A cross-platform public domain PC image-analysis program for the comet assay

The single-cell gel electrophoresis, also known as the comet assay, has gained wide-spread popularity as a simple and reliable method to measure genotoxic and cytotoxic effects of physical and chemical agents as well as kinetics of DNA repair. Cells are generally stained with fluorescent dyes. The analysis of comets--damaged cells which form a typical comet-shaped pattern--is greatly facilitated by the use of a computer image-analysis program. Although several image-analysis programs are available commercially, they are expensive and their source codes are not provided. For Macintosh computers a cost-free public domain macro is available on the Internet. No ready for use, cost-free program exists for the PC platform. We have, therefore, developed such a public domain program under the GNU license for PC computers. The program is called CASP and can be run on a variety of hardware and software platforms. Its practical merit was tested on human lymphocytes exposed to gamma-rays and found to yield reproducible results. The binaries for Windows 95 and Linux, together with the source code can be obtained from: http://www.casp.of.pl.     

On the specificity of 4-amino-5-methylamino-2',7'-difluorofluorescein as a probe for nitric oxide

The specificity of 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) for nitric oxide was evaluated in in vitro systems. The probe was found fairly specific for nitric oxide. Potential sources of artifacts include the autoxidation of DAF-FM, potentiated by light, and its oxidation by sources of superoxide and peroxyl radicals, leading to fluorescence spectra indistinguishable from those of the nitric oxide adduct. Although DAF-FM reacts with peroxynitrite, this reaction seems to be of secondary importance under quasi-physiological conditions. On the other hand, a simultaneous presence of a nitric oxide source and a superoxide or hydrogen peroxide decreases or increases the fluorescence of DAF-FM, respectively, resulting in biased estimates of nitric oxide production.     

Occurrence of Wolbachia in central European weevils: correlations with host systematics,ecology, and biology

We studied the occurrence of Wolbachia in relation to the systematics, ecology, and biology of 40 weevil species from central Europe. Identification of Wolbachia supergroups and phylogeny was performed on the basis of 16S rDNA, ftsZ, wsp, and hcpA sequences. Sixteen species (40%) were infected by Wolbachia. Six of these possess only supergroup A (15% of all studied species, 37.5% of the infected species), and four harbored only supergroup B (10 and 25%, respectively). Six species were infected by both supergroups A and B or their genomes harbored parts of these two supergroups (15 and 37.5%, respectively). No differences between Wolbachia supergroup frequencies were detected. There was almost no correlation between Wolbachia phylogeny and host systematics and phylogeny at the level of subfamily and tribe, because the representatives of both supergroups were detected in all the studied multi‐species tribes. Wolbachia strains were probably inherited from a common ancestor only in the case of the genus Strophosoma, where two of three analyzed species possessed bacteria which are genetically very close in all the studied genes. There was also only limited congruence between phylogenies obtained from the four studied genes. These results suggest horizontal transmission of Wolbachia strains between species and recombination events between different strains. A significant correlation was detected between infected and uninfected species in relation to mobility (flying species were 2× more frequently infected than non‐flying species), foraging (polyphagous species were 2.5× less frequently infected than mono‐ or oligophagous species), and reproductive mode (parthenogenetic weevils were infected nearly 2× as often as bisexuals). No differences were detected between mesophilous and xerothermophilous species, nor between those inhabiting open areas vs. arboreal species. However, these results might have been influenced by common ancestry among the studied weevils. Because weevils include many plant pests of economic importance, it is possible to use these data in developing alternative, biology‐based strategies for controlling them.     

Adeno-associated viruses (AAV)

Recombinant adeno-associated virus derived vectors (rAAV) a thought to be a most promising candidates for gene therapy applications. Their nonpathogenic nature as well as the encouraging capability to infect both proliferating and non proliferating cells are advantages for gene therapy applications. Here, we summarize the potential mechanisms responsible for AAV maintenance and site-specific integration to human genome. The role of Rep proteins, inverted terminal repeats and p5 promotor sequences for chromosomal incorporation of AAV are discussed. Making the site-specific integrative recombinant AAV vectors for gene therapy seems to be closely dependent on the development of viral vectorology.     

The effect of new non-cross resistant antitumour agents on the energy state of human erythrocytes

Multidrug resistance (MDR) of tumour cells is related to the overexpression of ATP-dependent pumps responsible for the active efflux of antitumour agents out of resistant cells. Benzoperimidine and anthrapyridone compounds exhibit comparable cytotoxic activity against sensitive and MDR tumour cells. They diffuse extremely rapidly across the plasma membrane and render the ATP-dependent efflux inefficient. Such uptake could disturb an energy metabolism of normal cells possessing an elevated level of ATP-dependent proteins, especially erythrocytes having a high level of the MRP1, MRP4 and MRP5 proteins. In this study the effect of five antitumour agents: benzoperimidine (BP1), anthrapyridones (CO1, CO7) and reference drugs used in the clinic: doxorubicin (DOX) and pirarubicin (PIRA), on the energetic state in human erythrocytes has been examined. These compounds have various types of structure and kinetics of cellular uptake (slow--DOX, CO7, moderate--PIRA, fast--BP1, CO1) resulting in their different ability to saturate ATP-dependent transporters. The energetic state of erythrocytes was examined by determination of purine nucleotide contents (ATP, ADP, AMP), NAD(+) and values of adenylate energy charge (AEC) using an HPLC method. It was found that the level of nucleotides as well as the AEC value of erythrocytes were not changed during 24 h of incubation with these agents independently of their structure and ability to saturate ATP-dependent pumps. This is a very promising result in view of their potential use in the clinic as antitumour drugs against multidrug resistant cancers.     

Recombinant VP60 in the form of virion-like particles as a potential vaccine against rabbit hemorrhagic disease virus

Rabbit hemorrhagic disease virus (RHDV) which causes a highly contagious disease of wild and domestic rabbits belongs to the family Caliciviridae. It is a small, positive single-stranded RNA virus with a genome of 7.5 kb and has a diameter of approximately 40 nm. In negatively stained electron micrographs the virus shows typical calicivirus morphology with regularly arranged cup-shaped structures on the surface. It is a major pathogen of rabbits in many countries. Vp60 - a coat protein of molecular mass around 60 kDa is the major antigen of RHDV. It is present as 90 dimeric units per virion particle. We have expressed VP60 gene in the baculovirus system with the aim to use it as a potential vaccine against RHDV and a diagnostic reagent in immunological tests. cDNA of the vp60 gene of strain SGM, was cloned into a baculovirus transfer vector as full-length gene, as well as truncated gene lacking 600 5'-terminal nucleotides. The sequence of SGM VP60 differed markedly from that of the reference strain. Full-length recombinant VP60 protein from the SGM strain self-assembled to form virus-like particles (VLPs). These particles observed by electron microscopy were morphologically similar to native virions and were able to agglutinate human group 0 erythrocytes. After immunization the recombinant particles induced RHDV-specific antibodies in rabbits and guinea pigs. Rabbits immunized with the VLPs were fully protected against challenge with a virulent RHDV.     

Macrophyte and macroinvertebrate patterns in unimpacted mountain rivers of two European ecoregions

Hydrobiologia - The aim of the study was to compare the patterns of development of macrophytes and macroinvertebrates in different types of reference mountain rivers. The study is based on...     

Absorption of the flavin dimers

The electronic absorption spectra of the flavomononucleotide (FMN) in aqueous solution and in glycerine-water solution with change of the dye concentration have been measured. The FMN dimer absorption spectrum, monomer absorption spectrum, dimerization constant K and molar fraction of the monomer were calculated. It was found that FMN dimerization constants in aqueous solution were K_a = 118.0 l/mol and in glycerine K_g = 20.5 l/mol. In the region of the monomer absorption band two dimer absorption bands appear, in accordance with the Kasha molecular exciton theory.