Membrane Potential and Catecholamine Secretion by Bovine Adrenal Chromaffin Cells: Use of Tetraphenylphosphonium Distribution and Carbocyanine Dye Fluorescence

Changes in plasma membrane potential of isolated bovine adrenal chromaffin cells were measured independently by two chemical probe methods and related to corresponding effects on catecholamine secretion. The lipophilic cation tetraphenylphosphonium (TPP+) and the carbocyanine dye 3,3'-dipropylthiadicarbocyanine [DiS-C3-(5)] were used. The necessity of evaluating the subcellular distribution of TPP+ among cytoplasmic, mitochondrial, secretory granule, and bound compartments was demonstrated and the resting plasma membrane potential determined to be -55 mV. The relationship between membrane potential and catecholamine secretion was determined in response to variations in extracellular K+ and to the presence of several secretagogues including cholinergic receptor ligands, veratridine, and ionophores for Na+ and K+. The dependence of potential on K+ concentration fit the Goldman constant field equation with a Na/K permeability ratio of 0.1. The dependence of both K+- and veratridine-evoked catecholamine secretion on membrane potential exhibited a potential threshold of about -40 mV before a significant rise in secretion occurred. This is likely related to the threshold for opening of voltage-sensitive Ca2+ channels. Acetylcholine and nicotine evoked a large secretory response without a sufficiently sustained depolarization to be detectable by the relatively slow potential sensitive chemical probes. Decamethonium induced a detectable depolarization of the chromaffin cells. Veratridine and gramicidin evoked both membrane depolarization and catecholamine release. By contrast the K ionophore valinomycin evoked significant levels of secretion without any depolarization. This is consistent with its utilization of an intracellular source of Ca2+ and the independence of its measured secretory response on extracellular Ca2+.     

Sea urchin Hox genes: insights into the ancestral Hox cluster

We describe the Hox cluster in the radially symmetric sea urchin and compare our findings to what is known from clusters in bilaterally symmetric animals. Several Hox genes from the direct-developing sea urchin Heliocidaris erythrogramma are described. CHEF gel analysis shows that the Hox genes are clustered on a < or = 300 kilobase (kb) fragment of DNA, and only a single cluster is present, as in lower chordates and other nonvertebrate metazoans. Phylogenetic analyses of sea urchin, amphioxus, Drosophila, and selected vertebrate Hox genes confirm that the H. erythrogramma genes, and others previously cloned from other sea urchins, belong to anterior, central, and posterior groups. Despite their radial body plan and lack of cephalization, echinoderms retain at least one of the anterior group Hox genes, an orthologue of Hox3. The structure of the echinoderm Hox cluster suggests that the ancestral deuterostome had a Hox cluster more similar to the current chordate cluster than was expected Sea urchins have at least three Abd-B type genes, suggesting that Abd-B expansion began before the radiation of deuterostomes.      

Microbial reductive dechlorination of trichloroethene to ethene with electrodes serving as electron donors without the external addition of redox mediators

In situ bioremediation of industrial chlorinated solvents, such as trichloroethene (TCE), is typically accomplished by providing an organic electron donor to naturally occurring dechlorinating populations. In the present study, we show that TCE dechlorinating bacteria can access the electrons required for TCE dechlorination directly from a negatively polarized (?450 mV vs. SHE) carbon paper electrode. In replicated batch experiments, a mixed dechlorinating culture, also containing Dehalococcoides spp., dechlorinated TCE to cis‐dichloroethene (cis‐DCE) and lower amounts of vinyl chloride (VC) and ethene using the polarized electrode as the sole electron donor. Conversely, neither VC nor ethene formation occurred when a pure culture of the electro‐active microorganism Geobacter lovleyi was used, under identical experimental conditions. Cyclic voltammetry tests, carried out on the filter‐sterilized supernatant of the mixed culture revealed the presence of a self‐produced redox mediator, exhibiting a midpoint potential of around ?400 mV (vs. SHE). This yet unidentified redox‐active molecule appeared to be involved in the extracellular electron transfer from the electrode to the dechlorinating bacteria. The ability of dechlorinating bacteria to use electrodes as electron donors opens new perspectives for the development of clean, versatile, and efficient bioremediation systems based on a controlled subsurface delivery of electrons in support of biodegradative metabolisms and provides further evidence on the possibility of using conductive materials to manipulate and control a range of microbial bioprocesses. Biotechnol. Bioeng. 2009;103: 85–91. © 2008 Wiley Periodicals, Inc.     

Zur elektronenmikroskopischen Untersuchung der juxtaglomerulären Spezialeinrichtungen der Niere

Summary In the mouse and the rat, the epithelioid (juxtaglomerular) cells in the media of the preglomerular part of the afferent arteriole are relatively large cells which are rich in more or less osmophilic granules and are embedded in a framework of basement membranes. These membranes are here and there interrupted while the cell membranes are always continuous.The nuclei are ellipsoid and may be indented by the secretory granules; in some sections nucleoli are seen.The most striking structures of the epithelioid cells are the spherical or polymorphous secretory granules which have a smooth contour and are surrounded by a smooth single membrane. As a rule their content is flocculent; often it contains smaller inclusions that are more or less electron dense and sometimes of a complex structure.The rather pale mitochondria show the usual structure; they are abundant but frequently somewhat concealed between the generally much larger secretory granules. The Golgi apparatus is very well developped; Golgi elements (parallel lamellae, vacuoles and vesicles) may be seen several times in the same cell section. In two epithelioid cells a centriole has been found near the nucleus.Moreover the cytoplasm contains a very rich ergastoplasm (endoplasmic reticulum) and is plentifully supplied with ribosomes. Microbodies and multivesicular bodies are rare. On the other hand, fibrillar structures (myofibrils ?) are often present.The ultrastructure of the epithelioid cells suggests that they have an active metabolism and, according to all evidence (see discussion), a secretory function. The nature of the elaborated substance has not yet been defined.

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Mit Unterstützung durch die Fritz Hoffmann-La Roche-Stiftung zur Förderung wissenschaftlicher Arbeitsgemeinschaften in der Schweiz.     

Histochemische Reaktion und „negative staining“ von Zellfraktionen

Zusammenfassung Ein mit Formvar-Kohle befilmtes Netz wird auf die Oberfläche einer suspendierten Zellfraktion gelegt. Ein Teil der Suspension wird somit aufgenommen. Für die Untersuchung von morphologischen Strukturen wird es sofort auf die Oberfläche einer Waschlösung übertragen, zur Identifizierung von histochemischen Zellstrukturen auf eine Inkubationslösung. Danach wird das Netz auf die Oberfläche der negative-staining-Lösung (PWS 10%, pH 6,5 mit 1 n NaOH) gebracht und anschließend nochmals gewaschen. Morphologische und histochemische (Säurephosphatase nach Gomori) Befunde an Membranen und Partikeln einer Fraktion der Rattenmilz werden diskutiert.

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Summary A formvar-carbon coated grid is placed on the surface of a suspension of a cell fraction. Part of the cellular material will adhere to the formvar-carbon film. For morphologic investigation the grid is immediately transferred to the surface of a washing solution; for histochemical identification of cell structures the grid is however placed on the surface of a incubation medium. After washing or incubation, the grid is negatively stained, i.e. transferred to the surface of the PTA solution (PTA 10%, pH=6,5 with 1 n NaOH), and finally again washed for a short time. Morphological and histochemical (Gomori method for the localisation of the acid phosphatase activity) findings on membranes and particles of a fraction of rat spleen are reported.

     

CCCTC-binding factor activates PARP-1 affecting DNA methylation machinery

Our previous data have shown that in L929 mouse fibroblasts the control of methylation pattern depends in part on poly(ADP-ribosyl)ation and that ADP-ribose polymers (PARs), both present on poly(ADP-ribosyl)ated PARP-1 and/or protein-free, have an inhibitory effect on Dnmt1 activity. Here we show that transient ectopic overexpression of CCCTC-binding factor (CTCF) induces PAR accumulation, PARP-1, and CTCF poly(ADP-ribosyl)ation in the same mouse fibroblasts. The persistence in time of a high PAR level affects the DNA methylation machinery; the DNA methyltransferase activity is inhibited with consequences for the methylation state of genome, which becomes diffusely hypomethylated affecting centromeric minor satellite and B1 DNA repeats. In vitro data show that CTCF is able to activate PARP-1 automodification even in the absence of nicked DNA. Our new finding that CTCF is able per se to activate PARP-1 automodification in vitro is of great interest as so far a burst of poly(ADP-ribosyl)ated PARP-1 has generally been found following introduction of DNA strand breaks. CTCF is unable to inhibit DNMT1 activity, whereas poly(ADP-ribosyl)ated PARP-1 plays this inhibitory role. These data suggest that CTCF is involved in the cross-talk between poly(ADP-ribosyl)ation and DNA methylation and underscore the importance of a rapid reversal of PARP activity, as DNA methylation pattern is responsible for an important epigenetic code.     

Zur elektronenmikroskopischen Untersuchung der juxtaglomerulären Spezialeinrichtungen der Niere

Summary After a general view of the constituents of the juxtaglomerular apparatus of the kidney, the authors are presently publishing on this subject some of their preliminary findings which have been obtained with the aid of the electron microscope:The cells of the macula densa are distinguished from the other cells of the distal convoluted tubule by a lesser development of the infolded basal plasma membranes as well as that of the chondriome which is generally found in a circumand supranuclear position.The cells of Goormaghtigh are in a close topographical relationship with the macula densa, although separated from it by a basement membrane; they are integrated in a complex system of basement membranes.The epithelioid cells of the afferent arteriole contain, in addition to ribosomes and ergastoplasmic structures, vesicles of which the size and the contrast of the content are different. paraportal cells of Becher have not as yet been positively identified with the electron microscope.The intertubular space is poor in cells; the various interstitial cells, often rich in ergastoplasm, are yet to be studied in detail.

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Mit Unterstützung durch die Fritz Hoffmann-La Roche-Stiftung zur Förderung wissenschaftlicher Arbeitsgemeinschaften in der Schweiz.     

Sparse Spectro-Temporal Receptive Fields Based on Multi-Unit and High-Gamma Responses in Human Auditory Cortex

Spectro-Temporal Receptive Fields (STRFs) were estimated from both multi-unit sorted clusters and high-gamma power responses in human auditory cortex. Intracranial electrophysiological recordings were used to measure responses to a random chord sequence of Gammatone stimuli. Traditional methods for estimating STRFs from single-unit recordings, such as spike-triggered-averages, tend to be noisy and are less robust to other response signals such as local field potentials. We present an extension to recently advanced methods for estimating STRFs from generalized linear models (GLM). A new variant of regression using regularization that penalizes non-zero coefficients is described, which results in a sparse solution. The frequency-time structure of the STRF tends toward grouping in different areas of frequency-time and we demonstrate that group sparsity-inducing penalties applied to GLM estimates of STRFs reduces the background noise while preserving the complex internal structure. The contribution of local spiking activity to the high-gamma power signal was factored out of the STRF using the GLM method, and this contribution was significant in 85 percent of the cases. Although the GLM methods have been used to estimate STRFs in animals, this study examines the detailed structure directly from auditory cortex in the awake human brain. We used this approach to identify an abrupt change in the best frequency of estimated STRFs along posteromedial-to-anterolateral recording locations along the long axis of Heschl’s gyrus. This change correlates well with a proposed transition from core to non-core auditory fields previously identified using the temporal response properties of Heschl’s gyrus recordings elicited by click-train stimuli.