Stress adaptation and low-frequency impedance of rat lungs

At transpulmonary pressures (Ptp) of 7-12 cmH2O, pressure-volume hysteresis of isolated cat lungs has been found to be 20-50% larger than predicted from their amount of stress adaptation (J. Hildebrandt, J. Appl. Physiol. 28: 365-372, 1970). This behavior is inconsistent with linear viscoelasticity and has been interpreted in terms of plastoelasticity. We have reinvestigated this phenomenon in isolated lungs from 12 Wistar rats by measuring 1) the changes in Ptp after 0.5-ml step volume changes (initial Ptp of 5 cmH2O) and 2) their response to sinusoidal pressure forcing from 0.01 to 0.67 Hz (2 cmH2O peak to peak, mean Ptp of 6 cmH2O). Stress adaptation curves were found to fit approximately Hildebrandt's logarithmic model [delta Ptp/delta V = A - B.log(t)] from 0.2 to 100 s, where delta V is the step volume change, A and B are coefficients, and t is time. A and B averaged 1.06 +/- 0.11 and 0.173 +/- 0.019 cmH2O/ml, respectively, with minor differences between stress relaxation and stress recovery curves. The response to sinusoidal forcing was characterized by the effective resistance (Re) and elastance (EL). Re decreased from 2.48 +/- 0.41 cmH2O.ml-1.s at 0.01 Hz to 0.18 +/- 0.03 cmH2O.ml-1.s at 0.5 Hz, and EL increased from 0.99 +/- 0.10 to 1.26 +/- 0.20 cmH2O/ml on the same frequency range. These data were analyzed with the frequency-domain version of the same model, complemented by a Newtonian resistance (R) to account for airway resistance: Re = R + B/ (9.2f) and EL = A + 0.25B + B . log 2 pi f, where f is the frequency.(ABSTRACT TRUNCATED AT 250 WORDS)     

Breath holding during intense exercise: arterial blood gases, pH, and lactate

Seven healthy endurance-trained [maximal O2 uptake (VO2max) = 57.1 +/- 4.1 ml.kg-1.min-1)] female volunteers (mean age 24.4 +/- 3.6 yr) served as subjects in an experiment measuring arterial blood gases, acid-base status, and lactate changes while breath holding (BH) during intense intermittent exercise. By the use of a counterbalance design, each subject repeated five intervals of a 15-s on:30-s off treadmill run at 125% VO2max while BH and while breathing freely (NBH). Arterial blood for pH, PO2, PCO2, O2 saturation (SO2) HCO3, and lactate was sampled from a radial arterial catheter at the end of each work and rest interval and throughout recovery, and the results were analyzed using repeated-measures analysis of variance. Significant reductions in pHa (delta mean = 0.07, P less than 0.01), arterial PO2 (delta mean = 24.2 Torr, P less than 0.01), and O2 saturation (delta mean = 4.6%, P less than 0.01) and elevations in arterial PCO2 (delta mean = 8.2 Torr, P less than 0.01) and arterial HCO3 (delta mean = 1.3 meq/l, P = 0.05) were found at the end of each exercise interval in the BH condition. All of the observed changes in arterial blood gases and acid-base status induced by BH were reversed during the rest intervals. During recovery, significantly (P less than 0.025) greater levels of arterial lactate were found in the BH condition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of inorganic phosphate on photosynthesis in bean leaves

It was shown that raising pod seedlings by the hydroponics method on KH2PO4 solutions at concentrations between 10(-7) and 10(-5) M leads to an increase in the rate of oxygen release (delta O2/delta t), with the chlorophyll content in leaves being unchanged. The values of the parameters FM/FT of slow fluorescence induction and B/A of photoinduced changes in ESR1 signals from pod leaves correlate with the delta O2/delta t value.     

Arterial blood gases and acid-base status of dogs during graded dynamic exercise

The objective of this study was to determine whether arterial PCO2 (PaCO2) decreases or remains unchanged from resting levels during mild to moderate steady-state exercise in the dog. To accomplish this, O2 consumption (VO2) arterial blood gases and acid-base status, arterial lactate concentration ([LA-]a), and rectal temperature (Tr) were measured in 27 chronically instrumented dogs at rest, during different levels of submaximal exercise, and during maximal exercise on a motor-driven treadmill. During mild exercise [35% of maximal O2 consumption (VO2 max)], PaCO2 decreased 5.3 +/- 0.4 Torr and resulted in a respiratory alkalosis (delta pHa = +0.029 +/- 0.005). Arterial PO2 (PaO2) increased 5.9 +/- 1.5 Torr and Tr increased 0.5 +/- 0.1 degree C. As the exercise levels progressed from mild to moderate exercise (64% of VO2 max) the magnitude of the hypocapnia and the resultant respiratory alkalosis remained unchanged as PaCO2 remained 5.9 +/- 0.7 Torr below and delta pHa remained 0.029 +/- 0.008 above resting values. When the exercise work rate was increased to elicit VO2 max (96 +/- 2 ml X kg-1 X min-1) the amount of hypocapnia again remained unchanged from submaximal exercise levels and PaCO2 remained 6.0 +/- 0.6 Torr below resting values; however, this response occurred despite continued increases in Tr (delta Tr = 1.7 +/- 0.1 degree C), significant increases in [LA-]a (delta [LA-]a = 2.5 +/- 0.4), and a resultant metabolic acidosis (delta pHa = -0.031 +/- 0.011). The dog, like other nonhuman vertebrates, responded to mild and moderate steady-state exercise with a significant hyperventilation and respiratory alkalosis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamics of hydrolysis of oligosaccharides

Microcalorimetry has been used to determine enthalpy changes for the hydrolysis of a series of oligosaccharides. High-pressure liquid chromatography was used to determine the extents of reaction and to check for any possible side reactions. The enzyme glucan 1,4-alpha-glucosidase was used to bring about the following hydrolysis reactions: (A) maltose(aq) + H2O(liq) = 2D-glucose(aq); (B) maltotriose(aq) + 2H2O(liq) = 3D-glucose(aq); (C) maltotetraose(aq) + 3H2O(liq) = 4D-glucose(aq); (D) maltopentaose(aq) + 4H2O(liq) = 5D-glucose(aq); (E) maltohexaose(aq) + 5H2O(liq) = 6D-glucose(aq); (F) maltoheptaose(aq) + 6H2O(liq) = 7D-glucose(aq); (G) amylose(aq) + nH2O(liq) = (n + 1) D-glucose(aq); and (H) panose(aq) + 2H2O(liq) = 3D-glucose(aq); (J) isomaltotriose(aq) + 2H2O(liq) = 3D-glucose(aq). The enzyme beta-fructofuranosidase was used for the reactions: (K) raffinose(aq) + H2O(liq) = alpha-D-melibiose(aq) + D-fructose(aq); and (L) stachyose(aq) + H2O(liq) = o-alpha-D-galactopyranosyl-(1----6)- alpha-o-D-galactopyranosyl-(1----6)-alpha-D-glucopyranose + D-fructose(aq). The results of the calorimetric measurements (298.15 K, 0.1 M sodium acetate buffer, pH 4.44-6.00) are: delta H0A = -4.55 +/- 0.10, delta H0B = -9.03 +/- 0.10, delta H0C = -13.79 +/- 0.15, delta H0D = -18.12 +/- 0.10, delta H0E = -22.40 +/- 0.15, delta H0F = -26.81 +/- 0.20, delta H0H = 1.46 +/- 0.40, delta H0J = 11.4 +/- 2.0, delta H0K = -15.25 +/- 0.20, and delta H0L = -14.93 +/- 0.20 kJ mol-1. The enthalpies of hydrolysis of two different samples of amylose were 1062 +/- 20 and 2719 +/- 100 kJ mol-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of oxygen consumption after a single flash of light in photoreceptors of the drone (Apis mellifera)

The time course of the rate of oxygen consumption (QO2) after a single flash of light has been measured in 300-micrometers slices of drone retina at 22 degrees C. To measure delta QO2(t), the change in QO2 from its level in darkness, the transients of the partial pressure of O2 (PO2) were recorded with O2 microelectrodes simultaneously in two sites in the slice and delta QO2 was calculated by a computer using Fourier transforms. After a 40-ms flash of intense light, delta QO2, reached a peak of 40 microliters O2/g.min and then declined exponentially to the baseline with a time constant tau 1 = 4.96 +/- 0.49 s (SD, n = 10). The rising phase was characterized by a time constant tau 2 = 1.90 +/- 0.35 s (SD, n = 10). The peak amplitude of delta QO2 increased linearly with the log of the light intensity. Replacement of Na+ by choline, known to decrease greatly the light-induced transmembrane current, caused a 63% decrease of delta QO2. With these changes, however, the kinetics of delta QO2 (t) were unchanged. This suggest that the recovery phase is rate-limited by a single reaction with apparent first-order kinetics. Evidence is provided that suggests that this reaction may be the working of the sodium pump. Exposure of the retina to high concentrations of ouabain or strophanthidin (inhibitors of the sodium pump) reduced the peak amplitude of delta QO2 by approximately 80% and increased tau 1. The increase of tau 1 was an exponential function of the time of exposure to the cardioactive steroids. Hence, it seems likely that the greatest part of delta QO2 is used for the working of the pump, whose activity is the mechanism underlying the rate constant of the descending limb of delta QO2 (t).     

Independent cerebral vasoconstrictive effects of hyperoxia and accompanying arterial hypocapnia at 1 ATA.

Breathing 100% O2 at 1 atmosphere absolute (ATA) is known to be associated with a decrease in cerebral blood flow (CBF). It is also accompanied by a fall in arterial Pco2 leading to uncertainty as to whether the cerebral vasoconstriction is totally or only in part caused by arterial hypocapnia. We tested the hypothesis that the increase in arterial Po2 while O2 was breathed at 1.0 ATA decreases CBF independently of a concurrent fall in arterial Pco2. CBF was measured in seven healthy men aged 21-62 yr by using noninvasive continuous arterial spin-labeled-perfusion MRI. The tracer in this technique, magnetically labeled protons in blood, has a half-life of seconds, allowing repetitive measurements over short time frames without contamination. CBF and arterial blood gases were measured while breathing air, 100% O2, and 4 and 6% CO2 in air and O2 backgrounds. Arterial Po2 increased from 91.7 +/- 6.8 Torr in air to 576.7 +/- 18.9 Torr in O2. Arterial Pco2 fell from 43.3 +/- 1.8 Torr in air to 40.2 +/- 3.3 Torr in O2. CBF-arterial Pco2 response curves for the air and hyperoxic runs were nearly parallel and separated by a distance representing a 28.7-32.6% decrement in CBF. Regression analysis confirmed the independent cerebral vasoconstrictive effect of increased arterial Po2. The present results also demonstrate that the magnitude of this effect at 1.0 ATA is greater than previously measured.     

Hyperoxia increases H2O2 production by brain in vivo

Hyperoxia and hyperbaric hyperoxia increased the rate of cerebral hydrogen peroxide (H2O2) production in unanesthetized rats in vivo, as measured by the H2O2-mediated inactivation of endogenous catalase activity following injection of 3-amino-1,2,4-triazole. Brain catalase activity in rats breathing air (0.2 ATA O2) decreased to 75, 61, and 40% of controls due to endogenous H2O2 production at 30, 60, and 120 min, respectively, after intraperitoneal injection of 3-amino-1,2,4-triazole. The rate of catalase inactivation increased linearly in rats exposed to 0.6 ATA O2 (3 ATA air), 1.0 ATA O2 (normobaric 100% O2) and 3.0 ATA O2 (3 ATA 100% O2) compared with 0.2 ATA O2 (room air). Catalase inactivation was prevented by pretreatment of rats with ethanol (4 g/kg), a competitive substrate for the reactive catalase-H2O2 intermediate, compound I. This confirmed that catalase inactivation by 3-amino-1,2,4-triazole was due to formation of the catalase-H2O2 intermediate, compound I. The linear rate of catalase inactivation allows estimates of the average steady-state H2O2 concentration within brain peroxisomes to be calculated from the formula: [H2O2] = 6.6 pM + 5.6 ATA-1 X pM X [O2], where [O2] is the concentration of oxygen in ATA that the rats are breathing. Thus the H2O2 concentration in brains of rats exposed to room air is calculated to be about 7.7 pM, rises 60% when O2 tension is increased to 100% O2, and increases 300% at 3 ATA 100% O2, where symptoms of central nervous system toxicity first become apparent. These studies support the concept that H2O2 is an important mediator of O2-induced injury to the central nervous system.     

Effects of acute and chronic hypercapnia on oxygen tolerance in rats

Groups of 16-52 normal or CO2-adapted rats were exposed top 100% O2 or to O2 with 60 Torr PICO2 (O2-CO2) at pressures of 1.0, 1.5, 2.0, 3.0, and 4.0 ATA. Exposure durations for 50% mortality (LD50) in normal rats at 4.0, 3.0, 2.0, 1.5, and 1.0 ATA O2 were 6.3, 9.3, 17.2, 27.4, and 76.1 h, respectively. Corresponding LD50 values for normal rats exposed to O2-CO2 were 2.0, 2.9, 16.3, 24.8, and 74.8 h. Survival times of CO2-adapted rats exposed to O2 were nearly identical to those of normal rats. LD50 values for CO2-adapted rat exposed to O2-CO2 were 4.1, 7.5, 17.9, 23.6, and 65.4 h, respectively. These data confirm acceleration of O2 intoxication by acute hypercapnia at 4.0 and 3.0 ATA, but they show less prominent effects at 2.0, 1.5, and 1.0 ATA. Hypercapnia adaptation clearly has a protective effect in rats exposed to O2-CO2 at 4.0 and 3.0 ATA. At 2.0, 1.5, and 1.0 ATA, where acute hypercapnia has less effect, the effects of CO2 adaptation are also less prominent. The observed changes in oxygen tolerance can be explained by cerebral vasodilation with increased brain oxygenation in acute hypercapnia and by significant amelioration of this response during chronic hypercapnia.     

The Haber-Weiss cycle--70 years later

The chain reactions HO* + H2O2 --> H2O + O2*- + H+ and O2*- + H+ + H2O2 --> O2 + HO* + H2O, commonly known as the Haber-Weiss cycle, were first mentioned by Haber and Willst?tter in 1931. George showed in 1947 that the second reaction is insignificant in comparison to the fast dismutation of superoxide, and this finding appears to have been accepted by Weiss in 1949. In 1970, the Haber-Weiss reaction was revived by Beauchamp and Fridovich to explain the toxicity of superoxide. During the 1970s various groups determined that the rate constant for this reaction is of the order of 1 M(-1) s(-1) or less, which confirmed George's conclusion. The reaction of superoxide with hydrogen peroxide was dropped from the scheme of oxygen toxicity, and superoxide became the source of hydrogen peroxide, which yields hydroxyl radicals via the Fenton reaction, Fe2+ + H2O2 --> Fe3+ + HO- + HO*. In 1994, Kahn and Kasha resurrected the Haber-Weiss reaction again, but this time the oxygen was believed to be in the singlet (1delta(g)) state. As toxicity arises not from a Fenton-catalysed Haber-Weiss reaction, but from the Fenton reaction, the Haber-Weiss reaction should not be mentioned anymore.     

Similar but not the same: normobaric and hyperbaric pulmonary oxygen toxicity, the role of nitric oxide

Pulmonary manifestations of oxygen toxicity were studied and quantified in rats breathing >98% O(2) at 1, 1.5, 2, 2.5, and 3 ATA to test our hypothesis that different patterns of pulmonary injury would emerge, reflecting a role for central nervous system (CNS) excitation by hyperbaric oxygen. At 1.5 atmosphere absolute (ATA) and below, the well-recognized pattern of diffuse pulmonary damage developed slowly with an extensive inflammatory response and destruction of the alveolar-capillary barrier leading to edema, impaired gas exchange, respiratory failure, and death; the severity of these effects increased with time over the 56-h period of observation. At higher inspired O(2) pressures, 2-3 ATA, pulmonary injury was greatly accelerated but less inflammatory in character, and events in the brain were a prelude to a distinct lung pathology. The CNS-mediated component of this lung injury could be attenuated by selective inhibition of neuronal nitric oxide synthase (nNOS) or by unilateral transection of the vagus nerve. We propose that extrapulmonary, neurogenic events predominate in the pathogenesis of acute pulmonary oxygen toxicity in hyperbaric oxygenation, as nNOS activity drives lung injury by modulating the output of central autonomic pathways.     

Muscle maximal O2 uptake at constant O2 delivery with and without CO in the blood

In the present study we investigated the effects of carboxyhemoglobinemia (HbCO) on muscle maximal O2 uptake (VO2max) during hypoxia. O2 uptake (VO2) was measured in isolated in situ canine gastrocnemius (n = 12) working maximally (isometric twitch contractions at 5 Hz for 3 min). The muscles were pump perfused at identical blood flow, arterial PO2 (PaO2) and total hemoglobin concentration [( Hb]) with blood containing either 1% (control) or 30% HbCO. In both conditions PaO2 was set at 30 Torr, which produced the same arterial O2 contents, and muscle blood flow was set at 120 ml.100 g-1.min-1, so that O2 delivery in both conditions was the same. To minimize CO diffusion into the tissues, perfusion with HbCO-containing blood was limited to the time of the contraction period. VO2max was 8.8 +/- 0.6 (SE) ml.min-1.100 g-1 (n = 12) with hypoxemia alone and was reduced by 26% to 6.5 +/- 0.4 ml.min-1.100 g-1 when HbCO was present (n = 12; P less than 0.01). In both cases, mean muscle effluent venous PO2 (PVO2) was the same (16 +/- 1 Torr). Because PaO2 and PVO2 were the same for both conditions, the mean capillary PO2 (estimate of mean O2 driving pressure) was probably not much different for the two conditions, even though the O2 dissociation curve was shifted to the left by HbCO. Consequently the blood-to-mitochondria O2 diffusive conductance was likely reduced by HbCO.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tissue oxygen extraction during hypovolemia: role of hemoglobin P50

When the delivery of O2 to tissues (QO2 = blood flow X O2 content) falls below a critical threshold, tissue O2 uptake (VO2) becomes limited by QO2. The mechanism responsible for this extraction limitation is not understood but may involve molecular diffusion limitation as mean capillary PO2 drops below a critical minimum level in some capillaries. We tested this hypothesis by measuring the critical QO2 necessary to maintain VO2 independent of QO2 in anesthetized, paralyzed normal dogs (n = 7) and in a second group in which PO2 at 50% saturation of hemoglobin (P50) was reduced by exchange transfusion with low-P50 erythrocytes (n = 7). QO2 was reduced in stages by removing blood volume to reduce blood flow while VO2 was measured by spirometry at each step. To the extent that O2 extraction was limited by a critical capillary PO2, we reasoned that the onset of diffusion limitation should occur at a higher QO2 with low P50, since a lower end-capillary PO2 is required to achieve the same O2 extraction. The critical QO2 (7.8 +/- 1.2 ml X min-1 X kg-1) and extraction ratio (0.63 +/- 0.06) in dogs with reduced P50 were not different from controls. At the critical delivery, mixed venous PO2 was lower in low P50 (16.1 +/- 2.9 Torr) than controls (29.9 +/- 2.3 Torr). We concluded that diffusion limitation does not initiate the early fall in VO2 below the critical QO2 and offer an alternative model to explain the onset of supply dependency.(ABSTRACT TRUNCATED AT 250 WORDS)     

Voltage and temperature dependence of single K+ channels isolated from canine cardiac sarcoplasmic reticulum.

The temperature and voltage dependence of gating and conductance of sarcoplasmic reticulum K+ channels (S-R K+) isolated from adult canine hearts were studied using the reconstituted bilayer technique. Fusion of vesicles from this preparation frequently resulted in the incorporation of a single channel. Only bilayers into which a single S-R K+ channel had fused were studied. The three conductance states of the channel, fully open (O2), substate conductance (O1), and closed (C) were studied as a function of voltage (-50 to +50 mV) and temperature (16 to 37 degrees C). Permeation through the O1 state showed the same temperature dependence as the O2 state corresponding to an enthalpy of permeation of 4.1-4.2 kcal/mol, which is similar to that for K+ diffusion through water. As expected, increased temperature increased the frequency of gating transitions and shortened the average dwell time spent in any conductance state. Over the range of 25 to 37 degrees C, the average dwell time spent in the O1, O2, and C states decreased by 44 +/- 11, 36 +/- 13, and 78 +/- 7% (n = 3 to 4 channels), respectively. The ratio of probabilities between the various conductance states was not strongly temperature sensitive. Analysis of the voltage dependence of this channel was carried out at 37 degrees C and revealed that the dwell times of the O1 and O2 states were voltage insensitive and the probability ratio (PO2:PO1) was approximately 7 and was voltage insensitive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anomalous hypoxic acidification of medullary ventral surface.

In castrated male goats, two flexible catheters, one open ended for reference and the other ending in a 1-mm-diam glass bulb pH electrode, were advanced ventrally through a left posterior fossa craniotomy into the subarachnoid space between the 9th and 10th cranial nerve roots, passing medially into cerebrospinal fluid (CSF) over the medullary ventral surface (MVS). They were anchored to dura and fascia, tunneled under the scalp, and terminated in connectors on the left horn. After several days for recovery, while the animals were awake, the effects of CO2 and hypoxia on pH of the film of CSF between the pia and arachnoid (pHMVS) were recorded along with end-tidal PCO2 and PO2 (mass spectrometer), ventilation (pneumotachometer) through a permanent tracheostomy, and, when possible, ear arterial O2 saturation (SaO2). High PCO2 acidified MVS as expected: delta pH MVS/delta log PCO2. = -0.64 +/- 0.14, producing a ventilatory response slope delta VI/delta pHMVS = 372 l/min. Hypoxia resulted in acid shifts even when PCO2 was allowed to fall. The development of hypoxic acidosis was related to the location of pH electrodes determined at necropsy. In isocapnic hypoxia, pH over putative chemoreceptor surfaces fell in proportion to desaturation: delta pHMVS = 0.0033(SaO2)-0.34, r = 0.80, Sy.x = 0.025. With uncontrolled arterial PCO2, similar acidosis occurred when SaO2 fell below 85-90%: delta pHMVS = 0.0039(SaO2)-0.34, r = 0.88, Sy.x = 0.032. With constant hypoxia, pH fell (tau = 3.7 +/- 2.2 min) to a plateau after 10-20 min and showed rapid recovery (tau = 2.0 +/- 1.3 min).(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxygen uptake dynamics during high-intensity exercise in children and adults

We hypothesized that the O2 uptake (Vo2) response to high-intensity exercise would be different in children than in adults. To test this hypothesis, 22 children (6-12 yr old) and 7 adults (27-40 yr old) performed 6 min of constant-work-rate cycle-ergometer exercise. Sixteen children performed a single test above their anaerobic threshold (AT). In a separate protocol, six children and all adults exercised at low and high intensity. Low-intensity exercise corresponded to the work rate at 80% of each subject's AT. High-intensity exercise (above the AT) was determined first by calculating the difference in work rate between the AT and the maximal Vo2 (delta). Twenty-five, 50, and 75% of this difference were added to the work rate at the subject's AT, and these work rates were referred to as 25% delta, 50% delta, and 75% delta. For exercise at 50% delta and 75% delta, Vo2 increased throughout exercise (O2 drift, linear regression slope of Vo2 as a function of time from 3 to 6 min) in all the adults, and the magnitude of the drift was correlated with increasing work rates in the above-AT range (r = 0.91, P less than 0.0001). In contrast, no O2 drift was observed in over half of the children during above-AT exercise. The O2 drifts were much higher in adults (1.76 +/- 0.63 ml O2.kg-1.min-2 at 75% delta) than in children (0.20 +/- 0.42, P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR studies of conformations and interactions of substrates and ribonucleotide templates bound to the large fragment of DNA polymerase I

The large fragment of DNA polymerase I (Pol I) effectively uses oligoribouridylates and oligoriboadenylates as templates, with kinetic properties similar to those of poly(U) and poly(A), respectively, and has little or no activity in degrading them. In the presence of such oligoribonucleotide templates, nuclear Overhauser effects (NOE's) were used to determine interproton distances within and conformations of substrates bound to the large fragment of Pol I, as well as conformations and interactions of the enzyme-bound templates. In the enzyme-oligo(rU)54 +/- 11-Mg2+dATP complex, the substrate dATP has a high anti-glycosidic torsional angle (chi = 62 +/- 10 degrees) and an O1'-endo/C3'-endo sugar pucker (delta = 90 +/- 10 degrees) differing only slightly from those previously found for enzyme-bound dATP in the absence of template [Ferrin, L.J., & Mildvan, A.S. (1985) Biochemistry 24, 4680-4694]. Both conformations are similar to those of deoxynucleotidyl units of B DNA but differ greatly from those of A or Z DNA. The conformation of the enzyme-bound substrate analogue AMPCPP (chi = 50 +/- 10 degrees, delta = 90 +/- 10 degrees) is very similar to that of enzyme-bound dATP and is unaltered by the binding of the template oligo(rU)54 +/- 11 or by the subsequent binding of the primer (Ap)9A. In the enzyme-oligo(rA)50-Mg2+TTP complex, the substrate TTP has an anti-glycosidic torsional angle (chi = 40 +/- 10 degrees) and an O1'-endo sugar pucker (delta = 100 +/- 10 degrees), indistinguishable from those found in the absence of template and compatible with those of B DNA but not with those of A or Z DNA. In the absence of templates, the interproton distances on enzyme-bound dGTP cannot be fit by a single conformation but require a 40% contribution from a syn structure (chi = 222 degrees) and a 60% contribution from one or more anti structures. The presence of the template oligo(rU)43 +/- 9 simplifies the conformation of enzyme-bound dGTP to a single structure with an anti-glycosyl angle (chi = 32 +/- 10 degrees) and an O1'-endo/C3'-endo sugar pucker (delta = 90 +/- 10 degrees), compatible with those of B DNA, possibly due to the formation of a G-U wobble base pair. However, no significant misincorporation of guanine deoxynucleotides by the enzyme is detected with oligo(rU) as template.(ABSTRACT TRUNCATED AT 400 WORDS)     

O2 exchange between blood and brain tissues studied with 18O2 indicator-dilution technique

A technique has been developed to record 18O2 dilution curves of an organ in vivo by use of 51Cr-labeled erythrocytes as a reference tracer. The technique employs anaerobic sampling of venous outflow following an intraarterial injection of tracer-laden blood and off-line determination of [18O2] and [51Cr] profiles in the venous outflow. O2 and reference indicator-dilution curves of cerebral circulation were recorded in eight experiments with six halothane-anesthetized dogs. Autologous blood labeled with the tracers was injected into a carotid artery, and brain venous outflow was sampled from the sagittal sinus. The total net extraction of O2 tracer was equal to the extraction of elemental O2. Instantaneous extraction of 18O2 along the outflow curve fell linearly with time, from an initial value of 0.6-0.7 to very small or even negative values toward the end of a pulse. This indicates that O2 undergoes a flow-limited distribution. In all experiments, the mean transit time of unmetabolized 18O2 was longer than the mean transit time of the Cr tracer. An index of the tissue O2 dilution space, hence the mean tissue PO2, is calculated from this data with the use of a modified central volume principle. This estimate of mean tissue PO2 increases as a linear function of sagittal sinus PO2 with a slope of 0.97. The method may provide an index of the critical PO2 of venous blood, the PO2 below which O2 diffusion from blood to tissue may limit its rate of metabolic uptake.     

Thermodynamics of hydrolysis of disaccharides. Lactulose, alpha-D-melibiose, palatinose, D-trehalose, D-turanose and 3-o-beta-D-galactopyranosyl-D-arabinose

High-pressure liquid chromatography and microcalorimetry have been used to study the thermodynamics of the hydrolysis reactions of a series of disaccharides. The enzymes used to bring about the hydrolyses were: beta-galactosidase for lactulose and 3-o-beta-D-galactopyranosyl-D-arabinose; beta-glucosidase for alpha-D-melibiose; beta-amylase for D-trehalose; isomaltase for palatinose; and alpha-glucosidase for D-turanose. The buffer used was sodium acetate (0.02-0.10 M and pH 4.44-5.65). For the following processes at 298.15 K: lactulose(aq) + H2O(liq) = D-galactose(aq) + D-fructose(aq), K0 = 128 +/- 10 and delta H0 = 2.21 +/- 0.10 kJ mol-1; alpha-D-melibiose(aq) + H2O(liq) = D-galactose(aq) + D-glucose(aq), K0 = 123 +/- 42 and delta H0 = -0.88 +/- 0.50 kJ mol-1; palatinose(aq) + H2O(liq) = D-glucose(aq) + D-fructose(aq), delta H0 = -4.44 +/- 1.1 kJ mol-1; D-trehalose(aq) + H2O(liq) = 2 D-glucose(aq), K0 = 119 +/- 10 and delta H0 = 4.73 +/- 0.41 kJ mol-1; D-turanose(aq) + H2O(liq) = D-glucose(aq) + D-fructose(aq), delta H0 = -2.68 +/- 0.75 kJ mol-1; and 3-o-beta-D-galactopyranosyl-D-arabinose(aq) + H2O(liq) = D-galactose(aq) + D- arabinose(aq),0H0 = 107 +/- 10 and delta H0 = 2.97 +/- 0.10 kJ mol-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of increased Hb-O2 affinity on VO2max at constant O2 delivery in dog muscle in situ

We investigated the effect of increasing hemoglobin- (Hb) O2 affinity on muscle maximal O2 uptake (VO2max) while muscle blood flow, [Hb], HbO2 saturation, and thus O2 delivery (muscle blood flow X arterial O2 content) to the working muscle were kept unchanged from control. VO2max was measured in isolated in situ canine gastrocnemius working maximally (isometric tetanic contractions). The muscles were pump perfused, in alternating order, with either normal blood [O2 half-saturation pressure of hemoglobin (P50) = 32.1 +/- 0.5 (SE) Torr] or blood from dogs that had been fed sodium cyanate (150 mg.kg-1.day-1) for 3-4 wk (P50 = 23.2 +/- 0.9). In both conditions (n = 8) arterial PO2 was set at approximately 200 Torr to fully saturate arterial blood, which thereby produced the same arterial O2 contents, and muscle blood flow was set at 106 ml.100 g-1.min-1, so that O2 delivery in both conditions was the same. VO2max was 11.8 +/- 1.0 ml.min-1.100 g-1 when perfused with the normal blood (control) and was reduced by 17% to 9.8 +/- 0.7 ml.min-1.100 g-1 when perfused with the low-P50 blood (P less than 0.01). Mean muscle effluent venous PO2 was also significantly less (26 +/- 3 vs. 30 +/- 2 Torr; P less than 0.01) in the low-P50 condition, as was an estimate of the capillary driving pressure for O2 diffusion, the mean capillary PO2 (45 +/- 3 vs. 51 +/- 2 Torr). However, the estimated muscle O2 diffusing capacity was not different between conditions.(ABSTRACT TRUNCATED AT 250 WORDS)