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Cell‐membrane sheets suitable for in‐vitro functional fluorescence studies have been prepared by direct detachment from cell membranes using poly‐L‐lysine‐coated glass slides. The resulting transferred planar membranes conserve the composition as well as most properties of the original plasma membrane; in particular, both membrane leaflets remain fluid, allowing the investigation of diffusion properties of different cellular membrane components. Measurements on membrane sheets offer several advantages as compared to those on living cells. First, access to the intracellular leaflet is obtained, in particular to the intracellular part of membrane proteins and to cytoplasmic membrane‐associated proteins, opening the possibility of labeling them and modulating their properties with membrane impermeable compounds. Second, the cytosolic autofluorescence of the cells is absent, allowing ultrasensitive measurements to be performed down to the single‐molecule level. Third, the complexity of cellular processes occurring at the plasma membrane can be reduced, allowing the sequential investigation of selected events from complex biochemical networks. These advantages are illustrated by ligand‐binding studies on the α1b‐adrenergic receptor. Our results indicate that supported membrane sheets might find a broad application as an ideal in‐vitro system for the elucidation of complex signaling pathways.

Low-light adapted B800 light-harvesting complex 4 (LH4) from Rhodopseudomonas palustris is a complex in which the arrangement of the bacteriochloropyll a pigments is very different from the well-known B800-850 LH2 complex. For bulk samples, the main spectroscopic feature in the near-infrared is the occurrence of a single absorption band at 802nm. Single-molecule spectroscopy can resolve the narrow bands that are associated with the exciton states of the individual complexes. The low temperature (1.2K) fluorescence excitation spectra of individual LH4 complexes are very heterogeneous and display unique features. It is shown that an exciton model can adequately reproduce the polarization behavior of the complex, the experimental distributions of the number of observed peaks per complex, and the widths of the absorption bands. The results indicate that the excited states are mainly localized on one or a few subunits of the complex and provide further evidence supporting the recently proposed structure model.

CdSe quantum dots (QDs) with a high fluorescence quantum yield of 25% and a narrow size distribution were synthesized in a single step in water using glutathione as a stabilizing molecule. The exceptional optical properties enabled for the first time the detection of in-water-prepared single quantum dots at room temperature. For application as fluorescent bioanalytical probes, the QDs were coated with streptavidin. These QDs self-assemble with high contrast on micropatterned biotin while preserving their optical properties and their capability to bind in addition biotinylated molecules, a prerequisite for the development of novel supramolecular structures and bioassays.

To study the clinical significance of metabolic alterations as measured in vivo with proton MRS in the striatum of patients with Huntington's disease (HD).

We have reported the purification and characterization of a protein that behaves as a liver growth factor, showing activity either in vivo or in vitro [Díaz-Gil et al. (1986) Biochem. J. 235, 49-55]. In the present paper, we identify this liver growth factor (LGF) as an albumin-bilirubin complex. This conclusion is supported by the results of chemical and spectroscopic characterization of this protein as well as by experiments in vivo. Incubation of albumin isolated from normal rats with bilirubin/albumin molar ratios (r) resulted (when r = 1 or 2) in a complex with liver DNA synthesis promoter activity identical with that of LGF. The exact amount of bilirubin bound to albumin was assessed by fluorescence and c.d. spectra. This albumin-bilirubin complex showed the same dose-dependence profile as LGF either at low or high dose of protein injected per mouse. Both LGF and albumin-bilirubin complex produced similar increases in the mitotic index of mouse hepatocytes in vivo. A new mechanism for the onset of the hepatic regenerative process is proposed.

Ribonuclease Inhibitor (RI) has been purified from pig testis. It contains 30 half-cystines whose oxidation affects its ability to bind and inhibit ribonuclease (RNase). By N-terminal sequence analyses testis RI showed to be identical to that from porcine liver, for which a characteristic all-or-none type of SH-oxidation by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) has been reported (Fominaya, J.M., and Hofsteenge, J. (1992) J. Biol. Chem. 257, 24655-24660). Under comparable reaction conditions, testis RI bound to RNase A did not exhibit this particular type of oxidation; instead, bound RI got intermediate oxidation degrees (up to 14 thiols oxidized per RI moiety) without dissociating from RNase. Moreover, RNase bound to partially oxidized RI was able to express some (15%) of its potential activity (active complex). Only when DTNB treatments accounted for complex dissociation (> 14 thiols oxidized per RI moiety) the released RI molecules exhibited the all-or-none oxidation behavior. By both kinetic and circular dichroism analyses, conformational changes have been evidenced for the transition from the inactive to the active form of RI-RNase complex. Relaxation of RI-RNase binding without major alterations in RI structure is proposed as responsible for complex activation. The results are discussed in terms of a model for the reversible regulation of RNase activity mediated by the redox status of RI.

A general treatment of very tight-binding inhibition is described. It was applied to purified endogenous RNAase inhibitor from rat testis. This treatment discriminates among the different types of inhibition and allows for calculation of the inhibition parameters. When very tight-binding inhibitions are studied at similar molar concentrations of both enzyme and inhibitor, a further approach is required. This is also described and applied to the RNAase inhibitor. A Ki value of 3.2 x 10(-12) M was found for this inhibitor protein. On the basis of this result, it was considered inappropriate to classify this type of inhibitor in terms of competitive or non-competitive, as has been done for such inhibitors so far. Functional consequences of this analysis are discussed for the RNAase-RNAase inhibitor system.

Summary: This paper outlines an analytical method for computing normal and shear stresses generated in a curved laminated beam under bending loads. Each cross section is assumed to be symmetrical and loads are applied in the plane of symmetry. We build a statically admissible stress field in order to plot normal and shear stress distributions.

The initial event in bacterial photosynthesis is the absorption of sunlight by an array of pigment-protein complexes, the so called light-harvesting complexes (LH). The absorbed light energy is then efficiently transferred to the reaction centre (RC), where the charge separation and thus the primary conversion into chemical energy takes place. The photosynthetic purple bacterium Rhodopseudomonas (Rps.) acidophila usually contains two types of light-harvesting complexes, light-harvesting complex 1 (LH1) and light-harvesting complex 2 (LH2). When grown under low-light and/or low temperature conditions, an additional spectroscopic variant of LH2 is expressed: the light-harvesting complex 3 (LH3) [1, 2]. The high-resolution crystal structure of LH3, which is also denoted as B800-820, has recently been resolved and revealed a C9-symmetry similar to that of LH2, thus with the bacteriochlorophyll a (BChl a) pigments arranged in two concentric rings. The outer ring of LH3 has nine well-separated BChl a pigments, that absorb at around 800 nm (B800 ring), while the eighteen closely interacting BChl a pigments in the inner ring absorb at around 820 nm (B820 ring). The B800 and B820 pigments are arranged with their molecular plane perpendicular and parallel to the symmetry axis of the complex, respectively. Since the overall structures of LH2 and LH3 are very similar, the interesting question to address is what causes the shift of the long-wavelength absorption band from 850 nm in LH2 down to 820 nm in LH3. It has previously been demonstrated that optical single-molecule spectroscopy at low temperature provides direct insight into relevant parameters determining the electronic structure of LH2 and LH1 complexes [5-8]. Measuring fluorescence-excitation spectra of single LH complexes avoids ensemble averaging and resolves bands otherwise masked by inhomogeneous line broadening. By applying this technique to individual LH3 complexes of Rps. acidophila (str. 7750), we were able to gain more insight into the electronic structure of LH3 as well as in the possible origin of the spectral shift from 850 to 820 nm. The spectra revealed a clear difference in the spectral features of the B800 and B820 band. In the B800 band several, relatively narrow, lines with bandwidths ranging from 2 to 13 cm^-1 full width half maximum (FWHM) were observed, whereas in the B820 band only a limited number of broad bands with spectral widths of 60 to 150 cm^-1 FWHM were present. These results clearly indicate a difference in electronic structure and dynamics of the bands. In the B800 band the excitations are mainly localized on individual pigments with picosecond dynamics within the band. In the B820 band the excitations are strongly delocalized over the ring with exciton relaxation in the femtosecond regime. Furthermore, studying the polarization dependence of the spectra revealed that about 60 % of the studied complexes show two broad, orthogonally polarized bands in the 820 nm region. These two bands were assigned to the k^circ= ±1 states of a circular exciton which have their degeneracy lifted by δE ± 1 = 160 cm^-1. The overall spectral behaviour of the studied LH3 complexes and the previously studied LH2 complexes [5-7] is very similar, even though LH3 exhibits more spectral heterogeneity than LH2. Taking these spectral similarities into account, especially with respect to the excitonic behaviour of the B820 (LH3) and B850 (LH2) bands, it is concluded that the spectral shift from 850 to 820 nm is not caused by changes in the interaction energy. Instead, the spectral shift appears to be induced by changes in the site energies of the pigments.