Chromatographic methods for the isolation of,and refolding of proteins from,Escherichia coli inclusion bodies

New methods for the chromatographic isolation of inclusion bodies directly from crude Escherichia coli homogenates and for the refolding of denatured protein are presented. The traditional method of differential centrifugation for the isolation of purified inclusion bodies is replaced by a single gel-filtration step. The principle is that the exclusion limit of the gel particles is chosen such that only the inclusion bodies are excluded, i.e., all other components of the crude homogenate penetrate the gel under the conditions selected. In the novel column refolding process, a decreasing gradient of denaturant (urea or Gu-HCl), combined with an increasing pH gradient, is introduced into a gel-filtration column packed with a gel medium that has an exclusion limit lower than the molecular mass of the protein to be refolded. A limited sample volume of the protein, dissolved in the highest denaturant concentration at the lowest pH of the selected gradient combination, is applied to the column. During the course of elution, the zone of denatured protein moves down the column at a speed approximately threefold higher than that of the denaturant. This means that the protein sample will gradually pass through areas of increasingly lower denaturant concentrations and higher pH, which promotes refolding into the native conformation. The shape and slope of the gradients, as well as the flow rate, will influence the refolding rate and can be adjusted for different protein samples. The principle is illustrated using a denatured recombinant scFv fusion protein obtained from E. coli inclusion bodies.     

Microbial community of a saline mud volcano at San Biagio-Belpasso,Mt. Etna (Italy)

In San Biagio of Belpasso, approximately 20 km south of Mt. Etna, in the area of contact between volcanic and sedimentary formations, a number of small (3- 60 cm in diameter) active mud eruptions discharge CO2-rich gases, mud and NaCl brines. They can be described as mini-volcanoes owing to their typical conic shapes and continuously bubbling peak craters. Samples were collected from the active peak craters at a depth of 20 cm and DNA was immediately extracted and amplified with universal 16S rRNA gene-specific primers, followed by cloning procedure. A total of 140 bacterial clones obtained were screened and clustered by restriction fragment length polymorphism (RFLP) analysis. The pool of 16S rRNA sequences representing each RFLP cluster was subjected to phylogenetic analysis. All of the 33 sequences analysed were affiliated with the kingdom of Eubacteria; 28 sequences (77% of all clones) affiliated with the Proteobacteria, two sequences (19% of all clones) were affiliated with Actinobacteria and three sequences (4% of all clones) were affiliated with the Flexibacter-Cytophaga-Bacteroides division. The data obtained suggest that the microorganisms phylogenetically affiliated to autotrophic methane oxidizers and heterotrophic hydrocarbon degraders belonging to the gamma-subclass of Proteobacteria are major constituents of the microbial communities of the saline volcanic muds. Overall, the composition of the microbial community of the San Biagio mud volcano resembles the compositions of marine microbial communities, which might indicate that wind-blown seawater vapour acted as an inoculum for microbial community described in present work.     

Glycopeptidolipids — a new class of artificial antigens with carbohydrate determinants. Synthesis of artificial antigen with type-specific oligosaccharide hapten fromNeisseria meningitidis group B

Synthetic lipopeptideN-palmitoyltyrosyl-seryl-seryl-asparaginyl-alanine, an analogue of B-mitogenic tripalmitoyl-pentapeptide fromEscherichia coli lipoprotein, was coupled with an oligosaccharide hapten fromNeisseria meningitidis lipooligosaccharide to give a glycopeptidolipid conjugate — the artificial antigen of a new type possessing the type-specific microbial determinant.Abbreviations iBu isobutyl - Bu^t t-butyl - Boc t-butoxycarbonyl - DCC N,N-dicyclohexylcarbodiimide - DMF N,N-dimethylformamide - DMSO dimethylsulfoxide - ONB N-hydroxy-5-norbornen-2,3-dicarboximide ester - ONp 4-nitrophenyl ester - Pal palmitoyl - TEMED N,N,N,N-tetramethylethylenediamine - Z benzyloxycarbonyl - KDO 2-keto-3-deoxyoctonic acid - Hep L-glycero-d-manno-heptose - TPP S-[2,3-bis(palmitoyloxy)-(2RS)propyl]-N-palmitoylcysteinyl-seryl-asparaginyl-alanine.     

Progenitor cells of the testosterone-producing Leydig cells revealed

The cells responsible for production of the male sex hormone testosterone, the Leydig cells of the testis, are post-mitotic cells with neuroendocrine characteristics. Their origin during ontogeny and regeneration processes is still a matter of debate. Here, we show that cells of testicular blood vessels, namely vascular smooth muscle cells and pericytes, are the progenitors of Leydig cells. Resembling stem cells of the nervous system, the Leydig cell progenitors are characterized by the expression of nestin. Using an in vivo model to induce and monitor the synchronized generation of a completely new Leydig cell population in adult rats, we demonstrate specific proliferation of vascular progenitors and their subsequent transdifferentiation into steroidogenic Leydig cells which, in addition, rapidly acquire neuronal and glial properties. These findings, shown to be representative also for ontogenetic Leydig cell formation and for the human testis, provide further evidence that cellular components of blood vessels can act as progenitor cells for organogenesis and repair.     

Mechanism of chaperone-like activity. Suppression of thermal aggregation of betaL-crystallin by alpha-crystallin

Thermal denaturation and aggregation of beta(L)-crystallin from bovine lens have been studied using differential scanning calorimetry (DSC) and dynamic light scattering (DLS). According to the DLS data, the distribution of the beta(L)-crystallin aggregates by their hydrodynamic radius (R(h)) remains monomodal to the point of precipitating aggregates (sodium phosphate, pH 6.8; 100 mM NaCl; 60 degrees C). The size of the start aggregates (R(h,0)) and duration of the latent stage (t(0)) leading to the formation of the start aggregates have been determined from the light scattering intensity versus the hydrodynamic radius plots and the dependences of R(h) on time. The R(h,0) value remains constant at variation of the beta(L)-crystallin concentration, whereas the t(0) value increases with diminishing beta(L)-crystallin concentration. The suppression of beta(L)-crystallin aggregation by alpha-crystallin is connected with the decrease in the R(h,0) value and increase in the t(0) value. In the presence of alpha-crystallin the aggregate population is split into two components. The first component is represented by stable aggregates whose size remains constant in time. The aggregates of the other kind grow until they reach the size characteristic of aggregates prone to precipitation. The DSC data show that alpha-crystallin has no appreciable influence on thermal denaturation of beta(L)-crystallin.     

Infections of Jute, Maize and Rice ot Revealed by the Standard Blotter Method of Seed Health Testing

In the standard blotter seed health testing of jute, maize and rice some ungerminated seeds, and seedlings with or without symptoms were eventually found to be infected by pathogenic fungi on prolonged incubation any by trans-planting seedlings from blotters to autoclaved soil in pots.     

Fenoprofen: Inhibitor of prostaglandin synthesis

Fenoprofen, an anti-inflammatory agent, is a potent inhibitor of prostaglandin synthesis at physiological substrate concentration. I₅₀'s of 1 μM to 100 μM were obtained when the concentration of arachidonate was increased from 1 μM to 82 μM. In contrast, a small change in I₅₀ was observed with indomethacin. Kinetic studies indicated that inhibition by fenoprofen was competitive while that of indomethacin was non-competitive.     

Accurate Prediction of Severe Allergic Reactions by a Small Set of Environmental Parameters (NDVI,Temperature)

Severe allergic reactions of unknown etiology,necessitating a hospital visit, have an important impact in the life of affected individuals and impose a major economic burden to societies. The prediction of clinically severe allergic reactions would be of great importance, but current attempts have been limited by the lack of a well-founded applicable methodology and the wide spatiotemporal distribution of allergic reactions. The valid prediction of severe allergies (and especially those needing hospital treatment) in a region, could alert health authorities and implicated individuals to take appropriate preemptive measures. In the present report we have collecterd visits for serious allergic reactions of unknown etiology from two major hospitals in the island of Crete, for two distinct time periods (validation and test sets). We have used the Normalized Difference Vegetation Index (NDVI), a satellite-based, freely available measurement, which is an indicator of live green vegetation at a given geographic area, and a set of meteorological data to develop a model capable of describing and predicting severe allergic reaction frequency. Our analysis has retained NDVI and temperature as accurate identifiers and predictors of increased hospital severe allergic reactions visits. Our approach may contribute towards the development of satellite-based modules, for the prediction of severe allergic reactions in specific, well-defined geographical areas. It could also probably be used for the prediction of other environment related diseases and conditions.