RSSDifferential in-gel electrophoresis (DIGE) analysis of CHO cells under hyperosmotic pressure: osmoprotective effect of glycine betaine addition.
Source
Department of Biological Sciences, Graduate School of Nanoscience & Technology (WCU), KAIST, 373-1 Kusong-Dong, Yusong-Gu, Daejon 305-701, Korea.
Abstract
The use of glycine betaine combined with hyperosmolality is known to be an efficient means for achieving high protein production in recombinant Chinese hamster ovary (rCHO) cells. In order to understand the intracellular events and identify the key factors in rCHO cells cultivated with glycine betaine under hyperosmotic conditions, two-dimensional differential in-gel electrophoresis (2D-DIGE) followed by mass spectrometric analysis was applied. Differentially expressed 19 protein spots were selected and 16 different kinds of proteins were successfully identified. The identified proteins were associated with cellular metabolism (PEPCK, GAPDH, and PK), cellular architecture (β-tubulin and β-actin), protein folding (GRP78 and OSP94), mRNA processing (Rbm34, ACF, and IPMK), and protein secretion (γ-COP). 2D-Western blot analysis of β-tubulin, GAPDH, Peroxidoxin-1, and GRP78 confirmed the proteomic findings. The proteins identified from this study, which are related to cell growth and antibody production, can be applied to cell engineering for maximizing the efficacy of the use of glycine betaine combined with hyperosmolality in rCHO cells. Biotechnol. Bioeng. © 2012 Wiley Periodicals, Inc.
Copyright © 2012 Wiley Periodicals, Inc.
Classic 18.5- and 21.5-kDa Myelin Basic Protein Isoforms Associate with Cytoskeletal and SH3-Domain Proteins in the Immortalized N19-Oligodendroglial Cell Line Stimulated by Phorbol Ester and IGF-1.
Source
Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road East, Guelph, ON, N1G 2W1, Canada.
Abstract
The 18.5-kDa classic myelin basic protein (MBP) is an intrinsically disordered protein arising from the Golli (Genes of Oligodendrocyte Lineage) gene complex and is responsible for compaction of the myelin sheath in the central nervous system. This MBP splice isoform also has a plethora of post-translational modifications including phosphorylation, deimination, methylation, and deamidation, that reduce its overall net charge and alter its protein and lipid associations within oligodendrocytes (OLGs). It was originally thought that MBP was simply a structural component of myelin; however, additional investigations have demonstrated that MBP is multi-functional, having numerous protein-protein interactions with Ca(2+)-calmodulin, actin, tubulin, and proteins with SH3-domains, and it can tether these proteins to a lipid membrane in vitro. Here, we have examined cytoskeletal interactions of classic 18.5-kDa MBP, in vivo, using early developmental N19-OLGs transfected with fluorescently-tagged MBP, actin, tubulin, and zonula occludens 1 (ZO-1). We show that MBP redistributes to distinct 'membrane-ruffled' regions of the plasma membrane where it co-localizes with actin and tubulin, and with the SH3-domain-containing proteins cortactin and ZO-1, when stimulated with PMA, a potent activator of the protein kinase C pathway. Moreover, using phospho-specific antibody staining, we show an increase in phosphorylated Thr98 MBP (human sequence numbering) in membrane-ruffled OLGs. Previously, Thr98 phosphorylation of MBP has been shown to affect its conformation, interactions with other proteins, and tethering of other proteins to the membrane in vitro. Here, MBP and actin were also co-localized in new focal adhesion contacts induced by IGF-1 stimulation in cells grown on laminin-2. This study supports a role for classic MBP isoforms in cytoskeletal and other protein-protein interactions during membrane and cytoskeletal remodeling in OLGs.
Neutralization of LINGO-1 during In Vitro Differentiation of Neural Stem Cells Results in Proliferation of Immature Neurons.
Source
Department of Neuroscience, Uppsala University, Uppsala, Sweden.
Abstract
Identifying external factors that can be used to control neural stem cells division and their differentiation to neurons, astrocytes and oligodendrocytes is of high scientific and clinical interest. Here we show that the Nogo-66 receptor interacting protein LINGO-1 is a potent regulator of neural stem cell maturation to neurons. LINGO-1 is expressed by cortical neural stem cells from E14 mouse embryos and inhibition of LINGO-1 during the first days of neural stem cell differentiation results in decreased neuronal maturation. Compared to neurons in control cultures, which after 6 days of differentiation have long extending neurites, neurons in cultures treated with anti-LINGO-1 antibodies retain an immature, round phenotype with only very short processes. Furthermore, neutralization of LINGO-1 results in a threefold increase in βIII tubulin-positive cells compared to untreated control cultures. By using BrdU incorporation assays we show that the immature neurons in LINGO-1 neutralized cultures are dividing neuroblasts. In contrast to control cultures, in which no cells were double positive for βIII tubulin and BrdU, 36% of the neurons in cultures treated with anti-LINGO-1antibodies were proliferating after three days of differentiation. TUNEL assays revealed that the amount of cells going through apoptosis during the early phase of differentiation was significantly decreased in cultures treated with anti-LINGO-1 antibodies compared to untreated control cultures. Taken together, our results demonstrate a novel role for LINGO-1 in neural stem cell differentiation to neurons and suggest a possibility to use LINGO-1 inhibitors to compensate for neuronal cell loss in the injured brain.
An Obligatory Role for Lung Infiltrating B Cells in the Immunopathogenesis of Obliterative Airway Disease Induced by Antibodies to MHC Class I Molecules.
Source
Departments of SurgeryPathology and Immunology, Washington University School of Medicine, St. Louis, MO.
Abstract
Using a murine model, we demonstrated that endobronchial administration of antibodies (Abs) to major histocompatibility complex (MHC) class I results in cellular infiltration, epithelial metaplasia, fibrosis and obstruction of the small airways (obliterative airway disease [OAD]) mediated predominantly by Th17 responses to self-antigens. This resembles bronchiolitis obliterans syndrome developed following human lung transplantation. Since B cells play a crucial role in induction of autoimmune responses, we defined the role of B cells and its antigen presenting properties in induction of OAD in this study. Anti-MHC class I was administered endobronchially in B(-/-) and wild-type mice. In contrast to wild type, B(-/-) animals did not demonstrate cellular infiltration, epithelial metaplasia and obstruction of airways following anti-MHC. Frequency of K-α1 tubulin and CollagenV-specific IL-17 cells was significantly decreased in B(-/-) mice. As expected, Abs against self-antigens and germinal center formation were not developed in B(-/-) mice. Thus, we conclude that B cells and its antigen presenting capacity play an important role in induction of immune responses to self-antigens and immunopathogenesis of OAD following the administration of anti-MHC. Therefore, strategies to block B-cell and its antigen presenting functions should be considered for preventing the development of chronic rejection.
© Copyright 2012 The American Society of Transplantation and the American Society of Transplant Surgeons.
Variations of chromatin, tubulin and actin structures in primate oocytes arrested during in vitro maturation and fertilization-what is this telling us about the relationships between cytoskeletal and chromatin meiotic defects?
Source
Department of Reproductive Biology, German Primate Center, Goettingen, Germany; Department of Biology, Medical University of Sofia, Sofia, Bulgaria.
Abstract
A nonhuman primate model was applied to investigate the relationships between variations in the organization of microtubules, microfilaments, and chromatin in metaphase I and metaphase II oocytes. Marmoset oocytes were subjected to in vitro maturation and coincubation with sperm. Oocytes which failed to cleave were investigated for chromatin, tubulin, and actin using Hoechst 33258, fluorescein isothiocyanate (FITC)-labeled alpha-tubulin antibody and rhodamine-labeled phalloidin, respectively. Spindles were categorized according to size, shape and microtubule organization: normal, large, multipolar, disorganized, absent spindle, and spindles with broad poles. Actin caps were categorized as: normal, small, split, and disorganized. Chromosomal condensation and alignment were described as normal or abnormal. Improper chromosomal condensation was associated with both abnormal microfilament and microtubule arrangement. This was further associated with abnormal actin organization, disorientation and late stabilization of microtubules, but not related to abnormal organization of spindle poles. Chromosomal misalignment was associated with disorientation and late stabilization of tubulin, but not to broad spindle pole. Additionally, abnormal actin polarization appeared not to be related to abnormal spindle poles. The model system presented in this study could be used as an experimental platform for studying the contribution of different factors to the exactness of late meiotic events in primate oocytes. The present study provides basic information on spindle, chromosome, and actin normal and abnormal organization, which can be observed in in vitro matured, but failed to cleave primate oocytes.
Copyright © 2011 Elsevier Inc. All rights reserved.
Composition and dynamics of the nucleolinus, a link between the nucleolus and cell division apparatus in surf clam (Spisula) oocytes.
Source
Marine Biological Laboratory, United States;
Abstract
The nucleolinus is a little-known cellular structure, discovered over 150 years ago (1) and thought by some investigators in the late 19th to mid-20th century to function in the formation of the centrosomes or spindle. A role for the nucleolinus in formation of the cell division apparatus has recently been confirmed in oocytes of the surf clam, Spisula solidissima (2). However, we know so little about the composition and dynamics of this compartment, it is difficult to construct mechanistic hypotheses or even to be sure that prior reports were describing analogous structures in the cells of mammals, amphibians, plants, and other organisms where it was observed. Surf clam oocytes are an attractive model to approach this problem because the nucleolinus is easily visible by light microscopy, making it accessible by laser microsurgery as well as isolation by common cell fractionation techniques. In this report we analyze the macromolecular composition of isolated Spisula nucleolini and examine the relationship of this structure to the nucleolus and cell division apparatus. Analysis of nucleolinar RNA and protein revealed a set of molecules that overlaps with, but is nevertheless distinct from the nucleolus. The proteins identified were primarily ones involved in nucleic acid metabolism and cell cycle regulation. Monoclonal antibodies generated against isolated nucleolini revealed centrosomal forerunners in the oocyte cytoplasm. Finally, induction of damage to the nucleolinus by laser microsurgery altered the trafficking of α- and γ-tubulin after fertilization. These observations strongly support a role for the nucleolinus in cell division and represent our first clues regarding mechanism.
Disruption of tubulin polymerization and cell proliferation by 1-Naphthylarsonic acid.
Abstract
Recent studies suggest that arsenical compounds exhibit a differential toxicity to cancer cells. Microtubules are dynamic polymers of tubulin dimers involved in cell proliferation, morphogenesis and organelle transport, which are a primary target of a number of anticancer drugs, such as arsenical compounds. During the present study, the interaction of 1-naphthylarsonic acid (1-NAA) was evaluated on microtubule polymerization under in vitro and cellular conditions. Microtubules were extracted from sheep brain. Transmission electron microscopy was used to show microtubule structure in the presence of 1-NAA. Computational docking method was applied for the discovery of ligand binding sites on the microtubular proteins. The proliferation of HeLa cell line and human foreskin fibroblast (HF2) was observed following their incubation with 1-NAA and detection via MTT [3-(4,5-dimethylthiazolyl)-2,5-diphenyl-tetrazolium bromide] assay. Fluorescence microscopic labeling was done with the help of α-tubulin monoclonal antibody. Tunnel kit was used to investigate the apoptotic effects of 1-NAA on the HeLa cells. It is concluded that 1-NAA inhibits the tubulinpolymerization by the formation of abnormal polymers having high affinity to the inner cell wall.
HIF-1α signaling by airway epithelial cell K-α1-tubulin: Role in fibrosis and chronic rejection of human lung allografts.
Source
Department of Surgery, Washington University School of Medicine, St. Louis, MO, USA.
Abstract
Long term survival of the human lung allografts are hindered by chronic rejection, manifested clinically as bronchiolitis obliterans syndrome (BOS). We previously demonstrated significant correlation between the development of antibodies(Abs) to K-α1-tubulin (Kα1T) and BOS. In this study, we investigated the molecular basis for fibrinogenesis mediated by ligation of Kα1T expressed on airway epithelial cells by its specific Abs. Using RT-PCR we demonstrate that normal human bronchial epithelial (NHBE) cells upon ligation of Kα1T with specific Abs caused upregulation of pro-fibrotic growth factors. Western blot analysis of NHBE incubated with Kα1T Abs increased hypoxia inducible factor (HIF-1α). Kα1T Ab-mediated growth factor expression is dependent on HIF-1α as inhibition of HIF-1α returned fibrotic growth factor expression to basal levels. In conclusion, we propose that HIF-1α -mediated upregulation of fibrogenic growth factors induced by ligation of Kα1T Abs is critical for development of fibrosis leading to chronic rejection of lung allograft.
Copyright © 2011 Elsevier Inc. All rights reserved.
New details indicated by different stainings during conjugation of ciliated protozoa Paramecium.
Source
The Nurturing Station for the State Key Laboratory of Subtropical Silviculture, Zhejiang A & F University, Lin'an 311300, China. xianyu_yang@hotmail.com.
Abstract
During conjugation of Paramecium caudatum, nuclear events occur in a scheduled program. Morphological studies on nuclear behavior during conjugation of P. caudatum have been performed since the end of the 19th century. Here we report on new details concerning the conjugation of P. caudatum through the staining of conjugating cells with protargol, carbol fuchsin solution, Hoechst 33342 and immunofluorescence labeling with monoclonal antibody of anti-α tubulin. 1) The crescent nucleus is a characteristic of the meiotic prophase of P. caudatum, has an unstained area. We stained this area with protargol, which was separated from the chromatin area and was not detected by the other stainings. 2) In regards to the four meiotic products, it has long been considered that only one product enters the paroral cone region (PC) and survives after meiosis. However, our protargol and immunofluorescence labeling results indicated that PC entrance of the meiotic product happened before the completion of meiosis instead of after. 3) In our previous study, protargol staining indicated the presence of a swollen structure around the central part of the "U" and "V" shaped spindles connecting the two types of prospective pronuclei. However, immunofluorescence labeling with anti-α tubulinantibodies gave a different image from protargol. All these observations form the basis for further studies of their molecular mechanisms.
Uncultivated microbial eukaryotic diversity: a method to link ssu rRNA gene sequences with morphology.
Source
Department of Microbiology, University of California Davis, Davis, California, United States of America.
Abstract
Protists have traditionally been identified by cultivation and classified taxonomically based on their cellular morphologies and behavior. In the past decade, however, many novel protist taxa have been identified using cultivation independent ssu rRNA sequence surveys. New rRNA "phylotypes" from uncultivated eukaryotes have no connection to the wealth of prior morphological descriptions of protists. To link phylogenetically informative sequences with taxonomically informative morphological descriptions, we demonstrate several methods for combining whole cell rRNA-targeted fluorescent in situ hybridization (FISH) with cytoskeletal or organellar immunostaining. Either eukaryote or ciliate-specific ssu rRNA probes were combined with an anti-α-tubulin antibody or phalloidin, a common actin stain, to define cytoskeletal features of uncultivated protists in several environmental samples. The eukaryote ssu rRNA probe was also combined with Mitotracker® or a hydrogenosomal-specific anti-Hsp70 antibody to localize mitochondria and hydrogenosomes, respectively, in uncultivated protists from different environments. Using rRNA probes in combination with immunostaining, we linked ssu rRNA phylotypes with microtubule structure to describe flagellate and ciliate morphology in three diverse environments, and linked Naegleria spp. to their amoeboid morphology using actin staining in hay infusion samples. We also linked uncultivated ciliates to morphologically similar Colpoda-like ciliates usingtubulin immunostaining with a ciliate-specific rRNA probe. Combining rRNA-targeted FISH with cytoskeletal immunostaining or stains targeting specific organelles provides a fast, efficient, high throughput method for linking genetic sequences with morphological features in uncultivated protists. When linked to phylotype, morphological descriptions of protists can both complement and vet the increasing number of sequences from uncultivated protists, including those of novel lineages, identified in diverse environments.
Therapeutic Mechanism and Efficacy of the Antibody Drug-Conjugate BAY 79-4620 Targeting Human Carbonic Anhydrase 9.
Source
1Oncology, Bayer HealthCare AG.
Abstract
Carbonic anhydrase 9 (CAIX, carbonic anhydrase 9) is a cell surface glycoprotein that is expressed in many different tumors and yet restricted in normal tissues to the gastrointestinal tract. It is upregulated by hypoxia, and correlates with tumor grade and poor survival in several tumor indications. Monoclonal antibodies with single digit nanomolar binding affinity for CAIX were derived by panning with the recombinant ecto-domain of CAIX against the MorphoSys HUCAL Gold library of human Fabs. Highest affinity Fabs were converted to full length IgGs and subjected to further characterization based upon their avidity and selectivity for CAIX, their capacity to undergo internalization in CAIX expressing cell lines and their selective localization to CAIX positive human xenografted tumors when administered to mice as fluorescent-conjugates. Through this selection process, the 3ee9 monoclonal antibody was identified which upon conjugation to Monomethyl Auristatin E (MMAE) through a self-imolative enzyme-cleavable linker yielded the potent and selective CAIXantibody drug conjugate CAIX-ADC (BAY 79-4620). In preclinical human xenograft models in mice representing several tumor indications, BAY 79-4620 showed potent anti-tumor efficacy and in some models demonstrated partial and complete tumor shrinkage even following a single dose. The mechanism of action was shown by histology to involve the sequelae of events typical of anti-tubulin agents. Efficacy in murine preclinical models correlated semi-quantitatively with CAIX expression levels as determined by IHC and ELISA. These preclinical data collectively support the development of BAY 79-4620 for the treatment of cancer patients with CAIX over-expressing tumors.
Congenital cataract causing mutants of αA-crystallin/sHSP form aggregates and aggresomes degraded through ubiquitin-proteasome pathway.
Source
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America.
Abstract
BACKGROUND:
Mutations of human αA-crystallin cause congenital cataract by protein aggregation. How mutations of αA-crystallin cause disease pathogenesis through protein aggregation is not well understood. To better understand the cellular events leading to protein aggregation, we transfected cataract causing mutants, R12C, R21L, R21W, R49C, R54C, R116C and R116H, of human αA-crystallin in HeLa cells and examined the formation of intracellular protein aggregates and aggresomes by confocal microscopy.
METHODOLOGY/PRINCIPAL FINDINGS:
YFP-tagged human αA-wild-type (αA-wt) was sub-cloned and the mutants were generated by site-directed mutagenesis. The αA-wt and the mutants were individually transfected or co-transfected with CFP-tagged αA-wt or αB-wild-type (αB-wt) in HeLa cells. Overexpression of these mutants forms multiple small dispersed cytoplasmic aggregates as well as aggresomes. Co-expression of αB-wt with these mutants significantly inhibited protein aggregates where as co-expression with αA-wt enhanced protein aggregates which seems to be due to co-aggregation of the mutants with αA-wt. Aggresomes were validated by double immunofluorescence by co-localization of γ-tubulin, a centrosome marker protein with αA-crystallin. Furthermore, increased ubiquitination was detected in R21W, R116C and R116H as assessed by western blot analyses. Immunostaining with an ubiquitin antibody revealed that ubiquitin inclusions in the perinuclear regions were evident only in R116C transfected cells. Pulse chase assay, after cycloheximide treatment, suggested that R116C degraded faster than the wild-type control.
CONCLUSIONS/SIGNIFICANCE:
Mutants of αA-crystallin form aggregates and aggresomes. Co-expression of αA-wt with the mutants increased aggregates and co-expression of αB-wt with the mutants significantly decreased the aggregates. The mutant, R116C protein degraded faster than wild-type control and increased ubiquitination was evident in R116C expressing cells.
Viability and neural differentiation of mesenchymal stem cells derived from the umbilical cord following perinatal asphyxia.
Source
Department of Newborn Services, George Washington University and Children's National Medical Center, Washington, DC, USA.
Abstract
Objective:Hypoxia-ischemia is the leading cause of neurological handicaps in newborns worldwide. Mesenchymal stem cells (MSCs) collected from fresh cord blood of asphyxiated newborns have the potential to regenerate damaged neural tissues. The aim of this study was to examine the capacity for MSCs to differentiate into neural tissue that could subsequently be used for autologous transplantation.Study Design:We collected cord blood samples from full-term newborns with perinatal hypoxemia (n=27), healthy newborns (n=14) and non-hypoxic premature neonates (n=14). Mononuclear cells were separated, counted, and then analyzed by flow cytometry to assess various stem cell populations. MSCs were isolated by plastic adherence and characterized by morphology. Cells underwent immunophenotyping and trilineage differentiation potential. They were then cultured in conditions favoring neural differentiation. Neural lineage commitment was detected using immunohistochemical staining for glial fibrillary acidic protein, tubulin III and oligodendrocyte marker O4 antibodies.Result:Mononuclear cell count and viability did not differ among the three groups of infants. Neural differentiation was best demonstrated in the cells derived from hypoxia-ischemia term neonates, of which 69% had complete and 31% had partial neural differentiation. Cells derived from preterm neonates had the least amount of neural differentiation, whereas partial differentiation was observed in only 12%.Conclusion:These findings support the potential utilization of umbilical cord stem cells as a source for autologous transplant in asphyxiated neonates.Journal of Perinatology advance online publication, 1 December 2011; doi:10.1038/jp.2011.174.
Investigating the deep supercooling ability of an Alaskan beetle, Cucujus clavipes puniceus, via high throughput proteomics.
Source
Department of Biological Sciences, University of Notre Dame, United States.
Abstract
Cucujus clavipes puniceus is a freeze avoiding beetle capable of surviving the long, extremely cold winters of the Interior of Alaska. Previous studies showed that some individuals typically supercool to mean values of approximately -40°C, with some individuals supercooling to as low as -58°C, but these non-deep supercooling (NDSC) individuals eventually freeze if temperatures drop below this. However, other larvae, especially if exposed to very cold temperatures, supercool even further. These deep supercooling (DSC) individuals do not freeze even if cooled to -100°C. In addition, the body water of the DSC larvae vitrifies (turns to a glass) at glass transition temperatures of -58 to -70°C. This study examines the proteomes of DSC and NDSC larvae to assess proteins that may contribute to or inhibit the DSC trait. Using high throughput proteomics, we identified 138 proteins and 513 Gene Ontology categories in the DSC group and 104 proteins and 573 GO categories in the NDSC group. GO categories enriched in DSC include alcohol metabolic process, cellular component morphogenesis, monosaccharide metabolic process, regulation of biological quality, extracellular region, structural molecule activity, and antioxidant activity. Proteins unique to DSC include alpha casein precursor, alpha-actinin, vimentin, tropomyosin, beta-lactoglobulin, immunoglobulins, tubulin, cuticle proteins and endothelins.
Copyright © 2011 Elsevier B.V. All rights reserved.
Involvement of EphB/Ephrin-B Signaling in Axonal Survival in Mouse Experimental Glaucoma.
Source
Departments of Ophthalmology and.
Abstract
Purpose. To examine the functional significance of EphB/ephrin-B upregulation in mouse experimental glaucoma. Methods. In a loss-of-function approach, mouse mutants lacking EphB2 (EphB2(-/-)) or EphB3 (EphB3(-/-)) protein, and mutants expressing EphB2 truncated in the C-terminus (EphB2(lacZ/lacZ)) were subjected to laser-induced ocular hypertension (LIOH), an experimental mouse model of glaucoma. The number of optic nerve axons was counted in paraphenylenediamine (PPD)-stained sections and compared between EphB mutants and wild type littermates. In a gain-of-function approach, retina/optic nerve explants obtained from LIOH-treated animals were exposed to EphB2-Fc recombinant proteins or Fc control proteins. Tissue sections through the optic nerve head (ONH) were labeled with neuron-specific anti-tubulin β-III antibody to determine axonal integrity. Results. Both EphB2 and EphB3 null mutant mice exhibited more severe axonal degeneration than wild type littermates after treatment with LIOH. Mutant mice in which the C-terminal portion of EphB2 is truncated had an intermediate phenotype. Application of EphB2-Fc recombinant protein to LIOH-treated optic nerve explants resulted in greater sparing of tubulin β-III-containing retinal ganglion cell (RGC) axons. Conclusions. These results provide genetic evidence in mice that both EphB/ephrin-B forward and reverse signaling feed into an endogenous pathway to moderate the effects of glaucomatous insult on RGC axons. LIOH-induced axon loss is maintained in retina/optic nerve explants after removal from an ocular hypertensive environment. Exogenous application of EphB2 protein enhances RGC axon survival in explants, suggesting that modulation of Eph/ephrin signaling may be of therapeutic interest.
Cytoskeletal analysis of human blastocysts by confocal laser scanning microscopy following vitrification.
Source
Section of Reproductive Medicine, First Department of Obstetrics & Gynaecology, Aristotle University Medical School, Papageorgiou General Hospital, Thessaloniki 56403, Greece. katerinachatzime@hotmail.com
Abstract
BACKGROUND:
Vitrification of human blastocysts is being used increasingly to cryopreserve supernumerary embryos following IVF. In this study, we investigate the effects of aseptic vitrification on the cytoskeleton and development of human blastocysts, by analysing survival rates and spindle and chromosome configurations by fluorescence and confocal laser scanning microscopy.
METHODS:
A total of 55 fresh blastocysts and 55 day 5 dimethylsulphoxide/ethylene glycol vitrified blastocysts, which were allowed to remain in culture for 24 h post-warming, were rapidly fixed in ice cold methanol, and immunostained with an a-tubulin antibody to visualize microtubules in combination with antibodies against acetylated tubulin (to visualize spindles, poles and mid bodies), gamma tubulin (to identify spindle poles) and 4(6-diamidino-2-phenylindole) to visualize DNA.
RESULTS:
In total, 213 spindles were analysed in the control (fresh) group of which 183/213 (85.9%) were normal, 20/213 (9.4%) were abnormally shaped, 9/213 (4.2%) were multipolar and 1/213 (0.5%) was monopolar. A total of 175 spindles were analysed in the vitrified group, of which 120/175 (68.6%) were normal, 39/175 (22.3%) were abnormally shaped, 10/175 (5.7%) were multipolar and 6/175 (3.4%) were monopolar. The incidence of multipolar spindles was similar in the two groups, but the level of abnormally shaped spindles, often associated with chromosome lagging, or congression failure, was significantly higher in the vitrified group compared with the fresh group (P< 0.05).
CONCLUSIONS:
The high survival rate following thawing and the large proportion of normal spindle/chromosome configurations suggests that vitrification at the blastocyst stage on Day 5 does not adversely affect the development of human embryos and the ability of spindles to form and continue normal cell divisions. However, there was a significantly higher incidence of abnormal spindles in the vitrified group compared with the fresh group, notably of spindles with a focused and an unfocused pole as well as chromosome bridging and disorganized middle spindle fibres at telophase. Further investigation is warranted to elucidate the mitotic stages that are more vulnerable to damage during vitrification, the fate of the abnormal spindles and any potential effects that may be reflected on the chromosomal constitution of the developing blastocysts.
[Antitubulin agents].
Source
Hospices civils de Lyon, université Lyon-I, centre de recherche en cancérologie de Lyon, France. charles.dumontet@chu-lyon.fr
Abstract
Microtubules are dynamic filamentous cytoskeletal proteins that are an important therapeutic target in patients with tumors. Microtubule binding agents have been part of the pharmacopoeia of cancer for decades, and until the advent of targeted therapy microtubules represented the only alternative to DNA as a therapeutic target in cancer. There are currently a variety of available vinca alkaloids and taxanes and other agents, such as ixabepilone and eribulin, have also been approved. Maytansinoids have been used for the production of immunoconjugates, monoclonal antibodiescovalently bound to antimitotic molecules. The screening of a variety of botanical species and marine organisms continues to yield promising new antitubulin agents with novel properties. Enhanced tumor specificity, reduced neurotoxicity, and insensitivity to chemoresistance mechanisms are the three main objectives in the current search for novel microtubule binding agents.
TEM8 Targeted Cancer Therapy.
Source
5701 South Airport Rd, Scott&White Cancer Research Institute, Temple, TX 76502, USA. afrankel@swmail.sw.org.
Abstract
Tumor growth depends upon access to host blood vessels. Many steps in tumor angiogenesis have been defined including tumor cell hypoxia, tumor cell secretion of pro-angiogenic growth factors, receptor activation on host endothelium and stroma, and establishment of new blood vessels feeding the tumor mass. Inhibitors for some of these steps have been synthesized and tested clinically. While modest improvements in response, progression-free survival and overall survival have been observed in metastatic colorectal carcinoma, non-small cell lung carcinoma, breast carcinoma, renal cell carcinoma, and glioblastoma, almost all patients ultimately relapse and die from metastatic disease. Explanations for the limited effects of anti-angiogenesis therapy include lack of activity on all the parallel angiogenic pathways, non-specific toxicities of some of the agents, induction of a pro-metastatic phenotype by the enhanced hypoxia from therapy, and lack of effect on already established tumor blood vessels. One solution is to directly attack the tumor vasculature rather than inhibit tumor vessel formation. The flavonoid ASA404 and the tubulin-binder combretastatin A-4 phosphate directly damage tumor endothelium by different mechanisms. Both compounds have shown minimal single agent disease activity and produce cardiac ischemia in clinical trials. Recently, ligand-directed vascular disrupting agents have been synthesized and tested. A promising member of this class of therapeutics targets the tumor endothelial marker-8 (TEM8). Anti-TEM8 antibody drug conjugate may facilitate selective destruction of tumor blood vessels yielding enhanced anti-cancer efficacy and reduced normal tissue toxicities. Advances in this field are described which should lead to clinical studies of TEM8 targeted cancer therapeutics.
Immunohistochemical localization of aggresomal proteins in glial cytoplasmic inclusions in multiple system atrophy.
Source
Department of Pathology, Institute for Developmental Research, Aichi Human Service Center, 713-8 Kamiya, Kasugai, Aichi 480-0392 Japan Department of Embryology, Institute for Developmental Research, Aichi Human Service Center, 713-8 Kamiya, Kasugai, Aichi 480-0392 Japan Department of Neuropathology, Neurological Institute, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582 Japan.
Abstract
Aims: Multiple system atrophy (MSA) is pathologically characterized by the formation of α-synuclein-containing glial cytoplasmic inclusions (GCIs) in oligodendrocytes. However, the mechanisms of GCI formation are not fully understood. Cellular machinery for the formation of aggresomes has been linked to the biogenesis of the Lewy body, a characteristic α-synuclein-containing inclusion of Parkinson's disease and dementia with Lewy bodies. Here we examined whether GCIs contain the components of aggresomes by immunohistochemistry. Methods: Sections from 5 patients with MSA were stained immunohistochemically with antibodies against aggresome-related proteins and analyzed in comparison with sections from 5 patients with no neurological disease. We evaluated the presence or absence of aggresome-related proteins in GCIs by double immunofluorescence and immuno-electron microscopy. Results: GCIs were clearly immunolabeled with antibodies against aggresome-related proteins, such as γ-tubulin, histone deacetylase 6 (HDAC6), and 20S proteasome subunits. Neuronal cytoplasmic inclusions (NCIs) were also immunopositive for these aggresome-related proteins. Double immunofluorescence staining and quantitative analysis demonstrated that the majority of GCIs contained these proteins, as well as other aggresome-related proteins, such as Hsp70, Hsp90, and 62 kDa protein/sequestosome 1 (p62/SQSTM1). Immuno-electron microscopy demonstrated immunoreactivities for γ-tubulinand HDAC6 along the fibrils comprising GCIs. Conclusions: Our results indicate that GCIs, and probably NCIs, share at least some characteristics with aggresomes in terms of their protein components. Therefore, GCIs and NCIs may be another manifestation of aggresome-related inclusion bodies observed in neurodegenerative diseases.
© 2011 The Authors. Neuropathology and Applied Neurobiology © 2011 British Neuropathological Society.
Targets of anti-endothelial cell antibodies in pulmonary hypertension and scleroderma.
Source
Institut Cochin, Paris, France.
Abstract
Anti-endothelial cell antibodies have been identified in patients with systemic sclerosis with and without pulmonary arterial hypertension and in patients with idiopathic pulmonary arterial hypertension. However, their target antigens remain poorly identified.Sera from 24 patients with systemic sclerosis without pulmonary arterial hypertension, 20 patients with systemic sclerosis with pulmonary arterial hypertension, 30 with idiopathic pulmonary arterial hypertension and 12 healthy controls were collected. Target antigens were identified by 2-D electrophoresis and immunoblotting in protein extracts of human umbilical vein endothelial cells. Targeted antigens were identified by mass spectrometry.Serum IgG from patients with systemic sclerosis with or without pulmonary arterial hypertension and patients with idiopathic pulmonary arterial hypertension specifically recognized 110, 82, and 37 protein spots respectively. Among others, target antigens of AECA included lamin A/C, tubulin beta chain and vinculin. One-dimension immunoblotting experiments confirmed the identification of lamin A/C and tubulin beta chain.In conclusion, our results confirm the presence of AECA in patients with systemic sclerosis with and without pulmonary arterial hypertension and in those with idiopathic pulmonary arterial hypertension and provide evidence for the identification of target antigens of these autoantibodies including lamin A/C and tubulin beta chain.
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