Cell-penetrating peptides as a promising tool for delivery of various molecules into the cells

Many biologically active compounds, including macromolecules that are used as various kinds of drugs, must be delivered to the interior of cell or organelles such as mitochondria or nuclei to achieve a therapeutic effect. However, very often, lipophilic cell membrane is impermeable for these molecules. A new method in the transport of macromolecules through the cell membrane is the one based on utilizing cell-penetrating peptides (CPPs). Invented 25 years ago, CPPs are currently the subject of intensive research in many laboratories all over the world. CPPs are short compounds comprising up to 30 amino acid residues, which penetrate the cell membrane but do not cause cell damage. Additionally, CPPs can transfer hydrophilic molecules (peptides, proteins, nucleic acids) which exceed their mass, and for which the cell membrane is generally impermeable. In this review, we concentrate on the cellular uptake mechanism of CPPs and a method of conjunction of CPPs to the transported molecules. We also highlight the potential of CPPs in delivering various kinds of macromolecules into cells, including compounds of therapeutic interest

Prawne aspekty wymiaru i egzekucji kary administracyjnej z tytułu niezarejestrowania pojazdu sprowadzonego z terytorium państwa członkowskiego Unii Europejskiej

The amendment to the Act on Road Traffic Law introduced a new legal institution related to failure to register a vehicle consisting of an administrative fine in the amount of PLN 200 to 1,000. The penalty is imposed by means of an administrative decision issued by a competent starost. With the introduction of the above-mentioned institution in legal circulation, new problems will arise in connection with conducting administrative proceedings to impose a penalty, as well as determining its amount. In addition, a starost, as a creditor, is obliged to initiate enforcement proceedings in the absence of voluntary execution of the decision imposing the penalty. The established body aims to ensure the implementation of Directive 2000/53/EC of the European Parliament and of the Council of 18 September 2000 on end-of-life vehicles, concerning, i.a., the absence of sanctions for not registering an imported vehicle and not registering an end-of-life vehicle. The following article discusses the main problems related to the application of the institution introduced with regard to the administrative procedure, the nature of the penalty imposed, as well as its enforcement. The considerations were carried out on the basis of a dogmatic method.Nowelizacja ustawy Prawo o ruchu drogowym wprowadziła nową instytucję prawną związaną z brakiem dokonania zarejestrowania pojazdu, jaką jest administracyjna kara pieniężna w wysokości od 200 do 1000 zł. Kara nakładana jest w drodze decyzji administracyjnej przez właściwego starostę. Wraz z wejściem do obrotu prawnego powyższej instytucji pojawią się nowe problemy związane z prowadzeniem postępowania administracyjnego w przedmiocie nałożenia kary, a także ustalenia jej wysokości. Ponadto starosta jako wierzyciel jest zobowiązany do wszczęcia postępowania egzekucyjnego w sytuacji braku dobrowolnego wykonania decyzji nakładającej karę. Wprowadzona instytucja ma na celu zapewnienie realizacji dyrektywy Parlamentu Europejskiego i Rady 2000/53/WE z dnia 18 września 2000 r. w sprawie pojazdów wycofanych z eksploatacji, dotyczącej m.in. braku sankcji za niezarejestrowanie importowanego pojazdu oraz niewyrejestrowanie pojazdu wycofanego z eksploatacji. W niniejszym artykule omówiono główne problemy związane ze stosowaniem wprowadzonej instytucji w zakresie procedury administracyjnej, charakterem nakładanej kary oraz jej egzekucji. Rozważania przeprowadzono w oparciu o metodę dogmatyczną

Protein and siRNA delivery by transportan and transportan 10 into colorectal cancer cell lines

Introduction. Cell penetrating peptides (CPPs) have the ability to translocate through cell membranes with high efficiency and therefore can introduce biological agents with pharmaceutical properties into the cell. Transportan (TP) and its shorter analog transportan 10 (TP10) are among the best studied CPPs, however, their effects on viability of and cargo introduction into colorectal cancer (CRC) cells have yet not been investigated. The aim of our study was to evaluate the cytotoxic effects of TP and TP10 on representative CRC lines and the efficiency of protein (streptavidin) and siRNA cargo delivery by TP-biotinylated derivatives (TP-biot). Material and methods. HT29 (early stage CRC model) and HCT116 (metastatic CRC model) cell lines were incubated with TP, TP10, TP-biot1, TP-biot13 and TP10-biot1. The effects of studied CPPs on cell viability and cell cycle were assessed by MTT and annexin V assays. The uptake of streptavidin-FITC complex into cells was determined by flow cytometry and fluorescence microscopy, with the inhibition of cellular vesicle trafficking by brefeldin A. The efficiency of siRNA for SASH1 gene delivery was measured by quantitative PCR (qPCR). Results. Since up to 10 µM concentrations of each CPP showed no significant cytotoxic effect, the concentrations of 0.5–5 µM were used for further analyses. Within this concentration range none of the studied CPPs affected cell viability and cell cycle. The efficient and endocytosis-independent introduction of streptavidin-FITC complex into cells was observed for TP10-biot1 and TP-biot1 with the cytoplasmic location of the fluorescent cargo; decreased SASH1 mRNA level was noticed with the use of siRNA and analyzed CPPs. Conclusions. We conclude that TP, TP10 and their biotinylated derivatives can be used as efficient delivery vehicles of small and large cargoes into CRC cells

PROPERTIES OF DIAMOND-LIKE CARBON COATINGS DEPOSITED ON CoCrMo ALLOYS

This paper presents results of the structure analysis and tribological testing of a-C:H type diamond-like carbon (DLC) coatings produced by the Plasma Assisted Chemical Vapour Deposition (PACVD) technology on CoCrMo specimens. The DLC coating structure was studied by observing the surface topography using a scanning electron microscope (SEM) in the SE and STEM modes and a profilometer. Raman spectroscopy provided information on hybridized covalent bonds. The structural analysis involved observing the cross-sections of the coatings using a scanning electron microscope (SEM) with an energy dispersive spectrometer (EDS) to determine the concentration and distribution of the constituent elements in the coating. Wear resistance was studied by applying a T-17 pin-on-plate tribological system: a polymeric pin made of ultra-high-molecular-weight polyethylene (UHMWPE) and a CoCrMo plate with a DLC coating. The testing was conducted under friction conditions using Ringer’s solution as a lubricant. The test results showed that the application of DLC coatings definitely improves the serviceability of a surface

Using capillary electrophoresis to study methylation effect on RNA-peptide interaction.

Methylation of RNA and proteins is one of a broad spectrum of post-transcriptional/translational mechanisms of gene expression regulation. Its functional signification is only beginning to be understood. A sensitive capillary electrophoresis mobility shift assay (CEMSA) for qualitative study of the methylation effect on biomolecules interaction is presented. Two RNA-peptide systems were chosen for the study. The first one consists of a 17-nucleotide analogue (+27-+43) of the yeast tRNAPhe anticodon stem and loop domain (ASLPhe) containing three of the five naturally occurring modifications (2'-O-methylcytidine (Cm32), 2'-O-methylguanine (Gm34) and 5-methylcytidine (m5C40)) (ASLPhe-Cm32,Gm34,m5C40) and a 15-amino-acid peptide (named tF2 : Ser1-Ile-Ser-Pro-Trp5-Gly-Phe-Ser-Gly-Leu10-Leu- Arg-Trp-Ser-Tyr15) selected from a random phage display library (RPL). A peptide-concentration-dependent formation of an RNA-peptide complex was clearly observable by CEMSA. In the presence of the peptide the capillary electrophoresis (CE) peak for triply methylated ASLPhe shifted from 18.16 to 20.90 min. Formation of the complex was not observed when an unmethylated version of ASLPhe was used. The second system studied consisted of the (+18)-(+44) fragment of the trans-activation response element of human immunodeficiency virus type 1 (TAR RNA HIV-1) and a 9-amino-acid peptide of the trans-activator of transcription protein (Tat HIV-1) Tat(49-57)-NH2 (named Tat1 : Arg49-Lys-Lys-Arg52-Arg-Gln-Arg-Arg- Arg57-NH2). In the presence of Tat(49-57)-NH2 a significant shift of migration time of TAR from 18.66 min to 20.12 min was observed. Methylation of a residue Arg52→Arg(Me)2, crucial for TAR binding, strongly disrupted formation of the complex. Only at a high micromolar peptide concentration a poorly shaped, broad peak of the complex was observed. CE was found to be an efficient and sensitive method for the analysis of methylation effects on interaction of biomolecules

Interaction of Arginine-Rich Cell-Penetrating Peptides with an Artificial Neuronal Membrane

Arginine-rich cell-penetrating peptides (RRCPPs) exhibit intrinsic neuroprotective effects on neurons injured by acute ischemic stroke. Conformational properties, interaction, and the ability to penetrate the neural membrane are critical for the neuroprotective effects of RRCCPs. In this study, we applied circular dichroism (CD) spectroscopy and coarse-grained molecular dynamics (CG MD) simulations to investigate the interactions of two RRCPPs, Tat(49–57)-NH2 (arginine-rich motif of Tat HIV-1 protein) and PTD4 (a less basic Ala-scan analog of the Tat peptide), with an artificial neuronal membrane (ANM). CD spectra showed that in an aqueous environment, such as phosphate-buffered saline, the peptides mostly adopted a random coil (PTD4) or a polyproline type II helical (Tat(49–57)-NH2) conformation. On the other hand, in the hydrophobic environment of the ANM liposomes, the peptides showed moderate conformational changes, especially around 200 nm, as indicated by CD curves. The changes induced by the liposomes were slightly more significant in the PTD4 peptide. However, the nature of the conformational changes could not be clearly defined. CG MD simulations showed that the peptides are quickly attracted to the neuronal lipid bilayer and bind preferentially to monosialotetrahexosylganglioside (DPG1) molecules. However, the peptides did not penetrate the membrane even at increasing concentrations. This suggests that the energy barrier required to break the strong peptide–lipid electrostatic interactions was not exceeded in the simulated models. The obtained results show a correlation between the potential of mean force parameter and a peptide’s cell membrane-penetrating ability and neuroprotective properties