Reduced levels of two modifiers of epigenetic gene silencing, Dnmt3a and Trim28, cause increased phenotypic noise

Background: Inbred individuals reared in controlled environments display considerable variance in many complex traits but the underlying cause of this intangible variation has been an enigma. Here we show that two modifiers of epigenetic gene silencing play a critical role in the process.Results: Inbred mice heterozygous for a null mutation in DNA methyltransferase 3a (Dnmt3a) or tripartite motif protein 28 (Trim28) show greater coefficients of variance in body weight than their wild-type littermates. Trim28 mutants additionally develop metabolic syndrome and abnormal behavior with incomplete penetrance. Genome-wide gene expression analyses identified 284 significantly dysregulated genes in Trim28 heterozygote mutants compared to wild-type mice, with Mas1, which encodes a G-protein coupled receptor implicated in lipid metabolism, showing the greatest average change in expression (7.8-fold higher in mutants). This gene also showed highly variable expression between mutant individuals.Conclusions: These studies provide a molecular explanation of developmental noise in whole organisms and suggest that faithful epigenetic control of transcription is central to suppressing deleterious levels of phenotypic variation. These findings have broad implications for understanding the mechanisms underlying sporadic and complex disease in humans

Efficient diagnosis of ratoon stunting disease of sugarcane by quantitative PCR on pooled leaf sheath biopsies

Ratoon stunting disease (RSD), caused by the bacterium Leifsonia xyli subsp. xyli, is arguably one of the most devastating diseases of sugarcane. Four diagnostic techniques were compared for 100 fields of sugarcane (Saccharum interspecific hybrids) of unknown infection status. These were quantitative polymerase chain reaction on pooled leaf sheath biopsies (LSB-qPCR), conventional PCR on the same templates (LSB-PCR), evaporative-binding immunoassay (EB-EIA) coupled with phase contrast microscopy (PCM) on expressed xylem sap from the same fields, and conventional PCR on the same xylem sap samples. LSB-qPCR and LSB-PCR detected the causal agent in 27 and 18 fields respectively, while from samples of expressed xylem sap from the same fields, conventional PCR identified 12 infections, and EB-EIA/PCM detected L. xyli subsp. xyli in 3 fields. The sensitivities of qPCR and PCR were approximately 103 CFU ml-1 and 104 CFU ml-1 respectively, determined from plate counts of a dilution series. Tests were conducted on a further 139 LSB samples from across the Australian industry, with qPCR and PCR diagnosing RSD in 31 and 25 fields respectively. Using qPCR and PCR on LSB samples, RSD was diagnosed in a range of cultivars throughout the year, and could detect L. xyli subsp. xyli in sugarcane ranging from 3 months to greater than 1 year old

Compounds from Geijera parviflora with prostaglandin E2 inhibitory activity may explain its traditional use for pain relief

Ethnopharmacological relevance: Australian Aboriginal people used crushed leaves of Geijera parviflora Lindl. both internally and externally for pain relief, including for toothache ( Cribb and Cribb, 1981). This study tested the hypothesis that this traditional use might be at least in part explained by the presence of compounds with anti-inflammatory activity.Materials and methods: A crude extract (95% EtOH) was prepared from powdered dried leaves. From the CH3Cl fraction of this extract compounds were isolated by bioassay-guided fractionation and tested for: (1) cytotoxicity in RAW 264.7 murine leukemic monocyte–macrophages, (2) prostaglandin E2 (PGE2) inhibitory activity in 3T3 Swiss albino mouse embryonic fibroblast cells, as well as (3) nitric oxide (NO) and (4) tumour necrosis factor alpha (TNFα) inhibitory activity in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Isolated compounds were also tested for (5) antibacterial activity against a panel of Gram-positive (Staphylococcus aureus ATCC 29213 and ATCC 25923, Staphylococcusepidermidis ATCC 35984, biofilm-forming) and Gram-negative (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853) strains by broth microdilution. Results: Eleven compounds were isolated, including one new flavone and one new natural product, with a further four compounds reported from this species for the first time. Some of the compounds showed good anti-inflammatory activity in vitro. In particular, flindersine (1) and N-(acetoxymethyl) flindersine (3) inhibited PGE2 release with IC50values of 5.0 μM and 4.9 μM, respectively, without any significant cytotoxicity. Several other compounds showed moderate inhibition of NO (5, 6, 7) and TNF-α (6), with IC50 in the low micromolar range; however much of this apparent activity could be accounted for by the cytotoxicity of these compounds. None of the compounds showed anti-bacterial activity. Conclusions: The inhibition of PGE2, an important mediator of inflammation and pain, by flindersine and a derivative thereof, along with the moderate anti-inflammatory activity shown by several other compounds isolated from Geijera parviflora leaf extract, support the traditional use of this plant for pain relief by Australian Aboriginal people

Pilidiostigmin, a novel bioactive dimeric acylphloroglucinol derivative isolated from Pilidiostigma glabrum

Pilidiostigmin, a novel dimeric acylphloroglucinol derivative, was isolated from the leaves of the Australian plant species Pilidiostigma glabrum (Myrtaceae). Pilidiostigmin exists in the plant in the form of a sodium salt, which is rare in natural products. The elucidation of the structure and relative configuration was achieved by spectroscopic measurements with special emphasis on 1D and 2D NMR techniques. Pilidiostigmin was found to display biological activity; it significantly and dose-dependently inhibited the synthesis of nitric oxide in LPS-stimulated RAW 264.7 macrophages at low micromolar concentrations

Pilidiostigmin, a novel bioactive dimeric acylphloroglucinol derivative isolated from Pilidiostigma glabrum

Pilidiostigmin, a novel dimeric acylphloroglucinol derivative, was isolated from the leaves of the Australian plant species Pilidiostigma glabrum (Myrtaceae). Pilidiostigmin exists in the plant in the form of a sodium salt, which is rare in natural products. The elucidation of the structure and relative configuration was achieved by spectroscopic measurements with special emphasis on 1D and 2D NMR techniques. Pilidiostigmin was found to display biological activity; it significantly and dose-dependently inhibited the synthesis of nitric oxide in LPS-stimulated RAW 264.7 macrophages at low micromolar concentrations.

Functional reversion of antigen-specific CD8(+) T cells from patients with Hodgkin lymphoma following in vitro stimulation with recombinant polyepitope

Recent studies on Hodgkin's lymphoma (HL) have indicated that patients with active disease display functional impairment of Ag-specific CD8(+) T cells due to expansion of regulatory T cells at sites of disease and in the peripheral blood. Adoptive cellular immunotherapy based on EBV-specific CD8(+) T cells has been explored with limited success to date. It has been proposed that improved targeting of these CD8(+) T cells toward viral Ags that are expressed in HL may enhance future therapeutic vaccine strategies. In this study, we have developed a novel replication-deficient adenoviral Ag presentation system that is designed to encode glycine alanine repeat-deleted EBV nuclear Ag 1 covalently linked to multiple CD8(+) T cell epitopes from latent membrane proteins 1 and 2. A single stimulation of CD8(+) T cells from healthy virus carriers, and patients with HL with this adenoviral construct in combination with IL-2, was sufficient to reverse the functional T cell impairment and restored both IFN-gamma production and cytolytic function. More importantly, these activated CD8(+) T cells responded to tumor cells expressing membrane proteins and recognized novel EBNA1 epitopes. Flow cytometric analysis revealed that a large proportion of T cells expanded from patients with HL were CD62L(high) and CD27(high), and CCR7(low), consistent with early to mid effector T cells. These findings provide an important platform for translation of Ag-specific adoptive immunotherapy for the treatment of EBV-associated malignancies such as HL and nasopharyngeal carcinoma

Biologically active dibenzofurans from Pilidiostigma glabrum, an endemic Australian myrtaceae

In an effort to identify new anti-inflammatory and antibacterial agents with potential application in wound healing, five new dibenzofurans, 1,3,7,9-tetrahydroxy-2,8-dimethyl-4,6-di(2-methylbutanoyl)dibenzofuran (1), 1,3,7,9-tetrahydroxy-2,8-dimethyl-4-(2-methylbutanoyl)-6-(2-methylpropionyl) dibenzofuran (2), 1,3,7,9-tetrahydroxy-2,8-dimethyl-4,6-di(2-methylpropionyl) dibenzofuran (3), 1,3,7,9-tetrahydroxy-4,6-dimethyl-2-(2-methylbutanoyl)-8-(2- methylpropionyl)dibenzofuran (4), and 1,3,7,9-tetrahydroxy-4,6-dimethyl-2,8- di(2-methylpropionyl)dibenzofuran (5), were isolated from the leaves of Pilidiostigma glabrum together with one previously described dibenzofuran. Structure elucidation was achieved by way of spectroscopic measurements including 2D-NMR spectroscopy. Compounds with 2,8-acyl substitutions had potent antibacterial activity against several Gram-positive strains (MIC in the low micromolar range), while compounds with 4,6-acyl substitutions were less active. All compounds except 3 inhibited the synthesis of nitric oxide in RAW264 macrophages with IC values in the low micromolar range. Compounds with 2,8-acyl substitutions also inhibited the synthesis of PGE in 3T3 cells, whereas 4,6-acyl-substituted compounds were inactive. None of the compounds inhibited the synthesis of TNF-α in RAW264 cells. The compounds showed variable but modest antioxidant activity in the oxygen radical absorbance capacity assay. These findings highlight that much of the Australian flora remains unexplored and may yet yield many new compounds of interest. Initial clues are provided on structure/activity relationships for this class of bioactives, which may enable the design and synthesis of compounds with higher activity and/or selectivity

Biologically Active Dibenzofurans from <i>Pilidiostigma glabrum</i>, an Endemic Australian Myrtaceae

In an effort to identify new anti-inflammatory and antibacterial agents with potential application in wound healing, five new dibenzofurans, 1,3,7,9-tetrahydroxy-2,8-dimethyl-4,6-di­(2-methylbutanoyl)­dibenzofuran (<b>1</b>), 1,3,7,9-tetrahydroxy-2,8-dimethyl-4-(2-methylbutanoyl)-6-(2-methylpropionyl)­dibenzofuran (<b>2</b>), 1,3,7,9-tetrahydroxy-2,8-dimethyl-4,6-di­(2-methylpropionyl)­dibenzofuran (<b>3</b>), 1,3,7,9-tetrahydroxy-4,6-dimethyl-2-(2-methylbutanoyl)-8-(2-methylpropionyl)­dibenzofuran (<b>4</b>), and 1,3,7,9-tetrahydroxy-4,6-dimethyl-2,8-di­(2-methylpropionyl)­dibenzofuran (<b>5</b>), were isolated from the leaves of <i>Pilidiostigma glabrum</i> together with one previously described dibenzofuran. Structure elucidation was achieved by way of spectroscopic measurements including 2D-NMR spectroscopy. Compounds with 2,8-acyl substitutions had potent antibacterial activity against several Gram-positive strains (MIC in the low micromolar range), while compounds with 4,6-acyl substitutions were less active. All compounds except <b>3</b> inhibited the synthesis of nitric oxide in RAW264 macrophages with IC₅₀ values in the low micromolar range. Compounds with 2,8-acyl substitutions also inhibited the synthesis of PGE₂ in 3T3 cells, whereas 4,6-acyl-substituted compounds were inactive. None of the compounds inhibited the synthesis of TNF-α in RAW264 cells. The compounds showed variable but modest antioxidant activity in the oxygen radical absorbance capacity assay. These findings highlight that much of the Australian flora remains unexplored and may yet yield many new compounds of interest. Initial clues are provided on structure/activity relationships for this class of bioactives, which may enable the design and synthesis of compounds with higher activity and/or selectivity

A physiological function of inflammation-associated SerpinB2 is regulation of adaptive immunity

SerpinB2 (plasminogen activator inhibitor-2) is widely described as an inhibitor of urokinase plasminogen activator; however, SerpinB2(-/-) mice show no detectable increase in urokinase plasminogen activator activity. In this study, we describe an unexpected immune phenotype in SerpinB2(-/-) mice. After immunization with OVA in CIA, SerpinB2(-/-) mice made approximate to 6-fold more IgG2c and generated approximate to 2.5-fold more OVA-specific IFN-gamma-secreting T cells than SerpinB2(+/+) littermate controls. In SerpinB2(+/+) mice, high inducible SerpinB2 expression was seen at the injection site and in macrophages low levels in draining lymph nodes and conventional dendritic cells, and no expression was seen in plasmacytoid dendritic, B, T, or NK cells. SerpinB2(-/-) macrophages promoted greater IFN-gamma secretion from wild-type T cells in vivo and in vitro and, when stimulated with anti-CD40/IFN-gamma or cultured with wild-type T cells in vitro, secreted more Th1-promoting cytokines than macrophages from littermate controls. Draining lymph node SerpinB2(-/-) myeloid APCs similarly secreted more Th1-promoting cytokines when cocultured with wild-type T cells. Regulation of Th1 responses thus appears to be a physiological function of inflammation-associated SerpinB2; an observation that may shed light on human inflammatory diseases like pre-eclampsia, lupus, asthma, scleroderma, and periodontitis, which are associated with SerpinB2 polymorphisms or dysregulated SerpinB2 expression. The Journal of Immunology, 2010, 184: 2663-2670

Galectin-1 mediated suppression of Epstein-Barr virus–specific T-cell immunity in classic Hodgkin lymphoma

In Hodgkin lymphoma (HL), the malignant Hodgkin Reed-Sternberg cells interact with the host microenvironment to create an immunosuppressive network that protects the lymphoma from immune attack. These mechanisms are not fully understood. We examined the role of the immunomodulatory protein galectin-1 (Gal-1) on Epstein-Barr virus-specific CD8+ T cell responses in HL. Initial studies indicated Gal-1 expression in all in vitro established Hodgkin Reed-Sternberg cell lines. In situ analysis revealed Gal-1 expression in 26 of 42 classic HL, whereas Gal-1 was uniformly negative in nodular lymphocyte predominant HL. Gal-1hi expression was associated with male gender, older patients, reduced CD8+ T cell infiltration at the tumor site, and most importantly, an impaired latent membrane protein 1 and 2-specific CD8+ T-cell responses. In vitro exposure to recombinant Gal-1 inhibited proliferation and interferon-γ expression by Epstein-Barr virus-specific T cells. These observations provide an important link between the Gal-1-mediated immunomodulatory networks and loss of antigen-specific T-cell function in classic HL