Intrinsic choroidal neurons in the duck eye receive sympathetic input: anatomical evidence for adrenergic modulation of nitrergic functions in the choroid

Intrinsic choroidal neurons (ICN) in the duck eye form an intramural ganglionic plexus that may subserve complex integrative functions. A key feature of such ganglia is an innervation by sympathetic postganglionic neurons. The present study was thus aimed at determining the sympathetic postganglionic innervation of ICN. Choroids were processed for double immunofluorescence labelling with the following markers: tyrosine-hydroxylase (TH)/nitric oxide synthase (nNOS), TH/galanin (GAL), dopamine-beta-hydroxylase (DBH)/vasoactive intestinal polypeptide (VIP), TH/DBH and DBH/alpha-smooth-muscle actin (alphaSMA), and for triple immunofluorescence labelling with VIP/DBH/TH. Epifluorescence and confocal laser scanning microscopy were used for evaluation. Immunoperoxidase staining for TH or DBH in combination with NADPH-diaphorase histochemistry was applied for electron microscopy. ICN spread over the entire choroid but were concentrated in an equatorial zone passing obliquely from naso-cranial to temporocaudal. More than 80% of nNOS-positive ICN showed close appositions of TH/DBH-immunoreactive varicose nerve fibres at the light-microscopic level, as could be confirmed by confocal laser scanning microscopy. Ultrastructurally, these appositions could be defined as both synapses or close contacts without synaptic specialisation. Vascular and non-vascular smooth muscle fibres also received TH/DBH-immunopositive innervation. Our findings suggest that most ICN receive a sympathetic input that might modulate their nitrergic effects upon vascular and non-vascular smooth muscle fibres in the choroid and that they may have more complex functions than merely being a simple parasympathetic relay.     

The Aspergillus nidulans homoaconitase gene lysF is negatively regulated by the multimeric CCAAT-binding complex AnCF and positively regulated by GATA sites

In beta-lactam-antibiotic-producing fungi, such as Aspergillus (Emericella) nidulans, L-alpha-aminoadipic acid is the branching point of the lysine and penicillin biosynthesis pathways. To obtain a deeper insight into the regulation of lysine biosynthesis genes, the regulation of the A. nidulans lysF gene, which encodes homoaconitase, was studied. Band-shift assays indicated that the A. nidulans multimeric CCAAT-binding complex AnCF binds to two of four CCAAT motifs present in the lysF promoter region. AnCF consists at least of three different subunits, designated HapB, HapC, and HapE. In both a delta hapB and a delta hapC strain, the expression of a translational lysF-lacZ gene fusion integrated in single copy at the chromosomal argB gene locus was two to three-fold higher than in a wild-type strain. These data show that AnCF negatively regulates lysF expression. The results of Northern blot analysis and lysF-lacZ expression analysis did not indicate a lysine-dependent repression of lysF expression. Furthermore, mutational analysis of the lysF promoter region revealed that two GATA sites matching the GATA consensus sequence HGATAR positively affected lysF-lacZ expression. Results of Northern blot analysis also excluded that the global nitrogen regulator AreA is the responsible trans-acting GATA-binding factor.     

Effect of Temperature on Carbon and Electron Flow and on the Archaeal Community in Methanogenic Rice Field Soil

Temperature is an important factor controlling CH₄ production in anoxic rice soils. Soil slurries, prepared from Italian rice field soil, were incubated anaerobically in the dark at six temperatures of between 10 to 37°C or in a temperature gradient block covering the same temperature range at intervals of 1°C. Methane production reached quasi-steady state after 60 to 90 days. Steady-state CH₄ production rates increased with temperature, with an apparent activation energy of 61 kJ mol⁻¹. Steady-state partial pressures of the methanogenic precursor H₂ also increased with increasing temperature from <0.5 to 3.5 Pa, so that the Gibbs free energy change of H₂ plus CO₂-dependent methanogenesis was kept at −20 to −25 kJ mol of CH₄⁻¹ over the whole temperature range. Steady-state concentrations of the methanogenic precursor acetate, on the other hand, increased with decreasing temperature from <5 to 50 μM. Simultaneously, the relative contribution of H₂ as methanogenic precursor decreased, as determined by the conversion of radioactive bicarbonate to ¹⁴CH₄, so that the carbon and electron flow to CH₄ was increasingly dominated by acetate, indicating that psychrotolerant homoacetogenesis was important. The relative composition of the archaeal community was determined by terminal restriction fragment length polymorphism (T-RFLP) analysis of the 16S rRNA genes (16S rDNA). T-RFLP analysis differentiated the archaeal Methanobacteriaceae, Methanomicrobiaceae, Methanosaetaceae, Methanosarcinaceae, and Rice clusters I, III, IV, V, and VI, which were all present in the rice field soil incubated at different temperatures. The 16S rRNA genes of Rice cluster I and Methanosaetaceae were the most frequent methanogenic groups. The relative abundance of Rice cluster I decreased with temperature. The substrates used by this microbial cluster, and thus its function in the microbial community, are unknown. The relative abundance of acetoclastic methanogens, on the other hand, was consistent with their physiology and the acetate concentrations observed at the different temperatures, i.e., the high-acetate-requiring Methanosarcinaceae decreased and the more modest Methanosaetaceae increased with increasing temperature. Our results demonstrate that temperature not only affected the activity but also changed the structure and the function (carbon and electron flow) of a complex methanogenic system.     

Development of facies and C/O-isotopes in transects through the Ludlow of Gotland: Evidence for global and local influences on a shallow-marine environment

Summary The Silurian of Gotland is characterized by repeated changes in depositional facies development. The deposition of uniform sequences of micritic limestones and marls was interrupted four times by the growth of reef complexes and the formation of expanded carbonate platforms. Coinciding with these, often abrupt, facies changes extinction events occurred which predominantly affected nektonic and planktonic organisms. Ratios of carbon- and oxygen-isotopes covary with the facies development. Periods in which the deposition of limestonemarl alternations prevailed are characterized by relatively low C- and O-isotope values. During periods of enhanced reef growth isotope values are high. For these changes,Bickert et al. (1997) assume climatic changes between humid “H-periods”, with estuarine circulation systems and cutrophic surface waters with decreased salinity in marginal seas, and arid “A-periods”, with an antiestuarine circulation and oligotrophic, stronger saline surface waters. In order to separate local and regional influences on the isotopic development from the global trend, the interactions between facies formation and isotope record have to be clarified. For this purpose, the patterns of isotope values in the upper part of the Silurian sequence on Gotland (upper Wenlock —upper Ludlow) has been determined and stratigraphically correlated along four transects through different facies areas. Facies formation during this time interval was investigated by differentiation and mapping of twelve facies complexes in the southern part of Gotland. These include shelf areas, reef complexes with patch reefs and biostromes, backreef facies, and marginal-marine deposits. The good correspondence between the carbon-isotope records of the four transects suggests that local environmental conditions in the different facies areas did not influence the δ¹³C values. Therefore, a supra-regional or even global mechanism for the C-isotope variations is likely. In contrast to carbon istopes, the oxygen-isotope values of the four transects generally show parallel trends, but higher variabilities and in parts distinctly deviating developments with a trend to more negative values. These are interpreted as an effect of local warming in small shallow-water areas which developed during arid periods in reef complexes and backreef areas. The boundaries between A-periods and H-periods, as defined by δ¹³C values, which are interpreted as isochrones, can be mapped. From the upper Homerian to the Pridolian six parastratigraphic isotope zones are defined which only partly match the stratigraphic division ofHede (1942, 1960). The isotope stratigraphy results in an improved correlation between the shallow and marginal-marine areas in the eastern part of Gotland and the uniform shelf areas at the west coast of the island. Furthermore, a detailed relationship between the development of carbon and oxygen isotope ratios, the carbonate facies formation, and the succession of palaeontological events could be observed. At the transition from H-periods to A-periods, major extinction events occurred prior to the first increase of δ¹³C and δ¹⁸O values. Extinction events affected conodonts, graptolites, acritarchs, chitinozoans, and vertebrates and resulted in impoverished nektonic and planktonic communities. The reef-building benthos was less affected. Parallel to a first slight increase of isotope values, facies began to change, and reefs developed in suitable locations. The subsequent rapid increase of C- and O-isotope values occurred contemporarily with strong facies changes and a short-term drop of sea-level. Oligotrophic conditions in the later stages of A-periods led to strong reef growth and to an expansion of carbonate platforms. The transitions from A-periods to H-periods were more gradual. The δ¹³C values decreased slowly, but reef growth continued. Later the reefs retreated and were covered by the prograding depositional facies of shelf areas. The diversity of planktonic and nektonic communities increased again. The close relationship between facies formation, palaeontological events and isotope records in the Silurian suggests common steering mechanisms but gives no indication of the causes for the repeated extincion events related to H-period/A-period transitions. Especially the observation, that strong extinctions occurred prior to changes of isotope values and facies, points to causes that left no signals in the geological record. Hypothetical causes like collapse of trophical nets, anoxias, or cooling events are not evident in the sediment record or do not fit into the regular succession of period transitions.     

Comparative evaluation of gene delivery devices in primary cultures of rat hepatic stellate cells and rat myofibroblasts

Background  

The cell surface undergoes continuous change during cell movement. This is characterized by transient protrusion and partial or complete retraction of microspikes, filopodia, and lamellipodia. This requires a dynamic actin cytoskeleton, moesin, components of Rho-mediated signal pathways, rearrangement of membrane constituents and the formation of focal adhesion sites. While the immunofluorescence distribution of endogenous moesin is that of a membrane-bound molecule with marked enhancement in some but not all microextensions, the C-terminal fragment of moesin co-distributes with filamentous actin consistent with its actin-binding activity. By taking advantage of this property we studied the spontaneous protrusive activity of live NIH3T3 cells, expressing a fusion of GFP and the C-terminal domain of moesin.     

The MaoP/maoS Site-Specific System Organizes the Ori Region of the E. coli Chromosome into a Macrodomain

The Ori region of bacterial genomes is segregated early in the replication cycle of bacterial chromosomes. Consequently, Ori region positioning plays a pivotal role in chromosome dynamics. The Ori region of the E. coli chromosome is organized as a macrodomain with specific properties concerning DNA mobility, segregation of loci and long distance DNA interactions. Here, by using strains with chromosome rearrangements and DNA mobility as a read-out, we have identified the MaoP/maoS system responsible for constraining DNA mobility in the Ori region and limiting long distance DNA interactions with other regions of the chromosome. MaoP belongs to a group of proteins conserved in the Enterobacteria that coevolved with Dam methylase including SeqA, MukBEF and MatP that are all involved in the control of chromosome conformation and segregation. Analysis of DNA rings excised from the chromosome demonstrated that the single maoS site is required in cis on the chromosome to exert its effect while MaoP can act both in cis and in trans. The position of markers in the Ori region was affected by inactivating maoP. However, the MaoP/maoS system was not sufficient for positioning the Ori region at the ¼–¾ regions of the cell. We also demonstrate that the replication and the resulting expansion of bulk DNA are localized centrally in the cell. Implications of these results for chromosome positioning and segregation in E. coli are discussed.