Effect of signal peptide changes on the extracellular processing of streptokinase from Escherichia coli : requirement for secondary structure at the cleavage junction

Streptokinase (SK), an extracellular protein from Streptococcus equisimilis, is secreted post-translationally by Escherichia coli using both its native and E. coli-derived transport signals. In this communication we report that cleavage specificity of signal peptidase I, and thus efficiency of secretion, varies in E. coli when SK export is directed by different transport signals. The native (+1) N-terminus of mature SK was retained when it was transported under the control of its own, PelB or LamB signal peptide. However, when translocation of SK was controlled by the OmpA or MalE signal peptide, Ala2 of mature SK was preferred as a cleavage site for the pre-SK processing. Our results indicate that compatibility of the leader peptide with the mature sequences of SK, which fulfils the requirement for a given secondary structure within the cleavage region, is essential for maintaining the correct processing of pre-SK. An OmpA-SK fusion, which results in the deletion of two N-terminal amino acid residues of mature SK, was further studied with respect to the recognition of alternative cleavage site in E. coli. The alanine at +2 in mature SK was changed to glycine or its relative position was changed to +3 by introducing a methionine residue at the +1 position. Both alterations resulted in the correct cleavage of pre-SK at the original OmpA fusion site. In contrast, introduction of an additional alanine at +4, creating three probable cleavage sites (Ala-x-Ala-x-Ala-x-Ala), resulted in the recognition of all three target sites for cleavage, with varying efficiency. The results indicate that the nature of the secondary structure generated at the cleavage junction of pre-SK, resulting from the fusion of different signal peptides, modulates the cleavage specificity of signal peptidase I during extracellular processing of SK. Based on these findings it is proposed that flexibility in the interaction of the active site of signal peptidase I with the cleavage sites of signal peptides may occur when it encounters two or more juxtaposed cleavage sites. Preference for one cleavage site over another, then, may depend on fulfillment of secondary structure requirements in the vicinity of the pre-protein cleavage junction. Received: 22 September 1997 / Accepted: 17 December 1997     

Analysis of the robustness of spectral indices during ventricular fibrillation

The spatiotemporal characteristics of cardiac fibrillation are often investigated by using indices extracted from the spectrum of cardiac signals. However different signal acquisition systems may produce signals of different spectra and affect the estimation of some spectral indices. In this study, we investigate the robustness of four spectral indices previously proposed for describing fibrillation, namely the dominant frequency (DF), the peak frequency (PF), the median frequency (MF) and the organization index (OI). The effects of different lead configurations on the values of the spectral indices are statistically quantified and further analyzed in a database consisting of unipolar and bipolar intracardiac electrograms (EGM), recorded by implantable cardioverter-defibrillators during ventricular fibrillation. Our analysis shows that the lead configuration significantly affects the PF, the MF and the OI, whereas the DF remains unaffected. We further explore the nature of cardiac spectrum and show that unipolar EGM concentrate power at lower frequencies than bipolar EGM. We conclude that indices that depend on the envelope of the spectrum of cardiac signals are in general sensitive to the lead configuration.     

第一和第二语言Stroop任务中EEG同步化分析

采用基于多元自回归的瞬时EEG相干方法研究了十位汉英双语者执行Stroop任务时脑神经电活动及其功能皮层区的协同作用。结果显示：在β1(13-18Hz)频段,无论是汉语(第一语言,L1)还是英语(第二语言,L2)呈现的刺激,不一致条件的EEG相干值明显大于一致条件的EEG相干值,表明β1频段对刺激类型敏感;与L2相比,L1的Stroop任务中,额一顶区的相干值显著增强。EEG相干值反映了不同脑皮层间的相互作用强度。因此研究结果表明：判断和处理冲突信息(如Stroop的不一致条件)时脑功能皮层区之间的协同作用增强;相对于第二语言,第一语言处理过程中额一顶区之间的通信协作增加。     

Selection for transport competence of C-terminal polypeptides derived from Escherichia coli hemolysin: the shortest peptide capable of autonomous HIyB/HIyD-dependent secretion comprises the C-terminal 62 amino acids of HlyA

Escherichia coli hemolysin (HlyA) is secreted by a specific export machinery which recognizes a topogenic secretion signal located at the C-terminal end of HlyA. This signal sequence has been variously defined as comprising from 27 to about 300 amino acids at the C-terminus of HlyA. We have used here a combined genetic and immunological approach to select for C-terminal HlyA peptides that are still secretion-component. A deletion library of HlyA mutant proteins was generated in vitro by successive degradation of hy1A from the 5 end with exonuclease III. Secretion competence was tested by immunoblotting of the supernatant of each clone with an antiserum raised against a C-terminal portion of hemolysin. It was found that the hemolysin secretion system has no apparent size limitation for HlyA proteins over a range from 1024 to 62 amino acids. The smallest autonomously secretable peptide isolated in this selection procedure consists of the C-terminal 62 amino acids of HlyA. This sequence is shared by all secretion-competent, truncated HlyA proteins, which suggests that secretion of the E.coli hemolysin is strictly post-translational. The capacity of the hemolysin secretion machinery was found to be unsaturated by the steady-state level of its natural HlyA substrate and large amounts of truncated HlyA derivatives could still be secreted in addition to full-length HlyA.     

Analysis of the carboxypeptidase D cytoplasmic domain: Implications in intracellular trafficking

Metallocarboxypeptidase D (CPD) is a type 1 transmembrane protein that functions in the processing of proteins that transit the secretory pathway. Previously, CPD was found to be enriched in the trans Golgi network (TGN) and to cycle between this compartment and the cell surface. In the present study, the roles of specific regions of the CPD cytosolic tail in intracellular trafficking were investigated in the AtT-20 cell line. When the CPD transmembrane region and cytosolic tail are attached to the C-terminus of albumin, this protein is retained in the TGN and cycles to the cell surface. Deletion analysis indicates that a C-terminal region functions in TGN-retention; removal of 10 amino acids from the C-terminus greatly increases the amount of fusion protein that enters nascent vesicles, which bud from the Golgi, but does not affect the half-life of the fusion protein or the ability of cell surface protein to return to the TGN. Because the 10-residue deletion disrupts a casein kinase 2 (CK2) consensus site, the two Thr in this site (TDT) were mutated to either Ala (ADA) or Glu (EDE). Neither mutation has an increased rate of budding from the TGN, although the ADA mutant has a shorter half-life than either the wild type sequence or the EDE mutant. Adaptor protein-1 and -2 bind to most of the deletion mutants, the EDE point mutant, and the CK2-phosphorylated CPD tail, but not to the wild type tail. Taken together, these results suggest that CPD localization to the TGN requires both static retention involving the C-terminal domain and phosphorylation at a CK2 site, which regulates the binding of adaptor proteins.     

Building time-budgets from bioacoustic signals to measure population-level changes in behavior: a case study with sperm whales in the Gulf of Mexico

Evaluating changes in the collective behavior of a population can be an indirect method for inferring organismal responses to changing environmental conditions. Apex predators, such as the sperm whale (Physeter macrocephalus), can provide valuable insights into the ecosystem processes of the deep sea, where little direct observation can be made. Sperm whales are often difficult to observe at sea, as they inhabit deep, offshore waters and spend most of their lives beneath the surface. However, sperm whales are extremely amenable to passive acoustic monitoring, as their vocalizations are well-studied, highly distinguishable, produced regularly, and can be detected at relatively long ranges (>10 km). Sperm whales produce distinct clicks in two behavioral contexts (social interaction or foraging/prey capture); thus, we can use acoustic detection of these vocalizations to infer patterns of large-scale, collective behavior, which is similar to studying calling frogs or insects indicating their reproductive phenology. We recorded behaviorally-specific sperm whale vocalizations at three sites in the Northern Gulf of Mexico in July 2010 and 2011. We used these recordings to construct population-level time budgets, an empirical collective metric of behavior, based on the ratio of hours in a day with social clicks to the hours in a day with foraging clicks, and represented this as an “acoustic activity index.” Our index showed significant differences in the proportions of social and foraging behavior across the range of sperm whales in the Northern Gulf of Mexico, and the proportion of social activity increased by more than a factor of two from 2010 to 2011. These differences support previous evidence of differential habitat use by sperm whales in the Gulf of Mexico, and suggest possible changes in environmental conditions between years. Thus, the acoustic activity index may provide a powerful way to evaluate changes in behavior and link them to changing ecological conditions. This novel application of bioacoustics to constructing time budgets and creating a behaviorally-based index at the population scale can serve as an indicator of ecological change, and greatly enhance our ability to understand the behavior and ecology of many acoustically active species.     

A new parameter for quantifying the variability of surface electromyographic signals during gait: The occurrence frequency

Natural variability of myoelectric activity during walking was recently analyzed considering hundreds of strides. This allowed assessing a parameter seldom considered in classic surface EMG (sEMG) studies: the occurrence frequency, defined as the frequency each muscle activation occurs with, quantified by the number of strides when a muscle is recruited with that specific activation modality. Aim of present study was to propose the occurrence frequency as a new parameter for assessing sEMG-signal variability during walking. Aim was addressed by processing sEMG signals acquired from Gastrocnemius Lateralis, Tibialis Anterior, Rectus Femoris and Biceps femoris in 40 healthy subjects in order to: (1) show that occurrence frequency is not correlated with ON/OFF instants (R_mean = 0.11 ± 0.07; P > 0.05) and total time of activation (R_mean = 0.15 ± 0.08; P > 0.05); (2) confirm the above results by two handy examples of application (analysis of gender and age) which highlighted that significant (P < 0.05) gender-related and age-related differences within population were detected in occurrence frequency, but not in temporal sEMG parameters. In conclusion, present study demonstrated that occurrence frequency is able to provide further information, besides those supplied by classical temporal sEMG parameters and thus it is suitable to complement them in the evaluation of variability of myoelectric activity during walking.     

Association of BMI with the β3‐Adrenergic Receptor Gene Polymorphism in Japanese: Meta‐Analysis

Objective: To assess the effect of the Trp64Arg polymorphism in the β3‐adrenergic receptor gene (ADRB3) on body mass index (BMI) in the Japanese population. Research Methods and Procedures: We selected studies that evaluated the association between BMI and ADRB3 polymorphism among Japanese, using MEDLINE and PubMed. After data collection, an extension of ANOVA was performed to assess the differences according to the genotype. Results: In a total of 35 subgroups including 2316 subjects with the Trp64Arg variant and 4266 subjects without this variant, the weighted mean difference in BMI was 0.26 kg/m² (95% confidence interval: 0.18 to 0.42; p < 0.01), indicating that variant carriers exhibited higher BMI than did normal homozygous subjects. Discussion: Although it is known that the allele frequency of the ADRB3 polymorphism differs among races, this study focuses on the Japanese population, which has a high allele frequency of ADRB3 polymorphism. We assumed that statistical errors would be prevented due to the sufficient number of subjects. In conclusion, the results support the hypothesis that ADRB3 gene polymorphism is associated with BMI.     

Spectral EEG indicator of pressure to enter into deep sleep: its responsiveness to closing the eyes for just a few minutes exhibits a pure exponential buildup during sleep deprivation

According to the two-process model of sleep–wake regulation, a homeostatic sleep pressure, i.e. a pressure to enter into deep non-rapid eyes movement (NREM) sleep, must exhibit a purely exponential buildup during prolonged wakefulness. However, this pressure is usually measured indirectly, i.e. during the following episode of actual deep NREM sleep. The purpose of this paper was to show that, despite a prominent circadian modulation of time course of any waking EEG index, the model-postulated purely exponential buildup of the homeostatic sleep pressure can be directly confirmed. During two days of sleep deprivation experiments, the EEG of healthy adults (N = 30) was recorded every other hour throughout 5-min eyes closed relaxation. Sixteen ln-transformed single-Hz power densities (from 1 to 16 Hz) were computed for each of 5 one-min intervals. Differences between these densities obtained for the first and the following intervals were calculated and averaged. The obtained 16 values were used as the frequency weighting curve for weighting densities of each set of 16 single-Hz power densities. Summing-up of these weighted densities provided a single measure that was found to co-vary with self-rated sleepiness throughout two-day interval of sleep deprivation, thus reflecting the joint influence of the circadian and homeostatic processes. However, two-day time course of responsiveness of this measure to closing the eyes for just a few minutes exhibited a purely exponential buildup. It was concluded that this result provided a direct experimental confirmation of the model-predicted exponential buildup of the homeostatic sleep pressure across prolonged episode of wakefulness.     

Selection and genomic differentiation during ecological speciation: isolating the contributions of host association via a comparative genome scan of Neochlamisus bebbianae leaf beetles

This study uses a comparative genome scan to evaluate the contributions of host plant related divergent selection to genetic differentiation and ecological speciation in maple- and willow-associated populations of Neochlamisus bebbianae leaf beetles. For each of 15 pairwise population comparisons, we identified "outlier loci" whose strong differentiation putatively reflects divergent selection. Of 447 AFLP loci, 15% were outliers across multiple population comparisons, and low linkage disequilibrium indicated that these outliers derived from multiple regions of the genome. Outliers were further classified as "host-specific" if repeatedly observed in "different-host" population comparisons but never in "same-host" comparisons. Outliers exhibiting the opposite pattern were analogously classified as "host-independent." Host-specific outliers represented 5% of all loci and were more frequent than host-independent outliers, thus revealing a large role for host-adaptation in population genomic differentiation. Evidence that host-related selection can promote divergence despite gene flow was provided by population trees. These were structured by host-association when datasets included host-specific outliers, but not when based on neutral loci, which united sympatric populations. Lastly, three host-specific outliers were highly differentiated in all nine different-host comparisons. Because host-adaptation promotes reproductive isolation in these beetles, these loci provide promising candidate gene regions for future molecular studies of ecological speciation.     

Analysis of Foraging Behaviour of the Whitefly Parasitoid Encarsia formosa on a plant: A Simulation Study

The foraging behaviour of Encarsia formosa was analyzed using a stochastic simulation model of the parasitoid's behaviour. Parasitoids were allowed to search during a day on a tomato plant infested with immatures of the greenhouse whitefly, Trialeurodes vaporariorum. The model simulates searching, host selection, host handling and patch leaving behaviour, and the physiological state of the parasitoid. The outputs of the model are the number of visited leaflets and the number of hosts encountered, parasitized or killed by host feeding. The simulation results agreed well with observations of parasitoids foraging on tomato plants. The number of encounters and ovipositions on the plant increased with host density according to a type II functional response. At a clustered host distribution over leaflets and low host densities, the most important parameters affecting the number of ovipositions were the leaf area, the parasitoid's walking speed and walking activity, the probability of oviposition after encountering a host, the initial egg load and the ratio of search times on both leaf sides. At high densities, the maximum egg load and the giving-up time on a leaflet since latest host encounter were the most essential parameters.     

Analysis of the Sarcocystis neurona microneme protein SnMIC10: protein characteristics and expression during intracellular development

Sarcocystis neurona, an apicomplexan parasite, is the primary causative agent of equine protozoal myeloencephalitis. Like other members of the Apicomplexa, S. neurona zoites possess secretory organelles that contain proteins necessary for host cell invasion and intracellular survival. From a collection of S. neurona expressed sequence tags, we identified a sequence encoding a putative microneme protein based on similarity to Toxoplasma gondii MIC10 (TgMIC10). Pairwise sequence alignments of SnMIC10 to TgMIC10 and NcMIC10 from Neospora caninum revealed approximately 33% identity to both orthologues. The open reading frame of the S. neurona gene encodes a 255 amino acid protein with a predicted 39-residue signal peptide. Like TgMIC10 and NcMIC10, SnMIC10 is predicted to be hydrophilic, highly alpha-helical in structure, and devoid of identifiable adhesive domains. Antibodies raised against recombinant SnMIC10 recognised a protein band with an apparent molecular weight of 24 kDa in Western blots of S. neurona merozoites, consistent with the size predicted for SnMIC10. In vitro secretion assays demonstrated that this protein is secreted by extracellular merozoites in a temperature-dependent manner. Indirect immunofluorescence analysis of SnMIC10 showed a polar labelling pattern, which is consistent with the apical position of the micronemes, and immunoelectron microscopy provided definitive localisation of the protein to these secretory organelles. Further analysis of SnMIC10 in intracellular parasites revealed that expression of this protein is temporally regulated during endopolygeny, supporting the view that micronemes are only needed during host cell invasion. Collectively, the data indicate that SnMIC10 is a microneme protein that is part of the excreted/secreted antigen fraction of S. neurona. Identification and characterisation of additional S. neurona microneme antigens and comparisons to orthologues in other Apicomplexa could provide further insight into the functions that these proteins serve during invasion of host cells.     

Thrombosis during pregnancy: Risks,prevention, and treatment for mother and fetus—harvesting the power of omic technology,biomarkers and in vitro or in vivo models to facilitate the treatment of thrombosis

Maternal thromboembolism and a spectrum of placenta‐mediated complications including the pre‐eclampsia syndromes, fetal growth restriction, fetal loss, and abruption manifest a shared etiopathogenesis and predisposing risk factors. Furthermore, these maternal and fetal complications are often linked to subsequent maternal health consequences that comprise the metabolic syndrome, namely, thromboembolism, chronic hypertension, and type II diabetes. Traditionally, several lines of evidence have linked vasoconstriction, excessive thrombosis and inflammation, and impaired trophoblast invasion at the uteroplacental interface as hallmark features of the placental complications. “Omic” technologies and biomarker development have been largely based upon advances in vascular biology, improved understanding of the molecular basis and biochemical pathways responsible for the clinically relevant diseases, and increasingly robust large cohort and/or registry based studies. Advances in understanding of innate and adaptive immunity appear to play an important role in several pregnancy complications. Strategies aimed at improving prediction of these pregnancy complications are often incorporating hemodynamic blood flow data using non‐invasive imaging technologies of the utero‐placental and maternal circulations early in pregnancy. Some evidence suggests that a multiple marker approach will yield the best performing prediction tools, which may then in turn offer the possibility of early intervention to prevent or ameliorate these pregnancy complications. Prediction of maternal cardiovascular and non‐cardiovascular consequences following pregnancy represents an important area of future research, which may have significant public health consequences not only for cardiovascular disease, but also for a variety of other disorders, such as autoimmune and neurodegenerative diseases. Birth Defects Research (Part C) 105:209–225, 2015. © 2015 Wiley Periodicals, Inc.     

Analysis of some morphological and biochemical characteristics of the egg of the predaceous bugMacrolophus caliginosus (Het.: Miridae) during embryogenesis

The predaceous bug,Macrolophus caliginosus Wagner (Heteroptera: Miridae) is a biological control agent used in greenhouses in the French Mediterranean region. The egg of this mirid is embedded in plant tissues and all embryonic development occurs in this environment. In order to define an artificial substrate for egg-laying, exchanges of chemical substances between the egg and the host plant were investigated by morphological and biochemical analyses. Egg wet weight doubled throughout embryonic development, while egg dry weight did not vary. The amino acid content of the egg remained stable during embryogenesis. Only aspartic acid, serine and alanine decreased during the first five days of development. It can be concluded that exchanges mainly involved uptake of water by the egg.     

The cortical actin cytoskeleton in a Dipteran embryo: Analysis of the spatial reorganization of F-actin aggregates during the early nuclear division cycles

Rhodamine phalloidin-staining was used to study the organization of the cortical actin cytoskeleton of the early Ceratitis capitata embryo. The dynamics of the actin aggregates and their changes in distribution during the formation of the syncytial blastoderm, were followed in detail. It was found that these aggregates formed a shell-like cluster around the interphase nuclei, and concentrated toward the poles of the mitotic apparatus when the nuclei divided. Laser scanning confocal microscopy revealed that aggregates not clustered at the poles of the mitotic apparatus were closely associated with fine fibers of a dense cytoplasmic network of actin filaments.