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 Toll-Like Receptor 4 (TLR4) Variant rs2149356 and Risk of Gout in European and Polynesian Sample Sets

Deposition of crystallized monosodium urate (MSU) in joints as a result of hyperuricemia is a central risk factor for gout. However other factors must exist that control the progression from hyperuricaemia to gout. A previous genetic association study has implicated the toll-like receptor 4 (TLR4) which activates the NLRP3 inflammasome via the nuclear factor-κB signaling pathway upon stimulation by MSU crystals. The T-allele of single nucleotide polymorphism rs2149356 in TLR4 is a risk factor associated with gout in a Chinese study. Our aim was to replicate this observation in participants of European and New Zealand Polynesian (Māori and Pacific) ancestry. A total of 2250 clinically-ascertained prevalent gout cases and 13925 controls were used. Non-clinically-ascertained incident gout cases and controls from the Health Professional Follow-up (HPFS) and Nurses Health Studies (NHS) were also used. Genotypes were derived from genome-wide genotype data or directly obtained using Taqman. Logistic regression analysis was done including age, sex, diuretic exposure and ancestry as covariates as appropriate. The T-allele increased the risk of gout in the clinically-ascertained European samples (OR = 1.12, P = 0.012) and decreased the risk of gout in Polynesians (OR = 0.80, P = 0.011). There was no evidence for association in the HPFS or NHS sample sets. In conclusion TLR4 SNP rs2143956 associates with gout risk in prevalent clinically-ascertained gout in Europeans, in a direction consistent with previously published results in Han Chinese. However, with an opposite direction of association in Polynesians and no evidence for association in a non-clinically-ascertained incident gout cohort this variant should be analysed in other international gout genetic data sets to determine if there is genuine evidence for association.     

Characterization of an insulin-like growth factor binding protein, analogous to human pregnancy-associated secreted endometrial alpha 1-globulin, in decidua of the baboon (Papio anubis) placenta

The major secreted protein of the human decidua (pregnancy-associated endometrial alpha 1-globulin [alpha 1-PEG]), is an insulin-like growth factor-binding protein (IGF-BP) that is immunologically and biochemically similar to placental protein 12 (PP12) extracted from term human placenta. Since previous studies have demonstrated that the baboon and human endometrium synthesize and release a number of biochemically and immunologically related polypeptides in culture, this study was undertaken to further characterize a related IGF-BP in baboon placental tissues. Decidua, chorio-amniotic membranes with adhering decidua (CAM-D), and placental villi were obtained from pregnant baboons between Days 134 and 160 of gestation by Cesarean sections. Portions of tissue were either cultured in the presence of 35S-methionine, fixed for immunocytochemistry, or frozen in liquid nitrogen for cytosol extraction. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of tissue culture media (TCM) revealed that the major secretory product of the decidua and CAM-D was an acidic polypeptide (Mr 33,000). Western blot analysis and immunoprecipitation of TCM with murine monoclonal antibody (B2H10) against human alpha 1-PEG demonstrated that this molecule, secreted by the baboon decidua and CAM-D, but not the placental villi, was immunologically identical to the human IGF-BP. Immunocytochemical localization of IGF-BP was intense in the cytoplasm of stromal cells in decidua and CAM-D and absent in the placenta. Gel filtration of TCM and cytosol followed by screening of eluates for 125I-IGF-I binding resolved two peaks (Mr greater than 100,000 and 35,000) of specific IGF-BP in decidua and CAM-D. The 35,000 peak had 100-200 times the binding capacity of the Mr greater than 100,000 peak and a Kd of 1.14-1.83 nM. The eluates contained in the Mr 35,000 peak were also immunoreactive to alpha 1-PEG, as accessed by a polyclonal radioimmunoassay. Affinity cross-linking with 125I-IGF-I followed by sodium dodecyl sulfate-PAGE revealed an immunoreactive complex of Mr 36,000, confirming that the baboon protein represents a high affinity IGF-BP. These studies indicate that the hypertrophied stromal cells of the baboon decidua and CAM-D synthesize and release an IGF-BP as their major secretory product, analogous to the situation in humans. The results of this study suggest that this protein may play a role in the regulation of IGF action during pregnancy.     

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     

Estrogen-induced differentiation of the oviductal epithelium in prepubertal dogs

24 prepubertal 6-8 week old Beagle dogs were injected intramuscularly every other day either with 150 mcg/kg estradiol-beta valerate (14), or progesterone respositol (5), or used as controls (5) to determine what effect estrogen and progesterone had on cytodifferentiation. Light and electron micrographs of the oviductal epithelium of the ampullae of the controls and hormone-treated dogs were studied. The epithelial cells in the controls were uniformly low cuboidal with a high nucleocytoplasmic ratio. After 36 hours of estradiol treatment there was nuclear and nucleolar hypertrophy; an increase in the numbers of cytoplasmic polyribosomes and numerous mitotic figues were apparent. Ciliogenesis began and 2 cell types were distinguishable after 3 days of estradiol treatment. Basal bodies in various stages of formation were randomly distributed throughout the apex of many cells. In others, more mature basal bodies were aligned at the apical border or cilia were evident. 6 days of estradiol resulted in complete ciliation of approximately 60% of all epithelial cells. The remaining cells possessed abundant cisternae of rough endoplasmic reticulum (RER) and appeared to be differentiating into mucus-secreting cells. After 10 days, these cells possessed a large Golgi apparatus, large dilated cisternae of RER, and abundant periodic-acid-Schiff positive granules at their apex. Progesterone treatment alone produced no hypertrophy of the oviductal epithelium. In fact, cytomorphometric measurements indicate that progesterone had an opposite effect, causing atrophy (p less than.05) and increasing the nucleocytoplasmic ratio. It is concluded that estrogen alone was capable of producing complete cytodifferentiation in the oviduct of the prepubertal dog.