FITC-conjugated Secondary Antibody Research Articles

Immunofluorescence Staining for IgG Subclass: Cause for Discrepancy in the Detection of IgG1

Immunofluorescence (IF) staining for IgG subclasses plays an important role in the classification of kidney disease. However, widely used IgG subclass-specific antibodies are now commercially unavailable. Thus, we compared alternative antibodies for performing IgG subclass staining. A total of 21 cases were stained by 3 different methods: direct IF using fluorescein isothiocyanate (FITC)-conjugated polyclonal antibodies against IgG1-4 (commercially unavailable method), direct IF using FITC-conjugated monoclonal antibodies (clones HP-6091, 6014, 6050, and 6025), indirect IF using monoclonal antibodies (clones HP-6069, 6002, 6050, and 6025), and FITC-conjugated polyclonal secondary antibody. For cases with discrepancy in IgG1 staining, additional direct IF using FITC-conjugated monoclonal antibody (clone 4E3) was performed. Of 21 cases, 11 (52%) had no staining for IgG1 by direct IF using the clone HP-6091 despite≥1+ staining by the direct IF using polyclonal antibodies. Similarly, direct IF for IgG1 using the clone 4E3 had negative result in all 10 cases with available tissue. However, indirect IF for IgG1 using the clone HP-6069 had similar staining intensity (within 1 order of magnitude) as direct IF using the polyclonal antibodies (10 of 10). Results of IF for IgG2, IgG3, and IgG4 were similar in most cases. The choice of antibodies influences the result of IgG subclass staining, especially for anti-IgG1 antibodies, in which 2 monoclonal antibodies (HP6091 and 4E3) appear less sensitive. Although this may be due to unaccounted variables and requires confirmation, our results may partially explain the difference in IgG1 staining in the literature and underscore the need for careful validation.

Cannabidiol: assessing activity in ovarian and endometrial carcinoma cell lines

<h3>Objectives:</h3> Cancer patients use cannabidiol (CBD) for chemotherapy and cancer symptoms, though recognition of CBD by and its effect on endometrial cancer (EnC) and epithelial ovarian cancer (EOC) are not established. We sought to test expression of cannabidiol receptor 1 (CB1) and cannabidiol receptor 2 (CB2) on EnC (ECC1) and EOC (Kuramochi, OVCar-3, and murine ID8 (p53/<i>BRCA2</i> negative)) cell lines. We further explore EnC (ECC1) and EOC (Kuramochi) cell proliferation when exposed to different concentrations of CBD alone, and with cytotoxic chemotherapies carboplatin (CP) and paclitaxel (PT). <h3>Methods:</h3> We assessed CB1 and CB2 receptor expression in the four cell lines by flow cytometry using primary mouse monoclonal antibodies against these receptors followed by detection using a FITC-conjugated secondary antibody. ECC1 and Kuramochi cells were kept in RPMI media with 10% bovine serum + 1% penicillin/streptomycin. We passaged cells when >90% confluent by adding trypsin-EDTA, incubating at 370 C for 3 minutes, then spinning down with media to harvest cell pellets. Cells were re-suspended in media, apportioned to 96 well plates, and incubated at 370C for 24 hours. CBD (Cayman Chemical) was suspended in DMSO per manufacturer then used to treat cells for72 hours at different concentrations (2.5-50 µM). We also treated cells with CBD and CP and/or PT, choosing concentration gradients for combinations based on assays with single agents. MTT was added and cells were incubated at 370 C for 3 hours. The media and MTT were removed and DMSO was added to dissolve formazan crystals. Optical density (OD) at 570 nm was calculated for plates using SoftMaxPro version 6.2.2. ODs were used to calculate inhibitory concentration for 50% cell death (IC50) for CBD alone, CBD+CP, CBD+PT and CBD+CP and PT. <h3>Results:</h3> All four cell lines tested had measurable CB1 but no CB2 receptor expression on flow cytometry. Kuramochi and ECC1 had decreased proliferation after 72 hours of CBD exposure. ECC1 IC50 was 2.5-5 µM CBD concentration; Kuramochi IC50 was 15-20 µM CBD. Nearly all ECC1 growth was inhibited at concentrations 10 µM or greater. Kuramochi proliferation was 15% that of controls at concentrations of 40 and 50 µM CBD. ECC1 had expected dose-dependent decreased in proliferation when treated with CBD in combination with CP or PT. Kuramochi as a platinum resistant cell line demonstrated decreased proliferation with CBD alone and in combo with PT but not with CP. <h3>Conclusions:</h3> ECC1 and Kuramochi demonstrated decreased proliferation in the presence of CBD, with combination studies having similar trends. CB1 likely mitigates proliferation effect of CBD on Kuramochi and ECC1. OVCAR3 and ID8 cells have CB1; further experimentation will establish CBD treatment effect on these lines. Our studies suggest a beneficial anti-proliferative effect of CBD as a single agent or when used in combination with CP or PT, an important finding considering the use of CBD by gynecologic cancer patients for relief from cancer and chemotherapy side effects.

Progression of Watermelon Bud Necrosis Virus Infection in Its Vector, Thrips palmi.

Thrips are important pests of agricultural, horticultural, and forest crops worldwide. In addition to direct damages caused by feeding, several thrips species can transmit diverse tospoviruses. The present understanding of thrips–tospovirus relationships is largely based on studies of tomato spotted wilt virus (TSWV) and Western flower thrips (Frankliniella occidentalis). Little is known about other predominant tospoviruses and their thrips vectors. In this study, we report the progression of watermelon bud necrosis virus (WBNV) infection in its vector, melon thrips (Thrips palmi). Virus infection was visualized in different life stages of thrips using WBNV-nucleocapsid protein antibodies detected with FITC-conjugated secondary antibodies. The anterior midgut was the first to be infected with WBNV in the first instar larvae. The midgut of T. palmi was connected to the principal salivary glands (PSG) via ligaments and the tubular salivary glands (TSG). The infection progressed to the PSG primarily through the connecting ligaments during early larval instars. The TSG may also have an ancillary role in disseminating WBNV from the midgut to PSG in older instars of T. palmi. Infection of WBNV was also spread to the Malpighian tubules, hindgut, and posterior portion of the foregut during the adult stage. Maximum virus-specific fluorescence in the anterior midgut and PSG indicated the primary sites for WBNV replication. These findings will help to better understand the thrips–tospovirus molecular relationships and identify novel potential targets for their management. To our knowledge, this is the first report of the WBNV dissemination path in its vector, T. palmi.

Regulatory T cells promote corneal endothelial cell survival following transplantation via interleukin-10.

The functional competence of corneal endothelial cells (CEnCs) is critical for survival of corneal allografts, but these cells are often targets of the immune response mediated by graft-attacking effector T cells. Although regulatory T cells (Tregs) have been studied for their role in regulating the host's alloimmune response towards the graft, the cytoprotective function of these cells on CEnCs has not been investigated. The aim of this study was to determine whether Tregs suppress effector T cell-mediated and inflammatory cytokine-induced CEnC death, and to elucidate the mechanism by which this cytoprotection occurs. Using 2 well-established models of corneal transplantation (low-risk and high-risk models), we show that Tregs derived from low-risk graft recipients have a superior capacity in protecting CEnCs against effector T cell-mediated and interferon-γ and tumor necrosis factor-α-induced cell death compared to Tregs derived from high-risk hosts. We further demonstrate that the cytoprotective function of Tregs derived from low-risk hosts occurs independently of direct cell-cell contact and is mediated by the immunoregulatory cytokine IL-10. Our study is the first to report that Tregs provide cytoprotection for CEnCs through secretion of IL-10, indicating potentially novel therapeutic targets for enhancing CEnC survival following corneal transplantation.

REGULATION OF Aβ- OR ROS-MEDIATED NEURITE DEGENERATION BY AK5

Adenylate kinases (AKs) play a key role in nucleotide metabolism via nucleotide phosphoryl exchange. A new member of this family, Ak5, has recently been identified, but its function remains unknown. In this study, we find that neuronal Ak5 mediates amyloid-beta (Ab) or ROS-mediated neurite degeneration and neurotoxicity. Cell-based functional screening was performed using cell-based tau aggregation assay and cDNA expression library. Primary cortical or hippocampal neurons from mouse embryos (E16 or E18) were prepared and seeded on poly-L-lysine-coated glass cover slips. For Immunocytochemistry, Cells were double-stained with Ak5 and beta-tubulin and washed with PBS before the incubation with FITC-conjugated or TRITC-conjugated secondary antibodies. In vitro microtubule assay was performed using purified AK5 and tubulins. We found that Ak5 is mainly expressed in the hippocampus and cortex of the mouse brains. Compared to AK1, purified Ak5 protein shows little kinase activity in vitro. From pull-down and LC/MS/MS analysis, we found the microtubules as an interaction partner of Ak5 in the mouse brains. Ak5 expression is significantly up-regulated in the neuronal cells exposed to Ab and hydrogen peroxides (H2O2). Also ectopic expression of Ak5 lead to neurite degeneration and neuronal death. Interestingly, Ak5 antagonizes the interaction of tau with microtubules in vitro and in cells. Furthermore, expression of Ak5 is increased in the brains of AD model mice and patients. We conclude that Ak5 plays a novel role in Ab- or ROS-mediated neurite degeneration via its competitive binding with tau to microtubules, providing insight into a mechanism for tau-mediated neurodegeneration in AD. Reference: Park H, Kam TI, Kim Y, Choi H, Gwon Y, Kim C, Koh JY, Jung YK (2012) Neuropathogenic role of adenylate kinase-1 in Aβ-mediated tau phosphorylation via AMPK and GSK3β. Hum Mol Genet 21: 2725–2737. Solaroli N, Panayiotou C, Johansson M, Karlsson A (2009) Identification of two active functional domains of human adenylate kinase 5. FEBS Lett 583: 2872–2876.

Quantitative Analysis of Endocytic Recycling of Membrane Proteins by Monoclonal Antibody-Based Recycling Assays.

In this report, we present an analysis of several recycling protocols based on labeling of membrane proteins with specific monoclonal antibodies (mAbs). We analyzed recycling of membrane proteins that are internalized by clathrin-dependent endocytosis, represented by the transferrin receptor, and by clathrin-independent endocytosis, represented by the Major Histocompatibility Class I molecules. Cell surface membrane proteins were labeled with mAbs and recycling of mAb:protein complexes was determined by several approaches. Our study demonstrates that direct and indirect detection of recycled mAb:protein complexes at the cell surface underestimate the recycling pool, especially for clathrin-dependent membrane proteins that are rapidly reinternalized after recycling. Recycling protocols based on the capture of recycled mAb:protein complexes require the use of the Alexa Fluor 488 conjugated secondary antibodies or FITC-conjugated secondary antibodies in combination with inhibitors of endosomal acidification and degradation. Finally, protocols based on the capture of recycled proteins that are labeled with Alexa Fluor 488 conjugated primary antibodies and quenching of fluorescence by the anti-Alexa Fluor 488 displayed the same quantitative assessment of recycling as the antibody-capture protocols. J. Cell. Physiol. 232: 463-476, 2017. © 2016 Wiley Periodicals, Inc.

Topical fentanyl stimulates healing of ischemic wounds in diabetic rats.

Topically applied opioids promote angiogenesis and healing of ischemic wounds in rats. We examined if topical fentanyl stimulates wound healing in diabetic rats by stimulating growth-promoting signaling, angiogenesis, lymphangiogenesis and nerve regeneration. We used Zucker diabetic fatty rats that develop obesity and diabetes on a high fat diet due to a mutation in the Leptin receptor. Fentanyl blended with hydrocream was applied topically on ischemic wounds twice daily, and wound closure was analyzed regularly. Wound histology was analyzed by hematoxylin and eosin staining. Angiogenesis, lymphangiogenesis, nerve fibers and phospho-platelet derived growth factor receptor-β (PDGFR-β) were visualized by CD31-, lymphatic vessel endothelium-1, protein gene product 9.5- and anti-phospho PDGFR-β-immunoreactivity, respectively. Nitric oxide synthase (NOS) and PDGFR-β signaling were analyzed using Western immunoblotting. Fentanyl significantly promoted wound closure as compared to phosphate-buffered saline (PBS). Histology scores were significantly higher in fentanyl-treated wounds, indicative of increased granulation tissue formation, reduced edema and inflammation, and increased matrix deposition. Fentanyl treatment resulted in increased wound angiogenesis, lymphatic vasculature, nerve fibers, nitric oxide, NOS and PDGFR-β signaling as compared to PBS. Phospho-PDGFR-β co-localized with CD31 co-staining for vasculature. Topically applied fentanyl promotes closure of ischemic wounds in diabetic rats. Increased angiogenesis, lymphangiogenesis, peripheral nerve regeneration, NO and PDGFR-β signaling are associated with fentanyl-induced tissue remodeling and wound healing.

Apolipoprotein A-IV Reduces Hepatic Gluconeogenesis through Nuclear Receptor NR1D1

We showed recently that apoA-IV improves glucose homeostasis by enhancing pancreatic insulin secretion in the presence of elevated levels of glucose. Therefore, examined whether apolipoprotein A-IV (apoA-IV) also regulates glucose metabolism through the suppression of hepatic gluconeogenesis. The ability of apoA-IV to lower gluconeogenic gene expression and glucose production was measured in apoA-IV(-/-) and wild-type mice and primary mouse hepatocytes. The transcriptional regulation of Glc-6-Pase and phosphoenolpyruvate carboxykinase (PEPCK) by apoA-IV was determined by luciferase activity assay. Using bacterial two-hybrid library screening, NR1D1 was identified as a putative apoA-IV-binding protein. The colocalization and interaction between apoA-IV and NR1D1 were confirmed by immunofluorescence, in situ proximity ligation assay, and coimmunoprecipitation. Enhanced recruitment of NR1D1 and activity by apoA-IV to Glc-6-Pase promoter was verified with ChIP and a luciferase assay. Down-regulation of apoA-IV on gluconeogenic genes is mediated through NR1D1, as illustrated in cells with NR1D1 knockdown by siRNA. We found that apoA-IV suppresses the expression of PEPCK and Glc-6-Pase in hepatocytes; decreases hepatic glucose production; binds and activates nuclear receptor NR1D1 and stimulates NR1D1 expression; in cells lacking NR1D1, fails to inhibit PEPCK and Glc-6-Pase gene expression; and stimulates higher hepatic glucose production and higher gluconeogenic gene expression in apoA-IV(-/-) mice. We conclude that apoA-IV inhibits hepatic gluconeogenesis by decreasing Glc-6-Pase and PEPCK gene expression through NR1D1. This novel regulatory pathway connects an influx of energy as fat from the gut (and subsequent apoA-IV secretion) with inhibition of hepatic glucose production.

Leukemic Blasts With The PNH Phenotype: Correlation With Cytogenetics In ALL

It has been proposed that hypermutability is essential for the generation of malignancies that require more than a few oncogenic mutations and may also promote the emergence of drug-resistant clones by contributing to intratumoral diversity. However, most genes are not suitable as a reporter for quantitating the frequency of spontaneous somatic inactivating mutations. A notable exception is the PIG-A gene, which encodes an enzyme catalyzing an initial step in the biosynthesis of glycosylphosphatidylinositol (GPI), which links certain membrane proteins to the cell surface. Of note, the PIG-A-mutant phenotype can be readily demonstrated by flow cytometry, using antibodies specific for GPI-linked proteins (e.g., CD55 and CD59), as well as the FLAER reagent (which binds to GPI directly), enabling the rapid screening of large populations for rare variants that are GPI-negative. It is known from the condition paroxysmal nocturnal hemoglobinuria that a very broad spectrum of PIG-A mutations can produce this “PNH phenotype”, and because the gene is X-linked, this requires only a single mutation. Apart from patients with PNH, there is evidence that PIG-A mutations are growth-neutral in most all other circumstances. We have previously demonstrated rare spontaneously arising cells with the PNH phenotype and genotype in blood cells of normal donors and in B-lymphoblastoid cell lines (BLCLs); similar populations can be demonstrated in mice, with marked increases after mutagen exposure. We have also shown that malignant cell lines often demonstrate an elevated frequency (f) of spontaneously arising cells with the PNH phenotype. Recently, we have reported similar findings in ex vivo leukemic blasts derived from patients with ALL: in samples from 10 of 19 patients, the median frequency of blasts with the PNH phenotype was low, with a median f value of 13 per million, which was similar to that of BLCLs from normal donors. The remaining 9 samples demonstrated an elevated f value (median 566 per million). Based on our preliminary subset analysis, along with published data demonstrating a higher mutation burden in leukemias with the ETV6-RUNX1 (TEL-AML1) translocation compared with hyperdiploid samples, we hypothesized that we would find a higher mutant frequency in the former group. Therefore, we have now analyzed in a blinded manner a new panel of blast cells derived from ficolled pre-treatment marrow samples from patients with B-lineage ALL obtained from the Children's Oncology Group ALL Cell Bank. To determine f, samples were stained sequentially with an Alexa-488 conjugate of the FLAER reagent and then a mixture of mouse anti-CD55 and anti-CD59 antibodies, followed by FITC-conjugated rabbit anti-mouse immunoglobulin secondary antibody (registering on FL1), and then PE-conjugated anti-CD45 antibody (registering on FL2). The blast population was identified based on forward and side scatter, expression of CD45, and exclusion of propidium iodide. GPI-negative cells were defined as expressing <4% of the mean level of FL1 fluorescence as the GPI-positive population, and >10% of its mean FL2 fluorescence. Overall, among 26 ALL samples, the f values spanned 5 orders of magnitude and had a median value of 49 per million. For the 8 ETV6-RUNX1 samples, the median f value was 2243 per million, compared with 18 per million for the 11 hyperdiploid samples (p <0.003). Among 7 samples with neither abnormality, the median f value was 60 per million. The one sample with a t(1;19) translocation that we analyzed demonstrated an f value of 330 per million. In comparison, the median f value among 11 BLCLs from normal donors was ∼ 5 per million, and f was ∼1850 per million for HBL2A, a mantle cell lymphoma line that exhibits hypermutability. Based on the data for BLCLs, we used a value of 50 per million as a cutoff: 7 of 8 ETV6-RUNX1 samples demonstrated hypermutabilty, compared with 2 of 11 hyperdiploid samples, and 4 of 7 samples with neither abnormality. These data demonstrate that hypermutability is frequently seen in ALL and is associated with, but not restricted to samples with the ETV6-RUNX1 fusion. These data also confirm that hypermutability—at least as detected by this assay, is not always a requirement for leukemogenesis. We predict that it will be possible to find associations between other cytogenetic subsets and hypermutability, which may correlate with the number of mutations required to produce the malignancy. Disclosures:No relevant conflicts of interest to declare.

Regulation of integrin αV subunit expression by sulfatide in hepatocellular carcinoma cells

Integrin is important in migration and metastasis of tumor cells. Changes of integrin expression and distribution will cause an alteration of cellular adhesion and migration behaviors. In this study, we investigated sulfatide regulation of the integrin αV subunit expression in hepatoma cells and observed that either exogenous or endogenous sulfatide elicited a robust upregulation of integrin αV subunit mRNA and protein expression in hepatoma cells. This regulatory effect occurred with a corresponding phosphorylation (T739) of the transcription factor Sp1. Based on the electrophoretic mobility shift assay, sulfatide enhanced the integrin αV promoter activity and strengthened the Sp1 complex super-shift. The results of chromatin immunoprecipitation analysis also indicated that sulfatide enhanced Sp1 binding to the integrin αV promoter in vivo. Silence of Sp1 diminished the stimulation of integrin αV expression by sulfatide. In the early stage of sulfatide stimulation, phosphorylation of Erk as well as c-Src was noted, and inhibition of Erk activation with either U0126 or PD98059 significantly suppressed Sp1 phosphorylation and integrin αV expression. We demonstrated that sulfatide regulated integrin αV expression and cell adhesion, which was associated with Erk activation.

213 HOXB9 PROTEIN IS PRESENT THROUGHOUT EARLY EMBRYO DEVELOPMENT IN THE BOVINE

Hox genes encode for homeodomain transcription factors well known to be involved in developmental control after gastrulation. However, the expression of some of these genes has been detected during oocyte maturation and early embryo development. An interesting expression profile has been obtained for HOXB9 in the bovine (Paul et al. 2011 Mol. Reprod. Dev. 78, 436): its relative expression increases between the immature oocyte and the zygote, further increases at the 5- to 8-cell stage to peak at the morula stage before decreasing at the blastocyst stage. The main objective of this work is to establish the HOXB9 protein profile from the immature oocyte to the blastocyst in the bovine. Bovine embryos were produced in vitro from immature oocytes obtained from slaughterhouse ovaries. Embryos were collected at the following stages: immature oocyte, mature oocyte, zygote (18 h post-insemination, hpi), 2-cell (26 hpi), 5 to 8 cell (48 hpi), 9 to 16 cell (96 hpi), morula (120 hpi), and blastocyst (180 hpi). The presence and distribution of HOXB9 proteins were detected by whole-mount immunofluorescence followed by confocal microscopy using an anti-human HOXB9 polyclonal antibody directed against a sequence showing 100% homology with the bovine protein. Its specificity to the bovine protein was controlled by Western blot on total protein extract from the bovine uterus and revealed, among a few bands of weak intensities, 2 bands of high intensity corresponding to the expected size. Oocytes or embryos were fixed and incubated overnight with rabbit anti-HOXB9 (Sigma, St. Louis, MO, USA) and mouse anti-E-cadherin (BD Biosciences, Franklin Lakes, NJ, USA) primary antibodies and then for 1 h with goat anti-rabbit Alexafluor 555 conjugated (Cell Signaling Technology, Beverly, MA, USA) and goat anti-mouse FITC-conjugated (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) secondary antibodies. Embryos were then mounted in Vectashield containing DAPI. HOXB9 is detected from the immature oocyte to the blastocyst stage. At the immature oocyte stage, it is mainly localised in the germinal vesicle with a weak signal in the cytoplasm. At the mature oocyte stage, HOXB9 labelling is present in the cytoplasm. At the zygote stage, a stronger immunoreactivity is observed in the pronuclei than in the cytoplasm. From the 2-cell stage to the morula stage, the presence of HOXB9 is also more important in the nuclei than in the cytoplasm. HOXB9 is also observed at the blastocyst stage where it is localised in the nuclei of the trophectoderm cells, whereas an inconstant or weaker labelling is observed in the inner cell mass cells. In conclusion, we have shown for the first time the presence of the HOXB9 protein throughout early bovine embryo development. The results obtained suggest the presence of the maternal HOXB9 protein because it is already detected before the maternal to embryonic transition that occurs during the fourth cell cycle in the bovine. Finally, the pattern obtained at the blastocyst stage suggests a differential role of HOXB9 in the inner cell mass and trophectoderm cells. C. Sauvegarde holds a FRIA PhD grant from the Fonds National de la Recherche Scientifique (Belgium).

Effect of Supplementation of Taurine or Trehalose in Extender on Immunolocalization of Tyrosine Phosphoproteins in Buffalo and Cattle (Karan Fries) Cryopreserved Spermatozoa

The present study assessed the effects of incorporation of Taurine or Trehalose in extender on immunolocalization of tyrosine phosphoproteins, Cryocapacitation and other sperm quality parameters (motility, viability and membrane integrity) in post-thawed sperm from Buffalo (Murrah) and Cattle (Karan Fries). Six ejaculates from six individual bulls from both species were chosen at random and split into four aliquots: one aliquot without dilution (fresh sample), another diluted in egg yolk tris-citrate (EYTC) extender and the rest of aliquots with EYTC dilution supplemented with taurine (50mm) or trehalose (100mm), respectively, and cryopreserved. Following cryopreservation, semen were thawed and assessed for standard semen quality parameters. Extent of capacitation in cryopreserved spermatozoa was measured by inducing in vitro acrosome reaction followed by dual staining. Immunolocalization of tyrosine phosphoproteins was carried out by immunocytochemistry using primary antibody clone pT-154 (anti-phosphotyrosine antibody) and FITC-conjugated secondary antibody. Immunofluorescent signals were analysed for level of protein tyrosine phosphorylation in spermatozoa. Post-thaw semen evaluation showed supplementation of taurine or trehalose to EYTC extender significantly (p<0.05) increased motility, viability and membrane integrity of spermatozoa in both species. Percentage of cryocapacitated spermatozoa was significantly (p<0.05) higher in cattle as compared to buffalo and degree of cryocapacitaion of spermatozoa decreased significantly (p<0.05) upon supplementation of additives in both the species. It was also found that tyrosine phosphoproteins were localized differentially in fresh and cryopreserved spermatozoa. Supplementation of taurine or trehalose to freezing extender changed the localization of tyrosine phosphoproteins in cryopreserved spermatozoa similar to fresh in both the species. The results obtained clearly indicated that supplementation of taurine or trehalose to EYTC prior to cryopreservation improves Buffalo and Cattle sperm quality in terms of cryocapacitation and immunolocalization of tyrosine phosphoproteins during freezing-thawing process.

Protein Implicated in Nonsyndromic Mental Retardation Regulates Protein Kinase A (PKA) Activity

Mutation of the coiled-coil and C2 domain-containing 1A (CC2D1A) gene, which encodes a C2 domain and DM14 domain-containing protein, has been linked to severe autosomal recessive nonsyndromic mental retardation. Using a mouse model that produces a truncated form of CC2D1A that lacks the C2 domain and three of the four DM14 domains, we show that CC2D1A is important for neuronal differentiation and brain development. CC2D1A mutant neurons are hypersensitive to stress and have a reduced capacity to form dendrites and synapses in culture. At the biochemical level, CC2D1A transduces signals to the cyclic adenosine 3',5'-monophosphate (cAMP)-protein kinase A (PKA) pathway during neuronal cell differentiation. PKA activity is compromised, and the translocation of its catalytic subunit to the nucleus is also defective in CC2D1A mutant cells. Consistently, phosphorylation of the PKA target cAMP-responsive element-binding protein, at serine 133, is nearly abolished in CC2D1A mutant cells. The defects in cAMP/PKA signaling were observed in fibroblast, macrophage, and neuronal primary cells derived from the CC2D1A KO mice. CC2D1A associates with the cAMP-PKA complex following forskolin treatment and accumulates in vesicles or on the plasma membrane in wild-type cells, suggesting that CC2D1A may recruit the PKA complex to the membrane to facilitate signal transduction. Together, our data show that CC2D1A is an important regulator of the cAMP/PKA signaling pathway, which may be the underlying cause for impaired mental function in nonsyndromic mental retardation patients with CC2D1A mutation.

Malin Regulates Wnt Signaling Pathway through Degradation of Dishevelled2

Using yeast-two hybrid screening followed by co-immunoprecipitation assay, we have found that the Lafora disease ubiquitin ligase malin interacts with dishevelled2, a key mediator of Wnt signaling pathway. Overexpression of malin enhances the degradation of dishevelled2 and inhibits Wnt signaling, which is evident from the down-regulation of β-catenin target genes and the decrease in β-catenin-mediated transcriptional activity. Partial knockdown of malin significantly increases the level of dishevelled2 and up-regulates Wnt signaling. Several malin mutants are found to be ineffective in degrading dishevelled2 and regulating the Wnt pathway. We have also found that malin enhances K48- and K63-linked ubiquitination of dishevelled2 that could lead to its degradation through both proteasome and autophagy. Altogether, our results indicate that malin regulates Wnt signaling pathway through the degradation of dishevelled2 and suggest possible deregulation of Wnt signaling in Lafora disease.

Combination Therapy with FTY720 (Fingolimod) and Bispecific Anti-CD20/CD74 Antibodies in Mantle Cell Lymphoma,

Abstract 3736 Introduction:Mantle cell lymphoma (MCL) is one of the most challenging B-cell lymphomas to treat. Although the response rates with first-line conventional or high-dose chemotherapy, with or without stem-cell transplantation, are high, most patients relapse, after which their prognosis is considered poor. Thus, new therapeutic interventions in MCL are needed, among which targeted therapy with a variety of monoclonal antibodies (mAbs), either alone or in combination with other biological agents or drugs, is a major focus of ongoing clinical studies. FTY720 (fingolimod), an immunosuppressive agent approved by the FDA as a frontline treatment for relapsing multiple sclerosis, shows promising pre-clinical activity in chronic lymphocytic leukemia and MCL. More recently, FTY720 was also found to increase CD74 expression and sensitize MCL cells to milatuzumab (anti-CD74 humanized antibody)-mediated cell death (Alinari L, et al. ASH 2011 Abstract submitted). Here we report that the in vitro cytotoxicity of two novel bispecific anti-CD20-/CD74 antibodies to MCL lines can be enhanced significantly by combining with FTY720. Methods: The two bispecific, anti-CD20/CD74, hexavalent antibodies (HexAbs), designated 20-(74)-(74) and 74-(20)-(20), were generated from veltuzumab (anti-CD20 humanized mAb) and milatuzumab with the Dock-and-Lock (DNL) method. The effect of FTY720 on cell-surface expression of CD20, CD22, CD74, and HLA-DR was assessed by flow cytometry after treating JeKo-1, Granta-519 and Mino MCL cell lines with FTY720 for 18 h followed by labeling the cells with primary antibody and FITC-conjugated secondary antibody at 4°C. The in vitro cytotoxicity of the combination treatment was determined by Annexin V /PI binding assays and the data analyzed by the Chou-Talalay equation to calculate the combination index (CI). Results: In the three MCL lines tested, FTY720 at 12.5 μM induced almost 9-fold increase of CD74, a modest decrease of CD20, and no apparent change in CD22 and HLA-DR, when compared to untreated controls. Further studies in JeKo-1 incubated with varying doses of FTY720 revealed a 5-fold enhanced expression of CD74 could be achieved at 4 μM, but no notable changes were observed with FTY720 at 2.5 μM, or below. The viability of JeKo-1 cells treated with a single agent for 16 h was determined by the Annexin V/PI staining assay to be 45% for FTY720 at 4 μM and 65–70 % for either 20-(74)-(74) or 74-(20)-(20) at 33 nM. Combined treatments with FTY720 and 20-(74)-(74) at 4 μM and 33 nM, respectively, resulted in about 15% live cells (Annexin V-negative/PI-negative) with statistically significant P values (<0.01) compared to 20-(74)-(74) and FTY720 alone. For FTY720 and 74-(20)-(20), the combination treatment resulted in 21% live cells, also with statistically significant P values (<0.01) compared to each agent alone. Initial analysis of these data yielded a CI of <0.87, suggesting a synergistic effect with the combination therapy. Conclusion: The anti-MCL activity of 20-(74)-(74) and 74-(20)-(20) can be further enhanced by FTY720, as demonstrated for JeKo-1. Studies are underway to evaluate similar cytotoxic effects in xenograft mouse models of MCL and to define the signaling pathways triggered by combining FTY720 with the bispecific anti-CD20/CD74 HexAbs. Disclosures:Gupta:Immunomedics, Inc.: Employment. Michel:Immunomedics, Inc.: Employment. Goldenberg:Immunomedics, Inc.: Consultancy, Employment, Equity Ownership, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding. Chang:Immunomedcis, Inc.: Employment, Patents & Royalties, Stock Options.

Histone Deacetylase 9 Is a Negative Regulator of Adipogenic Differentiation

Differentiation of preadipocytes into mature adipocytes capable of efficiently storing lipids is an important regulatory mechanism in obesity. Here, we examined the involvement of histone deacetylases (HDACs) and histone acetyltransferases (HATs) in the regulation of adipogenesis. We find that among the various members of the HDAC and HAT families, only HDAC9 exhibited dramatic down-regulation preceding adipogenic differentiation. Preadipocytes from HDAC9 gene knock-out mice exhibited accelerated adipogenic differentiation, whereas HDAC9 overexpression in 3T3-L1 preadipocytes suppressed adipogenic differentiation, demonstrating its direct role as a negative regulator of adipogenesis. HDAC9 expression was higher in visceral as compared with subcutaneous preadipocytes, negatively correlating with their potential to undergo adipogenic differentiation in vitro. HDAC9 localized in the nucleus, and its negative regulation of adipogenesis segregates with the N-terminal nuclear targeting domain, whereas the C-terminal deacetylase domain is dispensable for this function. HDAC9 co-precipitates with USF1 and is recruited with USF1 at the E-box region of the C/EBPα gene promoter in preadipocytes. Upon induction of adipogenic differentiation, HDAC9 is down-regulated, leading to its dissociation from the USF1 complex, whereas p300 HAT is up-regulated to allow its association with USF1 and accumulation at the E-box site of the C/EBPα promoter in differentiated adipocytes. This reciprocal regulation of HDAC9 and p300 HAT in the USF1 complex is associated with increased C/EBPα expression, a master regulator of adipogenic differentiation. These findings provide new insights into mechanisms of adipogenic differentiation and document a critical regulatory role for HDAC9 in adipogenic differentiation through a deacetylase-independent mechanism.

Identification of a Negative Regulatory Region for the Exchange Activity and Characterization of T332I Mutant of Rho Guanine Nucleotide Exchange Factor 10 (ARHGEF10)

The T332I mutation in Rho guanine nucleotide exchange factor 10 (ARHGEF10) was previously found in persons with slowed nerve conduction velocities and thin myelination of peripheral nerves. However, the molecular and cellular basis of the T332I mutant is not understood. Here, we show that ARHGEF10 has a negative regulatory region in the N terminus, in which residue 332 is located, and the T332I mutant is constitutively active. An N-terminal truncated ARHGEF10 mutant, ARHGEF10 ΔN (lacking amino acids 1-332), induced cell contraction that was inhibited by a Rho kinase inhibitor Y27632 and had higher GEF activity for RhoA than the wild type. The T332I mutant also showed the phenotype similar to the N-terminal truncated mutant. These data suggest that the ARHGEF10 T332I mutation-associated phenotype observed in the peripheral nerves is due to activated GEF activity of the ARHGEF10 T332I mutant.

TRAF7 Protein Promotes Lys-29-linked Polyubiquitination of IκB Kinase (IKKγ)/NF-κB Essential Modulator (NEMO) and p65/RelA Protein and Represses NF-κB Activation

TRAF7 Protein Promotes Lys-29-linked Polyubiquitination of IκB Kinase (IKKγ)/NF-κB Essential Modulator (NEMO) and p65/RelA Protein and Represses NF-κB Activation

Glucosamine inhibits epithelial-to-mesenchymal transition and migration of retinal pigment epithelium cells in culture and morphologic changes in a mouse model of proliferative vitreoretinopathy

To explore the effect of glucosamine (GlcN) on transforming growth factor (TGF)-β signalling and several processes involved in proliferative vitreoretinopathy (PVR). We evaluated the surface levels of TGF-β receptor and its binding of TGF-β in ARPE-19 cells. Release of cytokines and collagen, and expression of signalling intermediates were quantified. Migration was qualitatively and quantitatively examined. The morphology of cells undergoing PVR in vitro and in a mouse PVR model was observed. Glucosamine reduced the surface levels of TGF-β receptor and the ability of ARPE-19 cells to bind TGF-β. In ARPE-19 cells, TGF-β1 plus epidermal growth factor (EGF) or TGF-β2 increased the expression of alpha-smooth muscle actin (α-SMA) and decreased the expression of zona occludens protein (ZO-1). Transforming growth factor-(β2) also caused the release of platelet-derived growth factor (PDGF), connective tissue growth factor (CTGF) and type 1 collagen and increased the phosphorylation of SMAD2 and SMAD3. Platelet-derived growth factor and CTGF stimulated cell migration, and TGF-β2 stimulated wound closure, contraction of collagen and changes in cell morphology. Treatment with GlcN counteracted all of these effects, and its administration in the mouse model reduced the morphologic appearance of PVR. Glucosamine could inhibit the TGF-β signalling pathway in retinal pigment epithelium cells and several of the downstream events associated with epithelial-mesenchymal transition and PVR.

Attenuation of host NO production by MAMPs potentiates development of the host in the squid-vibrio symbiosis

Bacterial pathogens typically upregulate the host's production of nitric oxide synthase (NOS) and nitric oxide (NO) as antimicrobial agents, a response that is often mediated by microbe-associated molecular patterns (MAMPs) of the pathogen. In contrast, previous studies of the beneficial Euprymna scolopes/Vibrio fischeri symbiosis demonstrated that symbiont colonization results in attenuation of host NOS/NO, which occurs in high levels in hatchling light organs. Here, we sought to determine whether V. fischeri MAMPs, specifically lipopolysaccharide (LPS) and the peptidoglycan derivative tracheal cytotoxin (TCT), attenuate NOS/NO, and whether this activity mediates the MAMPs-induced light organ morphogenesis. Using confocal microscopy, we characterized levels of NOS with immunocytochemistry and NO with a NO-specific fluorochrome. When added exogenously to seawater containing hatchling animals, V. fischeri LPS and TCT together, but not individually, induced normal NOS/NO attenuation. Further, V. fischeri mutants defective in TCT release did not. Experiments with NOS inhibitors and NO donors provided evidence that NO mediates apoptosis and morphogenesis associated with symbiont colonization. Attenuation of NOS/NO by LPS and TCT in the squid-vibrio symbiosis provides another example of how the host's response to MAMPs depends on the context. These data also provide a mechanism by which symbiont MAMPs regulate host development.