Regulatory role of membrane fluidity in gene expression and physiological functions

Plants, algae, and photosynthetic bacteria experience frequent changes in environment. The ability to survive depends on their capacity to acclimate to such changes. In particular, fluctuations in temperature affect the fluidity of cytoplasmic and thylakoid membranes. The molecular mechanisms responsible for the perception of changes in membrane fluidity have not been fully characterized. However, the understanding of the functions of the individual genes for fatty acid desaturases in cyanobacteria and plants led to the directed mutagenesis of such genes that altered the membrane fluidity of cytoplasmic and thylakoid membranes. Characterization of the photosynthetic properties of the transformed cyanobacteria and higher plants revealed that lipid unsaturation is essential for protection of the photosynthetic machinery against environmental stresses, such as strong light, salt stress, and high and low temperatures. The unsaturation of fatty acids enhances the repair of the damaged photosystem II complex under stress conditions. In this review, we summarize the knowledge on the mechanisms that regulate membrane fluidity, on putative sensors that perceive changes in membrane fluidity, on genes that are involved in acclimation to new sets of environmental conditions, and on the influence of membrane properties on photosynthetic functions.     

Tumor-suppressor Gene Promoter Hypermethylation in Saliva of Head and Neck Cancer Patients

Head and neck squamous cell carcinoma (HNSCC) accounts for a bulk of the oral and laryngeal cancers, the majority (70%) of which are associated with smoking and excessive drinking, major known risk factors for the development of HNSCC. In contrast to reports that suggest an inverse relationship between smoking and global DNA CpG methylation, hypermethylation of promoters of a number of genes was detected in saliva collected from patients with HNSCC. Using a sensitive methylation-specific polymerase chain reaction (MSP) assay to determine specific methylation events in the promoters of RASSF1A, DAPK1, and p16 genes, we demonstrate that we can detect tumor presence with an overall accuracy of 81% in the DNA isolated from saliva of patients with HNSCC (n = 143) when compared with the DNA isolated from the saliva of healthy nonsmoker controls (n = 31). The specificity for this MSP panel was 87% and the sensitivity was 80% (with a Fisher exact test P < .0001). In addition, the test panel performed extremely well in the detection of the early stages of HNSCCs, with a sensitivity of 94% and a specificity of 87%, and a high κ concordance value of 0.8, indicating an excellent overall agreement between the presence of HNSCC and a positive MSP panel result. In conclusion, we demonstrate that the promoter methylation of RASSF1A, DAPK1, and p16 MSP panel is useful in detecting hypermethylation events in a noninvasive manner in patients with HNSCC.     

The identification of the acid-base catalyst of alpha-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycoside hydrolase

The alpha-L-arabinofuranosidase from Geobacillus stearothermophilus T-6 (AbfA T-6) belongs to the retaining family 51 glycoside hydrolases. The conserved Glu175 was proposed to be the acid-base catalytic residue. AbfA T-6 exhibits residual activity towards aryl beta-D-xylopyranosides. This phenomenon was used to examine the catalytic properties of the putative acid-base mutant E175A. Data from kinetic experiments, pH profiles, azide rescue, and the identification of the xylopyranosyl azide product provide firm support to the assignment of Glu175 as the acid-base catalyst of AbfA T-6.     

Ca is involved in phytochrome A-dependent regulation of the succinate dehydrogenase gene sdh1-2 in Arabidopsis

The mechanism of transduction of the phytochrome signal regulating the expression of succinate dehydrogenase in Arabidopsis has been investigated. Using the phytochrome mutants of Arabidopsis, it is demonstrated that the inhibition of succinate dehydrogenase in the light may result from the phytochrome A-dependent modulation of Ca²⁺ amount in the nuclear fraction of leaves. This leads to the activation of expression of the gene pif3 encoding the phytochrome-interacting factor PIF3, which binds to the promoter of the gene sdh1-2 encoding the SDHA subunit of succinate dehydrogenase and suppresses its expression. It is concluded that Ca²⁺ ions are involved in the phytochrome A-mediated inhibition of succinate dehydrogenase activity in the light.     

Inflammatory pre-conditioning of mesenchymal multipotent stromal cells improves their immunomodulatory potency in acute pyelonephritis in rats

Background aimsAcute pyelonephritis is one of the most frequent infectious diseases of the urinary tract and a leading cause of kidney failure worldwide. One strategy for modulating excessive inflammatory responses in pyelonephritis is administration of mesenchymal multipotent stromal cells (MMSCs).MethodsThe putative protective effect of injection of MMSCs against experimental acute pyelonephritis was examined. We used in vivo experimental model of APN where bacteria are introduced in the bladder of rat. Three days after, intravenous injection of MMSCs was done. On the 7th day blood samples and kidneys were taken for further analysis.ResultsWe found obvious signs of oxidative stress and inflammation in the kidney in acute pyelonephritis in rats. Particularly, pro-inflammatory cytokine tumor necrosis factor-α levels, malondialdehyde, nitrite and myeloperoxidase activity were significantly increased. Histologic evaluation revealed numerous attributes of inflammation and tissue damage in the kidney. Treatment with MMSCs caused a remarkable decrease of all of these pathologic signs in renal tissue. Also, activated leukocytes induced pre-conditioning-like signaling in MMSCs. We showed alterations of expression or activity of inducible nitric oxide synthase, transforming growth factor-β, matrix metalloproteinase-2 and glycogen synthase kinase-3β, which could mediate immunomodulation and protective effects of MMSCs. This signaling could be characterized as inflammatory pre-conditioning.ConclusionsThe beneficial capacity of MMSCs to alleviate renal inflammation was more pronounced when pre-conditioned MMSCs were used. This approach could be used to prime MMSCs with different inflammatory modulators to enhance their engraftment and function in an immunoprotected fashion.     

Subunit-selective N-terminal domain associations organize the formation of AMPA receptor heteromers

The assembly of AMPA-type glutamate receptors (AMPARs) into distinct ion channel tetramers ultimately governs the nature of information transfer at excitatory synapses. How cells regulate the formation of diverse homo- and heteromeric AMPARs is unknown. Using a sensitive biophysical approach, we show that the extracellular, membrane-distal AMPAR N-terminal domains (NTDs) orchestrate selective routes of heteromeric assembly via a surprisingly wide spectrum of subunit-specific association affinities. Heteromerization is dominant, occurs at the level of the dimer, and results in a preferential incorporation of the functionally critical GluA2 subunit. Using a combination of structure-guided mutagenesis and electrophysiology, we further map evolutionarily variable hotspots in the NTD dimer interface, which modulate heteromerization capacity. This 'flexibility' of the NTD not only explains why heteromers predominate but also how GluA2-lacking, Ca(2+)-permeable homomers could form, which are induced under specific physiological and pathological conditions. Our findings reveal that distinct NTD properties set the stage for the biogenesis of functionally diverse pools of homo- and heteromeric AMPAR tetramers.     

NAADP triggers the fertilization potential in starfish oocytes

In invertebrates oocytes or eggs, the fertilization or activation potential establishes the fast electrical block to polyspermy and, in some species, provides the Ca2+ influx which contributes to the following intracellular Ca2+ wave. In echinoderms, the molecule triggering the activation potential is still unknown. The aim of this study was to assess whether nicotinic acid-adenine dinucleotide phosphate (NAADP) elicited the fertilization potential in starfish oocytes. The changes in membrane potential induced by the sperm were measured in oocytes held at a low resting potential, so that the Ca2+-action potential was inactivated and only the initial slower depolarization caused by the sperm could be studied. Decreasing extracellular Na+ concentration did not prevent the onset of the fertilization potential, while removal of external Ca2+ abolished it. The pre-incubation with SK&F 96365 and verapamil and the pre-injection of BAPTA inhibited the fertilization potential, while the injection of heparin only reduced its duration. The biophysical and pharmacological properties of the sperm-elicited depolarization were similar to those displayed by the NAADP-activated Ca2+-mediated current recently described in starfish oocytes. Indeed, the desensitization of NAADP-receptors prevented the onset of the fertilization potential. Taken together, these data suggest that NAADP could trigger the fertilization potential in starfish oocytes.     

Non-stressful death of 23S rRNA mutant G2061C defective in puromycin reaction

Catalysis of peptide bond formation in the peptidyl transferase center is a major enzymatic activity of the ribosome. Mutations limiting peptidyl transferase activity are mostly lethal. However, cellular processes triggered by peptidyl transferase deficiency in the bacterial cell are largely unknown. Here we report a study of the lethal G2061C mutant of Escherichia coli 23S ribosomal RNA (rRNA). The G2061C mutation completely impaired the puromycin reaction and abolished formation of the active firefly luciferase in an in vitro translation system, while poly(U)- and short synthetic mRNA-directed peptidyl transferase reaction with aminoacylated tRNAs in vitro was seemingly unaffected. Study of the cellular proteome upon expression of the 23S rRNA gene carrying the G2061C mutation compared to cells expressing wild-type 23S rRNA gene revealed substantial differences. Most of the observed effects in the mutant were associated with reduced expression of stress response proteins and particularly proteins associated with the ppGpp-mediated stringent response.