Australia antigen (a hepatitis-associated antigen): purification and physical properties.

Australia antigen [Au(1)], a particle associated with viral hepatitis, was isolated from the plasma of a patient with chronic anicteric hepatitis and leukemia who had received radioactive phosphorus. We have found that the immunoreactivity and appearance of Au(1) in the electron microscope were not altered by treatment with enzymes including trypsin, pronase, lipase, phospholipase C, ribonuclease, deoxyribonuclease, amylase, and neuraminidase. In contrast, other serum constituents were degraded by these enzymes. Therefore, treatment of the patient's plasma with many enzymes was exploited as an initial step for the isolation of Au(1). Subsequently, Au(1) was purified from the enzyme-treated (32)P-labeled plasma by gel filtration through Sephadex G-200 and centrifugation through sucrose and in cesium chloride gradients. There were no detectable human serum components in the purest fractions, as tested by immunoelectrophoresis and immunodiffusion. The density of the purified Au(1) was 1.21 in CsCl. The particle measured about 200 A in diameter, was predominantly spherical in shape and appeared to be composed of subunits. Nucleic acids were not detected by spectrophotometric, radiochemical, and chemical analyses. Immunoreactivity of purified Au(1) was destroyed by heating for 1 hr at 85 degrees C but was stable at 56 degrees C. Treatment with Carnoy's solution (3 parts ethanol:1 part glacial acetic acid) followed by pronase disrupted the particles as seen with the electron microscope. These findings, combined with other published information on Australia antigen and viral hepatitis, suggest that the bulk of Australia antigen in the blood of this patient is an incomplete virus or virus capsid.

Characterization of a new class of DNA delivery complexes formed by the local anesthetic bupivacaine.

Bupivacaine, a local anesthetic and cationic amphiphile, forms stable liposomal-like structures upon direct mixing with plasmid DNA in aqueous solutions. These structures are on the order of 50-70 nm as determined by scanning electron microscopy, and are homogeneous populations as analyzed by density gradient centrifugation. The DNA within these structures is protected from nuclease degradation and UV-induced damage in vitro. Bupivacaine:DNA complexes have a negative zeta potential (surface charge), homogeneous nature, and an ability to rapidly assemble in aqueous solutions. Bupivacaine:DNA complexes, as well as similar complexes of DNA with other local anesthetics, have the potential to be a novel class of DNA delivery agents for gene therapy and DNA vaccines.

Visualization of the bacterial polysaccharide capsule.

The highly hydrated capsule of E. coli strains is composed of a large number of polysaccharide fibers of which the thinnest measure about 2 nm in width. The fibers may span the entire distance from the outer membrane to the outer rim of the capsule and show a propensity to associate with each other to form thicker filaments. Presence of thick filaments may also indicate a partial collapse of the capsular organization due to removal of water. The in vivo capsule represents a relatively open structure with the negatively charged polysaccharide fibers permitting the binding of large quantities of water and ions, and providing intracellular space for diffusing molecules to access the envelope membranes even in conditions of high cell density. Negative charge and steric hindrance of the polysaccharide strands protect the cells against attack by a large variety of harmful macromolecules and against infection by most bacteriophages. Two types of procedure have been most successful in maintaining the size and overall structure of the capsule: (a) the interaction of cationic molecules with the in vivo capsule, and (b) the use of antibody to stabilize capsules for subsequent dehydration and plastic embedding. A further type of potentially useful procedure, cryofixation and cryosubstitution, has shown interesting results in a number of cases. These techniques are expected to play a significant role in structural studies in the near future. The sites of export of capsular antigen have been described in earlier conventional electron microscopic studies. Data obtained from the recent technique of "on-section" labeling support the model that both the capsular antigen and the O antigen are assembled at junctions of the inner and outer membrane. It is anticipated that one will be able to discern in greater ultrastructural detail the membranes at which the antigen is translocated. Novel membrane fixation and isolation techniques will have to be established and employed in a combination of sensitive microscopic techniques and immuno- and enzyme localization methods. These developments will make it possible to explore questions pertaining to the maintenance and structural organization of microbial capsules and the functional interaction of polysaccharides with natural surfaces, man-made substances and drugs.

Measurement of the rotational behaviour of virus particles during adsorption to the host cell surface.

The rotational diffusion of bacteriophage epsilon 15 was measured before and after virus adsorption to outer membrane vesicles of the host Salmonella anatum. The virus capsid was labeled with eosin isothiocyanate, and the decay of transient dichroism following dye excitation by pulses of plane-polarized light was measured. From the data, the rotational diffusion constant of the unadsorbed virion and its hydrodynamic diameter were estimated and found to be consistent with electron microscopic measurements of the capsid dimensions. Addition of outer membrane vesicles of S. anatum to the virus suspension revealed the immobilization of the virus particles on the membrane surface.

Automated electrokinetic analysis; description and application in virology and cell biology.

The electrophoretic mobility (EPM) of selected macromolecules in solution was shown to be accurately determined using an automated electrokinetic analyzer, the PenKem S3000. In addition, the S3000 was used to monitor the effects of T4 phage infection on the EPM of Escherichia coli B. EPM, expressed as the ratio of velocity in microns/sec to field strength in V/cm, was measured for calf thymus DNA, for pneumococcal capsular polysaccharide serotype 3 (PCP-3), and for bovine serum albumin (BSA) unbound in solution; values of -3.05, -2.736 and -1.176, respectively, were obtained. The EPM of these macromolecules remained the same when they were bound to latex beads. The S3000 may therefore be suitable for measurement of the EPM of unbound macromolecules. The EPM of T4 phage in solution was measured to be -1.203. However, both the zwitterionic latex-bound T4 phage as well as T4 phage disrupted by ultrasonication exhibited an EPM of approximately -2.50, suggesting to us that binding to zwitterionic latex may cause release of phage DNA. The notion that phage DNA is responsible for the increased negative charge was supported by the observation that the EPM of E. coli B increased to the level of free DNA within 5 min when E. coli B (the host cell for phage T4) had been exposed to 10 phage particles per cell. Electronmicrographs of phage infected E. coli B cells showed numerous strands of free DNA at the bacterial surface. It is concluded that the S3000 not only measures the EPM of macromolecules in solution but that the instrument can be used also to monitor the behavior of the host cell surface in response to attachment of viral particles.

Zones of membrane adhesion in the cryofixed envelope of Escherichia coli.

The envelopes of Escherichia coli B and E. coli K29 were examined using cryofixation and freeze substitution. Emphasis was directed toward the question whether membrane adhesion zones (which connect inner membrane (IM) and outer membrane (OM) after plasmolysis in 10-20% sucrose) can be visualized with the use of cryotechniques. Plasmolysis in 10-20% sucrose was observed to have no effect on cell viability. We found that simple plunge-freezing methods preserve adhesion sites, whereas these sites were not observed after impact-freezing. Also, plasmolysis "bays," visible in light microscopic preparations of living cells, were seen to be maintained intact after plunge-freezing. Employment of photocrosslinking with UV-flashes before or after plasmolysis showed a significant increase in the number of adhesion areas compared to noncrosslinked specimens. To control the contact speed of the specimen during immersion into the cryogen, a hollow rotor was constructed in which the cryogenic liquid is moving at desired high speeds. Adhesion sites presented themselves in the plasmolyzed cell as sites of close contact of the outer and inner membrane, an arrangement that would leave very limited space for peptidoglycan layers at the contact site of the two membranes. Adhesion sites may occur either as single, isolated sites or within stretches of IM/OM apposition where they appear to function as "spot welds" between the two membranes. Exposure of cells to sucrose concentrations of 35% caused rupture of adhesions with cytoplasmic fragments remaining attached to the envelope. The cryofixation procedures described here do not presently yield the number of membrane adhesions obtainable with conventional aldehyde fixation. However, since the combination of millisecond photocrosslinking and cryofixation of plasmolyzed cells resulted in a higher membrane stabilization and in an increase of the number of adhesion sites, this combination appears to be a useful tool for the analysis of sensitive membrane structures.

Adsorption of bacteriophages to adhesions between wall and membrane of Escherichia coli.

In plasmolyzed Escherichia coli, wall and membrane adhered to one another at 200 to 400 localized areas. This number of specialized wall areas per cell was of the same order of magnitude as the total number of bacteriophage receptors. When bacteriophages T1 to T7 were adsorbed to the bacteria, they were seen to attach almost exclusively to these areas. Comparisons of the number of adsorbed phage particles observed in ultrathin sections and the expected number of phages per cell were in agreement. These results suggest a sharing of receptive areas by the various phages. Adsorption to the wall-membrane associations would permit the virus to release its nucleic acid at an area closest to the cell's protoplasmic contents.

Response of Cell Walls of Escherichia coli to a Sudden Reduction of the Environmental Osmotic Pressure.

The rate of survival after osmotic shocks was found to be dependent on the state of growth. When growing logarithmically, Escherichia coli was about 20 to 100 times more sensitive to an abrupt decrease of the environmental osmotic pressure than when it was in the stationary phase. Subjecting rapidly growing cells to such a treatment caused fingerlike extrusions to emerge from the bacterial wall. Our results suggest that underneath these extrusions the rigid layer of the wall contains weak areas which appear as discontinuities or gaps when viewed in an electron microscope. After exposure to osmotic shock, the gaps became wider. We concluded that the gaps represent sites of mucopolymer synthesis where the rigid structure has temporarily been opened by hydrolytic enzymes to allow for the insertion of new wall material into the older portions of the wall.

Tubular spinae are long-distance connectors between bacteria.

The marine pseudomonad D71 (NCMB 2018) ['Spinomonas maritima'] can be induced to produce long tubular surface appendages (spinae) in a growth medium of low osmolarity. In general, spina-carrying cells show these appendages with open distal ends. We examined cultured cells by scanning and transmission electron microscopy, using both critical-point drying and thin sectioning after embedding with agarose protection. By scanning electron microscopy, spinae were observed that connected cells over distances of several micrometers. Ultrathin sections often revealed an additional layer outside the outer membrane, resembling an S-layer. The inner and outer cell membranes were often joined at spina-insertion areas. Furthermore, evidence was found in ultrathin sections for uninterrupted tubes connecting two cells over a distance of up to 7 microns. We propose, therefore, that spinae form the framework for wide open cell clusters; we hypothesize that these spinae might also permit an exchange of cell-to-cell signals.

Anionic sites on the envelope of Salmonella typhimurium mapped with cationized ferritin.

Binding of either ferritin (F) or cationized ferritin (CF) was employed to indicate the surface charge of the envelope of mainly two Salmonella typhimurium strains (395 MR10, a Rd-mutant, and LT2-M1, a UDP-galactose-4-epimerase-less mutant). Lowering the pH from 7 to 4 decreased binding of CF, but increased binding of F. At low concentrations, the distribution of CF on S. typhimurium 395 MR10 was in general random, with individual ferritin molecules often forming clusters of two or three particles. At ionic strengths of 0.25M NaCl, ferritin produced distinctive, larger clusters at relatively few sites (10-50/cell). Addition of galactose to cultures of growing S. typhimurium, LT2-M1 reduced the binding of CF in 1-10 min, and numerous ferritin-free areas became visible. Possibly this is caused by a pluri-focal reduction in the negative cell surface charge that was generated at the multiple sites of export of new, smooth-type lipopolysaccharide, which either exhibits lesser charge or masks a preexisting surface charge. Dividing cells may show unequal charges on the prospective daughter cells, and the difference in the capacity for ferritin adsorption of both daughter cells is sharply separated at the division site.

Effects of bacteriophage fd infection on Escherichia coli HB11 envelope: a morphological and biochemical study.

Phage fd-infected host bacteria revealed three characteristic changes in their envelope. (i) The preferred cleavage plane during freeze-fracturing shifted from the inner to the outer membrane (OM). (ii) The total lipids of the OM of the infected cells increased by 25% without major alterations in the relative concentration of phospholipids. We propose that such an increase would to some extent contribute to the change in the freeze-fracture behavior of the OM; however, additional factors will have to play a role in the apparent fracture resistance of the inner membrane. (iii) Ultrathin sectioning and immunolabeling methods revealed that extrusion of fd phages takes place at membrane adhesion sites of the infected cells.

Lanthanide accumulation in the periplasmic space of Escherichia coli B.

Treatment of growing Escherichia coli B with lanthanide ions [lanthanum(III), terbium(III), and europium(III)] and subsequent aldehyde-OsO4 fixation caused areas of high contrast to appear within the periplasm (the space between inner and outer membrane of the cell envelope). X-ray microanalysis of ultrathin sections of Epon-embedded or acrylic resin-embedded cells revealed the presence of the lanthanide and of phosphorus in the areas, whose contrast greatly exceeded that of other stained structures. Comparatively small amounts of the lanthanide were also present in the outer membrane and in the cytoplasm. The distribution of the periplasmic areas of high contrast was found to be random and not clustered at areas of current or future septum formation. Irregular cell shapes were observed after lanthanide treatment before onset of fixation. In contrast to glutaraldehyde-OsO4 fixation, glutaraldehyde used as the sole fixer caused a scattered distribution of the lanthanide. Cryofixation (slam-freezing) and freeze substitution revealed a lanthanum stain at both the periplasm and the outer part of the outer membrane. Deenergization of the cell membrane by either phage T4 or carbonyl cyanide m-chlorophenylhydrazone abolished the metal accumulation. Furthermore, addition of excess calcium, administered together with the lanthanide solution, diminished the quantity and size of areas of high contrast. Cells grown in media of high NaCl concentration revealed strongly stained areas of periplasmic precipitates, whereas cells grown under low-salt conditions showed very few high-contrast patches in the periplasm. Terbium treatment (during fixation) enhanced the visibility of the sites of inner-outer membrane contact (the membrane adhesion sites) in plasmolized cells, possibly as the result of an accumulation of the metal at the adhesion domains. The data suggest a rapid interaction of the lanthanides with components of the cell envelope, the periplasm, and the energized inner membrane.

Structure of Escherichia coli after freeze-etching.

Survival of Escherichia coli, quick-frozen under conditions similar to those employed for freeze-etching, is close to 100%. For determination of cell shrinkage, the diameters of freeze-etched E. coli cells (average, 0.99 mum) were compared with those of preparations after negative staining and after ultrathin sectioning. Negatively stained cells measured from 0.65 to 1.0 mum in diameter, and ultrathin sections showed average cell diameters of 0.70 mum. Freeze-etched replicas of logarithmically growing, as well as stationary, E. coli B cells revealed a smooth, finely pitted cell surface in contrast to cell surfaces seen with other preparative methods. The frozen cell wall may cleave in two planes, exposing (i) a smooth fracture face within the lipid layer and (ii) in rare instances an ill-defined particulate layer. Most frequently, however, cleavage of the envelope occurred between wall and protoplasmic membrane; large areas of the membrane were then exposed and showed a surface studded with predominantly spherical particles, an appearance which did not significantly change when the cells were fixed in formaldehyde and osmium tetroxide before freeze-etching. The distribution of these particles differed between logarithmically growing cells and stationary cells.

Diffusion constant and dimension of bacteriophage phi X174 as determined by self-beat laser light spectroscopy and electron microscopy.

The diffusion constant of bacteriophage phiX174 was determined by laser light self-beat spectroscopy. The method allows one to establish the diffusion constant in comparatively short time and with high accuracy. From the diffusion constant D(37) = 1.96 +/- 0.08 x 10(-7) cm(2)/s, the size of the virus particles within their aqueous milieu can be calculated. For phiX174, a diffusional diameter of 31.4 +/- 1.0 nm was found, in good agreement with measurements of diameters of freezeetched (32.3 +/- 1.8 nm) and negatively stained particles (33.8 +/- 2.1 nm), provided that the entire spikes of the virion are included. Other isometric viruses may show a complex interaction of the virion with the surrounding water and its ions.

Phosphoglycerides and phospholipase C in membrane fractions of Escherichia coli B.

The phospholipid composition and the phospholipase C activity of envelope fractions of Escherichia coli B were determined with special consideration of fractions containing sites at which an attachment of inner and outer membranes had been observed in the electron microscope (Int.M). Phosphoglycerides labeled with [14C]palmitic acid and [3H]serine were extracted from membrane fractions and identified by two-dimensional thin-layer chromatography. The amount of phosphatidylethanolamine was highest in the outer membrane, whereas the amounts of phosphatidylglycerol and cardiolipin were highest in the inner membrane. The Int.M fractions were observed to have concentrations of phospholipids intermediate to those of the inner and outer membranes. This result supports the assumption that a concentration gradient of inner membrane-outer membrane lipids might exist at the membrane contact sites. The highest phospholipase C activity was detected in the inner membrane and Int.M fractions. The presence of phospholipase C and other lipolytic enzymes in the Int.M fractions suggests a possible involvement of adhesion sites in lipid metabolism, adding a further set of activities to the function of these domains.