The RNA replicase proteins of Semliki Forest virus (SFV) are translated as a P1234 polyprotein precursor that contains two putative autoproteases. Point mutations introduced into the predicted active sites of both proteases nsP2 (P2) and nsP4 (P4), separately or in combination, completely abolished virus replication in mammalian cells. The effects of these mutations on polyprotein processing were studied by in vitro translation and by expression of wild-type polyproteins P1234, P123, P23, P34 and their mutated counterparts in insect cells using recombinant baculoviruses. A mutation in the catalytic site of the P2 protease, C(478)A, (P2(CA)) completely abolished the processing of P12(CA)34, P12(CA)3 and P2(CA)3. Co-expression of P23 and P12(CA)34 in insect cells resulted in in trans cleavages at the P2/3 and P3/4 sites. Co-expression of P23 and P34 resulted in cleavage at the P3/4 site. In contrast, a construct with a mutation in the active site of the putative P4 protease, D(6)A, (P1234(DA)) was processed like the wild-type protein. P34 or its truncated forms were not processed when expressed alone. In insect cells, P4 was rapidly destroyed unless an inhibitor of proteosomal degradation was used. It is concluded that P2 is the only protease needed for the processing of SFV polyprotein P1234. Analysis of the cleavage products revealed that P23 or P2 could not cleave the P1/2 site in trans.

High-density cultures are widely used as an in vitro model for studies of embryonic cartilage formation. In the present study we investigated the suitability of high-density cultures for the redifferentiation of dedifferentiated chondrocytes. When primary human chondrocytes were cultured in alginate beads, some cells emigrated into Petri dishes. These cells were cultured for one to eight passages (each passage lasting about 3 days) in monolayer culture. At each passage, monolayer cells were removed and allowed to grow in high-density cultures at the medium-air interface and subsequently investigated with morphological, immunolocalization and biochemical methods for the production of cartilage-specific markers, i.e. collagen type II and cartilage-specific proteoglycans. When such dedifferentiated chondrocytes from monolayer passages P1-P4 were introduced in high-density culture, they regained a chondrocyte phenotype and formed cartilage nodules surrounded by fibroblast-like cells. Cells were interconnected by typical gap junctions and after a few days in culturing produced cartilage-specific extracellular matrix, notably collagen type II and cartilage-specific proteoglycans. In contrast, cells taken from monolayer passages P5-P8 did not produce these chondrocyte-specific extracellular materials when grown in high-density culture. We conclude that the growth of dedifferentiated chondrocytes in high-density culture promotes their redifferentiation and reveals their chondrogenic potential. Such high-density cultures might serve as a model system to initiate and promote the redifferentiation of chondrocytes and to provide sufficient quantities of differentiated chondrocytes for autologous chondrocyte transplantation.

Members of the P(4) subfamily of P-type ATPases catalyze phospholipid transport and create membrane lipid asymmetry in late secretory and endocytic compartments. P-type ATPases usually pump small cations and the transport mechanism involved appears conserved throughout the family. How this mechanism is adapted to flip phospholipids remains to be established. P(4)-ATPases form heteromeric complexes with CDC50 proteins. Dissociation of the yeast P(4)-ATPase Drs2p from its binding partner Cdc50p disrupts catalytic activity (Lenoir, G., Williamson, P., Puts, C. F., and Holthuis, J. C. (2009) J. Biol. Chem. 284, 17956-17967), suggesting that CDC50 subunits play an intimate role in the mechanism of transport by P(4)-ATPases. The human genome encodes 14 P(4)-ATPases while only three human CDC50 homologues have been identified. This implies that each human CDC50 protein interacts with multiple P(4)-ATPases or, alternatively, that some human P(4)-ATPases function without a CDC50 binding partner. Here we show that human CDC50 proteins each bind multiple class-1 P(4)-ATPases, and that in all cases examined, association with a CDC50 subunit is required for P(4)-ATPase export from the ER. Moreover, we find that phosphorylation of the catalytically important Asp residue in human P(4)-ATPases ATP8B1 and ATP8B2 is critically dependent on their CDC50 subunit. These results indicate that CDC50 proteins are integral part of the P(4)-ATPase flippase machinery.

In C. elegans, four asymmetric divisions, beginning with the zygote (P0), generate transcriptionally repressed germline blastomeres (P1-P4) and somatic sisters that become transcriptionally active. The protein PIE-1 represses transcription in the later germline blastomeres but not in the earlier germline blastomeres P0 and P1. We show here that OMA-1 and OMA-2, previously shown to regulate oocyte maturation, repress transcription in P0 and P1 by binding to and sequestering in the cytoplasm TAF-4, a component critical for assembly of TFIID and the pol II preinitiation complex. OMA-1/2 binding to TAF-4 is developmentally regulated, requiring phosphorylation by the DYRK kinase MBK-2, which is activated at meiosis II after fertilization. OMA-1/2 are normally degraded after the first mitosis, but ectopic expression of wild-type OMA-1 is sufficient to repress transcription in both somatic and later germline blastomeres. We propose that phosphorylation by MBK-2 serves as a developmental switch, converting OMA-1/2 from oocyte to embryo regulators.

Caenorhabditis elegans and C. briggsae have many parallels in terms of morphology, life history and breeding system. Both species also share similar low levels of molecular diversity, although the global sampling of natural populations has been limited and geographically biased. In this study, we describe the first cultured isolates of C. elegans and C. briggsae from sub-Saharan Africa. We characterize these samples for patterns of nucleotide polymorphism and vulva precursor cell lineage, and conduct a series of hybrid crosses in C. briggsae to test for genetic incompatibilities. The distribution of genetic diversity confirms a lack of geographic structure to C. elegans sequences but shows genetic differentiation of C. briggsae into three distinct clades that may correspond to three latitudinal ranges. Despite low levels of molecular diversity, we find considerable variation in cell division frequency in African C. elegans for the P3.p vulva precursor cell, and in African C. briggsae for P4.p, a variation that was not previously observed in this species. Hybrid crosses did not reveal major incompatibilities between C. briggsae strains from Africa and elsewhere, and there was some evidence of inbreeding depression. These new African isolates suggest that important ecological factors may be shaping the patterns of diversity in C. briggsae, and that despite many similarities between C. elegans and C. briggsae, there may be more subtle differences in their natural histories than previously appreciated.

Transcriptional control of the himA and the himD/hip genes coding for the two subunits of the integration host factor (IHF) was investigated. The promoters for the two genes were identified by the use of primer extension and S1 analysis. Expression from both promoters was found to increase as the cells enter stationary phase. Mutation in rpoS, known to be induced upon entry to stationary phase, dramatically reduced the growth-phase response of the himA P4 promoter but had only a small effect on the induction of the himD/hip promoter. The increased activity of both promoters required the presence of the relA and spoT genes, suggesting that ppGpp plays a major role in the response to stationary phase. An artificial increase in ppGpp in exponentially growing cells induced a rapid increase in himA P4 and himD/hip mRNA levels. Experiments with a mutant defective in rpoS showed that the response of the himA P4 promoter to high ppGpp levels was greatly reduced while that of himD/hip was only slightly affected. Therefore, it seems that different mechanisms involving RpoS and ppGpp regulate the growth-phase response of the two promoters. We propose that the effect of ppGpp on himA P4 is mediated via RpoS whereas the himD/hip promoter is affected by ppGpp independently of RpoS. Expression of the himD/hip and himA genes was found to be subject to negative autoregulation. IHF-binding sites, implicated in autoregulation, were found to overlap both the himD/hip and himA P4 promoters. An additional IHF-binding site was found upstream of the himD/hip promoter. All three sites show low binding affinity to IHF suggesting that autoregulation can take place only after sufficiently high levels of IHF accumulate in the cell.

Time-resolved small-angle X-ray scattering (SAXS) with millisecond time-resolution reveals two discrete phases of global compaction upon Mg2+-mediated folding of the Tetrahymena thermophila ribozyme. Electrostatic relaxation of the RNA occurs rapidly and dominates the first phase of compaction during which the observed radius of gyration (R(g)) decreases from 75 angstroms to 55 angstroms. A further decrease in R(g) to 45 angstroms occurs in a well-defined second phase. An analysis of mutant ribozymes shows that the latter phase depends upon the formation of long-range tertiary contacts within the P4-P6 domain of the ribozyme; disruption of the three remaining long-range contacts linking the peripheral helices has no effect on the 55-45 angstroms compaction transition. A better understanding of the role of specific tertiary contacts in compaction was obtained by concordant time-resolved hydroxyl radical (OH) analyses that report local changes in the solvent accessibility of the RNA backbone. Comparison of the global and local measures of folding shows that formation of a subset of native tertiary contacts (i.e. those defining the ribozyme core) can occur within a highly compact ensemble whose R(g) is close to that of the fully folded ribozyme. Analyses of additional ribozyme mutants and reaction conditions establish the generality of the rapid formation of a partially collapsed state with little to no detectable tertiary structure. These studies directly link global RNA compaction with formation of tertiary structure as the molecule acquires its biologically active structure, and underscore the strong dependence on salt of both local and global measures of folding kinetics.

We report here the x-ray crystal structure of a soluble catalytically active fragment of the Escherichia coli type I signal peptidase (SPase-(Delta2-75)) in the absence of inhibitor or substrate (apoenzyme). The structure was solved by molecular replacement and refined to 2.4 A resolution in a different space group (P4(1)2(1)2) from that of the previously published acyl-enzyme inhibitor-bound structure (P2(1)2(1)2) (Paetzel, M., Dalbey, R.E., and Strynadka, N.C.J. (1998) Nature 396, 186-190). A comparison with the acyl-enzyme structure shows significant side-chain and main-chain differences in the binding site and active site regions, which result in a smaller S1 binding pocket in the apoenzyme. The apoenzyme structure is consistent with SPase utilizing an unusual oxyanion hole containing one side-chain hydroxyl hydrogen (Ser-88 OgammaH) and one main-chain amide hydrogen (Ser-90 NH). Analysis of the apoenzyme active site reveals a potential deacylating water that was displaced by the inhibitor. It has been proposed that SPase utilizes a Ser-Lys dyad mechanism in the cleavage reaction. A similar mechanism has been proposed for the LexA family of proteases. A structural comparison of SPase and members of the LexA family of proteases reveals a difference in the side-chain orientation for the general base lysine, both of which are stabilized by an adjacent hydroxyl group. To gain insight into how signal peptidase recognizes its substrates, we have modeled a signal peptide into the binding site of SPase. The model is built based on the recently solved crystal structure of the analogous enzyme LexA (Luo, Y., Pfuetzner, R. A., Mosimann, S., Paetzel, M., Frey, E. A., Cherney, M., Kim, B., Little, J. W., and Strynadka, N. C. J. (2001) Cell 106, 1-10) with its bound cleavage site region.

Levels of two types of lectin-reactive alpha-fetoprotein (AFP), designated AFP-L3 and AFP-P4+P5, were analyzed with Lens culinaris agglutinin A and AFP-P4+P5 with erythroagglutinating phytohemagglutinin, respectively, in an attempt to determine the utility and significance of these macromolecules as early indicators of hepatocellular carcinoma during the periodic follow-up of cirrhotic patients. The subjects were 51 of 190 consecutive cirrhotic patients in whom hepatocellular carcinoma developed during a 6-year follow-up period and 21 cirrhotic patients without hepatocellular carcinoma. Serum AFP levels were of limited value to diagnose and predict hepatocellular carcinoma. The relative levels of AFP-L3 and AFP-P4+P5 in patients with hepatocellular carcinoma at the time of tumor detection were significantly higher than those in patients with cirrhosis. The sensitivity was 61%, and the specificity was 90%. Fourteen patients (48%) of 29 patients with small hepatocellular carcinomas less than 2 cm in diameter showed elevated percentage of lectin-reactive AFP. Retrospective examination of 21 patients who were positive for lectin-reactive AFP at diagnosis of hepatocellular carcinoma showed that 41% of them had already expressed lectin-reactive AFP 12 months before the direct detection of hepatocellular carcinoma by diagnostic imaging. These results lead us to conclude that the level of lectin-reactive AFP is a suitable predictive marker for the early recognition of hepatocellular carcinoma in the follow-up of patients with cirrhosis, and that measurements of the level of lectin-reactive AFP should be added to the screening methods that are now in use.

Susceptibility to insulin-dependent diabetes mellitus is linked to MHC class II genes. The only MHC class II molecule expressed by nonobese diabetic (NOD) mice, I-Ag7, shares a common alpha-chain with I-Ad but has a peculiar beta-chain. As with most beta-chain alleles linked to diabetes susceptibility, I-Ag7 contains a nonaspartic residue at position beta57. We have produced large amounts of empty I-Ag7 molecules using a fly expression system to characterize its biochemical properties and peptide binding by phage-displayed peptide libraries. The identification of a specific binding peptide derived from glutamic acid decarboxylase (GAD65) has allowed us to crystallize and obtain the three-dimensional structure of I-Ag7. Structural information was critical in evaluating the binding studies. I-Ag7, like I-Ad, appears to be very promiscuous in terms of peptide binding. Their binding motifs are degenerate and contain small and/or small hydrophobic residues at P4 and P6 of the peptide, a motif frequently found in most globular proteins. The degree of promiscuity is increased for I-Ag7 over I-Ad as a consequence of a larger P9 pocket that can specifically accommodate negatively charged residues, as well as possibly residues with bulky side chains. So, although I-Ad and I-Ag7 are structurally closely related, stable molecules and good peptide binders, they differ functionally in their ability to bind significantly different peptide repertoires that are heavily influenced by the presence or the absence of a negatively charged residue at position 57 of the beta-chain. These characteristics link I-Ag7 with autoimmune diseases, such as insulin-dependent diabetes mellitus.

Many neutrophil functions are regulated by phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) that mediates protein membrane translocation via binding to pleckstrin homolog (PH) domains within target proteins. Here we show that inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), a cytosolic small molecule, bound the same PH domain of target proteins and competed for binding to PtdIns(3,4,5)P3. In neutrophils, chemoattractant stimulation triggered rapid elevation in Ins(1,3,4,5)P4 concentration. Depletion of Ins(1,3,4,5)P4 by deleting the gene encoding InsP3KB, which converts Ins(1,4,5)P3 to Ins(1,3,4,5)P4, enhanced membrane translocation of the PtdIns(3,4,5)P3-specific PH domain. This led to enhanced sensitivity to chemoattractant stimulation, elevated superoxide production, and enhanced neutrophil recruitment to inflamed peritoneal cavity. On the contrary, augmentation of intracellular Ins(1,3,4,5)P4 concentration blocked PH domain-mediated membrane translocation of target proteins and dramatically decreased the sensitivity of neutrophils to chemoattractant stimulation. These findings establish a role for Ins(1,3,4,5)P4 in cellular signal transduction pathways and provide another mechanism for modulating PtdIns(3,4,5)P3 signaling in neutrophils.

In rabbit portal vein myocytes noradrenaline activates a non-selective cation current (Icat) which involves a transient receptor potential protein (TRPC6). Previously we have shown that the diaylglycerol (DAG) analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) stimulates Icat via a protein kinase C (PKC)-independent mechanism, and in the present study we have investigated the interaction between inositol phosphates (InsPs) and OAG on Icat. With whole-cell recording of Icat from freshly isolated rabbit portal vein myocytes the amplitude and rate of activation of noradrenaline-evoked Icat were much greater than those of OAG-induced Icat. Inclusion of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) in the pipette solution did not evoke Icat but greatly potentiated the amplitude and rate of activation of OAG-induced Icat. With isolated outside-out patches Ins(1,4,5)P3 markedly increased the rate of activation and the open probability of OAG-evoked channel activity, with no change in unitary conductance, channel mean open times or burst durations. The effects of Ins(1,4,5)P3 were mimicked by Ins(2,4,5)P3, 3-F-Ins(1,4,5)P3 and Ins(1,4)P2 but not by Ins(1,3,4,5)P4 and the potentiating effects of InsPs were not inhibited by heparin. Therefore it is concluded that both DAG and InsPs are necessary for full activation of Icat by noradrenaline and the effect of InsPs is via a heparin-insensitive mechanism and represents a novel action of InsPs.

The mammalian cell cycle is regulated by the cyclin/cyclin-dependent kinase (CDK) phosphorylation of the retinoblastoma (pRB) family of proteins. Cyclin D1 with its CDK4/6 partners initiates the cell cycle and acts as the link between extracellular signals and the cell cycle machinery. Estradiol-17beta (E2) stimulates uterine epithelial cell proliferation, a process that is completely inhibited by pretreatment with progesterone (P4). Previously, we identified cyclin D1 localization as a key point of regulation in these cells with E2 causing its nuclear accumulation and P4 retaining it in the cytoplasm with the resultant inhibition of pRB phosphorylation. Here we show that E2 stimulates phosphoinositide 3-kinase to activate phosphokinase B/AKT to effect an inhibitory phosphorylation of glycogen synthase kinase (GSK-3beta). This pathway is suppressed by P4. Inhibition of the GSK-3beta activity in P4-treated uteri by the specific inhibitor, LiCl, reversed the nuclear accumulation of cyclin D1 and in doing so, caused pRB phosphorylation and the induction of downstream genes, proliferating cell nuclear antigen and Ki67. Conversely, inhibition of phosphoinositide 3 kinase by LY294002 or Wortmanin reversed the E2-induced GSK-3beta Ser9 inhibitory phosphorylation and blocked nuclear accumulation of cyclin D1. These data show the reciprocal actions of E2 and P4 on the phosphoinositide 3-kinase through to the GSK-3beta pathway that in turn regulates cyclin D1 localization and cell cycle progression. These data reveal a novel signaling pathway that links E2 and P4 action to growth factor-mediated signaling in the uterus.

Personalized medicine is a term for a revolution in medicine that envisions the individual patient as the central focus of healthcare in the future. The term "personalized medicine", however, fails to reflect the enormous dimensionality of this new medicine that will be predictive, preventive, personalized, and participatory-a vision of medicine we have termed P4 medicine. This reflects a paradigm change in how medicine will be practiced that is revolutionary rather than evolutionary. P4 medicine arises from the confluence of a systems approach to medicine and from the digitalization of medicine that creates the large data sets necessary to deal with the complexities of disease. We predict that systems approaches will empower the transition from conventional reactive medical practice to a more proactive P4 medicine focused on wellness, and will reverse the escalating costs of drug development an will have enormous social and economic benefits. Our vision for P4 medicine in 10 years is that each patient will be associated with a virtual data cloud of billions of data points and that we will have the information technology for healthcare to reduce this enormous data dimensionality to simple hypotheses about health and/or disease for each individual. These data will be multi-scale across all levels of biological organization and extremely heterogeneous in type - this enormous amount of data represents a striking signal-to-noise (S/N) challenge. The key to dealing with this S/N challenge is to take a "holistic systems approach" to disease as we will discuss in this article.

Recently, we identified extracellular ubiquitin as an endogenous CXC chemokine receptor (CXCR) 4 agonist. However, the receptor selectivity and molecular basis of the CXCR4 agonist activity of ubiquitin are unknown, and functional consequences of CXCR4 activation with ubiquitin are poorly defined. Here, we provide evidence that ubiquitin and the cognate CXCR4 ligand stromal cell-derived factor (SDF)-1α do not share CXCR7 as a receptor. We further demonstrate that ubiquitin does not utilize the typical two-site binding mechanism of chemokine-receptor interactions, in which the receptor N terminus is important for ligand binding. CXCR4 activation with ubiquitin and SDF-1α lead to similar Gα(i)-responses and to a comparable magnitude of phosphorylation of ERK-1/2, p90 ribosomal S6 kinase-l and Akt, although phosphorylations occur more transiently after activation with ubiquitin. Despite the similarity of signal transduction events after activation of CXCR4 with both ligands, ubiquitin possesses weaker chemotactic activity than SDF-lα in cell migration assays and does not interfere with productive entry of HIV-1 into P4.R5 multinuclear activation of galactosidase indicator cells. Unlike SDF-1α, ubiquitin lacks interactions with an N-terminal CXCR4 peptide in NMR spectroscopy experiments. Binding and signaling studies in the presence of antibodies against the N terminus and extracellular loops 2/3 of CXCR4 confirm that the ubiquitin CXCR4 interaction is independent of the N-terminal receptor domain, whereas blockade of extracellular loops 2/3 prevents receptor binding and activation. Our findings define ubiquitin as a CXCR4 agonist, which does not interfere with productive cellular entry of HIV-1, and provide new mechanistic insights into interactions between CXCR4 and its natural ligands.

Studying complex biological systems in a holistic rather than a "one gene or one protein" at a time approach requires the concerted effort of scientists from a wide variety of disciplines. The Institute for Systems Biology (ISB) has seamlessly integrated these disparate fields to create a cross-disciplinary platform and culture in which "biology drives technology drives computation." To achieve this platform/culture, it has been necessary for cross-disciplinary ISB scientists to learn one another's languages and work together effectively in teams. The focus of this "systems" approach on disease has led to a discipline denoted systems medicine. The advent of technological breakthroughs in the fields of genomics, proteomics, and, indeed, the other "omics" is catalyzing striking advances in systems medicine that have and are transforming diagnostic and therapeutic strategies. Systems medicine has united genomics and genetics through family genomics to more readily identify disease genes. It has made blood a window into health and disease. It is leading to the stratification of diseases (division into discrete subtypes) for proper impedance match against drugs and the stratification of patients into subgroups that respond to environmental challenges in a similar manner (e.g. response to drugs, response to toxins, etc.). The convergence of patient-activated social networks, big data and their analytics, and systems medicine has led to a P4 medicine that is predictive, preventive, personalized, and participatory. Medicine will focus on each individual. It will become proactive in nature. It will increasingly focus on wellness rather than disease. For example, in 10 years each patient will be surrounded by a virtual cloud of billions of data points, and we will have the tools to reduce this enormous data dimensionality into simple hypotheses about how to optimize wellness and avoid disease for each individual. P4 medicine will be able to detect and treat perturbations in healthy individuals long before disease symptoms appear, thus optimizing the wellness of individuals and avoiding disease. P4 medicine will 1) improve health care, 2) reduce the cost of health care, and 3) stimulate innovation and new company creation. Health care is not the only subject that can benefit from such integrative, cross-disciplinary, and systems-driven platforms and cultures. Many other challenges plaguing our planet, such as energy, environment, nutrition, and agriculture can be transformed by using such an integrated and systems-driven approach.

The properties of neonatal GABAergic synapses were investigated in neurones of the hippocampal CA3 region. GABA, acting on GABAA receptors, provides most of the excitatory drive on immature CA3 pyramidal neurones at an early stage of development, whereas glutamatergic synapses (in particular, those mediated by AMPA receptors) are mostly quiescent. Thus, during the first postnatal week of life, bicuculline fully blocked spontaneous and evoked depolarising potentials, and GABAA receptor agonists depolarised CA3 pyramidal neurones. GABAA mediated currents also had a reduced sensitivity to benzodiazepines. In the presence of bicuculline, between P0 and P4, increasing the stimulus strength reveals an excitatory postsynaptic potential which is mostly mediated by NMDA receptors. During the same developmental period, pre- (but not post) synaptic GABAB inhibition is present. Intracellular injections of biocytin showed that the axonal network of the GABAergic interneurones is well developed at birth, whereas the pyramidal recurrent collaterals are only beginning to develop. Finally, chronic bicuculline treatment of hippocampal neurones in culture reduced the extent of neuritic arborisation, suggesting that GABA acts as a trophic factor in that period. In conclusion, it is suggested that during the first postnatal week of life, when excitatory inputs are still poorly developed, GABAA receptors provide the excitatory drive necessary for pyramidal cell outgrowth. Starting from the end of the first postnatal week of life, when excitatory inputs are well developed, GABA (acting on both GABAA and GABAB receptors) will hyperpolarise the CA3 pyramidal neurones and, as in the adult, will prevent excessive neuronal discharges. Our electrophysiological and morphological studies have shown that hippocampal GABAergic interneurones are in a unique position to modulate the development of CA3 pyramidal neurones. Developing neurones require a certain degree of membrane depolarisation, and a consequent rise in intracellular calcium, for stimulating neurite outgrowth; the GABAergic network, which develops prior to the glutamatergic one, appears to provide this depolarisation. Starting from the end of the first postnatal week of life, at a time when excitatory pathways are developing, GABA (acting on both GABAA and GABAB receptors) would reverse its action, and start to play its well-known role as an inhibitory neurotransmitter.

METHODS

Auditory evoked potentials (AEPs) to the subject's own name and to seven other first names were recorded in ten normal adults during wakefulness, in both passive listening and active detection conditions, and during sleep stage II (SII) and paradoxical sleep (PS). All stimuli were disyllabic, equiprobable and presented in random order.

RESULTS

During wakefulness, a parietal positive 'P3' component, peaking at about 500 ms, probably equivalent to the endogenous P300 wave, was enhanced in response to the subject's own name, even in the passive condition. During SII, K-complexes (KCs) were evoked by all first names and were formed by two biphasic consecutive waveforms. While the amplitude of the late complex (N3/P4) was identical for both types of stimuli, the early portion of the KC (N2/P3), and notably the positive wave 'SII-P3' at about 600 ms, was selectively enhanced after the subject's own name. This supports the hypothesis that at least two distinct neuronal systems are activated in parallel in response to auditory stimuli during SII, one reflecting the detection of stimulus' salience and the other the processing of its intrinsic relevance. During PS, the AEP morphology was comparable to that observed in wakefulness. Notably, a posterior 'PS-P3' wave appeared exclusively in response to own names at about 550 ms, and was considered as an equivalent of the waking P300.

CONCLUSIONS

These results suggest that the sleeping brain, during SII and PS, elicits a differential cognitive response to the presentation of the subject's own name, comparable to that occurring during wakefulness, and therefore that the sleeping brain is able to detect and categorize some particular aspects of stimulus significance.

The high-pathogenicity island (HPI) is a genomic island essential for the mouse-virulence phenotype in Yersinia and indispensable for pathogenicity of Yersinia and certain pathotypes of Escherichia coli. In contrast to most genomic islands, the HPI is a functional island widely disseminated among members of the family of Enterobacteriaceae. The HPI-encoded phage P4-like integrase together with excisionase and recombination sites make up the genetic mobility module of the island, while the siderophore yersiniabactin biosynthesis and uptake system comprises its functional part with respect to fitness and pathogenicity. The HPI-integrase promotes integration of the island into attB sites represented by three to four asn tDNAs in Yersinia pestis and E. coli. An additional enzyme, excisionase, is essential for efficient excision of the HPI from the initial site of integration. Furthermore a unique type of HPI has been characterized in the E. coli strain ECOR31 carrying a functional conjugative mating pair formation (Mpf) and a DNA-processing system, both of which are characteristic of integrative and conjugative elements (ICE). A model of conjugative transfer for the dissemination of HPIs is proposed in which the excised HPI is mobilized to a new recipient either trapped by a transmissive asn tDNA-carrying plasmid or autonomously as an ICE named ICEEcl.

The opportunistic mycopathogen Aspergillus fumigatus expresses both glucosylceramide and galactosylceramide (GlcCer and GalCer), but their functional significance in Aspergillus species is unknown. We here identified and characterized a GlcCer from Aspergillus nidulans, a non-pathogenic model fungus. Involvement of GlcCer in fungal development was tested on both species using a family of compounds known to inhibit GlcCer synthase in mammals. Two analogs, D-threo-1-phenyl-2-palmitoyl-3-pyrrolidinopropanol (P4) and D-threo-3',4'-ethylenedioxy-P4, strongly inhibited germination and hyphal growth. Neutral lipids from A. fumigatus cultured in the presence of these inhibitors displayed a significantly reduced GlcCer/GalCer ratio. These results suggest that synthesis of GlcCer is essential for normal development of A. fumigatus and A. nidulans.

The effect of acupuncture stimulation applied to a Ximen point (P4) of a forearm on heart rate was studied in healthy volunteer human subjects. Acupuncture stimulation decreased heart rate, or gave no significant response. The decreased response of heart rate following acupuncture was attenuated by administration of atropine and propranolol. Therefore, the acupuncture-induced response of decrease in heart rate was concluded to be a result of a reciprocal coordination of an increase in cardiac vagal activity and a decrease in cardiac sympathetic activity.

Endophytic isolates of Trichoderma species are being considered as biocontrol agents for diseases of Theobroma cacao (cacao). Gene expression was studied during the interaction between cacao seedlings and four endophytic Trichoderma isolates, T. ovalisporum-DIS 70a, T. hamatum-DIS 219b, T. harzianum-DIS 219f, and Trichoderma sp.-DIS 172ai. Isolates DIS 70a, DIS 219b, and DIS 219f were mycoparasitic on the pathogen Moniliophthora roreri, and DIS 172ai produced metabolites that inhibited growth of M. roreri in culture. ESTs (116) responsive to endophytic colonization of cacao were identified using differential display and their expression analyzed using macroarrays. Nineteen cacao ESTs and 17 Trichoderma ESTs were chosen for real-time quantitative PCR analysis. Seven cacao ESTs were induced during colonization by the Trichoderma isolates. These included putative genes for ornithine decarboxylase (P1), GST-like proteins (P4), zinc finger protein (P13), wound-induced protein (P26), EF-calcium-binding protein (P29), carbohydrate oxidase (P59), and an unknown protein (U4). Two plant ESTs, extensin-like protein (P12) and major intrinsic protein (P31), were repressed due to colonization. The plant gene expression profile was dependent on the Trichoderma isolate colonizing the cacao seedling. The fungal ESTs induced in colonized cacao seedlings also varied with the Trichoderma isolate used. The most highly induced fungal ESTs were putative glucosyl hydrolase family 2 (F3), glucosyl hydrolase family 7 (F7), serine protease (F11), and alcohol oxidase (F19). The pattern of altered gene expression suggests a complex system of genetic cross talk occurs between the cacao tree and Trichoderma isolates during the establishment of the endophytic association.

The National Electrical Manufacturers Association (NEMA) standard NU 4-2008 for performance measurements of small-animal tomographs was recently published. Before this standard, there were no standard testing procedures for preclinical PET systems, and manufacturers could not provide clear specifications similar to those available for clinical systems under NEMA NU 2-1994 and 2-2001. Consequently, performance evaluation papers used methods that were modified ad hoc from the clinical PET NEMA standard, thus making comparisons between systems difficult.

METHODS

We acquired NEMA NU 4-2008 performance data for a collection of commercial animal PET systems manufactured since 2000: microPET P4, microPET R4, microPET Focus 120, microPET Focus 220, Inveon, ClearPET, Mosaic HP, Argus (formerly eXplore Vista), VrPET, LabPET 8, and LabPET 12. The data included spatial resolution, counting-rate performance, scatter fraction, sensitivity, and image quality and were acquired using settings for routine PET.

RESULTS

The data showed a steady improvement in system performance for newer systems as compared with first-generation systems, with notable improvements in spatial resolution and sensitivity.

CONCLUSIONS

Variation in system design makes direct comparisons between systems from different vendors difficult. When considering the results from NEMA testing, one must also consider the suitability of the PET system for the specific imaging task at hand.