Lack of nitric oxide synthases increases lipoprotein immune complex deposition in the aorta and elevates plasma sphingolipid levels in lupus

Systemic lupus erythematosus (SLE) patients display impaired endothelial nitric oxide synthase (eNOS) function required for normal vasodilatation. SLE patients express increased compensatory activity of inducible nitric oxide synthase (iNOS) generating excess nitric oxide that may result in inflammation. We examined the effects of genetic deletion of NOS2 and NOS3, encoding iNOS and eNOS respectively, on accelerated vascular disease in MRL/lpr lupus mouse model. NOS2 and NOS3 knockout (KO) MRL/lpr mice had higher plasma levels of triglycerides (23% and 35%, respectively), ceramide (45% and 21%, respectively), and sphingosine 1-phosphate (S1P) (21%) compared to counterpart MRL/lpr controls. Plasma levels of the anti-inflammatory cytokine interleukin 10 (IL-10) in NOS2 and NOS3 KO MRL/lpr mice were lower (53% and 80%, respectively) than counterpart controls. Nodule-like lesions in the adventitia were detected in aortas from both NOS2 and NOS3 KO MRL/lpr mice. Immunohistochemical evaluation of the lesions revealed activated endothelial cells and lipid-laden macrophages (foam cells), elevated sphingosine kinase 1 expression, and oxidized low-density lipoprotein immune complexes (oxLDL-IC). The findings suggest that advanced vascular disease in NOS2 and NOS3 KO MRL/lpr mice maybe mediated by increased plasma triglycerides, ceramide and S1P; decreased plasma IL-10; and accumulation of oxLDL-IC in the vessel wall. The results expose possible new targets to mitigate lupus-associated complications.     

Intra-specific variability in biomass partitioning and carbon isotopic discrimination under moderate drought stress in seedlings from four <Emphasis Type="Italic">Pinus pinaster</Emphasis> populations

The response to drought of 20 open-pollinated families from four Pinus pinaster Ait. populations covering a latitudinal cline (France, Central and Southern Spain, and Morocco) was assessed. The seedlings were cultivated in a greenhouse under controlled temperature and vapor pressure deficit for 120 days, and subjected to two watering regimes for 60 days. Different biomass partitioning variables, pre-dawn water potential, and isotopic discrimination of ¹³C in needles (Δ) as surrogate of long-term water use efficiency were estimated for each seedling at the end of the experiment. In response to the imposed drought, there was no change in the root biomass partitioning, but the overall Δ values decreased in response to water stress. All the families of the population from Morocco showed the highest investment in roots, regardless of the watering regime imposed. Inter-family differences within populations were also significant for most parameters as confirmed by the heritability values estimated (higher under the well-watered treatment). The studied P. pinaster populations showed different strategies of response to drought. This may represent an important mechanism by local populations in facing future climatic change. The results could be of value in forest conservation and breeding programs of maritime pine in the future.     

Low-dose hydrocortisone in patients with cirrhosis and septic shock: a randomized controlled trial

Background

Recent studies have reported a high prevalence of relative adrenal insufficiency in patients with liver cirrhosis. However, the effect of corticosteroid replacement on mortality in this high-risk group remains unclear. We examined the effect of low-dose hydrocortisone in patients with cirrhosis who presented with septic shock.

Methods

We enrolled patients with cirrhosis and septic shock aged 18 years or older in a randomized double-blind placebo-controlled trial. Relative adrenal insufficiency was defined as a serum cortisol increase of less than 250 nmol/L or 9 μg/dL from baseline after stimulation with 250 μg of intravenous corticotropin. Patients were assigned to receive 50 mg of intravenous hydrocortisone or placebo every six hours until hemodynamic stability was achieved, followed by steroid tapering over eight days. The primary outcome was 28-day all-cause mortality.

Results

The trial was stopped for futility at interim analysis after 75 patients were enrolled. Relative adrenal insufficiency was diagnosed in 76% of patients. Compared with the placebo group (n = 36), patients in the hydrocortisone group (n = 39) had a significant reduction in vasopressor doses and higher rates of shock reversal (relative risk [RR] 1.58, 95% confidence interval [CI] 0.98–2.55, p = 0.05). Hydrocortisone use was not associated with a reduction in 28-day mortality (RR 1.17, 95% CI 0.92–1.49, p = 0.19) but was associated with an increase in shock relapse (RR 2.58, 95% CI 1.04–6.45, p = 0.03) and gastrointestinal bleeding (RR 3.00, 95% CI 1.08–8.36, p = 0.02).

Interpretation

Relative adrenal insufficiency was very common in patients with cirrhosis presenting with septic shock. Despite initial favourable effects on hemodynamic parameters, hydrocortisone therapy did not reduce mortality and was associated with an increase in adverse effects. (Current Controlled Trials registry no. ISRCTN99675218.)Cirrhosis is a leading cause of death worldwide,¹ often with septic shock as the terminal event.^2–9 Relative adrenal insufficiency shares similar features of distributive hyperdynamic shock with both cirrhosis and sepsis^10,11 and increasingly has been reported to coexist with both conditions.^11,12 The effect of low-dose hydrocortisone therapy on survival of critically ill patients in general with septic shock remains controversial, with conflicting results from randomized controlled trials^13–17 and meta-analyses.^18,19 The effect of hydrocortisone therapy on mortality among patients with cirrhosis, who are known to be a group at high risk for relative adrenal insufficiency, has not been studied and hence was the objective of our study.     

The hydroxyl functional group of N-(4-hydroxyphenyl)retinamide mediates cellular uptake and cytotoxicity in premalignant and malignant human epithelial cells

In a previous study, we demonstrated that the anticancer synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) redox cycles at the mitochondrial enzyme dihydroorotate dehydrogenase to trigger anomalous reactive oxygen species (ROS) production and attendant apoptosis in transformed human epithelial cells. Furthermore, we speculated that the hydroxyl functional group of 4HPR was required for this pro-oxidant property. In this study, we investigated the role of the hydroxyl functional group in the in vitro cytotoxicity of 4HPR. Using 4HPR, its primary in vivo metabolite N-(4-methoxyphenyl)retinamide (4MPR), and the synthetic derivative N-(4-trifluoromethylphenyl)retinamide (4TPR), we examined the pro-oxidant and apoptotic effects, as well as the cellular uptake, of these three N-(4-substituted-phenyl)retinamides in premalignant and malignant human skin, prostate, and breast epithelial cells. Compared to 4HPR, both 4MPR and 4TPR were ineffective in promoting conspicuous cellular ROS production, mitochondrial disruption, or DNA fragmentation in these transformed cells. Interestingly, both 4MPR and 4TPR were not particularly cell permeative relative to 4HPR in skin or breast epithelial cells, which implied an additional role for the hydroxyl functional group in the cellular uptake of 4HPR. Moreover, the short-term uptake of 4HPR was directly proportional to cell size, but this characteristic, in obvious contrast to cellular bioenergetic status and/or dihydroorotate dehydrogenase expression, was not fundamentally influential in the overall sensitivity to the promotion of cellular ROS production and apoptosis induction by this agent. Together, these results strongly implicate the hydroxyl functional group in the cytotoxic effects of 4HPR.     

Influence of Seed‐borne Cochliobolus sativus (Anamorph Bipolaris sorokiniana) on Crown Rot and Root Rot of Barley and Wheat

The effect of seed‐borne pathogens of wheat and barley on crown and root rot diseases of seven barley cultivars (Jimah‐6, Jimah‐51, Jimah‐54, Jimah‐58, Omani, Beecher and Duraqi) and three wheat cultivars (Cooley, Maissani and Shawarir) was investigated. Bipolaris sorokiniana and Alternaria alternata were detected in seeds of at least eight cultivars, but Fusarium species in seeds of only two barley cultivars (Jimah‐54 and Jimah‐58). Crown rot and root rot symptoms developed on barley and wheat cultivars following germination of infected seeds in sterilized growing media. Bipolaris sorokiniana was the only pathogen consistently isolated from crowns and roots of the emerging seedlings. In addition, crown rot and root rot diseases of non‐inoculated barley cultivars correlated significantly with B. sorokiniana inoculum in seeds (P = 0.0019), but not with Fusarium or Alternaria (P > 0.05). These results indicate the role of seed‐borne inoculum of B. sorokiniana in development of crown rot and root rot diseases. Pathogenicity tests of B. sorokiniana isolates confirmed its role in inducing crown rot and root rot, with two wheat cultivars being more resistant to crown and root rots than most barley cultivars (P < 0.05). Barley cultivars also exhibited significant differences in resistance to crown rot (P < 0.05). In addition, black point disease symptoms were observed on seeds of three barley cultivars and were found to significantly affect seed germination and growth of some of these cultivars. This study confirms the role of seed‐borne inoculum of B. sorokiniana in crown and root rots of wheat and barley and is the first report in Oman of the association of B. sorokiniana with black point disease of barley.     

Stomatal action directly feeds back on leaf turgor: new insights into the regulation of the plant water status from non‐invasive pressure probe measurements

Uptake of CO₂ by the leaf is associated with loss of water. Control of stomatal aperture by volume changes of guard cell pairs optimizes the efficiency of water use. Under water stress, the protein kinase OPEN STOMATA 1 (OST1) activates the guard‐cell anion release channel SLOW ANION CHANNEL‐ASSOCIATED 1 (SLAC1), and thereby triggers stomatal closure. Plants with mutated OST1 and SLAC1 are defective in guard‐cell turgor regulation. To study the effect of stomatal movement on leaf turgor using intact leaves of Arabidopsis, we used a new pressure probe to monitor transpiration and turgor pressure simultaneously and non‐invasively. This probe permits routine easy access to parameters related to water status and stomatal conductance under physiological conditions using the model plant Arabidopsis thaliana. Long‐term leaf turgor pressure recordings over several weeks showed a drop in turgor during the day and recovery at night. Thus pressure changes directly correlated with the degree of plant transpiration. Leaf turgor of wild‐type plants responded to CO₂, light, humidity, ozone and abscisic acid (ABA) in a guard cell‐specific manner. Pressure probe measurements of mutants lacking OST1 and SLAC1 function indicated impairment in stomatal responses to light and humidity. In contrast to wild‐type plants, leaves from well‐watered ost1 plants exposed to a dry atmosphere wilted after light‐induced stomatal opening. Experiments with open stomata mutants indicated that the hydraulic conductance of leaf stomata is higher than that of the root–shoot continuum. Thus leaf turgor appears to rely to a large extent on the anion channel activity of autonomously regulated stomatal guard cells.     

Synthesis,structural elucidation and in vitro antiparasitic activity against Trypanosoma cruzi and Leishmania chagasi parasites of novel tetrahydro-1-benzazepine derivatives

Forty six new 1,4-epoxy-2-exo-aryl- and cis-2-aryl-4-hydroxytetrahydro-1-benzazepine derivatives were synthesized and fully characterized. All compounds were tested in vitro against both Trypanosoma cruzi and Leishmania chagasi parasites and also for cytotoxicity using Vero and THP-1 mammalian cell lines. Many of the evaluated compounds showed remarkable activity against the epimastigote and intracellular amastigote forms of T. cruzi, with IC₅₀ values comparable with that of control drug nifurtimox, a nitrofuran derivative currently used in the treatment of Chagas’ disease. Other derivatives were found to have good activity against L. chagasi promastigotes, with low toxicity against the mammalian cells, but neither of them was active on intracellular amastigotes of L. chagasi infecting THP-1 macrophages.     

Prediction of protein‐glucose binding sites using support vector machines

Glucose is a simple sugar that plays an essential role in many basic metabolic and signaling pathways. Many proteins have binding sites that are highly specific to glucose. The exponential increase of genomic data has revealed the identity of many proteins that seem to be central to biological processes, but whose exact functions are unknown. Many of these proteins seem to be associated with disease processes. Being able to predict glucose‐specific binding sites in these proteins will greatly enhance our ability to annotate protein function and may significantly contribute to drug design. We hereby present the first glucose‐binding site classifier algorithm. We consider the sugar‐binding pocket as a spherical spatio‐chemical environment and represent it as a vector of geometric and chemical features. We then perform Random Forests feature selection to identify key features and analyze them using support vector machines classification. Our work shows that glucose binding sites can be modeled effectively using a limited number of basic chemical and residue features. Using a leave‐one‐out cross‐validation method, our classifier achieves a 8.11% error, a 89.66% sensitivity and a 93.33% specificity over our dataset. From a biochemical perspective, our results support the relevance of ordered water molecules and ions in determining glucose specificity. They also reveal the importance of carboxylate residues in glucose binding and the high concentration of negatively charged atoms in direct contact with the bound glucose molecule. Proteins 2009. © 2009 Wiley‐Liss, Inc.