The quantitative histochemistry of multiple sclerosis plaques: acid proteinase and other acid hydrolases.

Abstract— Sensitive micromethods were used to study the plaques, adjacent white matter and remote, grossly normal white matter from two cases of multiple sclerosis and to compare them with white matter from normal controls. Lipid-free dry wt/unit of volume was found to be similar for plaques and for normal white matter, reflecting a high water content of plaque tissue and establishing a base for comparison of enzyme activities. Elevations of acid proteinase in and around plaques were confirmed, but they were far exceeded by the increases in acid phosphatase; other acid hydrolases (β-galactosidase, β-glucuronidase and dipeptidyl arylamidase II) showed no significant or consistent changes. However, an acid lipase-esterase hydrolysing 4-methylumbelliferyl oleate was about 30% as active in plaques as in normal-appearing white matter. Glucose-6-phosphate dehydrogenase was unchanged except in one plaque, but lactic dehydrogenase was markedly elevated both in plaques and adjacent white matter. The grossly normal white matter of MS patients, although histologically far from normal (showing gliosis, perivascular infiltrations and small plaques), did not differ significantly from controls with regard to the activity of any of the enzymes studied. DNA levels were much reduced in plaques, but comparisons were difficult because of the apparent gliosis in normal white matter. Decreases in dry wt/unit vol, reflecting partial demyelination, could be shown to extend in a gradient to a distance of about 2 mm. from the edge of certain plaques.     

The quantitative histochemistry of a chemically induced ependymoblastoma. I. Enzymes

Enzymes from several metabolic pathways were studied quantitatively in homogenates and in homogeneous areas of frozen-dried cryostat sections of an experimental, mouse ependymoblastoma, mouse mammary carcinoma, and mouse melanoma, growing as transplants in mouse brain. Micro analyses were performed in fiveto sixfold replicates on portions of tumour with a dry weight of 0·03-0·2 μg. A close resemblance of the enzyme spectrum of the ependymoblastoma to that of immature brain was noted. Hexokinase and malate dehydrogenase were lower and lactate, glucose-6-phosphate, and NADP⁺-linked isocitrate dehydrogenases, and β-glucuronidase higher in the ependymoblastoma than in whole, adult mouse brain. Mouse mammary carcinoma had a higher level of hexokinase and lower levels of lactate and glucose-6-phosphate dehydrogenases and β-glucuronidase than ependymoblastoma. The concentration of malate dehydrogenase was lower and that of lactate, glucosed-6-phosphate, and NADP⁺-linked isocitric dehydrogenases was higher in the melanoma than in the ependymoblastoma. β-Glucuronidase levels were similar in these two neoplasms. It is suggested that the relatively high levels of several NADP⁺-linked enzymes in the ependymoblastoma may be related to increased capacity for lipid synthesis.     

The aminoacylation of transfer ribonucleic acid. Recognition of methionine by Escherichia coli methionyl-transfer ribonucleic acid synthetase.

The mechanism of the recognition of methionine by Escherichia coli methionyl-tRNA synthetase was examined by a kinetic study of the recognition of methionine analogues in the ATP-PPi exchange reaction and the tRNA-aminoacylation reaction. The results show that the recognition mechanism consists of three parts: (1) the recognition of the size, shape and chemical nature of the amino acid side chain at the methionine-binding stage of the reaction; (2) the recognition of the length of the side chain at the stage of aminoacyl-adenylate complex-formation; (3) the recognition of the sulphur atom in the side chain at the stage of methionyl-tRNA formation. It is proposed that the sulphur atom interacts with the enzyme to induce a conformational change. A model of the active site incorporating the mechanism of methionine recognition is presented.     

The postnatal methylation of transfer ribonucleic acid in brain. Evidence for the methylation of precursor transfer ribonucleic acid.

Incubation of 3-day-old rat brain with L-[methyl-3H]methionine resulted in the rapid labeling of low-molecular-weight cytoplasmic RNA. Electrophoresis in 15% polyacrylamide gels provided evidence for the methylation of precursor tRNA molecules, and high-performance liquid chromatography demonstrated N2-methylguanine to be the predominant methylated base formed during the first 2 min of labelling.     

The dissociation of sigma-factor from ribonucleic acid polymerase.

The sigma-factor of Escherichia coli RNA polymerase was shown to dissociate from the core enzyme as a function of absolute concentration. The association constant is in the range 10(6)-10(8) litre/mol. This implies that the amount of holoenzyme, core enzyme and sigma-factor in RNA polymerase assays may vary according to the absolute concentration of the enzyme.     

Kinetics and template-dependency of ribonucleic acid synthesis by bacterial ribonucleic acid polymerase.

The rate of RNA synthesis catalysed by DNA-dependent RNA polymerase shows a Michealis-Menten-type saturation curve with increasing template concentration. However, the apparent Km is proportional to enzyme concentration, indicating that the reaction does not obey a simple kinetic scheme. The action of inhibitors also indicates a more complex interaction between the enzyme and the DNA template; many inhibitors of RNA synthesis either decrease Vmax. without affecting Km, or increase Km without affecting Vmax. All of these observations can be accounted for quantitatively by a reaction pathway in which the non-specific binding sites of the viral DNA template inhibit competitively the binding of the enzyme to the initiation sites. In terms of this pathway the two classes of inhibitors of RNA synthesis must then act predominantly either on the rate of elongation or on the availability of the binding sites respectively.