scholarly journals Expression and tissue distribution of rat sulfated glycoprotein-1 (prosaposin).

1996 ◽  
Vol 44 (4) ◽  
pp. 327-337 ◽  
Author(s):  
C R Morales ◽  
M El-Alfy ◽  
Q Zhao ◽  
S A Igdoura
Keyword(s):  
Epithelial Cells ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Biological Fluids ◽  
Cell Types ◽  
Rat Tissues ◽  
Fluid Compartments ◽  
Rat Tissue ◽  
Sulfated Glycoprotein

Sulfated glycoprotein-1 (SGP-1/prosaposin) exists as a sulfated secreted protein or as a lysosomal precursor of four smaller saposin molecules. The protein exhibits ubiquitous expression, evolutionary conservation, and diverse tissue inducibility. The lysosomal form of SGP-1 plays a role in the hydrolysis of glycolipids and sphingomyelin. The function of the secreted form of SGP-1 is still unclear. However, it could act as a glycolipid transfer protein, since several gangliosides (a series) were found to bind with high affinity to prosaposin. To identify cell types that produce SGP-1 mRNA, we constructed an SGP-1 cDNA and used for screening of different rat tissues by Northern blot analysis. To localize the translation product of SGP-1 transcripts, we immunostained the same tissues with an anti-SGP-1 antibody. The SGP-1 cDNA construct was generated by amplifying a rat testicular Zap cDNA library by PCR (polymerase chain reaction) with two synthetic oligonucleotide primers. A positive signal of 1.7 KB was isolated, subcloned into the pGEM-7Zf (+). Sequence analysis showed a near-identical nucleotide and amino acid similarity to a previous rat SGP-1 cDNA. The majority of the heterogeneites were conservative substitutions. Northern blot analysis demonstrated that all examined rat tissue and organs have SGP-1 mRNA. Immunocytochemistry identified two staining patterns in the cytoplasm of positive cells: (a) a granular reaction characteristic of lysosomes in the supranuclear and basal regions of epithelial cells and in the perinuclear region of neurons; and (b) a homogeneous reaction in the cytoplasm of Sertoli cells, Type II pneumocytes, macrophages, and epithelial cells lining the choroid plexus. The latter staining pattern could be characteristic of cells that exhibit a secretory routing of SGP- 1. The production of SGP-1 by a variety of specialized cells lining fluid compartments suggests that its secreted form has a role in the transport of lipids in biological fluids, possibly by the formation of soluble complexes with glycolipids. Similarly, the lysosomal form of SGP-1/prosaposin and their derived saposins also solubilizes certain glycolipids to promote their degradation by specific hydrolases.

Expression of Basic Fibroblast Growth Factor in Nasal Polyps

1998 ◽  
Vol 107 (10) ◽  
pp. 891-897 ◽  
Author(s):  
Michael R. Powers ◽  
Janice M. Liebler ◽  
Zhenhong Qu ◽  
Michael A. Wall ◽  
Philip C. Lagesse ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epithelial Cells ◽  
Fibroblast Growth Factor ◽  
Basic Fibroblast Growth Factor ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Mononuclear Cells ◽  
Fibroblast Growth ◽  
Polyp Tissue

Basic fibroblast growth factor (bFGF) is a polypeptide that is mitogenic for a wide variety of cell types. We used Northern blot analysis and immunohistochemistry to determine if bFGF is expressed in the nasal polyp tissue; bFGF messenger RNA was detectable in the polyps examined by Northern blot analysis. Strong immunostaining for bFGF was found in blood vessels and along the basement membrane of the epithelial cell layers. Basal epithelial cells and some infiltrating mononuclear cells also stained for bFGF. Proliferating cell nuclear antigen colocalized with bFGF to basal epithelial cells, endothelial cells, and areas of focal epithelial metaplasia. The polyp tissue was double-labeled with a mouse monoclonal antitryptase, a specific mast cell marker, and anti-bFGF. A significant number (65% ± 19%) of the bFGF-positive mononuclear cells in the polyp tissues were positive for tryptase. These findings suggest that bFGF may contribute to the endothelial and epithelial proliferation in nasal polyp tissues and that mast cells are one source of this growth factor.

scholarly journals Tissue distribution of rat glutathione transferase subunit 7, a hepatoma marker

1986 ◽  
Vol 240 (3) ◽  
pp. 885-889 ◽  
Author(s):  
S E Pemble ◽  
J B Taylor ◽  
B Ketterer
Keyword(s):  
Northern Blot ◽  
Blot Analysis ◽  
Genomic Dna ◽  
Southern Blot Analysis ◽  
Northern Blot Analysis ◽  
Glutathione Transferase ◽  
Rat Tissues ◽  
Polyadenylated Rna ◽  
Normal Rat ◽  
Rat Hepatoma

Polyadenylated RNA isolated from NN-dimethyl-4-aminoazobenzene-induced rat hepatoma was used to prepare a cDNA library in lambda gt10. Full-length clones complementary to mRNA coding for glutathione transferase subunit 7 were isolated and one of these clones (pGSTr7) was fully characterized. In Northern blot analysis, mRNA hybridizing to 32P-labelled pGSTr7 was found in poly(A)-containing RNA isolated from seven normal rat tissues but not from testis and liver. A similar hybridizing mRNA species was also detected in human placental mRNA. The same probe, used in a Southern blot analysis of genomic DNA, suggests the presence of a multigene family in the rat.

scholarly journals IGF-I differentially regulates IGF-binding protein expression in primary mammary fibroblasts and epithelial cells

2005 ◽  
Vol 186 (1) ◽  
pp. 165-178 ◽  
Author(s):  
J M Fleming ◽  
B J Leibowitz ◽  
D E Kerr ◽  
W S Cohick
Keyword(s):  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Cell Types ◽  
Mammary Epithelial ◽  
Mrna Levels ◽  
Protein Levels ◽  
Qrt Pcr ◽  
Igf I ◽  
Igfbp 3

Elucidating how mitogens facilitate epithelial/stromal interactions is critical given that mitogens regulate mammary gland development and function. IGF-I is a potent mammary cell mitogen that is locally produced in the mammary gland. Since IGF-binding proteins (IGFBPs) regulate IGF-I bioavailability, we characterized the cell-type-specific production of IGFBP in primary bovine mammary epithelial (BME) and fibroblast (BMF) cells. Cells were treated with IGF-I and mRNA levels were analyzed via quantitative real-time (qRT)-PCR and Northern blot analysis. Media conditioned by cells treated with IGF-I for 48 h were analyzed via ligand blotting with 125I-labeled IGF-I and -II and immunoblotting with specific IGFBP antibodies. A reciprocal regulation of IGFBP-3 and -5 by IGF-I was observed between the two cell types. IGF-I induced large dose-dependent increases in IGFBP-3 mRNA and protein levels in BME cells, while IGFBP-5 protein was barely detectable and mRNA levels were detectable only by qRT-PCR. In BMFs, IGF-I induced large increases in IGFBP-5 mRNA and protein while IGFBP-3 mRNA was only slightly increased by IGF-I treatment and the protein was difficult to detect. IGFBP-6 mRNA was detected by Northern blot analysis in both cell types but was not regulated by IGF-I. In BME cells, IGFBP-6 protein levels were readily detectable under basal conditions and were increased by IGF-I. Interestingly, IGFBP-6 protein could not be detected in media conditioned by BMFs. IGFBP-4 mRNA was readily seen by Northern blot analysis in BMFs, however qRT-PCR was required to detect IGFBP-4 mRNA in BME cells. IGF-I increased IGFBP-4 mRNA levels by 2-fold in both cell types. IGFBP-4 protein was only detectable in media conditioned by BME cells when stimulated by IGF-I. In contrast, IGFBP-4 was present in media conditioned by untreated BMFs but was not consistently increased by IGF-I treatment. This was explained by the finding that IGF-I stimulated proteolysis of IGFBP-4, as evidenced by the appearance of two immuno-responsive fragments of 18 and 14 kDa. This proteolysis was specific to IGFBP-4, and was not observed in BME cells. We confirmed the protease to be pregnancy-associated plasma protein A (PAPP-A) by immunoblotting with an antibody against human PAPP-A/proMBP (pro form of eosinophil major basic protein) complex. In vitro immuno-neutralization experiments showed that blocking PAPP-A prevented the ability of IGF-I to stimulate IGFBP-4 proteolysis. IGFBP-2 mRNA and protein levels were observed under basal conditions in both cell types, with no significant regulation by IGF-I. The analysis of cell-type-specific regulation of the IGF system in both primary mammary epithelial cells and stromal cells will assist in the characterization of the mechanisms behind the role of the IGF system in normal mammary physiology and ultimately breast cancer.

scholarly journals Tissue- and cell-specific expression of Ins(1,4,5)P3 3-kinase isoenzymes

1995 ◽  
Vol 306 (2) ◽  
pp. 429-435 ◽  
Author(s):  
V Vanweyenberg ◽  
D Communi ◽  
C S D'Santos ◽  
C Erneux
Keyword(s):  
Cell Lines ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Biological Data ◽  
Human Neuroblastoma Cell ◽  
Rat Tissues ◽  
Human Neuroblastoma ◽  
Mrna Species ◽  
Kinase A

The phosphorylation of Ins(1,4,5)P3 (InsP3) to Ins(1,3,4,5)P4 (InsP4) is catalysed by InsP3 3-kinase. Molecular-biological data have shown the presence of two human isoenzymes of InsP3 3-kinase, namely InsP3 3-kinases A and B. We have isolated from a rat thymus cDNA library a 2235 bp cDNA (clone B15) encoding rat InsP3 3-kinase B. Northern-blot analysis of mRNA isolated from rat tissues (thymus, testis, brain, spleen, liver, kidney, heart, lung and intestine) revealed that a rat InsP3 3-kinase B probe hybridized to a 6 kb mRNA in lung, thymus, testis, brain and heart. In contrast, Northern-blot analysis of the same tissues probed under stringent conditions with a rat InsP3 3-kinase A probe hybridized to a 2 kb mRNA only in brain and a 1.8-2.0 kb mRNA species in testis. Northern-blot analysis of three human cell lines (HL-60, SH-SY5Y and HTB-138) probed with a human InsP3 3-kinase B probe showed the presence of a 6 kb mRNA in all cell lines, except in the human neuroblastoma cell line (SH-SY5Y), where two mRNA species of 5.7 and 6 kb were detected. Using the same blot, no hybridization signal could be seen with a human InsP3 3-kinase A probe. Altogether, our data are consistent with the notion that the two InsP3 3-kinase isoenzymes, A and B, are specifically expressed in different tissues and cells.

scholarly journals IL-8 and MCP Gene Expression and Production by LPS-Stimulated Human Corneal Stromal Cells

2012 ◽  
Vol 2012 ◽  
pp. 1-4 ◽  
Author(s):  
Roni M. Shtein ◽  
Susan G. Elner ◽  
Zong-Mei Bian ◽  
Victor M. Elner
Keyword(s):  
Gene Expression ◽  
Northern Blot ◽  
Stromal Cells ◽  
Blot Analysis ◽  
Time Course ◽  
Northern Blot Analysis ◽  
Statistical Significance ◽  
Dependent Manner ◽  
Corneal Inflammation ◽  
Chemotactic Protein

Purpose. To determine time course of effect of lipopolysaccharide (LPS) on production of interleukin-8 (IL-8) and monocyte chemotactic protein (MCP) by cultured human corneal stromal cells.Methods. Human corneal stromal cells were harvested from donor corneal specimens, and fourth to sixth passaged cells were used. Cell cultures were stimulated with LPS for 2, 4, 8, and 24 hours. Northern blot analysis of IL-8 and MCP gene expression and ELISA for IL-8 and MCP secretion were performed. ELISA results were analyzed for statistical significance using two-tailed Student'st-test.Results. Northern blot analysis demonstrated significantly increased IL-8 and MCP gene expression after 4 and 8 hours of exposure to LPS. ELISA for secreted IL-8 and MCP demonstrated statistically significant increases (P<0.05) after corneal stromal cell stimulation with LPS.Conclusions. This paper suggests that human corneal stromal cells may participate in corneal inflammation by secreting potent leukocyte chemotactic and activating proteins in a time-dependent manner when exposed to LPS.

scholarly journals A rapid and accurate method for quantitating total RNA transferred during Northern blot analysis

1988 ◽  
Vol 16 (5) ◽  
pp. 2354-2354 ◽  
Author(s):  
Nathalie Denis ◽  
Daniel Corcos ◽  
Jacques Kruh ◽  
Alain Kitzis
Keyword(s):  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Accurate Method ◽  
Total Rna

The mouse prostaglandin E receptor EP2 subtype: cloning, expression, and Northern blot analysis

FEBS Letters ◽  
1995 ◽  
Vol 372 (2-3) ◽  
pp. 151-156 ◽  
Author(s):  
Masato Katsuyama ◽  
Nobuhiro Nishigaki ◽  
Yukihiko Sugimoto ◽  
Kimiko Morimoto ◽  
Manabu Negishi ◽  
...  
Keyword(s):  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Prostaglandin E
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