Low Concentrations Of Magnesium Ions Research Articles

Calcium Ion Deposition with Precipitated Calcium Carbonate: Influencing Factors and Mechanism Exploration

In order to apply precipitated calcium carbonate (PCC) in the detergent industry, its ability to deposit calcium ions in hard water is an important process. In this work, the calcium ion deposition in the presence of PCC from different sources is investigated to reveal the influencing factors and mechanism of nucleation and crystal growth of CaCO3. SEM, XRD, Malvern particle size analysis, and calcium electrodes are used to evaluate the effects of PCC morphology, saturation of Ca2+, and PCC additive amount on the deposition behavior of CaCO3. Through SEM and Malvern particle size analysis, it is found that the precipitation of calcium ions is obviously accelerated by PCC acting as seeds. Moreover, calcium ions are effectively adsorbed on (211) crystal facets, thus prismatic and scalenohedral PCC crystals exhibit better adsorption performance than irregular cubic PCC ones. In addition, XRD demonstrates that PCC reduces or even eliminates the formation of crystals such as vaterite, displaying high deposition capacity under complex water conditions (slightly acidic or highly alkaline pH, low magnesium ion concentration (<0.01 M), and temperatures of 0–60 °C), forming thermodynamically stable calcite in water, which significantly controls the instability of the washing process.

XdfA, a novel membrane-associated DedA family protein of Xanthomonas campestris, is required for optimum virulence, maintenance of magnesium, and membrane homeostasis.

Xanthomonas campestris is an important member of the Xanthomonas group of phytopathogens that causes diseases in crucifers. In X. campestris, several virulence-associated functions, including some belonging to unknown predicted functions, have been implicated in the colonization and disease processes. However, the role of many of these unknown predicted proteins in Xanthomonas-host interaction and their exact physiological function is not clearly known. In this study, we identified a novel membrane-associated protein belonging to the DedA super family, XdfA, which is required for virulence in X. campestris. The DedA family of proteins are generally ubiquitous in bacteria; however, their function and actual physiological role are largely elusive. Characterization of ∆xdfA by homology modeling, membrane localization, and physiological studies indicated that XdfA is a membrane-associated protein that plays a role in the maintenance of membrane integrity. Furthermore, functional homology modeling analysis revealed that the XdfA exhibits structural similarity to a CorA-like magnesium transporter and is required for optimum growth under low magnesium ion concentration. We report for the first time that a putative DedA family of protein in Xanthomonas is required for optimum virulence and plays a role in the maintenance of membrane-associated functions and magnesium homeostasis. IMPORTANCE Bacterial DedA family proteins are involved in a range of cellular processes such as ion transport, signal transduction, and cell division. Here, we have discussed about a novel DedA family protein XdfA in Xanthomonas campestris pv. campestris that has a role in membrane homeostasis, magnesium transport, and virulence. Understanding membrane and magnesium homeostasis will aid in our comprehension of bacterial physiology and eventually will help us devise effective antimicrobial strategies to safeguard horticulturally and agriculturally important crop plants.

New Nuclease from Extremely Psychrophilic Microorganism Psychromonas ingrahamii 37: Identification and Characterization

Nucleases are an important group of hydrolases that degrade nucleic acids, with broad spectrum of applications in science and industry. In this paper, we report the identification and characterization of the nuclease from extremely psychrophilic bacterium Psychromonas ingrahamii that grows exponentially at 5 °C, but may also grow at even lower temperatures (down to − 12 °C). The putative endonuclease I gene, identified in the genome of P. ingrahamii, was cloned and expressed in Pichia pastoris. The recombinant protein was purified and its nucleolytic features were studied. The new enzyme, named by us as PinNuc, displays the features characteristic for the nonselective endonucleases, and has the ability to degrade different forms of nucleic acids. It is very active at room temperature in low ion-strength buffer and in the presence of low concentrations of magnesium ions. The enzyme, which possesses six cysteine residues, the most likely all engaged in disulphide bridges, is active only in oxidized form, and can be efficiently inactivated by the addition of low amounts of a reducing agent. According to our knowledge, it is the first nuclease, belonging to endonuclease I family, isolated from such extremely psychrophilic organism.

A ribosomal strategy for magnesium deficiency

Under low-magnesium stress, bacteria suppress ribosome synthesis.

Expression, purification and identification of a thermolysin-like protease, neutral protease I, from Aspergillus oryzae with the Pichia pastoris expression system

Neutral proteases are widely used in the textile, food and medical industries. This study was designed to obtain high expression levels of neutral protease I from Aspergillus oryzae 3.042 by using Pichia pastoris GS115 as the host strain for industrial purposes. The coding sequence of the target gene was modified, synthesized, and then cloned into the expression vector pHBM905BDM, which harbored the d1+2 × 201 AOX1 promoter and the MF4I leader sequence. The recombinant plasmid was transformed into Pichia pastoris GS115. The recombinant strain was used for high-density fermentation in a 4-L fermenter. The yield of the target protein reached 12.87 mg/mL, and the enzyme activity was approximately 49370 U/mL, which indicated that this enzyme was expressed in Pichia pastoris at a high level. The target protein was purified and characterized. Its optimum temperature and pH were 55 °C and 8.0, respectively. This enzyme was extremely sensitive to EDTA, which is consistent with the previous reports that it is a zinc-dependent metalloprotease. Our results indicated that low concentrations of zinc, calcium and magnesium ions stimulated the enzyme activity, whereas high concentrations inhibited its activity. In addition, calcium and magnesium ions increased the thermostability of the enzyme. All of the evidence indicated that this protease is a thermolysin-like peptidase.

Intramolecular phenotypic capacitance in a modular RNA molecule

Phenotypic capacitance refers to the ability of a genome to accumulate mutations that are conditionally hidden and only reveal phenotype-altering effects after certain environmental or genetic changes. Capacitance has important implications for the evolution of novel forms and functions, but experimentally studied mechanisms behind capacitance are mostly limited to complex, multicomponent systems often involving several interacting protein molecules. Here we demonstrate phenotypic capacitance within a much simpler system, an individual RNA molecule with catalytic activity (ribozyme). This naturally occurring RNA molecule has a modular structure, where a scaffold module acts as an intramolecular chaperone that facilitates folding of a second catalytic module. Previous studies have shown that the scaffold module is not absolutely required for activity, but dramatically decreases the concentration of magnesium ions required for the formation of an active site. Here, we use an experimental perturbation of magnesium ion concentration that disrupts the folding of certain genetic variants of this ribozyme and use in vitro selection followed by deep sequencing to identify genotypes with altered phenotypes (catalytic activity). We identify multiple conditional mutations that alter the wild-type ribozyme phenotype under a stressful environmental condition of low magnesium ion concentration, but preserve the phenotype under more relaxed conditions. This conditional buffering is confined to the scaffold module, but controls the catalytic phenotype, demonstrating how modularity can enable phenotypic capacitance within a single macromolecule. RNA's ancient role in life suggests that phenotypic capacitance may have influenced evolution since life's origins.

Biochemical characterization of a new type of intracellular PHB depolymerase from Rhodospirillum rubrum with high hydrolytic activity on native PHB granules

A Rhodospirillum rubrum gene that is predicted to code for an extracellular poly(3-hydroxybutyrate) (PHB) depolymerase by the recently published polyhydroxyalkanoates (PHA) depolymerase engineering database was cloned. The gene product (PhaZ3( Rru )) was expressed in recombinant E. coli, purified and biochemically characterized. PhaZ3( Rru ) turned out, however, to share characteristics of intracellular PHB depolymerases and revealed a combination of properties that have not yet been described for other PHB depolymerases. A fusion of PhaZ3( Rru )with the enhanced cyan fluorescent protein was able to bind to PHB granules in vivo and supported the function as an intracellular PHB depolymerase. Purified PhaZ3( Rru ) was specific for short-chain-length polyhydroxyalkanoates (PHA(SCL)) and hydrolysed both untreated native PHB granules as well as trypsin-activated native PHB granules to a mixture of mono- and dimeric 3-hydroxybutyrate. Crystalline (denatured) PHB granules were not hydrolysed by PhayZ3( Rru ). Low concentrations of calcium or magnesium ions (1-5mM) reversibly (EDTA) inhibited the enzyme. Our data suggest that PhaZ3( Rru ) is the representative of a new type of the growing number of intracellular PHB depolymerases.

Structural Diversity of Triplet Repeat RNAs

Tandem repeats of various trinucleotide motifs are present in the human transcriptome, but the functions of these regular sequences, which likely depend on the structures they form, are still poorly understood. To gain new insight into the structural and functional properties of triplet repeats in RNA, we have performed a biochemical structural analysis of the complete set of triplet repeat transcripts, each composed of a single sequence repeated 17 times. We show that these transcripts fall into four structural classes. The repeated CAA, UUG, AAG, CUU, CCU, CCA, and UAA motifs did not form any higher order structure under any analyzed conditions. The CAU, CUA, UUA, AUG, and UAG repeats are ordered according to their increasing tendency to form semistable hairpins. The repeated CGA, CGU, and all CNG motifs form fairly stable hairpins, whereas AGG and UGG repeats fold into stable G-quadruplexes. The triplet repeats that formed the most stable structures were characterized further by biophysical methods. UV-monitored structure melting revealed that CGG and CCG repeats form, respectively, the most and least stable hairpins of all CNG repeats. Circular dichroism spectra showed that the AGG and UGG repeat quadruplexes are formed by parallel RNA strands. Furthermore, we demonstrated that the different susceptibility of various triplet repeat transcripts to serum nucleases can be explained by the sequence and structural features of the tested RNAs. The results of this study provide a comprehensive structural foundation for the functional analysis of triplet repeats in transcripts.

Thermodynamic and Kinetic Effects of Morpholino Modification on Pyrimidine Motif Triplex Nucleic Acid Formation under Physiological Condition

Due to instability of pyrimidine motif triplex nucleic acid under physiological pH and low magnesium ion concentration, stabilization of the triplex under the physiological condition is crucial in improving its therapeutic potential to artificially control gene expression in vivo. To this end, we investigated the thermodynamic and kinetic effects of morpholino (MOR) modification of triplex-forming oligonucleotide (TFO) on the triplex formation under the physiological condition. The thermodynamic analyses indicated that the MOR modification of TFO not only significantly increased the thermal stability of the triplex but also increased the binding constant for the triplex formation by nearly 2 orders of magnitude. The consideration of the observed thermodynamic parameters suggested that the increased rigidity of the MOR-modified TFO in the free state relative to the corresponding unmodified TFO may enable the significant increase in the binding constant. Kinetic data demonstrated that the observed increase in the binding constant resulted from the considerable increase in the association rate constant rather than the decrease in the dissociation rate constant. This information will be valuable for designing novel chemically modified TFO with higher binding affinity in the triplex formation under physiological conditions, leading to progress in therapeutic applications of the antigene strategy in vivo.

Structural Rearrangements in the Active Site of the Thermus thermophilus 16S rRNA Methyltransferase KsgA in a Binary Complex with 5′-Methylthioadenosine

Posttranscriptional modification of ribosomal RNA (rRNA) occurs in all kingdoms of life. The S-adenosyl- l-methionine-dependent methyltransferase KsgA introduces the most highly conserved rRNA modification, the dimethylation of A1518 and A1519 of 16S rRNA. Loss of this dimethylation confers resistance to the antibiotic kasugamycin. Here, we report biochemical studies and high-resolution crystal structures of KsgA from Thermus thermophilus. Methylation of 30S ribosomal subunits by T. thermophilus KsgA is more efficient at low concentrations of magnesium ions, suggesting that partially unfolded RNA is the preferred substrate. The overall structure is similar to that of other methyltransferases but contains an additional α-helix in a novel N-terminal extension. Comparison of the apoenzyme with complex structures with 5′-methylthioadenosine or adenosine bound in the cofactor-binding site reveals novel features when compared with related enzymes. Several mobile loop regions that restrict access to the cofactor-binding site are observed. In addition, the orientation of residues in the substrate-binding site indicates that conformational changes are required for binding two adjacent residues of the substrate rRNA.

Study of electrostatic energy contribution on monolayer domains size

Phospholipid organic monolayer at the air–water interface was studied in the presence of magnesium ions. For this reason the domain size by Brewster angle microscopy (BAM) as well as the Gibbs energy by thermodynamic analysis is evaluated. Domain size increase is discussed with respect to the shape equation on the basis of the electrostatic energy contribution. By coupling these techniques it was found that for ion adsorption at first it is preferred domain boundary and liquid expanded (LE) phase. Only after reaching of specific concentration (30 mM) ions are adsorbed also inside of the liquid condensed (LC) domain. Evaluation of BAM images and thermodynamic analysis reveals existence of pure repulsive forces; it is illustrated by gradual increase of average domain area. Additionally, in low magnesium ion concentration region it was successful to use Langmuir adsorption isotherm for fitting the observed domain area.

The roles of cosolutes on the hammerhead ribozyme activity

The hammerhead ribozyme is often used for gene regulations in a cell. One of the major differences between in vitro and in cell conditions is the molecular crowding. However, the influence of crowding conditions on the ribozyme activity is still unclear. Here, we investigated the activity of the hammerhead ribozyme under molecular crowding condition by ethylene glycol (EG), poly (ethylene glycol) (PEG), dextran, and Ficoll. These cosolutes enhanced the hammerhead ribozyme activity by 2 to approximately 7-times, and larger-sized PEGs exhibited the greater activity. More importantly, the rate constant at 1 mM Mg(2+) and 37 degrees C increased about 1000-times and 500-times upon the addition of 20 wt% PEG8000 and PEG200, respectively. Additionally, the ribozyme still retained the cleavage activity even at 50 degrees C in the presence of PEG8000. Our results suggest that the ribozyme activity can be enhanced by the addition of the crowding reagent even at a low magnesium ion concentration and even at a high temperature.

Substrate Shape Preference ofEscherichia coliRibonuclease P Ribozyme and Holo Enzyme Using Bottom-Half Part-Shifting Variants of Pre-tRNA

We showed previously that the bacterial ribonuclease P (RNase P) ribozyme has substrate shape preference depending on the concentrations of catalytically important magnesium ions. The ribozyme discriminates a canonical cloverleaf precursor tRNA from a hairpin RNA with a CCA-tag sequence at low concentrations of magnesium ions. By detailed analysis of the shape preference using the bottom-half part-shifting variants of a tRNA precursor, we showed that the RNAs in a T-shape structure can be substrates for the ribozyme reactions even at low concentrations of magnesium ions, and that the RNA in a natural L-shape is the best substrate for both the ribozyme and the holo enzyme. The results also showed that the position of the bottom-half part did not affect the cleavage site selection of a substrate by the enzyme. Our results are the first kinetic evidence to show the importance of the bottom-half part of tRNA molecule, and our result also showed that the holo enzyme can discriminate substrate shape as well as the ribozyme at low concentrations of metal ions.

Magnesium Ion-dependent Activation of the RecA Protein Involves the C Terminus

Optimal conditions for RecA protein-mediated DNA strand exchange include 6-8 mm Mg(2+) in excess of that required to form complexes with ATP. We provide evidence that the free magnesium ion is required to mediate a conformational change in the RecA protein C terminus that activates RecA-mediated DNA strand exchange. In particular, a "closed" (low Mg(2+)) conformation of a RecA nucleoprotein filament restricts DNA pairing by incoming duplex DNA, although single-stranded overhangs at the ends of a duplex allow limited DNA pairing to occur. The addition of excess Mg(2+) results in an "open" conformation, which can promote efficient DNA pairing and strand exchange regardless of DNA end structure. The removal of 17 amino acid residues at the Escherichia coli RecA C terminus eliminates a measurable requirement for excess Mg(2+) and permits efficient DNA pairing and exchange similar to that seen with the wild-type protein at high Mg(2+) levels. Thus, the RecA C terminus imposes the need for the high magnesium ion concentrations requisite in RecA reactions in vitro. We propose that the C terminus acts as a regulatory switch, modulating the access of double-stranded DNA to the presynaptic filament and thereby inhibiting homologous DNA pairing and strand exchange at low magnesium ion concentrations.

Novel spermine–Amino acid conjugates and basic tripeptides enhance cleavage of the hairpin ribozyme at low magnesium ion concentration

Combinations of the polyamine spermine and magnesium ions synergize to dramatically enhance cleavage of the hairpin ribozyme. Certain synthetic basic tripeptides stimulate hairpin cleavage significantly at limiting magnesium ion concentration, notably the tripeptide of l-diaminobutyric acid (Dab). Of a range of novel synthetic spermine–amino acid conjugates, l-Dab-spermine (but not d-Dab nor other amino acid conjugates) was more effective than spermine itself.

Solution structure of HI0257, a bacterial ribosome binding protein.

A novel bacterial ribosome binding protein, protein Y (also known as YfiA), was recently shown to reside at the 30S/50S subunit interface and to stabilize the ribosomal 70S complex against dissociation at low magnesium ion concentrations. We report here the three-dimensional NMR structure in solution of a homologue from Haemophilus influenzae, HI0257, that has 64% sequence identity to protein Y. The 107 residue protein has a beta-alpha-beta-beta-beta-alpha folding topology with two parallel alpha-helices packed against the same side of a four-stranded beta-sheet. The closest structural relatives are proteins with the double-stranded RNA-binding domain (dsRBD) motif although there is little (<10%) sequence homology. The most immediate differences between the dsRBD and HI0257 structures are that (1) HI0257 has a larger beta-sheet motif with an extra beta-strand at the N-terminus, (2) the helices are parallel in HI0257 but at an angle of about 30 degrees to each other in the dsRBD, and (3) HI0257 lacks the extended loop commonly seen between the first and second beta-strands of the dsRBD. Further, an analysis of the surface electrostatic potential in HI0257 and the dsRBD family reveals significant differences in the location of contiguous positively (and negatively) charged regions. The structural data, in combination with sequence analysis of HI0257 and its homologues, suggest that the most likely mode of RNA recognition for HI0257 may be distinct from that of the dsRBD family of proteins.

Low [Mg(2+)](o) induces contraction of cerebral arteries: roles of tyrosine and mitogen-activated protein kinases.

The present study was designed to investigate the mechanism of action of low extracellular magnesium ion concentration ([Mg(2+)](o)) on isolated canine basilar arteries and single cerebral vascular smooth muscle cells from these arteries. Low-[Mg(2+)](o) medium (0-0.6 mM) produces endothelium-independent contractions in isolated canine basilar arteries in a concentration-dependent manner; the lower the concentration of [Mg(2+)](o), the stronger the contractions. The low-[Mg(2+)](o) medium-induced contractions are significantly attenuated by pretreatment of the arteries with low concentrations of either SB-203580, U-0126, PD-98059, genistein, or an Src homology 2 (SH2) domain inhibitor peptide. IC(50) levels obtained for these five antagonists are consistent with reported inhibitor constant (K(i)) values for these tyrosine kinase and mitogen-activated protein kinase (MAPK) antagonists. Low-[Mg(2+)](o) medium (0-0.6 mM) produces transient intracellular calcium ion concentration ([Ca(2+)](i)) peaks followed by a slow, sustained, and elevated plateau of [Ca(2+)](i) in primary single smooth muscle cells from canine basilar arteries. Low-[Mg(2+)](o) medium induces rapid and stable increases in [Ca(2+)](i); these increases are inhibited markedly in the presence of either SB-203580, U-0126, PD-98059, genistein or a SH2 domain inhibitor peptide. Several specific antagonists of known endogenously formed vasoconstrictors do not inhibit or attenuate either the low-[Mg(2+)](o)-induced contractions or the elevation of [Ca(2+)](i). The present study suggests that activation of several cellular signaling pathways, such as protein tyrosine kinases (including the Src family) and MAPK, appears to play important roles in low-[Mg(2+)](o)-induced contractions and the elevation of [Ca(2+)](i) in smooth muscle cells from canine basilar arteries.

Cytotoxicity by stored human breast-milk: possible contribution of complement system.

Human milk stored over some period in vitro possesses certain cytotoxic properties, which require further studies. Cytolysis induced by stored human milk has now been further characterized, using rabbit red blood cells as targets, to determine the contribution of other components, particularly the complement system. Cytolysis was found to be temperature dependent, greatly enhanced by low concentrations of magnesium and calcium ions, but inhibited by moderate to excessive amounts of calcium ions, and by heating at 56 degrees C.

Global structure of four-way RNA junctions studied using fluorescence resonance energy transfer.

Four-way helical junctions are found widely in natural RNA species. In this study, we have studied the conformation of two junctions by fluorescence resonance energy transfer. We show that the junctions are folded by pairwise coaxial helical stacking, forming one predominant stacking conformer in both examples studied. At low magnesium ion concentrations, the helical axes of both junctions are approximately perpendicular. One junction undergoes a rotation into a distorted antiparallel structure induced by the binding of a single magnesium ion. By contrast, the axes of the four-way junction of the U1 snRNA remain approximately perpendicular under all conditions examined, and we have determined the stacking conformer adopted.

Allosteric modulation of pyrophosphatase activity of rat osseous plate alkaline phosphatase by magnesium ions

Pyrophosphatase activity of rat osseous plate alkaline phosphatase was studied at different concentrations of calcium and magnesium ions, with the aim of characterizing the modulation of enzyme activity by these metals. In the absence of metal ions, the enzyme hydrolysed pyrophosphate following “Michaelian” kinetics with a specific activity of 36.7 U/mg and K 0.5=88 μM. In the presence of low concentrations (0.1 mM) of magnesium (or calcium) ions, the enzyme also exhibited “Michaelian” kinetics for the hydrolysis of pyrophosphate, but a significant increase in specific activity (123 U/mg) was observed. K m values remained almost unchanged. Quite different behavior occurred in the presence of 2 mM magnesium (or calcium) ions. In addition to low-affinity sites ( K 0.5=40 and 90 μM, for magnesium and calcium, respectively), high-affinity sites were also observed with K 0.5 values 100-fold lower. The high-affinity sites observed in the presence of calcium ions represented about 10% of those observed for magnesium ions. This was correlated with the fact that only magnesium ions triggered conformational changes yielding a fully active enzyme. These results suggested that the enzyme could hydrolyse pyrophosphate, even at physiological concentrations (4 μM), since magnesium concentrations are high enough to trigger conformational changes increasing the enzyme activity. A model, suggesting the involvement of magnesium ions in the hydrolysis of pyrophosphate by rat osseous plate alkaline phosphatase is proposed.