Stress affects the male reproductive system and can cause sub-fertility or infertility. Although Phyllanthus emblica L. (PE) extract has been shown to have high antioxidant capacity and protective properties in damaged tissue, the preventive effects of PE extract on testicular function from stress-related impairment have never been demonstrated. This study aimed to investigate the effects of PE aqueous leaf extract on testicular impairment and protein marker changes in rats suffering from chronic stress. Adult male rats were divided into four groups: a control group, a chronic stress (CS) group, and two groups with CS that received different doses of PE extract (50 or 100 mg/kg body weight (BW)). In the treatment groups, the animals were given PE extract daily before stress induction for 42 consecutive days. Stress was induced through immobilization (4 h/d) followed by forced cold swimming (15 min/d). Sperm quality and the histology of the testes and caudal epididymis were examined, as were levels of serum corticosterone, testosterone, and malondialdehyde (MDA). The expressions of testicular steroidogenic acute regulatory (StAR) and tyrosine-phosphorylated proteins were investigated using immuno-Western blot analysis, as these proteins are assumed to play important roles in spermatogenesis and androgen synthesis. The results showed that PE (50 mg/kg BW) significantly increased sperm concentration and testosterone levels, while decreasing corticosterone levels, MDA levels, sperm head abnormalities, and acrosome-reacted sperm in CS rats. In addition, PE at both doses was found to diminish testicular histopathology in the CS rats. We also found that 50 mg/kg BW of PE significantly improved StAR protein expression and altered the intensities of some tyrosine-phosphorylated proteins in testis. We conclude that PE leaf extract at 50 mg/kg BW can prevent testicular damage in rats with CS.
Acrosome Reaction ; Animals ; Antioxidants/pharmacology* ; Corticosterone/blood* ; Epididymis/metabolism* ; Male ; Malondialdehyde/blood* ; Phosphoproteins/metabolism* ; Phosphorylation ; Phyllanthus emblica/chemistry* ; Plant Extracts/pharmacology* ; Plant Leaves/chemistry* ; Rats ; Rats, Sprague-Dawley ; Sperm Count ; Spermatogenesis/drug effects* ; Spermatozoa/drug effects* ; Stress, Physiological ; Testis/drug effects* ; Testosterone/blood* ; Tyrosine/chemistry*

OBJECTIVETo study the expression and significance of the mammalian target of rapamycin (mTOR)/eukaryote initiating factor 4E binding protein 1(4EBP1)/hypoxia inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF) signaling pathway in asthmatic mice.

METHODSForty SPF level 6-8 week-old female Balb/C mice were randomly divided into control, asthma, budesonide and mTOR inhibitor (rapamycin) intervention groups (n=10 each). The asthmatic mouse model was prepared via OVA induction and challenge test. The intervention groups were administered with rapamycin at the dosage of 3 mg/kg by an intraperitoneal injection or budesonide suspension at the dosage of l mg by aerosol inhalation respectively 30 minutes before the OVA challenge. The control and asthma groups were treated with normal saline instead. The concentrations of HIF-1α and VEGF in bronchoalveolar lavage fluid (BALF) were examined using ELISA 24 hours after the last challenge. The pathological changes of lung tissue were observed by hematoxylin-eosin (HE) staining. The p-mTOR and p-4EBP1 from the lung tissues were detected by immunohistochemistry and Western blot. Pearson analysis was used to study the correlation between p-mTOR, p-4EBP1, HIF-1α, and VEGF expression.

RESULTSCompared with the control group, inflammatory cell infiltration and secretions in the trachea increased in the asthma group. The levels of HIF-1α and VEGF in BALF and p-mTOR and p-4EBP1 expression in lung tissues also increased (P<0.01). Compared with the asthma group, inflammatory cell infiltration and secretions in the trachea were reduced in the two intervention groups, and the levels of HIF-1α and VEGF in BALF and p-mTOR and p-4EBP1 expression in lung tissues were also reduced (P<0.01). There were no significant differences in the above changes between the two intervention groups and control group (P>0.05). In the asthma group, there was a pairwise positive correlation between lung p-mTOR and p-4EBP1 expression and HIF-1α and VEGF levels in BALF (P<0.05). However, there were no correlations in the above indexes in the intervention groups and control group.

CONCLUSIONSp-mTOR, p-4EBP1, HIF-1α and VEGF together are involved in the pathogenesis of asthma. Rapamycin treatment can block this signaling pathway, suggesting that this pathway can be used as a novel target for asthma treatment.

Animals ; Asthma ; drug therapy ; metabolism ; Carrier Proteins ; analysis ; physiology ; Female ; Hypoxia-Inducible Factor 1, alpha Subunit ; analysis ; physiology ; Lung ; chemistry ; pathology ; Mice ; Mice, Inbred BALB C ; Phosphoproteins ; analysis ; physiology ; Signal Transduction ; physiology ; TOR Serine-Threonine Kinases ; analysis ; physiology ; Vascular Endothelial Growth Factor A ; analysis ; physiology

BACKGROUNDAnkylosing spondylitis (AS) is the most common rheumatic condition that is slowly progressive and predominantly affects adolescents. Pathological bone formation associated with AS is an important cause of disability. The aim of the study was to investigate the possible involvement of the genes related to endochondral ossification and ectopia ossification in genetic susceptibility to AS in a Chinese Han population.

METHODSSixty-eight single nucleotide polymorphisms (SNPs) from 13 genes were genotyped in discovery cohorts including 300 AS patients and 180 healthy controls. The rs10019009 in dentin matrix protein 1 (DMP1) gene shown as association with AS after multiple testing corrections in discovery cohorts was replicated in a validation independent cohort of 620 AS patients and 683 healthy controls. The rs10019009 was assessed with bioinformatics including phylogenetic context, F-SNP and FastSNP functional predictions, secondary structure prediction, and molecular modeling. We performed a functional analysis of rs10019009 via reverse transcription-polymerase chain reaction, alkaline phosphatase (ALP) activity in human osteosarcoma U 2 OS cells.

RESULTSInterestingly, the SNP rs10019009 was associated with AS in both the discovery cohort (P = 0.0012) and validation cohort (P = 0.0349), as well as overall (P = 0.0004) in genetic case-control association analysis. After a multivariate logistic regression analysis, the effect of this genetic variant was observed to be independent of linkage disequilibrium. Via bioinformatics analysis, it was found that the amino acid change of the rs10019009 led to changes of SNP function, secondary structure, tertiary conformation, and splice mode. Finally, functional analysis of rs10019009 in U 2 OS cells demonstrated that the risk T allele of the rs10019009 increased enzymatic activity of ALP, compared to that of the nonrisk allele (P = 0.0080).

CONCLUSIONSThese results suggested that the DMP1 gene seems to be involved in genetic predisposition to AS, which may contribute to the ectopic mineralization or ossification in AS. In addition, DMP1 gene may be a promising intervention target for AS in the future.

Adult ; China ; ethnology ; Extracellular Matrix Proteins ; chemistry ; genetics ; Female ; Genetic Predisposition to Disease ; Humans ; Logistic Models ; Male ; Phosphoproteins ; chemistry ; genetics ; Polymorphism, Single Nucleotide ; Spondylitis, Ankylosing ; etiology ; genetics

Reversible phosphorylation plays a crucial role in regulating protein activities and functions. Sexual reproduction directly affects yield of most agricultural crops. As the male reproductive organ, anther generates microspores (pollen), delivering gametes (sperms) to complete double fertilization in higher plants. Here, we took the advantage of Nano UHPLC-MS/MS to analyze maize (Zea mays, B73) early anthers at proteomic and phosphoproteomic levels, to explore the protein and phosphorylation modification regulatory networks controlling maize anther development. Our proteomic analysis identified 3 016 unique peptides, belonging to 1 032 maize proteins. MapMan analysis revealed variously potential proteins associated with maize anther development, such as receptor-like kinases (GRMZM2G082823_P01 and GRMZM5G805485_P01). Using phospho-peptides enriched by TiO2 affinity chromatography, our phosphoproteomic analysis detected 257 phospho-peptides from 210 phosphoproteins, discovering 223 phosphosites. Compared to the 86 maize phosphoproteins collected in the Plant Protein Phosphorylation Data Base (P3DB), we found that 203 phosphoproteins and 218 phosphosites were not revealed before. Further bioinformatics analysis revealed that phosphorylation of 14-3-3 proteins, kinases, phosphatases, transcription factors, cell cycle and chromatin structure related proteins might play important roles in regulating normal anther development in maize. Our findings not only enlarged the maize phosphoproteome data, but also provided information for analyzing the molecular mechanism controlling maize anther development at genetic and biochemical levels.
Crops, Agricultural ; chemistry ; Phosphoproteins ; chemistry ; Phosphorylation ; Plant Proteins ; chemistry ; Pollen ; chemistry ; Proteome ; Tandem Mass Spectrometry ; Zea mays ; chemistry

This work was mainly studied the effects of the four alkaloids from Coptidis Rhizoma on the mouse peritoneal macrophages in vitro and preliminarily discussed the regulating mechanisms. The effect of alkaloids from Coptidis Rhizoma on the vitality of macrophages was measured by the MTT assay. The effect of alkaloids on the phagocytosis of macrophages was determined by neutral red trial and respiratory burst activity was tested by NBT. The expressions of respiratory-burst-associated genes influenced by alkaloids were detected by qRT-PCR. The conformation change of membrane protein in macrophages by the impact of alkaloids was studied by fluorospectro-photometer. Results showed that the four alkaloids from Coptidis Rhizoma could increase the phagocytosis of macrophages in different level and berberine had the best effect. Berberine, coptisine and palmatine had up-regulation effects on respiratory burst activity of mouse peritoneal macrophages stimulated by PMA and regulatory activity on the mRNA expression of PKC, p40phox or p47phox, whereas the epiberberine had no significant influence on respiratory burst. Moreover, alkaloids from Coptidis Rhizoma could change the conformation of membrane protein and the berberine showed the strongest activity. The results suggested that the four alkaloids from Coptidis Rhizoma might activate macrophages through changing the conformation of membrane protein of macrophages and then enhanced the phagocytosis and respiratory burst activity of macrophages. Furthermore, the regulatory mechanism of alkaloids on the respiratory burst activity of macrophages may be also related to the expression level of PKC, p40phox and p47phox.
Alkaloids ; pharmacology ; Animals ; Cells, Cultured ; Coptis ; chemistry ; Drugs, Chinese Herbal ; pharmacology ; Female ; Gene Expression ; drug effects ; Macrophages, Peritoneal ; drug effects ; Mice ; Phosphoproteins ; genetics ; metabolism ; Protein Kinase C ; genetics ; metabolism ; Rhizome ; chemistry

OBJECTIVETo investigate the anti-atherosclerosis mechanism of Astragalus mongholicus polysaccharides (APS) by examining its effect on gene expression profiles of the dendritic cells (DCs) from healthy donors.

METHODSPeripheral blood DCs from healthy donors were incubated with 200 mg/L APS overnight, and changes in the gene expression profiles were investigated using microarray technique and RT-PCR.

RESULTSCompared with the control cells, APS-treated DCs showed significantly up-regulated expressions of CD36 (0.97 ± 0.23 vs 5.45 ± 1.14) and IL-27 (1.08 ± 0.22 vs 2.97 ± 0.61) and down-regulated expression of expression of IFI16 (0.98 ± 0.18 vs 0.46 ± 0.11).

CONCLUSIONSAPS can promote the maturation and differentiation of DCs by up-regulating CD36 and IL-27 and down-regulating IFI16, and thus positively affects the occurrence and progression of the atherosclerosis.

Astragalus Plant ; chemistry ; CD36 Antigens ; metabolism ; Cell Differentiation ; Dendritic Cells ; drug effects ; Humans ; Interleukins ; metabolism ; Nuclear Proteins ; metabolism ; Phosphoproteins ; metabolism ; Polysaccharides ; pharmacology ; Transcriptome

Endocytosis is differentially regulated by hypoxia-inducible factor-1alpha (HIF-1alpha) and phospholipase D (PLD). However, the relationship between HIF-1alpha and PLD in endocytosis is unknown. HIF-1alpha is degraded through the prolyl hydroxylase (PHD)/von Hippel-Lindau (VHL) ubiquitination pathway in an oxygen-dependent manner. Here, we show that PLD1 recovers the decrease in epidermal growth factor receptor (EGFR) endocytosis induced by HIF-1alpha independent of lipase activity via the Rab5-mediated endosome fusion pathway. EGF-induced interaction of PLD1 with HIF-1alpha, PHD and VHL may contribute to EGFR endocytosis. The pleckstrin homology domain (PH) of PLD1 itself promotes degradation of HIF-1alpha, then accelerates EGFR endocytosis via upregulation of rabaptin-5 and suppresses tumor progression. These findings reveal a novel role of the PLD1-PH domain as a positive regulator of endocytosis and provide a link between PLD1 and HIF-1alpha in the EGFR endocytosis pathway.
Animals ; Blood Proteins/chemistry/*metabolism ; Endocytosis ; Female ; HEK293 Cells ; HT29 Cells ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism ; Mice, Nude ; Neoplasms/genetics/metabolism/pathology ; Phospholipase D/chemistry/*metabolism ; Phosphoproteins/chemistry/*metabolism ; Protein Structure, Tertiary ; Receptor, Epidermal Growth Factor/*metabolism ; Signal Transduction ; *Up-Regulation ; Vesicular Transport Proteins/*genetics/metabolism ; rab5 GTP-Binding Proteins/*metabolism

In recent years, human cancer genome projects provide unprecedented opportunities for the discovery of cancer genes and signaling pathways that contribute to tumor development. While numerous gene mutations can be identified from each cancer genome, what these mutations mean for cancer is a challenging question to address, especially for those from less understood putative new cancer genes. As a powerful approach, in silico bioinformatics analysis could efficiently sort out mutations that are predicted to damage gene function. Such an analysis of human large tumor suppressor genes, LATS1 and LATS2, has been carried out and the results support a role of hLATS1//2 as negative growth regulators and tumor suppressors.
Adaptor Proteins, Signal Transducing ; chemistry ; metabolism ; Animals ; Carrier Proteins ; chemistry ; metabolism ; Computational Biology ; Genes, Neoplasm ; Humans ; LIM Domain Proteins ; chemistry ; metabolism ; Mice ; Mutation ; Neoplasms ; genetics ; pathology ; Phosphoproteins ; chemistry ; metabolism ; Phosphorylation ; Protein Binding ; Protein Structure, Tertiary ; Protein-Serine-Threonine Kinases ; chemistry ; genetics ; metabolism ; Transferases (Other Substituted Phosphate Groups) ; chemistry ; metabolism ; Tumor Suppressor Proteins ; chemistry ; genetics ; metabolism

OBJECTIVETo evaluate the feasibility of monitoring clopidogrel resistance with flow cytometric analysis of platelet vasodilator stimulated phosphoprotein (VASP) phosphorylation.

METHODSTwenty patients with coronary heart disease (CHD) and 17 healthy volunteers were examined for platelet aggregation rate and phosphorylation of VASP (calculated as platelet reactivity index, PRI) using traditional optical nephelometry and flow cytometry before and after concurrent therapy of clopidogrel and aspirin.

RESULTSNo significant differences were found in PRI between CHD group and healthy control group [(89.45∓5.22)% vs (86.58∓4.35)%] before treatment. The PRI in CHD group was significantly lowered after treatment to (67.66∓19.77)% (P<0.05). Clopidogrel resistance was found in 6 (30%) cases in CHD group by flow cytometric analysis, which showed a higher sensitivity than optical nephelometry (10%).

CONCLUSIONFlow cytometric analysis of VASP phosphorylation is a more reliable test to specifically evaluate clopidogrel resistance.

Aged ; Aged, 80 and over ; Case-Control Studies ; Cell Adhesion Molecules ; chemistry ; Drug Resistance ; Female ; Flow Cytometry ; Humans ; Male ; Microfilament Proteins ; chemistry ; Middle Aged ; Phosphoproteins ; chemistry ; Phosphorylation ; Platelet Function Tests ; Ticlopidine ; analogs & derivatives ; pharmacology

OBJECTIVETo regenerate dentin-pulp complex by tissue engineering with human stem cells from apical papilla cells (SCAP) as the seed cells.

METHODSSCAP was separated from from normal human impacted third molars with immature roots by outgrowth culture. The cells were then cultured in the differentiation medium for 3 weeks or in normal medium for 60 days, and analyzed for mineralization potential by Alizarin red staining. The osteo/odontogenic markers including alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OC) and dentin sialoprotein (DSP) were investigated by immunofluorescence staining and reverse transcription-polymerase chain reaction. The co-cultured mixture of SCAP and HA/TCP, or HA/TCP alone was implanted subcutaneously on the back of nude mice for 8 weeks, and the implants were collected and examined by HE and immunohistochemical staining.

RESULTSRound alizarin red-positive nodules formed in the isolated cells after cell culture in the differentiation medium for 3 weeks or in normal medium for 60 days with positive staining for osteo/odontogenic markers. SCAP with HA/TCP could regenerate pulp-dentin complex-like tissue in nude mice. The cells near the dentin-like tissue were positive for DSP. No mineral tissue was found in mice receiving HA/TCP implantation.

CONCLUSIONSSCAP may serve as a promising seed cell for dentin-pulp complex tissue engineering.

Adolescent ; Adult ; Alkaline Phosphatase ; analysis ; Animals ; Cell Culture Techniques ; Cell Differentiation ; Coculture Techniques ; Dental Papilla ; cytology ; Dental Pulp ; cytology ; Extracellular Matrix Proteins ; analysis ; Female ; Humans ; Integrin-Binding Sialoprotein ; analysis ; Mice ; Mice, Nude ; Odontogenesis ; physiology ; Osteocalcin ; analysis ; Phosphoproteins ; analysis ; Sialoglycoproteins ; analysis ; Stem Cells ; chemistry ; physiology ; Tissue Engineering ; methods ; Young Adult