Increases in serum carbonylated protein levels of dogs with hypercortisolism.

Protein carbonylation is an irreversible and degenerative modification that can be used to evaluate oxidative stress caused by glucocorticoids. In this study, we focused on protein carbonylation in dogs with hypercortisolism (HC). Sera samples were collected from 14 dogs diagnosed with HC and treated with trilostane, 12 dogs with inflammatory diseases (disease control group), and eight clinically healthy dogs. When the carbonylated protein levels were detected by the immunoblot analysis, one band of approximately 40 kDa was predominantly increased in the dogs with HC. The band was identified as haptoglobin using the liquid chromatography tandem mass spectrometry method. Furthermore, haptoglobin immune reactivity was higher in the dogs with HC. Although the average protein carbonylation level of the HC group was not significantly different from that of the other groups, the carbonylation level was significantly higher for the poorly controlled HC cases than for the well-controlled HC group. Additionally, the primary culture of canine hepatocytes was used to clarify the direct effect of glucocorticoids on protein carbonylation in dog livers. Both the carbonylated protein and haptoglobin clearly increased after 72 h. These findings suggest that haptoglobin and its carbonylated form are increased with canine HC, and that the protein carbonylation ratio and/or haptoglobin level could be related to disease management. These factors could be useful as biomarkers for an oxidative stress reaction, at least in the liver, and for treatment monitoring of HC.

Neuronal ceroid lipofuscinosis in Border Collie dogs in Japan: clinical and molecular epidemiological study (2000-2011).

Neuronal ceroid lipofuscinosis (NCL) is an inherited, neurodegenerative lysosomal disease that causes premature death. The present study describes the clinical and molecular epidemiologic findings of NCL in Border Collies in Japan for 12 years, between 2000 and 2011. The number of affected dogs was surveyed, and their clinical characteristics were analyzed. In 4 kennels with affected dogs, the dogs were genotyped. The genetic relationships of all affected dogs and carriers identified were analyzed. The survey revealed 27 affected dogs, but there was a decreasing trend at the end of the study period. The clinical characteristics of these affected dogs were updated in detail. The genotyping survey demonstrated a high mutant allele frequency in examined kennels (34.8%). The pedigree analysis demonstrated that all affected dogs and carriers in Japan are related to some presumptive carriers imported from Oceania and having a common ancestor. The current high prevalence in Japan might be due to an overuse of these carriers by breeders without any knowledge of the disease. For NCL control and prevention, it is necessary to examine all breeding dogs, especially in kennels with a high prevalence. Such endeavors will reduce NCL prevalence and may already be contributing to the recent decreasing trend in Japan.

Clinical assessment of testosterone analogues for urethral sphincter mechanism incompetence in ten spayed female dogs.

Urethral sphincter mechanism incompetence (USMI) is a common cause of urinary incontinence in dogs. Although estrogen is often prescribed for the medical therapy of USMI for spayed female dogs, they are known to have limited effectiveness and potential adverse effects. In castrated male dogs with USMI, testosterone reagents have been attempted besides estrogen. In this study, the effect of testosterone drugs, mainly methyltestosterone, on spayed female dogs with USMI was retrospectively evaluated. Ten spayed female dogs with USMI were included. Diagnosis of USMI was based on the results of the dogs' medical history, clinical signs, and no abnormalities in physical examinations, urinalysis, ultrasonography, X-ray imaging, and neurological examinations. Methyltestosterone was administered at doses of 0.32-1.27 mg/kg BW p.o. semel in die (sid.) to twice a week. Nine of the ten dogs had good or excellent responses 2 to 4 weeks after the start of treatment. The minimum effective dose was 0.32 mg/kg/day. Although no severe adverse symptoms occurred in any dog, a mild increase in alanine aminotransferase was temporally observed at doses of 1.0 and 1.1 mg/kg/day in the two dogs. After dose reduction or withdrawal, two of eight dogs had recurrence of urinary incontinence. Resumption of testosterone treatment clearly improved the symptoms in the two dogs. These results indicate that testosterone reagents might be an option for treating USMI in spayed female dogs as well.

Parallel reductions in stomatin and Na,K-ATPase through the exosomal pathway during reticulocyte maturation in dogs: stomatin as a genotypic and phenotypic marker of high K(+) and low K(+) red cells.

Dogs can be divided into two genetic groups (a minor HK phenotype and a major LK phenotype) based on erythrocyte monovalent cation concentrations, which are controlled by the putative hk and lk allelic genes. HK dogs retain Na,K-ATPase in their erythrocytes due to the high activity of the enzyme in their precursor cells, whereas total loss of reticulocyte Na,K-ATPase occurs in LK dogs. Here, we report that the levels of the lipid raft-associated membrane protein stomatin decrease in parallel with those of Na,K-ATPase during reticulocyte maturation due to its extrusion in exosomes. The stomatin content of HK reticulocytes is higher than that of LK reticulocytes, and remains in the erythrocytes at levels compatible with that in human erythrocytes. However, it is almost absent from LK erythrocytes with the lk/lk genotype; similar to the deficiency seen in human red cells with overhydrated stomatocytosis. LK erythrocytes from hk/lk genotype dogs show reduced, but not negligible, levels of stomatin. These results indicate that the erythrocyte stomatin level is a suitable genotypic marker for the HK/LK red cell phenotype, and suggests a functional association between stomatin and Na,K-ATPase. The absence of morphological abnormalities in the erythrocytes of stomatin-deficient LK dogs also confirms that stomatin deficiency and stomatocytic shape change are independent from each other.

Serial examinations of anti-GFAP autoantibodies in cerebrospinal fluids in canine necrotizing meningoencephalitis.

Canine necrotizing meningoencephalitis (NME) is characterized by autoantibodies against glial fibrillary acidic protein (GFAP) in cerebrospinal fluids (CSFs). To clarify the time-course changes in autoantibodies, serial examinations were conducted in three dogs with NME (two Pugs and a Pomeranian) that were treated by immunosuppressive therapy. The Pugs retained high autoantibody titers throughout the observation periods (146 and 813 days) and died with neurological signs. On the other hand, the Pomeranian switched from being positive for autoantibody to negative after day 580, and its NME seemed to be in clinical remission until death on day 1238. Therefore, the anti-GFAP autoantibodies can be detected over time in canine NME even during immunosuppressive therapies. However, the autoantibodies can also disappear within a certain period after onset.

A novel sandwich enzyme-linked immunosorbent assay for feline insulin.

A novel sandwich enzyme-linked immunosorbent assay (ELISA) was established to determine the serum insulin concentrations in domestic cats. By using a solid-phase mouse anti-bovine insulin monoclonal antibody and a peroxidase-conjugated guinea pig anti-rat insulin polyclonal antibody, feline serum insulin concentrations in the range of 0.1 to 3.6 ng/ml could be measured. The intraassay CV and interassay CV were less than 6% and less than 10%, respectively. The present insulin assay will strongly help studies on feline diabetes mellitus.

Impaired expression of excitatory amino acid transporter 2 (EAAT2) and glutamate homeostasis in canine necrotizing meningoencephalitis.

To clarify the involvement of excitatory and inhibitory amino acids in canine necrotizing meningoencephalitis (NME), glutamate, aspartate, taurine and gamma-aminobutylic acid (GABA) were determined in the cerebrospinal fluids (CSF) from eight NME cases and ten healthy controls. NME dogs exhibited significantly higher concentrations of glutamate and aspartate than those in controls (p<0.001 and p<0.001, respectively), while there was no difference in taurine or GABA between the two groups. When fetal canine astrocytes were cultured for 24 hr in the presence of NME-CSF, supernatant concentrations of glutamate, aspartate and taurine were significantly elevated. Simultaneously, expression of excitatory amino acid transporter 2 (EAAT2) mRNA was significantly reduced in the astrocytes without change in EAAT1 mRNA. Hence, reduced expression of EAAT2 and impaired glutamate homeostasis may contribute to the pathogenesis of NME.

In vitro differentiation of canine celiac adipose tissue-derived stromal cells into neuronal cells.

To investigate in vitro differentiation of canine adipose tissue-derived stromal cells (ATSCs) into neuronal cells, ATSCs from celiac adipose tissue in clinically healthy beagle dogs were treated with 100 muM dibutyryl cyclic adenosine monophosphate (dbcAMP) and 125 muM isobuthylmethylxanthine (IBMX). ATSCs were morphologically changed into differentiated ATSCs from spindle-shaped cells to neuron-like cells with numerous processes after the treatment. Expression of neuron-specific enolase (NSE) as an early neuron specific marker protein was detected in both ATSCs and differentiated ATSCs, however diachronic increase of NSE expression was observed in differentiated ATSCs after the treatment with dbcAMP/IBMX. In addition, neurofilament-68 (NF-68) as an early to mature neuron specific marker protein was weakly expressed in differentiated ATSCs. Neuron specific glutamate and glucose transporter (EAAC1 and GLUT-3, respectively) mRNAs were strongly expressed in differentiated ATSCs compared with those in ATSCs, although glia specific glutamate transporter mRNA (GLT-1) was also detected in differentiated ATSCs. ATSCs can differentiate into early to mature neuronal cells and are candidate cells for autologous nerve regeneration therapy, although additional research is needed to examine functional characteristics of differentiated ATSCs.

Identification of genes for two major sialoglycoproteins, glycophorin A and glycophorin C in canine red cell membranes.

Glycophorins are the major sialoglycoproteins in red blood cell membranes, possessing various physiological and pathological roles. We examined membrane glycoproteins in canine red cells and cloned cDNAs for two major glycophorins, glycophorins A (GPA) and C (GPC) from bone marrow cells. Periodic acid-Schiff staining and immunoblotting analyses showed that canine red cell membranes contained several glycoproteins immunoreactive to an anti-bovine GPC antibody, whereas the most abundant sialoglycoproteins, the candidates for GPA, did not react with an anti-human GPA antibody. The amino acid sequences of the extracellular domains of GPA and GPC had no significant homology to those from other mammalian species, including humans, and had O-linked and/or N-linked glycosylation sites. On the other hand, the C-terminal cytoplasmic domain and/or the transmembrane helices of GPA and GPC were conserved among species, indicating some functional significance of those regions in red cell membranes that include dimerization of GPA in the membrane-spanning region, and association of GPC with membrane skeletal proteins through binding with protein 4.1 and p55 in the cytoplasmic domain. These findings provide insights for clinical studies to evaluate the involvement of GPA and GPC in the pathogenesis of red cell diseases.

Autoantibodies against glial fibrillary acidic protein (GFAP) in cerebrospinal fluids from Pug dogs with necrotizing meningoencephalitis.

Cerebrospinal fluids (CSFs) from 9 Pug dogs with necrotizing meningoencephalitis (NME: Pug dog encephalitis) were examined to identify the antigens for anti-astrocyte autoantibodies. Each CSF exhibited a positive reaction to the cytoplasm of cultured canine astrocytes by an indirect fluorescent antibody test. In an immunoblotting analysis on normal canine brain proteins, eight of 9 CSFs showed a common band of 52 kDa, corresponding to glial fibrillary acidic protein (GFAP), and all of 9 CSFs reacted with purified bovine GFAP. From these results, GFAP is one of the common autoantigens in Pug dogs with NME. On the other hand, the reactivity of CSFs to chymotrypsin-digested bovine GFAP fragments were variable among dogs, indicating that the antibodies in the CSFs recognized different epitopes on GFAP.

Age-associated changes of flash visual evoked potentials in dogs.

Age-associated changes of visual evoked potentials by flash stimulation (flash VEP) were evaluated in 53 beagle dogs aged from 1- to 15-year-old. Among the components of flash VEP consisted of 3 positive (P1, P2 and P3) and 2 negative (N1 and N2) peaks by 150 msec, the latency of P2 and the later peaks (N2 and P3) were significantly delayed with aging. Both amplitudes of the P2-N2 and N2-P3 also showed a significant correlation with aging. The flash VEP is considered to be an available and useful technique to evaluate not only for visual pathway, but also some disturbance of neurological functions, like as those reported in demented human.

Establishment and biological characterization of canine histiocytic sarcoma cell lines.

Seven novel cell lines from canine histiocytic sarcoma (HS), three of which were disseminated cutaneous HS and four of which were synovial HS, were established. All of the established cell lines had the same morphological (by light and electron microscopic findings), cytochemical (alpha-naphthyl butyrate esterase-positive), and immunohistochemical (vimentin- and lysozyme-positive, and cyto-keratin-negative) characteristics as the original HS tumor cells. All of the established cell lines injected into nude mice subcutaneously produced solid tumors. Because the established cell lines also showed phagocytic and processing activities, the HS tumor cells appear to originate from the mononuclear phagocytic system cells, despite their differences in locations or organs.

Apoptosis inhibitor of macrophage (AIM) reduces cell number in canine histiocytic sarcoma cell lines.

Apoptosis inhibitor of macrophage (AIM) is initially reported to protect macrophages from apoptosis. In this study, we determined the effect of AIM on the macrophage-derived tumor, histiocytic sarcoma cell lines (HS) of dogs. Five HS and five other tumor cell lines were used. When recombinant canine AIM was applied to non-serum culture media, cell numbers of all the HS and two of other tumor cell lines decreased dose-dependently. The DNA fragmentation, TUNEL staining and flow cytometry tests revealed that AIM induced both of apoptosis and cell cycle arrest in the HS. Although AIM is known as an apoptosis inhibitor, these results suggest that a high dose of AIM could have an opposite function in HS and some tumor cell lines.

Topographic analysis of flash visual evoked potentials in dogs.

Visual evoked potentials by flash stimulation (flash VEP) were analyzed in dogs using a topographic method. The flash VEP consisted of 3 positive (P1, P2 and P3) and 2 negative (N1 and N2) components by 150 msec after the flash stimuli. On the topographic mappings, a negative response area was observed in the frontal region of the scalp in the stimulated site followed by the shifting of the area to the contralateral frontal region and occipital region, during the first 100 msec. The negative response area in the frontal region in the stimulated site, contralateral frontal and temporal region, and occipital region were corresponded to N1, P2, and N2 on the flash VEP, respectively, according to their latencies. In the dogs with experimentally impaired the right lateral geniculate body, the latency of P2 was prolonged, and N2 and P3 were disappeared after the left eye stimulation. On the topographic mapping, only the early negative response area was detected on the stimulated site of the frontal region of the brain. Therefore, it is concluded that P1 and N1, P2, and N2 are referred to the retinal potentials, the potentials from the retina to the brainstem included the lateral geniculate body, and those from the brainstem to the visual cortex, respectively.

Changes of magnetic resonance imaging on the brain in beagle dogs with aging.

Age-associated changes of magnetic resonance imaging (MRI) on the brain were evaluated in 19 beagle dogs aged from 8-month- to 16-year-old. A significant correlation of the volume of lateral ventricle space was observed in the dogs with age advanced, however, no correlation was found between hippocampus size and the aging. The hypo-intensity areas on T2-weighted MRI were detected in globus pallidus and substantia nigra with a significant correlation of both intensity ratios to lateral ventricle with age advanced. These areas were coincided with the accumulation of iron in the slice of the brain with Perls' staining. In addition, hyper-intensity area, suggesting perivascular demyelination with fluid-filled space, was also observed in white matter surrounding the lateral ventricle on T2-weighted MRI. These results suggested that age-associated changes of T2-weighted MRI were developed in the dog brain, especially in globus pallidus, substantia nigra, and white matter surrounding lateral ventricle, like as those reported in the human brain.

Glucose uptake activity in murine red blood cells infected with Babesia microti and Babesia rodhaini.

The glucose uptake activity in Babesia rodhaini and B. microti - infected red blood cell (IRBC) was investigated in mice using 2-deoxy-D-glucose (2DOG) and L-glucose (L-Glc), a non-metabolizable analogue of D-glucose and non-incorporative glucose to non-infected RBC (NRBC), respectively. The uptake activities of both DOG and L-Glc were higher in IRBCs than those in NRBC. The concentration dependent uptake of 2DOG and L-Glc in both IRBC revealed a linear curve, indicating non-transporter mediated uptake. In addition, B. microti IRBC showed higher 2DOG uptake than B. rodhaini IRBC, whereas no difference was observed in L-Glc uptake. These results indicated that some new glucose uptake system, at least two systems, developed in both IRBC. The new systems were sodium independent, non-competitive to L-Glc, and sensitive to temperature. One of two systems had no kinetical difference between B. rodhaini and B. microti IRBC, however another one might have higher uptake activity in B. microti IRBC compared to that in B. rodhaini IRBC.

Prevalence of autoantibody in cerebrospinal fluids from dogs with various CNS diseases.

To examine the prevalence of autoantibody in canine cerebrospinal fluids (CSFs), CSFs were collected from 14 healthy controls and 88 clinical cases with various diseases in the central nervous system (CNS), and were analyzed by an indirect fluorescence antibody test on frozen sections of the cerebrum from normal Beagle dogs. An anti-astrocyte autoantibody was detected in 31 clinical cases with titers ranging from 1:1 to >/=1:100. All tested cases with necrotizing meningoencephalitis (NME: n=22) and granulomatous meningoencephalitis (GME: n=3) possessed the anti-astrocyte autoantibody, while the autoantibody was negative in most cases with other inflammatory CNS diseases. The autoantibody was also detected in 4 of 12 cases with brain tumors. Hence, examination of the autoantibody in the canine CFS would be significant for diagnosing NME and/or GME, as well as for understanding peritumoral events in cases with brain tumors.

Molecular cloning and gene expression of canine apoptosis inhibitor of macrophage.

Apoptosis inhibitor of macrophage (AIM) plays roles in survival of macrophages. In this study, we cloned canine AIM cDNA and observed its transcriptional expression levels in various tissues. The coding sequence of canine AIM was 1,023 bp encoding 340 amino acid residues, which had around 65% homology with those of the human, mouse and rat. Transcriptional expression of AIM was observed in the spleen, lung, liver and lymph node, which confirmed the expression of canine AIM in tissue macrophages. Moreover, AIM was highly expressed in one of the canine histiocytic sarcoma cell lines. CD36, the receptor of AIM, was also expressed in various tissues and these cell lines. These findings are useful to reveal the actual functions of canine AIM.

Transglutaminase 2: a novel autoantigen in canine idiopathic central nervous system inflammatory diseases.

Necrotizing meningoencephalitis (NME), necrotizing leukoencephalitis (NLE) and granulomatous meningoencephalomyelitis (GME) are common idiopathic inflammatory central nervous system (CNS) diseases with unknown etiology in dogs. We previously showed that IgG autoantibodies in the cerebrospinal fluid (CSF) of NME cases reacted to unknown brain proteins as well as to glial fibrillary acidic protein (GFAP). In the present report, we evaluated the autoantibodies against transglutaminase2 (TG2) in the canine CNS diseases. CSF samples obtained from dogs with NME (n=19), NLE (n=7), GME (n=11) and miscellaneous CNS diseases (n=12) were subjected. CSFs from 20 healthy dogs were used as controls. Indirect fluorescent antibody test on the canine cerebrum revealed astrocyte-binding IgG in the CSF of NME. After absorption of the CSF with bovine GFAP, the CSF still possessed the reactivity to astrocytes. Double-color staining showed clear colocalization of the autoantibodies and anti-human TG2 rabbit polyclonal IgG. An immunoblot assay against human recombinant TG2 revealed anti-TG2 IgG in the CSF from dogs with NME, NLE and GME. The CSF of canine idiopathic encephalitis cases, notably of NME, tended to show high ELISA OD values against human recombinant TG2 compared to healthy controls. The presence of anti-TG2 autoantibodies in the CSF may contribute to the elucidation of the etiology of canine NME, NLE and GME.

Ubiquitylation-independent ER-associated degradation of an AE1 mutant associated with dominant hereditary spherocytosis in cattle.

Various mutations in the AE1 (anion exchanger 1, band 3) gene cause dominant hereditary spherocytosis, a common congenital hemolytic anemia associated with deficiencies of AE1 of different degrees and loss of mutant protein from red blood cell membranes. To determine the mechanisms underlying decreases in AE1 protein levels, we employed K562 and HEK293 cell lines and Xenopus oocytes together with bovine wild-type AE1 and an R664X nonsense mutant responsible for dominant hereditary spherocytosis to analyze protein expression, turnover, and intracellular localization. R664X-mutant protein underwent rapid degradation and caused specifically increased turnover and impaired trafficking to the plasma membrane of the wild-type protein through hetero-oligomer formation in K562 cells. Consistent with those observations, co-expression of mutant and wild-type AE1 reduced anion transport by the wild-type protein in oocytes. Transfection studies in K562 and HEK293 cells revealed that the major pathway mediating degradation of both R664X and wild-type AE1 employed endoplasmic reticulum (ER)-associated degradation through the proteasomal pathway. Proteasomal degradation of R664X protein appeared to be independent of both ubiquitylation and N-glycosylation, and aggresome formation was not observed following proteasome inhibition. These findings indicate that AE1 R664X protein, which is associated with dominant hereditary spherocytosis, has a dominant-negative effect on the expression of wild-type AE1.