Warmblood Fragile Foal Syndrome causative single nucleotide polymorphism frequency in Warmblood horses in Brazil.

Warmblood Fragile Foal Syndrome (WFFS) is an autosomal recessive genetic disorder caused by a mutation in the procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (PLOD1) gene, associated with collagen biosynthesis. WFFS causes lesions and malformations of the skin in neonatal foals, and abortion. The objective of this study was to investigate the allelic frequency of the single nucleotide polymorphism (SNP) c.2032G>A in the PLOD1 gene in warmblood samples from Brazil. Of the 374 Warmblood horses tested, 41 animals (11%) were identified as heterozygous for the WFFS SNP and 333 (89%) were homozygous for the wild-type allele (N/N), and therefore, the allele frequency was 5.5%. This study highlights the importance of control measures to prevent an increase in the incidence of WFFS in Warmblood horses worldwide.

Emergent presentation of a cat with dystrophin-deficient muscular dystrophy.

This report describes a case of feline dystrophin-deficient muscular dystrophy (DDMD) with an atypical clinical presentation. A novel gene mutation is reported to be responsible for dystrophin-deficient hypertrophic muscular dystrophy. In an emergency setting, clinicians should be aware of muscular dystrophy in young cats and the importance of elevated creatine kinase (CK) activity. Muscular dystrophy is rare but can present both a diagnostic and therapeutic challenge in an emergency setting. Patients with muscular dystrophy have a progressive disease with no specific treatment and have an increased risk for death during their hospital stay.

Inflamação induzida por Adjuvante de Freund: achados clínicos e efeitos sobre a expressão do RNAm da hepcidina em equinos / Freund's adjuvant-induced inflammation: clinical findings and its effect on hepcidin mRNA expression in horses

Hypoferremia observed during systemic inflammatory disorders is regulated by hepcidin. Hepcidin up-regulation is particularly important during acute inflammation, as it restricts the availability of iron, which is necessary for pathogenic microorganism growth before adaptive immunity occurs. The aim of this study was to evaluate the clinical findings and hepatic hepcidin mRNA expression in horses using a Freund's complete adjuvant (FCA) model of inflammation. The expression of hepcidin mRNA in the liver was determined in healthy horses following two intramuscular injections of FCA at 0 h and 12 h. Plasma iron and fibrinogen concentrations were measured at multiple time points between 0 h and 240 h post-FCA injection (PI). Hepcidin mRNA expression was determined by RT-qPCR using liver biopsy samples performed at 0 h (control), 6 h and 18 h PI. The mean plasma fibrinogen level was significantly different from the control values only between 120 and 216 h PI. The mean plasma iron level was significantly lower than the control between 16 and 72 h PI, reaching the lowest levels at 30 h PI (33 % of the initial value), and returned to the reference value from 96 h PI to the end of the experiment. Hepcidin mRNA expression increased at 6 h PI and remained high at 18 h PI. The iron plasma concentration was an earlier indicator of inflammatory processes in horses when compared with fibrinogen and might be useful for the early detection of inflammation in the horse. FCA administration caused the rapid onset of hypoferremia, and this effect was likely the result of up-regulated hepatic hepcidin gene expression. This study emphasizes the importance of hepcidin and iron metabolism during inflammation in horses.

A hipoferremia observada durante os processos inflamatórios sistêmicos é mediada pela hepcidina. O aumento da expressão da hepcidina é particularmente importante durante a inflamação aguda, por restringir a disponibilidade de ferro necessária para o crescimento de microrganismos patogênicos antes que a imunidade adaptativa ocorra. O objetivo deste estudo foi avaliar os achados clínicos e a expressão hepática do RNA mensageiro (RNAm) da hepcidina em cavalos após a indução da inflamação com Adjuvante completo de Freund (FCA). A expressão hepática do RNAm da hepcidina foi determinada em cavalos sadios após duas administrações intramusculares de FCA às 0 h (M0) e 12 h (M12). As concentrações plasmáticas de ferro e fibrinogênio foram mensuradas em múltiplos momentos entre 0 h e 240 h (M240) após a primeira administração de FCA (PI). A expressão do RNAm da hepcidina foi determinada por RT-qPCR usando amostras de biopsias hepáticas colhidas as 0 h (controle), 6 h (M6) e 18 h (M18) PI. A concentração plasmática média de fibrinogênio foi estatisticamente diferente do M0 entre 120 h e 216 h PI. A concentração plasmática média de ferro foi significantemente menor que o controle entre 16 h e 72 h PI, alcançou o nível mais baixo às 30 h PI (33% do valor inicial) e retornou aos valores de referência entre 96 h PI e até o final do experimento. A expressão do RNAm da hepcidina aumentou no M6 e permaneceu alta no M18. A concentração plasmática de ferro foi um indicador precoce da inflamação quando comparada com o fibrinogênio e pode ser útil na detecção precoce da inflamação em cavalos. A administração do FCA causou um rápido início da hipoferremia, e isto foi resultante do aumento da expressão hepática da hepcidina. Estes resultados enfatizam a importância da hepcidina e do metabolismo do ferro durante a inflamação em cavalos.

Allele frequency of hereditary equine regional dermal asthenia in American Quarter horses in Brazil determined by quantitative real-time PCR with high resolution melting analysis.

Hereditary equine regional dermal asthenia (HERDA) is a genetic disorder that occurs in the American Quarter horse (AQH) and is caused by a c.115G>A missense mutation in the peptidylprolyl isomerase B (PPIB) gene. Using a quantitative real-time PCR high resolution melting analysis genotyping assay for the PPIB mutation, the estimated HERDA allele and carrier frequencies in a sample of Brazilian AQHs were 2.9% and 5.8%, respectively.

Clinical and molecular study of a new form of hereditary myotonia in Murrah water buffalo.

Hereditary myotonia caused by mutations in CLCN1 has been previously described in humans, goats, dogs, mice and horses. The goal of this study was to characterize the clinical, morphological and genetic features of hereditary myotonia in Murrah buffalo. Clinical and laboratory evaluations were performed on affected and normal animals. CLCN1 cDNA and the relevant genomic region from normal and affected animals were sequenced. The affected animals exhibited muscle hypertrophy and stiffness. Myotonic discharges were observed during EMG, and dystrophic changes were not present in skeletal muscle biopsies; the last 43 nucleotides of exon-3 of the CLCN1 mRNA were deleted. Cloning of the genomic fragment revealed that the exclusion of this exonic sequence was caused by aberrant splicing, which was associated with the presence of a synonymous SNP in exon-3 (c.396C>T). The mutant allele triggered the efficient use of an ectopic 5' splice donor site located at nucleotides 90-91 of exon-3. The predicted impact of this aberrant splicing event is the alteration of the CLCN1 translational reading frame, which results in the incorporation of 24 unrelated amino acids followed by a premature stop codon.

Mutation in cyclophilin B that causes hyperelastosis cutis in American Quarter Horse does not affect peptidylprolyl cis-trans isomerase activity but shows altered cyclophilin B-protein interactions and affects collagen folding.

The rate-limiting step of folding of the collagen triple helix is catalyzed by cyclophilin B (CypB). The G6R mutation in cyclophilin B found in the American Quarter Horse leads to autosomal recessive hyperelastosis cutis, also known as hereditary equine regional dermal asthenia. The mutant protein shows small structural changes in the region of the mutation at the side opposite the catalytic domain of CypB. The peptidylprolyl cis-trans isomerase activity of the mutant CypB is normal when analyzed in vitro. However, the biosynthesis of type I collagen in affected horse fibroblasts shows a delay in folding and secretion and a decrease in hydroxylysine and glucosyl-galactosyl hydroxylysine. This leads to changes in the structure of collagen fibrils in tendon, similar to those observed in P3H1 null mice. In contrast to cyclophilin B null mice, where little 3-hydroxylation was found in type I collagen, 3-hydroxylation of type I collagen in affected horses is normal. The mutation disrupts the interaction of cyclophilin B with the P-domain of calreticulin, with lysyl hydroxylase 1, and probably other proteins, such as the formation of the P3H1·CypB·cartilage-associated protein complex, resulting in less effective catalysis of the rate-limiting step in collagen folding in the rough endoplasmic reticulum.

Lipopolysaccharide infusion up-regulates hepcidin mRNA expression in equine liver.

Hepcidin has been found to be the key regulator of iron metabolism that leads to hypoferremia during inflammation. Recent work has shown that equine hepcidin is predominantly expressed in the liver of horses. In this study, hepcidin gene expression was determined in the liver and bone marrow of six healthy horses after iv infusion of Escherichia coli O55:B5 LPS. The IL-6 gene expression was also determined in liver and bone marrow samples. Clinical and laboratory evaluations were measured at multiple time points between 0 and 240 h post-LPS infusion (PI). Liver and bone marrow biopsies were taken immediately before (baseline) and at 6 and 18 h PI. In response to endotoxin infusion, all horses showed characteristic clinical signs of endotoxemia. Plasma iron concentration was decreased significantly from the pre-infusion level at 8 h PI. Hypoferremia peak was observed at 12 h and returned to normal levels at 30 h PI. Relative real-time RT-PCR analysis showed that liver hepcidin and IL-6 mRNA expression was up-regulated at 6 h PI. Bone marrow hepcidin relative expression was not influenced by LPS infusion. In another experiment, equine monocyte cultures were stimulated with LPS (1 µg/ml). Monocyte hepcidin and IL-6 gene expression was significantly induced after 2 h of LPS stimulus and returned to baseline levels thereafter. The present study describes that, in horses, LPS infusion up-regulates hepatic hepcidin mRNA expression resulting in early observed hypoferremia and suggests that hepcidin may act as an acute-phase protein in horses.

Influence of experimental inflammatory response on hepatic hepcidin gene expression and plasma iron concentration in sheep.

Hepcidin is a highly conserved disulfide-bonded peptide that plays a central role in iron homeostasis. During systemic inflammation, hepcidin up-regulation is responsible for hypoferremia. This study aimed to analyze the influence of the inflammatory process induced by complete Freund's adjuvant (CFA) or lipopolysaccharide (LPS) on the liver expression of hepcidin mRNA transcripts and plasma iron concentration of sheep. The expression levels of hepcidin transcripts were up-regulated after CFA or LPS. Hypoferremic response was observed at 12 h (15.46 ± 6.05 µmol/L) or 6h (14.59 ± 4.38µmol/L) and iron reached its lowest level at 96 h (3.08 ± 1.18 µmol/L) or 16h (4.06 ± 1.58 µmol/L) after CFA administration or LPS infusion, respectively. This study demonstrated that the iron regulatory hormone hepcidin was up-regulated in sheep liver in response to systemic inflammation. These findings extend our knowledge on the relationship between the systemic inflammatory response, hepcidin and iron, and provide a starting point for additional studies on iron metabolism and the inflammatory process in sheep.

Identification, characterization and expression analysis of hepcidin gene in sheep.

Hepcidin is part of the innate immune system, and it plays a central role in the regulation of iron homeostasis. This peptide has been previously characterized in man, non-human primates, rat, mouse, dog, swine, cattle, horse, fishes, reptiles and birds but until now not in sheep. The aim of this study was to sequence, characterize and perform hepcidin expression analysis in different tissues collected from healthy sheep. The resulting open reading frame consisted of 249 bp predicted to encode an 82 aa peptide with a putative 23 aa signal peptide, a 34 aa pro-region and the 25 aa mature hepcidin. The deduced sequence of the sheep hepcidin precursor was most homologous to Bos taurus and Bubalus bubalis. Hepcidin was predominantly expressed in liver, although high expression was present in abomasum and lower level expression occurred in other tissues. These findings extend our comparative knowledge showing the relationship of sheep hepcidin to other mammalian hepcidins and will be helpful for additional studies on iron metabolism and inflammatory processes in sheep.

Cloning, sequencing and expression analysis of the equine hepcidin gene by real-time PCR.

Equine serum or plasma iron concentration drops quickly during inflammation. Accumulation of iron inside macrophages and reduction of the intestinal absorption of this element cause hypoferremia during systemic inflammatory processes. These mechanisms are mediated by hepcidin, a 25 amino acids peptide synthesized mainly in the liver in response to iron stores and inflammation. Hepcidin is an important peptide for systemic iron homeostasis and also has antibacterial and antifungal activities. Hepcidin up-regulation is particularly useful during acute inflammation, especially before adaptive immunity occurs, restricting iron availability necessary for pathogenic microorganism growth. Hepcidin gene products have been previously characterized in man, non-human primates, rat, mouse, dog swine, cattle, fishes, reptiles and birds; but until now not in the horse. We have cloned and sequenced equine hepcidin mRNA and performed hepcidin expression analysis in different tissues collected from four healthy horses. The deduced precursor of equine hepcidin was most homologous to Bos taurus and Sus scrofa. The expressed profile of equine hepcidin in liver was very high. Expression in cervical spinal cord and cerebral cortex was much lower than liver but higher than lung, duodenum, stomach, spleen, kidney, skeletal muscle and bladder. This sequence will be helpful for additional studies on iron metabolism and inflammatory process in horses.

Isolation, molecular cloning and functional characterization of a novel beta-toxin from the Venezuelan scorpion, Tityus zulianus.

Sting in children by Tityus zulianus scorpions (western Venezuela) often produces cardiorespiratory arrest and death by pulmonary oedema. To assess its toxicity, lethality in mice of T. zulianus soluble venom was determined. Toxin composition was studied by fractionating the crude venom through reversed-phase HPLC. The most abundant peptide, Tz1, was purified further and its N-terminal sequence, amino acid composition and molecular mass (by electron-spray ionization mass spectrometry) determined. In the presence of Tz1, activation of recombinant rat skeletal muscle sodium channels (Na(V)1.4) was shifted about 35 mV in the hyperpolarizing direction in a prepulse-dependent manner. This typical beta-toxin effect had an apparent EC50 of 3.5 microM A cDNA sequence encoding Tz1 was isolated from T. zulianus venom gland RNA using a combination of 5'- and 3'-RACE PCR. Analysis of the encoded sequence indicated that Tz1 is the processed product of a precursor containing: (i) a 20-residue long leader peptide; (ii) the amino acid sequence of the mature toxin (64 residues); and (iii) an extra Gly-Lys tail at the C-terminus, probably removed post-translationally. A comparison of Tz1 with Tityus serrulatus beta-toxin Ts1 revealed that some of the non-conservative replacements in Tz1 lie in regions potentially involved in receptor recognition.

Cloning and sequencing of the canine and feline cardiac troponin I genes.

OBJECTIVE: To determine the gene sequences of canine and feline cardiac troponin I (cTnI), express the protein from the cloned gene in vitro, and validate the use of a commercial cTnI serum analyzer in these species via detection of the expressed protein or comparison of sequence homology. SAMPLE POPULATION: Samples of ventricular myocardium from 5 healthy adult mixed-breed dogs and 5 healthy adult domestic shorthair cats. PROCEDURE: The RNA was extracted from myocardial samples, and cDNA was synthesized via reverse transcriptase polymerase chain reaction and sequenced. The canine cDNA for the coding region was expressed in cell culture and analyzed by western blot and sandwich enzyme-linked immunosorbent assays. RESULTS: Canine and feline cTnI genes were cloned and sequenced. Homology of the nucleotide and amino acid sequences of the canine and feline cTnI genes with human and rodent cTnI genes were high; the greatest homology was detected between canine and feline genes (95% and 96%, respectively). Recombinant canine cTnI protein was detected by a commercial serum cTnI analyzer and by western blot analysis. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that commercial cTnI analyzers can be used to measure serum cTnI concentration from dogs and cats. Additionally, our preliminary characterization of the feline cTnI gene may facilitate further investigation of cTnI and its role in familial hypertrophic cardiomyopathy in cats.

Dual prenylation is required for Rab protein localization and function.

The majority of Rab proteins are posttranslationally modified with two geranylgeranyl lipid moieties that enable their stable association with membranes. In this study, we present evidence to demonstrate that there is a specific lipid requirement for Rab protein localization and function. Substitution of different prenyl anchors on Rab GTPases does not lead to correct function. In the case of YPT1 and SEC4, two essential Rab genes in Saccharomyces cerevisiae, alternative lipid tails cannot support life when present as the sole source of YPT1 and SEC4. Furthermore, our data suggest that double geranyl-geranyl groups are required for Rab proteins to correctly localize to their characteristic organelle membrane. We have identified a factor, Yip1p that specifically binds the di-geranylgeranylated Rab and does not interact with mono-prenylated Rab proteins. This is the first demonstration that the double prenylation modification of Rab proteins is an important feature in the function of this small GTPase family and adds specific prenylation to the already known determinants of Rab localization.

Identification of the novel proteins Yip4p and Yip5p as Rab GTPase interacting factors.

The Rab GTPases are key regulators of membrane traffic. Yip1p is a membrane protein of unknown function that has been reported to interact with the Rabs Ypt1p and Ypt31p. In this study we identify Yif1p, and two unknown open reading frames, Ygl198p and Ygl161p, which we term Yip4p and Yip5p, as Yip1p-related sequences. We demonstrate that the Yip1p-related proteins possess several features: (i) they have a common overall domain topology, (ii) they are capable of biochemical interaction with a variety of Rab proteins in a manner dependent on C-terminal prenylation, and (iii) they share an ability to physically associate with other members of the YIP1 family.