The effect of retinoic acid on protein phosphorylation in mouse melanoma cells

Vitamin A inhibits growth and increases the activity of cAMP-dependent protein kinase in B16 mouse melanoma cells. In this report we show that retinoic acid (RA) treatment of intact cells alters their subsequent in vitro protein phosphorylation, but we could not demonstrate any changes in in vivo protein phosphorylation. A 48-h treatment with RA results in a concentration-dependent decrease of protein phosphorylation of a 95K molecular weight (MW) protein in both supernatant and particulate fractions. The phosphorylation of this protein does not appear to be regulated by cAMP. Proteins at 92K and 82K MW in the supernatant fraction are increased in phosphorylation. The former (but not the latter) is regulated by cAMP. In the particulate fraction a variety of proteins 12K-68K MW are increased in phosphorylation, as the cells are treated with increasing amounts of RA. The phosphorylation of most of these proteins is regulated by cAMP. Another inhibitor of B16 cell growth, melanocyte-stimulating hormone (MSH) also alters protein phosphorylation. At short incubation periods (1 h), this hormone stimulates phosphorylation of a number of proteins (17-40K MW), while in longer incubation periods (48 h) phosphorylation is inhibited. All of these phosphorylations appear to be regulated by cAMP. We attempted to repeat these observations using intact-cell phosphorylation with 32PO4. In two experiments we saw small changes in the phosphorylation of proteins. In most experiments, however, we could find no change in the phosphoproteins. Further experiments have led us to question the in vivo phosphorylation, since treatment of the cells with MSH, cholera toxin, or db-cAMP also did not affect intact-cell protein phosphorylation. We have previously documented that under these latter conditions cAMP levels are greatly elevated and cAMP-dependent protein kinase is activated. The in vitro phosphorylation results suggests that in RA-treated cells, kinase activities and/or protein substrate levels are changing. However, the physiological significance of the particular MW phosphoproteins changes we have described must await resolution of the in vivo phosphorylation data.     

Context matters: On the road to responsible biosafety technologies in synthetic biology

Biosafety is a major challenge for developing for synthetic organisms. An early focus on application and their context could assist with the design of appropriate genetic safeguards. Subject Categories: Synthetic Biology & Biotechnology, S&S: Economics & Business

One of the goals of synthetic biology is the development of robust chassis cells for their application in medicine, agriculture, and the food, chemical and environmental industries. These cells can be streamlined by removing undesirable features and can be augmented with desirable functionalities to design an optimized organism. In a direct analogy with a car chassis, they provide the frame for different modules or “plug‐in” regulatory networks, metabolic pathways, or safety elements. In an effort to ensure a safe microbial chassis upfront, safety measures are implemented as genetic safeguards to limit risks such as unwanted cellular proliferation or horizontal gene transfer. Examples of this technology include complex genetic circuits, sophisticated metabolic dependencies (auxotrophies), and altered genomes (Schmidt & de Lorenzo, 2016; Asin‐Garcia et al, 2020). Much like seat belts or airbags in cars, these built‐in measures increase the safety of the chassis and of any organisms derived from it. Indeed, when it comes to safety, synthetic biology can still learn from a century‐old technology such as cars about the significance of context for the development of biosafety technologies.Every car today has seat belts installed by default. Yet, seat belts were not always a standard component; in fact, they were not even designed for cars to begin with. The original 2‐point belts were first used in aviation and only slowly introduced for motorized vehicles. Only after some redesign, the now‐common 3‐point car seat belts would become the life‐saving equipment that they are today. A proper understanding of the context of their application was therefore one of the crucial factors for their success and wide adoption. Context matters: It provides meaning for and defines what a technological application is best suited for. What was true for seat belts may be also true for biosafety technologies such as genetic safeguards.

… when it comes to safety, synthetic biology can still learn from a century‐old technology such as cars about the significance of context for the development of biosafety technologies.

Society has a much higher awareness of technology’s risks compared to the early days of cars. Society today requires that technological risks are anticipated and assessed before an innovation or its applications are widely deployed. In addition, society increasingly demands that research and innovation take into account societal needs and values. This has led to, among others, the Responsible Research and Innovation (RRI; von Schomberg, 2013) concept that has become prominent in European science policy. In a nutshell, RRI requires that innovative products and processes align with societal needs, expectations, and values in consultation with stakeholders. RRI and similar frameworks suggest that synthetic biology must anticipate and respond not only to risks, but also to societal views that frame its evaluation and risk assessment.     

A surface net on parasporal inclusions of Bacillus thuringiensis

Protease digestion of parasporal inclusions from several subspecies of Bacillus thuringiensis revealed by electron microscopy a delicate protein net residue on the crystal surfaces. Pretreatment of inclusions with a catechol-ascorbic acid reagent potentiated the subsequent digestion of the crystals and the sharpness of the nets. The net structure maintained the overall shape of the digested crystal as a hexagonally assembled sheet but expanded somewhat from the original crystal size. Each hexagon of the expanded net was some 20 nm in diameter. A mesh size of some 10 nm was occasionally seen in the small amounts of net residue remaining after dissolution of crystals at elevated pH in the presence of thiol reducing agents. The net could also be seen in thin sections and on freeze-etched crystals. Net formation appeared to be associated with the sporulation (stage VI) uptake of cystine. They were rich in hexose (11%) and absent on developing crystals as well as on those in a Sp^? Cry⁺ mutant blocked at stage II of sporulation. Toxicity analyses, with silkworm (Bombyx mori) larvae, indicated that the net residue was more toxic than the protease-digested contents but less toxic than the original crystal. Crystal and net protein showed complete identity against crystal antiserum.     

The effect of retinoic acid on cyclic-AMP-binding proteins in mouse melanoma cells

We have previously reported [(1980) J. Biol. Chem. 255, 5999-6002] that retinoic acid inhibited growth and increased cyclic-AMP-dependent protein kinase activity in mouse melanoma cells. A variant melanoma line having depressed levels of cyclic-AMP-dependent protein kinase was not growth-inhibited by retinoic acid. In this report we describe the effect of retinoic acid on cyclic AMP binding proteins in B16 mouse melanoma cells. Using the technique of photoaffinity labeling, we found three major proteins of Mr 49 000, 52 000, and 55 000 which were specifically labeled with 8-N3-[32P]AMP in both control and treated cells. Based upon their molecular weight, relative affinity for 8-N3-[32P]AMP and comigration with standards, we have designated the 49 000-Mr and 55 000-Mr species as RI and RII respectively. The position of the intermediate band (Mr 52 000) was not affected by pre-incubation with ATP or alkaline phosphatase, and two-dimensional gel analysis indicated that it had the same pI as RI. Retinoic acid increased the 8-N3-[32P]AMP labeling of RI within 24 h, reaching a maximal six fold increase by 48 h. These increases were limited to the 40 000 X g supernatant fraction and occurred prior to any growth inhibition. By using increasing concentrations of 8-N3-cAMP we were able to construct a saturation curve for RI binding. Calculation of apparent Kd values from these curves showed nearly identical affinities for RI binding of 8-N3-cAMP from control and retinoic-acid-treated cells. Therefore we conclude that retinoic acid is increasing the amount of RI rather than altering its properties. Corroboration of these results was obtained by DEAE-cellulose chromatography. Peak I (corresponding to type I protein kinase) from retinoid-treated cells was increased about six fold in binding activity.     

The neuroanatomical basis of central cardiovascular control

A brief review is given of some of the recent neuroanatomical studies of the central autonomic pathways. Two major points are discussed. 1) There are several descending inputs to the intermediolateral cell column that have recently been demonstrated; these include the A5 catecholamine cell group, certain of the raphe nuclei, the nucleus of the solitary tract, the K?lliker Fuse nucleus, and the paraventricular nucleus of the hypothalamus. 2) Certain nuclei of the brain that function as autonomic centers are extensively interconnected: the nucleus of the solitary tract, the parabrachial nucleus, the paraventricular nucleus of the hypothalamus, the central nucleus of the amygdala, and the bed nucleus of the stria terminalis. This network may play an important role in cardiovascular regulation and related neuroendocrine functions.     

Endogenous inhibition of cyclic nucleotide phosphodiesterase in cultured human epithelial cells

We have identified an endogenous inhibitor of cyclic nucleotide phosphodiesterase (PDE) activity in cultured human epithelial cells. The inhibitor was non-dialyzable, inactivated by trypsin and boiling, but stable to a 60° C, 30 min. treatment. Separation of inhibitor from PDE was achieved by blue dextran affinity chromatography. PDE was eluted from this column by EDTA, while the inhibitor remained bound and was subsequently eluted with buffer containing cyclic GMP. The inhibitor was active against PDE from several sources including both Ca⁺⁺ dependent and Ca⁺⁺ independent forms from bovine brain and retina respectively. These characteristics differentiate the PDE inhibitor from human epithelial cells from those previously described from various bovine tissues.