Application of magnetic techniques in the field of drug discovery and biomedicine

Magnetic separation technology, using magnetic particles, is quick and easy method for sensitive and reliable capture of specific proteins, genetic material and other biomolecules. The technique offers an advantage in terms of subjecting the analyte to very little mechanical stress compared to other methods. Secondly, these methods are non-laborious, cheap and often highly scalable. Moreover, techniques employing magnetism are more amenable to automation and miniaturization. Now that the human genome is sequenced and about 30,000 genes are annotated, the next step is to identify the function of these individual genes, carrying out genotyping studies for allelic variation and SNP analysis, ultimately leading to identification of novel drug targets. In this post-genomic era, technologies based on magnetic separation are becoming an integral part of todays biology laboratory. This article briefly reviews the selected applications of magnetic separation techniques in the field of biotechnology, biomedicine and drug discovery.     

Side effects of genome structural changes

The first extensive catalog of structural human variation was recently released. It showed that large stretches of genomic DNA that vary considerably in copy number were extremely abundant. Thus it is conceivable that they play a major role in functional variation. Consistently, genomic insertions and deletions were shown to contribute to phenotypic differences by modifying not only the expression levels of genes within the aneuploid segments but also of normal copy-number neighboring genes. In this report, we review the possible mechanisms behind this latter effect.     

Mosaic pbpX genes of major clones of penicillin-resistant Streptococcus pneumoniae have evolved from pbpX genes of a penicillin-sensitive Streptococcus oralis

Penicillin-resistant clinical isolates of Streptococcus pneumoniae contain mosaic penicillin-binding protein (PBP) genes that encode PBPs with decreased affinity for β-lactam antibiotics. The mosaic blocks are believed to be the result of gene transfer of homologous PBP genes from related penicillin-resistant species. We have now identified a gene homologous to the pneumococcal PBP2x gene (pbpX) in a penicillin-sensitive Streptococcus oralis isolate M3 from South Africa that diverged by almost 20% from pbpX of penicillin-sensitive pneumococci, and a central sequence block of a mosaic pbpX gene of Streptococcus mitis strain NCTC 10712. In contrast, it differed by only 2-4% of the 1 to 1.5 kb mosaic block in pbpX genes of three genetically unrelated penicillin-resistant S. pneumoniae isolates, two of them representing clones of serotype 6B and 23F, which are prevalent in Spain and are also already found in other countries. With low concentrations of cefotaxime, transformants of the sensitive S. pneumoniae R6 strain could be selected containing pbpX genes from either S. mitis NCTC 10712 or S. oralis M3, demonstrating that genetic exchange can already occur between β-lactam-sensitive species. These data are in agreement with the assumption that PBPs as penicillin-resistance determinants have evolved by the accumulation of point mutations in genes of sensitive commensal species.     

The use of invertebrate functional groups to characterize ecosystem attributes in selected streams and rivers in south Brazil

An analysis conducted at nine stream/river sites in the Atlantic Forest region in the State of Paraná, Brazil used macroinvertebrate functional feeding group (FFG) assessments to evaluate ecological condition of the sites. The FFG approach categorizes qualitative macroinvertebrate collections according to their morphological-behavioral adaptations for food acquisition (e.g. scrapers that harvest non-filamentous, attached algae from stable surfaces in flowing water). FFG ratios were employed as surrogates for stream/river ecosystem attributes: balance between autotrophy and heterotrophy; linkage between riparian inputs of coarse particulate organic matter and in-stream food webs; relative dominance of fine particulate organic matter in transport (suspended load) compared to that deposited in the sediments; and geomorphic stability of the channel. The analyses indicated that all nine sites were heterotrophic, six of the nine carried expected levels of suspended organic load and showed below expected linkage with riparian inputs, and in only two were stable substrates limiting. The implications of the findings and recommendations for further analysis and modifications of the protocol are discussed.     

Docking studies on novel analogues of 8 methoxy fluoroquinolones against GyrA mutants of Mycobacterium tuberculosis

Background

Modeling of transmembrane domains (TMDs) requires correct prediction of interfacial residues for in-silico modeling and membrane insertion studies. This implies the defining of a target sequence long enough to contain interfacial residues. However, too long sequences induce artifactual polymorphism: within tested modeling methods, the longer the target sequence, the more variable the secondary structure, as though the procedure were stopped before the end of the calculation (which may in fact be unreachable). Moreover, delimitation of these TMDs can produce variable results with sequence based two-dimensional prediction methods, especially for sequences showing polymorphism. To solve this problem, we developed a new modeling procedure using the PepLook method. We scanned the sequences by modeling peptides from the target sequence with a window of 19 residues.

Results

Using sequences whose NMR-structures are already known (GpA, EphA1 and Erb2-HER2), we first determined that the hydrophobic to hydrophilic accessible surface area ratio (ASAr) was the best criterion for delimiting the TMD sequence. The length of the helical structure and the Impala method further supported the determination of the TMD limits. This method was applied to the IL-2Rβ and IL-2Rγ TMD sequences of Homo sapiens, Rattus norvegicus, Mus musculus and Bos taurus.

Conclusions

We succeeded in reducing the variation in the TMD limits to only 2 residues and in gaining structural information.     

Glucose lowering effect of transgenic human insulin-like growth factor-I from rice: <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis>studies

Background  

Human insulin-like growth factor-I (hIGF-I) is a growth factor which is highly resemble to insulin. It is essential for cell proliferation and has been proposed for treatment of various endocrine-associated diseases including growth hormone insensitivity syndrome and diabetes mellitus. In the present study, an efficient plant expression system was developed to produce biologically active recombinant hIGF-I (rhIGF-I) in transgenic rice grains.     

Disease-associated glycosylated molecular variants of human C-reactive protein activate complement-mediated hemolysis of erythrocytes in tuberculosis and Indian visceral leishmaniasis

Human C-reactive protein (CRP), as a mediator of innate immunity, removed damaged cells by activating the classical complement pathway. Previous studies have successfully demonstrated that CRPs are differentially induced as glycosylated molecular variants in certain pathological conditions. Affinity-purified CRPs from two most prevalent diseases in India viz. tuberculosis (TB) and visceral leishmaniasis (VL) have differential glycosylation in their sugar composition and linkages. As anemia is a common manifestation in TB and VL, we assessed the contributory role of glycosylated CRPs to influence hemolysis via CRP-complement-pathway as compared to healthy control subjects. Accordingly, the specific binding of glycosylated CRPs with erythrocytes was established by flow-cytometry and ELISA. Significantly, deglycosylated CRPs showed a 7–8-fold reduced binding with erythrocytes confirming the role of glycosylated moieties. Scatchard analysis revealed striking differences in the apparent binding constants (10⁴–10⁵ M⁻¹) and number of binding sites (10⁶–10⁷sites/erythrocyte) for CRP on patients’ erythrocytes as compared to normal. Western blotting along with immunoprecipitation analysis revealed the presence of distinct molecular determinants on TB and VL erythrocytes specific to disease-associated CRP. Increased fragility, hydrophobicity and decreased rigidity of diseased-erythrocytes upon binding with glycosylated CRP suggested membrane damage. Finally, the erythrocyte-CRP binding was shown to activate the CRP-complement-cascade causing hemolysis, even at physiological concentration of CRP (10 μg/ml). Thus, it may be postulated that CRP have a protective role towards the clearance of damaged-erythrocytes in these two diseases.     

Cereulide, the emetic toxin of Bacillus cereus, is putatively a product of nonribosomal peptide synthesis

AIMS: To determine if cereulide, the emetic toxin produced by Bacillus cereus, is produced by a nonribosomal peptide synthetase (NRPS). METHODS AND RESULTS: NC Y, an emetic strain of Bacillus cereus, was examined for a NRPS gene using PCR with primers recognizing a fragment of a NRPS gene from the cyanobacterium Microcystis. The amplicon was sequenced and compared with other gene sequences using BLAST analysis, which showed that the amplicon from strain NC Y was similar in sequence to peptide synthetase genes in other micro-organisms, including Bacillus subtilis and B. brevis, while no such sequence was found in the complete genome sequence of a nonemetic strain of B. cereus. Specific PCR primers were then designed and used to screen 40 B. cereus isolates previously implicated in outbreaks of foodborne illness. The isolates were also screened for toxin production using the MTT cell cytotoxicity assay. PCR and MTT assay screening of the B. cereus isolates revealed a high correlation between the presence of the NRPS gene and cereulide production. CONCLUSIONS: The results indicate that cereulide is produced by a NRPS complex. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to provide evidence identifying the mechanism of production of cereulide, the emetic toxin of B. cereus. The PCR primers developed in the study allow determination of the potential for cereulide production among isolates of B. cereus.     

The electronic fight against the schistosomes.

A DOS-compatible model for use on personal computers has been constructed for teaching in parasite epidemiology. The program is based on the Macdonald model of schistosome dynamics and enables simulation of the effect of a control campaign on a human worm load by reducing four transmission factors in the parasite life-cycle: (1) egg contamination, (2) snail lifetime, (3) exposure to cercariae and (4) adult worm lifetime.