Construction of a screening system for selecting lysozyme mutants unable to form a stable structure from random mutants.

To collect folding information, we screened and analyzed the recombinant hen lysozyme mutants which were not secreted from yeast. As model mutants, Leu8Arg, Ala10Gly, and Met12Arg were prepared by site-directed mutagenesis and analyzed as to whether they were secreted from yeast or not. Consequently, Ala10Gly was found to be secreted from yeast, but Leu8Arg and Met12Arg were not. Next, these mutants were expressed in Escherichia coli and refolded in vitro. As a result, Ala10Gly folded as the wild-type did. Leu8Arg efficiently refolded in renaturation buffer containing glycerol. Met12Arg did not refold even in the presence of glycerol. These results show that the Ala10Gly mutation does not affect folding or stability, that Leu8Arg is too unstable to be secreted from yeast, and that Met12Arg may be very unstable or the mutation affects the folding pathway. We screened the mutants that were not secreted by yeast from a randomly mutated lysozyme library, and obtained Asp18His/Leu25Arg and Ala42Val/Ser50Ile/Leu56Gln. These two mutants were expressed in E. coli and then refolded in the presence of urea or glycerol. These mutants were refolded only in the presence of glycerol. Each single mutant of Asp18His/Leu25Arg and Ala42Val/Ser50Ile/Leu56Gln was independently prepared and folded in vitro. The results showed that Leu25Arg and Leu56Gln were the dominant mutations, respectively, which cause destabilization. These results show that the mutant lysozymes which were not secreted from yeast may be unstable or have a defect in the folding pathway. Thus, we established a screening system for selecting mutants which are unable to form a stable structure from random mutants.

Evaluation of cytology and tumor markers of pure pancreatic juice for the diagnosis of pancreatic cancer at early stages.

To evaluate the value of cytology of pure pancreatic juice (PPJ) and tumor marker determination in PPJ and serum for the diagnosis of early pancreatic cancer (EPC), PPJ was obtained endoscopically from 16 patients with EPC (< 20 mm, confined to pancreas), 16 patients with chronic pancreatitis (CP), and 20 controls. Cutoff levels of CEA, CA19-9, and POA in PPJ were set from ROC curves at 40 ng/ml, 7,500 U/ml, and 1.5 U/ml, respectively. For the differentiation of EPC from CP, the sensitivity of CEA, CA19-9, and POA was 71.4, 42.9, and 50%, respectively; specificity was 93.3, 46.7, and 80%, respectively; and diagnostic accuracy was 82.8, 44.8, and 65.5%, respectively. Determination of serum tumor markers was useless. Sensitivity of cytology was 75%, specificity was 93.8%, and diagnostic accuracy was 84.4%. Combined cytology and CEA determination in PPJ increased the diagnostic accuracy to 93.1%. The combination was useful in supporting and supplementing endoscopic retrograde cholangiopancreatography (ERCP) findings for the correct diagnosis in 11 and 4 patients, respectively, with EPC; in one patient EPC was correctly diagnosed on ERCP findings alone. One of 16 patients with CP showed false-positive results. We conclude that cytology and CEA determination in PPJ with ERCP is a useful combination for the diagnosis of pancreatic cancer even in early stages.

Prognosis and prognostic factors in chronic pancreatitis.

To evaluate the prognosis and prognostic factors of chronic pancreatitis, 84 patients with alcoholic chronic pancreatitis and 51 with nonalcoholic chronic pancreatitis have been followed for 1-21 years (average of 7.1 years). The follow-up period was defined as the period from diagnosis to death in those who died and to the present in those still alive. The following conclusions were obtained. (1) Patients with alcoholic chronic pancreatitis showed a significantly higher mortality rate (26.2%) and cancer death rate (8.3%) than the age- and sex-matched population. In patients with nonalcoholic chronic pancreatitis, however, the difference did not reach the level of statistical significance, although both rates tended to be higher. (2) Patients with alcoholic chronic pancreatitis showed a significantly poorer prognosis than those with nonalcoholic chronic pancreatitis. (3) Frequent causes of death in chronic pancreatitis were cancer (11 cases) and diabetes-associated conditions (renal failure in three cases, intractable pneumonia in one, hypoglycemic shock in two, and myocardial infarction in two). Death directly from pancreatitis was observed in four. (4) Unfavorable prognostic factors in alcoholic chronic pancreatitis included heavy drinking, continuance of drinking after diagnosis, smoking, insulin-dependent diabetes, and an advanced age. In nonalcoholic chronic pancreatitis, however, patients' age was the only significant prognostic factor; smoking did not reach the level of statistical significance, although it tended to lead to a poorer prognosis.

Clinical course and prognosis of chronic pancreatitis.

Course and prognosis of 125 patients with chronic pancreatitis (CP) were evaluated. Follow-up period ranged from 1-20 years with a median of 6.3 years. The following conclusions were obtained. Recent increase of CP in our clinics was ascribed to alcoholic CP and idiopathic CP in the aged. Of 106 patients with pain, 74 showed improvement or disappearance of pain. Drinking habit and observation period were the main factors determining the rate of pain relief. Serial endoscopic retrograde pancreatography (ERP) showed aggravation in 17/47 patients, cholecystokinin-pancreozymin (CCK-PZ) secretin test in 4/40 patients, and oral glucose tolerance test (OGTT) in 7/25 patients. Exocrine function showed improvement in five patients, whereas endocrine function showed none. Improvement or aggravation of exocrine function was closely related to drinking habit. Main complications included 15 cases of peptic ulcer, 19 of pancreatic pseudocyst, and 15 of bile duct stenosis. Twenty-six patients died, often due to malignant neoplasms and diabetic complications. Those who continued drinking as much showed a lower survival rate than those who discontinued or decreased alcohol intake. The socioeconomic status deteriorated often due to pain or alcoholism. Three patients had to degrade jobs and six fell into inactive social life.

Histochemical studies on protein plugs obtained by endoscopic retrograde catheterization of the papilla.

In order to elucidate mechanisms of protein plug formation, histochemical studies were performed on aggregates and protein plugs present in pancreatic juice. Pancreatic juice was obtained from three control subjects and five patients with chronic pancreatitis through endoscopic retrograde catheterization of the papilla. Specimens for staining were prepared in two ways: (1) fixed with 10 per cent formaldehyde, embedded in paraffin and sectioned, and (2) placed on slide glass and fixed with isopropylalcohol. Staining included hematoxylin-eosin, periodic-acid Schiff, von Kossa, alcian blue, toluidine blue and double staining with PAS and AB. The process of protein plug formation can be as follows: (1) a prerequisite for aggregate formation, consisting of clusters of desquamated epithelial cells, highly concentrated sulfated acidic mucopolysaccharide and neutral mucopolysaccharide, (2) formation of aggregates in which epithelial cells and amorphous substance are interlaced with developing fine reticular substance, (3) enlargement of aggregates by fusion with adjacent aggregates through bridging action of the reticular substance sprouting, like prickles, from their surface, and (4) "maturity" of aggregates, taking a three-dimensional form which result in a spherical, spheroidal or cylindrical protein plug.

The calcium concentration in human pure pancreatic juice in chronic pancreatitis.

Calcium concentration in human pure pancreatic juice was determined during wash-out period, secretin stimulation and pancreozymin stimulation. Specimens were collected by endoscopic retrograde catheterization of the papilla at one minute intervals for 20 minutes after intravenous injection of secretin (Eisai, 1 U/kg) and for 10 minutes after pancreozymin injection (Boots, 1 U/kg). The determination was also made in duodenal juice obtained during the ordinary pancreozymin-secretin test. Calcium concentration in duodenal juice was significantly raised in patients with chronic pancreatitis (suspected, definite or calcifying), confirming the results of previous investigators. Calcium concentration and calcium per mg of protein in human pure pancreatic juice were significantly raised in patients with chronic pancreatitis (suspected, definite or calcifying). Calcium per mg of protein in normal human pure pancreatic juice was quite similar to that reported in zymogen granules of the guinea-pig pancreas. Calcium output in human pure pancreatic juice was also significantly raised in patients with suspected chronic pancreatitis; it was within normal limits in patients with definite or calcifying chronic pancreatitis due to decreased volume output. Significance of these findings were discussed in relation to pathogenesis and early detection of chronic pancreatitis.

The hexosamine concentration and output in human pure pancreatic juice in chronic pancreatitis.

Hexosamine concentration in human pure pancreatic juice was determined during wash-out phase and secretin stimulation phase. Specimens were collected by endoscopic retrograde catheterization of the papilla at one minute intervals for 20 minutes after intravenous injection of secretin (Eisai, 1 U/kg) and for 10 minutes after pancreozymin injection (Boots, 1 U/kg). In suspected and established chronic pancreatitis (calcifying or non-calcifying), hexosamine concentration was significantly raised during both wash-out phase and secretin phase. Hexosamine output was significantly raised in suspected chronic pancreatitis and non-calcifying chronic pancreatitis during both wash-out phase and secretin phase; in calcifying chronic pancreatitis, no significant increase in hexosamine output was noted during both phases because of decreased secretory volume. Significance of these findings was discussed in relation to the pathogenesis of chronic pancreatitis.