An insight into the mechanism of inhibition of unusual bi-subunit topoisomerase I from Leishmania donovani by 3,3'-di-indolylmethane, a novel DNA topoisomerase I poison with a strong binding affinity to the enzyme

DIM (3,3'-di-indolylmethane), an abundant dietary component of cruciferous vegetables, exhibits a wide spectrum of pharmacological properties. In the present study, we show that DIM is a potent inhibitor of Leishmania donovani topoisomerase I with an IC50 of 1.2 microM. Equilibrium dialysis shows that DIM binds strongly to the free enzyme with a binding constant of 9.73x10(-9) M. The binding affinity of DIM to the small subunit is 8.6-fold more than that of the large subunit of unusual LdTOP1LS (bi-subunit L. donovani topoisomerase I). DIM stabilizes topoisomerase I-DNA cleavage complexes in vitro and also in vivo. Like CPT (camptothecin), DIM inhibits the religation step when the drug was added to preformed topoisomerase I-DNA binary complex. Hence, DIM is similar to CPT with respect to its ability to form the topoisomerase I-mediated 'cleavable complexes' in vitro and in vivo. But unlike CPT, DIM interacts with both free enzyme and substrate DNA. Therefore DIM is a non-competitive class I inhibitor of topoisomerase I. DIM also inhibits the relaxation activity of the CPT-resistant mutant enzyme LdTOP1Delta39LS (N-terminal deletion of amino acids 1-39 of LdTOP1LS). The IC50 values of DIM in simultaneous and enzyme pre-incubation relaxation assays were 3.6 and 2.9 muM respectively, which are higher than that of wild-type topoisomerase I (LdTOP1LS), indicating that the affinity of DIM to LdTOP1Delta39LS is less than that for LdTOP1LS. This is the first report on DIM as an L. donovani topoisomerase I poison. Our study illuminates a new mode of action of enzyme inhibition by DIM that might be exploited for rational drug design in human leishmaniasis.     

Assessment of the efficacy of amino acids and polyamines on regeneration of watermelon (<Emphasis Type="Italic">Citrullus lanatus</Emphasis> Thunb.) and analysis of genetic fidelity of regenerated plants by SCoT and RAPD markers

A simple and efficient regeneration protocol was developed for watermelon from cotyledonary node explants excised from 7-day-old in vitro grown seedlings. This study describes the effect of amino acids and polyamines (PAs) along with plant growth regulators (PGRs) on multiple shoot induction and rooting. The highest number of multiple shoots (46.43 shoots/explant) was obtained from cotyledonary node and they were also elongated (6.3 cm/shoot) on MS medium supplemented with 1 mg l^??1 N ⁶ –Benzyladenine (BA), 5 mg l^??1 leucine, and 10 mg l^??1 spermidine. The elongated shoots developed profuse roots (23.03 roots/shoot) in MS medium containing 1 mg l^??1 indole-3-butyric acid (IBA), 5 mg l^??1 isoleucine, and 10 mg l^??1 putrescine. All the rooted plantlets were successfully hardened and acclimatized in the greenhouse with a survival rate of 98%. The present study described an efficient method to obtain a 1.5-fold increase in the number of shoots, compared with the available regeneration protocols for watermelon. The plants developed in this study showed fivefold higher photosynthetic pigments compared to the control plants. The genetic fidelity of the regenerated plants was evaluated by SCoT and RAPD marker analyses, and banding patterns confirmed the true-to-type nature of in vitro regenerated plants.     

A rapid protocol for somatic embryogenesis from immature leaflets of groundnut (Arachis hypogaea L.)

Summary Plant regeneration via somatic embryogenesis was developed in two groundnut varieties. Somatic embryogenesis was induced from immature leaflets on MS medium with different concentrations of the auxins 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthaleneacetic acid (NAA) in combination with 0.5 mg/l of the cytokinin BA. The highest frequency of somatic embryo formation occurred on MS medium fortified with 20 mg 2,4-D per l. Of the two auxins tested individually 2,4-D was more effective for induction of embryogenesis as well as production of embryos. Embryo development and maturation was achieved on MS medium supplemented with N⁶-benzyladenine (BA) (0.5–2.0 mg/l) and 2,4-D (0.5 mg/l). Plant conversion frequency from somatic embryos was highest in presence of 2.0 mg BA per l and 0.5 mg NAA per l. The frequency of embryogenesis and plant regeneration was higher in the VRI-2 cultivar than in the other cultivar tested. Regenerated plants were transferred to soil, grown to maturity, and produced viable seeds.     

Chemical modification of horseradish peroxidase. Preparation and characterization of tracer enzymes with different isoelectric points.

Horseradish peroxidase (HRP), a plant glycoprotein with a molecular weight of 40,000 D and a molecular radius (ae) of 30 A, has been modified chemically to prepare tracer molecules with different molecular charge. Modification of free carboxyl groups on the enzyme is achieved by carbodiimide activation and subsequent reaction of activated carboxyl groups with a nucleophile; uncharged groups or radicals containing additional positively charged moieties are introduced into the protein molecule resulting in an increased net positive charge of the tracer. Amino groups in the protein molecule are modified by acetylation or succinylation; this reaction will increase the net negative charge of the enzyme by either introducing an uncharged group or an additional carboxyl radical. The tracer molecules so obtained are then characterized in terms of molecular size and charge by column chromatography and isoelectric focusing respectively. The enzymatic activity as measured by 3,3'-diaminobenzidine reaction, the pH optimum and the absorption spectra for the modified enzymes remain virtually unchanged.     

Vocal Fry May Undermine the Success of Young Women in the Labor Market

Vocal fry is speech that is low pitched and creaky sounding, and is increasingly common among young American females. Some argue that vocal fry enhances speaker labor market perceptions while others argue that vocal fry is perceived negatively and can damage job prospects. In a large national sample of American adults we find that vocal fry is interpreted negatively. Relative to a normal speaking voice, young adult female voices exhibiting vocal fry are perceived as less competent, less educated, less trustworthy, less attractive, and less hirable. The negative perceptions of vocal fry are stronger for female voices relative to male voices. These results suggest that young American females should avoid using vocal fry speech in order to maximize labor market opportunities.     

Stereochemical criteria for polypeptides and proteins. IV. Standard dimensions for the cis-peptide unit and conformation of cis-polypeptides

Using potential energy formulas for variation of bond angles and for ω-distortion, the conformation of minimum energy for the cisform of the petide unit has been worked out. This agrees very well with the corresponding set of atoms in the crystal structure of Leu-Pro-Gly and the dimensions of a standard cispeptide unit are given based on these. The conformational (?,Ψ) map for a cispeptide unit has been worked out from contact criteria, both for a pair of linked units as well as for-helices having constant (?,Ψ). The small allowed region of the helical map contains the conformation experimentally observed for poly-L -proline I .     

Focus Issue on Roots: Functional Soil Microbiome: Belowground Solutions to an Aboveground Problem

There is considerable evidence in the literature that beneficial rhizospheric microbes can alter plant morphology, enhance plant growth, and increase mineral content. Of late, there is a surge to understand the impact of the microbiome on plant health. Recent research shows the utilization of novel sequencing techniques to identify the microbiome in model systems such as Arabidopsis (Arabidopsis thaliana) and maize (Zea mays). However, it is not known how the community of microbes identified may play a role to improve plant health and fitness. There are very few detailed studies with isolated beneficial microbes showing the importance of the functional microbiome in plant fitness and disease protection. Some recent work on the cultivated microbiome in rice (Oryza sativa) shows that a wide diversity of bacterial species is associated with the roots of field-grown rice plants. However, the biological significance and potential effects of the microbiome on the host plants are completely unknown. Work performed with isolated strains showed various genetic pathways that are involved in the recognition of host-specific factors that play roles in beneficial host-microbe interactions. The composition of the microbiome in plants is dynamic and controlled by multiple factors. In the case of the rhizosphere, temperature, pH, and the presence of chemical signals from bacteria, plants, and nematodes all shape the environment and influence which organisms will flourish. This provides a basis for plants and their microbiomes to selectively associate with one another. This Update addresses the importance of the functional microbiome to identify phenotypes that may provide a sustainable and effective strategy to increase crop yield and food security.In recent years, the term plant microbiome has received substantial attention, since it influences both plant health and productivity. The plant microbiome encompasses the diverse functional gene pool, originating from viruses, prokaryotes, and eukaryotes, associated with various habitats of a plant host. Such plant habitats range from the whole organism (individual plants) to specific organs (e.g. roots, leaves, shoots, flowers, and seeds, including zones of interaction between roots and the surrounding soil, the rhizosphere; Rout and Southworth, 2013). The rhizosphere is the region of the soil being continuously influenced by plant roots through the rhizodeposition of exudates, mucilages, and sloughed cells (Uren, 2001; Bais et al., 2006; Moe, 2013). Thus, plant roots can influence the surrounding soil and inhabiting organisms. Mutually, the rhizosphere organisms can influence the plant by producing regulatory compounds. Thus, the rhizospheric microbiome acts as a highly evolved external functional milieu for plants (for review, see Bais et al., 2006; Badri et al., 2009b; Pineda et al., 2010; Shi et al., 2011; Philippot et al., 2013; Spence and Bais, 2013; Turner et al., 2013a; Spence et al., 2014). In another sense, it is considered as a second genome to a plant (Berendsen et al., 2012). Plant rhizospheric microbiomes have positive or negative influence on plant growth and fitness. It is influenced directly by beneficial mutualistic microbes or pathogens and indirectly through decomposition, nutrient solubilization, nutrient cycling (Glick 1995), secretion of plant growth hormones (Narula et al., 2006; Ortíz-Castro et al., 2008; Ali et al., 2009; Mishra et al., 2009), antagonism of pathogens (Kloepper et al., 2004), and induction of the plant immune system (Pieterse et al., 2001; Ramamoorthy et al., 2001; Vessey, 2003; Rudrappa et al., 2008, 2010). The establishment of plant and rhizospheric microbiome interaction is a highly coordinated event influenced by the plant host and soil. Recent studies show that plant host and developmental stage has a significant influence on shaping the rhizospheric microbiome (Peiffer et al., 2013; Chaparro et al., 2014).There are various factors involved in the establishment of the rhizospheric and endophytic microbiome. They are greatly affected by soil and host type (Bulgarelli et al., 2012; Lundberg et al., 2012). Apart from these factors, other external factors such as biotic/abiotic stress, climatic conditions, and anthropogenic effects also can impact the microbial population dynamics in particular plant species. Plant host species differences can mainly be perceived from the secretory exudates by microbes. The root exudates act as a crucial driving force for multitrophic interactions in the rhizosphere involving microbes, neighboring plants, and nematodes (Bais et al., 2006). Thus, it is important to understand root exudate-shaped microbial community profiling in establishing phenotypes involved in plant health. Microbial components associated with plant hosts have to respond to these exudates along with utilizing them in order to grow competitively in a complex interactive root environment. Commonly, there are three groups of microbes present in the rhizosphere, commensal, beneficial, and pathogenic microbes, and their competition for plant nutrition and interactions confer the overall soil suppressiveness against pathogens and insects (Berendsen et al., 2012).Traditionally, the components of the plant microbiome were characterized by isolating and culturing microbes on different media and growth conditions. These culture-based techniques missed the vast majority of microbial diversity in an environment or in plant-associated habitats, which is now detectable by modern culture-independent molecular techniques for analyzing whole environmental metagenomes (comprising all organisms’ genomes). Over the last 5 years, these culture-independent techniques have dramatically changed our view of the microbial diversity in a particular environment, from which only less than 1% are culturable (Hugenholtz et al., 1998). After discovering the importance of the conserved 16S ribosomal RNA (rRNA) sequence (Woese and Fox, 1977) and the first use of denaturing gradient gel electrophoresis (DGGE) of the amplified 16S rRNA gene in the analysis of a microbial community (Muyzer et al., 1993), there was a sudden explosion of research toward microbial ecology using various molecular fingerprinting techniques. Apart from DGGE, thermal gradient gel electrophoresis, and fluorescence in situ hybridization, clone library construction of microbial community-amplified products and sequencing emerged as other supporting techniques for better understanding of microbial ecology (Muyzer, 1999). Furthermore, there are many newer techniques used to understand the microbiome, from metagenomics to metaproteomics (Friedrich, 2006; Mendes et al., 2011; Knief et al., 2012; Rincon-Florez et al., 2013; Schlaeppi et al., 2014; Yergeau et al., 2014). These techniques cover the whole microbiome, instead of selecting particular species, unlike conventional microbial analysis. However, their presence was not yet correlated well with the phenotypic manifestation (phenome) they establish in the host plant.As a consequence of population growth, food consumption is also increasing. On the other hand, cultivable agricultural land and productivity are significantly reduced due to global industrialization, drought, salinity, and global warming (Gamalero et al., 2009). This problem is only addressed by practicing the sustainable agriculture that protects the health of the ecosystem. The basic principle of sustainable agriculture is to significantly reduce the chemical input, such as fertilizers, insecticides, and herbicides, while reducing the emission of greenhouse gas. Manipulation of the plant microbiome has great potential in reducing the incidence of pests and diseases (van Loon et al., 1998; Kloepper et al., 2004; Van Oosten et al., 2008), promoting plant growth and plant fitness, and increasing productivity (Kloepper and Schroth 1978; Lugtenberg and Kamilova, 2009; Vessey, 2003). Single strains or mixed inoculum treatments induced resistance to multiple plant diseases (Jetiyanon and Kloepper, 2002). In recent years, several microbial biofertilizers and inoculants were formulated, produced, marketed, and successfully used by farmers worldwide (Bhardwaj et al., 2014). Although plants are being considered as a metaorganism (East, 2013), our understanding of the exact manifestation of this microbiome on plant health in terms of phenotypes is insufficient. Of late, there is a surge to understand and explore the genomic wealth of rhizosphere microbes. Hence, this Update will focus mainly on existing knowledge based on the root microbiome, its functional importance, and its potential relationship to the establishment of a host phenome, toward achieving sustainable agriculture.     

Drosophila TRPML Forms PI(3,5)P2-activated Cation Channels in Both Endolysosomes and Plasma Membrane

Transient Receptor Potential mucolipin (TRPML) channels are implicated in endolysosomal trafficking, lysosomal Ca²⁺ and Fe²⁺ release, lysosomal biogenesis, and autophagy. Mutations in human TRPML1 cause the lysosome storage disease, mucolipidosis type IV (MLIV). Unlike vertebrates, which express three TRPML genes, TRPML1–3, the Drosophila genome encodes a single trpml gene. Although the trpml-deficient flies exhibit cellular defects similar to those in mammalian TRPML1 mutants, the biophysical properties of Drosophila TRPML channel remained uncharacterized. Here, we show that transgenic expression of human TRPML1 in the neurons of Drosophila trpml mutants partially suppressed the pupal lethality phenotype. When expressed in HEK293 cells, Drosophila TRPML was localized in both endolysosomes and plasma membrane and was activated by phosphatidylinositol 3,5-bisphosphate (PI(3,5)P₂) applied to the cytoplasmic side in whole lysosomes and inside-out patches excised from plasma membrane. The PI(3,5)P₂-evoked currents were blocked by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂), but not other phosphoinositides. Using TRPML A487P, which mimics the varitint-waddler (Va) mutant of mouse TRPML3 with constitutive whole-cell currents, we show that TRPML is biphasically regulated by extracytosolic pH, with an optimal pH about 0.6 pH unit higher than that of human TRPML1. In addition to monovalent cations, TRPML exhibits high permeability to Ca²⁺, Mn²⁺, and Fe²⁺, but not Fe³⁺. The TRPML currents were inhibited by trivalent cations Fe³⁺, La³⁺, and Gd³⁺. These features resemble more closely to mammalian TRPML1 than TRPML2 and TRPML3, but with some obvious differences. Together, our data support the use of Drosophila for assessing functional significance of TRPML1 in cell physiology.