scholarly journals Development and Characterization of Novel Rat Anti-mERβ Sera

Endocrinology ◽  
2016 ◽  
Vol 157 (7) ◽  
pp. 2844-2852 ◽  
Author(s):  
Horacio J. Novaira ◽  
J. B. Graceli ◽  
S. Capellino ◽  
A. Schoeffield ◽  
G. E. Hoffman ◽  
...  
Keyword(s):  
Physiological Role ◽  
Molecular Size ◽  
Keyhole Limpet Hemocyanin ◽  
Protein Band ◽  
Sprague Dawley ◽  
Amino Acid Peptide ◽  
Nuclear Extracts ◽  
Protein Partners ◽  
Mouse Tissues

Estrogens regulate normal sexual and reproductive development in females. Their actions are mediated mainly by estrogen receptor (ER)α and ERβ. Understanding the function of ERs necessitates knowing their cellular location and protein partners, which, in turn, requires reliable and specific antibodies. Several antibodies are available for ERα; however, discrepancies in immunoreactivity have been reported for ERβ. Here, we have developed antisera for mouse ERβ (mERβ) using a specific C-terminal 18-amino acid peptide conjugated to mariculture keyhole limpet hemocyanin. Sprague Dawley rats were immunized, and the resulting antisera were characterized by Western blot analysis of nuclear extracts from tissues of wild-type (WT) mice, and mice genetically modified to lack either ERα (CERαKO) or ERβ (CERβKO). An approximately 56-kDa protein was detected in the hypothalamus, uterus, ovary, mammary gland, testes, and epididymis of WT mice, consistent with the predicted molecular size of ERβ. In addition, the same protein band was identified in in vitro synthesized mERβ protein and in the mammary glands of CERαKO mice. The approximately 56-kDa protein was not observed in in vitro synthesized mERα protein or in any tissue examined in the CERβKO mice. Immunohistochemistry using the antisera revealed ERβ staining in the granulosa cells of WT ovaries and in the mediobasal hypothalamus, paraventricular nucleus, and cerebral cortex in the WT adult mouse brain. These data suggest that the novel rat anti-mERβ sera are specific to ERβ to allow investigators to explore to cellular and physiological role of ERβ in the brain and other mouse tissues.

Expression in non-lens tissues of an enzyme activity related to the ‘lens-specific’ protein, delta crystallin

Development ◽  
1991 ◽  
Vol 111 (1) ◽  
pp. 181-190
Author(s):  
D.I. de Pomerai ◽  
W.K. Ip ◽  
M. McLaughlin ◽  
K.C. Perry
Keyword(s):  
Ectopic Expression ◽  
Molecular Size ◽  
Protein Band ◽  
Major Constituent ◽  
Lens Protein ◽  
Related Protein ◽  
Structural Protein ◽  
Cycle Enzyme ◽  
Published Evidence

When chick embryo neutral retina (NR) cells are cultured for long periods in vitro, they undergo extensive transdifferentiation into lens and express the lens protein, delta crystallin. We now demonstrate that this process is accompanied by a change in the chromatin conformation of the delta-gene locus from DNAase1-resistant to DNAase1-sensitive in the nuclei of most cells. Transcripts hybridising to a delta probe are also much more prevalent among the in vitro transcription products from lens or transdifferentiated NR culture nuclei, as compared to nuclei from fresh NR tissue. Published evidence indicates that the chick delta 1 crystallin gene encodes the major structural protein of embryonic lens fibres, whereas the closely related delta 2 gene may encode the urea-cycle enzyme argininosuccinate lyase (ASL). Our present data lends further support to this view. Both immunodetectable delta-related protein(s) and ASL activity are present in fresh embryonic NR tissue, as well as in mouse and Rana liver, and in Rana lens. Our polyclonal anti-delta antibody also cross-reacts with a major constituent of commercial bovine ASL, of the same molecular size as chick delta crystallin. Immunoselection studies suggest that the ASL activity in chick embryonic NR is conferred mainly by the delta-related protein band. So-called ‘ectopic’ expression of delta crystallin in embryonic NR (and other tissues) may thus involve the delta 2/ASL gene, and could reflect some metabolic requirement for ASL activity.

scholarly journals Characterization and purification of H1TF2, a novel CCAAT-binding protein that interacts with a histone H1 subtype-specific consensus element.

1989 ◽  
Vol 9 (4) ◽  
pp. 1566-1575 ◽  
Author(s):  
P Gallinari ◽  
F La Bella ◽  
N Heintz
Keyword(s):  
Cell Cycle ◽  
Biochemical Characterization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gene Promoter ◽  
Molecular Size ◽  
Histone H1 ◽  
Nuclear Extracts ◽  
Definition Of

Definition of mechanisms regulating human histone H1 gene transcription during the cell cycle requires the isolation and biochemical characterization of protein factors which interact with specific promoter elements. Two distinct binding activities have been identified in nuclear extracts from HeLa cells and mapped within a 180-base-pair (bp) region of a cell cycle-regulated H1 gene promoter. H1TF1 bound to an H1-specific A + C-rich sequence (AC box), 100 bp upstream of the cap site; H1TF2 interacted with the H1 subtype-specific consensus element and was dependent on the presence of an intact CCAAT box for binding. H1TF2 was purified through a combination of ion-exchange and oligonucleotide affinity chromatographies. Analysis of purified fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and UV crosslinking showed that H1TF2 was a single polypeptide of 47 kilodaltons. This factor was distinct from previously characterized CCAAT-binding proteins in both molecular size and binding properties. Fractions containing H1TF2 activity activated transcription in vitro only if programmed with an H1 DNA template carrying an intact H1TF2-binding site.

Accumulation of Unphosphorylated Calponin in the Submembranous Cytoskeletons of Arachidonic Acid-stimulated Human Platelets

1996 ◽  
Vol 75 (04) ◽  
pp. 617-622 ◽  
Author(s):  
Thomas Meyer ◽  
Christina Unterberg ◽  
Heinrich Kreuzer ◽  
Arnd B Buchwald
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase ◽  
Muscle Protein ◽  
Physiological Role ◽  
Protein Band ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Calpain Inhibitor ◽  
Dependent Protein

SummaryCalponin, a basic smooth-muscle protein capable of binding to F-actin, tropomyosin and calmodulin in vitro, was tested for its expression and subcellular localization in resting and stimulated human platelets. Using immunoblotting techniques calponin was revealed as a single protein band with a molecular weight of 34 kDa. Although calponin has been shown to be proteolytically degraded by calpain, in the presence of the calpain inhibitor E-64 and EGTA a significant hydrolysis of calponin could not be detected. Upon stimulation with 10 μM arachidonic acid calponin became increasingly incorporated into Triton X-100 insoluble cytoskeletal fractions reaching a plateau after 15 s. The accumulation of calponin in the cytoskeletons of stimulated platelets paralleled the polymerization of actin into newly formed microfilaments. Immunofluorescence microscopy revealed a sub-membranous co-localization of calponin and actin in aggregated platelets. Since isolated calponin is phosphorylated by protein kinase C and Ca2+/calmodulin-dependent protein kinase II thereby losing its inhibitory effect on the actomyosin MgATPase activity, we examined whether changes in cell shape due to platelet stimulation are accompanied by a phosphorylation of calponin. By performing immunoblotting analysis on either resting or stimulated platelets phosphorylation of calponin on tyrosine, serine or threonine residues could not be demonstrated. In line, [32P]radiolabeling experiments were unable to detect phosphate incorporation into calponin. These observations support the hypothesis that calponin plays a physiological role in regulating contraction and secretion of human platelets even in the absence of its phosphorylation.

Elevated IL-1β contributes to antibody suppression produced by stress

2002 ◽  
Vol 93 (1) ◽  
pp. 207-215 ◽  
Author(s):  
Albert Moraska ◽  
Jay Campisi ◽  
Kien T. Nguyen ◽  
Steven F. Maier ◽  
Linda R. Watkins ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Acute Stress ◽  
Ex Vivo ◽  
Interleukin 1 ◽  
Keyhole Limpet Hemocyanin ◽  
Acquired Immunity ◽  
Sprague Dawley

Acute stressor exposure can facilitate innate immunity and suppress acquired immunity. The present study further characterized the potentiating effect of stress on innate immunity, interleukin-1β (IL-1β), and demonstrated that stress-induced potentiation of innate immunity may contribute to the stress-induced suppression of acquired immunity. The long-term effect of stress on IL-1β was measured by using an ex vivo approach. Sprague-Dawley rats were challenged with lipopolysaccharide (LPS) in vivo, and the IL-1β response was measured in vitro. Splenocytes, mesenteric lymphocytes, and peritoneal cavity cells had a dose- and time-dependent ex vivo IL-1β response to LPS. Rats that were exposed to inescapable shock (IS, 100 1.6 mA, 5-s tail shocks, 60-s intertrial interval) and challenged with a submaximal dose of LPS 4 days later had elevated IL-1β measured ex vivo. To test whether the acute stress-induced elevation in IL-1β contributes to the long-term suppression in acquired immunity, IL-1β receptors were blocked for 24 h after stress. Serum anti-keyhole limpet hemocyanin (KLH) immunoglobulin (Ig) was measured. In addition, the acute elevation (2 h post-IS) of splenic IL-1β in the absence of antigen was verified. Interleukin-1 receptor antagonist prevented IS-induced suppression in anti-KLH Ig. These data support the hypothesis that stress-induced increases in innate immunity (i.e., IL-1β) may contribute to stress-induced suppression in acquired immunity (i.e., anti-KLH Ig).

Hepatic portal infusion of glucagon antibodies increases spontaneous meal size in rats

1991 ◽  
Vol 261 (1) ◽  
pp. R162-R165 ◽  
Author(s):  
J. Le Sauter ◽  
U. Noh ◽  
N. Geary
Keyword(s):  
Physiological Role ◽  
Meal Size ◽  
Dark Phase ◽  
Sprague Dawley ◽  
Nocturnal Feeding ◽  
Sprague Dawley Rats ◽  
Ad Libitum ◽  
Hepatic Portal ◽  
Portal Infusion

Specific antibodies to pancreatic glucagon in a dose sufficient to neutralize 1.5 ng glucagon in vitro were intraportally infused during the first 2 min of spontaneous meals in ad libitum-fed male Sprague-Dawley rats. In separate tests, glucagon antibodies stimulated feeding during the first spontaneous meal of the dark phase (73% mean increase in meal size) and during spontaneous meals in the last quarter of the dark phase (58% increase). These results indicate that a glucagon-sensitive satiety mechanism has a physiological role in the control of nocturnal feeding in rats.

scholarly journals A cadherin-like protein in eggs and cleaving embryos of Xenopus laevis is expressed in oocytes in response to progesterone.

1990 ◽  
Vol 110 (5) ◽  
pp. 1575-1582 ◽  
Author(s):  
Y S Choi ◽  
R Sehgal ◽  
P McCrea ◽  
B Gumbiner
Keyword(s):  
Xenopus Laevis ◽  
Cleavage Stage ◽  
Amino Acid Peptide ◽  
Cell Surface Biotinylation ◽  
Mouse Tissues ◽  
Junction Formation ◽  
Brain Extracts ◽  
Antipeptide Antibody ◽  
Relationship Of

A new cadherin-like protein (CLP) was identified in oocytes, eggs, and cleavage stage embryos of Xenopus laevis. As a probe for detecting new cadherin proteins, an antiserum was raised to a 17 amino acid peptide derived from a highly conserved region in the cytoplasmic domain of all cadherins which have been sequenced to date. This antipeptide antibody recognized Xenopus E-cadherin and a polypeptide in Xenopus brain extracts similar to N-cadherin, which were independently identified by specific mAbs. In extracts of eggs and midblastula stage embryos the antipeptide antibody recognized specifically a 120-kD glycoprotein that migrated faster on SDS gels than the 140-kD E- and N-cadherin polypeptides. This 120-kD polypeptide was not recognized by the mAbs specific to E- and N-cadherin. In fact, E- and N-cadherin were not detectable in eggs or midblastula stage embryos. The possible relationship of CLP to P-cadherin, which has been identified in mouse tissues, has not yet been determined. CLP was synthesized by large, late stage oocytes. When oocytes were induced to mature in vitro with progesterone it accumulated to the same level found in normally laid eggs. It did not accumulate further to any significant extent during the early cleavage stages. CLP was detected on the surface of stage 8 blastomeres by cell surface biotinylation, but only after the tight junctions of the blastula epithelium were opened by removal of Ca2+. We conclude that CLP is a maternally encoded protein that is the major, if not only, cadherin-related protein present in the earliest stages of Xenopus development, and we propose that it may play a role in the Ca2(+)-dependent adhesion and junction formation between cleavage stage blastomeres.

Characterization and purification of H1TF2, a novel CCAAT-binding protein that interacts with a histone H1 subtype-specific consensus element

1989 ◽  
Vol 9 (4) ◽  
pp. 1566-1575
Author(s):  
P Gallinari ◽  
F La Bella ◽  
N Heintz
Keyword(s):  
Cell Cycle ◽  
Biochemical Characterization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gene Promoter ◽  
Molecular Size ◽  
Histone H1 ◽  
Nuclear Extracts ◽  
Definition Of

Definition of mechanisms regulating human histone H1 gene transcription during the cell cycle requires the isolation and biochemical characterization of protein factors which interact with specific promoter elements. Two distinct binding activities have been identified in nuclear extracts from HeLa cells and mapped within a 180-base-pair (bp) region of a cell cycle-regulated H1 gene promoter. H1TF1 bound to an H1-specific A + C-rich sequence (AC box), 100 bp upstream of the cap site; H1TF2 interacted with the H1 subtype-specific consensus element and was dependent on the presence of an intact CCAAT box for binding. H1TF2 was purified through a combination of ion-exchange and oligonucleotide affinity chromatographies. Analysis of purified fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and UV crosslinking showed that H1TF2 was a single polypeptide of 47 kilodaltons. This factor was distinct from previously characterized CCAAT-binding proteins in both molecular size and binding properties. Fractions containing H1TF2 activity activated transcription in vitro only if programmed with an H1 DNA template carrying an intact H1TF2-binding site.

scholarly journals Paraspeckles are subpopulation-specific nuclear bodies that are not essential in mice

2011 ◽  
Vol 193 (1) ◽  
pp. 31-39 ◽  
Author(s):  
Shinichi Nakagawa ◽  
Takao Naganuma ◽  
Go Shioi ◽  
Tetsuro Hirose
Keyword(s):  
Adult Mouse ◽  
Expression Patterns ◽  
Physiological Role ◽  
Growth Conditions ◽  
Nuclear Bodies ◽  
Cellular Functions ◽  
Mouse Tissues

Nuclei of higher organisms are well structured and have multiple, distinct nuclear compartments or nuclear bodies. Paraspeckles are recently identified mammal-specific nuclear bodies ubiquitously found in most cells cultured in vitro. To investigate the physiological role of paraspeckles, we examined the in vivo expression patterns of two long noncoding RNAs, NEAT1_1 and NEAT1_2, which are essential for the architectural integrity of nuclear bodies. Unexpectedly, these genes were only strongly expressed in a particular subpopulation of cells in adult mouse tissues, and prominent paraspeckle formation was observed only in the cells highly expressing NEAT1_2. To further investigate the cellular functions of paraspeckles, we created an animal model lacking NEAT1 by gene targeting. These knockout mice were viable and fertile under laboratory growth conditions, showing no apparent phenotypes except for the disappearance of paraspeckles. We propose that paraspeckles are nonessential, subpopulation-specific nuclear bodies formed secondary to particular environmental triggers.

Glomerular permselectivity in the isolated perfused rat kidney

1985 ◽  
Vol 249 (5) ◽  
pp. F780-F784 ◽  
Author(s):  
N. W. Boyce ◽  
S. R. Holdsworth
Keyword(s):  
Negative Charge ◽  
Rat Kidney ◽  
Molecular Size ◽  
In Vitro Models ◽  
Sprague Dawley ◽  
Filtration Barrier ◽  
Sprague Dawley Rat ◽  
Intermediate Size

Glomerular permselectivity characteristics were studied in the Sprague-Dawley rat in vivo and in the isolated rat kidney perfused with an erythrocyte-free Krebs-Henseleit buffered 5% albumin solution (IPK). IPK permselectivity in vitro, assessed by fractional clearances of neutral dextran (FCND) and dextran sulfate (FCDS) with molecular radii 18-43 A, was essentially similar to that of the Sprague-Dawley rat in vivo. The negative charge barrier of the IPK glomerular filter was intact [e.g., FCND of 36 A = 0.10 +/- 0.01 (SE) vs. FCDS of 36 A = 0.01 +/- 0.00 (P less than 0.01)]. Dextrans of an intermediate size (26-34 A) had lower fractional clearances in the IPK than in vivo [e.g., FCND of 30 A in IPK = 0.23 +/- .04 vs. FCND of 30 A in vivo 0.40 +/- 0.01 (P less than 0.01)]. This decreased clearance of dextrans of an intermediate molecular size is predicted by pore theory, since the IPK has an increased afferent glomerular plasma flow rate. As glomerular permselectivity characteristics in the IPK simulate in vivo characteristics, such preparations are suitable in vitro models in which to study factors that modulate permselectivity. The demonstration that the glomerular filter in the IPK has a normal negative charge barrier indicates that the increased protein excretion in IPK systems cannot be attributed to abnormalities of this component of the filtration barrier.

scholarly journals THE IMMUNOGENICITY OF ANTIGEN BOUND TO THE PLASMA MEMBRANE OF MACROPHAGES

1970 ◽  
Vol 131 (4) ◽  
pp. 711-725 ◽  
Author(s):  
Emil R. Unanue ◽  
Jean-Charles Cerottini
Keyword(s):  
Plasma Membrane ◽  
Low Temperature ◽  
Immune Responses ◽  
Great Part ◽  
Molecular Size ◽  
Keyhole Limpet Hemocyanin ◽  
Antibody Fragments ◽  
Membrane Bound

Macrophages were cultured for several hours after a brief exposure to radio-iodinated keyhole limpet hemocyanin. Most of the hemocyanin taken up by the macrophages was rapidly catabolized and eliminated from the cell. A few molecules were retained on the plasma membrane of the cells for prolonged periods and were not subject to endocytosis and catabolism. These few molecules of hemocyanin bound to the plasma membrane were identified by observing the fixation of antibody fragments to macrophages at low temperature. The membrane-bound antigen, which could be removed by trypsin or EDTA, was of large molecular size, though heterogeneous. A great part of the immune responses of mice to hemocyanin bound to live macrophages could be abrogated by treatment of the macrophages in vitro with antibody or trypsin. Hence, most of the immunogenicity of hemocyanin bound to macrophages was attributed to the few molecules of antigen bound to the plasma membrane.

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