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.
This study has focused on enhancing the signal generated from the interaction between a G-protein-coupled receptor (GPCR) and beta-arrestin 2 (beta-arr2), measured by the bioluminescence resonance ...energy transfer (BRET(2)) technology. Both class A (beta(2)-adrenergic receptor beta(2)-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 BRET(2) signal can be enhanced by using (1) beta-arr2 phosphorylation-independent mutant (beta-arr2 R169E) and (2) beta-arr2 mutants deficient in their ability to interact with the components of the clathrin-coated vesicles (beta-arr2 R393E, R395E and beta-arr2 373 stop). For the class B receptor, there was no major difference in the agonist-promoted BRET(2) signal when comparing results obtained with wild-type (wt) and mutant beta-arr2. However, with the class A receptor, a more than 2-fold increase in the BRET(2) signal was observed with beta-arr2 mutants lacking the AP-2 or both AP-2 and clathrin binding sites. This set of data suggests that the inability of these beta-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 BRET(2) signal. The beta-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 BRET(2)-based agonist and antagonist screening assays.
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.
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.
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.
The distribution of selected lipophilic organochlorine pollutants, including two pairs of tetra- and hexa-chlorobiphenyl isomers (PCB-54, -80, -155, -169) and organochlorine pesticides ...hexachlorobenzene (HCB) and 1,1-bis(4-chlorophenyl)-2,2-dichloroethene (4,4′-DDE), in ovine dental pulp, dentine, enamel and mandibular bone was examined. Sheeps were given a single dose of individual organochlorine (1–4
μmol/kg) in olive oil by intramuscular injection and sacrificed 2 months later. Organochlorine residues were determined by gas chromatography. The highest levels of organochlorines were found in bone. The lipid adjusted levels varied significantly between tissues. Lower chlorinated, metabolically unstable, non-planar PCB-54 was enriched in enamel, while higher chlorinated, metabolically resistant, planar PCB-169 was preferentially retained in pulp. The ratio of planar to non-planar PCB homologues and pesticides (PCB-169/-155, PCB-80/-54, HCB/4,4′-DDE) decreased in the order pulp
>
dentine
>
enamel, suggesting that besides compound's lipophilicity and the tissue's lipid content, other physicochemical properties of organochlorines and tissue specific characteristics affect their distribution.
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.
The cremaster muscle is formed by muscle fibres that are descended from the caudal border of m. obliquus internus abdominis in males. The extent and functioning of the cremaster muscle differs among ...mammals. In rodents and lagomorphs testes migrate between scrotum and abdominal cavity and therefore the cremaster is extensive and highly active. There were hypotheses that the functioning of cremaster muscle is correlated with the seasonal testicular regression in brown hare. We previously established that position of the testes in hares is strongly correlated to the ambient temperature and not to the reproductive activity. Our present aim was to establish fibre type profile of the cremaster muscle in comparison with its origin, the internal oblique abdominal muscle, in both brown hare and laboratory rabbits. Muscle samples from 6 hares (3 in quiescent and 3 in reproductively active period) and 6 rabbits were collected and subjected to enzyme histochemistry (fibre typing according to mATPase, SDH and _´-GPDH activity and glycogen content) and immunohistochemistry directed to different myosin heavy chain isoforms. The cremaster muscle completely surrounded the testicle and epididymis in both species. While enzyme- and immunohistology revealed m. obliquus internus abdominis as a predominantly fast muscle, central bundles of cremaster muscle contained mainly slow fibre types. However, the peripheral parts of cremaster were predominantly composed of fast IIA fibre type indicating functionally important intra-muscular variability. In rabbits, which were housed in constant ambient temperature, the central 'slow' bundle was substantially smaller. Furthermore m. cremaster had high glycogen content and high glycolitic capacity, including the presence of uncommon SOG fibres. No prominent differences in fibre types between breeding and non-breeding seasons were found.
The effects of oxalic (OA) and formic acids (FA) on honeybee larvae in colonies were assessed and evaluated. Cell death was detected by the TUNEL technique for DNA labelling. In 3- and 5-day-old ...larvae exposed to OA, cell death was found in 25% of midgut epithelial cells 5 h after the treatment, using an "In situ cell death detection kit, AP" (Roche). The level of cell death increased to 70% by the 21st hour and the morphology of the epithelium remained unchanged. Fifty hours after the application, cell death was established in 18% of the epithelial cells of the 3-day-old larvae and had increased to 82% in the 5-day-old larvae. A "DeadEnd" apoptosis detection kit (Promega) showed sporadic cell death mainly in the larval fat body 5 h after treatment. Twenty-one hours after the OA application cell death was found in 4% of the larval midgut epithelial cells. Evaporated formic acid induced extensive apoptotic cell death in the peripheral, cuticular and subcuticular tissues that preceded the cell death of the entire larval body.
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