Morphometry of feline neutrophil granule genesis

During neutrophil granule genesis, the formation of primary granules is generally thought to be limited to the promyelocyte stage; whereas synthesis of secondary granules is thought to occur only at the myelocyte stage. This hypothesis was tested morphometrically in feline neutrophils that are known to contain both granule types. Marrow specimens obtained from six cats were stained with peroxidase for identification of neutrophil primary granules and counterstained with periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) for identification of secondary granules. By regression analysis using arithmetic models, numbers of cytoplasmic granules in 311 cells were correlated with the degree of nuclear chromatin condensation, which was shown to be an adequate parameter for cell maturation. Promyelocytes and myelocytes had similar mean numbers of peroxidase-positive granules per unit area. A significant increase (p less than or equal to 0.0001) in the numbers of peroxidase-positive granules was noted between the metamyelocyte and the mature neutrophil stage, despite the lack of peroxidase activity in endoplasmic reticulum and Golgi lamellae. By contrast, a significant increase of peroxidase-negative granules between the metamyelocyte and the mature neutrophil stage was not clearly established with these methods. The increase in peroxidase-positive granules may indicate continued production of peroxidase-containing granules and/or redistribution of peroxidase among lysosomal organelles in late feline neutrophils.     

Oscillations of cAMP with the cardiac cycle

Oscillations of cAMP with the cardiac cycle were demonstrated in the rat heart using a stimulator-triggered rapid freeze-clamp to decrease the temperature of the heart from 37 degrees C to -80 degrees C in 5 msec (20,000 degrees/sec) at a predetermined phase of the cardiac cycle. The nucleotide, cAMP, oscillated 60% with the cardiac cycle during normal working conditions, the higher cAMP value occurring during systole.     

Ferrocyanide staining of transferrin and ferritin-conjugated antibody to transferrin.

To evaluate the ultrastructural distribution of transferrin on the surface of L1210 ascites tumor cells, we used ferrocyanide to stain ferric iron (Prussian blue reaction) in transferrin, as well as in ferritin conjugated to antibody that was immunologically attached to the transferrin. Small deposits averaging 5 nm in diameter identified transferrin iron, whereas large cuboidal deposits averaging 50 nm in diameter stained ferritin conjugated-antibody that was bound to both transferrin and apotransferrin on the cell surface. The ability of transferrin to deliver iron to ascites tumor cells was confirmed by kinetic studies of transferrin labeled with 59Fe and 125I. These preliminary results are consistent with release of transferrin iron at the cell surface and demonstrate additional uses for ferrocyanide in ultrastructural cytochemical techniques.     

Growth hormone alters metabolic effects and proteolysis of insulin in adipose tissue during lactation.

To study the regulation of lipogenesis in adipose tissue by insulin and growth hormone during lactation, tissue was biopsied from primiparous bovines at 30 days antepartum and 60 days postpartum. Tissue was cultured for 24 hr or 48 hr in M199 with acetate and glucose, with a change of medium at 24 hr. The three in vitro treatments were: insulin and hydrocortisone at 10 and 50 ng/ml, respectively (IH); IH + 10 ng/ml of growth hormone (G10); and IH + 100 ng/ml of growth hormone (G100). IH allowed lipogenesis rates from 50% to 85% of those in fresh tissue. Addition of 10 ng/ml of growth hormone reduced (P less than 0.05) lipogenesis; at 100 ng/ml, the effect was only slightly greater. The hypothesis that insulin and growth hormone could be degraded by bovine adipose tissue was tested. Adipose tissue cell-free extracts degraded 125I-labeled insulin, but did not degrade labeled growth hormone. The insulin protease activity was further characterized and had a pH optimum of 7.1, a maximum hydrolysis of approximately 70%, and a hydrated molecular mass of approximately 23,000 daltons. Insulin proteolysis was inhibited by specific insulin protease inhibitors and stimulated by disulfide reducing agents. Bovine growth hormone, prolactin, and histone inhibited (P less than 0.05) the proteolysis of insulin, while bovine serum albumin, egg albumin, trypsin inhibitor, and lysozyme did not. Adipose tissue from pregnant and lactating bovines was sensitive to insulin and growth hormone, and growth hormone may modulate activity of an insulin-specific protease.     

Induction of tumor necrosis factor-alpha and inducible nitric oxide synthase fails to prevent toxoplasmic encephalitis in the absence of interferon-gamma in genetically resistant BALB/c mice

Following infection with Toxoplasma gondii, certain strains of mice, such as BALB/c, are genetically resistant to development of toxoplasmic encephalitis (TE) and establish a latent chronic infection as do humans. Thus, these animals appear to be a suitable model to analyze the mechanism of resistance to TE. Since the mechanism for their genetic resistance is unknown, we examined the role of interferon-gamma (IFN-gamma) tumor necrosis factor-alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS) in the resistance using BALB/c-background IFN-gamma-deficient (IFN-gamma(-/-)) mice. IFN-gamma(-/-) and control mice were infected with the ME49 strain of T. gondii and treated with sulfadiazine to establish chronic infection. After discontinuing sulfadiazine, the IFN-gamma(-/-) mice all died, whereas the control mice all survived. Histological studies revealed remarkable inflammatory changes associated with large numbers of tachyzoites in brains of the IFN-gamma(-/-) mice but not in the control mice after discontinuation of sulfadiazine. Large amounts of mRNA for tachyzoite-specific SAG1 were detected in brains of only the IFN-gamma(-/-) mice. IFN-gamma mRNA was detected in brains of only the control mice, whereas mRNA for TNF-alpha and iNOS were detected in brains of both strains of mice. The amounts of the mRNA for TNF-alpha and iNOS did not differ between these mice. Treatment of IFN-gamma(-/-) mice with recombinant IFN-gamma prevented development of TE. These results demonstrate that IFN-gamma is crucial for genetic resistance of BALB/c mice against TE and that TNF-alpha and iNOS are insufficient to prevent TE in the absence of IFN-gamma.     

Strain typing of Toxoplasma gondii: comparison of antigen-coding and housekeeping genes

Molecular characterization of Toxoplasma gondii isolates is central for understanding differences in disease transmission and manifestations. Only 3 subgroups (lineages) have been discerned with subtle within-lineage variation, permitting low-resolution classification of isolates. Because proteins, coding sequences, and especially antigen-coding genes have been used extensively in previous studies, we focused on sequence variation in introns of housekeeping genes, which may be more informative for phylogenetic analysis because they evolve under lower selection. We compared sequence variation in introns of 5 housekeeping genes with 2 antigen-coding genes. Introns of housekeeping genes were slightly more polymorphic than coding and noncoding regions of antigen-coding genes and only the former showed intralineage variation. Intragenic linkage disequilibrium was complete, but intergenic linkage, although highly significant, was incomplete, suggesting that genes are partially uncoupled. Six of 7 substitutions found within the region coding for the tachyzoite surface antigen, SAG2, were nonsynonymous, indicating that diversifying selection acts on this locus. Typing isolates on the basis of housekeeping and antigen-coding genes was consistent, but the phylogenetic relationships among the resulting groups was inconsistent. A cougar isolate typed as lineage II using a restriction fragment length polymorphism assay possessed multiple unique polymorphisms, suggesting that it represents a new lineage. We concluded that introns of housekeeping genes are preferred markers for phylogenetic study, and that multilocus genotyping is preferred for typing parasites, especially from feral or unstudied environments.