Changes In Magnesium Concentration Research Articles

MODERN ASPECTS OF THE ETIOPATHOGENESIS OF ISTMIC-CERVICAL INSUFFICIENCY

Isthmic-cervical insufficiency (ICI), which results in the failure of the circular muscles of the internal os, isthmus and cervix of the uterus is important in the pathogenesis of preterm birth. The existing types are anatomical, functional and congenital ICI. ICI is a pathological condition of the cervix and the isthmus, in which they are unable to withstand the intrauterine pressure and keep a fertilized egg in the uterine cavity until birth. ICI is a serious complication of pregnancy. Genetic, hormonal, inflammatory-infectious, immunological, traumatic factors play a role in the occurrence of ICI. The aim of the study is to study the peculiarities of reproductive health and gestational process of pregnant women with ICI, according to the literature. For the study, we analyzed domestic and foreign publications on this topic in professional journals, as well as data from Internet resources. Results of the research show that ICI plays a special role among many causes that lead to preterm termination of pregnancy and premature birth in the II-III trimesters. The properties of the cervix depend on the ratio of connective and muscle tissue. Today, most authors distinguish between congenital, acquired, organic and functional isthmic-cervical insufficiency.Organic (post-traumatic, secondary) ICI occurs due to mechanical damage to the cervix. Functional ICI is the result of a violation of the relationship between the muscular and connective tissues of the cervix, as well as the result of changes in the response of its structural elements to neurohumoral stimuli. Functional ICI most often occurs as a consequence of a violation of the hormonal background of women (ovarian hypofunction, hyperandrogenism). Congenital ICI occurs in malformations of the uterus, genital infantilism. Histological examination of cervical tissue in women with ICI reveals an increase in muscle tissue up to 50%, which leads to early softening of the cervix of the uterus and development of its functional insufficiency. According to Kaufman KP (2021), the role of connective tissue in the development of ICI is confirmed by the fact, that this pathological condition of the cervix is found in women with Marfan syndrome and Ehlers-Danlos syndrome. The role of undifferentiated connective tissue dysplasia (UCTD) as a risk factor for complicated pregnancy, including ICI.Research of Huang J and co-authors (2021) found the presence of nutritional imbalance in patients with UCTD, which was manifested by changes in serum concentrations of magnesium, calcium, zinc, iron, copper, selenium, inorganic phosphorus. The relationship between changes in the microcirculation system, tissue perfusion and cervical condition is currently being actively discussed and confirmed in numerous studies.Fettweis JM (2019) believes that the mechanism of abortion at different stages of gestation is not the same in pregnant women with ICI. At the same time, all pregnant women with ICI at all stages of gestation have increased cytokine levels. However, an important factor in the premature termination of pregnancy in ICI is assigned to the infectious factor. The results of the study of the pathogenetic role of local immune responses in women with habitual miscarriage are often contradictory and dictate the need for further researches.Eventually, isthmic-cervical insufficiency is a multifactorial complication of pregnancy, in which genetic, hormonal, inflammatory-infectious, immunological or traumatic factors play a role.

The Relationship between the Concentration of Magnesium and the Presence of Depressive Symptoms and Selected Metabolic Disorders among Men over 50 Years of Age.

Background: changes in the concentration of magnesium influence numerous processes in the body, such as hormone and lipid metabolism, nerve conduction, a number of biochemical pathways in the brain, and metabolic cycles. As a result, changes in magnesium concentration may contribute to the emergence of such pathologies as depressive and metabolic disorders, including hypertension, diabetes, and dyslipidemia. Methods: blood samples were taken from 342 men whose mean age was 61.66 ± 6.38 years. The concentrations of magnesium, lipid parameters, and glucose were determined using the spectrophotometric method. Anthropometric measurements were performed to determine each participant’s body mass index (BMI). Additionally, all participants completed two questionnaires: the Beck Depression Inventory and the author’s questionnaire. Results: abnormal levels of magnesium were found in 78 people. The analysis showed that these subjects more often suffered from metabolic disorders such as diabetes mellitus (p < 0.001), hypertension (p < 0.001), and depressive symptoms (p = 0.002) than participants with normal magnesium levels. Conclusion: our research showed that there is a relationship between abnormal levels of magnesium and the presence of self-reported conditions, such as diabetes, hypertension, and depressive symptoms among aging men. These findings may contribute to the improvement of the diagnosis and treatment of patients with these conditions.

Identification of SNPs associated with magnesium and sodium uptake and the effect of their accumulation on micro and macro nutrient levels in Vitis vinifera.

Macro and micro nutrient accumulation affects all stages of plant growth and development. When nutrient deficiencies or excesses occur, normal plant growth is altered resulting in symptoms such as leaf chlorosis, plant stunting or death. In grapes, few genomic regions associated with nutrient accumulation or deficiencies have been identified. Our study evaluated micro and macro nutrient concentrations in Vitis vinifera L. to identify associated SNPs using an association approach with genotype by sequencing data. Nutrient concentrations and foliar symptoms (leaf chlorosis and stunting) were compared among 249 F1 Vitis vinifera individuals in 2015 and 2016. Foliar symptoms were consistent (≥90%) between years and correlated with changes in nutrient concentrations of magnesium (r = 0.65 and r = 0.38 in 2015 and 2016, respectively), aluminum (r = 0.24 and r = 0.49), iron (r = 0.21 and r = 0.49), and sodium (r = 0.32 and r = 0.21). Single nucleotide polymorphisms associated with symptoms, sodium, and magnesium were detected on each chromosome with the exception of 5, 7 and 17 depending on the trait and genome used for analyses explaining up to 40% of the observed variation. Symptoms and magnesium concentration were primarily associated with SNPs on chromosome 3, while SNPs associated with increased sodium content were primarily found on chromosomes 11 and 18. Mean concentrations for each nutrient varied between years in the population between symptomatic and asymptomatic plants, but relative relationships were mostly consistent. These data suggest a complex relationship among foliar symptoms and micro and macro nutrients accumulating in grapevines.

A Highly Selective Green Fluorescent Magnesium Indicator for Intracellular Magnesium Ion Analysis

Intracellular magnesium ion is a crucial ion for meditating enzymatic reactions, DNA synthesis, hormonal secretion and muscle contractions. There are a few commercial fluorescent indicators used for the measurement of Magnesium ion in cells. However, all the commercially available indicators are based on tricarboxylate APTRA chelator derived from the popular tetracarboxylate BAPTA calcium ion chelator, including mag-fura-2, mag-indo-1, mag-fluo-4 and mag-rhod-2. Measurement of magnesium ion using these fluorescent indicators is challenging since mag-fura-2, mag-indo-1, mag-fluo-4 and mag-rhod-2 have higher affinity to calcium than magnesium. Magnesium and calcium ion-induced cellular signals are often intertwined, thus the poor selectivity of the existing indicators caused severe artifacts and interferences by calcium ion-induced signals. In addition, the physiological changes in magnesiumconcentrations are generally not as great as calcium concentration change upon biological stimulations, thus demanding more sensitive and selective magnesium indicators. Mag-520 has been developed to address the drawbacks of the existing magnesium ion indicators. We demonstrated that Mag-520 can detect the magnesium from 0.1 to10 mM. It has 5 times higher selectivity for magnesium than calcium while mag-fluo-4 has higher affinity to calcium than magnesium. Mag-520, AM was further evaluated for intracellular changes in magnesium using several cell lines such as HL-60, HEK-293, and HeLa cells with microplate reader, flow cytometry and fluorescence microscope. The intracellular magnesium ion level changes from forskolin stimulation can be readily detected with Mag-520, AM. In conclusion, Mag-520 is a highly selective magnesium ion indicator and can be applied in the detection of cellular Magnesium ion influx with very low interference from Calcium ion signaling. Mag-520 can be easily used with widely available FITC filter set. Compare to other magnesium ion detection dyes, Mag-520 has better performance inside the cells.

Magnesium and liver disease.

Magnesium is a vital cation that takes part in many cellular processes. Magnesium balance can be disturbed in multiple conditions, and differences in magnesium concentration can be responsible for numerous physiological and pathological processes. Magnesium deficiency is commonly associated with liver diseases, and may result from low nutrient uptake, greater urinary secretion, low serum albumin concentration, or hormone inactivation. In turn, low magnesium content in serum and liver tissue can lead to the progression of these diseases, due to a disruption in mitochondrial function, defective protein kinase C (PKC) translocation, inflammatory responses, oxidative stress, or metabolic disorders. Furthermore, magnesium supplementation can improve liver function in certain liver diseases. This paper comprehensively reviews the changes in magnesium concentrations associated with liver cirrhosis, alcoholic liver disease (ALD), liver cancer, and viral hepatitis, and explains how such changes may in turn impact these disease processes.

Long-term and season-specific changes in the body concentrations of magnesium, potassium and calcium by means of quantitative analyses of beard samples collected every day: A case study

More than 4000 beard samples were collected from a single person every morning over a 12-year period and analyzed using a standard-free method. We detected significant annual variations in the concentrations of magnesium and calcium in the study period, although significant monthly and seasonal variations were not noted. Calcium tends not to be obtained from season-specific foods or drinks, so its variations may be affected by changes in the metabolism of the body. In contrast, however, potassium showed marked long-term variations with significant seasonal differences. As our previous studies confirmed that green vegetables contain large quantities of potassium and that their supply and consumption increase from May to July, the seasonal variations in potassium concentration were attributed to the ingestion of green vegetables. Although magnesium is also contained in green vegetables in large quantities, its principle supply sources are diverse and its concentration did not show any clear seasonal changes.

Change in magnesium concentration in erythrocytes in fish under stress

At present, the role of erythrocyte magnesium in the respiratory processes of fish (and other animals) under conditions of stress load is not known. This article presents the results of research on change in the concentration of magnesium in erythrocytes under the action of stressors of different quality and quantity for bream (Abramis brama L.), silver crucian carp (Carassius auratus gabelio Bloch) and tench, (Tinca tinca L.) in Rybinsk Reservoir. The concentration of magnesium ions was analyzed on an atomic absorption spectrometer AAS-1 manufactured by Carl Zeiss (Germany) in the absorption regime in an air-acetylene flame. For the first time, the dependence of the change in magnesium concentration in erythrocytes on the intensity of the stress load of different nature on the example of fishes was revealed. Weak and moderate strength effects (low doses of epinephrine, norepinephrine, small changes in water temperature, saline injection, prick into the abdominal cavity, short-term removal of fish from the water, short-term keeping of fish in a limited volume of water) increased the concentration of magnesium in erythrocytes up to 2.5 times. At the same time, an increase in the affinity of hemoglobin for oxygen and a decrease in oxygen consumption were observed. However, strong stressors such as catching, transporting fish to the laboratory (representing complex effects of hypoxia, limited water volume, mechanical effects, etc.), as well as a sharp and large change in water temperature, high doses of adrenaline reduced the concentration of magnesium in erythrocytes 3 times. At the same time, the hemoglobin affinity for oxygen decreased and oxygen absorption increased. However, before the death of fish (from exhaustion), with a low level of magnesium in erythrocytes the affinity of hemoglobin for oxygen increased and the intensity of gas exchange decreased. The research conducted allow us to consider the change in the concentration of magnesium ions in erythrocytes – which are positive modulators of the affinity of hemoglobin for oxygen – in fish exposed to stress as one of the mechanisms for reducing the gas exchange intensity for weak and medium short-term stress effects and increasing it for strong short-term ones. Especially important is the role of erythrocyte magnesium as a molecular mechanism for reducing oxygen uptake and, consequently, increasing anabolism and, thus, increasing the growth and development of animals under the action of mild, short-term stressors, i.e. with eustress. In addition, the concentration of magnesium in erythrocytes can serve as an indicator of the state of fish. A high level of this cation in erythrocytes (1.5–2.0 times higher and more than normal) is characteristic for strong, healthy animals in a state of eustress or physiological stress, and extremely low values of this indicator (1.5–2.0 and more times below the norm) are an indicator of reversible or permanent ill-being (distress or pathological stress). Weakened, exhausted animals are not capable of a response to eustress or physiological stress. The possible causes of low magnesium concentrations in human erythrocytes are discussed.

Mechanism of Action Potential Prolongation During Metabolic Inhibition in the Whole Rabbit Heart.

Myocardial ischemia is associated with significant changes in action potential (AP) duration, which has a biphasic response to metabolic inhibition. Here, we investigated the mechanism of initial AP prolongation in whole Langendorff-perfused rabbit heart. We used glass microelectrodes to record APs transmurally. Simultaneously, optical AP, calcium transient (CaT), intracellular pH, and magnesium concentration changes were recorded using fluorescent dyes. The fluorescence signals were recorded using an EMCCD camera equipped with emission filters; excitation was induced by LEDs. We demonstrated that metabolic inhibition by carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) resulted in AP shortening preceded by an initial prolongation and that there were no important differences in the response throughout the wall of the heart and in the apical/basal direction. AP prolongation was reduced by blocking the ICaL and transient outward potassium current (Ito) with diltiazem (DTZ) and 4-aminopyridine (4-AP), respectively. FCCP, an uncoupler of oxidative phosphorylation, induced reductions in CaTs and intracellular pH and increased the intracellular Mg2+ concentration. In addition, resting potential depolarization was observed, clearly indicating a decrease in the inward rectifier K+ current (IK1) that can retard AP repolarization. Thus, we suggest that the main currents responsible for AP prolongation during metabolic inhibition are the ICaL, Ito, and IK1, the activities of which are modulated mainly by changes in intracellular ATP, calcium, magnesium, and pH.

A Pilot Randomized Trial of Oral Magnesium Supplementation on Supraventricular Arrhythmias.

Low magnesium may increase the risk of atrial fibrillation. We conducted a double-blind pilot randomized trial to assess adherence to oral magnesium supplementation (400 mg of magnesium oxide daily) and a matching placebo, estimate the effect on circulating magnesium concentrations, and evaluate the feasibility of using an ambulatory heart rhythm monitoring device (ZioPatch) for assessing premature atrial contractions. A total of 59 participants were randomized; 73% were women, and the mean age was 62 years. A total of 98% of the participants completed the follow-up. In the magnesium supplement group, 75% of pills were taken, and in the placebo group, 83% were taken. The change in magnesium concentrations was significantly greater for those given the magnesium supplements than for those given the placebo (0.07; 95% confidence interval: 0.03, 0.12 mEq/L; p = 0.002). The ZioPatch wear time was approximately 13 of the requested 14 days at baseline and follow-up. There was no difference by intervention assignment in the change in log premature atrial contractions burden, glucose, or blood pressure. Gastrointestinal changes were more common among the participants assigned magnesium (50%) than among those assigned the placebo (7%), but only one person discontinued participation. In sum, compliance with the oral magnesium supplementation was very good, and acceptance of the ZioPatch monitoring was excellent. These findings support the feasibility of a larger trial for atrial fibrillation (AF) prevention with oral magnesium supplementation.

Response to Comment by Zeebe and Tyrrell on “The Effects of Secular Calcium and Magnesium Concentration Changes on the Thermodynamics of Seawater Acid/Base Chemistry: Implications for the Eocene and Cretaceous Ocean Carbon Chemistry and Buffering”

Key Points Suspect parameters were changed to new reference The main results of original study hold true We demonstrate skill of MyAMI model, which is superior to existing correction factors

Comment on “The Effects of Secular Calcium and Magnesium Concentration Changes on the Thermodynamics of Seawater Acid/Base Chemistry: Implications for Eocene and Cretaceous Ocean Carbon Chemistry and Buffering” by Hain et al. (2015)

Hain et al. (2015, https://doi.org/10.1002/2014GB004986, hereafter H15) calculated effects of changing [Ca2+] and [Mg2+] on the equilibrium constants of the CO2‐CaCO3 system in seawater based on a Pitzer ionic interaction model. We show here that critical results presented in H15 are fundamentally wrong. We therefore do not recommend using H15's stoichiometric equilibrium constants to correct for past changes in major‐ion seawater composition.

Determination of the center composition of gradient-activated lithium niobate crystals doped with magnesium and chromium

Investigations of the defect structure of a gradient-activated stoichiometric LiNbO3:Mg2+, Cr3+ crystal, in which the chromium concentration has a constant value of 0.1 at. % along the crystal growth axis, and the magnesium concentration changes smoothly from 5.5 to 2.0 al. %. The threshold concentration of Mg2+ ions (2.35 at. %) in these crystals with a concentration width of change in the center composition (Cr optical center and its nearest neighbors) of 0.09 at. % Mg2+ ions is established..

The effects of secular calcium and magnesium concentration changes on the thermodynamics of seawater acid/base chemistry: Implications for Eocene and Cretaceous ocean carbon chemistry and buffering

AbstractReconstructed changes in seawater calcium and magnesium concentration ([Ca2+], [Mg2+]) predictably affect the ocean's acid/base and carbon chemistry. Yet inaccurate formulations of chemical equilibrium “constants” are currently in use to account for these changes. Here we develop an efficient implementation of the MIAMI Ionic Interaction Model to predict all chemical equilibrium constants required for carbon chemistry calculations under variable [Ca2+] and [Mg2+]. We investigate the impact of [Ca2+] and [Mg2+] on the relationships among the ocean's pH, CO2, dissolved inorganic carbon (DIC), saturation state of CaCO3 (Ω), and buffer capacity. Increasing [Ca2+] and/or [Mg2+] enhances “ion pairing,” which increases seawater buffering by increasing the concentration ratio of total to “free” (uncomplexed) carbonate ion. An increase in [Ca2+], however, also causes a decline in carbonate ion to maintain a given Ω, thereby overwhelming the ion pairing effect and decreasing seawater buffering. Given the reconstructions of Eocene [Ca2+] and [Mg2+] ([Ca2+]~20 mM; [Mg2+]~30 mM), Eocene seawater would have required essentially the same DIC as today to simultaneously explain a similar‐to‐modern Ω and the estimated Eocene atmospheric CO2 of ~1000 ppm. During the Cretaceous, at ~4 times modern [Ca2+], ocean buffering would have been at a minimum. Overall, during times of high seawater [Ca2+], CaCO3 saturation, pH, and atmospheric CO2 were more susceptible to perturbations of the global carbon cycle. For example, given both Eocene and Cretaceous seawater [Ca2+] and [Mg2+], a doubling of atmospheric CO2 would require less carbon addition to the ocean/atmosphere system than under modern seawater composition. Moreover, increasing seawater buffering since the Cretaceous may have been a driver of evolution by raising energetic demands of biologically controlled calcification and CO2 concentration mechanisms that aid photosynthesis.

Magnesium in drug-naïve patients with a short-duration, first episode of major depressive disorder: impact on psychopathological features

Plasma magnesium concentration changes are seen in major depressive disorder (MDD). Dysregulation of magnesium homeostasis is associated with the severity of depression and specific psychopathological features including apathy, anxiety, irritability, fatigue and weakness. Results from studies on magnesium concentrations in MDD patients are inconsistent, and the evidence for magnesium ions being associated with specific features of depression and anxiolytic effects is unclear. This study was designed to examine whether and to what extent plasma magnesium is related to the psychopathological features of depression including severity of symptoms and specific psychopathological aspects. Plasma magnesium levels were studied in this cross-sectional, case-control study and involved 20, non-late-life adults who were treatment-naïve, short- duration, first episode, MDD patients, and 20 age- and sex-matched, healthy controls. Psychometric evaluations were performed using the Hamilton Rating Scale for Depression (HAMD-17) and the Spielberger State-Trait Anxiety Inventory (STAI). A significantly higher magnesium (p = 0.016) concentration was observed in MDD patients compared to controls. No significant correlations were observed between magnesium concentrations and the total HAMD-17 score or with regard to the specific core depression, insomnia, anxiety or somatic HAM-D psychopathological features. In addition, no significant correlations were found between magnesium concentrations and STAIX-1 and STAIX-2 scores. The present study provides evidence of hypermagnesaemia in drug-naïve patients with a short-duration, first episode of MDD. A cross-sectional analysis adds to the evidence linking plasma magnesium concentrations with psychopathology of MDD during early stages of the disease although with no correlations between plasma magnesium concentrations and psychopathology including severity of symptoms and specific psychopathological features.

Ageing, hippocampal synaptic activity and magnesium.

Ageing is associated with a general decline in physiological functions. Amongst the different aspects of body deterioration, cognitive impairments, and particularly defects in learning and memory, represent one of the most frequent features in the elderly. However, a great variability exists among aged subjects. Clinical reports and experimental data in animal models of ageing have shown that age-associated memory deficits are broadly identical to those induced by damage to the hippocampus. It is therefore not surprising that many functional properties of hippocampal neuronal networks are particularly altered with ageing. Whereas passive membrane properties of neurons are conserved with age, neuronal excitability is altered, in keeping with weaker performances of aged subjects in memory tasks. Synaptic transmission within hippocampal networks also decreases in brain ageing. Deficits concern both glutamatergic and cholinergic pathways, which represent the main excitatory neurotransmitter systems responsible for neuronal communication in the hippocampus. In addition, long-term changes in synaptic transmission, possible cellular substrates for learning and memory, are also impaired in ageing in correlation with cognitive impairments. Neuronal properties and synaptic plasticity closely depend on ion exchanges between intra- and extracellular compartments. Changes in ion regulation during ageing may therefore participate in altering functional properties of neuronal networks. Calcium dysregulation has been extensively investigated in brain ageing but the role of magnesium has received less attention though ageing constitutes a risk factor for magnesium deficit. One of general properties of magnesium at presynaptic fibre terminals is to reduce transmitter release. At the postsynaptic level, it closely controls the activation of the N-methyl-D-aspartate receptor, a subtype of glutamate receptor, which is critical for the expression of long-term changes in synaptic transmission. In addition, magnesium is a cofactor of many enzymes localized either in neurons or in glial cells that control neuronal properties and synaptic plasticity such as protein-kinase C, calcium/calmodulin-dependent protein kinase II and serine racemase. It is therefore likely that a change in magnesium concentration would significantly impair synaptic functions in the aged hippocampus. Experiments addressing this question remain too scarce but recent data indicate that magnesium is involved in age-related deficits in transmitter release, neuronal excitability and in some forms of synaptic plasticity such as long-term depression of synaptic transmission. Further studies are still necessary to better delineate to what extent magnesium contributes to the impaired cellular mechanisms of cognitive functions in the elderly which will help to develop new strategies to minimize age-related memory declines.

Changes in magnesium concentration in the serum and cerebrospinal fluid of neuropathic rats

Central sensitization of neuropathic pain is associated with an influx of extracellular calcium via the opening of N-methyl-D-aspartate (NMDA) receptor-gated ion channels, which are usually blocked by magnesium plugs. As magnesium-deficient rats develop a mechanical hyperalgesia and intrathecal or intraperitoneal magnesium suppresses neuropathic pain, the magnesium concentrations in serum and cerebrospinal fluid may be altered in neuropathic pain. We therefore compared the magnesium concentrations in serum and cerebrospinal fluid of neuropathic rats with those in injured rats without symptoms of neuropathic pain and normal rats. Mechanical allodynia was induced in male Sprague-Dawley rats by tight ligature of the left lumbar fifth and sixth spinal nerves. The threshold of paw withdrawal was evaluated by the up-down method using withdrawal response to stimulus with a von Frey filament on the third, seventh and 14th days. Rats with a threshold of less than 4 g were selected as the symptomatic group and compared with an asymptomatic group, an unoperated control group and a sham-operated group. On the 16th day, the Mg2+ concentrations in serum and cerebrospinal fluid were measured. The magnesium concentrations in the serum and cerebrospinal fluid of symptomatic neuropathic rats did not differ from those in the injured rats without symptoms of neuropathic pain, sham-operated rats and normal rats. Our results suggest that physiologic homeostasis is maintained by active transport through the blood-brain barrier despite the activation of NMDA receptor-gated ion channels. However, rats with neuropathic pain may be in a magnesium-deficient condition at the effector site, such that magnesium treatment can decrease neuropathic pain.

Modelling Al 3Zr dispersoid precipitation in multicomponent aluminium alloys

An integrated modelling approach has been used to predict the precipitation of metastable L1 2 Al 3Zr dispersoids in multicomponent aluminium alloys, during homogenization heat treatment. The Scheil–Gulliver method has been used to predict the segregation of the alloying elements after casting. A coupled model was then developed to predict the simultaneous precipitation of Al 3Zr dispersoids and diffusional redistribution of the alloying elements during subsequent homogenization. The effect of the concentration of copper, magnesium and zinc solute on dispersoid precipitation has been predicted for 7xxx aluminium alloys. These elements are predicted to accelerate Al 3Zr precipitation kinetics, with changes in magnesium concentration showing the strongest effect. The effect of the solute concentration on the width of the dispersoid free zone towards the edge of the dendrites has also been calculated. Increasing solute concentration is predicted to reduce this width, and the possible consequences of this on the fraction of recrystallization have been investigated.

Magnesium is required for specific DNA binding of the CREB B-ZIP domain.

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.

Subcellular localization of glutamate-stimulated intracellular magnesium concentration changes in cultured rat forebrain neurons using confocal microscopy

Glutamate can stimulate increases in intracellular magnesium concentration ([Mg 2+] i) and induce neurotoxicity, both independent of Ca 2+ changes. Although Mg 2+ is essential within the cell, very little is known about how it is regulated, especially in neurons. Therefore we used the fluorescent indicator, magindo-1 and confocal microscopy to examine possible intracellular pools of Mg 2+ in cultured neurons that can be dynamically regulated by glutamate. The magindo-1 fluorescence signal was present throughout the cell body and extends into the neuronal processes. The magindo-1 405 nm/490 nm ratio signal was similar in the cytoplasm and nucleus, suggesting that resting [Mg 2+] i is uniform across the neuron. The addition of 100 μM glutamate/10 μM glycine in an extracellular Ca 2+- and Na +-free buffer stimulated an increase in [Mg 2+] i in both the nuclear and cytoplasmic regions of similar magnitude and duration. This glutamate exposure also stimulated a [Mg 2+] i increase in neuronal processes which was inhibited by the N-methyl- d-aspartate receptor antagonist, MK-801 (10 μM). The glutamate-stimulated [Mg 2+] i increase in both the cell body and neuronal processes was dependent on the extracellular Mg 2+ concentration. These findings suggest glutamate-stimulated [Mg 2+] i changes may not only impact cytoplasmic processes, but also directly trigger nuclear events involved, for example, in neuronal injury.

Changes in Magnesium Content and Subcellular Distribution during Retinoic Acid-Induced Differentiation of HL60 Cells

Magnesium (Mg) is required for cellular proliferation; however, the differences in subcellular regulation of Mg between proliferating and differentiated cells has not been determined. We used electron probe microanalysis (EPMA) to investigate the subcellular distribution of Mg in HL60 cells (a promyelocytic leukemia cell line) before and after retinoic acid (RA)-induced differentiation. Most intracellular Mg is bound to ATP and the Mg–ATP complex regulates several metabolic enzymes. We also compared alterations in Mg content following differentiation with the changes in ATP and ADP levels. Using atomic absorption spectrophotometry, we observed a significant decrease (−20%) in cellular Mg content in RA-differentiated HL60 cells. To investigate which intracellular compartments were involved in these changes, we analyzed subcellular elemental composition in freeze-dried cryosections of rapidly frozen undifferentiated and differentiated HL60 cells by EPMA. Following differentiation of HL60 cells, we observed an 18% decrease in Mg content in both the cytoplasm (regions of the cell excluding mitochondria and nuclei) and mitochondria. There was also a significant (40%) decrease in cytoplasmic Ca content after RA-induced differentiation. Nuclear Mg concentration was not significantly different between differentiated and undifferentiated HL60 cells, although differentiation was accompanied by a 30% decrease in the nuclear K/Na ratio. After differentiation, cellular ATP and ADP content decreased by 31 and 40%, respectively. We conclude that during exit from the cell cycle, Mg redistributes within cells and that the decrease in cytoplasmic and mitochondrial Mg is accompanied by a decrease in ATP and ADP content.