Modulation of prolactin binding sites in vitro by membrane fluidizers II. Age-dependent effects on rat ventral prostatic membranes

The objectives of this study were to determine (i) if the age-related changes in ¹²⁵I-labeled ovine prolactin specific binding of rat ventral prostate was correlated with changes in membrane lipid microviscosity and (ii) if membrane fluidizers produced age-dependent effects on prolactin binding of prostatic membranes. The degree of fluidization was monitored by a fluorescence polarization method using 1,6-diphenylhexatriene. Membrane preparations of ventral prostate glands obtained from immature (24–25 days old), young-adult (80–90 days old) and aged (550–610 days old) male rats were used for prolactin binding and membrane lipid microviscosity measurements. Relative to immature rats, prostatic prolactin binding decreased approximately 50% in young-adult rats and 75% in aged rats. Membrane lipid microviscosity, relative to immature rats, was increased 72% in young-adult rats and 140% in aged rats. Prostatic membranes obtained from immature animals exhibited no significant effects of in vitro alcohol treatment on prolactin binding, whereas, those obtained from aged animals exhibited maximal increase in prolactin binding. The value of the microviscosity parameter, after in vitro alcohol exposure, exhibited no significant changes in immature animals, whereas, this parameter was decreased approximately 15% in young-adults and approximately 30% in aged animals. These data suggest that in vitro fluidization of prostatic membrane exhibits an age-dependent modification of prolactin binding.     

Arachidonic acid, bradykinin and phospholipase A2 modify both prolactin binding capacity and fluidity of mouse hepatic membranes

The objective of this study was to determine if arachidonic acid, a precursor of prostaglandin synthesis, bradykinin, a decapeptide known to stimulate membrane phospholipid methylation, arachidonic acid release and prostacyclin synthesis, and enzyme phospholipase A₂, capable of liberating arachidonic acid, alter the fluidity of hepatic membranes which could in turn modify the functionality of prolactin receptors. Liver homogenates of adult C₃H female mice incubated at 28°C for various times with 1–20 μg/ml arachidonic acid, 1–100 μg/ml bradykinin or 0.26–0.00026 U/ml phospholipase A₂ provided the 100,000 × g membrane pellets for subsequent ovine prolactin binding and membrane fluidity studies. Membrane microviscosity was determined by fluorescence polarization techniques using the lipid probe 1,6 diphenylhexatriene. Arachidonic acid, bradykinin and phospholipase A₂ stimulated specific oPRL binding, in a dose-related fashion, with maximum increases of 73%, 21% and 46%, at 4 μg/ml arachidonic acid, 5 μg/ml bradykinin and 0.026 U/ml PLA₂, respectively. This induction, occurring within 30 min of incubation, was found to be due to an increase in the number of receptor sites. Under the same conditions, arachidonic acid, bradykinin and PLA₂ induced 22%, 16%, and 18% decreases in membrane microviscosity, respectively. These data suggest that prostaglandin synthesis modifying agents may modulate the number of prolactin receptors $\text{in vivo}$ by changing the lipid fluidity of the target cell membranes by either of their known effects: arachidonic acid release from the phospholipid matrix, synthesizing appropriate prostaglandins at correct concentration or methylation of membrane phospholipids.     

DNA synthesis in nuclei and nuclear matrices of regenerating rat liver: Effect of whole-body gamma irradiation

Summary Partial hepatectomy (PH) of rats (Wistar strain) resulted in acceleration of DNA synthesis in liver which reached a maximum at 36 h after PH. Whole-body radiation exposure (10 Gy) of the rats at 12 h after PH completely arrested this stimulation in DNA synthesis. The elevation of DNA synthetic rate in response to PH and complete obliteration of this stimulation by whole-body radiation exposure were found to be the reflection of levels of DNA polymerase-alpha in nuclei and nuclear matrices isolated from the rat livers. Studies based on assays of DNA polymerase in nuclei and nuclear matrices, with and without exogenous DNA template (activated calf thymus DNA), revealed that whole-body irradiation blocked induction of DNA polymerase-alpha and, in turn, assembling of DNA polymerizing apparatus. Irradiation of nuclei (suspended in buffer) in vitro at doses as high as 500 Gy did not have any inhibitory effect on DNA polymerase-alpha activitiy.     

Expression of an inwardly rectifying K channel from rat basophilic leukemia cell mRNA in Xenopus oocytes

Rat basophilic leukemia cells (RBL-2H3) have previously been shown to contain a single type of voltage-activated channel, namely an inwardly rectifying K⁺ channel, under normal recording conditions. Thus, RBL-2H3 cells seemed like a logical source of mRNA for the expression cloning of inwardly rectifying K⁺ channels. Injection of mRNA isolated from RBL-2H3 cells into Xenopus oocytes resulted in the expression of an inward current which (1) activated at potentials negative to the K⁺ equilibrium potential (E_K), (2)decreased in slope conductance near E_K, (3) was dependent on [K⁺]_o and (4) was blocked by external Ba²⁺ and Cs⁺. These properties were similar to those of the inwardly rectifying K⁺ current recorded from RBL-2H3 cells using whole-cell voltage clamp. Injection of size-fractionated mRNA into Xenopus oocytes revealed that the current was most strongly expressed from the fraction containing mRNA of approximately 4–5 kb. Expression of this channel represents a starting point for the expression cloning of a novel class of K⁺ channels.     

Production of tumor necrosis factor α and interferon γ in interleukin-2-treated melanoma patients: Correlation with clinical toxicity

Summary Interleukin-2 (IL-2)-based immunotherapy regimens are accompanied by dose-limiting toxicity consisting of fever, tachycardia, chills and capillary leak syndrome. We hypothesized that the toxicity was caused by the induction and release of endogenous cytokines such as tumor necrosis factor (TNF) and interferon (IFN). We measured the serum levels of TNF and IFN in IL-2-treated melanoma patients and attempted a correlation with clinical toxicity. A total of 23 patients received either 6 × 10⁶ IU or 12 × 10⁶ IU Cetus IL-2/m² by i. v. bolus daily for 5 consecutive days on weeks 1, 3 and 5. Serum TNF and IFN levels were measured by enzyme-linked immunosorbent assay. Clinical toxicity was scored each day by objective measurements of hypotension, tachycardia, fever and chills/rigors. Clinical toxicity and IFN levels correlated nicely, peaking on the 5th day of each treatment cycle. The kinetics and magnitude of TNF production, however, were not predictable and did not correlate with either IFN or toxicity. Some patients had modest increases in TNF production while others had markedly increased levels during the second and third treatment weeks. Remarkably, these high levels persisted during nontreatment weeks and after completion of therapy. This clinical study demonstrates novel kinetics for immunoreactive TNF in IL-2 cancer patients, which do not correlate well with toxicity.This work was supported by NIH Grants CA 50 780 (J. E.) and CA 29 605, CA 12 582 (D. L. M.) and the U. C. Tobacco-Related Disease Research Program RT-62 (J. E.). J. E. is the recipient of an NCI Clinical Investigator Award (KO8-01360) and is a Dorothy and Leonard Straus Scholar at UCLA     

Basal song rate variation in male red-winged blackbirds: sound and fury signifying nothing?

Male red-winged blackbirds (Agelaius phoeniceus) vary substantiallyin how much they sing. We tested whether song rate reflectedthe male’s quality, the quality of his territory, or hisfrequency of interaction with potential competitors. Song ratedeclined with the seasonal decline in nest initiations, suggestingthat song is used to attract females. However, males with highersong rates did not obtain larger harems. This suggests thatfemales do not use male song rate to assess either male qualityor territory quality. If song rate signals male quality to malerivals, and if territory owners are of higher quality than floaters,then owners' song rates should be higher than those of floaters.However, when we removed territory owners temporarily, the songrate of their floater replacements was similar to that of theoriginal owners. Finally, a male’s song rate was not affectedby how frequently he chased intruders, or by the proximity ornumber of neighbors he had. In short, the only information thatbasal song rate of redwinged blackbirds appears to provide toconspecifics is that the territory is occupied. In simulatedterritory takeover attempts, however, song rate was significantlyhigher than the basal rate. Thus, more may be revealed by thesinger in these situations. [Behav Ecol 1991;2:123-132]     

Effects of copper on growth, reproduction, survival and haemoglobin in Daphnia magna

The effects of additions of CuSO4 X 5H2O to final concentrations between 0.0004 and 105 micrograms Cu l-1 on growth, reproduction, survival and haemoglobin content of Daphnia magna were studied in hard reconstituted water and compared to the response in the dilution water without addition of copper. Concentrations of copper are nominal values. The 48-hr EC50 (immobilization) for unfed neonates was 6.5 micrograms Cu l-1 and the 48-hr and 21-day LC50 for fed neonates were 18.5 and 1.4 microgram Cu l-1, respectively. Growth expressed as body length of juveniles after 7 days and adult females after 21 days was only reduced in survivors at the highest non-lethal concentration (6.6 micrograms Cu l-1). Reproduction was stimulated by low concentrations of copper. Optimal reproduction after 21 days was found between 0.001 and 0.1 microgram Cu l-1. Higher concentrations were partially inhibitory (0.4 microgram Cu l-1), stimulatory (0.8 and 1.6 microgram Cu l-1) or completely inhibitory (3.2 micrograms Cu l-1 and above). The stimulatory peak around 1 microgram Cu l-1 was accompanied by a reduced survival (above 0.4 microgram Cu l-1). The Zero Equivalent Point (ZEP) for reproduction at non-reduced survival was 0.23 microgram Cu l-1. This concentration should be "safe" for D. magna under prevailing conditions (reconstituted water with a hardness of 250 mg l-1 as CaCo3 and a synthetic diet based on fish food and baby gruel). The haemoglobin content was affected by copper in a complex pattern which was not related to growth, reproduction or survival.