Silent mutations in secondary Shine-Dalgarno sequences in the cDNA of human serum amyloid A4 promotes expression of recombinant protein in Escherichia coli.

The serum amyloid A (SAA) superfamily comprises a number of differentially expressed genes with a high degree of homology in mammalian species. SAA4, an apolipoprotein constitutively expressed only in humans and mice, is associated almost entirely with lipoproteins of the high-density range. The presence of SAA4 mRNA and protein in macrophage-derived foam cells of coronary and carotid arteries suggested a specific role of human SAA4 during inflammation including atherosclerosis. Here we underline the importance of ribosome binding site (rbs)-like sequences (also known as Shine-Dalgarno sequences) in the SAA4 cDNA for expression of recombinant SAA4 protein in Escherichia coli. In contrast to rbs sequences coded by the expression vectors, rbs-like sequences in the cDNA of target protein(s) are known to interfere with protein translation via binding to the small 16S ribosome subunit, yielding low or even no expression. Here we show that PCR mutations of two rbs-like sequences in the human SAA4 cDNA promote expression of considerable amounts of recombinant SAA4 in E.coli.     

GISHGenomic in situ hybridization reveals cryptic genetic differences between maize and its putative wild progenitor Zea mays subsp. parviglumis.

The aim of this paper is to test with genomic in situ hybridization the genomic affinities between maize and its putative progenitor Zea mays subsp. parviglumis. Blocking procedures were applied for the purpose of improving discrimination among chromosome regions. Unlabeled genomic DNA from Z. mays subsp. parviglumis as a blocking agent and labeled genomic DNA from maize were hybridized on maize chromosomes. On the other hand, mitotic metaphases from Z. mays subsp. parviglumis were blocked with unlabeled genomic DNA of maize and hybridized with labeled genomic DNA from Z. mays subsp. parviglumis. Both experiments showed that either maize or Z. mays subsp. parviglumis chromosomes have their own unique sequences. This means an unexpected degree of divergence if Z. mays subsp. parviglumis is the only progenitor of maize, a result that is discussed in relation to our previous genomic in situ hybridization observations and to the different scenarios proposed about the origin of maize.     

Effect of Temperature on the Formation of Microsclerotia of Verticillium dahliae

Microsclerotium formation by six isolates of Verticillium dahliae was studied at different temperatures both in vitro and in Arabidopsis thaliana . In vitro mycelial growth was optimal at 25°C, but microsclerotium formation was greatest at 20°C (two isolates) or 15–20°C (one isolate). Seedlings of A. thaliana were root-dipped in a conidial suspension, planted, and either placed at 5, 10, 15, or 25°C, or left at 20°C until the onset of senescence, after which some of the plants were placed at 5, 10, 15, or 25°C. The amount of microsclerotia per unit of shoot weight was assessed in relation to isolate and temperature. The optimal temperature for production of microsclerotia was 15–25°C. Two isolates each produced about 10 times more microsclerotia than each of the other four isolates. For these isolates, high R ²_adj.-values of 0.77 and 0.66 were obtained, with temperature and its square as highly significant (P   < 0.001) independent variables. R ²_adj.-values for the other isolates varied between 0.28 and 0.39. Moving plants to different temperatures at the onset of senescence led to microsclerotial densities that were intermediate between densities on plants that had grown at constantly 20°C and plants grown at other temperatures. This suggests that vascular colonization rate and rate of microsclerotium formation are similarly affected by temperature. The senescence rate of plants appeared unimportant except for plants grown at 25°C, which showed the highest amounts of microsclerotia per unit of plant weight in the most rapidly senescing plants.     

Different receptor subtypes are involved in the serotonin-induced modulation of epileptiform activity in rat frontal cortex in vitro.

The frontal cortex is innervated by serotonergic terminals from the raphe nuclei and it expresses diverse 5-HT receptor subtypes. We investigated the effects of 5-HT and different 5-HT receptor subtype-selective agonists on spontaneous discharges which had developed in rat cortical slices perfused with a Mg2+-free medium and the GABA(A) receptor antagonist picrotoxin. The frequency of synchronous discharges, recorded extracellularly in superficial layers (II/III) of the frontal cortex, was dose-dependently enhanced by 5-HT (2.5-40 microM). That excitatory effect was blocked by the 5-HT2 receptor selective antagonist ketanserin. The 5-HT2A/2C receptor-selective agonist DOI and the 5-HT4 receptor agonist zacopride also increased the frequency of spontaneous discharges. In the presence of ketanserin, 5-HT decreased the discharge rate; a similar effect was observed when the 5-HT1A receptor agonist 8-OH-DPAT or the 5-HT1B receptor agonist CGS-12066B was applied. The 5-HT3 receptor agonist m-CPBG was ineffective. In conclusion, 5-HT produces multiple effects on epileptiform activity in the frontal cortex via activation of various 5-HT receptor subtypes. The excitatory action of 5-HT, which predominates, is mediated mainly by 5-HT2 receptors. The inhibitory effects can be attributed to activation of 5-HT1A and 5-HT1B receptors.     

Microelements preparations obtained during processing of natural flint affect the physiology and biochemistry of bifidobacteria]

Microelement preparations obtained in the course of processing of flint powder stimulate the biological activity of Bifidobacterium adolescentis 94 BIM, grown on complex and synthetic nutritive media. The composition of the microelement preparations differed in the content of cations and anions. Introduction of the preparations into the cultures of physiologically active or anabiotic forms of bifidobacteria changed the parameters of exponential growth: compared to controls, the cultures were characterized by increased specific growth rate and decreased generation time. In the presence of microelements, the development of populations of bifidobacteria was associated with more pronounced accumulation of metabolic products (acetate, lactate, and ethanol). Introduction of microelement preparations increased the rate of synthesis of the extracellular proteinase (maximum content of the enzyme was observed after 3 h, whereas control cultures attained this level only after 6 h).     

Cervical screening in Luxembourg: 1990–1999

For quality assurance purposes, the results of the 1990's obtained by the National Cervical Cancer Screening Programme (NCCSP) launched in 1962 were reviewed. The positive cytodiagnosis, the histologically verified in situ and invasive cervical cancers and the mortality rates were reported.     

Exopolysaccharide-producing strains of thermophilic lactic acid bacteria cluster into groups according to their EPS structure

AIMS: To compare galactose-negative strains of Streptococcus thermophilus and Lactobacillus delbrueckii subspecies bulgaricus isolated from fermented milk products and known to produce exopolysaccharides (EPSs). METHODS AND RESULTS: The structures of the EPSs were determined using nuclear magnetic resonance (NMR) and their genetic relationships determined using restriction endonuclease analysis (REA) and random amplification of polymorphic DNA (RAPD). Similar groupings were apparent by REA and RAPD, and each group produced an EPS with a particular subunit structure. CONCLUSION: Although none of the strains assimilated galactose, all inserted a high proportion of galactose into their EPS when grown in skimmed milk, and fell into three distinct groups. Significance and Impact of the Study: This information should help in an understanding of genetic exchanges in lactic acid bacteria.     

Three C. elegans Rac proteins and several alternative Rac regulators control axon guidance, cell migration and apoptotic cell phagocytosis.

The Caenorhabditis elegans genome contains three rac-like genes, ced-10, mig-2, and rac-2. We report that ced-10, mig-2 and rac-2 act redundantly in axon pathfinding: inactivating one gene had little effect, but inactivating two or more genes perturbed both axon outgrowth and guidance. mig-2 and ced-10 also have redundant functions in some cell migrations. By contrast, ced-10 is uniquely required for cell-corpse phagocytosis, and mig-2 and rac-2 have only subtle roles in this process. Rac activators are also used differentially. The UNC-73 Trio Rac GTP exchange factor affected all Rac pathways in axon pathfinding and cell migration but did not affect cell-corpse phagocytosis. CED-5 DOCK180, which acts with CED-10 Rac in cell-corpse phagocytosis, acted with MIG-2 but not CED-10 in axon pathfinding. Thus, distinct regulatory proteins modulate Rac activation and function in different developmental processes.