Green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its homologs from diverse marine animals are widely used as universal genetically encoded fluorescent labels. Many laboratories have focused their efforts on identification and development of fluorescent proteins with novel characteristics and enhanced properties, resulting in a powerful toolkit for visualization of structural organization and dynamic processes in living cells and organisms. The diversity of currently available fluorescent proteins covers nearly the entire visible spectrum, providing numerous alternative possibilities for multicolor labeling and studies of protein interactions. Photoactivatable fluorescent proteins enable tracking of photolabeled molecules and cells in space and time and can also be used for super-resolution imaging. Genetically encoded sensors make it possible to monitor the activity of enzymes and the concentrations of various analytes. Fast-maturing fluorescent proteins, cell clocks, and timers further expand the options for real time studies in living tissues. Here we focus on the structure, evolution, and function of GFP-like proteins and their numerous applications for in vivo imaging, with particular attention to recent techniques.

Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues

Many types of cells methylate phospholipids using two methyltransferase enzymes that are asymmetrically distributed in membranes. As the phospholipids are successively methylated, they are translocated from the inside to the outside of the membrane. When catecholamine neurotransmitters, lectins, immunoglobulins or chemotaxic peptides bind to the cell surface, they stimulate the methyltransferase enzymes and reduce membrane viscosity. The methylation of phospholipids is coupled to Ca2+ influx and the release of arachidonic acid, lysophosphatidylcholine, and prostaglandins. These closely associated biochemical changes facilitate the transmission of many signals through membranes, resulting in the generation of adenosine 3',5'-monophophate in many cell types, release of histamine in mast cells and basophils, mitogenesis in lymphocytes, and chemotaxis in neutrophils.

https://science.sciencemag.org/content/sci/209/4461/1082.full.pdf

Phospholipid methylation and biological signal transmission

Anthrax toxin is composed of three proteins: protective antigen (PA), lethal factor (LF), and edema factor (EF). These proteins individually cause no known physiological effects in animals but in pairs produce two toxic actions. Injection of PA with LF causes death of rats in 60 min, whereas PA with EF causes edema in the skin of rabbits and guinea pigs. The mechanisms of action of these proteins have not been determined. It is shown here that EF is an adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] produced by Bacillus anthracis in an inactive form. Activation occurs upon contact with a heat-stable eukaryotic cell material. The specific activity of the resulting adenylate cyclase nearly equals that of the most active known cyclase. In Chinese hamster ovary cells exposed to PA and EF, cAMP concentrations increase without a lag to values about 200-fold above normal, remain high in the continued presence of toxin, and decrease rapidly after its removal. The increase in cAMP is completely blocked by excess LF. It is suggested that PA interacts with cells to form a receptor system by which EF and perhaps LF gain access to the cytoplasm.

https://www.pnas.org/content/pnas/79/10/3162.full.pdf

Anthrax toxin edema factor: a bacterial adenylate cyclase that increases cyclic AMP concentrations of eukaryotic cells.

http://www.biblioteca.uma.es/bbldoc/articulos/16730379.pdf

Mutation in the follicle-stimulating hormone receptor gene causes hereditary hypergonadotropic ovarian failure

To investigate whether polymorphic forms of the human dopamine D4 receptor have different functional characteristics, we have stably expressed cDNAs of the D4.2, D4.4, and D4.7 isoforms in several cell lines. Chinese hamster ovary CHO-K1 cell lines expressing D4 receptor variants displayed pharmacological profiles that were in close agreement with previous data from transiently expressed D4 receptors in COS-7 cells. Dopamine stimulation of the D4 receptors resulted in a concentration-dependent inhibition of the forskolin-stimulated cyclic AMP (cAMP) levels. The potency of dopamine to inhibit cAMP formation was about twofold reduced for D4.7 (EC50 of approximately 37 nM) compared with the D4.2 and D4.4 variants (EC50 of approximately 16 nM). Antagonists block the dopamine-mediated inhibition of cAMP formation with a rank order of potency of emonapride > haloperidol = clozapine >> raclopride. There was no obvious correlation between the efficacy of inhibition of forskolin-stimulated cAMP levels and the D4 subtypes. Dopamine could completely reverse prostaglandin E2-stimulated cAMP levels for all three D4 receptor variants. Deletion of the repeat sequence does not affect functional activity of the receptor. The data presented indicate that the polymorphic repeat sequence causes only small changes in the ability of the D4 receptor to block cAMP production in CHO cells.

Modulation of Intracellular Cyclic AMP Levels by Different Human Dopamine D4 Receptor Variants

Radioimmunoassay of cyclic AMP and cyclic GMP.

https://science.sciencemag.org/content/sci/194/4262/270.full.pdf

Gammaflow: a completely automated radioimmunoassay system

Involvement of cyclic amp and protein synthesis in catecholamine refractoriness.

Ornithine decarboxylase (ODCase; L-ornithine carboxy-lyase; EC 4.1.1.17) activity was increased 5-20 fold above basal activity by N6,O2′-dibutyryl cyclic AMP, isoproterenol, epinephrine, or fetal calf serum in confluent C6-2B rat astrocytoma cells. Serum increased ODCase activity by a mechanism apparently independent of cyclic AMP because it decreased intracellular cyclic AMP. Calcium ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) buffers (pCa = 6.4) reduced basal ODCase activity and blocked the increase in activity by β-adrenergic agonists, dibutyryl cyclic AMP, or serum. Readdition of calcium to pCa = 4.0 restored the ability of the cells to respond to these agents. The rise in ODCase activity stimulated by isoproterenol, dibutyryl cyclic AMP, or serum was also blocked by the microtubule disrupting agents, vinblastine and colchicine, and by the microfilament disrupting agent, cytochalasin B. Lumicolchicine, an analog of colchicine that does not disrupt microtubules, was ineffective. The microtubule and microfilament disrupting agents and EGTA had no effect on the intracellular accumulation of cyclic AMP. Cycloheximide inhibited protein synthesis >95% within 30 min, and caused an immediate decline of stimulated ODCase activity with a half-time of 20-30 min. The inhibition of ODCase activity by colchicine or EGTA was distinct from that seen with cycloheximide and could not be correlated to their effects on general protein synthesis. Colchicine or EGTA each caused identical rates of decline in ODCase activity with a half-time of 20-30 min after an initial lag period of about 60 min.

Calcium and microtubule dependence for increased ornithine decarboxylase activity stimulated by β-adrenergic agonists, dibutyryl cyclic AMP, or serum in a rat astrocytoma cell line

The aminobenzyl analog of propranolol, 1- (p-amino-alpha,alpha-dimethylphenethylamino)-3-(1-naphthoxy)-2- propanol, was synthesized and found to be a potent beta-adrenergic blocking agent. The beta-adrenergic receptors of cultured rat C6 glioma cells (2B clone) as assessed by [(125)I]iodohydroxybenzylpindolol binding were decreased 50 and >95% after pretreatment with 8 nM and 1 muM aminobenzylpropranolol, respectively. Unlike propranolol, aminobenzylpropranolol displayed a prolonged blockade of receptors that was maintained during several hours of washing. [(125)I]Iodohydroxybenzylpindolol saturation binding experiments in cells exposed to aminobenzylpropranolol and subsequently washed indicated that the compound effectively diminished receptor number with no change in the affinity of the remaining receptors for iodohydroxybenzylpindolol. Aminobenzylpropranolol inhibited catecholamine-stimulated intracellular cyclic AMP accumulation; with increasing blockade, isoproterenol dose-response curves became progressively shifted to the right but the maximal response was unaltered. Aminobenzylpropranolol inhibited the beta-adrenergic contractile response in atria isolated from rats and guinea pigs. Treatment with 0.1 and 10 muM aminobenzylpropranolol produced decreases of 0.5 and 2 orders of magnitude in the contractile potency of isoproterenol. As in glioma cells, aminobenzylpropranolol failed to decrease the maximal response to isoproterenol. The effects of aminobenzylpropranolol persisted during extensive washing of atria (up to 17 hr). Repeated exposures to isoproterenol at concentrations sufficient to produce maximal tension development also failed to alleviate the blockade. The inotropic potency of histamine in guinea pig atria was not affected by aminobenzylpropranolol. These data suggest that catecholamines are capable of eliciting full biological responses in glioma cells and isolated atria even though the great majority of beta-adrenergic receptors are persistently blocked.

Wesley L. Terasaki

Papers

Radioimmunoassay of cyclic AMP and cyclic GMP.

Gammaflow: a completely automated radioimmunoassay system

Involvement of cyclic amp and protein synthesis in catecholamine refractoriness.

Calcium and microtubule dependence for increased ornithine decarboxylase activity stimulated by β-adrenergic agonists, dibutyryl cyclic AMP, or serum in a rat astrocytoma cell line

Quantitative Relationship between β -adrenergic Receptor Number and Physiologic Responses as Studied with a Long-Lasting β -adrenergic Antagonist