N-Acetyl-L-glutamate Kinase of Chlamydomonas reinhardtii: In Vivo Regulation by PII Protein and Beyond.

N-Acetyl-L-glutamate kinase (NAGK) catalyzes the rate-limiting step in the ornithine/arginine biosynthesis pathway in eukaryotic and bacterial oxygenic phototrophs. NAGK is the most highly conserved target of the PII signal transduction protein in Cyanobacteria and Archaeplastida (red algae and Chlorophyta). However, there is still much to be learned about how NAGK is regulated in vivo. The use of unicellular green alga Chlamydomonas reinhardtii as a model system has already been instrumental in identifying several key regulation mechanisms that control nitrogen (N) metabolism. With a combination of molecular-genetic and biochemical approaches, we show the existence of the complex CrNAGK control at the transcriptional level, which is dependent on N source and N availability. In growing cells, CrNAGK requires CrPII to properly sense the feedback inhibitor arginine. Moreover, we provide primary evidence that CrPII is only partly responsible for regulating CrNAGK activity to adapt to changing nutritional conditions. Collectively, our results suggest that in vivo CrNAGK is tuned at the transcriptional and post-translational levels, and CrPII and additional as yet unknown factor(s) are integral parts of this regulation.

Truncated hemoglobin 2 modulates phosphorus deficiency response by controlling of gene expression in nitric oxide-dependent pathway in Chlamydomonas reinhardtii.

MAIN CONCLUSION: Truncated hemoglobin 2 is involved in fine-tuning of PSR1-regulated gene expression during phosphorus deprivation. Truncated hemoglobins form a large family found in all domains of life. However, a majority of physiological functions of these proteins remain to be elucidated. In the model alga Chlamydomonas reinhardtii, macro-nutritional deprivation is known to elevate truncated hemoglobin 2 (THB2). This study investigated the role of THB2 in the regulation of a subset of phosphorus (P) limitation-responsive genes in cells suffering from P-deficiency. Underexpression of THB2 in amiTHB2 strains resulted in downregulation of a suite of P deprivation-induced genes encoding proteins with different subcellular location and functions (e.g., PHOX, LHCSR3.1, LHCSR3.2, PTB2, and PTB5). Moreover, our results provided primary evidence that the soluble guanylate cyclase 12 gene (CYG12) is a component of the P deprivation regulation. Furthermore, the transcription of PSR1 gene for the most critical regulator in the acclimation process under P restriction was repressed by nitric oxide (NO). Collectively, the results indicated a tight regulatory link between the THB2-controlled NO levels and PSR1-dependent induction of several P deprivation responsive genes with various roles in cells during P-limitation.

Effects of arginine on Polytomella parva growth, PII protein levels and lipid body formation.

MAIN CONCLUSION: L-Arginine supports growth and resulted in increased PII signaling protein levels and lipid droplet accumulation in the colorless green alga Polytomella parva. Polytomella parva, a model system for nonphotosynthetic green algae, utilizes ammonium and several carbon sources, including ethanol and acetate. We previously reported that P. parva accumulates high amounts of arginine with the key enzyme of the ornithine/arginine biosynthesis pathway, N-acetyl-L-glutamate kinase, exhibiting high activity. Here we demonstrate that L-arginine can be used by this alga as a nitrogen source. Externally supplied arginine directly influenced the levels of PII signaling protein and formation of triacylglycerol (TAG)-filled lipid bodies (LBs). Our results suggest that the nitrogen source, but not nitrogen starvation, may be critical for the accumulation of LBs in a PII-independent manner in P. parva.

Herbal Preparation STW 5 for Functional Gastrointestinal Disorders: Clinical Experience in Everyday Practice.

BACKGROUND: The most common functional gastrointestinal disorders (FGID) are functional dyspepsia (FD) and irritable bowel syndrome (IBS), with a prevalence in the general population of 15-20% (FD) and 10% (IBS), respectively. The complexity of pathophysiologic mechanisms and limitations in therapeutic options make the management of FD and IBS patients a challenge in routine clinical practice. SUMMARY: Syndromes classified as FGID frequently overlap, and coexist with gastroesophageal reflux disease (GERD). Patients with overlapping symptoms are more likely to seek medical care. The challenge for routine clinical practice is to find the best approach for treatment of multiple symptoms. STW 5, a combination of 9 herbal extracts, was shown to have multi-target effects: it normalizes the disturbed gastrointestinal motility, alleviates hypersensitivity, inhibits inflammation, suppresses gastric hypersecretion, and modulates the microbiota. Controlled randomized studies proved STW 5 to be efficacious both in FD and IBS, with control over the full spectrum of upper and lower gastrointestinal symptoms. STW 5 reduced concomitant heartburn in FD patients. STW 5 was well tolerated in the examined populations, independent of concomitant diseases and concomitant medication. Key Messages: The clinical use of the herbal preparation STW 5 in FD and IBS is evidence-based. STW 5 is an example for the concept of multi-target therapy. It offers treatment opportunities in routine clinical practice with high prevalence of overlap of FGID and concomitant GERD. Considering that FD and IBS are typically chronic and recurrent conditions, the clinically observed good tolerability and safety of STW 5 is an advantage.

Efficacy and Safety of Postbiotic Contained Inactivated Lactobacillus reuteri ( Limosilactobacillus reuteri ) DSM 17648 as Adjuvant Therapy in the Eradication of Helicobacter pylori in Adults With Functional Dyspepsia: A Randomized Double-Blind Placebo-Controlled Trial.

INTRODUCTION: Increasing the effectiveness of eradication therapy is an important task in gastroenterology. The aim of this study was to evaluate the efficacy and safety of postbiotic containing inactivated (nonviable) Limosilactobacillus (Lactobacillus) reuteri DSM 17648 (Pylopass) as adjuvant treatment of Helicobacter pylori eradication in patients with functional dyspepsia (FD). METHODS: This randomized, double-blind, placebo-controlled, multicenter, parallel study included H. pylori -positive patients with FD. The postbiotic group received Pylopass 200 mg bid for 14 days in combination with eradication therapy (esomeprazole 20 mg bid + amoxicillin 1,000 mg bid + clarithromycin 500 mg bid for 14 days) and another 14 days after the completion of eradication therapy. The study was registered in the ISRCTN registry (ISRCTN20716052). RESULTS: Eradication efficiency was 96.7% for the postbiotic group vs 86.0% for the placebo group ( P = 0.039). Both groups showed significant improvements in quality of life and reduction of most gastrointestinal symptoms with no significant differences between groups. The overall number of digestive adverse effects in the postbiotic group was lower than in the placebo group. Serious adverse effects were not registered. DISCUSSION: The postbiotic containing inactivated L. reuteri DSM 17648 significantly improves the effectiveness of H. pylori eradication therapy in FD and decreases overall number of digestive adverse effects of this therapy.

Arginine-Dependent Nitric Oxide Generation and S-Nitrosation in the Non-Photosynthetic Unicellular Alga Polytomella parva.

Nitric oxide (NO) acts as a key signaling molecule in higher plants, regulating many physiological processes. Several photosynthetic algae from different lineages are also known to produce NO. However, it remains unclear whether this messenger is produced by non-photosynthetic algae. Among these organisms, the colorless alga Polytomella parva is a special case, as it has lost not only its plastid genome, but also nitrate reductase and nitrite reductase. Up to now, the question of whether NO synthesis occurs in the absence of functional nitrate reductase (NR) and the assimilation of nitrates/nitrites in P. parva has not been elucidated. Using spectrofluorometric assays and confocal microscopy with NO-sensitive fluorescence dye, we demonstrate L-arginine-dependent NO synthesis by P. parva cells. Based on a pharmacological approach, we propose the existence of arginine-dependent NO synthase-like activity in this non-photosynthetic alga. GC-MS analysis provides primary evidence that P. parva synthesizes putrescine, which is not an NO source in this alga. Moreover, the generated NO causes the S-nitrosation of protein cysteine thiol groups. Together, our data argue for NR-independent NO synthesis and its active role in S-nitrosation as an essential post-translational modification in P. parva.

The HSP70 chaperone machines of Chlamydomonas are induced by cold stress.

The responses of Chlamydomonas reinhardtii cells to low temperatures have not been extensively studied compared with other stresses. Like other organisms, this green alga has heat shock protein 70s (HSP70s) that are located in chloroplast, mitochondrion and cytosol. To test whether temperature downshifts affected HSP70s synthesis, we used real-time PCR and protein gel blot analysis. C. reinhardtii cells exposed to cold stress show increased HSP70s mRNA levels. Genes encoding other components of HSP70 chaperone machines (e.g. CGE1, CDJ1, HSP90C and HSP90A) are also up-regulated in response to decreased temperature. We demonstrated that the accumulation of all analyzed mRNA occur more slowly and with reduced amplitude in cells exposed to cold than in cells treated with heat. Furthermore, C. reinhardtii cells display the splicing of the CGE1 transcript that was dependent on low temperature. Finally, the transcription regulator of C. reinhardtii HSF1 is also cold-responsive, suggesting its role in the transcriptional regulation of HSP genes at low temperature.

The Chlamydomonas reinhardtii alternative oxidase 1 is regulated by heat stress.

The alternative oxidase (AOX) is a non-energy conserving terminal oxidase that has emerged as an important mitochondrial component of the cell stress responses. Although the most studied abiotic condition in relation to Chlamydomonas reinhardtii is high temperature, changes in AOX capacity of the alga were studied only under oxidative stress and cold. To examine whether elevated temperatures affected AOX1 expression, we applied quantitative real-time PCR and pharmaceutical approaches. In this work, we demonstrated a sharp increase in AOX1 transcript and protein abundance under heat stress. Furthermore, C. reinhardtii cells displayed a large increase in alternative respiration in response to high temperature. Feeding with the protein kinase inhibitor staurosporine strongly retarded the AOX1 transcription. Finally, the addition of the calcium chelator EGTA prevented heat-induced AOX1 expression. Together, our results imply that heat-inducible Ca(2+) influx and protein kinase(s) may mediate AOX1 expression at elevated temperatures. Characterization of heat-induced AOX1 regulation in the green alga C. reinhardtii provides a framework for a more complete understanding of the function of this conserved protein.

Azithromycin in a triple therapy for H.pylori eradication in active duodenal ulcer.

AIM: To assess and compare the efficacy and safety of two triple regimes: A) metronidazole, amoxicillin and omeprazole, which is still widely used in Russia, and B) azithromycin, amoxicillin and omeprazole in healing active duodenal ulcer and H.pylori eradication. METHODS: 100 patients with active duodenal ulcer were included in the open, multicentre, randomized study with comparative groups. Patients were randomly assigned to one of the following one-week triple regimes: A) metronidazole 500 mg bid, amoxicillin 1 g bid and omeprazole 20 mg bid (OAM, n=50) and B) azithromycin 1 g od for the first 3 days (total dose 3 g), amoxicillin 1 g bid and omeprazole 20 mg bid (OAA, n=50). Omeprazole 20 mg od was given after the eradication course as a monotherapy for three weeks. The control endoscopy was performed 8 weeks after the entry. H.pylori infection was determined in the entry of the study and four weeks after the cessation of treatment by means of histology and CLO-test. RESULTS: 97 patients completed the study according to the protocol (1 patient of the OAM group did not come to the control endoscopy, 2 patients of the OAA group stopped the treatment because of mild allergic urticaria). Duodenal ulcers were healed in 48 patients of the OAM group (96 %; CI 90.5-100 %) and in 46 patients of the OAA group (92 %; CI 89.5-94.5 %) (p=ns). H.pylori infection was eradicated in 15 out of 50 patients with OAM (30 %; CI 17-43 %) and in 36 out of 50 patients treated with OAA (72 %; CI 59-85 %) (P<0.001)- ITT analysis. CONCLUSION: The triple therapy with omeprazole, amoxicillin and metronidazole failed to eradicate H.pylori in the majority of patients, which is an essential argument to withdraw this regimen out of the national recommendations. Macrolide with amoxicillin are preferable to achieve higher eradication rates. Azithromycin (1 g od for the first 3 days) can be considered as a successful component of the triple PPI-based regimen.