Effect of fungal interactions on the numbers of the pinewood nematode, Bursaphelenchus xylophilus (Nematoda : Aphelenchoididae), carried by the japanese pine sawyer, Monochamus alternatus (Coleoptera : Cerambycidae)

#Monochamus alternatus sortant de blocs de bois préalablement inoculés avec #Ophiostoma minus et ensuite avec #Trichoderma sp. (O + T) transporte un plus grand nombre de #Bursaphelenchus xylophilus (PWN) que ceux sortant de blocs inoculés simultanément avec ces deux champignons (O, T) ou inoculés avec #Trichoderma sp. puis avec #O. minus (T + O). La raison est que : les populations de PWN sont plus élevées dans les blocs O + T que dans ceux des autres traitements, et que le pourcentage de juvéniles de dispersion de 3ème stade, les "dauer" juvéniles et les PWN qui passent réellement dans l'insecte sont plus nombreux dans les blocs O + T. En contraste, le nombre de PWN transportés par l'insecte sortant de blocs inoculés avec #O. minus et #Verticillium sp. est beaucoup moins élevé quelle que soit la séquence d'innoculum parce que les populations de PWN ne s'établissent pas. Il est conclu que les espèces de champignon les plus abondantes dans les pins tués par le dessèchement concourent à déterminer le nombre de PWN transportés par l'insecte sortant du bois. (Résumé d'auteur

PCR-RFLP and sequencing analysis of ribosomal DNA of Bursaphelenchus nematodes related to pine wilt disease

La réaction en chaîne des polymérases/polymorphisme des fragments de restriction (PCR-RFLP) a été utilisée pour séparer des isolats du nématode #Bursaphelenchus. Les isolats de #B. xylophilus examinés provenaient du Japon, des USA, de Chine et du Canada, et ceux de #B. mucronatus du Japon, de Chine et de la France. L'ADN ribosomal contenant le gène 5.8S, les segments de transciption interne 1 et 2, et les segments partiels des gènes 18S et 28S ont été amplifiés par PCR. La digestion des produits amplifiés provenant de chaque isolat à l'aide de douze endonucléases de restriction et l'examen des données en RFLP qui en découlent révèlent, par une analyse en grappe, une séparation significative entre #B. xylophilus et #B. mucronatus. Parmi les isolats de #B. xylophilus examinés, les isolats pathogènes du Japon, ceux de Chine et des USA étaient tous identiques, tandis que les isloats non pathogènes du Japon étaient légèrement distincts et que ceux du Canada formaient une grappe séparée. Parmi les isolats de #B. mucronatus, deux isolats provenant du Japon étaient très semblables ; de même un autre isolat du Japon et un isolat de Chine étaient identiques. Les données provenant des séquences d'ADN montrent 98 différences (substitution nucléotidiques ou séparations) dans les 884 paires de bases examinées chez les isolats de #B. xylophilus et #B. mucronatus. Les données provenant des séquences d'ADN chez #Aphelenchus avenae et #Aphelenchoides fragariae diffèrent non seulement de celles des #Bursaphelenchus mais aussi entre elles. Afin de préciser les relations phylogéniques de ces espèces, les données séquentielles du gène 5.8S provenant de l'ADN ribosomal ont été examinées... (D'après résume d'auteur

Characterization of the L-Lactate Dehydrogenase from Aggregatibacter actinomycetemcomitans

Aggregatibacter actinomycetemcomitans is a Gram-negative opportunistic pathogen and the proposed causative agent of localized aggressive periodontitis. A. actinomycetemcomitans is found exclusively in the mammalian oral cavity in the space between the gums and the teeth known as the gingival crevice. Many bacterial species reside in this environment where competition for carbon is high. A. actinomycetemcomitans utilizes a unique carbon resource partitioning system whereby the presence of L-lactate inhibits uptake of glucose, thus allowing preferential catabolism of L-lactate. Although the mechanism for this process is not fully elucidated, we previously demonstrated that high levels of intracellular pyruvate are critical for L-lactate preference. As the first step in L-lactate catabolism is conversion of L-lactate to pyruvate by lactate dehydrogenase, we proposed a model in which the A. actinomycetemcomitans L-lactate dehydrogenase, unlike homologous enzymes, is not feedback inhibited by pyruvate. This lack of feedback inhibition allows intracellular pyruvate to rise to levels sufficient to inhibit glucose uptake in other bacteria. In the present study, the A. actinomycetemcomitans L-lactate dehydrogenase was purified and shown to convert L-lactate, but not D-lactate, to pyruvate with a Km of approximately 150 µM. Inhibition studies reveal that pyruvate is a poor inhibitor of L-lactate dehydrogenase activity, providing mechanistic insight into L-lactate preference in A. actinomycetemcomitans

Phase-Locked Signals Elucidate Circuit Architecture of an Oscillatory Pathway

This paper introduces the concept of phase-locking analysis of oscillatory cellular signaling systems to elucidate biochemical circuit architecture. Phase-locking is a physical phenomenon that refers to a response mode in which system output is synchronized to a periodic stimulus; in some instances, the number of responses can be fewer than the number of inputs, indicative of skipped beats. While the observation of phase-locking alone is largely independent of detailed mechanism, we find that the properties of phase-locking are useful for discriminating circuit architectures because they reflect not only the activation but also the recovery characteristics of biochemical circuits. Here, this principle is demonstrated for analysis of a G-protein coupled receptor system, the M3 muscarinic receptor-calcium signaling pathway, using microfluidic-mediated periodic chemical stimulation of the M3 receptor with carbachol and real-time imaging of resulting calcium transients. Using this approach we uncovered the potential importance of basal IP3 production, a finding that has important implications on calcium response fidelity to periodic stimulation. Based upon our analysis, we also negated the notion that the Gq-PLC interaction is switch-like, which has a strong influence upon how extracellular signals are filtered and interpreted downstream. Phase-locking analysis is a new and useful tool for model revision and mechanism elucidation; the method complements conventional genetic and chemical tools for analysis of cellular signaling circuitry and should be broadly applicable to other oscillatory pathways

Reliability of Synaptic Transmission at the Synapses of Held In Vivo under Acoustic Stimulation

BACKGROUND:The giant synapses of Held play an important role in high-fidelity auditory processing and provide a model system for synaptic transmission at central synapses. Whether transmission of action potentials can fail at these synapses has been investigated in recent studies. At the endbulbs of Held in the anteroventral cochlear nucleus (AVCN) a consistent picture emerged, whereas at the calyx of Held in the medial nucleus of the trapezoid body (MNTB) results on the reliability of transmission remain inconsistent. In vivo this discrepancy could be due to the difficulty in identifying failures of transmission. METHODS/FINDINGS:We introduce a novel method for detecting unreliable transmission in vivo. Based on the temporal relationship between a cells' waveform and other potentials in the recordings, a statistical test is developed that provides a balanced decision between the presence and the absence of failures. Its performance is quantified using simulated voltage recordings and found to exhibit a high level of accuracy. The method was applied to extracellular recordings from the synapses of Held in vivo. At the calyces of Held failures of transmission were found only rarely. By contrast, at the endbulbs of Held in the AVCN failures were found under spontaneous, excited, and suppressed conditions. In accordance with previous studies, failures occurred most abundantly in the suppressed condition, suggesting a role for inhibition. CONCLUSIONS/SIGNIFICANCE:Under the investigated activity conditions/anesthesia, transmission seems to remain largely unimpeded in the MNTB, whereas in the AVCN the occurrence of failures is related to inhibition and could be the basis/result of computational mechanisms for temporal processing. More generally, our approach provides a formal tool for studying the reliability of transmission with high statistical accuracy under typical in vivo recording conditions

NAD-Independent L-Lactate Dehydrogenase Is Required for L-Lactate Utilization in Pseudomonas stutzeri SDM

BACKGROUND: Various Pseudomonas strains can use L-lactate as their sole carbon source for growth. However, the L-lactate-utilizing enzymes in Pseudomonas have never been identified and further studied. METHODOLOGY/PRINCIPAL FINDINGS: An NAD-independent L-lactate dehydrogenase (L-iLDH) was purified from the membrane fraction of Pseudomonas stutzeri SDM. The enzyme catalyzes the oxidation of L-lactate to pyruvate by using FMN as cofactor. After cloning its encoding gene (lldD), L-iLDH was successfully expressed, purified from a recombinant Escherichia coli strain, and characterized. An lldD mutant of P. stutzeri SDM was constructed by gene knockout technology. This mutant was unable to grow on L-lactate, but retained the ability to grow on pyruvate. CONCLUSIONS/SIGNIFICANCE: It is proposed that L-iLDH plays an indispensable function in Pseudomonas L-lactate utilization by catalyzing the conversion of L-lactate into pyruvate

Presenilin/γ-Secretase Regulates Neurexin Processing at Synapses

Neurexins are a large family of neuronal plasma membrane proteins, which function as trans-synaptic receptors during synaptic differentiation. The binding of presynaptic neurexins to postsynaptic partners, such as neuroligins, has been proposed to participate in a signaling pathway that regulates synapse formation/stabilization. The identification of mutations in neurexin genes associated with autism and mental retardation suggests that dysfunction of neurexins may underlie synaptic defects associated with brain disorders. However, the mechanisms that regulate neurexin function at synapses are still unclear. Here, we show that neurexins are proteolytically processed by presenilins (PS), the catalytic components of the γ-secretase complex that mediates the intramembraneous cleavage of several type I membrane proteins. Inhibition of PS/γ-secretase by using pharmacological and genetic approaches induces a drastic accumulation of neurexin C-terminal fragments (CTFs) in cultured rat hippocampal neurons and mouse brain. Neurexin-CTFs accumulate mainly at the presynaptic terminals of PS conditional double knockout (PS cDKO) mice lacking both PS genes in glutamatergic neurons of the forebrain. The fact that loss of PS function enhances neurexin accumulation at glutamatergic terminals mediated by neuroligin-1 suggests that PS regulate the processing of neurexins at glutamatergic synapses. Interestingly, presenilin 1 (PS1) is recruited to glutamatergic terminals mediated by neuroligin-1, thus concentrating PS1 at terminals containing β-neurexins. Furthermore, familial Alzheimer's disease (FAD)-linked PS1 mutations differentially affect β-neurexin-1 processing. Expression of PS1 M146L and PS1 H163R mutants in PS−/− cells rescues the processing of β-neurexin-1, whereas PS1 C410Y and PS1 ΔE9 fail to rescue the processing defect. These results suggest that PS regulate the synaptic function and processing of neurexins at glutamatergic synapses, and that impaired neurexin processing by PS may play a role in FAD

CK2 Phosphorylates Sec31 and Regulates ER-To-Golgi Trafficking

Protein export from the endoplasmic reticulum (ER) is an initial and rate-limiting step of molecular trafficking and secretion. This is mediated by coat protein II (COPII)-coated vesicles, whose formation requires small GTPase Sar1 and 6 Sec proteins including Sec23 and Sec31. Sec31 is a component of the outer layer of COPII coat and has been identified as a phosphoprotein. The initiation and promotion of COPII vesicle formation is regulated by Sar1; however, the mechanism regulating the completion of COPII vesicle formation followed by vesicle release is largely unknown. Hypothesizing that the Sec31 phosphorylation may be such a mechanism, we identified phosphorylation sites in the middle linker region of Sec31. Sec31 phosphorylation appeared to decrease its association with ER membranes and Sec23. Non-phosphorylatable mutant of Sec31 stayed longer at ER exit sites and bound more strongly to Sec23. We also found that CK2 is one of the kinases responsible for Sec31 phosphorylation because CK2 knockdown decreased Sec31 phosphorylation, whereas CK2 overexpression increased Sec31 phosphorylation. Furthermore, CK2 knockdown increased affinity of Sec31 for Sec23 and inhibited ER-to-Golgi trafficking. These results suggest that Sec31 phosphorylation by CK2 controls the duration of COPII vesicle formation, which regulates ER-to-Golgi trafficking

Association of the Chromosome Replication Initiator DnaA with the Escherichia coli Inner Membrane In Vivo: Quantity and Mode of Binding

DnaA initiates chromosome replication in most known bacteria and its activity is controlled so that this event occurs only once every cell division cycle. ATP in the active ATP-DnaA is hydrolyzed after initiation and the resulting ADP is replaced with ATP on the verge of the next initiation. Two putative recycling mechanisms depend on the binding of DnaA either to the membrane or to specific chromosomal sites, promoting nucleotide dissociation. While there is no doubt that DnaA interacts with artificial membranes in vitro, it is still controversial as to whether it binds the cytoplasmic membrane in vivo. In this work we looked for DnaA-membrane interaction in E. coli cells by employing cell fractionation with both native and fluorescent DnaA hybrids. We show that about 10% of cellular DnaA is reproducibly membrane-associated. This small fraction might be physiologically significant and represent the free DnaA available for initiation, rather than the vast majority bound to the datA reservoir. Using the combination of mCherry with a variety of DnaA fragments, we demonstrate that the membrane binding function is delocalized on the surface of the protein’s domain III, rather than confined to a particular sequence. We propose a new binding-bending mechanism to explain the membrane-induced nucleotide release from DnaA. This mechanism would be fundamental to the initiation of replication

Natural Selection and Adaptive Evolution of Leptin in the Ochotona Family Driven by the Cold Environmental Stress

BACKGROUND: Environmental stress can accelerate the evolutionary rate of specific stress-response proteins and create new functions specialized for different environments, enhancing an organism's fitness to stressful environments. Pikas (order Lagomorpha), endemic, non-hibernating mammals in the modern Holarctic Region, live in cold regions at either high altitudes or high latitudes and have a maximum distribution of species diversification confined to the Qinghai-Tibet Plateau. Variations in energy metabolism are remarkable for them living in cold environments. Leptin, an adipocyte-derived hormone, plays important roles in energy homeostasis. METHODOLOGY/PRINCIPAL FINDINGS: To examine the extent of leptin variations within the Ochotona family, we cloned the entire coding sequence of pika leptin from 6 species in two regions (Qinghai-Tibet Plateau and Inner Mongolia steppe in China) and the leptin sequences of plateau pikas (O. curzonia) from different altitudes on Qinghai-Tibet Plateau. We carried out both DNA and amino acid sequence analyses in molecular evolution and compared modeled spatial structures. Our results show that positive selection (PS) acts on pika leptin, while nine PS sites located within the functionally significant segment 85-119 of leptin and one unique motif appeared only in pika lineages-the ATP synthase alpha and beta subunit signature site. To reveal the environmental factors affecting sequence evolution of pika leptin, relative rate test was performed in pikas from different altitudes. Stepwise multiple regression shows that temperature is significantly and negatively correlated with the rates of non-synonymous substitution (Ka) and amino acid substitution (Aa), whereas altitude does not significantly affect synonymous substitution (Ks), Ka and Aa. CONCLUSIONS/SIGNIFICANCE: Our findings support the viewpoint that adaptive evolution may occur in pika leptin, which may play important roles in pikas' ecological adaptation to extreme environmental stress. We speculate that cold, and probably not hypoxia, may be the primary environmental factor for driving adaptive evolution of pika leptin