Report from the European Conference on the Role of Research in Combating Antibiotic Resistance, 2003.

Europe has been at the forefront of efforts to control antibiotic resistance, and this globally important health care problem has prompted numerous recommendations for action at both the national and international levels. Starting in 2002, research on antimicrobial resistance has been considered to be one of the specific objectives of the Sixth Framework Programme (FP6) within the European Union. This report summarises the plenary presentations, as well as the findings of six Working Groups covering specific areas of antibiotic resistance, given at a conference in November 2003 entitled 'The Role of Research in Combating Antibiotic Resistance', co-organised by the European Union and the European Society for Clinical Microbiology and Infectious Diseases, and held in Rome under the patronage of the Italian government.

The European Community strategy against antimicrobial resistance.

In 2001 the European Commission presented a 'Community strategy against Antimicrobial Resistance'. In previous years, the problem was addressed through an increasing number of isolated measures, but in this strategy the Commission outlined a comprehensive European Community approach across all sectors. The strategy consists of fifteen actions in four key areas: surveillance, prevention, research and product development, and international cooperation. An important part of this strategy is the 'Council Recommendation on the prudent use of antimicrobial agents in human medicine'. The Recommendation provides a detailed set of public health actions to contain antimicrobial resistance. This paper presents the eleven points of action of the strategy that are directly related to human medicine, and discusses related European Community activities. Under the new public health programme as well as under the research programme of the European Union, antimicrobial resistance is a key priority.

The adsorption staining technique applied to isolated premessenger ribonucleoprotein particles: a comparison with conventional techniques using electron microscope tomography.

A specific type of premessenger RNP particle, Balbiani ring granules from the dipteran Chironomus tentans, was biochemically isolated and visualized in three dimensions with electron microscope tomography. The particles were prepared for electron microscopy in three different ways: positively stained, negatively stained and adsorption-stained (embedded in polyvinyl alcohol, PVA, and concomitantly stained). The results were compared with those obtained for RNP particles studied in situ in ultrathin sections of plastic-embedded cells. The positively stained particles were compacted and heavily deformed with little or no internal structure. The negatively stained and the adsorption-stained particles were well preserved; the outer contours and the central cavities of the particles were outlined. The internal structure, i.e. the folded 7-nm elementary fibre, could not be recognized in the negatively stained particles. In the adsorption-stained particles, however, the fibre was discernable, although not quite as distinctly demarcated as in the plastic-embedded samples. We conclude that embedding in PVA with concomitant staining with uranyl acetate is a rapid method to obtain both good preservation and staining of isolated RNP particles. The PVA-embedded particles were also found to be sufficiently resistant to irradiation to permit a comprehensive tilt-series to be taken for electron microscope tomography.

The elementary RNP fiber--not the higher order structure--determines the all-or-none disintegration behaviour of Balbiani ring pre-messenger RNP particles upon RNase A treatment.

Balbiani ring premessenger ribonucleoprotein (RNP) particles are built from a 7-nm RNP fiber which is tightly folded into a ring-shaped RNP ribbon. Isolated particles are known to disintegrate in all-or-none fashion upon RNase A treatment. In the present study we investigated whether this mode of disintegration is dependent on an intact particle structure or is inherent in the 7-nm fiber. When treated at low ionic strength, the Balbiani ring (BR) particles lost their higher order structure and the 7-nm fiber was unpacked, as evidenced by sucrose gradient sedimentation and electron microscopy. When treated with RNase A, unfolded as well as intact particles disintegrated in the all-or-none fashion, with similar kinetics and without apparent intermediates. Proteinase K treatment, however, obliterated this pattern: the protein-free particle RNA degraded progressively. As the typical disintegration pattern of the particles was not altered by unfolding, but was lost by deproteinization, the all-or-none mode of disintegration is likely to be a property of the 7-nm RNP fiber.

Demonstration of a 7-nm RNP fiber as the basic structural element in a premessenger RNP particle.

Balbiani ring granules in Chironomus salivary glands represent premessenger ribonucleoprotein (RNP) particles, each containing a 35- to 40-kb message for a secretory polypeptide. Their gross structure can be described as an RNP ribbon bent into a toroid. We now demonstrate that an unfolded, thin RNP fiber is observed after low salt treatment of isolated Balbiani ring granules. Moreover, the thin RNP fiber, 7 nm in diameter, can be revealed as the main structural element in Balbiani ring granules studied in situ in 3-D with electron microscope tomography. It is proposed that the thin RNP fiber consists of a premessenger RNA molecule coiled around a filamentous core of polymeric proteins, which has functional implications for processes such as assembly of RNP, intranuclear degradation of RNA, and delivery of RNA through the nuclear pores.

Higher order structure of Balbiani ring premessenger RNP particles depends on certain RNase A sensitive sites.

Specific premessenger ribonucleoprotein (RNP) particles, the Balbiani ring (BR) granules from Chironomus tentans salivary glands, were treated with RNase A to study the effect of RNA strand breaks on the higher order structure of the particles. Isolated, radioactively labeled BR granules, known to sediment at 300 S, were digested with RNase A and centrifuged in sucrose gradients. The fractionated particles were subsequently analyzed using electron microscopy and caesium chloride centrifugation. At a low RNase concentration, most of the 300 S particles disintegrated completely, and no metastable degradation products were observed. At intermediate RNase concentrations, no 300 S particles were left, but a minor fraction of the BR granules had unfolded and sedimented at 160 S. These granules could represent particles modified during the RNase treatment or represent a more slowly degrading subfraction of the particles. At a high RNase concentration, no RNP particles at all remained in the gradient. The rapid disintegration of the majority of the BR granules was investigated further by electrophoretic analysis of RNA in the remaining particles. During the RNase treatment BR granules, still sedimenting at 300 S, accumulated strand breaks; in fact, as many as 50 to 100 nicks in the 37 kb RNA could be tolerated. It was concluded from RNA analyses that the disintegration of the BR granules was not dependent on any single nick in the RNA, nor on the accumulation of a certain number of nicks, but rather on one or a few critical strand breaks. We propose that there are organizing sequences essential for particle integrity; once these sequences are nicked, the premessenger RNP particles are rapidly and completely degraded.

Isolation and initial characterization of a specific premessenger ribonucleoprotein particle.

A specific type of premessenger ribonucleoprotein (RNP) particle, Balbiani ring (BR) granules, has been isolated from heterogeneous nuclear RNP (hnRNP) in the salivary glands of the dipteran Chironomus tentans. A BR granule contains a single 75S RNA molecule coding for a large secretory protein (Sp1). The isolation procedure is based on the abundance and exceptional size of the BR granules: in EDTA-containing sucrose gradients they sediment as a sharp 300S peak ahead of the remainder of the hnRNP population. The isolated BR granules were identified on the basis of both ultrastructural and biochemical criteria: large spherical particles that contain 75S RNA and BR sequences. A three-dimensional reconstruction of isolated particles by electron microscope tomography further supported the identification of the isolated particles as BR granules. In contrast to the entire hnRNP population, the BR granules exhibited a sharp peak in CsCl gradients with a buoyant density of 1.45 g/cm3. This result indicates that a BR granule consists of 40% RNA and 60% protein by weight, corresponding to a 75S RNA molecule of 12 megadaltons and a total protein content of 18 megadaltons, or about 500 average-sized protein molecules.