Mechanically Mediated Microwave Frequency Conversion in the Quantum Regime.

We report the observation of efficient and low-noise frequency conversion between two microwave modes, mediated by the motion of a mechanical resonator subjected to radiation pressure. We achieve coherent conversion of more than 10^{12} photons/s with a 95% efficiency and a 14 kHz bandwidth. With less than 10^{-1} photons·s^{-1}·Hz^{-1} of added noise, this optomechanical frequency converter is suitable for quantum state transduction. We show the ability to operate this converter as a tunable beam splitter, with direct applications for photon routing and communication through complex quantum networks.

The impact of cerebral embolization during infant cardiac surgery on neurodevelopmental outcomes at intermediate follow-up.

Cerebral embolization during pediatric cardiac surgery may be an underappreciated source of subsequent neurodevelopmental impairment. Transcranial Doppler ultrasound is a neuromonitoring tool that can provide intraoperative surveillance for cerebral embolization. We hypothesized that increased cerebral embolic signals detected during infant cardiac surgery would be associated with worse neurodevelopmental outcomes at follow-up. A study group of 24 children who underwent infant cardiac surgery with transcranial Doppler detection of cerebral embolic signals returned at intermediate follow-up for standardized neurodevelopmental assessment. The children were evaluated using two neurocognitive tests and the parents completed two questionnaires regarding observed behavior. Statistical analysis assessed for correlation between the number of cerebral embolic signals at surgery and the results of the neurodevelopmental assessment. Of the 67 test parameters analyzed, five showed a significant association with the number of embolic signals, yet, all in the contrary direction of the clinical hypothesis, likely representing a Type I error. Thus, in this small cohort of patients, the number of cerebral embolic signals detected during infant cardiac surgery was not shown to be associated with worse neurodevelopmental outcomes at intermediate follow-up. A larger study is probably necessary to ascertain the potential influence of cerebral embolic signals on eventual neurologic outcomes in children. The clinical relevance of cerebral embolic signals during pediatric cardiac surgery remains undetermined and deserves further investigation.

Hemodynamic evaluation of arterial and venous cannulae performance in a simulated neonatal extracorporeal life support circuit.

OBJECTIVE: To construct an ideal extracorporeal life support (ECLS) circuit in terms of hemodynamic performance, each component of the circuit should be evaluated. Most cannulae manufacturers evaluate their products using water as the priming solution. We conducted this study to evaluate the different sizes of arterial and venous cannulae in a simulated neonatal ECLS circuit primed with human blood. METHODS: The simulated neonatal ECLS circuit was composed of a Capiox Baby RX05 oxygenator, a Rotaflow centrifugal pump and a heater & cooler unit. Three Medtronic Bio-Medicus arterial cannulae (8Fr, 10Fr, 12Fr) and three venous cannulae (10Fr, 12Fr, 14Fr) were tested in seven combinations (8A-10V, 8A-12V, 10A-10V, 10A-12V, 10A-14V, 12A-12V, 12A-14V). All the experiments were conducted using human blood at a hematocrit of 40% and at a constant temperature of 37°C. The "tip to tip" priming volume of the entire circuit was 135ml. The blood volume of the pseudo patient was 500ml. RESULTS: Flow rates increased linearly with increasing size in both venous and arterial cannulae at the same pump speeds. The increase in flow rate was greater when changing the arterial cannulae (next size larger) compared to changing the venous cannulae (next size larger). The pressure drops of the arterial cannula were correlated with the flow rates, regardless of the pseudo patient pressure and the venous cannula used simultaneously. CONCLUSIONS: The results show the difference in flow ranges and pressure drops of seven combinations of arterial and venous cannulae. It also suggests that the arterial cannula, not the venous cannula, has greater impact on the flow rate when a centrifugal pump is used in a neonatal ECLS circuit. The results of this study have been translated to further advancing the clinical practice in our institution.

Benefits of pulsatile flow in pediatric cardiopulmonary bypass procedures: from conception to conduction.

This review on the benefits of pulsatile flow includes not only experimental and clinical data, but also attempts to further illuminate the major factors as to why this debate has continued during the past 55 years. Every single component of the cardiopulmonary bypass (CPB) circuitry is equally important for generating adequate quality of pulsatility, not only the pump. Therefore, translational research is a necessity to select the best components for the circuit. Generation of pulsatile flow depends on an energy gradient; precise quantification in terms of hemodynamic energy levels is, therefore, a necessity, not an option. Comparisons between perfusion modes should be done after these basic steps have been taken. We have also included experimental and clinical data for direct comparisons between the perfusion modes. In addition, we included several suggestions for future clinical trials for other interested investigators.

Possible horizontal transfer of Drosophila genes by the mite Proctolaelaps regalis.

There is strong inferential evidence for recent horizontal gene transfer of the P (mobile) element to Drosophila melanogaster from a species of the Drosophila willistoni group. One potential vector of this transfer is a semiparasitic mite, Proctolaelaps regalis DeLeon, whose morphology, behavior, and co-occurrence with Drosophila are consistent with the properties necessary for such a vector. Southern blot hybridization, polymerase chain reaction (PCR) amplification, and DNA sequencing showed that samples of P. regalis associated with a P strain of D. melanogaster carried P element sequences. Similarly, Drosophila ribosomal DNA sequences were identified in P. regalis samples that had been associated with Drosophila cultures. These results have potentially important evolutionary implications, not only for understanding the mechanisms by which genes may be transferred between reproductively isolated species, but also for improved detection of some host-parasite and predator-prey relationships.

Interrelationships between astrocyte function, oxidative stress and antioxidant status within the central nervous system.

Astrocytes have, until recently, been thought of as the passive supporting elements of the central nervous system. However, recent developments suggest that these cells actually play a crucial and vital role in the overall physiology of the brain. Astrocytes selectively express a host of cell membrane and nuclear receptors that are responsive to various neuroactive compounds. In addition, the cell membrane has a number of important transporters for these compounds. Direct evidence for the selective co-expression of neurotransmitters, transporters on both neurons and astrocytes, provides additional evidence for metabolic compartmentation within the central nervous system. Oxidative stress as defined by the excessive production of free radicals can alter dramatically the function of the cell. The free radical nitric oxide has attracted a considerable amount of attention recently, due to its role as a physiological second messenger but also because of its neurotoxic potential when produced in excess. We provide, therefore, an in-depth discussion on how this free radical and its metabolites affect the intra and intercellular physiology of the astrocyte(s) and surrounding neurons. Finally, we look at the ways in which astrocytes can counteract the production of free radicals in general by using their antioxidant pathways. The glutathione antioxidant system will be the focus of attention, since astrocytes have an enormous capacity for, and efficiency built into this particular system.

Mitochondrial/cytosolic carbon transfer in the developing rat brain.

The rates of citrate and acetoacetate efflux from rat brain mitochondria (synaptic and free) utilizing different substrates (pyruvate, 3-hydroxybutyrate or acetoacetate) under different conditions have been studied as a function of development. In general there were no marked differences in the acetoacetate efflux rates between 'free' and 'synaptic' brain mitochondria whereas citrate efflux rates were usually higher in 'free' mitochondria. Developmental studies with brain mitochondria utilizing 3-hydroxybutyrate + malate showed a profile for acetoacetate efflux which was at a peak at weaning (21 days) and then decreased by 50% in the adult state. Similar studies measuring citrate efflux showed little change as the brain developed, but when pyruvate + malate were used as substrates the citrate efflux doubled during the period 5--20 days and was then maintained in the adult state. Phenylpyruvate was found to inhibit both acetoacetate and citrate efflux from 21-day-old and adult rat brain mitochondria when they used either 3-hydroxybutyrate or pyruvate as substrate. It is concluded that ketone bodies may be potentially as effective, if not better, than glucose in the brain of the suckling rat as precursors of cytosolic biosynthetic activities whereas in the adult rat brain, ketone bodies are relatively poor precursors of these activities.

Characterization of cDNAs encoding the rat testis-specific E1 alpha subunit of the pyruvate dehydrogenase complex: comparison of expression of the corresponding mRNA with that of the somatic E1 alpha subunit.

cDNA clones encoding the testis-specific form of the rat pyruvate dehydrogenase complex E1 alpha subunit have been isolated. Comparison of the predicted amino acid sequence with those of the somatic and testis-specific E1 alpha forms of man and mouse and the somatic E1 alpha form of rat indicates the change of a serine residue, believed to be phosphorylated in vivo by pyruvate dehydrogenase E1 alpha-specific kinase, to an alanine at position 233. The implications of this change are discussed. Northern blot analysis and RNase protection assays indicate that the expression of mRNA encoding testis-specific E1 alpha subunit is restricted to testis whereas mRNA for the somatic form is found in all tissues analyzed, albeit in very small amounts in testis.

Fluorocarbon perfusion of the isolated rat brain: measurement of tissue spaces, EEG and oxygen uptake.

Previously, we have used the isolated perfused rat brain (IPRB) to demonstrate authentic cerebral synthesis of the lipid mediator platelet-activating-factor (Kumar, R., Harvey, S.A.K., Kester, M., Hanahan, D.J. and Olson, M.S. (1988) Biochim. Biophys. Acta 963, 375-383). The present study demonstrates that this fluorocarbon perfusion technique maintains the integrity of the blood-brain barrier (BBB), as evidenced by the small volume (1.77-3.33%) accessible to [carboxyl-14C]inulin. 51-66% of the brain was accessible to 3H2O, except for the spinal cord which is poorly perfused (16% accessible to 3H2O). There is no effective perfusion of muscle tissue associated with the preparation (less than 6% accessible to 3H2O). Fast Fourier Transform analysis of digitized EEG data showed that in low frequency bands (less than 7.5 Hz) the IPRB had reduced electrical activity relative to the whole conscious animal. The GABA antagonist bicuculline, which has convulsant effects in vivo, causes a 3-4-fold increase in overall (root-mean-square) electrical activity, but decreases further the relative amplitude of low frequencies. With appropriate corrections, measurement of the oxygen consumption of the IPRB can be made without the necessity for venous cannulation. Oxygen consumption of the IPRB is flow-dependent. At a perfusion rate of 1.54 ml/min per g, unstimulated oxygen consumption of the IPRB is 2.07-2.23 mumol/min per g, or 67-72% of the consumption of the brain in vivo. Administration of bicuculline to the IPRB causes a 31% increase in lactate efflux, but only a 15% increase in oxygen uptake, suggesting that the preparation becomes functionally ischemic. Measurement of ATP/ADP levels in control and bicuculline-treated IPRBs confirms this. Other workers have used the IPRB as a model for the cerebral effects of pharmacological agents and of metabolic insult. The present study shows that under various experimental conditions oxygen uptake, analytical EEG measurements, and the integrity of the blood-brain barrier all can be monitored.

The molecular pathology of respiratory-chain dysfunction in human mitochondrial myopathies.

Some of the different molecular pathologies of respiratory-chain dysfunction in human mitochondrial myopathies will be reviewed in relation to the findings in 58 cases. Deletions of mitochondrial DNA were identified in 21 cases [36%]. There was some correlation between the sites of the deletion and the mitochondrial biochemistry in patients with defects of Complex I but not in cases with more extensive loss of respiratory chain activity. Complex I and Complex IV polypeptides were usually normal in deleted cases. Non-deleted cases, however, often showed specific subunit deficiencies which involved the products of both nuclear and mitochondrial genes. Immunoblots of respiratory-chain polypeptides in one case pointed to defective translocation of the Rieske precursor from the cytosol into the mitochondria. The pathogenic role of circulating autoantibodies to specific matrix proteins and the nature of the target antigens in two patients with mitochondrial encephalomyopathies and respiratory-chain dysfunction will also be discussed.

Nitric oxide, mitochondria and neurological disease.

Damage to the mitochondrial electron transport chain has been suggested to be an important factor in the pathogenesis of a range of neurological disorders, such as Parkinson's disease, Alzheimer's disease, multiple sclerosis, stroke and amyotrophic lateral sclerosis. There is also a growing body of evidence to implicate excessive or inappropriate generation of nitric oxide (NO) in these disorders. It is now well documented that NO and its toxic metabolite, peroxynitrite (ONOO-), can inhibit components of the mitochondrial respiratory chain leading, if damage is severe enough, to a cellular energy deficiency state. Within the brain, the susceptibility of different brain cell types to NO and ONOO- exposure may be dependent on factors such as the intracellular reduced glutathione (GSH) concentration and an ability to increase glycolytic flux in the face of mitochondrial damage. Thus neurones, in contrast to astrocytes, appear particularly vulnerable to the action of these molecules. Following cytokine exposure, astrocytes can increase NO generation, due to de novo synthesis of the inducible form of nitric oxide synthase (NOS). Whilst the NO/ONOO- so formed may not affect astrocyte survival, these molecules may diffuse out to cause mitochondrial damage, and possibly cell death, to other cells, such as neurones, in close proximity. Evidence is now available to support this scenario for neurological disorders, such as multiple sclerosis. In other conditions, such as ischaemia, increased availability of glutamate may lead to an activation of a calcium-dependent nitric oxide synthase associated with neurones. Such increased/inappropriate NO formation may contribute to energy depletion and neuronal cell death. The evidence available for NO/ONOO--mediated mitochondrial damage in various neurological disorders is considered and potential therapeutic strategies are proposed.

Mitochondrial DNA (mtDNA) diseases: correlation of genotype to phenotype.

This study examines the relationship of genotype to phenotype in 14 unselected patients who were found to harbour the A3243G transition in the mitochondrial transfer RNALeu(UUR) gene commonly associated with the syndrome of mitochondrial encephalopathy, lactic acidosis and strokes (MELAS). Only 6 of the 14 cases (43%) had seizures and recurrent strokes, the core clinical features of the MELAS phenotype. Of the remaining cases, four had an encephalomyopathy with deafness, ataxia and dementia, two had syndromes with progressive external ophthalmoplegia and two had limb weakness alone. Even within the MELAS subgroup, the majority of patients had one or more clinical manifestations considered to be atypical of the MELAS syndrome. They included developmental delay, ophthalmoparesis, pigmentary retinopathy and intestinal pseudo-obstruction. The proportion of mutant mitochondrial DNA (mtDNA) in muscle was generally higher in patients with recurrent strokes than in those without strokes, the highest levels being observed in MELAS cases with early onset disease. Studies of isolated muscle mitochondria identified a range of respiratory chain abnormalities mostly involving Complex I; immunoblots of Complex I in 3 of 10 cases showed selective loss of specific subunits encoded by nuclear genes. In the group as a whole, however, no clear correlations were observed between the severity or extent of the respiratory chain abnormality and clinical phenotype or the proportion of mutant mtDNA in biopsied skeletal muscle. These discrepancies suggest that, in patients harbouring the common MELAS3243 mutation, differences in heteroplasmy and the proportions of mutant mtDNA may not be the sole determinants of disease expression and that additional genetic mechanisms are involved in defining the range of clinical and biochemical phenotypes associated with this aberrant mitochondrial genome.

Studies on the regulation of insulin binding by liver plasma membranes from Zucker fatty rats.

The hyperinsulinemia of obese rodents has been associated with a reduced number of hepatic insulin receptors except in hepatocytes from fatty Zucker rats. We isolated liver plasma membranes from 10-11-wk-old lean and fatty Zucker rats, some of which were injected with streptozotocin 2--4 wk earlier. We have determined that although the number of hepatic insulin receptors is not reduced in young hyperinsulinemic fatty Zucker rats, the number of receptors can be increased when the hyperinsulinemia of the fatty rats is reduced by treatment with streptozotocin. In the fatty rats, this reduction in circulating insulin is accompanied by a reduction in plasma triglyceride concentration, consistent with a decreased stimulation of hepatic lipogenesis. Competitive binding curves for insulin were obtained with isolated liver plasma membranes and 125I-insulin. Analysis of these curves for affinity and number of receptors indicated that the number of insulin receptors was unchanged for the fatty control rats relative to the lean control rats but was increased in streptozotocin-treated animals. These data indicate that the regulation of hepatic insulin receptors is altered in the young fatty Zucker rat as characterized by a lack of downregulation of hepatic insulin receptors by hyperinsulinemia and an upregulation of hepatic insulin receptors at insulin concentrations higher than those found in lean rats. An altered state of hepatic insulin receptor regulation may be characteristic of developing obesity.

Co-culture of neurones with glutathione deficient astrocytes leads to increased neuronal susceptibility to nitric oxide and increased glutamate-cysteine ligase activity.

The antioxidant glutathione (GSH) plays an important role in protecting the mitochondrial electron transport chain (ETC) from damage by oxidative stress in astrocytes and neurones. Neurones co-cultured with astrocytes have greater GSH levels, compared to neurones cultured alone, leading to the hypothesis that astrocytes play a key role in brain GSH metabolism by supplying essential GSH precursors to neurones. A previous study has postulated that damage to the ETC following exposure to reactive nitrogen species (RNS) is less in co-cultured neurones, compared to neurones cultured alone, because of the greater GSH levels in the former cells. To investigate this further, primary culture rat neurones were co-cultured with either rat astrocytes activated with IFN-gamma and LPS to produce NO, or NO-generating astrocytes that had been depleted of intracellular GSH by 87% following incubation with the GSH synthesis inhibitor L-buthionine-S,R-sulfoximine (L-BSO). Neurones incubated with NO-generating astrocytes depleted of GSH were unable to elevate GSH levels, unlike neurones co-cultured with NO-generating astrocytes. Complexes II + III and IV of the neuronal ETC were significantly inhibited following exposure to NO-generating astrocytes depleted of GSH. No ETC damage was observed in neurones co-cultured with NO-generating astrocytes. Although neurones co-cultured with GSH depleted astrocytes did not increase cellular GSH levels, the activity of glutamate cysteine ligase (GCL), the rate-limiting enzyme of GSH synthesis, was increased by 218%, compared to neurones cultured with control astrocytes. This suggests that neuronal GCL activity could be modulated when GSH metabolism is inhibited in neighboring astrocytes.

Quantum Nondemolition Measurement of a Nonclassical State of a Massive Object.

By coupling a macroscopic mechanical oscillator to two microwave cavities, we simultaneously prepare and monitor a nonclassical steady state of mechanical motion. In each cavity, correlated radiation pressure forces induced by two coherent drives engineer the coupling between the quadratures of light and motion. We, first, demonstrate the ability to perform a continuous quantum nondemolition measurement of a single mechanical quadrature at a rate that exceeds the mechanical decoherence rate, while avoiding measurement backaction by more than 13 dB. Second, we apply this measurement technique to independently verify the preparation of a squeezed state in the mechanical oscillator, resolving quadrature fluctuations 20% below the quantum noise.

Age-related changes in insulin receptor regulation in liver membranes from Zucker fatty rats.

Hyperinsulinemic obese rodents have been shown to have reduced insulin binding to liver plasma membranes, a finding consistent with the model of down-regulation of insulin receptors by hyperinsulinemia. In contrast, hepatocytes from the hyperinsulinemic Zucker fatty rat were reported to have insulin binding equivalent to that of lean littermates. We have isolated liver plasma membranes from Zucker fatty rats 10 weeks, 5 months, and 1 yr of age and from fatty rats diet restricted until 5 months and 1 yr of age. We found no evidence for down-regulation of hepatic insulin receptors at 10 weeks of age. However, at 5 months and 1 yr of age there was marked down-regulation evident in isolated liver plasma membranes. Scatchard plots of the data are consistent with a decreased number of receptors rather than changes in receptor affinity accounting for the decreased binding. Diet restriction of the fatty rats resulted in lower plasma insulin levels and less marked down-regulation of hepatic insulin receptors. Our results are consistent with the hypothesis that in developing obesity there is a pattern of developing insulin resistance, first seen in muscle, later in fat, and finally in liver. Regulation of insulin receptors by hyperinsulinemia is probably less important than prior alterations in tissue metabolism by hyperinsulinemia in accounting for insulin resistance.

Activity of mitochondrial respiratory chain enzymes after transient focal ischemia in the rat.

Previous results demonstrated that after 2-hour middle cerebral artery occlusion (MCAO) in the rat, 1- to 2-hour recirculation temporarily restored the bioenergetic state and mitochondrial function, but secondary deterioration took place after 4 hours. The authors measured the activity of mitochondrial respiratory chain complexes, citrate synthase, and glutamate dehydrogenase as possible targets of secondary damage. Focal and penumbral tissues were sampled in the control condition, after 2 hours of MCAO, and after 1, 2, or 4 hours of postischemic recirculation; two groups were treated with alpha-phenyl-N-tert-butyl-nitrone (PBN). Complex IV activity transiently decreased after MCAO, but after recirculation all measured activities returned to control values.

beta-Amyloid fragment 25-35 selectively decreases complex IV activity in isolated mitochondria.

Defects in mitochondrial oxidative metabolism, in particular decreased activity of cytochrome c oxidase, have been demonstrated in Alzheimer's disease, and after the expression of the amyloid precursor protein (APP) in cultured cells, suggesting that mitochondria might be involved in beta-amyloid toxicity. Recent evidence suggests that the proteolysis of APP to generate beta-amyloid is at least in part intracellular, preceding the deposition of extracellular fibrils. We have therefore investigated the effect of incubation of isolated rat brain mitochondria with the beta-amyloid fragment 25-35 (100 microM) on the activities of the mitochondrial respiratory chain complexes I, II-III, IV (cytochrome c oxidase) and citrate synthase. The peptide caused a rapid, dose-dependent decrease in the activity of complex IV, white it had no effect on the activities on any of the other enzymes tested. The reverse sequence peptide (35-25) had no effect on any of the activities measured. We conclude that inhibition of mitochondrial complex IV might be a contributing factor to the pathogenesis of Alzheimer's disease.

Stimulation of glyceraldehyde-3-phosphate dehydrogenase by oxyhemoglobin.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key glycolytic enzyme regulated by many diverse mechanisms. In this study we present evidence that GAPDH activity is stimulated in the presence of oxyhemoglobin (2.3-fold, P < 0.005). No stimulation was seen by myoglobin, and only slight stimulation (1.2-fold, not significant) by methemoglobin was observed. Such stimulation may have physiological significance as 1,3-bis-phosphoglycerate, the product of GAPDH, isomerises to 2,3-bis-phosphoglycerate, an allosteric effector that decreases the oxygen affinity of hemoglobin, thus providing a feedback loop. The results suggest that when assaying GAPDH activity in biological samples, hemoglobin content should be taken into account.

Nitric oxide-mediated mitochondrial damage: a potential neuroprotective role for glutathione.

In this study we have investigated the mechanisms leading to mitochondrial damage in cultured neurons following sustained exposure to nitric oxide. Thus, the effects upon neuronal mitochondrial respiratory chain complex activity and reduced glutathione concentration following exposure to either the nitric oxide donor, S-nitroso-N-acetylpenicillamine, or to nitric oxide releasing astrocytes were assessed. Incubation with S-nitroso-N-acetylpenicillamine (1 mM) for 24 h decreased neuronal glutathione concentration by 57%, and this effect was accompanied by a marked decrease of complex I (43%), complex II-III (63%), and complex IV (41%) activities. Incubation of neurons with the glutathione synthesis inhibitor, L-buthionine-[S,R]-sulfoximine caused a major depletion of neuronal glutathione (93%), an effect that was accompanied by a marked loss of complex II-III (60%) and complex IV (41%) activities, although complex I activity was only mildly decreased (34%). In an attempt to approach a more physiological situation, we studied the effects upon glutathione status and mitochondrial respiratory chain activity of neurons incubated in coculture with nitric oxide releasing astrocytes. Astrocytes were activated by incubation with lipopolysaccharide/interferon-gamma for 18 h, thereby inducing nitric oxide synthase and, hence, a continuous release of nitric oxide. Coincubation for 24 h of activated astrocytes with neurons caused a limited loss of complex IV activity and had no effect on the activities of complexes I or II-III. However, neurons exposed to astrocytes had a 1.7-fold fold increase in glutathione concentration compared to neurons cultured alone. Under these coculture conditions, the neuronal ATP concentration was modestly reduced (14%). This loss of ATP was prevented by the nitric oxide synthase inhibitor, NG-monomethyl-L-arginine. These results suggest that the neuronal mitochondrial respiratory chain is damaged by sustained exposure to nitric oxide and that reduced glutathione may be an important defence against such damage.