Fluorometric determination of histamine with OPT: optimum reaction conditions and tests of identity

Histamine reacts with OPT at an alkaline pH giving fluorescent conjugation products. Optimum fluorophore formation was observed at pH 12.5 after 40 min reaction at 0°C under continuous gassing with nitrogen. After acidification with sulfuric acid to pH 2–5 the fluorescence was stable for hours. Reagent blanks were reduced by the lowering of the reaction temperature and by decreasing the amount of OPT added. These modifications permitted the determination of 2 ng histamine/ml and gave far better reproducibility than the original procedure of Shore, Burkhalter, and Cohn. The fluorometric assay for histamine is nonspecific; the major interfering OPT-reactive tissue component is believed to be spermidine. Specificity was secured by adding formaldehyde before acidification, thus abolishing the fluorescence of histamine but not that of spermidine, or by adding CdCl₂ or SrCl₂ together with OPT, thus preventing the formation of the spermidine fluorophore but not that of the histamine fluorophore.     

Muscarinic acetylcholine receptor activation causes inhibition of cyclic AMP accumulation, prolactin and growth hormone secretion in GH3 rat anterior pituitary tumour cells

Acetylcholine, oxotremorine and carbachol, compounds that exhibit muscarinic agonist activity, maximally inhibited basal prolactin secretion from GH3 cells by approx. 50% and intracellular cyclic AMP levels by approx. 20%. Both parameters were inhibited with similar potencies by each agonist. These inhibitory effects were blocked by a muscarinic but not by a nicotinic receptor antagonist. In the presence of VIP or IBMX, which raise intracellular cyclic AMP levels and stimulate hormone release, the degree of muscarinic inhibition was increased, but the potency remained unchanged. Similar changes in the secretory rate of prolactin and growth hormone occurred in these and in cell perifusion experiments. These results suggest that the inhibition of hormone secretion from GH3 cells by muscarinic agonists is mediated by a decrease in intracellular cyclic AMP levels.     

The binding of 1,N6-ethenoNAD to bovine liver glutamate dehydrogenase: studies using the time-correlated single photon counting fluorescence technique

The time-correlated single photon counting (TCPC) fluorescence technique has been used as a novel approach to investigate ligand-protein interaction, for the case of the binding of the fluorescent coenzyme analogue 1,N6-ethenoNAD (epsilon NAD) to bovine liver glutamate dehydrogenase in the presence of glutarate, a substrate analogue which stabilizes the complex. System calibration was performed using solutions of epsilon ADP and carefully purified epsilon NAD mixed at variable molar ratios (pH 7.0, 0.05 M sodium phosphate buffer, 20 degrees C). The fluorescence lifetimes obtained after deconvolution were 2.4 ns (for epsilon NAD) and 23 ns (for epsilon ADP), in good agreement with literature values obtained under similar conditions. epsilon NAD binds to glutamate dehydrogenase in the presence of 50 mM glutarate, with a fluorescence quantum yield enhancement factor, Q, of about 17-fold, as previously reported (Favilla, R. and Mazzini, A. (1984) Biochim. Biophys. Acta 48-57). For this system, fluorescence lifetime values were obtained after deconvolution as 2.4 ns for free epsilon NAD and 21 ns for bound epsilon NAD. These values did not vary appreciably with enzyme concentration nor with degree of saturation, thus reflecting the existence of only one spectroscopically relevant type of complex. Addition of either GTP or ADP did not affect the lifetime of epsilon NAD bound to the enzyme, but only its affinity, thus allowing calculations of binding strengths. In the case of a simple binding (i.e., in the absence of GTP) the dissociation constant of the complex could be derived from a simple relationship, in which only the ratio between the pre-exponential factors and the parameter gamma, which represents the molar fraction of epsilon NAD molecules free in solution in the open conformation, are to be taken into account. The results are in good agreement with those reported by some of us (reference above) using a steady-state fluorescence technique, which by itself is, however, unable to resolve the number of relevant species present in the system.     

Degradation of aromatic amino acids by Rhodococcus erythropolis

A Gram-positive Rhodococcus erythropolis strain S1 was shown to assimilate aromatic amino acids such as L-phenylalanine, L-tyrosine, L-tryptophan, D-phenylalanine, D-tyrosine and D-tryptophan, which were utilized not only as the sole carbon source but also as a suitable nitrogen source. The highest growth on these aromatic amino acids occurred at a temperature of 30°C. L-Phenylalanine, L-tyrosine and L-tryptophan degradative pathways would appear to be independent, and to be induced alternatively. The strain S1 also showed the ability to assimilate peptides which consisted of only L-phenylalanine and L-tyrosine.     

Macrophage catabolism of lipid A is regulated by endotoxin stimulation

Lipopolysaccharide (LPS) is a Gram-negative bacterial glycolipid that is believed to cause, by virtue of its stimulatory actions on macrophages and other eukaryotic cells, the life-threatening symptoms associated with Gram-negative infections. Macrophages both respond to and catabolically deactivate LPS. The lipid A moiety of LPS is responsible for the stimulatory actions of LPS on macrophages. We have previously developed methods employing a radiolabeled bioactive lipid A precursor, 4'-32P-lipid IVA, to study the interaction of this class of lipids with animal cells (Hampton, R. Y., Golenbock, D. T., and Raetz, C. R. H. (1988). J. Biol. Chem. 263, 14802-14807). In the current work, we have examined the uptake and catabolism of 4'-32P-lipid IVA by the RAW 264.7 cell line in serum-containing medium at physiological temperatures and have studied the effect of LPS stimulation on the ability of these cells to catabolize lipid IVA. RAW 264.7 macrophage-like cells avidly take up 4'-32P-lipid IVA under cell culture conditions at nanomolar concentrations. Uptake of lipid IVA was accompanied by lysosomal dephosphorylation of a fraction of the lipid to yield 4'-monophosphoryl lipid IVA. Chemically generated 4'-monophosphoryl lipid IVA was found to be substantially less bioactive than lipid IVA in the RAW cell, indicating that this catabolic dephosphorylation results in detoxification. In uptake experiments of 3-4 h duration, all metabolism of lipid IVA is blocked by ligands of the macrophage scavenger receptor. In longer experiments (24 h), both scavenger receptor-dependent and -independent uptake are responsible for the lysosomal catabolism of lipid IVA. Preincubation of RAW 264.7 cells with LPS caused dose-dependent inhibition of lipid IVA dephosphorylation. Sufficient LPS stimulation resulted in essentially complete inhibition of lipid IVA catabolism in both short- and long-term uptake experiments. This effect occurred at physiologically relevant concentrations of LPS (IC50 less than 1 ng/ml), and our data indicate that LPS-induced blockade of lipid IVA catabolism was due to the resultant physiological stimulation of the cells, and not inhibition of dephosphorylation by competition for uptake or enzymatic sites or by simple sequestration of labeled lipid IVA by LPS aggregates. We suggest that in the macrophage, LPS can modulate its own catabolism by virtue of its pharmacological properties. This effect of LPS could play a role in LPS pathophysiology as well as in macrophage biology.     

The Energy Expenditure of Stair Climbing One Step and Two Steps at a Time: Estimations from Measures of Heart Rate

Stairway climbing provides a ubiquitous and inconspicuous method of burning calories. While typically two strategies are employed for climbing stairs, climbing one stair step per stride or two steps per stride, research to date has not clarified if there are any differences in energy expenditure between them. Fourteen participants took part in two stair climbing trials whereby measures of heart rate were used to estimate energy expenditure during stairway ascent at speeds chosen by the participants. The relationship between rate of oxygen consumption () and heart rate was calibrated for each participant using an inclined treadmill. The trials involved climbing up and down a 14.05 m high stairway, either ascending one step per stride or ascending two stair steps per stride. Single-step climbing used 8.5±0.1 kcal min⁻¹, whereas double step climbing used 9.2±0.1 kcal min⁻¹. These estimations are similar to equivalent measures in all previous studies, which have all directly measured The present study findings indicate that (1) treadmill-calibrated heart rate recordings can be used as a valid alternative to respirometry to ascertain rate of energy expenditure during stair climbing; (2) two step climbing invokes a higher rate of energy expenditure; however, one step climbing is energetically more expensive in total over the entirety of a stairway. Therefore to expend the maximum number of calories when climbing a set of stairs the single-step strategy is better.