On the highest chromosome number in mammals

The mitotic and meiotic chromosomes of the semiaquatic rodent Ichthyomys pittieri (Rodentia, Cricetinae) from Venezuela were analyzed by means of conventional staining and several banding techniques. The diploid chromosome number of this rare species is 2n = 92, which is the highest value known for mammals. It is assumed that this exceptionally high chromosome number is the result of repeated centric fissions. The karyotype of I. pittieri was compared with that of Anotomys leander, for which a diploid number of 2n = 92 has also been reported. The karyological relationships existing within the Neotropical Cricetidae are summarized.     

Cell biologists sort things out: Analysis and purification of intracellular organelles by flow cytometry

Flow cytometry was established originally for measuring DNA content and for the analysis of cell-surface markers in combination with cell sorting. During the past two decades, it has added new dimensions to various areas of immunology and medicine. Increased sensitivity and precision of flow cytometers, accompanied by the development of new fluorescent dyes and probes, has led to new applications in molecular cell biology and genetics. This article focuses on applications of flow cytometry in analysis and sorting of intracellular organelles.     

Morphological studies in a model for dengue-2 virus infection in mice

One of the main difficulties in studying dengue virus infection in humans and in developing a vaccine is the absence of a suitable animal model which develops the full spectrum of dengue fever, dengue haemorrhagic fever, and dengue shock syndrome. It is our proposal to present morphological aspects of an animal model which shows many similarities with the dengue infection in humans. BALB/c mice were intraperitoneally infected with non-neuroadapted dengue virus serotype 2 (DENV-2). Histopathological and morphometrical analyses of liver tissue revealed focal alterations along the infection, reaching wide-ranging portal and centrolobular veins congestion and sinusoidal cell death. Additional ultrastructural observations demonstrated multifocal endothelial injury, platelet recruitment, and alterated hepatocytes. Dengue virus antigen was detected in hepatocytes and in the capillar endothelium of the central lobular vein area. Liver function tests showed high levels of aspartate transaminase and alanine transaminase enzyme activity. Lung tissue showed interstitial pneumonia and mononuclear cells, interseptal oedema, hyperplasia, and hypertrophy of the bronchiolar epithelial cells. DENV-2 led to a transient inflammatory process, but caused focal alterations of the blood-exchange barrier. Viremia was observed from 2nd to 11th day p.i. by isolation of DENV-2 in C6/36 mosquito cell line inoculated with the supernatant of macerated liver, lung, kidney, and cerebellum tissues of the infected mice.     

Assessing Conservation Action for Substitution of Missing Dynamics on Former Military Training Areas in Central Europe

Disproportionately large numbers of threatened and endangered species and unusually high biodiversity have been documented on former military training areas. However, throughout the world, nature protection areas on former military grounds are faced with the dilemma of preserving ecosystems that must change. In central Europe, many of these areas are characterized by dry acidic grasslands on sandy soils. However, these resource-limited ecosystems are faced with two interacting threats: missing disturbance dynamics and increasing atmospheric nitrogen deposition from urban agglomerations. The goal of this study was to assess the effectiveness of conservation action for substitution of missing dynamics. Impacts on plant species assembly, performance of functional groups, soil nutrient availability, and successional trajectory are analyzed. Of particular concern is mimicking beneficial effects of military maneuver on pioneer species such as Corynephorus canescens , which is endangered in many countries. In a controlled field experiment, we manipulated mechanical disturbances representing conservation measures—harrowing, topsoil removal, tilling, and tank driving. Our findings suggest that only topsoil removal and tank driving effectively remove organic material and create self-sustaining vegetation dynamics. Topsoil removal affects both filters in community assembly: the abiotic filter related to soil resources and the biotic filter related to competition between species. We further demonstrate that plant functional groups based on growth form show different rates of recruitment after disturbance with graminoids-dominating initial dynamics. This study provides basic information on effective conservation strategies for dry acidic grasslands on former military training areas.     

The structures of the human neuronal nicotinic acetylcholine receptor β2- and α3-subunit genes (CHRNB2 and CHRNA3)

The α4-subunit gene (CHRNA4) of the neuronal nicotinic acetylcholine receptor (nAChR) subunit family has recently been identified in two families as the gene responsible for autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), a rare monogenic idiopathic epilepsy. As a result of this finding, other subunits of the neuronal nAChR gene family are being considered as candidate genes for ADNFLE in families not linked to CHRNA4 and for other idiopathic epilepsies. α4-subunitsoften assemble together with β2-subunits (gene symbol CHRNB2) to build heteromeric nAChRs. The gene encoding another abundant AChR subunit, the α3-subunit gene (CHRNA3), is present with those encoding two other subunits, CHRNB4 and CHRNA5, in a gene cluster whose functional role is still unclear. Here we provide the information on the genomic structures of both the CHRNB2 and the CHRNA3 genes that is necessary for comprehensive mutational analyses, and we refine the genomic assignment of CHRNB2 on chromosome 1. Received: 5 August 1998 / Accepted: 13 October 1998     

Intrinsic Indicator of Photodamage during Label-Free Multiphoton Microscopy of Cells and Tissues

Multiphoton imaging has evolved as an indispensable tool in cell biology and holds prospects for clinical applications. When addressing endogenous signals such as coherent anti-Stokes Raman scattering (CARS) or second harmonic generation, it requires intense laser irradiation that may cause photodamage. We report that increasing endogenous fluorescence signal upon multiphoton imaging constitutes a marker of photodamage. The effect was studied on mouse brain in vivo and ex vivo, on ex vivo human brain tissue samples, as well as on glioblastoma cells in vitro, demonstrating that this phenomenon is common to a variety of different systems, both ex vivo and in vivo. CARS microscopy and vibrational spectroscopy were used to analyze the photodamage. The development of a standard easy-to-use model that employs rehydrated cryosections allowed the characterization of the irradiation-induced fluorescence and related it to nonlinear photodamage. In conclusion, the monitoring of endogenous two-photon excited fluorescence during label-free multiphoton microscopy enables to estimate damage thresholds ex vivo as well as detect photodamage during in vivo experiments.     

Assessing the efficacy of coherent anti‐Stokes Raman scattering microscopy for the detection of infiltrating glioblastoma in fresh brain samples

Coherent anti‐Stokes Raman scattering (CARS) microscopy is an emerging technique for identification of brain tumors. However, tumor identification by CARS microscopy on bulk samples and in vivo has been so far verified retrospectively on histological sections, which only provide a gross reference for the interpretation of CARS images without matching at cellular level. Therefore, fluorescent labels were exploited for direct assessment of the interpretation of CARS images of solid and infiltrative tumors. Glioblastoma cells expressing green fluorescent protein (GFP) were used for induction of tumors in mice (n = 7). The neoplastic nature of cells imaged by CARS microscopy was unequivocally verified by addressing two‐photon fluorescence of GFP on fresh brain slices and in vivo. In fresh unfixed biopsies of human glioblastoma (n = 10), the fluorescence of 5‐aminolevulinic acid‐induced protoporphyrin IX was used for identification of tumorous tissue. Distinctive morphological features of glioblastoma cells, i.e. larger nuclei, evident nuclear membrane and nucleolus, were identified in the CARS images of both mouse and human brain tumors. This approach demonstrates that the chemical contrast provided by CARS allows the localization of infiltrating tumor cells in fresh tissue and that the cell morphology in CARS images is useful for tumor recognition.

Experimental glioblastoma expressing green fluorescent protein.     

Soil crusts and disturbance benefit plant germination, establishment and growth on nutrient deficient sand

The influence of cyanobacterial soil crusts combined with mechanical soil disturbance on germination, establishment, growth and nutrition of Lepidium sativum seedlings on nutrient poor sand was analyzed under controlled conditions in a growth chamber. Marked release of nitrate and ammonia into the soil was only found in established and older (aging) crusts. Analyses of the ¹⁵N signatures of the crust and the seedling biomass revealed, that there was clear evidence that the established seedlings took up considerable amounts of the released nitrogen. This positively affected plant performance. Mechanical disturbance of the crust led to a temporal increase of nitrogen release but later to a considerable decrease of the released nitrogen components. Undisturbed crusts had a low stimulating effect on seed germination. In contrast and independent of the presence or absence of crusts mechanical soil disturbance led to significantly higher seed germination rates. It is concluded that the occurrence of small-scale disturbance events of moderate intensity at an intermediate frequency, which is the typical disturbance regime in many natural soil crust habitats, seems to be necessary for the successful coexistence of crust organisms and higher plants in nutrient poor environments.