scholarly journals Identification and initial characterization of stathmin by the differential display method in nerve growth factor-treated PC12 cells

1998 ◽  
pp. 707-712 ◽  
Author(s):  
K Takekoshi ◽  
F Nomura ◽  
K Isobe ◽  
M Motooka ◽  
T Nammoku ◽  
...  
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Pc12 Cells ◽  
Differential Display ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Differentially Expressed ◽  
Rt Pcr ◽  
Differential Display Method

The differential display of mRNA is a new strategy to identify genes that are differentially expressed under altered conditions. We applied this method to determine differential gene expression in the rat pheochromocytoma cell line during differentiation induced by nerve growth factor (NGF). Three different mRNA species were isolated, and their differential expression was confirmed by RT-PCR. One of the mRNA species was identified as stathmin, a 19 kDa cytosolic protein attracting increasing interest for its role in signal transduction. In the NGF-treated PC12 cells, the expression of stathmin mRNA increased in a time-dependent manner, as assessed by northern blot analysis and RT-PCR. We also assessed by northern blot analysis how the expression of stathmin mRNA was altered in human pheochromocytomas (n = 5) compared with that in normal adrenal medulla tissue (n = 5). The mRNA concentrations were found to be significantly greater in the pheochromocytomas than in the normal tissues. It has been shown that stathmin mRNA concentrations are increased in various tumor cells. As pheochromocytomas are well-differentiated tumors of neural origin, it is not unexpected that stathmin mRNA is overexpressed in these tumors. Stathmin was isolated and identified as a differentially expressed gene by the differential display method in PC12 cells during differentiation induced by NGF. In addition, stathmin mRNA was found to be overexpressed in human pheochromocytomas. The mechanisms responsible for the up-regulation of stathmin mRNA during differentiation of PC12 cells and the significance of its overexpression in human pheochromocytomas remain to be determined.

Nerve Growth Factor Expression Correlates With Perineural Invasion and Pain in Human Pancreatic Cancer

1999 ◽  
Vol 17 (8) ◽  
pp. 2419-2419 ◽  
Author(s):  
Zhaowen Zhu ◽  
Helmut Friess ◽  
Fabio F. diMola ◽  
Arthur Zimmermann ◽  
Hans U. Graber ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
In Situ Hybridization ◽  
Nerve Growth Factor ◽  
Growth Factor ◽  
Northern Blot ◽  
Blot Analysis ◽  
Perineural Invasion ◽  
Northern Blot Analysis ◽  
Human Pancreatic Cancer

PURPOSE: The reasons for the high frequency of perineural invasion and the presence of pain in pancreatic cancer are still not clear. Nerve growth factor (NGF) and its high-affinity receptor TrkA are involved in stimulating epithelial cancer cell growth and perineural invasion, as well as in pain generation in chronic benign disorders. PATIENTS AND METHODS: NGF and TrkA were examined by Northern blot analysis, in situ hybridization, and immunohistochemistry in 27 normal and 37 pancreatic cancer tissue samples. The molecular findings were correlated with the degree of perineural invasion, pain, and histopathologic tumor characteristics. RESULTS: Northern blot analysis indicated that NGF and TrkA mRNA levels were increased 2.7-fold and 5.6-fold, respectively (P < .05 and P < .05), in pancreatic cancer tissues compared with the normal pancreas tissue. As shown by in situ hybridization and immunohistochemistry, NGF was strongly present in the cytoplasm of pancreatic cancer cells. TrkA was intensely present in the perineurium of pancreatic nerves but not in the cancer cells. There was no difference in NGF and TrkA expression between early (stages I and II) and advanced (stage III) tumor stages and between well-/moderately differentiated (grades 1 and 2) and poorly differentiated (grade 3) tumors. However, tumors with high NGF/TrkA expression levels exhibited more frequent perineural invasion (P < .01). Furthermore, increased NGF/TrkA expression levels were associated with a higher degree of pain (P < .01). CONCLUSION: Enhanced expression of the NGF/TrkA system may influence perineural invasion and may contribute to the pain syndrome in human pancreatic cancer.

scholarly journals RT-PCR and Northern blot analysis in search for a putative Paramecium beta-adrenergic receptor.

1999 ◽  
Vol 46 (3) ◽  
pp. 813-821
Author(s):  
A Płatek ◽  
J Wiejak ◽  
E Wyroba
Keyword(s):  
Northern Blot ◽  
Blot Analysis ◽  
Adrenergic Receptor ◽  
Northern Blot Analysis ◽  
Molecular Probes ◽  
Northern Hybridization ◽  
Rt Pcr ◽  
Beta Adrenergic Receptor ◽  
Beta Adrenergic ◽  
Sequence Tagged Sites

RT-PCR and Northern blot analysis were performed in order to search for a putative beta-adrenergic receptor (beta-AR) in Paramecium using several beta2-adrenergic-specific molecular probes. Under strictly defined RT-PCR conditions DNA species of expected molecular size about 360 bp were generated with the primers corresponding to the universal mammalian beta2-AR sequence tagged sites (located within the 4th and the 6th transmembrane regions of the receptor). This RT-PCR product hybridized in Southern blot analysis with the oligonucleotide probe designed to the highly conservative beta2-AR region involved in G-proteins interaction and located within the amplified region. Northern hybridization was performed on Paramecium total RNA and mRNA with human beta2-AR cDNA and two oligonucleotide probes: the first included Phe 290 involved in agonist binding (Strader et al., 1995) and the second was the backward RT-PCR primer. All these probes revealed the presence of about 2 kb mRNA which is consistent with the size of beta2-AR transcripts found in higher eukaryotes.

scholarly journals rRNA Stability in Heat-Killed and UV-Irradiated EnterotoxigenicStaphylococcus aureus and Escherichia coliO157:H7†

1998 ◽  
Vol 64 (11) ◽  
pp. 4264-4268 ◽  
Author(s):  
John L. McKillip ◽  
Lee-Ann Jaykus ◽  
Maryanne Drake
Keyword(s):  
Uv Irradiation ◽  
Foodborne Pathogens ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Brain Heart Infusion ◽  
Northern Blotting ◽  
Extreme Heat ◽  
Heat Inactivation ◽  
Rt Pcr

ABSTRACT Differentiation of viable cells from nonviable cells is of considerable importance in the development of methods to detect foodborne pathogens. To study the suitability of 16S rRNA as an indicator of cell viability in nucleic acid-based detection assays, we examined rRNA stability in two representative foodborne pathogens,Escherichia coli O157:H7 and enterotoxigenicStaphylococcus aureus, which were inactivated by extreme heat, moderate heat, and UV irradiation. Cell death under all conditions was confirmed by a failure to grow in brain heart infusion broth after incubation for 48 h at 37°C. rRNA stability was monitored by a Northern blot analysis, and detection was evaluated by using reverse transcription (RT)-PCR performed with two primer sets (which produced 325- and 1,400-bp amplicons). rRNA of neither pathogen was detected by Northern blot analysis and RT-PCR after cells were killed by autoclaving at 121°C for 15 min. In contrast, intact rRNA of both pathogens were detected by Northern blotting and could be amplified by RT-PCR up to 48 h after cells were killed by heat treatment at 80°C and UV irradiation at 254 nm. rRNA was a suitable target molecule for monitoring bacterial viability under extreme heat conditions, but the presence of rRNA was not correlated with viability following moderate heat inactivation or UV irradiation of cells.

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 Insulin-like growth factor 1 (IGF-1) gene expression in porcine ovarian tissue

1993 ◽  
Vol 73 (2) ◽  
pp. 253-257 ◽  
Author(s):  
S. T. Charlton ◽  
B. J. Cameron ◽  
D. R. Glimm ◽  
G. R. Foxcroft ◽  
J. J. Kennelly
Keyword(s):  
Gene Expression ◽  
Growth Factor ◽  
Northern Blot ◽  
Blot Analysis ◽  
Ribonucleic Acid ◽  
Northern Blot Analysis ◽  
Ovarian Tissue ◽  
Messenger Ribonucleic Acid ◽  
Polyadenylated Rna ◽  
Mrna Transcripts

A study was conducted to demonstrate IGF-1 gene expression in porcine ovarian tissue. Using northern blot analysis, IGF-1 messenger ribonucleic acid (mRNA) transcripts could not be consistently detected in total RNA. Authentic mRNA transcripts were detected in polyadenylated RNA-enriched RNA with sizes of 8.0, 3.6 and 2.3 bases, and possibly 0.8–1.1 kbases. Key words: Ovary, IGF-1, pig, mRNA

scholarly journals Absence of c-kit receptor and absent proliferative response to stem cell factor in childhood Burkitt's lymphoma cells

Blood ◽  
1995 ◽  
Vol 86 (4) ◽  
pp. 1469-1480 ◽  
Author(s):  
J Tomeczkowski ◽  
A Beilken ◽  
D Frick ◽  
B Wieland ◽  
A Konig ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Northern Blot ◽  
Blot Analysis ◽  
Stem Cell Factor ◽  
Northern Blot Analysis ◽  
Thymidine Incorporation ◽  
Rt Pcr ◽  
Low Level

The cytokine stem cell factor (SCF) synergizes with interleukin-7 (IL- 7) to enhance the proliferation of pre-B cells. To examine the role of SCF and its receptor, c-kit, in the pathogenesis of pediatric Burkitt's lymphomas (BL), we investigated the expression of SCF and c-kit in BL cells and the mitogenic activity of SCF on BL cells. A panel of 13 BL cell lines and 7 fresh biopsy tumors was investigated. BL cells were stimulated either by Epstein-Barr virus (EBV) infection or by different reagents and cytokines, and expression of SCF and c-kit was studied on the mRNA level by Northern blot analysis and reverse-transcriptase polymerase chain reaction (RT-PCR), followed by Southern blotting. c- kit expression was also studied by fluorescence-activated cell sorting and by crosslinking of digoxigenin-labeled recombinant human SCF to the cell surface. Proliferation of BL cell lines was measured by 3H- thymidine incorporation. Low-level expression of c-kit mRNA was detected in 2 of 13 unstimulated BL cell lines and in 1 fresh BL tumor. One cell line showed upregulation of c-kit mRNA with A23187 and downregulation with phorbol myristate acetate. Neither c-kit nor SCF could be detected in any other cell line under any condition of stimulation as analyzed by Northern blot analysis, RT-PCR followed by Southern blot analysis, crosslinking, and immunofluorescence. No response to SCF was seen in 3H-thymidine incorporation assays. We conclude that most BL cells express neither SCF nor c-kit and that the low-level expression of c-kit in some BL cells most likely has no biologic significance.

A comparison of RT-PCR and Northern blot analysis in quantifying metallothionein mRNA levels in killifish exposed to waterborne cadmium

1995 ◽  
Vol 39 (1-4) ◽  
pp. 137-141 ◽  
Author(s):  
Lisa Ann Elizabeth Kaplan ◽  
Kathleen Van Cleef ◽  
Isaac Wirgin ◽  
Joseph F. Crivello
Keyword(s):  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Waterborne Cadmium
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