M. Vrecl - Academia.edu (original) (raw)

Papers by M. Vrecl

animal, 2021

Immunocastrated pigs (IC) exhibit intensive fat deposition after immunisation, but the underlying... more Immunocastrated pigs (IC) exhibit intensive fat deposition after immunisation, but the underlying mechanisms of intensified fat metabolism and deposition are not yet fully understood. Moreover, there is also a lack of comparative studies performed on IC, entire males (EM) and surgical castrates (SC). The main objective of our research was, therefore, to characterise the adipose tissue from the quantitative, histo-morphological and biochemical perspectives in IC 5 weeks after their immunisation in comparison to EM and SC. Immunocastrated pigs had an intermediate position in carcass fatness traits between EM (the leanest) and SC (the fattest). The histo-morphological traits of the subcutaneous adipose tissue of IC were similar to those of SC and differed from those of EM; i.e., they exhibited larger adipocytes in the outer backfat and a larger lobulus surface area in both backfat layers than EM. Intensive fat tissue development in IC was corroborated with higher activities of lipogenic enzymes (i.e., fatty acid synthase, malic enzyme, glucose 6-phosphate dehydrogenase, citrate cleavage enzyme), which was especially pronounced in the subcutaneous adipose tissue of IC (1.5-to 2.7-fold higher activity than in EM or SC). The fatty acid composition of the backfat in IC was similar to that in EM pigs. Both IC and EM exhibited less saturated and more polyunsaturated fatty acids than SC. In contrast, the fatty acid composition of the intramuscular fat of longissimus dorsi muscle in IC pigs was more similar to SC than to EM (higher monounsaturated and lower polyunsaturated fatty acid content in IC and SC than EM). In this study, it was demonstrated that immunocastration notably influenced lipid metabolism. This was shown by increased quantity of lipid depots and with changes in adipose tissue cellularity compared to EM, with changes in the fatty acid composition of the intramuscular fat and enhanced lipogenic activity compared to both EM and SC. These results provide new insights into the specificity of adipose tissue development and deposition in IC compared to EM and SC.

Front. Endocrin., 2012

The idea that seven transmembrane receptors (7TMRs; also designated G-protein coupled receptors, ... more The idea that seven transmembrane receptors (7TMRs; also designated G-protein coupled receptors, GPCRs) might form dimers or higher order oligomeric complexes was formulated more than 20 years ago and has been intensively studied since then. In the last decade, bioluminescence resonance energy transfer (BRET) has been one of the most frequently used biophysical methods for studying 7TMRs oligomerization. This technique enables monitoring physical interactions between protein partners in living cells fused to donor and acceptor moieties. It relies on non-radiative transfer of energy between donor and acceptor, depending on their intermolecular distance (1-10 nm) and relative orientation. Results derived from BRET-based techniques are very persuasive; however, they need appropriate controls and critical interpretation. To overcome concerns about the specificity of BRET-derived results, a set of experiments has been proposed, including negative control with a non-interacting receptor or protein, BRET dilution, saturation, and competition assays. This article presents the theoretical background behind BRET assays, then outlines mathematical models for quantitative interpretation of BRET saturation and competition assay results, gives examples of their utilization and discusses the possibilities of quantitative analysis of data generated with other RET-based techniques.

European Journal of Cancer Supplements, 2010

a delay in the transition from G 2-to M-phase, subsequent blockage of the transition from G 1-to ... more a delay in the transition from G 2-to M-phase, subsequent blockage of the transition from G 1-to S-phase, and apoptosis through caspase activation. Under these specific experimental conditions Tac did not affect MARKCS or ERK1/2 protein levels and phosphorylation state but did alter RSK's activity as this was depicted by the eEF2 phosphorylation levels. Intraperitoneal (ip) administration of Tac at the maximum tolerated dose (MTD), following a [(Q1D5)×2] schedule significantly suppressed growth of HCT116 tumours in xenografts. Conclusions: The results indicate that Tac induced apoptotic cell death to colon cancer cells by a mechanism involving MAPK pathway and more specifically RSK. COMPARE analysis further revealed similarities of the mechanism of action of Tac to that of DNA damaging agents thus linking RSK to DNA damage. In conclusion, the in vitro and in vivo results taken together suggest RSK may be an important novel target for the development of new anticancer therapies.

Journal of Veterinary Medicine Series A, 2006

Clinicopathological and electron microscopical findings of eight cases of enzootic nasal adenocar... more Clinicopathological and electron microscopical findings of eight cases of enzootic nasal adenocarcinoma of sheep, diagnosed solely in one big flock in Slovenia between years 2001 and 2003 are described. All affected sheep were female, their mean age was 4.5 ± 1.5 years and they either belonged to the Istrian pramenka breed (five sheep) or were crossbreeds (three sheep). Tumours that arose from the ethmoid area of the nasal cavity were unilateral in six cases (75%) and bilateral in two cases (25%). All tumours were classified as adenocarcinomas by histopathological examination and they displayed either a combination of tubular and papillary growth or less often solely tubular proliferation. No metastases were detected in regional lymph nodes, brain or other organs. Electron microscopical studies performed on the reprocessed paraffin-embedded tissues revealed the presence of the virus-like particles with an average diameter between 70 and 90 nm.

Nutrition, 2007

The aim of this study was to determine if protein metabolism was altered in small and large intes... more The aim of this study was to determine if protein metabolism was altered in small and large intestines by feeding pectin, a soluble fiber known to stimulate cecal production of short-chain fatty acids (SCFAs) and to have a trophic effect in these tissues. Methods: Twenty-four weanling male Sprague-Dawley rats were fed ad libitum for 14 d with a balanced control diet or an isoproteic, isocaloric pectin (citrus) diet (80 g/kg). SCFA production, intestinal histomorphometry, and protein synthesis were determined in the proximal and distal parts of the small intestine, the cecum, and the colon. Protein synthesis rates were determined by measuring the 13 C valine incorporation rate in tissue proteins. Results: Pectin feeding slightly decreased food intake and growth rate. It increased the acetate, propionate, and butyrate pools in the cecum. Pectin feeding resulted in heavier intestinal tissues corresponding to higher villus height in the small intestine and crypt depth in the small and large intestines compared with feeding of the control diet. Compared with the control group, the rats fed the pectin diet had significantly higher protein synthesis rates in all the parts of their intestines. Conclusion: Supplementation of pectin, as a soluble fiber, in the diets, stimulated SCFA production, had a trophic effect on the different parts of the intestines, and greatly stimulated protein synthesis in those tissues.

SLAS Discovery, 2009

CB1 receptor (CB1R) antagonists have been demonstrated to be effective in treating obesity and re... more CB1 receptor (CB1R) antagonists have been demonstrated to be effective in treating obesity and related disorders. This study has been focused on establishing a β-arrestin 2—based screening assay for the CB1R using BRET2 technology. When the existing BRET2 screening platform was applied to the CB1R, the authors discovered that the receptor interacted weakly with β-arrestin 2, resulting in unsatisfactory assay performance. To enhance the β-arrestin binding capacity, they replaced the C-terminal tail of the CB1R with tails from either the V2 or BRS3 receptors, both of which interact strongly with β-arrestin 2. Using this chimeric approach, the authors screened a small compound library and identified 21 antagonist and inverse agonist hits with IC50 and EC50 values ranging from 0.3 nM to 7.5 µM. Both primary and secondary screening were performed with Z' > 0.5, suggesting that the assay is a robust and cost-effective alternative to existing cell-based assays. ( Journal of Biomolec...

SLAS Discovery, 2004

This study has focused on enhancing the signal generated from the interaction between a G-protein... more This study has focused on enhancing the signal generated from the interaction between a G-protein-coupled receptor (GPCR) and β-arrestin 2 (β-arr2), measured by the bioluminescence resonance energy transfer (BRET2) technology. Both class A (β2-adrenergic receptor [β2-AR]) and class B (neurokinin-type 1 receptor [NK1-R]) GPCRs, classified based on their internalization characteristics, have been analyzed. It was evaluated whether the BRET2 signal can be enhanced by using (1) β-arr2 phosphorylation-independent mutant (β-arr2 R169E) and (2) β-arr2 mutants deficient in their ability to interact with the components of the clathrin-coated vesicles (β-arr2 R393E, R395E and β-arr2 373 stop). For the class B receptor, there was no major difference in the agonist-promoted BRET2 signal when comparing results obtained with wild-type (wt) and mutant β-arr2. However, with the class A receptor, a more than 2-fold increase in the BRET2 signal was observed with β-arr2 mutants lacking the AP-2 or bot...

Journal of Biological Chemistry, 1998

The mammalian gonadotropin-releasing hormone receptor (GnRH-R) is the only G-protein-coupled rece... more The mammalian gonadotropin-releasing hormone receptor (GnRH-R) is the only G-protein-coupled receptor (GPCR) in which the intracellular C-terminal tail is completely absent. In contrast to other GPCRs, the GnRH-R does not show rapid desensitization of total inositol (IP) production, and the rates of internalization are exceptionally slow. We investigated whether the incorporation of a cytoplasmic tail into the C terminus of the GnRH-R affects desensitization events and receptor internalization rates. A GnRH-R/TRH-R chimera was created where the intracellular tail of the rat thyrotropin-releasing hormone receptor (TRH-R) was engineered into the C terminus of the rat GnRH-R. Three different rat GnRH-R cDNA stop codon mutations (one for each reading frame) were also made. The GnRHstimulated IP production of the wild-type rat GnRH-R expressed in either COS-7 or HEK 293 cells did not desensitize even after prolonged stimulation with GnRH. In contrast, the catfish GnRH-R (which does possess an intracellular tail) and the TRH-R rapidly (<10 min) desensitized following agonist stimulation. The GnRH-R/TRH-R chimera also desensitized following treatment with GnRH, resembling the pattern shown by the TRH-R and the catfish GnRH-R. Two of the stop codon mutants did not show desensitization of IP production, and the third mutant with the longest tail was not functional. Internalization experiments showed that the rat GnRH-R had the slowest endocytosis and recycling rates compared with the TRH-R, the catfish GnRH-R, and the chimeric GnRH/TRH-R. This study demonstrates that the addition of a functional intracellular C-terminal tail to the GnRH-R produces rapid desensitization of IP production and significantly increases internalization rates. Gonadotropin-releasing hormone (GnRH) 1 is a decapeptide released by the hypothalamus which acts on specific receptors in the anterior pituitary. GnRH and its receptor (GnRH-R) are

Cell and Tissue Research, 2007

This study was focused on the relationship between the plasma-membrane localization of neurokinin... more This study was focused on the relationship between the plasma-membrane localization of neurokinin-1 receptor (NK1-R) and its endocytic and signaling properties. First, we employed electron paramagnetic resonance (EPR) to study the domain structure of HEK-293 cells and NK1-R microlocalization. EPR spectra and the GHOST condensation routine demonstrated that NK1-R was distributed in a well-ordered domain of HEK-293 cells possibly representing lipid raft/caveolae microdomains, whereas the impairment of caveolae changed the NK1-R plasma-membrane distribution. Internalization and second messenger assays combined with bioluminescence resonance energy transfer were employed subsequently to evaluate the functional importance of the NK1-R microlocalization in lipid raft/caveolae microdomains. The internalization pattern was delineated through the use of dominant-negative mutants (DNM) of caveolin-1 S80E (Cav1 S80E), dynamin-1 K44A (Dyn K44A), and beta-arrestin (beta-arr 319-418) and by means of cell lines that expressed various endogenous levels of beta-arrestins. NK1-R displayed rapid internalization that was substantially reduced by DNMs of dynamin-1 and beta-arrestin and even more profoundly in cells lacking both beta-arrestin1 and beta-arrestin2. These internalization data were highly suggestive of the predominant use of the clathrin-mediated pathway by NK1-R, even though NK1-R tended to reside constitutively in lipid raft/caveolae microdomains. Evidence was also obtained that the proper clustering of the receptor in these microdomains was important for effective agonist-induced NK1-R signaling and for its interaction with beta-arrestin2.

Anatomia, Histologia, Embryologia: Journal of Veterinary Medicine Series C, 2002

This study examined the frequency, morphological and immunohistochemical characteristics of the g... more This study examined the frequency, morphological and immunohistochemical characteristics of the giant fibres in the longissimus muscle of local Krsˇko polje pigs with different Ryr1 genotypes. Giant fibres were round-shaped and had significantly increased cross-sectional area compared with normal muscle fibres. Only fast-twitch glycolytic fibres were affected, usually showing enhanced succinate dehydrogenase activity. On the ultrastructural level, the dilation of the sarcoplasmic reticulum, swelling of mitochondria and destruction of myofilaments was observed. The incidence of giant fibres was the highest in Ryr1 dimutant pigs (Ryr1 nn), which also exhibited lower muscle pH1 than heterozygous (Ryr1 Nn) or pigs with the wild Ryr1 gene (Ryr1 NN). However, the giant fibres were also present in pigs free of Ryr1 gene mutation. Our results suggest that the giant fibre syndrome depends mostly upon the rate and intensity of early post-mortem glycolysis, which results in acidity of muscle tissue. We suppose that the giant fibre formation is a result of excessive intracellular lactate accumulation in some fast-twitch glycolytic fibres. This process could also explain the ultrastructural alterations and the consequent changes in the oxidative enzymes and myofibrillar ATPase staining pattern observed in our and some previous studies.

Molecular and Cellular Endocrinology, 2008

H2 relaxin, a member of the insulin superfamily, binds to the G-protein-coupled receptor RXFP1 (r... more H2 relaxin, a member of the insulin superfamily, binds to the G-protein-coupled receptor RXFP1 (relaxin family peptide 1), a receptor that belongs to the leucine-rich repeat (LRR)-containing subgroup (LGRs) of class A GPCRs. We recently demonstrated negative cooperativity in INSL3 binding to RXFP2 and showed that this subgroup of GPCRs functions as constitutive dimers. In this work, we investigated whether the binding of H2 relaxin to RXFP1 also shows negative cooperativity, and whether this receptor functions as a dimer using BRET 2. Both binding and dissociation were temperature dependent, and the pH optimum for binding was pH 7.0. Our results showed that RXFP1 is a constitutive dimer with negative cooperativity in ligand binding, that dimerization occurs through the 7TM domain, and that the ectodomain has a stabilizing effect on this interaction. Dimerization and negative cooperativity appear to be general properties of LGRs involved in reproduction as well as other GPCRs.

Toxicological Sciences, 2015

Ostreolysin A (OlyA) and pleurotolysin B (PlyB), isolated from edible oyster mushrooms, form a cy... more Ostreolysin A (OlyA) and pleurotolysin B (PlyB), isolated from edible oyster mushrooms, form a cytolytic complex (OlyA/PlyB) in membrane cells that causes respiratory arrest. This study evaluated the mechanisms underlying cytotoxic OlyA/PlyB activity in neuroblastoma NG108-15 cells. Confocal microscopy with morphometric analysis revealed that OlyA/PlyB increased the 3-dimensional projected area of differentiated cells. Iso-osmotic replacement of NaCl by sucrose or Na-isethionate prevented the cellular swelling. This suggests that formation of cellular edema requires the presence of Na(+) and/or Cl(-) in the extracellular space and may be related to an influx of Na(+) and/or a shift in Cl(-), which induce a marked influx of water that is ultimately responsible for cellular swelling. In addition, extracellular Ca(2+) moderately contributed to the swelling because benzamil (10 µM), a 3Na(+)/Ca(2+) exchange (NCX) inhibitor, and Ca(2+)-free medium partially prevented this response. Fluorometric measurements revealed that OlyA/PlyB, at approximately 15-fold higher concentrations, increased the intracellular Ca(2+) activity [Ca(2+)]i. This increase was dependent on the presence of Na(+) and Ca(2+) in the external medium and was sensitive to benzamil. It is thus likely that a switch in the NCX mode, associated with the de novo formation of non-selective ion pores by OlyA/PlyB in cellular plasma membranes, plays an important role in this effect. Overall, OlyA/PlyB affects neuroblastoma cell morphology and Ca(2+) homeostasis to influence the toxin-induced respiratory arrest.

Enzyme and Microbial Technology, 2005

A keratinase, produced biotechnologically by Doratomyces microsporus, was used to treat porcine s... more A keratinase, produced biotechnologically by Doratomyces microsporus, was used to treat porcine skin in vitro under different experimental conditions. The amount of soluble proteins, their electrophoretic profile and the histological appearance of the skin cuttings were followed. The amount of soluble protein released by the keratinase was maximal after 6 h of incubation in neutral or alkaline environments up to pH 9. With prolonged incubation, the amount of protein released due to the enzyme decreased on account of further hydrolysis of soluble proteins into smaller units. As shown by SDS-PAGE, the proteins of 45-70 kDa detected in controls were hydrolysed by the enzyme to fragments of 14 kDa or less after 3 h incubation in the optimal pH range. Histological examination of the skin cuttings suggested that the enzyme was active throughout the incubation period, since the effect on epidermis progressed with time. Stratum corneum was detached from the underlying layers of epidermis that were severely disintegrated and separated from the unaffected dermis at the epidermal-dermal zone. Keratinase also hydrolysed the outer epithelial sheath of hair roots provoking depilation. These data suggest the potential of this enzyme for application in ecologically-friendly leather processing.

Endocrinology, 2000

This study examined the mechanism underlying the rat GnRH receptor (GnRH-R) internalization pathw... more This study examined the mechanism underlying the rat GnRH receptor (GnRH-R) internalization pathway by investigating the role of added/extended C-terminal tails and the effect of beta-arrestins and dynamin. The internalization of the wild-type (WT) rat GnRH-R, stop codon mutants, GnRH-R/TRH receptor (TRH-R) chimera, rat TRH-R, and catfish GnRH-R was examined using radioligand binding assay. Overexpression of beta-arrestin in COS-7 cells expressing each of the receptor constructs substantially increased endocytosis rate constants (k(e)) of the TRH-R, catfish GnRH-R, and GnRH-R/TRH-R chimera, but not of the WT rat GnRH-R and stop codon mutants. The beta-arrestin-promoted increase in the k(e) value was diminished by cotransfecting cells with the dominant negative beta-arrestin-(319-418) mutant, whereas WT GnRH-R and stop codon mutant internalization were unaffected. Additionally, confocal microscopy showed that activated GnRH-Rs failed to induce time-dependent redistribution of either beta-arrestin-1- or beta-arrestin-2-green fluorescent protein conjugate to the plasma membrane. However, the dominant negative dynamin (DynK44A) mutant impaired internalization of all of the receptors regardless of their beta-arrestin dependency, indicating that they internalize via a clathrin-mediated pathway. We conclude that the mammalian GnRH-R uses a beta-arrestin-independent, dynamin-dependent internalization mechanism distinct from that employed by the other receptors studied.

Bioluminescence - Recent Advances in Oceanic Measurements and Laboratory Applications, 2012

animal, 2016

Dynamics of myofiber differentiation/maturation in porcine skeletal muscle is associated with dom... more Dynamics of myofiber differentiation/maturation in porcine skeletal muscle is associated with domestication, breeding and rearing conditions. This study was aimed to comparatively elucidate the age-dependent myosin heavy chain (MyHC) isoform expression and transition pattern in domestic and wild pig (WP) skeletal muscle from birth until adulthood. Domestic pigs (DPs) of Large White breed raised in conventional production system were compared with WPs reared in a large hunting enclosure. Muscle samples for immuno/enzyme histochemistry were taken from the longissimus dorsi muscle within 24 h postmortem at 24 to 48 h, 21 to 23 days, 7 months and ~2 years postpartum. Based on the antibody reactivity to MyHCs (NCL-MHCs, A4.74, BF-F3) and succinate dehydrogenase activity, myofibers were classified into I, I/IIa, IIa, IIx and IIb types. In addition, foetal MyHC expression was determined with the use of F158.4C10 antibody. Maturation of the longissimus dorsi muscle in the WP was characteriz...

Biochemical Society Transactions, 1999

Microbiology (Reading, England), 1997

The upper pathway operon of the toluene catabolic pathway of TOL plasmid pWW0 was shown to carry ... more The upper pathway operon of the toluene catabolic pathway of TOL plasmid pWW0 was shown to carry two open reading frames between the start of transcription and xylC (encoding benzaldehyde dehydrogenase), the first previously reported gene of the operon. These were designated xylUW: xylU encoded a protein of 131 amino acid residues (M(r) 14,244) which bore no relationship with any protein in the databases, and xylW encoded a protein of 348 residues (M(r) 36,992) which was strongly homologous to other long-chain Zn-containing alcohol dehydrogenases. Extracts of Escherichia coli carrying xylUW in expression vector pTrc99A contained a novel protein corresponding to XylW, but no NAD(+)-dependent dehydrogenase activity against benzyl alcohol, mandelate or bezylamine. A mini-Tn5 transposon carrying the meta pathway operon was constructed and from it two strains of Pseudomonas putida were constructed with the normally plasmid-encoded catabolic operons integrated into the chromosome. Three d...