Lipopolysaccharide-induced pulmonary inflammation is not accompanied by a release of anandamide into the lavage fluid or a down-regulation of the activity of fatty acid amide hydrolase

The effect of lipopolysaccharide inhalation upon lung anandamide levels, anandamide synthetic enzymes and fatty acid amide hydrolase has been investigated. Lipopolysaccharide exposure produced a dramatic extravasation of neutrophils and release of tumour necrosis factor alpha into the bronchoalveolar lavage (BAL) fluid, which was not accompanied by epithelial cell injury. The treatment, however, did not change significantly the levels of anandamide and the related compound palmitoylethanolamide in the cell-free fraction of the BAL fluid. The activities of the anandamide synthetic enzymes N-acyltransferase and N-acylphosphatidylethanolamine phospholipase D and the activity of fatty acid amide hydrolase in lung membrane fractions did not change significantly following the exposure to lipopolysaccharide. The non-selective fatty acid amide hydrolase inhibitor phenylmethylsulfonyl fluoride was a less potent inhibitor of lung fatty acid amide hydrolase than expected from the literature, and a dose of 30 mg/kg i.p. of this compound, which produced a complete inhibition of brain anandamide metabolism, only partially inhibited the lung metabolic activity.     

Molecular imaging of the transcription factor NF-kappaB, a primary regulator of stress response

A wide range of environmental stress and human disorders involves inappropriate regulation of NF-kappaB, including cancers and numerous inflammatory conditions. We have developed transgenic mice that express luciferase under the control of NF-kappaB, enabling real-time non-invasive imaging of NF-kappaB activity in intact animals. We show that, in the absence of stimulation, strong, intrinsic luminescence is evident in lymph nodes in the neck region, thymus, and Peyer's patches. Treating mice with stressors, such as TNF-alpha, IL-1alpha, or lipopolysaccharide (LPS) increases the luminescence in a tissue-specific manner, with the strongest activity observable in the skin, lungs, spleen, Peyer's patches, and the wall of the small intestine. Liver, kidney, heart, muscle, and adipose tissue exhibit less intense activities. Exposure of the skin to a low dose of UV-B radiation increases luminescence in the exposed areas. In ocular experiments, LPS- and TNF-alpha injected NF-kappaB-luciferase transgenic mice exhibit a 20-40-fold increase in lens NF-kappaB activity, similar to other LPS- and TNF-alpha-responsive organs. Peak NF-kappaB activity occurs 6h after injection of TNF-alpha and 12h after injection of LPS. Peak activities occur, respectively, 3 and 6h later than that in other tissues. Mice exposed to 360J/m(2) of UV-B exhibit a 16-fold increase in NF-kappaB activity 6h after exposure, characteristically similar to TNF-alpha-exposed mice. Thus, in NF-kappaB-luciferase transgenic mice, NF-kappaB activity also occurs in lens epithelial tissue and is activated when the intact mouse is exposed to classical stressors. Furthermore, as revealed by real-time non-invasive imaging, induction of chronic inflammation resembling rheumatoid arthritis produces strong NF-kappaB activity in the affected joints. Finally, we have used the model to demonstrate NF-kappaB regulation by manipulating the Vitamin A status in mice. NF-kappaB activity is elevated in mice fed a Vitamin A deficient (VAD) diet, and suppressed by surplus doses of retinoic acid (RA). We thus demonstrate the development and use of a versatile model for monitoring NF-kappaB activation both in tissue homogenates and in intact animals after the use of classical activators, during disease progression and after dietary intervention.     

Recombinant water-soluble chlorophyll protein from Brassica oleracea var. Botrys binds various chlorophyll derivatives

A gene coding for water-soluble chlorophyll-binding protein (WSCP) from Brassica oleracea var. Botrys has been used to express the protein, extended by a hexahistidyl tag, in Escherichia coli. The protein has been refolded in vitro to study its pigment binding behavior. Recombinant WSCP was found to bind two chlorophylls (Chls) per tetrameric protein complex but no carotenoids in accordance with previous observations with the native protein [Satoh, H., Nakayama, K., Okada, M. (1998) J. Biol. Chem. 273, 30568-30575]. WSCP binds Chl a, Chl b, bacteriochlorophyll a, and the Zn derivative of Chl a but not pheophytin a, indicating that the central metal ion in Chl is essential for binding. WSCP also binds chlorophyllides a and b and even the more distant Chl precursor Mg-protoporphyrin IX; however, these pigments fail to induce oligomerization of the protein. We conclude that the phytol group in bound Chl plays a role in the formation of tetrameric WSCP complexes. If WSCP in fact binds Chl or its derivative(s) in vivo, the lack of carotenoids in pigmented WSCP raises the question of how photooxidation, mediated by triplet-excited Chl and singlet oxygen, is prohibited. We show by spin-trap electron-paramagnetic resonance that the light-induced singlet-oxygen formation of WSCP-bound Chl is lower by a factor of about 4 than that of unbound Chl. This as-yet-unknown mechanism of WSCP to protect its bound Chl against photooxidation supports the notion that WSCP may function as a transient carrier of Chl or its derivatives.     

Dimerization and inter-chromophore distance of Cph1 phytochrome from Synechocystis,as monitored by fluorescence homo and hetero energy transfer

We investigated the dimerization of phytochrome Cph1 from the cyanobacterium Synechocystis by fluorescence resonance energy transfer (FRET). As donor we used the chromophore analogue phycoerythrobilin (PEB) and as acceptor either the natural chromophore phycocyanobilin (PCB; hetero transfer) or PEB (homo transfer). Both chromophores bind in a 1:1 stoichiometry to apo-monomers expressed in Escherichia coli. Energy transfer was characterized by time-resolved fluorescence intensity and anisotropy decay after excitation of PEB by picosecond pulses from a tunable Ti-sapphire laser system. ApoCph1 was first assembled with PEB at a low stoichiometry of 0.1. The remaining sites were then sequentially titrated with PCB. In the course of this titration, the mean lifetime of PEB decreased from 3.33 to 1.25 ns in the P(r) form of Cph1, whereas the anisotropy decay was unaffected. In the P(fr)/P(r) photoequilibrium (about 65% P(fr)), the mean lifetime decreased significantly less, to 1.67 ns. These observations provide strong support for inter-chromophore hetero energy transfer in mixed PEB/PCB dimers. The reduced energy transfer in P(fr) may be due to a structural difference but is at least in part due to the difference in spectral overlap, which was 4.1 x 10(-13) and 1.6 x 10(-13) cm(3) M(-1) in P(r) and P(fr), respectively. From the changes in the mean lifetime, rates of hetero energy transfer of 0.68 and 0.37 ns(-1) were calculated for the P(r) form and the P(fr)/P(r) photoequilibrium, respectively. Sequential titration of apo Cph1 with PEB alone to full occupancy did not affect the intensity decay but led to a substantial increase in depolarization. This is the experimental signature of homo energy transfer. Values for the rate of energy transfer k(HT) (0.47 ns(-1)) and the angle 2theta between the transition dipole moment directions (2theta = 45 +/- 5 degrees) were determined from an analysis of the concentration dependence of the anisotropy at five different PEB/Cph1 stoichiometries. The independently determined rates of hetero and homo energy transfer are thus of comparable magnitude and consistent with the energy transfer interpretation. Using these results and exploiting the 2-fold symmetry of the dimer, the chromophore-chromophore distance R(DA) was calculated and found to be in the range 49 A < R(DA) < 63 A. Further evidence for energy transfer in Cph1 dimers was obtained from dilution experiments with PEB/PEB dimers: the lifetime was unchanged, but the anisotropy increased as the dimers dissociated with increasing dilution. These experiments allowed a rough estimate of 5 +/- 3 microM for the dimer dissociation constant. With the deletion mutant Cph1Delta2 that lacks the carboxy terminal histidine kinase domain less energy transfer was observed suggesting that in this mutant dimerization is much weaker. The carboxy terminal domain of Cph1 that is involved in intersubunit trans-phosphorylation and signal transduction thus plays a dominant role in the dimerization. The FRET method provides a sensitive assay to monitor the association of Cph1 monomers.     

Chlorophylls of the c family: absolute configuration and inhibition of NADPH:protochlorophyllide oxidoreductase

Using circular dichroism (CD) spectroscopy, the stereochemistry at C-13(2) of members of the chlorophyll (Chl) c family, namely Chls c(1), c(2), c(3) and [8-vinyl]-protochlorophyllide a (Pchlide a) was determined. By comparison with spectra of known enantiomers, all Chl c members turned out to have the (R) configuration, which is in agreement with considerations drawn from chlorophyll biosynthesis. Except for a double bond in the side chain at C-17, the chemical structure of Chl c(1) is identical with Pchlide a, the natural substrate of the light-dependent NADPH:protochlorophyllide oxidoreductase (POR). Thus, lack of binding to the active site due to the wrong configuration at C-13(2), which had been proposed previously, cannot be an explanation for inactivity of Chl c in this enzymic reaction. Our results show rather that Chl c(1) is a competitive inhibitor for this enzyme, tested with Pchlide a and Zn-protopheophorbide a (Zn-Ppheide a) as substrates.     

SC1/hevin. An extracellular calcium-modulated protein that binds collagen I

SC1, a member of the BM-40 family of extracellular matrix proteins, was recombinantly expressed in a eukaryotic expression system. The full-length protein as well as truncated versions were purified to homogeneity under non-denaturing conditions. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry of full-length SC1 revealed a mass of 87.8 kDa of which 16.8 kDa is contributed by posttranslational modifications. In electron microscopy, after negative staining, SC1 was revealed as a globule attached to a thread-like structure. A calcium dependence of the SC1 conformation could be demonstrated by fluorescence spectroscopy. In the extracellular matrix of cultured osteosarcoma cells SC1 was found associated with collagen I-containing fibrils, and binding of SC1 to reconstituted collagen I fibrils could be demonstrated by immunogold labeling and electron microscopy. SC1 showed a broad expression in a variety of tissues.     

The core of the tetrameric mycobacterial porin MspA is an extremely stable beta-sheet domain

MspA is the major porin of Mycobacterium smegmatis mediating the exchange of hydrophilic solutes across the cell wall and is the prototype of a new family of tetrameric porins with a single central pore of 10 nm in length. Infrared and circular dichroism spectroscopy revealed that MspA consists mainly of antiparallel beta-strands organized in a coherent domain. Heating to 92 and 112 degrees C was required to dissociate the MspA tetramer and to unfold the beta-sheet domain in the monomer, respectively. The stability of the MspA tetramer exceeded the remarkable stability of the porins of Gram-negative bacteria for every condition tested and was not reduced in the presence of 2% SDS and at any pH from 2 to 14. These results indicated that the interactions between the MspA subunits are different from those in the porins of Gram-negative bacteria and are discussed in the light of a channel-forming beta-barrel as a core structure of MspA. Surprisingly, the channel activity of MspA in 2% SDS and 7.6 m urea at 50 degrees C was reduced 13- and 30-fold, respectively, although the MspA tetramer and the beta-sheet domain were stable under those conditions. Channel closure by conformational changes of extracellular loops under those conditions is discussed to explain these observations. This study presents the first experimental evidence that outer membrane proteins not only from Gram-negative bacteria but also from mycobacteria are beta-sheet proteins and demonstrates that MspA constitutes the most stable transmembrane channel protein known so far. Thus, MspA may be of special interest for biotechnological applications.     

BCL-2 and BCL-XL restrict lineage choice during hematopoietic differentiation

Differentiation of hematopoietic cells from multipotential progenitors is regulated by multiple growth factors and cytokines. A prominent feature of these soluble factors is promotion of cell survival, in part mediated by expression of either of the anti-apoptotic proteins, BCL-2 and BCL-XL. The complex expression pattern of these frequently redundant survival factors during hematopoiesis may indicate a role in lineage determination. To investigate the latter possibility, we analyzed factor-dependent cell-Patersen (FDCP)-Mix multipotent progenitor cells in which we stably expressed BCL-2 or BCL-XL. Each factor maintained complete survival of interleukin-3 (IL-3)-deprived FDCP-Mix cells but, unexpectedly, directed FDCP-Mix cells along restricted and divergent differentiation pathways. Thus, IL-3-deprived FDCP-Mix BCL-2 cells differentiated exclusively to granulocytes and monocytes/macrophages, whereas FDCP-Mix BCL-XL cells became erythroid. FDCP-Mix BCL-2 cells grown in IL-3 were distinguished from FDCP-Mix and FDCP-Mix BCL-XL cells by a striking reduction in cellular levels of Raf-1 protein. Replacement of the BCL-2 BH4 domain with the related BCL-XL BH4 sequence resulted in a switch of FDCP-Mix BCL-2 cells to erythroid fate accompanied by persistence of Raf-1 protein expression. Moreover, enforced expression of Raf-1 redirected FDCP-Mix BCL-2 cells to an erythroid fate, and prohibited generation of myeloid cells. These results identify novel roles for BCL-2 and BCL-XL in cell fate decisions beyond cell survival. These effects are associated with differential regulation of Raf-1 expression, perhaps involving the previously identified interaction between BCL-2-BH4 and the catalytic domain of Raf-1.     

STAM and Hrs are subunits of a multivalent ubiquitin-binding complex on early endosomes

STAM1 and STAM2, which have been identified as regulators of receptor signaling and trafficking, interact directly with Hrs, which mediates the endocytic sorting of ubiquitinated membrane proteins. The STAM proteins interact with the same coiled-coil domain that is involved in the targeting of Hrs to endosomes. In this work, we show that STAM1 and STAM2, as well as an endocytic regulator protein, Eps15, can be co-immunoprecipitated with Hrs both from membrane and cytosolic fractions and that recombinant Hrs, STAM1/STAM2, and Eps15 form a ternary complex. We find that overexpression of Hrs causes a strong recruitment of STAM2 to endosome membranes. Moreover, STAM2, like Hrs and Eps15, binds ubiquitin, and Hrs, STAM2, and Eps15 colocalize with ubiquitinated proteins in clathrin-containing endosomal microdomains. The localization of Hrs, STAM2, Eps15, and clathrin to endosome membranes is controlled by the AAA ATPase mVps4, which has been implicated in multivesicular body formation. Depletion of cellular Hrs by small interfering RNA results in a strongly reduced recruitment of STAM2 to endosome membranes and an impaired degradation of endocytosed epidermal growth factor receptors. We propose that Hrs, Eps15, and STAM proteins function in a multivalent complex that sorts ubiquitinated proteins into the multivesicular body pathway.