Evaluation of the relative cell surface charge by using microbial adhesion to hydrocarbon

A simple and rapid method, Microbial adhesion to hexadecane, for estimating the cell surface charge is proposed. This method is based on the determination of cell affinity to hexadecane at low ionic strength and at high ionic strength. The difference between these two affinities can provide the relative cell surface charge. The application of this method for Staphylococcus aureus and Escherichia coli show that the profile of surface charge evolution as a function of pH was similar to these obtained by microelectrophoresis method.     

Angiotensin II‐induced redox‐sensitive SGLT1 and 2 expression promotes high glucose‐induced endothelial cell senescence

High glucose (HG)‐induced endothelial senescence and dysfunction contribute to the increased cardiovascular risk in diabetes. Empagliflozin, a selective sodium glucose co‐transporter2 (SGLT2) inhibitor, reduced the risk of cardiovascular mortality in type 2 diabetic patients but the protective mechanism remains unclear. This study examines the role of SGLT2 in HG‐induced endothelial senescence and dysfunction. Porcine coronary artery cultured endothelial cells (ECs) or segments were exposed to HG (25 mmol/L) before determination of senescence‐associated beta‐galactosidase activity, protein level by Western blot and immunofluorescence staining, mRNA by RT‐PCR, nitric oxide (NO) by electron paramagnetic resonance, oxidative stress using dihydroethidium and glucose uptake using 2‐NBD‐glucose. HG increased ECs senescence markers and oxidative stress, down‐regulated eNOS expression and NO formation, and induced the expression of VCAM‐1, tissue factor, and the local angiotensin system, all these effects were prevented by empagliflozin. Empagliflozin and LX‐4211 (dual SGLT1/2 inhibitor) reduced glucose uptake stimulated by HG and H₂O₂ in ECs. HG increased SGLT1 and 2 protein levels in cultured ECs and native endothelium. Inhibition of the angiotensin system prevented HG‐induced ECs senescence and SGLT1 and 2 expression. Thus, HG‐induced ECs ageing is driven by the local angiotensin system via the redox‐sensitive up‐regulation of SGLT1 and 2, and, in turn, enhanced glucotoxicity.     

Significance of neutrophil microparticles in ischaemia‐reperfusion: Pro‐inflammatory effectors of endothelial senescence and vascular dysfunction

Endothelial senescence is an emerging cause of vascular dysfunction. Because microparticles are effectors of endothelial inflammation and vascular injury after ischaemia‐reperfusion, we examined leucocyte‐derived microparticles of spleen origin as possible contributors. Microparticles were generated from primary rat splenocytes by either lipopolysaccharide or phorbol‐myristate‐acetate/calcium ionophore, under conditions mimicking innate and adaptive immune responses. Incubation of primary porcine coronary endothelial cells with either type of microparticles, but not with those from unstimulated splenocytes, leads to a similar threefold raise in senescence‐associated β‐galactosidase activity within 48 hours, indicating accelerated senescence, to endothelial oxidative stress, and a fivefold and threefold increase in p21 and p16 senescence markers after 24 hours. After 12‐hour incubation, the endothelial‐dependent relaxation of coronary artery rings was reduced by 50%, at distinct optimal microparticle concentration. In vitro, microparticles were pro‐thrombotic by up‐regulating the local angiotensin system, by prompting tissue factor activity and a secondary generation of pro‐coagulant endothelial microparticles. They initiated an early pro‐inflammatory response by inducing phosphorylation of NF‐κB, MAP kinases and Akt after 1 hour, and up‐regulated VCAM‐1 and ICAM‐1 at 24 hours. Accordingly, VCAM‐1 and COX‐2 were also up‐regulated in the coronary artery endothelium and eNOS down‐regulated. Lipopolysaccharide specifically favoured the shedding of neutrophil‐ and monocyte‐derived microparticles. A 80% immuno‐depletion of neutrophil microparticles reduced endothelial senescence by 55%, indicating a key role. Altogether, data suggest that microparticles from activated splenocytes prompt early pro‐inflammatory, pro‐coagulant and pro‐senescent responses in endothelial cells through redox‐sensitive pathways. The control of neutrophil shedding could preserve the endothelium at site of ischaemia‐reperfusion–driven inflammation and delay its dysfunction.     

Evaluation of stem aqueous extract and synthesized silver nanoparticles using Cissus quadrangularis against Hippobosca maculata and Rhipicephalus (Boophilus) microplus

The present study was to determine the efficacies of anti-parasitic activities of synthesized silver nanoparticles (Ag NPs) using stem aqueous extract of Cissus quadrangularis against the adult of hematophagous fly, Hippobosca maculata (Diptera: Hippoboscidae), and the larvae of cattle tick, Rhipicephalus (Boophilus) microplus (Acari: Ixodidae). Contact toxicity method was followed to determine the potential of parasitic activity. Twelve milliliters of stem aqueous extract of C. quadrangularis was treated with 88ml of 1mM silver nitrate (AgNO(3)) solution at room temperature for 30min and the resulting solution was yellow-brown color indicating the formation extracellular synthesis of Ag NPs. The synthesized Ag NPs were characterized with UV-visible spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), Field emission scanning electron microscope (FESEM) and energy dispersive X-ray (EDX) spectroscopy. The synthesized Ag NPs were recorded by UV-visible spectrum at 420nm and XRD patterns showed the nanoparticles crystalline in nature. FTIR analysis confirmed that the bioreduction of Ag((+)) ions to Ag NPs were due to the reduction by capping material of plant extract. FESEM image of Ag NPs showed spherical and oval in shape. By using the Bragg's Law and Scherrer's constant, the average mean size of synthesized Ag NPs was 42.46nm. The spot EDX analysis showed the complete chemical composition of the synthesized Ag NPs. The mortality obtained by the synthesized Ag NPs from the C. quadrangularis was more effective than the aqueous extract of C. quadrangularis and AgNO(3) solution (1mM). The adulticidal activity was observed in the aqueous extract, AgNO(3) solution and synthesized Ag NPs against the adult of H. maculata with LC(50) values of 37.08, 40.35 and 6.30mg/L; LC(90) values of 175.46, 192.17 and 18.14mg/L and r(2) values of 0.970, 0.992 and 0.969, respectively. The maximum efficacy showed in the aqueous extract, AgNO(3) solution and synthesized Ag NPs against the larvae of R. (B.) microplus with LC(50) values of 50.00, 21.72 and 7.61mg/L; LC(90) values of 205.12, 82.99 and 22.68mg/L and r(2) values of 0.968, 0.945and 0.994, respectively. The present study is the first report on antiparasitic activity of the experimental plant extract and synthesized Ag NPs. This is an ideal eco-friendly and inexpensive approach for the control of H. maculata and R. (B.) microplus.     

Comparative effectiveness of <Emphasis Type="Italic">Pseudomonas</Emphasis> and <Emphasis Type="Italic">Serratia</Emphasis> sp. containing ACC-deaminase for improving growth and yield of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) under salt-stressed conditions

Ethylene synthesis is accelerated in response to various environmental stresses like salinity. Ten rhizobacterial strains isolated from wheat rhizosphere taken from different salt affected areas were screened for growth promotion of wheat under axenic conditions at 1, 5, 10 and 15 dS m⁻¹. Three strains, i.e., Pseudomonas putida (N21), Pseudomonas aeruginosa (N39) and Serratia proteamaculans (M35) showing promising performance under axenic conditions were selected for a pot trial at 1.63 (original), 5, 10 and 15 dS m⁻¹. Results showed that inoculation was effective even in the presence of higher salinity levels. P. putida was the most efficient strain compared to the other strains and significantly increased the plant height, root length, grain yield, 100-grain weight and straw yield up to 52, 60, 76, 19 and 67%, respectively, over uninoculated control at 15 dS m⁻¹. Similarly, chlorophyll content and K⁺/Na⁺ of leaves also increased by P. putida over control. It is highly likely that under salinity stress, 1-aminocyclopropane-1-carboxylic acid-deaminase activity of these microbial strains might have caused reduction in the synthesis of stress (salt)-induced inhibitory levels of ethylene. The results suggested that these strains could be employed for salinity tolerance in wheat; however, P. putida may have better prospects in stress alleviation/reduction.     

Effect of plant growth promoting rhizobacteria containing ACC-deaminase on maize (Zea mays L.) growth under axenic conditions and on nodulation in mung bean (Vigna radiata L.)

AIMS: This study was conducted to test the hypothesis that the bacterial strains possessing 1-aminocyclopropane-1-carboxylic acid (ACC)-deaminase activity may also promote growth of inoculated plants and could increase nodulation in legumes upon co-inoculation with rhizobia. METHODS AND RESULTS: Several rhizobacteria were isolated from maize rhizosphere through enrichment on ACC as a sole N source. Purified isolates were screened for growth promotion in maize under axenic conditions and for in vitro ACC-deaminase activity. A significant positive correlation was observed between in vitro ACC-deaminase activity of bacterial cells and root elongation. None of the isolates produced auxins. Bradyrhizobium japonicum produced less amount of auxins but did not carry ACC-deaminase activity. Results of pot experiment revealed that co-inoculation with Bradyrhizobium and plant growth promoting rhizobacteria (PGPR) isolates enhanced the nodulation in mung bean compared with inoculation with Bradyrhizobium alone. CONCLUSIONS: It is highly expected that inoculation with rhizobacteria containing ACC-deaminase hydrolysed endogenous ACC into ammonia and alpha-ketobutyrate instead of ethylene. Consequently, root and shoot growth as well as nodulation were promoted. SIGNIFICANCE AND IMPACT OF THE STUDY: The ACC-deaminase trait could be employed as an efficient tool to screen effective PGPR, which could be successfully used as biofertilizers to increase the growth of inoculated plants as well as nodulation in legumes.     

Fertilizer-dependent efficiency of Pseudomonads for improving growth,yield, and nutrient use efficiency of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Acquisition of nutrients by plants is primarily dependent on root growth and bioavailability of nutrients in the rooting medium. Most of the beneficial bacteria enhance root growth, but their effectiveness could be influenced by the nutrient status around the roots. In this study, two 1-aminocyclopropane-1-carboxylate (ACC)-deaminase containing plant-growth-promoting rhizobacteria (PGPR), Pseudomonas fluorescens and P. fluorescens biotype F were tested for their effect on growth, yield, and nutrient use efficiency of wheat under simultaneously varying levels of all the three major nutrients N, P, and K (at 0%, 25%, 50%, 75%, and 100% of recommended doses). Results of pot and field trials revealed that the efficacy of these strains for improving growth and yield of wheat reduced with the increasing rates of NPK added to the soil. In most of the cases, significant negative linear correlations were recorded between percentage increases in growth and yield parameters of wheat caused by inoculation and increasing levels of applied NPK fertilizers. It is highly likely that under low fertilizer application, the ACC-deaminase activity of PGPR might have caused reduction in the synthesis of stress (nutrient)-induced inhibitory levels of ethylene in the roots through ACC hydrolysis into NH₃ and α-ketobutyrate. The results of this study imply that these Pseudomonads could be employed in combination with appropriate doses of fertilizers for better plant growth and savings of fertilizers.     

Phytoremediation of Light Crude Oil by Maize (Zea mays L.) Bio-Augmented with Plant Growth Promoting Bacteria

The aim of this study was to evaluate the converged effect of maize and plant growth promoting bacteria on degradation of petroleum hydrocarbons under axenic conditions. Artificially spiked sand with 10 g kg^?1 light crude oil was planted with maize alone and in combination with eight bacterial isolates having plant growth promotion and bioremediation potential to observe the dissipation of petroleum hydrocarbons. Results showed remarkable suppression of maize growth and biomass production due to phytotoxicity of the crude oil contamination. However, bio-augmentation of plants with bacteria having ACC-deaminase activity significantly compensated the reduction in plant growth compared to uninoculated plants. The results revealed that plants bio-augmented with PM32Y exhibited significant increase in root length (75%), plant height (74%), and biomass (67%) as compared to uninoculated plants after 60 days of planting. The same bacterium in convergence with maize caused 43% degradation of petroleum hydrocarbons as compared to the unplanted and uninoculated control. Amplification, sequencing and phylogenetic analysis of 16S rRNA gene sequence identified PM32Y bacterium as Bacillus subtilis strain. It is concluded that bio-augmentation of plants with plant growth promoting bacteria having bioremediation potential and ACC-deaminase activity can successfully be used in phytoremediation of petroleum hydrocarbons.     

Autocrine laminin-5 ligates alpha6beta4 integrin and activates RAC and NFkappaB to mediate anchorage-independent survival of mammary tumors

Invasive carcinomas survive and evade apoptosis despite the absence of an exogenous basement membrane. How epithelial tumors acquire anchorage independence for survival remains poorly defined. Epithelial tumors often secrete abundant amounts of the extracellular matrix protein laminin 5 (LM-5) and frequently express alpha6beta4 integrin. Here, we show that autocrine LM-5 mediates anchorage-independent survival in breast tumors through ligation of a wild-type, but not a cytoplasmic tail-truncated alpha6beta4 integrin. alpha6beta4 integrin does not mediate tumor survival through activation of ERK or AKT. Instead, the cytoplasmic tail of beta4 integrin is necessary for basal and epidermal growth factor-induced RAC activity, and RAC mediates tumor survival. Indeed, a constitutively active RAC sustains the viability of mammary tumors lacking functional beta1 and beta4 integrin through activation of NFkappaB, and overexpression of NFkappaB p65 mediates anchorage-independent survival of nonmalignant mammary epithelial cells. Therefore, epithelial tumors could survive in the absence of exogenous basement membrane through autocrine LM-5-alpha6beta4 integrin-RAC-NFkappaB signaling.