George Scheele - Academia.edu (original) (raw)
Papers by George Scheele
Molecular and Cellular Biology, Oct 1, 1985
In the absence of changes in functional mRNA levels, stimulation of the pancreas with caerulein, ... more In the absence of changes in functional mRNA levels, stimulation of the pancreas with caerulein, a peptide analog of cholecystokinin, has been previously shown to increase the synthesis of anionic but not cationic trypsinogen. To look for structure-function correlations, a high-yield, full-length cDNA library has been constructed from canine pancreatic poly(A)+ mRNA. Full-length clones coding for the two major trypsinogen isoenzyme forms have been identified by colony hybridization and verified by in vitro translation of hybrid-selected mRNA in the presence of microsomal membranes and an optimal redox potential. Disulfidebonded translation products were separated and identfied by two-dimensional isoelectric focusing-sodium dodecyl sulfate-gel electrophoresis. Nucleotide sequence analysis allowed us to deduce the amino acid sequences for the anionic and cationic forms of canine trypsinogen, which contain 232 and 231 residues, respectively (77% amino acid identity), and the 15-residue amino terminal signal sequences (53% amino acid identity) associated with the two presecretory forms. Measurements of relative and absolute mRNA levels, when related to relative protein synthesis values, indicated that the translational efficiency of anionic trypsinogen mRNA exceeded that of cationic trypsinogen mRNA by 1.5to 2.9-fold under basal conditions. Analysis of the 5' noncoding regions of trypsinogen mRNAs revealed a striking conservation of sequence (10 of 12 bases) between dog and rat anionic trypsinogen forms. This contrasted markedly with the divergence of the 5' noncoding regions observed between dog anionic and cationic trypsinogen mRNAs.
S. Karger AG eBooks, Apr 16, 2015
American Journal of Physiology-gastrointestinal and Liver Physiology, Jun 1, 1980
Recent findings that contribute to an understanding of the secretory process in the pancreatic ex... more Recent findings that contribute to an understanding of the secretory process in the pancreatic exocrine cell are reviewed. These include 1) the separation, identification, and characterization of secreted proteins by two-dimensional gel electrophoresis, 2) the intracellular route and timetable of movement of secretory proteins from one intracellular compartment to another, 3) the evidence that suggests that all enzymes and zymogens secreted by the pancreas are segregated and transported within a single intracellular pathway, and 4) the mechanism by which presecretory proteins are thought to be transported across the lipid bilayer of the rough endoplasmic reticulum (the transport peptide hypothesis).
Methods in Enzymology, 1983
Publisher Summary This chapter discusses the methods for the preparation and study of pancreatic ... more Publisher Summary This chapter discusses the methods for the preparation and study of pancreatic lobules. When properly prepared, pancreatic lobules and dissociated acini, show similar in vitro responses to varying levels of exogenously added secretagogues. The use of pancreatic lobules has two advantages over the use of dispersed acini: (1) the ease of preparation and (2) the omission of degradative enzymes and chelators during tissue preparation. The use of dispersed acini shows two advantages over the use of lobules: (1) the increased access of labeled probes to the plasma membrane, and (2) the apparent removal of nerve endings from isolated acini. Purified preparations of secretory proteins can be extracted from a crude fraction of zymogen granules or obtained physiologically after carbamylcholine-induced secretion of proteins into the incubation medium. The high resolution achieved in the separation of exocrine pancreatic proteins by two-dimensional isoelectric focusing/sodium dodecyl sulfate (IEF/SDS)-gel electrophoresis allows the precise measurement of biosynthetic rates of individual secretory products.
Cell and Tissue Research, 1987
Previous studies from our laboratory indicate that the adaptive response of the exocrine pancreas... more Previous studies from our laboratory indicate that the adaptive response of the exocrine pancreas of the rat to prolonged stimulation with optimal doses of caerulein (0.25 microgram X kg-1 X h-1) follows a characteristic time course in which each step in the secretory pathway is activated. The immediate response is the depletion of zymogen-granule stores followed by coordinate and anticoordinate changes in individual rates of (pro-)enzyme synthesis after a lag period of 2 h. The sum of such changes leads to an increase in total rate of protein synthesis by 3 h which is combined with acceleration of intracellular transport packaging and granule discharge. In the present study the time course of DNA synthesis and the labeling index of five populations of pancreatic cells have been analyzed after caerulein stimulation for periods ranging from 6 to 72 h, using in vivo labeling with 1 mu Ci/g 3H-thymidine 1 h prior to sacrifice of the animals. DNA synthesis did not change during the initial 18 h in spite of persistent stimulation indicated by a 80% reduction on enzyme content. Following this lag period a sharp rise in DNA synthesis 20- to 25-fold above control levels was observed, which decreased by 48 h to reach control levels by 72 h. Increase in DNA synthesis was most pronounced in animals with lowest enzyme content in the pancreas. From the five cell populations studied by autoradiography interlobular duct cells and islet cells had no significant increase in labeling index at any time of stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)
Elsevier eBooks, 1983
Publisher Summary This chapter presents the methods developed to elucidate this mechanism for the... more Publisher Summary This chapter presents the methods developed to elucidate this mechanism for the transport of pancreatic exocrine proteins across the rough endoplasmic reticulum (RER) membrane. The methods are optimized for the simultaneous study of 17 discrete pancreatic secretory proteins, which include individual members of several families of enzymes and proenzymes. The results show that secretory proteins synthesized by dog pancreas mRNA in the presence of nuclease treated microsomal membranes were similar to those synthesized in vivo . The findings indicate that microsomal membranes isolated from dog pancreas and added to the reticulocyte lysate system are unstable in the presence of 50 μg/ml each of trypsin and chymotrypsin. Using polyacrylamide gel electrophoresis in SDS, the chapter demonstrates that the addition of micrococcal nuclease-treated rough microsomes from dog pancreas results in complete proteolytic processing of pancreatic presecretory proteins to polypeptide chains with apparent molecular weights identical with authentic secretory enzymes and zymogens.
Pancreas, 1996
Recent progress in understanding the luminal biochemistry of regulated pancreatic exocrine secret... more Recent progress in understanding the luminal biochemistry of regulated pancreatic exocrine secretion, including acid-base interactions between acinar and duct cells and pH-dependent processes that regulate membrane trafficking (endocytosis) at the apical plasma membrane, have led to the development of in vitro models of cystic fibrosis in the rat exocrine pancreas. Based on investigations in these model systems, a unifying hypothesis is presented that proposes that pancreatic dysfunction in cystic fibrosis occurs as a result of progressive acidification of the acinar and duct lumen, which leads to secondary defects in (i) apical trafficking of zymogen granule membranes and (ii) solubilization of secretory (pro)en-zymes. By directly acidifying the pH of the acinar lumen in cholescystokinin-stimulated acini, the early cytological findings observed in cystic fibrosis, including (i) massive dilatation of the acinar lumen, (ii) decreased appearance of zymogen granules, (iii) loss of the apical pole of the acinar cell, and (iv) persistent aggregation of secretory (pro)enzymes released into the luminal space, have been reproduced in primary cultures of pancreatic tissue.
Gastroenterology, Feb 1, 1987
Journal of Cell Biology, Nov 1, 1984
24-h intravenous caerulein infusion studies in the rat were combined with in vitro amino acid inc... more 24-h intravenous caerulein infusion studies in the rat were combined with in vitro amino acid incorporation studies followed by high-resolution separation of proteins by twodimensional isoelectric focusing and SDS gel electrophoresis to study the extent to which persistent changes in the biosynthesis of exocrine pancreatic proteins are regulated by cholecystokinin-like peptides. Beginning in the third hour of optimal hormone infusion at 0.25 /~g kg-1 h-1, changes were observed in the synthetic rates of 12 proteins, which progressed over the course of the 24-h study. Based on coordinate response patterns, exocrine proteins could be classified into four distinct groups. Group I (trypsinogen forms 1 and 2) showed progressive increases in synthetic rates reaching a combined 4.3-fold increase over control levels. Group II (amylase forms 1 and 2) showed progressive decreases in synthesis to levels 7.1-and 14.3-fold lower than control levels, respectively. Group III proteins (ribonuclease, chymotrypsinogen forms 1 and 2, procarboxypeptidase forms A and B, and proelastase 1) showed moderate increases in synthesis, 1.4-2.8-fold, and group IV proteins (trypsinogen 3, lipase, proelastase 2, and unidentified proteins 1-4) did not show changes in synthesis with hormone stimulation. Regulation of protein synthesis in response to caerulein infusion was specific for individual isoenzymic forms in the case of both trypsinogen and proelastase. The ratio of biosynthetic rates of trypsinogen forms 1 + 2 to amylase forms 1 + 2 increased from a control value of 0.56 to 24.4 after 24 h of hormonal stimulation (43.5-fold increase). Biosynthetic rates for an unidentified protein (P23) with an Mr = 23,000 and isoelectric point of 6.2 increased 14.2-fold, and the ratio of synthesis of P23 to amylase 2 increased 200-fold during caerulein infusion. During hormone stimulation the anticoordinate response in the synthesis of pancreatic glycosidases (decreased synthesis) and serine protease zymogens (increased synthesis) explain previous observations that showed little change in rates of total protein synthesis under similar conditions.
Cancer, Mar 15, 1981
Nineteen discrete proteins contained in normal human pancreatic juice were separated by two‐dimen... more Nineteen discrete proteins contained in normal human pancreatic juice were separated by two‐dimensional isoelectric focusing/SDS gel electrophoresis. Enzyme or proenzyme activity was assigned to 15 of these proteins. Analysis of pancreatic juice obtained from a patient with pancreatic cancer showed a number of additional proteins not observed in pancreatic juice obtained from normals.
J.F. Bergmann-Verlag eBooks, 1981
Biochimie, Sep 1, 1988
m Cotranslational translocation of exportable proteins across the RER membrane prior to their rel... more m Cotranslational translocation of exportable proteins across the RER membrane prior to their release into the extracellular space has been essentially described by use of canine pancreatic microsomal membranes. Intracisternal segregation of nascent secretory proteins was observed to be irreversible and proteolytic removal of signal sequences resulted in conformationally mature and stable proteins. Structural studies on various translocation peptides from both eukaryotic and prokaryotic preparations showed that many of them have a comparable three-domain organization. A hydrophilic amino-terminal domain is followed by a core region of hydrophobic amino acids and by the region in which the proteolytic cleavage occurs. Membrane components involved in the translocation process namely the signal recognition particle and the SRP receptor as well as the way the vectorial transport mechanism of nascent secretory proteins occurs are also discussed. secretory proteins / translocation peptides / vectorial transport / microsomai membranes / canine pancreas
Nucleic Acids Research, 1991
Previous attempts to obtain full-length double-stranded (ds) cDNA transcripts have relied on deox... more Previous attempts to obtain full-length double-stranded (ds) cDNA transcripts have relied on deoxy cytosine (dC)-deoxy guanine (dG) tailing methods. In this approach first strand cDNA transcripts are tailed with either dC or dG using terminal deoxynucleotidyl transferase (tdt) and second strand cDNA is primed with oligo-dG or-dC, respectively, and synthesized using DNA polymerase I or Klenow fragment (1). Modifications of this method have used cDNA transcripts tailed with dC and plasmid vectors tailed with dG (2, 3). By use of these tailing methods homopolymeric dC-dG regions (10-30 bp) are inserted upstream of cDNA constructs. We have observed (H.Lutcke and G.A.Scheele, unpublished observations) that such dC-dG regions inhibit (a) in vitro transcription of DNA using RNA polymerases (75% inhibition in the case of anionic trypsinogen cDNA), (b) translation of resulting RNA transcripts in wheat germ or reticulocyte lysate protein synthesizing systems (complete inhibition) and (c) sequencing of single stranded (ss) or ds DNA. Synthesis of ds cDNA by self-priming (4) or RNase-H (5) methods avoid the introduction of dC-dG tails but do not preserve the full-length character of cDNA constructs and therefore may not reveal the transcriptional start-site (cap site) or the complete 5' nontranslational sequence. In the novel method described here, a dC homopolymer tail (-15 bases) is added to first cDNA transcripts using tdt. Synthetic oligo ribo-G (rG, 15 mer, synthesized by Peninsula Laboratories, Belmont, CA) is used to prime the synthesis of full-length second strand cDNA. After completion of synthesis of ds cDNA, rG nucleotides in the RNA-DNA heteroduplex tail are removed with RNase H and the exposed ss dC homopolymer tail is removed with T4 DNA polymerase. The resulting full-length ds cDNA transcripts, devoid of C/G tails, are then inserted into any desirable vector, including expression vectors, using adapter-linker assisted ligation. To demonstrate the fidelity of our method we employed dog pancreas poly(A)+ RNA, which contains three well-defined size classes of high-abundance mRNA (-1.7 kb transcripts encode alpha-amylase [55 kDa] and procarboxypeptidase A and B forms [40-44 kDa],-1.0 kb transcripts encode serine protease zymogens [trypsinogen, chymotrypsinogen, and proelastase forms, 23-27 kDa], and-0.6-0.8 kb transcripts encode colipase and prophospholipase A2 forms [10-16 kDa].
European journal of biochemistry, Jun 1, 1990
A genomic library has been constructed in EMBL3 2 phage using high molecular mass DNA isolated fr... more A genomic library has been constructed in EMBL3 2 phage using high molecular mass DNA isolated from canine spleen. A cDNA clone, shown to code for preprophospholipase A2 which is processed to the prosecretory form prior to release from secretory cells, was used to identify a 1-clone which contains the complete phospholipase A2 gene. Restriction enzyme and DNA sequence analysis indicate that the primary transcriptional unit for the phospholipase gene, % 9.0 kb, is organized into four exon sequences. Exon 1 encodes the 5' nontranslated sequence, the ATG initiation codon, and the hydrophobic core of the signal peptide. Exons 2-4 encode regions of the peptide of residues-11 to 43, 43 to 86 and 86 to 124, respectively. The 5' flanking region shows a TATA box at position-29 and multiple CAAT boxes at positions-279,-206,-183 and-159. Regions of the 5' flanking sequence in the canine sequence, from nucleotides-47 to-74 and-91 to-129, show high similarity to similar regions in the human gene. However, an analysis of 400 nucleotides of the 5' flanking sequence in transient expression studies was unable to identify tissue-specific promoter or enhancer sequences. Within 5' nontranslated regions the canine and human genes share a pyrimidine-rich sequence which may be involved in differential regulation of mRNA translation.
Analytical Biochemistry, Dec 1, 1980
ABSTRACT
International journal of pancreatology, 1994
Methods: Sere of 130 patients suffering from pancreatic carcinoma were tested for anti-p53-Ab via... more Methods: Sere of 130 patients suffering from pancreatic carcinoma were tested for anti-p53-Ab via ELISA and immunoblorting using recombinant p53-protein as a target. These patients were classified according to UICC-criteria: stage I: 14%, stage II: 5%, stage III: 48% and ...
Journal of Biological Chemistry, 1985
The path and synchrony of intracellular transport of 12 secretory proteins of the guinea pig exoc... more The path and synchrony of intracellular transport of 12 secretory proteins of the guinea pig exocrine pancreas have been studied in pulse-chase amino acid labeling experiments by quantitative analysis of the individual proteins recovered in subcellular fractions and extracellular samples. Protein fractionation was accomplished by two-dimensional isoelectric focusing/ SDS-gel electrophoresis. Use of a double-label protocol allowed correction of the data on a protein-by-protein basis for leakage and adsorption artifacts which accompany tissue homogenization. All the labeled secretory (pro)enzymes, including their isoenzymic forms, were recovered in rough microsomal, Golgi-enriched and granule fractions during their transport to the cell surface. However, major asynchrony was observed at four levels: exit from the rough endoplasmic reticulum; transit through the Golgi complex; entry into granules; and discharge from the cell. Rapid transport rates were observed for trypsinogen, chymotrypsinogen 2, procarboxypeptidase A2, and lipase 2. Slow transport rates were observed for amylase and procarboxypeptidase B. In the presence of carbamylcholine or cholecystokinin stimulation, the times required for 40% discharge of labeled chymotrypsinogen 2, trypsinogen, amylase, and procarboxypeptidase B were 98, 102, 148, and 180 min, respectively. Transport rates did not correlate with isoelectric point, molecular weight, or the presence of carbohydrate. These data suggest that interactions occur within the rough endoplasmic reticulum, either between secretory (nong1yco)proteins themselves or between such proteins and the cisternal face of the rough endoplasmic reticulum.
Journal of Biological Chemistry, 1975
The discharge and the intracellular transport of the exocrine proteins produced by the guinea pig... more The discharge and the intracellular transport of the exocrine proteins produced by the guinea pig pancreas have been investigated in the in vitro lobule system described by SCHEELE, G. A., AND PALADE, G. E. (1975) J. Biol. Chem. 250, 2660-2670. The inquiry was carried out on a protein-specific basis by using the sodium dodecyl sulfate polyacrylamide gel electrophoretic procedures worked out by TARTAKOFF, A. M., GREENE, L. J., AND PALADE, G. E. (1974) J. Biol; Chem. 249, 7420-7431. The results show that the same protein mixture is discharged by the lobules regardless of whether stimulation is by carbamylcholine (which mimics the action of acetylcholine), caerulein (which mimics the action of pancreozymin), or by 75 mM KCl. When no stimulant is present, the small quantity of discharged protein (resting secretion) also has the same composition. Analysis of successive secretion aliquots collected over a period of 2 hours of continuous timulation by carbamylcholine showed that the composition of the secretory output remains quasi-constant with time; therefore, the exocrine proteins appear to be discharged in parallel and in constant proportions, irrespective of stimulant and time under stimulation (for carbamylcholine). The analysis of a series of zymogen granule fractions prepared from lobules pulse-labeled with radioactive amino acids and chased for periods of 15 to 155 min shows that all major secretory proteins enter the condensing vacuoles of the Golgi complex and appear in zymogen granules in apparent synchrony.
Molecular and Cellular Biology, Oct 1, 1985
In the absence of changes in functional mRNA levels, stimulation of the pancreas with caerulein, ... more In the absence of changes in functional mRNA levels, stimulation of the pancreas with caerulein, a peptide analog of cholecystokinin, has been previously shown to increase the synthesis of anionic but not cationic trypsinogen. To look for structure-function correlations, a high-yield, full-length cDNA library has been constructed from canine pancreatic poly(A)+ mRNA. Full-length clones coding for the two major trypsinogen isoenzyme forms have been identified by colony hybridization and verified by in vitro translation of hybrid-selected mRNA in the presence of microsomal membranes and an optimal redox potential. Disulfidebonded translation products were separated and identfied by two-dimensional isoelectric focusing-sodium dodecyl sulfate-gel electrophoresis. Nucleotide sequence analysis allowed us to deduce the amino acid sequences for the anionic and cationic forms of canine trypsinogen, which contain 232 and 231 residues, respectively (77% amino acid identity), and the 15-residue amino terminal signal sequences (53% amino acid identity) associated with the two presecretory forms. Measurements of relative and absolute mRNA levels, when related to relative protein synthesis values, indicated that the translational efficiency of anionic trypsinogen mRNA exceeded that of cationic trypsinogen mRNA by 1.5to 2.9-fold under basal conditions. Analysis of the 5' noncoding regions of trypsinogen mRNAs revealed a striking conservation of sequence (10 of 12 bases) between dog and rat anionic trypsinogen forms. This contrasted markedly with the divergence of the 5' noncoding regions observed between dog anionic and cationic trypsinogen mRNAs.
S. Karger AG eBooks, Apr 16, 2015
American Journal of Physiology-gastrointestinal and Liver Physiology, Jun 1, 1980
Recent findings that contribute to an understanding of the secretory process in the pancreatic ex... more Recent findings that contribute to an understanding of the secretory process in the pancreatic exocrine cell are reviewed. These include 1) the separation, identification, and characterization of secreted proteins by two-dimensional gel electrophoresis, 2) the intracellular route and timetable of movement of secretory proteins from one intracellular compartment to another, 3) the evidence that suggests that all enzymes and zymogens secreted by the pancreas are segregated and transported within a single intracellular pathway, and 4) the mechanism by which presecretory proteins are thought to be transported across the lipid bilayer of the rough endoplasmic reticulum (the transport peptide hypothesis).
Methods in Enzymology, 1983
Publisher Summary This chapter discusses the methods for the preparation and study of pancreatic ... more Publisher Summary This chapter discusses the methods for the preparation and study of pancreatic lobules. When properly prepared, pancreatic lobules and dissociated acini, show similar in vitro responses to varying levels of exogenously added secretagogues. The use of pancreatic lobules has two advantages over the use of dispersed acini: (1) the ease of preparation and (2) the omission of degradative enzymes and chelators during tissue preparation. The use of dispersed acini shows two advantages over the use of lobules: (1) the increased access of labeled probes to the plasma membrane, and (2) the apparent removal of nerve endings from isolated acini. Purified preparations of secretory proteins can be extracted from a crude fraction of zymogen granules or obtained physiologically after carbamylcholine-induced secretion of proteins into the incubation medium. The high resolution achieved in the separation of exocrine pancreatic proteins by two-dimensional isoelectric focusing/sodium dodecyl sulfate (IEF/SDS)-gel electrophoresis allows the precise measurement of biosynthetic rates of individual secretory products.
Cell and Tissue Research, 1987
Previous studies from our laboratory indicate that the adaptive response of the exocrine pancreas... more Previous studies from our laboratory indicate that the adaptive response of the exocrine pancreas of the rat to prolonged stimulation with optimal doses of caerulein (0.25 microgram X kg-1 X h-1) follows a characteristic time course in which each step in the secretory pathway is activated. The immediate response is the depletion of zymogen-granule stores followed by coordinate and anticoordinate changes in individual rates of (pro-)enzyme synthesis after a lag period of 2 h. The sum of such changes leads to an increase in total rate of protein synthesis by 3 h which is combined with acceleration of intracellular transport packaging and granule discharge. In the present study the time course of DNA synthesis and the labeling index of five populations of pancreatic cells have been analyzed after caerulein stimulation for periods ranging from 6 to 72 h, using in vivo labeling with 1 mu Ci/g 3H-thymidine 1 h prior to sacrifice of the animals. DNA synthesis did not change during the initial 18 h in spite of persistent stimulation indicated by a 80% reduction on enzyme content. Following this lag period a sharp rise in DNA synthesis 20- to 25-fold above control levels was observed, which decreased by 48 h to reach control levels by 72 h. Increase in DNA synthesis was most pronounced in animals with lowest enzyme content in the pancreas. From the five cell populations studied by autoradiography interlobular duct cells and islet cells had no significant increase in labeling index at any time of stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)
Elsevier eBooks, 1983
Publisher Summary This chapter presents the methods developed to elucidate this mechanism for the... more Publisher Summary This chapter presents the methods developed to elucidate this mechanism for the transport of pancreatic exocrine proteins across the rough endoplasmic reticulum (RER) membrane. The methods are optimized for the simultaneous study of 17 discrete pancreatic secretory proteins, which include individual members of several families of enzymes and proenzymes. The results show that secretory proteins synthesized by dog pancreas mRNA in the presence of nuclease treated microsomal membranes were similar to those synthesized in vivo . The findings indicate that microsomal membranes isolated from dog pancreas and added to the reticulocyte lysate system are unstable in the presence of 50 μg/ml each of trypsin and chymotrypsin. Using polyacrylamide gel electrophoresis in SDS, the chapter demonstrates that the addition of micrococcal nuclease-treated rough microsomes from dog pancreas results in complete proteolytic processing of pancreatic presecretory proteins to polypeptide chains with apparent molecular weights identical with authentic secretory enzymes and zymogens.
Pancreas, 1996
Recent progress in understanding the luminal biochemistry of regulated pancreatic exocrine secret... more Recent progress in understanding the luminal biochemistry of regulated pancreatic exocrine secretion, including acid-base interactions between acinar and duct cells and pH-dependent processes that regulate membrane trafficking (endocytosis) at the apical plasma membrane, have led to the development of in vitro models of cystic fibrosis in the rat exocrine pancreas. Based on investigations in these model systems, a unifying hypothesis is presented that proposes that pancreatic dysfunction in cystic fibrosis occurs as a result of progressive acidification of the acinar and duct lumen, which leads to secondary defects in (i) apical trafficking of zymogen granule membranes and (ii) solubilization of secretory (pro)en-zymes. By directly acidifying the pH of the acinar lumen in cholescystokinin-stimulated acini, the early cytological findings observed in cystic fibrosis, including (i) massive dilatation of the acinar lumen, (ii) decreased appearance of zymogen granules, (iii) loss of the apical pole of the acinar cell, and (iv) persistent aggregation of secretory (pro)enzymes released into the luminal space, have been reproduced in primary cultures of pancreatic tissue.
Gastroenterology, Feb 1, 1987
Journal of Cell Biology, Nov 1, 1984
24-h intravenous caerulein infusion studies in the rat were combined with in vitro amino acid inc... more 24-h intravenous caerulein infusion studies in the rat were combined with in vitro amino acid incorporation studies followed by high-resolution separation of proteins by twodimensional isoelectric focusing and SDS gel electrophoresis to study the extent to which persistent changes in the biosynthesis of exocrine pancreatic proteins are regulated by cholecystokinin-like peptides. Beginning in the third hour of optimal hormone infusion at 0.25 /~g kg-1 h-1, changes were observed in the synthetic rates of 12 proteins, which progressed over the course of the 24-h study. Based on coordinate response patterns, exocrine proteins could be classified into four distinct groups. Group I (trypsinogen forms 1 and 2) showed progressive increases in synthetic rates reaching a combined 4.3-fold increase over control levels. Group II (amylase forms 1 and 2) showed progressive decreases in synthesis to levels 7.1-and 14.3-fold lower than control levels, respectively. Group III proteins (ribonuclease, chymotrypsinogen forms 1 and 2, procarboxypeptidase forms A and B, and proelastase 1) showed moderate increases in synthesis, 1.4-2.8-fold, and group IV proteins (trypsinogen 3, lipase, proelastase 2, and unidentified proteins 1-4) did not show changes in synthesis with hormone stimulation. Regulation of protein synthesis in response to caerulein infusion was specific for individual isoenzymic forms in the case of both trypsinogen and proelastase. The ratio of biosynthetic rates of trypsinogen forms 1 + 2 to amylase forms 1 + 2 increased from a control value of 0.56 to 24.4 after 24 h of hormonal stimulation (43.5-fold increase). Biosynthetic rates for an unidentified protein (P23) with an Mr = 23,000 and isoelectric point of 6.2 increased 14.2-fold, and the ratio of synthesis of P23 to amylase 2 increased 200-fold during caerulein infusion. During hormone stimulation the anticoordinate response in the synthesis of pancreatic glycosidases (decreased synthesis) and serine protease zymogens (increased synthesis) explain previous observations that showed little change in rates of total protein synthesis under similar conditions.
Cancer, Mar 15, 1981
Nineteen discrete proteins contained in normal human pancreatic juice were separated by two‐dimen... more Nineteen discrete proteins contained in normal human pancreatic juice were separated by two‐dimensional isoelectric focusing/SDS gel electrophoresis. Enzyme or proenzyme activity was assigned to 15 of these proteins. Analysis of pancreatic juice obtained from a patient with pancreatic cancer showed a number of additional proteins not observed in pancreatic juice obtained from normals.
J.F. Bergmann-Verlag eBooks, 1981
Biochimie, Sep 1, 1988
m Cotranslational translocation of exportable proteins across the RER membrane prior to their rel... more m Cotranslational translocation of exportable proteins across the RER membrane prior to their release into the extracellular space has been essentially described by use of canine pancreatic microsomal membranes. Intracisternal segregation of nascent secretory proteins was observed to be irreversible and proteolytic removal of signal sequences resulted in conformationally mature and stable proteins. Structural studies on various translocation peptides from both eukaryotic and prokaryotic preparations showed that many of them have a comparable three-domain organization. A hydrophilic amino-terminal domain is followed by a core region of hydrophobic amino acids and by the region in which the proteolytic cleavage occurs. Membrane components involved in the translocation process namely the signal recognition particle and the SRP receptor as well as the way the vectorial transport mechanism of nascent secretory proteins occurs are also discussed. secretory proteins / translocation peptides / vectorial transport / microsomai membranes / canine pancreas
Nucleic Acids Research, 1991
Previous attempts to obtain full-length double-stranded (ds) cDNA transcripts have relied on deox... more Previous attempts to obtain full-length double-stranded (ds) cDNA transcripts have relied on deoxy cytosine (dC)-deoxy guanine (dG) tailing methods. In this approach first strand cDNA transcripts are tailed with either dC or dG using terminal deoxynucleotidyl transferase (tdt) and second strand cDNA is primed with oligo-dG or-dC, respectively, and synthesized using DNA polymerase I or Klenow fragment (1). Modifications of this method have used cDNA transcripts tailed with dC and plasmid vectors tailed with dG (2, 3). By use of these tailing methods homopolymeric dC-dG regions (10-30 bp) are inserted upstream of cDNA constructs. We have observed (H.Lutcke and G.A.Scheele, unpublished observations) that such dC-dG regions inhibit (a) in vitro transcription of DNA using RNA polymerases (75% inhibition in the case of anionic trypsinogen cDNA), (b) translation of resulting RNA transcripts in wheat germ or reticulocyte lysate protein synthesizing systems (complete inhibition) and (c) sequencing of single stranded (ss) or ds DNA. Synthesis of ds cDNA by self-priming (4) or RNase-H (5) methods avoid the introduction of dC-dG tails but do not preserve the full-length character of cDNA constructs and therefore may not reveal the transcriptional start-site (cap site) or the complete 5' nontranslational sequence. In the novel method described here, a dC homopolymer tail (-15 bases) is added to first cDNA transcripts using tdt. Synthetic oligo ribo-G (rG, 15 mer, synthesized by Peninsula Laboratories, Belmont, CA) is used to prime the synthesis of full-length second strand cDNA. After completion of synthesis of ds cDNA, rG nucleotides in the RNA-DNA heteroduplex tail are removed with RNase H and the exposed ss dC homopolymer tail is removed with T4 DNA polymerase. The resulting full-length ds cDNA transcripts, devoid of C/G tails, are then inserted into any desirable vector, including expression vectors, using adapter-linker assisted ligation. To demonstrate the fidelity of our method we employed dog pancreas poly(A)+ RNA, which contains three well-defined size classes of high-abundance mRNA (-1.7 kb transcripts encode alpha-amylase [55 kDa] and procarboxypeptidase A and B forms [40-44 kDa],-1.0 kb transcripts encode serine protease zymogens [trypsinogen, chymotrypsinogen, and proelastase forms, 23-27 kDa], and-0.6-0.8 kb transcripts encode colipase and prophospholipase A2 forms [10-16 kDa].
European journal of biochemistry, Jun 1, 1990
A genomic library has been constructed in EMBL3 2 phage using high molecular mass DNA isolated fr... more A genomic library has been constructed in EMBL3 2 phage using high molecular mass DNA isolated from canine spleen. A cDNA clone, shown to code for preprophospholipase A2 which is processed to the prosecretory form prior to release from secretory cells, was used to identify a 1-clone which contains the complete phospholipase A2 gene. Restriction enzyme and DNA sequence analysis indicate that the primary transcriptional unit for the phospholipase gene, % 9.0 kb, is organized into four exon sequences. Exon 1 encodes the 5' nontranslated sequence, the ATG initiation codon, and the hydrophobic core of the signal peptide. Exons 2-4 encode regions of the peptide of residues-11 to 43, 43 to 86 and 86 to 124, respectively. The 5' flanking region shows a TATA box at position-29 and multiple CAAT boxes at positions-279,-206,-183 and-159. Regions of the 5' flanking sequence in the canine sequence, from nucleotides-47 to-74 and-91 to-129, show high similarity to similar regions in the human gene. However, an analysis of 400 nucleotides of the 5' flanking sequence in transient expression studies was unable to identify tissue-specific promoter or enhancer sequences. Within 5' nontranslated regions the canine and human genes share a pyrimidine-rich sequence which may be involved in differential regulation of mRNA translation.
Analytical Biochemistry, Dec 1, 1980
ABSTRACT
International journal of pancreatology, 1994
Methods: Sere of 130 patients suffering from pancreatic carcinoma were tested for anti-p53-Ab via... more Methods: Sere of 130 patients suffering from pancreatic carcinoma were tested for anti-p53-Ab via ELISA and immunoblorting using recombinant p53-protein as a target. These patients were classified according to UICC-criteria: stage I: 14%, stage II: 5%, stage III: 48% and ...
Journal of Biological Chemistry, 1985
The path and synchrony of intracellular transport of 12 secretory proteins of the guinea pig exoc... more The path and synchrony of intracellular transport of 12 secretory proteins of the guinea pig exocrine pancreas have been studied in pulse-chase amino acid labeling experiments by quantitative analysis of the individual proteins recovered in subcellular fractions and extracellular samples. Protein fractionation was accomplished by two-dimensional isoelectric focusing/ SDS-gel electrophoresis. Use of a double-label protocol allowed correction of the data on a protein-by-protein basis for leakage and adsorption artifacts which accompany tissue homogenization. All the labeled secretory (pro)enzymes, including their isoenzymic forms, were recovered in rough microsomal, Golgi-enriched and granule fractions during their transport to the cell surface. However, major asynchrony was observed at four levels: exit from the rough endoplasmic reticulum; transit through the Golgi complex; entry into granules; and discharge from the cell. Rapid transport rates were observed for trypsinogen, chymotrypsinogen 2, procarboxypeptidase A2, and lipase 2. Slow transport rates were observed for amylase and procarboxypeptidase B. In the presence of carbamylcholine or cholecystokinin stimulation, the times required for 40% discharge of labeled chymotrypsinogen 2, trypsinogen, amylase, and procarboxypeptidase B were 98, 102, 148, and 180 min, respectively. Transport rates did not correlate with isoelectric point, molecular weight, or the presence of carbohydrate. These data suggest that interactions occur within the rough endoplasmic reticulum, either between secretory (nong1yco)proteins themselves or between such proteins and the cisternal face of the rough endoplasmic reticulum.
Journal of Biological Chemistry, 1975
The discharge and the intracellular transport of the exocrine proteins produced by the guinea pig... more The discharge and the intracellular transport of the exocrine proteins produced by the guinea pig pancreas have been investigated in the in vitro lobule system described by SCHEELE, G. A., AND PALADE, G. E. (1975) J. Biol. Chem. 250, 2660-2670. The inquiry was carried out on a protein-specific basis by using the sodium dodecyl sulfate polyacrylamide gel electrophoretic procedures worked out by TARTAKOFF, A. M., GREENE, L. J., AND PALADE, G. E. (1974) J. Biol; Chem. 249, 7420-7431. The results show that the same protein mixture is discharged by the lobules regardless of whether stimulation is by carbamylcholine (which mimics the action of acetylcholine), caerulein (which mimics the action of pancreozymin), or by 75 mM KCl. When no stimulant is present, the small quantity of discharged protein (resting secretion) also has the same composition. Analysis of successive secretion aliquots collected over a period of 2 hours of continuous timulation by carbamylcholine showed that the composition of the secretory output remains quasi-constant with time; therefore, the exocrine proteins appear to be discharged in parallel and in constant proportions, irrespective of stimulant and time under stimulation (for carbamylcholine). The analysis of a series of zymogen granule fractions prepared from lobules pulse-labeled with radioactive amino acids and chased for periods of 15 to 155 min shows that all major secretory proteins enter the condensing vacuoles of the Golgi complex and appear in zymogen granules in apparent synchrony.