Elucidating cell-penetrating peptide mechanisms of action for membrane interaction,cellular uptake,and translocation utilizing the hydrophobic counter-anion pyrenebutyrate

Cell-penetrating peptides (CPPs) are membrane permeable vectors recognized for their intrinsic ability to gain access to the cell interior. The hydrophobic counter-anion, pyrenebutyrate, enhances cellular uptake of oligoarginine CPPs. To elucidate CPP uptake mechanisms, the effect of pyrenebutyrate on well-recognized CPPs with varying hydrophobicity and arginine content is investigated. The cellular CPP uptake and CPP-mediated oligonucleotide delivery is analyzed by fluorescence activated cell sorting, confocal microscopy, and a cell-based splice-switching assay. The splice-switching oligonucleotide is a mixmer of 2′-O-methyl RNA and locked nucleic acids delivered as a non-covalent complex with 10-fold molar CPP excess. CPP-induced membrane perturbation on large unilamellar vesicles is investigated in calcein release experiments. We observed that pyrenebutyrate facilitates cellular uptake and translocation of oligonucleotide mediated by oligoarginine nonamer while limited effect of pyrenebutyrate on more hydrophobic CPPs was observed. By combining the different experimental results we conclude that the pathway for cellular uptake of oligoarginine is dominated by direct membrane translocation, whereas the pathway for oligoarginine-mediated oligonucleotide translocation is dominated by endocytosis. Both mechanisms are promoted by pyrenebutyrate and we suggest that pyrenebutyrate has different sites of action for the two uptake and translocation mechanisms.     

Norepinephrine-dependently released Dr fimbriae of diffusely adhering Escherichia coli strain IH11128 promotes a mitogen-activated protein kinase ERK1/2-dependent production of pro-inflammatory cytokine, IL-8 in human intestinal Caco-2/TC7 cells

The diffusely adhering Escherichia coli (Afa/Dr DAEC) are associated with recurrent urinary tract infections in adults as well as with diarrheal disease in infants. We previously demonstrated that in wild-type strain IH11128, the Dr fimbriae is released in the extracellular medium in response to multiple environmental signals such as temperature, low aeration and rich medium. A number of molecules of eukaryotic origin, such as catecholamines, have been reported to stimulate bacterial growth and virulence factor production. We show that norepinephrine affects the production and release of Dr fimbriae in Afa/Dr DAEC WT-IH11128 bacteria. The regulatory mechanism involved with norepinephrine-induced Dr fimbriae liberation was apparently due to a differential induction of genes draC, encoding the usher, and draE, encoding the major fimbrial subunit. In addition, we show that the released Dr fimbriae induces the phosphorylation of the mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2 (ERK1/2) and the production of the pro-inflammatory cytokine, IL-8 in fully differentiated cultured human intestinal Caco-2/TC7 cells.     

Impact of mineral fertilizers on mineral nutrients in the ginger rhizome and on soil enzymes activities and soil properties

Ginger is used as one of the important ingredients in traditional as well as modern medicine besides as a spice. It boosts immunity and is a rich source of many biologically active substances and minerals. Although it is a medicinally important crop, its productivity is, however, affected due to poor nutrient management and therefore it requires an adequate supply of nutrients in the form of inorganic fertilizers or organic manuring, or a mixture of both. In this context, the present study was aimed to investigate the effect of mineral fertilizers on the content of mineral elements in the ginger rhizome, on soil enzyme activity, and soil properties. Lysimeter experiments were conducted at the Institute of Genetics and Plant Experimental Biology, Kibray, Tashkent region, Uzbekistan. The experiment comprised of four treatments T1 – Control, T2 - N₇₅P₅₀K₅₀ kg/ha, T3 - and T4 - N₁₀₀P₇₅K₇₅ + B₃Zn₆Fe₆ kg/ha. The results showed that the application of N₁₂₅P₁₀₀K₁₀₀ kg/ha increased rhizome K content by 49%, P content by 20%, and Na content by 58% as compared to control without fertilizer. While the application of N₁₀₀P₇₅K₇₅ + B₃Zn₆Fe₆ kg/ha showed a significant enhancement in rhizome K, Ca, P, Mg, Na, Fe, Mn, Zn, Cu, Cr, Mo, and Si contents over the control. This treatment also improved active P content by 29%, total P content by 80%, total K content 16%, and N content by 33% content, and the activities of urease, invertase, and catalase activities as compared to control of without mineral fertilizer and control respectively. Thus the application of NPK + BZnFe at the rate of 100:75:75:3:6:6 kg/ha helps in improving macroelements and microelements in the ginger rhizome and activities of soil enzymes that helps in mineral nutrition of the rhizome.     

Leaf temperature of desert sand dune plants: perspectives on the adaptability of leaf morphology

The leaf temperature of six annual and six perennial plant species was monitored during spring and summer on a sand dune ecosystem in the delta Mediterranean coast of Egypt. During winter, leaves of all tested perennial species attained temperatures higher than the air temperature at night and shortly after sunrise, with maximum leaf–air temperature differences reaching up to 8°C. The lowest differences were less than 1°C. Around noon, the leaves of several species attained temperatures lower than that of the air whereas others showed higher temperatures. The opposite was true during summer, when leaf temperatures were lower than air temperature. The maximum leaf–air temperature differences occurred after midnight towards sunrise and reached up to 10°C. The lowest differences were found around noon and were of less than 5°C. The annual plant species have more pronounced variations than perennials in their leaf temperatures during the night and for most of the day. The leaves were heated or cooled a few degrees above or below the air temperature. The results are discussed in relation to the morphological characters of the leaves. The variation in leaf temperature at different times of the day was significantly related to leaf morphology, specific leaf area, thickness, volume, leaf area index and the surrounding environment.     

Incorporation of microalgae sterols by scallop Pecten maximus (L.) larvae

Changes in sterol composition of Pecten maximus larvae during the larval development stage with standard algal mixtures and unialgal diets were analysed. The sterol composition of four microalgae currently used in mollusc hatchery were also examined. Under standard algal conditions, the larvae quickly use the steryl ester from larvae reserves during the endotrophic and the mixotrophe phases. The preferential incorporation of Pavlova lutheri and T-Isochrysis sterols, rather than Skeletonema costatum sterols, during the larval development stage would indicate that S. costatum cells were poorly ingested and digested by larvae. Among the ingested sterols, cholesterol and stigmasterol were preferentially incorporated by the larvae. Conversely, the larvae appeared able to limit the incorporation of methylpavlovol, ethylpavlovol, and 4alpha-methylporiferasterol. In the unialgal experiment, the best growths were obtained with the diet richest in cholesterol (Chaetoceros calcitrans) and the best compromise of good growth and settlement rate was observed with the diet richest in C24 ethyl sterol. The selective incorporation of the cholesterol was confirmed by the larval rearing with C. calcitrans. The strong sterol dietary imprint in larvae corroborated the absence of an important capacity in P. maximus larvae to convert or biosynthesise sterol.     

Differential effects of inescapable footshocks and of stimuli previously paired with inescapable footshocks on dopamine turnover in cortical and limbic areas of the rat

The effect of electric footshocks and of exposure to environmental stimuli paired with electrical shocks upon the dopaminergic activity in various cortical and limbic areas of the rat were evaluated by measuring dihydroxyphenylacetic acid (DOPAC) levels in these areas. In animals exposed to a 20 min electric footshock session DOPAC concentrations were significantly increased in the antero-medial and sulcal frontal cortices, olfactory tubercle, nucleus accumbens and amygdaloid complex (by 66, 37, 28, 55 and 90% respectively). Re-exposure of rats to an environment where they had been shocked 24 h earlier induced an elevation of DOPAC content only in the anteromedial frontal cortex (by 47%). Plasma corticosterone levels were elevated in both situations. No change in serotonin or 5-hydroxyindolacetic acid content of these areas could be detected in either situation. The results show that electric footshocks and environmental stimuli associated to previous shocks both activate central dopaminergic systems, although the patterns of activation are different.     

Incorporation and modification of dietary fatty acids in gill polar lipids by two bivalve species Crassostrea gigas and Ruditapes philippinarum

Two bivalve species Crassostrea gigas and Ruditapes philippinarum were fed eight weeks with three mono-specific algae diets: T-Isochrysis galbana, Tetraselmis suecica, Chaetoceros calcitrans, selected on the basis of their polyunsaturated fatty acid (PUFA) composition. The incorporation and the modification of dietary fatty acids in C. gigas and R. philippinarum gill lipids were analysed and compared. Essential PUFA (20:4n-6, 20:5n-3 and 22:6n-3) and non-methylene interrupted PUFAs (known to be synthesised from monounsaturated precursors) contents of gill polar lipid of both species were greatly influenced by the dietary conditioning. Interestingly, oysters and clams responded differentially to the mono-specific diets. Oysters maintained higher 20:5n-3 level and higher 22:2j/22:i and n-7/n-9 ratio in gill polar lipids than clams. To better discriminate dietary and species influences on the fatty acid composition, a Principal Component Analysis followed by a MANOVA on the two most explicative components was performed. These statistical analyses showed that difference in fatty acid compositions attributable to species were just as significant as the diet inputs. The differences of gill fatty acid compositions between oysters and clams are speculated to result of an intrinsic species characteristic and perhaps of a group characteristic: Fillibranch vs. Eulamellibranch.