Alteration of cylindrospermopsin production in sulfate- or phosphate-starved cyanobacterium Aphanizomenon ovalisporum

The effect of sulfate and phosphate deprivation on cell growth and cylindrospermopsin level was studied in Aphanizomenon ovalisporum ILC-164. Sulfate starvation induced a characteristic reduction of cylindrospermopsin pool size on the basis of cell number and unit of dry mass of culture. Phosphorous starvation of A. ovalisporum cultures induced a lesser reduction of cylindrospermopsin pool size. This divergence in the pool size of cylindrospermopsin may be the consequence of different growth rate. To show the metabolic changes concomitant with reduction of cylindrospermopsin pool size were obtained by measurement of ATP sulfurylase and alkaline phosphatase activity. The present study is the first concerning the cylindrospermopsin content under sulfate starvation and discusses it in relation to phosphorous starvation.     

Ascorbate accumulation during sulphur deprivation and its effects on photosystem II activity and H2 production of the green alga Chlamydomonas reinhardtii

In nature, H₂ production in Chlamydomonas reinhardtii serves as a safety valve during the induction of photosynthesis in anoxia, and it prevents the over‐reduction of the photosynthetic electron transport chain. Sulphur deprivation of C. reinhardtii also triggers a complex metabolic response resulting in the induction of various stress‐related genes, down‐regulation of photosynthesis, the establishment of anaerobiosis and expression of active hydrogenase. Photosystem II (PSII) plays dual role in H₂ production because it supplies electrons but the evolved O₂ inhibits the hydrogenase. Here, we show that upon sulphur deprivation, the ascorbate content in C. reinhardtii increases about 50‐fold, reaching the mM range; at this concentration, ascorbate inactivates the Mn‐cluster of PSII, and afterwards, it can donate electrons to tyrozin Z⁺ at a slow rate. This stage is followed by donor‐side‐induced photoinhibition, leading to the loss of charge separation activity in PSII and reaction centre degradation. The time point at which maximum ascorbate concentration is reached in the cell is critical for the establishment of anaerobiosis and initiation of H₂ production. We also show that ascorbate influenced H₂ evolution via altering the photosynthetic electron transport rather than hydrogenase activity and starch degradation.     

Rosuvastatin improves impaired endothelial function,lowers high sensitivity CRP,complement and immuncomplex production in patients with systemic sclerosis - a prospective case-series study

Introduction

We studied the effect of rosuvastatin on endothelial and macrovascular function, cardiovascular risk factors and the complement pathway in patients with systemic sclerosis (SSc).

Methods

Altogether 28 patients with SSc underwent laboratory and complex vascular assessments before and after six months of 20 mg rosuvastatin treatment. Flow-mediated dilation (FMD) of the brachial artery, as well as carotid artery intima-media thickness (ccIMT), carotid-femoral and aorto-femoral pulse wave-velocity (PWV) were analyzed by ECG-synchronized ultrasound. Ankle-brachial index (ABI) was determined by Doppler, and forearm skin microcirculation was assessed by Laser Doppler perfusion monitoring.

Results

Brachial artery FMD significantly improved upon rosuvastatin therapy (2.2% ± 3.3% before versus 5.7% ± 3.9% after treatment, P = 0.0002). With regard to patient subsets, FMD significantly improved in the 21 lcSSc patients (from 2.1% to 5.6%, P = 0.001). In the seven dcSSc patients, we observed a tendency of improvement in FMD (from 3% to 6%, P = 0.25). Changes in PWV, ccIMT and ABI were not significant. Mean triglyceride (1.7 ± 0.97 versus 1.3 ± 0.46 mmol/l, P = 0.0004), total cholesterol (5.3 ± 1.6 mmol/l versus 4.2 ± 1.3 mmol/l, P = 0.0003), low density lipoprotein cholesterol (3.0 ± 1.3 versus 2.2 ± 1.0 mmol/l, P = 0.005) and C-reactive protein levels (CRP) (5.1 ± 5.2 versus 3.4 ± 2.7, P = 0.01) levels significantly decreased after rosuvastatin treatment. Mean C3, C4 and IC levels also decreased significantly as compared to pretreatment values.

Conclusions

Six-month rosuvastatin therapy improves endothelial function and lowers CRP, C3, C4 and IC levels indicating possible favourable effects of this statin on the cardiovascular and immune system in SSc.     

Retinoic acid and hydrocortisone strengthen the barrier function of human RPMI 2650 cells, a model for nasal epithelial permeability

The nasal pathway represents an alternative route for non-invasive systemic administration of drugs. The main advantages of nasal drug delivery are the rapid onset of action, the avoidance of the first-pass metabolism in the liver and the easy applicability. In vitro cell culture systems offer an opportunity to model biological barriers. Our aim was to develop and characterize an in vitro model based on confluent layers of the human RPMI 2650 cell line. Retinoic acid, hydrocortisone and cyclic adenosine monophosphate, which influence cell attachment, growth and differentiation have been investigated on the barrier formation and function of the nasal epithelial cell layers. Real-time cell microelectronic sensing, a novel label-free technique was used for dynamic monitoring of cell growth and barrier properties of RPMI 2650 cells. Treatments enhanced the formation of adherens and tight intercellular junctions visualized by electron microscopy, the presence and localization of junctional proteins ZO-1 and β-catenin demonstrated by fluorescent immunohistochemistry, and the barrier function of nasal epithelial cell layers. The transepithelial resistance of the RPMI 2650 cell model reached 50 to 200 Ω × cm², the permeability coefficient for 4.4 kDa FITC-dextran was 9.3 to 17 × 10⁻⁶ cm/s, in agreement with values measured on nasal mucosa from in vivo and ex vivo experiments. Based on these results human RPMI 2650 cells seem to be a suitable nasal epithelial model to test different pharmaceutical excipients and various novel formulations, such as nanoparticles for toxicity and permeability.

Electronic supplementary material

The online version of this article (doi:10.1007/s10616-012-9493-7) contains supplementary material, which is available to authorized users.     

Synthetic antisense oligodeoxynucleotides to transiently suppress different nucleus- and chloroplast-encoded proteins of higher plant chloroplasts

Selective inhibition of gene expression by antisense oligodeoxynucleotides (ODNs) is widely applied in gene function analyses; however, experiments with ODNs in plants are scarce. In this work, we extend the use of ODNs in different plant species, optimizing the uptake, stability, and efficiency of ODNs with a combination of molecular biological and biophysical techniques to transiently inhibit the gene expression of different chloroplast proteins. We targeted the nucleus-encoded phytoene desaturase (pds) gene, encoding a key enzyme in carotenoid biosynthesis, the chlorophyll a/b-binding (cab) protein genes, and the chloroplast-encoded psbA gene, encoding the D1 protein. For pds and psbA, the in vivo stability of ODNs was increased by phosphorothioate modifications. After infiltration of ODNs into juvenile tobacco (Nicotiana benthamiana) leaves, we detected a 25% to 35% reduction in mRNA level and an approximately 5% decrease in both carotenoid content and the variable fluorescence of photosystem II. In detached etiolated wheat (Triticum aestivum) leaves, after 8 h of greening, the mRNA level, carotenoid content, and variable fluorescence were inhibited up to 75%, 25%, and 20%, respectively. Regarding cab, ODN treatments of etiolated wheat leaves resulted in an up to 59% decrease in the amount of chlorophyll b, a 41% decrease of the maximum chlorophyll fluorescence intensity, the cab mRNA level was reduced to 66%, and the protein level was suppressed up to 85% compared with the control. The psbA mRNA and protein levels in Arabidopsis (Arabidopsis thaliana) leaves were inhibited by up to 85% and 72%, respectively. To exploit the potential of ODNs for photosynthetic genes, we propose molecular design combined with fast, noninvasive techniques to test their functional effects.     

In vivo and in situ visualization of early physiological events induced by heavy metals in pea root meristem

Heavy metals (HMs) are toxic pollutants, which can negatively affect the physiological processes of plants; moreover, HMs can be present in the food chain endangering people’s health. The aim of this study was to investigate the early physiological events during HM exposure in the root tips of the food plant Pisum sativum L. Ten-day-old pea plants were treated with 100 μM CdCl₂ or CuSO₄, in nutrient solution for 48 h. We studied the rapid formation of different reactive oxygen species (hydrogen peroxide H₂O₂ and superoxide radical O₂^·−) and reactive nitrogen species (nitric oxide NO· and peroxynitrite ONOO⁻) together with membrane damage and cell death in the meristem cells of pea roots using in vivo and in situ microscopic methods. In our experimental system, copper and cadmium induced the formation of H₂O₂ and NO. Two hours of heavy metal treatments resulted in an increased O₂^·− formation; however, later the level of this reactive molecule dramatically decreased. We found that high levels of NO were needed for ONOO⁻ production under HM exposure. A fast loss of membrane integrity and decreased cell viability were detected in root tips of copper-treated plants. The effects of cadmium seemed to be slower compared to copper, but this non-essential metal also caused cell death. We concluded that viability decreased when NO and H₂O₂ levels were simultaneously high in the same tissues. Using the NO scavenger it was also evidenced that NO generation is essential for cell death induction under copper or cadmium stress.     

Complex contribution of cyclophilin D to Ca2+-induced permeability transition in brain mitochondria, with relation to the bioenergetic state

Cyclophilin D (cypD)-deficient mice exhibit resistance to focal cerebral ischemia and to necrotic but not apoptotic stimuli. To address this disparity, we investigated isolated brain and in situ neuronal and astrocytic mitochondria from cypD-deficient and wild-type mice. Isolated mitochondria were challenged by high Ca(2+), and the effects of substrates and respiratory chain inhibitors were evaluated on permeability transition pore opening by light scatter. In situ neuronal and astrocytic mitochondria were visualized by mito-DsRed2 targeting and challenged by calcimycin, and the effects of glucose, NaCN, and an uncoupler were evaluated by measuring mitochondrial volume. In isolated mitochondria, Ca(2+) caused a large cypD-dependent change in light scatter in the absence of substrates that was insensitive to Ruthenium red or Ru360. Uniporter inhibitors only partially affected the entry of free Ca(2+) in the matrix. Inhibition of complex III/IV negated the effect of substrates, but inhibition of complex I was protective. Mitochondria within neurons and astrocytes exhibited cypD-independent swelling that was dramatically hastened when NaCN and 2-deoxyglucose were present in a glucose-free medium during calcimycin treatment. In the presence of an uncoupler, cypD-deficient astrocytic mitochondria performed better than wild-type mitochondria, whereas the opposite was observed in neurons. Neuronal mitochondria were examined further during glutamate-induced delayed Ca(2+) deregulation. CypD-knock-out mitochondria exhibited an absence or a delay in the onset of mitochondrial swelling after glutamate application. Apparently, some conditions involving deenergization render cypD an important modulator of PTP in the brain. These findings could explain why absence of cypD protects against necrotic (deenergized mitochondria), but not apoptotic (energized mitochondria) stimuli.     

Identification and characterization of a stable intermediate in photosystem I assembly in tobacco

Photosystem I (PSI) is the most efficient bioenergetic nanomachine in nature and one of the largest membrane protein complexes known. It is composed of 18 protein subunits that bind more than 200 co‐factors and prosthetic groups. While the structure and function of PSI have been studied in great detail, very little is known about the PSI assembly process. In this work, we have characterized a PSI assembly intermediate in tobacco plants, which we named PSI*. We found PSI* to contain only a specific subset of the core subunits of PSI. PSI* is particularly abundant in young leaves where active thylakoid biogenesis takes place. Moreover, PSI* was found to overaccumulate in PsaF‐deficient mutant plants, and we show that re‐initiation of PsaF synthesis promotes the maturation of PSI* into PSI. The attachment of antenna proteins to PSI also requires the transition from PSI* to mature PSI. Our data could provide a biochemical entry point into the challenging investigation of PSI biogenesis and allow us to improve the model for the assembly pathway of PSI in thylakoid membranes of vascular plants.