Extraneous bodily movements and irrelevant vocalizations by dyslexic and non-dyslexic boys during calculation tasks.

Thirty dyslexic boys, aged between 9 and 15 years, and 30 age-matched controls were tested on a series of sums involving division, subtraction and addition. During the testing a record was kept of any bodily movements or verbal utterances (vocalizations) irrelevant to the task in hand. It was found that the dyslexics produced many more extraneous bodily movements and many more irrelevant vocalizations than did the controls. Possible reasons for these findings are tentatively suggested.

Stimulation of cardiac glucose transport by inhibitors of oxidative phosphorylation.

Previously we showed that hypoxia in heart stimulates glucose transport via translocation of glucose transporters from intracellular membranes to the plasma membrane. We later showed that rotenone, an inhibitor of oxidative phosphorylation, also decreased intracellular transporters. Here, using another membrane fractionation technique, we show that rotenone increases plasma membrane transporters, and that another respiratory chain inhibitor, azide, acts similarly. Thus, they likely activate the same signaling pathway as hypoxia. Genistein, a tyrosine kinase inhibitor, inhibited insulin- and azide-stimulated 3-O-methylglucose transport similarly in cardiac myocytes. It also increased glucose transporters in the plasma membranes of perfused hearts even though it inhibited glucose uptake, suggesting effects on membrane trafficking. Another tyrosine kinase inhibitor, lavendustin A, and the cyclic nucleotide-dependent protein kinase inhibitors H-8 and H-7 had little effect on basal or azide-stimulated transport. Polymyxin B was a weak inhibitor of basal, insulin-stimulated, and azide-stimulated transport. A nitric oxide donor and a nitric oxide synthase inhibitor had no effect on basal and azide-stimulated transport. The results indicate that tyrosine kinases; protein kinases A, G, and C; and nitric oxide are not involved in the hypoxic activation of cardiac glucose transport.

Characterization of glucose transport activity reconstituted from heart and other tissues.

We examined several aspects of glucose transport reconstituted in liposomes, with emphasis on transporters of rat heart (mostly GLUT4) compared to those of human erythrocytes (GLUT1), and on effects of agents that modulate transport in intact cells. Several types of samples gave higher reconstituted activity using liposomes of egg lipids rather than soybean lipids. Diacylglycerol, proposed to activate transporters directly as part of the mechanism of insulin action, increased the intrinsic activity of heart transporters by only 25%, but increased the size of the reconstituted liposomes by 90%. The dipeptide Cbz-Gly-Phe-NH2 inhibited GLUT4 with a Ki of 0.2 mM, compared to 2.5 mM for GLUT1, which explains its preferential inhibition of insulin-stimulated glucose transport in adipocytes. Verapamil, which inhibits insulin- and hypoxia-stimulated glucose transport in muscle, had no effect on reconstituted transporters. Heart transporters had a higher Km for glucose uptake (13.4) than did GLUT1 (1.6 mM), in agreement with a recent study of GLUT1 and GLUT4 expressed in yeast and reconstituted in liposomes. Transporters reconstituted from heart and adipocytes were 40-70% inactivated by external trypsin, suggesting the presence of trypsin-sensitive sites on the cytoplasmic domain of GLUT4. NaCl and KCl both reduced reconstituted transport activity, but KCl had a much smaller effect on the size of the liposomes.

Pentobarbital sedation for patients in the pediatric intensive care unit.

We present our experience with pentobarbital for sedation during mechanical ventilation in six infants when fentanyl and midazolam failed. The patients ranged in age from 2 to 17 months and in weight from 3.0 to 11.4 kg. Before the switch to pentobarbital, the maximum doses of fentanyl ranged from 7 to 13 micrograms/kg/hr and the midazolam infusions, from 0.2 to 0.4 mg/kg/hr. Pentobarbital was administered as a bolus dose followed by a continuous infusion. The hourly infusion rates ranged from 1 to 4 mg/kg. Adequate sedation was achieved in all six patients. In the four patients who required neuromuscular blocking agents, their use was discontinued after pentobarbital was given. The antihypertensive agents (diazoxide and nitroprusside) required by the two patients receiving extracorporeal membrane oxygenation were also discontinued after pentobarbital administration. Although we continue to use fentanyl and benzodiazepines as first-line drugs for sedation, pentobarbital may be an effective alternative when these agents fail.

Translocation of two glucose transporters in heart: effects of rotenone, uncouplers, workload, palmitate, insulin and anoxia.

Our previous studies on the acute regulation of glucose transport in perfused rat hearts were extended to explore further the mechanism of regulation by anoxia; to test the effects of palmitate, a transport inhibitor; and to compare the translocation of two glucose transporter isoforms (GLUT1 and GLUT4). Following heart perfusions under various conditions, glucose transporters in intracellular membranes were quantitated by reconstitution of transport activity and by Western blotting. Rotenone stimulated glucose uptake and decreased the intracellular contents of glucose transporters. This indicates that it activates glucose transport via net outward translocation, similarly to anoxia. However, two uncouplers of oxidative phosphorylation produced little or no effect. Increased workload (which stimulates glucose transport) reduced the intracellular contents of transporters, while palmitate increased the contents, indicating that these factors cause net translocation from or to the intracellular pool, respectively. Relative changes in GLUT1 were similar to those in GLUT4 for most factors tested. A plot of changes in total intracellular transporter content vs. changes in glucose uptake was roughly linear, with a slope of -0.18. This indicates that translocation accounts for most of the changes in glucose transport, and the basal pool of intracellular transporters is five times as large as the plasma membrane pool.

Noninvasive monitoring of end-tidal CO2 via nasal cannulas in spontaneously breathing children during the perioperative period.

OBJECTIVE: To determine the correlation between end-tidal CO2 and PaCO2 values measured via nasal cannulas in spontaneously breathing children during the perioperative period. DESIGN: Prospective evaluation. SETTING: Pediatric intensive/intermediate care unit in a tertiary care referral center. PATIENTS: Thirty postoperative surgical and trauma patients aged < or = 18 yrs (average age 7.8 yrs [range 6 months to 16 yrs] and average weight 28.3 kg (range 8.5 to 69). MEASUREMENTS AND MAIN RESULTS: Spontaneously breathing, nonintubated patients with an arterial cannula in place were selected for study. End-tidal CO2 was sampled from nasal cannulas by a sidestream aspirator and was estimated by infrared spectroscopy. The difference between PaCO2 and end-tidal CO2 was compared using linear regression analysis. A total of 55 blood gas measurements were obtained on the 30 patients. The PaCO2 to end-tidal CO2 gradient was < or = 4 torr in 54 of the 55 samples. The mean PaCO2 was 39.5 +/- 3.3 torr (5.27 +/- 0.44 kPa) with a mean end-tidal CO2 value of 39.7 +/- 3.8 torr (5.29 +/- 0.51 kPa). Linear regression analysis of arterial vs. end-tidal CO2 yielded a slope of 0.992 and p = .0001. CONCLUSIONS: End-tidal CO2 measurement by infrared spectroscopy provided an accurate estimation of PaCO2 in this patient population. Its use may limit the need for invasive monitoring and/or repeated arterial blood gas analysis.

Complications of autotransfusion with salvaged blood.

As a means of limiting homologous transfusions, many centers are using autotransfusion devices during the postoperative period. Although their use may limit the risks associated with homologous blood administration, various adverse effects have been reported including sepsis, disseminated intravascular coagulation, and renal insufficiency. The authors present the case of a 9-year-old girl who developed acute cardiorespiratory dysfunction after reinfusion of salvaged blood. The use of autotransfusion devices and the probable mechanisms responsible for adverse effects are discussed.

Accelerated net efflux of 3-O-methylglucose from rat adipocytes: a reevaluation.

In a study of 3-O-methylglucose transport in insulin-stimulated rat adipocytes (catalyzed primarily by the GLUT4 isoform), it was reported that at 37 degrees C the Km and Vmax were 2.8-fold higher for net efflux than for equilibrium exchange (Vinten, J. (1984) Biochim. Biophys. Acta 772, 244-250). Because of its implications for the relative sizes of steps in the transport cycle, we reinvestigated this phenomenon. Accelerated net efflux was apparent when the extracellular methylglucose was diluted 26-fold but not when it was diluted 11-fold. When analyzed according to the one-site alternating conformation model, the data indicate about a 1.7-fold higher Vmax for efflux than for exchange, only about 40% of the difference reported previously. Together with other results in the literature, the accelerated net flux indicates that the conformational change of the loaded transporter from its outward-facing to its inward-facing form is likely the slowest step in the transport cycle, in contrast to the case for GLUT1. Experiments at 25 degrees C indicate a lower degree of accelerated net flux than at 37 degrees C. This is consistent with the above conformational change being the step with the lowest activation energy, as for GLUT1.

ATP does not regulate the reconstituted glucose transporter.

ATP has been reported to affect glucose transport in human erythrocytes and resealed erythrocyte ghosts [Jacquez, J. A. (1983) Biochim. Biophys. Acta 727, 367-378; Jensen, M. R., & Brahm, J. (1987) Biochim. Biophys. Acta 900, 282-290]. In more detailed studies, effects of micromolar levels of ATP on transport in ghosts and inside-out vesicles, and on the fluorescence of ghosts and the purified glucose transporter [Carruthers, A. (1986) Biochemistry 25, 3592-3602; Hebert, D. N., & Carruthers, A. (1986) J. Biol. Chem. 261, 10093-10099; Carruthers, A. (1986) J. Biol. Chem. 261, 11028-11037], have been interpreted as supporting a model in which ATP regulates the catalytic properties of the transporter. Both allosteric and covalent effects of ATP were proposed; among the allosteric effects was a 60% reduction in the Km for zero-trans uptake. In order to test whether allosteric ATP regulation of the transporter occurs, we reconstituted glucose transport activity into liposomes using erythrocyte membranes without detergent treatment. The effects of ATP, present either outside, inside, or both inside and outside the liposomes, on the transport activity were examined. Effects of ATP on trypsin-treated liposomes, which have only a single orientation of active transporters, were also tested. While the model predicts activation by ATP, only inhibition was observed. This was significant only at millimolar concentrations of ATP, in contrast to the previously reported effects at micromolar levels, and was primarily on the extracellular surface of the transporter. In addition, the ATP effects on reconstituted transport were nonspecific, with similar effects produced by tripolyphosphate.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of three proposed inhibitors of adipocyte glucose transport on the reconstituted transporter.

Three compounds which inhibit glucose transport in rat adipocytes have been proposed to act directly on the glucose transporter protein. We tested these proposals by examining the effects of the compounds on the stereospecific glucose uptake catalyzed by adipocyte membrane proteins after reconstitution into liposomes. Effects on the transport activity reconstituted from human erythrocyte membranes were also examined. Glucose 6-phosphate, which was suggested to inhibit the transporter noncompetitively (Foley, J.E. and Huecksteadt, T.P. (1984) Biochim. Biophys. Acta 805, 313-316), had no effect on either type of reconstituted transporter, even when present at 5 mM on both sides of the liposomal membranes. Thus, it is unlikely to act directly on the transporter. The metalloendoproteinase substrate dipeptide Cbz-Gly-Phe-NH2, which inhibited insulin-stimulated but not basal glucose uptake in adipocytes (Aiello, L.P., Wessling-Resnick, M. and Pilch, P.F. (1986) Biochemistry 25, 3944-3950), inhibited the reconstituted erythrocyte transporter noncompetitively with a Ki of 1.5-2 mM. The inhibition of the erythrocyte transporter was identical in liposomes of soybean and egg lipid. Transport reconstituted using adipocyte membrane fractions was also inhibited by the dipeptide, with the activity from basal microsomes more sensitive than that from insulin-stimulated plasma membranes. These results indicate that the dipeptide interacts directly with the transporter, and may be a potentially useful probe for changes in transporter structure accompanying insulin action. Phenylarsine oxide, which was suggested to act directly on the adipocyte transporter (Douen, A.G., and Jones, M.N. (1988) Biochim. Biophys. Acta 968, 109-118), produced only slight (about 10%) inhibition of the reconstituted adipocyte and erythrocyte transporters, even when present at 100-200 microM and after 30 min of pretreatment. These results suggest that the major actions of phenylarsine oxide observed in adipocytes are not direct effects on the transporter, but rather effects on the pathways by which insulin regulates glucose transport activity (Frost, S.C. and Lane, M.D. (1985) J. Biol. Chem. 260, 2646-2652).

Translocation of glucose transporters in response to anoxia in heart.

We tested whether translocation of glucose transporters between subcellular membrane fractions is involved in the stimulation of glucose transport by anoxia by perfusing rat hearts in the presence or absence of oxygen. The hearts were then fractionated by a modification of the procedures of Watanabe, et al. (Watanabe, T., Smith, M. M., Robinson, F. W., and Kono, T. (1984) J. Biol. Chem. 259, 13117-13122), who previously demonstrated translocation in response to insulin in heart, to give plasma membrane and high-speed pellet fractions. The contents of glucose transporters in the two fractions were determined by reconstitution of transport activity, D-glucose-reversible binding of cytochalasin B, and labeling with antibodies against the erythrocyte transporter. The heart transporter was also recognized by antibodies against the COOH-terminal peptide of the glucose transporter. All three types of assays revealed a decrease (20-30%) in the high-speed pellet fraction and an increase (20-70%) in the plasma membranes in response to anoxia. Treatment of hearts with insulin produced a similar extent of translocation and a similar stimulation (about 2-fold) of glucose uptake, indicating that translocation plays a role of similar importance in the stimulation of transport by both of these effectors.

Infinite-cis kinetics support the carrier model for erythrocyte glucose transport.

It has been claimed that the Km for infinite-cis uptake of glucose in human erythrocytes is so low that the carrier model for transport must be rejected. We redetermined this parameter for three experimental conditions and found instead that the Km values were in good agreement with the model. For each of a variety of cis glucose concentrations, cells were preequilibrated with various concentrations of glucose, and the apparent Km was determined as the intracellular concentration reducing the initial rate of net uptake by half. The dependence of the apparent Km values on the cis glucose was as predicted by the carrier model; the infinite-cis Km was determined from both this concentration dependence and the extrapolated value at infinite cis glucose. The resulting values were 15 mM for fresh blood at 0 degrees C, 39 mM for outdated blood at 0 degrees C, and 11 mM for outdated blood at 25 degrees C. Previous measurements of the Km at room temperature yielded values of 2-3 mM. These earlier studies used a time course procedure that indicated rapid changes in rates during the initial 10 s of uptake but did not directly measure such changes. We examined the uptake of 60 mM glucose at 20 degrees C into cells containing 0 and 5 mM glucose; rapid changes in rates were not observed in the first few seconds, and the time courses were more consistent with our higher Km values. Our new values, together with other initial rate measurements in the literature, support the adequacy of the carrier model to account for the kinetics of glucose transport in human erythrocytes.

Reconstitution of the glucose transporter from rat skeletal muscle.

As a step in the purification and characterization of the glucose transporter from rat skeletal muscle, we have reconstituted glucose transport activity in liposomes. Plasma membranes were prepared from skeletal muscle which display D-glucose reversible binding of cytochalasin B (10 pmol sites/mg protein; KD = 0.3 microM). Older rats gave a slightly lower specific activity and much lower yield of sites per g muscle than young rats. Glucose transport activity was reconstituted into liposomes by the freeze-thaw procedure using either plasma membranes directly or cholate-extracted membrane proteins; the latter gave a 50% higher specific activity. The reconstituted transport activity was stereospecific, saturable, and inhibited by cytochalasin B, phloretin, and mercuric chloride. The optimum cholate concentration for extraction and reconstitution of transport activity was about 1.5%, and the highest specific activity of reconstituted transport was seen only at low ratios of protein to lipid in the reconstitution. Chromatography on agarose lentil lectin and agarose ethanethiol doubled both the specific activity of reconstituted transport and the fraction of glucose uptake which was stereospecific. In all of these respects the results were similar to our results with the bovine heart transporter (T. J. Wheeler and M. A. Hauck, Biochim. Biophys. Acta 818, 171-182 (1985)). Our findings suggest that further purification procedures developed for the heart transporter may be applicable to the skeletal muscle transporter as well.

Kinetics of glucose transport in human erythrocytes: zero-trans efflux and infinite-trans efflux at 0 degree C.

The kinetic features of glucose transport in human erythrocytes have been the subject of many studies, but no model is consistent with both the kinetic observations and the characteristics of the purified transporter. In order to reevaluate some of the kinetic features, initial rate measurements were performed at 0 degree C. The following kinetic parameters were obtained for fresh blood: zero-trans efflux Km = 3.4 mM, Vmax = 5.5 mM/min; infinite-trans efflux Km = 8.7 mM, Vmax = 28 mM/min. For outdated blood, somewhat different parameters were obtained: zero-trans efflux Km = 2.7 mM, Vmax = 2.4 mM/min; infinite-trans efflux Km = 19 mM, Vmax = 23 mM/min. The Km values for fresh blood differ from the previously reported values of 16 mM and 3.4 mM for zero-trans and infinite-trans efflux, respectively (Baker, G.F. and Naftalin, R.J. (1979) Biochim. Biophys. Acta 550, 474-484). The use of 50 mM galactose rather than 100 mM glucose as the infinite-trans sugar produced no change in the infinite-trans efflux Km values but somewhat lower Vmax values. Simulations indicate that initial rates were closely approximated by the experimental conditions. The observed time courses of efflux are inconsistent with a model involving rate-limiting dissociation of glucose from hemoglobin (Naftalin, R.J., Smith, P.M. and Roselaar, S.E. (1985) Biochim. Biophys. Acta 820, 235-249). The results presented here support the adequacy of the carrier model to account for the kinetics.

Reconstitution of glucose transport activity from erythrocyte membranes without detergent and its use in studying effects of ATP depletion.

The direct reconstitution of unsolubilized membrane proteins by the freeze-thaw procedure avoids possible changes in properties produced by detergent solubilization and fractionation. Glucose transport activity was reconstituted using human erythrocyte membranes, with about 2/3 of the glucose uptake being stereo-specific. The highest specific activity occurred at low ratios of protein to lipid in the reconstitution, where most transport was due to liposomes containing single transporter molecules. Transporters were reconstituted with a scrambling of orientations, indicated by a 50% inactivation by added trypsin. Separation of unreconstituted protein doubled the specific activity. Similar results were obtained using the purified transporter (Wheeler, T.J. and Hinkle, P.C. (1981) J. Biol. Chem. 256, 8907-8914). The same ratio of net uptake to equilibrium exchange was observed for the two preparations. Their relative reconstituted transport activities and cytochalasin B binding activities were equal, indicating that the two were reconstituted with similar efficiencies. The decrease in glucose transport in erythrocytes produced by ATP depletion and the stimulation produced by resealing with ATP (Jacquez, J.A. (1983) Biochim. Biophys. Acta 727, 367-378) were confirmed. However, no difference was observed in reconstituted transport activity using ghosts resealed with or without ATP, indicating that ATP produces indirect effects rather than modifications of the transporter.