Multiplication of Legionella pneumophila Philadelphia-1 in cultured peritoneal macrophages and its correlation to susceptibility of animals

Intracellular growth of Legionella pneumophila Philadelphia-1 strain in peritoneal macrophages (PMP) from various rodents was measured and its correlation to the level of susceptibility of the animal was examined. In guinea pig PMP, the organism grew well and the guinea pig was very susceptible to it (50% lethal dose, LD50 = 7.6 X 10(4)). On the other hand, the bacteria hardly multiplied in mouse PMP and the animal was resistant to infection (LD50 = 6.7 X 10(7)). Intracellular growth rate correlated well with susceptibility in these animals. In golden hamsters, a discrepancy between intracellular growth and susceptibility was found. The organism grew intracellularly as rapid as in guinea pig PMP, but the golden hamster was very resistant to infection (LD50 = 2.2 X 10(8)). In rat PMP, the organism did not grow intracellularly during a 24-h period of infection, but started to grow after that and the growth rate thereafter was as rapid as in guinea pig PMP. WKA rats were resistant and the LD50 in the animal was 1.9 X 10(7). In vivo natural resistance of rats and golden hamsters to the organism was considered to be a result of other factors than macrophages.     

Plasma lipoprotein cholesterol and triglycerides and lipoprotein lipase activity in epididymal white adipose tissue of rats fed high sucrose or high corn oil diets

The present study was undertaken to compare plasma lipoprotein lipid composition, as well as white adipose tissue lipoprotein lipase activity, in rats fed purified diets high in either sucrose or corn oil. The experimental diets (65% of calories as sucrose or corn oil, 15% as the opposite nutrient, and 20% as casein) were given ad libitum for 4 weeks. An additional group was fed a nonpurified diet as a reference diet. Both sucrose and oil diets were spontaneously consumed in isocaloric amounts by the animals. Despite energy intakes that were 35% lower than that of the reference group, the sucrose and oil groups exhibited final body weights that were only 6 and 9% lower, respectively, than that of the reference group, and accumulated more fat in the epididymal depots. Postprandial as well as fasting total cholesterol levels were similar in the sucrose and oil groups, while the high-density lipoprotein to total cholesterol ratio was highest in the animals fed corn oil. In both the fasted and fed states, plasma total triglyceride levels were 73% higher in the sucrose group than in the corn oil group. The largest triglyceride differences due to diet were observed in the chylomicron + very-low-density lipoprotein fraction. The oil-fed rats accumulated large amounts of triglycerides in their livers. Postprandial lipoprotein lipase activity in epididymal adipose tissue was almost twice as high in the sucrose group as in the oil group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Secretion of a suppressor cell inducing factor by an interleukin-3 dependent cell line with natural cytotoxic activity

This report describes the morphology, surface markers, growth requirements, and functional activity of the M1-A5 cell line, which was established by the limiting dilution of spleen cells from a mouse bearing a large methylcholanthrene-induced fibrosarcoma. The M1-A5 cells share many of the morphological features of large granular lymphocytes and, in addition, express asialo GM1 and Ly-5 surface markers which are commonly found on natural killer cells (NK) cells. There is no expression of T-cell differentiation antigens, surface immunoglobulin, or the granulocyte/macrophage marker, MAC-1. M1-A5 cells are dependent on exogenous growth factor(s) for survival and will proliferate if cultured in interleukin 3 (IL-3), but not in interleukin 1 (IL-1), interleukin 2 (IL-2), or granulocyte/macrophage colony stimulating factor (GM-CSF). In addition, the M1-A5 cells do not absorb IL-2. Despite their morphology and surface characteristics, the M1-A5 cells do not lyse NK targets such as YAC-1 and RLM1 in 4- or 18-hr cytotoxic assays but do lyse the natural cytotoxic (NC) susceptible target, WEHI-164, and to a very small extent, the M-1 fibrosarcoma cells, in an 18-hr assay. Thus they exhibit NC-like cytotoxic activity. In addition, the M1-A5 cells secrete a small molecular weight factor which activates suppressor cells capable of inhibiting antibody synthesis by cocultured syngeneic spleen cells.     

Chromosome condensation activity in the cytoplasm of anucleate and nucleate fragments of mouse oocytes

The activity of maturation promoting factor (MPF) which causes chromosome condensation and subsequent oocyte maturation was investigated in mouse oocytes using polyethylene-glycol-mediated cell fusion technique. Fully grown oocytes were bisected at germinal vesicle (GV) stage or shortly after germinal vesicle breakdown (GVBD) into anucleate and nucleate fragments. After 2-3 or 15-17 hr of culture these fragments were fused with interphase blastomeres from two-cell embryos. It was found that almost all the anucleate oocyte fragments cultured for a short term (2-3 hr), regardless of whether they were produced at GV stage or after GVBD, induced premature chromosome condensation in the blastomere nuclei, whereas only about 20% of those cultured for a long term (15-17 hr) could do so. On the other hand, the nucleate fragments always retain the cytoplasmic activity to induce chromosome condensation. Thus we suggested that the MPF initially could appear in mouse oocytes independently of the GV, that the mixing of GV material with the oocyte cytoplasm following GVBD had no effect on the activity of MPF in anucleate fragments, and that oocyte chromosomes or some components associated with them could play a significant role in maintaining the MPF activity.     

Immunohistochemical evidence of progestin target cells in the pituitary gland of ovariectomized rat

Progestin (P) target cells were identified in the pituitary gland of gonadectomized female rats which had been primed with estrogen (E). P staining was localized using the immunohistochemical avidin-biotin-peroxidase (ABP) complex method. Dark brown precipitates were primarily found over the cytoplasm of cells in the pars distalis, but not in the pars intermedia nor in the pars nervosa. The majority of P-sensitive cells in the pars distalis were identical with luteotrophs, a few being lactotrophs. These observations suggest a role of P in the regulation of production and secretion of gonadotrophins in the pituitary glands of female rats.     

Comparison of plasma prostanoid levels in the human cord artery in normal and fetal distressed deliveries

Prostaglandin E2 (PGE2), thromboxane B2 (TXB2; as a stable metabolite of TXA2), prostaglandin F2 alpha (PGF2 alpha) and 6-keto-PGF1 alpha (as a stable end product of prostacyclin) have been measured by using specific radioimmunoassay in the plasma of the cord artery immediately after delivery before the cord was clamped. Plasma prostanoid concentrations in normal deliveries (n = 8, as controls) were 24.8 +/- 2.6 (PGE2), 246.8 +/- 37.0 (TXB2), 122.2 +/- 13.3 (PGF2 alpha) and 82.1 +/- 7.7 (6-keto-PGF1 alpha) respectively (pg/ml, mean +/- s.e). On the other hand, in fetal distressed deliveries showing continuous bradycardia (n = 6), they increased significantly to 275.4 +/- 20.1 (PGE2), 948.6 +/- 102.5 (TXB2), 218.0 +/- 21.4 (PGF2 alpha) and 1498.6 +/- 298.4 (6-keto-PGF1 alpha) respectively (pg/ml, mean +/- s.e, p less than 0.005). However, both PGF2 alpha/PGE2 and TXB2/6-keto-PGF1 alpha ratios declined significantly from 4.70 +/- 0.33 to 0.68 +/- 0.05 and from 3.07 +/- 0.37 to 0.68 +/- 0.12 respectively (mean +/- s.e, p less than 0.005) in the fetal distressed group compared with those of the controls. From these results, it may be concluded that the cord artery, which is known as the patent source for the production of PGE2 and prostacyclin, did exert a sufficiently strong reaction to overcome the undesirable haemodynamic changes to maintain the fetal well-being in utero.     

Concentrations and origin of oxytocin in breast milk

Samples of human milk obtained from lactating women in the early postpartum period were assayed for oxytocin concentrations by specific RIA, following extraction procedures with Florisil. Mean oxytocin concentrations in human milk at postpartum day 1 to 5 were 4.5 +/- 1.1, 4.7 +/- 1.1, 4.0 +/- 1.3, 3.2 +/- 0.4, 3.3 +/- 0.6 microunits/ml (+/- SE), respectively. Oxytocin levels in milk were significantly increased by nursing (3.1 +/- 0.6, 5.3 +/- 1.0 microunits/ml, respectively). 3H-oxytocin in human milk was stable even after incubation at 37 degrees C for 2 hours. The dilution curve for milk was parallel to the curve for the standard oxytocin. The chromatographic fraction of immunoreactive oxytocin was identical to that of 3H-oxytocin. 3H-oxytocin was administered to lactating rats. Radioactivity in the neonatal gastric contents and plasma were 12.8% and 4.4% of the counts in the maternal plasma. It was made clear that oxytocin is stable in milk and that oxytocin in maternal blood can be transferred to mik and then to neonates.     

Effect of changes in thyroid state on metabolism of thyroxine by rat placenta

We studied the effect of the state of the thyroid on T4 monodeiodination in the rat placenta, and it was compared with those in the liver and kidney. The tissues, maternal serum, and amniotic fluid were obtained from pregnant rats. The tissues were homogenized in cold 50 mM Tris-HCl buffer, pH 7.5. The homogenate (1 mg protein) was incubated at 37 degrees C for 60 min with 1 microgram T4 in the presence of 5 mM DTT. The T3 and reverse T3 generated in the reaction mixture were extracted into cold ethanol and measured by RIAs. The conversion of T4 to reverse T3 in rat placenta was not significantly changed in MMI-induced hypothyroidism or T4 induced hyperthyroidism. On the other hand, conversion of T4 to T3 in the liver and kidney were changed in parallel with the thyroid state. The concentration of reverse T3 in the amniotic fluid was increased in accordance with the increase in the maternal serum T4 concentration. These results indicate that the placental T4 inner ring deiodination is not affected by the thyroid state, and that the change in the amniotic fluid reverse T3 concentration in this study is mainly dependent upon the change in maternal thyroid function.     

Properties of different Ca2+ pools in permeabilized rat thymocytes

The regulation of free Ca2+ concentration by intracellular pools and their participation in the mitogen-induced changes of the cytosolic free Ca2+ concentration, [Ca2+]i, was studied in digitonin-permeabilized and intact rat thymocytes using a Ca2+-selective electrode, chlortetracycline fluorescence and the Ca2+ indicator quin-2. It is shown that in permeabilized thymocytes Ca2+ can be accumulated by two intracellular compartments, mitochondrial and non-mitochondrial. Ca2+ uptake by the non-mitochondrial compartment, presumably the endoplasmic reticulum, is observed only in the presence of MgATP, is increased by oxalate and inhibited by vanadate. The mitochondria do not accumulate calcium at a free Ca2+ concentration below 1 microM. The non-mitochondrial compartment has a greater affinity for calcium and is capable of sequestering Ca2+ at a free Ca2+ concentration less than 1 microM. At free Ca2+ concentration close to the cytoplasmic (0.1 microM) the main calcium pool in permeabilized thymocytes is localized in the non-mitochondrial compartment. Ca2+ accumulated in the non-mitochondrial pool can be released by inositol 1,4,5-triphosphate (IP3) which has been inferred to mediate Ca2+ mobilization in a number of cell types. Under experimental conditions in which ATP-dependent Ca2+ influx is blocked, the addition of IP3 results in a large Ca2+ release from the non-mitochondrial pool; thus IP3 acts by activation of a specific efflux pathway rather than by inhibiting Ca2+ influx. SH reagents do not prevent IP3-induced Ca2+ mobilization. Addition of the mitochondrial uncouplers, FCCP or ClCCP, to intact thymocytes results in no increase in [Ca2+]i measured with quin-2 tetraoxymethyl ester whereas the Ca2+ ionophore A23187 induces a Ca2+ release from the non-mitochondrial store(s). Thus, the data obtained on intact cells agree with those obtained in permeabilized thymocytes. The mitogen concanavalin A increases [Ca2+]i in intact thymocytes suspended in both Ca2+-containing an Ca2+-free medium. This indicates a mitogen-induced mobilization of an intracellular Ca2+ pool, probably via the IP3 pathway.     

Mechanism of renal peritubular extraction of plasma glutathione. The catalytic activity of contralumenal gamma-glutamyltransferase is prerequisite to the apparent peritubular extraction of plasma glutathione

To clarify the peritubular mechanism for renal handling of plasma glutathione (GSH), variation of GSH levels in plasma, urine, kidney and liver was examined after intravenous administration of GSH to three groups of animals; control, acivicin-treated and rats treated with buthionine sulfoximine (BSO). Treatment of animals with BSO, a potent inhibitor of de novo GSH synthesis, markedly reduced hepatorenal GSH levels. Acivicin did not affect these levels. Upon intravenous injection of GSH (0.1 mmol/kg), renal GSH levels did not appreciably change in any of three animal groups. The rate of GSH disappearance from the circulation was rapid in control and BSO-treated rats, while it was markedly retarded in animals whose renal gamma-glutamyltransferase was extensively inactivated by acivicin. At 30 min after administration a significant amount of injected GSH was localized extracellularly (urine and plasma) in acivicin-treated animals. By contrast, most of the GSH rapidly disappeared from the extracellular space in control and BSO-treated animals. Together with the immunocytochemical evidence for the peritubular gamma-glutamyltransferase [Spater, H.W., Poruchynsky, M.S., Quintana, N., Inoue, M. & Novikoff, A.B. (1982) Proc. Natl Acad. Sci. USA 79, 3547-3550] the present results are fully consistent with the contention that the catalytic function of this enzyme is principally responsible for the peritubular mechanism for the renal handling of plasma GSH.