Astrocyte control of fetal cortical neuron glutathione homeostasis: up-regulation by ethanol

Ethanol increases apoptotic neuron death in the developing brain and at least part of this may be mediated by oxidative stress. In cultured fetal rat cortical neurons, Ethanol increases levels of reactive oxygen species (ROS) within minutes of exposure and reduces total cellular glutathione (GSH) shortly thereafter. This is followed by onset of apoptotic cell death. These responses to Ethanol can be blocked by elevating neuron GSH with N-acetylcysteine or by co-culturing neurons with neonatal cortical astrocytes. We describe here mechanisms by which the astrocyte-neuron gamma-glutamyl cycle is up-regulated by Ethanol, enhancing control of neuron GSH in response to the pro-oxidant, Ethanol. Up to 6 days of Ethanol exposure had no consistent effects on activities of gamma-glutamyl cysteine ligase or glutathione synthetase, and GSH content remained unchanged (p < 0.05). However, glutathione reductase was increased with 1 and 2 day Ethanol exposures, 25% and 39% for 2.5 and 4.0 mg/mL Ethanol by 1 day, and 11% and 16% for 2.5 and 4.0 mg/mL at 2 days, respectively (p < 0.05). A 24 h exposure to 4.0 mg/mL Ethanol increased GSH efflux from astrocyte up to 517% (p < 0.05). Ethanol increased both gamma-glutamyl transpeptidase expression and activity on astrocyte within 24 h of exposure (40%, p = 0.05 with 4.0 mg/mL) and this continued for at least 4 days of Ethanol treatment. Aminopeptidase N activity on neurons increased by 62% and 55% within 1 h of Ethanol for 2.5 and 4.0 mg/mL concentration, respectively (p < 0.05), remaining elevated for 24 h of treatment. Thus, there are at least three key points of the gamma-glutamyl cycle that are up-regulated by Ethanol, the net effect being to enhance neuron GSH homeostasis, thereby protecting neurons from Ethanol-mediated oxidative stress and apoptotic death.     

Active metabolites from Dunalia spinosa resinous exudates

Dunalia spinosa, a plant used in folk medicine for toothaches, breathing problems and cleansing wounds, was found active as antimicrobial and antioxidant. A new (E)-aurone rutinoside (dunaurone) has been isolated from the aerial parts of the plant, and its structure was determined by spectroscopic means. Lupeol, beta-sitosterol, scopoletin, quercetin and withaferin A were also found. All the extracts exhibited strong antimicrobial activity while dunaurone showed only weak antimicrobial inhibition against Klebsiella pneumoniae; in addition it presented a significant free radical scavenging activity.     

Attenuation of oxalate-induced nephrotoxicity by eicosapentaenoate-lipoate (EPA-LA) derivative in experimental rat model

Hyperoxaluria is one of the major risk factors for the formation of urinary calcium oxalate stones. Calcium oxalate crystals and their deposition have been implicated in inducing renal tubular damage. Lipoic acid (LA) and eicosapentaenoic acid (EPA) have been shown to ameliorate the changes associated with hyperoxaluria. This prompted us to investigate the nephroprotectant role of EPA-LA, a new derivative, in vivo in hyperoxaluric rats. Elevation in the levels of calcium, oxalate and phosphorus, the stone-forming constituents, were observed in calculogenic rats as a manifestation of crystal deposition.Tubular damage to the renal tissue was assessed byassaying the excretion of marker enzymes in the urine. Damage to the tubules was indicated by increased excretion of alkaline phosphatase (ALP), lactate dehydrogenase (LDH), gamma-glutamyl transferase (gamma-GT), beta-Glucuronidase (beta-GLU) and N-Acetyl beta-D glucosaminidase (NAG). Fibrinolytic activity was found to be reduced. Administration of EPA, LA and EPA-LA reduced the tubular damage and decreased the markers of crystal deposition markedly, which was substantiated by the reduction in weight of bladder stone formed. Our results highlight that EPA-LA is the most effective drug in inhibiting stone formation and mitigating renal damage caused by oxalate toxicity, thus confirming it as a nephroprotectant. Further work in this direction is warranted to establish the therapeutic effectiveness of this new derivative.     

Expression of a cry2Aa gene in an acrystalliferous Bacillus thuringiensis strain and toxicity of Cry2Aa against Helicoverpa armigera

In the recent past research has been mainly focused on the expression of cry1 genes of Bacillus thuringiensis (Bt) to engineer lepidopteran insect resistance in plants. Search for structurally different toxins is necessary for the management of resistance development in insects. The intact cry2Aa operon (3.95 kb) of a new isolate of Bt, 47-8, was subcloned into a Bt shuttle vector, pHT3101 (6.7 kb). Recombinant pHT3101 containing the cry2Aa operon of Bt strain 47-8 was named as pTN2Aa and used to transform acrystalliferous Bt strain 4Q7 by electroporation. Phase contrast microscopic observation revealed the presence of crystalline inclusions in the transformants of Bt strain 4Q7 harbouring pTN2Aa. SDS–PAGE of a spore–crystal mixture prepared from transformants of acrystalliferous Bt strain 4Q7 harbouring pTN2Aa showed a single band of about 65 kDa alone confirming the expression of the cloned cry2Aa. Bioassay with Helicoverpa armigera showed 71.4% mortality caused by the proteins encoded by the newly cloned cry2Aa gene (at the concentration of 2.3 g/l) on the seventh day and all the survivors that escaped from Cry2Aa toxicity showed severe (81–99%) inhibition in larval growth.     

A new cucurbitacin glycoside from Kageneckia oblonga (Rosaceae)

A novel cucurbitacin glycoside has been isolated from aerial parts of Kageneckia oblonga R. et P. and shown to be 3beta-(beta-D-glucosyloxy)-16alpha,23alpha-epoxycuc urbita-5,24-dien-11-one. The structure was established by usual spectroscopic and two-dimensional (2D) NMR techniques. This compound has found to be nontoxic when tested in-vivo cell culture assays. In previous investigations we reported 23,24-dihydrocucurbitacin F and prunasine. This was the first report on cucurbitacins from the genus Kageneckia (Rosaceae).     

A novel genetic engineering platform for the effective management of biological contaminants for the production of microalgae

Microalgal cultivation that takes advantage of solar energy is one of the most cost‐effective systems for the biotechnological production of biofuels, and a range of high value products, including pharmaceuticals, fertilizers and feed. However, one of the main constraints for the cultivation of microalgae is the potential contamination with biological pollutants, such as bacteria, fungi, zooplankton or other undesirable microalgae. In closed bioreactors, the control of contamination requires the sterilization of the media, containers and all materials, which increases the cost of production, whereas open pond systems severely limits the number of species that can be cultivated under extreme environmental conditions to prevent contaminations. Here, we report the metabolic engineering of Chlamydomonas reinhardtii to use phosphite as its sole phosphorus source by expressing the ptxD gene from Pseudomonas stutzeri WM88, which encodes a phosphite oxidoreductase able to oxidize phosphite into phosphate using NAD as a cofactor. Engineered C. reinhardtii lines are capable of becoming the dominant species in a mixed culture when fertilized with phosphite as a sole phosphorus source. Our results represent a new platform for the production of microalgae, potentially useful for both closed photobioreactors and open pond systems without the need for using sterile conditions nor antibiotics or herbicides to prevent contamination with biological pollutants.     

High-mass-resolution MALDI mass spectrometry imaging reveals detailed spatial distribution of metabolites and lipids in roots of barley seedlings in response to salinity stress

Introduction

Mass spectrometry imaging (MSI) is a technology that enables the visualization of the spatial distribution of hundreds to thousands of metabolites in the same tissue section simultaneously. Roots are below-ground plant organs that anchor plants to the soil, take up water and nutrients, and sense and respond to external stresses. Physiological responses to salinity are multifaceted and have predominantly been studied using whole plant tissues that cannot resolve plant salinity responses spatially.

Objectives

This study aimed to use a comprehensive approach to study the spatial distribution and profiles of metabolites, and to quantify the changes in the elemental content in young developing barley seminal roots before and after salinity stress.

Methods

Here, we used a combination of liquid chromatography–mass spectrometry (LC–MS), inductively coupled plasma mass spectrometry (ICP–MS), and matrix-assisted laser desorption/ionization (MALDI–MSI) platforms to profile and analyze the spatial distribution of ions, metabolites and lipids across three anatomically different barley root zones before and after a short-term salinity stress (150 mM NaCl).

Results

We localized, visualized and discriminated compounds in fine detail along longitudinal root sections and compared ion, metabolite, and lipid composition before and after salt stress. Large changes in the phosphatidylcholine (PC) profiles were observed as a response to salt stress with PC 34:n showing an overall reduction in salt treated roots. ICP–MS analysis quantified changes in the elemental content of roots with increases of Na⁺ and decreases of K⁺ content.

Conclusion

Our results established the suitability of combining three mass spectrometry platforms to analyze and map ionic and metabolic responses to salinity stress in plant roots and to elucidate tolerance mechanisms in response to abiotic stress, such as salinity stress.

     

CD4+ T cell immunity to Salmonella is transient in the circulation

While Salmonella enterica is seen as an archetypal facultative intracellular bacterial pathogen where protection is mediated by CD4⁺ T cells, identifying circulating protective cells has proved very difficult, inhibiting steps to identify key antigen specificities. Exploiting a mouse model of vaccination, we show that the spleens of C57BL/6 mice vaccinated with live-attenuated Salmonella serovar Typhimurium (S. Typhimurium) strains carried a pool of IFN-γ⁺ CD4⁺ T cells that could adoptively transfer protection, but only transiently. Circulating Salmonella-reactive CD4⁺ T cells expressed the liver-homing chemokine receptor CXCR6, accumulated over time in the liver and assumed phenotypic characteristics associated with tissue-associated T cells. Liver memory CD4⁺ T cells showed TCR selection bias and their accumulation in the liver could be inhibited by blocking CXCL16. These data showed that the circulation of CD4⁺ T cells mediating immunity to Salmonella is limited to a brief window after which Salmonella-specific CD4⁺ T cells migrate to peripheral tissues. Our observations highlight the importance of triggering tissue-specific immunity against systemic infections.