Receptor-mediated regulation of calcium mobilization and cyclic GMP synthesis in neuroblastoma cells

In neuroblastoma N1E 115 cells, carbachol, histamine and PGE1 elevated cyclic GMP content and, induced the efflux of preloaded 45Ca2+, the release of membrane-bound Ca2+ measured by fluorescent CTC, and the increase in [Ca2+]i as measured by Quin 2 fluorescence. The time course of the responses, the absolute requirement of extracellular Ca2+, the inhibition by receptor blockers, and the concentration dependency on histamine were all similar between these responses. The observation indicates that the mobilization of Ca2+, especially the increase of [Ca2+]i, may be intimately linked to the synthesis of cyclic GMP in the cells.     

Phosphatidylinositol 4,5-bisphosphate: Light-mediated breakdown in the vertebrate retina

Isolated frog ($\text{Rana}\text{Pipiens}$) retinas were labeled in the dark with either [³²P]PO₄-orthophosphate or $\text{myo}$-[2-³H]inositol for 2.5–4 hrs. After washing the retinas with cold buffer, they were exposed to brief flashes of light (5 secs or 15 secs) and their rod outer segments isolated. Upon separation of labeled phospholipids, a specific decrease in label in phosphatidylinositol 4,5-bisphosphate was observed, whereas there was no significant effect on the labeling of phosphatidylinositol 4-phosphate, phosphatidylinositol, or phosphatidic acid. These results are indicative of a light-activated phosphatidylinositol 4,5-bisphosphate-specific phospholipase C in frog rod outer segments.     

Forward mutation assay of V79 cells to 6-thioguanine resistance in a soft agar technique that eliminates effects of metabolic co-operation

A technique involving culture in soft agar was used for the assay of forward mutation of V79 cells to 6-thioguanine (6TG) resistance. The main reason for the use of soft agar was to prevent reduction in recovery of mutants depending on the cell density plated for mutation selection, which is the chief problem in the liquid method, and which results mainly from metabolic co-operation due to cell-to-cell contact.V79 cells grew well in fortified soft agar medium (DMEM + 20% FBS) showing cloning efficiencies (>80%) as high as in liquid culture. Therefore, V79/HGPRT mutagenesis could be assayed quantitatively in soft agar culture.The frequency of 6TG-resistant colonies in agar selective medium increased linearly with increase in concentration of EMS. Toxicity and mutagenic responses were greater in soft agar than in liquid culture.In cultures of untreated and EMS-treated cells, more than 95% of the 6TG-resistant colonies isolated were aminopterin-sensitive.Use of soft agar for selection prevented the reduction in the number of mutants with increase in the size of incula on plating up to 1?2 × 10⁶ cells per 9-cm dish: in liquid culture, even with a lower plating number (2 × 10⁵ cells per 9-cm dish), a notable reduction in numbers of mutants was observed. This character was re-examined in a reconstruction experiment. The results show that, when up to 2 × 10⁶ cells were plated per 9-cm dish, 6TG-resistant cells were almost completely recovered from the soft agar medium, whereas only 10% were recovered from liquid culture.     

Inhibition of ornithine decarboxylase induces embryonal carcinoma cell differentiation

Murine embryonal carcinoma cells can be induced to differentiate in vitro by various physical and chemical means. We report here that inhibition of ornithine decarboxylase activity with a specific enzyme-activated inhibitor, alpha-difluoromethylornithine, can induce differentiation in embryonal carcinoma cells. The differentiated phenotype can be distinguished from undifferentiated embryonal carcinoma cells by altered cellular morphology, biochemical and cell surface antigenic properties. These results suggest that alterations in the levels of cellular polyamines may play a role in embryonal carcinoma cell differentiation.     

Exposure of dog thyroid slices to acetylcholine induces refractoriness to its subsequent stimulation of glucose oxidation

An initial incubation of dog thyroid slices with 0.1 or 1 microM acetylcholine (ACH) for at least 2 h decreases its subsequent stimulation of [1-14C]glucose oxidation. Refractoriness persists for as long as 6 h in the absence of ACH. While new protein synthesis is essential for recovery, it is not necessary for its induction. Refractoriness is prevented when 25 microM tropicamide, an atropine-like drug, is present from the beginning of the initial incubation, but not when it is added after 2 h of incubation of slices with ACH, indicating that at this time ACH is no longer necessary for refractoriness. During refractoriness induced by ACH, stimulation of glucose oxidation by thyroid-stimulating hormone, prostaglandin E1, dibutyryl cyclic AMP, and cholera toxin, but not menadiol, is also significantly diminished. Incubation of thyroid slices with ACH does not modify its stimulation of iodide organification or 32Pi incorporation into phospholipids. These results suggest that the desensitization is not due to changes in the ACH receptor but rather to intracellular metabolic effects. This phenomenon may be important in the regulation of cholinergic effects on the thyroid.     

Trypanosoma cruzi: 4-aminopyrazolopyrimidine in the treatment of experimental Chagas' disease

An allopurinol metabolite, 4-aminopyrazolopyrimidine, was tested on two different strains of mice (NMRI-IVIC and C57Bl/6J) that had been infected 4 days earlier with the virulent Ya strain of Trypanosoma cruzi. Low doses of 4-aminopyrazolopyrimidine (0.125-0.500 mg/kg body wt/day) for 10 days induced a significant reduction in parasitemia (direct counts and subinoculation experiments) and increased survival time (without any evidence of toxicity) compared with untreated animals. When tested in vitro, 4-aminopyrazolopyrimidine was sixfold more active than allopurinol as a trypanostatic drug. The low therapeutic doses of 4-aminopyrazolopyrimidine suggest that this drug may be useful in the treatment of acute Chagas' disease.     

Regulation of insulin receptor kinase activity by insulin mimickers and an insulin antagonist

Three agents which mimic insulin action in intact cells (concanavalin A, wheat germ agglutinin, and polyclonal insulin receptor antibody), mimicked insulin's ability to stimulate the kinase activity of purified insulin receptors. In contrast, monoclonal insulin receptor antibody, an antagonist of insulin action, did not stimulate the phosphorylation of the insulin receptor either in intact IM-9 cells or in purified receptor preparations. This antibody, however, antagonized the ability of insulin to stimulate the phosphorylation of the receptor both in intact cells and in the purified receptor. These studies with insulin mimickers and an insulin antagonist are consistent with a role for the kinase activity of the receptor mediating the actions of insulin.     

Monoclonal antibody-directed immunopurification and identification of cytochromes P-450

A 28 amino acid peptide with diuretic and natriuretic activity has been purified from rat atrial muscle. The primary structure of this atrial peptide is H-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly- (sequence in text) Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-(Arg)-Tyr-OH. The biological activity of this peptide is identical to that of atrial natriuretic factor and cardionatrin I isolated from rat atria.     

Effect of thyrotropin on glycosaminoglycans synthesized by primocultured thyroid cells

The synthesis of glycosaminoglycans (GAGs) was investigated in porcine thyroid cells under the influence or not of thyrotropin. After labelling with [³H] glucosamine and [³⁵S] $\text{SO}{}_4{}^{\mathrm{2?}}$, enriched GAG-fractions prepared from culture media, cells, and eventually substrate adhering materials, were analyzed by cellulose acetate electrophoresis combined with specific degradations. They comprised heparan sulfate and hyaluronic acid together with an unknown sulfated component labile to endo-β-galactosidase. Whereas global labellings of newly made GAGs were not significantly modified by thyrotropin, we reproducibly observed with the hormone a substantial increase in the proportion of hyaluronic acid [³H] label and, when cells organized into follicles, of the proportion of cell-associated [³H] GAGs. This system thus offers an interesting model to study how the responsiveness to an hormone and the reorganization that follows might implicate specific glycoconjugates.     

Ammoniacal silver staining of proteins: mechanism of glutaraldehyde enhancement

When the conditions for detecting proteins by ammoniacal silver staining (B. R. Oakley, D. R. Kirsch, and N. R. Morris (1980) Anal. Biochem. 105, 361-363.) following gel electrophoresis were varied, it was noted that glutaraldehyde pretreatment was necessary for maximal staining, which could not be explained simply as the result of "fixation." Further studies indicated that glutaraldehyde enhancement of protein staining with this silver reagent was probably due to oxidation of the aldehyde groups by silver ions, resulting in metallic silver depositions within the gel which act as nucleation sites for additional metallic silver localization in the protein bands upon the addition of formaldehyde developer. This proposed mechanism is consistent with the Tollen's reaction, as well as some aspects of the photographic process. Consistent with this notion, silver-staining intensities are directly related to mole percentage lysine of various standard proteins.