Structural, chemical, and magnetic investigation of a graphene/cobalt/platinum multilayer system on silicon carbide.

We investigate the magnetic interlayer coupling and domain structure of ultra-thin ferromagnetic (FM) cobalt (Co) layers embedded between a graphene (G) layer and a platinum (Pt) layer on a silicon carbide (SiC) substrate (G/Co/Pt on SiC). Experimentally, a combination of x-ray photoemission electron microscopy with x-ray magnetic circular dichroism has been carried out at the Co L-edge. Furthermore, structural and chemical properties of the system have been investigated using low energy electron diffraction (LEED) and x-ray photoelectron spectroscopy (XPS).In situLEED patterns revealed the crystalline structure of each layer within the system. Moreover, XPS confirmed the presence of quasi-freestanding graphene, the absence of cobalt silicide, and the appearance of two silicon carbide surface components due to Pt intercalation. Thus, the Pt-layer effectively functions as a diffusion barrier. The magnetic structure of the system was unaffected by the substrate's step structure. Furthermore, numerous vortices and anti-vortices were found in all samples, distributed all over the surfaces, indicating Dzyaloshinskii-Moriya interaction. Only regions with a locally increased Co-layer thickness showed no vortices. Moreover, unlike in similar systems, the magnetization was predominantly in-plane, so no perpendicular magnetic anisotropy was found.

Structural, chemical, and magnetic properties of cobalt intercalated graphene on silicon carbide.

We report on a study of the Co intercalation process underneath the [Formula: see text] R30° reconstructed 6H-SiC(0001) surface for Co film-thicknesses in a range of 0.4-12 nm using a combination of surface sensitive imaging, diffractive, and spectroscopic methods. In situ photoemission electron microscopy reveals a dependence of the intercalation temperature on the Co film-thickness. Using low energy electron diffraction and photoemission spectroscopy (XPS), we find that the SiC surface reconstruction is partially lifted and transformed. We show that the [Formula: see text] R30° reconstruction does not prevent silicide formation for Co film-thicknesses  ≥0.4 nm according to XPS and x-ray absorption spectra. Our results indicate that the silicide formation is self-limited to a thin interface region and is followed by Co intercalation between graphene and silicide. Furthermore, we analyze the magnetic properties using x-ray magnetic circular dichroism at the Co L-edge. In-plane magnetization is observed for all analyzed film-thicknesses. For ultra-thin Co films, self-assembled magnetic wires with a width of the order of 100 nm form at the step-edges.

atm and p53 cooperate in apoptosis and suppression of tumorigenesis, but not in resistance to acute radiation toxicity.

Mutations in atm and p53 cause the human cancer-associated diseases ataxia-telangiectasia and Li-Fraumeni syndrome, respectively. The two genes are believed to interact in a number of pathways, including regulation of DNA damage-induced cell-cycle checkpoints, apoptosis and radiation sensitivity, and cellular proliferation. Atm-null mice, as well as those null for p53, develop mainly T-cell lymphomas, supporting the view that these genes have similar roles in thymocyte development. To study the interactions of these two genes on an organismal level, we bred mice heterozygous for null alleles of both atm and p53 to produce all genotypic combinations. Mice doubly null for atm and p53 exhibited a dramatic acceleration of tumour formation relative to singly null mice, indicating that both genes collaborate in a significant manner to prevent tumorigenesis. With respect to their roles in apoptosis, loss of atm rendered thymocytes only partly resistant to irradiation-induced apoptosis, whereas additional loss of p53 engendered complete resistance. This implies that the irradiation-induced atm and p53 apoptotic pathways are not completely congruent. Finally-and in contrast to prior predictions-atm and p53 do not appear to interact in acute radiation toxicity, suggesting a separate atm effector pathway for this DNA damage response and having implications for the prognosis and treatment of human tumours.

A therapeutic role for sirtuins in diseases of aging?

The sirtuins are a group of proteins linked to aging, metabolism and stress tolerance in several organisms. Among the many genes that have been shown to affect aging in model organisms, sirtuin genes are unique in that their activity level is positively correlated with lifespan (i.e. they are anti-aging genes). Sirtuins are a druggable class of enzymes (i.e. amenable to intervention by small molecules) that could have beneficial effects on a variety of human diseases. In view of the many functions of Sirtuin 1 (SIRT1) in cells, this review focuses on its role in regulating important aspects of mitochondrial biology. Mitochondria have been linked to aging, and also to diseases of aging. Thus, sirtuins might provide a key link between mitochondrial dysfunction, aging and metabolic disease.

Establishment of a cross-European field site network in the ALARM project for assessing large-scale changes in biodiversity.

The field site network (FSN) plays a central role in conducting joint research within all Assessing Large-scale Risks for biodiversity with tested Methods (ALARM) modules and provides a mechanism for integrating research on different topics in ALARM on the same site for measuring multiple impacts on biodiversity. The network covers most European climates and biogeographic regions, from Mediterranean through central European and boreal to subarctic. The project links databases with the European-wide field site network FSN, including geographic information system (GIS)-based information to characterise the test location for ALARM researchers for joint on-site research. Maps are provided in a standardised way and merged with other site-specific information. The application of GIS for these field sites and the information management promotes the use of the FSN for research and to disseminate the results. We conclude that ALARM FSN sites together with other research sites in Europe jointly could be used as a future backbone for research proposals.

Cytoskeleton-secretory vesicle interactions during the docking of secretory vesicles at the cell membrane in Paramecium tetraurelia cells.

Stationary-phase cells of Paramecium tetraurelia have most of their many secretory vesicles ("trichocysts") attached to the cell surface. Log-phase cells contain numerous unoccupied potential docking sites for trichocysts and many free trichocysts in the cytoplasm. To study the possible involvement of cytoskeletal elements, notably of microtubules, in the process of positioning of trichocysts at the cell surface, we took advantage of these stages. Cells were stained with tannic acid and subsequently analyzed by electron microscopy. Semithin sections allowed the determination of structural connections over a range of up to 10 micrometer. Microtubules emanating from ciliary basal bodies are seen in contact with free trichocysts, which appear to be transported, with their tip first, to the cell surface. (This can account for the saltatory movement reported by others). It is noteworthy that the "rails" represented by the microtubules do not directly determine the final attachment site of a trichocyst. Unoccupied attachment sites are characterized by a "plug" of electron-dense material just below the plasma membrane; the "plug" seems to act as a recognition or anchoring site; this material is squeezed out all around the trichocyst attachment zone, once a trichocyst is inserted (Westphal and Plattner, in press. [53]). Slightly below this "plug" we observed fasciae of microfilaments (identified by immunocytochemistry using peroxidase labeled F(ab) fragments against P. tetraurelia actin). Their arrangement is not altered when a trichocyst is docked. These fasciae seem to form a loophole for the insertion of a trichocyst. Trichocyst remain attached to the microtubules originating from the ciliary basal bodies--at least for some time--even after they are firmly installed in the preformed attachment sites. Evidently, the regular arrangement of exocytotic organelles is controlled on three levels: one operating over a long distance from the exocytosis site proper (microtubules), one over a short distance (microfilament bundles), and one directly on the exocytosis site ("plug").

Integrin VLA-3: ultrastructural localization at cell-cell contact sites of human cell cultures.

The integrin VLA-3 is a cell surface receptor, which binds to fibronectin, laminin, collagen type I and VI (Takada, Y., E. A. Wayner, W. G. Carter, and M. E. Hemler. 1988. J. Cell. Biochem. 37:385-393) and is highly expressed in substrate adherent cultures of almost all human cell types. The ligand specificity of VLA-3 and the inhibition of cell adhesion by anti-VLA-3 monoclonal antibodies suggest its involvement in cell-substrate interaction. In normal tissues, VLA-3 is restricted to few cell types, notably the kidney glomeruli and basal cells of the epidermis. In the epidermis, VLA-3 is generally strongly expressed on the entire plasma membrane of basal cells and is not polarized towards the basement membrane (Klein, C. E., C. Cardon-Cardo, R. Soehnchen, R. J. Cote, H. F. Oettgen, M. Eisinger, and L. J. Old. 1987. J. Invest. Dermatol. 89:500-507). Based on this finding we speculated that, in addition to a role of VLA-3 for adhesion of cells to substrate, it could also be relevant for cell-cell interaction. To investigate this, we ultrastructurally localized VLA-3 on the surface of cultured cells by immunoelectron microscopy. In accordance with our concept, we found VLA-3 strongly associated with intercellular contact sites. Interestingly, very little immunoreactivity was detected at the under-surface of cells which had been cultured for 18-32 h. This observation was unexpected but is consistent with previous findings (Kantor, R. R. S., M. J. Mattes, K. D. Lloyd, L. J. Old, and A. P. Albino. 1987. J. Biol. Chem. 262:15158-15165) which suggest that the association of VLA-3 with the basal surface of substrate adherent tumor cells is a late event occurring after days of culture under confluent conditions. However, we cannot formally rule out VLA-3 expression at the undersurface of cells under our experimental conditions, since VLA-3 molecules at this location could be inaccessible for in situ labeling of unfixed cells because of spatial interferences. In conclusion, our results demonstrate the expression of VLA-3 at intercellular contact sites of cultured cells supporting the concept that it may be relevant for intercellular interactions also.

Cell-cycle signaling: Atm displays its many talents.

The discovery of multiple signaling cascades downstream of Atm may lead to a clearer understanding of the diverse defects seen in ataxia-telangiectasia. These pathways - which include evolutionarily conserved Chk1 and Atr, and non-conserved p21, p53 and AbI - guard genomic integrity after DNA damage.

Transposon-generated 'knock-out' and 'knock-in' gene-targeting constructs for use in mice.

The conventional technique for targeted mutation of mouse genes entails placing a genomic DNA fragment containing the gene of interest into a vector for fine mapping, followed by cloning of two genomic arms around a selectable neomycin-resistance cassette in a vector containing thymidine kinase [1]; this generally requires 1-2 months of work for each construct. The single 'knock-out' construct is then transfected into mouse embryonic stem (ES) cells, which are subsequently subjected to positive selection (using G418 to select for neomycin-resistance) and negative selection (using FIAU to exclude cells lacking thymidine kinase), allowing the selection of cells which have undergone homologous recombination with the knockout vector. This approach leads to inactivation of the gene of interest [2]. Recently, an in vitro reaction was developed, on the basis of the yeast Ty transposon, as a useful technique in shotgun sequencing [3]. An artificial transposable element, integrase enzyme and the target plasmid are incubated together to engender transposition. The DNA is then purified, and subsequently electroporated into bacteria. The transposon and the target plasmid bear distinct antibiotic resistance markers (trimethoprim and ampicillin, respectively), allowing double selection for transposition events. In the present study, we have modified this system to allow the rapid, simultaneous generation of a palette of potential gene targeting constructs. Our approach led from genomic clone to completed construct ready for transfection in a matter of days. The results presented here indicate that this technique should also be applicable to the generation of gene fusion constructs [4-8], simplifying this technically demanding method.

Atm-dependent interactions of a mammalian chk1 homolog with meiotic chromosomes.

BACKGROUND: Checkpoint pathways prevent cell-cycle progression in the event of DNA lesions. Checkpoints are well defined in mitosis, where lesions can be the result of extrinsic damage, and they are critical in meiosis, where DNA breaks are a programmed step in meiotic recombination. In mitotic yeast cells, the Chk1 protein couples DNA repair to the cell-cycle machinery. The Atm and Atr proteins are mitotic cell-cycle proteins that also associate with chromatin during meiotic prophase I. The genetic and regulatory interaction between Atm and mammalian Chk1 appears to be important for integrating DNA-damage repair with cell-cycle arrest. RESULTS: We have identified structural homologs of yeast Chk1 in human and mouse. Chk1(Hu/Mo) has protein kinase activity and is expressed in the testis. Chk1 accumulates in late zygotene and pachytene spermatocytes and is present along synapsed meiotic chromosomes. Chk1 localizes along the unsynapsed axes of X and Y chromosomes in pachytene spermatocytes. The association of Chk1 with meiotic chromosomes and levels of Chk1 protein depend upon a functional Atm gene product, but Chk1 is not dependent upon p53 for meiosis I functions. Mapping of CHK1 to human chromosomes indicates that the gene is located at 11q22-23, a region marked by frequent deletions and loss of heterozygosity in human tumors. CONCLUSIONS: The Atm-dependent presence of Chk1 in mouse cells and along meiotic chromosomes, and the late pachynema co-localization of Atr and Chk1 on the unsynapsed axes of the paired X and Y chromosomes, suggest that Chk1 acts as an integrator for Atm and Atr signals and may be involved in monitoring the processing of meiotic recombination. Furthermore, mapping of the CHK1 gene to a region of frequent loss of heterozygosity in human tumors at 11q22-23 indicates that the CHK1 gene is a candidate tumor suppressor gene.

Atm inactivation results in aberrant telomere clustering during meiotic prophase.

A-T (ataxia telangiectasia) individuals frequently display gonadal atrophy, and Atm-/- mice show spermatogenic failure due to arrest at prophase of meiosis I. Chromosomal movements take place during meiotic prophase, with telomeres congregating on the nuclear envelope to transiently form a cluster during the leptotene/zygotene transition (bouquet arrangement). Since the ATM protein has been implicated in telomere metabolism of somatic cells, we have set out to investigate the effects of Atm inactivation on meiotic telomere behavior. Fluorescent in situ hybridization and synaptonemal complex (SC) immunostaining of structurally preserved spermatocytes I revealed that telomere clustering occurs aberrantly in Atm-/- mice. Numerous spermatocytes of Atm-/- mice displayed locally accumulated telomeres with stretches of SC near the clustered chromosome ends. This contrasted with spermatogenesis of normal mice, where only a few leptotene/zygotene spermatocytes I with clustered telomeres were detected. Pachytene nuclei, which were much more abundant in normal mice, displayed telomeres scattered over the nuclear periphery. It appears that the timing and occurrence of chromosome polarization is altered in Atm-/- mice. When we examined telomere-nuclear matrix interactions in spermatocytes I, a significant difference was observed in the ratio of soluble versus matrix-associated telomeric DNA sequences between meiocytes of Atm-/- and control mice. We propose that the severe disruption of spermatogenesis during early prophase I in the absence of functional Atm may be partly due to altered interactions of telomeres with the nuclear matrix and distorted meiotic telomere clustering.

Genetic interactions between atm and p53 influence cellular proliferation and irradiation-induced cell cycle checkpoints.

Ataxia-telangiectasia and Li-Fraumeni syndrome, pleiotropic disorders caused by mutations in the genes atm and p53, share a marked increase in cancer rates. A number of studies have argued for an interaction between these two genes (for comprehensive reviews, see M. S. Meyn, Cancer Res., 55: 5991-6001, 1995, and M. F. Lavin and Y. Shiloh, Annu. Rev., Immunol., 15: 177-202, 1996). Specifically, atm is placed upstream of p53 in mediating G1-S cell cycle checkpoint control, and both atm and p53 are believed to influence cellular proliferation. To analyze the genetic interactions of atm and p53, mouse embryonic fibroblasts (MEFs) homozygously deficient for both atm and p53 were used to assess cell cycle and growth control. These double-null fibroblasts proliferate rapidly and fail to exhibit the premature growth arrest seen with atm-null MEFs. MEFs null for both atm and p53 do not express any p21(cipl/wafl), showing that p53 is required for p21(cipl/wafl) expression in an atm-null background. By contrast, homozygous loss of either atm, p53, or both results in similar abnormalities of the irradiation-induced G1-S cell cycle checkpoint. Our results suggest two separate pathways of interaction between atm and p53, one linear, involving G1-S cell cycle control, and another more complex, involving aspects of growth regulation.

Loss of atm radiosensitizes multiple p53 null tissues.

An unusual clinical finding in ataxia-telangiectasia, a human disorder caused by mutations in atm, is exquisite sensitivity to gamma irradiation. By contrast, homozygous deletion of p53 is marked by radiation resistance in certain tissue compartments. Previous studies (A. J. Levine, Cell, 88: 323-331, 1997) have shown that, in vitro, p53-deficient bone marrow cells are resistant to gamma irradiation. Furthermore, the gastrointestinal radiosensitization engendered by the loss of atm has recently been shown (C. H. Westphal et al., Nat. Genet., 16: 397-401, 1997) to be independent of p53. Expanding on previous work, we have looked at in vivo bone marrow resistance in p53-deficient mice. Our results indicate that inbred FVB strain p53 null mice survive lethal irradiation doses because of bone marrow resistance. Moreover, the deletion of atm radiosensitizes even p53 null bone marrow and mouse embryonic fibroblast cells. The results presented here argue that the loss of atm radiosensitizes multiple tissues in a p53-independent manner. Hence, functional inhibition of atm in p53 null and p53 wild-type human tumors may be a useful adjunct to gamma irradiation-based antitumor therapy.

The mammalian mid-pachytene checkpoint: meiotic arrest in spermatocytes with a mutation in Atm alone or in combination with a Trp53 (p53) or Cdkn1a (p21/cip1) mutation.

ATM, the protein product of the gene mutated in the human autosomal recessive disorder ataxia telangiectasia, is involved in detection of double strand breaks (DSBs) and is a key component of the damage surveillance network of cell cycle proteins. In somatic cells ATM phosphorylates many other proteins including p53, an important regulator of cell cycle control. Mice deficient for Atm are male sterile with arrest and apoptosis occurring at testis epithelial stage IV, which in normal spermatocytes corresponds to mid-pachynema. Unlike the situation in somatic cells, we find no evidence that disruption of the Trp53 (p53) gene, or its down-stream target Cdkn1a (p21/Cip1) results in even a partial rescue of the Atm defect.

Improved preservation of glycogen in unfixed cyanobacteria embedded at -82 degrees C in nanoplast.

A technique is presented for embedding unfixed cells at -82 degrees C in Nanoplast, a water-soluble melamine resin. By use of this procedure, improved ultrastructural preservation is demonstrated in cyanobacteria. Sections treated in this way are suitable for histochemical techniques such as Thiéry's silver staining method for glycogen.

Glycogen staining on sections of aqueous-embedded cyanobacteria and muscle.

We offer histochemical evidence that glycogen is preserved in the cytoplasm of cyanobacteria and muscle specimens that have been embedded in the water-soluble melamine resin Nanoplast for electron microscopy.

The secretory contents of Paramecium tetraurelia trichocysts: ultrastructural--cytochemical characterization.

The secretory contents ("matrix") of Paramecium tetraurelia trichocysts expand by a factor of 4.5 when they undergo a Ca2+-mediated decondensation in the course of exocytosis. This is paralleled by a concomitant increase in the interval of the periodic banding of the matrix from 12 nm to 45-51 nm, which becomes visible with different electron stains for proteins and negatively charged groups. Recent reports of actin in secretory contents led us to investigate its redistribution and artifactual adsorption to the trichocyst contents upon their expansion. To visualize this effect we used peroxidase-labeled F(ab) fragments from an IgG directed against Paramecium actin, a DNAase I-gold complex, and the induction of F-actin polymerization. The trichocysts were analyzed in situ as well as after isolation by density-gradient centrifugation. Additionally, in response to current reports in the literature, we reanalyzed trichocyst contents for any possible presence of calmodulin. We applied three independent in situ methods for this: autofluorescence after trifluoperazine affinity labeling, calmodulin-fluorescence affinity labeling, and an electron microscopic immunocytochemical method. All three methods failed to reveal any significant labeling of structurally intact trichocysts in situ, although we also showed that discharged trichocysts avidly adsorb calmodulin from the culture medium. From the present data we conclude that the decondensation of trichocysts during exocytosis is mediated by a sudden conformational rearrangement of secretory proteins in the trichocyst contents, without the involvement of any other regulatory or contractile proteins, which occur only in the cytoplasm. Trichocyst contents are not significantly--if at all--glycosylated.

A determination of thickness and surface relief in reembedded sections of an epoxy- and a melamine-resin containing ferritin as size standard.

Chemical and physical data of two electron microscopic embedding media (the non-polar epoxy resin Epon 812 and the polar melamine resin Nanoplast FB 101) suggest that less kinetic energy must be applied for cutting a section from a Nanoplast block than from an Epon block of the same hardness and that, consequently, the cutting qualities of Nanoplast are better. To test this hypothesis, normal and extremely thin sections of Epon- and Nanoplast-embedded horse spleen ferritin micropellets were reembedded and resectioned for a determination of thickness and surface roughness. The ease with which extremely thin sections can be cut from the Nanoplast resin (8 nm versus 15 nm in Epon) and the smooth surface of these sections support the hypothesis that the cutting quality of an embedding material is determined primarily by its energy balance, i.e. by the kinetic energy which must be introduced for sectioning and the bonding energy which is released exothermically from a polymer while being sectioned.

Application of nuclear analytical methods in the investigation and identification of new selenoproteins.

Nuclear methods have been applied in the investigation of selenium-containing proteins in rat tissues. Selenium was determined in tissues, cells, and cellular compartments by instrumental neutron activation analysis via 77mSe or 75Se. For tracer studies, the selenium compounds were labeled in vivo by administering 75Se with a high specific activity to rats. Quantitative determination of very small amounts of the element in protein fractions was achieved by measurement of the tracer after replenishment of selenium-depleted animals with the labeled element. The application of the nuclear methods in the detection, characterization, and identification of new selenium-containing proteins is shown with the help of some examples.

Newly found selenium-containing proteins in the tissues of the rat.

The Se-containing proteins in 27 tissues of the rat were investigated by in vivo labeling with 75Se-selenite, separation of the tissue homogenate proteins by SDS-polyacrylamide gel electrophoresis, and determination of the labeled proteins by autoradiography. By using Se-depleted rats and a 75Se-tracer with a high specific activity, Se compounds present at only very low concentrations could be detected. Besides the 13 Se-containing proteins previously described, for which apparent molecular masses of 12, 15, 18, 20, 22, 25, 28, 34, 56, 60, 65, 70, and 75 kD have been found here, a further 15 75Se-labeled bands, with apparent molecular masses of 8, 10, 15.5, 16.5, 24, 32, 34.5, 38, 40, 41, 44, 45, 46.5, 53 and 116 kD could be distinguished. Two-dimensional separation of the kidney homogenate proteins showed that some of the Se-containing bands could be resolved into several labeled spots. Most of the newly found compounds were present in various tissues, but with some the enrichment in certain tissues suggested specific sites of action.