Membrane fluidity in glutamic acid-producing bacteria Brevibacterium sp. ATCC 13869

The bacterial secretion of glutamate was studied through plasma membrane fluidity, measured by anisotropy using the fluorophore TMA-DPH incorporated in the lipid part of the cell membrane. Cells of Brevibacterium sp. ATCC 13869 (wild type) were switched from the biotin-limited, producing state to the biotin-supplemented, non-producing state, and back. The following conclusions could be drawn: 1. It was not possible to detect any change in anisotropy by switching the cells from biotin-limited biotin-supplemented, as well as from biotin-supplemented, to biotin-limited, media. 2. The anisotropy value in the glutamic acid fermentation remains constant during the lag, exponential, growth, production and stationary phases. 3. The treatment of cells with a neutral synthetic polyester of ethylene-and propyleneoxide with soya oil-fatty acids increased the anisotropy values, indicating incorporation of the surfactant. 4. Glutamate secretion is not coupled with membrane fluidity, so a leak providing a general fluidization of the membrane could not be detected.     

Papilionoid inflorescences revisited (Leguminosae-Papilionoideae)

Background and Aims

The inflorescence structure determines the spatiotemporal arrangement of the flowers during anthesis and is therefore vital for reproductive success. The Leguminosae are among the largest angiosperm plant families and they include some important crop plants. In papilionoid legumes, the raceme is the most common type of inflorescence. However, a range of other inflorescence types have evolved via various developmental processes. A (re-)investigation of inflorescences in Swainsona formosa, Cicer arietinum, Abrus precatorius, Hardenbergia violacea and Kennedia nigricans leads to new insights into reduction mechanisms and to a new hypothesis on the evolution of the papilionoid pseudoraceme.

Methods

Inflorescence morphology and ontogeny were studied using scanning electron microscopy (SEM).

Key Results

The inflorescence in S. formosa is an umbel with a rare type of pendulum symmetry which may be triggered by the subtending leaf. Inflorescences in C. arietinum are reduced to a single flower. An early formed adaxial bulge is the sterile apex of the inflorescence (i.e. the inflorescence is open and not terminated by a flower). In partial inflorescences of A. precatorius, the axis is reduced and its meristem is relocated towards the main inflorescence. Flower initiation follows a peculiar pendulum pattern. Partial inflorescences in H. violacea and in K. nigricans show reduction tendencies. In both taxa, initiated but early reduced bracteoles are present.

Conclusions

Pendulum symmetry in S. formosa is probably associated with distichous phyllotaxis. In C. arietinum, strong reduction tendencies are revealed. Based on studies of A. precatorius, the papilionoid pseudoraceme is reinterpreted as a compound raceme with condensed lateral axes. From an Abrus-like inflorescence, other types can be derived via reduction of flower number and synchronization of flower development. A plea is made for uniform usage of inflorescence terminology.Key words: Abrus precatorius, Cicer arietinum, Hardenbergia violacea, Kennedia nigricans, inflorescence, Leguminosae, Papilionoideae, pseudoraceme, Swainsona formosa     

Cholesterol attenuates the interaction of the antimicrobial peptide gramicidin S with phospholipid bilayer membranes

We have investigated the effect of the presence of 25 mol percent cholesterol on the interactions of the antimicrobial peptide gramicidin S (GS) with phosphatidylcholine and phosphatidylethanolamine model membrane systems using a variety of methods. Our circular dichroism spectroscopic measurements indicate that the incorporation of cholesterol into egg phosphatidylcholine vesicles has no significant effect on the conformation of the GS molecule but that this peptide resides in a range of intermediate polarity as compared to aqueous solution or an organic solvent. Our Fourier transform infrared spectroscopic measurements confirm these findings and demonstrate that in both cholesterol-containing and cholesterol-free dimyristoylphosphatidylcholine liquid-crystalline bilayers, GS is located in a region of intermediate polarity at the polar--nonpolar interfacial region of the lipid bilayer. However, GS appears to be located in a more polar environment nearer the bilayer surface when cholesterol is present. Our (31)P-nuclear magnetic resonance studies demonstrate that the presence of cholesterol markedly reduces the tendency of GS to induce the formation of inverted nonlamellar phases in model membranes composed of an unsaturated phosphatidylethanolamine. Finally, fluorescence dye leakage experiments indicate that cholesterol inhibits the GS-induced permeabilization of phosphatidylcholine vesicles. Thus in all respects the presence of cholesterol attenuates but does not abolish the interactions of GS with, and the characteristic effects of GS on, phospholipid bilayers. These findings may explain why it is more potent at disrupting cholesterol-free bacterial than cholesterol-containing eukaryotic membranes while nevertheless disrupting the integrity of the latter at higher peptide concentrations. This additional example of the lipid specificity of GS may aid in the rational design of GS analogs with increased antibacterial but reduced hemolytic activities.     

New aspects in floral development of Papilionoideae: initiated but suppressed bracteoles and variable initiation of sepals

BACKGROUND AND AIMS: The increase of molecular data and the resulting insights into legume systematics make the search for new morphological characters and a careful re-investigation of already stated characters necessary. Bracteoles are small, reduced leaves borne close to the base of lateral branches. Although they seem unimportant in older buds, they have an ecological function in protecting the sepal primordia. Furthermore, a morphogenetic function in mediating the onset of sepal initiation is suspected in the literature. The occurrence of bracteoles varies within Papilionoideae, and their distribution is used in legume systematics. But this is open to criticism, because there is a tendency to use 'absent' for 'caducous'. Thus attention here was paid to the initiation of bracteoles as well as to the sequence of sepal initiation. METHODS: The floral development of 30 taxa out of 15 tribes of Papilionoideae was investigated using scanning electron microscopy (SEM). KEY RESULTS: In five taxa the bracteoles initiated, but suppressed early. Furthermore, a broad variability of sepal initiation was found. Besides the widely stated unidirectional pattern, modified unidirectionality, tendencies towards whorled, fully whorled, bidirectional and successive initiation of sepals were all found. CONCLUSION: Initiated but suppressed bracteoles are presented as a 'new' character in Papilionoideae. Considering the presence of bracteoles as a plesiomorphy, their suppression can be seen as a step towards completely reduced bracteoles. The remarkable variability of the sequence of sepal initiation questions the widely stated unidirectionality of organ initiation in Papilionoideae. The different modes of sepal initiation are deducible from the helical pattern of some caesalpinioids, which is seen as a developmental link of the flowers of Papilionoideae and Caesalpinioideae. The bidirectional sepal initiation is possibly a consequence of the morphogenetic function of bracteoles, although bidirectionality is not found in all taxa with reduced bracteoles.     

Protocol of a prospective study on the diagnostic value of transcranial duplex scanning of the substantia nigra in patients with parkinsonian symptoms

Background  

Parkinson's disease (PD) is the second most common neurodegenerative disorder. As there is no definitive diagnostic test, its diagnosis is based on clinical criteria. Recently transcranial duplex scanning (TCD) of the substantia nigra in the brainstem has been proposed as an instrument to diagnose PD. We and others have found that TCD scanning of substantia nigra duplex is a relatively accurate diagnostic instrument in patients with parkinsonian symptoms. However, all studies on TCD so far have involved well-defined, later-stage PD patients, which will obviously lead to an overestimate of the diagnostic accuracy of TCD.     

Dynamics of beta2-adrenergic receptor-ligand complexes on living cells

The agonist-induced dynamic regulation of the beta(2)-adrenergic receptor (beta(2)-AR) on living cells was examined by means of fluorescence correlation spectroscopy (FCS) using a fluorescence-labeled arterenol derivative (Alexa-NA) in hippocampal neurons and in alveolar epithelial type II cell line A549. Alexa-NA specifically bound to the beta(2)-AR of neurons with a K(D) value of 1.29 +/- 0.31 nM and of A549 cells with a K(D) of 5.98 +/- 1.62 nM. The receptor density equaled 4.5 +/- 0.9 microm(-2) in neurons (rho(N)) and 19.9 +/- 2.0 microm(-2) in A549 cells (rho(A549)). Kinetic experiments revealed comparable on-rate constants in both cell types (k(on) = 0.49 +/- 0.03 s(-1) nM(-1) in neurons and k(on) = 0.12 +/- 0.02 s(-1) nM(-1) in A549 cells). In addition to the free ligand diffusing with a D(free) of (2.11 +/- 0.04) x 10(-6) cm(2)/s, in both cell types receptor-ligand complexes with two distinct diffusion coefficients, D(bound1) (fast lateral mobility) and D(bound2) (hindered mobility), were observed [D(bound1) = (5.23 +/- 0.64) x 10(-8) cm(2)/s and D(bound2) = (6.05 +/- 0.23) x 10(-10) cm(2)/s for neurons, and D(bound1) = (2.88 +/- 1.72) x 10(-8) cm(2)/s and D(bound2) = (1.01 +/- 0.46) x 10(-9) cm(2)/s for A549 cells]. Fast lateral mobility of the receptor-ligand complex was detected immediately after addition of the ligand, whereas hindered mobility (D(bound2)) was observed after a delay of 5 min in neurons (up to 38% of total binding) and of 15-20 min in A549 cells (up to 40% of total binding). Thus, the receptor-ligand complexes with low mobility were formed during receptor regulation. Consistently, stimulation of receptor internalization using the adenylate cyclase activator forskolin shifted the ratio of receptor-ligand complexes toward D(bound2). Intracellular FCS measurements and immunocytochemical studies confirmed the appearance of endocytosed receptor-ligand complexes in the cytoplasm subjacent to the plasma membrane after stimulation with the agonist terbutaline (1 microM). This regulatory receptor internalization was blocked after preincubation with propranolol and with a cholesterol-complexing saponin alpha-hederin.     

Real-time imaging of lipid domains and distinct coexisting membrane protein clusters

A detailed understanding of biomembrane architecture is still a challenging task. Many in vitro studies have shown lipid domains but much less information is known about the lateral organization of membrane proteins because their hydrophobic nature limits the use of many experimental methods. We examined lipid domain formation in biomimetic Escherichia coli membranes composed of phosphatidylethanolamine and phosphatidylglycerol in the absence and presence of 1% and 5% (mol/mol) membrane multidrug resistance protein, EmrE. Monolayer isotherms demonstrated protein insertion into the lipid monolayer. Subsequently, Brewster angle microscopy was applied to image domains in lipid matrices and lipid-protein mixtures. The images showed a concentration dependent impact of the protein on lipid domain size and shape and more interestingly distinct coexisting protein clusters. Whereas lipid domains varied in size (14-47μm), protein clusters exhibited a narrow size distribution (2.6-4.8μm) suggesting a non-random process of cluster formation. A 3-D display clearly indicates that these proteins clusters protrude from the membrane plane. These data demonstrate distinct co-existing lipid domains and membrane protein clusters as the monofilm is being compressed and illustrate the significant mutual impact of lipid-protein interactions on lateral membrane architecture.     

Thermodynamics of the interactions of tryptophan-rich cathelicidin antimicrobial peptides with model and natural membranes

Tritrpticin and indolicidin are short 13-residue tryptophan-rich antimicrobial peptides that hold potential as future alternatives for antibiotics. Isothermal titration calorimetry (ITC) has been applied as the main tool in this study to investigate the thermodynamics of the interaction of these two cathelicidin peptides as well as five tritrpticin analogs with large unilamellar vesicles (LUVs), representing model and natural anionic membranes. The anionic LUVs were composed of (a) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPE/POPG) (7:3) and (b) natural E. coli polar lipid extract. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was used to make model zwitterionic membranes. Binding isotherms were obtained to characterize the antimicrobial peptide binding to the LUVs, which then allowed for calculation of the thermodynamic parameters of the interaction. All peptides exhibited substantially stronger binding to anionic POPE/POPG and E. coli membrane systems than to the zwitterionic POPC system due to strong electrostatic attractions between the highly positively charged peptides and the negatively charged membrane surface, and results with tritrpticin derivatives further revealed the effects of various amino acid substitutions on membrane binding. No significant improvement was observed upon increasing the Tritrp peptide charge from +4 to +5. Replacement of Arg residues with Lys did not substantially change peptide binding to anionic vesicles but moderately decreased the binding to zwitterionic LUVs. Pro to Ala substitutions in tritrpticin, allowing the peptide to adopt an alpha-helical structure, resulted in a significant increase of the binding to both anionic and zwitterionic vesicles and therefore reduced the selectivity for bacterial and mammalian membranes. In contrast, substitution of Trp with other aromatic amino acids significantly decreased the peptide's ability to bind to anionic LUVs and essentially eliminated binding to zwitterionic LUVs. The ITC results were consistent with the outcome of fluorescence spectroscopy membrane binding and perturbation studies. Overall, our work showed that a natural E. coli polar lipid extract as a bacterial membrane model was advantageous compared to the simpler and more widely used POPE/POPG lipid system.