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71.
The SOS response master regulator LexA is associated with sporulation, motility and biofilm formation in Clostridium difficile
Beata Maria Walter, Stephen Cartman, Nigel Peter Minton, Matej Butala, Maja Rupnik, 2015, izvirni znanstveni članek

Opis: The LexA regulated SOS network is a bacterial response to DNA damage of metabolic or environmental origin. In Clostridium difficile, a nosocomial pathogen causing a range of intestinal diseases, the in-silico deduced LexA network included the core SOS genes involved in the DNA repair and genes involved in various other biological functions that vary among different ribotypes. Here we describe the construction and characterization of a lexA ClosTron mutant in C. difficile R20291 strain. The mutation of lexA caused inhibition of cell division resulting in a filamentous phenotype. The lexA mutant also showed decreased sporulation, a reduction in swimming motility, greater sensitivity to metronidazole, and increased biofilm formation. Changes in the regulation of toxin A, but not toxin B, were observed in the lexA mutant in the presence of sub-inhibitory concentrations of levofloxacin. C. difficile LexA is, therefore, not only a regulator of DNA damage but also controls many biological functions associated with virulence.
Ključne besede: toxins, bacterial sporulation, DNA damage, antibiotics
Objavljeno: 19.06.2017; Ogledov: 86; Prenosov: 1
.pdf Celotno besedilo (6,06 MB)

72.
The relationship between membrane potential and calcium dynamics in glucose-stimulated beta cell syncytium in acute mouse pancreas tissue slices
Jurij Dolenšek, Andraž Stožer, Maša Skelin, Evan Miller, Marjan Rupnik, 2013, izvirni znanstveni članek

Opis: Oscillatory electrical activity is regarded as a hallmark of the pancreatic beta cell glucose-dependent excitability pattern. Electrophysiologically recorded membrane potential oscillations in beta cells are associated with in-phase oscillatory cytosolic calcium activity ([Ca2+]i) measured with fluorescent probes. Recent high spatial and temporal resolution confocal imaging revealed that glucose stimulation of beta cells in intact islets within acute tissue slices produces a [Ca2+]i change with initial transient phase followed by a plateau phase with highly synchronized [Ca2+]i oscillations. Here, we aimed to correlate the plateau [Ca2+]i oscillations with the oscillations of membrane potential using patch-clamp and for the first time high resolution voltage-sensitive dye based confocal imaging. Our results demonstrated that the glucose-evoked membrane potential oscillations spread over the islet in a wave-like manner, their durations and wave velocities being comparable to the ones for [Ca2+]i oscillations and waves. High temporal resolution simultaneous records of membrane potential and [Ca2+]i confirmed tight but nevertheless limited coupling of the two processes, with membrane depolarization preceding the [Ca2+]i increase. The potassium channel blocker tetraethylammonium increased the velocity at which oscillations advanced over the islet by several-fold while, at the same time, emphasized differences in kinetics of the membrane potential and the [Ca2+]i. The combination of both imaging techniques provides a powerful tool that will help us attain deeper knowledge of the beta cell network.
Ključne besede: glucose, pancreas, mice
Objavljeno: 19.06.2017; Ogledov: 104; Prenosov: 2
.pdf Celotno besedilo (2,57 MB)

73.
The analysis of intracellular and intercellular calcium signaling in human anterior lens capsule epithelial cells with regard to different types and stages of the cataract
Marko Gosak, Rene Markovič, Aleš Fajmut, Marko Marhl, Marko Hawlina, Sofija Andjelić, 2015, izvirni znanstveni članek

Opis: In this work we investigated how modifications of the Ca2+ homeostasis in anterior lens epithelial cells (LECs) are associated with different types of cataract (cortical or nuclear) and how the progression of the cataract (mild or moderate) affects the Ca2+ signaling. We systematically analyzed different aspects of intra- and inter-cellular Ca2+ signaling in the human LECs, which are attached to surgically isolated lens capsule (LC), obtained during cataract surgery. We monitored the temporal and spatial changes in intracellular Ca2+ concentration after stimulation with acetylcholine by means of Fura-2 fluorescence captured with an inverted microscope. In our analysis we compared the features of Ca2+ signals in individual cells, synchronized activations, spatio-temporal grouping and the nature of intercellular communication between LECs. The latter was assessed by using the methodologies of the complex network theory. Our results point out that at the level of individual cells there are no significant differences when comparing the features of the signals with regard either to the type or the stage of the cataract. On the other hand, noticeable differences are observed at the multicellular level, despite inter-capsule variability. LCs associated with more developed cataracts were found to exhibit a slower collective response to stimulation, a less pronounced spatio-temporal clustering of LECs with similar signaling characteristics. The reconstructed intercellular networks were found to be sparser and more segregated than in LCs associated with mild cataracts. Moreover, we show that spontaneously active LECs often operate in localized groups with quite well aligned Ca2+ activity. The presence of spontaneous activity was also found to affect the stimulated Ca2+ responses of individual cells. Our findings indicate that the cataract progression entails the impairment of intercellular signaling thereby suggesting the functional importance of altered Ca2+ signaling of LECs in cataractogenesis.
Ključne besede: intracellular calcium signaling, anterior lens, epithelial cells, cataract
Objavljeno: 19.06.2017; Ogledov: 104; Prenosov: 1
.pdf Celotno besedilo (1,45 MB)

74.
Sequence similarity of Clostridium difficile strains by analysis of conserved genes and genome content is reflected by their ribotype affiliation
Hedwig Kurka, Armin Ehrenreich, Wolfgang Ludwig, Marc Monot, Maja Rupnik, Frédéric Barbut, Alexander Indra, Bruno Dupuy, Wolfgang Liebl, 2014, izvirni znanstveni članek

Opis: PCR-ribotyping is a broadly used method for the classification of isolates of Clostridium difficile, an emerging intestinal pathogen, causing infections with increased disease severity and incidence in several European and North American countries. We have now carried out clustering analysis with selected genes of numerous C. difficile strains as well as gene content comparisons of their genomes in order to broaden our view of the relatedness of strains assigned to different ribotypes. We analyzed the genomic content of 48 C. difficile strains representing 21 different ribotypes. The calculation of distance matrix-based dendrograms using the neighbor joining method for 14 conserved genes (standard phylogenetic marker genes) from the genomes of the C. difficile strains demonstrated that the genes from strains with the same ribotype generally clustered together. Further, certain ribotypes always clustered together and formed ribotype groups, i.e. ribotypes 078, 033 and 126, as well as ribotypes 002 and 017, indicating their relatedness. Comparisons of the gene contents of the genomes of ribotypes that clustered according to the conserved gene analysis revealed that the number of common genes of the ribotypes belonging to each of these three ribotype groups were very similar for the 078/033/126 group (at most 69 specific genes between the different strains with the same ribotype) but less similar for the 002/017 group (86 genes difference). It appears that the ribotype is indicative not only of a specific pattern of the amplified 16S23S rRNA intergenic spacer but also reflects specific differences in the nucleotide sequences of the conserved genes studied here. It can be anticipated that the sequence deviations of more genes of C. difficile strains are correlated with their PCR-ribotype. In conclusion, the results of this study corroborate and extend the concept of clonal C. difficile lineages, which correlate with ribotypes affiliation.
Ključne besede: clostridium difficile, comparative genomics, RNA polymerase
Objavljeno: 19.06.2017; Ogledov: 91; Prenosov: 3
.pdf Celotno besedilo (1,35 MB)

75.
Recombination drives evolution of the Clostridium difficile 16S-23S rRNA intergenic spacer region
Sandra Janežič, Alexander Indra, Thomas Rattei, Thomas Weinmaier, Maja Rupnik, 2014, izvirni znanstveni članek

Opis: PCR-ribotyping, a typing method based on size variation in 16S-23S rRNA intergenic spacer region (ISR), has been used widely for molecular epidemiological investigations of C. difficile infections. In the present study, we describe the sequence diversity of ISRs from 43 C. difficile strains, representing different PCR-ribotypes and suggest homologous recombination as a possible mechanism driving the evolution of 16S-23S rRNA ISRs. ISRs of 45 different lengths (ranging from 185 bp to 564 bp) were found among 458 ISRs. All ISRs could be described with one of the 22 different structural groups defined by the presence or absence of different sequence modules; tRNAAla genes and different combinations of spacers of different lengths (33 bp, 53 bp or 20 bp) and 9 bp direct repeats separating the spacers. The ISR structural group, in most cases, coincided with the sequence length. ISRs that were of the same lengths had also very similar nucleotide sequence, suggesting that ISRs were not suitable for discriminating between different strains based only on the ISR sequence. Despite large variations in the length, the alignment of ISR sequences, based on the primary sequence and secondary structure information, revealed many conserved regions which were mainly involved in maturation of pre-rRNA. Phylogenetic analysis of the ISR alignment yielded strong evidence for intra- and inter-homologous recombination which could be one of the mechanisms driving the evolution of C. difficile 16S-23S ISRs. The modular structure of the ISR, the high sequence similarities of ISRs of the same sizes and the presence of homologous recombination also suggest that different copies of C. difficile 16S-23S rRNA ISR are evolving in concert
Ključne besede: clostridium difficile, sequence analysis, ribosomal RNS
Objavljeno: 19.06.2017; Ogledov: 64; Prenosov: 3
.pdf Celotno besedilo (846,32 KB)

76.
Rab3a is critical for trapping alpha-MSH granules in the high Ca2+-affinity pool by preventing constitutive exocytosis
Simon Sedej, Maša Skelin, Oliver Schlüter, Marjan Rupnik, 2013, izvirni znanstveni članek

Opis: Rab3a is a small GTPase of the Rab3 subfamily that acts during late stages of Ca2+-regulated exocytosis. Previous functional analysis in pituitary melanotrophs described Rab3a as a positive regulator of Ca2+-dependent exocytosis. However, the precise role of the Rab3a isoform on the kinetics and intracellular [Ca2+] sensitivity of regulated exocytosis, which may affect the availability of two major peptide hormones, -melanocyte stimulating hormone (-MSH) and -endorphin in plasma, remain elusive. We employed Rab3a knock-out mice (Rab3a KO) to explore the secretory phenotype in melanotrophs from fresh pituitary tissue slices. High resolution capacitance measurements showed that Rab3a KO melanotrophs possessed impaired Ca2+-triggered secretory activity as compared to wild-type cells. The hampered secretion was associated with the absence of cAMP-guanine exchange factor II/ Epac2-dependent secretory component. This component has been attributed to high Ca2+-sensitive release-ready vesicles as determined by slow photo-release of caged Ca2+. Radioimmunoassay revealed that -MSH, but not -endorphin, was elevated in the plasma of Rab3a KO mice, indicating increased constitutive exocytosis of -MSH. Increased constitutive secretion of -MSH from incubated tissue slices was associated with reduced -MSH cellular content in Rab3a-deficient pituitary cells. Viral re-expression of the Rab3a protein in vitro rescued the secretory phenotype of melanotrophs from Rab3a KO mice. In conclusion, we suggest that Rab3a deficiency promotes constitutive secretion and underlies selective impairment of Ca2+-dependent release of -MSH.
Ključne besede: melanotrophs, exocytosis
Objavljeno: 19.06.2017; Ogledov: 47; Prenosov: 1
.pdf Celotno besedilo (1,90 MB)

77.
Platelet-rich plasma, especially when combined with a TGF-ß inhibitor promotes proliferation, viability and myogenic differentiation of myoblasts in vitro
Robi Kelc, Martin Trapečar, Lidija Gradišnik, Marjan Rupnik, Matjaž Vogrin, 2015, izvirni znanstveni članek

Opis: Regeneration of skeletal muscle after injury is limited by scar formation, slow healing time and a high recurrence rate. A therapy based on platelet-rich plasma (PRP) has become a promising lead for tendon and ligament injuries in recent years, however concerns have been raised that PRP-derived TGF-β could contribute to fibrotic remodelling in skeletal muscle after injury. Due to the lack of scientific grounds for a PRP -based muscle regeneration therapy, we have designed a study using human myogenic progenitors and evaluated the potential of PRP alone and in combination with decorin (a TGF-β inhibitor), to alter myoblast proliferation, metabolic activity, cytokine profile and expression of myogenic regulatory factors (MRFs). Advanced imaging multicolor single-cell analysis enabled us to create a valuable picture on the ratio of quiescent, activated and terminally committed myoblasts in treated versus control cell populations. Finally high-resolution confocal microscopy validated the potential of PRP and decorin to stimulate the formation of polynucleated myotubules. PRP was shown to down-regulate fibrotic cytokines, increase cell viability and proliferation, enhance the expression of MRFs, and contribute to a significant myogenic shift during differentiation. When combined with decorin further synergistc effects were identified. These results suggest that PRP could not only prevent fibrosis but could also stimulate muscle commitment, especially when combined with a TGF-β inhibitor.
Ključne besede: muscles, skeletal, injuries, TGF-beta, plasma, thrombocytes, myoblasts, fibrosis, prevention, regeneration
Objavljeno: 19.06.2017; Ogledov: 61; Prenosov: 1
.pdf Celotno besedilo (2,07 MB)

78.
Novel insights into the downstream pathways and targets controlled by transcription factors CREM in the testis
Rok Košir, Peter Juvan, Martina Perše, Tomaž Büdefeld, Gregor Majdič, Martina Fink, Paolo Sassone-Corsi, Damjana Rozman, 2012, izvirni znanstveni članek

Opis: The essential role of the Crem gene in normal sperm development is widely accepted and is confirmed by azoospermia in male mice lacking the Crem gene. The exact number of genes affected by Crem absence is not known, however a large difference has been observed recently between the estimated number of differentially expressed genes found in Crem knock-out (KO) mice compared to the number of gene loci bound by CREM. We therefore re-examined global gene expression in male mice lacking the Crem gene using whole genome transcriptomeanalysis with Affymetrix microarrays and compared the lists of differentially expressed genes from Crem-/- mice to a dataset of genes where binding of CREM was determined by Chip-seq. We determined the global effect ofCREM on spermatogenesis as well as distinguished between primary and secondary effects of the CREM absence. We demonstrated that the absence of Crem deregulates over 4700 genes in KO testis. Among them are 101 genes associated with spermatogenesis 41 of which are bound by CREM and are deregulated in Crem KO testis. Absence of several of these genes in mouse models has proven their importance for normal spermatogenesis and male fertility. Our study showed that the absence of Crem plays a more important role on different aspects of spermatogenesis as estimated previously, with itsimpact ranging from apoptosis induction to deregulation of major circadian clock genes, steroidogenesis and the cell-cell junction dynamics. Several new genes important for normal spermatogenesis and fertility are down-regulated inKO testis and are therefore possible novel targets of CREM.
Ključne besede: spermatogenesis, genetics, Crem, mice
Objavljeno: 19.06.2017; Ogledov: 28; Prenosov: 1
.pdf Celotno besedilo (1,77 MB)

79.
Malpighian tubule cells in overwintering cave crickets Troglophilus cavicola (Kollar, 1833) and T. neglectus Krauss, 1879 (Rhaphidophoridae, Ensifera)
Saška Lipovšek Delakorda, Tone Novak, Franc Janžekovič, Nina Weiland, Gerd Leitinger, 2016, izvirni znanstveni članek

Opis: During winter, cave cricket larvae undergo dormancy in subterranean habitats; this dormancy is termed diapause in second year Troglophilus cavicola larvae because they mature during this time, and termed quiescence in T. neglectus, because they mature after dormancy. Here we used electron microscopy to analyze ultrastructural changes in the epithelial cells in the Malpighian tubules (MTs) of T. cavicola during diapause, in order to compare them with previous findings on T. neglectus. Moreover, the autophagosomes were studied with immunofluorescence microscopy in both species. Although the basic ultrastructure of the cells was similar, specific differences appeared during overwintering. During this natural starvation period, the nucleus, rER, the Golgi apparatus and mitochondria did not show structural changes, and the spherites were exploited. The abundances of autophagic structures in both species increased during overwintering. At the beginning of overwintering, in both species and sexes, the rates of cells with autophagic structures (phagophores, autophagosomes, autolysosomes and residual bodies) were low, while their rates increased gradually towards the end of overwintering. Between sexes, in T. cavicola significant differences were found in the autophagosome abundances in the middle and at the end, and in T. neglectus at the end of overwintering. Females showed higher rates of autophagic cells than males, and these were more abundant in T. cavicola. Thus, autophagic processes in the MT epithelial cells induced by starvation are mostly parallel in diapausing T. cavicola and quiescent T. neglectus, but more intensive in diapausing females.
Ključne besede: cave cricket, overwintering, Troglophilus cavicola, Troglophilus neglectus
Objavljeno: 19.06.2017; Ogledov: 35; Prenosov: 2
.pdf Celotno besedilo (9,29 MB)

80.
Highly divergent Clostridium difficile strains isolated from the environment
Sandra Janežič, Mojca Potočnik, Valerija Tkalec, Maja Rupnik, 2016, izvirni znanstveni članek

Opis: Clostridium difficile is one of the most important human and animal pathogens. However, the bacterium is ubiquitous and can be isolated from various sources. Here we report the prevalence and characterization of C. difficile in less studied environmental samples, puddle water (n = 104) and soil (n = 79). C. difficile was detected in 14.4% of puddle water and in 36.7% of soil samples. Environmental strains displayed antimicrobial resistance patterns comparable to already published data of human and animal isolates. A total of 480 isolates were grouped into 34 different PCR ribotypes. More than half of these (52.9%; 18 of 34) were already described in humans or animals. However, 14 PCR ribotypes were new in our PCR ribotype library and all but one were non-toxigenic. The multilocus sequence analysis of these new PCR ribotypes revealed that non-toxigenic environmental isolates are phylogenetically distinct and belong to three highly divergent clades, two of which have not been described before. Our data suggest that environment is a potential reservoir of genetically diverse population of C. difficile.
Ključne besede: clostridium difficile, environment, isolation
Objavljeno: 19.06.2017; Ogledov: 60; Prenosov: 4
.pdf Celotno besedilo (1,13 MB)

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