Preliminary Studies of Antimicrobial Activity of New Synthesized Hybrids of 2-Thiohydantoin and 2-Quinolone Derivatives Activated with Blue Light.

Thiohydantoin and quinolone derivatives have attracted researchers' attention because of a broad spectrum of their medical applications. The aim of our research was to synthesize and analyze the antimicrobial properties of novel 2-thiohydantoin and 2-quinolone derivatives. For this purpose, two series of hybrid compounds were synthesized. Both series consisted of 2-thiohydantoin core and 2-quinolone derivative ring, however one of them was enriched with an acetic acid group at N3 atom in 2-thiohydantoin core. Antibacterial properties of these compounds were examined against bacteria: Staphylococcus aureus, Bacillus subtilis, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae. The antimicrobial assay was carried out using a serial dilution method to obtain the MIC. The influence of blue light irradiation on the tested compounds was investigated. The relative yield of singlet oxygen (1O2*, 1Δg) generation upon excitation with 420 nm was determined by a comparative method, employing perinaphthenone (PN) as a standard. Antimicrobial properties were also investigated after blue light irradiation of the suspensions of the hybrids and bacteria placed in microtitrate plates. Preliminary results confirmed that some of the hybrid compounds showed bacteriostatic activity to the reference Gram-positive bacterial strains and a few of them were bacteriostatic towards Gram-negative bacteria, as well. Blue light activation enhanced bacteriostatic effect of the tested compounds.

Genetic Diversity of Symbiotic Green Algae of Paramecium bursaria Syngens Originating from Distant Geographical Locations.

Paramecium bursaria (Ehrenberg 1831) is a ciliate species living in a symbiotic relationship with green algae. The aim of the study was to identify green algal symbionts of P. bursaria originating from distant geographical locations and to answer the question of whether the occurrence of endosymbiont taxa was correlated with a specific ciliate syngen (sexually separated sibling group). In a comparative analysis, we investigated 43 P. bursaria symbiont strains based on molecular features. Three DNA fragments were sequenced: two from the nuclear genomes-a fragment of the ITS1-5.8S rDNA-ITS2 region and a fragment of the gene encoding large subunit ribosomal RNA (28S rDNA), as well as a fragment of the plastid genome comprising the 3'rpl36-5'infA genes. The analysis of two ribosomal sequences showed the presence of 29 haplotypes (haplotype diversity Hd = 0.98736 for ITS1-5.8S rDNA-ITS2 and Hd = 0.908 for 28S rDNA) in the former two regions, and 36 haplotypes in the 3'rpl36-5'infA gene fragment (Hd = 0.984). The following symbiotic strains were identified: Chlorella vulgaris, Chlorella variabilis, Chlorella sorokiniana and Micractinium conductrix. We rejected the hypotheses concerning (i) the correlation between P. bursaria syngen and symbiotic species, and (ii) the relationship between symbiotic species and geographic distribution.

The Evolutionary Relationships between Endosymbiotic Green Algae of Paramecium bursaria Syngens Originating from Different Geographical Locations.

Paramecium bursaria (Ehrenberg 1831), a freshwater ciliate, typically harbors hundreds of green algal symbionts inside the cell. The aim of present study was the molecular identification of newly analyzed P. bursaria symbionts. The second aspect of the present survey was testing a hypothesis whether endosymbionts prefer the specified syngen of the host, and the specified geographical distribution. Ten strains of endosymbionts isolated from strains of P. bursaria originating from different geographical locations were studied. We analyzed for the first time, both the fragment of plastid genome containing 3'rpl36-5' infA genes and a fragment of a nuclear gene encoding large subunit ribosomal RNA (LSU rDNA). The analysis of the LSU rDNA sequences showed the existence of 3 haplotypes and the haplotype diversity of 0.733, and 8 haplotypes for the 3'rpl36-5' infA gene fragment and haplotype diversity of 0.956. The endosymbionts isolated from P. bursaria strains were identified as Chlorella vulgaris, Ch. variabilis and Micractinium conductrix. There was no correlation between the syngen of P. bursaria and the species of endosymbiont.

Molecular Identification of Paramecium bursaria Syngens and Studies on Geographic Distribution using Mitochondrial Cytochrome C Oxidase Subunit I (COI).

Paramecium bursaria is composed of five syngens that are morphologically indistinguishable but sexually isolated. The aim of the present study was to confirm by molecular methods (analyses of mitochondrial COI) the identification of P. bursaria syngens originating from different geographical locations. Phylograms constructed using both the neighbor-joining and maximum-likelihood methods based on a comparison of 34 sequences of P. bursaria strains and P. multimicronucleatum, P. caudatum and P.calkinsi strains used as outgroups revealed five clusters which correspond to results obtained previously by mating reaction. Our analysis shows the existence of 24 haplotypes for the COI gene sequence in the studied strains. The interspecies haplotype diversity was Hd = 0.967. We confirmed genetic differentiation between strains of P. bursaria and the occurrence of a correlation between geographical distribution and the correspondent syngen.

Identification of Paramecium bursaria syngens through molecular markers--comparative analysis of three loci in the nuclear and mitochondrial DNA.

This is the first attempt to resolve the phylogenetic relationship between different syngens of Paramecium bursaria and to investigate at a molecular level the intraspecific differentiation of strains originating from very distant geographical locations. Herein we introduce a new collection of five P. bursaria syngens maintained at St Petersburg State University, as the international collection of syngens was lost in the 1960s. To analyze the degree of speciation within Paramecium bursaria, we examined 26 strains belonging to five different syngens from distant and geographically isolated localities using rDNA (ITS1-5.8S-ITS2-5'LSU) fragments, mitochondrial cytochrome c oxidase subunit I (COI), and H4 gene fragments. It was shown that P. bursaria strains of the same syngens cluster together in all three inferred molecular phylogenies. The genetic diversity among the studied P. bursaria strains based on rDNA sequences was rather low. The COI divergence of Paramecium bursaria was also definitely lower than that observed in the Paramecium aurelia complex. The nucleotide sequences of the H4 gene analyzed in the present study indicate the extent of genetic differences between the syngens of Paramecium bursaria. Our study demonstrates the diagnostic value of molecular markers, which are important tools in the identification of Paramecium bursaria syngens.

Polymorphism of Paramecium pentaurelia (Ciliophora, Oligohymenophorea) strains revealed by rDNA and mtDNA sequences.

Paramecium pentaurelia is one of 15 known sibling species of the Paramecium aurelia complex. It is recognized as a species showing no intra-specific differentiation on the basis of molecular fingerprint analyses, whereas the majority of other species are polymorphic. This study aimed at assessing genetic polymorphism within P. pentaurelia including new strains recently found in Poland (originating from two water bodies, different years, seasons, and clones of one strain) as well as strains collected from distant habitats (USA, Europe, Asia), and strains representing other species of the complex. We compared two DNA fragments: partial sequences (349 bp) of the LSU rDNA and partial sequences (618 bp) of cytochrome B gene. A correlation between the geographical origin of the strains and the genetic characteristics of their genotypes was not observed. Different genotypes were found in Kraków in two types of water bodies (Opatkowice-natural pond; Jordan's Park-artificial pond). Haplotype diversity within a single water body was not recorded. Likewise, seasonal haplotype differences between the strains within the artificial water body, as well as differences between clones originating from one strain, were not detected. The clustering of some strains belonging to different species was observed in the phylogenies.

Molecular approach to the systematics of European Tortricini (Lepidoptera: Tortricidae).

Tortricini is a cosmopolitan tribe of the subfamily Tortricinae of the lepidopteran family Tortricidae. The most recent systematic of Tortricini are based on the external morphology of imagines and the structure of their genital organs. The present paper is the first comparative molecular study of the representatives of this tribe. We examined DNA variation in a 606 bp fragment of COI mtDNA obtained from 23 species of Tortricini and two representatives of other tribes (Archips podanus of Archipini and Aethes hartmanniana of Cochylini). The position of Spatalistis, Tortrix, Aleimma and Acleris, and some groupings of species within Acleris were confirmed by molecular data, including the synonymization of Croesia and Phylacophora with Acleris. The positions of a few groupings of the Acleris species remain unresolved.

Intra-specific differentiation of Paramecium bursaria strains by molecular methods--preliminary studies.

Ten strains of Paramecium bursaria and also P. caudatum, P. multimicronucleatum, P. tetraurelia strains (as outgroups) were characterized by using Random Amplified Polymorphic DNA (RAPD), Amplified Ribosomal DNA Restriction Analysis (ARDRA) and sequencing of the non-coding ribosomal internal transcribed spacer (ITS) regions. RAPD analysis revealed that all Paramecium bursaria strains possessed characteristic band patterns; there was a correlation between the degree of differentiation of DNA revealed by RAPD-fingerprinting and the geographic origin of a particular strain. ARDRA riboprinting (using a fragment of SSU-LSU rDNA, about 3085 bp) with restriction enzymes DraI, EcoRV, HhaI, HindIII, MspI, PstI distinguished groups of P. bursaria strains with characteristic band patterns originating from different sites. Comparison of the 550 bp ITS 1-5.8S-ITS2 fragment showed differentiation (0.9%) of the P. bursaria strains as three main groups of strains connected by site of origin in the constructed tree.

Molecular studies on intraspecific differentiation of Paramecium dodecaurelia, with description of new stands of the species (Protozoa, Ciliophora).

New stands of Paramecium dodecaurelia were found in Russia, Ukraine, Kazahstan, Poland, and Tenerife. Molecular studies (RAPD and sequencing of LSU rRNA and CO1 mtDNA gene fragments) of P. dodecaurelia strains depicted intraspecific polymorphism within the species, revealed as distinct differences between strains from the USA in comparison with strains from different regions of the Palearctic.

Polymorphism within Paramecium sexaurelia (Ciliophora, Oligohymenophorea) and description of a new stand of the species in China.

The presence of Paramecium sexaurelia from the Paramecium aurelia complex was recorded for the first time in China (Beijing). RAPD fingerprints (band patterns) of P. sexaurelia strains, the new strain from China and others from Asia, as well as from Europe and Puerto Rico, showed polymorphism within the species as several groups of genotypes characterized by different band patterns.

Strains of Paramecium decaurelia (Ciliophora, Protozoa) from Russia with molecular characteristics of other known strains of the species.

The presence of Paramecium decaurelia from the Paramecium aurelia species complex was demonstrated in Yaroslavl, Russia, (European part, northwestern Russia) and in the Altai Mts (Asiatic part of Russia, western Siberia). RAPD-PCR fingerprints of the newly identified strains of P. decaurelia, rare throughout the world, were compared to those characteristic for the other known strains ofthe species. P. decaurelia strains show some polymorphism within species, strains from Russia have 60% similarity of band patterns, and strains from USA and Japan about 70% similarity of band patterns.

Molecular analysis (RAPD-PCR) of inter-strain hybrids of the Paramecium aurelia species complex (Ciliophora, Protozoa).

RAPD-PCR analysis showed that species of the Paramecium aurelia complex possessed characteristic band patterns and that the majority were also polymorphic intra-specifically. A comparison of band patterns was performed for some inter-strain hybrids within P. primaurelia, P. tetraurelia, P. pentaurelia, P. septaurelia, P. octaurelia, P. decaurelia, P. dodecaurelia, P. tredecaurelia, and P. quadecaurelia to band patterns characteristic for the parental strains. The investigations, however, did not reveal a close correlation between the degree of inbreeding characteristic for the species and similarity of genotypes. A low similarity of hybrid and parental band patterns was observed in P. octaurelia, P. dodecaurelia, P. quadecaurelia and also P. primaurelia. A high similarity of band patterns of hybrid and parental strains was found in P. tetraurelia, P. septaurelia, P. decaurelia, and P. tredecaurelia.

New European stands of Paramecium pentaurelia, Paramecium septaurelia, and Paramecium dodecaurelia, genetic and molecular studies.

New stands of rare species of the Paramecium aurelia complex were found in Europe, i.e. P. pentaurelia and P. dodecaurelia in Italy and P. septaurelia in Germany. The species were identified by mating reactions with the standard strains of each species. Their relationships with some other known strains of particular species were studied by classical strain crosses (survival in F1 and F2 generations) and by comparison of RAPD-PCR fingerprints. The presence of the cosmopolitan species P. tetraurelia in Italy was also recorded.