Hereditary pancreatitis (HP) is a rare, early-onset genetic disorder characterized by epigastric pain and often more serious complications. We now report that an Arg-His substitution at residue 117 of the cationic trypsinogen gene is associated with the HP phenotype. This mutation was observed in all HP affected individuals and obligate carriers from five kindreds, but not in individuals who married into the families nor in 140 unrelated individuals. X-ray crystal structure analysis, molecular modelling, and protein digest data indicate that the Arg 117 residue is a trypsin-sensitive site. Cleavage at this site is probably part of a fail-safe mechanism by which trypsin, which is activated within the pancreas, may be inactivated; loss of this cleavage site would permit autodigestion resulting in pancreatitis.

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As part of a genetic analysis of the biogenesis and function of the vacuole (lysosome) in the yeast Saccharomyces cerevisiae, assays of vacuolar pH were developed and used to identify mutants defective in vacuolar acidification.Vacuoles were labeled with 6-carboxyfluorescein with the membrane-permeant precursor 6-carboxyfluorescein diacetate. Dual-excitation flow cytometry was used to calibrate the pH-dependence of 6-carboxyfluorescein fluorescence in vivo.Vacuoles in wild-type yeast were mildly acidic, pH 6.2, in cells grown under several different conditions. Cultures labeled with 6-carboxyfluorescein were screened by fluorescence-ratio microscopy to detect mutants that had defects related to vacuolar acidification. A recessive nuclear mutation, vphl-1, caused an abnormally high vacuolar pH of 6.9, as assayed by flow cytometry, and eliminated vacuolar uptake of the weak base quinacrine. Acidification in a pepl2::LEU2 mutant appeared defective by fluorescence-ratio microscopy and qulnacrineuptake assays, but the vacuolar pH in thepepl2::LEU2 mutant was nearly normal (pH 6.3) in flow cytometric assays.Vacuoles in Saccharomyces cerevisiae are analogous to lysosomes of other organisms. Both types of organelles contain similar sets of proteases and other hydrolytic enzymes (1, 2), and both are energized and acidified by similar membrane-bound, proton-translocating ATPases (H+-ATPase) (3-6). Vacuolar proteases are required for intracellular protein turnover induced by nitrogen starvation (7). Apart from that typically "lysosomal" activity, a variety of additional functions have been ascribed to the vacuole, including maintenance of cytosolic amino acid homeostasis (8, 9), regulation of cellular calcium metabolism (8, 10), and others (11). Genetic analysis of protease-deficient mutants has been instrumental in delimiting the physiological significance of the vacuolar proteases (7 METHODSStrains and Culture Conditions. The wild-type strain was the diploid formed by mating strains X2180-1A and X2180-1B. Mutant strains and genotypes were BJ4895, a/a vphl-J/ vphl-1 trpl/+ leu2/+ +/ura3-52 and BJ4984, a/a pepl2:: LEU2/pepl2::LEU2 ura3-52/ura3-52 leu2-1/leu2-1 hisl1+ ade6/+. Growth media YPD and SC have been described (12). Cells in the logarithmic-growth phase were prepared by overnight growth in YPD or SC (10 ml) at 30'C in roller tube cultures. Culture densities were maintained below 2 x 107 cell per ml by dilution with YPD or SC as required.Labeling with Fluorescent Dyes. Medium for labeling with 6-CF diacetate (6-CFDA; C1362; Molecular Probes) was prepared by adding 50 mM citric acid to YPD, adjusting the pH to 3.0 with 12 M HCl, autoclaving, and then filtering (0.2 gm membrane filter); 6-CFDA was added to the medium immediately before use by dilution from 5 mM stock solutions in dimethyl sulfoxide; unless otherwise noted, the final 6-CFDA concentration was 5 1LM. Logarithmic-phase cells were harvested by membrane filtration, resuspended at 2 X 107 cell per ml in the labeling medium, and incubated with shakin...

Mapping of an autosomal dominant gene for Dupuytren's contracture to chromosome 16q in a Swedish family.Dupuytren's contracture (DC) (OMIM 126900) is the most common connective tissue disease of mankind and has both heritable and sporadic forms. The inherited form is most frequently observed among the xanthochroi peoples of Northern Europe where its most common manifestations are thickening of the palmar fascia and contracture of the fingers. We ascertained a five-generation Swedish family in which DC is inherited in an autosomal dominant manner with high, but incomplete, penetrance by the end of the fifth decade. Blood was collected from all affected and informative unaffected family members for the performance of a genome-wide scan at a resolution of approximately 8 cM for all autosomes. Linkage was established to a single 6 cM region between markers D16S419 and D16S3032 on chromosome 16. A maximal two-point logarithm of odds (LOD) score of 3.18 was achieved at microsatellite marker D16S415 with four other markers in the region producing LODs of >1.5.