Characterization of two distinct immortalized endothelial cell lines, EA.hy926 and HMEC-1, for in vitro studies: exploring the impact of calcium electroporation, Ca2+ signaling and transcriptomic profiles.

BACKGROUND: Disruption of Ca2+ homeostasis after calcium electroporation (CaEP) in tumors has been shown to elicit an enhanced antitumor effect with varying impacts on healthy tissue, such as endothelium. Therefore, our study aimed to determine differences in Ca2+ kinetics and gene expression involved in the regulation of Ca2+ signaling and homeostasis, as well as effects of CaEP on cytoskeleton and adherens junctions of the established endothelial cell lines EA.hy926 and HMEC-1. METHODS: CaEP was performed on EA.hy926 and HMEC-1 cells with increasing Ca2+ concentrations. Viability after CaEP was assessed using Presto Blue, while the effect on cytoskeleton and adherens junctions was evaluated via immunofluorescence staining (F-actin, α-tubulin, VE-cadherin). Differences in intracellular Ca2+ regulation ([Ca2+]i) were determined with spectrofluorometric measurements using Fura-2-AM, exposing cells to DPBS, ionomycin, thapsigargin, ATP, bradykinin, angiotensin II, acetylcholine, LaCl3, and GdCl3. Molecular distinctions were identified by analyzing differentially expressed genes and pathways related to the cytoskeleton and Ca2+ signaling through RNA sequencing. RESULTS: EA.hy926 cells, at increasing Ca2+ concentrations, displayed higher CaEP susceptibility and lower survival than HMEC-1. Immunofluorescence confirmed CaEP-induced, time- and Ca2+-dependent morphological changes in EA.hy926's actin filaments, microtubules, and cell-cell junctions. Spectrofluorometric Ca2+ kinetics showed higher amplitudes in Ca2+ responses in EA.hy926 exposed to buffer, G protein coupled receptor agonists, bradykinin, and angiotensin II compared to HMEC-1. HMEC-1 exhibited significantly higher [Ca2+]i changes after ionomycin exposure, while responses to thapsigargin, ATP, and acetylcholine were similar in both cell lines. ATP without extracellular Ca2+ ions induced a significantly higher [Ca2+]i rise in EA.hy926, suggesting purinergic ionotropic P2X and metabotropic P2Y receptor activation. RNA-sequencing analysis showed significant differences in cytoskeleton- and Ca2+-related gene expression, highlighting upregulation of ORAI2, TRPC1, TRPM2, CNGA3, TRPM6, and downregulation of TRPV4 and TRPC4 in EA.hy926 versus HMEC-1. Moreover, KEGG analysis showed upregulated Ca2+ import and downregulated export genes in EA.hy926. CONCLUSIONS: Our finding show that significant differences in CaEP response and [Ca2+]i regulation exist between EA.hy926 and HMEC-1, which may be attributed to distinct transcriptomic profiles. EA.hy926, compared to HMEC-1, displayed higher susceptibility and sensitivity to [Ca2+]i changes, which may be linked to overexpression of Ca2+-related genes and an inability to mitigate changes in [Ca2+]i. The study offers a bioinformatic basis for selecting EC models based on research objectives.

Tff3 Deficiency Differentially Affects the Morphology of Male and Female Intestines in a Long-Term High-Fat-Diet-Fed Mouse Model.

Trefoil factor family protein 3 (Tff3) protects the gastrointestinal mucosa and has a complex mode of action in different tissues. Here, we aimed to determine the effect of Tff3 deficiency on intestinal tissues in a long-term high-fat-diet (HFD)-fed model. A novel congenic strain without additional metabolically relevant mutations (Tff3-/-/C57Bl6NCrl strain, male and female) was used. Wild type (Wt) and Tff3-deficient mice of both sexes were fed a HFD for 36 weeks. Long-term feeding of a HFD induces different effects on the intestinal structure of Tff3-deficient male and female mice. For the first time, we found sex-specific differences in duodenal morphology. HFD feeding reduced microvilli height in Tff3-deficient females compared to that in Wt females, suggesting a possible effect on microvillar actin filament dynamics. These changes could not be attributed to genes involved in ER and oxidative stress, apoptosis, or inflammation. Tff3-deficient males exhibited a reduced cecal crypt depth compared to that of Wt males, but this was not the case in females. Microbiome-related short-chain fatty acid content was not affected by Tff3 deficiency in HFD-fed male or female mice. Sex-related differences due to Tff3 deficiency imply the need to consider both sexes in future studies on the role of Tff in intestinal function.

Male Reproductive Organ Weight: Criteria for Detection of Androstenone-Positive Carcasses in Immunocastrated and Entire Male Pigs.

Immunocastration and rearing of entire males (EMs) are sustainable alternatives to surgical castration. However, these animal carcasses have variable risk of boar taint and should be identified at the slaughter line. We aimed to identify a simple and reliable indicator of androstenone-related boar taint by evaluating pelvic urogenital tract weight as a marker of boar-taint animals at the slaughter line. The pelvic urogenital tract, testes, and accessory sex glands of EMs and immunocastrates (ICs) were collected, dissected, and weighed, before colorimetric measurements of testicular tissue. Additionally, GnRH antibody titers and testosterone, androstenone, and skatole levels were determined. Our results showed that 81.8% of EMs had androstenone levels above the risk threshold (>0.5 µg/g fat; EM/Ahigh subgroup), whereas in ICs, the C/Ahigh subgroup with androstenone >0.5 µg/g fat accounted for only 4.3%. Androstenone levels correlated negatively with GnRH antibody titers and positively with testosterone levels and reproductive organ weights. Identification of ICs with androstenone levels above the threshold (IC/Ahigh subgroup) may be achieved via testes or pelvic urogenital tract weight measurements. However, in EMs, the latter is a more reliable parameter. A principal component analysis based on these variables and hierarchical clustering also distinguished the Ahigh from the Alow subgroup, irrespective of IC/EM. The findings highlight the possible use of pelvic urogenital tract weight along with testes weight as a simple, reliable, and efficient morphometric indicator for identifying androstenone-positive carcasses of different sex categories.

Patterns of human and porcine gammaherpesvirus-encoded BILF1 receptor endocytosis.

BACKGROUND: The viral G-protein-coupled receptor (vGPCR) BILF1 encoded by the Epstein-Barr virus (EBV) is an oncogene and immunoevasin and can downregulate MHC-I molecules at the surface of infected cells. MHC-I downregulation, which presumably occurs through co-internalization with EBV-BILF1, is preserved among BILF1 receptors, including the three BILF1 orthologs encoded by porcine lymphotropic herpesviruses (PLHV BILFs). This study aimed to understand the detailed mechanisms of BILF1 receptor constitutive internalization, to explore the translational potential of PLHV BILFs compared with EBV-BILF1. METHODS: A novel real-time fluorescence resonance energy transfer (FRET)-based internalization assay combined with dominant-negative variants of dynamin-1 (Dyn K44A) and the chemical clathrin inhibitor Pitstop2 in HEK-293A cells was used to study the effect of specific endocytic proteins on BILF1 internalization. Bioluminescence resonance energy transfer (BRET)-saturation analysis was used to study BILF1 receptor interaction with ß-arrestin2 and Rab7. In addition, a bioinformatics approach informational spectrum method (ISM) was used to investigate the interaction affinity of BILF1 receptors with ß-arrestin2, AP-2, and caveolin-1. RESULTS: We identified dynamin-dependent, clathrin-mediated constitutive endocytosis for all BILF1 receptors. The observed interaction affinity between BILF1 receptors and caveolin-1 and the decreased internalization in the presence of a dominant-negative variant of caveolin-1 (Cav S80E) indicated the involvement of caveolin-1 in BILF1 trafficking. Furthermore, after BILF1 internalization from the plasma membrane, both the recycling and degradation pathways are proposed for BILF1 receptors. CONCLUSIONS: The similarity in the internalization mechanisms observed for EBV-BILF1 and PLHV1-2 BILF1 provide a foundation for further studies exploring a possible translational potential for PLHVs, as proposed previously, and provides new information about receptor trafficking.

Experimental Exposure to Bisphenol A Has Minimal Effects on Bone Tissue in Growing Rams-A Preliminary Study.

Bisphenol A (BPA) is a well-known synthetic compound that belongs to the group of endocrine-disrupting chemicals. Although bone tissue is a target for these compounds, studies on BPA-related effects on bone morphology in farm animals are limited. In this preliminary study, we investigated the effects of short-term dietary BPA exposure on femoral morphology, metabolism, mineral content, and biomechanical behavior in rams aged 9-12 months. Fourteen rams of the Istrian Pramenka breed were randomly divided into a BPA group and a control group (seven rams/group) and exposed to 25 µg BPA/kg bw for 64 days in feed. Blood was collected for determination of bone turnover markers (procollagen N-terminal propeptide, C-terminal telopeptide), and femurs were assessed via computed tomography, histomorphometry, three-point bending test, and mineral analysis. BPA had no significant effects on most of the parameters studied. Only mineral analysis showed decreased manganese (50%; p ≤ 0.05) and increased copper content (25%; p ≤ 0.05) in the femurs of BPA-exposed rams. These results suggest that a 2-month, low-dose exposure to BPA in growing rams did not affect the histomorphology, metabolism, and biomechanical behavior of femurs; however, it affected the composition of microelements, which could affect the histometric and biophysical properties of bone in the long term.

Tff3 Deficiency Protects against Hepatic Fat Accumulation after Prolonged High-Fat Diet.

Trefoil factor 3 (Tff3) protein is a small secretory protein expressed on various mucosal surfaces and is involved in proper mucosal function and recovery via various mechanisms, including immune response. However, Tff3 is also found in the bloodstream and in various other tissues, including the liver. Its complete attenuation was observed as the most prominent event in the early phase of diabetes in the polygenic Tally Ho mouse model of diabesity. Since then, its role in metabolic processes has emerged. To elucidate the complex role of Tff3, we used a new Tff3-deficient mouse model without additional metabolically relevant mutations (Tff3-/-/C57BL/6NCrl) and exposed it to a high-fat diet (HFD) for a prolonged period (8 months). The effect was observed in male and female mice compared to wild-type (WT) counter groups (n = 10 animals per group). We monitored the animals' general metabolic parameters, liver morphology, ultrastructure and molecular genes in relevant lipid and inflammatory pathways. Tff3-deficient male mice had reduced body weight and better glucose utilization after 17 weeks of HFD, but longer HFD exposure (32 weeks) resulted in no such change. We found a strong reduction in lipid accumulation in male Tff3-/-/C57BL/6NCrl mice and a less prominent reduction in female mice. This was associated with downregulated peroxisome proliferator-activated receptor gamma (Pparγ) and upregulated interleukin-6 (Il-6) gene expression, although protein level difference did not reach statistical significance due to higher individual variations. Tff3-/-/C57Bl6N mice of both sex had reduced liver steatosis, without major fatty acid content perturbations. Our research shows that Tff3 protein is clearly involved in complex metabolic pathways. Tff3 deficiency in C57Bl6N genetic background caused reduced lipid accumulation in the liver; further research is needed to elucidate its precise role in metabolism-related events.

Immunocastration in adult boars as a model for late-onset hypogonadism.

BACKGROUND: While immunocastration has been studied in male pre-pubertal pigs, data on older, sexually mature animals are limited. To understand the physiological effects of androgen deprivation in the late sexual development phase, we compared mature immunocastrated boars (n = 19; average age = 480 days) to young male immunocastrated pigs (n = 6; average age = 183 days) and young entire males (n = 6; average age = 186 days) as positive and negative controls, respectively. OBJECTIVES: We hypothesized that the timing of gonadotropin-releasing hormone suppression (early or late sexual development phases) influences the extent of reproductive function inhibition, histological structure of testicular tissue, and expression levels of selected genes related to steroid metabolism. MATERIALS AND METHODS: Antibody titer, hormonal status, and histomorphometric analysis of testicular tissue were subjected to principal component analysis followed by hierarchical clustering to evaluate the immunocastration effectiveness in mature boars. RESULTS: Hierarchical clustering differentiated mature immunocastrated boars clustered with young immunocastrated pigs from those clustered with entire males. Although all mature immunocastrated boars responded to vaccination, as evidenced by the increased gonadotropin-releasing hormone antibody titers (p < 0.001), decreased serum luteinizing hormone concentrations (p = 0.002), and changes in testicular tissue vascularization (lighter and less red testicular parenchyma; p ≤ 0.001), the responses were variable. Sharp decreases in testes index (p < 0.001), Leydig cell volume density (p < 0.001), Leydig cell nucleus-to-cytoplasm ratio (p < 0.001), and testosterone concentration (p < 0.001) were observed in mature immunocastrated boars clustered with young immunocastrated pigs compared with those that clustered with entire males. Additionally, mature immunocastrated boars clustered with young immunocastrated pigs showed lower hydroxysteroid 17-beta dehydrogenase 7 expression than entire males (p < 0.05). The young immunocastrated pigs group showed higher follicle-stimulating hormone receptors than the entire males and mature immunocastrated boars, lower steroidogenic acute regulatory protein expression levels compared with entire males, and mature immunocastrated boars clustered with entire males (p < 0.01). CONCLUSION: The two-dose vaccination regime resulted in progressive but variable regression of testicular function in adult (post-pubertal) pigs; however, it was insufficient to induce a complete immunocastration response in all animals.

Epstein-Barr Virus-Encoded BILF1 Orthologues From Porcine Lymphotropic Herpesviruses Display Common Molecular Functionality.

Infection of immunosuppressed transplant patients with the human γ-herpesvirus Epstein-Barr virus (EBV) is associated with post-transplant lymphoproliferative disease (PTLD), an often fatal complication. Immunosuppressed miniature pigs infected with γ-herpesvirus porcine lymphotropic herpesvirus 1 (PLHV1) develop a similar disease, identifying pigs as a potential preclinical model for PTLD in humans. BILF1 is a G protein-coupled receptor (GPCR) encoded by EBV with constitutive activity linked to tumorigenesis and immunoevasive function downregulating MHC-I. In the present study, we compared BILF1-orthologues encoded by the three known PLHVs (PLHV1-3) with EBV-BILF1 to determine pharmacological suitability of BILF1 orthologues as model system to study EBV-BILF1 druggability. Cell surface localization, constitutive internalization, and MHC-I downregulation as well as membrane proximal constitutive Gαi signaling patterns were conserved across all BILFs. Only subtle differences between the individual BILFs were observed in downstream transcription factor activation. Using Illumina sequencing, PLHV1 was observed in lymphatic tissue from PTLD-diseased, but not non-diseased pigs. Importantly, these tissues showed enhanced expression of PLHV1-BILF1 supporting its involvement in PTLD infection.

Effects of Bioinsecticidal Aegerolysin-Based Cytolytic Complexes on Non-Target Organisms.

Aegerolysin proteins ostreolysin A6 (OlyA6), pleurotolysin A2 (PlyA2) and erylysin A (EryA) produced by the mushroom genus Pleurotus bind strongly to an invertebrate-specific membrane sphingolipid, and together with a protein partner pleurotolysin B (PlyB), form transmembrane pore complexes. This pore formation is the basis for the selective insecticidal activity of aegerolysin/PlyB complexes against two economically important coleopteran pests: the Colorado potato beetle and the western corn rootworm. In this study, we evaluated the toxicities of these aegerolysin/PlyB complexes using feeding tests with two ecologically important non-target arthropod species: the woodlouse and the honey bee. The mammalian toxicity of the EryA/PlyB complex was also evaluated after intravenous administration to mice. None of the aegerolysin/PlyB complexes were toxic against woodlice, but OlyA6/PlyB and PlyA2/PlyB were toxic to honeybees, with 48 h mean lethal concentrations (LC50) of 0.22 and 0.39 mg/mL, respectively, in their food. EryA/PlyB was also tested intravenously in mice up to 3 mg/kg body mass, without showing toxicity. With no toxicity seen for EryA/PlyB for environmentally beneficial arthropods and mammals at the tested concentrations, these EryA/PlyB complexes are of particular interest for development of new bioinsecticides for control of selected coleopteran pests.

Effect of Olive Leaves or Marigold Petal Extract on Oxidative Stress, Gut Fermentative Activity, and Mucosa Morphology in Broiler Chickens Fed a Diet Rich in n-3 Polyunsaturated Fats.

An experiment in broilers was conducted to investigate the effect of olive (Olea europea) leaves and marigold (Calendula officinalis) petal extract supplementation on oxidative stress, characteristics of intestinal contents, and on the morphology of the small intestine. Oxidative stress was induced by a n-3 polyunsaturated fatty acids rich diet. 1-day-old male broiler chickens, Ross 308, were housed in a deep litter system. After the first 21 days, animals were randomly divided into three groups of 16 animals in two replicates and fed, until slaughter on day 39, a diet that contained 7% linseed oil. Control diet (Cont) remained unsupplemented, while both experimental diets were supplemented with olive leaves (OliveEx) or marigold petal (MarigEx) extracts. Oxidative stress was evaluated in blood and liver by measuring markers of lipid peroxidation (malondialdehyde (MDA), isoprostanes), rate of DNA damage in lymphocytes and in blood (comet assay, 8-hydroxy-2'deoxyguanosine (8-OHdG)), and activity of antioxidant and liver enzymes in blood. In different parts of the intestine, levels of short chain fatty acids (SCFA), and viscosity of intestinal contents were measured, and the health of the gastrointestinal tract was assessed using histological measurements. OliveEx significantly (p<0.05) decreased the MDA and 8-OHdG concentration in plasma, and the level of ethanoic acid in small intestinal contents and total SCFA in caecum, indicating improved oxidative status and increased microbial activity in the intestine. MarigEx significantly (p<0.05) decreased the rate of lymphocyte DNA damage and the crypt depth in duodenum, indicating potentially beneficial effects on the immune system and the health of the small intestine. In conclusion, dietary OliveEx and MarigEx supplementation improved some markers of oxidative stress and intestinal health. However, positive effects could be more pronounced in more unfavorable environmental conditions or in cases of diseases, but further studies are needed.

Electron Paramagnetic Resonance Gives Evidence for the Presence of Type 1 Gonadotropin-Releasing Hormone Receptor (GnRH-R) in Subdomains of Lipid Rafts.

This study investigated the effect of type 1 gonadotropin releasing hormone receptor (GnRH-R) localization within lipid rafts on the properties of plasma membrane (PM) nanodomain structure. Confocal microscopy revealed colocalization of PM-localized GnRH-R with GM1-enriched raft-like PM subdomains. Electron paramagnetic resonance spectroscopy (EPR) of a membrane-partitioned spin probe was then used to study PM fluidity of immortalized pituitary gonadotrope cell line αT3-1 and HEK-293 cells stably expressing GnRH-R and compared it with their corresponding controls (αT4 and HEK-293 cells). Computer-assisted interpretation of EPR spectra revealed three modes of spin probe movement reflecting the properties of three types of PM nanodomains. Domains with an intermediate order parameter (domain 2) were the most affected by the presence of the GnRH-Rs, which increased PM ordering (order parameter (S)) and rotational mobility of PM lipids (decreased rotational correlation time (τc)). Depletion of cholesterol by methyl-ß-cyclodextrin (methyl-ß-CD) inhibited agonist-induced GnRH-R internalization and intracellular Ca2+ activity and resulted in an overall reduction in PM order; an observation further supported by molecular dynamics (MD) simulations of model membrane systems. This study provides evidence that GnRH-R PM localization may be related to a subdomain of lipid rafts that has lower PM ordering, suggesting lateral heterogeneity within lipid raft domains.

Adipose Tissue Gene Expression of Entire Male, Immunocastrated and Surgically Castrated Pigs.

Differences in adipose tissue deposition and properties between pig male sex categories, i.e., entire males (EM), immunocastrates (IC) and surgical castrates (SC) are relatively well-characterized, whereas the underlying molecular mechanisms are still not fully understood. To gain knowledge about the genetic regulation of the differences in adipose tissue deposition, two different approaches were used: RNA-sequencing and candidate gene expression by quantitative PCR. A total of 83 differentially expressed genes were identified between EM and IC, 15 between IC and SC and 48 between EM and SC by RNA-sequencing of the subcutaneous adipose tissue. Comparing EM with IC or SC, upregulated genes related to extracellular matrix dynamics and adipogenesis, and downregulated genes involved in the control of lipid and carbohydrate metabolism were detected. Differential gene expression generally indicated high similarity between IC and SC as opposed to EM, except for several heat shock protein genes that were upregulated in EM and IC compared with SC. The candidate gene expression approach showed that genes involved in lipogenesis were downregulated in EM compared with IC pigs, further confirming RNA-sequencing results.

Methods for Studying Endocytotic Pathways of Herpesvirus Encoded G Protein-Coupled Receptors.

Endocytosis is a fundamental process involved in trafficking of various extracellular and transmembrane molecules from the cell surface to its interior. This enables cells to communicate and respond to external environments, maintain cellular homeostasis, and transduce signals. G protein-coupled receptors (GPCRs) constitute a family of receptors with seven transmembrane alpha-helical domains (7TM receptors) expressed at the cell surface, where they regulate physiological and pathological cellular processes. Several herpesviruses encode receptors (vGPCRs) which benefits the virus by avoiding host immune surveillance, supporting viral dissemination, and thereby establishing widespread and lifelong infection, processes where receptor signaling and/or endocytosis seem central. vGPCRs are rising as potential drug targets as exemplified by the cytomegalovirus-encoded receptor US28, where its constitutive internalization has been exploited for selective drug delivery in virus infected cells. Therefore, studying GPCR trafficking is of great importance. This review provides an overview of the current knowledge of endocytic and cell localization properties of vGPCRs and methodological approaches used for studying receptor internalization. Using such novel approaches, we show constitutive internalization of the BILF1 receptor from human and porcine γ-1 herpesviruses and present motifs from the eukaryotic linear motif (ELM) resources with importance for vGPCR endocytosis.

Bone tissue morphology of rat offspring lactationally exposed to polychlorinated biphenyl 169 and 155.

Polychlorinated biphenyls (PCBs) are ubiquitous, persistent, organic pollutants also considered endocrine-disrupting chemicals. Our study examined the effects of lactational exposure to nondioxin-like PCB-155 and/or dioxin-like PCB-169 on longitudinal femur growth at the distal epiphyseal growth plate (EGP) in young rats at three different ages [postnatal days (PNDs) 9, 22, and 42]. After delivery, lactating rats were divided into four groups (PCB-169, PCB-155, PCB-155 + 169, and control) and administered PCBs intraperitoneally. The femurs of offspring were used to estimate growth rate (µm/day), and histomorphometric analysis on the distal femur included the thickness of the EGP and zones of proliferation and hypertrophy with calcification. Stereometry was used to determine trabecular bone volume density. In the PCB-169 and PCB-155 + 169 groups, PCB-169 affected longitudinal bone growth in the early postnatal period by interfering with chondrocytes in the EGP zone of proliferation and, to a lesser extent, the zone of hypertrophy. Morphometric alterations in EGP structure diminished until puberty. A slow growth rate persisted in the PCB-169 group until PND 42, while in the PCB-155 group, a fast growth rate between PNDs 9 to 22 was significantly reduced between PNDs 22 to 42. Sterometric assessment showed decreased trabecular bone volume in the PCB-155 + 169 group compared with that in the control on PND 9 and increased in the PCB-169 group compared with that in the PCB-155 group on PND 42. To summarize, studied PCB congeners exerted congener- and age-dependent effects on femur growth rate and its histomorphometric characteristics.

Expression of Myosin Heavy Chain and Some Energy Metabolism-Related Genes in the Longissimus Dorsi Muscle of Krskopolje Pigs: Effect of the Production System.

The Slovenian Krskopolje pig is the only preserved local autochthonous breed, appreciated mainly for its good meat quality and considered more appropriate for processing into dry-cured products. However, the biological characteristics of the skeletal myofibers of the Krskopolje breed, specifically the heavy myosin chain-based contractile and metabolic phenotypes that could affect meat quality, have not been established under different husbandry systems. The breed is generally maintained in either conventional indoor or organic systems. In the present study, the morphological, contractile, and metabolic properties of myofibers of the longissimus dorsi muscle were compared between animals reared in either an organic or a conventional indoor system. The myofibers were studied using immunohistochemical and succinate dehydrogenase (SDH) activity-based classification, histomorphometric assessment, and qPCR. Results revealed that the organic production system influenced the composition of the longissimus dorsi myofiber type, characterized by a smaller myofiber cross-sectional area, a shift toward oxidative (SDH-positive) myofiber types, increased relative expression of myosin heavy chain (MyHC) isoforms I, IIa, and IIx, and downregulation of MyHC IIb. On the contrary, no apparent effect was observed on the metabolic phenotype of the myofiber as assessed through relative mRNA expression of energy metabolism-related genes [peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1α), peroxisome proliferator-activated receptor gamma (PPARγ), lipoprotein-lipase (LPL), carnitine palmitoyltransferase 1B (CPT1B), glycogen synthase 1 (GYS1), hexokinase 2 (HK2), and fatty acid synthase (FASN)]. Differences in MyHC expression were largely corroborated by the histochemical classification, indicating that the contractile protein content is directly regulated by the MyHC genes. A correlation between the muscle contractile and metabolic phenotypes was not established, except for that between the HK2 and MyHC I genes. In conclusion, the present study showed an evident effect of rearing on the longissimus dorsi myofiber contractile phenotype but not the metabolic phenotype. Moreover, obtained data suggest that rearing the Krskopolje pig breed in a conventional system would result in an increased fiber size and a greater proportion of type IIb myofibers, which are known to be negatively correlated with some meat quality traits.

Critical Impact of Different Conserved Endoplasmic Retention Motifs and Dopamine Receptor Interacting Proteins (DRIPs) on Intracellular Localization and Trafficking of the D2 Dopamine Receptor (D2-R) Isoforms.

The type 2 dopamine receptor D2 (D2-R), member of the G protein-coupled receptor (GPCR) superfamily, exists in two isoforms, short (D2S-R) and long (D2L-R). They differ by an additional 29 amino acids (AA) in the third cytoplasmic loop (ICL3) of the D2L-R. These isoforms differ in their intracellular localization and trafficking functionality, as D2L-R possesses a larger intracellular pool, mostly in the endoplasmic reticulum (ER). This review focuses on the evolutionarily conserved motifs in the ICL3 of the D2-R and proteins interacting with the ICL3 of both isoforms, specifically with the 29 AA insert. These motifs might be involved in D2-R exit from the ER and have an impact on cell-surface and intracellular localization and, therefore, also play a role in the function of dopamine receptor signaling, ligand binding and possible homo/heterodimerization. Our recent bioinformatic data on potential new interaction partners for the ICL3 of D2-Rs are also presented. Both are highly relevant, and have clinical impacts on the pathophysiology of several diseases such as Parkinson's disease, schizophrenia, Tourette's syndrome, Huntington's disease, manic depression, and others, as they are connected to a variety of essential motifs and differences in communication with interaction partners.

Effect of immunocastration and housing conditions on pig carcass and meat quality traits.

The present study investigated the effects of immunocastration and housing conditions on carcass, meat, and fat quality traits. Immunocastrates (IC, n = 48), entire (EM, n = 48), and surgical castrates (SC, n = 48) male pigs were reared under three different housing conditions. The conditions were standard (n = 36), enriched (n = 36, twice as much space as standard and additional outdoor access), or standard with repeated social mixing (n = 72). Pigs of the IC group were vaccinated at the age of 12 and 22 wk. The animals were slaughtered in four batches, balanced for sex category and housing, at the age of 27 wk reaching 124.7 ± 1.0 kg. Immunocastration led to increased fat deposition (i.e., thicker subcutaneous fat at different anatomical locations, more leaf fat, fatter belly in IC than EM, P < 0.05) but did not affect muscularity traits. As a result, EM exhibited higher and SC lower (P < 0.05) carcass leanness than IC. Fatty acids composition of either subcutaneous or intramuscular fat (IMF) agreed with general adiposity, that is, IC were intermediate between EM and SC exhibiting the lowest and highest fat saturation (P < 0.05), respectively. Compared to SC, EM exhibited higher (P < 0.05) levels of muscle oxidation and collagen content than SC, with IC taking an intermediate position in the case of the level of peroxidation and collagen content, or closer to SC as regards to oxidation of muscle proteins (i.e., carbonyl groups). Meat quality (including marbling score, cooking loss, subjective color redness, and chroma) of IC was similar to EM, and both differed (P < 0.05) from SC. However, IC and SC had less (P < 0.05) tough meat than EM, consistent with protein oxidation. The effect of housing was less evident. Mixing of pigs resulted in lower (P < 0.05) carcass weight and fatness in all sex categories with lower (P < 0.05) oleic and higher (P < 0.05) arachidonic acid in IMF of EM.