Patterns of crystal organization and calcite twin formation in planktonic, rotaliid, foraminifera shells and spines.

The foraminiferal order Rotaliida represents one third of the extant genera of foraminifers. The shells of these organisms are extensively used to decipher characteristics of marine ecosystems and global climate events. It was shown that shell calcite of benthic Rotaliida is twinned. We extend our previous work on microstructure and texture characterization of benthic Rotaliida and investigate shell calcite organization for planktonic rotaliid species. Based on results gained from electron backscattered diffraction (EBSD) and field emission electron microscopy (FESEM) imaging of chemically etched/fixed shell surfaces we show for the planktonic species Globigerinoides sacculifer, Pulleniatina obliquiloculata, Orbulina universa (belonging to the two main planktonic, the globigerinid and globorotaliid, clades): very extensive 60°-{001}-twinning of the calcite and describe a new and specific microstructure for the twinned crystals. We address twin and crystal morphology development from nucleation within a biopolymer template (POS) to outermost shell surfaces. We demonstrate that the calcite of the investigated planktonic Rotaliida forms through competitive growth. We complement the structural knowledge gained on the clade 1 and clade 2 species with EBSD results of Globigerinita glutinata and Candeina nitida shells (clade 3 planktonic species). The latter are significantly less twinned and have a different shell calcite microstructure. We demonstrate that the calcite of all rotaliid species is twinned, however, to different degrees. We discuss for the species of the three planktonic clades characteristics of the twinned calcite and of other systematic misorientations. We address the strong functionalization of foraminiferal calcite and indicate how the twinning affects biocalcite material properties.

Calcite crystal orientation patterns in the bilayers of laminated shells of benthic rotaliid foraminifera.

Shells of calcifying foraminifera play a major role in marine biogeochemical cycles; fossil shells form important archives for paleoenvironment reconstruction. Despite their importance in many Earth science disciplines, there is still little consensus on foraminiferal shell mineralization. Geochemical, biochemical, and physiological studies showed that foraminiferal shell formation might take place through various and diverse mineralization mechanisms. In this study, we contribute to benthic foraminiferal shell calcification through deciphering crystallite organization within the shells. We base our conclusions on results gained from electron backscattered diffraction (EBSD) measurements and describe microstructure/texture characteristics within the laminated shell walls of the benthic, symbiontic foraminifera: Ammonia tepida, Amphistegina lobifera, Amphistegina lessonii. We highlight crystallite assembly patterns obtained on differently oriented cuts and discuss crystallite sizes, morphologies, interlinkages, orientations, and co-orientation strengths. We show that: (i) crystals within benthic foraminiferal shells are mesocrystals, (ii) have dendritic-fractal morphologies and (iii) interdigitate strongly. Based on crystal size, we (iv) differentiate between the two layers that comprise the shells and demonstrate that (v) crystals in the septa have different assemblies relative to those in the shell walls. We highlight that (vi) at junctions of different shell elements the axis of crystal orientation jumps abruptly such that their assembly in EBSD maps has a bimodal distribution. We prove (vii) extensive twin-formation within foraminiferal calcite; we demonstrate (viii) the presence of two twin modes: 60°/[001] and 77°/~[6 -6 1] and visualize their distributions within the shells. In a broader perspective, we draw conclusions on processes that lead to the observed microstructure/texture patterns.

Terebratulide brachiopod shell biomineralization by mantle epithelial cells.

To understand mineral transport pathways for shell secretion and to assess differences in cellular activity during mineralization, we imaged with TEM and FE-SEM ultrastructural characteristics of outer mantle epithelium (OME) cells. Imaging was carried out on Magellania venosa shells embedded/etched, chemically fixed/decalcified and high-pressure frozen/freeze-substituted samples from the commissure, central shell portions and from puncta. Imaging results are complemented with morphometric evaluations of volume fractions of membrane-bound organelles. At the commissure the OME consists of several layers of cells. These cells form oblique extensions that, in cross-section, are round below the primary layer and flat underneath fibres. At the commissure the OME is multi-cell layered, in central shell regions it is single-cell layered. When actively secreting shell carbonate extrapallial space is lacking, because OME cells are in direct contact with the calcite of the forming fibres. Upon termination of secretion, OME cells attach via apical hemidesmosomes to extracellular matrix membranes that line the proximal surface of fibres. At the commissure volume fractions for vesicles, mitochondria and lysosomes are higher relative to single-cell layered regions, whereas for endoplasmic-reticulum and Golgi apparatus there is no difference. FE-SEM, TEM imaging reveals the lack of extrapallial space between OME cells and developing fibres. In addition, there is no indication for an amorphous precursor within fibres when these are in active secretion mode. Accordingly, our results do not support transport of minerals by vesicles from cells to sites of mineralization, rather by transfer of carbonate ions via transport mechanisms associated with OME cell membranes.

In-depth knowledge of the low-temperature hydrothermal synthesis of nanocrystalline hydroxyapatite from waste green mussel shell (Perna Viridis).

ABSTRACTThis study presents the use of a low-temperature hydrothermal method for extracting calcium sources from green mussel shell (P. Viridis) wastes and converting them into synthetic nanosized hydroxyapatite (HA). In this study, raw mussel shells were washed, pulverised, and sieved to start producing a fine calcium carbonate-rich powder. XRD quantitative analysis confirmed that the powder contains 97.6 wt. % aragonite. This powder was then calcined for 5 h at 900 °C to remove water, salt, and mud, yielding a calcium-rich feedstock with major minerals of calcite (68.7 wt.%), portlandite (24.7 wt.%), and minor aragonite (6.5 wt.%). The calcined powders were dissolved in aqueous stock solutions of HNO3 and NH4OH before hydrothermally reacting with phosphoric acid [(NH4)2HPO4], yielding pure, nanoscale (16-18 nm) carbonated HA crystals, according to XRD, FT-IR, and SEM analyses. The use of a low-temperature hydrothermal method for a feedstock powder produced by the calcination of low-cost mussel shell wastes would be a valuable processing approach for the industry's development of large-scale hydroxyapatite nanoparticle production.

Biomineral crystallographic preferred orientation in Solenogastres molluscs (Aplacophora) is controlled by organic templating.

Aplacophoran molluscs are shell-less and have a worm-like body which is covered by biomineralized sclerites. We investigated sclerite crystallography and the sclerite mosaic of the Solenogastres species Dorymenia sarsii, Anamenia gorgonophila, and Simrothiella margaritacea with electron-backscattered-diffraction (EBSD), laser-confocal-microscopy and FE-SEM imaging. The soft tissue of the molluscs is covered by spicule-shaped, aragonitic sclerites. These are sub-parallel to the soft body of the organism. We find, for all three species, that individual sclerites are untwinned aragonite single crystals. For individual sclerites, aragonite c-axis is parallel to the morphological, long axis of the sclerite. Aragonite a- and b-axes are perpendicular to sclerite aragonite c-axis. For the scleritomes of the investigated species we find different sclerite and aragonite crystal arrangement patterns. For the A. gorgonophila scleritome, sclerite assembly is disordered such that sclerites with their morphological, long axis (always the aragonite c-axis) are pointing in many different directions, being, more or less, tangential to cuticle surface. For D. sarsii, the sclerite axes (equal to aragonite c-axes) show a stronger tendency to parallel arrangement, while for S. margaritacea, sclerite and aragonite organization is strongly structured into sequential rows of orthogonally alternating sclerite directions. The different arrangements are well reflected in the structured orientational distributions of aragonite a-, b-, c-axes across the EBSD-mapped parts of the scleritomes. We discuss that morphological and crystallographic preferred orientation (texture) is not generated by competitive growth selection (the crystals are not in contact), but is determined by templating on organic matter of the sclerite-secreting epithelial cells and associated papillae.

Calcination-based direct extraction of hydroxyapatite from bovine bone waste.

The main chemical components of waste cow bones are apatite minerals, especially those containing calcium and phosphorus. This study investigated whether this bone could produce extracted hydroxyapatite through calcining at 900° C for different holding times (1-6 h). An average mass loss of 45% occurred in this experiment during the preparation of bone powders, which involved crushing and further calcining at this temperature. The quantitative XRD analysis showed that 99.97 wt.% hydroxyapatite and over 0.3 wt.% calcite were present in the raw and as-calcined bone powders, with trace amounts of CaFe3O5 (calcium ferrite) phases appearing in the calcined product. Depending on the holding calcining times, SEM images of the calcined bovine powders revealed aggregate sizes ranging from 0.5-3 µm and crystallite (grain) sizes ranging from 70 to 340 nm in all calcium-phosphate powder products. Following EDX analysis of all sample surfaces, possible calcium-deficient hydroxyapatite instead of hydroxyapatite formed, as evidenced by the calcined product's Ca/P ratio exceeding 1.67. Additionally, calcining cow bones for 5-6 h at 900° C yielded a high-purity nano-crystalline hydroxyapatite powder precursor in biomedical applications.

Molluscs generate preferred crystallographic orientation of biominerals by organic templates, the texture and microstructure of Caudofoveata (Aplacophora) shells.

Caudofoveata are molluscs that protect their vermiform body with a scleritome, a mosaic of unconnected blade/lanceolate-shaped aragonite sclerites. For the species Falcidens gutturosus and Scutopus ventrolineatus we studied the crystallographic constitution and crystal orientation texture of the sclerites and the scleritome with electron-backscatter-diffraction (EBSD), laser-confocal-microscopy (LCM) and field-emission electron microscopy (FE-SEM) imaging. Each sclerite is an aragonite single crystal that is completely enveloped by an organic sheath. Adjacent sclerites overlap laterally and vertically are, however, not connected to each other. Sclerites are thickened in their central portion, relative to their periphery. Thickening increases also from sclerite tip towards its base. Accordingly, cross-sections through a sclerite are straight at its tip, curved and bent towards the sclerite base. Irrespective of curved sclerite morphologies, the aragonite lattice within the sclerite is coherent. Sclerite aragonite is not twinned. For each sclerite the crystallographic c-axis is parallel to the morphological long axis of the sclerite, the a-axis is perpendicular to its width and the b-axis is within the width of the sclerite. The single-crystalinity of the sclerites and their mode of organization in the scleritome is outstanding. Sclerite and aragonite arrangement in the scleritome is not given by a specific crystal growth mode, it is inherent to the secreting cells. We discuss that morphological characteristics of the sclerites and crystallographic preferred orientation (texture) of sclerite aragonite is not the result of competitive growth selection. It is generated by the templating effect of the organic substance of the secreting cells and associated extracellular biopolymers.

Interaction between undulated and Patch leads to an extreme form of spina bifida in double-mutant mice.

The aetiology of spina bifida involves genetic and environmental factors, which may be why major genes contributing to pathogenesis have not been identified. Here we report that undulated-Patch double-mutant mice have a phenotype reminiscent of an extreme form of spina bifida occulta in humans. This unexpected phenotype in double-mutant but not single-mutant mice shows that novel congenital anomalies such as spina bifida can result from interaction between products of independently segregating loci. This example of digenic inheritance may explain the often sporadic nature of spina bifida in humans.

The unique fibrilar to platy nano- and microstructure of twinned rotaliid foraminiferal shell calcite.

Diversification of biocrystal arrangements, incorporation of biopolymers at many scale levels and hierarchical architectures are keys for biomaterial optimization. The planktonic rotaliid foraminifer Pulleniatina obliquiloculata displays in its shell a new kind of mesocrystal architecture. Shell formation starts with crystallization of a rhizopodial network, the primary organic sheet (POS). On one side of the POS, crystals consist of blocky domains of 1 µm. On the other side of the POS crystals have dendritic-fractal morphologies, interdigitate and reach sizes of tens of micrometers. The dendritic-fractal crystals are twinned. At the site of nucleation, twinned crystals consist of minute fibrils. With distance away from the nucleation-site, fibrils evolve to bundles of crystallographically well co-oriented nanofibrils and to, twinned, platy-blade-shaped crystals that seam outer shell surfaces. The morphological nanofibril axis is the crystallographic c-axis, both are perpendicular to shell vault. The nanofibrillar calcite is polysynthetically twinned according to the 60°/[100] (= m/{001}) twin law. We demonstrate for the twinned, fractal-dendritic, crystals formation at high supersaturation and growth through crystal competition. We show also that c-axis-alignment is already induced by biopolymers of the POS and is not simply a consequence of growth competition. We discuss determinants that lead to rotaliid calcite formation.

T1, an immunoglobulin superfamily member, is expressed in H-ras-dependent epithelial tumours of mammary cells.

T1 is a glycosylated protein in the carcinoembryonic antigen (CEA) family of tumour marker molecules. It was originally identified by virtue of its transient induction after the expression of p21H-ras in NIH3T3 fibroblasts. Here we show that the T1 gene is activated in mammary adenocarcinomas of transgenic mice harbouring an H-ras transgene under the control of the mammary-specific whey acidic protein (WAP) promoter. By contrast, T1 mRNA was not, or only faintly, detectable in mammary carcinomas of transgenic mice bearing a WAP-myc transgene. Thus, T1 overexpression does not appear to be a general tumour-specific phenomenon. A dependence of T1 gene expression on the action of p21H-ras is suggested by the observation of T1 mRNA in nude mouse tumours generated from H-ras-transformed cultured mammary epithelial cells. Interestingly, activation of the T1 gene is also found during the maturation of the mammary gland (3-4 weeks after birth), whereas it is absent during its terminal differentiation in pregnancy and lactation. This expression pattern suggests a role for the secreted T1 glycoprotein in the phase of epithelial proliferation of the mammary gland. It appears that p21H-ras-induced transformation of mammary epithelial cells mimics the situation occurring in puberty. In both developmental stages the T1 glycoprotein might affect cell interactions of the proliferating epithelial cells with the surrounding stroma. It might thus promote ductal outgrowth in gland maturation as well as invasive growth of p21H-ras-transformed mammary epithelial cells.

Molecular characterization of Pax6(2Neu) through Pax6(10Neu): an extension of the Pax6 allelic series and the identification of two possible hypomorph alleles in the mouse Mus musculus.

Phenotype-based mutagenesis experiments will increase the mouse mutant resource, generating mutations at previously unmarked loci as well as extending the allelic series at known loci. Mapping, molecular characterization, and phenotypic analysis of nine independent Pax6 mutations of the mouse recovered in mutagenesis experiments is presented. Seven mutations result in premature termination of translation and all express phenotypes characteristic of null alleles, suggesting that Pax6 function requires all domains to be intact. Of major interest is the identification of two possible hypomorph mutations: Heterozygotes express less severe phenotypes and homozygotes develop rudimentary eyes and nasal processes and survive up to 36 hr after birth. Pax6(4Neu) results in an amino acid substitution within the third helix of the homeodomain. Three-dimensional modeling indicates that the amino acid substitution interrupts the homeodomain recognition alpha-helix, which is critical for DNA binding. Whereas cooperative dimer binding of the mutant homeodomain to a paired-class DNA target sequence was eliminated, weak monomer binding was observed. Thus, a residual function of the mutated homeodomain may explain the hypomorphic nature of the Pax6(4Neu) allele. Pax6(7Neu) is a base pair substitution in the Kozak sequence and results in a reduced level of Pax6 translation product. The Pax6(4Neu) and Pax6(7Neu) alleles may be very useful for gene-dosage studies.

Diaplacental carcinogenic effects of 5-azacytidine in NMRI-mice.

5-Azacytidine was applied to NMRI-mice (1-2 mg/kg) either on gestation day 12, 14, or 16. In the first case it mainly induced leukemias, while in the latter experiments leukemias, lung adenomas and soft tissue sarcomas represent the main effects. The experiments performed on gestation day 14 led to tumor rates below the spontaneously occurring tumor frequencies in NMRI-mice. There is a clear-cut inverse dose-response relationship in leukemia induction, as the higher dose principally shows a lower degree of effectiveness. This, as well as a reduction of tumor frequency below control levels after application of this drug on day 14, can be explained by an "overkill" effect. The cytotoxic and embryolethal efficacy of the agent thus surpasses the transformation effects at the cellular genome. While a negative correlation between the general embryotoxicity of azacytidine and the simultaneous tumor inducibility is to be observed, there is no correlation at the target organ level between the embryotoxic and the carcinogenic effects.

Expression of neu/c-erbB-2 in human brain tumors.

The neu/c-erbB-2 oncogene encodes a 185 kd transmembrane protein (p185). Here we have used the monoclonal antibody (mAb) 3B5 to determine the expression of p185 in a series of fixed biopsy specimens of 180 human brain tumors, including the most frequent entities and, in addition, 18 recurrent gliomas with malignant progression. In summary, 3B5 immunoreaction was most prominent in astrocytomas of different grades of malignancies and in meningiomas. In World Health Organization (WHO) grade II astrocytomas mab 3B5-immunoreaction was related to the cytomorphological phenotype. Fibrillary astrocytomas showed no or only a weak immunoreaction (four of five, 80%) in contrast with protoplasmic or gemistocytic astrocytomas, where a strong reaction was observed in most cases (six of nine, 66.6%, and four of five, 80%, respectively). In WHO grade II to WHO grade IV astrocytomas a trend towards higher scores with increasing grade was found. In a limited number of cases (18 gliomas and two meningiomas) of the tumor series tested other mAbs against neu/c-erbB-2 epitopes, especially the mabs 9G6 and CB11, gave qualitatively comparable results. In WHO grade I pilocytic astrocytomas a wide range of 3B5 immunoreactivity has been observed. The results of in situ hybridization using a 32P-labeled neu/erbB-2 RNA probe performed on four WHO grade I and II astrocytomas, seven WHO grade IV glioblastomas, one WHO grade II oligoastrocytoma, one WHO grade III anaplastic astrocytoma, and three WHO grade I meningiomas were consistent with these immunomorphological data, and Northern blot analysis also indicated an overexpression of neu/c-erbB-2 mRNA in gliomas of different grades of malignancy and in meningiomas. These elevated neu-erbB-2 transcript levels occurred in the absence of gene amplification. In a second series of recurrent gliomas with malignant progression (n = 18) the higher 3B5-immunoreaction scores were apparent in the more malignant recurrent gliomas. In this series the overexpression of neu/c-erbB-2 parallels glioma progression. In our cases it was not, however, correlated with the postoperative relapse-free interval or with the overall length of survival.

Ovary tumors in NMRI mice subjected to fractionated X-irradiation during fetal development.

Fractionated X-irradiation of pregnant mice was performed either during late organogenesis (gestational days 11-13), during the early fetal period (g.d. 14-16), or during both periods (g.d. 11-16). The offspring were observed for 39 months. A significant increase of ovary tumor frequency was observed with 3 X 1.2 Gy, applied either in late organogenesis or in the early fetal period. Lower X-irradiation doses were ineffective in these periods with respect to ovary tumor development. A sharp increase in ovary tumor frequency resulted after irradiation with 6 X 0.8 Gy or 6 X 1.2 Gy. The highest incidence of ovary cysts was observed after 3 X 1.0 Gy or 3 X 1.2 Gy on g.d. 11-13, while the frequency of these cysts was lowest in the animals irradiated six times, which, however, showed a high ovary tumor frequency. Autoradiography of the fetal ovaries either 1 or 6 days after irradiation at the late organogenesis stage revealed a persistent depression of this organ's proliferation rate throughout pregnancy. This may be consistent with the low tumor inducibility after X-irradiation in this period.

Can prenatal X-irradiation in mice act as an initiator stimulus in a modified 2-stage Berenblum/Mottram experiment with postnatal promotion with phorbol ester TPA?

Long-term animal experiments with prenatally X-irradiated offspring have so far not unequivocally settled the question of elevated tumor susceptibility. We have pursued this problem further in a modified 2-stage carcinogenesis-Berenblum/Mottram experiment. Prenatal X-irradiation of mice has thus been regarded as a possible initiator stimulus, with a postnatal promotion stimulus being given by applying the phorbol ester TPA to the offsprings' skin. This treatment has, however, not produced a higher tumor yield, neither of the skin nor of the internal organs, than that produced by X-irradiation in utero alone. This failure seems partly due to the dysplastic nature of the epidermis of prenatally X-irradiated mice, which also fails to respond to TPA application by way of hyperplasia or by an increased inflammation tendency and ulcer formation. We suggest that a decrease in prostaglandin synthesis after prenatal X-irradiation is an important factor for the unchanged tumor susceptibility, especially of the skin.

Radiation-induced meningeal and pituitary tumors in the rat after prenatal application of strontium-90.

Strontium-90 was inject i.v. into pregnant rats on day 18 post conception (p.c.). This caused a remarkable transplacental radioactivity uptake and accumulation in the ossification centers of the skull basis. The total radiation dose within the surface of these regions was consequently calculated to be 0.6-1.2 Gy within the entire lifespan. About 50% of it was delivered during the 7 days following the injection of the isotope. The pathologic examination of the offspring throughout their lifetime revealed a pituitary tumor frequency in the exposed groups which was about tenfold in the males and threefold in the females in comparison to the controls. A very outstanding result in the animals treated was the occurrence of metastasizing meningeal sarcomas in about 6% of all cases. In four cases (= 2%) the simultaneous occurrence of a pituitary adenoma and of a meningeal sarcoma could be observed.

Distribution of advanced glycation end products in the cerebellar neurons of dogs.

Nonenzymatically glycated proteins and their advanced stage, the 'advanced glycation end products' (AGEs), have been detected in long-lived proteins and protein deposits in human and animal tissues. They are thought to be associated with normal aging and particularly with the pathogenesis of diabetic complications and Alzheimer's disease. AGEs accumulate in human neurons in an age-dependent manner and, in Alzheimer's disease patients, particularly in amyloid plaques and neurofibrillary tangles. In this study, we demonstrate AGE immunoreactivity in the canine brain, particularly in cerebellar Purkinje cells and brainstem neurons. In addition, distinct AGE-positive granules can be detected in the Purkinje cells which accumulate in an age-dependent manner. Staining with PAS and oil-red suggests that these AGE-positive granules contain the protein, but not the lipid constituents associated with lipofuscin. Our results show that the pattern of AGE distribution in the canine cerebellum resembles the situation in the human brain, but that the time course of AGE formation is much faster in dogs reflecting their much shorter life span.

Long-lasting effects of naltrexone, an opioid receptor antagonist, on cell proliferation in developing rat forebrain.

Several studies have demonstrated in the past that endogenous opioid peptides and opioid receptors may be involved as mediators of brain tissue growth and function in the neonate. Applying histological and autoradiographic methods, we have examined the effect of the mu-receptor-specific antagonist, naltrexone, on the proliferation of the 4-12-week-old rat forebrain subependymal layer. We found that naltrexone, when given daily throughout the weaning period, evoked a long-lasting increase of the mitotic rate and the [3H]thymidine labelling index. This effect was most significant about 8-10 weeks after ending the naltrexone treatment. Although a direct influence of naltrexone on long-term subependymal cell proliferation cannot be excluded, we are discussing evidence of an indirect effect via suppression of noradrenergic activity in the forebrain.

Placental toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a well-known model substance for inducing in humans and monkeys a severe extrapyramidal syndrome similar to Parkinson's disease. The neurotoxic action of MPTP can be exerted not only in adult animals but also during fetal development by diaplacental passage. Here we show that, during the gestation period of mice, the placenta is another important target organ of MPTP cytotoxicity. Pregnant NMRI mice on gestation day 15 received a single intraperitoneal dose of 20, 40, or 60 mg/kg MPTP. Developmental parameters of the fetuses and the placentas were determined on gestation day 18. Placental weight was consistently reduced in all experimental groups. Histology showed conspicuous alterations of the labyrinth layer; at 20 mg/kg MPTP there was already a significant reduction of the trabecular diameters and from 40 mg/kg onwards, severe necrosis of the syncytial trophoblast cells. In addition, there were necrotic alterations of the cells of the visceral yolk sac. The toxic effects are confined to the placenta at the doses used in the present experiments, leading at just 60 mg/kg to a marked placental insufficiency syndrome.

The effect of fetal irradiation on the growth of postnatally xenotransplanted tumour cells.

Exposure of the mouse fetus (NMRI-strain) to 1.0 Gy X-irradiation has a marked effect on postnatally xenotransplanted glioma cells. In comparison to non-irradiated animals, irradiation on gestation day 14 resulted in: (a) a significantly higher rate of animals which failed to develop visible tumours growing from the inoculum; (b) a significant inhibition of the growth rate of solid gliomas; (c) a pronounced granulocytic and mast cell infiltration, and tissue necrosis, in the invading gliomas. The results suggest that irradiation in prenatal life exerts an amplifying effect on the antitumour response in postnatal life.