Technique for isolating plasmids from exopolysaccharide producing Lactobacillus spp.

Summary A large scale plasmid isolation technique is described for the isolation of plasmids from exopolysaccharide producing strains of Lactobacillus spp. Plasmids of 1.9 to 56 kb were isolated which were pure enough to be used for restriction analysis and cloning experiments.     

Role of FGFs in skeletal muscle and limb development

Fibroblast growth factors (FGFs) are a family of nine proteins that bind to three distinct types of cell surface molecules: (i) FGF receptor tyrosine kinases (FGFR-1 through FGFR-4); (ii) a cysteine-rich FGF receptor (CFR); and (iii) heparan sulfate proteoglycans (HSPGs). Signaling by FGFs requires participation of at least two of these receptors: the FGFRs and HSPGs form a signaling complex. The length and sulfation pattern of the heparan sulfate chain determines both the activity of the signaling complex and, in part, the ligand specificity for FGFR-1. Thus, the heparan sulfate proteoglycans are likely to play an essential role in signaling. We have recently identified a role for FGF in limb bud development in vivo. In the chick limb bud, ectopic expression of the 18 kDa form of FGF-2 or FGF-2 fused to an artificial signal peptide at its amino terminus causes skeletal duplications. These data, and the observations that FGF-2 is localized to the subjacent mesoderm and the apical ectodermal ridge in the early developing limb, suggest that FGF-2 plays an important role in limb outgrowth. We propose that FGF-2 is an apical ectodermal ridgederived factor that participates in limb outgrowth and patterning. © 1994 Wiley-Liss, Inc.     

A complex experimental assessment for objective description of hierarchical psychophysiological behavior as human regulatory phenotype

For the objective and valid identification of different human regulatory phenotypes it should be useful to analyze the behavior of different regulatory subsystems (Anochin 1976) in one multivariate design. Therefore in a DARA supported project a fully computerized and reliable laboratory assessment was developed and tested. We used a set of electrophysiological parameters that should indicate the activity of different functional regulation systems on different "behavioral levels". Skin conductance, skin temperature and voice pitch were used as indicators of sympathico-parasympathical activity. Breathing, heart rate variability and bloodpressure should indicate cardiovascular activity and electromyogram and mimic variablity were thought as indicators of locomotional external behavioral activity. To identify physiological reactions which are influenced by emotional stress we used voice stress measures. Even in the field of aviation and space medicine there exist data about the correlation of voice pitch with emotional excitation (Hecker et. al. 1968, Williams et.al. 1969, Friedrich, Vaic 1978, Vaic et.al. 1981,1982, Griffin, Williams 1987). In our former study (MOSAIC-study, Johannes 1990) the voice pitch and its variation range correlated with perceived emotional excitation but were independent of real bloodpressure variations. Two different types of pitch reaction to this experimental design were correlated to psychological personality scales and assigned subjects to "sensitizers" and "suppressors".     

Flow cytometry reveals different lag times in rapid cytoplasmic calcium elevations in human neutrophils in response to N-formyl peptide

Flow cytometric analyses were performed to study intracellular single-cell calcium transients ([Ca²⁺]_i) in suspended human neutrophils during the initial phase of N-formyl peptide stimulation. Thereby, two neutrophil populations became apparent. Early maximally Ca²⁺-responding (high fluorescence) neutrophils and not-yet Ca²⁺-responding (low fluorescence) neutrophils, but no neutrophils with intermediate levels of [Ca²⁺]_i, were detected. Within 7 s the number of low fluorescence neutrophils decreased and the number of high fluorescence neutrophils increased maximally. This suggests that [Ca²⁺]_i transients occurred abruptly in individual neutrophils within a time interval below 1 s. At lower N-formyl peptide concentrations the lag times of individual neutrophils and the interval time of maximal activation of the [Ca²⁺]_i-responding neutrophil population increased, however the percentage of [Ca²⁺]_i-responding cells decreased. Surprisingly, no influence of the N-formyl peptide concentration on the [Ca²⁺]_i-induced fluorescence signal of the individual cell was observed: it was always in an almost maximal range or not responding. In parallel, binding studies performed with fluorescein-labeled N-formyl peptide revealed that the heterogeneity of [Ca²⁺]_i-responding cells cannot be explained by different receptor occupancy. In summary, this study demonstrates that [Ca²⁺]_i transients induced by N-formyl peptides in suspended individual human neutrophils occur very rapidly in an almost “all-or-none manner” and that the mean increasing fluorescence signal of a calcium indicator within a whole neutrophil population results from varying lag times of the individual cells, rather than from the mean simultaneous progress of many cells. © 1993 Wiley-Liss, Inc.     

Kinetics of growth and sugar consumption in yeasts

An overview is presented of the steady- and transient state kinetics of growth and formation of metabolic byproducts in yeasts.Saccharomyces cerevisiae is strongly inclined to perform alcoholic fermentation. Even under fully aerobic conditions, ethanol is produced by this yeast when sugars are present in excess. This so-called Crabtree effect probably results from a multiplicity of factors, including the mode of sugar transport and the regulation of enzyme activities involved in respiration and alcoholic fermentation. The Crabtree effect inS. cerevisiae is not caused by an intrinsic inability to adjust its respiratory activity to high glycolytic fluxes. Under certain cultivation conditions, for example during growth in the presence of weak organic acids, very high respiration rates can be achieved by this yeast.S. cerevisiae is an exceptional yeast since, in contrast to most other species that are able to perform alcoholic fermentation, it can grow under strictly anaerobic conditions.Non-Saccharomyces yeasts require a growth-limiting supply of oxygen (i.e. oxygen-limited growth conditions) to trigger alcoholic fermentation. However, complete absence of oxygen results in cessation of growth and therefore, ultimately, of alcoholic fermentation. Since it is very difficult to reproducibly achieve the right oxygen dosage in large-scale fermentations, non-Saccharomyces yeasts are therefore not suitable for large-scale alcoholic fermentation of sugar-containing waste streams. In these yeasts, alcoholic fermentation is also dependent on the type of sugar. For example, the facultatively fermentative yeastCandida utilis does not ferment maltose, not even under oxygen-limited growth conditions, although this disaccharide supports rapid oxidative growth.     

Quantitative Determination of the Spatial Distribution of Nitrosomonas europaea and Nitrobacter agilis Cells Immobilized in κ-Carrageenan Gel Beads by a Specific Fluorescent-Antibody Labelling Technique

A novel technique, combining labelling and stereological methods, for the determination of spatial distribution of two microorganisms in a biofilm is presented. Cells of Nitrosomonas europaea (ATCC 19718) and Nitrobacter agilis (ATCC 14123) were homogeneously distributed in a κ-carrageenan gel during immobilization and allowed to grow out to colonies. The gel beads were sliced in thin cross sections after fixation and embedding. A two-step labelling method resulted in green fluorescent colonies of either N. europaea or N. agilis in the respective cross sections. The positions and surface areas of the colonies of each species were determined, and from that a biomass volume distribution for N. europaea and N. agilis in κ-carrageenan gel beads was estimated. This technique will be useful for the validation of biofilm models, which predict such biomass distributions.     

CAPITULUM PHYLLOTAXIS AND NUMERICAL CANALIZATION IN MICROSERIS PYGMAEA (ASTERACEAE: LACTUCEAE)

The positions at which floret primordia arise in developing capitulum buds of Microseris pygmaea D. Don have been mapped by computer-assisted light microscopy. The primordia can be assigned positions along a basic phyllotactic spiral with a divergence angle of about 137.5°. In addition, there are regular deviations from a spiral arrangement. Typically, the first 26 primordia in phyllotactic sequence are arranged in two concentric circles of 13 primordia with considerable deviations in the divergence angle and in the distances between primordia along a parastichy at positions 13 and 26. This arrangement can be simulated by geometric models that include nearest neighbor packing, together with spiral phyllotaxis. The circular arrangement of peripheral primordia at nearly equal radial distances from the center of the developing capitulum helps to explain the numerical constancy (canalization) of peripheral structures, especially the constant number of 13 inner phyllaries on heads with very different numbers of florets.