Unburdening evo-devo: ancestral attractions, model organisms, and basal baloney

Although flourishing, I argue that evo-devo is not yet a mature scientific discipline. Its philosophical foundation exhibits an internal inconsistency that results from a metaphysical confusion. In modern evolutionary biology, species and other taxa are most commonly considered as individuals. I accept this thesis to be the best available foundation for modern evolutionary biology. However, evo-devo is characterized by a remarkable degree of typological thinking, which instead treats taxa as classes. This metaphysical incompatibility causes much distorted thinking. In this paper, I will discuss the logical implications of accepting the individuality thesis for evo-devo. First, I will illustrate the degree to which typological thinking pervades evo-devo. This ranges from the relatively innocent use of typologically tainted language to the more serious misuse of differences between taxa as evidence against homology and monophyly, and the logically flawed concept of partial homology. Second, I will illustrate how, in a context of typological thinking, evo-devo's harmless preoccupation with distant ancestors has become transformed into a pernicious problem afflicting the choice of model organisms. I will expose the logical flaws underlying the common assumption that model organisms can be expected to represent the clades they are a part of in an unambiguous way. I will expose the logical flaws underlying the general assumption that basal taxa are the best available stand-ins for ancestors and that they best represent the clade of which they are a part, while also allowing for optimal extrapolation of results.     

Microarray screening reveals two non-conventional SUMO-binding modules linked to DNA repair by non-homologous end-joining

SUMOylation is critical for numerous cellular signalling pathways, including the maintenance of genome integrity via the repair of DNA double-strand breaks (DSBs). If misrepaired, DSBs can lead to cancer, neurodegeneration, immunodeficiency and premature ageing. Using systematic human proteome microarray screening combined with widely applicable carbene footprinting, genetic code expansion and high-resolution structural profiling, we define two non-conventional and topology-selective SUMO2-binding regions on XRCC4, a DNA repair protein important for DSB repair by non-homologous end-joining (NHEJ). Mechanistically, the interaction of SUMO2 and XRCC4 is incompatible with XRCC4 binding to three other proteins important for NHEJ-mediated DSB repair. These findings are consistent with SUMO2 forming a redundant NHEJ layer with the potential to regulate different NHEJ complexes at distinct levels including, but not limited to, XRCC4 interactions with XLF, LIG4 and IFFO1. Regulation of NHEJ is not only relevant for carcinogenesis, but also for the design of precision anti-cancer medicines and the optimisation of CRISPR/Cas9-based gene editing. In addition to providing molecular insights into NHEJ, this work uncovers a conserved SUMO-binding module and provides a rich resource on direct SUMO binders exploitable towards uncovering SUMOylation pathways in a wide array of cellular processes.     

Differential distribution of G-protein beta-subunits in brain: an immunocytochemical analysis.

Heterotrimeric G proteins play central roles in signal transduction of neurons and other cells. The variety of their alpha-, beta-, and gamma-subunits allows numerous combinations thereby confering specificity to receptor-G-protein-effector interactions. Using antisera against individual G-protein beta-subunits we here present a regional and subcellular distribution of Gbeta1, Gbeta2, and Gbeta5 in rat brain. Immunocytochemical specificity of the subtype-specific antisera is revealed in Sf9 cells infected with various G-protein beta-subunits. Since Gbeta-subunits together with a G-protein gamma-subunit affect signal cascades we include a distribution of the neuron-specific Ggamma2- and Ggamma3-subunits in selected brain areas. Gbeta1, Gbeta2, and Gbeta5 are preferentially distributed in the neuropil of hippocampus, cerebellum and spinal cord. Gbeta2 is highly concentrated in the mossy fibres of dentate gyrus neurons ending in the stratum lucidum of hippocampal CA3-area. High amounts of Gbeta2 also occur in interneurons innervating spinal cord alpha-motoneurons. Gbeta5 is differentially distributed in all brain areas studied. It is found in the pyramidal cells of hippocampal CA1-CA3 as well as in the granule cell layer of dentate gyrus and in some interneurons. In the spinal cord Gbeta5 in contrast to Gbeta2 concentrates around alpha-motoneurons. In cultivated mouse hippocampal and hypothalamic neurons Gbeta2 and Gbeta5 are found in different subcellular compartments. Whereas Gbeta5 is restricted to the perikarya, Gbeta2 is also found in processes and synaptic contacts where it partially colocalizes with the synaptic vesicle protein synaptobrevin. An antiserum recognizing Ggamma2 and Ggamma3 reveals that these subunits are less expressed in hippocampus and cerebellum. Presumably this antiserum specifically recognizes Ggamma2 and Ggamma3 in combinations with certain G alphas and/or Gbetas. The widespread but regionally and cellularly rather different distribution of Gbeta- and Ggamma2/3-subunits suggests that region-specific combinations of G-protein subunits mediate signal transduction in the central nervous system. The different subcellular distribution of Gbeta-subunits in cultivated neurons reflects that observed in tissue where Gbeta5 and Gbeta2 associate preferentially with the perikarya and the neuropil, respectively, and suggests an additional association of Gbeta2 with secretory vesicles.     

Potential artifacts in the measurement of DNA deamination

Attack on DNA by some reactive nitrogen species results in deamination of adenine and guanine, leading to the formation of hypoxanthine and xanthine, respectively. Published levels of these products in cellular DNA have varied widely. Although these two deamination products are often measured by GC-MS analysis, the procedure of acid hydrolysis to release DNA bases for derivatization poses a risk of artifactual deamination of the DNA. In this study, we demonstrated the artifactual formation of these two deamination products during acid hydrolysis and hence developed a method for detecting and measuring 2'-deoxyinosine, the nucleoside of hypoxanthine. Our assay for 2'-deoxyinosine employs nuclease P1 and alkaline phosphatase to achieve release of the nucleosides from DNA, followed by HPLC prepurification with subsequent GC-MS analysis of the nucleosides. This assay detected an increase in the levels of 2'-deoxyinosine in DNA when commercial salmon testis DNA was treated with nitrous acid. We also used it to measure levels in various rat tissues of both normal and endotoxin-treated rats, but could not find increased 2'-deoxyinosine formation in tissues even though *NO production was substantially increased.     

Differential Toxicity of 6-Hydroxydopamine in SH-SY5Y Human Neuroblastoma Cells and Rat Brain Mitochondria: Protective Role of Catalase and Superoxide Dismutase

Oxidative stress and mitochondrial dysfunction are two pathophysiological factors often associated with the neurodegenerative process involved in Parkinson's disease (PD). Although, 6-hydroxydopamine (6-OHDA) is able to cause dopaminergic neurodegeneration in experimental models of PD by an oxidative stress-mediated process, the underlying molecular mechanism remains unclear. It has been established that some antioxidant enzymes such as catalase (CAT) and superoxide dismutase (SOD) are often altered in PD, which suggests a potential role of these enzymes in the onset and/or development of this multifactorial syndrome. In this study we have used high-resolution respirometry to evaluate the effect of 6-OHDA on mitochondrial respiration of isolated rat brain mitochondria and the lactate dehydrogenase cytotoxicity assay to assess the percentage of cell death induced by 6-OHDA in human neuroblastoma cell line SH-SY5Y. Our results show that 6-OHDA affects mitochondrial respiration by causing a reduction in both respiratory control ratio (IC(50)?=?200?±?15?nM) and state 3 respiration (IC(50)?=?192?±?17?nM), with no significant effects on state 4(o). An inhibition in the activity of both complex I and V was also observed. 6-OHDA also caused cellular death in human neuroblastoma SH-SY5Y cells (IC(50)?=?100?±?9?μM). Both SOD and CAT have been shown to protect against the toxic effects caused by 6-OHDA on mitochondrial respiration. However, whereas SOD protects against 6-OHDA-induced cellular death, CAT enhances its cytotoxicity. The here reported data suggest that both superoxide anion and hydroperoxyl radical could account for 6-OHDA toxicity. Furthermore, factors reducing the rate of 6-OHDA autoxidation to its p-quinone appear to enhance its cytotoxicity.     

No evidence for increased oxidative damage to lipids, proteins, or DNA in Huntington's disease

It has been proposed that mitochondrial dysfunction and excitotoxic mechanisms lead to oxidative damage in the brain of Huntington;s disease patients. We sought evidence that increased oxidative damage occurs by examining postmortem brain material from patients who had died with clinically and pathologically diagnosed Huntington's disease. Oxidative damage was measured using methods that have already demonstrated the presence of increased oxidative damage in Parkinson's disease, Alzheimer's disease, and senile dementia of the Lewy body type. No alterations in the levels of lipid peroxidation (as measured by lipid peroxides and thiobarbituric acid-malondialdehyde adducts) were found in the caudate nucleus, putamen, or frontal cortex of patients with Huntington's disease compared with normal controls. Similarly, there were no elevations in the levels of 8-hydroxyguanine or of a wide range of other markers of oxidative DNA damage. Levels of protein carbonyls in these tissues were also unaltered. Our data suggest that oxidative stress is not a major component of the degenerative processes occurring in Huntington's disease, or at least not to the extent that occurs in other neurodegenerative disorders.     

The effect of prolonged oral contraceptive steroid use on erythrocyte glutathione peroxidase activity

A clinical study was undertaken to determine whether oral contraceptives (OCs) affect the activity of the enzyme glutathione peroxidase. OC users recruited for the study were volunteers attending the Redhill Family Planning Clinic in England. Their demographic characteristics were noted. Pre- and postmenopausal comparative subjects were also used. The laboratory procedures involved in the study are described. Findings are tabulated. The average erythrocyte glutathione peroxidase levels of women using OCs for more than 7 months were significantly higher than those of the pre- and postmenopausal subjects. These levels increased progressively with duration of OC use. These levels did not fluctuate with the menstrual cycle in either OC or non-OC users. Levels of erythrocyte selenium and plasma pyridoxal were not significantly altered by OC use. Riboflavin status, however, as estimated by glutathione reductase activity was substantially lower in OC users and was lowest in women who had used OCs for the longest amount of time. Riboflavin status was found to be directly correlated with erythrocyte glutathione peroxidase levels. These findings may be important because selenium is currently believed to offer protective benefits against carcinogenesis, especially breast cancer. All the OCs studied produced the same effects.