Molecular Basis of Substrate Polyspecificity of the Candida albicans Mdr1p Multidrug/H+ Antiporter

The molecular basis of polyspecificity of Mdr1p, a major drug/H⁺ antiporter of Candida albicans, is not elucidated. We have probed the nature of the drug-binding pocket by performing systematic mutagenesis of the 12 transmembrane segments. Replacement of the 252 amino acid residues with alanine or glycine yielded 2/3 neutral mutations while 1/3 led to the complete or selective loss of resistance to drugs or substrates transported by the pump. Using the GlpT-based 3D–model of Mdr1p, we roughly categorized these critical residues depending on their type and localization, 1°/ main structural impact (“S” group), 2°/ exposure to the lipid interface (“L” group), 3°/ buried but not facing the main central pocket, inferred as critical for the overall H⁺/drug antiport mechanism (“M” group) and finally 4°/ buried and facing the main central pocket (“B” group). Among “B” category, 13 residues were essential for the large majority of drugs/substrates, while 5 residues were much substrate-specific, suggesting a role in governing polyspecificity (P group). 3D superposition of the substrate-specific MFS Glut1 and XylE with the MDR substrate-polyspecific MdfA and Mdr1p revealed that the B group forms a common substrate interaction core while the P group is only found in the 2 MDR MFS transporters, distributed into 3 areas around the B core. This specific pattern has let us to propose that the structural basis for polyspecificity of MDR MFS transporters is the extended capacity brought by residues located at the periphery of a binding core to accomodate compounds differing in size and type.     

Rationally Designed Transmembrane Peptide Mimics of the Multidrug Transporter Protein Cdr1 Act as Antagonists to Selectively Block Drug Efflux and Chemosensitize Azole-resistant Clinical Isolates of Candida albicans

Drug-resistant pathogenic fungi use several families of membrane-embedded transporters to efflux antifungal drugs from the cells. The efflux pump Cdr1 (Candida drug resistance 1) belongs to the ATP-binding cassette (ABC) superfamily of transporters. Cdr1 is one of the most predominant mechanisms of multidrug resistance in azole-resistant (AR) clinical isolates of Candida albicans. Blocking drug efflux represents an attractive approach to combat the multidrug resistance of this opportunistic human pathogen. In this study, we rationally designed and synthesized transmembrane peptide mimics (TMPMs) of Cdr1 protein (Cdr1p) that correspond to each of the 12 transmembrane helices (TMHs) of the two transmembrane domains of the protein to target the primary structure of the Cdr1p. Several FITC-tagged TMPMs specifically bound to Cdr1p and blocked the efflux of entrapped fluorescent dyes from the AR (Gu5) isolate. These TMPMs did not affect the efflux of entrapped fluorescent dye from cells expressing the Cdr1p homologue Cdr2p or from cells expressing a non-ABC transporter Mdr1p. Notably, the time correlation of single photon counting fluorescence measurements confirmed the specific interaction of FITC-tagged TMPMs with their respective TMH. By using mutant variants of Cdr1p, we show that these TMPM antagonists contain the structural information necessary to target their respective TMHs of Cdr1p and specific binding sites that mediate the interactions between the mimics and its respective helix. Additionally, TMPMs that were devoid of any demonstrable hemolytic, cytotoxic, and antifungal activities chemosensitize AR clinical isolates and demonstrate synergy with drugs that further improved the therapeutic potential of fluconazole in vivo.     

Relationship between BDNF expression in major striatal afferents,striatum morphology and motor behavior in the R6/2 mouse model of Huntington's disease

Patients with Huntington's disease (HD) and transgenic mouse models of HD show neuronal loss in the striatum as a major feature, which contributes to cognitive and motor manifestations. Reduced expression of the neurotrophin brain‐derived neurotrophic factor (BDNF) in striatal afferents may play a role in neuronal loss. How progressive loss of BDNF expression in different cortical or subcortical afferents contributes to striatal atrophy and behavioral dysfunction in HD is not known, and may best be determined in animal models. We compared age‐dependent alterations of BDNF mRNA expression in major striatal afferents from the cerebral cortex, thalamus and midbrain in the R6/2 transgenic mouse model of HD. Corresponding changes in striatal morphology were quantified using unbiased stereology. Changes in motor behavior were measured using an open field, grip strength monitor, limb clasping and a rotarod apparatus. BDNF expression in cortical limbic and midbrain striatal afferents is reduced by age 4 weeks, prior to onset of motor abnormalities. BDNF expression in motor cortex and thalamic afferents is reduced by 6 weeks, coinciding with early motor dysfunction and reduced striatum volume. BDNF loss in afferents progresses until death at 13–15 weeks, correlating with progressive striatal neuronal loss and motor abnormalities. Mutant huntingtin protein expression in R6/2 mice results in progressive loss of BDNF in both cortical and subcortical striatal afferents. BDNF loss in limbic and dopaminergic striatal inputs may contribute to cognitive/psychiatric dysfunction in HD. Subsequent BDNF loss in cortical motor and thalamic afferents may accelerate striatal degeneration, resulting in progressive involuntary movements.     

Molecular docking uncovers TSPY binds more efficiently with eEF1A2 compared to eEF1A1

Testis-specific protein, Y-encoded (TSPY) binds to eukaryotic translation elongation factor 1 alpha (eEF1A) at its SET/NAP domain that is essential for the elongation during protein synthesis implicated with normal spermatogenesis. The eEF1A exists in two forms, eEF1A1 (alpha 1) and eEF1A2 (alpha 2), encoded by separate loci. Despite critical interplay of the TSPY and eEF1A proteins, literature remained silent on the residues playing significant roles during such interactions. We deduced 3D structures of TSPY and eEF1A variants by comparative modeling (Modeller 9.13) and assessed protein–protein interactions employing HADDOCK docking. Pairwise alignment using EMBOSS Needle for eEF1A1 and eEF1A2 proteins revealed high degree (~92%) of homology. Efficient binding of TSPY with eEF1A2 as compared to eEF1A1 was observed, in spite of the occurrence of significant structural similarities between the two variants. We also detected strong interactions of domain III followed by domains II and I of both eEF1A variants with TSPY. In the process, seven interacting residues of TSPY’s NAP domain namely, Asp 175, Glu 176, Asp 179, Tyr 183, Asp 240, Glu 244, and Tyr 246 common to both eEF1A variants were detected. Additionally, six lysine residues observed in eEF1A2 suggest their possible role in TSPY–eEF1A2 complex formation essential for germ cell development and spermatogenesis. Thus, more efficient binding of TSPY with eEF1A2 as compared to that of eEF1A1 established autonomous functioning of these two variants. Studies on mutated protein following similar approach would uncover the causative obstruction, between the interacting partners leading to deeper understanding on the structure–function relationship.     

Invasive brown trout Salmo trutta induce differential growth strategies in the native snow trout Schizothorax richardsonii of Himalaya: Are natives in unaltered rivers better at picking the gauntlet of invasion?

Brown trout Salmo trutta is a potent global invader and its establishments have progressively altered physiologies, life-histories and niche-availabilities for native fish species. River impoundments further escalate its invasion potential. The Himalayan rivers however, stay uncharted for the effects of brown trout interactions with the native fish fauna. Snow trout Schizothorax richardsonii a Himalayan cold-water native, concerningly overlaps its range with brown trout. To understand its responses to invasion pressures, we investigated brown trout effects on the age and growth of snow trout populations in three rivers with varying levels of perturbation: (a) a dammed and (b) an undammed river with the invasive brown trout in comparison to (c) an undammed river without invasion pressures. We found sympatric snow trout in the undammed river to respond to brown trout invasion with fast life history responses, showing an early age-at-maturity (A₅₀ = 1.2 years) and fast growth with a higher growth constant (K = 0.40 yr⁻¹) and specific rate of linear growth across life. On the contrary, sympatric snow trout in the dammed river showed an explicitly slow life-history by maturing at a higher age (A₅₀ = 2.9 years) and a slow growth, with a lower growth constant (K = 0.26 yr⁻¹) and specific linear growth rates. Our findings suggest that, the snow trout appear to present stronger response to brown trout invasions when the river is unaltered and free from hydropower operations and damming. Further research is strongly warranted from other high-altitude Himalayan basins to delineate the variation in growth strategies exhibited by snow trout in sympatry with the invasives.     

Associations between thyroid hormones and serum metabolite profiles in an euthyroid population

Metabolomics - The aim was to characterise associations between circulating thyroid hormones—free thyroxine (FT4) and thyrotropin (TSH)—and the metabolite profiles in serum samples from...     

Evaluation of arbuscular mycorrhiza and other biocontrol agents in managing Fusarium oxysporum f. sp. Cubense infection in banana cv. Neypoovan

Panama wilt of banana caused by Fusarium oxysporum f. sp. cubense (race 1) is a serious disease devastating the important cultivar Neypoovan (syn Elakki Bale AB) in southern India. Chemical control methods are not very effective in controlling the disease. The objective of this study was to evaluate biocontrol agents (BCAs) under controlled and field conditions for their efficacy against the pathogen and to detect and quantify the reduction in FOC population. Arbuscular mycorrhizal (AM) fungi, Trichoderma harzianum and Pseudomonas fluorescens were inoculated at the time of planting in single, dual and tripartite combinations allowing colonization up to 0, 45 and 90 days. Plants were challenged thereafter with 50 g of FOC inoculum multiplied on sorghum grains containing 1.5×10⁶ cfu g^?1. Uninoculated plants and those inoculated with pathogen only were controls. Plant growth parameters were measured and structural modifications in the roots were studied. FOC populations in the roots were determined by ELISA every month and final yield was recorded. At the end of 7 months, plants pre-inoculated with BCAs i.e., G. mosseae+T. harzianum and challenged with Fusarium under field conditions could sustain 61 and 70% improvement in plant height and girth, respectively, and 75% in bunch weight over plants not precolonised with BCAs but challenged with FOC which finally succumbed to the disease. ELISA study revealed Fusarium population was reduced to 0.58 OD in 7 months in G. mosseae and T. harzianum treatment compared to a level of 1.9 OD in Fusarium alone treated plants. Beneficial effect of BCAs may be due to the over all protection provided by them by causing physical modifications in the cell wall, growth promotion and through induction of disease resistance.     

Three-dimensional model of zeaxanthin binding PsbS protein associated with nonphotochemical quenching of excess quanta of light energy absorbed by the photosynthetic apparatus

A three-dimensional model of the PsbS protein was built with the help of homology-modeling methods. This protein is also known as CP22 and is associated with the protection of photosystem II of thylakoid from excess quanta of light energy absorbed by the photosynthetic apparatus. PsbS is reported to bind two molecules of zeaxanthin at low pH (<5.0) and is believed to be essential for rapid nonphotochemical quenching (qE) of chlorophyll a fluorescence in photosystem II. An attempt was made to explain the pH modulation of the conformation of protein through salt-bridges Glu⁻(122)-Lys⁺(113) and Glu⁻(226)-Lys⁺(217). Binding of two molecules of zeaxanthin in the three-dimensional model of PsbS is postulated. The molecular mechanism of photoprotection by PsbS is explained through the model. 1 Backbone structure of the PsbS protein with two molecules of all trans zeaxanthin (ZEX). Residues Glu 90, 122, 194, 226 and Lys 113, 217 are shown. The figure is drawn with RASMOL (Molecular Visualization Program, RasMol V2.6, Roger Sayle, Glaxo Wellcome Research and Development, Stevenage, Hertfordshire, UK) Electronic Supplementary Material Supplementary material is available for this article at     

Influence of Boron on Somatic Embryogenesis in Papaya

Influence of boron on somatic embryogenesis in papaya (Carica papaya L.) cv. Honey Dew was investigated. Immature zygotic embryos were grown in the induction medium containing Murashige and Skoog basal salts, with B₅ vitamins, picloram (1 mg dm⁻³) or 2,4-dichlorophenoxy acetic acid (2 mg dm⁻³) and different concentrations of boric acid (30 to 500 mg dm⁻³). Maximum somatic embryo initiation was observed at 62 mg dm⁻³ boric acid irrespective of the growth regulator used. The cotyledonary stage somatic embryos were germinated on MS basal medium devoid of growth regulators. The regenerated plantlets were hardened under greenhouse conditions and transferred to field. This revised version was published online in July 2006 with corrections to the Cover Date.