Expression of Blood Coagulation Factor X Is Not Liver-Restricted.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1939-1939
Author(s):  
Shing Jen Tai ◽  
Parker Hudson ◽  
Christian Furlan Freguia ◽  
Katherine A. High
Keyword(s):  
Blood Coagulation ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Factor X ◽  
Spatial And Temporal Patterns ◽  
Adult Mice ◽  
Immuno Histochemistry ◽  
The Brain

Abstract Factor X (FX) is a vitamin K-dependent clotting factor that plays a critical role in blood coagulation by catalyzing the conversion of prothrombin to thrombin. Although predominantly found in liver, expression of FX is not liver-restricted as shown in previous studies (JBC271:2323–2331, 1996; EMBO J.11:467–472, 1992). Expression of FX was also detected in whole mouse embryo extracts by RT-PCR as early as E7.5, prior to the formation of a liver bud (TH84:1023–1030, 2000). Other studies have suggested additional biological functions for FX that are independent of its role in blood coagulation, including stimulation of mitogenic activity in endothelial cells, enhancement of platelet-derived growth factor (PDGF) release from vascular smooth muscle cells, induction of cytokine production, and up-regulation of the early growth response-1 (egr-1) gene transcription. FX-knockout [FX (−/−)] mice generated by our group as well as by Dewerchin et al. showed partial embryonic lethality beginning as early as E10.5 and fatal perinatal bleeding in the remaining FX (−/−) mice surviving to term. However, as for several other coagulation-related knock-out mice, the exact cause of embryonic lethality observed in some FX (−/−) mice remains to be deciphered. In light of these observations, we sought to determine the spatial and temporal patterns of FX expression in both developing and adult mice. Our preliminary studies, utilizing the combined techniques of Northern blot analysis, immuno-histochemistry, and in situ hybridization revealed the following. Northern blot analysis of mRNA isolated from different tissues of wild-type (+/+) adult mice showed FX transcript in multiple tissues including liver, stomach, spleen, lung, colon, ovaries, placenta, and heart (in decreasing levels of FX expression). Results of immuno-histochemistry on selective adult mouse tissues were similar to the results of Northern blot analysis with the exception of kidney, in which we found FX protein in the cortical, but not in the medullary region. Specifically, we noted expression of FX protein in the bronchi/bronchioles of the lung, and selective cells in the myocardium and in pancreas. However, despite detection of FX transcript in spleen and placenta, we failed to detect FX protein in either of these tissues. In developing embryos, immuno-histochemistry revealed expression of FX protein in liver, small intestines, and thymus for wild-type E14.5 paraffin-embedded sagittal sections, and expression of FX protein in liver and selective cells within the brain for E15.5 sagittal sections. Additionally, we carried out in situ hybridization of paraffin-embedded sagittal sections, using digoxigenin-labeled Factor X antisense riboprobe constructed from a 1 kb fragment of the 5′ end of the murine FX cDNA (identical to the probe used in Northern blot). For E12.5 embryos, FX transcript was found predominantly in the liver. For E14.5 embryos, we detected FX transcript not only in the liver, but also in the kidney (specifically in the primitive glomeruli). For E15.5 embryos, we noted FX transcripts in liver, lung, and selective cells in the brain. Already in progress, additional studies including both FX (+/+) and FX (−/−) embryos at earlier stages of development (for example E9.5 to E13.5) and additional adult tissues should provide a more complete delineation of the spatial and temporal patterns of FX expression in mouse development and adulthood. In conclusion, expression of murine FX is not restricted to liver during embryonic development or during adulthood.

Cloning and widespread distribution of the rat rod-type cyclic nucleotide-gated cation channel

1997 ◽  
Vol 272 (4) ◽  
pp. C1335-C1344 ◽  
Author(s):  
C. Ding ◽  
E. D. Potter ◽  
W. Qiu ◽  
S. L. Coon ◽  
M. A. Levine ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
In Situ Hybridization ◽  
Pineal Gland ◽  
Northern Blot ◽  
Blot Analysis ◽  
Cyclic Nucleotide ◽  
Northern Blot Analysis ◽  
Chain Reaction ◽  
Polymerase Chain

We used Northern blot analysis, ribonuclease protection assay (RPA), reverse transcriptase-polymerase chain reaction, and in situ hybridization to investigate the hypothesis that the CNG1 isoform of the cyclic nucleotide-gated nonselective cation channel may be widely distributed in tissues of the rat. A cDNA encoding the CNG1 isoform was isolated from rat eye and human retina, and partial sequences were isolated from rat pineal gland and human kidney. Northern blot analysis revealed a 3.1-kilobase (kb) CNG1 transcript in rat eye, pineal gland, pituitary, adrenal gland, and spleen, and a larger transcript of 3.5 kb was found in testis. RPA confirmed the identity of CNG1 mRNA in rat eye, lung, spleen, and brain. Polymerase chain reaction-based detection of the mRNA for CNG1 indicates that the channel is expressed in lower abundance in many other tissues, including thymus, skeletal muscle, heart, and parathyroid gland. The cellular distribution of CNG1 was further studied by in situ hybridization, which demonstrated expression of mRNA in lung, thymus, pineal gland, hippocampus, cerebellum, and cerebral cortex but not in heart or kidney.

scholarly journals Acetylcholine receptor alpha-subunit mRNA is increased by ascorbic acid in cloned L5 muscle cells: Northern blot analysis and in situ hybridization.

1989 ◽  
Vol 108 (5) ◽  
pp. 1823-1832 ◽  
Author(s):  
O Horovitz ◽  
D Knaack ◽  
T R Podleski ◽  
M M Salpeter
Keyword(s):  
Ascorbic Acid ◽  
In Situ Hybridization ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Alpha Subunit ◽  
Mrna Levels ◽  
Surface Receptors ◽  
Subunit Mrna

Ascorbic acid is the major factor in brain extract responsible for increasing the average acetylcholine receptor (AChR) site density on the cloned muscle cell line L5. In the present study, we show that this effect of ascorbic acid requires mRNA synthesis, and that the mRNA level for the AChR alpha-subunit is increased to about the same level as are the surface receptors. We have found no increase in the mRNA levels of the beta-, gamma-, and delta-subunits, or in the mRNAs of other muscle-specific proteins, such as that of light chain myosin 2, alpha-actin, and creatine kinase. By in situ hybridization, we further show that the increase in alpha-mRNA in response to ascorbic acid is exclusively in myotubes and is located near clusters of nuclei. mRNA levels for the alpha-subunit in mononucleated cells are very low and do not significantly increase in response to ascorbic acid. The mononucleated cells are thus excluded as a possible source for the increase in alpha-subunit mRNA detected by Northern blot analysis. Our results indicate that there is a very specific action of ascorbic acid on the regulation of AChR alpha-mRNA in the L5 muscle cells, and that the expression of surface receptors in these cells is limited by the amount of AChR alpha-subunit mRNA.

scholarly journals Towards Quantitative In Situ Hybridization

1997 ◽  
Vol 45 (3) ◽  
pp. 413-423 ◽  
Author(s):  
Ard Jonker ◽  
Piet A. J. de Boer ◽  
Maurice J. B. van den Hoff ◽  
Wouter H. Lamers ◽  
Antoon F. M. Moorman
Keyword(s):  
In Situ Hybridization ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Photographic Emulsion ◽  
Mrna Levels ◽  
Intestinal Tissue ◽  
Tissue Sections ◽  
Silver Grains

In situ hybridization analysis of tissue mRNA concentrations remains to be accepted as a quantitative technique, even though exposure of tissue sections to photographic emulsion is equivalent to Northern blot analysis. Because of the biological importance of in situ quantification of RNA sequences within a morphological context, we evaluated the quantitative aspects of this technique. In calibrated microscopic samples, autoradiographic signal (density of silver grains) was proportionate to the radioactivity present, to the exposure time, and to time of development of the photographic emulsion. Similar results were obtained with tissue sections, showing that all steps of the in situ hybridization protocol, before and including the detection of the signal, can be reproducibly performed. Furthermore, the integrated density of silver grains produced in liver and intestinal sections by the in situ hybridization procedure using 35S-labeled riboprobes is directly proportionate to the signal obtained by quantitative Northern blot analysis. The significance of this finding is that in situ quantification of RNA can be realized with high sensitivity and with the additional advantage of the possibility of localizing mRNA within the cells of interest. Application of this procedure on fetal and adult intestinal tissue showed that the carbamoylphosphate synthetase (CPS)-expressing epithelial cells of both tissues accumulated CPS mRNA to the same level but that whole-organ CPS mRNA levels decreased four- to fivefold in the same period, owing to a comparable decrease in the number of CPS-expressing cells in total intestinal tissue. (J Histochem Cytochem 45:413–423, 1997)

Substance P receptor expression in intestinal epithelium inClostridium difficile toxin A enteritis in rats

1998 ◽  
Vol 275 (1) ◽  
pp. G68-G75 ◽  
Author(s):  
Charalabos Pothoulakis ◽  
Ignazio Castagliuolo ◽  
S. E. Leeman ◽  
Chi-Chung Wang ◽  
Hanzong Li ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Substance P ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Intestinal Epithelial Cells ◽  
Intestinal Epithelial ◽  
Rat Intestine ◽  
Toxin A

We previously reported that the inflammatory effects of Clostridium difficile toxin A on rat intestine can be significantly inhibited with a specific neurokinin-1 receptor (NK-1R) antagonist. In this study we investigated the localization and expression of NK-1R mRNA and protein in rat intestine by in situ hybridization, Northern blot analysis, and immunohistochemistry, respectively, after exposure to toxin A. Northern blot analysis showed increased mucosal levels of NK-1R mRNA starting 30 min after toxin A administration. In situ hybridization showed that toxin A increased NK-1R mRNA expression in intestinal epithelial cells after 30, 120, and 180 min. In rats pretreated with the NK-1R antagonist CP-96345 the increase in NK-1R mRNA levels after exposure to toxin A was inhibited, indicating that NK-1R upregulation is substance P (SP) dependent. One hour after exposure to toxin A many of the intestinal epithelial cells showed staining for NK-1R compared with controls. Specific 125I-SP binding to purified epithelial cell membranes obtained from ileum exposed to toxin A for 15 min was increased twofold over control and persisted for 4 h. This report provides evidence that NK-1R expression is increased in the intestinal epithelium shortly after exposure to toxin A and may be important in toxin A-induced inflammation.

Prolactin and growth hormone mRNA and protein characterization in SMtTW rat pituitary tumours

1995 ◽  
Vol 15 (3) ◽  
pp. 233-243 ◽  
Author(s):  
M Delhase ◽  
F Rajas ◽  
P Verdood ◽  
C Remy ◽  
P Chevallier ◽  
...  
Keyword(s):  
Western Blot ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Biological Significance ◽  
Protein Characterization ◽  
Pituitary Tumours ◽  
Rat Pituitary ◽  
Normal Rat

ABSTRACT We have combined different techniques to analyse passages of five different rat spontaneous pituitary tumours (SMtTW) that were transplanted under the kidney capsule. These tumours were secreting prolactin (PRL), GH or both hormones. RIA, immunocytochemistry (ICC) and Western blot analysis were applied to characterize the hormone(s) stored (ICC and Western blot) and secreted (RIA). mRNA content was analysed by PCR, Northern blot analysis and in situ hybridization. The data point not only to the reliability of the techniques used at both protein and RNA levels for each tumour studied but also to the complementarity of some techniques. For example, whereas Northern blot analysis demonstrates the presence and size of hormone mRNA, in situ hybridization indicates the percentage of cells expressing a given hormone mRNA and allows the presence of one population (or more) of cells in a given tumour to be identified. Moreover, the tumours were compared with normal rat pituitary. Although the PRL and GH mRNAs were identical in size, the amount of mRNA was lower in the tumours. At the protein level, the PRL and GH variants exhibited a different pattern of expression in tumours compared with the normal rat pituitary. The biological significance of these differences is discussed.

scholarly journals Expression and localization of mRNA for the 16 KD subunit of V-ATPase in the rat embryo.

1995 ◽  
Vol 43 (8) ◽  
pp. 761-769 ◽  
Author(s):  
M Numata ◽  
S Ohkuma ◽  
S Iseki
Keyword(s):  
In Situ Hybridization ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Mammalian Development ◽  
Mesenchymal Differentiation ◽  
Cell Proliferation And Differentiation ◽  
Proliferation And Differentiation

Vacuolar H(+)-ATPase (V-ATPase), an enzyme composed of multisubunits, is located in the membrane of intracellular organelles (e.g., lysosomes, and endosomes) and maintains the intraorganellar acidic pH by pumping protons across the membrane. Although there is growing evidence for some important role of V-ATPase in cell proliferation and differentiation, the functional significance of V-ATPase in vivo during mammalian development remains obscure. In the present study we investigated the expression and localization of mRNA for the 16 KD subunit of V-ATPase, an essential sector for enzymatic activity, in prenatal rat by Northern blot analysis and in situ hybridization with a specific oligonucleotide probe. With Northern blot analysis, consistent expression of the mRNA was observed in the embryos throughout the period examined (E14-E20). On in situ hybridization, mRNA signal was distributed with various intensities in both the epithelial and mesenchymal tissues at embryonic day 14 (E14). In E17 and E20 embryos, localization of strong signal became more restricted to distinct mesenchymal cells such as fibroblasts adjacent to the epithelia of skin, lung, and intestine, the cells of perichondrium, and myoblasts in the process of fusion. These results suggest that V-ATPase performs specific functions during the later stages of embryogenesis, especially at sites of mesenchymal differentiation and epithelium-mesenchyme interaction.

Thyrotropin receptor and leukocyte adhesion molecules in autoimmune thyroid disease: a study of their gene expression by Northern blot analysis and in situ hybridization

1994 ◽  
Vol 131 (5) ◽  
pp. 480-488 ◽  
Author(s):  
Frank Schuppert ◽  
Markus Reiser ◽  
Niels-E Heldin ◽  
Sülke Ede ◽  
Georg FW Scheumann ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Northern Blot ◽  
Blot Analysis ◽  
Thyroid Disease ◽  
Autoimmune Thyroid Disease ◽  
Northern Blot Analysis ◽  
Leukocyte Adhesion ◽  
Immunological Parameters ◽  
Autoimmune Thyroid

Schuppert F, Reiser M, Heldin N-E, Ede S, Scheumann GFW, Dralle H, von zur Mühlen A. Thyrotropin receptor and leukocyte adhesion molecules in autoimmune thyroid disease: a study of their gene expression by Northern blot analysis and in situ hybridization. Eur J Endocrinol 1994;131:480–8. ISSN 0804–4643 In order to characterize the role of leukocyte-activating antigens and other immunological parameters in autoimmune thyroid disease, mRNA levels of intercellular adhesion molecule 1 (ICAM-1), endothelial leukocyte adhesion molecule 1 (ELAM-1, E-selectin), invariant chain (Ii) and the thymic hormone thymosin β4 (Tβ4) were investigated in 18 human thyroid glands, including eight Graves' thyroids, two Hashimoto's thyroids, two endemic goiters and six healthy controls. Northern blot analysis showed that in autoimmune thyroid disease, expression of ICAM-1 and Tβ4 was correlated to transcript levels of Ii, whereas in the healthy controls, expression of Tβ4, ICAM-1 and ELAM-1 was low or nearly absent. ELAM-1 and TSH receptor (TSH-R) expression, the latter serving as a thyroid specific marker, was increased in some diseased glands but showed no relation to the immunological parameters mentioned above. Localization of the specific mRNAs by in situ hybridization demonstrated a cell-specific expression of TSH-R (thyrocytes), ELAM-1 (vascular endothelial cells) and Tβ4 (cells of hematopoietic origin). In contrast, transcripts of Ii and ICAM-1 were found in thyrocytes, leukocytes and endothelial cells. Our results implicate a coordinate expression of ICAM-1, Tβ4 and Ii in autoimmune thyroid disease, yielding distinct cellular expression patterns. Differential expression of ICAM-1. Ii and the TSH-R in thyroid epithelial cells indicates active regulatory events within the thyrocyte. Frank Schuppert, Department of Clinical Endocrinology, Medizinische Hochschule Hannover, Konstanty-Gutschow-Strasse 8, D-30625 Hannover, Germany

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