An eight-electrode conductance catheter previously developed by us and used to determine stroke volume in dogs was applied in human beings and dogs to measure absolute left ventricular volume quantitatively. For calibration we developed the formula V(t) = (l/a)(L2/Yb)G(t) -

From 15 May to 15 December 1994, 2943 patients suspected of having contact dermatitis (1955 women, 988 men) were patch tested with methyldibromoglutaronitrile 0.3%, 0.1% and 0.05% pet. 119 patients (4.0%; women 4.1%), men 3.8%) proved to be allergic. 71% of the reactions were considered to be relevant. In 2/3 of the patients, causative products were cosmetics, in 1/3 moistened toilet tissues. Testing with methyldibromoglutaronitrile at lower concentrations (0.05% and 0.1%) and with commercial allergens (Euxyl® K 400 and methyldibromoglutaronitrile, both containing methyldibromoglutaronitrile 0.1 % >), resulted in a number of false-negative reactions. All preserv atives in the European standard series had lower scores than the 4% positive reactions to methyldi bromoglutaronitrile (formaldehyde 2.0%), MCI/MI (Kathon® CG) 3.2%, parabens 1.0%o, quaternium-15 1.3%). It is concluded that methyldibromoglutaronitrile (present in the commercial pre servative Euxyl® K 400) is an important contact allergen in the Netherlands in cosmetics and moistened toilet tissues. It should be added to cosmetics series and to proctological series. The optimal test concentration is unknow, but may be 0.3% pet. The concentration o f 0.1% methyldi bromoglutaronitrile in the currently available commercial allergens appears to be too low, resulting in a number of false-negative reactions.

To estimate the frequency of false-negative reactions to the fragrance mix, the 8 constituents of the mix in concentrations of 5% (2% for cinnamic aldehyde) were added to the European standard series for routine testing. Patients with positive reactions to individual ingredients in the absence of a reaction to the mix were retested with serial dilutions. In a 4-month period, 677 patients were tested. 61 (9%) reacted to the mix and to 1 or more of the ingredients. 4 patients (0.6% of all patients tested and 6.2% of the patients allergic to fragrances) had false-negative reactions to the mix. They were allergic to cinnamic alcohol, geraniol, isoeugenol and oak moss (1 reaction each), in the absence of a reaction to the fragrance mix. It is concluded that the currently used concentration of the mix (8 x 1%) not infrequently results in false-negative reactions, and that further research should be done to overcome this problem.

No abstract

We have used site-directed mutagenesis in combination with a battery of biophysical techniques to probe the stability and folding behavior of a small globular protein, the histidine-containing phosphocarrier protein (HPr). Specifically, the four phenylalanine residues (2, 22, 29, and 48) of the wild-type protein were individually replaced by single tryptophans, thus introducing site-specific probes for monitoring the behavior of the protein. The folding of the tryptophan mutants was investigated by NMR, DSC, CD, intrinsic fluorescence, fluorescence anisotropy, and fluorescence quenching. The heat-induced denaturation of all four mutants, and the GdnHCl-induced unfolding curves of F2W, F29W, and F48W, can be fitted adequately to a two-state model, in agreement with the observations for the wild-type protein. The GdnHCl unfolding transitions of F22W, however, showed the accumulation of an intermediate state at low concentrations of denaturant. Kinetic refolding studies of F2W, F29W, and F48W showed a major single phase, independent of the probe used (CD, fluorescence, and fluorescence anisotropy) and similar to that of the wild-type protein. In contrast, F22W showed two phases in the fluorescence experiments corresponding to the two phases previously observed in ANS binding studies of the wild-type protein [Van Nuland et al. (1998) Biochemistry 37, 622-637]. Residue 22 was found from NMR studies to be part of the binding interface on HPr for ANS. These observations indicate that the second slow phase reflects a local, rather than a global, rearrangement from a well-structured highly nativelike intermediate state to the fully folded native state that has less hydrophobic surface exposed to the solvent. The detection of the second slow phase by the use of selective labeling of different regions of the protein with fluorophores illustrates the need for an integrated approach in order to understand the intricate details of the folding reactions of even the simplest proteins.