The Salinity Tolerance of Freshwater Cyanobacterium Synechococcus sp. PCC 7942 is Determined by Its Ability for Osmotic Adjustment and Presence of Osmolyte Sucrose

We investigated the factors that impose an upper limit of salinity tolerance to the unicellular freshwater cyanobacterium Synechococcus sp. PCC 7942. Above approx. 0.4 M NaCl, Synechococcus cells cease to proliferate, after having accumulated 0.3 M sucrose. Cells that pre-accumulated sucrose could tolerate up to 0.5 M NaCl, but not 0.6 M NaCl. After exposure to 0.5 M NaCl or higher, the cells were irreversibly modified becoming unable for osmotic volume adjustments. This revised version was published online in August 2006 with corrections to the Cover Date.     

A method to probe the cytoplasmic osmolality and osmotic water and solute fluxes across the cell membrane of cyanobacteria with chlorophyll a fluorescence: Experiments with Synechococcus sp. PCC7942

We present a method with which osmotic properties of the cytoplasm of cyanobacterial cells and the osmotic permeability of plasma membranes to water and solutes can be assessed from measurements of chlorophyll a fluorescence. When the electron transport of photosystem II is inhibited, the quantum yield of chlorophyll a fluorescence in cyanobacterial cells varied between a low yield limit that was attained after acclimation to darkness (state 2) and a high yield limit that was attained after acclimation to light (state 1). It was shown recently that the difference between chlorophyll a fluorescence of light‐acclimated and of dark‐acclimated cells relates quantitatively to the internal osmolality of cyanobacteria (G. C. Papageorgiou and A. Alygizaki‐Zorba. 1997. Biochim. Biophys. Acta 1335: 1‐4). In the present work we employed rapid mixing of Synechococcus sp. PCC7942 (strain PAMCOD) suspensions with solutions of defined osmolality in order to measure cell osmolality and turgor threshold, as well as water and solute fluxes across cell membranes. Concentration upshocks with sorbitol, glycine betaine, Na⁺ and K⁺ salts caused rapid (t_1/2 < 10 ms) depression of fluorescence that was correlated to osmotic water outflow from the cells. The fluorescence remained depressed in all cases except for NaCl. With NaCl, the depression was transient and fluorescence recovered with an apparent time constant of 200 ms. The fluorescence rise correlates to inflows of NaCl and water.     

LiDAR‐derived canopy structure supports the more‐individuals hypothesis for arthropod diversity in temperate forests

Despite considerable progress in the ability to measure the complex 3‐D structure of forests with the improvement of remote‐sensing techniques, our mechanistic understanding of how biodiversity is linked to canopy structure is still limited. Here we tested whether the increase in arthropod abundance and richness in beech forest canopies with increasing canopy complexity supports the more‐individuals hypothesis or the habitat‐heterogeneity hypothesis. We used fogging to collect arthropod samples from 80 standardized plots from canopies of single‐ to multi‐layered mature montane European beech stands. Tree height and an independent measure of vertical heterogeneity – the vertical distribution ratio – on each arthropod sampling plot were derived from high‐resolution full‐waveform airborne laser scanning data. Mixed‐model path analysis based on almost 20 000 specimens of 762 species from 11 orders provided support for the more‐individuals hypothesis, with higher arthropod abundance but not higher species richness in stands with a more equal vertical distribution of plant biomass. By contrast, we found no support for the habitat‐heterogeneity hypothesis. The increase in the number of individuals with increasing vertical distribution of biomass might be caused either by increasing leaf area, as indicated by higher space filling and productivity in multi‐layered stands, or by higher persistence of arthropod populations owing to better shelter, reduced competition and more refuges under harsh conditions, or by both. High‐resolution airborne laser scanning, with its ability to penetrate dense canopies under leaf‐on conditions, has proved suitable for measuring vertical structures as a predictor for canopy diversity. Expanding combinations of remote‐sensing and canopy‐biodiversity data opens many avenues for improving our understanding of the link between diversity and forest structures.     

Linearized oscillations in population dynamics

A linearized oscillation theorem due to Kulenović, Ladas and Meimaridou (1987,Quart. appl. Math. XLV, 155–164) and an extension of it are applied to obtain the oscillation of solutions of several equations which have appeared in population dynamics. They include the logistic equation with several delays, Nicholson's blowflies model as described by Gurney, Blythe and Nisbet (1980,Nature, Lond. 287, 17–21) and the Lasota-Wazewska model of the red blood cell supply in an animal. We also developed a linearized oscillation result for difference equations and applied it to several equations taken from the biological literature.     

Structure of tandem RNA recognition motifs from polypyrimidine tract binding protein reveals novel features of the RRM fold

Polypyrimidine tract binding protein (PTB), an RNA binding protein containing four RNA recognition motifs (RRMs), is involved in both pre-mRNA splicing and translation initiation directed by picornaviral internal ribosome entry sites. Sequence comparisons previously indicated that PTB is a non-canonical RRM protein. The solution structure of a PTB fragment containing RRMs 3 and 4 shows that the protein consists of two domains connected by a long, flexible linker. The two domains tumble independently in solution, having no fixed relative orientation. In addition to the betaalphabetabetaalphabeta topology, which is characteristic of RRM domains, the C-terminal extension of PTB RRM-3 incorporates an unanticipated fifth beta-strand, which extends the RNA binding surface. The long, disordered polypeptide connecting beta4 and beta5 in RRM-3 is poised above the RNA binding surface and is likely to contribute to RNA recognition. Mutational analyses show that both RRM-3 and RRM-4 contribute to RNA binding specificity and that, despite its unusual sequence, PTB binds RNA in a manner akin to that of other RRM proteins.     

Good manufacturing practice-compliant validation and preparation of BM cells for the therapy of acute myocardial infarction

BACKGROUND: Intracoronary application of BM-derived cells for the treatment of acute myocardial infarction (AMI) is currently being studied intensively. Simultaneously, strict legal requirements surround the production of cells for clinical studies. Thus good manufacturing practice (GMP)-compliant collection and preparation of BM for patients with AMI was established by the Cytonet group. METHODS: As well as fulfillment of standard GMP requirements, including a manufacturing license, validation of the preparation process and the final product was performed. Whole blood (n=6) and BM (n=3) validation samples were processed under GMP conditions by gelafundin or hydroxyethylstarch sedimentation in order to reduce erythrocytes/platelets and volume and to achieve specifications defined in advance. Special attention was paid to the free potassium (<6 mmol/L), some rheologically relevant cellular characteristics (hematocrit <0.45, platelets <450 x 10(6)/mL) and the sterility of the final product. RESULTS: The data were reviewed and GMP compliance was confirmed by the German authorities (Paul-Ehrlich Institute). Forty-five BM cell preparations for clinical use were carried out following the validated methodology and standards. Additionally three selections of CD34+ BM cells for infusion were performed. All specification limits were met. Discussion In conclusion, preparation of BM cells for intracoronary application is feasible under GMP conditions. As the results of sterility testing may not be available at the time of intracoronary application, the highest possible standards to avoid bacterial and other contaminations have to be applied. The increased expense of the GMP-compliant process can be justified by higher safety for patients and better control of the final product.     

The 5-HT1A receptor agonist buspirone improves esophageal motor function and symptoms in systemic sclerosis: a 4-week,open-label trial

BackgroundAcute administration of the oral 5-HT₁A receptor agonist buspirone, which is commonly used as an anxiolytic drug, may improve compromised lower esophageal sphincter function. In an open-label trial we assessed the effects of buspirone on esophageal motor function and symptoms in patients with esophageal involvement associated with systemic sclerosis (SSc).MethodsThirty consecutive patients with SSc and symptomatic esophageal involvement, despite treatment with proton pump inhibitors, underwent high resolution manometry and chest computed tomography for assessment of motor function and esophageal dilatation, respectively. Regurgitation, heartburn, dysphagia, and chest pain severity was subjectively scored by visual analog scales. Manometric parameters (primary endpoint) and symptom severity (secondary endpoint) were re-examined after 4-week daily administration of 20 mg buspirone. Other medications remained unchanged.ResultsEight patients did not complete the trial because of buspirone-associated dizziness (n = 2), or nausea (n = 2), or reluctancy to undergo final manometry. In the remaining 22 patients lower esophageal sphincter (LES) resting pressure increased from 7.7 ± 3.9 to 12.2 ± 4.6 mmHg (p = 0.00002) after buspirone administration; other manometric parameters did not change. Statistical analysis revealed negative correlation between individual increases in resting LES pressure and supra-aortic esophageal diameter (r = -0.589, p = 0.017), suggesting a more beneficial effect in patients with less severely affected esophageal function. Heartburn and regurgitation scores decreased at 4 weeks compared to baseline (p = 0.001, and p = 0.022, respectively).ConclusionOur findings warrant more conclusive evaluation with a double-blind controlled study; however, buspirone could potentially be given under observation for objective improvement in all patients with SSc who report reflux symptoms despite undergoing standard treatment.

Trial registration

ClinicalTrials.gov Identifier: {"type":"clinical-trial","attrs":{"text":"NCT02363478","term_id":"NCT02363478"}}NCT02363478 Registered: 21-02-2014.     

Cell turgor: A critical factor for the proliferation of cyanobacteria at unfavorable salinity

We employed chlorophyll a fluorometry in order to measure the evolution of turgor threshold (intracellular osmolality) during the adaptation of two genetic transformants of the freshwater cyanobacterium Synechococcus sp. PCC7942 to unfavorable external salinity: PAMCOD cells which oxidize imported choline and accumulate approx. 0.06–0.08 M glycine betaine; and PAM cells which do not oxidize choline [Deshnium et al. (1995a) Plant Mol Biol 29: 897–909]. Turgor thresholds increased linearly (a) with the NaCl concentration in the culture, and (b) with the molar sucrose/chlorophyll a ratio in the cell. PAMCOD cells could proliferate in culture medium containing 0.4 M NaCl (external osmolality, 0.815 Osm kg⁻¹), after a lag period, during which intracellular sucrose rose to 10 mol (mol Chl a)⁻¹, or more, and turgor threshold (cytoplasmic osmolality) exceeded 1 Osm kg⁻¹. At comparative conditions, PAM cells accumulated approx. half as much sucrose, and attained approx. half as high turgor thresholds as the PAMCOD cells, but they did not proliferate. These results indicate that glycine betaine improved the salinity tolerance of the PAMCOD cells synergistically, by means of two effects that implicate sucrose, the main organic osmolyte of Synechocccus: enhancement of sucrose biosynthesis, and/or alleviation of sucrose toxicity. This revised version was published online in June 2006 with corrections to the Cover Date.     

Facilitated water transport in cyanobacterium Synechococcus sp. PCC 7942 studied by phycobilisome-sensitized chlorophyll a fluorescence

Water transport across plant cell membranes is difficult to measure. We present here a model assay, based on chlorophyll (Chl) a fluorometry, with which net water transport across the cell membrane of freshwater cyanobacterium Synechococcus sp. PCC7942 (S7942) can be followed kinetically with millisecond-time resolution. In cyanobacteria, the phycobilisome (PBS)-sensitized Chl a fluorescence increases when cells expand (e.g., in hypo-osmotic suspension) and decreases when cells contract (e.g., in hyper-osmotic suspension). The osmotically-induced Chl a fluorescence changes are proportional to the reciprocal of the suspension osmolality (ΔF ∝ Osm⁻¹; Papageorgiou GC and Alygizaki-Zorba A (1997) Biochim Biophys Acta 1335: 1–4). In our model assay, S7942 cells were loaded with NaCl (passively penetrating solute) and shrunk in hyper-osmotic glycine betaine (nonpenetrating solute). Upon injecting these cells into hypo-osmotic medium, the PBS-sensitized Chl a fluorescence rose to a maximum due to the osmotically-driven water uptake. The rise of Chl a fluorescence (water uptake) was partially inhibited by HgCl₂, at micromolar concentrations. Arrhenius plots of the water uptake rates gave activation energies of E_A=4.9 kcal mol⁻¹, in the absence of HgCl₂, and E_A=11.9 kcal mol⁻¹ in its presence. These results satisfy the usual criteria for facilitated water transport through protein water pores of plasma membranes (aquaporins), namely sensitivity to Hg²⁺ ions and low activation energy.