Secondary metabolites of ponderosa lemon (Citrus pyriformis) and their antioxidant, anti-inflammatory, and cytotoxic activities

Column chromatography of the dichloromethane fraction from an aqueous methanolic extract of fruit peel of Citrus pyriformis Hassk. (Rutaceae) resulted in the isolation of seven compounds including one coumarin (citropten), two limonoids (limonin and deacetylnomilin), and four sterols (stigmasterol, ergosterol, sitosteryl-3-beta-D-glucoside, and sitosteryl-6'-O-acyl-3-beta-D-glucoside). From the ethyl acetate fraction naringin, hesperidin, and neohesperidin were isolated. The dichloromethane extract of the defatted seeds contained three additional compounds, nomilin, ichangin, and cholesterol. The isolated compounds were identified by MS (EI, CI, and ESI), 1H, 13C, and 2D-NMR spectral data. The limonoids were determined qualitatively by LC-ESI/MS resulting in the identification of 11 limonoid aglycones. The total methanolic extract of the peel and the petroleum ether, dichloromethane, and ethyl acetate fractions were screened for their antioxidant and anti-inflammatory activities. The ethyl acetate fraction exhibited a significant scavenging activity for DPPH free radicals (IC50 = 132.3 microg/mL). The petroleum ether fraction inhibited 5-lipoxygenase with IC50 = 30.6 microg/mL indicating potential anti-inflammatory properties. Limonin has a potent cytotoxic effect against COS7 cells [IC50 = (35.0 +/- 6.1) microM] compared with acteoside as a positive control [IC50 = (144.5 +/- 10.96) microM].     

Development of a pterin-based fluorescent probe for screening dihydropteroate synthase

Dihydropteroate synthase (DHPS) is the classical target of the sulfonamide class of antimicrobial agents, whose use has been limited by widespread resistance and pharmacological side effects. We have initiated a structure-based drug design approach for the development of novel DHPS inhibitors that bind to the highly conserved and structured pterin subsite rather than to the adjacent p-aminobenzoic acid binding pocket that is targeted by the sulfonamide class of antibiotics. To facilitate these studies, a robust pterin site-specific fluorescence polarization (FP) assay has been developed and is discussed herein. These studies include the design, synthesis, and characterization of two fluorescent probes, and the development and validation of a rapid DHPS FP assay. This assay has excellent DMSO tolerance and is highly reproducible as evidenced by a high Z' factor. This assay offers significant advantages over traditional radiometric or phosphate release assays against this target, and is suitable for site-specific high-throughput and fragment-based screening studies.     

Recombinant plant-expressed tumour-associated MUC1 peptide is immunogenic and capable of breaking tolerance in MUC1.Tg mice

The human epithelial mucin MUC1 is a heavily glycosylated transmembrane protein that is overexpressed and aberrantly glycosylated on over 90% of human breast cancers. The altered glycosylation of MUC1 reveals an immunodominant peptide along its tandem repeat (TR) that has been used as a target for tumour immunotherapy. In this study, we used the MUC1 TR peptide as a test antigen to determine whether a plant-expressed human tumour-associated antigen can be successfully expressed in a plant system and whether it will be able to break self-antigen tolerance in a MUC1-tolerant mouse model. We report the expression of MUC1 TR peptide fused to the mucosal-targeting Escherichia coli enterotoxin B subunit (LTB-MUC1) in a plant host. Utilizing a rapid viral replicon transient expression system, we obtained high yields of LTB-MUC1. Importantly, the LTB-MUC1 fusion protein displayed post-translational modifications that affected its antigenicity. Glycan analysis revealed that LTB-MUC1 was glycosylated and a MUC1-specific monoclonal antibody detected only the glycosylated forms. A thorough saccharide analysis revealed that the glycans are tri-arabinans linked to hydroxyprolines within the MUC1 tandem repeat sequence. We immunized MUC1-tolerant mice (MUC1.Tg) with transiently expressed LTB-MUC1, and observed production of anti-MUC1 serum antibodies, indicating breach of tolerance. The results indicate that a plant-derived human tumour-associated antigen is equivalent to the human antigen in the context of immune recognition.     

MGMT is a molecular determinant for potency of the DNA-EGFR-combi-molecule ZRS1

To enhance the potency of current EGFR inhibitors, we developed a novel strategy that seeks to confer them an additional DNA damaging function, leading to the design of drugs termed combi-molecules. ZRS1 is a novel combi-molecule that contains an EGFR tyrosine kinase targeting quinazoline arm and a methyltriazene-based DNA damaging one. We examined its effect on human tumor cell lines with varied levels of EGFR and O6-methylguanine DNA methyltransferase (MGMT). ZRS1 was more potent than the clinical methylating agent temozolomide in all cell lines, regardless of their MGMT status. However, its potency was in the same range as or less than that of Iressa, an EGFR inhibitor, against MGMT-proficient cells. In the MGMT-deficient or in MGMT-proficient cells exposed to the MGMT inhibitor O6-benzylguanine, its potency was superior to that of Iressa and temozolomide or a temozolomide+Iressa combination. Cell signaling analysis in A549 (MGMT(+)) and A427 (MGMT(-)) showed that ZRS1 strongly inhibited EGFR phosphorylation and related signaling pathways. In addition, the p53 pathway was activated by DNA damage in both cell lines, but apoptosis was significantly more pronounced in A427 cells. Using MGMT shRNA to block endogenous MGMT protein expression in A549 resulted in significant sensitization to ZRS1. Furthermore, transfection of MGMT into A427 greatly decreased the potency of ZRS1. These results conclusively show that MGMT is a critical molecular determinant for the full-blown potency of the dual EGFR-DNA targeting combi-molecule.     

Uncovering genomic causes of co-morbidity in epilepsy: gene-driven phenotypic characterization of rare microdeletions

Background

Patients with epilepsy often suffer from other important conditions. The existence of such co-morbidities is frequently not recognized and their relationship with epilepsy usually remains unexplained.

Methodology/Principal Findings

We describe three patients with common, sporadic, non-syndromic epilepsies in whom large genomic microdeletions were found during a study of genetic susceptibility to epilepsy. We performed detailed gene-driven clinical investigations in each patient. Disruption of the function of genes in the deleted regions can explain co-morbidities in these patients.

Conclusions/Significance

Co-morbidities in patients with epilepsy can be part of a genomic abnormality even in the absence of (known) congenital malformations or intellectual disabilities. Gene-driven phenotype examination can also reveal clinically significant unsuspected condition.     

Features of visual function in the naked mole-rat <Emphasis Type="Italic">Heterocephalus glaber</Emphasis>

The eyes and visual capacity of the naked mole-rat, Heterocephalus glaber, a subterranean rodent, were evaluated using anatomical, biochemical, and functional assays, and compared to other rodents of similar body size (mouse and gerbil). The eye is small compared to mouse, yet possesses cornea, lens, and retina with typical mammalian organization. The optic nerve cross-sectional area and fiber density are ~10% and ~50% that of gerbil, respectively. Levels per unit retinal area of 11-cis and all-trans retinal, derivatives of vitamin A associated with the visual cycle, are comparable to mouse. The corneal electroretinogram (ERG) exhibits early and late negative components that scale with flash strength; raising the body temperature of this poikilothermic animal from 30°C (normal for H. glaber ) to 37°C (normal for mouse) revealed an ERG response with typically mammalian features, but greatly attenuated and with slower kinetics. Leaving the nest chamber was a behavior correlated with light onset displayed preferentially by breeding females. Optical models of five mole-rat eyes suggest reasonable, but variable, image formation at the retina, possibly related to age. Results are consistent with amorphous light detection, possibly useful for circadian entrainment or escape behavior in the event of tunnel breeches.     

Protein kinase C epsilon induces systolic cardiac failure marked by exhausted inotropic reserve and intact Frank-Starling mechanism

Myofilament dysfunction is a common point of convergence for many forms of heart failure. Recently, we showed that cardiac overexpression of PKC epsilon initially depresses myofilament activity and then leads to a progression of changes characteristic of human heart failure. Here, we examined the effects of PKC epsilon on contractile reserve, Starling mechanism, and myofilament activation in this model of end-stage dilated cardiomyopathy. Pressure-volume loop analysis and echocardiography showed that the PKC epsilon mice have markedly compromised systolic function and increased end-diastolic volumes. Dobutamine challenge resulted in a small increase in contractility in PKC epsilon mice but failed to enhance cardiac output. The PKC epsilon mice showed a normal length-dependent tension development in skinned cardiac muscle preparations, although Frank-Starling mechanism appeared to be compromised in the intact animal. Simultaneous measurement of tension and ATPase demonstrated that the maximum tension and ATPase were markedly lower in the PKC epsilon mice at any length or Ca2+ concentration. However, the tension cost was also lower indicating less energy expenditure. We conclude 1) that prolonged overexpression of PKC epsilon ultimately leads to a dilated cardiomyopathy marked by exhausted contractile reserve, 2) that PKC epsilon does not compromise the Frank-Starling mechanism at the myofilament level, and 3) that the Starling curve excursion is limited by the inotropic state of the heart. These results reflect the significance of the primary myofilament contractilopathy induced by phosphorylation and imply a role for PKC epsilon-mediated phosphorylation in myofilament physiology and the pathophysiology of decompensated cardiac failure.     

The relA homolog of Mycobacterium smegmatis affects cell appearance, viability, and gene expression

The modification of metabolic pathways to allow for a dormant lifestyle appears to be an important feature for the survival of pathogenic bacteria within their host. One regulatory mechanism for persistent Mycobacterium tuberculosis infections is the stringent response. In this study, we analyze the stringent response of a nonpathogenic, saprophytic mycobacterial species, Mycobacterium smegmatis. The use of M. smegmatis as a tool for studying the mycobacterial stringent response was demonstrated by measuring the expression of two M. tuberculosis genes, hspX and eis, in M. smegmatis in the presence and absence of rel(Msm). The stringent response plays a role in M. smegmatis cellular and colony formation that is suggestive of changes in the bacterial cell wall structure.     

Common features in the functional surface of scorpion beta-toxins and elements that confer specificity for insect and mammalian voltage-gated sodium channels

Scorpion beta-toxins that affect the activation of mammalian voltage-gated sodium channels (Navs) have been studied extensively, but little is known about their functional surface and mode of interaction with the channel receptor. To enable a molecular approach to this question, we have established a successful expression system for the anti-mammalian scorpion beta-toxin, Css4, whose effects on rat brain Navs have been well characterized. A recombinant toxin, His-Css4, was obtained when fused to a His tag and a thrombin cleavage site and had similar binding affinity for and effect on Na currents of rat brain sodium channels as those of the native toxin isolated from the scorpion venom. Molecular dissection of His-Css4 elucidated a functional surface of 1245 A2 composed of the following: 1) a cluster of residues associated with the alpha-helix, which includes a putative "hot spot" (this cluster is conserved among scorpion beta-toxins and contains their "pharmacophore"); 2) a hydrophobic cluster associated mainly with the beta2 and beta3 strands, which is likely to confer the specificity for mammalian Navs; 3) a single bioactive residue (Trp-58) in the C-tail; and 4) a negatively charged residue (Glu-15) involved in voltage sensor trapping as inferred from our ability to uncouple toxin binding from activity upon its substitution. This study expands our understanding about the mode of action of scorpion beta-toxins and illuminates differences in the functional surfaces that may dictate their specificities for mammalian versus insect sodium channels.