Characterization and visualization of cholecystokinin receptors in rat brain using [3H]pentagastrin

[³H]Pentagastrin binds specifically to an apparent single class of CCK receptors on slide-mounted sections of rat brain (K_D=5.6 nM; B_max=36.6 fmol/mg protein). This specific binding is temperature-dependant and regulated by ions and nucleotides. The relative potencies of C-terminal fragments of CCK-8(SO₃H), benzotript and proglumide in inhibiting specific [³H]pentagastrin binding to CCK brain receptors reinforce the concept of different brain and pancreas CCK receptors. CCK receptors were visualized by using tritium-sensitive LKB film analyzed by computerized densitometry. CCK receptors are highly concentrated in the cortex, dentate gyrus, granular and external plexiform layers of the olfactory bulb, anterior olfactory nuclei, olfactory tubercle, claustrum, accumbens nucleus, some nuclei of the amygdala, thalamus and hypothalamus.     

Scaling up for mass cultivation of the temperature-sensitive mutant Tetrahymena thermophila SJ180

Tetrahymena thermophila SJ180 growth was investigated in five complex media,cultivated in static flasks or in fermenter. In flask, the best maximum populations were obtained in the yeast extract and glucose medium. In fermentor, the best results were observed with the yeast extract and skimmed milk medium. Protease secretion was also studied.     

Hazard Inherent in Microbial Tracers: Reduction of Risk by the Use of Bacillus stearothermophilus Spores in Aerobiology

The use of a „biological tracer” forms an essential part of many aerobiological experiments. Where release of such tracers is likely to result in deliberate or inadvertent human exposure, safety becomes a primary consideration in the selection of the tracer organism. Of the three most commonly used organisms, namely Bacillus subtilis, Escherichia coli, and Serratia marcescens, only the first comes near to satisfying the need for nonpathogenicity and even it has been incriminated as a cause of human infection, sometimes with a fatal outcome. The relevant characteristics of B. stearothermophilus were, therefore, investigated. Because it can grow only at elevated temperatures (minimum 41 C; optimum 56 C), it should not pose a threat to human health and this view is supported by experimental evidence to be presented. It is extremely easy to grow and maintain in the laboratory, and spore suspensions are easily prepared and stored. It withstands the stresses of aerosolization and sampling and its stability in the aerosol state compares favorably with that of B. subtilis var. niger.     

Botrytis cinerea virulence is drastically reduced after disruption of chitin synthase class III gene (Bcchs3a)

Botrytis cinerea is an important phytopathogenic fungus requiring new methods of control. Chitin biosynthesis, which involves seven classes of chitin synthases, could be an attractive target. A fragment encoding one of the class III enzymes was used to disrupt the corresponding Bcchs3a gene in the B. cinerea genome. The resulting mutant exhibited a 39% reduction in its chitin content and an 89% reduction in its in vitro chitin synthase activity, compared with the wild-type strain. Bcchs3a mutant was not affected in its growth in liquid medium, neither in its production of sclerotia, micro- and macroconidia. In contrast, the mutant Bcchs3a was severely impaired in its growth on solid medium. Counterbalancing this defect in radial growth, Bcchs3a mutant presented a large increase in hyphal ramification, resulting in an enhanced aerial growth. Observations by different techniques of microscopy revealed a thick extracellular matrix around the hyphal tips. Moreover, Bcchs3a mutant had a largely reduced virulence on Vitis vinifera and Arabidopsis thaliana leaves.     

Hemoprotein models based on a covalent helix-heme-helix sandwich. 3. Coordination properties, reactivity and catalytic application of Fe(III)- and Fe(II)-mimochrome I

 The coordination state of Fe(III)- and Fe(II)-mimochrome I, a covalent peptide-deuteroheme sandwich involving two nonapeptides bearing a histidine residue in a central position, was studied by UV-visible, EPR, and resonance Raman spectroscopy. The ferric and ferrous states of this new iron species mainly exist, at pH 7, in a low-spin hexacoordinate form with two axial histidine ligands coming from the peptide chains. A minor amount of high-spin form for the ferric state is also present at pH 7. However, it is mainly high-spin at pH 2 or in DMSO. Fe(II)-mimochrome I binds CO with an affinity comparable to that of myoglobin and hemoglobin. Fe(III)-mimochrome I reacts with alkylhydroxylamine and arylhydrazines, leading to the corresponding Fe(II)-nitrosoalkyl and Fe(III)-σ-aryl complexes, respectively. These reactions were greatly dependent on the solvent used and on the pH, and were much slower than the corresponding reactions performed by deuterohemin in the presence of excess imidazole. All these results indicate that the reactivity of iron-mimochrome I is controlled by the binding of the peptide chains to the iron. The reactivity shown by this complex at neutral pH is intermediate between that observed for iron porphyrins in the presence of excess imidazole and that of hemoproteins characterized by a strong bis-histidine axial coordination, such as cytochrome b ₅. Fe(III)-mimochrome I is able to catalyze styrene epoxidation by using a [Fe(III)-mimochrome I]/[H₂O₂]/[stryrene] ratio of 1 : 10 : 2000 in phosphate buffer solution (pH 7.2) containing 2% CTAB both under strictly anaerobic conditions and in the presence of oxygen, at 0  °C. Received: 26 May 1998 / Accepted: 20 August 1998     

Abnormal accumulation of sugars and phenolics in tobacco roots expressing the Agrobacterium T-6b oncogene and the role of these compounds in 6b-induced growth

The Agrobacterium T-DNA oncogene 6b induces tumors and modifies the growth of transgenic plants by an unknown mechanism. We have investigated changes in roots of tobacco seedlings that express a dexamethasone-inducible T-6b (dex-T-6b) gene. On induction medium with sucrose, intact or isolated dex-T-6b roots accumulated sucrose, glucose, and fructose and changed their growth, contrary to noninduced roots. Root fragments bridging agar blocks with or without sucrose accumulated sugars at the site of sucrose uptake, resulting in local growth. Induced root fragments showed enhanced uptake of 14C-labeled sucrose, glucose, and fructose. When seedlings were placed on sucrose-free induction medium, sugar levels strongly decreased in roots and increased in cotyledons. Collectively, these results demonstrate that 6b stimulates sugar uptake and retention with drastic effects on growth. Apart from sugars, phenolic compounds also have been found to accumulate in 6b tissues and have been proposed earlier to play a role in 6b-induced growth. Induced dex-T-6b roots accumulated high levels of 5-caffeoylquinic acid (or chlorogenic acid [CGA]), but only under conditions where endogenous sugars increased. Inhibition of phenylalanine ammonia-lyase with the competitive inhibitor 2-aminoindan-2-phosphonic acid (AIP) abolished CGA accumulation without modifying sugar accumulation or affecting the 6b phenotype. We conclude that the absorption, retention, and abnormal accumulation of sugars are essential factors in 6b-induced growth changes, whereas phenylpropanoids only marginally contribute to the 6b seedling phenotype.     

Analysis of TETRAKETIDE α-PYRONE REDUCTASE function in Arabidopsis thaliana reveals a previously unknown, but conserved, biochemical pathway in sporopollenin monomer biosynthesis

The precise structure of the sporopollenin polymer that is the major constituent of exine, the outer pollen wall, remains poorly understood. Recently, characterization of Arabidopsis thaliana genes and corresponding enzymes involved in exine formation has demonstrated the role of fatty acid derivatives as precursors of sporopollenin building units. Fatty acyl-CoA esters synthesized by ACYL-COA SYNTHETASE5 (ACOS5) are condensed with malonyl-CoA by POLYKETIDE SYNTHASE A (PKSA) and PKSB to yield α-pyrone polyketides required for exine formation. Here, we show that two closely related genes encoding oxidoreductases are specifically and transiently expressed in tapetal cells during microspore development in Arabidopsis anthers. Mutants compromised in expression of the reductases displayed a range of pollen exine layer defects, depending on the mutant allele. Phylogenetic studies indicated that the two reductases belong to a large reductase/dehydrogenase gene family and cluster in two distinct clades with putative orthologs from several angiosperm lineages and the moss Physcomitrella patens. Recombinant proteins produced in bacteria reduced the carbonyl function of tetraketide α-pyrone compounds synthesized by PKSA/B, and the proteins were therefore named TETRAKETIDE α-PYRONE REDUCTASE1 (TKPR1) and TKPR2 (previously called DRL1 and CCRL6, respectively). TKPR activities, together with those of ACOS5 and PKSA/B, identify a conserved biosynthetic pathway leading to hydroxylated α-pyrone compounds that were previously unknown to be sporopollenin precursors.     

Species-Specific Contribution of the Four C-Terminal Amino Acids of Influenza A Virus NS1 Protein to Virulence

Large-scale sequence analyses of influenza viruses revealed that nonstructural 1 (NS1) proteins from avian influenza viruses have a conserved C-terminal ESEV amino acid motif, while NS1 proteins from typical human influenza viruses have a C-terminal RSKV motif. To test the influence of the C-terminal domains of NS1 on the virulence of an avian influenza virus, we generated a wild-type H7N1 virus with an ESEV motif and a mutant virus with an NS1 protein containing a C-terminal RSKV motif by reverse genetics. We compared the phenotypes of these viruses in vitro in human, mouse, and duck cells as well as in vivo in mice and ducks. In human cells, the human C-terminal RSKV domain increased virus replication. In contrast, the avian C-terminal ESEV motif of NS1 increased virulence in mice. We linked this increase in pathogenicity in mice to an increase in virus replication and to a more severe lung inflammation associated with a higher level of production of type I interferons. Interestingly, the human C-terminal RSKV motif of NS1 increased viral replication in ducks. H7N1 virus with a C-terminal RSKV motif replicated to higher levels in ducks and induced higher levels of Mx, a type I interferon-stimulated gene. Thus, we identify the C-terminal domain of NS1 as a species-specific virulence domain.Interspecies transmission of influenza viruses can lead to the introduction of new subtypes of influenza virus into the human population (31). The emergence of a new influenza virus that is able to spread efficiently between humans can cause a pandemic, as evidenced by the recent introduction of the swine-origin 2009 A/H1N1 virus to humans (10). The spread of avian influenza A viruses from birds to humans could also lead to the introduction of a new viral subtype with pandemic potential (22). Fortunately, the efficient replication of avian influenza A viruses in humans and interhuman transmission are generally limited and require further adaptations of the virus to humans. One determinant of host adaptation lies in the receptor binding specificity of hemagglutinin (HA) (52). In addition, several reports have underlined the role of amino acid 627 of the PB2 polymerase subunit in determining viral host range and virulence (15, 36, 44, 45). Large-scale sequence analyses of viruses isolated from different bird and mammalian species have been performed in order to identify previously unrecognized determinants of host adaptation and virulence (2, 32). Those studies have identified a 4-amino-acid motif in the C-terminal domain of NS1 that could represent a previously unnoticed host adaptation motif. Indeed, the vast majority of avian influenza viruses have an NS1 protein with a C-terminal ESEV domain, while typical human viruses have a conserved RSKV domain. The conservation of these species-specific motifs in the NS1 protein despite important sequence variability in the rest of the protein suggests that these four C-terminal amino acids are under strong selection pressure in their respective natural hosts (3, 5, 25).NS1 is a multifunctional protein implicated in the regulation of viral gene expression and in the inhibition of the host antiviral response (12). In order to test the role of these newly identified NS1 domains, Jackson et al. previously introduced various C-terminal motifs into NS1 of the mouse-adapted human influenza virus A/WSN/33 strain by use of reverse genetics (24). Mice inoculated with a virus containing an avian C-terminal ESEV NS1 domain had high viral loads in the lungs and decreased survival compared to mice inoculated with a virus containing a C-terminal RSKV domain. These results showed that the C-terminal ESEV motif found in avian NS1 proteins increases virulence in mice when introduced into a human strain of influenza virus. Whether this finding also applies to avian influenza viruses remains unknown. Moreover, whether the C-terminal ESEV domain of NS1 increases replication in human cells remains unknown. Finally, how the C-terminal domains of NS1 modulate virulence in nonmammalian hosts, such as birds, is also unknown.Here, we assessed the contribution of the C-terminal domains of NS1 to the pathogenicity of an avian influenza virus. By using reverse genetics, we generated H7N1 viruses containing an NS1 protein with a C-terminal avian ESEV domain or a C-terminal human RSKV domain. The replications of these viruses in human, mouse, and duck cell were compared. In addition, we assessed their pathogenicity in mice and ducks. Our results show that the C-terminal RSKV domain increases the replication of an avian influenza virus in human cells. To our surprise, we observed that the C-terminal RSKV domain increases replication in ducks. In contrast, the C-terminal ESEV domain increases virulence in mice. Thus, we identify the C-terminal domain of NS1 as a species-specific virulence domain.     

Heterogeneity of persistence of Salmonella enterica serotype Senftenberg strains could explain the emergence of this serotype in poultry flocks

Salmonella enterica serotype Senftenberg (S. Senftenberg) has recently become more frequent in poultry flocks. Moreover some strains have been implicated in severe clinical cases. To explain the causes of this emergence in farm animals, 134 S. Senftenberg isolates from hatcheries, poultry farms and human clinical cases were analyzed. Persistent and non-persistent strains were identified in chicks. The non-persistent strains disappeared from ceca a few weeks post inoculation. This lack of persistence could be related to the disappearance of this serotype from poultry farms in the past. In contrast, persistent S. Senftenberg strains induced an intestinal asymptomatic carrier state in chicks similar to S. Enteritidis, but a weaker systemic infection than S. Enteritidis in chicks and mice. An in vitro analysis showed that the low infectivity of S. Senftenberg is in part related to its low capacity to invade enterocytes and thus to translocate the intestinal barrier. The higher capacity of persistent than non-persistent strains to colonize and persist in the ceca of chickens could explain the increased persistence of S. Senftenberg in poultry flocks. This trait might thus present a human health risk as these bacteria could be present in animals before slaughter and during food processing.