Immunoelectron microscopic studies on the localization of peptidoglycan peptide subunit pentapeptides in bacterial cell walls

The peptide subunit pentapeptide H-L-Ala-D-Glu(L-Lys-D-Ala-D-Ala-OH)-NH₂ of peptidoglycan was localized in the cell walls of several Gram-positive bacteria employing the indirect immunoferritin technique. Specific antibodies to the D-alanyl-D-alanine moiety of non-crosslinked peptide subunit pentapeptide were raised in rabbits by immunization with synthetic immunogen albumin-(CH₂CO-Gly-L-Ala-L-Ala-D-Ala-D-Ala-OH)₃₉. Specificity of these antibodies for the peptide subunit pentapeptide and not for the peptide subunit tetrapeptide was corroborated in a Farr-type radio-active hapten binding assay. Specificity of labelling with ferritin was established by immunoelectron microscopic controls, and by the excellent correlation between specific labelling of cells with ferritin and the particular peptidoglycan primary structure of bacterial strains investigated. Cells of Lactobacillus gasseri, Streptococcus pyogenes and Staphylococcus aureus revealing non-crosslinked peptide subunit pentapeptides in their peptidoglycans could specifically be labelled. Lactobacillus acidophilus and Bacillus subtilis, on the contrary, missing such pentapeptides, failed in labelling.The implication of this method to possibly localize the points of attack of penicillin or cycloserine is discussed.Abbreviations used meso-A₂pm meso-diaminopimelic acid - DSM Deutsche Sammlung für Mikroorganismen, Göttingen, FRG This paper is dedicated to Professor Gerhart Drews on the occasion of his 60th birthday     

Functional CD169 on Macrophages Mediates Interaction with Dendritic Cells for CD8+ T Cell Cross-Priming

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Serum Calcium Response Following Oral Zinc Oxide Administrations in Dairy Cows

Six non-pregnant cows were allocated into 3 groups. Group 1 comprised a pair of lactating cows, whereas groups 2 and 3 each comprised a pair of non-lactating cows. The cows in groups 1 and 2 were dosed intraruminally by stomach tube with zinc oxide at 120 mg Zn per kg of bodyweight at weekly intervals for a period of 33 days. Each cow received a total of 4 doses of zinc oxide. Group 3 served as non-treated control group. Blood samples were collected from all 6 cows daily. Serum was analysed for concentration of calcium. Within 12–24 h of each zinc oxide administration the serum calcium of the lactating cows dropped dramatically indicating the existence of an antagonistic effect between Zn and Ca. The first Zn induced hypocalcaemic episode in the lactating cows was followed by a rise in serum calcium to a level above the pre-dosing level and above the mean value of the control group. The depth of the hypocalcaemic response decreased with the number of zinc oxide dosings. This effect was explained as a response from the stimulation of the calcium homeostatic mechanisms. In the Zn dosed non-lactating cows responses were similar but less clear. The perspective of these findings is discussed in relation to resistance towards parturient hypocalcaemia.     

RNA interference in Agrobacterium rhizogenes-transformed roots of Arabidopsis and Medicago truncatula

RNA interference (RNAi) is a powerful reverse genetic tool to study gene function. The data presented here show that Agrobacterium rhizogenes-mediated RNAi is a fast and effective tool to study genes involved in root biology. The Arabidopsis gene KOJAK, involved in root hair development, was efficiently knocked down. A. rhizogenes-mediated root transformation is a fast method to generate adventitious, genetically transformed roots. In order to select for co-transformed roots a binary vector was developed that enables selection based on DsRED1 expression, with the additional benefit that chimaeric roots can be discriminated. The identification of chimaeric roots provided the opportunity to examine the extent of systemic spread of the silencing signal in the composite plants of both Arabidopsis and Medicago truncatula. It is shown that RNA silencing does not spread systemically to non-co-transformed (lateral) roots and only inefficiently to the non-transgenic shoot. Furthermore, evidence is presented which shows that RNAi is cell autonomous in the root epidermis.     

Granule-bound starch synthase: structure, function, and phylogenetic utility

Interest in the use of low-copy nuclear genes for phylogenetic analyses of plants has grown rapidly, because highly repetitive genes such as those commonly used are limited in number. Furthermore, because low- copy genes are subject to different evolutionary processes than are plastid genes or highly repetitive nuclear markers, they provide a valuable source of independent phylogenetic evidence. The gene for granule-bound starch synthase (GBSSI or waxy) exists in a single copy in nearly all plants examined so far. Our study of GBSSI had three parts: (1) Amino acid sequences were compared across a broad taxonomic range, including grasses, four dicotyledons, and the microbial homologs of GBSSI. Inferred structural information was used to aid in the alignment of these very divergent sequences. The informed alignments highlight amino acids that are conserved across all sequences, and demonstrate that structural motifs can be highly conserved in spite of marked divergence in amino acid sequence. (2) Maximum-likelihood (ML) analyses were used to examine exon sequence evolution throughout grasses. Differences in probabilities among substitution types and marked among-site rate variation contributed to the observed pattern of variation. Of the parameters examined in our set of likelihood models, the inclusion of among-site rate variation following a gamma distribution caused the greatest improvement in likelihood score. (3) We performed cladistic parsimony analyses of GBSSI sequences throughout grasses, within tribes, and within genera to examine the phylogenetic utility of the gene. Introns provide useful information among very closely related species, but quickly become difficult to align among more divergent taxa. Exons are variable enough to provide extensive resolution within the family, but with low bootstrap support. The combined results of amino acid sequence comparisons, maximum-likelihood analyses, and phylogenetic studies underscore factors that might affect phylogenetic reconstruction. In this case, accommodation of the variable rate of evolution among sites might be the first step in maximizing the phylogenetic utility of GBSSI.      

Effect of Silt, Water and Periphyton Quality on Survival and Growth of the Mayfly Heptagenia Sulphurea

The herbivorous mayfly Heptagenia sulphurea is characteristic of rivers with stony bottoms. Records from the 20th century showed that this species had disappeared from the Common Meuse in the Netherlands, probably due to river regulation or changes in water quality. A field survey in 2003 showed that H. sulphurea was present in the Geul tributary, approximately 300 m upstream of its confluence with the Common Meuse. H. sulphurea has not recolonized the Common Meuse despite improvements in water quality over the last decades. Concentration of suspended sediments in the River Meuse, however, is still high, much higher than in the beginning of the 20th century. The presence of a silt layer may limit the return of H. sulphurea in this river by reducing availability and quality of its food. The prime objective of this study was to investigate the impact of silt on survival and growth of H. sulphurea in a laboratory experiment. Furthermore, the impact of water and periphyton quality from the Common Meuse on survival and growth of this mayfly was also investigated. Results showed that neither water quality nor cultured periphyton from the Common Meuse reduced survival and growth of H. sulphurea. The presence of a silt layer resulted in a significantly lower growth rate of the mayfly larvae. It is concluded that the silt layer reduces the accessibility of the food. Covering of food is possibly one of the main factors limiting the recolonization of H. sulphurea and probably other benthic grazers in the Common Meuse.     

RT-qPCR reveals opsin gene upregulation associated with age and sex in guppies (Poecilia reticulata) - a species with color-based sexual selection and 11 visual-opsin genes

Background  

PCR-based surveys have shown that guppies (Poecilia reticulata) have an unusually large visual-opsin gene repertoire. This has led to speculation that opsin duplication and divergence has enhanced the evolution of elaborate male coloration because it improves spectral sensitivity and/or discrimination in females. However, this conjecture on evolutionary connections between opsin repertoire, vision, mate choice, and male coloration was generated with little data on gene expression. Here, we used RT-qPCR to survey visual-opsin gene expression in the eyes of males, females, and juveniles in order to further understand color-based sexual selection from the perspective of the visual system.     

Interaction of brassinosteroid functions and sucrose transporter SlSUT2 regulate the formation of arbuscular mycorrhiza

Transgenic tomato plants with reduced expression of the sucrose transporter SlSUT2 showed higher efficiency of mycorrhization suggesting a sucrose retrieval function of SlSUT2 from the peri-arbuscular space back into the cell cytoplasm plant cytoplasm thereby limiting mycorrhiza fungal development. Sucrose uptake in colonized root cells requires efficient plasma membrane-targeting of SlSUT2 which is often retained intracellularly in vacuolar vesicles. Protein-protein interaction studies suggested a link between SISUT2 function and components of brassinosteroid biosynthesis and signaling. Indeed, the tomato DWARF mutant d^x defective in BR synthesis¹ showed significantly reduced mycorrhization parameters.² The question has been raised whether the impact of brassinosteroids on mycorrhization is a general phenomenon. Here, we include a rice mutant defective in DIM1/DWARF1 involved in BR biosynthesis to investigate the effects on mycorrhization. A model is presented where brassinolides are able to impact mycorrhization by activating SUT2 internalization and inhibiting its role in sucrose retrieval.