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.

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.

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 Cytochrome bd Biosynthesis inBacillus subtilis: Characterization of thecydABCD Operon

1998 ◽  
Vol 180 (24) ◽  
pp. 6571-6580 ◽  
Author(s):  
Lena Winstedt ◽  
Ken-Ichi Yoshida ◽  
Yasutaro Fujita ◽  
Claes von Wachenfeldt
Keyword(s):  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Structural Genes ◽  
Low Oxygen ◽  
Terminal Oxidases ◽  
Cytochrome Bd ◽  
Transport Chain ◽  
Isolated Cell

ABSTRACT Under aerobic conditions Bacillus subtilis utilizes a branched electron transport chain comprising various cytochromes and terminal oxidases. At present there is evidence for three types of terminal oxidases in B. subtilis: acaa 3-, an aa 3-, and abd-type oxidase. We report here the cloning of the structural genes (cydA and cydB) encoding the cytochrome bd complex. Downstream of the structural genes,cydC and cydD are located. These genes encode proteins showing similarity to bacterial ATP-binding cassette (ABC)-type transporters. Analysis of isolated cell membranes showed that inactivation of cydA or deletion ofcydABCD resulted in the loss of spectral features associated with cytochrome bd. Gene disruption experiments and complementation analysis showed that the cydC andcydD gene products are required for the expression of a functional cytochrome bd complex. Disruption of thecyd genes had no apparent effect on the growth of cells in broth or defined media. The expression of the cydABCDoperon was investigated by Northern blot analysis and by transcriptional and translational cyd-lacZ fusions. Northern blot analysis confirmed that cydABCD is transcribed as a polycistronic message. The operon was found to be expressed maximally under conditions of low oxygen tension.

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

A procedure for Northern blot analysis of native RNA

1986 ◽  
Vol 159 (1) ◽  
pp. 227-232 ◽  
Author(s):  
Edouard W. Khandjian ◽  
Claude Méric
Keyword(s):  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis

The expression of NG2 proteoglycan in the developing rat limb

Development ◽  
1991 ◽  
Vol 111 (4) ◽  
pp. 933-944 ◽  
Author(s):  
A. Nishiyama ◽  
K.J. Dahlin ◽  
W.B. Stallcup
Keyword(s):  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Core Protein ◽  
Staining Intensity ◽  
Limb Bud ◽  
Protein Size ◽  
Glial Progenitor Cells ◽  
Ng2 Proteoglycan ◽  
Developing Rat

NG2 is a chondroitin sulfate proteoglycan previously found to be expressed by glial progenitor cells of the O2A lineage. We have examined the expression of NG2 in the developing rat limb by immunohistochemistry and northern blot analysis. Staining of embryonic day 14 (E14) rat limb bud sections with polyclonal and monoclonal anti-NG2 antibodies reveals reactivity in the precartilaginous mesenchymal condensation. The staining intensity increases with the differentiation of chondrocytes until E16. NG2 staining is not detected in the mature hypertrophic chondrocytes of E17 and postnatal day 3 (P3) limbs even after treatment of the sections with hyaluronidase or collagenase. Immuno-precipitations with anti-NG2 antibody using 125I-labeled limb cells in culture showed a 400 to 800 × 10(3) Mr proteoglycan species with a core protein size of 300 × 10(3) Mr, comparable to NG2 from O2A cells and neural cell lines. Northern blot analysis reveals the expression of an 8.9 kb mRNA in E16 limbs and at a lower level in P1 cartilage. The northern blot analyses also show that NG2 is distinct from the large aggregating proteoglycan of the cartilage. Our results indicate that in the developing limb cartilage, as in the differentiating oligodendrocytes, NG2 is present on immature cells in the process of differentiating, but its expression is downregulated as terminal differentiation of chondrocytes takes place.

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