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Riboswitches are cis-acting regulatory RNA biosensors that rival the efficiency of those found in proteins. At the heart of their regulatory function is the formation of a highly specific aptamer-ligand complex. Understanding how these RNAs recognize the ligand to regulate gene expression at physiological concentrations of Mg2+ ions and ligand is critical given their broad impact on bacterial gene expression and their potential as antibiotic targets. In this work, we used single-molecule FRET and biochemical techniques to demonstrate that Mg2+ ions act as fine-tuning elements of the amino acid-sensing lysC aptamer's ligand-free structure in the mesophile Bacillus subtilis. Mg2+ interactions with the aptamer produce encounter complexes with strikingly different sensitivities to the ligand in different, yet equally accessible, physiological ionic conditions. Our results demonstrate that the aptamer adapts its structure and folding landscape on a Mg2+-tunable scale to efficiently respond to changes in intracellular lysine of more than two orders of magnitude. The remarkable tunability of the lysC aptamer by sub-millimolar variations in the physiological concentration of Mg2+ ions suggests that some single-aptamer riboswitches have exploited the coupling of cellular levels of ligand and divalent metal ions to tightly control gene expression., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Divalent metal ions, usually Mg2+, are required for both DNA synthesis and proofreading functions by DNA polymerases (DNA Pol). Although used as a non-reactive cofactor substitute for binding and crystallographic studies, Ca2+ supports DNA polymerization by only one DNA Pol, Dpo4. Here, we explore whether Ca2+-driven catalysis might apply to high-fidelity (HiFi) family B DNA Pols. The consequences of replacing Mg2+ by Ca2+ on base pairing at the polymerase active site as well as the editing of terminal nucleotides at the exonuclease active site of the archaeal Pyrococcus abyssi DNA Pol (PabPolB) are characterized and compared to other (families B, A, Y, X, D) DNA Pols. Based on primer extension assays, steady-state kinetics and ion-chased experiments, we demonstrate that Ca2+ (and other metal ions) activates DNA synthesis by PabPolB. While showing a slower rate of phosphodiester bond formation, nucleotide selectivity is improved over that of Mg2+. Further mechanistic studies show that the affinities for primer/template are higher in the presence of Ca2+ and reinforced by a correct incoming nucleotide. Conversely, no exonuclease degradation of the terminal nucleotides occurs with Ca2+. Evolutionary and mechanistic insights among DNA Pols are thus discussed., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Study Objectives: The correlated activation of pre- and postsynaptic neurons is essential for the NMDA receptor-mediated Ca2+ influx by removing Mg2+ from block site and NMDA receptors have been implicated in phase resetting of circadian clocks. So we assessed rest:activity rhythms in Mg2+ block defective animals., Methods: Using Drosophila locomotor monitoring system, we checked circadian rest:activity rhythms of different mutants under constant darkness (DD) and light:dark (LD) conditions. We recorded NMDA receptor-mediated currents or Ca2+ increase in neurons using patch-clamp and Ca2+ imaging techniques., Results: We found that Mg2+ block defective mutant flies were completely arrhythmic under DD. To further understand the role of Mg2+ block in daily circadian rest:activity, we observed the mutant files under LD cycles, and we found severely reduced morning anticipation and advanced evening peak compared to control flies. We also used tissue-specific expression of Mg2+ block defective NMDA receptors and demonstrated pigment-dispersing factor receptor (PDFR)-expressing circadian neurons were implicated in mediating the circadian rest:activity deficits. Endogenous functional NMDA receptors are expressed in most Drosophila neurons, including in a subgroup of dorsal neurons (DN1s). Subsequently, we determined that the uncorrelated extra Ca2+ influx may act in part through Ca2+/Calmodulin (CaM)-stimulated PDE1c pathway leading to morning behavior phenotypes., Conclusions: These results demonstrate that Mg2+ block of NMDA receptors at resting potential is essential for the daily circadian rest:activity rhythms and we propose that Mg2+ block functions to suppress CaM-stimulated PDE1c activation at resting potential, thus regulating Ca2+ and cyclic AMP oscillations in circadian and sleep circuits., (© Sleep Research Society 2017. Published by Oxford University Press on behalf of the Sleep Research Society. All rights reserved. For permissions, please e-mail journals.permissions@oup.com.)

Small RNA silencing is mediated by the effector RNA-induced silencing complex (RISC) that consists of an Argonaute protein (AGOs 1-4 in humans). A fundamental step during RISC assembly involves the separation of two strands of a small RNA duplex, whereby only the guide strand is retained to form the mature RISC, a process not well understood. Despite the widely accepted view that 'slicer-dependent unwinding' via passenger-strand cleavage is a prerequisite for the assembly of a highly complementary siRNA into the AGO2-RISC, here we show by careful re-examination that 'slicer-independent unwinding' plays a more significant role in human RISC maturation than previously appreciated, not only for a miRNA duplex, but, unexpectedly, for a highly complementary siRNA as well. We discovered that 'slicer-dependency' for the unwinding was affected primarily by certain parameters such as temperature and Mg(2+). We further validate these observations in non-slicer AGOs (1, 3 and 4) that can be programmed with siRNAs at the physiological temperature of humans, suggesting that slicer-independent mechanism is likely a common feature of human AGOs. Our results now clearly explain why both miRNA and siRNA are found in all four human AGOs, which is in striking contrast to the strict small-RNA sorting system in Drosophila., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Toxin-antitoxin (TA) systems play important roles in bacterial physiology, such as multidrug tolerance, biofilm formation, and arrest of cellular growth under stress conditions. To develop novel antimicrobial agents against tuberculosis, we focused on VapBC systems, which encompass more than half of TA systems in Mycobacterium tuberculosis. Here, we report that theMycobacterium tuberculosis VapC30 toxin regulates cellular growth through both magnesium and manganese ion-dependent ribonuclease activity and is inhibited by the cognate VapB30 antitoxin. We also determined the 2.7-Å resolution crystal structure of the M. tuberculosis VapBC30 complex, which revealed a novel process of inactivation of the VapC30 toxin via swapped blocking by the VapB30 antitoxin. Our study on M. tuberculosis VapBC30 leads us to design two kinds of VapB30 and VapC30-based novel peptides which successfully disrupt the toxin-antitoxin complex and thus activate the ribonuclease activity of the VapC30 toxin. Our discovery herein possibly paves the way to treat tuberculosis for next generation., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Context: ATP-sensitive potassium (KATP) channels regulate insulin secretion by coupling glucose metabolism to β-cell membrane potential. Gain-of-function mutations in the sulfonylurea receptor 1 (SUR1) or Kir6.2 channel subunit underlie neonatal diabetes., Objective: The objective of the study was to determine the mechanisms by which two SUR1 mutations, E208K and V324M, associated with transient neonatal diabetes affect KATP channel function., Design: E208K or V324M mutant SUR1 was coexpressed with Kir6.2 in COS cells, and expression and gating properties of the resulting channels were assessed biochemically and electrophysiologically., Results: Both E208K and V324M augment channel response to MgADP stimulation without altering sensitivity to ATP4- or sulfonylureas. Surprisingly, whereas E208K causes only a small increase in MgADP response consistent with the mild transient diabetes phenotype, V324M causes a severe activating gating defect. Unlike E208K, V324M also impairs channel expression at the cell surface, which is expected to dampen its functional impact on β-cells. When either mutation was combined with a mutation in the second nucleotide binding domain of SUR1 previously shown to abolish Mg-nucleotide response, the activating effect of E208K and V324M was also abolished. Moreover, combination of E208K and V324M results in channels with Mg-nucleotide sensitivity greater than that seen in individual mutations alone., Conclusion: The results demonstrate that E208K and V324M, located in distinct domains of SUR1, enhance transduction of Mg-nucleotide stimulation from the SUR1 nucleotide binding folds to Kir6.2. Furthermore, they suggest that diabetes severity is determined by interplay between effects of a mutation on channel expression and channel gating.

About 60% of adults in the United States do not consume the estimated average requirement for magnesium, but widespread pathological conditions attributed to magnesium deficiency have not been reported. Nevertheless, low magnesium status has been associated with numerous pathological conditions characterized as having a chronic inflammatory stress component. In humans, deficient magnesium intakes are mostly marginal to moderate (approximately 50% to <100% of the recommended dietary allowance). Animal experiments indicate that signs of marginal-to-moderate magnesium deficiency can be compensated or exacerbated by other factors influencing inflammatory and oxidative stress; recent studies suggest a similar happening in humans. This suggestion may have significance in obesity, which is characterized as having a chronic low-grade inflammation component and an increased incidence of a low magnesium status. Marginal-to-moderate magnesium deficiency through exacerbating chronic inflammatory stress may be contributing significantly to the occurrence of chronic diseases such as atherosclerosis, hypertension, osteoporosis, diabetes mellitus, and cancer.

Voltage-dependent inward-rectifying (K(in)) and outward-rectifying (K(out)) K(+) channels are capable of mediating K(+) fluxes across the plasma membrane. Previous studies on guard cells or heterologously expressed K(+) channels provided evidence for the requirement of ATP to maintain K(+) channel activity. Here, the nucleotide and Mg(2+) dependencies of time-dependent K(in) and K(out) channels from maize subsidiary cells were examined, showing that MgATP as well as MgADP function as channel activators. In addition to K(out) channels, these studies revealed the presence of another outward-rectifying channel type (MgC) in the plasma membrane that however gates in a nucleotide-independent manner. MgC represents a new channel type distinguished from K(out) channels by fast activation kinetics, inhibition by elevated intracellular Mg(2+) concentration, permeability for K(+) as well as for Na(+) and insensitivity towards TEA(+). Similar observations made for guard cells from Zea mays and Vicia faba suggest a conserved regulation of channel-mediated K(+) and Na(+) transport in both cell types and species.

Although the symptoms of magnesium deficiency are well documented in plants, the primary physiological effects of low Mg availability remain largely unknown. This paper describes the physiological responses of Mg starvation in Arabidopsis thaliana. Growth characteristics, Mg and sugar concentration, and photochemical performance were measured at regular intervals during the induction of Mg deficiency. These data show that Mg deficiency increased the sugar concentration and altered sucrose export from young source leaves before any noticeable effect on photosynthetic activity was seen. The decline in photosynthetic activity might be elicited by increased leaf sugar concentrations. Transcript levels of Cab2 (encoding a chlorophyll a/b protein) were lower in Mg-deficient plants before any obvious decrease in the chlorophyll concentration. These transcriptional data suggest that the reduction of chlorophyll is a response to sugar levels, rather than a lack of Mg atoms for chelating chlorophyll.

AtMHX is an Arabidopsis tonoplast transporter that can exchange protons with Mg2+ and Zn2+ ions. This transporter, which may play a role in ion homeostasis of plants, is encoded by a single gene in Arabidopsis. The molecular mechanisms that regulate the expression of this transporter are practically unknown. While AtMHX transcript can be easily visualized, expression of the corresponding protein is apparently low. To understand whether AtMHX expression is repressed at the translational level, the 5' untranslated region (5' UTR) of this gene was fused to reporter genes. In vitro analyses showed that the 5' UTR of AtMHX can repress the translation of downstream coding sequences. The major cause of the repression was efficient initiation at an upstream open-reading-frame (uORF) included in the 5' UTR. Although the sequence context of the upstream AUG (uAUG) codon was highly unfavourable, it was recognized by over 90% of the scanning ribosomes both in vitro and in vivo. The inhibitory effect of the uORF was mediated by imposing the need for reinitiation and not by ribosome stalling, as the inhibition was not dependent on the amino-acid sequence of the uORF peptide. The efficiency of reinitiation was low. The in vivo studies, carried out with transiently transformed tobacco plants, indicated that alternations in the Mg2+ or Zn2+ levels did not affect the rate of translation. These data suggest that AtMHX expression is repressed by the 5' UTR of its gene.

Available evidence suggests that Mg2+ ions are involved in reactions catalyzed by hammerhead ribozymes. However, the activity in the presence of exclusively monovalent ions led us to question whether divalent metal ions really function as catalysts when they are present. We investigated ribozyme activity in the presence of high levels of Mg2+ ions and the effects of Li+ ions in promoting ribozyme activity. We found that catalytic activity increased linearly with increasing concentrations of Mg2+ ions and did not reach a plateau value even at 1 M Mg2+ ions. Furthermore, this dependence on Mg2+ ions was observed in the presence of a high concentration of Li+ ions. These results indicate that the Mg2+ ion is a very effective cofactor but that the affinity of the ribozyme for a specific Mg2+ ion is very low. Moreover, cleavage by the ribozyme in the presence of both Li+ and Mg2+ ions was more effective than expected, suggesting the existence of a new reaction pathway-a cooperative pathway-in the presence of these multiple ions, and the possibility that a Mg2+ ion with weak affinity for the ribozyme is likely to function in structural support and/or act as a catalyst.

Theoretical considerations based on nutrition experiments suggest that nutrient ratios of terrestrial plants are similar to the Redfield ratio found in marine phytoplankton. Laboratory experiments have shown that seedlings of many different plant species have similar nutrient concentration ratios when supplied with nutrients at free access. However, at free access, nutrients are likely to be taken up in amounts in excess of a plant's requirements for growth. In further experiments, therefore, the supply rate of each nutrient was reduced so that excessive uptake did not occur. Again, similar nutrient ratios were found among the plant species tested, although the ratios differed from those found in plants given free access to nutrients. Based on the law of the minimum, we suggest that optimum nutrient ratios be defined as the ratios found in plants when all nutrients are limiting growth simultaneously. The literature on nutrient concentrations was surveyed to investigate nutrient ratios in terrestrial ecosystems. Nutrients taken into consideration were nitrogen, phosphorus, potassium, calcium and magnesium. Based on the assumption that nitrogen is either the limiting nutrient or, when not limiting, is taken up only in small excess amounts, we calculated nutrient ratios from published data. The calculated ratios corresponded closely to the ratios determined in laboratory and field experiments.

Background: Cyclosporin A (CsA) nephrotoxicity has been attributed primarily to renal haemodynamic alterations caused by afferent arteriolar vasoconstriction. However, CsA nephropathy is also characterized by CsA-induced pre-glomerular disturbances and interstitial injury that may occur independently of haemodynamic changes. Given the high lipophilic activity of CsA, we hypothesized that direct tubular injury is likely to contribute to nephrotoxicity., Methods: To investigate tubular toxicity of CsA, increasing concentrations of CsA (1, 2.5, 10, 25, 50 and 100 micro g/ml) and its vehicle (cremophor) were added to isolated rat proximal tubules (PT). Cell injury was assessed by lactate dehydrogenase (LDH) release. The role of Ca(2+) ions in tubular toxicity and the effect of calcium channel blockers on CsA toxicity were evaluated by measuring intracellular calcium using the fluorescent dye Fura-2 AM. The role of Mg(2+) ions was assessed using high extracellular Mg(2+) medium (2 mM)., Results: Whereas cremophor alone was not toxic to PT, CsA caused PT injury but only at the highest concentration (100 micro g/ml). After 90 min incubation, LDH was 22.5% in control PT and 41.9% in PT treated with 100 micro g/ml CsA (P < 0.001, n = 11). There was a transient increase in intracellular calcium ([Ca(2+)](i)) after CsA administration. A low calcium medium (100 nM) prevented CsA injury to renal tubules. However, verapamil, but not nifedipine, enhanced cell damage. Only nifedipine completely prevented [Ca(2+)](i) increases following CsA. Finally, a high Mg(2+) medium attenuated CsA-induced injury., Conclusion: We found that high CsA concentrations caused Ca(2+)- and Mg(2+)-dependent PT injury. Thus, low extracellular Ca(2+) and high Mg(2+) media attenuated CsA-induced tubular injury. Verapamil, but not nifedipine, enhanced CsA tubular toxicity. Therefore, CsA-induced tubular injury may contribute to CsA nephrotoxicity independently of haemodynamic disturbances.

During epididymal transit, spermatozoa acquire selected proteins secreted by epithelial cells. We recently showed that P25b, a protein with predictive properties for bull fertility, is transferred from prostasome-like particles present in the cauda epididymal fluid (PLPCd) to the sperm surface. To further characterize the interactions between PLPCd and epididymal spermatozoa, PLPCd were prepared by ultracentrifugation of bull epididymal fluid, then surface-exposed proteins were biotinylated and coincubated in different conditions with caput epididymal spermatozoa. Western blot analysis revealed that only selected proteins are transferred from PLPCd to spermatozoa. MALDI-TOF analysis revealed that these transferred proteins are closely related. The pattern of distribution of the PLPCd transferred varied from one sperm cell to the other, with a bias toward the acrosomal cap. This transfer appeared to be temperature sensitive, being more efficient at 32-37 degrees C than at 22 degrees C. Transfer of PLPCd proteins to spermatozoa was also pH dependant, the optimal pH for transfer being 6.0-6.5. The effect of divalent cations on PLPCd protein transfer to caput spermatozoa was investigated. Whereas Mg(2+) and Ca(2+) have no effect on the amount of proteins remaining associated with spermatozoa following coincubation, Zn(2+) had a beneficial effect. These results are discussed with regard to the function of PLPCd in epididymal sperm maturation.

We have examined binding of the CREB B-ZIP protein domain to double-stranded DNA containing a consensus CRE sequence (5'-TGACGTCA-3'), the related PAR, C/EBP and AP-1 sequences and the unrelated SP1 sequence. DNA binding was assayed in the presence or absence of MgCl2 and/or KCl using two methods: circular dichroism (CD) spectroscopy and electrophoretic mobility shift assay (EMSA). The CD assay allows us to measure equilibrium binding in solution. Thermal denaturation in 150 mM KCl indicates that the CREB B-ZIP domain binds all the DNA sequences, with highest affinity for the CRE site, followed by the PAR (5'-TAACGTTA-3'), C/EBP (5'-TTGCGCAA-3') and AP-1 (5'-TGAGTCA-3') sites. The addition of 10 mM MgCl2 diminished DNA binding to the CRE and PAR DNA sequences and abolished binding to the C/EBP and AP-1 DNA sequences, resulting in more sequence-specific DNA binding. Using 'standard' EMSA conditions (0.25x TBE), CREB bound all the DNA sequences examined. The CREB-CRE complex had an apparent Kd of approximately 300 pM, PAR of approximately 1 nM, C/EBP and AP-1 of approximately 3 nM and SP1 of approximately 30 nM. The addition of 10 mM MgCl2 to the polyacrylamide gel dramatically altered sequence-specific DNA binding. CREB binding affinity for CRE DNA decreased 3-fold, but binding to the other DNA sequences decreased >1000-fold. In the EMSA, addition of 150 mM KCl to the gels had an effect similar to MgCl2. The magnesium concentration needed to prevent non-specific electrostatic interactions between CREB and DNA in solution is in the physiological range and thus changes in magnesium concentration may be a cellular signal that regulates gene expression.

Triplex-forming oligonucleotides (TFOs) are good candidates to be used as site-specific DNA-binding agents. Two obstacles encountered with TFOs are susceptibility to nuclease activity and a requirement for magnesium for triplex formation. Morpholino oligonucleotides were shown in one study to form triplexes in the absence of magnesium. In the current study, we have compared phosphodiester and morpholino oligonucleotides targeting a homopurine-homopyrimidine region in the human HER2/neu promoter. Using gel mobility shift analysis, our data demonstrate that triplex formation by phosphodiester oligonucleotides at the HER-2/neu promoter target is possible with pyrimidine-parallel, purine-antiparallel and mixed sequence (GT)-antiparallel motifs. Only the pyrimidine-parallel motif morpholino TFO was capable of efficient triple helix formation, which required low pH. Triplex formation with the morpholino TFO was efficient in low or no magnesium. The pyrimidine motif TFOs with either a phosphodiester or morpholino backbone were able to form triple helices in the presence of potassium ions, but required low pH. We have rationalized the experimental observations with detailed molecular modeling studies. These data demonstrate the potential for the development of TFOs based on the morpholino backbone modification and demonstrate that the pyrimidine motif is the preferred motif for triple helix formation by morpholino oligonucleotides.

A stable conformer of Escherichia coli tRNA(Glu), obtained in the absence of Mg(2+), is inactive in the aminoacylation reaction. Probing it with diethylpyrocarbonate, dimethyl sulfate and ribonuclease V1 revealed that it has a hairpin structure with two internal loops; the helical segments at both extremities have the same structure as the acceptor stem and the anticodon arm of the native conformer of tRNA(Glu)and the middle helix is formed of nucleotides from the D-loop (G15-C20:2) and parts of the T-loop and stem (G51-C56), with G19 bulging out. This model is consistent with other known properties of this inactive conformer, including its capacity to dimerize. Therefore, this tRNA requires magnesium to acquire a conformation that can be aminoacylated, as others require a post-transcriptional modification to reach this active conformation.

Two-thirds of total body magnesium content is located in the skeleton. Recently, there have been reports that high dietary magnesium intakes are associated with higher bone mineral density. The effect of magnesium supplementation on bone turnover has recently been investigated in young adults. However, the findings from these studies are conflicting. More studies are necessary to better elucidate the role of magnesium in bone health.

IgG purified from sera of several patients with systemic lupus erythematosus and hepatitis B are shown to present RNA hydrolyzing activities that are different from the weak RNase A-type activities found in the sera of healthy donors. Further investigation brings evidence for two intrinsic activities, one observed in low salt conditions and another specifically stimulated by Mg2+ions and distinguishable from human sera RNases. Cleavage of RNA substrates by the latter activity is not sequence-specific but sensitive to both subtle conformational and/or drastic folding changes, as evidenced by comparative analysis of couples of structurally well-studied RNA substrates. These include yeast tRNAAsp and its in vitro transcript and human mitochondrial tRNALys-derived in vitro transcripts. The discovery of catalytic antibodies with RNase activities is a first step towards creation of a new generation of tools for the investigation of RNA structure.

Magnesium is one of the most abundant ions present in living cells and its plasma concentration is remarkably constant in healthy subjects. Plasma and intracellular magnesium concentrations are tightly regulated by several factors. Among them, insulin seems to be one of the most important. In fact, in vitro and in vivo studies have demonstrated that insulin may modulate the shift of magnesium from extracellular to intracellular space. Intracellular magnesium concentration has also been shown to be effective on modulating insulin action (mainly oxidative glucose metabolism), offset calcium-related excitation-contraction coupling, and decrease smooth cell responsiveness to depolarizing stimuli, by stimulating Ca2+-dependent K+ channels. A poor intracellular magnesium concentration, as found in non-insulin-dependent diabetes mellitus (NIDDM) and in hypertensive (HP) patients, may result in a defective tyrosine-kinase activity at the insulin receptor level and exaggerated intracellular calcium concentration. Both events are responsible for the impairment in insulin action and a worsening of insulin resistance in non-insulin-dependent diabetic and hypertensive patients. By contrast, in NIDDM patients daily magnesium administration, restoring a more appropriate intracellular magnesium concentration, contributes to improve insulin-mediated glucose uptake. Similarly, in HP patients magnesium administration may be useful in decreasing arterial blood pressure and improving insulin-mediated glucose uptake. The benefits deriving from daily magnesium supplementation in NIDDM and HP patients are further supported by epidemiological studies showing that high daily magnesium intake to be predictive of a lower incidence of NIDDM and HP. In conclusion, a growing body of studies suggest that intracellular magnesium may play a key role on modulating insulin-mediated glucose uptake and vascular tone. We further suggest that a reduced intracellular magnesium concentration might be the missing link helping to explain the epidemiological association between NIDDM and hypertension.

Guinea pig peritoneal eosinophils stimulated by platelet-activating factor (PAF), leukotriene B4 (LTB4), and human recombinant C5a (C5a) undergo a rapid concentration-dependent and partially reversible homotypic aggregation as assessed by changes in light transmission. The phorbol ester phorbol myristate acetate similarly induces a concentration-dependent aggregation, which is, however, slower in onset, takes longer to reach maximal aggregation, and is irreversible. In addition, we confirmed, using light microscopy, that these agonist-induced changes in light transmission do indeed represent true homotypic aggregation. We further characterized the aggregation response and showed that there is homologous but little heterologous desensitization when PAF and LTB4 are used as stimuli. A requirement for both Ca2+ and Mg2+ for full manifestation of agonist-induced aggregation was observed. LTB4- and PAF-induced superoxide anion generation is enhanced by the diacyglycerol kinase inhibitor R59022, whereas aggregation induced by LTB4, but not PAF, is augmented. Lastly, we show that eosinophil aggregation is partially dependent on the adhesion glycoprotein CD18. In summary, therefore, we believe that eosinophil aggregation provides a useful and reliable measure of eosinophil activation.

To study the interaction and the role of the metal ion in the reaction catalyzed by Escherichia coli ribonuclease HI (E. coli RNase HI), substrate analogues containing a phosphorothioate linkage or 2'-modified nucleosides at the cleavage site were used. In the presence of Mg2+, Mn2+, Co2+, Zn2+, or Cd2+, the phosphorothioate linkage with the RP-configuration was cleaved, while the SP-isomer was not. Kinetic studies showed that Mn2+ and Cd2+ facilitated the cleavage of the phosphorothioate to only a small extent, which indicated the absence of an interaction between the metal ion and this phosphate residue. The interaction of the metal ion with the 2'-functional group was analyzed by Mg(2+)-titration experiments using the -OH, -NH2, and -F substrates. From Hill plots, it was found that the KMg values were almost the same. These results are evidence of an interaction between Mg2+ and the 2'-functional group by the formation of an outer-sphere complex with a water molecule. The Hill coefficient of 1.0 for the -OH substrate indicated that a single Mg2+ ion is required for the catalysis.

Magnesium (Mg) is the second most abundant intracellular cation and is a cofactor in more than 300 enzymatic reactions involving energy metabolism and protein and nucleic acid synthesis. Ionized Mg is the physiologically active form of the element. Protein-bound and chelated Mg buffer the ionized pool. Approximately half the total Mg in the body is present intracellularly in soft tissue, and the other half is present in bone. Less than 1% of the total body Mg is present in blood. However, the majority of our clinical laboratory information comes from the determination of total Mg in serum. Currently, the clinical laboratory evaluation of Mg status is limited primarily to the total serum Mg concentration and a 24-hour urinary excretion. Instrumentation to determine ionized Mg in serum (ion-selective electrode) and in soft tissue (nuclear magnetic resonance spectroscopy) should be available in the near future. Magnesium may be a factor in the treatment of acute myocardial infarction and the rate of atherosclerosis. Chronic changes of Mg status, that may be latent, are poorly understood and require a better knowledge of ionized Mg metabolism.

Acute withdrawal of external Mg2+ increased basal tone of rat isolated aorta incubated in the presence of Ca2+. Above normal levels of Mg2+ (1-4 mM) inhibited basal tone while much higher levels of the divalent cation (64-256 nM) evoked contractile responses regardless of the presence of Ca2+. Contractile responses to noradrenaline (1 microM) and KCl (80 mM) were inhibited by addition of cumulative concentrations of Mg2+. Acetylcholine-induced contractions in the presence of physiological concentrations of Mg2+ (1 mM) decreased gradually to the basal tone, but a sustained contraction was observed in the absence of this ion. In Ca(2+)-free medium, acetylcholine-induced phasic responses indicate the existence of an acetylcholine-sensitive Ca2+ store. KCl induced contraction only in Krebs solution, although a small residual contraction could be observed in Ca(2+)-free medium in some experiments. Mg(2+)-depletion in the extracellular medium increased contractile responses induced by acetylcholine and KCl in Ca(2+)-free medium. These results suggest that extracellular Mg2+ modulates basal tone, Ca2+ channels and responsiveness to various agents in the absence of Ca2+.

Using a new ion selective electrode (ISE) to measure ionized magnesium (IMg2+), we compared levels of IMg2+ with total magnesium (TMg) and serum ionized calcium levels (ICa2+) in umbilical arterial and venous serum of 13 parturients who developed transient hypertension in labor with levels in 38 who remained normotensive. Ionized, but not total magnesium levels were significantly lower in the umbilical veins of the hypertensive compared to the normotensive subjects (0.46 +/- 0.01 v 0.51 +/- 0.01 mmol/L), and these were at least 15% lower than maternal levels found in nonpregnant women (0.60 +/- 0.005 mmol/L, P < .001). If umbilical vein samples reflect maternal levels as well, then these results suggest that lower IMg2+ levels may play a role in the development of transient hypertension in labor, and the potential utility of an ion selective electrode for ionized magnesium in the diagnosis of subtle magnesium deficient states.

The stimulating activity of several preparations isolated from a membrane proteoglycan of a nonencapsulated smooth strain of Klebsiella pneumoniae (Kp-MPG) on the oxidative burst of human blood monocytes was assessed by luminol-enhanced chemiluminescence (CL). Five Kp derivatives were studied: a 34-kd acylpoly(1,3)galactoside (APG), obtained by drastic alkaline hydrolysis and purified by chromatography; an APG preparation subjected to acid hydrolysis that removed the core part and all fatty acids, leaving intact the galactose chain of APG (GC-APG); an APG preparation subjected to mild oxidation (ox APG); a preparation obtained by mild alkaline hydrolysis of Kp-MPG, containing additional ester-linked C14 and C16 fatty acids bound to the APG molecule (EFA-APG); and a polymer of the latter compound, APG pol. EFA-APG directly stimulated monocyte CL, whereas Kp-MPG, APG pol, and the whole bacterial cells had little or no activity. APG itself and ox APG induced a weaker response than EFA-APG. Polymyxin B sulfate completely inhibited the CL response to bacterial lipopolysaccharide (LPS) but not to EFA-APG. The stimulating action of EFA-APG on blood monocytes was dependent on the extracellular levels of both calcium and magnesium. Preincubation of monocytes with monoclonal antibody anti-Mac-1 directed against CD11b, the alpha chain of complement receptor type 3 (CR3; CD11b/CD18), strongly inhibited CL activation by EFA-APG and to a lesser extent CL activation by unopsonized zymosan and rough LPS. Altogether, these findings provide indirect evidence for the contribution of the CD11b/CD18 integrin in the functional interaction of EFA-APG with monocyte membranes. They demonstrate the role of fatty acids in the triggering of monocyte oxidative burst, while the polysaccharide chain itself does not contribute to induction of the CL response in this model. In keeping with the effects of EFA-APG and APG, we show that the monocyte CL response was triggered by bacterial LPS from the rough strain of Salmonella minnesota Re 595 and its lipid A, but not by LPS from smooth strains, again suggesting a critical role for the lipid moiety.

Human neutrophils were activated with soluble stimuli, formyl-methionyl-leucyl-phenylalanine (fMLP) or ionophore A23187, and with opsonized particles, zymosan or Streptococcus pneumoniae bacteria. Monoclonal antibodies and flow cytometry were used to assess the expression of Fc-gamma receptors (FcRI, FcRII, FcRIII) and complement receptors (CR1, CR3). The role of extracellular calcium and magnesium in the modulation of receptor expression was also examined. The low-level expression of FcRI was not affected by any activator tested. fMLP and A23187 did not alter the expression of FcRII, whereas a significant, Ca(2+)- and Mg(2+)-independent down-modulation was observed upon activation with opsonized particles. All activators clearly decreased the surface expression of FcRIII in the presence of Ca2+ and Mg2+, probably as a consequence of shedding of the phosphatidylinositol-glycan-anchored receptor protein. The removal of calcium and magnesium blocked the shedding of FcRIII caused by soluble stimuli, whereas it retarded but did not abolish the fall in FcRIII expression when cells were incubated with opsonized particles. This fall was likely due to internalization of the receptor molecules while the shedding was blocked. A rapid increase in CR1 and CR3 expression was seen upon activation with soluble stimuli. The change in CR1 expression was independent of extracellular Ca2+ and Mg2+. The increase in CR3 number required an influx of divalent cations. No total up-modulation of complement receptors occurred when neutrophils were activated with opsonized particles. However, the kinetic analysis revealed a temporary up-modulation that was followed by a down-modulation. The results indicate that the expression of both Fc-gamma and complement receptors on human neutrophils is changed upon activation and that the up- and down-modulation of these receptors depends on the nature of activator. We also suggest that in neutrophils the FcRIII down-modulation is the result of both receptor shedding and internalization, while FcRII is down-modulated by receptor internalization.

Changes in cytoplasmic magnesium concentration in the millimolar range are known to evoke directionally opposite changes in the amplitude of cardiac L-type calcium current, but the effects of magnesium changes in the micromolar range are not known. This concentration range is particularly interesting for two reasons: open-channel block of some ion channels (eg, the inward rectifier) occurs with a micromolar Kd; and magnesium at such low levels may limit the availability of magnesium adenosine triphosphate (MgATP) and magnesium guanidine triphosphate (MgGTP) for a variety of enzymatic processes, including calcium channel phosphorylation. To investigate the effects of critical magnesium depletion and its sudden reversal, we used flash photolysis to liberate magnesium from intracellular DM-nitrophen in guinea pig heart cells. Under our conditions, intracellular [Mg2+] changed from 0.25 mumol/L before the flash to less than or equal to 200 mumols/L afterwards. Calcium channel currents carried by either calcium or barium consistently increased after flash, a directional change opposite to the reported effects of elevated [Mg2+] in the millimolar range. Our data do not yet enable us to understand the basis of the magnesium effect, but we are particularly interested in the possibility that the sudden appearance of magnesium enables calcium channel phosphorylation.

In the previous studies with endonucleases specific for single-stranded DNA, we have indicated that the nonhistone chromosomal protein HMG(1 + 2) prepared from pig thymus has an activity to unwind DNA partially at low protein-to-DNA weight ratios (Yoshida, M. & Shimura, K. (1984) J. Biochem. 95, 117-124). In the present work, we have pursued the unwinding reaction by HMG(1 + 2) by thermal melting temperature analysis of DNA, and by investigating the effect of Mg2+ on the reaction. The melting temperature of DNA in the presence of HMG(1 + 2) at low protein weight ratios decreased in 2 mM Tris-HCl, pH 7.8, whereas it increased at higher ratios. The depressions of melting temperature by HMG(1 + 2) at low ratios were not observed either in the system of 2 mM Tris-HCl, pH 7.8, containing EDTA or in the system containing samples treated in advance with EDTA. An addition of Mg2+ to the system reproduced the depression of melting temperature at low protein-to-DNA ratios as well as the increase at higher ratios. Analysis by Mg2+-equilibrated gel filtration revealed that HMG(1 + 2) is a Mg2+-binding protein. However, the depression of melting temperature at low protein-to-DNA ratios was not due to removal of Mg2+ from DNA by HMG(1 + 2). From these results, it is concluded that HMG(1 + 2) causes a partial DNA unwinding detectable by thermal melting temperature analysis of DNA, and that Mg2+ is necessary for the unwinding reaction.

We identified a Mg2+ dependent 5' exo-ribonuclease and an RNA ligase in cell-free extracts of Trypanosome brucei. The exo-ribonuclease in S100 or nuclear extracts, removes about 20 nts from the 5' end of SP6 derived capped as well as uncapped RNA and then stops. In contrast to the activity of the exo-ribonuclease on capped SP6 mini-exon transcripts, the exonuclease cannot degrade trypanosome-derived mini-exon transcripts or the mini-exon located at hsp 70 mRNAs. We therefore assume that the four secondary base modifications adjacent to the mini-exon cap, generated in vivo, confer resistance to the exo-ribonuclease. After exonuclease shortening of SP6 transcripts, an RNA ligase catalizes intramolecular ligation, generating a 3'-5' phosphodiester bond in a Mg2+ and ATP dependent reaction.

Human cyclin/PCNA (proliferating cell nuclear antigen) is structurally, functionally, and immunologically homologous to the calf thymus auxiliary protein for DNA polymerase delta. This auxiliary protein has been investigated as a stimulatory factor for the nuclear DNA polymerases from S. cerevisiae. Calf cyclin/PCNA enhances by more than ten-fold the ability of DNA polymerase III to replicate templates with high template/primer ratios, e.g. poly(dA).oligo(dT) (40:1). The degree of stimulation increases with the template/primer ratio. At a high template/primer ratio, i.e. low primer density, cyclin/PCNA greatly increases processive DNA synthesis by DNA polymerase III. At low template/primer ratios (e.g. poly(dA).oligo(dT) (2.5:1), where addition of cyclin/PCNA only minimally increases the processivity of DNA polymerase III, a several-fold stimulation of total DNA synthesis is still observed. This indicates that cyclin/PCNA may also increase productive binding of DNA polymerase III to the template-primer and stabilize the template-primer-polymerase complex. The activity of yeast DNA polymerases I and II is not affected by addition of cyclin/PCNA. These results strengthen the hypothesis that yeast DNA polymerase III is functionally analogous to the mammalian DNA polymerase delta.

A single class of high affinity, low capacity, specific binding sites for [3H]arginine8 vasopressin (AVP) has been characterized in a plasma membrane-enriched microsomal fraction of the rat liver. Specific binding was saturable, linear with protein concentration, reversible, and 40-65% of the total binding. Binding at 25 C achieved a plateau after 30 min of incubation, whereas at 4 C, equilibrium was reached more slowly, and the level of binding was reduced. The presence of magnesium (Mg2+) in the assay medium enhanced the affinity of specific binding, while calcium and higher levels of sodium and potassium decreased binding. Scatchard analysis of binding in the presence of Mg2+ (5 mM) revealed an apparent mean +/- SE equilibrium dissociation constant (Kd) of 0.29 +/- 0.08 nM, with a maximal site density (Bmax) of 150.4 +/- 25.0 fmol/mg; in contrast, the Kd was 1.93 +/- 0.33 nM and the Bmax was 113.4 +/- 40.0 fmol/mg in the absence of Mg2+. No significant differences in Kd and Bmax were observed among membrane fractions derived from spontaneously hypertensive rats, Wistar-Kyoto rats, Long-Evans rats, and Brattleboro rats. Plasma levels of AVP were similar in spontaneously hypertensive, Wistar-Kyoto, and Long-Evans rats, but AVP was not detectable in the plasma from DI rats. Competitive inhibition of specific [3H]AVP binding by unlabeled AVP and related peptides showed the following Ki values: AVP, 0.19 nM; LVP, 1.7 nM; oxytocin, 41.4 nM; desamino AVP, 0.38 nM; [1-(beta-mercapto-beta, beta-cyclopentamethylene propionic acid) 4-Val,8-D-Arg] VP2-(O-methyl)tyrosine]AVP, 1.8 nM; desglycinamide AVP, 2.2 microM. The neuropeptide metabolite of AVP [[pGlu4,Cyt6] AVP-(4-9)], angiotensin II, and other unrelated peptides did not displace [3H]AVP, demonstrating the specificity of AVP and its related biologically active peptides for this binding site. Moreover, the rank order of potency for displacement of [3H]AVP binding by these various peptides parallels their reported glycogenolytic activity in liver and/or their agonistic or antagonistic potency in vascular smooth muscle. Finally, the Mg2+-induced increase in the affinity of [3H]AVP for this liver binding site is similar to the reported effect of Mg2+ on the contractile responses of vascular smooth muscle to AVP (i.e. increased affinity). The results are consistent with the interpretation that the high affinity receptor site characterized in rat liver microsomes is of the V1 type.

Pseudomonas aeruginosa strain PA01, which is normally sensitive to 10 units/ml of polymyxin, was adapted in eight successive steps to be resistant to polymyxin at 6000 units/ml. The polymyxin-resistant variant was very sensitive to all other antibiotics with which it was challenged, including hydrophobic antibiotics such as erythromycin. This increased sensitivity implies that the acquired polymyxin resistance had disrupted the permeability barrier of the outer membrane. Changes in the outer membrane protein profile of PA01 were studied at each adaption step using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). At the third step (resistant to 80 units/ml) protein H1 was absent. At succeeding steps up to 6000 units/ml of polymyxin, protein H1 was still absent and the levels of OM proteins D, E, F (major porin) and G were all considerably reduced compared with the wild type. Overproduction of protein H1 by wild type P. aeruginosa under conditions of magnesium depletion has been proposed as a mechanism to explain polymyxin resistance. However, protein H1 remained absent from the outer membrane of the polymyxin-resistant variants even under magnesium depletion. Furthermore, protein H1 could not be induced by magnesium depletion when the resistant variant was repeatedly grown in concentrations of polymyxin as low as 10 units/ml yet, at this low concentration, the variant retained its resistance to 6000 units/ml. These observations make it unlikely that induction of protein H1 alone is a mechanism of polymyxin resistance in P. aeruginosa.

The effects of intra- and extravesicular calcium and magnesium ions on the hydrolysis of the phosphoenzyme (EP) intermediate formed in the reaction of Ca2+,Mg2+-dependent ATPase of the sarcoplasmic reticulum were investigated. The rate constants of EP hydrolysis were measured under conditions that allowed a single turnover of ATP hydrolysis to minimize the increase in calcium concentration inside the vesicles. The EP formed during a single turnover was hydrolyzed biphasically and could be resolved into fast- and slow-decomposing components. When free Mg2+ outside the vesicles was chelated by adding excess EDTA, EP could also be kinetically resolved into two components; EDTA-sensitive EP, which could be quickly decomposed by adding EDTA, and EDTA-insensitive EP, which could be prevented from decomposing by adding EDTA. The amount of EDTA-sensitive EP decreased rapidly during the initial phase of the reaction, while that of EDTA-insensitive EP decreased slowly with the same rate constant as that of the slow-decomposing EP. These results showed that the biphasic time course of EP hydrolysis was caused by the formation of EDTA-sensitive and -insensitive EP during the reaction. The time course of EP hydrolysis could be quantitatively analyzed in terms of the following reaction mechanism. (formula; see text) The decomposition of EDTA-insensitive EP required Mg2+ outside the vesicles and was competitively inhibited by extravesicular Ca2+. The decomposition of EDTA-sensitive EP was inhibited by Ca2+ inside the vesicles but not by external Ca2+. The linear relationships between the inverse of the rate constants of EP decomposition during the initial phase and the intravesicular CaCl2 concentrations suggested that decomposition of EDTA-sensitive EP was inhibited by the binding of 1 mol of intravesicular Ca2+ to 1 mol of EP. Furthermore, Mg2+ inside the vesicles scarcely affected the inhibition of EP hydrolysis by intravesicular Ca2+. These results suggested that magnesium ions are not counter-transported during the active transport of calcium by SR vesicles.

Ribosomes unfolded by the removal of Mg2+ at 25 degrees C were studied by Raman spectroscopy and electron microscopy. Raman spectra showed a reduction in the 813 cm-1 phosphodiester signal of 30S and 50S ribosomes compared to intact ribosomes, suggesting that a fraction of the ribose moieties had shifted from the 3' endo (ordered) to the 3' exo (disordered) conformation. The maximum diameters of unfolded 30S and 50S ribosomes, judged by electron microscopy, were 1.8 and 2.5-fold greater, respectively, than those of intact ribosomes. Most unfolded 30S ribosomes had three distinct structural domains and appeared "Y-shaped"; whereas most unfolded 50S ribosomes had four distinct domains and appeared "X-shaped". When ribosomes were partially unfolded (by brief exposure to 0.04 mM Mg2+ or EDTA), several possible intermediates in the unfolding process were observed. Both the shapes of particles and their Raman spectra reached the same final state in 0.04 mM Mg2+, where more than 50% of the rRNA phosphates are discharged by Mg2+, as in 10 mM EDTA, where less than 1% are discharged.

Translation of the foot and mouth disease virus genome in vitro and in vivo indicated that all seven serotypes initiate protein synthesis at two separate AUGs. Sequence analysis of the region surrounding these AUGs has shown that the efficiency with which the initiating AUG is recognized is dependent on the flanking nucleotides. However, in vitro, the major factor determining which AUG is used is the concentration of Mg2+.

The necessity of calcium (Ca2+) and the Ca2+-calmodulin complex for resumption and completion of meiosis, expansion of cumulus cells, viability and hyaluronidase sensitivity of in vitro cultured bovine cumulus-oocyte complexes was examined by inhibition of the Ca2+-calmodulin complex with eight graduated doses of trifluoperazine (TFP) and by Ca2+ deficiency or depletion. Doses of TFP greater than 2.5 microM decreased the percent of cumulus complexes surviving culture and oocytes completing meiosis, whereas cumulus expansion was unaffected until the cultures contained a near lethal dose (greater than 10 microM). Hyaluronidase caused dispersion of cumulus cells whenever they were expanded regardless of TFP dose. In TC-199 media the completion of meiosis I was suppressed by 0.1 to 1 mM ethylenediaminotetraacetic acid (EDTA) (P less than 0.05) and drastically reduced by 1.0 mM (P less than 0.05). Viability of the cumulus-oocyte complex was not reduced until the dose of EDTA was increased to 1.0 mM (P less than 0.0001). Cumulus expansion was also not suppressed until the dose of EDTA reached 1.0 mM (P less than 0.05). In Ca2+-free (CF) basal media Eagles, completion of meiosis I was reduced by all doses of EDTA (P less than 0.05), whereas viability of the cumulus-oocyte complex was decreased by Ca2+ deficiency or by EDTA addition to basal media Eagles (P less than 0.01). Cumulus expansion was unaffected by Ca2+ removal or chelation. In all experiments, oocytes which were not degenerate underwent germinal vesicle breakdown regardless of treatment.

A sphingomyelinase, which specifically hydrolyzes sphingomyelin into ceramide and phosphocholine, was solubilized from nuclear matrix fraction of rat ascites hepatoma, AH7974 cells. The solubilized enzyme was subjected to Mono Q column chromatography in an FPLC system. The sphingomyelinase which was adsorbed on the column and eluted at 0.25-0.5 M NaCl was characterized. The enzyme required 10 mM MgCl2, 0.01% Triton X-100, 1 mM dithiothreitol, and a higher concentration of buffer than 1 M for its maximal activity, and the optimal pH was 6.7-7.2 in 2 M Tris/acetic acid or 7.5 in 2 M potassium acetate/acetic acid. N-Ethylmaleimide completely inhibited the enzyme activity at 0.2 mM. Therefore, this enzyme is classified as a Mg2+-dependent, neutral sphingomyelinase. The sphingomyelinase sedimented at 4.3S through a 10-30% glycerol gradient containing 2 M potassium acetate. This enzyme was highly specific to sphingomyelin and did not hydrolyze phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol. Various characteristics of the nuclear sphingomyelinase were similar to those of the plasma membrane enzyme except its requirement for a high concentration of buffer and SH-reagent.

The characteristics of ATP synthesis in cell envelope vesicles of Halobacterium halobium were further studied. The results confirmed the previous conclusion (Mukohata et al. (1986) J. Biochem. 99, 1-8) that the ATP synthase in this extremely halophilic archaebacterium can not be an ordinary type of F0F1-ATPase, which has been thought to be ubiquitous among all the aerobic organisms on our biosphere. The ATP synthesis was activated most in 1 M NaCl and/or KCl, and at 40 degrees C, and at 80 mM MgCl2 where F0F1-ATPase loses its activity completely. The synthesis was negligible at 10 degrees C, and at 5 mM MgCl2. The Km for ADP was about 0.3 mM in the presence of 20 mM Pi, 1 M NaCl, 80 mM MgCl2, and 10 mM PIPES at pH 6.8 and 20 degrees C. The ATP synthesis was not inhibited by NaN3 and quercetin (specific inhibitors for F0F1-ATPase) or vanadate (for E1E2-ATPase) or ouabain (for Na+,K+-ATPase) or P1,P5-di(adenosine-5')pentaphosphate (AP5A, for adenylate kinase). The ATP synthesis was not inhibited by modification (pretreatment) with NaN3 or 5'-p-fluorosulfonylbenzoyladenosine (FSBA). On the contrary, the ATP synthesis was rather non-specifically inhibited by N-ethylmaleimide (NEM), trinitrobenzenesulfonate (TNBS), phenylglyoxal, and pyridoxal phosphate. 7-Chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) as well as N,N'-dicyclohexylcarbodiimide (DCCD) was found to be a specific inhibitor at least partly, because the NBD-Cl inhibition was partly prevented by ADP added to the modification mixture.

The equilibrium for the binding reaction between the 30 S and 50 S ribosomal subunits of E. coli is shifted towards formation of 70 S ribosomes in the presence of a variety of polymers. The polymers also increase a further interaction between 70 S particles to form the 100 S dimer. The requirement for relatively high concentrations of non-specific polymers indicates that the shifts in equilibria arise from excluded volume effects. Analysis using scaled particle theory is consistent with this mechanism. The effects of high concentrations of polymers on the interactions between ribosomal species may make important changes in the function of ribosomes under the crowded conditions which occur in vivo.