In vitro micrografting for production of Indian citrus ringspot virus (ICRSV)-free plants of kinnow mandarin (Citrus nobilis Lour × C. deliciosa Tenora)

Production of Indian citrus ringspot virus (ICRSV)-free plants from an infected plant of kinnow mandarin (Citrus nobilis Lour × C. deliciosa Tenora) is reported. The shoot apices of different sizes (0.2–1.0 mm) excised from the ICRSV-infected plant were micrografted onto decapitated rootstock seedlings of rough lemon (C. jambhiri). Micrograft survival depended on the size of shoot apex and the sucrose concentration of the culture medium. Increase in scion size from 0.2 to 0.7 mm resulted in an increase in micrografting success rate from 30.55 to 51.88%. Further, micrograft survival obtained with 0.2 mm was improved from 30.55 to 38.88% by increasing sucrose concentration in the culture media from 5 to 7.5%. The micrografted plants were tested for ICRSV using ELISA and RT-PCR. All plants raised from 0.2-mm scion were found negative with both ELISA and RT-PCR whereas only 20% of the ELISA negative plants raised from 0.3-mm scion were found negative for ICRSV with RT-PCR. The outcome of this research is the successful establishment, acclimatization and virus testing of micrografted plants.     

Phosphorylation of slit diaphragm proteins NEPHRIN and NEPH1 upon binding of HGF promotes podocyte repair

Phosphorylation (activation) and dephosphorylation (deactivation) of the slit diaphragm proteins NEPHRIN and NEPH1 are critical for maintaining the kidney epithelial podocyte actin cytoskeleton and, therefore, proper glomerular filtration. However, the mechanisms underlying these events remain largely unknown. Here we show that NEPHRIN and NEPH1 are novel receptor proteins for hepatocyte growth factor (HGF) and can be phosphorylated independently of the mesenchymal epithelial transition receptor in a ligand-dependent fashion through engagement of their extracellular domains by HGF. Furthermore, we demonstrate SH2 domain–containing protein tyrosine phosphatase-2–dependent dephosphorylation of these proteins. To establish HGF as a ligand, purified baculovirus-expressed NEPHRIN and NEPH1 recombinant proteins were used in surface plasma resonance binding experiments. We report high-affinity interactions of NEPHRIN and NEPH1 with HGF, although NEPHRIN binding was 20-fold higher than that of NEPH1. In addition, using molecular modeling we constructed peptides that were used to map specific HGF-binding regions in the extracellular domains of NEPHRIN and NEPH1. Finally, using an in vitro model of cultured podocytes and an ex vivo model of Drosophila nephrocytes, as well as chemically induced injury models, we demonstrated that HGF-induced phosphorylation of NEPHRIN and NEPH1 is centrally involved in podocyte repair. Taken together, this is the first study demonstrating a receptor-based function for NEPHRIN and NEPH1. This has important biological and clinical implications for the repair of injured podocytes and the maintenance of podocyte integrity.     

Biogenic synthesis of metal nanoparticles from actinomycetes: biomedical applications and cytotoxicity

Biogenic synthesis of metal nanoparticles has been well proved by using bacteria, fungi, algae, actinomycetes, plants, etc. Among the different microorganisms used for the synthesis of metal nanoparticles, actinomycetes are less known. Although, there are reports, which have shown that actinomycetes are efficient candidates for the production of metal nanoparticles both intracellularly and extracellularly. The nanoparticles synthesized by the members of actinomycetes present good polydispersity and stability and possess significant biocidal activities against various pathogens. The present review focuses on biological synthesis of metal nanoparticles and their application in medicine. In addition, the toxicity of these biogenic metal nanoparticles to human beings and environment has also been discussed.     

Technical report: exploring the basis of congenital myasthenic syndromes in an undergraduate course, using the model organism, Caenorhabditis elegans

Mutations affecting acetylcholine receptors have been causally linked to the development of congenital myasthenic syndromes (CMS) in humans resulting from neuromuscular transmission defects. In an undergraduate Molecular Neurobiology course, the molecular basis of CMS was explored through study of a Caenorhabditis elegans model of the disease. The nicotinic acetylcholine receptor (nAChR), located on the postsynaptic muscle cell membrane, contains a pentameric ring structure comprised of five homologous subunits. In the nematode C. elegans, unc-63 encodes an α subunit of nAChR. UNC-63 is required for the function of nAChR at the neuromuscular junction. Mutations in unc-63 result in defects in locomotion and egg-laying and may be used as models for CMS. Here, we describe the responses of four unc-63 mutants to the cholinesterase inhibitor pyridostigmine bromide (range 0.9–15.6 mM in this study), a treatment for CMS that mitigates deficiencies in cholinergic transmission by elevating synaptic ACh levels. Our results show that 15.6 mM pyridostigmine bromide enhanced mobility in two alleles, depressed mobility in one allele and in N2, while having no effect on the fourth allele. This indicates that while pyridostigmine bromide may be effective at ameliorating symptoms of CMS in certain cases, it may not be a suitable treatment for all individuals due to the diverse etiology of this disease. Students in the Molecular Neurobiology course enhanced their experience in scientific research by conducting an experiment designed to increase understanding of genetic defects of neurological function.     

Evaluation of the Stability,Bioavailability, and Hypersensitivity of the Omega-3 Derived Anti-Leukemic Prostaglandin: Δ12-Prostaglandin J3

Previous studies have demonstrated the ability of an eicosapentaenoic acid (EPA)-derived endogenous cyclopentenone prostaglandin (CyPG) metabolite, Δ¹²-PGJ₃, to selectively target leukemic stem cells, but not the normal hematopoietic stems cells, in in vitro and in vivo models of chronic myelogenous leukemia (CML). Here we evaluated the stability, bioavailability, and hypersensitivity of Δ¹²-PGJ₃. The stability of Δ¹²-PGJ₃ was evaluated under simulated conditions using artificial gastric and intestinal juice. The bioavailability of Δ¹²-PGJ₃ in systemic circulation was demonstrated upon intraperitoneal injection into mice by LC-MS/MS. Δ¹²-PGJ₃ being a downstream metabolite of PGD₃ was tested in vitro using primary mouse bone marrow-derived mast cells (BMMCs) and in vivo mouse models for airway hypersensitivity. ZK118182, a synthetic PG analog with potent PGD₂ receptor (DP)-agonist activity and a drug candidate in current clinical trials, was used for toxicological comparison. Δ¹²-PGJ₃ was relatively more stable in simulated gastric juice than in simulated intestinal juice that followed first-order kinetics of degradation. Intraperitoneal injection into mice revealed that Δ¹²-PGJ₃ was bioavailable and well absorbed into systemic circulation with a C_max of 263 µg/L at 12 h. Treatment of BMMCs with ZK118182 for 12 h resulted in increased production of histamine, while Δ¹²-PGJ₃ did not induce degranulation in BMMCs nor increase histamine. In addition, in vivo testing for hypersensitivity in mice showed that ZK118182 induces higher airways hyperresponsiveness when compared Δ¹²-PGJ₃ and/or PBS control. Based on the stability studies, our data indicates that intraperitoneal route of administration of Δ¹²-PGJ₃ was favorable than oral administration to achieve effective pharmacological levels in the plasma against leukemia. Δ¹²-PGJ₃ failed to increase histamine and IL-4 in BMMCs, which is in agreement with reduced airway hyperresponsiveness in mice. In summary, our studies suggest Δ¹²-PGJ₃ to be a promising bioactive metabolite for further evaluation as a potential drug candidate for treating CML.     

Suppression of the ndv mutant phenotype of Rhizobium meliloti by cloned exo genes

The ndvA and ndvB genes of Rhizobium meliloti are involved in the export and synthesis, respectively, of the small cyclic polysaccharide beta(1,2)glucan. We have previously shown that spontaneous symbiotic pseudorevertants of ndv mutants do not produce periplasmic beta(1,2)glucan. Here we show that the pseudorevertants also do not produce extracellular beta(1,2)glucan, but do show alterations in the amount of the major acidic exopolysaccharide produced. This exopolysaccharide is not detectably different from that produced by the wild type or by the ndv mutants. A cosmid which suppresses the symbiotic defect of both ndvA and ndvB mutants was isolated from a gene bank prepared from DNA of an ndvA pseudorevertant. This cosmid contains a number of exo genes, including exoH and exoF. Subcloning and Tn5 mutagenesis were used to show that the widely separated exoH and exoF genes are both involved in suppression of the ndv mutant phenotype and that the 3.5 kb DNA fragment which contains the exoH gene does not carry the mutation responsible for second site suppression.     

Fluorescence and thermotropic studies of the interactions of PEI-cholesterol based PEI-chol lipopolymers with dipalmitoyl phosphatidylcholine membranes

Numerous PEI derived polymers have been explored for their use in gene delivery. Nine PEI-chol lipopolymers based on cholesterol grafting on three polyethyleneimines (PEI) of different molecular weights have been synthesized. Firstly their aggregation behavior has been studied using transmission electron microscopy and then their interactions with l-α-dipalmitoyl phosphatidylcholine (DPPC) membranes have been examined using fluorescence anisotropy and differential scanning calorimetry (DSC). These PEI-chol lipopolymers are found to quench the chain motion of the acyl chains of DPPC, when incorporated in membranes, depending upon the cholesterol grafting on PEI. These PEI-chol lipopolymers act as filler molecules in membranes. Electron microscopy shows the different aggregation behavior of these new PEI-chol lipopolymers depending upon the molecular weight of PEI and percentage of cholesterol grafting. Detailed analysis of the fluorescence anisotropy and DSC data indicate that the nature of perturbation induced by PEI-chol lipopolymers is dependent upon the molecular weight of the PEI used and the % of cholesterol grafting on PEI. In general, PEI-chol lipopolymers rigidify the liquid-crystalline phase of the membranes without any noticeable effect on the gel phase unlike natural cholesterol, which is known to fluidize the gel phase of the membranes.