Transcriptional status of known and novel genes tagged with consensus of 33.15 repeat loci employing minisatellite-associated sequence amplification (MASA) and real-time PCR in water buffalo, Bubalus bubalis

We conducted minisatellite-associated sequence amplification (MASA) with an oligo (5' CACCTCTCCACCTGCC 3') based on consensus of 33.15 repeat loci using cDNA from the testis, ovary, spleen, kidney, heart, liver, and lung of water buffalo Bubalus bubalis and uncovered 25 amplicons of six different sizes (1,263, 846/847, 602, 576, 487, and 324 base pairs). These fragments, cloned and sequenced, were found to represent several functional, regulatory, and structural genes. Blast search of all the 25 amplicons showed homologies with 43 transcribing genes across the species. Of these, the 846/847-bp fragment, having homology with the adenylate kinase gene, showed nucleotide changes at six identical places in the ovary and testis. The 1,263; 324; and 487-bp fragments showed homology with the secreted modular calcium binding protein (SMOC-1), leucine-rich repeat neuronal 6A (LRRN6A) mRNA, and human TTTY5 mRNA, respectively. Real-time PCR showed maximum expression of AKL, LRRN6A, and T-cell receptor gamma (TCR-gamma)-like genes in the testis, SMOC-1 in the liver, and the T-cell receptor-like (TCRL) gene in the spleen compared to those used as endogenous control. We construe that these genes have evolved from a common progenitor and conformed to various biological functions during the course of evolution. MASA approach coupled with real-time PCR has potentials to uncover accurate expression of a large number of genes within and across the species circumventing the screening of cDNA library.     

Metal Deficiency Increases Aberrant Hydrophobicity of Mutant Superoxide Dismutases That Cause Amyotrophic Lateral Sclerosis

The mechanisms by which mutant variants of Cu/Zn-superoxide dismutase (SOD1) cause familial amyotrophic lateral sclerosis are not clearly understood. Evidence to date suggests that altered conformations of amyotrophic lateral sclerosis mutant SOD1s trigger perturbations of cellular homeostasis that ultimately cause motor neuron degeneration. In this study we correlated the metal contents and disulfide bond status of purified wild-type (WT) and mutant SOD1 proteins to changes in electrophoretic mobility and surface hydrophobicity as detected by 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence. As-isolated WT and mutant SOD1s were copper-deficient and exhibited mobilities that correlated with their expected negative charge. However, upon disulfide reduction and demetallation at physiological pH, both WT and mutant SOD1s underwent a conformational change that produced a slower mobility indicative of partial unfolding. Furthermore, although ANS did not bind appreciably to the WT holoenzyme, incubation of metal-deficient WT or mutant SOD1s with ANS increased the ANS fluorescence and shifted its peak toward shorter wavelengths. This increased interaction with ANS was greater for the mutant SOD1s and could be reversed by the addition of metal ions, especially Cu²⁺, even for SOD1 variants incapable of forming the disulfide bond. Overall, our findings support the notion that misfolding associated with metal deficiency may facilitate aberrant interactions of SOD1 with itself or with other cellular constituents and may thereby contribute to neuronal toxicity.The sequence of events by which more than 100 mutations in the gene encoding Cu/Zn-superoxide dismutase (SOD1)³ cause familial forms of amyotrophic lateral sclerosis (ALS) is unknown. Studies of purified SOD1 proteins and cellular or rodent models of SOD1-linked ALS suggest that impaired metal ion binding or misfolding of mutant SOD1 proteins in the cellular environment may be related to their toxicity (1–10). Available evidence suggests that partially unfolded mutant SOD1 species could contribute to motor neuron death by promoting abnormal interactions that produce cellular dysfunction (11–16).In previous studies we characterized physicochemical properties of 14 different biologically metallated ALS SOD1 mutants (17) and demonstrated altered thermal stabilities of these mutants compared with wild-type (WT) SOD1 (18). These “as-isolated” SOD1 proteins, which contain variable amounts of copper and zinc, were broadly grouped into two classes based on their ability to incorporate and retain metal ions with high affinity. WT-like SOD1 mutants retain the ability to bind copper and zinc ions and exhibit dismutase activity similar to the normal enzyme, whereas metal binding region (MBR) mutants are significantly deficient in copper and/or zinc (17, 19). We also observed that ALS-associated SOD1 mutants were more susceptible than the WT enzyme to reduction of the intrasubunit disulfide bond between Cys-57 and Cys-146 (20). The significance of these results is that even WT-like mutants, which exhibit a nearly normal backbone structure (21–23), may be vulnerable to destabilizing influences in vivo. Our group and others subsequently showed that the mutant SOD1 proteins share a susceptibility to increased hydrophobicity under conditions that reduce disulfide bonds and/or chelate metal ions (5) and that similar hydrophobic species exist in tissue lysates from mutant SOD1 transgenic mice (4–6). One consequence of such hydrophobic exposure could be the facilitation of abnormal interactions between the mutant enzymes and other cellular constituents (e.g. chaperones, mitochondrial components, or other targets), which might influence pathways leading to motor neuron death (15, 16, 24–27).Accumulating evidence suggests that metal deficiency of SOD1 is an important factor that can influence SOD1 aggregation or neurotoxicity (4, 28–33), but the metal-deficient states of SOD1 that are most relevant to ALS remain unclear. Zinc-deficient, copper-replete SOD1 species, which can be produced in vitro by adding copper to SOD1 that has been stripped of its metal ions at acidic pH, were shown to be toxic to motor neurons in culture (28). However, it has not been shown that zinc-deficient, copper-replete SOD1 is produced in vivo as a consequence of ALS mutations, and loading of copper into SOD1 by the copper chaperone for SOD1 (CCS) is not required for toxicity (34, 35). Furthermore, the MBR mutants have a disrupted copper site and have been found to be severely deficient in both zinc and copper (17, 30), yet expression of these SOD1s still produces motor neuron disease (1, 2, 30, 34, 36, 37).When recombinant human SOD1 was overexpressed in insect cells, we instead observed zinc-replete but copper-deficient species for most WT-like mutants, probably because the capacity of the copper-loading mechanism was exceeded (17). These preparations indicate that zinc can be efficiently incorporated into many WT-like mutants in vivo, and much of it is retained after purification. Furthermore, these copper-deficient biologically metallated proteins may be useful reagents to assess the influence of copper binding upon other properties of SOD1 mutants that may be relevant to their neurotoxicity.We previously observed that reduction of the Cys-57—Cys-146 disulfide bond facilitates the ability of metal chelators to alter the electrophoretic mobility and to increase the hydrophobicity of SOD1 mutants (5). This is consistent with the known properties of this linkage to stabilize the dimeric interface, to orient Arg-143 via a hydrogen bond from the carbonyl oxygen of Cys-57 to Arg-143-NH₂, and to prevent metal ion loss (38–40). However, it remains unclear whether the Cys-57—Cys-146 bond is required to prevent abnormal SOD1 hydrophobic exposure or whether the aberrant conformational change primarily results from metal ion loss. Ablation of the disulfide bond by the experimental (non-ALS) mutants C57S and C146S provides useful reagents to test the relative influence of the disulfide bond and copper binding upon SOD1 properties.In this study we sought to correlate the consequences of copper deficiency, copper and zinc deficiency, and disulfide reduction upon the hydrodynamic behavior and surface hydrophobicity of WT and representative mutant SOD1 enzymes (Fig. 1A). We quantitated the metal contents of as-isolated SOD1 proteins, detected changes in conformation or metal occupancy using native PAGE to assess their electrophoretic mobility, a measure of global conformational change, and correlated these changes to hydrophobic exposure using 1-anilinonaphthalene-8-sulfonic acid (ANS), which is very sensitive to local conformational changes. ANS is a small amphipathic dye (Fig. 1B) that has been used as a sensitive probe to detect hydrophobic pockets on protein surfaces (41–44). Free ANS exhibits only weak fluorescence that is maximal near 520 nm, but when ANS binds to a hydrophobic site in a partially or fully folded protein, the fluorescence peak increases in amplitude and shifts to a shorter wavelength (42). ANS also has an anionic sulfonate group that can interact with cationic groups (e.g. Arg or Lys residues) through ion-pair formation which may be further strengthened by hydrophobic interactions (43–46).Open in a separate windowFIGURE 1.A, WT SOD1 structure showing the position of the C57-C146 intrasubunit disulfide bond (S–S, yellow), bound copper and zinc ions, and ALS mutant residues. The residues altered in A4V, G85R, G93A, D124V, and S134N SOD1s are indicated as green spheres. The backbone of the β-barrel core and the loops is shown in a rainbow color, from blue at the amino terminus to red at the carboxyl terminus. The figure was generated using PyMOL (84) and PDB entry 1HL5 (22). B, chemical structure of ANS fluorophore.To evaluate further the importance of metal ion binding, we measured spectral changes related to the binding of cobalt and copper to the same SOD1 proteins. We observed that as-isolated WT-like mutants containing zinc could interact with copper ions to produce an electrophoretic mobility and decreased hydrophobicity resembling that of the fully metalated holo-WT SOD1. In contrast, we saw no evidence for copper binding to MBR mutants in a manner that alters their hydrodynamic properties or their hydrophobicity. Our data suggest that binding of both copper and zinc are important determinants of SOD1 conformation and that perturbation of such binding may be relevant to the ALS disease process.     

Inter and intraspecific genetic diversity (RAPD) among three most frequent species of macrofungi (Ganoderma lucidum,Leucoagricus sp. and Lentinus sp.) of Tropical forest of Central India

In present study seven RAPD primers were used to access the diversity within and among twelve populations of three mushroom species Ganoderma lucidum, leucoagaricus sp. and Lentinus sp. Total of 111 bands were scored by 7 RAPD primers in 30 accessions of three mushroom species collected from different sampling sites of central India. Total 111 bands were generated using seven primers which were F-1, OPG-06, OPC-07, OPD-08, OPA-02, OPD-02, OPB-10. All 111 bands were polymorphic in nature (100%). Therefore, it revealed that the used primers had sufficient potency for population studies and 30 accessions had higher genetic differences among each other. In best of the knowledge, this is the first report, which accesses the genetic diversity between three mushroom species (Gd Ganoderma lucidum, Lg Leucoagaricus sp., Ls Lentinus). The polymorphic percentage ranged from 3.60 to 23% within twelve populations, while polymorphic percentage among group was 40.56, among population within groups was 41.12 and within population was 18.32. This indicated that the genetic diversity within the population was very low, but slightly higher in the populations of three species. Among three groups representing Gd., Lg and Ls, Among populations within groups shown highest percentage of variation (Pv?=?41.12) while within populations, the lowest percentage of variation (18.32) was observed. This result also support that the highest genetic variation was present among groups in comparison to among the population within a species and lowest genetic variation was observed within the population.     

Restriction enzyme digestion of heterochromatin inDrosophila nasuta

In situ digestion of metaphase and polytene chromosomes and of interphase nuclei in different cell types ofDrosophila nasuta with restriction enzymes revealed that enzymes like AluI, EcoRI, HaeIII, Sau3a and SinI did not affect Giemsa-stainability of heterochromatin while that of euchromatin was significantly reduced; TaqI and SalI digested both heterochromatin and euchromatin in mitotic chromosomes. Digestion of genomic DNA with AluI, EcoRI, HaeIII, Sau3a and KpnI left a 23 kb DNA band undigested in agarose gels while withTaqI, no such undigested band was seen. TheAluI resistant 23 kb DNA hybridized insitu specifically with the heterochromatic chromocentre. It appears that the digestibility of heterochromatin region in genome ofDrosophila nasuta with the tested restriction enzymes is dependent on the availability of their recognition sites.     

Genetic similarity among Trifolium species based on isozyme banding pattern

The study deals with similarity among 25 species of the genus Trifolium represented by 134 accessions. Clustering of the species based on isozyme banding pattern of five enzymes revealed that T. repens was distinctly different from other species. T. repens and T. retusum formed independent clusters. The group of species comprising of T. pratense, T. cherleri, T. spumosum, T. subterraneum, T. resupinatum, T. alexandrinum, T. echinatum, T. constantinopolitanum and T. tembense exhibited considerable similarity to the second cluster. This group joined another group of five species, i.e. T. nigrescens, T. glomeratum, T. apertum, T. alpestre and T. hybridum with nearly 50% similarity. T. purpureum, T. hirtum, T. campestre, T. incarnatum, and T. argutum grouped separately. There was no marked difference for banding pattern among T. alexandrinum genotypes. T. alexandrinum showed close affinity with T. subterraneum and T. resupinatum. T. lappaceum, T. diffusum, T. campestre, T. incarnatum and T. argutum showed only 44.8% similarity with other Trifolium species. Grouping together of accessions belonging to individual species indicated that incompatibility among species under study had restricted interspecific hybridization. Species belonging to subgenus Lotoidea clustered with species of subgenus Trifolium. Chonosemium species T. campestre formed one cluster with two Trifolium species T. hirtum and T. incarnatum. T. nigrescens was placed quite apart from the T. repens.     

The Saccharomyces cerevisiae YLL012/YEH1, YLR020/YEH2, and TGL1 genes encode a novel family of membrane-anchored lipases that are required for steryl ester hydrolysis

Sterol homeostasis in eukaryotic cells relies on the reciprocal interconversion of free sterols and steryl esters. The formation of steryl esters is well characterized, but the mechanisms that control steryl ester mobilization upon cellular demand are less well understood. We have identified a family of three lipases of Saccharomyces cerevisiae that are required for efficient steryl ester mobilization. These lipases, encoded by YLL012/YEH1, YLR020/YEH2, and TGL1, are paralogues of the mammalian acid lipase family, which is composed of the lysosomal acid lipase, the gastric lipase, and four novel as yet uncharacterized human open reading frames. Lipase triple-mutant yeast cells are completely blocked in steryl ester hydrolysis but do not affect the mobilization of triacylglycerols, indicating that the three lipases are required for steryl ester mobilization in vivo. Lipase single mutants mobilize steryl esters to various degrees, indicating partial functional redundancy of the three gene products. Lipase double-mutant cells in which the third lipase is expressed from the inducible GAL1 promoter have greatly reduced steady-state levels of steryl esters, indicating that overexpression of any of the three lipases is sufficient for steryl ester mobilization in vivo. The three yeast enzymes constitute a novel class of membrane-anchored lipases that differ in topology and subcellular localization.     

In silico high-throughput virtual screening and molecular dynamics simulation study to identify inhibitor for AdeABC efflux pump of Acinetobacter baumannii

Emergence of multi-drug resistant strains of Acinetobacter baumannii has caused significant health problems and is responsible for high morbidity and mortality. Overexpression of AdeABC efflux system is one of the major mechanisms. In this study, we have focused on overcoming the drug resistance by identifying inhibitors that can effectively bind and inhibit integral membrane protein, AdeB of this efflux pump. We performed homology modeling to generate structure of AdeB using MODELLER v9.16 followed by model refinement using 3D-Refine tool and validated using PSVS, ProsaWeb, ERRAT, etc. The energy minimization of modeled protein was done using Protein preparation wizard application included in Schrodinger suite. High-throughput virtual screening of 159,868 medicinal compounds against AdeB was performed using three sequential docking modes (i.e. HTVS, SP and XP). Furthermore, absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis was done using QIKPROP. The selected 123 compounds were further analyzed for binding free energy by molecular mechanics (using prime MM-GBSA). We have also performed enrichment study (ROC curve analysis) to validate our docking results. The selected molecule and its interaction with AdeB were validated by molecular dynamics simulation (MDS) using GROMACS v5.1.4. In silico high-throughput virtual screening and MDS validation identified ZINC01155930 ((4R)-3-(cycloheptoxycarbonyl)-4-(4-etochromen-3-yl)-2-methyl-4,6,7,8-tetrahydroquinolin-5-olate) as a possible inhibitor for AdeB. Hence, it might be a suitable efflux pump inhibitor worthy of further investigation in order to be used for controlling infections caused by Acinetobacter baumannii.