Protection of Salvia miltiorrhizae to the Spleen and Thymus of Rats with Severe Acute Pancreatitis or Obstructive Jaundice

Objective. To investigate the therapeutic effects and mechanism of Salvia miltiorrhizae in the treatment of SAP and OJ. Methods. A total of 288 rats were used for SAP- and OJ-associated experiments. The rats were randomly divided into sham-operated group, model control group and treated group. The mortality rates of rats, contents of endotoxin and PLA2 in blood, patholodgical changes of different indexes in spleen and thymus were observed. Results. The contents of endotoxin and PLA2 in treated group were significantly lower than those in model control group.The pathological severity scores of spleen and thymus of SAP rats as well as that of spleen of OJ rats in treated groups were significantly lower than those in model control groups (P < .05). The staining intensity as well as the product of the staining intensity and positive rate of Bax protein of spleen in model control group were significantly higher than those in treated groups (P < .01) , and the apoptosis index of spleen in treated group was significantly lower than that in model control group (P < .01). Conclusion. Salvia miltiorrhizae exerts protective effects on the spleen and thymus of SAP rats and spleen of OJ rats

Selection of reference genes for studies of human retinal endothelial cell gene expression by reverse transcriptionquantitative real-time polymerase chain reaction

© 2017 Elsevier. This manuscript version is made available under the CC-BY-NC-ND 4.0 license: http://creativecommons.org/licenses/by-nc-nd/4.0/ This author accepted manuscript is made available following 12 month embargo from date of publication (Nov 2017) in accordance with the publisher’s archiving policyBackground Human retinal endothelial cells are employed increasingly for investigations of retinal vascular diseases. Analysis of gene expression response to disease-associated stimuli by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) is common. However, most reported work does not follow the minimum information for publication of qPCR experiments (MIQE) recommendation that multiple, stably expressed reference genes be used for normalization. Methods Two human retinal endothelial cell lines were treated with medium alone or containing stimuli that included: glucose at supraphysiological concentration, dimethyloxalyl-glycine, vascular endothelial growth factor, tumor necrosis factor-α, lipopolysaccharide and Toxoplasma gondii tachyzoites. Biological response of cells was confirmed by measuring significant increase in a stimulus-relevant transcript. Total RNA was reverse transcribed and analyzed by commercial PCR arrays designed to detect 28 reference genes. Stability of reference gene expression, for each and both cell lines, and for each and all conditions, was judged on gene-stability measure (M-value) < 0.2 and coefficient of variation (CV-value) < 0.1. Results Reference gene expression varied substantially across stimulations and between cell lines. Of 27 detectable reference genes, 11–21 (41–78%) maintained expression stability across stimuli and cell lines. Ranking indicated substantial diversity in the most stable reference genes under different conditions, and no reference gene was expressed stably under all conditions of stimulation and for both cell lines. Four reference genes were expressed stably under 5 conditions: HSP90AB1, IPO8, PSMC4 and RPLPO. Conclusions We observed variation in stability of reference gene expression with different stimuli and between human retinal endothelial cell lines. Our findings support adherence to MIQE recommendations regarding normalization in RT-qPCR studies of human retinal endothelial cells

ICAM-1-related long non-coding RNA: promoter analysis and expression in human retinal endothelial cells

© The Author(s) 2018 This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Abstract Objective Regulation of intercellular adhesion molecule (ICAM)-1 in retinal endothelial cells is a promising druggable target for retinal vascular diseases. The ICAM-1-related (ICR) long non-coding RNA stabilizes ICAM-1 transcript, increasing protein expression. However, studies of ICR involvement in disease have been limited as the promoter is uncharacterized. To address this issue, we undertook a comprehensive in silico analysis of the human ICR gene promoter region. Results We used genomic evolutionary rate profiling to identify a 115 base pair (bp) sequence within 500 bp upstream of the transcription start site of the annotated human ICR gene that was conserved across 25 eutherian genomes. A second constrained sequence upstream of the orthologous mouse gene (68 bp; conserved across 27 Eutherian genomes including human) was also discovered. Searching these elements identified 33 matrices predictive of binding sites for transcription factors known to be responsive to a broad range of pathological stimuli, including hypoxia, and metabolic and inflammatory proteins. Five phenotype-associated single nucleotide polymorphisms (SNPs) in the immediate vicinity of these elements included four SNPs (i.e. rs2569693, rs281439, rs281440 and rs11575074) predicted to impact binding motifs of transcription factors, and thus the expression of ICR and ICAM-1 genes, with potential to influence disease susceptibility. We verified that human retinal endothelial cells expressed ICR, and observed induction of expression by tumor necrosis factor-α

Effect of NADPH oxidase 1 and 4 blockade in activated human retinal endothelial cells

© 2018 Royal Australian and New Zealand College of Ophthalmologists. This author accepted manuscript is made available following 12 month embargo from date of publication (January 2018) in accordance with the publisher's archiving policy.Background Over‐production of reactive oxygen species (ROS) and resulting oxidative stress contribute to retinal damage in vascular diseases that include diabetic retinopathy, retinopathy of prematurity and major retinal vessel occlusions. NADPH oxidase (Nox) proteins are professional ROS‐generating enzymes, and therapeutic targeting in these diseases has strong appeal. Pharmacological inhibition of Nox4 reduces the severity of experimental retinal vasculopathy. We investigated the potential application of this drug approach in humans. Methods Differential Nox enzyme expression was studied by real‐time‐quantitative polymerase chain reaction in primary human retinal endothelial cell isolates and a characterized human retinal endothelial cell line. Oxidative stress was triggered chemically in endothelial cells, by treatment with dimethyloxalylglycine (DMOG; 100 μM); Nox4 and vascular endothelial growth factor (VEGFA) transcript were measured; and production of ROS was detected by 2′,7′‐dichlorofluorescein. DMOG‐stimulated endothelial cells were treated with two Nox1/Nox4 inhibitors, GKT136901 and GKT137831; cell growth was monitored by DNA quantification, in addition to VEGFA transcript and ROS production. Results Nox4 (isoform Nox4A) was the predominant Nox enzyme expressed by human retinal endothelial cells. Treatment with DMOG significantly increased endothelial cell expression of Nox4 over 72 h, accompanied by ROS production and increased VEGFA expression. Treatment with GKT136901 or GKT137831 significantly reduced DMOG‐induced ROS production and VEGFA expression by endothelial cells, and the inhibitory effect of DMOG on cell growth. Conclusions Our findings in experiments on activated human retinal endothelial cells provide translational corroboration of studies in experimental models of retinal vasculopathy and support the therapeutic application of Nox4 inhibition by GKT136901 and GKT137831 in patients with retinal vascular diseases

Protein kinase activity of phosphoinositide 3-kinase regulates cytokine-dependent cell survival

Extent: 14 p.The dual specificity protein/lipid kinase, phosphoinositide 3-kinase (PI3K), promotes growth factor-mediated cell survival and is frequently deregulated in cancer. However, in contrast to canonical lipid-kinase functions, the role of PI3K protein kinase activity in regulating cell survival is unknown. We have employed a novel approach to purify and pharmacologically profile protein kinases from primary human acute myeloid leukemia (AML) cells that phosphorylate serine residues in the cytoplasmic portion of cytokine receptors to promote hemopoietic cell survival. We have isolated a kinase activity that is able to directly phosphorylate Ser585 in the cytoplasmic domain of the interleukin 3 (IL-3) and granulocyte macrophage colony stimulating factor (GM-CSF) receptors and shown it to be PI3K. Physiological concentrations of cytokine in the picomolar range were sufficient for activating the protein kinase activity of PI3K leading to Ser585 phosphorylation and hemopoietic cell survival but did not activate PI3K lipid kinase signaling or promote proliferation. Blockade of PI3K lipid signaling by expression of the pleckstrin homology of Akt1 had no significant impact on the ability of picomolar concentrations of cytokine to promote hemopoietic cell survival. Furthermore, inducible expression of a mutant form of PI3K that is defective in lipid kinase activity but retains protein kinase activity was able to promote Ser585 phosphorylation and hemopoietic cell survival in the absence of cytokine. Blockade of p110α by RNA interference or multiple independent PI3K inhibitors not only blocked Ser585 phosphorylation in cytokine-dependent cells and primary human AML blasts, but also resulted in a block in survival signaling and cell death. Our findings demonstrate a new role for the protein kinase activity of PI3K in phosphorylating the cytoplasmic tail of the GM-CSF and IL-3 receptors to selectively regulate cell survival highlighting the importance of targeting such pathways in cancer.Daniel Thomas, Jason A. Powell, Benjamin D. Green, Emma F. Barry, Yuefang Ma, Joanna Woodcock, Stephen Fitter, Andrew C.W. Zannettino, Stuart M. Pitson, Timothy P. Hughes, Angel F. Lopez, Peter R. Shepherd, Andrew H. Wei, Paul G. Ekert and Mark A. Guthridg

The role of PKCzeta in cord blood T-cell maturation towards Th1 cytokine profile and its epigenetic regulation by fish oil

While immunodeficiency of immaturity of the neonate has been considered important as the basis for unusual susceptibility to infection, it has also been recognized that the ability to progress from an immature Th2 cytokine predominance to a Th1 profile has relevance in determining whether children will develop allergy, providing an opportunity for epigenetic regulation through environmental pressures. However, this notion remains relatively unexplored. Here, we present evidence that there are two major control points to explain the immunodeficiency in cord blood (CB) T-cells, a deficiency in interleukin (IL)-12 (IL-12) producing and IL-10 overproducing accessory cells, leading to a decreased interferon γ (IFNγ) synthesis and the other, an intrinsic defect in T-cell protein kinase C (PKC) ζ (PKCζ) expression. An important finding was that human CB T-cells rendered deficient in PKCζ, by shRNA knockdown, develop into low tumour necrosis factor α (TNFα) and IFNγ but increased IL-13 producing cells. Interestingly, we found that the increase in PKCζ levels in CB T-cells caused by prenatal supplementation with fish oil correlated with modifications of histone acetylation at the PKCζ gene (PRKCZ) promoter. The data demonstrate that PKCζ expression regulates the maturation of neonatal T-cells into specific functional phenotypes and that environmental influences may work via PKCζ to regulate these phenotypes and disease susceptibility.Hani Harb, James Irvine, Manori Amarasekera, Charles S. Hii, Dörthe A. Kesper, YueFang Ma, Nina D′Vaz, Harald Renz, Daniel P. Potaczek, Susan L. Prescott and Antonio Ferrant

Immunological Molecular Responses of Human Retinal Pigment Epithelial Cells to Infection With Toxoplasma gondii

Ocular toxoplasmosis is the commonest clinical manifestation of infection with obligate intracellular parasite, Toxoplasma gondii. Active ocular toxoplasmosis is characterized by replication of T. gondii tachyzoites in the retina, with reactive inflammation. The multifunctional retinal pigment epithelium is a key target cell population for T. gondii. Since the global gene expression profile is germane to understanding molecular involvements of retinal pigment epithelial cells in ocular toxoplasmosis, we performed RNA-Sequencing (RNA-Seq) of human cells following infection with T. gondii tachyzoites. Primary cell isolates from eyes of cadaveric donors (n = 3), and the ARPE-19 human retinal pigment epithelial cell line, were infected for 24 h with GT-1 strain T. gondii tachyzoites (multiplicity of infection = 5) or incubated uninfected as control. Total and small RNA were extracted from cells and sequenced on the Illumina NextSeq 500 platform; results were aligned to the human hg19 reference sequence. Multidimensional scaling showed good separation between transcriptomes of infected and uninfected primary cell isolates, which were compared in edgeR software. This differential expression analysis revealed a sizeable response in the total RNA transcriptome—with significantly differentially expressed genes totaling 7,234 (28.9% of assigned transcripts)—but very limited changes in the small RNA transcriptome—totaling 30 (0.35% of assigned transcripts) and including 8 microRNA. Gene ontology and pathway enrichment analyses of differentially expressed total RNA in CAMERA software, identified a strong immunologic transcriptomic signature. We conducted RT-qPCR for 26 immune response-related protein-coding and long non-coding transcripts in epithelial cell isolates from different cadaveric donors (n = 3), extracted by a different isolation protocol but similarly infected with T. gondii, to confirm immunological activity of infected cells. For microRNA, increases in miR-146b and miR-212 were detected by RT-qPCR in 2 and 3 of these independent cell isolates. Biological network analysis in the InnateDB platform, including 735 annotated differentially expressed genes plus 2,046 first-order interactors, identified 10 contextural hubs and 5 subnetworks in the transcriptomic immune response of cells to T. gondii. Our observations provide a solid base for future studies of molecular and cellular interactions between T. gondii and the human retinal pigment epithelium to illuminate mechanisms of ocular toxoplasmosis

Characterisation and molecular manipulation of barley B-amylase / by Yuefang Ma.

On title page "B" is superscript.Bibliography: leaves 159-171.iii, 171 leaves : ill. (some col.) ; 30 cm.Thesis (Ph.D.)--Adelaide University, Dept. of Plant Science, 200