Expression of mdr49 and mdr65 multidrug resistance genes in larval tissues of Drosophila melanogaster under normal and stress conditions

In situ expression of 2 multidrug resistance genes, mdr49 and mdr65, of Drosophila melanogaster was examined in wild-type third instar larval tissues under physiological conditions and after heat shock or colchicine feeding. Expression of these 2 genes was also examined in tumorous tissues of lethal (2) giant larvae I(2)gl4 mutant larvae. These 2 mdr genes show similar constitutive expression in different larval tissues under physiological conditions. However, they are induced differentially by endogenous (tumorous growth) and exogenous stresses (colchcine feeding or heat shock): whereas heat shock and colchicine feeding induce mdr49, tumorous condition is accompanied by enhanced expression of mdr49 and mdr65 genes.     

A novel method of estimation of lipophilicity using distance-based topological indices: dominating role of equalized electronegativity

Attempt is made to propose yet another method of estimating lipophilicity of a heterogeneous set of 223 compounds. The method is based on the use of equalized electronegativity along with topological indices. It was observed that excellent results are obtained in multiparametric regression upon introduction of indicator parameters. The results are discussed critically on the basis various statistical parameters.     

Survey and analysis of microsatellites in the silkworm, Bombyx mori: frequency, distribution, mutations, marker potential and their conservation in heterologous species

We studied microsatellite frequency and distribution in 21.76-Mb random genomic sequences, 0.67-Mb BAC sequences from the Z chromosome, and 6.3-Mb EST sequences of Bombyx mori. We mined microsatellites of >/=15 bases of mononucleotide repeats and >/=5 repeat units of other classes of repeats. We estimated that microsatellites account for 0.31% of the genome of B. mori. Microsatellite tracts of A, AT, and ATT were the most abundant whereas their number drastically decreased as the length of the repeat motif increased. In general, tri- and hexanucleotide repeats were overrepresented in the transcribed sequences except TAA, GTA, and TGA, which were in excess in genomic sequences. The Z chromosome sequences contained shorter repeat types than the rest of the chromosomes in addition to a higher abundance of AT-rich repeats. Our results showed that base composition of the flanking sequence has an influence on the origin and evolution of microsatellites. Transitions/transversions were high in microsatellites of ESTs, whereas the genomic sequence had an equal number of substitutions and indels. The average heterozygosity value for 23 polymorphic microsatellite loci surveyed in 13 diverse silkmoth strains having 2-14 alleles was 0.54. Only 36 (18.2%) of 198 microsatellite loci were polymorphic between the two divergent silkworm populations and 10 (5%) loci revealed null alleles. The microsatellite map generated using these polymorphic markers resulted in 8 linkage groups. B. mori microsatellite loci were the most conserved in its immediate ancestor, B. mandarina, followed by the wild saturniid silkmoth, Antheraea assama.     

Isolation of mannose-binding C-type lectin from Heliothis virescens pupae

A mannose-binding C-type lectin (MBL) was isolated by affinity chromatography from Heliothis virescens immune pupal hemolymph. The immune pupal hemolymph was obtained after bacterial injection of live Enterobacter cloacae bacteria. MBL in mammals acts as an opsonin for phagocytosis and activates the lectin complement pathway of the innate immune response, which leads to killing of gram-negative bacteria and enveloped viruses. The affinity-purified and reduced pupal MBL showed a single band of 36 kDa by SDS-PAGE (12% gel). A dot-immunoblot ELISA (using guinea pig anti-MBL IgG as primary antibody) demonstrated specificity of the antibody for the affinity-purified pupal MBL. The immune pupal hemolymph contained 21 microg of MBL per ml of hemolymph. The amino acid composition of the purified pupal MBL was determined with high amounts of arginine and histidine detected. The presence of MBL in insect pupae has not before been reported and could be important in pupal innate immunity to bacterial infection.     

Platelet storage under in vitro condition is associated with calcium-dependent apoptosis-like lesions and novel reorganization in platelet cytoskeleton

Platelets are cleared from circulation after a life span of 8-10 days. The molecular mechanisms underlying platelet senescence remain poorly characterized. Here we report that, progressive functional impairment in the platelets incubated in vitro in a plasma-free isotonic medium for up to 24 h at 37 degrees C is associated with release of cytochrome c from platelet mitochondria and cleavage of procaspase-9, but without evidence of caspase-3 activation. Concomitantly, there was proteolysis of survival proteins like focal adhesion kinase, Src, gelsolin, and specific cytoskeleton-associated peptides, in a manner regulated by extracellular calcium and calpain activity. Cytoskeleton played a critical role as evidenced from the association of these proteins and their degradation products, as well as procaspase-3 and the actin regulatory small GTPase, CDC42Hs, with the cytoskeleton of the stored platelets. The cytoskeletal enrichment with specific proteins was not associated with increase in the content of F-actin and was cytochalasin-resistant, thus signifying a novel mechanism of interaction of the translocating proteins with the pre-existing cytoskeleton. There was progressive exposure of phosphatidylserine on the outer leaflet of platelet membrane and specific electron microscopic changes suggestive of apoptotic lesions. Based on these observations we discuss the caspase-independent but calpain-mediated signaling events in the stored platelets resembling the features of apoptosis in the nucleated cells.     

Evidences showing ultraviolet-B radiation-induced damage of DNA in cyanobacteria and its detection by PCR assay

Impact of ultraviolet-B radiation in causing the damages to the DNA of the cyanobacterium, Anabaena strain BT2 has been investigated. Exposure of genomic DNA (in vitro) to UV-B radiation for 1 h did not cause any shift in the absorption peak (lambda(max)) but more than 30% increase in absorbance was noticed in comparison to untreated control DNA (no exposure to UV-B). This increase in absorbance in a way may be comparable to typical hypochromic effect but there was no decrease in absorbance following transfer of UV-B-treated DNA to fluorescent light or in the dark. That the damaging effect of UV-B radiation on native structure of DNA is indeed real was also evident from the PCR-based assay such as RAPD, rDNA amplification, and ARDRA. Template activity of UV-B-treated genomic DNA was drastically inhibited, there was no amplification in RAPD assay after prior exposure of DNA to UV-B for 60 min. Only one band of approximately 400 bp was observed even after 60 min of exposure which suggests that certain segment of DNA strand is resistant to UV-B effects. Similar to the effects on RAPD profile, amplification of rDNA was significantly inhibited following exposure of genomic DNA to UV-B. Our findings clearly demonstrate that UV-B does affect the DNA of cyanobacteria and the killings of these microbes might be due to the irreversible damages caused to DNA by this high energy radiation. It is felt that PCR assay may be conveniently used for screening the damages caused to DNA by UV-B radiation in cyanobacteria and other microorganisms.     

Nucleic acid amplification of Mycobacterium tuberculosis complex DNA from archival fine needle aspiration smear scrapings vs. fresh fine needle aspirates of tuberculous lymphadenitis

OBJECTIVE: To assess the efficacy of the nucleic acid amplification (NAA) technique for Mycobacterium tuberculosis (MTB) complex from archival fine needle aspirate (FNA) smear scrapings of confirmed cases of extrapulmonary tuberculosis (EPTB) for a retrospective diagnosis of EPTB as compared to NAA from fresh FNA material from the same cases. STUDY DESIGN: Smear scrapings from 51 cases; 33 cases of tuberculous lymphadenitis (from patients who had undergone NAA 1 year before for MTB from fresh FNA material); 13 negative controls from nontuberculous, archival FNA smears; and 5 known acid-fast bacilli (AFB)-positive sputum smears, were subjected to NAA using the IS6110 primer sequence of M tuberculosis. Ziehl-Neelsen staining was done in all the smears. RESULTS: Of the 33 cases of tuberculous lymphadenitis, 15 (45.4%) were AFB positive and 18 (64.5%) AFB negative. MTB NAA was positive in 73.3% (11 of 15 AFB-positive cases) in the freshly aspirated material and was observed in 60% (9 of 15 AFB-positive cases) when done on DNA extracted from the archival smear scrapings of the same cases. Similarly, in the 18 AFB-negative cases, MTB NAA positivity was 72.2% (13 of 18) on fresh material and 44.4% (8 of 18) on archival smear scrapings from the same AFB-negative cases. Overall NAA positivity was 51.5% for archival smear scrapings as compared to 71% for fresh FNA of the same cases. CONCLUSION: Low NAA sensitivity of MTB DNA in archival material of known tuberculous cases limits the routine use of NAA based retrospective molecular diagnosis of MTB complex.     

Crystal structure of a highly acidic neurotoxin from scorpion Buthus tamulus at 2.2A resolution reveals novel structural features

The crystal structure of a highly acidic neurotoxin from the scorpion Buthus tamulus has been determined at 2.2A resolution. The amino acid sequence determination shows that the polypeptide chain has 64 amino acid residues. The pI measurement gave a value of 4.3 which is one of the lowest pI values reported so far for a scorpion toxin. As observed in other alpha-toxins, it contains four disulphide bridges, Cys12-Cys63, Cys16-Cys36, Cys22-Cys46, and Cys26-Cys48. The crystal structure reveals the presence of two crystallographically independent molecules in the asymmetric unit. The conformations of two molecules are identical with an r.m.s. value of 0.3A for their C(alpha) tracings. The overall fold of the toxin is very similar to other scorpion alpha-toxins. It is a betaalphabetabeta protein. The beta-sheet involves residues Glu2-Ile6 (strand beta1), Asp32-Trp39 (strand beta3) and Val45-Val55 (strand beta4). The single alpha-helix formed is by residues Asn19-Asp28 (alpha2). The structure shows a trans peptide bond between residues 9 and 10 in the five-membered reverse turn Asp8-Cys12. This suggests that this toxin belongs to classical alpha-toxin subfamily. The surface features of the present toxin are highly characteristic, the first (A-site) has residues, Phe18, Trp38 and Trp39 that protrude outwardly presumably to interact with its receptor. There is another novel face (N-site) of this neurotoxin that contains several negatively charged residues such as, Glu2, Asp3, Asp32, Glu49 and Asp50 which are clustered in a small region of the toxin structure. On yet another face (P-site) in a triangular arrangement, with respect to the above two faces there are several positively charged residues, Arg58, Lys62 and Arg64 that also protrude outwardly for a potentially potent interaction with other molecules. This toxin with three strong features appears to be one of the most toxic molecules reported so far. In this sense, it may be a new subclass of neurotoxins with the largest number of hot spots.     

MOLECULAR PHYLOGENY OF GRACILARIA SPECIES INFERRED FROM MOLECULAR MARKERS BELONGING TO THREE DIFFERENT GENOMES1

The nucleotide sequence data of molecular markers 18S rRNA, RUBISCO spacer, and cox2‐3 intergenic spacer were integrated to infer the phylogeny of Gracilaria species, collected from the western coast of India, reducing the possibility of misidentification and providing greater phylogenetic resolution. A phylogenetic tree was constructed using cox2‐3 and RUBISCO spacer sequences, exhibiting the same clustering but differing slightly from that of the rRNA‐based phylogenetic tree. The phylogeny inferred from the combined data set confers an analogous pattern of clustering, compared with those of trees constructed from individual data sets. The combined data set resulted in a phylogeny with better resolution, which supported the clade with higher consistency index, retention index, and bootstrap values. It was observed that Gracilaria foliifera (Forssk.) Børgesen is closer to G. corticata (J. Agardh) J. Agardh varieties, while G. salicornia (C. Agardh) E. Y. Dawson and G. fergusonii J. Agardh both originated from the same clade. The position of G. textorii (Suringar) De Toni faltered and toppled between G. salicornia and G. dura (C. Agardh) J. Agardh; however, G. gracilis (Stackh.) M. Steentoft, L. M. Irvine et W. F. Farnham was evidently distant from the rest of the species.