Neuron specific metabolic adaptations following multi-day exposures to oxygen glucose deprivation

Prior exposure to sub toxic insults can induce a powerful endogenous neuroprotective program known as ischemic preconditioning. Current models typically rely on a single stress episode to induce neuroprotection whereas the clinical reality is that patients may experience multiple transient ischemic attacks (TIAs) prior to suffering a stroke. We sought to develop a neuron-enriched preconditioning model using multiple oxygen glucose deprivation (OGD) episodes to assess the endogenous protective mechanisms neurons implement at the metabolic and cellular level. We found that neurons exposed to a five minute period of glucose deprivation recovered oxygen utilization and lactate production using novel microphysiometry techniques. Using the non-toxic and energetically favorable five minute exposure, we developed a preconditioning paradigm where neurons are exposed to this brief OGD for three consecutive days. These cells experienced a 45% greater survival following an otherwise lethal event and exhibited a longer lasting window of protection in comparison to our previous in vitro preconditioning model using a single stress. As in other models, preconditioned cells exhibited mild caspase activation, an increase in oxidized proteins and a requirement for reactive oxygen species for neuroprotection. Heat shock protein 70 was upregulated during preconditioning, yet the majority of this protein was released extracellularly. We believe coupling this neuron-enriched multi-day model with microphysiometry will allow us to assess neuronal specific real-time metabolic adaptations necessary for preconditioning.     

Quantitative and molecular analyses of ethyl methanesulfonate- and ICR 191-induced mutation in AS52 cells

A pSV2gpt-transformed Chinese hamster ovary (CHO) cell line has been used to study mutation at the molecular level. This cell line, designated AS52, was constructed from a hypoxanthine-guanine phosphoribosyl transferase (HPRT)-deficient CHO cell line, and has been previously shown to contain a single, functional copy of the E. coli xanthine-guanine phosphoribosyl transferase (XPRT) gene (gpt) stably integrated into the Chinese hamster genome. In this study, conditions for its use in the study of mammalian cell mutagenesis have been stringently defined. The spontaneous mutation rate (2 X 10(-6)/cell division) and phenotypic expression time (7 days) of the gpt locus compare favorably with those of the hprt locus in wild-type CHO-K1-BH4 cells. While both cell lines exhibit similar cytotoxic responses to ethyl methanesulfonate (EMSO and ICR 191, significant differences in mutation induction were observed. Ratios of XPRT to HPRT mutants induced per unit dose of EMS and ICR 191 are 0.70 and 1.6, respectively. Southern blot hybridization analyses revealed that most XPRT mutant cell lines which arose following treatment with EMS (20/22) or ICR 191 (20/24) exhibited no alterations of the gpt locus detectable by this technique. Similar observations were made for the hprt locus in EMS-(21/21) and ICR 191-induced (22/22) HPRT mutants. In contrast, most spontaneous gpt mutants (14/23) contained deletions, while most spontaneous hprt mutants (18/23) exhibited no detectable alterations. Results of this study indicate that the AS52 cell line promises to be useful for future study of mutation in mammalian cells at the DNA sequence level.     

Structural origins of fibrin clot rheology

The origins of clot rheological behavior associated with network morphology and factor XIIIa-induced cross-linking were studied in fibrin clots. Network morphology was manipulated by varying the concentrations of fibrinogen, thrombin, and calcium ion, and cross-linking was controlled by a synthetic, active-center inhibitor of FXIIIa. Quantitative measurements of network features (fiber lengths, fiber diameters, and fiber and branching densities) were made by analyzing computerized three-dimensional models constructed from stereo pairs of scanning electron micrographs. Large fiber diameters and lengths were established only when branching was minimal, and increases in fiber length were generally associated with increases in fiber diameter. Junctions at which three fibers joined were the dominant branchpoint type. Viscoelastic properties of the clots were measured with a rheometer and were correlated with structural features of the networks. At constant fibrinogen but varying thrombin and calcium concentrations, maximal rigidities were established in samples (both cross-linked and noncross-linked) which displayed a balance between large fiber sizes and great branching. Clot rigidity was also enhanced by increasing fiber and branchpoint densities at greater fibrinogen concentrations. Network morphology is only minimally altered by the FXIIIa-catalyzed cross-linking reaction, which seems to augment clot rigidity most likely by the stiffening of existing fibers.     

Sparse balance: Excitatory-inhibitory networks with small bias currents and broadly distributed synaptic weights

Cortical circuits generate excitatory currents that must be cancelled by strong inhibition to assure stability. The resulting excitatory-inhibitory (E-I) balance can generate spontaneous irregular activity but, in standard balanced E-I models, this requires that an extremely strong feedforward bias current be included along with the recurrent excitation and inhibition. The absence of experimental evidence for such large bias currents inspired us to examine an alternative regime that exhibits asynchronous activity without requiring unrealistically large feedforward input. In these networks, irregular spontaneous activity is supported by a continually changing sparse set of neurons. To support this activity, synaptic strengths must be drawn from high-variance distributions. Unlike standard balanced networks, these sparse balance networks exhibit robust nonlinear responses to uniform inputs and non-Gaussian input statistics. Interestingly, the speed, not the size, of synaptic fluctuations dictates the degree of sparsity in the model. In addition to simulations, we provide a mean-field analysis to illustrate the properties of these networks.     

Purification of galectin-3 from ovine placenta: developmentally regulated expression and immunological relevance

Galectins, beta-galactoside-binding lectins, are extensively distributed in the animal kingdom and share some basic molecular properties. Galectin-3, a member of this family, is generally associated with differentiation, morphogenesis, and metastasis. In this study, galectin-3 was isolated from ovine placental cotyledons round the middle of the gestation period by lactose extraction followed by affinity chromatography on lactosyl-agarose, and separated from galectin-1 by size exclusion chromatography on a Superose 12 column. Under native conditions this lectin behaved as a monomer with an apparent molecular weight of approximately 29,000 and an isoelectric point of 9.0. The partial amino acid sequence of the peptides obtained by tryptic digestion of this protein followed by HPLC separation showed striking homology with other members of the galectin-3 subfamily. Furthermore, ovine placental galectin-3 exhibited specific mitogenic activity toward rat spleen mononuclear cells. Besides, this protein strongly reacted with a rabbit antiserum raised against a chicken galectin. Results obtained by Western blot analysis showed that its expression was greatly decreased in term placenta with respect to the middle of the gestation period, suggesting a regulated expression throughout development.      

Analyses of mutation in pSV2gpt-transformed CHO cells

We have developed a system to study mutations which affect expression of the E. coli xanthine-guanine phosphoribosyl transferase (XPRT) gene (gpt) in hypoxanthine-guanine phosphoribosyl transferase-deficient (HPRT-) Chinese hamster ovary (CHO) cells that have been transformed by the plasmid pSV2gpt. Several gpt-transformed cell lines have been isolated and characterized with respect to integrated pSV2gpt sequences, expression of the gpt gene, and cytotoxic and mutagenic responses to UV light. While the gpt-transformed CHO and wild-type CHO-K1-BH4 cell lines have similar cytotoxic responses to UV light, the gpt-transformed cell lines respond differently from the parental CHO-K1-BH4 cell line in terms of mutation induction. As with CHO-K1-BH4 HPRT mutants, spontaneous or induced XPRT mutants derived from the gpt+ cell lines can be selected for 6-thioguanine resistance (TGr). Analysis of cell-free extracts from a number of these TGr clones indicates that the mutant phenotype is due to the absence of XPRT activity. One transformant, designated AS52, has previously been described in limited detail. Here we describe additional characteristics of this cell line, as well as several related transformants.     

Voltage-dependent pore activity of the peptide alamethicin correlated with incorporation in the membrane: salt and cholesterol effects

Strong aggregation of incorporated alamethicin in the bilayer of lipid vesicles has been observed spectroscopically at aqueous peptide concentrations above a critical value c*. On the other hand, in conventional gating studies with planar lipid films, the onset of conducting pore formation can be characterized by a threshold voltage V.. We present experimental evidence of a direct correspondence between the effects on c* and V. when these parameters are modulated by adding NaCl (to the aqueous medium) or cholesterol (to the lipid moiety). A quantitative analysis supports the idea that the measured aggregation actually results in pore formation, the voltage-dependence being due to an electric field effect on the partition equilibrium of the peptide between the aqueous and the lipid phases.     

Quantitative and molecular analyses of radiation-induced mutation in AS52 cells

pSV2gpt-Transformed and wild-type Chinese hamster ovary (CHO) cell lines have been used to study radiation-induced mutation at the molecular level. The transformant, designated AS52, was constructed from a hypoxanthine-guanine phosphoribosyl transferase (HPRT)-deficient CHO cell line and contains a single, functional copy of the Escherichia coli xanthine-guanine phosphoribosyl transferase (XPRT) gene (gpt) stably integrated into the Chinese hamster genome. AS52 and wild-type CHO-K1-BH4 cells exhibit similar cytotoxic responses to uv light and X rays; however, significant differences occur in mutation induction at the gpt and hprt loci. A number of HPRT and XPRT mutants which arose following irradiation were analyzed by Southern-blot hybridization. Most XPRT (21/26) and all HPRT (23/23) mutants induced by uv light exhibited hybridization patterns indistinguishable from their parental cell lines. In contrast, all XPRT (26/26) and most HPRT mutants (15/21) induced by X irradiation contained deletion mutations affecting some or all of the gpt and hprt loci, respectively. These results indicate that X rays induce predominantly deletion mutations, while uv light is likely to induce point mutations at both loci.     

(Patho‐)physiological relevance of PINK1‐dependent ubiquitin phosphorylation

Mutations in PINK1 and PARKIN cause recessive, early‐onset Parkinson's disease (PD). Together, these two proteins orchestrate a protective mitophagic response that ensures the safe disposal of damaged mitochondria. The kinase PINK1 phosphorylates ubiquitin (Ub) at the conserved residue S65, in addition to modifying the E3 ubiquitin ligase Parkin. The structural and functional consequences of Ub phosphorylation (pS65‐Ub) have already been suggested from in vitro experiments, but its (patho‐)physiological significance remains unknown. We have generated novel antibodies and assessed pS65‐Ub signals in vitro and in cells, including primary neurons, under endogenous conditions. pS65‐Ub is dependent on PINK1 kinase activity as confirmed in patient fibroblasts and postmortem brain samples harboring pathogenic mutations. We show that pS65‐Ub is reversible and barely detectable under basal conditions, but rapidly induced upon mitochondrial stress in cells and amplified in the presence of functional Parkin. pS65‐Ub accumulates in human brain during aging and disease in the form of cytoplasmic granules that partially overlap with mitochondrial, lysosomal, and total Ub markers. Additional studies are now warranted to further elucidate pS65‐Ub functions and fully explore its potential for biomarker or therapeutic development.     

Speciation in marine environments: Diving under the surface

Marine environments are inhabited by a broad representation of the tree of life, yet our understanding of speciation in marine ecosystems is extremely limited compared with terrestrial and freshwater environments. Developing a more comprehensive picture of speciation in marine environments requires that we 'dive under the surface' by studying a wider range of taxa and ecosystems is necessary for a more comprehensive picture of speciation. Although studying marine evolutionary processes is often challenging, recent technological advances in different fields, from maritime engineering to genomics, are making it increasingly possible to study speciation of marine life forms across diverse ecosystems and taxa. Motivated by recent research in the field, including the 14 contributions in this issue, we highlight and discuss six axes of research that we think will deepen our understanding of speciation in the marine realm: (a) study a broader range of marine environments and organisms; (b) identify the reproductive barriers driving speciation between marine taxa; (c) understand the role of different genomic architectures underlying reproductive isolation; (d) infer the evolutionary history of divergence using model‐based approaches; (e) study patterns of hybridization and introgression between marine taxa; and (f) implement highly interdisciplinary, collaborative research programmes. In outlining these goals, we hope to inspire researchers to continue filling this critical knowledge gap surrounding the origins of marine biodiversity.