Acute toxoplasma infection of mice induces spleen NK cells that are cytotoxic for T. gondii in vitro

Murine spleen natural killer (NK) cells from normal and Toxoplasma-infected BALB/c mice were examined for their reactivity against RH strain tachyzoites in vitro. First, the effect of suspending medium on survival of extracellular RH tachyzoites was determined. Optimal parasite viability (by ethidium bromide-acridine orange staining) was observed when tachyzoites were incubated in phosphate-buffered saline (PBS) containing 10% horse serum (HS) for as long as 5 hr. In addition, parasite viability in PBS-HS correlated with subsequent infectivity, because freshly harvested and PBS-HS-incubated tachyzoites were equivalent in their ability to cause lethal infections in normal mice and to survive within normal mouse macrophages. Furthermore, viability and tumoricidal capacity of murine spleen NK cells incubated in PBS-HS was comparable to that of NK cells incubated in a standard cytotoxicity medium. Incubation of effector NK cells and target tachyzoites in PBS-HS in vitro revealed that spleen NK cells from 3-day Toxoplasma-infected mice had significantly greater cytotoxicity for extracellular RH tachyzoites than did control cells from uninfected mice. Moreover, Toxoplasma gondii-induced spleen NK cell toxoplasmacidal activity was significant at all effector to target cell ratios tested, and appeared to be mediated by direct contact between the host cell and the parasite. These in vitro results suggest that NK cells may be important in host defense against T. gondii.     

Retroperitoneal fibromatosis and acquired immunodeficiency syndrome in macaques: clinical and immunologic studies

A simian acquired immunodeficiency syndrome (SAIDS) associated with retroperitoneal fibromatosis (RF) has been observed in several species of macaque at the Washington Regional Primate Research Center. Clinical signs were recurrent diarrhea, weight loss, mesenteric lymphadenopathy, and opportunistic infections. Most affected macaques in the later stages of illness showed marked immunodeficiency. Response of peripheral blood mononuclear cells to mitogens was impaired significantly. There was sharply depressed primary and secondary antibody response to the T-cell dependent antigen, bacteriophage phi X174. Affected monkeys did not switch from IgM to IgG antibody following a secondary immunization, as did normal macaques. Twenty-four (67%) of 36 affected animals with progressive RF or deteriorated stages of illness had hypoproteinemia and hypoalbuminemia. Quantitative serum immunoglobulins of 23 cases showed that eight (35%) had hypogammaglobulinemia, six (26%) had hypergammaglobulinemia, and the remainder (39%) were within the normal range. Opportunistic infections were predominantly bacterial pathogens. Type D retrovirus appeared to be closely associated with RF-affected macaques (12/12 or 100%). The case fatality rate (including animals sacrificed after prolonged illness) was 98%. The leading cause of death was due directly to RF lesions in 43%, to enterocolitis in 36%, septicemia in 12%, amyloidosis in 5%, and malignant lymphoma (2%). Clinical, immunologic and pathologic changes reveal an acquired immunodeficiency syndrome that has many similarities to human AIDS. SAIDS and RF may be a useful model for studying human AIDS.     

Expression and secretion of biologically active human atrial natriuretic peptide in Saccharomyces cerevisiae

A hybrid gene was constructed containing a fusion between the DNA sequences encoding the secretory precursor of the yeast mating pheromone alpha-factor and a synthetic sequence encoding a biologically active 24-amino acid carboxyl-terminal portion of the human atrial natriuretic peptide (hANP) precursor. Transformation of Saccharomyces cerevisiae with the hybrid gene resulted in the yeast cells secreting biologically active hANP into the extracellular medium. The secreted hANP was purified and found to be accurately processed at the junction in the chimeric alpha-factor/hANP protein, producing the desired mature hANP amino terminus. The secreted product was also folded correctly with respect to the single disulfide bond. However, the carboxyl terminus of the secreted hANP material was heterogeneous such that the major form lacked the last two amino acids of the peptide while the minor form was the full length material. The observed processing at the carboxyl terminus of the secreted hANP may reflect a normal processing event involved in alpha-factor peptide maturation.     

Stimulation of facilitated [3H]uridine transport by thyroid hormone in GH1 cells. Evidence for regulation by the thyroid hormone nuclear receptor

We have previously shown that 3,5,3'-triiodo-L-thyronine (L-T3) stimulates cell growth and a 4- to 8-fold increase in growth hormone mRNA in GH1 cells. These effects appear to be mediated by a thyroid hormone nuclear receptor with an equilibrium dissociation constant for L-T3 of 0.2 nM and an abundance of about 10,000 receptors per cell nucleus. In this report, we show that L-T3 exerts a pleiotypic effect on GH1 cells to rapidly (within 2 h) stimulate [3H]uridine uptake to a maximal value of 2.5- to 3-fold after 24 h. This results from an increase in the number of functional uridine "transport sites" as shown by studies documenting an increase in the apparent Vmax with no change in the Km, 17 microM. Although the labeling of the cellular uridine pool and pools of all phosphorylated uridine derivatives was increased by L-T3, there was no change in the relative amounts of the individual pools in cells incubated with or without hormone. The intracellular concentration of [3H]uridine was estimated to be similar to that of the medium, suggesting that facilitated transport mediates [3H]uridine uptake. That this increase in [3H]uridine transport was nuclear receptor-mediated is supported by the excellent correspondence of the L-T3 dose-response curve for [3H]uridine uptake and that for L-T3 binding to receptor. Finally, inhibition of protein synthesis by cycloheximide and RNA synthesis by actinomycin D demonstrated that the L-T3 effect required continuing protein and RNA synthesis. These results are consistent with an effect of the L-T3-nuclear receptor complex to increase uridine uptake in GH1 cells by altering the expression of gene(s) essential for the transport process.     

Isolation of a human stromal cell strain secreting hemopoietic growth factors

A diploid fibroblastoid cell strain, termed "ST-1," has been established from a long-term liquid culture of human fetal liver cells. ST-1 cells are nonphagocytic, nonspecific esterase negative and do not possess factor VIII-related antigen but stain with antibodies specific for fibronectin and type I collagen. The ST-1 cells produce nondialyzable hemopoietic growth factors capable of stimulating the development of erythroid bursts, mixed granulocyte-macrophage colonies, pure granulocyte colonies, and pure macrophage colonies. These factors are active on both human fetal liver and human adult bone marrow progenitors. When liquid cultures of human fetal liver hemopoietic progenitors are established with a preformed monolayer of ST-1 cells, the yields of nonadherent cells, erythroid progenitors, and myeloid progenitors are greatly increased. These studies demonstrate that the fibroblastoid ST-1 cells support hemopoiesis in vitro and may be a critical element in the stromal microenviroment in vivo.     

Kinetic and spectroscopic studies of transhydrogenase activity and nucleotide site of lipoamide dehydrogenase

The kinetic behavior and spectroscopic characteristics of the nucleotide site(s) of lipoamide dehydrogenase have been investigated. Both subunits of the dimeric enzyme interact with NAD+. The binding of NAD+ is associated with a negative trough around 420-450 nm and a positive peak at 507 nm of the difference spectrum. The transhydrogenation between NADH and thionicotinamide nucleotide or acetylpyridine nucleotide is shown to proceed via a Ping Pong or an ordered Bi Bi mechanism, respectively, at pH above 7.0. Lowering pH or acetamidation lose the spectral characteristic of the positive peak of the enzyme-NAD+ complex with a concurrent change in the kinetic mechanism in the NADH+-acetylpyridine nucleotide transhydrogenation.     

Dye-sensitized photo-oxidation of enzymes.

Heart lipoamide dehydrogenase, liver alcohol dehydrogenase and egg-white lysozyme are photo-oxidized in the presence of various dye sensitizers. The photodynamic process is preceded by the binding between the enzyme and the sensitizers. Among the commonly used dyes, halogenated xanthines and thiazine are effective sensitizers for the photo-inactivation of these three enzymes. Histidine residues are the primary target for the sensitized photo-oxidation that inactivates lipoamide dehydrogenase and alcohol dehydrogenase. However, the destruction of tryptophan residues is responsible for the photo-inactivation of lysozyme. The deuterium medium effect and the quenching effect by various scavengers of the potential photo-oxidative intermediates implicate the participation of the mixed type I-type II mechanism, with the involvement of singlet oxygen being of greater importance, in the photo-inactivation of the enzymes.     

Detailed profile of prolactin secretion in the immature female rat: evidence for the existence of an ultradian rhythm

The detailed profile of prolactin (PRL) secretion in 22-24 and 29-31 days old female rats was investigated by serial blood sampling through an intracardiac cannula at 15-min intervals for each of the 9 or 10-h periods beginning at 09.00 or 10.00 and 22.00 h. By analysis of the power spectrum and the least squares method the time series of PRL concentrations which were measured by RIA were found to have approximately a 3-h period ultradian rhythm in either sampling period of both the 22-24 and 29-31 days old rats. The peak times calculated based on the acrophase estimated through the calculation of periodicity were concentrated around 12.00, 15.00 and 18.00 h for the sampling period 10.00-19.00, and 24.00, 03.00 and 06.00 h for the sampling period 22.00-07.00 h. However, in more than half of the animals at 22-24 days of age, one secretory episode around 12.00 h, and two secretory episodes around 24.00 and 03.00 h had markedly small amplitudes, making the remaining secretory episodes distinct diurnal and nocturnal surges, respectively. In the animals at 29-31 days of age, the amplitudes of the PRL episodes occurring around 12.00 h were markedly small, making the remaining two episodes as diurnal surges, whereas the amplitudes of PRL secretory episodes during the period 22.00-07.00 h were analogous to each other. These findings indicate that the semicircadian rhythm of PRL secretion is established on the basis of PRL secretion with the 3.0-h period ultradian rhythm.     

Bifunctionality and pseudoisozymes of pyruvate kinase from codfish muscle

Pyruvate kinase has been purified from codfish muscle. The ratio of phosphotransferase and oxalacetate decarboxylase activities remains relatively constant throughout purification steps. These two activities are dependent as well as sensitive to sulfhydryl reagents. In the presence of dithioerythritol, only one molecular form of pyruvate kinase is detected. However, the enzyme exists as four pseudoisozymes in the presence of 2-mercaptoethanol. The pseudoisozymes of codfish pyruvate kinase are interconvertible under the influence of sulfhydryl reagents.