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 two-dimensional 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 microgram 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.
Rates of synthesis of 16 individual pancreatic exocrine proteins; tissue concentrations of amylase, trypsinogen, and chymotrypsinogen; and morphological assessment of pancreatic acinar cells were studied in the exocrine pancreas in response to inverse changes in protein and carbohydrate in the diet, administered for 12 days. Two distinct patterns of response were observed. During adaptation to diets containing normal protein (22%) or increased levels of protein (30, 45, 64, and 82% protein) and correspondingly decreased levels of carbohydrate, amylase and the majority of protease zymogens were synthesized in direct proportion to the nutritional substrates carbohydrate and protein, respectively, in the diet. With increases in dietary protein, anticoordinate patterns of response in the synthesis of exocrine isoenzymes were observed: 0.4- to 2.0-fold increases in trypsinogen forms 1 and 2, chymotrypsinogen forms 1 and 2, proelastase 1, and procarboxypeptidases A and B; 5- to 7-fold decreases in amylase forms 1 and 2; and insignificant changes in trypsinogen 3, proelastase 2, lipase, and ribonuclease. During adaptation to diets containing normal protein (22%) or decreased levels of protein (0 or 10% protein) and correspondingly increased levels of carbohydrate, amylase and the majority of protease zymogens were synthesized in inverse proportion to nutritional substrates in the diet.(ABSTRACT TRUNCATED AT 250 WORDS)
In KB cells productively infected with adenovirus type 2, alkali-stable >100S and 40-1OOS viral DNAs are synthesized starting 2-4 hr postinfection, i.e., before unit length (34 S) In previous publications (1, 2) an alkali-stable, high-molecular-weight (50-90 S) form of viral DNA was described in productively infected cells, and evidence was presented that this DNA represented viral sequences integrated into cellular DNA. Integrated viral sequences in productively infected cells were also described in other virus systems (3). With adenovirus, integrated viral genomes were previously found in abortively infected cells (4-6), and it was shown that in adenovirus-transformed cells specific fragments of the viral genome persisted (7), probably in an integrated form (8).In for 30-60 min. The extract was then made 0.5 M in NaOH with 6 M NaOH and was finally layered on top of an alkaline sucrose density gradient. In other experiments the Ad2-infected or mock-infected KB cells were first incubated at 370 with 250 ,ug of Pronase or proteinase K per ml for 1 or 18 hr at neutral pH, and incubation was subsequently continued in 0.5 M NaOH at 40 for 18 hr (in the case of 1 hr incubation with Pronase) or the lysate was layered on an alkaline sucrose gradient immediately after adding alkali (in the case of 18 hr incubation with Pronase).Resedimentation (14), and the peak of the cellular DNA was isolated on a neutral sucrose gradient (Fig. 7a). Samples of 0.5-1.0 ml of cellular DNA (1-2 jig) were dialyzed into 0.01 M Tris-HCl, pH 7.5, 0.001 M EDTA. Subsequently, 2-5 Mg of 14C-labeled Ad2 marker DNA was added, and the DNA was cleaved with the EcoRI restriction endonuclease under conditions outlined elsewhere (2, 12). The digestion products were analyzed by electrophoresis on 13 cm long, cylindrical, 1.5% polyacrylamide-0.8% agarose gels at 40 V for 10-12 hr (12); the gels were cut into 1 mm slices; and the DNA was eluted from each slice by ultrasonic treatment (microtip) for 3 min in 1.0 ml of 0.45 M NaCl, 0.045 M sodium citrate, pH 7.6 (3 X SSC) (16). The 3H-and "4C-labeled DNA from each slice was hybridized to unlabeled Ad2 DNA fixed to nitrocellu-
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