The role of topoisomerase II in the excision of DNA loop domains during apoptosis

Disintegration of nuclear DNA into high molecular weight (HMW) and oligonucleosomal DNA fragments represents two major periodicities of DNA fragmentation during apoptosis. These are thought to originate from the excision of DNA loop domains and from the cleavage of nuclear DNA at the internucleosomal positions, respectively. In this report, we demonstrate that different apoptotic insults induced apoptosis in NB-2a neuroblastoma cells that was invariably accompanied by the formation of HMW DNA fragments of about 50-100 kb but proceeded either with or without oligonucleosomal DNA cleavage, depending on the type of apoptotic inducer. We demonstrate that differences in the pattern of DNA fragmentation were reproducible in a cell-free apoptotic system and develop conditions that allow in vitro separation of the HMW and oligonucleosomal DNA fragmentation activities. In contrast to apoptosis associated with oligonucleosomal DNA fragmentation, the HMW DNA cleavage in apoptotic cells was accompanied by down-regulation of caspase-activated DNase (CAD) and was not affected by z-VAD-fmk, suggesting that the caspase/CAD pathway is not involved in the excision of DNA loop domains. We further demonstrate that nonapoptotic NB-2a cells contain a constitutively present nuclease activity located in the nuclear matrix fraction that possessed the properties of topoisomerase (topo) II and was capable of reproducing the pattern of HMW DNA cleavage that occurred in apoptotic cells. We demonstrate that the early stages of apoptosis induced by different stimuli were accompanied by activation of topo II-mediated HMW DNA cleavage that was reversible after removal of apoptotic inducers, and we present evidence of the involvement of topo II in the formation of HMW DNA fragments at the advanced stages of apoptosis. The results suggest that topo II is involved in caspase-independent excision of DNA loop domains during apoptosis, and this represents an alternative pathway of apoptotic DNA disintegration from CAD-driven caspase-dependent oligonucleosomal DNA cleavage.     

Historical biogeography and the origin of stomatal distributions in Banksia and Dryandra (Proteaceae) based on their cpDNA phylogeny

Banksia and Dryandra have undergone extensive speciation and adaptive radiation, especially in Australia's isolated Southwest Botanical Province. We derive a phylogeny for these groups based on cpDNA sequences and use it to reconstruct their historical biogeography and evolution of leaf traits thought to be adapted to drought and/or nutrient poverty. Slowly evolving regions (trnL intron, trnL/trnF spacer) are used to resolve large-scale relationships; faster evolving regions (rp116 intron, psbA/trnH and trnT/trnL spacers) are used to resolve relationships among closely related species. Banksia is paraphyletic with respect to Dryandra. The lineage underwent a basal split into two clades (here named /Cryptostomata and /Phanerostomata), and four infrageneric taxa supported by morphological cladistic analyses (series Spicigerae, Abietinae, Tetragonae, and Banksia) are not monophyletic. Dispersal-vicariance analysis resolves a southwestern Australian origin for the lineage, with two later expansions to the east followed by vicariance events. Stomatal crypts arose with the /Cryptostomata, which is characterized by tough, long-lived leaves and common in southwestern Australia. Sequestering of stomata also arose multiple times in /Phanerostomata, which is characterized by softer, short-lived leaves and common in moister coastal areas, via inrolling of the margins of narrow leaves and restricting stomata to shallow pits. The hypothesis that sclerophylly preadapted the plants to xeromorphy is supported in the case of shallow stomatal pits and deep stomatal crypts, but not narrow, needle-like leaves.     

Opposite Effects of Lithium on Proximal and Distal Caspases of Immature and Mature Primary Neurons Correlate with Earlier Paradoxical Actions on Viability

To provide an explanation for earlier paradoxical findings of lithium on survival of mature and immature neurons, this study monitors changes in cytosolic caspases in rat cerebellar granule cells (CGC) grown 2–7 days in vitro (DIV), or in murine E-17 cortical neurons. Data show Li⁺ protects mature 7-DIV CGC parallel to a decrease in proximal and distal caspases but increases levels for immature 2-DIV-CGC or E-17 cortical neurons. Caspases mirror viability based on morphological analyses (dye uptake, phase-contrast, DNA fragmentation), and suggest protection occurs by suppressing activation of a cascade resulting in distal effectors that destroy proteins essential for neuronal survival. Protection was dose-dependent with EC₅₀ 3.0 mM and extended to 64 h in K⁺-serum deprived apoptotic media. Neuronal extracts contain a spectrum of proximal (-2, -8, -9) and distal (-3, -6) caspases sensitive to Li⁺ on assay with preferred peptide substrates and by immunobloting. The lack of direct effect on activated cytosols indicates Li⁺ acts upstream only on intact cells, at sites for recruitment of pivotal procaspases. Alterations of procaspase-9 p46 and membrane-bound cytochrome c (Apaf-1) point to interaction with an intrinsic Mt-mediated pathway as one of the targets. The opposite effects on caspases and viability of immature or embryological neurons point to existence of alternative pathways that alter during neurite outgrowth suggesting the use of Li⁺ as a probe to unravel events relevant to neurogenesis.     

Dynamic relationship between sympathetic nerve activity and renal blood flow: a frequency domain approach

Blood pressure displays an oscillation at 0.1 Hz in humans that is well established to be due to oscillations in sympathetic nerve activity (SNA). However, the mechanisms that control the strength or frequency of this oscillation are poorly understood. The aim of the present study was to define the dynamic relationship between SNA and the vasculature. The sympathetic nerves to the kidney were electrically stimulated in six pentobarbital-sodium anesthetized rabbits, and the renal blood flow response was recorded. A pseudo-random binary sequence (PRBS) was applied to the renal nerves, which contains equal spectral power at frequencies in the range of interest (<1 Hz). Transfer function analysis revealed a complex system composed of low-pass filter characteristics but also with regions of constant gain. A model was developed that accounted for this relationship composed of a 2 zero/4 pole transfer function. Although the position of the poles and zeros varied among animals, the model structure was consistent. We also found the time delay between the stimulus and the RBF responses to be consistent among animals (mean 672 +/- 22 ms). We propose that the identification of the precise relationship between SNA and renal blood flow (RBF) is a fundamental and necessary step toward understanding the interaction between SNA and other physiological mediators of RBF.     

Modes of Evolution in the Protease and Kringle Domains of the Plasminogen–Prothrombin Family

Phylogenetic analysis of protease domains of the vertebrate plasminogen–prothrombin family revealed two major subfamilies: (1) a subfamily containing macrophage-stimulating protein (MSP), hepatocyte growth factor (HGF), plasminogen, and apolipoprotein(a) (APOA); and (2) a subfamily containing prothrombin, HGF activator, and plasminogen activators. There was evidence that these two subfamilies diverged prior to the divergence of amphibians and amniotes. The phylogeny indicated a close relationship of APOA from the European hedgehog, rhesus monkey, and human with plasminogen. Phylogenetic analysis of repeated kringle domains supported the hypothesis that APOA evolved independently in hedgehog and primates through numerous duplications of different kringle domains of the ancestral plasminogen. Phylogenies of kringle domains revealed two modes of evolution: (1) a conservative mode, whereby duplication of kringle domains occurred prior to cladogenesis and the same kringle structure has been maintained in different lineages (exemplified by plasminogen and prothrombin); and (2) a concerted mode, whereby kringle domains have duplicated since cladogenesis and thus orthologous relationships do not exist between kringles of different lineages (exemplified by APOA).     

Ecological Indicators in Risk Assessment: Workshop Summary

Ecological indicators can be defined as relatively simple measurements that relay scientific information about complex ecosystems. Such indicators are used to characterize risk in ecological risk assessment (ERA) and to mark progress toward resource management goals. In late 1997, scientists from the U.S. Environmental Protection Agency and from the Chemical Manufacturers Association (CMA) held a workshop to explore opportunities for collaborative research and scientific exchange on the development and application of ecological indicators. Several scientific challenges were identified as they relate to problem formulation, exposure and effects assessment, and risk characterization. Chief among these were a better understanding of multiple stressors (both chemical and non-chemical), characterization of reference sites and natural variability, extrapolation of measures to ecologically relevant scales, development of comprehensive, ecosystem-based models that incorporate multiple stressors and receptors, and a consistent system for evaluating ecological indicators.     

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.     

Frontal Lobe Contusion in Mice Chronically Impairs Prefrontal-Dependent Behavior

Traumatic brain injury (TBI) is a major cause of chronic disability in the world. Moderate to severe TBI often results in damage to the frontal lobe region and leads to cognitive, emotional, and social behavioral sequelae that negatively affect quality of life. More specifically, TBI patients often develop persistent deficits in social behavior, anxiety, and executive functions such as attention, mental flexibility, and task switching. These deficits are intrinsically associated with prefrontal cortex (PFC) functionality. Currently, there is a lack of analogous, behaviorally characterized TBI models for investigating frontal lobe injuries despite the prevalence of focal contusions to the frontal lobe in TBI patients. We used the controlled cortical impact (CCI) model in mice to generate a frontal lobe contusion and studied behavioral changes associated with PFC function. We found that unilateral frontal lobe contusion in mice produced long-term impairments to social recognition and reversal learning while having only a minor effect on anxiety and completely sparing rule shifting and hippocampal-dependent behavior.