Unexpected transcellular protein crossover occurs during canonical DNA transfection.

Transfection of DNA has been invaluable for biological sciences, yet the effects upon membrane homeostasis are far from negligible. Here, we demonstrate that Neuro2A cells transfected using Lipofectamine LTX with the fluorescently coupled Botulinum serotype A holoenzyme (EGFP-LcA) cDNA express this SNAP25 protease that can, once translated, escape the transfected host cytosol and become endocytosed into untransfected cells, without its innate binding and translocation domains. Fluorescent readouts revealed moderate transfection rates (30–50%) while immunoblotting revealed a surprisingly total enzymatic cleavage of SNAP25; the transgenic protein acted beyond the confines of its host cell. Using intracellular dyes, no important cytotoxic effects were observed from reagent treatment alone, which excluded the possibility of membrane ruptures, though noticeably, intracellular acidic organelles were redistributed towards the plasma membrane. This drastic, yet frequently unobserved, change in protein permeability and endosomal trafficking following reagent treatment highlights important concerns for all studies using transient transfection

Botulinum neurotoxin type C protease induces apoptosis in differentiated human neuroblastoma cells

Neuroblastomas constitute a major cause of cancer-related deaths in young children. In recent years, a number of translation-inhibiting enzymes have been evaluated for killing neuroblastoma cells. Here we investigated the potential vulnerability of human neuroblastoma cells to protease activity derived from botulinum neurotoxin type C. We show that following retinoic acid treatment, human neuroblastoma cells, SiMa and SH-SY5Y, acquire a neuronal phenotype evidenced by axonal growth and expression of neuronal markers. Botulinum neurotoxin type C which cleaves neuron-specific SNAP25 and syntaxin1 caused apoptotic death only in differentiated neuroblastoma cells. Direct comparison of translation-inhibiting enzymes and the type C botulinum protease revealed one order higher cytotoxic potency of the latter suggesting a novel neuroblastoma-targeting pathway. Our mechanistic insights revealed that loss of ubiquitous SNAP23 due to differentiation coupled to SNAP25 cleavage due to botulinum activity may underlie the apoptotic death of human neuroblastoma cells

Stopping of relativistic ions in multicomponent plasmas

Investigation of the processes of stopping of charged particles moving in different media is of significant interest for many realms of Physics, such that Nuclear Physics, Condensed Matter Physics, Plasma Physics, etc. The problem of evaluation of energy losses of relativistic protons has acquired special importance recently [1] and, due to the experimental conditions, it is necessary to estimate relativistic corrections to the asymptotic form of energy losses in non-ideal multicomponent plasmas..

Stopping of relativistic ions in multicomponent plasmas

Investigation of the processes of stopping of charged particles moving in different media is of significant interest for many realms of Physics, such that Nuclear Physics, Condensed Matter Physics, Plasma Physics, etc. The problem of evaluation of energy losses of relativistic protons has acquired special importance recently [1] and, due to the experimental conditions, it is necessary to estimate relativistic corrections to the asymptotic form of energy losses in non-ideal multicomponent plasmas..

DOC2B acts as a calcium switch and enhances vesicle fusion

Calcium-dependent exocytosis is regulated by a vast number of proteins. DOC2B is a synaptic protein that translocates to the plasma membrane (PM) after small elevations in intracellular calcium concentration. The aim of this study was to investigate the role of DOC2B in calcium-triggered exocytosis. Using biochemical and biophysical measurements, we demonstrate that the C2A domain of DOC2B interacts directly with the PM in a calcium-dependent manner. Using a combination of electrophysiological, morphological, and total internal reflection fluorescent measurements, we found that DOC2B acts as a priming factor and increases the number of fusion-competent vesicles. Comparing secretion during repeated stimulation between wild-type DOC2B and a mutated DOC2B that is constantly at the PM showed that DOC2B enhances catecholamine secretion also during repeated stimulation and that DOC2B has to translocate to the PM to exert its facilitating effect, suggesting that its activity is dependent on calcium. The hypothesis that DOC2B exerts its effect at the PMwas supported by the finding that DOC2B affects the fusion kinetics of single vesicles and interacts with the PM SNAREs (soluble NSF attachment receptors). We conclude that DOC2B is a calcium-dependent priming factor and its activity at the PM enables efficient expansion of the fusion pore, leading to increased catecholamine release. Copyright © 2008 Society for Neuroscience

The stopping power and straggling of strongly coupled electron fluids revisited

Measuring energy losses of beams of charged particles is an important diagnostic tool in both modern condensed matter and plasma physics..

Genetic diversity assessment in pea cultivars and lines using the SSR analysis

Background. Pea is the main leguminous crop in the Republic of Bashkortostan and widespread all over the world. The key role in the breeding of new pea cultivars is played by source material representing the phenotypic and genotypic diversity of Pisum sativum L., searched for in plant genetic resources collections. SSR markers are successfully used to study the DNA polymorphism of various genetic objects, including pea. However, the distribution of a number of microsatellite alleles in the genotypes of specific lines and cultivars of this valuable pulse crop remains practically unexplored.Materials and methods. Molecular genetic polymorphism was studied in 40 pea cultivar accessions of different ecological and geographical origin from the Vavilov Institute’s genebank of plant genetic resources or developed at regional breeding centers. Microsatellite analysis was performed using 5 SSR markers from the genomic library of microsatellites (Agrogene®, France).Results. All markers delivered good electrophoretic profiles and helped to amplify a number of alleles per locus varying from 2 (AB53) to 9 (AA355). The total number of alleles was 26, while the average number of alleles per locus was 5.2. The polymorphism information content (PIC) varied from 0.39 for locus AB53 to 0.82 for locus AA355, with the mean value of 0.60. The set of SSR markers used in the work made it possible to individualize each of the studied pea genotypes. The measured genetic distances were used to draw a dendrogram showing the distribution of genotypes according to their genetic relationship.Conclusion. Through studying the source material for pea breeding by the SSR analysis the data were obtained that provide additional information about the genetic structure of the collection and the polymorphism of the studied cultivar accessions. The results of genotyping pea cultivars and lines can be used for their genetic identification or to select parental pairs for hybridization

Vesicle fusion as a target process for the action of sphingosine and its derived drugs

The fusion of membranes is a central part of the physiological processes involving the intracellular transport and maturation of vesicles and the final release of their contents, such as neurotransmitters and hormones, by exocytosis. Traditionally, in this process, proteins, such SNAREs have been considered the essential components of the fusion molecular machinery, while lipids have been seen as merely structural elements. Nevertheless, sphingosine, an intracellular signalling lipid, greatly increases the release of neurotransmitters in neuronal and neuroendocrine cells, affecting the exocytotic fusion mode through the direct interaction with SNAREs. Moreover, recent studies suggest that FTY-720 (Fingolimod), a sphingosine structural analogue used in the treatment of multiple sclerosis, simulates sphingosine in the promotion of exocytosis. Furthermore, this drug also induces the intracellular fusion of organelles such as dense vesicles and mitochondria causing cell death in neuroendocrine cells. Therefore, the effect of sphingosine and synthetic derivatives on the heterologous and homologous fusion of organelles can be considered as a new mechanism of action of sphingolipids influencing important physiological processes, which could underlie therapeutic uses of sphingosine derived lipids in the treatment of neurodegenerative disorders and cancers of neuronal origin such neuroblastoma

Development of source material for pea breeding through chemical mutagenesis and evaluation of its genetic diversity using SSR markers

Background. Pea (Pisum sativum L.) is a valuable leguminous crop of worldwide importance. The main problem of modern plant breeding is a decrease in the genetic diversity of crops, including pea. One of the ways to increase genetic polymorphism is the use of chemically induced mutagenesis. Sodium azide (NaN3) is a highly effective chemical mutagen successfully used in mutation breeding to increase the productivity of cultivated plants and enrich them with new useful traits. We used it to obtain new pea breeding material.Materials and methods. Experiments were carried out to obtain pea mutants using sodium azide at the concentrations of 1, 5 and 10 mM and the exposure time of 3 and 9 h. Molecular genetic polymorphism of the М2 plants and the original cultivar was assessed using 10 SSR markers from the microsatellite genomic library (Agrogene®, France).Results. Optimal concentrations of sodium azide and the duration of seed treatment with it were identified: 1–5 mM for 3 h. Sixteen mutant populations were obtained; in ten of them a change in the leaf type was found. An analysis of the yield structure components revealed a significant superiority (p < 0.05) over the initial cultivar ‘Pamyati Khangildina’ in the mutant populations No. 1, No. 5, No. 9, No. 10, No. 15 and No. 16 in the number of seeds per pod, No. 9 and No. 16 in the weight of 1000 seeds, and No. 16 in the weight of seeds per plant. A dendrogram constructed on the basis of the SSR analysis data showed the degree of differences between the M2 populations of pea plants and the initial cultivar ‘Pamyati Khangildina’.Conclusion. The obtained mutant populations are planned to be used in pea breeding as sources of high seed numbers in pods, seed yield, seed weight per plant, and large seed size. A microsatellite analysis with 10 SSR markers revealed differences among the M2 mutant populations at the genetic level and made it possible to identify them

Two complementary approaches for intracellular delivery of exogenous enzymes.

Intracellular delivery of biologically active proteins remains a formidable challenge in biomedical research. Here we show that biomedically relevant enzymes can be delivered into cells using a new DNA transfection reagent, lipofectamine 3000, allowing assessment of their intracellular functions. We also show that the J774.2 macrophage cell line exhibits unusual intracellular uptake of structurally and functionally distinct enzymes providing a convenient, reagent-free approach for evaluation of intracellular activities of enzymes