Purification and biochemical properties of complex flagella isolated from Rhizobium lupini H13-3

1. The complex flagella of Rhizobium lupini H13-3 differ from plain bacterial flagella in the fine structure of their filaments dominated by conspicuous helical bands, in their fragility and their resistance against heat decomposition. To elucidate the basis of these differences, the composition of complex filaments and their subunits was analysed. 2. Isolated complex flagella containing the filament and hook protions were purified by differential centrifugation. Hooks were separated by ultracentrifugation after acid degradation of filaments at pH 2. The complex filaments consist of 43 000 dalton monomers (cx-flagellin), the hooks are composed of 41 000 dalton subunits. 3. Amino acid analysis of cx-flagellin indicated the presence of approx. 417 amino acid residues. These comprise 47% hydrophobic residues and 21% Asp and Glu (or amides), but no Cys, His, Pro and Trp. No carbohydrate, phosphate or lipid moieties have been detected. Fingerprint analysis after tryptic digestion yields approx. 36 peptides, about half of them clustered in the neutral region. A comparison with the composition of varous known flagellins from plain flagella indicates a 7% higher content of hydrophobic amino acid residues in complex filaments; this is largely compensated for by the higher content of Glu and Asp (presumably as Gln and Asn) in plain filaments. 4. Immunodiffusion and immunoelectrophoresis of cx-flagellin yield single precipitin bands indicating homogeneity. In contrast, isoelectric focusing lead to three close-running bands around pH4.7. When isolated, the two major bands again produced an "isoelectric spectrum" suggesting that it reflects an allomorphism of cx-flagellin. 5. Self-assembly experiments with cx-flagellin lead to coiled fibres including helical regions, but not to intact filaments. The products resemble heat-denatured complex filaments and may represent intermediates between monomers and complete polymers.     

A novel reaction of S-adenosyl-L-methionine correlated with the activation of pyruvate formate-lyase

Conversion of the inactive form of pyruvate formate-lyase to the catalytically active enzyme is accomplished by the Fe-dependent ‘enzyme II’; reduced flavodoxin, S-adenosyl-L-methionine and the effector pyruvate are required. It was found that adenosylmethionine is reductively processed during activation of pyruvate formate-lyase to yield methionine, adenine and 5-deoxyribose. We suggest that transient adenosylation of enzyme II is required for its function as a converter enzyme.     

Oxidase (donor: oxygen oxidoreductase) activity by peroxidase and alpha2-macroglobulin interaction.

The interaction between peroxidase (donor: hydrogenperoxide oxidoreductase, EC 1.11.1.7) and human alpha2-macroglobulin has been studied by employing starch gel electrophoresis and spectrophotometric assay analysis.     

Electron microscopy of ageing cells of Pseudomonas rhodos: fine structure of native and isolated tubular membranes (author's transl)]

During a 10 day-incubation on agar surfaces at 30 degrees C, cells of the gram-negative soil bacterium Pseudomonas rhodos pass through three phases distinguishable by physiological and morphological criteria. When viewed by electron microscopy, typically "rolled" mesosomes could frequently be observed in young cells. In aged cells instead, loosely rolled or stretched-out, flattened tubules could be discerned, presumed to be degenerate mesosomes. Tubular flattened structures have been isolated from these cells by lysozyme treatment or sonication and were concentrated by differential centrifugation. Electron micrographs of these preparations showed long, straight tubules which sometimes appeared sealed at one end. Their width was 34 +/- 5 nm. They contained a lining of material, which could be digested by trypsin leaving behind an electron-transparent matrix. In rare cases, isolated tubules showed a periodic fine structure composed of ellipsoidal subunits. Optical diffraction analysis yielded a lattice consisting of subunits arranged in helices of pitch-angle 27 degrees; the unit cell dimensions were shown to be 112 X 56 A. Owing to their sensitivity to trypsin, components of the regular lattice are supposed to consist of protein. It is postulated that these protein components are layered onto a tubular membrane. These tubules are clearly distinguishable by their shape and fine structure from the periodic structure of a P. rhodos cell wall layer, which exhibits a tetragonal pattern, and also from polyheads and polysheaths of defective bacteriophages. Their possible origin from intact mesosomes in discussed.     

Intraspecific nestmate recognition in two parabiotic ant species: acquired recognition cues and low inter-colony discrimination

Parabiotic ants—ants that share their nest with another ant species—need to tolerate not only conspecific nestmates, but also nestmates of a foreign species. The parabiotic ants Camponotus rufifemur and Crematogaster modiglianii display high interspecific tolerance, which exceeds their respective partner colony and extends to alien colonies of the partner species. The tolerance appears to be related to unusual cuticular substances in both species. Both species possess hydrocarbons of unusually high chain lengths. In addition, Cr. modiglianii carries high quantities of hereto unknown compounds on its cuticle. These unusual features of the cuticular profiles may affect nestmate recognition within both respective species as well. In the present study, we therefore examined inter-colony discrimination within the two parabiotic species in relation to chemical differentiation. Cr. modiglianii was highly aggressive against workers from alien conspecific colonies in experimental confrontations. In spite of high inter-colony variation in the unknown compounds, however, Cr. modiglianii failed to differentiate between intracolonial and allocolonial unknown compounds. Instead, the cuticular hydrocarbons functioned as recognition cues despite low variation across colonies. Moreover, inter-colony aggression within Cr. modiglianii was significantly influenced by the presence of two methylbranched alkenes acquired from its Ca. rufifemur partner. Ca. rufifemur occurs in two varieties (‘red’ and ‘black’) with almost no overlap in their cuticular hydrocarbons. Workers of this species showed low aggression against conspecifics from foreign colonies of the same variety, but attacked workers from the respective other variety. The low inter-colony discrimination within a variety may be related to low chemical differentiation between the colonies. Ca. rufifemur majors elicited significantly more inter-colony aggression than medium-sized workers. This may be explained by the density of recognition cues: majors carried significantly higher quantities of cuticular hydrocarbons per body surface.     

Design and validation of a homogeneous time-resolved fluorescence cell-based assay targeting the ligand-gated ion channel 5-HT3A

Ligand-gated ion channels (LGICs) are considered as attractive protein targets in the search for new therapeutic agents. Nowadays, this strategy involves the capability to screen large chemical libraries. We present a new Tag-lite ligand binding assay targeting LGICs on living cells. This technology combines the use of suicide enzyme tags fused to channels of interest with homogeneous time-resolved fluorescence (HTRF) as the detection readout. Using the 5-HT3 receptor as system model, we showed that the pharmacology of the HALO-5HT3 receptor was identical to that of the native receptor. After validation of the assay by using 5-HT3 agonists and antagonists of reference, a pilot screen enabled us to identify azelastine, a well-known histamine H1 antagonist, as a potent 5-HT3 antagonist. This interesting result was confirmed with electrophysiological experiments. The method described here is easy to implement and could be applicable for other LGICs, opening new ways for the screening of chemical libraries.     

Real‐time Raman and SRS imaging of living human macrophages reveals cell‐to‐cell heterogeneity and dynamics of lipid uptake

Monitoring living cells in real‐time is important in order to unravel complex dynamic processes in life sciences. In particular the dynamics of initiation and progression of degenerative diseases is intensely studied. In atherosclerosis the thickening of arterial walls is related to high lipid levels in the blood stream, which trigger the lipid uptake and formation of droplets as neutral lipid reservoirs in macrophages in the arterial wall. Unregulated lipid uptake finally results in foam cell formation, which is a hallmark of atherosclerosis. In previous studies, the uptake and storage of different fatty acids was monitored by measuring fixed cells. Commonly employed fluorescence staining protocols are often error prone because of cytotoxicity and unspecific fluorescence backgrounds. By following living cells with Raman spectroscopic imaging, lipid uptake of macrophages was studied with real‐time data acquisition. Isotopic labeling using deuterated palmitic acid has been combined with spontaneous and stimulated Raman imaging to investigate the dynamic process of fatty acid storage in human macrophages for incubation times from 45 min to 37 h. Striking heterogeneity in the uptake rate and the total concentration of deuterated palmitic acid covering two orders of magnitude is detected in single as well as ensembles of cultured human macrophages.

SRS signal of deuterated palmitic acid measured at the CD vibration band after incorporation into living macrophages.     

Multiple differentiation centres of a non-Mediterranean butterfly species in south-eastern Europe

Aim  The analysis of the phylogeographical structures of many European species reveals the importance of Mediterranean glacial refugia for many thermophilic species, but also underlines the relevance of extra-Mediterranean glacial differentiation centres for a number of temperate species. In this context, phylogeographical analyses of species from south-eastern Europe are highly important for a comprehensive understanding of Europe as a whole.
Location  Romania and Bulgaria.
Methods  We analysed 19 allozyme loci for 615 individuals of the temperate butterfly species Erebia medusa from 28 populations.
Results  These populations had an intermediate genetic diversity, but the Bulgarian populations were significantly more diverse than the ones north of the Danube in Romania. The differentiation among populations was strong, and 52.1% of the genetic variance among populations was distributed between these two countries. The genetic differentiation was considerably stronger in Romania than in Bulgaria, but several sublineages were distinguished within each of these countries.
Main conclusions  The observed genetic structure is so strong that it is most probably the result of glacial differentiation processes in south-eastern Europe and not a post-glacial structure. The strong differentiation into the two groups north and south of the Danube suggests a separating effect by this river valley. The strong differentiation accompanied with genetic impoverishment in Romania suggests the existence of several differentiation centres: at least two small ones on the southern slopes of the southern Carpathians and one in the eastern Carpathian Basin. The considerably weaker differentiation among the Bulgarian samples and their significantly higher genetic diversity imply that gene flow occurred among different regions of Bulgaria during the last ice age.