Conjugational fertility and location of chloramphenicol biosynthesis genes on the chromosomal linkage map of Streptomyces venezuelae

In Streptomyces venezuelae fertility, defined as chromosomal gene recombination, was enhanced over 1000-fold when one parent in a biparental conjugational cross lacked the physically-undetected plasmid SVP1, as compared with crosses in which both parents carried SVP1. The existence of SVP1 and at least two other fertility plasmids, SVP2 and SVP3, was detected in S. venezuelae by 'lethal zygosis' elicited by a plasmid-plus mycelium in contact with a plasmid-minus mycelium. Conjugational crosses were used to construct a linkage map of S. venezuelae which was highly consistent with the map of analogous loci in S. coelicolor A3(2). A cluster of genes governing chloramphenicol biosynthesis was located near arg, cys and pdxB genes at a position roughly equivalent to the 1-2 o'clock region of the S. coelicolor A3(2) map.     

Bispidine-Amino Acid Conjugates Act as a Novel Scaffold for the Design of Antivirals That Block Japanese Encephalitis Virus Replication

Background

Japanese encephalitis virus (JEV) is a major cause of viral encephalitis in South and South-East Asia. Lack of antivirals and non-availability of affordable vaccines in these endemic areas are a major setback in combating JEV and other closely related viruses such as West Nile virus and dengue virus. Protein secondary structure mimetics are excellent candidates for inhibiting the protein-protein interactions and therefore serve as an attractive tool in drug development. We synthesized derivatives containing the backbone of naturally occurring lupin alkaloid, sparteine, which act as protein secondary structure mimetics and show that these compounds exhibit antiviral properties.

Methodology/Principal Findings

In this study we have identified 3,7-diazabicyclo[3.3.1]nonane, commonly called bispidine, as a privileged scaffold to synthesize effective antiviral agents. We have synthesized derivatives of bispidine conjugated with amino acids and found that hydrophobic amino acid residues showed antiviral properties against JEV. We identified a tryptophan derivative, Bisp-W, which at 5 µM concentration inhibited JEV infection in neuroblastoma cells by more than 100-fold. Viral inhibition was at a stage post-entry and prior to viral protein translation possibly at viral RNA replication. We show that similar concentration of Bisp-W was capable of inhibiting viral infection of two other encephalitic viruses namely, West Nile virus and Chandipura virus.

Conclusions/Significance

We have demonstrated that the amino-acid conjugates of 3,7-diazabicyclo[3.3.1]nonane can serve as a molecular scaffold for development of potent antivirals against encephalitic viruses. Our findings will provide a novel platform to develop effective inhibitors of JEV and perhaps other RNA viruses causing encephalitis.     

Japanese Encephalitis Virus Disrupts Cell-Cell Junctions and Affects the Epithelial Permeability Barrier Functions

Japanese encephalitis virus (JEV) is a neurotropic flavivirus, which causes viral encephalitis leading to death in about 20–30% of severely-infected people. Although JEV is known to be a neurotropic virus its replication in non-neuronal cells in peripheral tissues is likely to play a key role in viral dissemination and pathogenesis. We have investigated the effect of JEV infection on cellular junctions in a number of non-neuronal cells. We show that JEV affects the permeability barrier functions in polarized epithelial cells at later stages of infection. The levels of some of the tight and adherens junction proteins were reduced in epithelial and endothelial cells and also in hepatocytes. Despite the induction of antiviral response, barrier disruption was not mediated by secreted factors from the infected cells. Localization of tight junction protein claudin-1 was severely perturbed in JEV-infected cells and claudin-1 partially colocalized with JEV in intracellular compartments and targeted for lysosomal degradation. Expression of JEV-capsid alone significantly affected the permeability barrier functions in these cells. Our results suggest that JEV infection modulates cellular junctions in non-neuronal cells and compromises the permeability barrier of epithelial and endothelial cells which may play a role in viral dissemination in peripheral tissues.     

Heavy metal-induced selection and proliferation of antibiotic resistance: A review

Antibiotic resistance is recognized as a global threat to public health. The selection and evolution of antibiotic resistance in clinical pathogens were believed to be majorly driven by the imprudent use of antibiotics. However, concerns regarding the same, through selection pressure by a multitude of other antimicrobial agents, such as heavy metals, are also growing. Heavy metal contamination co-selects antibiotic and metal resistance through numerous mechanisms, such as co-resistance and cross-resistance. Here, we have reviewed the role of heavy metals as antimicrobial resistance driving agents and the underlying concept and mechanisms of co-selection, while also highlighting the scarcity of studies explicitly inspecting the process of co-selection in clinical settings. Prospective strategies to manage heavy metal-induced antibiotic resistance have also been deliberated, underlining the need to find specific inhibitors so that alternate medicinal combinations can be added to the existing therapeutic armamentarium.     

Neuronal Control of Exocytosis and Calcium Homeostasis

We showed that 5 M acetylcholine (ACh) and 100 M norepinephrine (NE) cause increases in the total Ca²⁺ content in acinar cells by 30 and 87% and in the exocytosis intensity by 15 and 20%, respectively. Application of 5 M ACh and 100 M NE increased the free cytosolic Ca²⁺ concentration ([Ca²⁺] _i ) by 87 ± 2 and 140 ± 7 nM, respectively. Application of ACh and NE in a Ca²⁺-free external solution caused a [Ca²⁺] _i increase that was 40 and 67% lower than in physiological solution. We postulate that the exocytosis developing upon neural stimulation of the gland results from generation of Ca²⁺ transients that are spreading from the basal to the apical region of the exocrine cell, where secretory granules are concentrated.     

Photosynthetic Response to Irradiance in Valeriana jatamansi Jones,a Threatened Understorey Medicinal Herb of Western Himalaya

Net photosynthetic rate (P _N) of Valeriana jatamansi plants, grown under nylon net shade or under different tree canopies, was saturated with photons at 1 000 mol m^–2 s^–1 photosynthetic photon-flux-density (PPFD), whereas open-grown plants were able to photosynthesise even at higher PPFD, e.g. of 2 000 mol m^–2 s^–1. Plants grown under net shade had higher total chlorophyll (Chl) content per unit area of leaf surface. However, Chl a/b ratio was maximal in open-grown plants, but remained unchanged in plants grown in nylon net shade and under different tree canopies. Sun-grown plants had thicker leaves (higher leaf mass per leaf area unit), higher wax content, and higher P _N than shade grown plants. Thus V. jatamansi is able to acclimate to high PPFD and therefore this Himalayan species may be cultivated in open habitat to meet the ever-increasing industrial demand.     

Improving the accuracy of a solid spherical source radius and depth estimation using the diffusion equation in fluorescence reflectance mode

Background  

Non-invasive planar fluorescence reflectance imaging (FRI) is used for accessing physiological and molecular processes in biological tissue. This method is efficiently used to detect superficial fluorescent inclusions. FRI is based on recording the spatial radiance distribution (SRD) at the surface of a sample. SRD provides information for measuring structural parameters of a fluorescent source (such as radius and depth). The aim of this article is to estimate the depth and radius of the source distribution from SRD, measured at the sample surface. For this reason, a theoretical expression for the SRD at the surface of a turbid sample arising from a spherical light source embedded in the sample, was derived using a steady-state solution of the diffusion equation with an appropriate boundary condition.     

Dissecting the mechanism and assembly of a complex virulence mycobacterial lipid

Mycobacterium tuberculosis cell envelope is a treasure house of biologically active lipids of fascinating molecular architecture. Although genetic studies have alluded to an array of genes in biosynthesis of complex lipids, their mechanistic, structural, and biochemical principles have not been investigated. Here, we have dissected the molecular logic underlying the biosynthesis of a virulence lipid phthiocerol dimycocerosate (PDIM). Cell-free reconstitution studies demonstrate that polyketide synthases, which are usually involved in the biosynthesis of secondary metabolites, are responsible for generating complex lipids in mycobacteria. We show that PapA5 protein directly transfers the protein bound mycocerosic acid analogs on phthiocerol to catalyze the final esterification step. Based on precise identification of biological functions of proteins from Pps cluster, we have rationally produced a nonmethylated variant of mycocerosate esters. Apart from elucidating mechanisms that generate chemical heterogeneity with PDIMs, this study also presents an attractive approach to explore host-pathogen interactions by altering mycobacterial surface coat.     

Biochemical characterisation of extracellular phytase (myo-inositol hexakisphosphate phosphohydrolase) from a hyper-producing strain of Aspergillus niger van Teighem

Aspergillus niger van Teighem, isolated in our laboratory from samples of rotten wood logs, produced extracellular phytase having a high specific activity of 22,592 units (mg protein)^–1 . The enzyme was purified to near homogeneity using ion-exchange and gel-filtration chromatography. The molecular properties of the purified enzyme suggested the native phytase to be oligomeric, with a molecular weight of 353 kDa, the monomer being 66 kDa. The purified enzyme exhibited maximum activity at pH 2.5 and 52–55°C. The enzyme retained 97% activity after a 24-h incubation at 55°C in the presence of 10 mM glycine, while 87% activity was retained when no thermoprotectant was added. Phytase activity was not affected by most metal ions, inhibitors and organic solvents. Non-ionic and cationic detergents (0.1–5%) stabilise the enzyme, while the anionic detergent (SDS), even at a 0.1% level, severely inhibited enzyme activity. The chaotropic agents guanidinium hydrochloride, urea, and potassium iodide (0.5–8 M), significantly affected phytase activity. The maximum hydrolysis rate (V_max) and apparent Michaelis-Menten constant (K_m) were 1,074 IU/mL and 606 M, respectively, with a catalytic turnover number of 3×10⁵ s^–1 and catalytic efficiency of 3.69×10⁸ M^–1 s^–1.     

Photosynthetic response of Podophyllum hexandrum Royle from different altitudes in Himalayan ranges

Plants of Podophyllum hexandrum, collected from lower, mid, and upper distribution limits in alpine Himalaya were studied under greenhouse conditions to evaluate the photosynthetic response. Net photosynthetic rates (P _N), stomatal conductance (g _s), and efficiency of carbon uptake increased with altitude. The maximum P _N and g _s were measured in the considered population during the 3–6^th week of development. P _N and g _s decreased on an average by 58 and 48 % from maximum rates reached around 4^th week to the 10^th week of growth, respectively. The photosynthetic response in the three ecotypes appeared to be genetically controlled.