Total Protein Expression Research Articles

Bile Duct Ligation Upregulates Expression and Function of L-Amino Acid Transporter 1 at Blood-Brain Barrier of Rats via Activation of Aryl Hydrocarbon Receptor by Bilirubin.

Liver failure is associated with increased levels of brain aromatic amino acids (AAAs), whose transport across the blood–brain barrier (BBB) is mainly mediated by L-amino acid transporter 1 (LAT1). We aimed to investigate whether liver failure induced by bile duct ligation (BDL) increases levels of brain AAAs by affecting the expression and function of LAT1. The LAT1 function was assessed using the brain distribution of gabapentin. It was found that BDL significantly increased levels of gabapentin, phenylalanine, and tryptophan in the cortex, hippocampus, and striatum of rats, and upregulated the expression of total LAT1 protein in hippocampus and striatum as well as cortex membrane LAT1 protein. HCMEC/D3 served as in vitro BBB model, and the data showed that both the serum of BDL rats and bilirubin induced LAT1 expression and function, while bilirubin oxidase almost abolished the upregulation of LAT1 protein by bilirubin and the serum of BDL rats. The enhanced function and expression of LAT1 were also observed in the hippocampus and striatum of hyperbilirubinemia rats. Both aryl hydrocarbon receptor (AhR) antagonist α-naphthoflavone and AhR silencing obviously attenuated the upregulation of LAT1 protein by bilirubin or omeprazole. This study provides the first evidence that BDL upregulates LAT1 at the rat BBB, attributed to the activation of AhR by the increased plasma bilirubin. The results highlight the mechanisms causing BDL-increased levels of brain AAAs and their physiological significance.

Interaction of epigallocatechin-gallate and chlorhexidine with Streptococcus mutans stimulated odontoblast-like cells: Cytotoxicity, Interleukin-1β and co-species proteomic analyses

ObjectivesThe dentin therapeutic agent chlorhexidine has inflammatory and cytotoxic characteristics urging investigation of alternatives like the natural compound epigallocatechin-gallate. The aim is to verify the effect of epigallocatechin-gallate and chlorhexidine on viability, interleukin-1β (IL-1β) and differential protein expression of MDPC-23 odontoblast-like cells stimulated by Streptococcus mutans. DesignCells were stimulated with heat-killed S. mutans at multiplicity of infection (MOI) of 100–1000 and subsequently treated with 100–1 µM of epigallocatechin-gallate. Cells with no treatment or chlorhexidine were controls. Combined stimulated/treated cells were tested for cytotoxicity (Alamar-Blue, N = 3, n = 3), total protein (N = 3, n = 3), IL-1β (ELISA, N = 3, n = 3), and differential protein expression by liquid chromatography-tandem mass spectrometry (LC-MS/MS, n = 2). ResultsCells stimulated at MOI 100/1000 and treated with 10 µM epigallocatechin-gallate and chlorhexidine did not present cytotoxicity. IL-1β significantly increased in both un-stimulated and stimulated chlorhexidine 10 µM groups when compared to un-treated control (p < 0.05). MOI 100 chlorhexidine 10 µM group significantly increased IL-1β compared to un-stimulated chlorhexidine 10 µM and epigallocatechin-gallate 10 µM groups, as well as to MOI 100 epigallocatechin-gallate 10 µM group (p < 0.05). LC-MS/MS revealed S. mutans and mammalian proteins, with tooth-specific proteins exhibiting different abundance levels, depending on the tested condition. ConclusionsOdontoblast-like cells stimulated with S. mutans at different MOI combined with epigallocatechin-gallate treatment did not cause cytotoxicity. S. mutans stimulation combined with chlorhexidine 100 µM treatment decreased cell viability, while treatment with chlorhexidine 10 µM concentration significantly increased IL-1β. S. mutans stimulation and treatment of cells resulted in varied protein expression.

Abstract MP17: Neuropilin-1 Is A Novel Regulator Of Vascular Tone And Blood Pressure

Introduction: Neuropilin-1 (NRP1) is a transmembrane receptor present in vascular smooth muscle cells (VSMC) that mediates the inhibition of Rho signaling by binding the Class 3 Semaphorin (SEMA) ligand SEMA3A. Hypothesis: We hypothesize that loss of NRP1 in VSMC mitigates SEMA3A-induced Rho inhibition, thereby increasing vascular tone and blood pressure in vivo . Methods: Male and female adult mice (8-12 weeks) with inducible, smooth muscle cell-specific deletion of NRP1 (SM22a-Cre ERT2 X Nrp1 flox/flox ) were examined. Following recombination using 4-hydroxy tamoxifen (SM- NRP1 KO), systolic blood pressure (SBP) was measured using a tail cuff and compared to age- and sex-matched mice that did not receive tamoxifen (control). Aortic vascular reactivity and expression of key proteins in the Rho signaling cascade were measured using ex vivo tension myography and western blotting, respectively. Results: SBP was significantly increased in SM- NRP1 KO mice following recombination compared to control mice (SBP: 136.5 ± 10.9 vs 112.9 ± 5.6 mmHg; p=0.0006). Contractile responses in aortas of SM- NRP1 KO mice to phenylephrine (p=0.025), KCl (p=0.012), and the thromboxane agonist U44619 (p=0.019) were significantly enhanced compared to controls. Expression of total myosin light chain and LIMK-2 proteins were increased in SM- NRP1 KO compared to control aortas. In vitro , treatment of murine primary VSMC expressing NRP1 with SEMA3A decreased angiotensin II-induced Rho-GTP activation. Additionally, control and SM- NRP1 KO mice (starting at 2 weeks post-recombination) were administered angiotensin II (490 ng/kg/day) for 4 weeks. While there was no significant difference in SBP at weeks 1 and 2, SM- NRP1 KO mice had significantly lower SBP at weeks 3 and 4 following angiotensin II infusion compared to controls (Week 4 SBP: 150 ± 1.4 vs 130.5 ± 2.5 mmHg; p=0.02), suggesting a low ejection fraction and cardiac dysfunction in these mice. In support of this observation, mRNA expression of atrial natriuretic peptide was increased in hearts of angiotensin II-infused SM- NRP1 KO mice. Conclusion: Our data suggest that VSMC NRP1 regulates basal tone and blood pressure, and that loss of NRP1 causes hypertension and exacerbates cardiac dysfunction.

Effects of Simvastatin on Breast Carcinoma Cell Proliferation and Apoptosis via Transforming Growth Factor-β1/Smad3 Pathway

Objective: To investigate the function and causative role of simvastatin (Sim) in breast carcinoma cell apoptosis as well as proliferation. Methods: 20 breast carcinoma patients requiring surgery were treated with Sim (20 days, 30 mg), and samples of pre-treatment (pre) and post-treatment (post) were acquired. We detected tissue cell proliferation and apoptosis changes and used functional experiments to detect cell proliferation and apoptosis changes after treating not only estrogen receptor (ER)-positive (MCF-7) but also ER-negative cells (MDA-MB-231) with Sim or TGF-β1. Detection of p-Smad3 and total Smad3 protein expression changes was conducted, and we finally used in vivo experiments to assess the influence of Sim on breast tumor growth and drug safety. Results: Immunohistochemistry and TUNEL staining results showed that after treatment with Sim, breast carcinoma cell proliferation decreased and apoptosis increased. Functional experiments results showed that Sim notedly promoted the MDA-MB-231 and MCF-7 cell apoptosis, inhibiting migration, proliferation and epithelial mesenchymal transition. Moreover, TGF-β1 protein expression was strikingly lower in Sim group than that in DMSO group. When TGF-β1 and Sim were combined to use, the inhibitory ability of Sim on breast cancer cell proliferation markedly increased and the capability of TGF-β1 protein inducing p-Smad3 protein increased. In addition, after Sim treatment in mice, the tumor volume became smaller, the pathological changes weakened, and there was no significant effect on liver function and kidney function. Conclusion: Sim participates in breast cancer cell apoptosis and proliferation via regulating TGF-β1/Smad3 signal pathway.

Potential Significance and Clinical Value Explorations of Calmin (CLMN) in Breast Invasive Carcinoma.

ObjectiveFunction of calmin (CLMN) was rarely reported in human diseases, especially in tumor. Present study initially assessed the significance of CLMN in breast invasive carcinoma (BRCA).MethodsExpressions of CLMN containing mRNA and protein in BRCA was firstly assessed, and association of CLMN mRNA expression with clinical phenotypes of BRCA patients was analyzed as well. Prognostic value of CLMN in BRCA was subsequently predicted based on the clinical characteristics of patients. Finally, the potential biological function associated with CLMN involved in BRCA was revealed.Results(1) The mRNA expression of CLMN was lower in BRCA compared with that in normal patients (P<0.001). However, result of CLMN total protein expression was opposite (P<0.05). (2) The mRNA expression of CLMN was statistically associated with BRCA patient’s age, gender, PR status, ER status, histological type, tumor stage, copy number, and methylation level (all P<0.05). (3) Compared with low expression group, high expression of CLMN was conducive to the overall survival of BRCA patients (P=0.0011). Detailed, survival difference between CLMN high and low expression groups was observed in patients with stage 1 (P=0.0250), positive ER status (P=0.0042), negative HER status (P=0.0433), luminal A (P=0.0065), luminal B (P=0.0123) and positive lymph node status (P=0.0069). Pathway analysis suggested that CLMN mainly participated in cell cycle process (P<0.05) and exerted inhibition effect on the cell cycle involved in BRCA (P<0.05).ConclusionCLMN mRNA high expression prolonged the survival time of patients and caused a favorable prognosis. The positive function of CLMN in BRCA required further investigation in future work.

Oxidative stress in the skin: Impact and related protection.

Skin, our first interface to the external environment, is subjected to oxidative stress caused by a variety of factors such as solar ultraviolet, infrared and visible light, environmental pollution, including ozone and particulate matters, and psychological stress. Excessive reactive species, including reactive oxygen species and reactive nitrogen species, exacerbate skin pigmentation and aging, which further lead to skin tone unevenness, pigmentary disorder, skin roughness and wrinkles. Besides these, skin microbiota are also a very important factor ensuring the proper functions of skin. While environmental factors such as UV and pollutants impact skin microbiota compositions, skin dysbiosis results in various skin conditions. In this review, we summarize the generation of oxidative stress from exogenous and endogenous sources. We further introduce current knowledge on the possible roles of oxidative stress in skin pigmentation and aging, specifically with emphasis on oxidative stress and skin pigmentation. Meanwhile, we summarize the science and rationale of using three well-known antioxidants, namely vitamin C, resveratrol and ferulic acid, in the treatment of hyperpigmentation. Finally, we discuss the strategy for preventing oxidative stress-induced skin pigmentation and aging.

Design, synthesis and investigation of procaine based new Pd complexes as DNA methyltransferase inhibitor on gastric cancer cells

Procaine is a specific inhibitor of DNA methyltransferase (DNMT). In the present work, the Schiff base ligands were synthesized from procaine with the addition of salicylaldehyde and naphthaldehyde as metal precursor for procaine-based transition metal complexes (L1 and L2) with palladium (Pd). The compounds were checked and characterized for identity and purity using elemental analysis, SEM, melting points, FT-IR, and NMR spectral data. Among the compounds, L1-Pd and L2-Pd were found to display significant cytotoxicity with IC50 values of 10.21 µM and 10.79 µM against human gastric cancer cell line MKN-45. Novel synthesized procaine derivatives target molecules and their palladium complexes had an inhibitory effect on colony formation and wound healing on MKN-45 cells. Furthermore, these compounds were evaluated for their apoptotic activity and DNMT activity in MKN-45 cells. Western blotting and qPCR studies demonstrated that compound L1-Pd and L2-Pd induced apoptosis and a slight decrease in anti-apoptotic Bcl-2 protein/gene expression and highly increased pro-apoptotic Bax protein/gene expression in MKN-45 cells treated. Also, total DNMT enzyme activity and protein expression (DNMT1, DNMT3a, and DNMT3b) levels were decreased in L1-Pd and L2-Pd treated cells. The L1-Pd and L2-Pd complexes showed that they are potential new DNMT inhibitors to prevent cancer-induced DNA hypermethylation and can be used in the treatment of gastric cancer.

Inhibition of FLT3-ITD and Melk By Small Molecule Inhibitor OTS167 in FLT3 mutant Acute Myeloid Leukemia (AML)

Background and Rationale: Maternal embryonic leucine zipper kinase (MELK) is a Ser/Thr protein kinase that is aberrantly expressed in many tumor types and demonstrated to be important in formation and maintenance of cancer stem cells. We have previously reported that MELK is also aberrantly expressed in AML cell lines and primary patient leukemia cells and is associated with a poor prognosis (Alachkar et. al., Oncotarget, 2014). MELK knockdown resulted in leukemia cell growth inhibition and apoptosis. Based on these preclinical insights, a small molecule inhibitor of MELK kinase activity, OTS167, (OncoTherapy Sciences, Tokyo, Japan) has been developed and is being tested in an early phase clinical trial in advanced myeloid malignancies at the University of Chicago (IRB#15-1373). Here, we report the activity of MELK signaling blockade in AML with FLT3 mutations, which are associated with drug resistance and poor clinical outcomes, and demonstrate synergy with known FLT3 kinase inhibitors.Methods: Both MELK and FLT3-ITD activity were targeted by treating the human AML cell lines MV4:11, MOLM-13, KOCL-48 and KG-1 which aberrantly express MELK as well as a FLT3-ITD, FLT3-TKD or FLT3-WT with the small molecule MELK inhibitor OTS167 alone or in combination with FLT3 kinase inhibitors Crenolanib or Gilteritinib.Results: OTS167 induced cell death of FLT3 mutant cell lines in a dose dependent manner and with IC50 similar to known FLT3 kinase inhibitors (OTS167 IC50 1.8-18.2nM; Crenolanib IC50 2.31-13.8nM; Gilteritinib IC50 4.9-36.3nM). Using isobologram analysis, OTS167 in combination with either Gilteritinib or Crenolanib induced synergistic cell death in FLT3 mutant leukemia cells at 48hrs (Figure 1A), but not in FLT3-WT leukemia cells. We found that OTS167 alone had the effect of inhibiting auto-phosphorylation of FLT3-ITD, as well as inhibiting MELK activity resulting in downregulation of MELK protein expression. Treatment of cells with 50nM OTS167 resulted in loss of FLT3-ITD (Y591) phosphorylation within 1hr, and total FLT3-ITD protein expression within 24 hours (Figure 1B). Loss of FLT3-ITD protein expression occurred in the presence of FLT3 mRNA and was not due to decreased stability of the protein, as treatment with OTS167 in the presence of cycloheximide did not decrease FLT3-ITD lifespan. Notably, the loss of FLT3-ITD protein expression observed with OTS167 treatment was not observed with the same concentration of FLT3 inhibitors Crenolanib or Gilteritinib. In vitro kinase assays using recombinant FLT3-ITD confirmed that OTS167 could act directly to inhibit FLT3-ITD kinase activity at an IC50 of 4.9nM, compared to a reported MELK IC50 of 0.11-1.1nM (Chung, et. al., Oncotarget, 2012). In MV4:11 cells, the constitutive activation of the MAPK, PI3K, and STAT5 signaling pathways by FLT3-ITD is inhibited by 50nM OTS167, similar to inhibition by 50nM FLT3 inhibitors Crenolanib and Gilteritinib (Figure 1C).In addition, treatment with either OTS167 or FLT3 inhibitor resulted in loss of MYC mRNA and protein expression at 3hr, suggesting that more effective downregulation of MYC expression by the combination may increase the susceptibility of FLT3 mutant AML to apoptosis. The inhibition of FLT3-ITD activation and expression after OTS167 treatment was not observed when MELK protein activity was downregulated using siRNA, suggesting the inhibitor may simultaneously target two independent signaling pathways active in cellular transformation. These results indicate that OTS167 may have a dual specificity in FLT3 mutant AML through targeting both aberrant MELK and FLT3 activity.Conclusion: In summary, the MELK inhibitor, OTS167, may be an attractive therapeutic agent for FLT3 mutant AML, with dual targeting resulting in decreased levels of FLT3-ITD protein and kinase activity. These data provide a rationale for testing dual MELK and FLT3 kinase inhibitors in patients with FLT3 mutant AML. [Display omitted] DisclosuresOdenike:CTI/Baxalta: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria. Stock:Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Customized Induced Pluripotent Stem Cell-Derived Red Cell Reagents

Red blood cell transfusion is an essential therapy for patients with sickle cell disease (SCD). A major problem is the complexity of antibodies found in patients with SCD, in large part due to RH gene variation. Rh antibodies are common despite provision of serologic Rh-matched transfusions. The antibodies may be mistaken for autoantibodies when in fact they are alloantibodies to high prevalence Rh antigens that are absent or have altered Rh epitopes on the patient's RBCs. Finding compatible red cell units is often complicated by a lack of reagent red cells to properly identify the fine antibody specificity. This can delay patient care, increase costs, and make transfusion therapy impossible for some patients. We hypothesized that human induced pluripotent stem cells (iPSCs) could be reprogrammed from rare donors or genetically engineered to produce standard and reliable red cell reagents to aid identification for patients with complex antibodies. However, most human iPSC-derived red blood cells (iRBCs) are developmentally primitive red cells that differ from donor-derived red cells with high levels of embryonic globin expression, are larger in size and fail to enucleate in culture. We aimed to improve iPSC hematopoietic differentiation protocols to generate iRBCs compatible with standard blood bank assays.We designed a panel of customized iPSCs that included 1) Rh null cells, 2) cells lacking the high prevalence Rh antigen termed hrS, 3) cells expressing a partial C antigen and lacking the high prevalence Rh antigen termed hrB and, 4) cells expressing the low prevalence Rh antigens V, VS and lacking hrB. For the Rh null line, we identified wild type iPSC lines that were group O, RhD- by homozygous RHD deletion. We used CRISPR/Cas9 genetic engineering to disrupt the RHCE alleles either by a large deletion from exon 1 to exon 2 and/or via insertion-deletion mutations resulting in a frameshift and early stop codon. For lines lacking high prevalence Rh antigens, we reprogrammed group O donor cells whose genotypes were heterozygous for RHD*DAU0/RHCE*ceMO and RHD*DOL/RHCE*ceBI, homozygous for RHD*DIIIa-CE(4-7)-D/RHCE*ceS, and homozygous for RHD/RHCE*ce733G, respectively. At least 2 independent clones from each customized line were characterized for pluripotency by standard flow cytometry and gene expression markers, and a normal karyotype confirmed.We performed hematopoietic differentiation of customized iPSCs by a 3-step embryoid body protocol in defined, serum-free media with combinations of hematopoietic cytokines. We obtained CD41+235+ primitive hematopoietic progenitors on day 8 and cultured these cells in liquid culture with erythroid specific cytokines to generate iRBCs. We assessed erythroid cell maturation by morphology on cytospin preparations and by flow cytometry. We found an almost pure population of CD71+235+ iRBCs with erythroblast morphology by day 6 of culture, and progressive maturation to Band 3 high, alpha integrin low iRBCs that were smaller and had condensed nuclei that resemble erythroid cultures of primary CD34+ cells. Comparison of total Rh protein expression by flow cytometry on untargeted group O, D+ iRBCs compared to donor-derived RBCs demonstrated comparable cell surface Rh expression. As proof of principal, we assessed RBC agglutination by gel card assay of untargeted group O, D+, E-, e+ and the genetically engineered Rh null iRBCs using standard anti-D, -E, and -e reagents for Rh typing (Ortho Bioclone). As expected, the untargeted O, D+ iRBCs agglutinated with anti-D and anti-e while the engineered Rh-null iRBCs showed no agglutination with all 3 antibodies. Only 0.5 e6 iRBCs were required per gel assay.We have designed a panel of customized iPSCs reprogrammed from rare donors or genetically engineered to express rare blood group antigen phenotypes or combinations that are difficult or impossible to find as donor red cells. Any number of combinations not found in natural populations can be produced and generated in quantities sufficient for reagents. iRBC produced from these customized iPSCs can be used with standard blood bank assays and potentially provide the means to streamline and standardize antibody identification in alloimmunized patients with complex antibody specificities. In the future, when technology for scale-up is available, Rh null iRBCs could be used as “universal” donor cells for future therapeutic applications. DisclosuresNo relevant conflicts of interest to declare.

PCT3, a Combination of Active Steroidal Saponins from the Paris Forrestii (Takht.) H. Li, Displays Potent Activity Against Acute Myeloid Leukemia By Suppressing the PBX1/RNF6/AKT/mTOR Signaling Pathway

Acute myelogenous leukemia (AML) is a class of malignancy derived from the myeloid line of aberrant progenitor/stem cells from bone marrow. AML is incurable and the five year rate of the event free survival (EFS) remains less than 20% even with the intensive chemotherapy in combination with autologous hematopoietic stem cell transplantation. Therefore, it is urgent to find novel drugs to improve the outcome of AML patients. Paris forrestii (Takht.) H. Li is a traditional herb medicine specifically produced in the Yu'nan and Tibet regions of China and it is widely used for the inflammation and coagulation among local people. In the study to identify natural products for cancer treatment, we isolated and evaluated the active ingredients from Paris forrestii (Takht.) H. Li and found that PCT3, a combination of steroidal saponins, displayed potent anti-AML activity. The high performance liquid chromatography/mass spectrometry (HPLC/MS) assay isolated and identified 10 steroidal saponins (SPs). In the preliminary study, each individual SP displayed limited cytotoxicity to AML cell lines K562 and HL-60. However, when a random combinations of the SPs were tested, it turned out that PCT3 (mainly composed of Polyphyllin I and III) was the most potent one in inhibiting AML cell prolipferation. The IC50 of PCT3 on K562 was 2.935 ± 0.772 μg/ml within 24 hrs. Moreover, PCT3 induces potent AML cell apoptosis in a concentration-dependent manner. At the ranges of 0 to 8μg/ml, PCT3 induces apoptosis of selected AML cell lines, including K562, HL-60, HT-93 and KG-1 as evidenced of the cleavage and activation of Caspase-3 and PARP. Consistent with this finding, PCT3 downregulated oncoproteins such as Bcl-2 and Mcl-1 but upregulated pro-apoptotic signaling proteins including Bax. The anti-AML activity of PCT3 was further evaluated in AML xenografts in nude mice derived from K562 cells. Oral administration of PCT3 at 100 mg/kg for a continuous 14 days markedly delayed AML tumor growth but had no effects on mice body weight. These results thus suggested that PCT3 displayed potent anti-AML activity in both molecular, cellular and nude mice levels. To understand the underlying molecular mechanisms PCT3 kills AML cells, we first evaluated the AKT/mTOR signaling pathway, a central node of AML cell proliferation and AML cell survival. To our expectation, PCT3 suppressed the activation of the AKT/mTOR signals. PCT3 inhibited the phosphorylation of AKT and mTOR, but had no effects on their total protein expression levels. Consistently, 4E-BP1 and p70S6K, two typical downstream proteins of the AKT/mTOR pathway, were also inhibited by PCT3 in a concentration-dependent manner. Moreover, we found that PCT3 inhibited the expression of the PBX1, a key transcription factor in AML (Xu X et al, J Biol Chem, 2016). PBX1 promotes leukemia cell proliferation by promoting the transcription of RNF6, a ring finger protein. Interestingly, the PBX1/RNF6 axle upregulated the activation of the AKT/mTOR signaling pathway. Therefore, we found that PCT3 displayed potent anti-AML activity by suppression the PBX1/RNF6/AKT/mTOR signaling pathway. Given its tolerable toxicity and anti-AML activity, PCT3 could be developed for AML treatment. DisclosuresNo relevant conflicts of interest to declare.

Roles of adenosine A1 receptors in the regulation of SFK activity in the rat forebrain.

Adenosine A1 receptors are widely expressed in the mammalian brain. Through interacting with Gαi/o‐coupled A1 receptors, the neuromodulator adenosine modulates a variety of cellular and synaptic activities. To determine the linkage from A1 receptors to a key intracellular signaling pathway, we investigated the impact of blocking A1 receptors on a subfamily of nonreceptor tyrosine kinases, that is, the Src family kinase (SFK), in different rat brain regions in vivo. We found that pharmacological blockade of A1 receptors by a single systemic injection of the A1 selective antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX) induced an increase in autophosphorylation of SFKs at a consensus activation site, tyrosine 416 (Y416), in the two subdivisions of the striatum, the caudate putamen and nucleus accumbens. DPCPX also increased SFK Y416 phosphorylation in the medial prefrontal cortex (mPFC) but not the hippocampus. The DPCPX‐induced Y416 phosphorylation was time dependent and reversible. In immunopurified Fyn and Src proteins from the striatum, DPCPX elevated SFK Y416 phosphorylation and tyrosine kinase activity in Fyn but not in Src proteins. In the mPFC, DPCPX enhanced Y416 phosphorylation and tyrosine kinase activity in both Fyn and Src immunoprecipitates. DPCPX had no effect on expression of total Fyn and Src proteins in the striatum, mPFC, and hippocampus. These results demonstrate a tonic inhibitory linkage from A1 receptors to SFKs in the striatum and mPFC. Blocking this inhibitory tone could significantly enhance constitutive SFK Y416 phosphorylation in the rat brain in a region‐ and time‐dependent manner.

Clinicopathological significance of the EMT-related proteins and their interrelationships in prostate cancer. An immunohistochemical study.

The chronic inflammation influences a microenvironment, where as a result of losing control over tissue homeostatic mechanisms, the carcinogenesis process may be induced. Inflammatory response cells can secrete a number of factors that support both initiation and progression of cancer and also they may consequently induct an epithelial-mesenchymal transition (EMT), the process responsible for development of distant metastasis. Macrophage migration inhibitory factor (MIF) acts as a pro-inflammatory cytokine that is considered as a link between chronic inflammation and tumor development. MIF can function as a modulator of important cancer-related genes expression, as well as an activator of signaling pathways that promotes the development of prostate cancer. The study was performed on FFPE tissues resected from patients who underwent radical prostatectomy. To investigate the relationship of studied proteins with involvement in tumor progression and initiation of epithelial-to-mesenchymal transition (EMT) process, we selected clinicopathological parameters related to tumor progression. Immunohistochemical analyses of MIF, SOX-4, β-catenin and E-cadherin were performed on TMA slides. We found a statistically significant correlation of overall β-catenin expression with the both lymph node metastasis (p<0.001) and presence of angioinvasion (p = 0.012). Membrane β-catenin expression was associated with distant metastasis (p = 0.021). In turn, nuclear MIF was correlated with lymph node metastasis (p = 0.003). The positive protein-protein correlations have been shown between the total β-catenin protein expression level with level of nuclear SOX-4 protein expression (r = 0.27; p<0.05) as well as negative correlation of β-catenin expression with level of nuclear MIF protein expression (r = -0.23; p<0.05). Our results seem promising and strongly highlight the potential role of MIF in development of nodal metastases as well as may confirm an involvement of β-catenin in disease spread in case of prostate cancer.

Functional Analysis of the Promoter Regions of Two Apoptosis-Related Genes (Bcl-2 and Cycs) and Their Regulation by Zn in Yellow Catfish.

B-cell lymphoma 2 (Bcl-2) and cytochrome c (Cycs) are two important proteins relevant to cellular apoptosis. In this study, we characterized the functions of the promoter regions of two apoptosis-related genes, Bcl-2 and Cycs, in yellow catfish Pelteobagrus fulvidraco. We obtained a 1989 bp Bcl-2 promoter and an 1830 bp Cycs promoter and predicted several key transcription factor binding sites (TFBSs) on the promoters, such as Kruppel-like factor 4 (KLF4), signal transducer and activator of transcription factor 3 (STAT3), forkhead box O (FOXO), metal-responsive element (MRE) and hepatocyte nuclear factor 1α (HNF-1α). Zinc (Zn) increased the activities of the Bcl-2 promoter but decreased the activities of the Cycs promoter. Metal-responsive transcription factor 1 (MTF-1) and HNF-1α directly bound with Bcl-2 and Cycs promoters, and they positively regulated the activity of the Bcl-2 promoter but negatively regulated the activity of the Cycs promoter. Zn promoted the binding ability of HNF-1α to the Bcl-2 promoter but decreased its binding ability to the Cycs promoter. However, Zn had no significant effect on the binding capability of MTF-1 to the regions of Bcl-2 and Cycs promoters. Zn upregulated the mRNA and total protein expression of Bcl-2 but downregulated the mRNA and total protein expression of Cycs. At the same time, Annexin V–FITC/PI staining showed that Zn significantly reduced the apoptosis of primary hepatocytes. For the first time, our study provides evidence for the MRE and HNF-1α response elements on the Bcl-2 and Cycs promoters, offering new insight into the mechanism by which Zn affects apoptosis in vertebrates.

Expression of membrane protein disulphide isomerase A1 (PDIA1) disrupt a reducing microenvironment in endometrial epithelium for embryo implantation

Various proteins in the endometrial epithelium are differentially expressed in the receptive phase and play a pivotal role in embryo implantation. The Protein Disulphide Isomerase (PDI) family contains 21 members that function as chaperone proteins through their redox activities. Although total PDIA1 protein expression was high in four common receptive (Ishikawa and RL95-2) and non-receptive (HEC1-B and AN3CA) endometrial epithelial cell lines, significantly higher membrane PDIA1 expression was found in non-receptive AN3CA cells. In Ishikawa cells, oestrogen up-regulated while progesterone down-regulated membrane PDIA1 expression. Moreover, mid-luteal phase hormone treatment down-regulated membrane PDIA1 expression. Furthermore, oestrogen at 10 nM reduced spheroid attachment on Ishikawa cells. Interestingly, inhibition of PDIA1 function by bacitracin or 16F16 increased the spheroid attachment rate onto non-receptive AN3CA cells. Over-expression of PDIA1 in receptive Ishikawa cells reduced the spheroid attachment rate and significantly down-regulated integrin β3 levels, but not integrin αV and E-cadherin. Addition of reducing agent TCEP induced a sulphydryl-rich microenvironment and increased spheroid attachment onto AN3CA cells and human primary endometrial epithelial cells collected at LH+7/8 days. The luminal epithelial cells from human endometrial biopsies had higher PDIA1 protein expression in the proliferative phase than in the secretory phase. Our findings suggest oestrogen and progesterone regulate PDIA1 expression, resulting in the differential expressions of membrane PDIA1 protein to modulate endometrial receptivity. This suggests that membrane PDIA1 expression prior to embryo transfer could be used to predict endometrial receptivity and embryo implantation in women undergoing assisted reproduction treatment.

Transcriptional responses of four slc30a/znt family members and their roles in Zn homeostatic modulation in yellow catfish Pelteobagrus fulvidraco

The study characterized their regulatory functions of four znt members (znt1, znt2, znt6 and znt8) in Zn homeostasis in vertebrates. We found that the −1281/−1296 bp locus on the znt1 promoter, the −1/−16 bp locus on the znt2 promoter, the −825/−839 bp locus on the znt6 promoter, the −165/−180 bp locus and the −274/−292 bp STAT3 locus on the znt8 promoter were functional MTF-1 binding sites and had metal responsive element (MRE). Zn incubation increased activities of four znt promoters, which was mediated by MRE sites on znt1, znt2, znt6 and znt8 promoters and by STAT3 binding site on znt8 promoter. Moreover, Zn activated the transcription of these znts genes through MTF-1-MRE-dependent pathway. Zn incubation up-regulated the mRNA and total protein expression of ZnT1, ZnT2 and ZnT8 at both 24 h and 48 h. Overall, for the first time, this study offered novel insights for regulatory mechanism of Zn homeostasis in vertebrates.

Aerobic Exercise During Advance Stage of Uncontrolled Arterial Hypertension.

AimTo evaluate the influence of physical training on myocardial function, oxidative stress, energy metabolism, and MAPKs and NF-κB signaling pathways in spontaneously hypertensive rats (SHR), at advanced stage of arterial hypertension, which precedes heart failure development.MethodsWe studied four experimental groups: normotensive Wistar rats (W, n = 27), trained W (W-EX, n = 31), SHR (n = 27), and exercised SHR (SHR-EX, n = 32). At 13 months old, the exercise groups underwent treadmill exercise 5 days a week for 4 months. In vitro myocardial function was analyzed in left ventricular (LV) papillary muscle preparations. Antioxidant enzyme activity and energy metabolism were assessed by spectrophotometry. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity was analyzed by lucigenin reduction and protein expression by Western blot. Statistical analyzes: ANOVA and Tukey or Kruskal–Wallis and Dunn tests.ResultsSHR-EX had a lower frequency of heart failure features than SHR. Myocardial function and antioxidant enzyme activity were better in SHR-EX than SHR. Lipid hydroperoxide concentration, and phosphorylated JNK and total IkB protein expression were higher in hypertensive than control groups. Malondialdehyde, NADPH oxidase activity, total JNK, phosphorylated p38, phosphorylated and total p65 NF-κB, and phosphorylated IkB did not differ between groups. Protein expression from total p38, and total and phosphorylated ERK were higher in SHR than W. Lactate dehydrogenase and phosphorylated ERK were lower and citrate synthase and β-hydroxyacyldehydrogenase were higher in SHR-EX than SHR.ConclusionExercise improves physical capacity, myocardial function, and antioxidant enzyme activity; reduces the frequency of heart failure features and ERK phosphorylation; and normalizes energy metabolism in SHR.

HspB4/αA-Crystallin Modulates Neuroinflammation in the Retina via the Stress-Specific Inflammatory Pathways.

Purpose: We have previously demonstrated that HspB4/αA-crystallin, a molecular chaperone, plays an important intrinsic neuroprotective role during diabetes, by its phosphorylation on residue 148. We also reported that HspB4/αA-crystallin is highly expressed by glial cells. There is a growing interest in the potential causative role of low-grade inflammation in diabetic retinopathy pathophysiology and retinal Müller glial cells’ (MGCs’) participation in the inflammatory response. MGCs indeed play a central role in retinal homeostasis via secreting various cytokines and other mediators. Hence, this study was carried out to delineate and understand the regulatory function of HspB4/αA-crystallin in the inflammatory response associated with metabolic stresses. Methods: Primary MGCs were isolated from knockout HspB4/αA-crystallin mice. These primary cells were then transfected with plasmids encoding either wild-type (WT), phosphomimetic (T148D), or non-phosphorylatable mutants (T148A) of HspB4/αA-crystallin. The cells were exposed to multiple metabolic stresses including serum starvation (SS) or high glucose with TNF-alpha (HG + T) before being further evaluated for the expression of inflammatory markers by qPCR. The total protein expression along with subcellular localization of NF-kB and the NLRP3 component was assessed by Western blot. Results: Elevated levels of IL-6, IL-1β, MCP-1, and IL-18 in SS were significantly diminished in MGCs overexpressing WT and further in T148D as compared to EV. The HG + T-induced increase in these inflammatory markers was also dampened by WT and even more significantly by T148D overexpression, whereas T148A was ineffective in either stress. Further analysis revealed that overexpression of WT or the T148D, also led to a significant reduction of Nlrp3, Asc, and caspase-1 transcript expression in serum-deprived MGCs and nearly abolished the NF-kB induction in HG + T diabetes-like stress. This mechanistic effect was further evaluated at the protein level and confirmed the stress-dependent regulation of NLRP3 and NF-kB by αA-crystallin. Conclusions: The data gathered in this study demonstrate the central regulatory role of HspB4/αA-crystallin and its modulation by phosphorylation on T148 in retinal MGCs. For the first time, this study demonstrates that HspB4/αA-crystallin can dampen the stress-induced expression of pro-inflammatory cytokines through the modulation of multiple key inflammatory pathways, therefore, suggesting its potential as a therapeutic target for the modulation of chronic neuroinflammation.

Differential Membrane Protein Profile in Bovine X- and Y-Sperm.

The aim of this study was to understand the molecular mechanisms behind the biological differences of X- and Y-sperm and to screen the sex-specific candidate antigen proteins for sexed semen production. To this end, we investigated differential expression of total membrane proteins of the two sperm types by using high-purity X- and Y-sperm from 20 Holstein bulls and applying the label-free proteomic technique; 1521 proteins were identified. In the X-sperm group, 8 and 23 proteins were significantly up- and down-regulated, respectively. In the X- and the Y-sperm group, 151 and 88 proteins were specifically expressed, respectively. These were overexpressed in the dynamic changes of the actin cytoskeleton, and cell senescence/apoptosis induced by the immune response, and could result in differences in the state, size, and immune sensitivity of the X-/Y-sperm membranes. The prediction of transmembrane structure, subcellular localization, and Western blotting validation results showed that the CLRN3 and SCAMP1 proteins were cell surface specific antigens of X- and Y-sperm, respectively. Our findings help explain the molecular mechanism behind the biological differences of X-/Y-sperm and lay the foundation for application of immunological methods to produce sex-sorted semen and control livestock sex. Data are available via ProteomeXchange with identifier PXD019435.

Impact of short-term Dutasteride treatment on prostate-specific membrane antigen expression in a mouse xenograft model.

BackgroundDutasteride has been shown to increase expression of the prostate‐specific membrane antigen (PSMA) in prostate cancer cells in previous in vitro studies. This 5‐alpha‐reductase inhibitor is commonly used for the treatment of symptomatic benign prostatic enlargement. The modulation of PSMA expression might affect PSMA‐based prostate cancer imaging and therapy.AimThe purpose of this work was to further analyze concentration‐dependent effects of Dutasteride on PSMA expression in a mouse xenograft model.Methods and resultsFour groups of mice bearing LNCaP xenografts were treated for 14 days with daily intraperitoneal injections of either vehicle control or different concentrations of Dutasteride (0.1, 1, 10 mg/kg). Total expression of PSMA, androgen receptor (AR), and caspase‐3 protein was analyzed using immunoblotting (WES). In addition, PSMA, cleaved caspase‐3 and Ki‐67 expression was assessed and quantified by immunohistochemistry. Tumor size was measured by caliper on day 7 and 14, tumor weight was assessed following tissue harvesting.The mean PSMA protein expression in mice increased significantly after treatment with 1 mg/kg (10‐fold) or 10 mg/kg (sixfold) of Dutasteride compared to vehicle control. The mean fluorescence intensity significantly increased by daily injections of 0.1 mg/kg Dutasteride (1.6‐fold) as well as 1 and 10 mg/kg Dutasteride (twofold). While the reduction in tumor volume following treatment with high concentrations of 10 mg/kg Dutasteride was nonsignificant, no changes in AR, caspase‐3, cleaved caspase‐3, and Ki‐67 expression were observed.ConclusionShort‐term Dutasteride treatments with concentrations of 1 and 10 mg/kg significantly increase the total PSMA protein expression in a mouse LNCaP xenograft model. PSMA fluorescence intensity increases significantly even using lower daily concentrations of 0.1 mg/kg Dutasteride. Further investigations are needed to elucidate the impact of Dutasteride treatment on PSMA expression in patients.

Methane Inhalation Protects Against Lung Ischemia-Reperfusion Injury in Rats by Regulating Pulmonary Surfactant via the Nrf2 Pathway.

Methane (CH4) exerted protective effects against lung ischemia-reperfusion (I/R) injury, but the mechanism remains unclear, especially the role of pulmonary surfactant. Therefore, this study aimed to explore the effects of CH4 inhalation on pulmonary surfactant in rat lung I/R injury and to elucidate the mechanism. Rats were randomly divided into three groups (n = 6): the sham, I/R control, and I/R CH4 groups. In the sham group, only thoracotomy was performed on the rats. In the I/R control and I/R CH4 groups, the rats underwent left hilum occlusion for 90 min, followed by reperfusion for 180 min and ventilation with O2 or 2.5% CH4, respectively. Compared with those of the sham group, the levels of large surfactant aggregates (LAs) in pulmonary surfactant, lung compliance, oxygenation decreased, the small surfactant aggregates (SAs), inflammatory response, oxidative stress injury, and cell apoptosis increased in the control group (P < 0.05). Compared to the control treatment, CH4 increased LA (0.42 ± 0.06 vs. 0.31 ± 0.09 mg/kg), oxygenation (201 ± 11 vs. 151 ± 14 mmHg), and lung compliance (16.8 ± 1.0 vs. 11.5 ± 1.3 ml/kg), as well as total antioxidant capacity and Nrf2 protein expression and decreased the inflammatory response and number of apoptotic cells (P < 0.05). In conclusion, CH4 inhalation decreased oxidative stress injury, inflammatory response, and cell apoptosis, and improved lung function through Nrf2-mediated pulmonary surfactant regulation in rat lung I/R injury.