Mechanisms of respiratory rhythm generation.

In mammals, a three-phasic respiratory rhythm is generated by a network of various types of neurons in the lower brainstem. The cellular mechanisms of rhythmogenesis involve cooperative interactions between synaptic processes and specific membrane properties. The network seems to be driven by extrinsic sources in mature animals, whereas in the immature network pacemaker neurons might be involved.     

The neuronal mechanisms of respiratory rhythm generation

New, improved in vivo and in vitro approaches have led to a better understanding of the mechanisms that generate respiratory rhythm, which depends on a complex interaction between network and intrinsic membrane properties. The pre-Bötzinger complex in the ventrolateral medulla is particularly important for respiratory rhythm generation. This complex can be studied in isolation, and it contains all the known types of respiratory neurons that are now amenable to detailed cellular and molecular analyses.     

Opioid-induced quantal slowing reveals dual networks for respiratory rhythm generation

Current consensus holds that a single medullary network generates respiratory rhythm in mammals. Pre-B?tzinger Complex inspiratory (I) neurons, isolated in transverse slices, and preinspiratory (pre-I) neurons, found only in more intact en bloc preparations and in vivo, are each proposed as necessary for rhythm generation. Opioids slow I, but not pre-I, neuronal burst periods. In slices, opioids gradually lengthened respiratory periods, whereas in more intact preparations, periods jumped nondeterministically to integer multiples of the control period (quantal slowing). These findings suggest that opioid-induced quantal slowing results from transmission failure of rhythmic drive from pre-I neurons to preB?tC I networks, depressed below threshold for spontaneous rhythmic activity. Thus, both I (in the slice), and pre-I neurons are sufficient for respiratory rhythmogenesis.     

Mechanism of the generation of various forms of respiratory rhythm

The respiratory muscles and neurons activity in the transitional process from rhythmic respiration to its cessation and reappearance of the usual rhythmic breathing after the apnea was registered in the acute experiments on the anesthetized cats and rabbits under the action of extra intrapulmonary oxygen pressure or intravenous injection of sodium cyanide. Different forms of disturbances of respiratory rhythm (apneusis, hasping, the combination of hasping with apneusis and respiratory movements of usual form - eupnea) observed in the critical states of the organism are considered to be the result of changes in the character of activity of the medulla oblongata respiratory neurons which occur at a definite stage of hypoxia. Hasping mechanism differs essentially from the generation of eupnea and apneusis.     

Neural network implementation of a three-phase model of respiratory rhythm generation

A mathematical model of the central neural mechanisms of respiratory rhythm generation is developed. This model assumes that the respiratory cycle consists of three phases: inspiration, post-inspiration, and expiration. Five respiratory neuronal groups are included: inspiratory, late-inspiratory, post-inspiratory, expiratory, and early-inspiratory neurons. Proposed interconnections among these groups are based substantially on previous physiological findings. The model produces a stable limit cycle and generally reproduces the features of the firing patterns of the 5 neuronal groups. When simulated feedback from pulmonary stretch receptors is made to excite late-inspiratory neurons and inhibit early-inspiratory neurons, the model quantitatively reproduces previous observations of the expiratory-prolonging effects of pulses and steps of vagal afferent activity presented in expiration. In addition the model reproduces expected respiratory cycle timing and amplitude responses to change of chemical drive both in the absence and in the presence of simulated stretch receptor feedback. These results demonstrate the feasibility of generating the respiratory rhythm with a simple neural network based on observed respiratory neuronal groups. Other neuronal groups not included in the model may be more important for shaping the waveforms than for generating the basic oscillation.     

Unraveling the mechanism of the farnesyltransferase enzyme

Farnesyltransferase enzyme (FTase) is currently one of the most fascinating targets in cancer research. Studies in other areas, namely in the fight against parasites and viruses, have also led to very promising results. However, in spite of the thrilling achievements in the development of farnesyltransferase inhibitors (FTIs) over the past few years, the farnesylation mechanism remains, to some degree, a mystery. This review tries to shed some light on this puzzling enzyme by analyzing seven key mechanistic dilemmas, based on recent studies that have dramatically changed the way this enzyme is currently perceived.     

Unraveling the catalytic mechanism of nitrile hydratases

To elucidate a detailed catalytic mechanism for nitrile hydratases (NHases), the pH and temperature dependence of the kinetic constants k(cat) and K(m) for the cobalt-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) were examined. PtNHase was found to exhibit a bell-shaped curve for plots of relative activity versus pH at pH 3.2-11 and was found to display maximal activity between pH 7.2 and 7.8. Fits of these data provided pK(E)(S1) and pK(E)(S2) values of 5.9 +/- 0.1 and 9.2 +/- 0.1 (k(cat)' = 130 +/- 1 s(-1)), respectively, and pK(E)(1) and pK(E)(2) values of 5.8 +/- 0.1 and 9.1 +/- 0.1 (k(cat)'/K(m)' = (6.5 +/- 0.1) x 10(3) s(-1) mm(-1)), respectively. Proton inventory studies indicated that two protons are transferred in the rate-limiting step of the reaction at pH 7.6. Because PtNHase is stable at 60 degrees C, an Arrhenius plot was constructed by plotting ln(k(cat)) versus 1/T, providing E(a) = 23.0 +/- 1.2 kJ/mol. The thermal stability of PtNHase also allowed DeltaH(0) ionization values to be determined, thus helping to identify the ionizing groups exhibiting the pK(E)(S1) and pK(E)(S2) values. Based on DeltaH(0)(ion) data, pK(E)(S1) is assigned to betaTyr(68), whereas pK(E)(S2) is assigned to betaArg(52), betaArg(157), or alphaSer(112) (NHases are alpha(2)beta(2)-heterotetramers). A combination of these data with those previously reported for NHases and synthetic model complexes, along with sequence comparisons of both iron- and cobalt-type NHases, allowed a novel catalytic mechanism for NHases to be proposed.     

Phase resetting and fixed-delay stimulation of a simple model of respiratory rhythm generation.

In a previous study (Lewis et al., 1990), the response of the respiratory rhythm to a perturbing stimulus was investigated using two different stimulus protocols: phase resetting and fixed-delay stimulation. The first protocol consists of measuring the effects of perturbing an oscillator at different phases of the cycle on the duration of the perturbed cycle. The resulting phase response curves (PRCs) can be used to characterize the properties of the oscillator (Winfree, 1980). A second protocol, fixed-delay stimulation, involves perturbing an oscillator at a fixed latency from the onset of the cycle, repeated every n-th cycle. If a single stimulus produces an effect that lasts longer than a single cycle, complicated responses can be expected from fixed-delay stimulation (Lewis et al., 1987). A simple three-phase model for respiratory rhythm generation based on a hypothesis by Richter and coworkers (1982, 1983, 1986) was investigated in the context of these experimental studies. Phase resetting and fixed-delay stimulation protocols were simulated in the model. PRCs of the model resemble those obtained experimentally: a phase-dependent prolongation or shortening of the inspiratory phase depending on the stimulus magnitude, and a slight prolongation of the expiratory phase. Stimuli delivered to the model repetitively during successive inspiratory periods at a fixed-delay produced various combinations of shortened and prolonged cycles, similar to those observed in the experiments. However, the marked increases in cycle duration observed in the experiments during, as well as after, stimulation were not evident in the model. These comparisons suggest that (1) PRCs may not be an adequate way to evaluate certain models of rhythmogenesis, and (2) to improve the present simplified formulation of the three-phase model of the respiratory oscillator, time-varying stimulus dependent effects should be incorporated.     

Developmental changes in the modulation of respiratory rhythm generation by extracellular K+ in the isolated bullfrog brainstem

This study tested the hypothesis that voltage-dependent, respiratory-related activity in vitro, inferred from changes in [K(+)](o), changes during development in the amphibian brainstem. Respiratory-related neural activity was recorded from cranial nerve roots in isolated brainstem-spinal cord preparations from 7 premetamorphic tadpoles and 10 adults. Changes in fictive gill/lung activity in tadpoles and buccal/lung activity in adults were examined during superfusion with artificial CSF (aCSF) with [K(+)](o) ranging from 1 to 12 mM (4 mM control). In tadpoles, both fictive gill burst frequency (f(gill)) and lung burst frequency (f(lung)) were significantly dependent upon [K(+)](o) (r(2) > 0.75; p < 0.001) from 1 to 10 mM K(+), and there was a strong correlation between f(gill) and f(lung) (r(2) = 0.65; p < 0.001). When [K(+)](o) was raised to 12 mM, there was a reversible abolition of fictive breathing. In adults, fictive buccal frequency (f(buccal)), was significantly dependent on [K(+)](o) (r(2) = 0.47; p < 0.001), but [K(+)](o) had no effect on f(lung) (p > 0.2), and there was no significant correlation between f(buccal) and f(lung). These data suggest that the neural networks driving gill and lung burst activity in tadpoles may be strongly voltage modulated. In adults, buccal activity, the proposed remnant of gill ventilation in adults, also appears to be voltage dependent, but is not correlated with lung burst activity. These results suggest that lung burst activity in amphibians may shift from a "voltage-dependent" state to a "voltage-independent" state during development. This is consistent with the hypothesis that the fundamental mechanisms generating respiratory rhythm in the amphibian brainstem change during development. We hypothesize that lung respiratory rhythm generation in amphibians undergoes a developmental change from a pacemaker to network-driven process.     

Unraveling the catalytic mechanism of lactoperoxidase and myeloperoxidase.

Although belonging to the widely investigated peroxidase superfamily, lactoperoxidase (LPO) and myeloperoxidase (MPO) share structural and functional features that make them peculiar with respect to other enzymes of the same group. A survey of the available literature on their catalytic intermediates enabled us to ask some questions that remained unanswered. These questions concern controversial features of the LPO and MPO catalytic cycle, such as the existence of Compound I and Compound II isomers and the identification of their spectroscopic properties. After addressing each of these questions, we formulated a hypothesis that describes an integrated vision of the catalytic mechanism of both enzymes. The main points are: (a) a re-evaluation of the role of superoxide as a reductant in the catalytic cycle; (b) the existence of Cpd I isomers; (c) reciprocal interactions between catalytic intermediates and (d) a mechanistic explanation for catalase activity in both enzymes.     

Unraveling the secretion mechanism of the curious nectaries in Gentianaceae

Unusual nectaries were anatomically described as being usual traits for Gentianaceae. They are small, avascularized, and formed by 3 to 5 rosette cells with labyrinthine walls around one central cell. Such as nectaries have been reported for stems, petals, and sepals of different species of the family, however, there is no information on the mechanisms involved with the synthesis and release of secretion. Thus, this work aimed to unravel the mechanism of secretion and exudation of nectar for these curious nectaries using Calolisianthus speciosus as a model. Samples were processed according to standard methods for light and electron microscopy. Leaf and sepal nectaries were described, as were those of the apex of petals where ants were observed patrolling a darkened region. The enzymatic method was used for the detection of sugars, proteins, and amino acids in leaf and sepal exudates. The nectaries of petals of C. speciosus are similar to those of its leaves, sepals, and stem, although their activities are asynchronous. Polysaccharides were detected on the labyrinthine walls of rosette cells and protein in the opposite region of the cytoplasm. Labyrinthine walls increase the contact surface between rosette cells and the central cell, allowing for the transfer of secretion. After accumulation, the secretion is released to the subcuticular space of the central cell through disruption of the cuticle. The secretion and exudation of nectar were elucidated and involve distinct organelles.