The ferredoxin-NADP+ oxidoreductase-binding protein is not the 17-kDa component of the cytochrome b/f complex

The complex between ferredoxin-NADP+ oxidoreductase and its proposed membrane-binding protein (Vallejos, R. H., Ceccarelli, E., and Chan, R. (1984) J. Biol. Chem. 259, 8048-8051) was isolated from spinach thylakoids and compared with isolated cytochrome b/f complex containing associated ferredoxin NADP+ oxidoreductase (Clark, R. D., and Hind, G. (1983) J. Biol. Chem. 258, 10348-10354). There was no immunological cross-reactivity between the 17.5-kDa binding protein and an antiserum raised against the 17-kDa polypeptide of the cytochrome complex. Association of ferredoxin-NADP+ oxidoreductase with the binding protein or with the thylakoid membrane gave an allotopic shift in the pH profile of diaphorase activity, as compared to the free enzyme. This effect was not seen in enzyme associated with the cytochrome b/f complex. Identification of the 17.5-kDa binding protein as the 17-kDa component of the cytochrome b/f complex is ruled out by these results.     

KREMEN1 Is a Host Entry Receptor for a Major Group of Enteroviruses

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Rhodopsin(s) in eubacteria

While the biochemical basis of photosynthesis by bacteriochlorophyll-based reaction centres in purple phototrophic Eubacteria and retinal-based bacteriorhodopsin in the Archaebacterium Halobacterium salinarium has been elucidated in great detail, much less is known about photosensory signal transduction; this is especially the case for Eubacteria. Recent findings on two different photosensory proteins in two different Eubacteria, which both show clear resemblances to the rhodopsins, will be presented. The photoactive yellow protein (PYP) from the purple phototrophic organism Ectothiorhodospira halophila probably functions as the photoreceptor for a new type of negative phototaxis response and has been studied in some detail with respect to its structural and photochemical characteristics. On basis of crystallographic an photochemical data it has been proposed that PYP contains retinal as a chromophore. However, we have unambiguously demonstrated that the PYP chromophore is different from retinal, in spite of the fact that PYP's photochemical properties show striking similarities with the rhodopsins. The cyanobacterium Calothrix sp. displays complementary chromatic adaptation, a process in which the pigment composition of the phycobilisomes is adjusted to the spectral characteristics of the incident light. In orange light the blueish chromophore phycocyanin is present, in green light the reddish phycoerythrin is synthesized. On the basis of the action spectrum of this adaptation process, we hypothesized that a rhodopsin is the photosensor in this process. In line with this, we found that nicotine, an inhibitor of the biosynthesis of beta-carotene (which is the precursor of retinal), abolishes chromatic adaptation. Direct proof of the involvement of a photosensory rhodopsin was obtained in experiments in which the chromatic adaptation response was restored by the addition of retinal to the cultures. The two photosensory proteins mentioned above represent the first examples of eubacterial photoreceptors that can be studied at a molecular level. Our current knowledge on these two proteins and their status as retinal proteins will be reviewed.     

Presynaptic inhibition upon CB1 or mGlu2/3 receptor activation requires ERK/MAPK phosphorylation of Munc18‐1

Presynaptic cannabinoid (CB1R) and metabotropic glutamate receptors (mGluR2/3) regulate synaptic strength by inhibiting secretion. Here, we reveal a presynaptic inhibitory pathway activated by extracellular signal‐regulated kinase (ERK) that mediates CB1R‐ and mGluR2/3‐induced secretion inhibition. This pathway is triggered by a variety of events, from foot shock‐induced stress to intense neuronal activity, and induces phosphorylation of the presynaptic protein Munc18‐1. Mimicking constitutive phosphorylation of Munc18‐1 results in a drastic decrease in synaptic transmission. ERK‐mediated phosphorylation of Munc18‐1 ultimately leads to degradation by the ubiquitin–proteasome system. Conversely, preventing ERK‐dependent Munc18‐1 phosphorylation increases synaptic strength. CB1R‐ and mGluR2/3‐induced synaptic inhibition and depolarization‐induced suppression of excitation (DSE) are reduced upon ERK/MEK pathway inhibition and further reduced when ERK‐dependent Munc18‐1 phosphorylation is blocked. Thus, ERK‐dependent Munc18‐1 phosphorylation provides a major negative feedback loop to control synaptic strength upon activation of presynaptic receptors and during intense neuronal activity.     

Pyoverdine and histicorrugatin-mediated iron acquisition in <Emphasis Type="Italic">Pseudomonas thivervalensis</Emphasis>

The genome of Pseudomonas thivervalensis LMG 21626^T has been sequenced and a genomic, genetic and structural analysis of the siderophore mediated iron acquisition was undertaken. Pseudomonas thivervalensis produces two structurally new siderophores, pyoverdine PYO_thi which is typical for P. thivervalensis strains and a closely related strain, and the lipopeptidic siderophore histicorrugatin which is also detected in P. lini. Histicorrugatin consists out of an eight amino acid long peptide which is linked to octanoic acid. It is structurally related to the siderophores corrugatin and ornicorrugatin. Analysis of the proteome for TonB-dependent receptors identified 25 candidates. Comparison of the TonB-dependent receptors of P. thivervalensis with the 17 receptors of its phylogenetic neighbor, P. brassicacearum subsp. brassicacearum NFM 421, showed that NFM 421 shares the same set of receptors with LMG 21626^T, including the histicorrugatin receptor. An exception was found for their cognate pyoverdine receptor which can be explained by the observation that both strains produce structurally different pyoverdines. Mass analysis showed that NFM 421 did not produce histicorrugatin, but the analogue ornicorrugatin. Growth stimulation assays with a variety of structurally distinct pyoverdines produced by other Pseudomonas species demonstrated that LMG 21626^T and NFM 421 are able to utilize almost the same set of pyoverdines. Strain NFM 421 is able utilize two additional pyoverdines, pyoverdine of P. fluorescens Pf0–1 and P. citronellolis LMG 18378^T, these pyoverdines are probably taken up by the FpvA receptor of NFM 421.     

The role of water retaining substrata on the photosynthetic response of three drought tolerant phototrophic micro-organisms isolated from a terrestrial habitat

The water prerequisites of two drought tolerant Oscillatoria type cyanobacteria and one green alga were estimated by their ability to accomplish photosynthesis (carbon dioxide fixation) at conditions of subsaturating water supply. Fixation was zero in desiccated samples. Equilibration with solely water-saturated air did not enable any photosynthesis. However, granted properties of the physical environment of the samples could re-establish photosynthesis activity. These properties were elected by chosing membrane filters with different water retention characteristics as supporting substrata for the test samples in the de-and rehydration steps. Rehydration enabled the recovery of photosynthesis of desiccated samples only on the filters with good water retention, the filters with bad water retention were found ineffective. The Oscillatoria strains showed photosynthesis instantaneously and revealed nearly 100% viability. In contrast, rewetted cells of the green alga showed only 35% viability and the recovery of photosynthesis occurred only after 5 h. These differences reflect the natural environmental conditions: cyanobacteria are the first colonizers in the barren sand, whereas green algae can only start to colonize after progressing improvement of the water retention properties brought about by the pioneering cyanobacteria. The results will be discussed in the light of different specific mechanisms available to organisms which endeavour osmotic and matric water stress.Abbreviations DCMU 3(3,4-dichlorophenyl)-1,1-dimethyl-urea - DMF dimethylformamide - PFD photon flux density - TAPS N-tris[hydroxymethyl]methyl-3-amino-propanesulfonic acid     

Cytoplasmic free calcium, myosin light chain phosphorylation, and force in phasic and tonic smooth muscle

The time course of [Ca2+]i, tension, and myosin light chain phosphorylation were determined during prolonged depolarization with high K+ in intact tonic (rabbit pulmonary artery) and phasic (longitudinal layer of guinea pig ileum) smooth muscles. [Ca2+]i was monitored with the 340 nm/380 nm signal ratio of the fluorescent indicator fura-2. The fluorescence ratio had a similar time course in both muscle types during depolarization with 109 mM [K+]o; after a transient peak, there was a decline to 70% of its peak value in tonic smooth muscle, and to 60% in phasic smooth muscle. Tension, however, continued to increase in the pulmonary artery, while in the ileum it declined in parallel with the [Ca2+]i. On changing [K+]o from 109 to 20 mM, tension and [Ca2+]i either remained unchanged or declined in parallel in the pulmonary artery. Phosphorylation of the 20-kD myosin light chain, measured during stimulation of muscle strips with 109 mM [K+]o in another set of experiments, increased from 3% to a peak of 50% in the intact pulmonary artery, and then declined to a steady state value of 23%. In the intact ileum, a very rapid, early transient phosphorylation (up to 50%) at 2-3 s was seen. This transient declined by 30 s to a value that was close to the resting level (7%), while tension remained at 55% of its peak force. A quick release during maintained stimulation induced no detectable change in the [Ca2+]i in either type of smooth muscle. We discuss the possibility that the slowly rising tonic tension in pulmonary artery could be due to cooperativity between phosphorylated and nonphosphorylated crossbridges.     

Glucose ingestion during exercise blunts exercise-induced gene expression of skeletal muscle fat oxidative genes

Ingestion of carbohydrate during exercise may blunt the stimulation of fat oxidative pathways by raising plasma insulin and glucose concentrations and lowering plasma free fatty acid (FFA) levels, thereby causing a marked shift in substrate oxidation. We investigated the effects of a single 2-h bout of moderate-intensity exercise on the expression of key genes involved in fat and carbohydrate metabolism with or without glucose ingestion in seven healthy untrained men (22.7 +/- 0.6 yr; body mass index: 23.8 +/- 1.0 kg/m(2); maximal O(2) consumption: 3.85 +/- 0.21 l/min). Plasma FFA concentration increased during exercise (P < 0.01) in the fasted state but remained unchanged after glucose ingestion, whereas fat oxidation (indirect calorimetry) was higher in the fasted state vs. glucose feeding (P < 0.05). Except for a significant decrease in the expression of pyruvate dehydrogenase kinase-4 (P < 0.05), glucose ingestion during exercise produced minimal effects on the expression of genes involved in carbohydrate utilization. However, glucose ingestion resulted in a decrease in the expression of genes involved in fatty acid transport and oxidation (CD36, carnitine palmitoyltransferase-1, uncoupling protein 3, and 5'-AMP-activated protein kinase-alpha(2); P < 0.05). In conclusion, glucose ingestion during exercise decreases the expression of genes involved in lipid metabolism rather than increasing genes involved in carbohydrate metabolism.