Intraoperative hypertensive crisis in a dog with functional paraganglioma of the gall bladder.

A 15-year-old spayed female mongrel presented with anorexia and an abdominal mass. The mass originated from the gall bladder and was surgically resected along with divisionectomy of the central hepatic division. Paroxysmal hypertension and tachycardia were noted during manipulation of the mass. Following resection, arterial blood pressure decreased significantly. Histopathological analysis confirmed a diagnosis of neuroendocrine neoplasm. Immunohistochemical staining for synaptophysin and chromogranin A yielded diffuse and strong positive results, while gastrin was positive in only 10% of the cells. The preoperative elevated concentrations of catecholamine in the urinalysis showed a marked decrease after surgery. Based on these findings, the tumour was diagnosed as a functional paraganglioma of the gall bladder. The patient has undergone regular thoracic radiographs and ultrasound examinations and, until 431 days after surgery, has shown no signs of metastases or recurrences. Based on our literature search, we report the first case of functional paraganglioma of the gall bladder in a dog.

Detection of Merkel cell polyomavirus with a tumour-specific signature in non-small cell lung cancer.

BACKGROUND: We searched for a viral aetiology for non-small cell lung cancer (NSCLC), focusing on Merkel cell polyomavirus (MCPyV). METHODS: We analysed 112 Japanese cases of NSCLC for the presence of the MCPyV genome and the expressions of RNA transcripts and MCPyV-encoded antigen. We also conducted the first analysis of the molecular features of MCPyV in lung cancers. RESULTS: PCR revealed that 9 out of 32 squamous cell carcinomas (SCCs), 9 out of 45 adenocarcinomas (ACs), 1 out of 32 large-cell carcinomas, and 1 out of 3 pleomorphic carcinomas were positive for MCPyV DNA. Some MCPyV DNA-positive cancers expressed large T antigen (LT) RNA transcripts. Immunohistochemistry showed that MCPyV LT antigen was expressed in the tumour cells. The viral integration sites were identified in one SCC and one AC. One had both episomal and integrated/truncated forms. The other carried an integrated MCPyV genome with frameshift mutations in the LT gene. CONCLUSION: We have demonstrated the expression of a viral oncoprotein, the presence of integrated MCPyV, and a truncated LT gene with a preserved retinoblastoma tumour-suppressor protein-binding domain in NSCLCs. Although the viral prevalence was low, the tumour-specific molecular signatures support the possibility that MCPyV is partly associated with the pathogenesis of NSCLC in a subset of patients.

Role of hyperfine coupling in magnetic and quadrupolar ordering of Pr3Pd20Si6.

We study the ternary clathrate Pr3Pd20Si6 in specific heat and ac susceptibility measurements on a high-quality single crystal, distinguishing antiferromagnetic and antiferroquadrupolar ordering, as well as a hitherto unknown magnetic low-temperature transition. The specific heat shows the direct involvement of nuclear spin degrees of freedom in the antiferromagnetic ordering, which is well supported by our calculation of the hyperfine level scheme without adjustable parameters. Pr3Pd20Si6 is, therefore, one of the rare materials where the nuclear moments are involved in the formation of the magnetic ground state.

Involvement of geranylgeranylation of Rho and Rac GTPases in adipogenic and RANKL expression, which was inhibited by simvastatin.

Simvastatin suppresses myoblast differentiation via inhibition of Rac GTPase, which is involved in the mevalonic acid pathway that produces cholesterol. Statins also inhibit adipogenic differentiation and receptor activator of NFκB ligand (RANKL) expression, possibly through the mevalonic acid pathway, although the involvement of that pathway and effector proteins in these cellular events has not been fully clarified. In the present study, we aimed to elucidate the mechanism of the effects of simvastatin on adipogenic differentiation and calcitriol-induced RANKL expression in bone marrow stromal ST2 cells. Adipogenesis and mRNA up-regulation of peroxisome proliferator-activated receptor γ and adipocyte fatty acid-binding protein were induced by troglitazone, and those events were efficiently inhibited by simvastatin. In addition, RANKL expression induced by calcitriol was abrogated by simvastatin in ST2 cells. The inhibitory effects of simvastatin were adequately compensated by the addition of either mevalonic acid or an intermediate of the mevalonic acid pathway, geranylgeranyl pyrophosphate, but not by another intermediate, farnesyl pyrophosphate. These findings suggest that protein geranylgeranylation is related to cellular differentiation in those two directions. Furthermore, inhibitor analysis demonstrated that Rac GTPase is involved in adipogenic differentiation, whereas Rho GTPase was found to be involved in RANKL expression. Taken together, the present findings suggest that geranylgeranylation of Rho family GTPase is involved in both adipogenesis and RANKL expression of stromal cells, while Rac GTPase is involved in adipogenesis and Rho GTPase in RANKL expression.

Propeptide processing and proteolytic activity of proenzymes of the staphylococcal and enterococcal GluV8-family protease.

Proenzymes with various lengths of propeptides have been observed in GluV8 from Staphylococcus aureus and GluSE from S. epidermidis. However, the production mechanism of these proenzymes and roles of truncated propeptides have yet to be elucidated. Here we demonstrate that shortening of propeptide commonly occurs in an auto-catalytic manner in GluV8-family members, including those from coagulase negative Staphylococci and Enterococcus faecalis. Accompanied with propeptide shortening, the pro-mature junction (Asn/Ser_1-Val1) becomes more susceptible towards the hetero-catalytic maturation enzymes. The auto-catalytic propeptide truncation is not observed in Ser169Ala inert molecules of GluV8-family members. A faint proteolytic activity of proenzymes from Staphylococcus caprae and E. faecalis is detected. In addition, proteolytic activity of proenzyme of GluV8 carrying Arg-3AlaAsn.1 is demonstrated with synthetic peptide substrates LLE/Q-MCA. These results suggest that GluV8-family proenzymes with shortened propeptides intrinsically possess proteolytic activity and are involved in the propeptide shortening that facilitates the final hetero-catalytic maturation.

Clinical relationship between histopathological necrotic/partial necrotic findings and disease condition of gallbladder mucoceles in dogs.

Gallbladder mucocele (GM) is a common extrahepatic biliary disease recognized in dogs and is defined as the expansion and extension of the gallbladder by an accumulation of semi-solid bile or bile acid. Histopathological diagnosis of necrotizing cholecystitis and transmural coagulative necrosis of the gallbladder wall shows poor prognosis. Conversely, histopathological diagnosis with partial necrotic findings is often achieved. We hypothesized that histopathological partial necrosis of the gallbladder wall is the primary lesion of necrotic cholecystitis or transmural ischemic necrosis. Therefore, we investigated the relationship between histopathological necrosis/ partial necrosis findings and their clinical conditions. We retrospectively analyzed 55 dogs diagnosed with GM that had undergone cholecystectomy at the Yamaguchi University Animal Medical Center. The group with histopathological necrosis/partial necrosis of the gallbladder wall showed elevated levels of preoperative white blood cells, alanine transaminase, alkaline phosphatase, γ-glutamyltransferase, total bilirubin, and C-reactive protein compared to the non-necrotic group. Partial necrosis of the gallbladder wall may affect the progression of the disease and hematological abnormalities. Additionally, all death cases until 2 weeks were included in the histopathological necrosis/partial necrosis group. In this study, we found that poor prognosis factors were associated with partial necrosis of the gallbladder wall. Furthermore, these cases of partial necrosis showed elevated levels of blood test parameters. These results suggest that necrosis of the gallbladder wall is associated with poor prognosis and poor pathophysiological conditions.

Gene silencing of STAT6 with siRNA ameliorates contact hypersensitivity and allergic rhinitis.

BACKGROUND: Silencing of genes using small interfering RNA (siRNA) is a recently developed strategy to regulate the synthesis of target molecules. Signal transducer and activator of transcription 6 (STAT6) is a nuclear transcription factor that mediates Th2-type immunity. METHODS: To elucidate the therapeutic potential of using siRNA to inhibit STAT6 in allergic reactions, we determined the nucleotide sequences of siRNA specific for STAT6. RESULTS: The selected sequences of STAT6 siRNA specifically inhibited the generation of STAT6 synthesis in dermal fibroblasts and eotaxin (CCL11) production in response to IL-4/TNF-α in vitro. Local administration of STAT6 siRNA in vivo alleviated contact hypersensitivity responses to chemical haptens. This was accompanied by reduced local production of IL-4, IL-13, eotaxin (CCL11), TARC (CCL17) and MDC (CCL22). Similarly, consecutive intranasal instillation of STAT6 siRNA markedly inhibited inflammatory cellular infiltration of mucosal tissues in allergic rhinitis responses in association with reduced IL-4 and IL-5 production from regional lymph node cells. Immediate responses, such as sneezing and nasal rubbing behaviors, were also improved by STAT6 siRNA. CONCLUSIONS: Local administration of STAT6 siRNA is thus a promising therapeutic strategy for both Th2-mediated cutaneous diseases and allergic rhinitis.

Identification of cancer stem cells derived from a canine lung adenocarcinoma cell line.

Accumulating evidence supporting the cancer stem cell (CSC) hypothesis is based on the finding that tumors contain a small population of self-renewing cells that generate differentiated progeny and thereby contribute to tumor heterogeneity. CSCs are reported to exist in several human cancers, yet only a few reports demonstrate the existence of CSCs in primary lung cancer in dogs. In this study, the authors established a cancer cell line derived from a canine primary lung adenocarcinoma and identified a side population (SP) of cells that displayed drug-resistant features. To confirm the characteristics of these SP cells, the authors investigated the tumorigenicity of the cells in vivo by using a nude mouse xenograft model. Only 100 SP cells were able to give rise to new tumors, giving a 10-fold enrichment over the main population (MP) of cells, suggesting that these cells have the cancer-initiating ability of CSCs. Further studies characterizing CSCs in canine lung adenocarcinoma might contribute to the elucidation of the mechanisms of tumorigenesis and to the establishment of novel therapeutic strategies.

R2D5 antigen: a calcium-binding phosphoprotein predominantly expressed in olfactory receptor neurons.

R2D5 is a mouse monoclonal antibody that labels rabbit olfactory receptor neurons. Immunoblot analysis showed that mAb R2D5 recognizes a 22-kD protein with apparent pI of 4.8, which is abundantly contained in the olfactory epithelium and the olfactory bulb. We isolated cDNA for R2D5 antigen and confirmed by Northern analysis and neuronal depletion technique that R2D5 antigen is expressed predominantly, but not exclusively, in olfactory receptor neurons. Analysis of the deduced primary structure revealed that R2D5 antigen consists of 189 amino acids with calculated M(r) of 20,864 and pI of 4.74, has three calcium-binding EF hands, and has possible phosphorylation sites for Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and cAMP-dependent protein kinase (A kinase). Using the bacterially expressed protein, we directly examined the biochemical properties of R2D5 antigen. R2D5 antigen binds Ca2+ and undergoes a conformational change in a manner similar to calmodulin. R2D5 antigen is phosphorylated in vitro by CaM kinase II and A kinase at different sites, and 1.81 and 0.80 mol of Pi were maximally incorporated per mol of R2D5 antigen by CaM kinase II and A kinase, respectively. Detailed immunohistochemical study showed that R2D5 antigen is also expressed in a variety of ependymal cells in the rabbit central nervous system. Aside from ubiquitous calmodulin, R2D5 antigen is the first identified calcium-binding protein in olfactory receptor neurons that may modulate olfactory signal transduction. Furthermore our results indicate that olfactory receptor neurons and ependymal cells have certain signal transduction components in common, suggesting a novel physiological process in ependymal cells.

An ICAM-related neuronal glycoprotein, telencephalin, with brain segment-specific expression.

Telencephalin (TLN) is a 130 kd glycoprotein expressed exclusively in neurons of the telencephalon, the most rostral brain segment. In the neurons, TLN is localized to soma-dendritic membrane but not to axonal membrane. In this study, we have cloned cDNA encoding rabbit and mouse TLN. The cDNA-derived primary structure of TLN predicts an integral membrane protein with nine tandem immunoglobulin-like domains in an extra-cellular region, a transmembrane domain, and a short cytoplasmic tail. The distal eight immunoglobulin-like domains of TLN show highest homology with the immunoglobulin-like domains of intracellular adhesion molecules (ICAMs) 1, 2, and 3/R. The structural similarity of TLN with ICAMs provides a new and strong link between immunoglobulin superfamily molecules in the nervous and immune systems. TLN is an example of a dendrite-associated cell adhesion molecule involved in the brain's segmental organization, cell-cell interactions during dendritic development, and maintenance of functional neuronal networks.

Fission and uncoating of synaptic clathrin-coated vesicles are perturbed by disruption of interactions with the SH3 domain of endophilin.

Coordination between sequential steps in synaptic vesicle endocytosis, including clathrin coat formation, fission, and uncoating, appears to involve proteinprotein interactions. Here, we show that compounds that disrupt interactions of the SH3 domain of endophilin with dynamin and synaptojanin impair synaptic vesicle endocytosis in a living synapse. Two distinct endocytic intermediates accumulated. Free clathrin-coated vesicles were induced by a peptide-blocking endophilin's SH3 domain and by antibodies to the proline-rich domain (PRD) of synaptojanin. Invaginated clathrin-coated pits were induced by the same peptide and by the SH3 domain of endophilin. We suggest that the SH3 domain of endophilin participates in both fission and uncoating and that it may be a key component of a molecular switch that couples the fission reaction to uncoating.

Occurrence of staphylococci in the oral cavities of healthy adults and nasal oral trafficking of the bacteria.

To investigate a possible peroral route of infective endocarditis (IE), the occurrence of staphylococci in the oral cavity was examined using saliva and supragingival plaque specimens from 56 systemically and periodontally healthy adults aged 22-43 years old (27.1+/-5.3). Nine Staphylococcus species and 334 isolates were identified. In saliva, the total occurrence rate was 83.9 % and the total number of bacteria was 10(2)-10(4) c.f.u. ml(-1). Staphylococcus aureus was the most frequent species (46.4 %), followed by Staphylococcus epidermidis (41.1 %) and others (Staphylococcus hominis, Staphylococcus warneri, Staphylococcus intermedius, Staphylococcus capitis, Staphylococcus haemolyticus, Staphylococcus lugdunensis and Staphylococcus gallinarum, isolation frequencies ranging in order from 12.5 to 1.8 %). A similar isolation tendency was observed in supragingival plaque, with a total occurrence rate of 73.2 % and amounts of bacteria ranging from 10(2) to 10(5) c.f.u. g(-1). Four common Staphylococcus species (S. aureus, S. epidermidis, S. lugdunensis and S. hominis) were isolated from nasal swab samples taken from the oral staphylococci-positive subjects. Genotyping of all 18 combinations of oral- and nasal-derived isolates by PFGE indicated that identical clones or close relatives were commonly distributed in these two cavities. Since the provision of micro-organisms from the nasal cavity was shown and occurrence rates in the oral cavity were adequate, these results suggest a possible peroral route of staphylococcal IE, as in cases of viridans streptococcal IE.

Oncolytic reovirus therapy: Pilot study in dogs with spontaneously occurring tumours.

Oncolytic virotherapy is a novel treatment involving replication-competent virus in the elimination of cancer. We have previously reported the oncolytic effects of reovirus in various canine cancer cell lines. This study aims to establish the safety profile of reovirus in dogs with spontaneously occurring tumours and to determine a recommended dosing regimen. Nineteen dogs with various tumours, mostly of advanced stages, were treated with reovirus, ranging from 1.0 × 108 to 5.0 × 109 TCID50 given as intratumour injection (IT) or intravenous infusion (IV) daily for up to 5 consecutive days in 1 or multiple treatment cycles. Adverse events (AEs) were graded according to the Veterinary Cooperative Oncology Group- Common Terminology Criteria for Adverse Events (VCOG-CTCAE) v1.1 guidelines. Viral shedding, neutralizing anti-reovirus antibody (NARA) production and immunohistochemical (IHC) detection of reovirus protein in the tumours were also assessed. AE was not observed in most dogs and events were limited to Grade I or II fever, vomiting, diarrhoea and inflammation of the injected tumour. No infectious virus was shed and all dogs had elevated NARA levels post-treatment. Although IHC results were only available in 6 dogs, 4 were detected positive for reovirus protein. In conclusion, reovirus is well-tolerated and can be given safely to tumour-bearing dogs according to the dosing regimen used in this study without significant concerns of viral shedding. Reovirus is also potentially effective in various types of canine tumours.

Altered expression of fatty acid-metabolizing enzymes in aromatase-deficient mice.

Hepatic steatosis is a frequent complication in nonobese patients with breast cancer treated with tamoxifen, a potent antagonist of estrogen. In addition, hepatic steatosis became evident spontaneously in the aromatase-deficient (ArKO) mouse, which lacks intrinsic estrogen production. These clinical and laboratory observations suggest that estrogen helps to maintain constitutive lipid metabolism. To clarify this hypothesis, we characterized the expression and activity in ArKO mouse liver of enzymes involved in peroxisomal and mitochondrial fatty acid beta-oxidation. Northern analysis showed reduced expression of mRNAs for very long fatty acyl-CoA synthetase, peroxisomal fatty acyl-CoA oxidase, and medium-chain acyl-CoA dehydrogenase, enzymes required in fatty acid beta-oxidation. In vitro assays of fatty acid beta-oxidation activity using very long (C24:0), long (C16:0), or medium (C12:0) chain fatty acids as the substrates confirmed that the corresponding activities are also diminished. Impaired gene expression and enzyme activities of fatty acid beta-oxidation were restored to the wild-type levels, and hepatic steatosis was substantially diminished in animals treated with 17beta-estradiol. Wild-type and ArKO mice showed no difference in the binding activities of the hepatic nuclear extracts to a peroxisome proliferator response element. These findings demonstrate the pivotal role of estrogen in supporting constitutive hepatic expression of genes involved in lipid beta-oxidation and in maintaining hepatic lipid homeostasis.

Radioresistance of cancer stem-like cell derived from canine tumours.

Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are a small subpopulation of cancer cells that are responsible for the initiation, recurrence and metastasis of cancer. We previously demonstrated that, using the Hoechst 33342 dye-based side population technique, CSCs/CICs in canine lung adenocarcinoma cell line exist. In this study, as CSCs/CICs are known to form spheres in anchorage-independent environment in vitro, we evaluated the stemness of spheroid cells derived from canine lung adenocarcinoma and osteosarcoma cells by expression of stemness markers, and investigated radioresistance. Spheroid cells showed greater expression of stemness markers Oct-4 and CD133 gene than those of adherent-cultured cells. In nude mouse xenograft models, spheroid cells showed higher tumourigenic ability than adherent-cultured cells. In addition, spheroid cells showed significantly resistant against radioactivity as compared with adherent-cultured cells. These results suggest that spheroid cells could possess stemness and provide a CSCs/CICs research tool to investigate CSCs/CICs of canine tumour cells.

Direct association of YY-1 with c-Myc and the E-box binding protein in regulation of glycophorin gene expression.

We previously reported that YY-1, a versatile transcription factor, regulates expression of glycophorin gene by binding to its locus control region-like region (Gp-LCR) in combination with E-box binding protein during murine erythroleukemia (MEL) cell differentiation. In the present work, we demonstrated that YY-1 and c-Myc, a nuclear oncoprotein, were physically associated in vivo and that down regulation of c-Myc liberated free YY-1 from its complex, resulting in the functional binding of YY-1 to the Gp-LCR. We also showed that the E-box binding protein (EBP) which bound to E-box was physically associated with YY-1, facilitated binding of YY-1 to the neighboring site and their combinatorial binding may stimulate the GpLCR mediated enhancement of erythroid-specific transcription of glycophorin gene in MEL cells.

Development of a flow system for the microscopic observation of cultured nerve cells and its application to the neurite growth control of PC12h-R.

A flow system has been developed for the continual microscopic observation of cultured nerve cells. The undefined effects of coexisting cells could be efficiently reduced by the continuous flow of a medium. The present flow system was useful also for the experiments requiring medium exchanges. Its performance was demonstrated by the repeated actions of NGF or db-cAMP to PC12h-R cells. The neurite projection and its retraction were repeatedly observed by the supply and the removal of those factors.

Fluorescence polarization studies on the conformational transition of bovine plasma albumin in acidic solutions.

The acid-induced isomerization (the N-F transition) and expansion of bovine plasma albumin were studied by measuring fluorescence polarization and lifetime of the excited state of tryptophyl fluorophors. Most of the changes (decreases) in the reciprocal of fluorescence polarization and lifetime of the excited state correlated exactly with the N-F1 transition and/or the initial part of the N-F transition. These findings suggest that though the N-F transition is the cooperative pH-dependent conformational transition, the N-F transition clearly involves an intermediate step, such as the N-F1 and F1-F2 transitions. Rotational relaxation times for the N- and F-forms obtained by Perrin plot of tryptophyl fluorescence polarization were approximately 75 and 120-180 ns, respectively. The unexpected short rotational relaxation time of 75 ns of the N-form might be due to the rotational freedom of the tryptophyl side chain itself and/or of small flexible loci where tryptophyl fluorophors attach.

A novel fluorescent derivative of glucose applicable to the assessment of glucose uptake activity of Escherichia coli.

A novel fluorescent derivative of glucose was synthesized by reacting D-glucosamine and NBD-Cl. The TLC analysis of the reaction mixture showed the generation of a single spot with intense fluorescence (lambda Ex = 475 nm, lambda Em = 550 nm). The obtained novel fluorescent product, which was identified as 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) by 1H-NMR and FAB-MS spectrometries, was applied to the assessment of the glucose uptake activity of Escherichia coli B. 2-NBDG accumulated in living cells and not in dead cells. The uptake of 2-NBDG was competitively inhibited by D-glucose and not by L-glucose, which suggested the involvement of the glucose transporting system in the uptake of 2-NBDG. 2-NBDG taken into the cytoplasma of E. coli cells was supposedly converted into another derivative in the glucose metabolic pathway.

Binding of [3H]methyltrienolone to androgen receptor in rat liver.

The synthetic androgen methyltrienolone is superior to testosterone and androstenedione for the measurement of androgen receptor in tissues where the native ligands are metabolized into inactive derivatives. [3H]Methyltrienolone binds with a high affinity to androgen receptor in cytosol prepared from male rat livers, as the Scatchard analysis revealed that the Kd value was 3.3 X 10(-8) M and the number of binding sites was 35.5 fmol/mg protein. Since methyltrienolone also binds glucocorticoid receptor which exists in rat liver, the apparent binding of androgen receptor is faulty when measured in the presence of glucocorticoid receptor. The binding of methyltrienolone to glucocorticoid receptor can be blocked by the presence of a 100-fold molar excess of unlabeled synthetic glucocorticoid, triamcinolone acetonide, without interfering in its binding to androgen receptor, because triamcinolone does not bind to androgen receptor. Triamcinolone-blocked cytosol exhibited that the Kd value was 2.5 X 10(-8) M and the number of binding sites was 26.3 fmol/mg protein, indicating a reduction to 3/4 of that in the untreated cytosol. The profile of glycerol gradient centrifugation indicated that [3H]methyltrienolone-bound receptor migrated in the 8-9 S region in both untreated and triamcinolone-blocked cytosols, but the 8-9 S peak in triamcinolone-blocked cytosol was reduced to about 3/4 of that of untreated cytosol.