Nonlinear acoustic approach to material characterisation of polymers and composites in tensile tests.

The paper reports on experimental study of elastic nonlinearity of polymers and glass fibre-reinforced (GFR-) composites in a wide range of tensile stress applied (up to a fracture limit). Focused slanted transmission mode (FSTM) of air-coupled ultrasound is adapted for remote generation and detection of flexural waves in the samples of plastics. Local noncontact measurements of flexural wave velocity as a function of static strain are used to calculate the second-order nonlinearity parameters beta2 and study their behaviour through a loading cycle. Molecular untangling and crazing phenomena are identified, respectively, with maxima of positive and negative beta2 in thermoplastics. In composites, mechanics of fibre-matrix interaction is considered for brittle and plastic fractures. Hysteresis in velocity variation during loading-unloading cycle is used as an indicator of residual defect accumulation.

Nonlinear modulation technique for NDE with air-coupled ultrasound.

The present study is aimed at expanding flexibility and application area of nonlinear acoustic modulation (NAM-) technique by combining the benefits of noncontact ultrasound excitation (remote locating and imaging of defects) with sensitivity of nonlinear methods in a new air-coupled NAM-version. A pair of focused air-coupled transducers was used to generate and receive (high-frequency) longitudinal or flexural waves in plate-like samples. Low-frequency (LF-) vibrations were excited with a shaker or a loudspeaker. Temporal and spectral analysis of the output signal revealed an extremely efficient nonlinear amplitude modulation and multiple frequency side-bands for sound transmission and flexural wave propagation through cracked defects. On the contrary, a negligible modulation was observed for large and medium scale inclusions and material inhomogeneities (linear defects). A new subharmonic mode of the NAM was observed at high excitation levels. It was also shown for the first time that nonlinear vibrations of cracks resulted in radiation of a very high-order harmonics (well above 100) of the driving excitation in air that enabled imaging of cracks remotely by registration their highly nonlinear "acoustic emission" with air-coupled transducers.

Air-coupled ultrasound inspection of various materials.

Conventional ultrasound inspection, a standard non-destructive testing method, uses a coupling medium (e.g. water) because of impedance mismatch. This liquid contact is a drawback because it prevents inspection of many materials. There is a need, then, for air-coupled ultrasound testing, which is now feasible because of low impedance focused narrow band transducers and sensitive electronics, both of which improve the signal-to-noise ratio. We present results obtained on fibre-reinforced plastics, water sensitive materials (e.g. reinforced ceramics), and "shape adaptive" structures to reveal delaminations, impacts, and growth of internal defects. Actuators embedded in "adaptive" structures are used as transmitters while the receiver records the signals. Thus it is possible to image defect areas and non-linear behaviour of potential defects.

Identification of a stability determinant on the edge of the Tet repressor four-helix bundle dimerization motif.

Isofunctional tetracycline repressor (TetR) proteins isolated from different bacteria show a sequence identity between 38 and 88% of the residues. Their active state is a homodimer formed by a four-alpha-helix bundle as the main interaction motif. We utilize this sequence variation of isofunctional proteins to determine residues contributing to the stability of the four-helix bundle. The thermodynamic stabilities of two TetR proteins with 63% sequence identity were determined by urea-induced reversible denaturation followed by fluorescence and circular dichroism. Both methods yield identical results. The deltaG(o)U (H2O) values are 60 and 75 kJ x mol(-1). We have constructed TetR hybrid proteins derived from these wild types to identify the determinant leading to the 15 kJ x mol(-1) stability difference. Successive size reduction of the exchanged portion yielded two single residues affecting the overall protein stability. The P184Q exchange leads to a more stable protein, whereas the G181D exchange located at the solvent's exposed edge of the four-helix bundle is solely responsible for the reduced stability. Additional mutants based on crystal structures of TetR do not reveal any hint for steric interference of the Asp181 side chain with neighboring residues. Thus, this is an example for the role played by surface-exposed turn residues for the stability of four-helix bundles. We assume that the larger conformational flexibility of Gly and the reduction of the negative surface charge could favor formation of the turn on the edge of the four-helix bundle.

Solvent-exposed residues in the Tet repressor (TetR) four-helix bundle contribute to subunit recognition and dimer stability.

Dimerization specificity of Tet repressor (TetR) can be altered by changes in the core of the four-helix bundle that mediates protein-protein recognition. We demonstrate here that the affinity of subunit interaction depends also on the solvent-exposed residues at positions 128 and 179'-184', which interact across the dimerization surface. TetR(B) and (D), two naturally occurring sequence variants, differ at position 128 with respect to the monomer-monomer distances in the crystal structures and the charge of the amino acids, being glutamate in TetR(B) and arginine in TetR(D). In vivo analysis of chimeric TetR(B/D) variants revealed that the single E128R exchange does not alter the dimerization specificity of TetR(B) to the one of TetR(D). When combined with specificity mutations in alpha10, it is, however, able to increase dimerization efficiency of the TetR(B/D) chimera with TetR(D). A loss of contact analysis revealed a positive interaction between Arg-128 and residues located at positions 179'-184' of the second monomer. We constructed a hyperstable TetR(B) variant by replacing residues 128 and 179-184 by the respective TetR(D) sequence. These results establish that in addition to a region in the hydrophobic core residues at the solvent-exposed periphery of the dimerization surface participate in protein-protein recognition in the TetR four-helix bundle.

Determinants of protein-protein recognition by four helix bundles: changing the dimerization specificity of Tet repressor.

Homo- and heterodimerization is essential for the activity of many proteins, particularly transcription factors. One widely distributed structural motif for protein recognition is the four helix bundle. To understand the molecular details determining specificity of subunit recognition in a dimer formed by a four helix bundle, we investigated Tet repressor (TetR) sequence variants TetR(B) and TetR(D), which do not form heterodimers. We used molecular modeling to identify residues with the potential to determine recognition of subunits. Directed mutagenesis of these residues in TetR(B) by the TetR(D) sequence resulted in chimeric TetR(B/D) repressors with new subunit recognition specificities. The single LS192 exchange in TetR(B/D)192 in the center of the helix bundle leads to a relaxed specificity since this variant dimerizes with TetR(B) and (D). To construct a variant with a new specificity it was not sufficient to mutate the contacting residue, F197, in the other subunit. Instead, it was necessary to exchange two more residues in the vicinity of F197 and S192. The resulting TetR(B/D)188, 192,193,197 forms dimers with TetR(D) but not with TetR(B), indicating that four amino acid exchanges are sufficient to change subunit recognition. These results establish that targeted alterations in the structural complementarity of protein-protein interaction surfaces can be used to construct new recognition specificities. However, it is not sufficient to adjust the complementary residues since the surrounding amino acids contribute essentially to protein-protein recognition.

Fast large-scale purification of tetracycline repressor variants from overproducing Escherichia coli strains.

We constructed a plasmid for overexpression of Tn10 Tet repressor (TetR) by placing a synthetic tetR gene under control of the Pc promoter. Active TetR is expressed up to 30% of the total soluble cell protein. A protocol containing anion-exchange, cation-exchange, and size-exclusion chromatography steps is described for the large-scale purification of milligram amounts of TetR in three days. Cation-exchange chromatography already yields almost homogenous TetR. Purification of about fifty TetR mutants demonstrates that this protocol is generally applicable. No correlation between net charge of TetR variants and elution behaviour was detected for the anion-exchange column. On the other hand, TetR mutants with increased negative charge in their DNA binding domain eluted at lower NaCl concentration from the cation-exchange column. The applicability of this purification protocol to the wide variety of TetR variants suggests that it can be used for the rapid purification of other DNA binding proteins as well.

Deletion mutagenesis of Tn 10 Tet repressor--localization of regions important for dimerization and inducibility in vivo.

The gene for the Tn 10 Tet repressor (TetR) was subjected to deletion mutagenesis. Screening for a transdominant operator-binding negative phenotype yielded 10 mutants with internal deletions. Three deletions extend from residue D5 to residues L41, W75, or Q76, respectively, and two contain deletions of the alpha-helix-turn-alpha-helix DNA-binding motif. Five deletions range from residue K84 to residues between R87 and K98. Since residues from the N-terminus up to position 98 are not necessary for dimerization, this must take place in the C-terminal half of the protein. Ability to dimerize was probed by introducing ochre nonsense codons (oc) at residues G138, H151, E159, I174, or K202. Koc202 shows wild-type in vivo operator-binding and inducibility by tetracycline indicating that the six C-terminal residues of TetR are not important for activity. Mutants with longer C-terminal truncations are inactive and not transdominant. They show reduced steady-state protein levels and are probably impaired in folding and degraded in vivo. Two mutants (delta151-166, delta164-166) with deletions in a region variable in primary structure and length among Tet repressors from different resistance determinants bind tet operator efficiently, but are not inducible by tetracycline. This result indicates that these residues are not important for dimer formation in the operator-binding form.

[Coping with bone marrow transplantation. A contribution to adaptive vs. maladaptive coping]. / Coping bei Knochenmarktransplantation. Ein Beitrag zur Frage des "geeigneten" vs. "ungeeigneten Copings".

The coping strategies of 36 adult leukemia patients admitted for bone marrow transplantation have been investigated systematically by trained raters using audio-taped material from semi-structured interviews. So-called long term survivors with a median survival time of 978 days after BMT displayed significantly lower passive-stoic acceptance and resignation than non-survivors who died within a median time of 229 days after BMT. These copings were of high prognostic value prior to all treatment regimes in strong correlation with a negative hematological prognosis and age. Non-survivors showed significantly higher passive-stoic acceptance and resignation coping. Form of leukemia and other sociodemographic variables or stress had neither a significant impact on coping intensity nor on coping quality. Consequences for further coping research and possible treatment interventions prior to or immediately after BMT are being discussed.

Amino acids determining operator binding specificity in the helix-turn-helix motif of Tn10 Tet repressor.

Each of 22 amino acids in the proposed alpha-helix-turn-alpha-helix operator binding motif of the Tn10 encoded Tet repressor was replaced by alanine and one residue was replaced by valine to determine their role in tet operator recognition by a 'loss of contact' analysis with 16 operator variants. One class of amino acids consisting of T27 and R28 in the first alpha-helix and L41, Y42, W43 and H44 in the recognition alpha-helix are quantitatively most important for wild-type operator binding. These residues are probably involved in the structural architecture of the motif. A second class of residues is quantitatively less important for binding, but determines specificity by forming base pair specific contacts to three positions in tet operator. This property is most clearly demonstrated for Q38 and P39 and to a lesser extent for T40 at the N-terminus of the recognition alpha-helix. The contacted operator base pairs indicate that the N-terminus of the recognition alpha-helix is located towards the palindromic center in the repressor-operator complex. Although the orientation of the recognition alpha-helix in the Tet repressor-tet operator complex is inversed as compared with the lambda- and 434 repressor-operator complexes, the reduced operator binding of the TA27 mutation in the first alpha-helix suggests that the hydrogen bonding networks connecting the two alpha-helices may be similar in these proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

A threonine to alanine exchange at position 40 of Tet repressor alters the recognition of the sixth base pair of tet operator from GC to AT.

The tet operators of two naturally evolved tetracycline resistance determinants differ by a G.C to A.T transition at the sixth base pair. This mutation prevents heterologous recognition of these tet operators by their respective two Tet repressor proteins. The amino acid side chains responsible for this sequence-specific distinction of operators were determined. For this purpose in vitro recombinants of the two tetR genes were constructed. Restriction sites were introduced by oligonucleotide-directed mutagenesis in both genes followed by the exchange of different coding segments between them. The encoded chimeric Tet repressor proteins were expressed and their operator recognition specificity was scored in vivo. Exchanging gradually smaller coding segments led finally to a single amino acid exchange in both genes at position 40 of the primary structures. Each Tet repressor containing Thr at this position recognizes the G.C operator while those with Ala recognize the A.T operator regardless of the rest of the sequences. This result demonstrates clearly that the amino acid 40 of Tet repressor contacts and recognizes base pair 6 of tet operator. Sterical interference of the large Thr side chain with the methyl group of A.T and a possible involvement of the hydroxyl in hydrogen bonding to the operator are discussed as the molecular basis of this differentiation between A.T and G.C base pairs.