Anomeric and other substrate specificity studies with myo-inositol-1-P synthase

L-myo-Inositol-1-phosphate synthase has been found to have at least a 5-fold preference for the beta-anomer of its natural substrate D-Glc-6-P. The alpha-anomer appears to be an inhibitor of the reaction and may be converted to product as well. As well as showing an enzymatic preference for the equatorial C-1 hydroxyl of D-Glc-6-P, our results suggest that it is the pyranose form of D-Glc-6-P that binds to the enzyme and that ring-opening is an enzymatic step. We have also found D-2-dGlc-6-P, D-2-F-2-dGlc-6-P, and D-Man-6-P each to be both competitive inhibitors and substrates that are converted to inositol phosphates by the synthase. D-Allose-6-P is a weak inhibitor of the enzyme, but not a substrate. D-Gal-6-P is neither substrate nor inhibitor. Thus the specificity of the synthase with respect to single position epimers of D-Glc-6-P increases in the order C1 less than C2 much less than C3 less than C4.     

The existence of an optimal range of cytosolic free calcium for insulin-stimulated glucose transport in rat adipocytes

We have examined the effects of extracellular and intracellular Ca2+ concentrations upon basal and insulin-stimulated 2-deoxyglucose uptake in isolated rat adipocytes. In the absence of extracellular Ca2+, both basal and insulin-stimulated glucose uptake were significantly reduced. Insulin-stimulated glucose transport was optimal at 1 and 2 mM Ca2+. Further increases in extracellular Ca2+ concentration (3 mM) significantly diminished insulin-stimulated glucose uptake. When intracellular Ca2+ concentrations were augmented by ionomycin (1 microM), insulin-stimulated glucose uptake was significantly reduced at extracellular Ca2+ concentrations of 2 and 3 mM. The levels of intracellular free Ca2+ concentrations were then measured with Ca2+ indicator fura-2. The correlation between the levels of intracellular free Ca2+ and the magnitude of insulin-stimulated glucose uptake revealed that the optimal effect of insulin is observed at Ca2+ levels between 140 and 370 nM. At both extremes outside of this window, both low and high levels of intracellular Ca2+ result in diminished cellular responsiveness to insulin. These data suggest that intracellular calcium concentrations may exert a dual role in the regulation of cellular sensitivity to insulin. First, there must exist a minimal concentration of intracellular calcium to promote insulin action. Second, increased levels of intracellular calcium may provide a critical signal for diminution of insulin action.     

Genome-wide CRISPR Screens Reveal Host Factors Critical for SARS-CoV-2 Infection

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Environmentally constrained mutation and adaptive evolution in Salmonella

The relationship between environment and mutation is complex [1]. Claims of Lamarkian mutation [2] have proved unfounded [3], [4] and [5]; it is apparent, however, that the external environment can influence the generation of heritable variation, through either direct effects on DNA sequence [6] or DNA maintenance and copying mechanisms [7], [8], [9] and [10], or as a consequence of evolutionary processes [11], [12], [13], [14], [15] and [16]. The spectrum of mutational events subject to environmental influence is unknown [6] and precisely how environmental signals modulate mutation is unclear. Evidence from bacteria suggests that a transient recombination-dependent hypermutational state can be induced by starvation [5]. It is also apparent that chnages in the mutability of specific loci can be influenced by alterations in DNA topology [10] and [17]. Here we describe a remarkable instance of adaptive evolution in Salmonella which is caused by a mutation that occurs in intermediate-strength osmotic environments. We show that the mutation is not ‘directed’ and describe its genetic basis. We also present compelling evidence in support of the hypothesis that the mutational event is constrained by signals transmitted from the external environment via changes in the activity of DNA gyrase.