Inhibition of tissue repair by spironolactone: Role of mineralocorticoids in fibrous tissue formation

Mineralocorticoids have been implicated in promoting fibrous tissue formation in various organs. In the present study, we sought to address the potential contribution of mineralocorticoids to fibrous tissue formation using a skin pouch model which has proved valuable for the analysis of inflammatory and wound healing responses. Skin pouches were induced in rats by administration of a phorbol ester, croton oil (0.5 ml of a 1% solution). After 2 weeks, rats were killed and intact pouch tissue collected. Pouch weights of control and aldosterone-treated (0.75 g/h via osmotic minipump) rats were similar (3.33 ± 0.44 g vs. 3.70 ± 0.28 g respectively). However, pouch weights were reduced by more than 50% in spironolactone-treated (25 mg/day powdered in food) animals (1.62 ± 0.22 g and 1.27 ± 0.23 g respectively in aldosterone and spironolactone alone groups). To ascertain the effects of different treatments on collagen accumulation, hydroxyproline concentration was measured. Compared with controls, hydroxyproline concentration was significantly reduced following spironolactone treatment (17.1 ± 0.08 vs. 7.5 ± 2.0 g/mg dry wt, respectively, p < 0.01). This response to spironolactone was negated by coadministration of aldosterone (hydroxyproline concentration was 18.6 ± 2.1 g/mg dry wt). Following bilateral adrenalectomy, spironolactone reduced pouch weight and hydroxyproline concentration, which was not the case for adrenalectomy alone. Two week aldosterone administration in uninephrectomized rats on high salt diet was deemed ineffective in modulating pouch development (pouch wet wts were 3.48 ± 0.4 g vs. 3.00 ± 0.19 g in controls and aldosterone-treated rats, respectively). Mineralocorticoid receptor expression in pouch tissue was demonstrated by RT/PCR. Furthermore, NADP+-dependent 11-hydroxysteroid dehydrogenase 1 (11-HSD1) activity was detected in pouch tissue, together with lower levels of NAD+-dependent 11-HSD2. Spironolactone (p < 0.05) significantly reduced 11-HSD1 activity compared with controls. Thus, fibrous tissue possesses requisite components of MC action, and antagonism of mineralocorticoid receptors by spironolactone attenuates its formation. Pouch formation is under the influence of circulating MC and, we would like to propose, is also mediated through corticosteroids generated de novo at the site of tissue repair.     

Evaluating the efficiency of phiC31 integrase‐mediated monoclonal antibody expression in CHO cells

Traditional methods to generate CHO cell lines rely on random integration(s) of the gene of interest and result in unpredictable and unstable protein expression. In comparison, site‐specific recombination methods increase the recombinant protein expression by inserting transgene at a locus with specific expression features. PhiC31 serine integrase, catalyze unidirectional integration that occurs at higher frequency in comparison with the reversible integration carried out by recombinases such as Cre. In this study, using different ratios of phiC31 serine integrase, we evaluated the phiC31 mediated gene integration for expression of a humanized IgG1 antibody (mAb0014) in CHO‐S cells. Light chain (LC) and heavy chain (HC) genes were expressed in one operon under EF1α promoter and linked by internal ribosome entry site (IRES) element. The clonal selection was carried out by limiting dilution. Targeted integration approach increased recombinant protein yield and stability in cell pools. The productivity of targeted cell pools was about 4 mg/L and about 40 µg/L in the control cell pool. The number of integrated transgenes was about 19 fold higher than the control cells pools. Our results confirmed that the phiC31 integrase leads to mAb expression in more than 90% of colonies. The productivity of the PhiC31 integrated cell pools was stable for three months in the absence of selection as compared with conventional transfection methods. Hence, utilizing PhiC31 integrase can increase protein titer and decrease the required time for protein expression. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1570–1576, 2016     

Genetic and virulence variation in Fusarium oxysporum f. sp. cepae causing root and basal rot of common onion in Iran

Root and basal rot of common onion (Allium cepae L.) caused by Fusarium oxysporum f. sp. cepae is one of the most important diseases causing tremendous losses in onion‐growing areas worldwide. In this study, random amplified polymorphic DNA (RAPD), intersimple sequence repeats (ISSR) and virulence studies were conducted to analyse 26 F. oxysporum f. sp. cepae isolates obtained from the main onion‐growing regions of Iran, including Fars, Azerbaijan and Isfahan states. Cluster analysis using UPGMA method for both RAPD and ISSR markers revealed no clear grouping of the isolates obtained from different geographical regions, and the isolates were observed to derive probably from the same clonal lineage. Pathogenicity test indicated that all F. oxysporum f. sp. cepae isolates were pathogenic on onion; however, virulence variability was observed among the isolates. The grouping based on virulence variability was not correlated with the results of RAPD and ISSR analyses.     

Temperature effects on oxygen consumption of two nocturnal geckos,Ptyodactylus hasselquistii (Donndorff) andBunopus tuberculatus (Blanford) (Reptilia: Gekkonidae) in Saudi Arabia

Summary Oxygen consumption rates of the lizardsPtyodactylus hasselquistii (adult and subadults) andBunopus tuberculatus (adult) were determined in relation to ambient temperatures ranging from 10 to 35°C using a double-chamber, volumetric closed system. The metabolic rate-temperature curves for both species were triphasic and similar in shape, but O₂ consumption differed between species.The low thermal dependence at temperatures between 15 and 25°C, common to both species, was correlated with the lizards' dual mode of thermoregulatory activity and ecology.     

S-Nitrosoglutathione Reductase Inhibition Regulates Allergen-Induced Lung Inflammation and Airway Hyperreactivity

Allergic asthma is characterized by Th2 type inflammation, leading to airway hyperresponsivenes, mucus hypersecretion and tissue remodeling. S-Nitrosoglutathione reductase (GSNOR) is an alcohol dehydrogenase involved in the regulation of intracellular levels of S-nitrosothiols. GSNOR activity has been shown to be elevated in human asthmatic lungs, resulting in diminished S-nitrosothiols and thus contributing to increased airway hyperreactivity. Using a mouse model of allergic airway inflammation, we report that intranasal administration of a new selective inhibitor of GSNOR, SPL-334, caused a marked reduction in airway hyperreactivity, allergen-specific T cells and eosinophil accumulation, and mucus production in the lungs in response to allergen inhalation. Moreover, SPL-334 treatment resulted in a significant decrease in the production of the Th2 cytokines IL-5 and IL-13 and the level of the chemokine CCL11 (eotaxin-1) in the airways. Collectively, these observations reveal that GSNOR inhibitors are effective not only in reducing airway hyperresponsiveness but also in limiting lung inflammatory responses mediated by CD4⁺ Th2 cells. These findings suggest that the inhibition of GSNOR may provide a novel therapeutic approach for the treatment of allergic airway inflammation.     

Cultural and physiological characteristics of Stemphylium lycopersici causing leaf blight disease on vegetable crops

During 2011–2012, an extensive leaf spot disease caused by Stemphylium lycopersici was observed on vegetable crops including, tomato, eggplant, pepper and lettuce in major vegetable-growing regions of Malaysia. Four isolates of S. lycopersici obtained from each vegetable crop were used to determine cultural and physiological characteristics. The variations were found in colony colour (pale to light grey or light as well as the brown), texture (cottony or mycelium flat), shape (regular with concentric growth rings or irregular) and pigmentation (yellow or deep red) of the cultures. The optimum temperature for the conidial germination and mean radial growth of the isolates was 25?°C, and the radial growth of the isolates was maximal on V-8 juice agar followed by potato carrot agar. The maximum sporulation of S. lycopersici isolates was observed on V-8 juice agar media under 12/12 h light/darkness photoperiod at 25?°C.     

Characterization of cholesterol transport from midgut to fat body in Manduca sexta larvae

Using in vitro methods, we investigated the transfer of cholesterol from larval Manduca sexta midgut to the hemolymph lipoprotein, lipophorin, and the transfer of cholesterol from lipophorin to larval fat body. In the midgut, transfer of free cholesterol shows saturation kinetics, but the apparent Km is higher than the measured Kd for the midgut lipophorin-receptor complex. In addition, the transfer is unaffected by suramin, which binds to the receptor and inhibits lipophorin binding, and by antibodies to the lipid transfer particle, which is required for export of diacylglycerol from the midgut to lipophorin. In the fat body, transfer of free cholesterol also shows saturation kinetics, and the apparent Km is higher than the measured Kd for the fat body lipophorin-receptor complex. Suramin and anti-lipid transfer particle antibodies exert only a small (20%) inhibitory effect. In both tissues it seems that the most likely mode of cholesterol transfer is via aqueous diffusion, which is also an important mechanism in vertebrate cells. Based on these results, we propose that cholesterol homeostasis in larval M. sexta is maintained by a mass action mechanism in which cholesterol is freely transferred between lipophorin and tissues depending on the needs of the tissues. This simple mechanism is ideally suited to insects, which can neither make cholesterol nor internalize lipophorin, the two mechanisms that vertebrate cells use to control their cholesterol content.