Effect of docosahexaenoic acid on rate of differentiation of HL-60 human leukemia.

We have utilized an experimental model of cell lipid modification that allows study of the effect of a polyunsaturated fatty acid on the linked processes of cellular differentiation and growth arrest. HL-60 human leukemia cells were grown in media supplemented with 10 microM concentrations of the fatty acid docosahexaenoic acid (22:6) or oleic acid (18:1) or in unsupplemented media. Gas chromatographic analysis of phospholipid extracts from HL-60 cells grown in unmodified or 18:1-supplemented media revealed 39% and 36% 18:1, 13 and 12% polyenoics, and 2 and 3% 22:6, respectively. In contrast, cells from 22:6-supplemented cultures had 22% 18:1, 18% total polyunsaturated fatty acids, and 10% 22:6. Retinoic acid was added to cells grown in the various media, and phorbol ester-induced superoxide generation, nitroblue tetrazolium reduction, and growth arrest were determined as measures of differentiation. Unmodified and 18:1-enriched cells showed inducible oxidative burst activity beginning at 48 h after the addition of retinoic acid and continuing to increase for 5 days. In marked contrast, the 22:6-enriched leukemia cells exhibited an increased oxidative activity as early as 24 h which is equivalent to about one division cycle time. G1/0-specific growth arrest was associated with the oxidative phenotypic differentiation in all three cell types. However, cells enriched with 22:6 demonstrated early growth arrest and differentiation considerably in advance of 18:1-modified or unmodified cells. An effect on the cellular differentiation process could be detected after even a brief 1-h exposure of the cells to 22:6. Therefore, a highly polyunsaturated fatty acid which is actively incorporated into membrane structures appreciably accelerates the differentiation process of this human neoplastic cell.

Membrane lipid modification and sensitivity of leukemic cells to the thioether lipid analogue BM 41.440.

Since the ether lipid anticancer drugs are membrane targeted, we examined the effect of membrane lipid structural alteration on their cytotoxicity. Enrichment with docosahexaenoic acid increased the sensitivity to the thioether lipid BM 41.440, compared to control cells enriched with oleic acid. The effect was dependent upon drug concentration, time, and the extent of cellular fatty acid enrichment. Other polyunsaturated fatty acids had a similar effect, which was proportional to the degree of unsaturation of the molecule inserted. Depletion of cellular glutathione with buthionine sulfoximine increased the sensitivity to ether lipid, but prooxidants such as Fe2+ and antioxidants such as vitamin E had little effect. The addition of serum to the incubation medium markedly diminished the cytotoxicity of ether lipids for cells modified with both docosahexaenoic acid and oleic acid, probably due to binding of the drug to serum components. The toxicity of another ether lipid, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, was not affected appreciably by membrane alteration. Drug uptake studies with a radiolabeled BM 41.440 analogue, 1-[3H]hexadecylthio-2-ethyl-rac- glycero-3-phosphocholine, demonstrated no difference in transport at early time points and no difference in accumulation up to 60 min. We conclude that increases in cellular and/or membrane fatty acid polyunsaturation heighten the cytotoxic effect of a membrane-active ether lipid. The effect is not due to a change in drug transport or accumulation. It may be related to a change in oxidative events. These observations provide further confirmation of the membrane being the target of ether lipid action, using biochemical rather than morphological techniques. Most importantly, this observation offers a potential innovative approach to therapy.

Effects of beta-adrenergic blockade on the ventilatory responses to hypoxic and hyperoxic exercise in man.

1. The ventilatory responses to step changes from rest to 100 W cycling exercise were studied in five healthy human subjects. Exercise was performed in hypoxia (end-tidal O2 pressure, PET,O2, 50-55 mmHg), a condition characterized by a marked enhancement of arterial chemoreceptor activity, and in hyperoxia (PET,O2 greater than 250 mmHg), a condition in which arterial chemoreceptor activity is largely suppressed. The subjects were studied at each O2 level after placebo and after an oral dose of 120 mg propranolol. 2. The magnitude of phase 1, the immediate, rapid ventilatory response at the onset of work, was unaffected by hypoxia and at both oxygen levels it was also unaffected by propranolol. 3. Phase 2, analysed from 20 to 120 s after the onset of exercise, was significantly affected by both O2 level and beta-blockade. The kinetics of the ventilatory changes in this phase were well described in all four conditions by a simple exponential function. The overall mean time constants after placebo were shorter in hypoxia (31.0 s) than in hyperoxia (40.2 s), and at each O2 level longer after propranolol, in hypoxia 61.3 s and in hyperoxia 106.0 s. 4. Continuous analysis of gas sampled at the mouth with a mass spectrometer showed constancy of end-tidal PCO2 throughout the step change in hypoxia both with and without beta-blockade. In contrast, in both hyperoxic conditions PET,CO2 rose, mainly in phase 2, to a value 5-6 mmHg higher than the starting value. 5. The steady-state ventilation was higher in hypoxia than in hyperoxia, and end-tidal CO2 pressure, PET,CO2, correspondingly lower. Neither ventilation nor PCO2 were, however, affected by propranolol in either condition. 6. It is concluded that the arterial chemoreceptors are important for both the rate of adaptation of ventilation to a new rate of metabolism during a step change of work rate, and for the matching of ventilation to CO2 flow which normally ensures isocapnia. The further slowing of the dynamics of the ventilatory response in hyperoxia as well as the preserved isocapnia in hypoxia after beta-blockade argue against any major role of beta-adrenergic mechanisms for these functions of the arterial chemoreceptors. The observed effects are considered to be secondary to the reduced cardiac output and an increased CO2 storage initially during exercise following beta-adrenergic blockade.

Effects of body temperature on ventilatory response to hypoxia and breathing pattern in man.

The ventilatory response to hypoxia (PAO2 55 and 45 Torr) at each of four levels of PACO2 was studied in five healthy subjects before and after a rise in rectal temperature of 1.4 degrees C had been induced by means of a heated flying suit. At a given level of chemical drive both ventilation and mean inspiratory flow increased after heating, frequency relatively more than tidal volume. In isoventilation comparisons mean inspiratory flow was identical in normo- and hyperthermia, whereas the durations of inspiration (TI) and expiration (TE) were proportionately shortened. It is suggested that a rise in temperature shortens TI by affecting a central "clock" and that TE changes are secondary to changes in end-inspiratory volume. The euoxic CO2 response in hyperthermia was suggestive of multiplication between CO2 and temperature. Hypoxic sensitivity was significantly increased, indicating a temperature effect on the arterial chemoreceptors. The breathing pattern was in either temperature condition identical in euoxia and in hypoxia.

Effect of butylated hydroxytoluene on bilineage differentiation of the human HL-60 myeloblastic leukemia cell.

Butylated hydroxytoluene (BHT), which has both antioxidant and membrane active properties, has been reported to affect cellular differentiation. We studied its effect on the bipotent lineage differentiation of the important HL-60 human myeloblastic leukemia cell line using reduction of nitroblue tetrazolium, cell cycle analysis, population growth rate, monoclonal antibodies, and morphology. BHT markedly accelerated retinoic acid-induced myelocytic differentiation and dihydroxyvitamin D3-induced monocytic differentiation in a concentration and time-dependent manner. Butylated hydroxyanisole (BHA) had a comparable effect. Preincubation with the compounds was not necessary to evoke the acceleration Other antioxidants and inhibitors of eicosanoid synthesis were inactive. We conclude that the important food preservatives BHT and BHA accelerate the kinetics of terminal differentiation of human leukemia and that this effect is likely due at least in part to their membrane active properties.

Arterial chemoreceptors, ventilation and heart rate in man.

1. Transient changes of heart rate (HR) and ventilation were recorded following step changes in alveolar gas composition in three healthy subjects. From a steady state of normo- or slightly hypercapnic hypoxia (PA,CO2 38-46 torr, PA,O2 50-60 torr) arterial chemoreceptor stimulation was transiently relieved by breathing a CO2-free mixture for two breaths, either pur O2 (causing a fall in PA,CO2 and a rise in PA,O2; O2 test) or a low O2 mixture (causing a fall in PA,CO2 without any change in PA, O2; CO2 test). For both test types ventilation was either allowed to change freely ('free-breathing' tests) or was consciously maintained at the pre-test level by the subjects ('controlled-breathing tests). The circulatory delay from the lungs to the ear was measured with a sensitive ear oximeter. 2. In all 'free-breathing' tests ventilation decreased significantly after a mean latency of 5.2 sec; the average lung-ear circulation time was 4.9 sec. HR increased slightly above pre-test levels in eighty-one of one hundred and four tests of all types, the changes being significant after a latency identical to that of the ventilatory changes. Except in the 'controlled-breathing' CO2 tests this early tachycardia was followed by a decrease in HR within the following 5-6 sec. 3. These findings indicate that the primary effect of withdrawal of arterial chemoreceptor stimulation in conscious man as in the anesthetized animal is tachycardia. The secondary development of bradycardia in 'free-breathing' CO2 tests is probably due to the operation of a lung reflex sensing changes in ventilation. The absence of bradycardia in 'controlled-breathing' CO2 tests and its presence in 'controlled-breathing' O2 tests, finally, suggest that relief of systemic hypoxia causes a slowing of the heart not due to lung reflexes but to some other mechanism which operates with a latency nearly twice as long as the arterial chemoreflex.

Undergraduate medical education.

There were 31,323 applicants to US medical schools for the 1986-1987 academic year. Of this number, 17,092 were accepted by at least one school. First-year enrollment equaled 16,779. Because some students were repeating the first year, the number of first-time enrolled students was 16,206. This represents a decrease of 131 from the previous year. The number of students enrolled in 127 US medical schools in 1986-1987 was 66,142; of this number 22,082 (33.4%) were women. The estimated number of graduates in 1986-1987 was 15,872. The total enrollment of students from underrepresented ethnic/racial groups was 6650 (10.1%), of which 3853 (5.8%) were blacks not of Hispanic origin. The number of new entry first-year students from underrepresented groups was 1679 (10.4%), of which 987 (6.1%) were blacks. The number of full-time medical school faculty was 63,991; another 130,379 were part-time and volunteer faculty. Medical school faculties also have teaching responsibility for a variety of other students, in addition to patient care and research responsibility. The average time needed to complete the curriculum requirements leading to the MD degree is 152 weeks. Twenty-two medical schools offered a combined college-medical school program. The length of these combined programs averaged 254 weeks. More than 93% of students entering medical school in 1986-1987 had completed at least four years of college. More than two fifths of students had a premedical grade point average of 3.50 or higher. The number of schools offering a Fifth Pathway Program has decreased and the number of applicants for these programs has also declined. The net attrition rate, which excludes students who withdraw temporarily to pursue advanced study or research, has remained at about 2%. Only half of 1% of students were dismissed because of poor academic standing.

Undergraduate medical education.

There were 32,893 applicants for the 1985-1986 academic year in US medical schools. Of this number, 17,228 were accepted by at least one school. First-year enrollment equaled 16,929. Because some students were repeating the first year, the number of first-time enrolled students was 16,337. This represents a decrease of 122 students from the previous year. The number of students enrolled in 127 US medical schools in 1985-1986 was 66,604; of this number, 21,624 (32.5%) were women. The estimated number of graduates in 1985-1986 was 16 191. The total enrollment of students of minority groups was 10,964 (16.5%), of which 3556 (5.3%) were black (not of Hispanic origin). The number of full-time medical school faculty was 61,372; another 124,466 were part-time or volunteer faculty. Medical school faculties also have teaching responsibility for a variety of other students, in addition to patient care and research responsibility. The average time needed to complete the curriculum requirements leading to the MD degree is 156 weeks. Twenty-four medical schools offered a combined college-medical school program. The length of these combined programs averaged 265 weeks. Ninety-three percent of students entering medical school in 1985-1986 had completed at least four years of college. More than two fifths of students had a premedical GPA of 3.6 or higher.

Breathing in man during steady-state exercise on the bicycle at two pedalling frequencies, and during treadmill walking.

1. The breathing pattern, that is the changes in tidal volume (VT), and in inspiratory (TI) and expiratory (TE) durations, has been studied as ventilation increases in exercise. 2. Five healthy subjects were studied in steady-state exercise on a bicycle ergometer, breathing air, at two speeds of pedalling and at six different loads. The pattern was recorded for single breaths. Two of the subjects were also studied while walking on a treadmill with four combinations of speed and gradient. 3. In bicycle exercise, as the CO2 output increased mean VT increased, and mean TI and TE decreased, the absolute decrease in TI being small. The pedalling speed did not affect these relationships. 4. Individual breath durations showed no tendency to group around multiples of the period of rotation of the pedals. 5. In treadmill exercise, no clear influence of stride rate on respiratory rate could be found. The pattern was similar to that found in bicycle exercise. Again no grouping could be found. 6. No evidence of an effect of frequency of limb movement on breathing pattern in submaximal exercise has been found. The selection of breathing pattern seems to be unrelated to the nature of the stimulus but closely geared to the metabolic needs of the body.

Mean and breath-by-breath pattern of breathing in man during steady-state exercise.

1. The breathing pattern, that is the relation between tidal volume (VT) and the inspiratory (TI) and expiratory (TE) durations, has been studied for individual breaths (forty in each steady state). 2. Five healthy subjects were studied in steady-state exercise on a bicycle ergometer breathing air; three of them were also studied in hypercapnia, at rest and during exercise, and two of them also during exercise on a treadmill. 3. Tidal volume and respiratory frequency both increased with work load. The increase in frequency was largely due to a progressive decrease in TE; TI also decreased. 4. At any constant level of respiratory drive (constant work load or chemical load) VT was positively correlated with both TI and TE in more than 95% of cases. 5. A simple model of the respiratory cycle which fits both the observed mean and breath-by-breath patterns and which involves no new assumptions is presented.

Short-latency ventilatory responses to sudden withdrawal of hypoxia at normal and raised body temperature in man.

Approximately isopnoeic conditions (VE=40 l/min) were achieved by the inhalation of asphyxial gas mixtures (PA,O2 60 torr, PA,CO2 40-45 torr) in normothermia after a rise in rectal temperature of 1.6 degrees C had been induced by a heated flying suit. Arterial chemoreceptor drive was transiently reduced by either isocapnic removal of hypoxia (type (1) tests: two breaths of CO2 in O2) or simultaneous withdrawal of both hypercapnia and hypoxia (type (2) tests: two breaths of O2). 8-13 tests of each type were performed at both temperature conditions in 6 expts. on 4 healthy human subjects. Expired volume, total breath duration and inspiratory time were recorded, and minute ventilation and expiratory time subsequently computed breath by breath. In hyperthermia the steady-state ventilation of 40 l/min (at a relatively higher respiratory frequency and a correspondingly lower tidal volume) was achieved at a PA,CO2 which was 5 torr lower than in normothermia. Ventilation decreased significantly in all tests. Tested with a 3-way analysis of variance significant differences between the ventilatory responses at the two temperature conditions, and between the two test types were found. The rate of change of ventilation was greater in hyperthermia than in normothermia, and also greater in type (2) tests than in type (1) tests. Since isopnoeic conditions existed prior to the tests, this implies that the arterial chemoreceptor contribution to the total ventilatory drive is increased in hyperthermia. In type (2) tests a significant lengthening of expiratory time was observed in the first test breath. This finding confirms the effect in man of changes in airway PCO2 on lung stretch receptor discharge.

Undergraduate medical education.

There were 35,944 applicants for the 1984-1985 year in US medical schools. Of this number, 17,194 were accepted by at least one school. First-year enrollment equaled 16,992. Be-accepted by at least one school. First-year enrollment equaled 16,992. Because some students were repeating the first year, the number of first-time enrolled students was 16,459. This represents a decrease of 113 from the previous year. The number of students enrolled in 127 US medical schools in 1984-1985 was 67,090; of this number, 21,287 (31.7%) were women. The estimated number of graduates in 1984-1985 was 16,347. The total enrollment of students of minority groups was 10,552 (15.7%), of which 3,663 (5.5%) were black not of Hispanic origin. The number of full-time medical school faculty was 58,767, another 123,002 were part-time or volunteer faculty. Medical school faculties also have teaching responsibility for a variety of other students, in addition to patient care and research responsibility. The average time needed to complete the curriculum requirements leading to the MD degree is 153 weeks. Twenty-one medical schools offered a combined college-medical school program. The length of these combined programs averaged 262 weeks. Ninety-six percent of students entering medical school in 1984-1985 had completed at least four years of college. More than two fifths of students had a premedical GPA of 3.6 or higher.

Patterns of breathing in response to alternating patterns of alveolar carbon dioxide pressures in man.

The time profile of alveolar PCO2 within the respiratory cycle has been forced to follow contrasting patterns in alternate breaths, in two different ways. Within-breath changes (w.b.c., with a CO2-rich inspirate supplied early or late in alternate inspirations) involved minimal alternation of end-tidal PCO2. Between-breath changes (b.b.c., with whole inspirates of CO2-free or CO2-rich gas) involved large swings of end-tidal PCO2. As previously reported (Metias, Cunningham, Howson, Petersen & Wolff, 1981), both patterns of forcing were associated with alternation of ventilation, but only when hypoxia was present. The patterns of the alternating reflex responses in 118 runs on four human subjects in steady hypoxia are described in terms of alternation of inspiratory and expiratory tidal volume, time and mean flow. These patterns often disappeared, or changed unpredictably in mid-run. The inspiratory pattern of reflex alternation depended in part on the type of forcing, but alternation of inspiratory tidal volume was usually observed with both types. No single pattern of expiratory alternation emerged as predominant. The pattern of reflex expiratory alternation was surprisingly independent of the pattern of inspiratory alternation: indeed, in w.b.c., but not in b.b.c., alternation of mean expiratory flow and of mean inspiratory flow were mutually exclusive. It is concluded that in man, as in cats and dogs, the arterial chemoreceptor pathway has access to various parts of the respiratory pattern generator, the exact response depending to some extent on the timing within the respiratory cycle. In particular, expiratory variables may be influenced directly through the arterial chemoreceptor pathway, i.e. without any supposedly mediating inspiratory alternation being demonstrable. The results are discussed briefly in relation to some current views on the organization of respiratory control.

Subcellular distribution of doxorubicin: comparison of fatty acid-modified and unmodified cells.

We have examined the subcellular localization of doxorubicin and evaluated the effect of fatty acid modification on specific intracellular localization. L1210 leukemia cells enriched with docosahexaenoic acid (22:6) or oleic acid (18:1) were incubated with radiolabeled or unlabeled doxorubicin. After equilibration the cells were ruptured and the subcellular fractions were isolated by differential centrifugation and sucrose gradient separation. The doxorubicin localized primarily in nuclei, as expected, but appreciable amounts were also detected in mitochondria and smaller amounts in plasma membranes, microsomes, and cytoplasm. Subcellular distribution of another anticancer drug which binds to DNA, mitoxantrone, was similar. There were increased amounts of doxorubicin contained in the nuclei and all organelles of the 22:6-enriched cells. Although polyunsaturated fatty acid modification influenced the total amount of doxorubicin in fractions, the relative distribution of drug among the fractions was not different from that of the 18:1-enriched and unmodified cells. We conclude that enrichment with polyunsaturates influences total drug uptake but not proportional distribution of doxorubicin.

Inspiratory-expiratory responses to alternate-breath oscillation of PACO2 and PAO2.

Breath-by-breath respiratory responses of three healthy adults to imposed alternate-breath oscillation of end-tidal PCO2 (between +5 and +15 torr above the eupnoeic level) and/or PO2 (between 80 and 45 torr) were studied at rest and during mild cycle ergometer exercise. There was often alternation in inspiratory and expiratory tidal volumes and mean flows, and in expiratory duration, but not in inspiratory duration. The latency of responses, estimated by cross-correlation, corresponded closely to the lung-ear transport delay (measured by oximetry). There were two general patterns of response: in-phase, with inspiratory responses leading expiratory, and, more often, out-of-phase, with expiratory responses leading inspiratory. These patterns were associated with arrival of the onset of the alternating signal at the ear in inspiration and expiration, respectively. It is concluded that the timing of alternating humoral signals at the carotid bodies in relation to the phase of respiration determines the pattern of inspiratory-expiratory response, and that expiratory events can be independent of the previous inspiration.

Respiratory mechanics determined by flow interruption during passive expiration in cats.

We used the interrupter technique to measure the resistance Rinit (equal to the initial change delta Pinit in tracheal pressure divided by flow at interruption) during expiration in six normal anaesthetized-paralyzed cats. By performing interruptions at different points in expiration we found Rinit in each cat to be linearly dependent on flow. By allowing the cats to expire through two different resistances we were also able to demonstrate a volume dependence of Rinit in four of the cats. In addition, we obtained a secondary pressure change delta Pdif in each cat, as the magnitude of the slow change in tracheal pressure in the 2 sec following interruption of flow. delta Pdif was approximately constant over most of the expired volume range, and represented the difference between the static elastic recoil pressure of the respiratory system and the pressure driving flow at any volume during a passive expiration. delta Pdif became larger than delta Pinit towards the end of expiration. Since previously used methods for measuring respiratory system resistance have employed varying combinations of delta Pinit and delta Pdif as the resistive pressure drop, it is clear that measurements of resistance must be made with standard techniques under standard conditions if they are to be compared.

Effects of beta-adrenergic blockade on ventilation and gas exchange during exercise in humans.

The effects of beta-adrenergic blockade induced by intravenous propranolol hydrochloride (0.2 mg/kg) on ventilatory and gas exchange responses to exercise were studied during tests in which the work rate was either increased progressively or maintained at a constant load in six healthy young male subjects. Heart rate during exercise decreased by about 20% and cardiac output, as estimated by a modification of the method of Kim et al. (J. Appl. Physiol. 21: 1338-1344, 1966), by about 15%. The relation between work rate and O2 uptake (VO2) was unaffected by propranolol, whereas maximal O2 uptake (VO2max) decreased by 5% and the anaerobic threshold, estimated noninvasively, was lowered by 23%. The relations between CO2 output (VCO2) and end-tidal CO2 partial pressure (PCO2) and between VCO2 and minute ventilation (VE) were both unaffected. The time constants for changes of VO2, VCO2, and VE during on-transients from unloaded pedaling to either a moderate (ca. 50% VO2max) or a heavy (ca. 67% VO2max) work rate in the control studies were in agreement with previously reported values, i.e., 42, 60, and 69 s, respectively. beta-Blockade was associated with a significantly increased time constant for VO2 of 61 s but with less consistent and insignificant changes for VCO2 and VE. There was a small but significant increase of the time constant for heart rate from 40 to 45 s. It is concluded that propranolol exerts its primary influence during exercise on the cardiovascular system without any discernible effect on ventilatory control.

Reflex effects on human breathing of breath-by-breath changes of the time profile of alveolar PCO2 during steady hypoxia.

The respiratory effects of forced changes of alveolar PCO2 were studied in four healthy human subjects and in one anaesthetized cat. Solenoid valves, triggered by changes in mouth pressure, allowed changes from one inspiratory gas mixture to another, either during expiration (between-breath changes, BBC) or in the middle of inspiration (within-breath changes, WBC). In BBC the subject breathed CO2-free gas in one inspiration, CO2-rich gas in the next, and so on; end-tidal PCO2 alternated regularly from breath to breath by 1.1 kPa. In WBC CO2-free gas was given early in one inspiration and late in the next, with CO2-rich gas late in the former and early in the latter, and so on end-tidal PCO2 was nearly constant from breath to breath. Eight respiratory output variables were analysed. WBC induced small but significant alternation in most of the variables; these effects occurred almost exclusively in runs in hypoxia. The responses were not very different from those seen in BBC. The experiment on the cat showed that the alveolar PCO2 changes predicted during WBC are reflected by changes in pH in the arterial blood. The results confirm predictions based upon observations in the steady state of tube- and reversed-tube breathing in man. It seems likely that the responses are mediated by the arterial chemoreceptors responding to small changes in the profile of the (CO2, H+) oscillation.