Affinity labeling of cysteine-mutants evidences contact residues in modeled receptor binding sites

To investigate the topology of binding sites in two ionotropic receptors, we have initiated a strategy combining affinity labeling with cysteine-scanning mutagenesis. For the GABAA receptor we have used reactive derivatives of non-competitive blockers (NCBs) to explore interacting positions in its channel. The polypeptide positions of the M2 segment of the alpha1 subunit which we mutated into cysteine were selected for their established accessibility, as determined by the substituted-cysteine accessibility method (SCAM). Using the Xenopus oocyte expression system, we show that receptors containing mutations V257C and S272C are inactivated by several reactive NCBs. These position-selective inactivations lead to an analysis of NCB binding in the channel. For the NMDA receptor glycine-binding site, the prototype antagonist L-701,324 was derivatized at different positions with different reactive groups. The receptor positions to mutate into cysteine were selected after a 3-D homology model. The observed receptor inactivations are mutant- and probe-selective, leading to an unambiguous chemical docking of the antagonist pharmacophore and supporting the model. The site-specificity of the inactivating reactions is assessed by protection experiments and by mutant to wild-type (WT) comparisons. The scope and limitations of the method are briefly discussed.     

Spatial preservation of nuclear chromatin architecture during three-dimensional fluorescence in situ hybridization (3D-FISH)

3D-FISH has become a major tool for studying the higher order chromatin organization in the cell nucleus. It is not clear, however, to what extent chromatin arrangement in the nucleus after fixation and 3D-FISH still reflects the order in living cells. To study this question, we compared higher order chromatin arrangements in living cells with those found after the 3D-FISH procedure. For in vivo studies we employed replication labeling of DNA with Cy3-conjugated nucleotides and/or chromatin labeling by GFP-tagged histone 2B. At the light microscope level, we compared the intranuclear distribution of H2B-GFP-tagged chromatin and the positions of replication-labeled chromatin domains in the same individual cells in vivo, after fixation with 4% paraformaldehyde, and after 3D-FISH. Light microscope data demonstrate a high degree of preservation of the spatial arrangement of approximately 1-Mb chromatin domains. Subsequent electron microscope investigations of chromatin structure showed strong alterations in the ultrastructure of the nucleus caused mainly by the heat denaturation step. Through this step chromatin acquires the appearance of a net with mesh size of 50-200 nm roughly corresponding to the average displacement of the chromatin domains observed at light microscope level. We conclude that 3D-FISH is a useful tool to study chromosome territory structure and arrangements down to the level of approximately 1-Mb chromatin domain positions. However, important ultrastructural details of the chromatin architecture are destroyed by the heat denaturation step, thus putting a limit to the usefulness of 3D-FISH analyses at nanometer scales.     

DNA binding induces conformational transition within human DNA topoisomerase I in solution

We employed Raman and circular dichroism (CD) spectroscopy to probe the molecular structure of 68-kDa recombinant human DNA topoisomerase I (TopoI) in solution, in a complex with a 16-bp DNA fragment containing a camptothecin-enhanced TopoI cleavage site, and in a ternary complex with this oligonucleotide and topotecan. Raman spectroscopy reveals a TopoI secondary structure transition and significant changes in the hydrogen bonding of the tyrosine residues induced by the DNA binding. CD spectroscopy confirms the Raman data and identifies a DNA-induced (>7%) decrease of the TopoI alpha helix accompanied by at least a 6% increase of the beta structure. The Raman DNA molecular signatures demonstrated a bandshift that is expected for a net change in the environment of guanine C6 [double bond] O groups from pairing to solvent exposure. The formation of a ternary cleavage complex with TopoI, DNA, and topotecan as probed by CD spectroscopy reveals neither additional modifications of the TopoI secondary structure nor of the oligonucleotide structure, compared to the TopoI-oligonucleotide complex.     

Structural consequences of carboxyamidation of dermaseptin S3

Animal-derived antimicrobial peptides are gaining increasing interest for their role in the innate immune system and for their potential applications in the antimicrobial field. Defining the factors that affect potency and selectivity is presently a major challenge to their effective and safe use. Since amidating the C-terminal carboxyl is one of the means of enhancing antimicrobial activity, we report here our comparative study of the solution structures of the antimicrobial peptide dermaseptin S3 and its amidated analogue. Circular dichroism measurements suggested that the peptides are basically found in an alpha-helical structure. In contrast, NMR measurements revealed the complete absence of alpha-helical elements in S3 and a single four-residue helix in the amidated analogue. Whereas the native peptide was found to be flexible, containing a hydrogen-bonded turn and bends, the amidated analogue exhibited a defined alpha-helix at the C-terminal region, causing the latter to be significantly elongated and more structured. Hence, although the increased potency in amidated antimicrobial peptides can be attributed to the increased overall positive charge, in this case, amidation has had additional effects beyond modifying the net positive charge. It has induced and/or stabilized a helical conformation, causing the amidated dermaseptin to be more rigid and more extended than its nonamidated analogue. The possible implications on the mode of action are discussed herein.     

A proteomic approach for identification of secreted proteins during the differentiation of 3T3-L1 preadipocytes to adipocytes

We have undertaken a systematic proteomic approach to purify and identify secreted factors that are differentially expressed in preadipocytes versus adipocytes. Using one-dimensional gel electrophoresis combined with nanoelectrospray tandem mass spectrometry, proteins that were specifically secreted by 3T3-L1 preadipocytes or adipocytes were identified. In addition to a number of previously reported molecules that are up- or down-regulated during this differentiation process (adipsin, adipocyte complement-related protein 30 kDa, complement C3, and fibronectin), we identified four secreted molecules that have not been shown previously to be expressed differentially during the process of adipogenesis. Pigment epithelium-derived factor, a soluble molecule with potent antiangiogenic properties, was found to be highly secreted by preadipocytes but not adipocytes. Conversely, we found hippocampal cholinergic neurostimulating peptide, neutrophil gelatinase-associated lipocalin, and haptoglobin to be expressed highly by mature adipocytes. We also used liquid chromatography-based separation followed by automated tandem mass spectrometry to identify proteins secreted by mature adipocytes. Several additional secreted proteins including resistin, secreted acidic cysteine-rich glycoprotein/osteonectin, stromal cell-derived factor-1, cystatin C, gelsolin, and matrix metalloprotease-2 were identified by this method. To our knowledge, this is the first study to identify several novel secreted proteins by adipocytes by a proteomic approach using mass spectrometry.     

Evidence that intrinsic iron but not intrinsic copper determines S-nitrosocysteine decomposition in buffer solution.

The present experiments were designed to analyze the influence of copper and iron ions on the process of decomposition of S-nitrosocysteine (cysNO), the most labile species among S-nitrosothiols (RSNO). CysNO fate in buffer solution was evaluated by optical and electron paramagnetic resonance (EPR) spectroscopy, and the consequences on its vasorelaxant effect were studied on noradrenaline-precontracted rat aortic rings. The main results are the following: (i) copper or iron ions, especially in the presence of the reducing agent ascorbate, accelerated the decomposition of cysNO and markedly attenuated the amplitude and duration of the relaxant effect of cysNO; (ii) by contrast, the iron and copper chelators bathophenantroline disulfonic acid (BPDS) and bathocuproine disulfonic acid (BCS) exerted a stabilizing effect on cysNO, prolonged its vasorelaxant effect, and abolished the influence of ascorbate; (iii) in the presence of ascorbate, BPDS displayed a selective inhibitory effect toward the influence of iron ions (but not toward copper ions) on cysNO decomposition and vasorelaxant effect, while BCS prevented the effects of both copper and iron ions; (iv) L-cysteine enhanced stability and prolonged the relaxant effect of cysNO; (v) the process of iron-induced decomposition of cysNO was associated with the formation of EPR-detectable dinitrosyl-iron complexes (DNIC) either with non-thiol- or thiol-containing ligands (depending on the presence of L-cysteine), both of which exhibiting vasorelaxant properties. From these data, it is concluded that the amount of intrinsic copper was probably too low to produce a destabilizing effect even on the most labile RSNO, cysNO, and that only intrinsic iron, through the formation of DNIC, was responsible for the process of cysNO decomposition and thus influenced its vasorelaxant properties.     

Carbon dioxide exchange of a Russian boreal forest after disturbance by wind throw

The exchange of carbon dioxide (CO₂) between the atmosphere and a forest after disturbance by wind throw in the western Russian taiga was investigated between July and October 1998 using the eddy covariance technique. The research area was a regenerating forest (400 m × 1000 m), in which all trees of the preceding generation were uplifted during a storm in 1996. All deadwood had remained on site after the storm and had not been extracted for commercial purposes. Because of the heterogeneity of the terrain, several micrometeorological quality tests were applied. In addition to the eddy covariance measurements, carbon pools of decaying wood in a chronosequence of three different wind throw areas were analysed and the decay rate of coarse woody debris was derived. During daytime, the average CO₂ uptake flux was ?3 µmol m^?2s^?1, whereas during night‐time characterised by a well‐mixed atmosphere the rates of release were typically about 6 µmol m^?2s^?1. Suppression of turbulent fluxes was only observed under conditions with very low friction velocity (u* ≤ 0.08 ms^?1). On average, 164 mmol CO₂ m^?2d^?1 was released from the wind throw to the atmosphere, giving a total of 14.9 mol CO₂ m^?2 (180 g CO₂ m^?2) released during the 3‐month study period. The chronosequence of dead woody debris on three different wind throw areas suggested exponential decay with a decay coefficient of ?0.04 yr^?1. From the magnitude of the carbon pools and the decay rate, it is estimated that the decomposition of coarse woody debris accounted for about a third of the total ecosystem respiration at the measurement site. Hence, coarse woody debris had a long‐term influence on the net ecosystem exchange of this wind throw area. From the analysis performed in this work, a conclusion is drawn that it is necessary to include into flux networks the ecosystems that are subject to natural disturbances and that have been widely omitted into considerations of the global carbon budget. The half‐life time of about 17 years for deadwood in the wind throw suggests a fairly long storage of carbon in the ecosystem, and indicates a very different long‐term carbon budget for naturally disturbed vs. commercially managed forests.     

Sensory cells in the sucker of Craspedella pedum (Plathelminthes, Temnocephalida): in vivo staining with DiO and SEM and TEM observations

 Two pairs of identified sensory neurons innervating the sucker of Craspedella pedum (designated ADS1, ADS2) were found to accumulate DiO (3,3′-dioctadecyloxacarbocyanine perchlorate) in vivo. The number, position and morphology of these neurons do not change throughout the postembryonic period of life. The axons of the ADS cells run forward within the ventral cords and their dendrites are parallel. They enter the sucker, ramify and terminate in numerous sensory endings in a wide peripheral zone of the disc. SEM reveals a single type of sensilla: small spot-like structures with several short cilia. They are scattered within the zone accommodating the openings of the adhesive glands and their distribution corresponds to that of the stained terminals. TEM observations (including about 20 full reconstructions from serial ultrathin sections) show five types of sensory endings on the disc with the following structures: (1) a short cilium and thin rootlet, (2) aciliary with a normal rootlet and a club-shaped apical portion, (3) aciliary with a club-shaped apical portion and a body similar to the apical part of the rootlet, (4) aciliary with large apical granule and (5) aciliary with small apical granules. Type 2–5 receptors form a morphological series suggesting that they are stages of formation of the common type 4 receptor. Not fully formed type 1 receptors have been found within the epidermis in adult animals. This suggests that, although ADS perikarya persist throughout the life of the animal, the nerve endings they form might be constantly renewed. Judging from the morphological and behavioural data, the functions of the ADS neurons might include: (1) monitoring of the close contact between the surface of the sucker and the substratum prior to adhesion and (2) checking the viscosity of the adhesive secretion prior to release of the sucker. Accepted: 16 December 1997