Effect of Triton WR-1339 on lecithin-cholesterol acyltransferase in the rat

The effect of Triton WR-1339 on activity of lecithin-cholesterol acyltransferase was measured in rat serum following addition of Triton to the serum in vitro or after intravenous injection of the detergent. The inhibitory effect of Triton WR-1339 on activity of lecithin-cholesterol acyltransferase when the detergent was added in vitro was dose dependent and appeared to result from a direct action on the enzyme rather than from a physical modification of the substrate by the detergent. The serum half-life ($\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) of Triton WR-1339 injected intravenously in the rat was 23.1 ± 1.0 h. The inhibitory effect of Triton on serum LCAT activity when the detergent was given intravenously was also dose dependent and was reversed when the serum concentration of Triton decreased; under specific conditions, LCAT activity reached values higher than control. This behavior after treatment of the animal may be explained by increased concentration of the enzyme in the plasma, by stimulation of LCAT activity by the very low density lipoprotein or metabolites accumulating in the plasma of rats treated with Triton WR-1339, or by a combination of these factors.     

Sedimentation velocity studies on microgram quantities of rat intestinal goblet cell mucin.

A procedure is outlined by which sedimentation analyses of small quantities of mucin glycoproteins can be performed. Rat intestinal goblet cell mucin was stained with periodic acid-Schiff reagent to permit detection by light absorption at 555 nm. PAS treatment resulted in chemical modification of sialic acid and 55% of fucose residues in the mucin. No other chemical or physical alterations were detected. The stained mucus was subjected to band ultracentrifugation using D₂O-containing solvents. Sedimentation was monitored by scanning at 555 nm. Results compared favorably with those reported earlier for conventional boundary ultracentrifugation of intact goblet cell mucin. Because of the low concentrations of mucin used in band ultracentrifugation (0.2–1.5 μg protein/ml), S_20,w values are comparable to sedimentation coefficients at zero concentration (S^o values), determined by conventional means.     

Amylase secretion by rabbit parotid gland role of cyclic AMP and cyclic GMP

Amylase secretion and changes in the levels of cyclic AMP and GMP were studied in rabbit parotid gland slices incubated in vitro with a variety of neurohumoral transmitters, their analogs and inhibitors. Cyclic GMP levels increased 8-fold 5 min after exposure to carbachol (10⁻⁴ M), without a change in cyclic AMP levels; amylase output also rose. These effects were completely inhibited by muscarinic blockade with atropine, but were unaffected by α-adrenergic blockade with phenoxybenzamine. Epinephrine (4 · 10⁻⁵ M) produced a rapid increase in the levels of both cyclic nucleotides and in amylase release. The increase in cyclic GMP level was inhibited by previous exposure of the slices to phenoxybenzamine, while the cyclic AMP rise was prevented by the β-blocking agent, propranolol. Pure α-adrenergic stimulation with methoxamine (4 · 10⁻⁴ M) produced modest elevations in cyclic GMP content and amylase output, effects blocked by pre-treatment of slices with either atropine or phenoxybenzamine. At a concentration of 4 · 10^{−6 M}, isoproterenol (a β-agonist) failed to affect cyclic GMP levels, but promptly stimulated increases in cyclic AMP levels, and after a short lag, amylase secretion. At a higher dose (4 · 10⁻⁵ M) isoproterenol produced elevations in the levels of both nucleotides. The carbachol-induced effects on cylcic GMP content and amylase release were greatly potentiated by the addition of isoproterenol (4 · 10⁻⁶ M).These data strongly suggest that cholinergic muscarinic agonists and α-adrenergic agonist stimulate amylase output in rabbit parotid gland by mechanisms involving cyclic GMP. The atropine-sensitive intracellular events effected by α-stimulation may be dependent upon endogenous generation of acetylcholine. Both cyclic nucleotides seem to be required for the early rapid secretion of amylase. The unique responses achieved by the combination of carbachol and isoproterenol suggest that isoproterenol may increase the sensitivity of this issue to the effects of cholinergic stimuli.     

An automatic enzymatic determination of glycogen in a continuous-flow system without interference of free glucose.

An autoanalyzer system for enzymatic determination of glycogen is described. Free glucose is eliminated by hexokinase/glucose-6-phosphate dehydrogenase. Glycogen is hydrolyzed by amylo-1,6-glycosidase at pH 4,8, and the resulting glucose is dialyzed and determined by the hexokinase/glucose-6-phosphate dehydrogenase reaction.     

Electrophysiology of phagocytic membranes II. Membrane potential and induction of slow hyperpolarizations in activated macrophages

The potential differences measured on the cell surface and after penetration into the cytoplasm of activated macrophages are described. Linear regressions are made of the measured potential differences as functions of the tip potential of each microelectrode. The surface potential of the macrophage is not significantly different from zero.Mouse macrophages have a transmembrane potential of ?26 mV, whereas in guinea-pig cells this value is ?18 mV. The input resistances of guinea-pig cells are higher than those of mouse macrophages. The cytoplasmic location of the electrode was characterized both by fluorescent dye injection and by electric criteria.Slow membrane hyperpolarizations are directly elicited by mechanical stimulation. Electric responses evoked by current pulses were further characterized.Our results lead to the establishment of objective criteria to validate intracellular recordings from macrophage.     

Reactivation of herpes simplex virus in a cell line inducible for simian virus 40 synthesis

The reactivation of UV-irradiated herpes simplex virus (HSV) was investigated in irradiated and unirradiated transformed hamster cells in which infectious simian virus 40 (SV40) can be induced. Reactivation was enhanced when the cells were treated with UV light or mitomycin C prior to infection with HSV. The IV dose-response curve of this enhanced reactivation was strikingly similar to that found for induction of SV40 virus synthesis in cells treated under identical condictions. This is the first time that two SOS functions described in bacteria have been demonstrated in a single mammalian cell line.     

Mutagenicity testing in mammalian cells: I. Derivation of a chinese hamster ovary cell line heterozygous for the adenine phosphoribosyltransferase and thymidine kinase loci

As a first step in the development of a multiple-marker, mammalian cell mutagenesis assay system, we have isolated a Chinese hamster ovary (CHO) cell line that is heterozygous for both the adenine phosphoribosyltransferase (aprt) and thymidine kinase (tk) loci. Presumptive aprt^+/? heterozygotes with intermediate levels of APRT activity were selected from unmutagenized CHO cell populations on the basis of resistance to low concentrations of the adenine analog, 8-azaadenine. A functional aprt^+/? heterozygote with ～50% wild-type APRT activity was subsequently used to derive sublines that were also heterozygous for the tk locus. Biochemical and genetic characterization of one such subline, CHO-AT3-2, indicated that it was indeed heterozygous at both the aprt and tk loci. CHO-AT3-2 cells permitted single-step selection of mutants resistant to 8-azaadenine or 5-fluorodeoxyuridine, allowing quantitation and direct comparison of mutation induction at the autosomal aprt or tk loci, as well as in the gene involved in ouabain resistance or at the X-linked, hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus. Significant dose-dependent increases in mutation frequency were observed for all 4 genetic markers after treatment of CHO-AT3-2 cells with ethyl methanesulfonate.     

Bicarbonate measurements in rat liver, brain, heart, and skeletal muscle.

A method is described for measuring total CO₂ and HCO₃^? in tissues rapidly frozen in liquid nitrogen. The method is a modification of the procedure of D. D. Van Slyke and J. M. Neill (1924, J. Biol. Chem.61, 523–573) for use with freeze-clamped tissue where anoxic changes have not occurred. The HCO₃^? content exclusive of tissue CO₂ in fed rats was found to be: liver, 19.4 ± 1.0; brain, 20.2 ± 0.9; thigh, 16.2 ± 0.8; and heart, 15.4 ± 1.4 μmol/g.     

Isolation and characterization of plamsa membrane from monolayer cultures of epitherlial type II lung cells.

Pneumocyte type II produces a phospholipid, dipalmitoyl lecithin, which is stored in and secreted from the cell's inclusion bodies and is indispensable for alveolar stability. Cloned rat lung type II cells were harvested at monolayer confluence and homogenized in swelling buffer. After sequential differential centrifugations, the crude membrane fraction was subjected to discontinuous sucrose density gradient centrifugation at 65,000 × g. Quality of the relevant fractions was monitored by enzyme activities and phase contrast and electron microscopy of two major bands at densities 1.16 and 1.18, respectively. The less dense band contained only small quantities of organelles, little cytochrome c oxidase, and some glucose 6-phosphatase, but had a significant (Na⁺, K⁺)-ATPase activity; this and ultrastructural evidence certified the product as a suitable plasma membrane preparation. Upon sodium dodecyl sulfate-poly acrylamide gel electrophoresis, the protein pattern consisted of 11 major protein bands between 13,000 and 68,000 M_r ranges, and several minor ones. The lipid pattern was studied by two-dimensional thin layer chromatograpy, followed by various group reactions (e.g., amine, unsaturation, phosphorus, sugars). In the two major phospholipids, phosphatidyl choline and phosphatidyl ethanolamine, palmitic acid was the least abundant of four major fatty acids, accounting for 14.20% in phosphatidyl choline and 5.70% in phosphatidyl ethanolamine, whereas the most abundant were stearic and palmitoleic with about 28% each in phosphatidyl choline, and palmitoleic (29.90%) and oleic (23.05%) in the ethanolamine phosphatide. Apparently, the palmitic acid containing phosphatidyl choline must be in the lamellar inclusion bodies of type II cells and not in their plasma membranes.