For a number of years considerable interest has been centered in the role of nucleic acids in cellular processes. Recently desoxyribose nucleic acid (DNA) has been shown to possess interesting characteristics that have led several workers to consider it an essential component of the gene.'-3 DNA is probably a universal constituent of plant and animal nuclei. Its low turnover rate to radioactive phosphorus and nitrogen, in non-dividing tissues, is evidence for a chemical stability considerably greater than that of other cell components.4 6 Moreover, recent analytic data on the actual amounts of DNA within nuclei have suggested that it possesses a quantitative stability as well.Computations on the amount of DNA per nucleus have been made in two ways. Chemical analyses of large numbers of cells, with the number present estimated by sample counts, have given the average amount of VOL. 36, 1950 643

. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 6646 -6651). Here we report that these PC2 mutant mice have elevated circulating proinsulin, comprising 60% of immunoreactive insulin-like components. Acid ethanol extractable proinsulin from pancreas is also significantly elevated, representing about 35% of total immunoreactive insulin-like components. These increased amounts of proinsulin are mainly stored in secretory granules, giving rise to an altered appearance on electron microscopy. In pulsechase experiments, the mutant islets incorporate lesser amounts of isotopic amino acids into insulin-related components than normal islets. In both wild-type and mutant islets, proinsulin I was processed more rapidly to insulin, reflecting the preference of both PC2 and PC3 for substrates having a basic amino acid positioned four residues upstream of the cleavage site. The overall halftime for the conversion of proinsulin to insulin is increased approximately 3-fold in the mutant islets and is associated with a 4 -5-fold greater elevation of des-31,32 proinsulin, an intermediate that is formed by the preferential cleavage of proinsulin at the B chain-C-peptide junction by PC3 and is C-terminally processed to remove Arg 31 and Arg 32 by carboxypeptidase E. The constitutive release of newly synthesized proinsulin from both mutant and wild-type islets during the first 1-2 h of chase was normal (<2% of total). These results demonstrate that PC2 plays an essential role in proinsulin processing in vivo, but is quantitatively less important in this regard than PC3, and that its absence does not influence the efficient sorting of proinsulin into the regulated secretory pathway.The efficient production of insulin in the pancreatic beta cell requires that it be processed as completely as possible from its precursor form, proinsulin, into fully active hormone before it is stored and secreted. Conversion of proinsulin to insulin involves cleavages at both junctions of the connecting segment that links the B and A chains in the prohormone to liberate insulin and C-peptide (1). The recognition sites for cleavage by the converting endoproteases PC3 1 and PC2 include minimally six residues (2) at the B chain-C-peptide junction (residues 29 through 35) and at the C-peptide-A chain junction (residues 62 through 68) of proinsulin. Initial cleavage occurs within these sites between residues 32 and 33 and residues 65 and 66, respectively, and the resulting C-terminal basic residue pairs Arg 31 -Arg 32 and Lys 64 -Arg 65 are then removed by the action of carboxypeptidase E to complete the formation of the native beta cell products. In the islet beta cells, both PC2 and PC3 are present in the secretory granules, and these two enzymes are believed to cooperate in processing proinsulin (3-5). Earlier studies from this laboratory showed that PC3 cleaves preferentially at the B-C junction, while PC2 prefers the C-A junction (4). This was in keeping with the assignment of these two convertases as the Type 1 and Type 2 calcium-dependent proinsulin proc...

Renal tubular fluid is supersaturated with calcium and oxalate ions, which can nucleate to form crystals of calcium oxalate monohydrate (COM), the most abundant constituent of kidney stones. However, the mechanisms by which nascent crystals are retained in the nephron and then grow into kidney stones are unclear. An interaction of COM crystals with the surface of renal epithelial cells could be a critical initiating event in nephrolithiasis. To investigate this possibility we used cultures of monkey kidney epithelial cells (BSC-1 line) as a model system and found that [14CJCOM crystals bound to the cell surface within seconds. Scanning electron microscopy revealed that crystals bind first to apical microvilli, which subsequently migrate over the crystalline surface. When visualized by transmission electron microscopy, intracellular crystals were located within vesicles. Cytoskeletal responses to crystal uptake were sought by immunofuorescence microscopy, which revealed concentration of (3). Before use, crystals were sterilized by heating to 180TC overnight and then suspended in distilled water to form a slurry from which they were added to the culture medium (3). X-ray crystallography, performed by S. Deganello (University of Chicago), demonstrated that heating did not alter the structure of COM crystals.[14C]COM crystals (10-300 Mig/ml; 2.4-70.8 pg/cm2 cell surface) were added to high-density, quiescent cultures in 60-mm dishes (Nunc) to define the kinetics of association between a cell and crystal. After a specified period, the medium was aspirated, and the monolayer was washed three times with phosphate-buffered saline (PBS; 5 ml). Each culture was inspected under a microscope and the number of cells with adherent crystals was counted in five separate fields. Subsequently, the cell monolayer with adherent crystals was scraped directly into a scintillation vial containing 6 M HC (0.5 ml), 4.5 ml of Ecoscint (National Diagnostics) was added, and the amount of radioactivity was measured. To investigate the effect of crystal exposure for up to 15 days on renal epithelial cells, COM crystals (50 pg/ml) were added to near-confluent cultures of BSC-1 cells (106 cells per 60-mm dish) containing 0.5% calf serum. Every 4 days thereafter, the medium was aspirated and replaced with fresh medium containing 0.5% calf serum with no additional crystals. At 1, 8, and 15 days after addition of crystals, the medium was aspirated, and a solution of crystalline trypsin was used to detach the cells, which were then inspected under a microscope as described (4). The total number of cells in each culture was counted with a hemocytometer, and 100 cells from each culture were scored for the presence of internalized crystals.Abbreviations: COM, calcium oxalate monohydrate; TEM, transmission electron microscopy; SEM, scanning electron microscopy. tTo whom reprint requests should be addressed. 6987The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "adv...

The fine structure and cytochemistry of the extremely large RNA puffs, or Balbiani rings, in salivary gland nuclei of midge, Chironomus thummi, larvae have been investigated. The Balbiani rings are composed of a diffuse mass of electron-opaque 400 to 500 A granules, short threads about 180 to 220 A in diameter and associated fine chromatin fibrils. These components appear to be organized into brushlike elements which form the ring. Electron microscope cytochemistry has shown that the granules and short threads contain RNA. After ribonuclease digestion, only 50 to 100 A chromatin fibrils were apparent in the Balbiani ring, and the granules were no longer demonstrable. Deoxyribonuclease digestion had no apparent effect on these structures. Observations indicate that the granules are formed from the short threads and released into the nucleoplasm in which they are evenly distributed. At the nuclear envelope, many granules have been observed partially or completely within the nuclear pores. These granules become elongated and are shown to penetrate the center of the pore in a rodlike form, about 200 A in diameter. The Balbiani ring granules are not normally visible within the cytoplasm adjacent to the nuclear envelope, but have been rarely found in this region. It is suggested that the granules represent the product of the Balbiani ring, possibly a messenger RNA bound to protein, and that they regularly pass into the cytoplasm through a narrow central channel in the pores of the nuclear envelope.

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Adhesion of microcrystals to the apical surface of renal tubular cells could be a critical step in the formation of kidney stones. The role of membrane surface charge as a determinant of the interaction between renal epithelial cells (BSC-1 line) and the most common crystal in kidney stones, calcium oxalate monohydrate (COM), was studied in a tissue culture model system. Adhesion of COM crystals to cells was blocked by cationized ferritin. Other cations that bind to cells including cetylpyridinium chloride and polylysine, as well as cationic dyes such as Alcian blue, also inhibited adhesion of COM crystals, but not all polycations shared this effect. Specific lectins including Triticum vulgaris (wheat germ agglutinin) blocked crystal binding to the cells. Furthermore, treatment of cells with neuraminidase inhibited binding of crystals. Therefore, anionic cell surface sialic acid residues appear to function as COM crystal receptors that can be blocked by specific cations or lectins. In vivo, alterations in the structure, function, quantity, or availability of these anionic cell surface molecules could lead to crystal retention and formation of renal calculi.

A coding mutation in the human insulin gene (His-B10 -* Asp) is associated with familial hyperproinsulinemia. To model this syndrome, we have produced transgenic mice that express high levels of the mutant prohormone in their islets of Langerhans. Strain 24-6 mice, containing about 100 copies of the mutant gene, were normoglycemic but had marked increases of serum human proinsulin immunoreactive components. Biosynthetic studies on isolated islets revealed that =65% of the proinsulin synthesized in these mice was the human mutant form. Unlike the normal endogenous mouse proinsulin, which was almost exclusively handled via a regulated secretory pathway, up to 15% of the human [Aspl'proinsulin was rapidly secreted after synthesis via an unregulated or constitutive pathway, and =20% was degraded within the islet cells. The secreted human [Aspl'lproinsulin was not processed proteolytically. However, the processing ofthe normal mouse and human mutant proinsulins within the islets from transgenic mice was not sigiicantly impaired. These findings suggest that the hyperproinsulinemia of the patients is the result of the continuous secretion of unprocessed mutant prohormone from the islets via this alternative unregulated pathway.In earlier studies we defined a mutation at position B10 (His Asp) in the coding region of an insulin allele that cosegregated with affected members of a kindred with hyperproinsulinemia (1, 2). The predicted amino acid substitution does not involve the dibasic processing sites for conversion of proinsulin to insulin, as has been found in two other families with hyperproinsulinemia (3, 4). However, replacement of the histidine residue at position B10 with aspartic acid would be expected to inhibit the association of this proinsulin into hexamers in the presence of zinc, inasmuch as coordination of zinc by this residue stabilizes insulin hexamers (5). The isoelectric point of the mutant (pro)insulin is reduced, while its receptor binding affinity and biological potency are increased by about 4.5-fold (6). Although none of these changes in the properties ofthe [Asplg0proinsulin would necessarily be expected to alter its proteolytic processing to insulin, the clinical phenotype of this mutation is one of greatly increased circulating proinsulin levels with mild carbohydrate intolerance (1).To explore the mechanism leading to the hypersecretion of proinsulin in these patients, we have introduced the mutant human insulin gene into mouse embryos to create transgenic animals in which the abnormal proinsulin is expressed in the islets of Langerhans along with the two normal mouse proinsulins. The results indicate that the mutant proinsulin is selectively secreted in increased amounts via an unregulated pathway, bypassing proteolytic conversion and leading to hyperproinsulinemia. METHODSProduction of Transgenic Mice. A 12.7-kilobase (kb) HindIII DNA fragment containing the human [AspBlOIinsulin gene was isolated from recombinant clone AMD41 by electrophoretic elution from an agarose gel (7). Ferti...

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