Phosphatidic acid-mediated binding and mammalian cell internalization of the Vibrio cholerae cytotoxin MakA

Vibrio cholerae is a noninvasive intestinal pathogen extensively studied as the causative agent of the human disease cholera. Our recent work identified MakA as a potent virulence factor of V. cholerae in both Caenorhabditis elegans and zebrafish, prompting us to investigate the potential contribution of MakA to pathogenesis also in mammalian hosts. In this study, we demonstrate that the MakA protein could induce autophagy and cytotoxicity of target cells. In addition, we observed that phosphatidic acid (PA)-mediated MakA-binding to the host cell plasma membranes promoted macropinocytosis resulting in the formation of an endomembrane-rich aggregate and vacuolation in intoxicated cells that lead to induction of autophagy and dysfunction of intracellular organelles. Moreover, we functionally characterized the molecular basis of the MakA interaction with PA and identified that the N-terminal domain of MakA is required for its binding to PA and thereby for cell toxicity. Furthermore, we observed that the ΔmakA mutant outcompeted the wild-type V. cholerae strain A1552 in the adult mouse infection model. Based on the findings revealing mechanistic insights into the dynamic process of MakA-induced autophagy and cytotoxicity we discuss the potential role played by the MakA protein during late stages of cholera infection as an anti-colonization factor.     

Recent advances in bio‐chemical,molecular and physiological aspects of membrane lipid derivatives in plant pathology

Plant pathogens pose a significant threat to the food industry and food security accounting for 10–40% crop losses annually on a global scale. Economic losses from plant diseases are estimated at $300B for major food crops and are associated with reduced food availability and accessibility and also high food costs. Although strategies exist to reduce the impact of diseases in plants, many of these introduce harmful chemicals to our food chain. Therefore, it is important to understand and utilize plants' immune systems to control plant pathogens to enable more sustainable agriculture. Lipids are core components of cell membranes and as such are part of the first line of defense against pathogen attack. Recent developments in omics technologies have advanced our understanding of how plant membrane lipid biosynthesis, remodelling and/or signalling modulate plant responses to infection. Currently, there is limited information available in the scientific literature concerning lipid signalling targets and their biochemical and physiological consequences in response to plant pathogens. This review focusses on the functions of membrane lipid derivatives and their involvement in plant responses to pathogens as biotic stressors. We describe major plant defense systems including systemic‐acquired resistance, basal resistance, hypersensitivity and the gene‐for‐gene concept in this context.     

Human colon tissue in organ culture: preservation of normal and neoplastic characteristics

Normal and neoplastic human colon tissue obtained at surgery was used to establish conditions for organ culture. Optimal conditions included an atmosphere of 5% CO₂ and 95% O₂; tissue partially submerged with mucosa at the gas interface; and serum-free medium with 1.5 mM Ca²⁺ and a number of growth supplements. Histological, histochemical, and immunohistochemical features that distinguish normal and neoplastic tissue were preserved over a 2-d period. With normal tissue, this included the presence of elongated crypts with small, densely packed cells at the crypt base and mucin-containing goblet cells in the upper portion. Ki67 staining, for proliferating cells, was confined to the lower third of the crypt, while expression of extracellular calcium-sensing receptor was seen in the upper third and surface epithelium. E-cadherin and β-catenin were expressed throughout the epithelium and confined to the cell surface. In tumor tissue, the same disorganized, abnormal glandular structures seen at time zero were present after 2 d. The majority of cells in these structures were mucin-poor, but occasional goblet cells were seen and mucin staining was present. Ki67 staining was seen throughout the abnormal epithelium and calcium-sensing receptor expression was weak and variable. E-cadherin was seen at the cell surface (similar to normal tissue), but in some places, there was diffuse cytoplasmic staining. Finally, intense cytoplasmic and nuclear β-catenin staining was observed in cultured neoplastic tissue.     

Towards thermally stable cyclophanediene-dihydropyrene photoswitches

Cyclophanediene dihydropyrenes (CPD-DHP) are photochromic compounds because they change their color by irradiation with lights of different color. Potential use of CPD-DHP photoswitch in memory devices requires a very slow thermal return in the dark in the absence of any side reaction. Herein, thermal return of CPDs to DHPs, and an unwanted sigmatropic shift in DHP is studied through density functional theory calculations at (U)B3LYP/6-31+G(d). The thermal return occurs through symmetry forbidden conrotatory electrocyclic reaction. Dimethyl amino CPD-DHP photoswitch pair has the highest activation barrier for electrocyclization and sigmatropic shifts. The lowest activation barrier for symmetry forbidden electrocyclization is observed for GeBr₃ functionalized CPD. An unprecedented decomposition pathway involving elimination of the internal substituents is predicted for Cl, Br and SMe functionalized DHPs. This study shows great promise in understanding the Woodward Hoffmann forbidden processes and, in reducing the synthetic efforts toward robust photochromes for memory applications.     

The effects of Efaproxyn (efaproxiral) on subcutaneous RIF-1 tumor oxygenation and enhancement of radiotherapy-mediated inhibition of tumor growth in mice

Efaproxiral, an allosteric modifier of hemoglobin, reduces hemoglobin-oxygen binding affinity, facilitating oxygen release from hemoglobin, which is likely to increase tissue pO(2). The purpose of this study was to determine the effect of efaproxiral on tumor oxygenation and growth inhibition of RIF-1 tumors that received X radiation (4 Gy) plus oxygen breathing compared to radiation plus oxygen plus efaproxiral daily for 5 days. Two lithium phthalocyanine (LiPc) deposits were implanted in RIF-1 tumors in C3H mice for tumor pO(2) measurements using EPR oximetry. Efaproxiral significantly increased tumor oxygenation by 8.4 to 43.4 mmHg within 5 days, with maximum increases at 22-31 min after treatment. Oxygen breathing alone did not affect tumor pO(2). Radiation plus oxygen plus efaproxiral produced tumor growth inhibition throughout the treatment duration, and inhibition was significantly different from radiation plus oxygen from day 3 to day 5. The results of this study provide unambiguous quantitative information on the effectiveness of efaproxiral to consistently and reproducibly increase tumor oxygenation over the course of 5 days of treatment, modeling the clinical use of efaproxiral. Also, based on the tumor growth inhibition, the study shows the efaproxiral-enhanced tumor oxygenation was radiobiologically significant. This is the first study to demonstrate the ability of efaproxiral to increase tumor oxygenation and to increase the tumor growth inhibition of radiotherapy over 5 days of treatment.     

Prion protein gene polymorphisms in four goat breeds of Pakistan

Four different goat breeds (Pak-Angora, Dera Din Panah, Naachi and Teddy) of Pakistan were selected to investigate polymorphism in the prion protein gene (PrP gene) responsible for scrapie disease resistance in goats. Initially, genotyping of 187 animals of these four breeds by restriction fragment length polymorphism (RFLP) was done to see the genotype for codon 136 and 154. All the animals were monomorphic with a genotype of AARR except one animal of Teddy breed having the genotype of AARH. Sequencing of PrP gene of twenty animals representing these four goat breeds revealed two genotypes PPSSSS and PPSSPS with haplotypes PSS and PSP of PrP gene at the codon numbers 42, 138, and 240. All four breeds showed both wild type monomorphic sequence and mutant polymorphic sequences of these codons. The mutants of 42 and 138 codons translate the same amino acids as with the wild type sequences, while the mutant of codon 240 is responsible for a different amino acid translation i.e., serine to proline. In short, this study provides preliminary information about alleles and genotypes of PrP gene in four goat breeds of Pakistan.     

Molecular responses of Campylobacter jejuni to cadmium stress

Cadmium ions are a potent carcinogen in animals, and cadmium is a toxic metal of significant environmental importance for humans. Response curves were used to investigate the effects of cadmium chloride on the growth of Camplyobacter jejuni. In vitro, the bacterium showed reduced growth in the presence of 0.1 mm cadmium chloride, and the metal ions were lethal at 1 mm concentration. Two-dimensional gel electrophoresis combined with tandem mass spectrometry analysis enabled identification of 67 proteins differentially expressed in cells grown without and with 0.1 mm cadmium chloride. Cellular processes and pathways regulated under cadmium stress included fatty acid biosynthesis, protein biosynthesis, chemotaxis and mobility, the tricarboxylic acid cycle, protein modification, redox processes and the heat-shock response. Disulfide reductases and their substrates play many roles in cellular processes, including protection against reactive oxygen species and detoxification of xenobiotics, such as cadmium. The effects of cadmium on thioredoxin reductase and disulfide reductases using glutathione as a substrate were studied in bacterial lysates by spectrophotometry and nuclear magnetic resonance spectroscopy, respectively. The presence of 0.1 mm cadmium ions modulated the activities of both enzymes. The interactions of cadmium ions with oxidized glutathione and reduced glutathione were investigated using nuclear magnetic resonance spectroscopy. The data suggested that, unlike other organisms, C. jejuni downregulates thioredoxin reductase and upregulates other disulfide reductases involved in metal detoxification in the presence of cadmium.     

Microbial ACC-Deaminase: Prospects and Applications for Inducing Salt Tolerance in Plants

Salinity is one of the most important stresses that hamper agricultural productivity in nearly every part of the world. Enhanced biosynthesis of ethylene in plants under salinity stress is well established. Higher ethylene concentration inhibits root growth and ultimately affects the overall plant growth. Overcoming this ethylene-induced root inhibition is a prerequisite for successful crop production. Recent studies have shown that ethylene level in plants is regulated by a key enzyme 1-aminocyclopropane-1-carboxylicacid (ACC)-deaminase. This enzyme is present in plant growth-promoting bacteria (PGPR) and lowers the ethylene level by metabolizing its precursor ACC into α-ketobutyrate and ammonia (NH₃). Inoculation of plants under salinity stress with PGPR having ACC-deaminase activity mitigates the inhibitory effects of salinity on root growth by lowering the ethylene concentration in the plant. This in turn results in prolific root growth, which is beneficial for the uptake of nutrients and maintenance of growth under stressful environment. The present review critically discusses the effects of salinity stress on plant growth with special reference to ethylene production and the effects of rhizobacteria containing ACC-deaminase on crop improvement under salinity stress. It also discusses how much progress has been made in producing transgenic lines of different crops over-expressing the gene encoding ACC-deaminase and how far such transformed lines can tolerate salinity stress.     

A triterpenoidal saponin and sphingolipids from Pteleopsis hylodendron

From the stem bark of Pteleopsis hylodendron, a triterpenoidal saponin bellericagenin [B 3-O-[beta-D-glucopyranosyl-(1-->2)-alpha-D-glucopyranoside] (1) (Pteleopsoside)] and two sphingolipids, hylodendroside-I (2), and hylodendroside-II (3) were isolated, along with a synthetically known compound, [2alpha, 3beta, 23-triacetoxy-19alpha-hydroxyolean-12-en-28-oic acid (4)]. Other known compounds, friedelin (5), beta-carotene (6), lupeol (7), sitosterol (8), and stigmasterol (9), were also obtained. Their structures were deduced with the help of detailed spectroscopic studies.