Endothelin-1, endothelin converting enzyme-1 and endothelin receptors in the porcine corpus luteum

Porcine corpora lutea (CL) fail to show a luteolytic response to prostaglandin-F-2α (PGF-2α) (ie, luteolytic sensitivity [LS]) until about day 12-13 of the estrous cycle. Although little is known of the control of LS in any species, endothelin-1 (EDN1) is believed to play a role in LS control in ruminants. Therefore, we measured mRNA and protein expression and examined the cellular localization of EDN1 precursor (pre-pro EDN1, or ppEDN1), EDN-converting enzyme-1 (ECE1), and EDN receptors (A, EDNRA and B, EDNRB) in porcine CLs collected on days 4, 7, 10, 13, and 15 of the estrous cycle to look for differences between CLs displaying (days 13-15) versus those lacking (days 4-10) LS. Abundance of ppEDN1 mRNA was greatest (and significant vs all other days) on day 7 of the cycle, whereas EDN1 protein expression did not vary during the cycle and was localized exclusively to endothelial cells (EC). Abundance of ECE1 mRNA was also greatest on day 7 (vs all other days), but ECE1 protein was significantly elevated on day 10 (vs day 4) and was immunolocalized to ECs and large luteal cells (LLC). Abundance of EDNRA mRNA was also maximal on day 7 (vs all other days) of the cycle, whereas EDNRA protein expression was not significantly changed during the cycle and was observed in LLCs, ECs, and small luteal cells (SLC). On day 13, EDNRB mRNA was significantly decreased (versus day 7). Expression of EDNRB protein was decreased on day 10 (versus all other days), and on days 13-15 (vs day 4), and was primarily localized to ECs. In conclusion, the observed elevation in ECE1 protein concentrations on day 10 and the presence of EDNRA on LLC suggests a possible role for EDN1 (resulting from the actions of ECE1) acting via EDNRA in the control of LS in the pig.     

Short-term performance of mitral valve replacement with porcine bioprosthetic valves in dogs

Porcine bioprosthetic valves cross-linked with glutaraldehyde and polyepoxy compound were newly developed for mitral valve replacement (MVR) in dogs. Five beagle dogs were performed a left thoracotomy and underwent MVR using the porcine bioprosthetic valves during cardiopulmonary bypass (CPB). A vein catheter inserted into right atrium and a vent catheter inserted into the right ventricle to drain. The hemodynamic conditions of CPB were excellent during surgery. The left atrial pressure was measured before and after MVR; there was no significant difference and it was normal. Thrombosis and the prosthetic valve regurgitation were not observed one week after MVR. Pressure half time (PHT) prolonged significantly (P<0.05) from 31.40 +/- 4.0 msec presurgery to 99.20 +/- 19.4 msec at seven days after MVR, although it indicated the normal range as the bioprosthetic valve. The symptom of the prosthetic valve failure was not observed. This study indicated that the MVR using porcine bioprosthetic valves under CPB might have been effective in dogs as a short-term evaluation.     

Long-term clinical evaluation of mitral valve replacement with porcine bioprosthetic valves in dogs

This study evaluated the long-term clinical performance of newly developed porcine bioprosthetic valves cross-linked with glutaraldehyde and polyepoxy compound for mitral valve replacement (MVR) in dogs. Five beagle dogs underwent MVR using the porcine bioprosthetic valves during cardiopulmonary bypass. Antithrombotic drugs were administered only for one month after MVR. Six months after MVR, transvalvular regurgitation was not observed in all dogs, paravalvular leakage was seen only in one dog. Twelve months after MVR, mild transvalvular regurgitations were observed in two dogs. Although diastolic atrioventricular pressure gradient was increased gradually, no significant differences were observed. Pressure half-time and valve area were within normal ranges as the bioprosthetic value. There was no clinical symptom of the thrombosis and the thrombogenesis was not observed in the porcine bioprosthetic valve and the annulus in all dogs for twelve months after MVR. The clinical findings suggest that antithrombogenicity of the valves were maintained, though the duability might not be enough in the long-term period.     

Tissue Doppler imaging and echo-Doppler findings associated with a mitral valve stenosis with an immobile posterior valve leaflet in a bull terrier

A mitral valve stenosis was diagnosed in a 2-year-old female Bull Terrier by use of two-dimensional (2-D) and M-mode echocardiography, colour-flow imaging and spectral Doppler examinations. Tissue Doppler Imaging was also performed to assess the segmental radial myocardial motion. The mitral valve stenosis was characterized by a decreased mitral orifice area/left ventricle area ratio (0.14), an increased early diastolic flow velocity (E wave = 1.9 m/s), a prolonged pressure half-time (106 ms) and a decreased E-F slope (4.5 cm/s) on pulsed-wave Doppler examination. This mitral stenosis was associated with an immobile posterior leaflet, as seen on 2-D and M-mode echocardiography. Immobility of the posterior mitral leaflet is considered to be a rare finding in humans and, to our knowledge, has not been precisely documented in dogs with mitral valve stenosis.     

Direct measurements of nitric oxide release in relation to expression of endothelial nitric oxide synthase in isolated porcine mitral valves

The aim of this study was to measure the direct release of nitric oxide (NO) from the porcine mitral valve using a NO microelectrode. Furthermore, the expression and localization of endothelial nitric oxide synthase (eNOS) in the mitral valve was studied using immunohistochemistry, Western blotting and RT-PCR. Results show that bradykinin increases NO release from mitral valves (DeltaBradykinin: 33.71 +/- 10.41 nm NO, P < 0.001, n = 10), whereas N-nitro-l-arginine methyl esther (l-NAME) decreases NO release when compared with basal level (Deltal-NAME: 82.69 +/- 15.66 nm NO, P < 0.005, n = 4). Both protein and mRNA expression of eNOS in mitral valves and in isolated valvular endothelial cells suggest that the NO release is mainly associated with the mitral valve endothelium. It is concluded that direct NO release from porcine mitral valves coincides with eNOS expression. This study documents useful techniques for investigations into the role of local NO release in mitral valve diseases.     

肉鸡肺脏内皮素-1及其受体基因在高原低氧环境下的表达

为了研究高海拔缺氧环境对肉鸡肺部内皮素-1(ET-1)其受体ET-A和ET-B的影响,从低海拔地区引进1日龄AA肉仔鸡600羽,饲养在西藏大学农牧学院附属试验畜牧场。实验动物随机均分为2组,A组为对照组,饲喂于增氧房间内;B组暴露于高原低氧环境下。试验周期为28d,试验开始后每周随机扑杀20羽,测定心脏指数,统计腹水发病率。每个时间段每组肉仔鸡采集一部分肺组织制作石蜡切片,采用免疫组化的方法检测ET-1及其受体的表达,并应用image-pro plus图像分析系统对免疫组化切片进行半定量分析。试验结果显示:与对照组相比,高原低氧组肉鸡肺组织ET-1和ET-A基因在21,28d出现上调;ET-B表达水平在28d出现显著下降,其他时间段未见明显变化。此外,高原低氧环境对肉鸡增重和腹水发病率均有不利影响。本研究结果提示,ET-1、ET-A和ET-B表达于肉鸡肺脏;ET-1和ET-A表达水平上升和ET-B下调可能与肉鸡腹水发病紧密相关。     

Physiologic responses and plasma endothelin-1 concentrations associated with abrupt cessation of nitric oxide inhalation in isoflurane-anesthetized horses

OBJECTIVE: To assess physiologic responses and plasma endothelin (ET)-1 concentrations associated with abrupt cessation of nitric oxide (NO) inhalation in isoflurane-anesthetized horses. ANIMALS: 6 healthy adult Standardbreds. PROCEDURES: Horses were anesthetized with isoflurane in oxygen and placed in dorsal recumbency. Nitric oxide was pulsed into the respiratory tract for 2.5 hours, and then administration was abruptly discontinued. Just prior to commencement and at cessation of NO administration, and at intervals during a 30-minute period following cessation of NO inhalation, several variables including PaO(2), mean pulmonary artery pressure, venous admixture or pulmonary shunt fraction (Qs/Qt), and plasma ET-1 concentration were recorded or calculated. RESULTS: After cessation of NO inhalation, PaO(2) decreased slowly but significantly (172.7 +/- 29.8 mm Hg to 84.6 +/- 10.9 mm Hg) and Qs/Qt increased slowly but significantly (25 +/- 2% to 40 +/- 3%) over a 30-minute period. Mean pulmonary artery pressure increased slightly (14.0 +/- 1.3 mm Hg to 16.8 +/- 1 mm Hg) over the same time period. No change in serum ET-1 concentration was detected, and other variables did not change or underwent minor changes. CONCLUSIONS AND CLINICAL RELEVANCE: The improvement in arterial oxygenation during pulsed inhalation of NO to healthy isoflurane-anesthetized horses decreased only gradually during a 30-minute period following cessation of NO inhalation, and serum ET-1 concentration was not affected. Because a rapid rebound response did not develop, inhalation of NO might be clinically useful in the treatment of hypoxemia in healthy isoflurane-anesthetized horses.     

Increased annexin A1 and A2 levels in bronchoalveolar lavage fluid are associated with resistance to respiratory disease in beef calves

Strategies to control bovine respiratory disease depend on accurate classification of disease risk. An objective method to refine the risk classification of beef calves could be economically beneficial, improve welfare by preventing unexpected disease occurrences, refine and reduce the use of antibiotics in beef production, and facilitate alternative methods of disease control. The objective of this study was to identify proteins in bronchoalveolar lavage fluid (BALF) of stressed healthy calves that predict later disease outcome, serve as biomarkers of susceptibility to pneumonia, and play a role in pathogenesis. BALF was collected from 162 healthy beef calves 1–2 days after weaning and transportation. Difference in gel electrophoresis (DIGE) and mass spectrometry were used to compare proteins in samples from 7 calves that later developed respiratory disease compared to 7 calves that remained healthy. Calves that later developed pneumonia had significantly lower levels of annexin A1, annexin A2, peroxiredoxin I, calcyphosin, superoxide dismutase, macrophage capping protein and dihydrodiol dehydrogenase 3. Differences in annexin levels were partially confirmed by western blot analysis. Thus, lower levels of annexins A1 and A2 are potential biomarkers of increased susceptibility to pneumonia in recently weaned and transported feedlot cattle. Since annexins are regulated by glucocorticoids, this finding may reflect individual differences in the stress response that predispose to pneumonia. These findings also have implications in pathogenesis. Annexins A1 and A2 are known to prevent neutrophil influx and fibrin deposition respectively, and may thus act to minimize the harmful effects of the inflammatory response during development of pneumonia.     

DECR1 and ME1 genotypes are associated with lipid composition traits in Duroc pigs

Variation at the porcine DECR1 and ME1 genes has been associated with meat quality traits and backfat thickness in Landrace pigs, respectively. However, it has not been investigated yet whether DECR1 and ME1 genotypes influence lipid composition. With this aim, we have genotyped two missense DECR1 substitutions (c.160G>C and c.437G>C) and one silent ME1 (c.576C>T) polymorphism in 361 Duroc barrows distributed in five half‐sib families and phenotyped for serum lipid concentrations and intramuscular fat content and composition traits. At the whole‐population level, relevant associations, that is, with a posterior probability of the allele substitution effect to be over or below zero (PPN0) > 0.90, were observed between DECR1 genotype and serum cholesterol (CHOL) (PPN0 = 0.932) and LDL concentrations (PPN0 = 0.945) at 190 days, as well as between ME1 genotype and longissimus dorsi saturated fatty acid content (PPN0 = 0.924). At the within‐family level, we found relevant associations between DECR1 and ME1 genotypes and diverse lipid composition traits, but most of them were family‐specific. Discrepancies in allele substitution effects estimated in half‐sib families might be produced by many factors such as number of individuals, marker allele frequencies and informativeness in each family, unaccounted random genetic and environmental effects, epistasis and family‐specific differences in the linkage phase or amount of linkage disequilibrium between causal and marker mutations. This lack of consistency across families, combined with the fact that the ME1 mutation is synonymous and that the two DECR1 polymorphisms are conservative, suggests that the associations found are not causative.     

Presumed cholesterinic granulomas detected on CT in horses are associated with increased lateral ventricle height and age

Cholesterinic granulomas are mass‐like lesions that form at the choroid plexus of the ventricular system. Large cholesterinic granulomas within the lateral ventricles have been reported to cause severe neurological signs. However, little data are available about their prevalence or appearance in the overall population. The objective was to report the prevalence of presumed cholesterinic granulomas on CT in a population of horses, and investigate associations between presumed cholesterinic granuloma presence, lateral ventricle size, age, and neurological signs. The study was cross sectional, CT scans of the head were assessed for presumed cholesterinic granuloma presence and size, and lateral ventricle height. Computed tomography findings and clinical information were compared using nonparametric testing. Computed tomography scans of 139 horses were included. Presumed cholesterinic granulomas were found in 22 horses (15.8%), nine were unilateral and 13 bilateral. A significant increase in prevalence was observed with age (P < .0001), with 38% of horses over 15 years old affected. The median volume of presumed cholesterinic granulomas was 242 mm³ with a range from 51 to 2420 mm³. The mean lateral ventricle height was significantly increased in horses with presumed cholesterinic granulomas present (P = .004), with a median of 7.3 mm compared to 4.9 mm without. Neurological signs were not associated with presumed cholesterinic granuloma presence or lateral ventricle height. Fourth ventricle mineralizations were found in seven horses, which may represent cholesterinic granulomas. In conclusion, presumed cholesterinic granulomas occurred in a large proportion of the examined population and are associated with increased lateral ventricle dilation and advanced age.     

TGIF1 and SF1 polymorphisms are associated with litter size in Small Tail Han sheep

TGF-β induced factor homeobox 1 (TGIF1) and splicing factor 1 (SF1) are important for mammalian reproduction; however, the effects of these genes on litter size in sheep remain unexplored. In this study, we genotyped 768 ewes from seven sheep breeds at two loci: g.37871539C>T, a synonymous mutation of TGIF1; and g.42314637T>C, a 3′UTR variant of SF1. Our analysis of polymorphism revealed only two genotypes at locus g.37871539C>T in TGIF1, with most sheep populations being moderately polymorphic (0.25 < PIC < 0.5) at this site. In contrast, most breeds exhibited low polymorphism (PIC ≤0.25) at the SF1 locus g.42314637T>C. The association analysis revealed that a synonymous mutation at g.37871539C>T in TGIF1 was highly associated with litter size in Small Tail Han sheep, in which it causes a significant decrease in litter size. Conversely, while the SF1 3′UTR variant g.42314637T>C was also highly associated with litter size in sheep, it causes a significant increase in the number of litter size. Combined, these data provide valuable information regarding candidate genetic markers for sheep breeding programs.     

Effect of selenium intake and fetal age on mRNA levels of two selenoproteins in porcine fetal and maternal liver

The objective of this study was to determine if levels of mRNA encoding cytosolic glutathione peroxidase (cGPx) and thioredoxin reductase (TrxR-1) change during fetal development, and if maternal Se intake during gestation affects the mRNA levels of these proteins. Prepubertal gilts (n = 24) were randomly assigned to either Se-adequate (0.39 ppm of Se; n = 12) or Se-deficient (0.05 ppm of Se; n = 12) diets, 6 wk before breeding. Maternal liver was collected at d 10, 45, 70, and 114 of pregnancy, and fetal liver samples were collected at the same times except d 10. Complementary DNA sequences encoding cGPx and TrxR-1 were cloned and sequenced. Quantitative real-time PCR analysis indicated that levels of mRNA for cGPx in fetal liver decreased more than 3-fold between d 45 and 114 of gestation. Although the gilts were only marginally deficient in Se, and maternal Se intake did not affect cGPx mRNA levels in fetal liver, the low-Se diet tended (P = 0.1) to reduce fetal TrxR-1 mRNA levels. In the liver of the dams, the low Se intake did not affect mRNA levels for either cGPx or TrxR-1. Compared with the liver of the dams, mRNA levels for cGPx were about 3.5 times lower in fetal liver. Results of this study support the hypothesis that neonatal pigs are born with reduced cGPx corresponding to reduced cGPx mRNA levels during late gestation.     

Gene expression of endothelin-1 and its receptors in the heart of broiler chickens with T(3)-induced pulmonary hypertension

To investigate the effect of T₃-induced pulmonary hypertension on endothelin (ET) production and genes expression of ET-1, ET_A and ET_B receptors (ET_AR and ET_BR) during rearing, semiquantitative RT-PCR and enzyme immunometric assay were performed in the heart ventricles and serum, respectively. The ET-1 and its receptor genes were expressed in the right and left ventricles of control and T₃-treated broilers at 12, 28 and 49 days of age. There were significant (P < 0.05) reductions of the relative amounts of ET-1 (in both ventricles) and ET_AR (in the right ventricle) mRNAs at 28 and 49 days of age, in T₃-treated broilers compared to controls. The relative amounts of ET_BR mRNA in the right and left ventricles did not significantly differ between control and T₃-treated broilers at any age. The serum level of ET was significantly (P < 0.05) increased in T₃-treated chickens at 28 and 49 days of age when compared with that of the control. It is concluded that ET-1, ET_AR and ET_BR genes are normally expressed in the heart ventricles of broilers. It is likely that increased serum level of ET and decreased ET-1/ET_AR genes expression in the ventricles are involved in the heart dysfunction of broiler chickens with developmental pulmonary hypertension.     

Fumonisin B1 levels associated with an epizootic of equine leukoencephalomalacia

During the fall of 1989, an episode of equine leukoencephalomalacia involved 18 of 66 purebred Arabian horses at a breeding/training stable in Arizona. Of the 18 horses affected, the condition was fatal in 14. These horses, as well as 48 unaffected horses, had been fed a diet containing a substantial amount of white corn screenings. Gross pathologic findings included liquefactive necrosis in parts of the cerebral white matter and hemorrhagic foci of various sizes in the brain stem. Histopathologic findings included rarefied white matter with pyknotic nuclei and eosinophilic cytoplasm. Thin-layer chromatography, high-performance liquid chromatography, and gas chromatography/mass spectroscopy were utilized to identify and quantitate fumonisin B1 in 3 samples of corn from the farm. Concentrations of fumonisin B1 range from 37 to 122 ppm. Fumonisin B2 was also detected. Using information on diet, animal weights, and feeding practices, estimates of total fumonisin B1 dosage were determined. This is the first definitive report on equine leukoencephalomalacia and associated fumonisin B1 concentrations.     

Effects of leptin and adiponectin on the growth of porcine myoblasts are associated with changes in p44/42 MAPK signaling

We hypothesized that both adiponectin and leptin affect the growth of porcine skeletal muscle cells, with fatty acids acting as modifiers in adipokine action and that both adipokines influence the gene expression of their receptors. Therefore, the objective of this study was to investigate the effects of recombinant adiponectin and leptin on cell number (DNA) and DNA synthesis rate with and without oleic acid supplementation, on cell death, and on key intracellular signaling molecules of proliferating porcine myoblasts in vitro. Moreover, the mRNA expression of genes encoding for the leptin and adiponectin receptors (LEPR, ADIPOR1, ADIPOR2) as affected by leptin or adiponectin was examined. Recombinant porcine adiponectin (40 μg/mL) and leptin (20 ng/mL) increased DNA synthesis rate, measured as [³H]-thymidine incorporation (P < 0.01), reduced cell viability in terms of lactate dehydrogenase release (P < 0.05), or lowered DNA content after 24 h (P < 0.05). In adiponectin-treated cultures, oleic acid supplementation increased DNA synthesis rate and reduced cell number in a dose-dependent manner (P < 0.05). Both adiponectin (P = 0.07) and leptin (P < 0.05) induced a transient activation of p44/42 mitogen-activated protein kinase (MAPK) after 15 min, followed by decreases after 60 and 180 min (P < 0.05). Adiponectin tended to increase c-fos activation (P = 0.08) and decreased p53 activation at 180 min (P = 0.03). Both adiponectin and leptin down-regulated the abundance of ADIPOR2 mRNA and, transiently, of LEPR mRNA (P < 0.05). In conclusion, adiponectin and leptin may adversely affect the growth of porcine myoblasts, which is related to p44/42 MAPK signaling and associated with changes in ligand receptor gene expression.     

Fasting influences steroidogenesis, vascular endothelial growth factor (VEGF) levels and mRNAs expression for VEGF, VEGF receptor type 2 (VEGFR-2), endothelin-1 (ET-1), endothelin receptor type A (ET-A) and endothelin converting enzyme-1 (ECE-1) in newly formed pig corpora lutea

This study was designed to verify whether fasting influences vascular endothelial growth factor (VEGF) production and VEGF, VEGF receptor-2 (VEGFR-2) as well as endothelin (ET) system members (endothelin converting enzyme-1, ECE-1; ET-1; endothelin receptor type A, ET-A) mRNA expression in pig corpora lutea; furthermore, we wanted to assess whether fasting affects steroidogenesis in luteal cells. Eight prepubertal gilts were induced to ovulate and were randomly assigned to two groups: (A) n = 4, normally fed; and (B) n = 4, fasted for 72 h starting 3 days after ovulation. At the end of fasting, ovaries were removed from all the animals and corpora lutea (CLs) were collected. VEGF and steroid levels in luteal tissue were determined by ELISA and RIA, respectively; VEGF, VEGFR-2, ET-1, ET-A and ECE-1 mRNAs expression was measured by real-time PCR. VEGF protein levels were similar in the two groups, while all steroid (progesterone, testosterone, estradiol 17beta) concentrations were significantly (P < 0.001) higher in CLs collected from fasted animals compared with those from normally fed gilts. VEGF, VEGFR-2, ET-1 and ECE-1 (but not ET-A) mRNA expression was significantly lower (P < 0.05) in fasted versus normally fed animals. The overall conclusion is that all the parameters studied are affected by feed restriction, but the mechanisms activated at luteal level are possibly not fully adequate to compensate for nutrient shortage.     

Analyses of the regulatory mechanism of porcine WEE1B: the phosphorylation sites of porcine WEE1B and mouse WEE1B are different

WEE1B, an oocyte-specific kinase, phosphorylates the CDC2 inhibitory site and maintains the meiotic arrest of oocytes at the first meiotic prophase in several mammalian species. However, the molecular mechanisms controlling WEE1B activity have not been fully examined in species other than mice. In the present study, we analyzed the regulation mechanisms of porcine WEE1B (pWEE1B), focusing on the cAMP-dependent protein kinase (PKA) phosphorylation site and intracellular localization. As the PKA phosphorylation site in mouse WEE1B (mWEE1B) was not conserved in pWEE1B, we predicted that four serine residues would be phosphorylatable by PKA in pWEE1B (Ser77, Ser118, Ser133 and Ser149) and constructed FLAG-tagged replaced-pWEE1Bs, in which each of the PKA-phosphorylatable serines was mutated into a non-phosphorylatable alanine. We injected one of their mRNAs into porcine immature oocytes and found that the Ser77-replaced pWEE1B lost the WEE1B function, whereas the wild-type and other replaced-pWEE1Bs could maintain the meiotic arrest of oocytes. Next, the localization of pWEE1B was examined by immunohistochemistry, and exclusive nuclear localization was revealed in the fully grown oocytes. We generated a nuclear localization signal (NLS)-deleted pWEE1B (ΔNLS-pWEE1B) and then overexpressed it in porcine immature oocytes. We found that ΔNLS-pWEE1B was distributed uniformly in the cytoplasm and could not maintain the meiotic arrest of porcine oocytes. These results suggest that pWEE1B is activated after phosphorylation of the Ser77 residue, which is different from the phosphorylation site that activates mWEE1B; that pWEE1B is localized in the nucleus; and that the nuclear localization is essential for its function.