Bacterial Quorum-Sensing Regulation Induces Morphological Change in a Key Host Tissue during the Euprymna scolopes-Vibrio fischeri Symbiosis.

Microbes colonize the apical surfaces of polarized epithelia in nearly all animal taxa. In one example, the luminous bacterium Vibrio fischeri enters, grows to a dense population within, and persists for months inside, the light-emitting organ of the squid Euprymna scolopes. Crucial to the symbiont's success after entry is the ability to trigger the constriction of a host tissue region (the "bottleneck") at the entrance to the colonization site. Bottleneck constriction begins at about the same time as bioluminescence, which is induced in V. fischeri through an autoinduction process called quorum sensing. Here, we asked the following questions: (i) Are the quorum signals that induce symbiont bioluminescence also involved in triggering the constriction? (ii) Does improper signaling of constriction affect the normal maintenance of the symbiont population? We manipulated the presence of three factors, the two V. fischeri quorum signal synthases, AinS and LuxI, the transcriptional regulator LuxR, and light emission itself, and found that the major factor triggering and maintaining bottleneck constriction is an as yet unknown effector(s) regulated by LuxIR. Treating the animal with chemical inhibitors of actin polymerization reopened the bottlenecks, recapitulating the host's response to quorum-sensing defective symbionts, as well as suggesting that actin polymerization is the primary mechanism underlying constriction. Finally, we found that these host responses to the presence of symbionts changed as a function of tissue maturation. Taken together, this work broadens our concept of how quorum sensing can regulate host development, thereby allowing bacteria to maintain long-term tissue associations. IMPORTANCE Interbacterial signaling within a host-associated population can have profound effects on the behavior of the bacteria, for instance, in their production of virulence/colonization factors; in addition, such signaling can dictate the nature of the outcome for the host, in both pathogenic and beneficial associations. Using the monospecific squid-vibrio model of symbiosis, we examined how quorum-sensing regulation by the Vibrio fischeri population induces a biogeographic tissue phenotype that promotes the retention of this extracellular symbiont within the light organ of its host, Euprymna scolopes. Understanding the influence of bacterial symbionts on key sites of tissue architecture has implications for all horizontally transmitted symbioses, especially those that colonize an epithelial surface within the host.

Mechanisms of toxicity by and resistance to ferrous iron in anaerobic systems.

Iron is an essential element for nearly all life on Earth, primarily for its value as a redox active cofactor. Iron exists predominantly in two biologically relevant redox states: ferric iron, the oxidized state (Fe3+), and ferrous iron, the reduced state (Fe2+). Fe2+ is well known to facilitate electron transfer reactions that can lead to the generation of reactive oxygen species. Less is known about why iron is toxic to cells in the absence of oxygen, yet this phenomenon is critically important for our understanding of life on early Earth and in iron-rich ecosystems today. In this brief review, we will highlight our current understanding of anaerobic Fe2+ toxicity, focusing on molecular mechanistic studies in several model systems.

INO80 is required for oncogenic transcription and tumor growth in non-small cell lung cancer.

Epigenetic regulators are attractive targets for the development of new cancer therapies. Among them, the ATP-dependent chromatin remodeling complexes control the chromatin architecture and have important roles in gene regulation. They are often found to be mutated and de-regulated in cancers, but how they influence the cancer gene expression program during cancer initiation and progression is not fully understood. Here we show that the INO80 chromatin remodeling complex is required for oncogenic transcription and tumor growth in non-small-cell lung cancer (NSCLC). Ino80, the SWI/SNF ATPase in the complex, is highly expressed in NSCLC cells compared with normal lung epithelia cells. Further, its expression, as well as that of another subunit Ino80B, negatively correlates with disease prognosis in lung cancer patients. Functionally, INO80 silencing inhibits NSCLC cell proliferation and anchorage-independent growth in vitro and tumor formation in mouse xenografts. It occupies enhancer regions near lung cancer-associated genes, and its occupancy correlates with increased genome accessibility and enhanced expression of downstream genes. Together, our study defines a critical role of INO80 in promoting oncogenic transcription and NSCLC tumorigenesis, and reveals a potential treatment strategy for inhibiting the cancer transcription network by targeting the INO80 chromatin remodeling complex.

Photopolymerized sol-gel monoliths for capillary electrochromatography.

A solution of methacryloxypropyltrimethoxysilane in the presence of an acid catalyst, water, toluene, and a photoinitiator was irradiated at 365 nm for 5 min in a 75-microm i.d. capillary to prepare a porous monolithic sol-gel column by a one-step, in situ, process. The photopolymerized sol-gel (PSG) column shows reversed-phase behavior. Using this column, a variety of low-molecular-weight neutral compounds, including polycyclic aromatic hydrocarbons, alkyl benzenes, alkyl phenyl ketones, and steroids are separated from mixtures. Various different operational parameters, such as buffer composition, field strength, and column temperature, were varied to assess their influence on column performance. Use of PSG as a stationary phase for a pressure-driven separation is also demonstrated.

Photopolymerized sol-gel frits for packed columns in capillary electrochromatography.

Porous sol-gel frits are fabricated in a capillary column by filling it with a solution of 3-(trimethoxysilyl)propyl methacrylate, hydrochloric acid, water, toluene (porogen), and a photoinitiator (Irgacure 1800) and exposing it to UV light at 365 nm for 5 min. The separation column (30 cm x 75 microm I.D.) contains between the inlet and outlet frits a 15-cm packed segment filled with 5-microm silica particles modified with the chiral compound (S)-N-3,5-dinitrobenzoyl-1-naphthylglycine. A detection window (1 mm long) is placed immediately after the outlet frit. To demonstrate the performance of this chiral separation column, mixtures of 16 different amino acids (three of which are not naturally occurring) derivatized with the fluorogenic reagent 4-fluoro-7-nitro-2,1,3-benzoxadiazole were separated by capillary chromatography. The enantiomeric separation of the column results in a resolution ranging from 1.21 to 8.29, and a plate height ranging from 8.7 to 39 microm.

Receptor-operated osteoclast calcium sensing.

Osteoclasts "sense" elevated extracellular calcium, which leads to cytoskeletal changes that may be linked to phospholipase C (PLC) activation and the associated rise in intracellular calcium ([Ca(2+)](i)). Since PLC is linked to transient receptor potential channels (trp), we hypothesized that receptor activated calcium influx due to this channel type would be activated by osteoclasts sensing [Ca(2+)](e). We found that high [Ca(2+)](e) induced similar intracellular Ca(2+) rises in chicken osteoclasts with or without intracellular Ca(2+) store depletion by either TPEN or thapsigargin, thus defining store-insensitive Ca(2+) influx. This store-insensitive calcium sensing component was blocked by the PLC antagonist U73122. Also, the calcium channel inhibitor SKF 96365, a blocker of store-independent trp-like channels, was effective in inhibiting calcium sensing in the presence of thapsigargin. Thus, a store-independent component of calcium sensing was associated with ion channels linked to PLC. Since receptor activated transient receptor potential (trp) family cation channels open in a PLC-dependent and store-independent manner, we suggest that receptor operated channels are activated in osteoclasts stimulated by high extracellular Ca(2+).

Strategy for on-line preconcentration in chromatographic separations.

In chromatographic separations, the heights of peaks are proportional to the concentrations of sample components present in an injected mixture. In general, an increase in the peak height cannot be achieved by simply increasing the injection time or the sample plug length. An exception occurs if some form of on-line preconcentration is possible. We present a new strategy for achieving on-line preconcentration by the use of a porous chromatographic material that acts as a solid-phase extractor as well as a stationary-phase separator. We are able to realize significant on-line preconcentration using capillary columns filled with a photopolymerized sol-gel (PSG). More than 2-cm plugs of sample solution can be loaded into the capillary and concentrated using a running buffer that is the same as the injection buffer (to avoid solvent gradient effects). As a demonstration, mixtures of three different polycyclic aromatic hydrocarbons, eight different alkyl phenyl ketones, and five different peptides in solutions of aqueous acetonitrile have been injected onto the PSG column and separated by capillary electrochromatography. The preconcentration is marked in terms of peak heights, with up to 100-fold increase for the PAH mixture, 30-fold for the alkyl phenyl ketone mixture, and 20-fold for the peptide mixture. Preconcentration takes place because of the high mass-transfer rates possible in the highly porous structure, and the extent of preconcentration follows the retention factor k for a given analyte.

Intrinsic membrane properties underlying spontaneous tonic firing in neostriatal cholinergic interneurons.

Neostriatal cholinergic interneurons produce spontaneous tonic firing in the absence of synaptic input. Perforated patch recording and whole-cell recording combined with calcium imaging were used in vitro to identify the intrinsic membrane properties underlying endogenous excitability. Spontaneous firing was driven by the combined action of a sodium current and the hyperpolarization-activated cation current (I(h)), which together ensured that there was no zero current point in the subthreshold voltage range. Blockade of sodium channels or I(h) established a stable subthreshold resting membrane potential. A tetrodotoxin-sensitive region of negative slope conductance was observed between approximately -60 mV and threshold (approximately -50 mV) and the h-current was activated at all subthreshold voltages. Calcium imaging experiments revealed that there was minimal calcium influx at subthreshold membrane potentials but that action potentials produced elevations of calcium in both the soma and dendrites. Spike-triggered calcium entry shaped the falling phase of the action potential waveform and activated calcium-dependent potassium channels. Blockade of big-conductance channels caused spike broadening. Application of apamin, which blocks small-conductance channels, abolished the slow spike afterhyperpolarization (AHP) and caused a transition to burst firing. In the absence of synaptic input, a range of tonic firing patterns are observed, suggesting that the characteristic spike sequences described for tonically active cholinergic neurons (TANs) recorded in vivo are intrinsic in origin. The pivotal role of the AHP in regulating spike patterning indicates that burst firing of TANs in vivo could arise from direct or indirect modulation of the AHP without requiring phasic synaptic input.

A furin-like convertase mediates propeptide cleavage of BACE, the Alzheimer's beta -secretase.

The novel transmembrane aspartic protease BACE (for Beta-site APP Cleaving Enzyme) is the beta-secretase that cleaves amyloid precursor protein to initiate beta-amyloid formation. As such, BACE is a prime therapeutic target for the treatment of Alzheimer's disease. BACE, like other aspartic proteases, has a propeptide domain that is removed to form the mature enzyme. BACE propeptide cleavage occurs at the sequence RLPR downward arrowE, a potential furin recognition motif. Here, we explore the role of furin in BACE propeptide domain processing. BACE propeptide cleavage in cells does not appear to be autocatalytic, since an inactive D93A mutant of BACE is still cleaved appropriately. BACE and furin co-localize within the Golgi apparatus, and propeptide cleavage is inhibited by brefeldin A and monensin, drugs that disrupt trafficking through the Golgi. Treatment of cells with the calcium ionophore, leading to inhibition of calcium-dependent proteases including furin, or transfection with the alpha(1)-antitrypsin variant alpha(1)-PDX, a potent furin inhibitor, dramatically reduces cleavage of the BACE propeptide. Moreover, the BACE propeptide is not processed in the furin-deficient LoVo cell line; however, processing is restored upon furin transfection. Finally, in vitro digestion of recombinant soluble BACE with recombinant furin results in complete cleavage only at the established E46 site. Taken together, our results strongly suggest that furin, or a furin-like proprotein convertase, is responsible for cleaving the BACE propeptide domain to form the mature enzyme.

Characterization of Alzheimer's beta -secretase protein BACE. A pepsin family member with unusual properties.

The cerebral deposition of amyloid beta-peptide is an early and critical feature of Alzheimer's disease. Amyloid beta-peptide is released from the amyloid precursor protein by the sequential action of two proteases, beta-secretase and gamma-secretase, and these proteases are prime targets for therapeutic intervention. We have recently cloned a novel aspartic protease, BACE, with all the known properties of beta-secretase. Here we demonstrate that BACE is an N-glycosylated integral membrane protein that undergoes constitutive N-terminal processing in the Golgi apparatus. We have used a secreted Fc fusion-form of BACE (BACE-IgG) that contains the entire ectodomain for a detailed analysis of posttranslational modifications. This molecule starts at Glu(46) and contains four N-glycosylation sites (Asn(153), Asn(172), Asn(223), and Asn(354)). The six Cys residues in the ectodomain form three intramolecular disulfide linkages (Cys(216)-Cys(420), Cys(278)-Cys(443), and Cys(330)-Cys(380)). Despite the conservation of the active site residues and the 30-37% amino acid homology with known aspartic proteases, the disulfide motif is fundamentally different from that of other aspartic proteases. This difference may affect the substrate specificity of the enzyme. Taken together, both the presence of a transmembrane domain and the unusual disulfide bond structure lead us to conclude that BACE is an atypical pepsin family member.

A comparison of folding techniques in the chemical synthesis of the epidermal growth factor-like domain in neu differentiation factor alpha/beta.

The 52-residue alpha/beta chimera of the epidermal growth factor-like domain in neu differentiation factor (NDFealpha/beta) has been synthesized and folded to form a three disulfide bridge (Cys182-Cys196, Cys190-Cys210, Cys212-Cys221) containing peptide. We investigated two general strategies for the formation of the intramolecular disulfide bridges including, the single-step approach, which used fully deprotected and reduced peptide, and a sequential approach that relied on orthogonal cysteine protection in which specific pairs are excluded from the first oxidation step. Because there are 15 possible disulfide bridge arrangements in a peptide with six cysteines, the one-step approach may not always provide the desired disulfide pairing. Here, we compare the single-step approach with a systematic evaluation of the sequential approach. We employed the acetamidomethyl group to protect each pair of cysteines involved in disulfide bridges, i.e. Cys182 to Cys196, Cys190 to Cys210 and Cys212 to Cys221. This reduced the number of possible disulfide patterns from 15 to three in the first folding step. We compared the efficiencies of folding for each protected pair using RP-HPLC, mapped the disulfide connectivity of the predominant product and then formed the final disulfide from the partially folded intermediate via 12 oxidation. Only the peptide having the Cys182-Cys196 pair blocked with acetamidomethyl forms the desired disulfide isomer (Cys190-Cys210/Cys212-Cys221) as a single homogeneous product. By optimizing both approaches, as well as other steps in the synthesis, we can now rapidly provide large-scale syntheses of NDFealpha/beta and other novel EGF-like peptides.

Expression analysis of BACE2 in brain and peripheral tissues.

Beta-site amyloid precursor protein cleaving enzyme (BACE) is a novel transmembrane aspartic protease that possesses all the known characteristics of the beta-secretase involved in Alzheimer's disease (Vassar, R., Bennett, B. D., Babu-Khan, S., Kahn, S., Mendiaz, E. A., Denis, P., Teplow, D. B., Ross, S., Amarante, P., Loeloff, R., Luo, Y., Fisher, S., Fuller, J., Edenson, S., Lile, J., Jarosinski, M. A., Biere, A. L., Curran, E., Burgess, T., Louis, J. -C., Collins, F., Treanor, J., Rogers, G., and Citron, M. (1999) Science 286, 735-741). We have analyzed the sequence and expression pattern of a BACE homolog termed BACE2. BACE and BACE2 are unique among aspartic proteases in that they possess a carboxyl-terminal extension with a predicted transmembrane region and together they define a new family. Northern analysis reveals that BACE2 mRNA is expressed at low levels in most human peripheral tissues and at higher levels in colon, kidney, pancreas, placenta, prostate, stomach, and trachea. Human adult and fetal whole brain and most adult brain subregions express very low or undetectable levels of BACE2 mRNA. In addition, in situ hybridization of adult rat brain shows that BACE2 mRNA is expressed at very low levels in most brain regions. The very low or undetectable levels of BACE2 mRNA in the brain are not consistent with the expression pattern predicted for beta-secretase.

Beta-secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE.

Cerebral deposition of amyloid beta peptide (Abeta) is an early and critical feature of Alzheimer's disease. Abeta generation depends on proteolytic cleavage of the amyloid precursor protein (APP) by two unknown proteases: beta-secretase and gamma-secretase. These proteases are prime therapeutic targets. A transmembrane aspartic protease with all the known characteristics of beta-secretase was cloned and characterized. Overexpression of this protease, termed BACE (for beta-site APP-cleaving enzyme) increased the amount of beta-secretase cleavage products, and these were cleaved exactly and only at known beta-secretase positions. Antisense inhibition of endogenous BACE messenger RNA decreased the amount of beta-secretase cleavage products, and purified BACE protein cleaved APP-derived substrates with the same sequence specificity as beta-secretase. Finally, the expression pattern and subcellular localization of BACE were consistent with that expected for beta-secretase. Future development of BACE inhibitors may prove beneficial for the treatment of Alzheimer's disease.

Spontaneous activity of neostriatal cholinergic interneurons in vitro.

Neostriatal cholinergic interneurons fire irregularly but tonically in vivo. The summation of relatively few depolarizing potentials and their temporal sequence are thought to underlie spike triggering and the irregularity of action potential timing, respectively. In these experiments we used whole-cell, cell-attached, and extracellular recording techniques to investigate the role of spontaneous synaptic inputs in the generation and patterning of action potentials in cholinergic interneurons in vitro. Cholinergic cells were spontaneously active in vitro at 25 +/- 1 degrees C during whole-cell recording from 2 to 3 week postnatal slices and at 35 +/- 2 degrees C during cell-attached and extracellular recording from 3 to 4 week postnatal slices. A range of firing frequencies and patterns was observed including regular, irregular, and burst firing. Blockade of AMPA and NMDA receptors altered neither the firing rate nor the pattern, and accordingly, voltage-clamp data revealed a very low incidence of spontaneous EPSCs. GABAA receptor antagonists were also ineffective in altering the spiking frequency or pattern owing to minimal inhibitory input in vitro. Functional excitatory and inhibitory inputs to cholinergic cells were disclosed after application of 4-aminopyridine (100 microM), indicating that these synapses are present but not active in vitro. Blockade of D1 or D2 dopamine receptors or muscarinic receptors also failed to influence tonic activity in cholinergic cells. Together these data indicate that cholinergic interneurons are endogenously active and generate action potentials in the absence of any synaptic input. Interspike interval histograms and autocorrelograms generated from unit recordings of cholinergic cells in vitro were indistinguishable from those of tonically active neurons recorded in vivo. Irregular spiking is therefore embedded in the mechanism responsible for endogenous activity.

Synaptic regulation of action potential timing in neostriatal cholinergic interneurons.

Action potentials in neostriatal cholinergic interneurons recorded in vivo are triggered by summation of two or three discrete synaptic depolarizations (Wilson et al., 1990). The ability and precision with which EPSPs and IPSPs regulate action potential timing was therefore investigated in vitro. Cholinergic interneurons were identified on the basis of morphological and electrophysiological characteristics in neostriatal slices taken from 2- to 3-week-old postnatal rats recorded at 24-26 degreesC. During periods of induced regular firing, intrastriatal stimuli were used to evoke pharmacologically isolated monosynaptic AMPA receptor-mediated EPSPs or GABAA receptor-mediated IPSPs. EPSPs evoked during the interspike interval (ISI) produced a phase-dependent decrease in the ISI, whereas IPSPs produced a phase-independent prolongation of the ISI. Injection of brief depolarizing currents mimicked the action of EPSPs and revealed an alteration in the input resistance during the ISI. In contrast to IPSPs, the ability of brief hyperpolarizing current injections to delay spike generation was phase-dependent. After blockade of GABAergic and glutamatergic synaptic transmission, stimuli failed to produce a detectable conductance change but could still prolong the subsequent ISI primarily through a D1 dopamine receptor-mediated enhancement of the afterhyperpolarization (AHP). Hence, EPSPs are ideally suited to provide a precise regulation of spike timing in cholinergic cells, whereas IPSPs are more likely to influence the overall level of excitability. The D1-mediated modulation of the AHP may contribute to the prolonged ISI seen in tonically active neurons in vivo in monkeys trained to respond to a sensory cue.

Adrenergic modulation of GABAA receptor-mediated inhibition in rat sensorimotor cortex.

The effect of adrenoceptor activation on pharmacologically isolated monosynaptic inhibitory postsynaptic currents (IPSCs) detected in layer V pyramidal neurons was examined by using whole cell voltage-clamp in a slice preparation of rat sensorimotor cortex. Epinephrine (EPI; 10 muM) reversibly altered the amplitude of evoked IPSCs (eIPSCs) in slices from postnatal day 9-12 (P9-12) and P15-18 rats. The effects of EPI were heterogeneous in both age groups, and in individual cases an enhancement, a depression or no effect of eIPSCs was observed, although depression was observed more commonly in the younger age group. The effects of EPI on eIPSC amplitude were likely mediated through presynaptic mechanisms because they occurred in the absence of any alteration in the current produced by direct application of gamma-aminobutyric acid (GABA), or in input resistance. EPI always elicited an increase in the frequency of spontaneous IPSCs (sIPSCs) irrespective of whether or not it induced any change in the amplitude of eIPSCs in the same neuron. The increase in sIPSC frequency was blocked by phentolamine (10 muM) but not by propranolol (10 muM), supporting the conclusion that EPI-mediated effects on sIPSC frequency result from activation of alpha-adrenoceptors located on presynaptic inhibitory interneurons. In a subpopulation of neurons (3/9) from P15-18 rats, EPI increased both the amplitude and frequency of miniature IPSCs (mIPSCs) recorded in the presence of tetrodotoxin (TTX) and under conditions where postsynaptic EPI effects were blocked, suggesting activation of adrenoceptors on presynaptic terminals in these cells. Results of these experiments are consistent with an action of EPI at adrenoceptors located on presynaptic GABAergic interneurons. Adrenergic activation thus has multiple and complex influences on excitability in cortical circuits, some of which are a consequence of interactions that regulate the strength of GABAergic inhibition.

Cell-specific expression and regulation of a glucokinase gene locus transgene.

Transgenic mice containing one or more extra copies of the entire glucokinase (GK) gene locus were generated and characterized. The GK transgene, an 83-kilobase pair mouse genomic DNA fragment containing both promoter regions, was expressed and regulated in a cell-specific manner, and rescued GK null lethality when crossed into mice bearing a targeted mutation of the endogenous GK gene. Livers from the transgenic mice had elevated GK mRNA, protein, and activity levels, compared with controls, and the transgene was regulated in liver by dietary manipulations. The amount of GK immunoreactivity in hepatocyte nuclei, where GK binds to the GK regulatory protein, was also increased. Pancreatic islets displayed increased GK immunoreactivity and NAD(P)H responses to glucose, but only when isolated and cultured in 20 mM glucose, as a result of the hypoglycemic phenotype of these mice (Niswender, K. D., Shiota, M., Postic, C., Cherrington, A. D., and Magnuson, M. A. (1997) J. Biol. Chem. 272, 22604-22609). Together, these results indicate that the region of the gene from -55 to +28 kilobase pairs (relative to the liver GK transcription start site) contains all the regulatory sequences necessary for expression of both GK isoforms, thereby placing an upper limit on the size of the GK gene locus.

Adrenoceptor-mediated elevation of ambient GABA levels activates presynaptic GABA(B) receptors in rat sensorimotor cortex.

At inhibitory synapses in the mature neocortex and hippocampus in vitro, spontaneous action-potential-dependent and -independent release of gamma-aminobutyric acid (GABA) activates postsynaptic GABA(A) receptors but not pre- or postsynaptic GABA(B) receptors. Elevation of synaptic GABA levels with pharmacological agents or electrical stimulation can cause activation of GABA(B) receptors, but the physiological conditions under which such activation occurs need further elucidation. In rodent sensorimotor cortex, epinephrine produced a depression in the amplitude of evoked monosynaptic inhibitory postsynaptic currents (IPSCs) and a concomitant, adrenoceptor-mediated increase in the frequency of spontaneous IPSCs. Blockade of GABA(B) receptors prevented the depression of evoked IPSC amplitude by epinephrine but did not affect the increase in spontaneous IPSC frequency. These data show that adrenoceptor-mediated increases in spontaneous IPSCs can cause activation of presynaptic GABA(B) receptors and indirectly modulate impulse-related GABA release, presumably through elevation of synaptic GABA levels.