Comparative immunological characterization of various photosynthetic cytochromes c from pro- and eukaryotic algae

Photosynthetic c-type cytochromes isolated from various pro- and eukaryotic algae have been compared by an immunochemical method. Thereby the extent of cross-reactivity of several cytochromes with antisera to cytochrome c from Spirulina platensis, Bumilleriopsis filiformis, and Scenedesmus acutus was quantitatively determined by antigen-binding tests. When immunological relationship is taken as a measure of structural relationship, the following conclusions can be drawn: (1) c-type cytochromes from Anabaena variabilis, Nostoc muscorum, Calothrix membranacea, and Spirulina platensis show large differences in cross-reactivity. (2) The acidic Spirulina cytochrome c is fairly closely related to the two eukaryotic cytochromes assayed here.Abbreviations SAUG Sammlung von Algenkulturen am Pflanzenphysiologischen Institut der Universität Göttingen, FRG - PCC Pasteur Culture Collection     

Automated method for determination of glutardialdehyde residues in flexible endoscopes after disinfection

Glutardialdehyde (GDA) is the most commonly used disinfectant for flexible endoscopes. After inappropriate rinsing of endoscopes residual GDA in the narrow endoscope channels may lead to toxic effects in patients. Common methods for determination of aldehydes in water involve derivatization with 2,4-dinitrophenylhydrazine (DNPH), liquid-liquid or solid-phase extraction and HPLC determination. Since derivatization and extraction is both time-consuming and labor-intensive only a small number of samples can be measured. Thus, we developed a fully automated method which includes a conventional HPLC system, a programmable autosampler, and UV detection. After GDA derivatization using DNPH the samples remain in the aqueous phase and no preconcentration of the analyte is necessary. The samples are automatically derivatized through the autosampler. While derivatization in one sample takes place the previous sample is injected and measured by HPLC. Our method is well suited for screening residual GDA in endoscopes as it is both time- and labor-saving.     

Biosynthesis of calystegines: 15N NMR and kinetics of formation in root cultures of Calystegia sepium

Calystegines are nortropane alkaloids bearing between three and five hydroxyl groups in various positions. [15N]Tropinone was administered to root cultures of Calystegia sepium and the incorporation into calystegines was followed. Increase of label in calystegines was measured by one-dimensional 15N NMR and inverse-detected 2D NMR techniques. The results show that tropinone and pseudotropine are metabolites in the biosynthetic pathway of calystegines. The velocity of calystegine accumulation was followed kinetically by transfer of root cultures from 15N-enriched medium to 14N-medium and analysis by GC-MS. A constant calystegine formation with no interference by excretion or degradation was observed. A biosynthetic rate for individual calystegines at each time point was calculated, the maximum was 0.4 mg/day/g of biomass. This allowed the velocity of individual biosynthetic steps to be estimated.     

Development of human antibody fragments using antibody phage display for the detection and diagnosis of Venezuelan equine encephalitis virus (VEEV)

Background  

Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus group. Several species of this family are also pathogenic to humans and are recognized as potential agents of biological warfare and terrorism. The objective of this work was the generation of recombinant antibodies for the detection of VEEV after a potential bioterrorism assault or an natural outbreak of VEEV.     

Structural Requirements for Cub Domain Containing Protein 1 (CDCP1) and Src Dependent Cell Transformation

Cub domain containing protein 1 (CDCP1) is strongly expressed in tumors derived from lung, colon, ovary, or kidney. It is a membrane protein that is phosphorylated and then bound by Src family kinases. Although expression and phosphorylation of CDCP1 have been investigated in many tumor cell lines, the CDCP1 features responsible for transformation have not been fully evaluated. This is in part due to the lack of an experimental system in which cellular transformation depends on expression of exogenous CDCP1 and Src. Here we use retrovirus mediated co-overexpression of c-Src and CDCP1 to induce focus formation of NIH3T3 cells. Employing different mutants of CDCP1 we show that for a full transformation capacity, the intact amino- and carboxy-termini of CDCP1 are essential. Mutation of any of the core intracellular tyrosine residues (Y734, Y743, or Y762) abolished transformation, and mutation of a palmitoylation motif (C689,690G) strongly reduced it. Src kinase binding to CDCP1 was not required since Src with a defective SH2 domain generated even more CDCP1 dependent foci whereas Src myristoylation was necessary. Taken together, the focus formation assay allowed us to define structural requirements of CDCP1/Src dependent transformation and to characterize the interaction of CDCP1 and Src.     

Intestinal Microbiota Composition of Interleukin-10 Deficient C57BL/6J Mice and Susceptibility to Helicobacter hepaticus-Induced Colitis

The mouse pathobiont Helicobacter hepaticus can induce typhlocolitis in interleukin-10-deficient mice, and H. hepaticus infection of immunodeficient mice is widely used as a model to study the role of pathogens and commensal bacteria in the pathogenesis of inflammatory bowel disease. C57BL/6J Il10^−/− mice kept under specific pathogen-free conditions in two different facilities (MHH and MIT), displayed strong differences with respect to their susceptibilities to H. hepaticus-induced intestinal pathology. Mice at MIT developed robust typhlocolitis after infection with H. hepaticus, while mice at MHH developed no significant pathology after infection with the same H. hepaticus strain. We hypothesized that the intestinal microbiota might be responsible for these differences and therefore performed high resolution analysis of the intestinal microbiota composition in uninfected mice from the two facilities by deep sequencing of partial 16S rRNA amplicons. The microbiota composition differed markedly between mice from both facilities. Significant differences were also detected between two groups of MHH mice born in different years. Of the 119 operational taxonomic units (OTUs) that occurred in at least half the cecum or colon samples of at least one mouse group, 24 were only found in MIT mice, and another 13 OTUs could only be found in MHH samples. While most of the MHH-specific OTUs could only be identified to class or family level, the MIT-specific set contained OTUs identified to genus or species level, including the opportunistic pathogen, Bilophila wadsworthia. The susceptibility to H. hepaticus-induced colitis differed considerably between Il10^−/− mice originating from the two institutions. This was associated with significant differences in microbiota composition, highlighting the importance of characterizing the intestinal microbiome when studying murine models of IBD.     

Hyphal fusion during initial stages of trap formation in Arthrobotrys oligospora

Hyphal fusion during initial stages of trap formation by Arthrobotrys oligospora was studied by video-enhanced contrast and electron microscopy. Trap initials grew perpendicularly to the parent hypha, then curved around and anastomosed with a peg that developed on the hypha. Trap initials usually developed 40–140 m apart while the anastomosis occurred 20–25 m from the initial. Vigorous cytoplasmic movements in trap initials and developed traps corresponded to intense staining with fluorescein diacetate (FDA) of these cells. In addition, bundles of microfilaments were seen in developing loops of traps. On fusion organelle migration took place from the tip cell of the trap into the peg. Later on a septum was formed at the site of fusion.     

Dwarfism and low insulin-like growth factor-1 due to dopamine depletion in Pts-/- mice rescued by feeding neurotransmitter precursors and H4-biopterin

The tetrahydrobiopterin (BH4) cofactor is essential for the biosynthesis of catecholamines and serotonin and for nitric-oxide synthase (NOS). Alterations in BH4 metabolism are observed in various neurological and psychiatric diseases, and mutations in one of the human metabolic genes causes hyperphenylalaninemia and/or monoamine neurotransmitter deficiency. We report on a knockout mouse for the Pts gene, which codes for a BH4-biosynthetic enzyme. Homozygous Pts-/- mice developed with normal morphology but died after birth. Upon daily oral administration of BH4 and neurotransmitter precursors the Pts-/- mice eventually survived. However, at sexual maturity (6 weeks) the mice had only one-third of the normal body weight and were sexually immature. Biochemical analysis revealed no hyperphenylalaninemia, normal brain NOS activity, and almost normal serotonin levels, but brain dopamine was 3% of normal. Low dopamine leads to impaired food consumption as reflected by the severe growth deficiency and a 7-fold reduced serum insulin-like growth factor-1 (IGF-1). This is the first link shown between 6-pyruvoyltetrahydropterin synthase- or BH4-biosynthetic activity and IGF-1.     

Molecular characterization of the diurnal/circadian expression of the chlorophyll a/b-binding proteins in leaves of tomato and other dicotyledonous and monocotyledonous plant species

Diurnal oscillations of steady-state mRNA levels encoding the chlorophyll a/b-binding proteins were monitored inLycopersicon esculentum, Glycine max, Phaseolus vulgaris, P. aureus, P. coccineus, Pisum sativum, Sinapis alba, Hordeum vulgare, Triticum aestivum andZea mays. In these plant speciescab mRNA accumulation increases and decreases periodically indicating i) that the expression of the genes for chlorophyll a/b-binding proteins (cab genes) is controlled by a circadian rhythm, and ii) that the rhythm is widely distributed among monocotyledonous and dicotyledonous plant species. A detailed characterization of the pattern ofcab mRNA expression in tomato leaves shows that the amplitude of the oscillation is dependent on i) the developmental stage of the leaves, ii) the circadian phase and duration of light and iii) the circadian phase and duration of darkness. In addition to the chlorophyll a/b-binding proteins, genes coding for other cellular functions were examined for cyclic variations of their mRNA levels. The analysis includes genes involved in i) carbon metabolism (e.g. phosphoenolpyruvate carboxylase, pyruvate orthophosphate dikinase, alpha amylase, fructose-1,6-bisphosphate aldolase, triosephosphate isomerase), ii) photosynthesis (large and small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, Q_B-binding protein, reaction-center protein of photosystem I) and iii) other physiological or morphological reactions (e.g. ubiquitin, actin). However, no periodic fluctuation pattern was detected for the mRNA levels of these genes in tomato and maize leaves.