Personal model‐assisted identification of NAD+ and glutathione metabolism as intervention target in NAFLD

To elucidate the molecular mechanisms underlying non‐alcoholic fatty liver disease (NAFLD), we recruited 86 subjects with varying degrees of hepatic steatosis (HS). We obtained experimental data on lipoprotein fluxes and used these individual measurements as personalized constraints of a hepatocyte genome‐scale metabolic model to investigate metabolic differences in liver, taking into account its interactions with other tissues. Our systems level analysis predicted an altered demand for NAD⁺ and glutathione (GSH) in subjects with high HS. Our analysis and metabolomic measurements showed that plasma levels of glycine, serine, and associated metabolites are negatively correlated with HS, suggesting that these GSH metabolism precursors might be limiting. Quantification of the hepatic expression levels of the associated enzymes further pointed to altered de novo GSH synthesis. To assess the effect of GSH and NAD⁺ repletion on the development of NAFLD, we added precursors for GSH and NAD⁺ biosynthesis to the Western diet and demonstrated that supplementation prevents HS in mice. In a proof‐of‐concept human study, we found improved liver function and decreased HS after supplementation with serine (a precursor to glycine) and hereby propose a strategy for NAFLD treatment.     

Environmental heterogeneity does not affect levels of phenotypic plasticity in natural populations of three Drosophila species

Adaptation of natural populations to variable environmental conditions may occur by changes in trait means and/or in the levels of plasticity. Theory predicts that environmental heterogeneity favors plasticity of adaptive traits. Here we investigated the performance in several traits of three sympatric Drosophila species freshly collected in two environments that differ in the heterogeneity of environmental conditions. Differences in trait means within species were found in several traits, indicating that populations differed in their evolutionary response to the environmental conditions of their origin. Different species showed distinct adaptation with a very different role of plasticity across species for coping with environmental changes. However, geographically distinct populations of the same species generally displayed the same levels of plasticity as induced by fluctuating thermal regimes. This indicates a weak and trait‐specific effect of environmental heterogeneity on plasticity. Furthermore, similar levels of plasticity were found in a laboratory‐adapted population of Drosophila melanogaster with a common geographic origin but adapted to the laboratory conditions for more than 100 generations. Thus, this study does not confirm theoretical predictions on the degree of adaptive plasticity among populations in relation to environmental heterogeneity but shows a very distinct role of species‐specific plasticity.     

In situ T cells in melanoma

During the past decade new insights have been gained into the role of T lymphocytes in the host's immune response to cancer in general and to melanoma in particular. Several melanoma-associated antigens (MAA) recognized by T cells have been characterized, and a number of HLA class I- and class II-restricted peptides have been identified. These antigens can be divided into three different groups: tumor-associated testis-specific antigens, melanocyte differentiation antigens, and mutated or aberrantly expressed antigens. These proteins give rise to several antigenic peptides. The number of known melanoma-associated peptides that can induce killing by cytotoxic T-lymphocytes (CTL) exceeds 30 and is still increasing. In line with these findings, clinical data indicate that the immune system is essential in the control of tumor growth. A brisk infiltration of lymphocytes is associated with a favorable prognosis, and complete or partial regression of primary melanoma occurs quite frequently. Furthermore, immunomodulatory therapies have accomplished complete or partial tumor regression in a number of patients. However, the immune response is in most cases inadequate to control tumor growth as tumor progression often occurs. Hence, the coexistence of a cellular immune response in melanoma lesions, demonstrated by the presence of clonally expanded T cells, remains a major paradox of tumor immunology. In the present paper we review current knowledge regarding tumor infiltrating lymphocytes (TIL) in melanoma and discuss possible mechanisms of escape from immune surveillance. Received: 20 March 1999 / Accepted: 3 March 1999     

Root vole movement patterns: do ditches function as habitat corridors?

1. Ditches are often connected to root vole habitat patches (i.e. moist reed patches) in the Netherlands. Due to the linear structure of ditches and because ditch habitat is qualitatively similar to root vole habitat patches, we hypothesized that ditches could function as habitat corridors facilitating dispersal movement of root voles. In order to test this hypothesis, we radiotracked root voles released in a landscape novel to them, consisting of ditches and agricultural meadows.
2. Agricultural meadows often surround the marsh patches inhabited by root voles. As the meadows are mowed regularly, we included the length of the meadow vegetation as an experimental factor in the study.
3. Assuming that ditches function as habitat corridors, we expected root voles in the ditches to move faster and more unidirectionally than root voles in the meadows, and to prefer the ditches to meadows.
4. We found that the ditches did not facilitate faster movements than the meadows. Although the root voles moved back and forth within the ditches, they showed a more directional movement pattern than the root voles in the meadows. Furthermore, the root voles preferred the ditch habitat irrespective of the vegetative cover in the meadow.
5. We conclude that ditches could function as habitat corridors for root voles, as they preferred to move in ditches when in unfamiliar areas.     

Pre- and postsynaptically expressed spike-timing-dependent plasticity contribute differentially to neuronal learning

A plethora of experimental studies have shown that long-term synaptic plasticity can be expressed pre- or postsynaptically depending on a range of factors such as developmental stage, synapse type, and activity patterns. The functional consequences of this diversity are not clear, although it is understood that whereas postsynaptic expression of plasticity predominantly affects synaptic response amplitude, presynaptic expression alters both synaptic response amplitude and short-term dynamics. In most models of neuronal learning, long-term synaptic plasticity is implemented as changes in connective weights. The consideration of long-term plasticity as a fixed change in amplitude corresponds more closely to post- than to presynaptic expression, which means theoretical outcomes based on this choice of implementation may have a postsynaptic bias. To explore the functional implications of the diversity of expression of long-term synaptic plasticity, we adapted a model of long-term plasticity, more specifically spike-timing-dependent plasticity (STDP), such that it was expressed either independently pre- or postsynaptically, or in a mixture of both ways. We compared pair-based standard STDP models and a biologically tuned triplet STDP model, and investigated the outcomes in a minimal setting, using two different learning schemes: in the first, inputs were triggered at different latencies, and in the second a subset of inputs were temporally correlated. We found that presynaptic changes adjusted the speed of learning, while postsynaptic expression was more efficient at regulating spike timing and frequency. When combining both expression loci, postsynaptic changes amplified the response range, while presynaptic plasticity allowed control over postsynaptic firing rates, potentially providing a form of activity homeostasis. Our findings highlight how the seemingly innocuous choice of implementing synaptic plasticity by single weight modification may unwittingly introduce a postsynaptic bias in modelling outcomes. We conclude that pre- and postsynaptically expressed plasticity are not interchangeable, but enable complimentary functions.     

Three-Dimensional Structure of Aleutian Mink Disease Parvovirus: Implications for Disease Pathogenicity

The three-dimensional structure of expressed VP2 capsids of Aleutian mink disease parvovirus strain G (ADVG-VP2) has been determined to 22 A resolution by cryo-electron microscopy and image reconstruction techniques. A structure-based sequence alignment of the VP2 capsid protein of canine parvovirus (CPV) provided a means to construct an atomic model of the ADVG-VP2 capsid. The ADVG-VP2 reconstruction reveals a capsid structure with a mean external radius of 128 A and several surface features similar to those found in human parvovirus B19 (B19), CPV, feline panleukopenia virus (FPV), and minute virus of mice (MVM). Dimple-like depressions occur at the icosahedral twofold axes, canyon-like regions encircle the fivefold axes, and spike-like protrusions decorate the threefold axes. These spikes are not present in B19, and they are more prominent in ADV compared to the other parvoviruses owing to the presence of loop insertions which create mounds near the threefold axes. Cylindrical channels along the fivefold axes of CPV, FPV, and MVM, which are surrounded by five symmetry-related beta-ribbons, are closed in ADVG-VP2 and B19. Immunoreactive peptides made from segments of the ADVG-VP2 capsid protein map to residues in the mound structures. In vitro tissue tropism and in vivo pathogenic properties of ADV map to residues at the threefold axes and to the wall of the dimples.     

Surface mediated death of unconditioned Tetrahymena cells: Effect of physical parameters,growth factors,hormones, and surfactants

A new form of cell death has been observed. The death occurs at liquid-air interfaces when Tetrahymena cells are grown in a chemically defined medium (CDM) at low inocula. The cells die by lysis at the liquid-air interface (medium surface), which they reach due to negative gravitaxis as well as positive aerotaxis. When the cells are grown in a closed compartment, with no liquid-air interface, the death is not observed, and the cells proliferate. Cloning of cells in CDM is thus possible. The addition of effectors such as NGF (10⁻¹¹ M), EGF (10⁻¹⁰ M), PDGF (10⁻¹⁰ M), and insulin (10⁻¹⁰ M) to cells in CDM prevents the surface mediated death. Since detergents/surfactants like SDS (7 × 10⁻⁵ M), NP-40 (2 × 10⁻⁵ M), Tween 80 (10⁻⁴% w/v), Pluronic F-68 (10⁻⁷ M), and the biosurfactant surfactin (10⁻⁶ M) have the same effect, we suggest that the effectors act by stimulating the cells to exudate surfactant(s) of their own. Furthermore, lyzed cells and exudates from living cells (pre-conditioned medium) prevent the death. In conditions with liquid-air interfaces, certain physical parameters are of great importance for the survival of cells at low inocula. The parameters are the distance to the surface, the temperature, and the inoculum. By increasing the height of the medium, lowering the temperature, and increasing the inoculum of the culture, the survival can be greatly enhanced. There is no evidence for programmed cell death (PCD) or apoptosis. © 1996 Wiley-Liss, Inc.     

Phosphoproteome analysis of E. coli reveals evolutionary conservation of bacterial Ser/Thr/Tyr phosphorylation

Protein phosphorylation on serine, threonine, and tyrosine (Ser/Thr/Tyr) is generally considered the major regulatory posttranslational modification in eukaryotic cells. Increasing evidence at the genome and proteome level shows that this modification is also present and functional in prokaryotes. We have recently reported the first in-depth phosphorylation site-resolved dataset from the model Gram-positive bacterium, Bacillus subtilis, showing that Ser/Thr/Tyr phosphorylation is also present on many essential bacterial proteins. To test whether this modification is common in Eubacteria, here we use a recently developed proteomics approach based on phosphopeptide enrichment and high accuracy MS to analyze the phosphoproteome of the model Gram-negative bacterium Escherichia coli. We report 81 phosphorylation sites on 79 E. coli proteins, with distribution of Ser/Thr/Tyr phosphorylation sites 68%/23%/9%. Despite their phylogenetic distance, phosphoproteomes of E. coli and B. subtilis show striking similarity in size, classes of phosphorylated proteins, and distribution of Ser/Thr/Tyr phosphorylation sites. By combining the two datasets, we created the largest phosphorylation site-resolved database of bacterial phosphoproteins to date (available at www.phosida.com) and used it to study evolutionary conservation of bacterial phosphoproteins and phosphorylation sites across the phylogenetic tree. We demonstrate that bacterial phosphoproteins and phosphorylated residues are significantly more conserved than their nonphosphorylated counterparts, with a number of potential phosphorylation sites conserved from Archaea to humans. Our results establish Ser/Thr/Tyr phosphorylation as a common posttranslational modification in Eubacteria, present since the onset of cellular life.