Heinrich Maidhof

Nachweis gentechnischer Veränderungen in Mais mittels PCR

Ein PCR-Nachweis für gentechnisch veränderten Mais «Event 176» der Fa. Ciba-Geigy wurde etabliert. Der Mais enthält Gene, die Selbstschutz gegen den Maiszünsler (Delta-Endotoxin-Gen ausBacillus thuringiensis) und Toleranz gegen das Herbizid Basta (Phosphinothricin-Resistenz-Gen ausStreptomyces hygroscopicus) vermitteln. Zudem enthält der Mais ein Ampicillin-Resistenz-Gen. Für die Amplifikation von Bereichen aus allen drei Genen wurden PCR-Primer entworfen. Mit Hilfe dieser Primer und mit «Event 176»-Mais-DNA als Template konnten die entsprechenden Genbereiche in der PCR amplifiziert werden. Die PCR-Produkte wurden sequenziert, um ihre Identität zu bestätigen. Mit Hilfe der Delta-Endotoxin-PCR wurden, auch in Gegenwart von 104fachem Überschuß nicht gentechnisch veränderter Mais-DNA, fünf haploide Genome der «Event 176»-DNA nachgewiesen. A PCR-test for the genetically modified maize «Event 176» of Ciba-Geigy was established. The maize contains genes conferring resistance to the European corn borer (delta-endotoxin gene fromBacillus thuringiensis) and tolerance to the herbicide Basta (phosphinothricin resistance gene fromStreptomyces hygroscopicus). The maize contains also an ampicillin resistance gene. Primers were designed and using «Event 176»-maize-DNA as template internal regions of the three genes were amplified with PCR. The PCR products were sequenced to confirm their identity. Using the deltaendotoxin primers in PCR down to 5 haploid genomes of «Event 176»-DNA could be detected, even in the presence of a 104fold excess of DNA from non-modified maize.

Dual-Use Research and Technological Diffusion: Reconsidering the Bioterrorism Threat Spectrum

The global security community continues to view a potential bioterrorist event with concern. Kofi Annan, former Secretary General of the United Nations, stated “the most important under-addressed threat relating to terrorism…is that of terrorists using a biological weapon” [1]. The European Commission believes that biological weapons “may have particular attractions for terrorists” [2]. The United States Commission on the Prevention of Weapons of Mass Destruction Proliferation and Terrorism believes it is very likely that a weapon of mass destruction will be used in a terrorist attack by the end of 2013, and that an attack with a biological weapon is more likely than one with a nuclear weapon [3].

Chapter 2 Bacterial peptidoglycan: overview and evolving concepts

Publisher Summary This chapter presents an overview and evolving concepts of bacterial peptidoglycan. The essential cell wall polymer of most eubacteria, peptidoglycan, has attracted and fascinated scientists from many different disciplines as its distinct chemical composition, and its unique role as an “exoskeleton” were discovered more than 30 years ago. Because the mode of action of the most important group of antibacterial drugs, the β-lac-tam antibiotics, and the mechanisms that the bacteria have developed to survive in the presence of these antibiotics, are related to the biosynthesis, three-dimensional structure and morphogenesis of the peptidoglycan, even more efforts have been undertaken to gain a detailed knowledge of the chemistry, and structural features of the murein network. The peptidoglycan is a heteropolymer consisting of glycan strands crosslinked by peptides. It depicts a simple scheme of the primary structure of the building block of the peptidoglycan. By using four chemical linkages the giant network structure of the sacculus is formed.

A novel, “hidden” penicillin-induced death of staphylococci at high drug concentration, occurring earlier than murosome-mediated killing processes

In log-phase cells of staphylococci, cultivated under high, “non-lytic” concentrations of penicillin G, there occurred a novel killing process hitherto hidden behind seemingly bacteriostatic effects. Two events are essential for the apprearance of this “hidden death”: (i) the failure of the dividing cell to deposit enough fibrillar cross-wall material to be welded together, and (ii) a premature ripping up of incomplete cross walls along their splitting system. “Hidden death” started as early as 10–15 min after drug addition, already during the first division cycle. It was the consequence of a loss of cytoplasmic constituents which erupted through peripheral slit-like openings in the incomplete cross walls. The loss resulted either in more or less empty cells or in cell shrinkage. These destructions could be prevented by raising the external osmotic pressure. In contrast, the conventional “non-hidden death” occurred only much later and exclusively during the second division cycle and mainly in those dividing cells, whose nascent cross walls of the first division plane had been welded together. These welding processes at nascent cross walls, resulting in tough connecting bridges between presumptive individual cells, were considered as a morphogenetic tool which protects the cells, so that they can resist the otherwise fatal penicillin-induced damages for at least an additional generation time (“morphogenetic resistance system”). Such welded cells, in the virtual absence of underlying cross-wall material, lost cytoplasm and were killed via ejection through pore-like wall openings or via explosions in the second division plane and after liberation of their murosomes, as it was the case in the presence of low, “lytic” concentrations of penicillin. Bacteriolysis did not cause any of the hitherto known penicillin-induced killing processes.

Two alternative mechanisms of cell separation in staphylococci: one lytic and one mechanical

Abstract Electron microscopy studies revealed two different mechanisms of cell separation in Staphylococcus aureus. Both mechanisms were initiated by the centrifugal lytic action (directed outward from the center) of murosomes, which perforated the peripheral cell wall. Thereafter, during the first type of cell separation, murosomes also lysed large parts of the cross wall proper in the opposite, i.e., centripetal direction, forming spokelike lytic lesions (“separation scars”) next to the most prominent structure of the cross wall, the splitting system. This bidirectional lytic action of murosomes revealed that the staphylococcal cross wall is composed of permanent and transitory parts; transitory parts constituted about one-third of the volume of the total cross wall and seemed to be digested during cell separation. The second mechanism of cell separation was encountered within the splitting system, which has been regarded as the main control unit for lytic cell separation for more than 25 years. The splitting system, however, represents mainly a mechanical aid for cell separation and becomes effective when cell-wall autolytic activities are insufficient.

Biophysical and Biochemical Studies on the Fine Structure of the Sacculi from Escherichia coli and Staphylococcus aureus

The general principles of the chemical structure of the bacterial sacculus and the basic conformational features of its building bricks, — oligosaccharide chains bearing peptide side chains for crosslinking — are well known for a long time (for review see e.g. Schleifer and Kandler, 1972; Labischinski et al., 1985). However, these data gave only rough ideas how the real giant macromolecule murein (synonym: peptidoglycan) is organized to fulfill its functions like protecting the bacterial cell against its own osmotic pressure, maintaining shape, yet allowing growth and division of the cell. For these purposes the sacculus must confer rigidity, flexibility and structural dynamics at the same time. Obviously, the topology of the peptides to be crosslinked plays an important role for constructing a covalently linked sacculus. The enzymes engaged in this job are anchored in the cytoplasmic membrane and should crosslink peptides only, if these come in close spatial contact. For this reason, the helical nature of the murein glycan chains, which forces consecutive peptides along a sugar strand to point approximately to the left, downward, right and upward, largely limits the theoretical constructions of stress bearing, covalently linked networks (topotypes, see Labischinski et al., 1985; Labischinski and Johannsen, 1986; Koch, 1991). To differentiate between the still large number of possible arrangements, experimental data are urgently needed, but difficult to obtain for large, low-ordered macromolecular assemblies like the murein sacculus. This report will focus on two of the few experimental approaches available — small angle neutron solution scattering and HPLC-techniques to study enzymatically produced murein degradation products -, which recently contributed to our knowledge on the architecture of the gram-negative sacculus and on the interrelationships between peptidoglycan crosslinking fine structure and β-lactam resistance in staphylococci, respectively.

Unfinished Business: Efforts to Define Dual-Use Research of Bioterrorism Concern

Biotechnological research poses a special security problem because of the duality between beneficial use and misuse. In order to find a balance between regulating potentially dangerous research and assuring scientific advancement, a number of assessments have tried to define which types of research are especially open to misuse and should therefore be considered dual-use research of special concern requiring rigorous oversight. So far, there has been no common understanding of what such activities are. Here we present a review of 27 assessments focusing on biological dual-use issues published between 1997 and 2008. Dual-use research activities identified by these assessments as being of special concern were compiled and compared. Moreover, from these 27 assessments, the primary research publications explicitly identified as examples of concerning research activities were extracted and analyzed. We extracted a core list of 11 activities of special concern and show that this list does not match with the reasons why primary research publications were identified as being of special concern. Additionally, we note that the 11 activities identified are not easily conducted or replicated, and therefore the likelihood of their being used in a high-tech mass casualty bioterrorism event should be reevaluated.