Investigation of periodontal disease development and Porphyromonas gulae FimA genotype distribution in small dogs.

In dogs, Porphyromonas gulae is a major periodontal pathogen with 41-kDa proteins polymerizing to form a filamentous structure called fimbriae or pili, termed FimA. FimA is classified into three genotypes: A, B, and C, and there are combinations of types A, B, C, A/B, A/C, B/C, and A/B/C. Periodontal disease is the most common oral disease in small dogs, but the periodontal disease status and P. gulae colonization at each dog age and breed remain unclear. In this study, we stratified 665 small dogs and analyzed the periodontal status and distribution of P. gulae with each FimA genotype. Dogs with periodontal disease and FimA genotype tended to increase with age. The dogs with at least one FimA genotype had significantly more severe periodontal disease compared with P. gulae-negative dogs (P < 0.01). Additionally, periodontal status was significantly associated with specific FimA genotype distribution in Toy Poodles and Chihuahuas (P < 0.05), whereas there was no such association in Dachshunds. These results suggest that the onset of periodontal disease and P. gulae colonization are related and progress with age. The relationship between periodontal disease and FimA genotype may differ depending on the dog breeds.

Regulatory mechanisms underlying interleukin-6 expression in murine brown adipocytes.

Three types of adipocytes, white, brown, and beige, regulate the systemic energy balance through the storage and expenditure of chemical energy. In addition, adipocytes produce various bioactive molecules known as adipokines. In contrast to white adipocyte-derived molecules, less information is available on the adipokines produced by brown adipocytes (batokine). This study explored the regulatory expression of interleukin (IL)-6 in cell culture studies. Norepinephrine or a nonselective ß-adrenergic receptor agonist increased the expression of IL-6 in primary brown adipocytes and HB2 brown adipocytes. Treatment with forskolin (Fsk), an activator of the cAMP-dependent protein kinase (PKA) pathway (downstream signaling of the ß-adrenergic receptor), efficiently stimulated IL-6 expression in brown adipocytes and myotubes. Phosphorylated CREB and phosphorylated p38 MAP kinase levels were increased in Fsk-treated brown adipocytes within 5 min. In contrast, a long-term (∼60 min and ∼4 h) treatment with Fsk was required for increase in STAT3 phosphorylation and C/EBPß expression, respectively. The PKA, p38 MAP kinase, STAT3, and C/EBPß pathways are required for the maximal IL-6 expression induced by Fsk, which were verified by use of various inhibitors of these signal pathways. Vitamin C enhanced Fsk-induced IL-6 expression through the extracellular signal-regulated kinase activity. The present study provides basic information on the regulatory expression of IL-6 in activated brown adipocytes.

Weak response of bovine hepcidin induction to iron through decreased expression of Smad4.

Hepcidin negatively regulates systemic iron levels by inhibiting iron entry into the circulation. Hepcidin production is increased in response to an increase in systemic iron via the activation of the bone morphogenetic protein (BMP) pathway. Regulation of hepcidin expression by iron status has been proposed on the basis of evidence mainly from rodents and humans. We evaluated the effect of iron administration on plasma hepcidin concentrations in calves and the expression of bovine hepcidin by the BMP pathway in a cell culture study. Hematocrit as well as levels of blood hemoglobin and plasma iron were lower than the reference level in calves aged 1-4 weeks. Although intramuscular administration of iron increased iron-related parameters, plasma hepcidin concentrations were unaffected. Treatment with BMP6 increased hepcidin expression in human liver-derived cells but not in bovine liver-derived cells. A luciferase-based reporter assay revealed that Smad4 was required for hepcidin reporter transcription induced by Smad1. The reporter activity of hepcidin was lower in the cells transfected with bovine Smad4 than in those transfected with murine Smad4. The lower expression levels of bovine Smad4 were responsible for the lower activity of the hepcidin reporter, which might be due to the instability of bovine Smad4 mRNA. In fact, the endogenous Smad4 protein levels were lower in bovine cells than in human and murine cells. Smad4 also confers TGF-ß/activin-mediated signaling. Induction of TGF-ß-responsive genes was also lower after treatment with TGF-ß1 in bovine hepatocytes than in human hepatoma cells. We revealed the unique regulation of bovine hepcidin expression and the characteristic TGF-ß family signaling mediated by bovine Smad4. The present study suggests that knowledge of the regulatory expression of hepcidin as well as TGF-ß family signaling obtained in murine and human cells is not always applicable to bovine cells.

Possibility of uncoupling protein 1 expression in bovine fast-twitch muscle fibers.

Uncoupling protein 1 (UCP1) is responsible for non-shivering thermogenesis in brown/beige adipocytes in humans and rodents. Previously, we showed unexpected expression of UCP1 in bovine skeletal muscles. Here we evaluated Ucp1 mRNA levels in the muscle tissue of Japanese Black steers. Expression of Ucp1 was higher in 30-month-old cattle than in 26-month-old cattle. Levels of myosin heavy chain (Myh)1, an MYH predominantly expressed in fast-twitch muscles, were also significantly higher in cattle aged 30 months. A similar tendency was observed in the expression of other Myhs that are highly expressed in fast-twitch muscles, Myh2 and Myh4. Ucp1 expression was positively correlated with expression of Myh1, Myh2, and Myh4. Our results indicate the possibility of Ucp1 expression in fast-twitch muscle fibers.

Possible association of fimA genotype of Porphyromonas gulae with the severity of periodontal disease and the number of permanent teeth in dogs.

Previous research has demonstrated that Porphyromonas gulae (P. gulae) significantly contributes to the development of periodontal disease in dogs. Porphyromonas gulae is divided into three subtypes according to the 41-kDa filamentous appendage (fimA), defined as types A, B, and C. This study aimed to elucidate the association between fimA type of P. gulae with the number of permanent teeth, reflecting the severity of periodontal disease. Two hundred twenty-five dogs were categorized by P. gulae fimA type as negative, type A dominant, type B dominant, and type C dominant. The stage of periodontal disease in P. gulae-positive dogs increased with age, particularly in type C dominant dogs. Correspondingly, the number of permanent teeth in P. gulae fimA type C-dominant dogs was significantly lower than that of P. gulae-negative dogs, suggesting there is a significant association between fimA type of P. gulae and the number of permanent teeth resulting from the development of periodontal disease.

Transcriptional activation of hepcidin by the microphthalmia/transcription factor E family.

Hepcidin negatively regulates the circulating iron levels by inhibiting the intestinal absorption of iron as well as iron release from macrophages. Hepcidin activity is largely determined by its expression, which is regulated at the transcriptional level. Hepcidin transcription is induced not only by the iron status-related bone morphogenetic protein (BMP)-2/6, but also by inflammatory cytokines, such as interleukin (IL)-1ß and IL-6. The present study reveals that the microphthalmia (MiT)/transcription factor E (TFE) family members are novel regulators of hepcidin transcription. Melanocyte-inducing transcription factor (MITF)-A, a member of the MiT/TFE family, was identified as a positive regulator of hepcidin transcription via stimulus screening for transcription regulators. An E-box (5'-CATGTG-3') spanning nt-645 to nt-640 of the murine hepcidin promoter was identified as an MITF-A-responsive element. Responsiveness to MITF-A on hepcidin transcription decreased when the cells were stimulated with BMP2 or IL-1ß. These results suggest a functional interaction between the MITF pathway and BMP- or IL-1ß-mediated signaling. TFEB and TFE3, members of the MiT/TFE family, also stimulated hepcidin transcription, but the main region responsible for hepcidin transcription was distinct from that induced by MITF-A. The region spanning nt-581 to nt-526 was involved in TFEB/TFE3-mediated hepcidin transcription. Considering that members of the MiT/TFE family act as regulators of starvation-induced lysosomal biogenesis, hepcidin expression may be controlled by additional pathways apart from those identified so far.

Relationships between the expression of adipose genes and profiles of hospitalized dogs.

Obesity is one of the risk factors for the onset of various metabolic diseases in dogs. Energy expenditure in brown/beige adipocytes, which is partially regulated by the bone morphogenetic protein (BMP) pathway, is a key factor determining systemic energy balance. Here, we examined gene expression in the fat depots of 129 hospitalized dogs, and the relationship between the relative levels of gene expression and profiles of dogs. We evaluated the expression levels of 23 genes such as regulatory genes of adipocyte differentiation and function, adipokines, genes related to brown adipogenesis and uncoupling protein (Ucp), and genes involved in BMP signaling. A reliable equation of multiple regression was not obtained to explain the body condition score (BCS), which is an index of adiposity. Positive relationships were detected between the expression levels of many genes, except for Ucp1 or Ucp3. BCS was found to increase with age. BCS was negatively correlated to the expression levels of Pparγ and Fasn, and positively correlated to Leptin and Opn3 expression. Aging decreased the expression levels of genes related to adipocyte differentiation and function (Pparγ, Fabp4, Fasn, Hsl, and Insr) and Adipoq. In addition, age was negatively correlated with the expression of genes involved in brown adipogenesis and BMP signaling components (Prdm16, Bmp4, Alk3, Actr2a, and Actr2b). In contrast, the expression levels of Leptin and Ucp2 were found to increase with age. The present study clarifies BCS- and age-related gene expressions in the adipose tissue, which potentially contribute to elucidating the etiology of canine obesity.

Stimulation of uncoupling protein 1 expression by ß-alanine in brown adipocytes.

Carnosine, which is abundant in meat, is a dipeptide composed of ß-alanine and histidine, known to afford various health benefits. It has been suggested that carnosine can elicit an anti-obesity effect via induction and activation of brown/beige adipocytes responsible for non-shivering thermogenesis. However, the relationship between carnosine and brown/beige adipocytes has not been comprehensively elucidated. We hypothesized that ß-alanine directly modulates brown/beige adipogenesis and performed an in vitro assessment to test this hypothesis. HB2 brown preadipocytes were differentiated using insulin from day 0. Cells were treated with various concentrations of ß-alanine (12.5-100 µM) during adipogenesis (days 0-8) and differentiation (days 8-10). Then, cells were further stimulated with or without forskolin, an activator of the cAMP-dependent protein kinase pathway, on day 8 or day 10 for 4 h before harvesting. We observed that HB2 cells expressed molecules related to the transport and signal transduction of ß-alanine. Treatment with ß-alanine during brown adipogenesis dose-dependently enhanced forskolin-induced Ucp1 expression; this was not observed in differentiated brown adipocytes. Consistent with these findings, treatment with ß-alanine during days 0-8 increased phosphorylation levels of CREB in forskolin-treated HB2 cells. In addition, ß-alanine treatment during brown adipogenesis increased the expression of Pparα, known to induce brown/beige adipogenesis, in a dose-dependent manner. These findings revealed that ß-alanine could target HB2 adipogenic cells and enhance forskolin-induced Ucp1 expression during brown adipogenesis, possibly by accelerating phosphorylation and activation of CREB. Thus, ß-alanine, a carnosine-constituting amino acid, might directly act on brown adipogenic cells to stimulate energy expenditure.

Factors affecting the induction of uncoupling protein 1 in C2C12 myogenic cells.

Brown/beige adipocytes, which are derived from skeletal muscle/smooth muscle-lineage cells, consume excess energy as heat through the expression of mitochondrial uncoupling protein 1 (UCP1). Previous studies have shown that forced expression of PR/SET domain (PRDM)-16 or early B-cell factor (EBF)-2 induced UCP1-positive adipocytes in C2C12 myogenic cells. Here, we explored the culture conditions to induce Ucp1 expression in C2C12 cells without introducing exogenous genes. Treatment with rosiglitazone (a peroxisome proliferator-activated receptor (PPAR)-γ agonist), GW501516 (a PPARδ agonist), and bone morphogenetic protein (BMP)-7 for 8 days efficiently increased Ucp1 expression in response to treatment with forskolin, an activator of the protein kinase A pathway. BMP7 dose-dependently increased forskolin-induced Ucp1 expression in the presence of rosiglitazone and GW501516; however, GW501516 was not required for Ucp1 induction. Additionally, the structurally related proteins, BMP6 and BMP9, efficiently increased forskolin-induced Ucp1 expression in rosiglitazone-treated cells. UCP1 protein was localized in cells with lipid droplets, but adipocytes were not always positive for UCP1. Continuous treatment with BMP7 was needed for the efficient induction of Ucp1 by forskolin treatment. Significant expression of Prdm16 was not detected, irrespective of the treatment, and treatment with rosiglitazone, GW501516, and BMP7 did not affect the expression levels of Ebf2. Fibroblast growth factor receptor (Fgfr)-3 expression levels were increased by BMP9 in rosiglitazone-treated cells, and molecules that upregulate Fgfr3 transcription partly overlapped with those that stimulate Ucp1 transcription. The present results provide basic information on the practical differentiation of myogenic cells to brown adipocytes.

Isolation of the canine inhibin ßB subunit gene and characterization of signalling mediated by canine inhibin ßB.

Activin B, a homodimer of the inhibin ßB subunit, acts as a regulator of gonadal function and as an adipokine. To clarify the role of activin B in dogs, we characterized the canine inhibin ßB gene and signalling pathways regulated by the canine inhibin ßB. Using 5'- and 3'-rapid amplification of cDNA end (RACE) and RT-PCR on RNA isolated from the ovary of dogs, we identified short and long forms of the inhibin ßB gene. Immunoreactive inhibin ßB molecules were detected at ~25 and ~14 kDa under nonreducing and reducing conditions, respectively, in culture supernatants from HEK293 cells transfected with a plasmid containing the long form of the inhibin ßB gene, indicating activin B production and secretion. Similar to human and murine activin B, the canine activin B-stimulated transcriptions of reporter genes, CAGA-luc and Hepcidin-luc, regulated by the canonical activin/transforming growth factor-ß (TGF-ß) and bone morphogenetic protein (BMP) pathway, respectively. Activin B-induced CAGA-luc transcription was not detected in ALK7-deficient MDCK canine-derived cells; however, the forced expression of ALK7 resulted in the activin B-dependent expression in MDCK cells. Unexpectedly, the activin B-induced activation of the BMP pathway was partially blocked by the inhibition of endogenous activin/TGF-ß receptor activity. The present study identified an experimentally isolated long form of the canine inhibin ßB gene producing activin B that transactivates BMP- and activin/TGF-ß-regulated gene expression.

Diploid and aneuploid sperm in tetraploid ginbuna, Carassius auratus langsdorfii.

Ginbuna (Carassius auratus langsdorfii (Teleostei: Cyprinidae)) occur in diploid, triploid, and tetraploid forms in wild populations. Diploid females reproduce bisexually, whereas polyploid (triploid and tetraploid) females reproduce gynogenetically with no contribution from sperm nuclei. However, tetraploid males produce diploid sperm. The mechanism responsible for the differences in egg and sperm ploidy has not been elucidated as tetraploid males are rare in wild populations. Here, we aimed to characterize the types of sperm and elucidate the mechanism of spermatogenesis in ginbuna. In the present study, we artificially produced tetraploid males by crossbreeding triploid ginbuna females with diploid goldfish (Carassius auratusauratus) males via accidental incorporation of sperm nuclei. We then examined spermatogenesis to reveal the process by which reduced diploid sperm are generated from tetraploid germ cells. DNA fingerprinting by random amplified polymorphic DNA (RAPD)-PCR indicated that the tetraploid progeny had a paternally derived genome. For the tetraploid male sperm, there were narrow (N-type) and broad (B-type) flow cytometrical histograms. The N-type were determined to be diploid with a low coefficient of variation (CV) by flow cytometry. The B-type were found to be aneuploid (hypodiploid to hexaploid) with a high CV. The head sizes of B-type sperm were variable, whereas those of the N-type sperm were uniform. Computer-assisted sperm analysis (CASA) revealed that both the haploid and diploid B-type sperm were weakly motile compared with the haploid sperm of goldfish and the diploid N-type sperm of tetraploid males. Bivalents and various multivalents were observed in the meiotic configurations of diploid spermatogenesis. In aneuploid spermatogenesis, most of the chromosomes were unpaired univalents and there were very few bivalents. Our findings provide empirical evidence for two different types of spermatogenesis in tetraploid C. a. langsdorfii males. Meiotic synapses might explain the observed differences in the ploidy status of the two sperm types.

Efficacy of FimA antibody and clindamycin in silkworm larvae stimulated with Porphyromonas gulae.

Objective: Porphyromonas gulae, a major periodontal pathogen in animals, possesses fimbriae that have been classified into three genotypes (A, B, C) based on the diversity of fimA genes encoding fimbrillin protein (FimA). P. gulae strains with type C fimbriae were previously shown to be more virulent than other types. In this study, we further examined the host toxicity mediated by P. gulae fimbriae by constructing recombinant FimA (rFimA) expression vectors for each genotype and raised antibodies to the purified proteins. Methods and Results: All larvae died within 204 h following infection with P. gulae type C at the low-dose infection, whereas type A and B did not. Among fimA types, the survival rates of the larvae injected with rFimA type C were remarkably decreased, while the survival rates of the larvae injected with rFimA type A and type B were greater than 50%. Clindamycin treatment inhibited the growth of type C strains in a dose-dependent manner, resulting in an increased rate of silkworm survival. Finally, type C rFimA-specific antiserum prolonged the survival of silkworm larvae stimulated by infection with P. gulae type C strain or injection of rFimA type C protein. Conclusion: These results suggested that type C fimbriae have high potential for enhancement of bacterial pathogenesis, and that both clindamycin and anti-type C rFimA-specific antibodies are potent inhibitors of type C fimbriae-induced toxicity. This is the first report to establish a silkworm infection model using P. gulae for toxicity assessment.

FISH Identifies Chromosome Differentiation Between Contemporary Genomes of Wild Types and the Ancestral Genome of Unisexual Clones of Dojo Loach, Misgurnus anguillicaudatus.

In dojo loach (Misgurnus anguillicaudatus), although most wild types are gonochoristic diploids that are genetically differentiated into 2 groups, A and B, clonal lineages appear in certain localities. Clonal loaches have been considered to have hybrid origins between the 2 groups by a series of genetic studies. In this study, using FISH with a newly developed probe (ManDra-A), we identified 26 (1 pair of metacentric and 12 pairs of telocentric chromosomes) of 50 diploid chromosomes in contemporary wild-type group A loach. In contrast, ManDra-A signals were not detected on metacentric chromosomes derived from the ancestral group A of clonal loach. The FISH results clearly showed the presence of certain differentiations in metacentric chromosomes between ancestral and contemporary group A loach. Two-color FISH with ManDra-A and group B-specific ManDra (renamed ManDra-B) probes reconfirmed the hybrid origin of clones by identifying chromosomes from both groups A and B in metaphases. Our results showed the hybrid origin of clonally reproducing fish and the possibility that chromosomal differentiation between ancestral and contemporary fish can affect gametogenesis. In meiotic spermatocytes of sex-reversed clones, ManDra-A, and not ManDra-B, signals were detected in 12 out of 50 bivalents. Thus, the results further support the previous conclusion that clonal gametogenesis was assured by pairing between sister chromosomes duplicated from each ancestral chromosome from group A or B. Our study deepens the knowledge about the association between clonality and hybridity in unisexual vertebrates.

Regulatory expression of uncoupling protein 1 and its related genes by endogenous activity of the transforming growth factor-ß family in bovine myogenic cells.

Uncoupling protein 1 (UCP1) is responsible for non-shivering thermogenesis, with restricted expression in brown/beige adipocytes in humans and rodents. We have previously shown an unexpected expression of UCP1 in bovine skeletal muscles. This study evaluated factors affecting Ucp1 gene expression in cultured bovine myogenic cells. Myosatellite cells, which were isolated from the bovine musculus longissimus cervicis, were induced to differentiate into myotubes in the presence of 2% horse serum. Previous studies using murine brown/beige adipocytes revealed that Ucp1 expression levels are directly increased by forskolin and all-trans retinoic acid (RA). The transforming growth factor-ß (TGF-ß)/activin pathway negatively regulated Ucp1 expression, whereas activation of the bone morphogenetic protein (BMP) pathway indirectly increases Ucp1 expression through the stimulation of brown/beige adipogenesis. Neither forskolin nor RA significantly affected Ucp1 mRNA levels in bovine myogenic cells. A-83-01, an inhibitor of the TGF-ß/activin pathway, stimulated myogenesis in these cells. A-83-01 significantly increased the expression of some brown fat signature genes such as Pgc-1α, Cox7a1, and Dio2, with a quantitative but not significant increase in the expression of Ucp1. Treatment with LDN-193189, an inhibitor of the BMP pathway, did not affect the differentiation of bovine myosatellite cells. Rather, LDN-193189 increased Ucp1 mRNA levels without modulating the levels of other brown/beige adipocyte-related genes. The current results indicate that the regulation of Ucp1 expression in bovine myogenic cells is distinct from that in murine brown/beige adipocytes, which has been more intensely characterized. SIGNIFICANCE OF THE STUDY: We previously reported unexpected expression of Ucp1 in bovine muscle tissues; Ucp1 expression has been known to be detected predominantly in brown/beige adipocytes. This study examined regulatory expression of bovine Ucp1 in myogenic cells. Consistent with the changes in expression levels of brown/beige adipocyte-selective genes, Ucp1 expression tended to be increased by inhibition of endogenous TGF-ß activity. In contrast, inhibition of endogenous BMP significantly increased Ucp1 expression without affecting brown/beige adipocyte-selective gene expression. The current results indicate that regulatory expression of Ucp1 in bovine myogenic cells is distinct from that in murine brown/beige adipocytes that is more intensely characterized.

Response to iron overload in cultured hepatocytes.

Iron is essential for a variety of physiological processes. Hepatic iron overload acts as a trigger for the progression of hepatic steatosis to nonalcoholic steatohepatitis and hepatocellular carcinoma. In the present study, we aimed to study the effects of iron overload on cellular responses in hepatocytes. Rat primary hepatocytes (RPH), mouse primary hepatocytes (MPH), HepG2 human hepatoma cells and Hepa1-6 mouse hepatoma cells were treated with FeCl3. Treatment with FeCl3 effectively increased iron accumulation in primary hepatocytes. Expression levels of molecules involved in cellular signaling such as AMPK pathway, TGF-ß family pathway, and MAP kinase pathway were decreased by FeCl3 treatment in RPH. Cell viability in response to FeCl3 treatment was decreased in RPH but not in HepG2 and Hepa1-6 cells. Treatment with FeCl3 also decreased expression level of LC-3B, a marker of autophagy in RPH but not in liver-derived cell lines. Ultrastructural observations revealed that cell death resembling ferroptosis and necrosis was induced upon FeCl3 treatment in RPH. The expression level of genes involved in iron transport varied among different liver-derived cells- iron is thought to be efficiently incorporated as free Fe2+ in primary hepatocytes, whereas transferrin-iron is the main route for iron uptake in HepG2 cells. The present study reveals specific cellular responses in different liver-derived cells as a consequence of iron overload.

Factors affecting expression and transcription of uncoupling protein 2 gene.

Previous studies suggest a negative relationship between hepatic oxidative stress and productivity in beef cattle. Uncoupling protein 2 (UCP2) is involved in the disappearance of reactive oxygen species, suggesting the defensive role of UCP2 against oxidative stress. The present study examined the relationship between oxidative stress and expression levels of UCP2/Ucp2 in cultured human and mouse liver-derived cells. We also explored factors regulating bovine Ucp2 transcription. As oxidative stress inducers, hydrogen peroxide, ethanol, and cumene hydroperoxide (CmHP) were used. Expression levels of hemoxygenase 1 (HMOX1), a representative gene induced by oxidative stress, were not affected by any oxidative stress inducers in HepG2 human liver-derived cells. The levels of UCP2 mRNA were also unaffected by the oxidative stress inducers. Treatment with CmHP increased expression of Hmox1 in Hepa1-6 mouse liver-derived cells, but Ucp2 expression was not changed. Stimulus screening for regulator of transcription (SSRT) revealed that expression of p50 or p65, transcription factors conferring response to oxidative stress, did not stimulate bovine Ucp2 transcrition in HepG2 cells. SSRT also showed 11 molecules that induced Ucp2 transcription more than 4-fold; among them, endoplasmic reticulum (ER) stress-related transcription factors such as XBP1, c-JUN, JUNB, and C/EBPß were identified. However, treatment with ER stress inducers did not increase Ucp2 expression in HepG2 and Hepa1-6 cells. The present results suggest that 1) neither oxidative stress nor ER stress induces Ucp2 expression in liver-derived cells, and 2) Ucp2 transcription is stimulated by several transcription factors.

Unilateral diaphragmatic paralysis associated with surgical mitral valve repair in dogs.

OBJECTIVES: Unilateral diaphragmatic paralysis (UDP) due to phrenic nerve injury is a potential complication of thoracic surgery. This study evaluated the prevalence of UDP associated with surgical mitral valve repair (MVR) and its effect on surgical outcomes in dogs. ANIMALS, MATERIALS AND METHODS: Two hundred ninety-four dogs that underwent MVR were included in the study. A retrospective review of medical records was performed for dogs surviving surgery. Diagnosis of UDP was based on preoperative and postoperative thoracic dorsoventral radiographs. RESULTS: A total of 284 dogs survived until the day after surgery. The prevalence of UDP on the day after surgery, on the day of discharge, and after the first postoperative month was 30%, 24%, and 9%, respectively. One case of UDP was observed at 3 months after surgery. Unilateral diaphragmatic paralysis was exhibited by nine of the 21 patients that died in the hospital. The proportion of patients with UDP was higher in dogs that died of respiratory failure than in dogs that died of other causes (p = 0.002). Most dogs whose deaths were suspected to have been related to respiratory failure also had pre-existing respiratory diseases. The occurrence of UDP did not relate to the lengths of stay in the intensive care unit or the hospital. CONCLUSIONS: Our findings suggest that UDP is a common complication in dogs after MVR and that the prevalence of UDP decreases with time after surgery. Unilateral diaphragmatic paralysis is a risk factor for postoperative death, especially in patients with pre-existing respiratory disease.

Author Correction: Inhibition of Porphyromonas gulae and periodontal disease in dogs by a combination of clindamycin and interferon alpha.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Regulatory expression of bone morphogenetic protein 6 by 2,2'-dipyridyl.

BACKGROUND: Expression of hepcidin, a hormone produced by hepatocytes which negatively regulates the circulating iron levels, is known to be positively regulated by BMP6, a member of transforming growth factor (TGF)-ß family. Previous studies have shown that iron status is sensed by sinusoidal endothelial cells of hepatic lamina, leading to the modulation of BMP6 expression. METHODS: ISOS-1, HUVEC, F-2, and SK-HEP1 endothelial cells were treated with either iron or 2,2'-dipyridyl (2DP), a cell-permeable iron-chelator, and expression level of Bmp6 was examined. To identify factors affecting Bmp6 transcription, stimulus screening for regulator of transcription (SSRT) was developed. RESULTS: Treatment with iron slightly increased the expression levels of Bmp6, while 2DP unexpectedly increased Bmp6 expression in a dose-dependent manner. 2DP-induced Bmp6 expression was resistant to co-treatment with iron. 2DP-induced Bmp6 expression was also detected in HUVEC, F-2 cells, and SK-HEP1 cells. Luciferase-based reporter assays indicated that forced expression of JunB increased the transcription of Bmp6. 2DP induced phosphorylation of JunB; co-treatment with SP600125 blocked the 2DP-induced Bmp6 expression partially. JunB-induced Bmp6 transcription was not affected by mutations of putative JunB-responsive elements. Some endoplasmic reticulum stress inducers increased the expression of Bmp6. SSRT revealed pathways regulating Bmp6 transcription positively and negatively. Hepa1-6 liver cells and C2C12 myogenic cells were prone to 2DP induced Bmp6 expression. CONCLUSIONS: The present study reveals non­iron-regulated Bmp6 expression in endothelial cells. GENERAL SIGNIFICANCE: Regulatory expression of Bmp6 may be important as a key step for fine tuning of BMP activity.