Evidence for the hybrid origin of<Emphasis Type="Italic">potamogeton ×cooperi</Emphasis> (<Emphasis Type="Italic">Potamogetonaceae</Emphasis>): Traditional morphology-based taxonomy and molecular techniques in concert

The identity of plants morphologically intermediate betweenPotamogeton crispus andP. perfoliatus from two recently discovered sites, one in Moravia, Czech Republic and another in Wales, United Kingdom, was investigated with molecular markers. Evidence from restriction fragment length polymorphism analysis of the nuclear internal transcribed spacer region of ribosomal DNA and of thetrnK-trnQ chloroplast DNA intergenic spacer confirmed the morphology-based determination of two putative hybrid samples asP. ×cooperi. The hybrids showed the ITS variants of both parental taxa, consistent with the expected biparental inheritance of nuclear DNA. The chloroplast DNA markers indicateP. crispus as the female parent in both hybridization events. The hybrid origin of another dubious sample was excluded by the molecular data, in accordance with previous detailed morphological examination. This plant represented an extreme, narrow-leaved form ofP. perfoliatus, imitatingP. ×cooperi in some characters. The results of the molecular analyses are discussed in relation to the morphology of the plants. They underline that somePotamogeton hybrids could indeed be identified by careful and detailed morphological examination and also that these identifications were reliable and confirmed by molecular markers. This study exemplifies that long-term taxonomic expertise usually generates very well-founded specific questions suitable for straightforward treatment by appropriate molecular methods. The process and ecological implications of hybrid formation are also discussed     

Increased expression of Apaf-1 and procaspase-3 and the functionality of intrinsic apoptosis apparatus in non-small cell lung carcinoma

The intrinsic apoptosis apparatus plays a significant role in generating and amplifying cell death signals. In this study we examined whether there are differences in the expression of its components and in its functioning in non-small cell lung carcinoma (NSCLC) and the lung. We show that NSCLC cell lines express Apaf-1 and procaspase-9 and -3 proteins and that the expression of Apaf-1 and procaspase-3, but not of procaspase-9 and -7, is frequently up-regulated in NSCLC tissues as compared to the lung. NSCLC tissues and lungs and some NSCLC cell lines expressed also caspase-9S(b) and displayed a high caspase-9S(b)/procaspase-9 expression ratio. Procaspase-3 from NSCLCs and lungs was readily processed to caspase-3 by granzyme B or caspase-8, and the granzyme B-generated caspase-3-like activity was significantly higher in tumor tissues and cells than in lungs. By contrast, cytochrome c plus dATP could induce a significant increase of caspase-3-like activity in cytosol only in some NSCLC cell lines and in subsets of studied NSCLC tissues and lungs, while procaspase-3 and -7 were detectably processed only in NSCLC tissues which showed a high (cytochrome c+dATP)-induced caspase-3-like activity. Taken together, the present study provides evidence that the expression of Apaf-1 and procaspase-3 is up-regulated in NSCLCs and indicates that the tumors have a capability to suppress the apoptosome-driven caspase activation in their cytosol.     

Effect of 2-Chloro-Substitution of Adenine Moiety in Mixed-Ligand Gold(I) Triphenylphosphine Complexes on Anti-Inflammatory Activity: The Discrepancy between the In Vivo and In Vitro Models

A series of gold(I) triphenylphosphine (PPh₃) complexes (1–9) involving 2-chloro-N6-(substituted-benzyl)adenine derivatives as N-donor ligands was synthesized and thoroughly characterized by relevant methods, including electrospray-ionization (ESI) mass spectrometry and multinuclear NMR spectroscopy. The anti-inflammatory and antiedematous effects of three representatives 1, 5 and 9 were evaluated by means of in vitro model based on the expression of pro- and anti-inflammatory cytokines and influence of the complexes on selected forms of matrix metalloproteinases secreted by LPS-activated THP-1 monocytes and in vivo model evaluating the antiedematous effect of the complexes in the carrageenan-induced rat hind-paw edema model. In addition to the pharmacological observations, the affected hind paws were post mortem subjected to histological and immunohistochemical evaluations. The results of both in vivo and ex vivo methods revealed low antiedematous and anti-inflammatory effects of the complexes, even though the in vitro model identified them as promising anti-inflammatory acting compounds. The reason for this discrepancy lies probably in low stability of the studied complexes in biological environment, as demonstrated by the solution interaction studies with sulfur-containing biomolecules (cysteine and reduced glutathione) using the ESI mass spectrometry.     

Highly and Broad-Spectrum In Vitro Antitumor Active cis-Dichloridoplatinum(II) Complexes with 7-Azaindoles

The cis-[PtCl₂(naza)₂] complexes (1–3) containing monosubstituted 7-azaindole halogeno-derivatives (naza), showed significantly higher activity than cisplatin towards ovarian carcinoma A2780, its cisplatin-resistant variant A2780R, osteosarcoma HOS, breast carcinoma MCF7 and cervix carcinoma HeLa cell lines, with the IC₅₀ values of 3.8, 3.5, 4.5, 2.7, and 9.2 μM, respectively, obtained for the most active complex 3. As for 4 and 5 having disubstituted 7-azaindoles in their molecule, the significant cytotoxicity was detected only for 4 against A2780 (IC₅₀ = 4.8 μM), A2780R (IC₅₀ = 3.8 μM) and HOS (IC₅₀ = 4.3 μM), while 5 was evaluated as having only moderate antiproliferative effect against the mentioned cancer cell lines with IC₅₀ = 33.4, 24.7 and 46.7 μM, respectively. All the studied complexes 1–5 effectively avoided the acquired resistance of ovarian carcinoma cell line. On the other hand, the complexes did not reveal any inhibition activity on the purified 20S proteasome from the A2780 cells. The representative complexes 3 and 5 showed low ability to be hydrolysed, but their stability was markedly lowered in the presence of physiological sulphur-containing biomolecule glutathione (GSH), as proved by the ¹H NMR spectroscopy and mass spectrometry studies. A rate of interaction of the studied complexes with GSH was affected by an addition of another mechanistically relevant biomolecule guanosine monophosphate. The differences in interactions of 3 and 5 with GSH correlate well with their different cytotoxicity profiles.     

The diet of the spiny-cheek crayfish Orconectes limosus in the Czech Republic

The composition of the diet of the invasive spiny-cheek crayfish Orconectes limosus was studied using qualitative and quantitative analyses of stomach contents. A total of 368 specimens collected in 2003–2005 and 2008 in Czech localities were examined, predominantly from the Labe (Elbe) and Vltava River basins. Food components were compared for three size classes of crayfish and both sexes. The following conclusions were reached: (1) the spiny-cheek crayfish is an omnivorous species consuming plants, animals and detritus; (2) quantitatively, the main food component of O. limosus is detritus, while the plant component was second; (3) O. limosus may swallow whole food particles up to 4 mm in size, and the bodies of small animals may sometimes be found undamaged in their stomachs.     

Synthesis and Properties of ApA Analogues with Shortened Phosphonate Internucleotide Linkage

A complete series of the 2 ′–5 ′ and 3 ′–5 ′ regioisomeric types of r(ApA) and 2 ′-d(ApA) analogues with the α-hydroxy-phosphonate C3 ′-O-P-CH(OH)-C4 ″ internucleotide linkage, isopolar but non-isosteric with the phosphodiester one, were synthesized and their hybridization properties with polyU studied. Due to the chirality on the 5 ′-carbon atom of the modified internucleotide linkage bearing phosphorus and hydroxy moieties, each regioisomeric type of ApA dimer is split into epimeric pairs. To examine the role of the 5 ′-hydroxyl of the α-hydroxy-phosphonate moiety during hybridization, the appropriate r(ApA) analogues with 3 ′(2 ′)-O-P-CH₂-C4 ″ linkage lacking the 5 ′-hydroxyl were synthesized. Nuclear magnetic resonance (NMR) spectroscopy study on the conformation of the modified sugar-phosphate backbone, along with the hybridization measurements, revealed remarkable differences in the stability of complexes with polyU, depending on the 5 ′-carbon atom configuration. Potential usefulness of the α-hydroxy-phosphonate linkage in modified oligoribonucleotides is discussed.     

Acetylcholinesterases – the structural similarities and differences

Acetylcholinesterase (AChE) is a widely spread enzyme playing a very important role in nerve signal transmission. As AChE controls key processes, its inhibition leads to the very fast death of an organism, including humans. However, when this feature is to be used for killing of unwanted organisms (i.e. mosquitoes), one is faced with the question - how much do AChEs differ between species and what are the differences? Here, a theoretical point of view was utilized to identify the structural basis for such differences. The various primary and tertiary alignments show that AChEs are very evolutionary conserved enzymes and this fact could lead to difficulties, for example, in the search for inhibitors specific for a particular species.     

Long-run monitoring of bacteria,yeasts and other micromycetes in the air of an industrial conurbation

Three different methods were used for the monitoring of airborne microorganisms: (1). Cultivation of microbes trapped in a single-stage biological impactor directly on a solid agar nutrient medium (meat-pepton agar, Sabouraud's agar, blood agar) in Petri dishes. The repeated yearly course of concentrations of cultivable organisms, or colony-forming units (CFU), was obtained by long-run measurements. (2) Aeresol was trapped by impact on membrane filters, and the microorganisms were cultivated by placing the filters on the agar media as above. (3) Direct microorganism counting in a fluorescence microscope; air was sampled in a four-stage impactor where the aerosol was trapped on microscope slides, and the microorganisms were subsequently stained with fluorescent dyes (fluorescein diacctate, 4;6-diamidino-2-phenylindole and, particular, ethidium bromide).

The highest microorganism counts were obtained by using the fluorescence method, the direct cultivation method gave counts an order of magnitude lower, and the method of cultivation on filters gave values approximately 10 times lower than the conventional cultivation.

High variations in the airborne CFU concentrations over the year were observed in Prague. Over the winter season the variations in the amounts of airborne bacteria and other micromycetes as well as the amounts themselves were lower than in the remaining seasons. In the spring and in the summer, the concentrations of yeasts and other micromycetes were highest, whereas in the autumn the concentrations of the microorganisms decreased. Among the bacteria cultivated form the airborne aerosol, the genera Micrococcus, Bacillus, Neisseria and Corynebacterium predominated. The prevailing genera of micromycetes were Penicillium, Aspergillus and Cladosporium.

The concentrations of microorganisms in free air were also affected by the local weather conditions, temperature in particular, the overall air pollution by aerosols was of minor importance in this respect.