ULTRASTRUCTURAL LOCALIZATION OF MINERAL MATTER IN BACTERIAL SPORES BY MICROINCINERATION

The fine localization of mineral matter in spores of Bacillus megaterium and Bacillus cereus was studied by the technique of microincineration adapted for use with the electron microscope. The specimens, which included intact and thin-sectioned spores as well as shed spore coats, were burned either in the conventional way at high temperature or by a new technique using electrically excited oxygen at nearly room temperature. The ash residues were examined by bright field, dark field, and diffraction in the electron microscope and also with the phase contrast microscope. In some cases, the specimen was previewed in both microscopes before incineration. The results do not support a previous report that the mineral elements of the spore are confined to a peripheral layer, but rather indicate that the spore core as well as the coat are mineral-rich. The cortex may be deficient in minerals, but the possibility of artifact prevents a clear decision on this point. Incinerated B. megaterium spores show a highly ordered fine structure displaying 100 A periodicity in the ash of the middle layer of the coat. The nature of this structure is discussed, as is the technique which demonstrated it. The fine definition of the ash patterns, particularly those obtained with the low-temperature, excited-oxygen technique, suggests that microincineration may be generally useful in the study of fine structure.     

Subcellular Nutrient Element Localization and Enrichment in Ecto- and Arbuscular Mycorrhizas of Field-Grown Beech and Ash Trees Indicate Functional Differences

Mycorrhizas are the chief organ for plant mineral nutrient acquisition. In temperate, mixed forests, ash roots (Fraxinus excelsior) are colonized by arbuscular mycorrhizal fungi (AM) and beech roots (Fagus sylvatica) by ectomycorrhizal fungi (EcM). Knowledge on the functions of different mycorrhizal species that coexist in the same environment is scarce. The concentrations of nutrient elements in plant and fungal cells can inform on nutrient accessibility and interspecific differences of mycorrhizal life forms. Here, we hypothesized that mycorrhizal fungal species exhibit interspecific differences in mineral nutrient concentrations and that the differences correlate with the mineral nutrient concentrations of their associated root cells. Abundant mycorrhizal fungal species of mature beech and ash trees in a long-term undisturbed forest ecosystem were the EcM Lactarius subdulcis, Clavulina cristata and Cenococcum geophilum and the AM Glomus sp. Mineral nutrient subcellular localization and quantities of the mycorrhizas were analysed after non-aqueous sample preparation by electron dispersive X-ray transmission electron microscopy. Cenococcum geophilum contained the highest sulphur, Clavulina cristata the highest calcium levels, and Glomus, in which cations and P were generally high, exhibited the highest potassium levels. Lactarius subdulcis-associated root cells contained the highest phosphorus levels. The root cell concentrations of K, Mg and P were unrelated to those of the associated fungal structures, whereas S and Ca showed significant correlations between fungal and plant concentrations of those elements. Our results support profound interspecific differences for mineral nutrient acquisition among mycorrhizas formed by different fungal taxa. The lack of correlation between some plant and fungal nutrient element concentrations may reflect different retention of mineral nutrients in the fungal part of the symbiosis. High mineral concentrations, especially of potassium, in Glomus sp. suggest that the well-known influence of tree species on chemical soil properties may be related to their mycorrhizal associates.     

MICROINCINERATION, ELECTRON MICROSCOPY, AND ELECTRON DIFFRACTION OF CALCIUM PHOSPHATE-LOADED MITOCHONDRIA

Isolated rat liver mitochondria were incubated in vitro under conditions supporting the massive accumulation of calcium and phosphate. Samples were embedded, thin sectioned, and examined in the electron microscope. The intramitochondrial distribution of insoluble or structure-bound mineral substances was studied by electron microscopy coupled with recently developed techniques of high resolution microincineration. As shown previously, the ion-loaded mitochondria acquire large, internal granules which have inherent electron opacity indicative of high mineral content. Study of ash patterns in preselected areas of sections directly confirmed the high mineral content of the granules, and the appearance of the residues was consistent with the copresence in the granules of some organic material. Other mitochondrial structures were almost devoid of mineral. Thin sections of unincubated control mitochondria also were incinerated. They were found to contain appreciable amounts of intrinsic mineral, seemingly associated with membranes. The normal, dense matrix granules commonly seen in unaltered mitochondria could be seen in intact sections of these control preparations, but after burning no definite correspondence of any ash to the granules could be demonstrated. The normal granules perhaps do not contain mineral. Heating experiments on ash patterns of all the preparations demonstrated the thermal stability and crystallizability of the ash. The crystallized ash of the in vitro-produced dense granules was tentatively shown by electron diffraction to be β-tricalcium phosphate (whitlockite). This, together with evidence from the literature, suggests that the original, noncrystalline mineral may be a colloidal, subcrystalline precursor of calcium-deficient hydroxyapatite. Experiments were performed on synthetic calcium phosphates for comparison. Other possible applications of the microincineration techniques are briefly discussed.     

Contribution to the ecology ofStipa species in central Europe: Distribution of mineral elements in soils and phytomass

In 1963, 1964 and 1965, a comparative ecological study was made of the distribution of mineral elements in the soil and phytomass of some Stipa species, viz.Stipa capillata L., S. pulcherrima C. Koch,S. joannis ?elak.,S. dasyphylla ?erň.,and S. stenophylla ?erň. in various locations of Central Europe. In all species and sites studied, chemical analyses of the soil, fresh phytomass (shoots and roots) and dead phytomass were made, together with relevant statistical evaluation. IndividualStipa species differ according to their mineral composition. In comparison with fresh green plant material, the old dead plant material shows a marked increase in ash, calcium, and natrium content. Potassium, nitrogen, and phosphorus show a decrease in the dead plant material. The mineral composition of the roots differs considerably from that of green plant parts: the roots have much higher ash, phosphorus, calcium and natrium content, and lower organic matter, nitrogen, and potassium content. On the basis of the data collected, an attempt was made to estimate cycling of the individual mineral elements.     

Heat and radiofrequency plasma glow discharge pretreatment of a titanium alloy: Eveidence for enhanced osteoinductive properties

It is believed that orthopedic and implant longevity can be improved by optimizing fixation, or direct bone‐implant contact, through the stimulation of new bone formation around the implant. The purpose of this study was to determine whether heat (600°C) or radiofrequency plasma glow discharge (RFGD) pretreatment of Ti6Al4V stimulated calcium‐phosphate mineral formation in cultures of attached MC3T3 osteoprogenitor cells with or without a fibronectin coating. Calcium‐phosphate mineral was analyzed by flame atomic absorption spectrophotometry, scanning electron microscopy (SEM)/electron dispersive X‐ray microanalysis (EDAX) and Fourier transformed infrared spectroscopy (FTIR). RFGD and heat pretreatments produced a general pattern of increased total soluble calcium levels, although the effect of heat pretreatment was greater than that of RFGD. SEM/EDAX showed the presence of calcium‐and phosphorus‐containing particles on untreated and treated disks that were more numerous on fibronectin‐coated disks. These particles were observed earliest (1 week) on RFGD‐pretreated surfaces. FTIR analyses showed that the heat pretreatment produced a general pattern of increased levels of apatite mineral at 2–4 weeks; a greater effect was observed for fibronectin‐coated disks compared to uncoated disks. The observed findings suggest that heat pretreatment of Ti6Al4V increased the total mass of the mineral formed in MC3T3 osteoprogenitor cell cultures more than RFGD while the latter pretreatment hastened the early deposition of mineral. These findings help to support the hypothesis that the pretreatments enhance the osteoinductive properties of the alloy. J. Cell. Biochem. 114: 1917–1927, 2013. © 2013 Wiley Periodicals, Inc.     

Accuracy and reproducibility of lumbar bone mineral status determined by dual photon absorptiometry in live male cynomolgus macaques (Macaca fascicularis)

Dual photon absorptiometry (DPA) was used to determine the in vivo bone mineral content (BMC) and bone mineral density of lumbar vertebrae (L2-4) in feral adult male cynomolgus macaques (Macaca fascicularis, n = 20). Following in vivo DPA scans, all animals were euthanized and the lumbar spine segment excised. The excised lumbar vertebrae, plus spinal cord, were formalin-fixed and measured three times by ex vivo DPA. The first ex vivo scan matched the monkey's own in vivo soft tissue beam attenuation ratio (Rs, ranging from 1.38 to 1.45). The second ex vivo scan was made creating a constant obese-like condition (Rs = 1.38), and the third creating a constant lean-like condition (Rs = 1.45). All scans were taken at a 1.0 mm point resolution, 1.0 mm line spacing, a 6 cm width, and a scan speed of 5 mm/sec. The second lumbar vertebra (L2) was ashed and the ash weight was compared to the measured L2BMC of the in vivo and ex vivo DPA analyses. Noninvasive in vivo DPA proved to be adequate in measuring the lumbar bone mineral content in male cynomolgus macaques. Ex vivo DPA at a high and constant Rs value of 1.45 provided for the best accuracy of formalin-fixed wet bone tissue when compared to the bone ash weight.     

Bone mineralization proceeds through intracellular calcium phosphate loaded vesicles: a cryo-electron microscopy study

Bone is the most widespread mineralized tissue in vertebrates and its formation is orchestrated by specialized cells - the osteoblasts. Crystalline carbonated hydroxyapatite, an inorganic calcium phosphate mineral, constitutes a substantial fraction of mature bone tissue. Yet key aspects of the mineral formation mechanism, transport pathways and deposition in the extracellular matrix remain unidentified. Using cryo-electron microscopy on native frozen-hydrated tissues we show that during mineralization of developing mouse calvaria and long bones, bone-lining cells concentrate membrane-bound mineral granules within intracellular vesicles. Elemental analysis and electron diffraction show that the intracellular mineral granules consist of disordered calcium phosphate, a highly metastable phase and a potential precursor of carbonated hydroxyapatite. The intracellular mineral contains considerably less calcium than expected for synthetic amorphous calcium phosphate, suggesting the presence of a cellular mechanism by which phosphate entities are first formed and thereafter gradually sequester calcium within the vesicles. We thus demonstrate that in vivo osteoblasts actively produce disordered mineral packets within intracellular vesicles for mineralization of the extracellular developing bone tissue. The use of a highly disordered precursor mineral phase that later crystallizes within an extracellular matrix is a strategy employed in the formation of fish fin bones and by various invertebrate phyla. This therefore appears to be a widespread strategy used by many animal phyla, including vertebrates.     

Silver amplification of mercury sulfide and selenide: a histochemical method for light and electron microscopic localization of mercury in tissue

A method for light and electron microscopic demonstration of mercury sulfides and mercury selenides in mammalian tissue is presented. Silver ions adhering to the surface of submicroscopic traces of mercury sulfides or selenides in the tissue are reduced to metallic silver by hydroquinone. Physical development thereupon renders deposits of mercury sulfides or mercury selenide visible as spheres of solid silver. Examples of localization of mercury in the central nervous system and various organs from animals exposed to mercury chloride or methyl mercury chloride with or without additional sodium selenide treatment are presented. Selenium treatment results in a considerable increase in the amount of mercury that can be made visible by silver amplification. After mercury chloride treatment, most of the mercury is localized in lysosomes and is only rarely seen in secretory granules. After simultaneous selenium treatment, mercury is also found in nuclei of proximal tubule cells in the kidney and in macrophages. The "sulfide-osmium" method for ultrastructural localization of mercury suggested by Silberberg, Lawrence, and Leider (Arch Environ Health 19:7, 1969) and the light microscopic method using a photographic emulsion suggested by Umeda, Saito, and Saito (Jpn J Exp Med 39:17, 1969) have been experimentally analyzed and commented on.     

Effects of dietary calcium and phosphorus deficiency and subsequent recovery on broiler chicken growth performance and bone characteristics

The ability of birds to modify dietary phosphorus utilisation when fed with low-phosphorus and calcium (Ca) diets was studied using different sequences of dietary phosphorus and Ca restriction (depletion) and recovery (repletion) during the grower and the finisher phases. A total of 3600 Ross 708 broilers were randomly divided into 10 replicate pens per treatment (60 per pen, six pens per block). Chicks were fed a common starter diet from days 0 to 10, then a grower control diet (C: 0.90% Ca, 0.39% non-phytate phosphorus, nPP), mid-level diet (M: 0.71% Ca, 0.35% nPP) or low Ca and nPP diet (L: 0.60% Ca, 0.30% nPP) from days 11 to 21, followed by a finisher diet C, M or L containing, respectively, 0.85%, 0.57% or 0.48% Ca and 0.35%, 0.29% or 0.24% nPP from days 22 to 37. Six treatment sequences were tested: CC, MM, LL, ML, LC and LM. Bone mineral content by dual-energy X-ray, tibia ash, toe ash weight and tibia breaking strength were measured on days 21 and 37. No significant effect was observed on growth performance throughout the experiment. Diet L reduced bone mineral content, breaking strength, tibia and toe ash by 9%, 13%, 11% and 10%, respectively, on day 21 (compared with diet C, for linear effect, P<0.05). On day 37, bone mineral content, breaking strength, tibia and toe ash remained lower compared with control values (CC v. MM v. LL, P<0.05 for linear and quadratic effects). Mineral depletion duration (ML v. LL) did not affect bone mineral status. Replenishing with the C diet during the finisher phase (LC) restored bone mineral content, tibia ash and toe ash weight better than the M diet did, but not to control levels (CC v. LC v. LM, for linear effect, P<0.05). These results confirm that dietary Ca and nPP may be reduced in the grower phase without affecting final growth performance or breaking strength as long as the finisher diet contains sufficient Ca and nPP. The practical applications of this strategy require further study in order to optimise the depletion and repletion steps.     

CALCIUM PHOSPHATE GRANULES IN THE HEPATOPANCREAS OF THE BLUE CRAB CALLINECTES SAPIDUS

The hepatopancreas of the adult male blue crab Callinectes sapidus in intermolt was found to contain substantial amounts of calcium, magnesium, and inorganic phosphorus, averaging about 260, 20, and 250 µg-atoms per g wet tissue, respectively, accounting for over 10% of the tissue dry weight. Electron microscopy of the intact tissue showed three qualitatively different granular structures having electron densities suggestive of high mineral content. After fractionation of the tissue using centrifugal techniques, almost 95% of the total mineral was found to reside in a heavy, nonmitochondrial particulate fraction(s). The bulk of the low-speed pellet consisted of relatively dense, roughly spherical granules 1–5 µm in diameter, which could be considerably purified by repeated suspension in water and low-speed sedimentation. In the electron microscope the isolated granules appeared basically similar to one of the three characteristic types of electron-dense granules seen in the intact tissue. Although the freshly isolated granules lost approximately 50% of their wet weight when dried at 105°C, only 10% more was lost upon dry ashing at 450°C, suggesting a fairly low content of organic material. Chemical analysis revealed calcium, magnesium, and inorganic phosphate at 5.7, 2.1, and 4.4 µg-atoms per mg dried granules, respectively, accounting for 69% of the dry weight of the fraction. By specific enzymatic assays, the freshly isolated granules were found to contain ATP, ADP, and AMP at levels of 0.13, 0.03, and 0.01 µmol/mg, or 8% of their total dry weight. The remainder of the total phosphorus contributed an additional 3%, whereas carbonate, citrate, oxalate, and protein each constituted no more than 1%. The mineral granules of the crab hepatopancreas appear to function as storage forms of calcium and phosphate during the intermolt period. This tissue appears promising as a model for study of the cellular events associated with biological calcification, since conventional biochemical techniques can be employed. Furthermore, the major mineralized component of the tissue can be obtained in large amounts for direct study by a simple fractionation procedure.     

Mitochondrial granules of chondrocytes in cryosections of growth cartilage.

Fresh frozen specimens of growth cartilage have been dry-sectioned and examined in the electron microscope without any staining. Dense granules in the mitochondria of chondrocytes were observed near calcifying areas. This confirms the presence of these mineral granules in the original tissue because this technique avoids the preparative artefacts of conventional electron microscopy.     

Location of Elements in Ashed Spores of Bacillus megaterium

The structure of the skeleton of spores of Bacillus megaterium was examined after ashing in a plasma asher and the elemental composition of the ashed whole spores was determined with an analytical electron microscope. All spores were ashed in situ although they shrank by about 15%. Even P and S, in addition to metals, were recovered well from ashed samples. Ash was rich in the core and the coat, and poor in the cortex. Ca, P, S, and Mg were detected in the core and coat of the spore of B. megaterium QM B1551. Ca in the core was markedly decreased by germination or autoclaving. In the spore of B. megaterium ATCC 19213, almost all of the ash was detected in the core and its elemental composition was similar to that of the core of the strain QM B1551 spore. These results suggest strongly that the core is the site of Ca associated with dipicolinic acid.     

The proteolytic processing of amelogenin by enamel matrix metalloproteinase (MMP-20) is controlled by mineral ions

Background

Enamel synthesis is a highly dynamic process characterized by simultaneity of matrix secretion, assembly and processing during apatite mineralization. MMP-20 is the first protease to hydrolyze amelogenin, resulting in specific cleavage products that self-assemble into nanostructures at specific mineral compositions and pH. In this investigation, enzyme kinetics of MMP-20 proteolysis of recombinant full-length human amelogenin (rH174) under different mineral compositions is elucidated.

Methods

Recombinant amelogenin was cleaved by MMP-20 under various physicochemical conditions and the products were analyzed by SDS-PAGE and MALDI-TOF MS.

Results

It was observed that mineral ions largely affect cleavage pattern, and enzyme kinetics of rH174 hydrolysis. Out of the five selected mineral ion compositions, MMP-20 was most efficient at high calcium concentration, whereas it was slowest at high phosphate, and at high calcium and phosphate concentrations. In most of the compositions, N- and C-termini were cleaved rapidly at several places but the central region of amelogenin was protected up to some extent in solutions with high calcium and phosphate contents.

Conclusion

These in vitro studies showed that the chemistry of the protein solutions can significantly alter the processing of amelogenin by MMP-20, which may have significant effects in vivo matrix assembly and subsequent calcium phosphate mineralization.

General significance

This study elaborates the possibilities of the processing of the organic matrix into mineralized tissue during enamel development.     

Measurement of Total Calcium in Neurons by Electron Probe X-ray Microanalysis

In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique known as electron probe microanalysis (EPMA) are described. Intramitochondrial calcium is a particular focus because of the critical role that mitochondrial calcium overload plays in neurodegenerative diseases. The method is based on the analysis of X-rays generated in an electron microscope (EM) by interaction of an electron beam with the specimen. In order to maintain the native distribution of diffusible elements in electron microscopy specimens, EPMA requires "cryofixation" of tissue followed by the preparation of ultrathin cryosections. Rapid freezing of cultured cells or organotypic slice cultures is carried out by plunge freezing in liquid ethane or by slam freezing against a cold metal block, respectively. Cryosections nominally 80 nm thick are cut dry with a diamond knife at ca. -160 °C, mounted on carbon/pioloform-coated copper grids, and cryotransferred into a cryo-EM using a specialized cryospecimen holder. After visual survey and location mapping at ≤-160 °C and low electron dose, frozen-hydrated cryosections are freeze-dried at -100 °C for ~30 min. Organelle-level images of dried cryosections are recorded, also at low dose, by means of a slow-scan CCD camera and subcellular regions of interest selected for analysis. X-rays emitted from ROIs by a stationary, focused, high-intensity electron probe are collected by an energy-dispersive X-ray (EDX) spectrometer, processed by associated electronics, and presented as an X-ray spectrum, that is, a plot of X-ray intensity vs. energy. Additional software facilitates: 1) identification of elemental components by their "characteristic" peak energies and fingerprint; and 2) quantitative analysis by extraction of peak areas/background. This paper concludes with two examples that illustrate typical EPMA applications, one in which mitochondrial calcium analysis provided critical insight into mechanisms of excitotoxic injury and another that revealed the basis of ischemia resistance.     

Effects of wildfire ash on water chemistry and biota in South-Western U.S.A. streams

1. We monitored streams within the Gila River drainage in south‐western New Mexico, U.S.A., over a 5‐year period, to investigate the influence of ash input on water quality and stream biota following forest wildfires. 2. Nutrients [ammonium, nitrate, soluble reactive phosphate (SRP)], potassium and alkalinity were most affected by fires; all were increased in stream water following ash input. Concentrations of each returned to prefire conditions within 4 months. Ammonium and nitrate also increased in stream water as a result of atmospheric fallout (e.g. smoke) from fires outside the catchment. 3. Periphyton biomass was not affected significantly by wildfires, although there was a shift in diatom assemblage to smaller more adnate taxa. Cocconeis placentula was frequently the dominant postfire species. 4. The influence of wildfires on macroinvertebrates ranged from minimal to dramatic reductions in density depending upon the duration of ash flows and the characteristics of the ash material that entered each system. Macroinvertebrate densities returned to prefire conditions within 1 year. 5. An in‐situ ashing experiment was conducted on a first‐order stream in the Gila River drainage to monitor on‐site physiochemical and biotic responses during and after fire ash addition, for comparison with ash delivery from real wildfires on monitored streams. Physical–chemical parameters and algae and macroinvertebrates were monitored in an ashed and upstream reference reach for 13 months. Results generally substantiated findings from monitored streams. 6. Concentrations of major ions and nutrients, as well as turbidity, conductivity and pH, increased immediately in stream water below the point of ashing, while dissolved oxygen decreased. Changes in water chemistry were short‐lived (=24 h) except for SRP. The concentration of SRP in stream water was significantly higher in the ashed reach than the control reach for at least 1 month after ash input. 7. Periphyton biomass and diatom assemblages were not significantly altered in the ashing study, whereas macroinvertebrate density was measurably lower in the ashed reach for nearly a year. Macroinvertebrate drift was over 10‐fold greater in the ashed reach compared with the reference reach during ashing. Dissimilarity between macroinvertebrate communities in the reference and ashed reaches was significantly greater than variation within reaches for nearly a year.     

Simultaneous Raman Microspectroscopy and Fluorescence Imaging of Bone Mineralization in Living Zebrafish Larvae

Confocal Raman microspectroscopy and fluorescence imaging are two well-established methods providing functional insight into the extracellular matrix and into living cells and tissues, respectively, down to single molecule detection. In living tissues, however, cells and extracellular matrix coexist and interact. To acquire information on this cell-matrix interaction, we developed a technique for colocalized, correlative multispectral tissue analysis by implementing high-sensitivity, wide-field fluorescence imaging on a confocal Raman microscope. As a proof of principle, we study early stages of bone formation in the zebrafish (Danio rerio) larvae because the zebrafish has emerged as a model organism to study vertebrate development. The newly formed bones were stained using a calcium fluorescent marker and the maturation process was imaged and chemically characterized in vivo. Results obtained from early stages of mineral deposition in the zebrafish fin bone unequivocally show the presence of hydrogen phosphate containing mineral phases in addition to the carbonated apatite mineral. The approach developed here opens significant opportunities in molecular imaging of metabolic activities, intracellular sensing, and trafficking as well as in vivo exploration of cell-tissue interfaces under (patho-)physiological conditions.