Evaluation of bone marrow aspirates from multiple sites for staging of canine lymphoma and mast cell tumours.

This prospective study evaluated the utility of bone marrow aspirates (BMAs) obtained from multiple sites for staging of canine lymphoma (LSA) and mast cell tumours (MCTs). Forty dogs (LSA, n = 24; MCTs, n = 16) were enrolled, but only 33 (82.5%) had diagnostic bone marrow (BM) aspirates obtained from two sites for inclusion in the study. Nineteen dogs with LSA were included, and 6 (31.6%) had BM involvement. Neoplastic lymphocytes were present in BM from both sites in all of these dogs. Fourteen dogs with MCTs were included, and 3 (21.4%) had BM involvement. Neoplastic mast cells were present at both sites in two dogs and at only one site in the third. These results indicate that BMAs from multiple sites may not be needed for accurate staging of canine LSA patients, but more studies evaluating the pattern of BM infiltration in dogs with high-grade MCTs are warranted.

Multicentric cutaneous neuroendocrine (Merkel cell) carcinoma in a dog.

Multicentric cutaneous neuroendocrine (Merkel cell) carcinoma was diagnosed in a 5-year-old castrated male Keeshond dog with multiple firm nodular cutaneous masses. The neoplastic tissue locally effaced the periadnexal and deep dermis and consisted of densely cellular confluent clusters of round to polygonal cells supported by a delicate fibrovascular stroma. The cells were moderately immunoreactive with chromogranin A, synaptophysin, and cytokeratin. Ultrastructurally, the cells had characteristic membrane-bound dense-core neuroendocrine granules approximately 120 nm in diameter and randomly dispersed throughout the cytoplasm. Effacement of dermal structures and multicentric distribution suggested low-grade malignant phenotype. These findings contrast with the typical benign behavior of canine cutaneous neuroendocrine tumors.

Meningoencephalitis secondary to bacterial otitis media/interna in a dog.

Central nervous system (CNS) complications of bacterial otitis media/interna are an infrequent occurrence in human patients and have rarely been reported in the veterinary literature. Early recognition of CNS involvement and the use of appropriate diagnostic tests to characterize the nature of the lesion(s) are crucial in determining the best course of treatment. In this paper, the authors describe a dog with bacterial meningoencephalitis secondary to otitis media/interna.

Telomerase enzyme activity as a diagnostic tool to distinguish effusions of malignant and benign origin.

Telomerase enzyme activity is high in populations of cells that are dividing, and is low or undetectable in quiescent cell populations. Activation of telomerase in tissues that normally lack the capacity for self-renewal is strongly correlated with neoplasia. Telomerase activity can be detected in samples containing very small numbers of cells and studies of human patients suggest that measurement of telomerase activity may be useful for the evaluation of samples that can be obtained in a minimally invasive manner. This study compares the presence or absence of telomerase activity with cytologic evaluation of body cavity effusions, to determine if neoplasia is the underlying cause for the effusion in dogs and cats. Detection of telomerase in effusions was no more sensitive than cytologic evaluation for the identification of underlying neoplasia, and was less specific (telomerase assay: sensitivity = 50%, specificity = 83%; cytology: sensitivity = 50%, specificity = 100%). We conclude that although the telomerase assay may constitute a useful adjunctive test for the diagnosis of neoplasia in some dogs and cats with body cavity effusions, the results of this assay are not sufficiently reliable to be used as a sole diagnostic test.

Effect of human apoE4 on the clearance of chylomicron-like lipid emulsions and atherogenesis in transgenic mice.

Apolipoprotein (apo) E is a ligand for lipoprotein receptors and mediates the cellular uptake of several different lipoproteins. Human apoE occurs in three allelic forms designated E2, E3, and E4. The E2 isoform is associated with changes in lipoprotein metabolism, and the E4 isoform is associated with Alzheimer's disease and an increased risk of coronary heart disease. In this study transgenic mice were generated to assess the effect of a sustained increase in plasma apoE4 concentration. The transgenic animals had three- to sixfold increases in total plasma apoE, associated primarily with the non-high-density lipoprotein (HDL) fractions of plasma lipoproteins. In response to an atherogenic diet the transgenic mice developed hypercholesterolemia similar to that in nontransgenic mice but did not experience the decrease in HDL cholesterol normally observed in this strain of C57BL/6 mice. The rate of plasma clearance of a lipid emulsion mimicking lymph chylomicrons was measured in transgenic mice expressing the human apoE4 gene and compared with the clearance rate in nontransgenic control animals. In animals fed a low-fat diet the emulsion lipids were cleared significantly more rapidly from the plasma of transgenic than control mice. In animals adapted to a high-fat diet, the clearance of chylomicron remnants was slowed markedly in both transgenic and control mice and was not significantly accelerated in transgenic compared with control animals. We also investigated the effect of increasing the plasma concentration of apoE4 on the progression of atherosclerotic heart disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of the human zeta-globin gene promoter in transgenic mice.

zeta-Globin is the embryonic form of the alpha chain of hemoglobin. Transgenic mice generated with zeta-globin constructs containing the zeta-globin gene, 557 bp of 5' flanking sequence, and 2-kb of 3' flanking sequence linked to the beta-globin locus control region hypersensitive site 2 (HS2) expressed human zeta-globin only in embryonic yolk sac erythroid tissue, and not in definitive erythroid tissue in the fetal liver or in adult peripheral blood. To determine what sequences in the 5' flanking region of the zeta-globin gene might be important for developmental specificity, a series of 5' deletion constructs of the zeta-globin gene were made and used to generate transgenic mice. The 5' ends of these constructs were located 417, 207, and 128 bp 5' to the zeta-globin transcriptional start site, and HS2 was included to increase the level of erythroid-specific expression. In all lines of mice tested, human zeta-globin was expressed only in embryonic tissue, and not in fetal livers or in adult peripheral blood. Expression was independent of copy number and appeared to be dependent on the site of transgene insertion. These data suggest that the proximal 128 bp of the zeta-globin promoter is sufficient to properly regulate zeta-globin expression during development.

Inhibition of early atherogenesis in transgenic mice by human apolipoprotein AI.

Epidemiological surveys have identified a strong inverse relationship between the amount in the plasma of high density lipoproteins (HDL), apolipoprotein AI (ApoA-I), the major protein component of HDL, and the risk for atherosclerosis in humans. It is not known if this relationship arises from a direct antiatherogenic effect of these plasma components or if it is the result of other factors also associated with increases in ApoA-I and HDL levels. Because some strains of mice are susceptible to diet-induced formation of preatherosclerotic fatty streak lesions, and because of available techniques for the genetic manipulation of this organism, the murine system offers a unique setting in which to investigate the process of early atherogenesis. To test the hypothesis that induction of a high plasma concentration of ApoA-I and HDL would inhibit this process, we studied the effects of atherogenic diets on transgenic mice expressing high amounts of human ApoA-I. We report that transgenic mice with high plasma ApoA-I and HDL levels were significantly protected from the development of fatty streak lesions.

Developmental regulation of the human zeta globin gene in transgenic mice.

We have characterized the expression of the human zeta (zeta) gene, which encodes an embryonic alpha-like globin, in transgenic mice. We find that a 777 base pair fragment spanning erythroid specific hypersensitive site II (HSII) from the distal 5. region of the human beta globin gene cluster potentiates expression of the zeta globin gene. In the absence of the HSII fragment, no zeta expression is observed. Expression of the human zeta gene in mice parallels expression of a murine embryonic alpha-like globin gene (x). Thus, expression of the human zeta gene in mice requires linkage to an erythroid-specific enhancer sequence, but the presence of the enhancer does not affect the developmental regulation of the transgene. Our results indicate that the factors involved in switching from embryonic to adult alpha globin gene expression during development are evolutionarily conserved, and suggest that the transgenic mouse is an in vivo system in which the requirements for the developmental switch in alpha globin gene expression can be analyzed in detail.

Developmentally regulated telomere addition in Tetrahymena thermophila.

To investigate the developmentally programmed telomere addition that accompanies chromosome fragmentation during macronuclear differentiation in Tetrahymena thermophila, five representative telomeric regions from the macronucleus were cloned and characterized in detail. The sequences adjacent to the telomeric (C4A2:T2G4) repeats on these five macronuclear ends had no significant sequence homology or shared secondary structure. Two developmentally independent examples of one macronuclear telomere had a 5 base pair difference in the position of the junction between the telomeric repeats and the adjacent sequences. A telomere-adjacent sequence, in the form of a synthetic oligonucleotide, was unable to prime the addition of telomeric repeats in vitro. The implications of these results for the mechanisms underlying developmentally programmed chromosome fragmentation and telomere addition in Tetrahymena are discussed.

Dynamics of telomere length variation in Tetrahymena thermophila.

We have analyzed the mechanism and dynamics of telomere length variation in the macronucleus of Tetrahymena thermophila. In a newly differentiated macronucleus, the average length of the telomeric repeated sequence, (C4A2 X T2G4)n, is closely regulated. In contrast, in vegetatively dividing cells in log phase, all macronuclear telomeric sequences lengthen coordinately by 3-10 bp per generation until up to 1000 bp are added. In both elongated and short telomeres, characteristic single-stranded breaks on both strands are distally located. Reduction of elongated telomeres to their original length involves either the appearance of a novel type of variant cell, incapable of net telomere elongation, or, under stationary phase conditions, a reversible removal of telomeric sequences. The demonstration that telomeres are dynamic structures provides evidence for a model of telomere length regulation by activities that add and remove telomeric repeats.

The nucleotide sequence of the 17S ribosomal RNA gene of Tetrahymena thermophila and the identification of point mutations resulting in resistance to the antibiotics paromomycin and hygromycin.

We have determined the complete sequence of the nuclear gene encoding the small subunit (17 S) rRNA of the ciliated protozoan Tetrahymena thermophila. The gene encodes an RNA molecule which is 1753 nucleotides in length. The sequence of the Tetrahymena small subunit rRNA is homologous to those of other eukaryotes, and the predicted secondary structure for the molecule includes features which are characteristic of eukaryotic small subunit rRNAs. We have also determined the nature of two different mutations in the Tetrahymena 17 S gene which result in resistance to the aminoglycoside antibiotics paromomycin and hygromycin. In each case we have identified a single base change near the 3' end of the rRNA, within a region that is highly evolutionarily conserved in both sequence and secondary structure. Analysis of the effects of these mutations on rRNA structure, and of the impact of these drugs on translation, should help to elucidate the role of the small subunit ribosomal RNA in ribosome function.

Sendai virus-mediated lysis of liposomes requires cholesterol.

Vesicles were constituted with glycophorin, the Sendai virus receptor of human erythrocytes, and loaded with calcein, a polar derivative of fluorescein, at self-quenching concentrations. On exposure to Sendai virus and mild hypo-osmotic stress, vesicles of the appropriate composition released a significant portion of their internal contents, as indicated by an increase in calcein fluorescence. Susceptible liposomes were not induced to leak by heat-inactivated virus or by trypsin-treated virus. The response of the vesicles to virus attachment is thus analogous to virus-induced hemolysis and presumably involves fusion of the vesicle and virus membranes. In addition to glycophorin and phosphatidylcholine, cholesterol was absolutely required for the lytic response to the virus. The need for cholesterol was not attributable to inactivation of the virus by liposomes without cholesterol. The presence of gangliosides increased the encapsulated volume of the liposomes, but gangliosides did not effectively substitute for glycophorin. Thin-layer chromatography of lipid extracted from incubated virus and liposomes containing a small amount of a fluorescent phosphatidylcholine indicated that phosphatidylcholine in the vesicle is not chemically altered by functional interaction with the virus.