Kinetic modelling of coupled transport across biological membranes.

In this report, we have modelled a secondary active co-transporter (symport and antiport), based on the classical kinetics model. Michaelis-Menten model of enzyme kinetics for a single substrate, single intermediate enzyme catalyzed reaction was proposed more than a hundred years ago. However, no single model for the kinetics of co-transport of molecules across a membrane is available in the literature. We have made several simplifying assumptions and have followed the basic Michaelis-Menten approach. The results have been simulated using GNU Octave. The results will be useful in general kinetic simulations and modelling.

Promoters, toll like receptors and microRNAs: A strange association.

Toll-like receptors (TLRs) are proteins that play key role in the innate immune system. In the present study, ~1000 base pairs upstream are taken from the transcription start site of the various TLR genes (10 known) in human. About 40 microRNAs have been identified that share 12-19 nucleotide sequence similarity with the promoter regions of 10 TLRs. It is proposed that the microRNA performs potential role in identification of promoter sequence and initiation of transcription.

Signals in the promoter regions of several cancerous genes.

The eukaryotic core promoter regions are complex and fuzzy, usually lacking any conserved regions. However, they contain signals in the form of short stretches of nucleic acid sequences, for transcription start sites (TSS) that are recognized by the transcription factors (TFs). The core promoter region thus plays an important role in biological pathways (gene network and activation). It has been reported that these signals are composed of nucleotide hexamers in the promoter sequence (smaller sequences are likely to have too little information to be useful and longer sequences are too complex to be recognized by proteins) reasonably close to the TSS. The signals (nucleotide hexamers) have been identified by a similarity search on the eukaryotic promoter database (EPD, Homo sapiens). The signals have been classified, depending on their base composition. They have been have clustered using an algorithm, such that there are two and three nucleotide differences between the classes and a single nucleotide difference within a class. We have reclassified the hexamers taking the highest frequent hexamers present in the EPD (Homo sapiens) as the class representatives. Also we have tried to find whether the same composition is reflected on the miRNAs but found that they probably have other functions unrelated to promoter recognition. In this report melanoma carcinoma pathway has been chosen as the reference pathway and the promoters of the driver genes has been searched for the presence of the major classes. A few of these were found and are reported here. Several non-cancerous genes have also been studied as reference and comparison.

Short nucleotide sequences signal spliceosomal binding in nucleic acids.

We have explored the region around the splice sites of the human intron and exons from the exon-intron database (EID) and located a number of short 6-nucleotide and 7-nucleotide sequences that are relatively common in the regions. These short sequences, we expect play an important role in the selection of the appropriate splicing process. We propose that the external signals via short recognition sequences play the deterministic role in the actual splicing process. We have obtained 50 such sequences each from the exon and intron from the beginning and from the ending and noted a number of common features.

Electrochemical studies of glucose oxidase immobilized on glutathione coated gold nanoparticles.

Glutathione (L-gamma-glutamyl-L-cysteinyl-L-glycine; GSH) forms a surface monolayer on gold nanoparticles by tethering via sulfur bonds (Au:GSH). In the present study, glucose oxidase (GOx; EC 1.1.3.4) was immobilized by covalent chemical coupling reactions on to Au:GSH nanoparticles and the enzyme coupled nanoparticles formed a stable colloid (stable for several weeks) in water. The immobilized enzyme was investigated for electrochemical characteristics to monitor the FAD (prosthetic group of the GOx) redox potentials. Various concentrations of substrate (glucose) were added to check the oxidation characteristics. It was observed that with increase in substrate concentrations, the oxidation rate increased proportionally with the current. The present study demonstrated that GOx was effectively coupled to the gold nanoparticle (Au:GSH). The coupled nanoparticle system could be used in a potential biosensor application. Similarly, other enzymes (e.g., horseradish peroxidase) could be immobilized to the Au:GSH nanoparticles via the peptide arm (GSH) to achieve the desired characteristics needed for a specific application in biosensor.

1/f correlations in viral genomes--a Fast-Fourier Transformation (FFT) Study.

We have studied the presence of long-range correlations in the complete genomes of ten different dsDNA viruses and Saccharomyces cerevisiae (bakers' yeast) chromosome I. We have also studied the correlation between the distribution of the gene length and the domain of "1/f region" of their genomes. Linear regression analysis was done for the power-law region of these organisms and the slope values obtained were approximately -1, which signify the existence of "1/f noise" in the low and medium (intermediate) frequency regions. This suggests the presence of long-range correlations in their genomes. The presence of 1/f noise in a given frequency interval indicates the existence of a fractal (self-similar) structure in the corresponding range of wavelengths. The results of our study suggest that genes have correlations within themselves, and the correlations appear to be related with the scaling exponent alpha.

Covalent enzyme immobilization onto glassy carbon matrix-implications in biosensor design.

The aim of the present work is to design an electrode for biosensors by covalent immobilization of the redox enzyme. In the covalently modified electrode, the biocatalyst is located close to the electrode surface and this is expected to enhance the electron transfer rate from the enzyme to the electrode. Several methods of covalent immobilization of enzymes onto a glassy carbon surface are described. We have chosen horse radish peroxidase enzyme in our study but any other suitable enzyme can be immobilized depending on the intended use. A three step procedure that includes (i) heat treatment of matrix at lOO-l10°C to remove volatiles and absorbates, (ii) chemjcal pretreatment to introduce functional groups like -OH, -NO2, -Br etc. followed by (iii) glutaraldehyde coupling of the enzyme (for the nitrated mati x after subsequent reduction) or modification of the matrix by carboxymethylation and enzyme coupling using carbodiimide (for hydroxylated matrix) was followed. The amount of enzyme immobilized onto the carbon surface was estimated by spectrophotometric enzymatic activity assay, commonly used for the soluble enzyme. We found that simple nitration did not introduce any significant amount of functional groups and the matrix with hydrogen peroxide pretreatment showed the highest enzyme loading of 0.05 U/mg of carbon matrix. The HRP enzyme electrode was tested in a rotating disk experiment for its response with the substrate.

Proteins as special subsets of polypeptides.

A large protein sequence database with over 31,000 sequences and 10 million residues has been analysed. The pair probabilities have been converted to entropies using Boltzmann's law of statistical thermodynamics. A scoring weight corresponding to "mixing entropy" of the amino acid pairs has been developed from which the entropies of the protein sequences have been calculated. The entropy values of natural sequences are lower than their random counterparts of same length and similar amino acid composition. Based on the results it has been proposed that natural sequences are a special set of polypeptides with additional qualification of biological functionality that can be quantified using the entropy concept as worked out in this paper.

Correlation analysis of frequency distributions of residues in proteins.

Autocorrelation and spectrum analyses of amino acid residues along protein chains in a large data base has been performed. Results reveal the presence of general long range correlations. Similar analyses of simulated (random) peptides do not exhibit any such long range correlations. Based on the results of nur analysis, an attempt has been made to model the distribution of residues in protein sequences on a fractional Brownian motion and individual sequences as multi-fractals. For this purpose, the characteristics of an fractional Brownian motion namely, the scaling parameter H. the spectral exponent β and the fractal dimension D, have been described.

Periodicities in protein sequences.

The correlation between various amino acid residues (either same or different), along the polypeptide chain have been studied using a large data base. A table of preference values for pairs having strong correlations has been constructed, which can be used to study any sequence and by calculating the weight of these sequences based on these preference values, a rough distinction between a “natural” and a “random” sequence can be made, One can further comment on the evolutionary status of proteins based on these weights.

Protein sequences as random fractals.

The analysis of primary sequences from a protein sequence data base suggests that the sequences can be considered as examples of constrained random fractals. Fractal dimensions of the positional distributions of the 20 residues along the chain have been calculated. These fractal dimensions can be used as indices of intrinsic preferences of various residues.

Photoproduction of hydrogen by photosynthetic bacteria from sewage and waste water.

Numerous prokaryotes, belonging to physiologically and taxonomically different groups, are able to produce hydrogen. Some photosynthetic bacteria have the property of light-dependent production of hydrogen from organic substrates. We isolated several photosynthetic purple and green bacteria from enrichment cultures made from the water of a waste-water pond of a cool-drink refilling station. After testing them for their ability to use various organic compounds as carbon source, and sulphide, thiosulphate and organic compounds as electron donor, we selected the fastest-growing isolate, a Rhodopseudomonas, for a study of its ability to produce molecular hydrogen in presence of light. Immobilized cells of this isolate produced significant amounts of hydrogen from both sewage and waste water.