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.     

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.     

Micelle‐enhanced spectrofluorimetric method for determination of lacidipine in tablet form; application to content uniformity testing

A highly sensitive, simple and rapid spectrofluorimetric method was developed for the determination of lacidipine (LCP) in tablets. The proposed method is based on the investigation of the fluorescence spectral behavior of LCP in both sodium dodecyl sulphate (SDS) and the tween‐80 micellar system. In aqueous solutions of acetate buffer (pH 4.5), the fluorescence intensities of LCP were greatly enhanced (ca. 2.4 and 4.3 folds) in the presence of either SDS or tween‐80, respectively. The fluorescence intensity was measured at 444 nm after excitation at 277 nm using either SDS or tween‐80 as a surfactant. The fluorescence–concentration plots were rectilinear over the ranges of 50.0–500.0 ng/ml and 5.0–200.0 ng/ml with lower detection limits of 5.11 and 2.06 ng/ml and lower quantification limits of 17 and 6.87 ng/ml using SDS and tween‐80, respectively. The method was successfully applied to the analysis of LCP in commercial tablets and the results were in good agreement with those obtained with the comparison method. Furthermore, content uniformity testing of pharmaceutical tablets was also conducted. Copyright © 2014 John Wiley & Sons, Ltd.     

Evaluation of the genome stability and mitochondrial potential of tumor cells in vivo under the action of nanoferromagnetics with different sizes of nanoparticles

The transmembrane potential and genome stability of tumor cells under nanoferromagnetic action with different sizes of nanoparticles were studied in vivo. An evaluation of the genotoxic influence of nanoeferromagnetics on tumor cells and changes in their mitochondrial potential was carried out with the application of a micronuclei test and mitochondrial fluorescent probe JC-1. Nanoferromagnetics with nanoparticle diameters of 40 and 100 nm at a concentration of 3 mg/kg were not found to deminish the trasmembrane mitochondrial potential and the genome of tumor cells and increase the micronucleus frequency of tumor cells to only a small extent. Iron nanoparticles at a concentration of 6 mg/kg were noted to demonstrate an antidromonous character of the changing mitochondrial potential and micronuclei frequency depending on their size. Iron nanoparticles with a smaller diameter were found to provoke an essential rise in the transmembrane potential of tumor cell metachondria on the background of a constant number of micronuclei, and the opposite was true for those with a greater diameter. The data obtained can serve as an important criterion for further studies in experimental oncology to create an optimal system for delivering anticancer preparations to tissue targets.     

Termites Improve the Horizontal Movement of Carbonized Particles: A Step towards Sustainable Utilization of Biochar

Soil amendments containing carbonized materials increase the soil carbon reservoir, influence plant productivity, and, ultimately, help to clean the environment. There is data on the effect of such additions on soil physicochemical properties or plant growth, but few studies have focused on how these carbonized materials are distributed by termite species in the soil ecosystem. It is the first comprehensive study of the transportation of biochar (BC) by termite species under tropical environmental conditions in Pakistan. The present study was carried out to test the hypothesis that if termite species I) were involved in the distribution of biochar particles II) if yes, then how far these particles were transported during the study period (10 days) and III) check their preference between the enriched BC (EBC) and non-enriched BC. BC was enriched with the cattle slurry after its pyrolysis in the study. The results showed that EBC particles were significantly more widely distributed than non-enriched BC particles, but both types of BC were transported more than 4 cm (ring 4) within 10 days (at the end of the experiment). The current study also revealed that EBC was easily attached to the setae, cuticle, and legs of termites, implying that it could potentially be transported over a greater distance. Furthermore, transportation of EBC over larger distances indicated a potential preference of termite species between the EBC and BC particles. During the study, however, the preference among the termite species was also observed. Under the prevailing study conditions, the Coptotermes heimi and Heteroterme indicola species transported the EBC further than Microtermes obesi and Odontotermes obesus. These findings revealed that transportation preferences were observed among the four termite species. In conclusion, the current study found that termites were involved in the distribution of BC particles, with a preference for EBC and that these have the potential to transport BC particles more than 4 cm within 10 days. Furthermore, two species Coptotermes heimi and Heteroterme indicola may be more suitable candidates for EBC transpiration in Pakistani soils. It was necessary to conduct additional research into the effect of temperature on the transportation process.     

Promotion of crown-gall tumor initiation on primary pinto bean leaves by certain inorganic salts

Crown-gall tumor initiation by Agrobacterium tumefaciens (SMITHand TOWN.) CONN, strain B6, on Phaseolus vulgaris L. var. "Pinto"was found to be sensitive to the addition of various salts tothe freshly inoculated primary leaves. Calcium, magnesium, zincand ammonium sulfates, cobalt and nickel chlorides and potassiumnitrate increased the number of tumors on inoculated leavesby 100 to 200 percent. Maximal promotions were obtained at concentrationsof 10^–7 to 10^–5 M with all of these salts exceptpotassium nitrate which was most active at 10^–2M. Sodium,potassium, calcium, magnesium and aluminum chlorides, and sodium,potassium and nickel sulfates had little or no effect on tumorinitiation. The combination of sodium sulfate with magnesiumchloride gave promotions comparable to those obtained with magnesiumsulfate, indicating that both the magnesium and sulfate ionswere necessary for the promotion obtained with this latter salt.Combining any two of the active salts at their optimum concentrationsfor promotion resulted in a reciprocal inhibition of the promotionobtained with either salt alone, suggesting these salts to beactive by different and potentially antagonistic mechanisms.The possible nature of these promotions is discussed, but theyremain too obscure to warrant a specific proposal for theirmode of action. ¹Present adress: Department of Botany, University of Khartoum,Khartoum, Sudan.     

Ultrastructural Study of the Development of Pleistophora schubergi Zwölfer, 1927 (Protozoa,Microsporida) in Larvae of the Spruce Budworm,Choristoneura fumiferana and Its Subsequent Taxonomic Change to the Genus Endoreticulatus

This study demonstrates that Pleistophora schubergi Zwölfer, 1927, a microsporidium originally isolated from the midgut epithelium of Nygmia phaeorrhoea Don (Euproctis chrysorrhoea L.) and Porthetria dispar L., and subsequently reported in several other insects including the spruce budworm, Choristoneura fumiferana (the host used in this investigation), does not belong in the genus Pleistophora Gurley, 1893. Pleistophora schubergi lacks the major features that are characteristic of Pleistophora typicalis, the type species of this genus. A comparison of ultrastructural observations reported for the type species of the genus Pleistophora, P. typicalis, and our observations of P. schubergi revealed significant differences. A thick (0.5 μm) amorphous coat, derived from parasite secretions and deposited external to the parasite plasmalemma, surrounds all developmental stages in P. typicalis. Double membranes, derived from host rough endoplasmic reticulum cisternae encircle the parasite plasmalemma of all developmental stages in P. schubergi. The sporophorous vesicle encases the spores in P. typicalis, and originates from the parasite-secreted coat that is present around meronts. In P. schubergi, the host endoplasmic reticulum cisternae form the envelope that surrounds the meronts. Moreover, the sporophorous vesicle envelope in P. typicalis persists around groups of spores, while in P. schubergi this envelope breaks easily to release the spores in the host cytoplasm. By comparing the characteristics of the microsporidium found in the spruce budworm with those of the recently created polysporous genera that sporulate within a vesicle, we found that P. schubergi does belong in the new genus Endoreticulatus Brooks et al. 1988, and consequently rename it Endoreticulatus schubergi (Zwölfer, 1927) n. comb.