Folding,stability, and secondary structure of a new dimeric cysteine proteinase inhibitor

Clitocypin, a new type of cysteine proteinase inhibitor from the mushroom Clitocybe nebularis, is a 34-kDa homodimer lacking disulphide bonds, reported to have unusual stability properties. Sequence similarity is limited solely to certain proteins from mushrooms. Infrared spectroscopy shows that clitocypin is a high beta-structure protein which was lost at high temperatures. The far UV circular dichroism spectrum is not that of classical beta-structure, but similar to those of a group of small beta-strand proteins, with a peak at 189nm and a trough at 202nm. An aromatic peak at 232nm and infrared bands at 1633 and 1515cm(-1) associated with the peptide backbone and the tyrosine microenvironment, respectively, were used to characterize the thermal unfolding. The reversible transition has a midpoint at 67 degrees C, with DeltaG=34kJ/mol and DeltaH=300kJ/mol, and is, unusually, independent of protein concentration. The kinetics of thermal unfolding and refolding are slow, with activation energies of 167 and 44kJ/mol, respectively. A model for folding and assembly is discussed.     

A TOPRIM domain in the crystal structure of the catalytic core of Escherichia coli primase confirms a structural link to DNA topoisomerases

Primases synthesize short RNA strands on single-stranded DNA templates, thereby generating the hybrid duplexes required for the initiation of synthesis by DNA polymerases. We present the crystal structure of the catalytic unit of a primase enzyme, that of a approximately 320 residue fragment of Escherichia coli primase, determined at 2.9 A resolution. Central to the catalytic unit is a TOPRIM domain that is strikingly similar in its structure to that of corresponding domains in DNA topoisomerases, but is unrelated to the catalytic centers of other DNA or RNA polymerases. The catalytic domain of primase is crescent-shaped, and the concave face of the crescent is predicted to accommodate about 10 base-pairs of RNA-DNA duplex in a loose interaction, thereby limiting processivity.     

Morphological Response of the Halophilic Fungal Genus Wallemia to High Salinity

The basidiomycetous genus Wallemia is an active inhabitant of hypersaline environments, and it has recently been described as comprising three halophilic and xerophilic species: Wallemia ichthyophaga, Wallemia muriae, and Wallemia sebi. Considering the important protective role the fungal cell wall has under fluctuating physicochemical environments, this study was focused on cell morphology changes, with particular emphasis on the structure of the cell wall, when these fungi were grown in media with low and high salinities. We compared the influence of salinity on the morphological characteristics of Wallemia spp. by light, transmission, and focused-ion-beam/scanning electron microscopy. W. ichthyophaga was the only species of this genus that was metabolically active at saturated NaCl concentrations. W. ichthyophaga grew in multicellular clumps and adapted to the high salinity with a significant increase in cell wall thickness. The other two species, W. muriae and W. sebi, also demonstrated adaptive responses to the high NaCl concentration, showing in particular an increased size of mycelial pellets at the high salinities, with an increase in cell wall thickness that was less pronounced. The comparison of all three of the Wallemia spp. supports previous findings relating to the extremely halophilic character of the phylogenetically distant W. ichthyophaga and demonstrates that, through morphological adaptations, the eukaryotic Wallemia spp. are representative of eukaryotic organisms that have successfully adapted to life in extremely saline environments.Hypersaline habitats had long been considered to be populated almost exclusively by prokaryotic organisms and the research on hypersaline environments had consequently been monopolized by bacteriologists. In 2000, the first reports appeared showing that fungi are active inhabitants of solar salterns (20). Until then, fungi able to survive in environments with a low amount of biologically available water (low water activity [a_w]) were only known as contaminants of foods preserved with high concentrations of salt or sugar. Since their first discovery in salterns, many new species have been discovered in natural hypersaline environments around the world, including some species that were previously known only as food-borne contaminants. Due to these discoveries, fungi are now recognized as an integral part of indigenous halophilic microbial communities since they can grow and adjust across the whole salinity range, from freshwater to almost saturated NaCl solutions (49). Most fungi differ from the majority of halophilic prokaryotes (16): they tend to be extremely halotolerant rather than halophilic and do not require salt to remain viable, with the exception of Wallemia spp.The order Wallemiales (Wallemiomycetes, Basidiomycota) was only recently introduced to define the single genus Wallemia, a phylogenetic maverick in the Basidiomycota (49). Until 2005, this genus contained only the species W. sebi. However, taxonomic analyses of isolates from sweet, salty, and dried foods (41) and from hypersaline evaporation ponds in the Mediterranean Sea, the Caribbean, and the Dead Sea (45, 49) have resolved this genus into three species: W. ichthyophaga, W. muriae, and W. sebi. The first two of these three Wallemia spp. require additional solutes in the growth media, and W. ichthyophaga is the most halophilic eukaryote described to date, since it cannot grow without the addition of 9% NaCl (wt/vol), and it still shows growth at a_w of 0.77, equivalent to 30% NaCl (wt/vol) (49).The survival, and especially the growth, of microorganisms in highly saline environments requires numerous adaptations (6, 18, 21, 34). The dominant representatives and the most thoroughly investigated halophilic fungi in hypersaline waters of the salterns are the black yeasts, and particularly the model organism Hortaea werneckii (20). An important level of adaptation of the black yeasts to high salinity is seen in their extremophilic ecotype, which is characterized by a special meristematic morphology and changes in cell wall structure and pigmentation (27). Other fungal osmoadaptations include the accumulation of osmolytes (27, 28, 40), the extrusion of sodium (5), modification of the plasma membrane (44) and the cell wall, and even changes in fungal colony morphology (27).The fungal cell wall is the first line of defense against environmental stress; therefore, adaptation at the cell wall level is expected to have one of the most important roles for successful growth at a low a_w (24, 32). The cell wall is essential for maintaining the osmotic homeostasis of cells, since it protects them against mechanical damage as well as high concentrations of salts (7). The central fibrillar glycan network of the cell wall is embedded in highly flexible amorphous cement, which allows considerable stretching with changing osmotic pressure (14, 29). Its balance between a rigid and a dynamic structure influences the shape of cells (14) and enables growth and hyphal branching (11).Since the species within the genus Wallemia have been recognized only recently (49), little is known about their mechanisms of adaptation to high salinity. To investigate the effects of low and high NaCl concentrations on cell morphology, with particular emphasis on cell wall ultrastructure, we compared W. ichthyophaga, the most halophilic fungal species known thus far, with the related xerophilic W. muriae and W. sebi. Micrographs were prepared by using light, transmission, and scanning electron microscopy. The results reveal how this eukaryotic genus uses adaptations at the cell wall level for thriving in extremely saline environments.     

Semi-preparative chromatographic purification of the enantiomers S-(−)-amlodipine and R-(+)-amlodipine

Pharmacokinetic studies of optically pure compounds after single enantiomer administration are becoming increasingly important. The process of racemization in vivo can diminish all expected advantages of single enantiomer treatment. Amlodipine, one of the calcium channel blockers, currently used in therapy as a racemate, is one of such drugs under study. In order to administer single enantiomers of amlodipine to healthy volunteers both were chromatographically purified and characterised. The two optical isomers of amlodipine, active S-(−)- and non-active R-(+)-amlodipine, were purified using chromatographic procedure adopted from the analytical separation. Enantiomers were successfully converted to benzenesulphonic salt without any racemization. All semi-preparative purifications were monitored with complementary analytical methods, HPLC and CE, along with the determination of optical activity so that the final product was sufficiently defined for further in vivo studies. The analytical method developed for the determination of plasma concentrations of each enantiomer of amlodipine in these studies is also briefly described.     

Desiccation tolerance of the resurrection plant Ramonda serbica is associated with dehydration-dependent changes in levels of proteolytic activities

The unique response of desiccation-tolerant, or resurrection plants, to extreme drought is accompanied by major changes in the protein pool, raising the possibility of the involvement of proteases. We detected and characterized proteases present in their active state in leaf extracts of desiccated Ramonda serbica Pan?., a resurrection plant from the Balkan Peninsula. Plants desiccated under laboratory conditions and maintained in anhydrobiosis for 4 and 14 months revived upon rehydration. Protease activities were determined spectrophotometrically in solution and by zymography on gels. Several endo- and aminopeptidases were detected and characterized by their pH profiles. Their enzyme class was determined using specific inhibitors. Those with higher activities were a serine endopeptidase active against Bz-Arg-pNA with a pH optimum around 9, and aminopeptidases optimally active at pHs from 7 to 9 against Leu-pNA, Met-pNA, Phe-pNA, Pro-pNA and Ala-pNA. The levels of their activities in leaf extracts from desiccated plants were significantly higher than those from rehydrated plants and from regularly watered plants, implying their involvement in the recovery of vegetative tissues from desiccation.     

Agent-Based Mapping of Credit Risk for Sustainable Microfinance

By drawing analogies with independent research areas, we propose an unorthodox framework for mapping microfinance credit risk---a major obstacle to the sustainability of lenders outreaching to the poor. Specifically, using the elements of network theory, we constructed an agent-based model that obeys the stylized rules of microfinance industry. We found that in a deteriorating economic environment confounded with adverse selection, a form of latent moral hazard may cause a regime shift from a high to a low loan payment probability. An after-the-fact recovery, when possible, required the economic environment to improve beyond that which led to the shift in the first place. These findings suggest a small set of measurable quantities for mapping microfinance credit risk and, consequently, for balancing the requirements to reasonably price loans and to operate on a fully self-financed basis. We illustrate how the proposed mapping works using a 10-year monthly data set from one of the best-known microfinance representatives, Grameen Bank in Bangladesh. Finally, we discuss an entirely new perspective for managing microfinance credit risk based on enticing spontaneous cooperation by building social capital.     

Process-wide control and automation of an integrated continuous manufacturing platform for antibodies

Integrated continuous manufacturing is entering the biopharmaceutical industry. The main drivers range from improved economics, manufacturing flexibility, and more consistent product quality. However, studies on fully integrated production platforms have been limited due to the higher degree of system complexity, limited process information, disturbance, and drift sensitivity, as well as difficulties in digital process integration. In this study, we present an automated end-to-end integrated process consisting of a perfusion bioreactor, CaptureSMB, virus inactivation (VI), and two polishing steps to produce an antibody from an instable cell line. A supervisory control and data acquisition (SCADA) system was developed, which digitally integrates unit operations and analyzers, collects and centrally stores all process data, and allows process-wide monitoring and control. The integrated system consisting of bioreactor and capture step was operated initially for 4 days, after which the full end-to-end integrated run with no interruption lasted for 10 days. In response to decreasing cell-specific productivity, the supervisory control adjusted the loading duration of the capture step to obtain high capacity utilization without yield loss and constant antibody quantity for subsequent operations. Moreover, the SCADA system coordinated VI neutralization and discharge to enable constant loading conditions on the polishing unit. Lastly, the polishing was sufficiently robust to cope with significantly increased aggregate levels induced on purpose during virus inactivation. It is demonstrated that despite significant process disturbances and drifts, a robust process design and the supervisory control enabled constant (optimum) process performance and consistent product quality.     

A method for testing the stability of a steady-state system during the calculation of a response to large changes in regulator concentrations

A modification of the ‘finite decomposition’ method (Crabtree and Newsholme (1985) Curr. Top. Cell. Regul. 25, 21–76) for calculating physiological responses from sensitivities is described, to enable the system to be tested for stability at each step of the procedure. Instability is indicated by a change of sign of the determinant of the square matrix (N) in the governing equation for the system. The method cannot be used to predict responses beyond any step at which instability occurs.     

Abundance and Diversity of CO<Subscript>2</Subscript>-fixing Bacteria in Grassland Soils Close to Natural Carbon Dioxide Springs

Gaseous conditions at natural CO₂ springs (mofettes) affect many processes in these unique ecosystems. While the response of plants to extreme and fluctuating CO₂ concentrations ([CO₂]) is relatively well documented, little is known on microbial life in mofette soil. Therefore, it was the aim of this study to investigate the abundance and diversity of CO₂-fixing bacteria in grassland soils in different distances to a natural carbon dioxide spring. Samples of the same soil type were collected from the Stavešinci mofette, a natural CO₂ spring which is known for very pure CO₂ emissions, at different distances from the CO₂ releasing vents, at locations that clearly differed in soil CO₂ efflux (from 12.5 to over 200 μmol CO₂ m⁻² s⁻¹ yearly average). Bulk and rhizospheric soil samples were included into analyses. The microbial response was followed by a molecular analysis of cbbL genes, encoding for the large subunit of RubisCO, a carboxylase which is of crucial importance for C assimilation in chemolitoautotrophic microbes. In all samples analyzed, the “red-like” type of cbbL genes could be detected. In contrast, the “green-like” type of cbbL could not be measured by the applied technique. Surprisingly, a reduction of “red-like” cbbL genes copies was observed in bulk soil and rhizosphere samples from the sites with the highest CO₂ concentrations. Furthermore, the diversity pattern of “red-like” cbbL genes changed depending on the CO₂ regime. This indicates that only a part of the autotrophic CO₂-fixing microbes could adapt to the very high CO₂ concentrations and adverse life conditions that are governed by mofette gaseous regime. Urška Videmšek, Alexandra Hagn, Michael Schloter, and Dominik Vodnik contributed equally to this study.