Chlorophyll fluorescence induction and relaxation system for the continuous monitoring of photosynthetic capacity in photobioreactors

The development of high‐performance photobioreactors equipped with automatic systems for non‐invasive real‐time monitoring of cultivation conditions and photosynthetic parameters is a challenge in algae biotechnology. Therefore, we developed a chlorophyll (Chl) fluorescence measuring system for the online recording of the light‐induced fluorescence rise and the dark relaxation of the flash‐induced fluorescence yield (Qa^? ? re‐oxidation kinetics) in photobioreactors. This system provides automatic measurements in a broad range of Chl concentrations at high frequency of gas‐tight sampling, and advanced data analysis. The performance of this new technique was tested on the green microalgae Chlamydomonas reinhardtii subjected to a sulfur deficiency stress and to long‐term dark anaerobic conditions. More than thousand fluorescence kinetic curves were recorded and analyzed during aerobic and anaerobic stages of incubation. Lifetime and amplitude values of kinetic components were determined, and their dynamics plotted on heatmaps. Out of these data, stress‐sensitive kinetic parameters were specified. This implemented apparatus can therefore be useful for the continuous real‐time monitoring of algal photosynthesis in photobioreactors.     

Genetic polymorphism in Chondrilla (Asteraceae) in southern European Russia and the nature of Chondrilla juncea L

We studied genetic diversity in 54 populations of nine sexual and apomictic species of the genus Chondrilla (C. acantholepis, C. ambigua, C. brevirostris, C. canescens, C. graminea, C. juncea, C. laticoronata, C. latifolia and C. pauciflora) in SE European Russia and neighboring territories of NW Kazakhstan. We analysed the trnT–trnF region of plastid DNA and the internal transcribed spacer of ribosomal DNA (ITS1–5.8S–ITS2) using statistical parsimony, maximum likelihood and neighbor net methods. Two major evolutionary lineages, roughly corresponding to the two subgenera traditionally recognized in the region, were revealed. Within the first evolutionary lineage (subgenus Brachyrhynchus), the sexual diploid C. ambigua and its putatively hybrid apomictic derivatives C. brevirostris, C. laticoronata and C. pauciflora could be recognized. Their identity was also confirmed by analyses of ISSR markers. The second evolutionary lineage (subgenus Chondrilla) comprises C. juncea, C. acantholepis, C. canescens, C. graminea and C. latifolia in European Russia, but analyses of morphological variability and the genealogy of plastid and nuclear markers favor their treatment as the single facultatively apomictic species C. juncea. The results demonstrate that an apomictic mode of reproduction does not necessarily result in the formation of genetically separated microspecies.     

Extracellular oxidative enzyme production and PAH removal in soil by exploratory mycelium of white rot fungi

Selected strains of three species of white rot fungi, Pleurotus ostreatus, Phanerochaete chrysosporium and Trametes versicolor, were grown in sterilized soil from straw inocula. The respective colonization rates and mycelium density values decreased in the above mentioned order. Three- and four-ringed PAHs at 50 ppm inhibited growth of fungi in soil to some extent. The activities of fungal MnP and laccase (units per g dry weight of straw or soil), extracted with 50 mM succinate-lactate buffer (pH 4.5), were 5 to 20-fold higher in straw compared to soil. The enzyme activities per g dry soil in P. ostreatus and T. versicolor were similar, in contrast to P. chrysosporium, where they were extremely low. Compared to the aerated controls, P. ostreatus strains reduced the levels of anthracene, pyrene and phenanthrene by 81–87%, 84–93% and 41–64% within 2 months, respectively. During degradation of anthracene, all P. ostreatus strains accumulated anthraquinone. PAH removal rates in P. chrysosporium and T. versicolor soil cultures were much lower.     

Isoamyl alcohol-induced morphological change in Saccharomyces cerevisiae involves increases in mitochondria and cell wall chitin content

Isoamyl alcohol reduced growth and induced filament formation in Saccharomyces cerevisiae. Isoamyl alcohol-induced filamentation was accompanied by an almost threefold greater increase in the specific activity of succinate dehydrogenase than in untreated cells, which suggested that isoamyl alcohol treatment caused the cells to produce more mitochondria than in normal yeast form proliferation. This was supported by measuring the dry weight of purified, isolated mitochondria. Filaments have an increased chitin content which is distributed over the majority of their surface, and is not confined to bud scars and the chitin ring between mother and daughter cells as in yeast-form cells.     

Comparison of cytokinin-binding proteins from wheat and oat grains

Cytokinin-binding proteins (CBPs) isolated from mature grains of oat ( Avena sativa L.) and wheat ( Triticum aestivum L.) by acid precipitation, ion-exchange and affinity chromatography had similar characteristics, although they differed somewhat in apparent molecular weight of the native protein as determined by gel filtration (109 and 133 kDa, respectively) and subunit size as estimated by SDS-polyacrylamide gel electrophoresis (47 and 55 kDa, respectively). Highly purified oat CBP showed very weak but distinct immunochemical cross-reactivity with anti-wheat CBP IgG, indicating different immunogenic properties of the two CBPs. Nevertheless, both CBPs exhibited very similar binding of different cytokinins and were characterized by high affinity for N⁶-benzyladenine (BA)-type and by low affinity for zeatin-type cytokinins to both wheat and oat CBPs and by somewhat higher binding activities of oat CBP compared to wheat CBP (K_ds for BA: 4.6 × 10⁻⁷  M and 6.8 × 10⁻⁷  M , respectively). The potential role of CBPs in regulating free BA-type cytokinin levels during cereal grain development and germination is discussed.     

Activin and follicle-stimulating hormone signaling are required for long-term culture of functionally differentiated primary granulosa cells from the chicken ovary

Follicle-stimulating hormone, activin A, and transforming growth factor (TGF) alpha are important regulators of chicken granulosa cell (cGC) function. Hence, we aimed to test whether these growth factors are useful for establishing a suitable in vitro cell culture model system of primary cGC. Although cGC are easily isolated from distinct follicular stages, a long-term cGC culture system for in vitro studies has been unavailable. Here, we report a novel, long-term cell culture system that allows for cGC proliferation in vitro while maintaining the epithelial phenotype that granulosa cells exhibit in vivo. The cGC rapidly lose their epithelial morphology and acquire a mesenchymal or fibroblastoid phenotype when cultured in the absence of activin A. This process is strongly enhanced by TGFalpha, a well-known granulosa cell mitogen. However, FSH stimulates cGC proliferation without enhancing morphological changes and dedifferentiation. Interestingly, a combination of both activin A and FSH stimulates cGC proliferation and supports maintenance of differentiated epithelial morphology. Furthermore, activin A and FSH synergistically induce granulosa cell-specific differentiation markers such as inhibin alpha and chicken zona pellucida protein C, suggesting that cultured cGC resemble functionally differentiated granulosa cells. Our data demonstrate that activin signaling is necessary to sustain a morphologically differentiated phenotype of cGC in vitro. The results also suggest a pivotal importance of activin signaling for granulosa cell function in vivo.     

Ligand-receptor kinetics measured by total internal reflection with fluorescence correlation spectroscopy

Total internal reflection excitation used in combination with fluorescence correlation spectroscopy (TIR-FCS) is a method for characterizing the dynamic behavior and absolute concentrations of fluorescent molecules near or at the interface of a planar substrate and a solution. In this work, we demonstrate for the first time the use of TIR-FCS for examining the interaction kinetics of fluorescent ligands in solution which specifically and reversibly associate with receptors in substrate-supported planar membranes. Fluorescence fluctuation autocorrelation functions were obtained for a fluorescently labeled IgG reversibly associating with the mouse receptor FcgammaRII, which was purified and reconstituted into substrate-supported planar membranes. Data were obtained as a function of the IgG solution concentration, the Fc receptor surface density, the observation area size, and the incident intensity. Best fits of the autocorrelation functions to appropriate theoretical forms gave measures of the average surface density of bound IgG, the local solution concentration of IgG, the kinetic rate constant for surface dissociation, and the rate of diffusion through the depth of the evanescent field. The average number of observed fluorescent molecules, both in solution and bound to the surface, scaled with the solution concentration of IgG, observation area size, and Fc receptor surface density as expected. The dissociation rate constant and rate of diffusion through the evanescent field agree with previous results, and all measured parameters were independent of the incident intensity.     

The production and application of single-chain antibody fragments

This review discusses methods for the single-chain antibody fragment ($cFv) generation and scFv expression systems, and describes potential applications of scFv in the therapy of viral diseases and cancer, with emphasis on intracellularly expressed scFvs (intrabodies), application of scFvs in detection and diagnostics, and their use in proteomics.