Manfred E. Bayer

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.

Penetration of the polysaccharide capsule of Escherichia coli (Bi161/42) by bacteriophage K29

Abstract The interaction between the capsulated Escherichia coli strain of serotype K29 and a capsule K29-specific bacteriophage has been studied, using virus adsorption kinetics and immunological methods in combination with electron microscopy. The adsorption of the phage to capsulated wild-type (w.t.) E. coli is fast, with a rate constant “k” of 1 to 2.5 × 10−8 ml/min at 37°, and 4 × 10−9 ml/min at 0°. Mutant strains with temperature-sensitive defects in production of capsule antigen showed a much reduced virus adsorption rate when grown at permissive temperatures. The virus, being capable of enzymatically hydrolyzing the receptor polysaccharide, destroyed the macromolecular structure of both the isolated polysaccharides (p.s.) and the capsule in vivo. Phage adsorption occurred without release of virus DNA. Virus adsorption carried out with w.t. cells in the presence of isolated p.s. revealed receptor competition in which the isolated p.s. from mutant cells was about 103-fold less efficient than the isolated p.s. from the wild-type cells. Electron microscopy of ultrathin sections enabled us to follow the virion in its travel through the w.t. capsule. To observe this, the capsule had to be stabilized with anti-capsule IgG after phage adsorption. In the capsule of the infected cell, a tunnel-shaped penetration path of the virus became visible; the path of the virus was often but not exclusively unidirectional toward the outer membrane (OM) of the cell. A virus particle that had reached the OM might subsequently move along the surface of the OM or might turn back into the capsule. Movement of the adsorbed virion was halted by anti-capsule IgG which caused trapping of the virus particles. The virion was eventually found to be positioned over one of the adhesion sites at which inner and outer membrane are fused. After 4 min, the virus released its DNA, as judged from a decreased state of filling of the phage heads. The data support a multiple-step adsorption model.

The adsorption of bacteriophage ΦX174 and its interaction with Escherichia coli; a kinetic and morphological study

The dimensions of bacteriophage ΦX174 were measured in order to establish its diffusion constant and to examine the process of virus adsorption to host cells: In the electron microscope the capsid diameter of ΦX174 measured 33.8 ± 2.1 nm in negatively stained preparations (from tip to opposite tip of the capsid spikes), and 26.9 ± 2.1 nm without the spikes. In freeze-etched replicas ΦX174 revealed a capsid of 32.3 ± 1.8 nm diameter composed of 12 capsomeres. From these dimensions the diffusion constant was estimated to be 1.78 × 10−7 ± 0.10 cm2/see in nutrient broth at 37°. In stereomicrographs the orientation of the capsid bound to the host surface appears random, as does the distribution of the virus particles over the cell surface. After freeze-fracturing (avoiding etching) virus particles are not seen; instead, a “bumpy” structure of the fracture plane along the cell surface shows up; etching for 10–30 sec at -100°, however, produces smooth “cell surfaces” revealing the virus with its capsomeres. Freeze-etched intracellular virus particles are devoid of a well-defined capsid structure. In ultrathin sections the adsorbed virus particles measure 24.0 ± 1.5 nm; they appear to be randomly distributed over the cell surface. In order to examine the structure of the adsorption sites in ultrathin sections, a method was developed that kept ≥80% of the virus adsorbed to the cells during fixation, dehydration and embedding. In the ultrathin sections 74% of the adsorbed ΦX particles were seen to be positioned over wall membrane adhesions. The “maximal” adsorption rates were computed from the diffusion constant and compared to the adsorption rates obtained experimentally; these reached 70% of the “maximal” rate. Since the adhesion areas comprise only

The capsid structure of bacteriophage lambda

Abstract The structure and dimensions of bacteriophage λ have been studied with the freezeetching technique and were compared with those of negatively stained virus. The diameter of the phage head measured 58.0 ± 2.5 nm after negative staining, and 61.5 ± 5.5 nm after freeze-etching. In unfixed preparations, freeze-etching revealed a smooth surface of the head, whereas formaldehyde fixation prior to freeze-etching resulted in well-defined capsomeres. The diameter of the individual capsomeres measured 5.6 ± 0.8 nm, their center-to-center distance 7.4 ± 0.8 nm. Micrographs of λ heads in which two vertices were visible (with each vertex showing one central capsomere surrounded by five neighboring capsomeres), allowed one to study the capsomeric positioning on the capsid and to postulate a triangulation umber (T) of 21, with a right-handed skewness. A model was built accordingly. Our structural data together with biochemical and genetic evidence suggest the presence of several, possibly three, capsid protein units per capsomere. Since there was a surprisingly low incidence (⋍8%) of capsid views in our micrographs revealing simultaneously two vertices, a computer was used to draw capsids in random rotation. These idealized conditions increased the chance for seeing simultaneously two vertices to only about 13%. Stereo views of freezeetched particles as well as of the computer-generated models enhanced this chance slightly, mainly by noise reduction and by the three-dimensional perception of the capsid, thus affirming the relative capsomeric positioning. In a number of freezeetched phage tails helices of opposing handedness were observed, whereas the negatively stained tail revealed the image of “stacked discs.”

Effects of receptor destruction by Salmonella bacteriophages ϵ15 and c341

Abstract O-Antigen-specific bacteriophages ϵ15 and c341 cleave the lipopolysaccharide receptor of Salmonella anatum enzymatically before they undergo the final steps of infection, which end with the release of the viral DNA. Employing the electron microscope, we observed a number of effects which phage adsorption and subsequent desorption exert on host cell and virus: (1) After adsorption of high multiplicities of virus particle at 0–4°, the host bacteria can be covered with more than 10 3 virions of either phage ϵ15 or phage c341. (2) Temperature shift-up to 35° caused the majority of the ϵ15 virions to desorb. However, several hundred virus particles per cell remained attached, and most of these showed empty heads. (3) The heads of the desorbed virus particles appeared to remain filled. Cells from which the phages had desorbed failed to adsorb newly added phage. (4) When adsorption of high m.o.i. of ϵ15 was carried out right away at 35°, the number of adsorbed phages was comparable to the number of virions observed at (2) above. In contrast to phage ϵ15, phage c341 virions stayed attached to the host cell at 35° under both conditions (that is, after shift-up or primary adsorption at 35), and most of their heads remained full. When S. anatum was pretreated with the receptor-hydrolyzing enzyme from isolated adsorption organelles of phage E15, the subsequent adsorption of either type of phages was abolished. However, the enzyme treatment failed to release already adsorbed phage e341. The electron microscope data were supported by measurements of the distribution of radioactively labeled E15 before and after desorption. Desorption was accompanied by a 50 to 70% loss of infectivity. DNase treatment reduced to about half the infectious titer of the desorbed virions. We hypothesize that the striking difference in the desorption behavior of the two phages is caused by the differences in the substrate and binding site of the LPS receptors: phage ϵ15, hydrolyzing glycosidic linkages in the backbone of the oligosaccharide, will dislodge adjacent receptor strands with virions attached. In contrast, phage c341 hydrolyzes the O -acetyl side group in the O-antigen subunit, but maintains its binding to the backbone of the LPS.

Chapter 21 Periplasm

Publisher Summary This chapter discusses periplasm. It addresses current views on the structural and functional organization of the periplasm. Periplasm is a concept pertaining to the envelope of Gram-negative bacteria, and comprises the molecules and ions that are localized within the space between the inner membrane (IM) and outer membrane (OM). The periplasmic space can be considered as a trans-shipment region carrying out the traffic between the interior and exterior of the cell. The periplasmic space is accessible in the intact bacterium from the cells environment via three classes of transport pathways: (1) the non-specific protein assemblies, pores of the OM which allow passage of small, hydrophilic solutes; (2) via specific protein channels; and (3) larger molecular weight substrates bound with high affinity and specificity are actively transported across the OM. The periplasm contains a large number of proteins that have been classified in major groups: (1) binding proteins for amino acids, carbohydrates and vitamins; (2) enzymes involved in scavenging and detoxifying activities; and (3) a group of other diverse proteins.

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.