Proteomics of bronchoalveolar lavage fluid

Lung diseases are essentially multi-factorial diseases that require a global analysis, and thus, cannot be understood through the sole analysis of individual or small numbers of genes. Proteome analysis has rapidly developed in the post-genome era and is now widely accepted as the obligated complementary technology for genetic profiling. It has been shown to be a powerful tool for the study of human diseases and for identifying novel prognostic, diagnostic and therapeutic markers. During last years, proteomic approaches applied to lung diseases are centred on the analysis of proteins in samples, such as cell cultures, biopsies and physiological fluids like serum and, especially, bronchoalveolar lavage fluid (BALF). BALF is presently the most common way of sampling the components of the epithelial lining fluid (ELF) and the most faithful reflect of the protein composition of the pulmonary airways. This review focuses on the state of the investigations of BALF proteome and its powerful contribution both to a better knowledge of the lung structure at the molecular level and to the study of lung disorders at the clinical level.     

Recombinant interferon-γ secreted by chinese hamster ovary-320 cells cultivated in suspension in protein-free media is protected against extracellular proteolysis by the expression of natural protease inhibitors and by the addition of plant protein hydrolysates to the culture medium

Summary Biosafety requirements increasingly restrict the cultivation of mammalian cells producing therapeutic glycoproteins to conditions that are devoid of any compound of animal origin. On cultivation in serum-free media, the proteases inhibitors, usually found in serum, cannot protect secreted recombinant proteins against unwanted endogenous proteolysis. Chinese hamster ovary (CHO) cells, secreting recombinant human interferon-γ (CHO-320 cell line) and cultivated in suspension in an original protein-free medium, expressed at least two members of the matrix metalloproteinases (MMP), either at the cell surface (proMMP-14 and MMP-14) or secreted (proMMP-9). In addition, tissue- and urinary-type plasminogen activators were also secreted in such culture conditions. At the cell surface, dipeptidyl peptidase IV and tripeptidyl peptidase II (TPPII) activities were also detected, and their activities decreased during time course of batch cultures. The proteolytic activities of these proteins were counterbalanced by (1) their expression as zymogens (proMMP-9, proMMP-14), (2) the expression of their natural inhibitors, tissue inhibitors of metalloproteinases-1 and-2 and plasminogen activator inhibitor-1 (PAI-1), or (3) the addition of plant protein hydrolysates to the culture medium, acting as a nonspecific source of TPPII inhibitors. This study points out that, even in protein-free media, recombinant proteins secreted by CHO cells are actively protected against physiological and unwanted extracellular proteolysis either by endogenous or by exogenous inhibitors.     

Sulfate-reducing bacteria in floating macrophyte rhizospheres from an Amazonian floodplain lake in Bolivia and their association with Hg methylation

Five subgroups of sulfate-reducing bacteria (SRB) were detected by PCR in three macrophyte rhizospheres (Polygonum densiflorum, Hymenachne donacifolia, and Ludwigia helminthorriza) and three subgroups in Eichhornia crassipes from La Granja, a floodplain lake from the upper Madeira basin. The SRB community varied according to the macrophyte species but with different degrees of association with their roots. The rhizosphere of the C4 plant Polygonum densiflorum had higher frequencies of SRB subgroups as well as higher mercury methylation potentials (27.5 to 36.1%) and carbon (16.06 +/- 5.40%), nitrogen (2.03 +/- 0.64%), Hg (94.50 +/- 6.86 ng Hg g(-1)), and methylmercury (8.25 +/- 1.45 ng Hg g(-1)) contents than the rhizosphere of the C3 plant Eichhornia crassipes. Mercury methylation in Polygonum densiflorum and Eichhornia crassipes was reduced when SRB metabolism was inhibited by sodium molybdate.     

Database of bronchoalveolar lavage fluid proteins

Bronchoalveolar lavage during fiberoptic bronchoscopy is extensively used for investigating cellular and biochemical alterations of the epithelial lining fluid in various lung disorders. Two-dimensional electrophoresis (2-DE) offers the possibility to simultaneously display and analyze proteins contained in bronchoalveolar lavage fluid (BALF). We present the current status of 2-DE of BALF samples with an updated listing of the proteins already identified and of their level and/or posttranslational alterations in lung disorders. Alternatives to 2-DE of BALF samples and future prospects of proteomics to unravel lung functions and pathologies are discussed.     

Yersinia enterocolitica type III secretion-translocation system: channel formation by secreted Yops

'Type III secretion' allows extracellular adherent bacteria to inject bacterial effector proteins into the cytosol of their animal or plant host cells. In the archetypal Yersinia system the secreted proteins are called Yops. Some of them are intracellular effectors, while YopB and YopD have been shown by genetic analyses to be dedicated to the translocation of these effectors. Here, the secretion of Yops by Y.enterocolitica was induced in the presence of liposomes, and some Yops, including YopB and YopD, were found to be inserted into liposomes. The proteoliposomes were fused to a planar lipid membrane to characterize the putative pore-forming properties of the lipid-bound Yops. Electrophysiological experiments revealed the presence of channels with a 105 pS conductance and no ionic selectivity. Channels with those properties were generated by mutants devoid of the effectors and by lcrG mutants, as well as by wild-type bacteria. In contrast, mutants devoid of YopB did not generate channels and mutants devoid of YopD led to current fluctuations that were different from those observed with wild-type bacteria. The observed channel could be responsible for the translocation of Yop effectors.     

Structural modifications in the membrane-bound regions of the gastric H+/K+-ATPase upon ligand binding.

Extensive trypsin proteolysis was used to examine the accessibility of membrane bound segments of the gastric H+/K+-ATPase under different experimental conditions known to induce either the E1 or the E2 conformation. Membrane-anchored peptides were isolated after trypsinolysis and identified by sequencing. We show that several membrane bound segments are involved in the conformational change. In the N-terminal region, a M1-M2 peptide (12 kDa) was found to be associated with the membrane fraction after digestion in the presence of K+ or in the presence of vanadate (12 kDa and 15 kDa). In the M3 and M4 region, no difference was observed for the peptide obtained in E1 or E2-K conformations, but the peptide generated in the presence of vanadate begins 12 amino-acid residues earlier in the sequence. Cytoplasmic loop region: we show here that a peptide beginning at Asp574 and predicted to end at Arg693 is associated with the membrane for a vanadate-induced conformation. In the M5-M6 region, the membrane-anchored peptide obtained on E1 is 39 amino acids shorter than the E2 peptide. In the M7-M8 region, the same peptide encompassing the M7 and M8 transmembrane segments was produced for E1 and E2 conformations.     

The VO2 slow component for severe exercise depends on type of exercise and is not correlated with time to fatigue

The purpose ofthis study was to examine the influence of the type of exercise(running vs. cycling) on the O₂uptake (O₂) slow component.Ten triathletes performed exhaustive exercise on a treadmill and on acycloergometer at a work rate corresponding to 90% of maximalO₂ (90% work rate maximalO₂). The duration of thetests before exhaustion was superimposable for both type of exercises(10 min 37 s ± 4 min 11 s vs. 10 min 54 s ± 4 min 47 s forrunning and cycling, respectively). TheO₂ slow component (difference between O₂ atthe last minute and minute 3 ofexercise) was significantly lower during running compared with cycling(20.9 ± 2 vs. 268.8 ± 24 ml/min). Consequently, there was norelationship between the magnitude of theO₂ slow component and thetime to fatigue. Finally, because blood lactate levels at the end of the tests were similar for both running (7.2 ± 1.9 mmol/l) and cycling (7.3 ± 2.4 mmol/l), there was a clear dissociation between blood lactate and the O₂slow component during running. These data demonstrate that1) theO₂ slow component dependson the type of exercise in a group of triathletes and2) the time to fatigue isindependent of the magnitude of theO₂ slow component and bloodlactate concentration. It is speculated that the difference in muscularcontraction regimen between running and cycling could account for thedifference in theO₂ slow component.

     

Expression of a biologically active diphtheria toxin fragment B in Escherichia coli

The toxB gene of Corynebacterium diphtheriae bacteriophage β encoding the B fragment of diphtheria toxin was cloned into an inducible expression vector. When expressed In Escherichia coli, fragment B was not proteolysed and was indistinguishable, by immunological criteria, from wild-type C. diphthsriae derived fragment B. Soluble fragment B was partially purified from the cytoplasm by saline precipitation steps and was shown to compete with the wild-type diphtheria toxin for binding to receptors of sensitive eukaryotic cells. A complete diphtheria toxin was reconstituted by formation of the disulphide bridge between purified fragment A and recombinant fragment B, which migrates at the expected Mr on Western blots and which was able to block protein synthesis by ADP-ribosylation of elongation factor–2, thereby indicating that the recombinant fragment B had retained its biological activity.     

Secondary structure of diphtheria toxin and its fragments interacting with acidic liposomes studied by polarized infrared spectroscopy

We used infrared attenuated total reflection spectroscopy to study the structure of diphtheria toxin (DT) and its fragments A, B, CB1, and CB4 as a function of the pH in the absence and in the presence of phospholipid vesicles. Binding of DT to asolectin or DL-alpha-dipalmitoylphosphatidylcholine-DL-alpha-dipalmitoylphosphatidic acid liposomes at pH 7.3 results in a 10% increase in its alpha-helix content. At pH 4, in the presence of liposomes, the secondary structure of DT is characterized by the appearance of a beta-sheet structure with strengthened hydrogen bonds which did not exist before pH lowering. DT fragment B displays little conformational change upon pH lowering in the presence of liposomes. However, the alpha-helix content of CB1 increases by 10%, and polarization measurements indicate that the alpha-helices of CB1 at pH 4 are oriented parallel to the lipid acyl chains. On the other hand, the alpha-helix content of CB4 decreases dramatically while the low frequency beta-sheet content increases. Dichroism measurements demonstrate that this sheet lies close to a parallel to the bilayer surface. The fragment A of DT experiences a large conformational change upon pH lowering and binds to the liposome membrane even in the absence of DT fragment B. The conformational modification of DT fragment A is fully reversed when pH is brought back to 7.3.     

Enhancement of IGE-induced mediator release from rat peritoneal mast cells by serum-treated zymosan

Phagocytosis of zymosan (Z) treated with rat serum (ZX) by rat peritoneal mast cells caused only a small amount of [3H]serotonin release, and prior release of mediators from mast cells did not affect phagocytosis of sheep erythrocytes bearing IgG and C3b, indicating the independence of these two phenomena. When, however, mast cells were exposed to ZX, subsequent IgE-mediated release of histamine, [3H]serotonin, and beta-hexosaminidase was greatly enhanced. Prevention of complement activation by the presence of EDTA during the treatment of Z with the serum or prior heating of the serum at 56 degrees C for 30 min only slightly impaired the ability of ZX to augment mediator release, whereas prior absorption of the serum with zymosan at 0 degree C greatly diminished the enhancement. Exposure of fresh Z to variable amounts of either the acid or the high-salt eluate of ZX also generated ZX capable of enhancing [3H]serotonin release in a dose-dependent fashion. IgG, IgM, C3, and albumin were detected in the eluates by immunodiffusion. When IgG was depleted from the high-salt eluate by treating with Sepharose-anti-IgG, the enhancement was significantly reduced, indicating that IgG but not C3 or other immunoglobulins was required for the enhancement.