An annotated and illustrated identification guide to common mesophotic reef sponges (Porifera, Demospongiae, Hexactinellida, and Homoscleromorpha) inhabiting Flower Garden Banks National Marine Sanctuary and vicinities.

Sponges are recognized as a diverse and abundant component of mesophotic and deep-sea ecosystems worldwide. In Flower Garden Banks National Marine Sanctuary region within the northwestern Gulf of Mexico, sponges thrive among diverse biological and geological habitats between 16-200+ m deep (i.e., coral reefs and communities, algal nodules, and coralline algae reefs, mesophotic reefs, patch reefs, scarps, ridges, soft substrate, and rocky outcrops). A synoptic guide is presented, developed by studying common sponge species in the region, through direct sampling and in-situ photographic records. A total of 64 species is included: 60 are Demospongiae (14 orders), two are Hexactinellida (one order), and two are Homoscleromorpha (one order). Thirty-four taxa are identified to species and 13 were identified to have affinity with, but were not identical to, a known species. Fifteen taxa could only be identified to genus level, and the species remain as uncertain (incerta sedis), with the potential to represent new species or variants of known species. One specimen received only a family assignation. This study extends geographic or mesophotic occurrence data for eleven known species and includes several potentially new species. This work improves our knowledge of Gulf of Mexico sponge biodiversity and highlights the importance of the region for scientists and resource managers.

Imaging with neutral atoms: a new matter-wave microscope.

Matter-wave microscopy can be dated back to 1932 when Max Knoll and Ernst Ruska published the first image obtained with a beam of focussed electrons. In this paper a new step in the development of matter-wave microscopy is presented. We have created an instrument where a focussed beam of neutral, ground-state atoms (helium) is used to image a sample. We present the first 2D images obtained using this new technique. The imaged sample is a free-standing hexagonal copper grating (with a period of about 36 microm and rod thickness of about 8 microm). The images were obtained in transmission mode by scanning the focussed beam, which had a minimum spot size of about 2.0 microm in diameter (full width at half maximum) across the sample. The smallest focus achieved was 1.9 +/- 0.1 microm. The resolution for this experiment was limited by the speed ratio of the atomic beam through the chromatic aberrations of the zone plate that was used to focus. Ultimately the theoretical resolution limit is set by the wavelength of the probing particle. In praxis, the resolution is limited by the source and the focussing optics.

Effects of a combinations of emodepside and praziquantel on parasites of reptiles and rodents.

A new combination of two anthelmintic compounds containing emodepside and praziquantel (Profender, Bayer AG, Levekusen, Germany) was tested in pet rodents and reptiles. Topical application of the two compounds led to the quick disappearance of nematodes and cestodes from a broad spectrum of hosts including mice, jirds, snakes, anole lizards, turtles, monitor lizards, etc. In reptiles the dosage had to be increased, since the thick outer layer of the epidermis hinders the penetration of the compounds. In animals with an extremely thick epidermis (e.g. monitor lizards, leguans) the new product was applied under the armpits.

Efficacy of a combination of imidacloprid and moxidectin against parasites of reptiles and rodents: case reports.

A new compound containing imidacloprid 10% (w/v) and moxidectin 2.5% (w/v) (Advantage multi, Advocate) was applied as a spot-on treatment to mice experimentally infected with Trichuris muris. Case reports of reptiles found positive for nematode and mite infections following parasitological examination and treated with this compound are also discussed. The results demonstrated that the registered, recommended 2.5% moxidectin concentration for use in dogs was sufficient to eliminate nematodes and mites in reptiles. Infections with nematodes were successfully treated with a single application. Mite infestations in reptiles were eliminated using a treatment repeated on 3 consecutive days.

Low-grade tubular-mucinous renal neoplasms: morphologic, immunohistochemical, and genetic features.

The current classification system of renal tumors is based on morphologic criteria, as supported by genetic findings. We present a group of previously unclassified tumors with similar morphologic and genetic features, suggesting a new entity within renal neoplasms. Seven renal tumors from five patients (ages 31-67 years) were analyzed. All cases were stained with periodic acid-Schiff, Hale's colloidal iron (HCI), and Alcian blue (AB) at pH 2.5/1.0 with and without hyaluronidase (HA) digestion. Immunohistochemical (IHC) stains were performed for CK8, CK18, CK19, vimentin, villin, Tamm-Horsfall protein (THP), renal cell carcinoma marker (RCC), epithelial membrane antigen (EMA), ulex europaeus agglutinin (UEA-1), soy bean agglutinin (SBA), peanut agglutinin (PNA), and MIB-1. Comparative genomic hybridization (CGH) and loss of heterozygosity (LOH) studies were performed on all cases. All tumors showed circumscribed growth, a tubular growth pattern with focal solid areas, no significant nuclear atypia and absence of necrosis, desmoplasia, or inflammation. Abundant extracellular mucin was present. Immunohistochemistry stains support collecting duct origin (EMA+, PNA+, SBA+/-, CK 8/18/19+, vimentin+/-, UEA-1-, RCC-, villin-, THP-). The proliferative rate was low (<1%). CGH showed multiple consistent chromosomal losses (-1,-4, -6, -8, -9, -13, -14, -15, -22). Clinical outcome was favorable, with recurrences but no known distant metastases or death of disease. These findings are distinct from all previously classified renal neoplasms. Our data suggest the presence of a unique tumor entity within tumors of probable collecting duct origin: tubular-mucinous renal tumors of low malignant potential.

Computed tomography of cryogenic biological specimens based on X-ray microscopic images.

Soft X-ray microscopy employs the photoelectric absorption contrast between water and protein in the 2.34-4.38 nm wavelength region to visualize protein structures down to 30 nm size without any staining methods. Due to the large depth of focus of the Fresnel zone plates used as X-ray objectives, computed tomography based on the X-ray microscopic images can be used to reconstruct the local linear absorption coefficient inside the three-dimensional specimen volume. High-resolution X-ray images require a high specimen radiation dose, and a series of images taken at different viewing angles is needed for computed tomography. Therefore, cryo microscopy is necessary to preserve the structural integrity of hydrated biological specimens during image acquisition. The cryo transmission X-ray microscope at the electron storage ring BESSY I (Berlin) was used to obtain a tilt series of images of the frozen-hydrated green alga Chlamydomonas reinhardtii. The living specimens were inserted into borosilicate glass capillaries and, in this first experiment, rapidly cooled by plunging into liquid nitrogen. The capillary specimen holders allow image acquisition over the full angular range of 180 degrees. The reconstruction shows for the first time details down to 60 nm size inside a frozen-hydrated biological specimen and conveys a clear impression of the internal structures. This technique is expected to be applicable to a wide range of biological specimens, such as the cell nucleus. It offers the possibility of imaging the three-dimensional structure of hydrated biological specimens close to their natural living state.

X-ray microscopic studies of the Drosophila dosage compensation complex.

X-raymicroscopy is applied to detect specific proteins in whole cell nuclei of Drosophila melanogaster using immunogold labeling and silver enhancement. As a model for a small subnuclear structure the Drosophila dosage compensation protein MSL-1 was chosen. It associates with a number of other proteins to form a hetero-multiprotein complex, which elevates the transcriptional activity of the single X chromosome in males. This phenomenon is known as dosage compensation and is essential for the survival of male flies. The distribution of the Drosophila dosage compensation complex was studied by X-ray microscopy, because though the complex is expected to function by remodeling the structure of chromatin, its exact mode of action is not yet known. Many similar protein complexes are associated with different aspects of chromatin-mediated gene regulation in all eucaryotic organisms and can also be studied with the approach presented in this work. The distribution of MSL-1 protein in the nuclei of fixed D. melanogaster culture cells is visualized using the Göttingen X-ray microscope at the electron storage ring BESSY I. In addition to conventional and confocal laserscan fluorescence microscopy, X-ray microscopic investigations were performed at room as well as at cryogenic temperatures. The label can clearly be identified in the X-ray micrographs and shows detailed structure in the cell nuclei. Currently, X-ray micrographs show details in the cell nuclei about five times smaller than those in visible light micrographs.

Gene expression of the hematopoietic cell phosphatase in juvenile myelomonocytic leukemia.

We analyzed the relative expression of Hematopoietic cell phosphatase (HCP) in mononuclear cells (MNC) of peripheral blood (PB), bone marrow (BM) and spleen of patients with juvenile myelomonocytic leukemia (JMML) and normal donors. Two regions of HCP with alternative exon skipping of exon 6 or exon 12 are described. There was no difference in the expression of the amplified HCP cDNA regions in MNC of JMML patients compared to normal donors. The two forms of exon skipping were present in unstimulated MNC of JMML patients or normal donors. In contrast, phytohemagglutinin (PHA) stimulated MNC of normal donors, Epstein-Barr Virus (EBV) transformed B-cells of JMML patients, BFU-E and CFU-GM derived colonies of JMML patients, and the cell lines K562 and HEL did not or only barely express these two forms of exon skipping. These results may indicate that alternative HCP exon skipping may be associated with the proliferative state of the cell.

Expression of interferon regulatory factor 1 and 2 in hematopoietic cells of children with juvenile myelomonocytic leukemia.

Interferon regulatory factor 1 (IRF-1) is a transcriptional activator in the interferon system and acts as a tumor suppressor. The structurally related IRF-2 represses the effects of IRF-1 by competitive binding to the same DNA sequence elements. Changes in the relative balance between IRF-1 and IRF-2 lead to dysregulation of cell growth and may play a role in the development of neoplasias. The loss of functional IRF-1 has been observed in a number of patients with myelodysplastic syndrome (MDS) and leukemia, suggesting a potentially critical role of IRF-1 in leukemogenesis. We studied the expression of both transcription factors in peripheral blood (PB) and bone marrow (BM) cells of children with juvenile myelomonocytic leukemia (JMML) using RT-PCR and Southern blot hybridization. No significant difference between the expression levels of IRF-1 and IRF-2 could be detected in PB and BM of patients with JMML and normal donors. Although our results are preliminary they suggest that neither the tumor suppressor gene IRF-1 nor the oncogene IRF-2 is involved in the pathogenesis of JMML.

RAS mutations and clonality analysis in children with juvenile myelomonocytic leukemia (JMML).

Juvenile myelomonocytic leukemia (JMML) is a malignant hematopoietic disorder of early childhood with excessive proliferation of the myeloid and monocytic lineage. Deregulation of the RAS signal transduction pathway is thought to play a key role in its pathogenesis. We examined peripheral blood or bone marrow cells of 36 children with JMML for activating point mutations in codons 12, 13 and 61 of the NRAS and KRAS proto-oncogenes by allele-specific restriction assay, single-strand conformation polymorphism and/or direct sequencing. Codons 12, 13 and 61 of HRAS were examined in 26 of these patients. We detected RAS mutations in six cases (17%) located at N12 (n = 2), N13 (n = 3) and K13 (n = 1). In addition, we performed clonality studies on different cell lineages in four of these patients applying the RAS mutation, the karyotype and X-chromosome inactivation patterns as clonal markers. Erythroid cells carried mutant RAS, indicating clonal origin. In EBV B cell lines, one of three patients studied harbored a RAS mutation, while the other two patients had polyclonal B cells with wild-type RAS. T lymphocytes were examined in one patient; they were polyclonal and had wild-type RAS. It is likely that JMML is a heterogeneous disease with respect to clonal involvement of different lineages.

Treatment of fish parasites. 11. Effects of different benzimidazole derivatives (albendazole, mebendazole, fenbendazole) on Glugea anomala, Moniez, 1887 (Microsporidia): ultrastructural aspects and efficacy studies.

Three different benzimidazole derivatives, albendazole [methyl-5-(propylthio)-2-benzimidazolcarbamate], mebendazole (methyl-5-benzoyl-2-benzimidazolcarbamatic acid methyl ester), and fenbendazole [methyl-5-(phenylthio)-2-benzimidazolcarbamate] were tested in vivo against Glugea anomala parasitizing the connective tissue of sticklebacks (Gasterosteus aculeatus). Naturally infected sticklebacks were incubated in aerated plastic aquaria (10 1) at 22 degrees C in water containing 0, 1, 5, 10, or 50 micrograms of either albendazole, mebendazole or febendazole for 2 or 6 h. For intermittent treatment, 2 micrograms substance was administered three times for 6 h at intervals of 36 h. At the ultrastructural level, at all developmental stages of G. anomala there were no significant differences in the kind of damage caused by either albendazole, mebendazole, or febendazole. Starting with a dose of 1 microgram/ml for 2 h, each of the drugs irreversibly damaged uni- and multinucleate meronts, sporogonial plasmodia, and sporoblasts. Disorganized spores were also observed. Treatment with higher doses (10 micrograms/ml, 2 or 6 h) caused malformations of the merogonic and the sporogonic stages, a significant reduction in the number of ribosomes, and disruptions of the nuclear membranes. The first recognizable treatment effect was an enlargement of the smooth endoplasmic reticulum. In the sporogonial plasmodia, the membranes of the sporophorous vesicle envelopes were lumpy or even completely destroyed. After incubation with the highest dose (50 micrograms/ml, 6 h), microtubules were apparent within the karyoplasm of the uninucleate meronts. After interval treatment, all forms of damage were intensified, especially in the mature spores. When treatment was done three times at low doses (3 x 2 micrograms/ml, 6 h, 36-h intervals), spore infectivity was drastically lowered. Therefore, it seems likely that an intermittent regimen of medicinal baths can be successfully applied against susceptible Microsporidia in fish.

Studies on intracellular structures of COS cells by X-ray microscopy.

COS-7 cells, fixed with glutaraldehyde, were studied using the transmission X-ray microscope at the electron storage ring BESSY, Berlin. The border of the cell, the nucleus, nucleoli and mitochondria of the cells were clearly visualized with the X-ray microscope. In addition, we found many X-ray dense granules preferentially located around the nucleus. Electron microscopy showed that numerous multivesicular bodies, whose structures belong to the endosome-lysosomal system, were present around the nucleus. The size and localization patterns of the X-ray dense granules were quite similar to those of multivesicular bodies. These results strongly suggest that the X-ray dense granules are multivesicular bodies.

Expression of transcription factors during sodium phenylacetate induced erythroid differentiation in K562 cells.

During 15 days of treatment of K562 cells with sodium phenylacetate, we observed an increase in the cellular hemoglobin concentration with a similar increase in the expression of gamma-globin mRNA. Morphological studies demonstrated characteristic features of erythroid differentiation and maturation. At the same time there was no change in the level of expression of the cell surface antigenes CD33, CD34, CD45, CD71 and glycophorin A. Likewise, the level of expression of the erythroid transcription factors GATA-1, GATA-2, NF-E2, SCL and RBTN2, all expressed in untreated K562 cells, did not increase during sodium phenylacetate induced erythroid differentiation. The expression of the nuclear factors Evi-1 and c-myb, known to inhibit erythroid differentiation, did not decrease. We conclude that sodium phenylacetate treatment of K562 cells increases gamma-globin mRNA and induces cell maturation as judged by morphology without affecting the expression of the erythroid transcription factors, some of which are known to be involved in the regulation of beta-like globin genes.

Transmission X-ray microscopy of intact hydrated PtK2 cells during the cell cycle.

Transmission X-ray microscopy makes it possible to investigate biological specimens, i.e. cells and organelles, in their natural wet environment. The main processes determining the contrast in X-ray microscopy are photoelectric absorption and phase shift. X-ray microscopic experiments can therefore be carried out in both amplitude and phase contrast. The Göttingen X-ray microscope at the BESSY storage ring in Berlin is described. PtK2 cells were examined during different stages of the cell cycle. All major constituents of the mitotic apparatus, e.g. chromosomes, centromeres, microtubules and centrosomes, could be visualized, as well as the main structural compartments and organelles of the interphase cell, e.g. nuclear membrane, interphase chromatin, nucleolus and cytoplasmic mitochondria, as well as parts of the cytoskeletal apparatus. In this way new information can be obtained with regard to the ultrastructure of the constituents of intact and unstained cells at a resolution which bridges the gap between light microscopy and electron microscopy. The prospects for the future application of transmission X-ray microscopy in biomedical research are discussed.

Expression of the Evi-1 gene in haemopoietic cells of children with juvenile myelomonocytic leukaemia and normal donors.

Activation of the Evi-1 gene was first described to be associated with the transformation of murine myeloid leukaemias and has previously been detected in cases of human acute myeloid leukaemia (AML) and chronic myeloid leukaemia (CML) in blast crises and in myelodysplastic syndromes. In this study we determined the frequency and the level of Evi-1 expression in juvenile myelomonocytic leukaemia (JMML) and in normal haemopoiesis. Using RT-PCR and Southern blot hybridization mRNA of Evi-1 could be detected in bone marrow (BM) and peripheral blood (PB) mononuclear cells (MNC) of normal donors. In JMML 12/20 patients examined expressed elevated levels of Evi-1 compared to normal controls. In these samples over-expression of the gene was correlated with a higher percentage of blasts (P = 0.02). Expression levels in BFU-E and CFU-GM derived colonies from BM of JMML patients were lower than those in the corresponding MNC samples. Analysis of CD34+ and CD34- cells demonstrated that Evi-1 is primarily expressed in the CD34+ cell population of both JMML and normal donors. These findings suggest that Evi-1 expression is linked to the early stages of haemopoiesis. Studies on the regulation of Evi-1 expression in CD34+ cells will elucidate its function in progenitor cells and clarify its possible role in the pathogenesis of JMML.

Multiparameter microscopic analysis of nucleolar structure and ribosomal gene transcription.

A survey of novel microscopic approaches for structural and functional analysis of subnucleolar compartments will be presented. Research on nucleolar structure and function concentrates predominantly on two distinct types of nucleoli: (1) nucleoli present during the interphase of the cell cycle in somatic tissue culture cells and (2) nucleoli present in meiotic cells, e.g. oocytes of amphibians. These nucleoli are found during meiotic prophase of oogenesis and are functional during several months of the diplotene stage of oogenesis. A further characteristic is the fact that these nucleoli are extrachromosomal, since they originate by selective ribosomal DNA (rDNA) amplification during the early pachytene stage of oogenesis. Miller-type chromatin spread preparations using transcriptionally active nucleoli, to a major part, contributed to our understanding of the structural organization of polymerase I directed pre-rRNA transcription. Although the structural organization of the template-associated pre-rRNA transcript is known in some detail from chromatin spreads, relatively little is known about structural aspects of pre-rRNA processing. In order to investigate this intriguing question in more detail, we have developed a computer-based densitometry analysis of both template-associated and template-dissociated pre-rRNA transcripts in order to follow the structural modification of pre-rRNA transcripts during processing. Another line of experiments is devoted to the in situ structure of actively transcribing genes in the nucleolus. In order to bridge the gap between light microscopy and electron microscopy we started video-enhanced light microscopical analysis of actively transcribing genes. Although the dimensions of individual spread genes are critical for detection by optical microscopy, we succeeded in obtaining the first series of images of transcribing genes in their "native' hydrated state. An additional promising type of microscopy is transmission X-ray microscopy. Recent progress in instrumentation as well as in sample preparation has allowed us to obtain the first images of density distribution within intact, fully hydrated nucleoli using amplitude-contrast and/or phase-contrast X-ray microscopy of non-contrasted, fully hydrated nucleoli at different states of transcriptional activity. Whereas the above mentioned investigations using video microscopy and X-ray microscopy are predominantly applicable to the analysis of amplified nucleoli in amphibian oocytes, which are characterized by an extremely high transcription rate of 80-90% of rDNA genes per individual nucleolus, structural analysis of the in situ arrangement of actively transcribing genes in somatic nucleoli as present in the interphase nucleus is far more difficult to perform, mainly due to the much lower number of simultaneously transcribed active genes per individual nucleolus. Visualization of actively transcribed gene clusters is approached by an integrated experimental assay using video microscopy, confocal laser scan microscopy, and antibodies against specific nucleolar proteins.

A monochromator for scanning X-ray microscopy beamlines at third-generation synchrotron light sources.

A concept for a plane-grating monochromator for use at scanning X-ray microscopy beamlines at third-generation synchrotron light sources is presented. The design of the monochromator is optimized for a scanning transmission X-ray microscopy beamline at BESSY II. Ray-tracing calculations are presented which include geometric aberrations of the optics used in the beamline.

[X-ray microscopy]. / Röntgenmikroskopie.

Owing to the short wavelengths of X-radiation X-ray microscopes allow higher resolution than optical microscopes. In contrast to electron microscopes, X-radiation can be used to study relatively thick aqueous specimens in their natural environment. X-ray microscopes require intense X-radiation, which is best provided by electron storage rings, as well as efficient X-ray optics. X-ray microscopes with zone plate optics are installed at the storage ring BESSY in Berlin for studies in the fields of biology, medicine, biophysics, colloid chemistry, and soil sciences.