Effects of dimethyl sulphoxide on ATP content and protein synthesis inTetrahymena

Summary The content of ATP in cells exposed for 1 hour to 2.5% and 7.5% dimethyl sulphoxide (DMSO) was 90% and about 80%, respectively, of that in control cells. This difference of about 10% in the ATP content cannot explain the previously reported cessation of food vacuole formation in 7.5% DMSO and the uninhibited function in 2.5% DMSO (Nilsson 1974). However, DMSO had a dose-dependent effect on the rate of turnover of ATP in cell extracts, thus the amount of ATP expended per unit time in 7.5% DMSO is only 60% of that expended by extracts of control cells. The rate of protein synthesis was studied by electron microscope autoradiography which revealed considerably less labelled material in DMSO-treated cells than in control cells. Semi-quantitative estimates showed that cells in 2.5%, 5%, and 7.5% DMSO had a rate of incorporation of the labelled amino acid corresponding to 38%, 31%, and 51%, respectively, of that of control cells; the seemingly high rate of incorporation in 7.5% DMSO may reflect a low internal pool of amino acids in these cells. An additional fine structural detail is the induction of intranuclear fibrous bundles in all concentrations of DMSO. The findings are in accord with a random interference of DMSO, presumably by inducing conformational changes in some macromolecules which affect their cellular function.     

Effect of estrogen on tendon collagen synthesis, tendon structural characteristics, and biomechanical properties in postmenopausal women

The knowledge about the effect of estradiol on tendon connective tissue is limited. Therefore, we studied the influence of estradiol on tendon synthesis, structure, and biomechanical properties in postmenopausal women. Nonusers (control, n = 10) or habitual users of oral estradiol replacement therapy (ERT, n = 10) were studied at rest and in response to one-legged resistance exercise. Synthesis of tendon collagen was determined by stable isotope incorporation [fractional synthesis rate (FSR)] and microdialysis technique (NH(2)-terminal propeptide of type I collagen synthesis). Tendon area and fibril characteristics were determined by MRI and transmission electron microscopy, whereas tendon biomechanical properties were measured during isometric maximal voluntary contraction by ultrasound recording. Tendon FSR was markedly higher in ERT users (P < 0.001), whereas no group difference was seen in tendon NH(2)-terminal propeptide of type I collagen synthesis (P = 0.32). In ERT users, positive correlations between serum estradiol (s-estradiol) and tendon synthesis were observed, whereas change in tendon synthesis from rest to exercise was negatively correlated to s-estradiol. Tendon area, fibril density, fibril volume fraction, and fibril mean area did not differ between groups. However, the percentage of medium-sized fibrils was higher in ERT users (P < 0.05), whereas the percentage of large fibrils tended to be greater in control (P = 0.10). A lower Young's modulus (GPa/%) was found in ERT users (P < 0.05). In conclusion, estradiol administration was associated with higher tendon FSR and a higher relative number of smaller fibrils. Whereas this indicates stimulated collagen turnover in the resting state, collagen responses to exercise were negatively associated with s-estradiol. These results indicate a pivotal role for estradiol in maintaining homeostasis of female connective tissue.     

Identification and ecophysiological characterization of epiphytic protein-hydrolyzing saprospiraceae ("Candidatus Epiflobacter" spp.) in activated sludge

The identity and ecophysiology of a group of uncultured protein-hydrolyzing epiphytic rods attached to filamentous bacteria in activated sludge from nutrient removal plants were investigated by using the full-cycle rRNA approach combined with microautoradiography and histochemical staining. The epiphytic group consists of three closely related clusters, each containing 11 to 16 clones. The closest related cultured isolate is the type strain Haliscomenobacter hydrossis (ATCC 27775) (<87% similarity) in the family Saprospiraceae of the phylum Bacteroidetes. Oligonucleotide probes at different hierarchical levels were designed for each cluster and used for ecophysiological studies. All three clusters behaved similarly in their physiology and were specialized in protein hydrolysis and used amino acids as energy and carbon sources. They were not involved in denitrification. No storage of polyphosphate and polyhydroxyalkanoates was found. They all colonized probe-defined filamentous bacteria belonging to the phyla Chloroflexi, Proteobacteria, and candidate phylum TM7, with the exception of cluster 1, which did not colonize TM7 filaments. The three epiphytic clusters were all widespread in domestic and industrial wastewater treatment plants with or without biological phosphorus removal, constituting, in total, up to 9% of the bacterial biovolume. A new genus, "Candidatus Epiflobacter," is proposed for this epiphytic group in activated-sludge treatment plants, where it presumably plays an important role in protein degradation.     

Biofilm Dynamics and Kinetics during High-Rate Sulfate Reduction under Anaerobic Conditions

The sulfate kinetics in an anaerobic, sulfate-reducing biofilm were investigated with an annular biofilm reactor. Biofilm growth, sulfide production, and kinetic constants (K_m and V_max) for the bacterial sulfate uptake within the biofilm were determined. These parameters were used to model the biofilm kinetics, and the experimental results were in good agreement with the model predictions. Typical zero-order volume rate constants for sulfate reduction in a biofilm without substrate limitation ranged from 56 to 93 μmol of SO²₄-cm⁻³ h⁻¹ at 20°C. The temperature dependence (Q₁₀) of sulfate reduction was equivalent to 3.4 at between 9 and 20°C. The measured rates of sulfate reduction could explain the relatively high sulfide levels found in sewers and wastewater treatment systems. Furthermore, it has been shown that sulfate reduction in biofilms just a few hundred micrometers thick is limited by sulfate diffusion into biofilm at concentrations below 0.5 mM. This observation might, in some cases, be an explanation for the relatively poor capacity of the sulfate-reducing bacteria to compete with the methanogenic bacteria in anaerobic wastewater treatment in submerged filters.     

Identity and ecophysiology of uncultured actinobacterial polyphosphate-accumulating organisms in full-scale enhanced biological phosphorus removal plants

Microautoradiography combined with fluorescence in situ hybridization (MAR-FISH) was used to screen for potential polyphosphate-accumulating organisms (PAO) in a full-scale enhanced biological phosphorus removal (EBPR) plant. The results showed that, in addition to uncultured Rhodocyclus-related PAO, two morphotypes hybridizing with gene probes for the gram-positive Actinobacteria were also actively involved in uptake of orthophosphate (Pi). Clone library analysis and further investigations by MAR-FISH using two new oligonucleotide probes revealed that both morphotypes, cocci in clusters of tetrads and short rods in clumps, were relatively closely related to the genus Tetrasphaera within the family Intrasporangiaceae of the Actinobacteria (93 to 98% similarity in their 16S rRNA genes). FISH analysis of the community biomass in the treatment plant investigated showed that the short rods (targeted by probe Actino-658) were the most abundant (12% of all Bacteria hybridizing with general bacterial probes), while the cocci in tetrads (targeted by probe Actino-221) made up 7%. Both morphotypes took up P(i) aerobically only if, in a previous anaerobic phase, they had taken up organic matter from wastewater or a mixture of amino acids. They could not take up short-chain fatty acids (e.g., acetate), glucose, or ethanol under anaerobic or aerobic conditions. The storage compound produced during the anaerobic period was not polyhydroxyalkanoates, as for Rhodocyclus-related PAO, and its identity is still unknown. Growth and uptake of Pi took place in the presence of oxygen and nitrate but not nitrite, indicating a lack of denitrifying ability. A survey of the occurrence of these actinobacterial PAO in 10 full-scale EBPR plants revealed that both morphotypes were widely present, and in several plants more abundant than the Rhodocyclus-related PAO, thus playing a very important role in the EBPR process.     

High diversity and abundance of putative polyphosphate-accumulating Tetrasphaera-related bacteria in activated sludge systems

The diversity of the putative polyphosphate-accumulating genus Tetrasphaera in wastewater treatment systems with enhanced biological phosphorus removal (EBPR) was investigated using the full-cycle rRNA approach combined with microautoradiography and histochemical staining. 16S rRNA actinobacterial gene sequences were retrieved from different full-scale EBPR plants, and the sequences belonging to the genus Tetrasphaera (family Intrasporangiaceae) were found to form three clades. Quantitative FISH analyses of the communities in five full-scale EBPR plants using 10 new oligonucleotide probes were carried out. The results showed that the probe-defined Tetrasphaera displayed different morphologies and constituted up to 30% of the total biomass. It was shown that active uptake of orthophosphate and formation of polyphosphate took place in most of the probe-defined Tetrasphaera populations. However, aerobic uptake of orthophosphate only took place after uptake of certain carbon sources under anaerobic conditions and these were more diverse than hitherto assumed: amino acids, glucose, and for some also acetate. Tetrasphaera seemed to occupy a slightly different ecological niche compared with 'Candidatus Accumulibacter' contributing to a functional redundancy and stability of the EBPR process.     

The Use of RP-HPLC for measuring activation and cleavage of rFVIIa during purification

A reverse phase HPLC (RP-HPLC) method for analysis of recombinant factor VIIa (rFVIIa) has been developed. The method discriminates between different forms of recombinant FVII (rFVII). To obtain separation of these closely related molecules the method has been optimized with respect to gradient profile and temperature. The method has been used for optimization of purification processes and for kinetic studies. EVidence for autolytic cleavage was obtained. (c) 1995 John Wiley & Sons, Inc.     

Isotope labeling and microautoradiography of active heterotrophic bacteria on the basis of assimilation of 14CO(2)

Most heterotrophic bacteria assimilate CO(2) in various carboxylation reactions during biosynthesis. In this study, assimilation of (14)CO(2) by heterotrophic bacteria was used for isotope labeling of active microorganisms in pure cultures and environmental samples. Labeled cells were visualized by microautoradiography (MAR) combined with fluorescence in situ hybridization (FISH) to obtain simultaneous information about activity and identity. Cultures of Escherichia coli and Pseudomonas putida assimilated sufficient (14)CO(2) during growth on various organic substrates to obtain positive MAR signals. The MAR signals were comparable with the traditional MAR approach based on uptake of (14)C-labeled organic substrates. Experiments with E. coli showed that (14)CO(2) was assimilated during both fermentation and aerobic and anaerobic respiration. The new MAR approach, HetCO(2)-MAR, was evaluated by targeting metabolic active filamentous bacteria, including "Candidatus Microthrix parvicella" in activated sludge. "Ca. Microthrix parvicella" was able to take up oleic acid under anaerobic conditions, as shown by the traditional MAR approach with [(14)C]oleic acid. However, the new HetCO(2)-MAR approach indicated that "Ca. Microthrix parvicella," did not significantly grow on oleic acid under anaerobic conditions with or without addition of NO(2)(-), whereas the addition of O(2) or NO(3)(-) initiated growth, as indicated by detectable (14)CO(2) assimilation. This is a metabolic feature that has not been described previously for filamentous bacteria. Such information could not have been derived by using the traditional MAR procedure, whereas the new HetCO(2)-MAR approach differentiates better between substrate uptake and substrate metabolism that result in growth. The HetCO(2)-MAR results were supported by stable isotope analysis of (13)C-labeled phospholipid fatty acids from activated sludge incubated under aerobic and anaerobic conditions in the presence of (13)CO(2). In conclusion, the novel HetCO(2)-MAR approach expands the possibility for studies of the ecophysiology of uncultivated microorganisms.