Role of arachidonic acid and its metabolites in the regulation of progesterone and oxytocin release from the bovine corpus luteum.

We have examined the effects of arachidonic acid (AA) and some of its metabolites on progesterone (P4) and oxytocin (OT) release by corpora lutea obtained from Holstein heifers at day 8 of the estrous cycle (Day 0 = estrus). The luteal cells were dispersed with collagenase and small and large cells were separated by unit gravity sedimentation and flow cytometry. After an 18-hr preincubation period, the cells were incubated in the presence of various treatments for 1 hr, followed by a 23-hr incubation period with no treatment. OT was secreted by the large, but not by the small, luteal cells into the incubation medium. AA elicited a significant (P less than 0.05) release of OT from the large cells and P4 from both the large and small cells within 1 hr of incubation, having a specific effect at a concentration of 10 microM. Larger doses (25 and 100 microM) of AA adversely affected the cell viability. Phospholipases A2 (0.5 unit/ml) and C (0.05 unit/ml) and calcium ionophore A23187 (0.1 microM) stimulated OT release from the large cells to the same extent as AA (10 microM). Inhibition of the AA cyclooxygenase metabolic pathway by indomethacin did not affect AA-induced release of OT and P4, although exogenous prostaglandins F2 alpha and I2 (5-25 ng/ml) stimulated the release of OT. Lipoxygenase products of AA (hydroxyeicosatetraenoic acid and leukotrienes; 25 ng/ml) also stimulated OT release. Inhibition of the lipoxygenase metabolic pathway by nordihydroguaiaretic acid abolished AA-induced release of both OT and P4. These results suggest that intracellular accumulation of free AA may modulate secretory functions in the bovine corpora lutea, including OT and P4 release.     

Membrane electrical parameters in turtle bladder measured using impedance-analysis techniques

Summary Equivalent-circuit impedance analysis experiments were performed on the urinary bladders of freshwater turtles in order to quantify membrane ionic conductances and areas, and to investigate how changes in these parameters are associated with changes in the rate of proton secretion in this tissue. In all experiments, sodium reabsorption was inhibited thereby unmasking the electrogenic proton secretion process. We report the following: (1) transepithelial impedance is represented exceptionally well by a simple equivalent-circuit model, which results in estimates of the apical and basolateral membrane ionic conductances and capacitances; (2) when sodium transport is inhibited with mucosal amiloride and serosal ouabain, the apical and basolateral membrane conductances and capacitances exhibit a continual decline with time; (3) this decline in the membrane parameters is most likely caused by subtle time-dependent changes in cell volume, resulting in changes in the areas of the apical and basolateral membranes; (4) stable membrane parameters are obtained if the tissue is not treated with ouabain, and if the oncotic pressure of the serosal solution is increased by the addition of 2% albumin; (5) inhibition of proton secretion using acetazolamide in CO₂ and HCO ₃ ^– -free bathing solutions results in a decrease in the area of the apical membrane, with no significant change in its specific conductance; (6) stimulation of proton transport with CO₂ and HCO ₃ ^– -containing serosal solution results in an increase in the apical membrane area and specific conductance. These results show that our methods can be used to measure changes in the membrane electrophysiological parameters that are related to changes in the rate of proton transport. Notably, they can be used to quantify in the live tissue, changes in membrane area resulting from changes in the net rates of endocytosis and exocytosis which are postulated to be intimately involved in the regulation of proton transport.     

The effect of behavioural and morphological plasticity on foraging efficiency in the threespine stickleback (Gasterosteus sp.)

We conducted an experiment to assess the change in foraging efficiency resulting from diet-induced morphological and behavioural plasticity in a species of freshwater, threespine stickleback (Gasterosteus sp.). Different degrees of morphological and behavioural change were induced using two prey items commonly found in the diet of this species, allowing us to estimate the relative importance of each type of plasticity. The purpose of the experiment was twofold. First, earlier work had suggested that diet variability might be an important factor in the evolution of trophic morphological plasticity in sticklebacks. The present results extend this work by revealing the adaptive significance of morphological plasticity. The current experiment also qualitatively assessed the compatibility of the time scale of morphological change with that of the natural resource variability experienced by this species. The results indicate that diet-induced plasticity improves foraging efficiency continuously for up to 72 days of prey exposure. This is probably due in part to plasticity of the external trophic morphology but our results also suggest a complex interplay between morphology and behaviour. The time scale appears to be matched to that of natural diet variability although it is possible that some traits exhibit non-labile plasticity. Our discussion highlights the important distinction between conditions favouring the evolution of labile versus non-labile plasticity. The second objective of the experiment was to determine the relative importance of morphological and behavioural plasticity. Few studies have attempted to quantify the adaptive significance of morphological plasticity and no study to our knowledge has separated the effects of morphological and behavioural plasticity. Our experiment reveals that both behavioural and morphological plasticity are important and it also suggests a dichotomy between the two: behavioural plasticity predominately affects searching efficiency whereas morphological plasticity predominately affects handling efficiency.     

Ktaadn: Thoreau the anthropologist

Timothy Troy is a Librarian at the Center for Creative Photography, University of Arizona.     

Immunogenicity of a non-class I MHC expressing murine tumor transfected with the influenza virus hemagglutinin or murine interleukin-2 genes

Summary The transfection of murine SP1 tumor cells with the hemagglutinin (HA) gene of influenza virus results, after fluorescent-activated cell sorting (FACS), in the selection of high-HA-expressing cell lines called H4A and H4B. Both lines fail to grow in syngeneic animals at doses that result in 100% tumor take of non-transfected tumor cells. Both grow in immunosuppressed mice. SP1 and H4A or H4B cells express few class I major histocompatibility complex (MHC) antigens but do express class II IA^k antigens. H4A or H4B cells engender a cytotoxic T lymphocyte (CTL) response but cannot protect against a challenge with SP1 cells. This CTL response is inhibited by anti-CD4 but not anti-CD8 antibodies. Using FACS, we were able to select a population (called H5AK5) with high class-I MHC antigen expression. Like H4A and H4B, H5AK5 cells fail to grow in syngeneic animals but do grow in immunosuppressed mice. However, unlike H4A or H4B, H5AK5 can induce protection against a challenge with 1 × 10⁵ SP1 cells. These studies indicate that the immunogenicity ofHA-transfected SP1 cells may correlate with the cell-surface expression of class II MHC antigens. However, HA-expressing SP1 cells seem able to induce a protective response against a parent SP1 cell challenge only if they also express class I MHC antigens. This view is supported by the observations that SP1 cells expressing murine interleukin-2 do not express class I MHC antigens, fail to grow in syngeneic animals, do grow in immunosuppressed mice but do not protect against a challenge with parental SP1 cells.This work was supported by The Clayton Fund, The Sid W. Richardson Foundation and PHS grants CA 39853 and 41525. Toshiyuki Itaya is a visiting scientist supported by the Smith Education Fund of the Department of Cell Biology. Troy Fiesinger is a summer research investigator sponsored by The University of Texas M. D. Anderson Cancer Center Summer Program for College Students     

2H NMR investigation of the organization and dynamics of polyisoprenols in membranes

The polyisoprenols (PIs) dolichol and undecaprenol function as chemical carriers of glycosyl residues in the membrane-directed synthesis of glycoconjugates in prokaryotic and eukaryotic cells. The molecular details of how these lipid cofactors function is unknown. Presented here are results of deuterium NMR investigations of site specifically 2H-labeled PIs incorporated into model membranes. To complement previous omega-terminal PI labeling schemes, a simple synthesis of head group 2H-labeled PIs is presented in which a PI alcohol is esterified with deuterated acetyl chloride. The 2H-labeled PIs, when incorporated into multilamellar membranes composed of phosphatidylcholine, gave rise to 2H NMR powder patterns interpretable in terms of quadrupole splittings (delta vQ) and spin-lattice relaxation times (T1s). Pure isomers of head group 2H-labeled geraniol (C10) and solanesol (C45) gave rise to single splittings while farnesol (C15) gave rise to two sets of splittings due to cis-trans isomerization at the polar terminal double bond. Membranes containing C45 solanesol exhibited a large isotropic component, indicative of limited partitioning of this poly trans PI into the membrane. T1 measurements revealed high rates of motion for PIs relative to cholesterol in similar membrane hosts and revealed correlation times close to the fatty acyl methyl termini in phosphatidylcholine. The smaller PIs showed higher rates of motion but the T1s of head and tail labels were similar. These data indicate that both ends of the esterified PI molecules see similar environments, probably in the bilayer interior, and suggest that the esterified PIs studied here do not appear to adopt a conventional head group-at-interface orientation of lipids within the bilayer.     

Agarose slab-gel electrophoresis equipment.

Simple slab-gel molds which utilize the electrophoresis apparatus described by F. W. Studier (J. Mol. Biol.79, 237 (1973)) have been designed for pouring and running agarose slab-gels. Analytical gels in which many samples are run simultaneously facilitate the assay of many enzymes which lead to physical changes in DNA, whereas the preparative gels allow the separation of large quantities (1–20 mg) of DNA fragments.