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Quintilian (Marcus Fabius Quintilianus) was a prominent orator and teacher of rhetoric in Rome during the Flavian period. His best-known work is the Institutio Oratoria, “The Orator’s Education,” which presents an idealized curriculum for the training of orators, beginning with their birth and extending through their retirement in old age. It has been treated primarily as a storehouse of information about ancient rhetorical theory and Roman educational practices but is increasingly recognized as a literary work in its own right, marked in particular by Quintilian’s own deployment of the same rhetorical techniques that the Institutio teaches.

The roles of a trimeric GTP-binding regulatory protein, protein kinase A and mitochondria in the regulation of store-activated (thapsigargin-stimulated) Ca2+ inflow in freshly-isolated rat hepatocytes were investigated. Rates of Ca2+ inflow were estimated by measuring the increase in the fluorescence of intracellular fura-2 following the addition of extracellular Ca2+ (Ca2+o) to cells incubated in the absence of added Ca2+o. Guanosine 5ʹ-[γ-thio]-triphosphate (GTP[S]) and AlF4- inhibited the thapsigargin-stimulated Ca2+o-induced increase in cytoplasmic free Ca2+ concentration ([Ca2+]c) and this inhibition was prevented by the Rp diastereoisomer of adenosine 3ʹ,5ʹ-(cyclic)phosphoro[thioate]. cAMP, forskolin and glucagon (half-maximal effect at 10 nM) mimicked inhibition of the thapsigargin-stimulated Ca2+o-induced increase in [Ca2+]c by GTP[S], but had little effect on thapsigargin-induced release of Ca2+ from intracellular stores. Azide and carbonyl cyanide p-trifluoromethoxyphenylhydrazone inhibited the thapsigargin-stimulated Ca2+o-induced increase in [Ca2+]c in the presence of increased cAMP (induced by glucagon). In contrast, Ruthenium Red markedly enhanced the thapsigargin-stimulated Ca2+o-induced increase in [Ca2+]c in both the presence and absence of increased cAMP (induced by forskolin and dibutyryl cAMP). It is concluded that, in hepatocytes, protein kinase A regulates the disposition of Ca2+, which enters the cytoplasmic space through store-activated Ca2+ channels, by directing some of this Ca2+ to the mitochondria. The idea that caution should be exercised in using observed values of Ca2+o-induced increase in [Ca2+]c as estimates of rates of agonist-stimulated Ca2+ inflow is briefly discussed.

The roles of a monomeric GTP-binding regulatory protein in the activation of store-activated plasma membrane Ca2+ channels and in the release of Ca2+ from the smooth endoplasmic reticulum (SER) in rat liver parenchymal cells were investigated with the use of freshly isolated rat hepatocytes and rat liver microsomes. A low concentration (approx. 130 microM intracellular) of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) activated Ca2+ inflow in intact hepatocytes in the absence of an agonist, whereas a high concentration (approx. 530 microM intracellular) of GTP-S- or guanosine 5'-[betagamma-imido]triphosphate (p[NH]ppG) inhibited the Ca2+ inflow induced by inhibitors of the activity of the endoplasmic-reticulum Ca2+-ATPase (SERCA) and by vasopressin. GTP (530 microM) prevented the inhibition of Ca2+ inflow by GTP-S- and p[NH]ppG. Brefeldin A and the peptide human Arf-1-(2-17), which inhibit many functions of ADP ribosylation factor (Arf) proteins, inhibited the Ca2+ inflow induced by SERCA inhibitors and vasopressin, and altered the profile of Ca2+ release from the SER. These effects were observed at concentrations of Brefeldin A and Arf-1-(2-17) comparable with those that inhibit the functions of Arf proteins in other systems. Succinylated Arf-1-(2-17) had a negligible effect on Ca2+ inflow. GTP[S] and Arf-1-(2-17) completely inhibited the synergistic action of GTP and Ins(1,4,5)P3 in releasing 45Ca2+ from rat liver microsomes loaded with 45Ca2+. AlF4(-) (under conditions expected to activate trimeric G-proteins) and succinylated Arf-1-(2-17) had no effect on GTP/Ins(1,4,5))3-induced 45Ca2+ release, and a mastoparan analogue caused partial inhibition. Arf-1-(2-17) did not inhibit 45Ca2+ release induced by either thapsigargin or ionomycin. It is concluded that a low-molecular-mass G-protein, most probably a member of the Arf protein family, is required for store-activated Ca2+ inflow in rat hepatocytes. The idea that the role of this G-protein is to maintain a region of the SER in the correct intracellular location is discussed briefly.

In common with the F1-ATPase from other sources, yeast mitochondrial F1-ATPase was inhibited by 4-chloro-7-nitrobenzofurazan. Total inhibition of the F1-ATPase activity was compatible with the modification of a single tyrosine residue per F1-ATPase molecule. Radioactive labelling experiments localized this modification on a β-subunit. The inactive modified enzyme retained the capacity to bind the photoaffinity label 8-azido-1,N6-etheno-ATP, which has previously been shown to bind nucleotide sites of low affinity. As well, the inactive modified enzyme bound MgATP with high affinity, yielding a Kd of 14 μM. The results are consistent with the hypothesis of alternating, or cooperative, site catalysis by F1-ATPase.

Mitochondrial ATP synthase from yeast is an oligomeric enzyme consisting of a hydrophobic membrane part (Fe) and a hydrophilic part (Fr). Fr is composed of 5 different polypeptide chains (o to c) [Il. Lowand high-affinity nucleotide diand triphosphate binding sites have been characterized on the enzyme from various sources by different kinetic techniques [2,3]. Alternatively, binding sites have been located on specific subunits by photoaffinity labelling [4-81. Recently, the development of a fluorescent photoaffinity label (8-azido-I ,Ne-ethenoATP) was reported [9], which might allow the identification of a binding site with respect to the hydrolytic or synthetic function of Fr . We have therefore investigated the interaction of this compound with yeast Fr . It will be shown that the label interacts with a low-affinity nucleotide binding site, and that upon photolabelling it is bound to the P-subunit.