Recommendation for a test battery for the ecotoxicological evaluation of the environmental safety of construction products.

The European Construction Products Regulation allows Member States to adopt rules for evaluating the environmental impact of their buildings. The aim of the project was to develop recommendations for a test battery for the ecotoxicological assessment of the environmental impact of construction products for outdoor use and contribute to the European harmonization of test methods. From a shortlist of 39 products 20 products were included in the ecotoxicological testing program. Monolithic and plate-like construction products were eluted in the Dynamic Surface Leaching test (DSLT) in accordance with CEN/TS 16637-2, granular products were eluted in a one stage batch test in accordance with DIN EN 12457-1. The eluates were examined in four aquatic toxicity tests (algae, daphnia, luminescent bacteria, fish eggs), a genotoxicity test (umu test) and in the respirometer test (OECD 301 F). Here, low to very high ecotoxicity was observed (up to a dilution factor of 1536). Six out of 8 eluates, whose TOC exceeded 10 mg L-1 showed a good biodegradability above 75%. The intra-laboratory repeatability of the Lowest Ineffective Dilution (LID) usually was within ±1 dilution steps (ecotoxicity tests) and ±2 dilution steps (leaching and ecotoxicity tests). This is acceptable, when considering that the overall variability of sample preparation, leaching test, and bioassays add up. The conclusions lead to practical recommendations for a suitable combination of leaching and ecotoxicity tests.

Inhibin A is down-regulated during chemotherapy in patients with breast cancer.

BACKGROUND: Inhibins are dimeric glycoproteins, composed of an alpha-subunit (INH-α) and one of two possible beta-subunits (ßA or ßB), with substantial roles in human reproduction and in endocrine-responsive tumours. Aims of this study were to determine the serological measurement of inhibin A (α-ßA) in breast cancer patients during chemotherapy. PATIENTS AND METHODS: A series of 30 breast cancer patients who underwent standardised chemotherapy were prospectively evaluated before chemotherapeutic treatment as well as four weeks after chemotherapy and two years after chemotherapy for the serological expression of inhibin A. For statistical analysis the Wilcoxon rank sum test was used for paired samples. Statistical significance was assumed at p<0.05. RESULTS: The concentration of inhibin A showed a significant decrease between data obtained before chemotherapy and after chemotherapy (p<0.005) and two-year follow-up (p<0.001). Interestingly, there were no differences in inhibin A concentrations between the four-week and two-year follow-up (p=0.744). DISCUSSION: Chemotherapy significantly decreases inhibin A concentration during chemotherapy. This might reflect a suppression of ovarian function, being also a marker for chemotherapy-induced amenorrhoea. Moreover, it has been suggested that inhibin A might be a tumour marker for breast cancer, and therefore a sudden increase in its concentration might be indicative of breast cancer recurrence.

[Extradural lipomatosis after long-term treatment with steroids]. / Extradurale Lipomatose nach langjähriger Steroidbehandlung.

Epidural lipomatosis is a rare but severe complication of long-term corticoid treatment. A female who was treated with oral corticoids for more than 4 years developed progressive paraparesis over the course of 2 years. As causal for the clinical symptoms we found a massive epidural lipoma of the thoracic spine. Neurosurgical intervention was necessary.

Effect of buffer conditions on the position of tRNA on the 70 S ribosome as visualized by cryoelectron microscopy.

The effect of buffer conditions on the binding position of tRNA on the Escherichia coli 70 S ribosome have been studied by means of three-dimensional (3D) cryoelectron microscopy. Either deacylated tRNAfMet or fMet-tRNAfMet were bound to the 70 S ribosomes, which were programmed with a 46-nucleotide mRNA having AUG codon in the middle, under two different buffer conditions (conventional buffer: containing Tris and higher Mg2+ concentration [10-15 mM]; and polyamine buffer: containing Hepes, lower Mg2+ concentration [6 mM], and polyamines). Difference maps, obtained by subtracting 3D maps of naked control ribosome in the corresponding buffer from the 3D maps of tRNA.ribosome complexes, reveal the distinct locations of tRNA on the ribosome. The position of deacylated tRNAfMet depends on the buffer condition used, whereas that of fMet-tRNAfMet remains the same in both buffer conditions. The acylated tRNA binds in the classical P site, whereas deacylated tRNA binds mostly in an intermediate P/E position under the conventional buffer condition and mostly in the position corresponding to the classical P site, i. e. in the P/P state, under the polyamine buffer conditions.

Small angle scattering in ribosomal structure research: localization of the messenger RNA within ribosomal elongation states.

Besides EM and biochemical studies small angle scattering (SAS) examinations have contributed significantly to our current knowledge about the ribosomal structure. SAS does not only allow the validation of competing models but permits independent model building. However, the major contribution of SAS to ribosomal structure research derived from its ability to reveal the spatial distribution of the individual ribosomal components (57 in the E. coli ribosome) within the ribosomal structure. More recently, an improved scattering method (proton-spin contrast variation) made it possible also to address the question of mapping functional ligands in defined ribosomal elongation states. Here, we review the contributions of SAS to the current understanding of the ribosome. Furthermore we present the direct localization of a small mRNA fragment within 70S elongation complexes and describe its movement upon the translocation reaction. The successful mapping of this fragment comprising only about 0.6% of the total mass of the complex proves that proton-spin contrast-variation is a powerful tool in modern ribosome research.

Ribosomal protection from tetracycline mediated by Tet(O): Tet(O) interaction with ribosomes is GTP-dependent.

Tet(O) mediates tetracycline resistance by protecting the ribosome from inhibition. A recombinant Tet(O) protein with a histidine tag was purified and its activity in protein synthesis characterized. Tetracycline inhibited the rate of poly(Phe) synthesis, producing short peptide chains. Tet(O)-His was able to restore the elongation rate and processivity. 70S ribosomes bound tetracycline with high affinity. Tet(O)-His in the presence of GTP, but not GDP or GMP, reduced the affinity of the ribosomes for tetracycline. Non-hydrolyzable GTP analogs in the presence of the factor were also able to interfere with tetracycline binding. Ribosomes increased the affinity of Tet(O)-His for GTPgammaS. Tet(O), 70S ribosomes and GTPgammaS formed a complex that could be isolated by gel filtration. The GTP conformer is the active form of Tet(O) that interacts with the ribosome. GTP binding is necessary for Tet(O) activity.

Escherichia coli 70 S ribosome at 15 A resolution by cryo-electron microscopy: localization of fMet-tRNAfMet and fitting of L1 protein.

Cryo-electron microscopy of the ribosome in different binding states with mRNA and tRNA helps unravel the different steps of protein synthesis. Using over 29,000 projections of a ribosome complex in single-particle form, a three-dimensional map of the Escherichia coli 70 S ribosome was obtained in which a single site, the P site, is occupied by fMet-tRNAfMet as directed by an AUG codon containing mRNA. The superior resolution of this three-dimensional map, 14.9 A, has made it possible to fit the tRNA X-ray crystal structure directly and unambiguously into the electron density, thus determining the locations of anticodon-codon interaction and peptidyltransferase center of the ribosome. Furthermore, at this resolution, one of the distinctly visible domains corresponding to a ribosomal protein, L1, closely matches with its X-ray structure.

Ribosomal tRNA binding sites: three-site models of translation.

The first models of translation described protein synthesis in terms of two operationally defined tRNA binding sites, the P-site for the donor substrate, the peptidyl-tRNA, and the A-site for the acceptor substrates, the aminoacyl-tRNAs. The discovery and analysis of the third tRNA binding site, the E-site specific for deacylated tRNAs, resulted in the allosteric three-site model, the two major features of which are (1) the reciprocal relationship of A-site and E-site occupation, and (2) simultaneous codon-anticodon interactions of both tRNAs present at the elongating ribosome. However, structural studies do not support the three operationally defined sites in a simple fashion as three topographically fixed entities, thus leading to new concepts of tRNA binding and movement: (1) the hybrid-site model describes the tRNAs' movement through the ribosome in terms of changing binding sites on the 30S and 50S subunits in an alternating fashion. The tRNAs thereby pass through hybrid binding states. (2) The alpha-epsilon model introduces the concept of a movable tRNA-binding domain comprising two binding sites, termed alpha and epsilon. The translocation movement is seen as a result of a conformational change of the ribosome rather than as a diffusion process between fixed binding sites. The alpha-epsilon model reconciles most of the experimental data currently available.

Structure of the elongating ribosome: arrangement of the two tRNAs before and after translocation.

The ribosome uses tRNAs to translate the genetic information into the amino acid sequence of proteins. The mass ratio of a tRNA to the ribosome is in the order of 1:100; because of this unfavorable value it was not possible until now to determine the location of tRNAs within the ribosome by neutron-scattering techniques. However, the new technique of proton-spin contrast-variation improves the signal-to-noise ratio by more than one order of magnitude, thus enabling the direct determination of protonated tRNAs within a deuterated ribosome for the first time. Here we analyze a pair of ribosomal complexes being either in the pre- or post-translocational states that represent the main states of the elongating ribosome. Both complexes were derived from one preparation. The orientation of both tRNAs within the ribosome and their mutual arrangement are determined by using an electron microscopy model for the Escherichia coli ribosome and the tRNA structure. The mass center of gravity of the (tRNA)2mRNA complex moves within the ribosome by 12 +/- 4 A in the course of translocation as previously reported. The main results of the present analysis are that the mutual arrangement of the two tRNAs does not change on translocation and that the angle between them is, depending on the model used, 110 degrees +/- 10 degrees before and after translocation. The translocational movement of the constant tRNA complex within the ribosome can be described as a displacement toward the head of the 30S subunit combined with a rotational movement by about 18 degrees.

Solution scattering structural analysis of the 70 S Escherichia coli ribosome by contrast variation. II. A model of the ribosome and its RNA at 3.5 nm resolution.

Selectively deuterated 70 S E. coli ribosomes and isolated 30 S and 50 S subunits were analyzed by X-ray and neutron solution scattering. The resulting contrast variation data set (42 curves in total) was proven to be consistent in describing the ribosome as a four-phase system composed of the protein and rRNA moieties of both subunits. This data set thus provides ten times more information than a single scattering curve. A solid body four-phase model of the 70 S ribosome at low resolution was built from the envelope functions of the 30 S and 50 S subunits and of those of the corresponding RNA moieties. The four envelopes were parameterized at a resolution of 3.5 nm using spherical harmonics and taking into account interface layers between the phases. The initial approximation for the envelopes of the subunits was taken from electron microscopic data presented recently by J. Frank and co-workers (Albany); the rRNA envelopes were initially approximated by spheres. The optimization and the refinement of the model proceeded by non-linear least squares minimization fitting the available experimental data. The refined envelopes of the subunits differ by about 10% from the starting approximation and the shape of the final 70 S model lies between the outer envelopes of the models by Frank and by M. von Heel & R. Brimacombe (Berlin). The rRNA moiety in the 30 S subunit is more anisometric than the subunit itself, whereas the rRNA of the 50 S subunit forms a compact core. The rRNAs protrude to the surfaces of the subunits and occupy approximately 30 to 40% of the corresponding surface areas. X-ray scattering curves of the two main functional elongation 70 S complexes (pre- and post-translocational) differ only marginally from those of the non-programmed ribosomes, suggesting that the low resolution four-phase model is also valid for the elongating 70 S ribosome.

Solution scattering structural analysis of the 70 S Escherichia coli ribosome by contrast variation. I. Invariants and validation of electron microscopy models.

Solutions of selectively deuterated 70 S Escherichia coli ribosomes and of free 30 S and 50 S subunits were studied by neutron scattering using contrast variation. The integrity of the partially deuterated particles was controlled by parallel X-ray measurements. Integral parameters of the entire ribosome, of its subunits and of the protein and rRNA moieties were evaluated. The data allow an experimental validation of the two most recent electron microscopy reconstructions of the 70 S ribosome presented by the groups of J. Frank (Albany) and of M. van Heel & R. Brimacombe (Berlin). For each reconstruction, integral parameters and theoretical scattering curves from the 70 S and its subunits were calculated and compared with the experimental data. Although neither of the two models yields a comprehensive agreement with the experimental data, Frank's model provides a better fit. For the 50 S subunit of van Heel & Brimacombe's model the fit with the experimental data improves significantly when the internal channels and tunnels are filled up. The poorer fit of the latter model is thus caused by its "sponge"-like structure which may partly be due to an enhancement of high frequency contributions in some of the steps of the three-dimensional image reconstruction. It seems therefore unlikely that the ribosome has a "sponge"-like structure with a pronounced network of channels.

Large-scale isolation of proteins of the large subunit from Escherichia coli ribosomes.

A strategy has been developed and optimized that allows the isolation of proteins of the large subunit from Escherichia coli ribosomes and combines the following advantages: speed, applicability for the isolation of milligram amounts of a single protein, and preservation of the biological activity of the proteins. The method consists of the following steps: ion-exchange chromatography on MonoS and MonoQ, gel filtration on Sephadex 75, and salt washes. Eleven proteins can be purified by a single chromatographic step, and a combination of two steps enables the isolation of the other proteins.

Direct localization of the tRNAs within the elongating ribosome by means of neutron scattering (proton-spin contrast-variation).

A new technique for neutron scattering, the proton-spin contrast-variation, improves the signal-to-noise ratio more than one order of magnitude as compared to conventional techniques. The improved signal enables small RNA ligands within a large deuterated ribonucleic acid-protein complex to be measured. We used this technique to determine the positions of the two tRNAs within the elongating ribosome before and after translocation. Using a four-sphere model for each of the L-shaped tRNAs, unequivocal solutions were found for the localization of the mass centre of both tRNAs. The centre of gravity is located in the interface cavity separating the ribosomal subunits near the neck of the 30 S subunit. It moves during translocation by 12(+/-4) A towards the head of the 30 S subunit and slightly towards the L1 protuberance of the 50 S subunit.

The ribosome as affinity matrix': efficient purification scheme for translation factors.

A convenient method to purify each of the non-ribosomal proteins required to translate a native mRNA in vitro is described. In this scheme, the ribosome is used as an 'affinity' matrix to selectively elute the non-ribosomal proteins required for translation that are bound to these particles. Different sets of these proteins can be eluted with solutions of Mg2+ and NH4+ of various concentrations from either 70S, or 30S and 50S particles. A scheme for the purification of each initiation, elongation and release factor and 20 aminoacyl-tRNA synthetases is described. Specific examples of the purification of the initiation (IF-1, IF-2, IF-3) and elongation (EF-Tu and EF-G) factors and for a protein called 'rescue', which affects the association of native ribosomal subunits, are given. A scheme for the purification of EF-P, which stimulates peptide-bond synthesis and one of the W proteins, which permit reconstitution of translation is also described. The procedure markedly simplifies the isolation, in homogeneous form, of all the non-ribosomal proteins required to reconstruct translation.

The elongating ribosome: structural and functional aspects.

We determined the positions and arrangements of RNA ligands within the ribosome with a new neutron-scattering technique, the proton-spin contrast-variation. Two tRNAs were bound to the ribosome in the pre-translocational and the post-translocational state. The mass centre of gravity of both tRNAs resides at the subunit interface of the body of the 30S subunit. Both tRNAs are separated by an angle of 50-55 degrees, and their mutual arrangement does not change during translocation. The mass centre of gravity moves by 13 +/- 3 A (1A = 0.1 nm) during translocation, corresponding well with the length of one codon. Using an RNase-digestion technique, the length of the mRNA sequence covered by the ribosome was determined to be 39 +/- 3 nucleotides before and after translocation. The ribosome moves like a rigid frame along the mRNA during translocation. In contrast, both tRNAs seem to be located on a movable ribosomal domain, which carries the tRNAs before, during, and after translocation, leaving the microtopography of the tRNAs with the ribosome unaltered. This conclusion was derived from an analysis of the contract patterns of thioated tRNAs on the ribosome. The results have led to a new model of the elongation cycle, which reinterprets the features of the previous "allosteric three-sites model" in a surprisingly simple fashion. Finally, a mutational analysis has identified a single nucleotide of the 23S rRNA essential for the peptidyltransferase activity.

Direct shape determination of ribosomal proteins in solution and within the ribosome by means of neutron scattering.

Following the 'strategy of the glassy ribosome' single protonated ribosomal proteins (r-proteins) were reconstituted into deuterated 50S subunits of Escherichia coli. The deuteration of both rRNA and r-proteins were individually adjusted to such a degree that the ribosomal matrix appeared nearly homogeneous with respect to coherent neutron scattering and had a scattering density equivalent to a D2O solution of about 90%. Neutron scattering of ribosomal subunits was recorded in reconstitution buffer containing three different concentrations of D2O around 90% D2O (contrast variation). The signal-to-noise ratio achieved allowed us to make a direct determination of the radii of gyration of r-proteins within the 50S subunit and thus provides the first information relating to the shape of these proteins in situ. We present the radii of gyration of 11 r-proteins incorporated into 50S subunits and of 9 isolated r-proteins in solution. In addition, the data concerning the overall dimensions of the r-proteins we report on indicate that conformational changes of at least two individual r-proteins occur during the assembly process of the ribosome.

Large-scale preparation of fully deuterated cell components. Ribosomes from Escherichia coli with high biological activity.

Some applications of NMR and of neutron scattering require fully deuterated biological material which should be highly active and available in large quantities. These requirements are hardly compatible since full deuteration is achieved easily only if cells are grown in minimal media. This condition used in standard batch fermentation results in both low yields and reduced activities of the biological mass. Here we report a method which combines the apparently incompatible requirements taking advantage of a recent observation according to which the appearance of growth inhibiting extracellular products could be prevented. The method was applied for growing Escherichia coli cells, strain MRE600rif (resistance against high doses of rifampicin is used as selection marker) on partially deuterated media (76% and 84% D2O) with glucose as carbon source and on deuterated acetate and succinate with 100% D2O when full deuteration was to be achieved. The essential point for preserving the log-phase character of the cells is that the cultivation is carried out at substrate limiting conditions thus keeping the growth rate at low levels (for glucose the growth rate, mu < or = 0.35 h-1, for acetate/succinate mu < or = 0.1 h-1) which avoids the accumulation of the substrate or of by-products in the medium. Our data suggest that acetate is a main extracellular component for accompanying or triggering the transition from logarithmic growth to stationary phase of E. coli cells cultivated on glucose as carbon source. The cells were first grown in fed-batch to high cell densities (above 50 g wet cells/l) under conditions of substrate limitations. A steady-flow fermentation followed keeping the growth rate at about mu of 0.1 h-1. Cells were harvested in kg quantities, the extracted ribosomes showed a normal complement of proteins, contained intact rRNA and were fully active. The ribosomal protein and rRNA fractions could be efficiently reconstituted to highly active particles. In the case of full deuteration a matching point of 120% (tentative D2O scale) was achieved. The reported method facilitates the preparation of deuterated biological material for applications in NMR and neutron scattering analysis.