Planar cell polarity and vertebrate organogenesis

In addition to being polarized along their apical/basal axis, cells composing most (if not all) organs are also polarized in a plane vertical to the A/B axis. Recent studies indicate that this so-called planar cell polarity (PCP) plays an essential role in the formation of multiple organ systems regulating directed cell migrations, polarized cell division and proper differentiation. In this review we will discuss the molecular mechanisms regulating PCP, including the hypothesized roles for Wnt ligands in this process, and its roles in vertebrate organogenesis.     

Fluorescence-activated cell sorting and cloning of bona fide CD8+ CTL with reversible MHC-peptide and antibody Fab' conjugates

The isolation of subsets of Ag-specific T cells for in vitro and in vivo studies by FACS is compromised by the fact that the soluble MHC-peptide complexes and Abs used for staining, especially when combined, induce unwanted T cell activation and eventually apoptosis. This is especially a problem for CD8+ CTL, which are susceptible to activation-dependent cell death. In this study, we show that reversible MHC-peptide complexes (tetramers) can be prepared by conjugating MHC-peptide monomers with desthiobiotin (DTB; also called dethiobiotin) and multimerization by reaction with fluorescent streptavidin. While in the cold these reagents are stable and allow good staining, they rapidly dissociate in monomers at elevated temperatures, especially in the presence of free biotin. FACS cloning of Melan-A (MART-1)-specific CTL from a melanoma-infiltrated lymph node with reversible HLA-A2 Melan-A26-35 multimers yielded over two times more clones than when using the conventional biotin-containing multimers. CTL clones obtained by means of reversible multimers killed Melan-A-positive tumor cells more efficiently as compared with clones obtained with the stable multimers. Among the CTL obtained with the reversible multimers, but much less among those obtained with the stable multimers, a high proportion of clones exhibited high functional and physical avidity and died upon incubation with soluble MHC-peptide complexes. Finally, we show that Fab' of an anti-CD8 Ab can be converted in reversible DTB streptavidin conjugates the same way. These DTB reagents efficiently and reversibly stained murine and human CTL without affecting their viability.     

Interplay between T cell receptor binding kinetics and the level of cognate peptide presented by major histocompatibility complexes governs CD8+ T cell responsiveness

Through a rational design approach, we generated a panel of HLA-A*0201/NY-ESO-1(157-165)-specific T cell receptors (TCR) with increasing affinities of up to 150-fold from the wild-type TCR. Using these TCR variants which extend just beyond the natural affinity range, along with an extreme supraphysiologic one having 1400-fold enhanced affinity, and a low-binding one, we sought to determine the effect of TCR binding properties along with cognate peptide concentration on CD8(+) T cell responsiveness. Major histocompatibility complexes (MHC) expressed on the surface of various antigen presenting cells were peptide-pulsed and used to stimulate human CD8(+) T cells expressing the different TCR via lentiviral transduction. At intermediate peptide concentration we measured maximum cytokine/chemokine secretion, cytotoxicity, and Ca(2+) flux for CD8(+) T cells expressing TCR within a dissociation constant (K(D)) range of ～1-5 μM. Under these same conditions there was a gradual attenuation in activity for supraphysiologic affinity TCR with K(D) < ～1 μM, irrespective of CD8 co-engagement and of half-life (t(1/2) = ln 2/k(off)) values. With increased peptide concentration, however, the activity levels of CD8(+) T cells expressing supraphysiologic affinity TCR were gradually restored. Together our data support the productive hit rate model of T cell activation arguing that it is not the absolute number of TCR/pMHC complexes formed at equilibrium, but rather their productive turnover, that controls levels of biological activity. Our findings have important implications for various immunotherapies under development such as adoptive cell transfer of TCR-engineered CD8(+) T cells, as well as for peptide vaccination strategies.     

WNT-LRP5 Signaling Induces Warburg Effect through mTORC2 Activation during Osteoblast Differentiation

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Isolation and Identification of hydrocarbon degrading bacteria from Ennore creek

The widespread problem caused due to petroleum products, is their discharge and accidental spillage in marine environment proving to be hazardous to the surroundings as well as life forms. Thus remediation of these hydrocarbons by natural decontamination process is of utmost importance. Bioremediation is a non-invasive and cost effective technique for the clean-up of these petroleum hydrocarbons. In this study we have investigated the ability of microorganisms present in the sediment sample to degrade these hydrocarbons, crude oil in particular, so that contaminated soils and water can be treated using microbes. Sediments samples were collected once in a month for a period of twelve months from area surrounding Ennore creek and screened for hydrocarbon degrading bacteria. Of the 113 crude oil degrading isolates 15 isolates were selected and cultivated in BH media with 1% crude oil as a sole carbon and energy source. 3 efficient crude oil bacterial isolates Bacillus subtilis I1, Pseudomonas aeruginosa I5 and Pseudomonas putida I8 were identified both biochemically and phylogenetically. The quantitative analysis of biodegradation is carried out gravimetrically and highest degradation rate, 55% was recorded by Pseudomonas aeruginosa I5 isolate.     

The neurotransmitter serotonin interrupts α-synuclein amyloid maturation

Indolic derivatives can affect fibril growth of amyloid forming proteins. The neurotransmitter serotonin (5-HT) is of particular interest, as it is an endogenous molecule with a possible link to neuropsychiatric symptoms of Parkinson disease. A key pathomolecular mechanism of Parkinson disease is the misfolding and aggregation of the intrinsically unstructured protein α-synuclein. We performed a biophysical study to investigate an influence between these two molecules. In an isolated in vitro system, 5-HT interfered with α-synuclein amyloid fiber maturation, resulting in the formation of partially structured, SDS-resistant intermediate aggregates. The C-terminal region of α-synuclein was essential for this interaction, which was driven mainly by electrostatic forces. 5-HT did not bind directly to monomeric α-synuclein molecules and we propose a model where 5-HT interacts with early intermediates of α-synuclein amyloidogenesis, which disfavors their further conversion into amyloid fibrils.     

The influence of the different morphological changes on gastric mucosa on somatostatin cell number in antrum mucosa and serum somatostatin

The aim of our paper was to investigate the influence of the different morphological changes on gastric mucosa on somatostatin D-cell number in antral mucosa and serum Somatostatin. We analyzed according to Sydney classification to what extent the severity of gastritis affect the observed hormonal values. somatostatin D-cell number in antral mucosa and serum Somatostatin values were compared between three groups of patients; mild, moderate and severe chronic gastritis. The average number of somatostatin cell in biopsy sample of antrum mucosa was 30.41 +/- 35.38 (N = 17) in the case of middle form, 18.69 +/- 26.65 (N = 56) in moderate and in severe case of chronic gastritis 5.23 +/- 5.93 (N = 7) cells in mm2 of mucosa. The level of somatostatin in the serum of middle form gastritis were 26.43 +/- 28.76, moderate 19.95 +/- 35.93 and severe 17.88 +/- 17.66 pg/mL. In order to determine the number of somatostatin cells in antrum mucosa and serum somatostatin with present morphological changes of mucosa, it might helpful to exclude the patients with non-ulcer dyspepsia, but with the higher risk of premalignant and malignant changes.     

Protective effect of Nelumbo nucifera (Gaertn.) against H2O2-induced oxidative stress on H9c2 cardiomyocytes

Molecular Biology Reports - Ischemic heart disease (IHD), a severe condition of myocardium facing impediment in the supply of basic needs for cellular metabolism is caused by atherosclerosis....     

Superior In Vitro Stimulation of Human CD8+ T-Cells by Whole Virus versus Split Virus Influenza Vaccines

Pandemic and seasonal influenza viruses cause considerable morbidity and mortality in the general human population. Protection from severe disease may result from vaccines that activate antigen-presenting DC for effective stimulation of influenza-specific memory T cells. Special attention is paid to vaccine-induced CD8⁺ T-cell responses, because they are mainly directed against conserved internal influenza proteins thereby presumably mediating cross-protection against circulating seasonal as well as emerging pandemic virus strains. Our study showed that influenza whole virus vaccines of major seasonal A and B strains activated DC more efficiently than those of pandemic swine-origin H1N1 and pandemic-like avian H5N1 strains. In contrast, influenza split virus vaccines had a low ability to activate DC, regardless which strain was investigated. We also observed that whole virus vaccines stimulated virus-specific CD8⁺ memory T cells much stronger compared to split virus counterparts, whereas both vaccine formats activated CD4⁺ Th cell responses similarly. Moreover, our data showed that whole virus vaccine material is delivered into the cytosolic pathway of DC for effective activation of virus-specific CD8⁺ T cells. We conclude that vaccines against seasonal and pandemic (-like) influenza strains that aim to stimulate cross-reacting CD8⁺ T cells should include whole virus rather than split virus formulations.     

Cholesterol and Sphingomyelin Drive Ligand-independent T-cell Antigen Receptor Nanoclustering

The T-cell antigen receptor (TCR) exists in monomeric and nanoclustered forms independently of antigen binding. Although the clustering is involved in the regulation of T-cell sensitivity, it is unknown how the TCR nanoclusters form. We show that cholesterol is required for TCR nanoclustering in T cells and that this clustering enhances the avidity but not the affinity of the TCR-antigen interaction. Investigating the mechanism of the nanoclustering, we found that radioactive photocholesterol specifically binds to the TCRβ chain in vivo. In order to reduce the complexity of cellular membranes, we used a synthetic biology approach and reconstituted the TCR in liposomes of defined lipid composition. Both cholesterol and sphingomyelin were required for the formation of TCR dimers in phosphatidylcholine-containing large unilamellar vesicles. Further, the TCR was localized in the liquid disordered phase in giant unilamellar vesicles. We propose a model in which cholesterol and sphingomyelin binding to the TCRβ chain causes TCR dimerization. The lipid-induced TCR nanoclustering enhances the avidity to antigen and thus might be involved in enhanced sensitivity of memory compared with naive T cells. Our work contributes to the understanding of the function of specific nonannular lipid-membrane protein interactions.