Parametric and Nonparametric Analyses of Repeated Ordinal Categorical Data

We compare two models for the analysis of repeated ordinal categorical data: the classical parametric model for means of scores assigned to the categories of the response variable and a nonparametric model based on relative effects derived from the marginal distribution functions of the response. An example in the field of Dentistry is used to illustrate and to compare the models. We also consider a simulation study to evaluate the type‐I error rates and the power of tests under both models in a balanced design setup. The simulation results suggest that both approaches behave similarly for equally spaced scores but may perform differently otherwise. (© 2004 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Zonal variability and seasonal changes of the content of glycogen and glucose in the Mytilus mantle

Glycogen and free-glucose content in the ventral, central and dorsal parts, as well as glucose-6-phosphate phosphatase activity in mantle of Mytilus galloprovincialis Lmk. were examined. The glycogen content of mantle did not manifest asymmetrical distribution among the three parts. In the period studied, the typical glycogen content profile variation was found, being maximum in July. The tissue free-glucose content was similar in each part, and the obtained seasonal variation profile was opposite to the glycogen content, reaching the minimum in July. For every part of mantle, free-glucose/glycogen ratio showed similar monthly profiles. In each part the 50% point was found in July. Glucose-6-phosphate phosphatase activity was not found in the mantle tissue.     

Analysis of polyadenylated RNA from brain synaptosomes and mitochondria

We have isolated RNA from sheep brain synaptosomes and mitochondria separated by an aqueous two-phase system composed of dextran and poly(ethylene glycol). RNA was fractionated through oligo(dT)-cellulose columns and analyzed by electrophoresis through agarose slab gels containing methylmercuric hydroxide and stained with ethidium bromide. The electrophoretic patterns of the poly(A)-containing RNA fraction from synaptosomes and mitochondria are very similar although some high molecular weight RNA species, clearly visible in the synaptosomal fraction, are scarcely detected in the mitochondrial preparations. The electrophoretic analysis of a cleaner RNA preparation from digitonin-treated free mitochondria (mitoplasts) showed that all the poly (A)-RNA species of the synaptosomal preparation are also present in mitoplast. These results strongly suggest that all the discrete poly(A)-RNA species identified in brain synaptosomes are of mitochondrial origin.     

Biosynthesis of platelet-activating factor in glandular gastric mucosa. Evidence for the involvement of the ''de novo'' pathway and modulation by fatty acids.

The biosynthesis of platelet-activating factor (PAF), a phospholipid autocoid with potent ulcerogenic properties that is produced in secretory exocrine glands by physiological secretagogues, was assessed in microsomal preparations of glandular gastric mucosa. For this purpose, 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PAF):acetyl-CoA acetyltransferase (EC 2.3.1.67); the enzymes of the 'de novo' pathway: 1-O-alkyl-2-lyso-sn-glycero-3-phosphate (alkyl-lyso-GP):acetyl-CoA acetyltransferase and 1-O-alkyl-2-acetyl-sn-glycerol (alkylacetyl-G):CDP-choline cholinephosphotransferase (EC 2.7.8.16); and some enzymes involved in the catabolism of PAF and lyso-PAF were assayed. Only the enzymes of the 'de novo' pathway and small amounts of PAF acetylhydrolase, phospholipase A2 and a lysophospholipase D acting on either lipids could be detected in the gastric preparations, whereas lyso-PAF:acetyl-CoA acetyltransferase activity was undetectable. The specific activity of alkyl-lyso-GP:acetyl-CoA acetyltransferase in the gastric mucosa was about one-tenth of that found in spleen microsomes and its apparent Km for acetyl-CoA was 454 microM compared with 277 microM in spleen microsomes. Glandular mucosa homogenates contained preformed PAF at a concentration of 2.7 +/- 0.7 ng equivalents of PAF (hexadecyl)/mg of protein. When gastric microsomes were incubated with micromolar concentrations of fatty acids (arachidonic, palmitic and oleic) prior to the assay of dithiothreitol (DTT)-insensitive cholinephosphotransferase, a dose-dependent reduction in the formation of PAF was observed, arachidonic acid being the most potent inhibitor, followed by linoleic acid (only tested on spleen microsomes) and oleic acid. By contrast, 1,2-diolein and phosphatidylcholine (dipalmitoyl) showed no or little effect. These results indicate that glandular gastric mucosa can produce PAF through the 'de novo' pathway, and that fatty acids, especially unsaturated, can reduce that synthesis by modulating the expression of DTT-insensitive cholinephosphotransferase.     

Modulation of acetyl-CoA:1-alkyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PAF) acetyltransferase in human polymorphonuclears. The role of cyclic AMP-dependent and phospholipid sensitive, calcium-dependent protein kinases

In order to characterize the mechanism of activation of the enzyme 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase (EC 2.3.1.67) which is the limiting step in the regulation of the synthesis of the potent inflammatory mediator 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; homogenates from human polymorphonuclear leukocytes were incubated in the presence of the catalytic subunit of cyclic AMP-dependent protein kinase and in the presence of a partially purified phospholipid sensitive, calcium-dependent protein kinase (PrKC). The first kinase was found to enhance up to 3-fold acetyltransferase activity in a dose- and time-dependent manner. In homogenates from PMN previously stimulated with complement-coated zymosan particles, the decay of acetyltransferase activity was partially prevented by the addition of soybean trypsin inhibitor and almost completely inhibited when the homogenates were supplemented with inhibitors of alkaline phosphatase such as 50 mM KF and 100 microM paranitrophenylphosphate. Under these conditions it was possible to initiate the decay of acetyltransferase activity by adding an excess of alkaline phosphatase. Preincubation of PMN with 12-O-tetradecanoylphorbol-13-acetate previous or simultaneously to the addition of ionophore A23187 reduced the increase in acetyltransferase produced by ionophore A23187, whereas the generation of superoxide anions was enhanced. Addition of partially purified PrKC to homogenates from ionophore A23187-stimulated PMN, reduced acetyltransferase activity by 63%, whereas only a 16% inhibition was observed on homogenates from resting PMN. These data indicate the modulation of acetyltransferase activity in human polymorphonuclear leukocytes by a phosphorylation-dephosphorylation mechanism linked to cyclic AMP-dependent protein kinase. Phospholipid sensitive, calcium-dependent protein kinase seems not to be involved in the mechanism of activation, but, most probably, in the generation of negative activation signals.     

Subcellular localization and glycoprotein nature of the invertase from the fission yeast Schizosaccharomyces pombe

The subcellular localization of the enzyme invertase in Schizosaccharomyces pombe cells, both repressed and derepressed for synthesis of the enzyme, was studied. Most of the invertase was found to be located outside the plasma membrane and only a small percentage was found to be associated to membranes. A substantial portion of the external enzyme remained firmly bound to cell-wall material.All of the invertase recovered in soluble form from cellular extracts reacted with concanavalin A and with the lectin from Bandeiraea simplicifolia seeds, indicating the presence in the enzyme of a carbohydrate moiety which probably contains terminal mannosyl (or structurally related) and galactosyl residues.The possibility of the presence of two different forms of invertase in S. pombe was considered. An intracellular, soluble form of invertase, devoid of carbohydrate, similar to the small invertase of the budding yeast Saccharomyces cerevisiae, was not found in S. pombe. However, the Michaelis constant for sucrose of the enzyme present in repressed cells was smaller than that of the invertase synthesized under derepressing conditions, although this difference could also be the result of a different pattern of glycosylation of the invertase synthesized under different growth conditions.     

Tubulin and tubulin-colchicine complex bind to brain microsomal membrane in vitro

Brain tubulin was labeled in vitro by post-translational incorporation of [14C]-tyrosine or in vivo by intra-cranial injection of [3H]-leucine. The labeled protein was purified by ion-exchange chromatography. After incubating at 37 degrees C with a microsomal membrane preparation from rat brain, part of the labeled soluble tubulin became sedimentable at high-speed centrifugation. This was independent of the native configuration of tubulin, the state of tyrosination of the COOH-terminus, or the presence of 100 microM colchicine in the mixture. In addition, the double-labeled tubulin-colchicine complex obtained from the binding of [3H]-colchicine to [14C]-tyrosinated tubulin, bound to the membrane preparation to the same extent as [14C]-tyrosinated tubulin. The data show that either tubulin or the complex resulting from its binding to colchicine distributed between the soluble and the membrane fractions when mixed at 37 degrees C with a microsome preparation. Seemingly, the site for colchicine binding to tubulin needs not to be free for the protein-membrane association.