Unraveling molecular mechanisms involved in the development of leptin resistance using the pig as a model.

The increase in obesity worldwide underlines the need for research concerning its metabolic and genetic determinants. One of the most intriguing mechanisms regarding obesity involves leptin and its signaling cascade. Leptin is a key regulator contributing to the fine-tuned crosstalk between nutrient availability and appetite signaling in the central nervous system. Owing to ethical concerns, many human tissues are not readily available and pigs can serve as a good animal model owing to their comparable anatomy, metabolism and genetics. In the present study, we utilized the pig to investigate the possible impact of increased adiposity on the development of alterations within the leptin signaling pathway. Two divergent groups of pigs (High and Low) were defined based on a high and low amount of mesenteric fat. Cortex, cerebellum, hypothalamus, mesenteric, subcutaneous and retroperitoneal fat tissues were used to study changes in expression levels of 94 mRNA transcripts related to the leptin signaling pathway using the qPCR approach. No significant differences were found at the central nervous system, whereas the expression level of STAT1 was reduced in mesenteric fat and leptin (LEP) and interleukin 6 (IL6) were shown to be consistently increased in all analyzed fat compartments from pigs with a high amount of mesenteric fat. These results could imply the onset of leptin and pro-inflammatory cytokine overexpression at early stages of obesity in the analyzed pigs without affecting any key components in the central nervous system. Thus, these pigs showing a unique leptin deregulation in adipose tissues could be a useful translational resource for studies of obesity and leptin resistance phenotypes.

Validation of DNA test for hip dysplasia failed in Danish Labrador Retrievers.

Canine hip dysplasia is characterized by poor hip joint conformation and laxity. The disease is a complex trait influenced by both genetics and environment. Diagnosis and quantification of hip dysplasia are performed by radiographic examination of the hip joint and the diagnosis is used for making breeding decisions in many breeds. A prognostic genetic test (the Dysgen test) based on seven associated SNPs has been developed in a study based on Spanish Labrador Retrievers. In our study this test has been evaluated in 39 Danish Labrador Retrievers with known radiographic hip score: 14 with hip dysplasia (grade D or E) and 25 without hip dysplasia (grade A or B). There was no significant correlation between the Dysgen test results and the radiographic hip status (P = 0.3203) in these dogs, indicating that Dysgen test results obtained for Danish Labrador Retrievers have no prognostic value.

Inotropic and lusitropic effects of levosimendan and milrinone assessed by strain echocardiography-A randomised trial.

BACKGROUND: We compared the direct inotropic and lusitropic effects of two inodilators, milrinone and levosimendan in patients after aortic valve replacement for aortic stenosis. METHODS: In this randomised, blinded study, 31 patients with normal LV function, were randomised to either levosimendan (0.1 and 0.2 µg/kg/min, n = 15) or milrinone (0.4 and 0.8 µg/kg/min, n = 16) after aortic valve replacement. The effects on LV performance, LV strain, systolic (SR-S) and early diastolic (SR-E) strain rate were assessed by a pulmonary artery catheter and transoesophageal two-dimensional speckle tracking echocardiography of the LV inferior wall. To circumvent the inodilator-induced hemodynamic changes on LV systolic and diastolic deformation, central venous pressure (CVP), systolic artery pressure (SAP), and heart rate were maintained constant by colloid infusion, phenylephrine-induced vasoconstriction and atrial pacing, respectively, during drug infusion. RESULTS: Both inotropic agents induced a dose-dependent increase in cardiac index and stroke volume index by approximately 20% at the highest infusion rates with no differences between groups (P = .139 and .249, respectively). CVP, pulmonary capillary wedge pressure, SAP and heart rate were maintained constant in both groups. LV strain and SR-S increased with both agents, dose-dependently, by 17%-18% and 25%-30%, respectively, at the highest infusion rates, with no difference between groups (P = .434 and .284, respectively). Both agents improved early LV relaxation with no differences between groups (P = .637). At the higher doses, both agents increased SR-E by 30%. CONCLUSIONS: At clinically relevant infusion rates and a certain increase in LV performance the direct inotropic and lusitropic of milrinone and levosimendan were comparable.

Interbreed variation in serum serotonin (5-hydroxytryptamine) concentration in healthy dogs.

INTRODUCTION: Serotonin (5-hydroxytryptamine [5-HT]) has several biological functions. In different species, excessive 5-HT has been linked to valvular lesions, similar to those seen in dogs with myxomatous mitral valve disease. Previous studies suggest higher 5-HT in healthy Cavalier King Charles Spaniels (CKCSs), a breed highly affected by myxomatous mitral valve disease, compared to other breeds. OBJECTIVE: To investigate potential interbreed variation in serum 5-HT in healthy dogs. ANIMALS: 483 healthy dogs of nine breeds aged 1-7 years. METHODS: Dogs were examined at five European centers. Absence of cardiovascular, organ-related, or systemic diseases was ensured by thorough clinical investigations including echocardiography. Serum was frozen and later analyzed by enzyme-linked immunosorbent assay (ELISA). RESULTS: Median 5-HT concentration was 252.5 (interquartile range = 145.5-390.6) ng/mL. Overall breed difference was found (p<0.0001), and 42% of pairwise breed comparisons were significant. Univariate regression analysis showed association between serum 5-HT concentration and breed, center of examination, storage time, and sex, with higher 5-HT in females. In multiple regression analysis, the final model had an adjusted R2 of 0.27 with breed (p<0.0001), center (p<0.0001), and storage time (p=0.014) remaining significant. Within centers, overall breed differences were found at 3/5 centers (p≤0.028), and pairwise comparisons within those centers showed breed differences in 42% of comparisons. Among the included breeds, Newfoundlands, Belgian Shepherds and CKCSs had highest 5-HT concentrations. CONCLUSIONS: Interbreed variation in serum 5-HT concentration was found in healthy dogs aged 1-7 years. These differences should be taken into account when designing clinical studies.

Load-dependence of myocardial deformation variables - a clinical strain-echocardiographic study.

BACKGROUND: The effects of left ventricular (LV) loading on myocardial deformation variables are not well-studied in the clinical setting. In the present study, we evaluated the effects of isolated changes in preload, afterload and heart rate on LV longitudinal strain, systolic (SR-S) and early diastolic strain rate (SR-E) in post-cardiac surgery patients. METHODS: Twenty-one patients were studied early after cardiac surgery. Longitudinal myocardial strain and SR were analysed off-line using 2-D speckle echocardiography. The experimental protocol consisted of three consecutive interventions: (1) preload was increased by passive leg elevation, (2) afterload was increased by an infusion of phenylephrine to increase arterial blood pressure by 10-15% and (3) heart rate was increased 10% and 20% by atrial pacing. During both the preload and afterload challenges heart rate was kept constant by atrial pacing. Central venous pressure was kept constant during pacing by infusion of hetastarch/albumin. RESULTS: The increase in preload increased LV strain, SR-S and SR-E by 20%, 11% and 17%, respectively. The phenylephrine-induced increase in afterload, did not affect LV strain, SR-S or SR-E. LV strain was not affected while SR-S and SR-E increased by pacing-induced heart rate increase. CONCLUSION: After cardiac surgery, systolic and early diastolic strain rate are dependent on both preload and heart rate, while neither of these variables was afterload-dependent. LV strain was preload-dependent but not affected by atrial pacing. When evaluating the direct effects of various pharmacological or other interventions on myocardial contractility and relaxation, preload and heart rate must be controlled.

Circulating let-7g is down-regulated in Bernese Mountain dogs with disseminated histiocytic sarcoma and carcinomas - a prospective study.

Cancer is a prevalent cause of mortality in Bernese mountain dogs (BMDs). Circulating microRNAs (miRNAs) are found in blood and have been identified as promising biomarkers in various neoplastic diseases in humans. In the current study, the expression profile of different types of miRNAs was investigated in healthy BMDs and BMDs with cancer. Seven healthy and six non-treated BMDs with cancer [four with disseminated histiocytic sarcomas (DHS)] were enrolled in this study. Clinical evaluations including physical examination, blood analysis, urinalysis and diagnostic imaging were performed on all dogs. Twenty-four different miRNAs were profiled from RNA isolated from whole blood preserved in PAXgene® tubes using quantitative real-time PCR (qPCR). The miRNA let-7g was significantly down-regulated in dogs with cancer (P = 0.002) and dogs with DHS (P = 0.011) compared with healthy controls. This miRNA is a known tumour suppressor and further analyses are warranted to assess its value as a non-invasive biomarker for early detection of different types of cancer in BMDs.

Exonization of a LINE1 fragment implicated in X-linked hypohidrotic ectodermal dysplasia in cattle.

A case of X-linked hypohidrotic ectodermal dysplasia (XHED) was identified in a family of Danish Red Holstein cattle. The ectodysplasin-signalling protein (EDA) is known to be central in the normal development of ectodermal structures, and mutations in the ectodysplasin A (EDA) gene have been reported to cause XHED. In this study, we analysed different EDA transcript variants in affected and unaffected cattle and identified a new transcript variant including a LINE1-derived pseudoexon between EDA exons 1 and 2. The 161-bp-long pseudoexon introduces a shift in reading frame and a premature stop codon early in EDA exon 2 and is probably the cause of XHED in this Danish Red Holstein family.

Albinism in the American mink (Neovison vison) is associated with a tyrosinase nonsense mutation.

Albino phenotypes are documented in various species including the American mink. In other species the albino phenotypes are associated with tyrosinase (TYR) gene mutations; therefore TYR was considered the candidate gene for albinism in mink. Four microsatellite markers were chosen in the predicted region of the TYR gene. Genotypes at the markers Mvi6025 and Mvi6034 were found to be associated with the albino phenotype within an extended half-sib family. A BAC clone containing Mvi6034 was mapped to chromosome 7q1.1-q1.3 by fluorescent in situ hybridization. Subsequent analysis of genomic TYR sequences from wild-type and albino mink samples identified a nonsense mutation in exon 1, which converts a TGT codon encoding cysteine to a TGA stop codon (c.138T>A, p.C46X; EU627590). The mutation truncates more than 90% of the normal gene product including the putative catalytic domains. The results indicate that the nonsense mutation is responsible for the albino phenotype in the American mink.

Exocrine pancreatic insufficiency in the Eurasian dog breed--inheritance and exclusion of two candidate genes.

Exocrine pancreatic insufficiency is considered an inherited disease in several dog breeds. Affected dogs show polyphagia, weight loss and voluminous faeces of light colour due to the lack of pancreatic enzymes. In the study described herein, we performed a segregation analysis using the singles method for three families of the Eurasian dog breed. Our data were consistent with an autosomal recessive mode of inheritance. In addition, we performed a linkage analysis in these families using four microsatellite markers on CFA3 and two microsatellites on CFA23. Based on our results, we excluded the canine orthologs of the human cholecystokinin (CCK) and the cholecystokinin A receptor (CCKAR) genes as candidates for exocrine pancreatic insufficiency.

Porcine ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1/CD203a): cloning, transcription, expression, mapping, and identification of an NPP1/CD203a epitope for swine workshop cluster 9 (SWC9) monoclonal antibodies.

Swine workshop cluster 9 (SWC9) antibody identifying a porcine epitope on macrophages and thymocytes was used to precipitate and characterize the molecule from biotinylated macrophages and to obtain peptide sequence by mass spectrometry. The protein was identified as ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1/CD203a). The porcine NPP1/CD203a encoding gene was mapped to chromosome 1 using a radiation hybrid panel, and transcription was investigated by RT-PCR analysis of several tissues. The cDNA was cloned and introduced into COS7 cells resulting in expression of functionally active enzyme and verification of the specificity of an SWC9 reacting monoclonal antibody. The antibody was used for immunohistochemical examination of various porcine tissues. Most prominent expression of NPP1/CD203a was found in lung macrophages and liver sinusoids.

Molecular characterization of the porcine surfactant, pulmonary-associated protein C gene.

The surfactant, pulmonary-associated protein C (SFTPC) is a peptide secreted by the alveolar type II pneumocytes of the lung. We have characterized the porcine SFTPC gene at genomic, transcriptional, and protein levels. The porcine SFTPC is a single-copy gene on pig chromosome 14. Two transcripts were found in a newborn pig lung cDNA library: a full-length clone and a clone missing exon 5. cDNA sequence comparison revealed four synonymous and two nonsynonymous substitutions and in-frame insertions at the beginning of exon 5. Comparison of the SFTPC coding region between several mammals showed high levels of conservation. Northern blot studies showed lung-specific expression of the full-length SFTPC transcript, appearing in 50-day-old fetus and increasing during lung development. Both SFTPC transcripts were detected mainly in lung by real-time RT-PCR and they were significantly down-regulated in necrotic lungs of pigs infected with Actinobacillus pleuropneumoniae. Additionally, the protein levels were also decreased or absent in the necrotic tissue.

Epidemiology and inheritance of mitral valve prolapse in Dachshunds.

One hundred ninety consecutive Dachshunds >2 years of age, including 18 families consisting of both parents and 4 or more offspring, were examined clinically and echocardiographically to study the epidemiology and inheritance of mitral valve prolapse (MVP) and other signs of myxomatous mitral valve disease in the dog. From video-recorded echocardiograms, MVP severity, jet size (color Doppler), and leaflet thickness were assessed. With regard to murmur intensity and each of these 3 echocardiographic measurements. the inheritance and the influence of age, gender, coat type, body weight, degree of obesity, heart rate, and thorax dimensions were evaluated. MVP severity correlated positively with age (P < .0001) and heart rate (P = .002), negatively with thorax circumference (P = .0005), and was related to coat type (P = .006). MVP severity progressed faster in males than in females (P = .0002). The other measures of disease severity (jet size, leaflet thickness, and murmur intensity) also correlated positively with age (all P < .0001). When compared in pairs, all 4 measures of disease severity correlated significantly with one other. Pedigree analyses did not disclose agreement with simple Mendelian models, but high disease prevalence made interpretation difficult. Mean parental MVP severity correlated significantly with MVP severity in the offspring (P = .03). The epidemiology of MVP in Dachshunds resembles that of MVP in humans, MVP severity correlates significantly with other measures of the degree of myxomatous mitral valve disease, and MVP is an inherited condition in Dachshunds. A polygenic mode of inheritance is suggested.

A linkage group on pig chromosome 4 comprising the loci for blood group L, GBA, ATP1B1 and three microsatellites.

Restriction fragment length polymorphisms (RFLPs) were described for the porcine loci for beta-glucosidase (GBA) and the beta-polypeptide 1 of the Na+,K(+)-transporting ATPase (ATP1B1). Linkage analyses using a three-generation pedigree provided evidence for the assignment of ATP1B1, GBA and two microsatellite loci (S0001 and S0067) to a previously described linkage group comprising the loci for blood group L (EAL) and an anonymous microsatellite (S0097). The linear order of the six markers was determined with confidence by multipoint analyses and the length of the linkage group was estimated at 88cM. This linkage group was assigned to pig chromosome 4 on the basis of a previous physical localization of the ATP1B1 gene. In situ hybridization data for S0001 presented in this study were consistent with a localization on chromosome 4 and suggested a regional localization to 4p12-p13. The present study reveals conflicting data concerning the genetic localization of the K88 loci controlling the expression of the receptors for the E. coli pilus antigens. One group has reported data suggesting a loose linkage between K88 and EAL, now mapped to chromosome 4, whereas two other groups have found linkage between K88 and the transferrin locus (TF), mapped to chromosome 13 by in situ hybridization.

Conserved synteny between pig chromosome 8 and human chromosome 4 but rearranged and distorted linkage maps.

The porcine genes encoding interleukin 2, alcohol dehydrogenase (class I) gamma polypeptide, and osteopontin were mapped to chromosome 8 by linkage analysis. Together with previous assignments to this chromosome (the albumin, platelet-derived growth factor receptor A, and fibrinogen genes), an extensive syntenic homology with human chromosome 4 was discovered. Loci from about three-quarters of the q arm of human chromosome 4 are on pig chromosome 8. However, the linear order of the markers is not identical in the two species, and there are several examples of interspecific differences in the recombination fractions between adjacent markers. The conserved synteny between man and the pig gives strong support to a previous suggestion that a synteny group present in the ancestor of mammalian species has been retained on human chromosome 4q. Since loci from this synteny group are found on two cattle chromosomes, the bovine rearrangement must have occurred after the split of Suidae and Bovidae within Artiodactyla.

The dispersion of defective endogenous murine retroviral elements suggests retrotransposition-mediated amplification.

The dispersion of four replication-defective endogenous proviruses, originally detected in 129 strain mice and shown to have extensive deletions of gag, pol, and env gene regions, was investigated in 13 inbred strains and substrains of mice. Using probes to sequences flanking the integration sites in 129 mice, unique genomic Eco RI fragments were assigned to each of the four endogenous proviral elements. Analyses revealed that certain of these proviral elements are present both in strains closely related to strain 129 (i.e., strains 101 and LP/J) and in more distantly related strains (i.e., strains BALB/cJ, A/J, and C3H/HeJ). In mouse strains lacking proviral integration at a particular locus, the size of the corresponding Eco RI genomic fragment and absence of a characteristic Kpn I site indicated the lack of a residual solitary long terminal repeat. Hybridization of oligonucleotide probes that distinguish the specific deletions present within these elements identified additional analogous proviral integrations at many different sites in all strains investigated. These data indicate that the diversification of these proviral elements found in inbred strains is generated by integration of new copies, rather than excision through homologous recombination. Moreover, the results are consistent with other endogenous retroviruses providing the trans-acting proteins necessary to package the defective viral RNA.

Truncated gag products encoded by Gv-1-responsive endogenous retrovirus loci.

The conversion of endogenous or exogenous murine retroviruses to a leukemogenic phenotype involves recombination with retroviral sequences present in host genomic DNA. In the 129 Gix+ inbred strain, these endogenous sequences are replication defective but still express retroviral proteins under the apparent transcriptional control of the Gv-1 regulatory locus. To study the protein-coding potential of Gv-1-regulated endogenous retroviral loci, we used oligonucleotide probes directed to env deletion breakpoints identified in previously characterized cDNA clones. Four endogenous retroviral loci were isolated from a library of 129 Gix+ genomic DNA with these probes. Three loci cloned with the env deletion probe del env-1 had virtually identical proviral inserts by restriction analysis. A unique locus was identified and cloned with the del env-2 probe, which must therefore represent a Gv-1-responsive element. Restriction enzyme and nucleotide sequence analyses indicated that the del env-1 and del env-2 loci represented members of the polytropic and modified polytropic classes of endogenous retrovirus, respectively. Despite this divergence, members of both classes contained identical deletions of 19 nucleotides within p30gag and of 1,474 nucleotides from p10gag into the reverse transcriptase-coding region of pol, suggesting that a recombination event had occurred between these proviral sequences prior to insertion within the genome. The del env-1 and del env-2 loci retained coding capacity for truncated gag polyproteins, confirmed by in vitro translation and immunoprecipitation of the protein products. Nucleotide sequence comparison of the untranslated leader (L) regions of the del env-1 and del env-2 loci to a replication-competent ecotropic virus indicated regions that might be important to dispersion of these endogenous retroviral elements throughout the host genome.