Complex interactions on fig trees: ants capturing parasitic wasps as possible indirect mutualists of the fig–fig wasp interaction

Like other mutualisms, pollination mutualisms attract parasites, as well as opportunistic and specialist predators of the pollinators and parasites. These associated species influence the evolutionary dynamics of pairwise mutualisms. Predatory ants are frequent associates of pollination mutualisms, but their effects on the complex interactions between plants, pollinators and parasites have not yet been clearly established, even in the case of the well-described obligate interaction between figs and fig wasps. We attempted to quantify such effects for ants associated with three fig species, two dioecious ( Ficus condensa [Bruneï], F . carica [France]) and one monoecious ( F . racemosa [India]). In all these cases, ant presence on a fig tree strongly reduced the number of parasitic wasps on the figs. Experimental exclusion of ants resulted in an increase in the number of non-pollinating fig wasps on F . condensa and F . racemosa . Experimental ant supplementation led to a decrease in the number of non-pollinating fig wasps on F . carica . Moreover, on F . condensa , the level of reduction of the number of parasitic wasps depended on the number and identity of the ants. On F . carica , non-pollinating fig wasps even avoided trees occupied by the dominant predatory ant. The consistency of the effect of ants in these three cases, representing a geographically, ecologically, and taxonomically broad sample of figs, argues for the generality of the effect we observed. Because reduction of parasitism benefits the pollinator, ants may be considered as indirect mutualists of plants and pollinators in the network of complex interactions supported by fig trees.     

Identification of one of the apurinic/apyrimidinic lyase active sites of topoisomerase V by structural and functional studies

Topoisomerase V (Topo-V) is the only member of a novel topoisomerase subtype. Topo-V is unique because it is a bifunctional enzyme carrying both topoisomerase and DNA repair lyase activities within the same protein. Previous studies had shown that the topoisomerase domain spans the N-terminus of the protein and is followed by 12 tandem helix–hairpin–helix [(HhH)₂] domains. There are at least two DNA repair lyase active sites for apurinic/apyrimidinic (AP) site processing, one within the N-terminal region and the second within the C-terminal domain of Topo-V, but their exact locations and characteristics are unknown. In the present study, the N-terminal 78-kDa fragment of Topo-V (Topo-78), containing the topoisomerase domain and one of the lyase DNA repair domains, was characterized by structural and biochemical studies. The results show that an N-terminal 69-kDa fragment is the minimal fragment with both topoisomerase and AP lyase activities. The lyase active site of Topo-78 is at the junction of the fifth and sixth (HhH)₂ domains. From the biochemical and structural data, it appears that Lys571 is the most probable nucleophile responsible for the lyase activity. Our experiments also suggest that Topo-V most likely acts as a Class I AP endonuclease in vivo.     

Establishment of dedifferentiated callus of haploid origin from unfertilized ovaries of tea (Camellia sinensis (L.) O. Kuntze) as a potential source of total phenolics and antioxidant activity

This is the first report of induction of haploid callus with significant antioxidant activity from unpollinated ovary cultures of tea. Out of the five cultivars tested, TV18 gave the highest percentage of callus induction. Within 1 wk of induction, ovules swelled to almost double their original size, and white, friable callus emerged. A high cytokinin/auxin ratio, provided by 8.5 μM benzyl adenine and 4.5 μM 2,4-dichlorophenxyacetic acid, and high-temperature treatment (33°C) for 10 d in the dark promoted maximum callus induction. Callus was maintained on MS medium containing 22.2 μM benzyl adenine and 9.8 μM indolebutyric acid (callus line RM 1) in the light at 25°C. Well-developed tracheids were formed within 4 wk in callus subcultured on MS medium containing 1.8 μM thidiazuron and 5.0 μM 2,3,5-triiodobenzoic acid (line RM 2). Flow cytometric analysis revealed that most cells were haploid. Both RM 1 and RM 2 produced phenolic compounds with significant antioxidant capacity. Phenolic content showed a positive linear correlation with antioxidant activity. The total phenolic content of RM 1 was 3.47?±?0.21 gallic acid equivalents (GAE) mg/g dry weight and that of RM 2 was 2.39?±?0.12 GAE mg/g dry weight. Antioxidant activity was measured using IC₅₀, a measure of inhibitory concentration; a lower IC₅₀ value reflects greater antioxidant activity. The IC₅₀ value of RM 1 was 2,530 μg/ml and that of RM 2 was 3,170 μg/ml. The results suggested that the phenolic compounds contributed significantly to the antioxidant capacity of the in vitro cell lines.     

Estimation of Förster's distance between two ends of Dps protein from mycobacteria: distance heterogeneity as a function of oligomerization and DNA binding

Dps protein (DNA binding Protein from Starved Cells) from Mycobacterium smegmatis (Ms-Dps) is known to undergo an in vitro irreversible oligomeric transition from trimer to dodecamer. This transition helps the protein to provide for bimodal protection to the bacterial DNA from the free radical and Fenton mediated damages in the stationary state. The protein exists as a stable trimer, when purified from E. coli cells transformed with an over-expression plasmid. Both trimer as well as dodecamer are known to exhibit ferroxidation activity, thus removing toxic hydroxyl radicals in vivo, whereas iron accumulation and non-sequence specific DNA binding activity are found in dodecamer only. This seems to be aided by the positively charged long C-terminal tail of the protein. We used frequency domain phase-modulation fluorescence spectroscopy and Förster Resonance Energy Transfer (FRET) to monitor this oligomeric switch from a trimer to a dodecamer and to elucidate the structure of DNA–Dps dodecamer complex. As Ms-Dps is devoid of any Cysteine residues, a Serine is mutated to Cysteine (S169C) at a position adjacent to the putative DNA binding domain. This Cysteine is subsequently labeled with fluorescent probe and another probe is placed at the N-terminus, as crystal structure of the protein reveals several side-chain interactions between these two termini, and both are exposed towards the surface of the protein. Here, we report the Förster's distance distribution in the trimer and the dodecamer in the presence and absence of DNA. Through discrete lifetime analysis of the probes tagged at the respective regions in the macromolecule, coupled with Maximum Entropy Method (MEM) analysis, we show that the dodecamer, upon DNA binding shows conformational heterogeneity in overall structure, perhaps mediated by a non-specific DNA–protein interaction. On the other hand, the nature of DNA–Dps interaction is not known and several models exist in literature. We show here with the help of fluorescence anisotropy measurements of labeled DNA having different length and unlabeled native dodecameric protein that tandem occupation of DNA binding sites by a series of Dps molecules perhaps guide the tight packing of Dps over DNA backbone.     

Hypoglycosylation of dystroglycan due to T192M mutation: A molecular insight behind the fact

Abnormal glycosylation of dystroglycan (DG), a transmembrane glycoprotein, results in a group of diseases known as dystroglycanopathy. A severe dystroglycanopathy known as the limb girdle disease MDDGC9 [OMIM: 613818] occurs as a result of hypoglycosylation of alpha subunit of DG. Reasons behind this has been traced back to a point mutation (T192M) in DG that leads to weakening of interactions of DG protein with laminin and subsequent loss of signal flow through the DG protein. In this work we have tried to analyze the molecular details of the interactions between DG and laminin1 in order to propose a mechanism about the onset of the disease MDDGC9. We have observed noticeable changes between the modeled structures of wild type and mutant DG proteins. We also have employed molecular docking techniques to study and compare the binding interactions between laminin1 and both the wild type and mutant DG proteins. The docking simulations have revealed that the mutant DG has weaker interactions with laminin1 as compared to the wild type DG. Till date there are no previous reports that deal with the elucidation of the interactions of DG with laminin1 from the molecular level. Our study is therefore the first of its kind which analyzes the differences in binding patterns of laminin1 with both the wild type and mutant DG proteins. Our work would therefore facilitate analysis of the molecular mechanism of the disease MDDGC9. Future work based on our results may be useful for the development of suitable drugs against this disease.     

Influence of Formulation and Processing Factors on Stability of Levothyroxine Sodium Pentahydrate

Stability of formulations over shelf-life is critical for having a quality product. Choice of excipients, manufacturing process, storage conditions, and packaging can either mitigate or enhance the degradation of the active pharmaceutical ingredient (API), affecting potency and/or stability. The purpose was to investigate the influence of processing and formulation factors on stability of levothyroxine (API). The API was stored at long-term (25°C/60%RH), accelerated (40°C/75%RH), and low-humidity (25°C/0%RH and 40°C/0%RH) conditions for 28 days. Effect of moisture loss was evaluated by drying it (room temperature, N₂) and placed at 25°C/0%RH and 40°C/0%RH. The API was incubated with various excipients (based on package insert of marketed tablets) in either 1:1, 1:10, or 1:100 ratios with 5% moisture at 60°C. Commonly used ratios for excipients were used. The equilibrium sorption data was collected on the API and excipients. The API was stable in solid state for the study duration under all conditions for both forms (potency between 90% and 110%). Excipients effect on stability varied and crospovidone, povidone, and sodium laurel sulfate (SLS) caused significant API degradation where deiodination and deamination occurred. Moisture sorption values were different across excipients. Crospovidone and povidone were hygroscopic whereas SLS showed deliquescence at high RH. The transient formulation procedures where temperature might go up or humidity might go down would not have major impact on the API stability. Excipients influence stability and if possible, those three should either be avoided or used in minimum quantity which could provide more stable tablet formulations with minimum potency loss throughout its shelf-life.     

Rac function in epithelial tube morphogenesis

Epithelial cell migration and morphogenesis require dynamic remodeling of the actin cytoskeleton and cell-cell adhesion complexes. Numerous studies in cell culture and in model organisms have demonstrated the small GTPase Rac to be a critical regulator of these processes; however, little is known about Rac function in the morphogenic movements that drive epithelial tube formation. Here, we use the embryonic salivary glands of Drosophila to understand the role of Rac in epithelial tube morphogenesis. We show that inhibition of Rac function, either through loss of function mutations or dominant-negative mutations, disrupts salivary gland invagination and posterior migration. In contrast, constitutive activation of Rac induces motile behavior and subsequent cell death. We further show that Rac regulation of salivary gland morphogenesis occurs through modulation of cell-cell adhesion mediated by the E-cadherin/beta-catenin complex and that shibire, the Drosophila homolog of dynamin, functions downstream of Rac in regulating beta-catenin localization during gland morphogenesis. Our results demonstrate that regulation of cadherin-based adherens junctions by Rac is critical for salivary gland morphogenesis and that this regulation occurs through dynamin-mediated endocytosis.     

The intermembrane space domain of Tim23 is intrinsically disordered with a distinct binding region for presequences

Proteins targeted to the mitochondrial matrix are translocated through the outer and the inner mitochondrial membranes by two protein complexes, the translocase of the outer membrane (TOM) and one of the translocases of the inner membrane (TIM23). The protein Tim23, the core component of TIM23, consists of an N‐terminal, soluble domain in the intermembrane space (IMS) and a C‐terminal domain that forms the import pore across the inner membrane. Before translocation proceeds, precursor proteins are recognized by the N‐terminal domain of Tim23, Tim23N (residues 1–96). By using NMR spectroscopy, we show that Tim23N is a monomeric protein belonging to the family of intrinsically disordered proteins. Titrations of Tim23N with two presequences revealed a distinct binding region of Tim23N formed by residues 71–84. In a charge‐hydropathy plot containing all soluble domains of TOM and TIM23, Tim23N was found to be the only domain with more than 40 residues in the IMS that is predicted to be intrinsically disordered, suggesting that Tim23N might function as hub in the mitochondrial import machinery protein network.