SUBSURFACE CISTERNS AND THEIR RELATIONSHIP TO THE NEURONAL PLASMA MEMBRANE

Subsurface cisterns (SSC's) are large, flattened, membrane-limited vesicles which are very closely apposed to the inner aspect of the plasma membranes of nerve cell bodies and the proximal parts of their processes. They occur in a variety of vertebrate and invertebrate neurons of both the peripheral and central nervous systems, but not in the surrounding supporting cells. SSC's are sheet-like in configuration, having a luminal depth which may be less than 100 A and a breadth which may be as much as several microns. They are separated from the plasmalemma by a light zone of ~50 to 80 A which sometimes contains a faint intermediate line. Flattened, agranular cisterns resembling SSC's, but structurally distinct from both typical granular endoplasmic reticulum (ER) and from Golgi membranes, also occur deep in the cytoplasm of neurons. It is suggested that membranes which are closely apposed may interact, resulting in alterations in their respective properties. The patches of neuronal plasmalemma associated with subsurface cisterns may, therefore, have special properties because of this association, resulting in a non-uniform neuronal surface. The possible significance of SSC's in relation to neuronal electrophysiology and metabolism is discussed.     

IL-2 regulates expression of C-MAF in human CD4 T cells

Blockade of IL-2R with humanized anti-CD25 Abs, such as daclizumab, inhibits Th2 responses in human T cells. Recent murine studies have shown that IL-2 also plays a significant role in regulating Th2 cell differentiation by activated STAT5. To explore the role of activated STAT5 in the Th2 differentiation of primary human T cells, we studied the mechanisms underlying IL-2 regulation of C-MAF expression. Chromatin immunoprecipitation studies revealed that IL-2 induced STAT5 binding to specific sites in the C-MAF promoter. These sites corresponded to regions enriched for markers of chromatin architectural features in both resting CD4 and differentiated Th2 cells. Unlike IL-6, IL-2 induced C-MAF expression in CD4 T cells with or without prior TCR stimulation. TCR-induced C-MAF expression was significantly inhibited by treatment with daclizumab or a JAK3 inhibitor, R333. Furthermore, IL-2 and IL-6 synergistically induced C-MAF expression in TCR-activated T cells, suggesting functional cooperation between these cytokines. Finally, both TCR-induced early IL4 mRNA expression and IL-4 cytokine expression in differentiated Th2 cells were significantly inhibited by IL-2R blockade. Thus, our findings demonstrate the importance of IL-2 in Th2 differentiation in human T cells and support the notion that IL-2R-directed therapies may have utility in the treatment of allergic disorders.     

MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to knockdown gene expression levels in loss of gene function studies.There are two main methods for performing RNAi. The first is the use of small interfering RNAs (siRNAs) that are chemically synthesized, and the second utilizes short-hairpin RNAs (shRNAs) encoded within plasmids ¹. The latter can be transfected into cells directly or packaged into replication incompetent lentiviral particles. The main advantages of using lentiviral shRNAs is the ease of introduction into a wide variety of cell types, their ability to stably integrate into the genome for long term gene knockdown and selection, and their efficacy in conducting high-throughput loss of function screens. To facilitate this we have created the LentiPlex pooled shRNA library.The MISSION LentiPlex Human shRNA Pooled Library is a genome-wide lentiviral pool produced using a proprietary process. The library consists of over 75,000 shRNA constructs from the TRC collection targeting 15,000+ human genes ². Each library is tested for shRNA representation before product release to ensure robust library coverage. The library is provided in a ready-to-use lentiviral format at titers of at least 5 x 10⁸ TU/ml via p24 assay and is pre-divided into ten subpools of approximately 8,000 shRNA constructs each. Amplification and sequencing primers are also provided for downstream target identification.Previous studies established a synergistic antitumor activity of TRAIL when combined with Paclitaxel in A549 cells, a human lung carcinoma cell line ^{3, 4}. In this study we demonstrate the application of a pooled LentiPlex shRNA library to rapidly conduct a positive selection screen for genes involved in the cytotoxicity of A549 cells when exposed to TRAIL and Paclitaxel. One barrier often encountered with high-throughput screens is the cost and difficulty in deconvolution; we also detail a cost-effective polyclonal approach utilizing traditional sequencing.     

Elevated BCRP/ABCG2 expression confers acquired resistance to gefitinib in wild-type EGFR-expressing cells

Background

The sensitivity of non-small cell lung cancer (NSCLC) patients to EGFR tyrosine kinase inhibitors (TKIs) is strongly associated with activating EGFR mutations. Although not as sensitive as patients harboring these mutations, some patients with wild-type EGFR (wtEGFR) remain responsive to EGFR TKIs, suggesting that the existence of unexplored mechanisms renders most of wtEGFR-expressing cancer cells insensitive.

Methodology/Principal Findings

Here, we show that acquired resistance of wtEGFR-expressing cancer cells to an EGFR TKI, gefitinib, is associated with elevated expression of breast cancer resistance protein (BCRP/ABCG2), which in turn leads to gefitinib efflux from cells. In addition, BCRP/ABCG2 expression correlates with poor response to gefitinib in both cancer cell lines and lung cancer patients with wtEGFR. Co-treatment with BCRP/ABCG2 inhibitors enhanced the anti-tumor activity of gefitinib.

Conclusions/Significance

Thus, BCRP/ABCG2 expression may be a predictor for poor efficacy of gefitinib treatment, and targeting BCRP/ABCG2 may broaden the use of gefitinib in patients with wtEGFR.     

The evolutionary history of Melianthus (Melianthaceae)

The evolutionary origins of the morphological and taxonomic diversity of angiosperms is poorly known. We used the genus Melianthus to explore the diversification of the southern African flora. Melianthus comprises eight species, and a phylogeny based on one nuclear and two plastid genes, as well as a morphological data set, confirmed that the genus is monophyletic. The two earliest diverging lineages are found in relatively mesic habitats, whereas the two terminal clades (an eastern and a western clade), each with three species, favor more arid habitats. The eastern clade is largely restricted to the summer-rainfall parts of southern Africa, and the western clade is found in winter-rainfall region. Molecular dating indicates a mid-Tertiary origin of the genus, with diversification of the eastern and western clades coincident with the Late Miocene-Pliocene uplift of the Escarpment mountains and the establishment of summer aridity along the west coast. The remarkably complex flowers are indicative of sunbird pollination, but many smaller birds can also visit. Speciation may be the consequence of allopatric divergence into edaphic-climatic niches. Divergence in flower and inflorescence morphology might be in response to the divergent pressures for nectar conservation in arid regions coupled with the need for signaling to avian pollinators in generally shrubby vegetation.     

Engineering of betabellin 15D: Copper(II)-induced folding of a fibrillar β-sandwich protein

Summary The inverse protein-folding problem has been explored by designing de novo the betabellin target structure (a 64-residue β-sandwich protein), synthesizing a 32-residue peptide chain (HSLTAKIpkLTFSIAphTYTCAVpkYTAKVSH, wherep=DPro,k=DLys, andh=DHis) that might fold into this structure, and studying how its disulfide-bridged form (betabellin 15D) folds in 10 mM ammonium acetate with and without Cu²⁺. Circular dichroic spectropolarimetry indicated that at pH 5.8, 6.4, or 6.7 betabellin 15D exhibited β-sheet structure in the presence of Cu²⁺ but not in its absence. Electrospray mass spectrometry demonstrated that at pH 6.3 each molecule of betabellin 15D bound one or two Cu(II) ions. Electron microscopy showed that at pH 6.7 betabellin 15D formed short broad fibrils in the presence of Cu²⁺ but not in its absence. The observed width of the fibrils (7±2 nm) was consistent with the width (6.8nm) of a structural model of a fibril that contained two adjacent rows of betabellin 15D β-sandwiches joined lengthwise by multiple intersheet hydrogen bonds and widthwise by multiple Cu(II)-imidazole bonds. Electron paramagnetic resonance spectrometry revealed that some pairs of Cu(II) ions in a Cu(II)/betabellin 15D complex were magnetically coupled, which is consistent with the structural model of the Cu(II)/betabellin 15D fibril.     

A brief history of myelinated nerve fibers: one hundred and fifty years of controversy

The early controversies over myelinated nerve fibers focused on whether nerves are hollow or not, whether the fatty “marrow” (myelin) is inside the nerve fiber or around it, whether myelin is secreted by the axon or formed by another cell, whether nerve fibers are discrete or part of a syncytial network, whether nodes of Ranvier are present in central myelin or only in peripheral myelin. Since Geren's seminal discovery that peripheral myelin is formed by the Schwann cell plasma membrane wrapped around the axon, the focus has shifted. Myelin is clearly a living cell appendage, and the myelin sheath is dependent upon intercellular interactions not only during its formation, but throughout its lifetime and during pathological processes affecting either the axon or the myelin-forming cell. The myelinated fiber is a functional unit, an exquisite symbiosis, whose ability to perform optimally, in some cases whose very survival, depends on the effects the respective cells exert on one another. How are these interactions mediated? Which structures and functions depend on such interaction and which are independent of it? How do cells of the size and shape of myelin-forming cells cope with their metabolic demands and support their most distal components? What are the mechanisms and mutual consequences of demyelination or axonopathy? Relevant studies have burgeoned with the development of molecular biological and genetic engineering methods, and with improvements in microscopy, in vitro culture and specific immunostaining methods. This introductory essay provides an overview of the structural background and continuing controversies relevant to the articles that follow, which represent a sampling of current work and present new information on the molecular structure, function and pathology of myelin and axoglial interactions.

     

2-hydroxyglutarate detection by magnetic resonance spectroscopy in IDH-mutated patients with gliomas

Mutations in isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2) have been shown to be present in most World Health Organization grade 2 and grade 3 gliomas in adults. These mutations are associated with the accumulation of 2-hydroxyglutarate (2HG) in the tumor. Here we report the noninvasive detection of 2HG by proton magnetic resonance spectroscopy (MRS). We developed and optimized the pulse sequence with numerical and phantom analyses for 2HG detection, and we estimated the concentrations of 2HG using spectral fitting in the tumors of 30 subjects. Detection of 2HG correlated with mutations in IDH1 or IDH2 and with increased levels of D-2HG by mass spectrometry of the resected tumors. Noninvasive detection of 2HG may prove to be a valuable diagnostic and prognostic biomarker.     

Lagging strand synthesis in coordinated DNA synthesis by bacteriophage t7 replication proteins

The proteins of bacteriophage T7 DNA replication mediate coordinated leading and lagging strand synthesis on a minicircle template. A distinguishing feature of the coordinated synthesis is the presence of a replication loop containing double and single-stranded DNA with a combined average length of 2600 nucleotides. Lagging strands consist of multiple Okazaki fragments, with an average length of 3000 nucleotides, suggesting that the replication loop dictates the frequency of initiation of Okazaki fragments. The size of Okazaki fragments is not affected by varying the components (T7 DNA polymerase, gene 4 helicase-primase, gene 2.5 single-stranded DNA binding protein, and rNTPs) of the reaction over a relatively wide range. Changes in the size of Okazaki fragments occurs only when leading and lagging strand synthesis is no longer coordinated. The synthesis of each Okazaki fragment is initiated by the synthesis of an RNA primer by the gene 4 primase at specific recognition sites. In the absence of a primase recognition site on the minicircle template no lagging strand synthesis occurs. The size of the Okazaki fragments is not affected by the number of recognition sites on the template.     

A Proteomics Approach to Characterizing Tick Salivary Secretions

The saliva of ticks contains a complex mixture of bioactive molecules including proteins that modulate host responses ensuring successful feeding. The limited amount of saliva that can be obtained from ticks has hampered characterization of salivary proteins using traditional protein chemistry. Recent improvements in two-dimensional gel electrophoresis, mass spectrometry, and bioinformatics provide new tools to characterize small amounts of protein. These methods were employed to characterize salivary proteins from Amblyomma americanum and Amblyvomma maculatum. Salivation was induced by injection of dopamine and theophylline. It was necessary to desalt and concentrate saliva before analysis by 2-D electrophoresis. Comparison of 1-D and 2-D gel patterns revealed that the major protein component of saliva did not appear on 2-D gels. Characterization of this protein showed that it was identical to the major protein present in the hemolymph of both tick species. Protein profiles obtained by 1-D and 2-D gel electrophoresis were similar for both tick species, however, higher concentrations of lower molecular weight proteins were present in A. maculatum. Protein analysis by MALDI-TOF mass spectrometry and western blot analysis showed that except for the most abundant protein with a molecular weight of 95 kDa, all of the proteins detected were of host origin. It is not known if this is an artifact of the collection method or has physiological significance. In either case, in these species of ticks, host proteins will have to be removed from saliva samples prior to 2-D analysis in order to characterize lower abundance proteins of tick origin.