scholarly journals Erythrocyte adenosine deaminase overproduction in hereditary hemolytic anemia

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 448-453 ◽  
Author(s):  
EG Chottiner ◽  
D Ginsburg ◽  
AP Tartaglia ◽  
BS Mitchell
Keyword(s):  
Adenosine Deaminase ◽  
Hemolytic Anemia ◽  
Northern Blot ◽  
Blot Analysis ◽  
Molecular Basis ◽  
Northern Blot Analysis ◽  
Mrna Level ◽  
Untranslated Regions ◽  
Cdna Clones ◽  
Specific Increase

Abstract A marked tissue-specific increase in erythrocyte adenosine deaminase (ADA) activity is associated with an autosomal dominantly inherited hemolytic anemia. We investigated the molecular basis of ADA overproduction by studying reticulocyte ADA mRNA from affected individuals. Analysis of proband reticulocyte ADA cDNA clones revealed normal sequence. RNase mapping demonstrated that the amount of ADA mRNA in affected reticulocytes was greater than the amount in normal B lymphoblasts, whereas ADA mRNA was undetectable in normal reticulocytes. The 5′- and 3′-untranslated regions of reticulocyte and B-lymphoblast ADA mRNAs from affected individuals were structurally indistinguishable from those of normal B lymphoblasts. Northern blot analysis performed under stringent hybridization and washing conditions confirmed a markedly increased amount of reticulocyte ADA mRNA in affected individuals as compared with controls. We conclude that the RBC-specific overexpression of ADA in this disorder occurs at the mRNA level.

scholarly journals Erythrocyte adenosine deaminase overproduction in hereditary hemolytic anemia

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 448-453
Author(s):  
EG Chottiner ◽  
D Ginsburg ◽  
AP Tartaglia ◽  
BS Mitchell
Keyword(s):  
Adenosine Deaminase ◽  
Hemolytic Anemia ◽  
Northern Blot ◽  
Blot Analysis ◽  
Molecular Basis ◽  
Northern Blot Analysis ◽  
Mrna Level ◽  
Untranslated Regions ◽  
Cdna Clones ◽  
Specific Increase

A marked tissue-specific increase in erythrocyte adenosine deaminase (ADA) activity is associated with an autosomal dominantly inherited hemolytic anemia. We investigated the molecular basis of ADA overproduction by studying reticulocyte ADA mRNA from affected individuals. Analysis of proband reticulocyte ADA cDNA clones revealed normal sequence. RNase mapping demonstrated that the amount of ADA mRNA in affected reticulocytes was greater than the amount in normal B lymphoblasts, whereas ADA mRNA was undetectable in normal reticulocytes. The 5′- and 3′-untranslated regions of reticulocyte and B-lymphoblast ADA mRNAs from affected individuals were structurally indistinguishable from those of normal B lymphoblasts. Northern blot analysis performed under stringent hybridization and washing conditions confirmed a markedly increased amount of reticulocyte ADA mRNA in affected individuals as compared with controls. We conclude that the RBC-specific overexpression of ADA in this disorder occurs at the mRNA level.

Development and tissue distribution of sucrase-isomaltase mRNA in rats

1990 ◽  
Vol 258 (1) ◽  
pp. G52-G58 ◽  
Author(s):  
L. L. Leeper ◽  
S. J. Henning
Keyword(s):  
Enzyme Activity ◽  
Small Intestine ◽  
Northern Blot ◽  
Blot Analysis ◽  
Molecular Basis ◽  
Northern Blot Analysis ◽  
Sucrase Activity ◽  
Old Rats ◽  
Two Parameters ◽  
Precocious Development

Previous studies of sucrase-isomaltase (SI) activities have shown this complex to be absent in the suckling rat and to appear during the weaning period. We describe here the cloning of a heterologous SI cDNA and its use for the quantitation of SI mRNA as a first step toward understanding the molecular basis of SI development. A survey of RNA from 12 tissues of mature rats by Northern blot analysis showed a 6-kb band of SI mRNA only in the small intestine. Within the latter, both sucrase activity and SI mRNA peaked in the jejunum. Assay of jejunal tissue from developing rats showed sucrase activity and SI mRNA to be first detectable at 18 days, to rise in parallel through 24 days, and then to diverge a little (enzyme activity being lower) by 36 days. When glucocorticoid was administered to 10-day-old rats, neither sucrase activity nor SI mRNA was detectable 12 h later. Both parameters were readily detected 24 h postinjection, although the mRNA had risen relatively more than the enzyme activity. The two parameters increased in concert through 5 days postinjection and then plateaued. We conclude that, with respect to distribution along the intestine and to normal and precocious development, activities of SI in the rat are determined primarily by the abundance of its mRNA.

scholarly journals Human inositol 1,4,5-trisphosphate type-1 receptor, InsP3R1: structure, function, regulation of expression and chromosomal localization

1994 ◽  
Vol 302 (3) ◽  
pp. 781-790 ◽  
Author(s):  
N Yamada ◽  
Y Makino ◽  
R A Clark ◽  
D W Pearson ◽  
M G Mattei ◽  
...  
Keyword(s):  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Immunohistochemical Analysis ◽  
Predicted Amino Acid Sequence ◽  
Cdna Clones ◽  
Cellular Functions ◽  
Regulation Of Expression ◽  
Inositol 1,4,5 Trisphosphate

We have isolated cDNA clones encoding an inositol 1,4,5-trisphosphate receptor type 1 (InsP3R1) from human uteri and a leukaemic cell line, HL-60. Northern-blot analysis showed that approx. 10 kb of InsP3R1 mRNA is expressed in human uteri, oviducts and HL-60 cells. The predicted amino acid sequence of human InsP3R1 (2695 amino acids) has 99% identity with that of the mouse SI-/SII- splicing counterpart. Western-blot analysis with anti-(mouse InsP3R1) antibodies showed that InsP3R1 protein of human uteri and oviducts of approx 220 kDa is immunostained. Northern-blot analysis of HL-60 cell differentiation along the neutrophilic lineage induced by retinoic acid or dimethylsulphoxide showed an accompanying enhanced expression of InsP3R1 mRNA. Immunohistochemical analysis of the cerebella of spinocerebellar degeneration patients showed a variable loss of Purkinje cells with an altered pattern of immunostaining. The InsP3R1 gene (Insp3r1) was localized to the 3P25-26 region of human chromosome 3. The data presented here clearly show that InsP3R1 exists widely in human tissues and may play critical roles in various kinds of cellular functions.

Cloning and characterization of bovine cytosolic and mitochondrial PEPCK during transition to lactation

2002 ◽  
Vol 11 (2) ◽  
pp. 53-63 ◽  
Author(s):  
Cansu Agca ◽  
Randall B. Greenfield ◽  
Jennifer R. Hartwell ◽  
Shawn S. Donkin
Keyword(s):  
Dairy Cows ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Untranslated Regions ◽  
Transcript Expression ◽  
Early Lactation ◽  
Coding Sequence ◽  
Partial Clone

The cytosolic (C) and mitochondrial (M) forms of phospho enolpyruvate carboxykinase (PEPCK; EC 4.1.1.32 ) are encoded by two different nuclear genes in mouse, human, and chicken. Our objective was to clone the two forms of PEPCK for bovine and determine their expression during the immediate periparturient interval in dairy cows. Bovine PEPCK-C cDNA contains 2,592 nucleotides and contains 84% similarity to the coding sequence of human PEPCK-C cDNA. A 449-nt partial clone of the 3′ end of PEPCK-M is 76% similar to the corresponding sequence of human PEPCK-M. The coding sequence of bovine PEPCK-C and coding sequence of the partial PEPCK-M clone were 58% similar but the similarities in the 3′-untranslated regions were negligible. Northern blot analysis revealed single transcripts of 2.85 and 2.35 kb for PEPCK-C and PEPCK-M, respectively. The transition to lactation did not alter PEPCK-M transcript expression, but expression of PEPCK-C mRNA was transiently increased during early lactation, indicating that enhanced hepatic gluconeogenesis during this period may be tied to enhanced capacity for cytosolic rather than mitochondrial formation of phosphoenolpyruvate.

Activin inhibits but inhibin activates mouse placental lactogen-II secretion

1995 ◽  
Vol 146 (3) ◽  
pp. 469-474 ◽  
Author(s):  
M Yamaguchi ◽  
K Tasaka ◽  
K Ogura ◽  
M Sakata ◽  
J Mizuki ◽  
...  
Keyword(s):  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Mrna Level ◽  
Inhibitory Effect ◽  
Activin A ◽  
Placental Lactogen ◽  
Gene Expressions ◽  
Placental Cells ◽  
Dose Dependent

Abstract The regulation of mouse placental lactogen (mPL)-I and mPL-II secretion by activin and inhibin and the expression of activin and inhibin subunit mRNAs in the mouse decidua were examined. Activin-A at a concentration of 10 nm/l significantly inhibited mPL-II secretion by placental cells from days 9 and 12 of pregnancy. However, activin-A did not affect mPL-I secretion by cells from days 7 and 9 of pregnancy nor mPL-II secretion by cells from day 7 of pregnancy. By contrast, 10 nm/l inhibin activated mPL-II secretion by cells from day 12 of pregnancy. These effects of activin and inhibin on mPL-II secretion were dose-dependent. Follistatin, which binds to activin and blocks its bioactivity, completely eliminated the inhibitory effect of activin on mPL-II secretion. Incubation of placental cells from day 12 of pregnancy with activin-A resulted in a significant reduction of the mPL-II mRNA level assessed by Northern blot analysis. Northern blot analysis using poly(A)+ RNA extracted from the decidua indicated that mouse decidua, as well as the placenta, express all activin and inhibin subunits and that their gene expressions increased during gestation. The expression of these mRNAs in the decidua was much higher than those in the placenta. These findings suggest that activin and inhibin regulate mPL-II secretion and suggest the presence of an autocrine or paracrine regulation of mPL-II secretion in mouse placenta by activin and inhibin after mid-pregnancy in vivo. Journal of Endocrinology (1995) 146, 469–474

Immediate early genes of glucocorticoid action on the developing intestine

2005 ◽  
Vol 288 (5) ◽  
pp. G897-G906 ◽  
Author(s):  
Barbara M. Agbemafle ◽  
Thomas J. Oesterreicher ◽  
Chad A. Shaw ◽  
Susan J. Henning
Keyword(s):  
Microarray Analysis ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Primary Response ◽  
Cdna Clones ◽  
Secondary Effects ◽  
Additional Time ◽  
Glucocorticoid Treatment ◽  
Functional Maturation

Prior studies have demonstrated that glucocorticoid hormones elicit functional maturation of the small intestine as evidenced by their ability to induce increases in the expression of various digestive hydrolases, such as sucrase-isomaltase and trehalase. However, these increases have a lag time of ∼24 h, suggesting that they are secondary effects of hormone action. To identify candidate primary response genes, we performed microarray analysis on pooled RNA from jejunums of untreated postnatal day 8 mouse pups and from littermates who earlier received dexamethasone 2 h. Fluorescent dye-labeled samples were hybridized in quadruplicate to glass-spotted cDNA microarrays containing 15,000 cDNA clones from the National Institute of Aging cDNA clone set. Analysis of the resulting signals using relatively stringent criteria identified 66 transcripts upregulated and 36 downregulated by 2 h of glucocorticoid treatment. Among the upregulated transcripts, the magnitude of the increase detected by microarray ranged from 1.4- to 16-fold. Selected mRNAs from throughout the range were subsequently analyzed by Northern blot analysis. Of 11 mRNAs chosen all were confirmed, and there was a strong correlation between the magnitude of the increase observed from the microarray analysis and from Northern blot analysis. Additional time points showed that these transcripts peaked between 2 and 6 h and had returned to baseline by 24 h. Gene ontology analysis showed pleiotropic effects of dexamethasone on the developing intestine and pointed to genes in the development category as being likely candidates for mediation of glucocorticoid-induced maturation of intestinal function.

scholarly journals A new approach to understanding T cell development: the isolation and characterization of immature CD4-, CD8-, CD3- T cell cDNAs by subtraction cloning.

1992 ◽  
Vol 3 (7) ◽  
pp. 761-773 ◽  
Author(s):  
S L Orr ◽  
E Gese ◽  
L Hood
Keyword(s):  
T Cells ◽  
T Cell ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
T Cell Development ◽  
Cell Development ◽  
Cdna Clones ◽  
Immunoglobulin Gene ◽  
Sequence Determination

During T cell development in the mammalian thymus, immature T cells are observed that lack the cell surface markers CD4, CD8, and CD3. A subtracted cDNA library was constructed to isolate cDNAs that are specific for these immature T cells. Tissue-specific expression of 97 individual cDNAs were examined using different cell types by Northern blot analysis, and six cDNAs were analyzed by reverse transcriptase (RT) polymerase chain reaction (PCR) detection of RNA. Approximately 50% of the clones could not be detected on Northern blots, and 40% of the clones were expressed by at least one other cell-type including monocytes, mature T cells, and B cells. Eight cDNA clones appear to be specific for the CD4-, CD8-, CD3- T cell line, used to construct the library, as determined by Northern blot analysis. In addition, 330 cDNA clones were subjected to partial automated DNA sequence determination. Database searches, with both nucleotide and protein translations, revealed cDNAs that exhibit interesting similarities to human cell-cycle gene 1, platelet-derived growth factor receptor, c-fms oncogene (CSF-1) receptor, and members of the immunoglobulin gene superfamily. This approach of employing subtraction coupled with large scale partial cDNA sequence determination can be useful to identify genes that may be involved in early T cell growth, cellular recognition or differentiation.

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.

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