The Influence of Low Magnetic Fields and Magnesium Isotopes on <Emphasis Type="Italic">E. coli</Emphasis> Bacteria

The combined effects of external low static magnetic fields at 0–22 mT and magnesium isotopes on the growth and development of E. coli bacteria has been studied. The magnetic field and ²⁵Mg magnetic isotope effects were obtained in two ranges: 0.8–3.0 and 8–13 mT. The experimental values of the growth rate, the number of CFUs and the ATP pool of bacteria enriched in magnetic magnesium isotope ²⁵Mg (nuclear spin, I = 5/2) in the range of 0.8–3.0 mT are significantly higher compared to bacteria enriched in nonmagnetic isotopes ²⁴Mg, ²⁶Mg, or natural magnesium. The increase in the growth rate, colony-forming ability, and intracellular ATP concentration in bacteria in all groups cultivated under exposure to an external static magnetic field in the range of 0.8 to 3.0 mT confirms the existence of magnetosensitive stages of enzymatic reactions that proceed via the ion-radical mechanism. The combined influence of the magnetic field in the range of 8 to 13 mT and the magnesium magnetic isotope ²⁵Mg on the colony forming ability of E. coli bacteria is associated with processes that are responsible for cell division. The above-mentioned effects of bacterial magnetosensitivity (to magnetic fields and magnetic isotopes) are in good agreement with theoretical predictions of the theory of spin-dependent enzymatic reactions.     

Magnetic isotope of magnesium accelerates ATP hydrolysis catalyzed by myosin

In this paper, we present the results of experimental studies on the influence of different magnesium isotopes, magnetic ²⁵Mg and nonmagnetic ²⁴Mg or ²⁶Mg, on ATP-hydrolytic activity of the isolated myosin subfragment-1. The reaction rate in the presence of magnetic ²⁵Mg isotope turned out to be 2.0–2.5 times higher than that using non-magnetic ²⁴Mg or ²⁶Mg isotopes. In absence of the enzyme, as at spontaneous ATP hydrolysis in aqueous solution, no magnetic isotope effect was observed. Thus, a significant catalytic effect of the magnetic ²⁵Mg isotope (nuclear spin catalysis) was discovered in the enzymatic hydrolysis of ATP.     

Magnesium magnetic isotope effect: A key to the mechanochemistry of phosphorylating enzymes as molecular machines

The discovery of the powerful magnesium isotope effect on enzymatic ATP synthesis provides a new insight into the mechanochemistry of enzymes as molecular machines. The catalytic activities of ATPase, creatine kinase, and glycerophsphate kinase containing a Mg²⁺ ion with magnetic isotope nuclei (²⁵Mg) were found to be two to four times higher than those of the enzymes with spinless, nonmagnetic magnesium cation isotopes (²⁴Mg or ²⁶Mg). This demonstrates unambiguously that ATP synthesis is a spin-selective process involving Mg2+ as the electron-accepting reagent. ATP synthesis proceeds in an ion-radical pair consisting of an ADP oxyradical and Mg²⁺. In this process, the magnesium bivalent cation is the key agent that transforms the mechanics of a protein molecule into chemical processes. This ion is the crucial structural component of enzymes as mechanochemical molecular machines.     

Magnetosensitivity of <Emphasis Type="Italic">E. coli</Emphasis> Bacteria in the Presence of Zinc Isotopes

The combined effect of the zinc magnetic isotope ⁶⁷Zn and weak magnetic field 25–35 mT causes a 2–4-fold increase in the colony-forming ability of bacteria E. coli in comparison with the nonmagnetic isotopes ^{64, 66}Zn. The effects of magnetic field in the range of 2.2–8 mT were detected for all bacteria regardless of the zinc-isotope enrichment of the media. This indicates the sensitivity of intracellular processes to weak magnetic fields. An increase in the ATP concentration in E. coli cells was only detected for the bacteria grown on the medium with the magnetic zinc isotope in the range of 2.2–4.2 mT. The obtained data confirm the existence of stages of intracellular enzymatic processes that are sensitive to magnetic fields and magnetic moments of atomic nuclei.     

Spin biochemistry

The rates of adenosine triphosphate (ATP) production by isolated mitochondria and mitochondrial creatime kinase incubated in isotopically pure media containing, separately, ²⁴Mg²⁺, ²⁵Mg²⁺, and ²⁶Mg²⁺ ions were shown to be strongly dependent on the magnesium nuclear spin and magnetic moment. The rate of adenosine 5′-diphosphate phosphorylation in mitochondria with magnetic nuclei²⁵Mg is about twice higher than that with the spinless, nonmagnetic nuclei^24.26Mg. When mitochondrial oxidative phosphorylation was selectively blocked by treatment with 1-methylnicotine amide, ²⁵Mg²⁺ ions were shown to be nearly four times more active in mitochondrial ATP synthesis than ^24,26Mg²⁺ ions. The rate of ATP production associated with creatine kinase is twice higher for ²⁵Mg²⁺ than for ^24.26Mg and does not depend on the blockade of oxidative phosphorylation. There is no difference between ²⁴Mg²⁺ and ²⁶Mg²⁺ effects in both oxidative and substrate phophorylation. These observations demonstrate that the enzymatic phosphorylation is a nuclear spin selective process controlled by magnetic isotope effect. The reaction mechanism proposed includes a participation of intermediate ion-radical pairs with Mg⁺ cation as a radical partner. Therefore, the key mitochondrial phosphotransferases work as a magnesium nuclear spin mediated molecular machines.     

Magnetic isotope and magnetic field effects on the DNA synthesis

Magnetic isotope and magnetic field effects on the rate of DNA synthesis catalysed by polymerases β with isotopic ions ²⁴Mg²⁺, ²⁵Mg²⁺ and ²⁶Mg²⁺ in the catalytic sites were detected. No difference in enzymatic activity was found between polymerases β carrying ²⁴Mg²⁺ and ²⁶Mg²⁺ ions with spinless, non-magnetic nuclei ²⁴Mg and ²⁶Mg. However, ²⁵Mg²⁺ ions with magnetic nucleus ²⁵Mg were shown to suppress enzymatic activity by two to three times with respect to the enzymatic activity of polymerases β with ²⁴Mg²⁺ and ²⁶Mg²⁺ ions. Such an isotopic dependence directly indicates that in the DNA synthesis magnetic mass-independent isotope effect functions. Similar effect is exhibited by polymerases β with Zn²⁺ ions carrying magnetic ⁶⁷Zn and non-magnetic ⁶⁴Zn nuclei, respectively. A new, ion–radical mechanism of the DNA synthesis is suggested to explain these effects. Magnetic field dependence of the magnesium-catalysed DNA synthesis is in a perfect agreement with the proposed ion–radical mechanism. It is pointed out that the magnetic isotope and magnetic field effects may be used for medicinal purposes (trans-cranial magnetic treatment of cognitive deceases, cell proliferation, control of the cancer cells, etc).     

Magnetic isotope effect of magnesium <Superscript>25</Superscript>Mg on <Emphasis Type="Italic">E. coli</Emphasis> resistance to antibiotics

Effects of synergism and antagonism of antibacterial drugs and magnetic isotope of magnesium ²⁵Mg on antibiotic resistance of bacteria E. coli were discovered. Fourteen antibiotics from seven different groups were tested. The increase in antibiotic resistance in the presence of the ion ²⁵Mg²⁺ was discovered in E. coli cells incubated with quinolones/fluoroquinolones, indicating the inhibiting effect of the magnetic moments of nuclei ²⁵Mg on DNA synthesis. The change in antibiotic resistance was also detected in bacteria affected by magnesium ²⁵Mg and certain antibiotics from aminoglycoside and lincosamide groups.     

Magnetic-dependent ATP pool in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The ATP pool in Escherichia coli is a magnetic-dependent characteristic of microorganism vital activity. It depends on the values of the external static magnetic field and the existence of magnetic moment of magnesium isotopes nuclei added to the growth nutrient medium. The combined effects of the magnetic field 70–95 mT and magnesium magnetic isotope ²⁵Mg on E. coli bacteria leads to increase intracellular concentration of ATP. Magnetic-field effects in the range of 0.8–16 mT, registered for all bacteria regardless of the magnesium-isotopic enrichment of nutrient medium, evidence about the sensitivity of intracellular processes to weak magnetic fields.     

Efficiency of ATP synthase as a molecular machine

The experimentally measured effect of the odd magnetic isotope ²⁵Mg on the rate of ATP synthesis by the mitochondrial F₀F₁ enzyme is used to compare the rates of the main reactions in the catalytic site, in the framework of a radical-ion process. The rate-limiting step of synthesis is the addition of the ADP oxyradical to the phosphate P=O bond. The relationship of the rate constants deduced from the magnetic isotope effect shows that the mechanochemical efficiency of ATP synthase operating with spinless ²⁴Mg or ²⁶Mg nuclei will not exceed 50%. The performance is nearly doubled with magnetic ²⁵Mg.     

Mg isotopes and Mg/Ca values of coccoliths from cultured specimens of the species Emiliania huxleyi and Gephyrocapsa oceanica

Coccoliths from cultured specimens of two species of coccolithophores (Emiliania huxleyi and Gephyrocapsa oceanica) were sampled during two growth phases (late exponential and stationary), and their Mg isotope values (δ²⁶Mg) as well as Mg/Ca values were measured in order to investigate whether δ²⁶Mg can be used as a temperature proxy. Mg/Ca values were positively related with temperature (~ 0.002 mmol/mol/°C), without statistically significant differences between the two growth phases and the two species. Both species were depleted in heavier Mg isotopes relative to the culture medium, and δ²⁶Mg values were temperature dependent in both growth phases of E. huxleyi, although the δ²⁶Mg values differed in the two growth phases. In G. oceanica, a weak correlation between δ²⁶Mg values and temperature was seen in the late exponential growth phase only, and the δ²⁶Mg values differed between growth phases. The large differences between δ²⁶Mg values as measured in calcite formed during different growth phases indicate that Mg isotopes of coccoliths cannot be simply used as a temperature proxy. Our conclusions are preliminary and more data must be collected in order to fully evaluate the use of Mg isotopes of coccoliths as a temperature proxy.     

Magnesium isotope fractionation during microbially enhanced forsterite dissolution

Bacillus subtilis endospore‐mediated forsterite dissolution experiments were performed to assess the effects of cell surface reactivity on Mg isotope fractionation during chemical weathering. Endospores present a unique opportunity to study the isolated impact of cell surface reactivity because they exhibit extremely low metabolic activity. In abiotic control assays, ²⁴Mg was preferentially released into solution during forsterite dissolution, producing an isotopically light liquid phase (δ²⁶Mg = ?0.39 ± 0.06 to ?0.26 ± 0.09‰) relative to the initial mineral composition (δ²⁶Mg = ?0.24 ± 0.03‰). The presence of endospores did not have an apparent effect on Mg isotope fractionation associated with the release of Mg from the solid into the aqueous phase. However, the endospore surfaces preferentially adsorbed ²⁴Mg from the dissolution products, which resulted in relatively heavy aqueous Mg isotope compositions. These aqueous Mg isotope compositions increased proportional to the fraction of dissolved Mg that was adsorbed, with the highest measured δ²⁶Mg (?0.08 ± 0.07‰) corresponding to the highest degree of adsorption (~76%). The Mg isotope composition of the adsorbed fraction was correspondingly light, at an average δ²⁶Mg of ?0.49‰. Secondary mineral precipitation and Mg adsorption onto secondary minerals had a minimal effect on Mg isotopes at these experimental conditions. Results demonstrate the isolated effects of cell surface reactivity on Mg isotope fractionation separate from other common biological processes, such as metabolism and organic acid production. With further study, Mg isotopes could be used to elucidate the role of the biosphere on Mg cycling in the environment.     

Biological Fractionation of Lead Isotopes in Sprague-Dawley Rats Lead Poisoned via the Respiratory Tract

Objectives

It was considered that lead isotope ratios did not change during physical, chemical, or biological processes. Thus, lead isotope ratios have been used as fingerprints to identify possible lead sources. However, recent evidence has shown that the lead isotope ratios among different biological samples in human are not always identical from its lead origins in vitro. An animal experiment was conducted to explore the biological fractionation of lead isotopes in biological systems.

Methods

24 male Sprague-Dawley (SD) rats were divided into groups that received acute lead exposure (0, 0.02, 0.2, or 2 mg/kg body weight of lead acetate) via the respiratory route every day for 5 days. Biological samples (i.e., blood, urine, and feces) were collected for comparison with the lead acetate (test substance) and the low-lead animal feed (diet) administered to the rats. The lead isotope ratios were determined by inductively coupled plasma mass spectrometry (ICP-MS).

Results

There are significant differences (p<0.05) in lead isotope ratios between blood, urine, and feces. Moreover, a nonlinear relationship between the blood lead concentration and the blood lead isotope ratios was observed. There is also a threshold effect to the fractionation function. Only the blood isotope ratio of ²⁰⁴Pb/²⁰⁶Pb matches the test substance well. As for feces, when ²⁰⁴Pb/²⁰⁶Pb ratio is considered, there is no significant difference between feces-test substance pairs in medium and high dose group.

Conclusions

The biological fractionation of lead isotopes in SD rats was observed. Moreover, there might be a threshold for the biological fractionation of lead isotopes which is depending on whole blood lead level. It is considered to be more reliable that we compared the isotope ratios of potential lead hazards with both blood and feces lead fingerprints especially for ²⁰⁴Pb/²⁰⁶Pb ratio under high-dose exposure.     

Simultaneous quantification of free nucleotides in complex biological samples using ion pair reversed phase liquid chromatography isotope dilution tandem mass spectrometry

A new sensitive and accurate analytical method has been developed for quantification of intracellular nucleotides in complex biological samples from cultured cells of different microorganisms such as Saccharomyces cerevisiae, Escherichia coli, and Penicillium chrysogenum. This method is based on ion pair reversed phase liquid chromatography electrospray ionization isotope dilution tandem mass spectrometry (IP-LC-ESI-ID-MS/MS. A good separation and low detection limits were observed for these compounds using dibutylamine as volatile ion pair reagent in the mobile phase of the LC. Uniformly ¹³C-labeled isotopes of nucleotides were used as internal standards for both extraction and quantification of intracellular nucleotides. The method was validated by determining the linearity, sensitivity, and repeatability.     

Effects of nutritional restriction on nitrogen and carbon stable isotopes in growing seabirds

When using stable isotopes as dietary tracers it is essential to consider effects of nutritional state on isotopic fractionation. While starvation is known to induce enrichment of ¹⁵N in body tissues, effects of moderate food restriction on isotope signatures have rarely been tested. We conducted two experiments to investigate effects of a 50–55% reduction in food intake on δ¹⁵N and δ¹³C values in blood cells and whole blood of tufted puffin chicks, a species that exhibits a variety of adaptive responses to nutritional deficits. We found that blood from puffin chicks fed ad libitum became enriched in ¹⁵N and ¹³C compared to food-restricted chicks. Our results show that ¹⁵N enrichment is not always associated with food deprivation and argue effects of growth on diet–tissue fractionation of nitrogen stable isotopes (Δ¹⁵N) need to be considered in stable isotope studies. The decrease in δ¹³C of whole blood and blood cells in restricted birds is likely due to incorporation of carbon from ¹³C-depleted lipids into proteins. Effects of nutritional restriction on δ¹⁵N and δ¹³C values were relatively small in both experiments (δ¹⁵N: 0.77 and 0.41‰, δ¹³C: 0.20 and 0.25‰) compared to effects of ecological processes, indicating physiological effects do not preclude the use of carbon and nitrogen stable isotopes in studies of seabird ecology. Nevertheless, our results demonstrate that physiological processes affect nitrogen and carbon stable isotopes in growing birds and we caution isotope ecologists to consider these effects to avoid drawing spurious conclusions.     

Deuterium isotope effects in carbohydrates revisited. Cryoprobe studies of the anomerization and NH to ND deuterium isotope induced 13C NMR chemical shifts of acetamidodeoxy and aminodeoxy sugars

Complete ¹H and ¹³C NMR chemical shift assignments have been generated from a series of acetamidodeoxy and aminodeoxy sugar derivatives. For free sugars, the enhanced sensitivity of an NMR cryoprobe allowed simple 1D and 2D NMR spectra to be obtained from essentially single anomers, before significant mutarotation had occurred. The NMR assignments have been used to characterize deuterium isotope effects on ¹³C chemical shifts measured under conditions of slow NH to ND exchange in single solutions. Within a range of 0 to −0.138 ppm, β, γ, δ, and ζ deuterium isotope effects have been observed, thus providing additional reference data for assignment of the ¹³C NMR spectra of nitrogenous saccharides.     

Evaluation of the wick method for in situ 13C and 15N labelling of annual plants using sugar-urea mixtures

To investigate the amount and fate of root-derived C and N, often tracer techniques are used, where plants are labelled with isotopes. In the present study, we evaluated the suitability of the cotton wick method for in situ labelling of peas (Pisum sativum L.) and oats (Avena sativa L.) with ¹³C and ¹⁵N simultaneously. With two greenhouse experiments we investigated how the wick method and aqueous urea and sugar solutions at a variety of concentrations affected plant development. In addition, we investigated the distribution of ¹³C and ¹⁵N in plants from column experiments under outdoor conditions. Solution was taken up by the plant from a small vial connected to the stem by a cotton wick which was passed through a hole in the stem of the plants. Generally, solution uptake varied between individual plants and decreased with increasing sugar concentrations. Below-ground, above-ground and total plant dry matter, were not significantly affected by the wick method and the applied solutions. Mixtures of aqueous glucose solutions at 2 to 4% and aqueous urea solutions at 1% are useful carriers of ¹³C and ¹⁵N. However, in the investigated plants isotopes were not homogeneously distributed among plant parts. Above-ground plant biomass was preferentially enriched with ¹³C and ¹⁵N, whereas below-ground plant biomass was generally lower enriched. Moreover, isotope distribution ratio of individual plants varied considerably, independent of plant part or timing of labelling. This must be taken into account when estimating root-derived C and N. Future studies comparing labelling methods need to present the isotope distribution ratios among plant parts to allow a true comparison of the methods and the evaluation of their suitability for estimating rhizodeposition.     

Differences in nitrogen and carbon stable isotopes between planktonic and benthic microalgae

We compiled published data on the nitrogen and carbon stable isotope ratios of phytoplankton and benthic microalgae from lentic systems and explored the primary factors determining the isotope values among systems. Also, we investigated seasonal changes in nitrogen stable isotope ratios of phytoplankton and benthic microalgae in the strongly acidic lake, Lake Katanuma, which has only two dominant species, Pinnularia acidojaponica as a benthic diatom and Chlamydomonas acidophila, a planktonic green alga. From the published dataset, it may be concluded that δ¹³C of benthic diatoms were more enriched than those of phytoplankton at the same sites, although the nitrogen isotope of phytoplankton and benthic microalgae were similar. This differences in δ¹³C between benthic microalgae and phytoplankton could be explained by the boundary layer effect. On the other hand, nitrogen isotope values of both benthic microalgae and phytoplankton were primarily controlled by the same environmental factor, and boundary layer effects are not the primary factor determining the nitrogen isotope values of microalgae. Also, we showed temporal dynamics in nitrogen isotopes of benthic and planktonic microalgae species in Lake Katanuma, and the trends of nitrogen isotopes are similar between benthic and planktonic microalgae, as concluded from the published dataset.     

Depth variation in isotopic composition of benthic resources and assessment of sculpin feeding patterns in an oligotrophic Alaskan lake

Stable isotopes of carbon (C) and nitrogen (N) are commonly used to track resource flows through lake food webs. However, there remains a weak understanding of the spatial variation in isotopic composition of benthic resources and how this variation affects inference about energy flows among species. Boundary layers at the interface between benthic substrates and the overlying water column restrict diffusion of nutrients and carbon from the water column to benthic algae and may affect the isotopic composition of benthic algae as nutrient and CO₂ concentrations can become locally depleted in the benthic boundary layer. We quantified the variation in C and N stable isotope composition of benthic resources along a depth gradient in a large oligotrophic lake to assess the magnitude of change in stable isotope composition. Snails were increasingly depleted in ¹³C with depth, by about 10 ‰ from 0 to 20 m, while ¹⁵N in snails showed only subtle enrichment over this depth range. Sculpin (Cottas aleuticus) δ ¹³C and δ ¹⁵N signatures did not significantly change with depth and were more enriched in ¹⁵N than would be expected from consumption of snails alone. A comparison of δ ¹³C and δ ¹⁵N values from sculpins relative to shallow and deep snails, and alternative prey (marine-derived salmon resources), within a mixing model suggested sculpins feed selectively on deep grazers in this system in addition to marine-derived resources provided by migrating sockeye salmon. This study illustrates the importance of accounting for depth-related variation in isotope patterns when assessing benthic resource contributions to food webs using stable isotope data.     

Magnesium magnetic isotope effect: a key towards mechanochemistry of phosphorylating enzymes as molecular machines

A discovery of the huge magnesium isotope effect in enzymatic ATP synthesis provides a new insight into mechanochemistry of enzymes as the molecular machines. It has been found that the catalytic activity values of ATPase, creatine kinase and phosphoglycerate kinase are 2 to 4-fold higher once their active sites contain magnetic (25Mg) not spinless, non-magnetic (24Mg, 26Mg), magnesium cation isotopes. This clearly proves that the ATP synthesis is a spin-selective process involving Mg2+ as the electron accepting reagent. The formation of ATP takes place in an ion-radical pair resulted by two partners, ATP oxyradical and Mg+. The magnesium bivalent cation is a key player in this process, this ion transforms the protein molecule mechanics into a mere chemistry. This ion is a most critical detail of structure of the magnesium dependent phosphorylation enzymes as the mechanochemical molecular machines.