Repetitive self-limited acute pancreatitis induces pancreatic fibrogenesis in the mouse.

The role of repetitive acute injury in the pathogenesis of chronic pancreatitis remains unknown. To determine if repetitive injury induced by pancreatic hyperstimulation would reproduce the characteristic features of human chronic pancreatitis, acute reversible pancreatic injury was induced in mice by twice weekly cerulein treatment, 50 microg/kg/hr x 6 hr, for 10 weeks. Procollagen alpha1(I) mRNA was markedly increased by week 2. Sirius red staining of interstitial collagen demonstrated progressive accumulation of extracellular matrix surrounding acinar units and in interlobular spaces. Atrophy, transdifferentiation of acinar units to ductlike tubular complexes, and dilatation of intraacinar lumina also developed. Electron microscopy demonstrated the presence of stromal cells in areas of fibrosis with morphologic characteristics of pancreatic stellate cells. These findings demonstrate that, in a murine model, repetitive acute injury to the pancreas by hyperstimulation can reproduce the major morphological characteristics of human chronic pancreatitis.

Enzyme replacement in murine mucopolysaccharidosis type VII: neuronal and glial response to beta-glucuronidase requires early initiation of enzyme replacement therapy.

We have previously shown that mucopolysaccharidosis type VII (MPS VII) mice receiving six weekly injections of recombinant beta-glucuronidase from birth had improved cognitive ability and reduced central nervous system lysosomal storage. However, a single beta-glucuronidase injection at 5 wk of age did not correct neuronal storage. We define the age at which central nervous system storage in MPS VII mice becomes resistant to beta-glucuronidase therapy and determine the effect of enzyme on other tissues by comparing the histology of mice begun on therapy at various times after birth. MPS VII mice received injections on the day of birth and then weekly for 5 wk with 16,000U/g beta-glucuronidase had reduced lysosomal storage in brain. The same therapy begun on d 14 of life or thereafter failed to correct neuronal storage, even when treatment was continued for six doses. Glial responsiveness or accessibility to enzyme also depended on early treatment. In contrast, leptomeningeal, osteoblast, and retinal pigment epithelial storage reduction depended on enzyme dose rather than age at initiation of therapy. Fixed tissue macrophage storage was reduced in all treated MPS VII mice, even those receiving a single dose. These observations indicate that fixed tissue macrophages in MPS VII mice remain sensitive to enzyme replacement therapy well into adulthood although neurons are responsive or accessible to enzyme therapy early in life. Because early initiation of enzyme replacement is important to achieve a central nervous system response, these studies emphasize the importance of newborn screening for lysosomal storage diseases so that early treatment can maximize the likelihood of a favorable therapeutic response.

Intracellular localization and unique conserved sequences of three small nucleolar RNAs.

Three human small nucleolar RNAs (snoRNAs), E1, E2 and E3, were reported earlier that have unique sequences, interact directly with unique segments of pre-rRNA in vivo and are encoded in introns of protein genes. In the present report, human and frog E1, E2 and E3 RNAs injected into the cytoplasm of frog oocytes migrated to the nucleus and specifically to the nucleolus. This indicates that the nucleolar and nuclear localization signals of these snoRNAs reside within their evolutionarily conserved segments. Homologs of these snoRNAs from several vertebrates were sequenced and this information was used to develop RNA secondary structure models. These snoRNAs have unique phylogenetically conserved sequences.

Ultrastructural and immunocytochemical evidence for the presence of polarised plasma membrane H(+)-ATPase in two specialised cell types in the chick embryo chorioallantoic membrane.

The chick embryo, confined in the eggshell, has to dispose/buffer the acid generated by its metabolism, as well as to release calcium from the shell which is used for growth. To localise H(+)-ATPase, electron microscope and immunocytochemical studies were conducted on chorioallantoic membranes of 15-17 d chick embryos. Ultrastructural studies of the villus cavity (VC) cells in the chorionic epithelium demonstrated that their apical plasma membrane, juxtaposed with the shell membranes, contains microvilli as well as microplicae which possess 9-10 nm studs at a density of 16,700 particles/micron2, a characteristic feature of the polarised H(+)-ATPase pump. Immunocytochemical staining, using a monoclonal antibody to the 31 kDa subunit of H(+)-ATPase, confirmed the presence of large amounts of the vacuolar H(+)-ATPase in the VC shells with a distribution highly polarised towards the eggshell membranes. Immunoelectron-microscopic localisation studies using a rabbit antiserum to whole bovine H(+)-ATPase and immunogold technique, confirmed the localisation of H(+)-ATPase at the apical microvilli/microplicae as well as in the subapical vesicles. In the allantoic epithelium, the presence of mitochondria-rich (MR) cells was confirmed; it was shown that these cells extend through the full thickness of this epithelium. The MR cells also contained large numbers of 9-10 nm studs, typical of proton secreting cells, in their apical plasma membrane. This was confirmed by immunocytochemical staining which showed abundant localisation of H(+)-ATPase in these cells; this localisation was, however, diffuse rather than apical.(ABSTRACT TRUNCATED AT 250 WORDS)

Degenerative changes in epinephrine tonic vasomotor neurons in Alzheimer's disease.

The C-1 region in the rostral ventral lateral medulla contains mainly epinephrine (Epi) neurons. These neurons are the tonic vasomotor center of the brain. We previously demonstrated changes in the enzymatic activity of phenylethanolamine N-methyltransferase (PNMT) in axon terminals and cell bodies of Epi neurons from the medulla of Alzheimer's disease (AD) brains. In this study, we investigated the perikarya of C-1 neurons for the morphometric, immunohistochemical and histochemical changes that are seen in severely affected regions of Alzheimer brain. The mean areas and size distributions of C-1 neurons from 6 AD and 6 neurologically normal patients were compared using the Wilcoxon rank sum test and Kolmogorov-Smirnov z tests respectively. Additional brain sections from the C-1 region of AD and control individuals were stained with cresyl violet or immunostained with antibodies to the lysosomal hydrolase cathepsin D, Tau-2, Alz-50 and beta-amyloid protein. The average area of C-1 neurons in AD brains was decreased 18.3% (P < 0.001) compared to the areas of the same cell population in age-matched control brains. A shift toward smaller sized C-1 neurons was seen in the AD cases. Nissl stain demonstrated a central chromatolytic appearance in 3.7% of AD neurons sampled. No beta-amyloid deposits were detected histologically or immunocytochemically in the C-1 region of AD brains. Both Tau-2 and Alz-50 immunoreactivity was observed in occasional (1%) C-1 neurons from AD brains but not in controls. A small proportion (30%) of the C-1 neurons showing atrophy displayed increased cathepsin D immunoreactivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Pathology of the ear in murine mucopolysaccharidosis type VII. Morphologic correlates of hearing loss.

BACKGROUND: Patients with mucopolysaccharidosis commonly have hearing impairment but the morphologic alterations in the ear caused by these lysosomal storage diseases are incompletely defined. EXPERIMENTAL DESIGN: We studied a murine model of mucopolysaccharidosis VII with clinical features, including conductive hearing loss and biochemical, and pathologic features similar to those seen in human mucopolysaccharidoses. Gross morphology, radiography, light and electron microscopy were used to define the pathologic alterations in the ear that correlate with auditory dysfunction in mucopolysaccharidosis VII. RESULTS: Cerumen occluded the external auditory canal and there was a severe otitis media. The bone encasing the middle and inner ear was sclerotic and opaque and the temporal bone and the ossicles and their joints contained cells distended by enlarged lysosomes. Hair cell damage and multifocal lysosomal distention in endoneural fibroblasts and spiral ganglion neurons characterized the mucopolysaccharidosis VII cochlea. CONCLUSIONS: The external auditory canal obstruction, otitis media, and ossicle articular alterations in mucopolysaccharidosis VII mice cause a conductive hearing loss. The hair cell damage and neuronal storage may contribute to sensorineural deafness. This model allows investigation of the pathophysiology of auditory dysfunction in mucopolysaccharidosis and the effects of therapies on hearing loss.

Cloning and sequence analysis of murine cytomegalovirus protease and capsid assembly protein genes.

The murine cytomegalovirus UL80 open reading frame was cloned and the predicted amino acid sequence compared with those from other herpesviruses. The open reading frame encodes a fused protease-capsid assembly protein precursor and maintains conserved features including the active site serine, conserved regions CD1 through CD5, the release and maturation sites, and a potential internal cleavage site within the protease. However, the murine cytomegalovirus protease differs in comparison with the other proteases because it contains a unique 15-16 amino acid insertion between CD3 and CD1. The assembly protein sequences are relatively divergent, but they can be arranged into groups defined by herpesvirus subfamily, with each group possessing a conserved motif at its carboxyl terminus.

In Vitro Processing of Aleurain, a Barley Vacuolar Thiol Protease.

Aleurain, originally described from its cDNA as a thiol protease [Rogers, J.C., Dean, D., and Heck, G.R. (1985). Proc. Natl. Acad. Sci. USA 82, 6512-6516], is characterized here as a glycoprotein that is targeted to a distinct vacuolar compartment in aleurone cells. Monospecific antibodies to a bacterial trpE-aleurain fusion protein were used to show that aleurain is made as a 42-kilodalton (kD) proenzyme (proaleurain) that is proteolytically processed in a post-Golgi compartment in two steps to form a 32-kD protein. The first processing step is the discrete loss of 9 kD from proaleurain to yield a 33-kD intermediate that is further processed by the gradual loss of 1 kD resulting in mature 32-kD aleurain. Using proaleurain secreted from Xenopus oocytes as a substrate, we established an in vitro system using aleurone cell extracts that correctly processes proaleurain to a stable protein that is indistinguishable from native barley aleurain as judged by partial digestion with staphylococcal V8 protease. Proaleurain is not capable of self-cleavage in the absence of aleurone cell extracts and mature aleurain appears not to participate in processing in vitro.

Structure of human thrombospondin: complete amino acid sequence derived from cDNA.

Elucidation of the complete amino acid sequence of TSP has suggested plausible explanations for all of the earlier observations on TSP structure and has already suggested new and interesting avenues of investigation aimed at determining the precise function and mechanism of TSP action as a matrix protein. The potential for Ca++-regulated exposure of the RGDA sequence could represent a new level of control for this important recognition system. We have already shown that the binding of TSP to collagen is modulated by the binding of Ca++ to this region of TSP. That is, high Ca++ results in a lower affinity of TSP for collagen, whereas lower Ca++ concentrations enhance the affinity of this interaction. The highly conserved, although short, 15-residue segment, which is nearly identical to region II of the sporozoite malaria protein, may indicate that TSP interacts with a receptor on liver cells, which the malaria parasite uses to gain its initial toehold in the body. If so, this would be another example of pathogenic organisms using a preexisting host recognition system to gain entry to cells where it can multiply. The collagen propeptide-like segment occurs in the collagen-binding domain of TSP and thus may represent the site at which TSP interacts with the collagens. These speculations form the starting point for many exciting lines of experimentation, which will provide us with a better understanding of the role of TSP in hemostasis, in the matrix of a variety of cells, and in development.

Interaction of human thrombospondin with types I-V collagen: direct binding and electron microscopy.

Binding of thrombospondin (TSP) to types I-V collagen was examined by direct binding assays using 125I-TSP and by visualization of rotary-shadowed intermolecular complexes in the electron microscope. The binding of TSP was highest to type V collagen in the absence of Ca, while lower but significant levels of binding were observed to all other collagen types in the presence or absence of Ca. Unlike intact TSP, the trimeric collagen-binding domain of TSP composed of 70-kD chains showed no Ca dependence in its binding to type V collagen. Further evidence for binding of TSP to types I and III collagen was obtained by competition studies in which these soluble collagens effectively inhibited binding of 125I-TSP to immobilized type V collagen. The binding of TSP to type V collagen was inhibited by heparin and fucoidin, both high-affinity ligands of TSP's heparin-binding domain. mAb A6.1, which binds to the 70-kD domain of TSP, is also the best of a panel of anti-TSP mAbs at inhibiting the TSP-collagen interaction. Electron microscopy of rotary-shadowed replicas of TSP-collagen complexes revealed that all five types of collagen examined had a binding site for TSP at one end of the pepsinized, triple helical molecule. The specificity of this site was tested by examining the ability of BSA to form a complex with the end of the pepsinized collagens. Rotary-shadowed replicas revealed a low frequency of apparent BSA-collagen complexes, and histograms of these data showed no evidence for the preferential association of BSA with the end of the collagen molecules. In addition to the specific end site, type V collagen had an internal binding site for TSP located about two-thirds of the distance along the length of the collagen molecule from the end site. The internal binding site for TSP on type V collagen is apparently the site responsible for the higher affinity binding of TSP to that protein observed in direct binding assays. The trimeric 70-kD collagen-binding domain of TSP bound to the same sites on the collagens as did intact TSP.

Monoclonal antibodies that recognize calcium-dependent structures of human thrombospondin. Characterization and mapping of their epitopes.

Monoclonal antibodies (mAbs) raised against reduced and alkylated thrombospondin (TSP) were screened for the ability to react with Ca2+-replete TSP versus EDTA-treated TSP. Two mAbs designated A6.1 and D4.6 were found to react much more strongly with TSP after EDTA treatment. The dissociation constants for these mAbs were measured in 5 mM EDTA and found to be 6 X 10(-10) M for A6.1 and 7 X 10(-9) M for D4.6. Binding to A6.1 was undetectable in the presence of 1 mM Ca2+ while binding of D4.6 occurred with about 100-fold lower affinity. The Ca2+ concentration dependence of A6.1 binding was broad with a midpoint near 50 microM free Ca2+ while that of D4.6 showed a sharp transition below 0.1 microM. Upon dialysis of EDTA-treated TSP into Ca2+ containing buffer, the binding of the mAbs was prevented or decreased, indicating reversibility of the conformational transition induced by the initial removal of Ca2+ . Mg2+ can compete with the Ca2+ binding sites involved in mAb binding, but TSP dialyzed from Ca2+ into Mg2+ binds the two mAbs as well as EDTA-treated TSP, indicating that Mg2+ cannot maintain the Ca2+-replete structure of TSP. The proteolytic fragments of TSP with which the two mAbs react were determined by probing Western blots of digests of TSP with the mAbs. A6.1 reacts with the 70-kDa fragment generated by chymotrypsin in EDTA which contains the interchain disulfide bonds of TSP and the binding site(s) for type V collagen (Mumby, S. M., Raugi, G. J., and Bornstein, P. (1984) J. Cell Biol. 98, 646-652). D4.6 reacts with fragments of 140 and 120 kDa found in digests of Ca2+-replete TSP which are absent from digests in EDTA. Electron microscopy of rotary shadowed, carbon-coated replicas of TSP mAb complexes confirms the Ca2+ sensitivity of mAb binding and has been used to localize the epitopes for both mAbs on the three-dimensional structure of TSP.

Mapping of epitopes for monoclonal antibodies against human platelet thrombospondin with electron microscopy and high sensitivity amino acid sequencing.

A panel of monoclonal antibodies (Mab's) has been raised against human platelet thrombospondin (TSP). One Mab, designated A2.5, inhibits the hemagglutinating activity of TSP and immunoprecipitates the NH2 terminal 25 kD heparin binding domain of TSP (Dixit, V.M., D. M. Haverstick, K. M. O'Rourke, S. W. Hennessy, G. A. Grant, S. A. Santoro, and W. A. Frazier, 1985, Biochemistry, in press). Another Mab, C6.7, blocks the thrombin-stimulated aggregation of live platelets and immunoprecipitates an 18-kD fragment distinct from the heparin binding domain (Dixit, V. M., D. M. Haverstick, K. M. O'Rourke, S. W. Hennessy, G. A. Grant, S. A. Santoro, and W. A. Frazier, 1985, Proc. Natl. Acad. Sci. 82: 3472-3476). To determine the relative locations of the epitopes for these Mabs in the three-dimensional structure of TSP, we have examined TSP-Mab complexes by electron microscopy of rotary-shadowed proteins. The TSP molecule is composed of three 180-kD subunits, each of which consists of a small globular domain (approximately 8 nm diam) and a larger globular domain (approximately 16 nm diam) connected by a thin, flexible strand. The subunit interaction site is on the thin connecting strands, nearer the small globular domains. Mab A2.5 binds to the cluster of three small domains, indicating that this region contains the heparin binding domain and thus represents the NH2 termini of the TSP peptide chains. Mab C6.7 binds to the large globular domains on the side opposite the point at which the connecting strand enters the domain, essentially the maximum possible distance from the A2.5 epitope. Using high sensitivity automated NH2 terminal sequencing of TSP chymotryptic peptides we have ordered these fragments within the TSP peptide chain and have confirmed that the epitope for C6.7 in fact lies near the extreme COOH terminus of the peptide chain. In combination with other data, we have been able to construct a map of the linear order of the identified domains of TSP that indicates that to a large extent, the domains are arranged co-linearly with the peptide chain.

Association of the cyclic AMP chemotaxis receptor with the detergent-insoluble cytoskeleton of Dictyostelium discoideum.

Treatment of 6-h differentiated Dictyostelium discoideum cells with the nonionic detergent Triton X-100 dissolves away membranes and soluble components, as judged by marker enzyme distributions, leaving intact a cytoskeletal residue that contains approximately 10% of the cell protein and 50% of the actin. Nitrobenzooxadiazo-phallacidin staining for F-actin and electron microscopy of detergent-extracted whole-mounts indicate that the cytoskeletons retain the size and shape of intact cells and contain F-actin in cortical meshworks. The cytoskeletons contain little if any remaining membrane material by morphological criteria, and the plasma membrane enzymes cyclic nucleotide phosphodiesterase and alkaline phosphatase are absent from the insoluble residue, which retains only 15% of the membrane concanavalin A-binding glycoproteins. This detergent-insoluble residue retains a specific [3H]cAMP-binding site with the nucleotide specificity, rapid kinetics and approximate affinity of the cAMP receptor on intact cells. Upon detergent extraction of cells, the number of cAMP-binding sites increases 20-70%. The binding site is attached to the insoluble residue whether or not the cAMP receptor is occupied at the time of detergent addition. The pH dependence for recovery of the insoluble cAMP-binding site is much sharper than that on intact cells or membranes with an optimum at pH 6.1. Conditions of pH and ionic composition that lead to disruption of the cytoskeleton upon detergent treatment also result in the loss of cAMP binding. During differentiation, the detergent-insoluble cAMP binding increases in parallel with cell surface cAMP receptors and chemotaxis to cAMP.

The mode of termination of the hypothalamic projection to the dentate gyrus: an EM autoradiographic study.

It has been suggested, on electrophysiological grounds, that the projection from the hypothalamus to the dentate gyrus constitutes a long-axon, monosynaptic, inhibitory pathway. To clarify the mode of termination of this projection we have examined in EM autoradiographs the distribution and form of labeled synapses in the rat dentate gyrus following the injection of [3H]proline of high specific activity into the supramammillary region of the hypothalamus. As suggested by previous light microscopic studies the hypothalamo-dentate projection has been found to terminate in a narrow zone that extends from about the superficial half of the layer of granule cell somata to the inner one-fifth of the overlying molecular layer. Within this zone more than 80% of the silver grains observed were associated with vesicle-containing profiles, most of which could be identified as forming Type I, asymmetric synapses upon large dendritic shafts. A smaller number of labeled synapses was found upon granule cell somata or upon the sessile dendritic spines that occur on the proximal parts of the granule cell dendrites. Since all of the labeled synapses showed distinct asymmetric membrane specializations and contained spheroidal vesicles it is difficult to reconcile these morphological findings with the view that the hypothalamic afferents to the dentate granule cells are inhibitory.

Physiological and morphological evidence for coupling in mouse salivary gland acinar cells.

Three experimental techniques were employed to examine coupling between acinar cells of the mouse salivary gland. Passage of DC current pulses via intracellular microelectrodes between neighboring cells showed that small ions could be directly passed from one cell to another. Intracellular iontophoresis of the dye Lucifer Yellow CH into a single cell indicated that small molecules could spread by means of intercellular cytoplasmic bridges througout an acinus and, occasionally, into cells of adjacent acini. Freeze-fracture replicas of acinar cell membranes indicated the presence of gap junctions which were correlated with both electrical and dye coupling experiments. Suggestions are made for the function of direct intercellular exchange in salivary secretory cells. The role of electrical coupling in coordination of the activity of different secretory cell types is discussed as one possible function.