Intramuscular Grade 1 fibrosarcoma: Magnetic resonance imaging findings in 2 dogs

Two adult neutered male dogs were presented for evaluation of firm, painless masses arising within muscle: an 8-year-old German wirehaired pointer dog with an accessory tricipital growth, and a 3-year-old German shepherd dog with a gracilis muscle growth. Magnetic resonance imaging (MRI) characteristics suggested malignant behavior, with a central fluid-like portion with a hyperenhancing lining, a nidus of disorganized tissue, and an extensive reactive zone, whereas histopathology was consistent with low-grade fibrosarcoma. This report describes histologically low-grade, yet biologically high-grade intramuscular fibrosarcoma, in which MRI provided detailed information on tumor behavior and assisted with biopsy and surgical planning.     

Influence of estradiol-17β, testosterone, and 11-ketotestosterone on testicular development, serum steroid hormone, and gonadotropin secretion in male red sea bream Pagrus major

ABSTRACT:   In order to investigate the influence of estrogen and androgen on reproductive activities of male teleosts, male red sea bream were implanted with silicone capsules containing estradiol-17β (E2), testosterone (T) or 11-ketotestosterone (11-KT) in immature and early spermatogenic stages. One month after implantation of either E2 or T, the gonadosomatic index decreased in accordance with testicular regression in both stages. Implantation of E2 decreased circulating 11-KT levels but did not affect gonadotropin (GTH) subunits, follicle stimulating hormone-β (FSHβ), luteinizing hormone-β (LHβ), α glycoprotein subunit (αGSU) gene expression, and serum LH levels in both stages. Alternatively, T decreased serum 11-KT and LH levels, and FSHβ and LHβ mRNA levels in the early spermatogenic stage but not in the immature stage. These results suggest E2 may directly inhibit testicular development through the suppression of 11-KT production. Meanwhile, T may decrease serum 11-KT levels through the suppression of FSH and LH secretion, resulting to inhibition of testicular development in the early spermatogenic stage. Treatment with 11-KT did not affect the testis in either stage, whereas 11-KT increased LHβ and αGSU mRNA levels in immature, and decreased FSHβ mRNA levels in the early spermatogenic stage. These results suggest that 11-KT may have different effects on GTH subunit gene expression in each reproductive stage.     

Dough properties and bread-making quality-related characteristics of Yumechikara near-isogenic wheat lines carrying different Glu-B3 alleles

We investigated the relationships of three allelic variations in Glu-B3 (ab, g, and h) with dough properties and bread-making quality-related characteristics using near-isogenic lines (NILs) of ‘Yumechikara’ that commonly carry Glu-A1a, Glu-B1b, Glu-D1d, Glu-A3f, Glu-B3ab and Glu-D3a. Measurement of peak time (PT) in a 2-g mixograph indicated that Glu-B3g was the most effective for a strong dough property, followed by Glu-B3ab, with Glu-B3h being the least effective. The results of measurement of mixing time during bread-making were similar to those for PTs, i.e., the lines carrying Glu-B3g showed the longest mixing time, followed by those of Glu-B3ab, and those of Glu-B3h showed the shortest mixing time. Since two parameters of bread-making quality, loaf volume (LV) and specific loaf volume (SLV), were affected by flour protein contents in all groups of the Glu-B3 genotype, we compared the effects of the three Glu-B3 alleles on those parameters using analysis of covariance (ANCOVA) to remove the effect of protein content. The results indicated that the Glu-B3h group showed the largest SLV, followed by the Glu-B3ab group, and the Glu-B3g group showed the smallest SLV. These results suggest that the introduction of Glu-B3h into ‘Yumechikara’ makes it possible to breed varieties with good bread-making quality-related characteristics.     

Multiplex PCR and Genescan analysis to detect immunoglobulin heavy chain gene rearrangement in feline B-cell neoplasms

Lymphoid neoplasms are usually diagnosed on the basis of cytological and histopathological findings. However, in some cases, discrimination of lymphoid neoplasms from reactive lymphoid proliferation is difficult. PCR amplification of complementarity-determining region 3 (CDR3) of the immunoglobulin heavy-chain variable region (IGHV) gene can be used to assess clonality of B-cell populations as a supportive diagnostic tool for B-cell neoplasms. Because of the sequence variation and possible somatic hypermutation of the IGHV gene, sensitivity of the PCR-based assay to detect clonal IGHV gene rearrangement largely depends on the sequences and numbers of primer sets. Prior to the development of an efficient assay, we cloned and sequenced 97 IGHV complementary DNAs (48 IGHV-1 and 49 IGHV-3 clones) from normal cat spleens. On the basis of these sequences, we designed 6 forward primers at the variable region and 5 reverse primers at the joining region. Using each of 6 forward primers and a mixture of 5 reverse primers, we amplified CDR3 of IGHV genes and analyzed the PCR products by conventional PAGE and Genescan analyses using fluorescence-labeled primers. Twenty-six feline B-cell neoplasms diagnosed by histopathological and immunohistochemical examinations were subjected to the newly developed analysis of IGHV gene rearrangement. Clonal IGHV gene rearrangement was detected in 22 of 26 (84%) samples by both PAGE and Genescan analyses. To reduce the number of PCR reactions, we constructed a multiplex PCR analysis system using a mixture of IGHV-1- and IGHV-3-specific primers as forward primers and a mixture of 5 joining region reverse primers. Results of the multiplex PCR were 100% concordant with those obtained by each of the singleplex PCRs. The multiplex PCR-based assay and Genescan analysis developed in the present study would be useful and practical tools to detect clonal IGHV gene rearrangement in feline B-cell neoplasms.     

Binding of mammalian and avian ferritins with biotinylated hemin: demonstration of preferential binding of the H subunit to heme

The binding of ferritin to heme has been well studied using commercial horse spleen apoferritin, which is almost entirely composed of the L subunit, suggesting that mammalian ferritins bind heme. The present study revealed that both mammalian holoferritins (commercial horse spleen ferritin and purified horse spleen, bovine spleen and canine liver ferritins with L/H subunit ratios of 4.0, 1.1, and 2.3, respectively) and their apoferritins bound biotinylated hemin; apoferritins had higher binding activity than holoferritins, except for canine holo- and apoferritins, which showed the same binding. Bovine ferritin H subunit homopolymers expressed by a baculovirus expression system showed heme binding and had higher binding activity to biotinylated hemin than the L subunit homopolymer expressed by the same system. These bindings were inhibited by heme but not by iron-free or Zn-protoporphyrin IX (Zn-PPIX). Purified chicken liver holoferritin was found to be composed of only H subunits and showed the highest binding activity with biotinylated hemin compared with mammalian holoferritins. The binding of chicken liver holoferritin to biotinylated hemin was also inhibited by heme but not by PPIX or Zn-PPIX. These results indicate that mammalian and avian ferritins bind heme and that the H subunit preferentially recognizes heme.     

A rapid and simple method to obtain canine peripheral blood-derived macrophages

Macrophages play an important role in a variety of situations, including pathogen elimination, inflammation, and tissue repair. However, these cells are not fully studied in dogs, in part, due to the difficulty of efficiently isolating and culturing them in vitro. In this study, we cultured canine peripheral blood mononuclear cells (PBMCs) with 10 ng/ml of phorbol 12-myristate-13-acetate (PMA) for 5 days to obtain macrophages. A high number of round-adherent cells were obtained without the addition of any cytokine. These cells showed active phagocytic activity and a cell surface antigen profile different from dendritic cells. Our method facilitates a high yield of macrophages in a short cultivation period compared with previous studies. This method might be a powerful tool to study macrophage functions in dogs.     

Effects of trypsin on cytosolic calcium levels in the rat aortic endothelium

The effect of trypsin on vascular tone and the cytosolic calcium concentration ([Ca(2+)](i)) of endothelial and smooth muscle cells were examined in the rat aorta. A calcium indicator, fura-PE3, was used to measure [Ca(2+)](i) simultaneously with vascular tone. In the endothelium-intact rat aorta, carbachol and trypsin increased [Ca(2+)](i) in a dose-dependent manner. In the endothelium-denuded rat aorta, carbachol did not change [Ca(2+)](i), but trypsin slightly increased it. Addition of trypsin to the norepinephrine-stimulated rat aorta relaxed the muscle with an additional increase in [Ca(2+)](i). Under calcium-free conditions, trypsin induced a transient increase in [Ca(2+)](i). Trypsin-induced endothelium-dependent relaxation was inhibited by preincubation with l-NMMA, an endothelial NO synthase inhibitor, U-73122, a phospholipase C inhibitor, cyclopiazonic acid, a sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase blocker, and lanthanum, a nonselective Ca(2+) channel blocker. However, indomethacin, a nonselective cyclooxygenase inhibitor, and SKF-96365, a store-operated Ca(2+)-channel blocker, had no effect on the trypsin-induced relaxation. These results suggest that trypsin increases [Ca(2+)](i) in the endothelial cells through SKF-96365-insensitive Ca(2+) channels and regulates the release of NO, which results in relaxation of the rat aorta.     

Screening of therapeutic targets for canine mast cell tumors from a variety of kinase molecules

Options of systemic treatment for canine MCT have been still limited and most canine cases with MCTs eventually undergo relapses even after achievement of a remission. Thus additional therapies are required to establish for the tumor. To identify the novel candidate therapeutic targets for canine MCT, the mRNA expression and phosphorylation statuses of several receptor or non-receptor kinases as well as the inhibitory effect of 95 specific inhibitors on the growth were assessed in three canine MCT cell lines (HRMC, VIMC1 and CMMC1). Among the 14 targets, the mRNAs of 11, 7 and 7 kinases were amplified in HRMC, VIMC1 and CMMC1, respectively. The mRNAs of VEGFR3, PDGFRα, SRC, YES, LCK and FYN were detected in all cell lines. The phosphorylation of 12, 8 and 7 kinases was observed by using specific antibody arrays in HRMC, VIMC1 and CMMC1, respectively. DTK, EPHB6, AMPKα1, CREB, STAT5a and STAT5b were phosphorylated in all cell lines. The 10, 9 and 17 inhibitors exhibited the biological activity against the growth of HRMC, VIMC1 and CMMC1, respectively. Only three inhibitors such as SB218078 (for Chk1), PDGF RTK inhibitor IV (for PDGFR) and radicicol (for Hsp90) suppressed the growth of all three cell lines. The present study indicated that several kinases, such as Chk1, PDGFR and Hsp90, could be used as therapeutic targets in the treatment for canine MCT. Further studies and clinical trials are warranted to apply the inhibitors for the treatment of the tumor.     

Plasma concentration and cardiovascular effects of lidocaine during continuous epidural administration in dogs anesthetized with isoflurane

The cardiovascular effects of continuous epidural administration (CEA) of lidocaine were investigated in anesthetized dogs. Loading epidural injections of 2, 4, or 6 mg/kg of lidocaine were followed by CEA with 1, 2, or 3 mg/kg/hr lidocaine, respectively, for 2 hr under 2.0% isoflurane anesthesia. Heart rate, direct blood pressure, cardiac index, and stroke volume decreased dose-dependently during CEA, whereas systemic vascular resistance did not significantly differ with dose, and no characteristic changes were observed in any groups. Plasma lidocaine concentration reached a steady state during CEA and increased in a dose-dependent manner. Circulatory suppression caused by lidocaine CEA was not attributable to peripheral vasodilation, but rather to the direct cardiac action of systemic lidocaine absorption from the peridural space.     

Acute myeloid leukemia with mastocytic and megakaryocytic differentiation in a calf

A diagnosis of acute myeloid leukemia was made in a 10-month-old Holstein female calf. The leukemia was macroscopically characterized by great enlargement of the spleen and moderate enlargement of some lymph nodes. Histochemical and immunohistochemical examination disclosed the presence of neoplastic cells either containing metachromatic and tryptase-positive granules or expressing factor VIII-related antigen. The granules, which were positive for naphthol AS-D chloroacetate esterase and did not have particulate contents, were distinct from those of basophilic leukemia cells. This leukemia was thought to be derived from a common myeloid progenitor capable of giving rise to megakaryocyte-erythrocyte progenitors and granulocyte-monocyte progenitors with the ability to differentiate into mast cells.