Characteristics and DNA-Sequence of a Cryptic Haloalkanoic Acid Dehalogenase from Agrobacterium tumefaciens RS5

Agrobacterium tumefaciens RS5 harbors two different hydrolytic haloalkanoic acid dehalogenase genes, one coding for a nonstereospecific enzyme (DhlS5II) and a second for a cryptic L-isomer-specific dehalogenase (DhlS5I). The latter gene was cloned and expressed in Escherichia coli. Biochemical characterization and sequence analysis of dhlS5I shows its membership to the class of the L-isomer-specific hydrolytic dehalogenases. Highest homology of 72% was found to the dehalogenase LdexYL from Pseudomonas sp. YL. Both enzymes share an unusual high temperature optimum of 65°C. Controlled by a vector promoter, high specific dehalogenase activities up to 32 U mg⁻¹ protein were obtained in E. coli, and putatively, owing to its own σ⁷⁰-dependent promoter, a constitutive low-level expression of dhlS5I of 0.4 U mg⁻¹ protein was measured. Received: 27 June 1997 / Accepted: 4 August 1997     

Putative identification of an amphipathic alpha-helical sequence in hemolysin of Escherichia coli (HlyA) involved in transmembrane pore formation

Abstract Escherichia coli hemolysin is a pore-forming protein belonging to the RTX toxin family. Cysteine scanning mutagenesis was performed to characterize the putative pore-forming domain of the molecule. A single cysteine residue was introduced at 48 positions within the sequence spanning residues 170-400 and labeled with the polarity-sensitive dye badan. Spectrofluorimetric analyses indicated that several amino acids in this domain are inserted into the lipid bilayer during pore formation. An amphipathic alpha-helix spanning residues 272-298 was identified that may line the aqueous pore. The importance of this sequence was highlighted by the introduction of two prolines at positions 284 and 287. Disruption of the helix structure did not affect binding properties, but totally abolished the hemolytic activity of the molecule.     

Croton campanulatus (Euphorbiaceae s.s.), a new species from the Brazilian Atlantic rain forest

Croton campanulatus, a new species from southeastern Brazil in the states of Minas Gerais and Rio de Janeiro, is here described and illustrated. Morphological data indicate that this species belongs to Croton section Cleodora based on its arborescent habit, pistillate flowers with imbricate sepals, reduced petals, and multifid styles that are fused at the base.     

Fiber digestibility in juvenile Galapagos tortoises (Geochelone nigra) and implications for the development of captive animals

Digestive strategies have been recognized to be a key factor for healthy growth in juvenile Galapagos giant tortoises (Geochelone nigra). The aim of present study was to investigate digestive coefficients with special regard to fiber fractions. Four captive bred Galapagos giant tortoises 4–5 years of age were fed a controlled diet for 32 days. The diet consisted of 77% hay, 15% tortoise pellets, and 8% apples on a dry matter basis. On a dry matter basis diet analysis showed: 95.7% organic matter, 11.3% crude protein, 20.5% crude fiber, 22.6% acid detergent fiber, 5.0% acid detergent lignin, and 17.6% cellulose. Based on total fecal collection during 7 days average dry matter digestibilities were calculated: 65% for dry matter, 67% for organic matter, 63% for crude protein, 55% for crude fiber, 49% for acid detergent fiber, 41% for acid detergent lignin, 54% for cellulose. An increase in crude fiber content resulted in a reduced digestibility in comparative evaluations of data for different tortoise species, and in a comparison of tortoises and mammalian hindgut fermenters. Compared to some mammalian hindgut‐fermenting herbivore species (domestic horses, Asian elephants, Indian rhinoceroses) on a diet of hay and concentrates, the juvenile Galapagos giant tortoises showed a digestion of similar efficiency. If a reduction in dietary digestibility is warranted in juvenile Galapagos giant tortoises, it is concluded that dietary fiber levels should be increased and it is proposed that crude fiber levels of 30–40% on a dry matter basis should be achieved. Zoo Biol 24:185–191, 2005. © 2005 Wiley‐Liss, Inc.     

Toward a methodical framework for comprehensively assessing forest multifunctionality

Biodiversity–ecosystem functioning (BEF) research has extended its scope from communities that are short‐lived or reshape their structure annually to structurally complex forest ecosystems. The establishment of tree diversity experiments poses specific methodological challenges for assessing the multiple functions provided by forest ecosystems. In particular, methodological inconsistencies and nonstandardized protocols impede the analysis of multifunctionality within, and comparability across the increasing number of tree diversity experiments. By providing an overview on key methods currently applied in one of the largest forest biodiversity experiments, we show how methods differing in scale and simplicity can be combined to retrieve consistent data allowing novel insights into forest ecosystem functioning. Furthermore, we discuss and develop recommendations for the integration and transferability of diverse methodical approaches to present and future forest biodiversity experiments. We identified four principles that should guide basic decisions concerning method selection for tree diversity experiments and forest BEF research: (1) method selection should be directed toward maximizing data density to increase the number of measured variables in each plot. (2) Methods should cover all relevant scales of the experiment to consider scale dependencies of biodiversity effects. (3) The same variable should be evaluated with the same method across space and time for adequate larger‐scale and longer‐time data analysis and to reduce errors due to changing measurement protocols. (4) Standardized, practical and rapid methods for assessing biodiversity and ecosystem functions should be promoted to increase comparability among forest BEF experiments. We demonstrate that currently available methods provide us with a sophisticated toolbox to improve a synergistic understanding of forest multifunctionality. However, these methods require further adjustment to the specific requirements of structurally complex and long‐lived forest ecosystems. By applying methods connecting relevant scales, trophic levels, and above‐ and belowground ecosystem compartments, knowledge gain from large tree diversity experiments can be optimized.     

Expression profiling of metabolic genes in response to methyl jasmonate reveals regulation of genes of primary and secondary sulfur-related pathways in Arabidopsis thaliana

The treatment of Arabidopsis thaliana with methyl jasmonate was used to investigate the reaction of 2467 selected genes of primary and secondary metabolism by macroarray hybridization. Hierarchical cluster analysis allowed distinctions to be made between diurnally and methyl jasmonate regulated genes in a time course from 30 min to 24 h. 97 and 64 genes were identified that were up- or down-regulated more than 2–fold by methyl jasmonate, respectively. These genes belong to 18 functional categories of which sulfur-related genes were by far strongest affected. Gene expression and metabolite patterns of sulfur metabolism were analysed in detail, since numerous defense compounds contain oxidized or reduced sulfur. Genes encoding key reactions of sulfate reduction as well as of cysteine, methionine and glutathione synthesis were rapidly up-regulated, but none of the known sulfur-deficiency induced sulfate transporter genes. In addition, increased expression of genes of sulfur-rich defense proteins and of enzymes involved in glucosinolate metabolism was observed. In contrast, profiling of primary and secondary sulfur metabolites revealed only an increase in the indole glucosinolate glucobrassicin upon methyl jasmonate treatment. The observed rapid mRNA changes were thus regulated by a signal independent of the known sulfur deficiency response. These results document for the first time how comprehensively the regulation of sulfur-related genes and plant defense are connected. This interaction is discussed as a new approach to differentiate between supply- and demand-driven regulation of the sulfate assimilation pathway.     

Cross-presentation of the long-lived lymphocytic choriomeningitis virus nucleoprotein does not require neosynthesis and is enhanced via heat shock proteins

Many viral proteins that contain MHC class I-restricted peptides are long-lived, and it is elusive how they can give rise to class I epitopes. Recently, we showed that direct presentation of an epitope of the long-lived lymphocytic choriomeningitis virus nucleoprotein (LCMV-NP) required neosynthesis in accordance with the defective ribosomal products hypothesis. In this study, we report that LCMV-NP can be cross-primed in mice using either LCMV-NP-transfected human HEK293 or BALB/c-derived B8 cells as Ag donor cells. In addition, we establish that contrary to direct presentation, cross-presentation required accumulation of the mature LCMV-NP and could not be sustained by the newly synthesized LCMV-NP protein, intermediate proteasomal degradation products, or the minimal NP396 epitope. Nevertheless, NP cross-presentation was enhanced by heat shock and was blunted by inhibitors of heat shock protein 90 and gp96. We propose that cross-presentation has evolved to sustain the presentation of stable viral proteins when their neosynthesis has ceased in infected donor cells.     

Quantitative determination of bile salt hydrolase activity in bacteria isolated from the small intestine of chickens

A quantitative assay based on high-performance liquid chromatography analysis of bile salts and bacterial protein determination was established for investigating bile salt hydrolase (BSH) activity in bacteria isolated from the small intestine of chickens. Bacteria were isolated using various media and were subsequently grouped according to cell morphology, fermentation profile, and 16S ribosomal DNA sequence. Representative isolates from each bacterial group were assayed for BSH activity. The isolates differed in BSH activity with respect to the state of growth and preculturing with and without taurochenodeoxycholate. The highest levels of BSH activity were found with Enterococcus faecium and Clostridium perfringens.