Canine mastocytoma: report of 8 cases diagnosed by fine needle cytology and clinicopathologic correlations

BACKGROUND: Mast cell proliferations are commoner in dogs than in humans; mass forming lesions in the former are apt to fine needle sampling and the obtained cytopathological picture might be informing to enhance recognition of similar proliferations in humans. CASE: Clinical and cytopathologic data were collected from 8 cases of canine mastocytomas diagnosed by fine needle cytology. The cytopathologic presentation was correlated with the individual therapy performed and with the clinical stage. In all cases the cytopathological diagnosis was confirmed by histopathologic examination of the excised mass, by necropsy or by response to therapy. CONCLUSION: There are marked similarities between canine and human mastocytomas, despite possible differences in the clinical course of the disease in both species. Canine mastocytomas may hence be used as an animal model of a human disease and, as such, familiarity with their cytologic presentation may be useful for recognizing mast cell proliferations in humans.     

Drosophila FANCM Helicase Prevents Spontaneous Mitotic Crossovers Generated by the MUS81 and SLX1 Nucleases

Several helicases function during repair of double-strand breaks and handling of blocked or stalled replication forks to promote pathways that prevent formation of crossovers. Among these are the Bloom syndrome helicase BLM and the Fanconi anemia group M (FANCM) helicase. To better understand functions of these helicases, we compared phenotypes of Drosophila melanogaster Blm and Fancm mutants. As previously reported for BLM, FANCM has roles in responding to several types of DNA damage in preventing mitotic and meiotic crossovers and in promoting the synthesis-dependent strand annealing pathway for repair of a double-strand gap. In most assays, the phenotype of Fancm mutants is less severe than that of Blm mutants, and the phenotype of Blm Fancm double mutants is more severe than either single mutant, indicating both overlapping and unique functions. It is thought that mitotic crossovers arise when structure-selective nucleases cleave DNA intermediates that would normally be unwound or disassembled by these helicases. When BLM is absent, three nucleases believed to function as Holliday junction resolvases—MUS81-MMS4, MUS312-SLX1, and GEN—become essential. In contrast, no single resolvase is essential in mutants lacking FANCM, although simultaneous loss of GEN and either of the others is lethal in Fancm mutants. Since Fancm mutants can tolerate loss of a single resolvase, we were able to show that spontaneous mitotic crossovers that occur when FANCM is missing are dependent on MUS312 and either MUS81 or SLX1.     

ImmunoPET and biodistribution with human epidermal growth factor receptor 3 targeting antibody 89Zr-RG7116

The humanized monoclonal antibody with high affinity for the human epidermal growth factor receptor (HER) 3, RG7116, is a glycoengineered, IgG1 class antibody. By labeling RG7116 with zirconium-89 (⁸⁹Zr) we aimed to visualize in vivo HER3 expression and study the biodistribution of this antibody in human tumor-bearing mice. Biodistribution of ⁸⁹Zr-RG7116 was studied in subcutaneously xenografted FaDu tumor cells (HER3-positive). Dose-dependency of ⁸⁹Zr-RG7116 organ distribution and specific tumor uptake was assessed by administering doses ranging from 0.05 to 10 mg/kg RG7116 to SCID/Beige mice. Biodistribution was analyzed at 24 and 144 h after injection. MicroPET imaging was performed at 1, 3, and 6 days after injection of 1.0 mg/kg ⁸⁹Zr-RG7116 in the FaDu, H441, QG-56 and Calu-1 xenografts with varying HER3 expression. The excised tumors were analyzed for HER3 expression. Biodistribution analyses showed a dose- and time-dependent ⁸⁹Zr-RG7116 tumor uptake in FaDu tumors. The highest tumor uptake of ⁸⁹Zr-RG7116 was observed in the 0.05 mg/kg dose group with 27.5%ID/g at 144 h after tracer injection. MicroPET imaging revealed specific tumor uptake of ⁸⁹Zr-RG7116 in FaDu and H441 models with an increase in tumor uptake over time. Biodistribution data was consistent with the microPET findings in FaDu, H441, QG56 and Calu-1 xenografts, which correlated with HER3 expression levels. In conclusion, ⁸⁹Zr-RG7116 specifically accumulates in HER3 expressing tumors. PET imaging with this tracer provides real-time non-invasive information about RG7116 distribution, tumor targeting and tumor HER3 expression levels.     

Genomic instability and telomere fusion of canine osteosarcoma cells

Canine osteosarcoma (OSA) is known to present with highly variable and chaotic karyotypes, including hypodiploidy, hyperdiploidy, and increased numbers of metacentric chromosomes. The spectrum of genomic instabilities in canine OSA has significantly augmented the difficulty in clearly defining the biological and clinical significance of the observed cytogenetic abnormalities. In this study, eight canine OSA cell lines were used to investigate telomere fusions by fluorescence in situ hybridization (FISH) using a peptide nucleotide acid probe. We characterized each cell line by classical cytogenetic studies and cellular phenotypes including telomere associated factors and then evaluated correlations from this data. All eight canine OSA cell lines displayed increased abnormal metacentric chromosomes and exhibited numerous telomere fusions and interstitial telomeric signals. Also, as evidence of unstable telomeres, colocalization of γ-H2AX and telomere signals in interphase cells was observed. Each cell line was characterized by a combination of data representing cellular doubling time, DNA content, chromosome number, metacentric chromosome frequency, telomere signal level, cellular radiosensitivity, and DNA-PKcs protein expression level. We have also studied primary cultures from 10 spontaneous canine OSAs. Based on the observation of telomere aberrations in those primary cell cultures, we are reasonably certain that our observations in cell lines are not an artifact of prolonged culture. A correlation between telomere fusions and the other characteristics analyzed in our study could not be identified. However, it is important to note that all of the canine OSA samples exhibiting telomere fusion utilized in our study were telomerase positive. Pending further research regarding telomerase negative canine OSA cell lines, our findings may suggest telomere fusions can potentially serve as a novel marker for canine OSA.     

Relationship between muscle coordination and forehand drive velocity in tennis

This study aimed at investigating the relationship between trunk and upper limb muscle coordination and stroke velocity during tennis forehand drive. The electromyographic (EMG) activity of ten trunk and dominant upper limb muscles was recorded in 21 male tennis players while performing five series of ten crosscourt forehand drives. The forehand drive velocity ranged from 60% to 100% of individual maximal velocity. The onset, offset and activation level were calculated for each muscle and each player. The analysis of muscle activation order showed no modification in the recruitment pattern regardless of the velocity. However, the increased velocity resulted in earlier activation of the erector spinae, latissimus dorsi and triceps brachii muscles, as well as later deactivation of the erector spinae, biceps brachii and flexor carpi radialis muscles. Finally, a higher level of activation was observed with the velocity increase in the external oblique, latissimus dorsi, middle deltoid, biceps brachii and triceps brachii. These results might bring new knowledge for strength and tennis coaches to improve resistance training protocols in a performance and prophylactic perspective.     

Comparison between glycerol and ethylene glycol for dog semen cryopreservation

The aim of this study was to compare the effect of ethylene glycol versus glycerol for dog semen freezing, on post-thaw longevity, motility and motility parameters, and on plasma membrane functional integrity. Semen was diluted in two steps with an egg yolk TRIS extender containing a final concentration of either 5% glycerol or 5% ethylene glycol, and frozen in 0.5 ml straws, with 100 x 10(6) spermatozoa/ml, over nitrogen vapours. Semen motility was evaluated both under a light microscope and with a Computer Assisted Motility Analyser System, immediately after thawing and then hourly till 4h of incubation. Sperm membrane functional integrity was assessed with the hypoosmotic swelling test (60 mOsm fructose solution) applied at thawing and then hourly, for 4 h, on incubated samples. Motility (light microscope) and total and progressive motility (analyser) were significantly higher in ethylene glycol frozen samples at thawing (P < 0.01); from hour 1 onwards the effect of the cryoprotectant became not significant. Semen frozen with ethylene glycol showed higher path velocity and higher straight line velocity till 3 h after thawing; however, ethylene glycol semen samples also showed higher curvilinear velocity and higher lateral head displacement, which may indicate a capacitation-like condition affecting sperm membranes and possibly reducing post-thaw longevity. Functional integrity of plasma membrane was similar in glycerol and ethylene glycol samples till 3 h after thawing, then ethylene glycol samples showed a higher decline. The strong though short-lived positive effect of ethylene glycol is worth being evaluated further.     

Immortalization of human neural stem cells with the c-myc mutant T58A

Human neural stem cells (hNSC) represent an essential source of renewable brain cells for both experimental studies and cell replacement therapies. Their relatively slow rate of proliferation and physiological senescence in culture make their use cumbersome under some experimental and pre-clinical settings. The immortalization of hNSC with the v-myc gene (v-IhNSC) has been shown to generate stem cells endowed with enhanced proliferative capacity, which greatly facilitates the study of hNSCs, both in vitro and in vivo. Despite the excellent safety properties displayed by v-IhNSCs--which do not transform in vitro and are not tumorigenic in vivo--the v-myc gene contains several mutations and recombination elements, whose role(s) and effects remains to be elucidated, yielding unresolved safety concerns. To address this issue, we used a c-myc T58A retroviral vector to establish an immortal cell line (T-IhNSC) from the same hNSCs used to generate the original v-IhNSCs and compared their characteristics with the latter, with hNSC and with hNSC immortalized using c-myc wt (c-IhNSC). T-IhNSCs displayed an enhanced self-renewal ability, with their proliferative capacity and clonogenic potential being remarkably comparable to those of v-IhNSC and higher than wild type hNSCs and c-IhNSCs. Upon growth factors removal, T-IhNSC promptly gave rise to well-differentiated neurons, astrocytes and most importantly, to a heretofore undocumented high percentage of human oligodendrocytes (up to 23%). Persistent growth-factor dependence, steady functional properties, lack of ability to generate colonies in soft-agar colony-forming assay and to establish tumors upon orthotopic transplantation, point to the fact that immortalization by c-myc T58A does not bring about tumorigenicity in hNSCs. Hence, this work describes a novel and continuous cell line of immortalized human multipotent neural stem cells, in which the immortalizing agent is represented by a single gene which, in turn, carries a single and well characterized mutation. From a different perspective, these data report on a safe approach to increase human neural stem cells propagation in culture, without altering their basic properties. These T-IhNSC line provides a versatile model for the elucidation of the mechanisms involved in human neural stem cells expansion and for development of high throughput assays for both basic and translational research on human neural cell development. The improved proclivity of T-IhNSC to generate human oligodendrocytes propose T-IhNSC as a feasible candidate for the design of experimental and, perhaps, therapeutic approaches in demyelinating diseases.     

Hypothesis for a serine proteinase-like domain at the COOH terminus of Slowpoke calcium-activated potassium channels

Bovine pancreatic trypsin inhibitor (BPTI) is a 58-residue protein with three disulfide bonds that belongs to the Kunitz family of serine proteinase inhibitors. BPTI is an extremely potent inhibitor of trypsin, but it also specifically binds to various active and inactive serine proteinase homologs with KD values that range over eight orders of magnitude. We previously described an interaction of BPTI at an intracellular site that results in the production of discrete subconductance events in large conductance Ca2+ activated K+ channels (Moss, G.W.J., and E. Moczydlowski. 1996, J. Gen. Physiol, 107:47-68). In this paper, we summarize a variety of accumulated evidence which suggests that BPTI binds to a site on the KCa channel protein that structurally resembles a serine proteinase. One line of evidence includes the finding that the complex of BPTI and trypsin, in which the inhibitory loop of BPTI is masked by interaction with trypsin, is completely ineffective in the production of substate events in the KCa channel. To further investigate this notion, we performed a sequence analysis of the alpha-subunit of cloned slowpoke KCa channels from Drosophila and mammals. This analysis suggests that a region of approximately 250 residues near the COOH terminus of the KCa channel is homologous to members of the serine proteinase family, but is catalytically inactive because of various substitutions of key catalytic residues. The sequence analysis also predicts the location of a Ca(2+)-binding loop that is found in many serine proteinase enzymes. We hypothesize that this COOH-terminal domain of the slowpoke KCa channel adopts the characteristic double-barrel fold of serine proteinases, is involved in Ca(2+)-activation of the channel, and may also bind other intracellular components that regulate KCa channel activity.