Yoshitsugu Yamada

Practical Range of Effective Dose for Cre Recombinase-Expressing Recombinant Adenovirus without Cell Toxicity in Mammalian Cells

The site‐specific recombinase Cre is valuable for regulation of gene expression not only in vitro but also in vivo. We previously reported that replication‐deficient recombinant adenovirus (rAd) expressing Cre can mediate efficient and strict regulation in 100% of cultured cells. Recently, the constitutive‐expression of Cre using retrovirus or lentivirus vector reportedly inhibited cell‐growth, but the effect of transient Cre expression have not yet been examined. Here we showed that an excess amount of Cre produced from Cre‐expressing rAd caused a deleterious effect in cells even when Cre was transiently expressed. We used three rAds carrying promoters with different activities: the SV40 early promoter (AxSVENCre), the SRα promoter (AxSRCre) and the CAG promoter (AxCANCre). Cell toxicity was clearly caused by Cre itself and was distinguishable from that caused by rAd virions when the cytopathic effects of these rAds were compared with that of a control virus lacking the Cre expression unit. Cre toxicity was strongly correlated with the expression level of Cre. Importantly, AxSRCre and AxCANCre gave a 60‐fold range of effective MOIs (“effective range”) sufficient for gene activation without causing cell toxicity from either the rAd particles or Cre itself, while AxSVENCre failed to give such a range because the expression level of Cre was too low. When Cre was tagged with a nuclear localization signal (NLS), not only its activity but also Cre toxicity was increased fourfold, and the effective range was unchanged. Therefore, AxSRNCre might be more useful to control cell toxicity from the rAd virions than AxSRCre. Cre‐induced cell toxicity can be avoided by pre‐examining the “effective range” using the purpose cell lines before starting experiments utilizing the experiment of Cre‐expressing rAd.

Spectral analyses of electroencephalography and heart rate variability during sleep in normal subjects.

We investigated the relationship between electroencephalogram (EEG) activity and autonomic nervous system function using spectral analyses of EEG and heart rate variability (HRV) in healthy subjects during sleep. Eleven subjects were enrolled in this study. From EEG, the spectral edge frequencies (SEFs including SEF50, SEF90 and SEF95) were calculated. From electrocardiogram (ECG), the spectral powers of low-frequency band (LF: 0.04-0.15 Hz), high-frequency band (HF: 0.15-0.4 Hz) and the ratio of LF to HF (LF/HF) were calculated. During sleep, each set of data was obtained as the average of a 5-min measurement. We found that SEFs and LF/HF or LF decreased simultaneously and periodically, suggesting simultaneous depression of EEG activity and relative sympathetic activity, and SEFs significantly correlated with LF/HF and LF in all subjects during sleep, but not with HF. The existence of a clear correlation of SEFs with LF or LF/HF may offer a simple approach to estimate the relationship between EEG activity and autonomic nervous system function during sleep.

Automation of expiratory trigger sensitivity in pressure support ventilation.

Patients under pressure support ventilation sometimes encounter patient-ventilator asynchrony in the transition from inspiration to expiration, that is, expiratory asynchrony. This problem is caused by the incompatibility of the fixed level of expiratory trigger sensitivity termination criteria (i.e., flow termination criteria) in the ICU ventilators to various patient conditions. The user-adjustable expiratory trigger sensitivity implemented in some newly released ventilators has been experienced to be difficult to use and unable to adapt ever-changing patient conditions without user intervention, although it provides more flexibility. This article elucidates the rationale for automatic control of the expiratory trigger sensitivity and evaluates the automation system with a bench setup. The evaluation data suggest that good expiratory synchronies can be achieved through automatic adjustments of expiratory trigger sensitivity.

A1 adenosine receptor-mediated modulation of neuronal ATP-sensitive K channels in rat substantia nigra

ATP-sensitive K (K(ATP)) channels, widely expressed in cytoplasmic membranes of neurons, couple cell metabolism to excitability. They are considered to play important roles in controlling seizure activity during hypoxia and in neuroprotection against cell damage during hypoxia, ischemia and excitotoxicity. It is known that adenosine augments the opening of cardiac surface K(ATP) channels by reducing the sensitivity of these channels to ATP blockade. We investigated whether a similar modulation occurs in neuronal channels. Whole cell voltage-clamp recordings were made using rat midbrain slices to record the membrane current and conductance in principal neurons of the substantia nigra pars compacta (SNc). When the pipette solution contained 1 mM ATP, the membrane current at -60 mV and cellular conductance remained stable for at least 15 min. When slices were treated with (-)-N(6)-2-phenylisopropyl adenosine (R-PIA), a selective agonist for A(1) adenosine receptors, in the same condition, the outward current developed slowly to the amplitude of 109.9+/-26.6 pA, and conductance increased to 229+/-50% of the baseline. These changes were strongly inhibited by 200 microM tolbutamide, a K(ATP) channel blocker, suggesting that opening of K(ATP) channels mediated these changes. Pretreatment with 8-cyclopentyltheophylline (CPT), a selective A(1) adenosine receptor antagonist, abolished the outward current and conductance increases. Treatment of adenosine resulted in the similar changes sensitive to tolbutamide. These changes were abolished by CPT. These results suggest that activation of A(1) adenosine receptors promotes the opening of K(ATP) channels in principal neurons of the SNc by removing the blockade by ATP.

Effects of isoflurane and ketamine on ATP-sensitive K channels in rat substantia nigra.

Whole cell recordings were made using midbrain slices to examine the effects of two different anaesthetics on ATP-sensitive K (K(ATP)) channels in principle neurons of rat substantia nigra pars compacta. When neurons were dialyzed with an ATP-free pipette solution during perfusion with a glucose-free external solution, a hyperpolarization and an outward current developed slowly in a tolbutamide-inhibitable manner. The volatile anaesthetic 3% isoflurane slightly depolarised the neurons in the presence of ATP in the pipette solution and glucose in the external solution, but it did not affect the hyperpolarization or outward current in response to omission of ATP and glucose. Ketamine, an intravenous anaesthetic, did not change the membrane potential when ATP and glucose were included; however, it reversibly inhibited the hyperpolarization and outward current induced by intracellular ATP depletion in a dose-dependent manner. These effects of ketamine were not mimicked by AP-5, an NMDA receptor antagonist, or indatraline, an inhibitor of catecholamine uptake. These findings suggest that these anaesthetics have no stimulatory action on K(ATP) channels in these neurons when intracellular ATP is preserved and that ketamine but not isoflurane inhibits K(ATP) channels when the channels were activated by low intracellular ATP.Whole cell recordings were made using midbrain slices to examine the effects of two different anaesthetics on ATP-sensitive K (KATP) channels in principle neurons of rat substantia nigra pars compacta. When neurons were dialyzed with an ATP-free pipette solution during perfusion with a glucose-free external solution, a hyperpolarization and an outward current developed slowly in a tolbutamide-inhibitable manner. The volatile anaesthetic 3% isoflurane slightly depolarised the neurons in the presence of ATP in the pipette solution and glucose in the external solution, but it did not affect the hyperpolarization or outward current in response to omission of ATP and glucose. Ketamine, an intravenous anaesthetic, did not change the membrane potential when ATP and glucose were included; however, it reversibly inhibited the hyperpolarization and outward current induced by intracellular ATP depletion in a dose-dependent manner. These effects of ketamine were not mimicked by AP-5, an NMDA receptor antagonist, or indatraline, an inhibitor of catecholamine uptake. These findings suggest that these anaesthetics have no stimulatory action on KATP channels in these neurons when intracellular ATP is preserved and that ketamine but not isoflurane inhibits KATP channels when the channels were activated by low intracellular ATP.

PKC-independent inhibition of neuronal nicotinic acetylcholine receptors by diacylglycerol

Diacylglycerol modulates cell functions primarily through activation of protein kinase C (PKC). In a previous study, however, we found that a diacylglycerol analogue, 1-oleoyl-2-acetylglycerol (OAG), accelerated desensitization of neuronal nicotinic acetylcholine receptors (nAchRs) independently of PKC activation in PC12 cells. In the present study, we investigated whether other analogues and endogenous diacylglycerol exert similar effects on neuronal nAchRs and characterized the modulation by diacylglycerol. We measured the nicotine-induced whole-cell current in the absence and presence of diacylglycerol analogues in PC12 cells. We also investigated the effects of a blockade of metabolic pathways of diacylglycerol by inhibiting diacylglycerol lipase and kinase. We found that all four diacylglycerol analogues studied promoted desensitization and depressed the nondesensitized component of the nicotine-induced current. These effects seemed independent of PKC activation because they were not antagonized by the PKC inhibitors staurosporine or bisindolylmaleimide I; one analogue that lacks the PKC-stimulating action was also effective. The effects of diacylglycerol analogues were not antagonized by high doses of nicotine and were independent of the membrane potential. Similar modulatory effects were observed by treatment with RHC80267, a blocker of diacylglycerol lipase, and R59949, an inhibitor of diacylglycerol kinase, in the presence of staurosporine. These results suggest that diacylglycerol, both exogenously applied and endogenously produced, modulates neuronal nAchRs independently of PKC activation in PC12 cells; further, these effects seemed consistent with a noncompetitive and voltage-independent block. They raised the possibility that PKC-independent inhibition of neuronal nAchRs by diacylglycerol may be a novel modulatory process.

Neural mechanism of propofol anesthesia in severe depression: a positron emission tomographic study.

Background The precise neural mechanisms of propofol anesthesia in humans are still unknown. The authors examined the acute effects of propofol on regional cerebral blood flow (rCBF) using positron emission tomography in patients with severe depression. Methods In six severely depressed patients (mean age, 55.0 yr) scheduled for electroconvulsive therapy, anesthetic levels were monitored by electroencephalography, and rCBF was serially quantified in the awake, sedated, and anesthetized states. The authors used high-resolution positron emission tomography with 15O-labeled water and statistical parametric mapping 99 for imaging and analysis of the data. Results Global cerebral blood flow showed sharp decreases from the awake level during the administration of propofol, decreasing 26.8% in the sedated state and 54.4% in the anesthetized state. Moreover, a dose effect was seen in both parietal cortices and the left lateral prefrontal region with larger regions of relative decrease in rCBF at higher propofol doses. At the higher dose, the values of rCBF in the pulvinar nucleus of the thalamus, the pontine tegmentum, and the cerebellar cortex were also affected. Meanwhile, there were few changes of relative rCBF in the basal frontal lobes during both sedated and anesthetized states. Conclusions As in earlier studies using normal subjects, pronounced suppression in rCBF in the brain stem reticular formation, the thalamus, and the parietal association cortex occurred even in severely depressed patients. However, previously reported decreases in rCBF in the basal frontal lobe were absent in depressed patients.

Effects of barbiturates on ATP-sensitive K channels in rat substantia nigra

ATP-sensitive K channels are widely expressed in cytoplasmic membranes of neurons, and they couple cell metabolism to excitability. They are thought to be involved in neuroprotection against cell damage during hypoxia, ischemia and excitotoxicity by hyperpolarizing neurons and reducing excitability. Although barbiturates are often used in patients with brain ischemia, the effects of these agents on neuronal ATP-sensitive K channels have not been clarified. We studied the effects of thiopental and pentobarbital on surface ATP-sensitive K channels in principal neurons of rat substantia nigra pars compacta. Whole cell voltage- and current-clamp recordings were made using rat midbrain slices. ATP-sensitive K channels were activated by intracellular dialysis with an ATP-free pipette solution during perfusion with a glucose-free solution. When the pipette solution contained 4mM ATP and the perfusing solution contained 25 mM glucose, the membrane current at -60 mV remained stable. When intracellular ATP was depleted, hyperpolarization and an outward current developed slowly. Although thiopental did not affect the membrane current in the presence of ATP and glucose, it reversibly inhibited the hyperpolarization and outward current induced by intracellular ATP depletion at 100 and 300 microM. Thiopental reduced the ATP depletion-induced outward current by 4.7%, 36.7% and 87% at 30, 100 and 300 microM, respectively. The high dose of pentobarbital also exhibited similar effects on ATP-sensitive K channels. These results suggest that barbiturates at high concentrations but not at clinically relevant concentrations inhibit ATP-sensitive K channels activated by intracellular ATP depletion in rat substantia nigra.

An adult with ARDS managed with high-frequency oscillatory ventilation and prone position

tion. The preoperative chemotherapy had been started 11 days before the operation and had finished 9 days before the operation. On the first postoperative day, a chest radiograph revealed bilateral infiltrates, and he was diagnosed as having acute lung injury (ALI), or ARDS, according to the definition of the American-European consensus conference on ARDS [7]. On the second postoperative day, marked neutropenia was observed (neutrophils, 200/μl; leukocytes, 1800/μl), and granulocyte colonystimulating factor (G-CSF; 75μg/day) was administered subcutaneously for 2 days. On the third postoperative day, oxygenation and hemodynamics deteriorated after the second administration of G-CSF. One hour before the second administration of G-CSF, arterial oxygen tension (PaO2) was 73.3mmHg and arterial carbon dioxide tension (PaCO2) was 39.6 mmHg with pressure support ventilation; fractional inspired oxygen (FIO2) was 0.7, positive end-expiratory pressure (PEEP) was 7cmH2O, and pressure support, 18cmH2O (PaO2/FIO2 (P/F) ratio, 104.7). Two hours after the second G-CSF, PaO2 was 47.6mmHg and PaCO2 was 36.4mmHg with the same ventilator settings (P/F ratio, 68.0). Arterial blood pressure was 75/45mmHg, and central venous pressure was 15mmHg with dopamine, 5μg·kg 1·min 1 and dobutamine, 4 μg·kg 1·min 1. Although his hemodynamics improved with norepinephrine, 0.2μg·kg 1·min 1 and dopamine, 10μg·kg 1·min 1, urine output transiently decreased and continuous hemodiafiltration (CHDF) was started. Oxygenation did not improve and ventilation worsened in spite of the use of aggressive ventilator settings under conditions of sedation (continuous infusion of fentanyl and propofol) and muscle relaxation. PaO2 was 62.6 mmHg and PaCO2 was 52.4mmHg (pH 7.28) with pressure-controlled ventilation (PCV); FIO2, 1.0; PEEP, 10 cmH2O; distending pressure, 25 cmH2O; respiratory frequency, 30/min. This resulted in a tidal volume of 0.35 l, minute volume of 10.5 l/min, mean airway pressure (MAP) of 22.5 cmH2O,

Pre-anesthetic evaluation can play a crucial role in the determination of airway management in a child with oropharyngeal tumor.

We experienced a case of a huge hemangioma occupying the oropharyngeal space in an 11-year-old child. Although urgent surgical tracheostomy under local anesthesia was suggested initially, medical interview and findings of computerized tomography and fiberoptic laryngoscopy revealed that the airway of the patient was relatively stable when she was in the semi-left decubitus position. General anesthetic induction would have had potential risks of airway obstruction. Thus, after placing the patient in the semi-left decubutus position, we chose semi-awake induction to secure the airway. With a small dose of fentanyl, we accomplished orotracheal intubation. In this report, we discuss the importance of referring to an airway management algorithm when encountering a difficult airway.