The NATO project: nanoparticle-based countermeasures for microgravity-induced osteoporosis.

Recent advances in nanotechnology applied to medicine and regenerative medicine have an enormous and unexploited potential for future space and terrestrial medical applications. The Nanoparticles and Osteoporosis (NATO) project aimed to develop innovative countermeasures for secondary osteoporosis affecting astronauts after prolonged periods in space microgravity. Calcium- and Strontium-containing hydroxyapatite nanoparticles (nCa-HAP and nSr-HAP, respectively) were previously developed and chemically characterized. This study constitutes the first investigation of the effect of the exogenous addition of nCa-HAP and nSr-HAP on bone remodeling in gravity (1 g), Random Positioning Machine (RPM) and onboard International Space Station (ISS) using human bone marrow mesenchymal stem cells (hBMMSCs). In 1 g conditions, nSr-HAP accelerated and improved the commitment of cells to differentiate towards osteoblasts, as shown by the augmented alkaline phosphatase (ALP) activity and the up-regulation of the expression of bone marker genes, supporting the increased extracellular bone matrix deposition and mineralization. The nSr-HAP treatment exerted a protective effect on the microgravity-induced reduction of ALP activity in RPM samples, and a promoting effect on the deposition of hydroxyapatite crystals in either ISS or 1 g samples. The results indicate the exogenous addition of nSr-HAP could be potentially used to deliver Sr to bone tissue and promote its regeneration, as component of bone substitute synthetic materials and additive for pharmaceutical preparation or food supplementary for systemic distribution.

Structural and biochemical characterization of Rv0187, an O-methyltransferase from Mycobacterium tuberculosis.

Catechol O-methyltransferase (COMT) is widely distributed in nature and installs a methyl group onto one of the vicinal hydroxyl groups of a catechol derivative. Enzymes belonging to this family require two cofactors for methyl transfer: S-adenosyl-l-methionine as a methyl donor and a divalent metal cation for regiospecific binding and activation of a substrate. We have determined two high-resolution crystal structures of Rv0187, one of three COMT paralogs from Mycobacterium tuberculosis, in the presence and absence of cofactors. The cofactor-bound structure clearly locates strontium ions and S-adenosyl-l-homocysteine in the active site, and together with the complementary structure of the ligand-free form, it suggests conformational dynamics induced by the binding of cofactors. Examination of in vitro activities revealed promiscuous substrate specificity and relaxed regioselectivity against various catechol-like compounds. Unexpectedly, mutation of the proposed catalytic lysine residue did not abolish activity but altered the overall landscape of regiospecific methylation.

Association of soil selenium, strontium, and magnesium concentrations with Parkinson's disease mortality rates in the USA.

Among the 41 soil elements analyzed from 4856 sites across the contiguous 48 states, average Parkinson's disease (PD) mortality rates between 1999 and 2014 have the most significant positive correlation with the average soil strontium (Sr) concentrations (correlation r = 0.47, significance level p = 0.00), and average PD mortality rates have the most significant inverse correlation with the average soil selenium (Se) concentrations (r = -0.44, p = 0.00). Multivariate regression models indicate that soil Sr and Se concentrations can explain 35.4% of spatial disparities of the state average PD mortality rates between 1999 and 2014 (R 2 = 0.354). When the five outlier states were removed from the model, concentrations of soil Sr and Se can explain 62.4% (R 2 = 0.624) of the spatial disparities of PD mortality rates of the 43 remaining states. The results also indicate that high soil magnesium (Mg) concentrations suppressed the growth rate of the PD mortality rates between 1999 and 2014 in the 48 states (r = -0.42, p = 0.000). While both Se and Sr have been reported to affect the nervous system, this study is the first study that reported the statistically significant association between the PD mortality rates and soil concentrations of Se, Sr, and Mg in the 48 states. Given that soil elemental concentration in a region is broad indicator of the trace element intake from food, water, and air by people, implications of the results are that high soil Se and Mg concentrations helped reduce the PD mortality rates and benefited the PD patients in the 48 states.

The Stimulatory Effect of Strontium Ions on Phytoestrogens Content in Glycine max (L.) Merr.

The amount of secondary metabolites in plants can be enhanced or reduced by various external factors. In this study, the effect of strontium ions on the production of phytoestrogens in soybeans was investigated. The plants were treated with Hoagland's solution, modified with Sr(2+) with concentrations ranging from 0.5 to 3.0 mM, and were grown for 14 days in hydroponic cultivation. After harvest, soybean plants were separated into roots and shoots, dried, and pulverized. The plant material was extracted with methanol and hydrolyzed. Phytoestrogens were quantified by HPLC. The significant increase in the concentration of the compounds of interest was observed for all tested concentrations of strontium ions when compared to control. Sr(2+) at a concentration of 2 mM was the strongest elicitor, and the amount of phytoestrogens in plant increased ca. 2.70, 1.92, 3.77 and 2.88-fold, for daidzein, coumestrol, genistein and formononetin, respectively. Moreover, no cytotoxic effects were observed in HepG2 liver cell models after treatment with extracts from 2 mM Sr(2+)-stressed soybean plants when compared to extracts from non-stressed plants. Our results indicate that the addition of strontium ions to the culture media may be used to functionalize soybean plants with enhanced phytoestrogen content.

Compositional and material properties of rat bone after bisphosphonate and/or Strontium ranelate drug treatment.

PURPOSE: We investigated elemental strontium and/or bisphosphonate drug incorporation upon the compositional and biomechanical properties of vertebral bone, in a rat model of Osteoporosis secondary to ovariectomy. METHODS: Six month old female rats were ovariectomized (OVX) and divided into untreated OVX-Vehicle, OVX-RIS (Risedronate bisphosphonate [BP] treated), OVX-SrR (Strontium Ranelate [Protos®] treated), combination OVX-RIS+SrR, and sham-operated controls. After 16 weeks of treatment, rats were euthanized and lumbar vertebra were dissected. Micro-Computed Tomography (micro-CT), Electron Probe Micro-Analysis (EPMA), mechanical testing in compression and nano-indentation testing were then undertaken to evaluate bone morphometry, elemental composition, material properties and strength. RESULTS: Bone Volume was significantly reduced in the OVX-Vehicle (133±10 mm(3)) compared with OVX-RIS (169±22 mm(3)), OVX-SrR (145±2 mm(3)), and OVX-RIS+SrR (172±8 mm(3)). EPMA mapped elemental Sr deposition to the periosteal surface of cortical bone (50-100 µm thick), endosteal trabecular surfaces (20 µm thick), as well as to both vertebral growth plates. The atomic ratios of (Ca+Sr)/P were significantly reduced with SrR treatment (2.4%-6.6%), indicating Sr incorporation into bone mineral. No significant differences were measured in vertebral bone reduced modulus by nano-indentation. Conversely, all BP-dosed groups had significantly increased structural bone strength. CONCLUSIONS: Thus, we conclude that BP drugs dominate the conservation of trabecular geometry and structural strength in OP rats, whereas Sr drugs likely influence bone volume and material composition locally.

Accumulation of bone strontium measured by in vivo XRF in rats supplemented with strontium citrate and strontium ranelate.

Strontium ranelate is an approved pharmacotherapy for osteoporosis in Europe and Australia, but not in Canada or the United States. Strontium citrate, an alternative strontium salt, however, is available for purchase over-the-counter as a nutritional supplement. The effects of strontium citrate on bone are largely unknown. The study's objectives were 1) to quantify bone strontium accumulation in female Sprague Dawley rats administered strontium citrate (N=7) and compare these levels to rats administered strontium ranelate (N=6) and vehicle (N=6) over 8 weeks, and 2) to verify an in vivo X-ray fluorescence spectroscopy (XRF) system for measurement of bone strontium in the rat. Daily doses of strontium citrate and strontium ranelate were determined with the intention to achieve equivalent amounts of elemental strontium. However, post-hoc analyses of each strontium compound conducted using energy dispersive spectrometry microanalysis revealed a higher elemental strontium concentration in strontium citrate than strontium ranelate. Bone strontium levels were measured at baseline and 8 weeks follow-up using a unique in vivo XRF technique previously used in humans. XRF measurements were validated against ex vivo measurements of bone strontium using inductively coupled plasma mass spectrometry. Weight gain in rats in all three groups was equivalent over the study duration. A two-way ANOVA was conducted to compare bone strontium levels amongst the three groups. Bone strontium levels in rats administered strontium citrate were significantly greater (p<0.05) than rats administered strontium ranelate and vehicle. ANCOVA analyses were performed with Sr dose as a covariate to account for differences in strontium dosing. The ANCOVA revealed differences in bone strontium levels between the strontium groups were not significant, but that bone strontium levels were still very significantly greater than vehicle.

Age and growth of endangered smalltooth sawfish (Pristis pectinata) verified with LA-ICP-MS analysis of vertebrae.

Endangered smalltooth sawfish (Pristis pectinata) were opportunistically sampled in south Florida and aged by counting opaque bands in sectioned vertebrae (n=15). Small sample size precluded traditional age verification, but fish collected in spring and summer had translucent vertebrae margins, while fish collected in winter had opaque margins. Trends in Sr:Ca measured across vertebrae with laser ablation-inductively coupled plasma-mass spectrometry corresponded well to annual salinity trends observed in sawfish estuarine nursery habitats in south Florida, thus serve as a chemical marker verifying annual formation of opaque bands. Based on that finding and assumptions about mean birth date and timing of opaque band formation, estimated age ranged from 0.4 y for a 0.60 m total length (TL) male to 14.0 y for a 4.35 m TL female. Von Bertalanffy growth parameters computed from size at age data were 4.48 m for L(∞), 0.219 y(-1)for k, and -0.81 y for t(0). Results of this study have important implications for sawfish conservation as well as for inferring habitat residency of euryhaline elasmobranchs via chemical analysis of vertebrae.

Osteoclastogenesis and osteoclastic resorption of tricalcium phosphate: effect of strontium and magnesium doping.

Bone substitute materials are required to support the remodeling process, which consists of osteoclastic resorption and osteoblastic synthesis. Osteoclasts, the bone-resorbing cells, generate from differentiation of hemopoietic mononuclear cells. In the present study, we have evaluated the effects of 1.0 wt % strontium (Sr) and 1.0 wt % magnesium (Mg) doping in beta-tricalcium phosphate (ß-TCP) on the differentiation of mononuclear cells into osteoclast-like cells and its resorptive activity. In vitro osteoclast-like cell formation, adhesion, and resorption were studied using osteoclast precursor RAW 264.7 cell, supplemented with receptor activator of nuclear factor κß ligand (RANKL). Osteoclast-like cell formation was noticed on pure and Sr-doped ß-TCP samples at day 8, which was absent on Mg-doped ß-TCP samples indicating decrease in initial osteoclast differentiation due to Mg doping. After 21 days of culture, osteoclast-like cell formation was evident on all samples with osteoclastic markers such as actin ring, multiple nuclei, and presence of vitronectin receptor α(v)ß(3) integrin. After osteoclast differentiation, all substrates showed osteoclast-like cell-mediated degradation, however, significantly restricted for Mg-doped ß-TCP samples. Our present results indicated that substrate chemistry controlled osteoclast differentiation and resorptive activity, which can be used in designing TCP-based resorbable bone substitutes with controlled degradation properties.

Coal fly ash basins as an attractive nuisance to birds: parental provisioning exposes nestlings to harmful trace elements.

Birds attracted to nest around coal ash settling basins may expose their young to contaminants by provisioning them with contaminated food. Diet and tissues of Common Grackle (Quiscalus quiscala) nestlings were analyzed for trace elements to determine if nestlings were accumulating elements via dietary exposure and if feather growth limits elemental accumulation in other tissues. Arsenic, cadmium, and selenium concentrations in ash basin diets were 5× higher than reference diets. Arsenic, cadmium, and selenium concentrations were elevated in feather, liver, and carcass, but only liver Se concentrations approached levels of concern. Approximately 15% of the total body burden of Se, As, and Cd was sequestered in feathers of older (>5 days) nestlings, whereas only 1% of the total body burden of Sr was sequestered in feathers. Feather concentrations of only three elements (As, Se, and Sr) were correlated with liver concentrations, indicating their value as non-lethal indicators of exposure.

Screening of plant species for phytoremediation of uranium, thorium, barium, nickel, strontium and lead contaminated soils from a uranium mill tailings repository in South China.

The concentrations of uranium, thorium, barium, nickel, strontium and lead in the samples of the tailings and plant species collected from a uranium mill tailings repository in South China were analyzed. Then, the removal capability of a plant for a target element was assessed. It was found that Phragmites australis had the greatest removal capabilities for uranium (820 µg), thorium (103 µg) and lead (1,870 µg). Miscanthus floridulus had the greatest removal capabilities for barium (3,730 µg) and nickel (667 µg), and Parthenocissus quinquefolia had the greatest removal capability for strontium (3,920 µg). In this study, a novel coefficient, termed as phytoremediation factor (PF), was proposed, for the first time, to assess the potential of a plant to be used in phytoremediation of a target element contaminated soil. Phragmites australis has the highest PFs for uranium (16.6), thorium (8.68), barium (10.0) and lead (10.5). Miscanthus floridulus has the highest PF for Ni (25.0). Broussonetia papyrifera and Parthenocissus quinquefolia have the relatively high PFs for strontium (28.1 and 25.4, respectively). On the basis of the definition for a hyperaccumulator, only Cyperus iria and Parthenocissus quinquefolia satisfied the criteria for hyperaccumulator of uranium (36.4 µg/g) and strontium (190 µg/g), and could be the candidates for phytoremediation of uranium and strontium contaminated soils. The results show that the PF has advantage over the hyperaccumulator in reflecting the removal capabilities of a plant for a target element, and is more adequate for assessing the potential of a plant to be used in phytoremediation than conventional method.

Nano-crystalline hydroxyapatite bio-mineral for the treatment of strontium from aqueous solutions.

Hydroxyapatites were analysed using electron microscopy, X-ray diffraction (XRD) and X-ray fluorescence (XRF) analysis. Examination of a bacterially produced hydroxyapatite (Bio-HA) by scanning electron microscopy showed agglomerated nano-sized particles; XRD analysis confirmed that the Bio-HA was hydroxyapatite, with an organic matter content of 7.6%; XRF analysis gave a Ca/P ratio of 1.55, also indicative of HA. The size of the Bio-HA crystals was calculated as ~25 nm from XRD data using the Scherrer equation, whereas Comm-HA powder size was measured as ≤ 50 µm. The nano-crystalline Bio-HA was ~7 times more efficient in removing Sr(2+) from synthetic groundwater than Comm-HA. Dissolution of HA as indicated by the release of phosphate into the solution phase was higher in the Comm-HA than the Bio-HA, indicating a more stable biomaterial which has a potential for the remediation of contaminated sites.

Strontium ranelate decreases the incidence of new caudal vertebral fractures in a growing mouse model with spontaneous fractures by improving bone microarchitecture.

UNLABELLED: Young mice over-expressing Runx2 fail to gain bone relative to wild type mice with growth and present spontaneous fractures. It allows, for the first time in rodents, direct assessment of anti-fracture efficacy of strontium ranelate which was able to decrease caudal vertebrae fracture incidence through an improvement of trabecular and cortical architecture. INTRODUCTION: The aim was to investigate whether strontium ranelate was able to decrease fracture incidence in mice over-expressing Runx2, model of severe developmental osteopenia associated with spontaneous vertebral fractures. METHODS: Transgenic mice and their wild type littermates were treated by oral route with strontium ranelate or vehicle for 9 weeks. Caudal fracture incidence was assessed by repeated X-rays, resistance to compressive loading by biochemical tests, and bone microarchitecture by histomorphometry. RESULTS: Transgenic mice receiving strontium ranelate had significantly fewer new fractures occurring during the 9 weeks of the study (-60%, p < 0.05). In lumbar vertebrae, strontium ranelate improves resistance to compressive loading (higher ultimate force to failure, +120%, p < 0.05) and trabecular microarchitecture (higher bone volume and trabecular number, lower trabecular separation, +60%, +50%, -39%, p < 0.05) as well as cortical thickness (+17%, p < 0.05). In tibiae, marrow cavity cross-section area and equivalent diameter were lower (-39%, -21%, p < 0.05). The strontium level in plasma and bone was in the same range as the values measured in treated postmenopausal women. CONCLUSIONS: This model allows, for the first time, direct assessment of anti-fracture efficacy of strontium ranelate treatment in rodents. In these transgenic mice, strontium ranelate was able to decrease caudal vertebral fracture incidence through an improvement of trabecular and cortical architecture.

[Development of osteoporosis in prematurely aging OXYS rats].

The early osteoporosis in OXYS rats is the presentation of accelerated senescence and earlier positioned as senile. The present study shows the changes in metabolism detected in OXYS rats in the postnatal period. They lead to the development of osteoporosis in future and may underlie the formation of reduced peak bone mass. 90 males OXYS rats used in this study aged from 10 days to 24 months and the control group consisted of 90 male Wistar rats of the matched ages. No differences in BMD in OXYS and Wistar rats at the age of 10 days and 3 months was revealed. At the age of 10 days the OXYS rats showed the higher by 40% activity of ALH--the marker of osteoblast activity--than Wistar rats; but at the age of 3 months ALH activity in OXYS was lower than in Wistar rats. The peak bone mass and BMD in Wistar rats is formed by the age of 12 months, in OXYS rats already by 6, but it did not reach the level of Wistar. The content of Ca in the blood and bone tissue changes similarly: no difference in young age, but reduces in OXYS rats after 6 months to the background of enhanced Ca excretion in urine. However, changing the mineral composition of bone in OXYS rats did not affect the mechanical strength: the absolute strength of the long bones in OXYS at 12 months was lower than that of Wistar, but at the expense of decrease by 1,7 times the cross-sectional area. We suppose that genetically determined hypoplasia of the bone tissue in OXYS rats is the starts of pathogenetic mechanisms of idiopathic osteoporosis.

Analysis of trace elements during different developmental stages of somatic embryogenesis in Plantago ovata Forssk using energy dispersive X-ray fluorescence.

Energy dispersive X-ray fluorescence (ED-XRF) technique has been used for the determination of trace element profile during different developmental stages of somatic embryogenic callus of an economically important medicinal plant, Plantago ovata Forssk. Somatic embryogenesis is a plant tissue culture-based technique, which is used for plant regeneration and crop improvement. In the present investigation, elemental content was analysed using ED-XRF technique during different developmental stages and also determine the effect of additives--casein hydrolysate and coconut water on the trace elemental profile of embryogenic callus tissue of P. ovata. Subsequent experiments showed significant alteration in the concentration of K, Ca, Mn, Fe, Zn, Cu, Br, and Sr in both the embryogenic and non-embryogenic callus. Higher K, Ca, Fe, Cu, and Zn accumulation was in embryogenic tissue stage compared to other stages, suggesting these elements are crucial for successful embryogenesis. The results suggest that this information could be useful for formulating a media for in vitro embryo induction of P. ovata.

Reversal of osteoporotic changes of mineral composition in femurs of diabetic rats by insulin.

Insulin plays an important role in bone prevention of diabetic osteoporosis, but little is known about the relation between the bone mineral density (BMD) increase and the change of mineral element content after treated with insulin. To address this problem, male Wistar rats were randomly divided into three groups: normal group (n = 6), streptozotocin-induced diabetic group (n = 5), and streptozotocin-induced diabetic group with insulin treatment (n = 5). The femoral BMD was measured by dual energy X-ray absorptiometry, and the element content was determined by inductively coupled plasma atomic emission spectrometry (ICP-AES). The results showed that the femoral BMD in diabetic group was significantly lower than that in normal group (P < 0.01) but restored by insulin treatment (P < 0.01 vs diabetic group). ICP-AES analysis revealed that the element content of calcium (Ca), phosphorous (P), magnesium (Mg), strontium (Sr), and potassium (K) in diabetic group were remarkably lower than those in normal group (P < 0.01) but only Ca, P, and Mg content were significantly increased compared with diabetic group (P < 0.05) after insulin treatment. However, no significant differences were observed in element zinc (Zn) content among three groups. Our findings suggested that the loss of Ca, P, Mg, Sr, and K content accounted for the lower BMD in streptozotocin-induced diabetes rats, insulin treatment could restore BMD by increasing the content of Ca, P, and Mg.

The correction of BMD measurements for bone strontium content.

Strontium ranelate (SR) is a new oral treatment for osteoporosis associated with large increases in bone mineral density (BMD) compared with alternative therapies such as bisphosphonates. Much of the BMD increase during SR treatment is a physical effect caused by the increased attenuation of X-rays due to the accumulation of strontium in bone tissue. The aim of this study was to assess the contribution made by bone strontium content (BSC) to the overall BMD increase by evaluating the percentage F of the BMD change explained by the physical presence of strontium in bone. A value of F less than 100% would provide evidence of the anabolic effect of SR as an additional factor contributing to the overall BMD increase. Studies of mixtures of strontium hydroxyapatite (SrHA) and calcium hydroxyapatite (CaHA) scanned on a variety of dual-energy X-ray absorptiometry (DXA) systems show that a 1% molar ratio of SrHA/(CaHA+SrHA) causes a 10% overestimation of BMD. The correction of spine BMD measurements for the physical effects of strontium depends on knowledge of 2 further factors: (1) bone biopsy measurements of iliac crest BSC and (2) the ratio R of BSC at the DXA site to BSC at the iliac crest measured in animal studies. We used clinical trial data and values of R(spine) measured in studies of monkeys and beagle dogs to determine values of F(spine) for 1, 2, and 3 yr treatment with SR. Based on the average value of R(spine) approximately 0.7 for male and female monkeys, we found values for F(spine) approximately 75-80% for 1, 2, and 3 yr of treatment. Using the value of R(spine) approximately 1.0 from the beagle study gave values of F(spine) approximately 100%. Although values of F(spine) as low as 40% are possible, we conclude that the most likely figure is 75% or greater. However, it is apparent that there are large uncertainties in the correction of BMD results for the effect of bone strontium and that the most important of these is the inference of BSC values at DXA scan sites from measurements of iliac crest bone biopsy specimens.

Competitive inhibition and selectivity enhancement by Ca in the uptake of inorganic elements (Be, Na, Mg, K, Ca, Sc, Mn, Co, Zn, Se, Rb, Sr, Y, Zr, Ce, Pm, Gd, Hf) by carrot (Daucus carota cv. U.S. harumakigosun).

We investigated the uptake of inorganic elements (Be, Na, Mg, K, Ca, Sc, Mn, Co, Zn, Se, Rb, Sr, Y, Zr, Ce, Pm, Gd, and Hf) and the effect of Ca on their uptake in carrots (Daucus carota cv. U.S. harumakigosun) by the radioactive multitracer technique. The experimental results suggested that Na, Mg, K, and Rb competed for the functional groups outside the cells in roots with Ca but not for the transporter-binding sites on the plasma membrane of the root cortex cells. In contrast, Y, Ce, Pm, and Gd competed with Ca for the transporters on the plasma membrane. The selectivity, which was defined as the value obtained by dividing the concentration ratio of an elemental pair, K/Na, Rb/Na, Be/Sr, and Mg/Sr, in the presence of 0.2 and 2 ppm Ca by that of the corresponding elemental pair in the absence of Ca in the solution was estimated. The selectivity of K and Rb in roots was increased in the presence of Ca. The selectivity of Be in roots was not affected, whereas the selectivity of Mg was increased by Ca. These observations suggest that the presence of Ca in the uptake solution enhances the selectivity in the uptake of metabolically important elements against unwanted elements.

Elevated silver, barium and strontium in antlers, vegetation and soils sourced from CWD cluster areas: do Ag/Ba/Sr piezoelectric crystals represent the transmissible pathogenic agent in TSEs?

High levels of Silver (Ag), Barium (Ba) and Strontium (Sr) and low levels of copper (Cu) have been measured in the antlers, soils and pastures of the deer that are thriving in the chronic wasting disease (CWD) cluster zones in North America in relation to the areas where CWD and other transmissible spongiform encephalopathies (TSEs) have not been reported. The elevations of Ag, Ba and Sr were thought to originate from both natural geochemical and artificial pollutant sources--stemming from the common practise of aerial spraying with 'cloud seeding' Ag or Ba crystal nuclei for rain making in these drought prone areas of North America, the atmospheric spraying with Ba based aerosols for enhancing/refracting radar and radio signal communications as well as the spreading of waste Ba drilling mud from the local oil/gas well industry across pastureland. These metals have subsequently bioconcentrated up the foodchain and into the mammals who are dependent upon the local Cu deficient ecosystems. A dual eco-prerequisite theory is proposed on the aetiology of TSEs which is based upon an Ag, Ba, Sr or Mn replacement binding at the vacant Cu/Zn domains on the cellular prion protein (PrP)/sulphated proteoglycan molecules which impairs the capacities of the brain to protect itself against incoming shockbursts of sound and light energy. Ag/Ba/Sr chelation of free sulphur within the biosystem inhibits the viable synthesis of the sulphur dependent proteoglycans, which results in the overall collapse of the Cu mediated conduction of electric signals along the PrP-proteoglycan signalling pathways; ultimately disrupting GABA type inhibitory currents at the synapses/end plates of the auditory/circadian regulated circuitry, as well as disrupting proteoglycan co-regulation of the growth factor signalling systems which maintain the structural integrity of the nervous system. The resulting Ag, Ba, Sr or Mn based compounds seed piezoelectric crystals which incorporate PrP and ferritin into their structure. These ferrimagnetically ordered crystals multireplicate and choke up the PrP-proteoglycan conduits of electrical conduction throughout the CNS. The second stage of pathogenesis comes into play when the pressure energy from incoming shock bursts of low frequency acoustic waves from low fly jets, explosions, earthquakes, etc. (a key eco-characteristic of TSE cluster environments) are absorbed by the rogue 'piezoelectric' crystals, which duly convert the mechanical pressure energy into an electrical energy which accumulates in the crystal-PrP-ferritin aggregates (the fibrils) until a point of 'saturation polarization' is reached. Magnetic fields are generated on the crystal surface, which initiate chain reactions of deleterious free radical mediated spongiform neurodegeneration in surrounding tissues. Since Ag, Ba, Sr or Mn based piezoelectric crystals are heat resistant and carry a magnetic field inducing pathogenic capacity, it is proposed that these ferroelectric crystal pollutants represent the transmissible, pathogenic agents that initiate TSE.

Crystal structure of the leadzyme at 1.8 A resolution: metal ion binding and the implications for catalytic mechanism and allo site ion regulation.

The leadzyme is a small ribozyme, derived from in vitro selection, which catalyzes site specific, Pb(2+)-dependent RNA cleavage. Pb(2+) is required for activity; Mg(2+) inhibits activity, while many divalent and trivalent ions enhance it. The leadzyme structure consists of an RNA duplex interrupted by a trinucleotide bulge. Here, crystal structures determined to 1.8 A resolution, both with Mg(2+) as the sole divalent counterion and with Mg(2+) and Sr(2+) (which mimics Pb(2+) with respect to binding but not catalysis), reveal the metal ion interactions with both the ground state and precatalytic conformations of the leadzyme. Mg(H(2)O)(6)(2+) ions bridge complementary strands of the duplex at multiple locations by binding tandem purines of one RNA strand in the major groove. At one site, Mg(H(2)O)(6)(2+) ligates the phosphodiester backbone of the trinucleotide bulge in the ground state conformation, but not in the precatalytic conformation, suggesting (a) Mg(2+) may inhibit leadzyme activity by stabilizing the ground state and (b) metal ions which displace Mg(2+) from this site may activate the leadzyme. Binding of Sr(2+) to the presumed catalytic Pb(2+) site in the precatalytic leadzyme induces local structural changes in a manner that would facilitate alignment of the catalytic ribose 2'-hydroxyl with the scissile bond for cleavage. These data support a model wherein binding of a catalytic ion to a precatalytic conformation of the leadzyme, in conjunction with the flexibility of the trinucleotide bulge, may facilitate structural rearrangements around the scissle phosphodiester bond favoring configurations that allow bond cleavage.

Diethylstilbestrol and tetrahydrochrysenes are calcium channel blockers in human platelets: relationship to the stilbene pharmacophore.

The effects of compounds with the stilbene pharmacophore [diethylstilbestrol (DES), DES derivatives, tetrahydrochrysene (THC), and THC derivatives] were examined for their ability to inhibit thrombin-induced Ca(2+) influx in human platelets. DES derivatives (DES dimethyl ether, DES dipropionate, dienestrol, and hexestrol) had lower inhibitory activity than DES. Esterification of DES with the bulky monobenzyl group eliminated inhibitory activity. Unsubstituted THC diol had the lowest inhibitory activity in the series of the THC derivatives bearing substituents in the 5,11 positions. These derivatives, either diethyl or dipropyl, cis or trans, were potent inhibitors of thrombin-induced [Ca(2+)](i) elevation (near 100% inhibition at 10 microM). Therefore, stilbene pharmacophore having bulk out of the plane of the double bond (from the twisting of the two aromatic rings or from addition of all substituents) seems to be requirement for the inhibitory activity. Free hydroxyl groups are also required for inhibitory activity, most likely for hydrogen bonding, since trans-diethyl tetrahydrochrysene dimethyl ether was inactive. Compounds bearing ethyl substituents (DES and THC derivatives) inhibited thrombin-induced release of calcium from the endoplasmic reticulum. These compounds also inhibited thapsigargin-induced Ca(2+) influx. This result implies that these compounds also block store-operated Ca(2+) influx directly, as well as internal Ca(2+) release. Compounds without ethyl substituents (trans-resveratrol, genistein, daidzein, and THC diol) only inhibited calcium influx into platelets.