Salt Concentration-dependent Manner Research Articles

Salt-enhanced processing, proteolytic activity and stability of halophilic thermolysin-like proteinase, salilysin, isolated from a moderate halophile, Chromohalobacter salexigens DSM3043

Moderately halophilic bacterium, Chromohalobacter salexigens DSM3043, has a gene Csal_2537 encoding thermolysin-like M4 proteinase. This gene was cloned to pET expression vectors, resulting in high expression of recombinant proteinase, named as salilysin (salinity-dependent thermolysin-like proteinase), in Escherichia coli cytoplasm. This gene encodes precursor form of salilysin containing 348 amino acid residues (Pro-salilysin) consisting of 55 amino acids pro-sequence and following mature proteinase. Pro-sequence was cleaved three times to form intermediate 1, intermediate 2 and final mature salilysin. The processing rate was greatly accelerated in a salt concentration-dependent manner. Purified inactive mutant Pro-E167A-salilysin was correctly processed by purified mature salilysin, indicating that autolysis and inter-molecular processing occurred in its maturation processes. Proteolytic activity of mature salilysin against both peptide and protein substrates was also enhanced along with the addition of higher concentration of salt, 0–3.2 M NaCl, consistent with its halophilic origin. Mature salilysin was stabilized by ~8 °C in the presence of 1 M NaCl by thermal scanning using circular dichroism. One of the precursor form, intermediate 1, showed ~20 °C higher denaturation temperature than mature form, suggesting rigid and stable structure of this precursor form.

Multiple sensory neurons mediate starvation-dependent aversive navigation in Caenorhabditis elegans

Animals demonstrate flexible behaviors through associative learning based on their experiences. Deciphering the neural mechanisms for sensing and integrating multiple types of sensory information is critical for understanding such behavioral controls. The soil nematode Caenorhabditis elegans avoids salt concentrations it has previously experienced under starvation conditions. Here, we identify a pair of sensory neurons, the ASG neuron pair, which in cooperation with the ASER salt-sensing neuron generate starvation-dependent salt avoidance. Animals whose sensory input is restricted to only ASER failed to show learned avoidance due to inappropriately directed navigation behaviors. However, their navigation through a salt concentration gradient was improved by allowing sensory inputs to ASG in addition to ASER. Detailed behavioral analyses of these animals revealed that input from ASG neurons is required not only for controlling the frequency of initiating a set of sharp turns (called pirouettes) based on detected ambient salt concentrations but also adjusting the migration direction during pirouettes. Optogenetic activation of ASER by ChR2 induced turning behaviors in a salt concentration-dependent manner where presence of intact ASG was important for the starvation-dependent responses. Calcium imaging of the activity of ASG neurons in freely moving worms revealed that ASG is activated upon turning behavior. Our results indicate that ASG neurons cooperate with the ASER neuron to generate destination-directed reorientation in starvation-associated salt concentration avoidance.

Salt effect on bilayer phase transitions of dipalmitoylphosphatidylglycerol in saline water under high pressure

ABSTRACTThe phase transitions of dipalmitoylphosphatidylglycerol (DPPG) bilayer membrane at high NaCl concentrations under high pressure were investigated to construct the temperature–pressure phase diagram and to determine phase-transition properties. The constructed phase diagrams exhibited qualitative resemblance to that of the dipalmitoylphosphatidylcholine (DPPC) bilayer membrane: they showed the gel-phase polymorphism including the pressure-induced bilayer interdigitation. The phase-transition properties of the DPPG bilayer membrane changed in a salt concentration-dependent manner. We discussed the salt effect on the DPPG bilayer membrane from the variation in interactions between the polar head groups of the PG molecules.

Silicon improves salt tolerance of Glycyrrhiza uralensis Fisch. by ameliorating osmotic and oxidative stresses and improving phytohormonal balance.

Si has a beneficial effect on improving plant tolerance to salt stress. Nevertheless, the mechanisms of Si in mediating the stress responses are still poorly understood. Glycyrrhiza uralensis Fisch. (G. uralensis), a well-known medicinal plant, possesses vast therapeutic potentials. In the present study, a pot experiment was conducted to investigate the long-term effects of Si on growth and physiobiochemical characteristics in 2-year-old G. uralensis subjected to different levels of salinity. Si markedly affected G. uralensis growth in a salt concentration-dependent manner and had no effect on G. uralensis growth under 6g/kg NaCl. However, it partly reversed the reduction effect induced by 9g/kg NaCl. In addition, Si significantly increased the contents of soluble sugar and protein but deceased proline content and thus increased water relations; Si markedly increased the activities of SOD, peroxidase, and CAT and further resulted in decreased MDA content and membrane permeability. Moreover, Si altered the levels of phytohormones and their balances. With correlation analysis and principal component analysis (PCA), root biomass had a significant negative correlation with MDA and membrane permeability while a positive correlation with indole-3-acetic acid and GA3. The PCA partitioned the total variance into three PCs contributing maximum (88.234%) to the total diversity among the salt stress with or without Si due to the study of various traits. In conclusion, Si exerts a beneficial property on salt-induced harmful effects in G. uralensis by relieving osmotic stress, improving water relations, and alleviating oxidative stress; thus, altering the levels and balance of phytohormones results in improved growth of salt-stressed G. uralensis.

NMR elucidation of reduced B-Z transition activity of PKZ protein kinase at high NaCl concentration

A Z-DNA binding protein (ZBP)-containing protein kinase (PKZ) in fish species has an important role in the innate immune response. Previous structural studies of the Zα domain of the PKZ from Carassius auratus (caZαPKZ) showed that the protein initially binds to B-DNA and induces B-Z transition of double stranded DNA in a salt concentration-dependent manner. However, the significantly reduced B-Z transition activity of caZαPKZ at high salt concentration was not fully understood. In this study, we present the binding affinity of the protein for B-DNA and Z-DNA and characterize its extremely low B-Z transition activity at 250 mM NaCl. Our results emphasize that the B-DNA-bound form of caZαPKZ can be used as molecular ruler to measure the degree of B-Z transition.

Indigenous salt-tolerant rhizobacterium Pantoea dispersa (PSB3) reduces sodium uptake and mitigates the effects of salt stress on growth and yield of chickpea

Salt stress has multiple damaging effects on plants including physiological damage, reduced growth, and productivity. Plant growth-promoting rhizobacteria (PGPR) are one of the valuable options to mitigate the negative effects of this stress. In the present study, native bacteria from chickpea’s rhizosphere were isolated, and checked for their salt tolerance and plant growth-promoting attributes (phosphate (P) solubilization, siderophores, indole-3-acetic acid (IAA) production, and 1-aminocyclopropane-1-carboxylate (ACC) deaminase production). One isolate, subsequently identified as Pantoea dispersa, showed appreciable production of IAA (218.3 µg/ml) and siderophores (60.33% SU), P-solubilization (3.64 µg/ml) and ACC deaminase activity (207.45 nmol/mg/h) in the presence of 150 mM NaCl, under laboratory conditions. Salt stress in uninoculated chickpea (GPF2 cultivar) plants induced high accumulation of Na+ ions (3.86 mg g−1 dw) in the leaves, along with significant reduction in K+ uptake, membrane integrity, chlorophyll concentration, and leaf water content, thus resulting in impaired growth of the plant and yield (pods and seeds) in a salt concentration-dependent manner. The damage due to salt stress was restored significantly in plants inoculated with P. dispersa. A significant improvement in biomass (32–34%), pods number (31–34.5%), seeds number (32–35.7%), pods weight (30–32.6%), and seeds weight (27–35%) per plant occurred in salt stress-affected plants, which was associated with significant reduction in Na+ uptake, reduced membrane damage, significantly improved leaf water content, chlorophyll content, and K+ uptake. This study suggests for the first time that native P. dispersa strain PSB3 can be used to alleviate the negative effects of salt stress on chickpea plants and holds the potential to be used as a biofertilizer.

Silver(I) ions modulate the stability of DNA duplexes containing cytosine, methylcytosine and hydroxymethylcytosine at different salt concentrations.

Silver(I) ions can stabilize cytosine-cytosine, cytosine (C)-methylcytosine (5mC) and cytosine-hydroxymethylcytosine (5hmC) mismatched-base pairs. While cytosine modifications regulate DNA stability to regulate cellular functions, silver ions can modulate the stability of C-C, C-5mC and C-5hmC containing DNA duplexes in a salt concentration dependent manner.

Altered nucleic acid partitioning during phenol extraction or silica adsorption by guanidinium and potassium salts

Nucleic acids were found to partition into the phenol phase during phenol extraction in the presence of guanidinium at certain concentrations under acidic conditions. The guanidinium-concentration-dependent nucleic acid partitioning patterns were analogous to those of the nucleic acid adsorption/partitioning onto silica mediated by guanidinium, which implied that phenol and silica interact with nucleic acids through similar mechanisms. A competition effect was observed in which the nucleic acids that had partitioned into the phenol phase or onto the silica solid phase could be recovered to the aqueous phases by potassium in a molecular weight–salt concentration-dependent manner (the higher molecular weight nucleic acids needed higher concentrations of potassium to be recovered, and vice versa). Methods were developed based on these findings to isolate total RNA from Escherichia coli. By controlling the concentrations of guanidinium and potassium salts used before phenol extraction or silica adsorption, we can selectively recover total RNA but not the high molecular weight genomic DNA in the aqueous phases. Genomic DNA-free total RNA obtained by our methods is suitable for RT-PCR or other purposes. The methods can also be adapted to isolate small RNAs or RNA in certain molecular weight ranges by changing the salt concentrations used.

Ionic dependence of sulphur mustard cytotoxicity

The effect of ionic environment on sulphur mustard (bis 2-chloroethyl sulphide; HD) toxicity was examined in CHO-K1 cells. Cultures were treated with HD in different ionic environments at constant osmolar conditions (320 mOsM, pH 7.4). The cultures were refed with fresh culture medium 1 h after HD exposure, and viability was assessed. Little toxicity was apparent when HD exposures were carried out in ion-free sucrose buffer compared to LC 50 values of ~ 100–150 μM when the cultures were treated with HD in culture medium. Addition of NaCl to the buffer increased HD toxicity in a salt concentration-dependent manner to values similar to those obtained in culture medium. HD toxicity was dependent on both cationic and anionic species with anionic environment playing a much larger role in determining toxicity. Substitution of NaI for NaCl in the treatment buffers increased HD toxicity by over 1000%. The activity of the sodium hydrogen exchanger (NHE) in recovering from cytosolic acidification in salt-free and in different chloride salts did not correlate with the HD-induced toxicity in these buffers. However, the inhibition by HD of intracellular pH regulation correlated with its toxicity in NaCl, NaI and sucrose buffers. Analytical chemical studies and the toxicity of the iodine mustard derivative ruled out the role of chemical reactions yielding differentially toxic species as being responsible for the differences in HD toxicity observed. This work demonstrates that the early events that HD sets into motion to cause toxicity are dependent on ionic environment, possibly due to intracellular pH deregulation.

Salt Inactivates Endothelial Nitric Oxide Synthase in Endothelial Cells

There is a 1–4 mmol/L rise in plasma sodium concentrations in individuals with high salt intake and in patients with essential hypertension. In this study, we used 3 independent assays to determine whether such a small increase in sodium concentrations per se alters endothelial nitric oxide synthase (eNOS) function and contributes to hypertension. By directly measuring NOS activity in living bovine aortic endothelial cells, we demonstrated that a 5-mmol/L increase in salt concentration (from 137 to 142 mmol/L) caused a 25% decrease in NOS activity. Importantly, the decrease in NOS activity was in a salt concentration-dependent manner. The NOS activity was decreased by 25, 45, and 70%, with the increase of 5, 10, and 20 mmol/L of NaCl, respectively. Using Chinese hamster ovary cells stably expressing eNOS, we confirmed the inhibitory effects of salt on eNOS activity. The eNOS activity was unaffected in the presence of equal milliosmol of mannitol, which excludes an osmotic effect. Using an ex vivo aortic angiogenesis assay, we demonstrated that salt attenuated the nitric oxide (NO)-dependent proliferation of endothelial cells. By directly monitoring blood pressure changes in response to salt infusion, we found that in vivo infusion of salt induced an acute increase in blood pressure in a salt concentration-dependent manner. In conclusion, our findings demonstrated that eNOS is sensitive to changes in salt concentration. A 5-mmol/L rise in salt concentration, within the range observed in essential hypertension patients or in individuals with high salt intake, could significantly suppress eNOS activity. This salt-induced reduction in NO generation in endothelial cells may contribute to the development of hypertension.

Responsive modes of Medicago sativa proline dehydrogenase genes during salt stress and recovery dictate free proline accumulation

Free proline accumulation is an innate response of many plants to osmotic stress. To characterize transcriptional regulation of the key proline cycle enzymes in alfalfa (Medicago sativa), two proline dehydrogenase (MsPDH) genes and a partial sequence of Delta (1) -pyrroline-5-carboxylate dehydrogenase (MsP5CDH) gene were identified and cloned. The two MsPDH genes share a high nucleotide sequence homology and a similar exon/intron structure. Estimation of transcript levels during salt stress and recovery revealed that proline accumulation during stress was linearly correlated with a strong decline in MsPDH transcript levels, while Delta (1) -pyrroline-5-carboxylate synthetase (MsP5CS) and MsP5CDH steady-state transcript levels remained essentially unchanged. MsPDH transcript levels dramatically decreased in a fast, salt concentration-dependent manner. The extent of salt-induced proline accumulation also correlated with salt concentrations. Salt-induced repression of MsPDH1 promoter linked to the GUS reporter gene confirmed that the decline in MsPDH transcript levels was due to less transcription initiation. Contrary to the salt-dependent repression, a rapid induction of MsPDH transcription occurred at a very early stage of the recovery process, independently of earlier salt treatments. Hence our results suggest the existence of two different regulatory modes of MsPDH expression; the repressing mode that quantifies salt concentration in an as yet unknown mechanism and the "rehydration"-enhancing mode that responds to stress relief in a maximal induction of MsPDH transcription. As yet the components of salt sensing as well as those that might interact with MsPDH promoter to reduce transcription are still unknown.

Detergents Destabilize the Cubic Phase of Monoolein: Implications for Membrane Protein Crystallization

The in meso method for membrane protein crystallization uses a lipidic cubic phase as the hosting medium. The cubic phase provides a lipid bilayer into which the protein presumably reconstitutes and from which protein crystals nucleate and grow. The solutions used to spontaneously form the protein-enriched cubic phase often contain significant amounts of detergents that were employed initially to purify and to solubilize the membrane protein. By virtue of their surface activity, detergents have the potential to impact on the phase properties of the in meso system and, by extension, the outcome of the crystallization process. The purpose of this study was to quantify the effects that a popular series of nonionic detergents, the n-alkyl- β- d-glucopyranosides, have on the phase behavior of hydrated monoolein, the lipid upon which the in meso method is based. Phase identity and phase microstructure were characterized by small-angle x-ray diffraction on samples prepared to mimic in meso crystallization conditions. Measurements were made in the 0–40°C range. Samples prepared in the cooling direction allow for the expression of metastability, a feature of liquid crystalline phases that might be exploited in low-temperature crystallization. The results show that the cubic phase is relatively insensitive to small amounts of alkyl glucosides. However, at higher levels the detergents trigger a transition to the lamellar phase in a temperature- and salt concentration-dependent manner. These effects have important implications for in meso crystallization. A diffraction-based method for assaying detergents is presented.

Phosphoenolpyruvate-dependent Tubulin-Pyruvate Kinase Interaction at Different Organizational Levels

Evidence for the direct binding of pyruvate kinase to tubulin/microtubule and for the inhibitory effect of phosphoenolpyruvate on tubulin-enzyme hetero-association were provided by surface plasmon resonance and pelleting experiments. Electron microscopy revealed that pyruvate kinase induces depolymerization of paclitaxel-stabilized microtubules into large oligomeric aggregates and bundles the tubules in a salt concentration-dependent manner. The C-terminal "tail"-free microtubules did not bind pyruvate kinase, suggesting the crucial role of the C-terminal segments in the binding of kinase. Immunoblotting and polymerization experiments with cell-free brain extract revealed that pyruvate kinase specifically binds to microtubules, the binding of pyruvate kinase impedes microtubule assembly, and phosphoenolpyruvate counteracts the destabilization of microtubules induced by pyruvate kinase. We also showed by immunostaining the juxtanuclear localization of pyruvate kinase in intact L929 cells and that this localization was influenced by treatments with paclitaxel or vinblastine. These findings suggest that the distribution of the enzyme may be controlled by the microtubular network in vivo.

A model of PSI dimerization: destabilization of the C278-G303 stem-loop by the nucleocapsid protein (NCp10) of MoMuLV.

We have shown that at low ionic strength (i.e., 100 mM NaCl) a short autocomplementary sequence spanning nucleotides C283 to G298 of MoMuLV RNA genome is involved in the process of PSI dimerization in vitro [Girard, P.-M., Bonnet-Mathonière, B., Muriaux, D., & Paoletti, J. (1995) Biochemistry 34, 9785-9794]. In order to identify other contributions of the PSI structure to RNA dimerization, we studied the kinetics of dimerization as a function of salt concentration of short RNA transcripts comprising or not the autocomplementary sequence C283-G298. We propose that, apart from the crucial role of this sequence in RNA dimerization, the 364-565 domain of PSI can interfere, in vitro, with the initiation of dimer formation. Intermolecular loop-loop recognitions involving the 364-565 domain could stabilize, in a salt concentration-dependent manner, a transient RNA dimer built around the loop-loop U288-A293 interaction. This dimer evolves toward a more stable structure which mainly corresponds to the annealing of two C283-G298 sequences. We also show that chemically synthesized NCp10 does not modify these steps but rather helps the system to pass over the energy barriers associated with the transition to stable RNA structures comprising the stem-loop C278-G303. Data obtained in the presence of NCp10 suggest a binding site size of 9 +/- 1 nucleotides per protein at 37 degrees C and a 10-20-fold increase in the rate constant (i.e., k1 = 24 000 +/- 7000 M-1 s-1) of dimer formation.

Solubilization of matrix protein M1/M from virions occurs at different pH for orthomyxo- and paramyxoviruses

Enveloped viruses, of which the orthomyxo- and paramyxoviruses are members, are known to be uncoated by nonionic detergents in a salt concentration-dependent manner. In this study we have shown that detergent uncoating of myxoviruses depends not only on salt concentration but also on pH. Treatment of orthomyxoviruses with Nonidet-P40 or Triton N-101 at low salt concentrations results in solubilization of surface virion glycopolypeptides in alkaline and neutral pH (9.0-6.5), but in acidic pH (6.0-5.0) the viral matrix protein M1 is also removed, and the viral ribonucleoprotein complex is released. Conversely, the paramyxovirus matrix protein M is more completely solubilized in alkaline pH (pH 9.0) than in neutral and acidic pH 7.4-5.0 The described pH-dependent differences are discussed in terms of orthomyxo- and paramyxovirus uncoating in target cells.