BimS-induced apoptosis requires mitochondrial localization but not interaction with anti-apoptotic Bcl-2 proteins

Release of apoptogenic proteins such as cytochrome c from mitochondria is regulated by pro- and anti-apoptotic Bcl-2 family proteins, with pro-apoptotic BH3-only proteins activating Bax and Bak. Current models assume that apoptosis induction occurs via the binding and inactivation of anti-apoptotic Bcl-2 proteins by BH3-only proteins or by direct binding to Bax. Here, we analyze apoptosis induction by the BH3-only protein Bim(S). Regulated expression of Bim(S) in epithelial cells was followed by its rapid mitochondrial translocation and mitochondrial membrane insertion in the absence of detectable binding to anti-apoptotic Bcl-2 proteins. This caused mitochondrial recruitment and activation of Bax and apoptosis. Mutational analysis of Bim(S) showed that mitochondrial targeting, but not binding to Bcl-2 or Mcl-1, was required for apoptosis induction. In yeast, Bim(S) enhanced the killing activity of Bax in the absence of anti-apoptotic Bcl-2 proteins. Thus, cell death induction by a BH3-only protein can occur through a process that is independent of anti-apoptotic Bcl-2 proteins but requires mitochondrial targeting.     

The Morphological Identity of Insect Dendrites

Dendrite morphology, a neuron's anatomical fingerprint, is a neuroscientist's asset in unveiling organizational principles in the brain. However, the genetic program encoding the morphological identity of a single dendrite remains a mystery. In order to obtain a formal understanding of dendritic branching, we studied distributions of morphological parameters in a group of four individually identifiable neurons of the fly visual system. We found that parameters relating to the branching topology were similar throughout all cells. Only parameters relating to the area covered by the dendrite were cell type specific. With these areas, artificial dendrites were grown based on optimization principles minimizing the amount of wiring and maximizing synaptic democracy. Although the same branching rule was used for all cells, this yielded dendritic structures virtually indistinguishable from their real counterparts. From these principles we derived a fully-automated model-based neuron reconstruction procedure validating the artificial branching rule. In conclusion, we suggest that the genetic program implementing neuronal branching could be constant in all cells whereas the one responsible for the dendrite spanning field should be cell specific.     

Investigation of cellobiohydrolase from trichoderma reesei by small angle X-ray scattering

Summary Trichoderma reesei was grown on sulfite pulp and the major cellobiohydrolase of the culture filtrate was purified to homogeneity. The distance distribution function p(r) measured by the small angle X-ray scattering technique indicates that the enzyme molecule has a rather unusual tadpole like shape with an isotropic head and a long tail. The maximum length is 18 nm and the largest diameter is 4.4 nm.     

Control of echolocation pulses by neurons of the nucleus ambiguus in the rufous horseshoe bat,Rhinolophus rouxi

Horseradish peroxidase was applied by inotophoretic injections to physiologically identified regions of the laryngeal motor nucleus, the nucleus ambiguus in the CF/FM bat Rhinolophus rouxi. The connections of the nucleus ambiguus were analysed with regards to their possible functional significance in the vocal control system, in the respiration control system, and in mediating information from the central auditory system. The nucleus ambiguus is reciprocally interconnected with nuclei involved in the generation of the vocal motor pattern, i.e., the homonomous contralateral nucleus and the area of the lateral reticular formation. Similarly, reciprocal connections are found with the nuclei controlling the rhythm of respiration, i.e., medial parts of the medulla oblongata and the parabrachial nuclei. Afferents to the nucleus ambiguus derive from nuclei of the 'descending vocalization system' (periaqueductal gray and cuneiform nuclei) and from motor control centers (red nucleus and frontal cortex). Afferents to the nucleus ambiguus, possibly mediating auditory influence to the motor control of vocalization, come from the superior colliculus and from the pontine nuclei. The efferents from the pontine nuclei are restricted to rostral parts of the nucleus ambiguus, which hosts the motoneurons of the cricothyroid muscle controlling the call frequency.     

Photoaffinity labeling with [3H]RU 28362: a powerful tool for the study of rat brain glucocorticoid receptors

In order to study the receptor system for adrenocortical steroids in rat brain the synthetic glucocorticoid RU 28362 (11 beta, 17 beta-dihydroxy-6-methyl-17 alpha-(1-propynyl) androsta-1,4,6-trien-3-one) has been used for photoaffinity labeling. Competition and dissociation studies revealed a single class of binding sites for RU 28362 in rat brain cytosol. Photoaffinity labeling was performed by u.v.-irradiation for 2 min with a coupling efficiency of about 25%. The high efficiency permitted investigation of crude cytosolic preparations under denaturating conditions. Sodium dodecyl sulfate (SDS) and high resolution two-dimensional gel electrophoresis confirmed the high specificity of the photoaffinity labeling. The molecular weight (93 kD) as well as the isoelectric point (5.6) evaluated by these methods corresponded well to data reported for the classical glucocorticoid receptor in rat liver.     

Evaluation of the analytical sensitivity of a polymerase chain reaction assay for the detection of chicken infectious anemia virus in avian vaccines

Chicken infectious anemia virus (CAV) is a ubiquitous pathogen of chickens causing significant disease in commercial flocks worldwide. During CAV outbreaks, the Center for Veterinary Biologics requires manufacturers of veterinary biologicals to test materials derived from infected flocks for extraneous CAV by polymerase chain reaction (PCR). The analytical sensitivity of a PCR assay for detection of CAV was determined and the applicability of a CAV DNA standard as a positive control for assay validity was evaluated. The analytical sensitivity of the CAV PCR assay was assessed to be 100 copies per reaction for the DNA standard and 1 × 10^1.9 TCID₅₀/reaction for infectious virus. Establishing the analytical sensitivity of this CAV PCR assay and the inclusion of internal and external positive controls for validity provide a basis for determining whether suspect materials are safe for use in the production of veterinary biologics.     

Paratingia wudensis sp. nov., a whole noeggerathialean plant preserved in an earliest Permian air fall tuff in Inner Mongolia, China

Noeggerathiales are a little known group of Carboniferous and Permian plants of uncertain systematic position that have been variously considered to be ferns, sphenopsids, progymnosperms, or a separate group. These heterosporous plants carry adaxial sporangia on leaf-like or disk-shaped sporophylls that form cones. Leaves are pinnate with a rather stiff appearance, and pinnules can be attached in either two or four rows. In the present report, we present the top of a noeggerathialean plant with leaves and strobili attached, Paratingia wudensis Wang, Pfefferkorn et Bek sp. nov., from an earliest Permian volcanic ash fall tuff in Inner Mongolia. The excellent preservation allows the reconstruction of the whole plant, the complex three-dimensional leaves with anisophyllous pinnules, the heterosporous strobili, and the spores in situ. The homology of leaves and strobili can be elucidated and contributes to an understanding of the debated taxonomic position of Noeggerathiales. The "anisophyllous" leaves carry pinnules arranged in four rows. The strobili are bisporangiate and have disk-shaped sporophylls, each with one ring of 10-14 adaxial sporangia around the strobilus axis. Megaspores have an equatorial bulge. This new species expands the known diversity of Noeggerathiales. It grew in a peat-forming forest, thus changing earlier interpretations of the growth of noeggerathialean plants with anisophyllous pinnules.     

Sensitive detection of human growth hormone mRNA in routinely formalin-fixed,paraffin-embedded transgenic mouse tissues by non-isotopic in situ hybridization

A sensitive technique of non-isotopic in situ hybridization (NISH) is presented, which permits the detection of human growth hormone (hGH) mRNA in routinely formalin-fixed, paraffin-embedded transgenic mouse tissues and human post mortem pituitaries; the latter were used as positive tissue controls in this study. In addition, a double staining procedure combining NISH and immunohistochemistry for the visualization of both hGH and hGH mRNA in the same paraffin section is described. Digoxigenin-labelled antisense hGH RNA was used for NISH of hGH mRNA. The NISH protocol was based upon an established radioactive method. Alkaline phosphatase and horseradish peroxidase-based immunoenzymatic procedures for the detection of digoxigenin-labelled RNA probes using different chromogens [4-nitro blue tetrazolium chloride (NBT), Fast Blue BB, New Fuchsin, and 3,3-diaminobenzidine tetrahydrochloride (DAB) with or without intensification of the DAB staining] were compared. The proteolytic tissue pretreatment and the detection procedure were found to be the most critical steps for successful visualization of hGH mRNA. After optimization of the permeabilization conditions, hGH mRNA could be visualized in each case studied when alkaline phosphatase/NBT-based detection was employed. The NISH technique presented here, performed either separately or in combination with immunohistochemistry, permits retrospective analyses, of hGH (trans)gene expression in archival, paraffin-embedded specimens.     

Detection of apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and acridine orange in Drosophila embryos and adult male gonads

In Drosophila, vast numbers of cells undergo apoptosis during normal development. In addition, excessive apoptosis can be induced in response to a variety of stress or injury paradigms, including DNA damage, oxidative stress, nutrient deprivation, unfolded proteins and mechanical tissue damage. Two of the most commonly used methods to label apoptotic cells in Drosophila are terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) for fixed tissues and acridine orange (AO) staining for live embryos or tissues. Here, we describe protocols for labeling apoptotic cells in Drosophila embryos and adult male gonads. Slightly modified protocols can also be applied for other Drosophila tissues. The AO protocol is quick, simple and allows real-time imaging of doomed cells in live tissues. However, it is difficult to combine with conventional counterstains or Ab labeling. On the other hand, this functionality is readily afforded by the TUNEL protocol, which permits the detection of apoptotic cells in fixed tissues. These staining procedures can be completed in 1-2 d.