Essential role of Ca2+/calmodulin in Early Endosome Antigen-1 localization

Ca2+ is an essential requirement in membrane fusion, acting through binding proteins such as calmodulin (CaM). Ca2+/CaM is required for early endosome fusion in vitro, however, the molecular basis for this requirement is unknown. An additional requirement for endosome fusion is the protein Early Endosome Antigen 1 (EEA1), and its recruitment to the endosome depends on phosphatidylinositol 3-phosphate [PI(3)P] and the Rab5 GTPase. Herein, we demonstrate that inhibition of Ca2+/CaM, by using either chemical inhibitors or specific antibodies directed to CaM, results in a profound inhibition of EEA1 binding to endosomal membranes both in live cells and in vitro. The concentration of Ca2+/CaM inhibitors required for a full dissociation of EEA1 from endosomal membranes had no effect on the activity of phosphatidylinositol 3-kinases or on endogenous levels of PI(3)P. However, the interaction of EEA1 with liposomes containing PI(3)P was decreased by Ca2+/CaM inhibitors. Thus, Ca2+/CaM seems to be required for the stable interaction of EEA1 with endosomal PI(3)P, perhaps by directly or indirectly stabilizing the quaternary organization of the C-terminal FYVE domain of EEA1. This requirement is likely to underlie at least in part the essential role of Ca2+/CaM in endosome fusion.     

Conventional kinesin KIF5B mediates insulin-stimulated GLUT4 movements on microtubules

Insulin stimulates glucose uptake in muscle and adipose cells by mobilizing intracellular membrane vesicles containing GLUT4 glucose transporter proteins to the plasma membrane. Here we show in live cultured adipocytes that intracellular membranes containing GLUT4-yellow fluorescent protein (YFP) move along tubulin-cyan fluorescent protein-labeled microtubules in response to insulin by a mechanism that is insensitive to the phosphatidylinositol 3 (PI3)-kinase inhibitor wortmannin. Insulin increased by several fold the observed frequencies, but not velocities, of long-range movements of GLUT4-YFP on microtubules, both away from and towards the perinuclear region. Genomics screens show conventional kinesin KIF5B is highly expressed in adipocytes and this kinesin is partially co-localized with perinuclear GLUT4. Dominant-negative mutants of conventional kinesin light chain blocked outward GLUT4 vesicle movements and translocation of exofacial Myc-tagged GLUT4-green fluorescent protein to the plasma membrane in response to insulin. These data reveal that insulin signaling targets the engagement or initiates the movement of GLUT4-containing membranes on microtubules via conventional kinesin through a PI3-kinase-independent mechanism. This insulin signaling pathway regulating KIF5B function appears to be required for GLUT4 translocation to the plasma membrane.     

Nonlinear PCA: characterizing interactions between modes of brain activity

This paper presents a nonlinear principal component analysis (PCA) that identifies underlying sources causing the expression of spatial modes or patterns of activity in neuroimaging time-series. The critical aspect of this technique is that, in relation to conventional PCA, the sources can interact to produce (second-order) spatial modes that represent the modulation of one (first-order) spatial mode by another. This nonlinear PCA uses a simple neural network architecture that embodies a specific form for the nonlinear mixing of sources that cause observed data. This form is motivated by a second-order approximation to any general nonlinear mixing and emphasizes interactions among pairs of sources. By introducing these nonlinearities principal components obtain with a unique rotation and scaling that does not depend on the biologically implausible constraints adopted by conventional PCA. The technique is illustrated by application to functional (positron emission tomography and functional magnetic resonance imaging) imaging data where the ensuing first- and second-order modes can be interpreted in terms of distributed brain systems. The interactions among sources render the expression of any one mode context-sensitive, where that context is established by the expression of other modes. The examples considered include interactions between cognitive states and time (i.e. adaptation or plasticity in PET data) and among functionally specialized brain systems (using a fMRI study of colour and motion processing).     

Characterization of Peptidyl-Prolyl Cis-Trans Isomerase- and Calmodulin-Binding Activity of a Cytosolic Arabidopsis thaliana Cyclophilin AtCyp19-3

Cyclophilins, which bind to immunosuppressant cyclosporin A (CsA), are ubiquitous proteins and constitute a multigene family in higher organisms. Several members of this family are reported to catalyze cis-trans isomerisation of the peptidyl-prolyl bond, which is a rate limiting step in protein folding. The physiological role of these proteins in plants, with few exceptions, is still a matter of speculation. Although Arabidopsis genome is predicted to contain 35 cyclophilin genes, biochemical characterization, imperative for understanding their cellular function(s), has been carried only for few of the members. The present study reports the biochemical characterization of an Arabidopsis cyclophilin, AtCyp19-3, which demonstrated that this protein is enzymatically active and possesses peptidyl-prolyl cis-trans isomerase (PPIase) activity that is specifically inhibited by CsA with an inhibition constant (K_i) of 18.75 nM. The PPIase activity of AtCyp19-3 was also sensitive to Cu²⁺, which covalently reacts with the sulfhydryl groups, implying redox regulation. Further, using calmodulin (CaM) gel overlay assays it was demonstrated that in vitro interaction of AtCyp19-3 with CaM is Ca²⁺-dependent, and CaM-binding domain is localized to 35–70 amino acid residues in the N-terminus. Bimolecular fluorescence complementation assays showed that AtCyp19-3 interacts with CaM in vivo also, thus, validating the in vitro observations. However, the PPIase activity of the Arabidopsis cyclophilin was not affected by CaM. The implications of these findings are discussed in the context of Ca²⁺ signaling and cyclophilin activity in Arabidopsis.     

Cooperativity of Cdk4R24C and Ras in melanoma development

The importance of the Cdk4 protein in human cancer became evident following the identification of a germ line mutation in the Cdk4 locus that predisposes humans to melanoma. This mutation results in substitution of argininefirst with cysteine at position 24 (R24C). In an earlier study, we introduced the R24C mutation into the Cdk4 locus of mice using Cre-loxp-mediated “knock-in” technology and observed a very low incidence of spontaneous melanomas in Cdk4^R24C/R24C mice. This suggested that additional oncogenic mutations might be required for development of melanomas. Here we report an increased incidence of spontaneous cutaneous melanoma in mice expressing the oncogene HRAS(G12V) in melanocytes on a Cdk4^R24C background. Treatment of Tyr-HRas:Cdk4^R24C/R24C mice with the carcinogen, DMBA/TPA resulted in a further increase in the number of nevi and melanomas developed when compared with Tyr-HRas:Cdk4^+/+ mice. In summary, in Tyr-HRas:Cdk4^R24C/R24C mice, we observed that activated Cdk4 cooperates with the oncogenic HRAS(G12V) protein to increase the susceptibility of melanoma development in vivo.Key words: Cdk4^R24C, ras, melanoma, skin, carcinogen     

Docosanoids are multifunctional regulators of neural cell integrity and fate: significance in aging and disease

The identification of neuroprotectin D1 (NPD1), a biosynthetic product of docosahexaenoic acid (DHA), in brain and retina as well as the characterization of its bioactivity, is generating a renewed interest in the functional role and pathophysiological significance of omega-3 fatty acids in the central nervous system. Neurotrophins, particularly pigment epithelium-derived factor (PEDF), induce NPD1 synthesis and its polarized apical secretion, implying paracrine and autocrine bioactivity of this lipid mediator. Also, DHA and PEDF synergistically activate NPD1 synthesis and antiapoptotic protein expression and decreased proapoptotic Bcl-2 protein expression and caspase 3 activation during oxidative stress. In experimental stroke, endogenous NPD1 synthesis was found to be upregulated, and the infusion of the lipid mediator into the brain under these conditions revealed neuroprotective bioactivity of NPD1. The hippocampal CA1 region from Alzheimer's disease (AD) patients (rapidly sampled) shows a major reduction in NPD1. The interplay of DHA-derived neuroprotective signaling aims to counteract proinflammatory, cell-damaging events triggered by multiple, converging cytokine and amyloid peptide factors, as in the case of AD. Generation of NPD1 from DHA thereby appears to redirect cellular fate toward successful preservation of retinal pigment epithelial (RPE)-photoreceptor cell integrity and brain cell aging. The Bcl-2 pro- and antiapoptotic proteins, neurotrophins, and NPD1, lie along a cell fate-regulatory pathway whose component members are highly interactive, and have potential to function cooperatively in cell survival. Agents that stimulate NPD1 biosynthesis, NPD1 analogs, or dietary regimens may be useful as new preventive/therapeutic strategies for neurodegenerative diseases.     

Antioxidant activity of fractionated extracts of rhizomes of high-altitude Podophyllum hexandrum: Role in radiation protection

Whole extract of rhizomes of Podophyllum hexandrum has been reported earlier by our group to render whole-body radioprotection. High-altitude P. hexandrum (HAPH) was therefore fractionated using solvents of varying polarity (non-polar to polar) and the different fractions were designated as, n-hexane (HE), chloroform (CE), alcohol (AE), hydro-alcohol (HA) and water (WE). The total polyphenolic content (mg% of quercetin) was determined spectrophotometrically, while. The major constituents present in each fraction were identified and characterized using LC-APCI/MS/MS. In vitro screening of the individual fractions, rich in polyphenols and lignans, revealed several bioactivities of direct relevance to radioprotection e.g. metal-chelation activity, antioxidant activity, DNA protection, inhibition of radiation (250 Gy) and iron/ascorbate-induced lipid peroxidation (LPO). CE exhibited maximum protection to plasmid (pBR322) DNA in the plasmid relaxation assay (68.09% of SC form retention). It also showed maximal metal chelation activity (41.59%), evaluated using 2,2-bipyridyl assay, followed by AE (31.25%), which exhibited maximum antioxidant potential (lowest absorption unit value: 0.0389± 0.00717) in the reducing power assay. AE also maximally inhibited iron/ascorbate-induced and radiation-induced LPO (99.76 and 92.249%, respectively, at 2000 g/ml) in mouse liver homogenate. Under conditions of combined stress (radiation (250 Gy) + iron/ascorbate), at a concentration of 2000 g/ml, HA exhibited higher percentage of inhibition (93.05%) of LPO activity. HA was found to be effective in significantly (p < 0.05) lowering LPO activity over a wide range of concentrations as compared to AE. The present comparative study indicated that alcoholic (AE) and hydro-alcoholic (HA) fractions are the most promising fractions, which can effectively tackle radiation-induced oxidative stress.     

Production of islet-like structures from neonatal porcine pancreatic tissue in suspension bioreactors

The aim of this study was to develop a scaleable process to expand pancreatic endocrine tissue (i.e., aggregates or islet-like structures) in suspension bioreactors. Key issues addressed included (i) serum-free media, (ii) cell inoculation density, (iii) medium pH, and (iv) aggregate dissociation. Suspension bioreactors were inoculated with pancreatic neonatal tissue and operated under controlled conditions for a 9-day period. Medium studies showed that a new serum-free medium developed in our laboratory was capable of supporting endocrine cell expansion. An inoculation density of 127,000 cells/mL resulted in more than a 7.5-fold increase in the number of insulin-positive cells after 9 days. The resulting population consisted of single cells and many islet-like aggregates that contained all of the endocrine cell types (including insulin-positive, glucagon-positive, somatostatin-positive, and pancreatic polypeptide-positive cells). Furthermore, the cell aggregates exhibited a glucose-responsive behavior. This study represents a significant milestone on the path to the effective expansion of human islet-like tissue in bioreactors that may be used for cell therapy to treat Type 1 diabetes.