Drug modification of protein thiophosphorylation in permeabilized chromaffin cells

Treatment of permeabilized chromaffin cells with low concentrations of the ATP analog adenosine-5′-O-(3-thiotriphosphate)[³⁵S] results in ³⁵S incorporation into a small number of cellular proteins. Of these proteins, a 47 kilodalton protein is most heavily thiophosphorylated. Permeabilized cells were treated with various drugs known to influence cell functions, more specifically chromaffin granule function, to determine the kinase responsible for thiophosphorylation of the 47 kilodalton protein and if its thiophosphorylation is associated with a specific cell function.

Several drugs which influence the activity of cell kinases were examined for their effect on secretion and thiophosphorylation of the 47 kilodalton protein. There was no qualitative effect of cAMP, cGMP or trifluoperazine on thiophosphorylation of the protein. Both cyclic nucleotides slightly enhanced secretion, while trifluoperazine enhanced only unstimulated catecholamine release. Phorbol 12-myristate 13-acetate had no effect on secretion or ³⁵S incorporation into cell proteins. Only the free calcium concentration of the medium influenced thiophosphorylation of the 47 kilodalton protein, with increased calcium producing increased thiophosphorylation.

Drugs affecting chromaffin vesicle functions were used to assess the relationship between specific functions and thiophosphorylation of the protein. Inhibition of nucleotide translocation with atractyloside or 4,4′diisothiocyanatostilbene-2,2′disulfonic acid or inhibition of the proton translocating ATPase by N-ethylmaleimide inhibited thiophosphorylation of the 47 kilodalton protein, with little effect on secretion. Treatment with rotenone markedly enhanced secretion and thiophosphorylation of the protein. Calcium ionophores had no effect on thiophosphorylation of the protein. Dichloroacetic acid, which inhibits phosphorylation of mitochondrial pyruvate dehydrogenase, had no effect on secretion and a variable effect on thiophosphorylation of the 47 kilodalton protein. The data suggest that thiophosphorylation of the protein may be associated with nucleotide translocation across the vesicle membrane.     

Menstrual cycle phase and oral contraceptive effects on triglyceride mobilization during exercise.

We examined the effects of menstrual cycle phase and oral contraceptive (OC) use on triglyceride mobilization during 90 min of rest and 60 min of leg ergometry exercise at 45 and 65% peak O(2) uptake (Vo(2 peak)) in eight moderately physically active, eumenorrheic women (24.8 +/- 1.2 yr). Subjects were tested during the follicular phase (FP) and the luteal phase (LP) before OC use and during the inactive phase (IP) and high-dose phase (HP) after 4 complete mo of OC use. Glycerol rate of appearance (R(a)), a measure of triglyceride mobilization, was determined in a 3-h postabsorptive state using a primed constant infusion of [1,1,2,3,3-(2)H]glycerol. Before OC use (BOC), there were no significant differences between FP and LP in any of the variables studied. Dietary composition, exercise patterns, plasma glycerol concentrations, growth hormone concentrations, and exercise respiratory exchange ratio did not change with OC use. However, 4 mo of OC use significantly (P < 0.05) increased glycerol R(a) in HP during exercise at 45% Vo(2 peak) (6.2 +/- 0.2, 6.5 +/- 0.4, and 7.7 +/- 1.1 micromol.kg(-1).min(-1) for BOC, IP, and HP, respectively) and in IP and HP at 65% Vo(2 peak) (6.6 +/- 0.1, 8.2 +/- 0.6, and 8.1 +/- 0.7 micromol.kg(-1).min(-1) for BOC, IP, and HP, respectively). Plasma cortisol concentrations were significantly higher with OC use at rest and during exercise at 45 and 65% Vo(2 peak). In summary, although fluctuations of endogenous ovarian steroids have little effect on triglyceride mobilization, the synthetic ovarian steroids found in OCs increase triglyceride mobilization and plasma cortisol concentrations in exercising women. We conclude that the hierarchy of effects of ovarian steroids and their analogs on triglyceride mobilization in exercising women is as follows: energy flux > OC use > recent carbohydrate nutrition, menstrual cycle effects.     

Ready-to-use thermally stable mastermix pills for molecular biology applications

Rolling circle amplification (RCA), polymerase chain reaction (PCR), and loop-mediated isothermal amplification (LAMP), are powerful tools that can be used for gene manipulation, pathogen detection, and infectious disease diagnostics. However, these techniques require trained personnel, as the pipetting steps involved can lead to contamination and, consequently, erroneous results. Furthermore, many of the reagents used in molecular biology are thermally labile and must be kept within a cold-chain. In this article, we present a simple and cost-effective method that allows molecular biology reagents to be thermally stabilized into ready-to-use mastermixes via drying in pullulan and trehalose films. Our experimental results demonstrate that this method is capable of preserving the activity of RCA, PCR, LAMP, ligase, polynucleotide kinase, and Klenow fragment mastermixes for at least 3 months at ambient conditions. Thus, stabilizing reagents via drying in pullulan and trehalose film may allow for a drastic reduction in the number of pipetting steps and the elimination of the need for a cold chain. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2764, 2019.