Azra Pogačnik

Development of a BRET2 Screening Assay Using β-Arrestin 2 Mutants

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 both AP-2 and clathrin binding sites. This set of data suggests that the inability of these β-arr2 mutants to interact with the components of the clathrin-coated vesicle probably prevents their rapid dissociation from the receptor, thus yielding an increased and more stable BRET2 signal. The β-arr2 R393E, R395E mutant also enhanced the signal window with other members of the GPCR family (neuropeptide Y type 2 receptor [NPY2-R] and TG1019 receptor) and was successfully applied in full-plate BRET2-based agonist and antagonist screening assays.

Local and systemic antitumor effect of intratumoral and peritumoral IL-12 electrogene therapy on murine sarcoma

Soft tissue sarcomas pose a challenge for successful treatment with conventional therapeutic methods, therefore newer therapeutic approaches are considered. In this study, we evaluated the antitumor effect of IL-12 electrogene therapy (EGT) on murine SA-1 fibrosarcoma. The Therapeutic plasmid was injected either intratumorally into subcutaneous SA-1 nodules or intradermally into the peritumoral region. We achieved a remarkable local and systemic antitumor effect with both approaches after single plasmid DNA application, with significant intratumoral and systemic production of IL-12 and IFN-γ. Intratumoral IL-12 EGT resulted in over 90% complete response rate of the treated tumors with 60% of cured mice being resistant to challenge with SA-1 tumor cells. Peritumoral EGT resulted in a lower complete response rate (16%), with significant growth delay of remaining tumors. Both therapies also resulted in significant inhibition of growth of untreated tumors, growing simultaneously at a distant site. These data suggest that IL-12 EGT may be useful in the treatment of soft tissue sarcomas, exerting a local and systemic antitumor effect.

Efficient Electrotransfection into Canine Muscle

Two different types of electroporation protocols have been developed for efficient electro-transfer of plasmid DNA into skeletal muscle of experimental animals. At first, only low voltage electric pulses have been used, but lately, a combination of high and low voltage pulses has been suggested as more efficient. Up to date, in dogs, this type of electroporation protocol has never been used for muscle targeted plasmid DNA electrotransfection. In this study, we used two different DNA plasmids, one encoding green fluorescent protein and one encoding human interleukin-12. Five different electroporation protocols were evaluated. Three of them featured different combinations of high and low voltage pulses, and two were performed with delivery of low voltage pulses only. Our study shows that combination of 1 high voltage pulse (600 V/cm, 100 μs), followed by 4 low voltage pulses (80 V/cm, 100 ms, 1 Hz) yielded in the same transfection efficiency as the standard trains of low voltage pulses. However, this protocol is performed quicker and, thus, more suitable for potential use in clinical practice. In addition, it yielded in detectable systemic expression of human interleukin-12. Electrotransfer of either of the plasmids was associated with only mild and transitory local side effects, without clinically detectable systemic side effects. The results indicate that electrotransfection is a feasible, effective, and safe method for muscle targeted gene therapy in dogs, which could have potential for clinical applications in veterinary medicine of small animals.

Evidence for a role of caveolin-1 in neurokinin-1 receptor plasma-membrane localization, efficient signaling, and interaction with β-arrestin 2

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 β-arrestin (β-arr 319–418) and by means of cell lines that expressed various endogenous levels of β-arrestins. NK1-R displayed rapid internalization that was substantially reduced by DNMs of dynamin-1 and β-arrestin and even more profoundly in cells lacking both β-arrestin1 and β-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 β-arrestin2.

Giant muscle fibres in pigs with different Ryr1 genotype.

This study examined the frequency, morphological and immunohistochemical characteristics of the giant fibres in the longissimus muscle of local Krško 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.

Myosin heavy chain isoform transitions in canine skeletal muscles during postnatal growth

To gain a better understanding of the normal characteristics of developing canine muscles, myosin heavy chain (MHC) isoform expression was analysed in the axial and limb skeletal muscles of 18 young dogs whose ages ranged from the late prenatal stage to 6 months. We compared the results of immunohistochemistry using ten monoclonal antibodies, specific to different MHC isoforms, and enzyme‐histochemical reactions, which demonstrate the activity of myofibrillar ATPase, succinate dehydrogenase (SDH) and α‐glycerophosphate dehydrogenase (α‐GPDH). In the skeletal muscles of fetuses and neonatal dogs the developmental isoforms MHC‐emb and MHC‐neo were prevalent. In all muscles the primary fibres, located centrally in each muscle fascicle, strongly expressed the slow isoform MHC‐I. The adult fast isoform MHC‐IIa was first noted in some of the secondary fibres on fetal day 55. During the first 10 days after birth, the expression of MHC‐emb declined, as did that of MHC‐neo during the second and third weeks. Correspondingly, the expression of MHC‐IIa, and later, of MHC‐I increased in the secondary fibres. Between the sixth week and second month the expression of MHC‐IIx became prominent. The slow rhomboideus muscle exhibited an early expression of the slow isoform in the secondary fibres. Our results indicate that the timing of muscle maturation depends on its activity immediately following birth. The fastest developing muscle was the diaphragm, followed by the fast muscles. A pronounced changeover from developmental to adult isoforms was noted at 4–6 weeks of age, which coincides with the increased physical activity of puppies.

Growth dynamics of lipizzan horses and their comparison to other horse breeds.

Abstract A typical body format of Slovenian Lipizzan horse was investigated. The study included 6 foals (5 colts and 1 filly) at the Lipica stud farm. They were measured from birth to twenty-seven months and again at forty-four months of age. Measurements included body length, chest circumference, withers height and body mass. All those measurements were statistically evaluated and compared to some other horse breeds to determine the similarity of the growth dynamics of those horse breeds. It was concluded that the parameters of the head and neck reached their full growth at the age of 27 months and also exceeded the values of their parents. The same results were obtained with the length and heights of the body (body length, withers height, etc.) and with the parameters of the front and hind legs. However the girth width, hip width and some other parameters had not yet reached their full growth at 27 months. We observed different growth dynamics with the body weight. The parameter continued to grow after 27 months of age. The same growth dynamics also seem to apply to other investigated horse breeds although the actual measurements were slightly different.

Alterations in geometry, biomechanics, and mineral composition of juvenile rat femur induced by nonplanar PCB-155 and/or planar PCB-169

Exposure to widespread lipophilic and bioaccumulative polychlorinated biphenyls (PCBs) induces diverse biochemical and toxicological responses in various organs, including the bone. The aim of this study was to evaluate the changes in growth rate, geometry, serum, and bone biochemical parameters and biomechanics of juvenile rat femur induced by lactational exposure to nonplanar PCB‐155 and planar PCB‐169 individually and in combination. Fifteen lactating Wistar rats were divided into four groups (PCB‐169, PCB‐155, PCB‐155+169, and control), and PCBs were administered intraperitoneally at different time points after delivery. Femurs from 22‐day‐old offspring were analyzed by microCT, three‐point bending test and inductively coupled plasma‐mass spectrometry (ICP‐MS) to obtain data on bone geometry, biomechanics and mineral composition. The serum levels of calcium, phosphate and alkaline phosphatase were also determined. Lactational exposure to planar PCB‐169 resulted in shorter and thinner femurs, reduced endosteal and periosteal perimeters, smaller total cross‐sectional and medullary areas, and lowered serum bone marker levels and calcium levels in the bone, while femur mechanical properties were not significantly altered. The changes observed in the combination exposure (PCB‐155+169) group were similar to those observed in the PCB‐169 group but were less pronounced. In summary, our results demonstrate that alterations in lactationally exposed offspring were primarily induced by planar PCB‐169. The milder outcome in the combined group suggested that the PCB‐169‐mediated toxic effects on the bone might be reduced by a nonplanar PCB‐155 congener.

Establishing and functional characterization of an HEK-293 cell line expressing autofluorescently tagged β-actin (pEYFP-ACTIN) and the neurokinin type 1 receptor (NK1-R)

This study focused on establishing and making a comprehensive functional characterization of an HEK-293-transfected cell line that would coexpress the enhanced yellow fluorescent protein-actin (pEYFP-actin) construct and the neurokinin type 1 receptor (NK1-R), which is a member of the seven transmembrane (7TM) receptor family. In the initial selection procedure, the cloning ring technique was used alone, but failed to yield clones with homogenous pEYFP-actin expression. Flow cytometry sorting (FCS) was subsequently used to enrich the pEYFP-actin-expressing subpopulation of cells. The enzyme-linked immunosorbent assay (ELISA), FCS and quantitative real-time reverse transcription/polymerase chain reaction (RT-PCR) were then employed to monitor the passage-dependent effects on transgene expression and to estimate the total β-actin/pEYFP-actin ratio. NK1-R was characterized via radioactive ligand binding and the second messenger assay. The suitability of the pEYFP-actin as a marker of endogenous actin was assessed by colocalizing pEYFP-actin with rhodamine-phalloidine-stained F-actin and by comparing receptor- and jasplakinolide-induced changes in the actin cytoskeleton organization. These experiments demonstrated that: i) both constructs expressed in the generated transfected cell line are functional; ii) the estimated pEYFP-actin: endogenous β-actin ratio is within the limits required for the functional integrity of the actin filaments; and iii) pEYFP-actin and rhodamine-phalloidine-stained F-actin structures colocalize and display comparable reorganization patterns in pharmacologically challenged cells.

Dopamine D2 receptor mRNA measured in serial sections of the rat anterior pituitary.

Abstract Dopamine D2 receptors (D2-Rs) on lactotrophs in the pituitary gland are targets for dopamine to inhibit prolactin synthesis and release. The aim of our study was to examine if subpopulations of cells in the anterior pituitary that respond differently to dopamine show different pattern of D2-R mRNA expression. Therefore, we have used quantitative in situ hybridization technique to study the localisation of D2-R mRNA in the rat adenohypophysis. Pituitary tissue was obtained from mature and 18 days old rats. Riboprobe was transcribed from rat pituitary cDNA clone encoding D2-R and hybridized in situ with the serial sections of the pituitaries. Our results show that, although the anterior lobe of the pituitary gland contains a variety of cell types distributed in clusters, D2-R mRNA is relatively evenly distributed through the adenohypophysis. Level of expression of D2-R mRNA in the pituitary is slightly higher in mature than in young rats.