Development of a potent <Emphasis Type="Italic">in vitro</Emphasis> source of <Emphasis Type="Italic">Phylanthus amarus</Emphasis> roots with pronounced activity against surface antigen of the hepatitis B virus

Summary In vitro culture of hairy roots of Phyllanthus amarus induced by Agrobacterium rhizogenes was established. Their growth and ability for in vitro inactivation of hepatitis B virus surface antigen was studied and compared with adventitious roots grown in vitro. The selected hairy root clone HR-1 was capable of growing at a very fast rate, and an approximately 900-fold increase in weight of root biomass was achieved after 4 wk of culture in hormone-free quarter-strength liquid Murashige and Skoog medium with continuous agitation. Non-transformed roots cultured in the presence of 1.0 mg l⁻¹ (5.71 μM) indole-3-acetic acid increased by 330-fold. The immuno-inactive property of roots was maximal in the crude extract. The hairy roots were shown to possess 85% inhibition (in contrast to 15% in the control) in binding of hepatitis B surface antigen (HBsAg) to its antibody (anti-HBs) after 24 h of incubation with HbsAg-positive sera in vitro at 37°C. Out of three fractions selected on the basis of molecular weight components of the extract, the Fraction III containing comparatively lower molecular weight substances (≤3500) yielded the highest activity. The extract from non-transformed roots was found to possess similar efficiency (87% inhibition). The levels of activity in both types of in vitro-raised roots were higher than those of naturally occurring roots and leafy shoots. The ability of P. amarus hairy root cultures to yield high biomass with the anti-viral property at high levels may provide an alternative source of raw material for more detailed study in the field of pharmaceutical research.     

Lactobacillus acidophilus Alleviates Platelet-Activating Factor-Induced Inflammatory Responses in Human Intestinal Epithelial Cells

Probiotics have been used as alternative prevention and therapy modalities in intestinal inflammatory disorders including inflammatory bowel diseases (IBD) and necrotizing enterocolitis (NEC). Pathophysiology of IBD and NEC includes the production of diverse lipid mediators, including platelet-activating factor (PAF) that mediate inflammatory responses in the disease. PAF is known to activate NF-κB, however, the mechanisms of PAF-induced inflammation are not fully defined. We have recently described a novel PAF-triggered pathway of NF-κB activation and IL-8 production in intestinal epithelial cells (IECs), requiring the pivotal role of the adaptor protein Bcl10 and its interactions with CARMA3 and MALT1. The current studies examined the potential role of the probiotic Lactobacillus acidophilus in reversing the PAF-induced, Bcl10-dependent NF-κB activation and IL-8 production in IECs. PAF treatment (5 µM×24 h) of NCM460 and Caco-2 cells significantly increased nuclear p65 NF-κB levels and IL-8 secretion (2-3-fold, P<0.05), compared to control, which were blocked by pretreatment of the cells for 6 h with L. acidophilus (LA) or its culture supernatant (CS), followed by continued treatments with PAF for 24 h. LA-CS also attenuated PAF-induced increase in Bcl10 mRNA and protein levels and Bcl10 promoter activity. LA-CS did not alter PAF-induced interaction of Bcl10 with CARMA3, but attenuated Bcl10 interaction with MALT1 and also PAF-induced ubiquitination of IKKγ. Efficacy of bacteria-free CS of LA in counteracting PAF-induced inflammatory cascade suggests that soluble factor(s) in the CS of LA mediate these effects. These results define a novel mechanism by which probiotics counteract PAF-induced inflammation in IECs.     

Quantum Chemical Studies of Structures and Binding in Noncanonical RNA Base pairs: The Trans Watson-Crick:Watson-Crick Family

Abstract

The trans Watson-Crick/Watson-Crick family of base pairs represent a geometric class that play important structural and possible functional roles in the ribosome, tRNA, and other functional RNA molecules. They nucleate base triplets and quartets, participate as loop closing terminal base pairs in hair pin motifs and are also responsible for several tertiary interactions that enable sequentially distant regions to interact with each other in RNA molecules. Eleven representative examples spanning nine systems belonging to this geometric family of RNA base pairs, having widely different occurrence statistics in the PDB database, were studied at the HF/6–31G (d, p) level using Morokuma decomposition, Atoms in Molecules as well as Natural Bond Orbital methods in the optimized gas phase geometries and in their crystal structure geometries, respectively. The BSSE and deformation energy corrected interaction energy values for the optimized geometries are compared with the corresponding values in the crystal geometries of the base pairs. For non protonated base pairs in their optimized geometry, these values ranged from ?8.19 kcal/mol to ?21.84 kcal/mol and compared favorably with those of canonical base pairs. The interaction energies of these base pairs, in their respective crystal geometries, were, however, lesser to varying extents and in one case, that of A:A W:W trans, it was actually found to be positive. The variation in RMSD between the two geometries was also large and ranged from 0.32–2.19 Å. Our analysis shows that the hydrogen bonding characteristics and interaction energies obtained, correlated with the nature and type of hydrogen bonds between base pairs; but the occurrence frequencies, interaction energies, and geometric variabilities were conspicuous by the absence of any apparent correlation. Instead, the nature of local interaction energy hyperspace of different base pairs as inferred from the degree of their respective geometric variability could be correlated with the identities of free and bound hydrogen bond donor/acceptor groups present in interacting bases in conjunction with their tertiary and neighboring group interaction potentials in the global context. It also suggests that the concept of isostericity alone may not always determine covariation potentials for base pairs, particularly for those which may be important for RNA dynamics. These considerations are more important than the absolute values of the interaction energies in their respective optimized geometries in rationalizing their occurrences in functional RNAs. They highlight the importance of revising some of the existing DNA based structure analysis approaches and may have significant implications for RNA structure and dynamics, especially in the context of structure prediction algorithms.     

Studies on the transport of aliphatic glucosides by hamster small intestine in vitro

It has been found (1) that glucosides with a long alkyl chain (2–18 carbon atoms) as the aglycone can be transported by carrier-mediated processes in the hamster small intestine in vitro, (2) that these glucosides interact with the glucose carrier, and (3) that they compete with glucose and analogs for the binding to the carrier. There are Na⁺- and phlorizin-insensitive components of uptake for the long chain alkyl glucosides which suggest additional interactions or uptake processes.     

A novel plasma membrane-bound thioredoxin from soybean

Two thioredoxin cDNAs from soybean were isolated by screening an expression library using an anti-(plasma membrane) serum. The nucleotide sequences of the two cDNAs were found to be 89% identical. The polypeptides encoded by the two cDNAs, designated TRX1 and TRX2, contain a disulfide active site, as found in other thioredoxins. TRX1 was expressed as a fusion protein in Escherichia coli and shown to possess thiol-disufide interchange activity. Unlike other eukaryotic thioredoxins, these two soybean thioredoxins contain a putative transmembrane domain in their N-terminal regions. To determine subcellular location, the TRX1 was fused with a reporter epitope at its C-terminus and expressed in transgenic tobacco plants. The fusion protein was co-purified with plasma membrane markers 1,3-glucan synthase and vanadate-sensitive ATPase, indicating the plasma membrane location of TRX1. When the reporter epitope was inserted between the start codon and the transmembrane domain in the N-terminus, the fusion protein was found in the soluble fraction, possibly due to disruption of the transmembrane domain by the highly hydrophilic epitope sequence. Taken together, our results demonstrate that soybean TRX1 is a plasma membrane-bound thioredoxin, which is most likely anchored to the membrane through the N-terminal transmembrane domain. It is known that plant plasma membranes contain various proteins with thiol-disulfide interchange activity. The soybean thioredoxins reported here are the first group of such proteins to be characterized at the molecular level. However, the biological function of the plasma membrane-bound thioredoxin remains to be determined.     

A comparison of four different conformations adopted by human telomeric G‐quadruplex using computer simulations

The telomeric G‐quadruplexes for their unique structural features are considered as potential anticancer drug targets. These, however, exhibit structural polymorphism as different topology types for the intra‐molecular G‐quadruplexes from human telomeric G‐rich sequences have been reported based on NMR spectroscopy and X‐ray crystallography. These techniques provide detailed atomic‐level information about the molecule but relative conformational stability of the different topologies remains unsolved. Therefore, to understand the conformational preference, we have carried out quantum chemical calculations on G‐quartets; used all‐atom molecular dynamics (MD) simulations and steered molecular dynamics (SMD) simulations to characterize the four human telomeric G‐quadruplex topologies based on its G‐tetrad core‐types, viz., parallel, anti‐parallel, mixed‐(3 + 1)‐form1 and mixed‐(3 + 1)‐form2. We have also studied a non‐telomeric sequence along with these telomeric forms giving a comparison between the two G‐rich forms. The structural properties such as base pairing, stacking geometry and backbone conformations have been analyzed. The quantum calculations indicate that presence of a sodium ion inside the G‐tetrad plane or two potassium ions on both sides of the plane give it an overall planarity which is much needed for good stacking to form a helix. MD simulations indicate that capping of the G‐tetrad core by the TTA loops keep the terminal guanine bases away from water. The SMD simulations along with equilibrium MD studies indicate that the parallel and non‐telomeric forms are comparatively less stable. We could come to the conclusion that the anti‐parallel form and also the mixed‐(3 + 1)‐form1 topology are most likely to represent the major conformation., 2016. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 83–99, 2016     

A mouse model for mucopolysaccharidosis type III A (Sanfilippo syndrome)

Mucopolysaccharidosis type III A (MPS III A, Sanfilippo syndrome) is a rare, autosomal recessive, lysosomal storage disease characterized by accumulation of heparan sulfate secondary to defective function of the lysosomal enzyme heparan N- sulfatase (sulfamidase). Here we describe a spontaneous mouse mutant that replicates many of the features found in MPS III A in children. Brain sections revealed neurons with distended lysosomes filled with membranous and floccular materials with some having a classical zebra body morphology. Storage materials were also present in lysosomes of cells of many other tissues, and these often stained positively with periodic-acid Schiff reagent. Affected mice usually died at 7-10 months of age exhibiting a distended bladder and hepatosplenomegaly. Heparan sulfate isolated from urine and brain had nonreducing end glucosamine- N -sulfate residues that were digested with recombinant human sulfamidase. Enzyme assays of liver and brain extracts revealed a dramatic reduction in sulfamidase activity. Other lysosomal hydrolases that degrade heparan sulfate or other glycans and glycosaminoglycans were either normal, or were somewhat increased in specific activity. The MPS III A mouse provides an excellent model for evaluating pathogenic mechanisms of disease and for testing treatment strategies, including enzyme or cell replacement and gene therapy.     

Lens specification is the ground state of all sensory placodes, from which FGF promotes olfactory identity

The sense organs of the vertebrate head comprise structures as varied as the eye, inner ear, and olfactory epithelium. In the early embryo, these assorted structures share a common developmental origin within the preplacodal region and acquire specific characteristics only later. Here we demonstrate a fundamental similarity in placodal precursors: in the chick all are specified as lens prior to acquiring features of specific sensory or neurogenic placodes. Lens specification becomes progressively restricted in the head ectoderm, initially by FGF and subsequently by signals derived from migrating neural crest cells. We show that FGF8 from the anterior neural ridge is both necessary and sufficient to promote olfactory fate in adjacent ectoderm. Our results reveal that placode precursors share a common ground state as lens and progressive restriction allows the full range of placodal derivatives to form.