The transient receptor potential protein (Trp), a putative store-operated Ca2+ channel essential for phosphoinositide-mediated photoreception, forms a signaling complex with NorpA, InaC and InaD.

The transient receptor potential protein (Trp) is a putative capacitative Ca2+ entry channel present in fly photoreceptors, which use the inositol 1,4,5-trisphosphate (InsP3) signaling pathway for phototransduction. By immunoprecipitation studies, we find that Trp is associated into a multiprotein complex with the norpA-encoded phospholipase C, an eye-specific protein kinase C (InaC) and with the InaD protein (InaD). InaD is a putative substrate of InaC and contains two PDZ repeats, putative protein-protein interaction domains. These proteins are present in the photoreceptor membrane at about equimolar ratios. The Trp homolog analyzed here is isolated together with NorpA, InaC and InaD from blowfly (Calliphora) photoreceptors. Compared to Drosophila Trp, the Calliphora Trp homolog displays 77% amino acid identity. The highest sequence conservation is found in the region that contains the putative transmembrane domains S1-S6 (91% amino acid identity). As investigated by immunogold labeling with specific antibodies directed against Trp and InaD, the Trp signaling complex is located in the microvillar membranes of the photoreceptor cells. The spatial distribution of the signaling complex argues against a direct conformational coupling of Trp to an InsP3 receptor supposed to be present in the membrane of internal photoreceptor Ca2+ stores. It is suggested that the organization of signal transducing proteins into a multiprotein complex provides the structural basis for an efficient and fast activation and regulation of Ca2+ entry through the Trp channel.     

Inside or outside? Localization of the receptor relevant to auxin-induced growth

The localization of the auxin receptor relevant to the control of elongation growth is still a matter of controversy. Auxin-induced elongation of maize coleoptile segments was measured by means of a high resolution auxanometer. When indole-3-acetic acid (IAA) was removed from the bathing solution, a rapid cessation of auxin-induced elongation was detected. This decline was delayed when the auxin efflux carrier was blocked by the phytotropins naphthylphthalamic acid (NPA) and pyrenoylbenzoic acid (PBA) or by triiodobenzoic acid (TIBA). The IAA concentration in NPA-pretreated segments was 2–3 times higher than in NPA-free controls 35 min after the removal of IAA in the bathing medium.
A similar rapid drop of growth after removal of auxin was observed for the rapidly-transported synthetic auxin, naphthaleneacetic acid (NAA). When the auxin efflux was blocked, growth induced by NAA was sustained much longer than IAA-stimulated elongation.
In comparison with NAA, the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) is known to be excreted very slowly by the efflux carrier. 2,4-D-induced growth remained at a stimulated level when the auxin was washed off, even in the absence of any auxin efflux inhibitor. We conclude from these results that the presence of intracellular auxin is a necessary and sufficient condition for sustained auxin-induced elongation growth, at least for the phases during the 2 h after its application. Consequently, we postulate the existence of an intracellular auxin receptor relevant to the control of growth.     

Isolation of an anaerobic bacterium which reductively dechlorinates tetrachloroethene and trichloroethene

Strain TEA, a strictly anaerobic, motile rod with one to four lateral flagella and a crystalline surface layer was isolated from a mixed culture that completely reduces chlorinated ethenes to ethene. The organism coupled reductive dehalogenation of tetrachloroethene or trichloroethene to cis-1,2-dichloroethene to growth, using molecular hydrogen as the electron donor. It was unable to grow fermentatively or in the presence of tri- or tetrachloroethene with glucose, pyruvate, lactate, acetate or formate. The 16S rDNA sequence of strain TEA was 99.7% identical to that of Dehalobacter restrictus. The two organisms thus are representatives of the same species or the same genus within the Bacillus/Clostridium subphylum of the gram-positive bacteria.     

Stochastic resonance as a possible mechanism of amplification of weak electric signals in living cells

The most important but still unresolved problem in bioelectromagnetics is the interaction of weak electromagnetic fields (EMFs) with living cells. Thermal and other types of noise pose restrictions in cell detection of weak signals. As a consequence, some extant experimental results that indicate low-intensity field effects cannot be accounted for, and this renders the results themselves questionable. One way out of this dead end is to search for possible mechanisms of signal amplification. In this paper, we discuss a general mechanism in which a weak signal is amplified by system noise itself. This mechanism was discovered several years ago in physics and is known, in its simplest form, as a stochastic resonance. It was shown that signal amplification may exceed a factor of 1000, which renders existing estimations of EMF thresholds highly speculative. The applicability of the stochastic resonance concept to cells is discussed particularly with respect to the possible role of the cell membrane in the amplification process. © 1994 Wiley-Liss, Inc.     

Astrocytes alter their polarity in organotypic slice cultures of rat visual cortex

The ultrastructure of astrocytes in an organotypic slice culture of the rat visual cortex was investigated using ultrathin sections and freeze-fracture replicas. After a culture period of 9–15 days, a glial scaffold formed that separated the bulk of the slice neuropil from the medium and the underlying plasma clot. However, the glial cells and processes did not build a dense barrier but allowed the outgrowth of neurites. A basal lamina covering the medium-oriented surface of the astrocytes was not found. In freeze-fracture replicas, orthogonal arrays of particles (OAP) were characteristic components of astrocytic membranes. The OAP density in membranes bordering the medium was 35±13 OAP/m², corresponding to 2.5% of this membrane area; the OAP density in membranes within the slice neuropil was 22±12 OAP/², corresponding to 1.4% of this membrane area. Although the difference was significant, it was greatly reduced when comparing OAP densities in endfoot and non-endfoot membranes in vivo. Another mode of polarity was recognized in astrocytes of the organotypic slice culture. In membranes of astrocytes bordering upon the medium, the density of non-OAP intramembranous particles (IMP) was clearly higher (1130±136 IMP/ m²) than in membranes of astrocytes in the center of the slice (700±172 IMP/m²). This pronounced IMP-related polarity was observed neither in vivo nor in cultured astrocytes. The present study suggests, together with data from the literature, that the distribution of astrocytic OAP across the cell surface is influenced by the existence of a basal lamina and neuronal activity, and that astrocytes possess a more remarkable plasticity of membrane structure than previously suspected.     

Purification and PCR-based cDNA cloning of a plastidial n-6 desaturase

A plastidial membrane-bound n-6 desaturase from spinach (Spinacia oleracea) was purified from chloroplast envelope membranes by anion exchange, cation exchange and ferredoxin-affinity chromatography. The molecular mass of the protein was estimated by SDS-PAGE to be 40 kDa. The highest specific activity of the desaturase in the final preparation was 196 nmol/min per mg protein with free oleic acid as the substrate. The N-terminal amino acid sequence of the blotted protein was determined and used for the construction of a degenerated and inosine-containing oligonucleotide primer for PCR experiments with cDNA transcribed from leaf mRNA. A 3-RACE experiment with this primer amplified a single band of 1500 bp that after sequencing showed an open reading frame of 382 amino acids corresponding to a protein of 43 kDa. The 5 end of the cDNA was amplified by a 5-RACE experiment and isolated as a 500 bp fragment. Sequencing of this DNA revealed an additional 65 amino acids at the N-terminus of the native protein that are attributed to a plastidial leader peptide. With appropriate primers derived from these sequences a full-length clone was amplified by PCR and sequenced. Comparison of the plastidial oleate desaturase with the homologous enzyme from cyanobacteria showed about 50% amino acid homology. Comparison with other desaturases revealed three histidine boxes with the general sequence HXXXH that are highly conserved in all membrane-bound desaturases. These boxes might be involved in metal ion complexation required for reduction of oxygen.     

Sensitive detection of human growth hormone mRNA in routinely formalin-fixed,paraffin-embedded transgenic mouse tissues by non-isotopic in situ hybridization

A sensitive technique of non-isotopic in situ hybridization (NISH) is presented, which permits the detection of human growth hormone (hGH) mRNA in routinely formalin-fixed, paraffin-embedded transgenic mouse tissues and human post mortem pituitaries; the latter were used as positive tissue controls in this study. In addition, a double staining procedure combining NISH and immunohistochemistry for the visualization of both hGH and hGH mRNA in the same paraffin section is described. Digoxigenin-labelled antisense hGH RNA was used for NISH of hGH mRNA. The NISH protocol was based upon an established radioactive method. Alkaline phosphatase and horseradish peroxidase-based immunoenzymatic procedures for the detection of digoxigenin-labelled RNA probes using different chromogens [4-nitro blue tetrazolium chloride (NBT), Fast Blue BB, New Fuchsin, and 3,3-diaminobenzidine tetrahydrochloride (DAB) with or without intensification of the DAB staining] were compared. The proteolytic tissue pretreatment and the detection procedure were found to be the most critical steps for successful visualization of hGH mRNA. After optimization of the permeabilization conditions, hGH mRNA could be visualized in each case studied when alkaline phosphatase/NBT-based detection was employed. The NISH technique presented here, performed either separately or in combination with immunohistochemistry, permits retrospective analyses, of hGH (trans)gene expression in archival, paraffin-embedded specimens.     

Current Nitrogen Fixation Is Involved in the Regulation of Nitrogenase Activity in White Clover (Trifolium repens L.)

Previous studies have shown that nitrogenase activity decreases dramatically after defoliation, presumably because of an increase in the O2 diffusion resistance in the infected nodules. It is not known how this O2 diffusion resistance is regulated. The aim of this study was to test the hypothesis that current N2 fixation (ongoing flux of N2 through nitrogenase) is involved in the regulation of nitrogenase activity in white clover (Trifolium repens L. cv Ladino) nodules. We compared the nitrogenase activity of plants that were prevented from fixing N2 (by continuous exposure of their nodulated root system to an Ar:O2 [80:20] atmosphere) with that of plants allowed to fix N2 (those exposed to N2:O2, 80:20). Nitrogenase activity was determined as the amount of H2 evolved under Ar:O2. An open flow system was used. In experiment I, 6 h after complete defoliation and the continuous prevention of N2 fixation, nitrogenase activity was higher by a factor of 2 compared with that in plants allowed to fix N2 after leaf removal. This higher nitrogenase activity was associated with a lower O2 limitation (measured as the partial pressure of O2 required for highest nitrogenase activity). In experiment II, the nitrogenase activity of plants prevented from fixing N2 for 2 h before leaf removal showed no response to defoliation. The extent to which nitrogenase activity responded to defoliation was different in plants allowed to fix N2 and those that were prevented from doing so in both experiments. This leads to the conclusion that current N2 fixation is directly involved in the regulation of nitrogenase activity. It is suggested that an N feedback mechanism triggers such a response as a result of the loss of the plant's N sink strength after defoliation. This concept offers an alternative to other hypotheses (e.g. interruption of current photosynthesis, carbohydrate deprivation) that have been proposed to explain the immediate decrease in nitrogenase activity after defoliation.     

NPY- and C-PON-immunoreactive substances in the central nervous system of Helix pomatia

The distribution of neuropeptide-tyrosin (NPY)- and C-flanking peptide of neuropeptide-tyrosine (C-PON)-immunoreactivities in the central nervous system of the pulmonate gastropod, Helix pomatia, was investigated. NPY- and C-PON-like substances were localized in neuronal somata and neuntes, but were not co-localized within the same cells. NPY-immunoreactive substances were also found in endocrine/paracrine like cells located in the epineurium. C-PON and NPY, both reduced serotonin activated isometric contractions of Helix aorta, suggesting that they may act as modulators in the control of the vascular system.