Physical mapping of DNA base sequence homologies between an octopine and a nopaline Ti plasmid of Agrobacterium tumefaciens

A detailed physical map of the homologous and non-homologous regions between an octopine (pTiAch5) and a nopaline (pTiC58) Ti plasmid was determined by Southern type hybridization and by electron microscope heteroduplex analysis. This map was correlated with the functional maps of both plasmids. For the Southern type hybridizations, total labelled pTiAch5 DNA was hybridized to Southern blots of restriction fragments from a series of hybrid plasmids containing overlapping segments of the whole TiC58 plasmid. Reciprocal experiments were also carried out. The common sequences between the two plasmids (±30%) are restricted to four major stretches of homology. Analysis of heteroduplexes between pTiAch5 and several hybrid plasmids containing specific regions of pTiC58, and of heteroduplexes between hybrid plasmids derived from pTiC58 and pTiAch5 provided a detailed map of the fine structure of the four major homology regions. Two regions are distributed in the same relative order as compared to a common reference point, and two are inversed. Three regions contain a number of small, mostly asymmetrical substitution loops. Several regions distributed over the common DNA sequences were found to be partially homologous.     

A new method for C-terminal sequence analysis in the proteomic era

The overall study of post-translational modifications (PTMs) of proteins is gaining strong interest. Beside phosphorylation and glycosylation, truncations of the nascent polypeptide chain at the amino or carboxy terminus are by far the most common types of PTMs in proteins. In contrast to the analysis of phosphorylation and glycosylation sites, relatively little attention has been paid to the development of approaches for the systematic analysis of proteolytic processing events. Here we present a new mass spectrometry (MS)-based strategy that allows the identification of the C-terminal sequence of proteins. The method can be directly applied to proteins cleaved with cyanogen bromide (CNBr) and purified either by SDS-PAGE, by two-dimensional (2D) PAGE or in solution, and it therefore eliminates the need for specific isolation of the C-terminal peptide. Using Shewanella oneidensis as a model system, we have demonstrated that this approach can be used for C-terminal sequence analysis at a proteomic scale. We also applied the method to study the C-terminal proteolytic processing of procardosin A.     

Use of Fourier-transform infrared spectroscopy in the diagnosis of rheumatoid arthritis: a pilot study

Rheumatoid arthritis is an autoimmune inflammatory disease leading to joint cartilage, bone degradation and limitation of mobility. Diagnosis of RA is difficult and complex. There are also no effective methods for clear discrimination between RA patients and non-RA individuals. In this work we use IR spectroscopy to differentiate RA patients and blood donors’ sera. We found differences between investigated sera (RA and non-RA) in range of 3000–2800 and 1800–800 cm^?1 (W1–W5 regions). Based on mathematical analysis we developed a K-NN model characterized by 85?% of sensitivity and 100?% of specificity. Also we found that, wavenumber 1424 cm^?1, comprising in W3 region, was the most effective in human sera distinguishing. We conclude that IR spectroscopy may serve as a fast and easy method useful in RA serology.     

The ATPase activity of the <Emphasis Type="Italic">G2alt</Emphasis> gene encoding an aluminium tolerance protein from <Emphasis Type="Italic">Anoxybacillus gonensis</Emphasis> G2

The G2ALT gene was cloned and sequenced from the thermophilic bacterium Anoxybacillus gonensis G2. The gene is 666 bp long and encodes a protein 221 amino acids in length. The gene was overexpressed in E. coli and purified to homogeneity and biochemically characterized. The enzyme has a molecular mass of 24.5 kDa and it could be classified as a member of the family of bacterial aluminium resistance proteins based on homology searches. When this fragment was expressed in E. coli, it endowed E. coli with Al tolerance to 500 μM. The purified G2ALT protein is active at a broad pH range (pH 4.0–10.0) and temperature range (25°C–80°C) with optima of 6.0 and the apparent optimal temperature of 73°C respectively. Under optimal conditions, G2ALT exhibited a low ATPase activity with K _m ⁻ and V _max ⁻ values of 10±0.55 μM and 26.81±0.13 mg Pi released/min/mg enzyme, respectively. The ATPase activity of G2ALT requires Mg²⁺ and Na⁺ ions, while Zn²⁺ and Al³⁺ stimulate the activity. Cd²⁺ and Ag⁺ reduced the activity and Li⁺, Cu²⁺, and Co²⁺ inhibited the activity. Known inhibitors of most ATPases, like such as β-mercaptoethanol and ouabain, also inhibited the activity of the G2ALT. These biochemical characterizations suggested that G2ALT belongs to the PP-loop ATPase superfamily and it can be responsible for aluminium tolerance in A. gonensis G2.     

Disassembly and reassembly of amyloid fibrils in water-ethanol mixtures

This work presents the structural analysis of amyloid-like β-lactoglobulin fibrils incubated in ethanol-water mixtures after their formation in water. We observe for the first time the disassembly of semiflexible heat-denatured β-lactoglobulin fibrils and reassembly into highly flexible wormlike fibrils in ethanol-water solutions. Tapping mode atomic force microscopy is performed to follow structural changes. Our results show that in addition to their growth in length, there is a continuous nucleation process of new wormlike objects with time at the expense of the original β-lactoglobulin fibrils. The persistence length of wormlike fibrils (29.43 nm in the presence of 50% ethanol), indicative of their degree of flexibility, differs by 2 orders of magnitude from that of untreated β-lactoglobulin fibrils (2368.75 nm in pure water). Interestingly, wormlike fibrils do not exhibit a multiple strands nature like the pristine fibrils, as revealed by the lower maximum height and the lack of clear height periodicity along their contour length profile. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrates that the set of polypeptides obtained by ethanol degradation differs in some fractions from that present in pristine β-lactoglobulin fibrils. ATR-FTIR (attenuated total reflectance-Fourier transform infrared) spectroscopy also supports a different composition of the secondary structure of wormlike fibrils with a decreased amount of α-helix and increased random coils and turns content. These findings can contribute to deciphering the molecular mechanisms of protein aggregation into amyloid fibrils and their disassembly as well as enabling tailor-made production of protein fibrils.