Non-methylated Genomic Sites Coincidence Cloning (NGSCC): an approach to large scale analysis of hypomethylated CpG patterns at predetermined genomic loci

We have developed a new approach to the analysis of hypomethylated CpG patterns within predetermined, megabase long, genome regions. The approach, which we term Non-methylated Genomic Sites Coincidence Cloning (NGSCC), includes three main steps. First, total genomic DNA is digested with a methylation sensitive restriction endonuclease, such as Hpa II or Hha I. Then the fragments corresponding to the genomic area of interest are selected. To this end the fragmented genome DNA is hybridized with a mixture of clones (BACs, cosmids etc.) representing a given region and digested with the same restriction enzyme(s). A special version of the coincidence cloning procedure was developed to make this hybridization selection highly efficient and specific. Finally, fragments of the locus under study are mapped and sequenced. The technique proved to be efficient and specific. As a test, it was applied to the analysis of hypomethylated CpG patterns along the 1-Mb D19S208-COX7A1 (Chr 19q13.12) locus, on human chromosome 19, in normal testis and in seminoma tissues. Some differences in the distribution of hypomethylated CpGs between the two tissues were demonstrated. The methylation profiles in both tissues revealed a clear trend to clustering of non-methylated sites. We also analyzed the expression of genes located within hypomethylated clusters in both tissues. It was shown that, whereas the expression of some of the genes investigated was correlated with hypomethylation of the region, other genes were expressed regardless of their methylation status. NGSCC thus promises to be a useful approach for the analysis of the role of dynamic epigenetic factors in genome function.Communicated by G. P. Georgiev     

Ouabain is a potent promoter of growth and activator of ERK1/2 in ouabain-resistant rat renal epithelial cells

Endogenous cardiotonic steroids (ECS) are putative ligands of the inhibitory binding site of the membrane sodium pump (Na+, K+-ATPase). There is growing evidence that cardiotonic steroids may promote the growth of cardiac and vascular myocytes, including evidence indicating growth stimulation at concentrations in the same range as circulating ECS concentrations. We investigated four parameters to determine whether ouabain, a proposed ECS, promotes growth of immortalized rat proximal tubule epithelial cells: cell count by hemocytometer; metabolic activity as reflected in the mitochondrial conversion of the tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, to its formazan product (MA); DNA synthesis reflected as bromodeoxyuridine incorporation (DNA); and mitosis reflected as histone phosphorylation state detected using anti-phosphohistone 3 antibody (HP). Maximum stimulatory responses were observed at 1 nm ouabain (MA, 20.3% increase, p < 0.01; DNA, 28.4% increase, p < 0.001; HP, maximum response at 0.5 h, 50% increase, p < 0.001). We observed that growth stimulation was associated with stimulation of ERK1/2 phosphorylation (ERK-P), and both growth and ERK-P could be blocked by the MEK inhibitor (U0126, 100 nm). Western blot analysis revealed that the only alpha isoform of Na+, K+-ATPase that could be detected in these cultures was the highly ouabain-resistant alpha1 isoform. Measurement of ouabain inhibition of ion transport in these cultures using 86Rb+ uptake revealed the predominance of the expected ouabain-resistant isoform (IC50 = 24 microm) and an additional minor ( approximately 15%) ouabain-sensitive inhibition with IC50 approximately 30 pm. Similar bimodal transport inhibition curves were obtained in freshly dissected rat proximal tubules. These results indicate that renal epithelial cells may be a sensitive target of the ERK1/2-activating and growth-promoting effects of ouabain even in the presence of ouabain-resistant Na+, K+-ATPase.     

Molecular Evidence for the Existence of Two Species of Marteilia in Europe

Marteilia refringens is one of the most significant pathogens of bivalve molluscs. Previous sequencing of the small subunit ribosomal RNA gene of M. refringens isolates derived from the infected mussels (Mytilus edulis and Mytilus galloprovinciallis) and the oyster (Ostrea edulis) in Europe did not reveal genetic polymorphisms despite indications from epizootiological data that distinct types may exist. We investigated the existence of polymorphisms in the internal transcribed spacer region of the ribosomal RNA genes. The sequences of this region proved to be clearly dimorphic among Marteilia from five sampling sites. The distribution of the two genetic types, named "O" and "M", appeared to be linked to the host species, oysters and mussels, respectively. We therefore support the recognition of two species of Marteilia in Europe and propose that the "O" type corresponds to M. refringens and the "M" type to M. maurini.     

The peculiarities of repair DNA-polymerases in the loach embryogenesis

The current concept of eukaryotic DNA polymerases is considered, which are involved in nuclear DNA repair. The data are given on a new group of DNA polymerases that maintain the integrity of DNA structures without preliminary excision of damaged regions. A special attention is paid to specific features of the functioning of repair DNA polymerases in embryogenesis of the loach. A possible existence is discussed of the previously unknown pathway of DNA repair with participation of DNA polymerase delta as independent from the nuclear antigen of proliferating cells.     

Policlithrum ponticum sp. n. (Monogenea: Gyrodactylidae: Polyclithrinae) from Mugil cephalus from the Black Sea and problems of suprageneric systematics of the gydrodactylids

Polyclithrum ponticum sp. n. is described and P. mugilini Rogers, 1967 is redescribed. Both monogenean species are parasites of Mugil cephalus in the Black Sea. The new species differs from P. mugilini, P. alberti and P. boegeri by the lesser size of anchors, while it is distinguished from P. corallense by the larger size of these structures. P. ponticum sp. n. differs from all formerly described species by the greater length of dorsal connective bar. In both species from the Black Sea, "ear-like" structures situated near the external roots of anchors are described for the first time. It is suggested, that these structures take part in longitudinal, two-lobe folding of the haptor. The process of opening the haptor is probably performed by the additional bars of the haptor (bars 2 and 3 after: Rogers, 1967), joined to each other and with the anchors. The fifth pair of additional bars (Ernst e. a., 2000) derives from the "beard" of ventral connective bar and is united with its basal part. The sixth pair of additional bars (Ernst e. a., 2000) is considered as a typical "ribs" of the haptor, and therefore the "ribs" are represented by three pairs. Differences between marginal hooks of P. ponticum sp. n. and P. mugilini are insignificant, that probably depends on the presence of "ribs" of the haptor. Based on the subdivision of marginal hooks into two groups, the presence of additional supporting structure in the haptor, and the presence of the seminal receptacle, it is suggested that the subfamily Polyclithrinae Rogers, 1967 should include the genera Polyclithrum Rogers, 1967, Swingleus Rogers, 1969, Macrogyrodactylus Mamlberg, 1959, and probably Fundulotrema Hargis, 1955. Based on such characters as the lack of the anchors, the presence of suckers in the haptor, and ovipositing of eggs, it seems to be expedient to use the following taxa in systematics of gyrodactylids: Isancistrinae Fuhrmann, 1928 (genera Isancistrum, Anacanthocotyle); Gyrdicotylinae Vercammen-Grandjean, 1960 (Gyrdicotyle) and Ooegyrodactylinae Harris, 1983 (genera Phanerothecium, Ooegyrodactylus, Nothogyrodactylus, Hyperopletes).     

Cardiotonic steroids: potential endogenous sodium pump ligands with diverse function

The highly conserved cardiotonic steroid (CS) binding site present on the ubiquitous membrane sodium pump, sodium, potassium-ATPase, appears to have been conserved by no force other than its capacity to bind CS: a family that includes plant-derived cardiac glycosides and putative endogenous vertebrate counterparts. Binding of ligand is inhibited by increased extracellular potassium. This implies functional coordination because inhibition of the sodium pump would be counterproductive when extracellular potassium is elevated. The interesting biology of the CS binding site continues to stimulate investigations into the identity of endogenous ligands, their role as pump regulators at the cellular level, and as mediators of body fluid balance and blood pressure regulation. In addition to inhibition of sodium and potassium transport, there is considerable recent evidence suggesting that the sodium pump may act as a cell signaling receptor activated by CS binding and responding by coordination of intracellular signaling pathways that can be dependent on and also independent of the reduction in transmembrane ion flux resulting directly from pump inhibition. This signaling may influence cell survival, growth, and differentiation. Recent insight into the biology of pump regulation by CS is reviewed.     

Variability in the taxonomic characters of Black Sea gyrodactylids (Monogenea)

Variability of the haptoral hard parts of four Black Sea Gyrodactylus species (G. alviga Dmitrieva & Gerasev, 2000; G. crenilabri Zaika, 1966; G. flesi Malmberg, 1957; G. sphinx Dmitrieva & Gerasev, 2000) was studied in relation to season, host and geographical variability. A discriminant analysis was carried out on the measurements of 14 characters of the haptor of all four species. The haptoral hard parts of the investigated gyrodactylids decrease in size with an increase in water temperature. It is known that life-span of gyrodactylids is negatively correlated with water temperature and they exhibit peak intensities at low temperature. At the same time, life-span is considered to be a general indicator of a favourable environment. Therefore, high water temperature is not apparently advantageous for gyrodactylids. In the case of low marine salinity, in adapted species the sizes of the haptoral hard parts are larger at lower levels of salinity and vice versa for typical marine species. Lastly, in specimens parasitising the primary host, these features are larger than in those parasitising a secondary host. It is possible to say that, in general, the more favourable the environment for gyrodactylids the larger their haptoral hard parts. Unfavourable environmental conditions reduce the time of embryogenesis, which has a dual effect on gyrodactylids: positive in relation to their reproduction (by increasing the reproduction rate) and negative in relation to their development (by decreasing the size of the haptoral hard parts).     

Glycolate oxidase isoforms are distributed between the bundle sheath and mesophyll tissues of maize leaves

Glycolate oxidase (EC 1.1.3.15) activity was detected both in the bundle sheath (79%) and mesophyll (21%) tissues of maize leaves. Three peaks of glycolate oxidase activity were separated from maize leaves by the linear KCl gradient elution from the DEAE-Toyopearl column. The first peak corresponded to the glycolate oxidase isoenzyme located in the bundle sheath cells, the second peak had a dual location and the third peak was related to the mesophyll fraction. The mesophyll isoenzyme showed higher affinity for glycolate (Km 23 micromol x L(-1)) and a higher pH optimum (7.5-7.6) as compared to the bundle sheath isoenzyme (Km 65 micromol x L(-1), pH optimum 7.3). The bundle sheath isoenzyme was strongly activated by isocitrate and by succinate while the mesophyll isoenzyme was activated by isocitrate only slightly and was inhibited by succinate. It is concluded that although the glycolate oxidase activity is mainly attributed to the bundle sheath, conversion of glycolate to glyoxylate occurs also in the mesophyll tissue of C4 plant leaves.