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.     

Snake venom hyaluronidase: An evidence for isoforms and extracellular matrix degradation

The present study attempts to establish the isoforms of hyaluronidase enzyme and their possible role in the spreading of toxins during envenomation. Screening of venoms of 15 snakes belonging to three different families revealed varied hyaluronidase activity in ELISA-like assay, but with relatively similar pH and temperature optima. The zymograms of individual venoms showed varied activity banding patterns and indicated the presence of at least two molecular forms of the enzyme. During envenomation, activity of hyaluronidase is considered crucial for the spreading of toxins and is presumed to distort the integrity of extracellular matrix through the degradation of hyaluronic acid in it. This property has been addressed through localization of hyaluronic acid in human skin and muscle tissue sections using the probe, biotinylated hyaluronic acid binding protein. Faint and discontinuous staining pattern of hyaluronidase treated tissue sections over intense staining of untreated tissue sections confirm the selective degradation of hyaluronic acid in extracellular matrix and thus provide an evidence for the spreading property of the enzyme.     

Effects of mutations at tyrosine 66 and asparagine 123 in the active site pocket of Escherichia coli uracil DNA glycosylase on uracil excision from synthetic DNA oligomers: evidence for the occurrence of long-range interactions between the enzyme and substrate

Uracil DNA glycosylase (UDG), a highly conserved DNA repair enzyme, excises uracil from DNA. Crystal structures of several UDGs have identified residues important for their exquisite specificity in detection and removal of uracil. Of these, Y66 and N123 in Escherichia coli UDG have been proposed to restrict the entry of non-uracil residues into the active site pocket. In this study, we show that the uracil excision activity of the Y66F mutant was similar to that of the wild-type protein, whereas the activities of the other mutants (Y66C, Y66S, N123D, N123E and N123Q) were compromised approximately 1000-fold. The latter class of mutants showed an increased dependence on the substrate chain length and suggested the existence of long-range interactions of the substrate with UDG. Investigation of the phosphate interactions by the ethylation interference assay reaffirmed the key importance of the -1, +1 and +2 phosphates (with respect to the scissile uracil) to the enzyme activity. Interestingly, this assay also revealed an additional interference at the -5 position phosphate, whose presence in the substrate had a positive effect on substrate utilisation by the mutants that do not possess a full complement of interactions in the active site pocket. Such long-range interactions may be crucial even for the wild-type enzyme under in vivo conditions. Further, our results suggest that the role of Y66 and N123 in UDG is not restricted merely to preventing the entry of non-uracil residues. We discuss their additional roles in conferring stability to the transition state enzyme-substrate complex and/or enhancing the leaving group quality of the uracilate anion during catalysis.     

The MUR3 gene of Arabidopsis encodes a xyloglucan galactosyltransferase that is evolutionarily related to animal exostosins

Xyloglucans are the principal glycans that interlace cellulose microfibrils in most flowering plants. The mur3 mutant of Arabidopsis contains a severely altered structure of this polysaccharide because of the absence of a conserved alpha-L-fucosyl-(1-->2)-beta-D-galactosyl side chain and excessive galactosylation at an alternative xylose residue. Despite this severe structural alteration, mur3 plants were phenotypically normal and exhibited tensile strength in their inflorescence stems comparable to that of wild-type plants. The MUR3 gene was cloned positionally and shown to encode a xyloglucan galactosyltransferase that acts specifically on the third xylose residue within the XXXG core structure of xyloglucan. MUR3 belongs to a large family of type-II membrane proteins that is evolutionarily conserved among higher plants. The enzyme shows sequence similarities to the glucuronosyltransferase domain of exostosins, a class of animal glycosyltransferases that catalyze the synthesis of heparan sulfate, a glycosaminoglycan with numerous roles in cell differentiation and development. This finding suggests that components of the plant cell wall and of the animal extracellular matrix are synthesized by evolutionarily related enzymes even though the structures of the corresponding polysaccharides are entirely different from each other.     

Inward and outward potassium currents through the same chimeric human Kv channel

Voltage-gated ion channels are among the most intensely studied membrane proteins today and a variety of techniques has led to a basic mapping of functional roles onto specific regions of their structure. The architecture of the proteins appears to be modular and segments associated with voltage sensing and the pore lining have been identified. However, the means by which movement of the sensor is transduced into channel opening is still unclear. In this communication, we report on a chimeric potassium channel construct which can function in two distinct operating voltage ranges, spanning both inward and outward currents with a non-conducting intervening regime. The observed changes in operating range could be brought about by perturbing either the direction of sensor movement or the process of transducing movements of the sensor into channel opening and closing. The construct could thus provide a means to identify the machinery underlying these processes.     

Intracellular Ca2+ signaling and human disease: the hunt begins with Huntington's

Huntingtin, a protein altered by polyglutamine expansion in Huntington's disease (Httexp), forms a signaling complex with the InsP3R, an intracellular calcium channel, and Htt-associated protein 1A (HAP1A). The addition of Httexp increases the InsP3R sensitivity to InsP3, which subsequently makes neurons hyperresponsive to stimulation and presumably more prone to neurodegenerative processes.     

Production of Recombinant Human Bile Salt Stimulated Lipase and Its Variant inPichia pastoris

hBSSL and its truncated variant hBSSL-C cDNA clones were expressed inPichia pastorisusing two different signal peptides, native signal peptide and invertase signal peptide, respectively, to facilitate secretion of the recombinant proteins into the culture medium. Both recombinant proteins were secreted into the culture medium to a level of 45–50 mg/liter in shake flask cultures. Native signal peptide of hBSSL was recognized inP. pastorisand was cleaved at the same site as in humans. The level of expression of the hBSSL gene was found to be dependent on the number of its copies integrated into the host chromosome. The multicopy transformant clone was found to be very stable. When grown and induced in a fermentor, the level of accumulation of the recombinant hBSSL in the culture medium improved from 50 mg/liter in shake flask cultures to 300 mg/liter. The recombinant hBSSL purified from the culture supernatant was found to be similar to the native hBSSL in its biochemical properties except for the lectin-binding profile.     

Use of Mycobacterium smegmatis Deficient in ADP-Ribosyltransferase as Surrogate for Mycobacterium tuberculosis in Drug Testing and Mutation Analysis

Rifampicin (Rif) is a first line drug used for tuberculosis treatment. However, the emergence of drug resistant strains has necessitated synthesis and testing of newer analogs of Rif. Mycobacterium smegmatis is often used as a surrogate for M. tuberculosis. However, the presence of an ADP ribosyltransferase (Arr) in M. smegmatis inactivates Rif, rendering it impractical for screening of Rif analogs or other compounds when used in conjunction with them (Rif/Rif analogs). Rifampicin is also used in studying the role of various DNA repair enzymes by analyzing mutations in RpoB (a subunit of RNA polymerase) causing Rif resistance. These analyses use high concentrations of Rif when M. smegmatis is used as model. Here, we have generated M. smegmatis strains by deleting arr (Δarr). The M. smegmatis Δarr strains show minimum inhibitory concentration (MIC) for Rif which is similar to that for M. tuberculosis. The MICs for isoniazid, pyrazinamide, ethambutol, ciprofloxacin and streptomycin were essentially unaltered for M. smegmatis Δarr. The growth profiles and mutation spectrum of Δarr and, Δarr combined with ΔudgB (udgB encodes a DNA repair enzyme that excises uracil) strains were similar to their counterparts wild-type for arr. However, the mutation spectrum of ΔfpgΔarr strain differed somewhat from that of the Δfpg strain (fpg encodes a DNA repair enzyme that excises 8-oxo-G). Our studies suggest M. smegmatis Δarr strain as an ideal model system in drug testing and mutation spectrum determination in DNA repair studies.