scholarly journals Glucocorticoids increase amylase mRNA levels, secretory organelles, and secretion in pancreatic acinar AR42J cells.

1985 ◽  
Vol 100 (4) ◽  
pp. 1200-1208 ◽  
Author(s):  
C D Logsdon ◽  
J Moessner ◽  
J A Williams ◽  
I D Goldfine
Keyword(s):  
Secretory Granules ◽  
Tumor Cell Line ◽  
Volume Density ◽  
In Vitro Translation ◽  
Mrna Levels ◽  
Dexamethasone Treatment ◽  
Pancreatic Cells ◽  
Morphometric Analyses ◽  
Specific Mrnas

Previous studies have suggested a role for glucocorticoids in the differentiation of the acinar pancreas. We have now used the rat tumor cell line AR42J, derived from the acinar pancreas, to directly study this effect of glucocorticoids in vitro. The steroid hormones dexamethasone, corticosterone, aldosterone, and progesterone, but not estrogen, increased both the amylase content and the number of secretory granules of these cells. The potencies of the steroids were directly related to their effectiveness as glucocorticoids; dexamethasone was the most potent hormone and gave maximal effects at 100 nM. Morphometric analyses revealed that dexamethasone increased the volume density of granules 5.5-fold from 0.20 +/- 0.08 to 1.10 +/- 0.20% (n = 4) of the cytoplasmic volume. Dexamethasone treatment also increased the volume density of rough endoplasmic reticulum 2.4-fold from 1.20 +/- 0.09 to 2.86 +/- 0.30% (n = 5) of the cytoplasmic volume. After 48 h of dexamethasone treatment the cellular content of amylase increase eightfold from 2.8 +/- 0.4 to 22.6 +/- 3.8 U/mg protein (n = 6). This effect of dexamethasone was discernible after 12 h of incubation and approached maximal stimulation after 72 h of incubation. The increases in cellular amylase content were due to increased amylase synthesis as shown by specific immunoprecipitation of [35S]methionine-labeled proteins. Moreover, in vitro translation of cellular mRNA indicated that dexamethasone treatment increased amylase mRNA. Dexamethasone treatment also led to increased secretion of amylase in response to the secretagogue cholecystokinin. These data indicate, therefore, that glucocorticoids induce a more highly differentiated phenotype in AR42J pancreatic cells, and they suggest that glucocorticoids act via the enhanced transcription of specific mRNAs for acinar cell proteins.

Sheep antiluteolytic interferon: cDNA sequence and analysis of mRNA levels

1989 ◽  
Vol 2 (1) ◽  
pp. 65-70 ◽  
Author(s):  
H.J. Stewart ◽  
S.H.E. McCann ◽  
A.J. Northrop ◽  
G.E. Lamming ◽  
A.P.F. Flint
Keyword(s):  
Amino Acid ◽  
Northern Blotting ◽  
In Vitro Translation ◽  
Major Constituent ◽  
Interferon Α ◽  
Mrna Levels ◽  
Blastocyst Development ◽  
Cdna Sequences

ABSTRACT A cloned cDNA has been isolated by probing a sheep blastocyst cDNA library using a synthetic oligonucleotide representing the N-terminal amino acid sequence of the antiluteolytic protein, ovine trophoblast protein-1. Sequence analysis of the cDNA confirms the 70% homology between the antiluteolysin and the interferon-α family of proteins; however, the sequence reported here differs at several points from previously reported amino acid and cDNA sequences for the antiluteolysin. In-vitro translation of day-16 poly(A)+ RNA indicated that antiluteolysin mRNA is a major constituent of total mRNA at this stage of blastocyst development, and Northern blotting confirmed that antiluteolysin mRNA production occurred between days 13 and 22 after oestrus. This is consistent with the stage at which embryonic extracts are antiluteolytic on administration in vivo. These and other data confirm that the ovine trophoblast antiluteolysin is an interferon, and suggest that at least five isoforms of this protein may exist.

scholarly journals c-erbA encodes multiple proteins in chicken erythroid cells.

1988 ◽  
Vol 8 (10) ◽  
pp. 4155-4161 ◽  
Author(s):  
J Bigler ◽  
R N Eisenman
Keyword(s):  
Expression Vector ◽  
In Vitro Translation ◽  
Vector System ◽  
Mrna Levels ◽  
Erythroid Cells ◽  
Avian Erythroblastosis Virus ◽  
Related Proteins ◽  
Translational Mechanisms

To identify and characterize the proteins encoded by the erbA proto-oncogene, we expressed the C-terminal region of v-erbA in a bacterial trpE expression vector system and used the fusion protein to prepare antiserum. The anti-trp-erbA serum recognized the P75gag-erbA protein encoded by avian erythroblastosis virus and specifically precipitated six highly related proteins ranging in size from 27 to 46 kilodaltons from chicken embryonic erythroid cells. In vitro translation of a chicken erbA cDNA produced essentially the same pattern of proteins. Partial proteolytic maps and antigenicity and kinetic analyses of the in vivo and in vitro proteins indicated that they are related and that the multiple bands are likely to arise from internal initiations within c-erbA to generate a nested set of proteins. All of the c-erbA proteins are predominantly associated with chicken erythroblast nuclei. However, Nonidet P-40 treatment resulted in extraction of the three smaller proteins, whereas the larger proteins were retained. During differentiation of erythroid cells in chicken embryos, we found maximal levels of c-erbA protein synthesis at days 7 to 8 of embryogenesis. By contrast, c-erbA mRNA levels remained essentially constant from days 5 to 12. Together, our results indicate that posttranscriptional or translational mechanisms are involved in regulation of c-erbA expression and in the complexity of its protein products.

scholarly journals In vitro mutagenesis of trypsinogen: role of the amino terminus in intracellular protein targeting to secretory granules.

1987 ◽  
Vol 105 (2) ◽  
pp. 659-668 ◽  
Author(s):  
T L Burgess ◽  
C S Craik ◽  
L Matsuuchi ◽  
R B Kelly
Keyword(s):  
Signal Peptide ◽  
Secretory Pathway ◽  
Protein Targeting ◽  
Secretory Granules ◽  
Tumor Cell Line ◽  
Signal Peptidase ◽  
Secretory Proteins ◽  
In Vitro Mutagenesis ◽  
Regulated Secretory Pathway

The mouse anterior pituitary tumor cell line, AtT-20, targets secretory proteins into two distinct intracellular pathways. When the DNA that encodes trypsinogen is introduced into AtT-20 cells, the protein is sorted into the regulated secretory pathway as efficiently as the endogenous peptide hormone ACTH. In this study we have used double-label immunoelectron microscopy to demonstrate that trypsinogen colocalizes in the same secretory granules as ACTH. In vitro mutagenesis was used to test whether the information for targeting trypsinogen to the secretory granules resides at the amino (NH2) terminus of the protein. Mutations were made in the DNA that encodes trypsinogen, and the mutant proteins were expressed in AtT-20 cells to determine whether intracellular targeting could be altered. Replacing the trypsinogen signal peptide with that of the kappa-immunoglobulin light chain, a constitutively secreted protein, does not alter targeting to the regulated secretory pathway. In addition, deletion of the NH2-terminal "pro" sequence of trypsinogen has virtually no effect on protein targeting. However, this deletion does affect the signal peptidase cleavage site, and as a result the enzymatic activity of the truncated trypsin protein is abolished. We conclude that neither the signal peptide nor the 12 NH2-terminal amino acids of trypsinogen are essential for sorting to the regulated secretory pathway of AtT-20 cells.

scholarly journals Molecular cloning and sequence of a cholesterol-repressible enzyme related to prenyltransferase in the isoprene biosynthetic pathway.

1987 ◽  
Vol 7 (9) ◽  
pp. 3138-3146 ◽  
Author(s):  
C F Clarke ◽  
R D Tanaka ◽  
K Svenson ◽  
M Wamsley ◽  
A M Fogelman ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Molecular Cloning ◽  
In Vitro Transcription ◽  
In Vitro Translation ◽  
Mrna Levels ◽  
Hmg Coa Reductase ◽  
Transcription System ◽  
Coa Reductase

Differential hybridization and molecular cloning have been used to isolate CR39, a cDNA which hybridizes to a 1.2-kilobase (kb) mRNA in rat liver. The level of CR39 mRNA was increased seven- to ninefold over normal levels by dietary cholestyramine and mevinolin and decreased about fourfold compared with normal levels by cholesterol feeding or administration of mevalonate. Similar changes in the mRNA levels of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and HMG-CoA synthase were observed under the various conditions. In vitro translation of either CR39 hybrid selected RNA or 1.2-kb CR39 RNA generated by an SP6 in vitro transcription system produced a polypeptide of 39,000 daltons. As deduced from the nucleotide sequence of a full-length CR39 cDNA, the rat CR39 polypeptide contained 344 amino acids and had a molecular weight of 39,615. The predicted amino acid composition and submit molecular weight of the rat CR39 were very similar to those of prenyltransferases isolated from chicken, pig, and human. The sequence of amino acid residues 173 through 203 in the rat CR39 polypeptide showed that 17 out of 30 matched an active-site peptide of avian liver prenyltransferase. Thus, alterations in the rate of cholesterogenesis resulted in the coordinate regulation of three mRNAs encoding HMG-CoA reductase, HMG-CoA synthase, and CR39, the latter being tentatively identified as prenyltransferase.

Production of multiple forms of glucoamylase in Aspergillus awamori

1987 ◽  
Vol 65 (8) ◽  
pp. 762-765 ◽  
Author(s):  
Resham S. Bhella ◽  
Illimar Altosaar
Keyword(s):  
Molecular Weight ◽  
Northern Blot Analysis ◽  
De Novo ◽  
In Vitro Translation ◽  
Aspergillus Awamori ◽  
Multiple Forms ◽  
Molecular Weights ◽  
Specific Mrnas

The biosynthesis of glucoamylases in Aspergillus awamori was studied by in vivo protein labelling and analysis of glucoamylase-specific mRNAs. Two types of glucoamylases with molecular weights of 100 000 and 82 000 were shown to be synthesized de novo. Deglycosylation of the 100 000 molecular weight glucoamylase type resulted in the formation of another glucoamylase form with molecular weight of about 94 000. De novo synthesis of two types of glucoamylases was further confirmed by the existence of two types of glucoamylase-specific mRNAs, as demonstrated by in vitro translation and Northern blot analysis studies.

Molecular cloning and sequence of a cholesterol-repressible enzyme related to prenyltransferase in the isoprene biosynthetic pathway

1987 ◽  
Vol 7 (9) ◽  
pp. 3138-3146
Author(s):  
C F Clarke ◽  
R D Tanaka ◽  
K Svenson ◽  
M Wamsley ◽  
A M Fogelman ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Molecular Cloning ◽  
In Vitro Transcription ◽  
In Vitro Translation ◽  
Mrna Levels ◽  
Hmg Coa Reductase ◽  
Transcription System ◽  
Coa Reductase

Differential hybridization and molecular cloning have been used to isolate CR39, a cDNA which hybridizes to a 1.2-kilobase (kb) mRNA in rat liver. The level of CR39 mRNA was increased seven- to ninefold over normal levels by dietary cholestyramine and mevinolin and decreased about fourfold compared with normal levels by cholesterol feeding or administration of mevalonate. Similar changes in the mRNA levels of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and HMG-CoA synthase were observed under the various conditions. In vitro translation of either CR39 hybrid selected RNA or 1.2-kb CR39 RNA generated by an SP6 in vitro transcription system produced a polypeptide of 39,000 daltons. As deduced from the nucleotide sequence of a full-length CR39 cDNA, the rat CR39 polypeptide contained 344 amino acids and had a molecular weight of 39,615. The predicted amino acid composition and submit molecular weight of the rat CR39 were very similar to those of prenyltransferases isolated from chicken, pig, and human. The sequence of amino acid residues 173 through 203 in the rat CR39 polypeptide showed that 17 out of 30 matched an active-site peptide of avian liver prenyltransferase. Thus, alterations in the rate of cholesterogenesis resulted in the coordinate regulation of three mRNAs encoding HMG-CoA reductase, HMG-CoA synthase, and CR39, the latter being tentatively identified as prenyltransferase.

c-erbA encodes multiple proteins in chicken erythroid cells

1988 ◽  
Vol 8 (10) ◽  
pp. 4155-4161
Author(s):  
J Bigler ◽  
R N Eisenman
Keyword(s):  
Expression Vector ◽  
In Vitro Translation ◽  
Vector System ◽  
Mrna Levels ◽  
Erythroid Cells ◽  
Avian Erythroblastosis Virus ◽  
Related Proteins ◽  
Translational Mechanisms

To identify and characterize the proteins encoded by the erbA proto-oncogene, we expressed the C-terminal region of v-erbA in a bacterial trpE expression vector system and used the fusion protein to prepare antiserum. The anti-trp-erbA serum recognized the P75gag-erbA protein encoded by avian erythroblastosis virus and specifically precipitated six highly related proteins ranging in size from 27 to 46 kilodaltons from chicken embryonic erythroid cells. In vitro translation of a chicken erbA cDNA produced essentially the same pattern of proteins. Partial proteolytic maps and antigenicity and kinetic analyses of the in vivo and in vitro proteins indicated that they are related and that the multiple bands are likely to arise from internal initiations within c-erbA to generate a nested set of proteins. All of the c-erbA proteins are predominantly associated with chicken erythroblast nuclei. However, Nonidet P-40 treatment resulted in extraction of the three smaller proteins, whereas the larger proteins were retained. During differentiation of erythroid cells in chicken embryos, we found maximal levels of c-erbA protein synthesis at days 7 to 8 of embryogenesis. By contrast, c-erbA mRNA levels remained essentially constant from days 5 to 12. Together, our results indicate that posttranscriptional or translational mechanisms are involved in regulation of c-erbA expression and in the complexity of its protein products.

Development of bronchiolar epithelium in rats

1986 ◽  
Vol 250 (5) ◽  
pp. R783-R788 ◽  
Author(s):  
G. D. Massaro ◽  
D. Massaro
Keyword(s):  
Secretory Granules ◽  
Perinatal Period ◽  
Volume Density ◽  
Clara Cell ◽  
Numerical Density ◽  
Clara Cells ◽  
Ciliated Cells ◽  
Bronchiolar Epithelium ◽  
Conducting Airways

We used ultrastructural and morphometric means to examine aspects of the regulation of the maturation of rat bronchiolar Clara cells and the development of the epithelium of small conducting airways in the perinatal period. We found the nuclear numerical density per cubed centimeter (Nvn) of Clara cells fell and the Nvn of ciliated cells increased from birth to age 60 days, the prenatal administration of a glucocorticosteroid (dexamethasone) to dams caused a 35% decrease in the Nvn of ciliated cells present at birth but did not alter the Nvn of Clara cells, the administration of dexamethasone to pups from postnatal days 1 to 6 did not alter the Nvn of bronchiolar Clara or ciliated cells present at age 7 days but did diminish the total number of lung cells as assessed by measurements of lung DNA, the administration of dexamethasone to dams from gestation days 18 to 20 or from gestation days 20 to 22 did not alter the volume density of Clara cell glycogen, secretory granules, rough endoplasmic reticulum, or mitochondria of pups at gestation day 21.5 or on the day of birth, and glucagon, epinephrine, and 8-bromoadenosine 3',5'-cyclic monophosphate resulted in a decrease of Clara cell glycogen when individually incubated in vitro for 4 h with bronchiolar tissue from rat fetuses 21.5 days of age.

Cis-dichloro-diammine platinum (II): a stain for electron microscopic preparations

1978 ◽  
Vol 36 (2) ◽  
pp. 122-123
Author(s):  
Ernst Heinen
Keyword(s):  
Heart Muscle ◽  
Secretory Granules ◽  
Cell Types ◽  
Electron Microscopic ◽  
Antimitotic Agent ◽  
Dense Granules ◽  
Pancreatic Cells ◽  
Ultrathin Sections ◽  
Very High

We have previously reported that cis-dichloro-diammine platinum (II) (cis-Pt), an antimitotic agent discovered by Rosenberg and coworkers, can be used to contrast ultrathin sections. It was interesting to tend to increase the contrast given by this cis-Pt staining and to establish to which radicals cis-Pt binds in the cell.We have analysed various cell types (fibroblasts and Ehrlich tumour cells cultivated in vitro or various tissues : liver, pancreas, heart muscle, epididyme, intestine, etc.) fixed in glutaraldehyde, embedded in Epon and stained by immersion in a cis-Pt solution (1 mg/ml, 37°C, 2 or 3 days, in darkness).In all cases chromatin, especially heterochromatin, presents a very high contrast after cis-Pt staining and can easily be distinguished from the nucleolus (fig.). In the cytoplasm only ribosomes are well contrasted. The other organites are generally poorly stained, in some mitochondria, dense granules or filaments are apparent. Secretory granules containing proteins or mucopolysaccharides (basigranular or goblet cells of the intestine, mastocytes, pancreatic cells, etc.) are faintly stained by cis-Pt. In the heart muscle actin and myosin are only poorly contrasted. Cis-Pt suits thus for contrasting nucleic acids especially for differentiating DNA from RNA containing structures.

Translational regulation of light-induced ribulose 1,5-bisphosphate carboxylase gene expression in amaranth

1986 ◽  
Vol 6 (7) ◽  
pp. 2347-2353
Author(s):  
J O Berry ◽  
B J Nikolau ◽  
J P Carr ◽  
D F Klessig
Keyword(s):  
Translational Regulation ◽  
Small Subunit ◽  
In Vitro Translation ◽  
Mrna Levels ◽  
Mrna Accumulation ◽  
Bisphosphate Carboxylase ◽  
Genes Encoding ◽  
Translational Level

The regulation of the genes encoding the large and small subunits of ribulose 1,5-bisphosphate carboxylase was examined in amaranth cotyledons in response to changes in illumination. When dark-grown cotyledons were transferred into light, synthesis of the large- and small-subunit polypeptides was initiated very rapidly, before any increase in the levels of their corresponding mRNAs. Similarly, when light-grown cotyledons were transferred to total darkness, synthesis of the large- and small-subunit proteins was rapidly depressed without changes in mRNA levels for either subunit. In vitro translation or in vivo pulse-chase experiments indicated that these apparent changes in protein synthesis were not due to alterations in the functionality of the mRNAs or to protein turnover, respectively. These results, in combination with our previous studies, suggest that the expression of ribulose 1,5-bisphosphate carboxylase genes can be adjusted rapidly at the translational level and over a longer period through changes in mRNA accumulation.

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