Epigenetic natural variation in Arabidopsis thaliana

Cytosine methylation of repetitive sequences is widespread in plant genomes, occurring in both symmetric (CpG and CpNpG) as well as asymmetric sequence contexts. We used the methylation-dependent restriction enzyme McrBC to profile methylated DNA using tiling microarrays of Arabidopsis Chromosome 4 in two distinct ecotypes, Columbia and Landsberg erecta. We also used comparative genome hybridization to profile copy number polymorphisms. Repeated sequences and transposable elements (TEs), especially long terminal repeat retrotransposons, are densely methylated, but one third of genes also have low but detectable methylation in their transcribed regions. While TEs are almost always methylated, genic methylation is highly polymorphic, with half of all methylated genes being methylated in only one of the two ecotypes. A survey of loci in 96 Arabidopsis accessions revealed a similar degree of methylation polymorphism. Within-gene methylation is heritable, but is lost at a high frequency in segregating F₂ families. Promoter methylation is rare, and gene expression is not generally affected by differences in DNA methylation. Small interfering RNA are preferentially associated with methylated TEs, but not with methylated genes, indicating that most genic methylation is not guided by small interfering RNA. This may account for the instability of gene methylation, if occasional failure of maintenance methylation cannot be restored by other means.     

Loss of plasmids of Borrelia burgdorferi sensu lato during prolonged in vitro cultivation

In the present study we analyzed stability of plasmid content in 34 Borrelia strains of three different species (13 Borrelia afzelii, 10 Borrelia garinii and 11 Borrelia burgodorferi sensu stricto) using pulse field gel electrophoresis (PFGE). During long-term in vitro cultivation consisting of 50 passages, plasmid loss was established in 46% of B. afzelii, 40% of B. garinii and 36% of B. burgdorferi sensu stricto strains. Loss of plasmids occurred as early as between the 5th and 10th passage, affected only plasmids in the range 9-41 kb but not plasmids in the range 50-68 kb and manifested with the loss of one to up to three plasmids.     

Identification of proteins bearing beta1-6 branched N-glycans in human melanoma cell lines from different progression stages by tandem mass spectrometry analysis

The common structural alterations in the cell-surface glycoproteins concern the highly elevated expression of tri- and tetra-antennary beta1-6-N-acetylglucosamine (beta1-6 GlcNAc) bearing N-glycans, which are recognised by Phaseolus vulgaris agglutinin (PHA-L). In this report we identified proteins bearing beta1-6 GlcNAc branched N-glycans in three human melanoma cell lines: WM35--from the primary tumour site, as well as WM239 and WM9 from different metastatic sites: the skin and the lymph node, respectively, by tandem mass spectrometry (MS/MS) on PHA-L agarose bound material, followed by immunochemical identification. Our results show that melanoma cell lines differ from each other in the number of N-glycoproteins bearing beta1-6 GlcNAc branched oligosaccharides. Among identified proteins the largest group consists of integrin subunits. In addition, L1-CAM, Mac-2 binding protein, melanoma cell adhesion molecule, intercellular adhesion molecule, melanoma associated antigen, tumour rejection antigen-1, melanoma-associated chondroitin sulfate proteoglycan 4 and lysosome-associated membrane protein (LAMP-1) were found. It was indicated that WM35 cell line showed the lowest number of proteins possessing beta1-6 GlcNAc branched N-glycans in comparison to metastatic WM9 and WM239 cell lines. Our data suggest that changes in the number of proteins being a substrate for GlcNAc-TV are better correlated with melanoma development and progression than with expression of cell adhesion molecules.     

Effect of indole-3-butyric acid on phytoplasmas in infected Catharanthus roseus shoots grown in vitro

Phytoplasmas are noncultivable bacteria usually maintained in Catharanthus roseus shoots grown in vitro on MS medium with benzylaminopurine. The aim of our research was to examine the influence of indole-3-butyric acid (IBA) on C. roseus shoots infected with three different phytoplasma strains. Supplement of IBA in the medium supported plant growth, photosynthesis and remission of symptoms in all phytoplasma-infected shoots, but had no effect on the presence of EY-C and SA-I phytoplasma strains in tested tissue. However, HYDB phytoplasma was undetectable in approximately half of the tested shoots grown on the medium with IBA. After 1 year of IBA treatment, HYDB-infected periwinkle shoots were retransferred to the medium supplemented with benzylaminopurine. Some of the shoots showing remission of symptoms during the IBA treatment permanently escaped the infection and remained negative when tested for phytoplasma presence. This is the first report on the differential influence of plant growth regulators on phytoplasma-infected C. roseus shoots.     

Eimeria papillata: upregulation of specific miRNA-species in the mouse jejunum

Increasing evidence indicates miRNAs as critical regulators of gene expression, but little information is available for miRNAs in intestinal diseases. Here, we investigated intestinal infections of male Balb/c mice with the coccidian parasite Eimeria papillata. On day 4 after oral infection, mice were shedding 3150 ± 430 oocysts per gram feces. This was associated with a low inflammatory response of the jejunum of mice evidenced by histology, non-response of IL-1β mRNA, even slight downregulation of IL-6 mRNA, only slight increases in iNOS mRNA, nitrate/nitrate, malondialdehyde, and a small decrease in glutathione, respectively. Only IFNγ mRNA was strongly induced. Using miRNA microarray technology, there were significantly upregulated the four miRNA species miR-1959, MCMV-miR-M23-1-5P, miR-203, and miR-21 out of 634 miRNAs, which was also confirmed by quantitative RT-PCR. Our data provide evidence that E. papillata parasites are able to induce specific miRNA species in their host target organ.     

The use of scientometric parameters for the evaluation of scientific contributions

This paper deals with the application of scientometric parameters in the evaluation of scientists, either as individuals or in small formal groups. The parameters are divided into two groups: parameters of scientific productivity and citation parameters. The scientific productivity was further subdivided into three types of parameters: (i) total productivity, (ii) partial productivity, and (iii) productivity in scientific fields and subfields. These citation parameters were considered: (i) impact factors of journals, (ii) impact factors of scientific fields and subfields, (iii) citations of individual papers, (iv) citations of individual authors, (v) expected citation rates and relative citation rates, and (vi) self-citations, independent citations and negative citations. Particular attention was payed to the time-dependence of the scientometric parameters. If available, numeric values of the world parameters were given and compared with the data about the scientific output of Croatian scientists.     

Reproductive cycle of gilt sardine,Sardinella aurita,Valenciennes 1847, in the eastern middle Adriatic Sea

The annual reproduction cycle of gilt sardine, Sardinella aurita, based on gonad maturity stages, gonad weight and gonadosomatic index was the subject of this study. A total of 2033 gilt sardines (983 males, 1021 females and 29 undetermined) were analysed. Fish were collected monthly from commercial purse seiners between November 2007 and January 2009 in the eastern middle Adriatic Sea (mesh size 8 mm/bar length/; sampling: five boats per month). Based on the monthly evolution of gonad maturation stages, gonad weights and gonadosomatic index, the peak spawning season was determined to be from June to August. Variations in sea surface temperature (SST) coincided with monthly variations of the gonadosomatic index. Highest monthly average values for both analysed parameters were recorded in July (GSI = 3.38; T = 26.5°C). Fifty per cent (L₅₀) of males and females reached sexual maturity at TL 15.8 cm and at 16.6 cm, respectively. Absolute fecundity ranged from 8458 to 48 032 (mean 34 565 ± 10 310), whereas relative fecundity was from 171 to 722 (mean 385 ± 104.35). Mean value of the oocyte size was 0.53 ± 0.10 mm.     

ROS generation and multiple forms of mammalian mitochondrial glycerol-3-phosphate dehydrogenase

Overproduction of reactive oxygen species (ROS) has been implicated in a range of pathologies. Mitochondrial flavin dehydrogenases glycerol-3-phosphate dehydrogenase (mGPDH) and succinate dehydrogenase (SDH) represent important ROS source, but the mechanism of electron leak is still poorly understood. To investigate the ROS production by the isolated dehydrogenases, we used brown adipose tissue mitochondria solubilized by digitonin as a model. Enzyme activity measurements and hydrogen peroxide production studies by Amplex Red fluorescence, and luminol luminescence in combination with oxygraphy revealed flavin as the most likely source of electron leak in SDH under in vivo conditions, while we propose coenzyme Q as the site of ROS production in the case of mGPDH. Distinct mechanism of ROS production by the two dehydrogenases is also apparent from induction of ROS generation by ferricyanide which is unique for mGPDH. Furthermore, using native electrophoretic systems, we demonstrated that mGPDH associates into homooligomers as well as high molecular weight supercomplexes, which represent native forms of mGPDH in the membrane. By this approach, we also directly demonstrated that isolated mGPDH itself as well as its supramolecular assemblies are all capable of ROS production.     

Influence of Mannosylation on Immunostimulating Activity of Adamant‐1‐yl Tripeptide

The mannosylated derivative of adamant‐1‐yl tripeptide (D ‐(Ad‐1‐yl)Gly‐L ‐Ala‐D ‐isoGln) was prepared to study the effects of mannosylation on adjuvant (immunostimulating) activity. Mannosylated adamant‐1‐yl tripeptide (Man‐OCH₂CH(Me)CO‐D ‐(Ad‐1‐yl)Gly‐L ‐Ala‐D ‐isoGln) is a non‐pyrogenic, H₂O‐soluble, and non‐toxic compound. Adjuvant activity of mannosylated adamantyl tripeptide was tested in the mouse model with ovalbumin as an antigen and in comparison to the parent tripeptide and peptidoglycan monomer (PGM, β‐D ‐GlcNAc‐(1→4)‐D ‐MurNAc‐L ‐Ala‐D ‐isoGln‐mesoDAP(εNH₂)‐D ‐Ala‐D ‐Ala), a well‐known effective adjuvant. The mannosylation of adamantyl tripeptide caused the amplification of its immunostimulating activity in such a way that it was comparable to that of PGM.     

Homeostatic modulation of stimulation-dependent plasticity in human motor cortex

Since recently, it is possible, using noninvasive cortical stimulation, such as the protocol of paired associative stimulation (PAS), to induce the plastic changes in the motor cortex, in humans that mimic Hebb's model of learning. Application of TMS conjugated with peripheral electrical stimulation at strictly coherent temporal manner lead to convergence of inputs in the sensory-motor cortex, with the consequent synaptic potentiation or weakening, if applied repetitively. However, when optimal interstimulus interval (ISI) for induction of LTP-like effects is applied as a single pair, Motor evoked potential (MEP) amplitude inhibition is observed, the paradigm known as short-latency afferent inhibition (SLAI). Aiming to resolve this paradox, PAS protocols were applied, with 200 repetitions of TMS pulses paired with median nerve electrical stimulation, at ISI equal to individual latencies of evoked response of somatosensory cortex (N(20)) (PAS(LTP)), and at ISI of N(20) shortened for 5 msec (PAS(LTD)) - protocols that mimic LTP-like changes in the human motor cortex. MEP amplitudes before, during and after interventions were measured as an indicator based on output signals originating from the motor system. Post-intervention MEP amplitudes following the TMS protocols of PAS(LTP) and PAS(LTD) were facilitated and depressed, respectively, contrary to MEP amplitudes during intervention. During PAS(LTP) MEP amplitudes were significantly decreased in case of PAS(LTP), while in the case of PAS(LTD) an upward trend was observed. In conclusions, a possible explanation for the seemingly paradoxical effect of PAS can be found in the mechanism of homeostatic modulation of plasticity. Those findings indicate the existence of complex relationships in the development of plasticity induced by stimulation, depending on the level of the previous motor cortex excitability.