Mapping the Information Landscape: Discerning Peaks and Valleys for Ecological Monitoring

We investigate previously unreported phenomena that have a potentially significant impact on the design of surveillance monitoring programs for ecological systems. Ecological monitoring practitioners have long recognized that different species are differentially informative of a system’s dynamics, as codified in the well-known concepts of indicator or keystone species. Using a novel combination of analysis techniques from nonlinear dynamics, we describe marked variation among spatial sites in information content with respect to system dynamics in the entire region. We first observed these phenomena in a spatially extended predator–prey model, but we observed strikingly similar features in verified water-level data from a NOAA/NOS Great Lakes monitoring program. We suggest that these features may be widespread and the design of surveillance monitoring programs should reflect knowledge of their existence.     

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human FcγRIIIa and the rat α2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.     

Prasiolales (Trebouxiophyceae,Chlorophyta) of the Svalbard Archipelago: diversity,biogeography and description of the new genera Prasionella and Prasionema

Species of Prasiolales (Trebouxiophyceae, Chlorophyta) are among the most common terrestrial and freshwater algae in polar regions. Comprehensive molecular studies of this group are available for Antarctica, but not yet for Arctic regions. We examined the diversity of the Prasiolales in the Svalbard Archipelago combining morphological observations of field-collected material, culture studies, molecular data (plastid rbcL and tufA sequences) and literature records. We confirmed the widespread occurrence of Prasiola crispa and P. fluviatilis, species recorded from Spitsbergen since the 19th century. Molecular phylogenetic analyses led to the discovery of two new genera of Prasiolales. Prasionema payeri is morphologically identical to filamentous stages of P. crispa, but represents an early-diverging lineage in the order. Prasionella wendyae is a colonial alga reproducing by aplanospores; its phylogenetic position is among the basal lineages of the order, but it could not be reliably reconstructed due to weak statistical support. The inclusion of P. wendyae in the prasiolalean phylogeny determined the paraphyly of Rosenvingiella, requiring the establishment of the new genus Rosenvingiellopsis for R. constricta. A poorly known species described from Spitsbergen, Ulothrix discifera, is transferred here to Rosenvingiella. Whereas some species of Prasiolales have bipolar distribution (P. crispa), others appear to be restricted to one or other of the poles. Our results suggest that polar regions are still a major repository of unknown algal diversity and highlight the importance of continued field surveys and the use of molecular data.     

The discovery of 2-anilinothiazolones as 11beta-HSD1 inhibitors

A series of 2-anilinothiazolones were prepared as inhibitors of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). The most potent compounds contained a 2-chloro or 2-fluoro group on the aniline ring with an isopropyl substituent on the 5-position of the thiazolone ring (compounds 2 and 3, respectively). The binding mode was determined through the X-ray co-crystal structure of the enzyme with compound 3. This compound was also approximately 70-fold selective over 11beta-HSD2 and was orally bioavailable in rat pharmacokinetic studies. However, compound 3 was >580-fold less active in the 11beta-HSD1 cell assay when tested in the presence of 3% human serum albumin.     

The Giardia lamblia actin gene and the phylogeny of eukaryotes

The single-copy actin gene of Giardia lamblia lacks introns; it has an average of 58% amino acid identity with the actin of other species; and 49 of its amino acids can be aligned with the amino acids of a consensus sequence of heat shock protein 70. Analysis of the potential RNA secondary structure in the transcribed region of the G. lamblia actin gene and of the single-copy actin gene of nine other species did not reveal any conserved structures. The G. lamblia actin sequence was used to root the phylogenetic trees based on 65 actin protein sequences from 43 species. This tree is congruent with small-subunit rRNA trees in that it shows that oomycetes are not related to higher fungi; that kinetoplatid protozoans, green plants, fungi and animals are monophyletic groups; and that the animal and fungal lineages share a more recent common ancestor than either does with the plant lineage. In contrast to smalls-ubunit rRNA trees, this tree shows that slime molds diverged after the plant lineage. The slower rate of evolution of actin genes of slime molds relative to those of plants, fungi, and animals species might be responsible for this incongruent branching. Correspondence to: G. Drouin     

Signaling via cAMP in fungi: interconnections with mitogen-activated protein kinase pathways

The cAMP signal transduction pathway controls a wide variety of processes in fungi. For example, considerable progress has been made in describing the involvement of cAMP pathway components in the control of morphogenesis in Saccharomyces cerevisiae, Ustilago maydis, and Magnaporthe grisea. These morphological processes include the establishment of filamentous growth in S. cerevisiae and U. maydis, and the differentiation of an appressorial infection structure in M. grisea. The discovery that appressorium formation requires cAMP signaling provides an immediate connection to fungal virulence. This connection may have broader implications among fungal pathogens because recent work indicates that cAMP signaling controls the expression of virulence traits in the human pathogen Cryptococcus neoformans. In this fungus, cAMP also influences mating, as has been found for Schizosaccharomyces pombe and as may occur in U. maydis. Finally, cAMP and mitogen-activated protein kinase pathways appear to function coordinately to control the response of certain fungi, e.g., Saccharomyces cerevisiae and Schizosaccharomyces pombe, to environmental stress. There are clues that interconnections between these pathways may be common in the control of many fungal processes. Received: 5 June 1998 / Accepted: 11 September 1998     

Microbial production of fructosyltransferases for synthesis of pre-biotics

Fructooligosaccharides (FOS) are prebiotic substances found in several vegetable or natural foods. The main commercial production of FOS comes from enzymatic transformation of sucrose by the microbial enzyme fructosyltransferase. The development of more efficient enzymes, with high activity and stability, is required and this has attracted the interest of biotechnologists and microbiologists with production by several microorganisms being studied. This article reviews and discusses FOS chemical structure, enzyme characteristics, the nomenclature, producer microorganisms and enzyme production both in solid state fermentation and submerged cultivation.     

Enzymatic Basis for N-Glycan Sialylation: STRUCTURE OF RAT α2,6-SIALYLTRANSFERASE (ST6GAL1) REVEALS CONSERVED AND UNIQUE FEATURES FOR GLYCAN SIALYLATION*

Glycan structures on glycoproteins and glycolipids play critical roles in biological recognition, targeting, and modulation of functions in animal systems. Many classes of glycan structures are capped with terminal sialic acid residues, which contribute to biological functions by either forming or masking glycan recognition sites on the cell surface or secreted glycoconjugates. Sialylated glycans are synthesized in mammals by a single conserved family of sialyltransferases that have diverse linkage and acceptor specificities. We examined the enzymatic basis for glycan sialylation in animal systems by determining the crystal structures of rat ST6GAL1, an enzyme that creates terminal α2,6-sialic acid linkages on complex-type N-glycans, at 2.4 Å resolution. Crystals were obtained from enzyme preparations generated in mammalian cells. The resulting structure revealed an overall protein fold broadly resembling the previously determined structure of pig ST3GAL1, including a CMP-sialic acid-binding site assembled from conserved sialylmotif sequence elements. Significant differences in structure and disulfide bonding patterns were found outside the sialylmotif sequences, including differences in residues predicted to interact with the glycan acceptor. Computational substrate docking and molecular dynamics simulations were performed to predict and evaluate the CMP-sialic acid donor and glycan acceptor interactions, and the results were compared with kinetic analysis of active site mutants. Comparisons of the structure with pig ST3GAL1 and a bacterial sialyltransferase revealed a similar positioning of donor, acceptor, and catalytic residues that provide a common structural framework for catalysis by the mammalian and bacterial sialyltransferases.     

An automated,high‐throughput method for targeted quantification of intact insulin and its therapeutic analogs in human serum or plasma coupling mass spectrometric immunoassay with high resolution and accurate mass detection (MSIA‐HR/AM)

The detection and quantification of insulin and its therapeutic analogs is important for medical, sports doping, and forensic applications. Synthetic variants contain slight sequence variations to affect bioavailability. To reduce sample handling bias, a universal extraction method is required for simultaneous extraction of endogenous and variant insulins with subsequent targeted quantification by LC‐MS. A mass spectrometric immunoassay (MSIA), a multiplexed assay for intact insulin and its analogues that couples immunoenrichment with high resolution and accurate mass (HR/AM) spectrometric detection across the clinical range is presented in this report. The assay is sensitive, selective, semi‐automated and can potentially be applied to detect new insulin isoforms allowing their further incorporation into second or third generation assays.     

Two Arabidopsis proteins synthesize acetylated xylan in vitro

Xylan is the third most abundant glycopolymer on earth after cellulose and chitin. As a major component of wood, grain and forage, this natural biopolymer has far‐reaching impacts on human life. This highly acetylated cell wall polysaccharide is a vital component of the plant cell wall, which functions as a molecular scaffold, providing plants with mechanical strength and flexibility. Mutations that impair synthesis of the xylan backbone give rise to plants that fail to grow normally because of collapsed xylem cells in the vascular system. Phenotypic analysis of these mutants has implicated many proteins in xylan biosynthesis; however, the enzymes directly responsible for elongation and acetylation of the xylan backbone have not been unambiguously identified. Here we provide direct biochemical evidence that two Arabidopsis thaliana proteins, IRREGULAR XYLEM 10–L (IRX10‐L) and ESKIMO1/TRICOME BIREFRINGENCE 29 (ESK1/TBL29), catalyze these respective processes in vitro. By identifying the elusive xylan synthase and establishing ESK1/TBL29 as the archetypal plant polysaccharide O‐acetyltransferase, we have resolved two long‐standing questions in plant cell wall biochemistry. These findings shed light on integral steps in the molecular pathways used by plants to synthesize a major component of the world's biomass and expand our toolkit for producing glycopolymers with valuable properties.