm provado ser importante instrumento para sinalizar a impactação de poluentes ao longo de rios.