Natural Variation in Maize Aphid Resistance Is Associated with 2,4-Dihydroxy-7-Methoxy-1,4-Benzoxazin-3-One Glucoside Methyltransferase Activity

Plants differ greatly in their susceptibility to insect herbivory, suggesting both local adaptation and resistance tradeoffs. We used maize (Zea mays) recombinant inbred lines to map a quantitative trait locus (QTL) for the maize leaf aphid (Rhopalosiphum maidis) susceptibility to maize Chromosome 1. Phytochemical analysis revealed that the same locus was also associated with high levels of 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one glucoside (HDMBOA-Glc) and low levels of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside (DIMBOA-Glc). In vitro enzyme assays with candidate genes from the region of the QTL identified three O-methyltransferases (Bx10a-c) that convert DIMBOA-Glc to HDMBOA-Glc. Variation in HDMBOA-Glc production was attributed to a natural CACTA family transposon insertion that inactivates Bx10c in maize lines with low HDMBOA-Glc accumulation. When tested with a population of 26 diverse maize inbred lines, R. maidis produced more progeny on those with high HDMBOA-Glc and low DIMBOA-Glc. Although HDMBOA-Glc was more toxic to R. maidis than DIMBOA-Glc in vitro, BX10c activity and the resulting decline of DIMBOA-Glc upon methylation to HDMBOA-Glc were associated with reduced callose deposition as an aphid defense response in vivo. Thus, a natural transposon insertion appears to mediate an ecologically relevant trade-off between the direct toxicity and defense-inducing properties of maize benzoxazinoids.     

The role of adipose tissue-associated macrophages and T lymphocytes in the pathogenesis of inflammatory bowel disease

Inflammatory bowel diseases (IBDs) are chronic inflammatory disorders of the gastrointestinal tract that affect more than 3 million people worldwide, but the pathological etiology is still unknown. The overall purpose of our investigations was to elucidate the possibility of pathological causes of IBD, and therefore, we determined the difference of inflammatory cytokine profiles in adipose tissue macrophages (ATMs) and T lymphocytes (ATTs) obtained near active lesions of IBD; investigated whether the alteration in ATM activation induces genes involved in collagen formation; and evaluated the effects of fatty acid oxidation inhibitors on factors involved in inflammation and collagen production by ATMs in IBD. Adipose tissues (ATs) were collected near active lesions and also at the margin of resected segments of the bowel from IBD patients with ulcerative colitis (UC) and CD (n = 14/group). Normal appearing ATs from control subjects (n = 14) who had colon resection for adenocarcinoma were collected as far away from the cancer lesion as possible to rule out possible changes. Compared with inactive disease lesions, ATMs and ATTs from active lesions released more IL-6, IL-4 and IL-13. Treatments of cytokine IL-4 and/or IL-13 to ATMs reduced iNOS expression but increased Arg-I expression which were exacerbated when treated with T cell- and adipocyte-conditioned medium. However, fatty acid oxidation inhibitors prevented the effects of cytokines IL-4 and/or IL-13 on iNOS and Arg-I expressions. This study was the first to show the effect of IL-4 and IL-13 on collagen formation, through iNOS and Arg-I expressions, that was exacerbated in a condition that mimics in vivo condition of active lesions. Moreover, our study was the first to provide potential benefits of fatty acid oxidation inhibitors to ATMs on preventing collagen formation; thus, providing therapeutic implications for individuals with intestinal fibrosis and stricture lesions, although future study should be guaranteed to elucidate the underlying mechanisms.     

Development of Novel Elastic Vesicle-Based Topical Formulation of Cetirizine Dihydrochloride for Treatment of Atopic Dermatitis

Cetirizine is a piperazine-derived second-generation antihistaminic drug recommended for treatment of pruritus associated with atopic dermatitis. The present investigation encompasses development of a nanosized novel elastic vesicle-based topical formulation of cetirizine dihydrochloride using combination of Phospholipon® 90G and edge activators with an aim to have targeted peripheral H₁ antihistaminic activity. The formulation was optimized with respect to phospholipid/drug/charge inducer ratio along with type and concentration of edge activator. The optimized formulation was found to be satisfactory with respect to stability, drug content, entrapment efficiency, pH, viscosity, vesicular size, spreadability, and morphological characteristics. The ex vivo permeation studies through mice skin were performed using Franz diffusion cell assembly. It was found that the mean cumulative percentage amount permeated in 8 h was almost twice (60.001 ± 0.332) as compared to conventional cream (33.268 ± 0.795) and aqueous solution of drug (32.616 ± 0.969), suggesting better penetration and permeation of cetirizine from the novel vesicular delivery system. Further, therapeutic efficacy of optimized formulation was assessed against oxazolone-induced atopic dermatitis in mice. It was observed that the developed formulation was highly efficacious in reducing the itching score (4.75 itches per 20 min) compared to conventional cream (9.75 itches per 20 min) with profound reduction in dermal eosinophil count and erythema score. To conclude, a novel vesicular, dermally safe, and nontoxic topical formulation of cetirizine was successfully developed and may be used to treat atopic dermatitis after clinical investigation.KEY WORDS: atopic dermatitis, cetirizine, elastic vesicles, oxazolone, topical     

Pre-culturing islets with mesenchymal stromal cells using a direct contact configuration is beneficial for transplantation outcome in diabetic mice

Background aimsWe recently showed that co-transplantation of mesenchymal stromal cells (MSCs) improves islet function and revascularization in vivo. Pre-transplant islet culture is associated with the loss of islet cells. MSCs may enhance islet cell survival or function by direct cell contact mechanisms and soluble mediators. We investigated the capacity of MSCs to improve islet cell survival or β-cell function in vitro using direct and indirect contact islet-MSC configurations. We also investigated whether pre-culturing islets with MSCs improves islet transplantation outcome.MethodsThe effect of pre-culturing islets with MSCs on islet function in vitro was investigated by measuring glucose-stimulated insulin secretion. The endothelial cell density of fresh islets and islets cultured with or without MSCs was determined by immunohistochemistry. The efficacy of transplanted islets was tested in vivo using a syngeneic streptozotocin-diabetic minimal islet mass model. Graft function was investigated by monitoring blood glucose concentrations.ResultsIndirect islet-MSC co-culture configurations did not improve islet function in vitro. Pre-culturing islets using a direct contact MSC monolayer configuration improved glucose-stimulated insulin secretion in vitro, which correlated with superior islet graft function in vivo. MSC pre-culture had no effect on islet endothelial cell number in vitro or in vivo.ConclusionsPre-culturing islets with MSCs using a direct contact configuration maintains functional β-cell mass in vitro and the capacity of cultured islets to reverse hyperglycemia in diabetic mice.     

The Mode of Inhibitor Binding to Peptidyl-tRNA Hydrolase: Binding Studies and Structure Determination of Unbound and Bound Peptidyl-tRNA Hydrolase from Acinetobacter baumannii

The incidences of infections caused by an aerobic Gram-negative bacterium, Acinetobacter baumannii are very common in hospital environments. It usually causes soft tissue infections including urinary tract infections and pneumonia. It is difficult to treat due to acquired resistance to available antibiotics is well known. In order to design specific inhibitors against one of the important enzymes, peptidyl-tRNA hydrolase from Acinetobacter baumannii, we have determined its three-dimensional structure. Peptidyl-tRNA hydrolase (AbPth) is involved in recycling of peptidyl-tRNAs which are produced in the cell as a result of premature termination of translation process. We have also determined the structures of two complexes of AbPth with cytidine and uridine. AbPth was cloned, expressed and crystallized in unbound and in two bound states with cytidine and uridine. The binding studies carried out using fluorescence spectroscopic and surface plasmon resonance techniques revealed that both cytidine and uridine bound to AbPth at nanomolar concentrations. The structure determinations of the complexes revealed that both ligands were located in the active site cleft of AbPth. The introduction of ligands to AbPth caused a significant widening of the entrance gate to the active site region and in the process of binding, it expelled several water molecules from the active site. As a result of interactions with protein atoms, the ligands caused conformational changes in several residues to attain the induced tight fittings. Such a binding capability of this protein makes it a versatile molecule for hydrolysis of peptidyl-tRNAs having variable peptide sequences. These are the first studies that revealed the mode of inhibitor binding in Peptidyl-tRNA hydrolases which will facilitate the structure based ligand design.     

Structural and Functional Studies of a Phosphatidic Acid-Binding Antifungal Plant Defensin MtDef4: Identification of an RGFRRR Motif Governing Fungal Cell Entry

MtDef4 is a 47-amino acid cysteine-rich evolutionary conserved defensin from a model legume Medicago truncatula. It is an apoplast-localized plant defense protein that inhibits the growth of the ascomycetous fungal pathogen Fusarium graminearum in vitro at micromolar concentrations. Little is known about the mechanisms by which MtDef4 mediates its antifungal activity. In this study, we show that MtDef4 rapidly permeabilizes fungal plasma membrane and is internalized by the fungal cells where it accumulates in the cytoplasm. Furthermore, analysis of the structure of MtDef4 reveals the presence of a positively charged γ-core motif composed of β₂ and β₃ strands connected by a positively charged RGFRRR loop. Replacement of the RGFRRR sequence with AAAARR or RGFRAA abolishes the ability of MtDef4 to enter fungal cells, suggesting that the RGFRRR loop is a translocation signal required for the internalization of the protein. MtDef4 binds to phosphatidic acid (PA), a precursor for the biosynthesis of membrane phospholipids and a signaling lipid known to recruit cytosolic proteins to membranes. Amino acid substitutions in the RGFRRR sequence which abolish the ability of MtDef4 to enter fungal cells also impair its ability to bind PA. These findings suggest that MtDef4 is a novel antifungal plant defensin capable of entering into fungal cells and affecting intracellular targets and that these processes are mediated by the highly conserved cationic RGFRRR loop via its interaction with PA.     

Investigating the Antiproliferative Activity of High Affinity DNA Aptamer on Cancer Cells

Vascular endothelial growth factor (VEGF) is an angiogenic mitogen involved in promoting tumor angiogenesis inside the body. VEGF is a key protein required for progression of tumor from benign to malignant phenotype. In this study, we investigated the binding affinity of a previously selected 26-mer DNA aptamer sequence (SL₂-B) against heparin binding domain (HBD) of VEGF₁₆₅ protein. The SL₂-B was first chemically modified by introduction of phosphorothioate linkages (PS-linkages). Subsequently, surface plasmon resonance (SPR) spectroscopy and circular dichroism (CD) were used to determine the binding affinity, specificity and to deduce the conformation of PS-modified SL₂-B sequence. Finally, antiproliferative activity of the modified SL₂-B sequence on Hep G2 cancer cells was investigated. Our results demonstrate a marked enhancement in the biostability of the SL₂-B sequence after PS modification. The modified SL₂-B sequence also exhibits enhanced antiproliferative activity against Hep G2 cancer cells in hypoxia conditions. In addition, modified SL₂-B sequence inhibits the expression of Jagged-1 protein, which is one of the ligands to VEGF linked delta/jagged-notch signaling pathway.     

Functional properties and the oligomeric state of alkyl hydroperoxide reductase subunit F (AhpF) in Pseudomonas aeruginosa

Protoplasma - Alkyl hydroperoxide reductase subunit F (AhpF) is a well-known flavoprotein that transfers electrons from pyridine nucleotides to the peroxidase protein AhpC via redox-active...