Comparison of subcellular activation of the human neutrophil NADPH-oxidase by arachidonic acid, sodium dodecyl sulfate (SDS), and phorbol myristate acetate (PMA)

The phorbol myristate acetate (PMA) stimulation of the human neutrophil NADPH-oxidase has been demonstrated through the activation of protein kinase C (PK-C), using light membrane fractions from nitrogen-cavitated cells. Both arachidonic acid (AA) and sodium dodecyl sulfate (SDS) can also generate an active oxidase in cellfree systems. That the source of O2- with AA and SDS activation is the same NADPH-oxidase as previously studied was confirmed by the similar pH optima and Km values for NADPH as those previously described for the O2- -generating activity harvested from pre-stimulated human neutrophils. In contrast to the stimulation by PMA, however, the stimulation of the NADPH-oxidase by AA and SDS does not appear to require protein kinase C activation: the action of AA and SDS is independent of the addition of PK-C cofactors to the system, and the inhibitor of PK-C activity, H-7, had no effect on the stimulation by AA or SDS. AA and SDS activation are comparable, but the level of NADPH-oxidase expression is sixfold greater with each of these agents than that obtained with a reconstituted PK-C system. The basis of this difference in oxidase expression is unclear, but these findings suggest strongly that although activated PK-C is capable of stimulating a dormant NADPH-oxidase in a cellfree system, this is not the sole pathway for oxidase activation.     

DNA restriction-site polymorphisms associated with the alpha 1-antitrypsin gene.

Restriction-site variation in and around the alpha 1-antitrypsin gene has been studied using two genomic probes. With use of restriction enzymes SstI, MspI, and AvaII, three polymorphic sites have been described with a 4.6-kb probe in the 5' portion of the gene. With use of a 6.5-kb probe, polymorphisms in the coding and 3' regions of the gene have been detected with AvaII, MaeIII, and TaqI. All of these polymorphisms are of sufficiently high frequency to be useful in genetic mapping studies. The polymorphisms with AvaII and MaeIII (6.5-kb probe) are particularly useful for prenatal diagnosis. PI types and M subtypes tend to be associated with specific DNA haplotypes; there are two different types of DNA haplotypes associated with PI M1. The extent of linkage disequilibrium differs throughout the region of the alpha 1-antitrypsin gene.     

Mechanism of calmodulin inhibition of cAMP-dependent protein kinase activation of phosphorylation kinase

The activation of phosphorylase kinase (EC 2.7.1.38; ATP:phosphorylase b phosphotransferase) by the catalytic subunit of cAMP-dependent protein kinase (EC 2.7.1.37; ATP:protein phosphotransferase) is inhibited by calmodulin. The mechanism of that inhibition has been studied by kinetic measurements of the interactions of the three proteins. The binding constant for calmodulin with phosphorylase kinase was found to be 90 nM when measured by fluorescence polarization spectroscopy. Glycerol gradient centrifugation studies indicated that 1 mol of calmodulin was bound to each phosphorylase kinase. Phosphorylation of the phosphorylase kinase did not reduce the amount of calmodulin bound. Kinetic studies of the activity of the catalytic subunit of cAMP-dependent protein kinase on phosphorylase kinase as a function of phosphorylase kinase and calmodulin concentrations were performed. The results of those studies were compared with mathematical models of four different modes of inhibition: competitive, noncompetitive, substrate depletion, and inhibition by a complex between phosphorylase kinase and calmodulin. The data conform best to the model in which the inhibitory species is a complex of phosphorylase kinase and calmodulin. The complex apparently competes with the substrate, phosphorylase kinase, which does not have exogenous calmodulin bound to it. In contrast, the phosphorylation of the synthetic phosphate acceptor peptide, Kemptide, is not inhibited by calmodulin.     

The mouse homolog of the human amyloid beta protein (AD-AP) gene is located on the distal end of mouse chromosome 16: further extension of the homology between human chromosome 21 and mouse chromosome 16

The human amyloid beta protein is the major constituent of the brain amyloid plaques found in Alzheimer disease. The gene that encodes this protein is located on chromosome 21, and individuals with Down syndrome (trisomy 21) also exhibit an early onset form of Alzheimer disease. We have used the cloned human amyloid beta protein gene and a panel of somatic cell hybrids to map the location of the mouse homolog of this gene. We report here that the mouse gene is located on chromosome 16 within the region 16C3----ter, in common with three other genes which map within the Down syndrome region of human chromosome 21.     

A catalogue of some ecuadorean fermented beverages,with notes on their microflora

Summary A catalogue of indigenous fermented beverages produced by different ethnic groups in Ecuador has been compiled and the microflora of selected examples examined. A diversity of fermentation substrates was encountered depending on the climatic zone. The fermentations are typicallyLactobacillus spp.—yeast fermentations except for one which includes a mould fermentation by a mixed starter ofMoniha sitophila, Rhizopus stolonifer and aFusarium sp. A discussion is made of the role of these beverages in the human ecology of certain regions.

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Resumen Se ha confeccionado un catálogo de bebidas indígenas ecuatorianas producidas por distintos grupos étnicos, examinándose la microflora de algunos ejemplos seleccionados. Las fermentaciones son generalmente del tipoLactobacillus sp.—levaduras, excepto en un caso que incluye una fermentación fúngica iniciada de forma mixta porM. sitophila, R. stolonifer y unFusarium sp. Se discute el papel de estas bebidas en la ecologia humana de ciertas regiones.

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Résumé Un catalogue des boissons fermentées indigènes produites par divers groupes ethniques de l'Equateur a été compilé et les micro-flores des exemples sélectionnés ont été éxaminés. Les substrats de fermentation varient d'une région climatique à l'autre. Les fermentations sont généralement du typeLactobacillus sp — levures, sauf dans un cas qui comporte une fermentation par des moisissures, avec un mélange initial deMoniha sitophila, Rhizopus stolonifer et une espèce deFusarium. Le rôle de ces boissons dans l'écologie humaine de certaines régions est discuté.

     

Measurement variability in DNA flow cytometry of replicate samples

A Bladder Cancer Flow Cytometry Network study has been carried out aimed at identification of the sources of inter- and intralaboratory variability. Replicate "cocktail" samples containing a mixture of peripheral blood lymphocytes and an aneuploid cell line and samples of peripheral blood lymphocytes serving as a DNA reference standard were distributed to five network laboratories. The samples were stained for DNA using propidium iodide, with each laboratory using its own staining protocol. Sets of these samples were analyzed by flow cytometry to obtain cellular DNA distributions. DNA index and hyperdiploid fraction were calculated for each histogram using an automated technique. Results were evaluated by analysis of variance to identify sources of variability. Three important sources of variation were found that affect flow cytometry in general and- the transportability of flow cytometry results to routine clinical use in particular. The significant variation among laboratories that is constant across time most probably represents stable differences in instrumentation, instrument set-up, and laboratory techniques. This variation can be compensated for, if it is known and stable, to develop transportable classification criteria. The second type of variation, termed the interaction component, represents differences among laboratories that are not constant across time. Sources of this variation include inconsistency in sample preparation, staining, and analysis. The elimination of this type of variation is required for meaningful comparison of data within and among laboratories and the creation of interlaboratory data-bases. The third type of variation represents pure measurement variability and affects the sensitivity of the technique.