Prosomes and their multicatalytic proteinase activity.

Prosomes were first described as being mRNA-associated RNP (ribonucleoprotein) particles and subcomponents of repressed mRNPs (messenger ribonucleoprotein). We show here that prosomes isolated from translationally inactive mRNP have a protease activity identical to that described by others for the multicatalytic proteinase complex (MCP, 'proteasome'). By RNase or non-ionic detergent treatment, the MCP activity associated with repressed non-globin mRNP from avian erythroblasts, sedimenting at 35 S, could be quantitatively shifted on sucrose gradients to the 19-S sedimentation zone characteristic of prosomes, which were identified by monoclonal antibodies. The presence of small RNA in the enzymatic complex was shown by immunoprecipitation of the protease activity out of dissociated mRNP using a mixture of anti-prosome monoclonal antibodies; a set of small RNAs 80-120 nucleotides long was isolated from the immunoprecipitate. Furthermore, on CsCl gradients, colocalisation of the MCP activity with prosomal proteins and prosomal RNA was found, and no difference in the prosomal RNA pattern was observed whether the particles were fixed or not prior to centrifugation. These data indicate that the MCP activity is a property of prosomes, shown to be in part RNP and subcomplexes of in vivo untranslated mRNP. A hypothesis for the role of the prosome-MCP particles in maintaining homeostasis of specific protein levels is proposed.     

NADH-dependent dehydrogenase activity estimation by flow cytometric analysis of 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction.

MTT reduction is usually analysed by colorimetric assay to study mitochondrial dehydrogenase activity as a test of cytotoxicity. This enzymatic reaction produces dark-blue granules of formazan, which increase cell refringency. In this work, we define the conditions for MTT use in quantitative flow cytometric analysis. MTT reduction provides a non-fluorescent dye usable by this technique to study an intracellular NADH-dependent dehydrogenase activity in vital cells. We observe that formazan production increases asymptotically with cell concentration and that this temperature-dependent Michaelis enzymatic reduction is produced essentially by mitochondrial dehydrogenases. In isolated mitochondria from rat hepatocytes and in whole L1210 murine leukemia cells, the Michaelis constants (KM) observed in the presence of respiratory substrates were, respectively, 10 microM and 500 microM. The inhibition of mitochondrial protein synthesis by chloramphenicol, which induces a rise of MTT reduction due to the correlative stimulation of glycolysis (Pasteur effect), is a limit of the MTT assay as a cytotoxicity test.     

A comparison of the effects of sward improvement on invertebrates, sward establishment and herbage yield

Field experiments were done at two widely separated sites in England (Tadcaster, North Yorkshire and North Wyke, Devon). In each case an old permanent sward was re-seeded after ploughing or by direct drilling, or was left intact and fertilised. The carbamate pesticide aldicarb was applied to half of each experimental plot. The effects of sward improvement and pesticide on various soil invertebrate groups were assessed. Soil-dwelling macrofauna arthropods were scarce at both sites. Stem-boring dipterous larvae, however, were numerous at both sites. At Tadcaster, Oscinella vastator was the only species present in the original sward, but 0. frit was by far the most prevalent species in re-seeded areas. These can only have arisen from direct oviposition of eggs by adults on or near the emerging seedlings and not by migration of larvae from buried turf or the desiccated old pasture.
Mites and Collembola were abundant. Their numbers were reduced initially by cultivation and pesticide usage, but the effects of these practices were transient and their numbers recovered to previous levels usually within 10 wk.
Earthworms were present at both sites. Deep burrowing species, e.g. Lumbricus terrestris and Allolobophora longa were less affected by the treatments imposed than species such as A. chlorotica and L. rubelius which usually live close to the soil surface. Effects on their numbers were in any case, short-lived.
No improvement method consistently provided the greatest herbage dry matter yield. Pesticide application, however, greatly increased seedling stand and/or herbage yield at both sites and showed the benefits of using crop protectants, especially on direct-drilled re-seeds.     

EFFECTS OF ENVIRONMENTAL VARIATION ON SINKING RATES OF MARINE PHYTOPLANKTON1

The effects of environmental variables, particularly irradiance, on the sinking rates of phytoplankton were investigated using cultures of Chaetoceros gracilis Schütt and C. flexuosum Mangin in laboratory experiments; these data were compared with results from assemblages in the open ocean and marginal ice zone of the Greenland Sea. In culture experiments both the irradiance under which the diatom was grown and culture growth rate were positively correlated with sinking rates. Sinking rates (ψ) in the Greenland Sea were smallest when determined from chlorophyll (mean ψ_chl= 0.14 m · d^?1) and biogenic silica (ψ_si= 0.14 m · d^?1) and greatest when determined from particulate carbon (ψ_c= 0.55 m · d^?1) and nitrogen (ψ_N= 0.64 m · d^?1). Field measurements indicated that variations in sinking may be associated with changes in irradiance and nitrate concentrations. Because these factors do not directly affect water density, they must be inducing physiological changes in the cell which affect buoyancy. Although a direct response to a single environmental variable was not always evident, sinking rates were positively correlated with growth rates in the marginal ice zone, further indicating a connection to physiological processes. Estimats of carbon flux at stations with vertically mixed euphotic zones indicated that approximately 30% of the daily primary production sank from the euphotic zone in the form of small particulates. Calculated carbon flux tended to increase with primary productivity.