Entamoeba histolytica: rapid identification and differentiation of Indian isolates by riboprinting

Entamoeba histolytica infection still remains one of the major public health problem for developing countries like India. A rapid and accurate detection of this parasite is essential for prevention and control of amoebiasis. In this study, using the method of 'riboprinting' (PCR-RFLP of rRNA genes from amoeba) we have analysed 15 stool samples from symptomatic patients of amoebiasis. All 15 patients of clinical amoebiasis had E. histolytica in their stool and two of the samples also showed mixed infection of E. dispar. Apart from the known restriction enzyme sites within the amoeba SSU-rRNA genes, a new Sau3A site having a discriminatory value is identified in these E. histolytica isolates from India. Hence, it is possible to rapidly identify E. histolytica DNA and differentiating it from E. dispar using minute amounts of clinical stool samples, thus eliminating the laborious parasite culturing process. Thus, riboprinting is advantageous for clearcut identification of E. histolytica in order to decide an effective antiamoebic therapy.     

An insight into the possible mechanism of working of two-cistronic gene expression systems and rational designing of newer systems

The initial attempts at hyper-expressing buffalo/goat growth hormone (GH)-ORFs inEscherichia coli directly under various strong promoters were not successful despite the presence of a functional gene. High level expression of GH was achieved as a fusion protein with glutathione-S-transferase (GST). To produce native GH in an unfused state, we adapted an established strategy of two-cistronic approach in our system. In this strategy, utilizing one of the highly efficient reported sequences as the first cistron led to a nearly 1000-fold enhancement in the level of expression under anE. coli promoter (trc). In search of a newer first-cistron sequence as well as to see the generality of the two-cistronic approach, we explored the ability of different lengths of a highly expressing natural gene to act as an efficient first cistron. Surprisingly,GST, which is naturally highly expressible inE. coli, could not be fitted into a successful two-cistronic construct. In addition, placement of the entire two-cistronic expression cassette (which had earlier given high-level GH expression undertrc promoter) under theT7 promoter inE. coli failed to hyper-express GH. These results suggest that the successful exploitation of the two-cistron arrangement for hyper-expression of eukaryotic ORFs in bacteria is not as straightforward as was previously thought. It appears probable that factors such as the sequence context, together with the length and codons used in the first cistron are important as well.     

An intelligent biological information management system

MOTIVATION: As biomedical researchers are amassing a plethora of information in a variety of forms resulting from the advancements in biomedical research, there is a critical need for innovative information management and knowledge discovery tools to sift through these vast volumes of heterogeneous data and analysis tools. In this paper we present a general model for an information management system that is adaptable and scalable, followed by a detailed design and implementation of one component of the model. The prototype, called BioSifter, was applied to problems in the bioinformatics area. RESULTS: BioSifter was tested using 500 documents obtained from PubMed database on two biological problems related to genetic polymorphism and extracorporal shockwave lithotripsy. The results indicate that BioSifter is a powerful tool for biological researchers to automatically retrieve relevant text documents from biological literature based on their interest profile. The results also indicate that the first stage of information management process, i.e. data to information transformation, significantly reduces the size of the information space. The filtered data obtained through BioSifter is relevant as well as much smaller in dimension compared to all the retrieved data. This would in turn significantly reduce the complexity associated with the next level transformation, i.e. information to knowledge.     

Production of recombinant buffalo (Bubalus bubalis) and goat (Capra hircus) growth hormones from genetically modified E. coli strains

The growth hormone cDNAs of Indian reverine buffalo (Bubalus bubalis) and beetal goat (Capra hircus) were cloned in Escherichia coli through RT-PCR technique. Nucleotide sequencing revealed several silent mutations in both cDNAs and only one amino acid change in the case of goat when compared to reported bovine (Bos taurus) sequence. The high level expression of both the polypeptide hormones was achieved in E. coli (> or =30% of soluble intracellular proteins) through the construction of two-cistronic gene expression system. The solubilisation of recombinant growth hormones from inclusion bodies and subsequent oxidation to correctly folded monomeric form was also carried out. A combination of reverse-phase HPLC and non-reducing SDS-PAGE was successfully applied to distinguish between reduced and oxidised forms of growth hormones. A moderate yield ( approximately 40% of starting material, with potential for upscaling), two-step purification process comprising of hydrophobic interaction and ion-exchange chromatographies was developed. The process eliminates the need for costly, laborious and time-consuming steps of ultrafiltration and dialysis, as reported earlier for the purification of many recombinant animal growth hormones. The biophysical, biochemical and functional analyses of purified refolded polypeptides showed that the hormones produced in this study were identical to natural pituitary bovine growth hormone.     

Microbial arsenic: from geocycles to genes and enzymes

Arsenic compounds have been abundant at near toxic levels in the environment since the origin of life. In response, microbes have evolved mechanisms for arsenic resistance and enzymes that oxidize As(III) to As(V) or reduce As(V) to As(III). Formation and degradation of organoarsenicals, for example methylarsenic compounds, occur. There is a global arsenic geocycle, where microbial metabolism and mobilization (or immobilization) are important processes. Recent progress in studies of the ars operon (conferring resistance to As(III) and As(V)) in many bacterial types (and related systems in Archaea and yeast) and new understanding of arsenite oxidation and arsenate reduction by respiratory-chain-linked enzyme complexes has been substantial. The DNA sequencing and protein crystal structures have established the convergent evolution of three classes of arsenate reductases (that is classes of arsenate reductases are not of common evolutionary origin). Proposed reaction mechanisms in each case involve three cysteine thiols and S-As bond intermediates, so convergent evolution to similar mechanisms has taken place.     

Binding free energy calculations of galectin-3-ligand interactions

Galectins show remarkable binding specificity towards beta-galactosides. A recently developed method for calculating binding free energies between a protein and its substrates has been used to evaluate the binding specificity of galectin-3. Five disaccharides and a tetrasaccharide were used as the substrates. The calculated binding free energies agree quite well with the experimental data and the ranking of binding affinities is well reproduced. For all the six protein-ligand complexes it was observed that electrostatic interactions oppose binding whereas the non-polar contributions drive complex formation. The observed binding specificity of galectin-3 for galactosides rather than glucosides is discussed in light of our results.     

Growth of beta-amyloid(1-40) protofibrils by monomer elongation and lateral association. Characterization of distinct products by light scattering and atomic force microscopy

Amyloid plaques in brain tissue are a hallmark of Alzheimer's disease. Primary components of these plaques are 40- and 42-residue peptides, denoted A beta(1-40) and A beta(1-42), that are derived by proteolysis of cellular amyloid precursor protein. Synthetic A beta(1-40) and A beta(1-42) form amyloid fibrils in vitro that share many features with the amyloid in plaques. Soluble intermediates in A beta fibrillogenesis, termed protofibrils, have been identified previously, and here we describe the in vitro formation and isolation of A beta(1-40) protofibrils by size exclusion chromatography. In some experiments, the A beta(1-40) was radiomethylated to better quantify various A beta species. Mechanistic studies clarified two separate modes of protofibril growth, elongation by monomer deposition and protofibril-protofibril association, that could be resolved by varying the NaCl concentration. Small isolated protofibrils in dilute Tris-HCl buffers were directed along the elongation pathway by addition of A beta(1-40) monomer or along the association pathway by addition of NaCl. Multi-angle light scattering analysis revealed that protofibrils with initial molecular masses M(w) of (7-30) x 10(3) kDa grew to M(w) values of up to 250 x 10(3) kDa by these two growth processes. However, the mass per unit length of the associated protofibrils was about 2-3 times that of the elongated protofibrils. Rate constants for further elongation by monomer deposition with the elongated, associated, and initial protofibril pools were identical when equal number concentrations of original protofibrils were compared, indicating that the original number of protofibril ends had not been altered by the elongation or association processes. Atomic force microscopy revealed heterogeneous initial protofibrils that became more rodlike following the elongation reaction. Our data indicate that protofibril elongation in the absence of NaCl results from monomer deposition only at the ends of protofibrils and proceeds without an increase in protofibril diameter. In contrast, protofibril association occurs in the absence of monomer when NaCl is introduced, but this association involves lateral interactions that result in a relatively disordered fibril structure.     

Echinocyte shapes: bending, stretching, and shear determine spicule shape and spacing

We study the shapes of human red blood cells using continuum mechanics. In particular, we model the crenated, echinocytic shapes and show how they may arise from a competition between the bending energy of the plasma membrane and the stretching/shear elastic energies of the membrane skeleton. In contrast to earlier work, we calculate spicule shapes exactly by solving the equations of continuum mechanics subject to appropriate boundary conditions. A simple scaling analysis of this competition reveals an elastic length Lambda(el), which sets the length scale for the spicules and is, thus, related to the number of spicules experimentally observed on the fully developed echinocyte.