Fluorescent Secondary Antibodies Research Articles

Phosphonate-Anchored Monolayers for Antibody Binding to Magnetic Nanoparticles

Targeted delivery of magnetic iron oxide nanoparticles (IONPs) to a specific tissue can be achieved by conjugation with particular biological ligands on an appropriately functionalized IONP surface. To take best advantage of the unique magnetic properties of IONPs and to maximize their blood half-life, thin, strongly bonded, functionalized coatings are required. The work reported herein demonstrates the successful application of phosphonate-anchored self-assembled monolayers (SAMs) as ultrathin coatings for such particles. It also describes a new chemical approach to the anchoring of antibodies on the surface of SAM-coated IONPs (using nucleophilic aromatic substitution). This anchoring strategy results in stable, nonhydrolyzable, covalent attachment and allows the reactivity of the particles toward antibody binding to be activated in situ, such that prior to the activation the modified surface is stable for long-term storage. While the SAMs do not have the well-packed crystallinity of other such monolayers, their structure was studied using smooth model substrates based on an iron oxide layer on a double-side polished silicon wafer. In this way, atomic force microscopy, ellipsometry, and contact angle goniometry (tools that could not be applied to the nanoparticles' surfaces) could contribute to the determination of their monomolecular thickness and uniformity. Finally, the successful conjugation of IgG antibodies to the SAM-coated IONPs such that the antibodies retain their biological activity is verified by their complexation to a secondary fluorescent antibody.

Alveolar Epithelial Cells Undergo Epithelial-to-Mesenchymal Transition in Response to Endoplasmic Reticulum Stress

Expression of mutant surfactant protein C (SFTPC) results in endoplasmic reticulum (ER) stress in type II alveolar epithelial cells (AECs). AECs have been implicated as a source of lung fibroblasts via epithelial-to-mesenchymal transition (EMT); therefore, we investigated whether ER stress contributes to EMT as a possible mechanism for fibrotic remodeling. ER stress was induced by tunicamyin administration or stable expression of mutant (L188Q) SFTPC in type II AEC lines. Both tunicamycin treatment and mutant SFTPC expression induced ER stress and the unfolded protein response. With tunicamycin or mutant SFTPC expression, phase contrast imaging revealed a change to a fibroblast-like appearance. During ER stress, expression of epithelial markers E-cadherin and Zonula occludens-1 decreased while expression of mesenchymal markers S100A4 and α-smooth muscle actin increased. Following induction of ER stress, we found activation of a number of pathways, including MAPK, Smad, β-catenin, and Src kinase. Using specific inhibitors, the combination of a Smad2/3 inhibitor (SB431542) and a Src kinase inhibitor (PP2) blocked EMT with maintenance of epithelial appearance and epithelial marker expression. Similar results were noted with siRNA targeting Smad2 and Src kinase. Together, these studies reveal that induction of ER stress leads to EMT in lung epithelial cells, suggesting possible cross-talk between Smad and Src kinase pathways. Dissecting pathways involved in ER stress-induced EMT may lead to new treatment strategies to limit fibrosis.

The Effects of the c-kit Blocker Glivec on the Contractile Response of Urinary Bladder

To investigate the effects of imatinib (Glivec) on the urinary bladder contraction and excitation induced by neurostimulation, therefore to clarify the relationships between the bladder interstitial cell of Cajal (ICC) and the neural signals. In invivo experiments, pelvic nerves of rats were stimulated by square-wave pulses. The contractile response was recorded before and 40 min after the administration of medications (atropine, Glivec, and ketotifen). In invitro experiments, the bladder contractile response induced by acetylcholine with or without Glivec was evaluated. The space relationship between ICC and neural fibers were observed with double-labeled fluorescence using primary antibodies (anti-c-kit and anti-vesicular acetylcholine transferase) and secondary fluorescent antibodies (Alexa 488 and Alexa 594; Molecular Probes, Eugene, OR). Atropine and Glivec could significantly inhibit the bladder contractile response induced by the electrical stimulation in a dose-dependent manner, while ketotifen did not obviously affect bladder contractile response. In invitro experiments, Glivec did not affect acetylcholine-induced bladder contractile response. The location of ICC in close proximity to cholinergic nerve fibers was confirmed by double-labeled fluorescence. Bladder ICC play an important role as intermediaries in the transmission of cholinergic signals from nerve to smooth muscle cells.

Matrix Gla Protein Metabolism in Vascular Smooth Muscle and Role in Uremic Vascular Calcification

Matrix Gla protein (MGP) is an inhibitor of vascular calcification but its mechanism of action and pathogenic role are unclear. This was examined in cultured rat aortas and in a model of vascular calcification in rats with renal failure. Both carboxylated (GlaMGP) and uncarboxylated (GluMGP) forms were present in aorta and disappeared during culture with warfarin. MGP was also released into the medium and removed by ultracentrifugation, and similarly affected by warfarin. In a high-phosphate medium, warfarin increased aortic calcification but only in the absence of pyrophosphate, another endogenous inhibitor of vascular calcification. Although GlaMGP binds and inactivates bone morphogenic protein (BMP)-2, a proposed mediator of vascular calcification through up-regulation of the osteogenic transcription factor runx2, neither warfarin, BMP-2, nor the BMP-2 antagonist noggin altered runx2 mRNA content in aortas, and noggin did not prevent warfarin-induced calcification. Aortic content of MGP mRNA was increased 5-fold in renal failure but did not differ between calcified and noncalcified aortas. Immunoblots showed increased GlaMGP in noncalcified (5-fold) and calcified (20-fold) aortas from rats with renal failure, with similar increases in GluMGP. We conclude that rat aortic smooth muscle produces both GlaMGP and GluMGP in tissue-bound and soluble, presumably vesicular, forms. MGP inhibits calcification independent of BMP-2-driven osteogenesis and only in the absence of pyrophosphate, consistent with direct inhibition of hydroxyapatite formation. Synthesis of MGP is increased in renal failure and deficiency of GlaMGP is not a primary cause of medial calcification in this condition.

Receptor-like Protein-tyrosine Phosphatase α Enhances Cell Surface Expression of Neural Adhesion Molecule NB-3

Neural adhesion molecule NB-3 plays an important role in the apical dendrite development of layer V pyramidal neurons in the visual cortex, and receptor-like protein-tyrosine phosphatase α (PTPα) mediates NB-3 signaling in this process. Here we investigated the role of PTPα in regulating cell surface expression of NB-3. We found that cortical neurons from PTPα knock-out mice exhibited a lower level of NB-3 at the cell surface. When expressed in COS1 cells, NB-3 was enriched in the Golgi apparatus with a low level of cell surface expression. However, co-expression of PTPα increased the cell surface distribution of NB-3. Further analysis showed that PTPα facilitated Golgi exit of NB-3 and stabilized NB-3 protein at the cell surface by preventing its release from the plasma membrane. The extracellular region of PTPα but not its catalytic activity is necessary for its effect on NB-3 expression. Thus, the PTPα-mediated increase of NB-3 level at the cell surface represents a novel function of PTPα in NB-3 signaling in neural development.

Dissection and Staining of Drosophila Optic Lobes at Different Stages of Development

INTRODUCTIONThe Drosophila visual system is composed of the retina and the optic lobes, which are the ganglia where photoreceptors project and initial processing of visual inputs occur. This protocol outlines procedures for dissecting the optic lobes from Drosophila larvae, pupae, and adults. It also describes methods for visualizing the anatomy of brain neural circuits by staining with fluorescent secondary antibodies and primary antibodies specific for various neuronal populations and architectural features.

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

Identification of Novel Ryanodine Receptor 1 (RyR1) Protein Interaction with Calcium Homeostasis Endoplasmic Reticulum Protein (CHERP)

The ryanodine receptor type 1 (RyR1) is a homotetrameric Ca(2+) release channel located in the sarcoplasmic reticulum of skeletal muscle where it plays a role in the initiation of skeletal muscle contraction. A soluble, 6×-histidine affinity-tagged cytosolic fragment of RyR1 (amino acids 1-4243) was expressed in HEK-293 cells, and metal affinity chromatography under native conditions was used to purify the peptide together with interacting proteins. When analyzed by gel-free liquid chromatography mass spectrometry (LC-MS), 703 proteins were identified under all conditions. This group of proteins was filtered to identify putative RyR interacting proteins by removing those proteins found in only 1 RyR purification and proteins for which average spectral counts were enriched by less than 4-fold over control values. This resulted in 49 potential RyR1 interacting proteins, and 4 were selected for additional interaction studies: calcium homeostasis endoplasmic reticulum protein (CHERP), endoplasmic reticulum-Golgi intermediate compartment 53-kDa protein (LMAN1), T-complex protein, and phosphorylase kinase. Western blotting showed that only CHERP co-purified with affinity-tagged RyR1 and was eluted with imidazole. Immunofluorescence showed that endogenous CHERP co-localizes with endogenous RyR1 in the sarcoplasmic reticulum of rat soleus muscle. A combination of overexpression of RyR1 in HEK-293 cells with siRNA-mediated suppression of CHERP showed that CHERP affects Ca(2+) release from the ER via RyR1. Thus, we propose that CHERP is an RyR1 interacting protein that may be involved in the regulation of excitation-contraction coupling.

Lobe-specific Functions of Ca2+·Calmodulin in αCa2+·Calmodulin-dependent Protein Kinase II Activation

N-Methyl-d-aspartic acid receptor-dependent long term potentiation (LTP), a model of memory formation, requires Ca2+·calmodulin-dependent protein kinase II (αCaMKII) activity and Thr286 autophosphorylation via both global and local Ca2+ signaling, but the mechanisms of signal transduction are not understood. We tested the hypothesis that the Ca2+-binding activator protein calmodulin (CaM) is the primary decoder of Ca2+ signals, thereby determining the output, e.g. LTP. Thus, we investigated the function of CaM mutants, deficient in Ca2+ binding at sites 1 and 2 of the N-terminal lobe or sites 3 and 4 of the C-terminal CaM lobe, in the activation of αCaMKII. Occupancy of CaM Ca2+ binding sites 1, 3, and 4 is necessary and sufficient for full activation. Moreover, the N- and C-terminal CaM lobes have distinct functions. Ca2+ binding to N lobe Ca2+ binding site 1 increases the turnover rate of the enzyme 5-fold, whereas the C lobe plays a dual role; it is required for full activity, but in addition, via Ca2+ binding site 3, it stabilizes ATP binding to αCaMKII 4-fold. Thr286 autophosphorylation is also dependent on Ca2+ binding sites on both the N and the C lobes of CaM. As the CaM C lobe sites are populated by low amplitude/low frequency (global) Ca2+ signals, but occupancy of N lobe site 1 and thus activation of αCaMKII requires high amplitude/high frequency (local) Ca2+ signals, lobe-specific sensing of Ca2+-signaling patterns by CaM is proposed to explain the requirement for both global and local Ca2+ signaling in the induction of LTP via αCaMKII.

Human cadaveric tissue is suitable for histological, immunohistochemical and ultrastructural analysis

ObjectiveThis study examined the use of cadaveric material for morphological and pathological studies using histology, immunohistochemistry and TEM.MethodsParaffin‐embedded sections of liver, myocardium, kidney, testis and trachea were prepared from cadavers embalmed using the Dodge arterial injection method. Sections were either stained with H&E or incubated with antibodies against α‐smooth muscle actin (α‐SMA), acetylated α‐tubulin or connexin 43 and detected using either DAB or fluorescence. In addition, some samples were processed for TEM.ResultsAll tissues showed excellent histological morphology comparable to freshly‐fixed tissue; ultrastructural samples yielded moderate resolution. DAB‐positive labelling was observed for α‐SMA and α‐tubulin in all tissues. For immunofluorescence, all tissues were autofluorescent in the yellow‐green spectrum. However, we found that red fluorescent secondary antibodies positively labelled tubulin in tracheal cilia, sperm flagella and kidney primary cilia, as well as connexin 43 in myocardial intercalated disks and α‐SMA in all blood vessels.ConclusionAlthough it is often believed that there is poor microstructural preservation during embalming, we have shown that cadaveric tissues are an excellent source of ethically‐approved human tissue for histological and immunohistochemical studies in biomedical research. Funded by U of Auckland.

Molecular Characterization of the NPC1L1 Variants Identified from Cholesterol Low Absorbers

Niemann-Pick C1-like 1 (NPC1L1) is an essential protein for dietary cholesterol absorption. Nonsynonymous (NS) variants of NPC1L1 in humans have been suggested to associate with cholesterol absorption variations. However, information concerning the characteristics and mechanism of these variants in cholesterol uptake is limited. In this study, we analyzed the cholesterol uptake ability of the 19 reported NS variants of NPC1L1 identified from cholesterol low absorbers. Among these variants, L110F, R306C, A395V, G402S, T413M, R693C, R1214H, and R1268H could partially mediate cellular cholesterol uptake and were categorized as partially dysfunctional variants. The other 11 variants including T61M, N132S, D398G, R417W, G434R, T499M, S620C, I647N, G672R, S881L, and R1108W could barely facilitate cholesterol uptake, and were classified into the severely dysfunctional group. The partially dysfunctional variants showed mild defects in one or multiple aspects of cholesterol-regulated recycling, subcellular localization, glycosylation, and protein stability. The severely dysfunctional ones displayed remarkable defects in all these aspects and were rapidly degraded through the ER-associated degradation (ERAD) pathway. In vivo analyses using adenovirus-mediated expression in mouse liver confirmed that the S881L variant failed to localize to liver canalicular membrane, and the mice showed defects in biliary cholesterol re-absorption, while the G402S variant appeared to be similar to wild-type NPC1L1 in mouse liver. This study suggests that the dysfunction of the 19 variants on cholesterol absorption is due to the impairment of recycling, subcellular localization, glycosylation, or stability of NPC1L1.

Development and Characterization of High Affinity Leptins and Leptin Antagonists

Leptin is a pleiotropic hormone acting both centrally and peripherally. It participates in a variety of biological processes, including energy metabolism, reproduction, and modulation of the immune response. So far, structural elements affecting leptin binding to its receptor remain unknown. We employed random mutagenesis of leptin, followed by selection of high affinity mutants by yeast surface display and discovered that replacing residue Asp-23 with a non-negatively charged amino acid leads to dramatically enhanced affinity of leptin for its soluble receptor. Rational mutagenesis of Asp-23 revealed the D23L substitution to be most effective. Coupling the Asp-23 mutation with alanine mutagenesis of three amino acids (L39A/D40A/F41A) previously reported to convert leptin into antagonist resulted in potent antagonistic activity. These novel superactive mouse and human leptin antagonists (D23L/L39A/D40A/F41A), termed SMLA and SHLA, respectively, exhibited over 60-fold increased binding to leptin receptor and 14-fold higher antagonistic activity in vitro relative to the L39A/D40A/F41A mutants. To prolong and enhance in vivo activity, SMLA and SHLA were monopegylated mainly at the N terminus. Administration of the pegylated SMLA to mice resulted in a remarkably rapid, significant, and reversible 27-fold more potent increase in body weight (as compared with pegylated mouse leptin antagonist), because of increased food consumption. Thus, recognition and mutagenesis of Asp-23 enabled construction of novel compounds that induce potent and reversible central and peripheral leptin deficiency. In addition to enhancing our understanding of leptin interactions with its receptor, these antagonists enable in vivo study of the role of leptin in metabolic and immune processes and hold potential for future therapeutic use in disease pathologies involving leptin.

Multi-probe in situ hybridization to whole mount Arabidopsis seedlings

In situ RNA-RNA hybridization (ISH) is a molecular method for localization of gene transcripts at the cellular level and is widely used to provide spatial and temporal information regarding gene expression. However, standard protocols are complex and laborious to implement, restricting analysis to one or a few genes at any one time, each one observed on separate ISH preparations. Multi-probe whole-mount in situ hybridization is a powerful technique to compare the expression patterns of two or more genes simultaneously in the same tissue or organ. We describe for the first time in plants, the detection of three different mRNAs in a single fixed whole mount Arabidopsis seedling. A combination of bright fluorescent secondary antibodies was used for the detection of riboprobes differentially labeled by digoxigenin, biotin and fluorescein. The 3-D detection of each of the multiple fluorescent hybridization signals or in combination was obtained through confocal laser-scanning microscopy. The reliability of the method was tested in the root, using the PINFORMED (PIN) genes with non-overlapping temporal and spatial expression patterns. In the shoot, a class-I KNOTTED -like homeobox gene from Arabidopsis (KNAT1) with expression restricted to the shoot apical meristem was used in combination with ELONGATOR3 (ELO3) gene. In addition, the expression patterns of ELONGATOR complex gene (ELO2, ELO3) and HISTONE MONOUBIQUITINATION1 (HUB1) genes were analyzed in both shoot and root and a partial overlapping was observed. The whole procedure takes only 6 days.

Localization of αII Spectrin to Telomeres After DNA Interstrand Cross-Link Damage Is Deficient In Fanconi Anemia Group A Cells, Which May Lead to Telomere Dysfunction.

AbstractAbstract 1160A hallmark of the bone marrow failure disorder, Fanconi anemia (FA), is genomic instability which is characterized by both chromosome instability and defects in repair of DNA interstrand cross-links (ICLs). The chromosome instability in FA, in particular chromosome end fusions and breaks, suggests that there is telomere dysfunction. We have shown that the structural protein, nonerythroid a spectrin (aIISp), is present in the nucleus of normal human cells where it is critical in both repair of DNA ICLS and maintaining chromosome stability. aIISp is important in recruitment of the ICL repair protein, XPF, to sites of damage. We have shown that there is a deficiency in aIISp in FA cells lines, due to its increased breakdown, which correlates with reduced repair of DNA ICLs and increased chromosomal aberrations after ICL damage. The goal of the present study was to determine whether aIISp plays a role in telomere function after DNA ICL damage and whether telomere dysfunction in FA cells after damage can be corrected when levels of aIISp are restored to normal. Normal human and FA, complementation group A (FA-A) lymphoblastoid cells, which have telomerase, were used. Undamaged cells or cells treated with mitomycin C (MMC), a DNA ICL agent, were studied using immunofluorescence and in situ hybridization (IF-FISH). Cells were probed with anti-a-spectrin and either (C3TA2)3-Cy3 labeled peptide nucleic acid (PNA) telomeric probe or antibodies against two proteins, TRF1 or TRF2, which are components of the shelterin complex that is essential for protecting chromosome ends and preventing telomere dysfunction. The appropriate fluorescent secondary antibodies were used. The results showed that 47–50% of aIISp foci colocalized with the PNA probe, TRF1 or TRF2 in telomeres in MMC damaged but not undamaged normal cells. Levels of colocalization peaked at 16 hours and, by 24 hours, aIISp foci were no longer observed. This colocalization was specific for the S phase of the cell cycle. These results showed that in normal cells aIISp associates with telomeres in S phase after ICL damage and may play an important role in telomere function after damage. After ICLs are presumably unhooked/removed, aIISp dissociates from telomeres. The view that aIISp is involved in telomere function after ICL damage is strengthened by coimmunoprecipitation studies which show that aIISp has markedly enhanced binding for TRF1 and TFR2 after cells were damaged with MMC. IF-FISH also showed that the ICL repair protein, XPF, had enhanced association with telomeres after ICL damage, which further indicated that repair is taking place. In FA-A cells, in which aIISp levels are reduced, aIISp did not colocalize with the PNA probe, with TRF1 or with TRF2 after ICL damage; XPF also did not localize to telomeres after ICL formation. However, after aIISp levels were returned to normal in FA-A cells by siRNA knock down of m-calpain, a protease which cleaves aIISp and levels of which are elevated in FA cells, aIISp as well as XPF associated with telomeres after ICL damage. The chromosomal aberrations (i.e., breaks and end fusions) observed in FA-A cells after ICL damage were also corrected. ICLs can lead to stalled DNA replication forks in S phase. The present results suggest that, after damage, aIISp associates with telomeres in S phase at ICLs at stalled replication forks. Since aIISp is involved in ICL repair, this association could be of particular importance in repair of ICLs in replicating telomeres. In FA-A cells, failure of localization of aIISp and XPF to telomeres after ICL damage could have severe consequences. It could lead to unrepaired ICLs, which in replicating telomeres could lead to telomere dysfunction and to production of the chromosomal aberrations observed in these cells. These studies show that aIISp may play an important role in telomere function after DNA damage and during telomere replication. Correction of the deficiency in aIISp in FA cells by knocking down m-calpain could be significant in reversing telomere dysfunction and chromosomal aberrations after ICL damage and could potentially have important therapeutic relevance. Disclosures:No relevant conflicts of interest to declare.

A New Rapid Test for the Detection of PF4:Heparin Antibodies.

AbstractAbstract 3708Heparin Induced Thrombocytopenia (HIT), the most frequent drug-induced immune-mediated type of thrombocytopenia, is an important and sometimes life-threatening complication of heparin therapy. HIT can be triggered by standard therapeutic dose heparin, low-dose (prophylactic treatment), low molecular weight heparin, unfractionated heparin and even by minute quantities given to flush intravascular catheters.HIT is a clinicopathologic syndrome cause by platelet activating antibodies that recognize complexes of platelet factor 4 and heparin (PF4-heparin). Thrombocytopenia occurring within 5 – 10 days of administration of heparin is the most common clinical effect. The most severe complication of HIT is the occurrence of both venous and arterial thrombosis. Venous thrombosis can result in limb gangrene and the need for limb amputation.The diagnosis of HIT is based on clinical abnormalities including thrombocytopenia with or without thrombosis and the detection of antibodies to the PF4-heparin complex (termed PF4 dependant antibodies). There are two major types of assays for the detection of heparin dependant antibodies: functional assays and PF4 dependant antigen immunoassays. Functional assays include the serotonin release assay (SRA) and the heparin induced platelet activation assay (HIPA). Enzyme linked immunoassays (ELISAs) are the most frequently used PF4 dependent antigen immunoassay. Functional assays are technically difficult to perform and are considered to be complex specialty assays. ELISAs while not complex are not generally available on a STAT basis. There is a need for a simpler assay which retains or improves the sensitivity and specificity of the existing immunoassays that can be available on a STAT basis.GTI Diagnostics is developing a rapid test based on the proprietary technology used in GTI's PF4 Enhanced® ELISA. The PF4 Rapid test is a single use, semi-quantitative assay contained in a cartridge. It is designed to detect anti-PF4-heparin antibodies in human serum and/or plasma with a total assay time of 15 minutes. The assay uses only 5 μL of human serum and/or plasma along with a proprietary antigen and a proprietary fluorescent secondary antibody used for detection. The assay results are read using a fluorescent reader. A method comparison study was conducted which included an evaluation of 83 serum samples obtained from patients receiving heparin of which 36 were positive and 22 were negative in the PF4 Enhanced® assay. These samples were also tested in the PF4 Rapid Test assay. In addition 25 samples from normal individuals who were not receiving heparin were tested in both assays. Analysis of the data using a 2×2 table (shown below) resulted in a sensitivity of 100%, a specificity of 93.6% and an overall agreement of 96.4% of the PF4 Rapid Test compared to the PF4 Enhanced®.PF4 Enhanced® ELISAPositiveNegativeTotalPF4 Rapid TestPositive36339Negative04444Total364783Although HIT is a true clinicopathological syndrome, its diagnosis still rests primarily on clinical grounds since laboratory tests may not be available locally or may not be available in a sufficiently timely manner. Owing to the high risk of thrombosis associated with HIT, antithrombotic therapy with alternative anticoagulants should be started immediately when serologic assays confirm clinical suspicion. Readily available results, obtained from immunoassays for rapid detection of antibodies against PF4-heparin complexes, may be combined with the pretest estimation of clinical probability in order to exclude and/or confirm the diagnosis of HIT; therefore, assisting physicians in the time-sensitive clinical management of HIT. Disclosures:Ladvienka:GTI Diagnostics, Inc: Employment. Visentin:GTI Diagnostics, Inc: Employment. Chance:GTI Diagnostics, Inc: Employment.

Array Tomography: Immunostaining and Antibody Elution

Array tomography is a volumetric microscopy method based on physical serial sectioning. Ultrathin sections of a plastic-embedded tissue are cut using an ultramicrotome, bonded in an ordered array to a glass coverslip, stained as desired, and imaged. The resulting two-dimensional image tiles can then be reconstructed computationally into three-dimensional volume images for visualization and quantitative analysis. The minimal thickness of individual sections permits high-quality rapid staining and imaging, whereas the array format allows reliable and convenient section handling, staining, and automated imaging. Also, the physical stability of the arrays permits images to be acquired and registered from repeated cycles of staining, imaging, and stain elution, as well as from imaging using multiple modalities (e.g., fluorescence and electron microscopy). Array tomography makes it possible to visualize and quantify previously inaccessible features of tissue structure and molecular architecture. However, careful preparation of the tissue is essential for successful array tomography; these steps can be time-consuming and require some practice to perfect. In this protocol, tissue arrays are prepared for imaging by tagging with primary antibodies against specific cellular targets, followed by labeling with fluorescent secondary antibodies. Alternatively, fluorescent proteins that have been introduced into the tissue before dissection can be used.

Structural and Functional Characterization of the C-terminal Transmembrane Region of NBCe1-A

NBCe1-A and AE1 both belong to the SLC4 HCO(3)(-) transporter family. The two transporters share 40% sequence homology in the C-terminal transmembrane region. In this study, we performed extensive substituted cysteine-scanning mutagenesis analysis of the C-terminal region of NBCe1-A covering amino acids Ala(800)-Lys(967). Location of the introduced cysteines was determined by whole cell labeling with a membrane-permeant biotin maleimide and a membrane-impermeant 2-((5(6)-tetramethylrhodamine)carboxylamino) ethyl methanethiosulfonate (MTS-TAMRA) cysteine-reactive reagent. The results show that the extracellular surface of the NBCe1-A C-terminal transmembrane region is minimally exposed to aqueous media with Met(858) accessible to both biotin maleimide and TAMRA and Thr(926)-Ala(929) only to TAMRA labeling. The intracellular surface contains a highly exposed (Met(813)-Gly(828)) region and a cryptic (Met(887)-Arg(904)) connecting loop. The lipid/aqueous interface of the last transmembrane segment is at Asp(960). Our data clearly determined that the C terminus of NBCe1-A contains 5 transmembrane segments with greater average size compared with AE1. Functional assays revealed only two residues in the region of Pro(868)-Leu(967) (a functionally important region in AE1) that are highly sensitive to cysteine substitution. Our findings suggest that the C-terminal transmembrane region of NBCe1-A is tightly folded with unique structural and functional features that differ from AE1.

Enzymatic activity of the human 1-acylglycerol-3-phosphate-O-acyltransferase isoform 11: upregulated in breast and cervical cancers

The conversion of lysophosphatidic acid (LPA) to phosphatidic acid is carried out by the microsomal enzymes 1-acylglycerol-3-phosphate-O-acyltransferases (AGPATs). These enzymes are specific for acylating LPA at the sn-2 (carbon 2) position on the glycerol backbone and are important, because they provide substrates for the synthesis of phospholipids and triglycerides. At least, mutations in one isoform, AGPAT2, cause near complete loss of adipose tissue in humans. We cloned a cDNA predicted to be an AGPAT isoform, AGPAT11. This cDNA has been recently identified also as lysophosphatidylcholine acyltransferase 2 (LPCAT2) and lyso platelet-activating factor acetyltransferase. When AGPAT11/LPCAT2/lyso platelet-activating factor acetyltransferase cDNA was expressed in CHO and HeLa cells, the protein product localized to the endoplasmic reticulum. In vitro enzymatic activity using lysates of Human Embryonic Kidney-293 cells infected with recombinant AGPAT11/LPCAT2/lyso platelet-activating factor-acetyltransferase cDNA adenovirus show that the protein has an AGPAT activity but lacks glycerol-3-phosphate acyltransferase enzymatic activity. The AGPAT11 efficiently uses C18:1 LPA as acyl acceptor and C18:1 fatty acid as an acyl donor. Thus, it has similar substrate specificities for LPA and acyl-CoA as shown for AGPAT9 and 10. Expression of AGPAT11 mRNA was significantly upregulated in human breast, cervical, and colorectal cancer tissues, indicating its adjuvant role in the progression of these cancers. Our enzymatic assays strongly suggest that the cDNA previously identified as LPCAT2/lyso platelet-activating factor-acetyltransferase cDNA has AGPAT activity and thus we prefer to identify this clone as AGPAT11 as well.

One-step direct reconstitution of biomembranes onto cationic organic polymer bead supports

In this study, we addressed the straightforward reconstitution of red blood cell (RBC) membranes on the surface of cationic organic polymer beads. The RBC membrane–bead complex was obtained by the incubation of white, unsealed rat RBC ghost membranes with a nonporous quaternary ammonium-type anion-exchange polymer bead with a 350–550 μm diameter. Confocal microscopic observations using a fluorescence membrane probe revealed that the RBC membranes were reconstituted on the outer surface of the bead without any remarkable structural gaps in the membrane. The absence of activity of two peripheral enzymes that latently reside on the cytoplasmic face of the RBC membranes demonstrated that the orientation of the RBC membranes immobilized on the beads was asymmetric as well as that in the native state. The RBC membrane–polymer bead complex was incubated with a primary antibody that is directed against the amino-terminal extracellular domain of the integral protein glycophorin A (GPA). The resulting complex was further incubated with a fluorescent secondary antibody and then subjected to confocal microscopic observations. Fluorescence resulting in the binding of the secondary antibody was found on the surface of the complex, which indicates that the amino-terminal extracellular domain of GPA is exposed to the surface of the complex. In addition, the anion uptake function of the most abundant integral protein anion-exchanger 1 (AE1) immobilized on the polymer beads was inhibited by pretreatment with its specific inhibitor 4,4′-diisothiocyano-2,2′-stilbene disulfonate, as is observed for the intact RBCs. Based on all these results, the RBC membranes were thought to be reconstituted on the ionic polymer beads by our one-pot procedure while maintaining the orientation and functions of the membrane proteins to some extent.