Interaction between Nbp35 and Cfd1 Proteins of Cytosolic Fe-S Cluster Assembly Reveals a Stable Complex Formation in Entamoeba histolytica

Iron-Sulfur (Fe-S) proteins are involved in many biological functions such as electron transport, photosynthesis, regulation of gene expression and enzymatic activities. Biosynthesis and transfer of Fe-S clusters depend on Fe-S clusters assembly processes such as ISC, SUF, NIF, and CIA systems. Unlike other eukaryotes which possess ISC and CIA systems, amitochondriate Entamoeba histolytica has retained NIF & CIA systems for Fe-S cluster assembly in the cytosol. In the present study, we have elucidated interaction between two proteins of E. histolytica CIA system, Cytosolic Fe-S cluster deficient 1 (Cfd1) protein and Nucleotide binding protein 35 (Nbp35). In-silico analysis showed that structural regions ranging from amino acid residues (P33-K35, G131-V135 and I147-E151) of Nbp35 and (G5-V6, M34-D39 and G46-A52) of Cfd1 are involved in the formation of protein-protein complex. Furthermore, Molecular dynamic (MD) simulations study suggested that hydrophobic forces surpass over hydrophilic forces between Nbp35 and Cfd1 and Van-der-Waal interaction plays crucial role in the formation of stable complex. Both proteins were separately cloned, expressed as recombinant fusion proteins in E. coli and purified to homogeneity by affinity column chromatography. Physical interaction between Nbp35 and Cfd1 proteins was confirmed in vitro by co-purification of recombinant Nbp35 with thrombin digested Cfd1 and in vivo by pull down assay and immunoprecipitation. The insilico, in vitro as well as in vivo results prove a stable interaction between these two proteins, supporting the possibility of its involvement in Fe-S cluster transfer to target apo-proteins through CIA machinery in E. histolytica. Our study indicates that initial synthesis of a Fe-S precursor in mitochondria is not necessary for the formation of Cfd1-Nbp35 complex. Thus, Cfd1 and Nbp35 with the help of cytosolic NifS and NifU proteins can participate in the maturation of non-mitosomal Fe-S proteins without any apparent assistance of mitosomes.     

Antimicrobial efficacy,disease inhibition and phenolic acid-inducing potential of chloroform fraction of cow urine

High-performance liquid chromatographic (HPLC) analysis of chloroform fraction (CF) of cow urine showed rich pool of phenolic acids. Antifungal and antibacterial bioassays of CF have shown its tremendous efficacy against some fungal plant pathogens as well as human pathogenic bacteria at very low concentrations. The CF also inhibited powdery mildew (Erysiphe cichoracearum) of balsam (Impatiens balsamania) under field conditions during pre- and post-inoculation treatments. HPLC analysis of pre- and post-inoculation-treated plant leaves indicated that CF induced phenolic acid synthesis as compared to control. The results revealed that CF of cow urine has the potential for controlling some important human diseases. The result on balsam powdery mildew is a good signal that CF may also be effective against other plant diseases in the field.     

Hexafluoroisopropanol-induced helix–sheet transition of stem bromelain: Correlation to function

Stem bromelain is a proteolytic phytoprotein with a variety of therapeutic effects. Understanding its structural properties could provide insight into the mechanisms underlying its clinical utility. Stem bromelain was evaluated for its conformational and folding properties at the pH conditions it encounters when administered orally. It exists as a partially folded intermediate at pH 2.0. The conformational changes to this intermediate state were evaluated using fluorinated alcohols known to induce changes similar to those seen in vivo. Studies using circular dichroism, fluorescence emission spectroscopy, binding of the hydrophobic dye 1-anilino-8-naphthalene sulfonic acid and mass spectrometry indicate that treatment with 10–30% hexafluoroisopropanol induces the partially folded intermediate to adopt much of the native protein's secondary structure, but only a rudimentary tertiary structure, characteristic of the molten globule state. Addition of slightly higher concentrations of hexafluoroisopropanol caused transformation from an α-helix to a β-sheet and induced formation of a compact nonnative structure. This nonnative form was more inhibitory of cell survival than either the native or the partially folded intermediate forms, as measured by enhanced suppression of proliferative cues (e.g., extracellular-signal-regulated kinase) and initiation of apoptotic events. The nonnative form also showed better antitumorigenic properties, as evaluated using an induced two-stage mouse skin papilloma model. In contrast, the nonnative state showed only a fraction of the proteolytic activity of the native form. This study demonstrates that hexafluoroisopropanol can induce a conformational change in stem bromelain to a form with potentially useful therapeutic properties different from those of the native protein.     

Structure and Mechanical Characterization of DNA i-Motif Nanowires by Molecular Dynamics Simulation

We studied the structure and mechanical properties of DNA i-motif nanowires by means of molecular dynamics computer simulations. We built up to 230 nm-long nanowires, based on a repeated TC₅ sequence from crystallographic data, fully relaxed and equilibrated in water. The unusual C⋅C⁺ stacked structure, formed by four ssDNA strands arranged in an intercalated tetramer, is here fully characterized both statically and dynamically. By applying stretching, compression, and bending deformations with the steered molecular dynamics and umbrella sampling methods, we extract the apparent Young’s and bending moduli of the nanowire, as well as estimates for the tensile strength and persistence length. According to our results, the i-motif nanowire shares similarities with structural proteins, as far as its tensile stiffness, but is closer to nucleic acids and flexible proteins, as far as its bending rigidity is concerned. Furthermore, thanks to its very thin cross section, the apparent tensile toughness is close to that of a metal. Besides their yet to be clarified biological significance, i-motif nanowires may qualify as interesting candidates for nanotechnology templates, due to such outstanding mechanical properties.     

Assessment of genetic diversity in 31 species of mangroves and their associates through RAPD and AFLP markers

Random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers were used to assess the genetic diversity in 31 species of mangroves and mangrove associates. Four AFLP primer combinations resulted in the amplification of 840 bands with an average of 210 bands per primer combination and 11 RAPD primers yielded 319 bands with an average of 29 bands per primer. The percentage of polymorphism detected was too high indicating the high degree of genetic variability in mangrove taxa both at inter- and intra-generic levels. In the dendrogram, species belonging to a particular family/ genus, taxa inhabiting similar habitats or having similar adaptations tended to be together. There were exceptions too; as many unrelated species of mangroves form clusters. The intrafamilial classification and inter-relationships of genera in the family Rhizophoraceae could be confirmed by molecular analysis. Both the markers RAPD and AFLP were found equally informative and useful for a better understanding of the genetic variability and genome relationships among mangroves and their associated species.     

Development and survival of infective larvae of Haemonchus contortus and Trichostrongylus colubriformis on pasture in a tropical environment

A trial to determine the seasonal pattern of egg hatching and larval survival on pasture was carried out in representative wet and dry zones of Fiji. Fourteen plots were established on parasite-free pasture at each of two sites. One plot at each site was contaminated every month with faeces from naturally infected goats containing a known proportion of Haemonchus contortus and Trichostrongylus colubriformis eggs. Pasture was sampled at regular intervals after contamination and infective larvae identified and counted. Larvae of both species developed throughout the year in the wet zone but development was more sporadic in the dry zone. Larval counts generally declined to below detectable levels within 9 weeks of contamination between September and March but longevity increased during the cooler weather from April to August. The comparatively short larval survival times noted in this experiment may present opportunities for manipulation of parasite population dynamics in the wet tropics.     

Impermeant potential-sensitive oxonol dyes: III. The dependence of the absorption signal on membrane potential

Summary We have measured potential-dependent changes in the absorption of light by oxidized cholesterol bilayer lipid membranes in the presence of impermeant oxonol dyes. The magnitude of the absorption signal increased linearly with the size of potential steps over a range of 500 mV. The signal also increased when the offset voltage of the pulse train was increased from –150 to +150 mV. The data are consistent with the on-off mechanism proposed by E. B. George et al. (J. Membrane Biol.103:245–253, 1988) in which the probe undergoes potential-dependent movement between a binding site in the membrane and an aqueous region just off the surface of the membrane. An equilibrium thermodynamic analysis of the experimental data indicates that the negatively charged oxonol chromophore senses only 5–10% of the total membrane potential difference across the membrane when it is driven into a nonpolar binding site on the membrane.ASW, Carnegie-Mellon University     

Impermeant potential-sensitive oxonol dyes: II. The dependence of the absorption signal on the length of alkyl substituents attached to the dye

Summary We have measured the potential-dependent light absorption changes of 43 impermeant oxonol dyes with an oxidized cholesterol bilayer lipid membrane system. The size of the signal is strongly dependent on the chain length of alkyl groups attached to the chromophore. Dye molecules with intermediate chain lengths give the largest signals. To better understand the dependence of the absorbance signal on alkyl chain length, a simple equilibrium thermodynamic analysis has been derived. The analysis uses the free energy of dye binding to the membrane and the on-off model (E.B. George et al.,J. Membrane Biol.,103:245–253, 1988a) for the potential-sensing mechanism. In this model, a population of dye molecules in nonpolar membrane binding sites is in a potential-dependent equilibrium with a second population of dye that resides in an unstirred layer adjacent to the membrane. Dye in the unstirred layer is in a separate equilibrium with dye in the bulk bathing solution. The equilibrium binding theory predicts a sigmoidally shaped increase in signal with increasing alkyl chain length, even for very nonpolar dyes. We suggest that aggregation of the more hydrophobic dyes in the membrane bathing solution may be responsible for their low signals, which are not predicted by the theory.