Can heavy isotopes increase lifespan? Studies of relative abundance in various organisms reveal chemical perspectives on aging

Stable heavy isotopes co‐exist with their lighter counterparts in all elements commonly found in biology. These heavy isotopes represent a low natural abundance in isotopic composition but impose great retardation effects in chemical reactions because of kinetic isotopic effects (KIEs). Previous isotope analyses have recorded pervasive enrichment or depletion of heavy isotopes in various organisms, strongly supporting the capability of biological systems to distinguish different isotopes. This capability has recently been found to lead to general decline of heavy isotopes in metabolites during yeast aging. Conversely, supplementing heavy isotopes in growth medium promotes longevity. Whether this observation prevails in other organisms is not known, but it potentially bears promise in promoting human longevity.     

Dual-color bioluminescent assay using infected HepG2 cells sheds new light on Chlamydia pneumoniae and human cytomegalovirus effects on human cholesterol 7α-hydroxylase (CYP7A1) transcription

Human bones, recovered from excavations, are an important biological archive of information. In particular, the analysis of the collagen fraction is useful for paleodietary reconstruction, via light stable isotopes, and for (14)C dating. Generally, collagen extraction procedures do not prevent loss of integrity of proteins. As a consequence, information about the state-of-remains preservation is unavailable. Here we describe a "soft" nondestructive CH(3)COOH-based method to recover collagen from archaeological bones, and also to obtain material for successive isotopic analyses. Our isotopic measurements on the extracts indicate that the CH(3)COOH-based method of extraction may be routinely employed in the context of paleodiet studies. In addition, we propose that biochemical characterization by denaturant electrophoresis and Western blot on CH(3)COOH extracts may be used as a bone collagen quality indicator.     

Distribution of carbon isotopes ((13)C/(12)C) in cells and temporal organization of cellular processes]

Recent studies on fractionation of carbon isotopes in biological systems are reviewed. It follows that direct experimental proofs have been obtained that 1) basic fractionation of carbon isotopes in the cell is related to isotope effect in pyruvate decarboxylation; 2) fractionation of carbon isotopes in the above reaction in vivo proceeds with exhausting substrate pool. The latter provides natural relationship between metabolites isotope distribution and sequence of their synthesis in the cell cycle, or with the temporal organization of cellular metabolism. The non-steady and periodic pattern of pyruvate decarboxylation due to the exhausting substrate pool well agrees with the existing notions on reciprocal oscillations in the cell glycolytic chain. Experimental data are presented corroborating indirectly the existence of oscillations in bacterial cells. Earlier proposed model of the mechanism of carbon isotope fractionation based on the above principles can be used for analysing changes in isotopic characteristics of the organisms and interpreting their relations with metabolic processes.     

Cold-shock induced high-yield protein production in Escherichia coli

Overexpression of proteins in Escherichia coli at low temperature improves their solubility and stability. Here, we apply the unique features of the cspA gene to develop a series of expression vectors, termed pCold vectors, that drive the high expression of cloned genes upon induction by cold-shock. Several proteins were produced with very high yields, including E. coli EnvZ ATP-binding domain (EnvZ-B) and Xenopus laevis calmodulin (CaM). The pCold vector system can also be used to selectively enrich target proteins with isotopes to study their properties in cell lysates using NMR spectroscopy. We have cloned 38 genes from a range of prokaryotic and eukaryotic organisms into both pCold and pET14 (ref. 3) systems, and found that pCold vectors are highly complementary to the widely used pET vectors.     

Role of stable isotopes in life--testing isotopic resonance hypothesis

Stable isotopes of most important biological elements, such as C, H, N and O, affect living organisms. In rapidly growing species, deuterium and to a lesser extent other heavy isotopes reduce the growth rate. At least for deuterium it is known that its depletion also negatively impacts the speed of biological processes. As a rule, living organisms "resist" changes in their isotopic environment, preferring natural isotopic abundances. This preference could be due to evolutionary optimization; an additional effect could be due to the presence of the "isotopic resonance". The isotopic resonance phenomenon has been linked to the choice of earliest amino acids, and thus affected the evolution of genetic code. To test the isotopic resonance hypothesis, literature data were analyzed against quantitative and qualitative predictions of the hypothesis. Four studies provided five independent datasets, each in very good quantitative agreement with the predictions. Thus, the isotopic resonance hypothesis is no longer simply plausible; it can now be deemed likely. Additional testing is needed, however, before full acceptance of this hypothesis.     

Multiple sources of isotopic variation in a terrestrial arthropod community: challenges for disentangling food webs

Documenting trophic links in a food web has traditionally required complex exclusion experiments coupled with extraordinarily labor-intensive direct observations of predator foraging. Newer techniques such as stable isotope analysis (SIA) may facilitate relatively quick and accurate assessments of consumer feeding behavior. Ratios of N and C isotopes are thought to be useful for determining species' trophic position (e.g., 1 degrees consumer, 2 degrees consumer, or omnivore) and their original carbon source (e.g., C3 or C4 plants; terrestrial or marine nutrients). Thus far, however, applications of stable isotopes to terrestrial arthropod food webs have suggested that high taxon-specific variation may undermine the effectiveness of this method. We applied stable isotope analysis to a pear orchard food web, in which biological control of a dominant pest, pear psylla (Cacopsylla pyricola), involves primarily generalist arthropod predators with a high frequency of omnivory. We found multiple sources of isotopic variation in this food web, including differences among plant tissues; time, stage, and taxon-specific differences among herbivores (despite similar feeding modes); and high taxon-specific variation among predators (with no clear evidence of omnivory). Collectively, these multiple sources of isotopic variation blur our view of the structure of this food web. Idiosyncrasies in consumer trophic shifts make ad hoc application of SIA to even moderately complex food webs intractable. SIA may not be a generally applicable "quick and dirty" method for delineating terrestrial food web structure-not without calibration of specific consumer food trophic shifts.     

Martian Stable Isotopes: Volatile Evolution,Climate Change and Exobiological Implications

Measurements of the ratios of stable isotopes in the martian atmosphere and crust provide fundamental information about the evolution of the martian volatile and climate system. Current best estimates of the isotope ratios indicate that there has been substantial loss of gases to space and exchange of gases between the atmosphere and the crust throughout geologic time; exchange may have occurred through circulation of water in hydrothermal systems. Processes of volatile evolution and exchange will fractionate the isotopes in a manner that complicates the possible interpretation of isotopic data in terms of any fractionation that may have been caused by martian biota, and must be understood first. Key measurements are suggested that will enhance our understanding of the non-biological fractionation of the isotopes and of the evolution of the martian volatile system.     

Stable isotopes of carbon and nitrogen in soil ecological studies

The development of stable isotope techniques is one of the main methodological advances in ecology of the last decades of the 20th century. Many biogeochemical processes are accompanied by changes in the ratio between stable isotopes of carbon and nitrogen (¹²C/¹³C and ¹⁴N/¹⁵N), which allows different ecosystem components and different ecosystems to be distinguished by their isotopic composition. Analysis of isotopic composition makes it possible to trace matter and energy flows through biological systems and to evaluate the rate of many ecological processes. The main concepts and methods of stable isotope ecology and patterns of stable isotope fractionation during organic matter decomposition are considered with special emphasis on the fractionation of isotopes in food chains and the use of stable isotope studies of trophic relationships between soil animals in the field.     

Theoretical analysis of the relation between the cell state and pathways of carbon isotope fractionation in metabolic processes

On a dynamic model of fractionation of carbon isotopes in the living cell there are considered relationships between the distribution of carbon isotopes in the structures approximating basic biochemical fractions, their isotopic composition and parameters characterizing the dynamics of carbon metabolism, i.e. efficient carbon isotope separation factor in pyruvate enzymic decarboxylation, degree of its transformation at primary and secondary decarboxylation and ratios between the currents of carbon substrates. A wide range of variations of cell isotope parameters resulting from the change of its functional states was revealed. Possible applications of the relationships observed for studying biological systems are shown.     

Mathematical aspects of outer-product asynchronous content-addressable memories

Conclusions We have given evidence by mathematical analysis and by example that the construction of CAM's as quasineural networks based on the twin principles of an outer-product weight matrix and a random asynchronous single-neuron dynamics encounters two obstacles to good performance which appear to be inherent. It is desirable to have the stored memory vectors of a CAM as mutually far apart as possible in order to have unambiguous retrieval with as large a fraction of initial errors (minrad) as possible. For a given number,m, of memory vectors to be stored this requires that their dimension,n, be larger thanm and that the memory vectors be nearly mutually orthogonal (except for complementary pairs). In HOCAM's, it does not seem possible to have both a large minrad and an efficient ratiom/n. Attempts to increasem/n are likely to introduce extraneous fixed-points which reduce minrad appreciably. We have demonstrated this phenomenon in several cases for a particular mode of constructing CAM's of arbitrary size which have a desirable spacing between memory vectors. We conjecture that it is present also in HOCAM's having a random selection of memory vectors. (A mathematical proof of this conjecture now seems possible.) This may account for the rather pessimistic results on capacity obtained by mathematical analysis here and in Cottrell (1988), by a probabilistic analysis in Posner (1987) and by simulation in Hopfield (1982). Further, in Cottrell (1988) there is evidence that outer-product weights are near optimal with respect to minrad, so that otherW may not improve matters.We have left to another paper a study of other approaches to content-addressable memories of which we are aware, but which are not focused on asynchronous dynamics; e.g. computer CAM's as in Kohonen (1977) and biological memory models as in Little (1974); Palm (1980) and Little and Shaw (1978). We have not considered the learning, or adaptive, aspects of CAM's. However, insofar as learning is Hebbian and leads to outer-product weights, our analysis has implications for the effectiveness of learned weights, as may be inferred from our results on ambiguous retrieval.This research was partially supported by NSF Grant CCR-87121192 and AFOSR Grant 88-0245     

Intein-based biosynthetic incorporation of unlabeled protein tags into isotopically labeled proteins for NMR studies

Segmental isotopic labeling of proteins using protein ligation is a recently established in vitro method for incorporating isotopes into one domain or region of a protein to reduce the complexity of NMR spectra, thereby facilitating the NMR analysis of larger proteins. Here we demonstrate that segmental isotopic labeling of proteins can be conveniently achieved in Escherichia coli using intein-based protein ligation. Our method is based on a dual expression system that allows sequential expression of two precursor fragments in media enriched with different isotopes. Using this in vivo approach, unlabeled protein tags can be incorporated into isotopically labeled target proteins to improve protein stability and solubility for study by solution NMR spectroscopy.     

Zinc Isotope Ratios as Indicators of Diet and Trophic Level in Arctic Marine Mammals

Carbon and nitrogen stable isotope ratios of bone collagen are an established method for dietary reconstruction, but this method is limited by the protein preservation. Zinc (Zn) is found in bioapatite and the isotopic compositions of this element constitute a very promising dietary indicator. The extent of fractionation of Zn isotopes in marine environments, however, remains unknown. We report here on the measurement of zinc, carbon and nitrogen isotopes in 47 marine mammals from the archaeological site of Arvik in the Canadian Arctic. We undertook this study to test and demonstrate the utility of Zn isotopes in recent mammal bone minerals as a dietary indicator by comparing them to other isotopic dietary tracers. We found a correlation between δ⁶⁶Zn values and trophic level for most species, with the exception of walruses, which may be caused by their large seasonal movements. δ⁶Zn values can therefore be used as a dietary indicator in marine ecosystems for both modern and recent mammals.     

Stable sulphur isotopes in plants: a review

The determination of the natural abundances of stable isotopes has become a useful method by which to study the transformations of elements in biological and ecological studies as well as to investigate the mechanisms of chemical reactions. Unlike carbon and nitrogen isotopes, however, stable sulphur isotopes are used infrequently, and their potential as tracers in biochemical and physiological studies are only beginning to be realized. This review provides an overview of research involving stable sulphur isotopes in studies of plant metabolism and pollution. Topics discussed include the mechanisms and accompanying isotopic fractionations involved during the uptake and assimilation of inorganic sulphur compounds by plants, the utility of plants as bioindicators of environmental sulphur pollution, and the emission of isotopically light H₂S by plants in response to high concentations of sulphur. Future advances in the field are proposed.     

The confounding effects of source isotopic heterogeneity on consumer–diet and tissue–tissue stable isotope relationships

Stable isotope analysis of consumer tissues document patterns of resource use because data are linearly related to isotope compositions of their source(s) (i.e., food, water, etc.). Deviations in parameters estimated for these relationships can arise from variations in consumer tissue-diet spacing (Δ(TS)) and the level of isotopic heterogeneity in the source(s). We present a set of simple hypotheses that distinguish between the effects of Δ(TS) and source isotope heterogeneity. The latter may arise via mixed diets, during tissue turnover, or by isotopic routing of dietary components. We apply these concepts to stable carbon and nitrogen isotope relationships between gut contents and body tissues of large mammal herbivores from mixed C(3)/C(4) South African savannas and test predictions based on the compound- and/or time-specific data archived within each material. Predicted effects of source isotope heterogeneity are readily detected in carbon isotope relationships between materials representing different time periods or comprising bulk versus protein-only diet components. Differences in Δ(TS) of carbon isotopes across mammal herbivore species with very different feeding niches (and diet isotope compositions) are likely to be small or non-existent in these habitats. Variations in Δ(TS) estimated for nitrogen isotopes are much greater, leading to inconsistencies that cannot be explained by diet or trophic level effects alone. The effects of source heterogeneity on isotopic relationships generate numerical artefacts that have been misinterpreted as variations in Δ(TS). We caution against generalized application of hypotheses based on assumptions of source isotopic homogeneity, even for single diets commonly used in laboratory studies. More careful consideration of how heterogeneity affects consumer-diet relationships is needed for many field and laboratory systems.     

Tracing water sources of terrestrial animal populations with stable isotopes: laboratory tests with crickets and spiders

Fluxes of carbon, nitrogen, and water between ecosystem components and organisms have great impacts across levels of biological organization. Although much progress has been made in tracing carbon and nitrogen, difficulty remains in tracing water sources from the ecosystem to animals and among animals (the "water web"). Naturally occurring, non-radioactive isotopes of hydrogen and oxygen in water provide a potential method for tracing water sources. However, using this approach for terrestrial animals is complicated by a change in water isotopes within the body due to differences in activity of heavy and light isotopes during cuticular and transpiratory water losses. Here we present a technique to use stable water isotopes to estimate the mean mix of water sources in a population by sampling a group of sympatric animals over time. Strong correlations between H and O isotopes in the body water of animals collected over time provide linear patterns of enrichment that can be used to predict a mean mix of water sources useful in standard mixing models to determine relative source contribution. Multiple temperature and humidity treatment levels do not greatly alter these relationships, thus having little effect on our ability to estimate this population-level mix of water sources. We show evidence for the validity of using multiple samples of animal body water, collected across time, to estimate the isotopic mix of water sources in a population and more accurately trace water sources. The ability to use isotopes to document patterns of animal water use should be a great asset to biologists globally, especially those studying drylands, droughts, streamside areas, irrigated landscapes, and the effects of climate change.     

Dating of non-oak species in the United Kingdom historical buildings archive using stable oxygen isotopes

Stable oxygen isotope dendrochronology is an effective precision-dating method for fast grown, invariant (complacent) tree-rings and for trees growing in moist, temperate climatic regions where growth may not be strongly controlled by climate. The method works because trees preserve a strong common isotopic signal, from summer precipitation, and therefore do not need to be physiologically stressed to record a dating signal. This study explores the working hypothesis that whilst tree species may differ in their eco-physiology, leaf morphology and wood anatomy they will record an isotopic signal in their growth rings that is sufficiently similar to enable their precise dating against isotopic reference chronologies developed using dated oak tree rings from the same region. Modern and historical samples from six species (sweet chestnut, English elm, ash, alder, European beech and black poplar) were analysed and their oxygen isotopic variability was compared against an oak master chronology previously developed for central southern England. Whilst differences in the relative strength of the agreement between the different species and the master chronology are apparent, the potential for interspecies dating is demonstrated convincingly. The ability to date non-oak species using stable oxygen isotopes opens-up new opportunities for science-based archaeology and will improve understanding of a largely-unexplored, but significant part of the European historical buildings archive.     

What makes ecological systems reactive?

Although perturbations from a stable equilibrium must ultimately vanish, they can grow initially, and the maximum initial growth rate is called reactivity. Reactivity thus identifies systems that may undergo transient population surges or drops in response to perturbations; however, we lack biological and mathematical intuition about what makes a system reactive. This paper presents upper and lower bounds on reactivity for an arbitrary linearized model, explores their strictness, and discusses their biological implications. I find that less stable systems (i.e. systems with long transients) have a smaller possible range of reactivities for which no perturbations grow. Systems with more species have a higher capacity to be reactive, assuming species interactions do not weaken too rapidly as the number of species increases. Finally, I find that in discrete time, reactivity is determined largely by mean interaction strength and neither discrete nor continuous time reactivity are sensitive to food web topology.     

Unravelling complexities in benthic food webs using a dual stable isotope (hydrogen and carbon) approach

1. Stable carbon isotope studies have been an essential component of research regarding the contribution of methane (CH₄)-derived carbon to freshwater food webs and results have suggested that benthic macroinvertebrates in billabongs, streams and lakes may be partially, and in some instances, significantly 'fuelled' by methanotrophic biomass. However, the singular use of carbon isotopes can lead to ambiguous interpretations concerning the origin of carbon, especially in systems where phototrophs are likely to be using carbon respired sources and hence show more ¹³C-depleted values.
2. These uncertainties can be further resolved by the inclusion of additional isotopic data. Stable hydrogen isotopes are being increasingly used in food web studies with a marked advantage that sources may be isotopically distinct by one or two orders of magnitude greater than stable carbon or nitrogen, the isotopes most commonly used to delineate trophic interactions. By using hydrogen as a second biogeochemical tracer we provide further supportive evidence for the assimilation of methanotrophic microbial biomass by chironomid larvae.
3. Moreover, the hydrogen and carbon isotope values we found in chironomid tissues appear to reflect the original substrate used during methanogenesis; either acetate fermentation or carbonate reduction. Use of the former tends to result in relatively heavy carbon and light hydrogen isotope values due to kinetic isotope effects, whereas use of the latter results in relatively lighter carbon and heavier hydrogen isotope values.
4. We provide preliminary evidence to suggest that hydrogen and carbon isotope values in macroinvertebrates may be used to distinguish between CH₄ formation pathways and help to explain inter-depth and inter-specific differences between co-existing chironomid species found in the same lake.     

Solution NMR of large molecules and assemblies

Solution NMR spectroscopy represents a powerful tool for examining the structure and function of biological macromolecules. The advent of multidimensional (2D-4D) NMR, together with the widespread use of uniform isotopic labeling of proteins and RNA with the NMR-active isotopes, 15N and 13C, opened the door to detailed analyses of macromolecular structure, dynamics, and interactions of smaller macromolecules (< approximately 25 kDa). Over the past 10 years, advances in NMR and isotope labeling methods have expanded the range of NMR-tractable targets by at least an order of magnitude. Here we briefly describe the methodological advances that allow NMR spectroscopy of large macromolecules and their complexes and provide a perspective on the wide range of applications of NMR to biochemical problems.     

Biological activity and the Earth's surface evolution: Insights from carbon,sulfur, nitrogen and iron stable isotopes in the rock record

The search for early Earth biological activity is hindered by the scarcity of the rock record. The very few exposed sedimentary rocks have all been affected by secondary processes such as metamorphism and weathering, which might have distorted morphological microfossils and biogenic minerals beyond recognition and have altered organic matter to kerogen. The search for biological activity in such rocks therefore relies entirely on chemical, molecular or isotopic indicators. A powerful tool used for this purpose is the stable isotope signature of elements related to life (C, N, S, Fe). It provides key informations not only on the metabolic pathways operating at the time of the sediment deposition, but more globally on the biogeochemical cycling of these elements and thus on the Earth's surface evolution. Here, we review the basis of stable isotope biogeochemistry for these isotopic systems. Rather than an exhaustive approach, we address some examples to illustrate how they can be used as biosignatures of early life and as proxies for its environment, while keeping in mind what their limitations are. We then focus on the covariations among these isotopic systems during the Archean time period to show that they convey important information both on the evolution of the redox state of the terrestrial surface reservoirs and on co-occurring ecosystems in the Archean.