Development of dopamine-containing neurons and dopamine uptake in embryos of Hirudo medicinalis

The embryonic development of neurons which contain or take up dopamine was studied with glyoxylic acid histofluorescence in Hirudo medicinalis. Beginning at the time of the formation of the tail ganglion, one pair of dopamine-containing neurons was stained per segmental ganglion. The normal outgrowth of the cell bodies into the anterior roots was prevented in isolated and cultured chains of embryonic ganglia. Preincubation of intact embryos in dopamine led to the staining of additional neurons at certain developmental stages. These neurons presumably are the precursors of serotonin-containing cells, which have a temporary capability of taking up and storing dopamine.     

Protein expression patterns of identified neurons and of sprouting cells from the leech central nervous system

It has previously been shown that cephalic, segmental, and caudal ganglia from the medicinal leech show differences in their protein composition. Here we studied whether the neuronal reorganization that occurs in cultured segmental ganglia from the medicinal leech is accompanied by detectable changes in the protein expression pattern. Using silver-stained two-dimensional gels we showed that after 5 and 12 days in culture changes in the protein patterns can be detected in isolated ganglia. The changes observed in the two-dimensional gels occurred concomitantly with a sprouting of serotoninergic neurites and a decreased transmitter content of dopaminergic neurites as shown by using the glyoxylic acid condensation reaction. In addition, we present evidence that Retzius cells, which can be identified by their characteristic morphology and action potential waveform, exhibit biochemically unique properties with respect to their protein expression pattern.     

Evidence against a Looped Structure of the Inactive Human X-Chromosome Territory

Multicolor fluorescencein situhybridization with a whole chromosome composite probe for the X-chromosome and microdissection probes for the Xp and Xq arms, as well as for the Xp terminal, Xq terminal, and X centromer specific subregional probes, was applied to three-dimensional (3D) preserved human female amniotic fluid cell nuclei. Confocal laser scanning microscopy and three-dimensional image analysis demonstrated distinctly separated Xp arm and Xq arm domains. 3D distance measurements revealed a high variability of intrachromosomal distances between Xpter, Xcen, and Xqter specific probes within both X territories. A 3D distance measurement error of ±70 nm was found in control experiments using quartz glass microspheres labeled with different fluorochromes. Our data argue against the hypothesis of Walkeret al.(1991,Proc. Natl. Acad. Sci. USA88, 6191–6195) that a looped structure of the inactive X territory is formed by tight telomere–telomere associations.     

Quantitative comparison of DNA detection by GFP-lac repressor tagging,fluorescence in situ hybridization and immunostaining

Background  

GFP-fusion proteins and immunostaining are methods broadly applied to investigate the three-dimensional organization of cells and cell nuclei, the latter often studied in addition by fluorescence in situ hybridization (FISH). Direct comparisons of these detection methods are scarce, however.     

Regulation of neuronal excitability by release of proteins from glial cells

Effects of glial cells on electrical isolation and shaping of synaptic transmission between neurons have been extensively studied. Here we present evidence that the release of proteins from astrocytes as well as microglia may regulate voltage-activated Na⁺ currents in neurons, thereby increasing excitability and speed of transmission in neurons kept at distance from each other by specialized glial cells. As a first example, we show that basic fibroblast growth factor and neurotrophin-3, which are released from astrocytes by exposure to thyroid hormone, influence each other to enhance Na⁺ current density in cultured hippocampal neurons. As a second example, we show that the presence of microglia in hippocampal cultures can upregulate Na⁺ current density. The effect can be boosted by lipopolysaccharides, bacterial membrane-derived stimulators of microglial activation. Comparable effects are induced by the exposure of neuron-enriched hippocampal cultures to tumour necrosis factor-α, which is released from stimulated microglia. Taken together, our findings suggest that release of proteins from various types of glial cells can alter neuronal excitability over a time course of several days. This explains changes in neuronal excitability occurring in states of thyroid hormone imbalance and possibly also in seizures triggered by infectious diseases.     

Photosystem II supercomplex remodeling serves as an entry mechanism for state transitions in Arabidopsis

Within dense plant populations, strong light quality gradients cause unbalanced excitation of the two photosystems resulting in reduced photosynthetic efficiency. Plants redirect such imbalances by structural rearrangements of the photosynthetic apparatus via state transitions and photosystem stoichiometry adjustments. However, less is known about the function of photosystem II (PSII) supercomplexes in this context. Here, we show in Arabidopsis thaliana that PSII supercomplex remodeling precedes and facilitates state transitions. Intriguingly, the remodeling occurs in the short term, paralleling state transitions, but is also present in a state transition-deficient mutant, indicating that PSII supercomplex generation is independently regulated and does not require light-harvesting complex phosphorylation and movement. Instead, PSII supercomplex remodeling involves reversible phosphorylation of PSII core subunits (preferentially of CP43) and requires the luminal PSII subunit Psb27 for general formation and structural stabilization. Arabidopsis knockout mutants lacking Psb27 display highly accelerated state transitions, indicating that release of PSII supercomplexes is required for phosphorylation and subsequent movement of the antenna. Downregulation of PSII supercomplex number by physiological light treatments also results in acceleration of state transitions confirming the genetic analyses. Thus, supercomplex remodeling is a prerequisite and an important kinetic determinant of state transitions.     

Quantitative analysis of DNA-damage response factors after sequential ion microirradiation

Several proteins are known to form foci at DNA sites damaged by ionizing radiation. We study DNA damage response by immunofluorescence microscopy after microirradiation of cells with energetic ions. By using microirradiation, it is possible to irradiate different regions on a single dish at different time-points and to differentiate between cells irradiated earlier and later. This allows to directly compare immunofluorescence intensities in both subsets of cells with little systematic error because both subsets are cultivated and stained under identical conditions. In addition, by using irradiation patterns such as crossing lines, it is possible to irradiate individual cells twice and to differentiate between immunofluorescence signals resulting from the cellular response to the earlier and to the later irradiation event. Here, we describe the quantitative evaluation of immunofluorescence intensities after sequential irradiation.     

Energy dissipation mechanisms in the FCPb light-harvesting complex of the diatom Cyclotella meneghiniana

Transient absorption spectroscopy has been applied to investigate the energy dissipation mechanisms in the nonameric fucoxanthin-chlorophyll-a,c-binding protein FCPb of the centric diatom Cyclotella meneghiniana. FCPb complexes in their unquenched state were compared with those in two types of quenching environments, namely aggregation-induced quenching by detergent removal, and clustering via incorporation into liposomes. Applying global and target analysis, in combination with a fluorescence lifetime study and annihilation calculations, we were able to resolve two quenching channels in FCPb that involve chlorophyll-a pigments for FCPb exposed to both quenching environments. The fast quenching channel operates on a timescale of tens of picoseconds and exhibits similar spectral signatures as the unquenched state. The slower quenching channel operates on a timescale of tens to hundreds of picoseconds, depending on the degree of quenching, and is characterized by enhanced population of low-energy states between 680 and 710?nm. The results indicate that FCPb is, in principle, able to function as a dissipater of excess energy and can do this in vitro even more efficiently than the homologous FCPa complex, the sole complex involved in fast photoprotection in these organisms. This indicates that when a complex displays photoprotection-related spectral signatures in vitro it does not imply that the complex participates in photoprotection in vivo. We suggest that FCPa is favored over FCPb as the sole energy-regulating complex in diatoms because its composition can more easily establish the balance between light-harvesting and quenching required for efficient photoprotection.     

Large-scale chromatin structure and function.

Recent results in living cells have now established the existence of levels of chromatin folding above the 30 nm fiber within interphase chromosomes. We discuss the potential functional impact of this large-scale chromatin organization, including its possible role in regulating gene expression.