Iron and the liver: subcellular distribution of iron and decreased microsomal cytochrome P-450 in livers of iron-loaded rats.

To understand better the intracellular iron distribution and metabolic consequences of chronic hepatic iron overload, rats were given large doses of iron dextran or ferric citrate intraperitoneally. They accumulated large quantities of iron within Kupffer cells and hepatocytes. The relative subcellular iron distributions were similar in controls and iron-loaded rats, despite a ten- to 20-fold difference in hepatic iron concentration. Electron microscopy of whole liver and subcellular particulate fractions suggested that iron was present in highest concentration in lysosomes, which were rendered more labile by its presence. Nevertheless, quantitative iron determinations on all subcellular fractions, obtained by two preparative methods, showed that most of the iron was present in the "soluble" fraction. The amount of iron in the "microsomal" fraction varied, depending on the techniques used for preparation of this fraction. Cytochrome P-450 and total heme concentrations were decreased 40% to 50% in microsomes isolated from iron-loaded livers.     

Guidelines for the preparation and analysis of the fragile X chromosome in lymphocytes

The following guidelines were adopted by an Ad Hoc Committee convened at the Fourth International Workshop on the Fragile X Syndrome and X-Linked Mental Retardation to establish minimum cytogenetic standards for the preparation and analysis of the fragile X chromosome. The intention of the committee was to develop and provide practical standards for the routine cytogenetic detection of the fragile X. The guidelines describe reasonable criteria for effective tissue culture methods for eliciting the Xq27.3 fragile site in vitro and for the analysis of such chromosome preparations.     

Human granulocyte CD11b expression as a pharmacodynamic biomarker of inflammation

A method has been developed for the direct quantification of the CD11b integrin on granulocytes by flow cytometric analysis of whole blood specimens following either LTB₄ or lipopolysaccharide (LPS) stimulation. This method has utility in evaluating the pharmacodynamic action of either LTB₄ receptor antagonists or immune cell modulators in effecting CD11b integrin expression and granulocyte activation in human subjects administered such drugs. Previous studies using CD11b as a biomarker of granulocyte activation have faltered because of the difficulty in controlling the activation state of the granulocyte following removal of blood from subjects. The present study has made use of a newly validated method using either LTB₄ or LPS to stimulate CD11b expression on granulocytes and has been used, as one measure, in the evaluation of LPS activity when administered to normal human volunteers.     

Molecular epidemiology and genetic diversity of echovirus type 30 (E30): genotypes correlate with temporal dynamics of E30 isolation

Echovirus type 30 (E30) (genus, Enterovirus; family, Picornaviridae) has caused large outbreaks of aseptic meningitis in many regions of the world in the last 40 years. U.S. enterovirus surveillance data for the period 1961 to 1998 indicated that the annual proportion of E30 isolations relative to total enterovirus isolations has fluctuated widely, from a low of 0% in 1966 to a high of 42% in 1998. Peaks of E30 isolations occurred in the years 1968 to 1969, 1981 to 1984, 1990 to 1993, and 1997 to 1998, coincident with large nationwide outbreaks of E30-associated aseptic meningitis. Analysis of the complete VP1 sequence (876 nucleotides) of 136 E30 strains isolated in geographically dispersed regions of the United States and nine other countries between 1956 and 1998 indicated that the currently circulating E30 strains are genetically distinct from those isolated 30 to 40 years ago. Phylogenetic reconstruction demonstrated the existence of at least four distinct genetic groups, three of which have not been isolated in North America since 1981. Two of the three groups disappeared during periods when E30 was isolated infrequently. All North American E30 strains isolated after 1988 were closely related to one another, and all post-1993 isolates were of the same lineage within this group. Surveillance data indicate that E30 causes large national outbreaks of 2- to 4-year durations, separated by periods of relative quiescence. Our results show that shifts in the overall genetic diversity of E30 and the predominant genetic type correlate temporally with the dynamics of E30 isolation. The sequence data also provide a basis for the application of molecular techniques for future epidemiologic investigations of E30 disease.     

Receptors for excitatory amino acids on neurons in rat pyriform cortex

The actions of a variety of agonists and antagonists of the excitatory amino acids on rat pyriform cortex pyramidal neurons were studied in a submerged, perfused brain slice. The order of potency for the agonists, applied by ionophoresis, was kainate greater than quisqualate greater than N-methyl-D-aspartate greater than aspartate = glutamate. The endogenous monosynaptic excitation of pyramidal neurons upon stimulation of the lateral olfactory tract was blocked post-synaptically by DL-2-amino-4-phosphonobutyric acid, although this drug did not consistently block any of the exogenous responses. The synaptic excitation was not blocked, however, by antagonists presumed specific for the quisqualate (glutamate diethyl ester), kainate, (gamma-D-glutamylglycine), or N-methyl-D-aspartate (DL-2-amino-5-phosphonovaleric acid, DL-2-amino-7-phosphonohetaonic acid) receptors. Several antagonists blocked N-methyl-D-aspartate responses at lower concentrations than those to aspartate, and other antagonists distinguished between kainate and quisqualate responses. These results suggest that 1) pyriform neurons have a variety of receptors that have properties somewhat different from those found in other preparations and 2) the endogenous transmitter activates a receptor distinct from those activated by kainate, quisqualate, and N-methyl-D-aspartate.     

Heterogeneity of the MtTW15 mammosomatotropic tumor. I. Light microscopic evaluation of cell types by means of immunocytochemistry,morphometric quantitation,fluorescence cytophotometry and radioimmunoassay

Large MtTW₁₅ pituitary tumors produced 200- to 800-fold elevations in serum growth hormone (GH) and prolactin (PRL) levels. Female tumor hosts showed doubling in body weight, milk secretion, and a 2-fold hepatosplenomegaly. Pituitaries of host animals were reduced by about 50% in both weight and concentrations of GH and PRL. Large tumors were well-encapsulated, multinodular and showed variable amounts of necrosis and hemorrhage. Cytofluorometric analysis revealed a range of 100-fold in nuclear DNA content of tumor parenchymal cells which were chromophobic, pleomorphic and frequently mitotic. Concentrations of hormones in tumors were less than in normal pituitaries and highly variable with the ratio of GH/PRL ranging up to 30-fold within the same tumor. Immunostaining and linear scanning quantitation showed that about 50% of the tumor cells contained immunodetectable hormones. Comparison of immunostained adjacent sections showed that hormone-containing tumor cells were pleomorphic, unequally distributed within nodules, lacking in distinctive identifying morphological characteristics and that they contained GH or PRL but not both hormones simultaneously. Collectively our results show that large MtTW₁₅ tumors are comprised of a markedly heterogeneous population of tumor cells and they suggest that the hormone-containing cells are monohormonal secreting tumor cells which can produce GH or PRL but not both hormones.     

Characterization of primary cell cultures as potential target cells for analysis of bovine cytotoxic T lymphocytes

In domestic animal species, assessment of cell-mediated immune responses to virus infection is hampered by the requirement for class I MHC compatibility between target and effector cells. Additional complicating factors can include an inability to infect target cells in vitro, or virus-induced lysis of infected target cells. One way to circumvent these problems is to use virus-mediated gene transfer to deliver individual viral genes to autologous primary target cells. Several primary bovine cell cultures were assessed as potential target cells for cytotoxic T lymphocyte (CTL) assays by measuring their levels of class I MHC expression and susceptibilities to retroviral gene delivery. High levels in both class I MHC expression and susceptibility to gene delivery were seen in adherent cell cultures isolated from peripheral blood (PBAC). PBAC, which arose as an outgrowth of adherent peripheral blood mononuclear cell cultures, had morphology, protein expression patterns, and response to functional assays characteristic of high endothelial cells. Expression of viral vector-delivered genes in PBAC cells was confirmed with a recombinant retrovirus carrying the green fluorescent protein (GFP) gene. The use of vector-mediated delivery of viral genes to bovine high endothelial cells is a promising method for assessment of cell-mediated immunity in cattle.