Patterns of recombination activity on mouse chromosome 11 revealed by high resolution mapping

The success of high resolution genetic mapping of disease predisposition and quantitative trait loci in humans and experimental animals depends on the positions of key crossover events around the gene of interest. In mammals, the majority of recombination occurs at highly delimited 1-2 kb long sites known as recombination hotspots, whose locations and activities are distributed unevenly along the chromosomes and are tightly regulated in a sex specific manner. The factors determining the location of hotspots started to emerge with the finding of PRDM9 as a major hotspot regulator in mammals, however, additional factors modulating hotspot activity and sex specificity are yet to be defined. To address this limitation, we have collected and mapped the locations of 4829 crossover events occurring on mouse chromosome 11 in 5858 meioses of male and female reciprocal F1 hybrids of C57BL/6J and CAST/EiJ mice. This chromosome was chosen for its medium size and high gene density and provided a comparison with our previous analysis of recombination on the longest mouse chromosome 1. Crossovers were mapped to an average resolution of 127 kb, and thirteen hotspots were mapped to <8 kb. Most crossovers occurred in a small number of the most active hotspots. Females had higher recombination rate than males as a consequence of differences in crossover interference and regional variation of sex specific rates along the chromosome. Comparison with chromosome 1 showed that recombination events tend to be positioned in similar fashion along the centromere-telomere axis but independently of the local gene density. It appears that mammalian recombination is regulated on at least three levels, chromosome-wide, regional, and at individual hotspots, and these regulation levels are influenced by sex and genetic background but not by gene content.     

Markov Chain Monte Carlo Algorithm based metabolic flux distribution analysis on Corynebacterium glutamicum

MOTIVATION: Metabolic flux analysis via a (13)C tracer experiment has been achieved using a Monte Carlo method with the assumption of system noise as Gaussian noise. However, an unbiased flux analysis requires the estimation of fluxes and metabolites jointly without the restriction on the assumption of Gaussian noise. The flux distributions under such a framework can be freely obtained with various system noise and uncertainty models. RESULTS: In this paper, a stochastic generative model of the metabolic system is developed. Following this, the Markov Chain Monte Carlo (MCMC) approach is applied to flux distribution analysis. The disturbances and uncertainties in the system are simplified as truncated Gaussian multiplicative models. The performance in a real metabolic system is illustrated by the application to the central metabolism of Corynebacterium glutamicum. The flux distributions are illustrated and analyzed in order to understand the underlying flux activities in the system. AVAILABILITY: Algorithms are available upon request.     

F(ab)'2-mediated neutralization of C3a and C5a anaphylatoxins: a novel effector function of immunoglobulins

High-dose intravenous immunoglobulin (IVIG) prevents immune damage by scavenging complement fragments C3b and C4b. We tested the hypothesis that exogenous immunoglobulin molecules also bind anaphylatoxins C3a and C5a, thereby neutralizing their pro-inflammatory effects. Single-cell calcium measurements in HMC-1 human mast cells showed that a rise in intracellular calcium caused by C3a and C5a was inhibited in a concentration-dependent manner by IVIG, F(ab)2-IVIG and irrelevant human monoclonal antibody. C3a- and C5a-induced thromboxane (TXB2) generation and histamine release from HMC-1 cells and whole-blood basophils were also suppressed by exogenous immunoglobulins. In a mouse model of asthma, immunoglobulin treatment reduced cellular migration to the lung. Lethal C5a-mediated circulatory collapse in pigs was prevented by pretreatment with F(ab)2-IVIG. Molecular modeling, surface plasmon resonance (SPR) and western blot analyses suggested a physical association between anaphylatoxins and the constant region of F(ab)2. This binding could interfere with the role of C3a and C5a in inflammation.     

Exciting chaos with noise: unexpected dynamics in epidemic outbreaks

 In this paper, we identify a mechanism for chaos in the presence of noise. In a study of the SEIR model, which predicts epidemic outbreaks in childhood diseases, we show how chaotic dynamics can be attained by adding stochastic perturbations at parameters where chaos does not exist apriori. Data recordings of epidemics in childhood diseases are still argued as deterministic chaos. There also exists noise due to uncertainties in the contact parameters between those who are susceptible and those who are infected, as well as random fluctuations in the population. Although chaos has been found in deterministic models, it only occurs in parameter regions that require a very large population base or other large seasonal forcing. Our work identifies the mechanism whereby chaos can be induced by noise for realistic parameter regions of the deterministic model where it does not naturally occur. Received: 13 October 2000 / Revised version: 15 May 2001 / Published online: 7 December 2001     

Form and environment of Gryphaea arcuata

Gryphaea arcuata is one of the most studied fossils, but its detailed palaeoecology has been largely neglected. Specimens were collected within a short stratigraphic range (three ammonite zones) in the 'Calcaire à gryphées' of Xeuilley (Lorraine, France) dated Hettangian to Lower Sinemurian. As far as possible, they were sampled from each marly bed of the section. A biometric study and an isotopic analysis are compared in regard to organic matter measurements and palynological data, the results demonstrating a clear relationship between the shape of G. arcuata and environmental parameters. Factors responsible for the various shapes are temperature, oxygen levels on the sea floor and nutrient levels. Two main morphotypes can be related to two kinds of environment. In the first, controlled by a relatively hot and humid climate and tending towards eutrophication, the growth rate of Gryphaea was low, and the shells small, wide and thin. In the second environment, cooler than the first one and closer to the optimal living conditions of G. arcuata, the shell was large, thick and narrow, and exhibited a high growth rate.     

4-Iodotomoxetine: a novel ligand for serotonin uptake sites.

The tomoxetine analog, R-4-iodotomoxetine, binds in vitro to a single site of rat cortical membranes with high affinity (Kd = 0.03 +/- 0.01 nM, n = 4) and can be blocked by a selective serotonin reuptake site inhibitor, paroxetine. The [125I]R-4-iodotomoxetine binding at equilibrium is saturable and is temperature- and Na(+)-dependent. The number of specific [125I]R-4-iodotomoxetine binding sites (Bmax = 356 +/- 20 fmol/mg protein) is similar to that of [3H]citalopram (329 +/- 30 fmol/mg protein), a known serotonin uptake inhibitor. The binding of [125I]R-4-iodotomoxetine is selectively inhibited by several serotonin uptake blockers, and a good correlation is demonstrated between the potency of various drugs to inhibit in vitro binding of [125I]R-4-iodotomoxetine and [3H]citalopram. In addition, lesions performed with the neurotoxin p-chloroamphetamine, which destroys monoamine neurons, including serotonergic neuronal system, result in a 90% reduction of [125I]R-4-iodotomoxetine binding when compared to sham controls. These results indicate that the binding sites labeled by [125I]R-4-iodotomoxetine are associated with the neuronal serotonin uptake sites. However, the in vivo and ex vivo results do not show regional localization corresponding to the distribution of serotonin uptake sites. The nonspecific uptake may be related to this compound's high lipophilicity (octanol-buffer partition coefficient = 1100 - 1400 at pH 7). Although the in vivo properties of [125I]R-4-iodotomoxetine make it an unlikely candidate for mapping serotonin uptake sites with SPECT, the high affinity and selectivity should make it a useful tool for in vitro studies of the serotonin uptake sites.     

Membrane-associated nuclease activities in mycoplasmas.

Membrane-associated nucleases of various mycoplasmal species were investigated by using two nuclease assays. A lambda DNA assay was developed to measure nuclease activity associated with whole-cell suspensions, activity released from intact cells, and activity associated with detergent-disrupted cells. In most species, nuclease activities were entirely membrane associated, and disruption by a detergent had a stimulatory effect on these activities. All mycoplasmal species contained nuclease activity, but Mycoplasma capricolum was unusual because its activity was dependent upon magnesium and was inhibited by calcium. We developed a sodium dodecyl sulfate-polyacrylamide gel electrophoresis system that produced reproducible nuclease patterns, and this system was used to determine the apparent molecular weights of the nuclease proteins. An examination of 20 mycoplasmal species failed to identify common bands in their nuclease patterns. An examination of 11 Mycoplasma pulmonis strains, however, indicated that nuclease patterns on polyacrylamide gels may provide a means for categorizing strains within a species. Our results suggest that nucleases are important constituents of mycoplasmal membranes and may be involved in the acquisition of host nucleic acids required for growth.     

Phospholipase A2 activation in cultured mouse hepatocytes exposed to tumor necrosis factor-α

High concentrations of tumor necrosis factor α (TNFα) are cytotoxic to cultured hepatocytes. Impairment of energy metabolism and generation of an intracellular oxidant stress are important events in the pathogenesis of this toxicity (6). In the present study, we have examined the role of phospholipase A₂ activation in TNFα-in-duced toxicity in mouse hepatocytes, since it has been reported to play a key role in TNFα cytolytic activity in other cell types. Recombinant murine TNFα (0.1 μg/mL) caused a dose-dependent increase in PLA₂ activity in cultured mouse hepatocytes. The increase in PLA₂ activity was observed after only 0.5 hour of exposure (152 ± 10% of control), and continued to increased over the first 4 hours of exposure (292 ± 32%). However, TNFα-induced GSSG efflux and ATP depletion did not occur until after 2 hours of exposure. Furthermore, a small level of cytotoxicity was observed after a 24 hour incubation period. Putative PLA₂ inhibitors, chlorpromazine (CPZ) and 4-bromophenacyl bromide (BPB), both prevented the TNFα-induced increase in PLA₂ activity. They also reduced ATP depletion, GSSG efflux, and cytotoxicity. The PLA₂ inhibitor, manoalide (a natural marine product), completely prevented PLA₂ activation and cytotoxicity induced by TNFα. Pretreatment of hepatocytes with cycloheximide, to inhibit protein synthesis, increased TNFα-induced cytotoxicity. Cycloheximide pretreatment also potentiated PLA₂ activation, ATP depletion, and GSSG efflux. CPZ and BPB both reduced the extent of PLA₂ activation, ATP depletion, GSSG formation, and cytotoxicity in the cycloheximide pretreated cells exposed to TNFα. Taken together, these results demonstrate that TNFα activates PLA₂ which occurs prior to other deleterious events in hepatocytes, and that inhibition of PLA₂ activity reduces cell injury by TNFα. This suggests that PLA₂ activation may lead to impairment of energy metabolism, an oxidant stress, and cytotoxicity in cells exposed to TNFα. Additionally, protein synthesis inhibition potentiates TNFα induction of PLA₂ and toxicity, suggesting that there is a protein-synthesis-dependent protective mechanism in hepatocytes which ameliorates the effects induced by PLA₂. These findings provide strong evidence that PLA₂ activation plays an important role in the pathogenesis of toxicity induced by TNFα in cultured mouse hepatocytes.     

Rates and equilibria at the acetylcholine receptor of electrophorus electroplaques. A study of neurally evoked postsynaptic currents and of voltage-jump relaxations

Kinetic measurements are employed to reconstruct the steady-state activation of acetylcholine [Ach] receptor channels in electrophorus electroplaques. Neurally evoked postsynaptic currents (PSCs) decay exponentially; at 15 degrees C the rate constant, α, equals 1.2 ms(-1) at 0 mV and decreases e-fold for every 86 mV as the membrane voltage is made more negative. Voltage-jump relaxations have been measured with bath-applied ACh, decamethonium, carbachol, or suberylcholine. We interpret the reciprocal relaxation time 1/τ as the sum of the rate constant α for channel closing and a first-order rate constant for channel opening. Where measureable, the opening rate increases linearly with [agonist] and does not vary with voltage. The voltage sensitivity of small steady-state conductances (e- fold for 86 mV) equals that of the closing rate α, confirming that the opening rate has little or no additional voltage sensitivity. Exposure to α-bungarotoxin irreversibly decreases the agonist-induced conductance but does not affect the relaxation kinetics. Tubocurarine reversibly reduces both the conductance and the opening rate. In the simultaneous presence of two agonist species, voltage-jump relaxations have at least two exponential components. The data are fit by a model in which (a) the channel opens as the receptor binds the second in a sequence of two agonist molecules, with a forward rate constant to 10(7) to 2x10(8) M(-1)s(-1); and (b) the channel then closes as either agonist molecule dissociates, with a voltage-dependent rate constant of 10(2) to 3x10(3)s(-1).