Direct biochemical measurements of signal relay during Dictyostelium development

Upon starvation, individual Dictyostelium discoideum cells enter a developmental program that leads to collective migration and the formation of a multicellular organism. The process is mediated by extracellular cAMP binding to the G protein-coupled cAMP receptor 1, which initiates a signaling cascade leading to the activation of adenylyl cyclase A (ACA), the synthesis and secretion of additional cAMP, and an autocrine and paracrine activation loop. The release of cAMP allows neighboring cells to polarize and migrate directionally and form characteristic chains of cells called streams. We now report that cAMP relay can be measured biochemically by assessing ACA, ERK2, and TORC2 activities at successive time points in development after stimulating cells with subsaturating concentrations of cAMP. We also find that the activation profiles of ACA, ERK2, and TORC2 change in the course of development, with later developed cells showing a loss of sensitivity to the relayed signal. We examined mutants in PKA activity that have been associated with precocious development and find that this loss in responsiveness occurs earlier in these mutants. Remarkably, we show that this loss in sensitivity correlates with a switch in migration patterns as cells transition from streams to aggregates. We propose that as cells proceed through development, the cAMP-induced desensitization and down-regulation of cAMP receptor 1 impacts the sensitivities of chemotactic signaling cascades leading to changes in migration patterns.     

MicroRNA-21 orchestrates high glucose-induced signals to TOR complex 1, resulting in renal cell pathology in diabetes

Hyperglycemia induces a wide array of signaling pathways in the kidney that lead to hypertrophy and matrix expansion, eventually culminating in progressive kidney failure. High glucose-induced reduction of the tumor suppressor protein phosphatase and tensin homolog deleted in chromosome 10 (PTEN) contributes to renal cell hypertrophy and matrix expansion. We identified microRNA-21 (miR-21) as the molecular link between high glucose and PTEN suppression. Renal cortices from OVE26 type 1 diabetic mice showed significantly elevated levels of miR-21 associated with reduced PTEN and increased fibronectin content. In renal mesangial cells, high glucose increased the expression of miR-21, which targeted the 3'-UTR of PTEN mRNA to inhibit PTEN protein expression. Overexpression of miR-21 mimicked the action of high glucose, which included a reduction in PTEN expression and a concomitant increase in Akt phosphorylation. In contrast, expression of miR-21 Sponge, to inhibit endogenous miR-21, prevented down-regulation of PTEN and phosphorylation of Akt induced by high glucose. Interestingly, high glucose-stimulated miR-21 inactivated PRAS40, a negative regulator of TORC1. Finally, miR-21 enhanced high glucose-induced TORC1 activity, resulting in renal cell hypertrophy and fibronectin expression. Thus, our results identify a previously unrecognized function of miR-21 that is the reciprocal regulation of PTEN levels and Akt/TORC1 activity that mediate critical pathologic features of diabetic kidney disease.     

Human embryonic stem cell proliferation and differentiation as parameters to evaluate developmental toxicity

In vitro models based on embryonic stem cells (ESC) are highly promising for improvement of predictive toxicology screening in humans. After the successful validation of embryonic stem cell test (EST) in 2001; concerns have been raised on the usage of mouse ESC and also the morphological evaluation of beating cell clusters. This requires specialized skill-sets and is highly prone to misjudgement and false positive results. To overcome these limitations, we undertook the present study incorporating improvisations over the conventional EST. Here, we explored the potential of a human ESC (hESC)-based assay to evaluate the potential toxicity of penicillin-G, caffeine, and hydroxyurea. Drug treatment inhibited hESC adhesion and substantially altered the morphology and viability (～ 50%) of embryoid bodies (EBs). Flow cytometry analysis not only showed a significant increase of apoptotic cells in the highest doses but also induced a diverse pattern in DNA content and cell cycle distribution relative to control. Both semi-quantitative and quantitative RT-PCR studies revealed a selective down regulation of markers associated with stemness (Nanog, Rex1, SOX-2, and hTERT); cardiac mesoderm (Cripto1, MEF-2C, and Brachyury); hepatic endoderm (AFP, HNF-3β, HNF-4α, GATA-4, and SOX-17); and neuroectoderm (Nestin, SOX-1, NURR1, NEFH, Synaptophysin, TH, and Olig2) in a drug as well as dose dependent manner indicating abnormal differentiation. Furthermore, a decrease in the expression of AFP and GFAP proteins followed by a dose-dependent reduction in the levels of hCG-β, progesterone-II, and estradiol hormones was demonstrated by immunocytochemistry and ECLIA, respectively. This new and unique approach comprising of DNA cell cycle analysis, germ layer-specific marker expression and hormone levels as endpoints might offer a clinically relevant and commercially viable alternative for predicting in vivo developmental toxicity.     

Leaf micromorphology and leaf glandular hair ontogeny of Myoporum bontioides A. Gray

Myoporum bontioides A. Gray (Myoporaceae), a red list plant in Japan, is restricted to only a few East Asian countries like China, Japan and Taiwan, associated to some true mangroves. The leaf is isolateral and has a thick cuticle; stomata are anomocytic, sunken and have a beak‐shaped cuticular outgrowth at the inner and outer side of the stomatal pore (ledges); profuse glandular hairs are scattered on both leaf surfaces of young leaves. The mesophyll is compact with palisade and spongy parenchyma cells in the young stage, but at maturity profuse intercellular spaces can be observed. Secretory ducts occur in young leaves. Pear‐shaped glandular hairs protrude from the epidermal layer. Hair primordia are well distinct by their larger size and undergo divisions to produce two laterally placed basal cells, two stalk cells and four radiating terminal cells. The cuticle layers of the terminal cells are often separated from the cell wall to form a space, in which ions accumulate for excretion. Inner walls of the basal cells are connected with the mesophyll. Though ontogeny and structure of glandular hairs have resemblance to typical mangroves, considering leaf micromorphology, this plant is better termed as “mangrove associate” instead of “true mangrove”. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Anthelmintic efficacy of Flemingia vestita (Fabaceae): alterations in glucose metabolism of the cestode, Raillietina echinobothrida

The root-tuber peel of Flemingia vestita and its active component, genistein, were tested in respect of glucose metabolism in the cestode, Raillietina echinobothrida. Live R. echinobothrida, collected from the intestine of freshly slaughtered domestic fowl, were incubated at 39±1 °C in defined concentrations of the root-peel crude extract (5 mg/ml), genistein (0.2 mg/ml) and praziquantel (1 μg/ml) in phosphate buffered saline with 1% of dimethyl sulphoxide with simultaneous maintenance of controls. In the treated worms, there was a significant decrease in the glycogen concentration accompanied with the decrease of glucose by 14–32%, whereas the malate concentration increased by 49–134% as compared to controls. Both in controls and treated parasites, however, the pyruvate content was not measurable. While alanine and lactate contents showed a decline by 7–25% in the parasites exposed to all test materials, the lactate efflux into the incubation medium showed 37–71% increase in treatments indicating an overall increase of lactate production in comparison to controls. The results showing a decline in the glycogen and glucose contents and a significant rise in the malate content and lactate efflux under treatment conditions suggest that the energy demand in the parasites possibly got enhanced under stress, though it did not influence a switch over towards aerobic degradation of glucose in the parasites.     

Targeting internal ribosome entry site (IRES)-mediated translation to block hepatitis C and other RNA viruses

A number of RNA-containing viruses such as hepatitis C (HCV) and poliovirus (PV) that infect human beings and cause serious diseases use a common mechanism for synthesis of viral proteins, termed internal ribosome entry site (IRES)-mediated translation. This mode of translation initiation involves entry of 40S ribosome internally to the 5' untranslated region (UTR) of viral RNA. Cap-dependent translation of cellular mRNAs, on the other hand, requires recognition of mRNA 5' cap by the translation machinery. In this review, we discuss two inhibitors that specifically inhibit viral IRES-mediated translation without interfering with cellular cap-dependent translation. We present evidence, which suggest that one of these inhibitors, a small RNA (called IRNA) originally isolated from the yeast Saccharomyces cerevisiae, inhibits viral IRES-mediated translation by sequestering both noncanonical transacting factors and canonical initiation factors required for IRES-mediated translation. The other inhibitor, a small peptide from the lupus autoantigen La (called LAP), appears to block binding of cellular transacting factors to viral IRES elements. These results suggest that it might be possible to target viral IRES-mediated translation for future development of therapeutic agents effective against a number of RNA viruses including HCV that exclusively use cap-independent translation for synthesis of viral proteins.     

Identification of a rare point mutation at C-terminus of merozoite surface antigen-1 gene of Plasmodium falciparum in eastern Indian isolates

Merozoite surface antigen-1 (MSA-1) of Plasmodium falciparum is highly immunogenic in human. Several studies suggest that MSA-1 protein is an effective target for a protective immune response. Attempt has been made to find new point mutations by analyzing 244 bp [codon 1655(R) to 1735 (I)] relatively conserved C-terminus region of MSA-1 gene in 125 isolates. This region contains two EGF like domains, which are involved in generating protective immune response in human. Point mutations in this region are very much important in view of vaccine development. Searching of mutational hot spots in MSA-1 protein by sequencing method in a representative number of isolates is quite critical and expensive. Therefore, in this study slot blot and PCR-SSCP method have been used to find out new mutations in the individual isolates showing alterations in the mobility of DNA fragment. Sequencing of the altered bands from the SSCP gel shows a rare non-synonymous point mutation in 7 (5.6%) of the 125 isolates at amino acid position 1704 of MSA-1 gene where isoleucine is replaced by valine.     

Correlates of protective immune response in tuberculous pleuritis

Tuberculous pleuritis (TB) provides a good model to study the correlates of protective immune response at the site of infection. To study the in vivo correlates of immunity, cell subset profile and cytokine assay in plasma (BL) and pleural fluid (PF) of 82 patients were done. Lymphocyte proliferation and cytokine response to mycobacterial antigens were measured in 32 subjects to understand the in vitro correlates. Increase in CD4(+) cells and CD4(+)/CD8(+) ratio with selective concentration of interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha and interleukin (IL)-12 in PF suggests that the CD4(+) population may be of TH1 type. We observed an accelerated lymphoproliferative response to purified protein derivative (PPD) and heat killed Mycobacterium tuberculosis (MTB) in PF cells of both TB and non-TB (NTB) subjects. Interestingly, in in vitro studies, IL-4 levels together with IFN-gamma were significantly increased in the supernatants of PF mononuclear cells (PFMC) of TB patients and showed a shift in immune response towards TH0/TH2 type. PPD and MTB antigens induced an enhanced proliferation of PFMC and also increased in vitro IL-4 response together with apoptosis, thus eliciting a dual response.     

Benzene fused monocyclic enediynyl amides: synthesis, reactivity and DNA-cleavage activity in comparison to the corresponding sulfonamides

Monocyclic enediynyl amides 2a-2c have been synthesized via the corresponding free amine 5. Kinetic studies in chloroform revealed the reactivity of these amides towards Bergman cyclization to be less than that of the corresponding sulfonamides. However, differential scanning calorimetry (DSC) measurements in the solid state and DNA-cleavage studies in aqueous buffer showed higher reactivity for the amides than the sulphonamides.     

Thermodynamics of interaction of water vapour with 20 different poly-L-amino acids

The uptake of water vapour by 20 different polyaminoacids have been evaluated by an isopiestic vapour pressure technique in absence of solute at three different temperatures (22 degrees C, 30 degrees C and 37 degrees C). The water vapour adsorption isotherm for different polyaminoacids in the range of water activity varying between zero and unity apparently agreed with that expected from a type III BET isotherm. From the linear BET plots, obeyed in the lower range of water activity, the BET constants n(m) and Qm for different polyamines have been evaluated. The amount of water vapour adsorbed (n1) was calculated in moles/kg of polyaminoacids as well as in moles/mole of amino acid residue. Its value at unit water activity (deltan(o)1) has been evaluated by an extrapolation method and the results support that the multilayer adsorption of water vapour at the interface of polyaminoacids takes place. Values of deltan(o)1 are strictly comparable in terms of moles per kg rather than mole per mole unit. In case of beta lactoglobulin (betalg), lysozyme and BSA, theoretically obtained deltan(o)1 values were observed to be considerably larger than experimental values of deltan(o)1. This indicated that amino acid residues in the polypeptide release a large amount of water due to the formation of a globular structure. Using the Bull equation in the integrated form, standard free energies, deltaGo(w) for water-polyamino acid binding interaction at two different temperatures have been evaluated. Based on the Clausius-Clapeyron equation in an integrated form, the integral enthalpy for water-polyamino acid interaction has also been evaluated.