Ornibactins—a new family of siderophores from Pseudomonas

Novel linear hydroxamate/hydroxycarboxylate siderophores from strains of Pseudomonas cepacia were isolated and named ornibactins. The ornibactins represent modified tetrapeptide siderophores, possessing the sequence l-Orn¹(N -OH, N -acyl)-d-threo-Asp(-OH)-l-Ser-l-Orn⁴(N -OH, N -formyl)-1,4-diaminobutane. The N -acyl groups of Orn¹(N -OH, N -acyl) may vary and represent the three acids 3-hydroxybutanoic acid, 3-hydroxyhexanoic acid and 3-hydroxyoctanoic acid, leading to a mixture of three different ornibactins, designated according to their acyl chain length as ornibactin-C4, ornibactin-C6 and ornibactin-C8. Each of the siderophores is accompanied by a small amount of a more hydrophilic component with a 16 a.m.u. higher mass. The structure elucidation was based on results from gas chromatography amino acid analysis, electrospray mass spectrometry, and one- and two-dimensional nuclear magnetic resonance techniques.     

Effect of anoxia and reoxygenation on antioxidant enzyme activities in immortalized brain endothelial cells

Summary The effects of anoxia and reoxygenation on major antioxidant enzyme activities were investigatedin vitro in immortalized rat brain endothelial cells (RBE₄ cells). A sublethal anoxic period of 12 h was assessed for RBE₄ cells using the neutral red uptake test. Anoxia markedly influenced the specific activity of catalase and superoxide dismutase, with no major effect on glutathione peroxidase or glutathione reductase. After 24 h postanoxia, the superoxide dismutase activity modulated by the presence or absence of oxygen returned to control value. Damage and recovery of RBE₄ immortalized rat brain endothelial cells in culture after exposure to free radicals and other oxygen-derived species provides a usefulin vitro model to study anoxia-reoxygenation trauma at the cellular level.     

Investigation of the folding pathway of the TEM-1 β-lactamase

The TEM-1 β-lactamase is a globular protein containing 12 proline residues. The folding mechanism of this enzyme was investigated by kinetic and equilibrium experiments with the help of fluorescence spectroscopy and circular dichroism. The equilibrium denaturation of the protein induced by guanidine hydrochloride occurs in two discrete steps, indicating the existence of a thermodynamically stable intermediate state. Thisstate is 5.2 ± 0.4 kcal/mol less stable than the native conformation and 5.7 ± 0.2 kcal/mol more stable than the fully denaturedprotein. This intermediate state exhibits a high content of native secondary structure elements but is devoid of specific tertiary organization; its relation to the “molten globule” is discussed. Refolding kinetic experimentsrevealed the existence of a transient intermediate conformation between thethermodynamically stable intermediate and the native protein. This transient intermediate appears rapidly during the folding reaction. It exhibits a secondary structure content very similar to that of the native protein and has also recovered a significant amount of tertiary organisation. The final refolding step of the TEM-1 β-lactamase, leading to the native enzyme, is dominated by two major slow kinetic phases which probablyreflect a very complex process kinetically limited by proline cis/transisomerization. © 1995 Wiley-Liss, Inc.     

Saturation of penicillin-binding protein 1 by β-lactam antibiotics in growing cells of Bacillus licheniformis

With the help of a new highly sensitive method allowing the quantification of free penicillin-binding proteins (PBPs) and of an integrated mathematical model, the progressive saturation of PBP1 by various β-lactam antibiotics in growing cells of Bacillus licheniformis was studied. Although the results confirmed PBP1 as a major lethal target for these compounds, they also underlined several weaknesses in our present understanding of this phenomenon. In growing cells, but not in resting cells, the penicillin target(s) appeared to be somewhat protected from the action of the inactivators. In vitro experiments indicated that amino acids, peptides and depsipeptides mimicking the peptide moiety of the nascent peptidoglycan significantly interfered with the acylation of PBP1 by the antibiotics. In addition, the level of PBP1 saturation at antibiotic concentrations corresponding to the minimum inhibitory concentrations was not constant, suggesting that additional, presently undiscovered, factors might be necessary to account for the experimental observations.     

Sequence of ionophore conformational changes induced by a simulated membrane/water interface

We demonstrate how the progressive passage through an interface simulated by a linear increase in dielectric constant may mediate a reversible transconformation of Na⁺/X337A (lasalocid A) from a cyclic to an extended structure, During the passage through the interface, the complex adopts progressively a more extended conformation favorable to the Na⁺ complexation or decomplexation at the interface.     

Analysis of lambda receptor and beta-lactamase synthesis and export using cloned genes in a minicell system

Summary We have cloned lamB, the gene for receptor (an outer membrane protein), on a small plasmid which also carries the gene for -lactamase (a periplasmic protein). We have identified a promoter in the region of malK, the gene immediately preceding lamB, which is active in minicells but relatively inactive in vitro. Using a minicell system, we have found that both receptor and -lactamase are made as full length precursors which are subsequently processed. We also show that the receptor precursor can be exported to the outer membrane before it is processed. Mature -lactamase is found only in the periplasm, suggesting that processing may be a requirement for export to the periplasm.     

Genetic control of igG2a production in the response to sheep erythrocytes

A low IgG2a response in B10 mice during the primary response to sheep red blood cells (SRBC) is described. Analysis of the response in B10 × BALB/c hybrid progenies and in congenic strains indicates that this low response is a dominant phenotype placed under the control of a single Mendelian gene or a group of closely linked genes. This gene(s) is neither linked to CH allotypes orH2 haplotypes, nor is it sex-linked. It can be considered as an isotype- and antigenspecific regulatory gene of the immune response.     

Isolation of a cDNA fragment coding for Chlamydomonas reinhardtii ferredoxin and expression of the recombinant protein in Escherichia coli

A cDNA clone coding for mature C. reinhardtii ferredoxin has been isolated from a cDNA library using PCR and two oligonucleotide primers based on the N- and C-termini of the protein's amino acid sequence. The nucleotidic sequence of the PCR fragment (299 bp) agreed well with the amino acid sequence since a single conservative substitution (Thr-7 to Ser) could be deduced. The PCR fragment was inserted into the expression vector pTrc 99A, using the incorporated NcoI and BamHI restriction sites and the construction used to transform E. coli (DH5α F′). After subsequent large scale expression and purification of the recombinant protein, biochemical and biophysical analysis have indicated that the product isolated from E. coli is homologous to native ferredoxin isolated from green algae.     

Production of acetylcholine receptors from TE671 cells grown on microcarriers

Summary When grown in DMEM supplemented with 10 % fetal calf serum and using Cytodex 3® as microcarriers, TE671 cells entering the stationary phase optimally expressed acetylcholine receptors. These, receptors could be conveniently extracted from cell-saturated, microbeads or monodispersed cells obtained by trypsinization of microbeads. Typically, a 500 ml-batch gave 6–7 pmol of receptors which could be used as antigen to assay anti-acetylcholine receptor antibodies in the sera of myasthenic patients.