The use of whole-body sodium, potassium and calcium content to identify the nutrient status of first year salmon fry

Samples of fed and unfed hatchery reared Atlantic salmon fry and parr were analysed for body water and whole body Na, K and Ca content. From these body parameters it is possible to estimate the relative proportions of the skeletal mass, and intra- and extracellular spaces. These data can be applied to estimating the nutritional status of parr sampled at random from hatchery, and possibly from wild populations. When fish with a dry weight of about 25 and 70 mg were deprived of artificial food for 27 and 21 days, respectively, the Na/K ratio rose to 0.59 and 0.80 compared with 0.37 in the controls. With fish of about 475 mg in weight, percentage water content was a better indicator of undernourishment. The use of the K⁺: Ca²⁺ ratio to compare the amount of cellular material with the skeletal mass was a good indicator of nutritional depletion, especially in smaller fish.     

Novel structural and functional insights into the MoxR family of AAA+ ATPases

The MoxR family of AAA+ ATPases is widespread among bacteria and archaea, although their cellular functions are not well characterized. Based on recent studies, MoxR ATPases are proposed to have chaperone-like function for the maturation of specific protein complexes or for the insertion of cofactors into proteins. MoxR proteins have been found to be important modulators of multiple stress response pathways in different organisms. For example, the respective MoxR proteins have been found to play important roles in the cell envelope stress response in Rhizobium leguminosarum, in the oxidative stress, acid stress, and heat stress responses in Francisella tularensis, in the acid stress and stringent responses in Escherichia coli, in viral tail formation in the crenarchaeal Acidianus two-tailed virus, and in the utilization of carbon monoxide as the sole carbon source by the Gram-negative chemolithoautotrophe Oligotropha carboxidovorans. Recent structural studies on the MoxR proteins from E. coli and Cytophaga hutchinsonii show the unique spatial arrangement of the αβα and all-α subdomains of the AAA+ domain in these proteins compared to the typical arrangement found in canonical AAA+ proteins such as HslU. The spatial organization of the subdomains in the AAA+ domain of MoxR proteins is similar to that found in the ATPase component of the magnesium chelatase complexes, possibly suggesting a similar mechanism of function. In this review, we provide an overview of the newly identified functions and the newly obtained structures of MoxR AAA+ ATPases.     

Effect of the cations sodium,potassium and calcium on the interaction of hyaluronate chains: a light scattering and viscometric study

Light scattering and viscometric studies have been carried out on two preparations, A and B, of rooster comb hyaluronate. Sedimentation rate studies have also been performed with A. Light scattering measurements in 0.2 m KCl for preparation A gave a molecular weight of 3.3 × 10⁶ and for B, 1.0 × 10⁶. In (0.1–0.3) M NaCl similar measurements gave a particle weight for A of (4.4–6.4 × 10⁶ and for B (1.7–2.8 × 10⁶. In 0.066 m CaCl₂ molecular weight values of 9.5 × 10⁶ for A and 1.7 × 10⁶ for B were obtained. Thus in the presence of Na⁺ and Ca²⁺ ions aggregates of chains persisted into dilute solution. Measurements by light scattering on A and B in 4 m guanidinium chloride gave values in the same range as those obtained in 0.2 m KCl. Sedimentation rate studies on A gave values of 10.3 Svedbergs in 0.2 m KCl and 12.2 Svedbergs in 0.2 m NaCl and 0.066m CaCl₂. The shear dependence of the viscosity was studied using a conicylindrical viscometer at shear rates between 0.5 and 20 s^?1. Preparation A in 0.2 m KCl and NaCl yielded values for (ν_sp/cc→0 of 5000 and 7100 ml g^?1 respectively in keeping with the tendency to aggregate. The behaviour for preparation B was similar. In 0.066 m CaCl₂ there was a marked dependence of viscosity on shear speed below 10 s^?1 for all concentrations and the value of (ν_sp/c)→0 at 0 s^?1 for preparation A was 7700 ml g^?1 while at a shear rate of 8 s^?1 (ν_sp/c)c→0 ? 5000 ml g ^?1. Similar effects were found for preparation B and the data suggest associations of chains disruptable by weak shear forces. The increase in viscosity with concentration in the presence of 0.066 m CaCl₂ was much less than in the presence of KCl or NaCl, suggesting that the Ca²⁺ had a marked effect on the ”rigidity’ of the molecules in solution. A viscometric titration experiment with Ca^2? showed that a level of 0.02 m CaCl₂ in 0.2 m NaCl was sufficient to produce the change in viscosity presented above and that significant perturbations of the viscosity were present at 0.005?0.01 m CaCl₂.     

Recent Progress in Regulation of Aging by Insulin/IGF-1 Signaling in Caenorhabditis elegans

Caenorhabditis elegans has been used as a major model organism to identify genetic factors that regulate organismal aging and longevity. Insulin/insulin-like growth factor 1 (IGF-1) signaling (IIS) regulates aging in many species, ranging from nematodes to humans. C. elegans is a nonpathogenic genetic nematode model, which has been extensively utilized to identify molecular and cellular components that function in organismal aging and longevity. Here, we review the recent progress in the role of IIS in aging and longevity, which involves direct regulation of protein and RNA homeostasis, stress resistance, metabolism and the activities of the endocrine system. We also discuss recently identified genetic factors that interact with canonical IIS components to regulate aging and health span in C. elegans. We expect this review to provide valuable insights into understanding animal aging, which could eventually help develop anti-aging drugs for humans.     

The use of body water, sodium, potassium and calcium content to investigate the nutritional status of first year Atlantic salmon parr in two Scottish Highland streams

Water content of Atlantic salmon parr fell from about 84% at emergence (late May) to just under 79% in September but rose again towards March. Na⁺ content consequently rose from 3·3 mg g⁻¹ dry wt at the beginning of June to 6·2 mg g⁻¹ in early July. It then fell to 4·4 mg g⁻¹ in September, rising again towards March. K⁺ content rose to a maximum in July to stabilize at 16·6 mg g⁻¹ dry wt in September. The resultant Na⁺/K⁺ ratio peaked at 0·43: 1 in mid-June, falling to a minimum in mid-August but rising again in March reflecting changes in the relative proportions of intra and extracellular water. The changes in whole-body chemistry suggest a period of nutritional stress immediately after emergence and during the winter. In streams at higher altitude and of lower nutrient status, nutritional stress during the winter appears to be more severe.     

Increased mitochondrial calcium uptake and concomitant mitochondrial activity by presenilin loss promotes mTORC1 signaling to drive neurodegeneration

Metabolic dysfunction and protein aggregation are common characteristics that occur in age‐related neurodegenerative disease. However, the mechanisms underlying these abnormalities remain poorly understood. We have found that mutations in the gene encoding presenilin in Caenorhabditis elegans, sel‐12, results in elevated mitochondrial activity that drives oxidative stress and neuronal dysfunction. Mutations in the human presenilin genes are the primary cause of familial Alzheimer''s disease. Here, we demonstrate that loss of SEL‐12/presenilin results in the hyperactivation of the mTORC1 pathway. This hyperactivation is caused by elevated mitochondrial calcium influx and, likely, the associated increase in mitochondrial activity. Reducing mTORC1 activity improves proteostasis defects and neurodegenerative phenotypes associated with loss of SEL‐12 function. Consistent with high mTORC1 activity, we find that SEL‐12 loss reduces autophagosome formation, and this reduction is prevented by limiting mitochondrial calcium uptake. Moreover, the improvements of proteostasis and neuronal defects in sel‐12 mutants due to mTORC1 inhibition require the induction of autophagy. These results indicate that mTORC1 hyperactivation exacerbates the defects in proteostasis and neuronal function in sel‐12 mutants and demonstrate a critical role of presenilin in promoting neuronal health.     

Prolonged high-pressure treatments in mammalian skeletal muscle result in loss of functional sodium channels and altered calcium channel kinetics

Activation and inactivation of ion channels involve volume changes from conformational rearrangements of channel proteins. These volume changes are highly susceptible to changes in ambient pressure. Depending on the pressure level, channel function may be irreversibly altered by pressure. The corresponding structural changes persist through the post-decompression phase. High-pressure applications are a useful tool to evaluate the pressure dependence as well as pressure limits for reversibility of such alterations. Mammalian cells are only able to tolerate much lower pressures than microorganisms. Although some limits for pressure tolerance in mammalian cells have been evaluated, the mechanisms of pressure-induced alteration of membrane physiology, in particular of channel function, are unknown. To address this question, we recorded fast inward sodium (I(Na)) and slowly activating L-type calcium (I(Ca)) currents in single mammalian muscle fibers in the post-decompression phase after a prolonged 3-h, high-pressure treatment of up to 20 MPa. I(Na) and I(Ca) peak amplitudes were markedly reduced after pressure treatment at 20 MPa. This was not from a general breakdown of membrane integrity as judged from in situ high-pressure fluorescence microscopy. Membrane integrity was preserved even for pressures as high as 35 MPa at least for pressure applications of shorter durations. Therefore, the underlying mechanisms for the observed amplitude reductions have to be determined from the activation (time-to-peak [TTP]) and inactivation (tau(dec)) kinetics of I(Na) and I(Ca). No major changes in I(Na) kinetics, but marked increases, both in TTP and tau(dec) for I(Ca), were detected after 20 MPa. The apparent molecular volume changes (activation volumes) deltaV(double dagger) for the pressure-dependent irreversible alteration of channel gating approached zero for Na+ channels. For Ca2+ channels, deltaV(double dagger) was very large, with approx 2.5-fold greater values for channel activation than inactivation (approx 210 A3). We conclude, that in skeletal muscle, high pressure differentially and irreversibly affects the gating properties and the density of functional Na+ and Ca2+ channels. Based on these results, a model of high pressure-induced alterations to the channel conformation is proposed.     

Recent structural insights into the expanding world of carbohydrate-active enzymes

Enzymes that catalyse the synthesis and breakdown of glycosidic bonds account for 1-3% of the proteins encoded by the genomes of most organisms. At the current rate, over 12 000 glycosyltransferase and glycoside hydrolase open reading frames will appear during 2006. Recent advances in the study of the structure and mechanism of these carbohydrate-active enzymes reveal that glycoside hydrolases continue to display a wide variety of scaffolds, whereas nucleotide-sugar-dependent glycosyltransferases tend to be grafted onto just two protein folds. The past two years have seen significant advances, including the discovery of a novel NAD+-dependent glycosidase mechanism, the dissection of the reaction coordinate of sialidases and a better understanding of the expanding roles of auxiliary carbohydrate-binding domains.     

Molecular properties of sodium and calcium channels

Voltage-gated sodium and calcium channels are responsible for inward movement of sodium and calcium during electrical signals in cell membranes. Their principal subunits are members of a gene family and can function as voltage-gated ion channels by themselves. They are expressed in association with one or more auxiliary subunits which increase functional expression and modify the functional properties of the principal subunits. Structural elements which are required for voltage-dependent activation, selective ion conductance, and inactivation have been identified, and their mechanisms of action are being explored through mutagenesis, expression in heterologous cells, and functional analysis. These experiments reveal that these two channels are built on a common structural theme with variations appropriate for functional specialization of each channel type.     

Structural and mechanistic insights into MICU1 regulation of mitochondrial calcium uptake

Mitochondrial calcium uptake is a critical event in various cellular activities. Two recently identified proteins, the mitochondrial Ca²⁺ uniporter (MCU), which is the pore‐forming subunit of a Ca²⁺ channel, and mitochondrial calcium uptake 1 (MICU1), which is the regulator of MCU, are essential in this event. However, the molecular mechanism by which MICU1 regulates MCU remains elusive. In this study, we report the crystal structures of Ca²⁺‐free and Ca²⁺‐bound human MICU1. Our studies reveal that Ca²⁺‐free MICU1 forms a hexamer that binds and inhibits MCU. Upon Ca²⁺ binding, MICU1 undergoes large conformational changes, resulting in the formation of multiple oligomers to activate MCU. Furthermore, we demonstrate that the affinity of MICU1 for Ca²⁺ is approximately 15–20 μM. Collectively, our results provide valuable details to decipher the molecular mechanism of MICU1 regulation of mitochondrial calcium uptake.     

Strength of Cu-efflux response in Escherichia coli coordinates metal resistance in Caenorhabditis elegans and contributes to the severity of environmental toxicity

Without effective homeostatic systems in place, excess copper (Cu) is universally toxic to organisms. While increased utilization of anthropogenic Cu in the environment has driven the diversification of Cu-resistance systems within enterobacteria, little research has focused on how this change in bacterial architecture impacts host organisms that need to maintain their own Cu homeostasis. Therefore, we utilized a simplified host–microbe system to determine whether the efficiency of one bacterial Cu-resistance system, increasing Cu-efflux capacity via the ubiquitous CusRS two-component system, contributes to the availability and subsequent toxicity of Cu in host Caenorhabditis elegans nematode. We found that a fully functional Cu-efflux system in bacteria increased the severity of Cu toxicity in host nematodes without increasing the C. elegans Cu-body burden. Instead, increased Cu toxicity in the host was associated with reduced expression of a protective metal stress-response gene, numr-1, in the posterior pharynx of nematodes where pharyngeal grinding breaks apart ingested bacteria before passing into the digestive tract. The spatial localization of numr-1 transgene activation and loss of bacterially dependent Cu-resistance in nematodes without an effective numr-1 response support the hypothesis that numr-1 is responsive to the bacterial Cu-efflux capacity. We propose that the bacterial Cu-efflux capacity acts as a robust spatial determinant for a host’s response to chronic Cu stress.     

Photon radiation-induced structural and functional changes in the myocardium of hypertensive spontaneously hypertensive rats

Hypertensive SHR rates were irradiated with orange-red light using a Korobkov photon light-emitting diode matrix with a maximum radiation at 612 nm; irradiation was performed daily for 1 h for 13 days. After the course of irradiation, the rhythmoinothropic characteristics of the cardiac papillary muscle significantly improved. Morphological analysis revealed active rearrangement in myocytes, which were observed primarily in the structure of the sarcoplasmic reticulum (SR), whose relative area increased more than twice compared to the control. Apparently, photon therapy of hypertensive rats normalizes calcium homeostasis in myocytes and improves the calcium-transport function of SR. The normalization of structural and functional characteristics of the myocardium with hypertensive rates may result from an increase in the SR buffer capacity and activation of SR Ca²⁺-ATPase. Furthermore, qualitative and quantitative changes in the proportion of capillaries, myofibrils, and mitochondria in myocytes indicate the development of adaptive-compensatory processes leading to the activation of biosynthetic processes and an increase in the energy potential of the myocardium.     

Effects of calcium on growth and leaf ion concentrations of Gossypium hirsutum grown in saline hydroponic culture

The optimum Ca²⁺ concentration for growth of cotton (Gossypium hirsutum cv. Acala SJ-2) was in the range 1 to 15 mol m^–3 for plants growing in hydroponic culture with 100–150 mol m^–3 NaCl. Most saline (but not sodic) soils contain higher Ca²⁺ concentrations. CaCl₂ was inhibitory to the growth of cotton above 20–50 mol m^–3. Increasing concentrations of Ca²⁺ in the range 0–2 mol m^–2 drastically reduced Na⁺ accumulation in the leaves. As CaCl₂ concentrations were increased above the optimum for growth there was a further reduction in leaf Na⁺ accumulation, but this was more than offset by increased leaf Ca²⁺ and Cl^– concentrations. Leaf K⁺ concentrations were not much affected by changes in external CaCl₂ concentrations. The response of Mg²⁺ varied from an increase to a decrease with increasing external CaCl₂ and was influenced by nutritional status. There was no evidence that high Ca²⁺ caused a deficiency of Mg²⁺ in cotton. Except for Cl^–, whose concentrations tended to decrease initially and then increase as the CaCl₂ concentration increased, the anions were largely unaffected by changes in external CaCl₂.     

Role of a FMRFamide‐like family of neuropeptides in the pharyngeal nervous system of Caenorhabditis elegans

The nervous system of C. elegans has a remarkable abundance of flp genes encoding FMRFamide‐like (FLP) neuropeptides. To provide insight into the physiological relevance of this neuropeptide diversity, we have tested more than 30 FLPs (encoded by 23 flps) for bioactivity on C. elegans pharynx. Eleven flp genes encode peptides that inhibit pharyngeal activity, while eight flp genes encode peptides that are excitatory. Three potent peptides (inhibitory, FLP‐13A, APEASPFIRFamide; excitatory, FLP‐17A, KSAFVRFamide; excitatory, FLP‐17B, KSQYIRFamide) are encoded by flp genes, which, according to reporter gene constructs, are expressed in pharyngeal motoneurons. Thus, they may act through receptors localized on the pharyngeal muscle. The two other potent peptides, FLP‐8 (excitatory AF1, KNEFIRFamide,) and FLP‐11A (inhibitory, AMRNALVRFamide), appear to be expressed in extrapharyngeal neurons and are therefore likely to act either indirectly or as neurohormones. Intriguingly, a single neuron can express peptides that have potent but opposing biological activity in the pharynx. Only five flp genes encode neuropeptides that have no observable effect on the pharynx, but none of these have shown reporter gene expression in the pharyngeal nervous system. To examine the roles of multiple peptides produced from single precursors, a comparison was made between the bioactivity of different neuropeptides for five flp genes (flp‐3, flp‐13, flp‐14, flp‐17, and flp‐18). For all but one gene (flp‐14), the effects of peptides encoded by the same gene were similar. Overall, this study demonstrates the impressive neurochemical complexity of the simple circuit that regulates feeding in the nematode, C. elegans. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

Expression of the sodium/calcium exchanger in mammalian skeletal muscle cells in primary culture

Previous investigations have demonstrated molecular and functional expression, at early phases of development of skeletal muscle cells in primary culture, of cardiac isoforms of proteins involved in calcium transport and regulation, like the L-type calcium channel. Here the expression of the cardiac isoform of the Na(+)/Ca(2+) exchanger (NCX1) was studied in skeletal muscle cells developing in vitro, by using biochemical, immunological, and electrophysiological techniques. Northern and Western blot experiments revealed the presence of this cardiac exchanger and its increasing expression during the early phases of development. Confocal imaging of myotubes showed an NCX1 distribution that was predominantly sarcolemmal. The whole-cell patch-clamp technique allowed us to record ionic currents, the direction and the amplitude of which depended on extracellular sodium and calcium concentrations. The developmental changes of this functional expression could be correlated with the molecular NCX1 expression changes. Taken together these data demonstrate the presence of the NCX1 isoform of the Na(+)/Ca(2+) exchanger during in vitro myogenesis and reinforce the theory that significant levels of cardiac-type proteins are transiently expressed during the early phases of the skeletal muscle cell development.     

Structural and functional evolution of transthyretin and transthyretin-like proteins

Transthyretin (TTR) is a tetrameric protein involved in the distribution of thyroid hormones in vertebrates. The amino acid sequence of TTR is highly conserved across vertebrates. Hypothetical TTR-like proteins (TLPs) were inferred from the identification of genes in nonvertebrate species. Here, we identified five motifs defining TLPs and three motifs defining both TTRs and TLPs. These motifs were mapped onto structurally conserved and functionally important regions of TTRs. These motifs were used to build hidden Markov models for accurate identification of TLPs in other organisms. TLPs were divided into three main groups based on their N-terminal regions. Most TLPs are cytosolic, but in plants and slime mold, we predict they are peroxisomal. We verified that the TLPs from enterobacteria were periplasmic. We demonstrated that TLP genes are expressed in a bacterium (E. coli), an invertebrate animal (C. elegans), and a plant (A. thaliana). These TLPs have similar subunit molecular weights to TTRs, are tetramers, and are predicted to have similar three-dimensional (3D) structures to TTRs, but do not bind thyroid hormones or similar ligands. We suggest that like TTRs, the N-terminal and C-terminal regions of TLPs are integral in defining the function of TLPs in nonvertebrate species and that the TLP gene duplicated in primitive vertebrates to produce the TTR gene. TLP/TTR has retained its overall structure, but changed function and localization during evolution in bacteria, invertebrates, plants, and vertebrates.     

Effects of membrane potential on sodium-dependent calcium uptake by sarcolemma-enriched preparations from canine ventricle

Summary The effect of membrane potential on sodium-dependent calcium uptake by vesicles in an isolated cardiac sarcolemma preparation was examined. Initial time course studies showed that the reaction deviated from initial velocity conditions within minutes. This appeared to be due, in part, to loss of the sodium gradient. Assays carried out to 10 sec revealed a linear component of uptake (2 to 10 sec) and a faster component (complete by 2 sec). The latter was eliminated by loading the preparation with ethyleneglycol-bis-(-aminoethyl ether)N,N-tetraacetic acid (EGTA). This maneuver did not affect the slow component, and subsequent studies used preparations containing EGTA. Potassium Nernst potentials (E _K), established by potassium gradients in the presence of valinomycin, were varied from –100 to +30 mV by changing [K⁺] _o from 1.18 to 153.7mM ([K⁺] _i =50mM). The initial velocity of sodium-dependent calcium uptake was stimulated twofold by changingE _K from –100 to 0 mV and another twofold by raisingE _K from 0 to +30 mV. For the total range ofE _K and [K⁺] _o , 32 to 36% of the increase appeared to reflect stimulation by extravesicular potassium. The remainder appeared to be due to membrane potential. The profile of sodium-dependent calcium uptake versusE _K suggested that calcium influx through electrogenic sodium/calcium exchange may be much more affected by the positive region of the cardiac action potential than by the negative region.     

Recent insights into cotton functional genomics: progress and future perspectives

Functional genomics has transformed from futuristic concept to well‐established scientific discipline during the last decade. Cotton functional genomics promise to enhance the understanding of fundamental plant biology to systematically exploit genetic resources for the improvement of cotton fibre quality and yield, as well as utilization of genetic information for germplasm improvement. However, determining the cotton gene functions is a much more challenging task, which has not progressed at a rapid pace. This article presents a comprehensive overview of the recent tools and resources available with the major advances in cotton functional genomics to develop elite cotton genotypes. This effort ultimately helps to filter a subset of genes that can be used to assemble a final list of candidate genes that could be employed in future novel cotton breeding programme. We argue that next stage of cotton functional genomics requires the draft genomes refinement, re‐sequencing broad diversity panels with the development of high‐throughput functional genomics tools and integrating multidisciplinary approaches in upcoming cotton improvement programmes.     

Corrupted ER‐mitochondrial calcium homeostasis promotes the collapse of proteostasis

Aging and age‐related diseases are associated with a decline of protein homeostasis (proteostasis), but the mechanisms underlying this decline are not clear. In particular, decreased proteostasis is a widespread molecular feature of neurodegenerative diseases, such as Alzheimer's disease (AD). Familial AD is largely caused by mutations in the presenilin encoding genes; however, their role in AD is not understood. In this study, we investigate the role of presenilins in proteostasis using the model system Caenorhabditis elegans. Previously, we found that mutations in C. elegans presenilin cause elevated ER to mitochondria calcium signaling, which leads to an increase in mitochondrial generated oxidative stress. This, in turn, promotes neurodegeneration. To understand the cellular mechanisms driving neurodegeneration, using several molecular readouts of protein stability in C. elegans, we find that presenilin mutants have widespread defects in proteostasis. Markedly, we demonstrate that these defects are independent of the protease activity of presenilin and that reduction in ER to mitochondrial calcium signaling can significantly prevent the proteostasis defects observed in presenilin mutants. Furthermore, we show that supplementing presenilin mutants with antioxidants suppresses the proteostasis defects. Our findings indicate that defective ER to mitochondria calcium signaling promotes proteostatic collapse in presenilin mutants by increasing oxidative stress.