Non-invasive technique for examining the respiratory proprioceptor system in man

Our technique enables non-invasive experiments to be conducted on the proprioceptor part of respiratory control, while eliminating misleading responses due to interaction with the chemoreceptor system; interaction was prevented by stabilizing arterial P_O2 and P_CO2 with the aid of an optimal regulator based on a mini-computer which controlled the inspired gas mixture. The proprioceptor system in a human was disturbed by applying positive pressure pulses at the mouth, responses were derived from continuous air-flow measurement. The classical inflation inhibiting reflex and an effect akin to Head's paradoxical reflex were demonstrated.     

Fructose 2,6-bisphosphate and plant carbohydrate metabolism

The control of the fructose 2,6-bisphosphate (Fru2,6P₂) concentration and its possible role in controlling carbohydrate synthesis and degradation are discussed. This regulator metabolite is involved in the fine tuning of photosynthetic metabolism, and in controlling photosynthetic partitioning, and may also be involved in the response to hormones, wounding, and changing water relations. Study of the mechanisms controlling Fru2,6P₂ concentrations could reveal insights into how these responses are mediated. However, the detailed action of Fru2,6P₂ requires more attention, especially in respiratory metabolism where the background information about the compartmentation of metabolism between the plastid and cytosol is still inadequate, and the potential role of pyrophosphate has to be clarified.     

Mathematische Analyse des Respirationssystems

The respiratory system is described as a control system. The controller consists of the peripheral and central chemoreceptors, the respiratory centre in the medulla oblongata and the controlling signal “alveolar ventilation”. The controlled system comprise three compartments (lung, brain, tissue) connected by the circulating blood. The controlled values of the system are explicit the arterial O₂-pressure and the CO₂-pressure of the brain-compartment. Hypoxia, hyperoxia and hypercapnia are the disturbing signals, which are caused by changing concentrations in the inspired gas. In this research both dynamic and steady-state behavior are studied. The steady-state and transient data of the model generally approach the findings of the experiments. The analysis of the efficiency of the regulation states the quality of the control system. In the on-and off-transients the CO₂-fractions of the alveolar gas, and in the off-transient the alveolar ventilation deviate from the experimental results in hypercapnic disturbances. Reasons for these differences and others existing between simulation and experiment are discussed.

---

     

Banana Ripening: Implications of Changes in Internal Ethylene and CO(2) Concentrations, Pulp Fructose 2,6-Bisphosphate Concentration, and Activity of Some Glycolytic Enzymes

In ripening banana (Musa acuminata L. [AAA group, Cavandish subgroup] cv. Valery) fruit, the steady state concentration of the glycolytic regulator fructose 2,6-bisphosphate (Fru 2,6-P₂) underwent a transient increase 2 to 3 hours before the respiratory rise, but coincident with the increase in ethylene synthesis. Fru 2,6-P₂ concentration subsequently decreased, but increased again approximately one day after initiation of the respiratory climacteric. This second rise in Fru 2,6-P₂ continued as ripening proceeded, reaching approximately five times preclimacteric concentration. Pyrophosphate-dependent phosphofructokinase glycolytic activity exhibited a transitory rise during the early stages of the respiratory climacteric, then declined slightly with further ripening. Cytosolic fructose 1,6-bisphosphatase activity did not change appreciably during ripening. The activity of ATP-dependent phosphofructokinase increased approximately 1.6-fold concurrent with the respiratory rise. A balance in the simultaneous glycolytic and gluconeogenic carbon flow in ripening banana fruit appears to be maintained through changes in substrate levels, relative activities of glycolytic enzymes and steady state levels of Fru 2,6-P₂.     

Non-invasive tracking of peripheral ventilatory response to carbon dioxide

We propose a new method for quantifying the ventilatory sensitivity of the peripheral chemoreceptors to changes in CO₂ through analysis of the natural breath-to-breath variations in ventilation (y) and alveolar Pco₂ (x). This technique is truly non-invasive in that the need for administering CO₂-enriched mixtures is circumvented, and peripheral chemosensitivity is assessed while the respiratory control system operates in its normal eucapnic state, unperturbed by external interventions. The method is based on solution of the inverse problem relating the cross-correlation between alveolar Pco₂ and ventilation changes, and the autocorrelation of changes in alveolar Pco₂. Tests are performed using simulated data generated by a closed-loop respiratory control model. The impulse response of the controller and the convective delay between lungs and chemoreceptors are estimated from the data. Subsequently, the best exponential fit to the estimated impulse response yields values for the effective controller gain (G) and the associated time constant of the response. The estimates of G contain a small contribution from the control chemoreflex gain; however, the relation between changes in G and changes in peripheral gain remains unaltered. The effects of other details in the computation procedure, such as length of data sequence, maximum number of correlation lags and starting lag number, are also investigated.     

A model for control of breathing in mammals: coupling neural dynamics to peripheral gas exchange and transport

A new model for aspects of the control of respiration in mammals has been developed. The model integrates a reduced representation of the brainstem respiratory neural controller together with peripheral gas exchange and transport mechanisms. The neural controller consists of two components. One component represents the inspiratory oscillator in the pre-Bötzinger complex (pre-BötC) incorporating biophysical mechanisms for rhythm generation. The other component represents the ventral respiratory group (VRG), which is driven by the pre-BötC for generation of inspiratory (pre)motor output. The neural model was coupled to simplified models of the lungs incorporating oxygen and carbon dioxide transport. The simplified representation of the brainstem neural circuitry has regulation of both frequency and amplitude of respiration and is done in response to partial pressures of oxygen and carbon dioxide in the blood using proportional (P) and proportional plus integral (PI) controllers. We have studied the coupled system under open and closed loop control. We show that two breathing regimes can exist in the model. In one regime an increase in the inspiratory frequency is accompanied by an increase in amplitude. In the second regime an increase in frequency is accompanied by a decrease in amplitude. The dynamic response of the model to changes in the concentration of inspired O₂ or inspired CO₂ was compared qualitatively with experimental data reported in the physiological literature. We show that the dynamic response with a PI-controller fits the experimental data better but suggests that when high levels of CO₂ are inspired the respiratory system cannot reach steady state. Our model also predicts that there could be two possible mechanisms for apnea appearance when 100% O₂ is inspired following a period of 5% inspired O₂. This paper represents a novel attempt to link neural control and gas transport mechanisms, highlights important issues in amplitude and frequency control and sets the stage for more complete neurophysiological control models.     

A comparative meta-analysis of maximal aerobic metabolism of vertebrates: implications for respiratory and cardiovascular limits to gas exchange

Maximal aerobic metabolic rates (MMR) in vertebrates are supported by increased conductive and diffusive fluxes of O₂ from the environment to the mitochondria necessitating concomitant increases in CO₂ efflux. A question that has received much attention has been which step, respiratory or cardiovascular, provides the principal rate limitation to gas flux at MMR? Limitation analyses have principally focused on O₂ fluxes, though the excess capacity of the lung for O₂ ventilation and diffusion remains unexplained except as a safety factor. Analyses of MMR normally rely upon allometry and temperature to define these factors, but cannot account for much of the variation and often have narrow phylogenetic breadth. The unique aspect of our comparative approach was to use an interclass meta-analysis to examine cardio-respiratory variables during the increase from resting metabolic rate to MMR among vertebrates from fish to mammals, independent of allometry and phylogeny. Common patterns at MMR indicate universal principles governing O₂ and CO₂ transport in vertebrate cardiovascular and respiratory systems, despite the varied modes of activities (swimming, running, flying), different cardio-respiratory architecture, and vastly different rates of metabolism (endothermy vs. ectothermy). Our meta-analysis supports previous studies indicating a cardiovascular limit to maximal O₂ transport and also implicates a respiratory system limit to maximal CO₂ efflux, especially in ectotherms. Thus, natural selection would operate on the respiratory system to enhance maximal CO₂ excretion and the cardiovascular system to enhance maximal O₂ uptake. This provides a possible evolutionary explanation for the conundrum of why the respiratory system appears functionally over-designed from an O₂ perspective, a unique insight from previous work focused solely on O₂ fluxes. The results suggest a common gas transport blueprint, or Bauplan, in the vertebrate clade.     

Effect of butanedioic Acid mono (2,2-dimethylhydrazide) on the activity of membrane-bound succinate dehydrogenase

Mitochondria isolated from hypocotyls of five-day-old bean (Phaseolus vulgaris L. `Black Valentine') seedlings rapidly oxidized succinate, malate, and NADH. Oxidation rates, respiratory control, and ADP:O ratios obtained with saturating concentrations of all three substrates indicated that the mitochondria were tightly coupled. The mitochondrial preparation was then employed to investigate the respiration-inhibiting effects of butanedioic acid mono (2,2-dimethyl-hydrazide) (daminozide) a plant growth retardant having structural similarity to an endogenous respiratory substrate (succinate). Daminozide markedly inhibited the activity of membrane-bound succinate dehydrogenase. Inhibition was of the competitive type (apparent K_i, 20.2 millimolar) with respect to succinate. Although not excluding other hypotheses, the results support an active role for daminozide in the suppression of respiration as an important metabolic site of its action as a plant growth regulator.     

Effect of exercise-induced hyperventilation on airway resistance and cycling endurance

The purpose of the present study was to investigate the effect of exercise induced hyperventilation and hypocapnia on airway resistance (R _aw), and to try to answer the question whether a reduction of R _aw is a mechanism contributing to the increase of endurance time associated with a reduction of exercise induced hyperventilation as for example has been observed after respiratory training. Eight healthy volunteers of both sexes participated in the study. Cycling endurance tests (CET) at 223 (SD 47) W, i.e. at 74 (SD 5)% of the subject's peak exercise intensity, breathing endurance tests and body plethysmograph measurements of pre- and postexercise R _aw were carried out before and after a 4-week period of respiratory training. In one of the two CET before the respiratory training CO₂ was added to the inspired air to keep its end-tidal concentration at 5.4% to avoid hyperventilatory hypocapnia (CO₂-test); the other test was the control. The pre-exercise values of specific expiratory R _aw were 8.1 (SD 2.8), 6.8 (SD 2.6) and 8.0 (SD 2.1) cm H₂O · s and the postexercise values were 8.5 (SD 2.6), 7.4 (SD 1.9) and 8.0 (SD 2.7) cm H₂O · s for control CET, CO₂-CET and CET after respiratory training, respectively, all differences between these tests being nonsignificant. The respiratory training significantly increased the respiratory endurance time during breathing of 70% of maximal voluntary ventilation from 5.8 (SD 2.9) min to 26.7 (SD 12.5) min. Mean values of the cycling endurance time (t _cend) were 22.7 (SD 6.5) min in the control, 19.4 (SD 5.4) min in the CO₂-test and 18.4 (SD 6.0) min after respiratory training. Mean values of ventilation ( _E) during the last 3␣min of CET were 123 (SD 35.8) l · min⁻¹ in the control, 133.5 (SD 35.1) l · min⁻¹ in the CO₂-test and 130.9 (SD 29.1) l · min⁻¹ after respiratory training. In fact, six subjects ventilated more and cycled for a shorter time, whereas two subjects ventilated less and cycled for a longer time after the respiratory training than in the control CET. In general, the subjects cycled longer the lower the _E, if all three CET are compared. It is concluded that R _aw measured immediately after exercise is independent of exercise-induced hyperventilation and hypocapnia and is probably not involved in limiting t _cend, and that t _cend at a given exercise intensity is shorter when _E is higher, no matter whether the higher _E occurs before or after respiratory training or after CO₂ inhalation. Accepted: 11 September 1996     

Gill Morphometry in Growth Hormone Transgenic Atlantic Salmon

We show that many of the morphological features of the respiratory system of growth enhanced transgenic salmon are greater than those of similarly sized control salmon. Growth hormone transgenic Atlantic salmon, Salmo salar were the F₂ generation produced using eggs from a transgenic F₁ female and milt from a nontransgenic male. At the time the gill tissues were sampled, the transgenic salmon were growing 2.1 times more rapidly than the nontransgenic control salmon, and they had oxygen uptake rates that were about 1.6 times greater than control salmon. In the present study we show that the gill surface area available for respiratory exchange in the transgenic salmon is about 1.24 times that in control salmon which does not parallel the 1.6 elevation in oxygen uptake. The increase in gill exchange area was due largely to a relatively uniform increase in length of each gill filament.     

Aging-related changes in the sensitivity of the system of respiratory control and the intensity of free-radical processes in humans

Results of a comparative study of the sensitivity of the system of respiratory control to increases in the CO₂ concentration and the intensity of free-radical processes in young and elderly subjects are described. It is shown that normal (natural) aging is accompanied by a decrease in the sensitivity of the respiratory system to hypercapnic stimulation and a parallel significant decrease in the activity of catalase in the blood of examined subjects. Mechanisms responsible for the modifications of the sensitivity of the system of respiratory control to hypercapnia are discussed; these shifts can be at least partly related to changes in the intensity of production of free radicals observed in elderly subjects. Neirofiziologiya/Neurophysiology, Vol. 40, No. 1, pp. 53–57, January–February, 2008.     

THE EFFECT OF IONIZING RADIATION UPON MITOCHONDRIA OF THE CENTRAL NERVOUS SYSTEM

After irradiation of rats with a linear electron accelerator, the respiratory rate in rat brain mitochondria was studied in the presence of substrate + ADP and after the conversion of ADP → ATP. After 20,000 rads of irradiation to the head there was a transient diminution of mitochondrial respiratory control when glutamate was used as the substrate, but no changes were observed when succinate was the substrate. Irradiation with 10,000 rads had no effect upon respiratory control. The addition of NADH₂ to irradiated mitochondria had no effect upon mitochondrial respiration. Irradiation of the brain with 20,000 rads failed to produce mitochondrial peroxidation or swelling, even in the presence of FeNH₄(SO₄)₂ or ascorbate. The slight changes in respiratory control of brain mitochondria following irradiation is in marked contrast to the susceptibility of mitochondria from other organs. The comparative radioresistance of brain mitochondria may be the result of greatly diminished radiation-induced peroxidation of cerebral mitochondrial membranes.     

Conditional-Suicide Containment System for Bacteria Which Mineralize Aromatics

A model conditional-suicide system to control genetically engineered microorganisms able to degrade substituted benzoates is reported. The system is based on two elements. One element consists of a fusion between the promoter of the Pseudomonas putida TOL plasmid-encoded meta-cleavage pathway operon (P_m) and the lacI gene encoding Lac repressor plus xylS, coding for the positive regulator of P_m. The other element carries a fusion between the P_tac promoter and the gef gene, which encodes a killing function. In the presence of XylS effectors, LacI protein is synthesized, preventing the expression of the killing function. In the absence of effectors, expression of the P_tac::gef cassette is no longer prevented and a high rate of cell killing is observed. The substitution of XylS for XylSthr45, a mutant regulator with altered effector specificity and increased affinity for benzoates, allows the control of populations able to degrade a wider range of benzoates at micromolar substrate concentrations. Given the wide effector specificity of the key regulators, the wild-type and mutant XylS proteins, the system should allow the control of populations able to metabolize benzoate; methyl-, dimethyl-, chloro-, dichloro-, ethyl-, and methoxybenzoates; salicylate; and methyl- and chlorosalicylates. A small population of genetically engineered microorganisms became Gef resistant; however, the mechanism of such survival remains unknown.     

Superoxide anion production by the mitochondrial respiratory chain of hepatocytes of rats with experimental toxic hepatitis

The progression of toxic hepatitis is accompanied by the activation of oxidative processes in the liver associated with an enhancement of the mitochondrial respiratory chain activity and superoxide anion production (О₂^˙-). The purpose of this study was to examine our previously formulated assumption concerning the predominant contribution of the complex I to О₂^˙- production increase by the mitochondrial respiratory chain of hepatocytes in toxic hepatitis (Shiryaeva et al. Tsitologiia, 49, 125–132 2007). Toxic hepatitis was induced by a combined application of ССl₄ and ethanol. Respiratory chain function analysis was executed with submitochondrial particles (SP) in the presence of specific inhibitors. It was shown that the rate of О₂^˙- production by SP of animals with toxic hepatitis, when NADH was delivered, was 2.5-fold higher as compared with the control. The rates of О₂^˙- production by SP of rats with toxic hepatitis in the presence of NADH or NADH + rotenone were similar. The О₂^˙- production rate by control SP in the presence of NADH + rotenone corresponded to the О₂^˙- production rate by toxic hepatitis SP when only NADH was delivered. When NADH + myxothiazol were delivered to the incubation system, О₂^˙- production by toxic hepatitis SP was 72% higher than for the control. Conversely, in the presence of antimycin A, the production of О₂^˙- by toxic hepatitis SP was lower compared to the control. Collectively, the presented data indicate that the О₂^˙- production rate was enhanced by the complex I of the hepatocyte mitochondrial respiratory chain in experimental toxic hepatitis. Complex III contribution to the production of О₂^˙- was insignificant. We assume that the increase in О₂^˙- production by the respiratory chain may be considered not only as the mechanism of pathology progression, but also as a compensatory mechanism preserving the electron transport function of the mitochondrial respiratory chain when complex I functioning is blocked in part.     

Hydrogen peroxide-induced MAPK activation causes the increase of RCAN1 (DSCR1) protein expression

The Down syndrome critical region 1 (DSCR1) gene encodes a regulator of calcineurin 1 (RCAN1), which is overexpressed in the patients with Down syndrome. In this study, we found that the protein expression of RCAN1 was increased by the hydrogen peroxide (H₂O₂). The increase of RCAN1 expression by H₂O₂ was blocked by the treatment with anti-oxidants and inhibitors of mitogen-activated protein kinases (MAPKs), indicating that this increase was caused by the generation of reactive oxygen species and activation of MAPKs. In addition, we found that the phosphorylation of RCAN1 by H₂O₂ caused an increase of RCAN1 expression by increasing of the half-life of the protein. Our results provide the evidence that H₂O₂ acts as an important regulator in the control of RCAN1 protein expression through phosphorylation.     

Stomatin-Like Protein 2 Is Required for In Vivo Mitochondrial Respiratory Chain Supercomplex Formation and Optimal Cell Function

Stomatin-like protein 2 (SLP-2) is a mainly mitochondrial protein that is widely expressed and is highly conserved across evolution. We have previously shown that SLP-2 binds the mitochondrial lipid cardiolipin and interacts with prohibitin-1 and -2 to form specialized membrane microdomains in the mitochondrial inner membrane, which are associated with optimal mitochondrial respiration. To determine how SLP-2 functions, we performed bioenergetic analysis of primary T cells from T cell-selective Slp-2 knockout mice under conditions that forced energy production to come almost exclusively from oxidative phosphorylation. These cells had a phenotype characterized by increased uncoupled mitochondrial respiration and decreased mitochondrial membrane potential. Since formation of mitochondrial respiratory chain supercomplexes (RCS) may correlate with more efficient electron transfer during oxidative phosphorylation, we hypothesized that the defect in mitochondrial respiration in SLP-2-deficient T cells was due to deficient RCS formation. We found that in the absence of SLP-2, T cells had decreased levels and activities of complex I-III₂ and I-III₂-IV_1-3 RCS but no defects in assembly of individual respiratory complexes. Impaired RCS formation in SLP-2-deficient T cells correlated with significantly delayed T cell proliferation in response to activation under conditions of limiting glycolysis. Altogether, our findings identify SLP-2 as a key regulator of the formation of RCS in vivo and show that these supercomplexes are required for optimal cell function.