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Background: Desipramine a representative of tricyclic antidepressants (TCAs) promotes recovery of depressed patients by inhibition of reuptake of neurotransmitters serotonin (SER) and norepinephrine (NE) in the presynaptic membrane by directly blocking their respective transporters SERT and NET. Aims: To study the effect of desipramine on programmed erythrocyte death (eryptosis) and explore the underlying mechanisms. Methods: Phosphatidylserine (PS) exposure on the cell surface as marker of cell death was estimated from annexin-V-binding, cell volume from forward scatter in flow cytometry. Hemolysis was determined photometrically, and intracellular glutathione [GSH]i from high performance liquid chromatography. Results: Desipramine dose-dependently significantly enhanced the percentage of annexin-V-binding cells and didn´t impact glutathione (GSH) synthesis. Desipramine-induced eryptosis was significantly reversed by pre-treatment of erythrocytes with either nitric oxide (NO) donor sodium nitroprusside (SNP) or N-acetyl-L-cysteine (NAC). The highest inhibitory effect was obtained by using both inhibitors together. Calcium (Ca2+) depletion aggravated desipramine-induced eryptosis. Changing the order of treatment, i.e. desipramine first followed by inhibitors, could not influence the inhibitory effect of SNP or NAC. Conclusion: Antidepressants-caused intoxication can be treated by SNP and NAC, respectively. B) Patients with chronic hypocalcemia should not be treated with tricyclic anti-depressants or their dose should be noticeably reduced. [ABSTRACT FROM AUTHOR]

Glutamate-induced excitotoxicity is a pathological basis of many acute/chronic neurodegenerative diseases. Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2b) is a membrane-embedded P-type ATPase pump that manages the translocation of calcium ions (Ca2+) from cytosol into the lumen of the endoplasmic reticulum (ER) calcium stores. It participates in a wide range of biological functions in the central nervous system (CNS). However, the role of SERCA2b in glutamate-induced excitotoxicity and its mechanism must be elucidated. Herein, we demonstrate that SERCA2b mutants exacerbate the excitotoxicity of hypo-glutamate stimulation on HT22 cells. In this study, SERCA2b mutants accelerated Ca2+ depletion through loss-of-function (reduced pumping capacity) or gain-of-function (acquired leakage), resulting in ER stress. In addition, the occurrence of ER Ca2+ depletion increased mitochondria-associated membrane formation, which led to mitochondrial Ca2+ overload and dysfunction. Moreover, the enhancement of SERCA2b pumping capacity or inhibition of Ca2+ leakage attenuated Ca2+ depletion and impeded excitotoxicity in response to hypo-glutamate stimulation. In conclusion, SERCA2b mutants exacerbate ER Ca2+-depletion-mediated excitotoxicity in glutamate-sensitive HT22 cells. The mechanism of disruption is mainly related to the heterogeneity of SERCA2b mutation sites. Stabilization of SRECA2b function is a critical therapeutic approach against glutamate-induced excitotoxicity. These data will expand understanding of organelle regulatory networks and facilitate the discovery and creation of drugs against excitatory/inhibitory imbalance in the CNS. [ABSTRACT FROM AUTHOR]

Glutamate-induced excitotoxicity is a pathological basis of many acute/chronic neurodegenerative diseases. Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2b) is a membrane-embedded P-type ATPase pump that manages the translocation of calcium ions (Ca2+) from cytosol into the lumen of the endoplasmic reticulum (ER) calcium stores. It participates in a wide range of biological functions in the central nervous system (CNS). However, the role of SERCA2b in glutamate-induced excitotoxicity and its mechanism must be elucidated. Herein, we demonstrate that SERCA2b mutants exacerbate the excitotoxicity of hypo-glutamate stimulation on HT22 cells. In this study, SERCA2b mutants accelerated Ca2+ depletion through loss-of-function (reduced pumping capacity) or gain-of-function (acquired leakage), resulting in ER stress. In addition, the occurrence of ER Ca2+ depletion increased mitochondria-associated membrane formation, which led to mitochondrial Ca2+ overload and dysfunction. Moreover, the enhancement of SERCA2b pumping capacity or inhibition of Ca2+ leakage attenuated Ca2+ depletion and impeded excitotoxicity in response to hypo-glutamate stimulation. In conclusion, SERCA2b mutants exacerbate ER Ca2+-depletion-mediated excitotoxicity in glutamate-sensitive HT22 cells. The mechanism of disruption is mainly related to the heterogeneity of SERCA2b mutation sites. Stabilization of SRECA2b function is a critical therapeutic approach against glutamate-induced excitotoxicity. These data will expand understanding of organelle regulatory networks and facilitate the discovery and creation of drugs against excitatory/inhibitory imbalance in the CNS.

This study evaluates the effect of three calcium levels in the diet (normal, moderate, and severe calcium depletion) on bone metabolism of male Wistar rats during their growth period. Bone mineral density (BMD) and femur length were determined in vivo during the growth stage using a single X-ray transmission system. The apparent calcium absorption was calculated in the rat adolescent and adulthood stages. At the end of the experiment, calcium concentrations in serum and urine were analyzed. The bones were evaluated postmortem to corroborate in vivo analyses. Microstructural properties of cortical and trabecular tissues of femurs bones were assessed using scanning electron microscopy. Bone mineral contents (Mg, Ca, P, and K) were quantified by inductively coupled plasma. Severe calcium depletion in the diets in the development stage affects the bone quality parameters such as bone mineral density and mineral content. Moreover, it was found thinner cortical and trabecular bone areas. Additionally, it was found that severe calcium depletion increased the apparent absorption of calcium as a defense mechanism, but with the decrease of the BMD peak, and the thickness of cortical bone as well as trabecular bone porosity. The severe calcium depletion increased the efficiency of apparent absorption calcium as a defense mechanism, but, even so, decreases the BMD peak as well as the thickness of cortical bone and trabecular bone porosity. [ABSTRACT FROM AUTHOR]

As the global burden of cancer is on the rise it is essential to develop alternate therapeutics that confer zero or minimum side effects. In this experiment, we evaluated the anticancer efficacy of graphene oxide-functionalized gold nanoparticles (GO-AuNPs) in human breast cancer cell lines MCF7 and MDA-MB-231. MTT assay revealed a dose-dependent decline in cancer cell survival. Significant alterations were detected in normal cellular morphology after treatment. Two different cell staining methods viz. acridine orange-ethidium bromide staining and annexin-cy3.18/6-carboxyfluorescein diacetate staining confirmed that the GO-AuNPs rendered their detrimental effect in cancer cells through apoptosis. To decipher the mode of apoptosis Western blotting was performed in which expression pattern of several proteins (PARP1, P53, P21, Bcl2, Bax, Caspase 9 and Caspase 3) established the intrinsic apoptosis pathway in these cell lines after GO-AuNP exposure. Intracellular calcium level, as measured by energy dispersive X-ray fluorescence analysis, was present at non-detectable level following treatment that can be linked to the cell cycle arrest and apoptotic cell death in them. Thus, GO-AuNPs were found effective in both types of human breast cancer cells viz. hormone-responsive MCF7 and chemoresistant MDA-MB-231 and can be considered as a highly potential anticancer agent in breast cancer therapy. [ABSTRACT FROM AUTHOR]

Few environmental issues have resulted in such a heated policy-science controversy in Sweden as the 1990s acidification debate in the north of the country. The belief that exceptionally high stream acidity levels during hydrological events was caused by anthropogenic deposition resulted in a governmentally funded, multi-million dollar surface-water liming program. This program was heavily criticized by a large part of the scientific community arguing that the acidity of northern streams was primarily caused by naturally occurring organic acids. Here, we revisit the acid deposition legacy in northern Sweden two decades after the culmination of the controversy by examining the long-term water chemistry trends in the Svartberget/Krycklan research catchment that became a nexus for the Swedish debate. In this reference stream, trends in acidic episodes do show a modest recovery that matches declines in acid deposition to pre-industrial levels, although stream acidity continues to be overwhelmingly driven by organic acidity. Yet there are legacies of acid deposition related to calcium losses from soils, which are more pronounced than anticipated. Finally, assessment of these trends are becoming increasingly complicated by new changes and threats to water resources that must be recognized to avoid unnecessary, expensive, and potentially counterproductive measures to adapt and mitigate human influences. Here we make the argument that while the acidification era is ending, climate change, land-use transitions, and long-range transport of other contaminants warrant close monitoring in the decades to come.

Gammarus fossarum is an often-abundant crustacean detritivore that contributes importantly to leaf litter breakdown in oligotrophic, mainly heterotrophic, headwater streams. This species requires large amounts of Ca to moult, thus allowing growth and reproduction. Because resource quality is tightly coupled to the organism’s growth and physiological status, we hypothesised that low Ca concentration [Ca] and low food resource quality (low phosphorus [P] and/or reduced highly unsaturated fatty acid [HUFA] contents) would interactively impair molecular responses (gene expression) and reproduction of G. fossarum. To investigate the effects of food resources quality, we experimentally manipulated the P content of sycamore leaves and also used diatoms because they contain high amounts of HUFAs. Three resource quality treatments were tested: low quality (LQ, unmanipulated leaves: low P content), high quality 1 (HQ1; P-manipulated leaves: high P content), and high quality 2 (unmanipulated leaves supplemented with a pellet containing diatoms: high P and HUFA content). Naturally, demineralised stream water was supplemented with CaSO4 to obtain three Ca concentrations (2, 3.5, and 10.5 mg Ca L−1). For 21 days, pairs of G. fossarum were individually exposed to one of the nine treatments (3 [Ca] × 3 resource qualities). At the individual level, strong and significant delays in moult stage were observed in gammarids exposed to lower [Ca] and to lower resource quality, with additive effects lengthening the duration of the reproductive cycle. Effects at the molecular level were investigated by measuring expression of 12 genes involved in energy production, translation, or Ca or P homeostasis. Expression of ATP synthase beta (higher in HQ2), calcified cuticle protein (higher in HQ1 and HQ2), and tropomyosin (higher in HQ2 compared to HQ1) was significantly affected by resource quality, and significant additive effects on Ca transporting ATPase expression were induced by [Ca] and resource quality (higher for low [Ca] and higher resource quality). These results highlight the potential drastic deleterious effects of water [Ca] depletion on G. fossarum physiology, populations, and ecosystem functioning, especially in oligotrophic environments. [ABSTRACT FROM AUTHOR]

Milk protein concentrate (MPC) is a high-potential source of milk proteins with significant functional, nutritional, and market appeal. Although many factors affecting MPC's techno-functional properties have been studied, an updated review is still necessary to comprehensively understand MPC's diversification potential and identify research gaps. This review highlights the crucial factors, mechanisms, and strategies to enhance MPC's techno-functional properties, including physical, enzymatic, compositional, and chemical modifications. The review targets three major components for enhancing techno-functional properties: the ionic environment (Ca depletion – colloidal & soluble Ca), casein micelles (solubilization, interaction, association, & aggregation), and powder particle makeup. The partial acidification of skim milk, retentate pH restoration and physical treatment enhance hydration properties. This review provides valuable insights for future research and development of MPC. Caption: Various physical modifications and their effect on the techno-functional properties of milk protein concentrate (MPC). [Display omitted] • Review focuses on technological interventions for improving milk protein concentrate's rehydration, emulsification, gelation, drying & applications. • Ca depletion, micelle solubilization, particles' hydrophilization, & porosity significantly enhance rehydration properties. • Partial acidification of skim milk & pH restoration of retentate show great potential for higher solubility. • Ionic environment modification of buffalo milk results in >97% solubility & >88% Ca depletion in Buffalo-MPC 60. • High hydrostatic pressure preserves lactoferrin & α-lactalbumin bioactivity in human-MPC. [ABSTRACT FROM AUTHOR]

Milk protein concentrate (MPC) powders are increasingly utilized in manufacturing of protein fortified beverages. Thermal stability of the protein dispersions is of significant importance in such applications. It is known that a decrease in pH can induce partial dissociation of casein micelles and modify the natural equilibrium of calcium and phosphate between the micelles and the serum phase. The presence of soluble casein may improve the rehydration properties of MPC powders, and may impact their thermal stability. The objective of this work was to investigate the effects of partial acidification of milk prior to ultrafiltration on the heat stability of reconstituted MPC dispersions. Milk protein concentrate powders were prepared from skim milk acidified to pH 6.0 by addition of glucono-δ-lactone, and then concentrated using ultrafiltration (UF) and diafiltration (DF). The heat stability of the reconstituted MPC dispersions was studied, by determining heat coagulation time, particle size, turbidity, viscosity, soluble and colloidal calcium and phosphate, and non-sedimentable casein both before and after heating at 120 °C. Reconstituted MPC powders made with partially acidified skim milk contained lower soluble calcium and phosphate and exhibited very poor thermal stability compared to MPC powders made with skim milk at its natural pH. The thermal stability of the acidified MPC dispersions was not only recovered by restoration of pH and the serum composition through dialysis against skim milk, but it was improved compared to control MPC dispersions. All dialyzed samples had comparable pH, protein content and calcium and phosphate concentration, but the structure of the casein micelles was altered, causing differences in the type of soluble aggregates. It was concluded that the integrity of the casein micelles and the amount of dissociated, non-sedimentable caseins play a major role in determining the thermal stability of the MPC dispersions. [ABSTRACT FROM AUTHOR]

A soil resampling approach has detected an early stage of recovery in the cation chemistry of spruce forest soil due to reductions in acid deposition. That approach is limited by the lack of soil data and archived soil samples prior to major increases in acid deposition during the latter half of the 20th century. An alternative approach is the dendrochemical analysis of dated wood to detect temporal changes in base cations back into the 19th century. To infer environmental change from dendrochemical patterns of essential base cations, internal factors that affect cation chemistry such as the maturation of sapwood and the spread of wood infection need to be recognized. Potassium concentration was a useful marker of these internal maturation and infection that could affect the concentration of essential base cations in wood. Dendrochemical patterns in samples of red spruce in the eastern United States and Norway spruce in northwestern Russia were used to determine how internal changes in base cations can be separated from external changes in root-zone soil to date major changes in the availability of essential base cations associated with a changing environment. [ABSTRACT FROM AUTHOR]

Acidic deposition caused by fossil fuel combustion has degraded aquatic and terrestrial ecosystems in North America for over four decades. The only management option other than emissions reductions for combating the effects of acidic deposition has been the application of lime to neutralize acidity after it has been deposited on the landscape. For this reason, liming has been a part of acid rain science from the beginning. However, continued declines in acidic deposition have led to partial recovery of surface water chemistry, and the start of soil recovery. Liming is therefore no longer needed to prevent further damage, so the question becomes whether liming would be useful for accelerating recovery of systems where improvement has lagged. As more is learned about recovering ecosystems, it has become clear that recovery rates vary with watershed characteristics and among ecosystem components. Lakes appear to show the strongest recovery, but recovery in streams is sluggish and recovery of soils appears to be in the early stages. The method in which lime is applied is therefore critical in achieving the goal of accelerated recovery. Application of lime to a watershed provides the advantage of increasing Ca availability and reducing or preventing mobilization of toxic Al, an outcome that is beneficial to both terrestrial and aquatic ecosystems. However, the goal should not be complete neutralization of soil acidity, which is naturally produced. Liming of naturally acidic areas such as wetlands should also be avoided to prevent damage to indigenous species that rely on an acidic environment. [ABSTRACT FROM AUTHOR]

Genetic strategies such as gene disruption and fluorescent protein tagging largely contribute to understanding the molecular mechanisms of biological functions in bacteria. However, the methods for gene replacement remain underdeveloped for the filamentous bacteria Leptothrix cholodnii SP-6. Their cell chains are encased in sheath composed of entangled nanofibrils, which may prevent the conjugation for gene transfer. Here, we describe a protocol optimized for gene disruption through gene transfer mediated by conjugation with Escherichia coli S17-1 with details on cell ratio, sheath removal, and loci validation. The obtained deletion mutants for specific genes can be used to clarify the biological functions of the proteins encoded by the target genes. Graphical overview., Competing Interests: Competing interestsThe authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper., (Copyright © 2023 The Authors.)

Milk protein concentrate powders (MPC) with improved rehydration properties are often manufactured using processing steps, such as acidification and high-pressure processing, and with addition of other ingredients, such as sodium chloride, during their production. These steps are known to increase the amount of serum caseins or modify the mineral equilibrium, hence improving solubility of the retentates. The processing functionality of the micelles may be affected. The aim of this study was to investigate the effects of partial acidification by adding glucono-δ-lactone (GDL) to skim milk during membrane filtration on the structural changes of the casein micelles by observing their chymosin-induced coagulation behavior, as such coagulation is affected by both the supramolecular structure of the caseins and calcium equilibrium. Milk protein concentrates were prepared by preacidification with GDL to pH 6 using ultrafiltration (UF) and diafiltration (DF) followed by spray-drying. Reconstituted UF and DF samples (3.2% protein) treated with GDL showed significantly increased amounts of soluble calcium and nonsedimentable caseins compared with their respective controls, as measured by ion chromatography and sodium dodecyl sulfate-PAGE electrophoresis, respectively. The primary phase of chymosin-induced gelation was not significantly different between treatments as measured by the amount of caseino-macropeptide released. The rheological properties of the reconstituted MPC powders were determined immediately after addition of chymosin, both before and after dialysis against skim milk, to ensure similar serum composition for all samples. Reconstituted samples before dialysis showed no gelation (defined as tan δ = 1), and after re-equilibration only control UF and DF samples showed gelation. The gelation properties of reconstituted MPC powders were negatively affected by the presence of soluble casein, and positively affected by the amount of both soluble and insoluble calcium present after reconstitution. This work, testing the chymosin-induced gelation behavior of various reconstituted MPC samples, clearly demonstrated that a decrease in pH to 6.0 during membrane filtration affects the integrity of the casein micelles supramolecular structure with important consequences to their processing functionality. [ABSTRACT FROM AUTHOR]

An appetite for CaCl 2 and NaCl occurs in young rats after they are fed a diet lacking Ca or Na, respectively. Bilateral lesions of the parabrachial nuclei (PBN) disrupt normal taste aversion learning and essentially eliminate the expression of sodium appetite. Here we tested whether similar lesions of the PBN would disrupt the calcium-deprivation-induced appetite for CaCl 2 or NaCl. Controls and rats with PBN lesions failed to exhibit a calcium-deprivation-induced appetite for CaCl 2 . Nevertheless, both groups did exhibit a significant calcium-deprivation-induced appetite for 0.5 M NaCl. Thus, while damage to the second central gustatory relay in the PBN disrupts the appetite for 0.5 M NaCl induced by furosemide, deoxycorticosterone acetate, and polyethylene glycol, the sodium appetite induced by dietary CaCl 2 depletion remains intact. [ABSTRACT FROM AUTHOR]

Ecological evidence indicates a worldwide trend of dramatically decreased soil Ca2+ levels caused by increased acid deposition andmassive timber harvesting. Little is known about the genetic and cellularmechanismof plants' responses to Ca2+ depletion. In this study, transcriptional profiling analysis helped identify multiple extracellular Ca2+ ([Ca2+]ext) depletion-responsive genes in Arabidopsis thaliana L.,many ofwhich are involved in response to other environmental stresses. Interestingly, a group of genes encoding putative cytosolic Ca2+ ([Ca2+]cyt) sensors were significantly upregulated, implying that [Ca2+]cyt has a role in sensing [Ca2+]ext depletion. Consistent with this observation, [Ca2+]ext depletion stimulated a transient rise in [Ca2+]cyt that was negatively influenced by [K+]ext, suggesting the involvement of amembrane potential-sensitive component. The [Ca2+]cyt response to [Ca2+]ext depletion was significantly desensitized after the initial treatment, which is typical of a receptor-mediated signaling event. The response was insensitive to an animal Ca2+ sensor antagonist, but was suppressed by neomycin, an inhibitor of phospholipase C. Gd3+, an inhibitor of Ca2+ channels, suppressed the [Ca2+]ext-triggered rise in [Ca2+]cyt and downstream changes in gene expression. Taken together, this study demonstrates that [Ca2+]cyt plays an important role in the putative receptor-mediated cellular and transcriptional response to [Ca2+]ext depletion of plant cells. [ABSTRACT FROM AUTHOR]

Ecological evidence indicates a worldwide trend of dramatically decreased soil Ca2+ levels caused by increased acid deposition andmassive timber harvesting. Little is known about the genetic and cellularmechanismof plants' responses to Ca2+ depletion. In this study, transcriptional profiling analysis helped identify multiple extracellular Ca2+ ([Ca2+]ext) depletion-responsive genes in Arabidopsis thaliana L.,many ofwhich are involved in response to other environmental stresses. Interestingly, a group of genes encoding putative cytosolic Ca2+ ([Ca2+]cyt) sensors were significantly upregulated, implying that [Ca2+]cyt has a role in sensing [Ca2+]ext depletion. Consistent with this observation, [Ca2+]ext depletion stimulated a transient rise in [Ca2+]cyt that was negatively influenced by [K+]ext, suggesting the involvement of amembrane potential-sensitive component. The [Ca2+]cyt response to [Ca2+]ext depletion was significantly desensitized after the initial treatment, which is typical of a receptor-mediated signaling event. The response was insensitive to an animal Ca2+ sensor antagonist, but was suppressed by neomycin, an inhibitor of phospholipase C. Gd3+, an inhibitor of Ca2+ channels, suppressed the [Ca2+]ext-triggered rise in [Ca2+]cyt and downstream changes in gene expression. Taken together, this study demonstrates that [Ca2+]cyt plays an important role in the putative receptor-mediated cellular and transcriptional response to [Ca2+]ext depletion of plant cells. [ABSTRACT FROM AUTHOR]

Abstract: Calcium (Ca2+) has a crucial role in maintaining the intestinal protease activity and in forming the apical junctional complex (AJC) that preserves epithelial barrier function. Ethylene glycol tetraacetic acid (EGTA) is a Ca2+-specific chelating agent. To maintain the concentration of this chelator in areas where enzyme inhibition and paracellular permeation enhancement are needed, this study synthesized a poly(γ-glutamic acid)–EGTA conjugate (γPGA–EGTA) to form nanoparticles (NPs) with chitosan (CS) for oral insulin delivery. The results of our molecular dynamic (MD) simulations indicate that Ca2+ ions could be specifically chelated to the nitrogen atoms, ether oxygen atoms, and carboxylate oxygen atoms in [Ca(EGTA)]2− anions. By chelating Ca2+, γPGA–EGTA conferred a significant insulin protection effect against proteases in intestinal tracts isolated from rats. Additionally, calcium depletion by γPGA–EGTA could stimulate the endocytosis of AJC components in Caco-2 cell monolayers, which led to a reversible opening of AJCs and thus increased their paracellular permeability. Single-photon emission computed tomography images performed in the biodistribution study clearly show the 123I-insulin orally delivered by CS/γPGA–EGTA NPs in the heart, aorta, renal cortex, renal pelvis and liver, which ultimately produced a significant and prolonged hypoglycemic effect in diabetic rats. The above results confirm that this γPGA–EGTA conjugate is a promising candidate for oral insulin delivery. [Copyright &y& Elsevier]

Forest soil acidification and depletion of nutrient cations have been reported for several forested regions in North America, predominantly in the eastern United States, including the northeast and in the central Appalachians, but also in parts of southeastern Canada and the southern U.S. Continuing regional inputs of nitrogen and sulfur are of concern because of leaching of base cations, increased availability of soil Al, and the accumulation and ultimate transmission of acidity from forest soils to streams. Losses of calcium from forest soils and forested watersheds have now been documented as a sensitive early indicator and a functionally significant response to acid deposition for a wide range of forest soils in North America. For red spruce, a clear link has been established between acidic deposition, alterations in calcium and aluminum supplies and increased sensitivity to winter injury. Cation depletion appears to contribute to sugar maple decline on some soils, specifically the high mortality rates observed in northern Pennsylvania over the last decade. While responses to liming have not been systematically examined in North America, in a study in Pennsylvania, restoring basic cations through liming increased basal area growth of sugar maple and levels of calcium and magnesium in soil and foliage. In the San Bernardino Mountains in southern California near the west coast, the pH of the A horizon has declined by at least 2 pH units (to pH 4.0-4.3) over the past 30 years, with no detrimental effects on bole growth; presumably, because of the Mediterranean climate, base cation pools are still high and not limiting for plant growth.

Abstract: Extracellular calcium is crucial for functioning of the epithelial barrier. Compounds that bind calcium, reducing its extracellular levels, have therefore been investigated as mucosal absorption enhancers. However, the conditions under which calcium reduction sufficiently modulates the epithelial barrier to result in meaningful improvements in mucosal drug absorption are unclear. Present work investigated the settings in which calcium depletion leads to optimal epithelial barrier-modulating effects. Using Calu-3 and Caco-2 cell layers and inducing calcium depletion site-specifically (apically, basolaterally or on both sides) we demonstrate that apical calcium removal produces a modest effect on the tight junctions (the extent of the effect being dependent on the duration of apical calcium unavailability), whilst basolateral calcium exhaustion leads to a prominent effect on the epithelial barrier. However, using polyacrylic acid as an example, we show that polymeric calcium-binding agents proposed as mucosal absorption-enhancing excipients alter calcium levels exclusively on the apical side of the epithelium, which explains their modest effect on epithelial barrier modulation (also demonstrated in our work). Therefore the use of calcium-depleting agents, especially those based on macromolecular polymers, is a relatively inefficacious strategy to promote mucosal absorption of macromolecules. [Copyright &y& Elsevier]

Red spruce ( Picea rubens Sarg.) trees are uniquely vulnerable to foliar freezing injury during the cold season (fall and winter), but are also capable of photosynthetic activity if temperatures moderate. To evaluate the influence of calcium (Ca) addition on the physiology of red spruce during the cold season, we measured concentrations of foliar polyamines and free amino acids (putative stress-protection compounds), chlorophyll (a key photosystem component), and sapwood area (a proxy for foliar biomass), for trees in Ca-addition (CaSiO added) and Ca-depleted (reference) watersheds at the Hubbard Brook Experimental Forest (NH, USA). Ca-addition increased concentrations of the amino acids alanine and γ-aminobutyric acid (GABA) and the polyamines putrescine (Put) and spermidine (Spd) in November, and Put in February relative to foliage from the reference watershed. Consistent with increased stress protection, foliage from the Ca-addition watershed had higher total chlorophyll and chlorophyll a concentrations in February than foliage from the reference watershed. In contrast, foliage from the reference watershed had significantly lower glutamic acid (Glu) and higher alanine (Ala) concentrations in February than foliage from the Ca-addition watershed. Imbalances in Ala:Glu have been attributed to cold sensitivity or damage in other species. In addition to concentration-based differences in foliar compounds, trees from the Ca-addition watershed had higher estimated levels of foliar biomass than trees from the reference watershed. Our findings suggest that Ca-addition increased the stress tolerance and productive capacity of red spruce foliage during the cold season, and resulted in greater crown mass compared to trees growing on untreated soils. [ABSTRACT FROM AUTHOR]

Forest herbs account for greater species richness than any other plant type in deciduous forests and are the most vulnerable to anthropogenic disturbances. We examined whether the limited distribution of rare Appalachian forest herbs in Nova Scotia is related to edaphic specialization or a history of anthropogenic disturbance. Remnant populations are restricted to floodplain forest, where both habitat factors and disturbance history differ significantly from those of adjacent upland sugar maple forest. Contrasting soil and litter layers between floodplain stands and adjacent upland sites revealed the latter to be deficient in key cations (calcium, magnesium, boron); however, regression models for uplands and for floodplains showed that native herb richness was related to soil fertility in each case. Soil calcium accounted for most of the species richness variation among floodplains for native herbs and for a large seeded guild that contains most of the rare species on floodplains. Given the widespread anthropogenic decalcification of forest soils throughout eastern North America, conservation efforts must ( i) increase and connect deciduous forest floodplain ecosystems and ( ii) understand how to manage and create suitable cation-rich migration corridors in the forest landscape. [ABSTRACT FROM AUTHOR]

Aim: Cardiac basal metabolism upon extracellular calcium removal and its relationship with intracellular sodium and calcium homeostasis was evaluated. Methods: A mechano-calorimetric technique was used that allowed the simultaneous and continuous measurement of both heat rate and resting pressure in arterially perfused quiescent adult rat hearts. Using pharmacological tools, the possible underlying mechanisms related to sodium and calcium movements were investigated. Results: Resting heat rate (expressed in mW g−1dry wt) increased upon calcium withdrawal (+4.4 ± 0.2). This response was: (1) unaffected by the presence of tetrodotoxin (+4.3 ± 0.6), (2) fully blocked by both, the decrease in extracellular sodium concentration and the increase in extracellular magnesium concentration, (3) partially blocked by the presence of either nifedipine (+2.8 ± 0.4), KB-R7943 (KBR; +2.5 ± 0.2), clonazepam (CLO; +3.1 ± 0.3) or EGTA (+1.9 ± 0.3). The steady heat rate under Ca2+-free conditions was partially reduced by the addition of Ru360 (−1.1 ± 0.2) but not CLO in the presence of EGTA, KBR or Ru360. Conclusion: Energy expenditure for resting state maintenance upon calcium withdrawal depends on the intracellular rise in both sodium and calcium. Our data are consistent with a mitochondrial Ca2+ cycling, not detectable under normal calcium diastolic levels. The experimental condition here analysed, partially simulates findings reported under certain pathological situations including heart failure in which mildly increased levels of both diastolic sodium and calcium have also been found. Therefore, under such pathological conditions, hearts should distract chemical energy to fuel processes associated with sodium and calcium handling, making more expensive the maintenance of their functions. [ABSTRACT FROM AUTHOR]

In fall (November 2005) and winter (February 2006), we collected current-year foliage of native red spruce (Picea rubens Sarg.) growing in a reference watershed and in a watershed treated in 1999 with wollastonite (CaSiO3, a slowrelease calcium source) to simulate preindustrial soil calcium concentrations (Ca-addition watershed) at the Hubbard Brook Experimental Forest (Thornton, NH). We analyzed nutrition, soluble sugar concentrations, ascorbate peroxidase (APX) activity and cold tolerance, to evaluate the basis of recent (2003) differences between watersheds in red spruce foliar winter injury. Foliar Ca and total sugar concentrations were significantly higher in trees in the Ca-additionwatershed than in trees in the reference watershed during both fall (P = 0.037 and 0.035, respectively) and winter (P = 0.055 and 0.036, respectively). The Ca-addition treatment significantly increased foliar fructose and glucose concentrations in November (P = 0.013 and 0.007, respectively) and foliar sucrose concentrations in winter (P = 0.040). Foliar APX activity was similar in trees in both watersheds during fall (P = 0.28), but higher in trees in the Ca-addition watershed during winter (P = 0.063). Cold tolerance of foliage was significantly greater in trees in the Ca-addition watershed than in trees in the reference watershed (P < 0.001). Our results suggest that low foliar sugar concentrations and APX activity, and reduced cold tolerance in trees in the reference watershed contributed to their high vulnerability to winter injury in 2003. Because the reference watershed reflects forest conditions in the region, the consequences of impaired physiological function caused by soil Ca depletion may have widespread implications for forest health. [ABSTRACT FROM AUTHOR]

Measuring calcium (Ca2+) absorption, Ca2+ balance, serum, urinary and fecal Ca2+ levels, the aim of this work was to show if periodic hypokinesia (PHK) or continuous hypokinesia (CHK) could depress significantly more Ca2+ deposition leading to Ca2+ losses and thus Ca2+ imbalance. Studies were conducted during 30-day pre-hypokinesia (HK) and during 364-day PHK and CHK. Thirty male healthy volunteers 23.2 ± 5.0 years of age were chosen as subjects. They were equally divided into 3 groups: unrestricted active control subjects (UACS), continuously hypokinetic subjects (CHKS) and periodically hypokinetic subjects (PHKS). UACS were running average distances of 4.7 ± 1.2 km.day-1 for 364 days. CHKS were limited to average walking distances of 0.5 ± 0.2 km.day-1 for 364 days, while PHKS were limited to average walking distances of 0.5 ± 0.2 km.day-1 and running distances of 4.7 ± 1.2 km.day-1 for 5 days and 2 days/week for 364 days, respectively. Subjects consumed daily 15 mmol calcium lactate/kg body weight. Ca2+ absorption, fecal Ca2+ excretion, urinary Ca2+ and phosphate (P) excretion, serum Ca2+, P and ionized calcium (Cal) levels, Ca2+ balance and serum 1,25 dihydroxyvitamin D(1,25(OH)2D3) and intact parathyroid hormone (iPTH) levels did not change in UACS compared with their pre-HK values. Conversely, Ca2+ absorption, serum 1 ,25(OH)2D3 and iPTH levels decreased significantly (p <0.05) with time, while Ca2+ imbalance, fecal Ca2+ loss, urinary Ca2+ and P losses and serum Ca2+, Cal and P levels increased significantly (p < 0.05) with time in CHKS and PHKS compared with their pre-HK values and their active controls (UACS). However, Ca2+ absorption, serum 1,25(OH)2D3 and iPTH levels decreased significantly (p <0.05) more with time, and Ca2+ imbalance, fecal Ca2+ excretion, urinary Ca2+ and P excretion and serum Ca2+, Cal and P levels increased significant (p <0.05) more with time in PHKS than CHKS. Dissociation between Ca2+ excretion and Ca2+ imbalance showed decreased Ca2+ deposition as the mechanism for Ca2+ depletion during PHK and CHK. The lower Ca2+ absorption with higher Ca2+ imbalance in PHKS than CHKS showed that Ca2+ deposition decreased more during PHK than CHK. It was concluded that the higher the instability of muscular activity, the lower Ca2+ deposition and the higher Ca2+ losses and the greater Ca2+ imbalance. [ABSTRACT FROM AUTHOR]

Permeability estimates using Caco-2 cells do not accurately predict the absorption of hydrophilic drugs that are primarily absorbed via the paracellular pathway. The objective of this study was to investigate whether modulation of tight junctions would help differentiation of paracellularly absorbed compounds. Tight junctions in Caco-2 cell monolayers were manipulated using calcium depletion approaches to decrease the transepithelial electrical resistance (TEER) of the monolayers, and permeability of hydrophilic compounds were measured under these conditions. Permeability of these compounds were also measured in Calu-3 cells, which have tighter junctions than Caco-2 cells. Calcium depletion loosened the tight junctions of Caco-2 cells to varying levels as measured by the decrease in TEER values of the monolayers. While the absolute permeability of all the model compounds increased as the tight junctions were loosened, the ratios of their permeability relative to mannitol permeability were similar. The permeability of these compounds in the tighter Calu-3 cells were also found to be similar to each other. Altering the tight junctions of Caco-2 cells to obtain leakier cell monolayers, or using a cell line with tighter junctions like Calu-3 cells, did not improve differentiation between well absorbed and poorly absorbed hydrophilic drugs. Mere manipulation of the tight junctions to increase or decrease transepithelial electrical resistance does not appear to be a viable approach to predict human absorption for hydrophilic compounds that are primarily absorbed via the paracellular pathway. [ABSTRACT FROM AUTHOR]

Voltage-gated Ca2+ channels activated by action potentials evoke Ca2+ entry into presynaptic terminals thus briefly distorting the resting Ca2+ concentration. When this happens, a number of processes are initiated to re-establish the Ca2+ equilibrium. During the post-spike period, the increased Ca2+ concentration could enhance the presynaptic Ca2+ signalling. Some of the mechanisms contributing to presynaptic Ca2+ dynamics involve endogenous Ca2+ buffers, Ca2+ stores, mitochondria, the sodium–calcium exchanger, extraterminal Ca2+ depletion and presynaptic receptors. Additionally, subthreshold presynaptic depolarization has been proposed to have an effect on release of neurotransmitters through a mechanism involving changes in resting Ca2+. Direct evidence for the role of any of these participants in shaping the presynaptic Ca2+ dynamics comes from direct recordings of giant presynaptic terminals and from fluorescent Ca2+ imaging of axonal boutons. Here, some of this evidence is presented and discussed. [ABSTRACT FROM AUTHOR]

Abstract: Versatile peroxidase (VP) from Bjerkandera adusta, as other class II peroxidases, is inactivated by Ca2+ depletion. In this work, the spectroscopic characterizations of Ca2+-depleted VP at pH 4.5 (optimum for activity) and pH 7.5 are presented. Previous works on other ligninolytic peroxidases, such as lignin peroxidase and manganese peroxidase, have been performed at pH 7.5; nevertheless, at this pH these enzymes are inactive independently of their Ca2+ content. At pH 7.5, UV-Vis spectra indicate a heme-Fe3+ transition from 5-coordinated high-spin configuration in native peroxidase to 6-coordinated low-spin state in the inactive Ca2+-depleted form. This Fe3+ hexa-coordination has been proposed as the origin of inactivation. However, our results at pH 4.5 show that Ca2+-depleted enzyme has a high spin Fe3+. EPR measurements on VP confirm the differences in the Fe3+ spin states at pH 4.5 and at 7.5 for both, native and Ca2+-depleted enzymes. In addition, EPR spectra recorded after the addition of H2O2 to Ca2+-depleted VP show the formation of compound I with the radical species delocalized on the porphyrin ring. The lack of radical delocalization on an amino acid residue exposed to solvent, W170, as determined in native enzyme at pH 4.5, explains the inability of Ca2+-depleted VP to oxidize veratryl alcohol. These observations, in addition to a notorious redox potential decrease, suggest that Ca2+-depleted versatile peroxidase is able to form the active intermediate compound I but its long range electron transfer has been disrupted. [Copyright &y& Elsevier]

Abstract: The sulfur-containing amino acid taurine is an inhibitory neuromodulator in the brain of mammals, as well as a key substance in the regulation of cell volumes. The effect of Ca2+ on extracellular taurine concentrations is of special interest in the context of the regulatory mechanisms of taurine release. The aim of this study was to characterize the basal release of taurine in Ca2+-free medium using in vivo microdialysis of the striatum of anesthetized rats. Perfusion of Ca2+-free medium via a microdialysis probe evoked a sustained release of taurine (up to 180 % compared to the basal levels). The Ca2+ chelator EGTA (1mM) potentiated Ca2+ depletion-evoked taurine release. The substitution of CaCl2 by choline chloride did not alter the observed effect. Ca2+-free solution did not significantly evoke release of taurine from tissue loaded with the competitive inhibitor of taurine transporter guanidinoethanesulfonate (1mM), suggesting that in Ca2+ depletion taurine is released by the transporter operating in the outward direction. The volume-sensitive chloride channel blocker diisothiocyanostilbene-2,2′-disulfonate (1mM) did not attenuate the taurine release evoked by Ca2+ depletion. The non-specific blocker of voltage-sensitive Ca2+ channels NiCl2 (0.65mM) enhanced taurine release in the presence of Ca2+. CdCl2 (0.25mM) had no effect under these conditions. However, both CdCl2 and NiCl2 attenuated the effect of Ca2+-free medium on the release of taurine. The data obtained imply the involvement of both decreased influx of Ca2+ and increased non-specific influx of Na+ through voltage-sensitive calcium channels in the regulation of transporter-mediated taurine release in Ca2+ depletion. [Copyright &y& Elsevier]

Abstract: An experimental system was developed to assess and rank the performance of commercial antiscalants designed to inhibit mineral scale formation on reverse osmosis membrane surfaces. The study focused on calcium sulfate dihydrate (gypsum) precipitation to illustrate an approach to antiscalant ranking based on its ability to retard the observed onset (i.e., induction time) of gypsum crystallization from supersaturated solutions. The experimental method consisted of on-line monitoring solution turbidity rise and calcium ion concentration decline to signal the onset of gypsum crystallization. Significant differences were found among commercial antiscalants with respect to achievable levels of induction time retardation. Moreover, antiscalant effectiveness with respect to dosage also varied significantly among antiscalants. The present approach enables a relatively simple and robust initial screening of candidate antiscalants based on their induction time as a function of the applied dose. [Copyright &y& Elsevier]

Measuring calcium (Ca) absorption, Ca balance and Ca level in serum,feces and urine during HK (hypokinesia) with and without Ca loading, the aim of this study was to disclose if prolonged HK could reduce Ca deposition more with or without Ca load contributing to greater Ca imbalance. Studies were conducted during 30-days pre-HK and 364-days HK. Forty male normal volunteers 23.7 ± 6.0 years of age were chosen as subjects. They were divided into four groups: unloaded active control subjects (UACS), unloaded hypokinetic subjects (UHKS), loaded active control subjects (LACS), loaded hypokinetic subjects (LHKS). All hypokinetic subjects were walking average distances of 0.5 ± 0.2 km day−1, and active control subjects were running average distances of 6.6 ± 1.2 km day−1. LACS and LHKS were loaded with 1.3 mmol calcium lactate/kg body wt. Before Ca load, fecal Ca loss, urinary Ca and phosphate (P) losses, Ca imbalance, serum ionized calcium (Ca I), P and total Ca (Ca t) levels increased significantly. ( P < 0.05) with time, and serum intact parathyroid hormone (iPTH), 1.25 dihydroxyvitamin D (1.25(OH)2D3) levels and Ca absorption, decreased significantly ( P < 0.05) with time in LHKS and UHKS compared with their pre-HK values and their respective active controls (LACS and UACS). After Ca load, however, Ca absorption, serum iPTH and 1.25 (OH)2D3 levels decreased significantly ( P < 0.05) more with time, while fecal Ca loss, urinary Ca and P excretion and Ca imbalance increased significantly ( P < 0.05) more with time in LHKS than UHKS. Conversely, before and after Ca load, fecal Ca excretion, urinary P and Ca loss, serum CaI, P, Ca, iPTH and 1.25 (OH)2D3 levels, Ca absorption and Ca balance did not change in LACS and UACS compared with their pre-HK values. The greater Ca losses with than without Ca load have shown that the more Ca is consumed the more Ca is eliminated during HK and Ca imbalance. The significant increase of Ca loss with Ca imbalance demonstrated reduced Ca deposition. Dissociation between Ca loss and Ca imbalance demonstrated reduced Ca deposition as the mechanism of Ca imbalance development during HK. [ABSTRACT FROM AUTHOR]

Soil acidity and calcium (Ca) availability in the surface soil differ substantially beneath sugar maple ( Acer saccharum) and eastern hemlock ( Tsuga canadensis) trees in a mixed forest in northwestern Connecticut. We determined the effect of pumping of Ca from deep soil (rooting zone below 20-cm mineral soil) to explain the higher available Ca content in the surface soil beneath sugar maple. We measured the atmospheric input of Ca with bulk deposition collectors and estimated Ca weathering and Ca mineralization in the surface soil (rooting zone above 20-cm mineral soil) from strontium isotope measurements and observed changes in exchangeable Ca in soils during field incubation. Calcium leaching at 20 cm was calculated by combining modeled hydrology with measured Ca soil solution concentrations at 20-cm depth. We measured root length distribution with depth beneath both tree species. Calcium leaching from the surface soil was much higher beneath sugar maple than hemlock and was positively related with the amount of Ca available in the surface soil. Calcium leaching from the surface soil beneath sugar maple was higher than the combined Ca input from atmospheric deposition and soil weathering. Without Ca uptake in the deep soil, surface soils are being depleted in Ca, especially beneath sugar maple. More organically bound Ca was mineralized beneath sugar maple than beneath hemlock. A relatively small part of this Ca release was leached below the surface soil, suggesting that, beneath both tree species, most of the Ca cycling is occurring in the surface soil. Sugar maple had more fine roots in the deep soil than hemlock and a greater potential to absorb Ca in the deep soil. With a simple model, we showed that a relatively small amount of Ca uptake in the deep soil beneath sugar maple is able to sustain high amounts of available Ca in the surface soil. [ABSTRACT FROM AUTHOR]

Episodic acidification of streams, identified in the late 1980s as one of the most significant environmental problems caused by acidic deposition, had not been evaluated since the early 1990s despite decreasing levels of acidic deposition over the past decade. This analysis indicates that episodic acidification of streams in upland regions in the northeastern United States persists, and is likely to be much more widespread than chronic acidification. Depletion of exchangeable Ca in the mineral soil has decreased the neutralization capacity of soils and increased the role of the surface organic horizon in the neutralization of acidic soil water during episodes. Increased accumulation of N and S in the forest floor from decades of acidic deposition will delay the recovery of soil base status, and therefore, the elimination of acidic episodes, which is anticipated from decreasing emissions. [Copyright &y& Elsevier]

Glutamate-induced excitotoxicity is a pathological basis of many acute/chronic neurodegenerative diseases. Sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA2b) is a membrane-embedded P-type ATPase pump that manages the translocation of calcium ions (Ca 2+ ) from cytosol into the lumen of the endoplasmic reticulum (ER) calcium stores. It participates in a wide range of biological functions in the central nervous system (CNS). However, the role of SERCA2b in glutamate-induced excitotoxicity and its mechanism must be elucidated. Herein, we demonstrate that SERCA2b mutants exacerbate the excitotoxicity of hypo-glutamate stimulation on HT22 cells. In this study, SERCA2b mutants accelerated Ca 2+ depletion through loss-of-function (reduced pumping capacity) or gain-of-function (acquired leakage), resulting in ER stress. In addition, the occurrence of ER Ca 2+ depletion increased mitochondria-associated membrane formation, which led to mitochondrial Ca 2+ overload and dysfunction. Moreover, the enhancement of SERCA2b pumping capacity or inhibition of Ca 2+ leakage attenuated Ca 2+ depletion and impeded excitotoxicity in response to hypo-glutamate stimulation. In conclusion, SERCA2b mutants exacerbate ER Ca 2+ -depletion-mediated excitotoxicity in glutamate-sensitive HT22 cells. The mechanism of disruption is mainly related to the heterogeneity of SERCA2b mutation sites. Stabilization of SRECA2b function is a critical therapeutic approach against glutamate-induced excitotoxicity. These data will expand understanding of organelle regulatory networks and facilitate the discovery and creation of drugs against excitatory/inhibitory imbalance in the CNS., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhang, Ye, Pang, Kessi, Xiong, Chen, Peng, Yang and Yin.)

In a graded model (minimal, subtotal, total) of the calcium paradox phenomenon induced by a progressive increase in the flow rate and volume (5 ml, 10 ml, 45 ml) of calcium-free perfusion (5 min) the severity of tissue injury on calcium repletion was related to a potential elevation of cAMP during calcium depletion. In the subtotal and total models of the calcium paradox a 50% increase was found for tissue cAMP after calcium-free perfusion, but no such rise could be associated with the minimal calcium paradox. In the study tissue injury, as assessed by whole tissue and mitochondrial accumulation of calcium and by myocardial leakage of creatine kinase, could only partly be related to cAMP changes. It is concluded that calcium-free coronary perfusion induces a complex series of events favouring excessive calcium entry, only one of which may be related to a change in cAMP. [ABSTRACT FROM AUTHOR]

The changes in myocardial energy metabolism of isolated perfused Rana ridibunda hearts subjected to prolonged calcium depletion and reperfusion with calcium-containing medium were studied. Calcium-free perfusion resulted in an increase in the concentrations of glucose, glucose-6-phosphate, a-ketoglutarate and malate. The myocardial contents of high-energy phosphates were maintained while concentrations of key amino acids were significantly altered. During the reperfusion period the tissue high-energy phosphate content fell abruptly. A marked increase in glycolytic flux and lactate production was observed. The tissue contents of citric acid cycle intermediates and key amino acids decreased. Examination of the activities of marker enzymes during the calcium-free and reperfusion periods showed that only cytoplasmic enzymes are lost during reperfusion, while the activities of other enzymes remained unchanged. The results suggest that the fluxes of both glycolysis and the citric acid cycle are significantly altered during calcium depletion and following repletion in the amphibian heart. The major characteristics of calcium paradox-induced damage in Rana ridibunda heart are the depletion of high-energy stores, the impairment of mitochondrial oxidative metabolism, and a significant increase in anaerobic metabolism. [ABSTRACT FROM AUTHOR]

The present study examined the usefulness of the serum alkaline phosphatase (ALP) activity, osteocalcin, and tartrate-resistant acid phosphatase (TrACP) activity as bone turnover markers in a calcium depletion/repletion rat model. Weanling rats were fed calcium-deficient diet for 4 weeks, followed by 2 weeks of dietary calcium repletion. Serum phosphatases and osteocalcin were determined and compared with those of corresponding age-matched, pair-weighted controls. Rats were sacrificed at the end of each phase of the study, and bone phosphatase activities in tibiae and vertebrae were measured. During calcium depletion, rats developed hypocalcemia and lost significant bone calcium, which were reversed with dietary calcium repletion. During depletion when previously published histologic studies indicated a suppressed bone formation and stimulated bone resorption, serum ALP activity and osteocalcin levels were significantly elevated and serum TrACP activity reduced; at the same time, the bone ALP and TrACP activities were increased. Because the serum level of 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) which has been shown to stimulate the synthesis of skeletal ALP and of osteocalcin, was also significantly increased during depletion, the increased serum ALP and osteocalcin level could be indirect consequences of the hypocalcemia-mediated elevation in 1,25(OH)2D3 level. These effects were reversed upon calcium repletion, during which previously published histologic studies demonstrated a stimulated bone formation and a suppressed resorption in these rats. In conclusion, although there is increasing evidence for the usefulness of these serum proteins as markers of bone metabolism in humans, a great deal more work is required before we can understand the significance of these assays. Until such is accomplished, these assays should not be assumed to be validated. [ABSTRACT FROM AUTHOR]