Phenolic allelochemicals released by <Emphasis Type="Italic">Chenopodium murale</Emphasis> affect the growth,nodulation and macromolecule content in chickpea and pea

The present study was conducted to investigate the effect of the residue of Chenopodium murale L. on growth, nodulation and macromolecule content of two legume crops, viz., Cicer arietinum L. (chickpea) and Pisum sativum L. (pea). A significant reduction in root and shoot length as well as dry matter accumulation occurred when both the legumes were grown in the soil amended with 5, 10, 20 and 40 g residue kg⁻¹ soil. In general, a gradual decline in growth was associated with an increasing amount of residues in the soil. There was also a significant reduction in total chlorophyll content and the amounts of protein and carbohydrates (macromolecules) in plants growing in the residue-amended soil. The nodulation was completely absent in chickpea and pea when the plants were grown in the soil amended with 10 and 20 g residue kg⁻¹ soil, respectively. At a lower rate of residue amendment (5 g kg⁻¹ soil), a significant decline in nodule number and weight, and leghaemoglobin content was recorded. Root oxidizability, an indirect measure of tissue viability and cellular respiration, was adversely affected in both the legumes under various treatments of residue amendment. The observed growth reduction concomitant with increased proline accumulation indicated the presence of some inhibitory compounds in the residue-amended soil. It was rich in phenolics identified as protocatechuic, ferulic, p-coumaric and syringic acid with 12.8, 30.4, 20.2 and 33.6% relative content, respectively. The results suggest that the residue of C. murale releases phenolic allelochemicals, which deleteriously affect the growth, nodulation and macromolecule content of chickpea and pea.     

Protectin D1 is generated in asthma and dampens airway inflammation and hyperresponsiveness

Protectins are newly identified natural chemical mediators that counter leukocyte activation to promote resolution of inflammation. In this study, we provide the first evidence for protectin D1 (PD1, 10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid) formation from docosahexaenoic acid in human asthma in vivo and PD1 counterregulatory actions in allergic airway inflammation. PD1 and 17S-hydroxy-docosahexaenoic acid were present in exhaled breath condensates from healthy subjects. Of interest, levels of PD1 were significantly lower in exhaled breath condensates from subjects with asthma exacerbations. PD1 was also present in extracts of murine lungs from both control animals and those sensitized and aerosol challenged with allergen. When PD1 was administered before aeroallergen challenge, airway eosinophil and T lymphocyte recruitment were decreased, as were airway mucus, levels of specific proinflammatory mediators, including IL-13, cysteinyl leukotrienes, and PGD(2), and airway hyperresponsiveness to inhaled methacholine. Of interest, PD1 treatment after aeroallergen challenge markedly accelerated the resolution of airway inflammation. Together, these findings provide evidence for endogenous PD1 as a pivotal counterregulatory signal in allergic airway inflammation and point to new therapeutic strategies for modulating inflammation in asthmatic lung.     

Role of activated macrophages in Acanthamoeba keratitis

The purpose of this study was to determine whether activating the conjunctival macrophages would affect the course of Acanthamoeba spp. keratitis in a Chinese hamster model of this disease. Chinese hamster spleen cells were stimulated with concanavalin A (Con A), and interferon gamma (IFN-gamma) -containing supernatants were collected 24 hr later. The IFN-gamma-containing supernatants were loaded into liposomes, which were fed to peritoneal macrophages in vitro. Macrophage activation was assessed by testing for production of nitric oxide (NO) with the use of Griess reagent. Conjunctival macrophages were activated in situ by subconjunctival injection of liposomes containing Con A-activated spleen cell culture supernatants. Control liposomes were loaded with phosphate-buffered saline (PBS). Macrophages exposed to supernatants from Con A-stimulated spleen cells produced 4-fold-higher amounts of NO than unstimulated macrophages. Activation of macrophages via subconjunctival injection of liposomes containing supernatants from Con A-stimulated spleen cell cultures resulted in rapid resolution of the corneal infection. Approximately 80% of animals treated with PBS-containing liposomes demonstrated evidence of corneal disease at day 14 compared to 10% incidence of infection in the Con A-treated group. Moreover, at all time points examined, the clinical appearance of the keratitis in animals treated with liposomes containing Con A supernatant was significantly reduced compared to the group treated with liposomes containing PBS (P < 0.05). Macrophages stimulated with IFN-gamma-containing supernatants killed significant numbers of the trophozoites in vitro (P < 0.05). Killing was inhibited by cytochalasin D, but not by L-N6-1-iminoethyl-L-lysine dihydrochloride (L-NIL), which is a selective inhibitor of inducible NO synthase (INOS).     

Mitochondrial reactive oxygen species signal hepatocyte steatosis by regulating the phosphatidylinositol 3-kinase cell survival pathway

Abnormal dietary intake of macronutrients is implicated in the development of obesity and fatty liver disease. Steatosis develops in cultured hepatocytes exposed to medium containing either a high concentration of long chain free fatty acids (HFFA) or medium deficient in methionine and choline (MCD). This study examined the mitochondrial reactive oxygen species (ROS)-dependent regulation of the phosphoinositol (PI) 3-kinase pathway in steatosis induced by exposure of AML-12 mouse hepatocytes to MCD or HFFA medium. Exposure to either MCD or HFFA medium resulted in increased production of superoxide anions and H(2)O(2), transduction of the PI 3-kinase pathway and steatosis. Inhibition of PI 3-kinase with LY294002 prevented steatosis. Pharmacologically inhibiting electron transport chain complex III production of ROS prevented activation of PI 3-kinase during macronutrient perturbation, whereas pharmacologically promoting electron transport chain complex III ROS production activated PI 3-kinase independent of nutrient input. The data suggest that H(2)O(2) is the ROS species involved in signal transduction; promoting the rapid conversion of superoxide to H(2)O(2) does not inhibit PI 3-kinase pathway activation during nutrient perturbation, and exogenous H(2)O(2) activates it independent of nutrient input. In addition to transducing PI 3-kinase, the ROS-dependent signal cascade amplifies the PI 3-kinase signal by maintaining phosphatase and tensin homolog in its inactive phosphorylated state. Knockdown of phosphatase and tensin homolog by small interfering RNA independently activated the PI 3-kinase pathway. Our findings suggest a common path for response to altered nutrition involving mitochondrial ROS-dependent PI 3-kinase pathway regulation, leading to steatosis.     

Ion pairs in non-redundant protein structures

Ion pairs contribute to several functions including the activity of catalytic triads, fusion of viral membranes, stability in thermophilic proteins and solvent-protein interactions. Furthermore, they have the ability to affect the stability of protein structures and are also a part of the forces that act to hold monomers together. This paper deals with the possible ion pair combinations and networks in 25% and 90% non-redundant protein chains. Different types of ion pairs present in various secondary structural elements are analysed. The ion pairs existing between different subunits of multisubunit protein structures are also computed and the results of various analyses are presented in detail. The protein structures used in the analysis are solved using X-ray crystallography, whose resolution is better than or equal to 1.5 A and R-factor better than or equal to 20%. This study can, therefore, be useful for analyses of many protein functions. It also provides insights into the better understanding of the architecture of protein structure.     

Investigations on unconventional hydrogen bonds in RNA binding proteins: the role of CH...O=C interactions

We have investigated the roles played by C-H...O=C interactions in RNA binding proteins. There was an average of 78 CH...O=C interactions per protein and also there was an average of one significant CH...O=C interactions for every 6 residues in the 59 RNA binding proteins studied. Main chain-Main chain (MM) CH...O=C interactions are the predominant type of interactions in RNA binding proteins. The donor atom contribution to CH...O=C interactions was mainly from aliphatic residues. The acceptor atom contribution for MM CH...O=C interactions was mainly from Val, Phe, Leu, Ile, Arg and Ala. The secondary structure preference analysis of CH...O=C interacting residues showed that, Arg, Gln, Glu and Tyr preferred to be in helix, while Ala, Asp, Cys, Gly, Ile, Leu, Lys, Met, Phe, Trp and Val preferred to be in strand conformation. Most of the CH...O=C interacting polar amino acid residues were solvent exposed while, majority of the CH...O=C interacting non polar residues were excluded from the solvent. Long and medium-range CH...O=C interactions are the predominant type of interactions in RNA binding proteins. More than 50% of CH...O=C interacting residues had a higher conservation score. Significant percentage of CH...O=C interacting residues had one or more stabilization centers. Sixty-six percent of the theoretically predicted stabilizing residues were also involved in CH...O=C interactions and hence these residues may also contribute additional stability to RNA binding proteins.     

Biofilm Formation by and Antifungal Susceptibility of Candida Isolates from Urine

Biofilm formation (BF) in the setting of candiduria has not been well studied. We determined BF and MIC to antifungals in Candida spp. isolates grown from urine samples of patients and performed a retrospective chart review to examine the correlation with risk factors. A total of 67 Candida spp. isolates were grown from urine samples from 55 patients. The species distribution was C. albicans (54%), C. glabrata (36%), and C. tropicalis (10%). BF varied greatly among individual Candida isolates but was stable in sequential isolates during chronic infection. BF also depended on the growth medium and especially in C. albicans was significantly enhanced in artificial urine (AU) compared to RPMI medium. In nine of the C. albicans strains BF was 4- to 10-fold higher in AU, whereas in three of the C. albicans strains and two of the C. glabrata strains higher BF was measured in RPMI medium than in AU. Determination of the MICs showed that planktonic cells of all strains were susceptible to amphotericin B (AMB) and caspofungin (CASPO) and that three of the C. glabrata strains and two of the C. albicans strains were resistant to fluconazole (FLU). In contrast, all biofilm-associated adherent cells were resistant to CASPO and FLU. The biofilms of 14 strains (28%) were sensitive to AMB (MIC₅₀ of <1 μg/ml). Correlation between degree of BF and MIC of AMB was not seen in RPMI grown biofilms but was present when grown in AU. A retrospective chart review demonstrated no correlation of known risk factors of candiduria with BF in AU or RPMI. We conclude that BF is a stable characteristic of Candida strains that varies greatly among clinical strains and is dependent on the growth medium. Resistance to AMB is associated with higher BF in AU, which may represent the more physiologic medium to test BF. Future studies should address whether in vitro BF can predict treatment failure in vivo.     

Linkage and association analysis of candidate genes for TB and TNFα cytokine expression: evidence for association with IFNGR1, IL-10, and TNF receptor 1 genes

Tuberculosis (TB) is a growing public health threat globally and several studies suggest a role of host genetic susceptibility in increased TB risk. As part of a household contact study in Kampala, Uganda, we have taken a unique approach to the study of genetic susceptibility to TB by developing an intermediate phenotype model for TB susceptibility, analyzing levels of tumor necrosis factor-α (TNFα) in response to culture filtrate as the phenotype. In the present study, we analyzed candidate genes related to TNFα regulation and found that interleukin (IL)-10, interferon-gamma receptor 1 (IFNGR1), and TNFα receptor 1 (TNFR1) genes were linked and associated to both TB and TNFα. We also show that these associations are with progression to active disease and not susceptibility to latent infection. This is the first report of an association between TB and TNFR1 in a human population and our findings for IL-10 and IFNGR1 replicate previous findings. By observing pleiotropic effects on both phenotypes, we show construct validity of our intermediate phenotype model, which enables the characterization of the role of these genetic polymorphisms on TB pathogenesis. This study further illustrates the utility of such a model for disentangling complex traits. C. C. Whalen and S. K. Iyengar contributed equally as senior authors of this work.     

Unique helical conformation of the fourth cytoplasmic loop of the CB1 cannabinoid receptor in a negatively charged environment

The proximal portion of the C-terminus of the CB(1) cannabinoid receptor is a primary determinant for G-protein activation. A 17 residue proximal C-terminal peptide (rodent CB1 401-417), the intracellular loop 4 (IL4) peptide, mimicked the receptor's G-protein activation domain. Because of the importance of the cationic amino acids to G-protein activation, the three-dimensional structure of the IL4 peptide in a negatively charged sodium dodecyl sulfate (SDS) micellar environment has been studied by two-dimensional proton nuclear magnetic resonance (2D (1)H NMR) spectroscopy and distance geometry calculations. Unambiguous proton NMR assignments were carried out with the aid of correlation spectroscopy (DQF-COSY and TOCSY) and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The distance constraints were used in torsion angle dynamics algorithm for NMR applications (DYANA) to generate a family of structures which were refined using restrained energy minimization and dynamics. In water, the IL4 peptide prefers an extended conformation, whereas in SDS micelles, 3(10)-helical conformation is induced. The predominance of 3(10)-helical domain structure in SDS represents a unique difference compared with structure in alternative environments, which can significantly impact global electrostatic surface potential on the cytoplasmic surface of the CB(1) receptor and might influence the signal to the G-proteins.