The livestock photosensitizer, phytoporphyrin (phylloerythrin), is a substrate of the ATP-binding cassette transporter ABCG2

Hepatogenous photosensitization occurs in livestock following damage to the liver or biliary apparatus that results in impaired excretion of phytoporphyrin (phylloerythrin), a photosensitizer. Based on earlier observations that porphyrin-based photosensitizers are substrates of the ATP-binding cassette transporter ABCG2, we examined the ability of the hepatic transporters ABCB1 (P-glycoprotein) and ABCG2 to transport phytoporphyrin. Transport of phytoporphyrin was blocked by the ABCG2-specific inhibitor fumitremorgin C (FTC) in human embryonic kidney cells transfected with full length human ABCG2, while no transport by cells transfected with human ABCB1 was noted. FTC-inhibited transport of phytoporphyrin was also demonstrated in ABCG2-expressing LLC-PK1 pig kidney cells, consistent with the idea that the pig orthologue, like human ABCG2, transports the photosensitizer. ABCG2 expression was confirmed by immunohistochemistry in the hepatocytes of cow, pig and sheep livers. We conclude that phytoporphyrin is a substrate for ABCG2 and that the transporter is likely responsible for its biliary excretion.     

Rno‐miR‐425‐5p targets the DLST and SLC16A1 genes to reduce liver damage caused by excessive energy mobilization under cold stress

MicroRNAs (miRNAs) are a class of single‐stranded non‐coding small RNA molecules, which participate in the regulation of many physiological processes, and play a crucial role in cancer, metabolism and other processes. Rno‐miR‐425‐5p has been shown to play a role in the response to cold stress. To explore the mechanism by which rno‐miR‐425‐5p regulates the response to cold stress, we analysed the candidate target genes of rno‐miR‐425‐5p. After verification in rat hepatocyte BRL cells and in rat liver tissue, we identified several target genes that were altered in expression in response to cold stress. In rat liver tissue, the expression of rno‐miR‐425‐5p was significantly increased and the expression levels of target genes DLST and SLC16A1 were decreased under cold stress. The miRNA and mRNA levels were analysed by quantitative real‐time PCR and the protein levels were detected by Western blot analysis. Combined with the results of bioinformatic analysis, we concluded that rno‐miR‐425‐5p reduced the expression of DLST and SLC16A1, inhibiting energy release from the tricarboxylic acid cycle and preventing the liver from being injured by excessive energy mobilization.     

Polymorphisms in the ABCB1 gene in phenobarbital responsive and resistant idiopathic epileptic Border Collies

Background: Variation in the ABCB1 gene is believed to play a role in drug resistance in epilepsy. Hypothesis/Objectives: Variation in the ABCB1 gene encoding the permeability‐glycoprotein could have an influence on phenobarbital (PB) resistance, which occurs with high frequency in idiopathic epileptic Border Collies (BCs). Animals: Two hundred and thirty‐six client‐owned BCs from Switzerland and Germany including 25 with idiopathic epilepsy, of which 13 were resistant to PB treatment. Methods: Prospective and retrospective case‐control study. Data were collected retrospectively regarding disease status, antiepileptic drug (AED) therapy, and drug responsiveness. The frequency of a known mutation in the ABCB1 gene (4 base‐pair deletion in the ABCB1 gene [c.296_299del]) was determined in all BCs. Additionally, the ABCB1 coding exons and flanking sequences were completely sequenced to search for additional variation in 41 BCs. Association analyses were performed in 2 case‐control studies: idiopathic epileptic and control BCs and PB‐responsive and resistant idiopathic epileptic BCs. Results: One of 236 BCs (0.4%) was heterozygous for the mutation in the ABCB1 gene (c.296_299del). A total of 23 variations were identified in the ABCB1 gene: 4 in exons and 19 in introns. The G‐allele of the c.‐6‐180T > G variation in intron 1 was significantly more frequent in epileptic BCs resistant to PB treatment than in epileptic BCs responsive to PB treatment (P_raw= .0025). Conclusions and Clinical Importance: A variation in intron 1 of the ABCB1 gene is associated with drug responsiveness in BCs. This might indicate that regulatory mutations affecting the expression level of ABCB1 could exist, which may influence the reaction of a dog to AEDs.     

Myostatin down‐regulates the IGF‐2 expression via ALK‐Smad signaling during myogenesis in cattle

Myostatin (MSTN) is a negative regulator during muscle differentiation, whereas insulin‐like growth factors (IGFs) are essential for muscle development. MSTN and IGFs act oppositely during myogenesis, but there is little information on the mutual relationship of MSTN and IGFs. The present study was conducted to examine whether MSTN affects IGF expression during early myogenesis in cattle. IGF‐1 mRNA was similarly expressed in M. longissimus thoracis of double‐muscled (DM) and normal (NM) Japanese shorthorn cattle. IGF‐2 mRNA expression was consistently higher in the normal and regenerating muscle of DM cattle than those of NM cattle. When myoblasts were isolated from regenerating M. longissimus thoracis, IGF‐2 mRNA expression showed a significant increase in differentiating DM derived myoblasts (DM‐myoblasts) as compared with differentiating NM derived myoblasts (NM‐myoblasts). An addition of recombinant mouse myostatin (rMSTN) to myoblast cultures attenuated IGF‐2 mRNA expression and decreased myotube formation, but did not effect IGF‐1 mRNA expression. An activin‐like kinase (ALK) inhibitor, SB431542, mediates MSTN action, suppressed the translocation of Smad2/3 into the nucleus in DM‐myoblasts, and restored the attenuated IGF‐2 mRNA expression and the decreased myotube formation induced by rMSTN in myoblast cultures. The findings indicate that MSTN may negatively regulate myoblast differentiation by suppressing IGF‐2 expression via ALK‐Smad signaling.     

Grazing‐induced changes in muscle microRNA‐206 and ‐208b expression in association with myogenic gene expression in cattle

To investigate the roles of microRNAs (miRNAs) in muscle type conversion, the effects of 4 months of grazing on the expression levels of miRNAs and mRNAs associated with skeletal muscle development were analyzed by quantitative RT‐PCR using the Biceps femoris muscle of Japanese Shorthorn cattle. After 4 months of grazing, the expression of muscle fiber type‐associated miR‐208b was higher in the grazed cattle than in the housed. In concordance with the pattern in miR‐208b expression, the expression of MyoD, a myogenic regulatory factor associated with the shifting of muscle property to the fast type, was lower in the grazed cattle after 4 months of grazing than in the housed cattle. In addition, the expression of MyHC‐2x (a fast type) was higher in the housed cattle than in the grazed, after 4 months of grazing. During the grazing period, miR‐206 expression decreased in the housed cattle, whereas expression in the grazed cattle did not change, but rather remained higher than that of the housed cattle even at 3 months after the grazing ended. These miRNAs including miR‐206 persisting with muscles of grazed cattle may be associated with regulation of muscle gene expression during skeletal muscle adaptation to grazing.     

P‐glycoprotein in sheep liver and small intestine: gene expression and transport efflux activity

The role of the transporter P‐glycoprotein (P‐gp) in the disposition kinetics of different drugs therapeutically used in veterinary medicine has been demonstrated. Considering the anatomo‐physiological features of the ruminant species, the constitutive expression of P‐gp (ABCB1) along the sheep gastrointestinal tract was studied. Additionally, the effect of repeated dexamethasone (DEX) administrations on the ABCB1 gene expression in the liver and small intestine was also assessed. The ABCB1 mRNA expression was determined by real‐time quantitative PCR. P‐gp activity was evaluated in diffusion chambers to determine the efflux of rhodamine 123 (Rho 123) in the ileum from experimental sheep. The constitutive ABCB1 expression was 65‐fold higher in the liver than in the intestine (ileum). The highest ABCB1 mRNA expression along the small intestine was observed in the ileum (between 6‐ and 120‐fold higher). The treatment with DEX did not elicit a significant effect on the P‐gp gene expression levels in any of the investigated gastrointestinal tissues. Consistently, no significant differences were observed in the intestinal secretion of Rho 123, between untreated control (P_eff S‐M = 3.99 × 10^?6 ± 2.07 × 10^?6) and DEX‐treated animals (P_eff S‐M = 6.00 × 10^?6 ± 2.5 × 10^?6). The understanding of the efflux transporters expression and activity along the digestive tract may help to elucidate clinical implications emerging from drug interactions in livestock.     

Canine osteosarcoma cells exhibit resistance to aurora kinase inhibitors

We evaluated the effect of Aurora kinase inhibitors AZD1152 and VX680 on canine osteosarcoma cells. Cytotoxicity was seen in all four cell lines; however, half‐maximal inhibitory concentrations were significantly higher than in human leukaemia and canine lymphoma cells. AZD1152 reduced Aurora kinase B phosphorylation, indicating resistance was not because of failure of target recognition. Efflux mediated by ABCB1 and ABCG2 transporters is one known mechanism of resistance against these drugs and verapamil enhanced AZD1152‐induced apoptosis; however, these transporters were only expressed by a small percentage of cells in each line and the effects of verapamil were modest, suggesting other mechanisms contribute to resistance. Our results indicate that canine osteosarcoma cells are resistant to Aurora kinase inhibitors and suggest that these compounds are unlikely to be useful as single agents for this disease. Further investigation of these resistance mechanisms and the potential utility of Aurora kinase inhibitors in multi‐agent protocols is warranted.     

ABCG2 transporter: therapeutic and physiologic implications in veterinary species

Drug transporters significantly influence drug pharmacokinetics and pharmacodynamics. While P-glycoprotein, the product of the MDR1 (ABCB1) gene, is the most well-characterized ABC transporter, the pharmacological importance of a related transporter, ABCG2, is starting to be realized in veterinary medicine. Based primarily on human and rodent studies, a number of clinically relevant, structurally and functionally unrelated drugs are substrates for ABCG2. ABCG2 is expressed by a variety of normal tissues including the intestines, renal tubular cells, brain and retinal capillary endothelial cells, biliary canalicular cells, and others, where it functions to actively extrude substrate drugs. In this capacity, ABCG2 limits oral absorption of substrate drugs and restricts their distribution to privileged sites such as the brain and retina. ABCG2 is also expressed by tumor cells where it functions to limit the intracellular accumulation of cytotoxic agents, contributing to multidrug resistance. Several ABCG2 polymorphisms have been described in human patients, some of which result in altered drug disposition, increasing susceptibility to adverse drug reactions. Additionally, ABCG2 polymorphisms in humans have been associated with disease states such as gout. Feline ABCG2 has recently been demonstrated to have several amino acid differences at conserved sites compared with 10 other mammalian species. These amino acid differences adversely affect transport function of feline ABCG2 relative to that of human ABCG2. Furthermore, these differences appear to be responsible for fluoroquinolone-induced retinal toxicity in cats and may play a role in acetaminophen toxicity as well. Studies in rodents and sheep have determined that ABCG2 expressed in mammary tissue is responsible for the secretion of many compounds (both therapeutic and toxic) into milk. Finally, data in rodent models suggest that ABCG2 may play an important role in regulating a number of physiologic pathways involved in protecting erythrocytes from oxidative damage.     

Adenosine protects Sprague Dawley rats from high‐fat diet and repeated acute restraint stress‐induced intestinal inflammation and altered expression of nutrient transporters

This study investigated the effect of repeated acute restraint stress and high‐fat diet (HFD) on intestinal expression of nutrient transporters, concomitant to intestinal inflammation. The ability of adenosine to reverse any change was examined. Six‐week‐old male Sprague Dawley rats were divided into eight groups: control or non‐stressed (C), rats exposed to restraint stress for 6 h per day for 14 days (S), control rats fed with HFD (CHF) and restraint‐stressed rats fed with HFD (SHF); four additional groups received the same treatments and were also given 50 mg/l adenosine dissolved in drinking water. Fasting blood glucose, plasma insulin, adiponectin and corticosterone were measured. Intestinal expression of SLC5A1, SLC2A2, NPC1L1 and TNF‐α was analysed. Histological evaluation was conducted to observe for morphological and anatomical changes in the intestinal tissues. Results showed that HFD feeding increased glucose and insulin levels, and repeated acute restraint stress raised the corticosterone level by 22%. Exposure to both stress and HFD caused a further increase in corticosterone to 41%, while decreasing plasma adiponectin level. Restraint stress altered intestinal expression of SLC5A1, SLC2A2 and NPC1L1. These changes were enhanced in SHF rats. Adenosine was found to alleviate HFD‐induced increase in glucose and insulin levels, suppress elevation of corticosterone in S rats and improve the altered nutrient transporters expression profiles. It also prevented upregulation of TNF‐α in the intestine of SHF rats. In summary, a combination of stress and HFD exaggerated stress‐ and HFD‐induced pathophysiological changes in the intestine, and biochemical parameters related to obesity. Adenosine attenuated the elevation of corticosterone and altered expression of SLC5A1, NPC1L1 and TNF‐α.     

Decreased expression of L‐selectin (CD62L) on lymphocytes in enzootic bovine leukaemia

Expression of L‐selectin was determined by single‐ and two‐colour immunofluorescence on granulocytes, peripheral blood mononuclear cells (PBMC) and blasts of bovine origin by means of a monoclonal antibody IVA94 which recognizes bovine L‐selectin (CD62L). Cells were separated from peripheral blood of healthy cattle and colleagues infected with bovine leukaemia virus (BLV). BLV‐infected animals comprised lymphocytotic and non‐lymphocytotic cows. L‐selectin was expressed on 90–98 % of granulocytes in all tested animals. The percentage of PBMC expressing L‐selectin was lower in cattle with persistent lymphocytosis than in non‐lymphocytotic or BLV‐free cattle, and inversely correlated with lymphocyte counts. The ratio of B lymphocytes stained for L‐selectin was significantly decreased from 60.2 ± 1.9 % in BLV‐free cattle to 43.8 ± 3.6 and 22.5 ± 5.7 % in non‐lymphocytotic and lymphocytotic cattle, respectively. B‐lymphocytes stained for L‐selectin exhibited about 50 % reduction in L‐selectin expression in BLV‐infected cattle compared with BLV‐free cattle, as judged by the mean fluorescence intensity (MFI). The percentage of L‐selectin‐positive PBMC not bearing surface immunoglobulin M (predominantly T lymphocytes) was comparable in BLV‐free and BLV‐infected cattle. However, L‐selectin expression on T lymphocytes was reduced (about 50 %) in BLV‐infected cattle, as judged by the MFI. We suppose that BLV infection results in a decreased L‐selectin expression on lymphocytes, and accordingly, it may contribute to deregulation of the host immune system.     

Muscle type‐specific effect of myostatin deficiency on myogenic regulatory factor expression in adult double‐muscled Japanese Shorthorn cattle

To clarify muscle type‐specific effect of myostatin on myogenic regulatory factors (MRFs), we examined mRNA expression of MRFs in five skeletal muscles of normal (NM) and myostatin‐deficient double‐muscled (DM) adult Japanese Shorthorn cattle by quantitative reverse‐transcribed PCR. Among the four MRFs, namely, Myf5, MyoD, myogenin, and MRF4, MyoD expression was different among the muscles of the DM cattle (P < 0.01) but not of the NM cattle. Meanwhile, MyoD expression was significantly elevated only in masseter (MS) muscle in the DM cattle due to the myostatin deficiency (P < 0.05). Myf5 and MRF4 expression in semitendinosus (ST) was higher in the DM than in the NM cattle (P < 0.05). According to analysis of myosin heavy chain (MyHC) isoform expression, more MyHC‐2x and ‐2a and less ‐slow isoforms were expressed in the longissimus and ST muscles compared to the MS muscle in both cattle (P < 0.05), but no significant difference in MyHC expression was observed between the NM and DM cattle. Taken together, myostatin has influences on Myf5 and MRF4 expression in faster‐type muscles and on MyoD expression in slower‐type muscles, suggesting a possible muscle type‐specific effect of myostatin in skeletal muscle growth and maintenance.     

Loperamide‐induced expression of immune and inflammatory genes in Collies associated with ivermectin sensitivity

This study evaluated the impact of the ABCB1‐1Δ mutation in Collies which exhibited toxicity toward ivermectin, on changes in gene expression when given the unrelated ABCB1 substrate loperamide, to identify potential biomarkers predictive of drug safety. Thirty‐two healthy intact Collies consisting of dogs with either a wild‐type, heterozygous mutant, or homozygous mutant genotype were used. Whole blood samples were collected from Collies at 0 or 5 h following administration of loperamide at a dose of 0.10 mg/kg. Whole‐genome gene expression microarray was conducted to examine for changes in gene expression. Microarray analysis identified loperamide‐induced changes in gene expression which were specifically associated with ivermectin‐sensitive phenotypes in Collies possessing the ABCB1‐1Δ mutation. Gene pathway analysis further demonstrated that the altered genes are involved in immunological disease, cell death and survival, and cellular development. Thirteen genes, including CCL8 and IL‐8, were identified. Collie dogs harboring ABCB1‐1Δ mutation which also exhibited toxicity toward ivermectin demonstrated systematic responses following loperamide treatment exhibited by altered expression of genes involved in immune and inflammatory signaling pathways. Genes such as CCL8 and IL‐8 are potential biomarkers in whole blood that may predict the safety of loperamide in dogs with ABCB1‐1? mutation associated with ivermectin sensitivity.     

Expression of Mitochondria‐Associated Genes (PPARGC1A,NRF‐1, BCL‐2 and BAX) in Follicular Development and Atresia of Goat Ovaries

Most follicles undergo atresia during the developmental process. Follicular atresia is predominantly regulated by apoptosis of granulosa cells, but the mechanism underlying apoptosis via the mitochondria‐dependent apoptotic pathway is unclear. We aimed to investigate whether the mitochondria‐associated genes peroxisome proliferator‐activated receptor‐gamma, coactivator1‐alpha (PPARGC1A), nuclear respiratory factor‐1 (NRF‐1), B‐cell CLL/lymphoma 2 (BCL‐2) and BCL2‐associated X protein (BAX) played a role in follicular atresia through this pathway. The four mitochondria‐associated proteins (PGC‐1α, which are encoded by the PPARGC1A gene, NRF‐1, BCL‐2 and BAX) mainly expressed in granulosa cells. The mRNA and protein levels of PPARGC1A/PGC‐1α and NRF‐1 in granulosa cells increased with the follicular development. These results showed that these genes may play a role in the regulation of the follicular development. In addition, compared with healthy follicles, the granulosa cell in atretic follicles had a reduced expression of NRF‐1, increased BAX expression and increased ratio of BAX to BCL‐2 expression. These results suggested that changes of the mitochondria‐associated gene expression patterns in granulosa cells may lead to follicular atresia during goat follicle development.     

A functional model for feline P‐glycoprotein

P‐gp (ABCB1) belongs to the group of export transporters that is expressed in various species at biological barriers. Inhibition of P‐gp can lead to changes in pharmacokinetics of drugs (drug–drug interactions), which can lead to toxicity and adverse side effects. This study aimed to establish a functional assay to measure the inhibitory potential of veterinary drugs on feline P‐gp by means of fluorescence‐associated flow cytometry of feline lymphoma cells. In this model, PSC833 and ivermectin potently inhibited P‐gp function; cyclosporine and verapamil moderately inhibited P‐gp function, whereas ketoconazole, itraconazole, diazepam, and its metabolites had no effect on P‐gp function. This model can be used for testing the inhibitory potency of (new) drugs on feline P‐gp.     

Quantitative expression of candidate genes affecting eggshell color

There are three pigments that affect the color of an eggshell: protoporphyrin, biliverdin and biliverdin‐zinc chelate. Protoporphyrin is the main pigment in brown and light‐brown eggshells, whereas very little protoporphyrin is found in white eggshells. Eggshell protoporphyrin is derived from the heme formation in birds. Coproporphyrinogen III oxidase (CPOX) and ferrochelatase (FECH) represent rate‐limiting enzymes for the heme‐biosynthetic pathway. Breast cancer resistance protein (BCRP), feline leukemia virus receptor (FLVCR), and heme‐responsive gene‐1 (HRG1) serve as primary transporters for both protoporphyrinogen and heme. Finally, four organic anion transporting polypeptide family members (including solute carrier organic anion transporter family, SLCO1C1, SLCO1A2, SLCO1B3 and LOC418189) may affect pigment transport within eggshells. Here we measured gene expression levels in key tissues of egg‐producing hens. We analyzed three different types of hens that generated distinct eggshell colors: white, pink or brown. Our data revealed three ways in which eggshell color was genetically influenced. First, high‐level expression of CPOX generated more protoporphyrinogen and a brown eggshell color. In contrast, high expression of FECH likely converted more protoporphyrinogen into heme, reduced protoporphyrinogen levels within the eggshell and generated a light color. Second, heme transporters also affected eggshell color. High‐level expression of BCRP, HRG1 and FLVCR were associated with brown, white and generally lighter eggshell colors, respectively. Finally, protoporphyrin precipitation also affected eggshell color, as high expression of both SLCO1A2 and SLCO1C1 were associated with brown eggshell color. As such, we have identified seven genes in which expression levels in different tissues were associated with eggshell color.     

Cattle Infected with Bovine Leukaemia Virus may not only Develop Persistent B‐cell Lymphocytosis but also Persistent B‐cell Lymphopenia

We investigated the distribution of B and T cells in the peripheral blood of haematologically inconspicuous (non‐persistent lymphocytotic, PL^?) cattle infected with the bovine leukaemia virus (BLV). Flow cytometric data were obtained from six PL^? cattle and compared with six age‐matched animals with persistent lymphocytosis (PL⁺) and five non‐infected healthy controls (BLV^?). In the PL^? group, the percentage and number of surface immunoglobulin‐positive (sIg⁺) B cells were significantly reduced. Whereas in BLV^? cattle, about 40% of the peripheral blood lymphocytes (PBL) were sIg⁺ and 24% were sIgM⁺ B cells. In the PL^? group, less than 20% of the PBL were sIg⁺ and sIgM⁺ B cells. Only 5% of the PBL co‐expressed sIgM⁺ and CD5⁺ versus 16% in BLV^?. This decrease was persistent over 3 years and predominantly affected: (i) B cells that did not express sIgM; (ii) sIgM⁺ B cells co‐expressing CD5 and CD11b; and (iii) equally both λ‐ and κ‐type light chain B‐cell subpopulations. In contrast, the number of all circulating lymphocytes, CD5^? and CD11b^? sIgM⁺ B cells and CD2⁺ T cells did not differ. In PL⁺ animals, about 75% of the PBL were sIgM⁺CD5⁺ B cells. These cells were of polyclonal origin, as light chains of the λ‐ and κ‐type were expressed in a ratio of 4:1 (57.7% of PBL λ⁺, 14% κ⁺) as in BLV^? animals (33.6% of PBL λ⁺, 8.7% κ⁺). In PL⁺ cattle the absolute number of B‐cells and, therefore, their relative percentage is significantly increased. For this reason, even in case of absolutely increased T‐cell numbers, the relative percentage of T‐cells could be lower than in normal controls. The cause for the observed B cell decrease in PL^? cattle is unknown, but it can be assumed that cytotoxic T cells are involved in this B‐cell lymphopenia.