Increased amylosucrase activity and specificity, and identification of regions important for activity, specificity and stability through molecular evolution

Amylosucrase is a transglycosidase which belongs to family 13 of the glycoside hydrolases and transglycosidases, and catalyses the formation of amylose from sucrose. Its potential use as an industrial tool for the synthesis or modification of polysaccharides is hampered by its low catalytic efficiency on sucrose alone, its low stability and the catalysis of side reactions resulting in sucrose isomer formation. Therefore, combinatorial engineering of the enzyme through random mutagenesis, gene shuffling and selective screening (directed evolution) was applied, in order to generate more efficient variants of the enzyme. This resulted in isolation of the most active amylosucrase (Asn387Asp) characterized to date, with a 60% increase in activity and a highly efficient polymerase (Glu227Gly) that produces a longer polymer than the wild-type enzyme. Furthermore, judged from the screening results, several variants are expected to be improved concerning activity and/or thermostability. Most of the amino acid substitutions observed in the totality of these improved variants are clustered around specific regions. The secondary sucrose-binding site and beta strand 7, connected to the important Asp393 residue, are found to be important for amylosucrase activity, whereas a specific loop in the B-domain is involved in amylosucrase specificity and stability.     

Cutting edge: monovalency of CD28 maintains the antigen dependence of T cell costimulatory responses

CD28 and CTLA-4 are the major costimulatory receptors on naive T cells. But it is not clear why CD28 is monovalent whereas CTLA-4 is bivalent for their shared ligands CD80/86. We generated bivalent CD28 constructs by fusing the extracellular domains of CTLA-4 or CD80 with the intracellular domains of CD28. Bivalent or monovalent CD28 constructs were ligated with recombinant ligands with or without TCR coligation. Monovalent CD28 ligation did not induce responses unless the TCR was coligated. By contrast, bivalent CD28 ligation induced responses in the absence of TCR engagement. To extend these findings to primary cells, we used novel superagonistic and conventional CD28 Abs. Superagonistic Ab D665, but not conventional Ab E18, predominantly ligates CD28 bivalently at low CD28/Ab ratios and induces Ag-independent T cell proliferation. Monovalency of CD28 for its natural ligands is thus essential to provide costimulation without inducing responses in the absence of TCR engagement.     

Characterization of human complement receptor type 2 (CR2/CD21) as a receptor for IFN-alpha: a potential role in systemic lupus erythematosus

Human complement receptor type 2 (CR2/CD21) is a B lymphocyte membrane glycoprotein that plays a central role in the immune responses to foreign Ags as well as the development of autoimmunity to nuclear Ags in systemic lupus erythematosus. In addition to these three well-characterized ligands, C3d/iC3b, EBV-gp350, and CD23, a previous study has identified CR2 as a potential receptor for IFN-alpha. IFN-alpha, a multifunctional cytokine important in the innate immune system, has recently been proposed to play a major pathogenic role in the development of systemic lupus erythematosus in humans and mice. In this study, we have shown using surface plasmon resonance and ELISA approaches that CR2 will bind IFN-alpha in the same affinity range as the other three well-characterized ligands studied in parallel. In addition, we show that IFN-alpha interacts with short consensus repeat domains 1 and 2 in a region that serves as the ligand binding site for C3d/iC3b, EBV-gp350, and CD23. Finally, we show that treatment of purified human peripheral blood B cells with the inhibitory anti-CR2 mAb 171 diminishes the induction of IFN-alpha-responsive genes. Thus, IFN-alpha represents a fourth class of extracellular ligands for CR2 and interacts with the same domain as the other three ligands. Defining the role of CR2 as compared with the well-characterized type 1 IFN-alpha receptor 1 and 2 in mediating innate immune and autoimmune roles of this cytokine should provide additional insights into the biologic roles of this interaction.     

Reduced pulsatility does not explain renal hyporesponsiveness to cardiac natriuretic peptides in pulmonary hypertension

A pulsatile secretory pattern is assumed to improve hormonal efficiency. We examined the short-term time courses of circulating atrial (ANP) and brain natriuretic peptides (BNP) in patients with pulmonary hypertension (PH) and reduced renal efficiency of ANP-BNP, reflected by low natriuresis/ANP or BNP ratios. Compared to controls, we observed a persistence of ANP and BNP pulsatility in PH patients with a similar periodicity of 20min. Pulse amplitude increased proportionally to the rise in mean plasma level observed in patients (around 27%). In PH patients, the decrease in ANP-BNP renal efficiency is not attributable to a loss of the rhythmic pulsatility of these hormones.     

Comparison of different biological indices for the assessment of river quality: application to the upper river Moselle (France)

The aim of our study was to assess the water quality of the upper Moselle river by using biological indices. Simultaneous physico-chemical surveys were also undertaken from May 1999 to April 2000. Twelve sampling sites were selected in order to provide a wide range of potential pollution. Chemical analysis did not reveal any major problem of pollution. However a lower water quality resulting from domestic pollution was established for some sampling sites. A biological monitoring combining both macroinvertebrates and macrophytes was performed. Biological indices based on plant community structure and macrophyte composition were not pertinent tools, whereas simple indices based on taxonomic richness of particular groups of macroinvertebrates were strongly correlated with several chemical parameters, showing that such simple biological variables should represent powerful indicators of ecosystem degradation.     

Cytochrome b(5) is a major reductant in vivo of human indoleamine 2,3-dioxygenase expressed in yeast

The evolutionary relationship of indoleamine 2,3-dioxygenase (IDO) to some gastropod myoglobins suggests that IDO may undergo autoxidation in vivo such that one or more currently unidentified electron donors are required to maintain IDO heme iron in the active, ferrous state. To evaluate this hypothesis we have used yeast knockout mutants in combination with a recently developed yeast growth assay for IDO activity in vivo to demonstrate a role for cytochrome b(5) and cytochrome b(5) reductase in maintaining IDO activity in vivo.     

Whole-Genome Reciprocal BLAST Analysis Reveals that Planctomycetes Do Not Share an Unusually Large Number of Genes with Eukarya and Archaea

The genome sequences of Rhodopirellula baltica, formerly Pirellula sp. strain 1, Blastopirellula marina, Gemmata obscuriglobus, and Kuenenia stuttgartiensis were used in a series of pairwise reciprocal best-hit analyses to evaluate the contested evolutionary position of Planctomycetes. Contrary to previous reports which suggested that R. baltica had a high percentage of genes with closest matches to Archaea and Eukarya, we show here that these Planctomycetes do not share an unusually large number of genes with the Archaea or Eukarya, compared with other Bacteria. Thus, best-hit analyses may assign phylogenetic affinities incorrectly if close relatives are absent from the sequence database.     

Autonomic control of heart rate is virtually independent of temperature but seems related to the neuroanatomy of the efferent vagal supply to the heart in the bullfrog, Lithobathes catesbeianus

Bullfrogs, Lithobathes catesbeianus, bearing a femoral artery cannula were held at 3 temperatures (10, 20 and 30 °C) for 24 h. Changes in heart rate were recorded before and after injection of cholinergic and adrenergic antagonists. Normal heart rate doubled for each temperature increment. Adrenergic tone on the heart varied around 20% at all 3 temperatures but cholinergic tone increased from −5% to 10% between 10 and 30 °C. In contrast, cholinergic tone increased from 75% at 5 °C to 329% at 25 °C in Xenopus laevis. Injection of the neural tracer True Blue into the cervical vagus of the bullfrog revealed a single location for vagal preganglionic neurons (VPN) in the dorsal vagal motor nucleus (DVN), while Xenopus had 30% of its VPN in a ventro-lateral group outside the DVN. Broader comparative studies have suggested that differences in the extent of vagal tone may relate to the location of VPN in the brainstem and this may be the case in these amphibians.     

Leucine stimulates insulin secretion via down-regulation of surface expression of adrenergic α2A receptor through the mTOR (mammalian target of rapamycin) pathway: implication in new-onset diabetes in renal transplantation

The amino acid leucine is a potent secretagogue, capable of inducing insulin secretion. It also plays an important role in the regulation of mTOR activity, therefore, providing impetus to investigate if a leucine-sensing mechanism in the mTOR pathway is involved in insulin secretion. We found that leucine-induced insulin secretion was inhibited by both the mTOR inhibitor rapamycin as well as the adrenergic α2 receptor agonist clonidine. We also demonstrated that leucine down-regulated the surface expression of adrenergic α2A receptor via activation of the mTOR pathway. The leucine stimulatory effect on insulin secretion was attenuated in diabetic Goto-Kakizaki rats that overexpress adrenergic α2A receptors, confirming the role of leucine in insulin secretion. Thus, our data demonstrate that leucine regulates insulin secretion by modulating adrenergic α2 receptors through the mTOR pathway. The role of the mTOR pathway in metabolic homeostasis led us to a second important finding in this study; retrospective analysis of clinical data showed that co-administration of rapamycin and clonidine was associated with an increased incidence of new-onset diabetes in renal transplantation patients over those receiving rapamycin alone. We believe that inhibition of mTOR by rapamycin along with activation of adrenergic α2 receptors by clonidine represents a double-hit to pancreatic islets that synergistically disturbs glucose homeostasis. This new insight may have important implications for the clinical management of renal transplant patients.