The vascular Ca2+-sensing receptor regulates blood vessel tone and blood pressure.

The extracellular calcium-sensing receptor CaSR is expressed in blood vessels where its role is not completely understood. In this study, we tested the hypothesis that the CaSR expressed in vascular smooth muscle cells (VSMC) is directly involved in regulation of blood pressure and blood vessel tone. Mice with targeted CaSR gene ablation from vascular smooth muscle cells (VSMC) were generated by breeding exon 7 LoxP-CaSR mice with animals in which Cre recombinase is driven by a SM22α promoter (SM22α-Cre). Wire myography performed on Cre-negative [wild-type (WT)] and Cre-positive (SM22α)CaSR(Δflox/Δflox) [knockout (KO)] mice showed an endothelium-independent reduction in aorta and mesenteric artery contractility of KO compared with WT mice in response to KCl and to phenylephrine. Increasing extracellular calcium ion (Ca(2+)) concentrations (1-5 mM) evoked contraction in WT but only relaxation in KO aortas. Accordingly, diastolic and mean arterial blood pressures of KO animals were significantly reduced compared with WT, as measured by both tail cuff and radiotelemetry. This hypotension was mostly pronounced during the animals' active phase and was not rescued by either nitric oxide-synthase inhibition with nitro-l-arginine methyl ester or by a high-salt-supplemented diet. KO animals also exhibited cardiac remodeling, bradycardia, and reduced spontaneous activity in isolated hearts and cardiomyocyte-like cells. Our findings demonstrate a role for CaSR in the cardiovascular system and suggest that physiologically relevant changes in extracellular Ca(2+) concentrations could contribute to setting blood vessel tone levels and heart rate by directly acting on the cardiovascular CaSR.

Molecular and epidemiological analysis of methicillin-resistant Staphylococcus aureus otorrhoea: hospital- or community-acquired?

OBJECTIVES: (1) To identify newly diagnosed cases of methicillin-resistant Staphylococcus aureus ear infection in our local population; (2) to determine the risk factors involved in these patients' clinical courses, and (3) to type the bacterial strains isolated and thus identify whether they were hospital- or community-acquired. DESIGN AND SETTING: Retrospective review of case notes, together with laboratory-based molecular studies in the departments of otolaryngology and medical microbiology in a university teaching hospital in Scotland, UK. SUBJECTS: Over a two-year period, 35 patients were identified with ear swabs positive for methicillin-resistant Staphylococcus aureus infection. These cases came from both hospital and community settings. MAIN OUTCOME MEASURES: (1) Identification of primary methicillin-resistant Staphylococcus aureus otorrhoea in patients with no previously documented colonisation; and (2) molecular typing of the strains isolated, using spa technology, to identify whether they were hospital- or community-acquired. RESULTS: Of the 35 positive patients, 27 were previously known carriers of methicillin-resistant Staphylococcus aureus. The eight patients with newly diagnosed methicillin-resistant Staphylococcus aureus otorrhoea presented initially in the community. All of these patients had had contact with hospital staff (as in-patients or out-patients) in the weeks preceding development of their ear infection. Using the spa technique for molecular typing, we identified hospital-acquired ('epidemic') methicillin-resistant Staphylococcus aureus type 15 in all eight patients' isolates. All were sensitive to topical gentamicin. CONCLUSIONS: In our cohort, hospital-acquired methicillin-resistant Staphylococcus aureus type 15 was the commonest cause of methicillin-resistant Staphylococcus aureus otorrhoea, despite the fact that these patients all first presented in the community. We believe that contact with hospital staff or health care workers is a risk factor for acquiring methicillin-resistant Staphylococcus aureus otorrhoea in the community.

Neuromechanical simulation of the locust jump.

The neural circuitry and biomechanics of kicking in locusts have been studied to understand their roles in the control of both kicking and jumping. It has been hypothesized that the same neural circuit and biomechanics governed both behaviors but this hypothesis was not testable with current technology. We built a neuromechanical model to test this and to gain a better understanding of the role of the semi-lunar process (SLP) in jump dynamics. The jumping and kicking behaviors of the model were tested by comparing them with a variety of published data, and were found to reproduce the results from live animals. This confirmed that the kick neural circuitry can produce the jump behavior. The SLP is a set of highly sclerotized bands of cuticle that can be bent to store energy for use during kicking and jumping. It has not been possible to directly test the effects of the SLP on jump performance because it is an integral part of the joint, and attempts to remove its influence prevent the locust from being able to jump. Simulations demonstrated that the SLP can significantly increase jump distance, power, total energy and duration of the jump impulse. In addition, the geometry of the joint enables the SLP force to assist leg flexion when the leg is flexed, and to assist extension once the leg has begun to extend.

The Bacillus subtilis DivIVA protein has a sporulation-specific proximity to Spo0J.

The Bacillus subtilis DivIVA protein controls the positioning of the division site and the relocation of the chromosome during sporulation. By performing coimmunoprecipitation experiments, we demonstrated that a myc-DivIVA protein is in proximity to FtsZ and MinD during vegetative growth and Spo0J during the first 120 min of sporulation.

Identification of a polar targeting determinant for Bacillus subtilis DivIVA.

The Bacillus subtilis protein DivIVA controls both the positioning of the vegetative cell division site and the polar attachment of the chromosome during sporulation. In vegetative growth DivIVA attracts the bipartite cell division inhibitor MinCD away from the cell centre and towards the cell pole. This process ensures the inactivation of old polar division sites and leaves the cell centre free for the assembly of a new cell division complex. During sporulation MinCD and DivIVA levels fall, but DivIVA remains at the cell poles and becomes involved in the migration of the chromosomes to the pole. In order to investigate polar targeting of DivIVA, we undertook a mutational analysis of the 164-amino-acid protein. These studies identified one mutant (divIVA(R18C)) that could not localize to the cell pole but which retained the ability to support both vegetative growth and 50% sporulation efficiency. Further analysis revealed that, in the absence of polar targeting, DivIVA(R18C) localized to the nucleoid during vegetative growth in a Spo0J/Soj-dependent manner and required Spo0J/Soj and MinD to orientate the chromosomes correctly during sporulation. We demonstrate that polar targeting of DivIVA(R18C) is not essential during vegetative growth because the mutant can recognize the cell division site and influences the localization of MinD. Similarly we show that DivIVA(R18C) can function during sporulation because it can support the Spo0J/Soj orientation of the chromosome. In addition, we establish that both residues 18 and 19 constitute a DivIVA polar targeting determinant.

Shil'nikov homoclinic chaos is intimately related to type-III intermittency in isolated rabbit arteries: role of nitric oxide.

We provide experimental evidence for the existence of Shil'nikov homoclinic chaos in the fluctuations in flow which can be observed in isolated perfused rabbit ear arteries, and establish a close association between homoclinicity and type-III Pomeau-Manneville intermittent behavior. The transition between the homoclinic scenario and type-III intermittency is clarified by a mathematical model of the arterial smooth muscle cell. Simulations of the effects of nitric oxide (NO) by the vascular endothelium on these patterns of behavior closely match experimental observations.

Parallel changes in agonistic and non-agonistic behaviors during dominance hierarchy formation in crayfish.

Agonistic and non-agonistic behaviors were studied before, during, and after the formation of social status in crayfish. Differences in the expression of a non-agonistic behavior by dominant and subordinate crayfish developed in parallel with the differences in agonistic behaviors that indicate the animals' social status. An increase in burrowing behavior marked the ascendancy of the dominant animal, while an immediate suppression of burrowing paralleled the inhibition of aggressive behavior in the new subordinate. The strikingly similar changes in both agonistic and non-agonistic behaviors following the decision on status suggest related underlying neural mechanisms.

Universal scaling properties of type-I intermittent chaos in isolated resistance arteries are unaffected by endogenous nitric oxide synthesis.

Spontaneous fluctuations in flow in isolated rabbit ear resistance arteries may exhibit almost-periodic behavior interrupted by chaotic bursts that can be classified as type-I Pomeau-Manneville intermittency. This conclusion was supported by the construction of parabolic return maps and identification of the characteristic probability distributions for the number of oscillations per laminar segment (n) associated with the type-I scenario. Pharmacological inhibition of nitric oxide (NO) synthesis by the vascular endothelium modulated the dynamics of the reinjection mechanism, and thus the generic shape of the probability distribution for n. Nevertheless, average laminar length was related to a derived bifurcation parameter epsilon according to power-law scaling of the form approximately epsilon(beta), where the estimated critical exponent beta was close to the theoretical value of -0.5 both in the presence and absence of NO synthesis.

Dual and opposing modulatory effects of serotonin on crayfish lateral giant escape command neurons.

Serotonin modulates afferent synaptic transmission to the lateral giant neurons of crayfish, which are command neurons for escape behavior. Low concentrations, or high concentrations reached gradually, are facilitatory, whereas high concentrations reached rapidly are inhibitory. The modulatory effects rapidly reverse after brief periods of application, whereas longer periods of application are followed by facilitation that persists for hours. These effects of serotonin can be reproduced by models that involve multiple interacting intracellular signaling systems that are each stimulated by serotonin. The dependence of the neuromodulatory effect on dose, rate, and duration of modulator application may be relevant to understanding the effects of natural neuromodulation on behavior and cognition and to the design of drug therapies.

Gap junction-dependent increases in smooth muscle cAMP underpin the EDHF phenomenon in rabbit arteries.

We have investigated the role of cAMP in nitric oxide (NO)- and prostanoid-independent vascular relaxations evoked by acetylcholine (ACh) in isolated arteries and perfused ear preparations from the rabbit. These EDHF-type responses are shown to be associated with elevated cAMP levels specifically in smooth muscle and are attenuated by blocking adenylyl cyclase or protein kinase A (PKA). Relaxations are amplified by 3-isobutyl-1-methylxanthine, which prevents cAMP hydrolysis, while remaining susceptible to inhibition by the combination of two K(Ca) channel blockers, apamin and charybdotoxin. Analogous endothelium- and cAMP-dependent relaxations were evoked by cyclopiazonic acid (CPA) which stimulates Ca(2+) influx via channels linked to the depletion of Ca(2+) stores. Responses to ACh and CPA were both inhibited by interrupting cell-to-cell coupling via gap junctions with 18alpha-glycyrrhetinic acid and a connexin-specific Gap 27 peptide. The findings suggest that EDHF-type responses are initiated by capacitative Ca(2+) influx into the endothelium and propagated by direct intercellular communication to effect relaxation via cAMP/PKA-dependent phosphorylation events in smooth muscle.

Gap junctional communication underpins EDHF-type relaxations evoked by ACh in the rat hepatic artery.

Synthetic peptides homologous to the Gap 26 and Gap 27 domains of the first and second extracellular loops of the major vascular connexins (Cx37, Cx40, and Cx43) have been used to investigate the role of gap junctions in endothelium-derived hyperpolarizing factor (EDHF)-type relaxations of the rat hepatic artery. These peptides were designated 37,40Gap 26, 43Gap 26, 37,43Gap 27, and 40Gap 27, according to connexin specificity. When administered at 600 microM, none of the peptides individually affected maximal EDHF-type relaxations to ACh. By contrast, at 300 microM each, paired peptide combinations targeting more than one connexin subtype attenuated relaxation by up to 50%, and responses were abolished by the triple peptide combination 43Gap 26 + 40Gap 27 + 37,43Gap 27. In parallel experiments with A7r5 cells expressing Cx40 and Cx43, neither 43Gap 26 nor 40Gap 27 affected intercellular diffusion of Lucifer yellow individually but, in combination, significantly attenuated dye transfer. The findings confirm that functional cell-cell coupling may depend on more than one connexin subtype and demonstrate that direct intercellular communication via gap junctions constructed from Cx37, Cx40, and Cx43 underpins EDHF-type responses in the rat hepatic artery.

Patterns of neural circuit activation and behavior during dominance hierarchy formation in freely behaving crayfish.

Creation of a dominance hierarchy within a population of animals typically involves a period of agonistic activity in which winning and losing decide relative positions in the hierarchy. Among crayfish, fighting between size-matched animals leads to an abrupt change of behavior as the new subordinate retreats and escapes from the attacks and approaches of the dominant (Issa et al., 1999). We used high-speed videography and electrical recordings of aquarium field potentials to monitor the release of aggressive and defensive behavior, including the activation of neural circuits for four different tail-flip behaviors. We found that the sequence of tail-flip circuit excitation traced the development of their dominance hierarchy. Offensive tail flipping, attacks, and approaches by both animals were followed by a sharp rise in the frequency of nongiant and medial giant escape tail flips and a fall in the frequency of offensive tail flips of the new subordinate. These changes suggest that sudden, coordinated changes in the excitability of a set of neural circuits in one animal produce the changes in behavior that mark its transition to subordinate status.

Promiscuous targeting of Bacillus subtilis cell division protein DivIVA to division sites in Escherichia coli and fission yeast.

The Bacillus subtilis divIVA gene encodes a coiled-coil protein that shows weak similarity to eukaryotic tropomyosins. The protein is targeted to the sites of cell division and mature cell poles where, in B.subtilis, it controls the site specificity of cell division. Although clear homologues of DivIVA are present only in Gram-positive bacteria, and its role in division site selection is not conserved in the Gram-negative bacterium, Escherichia coli, a DivIVA-green fluorescent protein (GFP) fusion was targeted accurately to division sites and retained at the cell pole in this organism. Remarkably, the same fusion protein was also targeted to nascent division sites and growth zones in the fission yeast Schizosaccharomyces pombe, mimicking the localization of the endogenous tropomyosin-like cell division protein Cdc8p, and F-actin. The results show that a targeting signal for division sites is conserved across the eukaryote-prokaryote divide.

Integration of non-linear cellular mechanisms regulating microvascular perfusion.

It is becoming increasingly evident that interactions between the different cell types present in the vessel wall and the physical forces that result from blood flow are highly complex. This short article will review evidence that irregular fluctuations in vascular resistance are generated by non-linearity in the control mechanisms intrinsic to the smooth muscle cell and can be classified as chaotic. Non-linear systems theory has provided insights into the mechanisms involved at the cellular level by allowing the identification of dominant control variables and the construction of one-dimensional iterative maps to model vascular dynamics. Experiments with novel peptide inhibitors of gap junctions have shown that the coordination of aggregate responses depends on direct intercellular communication. The sensitivity of chaotic trajectories to perturbation may nevertheless generate a high degree of variability in the response to pharmacological interventions and altered perfusion conditions.

Minimal model of arterial chaos generated by coupled intracellular and membrane Ca2+ oscillators.

We have developed a mathematical model of arterial vasomotion in which irregular rhythmic activity is generated by the nonlinear interaction of intracellular and membrane oscillators that depend on cyclic release of Ca2+ from internal stores and cyclic influx of extracellular Ca2+, respectively. Four key control variables were selected on the basis of the pharmacological characteristics of histamine-induced vasomotion in rabbit ear arteries: Ca2+ concentration in the cytosol, Ca2+ concentration in ryanodine-sensitive stores, cell membrane potential, and the open state probability of Ca2+-activated K+ channels. Although not represented by independent dynamic variables, the model also incorporates Na+/Ca2+ exchange, the Na+-K+-ATPase, Cl- fluxes, and Ca2+ efflux via the extrusion ATPase. Simulations reproduce a wide spectrum of experimental observations, including 1) the effects of interventions that modulate the functionality of Ca2+ stores and membrane ion channels, 2) paradoxes such as the apparently unpredictable dual action of Ca2+ antagonists and low extracellular Na+ concentration, which can abolish vasomotion or promote the appearance of large-amplitude oscillations, and 3) period-doubling, quasiperiodic, and intermittent routes to chaos. Nonlinearity is essential to explain these diverse patterns of experimental vascular response.

Fifty years of a command neuron: the neurobiology of escape behavior in the crayfish.

Fifty years ago C.A.G. Wiersma established that the giant axons of the crayfish nerve cord drive tail-flip escape responses. The circuitry that includes these giant neurons has now become one of the best-understood neural circuits in the animal kingdom. Although it controls a specialized behavior of a relatively simple animal, this circuitry has provided insights that are of general neurobiological interest concerning matters as diverse as the identity of the neural substrates involved in making behavioral decisions, the cellular bases of learning, subcellular neuronal computation, voltage-gated electrical synaptic transmission and modification of neuromodulator actions that result from social experience. This work illustrates the value of studying a circuit of moderate, but tractable, complexity and known behavioral function.

Autoinhibition of serotonin cells: an intrinsic regulatory mechanism sensitive to the pattern of usage of the cells.

After periods of high-frequency firing, the normal rhythmically active serotonin (5HT)-containing neurosecretory neurons of the lobster ventral nerve cord display a period of suppressed spike generation and reduced synaptic input that we refer to as "autoinhibition." The duration of this autoinhibition is directly related to the magnitude and duration of the current injection triggering the high-frequency firing. More interesting, however, is that the autoinhibition is inversely related to the initial firing frequency of these cells within their normal range of firing (0.5-3 Hz). This allows more active 5HT neurons to resume firing after shorter durations of inhibition than cells that initially fired at slower rates. Although superfused 5HT inhibits the spontaneous firing of these cells, the persistence of autoinhibition in saline with no added calcium, in cadmium-containing saline, and in lobsters depleted of serotonin suggests that intrinsic membrane properties account for the autoinhibition. A similar autoinhibition is seen in spontaneously active octopamine neurons but is absent from spontaneously active gamma-aminobutyric acid cells. Thus, this might be a characteristic feature of amine-containing neurosecretory neurons. The 5HT cells of vertebrate brain nuclei share similarities in firing frequencies, spike shapes, and inhibition by 5HT with the lobster cells that were the focus of this study. However, the mechanism suggested to underlie autoinhibition in vertebrate neurons is that 5HT released from activated or neighboring cells acts back on inhibitory autoreceptors that are found on the dendrites and cell bodies of these neurons.

Polar localization of the MinD protein of Bacillus subtilis and its role in selection of the mid-cell division site.

Cell division in rod-shaped bacteria is initiated by formation of a ring of the tubulin-like protein FtsZ at mid-cell. Division site selection is controlled by a conserved division inhibitor MinCD, which prevents aberrant division at the cell poles. The Bacillus subtilis DivIVA protein controls the topological specificity of MinCD action. Here we show that DivIVA is targeted to division sites late in their assembly, after some MinCD-sensitive step requiring FtsZ and other division proteins has been passed. DivIVA then recruits MinD to the division sites preventing another division from taking place near the newly formed cell poles. Sequestration of MinD to the poles also releases the next mid-cell sites for division. Remarkably, this mechanism of DivIVA action is completely different from that of the equivalent protein MinE of Escherichia coli, even though both systems operate via the same division inhibitor MinCD.

Neuronal coincidence detection by voltage-sensitive electrical synapses.

Coincidence detection is important for functions as diverse as Hebbian learning, binaural localization, and visual attention. We show here that extremely precise coincidence detection is a natural consequence of the normal function of rectifying electrical synapses. Such synapses open to bidirectional current flow when presynaptic cells depolarize relative to their postsynaptic targets and remain open until well after completion of presynaptic spikes. When multiple input neurons fire simultaneously, the synaptic currents sum effectively and produce a large excitatory postsynaptic potential. However, when some inputs are delayed relative to the rest, their contributions are reduced because the early excitatory postsynaptic potential retards the opening of additional voltage-sensitive synapses, and the late synaptic currents are shunted by already opened junctions. These mechanisms account for the ability of the lateral giant neurons of crayfish to sum synchronous inputs, but not inputs separated by only 100 microsec. This coincidence detection enables crayfish to produce reflex escape responses only to very abrupt mechanical stimuli. In light of recent evidence that electrical synapses are common in the mammalian central nervous system, the mechanisms of coincidence detection described here may be widely used in many systems.