Production of rhamnolipids in solid-state cultivation using a mixture of sugarcane bagasse and corn bran supplemented with glycerol and soybean oil

Rhamnolipid biosurfactants are attracting attention due to their low toxicity, high biodegradability, and good ecological acceptability. However, production in submerged culture is made difficult by severe foaming problems. Solid-state cultivation (SSC) is a promising alternative production method. In the current work, we report the optimization of rhamnolipid production by Pseudomonas aeruginosa UFPEDA 614 on a solid substrate containing sugarcane bagasse and corn bran. The best rhamnolipid production, 45 g/l of impregnating solution used, was obtained with a 50:50 (m/m) mixture of sugarcane bagasse and corn bran supplemented with an impregnating solution containing 6% (v/v) of each of glycerol and soybean oil. This level is comparable with those of previous studies undertaken in solid-state cultivation; the composition of the biosurfactant is similar, but our medium is cheaper. Our work therefore provides a suitable basis for future studies of the development of an SSC-based process for rhamnolipid production.     

Endocytosis and degradation of alpha 1-antitrypsin-protease complexes is mediated by the serpin-enzyme complex (SEC) receptor

Alpha 1-Antitrypsin (alpha 1-AT) is similar to other members of the serine protease inhibitor (serpin) supergene family in that it undergoes structural rearrangement during the formation of a covalently stabilized inhibitory complex with its cognate enzyme, neutrophil elastase. We have recently demonstrated an abundant, high-affinity cell surface receptor on human hepatoma cells and human mononuclear phagocytes which recognizes a conformation-specific domain of the alpha 1-AT-elastase complex as well as of other serpin-enzyme complexes (Perlmutter, D. H., Glover, G. I., Rivetna, M., Schasteen, C. S., and Fallon, R. J. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3753-3757). Binding to this serpin-enzyme complex (SEC) receptor activates a signal transduction pathway for increased expression of the alpha 1-AT gene and may be responsible for clearance of serpin-enzyme complexes. In this study, we show that there is time-dependent and saturable internalization of alpha 1-AT-elastase and alpha 1-AT-trypsin complexes in human hepatoma HepG2 cells. Internalization is mediated by the SEC receptor as defined by inhibition by synthetic peptides corresponding to residues 359-374 of alpha 1-AT. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of intracellular radioactivity demonstrated that intact 75- and 66-kDa alpha 1-AT-trypsin complexes were internalized. Kinetic analysis of internalization at 37 degrees C showed that a single cohort of 125I-alpha 1-AT-trypsin complexes, prebound to cells at 4 degrees C, disappeared from the cell surface and accumulated intracellularly within 5-15 min at 37 degrees C. The intracellular concentration of radiolabeled complexes then decreased rapidly coincident with appearance of acid-soluble degradation products in the extracellular culture fluid. Intracellular degradation was inhibited by internalization at 18 degrees C or by internalization at 37 degrees C in the presence of weak bases ammonium chloride, primaquine, and chloroquine, indicating that degradation is lysosomal. These results indicate that in addition to its role in signal transduction the SEC receptor participates in internalization and delivery of alpha 1-AT-protease complexes to lysosome for degradation.     

The SEC receptor recognizes a pentapeptide neodomain of alpha 1-antitrypsin-protease complexes.

Formation of the covalently stabilized alpha 1-antitrypsin (alpha 1-AT)-neutrophil elastase complex, the archetype of serpin-enzyme complexes, results in a structurally rearranged alpha 1-AT molecule that possesses chemo-attractant activities, mediates an increase in synthesis of alpha 1-AT by mononuclear phagocytes and hepatocytes, and is more rapidly cleared from the circulation than is the native alpha 1-AT molecule. We have recently identified an abundant, high affinity cell surface receptor on human hepatoma HepG2 cells and human monocytes that binds alpha 1-AT-elastase complexes, mediates endocytosis and lysosomal degradation of alpha 1-AT-elastase complexes, and induces an increase in synthesis of alpha 1-AT. We have referred to this receptor as the serpin-enzyme complex, or SEC, receptor because it also recognizes complexes of serpins antithrombin III, alpha 1-antichymotrypsin, and C1 inhibitor with their cognate enzymes. In the current study, we show that a pentapeptide domain in the carboxyl terminal fragment of alpha 1-AT (amino acids 370-374, FVFLM) is sufficient for binding to the SEC receptor. A synthetic analog of this pentapeptide (peptide 105C, FVYLI) blocks binding and internalization of alpha 1-AT-125I-trypsin complexes by HepG2 cells. 125I-Peptide 105C binds specifically and saturably to HepG2 cells, and its binding is blocked by alpha 1-AT-trypsin or alpha 1-AT-elastase complexes. Alterations of this sequence introduced into synthetic peptides (mutations, deletions, or scrambling) demonstrate that binding of the pentapeptide domain is sequence-specific. Comparisons with the sequences of other serpins in the corresponding region indicate that this pentapeptide neodomain is highly conserved.     

Ancient papillomavirus-host co-speciation in Felidae

Background

Estimating evolutionary rates for slowly evolving viruses such as papillomaviruses (PVs) is not possible using fossil calibrations directly or sequences sampled over a time-scale of decades. An ability to correlate their divergence with a host species, however, can provide a means to estimate evolutionary rates for these viruses accurately. To determine whether such an approach is feasible, we sequenced complete feline PV genomes, previously available only for the domestic cat (Felis domesticus, FdPV1), from four additional, globally distributed feline species: Lynx rufus PV type 1, Puma concolor PV type 1, Panthera leo persica PV type 1, and Uncia uncia PV type 1.

Results

The feline PVs all belong to the Lambdapapillomavirus genus, and contain an unusual second noncoding region between the early and late protein region, which is only present in members of this genus. Our maximum likelihood and Bayesian phylogenetic analyses demonstrate that the evolutionary relationships between feline PVs perfectly mirror those of their feline hosts, despite a complex and dynamic phylogeographic history. By applying host species divergence times, we provide the first precise estimates for the rate of evolution for each PV gene, with an overall evolutionary rate of 1.95 × 10^-8 (95% confidence interval 1.32 × 10^-8 to 2.47 × 10^-8) nucleotide substitutions per site per year for the viral coding genome.

Conclusion

Our work provides evidence for long-term virus-host co-speciation of feline PVs, indicating that viral diversity in slowly evolving viruses can be used to investigate host species evolution. These findings, however, should not be extrapolated to other viral lineages without prior confirmation of virus-host co-divergence.