A first-generation EST RH comparative map of the porcine and human genome

We have constructed a first-generation EST radiation hybrid comparative map of the porcine genome by assigning 1058 markers to the IMpRH7000 panel. Chromosomal localization was determined with a 2pt LOD of 4.8 for 984 markers, using the IMpRH mapping tool. Annotated ESTs represent 46.2% or 489 of the markers. Marker distribution was not stochastic and ranged from 0.41 for SSC8 to 1.77 for SSC12, respectively. Two hundred fifty-one markers assigned to the physical map of the pig did not find a homologous sequence in V22 of the human genome assembly, indicative of gaps in the assembled human genome sequence. The comparative porcine/human map covers 3290 MB, or 98.3% of the presumed size of the human genome. However, 60 breakpoints were identified between chromosomes, as well as 90 micro-rearrangements within synteny groups. Six porcine chromosomes—SSC2, 5, 6, 7, 12, and 14—correspond to the three gene-richest human chromosomes, HSA17, 19, and 22, and show above average marker density. Porcine Chrs 1, 8, 11, and X display a low DNA/marker ratio and correspond to the 'genome deserts' on HSA 18, 4, 13, and X.     

Review on intermediate filaments of the nervous system and their pathological alterations

Intermediate filaments (IFs) of the nervous system, including neurofilaments, α-internexin, glial fibrillary acidic protein, synemin, nestin, peripherin and vimentin, are finely expressed following elaborated cell, tissue and developmental specific patterns. A common characteristic of several neurodegenerative diseases is the abnormal accumulation of neuronal IFs in cell bodies or along the axon, often associated with impairment of the axonal transport and degeneration of neurons. In this review, we also present several perturbations of IF metabolism and organization associated with neurodegenerative disorders. Such modifications could represent strong markers of neuronal damages. Moreover, recent data suggest that IFs represent potential biomarkers to determine the disease progression or the differential stages of a neuronal disorder. Finally, recent investigations on IF expression and function in cancer provide evidence that they may be useful as markers, or targets of brain tumours, especially high-grade glioma. A better knowledge of the molecular mechanisms of IF alterations, combined to neuroimaging, is essential to improve diagnosis and therapeutic strategies of such neurodegenerative diseases and glioma.     

The underdog invader: Breeding system and colony genetic structure of the dark rover ant (Brachymyrmex patagonicus Mayr)

Ants are among the most successful species at invading new environments. Their success undeniably comes from their various modes of reproduction and colony breeding structures, which influence their dispersal ability, reproductive potential, and foraging strategies. Almost all invasive ant species studied so far form supercolonies, a dense network of interconnected nests comprising numerous queens, without aggression toward non‐nestmates. This strategy results in invasive colonies that are able to grow extremely fast and large while avoiding intraspecific competition, allowing them to monopolize environmental resources and outcompete native species. Here, we developed and used 10 microsatellite markers to investigate the population structure and breeding system of the dark rover ant Brachymyrmex patagonicus Mayr in its introduced range. We determined whether this species exhibits a supercolonial structure by assessing whether different nests belonged to the same genetic colony. We inferred its dispersal ability by investigating isolation by distance and estimated the numbers of queens per colonies and mating per queen through parent‐offspring inferences. We found that most of the colonies of B. patagonicus were comprised of a single nest, headed by a single queen. Each nest was distinct from one another, without isolation by distance, which suggests strong dispersal ability through nuptial flights. These features are commonly observed in noninvasive and native ant species, but they are surprising for a successful invasive ant, as they strongly differ from other invasive ants. Overall, we discuss how this seemingly unfavorable strategy for an invasive ant might favor the invasive success of the dark rover ant in the United States.     

Clinical outcomes and kinetics of propanil following acute self-poisoning: a prospective case series

Background  

Propanil is an important cause of death from acute pesticide poisoning, of which methaemoglobinaemia is an important manifestation. However, there is limited information about the clinical toxicity and kinetics. The objective of this study is to describe the clinical outcomes and kinetics of propanil following acute intentional self-poisoning.     

Reactions of para-substituted nitrosobenzenes with human hemoglobin

Bio-monitoring the covalent binding of nitrosoarenes to the SH groups of human hemoglobin has been proposed as a reliable approach to get an integral parameter for exposure control and possibly risk assessment of persons exposed to aromatic amines and nitro compounds. Availability of nitrosoarenes to bind to the cysteine residues is greatly influenced by the competition of hemoglobin iron with nitrosoarenes. In contrast to earlier reports, we found that nitrosobenzene has a 14 fold higher affinity for "stripped" human hemoglobin than oxygen. The binding mode is similar to gaseous ligands and exhibits the same free energy of cooperation and sensitivity to heterotropic effectors like inositol hexaphosphate. To elucidate the electronic influence of para substituents, 4-chloronitrosobenzene, 4-nitrosotoluene and 4-nitrosophenetole were tested. A linear free energy relationship was found for all equilibrium parameters with a reaction constant rho = 3, when using Hammett sigma p constants. Similarly, the apparent second order rate constants for binding of para-substituted nitrosobenzenes to the cysteine residues (Cys beta 93) in hemoglobin followed the Hammett relationship with lg k-lg k0 = 1.7 X sigma p (r2 = 0.99). In case of 4-chloronitrosobenzene covalent binding proceeded biphasically and a "semimercaptal"-like intermediate was observed. The affinities for hemoglobin iron and for the SH groups were highest with 4-chloronitrosobenzene and lowest with 4-nitrosophenetole. All nitrosobenzenes were capable to produce ferrihemoglobin. In the absence of oxygen, 4-chloronitrosobenzene hemoglobin decayed with formation of ferrihemoglobin. Presumably the nitroxide radical anion is formed as an intermediate which comproportionates into the azoxy derivative. It is assumed that the efficiency of the microscopic compartmentation of nitrosoarenes by binding to hemoglobin iron has important impacts on the toxicokinetics of these compounds.     

N-WASp is required for Schwann cell cytoskeletal dynamics, normal myelin gene expression and peripheral nerve myelination

Schwann cells elaborate myelin sheaths around axons by spirally wrapping and compacting their plasma membranes. Although actin remodeling plays a crucial role in this process, the effectors that modulate the Schwann cell cytoskeleton are poorly defined. Here, we show that the actin cytoskeletal regulator, neural Wiskott-Aldrich syndrome protein (N-WASp), is upregulated in myelinating Schwann cells coincident with myelin elaboration. When N-WASp is conditionally deleted in Schwann cells at the onset of myelination, the cells continue to ensheath axons but fail to extend processes circumferentially to elaborate myelin. Myelin-related gene expression is also severely reduced in the N-WASp-deficient cells and in vitro process and lamellipodia formation are disrupted. Although affected mice demonstrate obvious motor deficits these do not appear to progress, the mutant animals achieving normal body weights and living to advanced age. Our observations demonstrate that N-WASp plays an essential role in Schwann cell maturation and myelin formation.     

Influence of the phosphorylation state of neurofilament proteins on the interactions between purified filaments in vitro.

Dephosphorylation of 1D-myo-inositol 1,4-bisphosphate [Ins(1,4)P2] in rat liver is catalysed by a cytosolic phosphatase that removes the 1-phosphate group. The Km for Ins(1,4)P2 is approx. 17 microM. Li+ (100 mM) causes 50% inhibition of Ins(1,4)P2 phosphatase activity when activity is measured at the very low substrate concentration of 10 nM, but on raising the substrate concentration to 100 microM there is a greater than 10-fold increase in sensitivity to Li+, suggesting that Li+ acts mainly, but not entirely, as an uncompetitive inhibitor of Ins(1,4)P2 phosphatase. In addition, rat liver cytosol shows Li+-sensitive phosphatase activity against 1D-myo-inositol 1-,3- and 4-monophosphates. The Ins(1,4)P2 1-phosphatase and inositol monophosphatase activities all share an apparent Mr of 47 x 10(3), as determined by gel-filtration chromatography. However, the Ins(1,4)P2 1-phosphatase is more sensitive to inactivation by heat, and can be separated from inositol monophosphatase activity by anion-exchange chromatography. We conclude that rat liver cytosol contains an Ins(1,4)P2 1-phosphatase that is distinct from, but in many ways similar to, inositol monophosphatase.     

Rapid estimation of the energy charge from cell lysates using matrix-assisted laser desorption/ionization mass spectrometry: Role of in-source fragmentation

Nucleotides are key players in the central energy metabolism of cells. Here we show how to estimate the energy charge from cell lysates by direct negative ion matrix-assisted laser desorption/ionization mass spectrometry (MALDI–MS) using 9-aminoacridine as matrix. We found a high level of in-source decay of all the phosphorylated nucleotides, with some of them producing considerable amounts of adenosine-5′-diphosphate (ADP) fragment ions. We investigated the behavior of adenosine-5′-monophosphate (AMP), ADP, and adenosine-5′-triphosphate (ATP) as well as the cofactors coenzyme A (CoA) and acetyl-coenzyme A (ACoA) and nicotinamide adenine dinucleotides (NAD⁺ and NADH) in detail. In-source decay of these compounds depends strongly on the applied laser power and on the extraction pulse delay. At standard instrument settings, the 9-aminoacridine (9-AA) matrix resulted in a much higher in-source decay compared with 2,4,6-trihydroxyacetophenone (2,4,6-THAP). By adding ¹³C-labeled ATP to a cell lysate, we were able to determine the degree of in-source decay during an experiment. Analyzing a cell extract of the monocytic cell line THP-1 with [¹³C]ATP as internal standard, we were able to obtain values for the energy charge that were similar to those determined by a reference liquid chromatography electrospray ionization coupled to mass spectrometry (LC–ESI–MS) method.     

Functional Interactions between Cytochromes P450 1A2 and 2B4 Require Both Enzymes to Reside in the Same Phospholipid Vesicle: EVIDENCE FOR PHYSICAL COMPLEX FORMATION*

Previous studies have shown that the combined presence of two cytochrome P450 enzymes (P450s) can affect the function of both enzymes, results that are consistent with the formation of heteromeric P450·P450 complexes. The goal of this study was to provide direct evidence for a physical interaction between P450 1A2 (CYP1A2) and P450 2B4 (CYP2B4), by determining if the interactions required both enzymes to reside in the same lipid vesicles. When NADPH-cytochrome P450 reductase (CPR) and a single P450 were incorporated into separate vesicles, extremely slow reduction rates were observed, demonstrating that the enzymes were anchored in the vesicles. Next, several reconstituted systems were prepared: 1) CPR·CYP1A2, 2) CPR·CYP2B4, 3) a mixture of CPR·CYP1A2 vesicles with CPR·CYP2B4 vesicles, and 4) CPR·CYP1A2·CYP2B4 in the same vesicles (ternary system). When in the ternary system, CYP2B4-mediated metabolism was significantly inhibited, and CYP1A2 activities were stimulated by the presence of the alternate P450. In contrast, P450s in separate vesicles were unable to interact. These data demonstrate that P450s must be in the same vesicles to alter metabolism. Additional evidence for a physical interaction among CPR, CYP1A2, and CYP2B4 was provided by cross-linking with bis(sulfosuccinimidyl) suberate. The results showed that after cross-linking, antibody to CYP1A2 was able to co-immunoprecipitate CYP2B4 but only when both proteins were in the same phospholipid vesicles. These results clearly demonstrate that the alterations in P450 function require both P450s to be present in the same vesicles and support a mechanism whereby P450s form a physical complex in the membrane.     

A 12,000-rad porcine radiation hybrid (IMNpRH2) panel refines the conserved synteny between SSC12 and HSA17

Reverse or bidirectional Zoo-FISH suggests that synteny between porcine chromosome 12 (SSC12) and human chromosome 17 (HSA17) is completely conserved. The construction of a high-resolution radiation hybrid (RH) map for SSC12 provides a unique opportunity to determine whether chromosomal synteny is reflected at the molecular level by comparative gene mapping of SSC12 and HSA17. We report an initial, high-resolution RH map of SSC12 on the 12,000-rad IMNpRH2 panel using CarthaGene software. This map contains a total of 320 markers, including 20 microsatellites and 300 ESTs/genes, covering approximately 4836.9 cR12,000. The markers were ordered in 16 linkage groups at LOD 6.0 using framework markers previously mapped on the IMpRH7000-rad SSC12 and porcine genetic maps. Ten linkage groups ordered more than 10 markers, with the largest containing 101 STSs. The resolution of the current RH map is approximately 15.3 kb/cR on SSC12, a significant improvement over the second-generation EST SSC12 RH7000-rad map of 103 ESTs and 15 framework markers covering approximately 2287.2 cR7000. Compared to HSA17, six distinct segments were identified, revealing macro-rearrangements within the apparently complete synteny between SSC12 and HSA17. Further analysis of the order of 245 genes (ESTs) on HSA17 and SSC12 also revealed several micro-rearrangements within a synteny segment. A high-resolution SSC12 RH12,000-rad map will be useful in fine-mapping QTL and as a scaffold for sequencing this chromosome.