Infectious pancreatic necrosis virus: identification of a VP3-containing ribonucleoprotein core structure and evidence for O-linked glycosylation of the capsid protein VP2.

Virions of infectious pancreatic necrosis virus (IPNV) were completely disintegrated upon dialysis against salt-free buffers. Direct visualization of such preparations by electron microscopy revealed 5.0- to 6.5-nm-thick entangled filaments. By using a specific colloidal gold immunolabeling technique, these structures were shown to contain the viral protein VP3. Isolation by sucrose gradient centrifugation of the filaments, followed by serological analysis, demonstrated that the entire VP3 content of the virion was recovered together with the radiolabeled genomic material forming the unique threadlike ribonucleoprotein complexes. In a sensitive blotting assay, the outer capsid component of IPNV, i.e., the major structural protein VP2, was shown to specifically bind lectins recognizing sugar moieties of N-acetylgalactosamine, mannose, and fucose. Three established metabolic inhibitors of N-linked glycosylation did not prevent addition of sugar residues to virions, and enzymatic deglycosylation of isolated virions using N-glycosidase failed to remove sugar residues of VP2 recognized by lectins. However, gentle alkaline beta elimination clearly reduced the ability of lectins to recognize VP2. These results suggest that the glycosylation of VP2 is of the O-linked type when IPNV is propagated in RTG-2 cells.

Differences in endothelial injury after balloon angioplasty, insertion of balloon-expanded stents or release of self-expanding stents: An electron microscopic experimental study.

PURPOSE: To evaluate which of six different commonly available stents inserted into an artery without percutaneous transluminal angioplasty (PTA) causes the least endothelial damage. To compare the degree of endothelial injury after insertion of such a stent with injury caused by PTA. METHODS: Twelve healthy pigs were used in the experiments. In the first part of the study six different types of stents were inserted into the common iliac arteries. In the second part of the study self-expanding stents with large spaces between the wires were used. PTA was performed in the contralateral iliac artery. The pigs were killed immediately after the procedure and resected specimens examined after fixation, using scanning electron microscopy. RESULTS: All procedures but two were accomplished successfully. More endothelium was preserved after insertion of self-expanding stents with large spaces between the wires, compared with stents with small spaces and balloon-expanded stents. After insertion of self-expanding stents with large spaces, 50.1% +/- 16.4% of the endothelium remained intact, compared with only 5.6% +/- 7.7% after PTA. The difference was statistically significant (p < 0.001). CONCLUSION: Self-expanding stents with large spaces between the wires, inserted without PTA, cause less damage to the endothelium than other stents and significantly less damage than PTA.

Apoptosis of renal cortical cells in the hemolytic-uremic syndrome: in vivo and in vitro studies.

This study examined apoptotic cell death associated with Shiga-like toxin (Stx)-producing Escherichia coli. Renal cortices from three children with postenteropathic hemolytic-uremic syndrome (HUS) and from mice infected with E. coli O157:H7 and pediatric renal tubular epithelial cells stimulated with Stx and E. coli O157:H7 extracts were examined for apoptotic changes. Apoptotic cells were detected by terminal dUTP nick end labeling of tubuli and glomeruli from HUS patients and from mice inoculated with Stx-2-positive and Stx-negative strains. Apoptosis was more extensive and severe ultramorphological nuclear and cytoplasmic changes were seen in the Stx-2-positive group. Stx caused DNA fragmentation and ultramorphological changes indicating apoptosis in cultured pediatric tubular cells. DNA fragmentation increased when cells were pre-stimulated with tumor necrosis factor alpha. Polymyxin extracts from Stx-2-positive and Stx-negative strains induced DNA fragmentation, but only extracts from Stx-2-positive strains caused ultramorphological changes and extensive DNA fragmentation. The results indicate that HUS is accompanied by increased apoptosis of kidney cells and that bacterial factors, possibly together with host cytokines in vivo, may activate apoptotic tissue injury.

B cell granule peptides affect human islet amyloid polypeptide (IAPP) fibril formation in vitro.

Islet amyloid polypeptide (IAPP) forms fibrils spontaneously. We examined whether other B cell granule peptides affect formation of beta-pleated sheet fibrils in vitro from human IAPP. Quantitative radioassay (radioactivity of soluble IAPP after adding 125I-IAPP) and thioflavine fluorescence spectroscopy showed that insulin, C-peptide, and pancreastatin inhibited fibril formation by 60-100% at ratio 100:1 (peptide:IAPP), whereas at 1:10 or 1:100, i.e., IAPP in excess, a potentiated IAPP fibril formation was induced by the peptides. Semi-quantitative analysis by electron microscopy yielded similar effects. Thus, spontaneous IAPP fibrillisation is influenced by other B cell secretory granule peptides in a molar ratio dependent manner.

Alpha-latrotoxin-induced transmitter release in feline oesophageal smooth muscle: focus on nitric oxide and vasoactive intestinal peptide.

1. The effects of alpha-latrotoxin (alpha LTX) on muscle tone, resting membrane potential, cyclic nucleotide content, and ultrastructure were examined in feline oesophageal smooth muscle, including the lower oesophageal sphincter (LOS). 2. In circular smooth muscle strips from LOS developing active tone alpha LTX (1 nM) induced a 94 +/- 3% (n = 16) relaxation. Intermittent treatment with alpha LTX for 4 h abolished the response. Pretreatment with NG-nitro-L-arginine (L-NOARG; 0.1 mM) attenuated the relaxation. 3. In carbachol-contracted circular smooth muscle strips from the LOS and oesophageal body (OB), alpha LTX induced a 95 +/- 5% (n = 6) and 73 +/- 9% (n = 8) relaxation, respectively. The relaxations were attenuated by L-NOARG, and in LOS strips, the relaxation was abolished by the combination of L-NOARG and vasoactive intestinal peptide (VIP)-antiserum (1:25). At resting tension in circular smooth muscle strips from the OB, alpha LTX induced a scopolamine sensitive contraction in the presence of L-NOARG. 4. In circular LOS and OB preparations, alpha LTX changed the resting membrane potential from -49 +/- 2mV to -59 +/- 3 mV (n = 4), and -62 +/- 2 mV to -71 +/- 3 mV (n = 4), respectively. 5. The alpha LTX-induced relaxation of LOS and OB muscle was associated with a 138% and 72% increase in cyclic GMP levels, respectively. No changes in cyclic AMP levels were observed. 6. Ultrastructural analysis of LOS and OB revealed a rich supply of nerve profiles containing small synaptic and large dense core vesicles. alpha LTX treatment resulted in a loss of both types of vesicle. 7. These results suggest that alpha LTX induces relaxation of oesophageal circular smooth muscle associated with NO-generation and transmitter release from synaptic vesicles. Beside NO, VIP seems to be involved in the relaxant effects of alpha LTX on the LOS. In addition, alpha LTX may have contractile effects by release of acetylcholine.

Inhibition of Alzheimer beta-peptide fibril formation by serum amyloid P component.

A 39-43-amino acid residue-long fragment (beta-peptide) from the amyloid precursor protein is the predominant component of amyloid deposits in the brain of individuals with Alzheimer's disease. Serum amyloid P component (SAP) is present in all types of amyloid, including that of Alzheimer's disease. We have used an in vitro model to study the effects of purified SAP on the fibril formation of synthetic Alzheimer beta-peptide 1-42. SAP was found to inhibit fibril formation and to increase the solubility of the peptide in a dose-dependent manner. At a 5:1 molar ratio of A beta 1-42 peptide to SAP, fibril formation was completely inhibited, and approximately 80% of the peptide remained in solution even after 4 days of incubation. At lower SAP concentrations, e.g. at peptide to SAP ratio of 1000:1, short fibrillar like structures, lacking amyloid characteristics, were formed. These structures frequently contained associated SAP molecules, suggesting that SAP binds to the polymerizing peptide in a reaction which prevented further fibril formation.

Escherichia coli induces transuroepithelial neutrophil migration by an intercellular adhesion molecule-1-dependent mechanism.

During bacterial infections at mucosal sites, neutrophils migrate to the mucosa and cross the epithelial barrier. We have examined neutrophil migration across Escherichia coli-stimulated uroepithelial cell layers in an attempt to more fully understand this process. Stimulation of uroepithelial cells with E. coli or interleukin-1 alpha (IL-1 alpha) induced transepithelial neutrophil migration in a time- and stimulant dose-dependent manner. Uroepithelial cell lines and nontransformed uroepithelial cells expressed intercellular adhesion molecule-1 (ICAM-1) but not ICAM-2, E-selectin, or P-selectin. Epithelial ICAM-1 expression was enhanced after stimulation with E. coli or IL-1 alpha. Anti-ICAM-1 antibody reduced transepithelial neutrophil migration by 61 to 85%, indicating that neutrophils bound ICAM-1 on the epithelial surface. Antibodies to CD18 and CD11b reduced migration by 70 to 79%, suggesting that CD11b/CD18 (Mac-1) was acting as the neutrophil receptor for ICAM-1 in this process. Anti-CD11a antibodies had no effect on neutrophil migration. In conclusion, E. coli induced ICAM-1- and Mac-1-dependent transepithelial neutrophil migration. Previous studies have shown that urinary tract epithelial cells secrete IL-8 when exposed to E. coli or IL-1 alpha. These observations suggest that epithelial cells play an active role in neutrophil migration during urinary tract infections.

In vitro fibril formation from alpha 1-antitrypsin-derived C-terminal peptides.

Fragments from various proteolytically degraded precursor proteins can form beta-amyloid fibrils. We studied, by electron microscopy and quantitative Congo red binding, the ability of three synthetic peptides, corresponding to residues 359-374 (C-36), 370-374 (C-5) and 375-394 (C-20) from the C-terminal part of alpha 1-antitrypsin (AAT) to form beta-amyloid fibrils in vitro. The peptides C-36 and C-5 had a pronounced tendency to form fibrils. C-20 lacked this property, suggesting that residues 359-375 and/or 370-374 are most critical for fibril formation. Native AAT added to peptide 125I-C-36 could bind and form complexes with the peptide, resulting in inhibition of amyloid fibril formation. Moreover, native AAT added to preformed fibrils induced disaggregation of fibrillar structures. The structural rearrangements of AAT that occurred during this 'autointeraction' included polymerization of the serpin, and an increase of its thermal stability. Also, following interaction, an increase (20-40%) of AAT's antielastase activity was noted. The demonstration of an in vitro beta-amyloid fibril formation from the AAT derived C-terminal peptides C-36 and C-5 and its regulation by the intact AAT molecule may have important in vivo implications.

In vitro amyloid fibril formation from alpha 1-antitrypsin.

We have previously shown that the interaction between alpha 1-antitrypsin (AAT) and lithocholic acid (LA) results in changes of AAT properties leading to its polymerization and inactivation. To define the structural rearrangements of AAT induced by such interaction, we studied the in vitro binding between AAT and LA at molar ratio 1:5 for varying time intervals at a physiological pH. Complex formation was shown by electrophoretic techniques and autoradiography. Studies of the AAT in complex with LA by using far-UV spectra circular dichroism and fluorescence measurements indicated an increase of beta-structure of AAT and pronounced changes in surroundings of the chromophores. In addition, complexed AAT showed increase in thermal stability, compatible with that after proteolytic cleavage. Characterization of the AAT-LA complexes by Congo red binding, polarization and negative staining electron microscopy provided clear evidence that AAT, under chosen experimental conditions, can self-assemble into amyloid fibrils, compatible with accepted models of fibrillar structures. This propensity of AAT to form stable beta-structures in a hydrophobic surrounding may contribute to improved characterization of various amyloid deposits occurring in vivo and be a guide for understanding details of structure-function relationships in the intact AAT-molecule.

Crystallization and preliminary X-ray investigation of the Escherichia coli molecular chaperone cpn60 (GroEL).

The Escherichia coli molecular chaperone, cpn60 (GroEL), has been purified from an overproducing E. coli strain and crystallized. Of the two crystal forms that were obtained, one was found to be suitable for crystallographic and structural studies at low resolution. Preliminary X-ray investigation of the crystals show unit cell dimensions: a = 143.3, b = 154.6 and c = 265 A, with alpha = 82 degrees, beta = 95 degrees and gamma = 107 degrees. The space group is P1 and the crystals diffract to a maximum of 7 A when using CuK alpha X-rays from a rotating anode. Both electron microscopy and non-denaturing electrophoretic analysis of redissolved cpn60 crystals show that cpn60 crystallizes in the native oligomeric form. Comparison between the dimensions of oligomeric cpn60 and the crystallographic unit cell volume suggests that the unit cell contains two oligomeric cpn60 molecules. The VM value for two cpn60 molecules per unit cell is 3.5 A3/Da, corresponding to a water content of 65%. Electrophoretic analysis under denaturing conditions shows that the cpn60 in crystals is heterogeneous, and this probably explains the limited resolution of the diffraction data.

Imaging of RNA-protein interactions in splicing complexes with dark-field STEM.

Splicing complexes were assembled in vitro on pre-messenger RNA. These complexes represent stages in the processing of pre-mRNA by snRNPs and accessory factors to remove the introns, thus forming messenger RNA. Complexes were purified using biotinylated oligonucleotide tags together with streptavidin-gold and simple centrifugation. The oligonucleotide tags with attached streptavidin-gold also serve as unambiguous labels of the splicing complexes for EM observation. The complexes were observed using dark-field scanning transmission electron microscopy (STEM). The high contrast and the high efficiency in detecting scattered electrons make it possible to visualize specimens at high resolution with low radiation dose. STEM visualization of the complexes allows a unique view of the events occurring in the splicing process. Three different classes of complex were identified. These complexes support the currently postulated steps in RNA splicing and demonstrate the dynamic nature of splicing complexes. This approach has broad application in high-resolution structural studies of nucleic acids and nucleic acid-protein interactions.

Streptococcal protein G-gold complex: comparison with staphylococcal protein A-gold complex for spot blotting and immunolabeling.

Protein G, a cell wall protein isolated from human group G streptococci strain G148, binds in a similar manner as protein A from Staphylococcus aureus to the Fc portion of IgG molecules. Indeed, protein G has been proposed as a superior Fc binding protein due to its broader species reactivity. Thus, we have prepared a complex of protein G with particles of colloidal gold and determined its applicability for spot-blot analysis and postembedding immunolabeling by comparing it with protein A-gold complex. By spot-blot analysis no difference in binding of protein G-gold or protein A-gold to IgG molecules from a whole spectrum of animal species was observed. Moreover, using rabbit, sheep, or goat anti-rat albumin antibodies to detect nitrocellulose-immobilized rat albumin or antigenic sites in paraffin and Lowicryl K4M thin sections from rat liver, no difference was found with protein G-gold or protein A-gold. Similarly, no difference in binding to protein G-gold or protein A-gold was observed with a battery of monoclonal antibodies. However, in contrast to expectations, protein A-gold reacted well with both sheep and goat IgG molecules; indeed, for the light and electron microscopic localization of albumin with sheep or goat antibodies it was as efficient as protein G-gold. These results demonstrate, therefore, that both protein G-gold and protein A-gold are useful second step reagents for immunolabeling and that protein G-gold was not a superior probe in the systems tested.

The efficiency of immunolabel on Lowicryl sections compared to theoretical predictions.

The surface of thin sections of aldehyde-fixed biological material shows a specimen-related relief of 2-6 nm with Lowicryl. Epon sections are about three times smoother. The relief is the consequence of thin-sectioning being in reality a cleavage. Epitopes are supposed to be laid open (or set free) because cleavage follows the interfaces between protein and Lowicryl. We have developed a simple theory on this basis and have theoretically estimated the efficiency of on-section labeling and compared it with experimental data. For randomly dispersed proteins in cytoplasm, Lowicryl sections will yield significant label only when the concentration of the antigen is about 10 microM or more. The complex situation of more compact proteins, as represented by fibers, sheets, and biological membranes is discussed and the difficulty of significant calculations is explained. Pre-embedding labeling and melted cryosections should give 10-30 times more label. The possible reasons for the observed much smaller gain of not more than two to three times are discussed.

Developments of new Lowicryl resins for embedding biological specimens at even lower temperatures.

Two new Lowicryl resins have been developed for embedding biological materials at temperatures down to 210 K (hydrophilic K11M) and to 190 K (hydrophobic HM23). They have similar properties to Lowicryl K4M and HM20. The new resins were first tested for low temperature applications by the 'progressive lowering of temperature' procedure and this shows that the low viscosity of K11M and HM23 is favourable for the infiltration of biological specimens. Hardening is achieved through photo-polymerization at these lower temperatures. These properties make K11M and HM23 suitable for cryosubstitution of rapidly frozen material and it is speculated that the preservation of antigenicity may be further improved.

Low temperature embedding with Lowicryl resins: two new formulations and some applications.

Lowicryl K4M and HM20 are methacrylate/acrylate based low temperature embedding resins for biological material which can be used in conjunction with either the progressive lowering of temperature (PLT) technique or with freeze-substitution. K4M and HM20 are applicable over a very extended temperature range, approximately 220 K to 340 K. With two new resins, K11M and HM23, one can reach even lower temperatures, c. 200 K. Freeze-substitution combined with low temperature embedding allows for very mild or no chemical fixation which seems to increase the sensitivity of immunocytochemical localization of antigens on sections.

Capsule of Escherichia coli K29: ultrastructural preservation and immunoelectron microscopy.

The polysaccharide capsule of Escherichia coli K29 fully surrounds the microorganism and thus occupies an extracellular space ca. 20 times larger in volume than that of the decapsulated cell. Since more than 95% of the capsule consists of water, dehydration for electron microscopy causes the material to collapse. We describe here a method for embedding the capsule in an uncollapsed form. Dehydration of gelatin-enrobed, glutaraldehyde-fixed cells was performed in dimethyl formamide. The cells were embedded in Lowicryl K4M with the "progressive lowering of temperature" method and UV polymerization. In ultrathin sections, the capsule can be identified by its low electron contrast. It occupies a layer 3/4 micron thick thick and shows fibrous strands embedded in a fine granular matrix. The thin strands extend radially from the cell wall and transverse the capsule. The entire capsule domain, as well as the outer membrane, binds specific anticapsular antibody, whereas the periplasmic space and most of the inner membrane lack capsule-specific immunostain.

Ultrastructural localization of keratin proteins in human skin using low-temperature embedding and the protein A-gold technique.

Human skin was embedded in Lowicryl K4M and keratin proteins were localized by incubation with antikeratin antisera, followed by protein A-gold. The antikeratin antisera labeled all intermediate filament (tonofilament) structures in all layers of the epidermis. The association of keratin filaments with hemidesmosomes, desmosomes, and keratohyaline granules was clearly visualized. Desmosomes and keratohyaline granules were not labeled by the antikeratin antisera. No nonfilamentous structures were labeled. The technique described is suitable for studying the distribution of keratin filaments in normal and diseased tissue.

Periplasmic gel: new concept resulting from the reinvestigation of bacterial cell envelope ultrastructure by new methods.

Bacterial cell envelope ultrastructure was investigated both by the progressive lowering of temperature embedding technique and freeze-substitution, using conventional and scanning transmission electron microscopy. Comparison with standard embedding procedures revealed a new aspect of cell envelope structure in specimens at low temperatures. The envelope was delimited by an electron-dark layer, beneath which was a uniform matter-containing layer lying between the outer and inner membranes. There was no empty periplasmic space. Buoyant densities of isolated peptidoglycan obtained in Percoll (1.02 to 1.07 g ml-1) and CsCl2 (1.44 g ml-1) led to a calculated hydration of the peptidoglycan which was more than was previously assumed. Peptidoglycan therefore possibly fills the entire space between the inner and outer membranes in the form of a periplasmic gel. The new model of cell envelope organization is discussed with respect to the current knowledge on bacterial cell wall structure and function.