Extracellular expression of glucose inhibition-resistant Cellulomonas flavigena PN-120 β-glucosidase by a diploid strain of Saccharomyces cerevisiae

The catalytic fraction of the Cellulomonas flavigena PN-120 oligomeric β-glucosidase (BGLA) was expressed both intra- and extracellularly in a recombinant diploid of Saccharomyces cerevisiae, under limited nutrient conditions. The recombinant enzyme (BGLA¹⁵) expressed in the supernatant of a rich medium showed 582 IU/L and 99.4 IU/g dry cell, with p-nitrophenyl-β-d-glucopyranoside as substrate. BGLA¹⁵ displayed activity against cello-oligosaccharides with 2–5 glucose monomers, demonstrating that the protein is not specific for cellobiose and that the oligomeric structure is not essential for β-d-1,4-bond hydrolysis. Native β-glucosidase is inhibited almost completely at 160 mM glucose, thus limiting cellobiose hydrolysis. At 200 mM glucose concentration, BGLA¹⁵ retained more than 50 % of its maximal activity, and even at 500 mM glucose concentration, more than 30 % of its activity was preserved. Due to these characteristics of BGLA¹⁵ activity, recombinant S. cerevisiae is able to utilize cellulosic materials (cello-oligosaccharides) to produce bioethanol.     

A Novel MicroRNA-132-Surtuin-1 Axis Underlies Aberrant B-cell Cytokine Regulation in Patients with Relapsing-Remitting Multiple Sclerosis

Clinical trial results demonstrating that B-cell depletion substantially reduces new relapses in patients with multiple sclerosis (MS) have established that B cells play a role in the pathophysiology of MS relapses. The same treatment appears not to impact antibodies directed against the central nervous system, which underscores the contribution of antibody-independent functions of B cells to disease activity. One mechanism by which B cells are now thought to contribute to MS activity is by over-activating T cells, including through aberrant expression of B cell pro-inflammatory cytokines. However, the mechanisms underlying the observed B cell cytokine dysregulation in MS remain unknown. We hypothesized that aberrant expression of particular microRNAs might be involved in the dysregulated pro-inflammatory cytokine responses of B cells of patients with MS. Through screening candidate microRNAs in activated B cells of MS patients and matched healthy subjects, we discovered that abnormally increased secretion of lymphotoxin and tumor necrosis factor α by MS B cells is associated with abnormally increased expression of miR-132. Over-expression of miR-132 in normal B cells significantly enhanced their production of lymphotoxin and tumor necrosis factor α. The over-expression of miR-132 also suppressed the miR-132 target, sirtuin-1. We confirmed that pharmacological inhibition of sirtuin-1 in normal B cells induces exaggerated lymphotoxin and tumor necrosis factor α production, while the abnormal production of these cytokines by MS B cells can be normalized by resveratrol, a sirtuin-1 activator. These results define a novel miR-132-sirtuin-1 axis that controls pro-inflammatory cytokine secretion by human B cells, and demonstrate that a dysregulation of this axis underlies abnormal pro-inflammatory B cell cytokine responses in patients with MS.     

DROSOPHILA S2 cell culture in a WAVE Bioreactor: potential for scaling up the production of the recombinant rabies virus glycoprotein

Applied Microbiology and Biotechnology - The transmembrane rabies virus glycoprotein (RVGP) is the main antigen of vaccine formulations used around the world to prevent rabies, the most lethal...     

Prolonged stability of the plantibody HB-01 directed against the hepatitis B surface antigen in cryo-preserved tobacco leaves

Since 1983, several recombinant antibodies have been expressed in important agronomic plant species. However, to date no evaluation has been published about prolonged antibody stability within plant tissues under cryo-preservation conditions. This current report presents an approach to the KDEL-plantibody HB-01 (PHB-01) stability in frozen tobacco leaves by presenting scientific evidence about the stability of a plantibody to a prolonged low temperature exposure in this biological source. Results clearly show that the PHB-01 amount is maintained during the storage of tobacco leaves at ?20 °C for 90 days. The PHB-01 recovery was not affected by any irreversible physical and/or chemical change produced in tobacco leaves after this cryo-preservation time. The amount of total soluble proteins in the clarified extract decreased in proportion with the storage time and the PHB-01 molecules isolated from frozen leaf extracts were highly pure, >95%, according to an SDS-PAGE assessment under reducing conditions. Low temperature exposure of tobacco leaves did not reveal visible changes in frozen leaves, which is essential for the further extractability of proteins. The PHB-01 is stable in tobacco leaves at ?20 °C during 90 days, which offers the possibility to overcome problems associated with detrimental climate conditions and optimize purification capabilities.     

The effects of diet mixing on consumer fitness: macroalgae, epiphytes, and animal matter as food for marine amphipods

Herbivores are thought to achieve adequate nutrition by consuming numerous species of plants or by occasionally consuming animal tissue. Although active selection of diverse foods is common in nature, the relationship between diet mixing and consumer fitness is poorly understood, especially in marine environments. We studied the fitness-based consequences of dietary mixing in the sympatric amphipods Ampithoe marcuzzii, A. valida, Cymadusa compta, and Gammarus mucronatus by measuring survivorship, growth, and fecundity of these amphipods when they were offered single species of algae, a single animal food, a mixture of algal species, or a combination of algae and animal matter. For the more sedentary, tube-building amphipods A. marcuzzii, A. valida, and C. compta, fitness on mixed algal diets was matched by fitness on at least one of the monospecific algal diets, suggesting that they could benefit from preferential feeding on those algae in the field. The more mobile amphipod, G. mucronatus, survived and grew similarly on the mixed diets and on the filamentous brown alga Ectocarpus siliculosus. However, its fecundity was significantly higher when feeding on the algal and animal mixture than on Ectocarpus alone. Additionally, for G. mucronatus, fitness on mixed algae, mixed algae plus animal matter, and animal matter alone was equivalent, although female growth (but not gonad production) was slightly lower on animal matter alone than on the mixed algae combined with animal food. Thus the more mobile amphipod, G. mucronatus, was the only species able to perform well on animal food alone. In contrast, A. valida and C. compta experienced large negative effects when limited to consuming animal matter alone. For these two species, combining algae and animal matter did not enhance fitness over combining only algae. Fitness effects of specific algal diets showed some general similarities, but also considerable variance among the amphipods. For example, E. siliculosus was generally better food than other algae for all four amphipods, whereas Sargassum filipendula was generally poor. However, A. marcuzzii did not suffer negative effects of consuming only Sargassum. The red alga Polysiphonia sp. and the green alga Enteromorpha flexuosa decreased fitness in A. marcuzzii, C. compta, and G. mucronatus, but not A. valida, and the negative effects of Polysiphonia were considerably larger for A. marcuzzii than for the other amphipods. Our data show that nutritional requirements, even among related species (e.g., A. marcuzzii and A. valida), can be dramatically different. Diet mixing may benefit more mobile consumers like Gammarus that are better able to search for different foods, and may be less important for more sedentary herbivores like Ampithoe and Cymadusa that consume, and live in close association with, individual host plants. Received: 14 September 1998 / Accepted: 22 October 1999     

A GDPase/UDPase bifunctional enzyme from Candida albicans: purification and biochemical characterization

The most frequently isolated human fungal pathogen is Candida albicans which is responsible for about 50% of all Candida infections. In healthy individuals, this organism resides as a part of the normal microbiota in equilibrium with the host. However, under certain conditions, particularly in immunocompromised patients, this opportunistic pathogen adheres to host cells causing serious systemic infections. Thus, much effort has been dedicated to the study of its physiology with emphasis on factors associated to pathogenicity. A representative analysis deals with the mechanisms of glycoprotein assembly as many cell surface antigens and other macromolecules that modulate the immune system fall within this chemical category. In this regard, studies of the terminal protein glycosylation stage which occurs in Golgi vesicles has led to the identification of nucleotidases that convert glycosyltransferase-generated dinucleotides into the corresponding mononucleotides, thus playing a double function: their activity prevent inhibition of further glycosyl transfer by the accumulation of dinucleotides and the resulting mononucleotides are exchanged by specific membrane transporters for equimolecular amounts of sugar donors from the cytosol. Here, using a simple protocol for protein separation we isolated a bifunctional nucleotidase from C. albicans active on GDP and UDP that was characterized in terms of its molecular mass, response to bivalent ions and other factors, substrate specificity and affinity. Results are discussed in terms of the similarities and differences of this nucleotidase with similar counterparts from other organisms thus contributing to the knowledge of a bifunctional diphosphatase not described before in C. albicans.