Pharmacological inhibition of PAR2 with the pepducin P2pal-18S protects mice against acute experimental biliary pancreatitis.

Pancreatic acinar cells express proteinase-activated receptor-2 (PAR2) that is activated by trypsin-like serine proteases and has been shown to exert model-specific effects on the severity of experimental pancreatitis, i.e., PAR2(-/-) mice are protected from experimental acute biliary pancreatitis but develop more severe secretagogue-induced pancreatitis. P2pal-18S is a novel pepducin lipopeptide that targets and inhibits PAR2. In studies monitoring PAR2-stimulated intracellular Ca(2+) concentration changes, we show that P2pal-18S is a full PAR2 inhibitor in acinar cells. Our in vivo studies show that P2pal-18S significantly reduces the severity of experimental biliary pancreatitis induced by retrograde intraductal bile acid infusion, which mimics injury induced by endoscopic retrograde cholangiopancreatography (ERCP). This reduction in pancreatitis severity is observed when the pepducin is given before or 2 h after bile acid infusion but not when it is given 5 h after bile acid infusion. Conversely, P2pal-18S increases the severity of secretagogue-induced pancreatitis. In vitro studies indicate that P2pal-18S protects acinar cells against bile acid-induced injury/death, but it does not alter bile acid-induced intracellular zymogen activation. These studies are the first to report the effects of an effective PAR2 pharmacological inhibitor on pancreatic acinar cells and on the severity of experimental pancreatitis. They raise the possibility that a pepducin such as P2pal-18S might prove useful in the clinical management of patients at risk for developing severe biliary pancreatitis such as occurs following ERCP.

Alcohol inhibits cell-cell adhesion mediated by human L1.

Mental retardation, hydrocephalus, and agenesis of the corpus callosum are observed both in fetal alcohol syndrome (FAS) and in children with mutations in the gene for the cell adhesion molecule L1. We studied the effects of ethanol on cell-cell adhesion in mouse fibroblasts transfected with human L1. L1-transfected fibroblasts exhibited increased cell-cell adhesion compared with wild-type or vector-transfected controls. Ethanol potently and completely inhibited L1-mediated adhesion both in transfected L cells and NIH/3T3 cells. Half-maximal inhibition was observed at 7 mM ethanol, a concentration achieved in blood and brain after ingesting one alcoholic beverage. In contrast, ethanol did not inhibit the adhesion of fibroblasts transfected with vector alone or with N-CAM-140. L1-mediated cell-cell adhesion was inhibited with increasing potency by n-propanol and n-butanol, but was not inhibited at all by n-alcohols of 5 to 8 carbons, acetaldehyde, or acetate, suggesting that ethanol interacts directly with a small hydrophobic pocket within L1. Phenylalanine, teratogenic anticonvulsants, and high concentrations of glucose did not inhibit L1-mediated cell-cell adhesion. Ethanol also inhibited potently the heterotypic adhesion of rat cerebellar granule cells to a monolayer of L1-transfected NIH/3T3 cells, but had no effect on their adhesion to N-CAM-140 or vector-transfected NIH/3T3 cells. Because L1 plays a role in both neural development and learning, ethanol inhibition of L1-mediated cell-cell interactions could contribute to FAS and ethanol-associated memory disorders.

Interaction of a brain extracellular matrix protein with hyaluronic acid.

A glial hyaluronate-binding protein (GHAP) was isolated from bovine spinal cord and partially characterized. Bovine GHAP consisted of three immunologically related polypeptides with molecular masses of 76, 64, and 54 kDa and isoelectric points of 4.1, 4.2, and 4.4, respectively. Peptide mapping and partial amino acid sequencing showed that all three polypeptides derive from the same protein. The protein was localized immunohistochemically with rabbit antisera in the white matter surrounding the myelinated axons. Sugar analyses indicated that the three polypeptides are glycosylated and the sugar residues account for at least 30% of their weight. After enzymatic deglycosylation, the apparent molecular mass of the bovine GHAP was reduced to 43 kDa. The biochemical properties of bovine GHAP were compared to those of human GHAP. Initial peptide mapping indicated similarities between bovine and human GHAP. Partial amino acid sequencing of bovine GHAP showed a striking identity (up to 90%) with human GHAP and with the hyaluronate binding domain of the large human fibroblast proteoglycan, versican. Bovine and human GHAP were demonstrated to bind specifically to hyaluronic acid (HA) with one protein molecule binding to an average 17 disaccharide repeating units. The binding of bovine and human GHAP was inhibited by oligosaccharides of HA and specifically by the octamer. Salt concentrations of up to 1 M NaCl had very little effect on the binding of the GHAP to HA. The GHAP-HA interaction was pH dependent. Dissociation only took place at low pH (less than 3.5). Analysis of several polypeptides derived from GHAP by limited proteolysis allowed us to conclude that one of the tandem repeated sequences is sufficient for HA binding and that the aminoterminal domain (which contains an immunoglobulin-like fold) is not involved in the GHAP-HA-binding event.

Influence of phospholipids on the formation and stability of vimentin-type intermediate filaments.

The interaction of vesicles produced from individual phospholipids and mixtures thereof with preformed vimentin filaments as well as the influence of these vesicles on filament assembly were investigated employing negative stain electron microscopy and sucrose density gradient equilibrium centrifugation. Liposomes with a phospholipid composition characteristic of Ehrlich ascites tumor cells were able to bind efficiently to vimentin filaments without significantly affecting their morphology at higher concentrations. However, in sucrose density gradient centrifugation partial disintegration of the filaments was observed. In addition, larger quantities of phospholipid mixture totally blocked intermediate filament (IF) formation. Using vesicles of individual phospholipids, these effects could be shown to be due to the presence of negatively charged lipid species in the phospholipid mixture. While these were highly active in preventing filament assembly and in dissociating preformed filaments, electrically uncharged phospholipids were virtually inactive. The highest efficiency was shown by phosphatidylinositol-4,5-diphosphate. These results demonstrate that a negative surface charge of liposomes is an essential prerequisite for their successful and tight association with vimentin filaments. However, the high susceptibility of these filaments to photoaffinity labeling with the membrane-penetrating reagent 1-azidopyrene in the presence of phospholipid vesicles, points to additional interactions between hydrophobic regions of both reactants. Finally, the data also suggest a direct relationship between IFs and the lipid bilayer as the active principle underlying the association of IFs with natural membranes as observed by electron and immunofluorescence microscopy.

An electron microscopic study of the interaction in vitro of vimentin intermediate filaments with vesicles prepared from Ehrlich ascites tumor cell lipids.

The interaction of intermediate filaments prepared from pure, delipidated vimentin with vesicles obtained from Ehrlich ascites tumor (EAT) cell lipids was studied employing sucrose density gradient centrifugation in combination with electron microscopy. In negative stain electron microscopy, preformed vimentin filaments were seen in lateral association with lipid vesicles; end-on contacts of filaments with liposomes were rarely detected. When the reaction of filaments with vesicles was carried out at 0 degree C, sucrose density gradient equilibrium centrifugation of the reaction products led to the banding of relatively light filament-vesicle meshworks in clear separation from free filaments and free vesicles. With certain vimentin and lipid preparations, occasionally partial breakdown of the filaments during centrifugation and banding of vesicle-free fragments in denser regions of the sucrose gradients was observed. However, when the reaction mixtures were incubated at 37 degrees C prior to sucrose gradient analysis, all filaments were released from vesicles and totally fragmented during centrifugation. Electron microscopy showed unraveling of the filament fragments into subfilament strands. Employing lipid vesicles labeled with [3H]cholesterol, a low but significant amount of radioactivity was found to be associated with the fragments in a non-vesicular form. Filament reconstitution experiments performed in the presence of EAT cell lipids revealed an inhibitory effect of vesicles on filament assembly, particularly at lower temperatures. The mechanical labilization of the filament structure by lipid vesicles might play a role in the redistribution of intermediate filaments in the course of certain cellular processes involving turnover and fragmentation of intracellular membrane systems.

Efficient interaction of nonpolar lipids with intermediate filaments of the vimentin type.

Based on the finding that vimentin isolated and purified from cultured mammalian cells is heavily contaminated by neutral lipids, the binding of a series of radioactively labeled nonpolar lipids to pure, delipidated vimentin was investigated. Employing gel permeation chromatography of the complexes on Sephacryl S-300, cholesterol, cholesteryl fatty acid esters and mono-, di- and triglycerides were found to efficiently associate with vimentin. These compounds also showed a strong tendency to bind to vimentin filaments. While the non-alpha-helical head piece of vimentin did not interact with neutral lipids under the above assay conditions, the alpha-helical rod domain was highly active. When cholesterol or 1,2-dioleoyl-glycerol was incorporated into phospholipid vesicles, the affinity of the liposomes for vimentin filaments was considerably increased. However, in sucrose density gradient equilibrium centrifugation the filament-vesicle adducts were only stable when the liposomes contained negatively charged phospholipids. These results suggest that the association of intermediate filaments with lipid vesicles is initiated by interaction of the arginine-rich N-termini of their subunit proteins with the negatively charged vesicle surface and stabilized by partial insertion of the protein molecules into the lipid bilayer, particularly at those sites where immiscible, nonpolar lipids create defects in phospholipid packing. Very likely, nonpolar lipids play a significant role in the interaction of intermediate filaments with natural membrane systems.

Probing of the structural stability of vimentin and desmin-type intermediate filaments with Ca2+-activated proteinase, thrombin and lysine-specific endoproteinase Lys-C.

Intermediate filaments (IFs) reconstituted from purified, delipidated vimentin and desmin as well as respective protofilaments were subjected to degradation by Ca2+-activated neutral thiol proteinase, thrombin and lysine-specific endoproteinase Lys-C, respectively. The breakdown products were analyzed by SDS-polyacrylamide gel electrophoresis and negative stain electron microscopy. While Ca2+-activated proteinase and thrombin caused rapid and complete degradation of IFs with kinetics not significantly different from those of the degradation of protofilaments, lysine-specific endoproteinase did not exert any electron microscopically detectable effect on filament structure. Although both types of subunit proteins were truncated at their non-alpha-helical, C-terminal polypeptides by this proteinase, they were still able to assemble into 10 nm filaments. Closer electron microscopic inspection of IFs treated with Ca2+-activated proteinase revealed numerous ruptures along the filaments already at very early stages of digestion. SDS-polyacrylamide gel electrophoresis of the processed filaments in conjunction with previous biochemical characterizations of the breakdown of protofilaments by Ca2+-activated proteinase showed that these inhomogeneities primarily arose from degradation of the arginine-rich, non-alpha-helical N-termini of the filament proteins. These findings demonstrate that, although the N-terminus of vimentin and desmin is essential for filament stability, it is still highly susceptible to proteolytic attack in particular and very likely to posttranslational modification in general. Such structural modifications of the N-termini of IF proteins might exert great influences on the intracellular distribution and molecular organization of IFs in various physiological and pathological conditions.

Glial hyaluronate-binding protein in polar spongioblastoma.

Glial hyaluronate-binding (GHA) protein is a 60 kDa glycoprotein isolated from human white matter by affinity chromatography on immobilized hyaluronate. It is localized in white matter astrocytes by immunofluorescence with monoclonal antibodies. Amino acid sequences have not revealed similarities with other proteins except cartilage extracellular matrix proteins, the region of similarity being located within the hyaluronate-binding region. Cryostat sections of 13 intracranial neoplasms removed at surgery were tested for the presence of GHA protein by indirect immunofluorescence with monoclonal antibodies. These included seven astrocytomas, one oligodendroglioma, one medulloblastoma and one spinal cord ependymoma. All tumors were negative with the exception of one astrocytoma in which the GHA protein-positive areas had the typical appearance of polar spongioblastoma, i.e. small cells palisading around blood vessels and very delicate glial fibrillary acidic (GFA) protein-positive fibrils. Conversely, neoplastic as well as reactive GFA protein-positive astrocytes were GHA protein-negative. We suggest that polar spongioblastoma derives from a GHA protein-positive glial precursor and pertinent to this suggestion is the observation that the periventricular germinal layer was found GHA protein-positive in a 22-week human fetus.

Tenacious binding of lipids to vimentin during its isolation and purification from Ehrlich ascites tumor cells.

Vimentin enriched in cytoskeletal frameworks by Triton X-100 extraction of Ehrlich ascites tumor cells and purified from a low ionic strength extract of the cell residues by (NH4)2SO4 precipitation and DEAE-Sepharose and ssDNA-cellulose chromatography in the presence of 6 M urea was highly contaminated with lipids. Thin-layer chromatography of a chloroform-methanol extract of the purified protein revealed, besides small amounts of phospholipids, the presence of large quantities of neutral lipids.

Extracellular matrix in regenerating rat sciatic nerve: a comparative study on the localization of laminin, hyaluronic acid, and chondroitin sulfate proteoglycans, including versican.

Using a glial hyaluronate-binding protein as a probe, monoclonal antibodies against versican and ABC digested chondroitin sulfate proteoglycans, and polyclonal antibodies against laminin, we localized these extracellular matrix (ECM) components in the endoneurium of the adult rat sciatic nerve. During Wallerian degeneration caused by nerve crushing, the staining pattern of these ECM elements changed dramatically. In the first stages and up to 5 days after injury, the tubular endoneurial structures remained the same as in control nerves. Ten days after crush, the bands of Büngner formed by proliferating Schwann cells in the distal stump of crushed nerves stained diffusely for hyaluronate, laminin, and chondroitin sulfate. Regenerating axons were demonstrated in this location by double-labeling experiments with neurofilament antibodies. Conversely, staining with antibodies against versican, a hyaluronate binding proteoglycan, was reduced and the bands of Büngner were not stained. After 30 days the endoneurial tubes made their reappearance and, as in normal nerve, they stained for hyaluronate, laminin, versican, and chondroitin sulfate. It is concluded that proliferating Schwann cells in peripheral nerve undergoing Wallerian degeneration are capable of producing several endoneurial ECM constituents, versican being a notable exception.

Effect of highly concentrated gels of sodium hyaluronate on early phases of regeneration in the transected and tubulized rat sciatic nerve.

Silicone regeneration chambers prefilled with sodium hyaluronate (HA) gels of different molecular weight (MW) and at different concentrations were used to tubulize the transected rat sciatic nerve. After two weeks, the tissue cables bridging the severed nerve ends within the silicone tubes were examined. Low MW HA gels had no significant effect while high MW HA gels reduced the diameter of the growing bridges.

Co-localization of hyaluronic acid and chondroitin sulfate proteoglycan in rat cerebral cortex.

The distribution of hyaluronate (HA) and chondroitin sulfate (CS) proteoglycan in the rat cerebral cortex was compared. For the localization of HA, the sections were incubated with human glial hyaluronate-binding protein (GHAP) and then reacted with monoclonal or polyclonal antibodies to GHAP. Polyclonal antibodies raised in rabbit were used for double-labeling experiments with monoclonal antibodies raised in mice and reacting with CS proteoglycans. Little reactivity was observed in rat cerebral cortex with polyclonal GHAP antibodies if the sections were not incubated with GHAP. Monoclonal antibodies to GHAP did not react with murine tissues. CS proteoglycans were localized in chondroitinase-digested sections with monoclonal antibodies reacting with the 4-sulfated oligosaccharide stubs formed by the digestion with chondroitinase ABC of CS side chains. In the rat cerebral cortex, the distribution of CS proteoglycans was similar to that reported by Bertolotto, A., Rocca, G. and Schiffer, D., J. Neurol. Sci., 100 (1990) 113-123, and his collaborators using the same antibodies. Many neurons mainly located in the upper and deep cortical layers were surrounded by CS immunoreactive material. Several (but not all) CS-positive neurons also stained for HA with an identical distribution except that in most instances the staining was confined to the periphery of the perikaryon and did not extend to the dendritic tree. The finding suggests that cerebral cortex CS proteoglycan is capable of interacting with HA.

Glial hyaluronate-binding protein (GHAP) in optic nerve and retina.

The distribution of glial fibrillary acidic protein (GFAP) and of glial hyaluronate-binding protein (GHAP) was studied by indirect immunofluorescence with monoclonal and polyclonal antibodies in dog, rat and rabbit optic nerve. In dog and rabbit, myelination extends into the optic nerve head inside the eye, while in the rat myelination of the optic nerve ceases abruptly at its entry into the eye. Outside the eye the distribution of the two proteins was similar. Both antigens formed a delicate mesh surrounding myelinated optic nerve axons. In all 3 species GFAP immunoreactivity continued uninterrupted into the optic nerve head inside the eye. Conversely, in both dog and rat, GHAP immunoreactivity ceased abruptly in the region of the lamina cribrosa, a sieve-like structure continuous with the sclera through which bundles of optic nerve axons pass. No staining was observed in the myelinated optic nerve head of the dog nor in the non-myelinated optic nerve head of the rat. In the rabbit lacking a lamina cribrosa, GHAP immunoreactivity did not cease abruptly at the optic nerve entry into the eye, but the staining intensity was reduced in the optic nerve head.

The extracellular matrix of cerebral gray matter: Golgi's pericellular net and Nissl's nervösen grau revisited.

Glial hyaluronate-binding protein (GHAP) and a large aggregating chondroitin sulfate proteoglycan (Ag-Pg) similar to a fibroblast proteoglycan (versican) were localized in bovine, dog and cat central nervous system (CNS) gray matter by indirect immunofluorescence. The distribution of the two hyaluronate-binding proteins was identical with that of hyaluronate, an extracellular glycosaminoglycan. All substances formed a finely reticulated mesh in the neuropil with a condensation of the stain around large neurons. It is concluded that in gray matter, as in white matter, the extracellular matrix (ECM) contains hyaluronate-protein aggregates. We suggest that the hyaluronate-protein aggregates correspond to the pericellular network first described by Golgi.

Neuroprotective effect of human osteogenic protein-1 in a rat model of cerebral hypoxia/ischemia.

Possible neuroprotective actions of osteogenic protein-1 (OP-1) were evaluated in a rat model of cerebral hypoxia/ischemia. Intraperitoneal injection of 50 micrograms of OP-1 prior to bilateral carotid ligation and transient hypoxia in 12-day-old rats reduced cerebral infarct area from 44.8 +/- 3.3% in vehicle-injected controls to 29 +/- 4.9%. Treatment of 14-day-old rats with 20 micrograms of OP-1 1 h after hypoxia reduced mortality from 45% to 13%. OP-1 may represent a novel class of neuroprotective agents.

Colchicine, colcemide and cytochalasin B do not affect translocation of the glucocorticoid hormone-receptor in rat thymocytes or Ehrlich ascites cells.

The involvement of intracellular cytoskeletal elements in the translocation of the dexamethasone-receptor complex from the cytoplasm to the nucleus was studied using the cytoskeleton-disrupting agents colcemide, colchicine and cytochalasin B. These compounds did not affect the translocation of the hormone-receptor complex. We conclude that microfilaments and microtubules do not play a role in the translocation of the glucocorticoid hormone-receptor complex from the cytoplasm to the nucleus.

Colocalization of tenascin with versican, a hyaluronate-binding chondroitin sulfate proteoglycan.

Rabbit antisera against tenascin, a large extracellular matrix protein, in conjunction with monoclonal antibodies of mouse origin against versican, a large hyaluronate-binding proteoglycan, were used to make a comparative study of the distribution of the two antigens in the same cryostat sections by double immunofluorescence. In the central nervous system, tenascin was invariably associated with versican, but the reverse was not true, in that versican was also found where tenascin was not detectable, particularly in gray matter. There were major species differences in the distribution of tenascin in the central nervous system. In the cow, tenascin was found in cerebral and spinal cord white matter and in the granule cell layer of the cerebellum. In the human brain, tenascin was found in cerebral white matter but not in the cerebellum. In the rat, tenascin was mainly confined to brain periventricular layer and spinal cord white matter. During the development of the cerebellum of the rat, the tenascin immunoreactivity decreased, and a lower molecular weight band appeared (J1-160/180/restrictin?) and persisted throughout adulthood. Tenascin expression was a relatively late event in the development of the rat central nervous system, immunoreactivity being first observed after birth. In the rat embryo, tenascin was found to co-localize with versican in precartilaginous mesenchyme and in connective tissue underlying epithelia. The colocalization of versican with tenascin suggests that versican may be the tenascin (cytotactin)-associated proteoglycan reported in the literature.

Cardiac conduction abnormalities in a mouse model of Lyme borreliosis.

BACKGROUND: Borrelia Burgdorferi (BB) induces cardiac conduction abnormalities in infected humans. Mice models of Lyme disease have been developed, however their electrophysiologic (EP) properties of conduction are unknown. METHODS: Seventy-six C3H/J mice (BB infected and age- and gender-matched controls) underwent blinded in vivo EP studies. In a first phase of the study, 40 male C3H/J mice were divided into 2 groups: Group (A) mice were infected at age 3 (weeks) and studied at 5, and Group (B) mice were infected at 9 and studied at 11. In a second phase, 36 female mice were divided into 2 groups: Group (C) mice were infected at 3 weeks and studied at 5, and Group (D) mice were infected at 3 and studied at 11. RESULTS: Infected mice of group (A) and (C) had wider QRS complexes (21.0+/-1.6 versus 17.3+/-1.3ms, p< or =0.0001 and 20.3+/-2.1 versus 18.5+/-1.7, p = 0.05, respectively) compared to the healthy controls (HC). Infected mice of group (B) and group (D) were similar to the HC. In all groups, the presence of conduction abnormalities correlated very closely with the amount of inflammation on pathology. CONCLUSION: This study describes the first EP mouse model of Lyme carditis. C3H/J mice exhibit conduction abnormalities that are reversible 8 weeks after inoculation, closely paralleling the resolution of inflammation on pathology. This model can be a valuable tool in the developing and testing of new modalities for the prevention and treatment of Lyme carditis.

Interaction in vitro of non-epithelial intermediate filament proteins with histones.

Non-epithelial intermediate filament (IF) subunit proteins show a high and specific affinity for core histones at physiological ionic strength. When IF proteins are titrated with a mixture of core histones and linker histone H1, in general the latter is totally excluded from complexation and in the adducts formed the moderately-arginine-rich histones H2A and H2B are progressively replaced by the very-arginine-rich histones H3 and H4. At histone saturation, 2 molecules of nonneuronal IF protein bind 1 histone H1 molecule or 8 core histone molecules, whereas due to its glutamic acid-rich, C-terminal extensions one dimer of the 68 kD neurofilament protein associates with 3 molecules of histone H1 or 24 molecules of core histones. The salt stability of the insoluble association products is dependent on the amount and arginine content of the constituent histone species. Removal of the non-alpha-helical N- and C-terminal polypeptides from IF proteins by partial chymotryptic digestion does not affect their histone-binding characteristics. Since core histones are only partially inactivated by limited tryptic digestion, they also appear to react through their alpha-helix-rich central domains; the limit peptide derived from histone H1 is completely inactive at physiological ionic strength. Affinity chromatography of rod domains of IF proteins on core histone-Sepharose 4B and of histones and their limit peptides on vimentin-Sepharose 4B has shown that the interactions involving fractions of histones H3 and H4 are extremely resistant to salt and can be dissociated only with arginine or salt under denaturing conditions. In general, the experimental results revealed close parallels between the association of histones with IF proteins and their interaction with DNA.