Fortimicins A and B, new aminoglycoside antibiotics. III. Structural identification.

The structures of fortimicins A and B have been determined by PMR, CMR, mass spectra and CD combined with chemical degradations. Both antibiotics are pseudodisaccharides and incorporate a novel aminocyclitol, fortamine. In contrast to the diaminocyclitol moieties of known aminoglycosides, fortamine is a 1,4-diamine, contains both N- and O-methyl groups and possesses chiro stereochemistry. Both antibiotics are glycosides of 6-epi-purpurosamine B, but fortimicin A differs from fortimicin B by being a glycyl amide.


BL-S786, a new parenteral cephalosporin. I. A collaborative in vitro susceptibility comparison to cephalothin against 5,762 clinical bacterial isolates.

The in vitro activity of Compound BL-S786 was compared with that of cephalothin against 5,762 clinical isolates by the microdilution broth method. BL-S786 demonstrated a broader spectrum and a significantly lower MIC against the Enterobacteriaceae. Although greater susceptibility to BL-S786 than to cephalothin was exhibited by Serratia marcescens, Proteus morganii and Proteus vulgaris, these three species were generally resistant to both drugs. By contrast, the staphylococci were significantly more susceptible to cephalothin than to BL-S786. Resistance to both drugs was demonstrated by Pseudomonas aeruginosa and other pseudomonads, enterococci and Bacteroides fragilis.


BL-S786, a new parenteral cephalosporin. II. In vitro antimicrobial activity comparison with six related cephalosporins.

BL-S786 was compared by in vitro studies with 6 other parenteral cephalosporins (cefamandole, cefazolin, cefoxitin, cephaloridine, cephalothin and cephradine). The following parameters were assessed: Comparative MICs against a wide variety of bacterial isolates, MIC/MBC comparisons and the effect of inoculum size on the MIC. BL-S786 showed the greatest antimicrobial activity against K. pneumoniae, C. diversus and Salmonella species; was equal to cefamandole against E. coli, E. agglomerans and P. mirabilis; and was second to cefamandole against Shigella, E. tarda, C. freundii, E. cloacae, E. aerogenes and the pathogenic Neisseriae. Essentially no activity against Serratia and Pseudomonas species was observed. Compared to the other cephalosporins tested BL-S786 showed poor activity against staphylococci and streptococci. For most species tested, the MBC of the various cephalosporins was the same or within one dilution of their respective MICs. However, for Enterobacter and indole-positive Proteus species, the MBC of BL-S786 and cefamandole was usually larger than or equal to 8-fold higher than the MICs. Cefoxitin, on the other hand, showed little MIC/MBC variations against indole-positive Proteus species. Inoculum size had only a small effect on the MICs against most gram-negative species--in some instances greater than 64-fold increases in MIC resulted by increasing inoculum size from 10(5) to 10(7) organisms per ml.


A comparison of pivmecillinam and cotrimoxazole in the treatment of simple cystitis in general practice.

Pivmecillinam was compared with cotrimoxazole in the treatment of uncomplicated bacterial cystitis in general practice. Similar bacteriological cure rates were obtained with the two treatments. Although micrococci were judged resistant to pivmecillinam by the disc sensitivity method used, cystitis attributed to these organisms and to resistant strains of Proteus mirabilis responded as readily as those due to apparently sensitive Gram-negative bacteria. Pivmecillinam was well tolerated.


Mecillinam serum levels following intravenous injection: a comparison with pivmecillinam.

Serum mecillinam concentrations have been obtained in 6 volunteers after intravenous injection of 200 mg mecillinam and two 200 mg tablets containing pivmecillinam hydrochloride. Initial concentrations were between 6 and 9 mg/1 and peak concentrations of about 2-0 mg/1 occurred at 1 to 1-5 h after the tablets. Analysis of the intravenous data shows the concentration/time curve to be biphasic and similar to that previously reported for penicillin G. The biexponential curves describing the data have been calculated using nonlin. Comparison of the areas under the serum mecillinam concentration/time curves suggests a bioavailability of 65 to 70% for the tablets. However, the two preparations contain therapeutically equivalent amounts of antibiotic, as two 200 mg tablets of pivmecillinam contain 30% more mecillinam than the 200 mg injection.


Cardiopulmonary baroreflexes: effects of pulmonary congestion and edema.

Systemic vasodepressor reflexes were initiated in pump-oxygenator perfused dogs by separately pressurizing the pulmonary vessels and the left cardiac chambers. Pulmonary vascular pressurization caused transient systemic vasodilation of a magnitude proportional to stimulus pressure over the range 0-65 cmH2O. The sensitivity of this reflex was sigificantly less than that of the left heart baroreflex. Mild pulmonary edema produced by a period of sustained congestion, and moderate edema, caused by sustained congestion in the presence of alloxan, had no discernible effect on systemic vasomotor tone or on subsequent pulmonary vascular baroreflexes. By comparison of these results with earlier studies in similar preparations I concluded that pulmonary arterial baroreflexes could alone produce the response obtained by pressurizing the entire pulmonary vascular bed. Although it was anticipated that type-J, irritant, and stretch receptors would be affected by congestion, no systemic vascular effects attributable to them were seen.


Effects of exercise on fluid exchange and body composition in man during 14-day bed rest.

To determine the cause of the body weight loss during bed rest (BR), fluid balance and anthropometric measurements were taken from seven men (19-21 yr) during three 2-wk BR periods which were separated by 3-wk ambulatory recovery periods. Caloric intake was 3,073 +/- 155 (SD) kcal/day. During two of the three BR periods they performed supine isotonic exercise at 68% of VO2max on the ergometer for 1 h/day; or supine isometric exercise at 21% of maximal leg extension force for 1 min followed by a 1-min rest for 1 h/day. No prescribed exercise was given during the other BR period. During BR, body weight decreased slightly with no exercise (-0.43 kg, NS), but decreased significantly (P less than 0.05) by -0.91 kg with isometric and by -1.77 kg with isotonic exercise. About one-third of the weight reduction with isotonic exercise was due to fat loss (-0.69 kg) and, the remainder, to loss of lean body mass (-0.98 kg). It is concluded that the reduction in body weight during bed rest has two major components: First, a loss of lean body mass caused by assumption of the horizontal body position that is independent of the metabolic rate. Second, a loss of body fat content that is proportional to the metabolic rate.


Skin blood flow and sweating changes following exercise training and heat acclimation.

Eight subjects underwent an exercise training program (10 days at 75% VO2max for 1 h/day at 25 degrees C db/13 degrees C wb) and a heat-acclimation program (10 days at 50% VO2max for 1 h/day at 35 degrees C db/32 degrees C wb). The relations of chest sweat rate and of forearm blood flow to internal temperature were determined for each subject at a 25 degrees C ambient temperature before training, between training and acclimation, and following acclimation. Training shifted the vasodilation and sweating thresholds toward lower internal temperatures, and acclimation further lowered these thresholds. All threshold shifts were statistically significant (P less than 0.05). Training and acclimation both appeared to increase the slope of the sweating relation, but these effects were not statistically significant. Changes in the slope of the blood flow relation were small and inconsistent. Since arm blood flow is higher at any given internal temperature after acclimation, the lower blood flow which is reported to accompany heat acclimation must result from the lower body temperatures.


Lung recoil and gas trapping during oxygen breathing at low lung volumes.

If airways are closed at lung volumes less than the closing volume (CV), there should be correlations among 1) the volume of trapped N2 (VTN) during N2-washout performed below CV, 2) the increase in static lung recoil pressure (delta P) while breathing below CV after denitrogenation compared with breathing air (due to absorption atelectasis distal to closed airways), and 3) the CV. Static inspiratory pressure-volume (PV) curves and CV were measured in 18 seated normal volunteers (ages 24-48 yr). Subjects then breathed air for 30 s and O2 for 2.5 min at RV + 0.6 liter (LVB-air), and an inspiratory PV curve and VTN were determined. While still breathing O2, the subjects repeated the 3 min of low-volume breathing (LVB-O2). There was a significant (P less than 0.001) delta P with LVB but no difference between delta P (LVB-air) and delta P (LVB-O2). CV was not related to VTN or to either delta P. VTN was not related to delta P (LVB-O2)--delta P (LVB-air) nor to delta P (LVB-air), but was related to delta P (LVB-O2). Evidence of airway closure could not be demonstrated in all subjects by LVB and when present showed no correlation with CV.


Effect of transpulmonary and vascular pressures on rate of pulmonary edema formation.

The effect of transpulmonary pressure (Ptp) on rate of extravascular fluid accumulation in isolated canine left lower lobes perfused at constant vascular pressures was investigated. Changes in rate of extravascular fluid accumulation were estimated by changes in rate of slow weight gain. Mean inflow pressure (Ppa) was constant at 34 cmH2O. Mean outflow pressure (Ppv) was constant at either 27.2 cmH2O or 13.6 cmH2O. When increasing Ptp is associated with a derecruitment of vascular beds as the lung changes from predominantly zone III toward zone II, or zone I, there is a decrease in rate of weight gain. When increasing Ptp is not associated with a derecruitment of vascular beds, it is impossible on the basis of these experiments to predict the change in rate of weight gain.


Specific airway conductance in relation to postconceptional age during infancy.

Thoracic gas volume (TGV) and airway conductance (Gaw) were measured in 69 healthy infants during the 1st yr of life, using an adaptation of the whole body plethysmographic technique in which a heated rebreathing system was used to eliminate temperature differences in respired gas. There was highly significant correlations between TGV and body wt (r=0.99), and between Gaw and TGV (r=0.97) in all the infants. However, the relationship between Gaw and TGV (i.e., specific conductance (SGaw)) was dependent on postconceptional age (PCA) in preterm infants, falling from 0.50 s-1-cmH2O-1 at 31 wk to 0.32 s-1-cmH2O-1 at 40 wk PCA. Gestational age and postnatal age per se did not affect this relationship, showing it to be a maturational event unaffected by the time of birth. After 40 wk, the decrease in SGaw was far more gradual throughout the 1st yr of life. Black and Chinese infants were found to have significantly higher values for SGaw than their White counterparts, which may be due to anatomical differences in nasal structure.


A new apparatus for the accurate measurement of airway resistance in infancy.

A new heated rebreathing system has been developed for the measurement of thoracic gas volume (TGV) and airway resistance (Raw) in infants by the plethysmographic technique. The apparatus has a linear response to flow rates between 0-160 ml-s-1, a dead space of 12 ml and a resistance of 5 cmH2O-1-1-S-1 at a flow rate of 60 ml-s-1. The inclusion of pneumatically operated valves in the apparatus is a major improvement over previous methods of occlusion for TGV measurements. The infant is allowed to rebreathe saturated gas at body temperature through the heated system, thus overcoming a potential source of error when measuring Raw in infants. Using this apparatus, the average coefficient of variation was 3.7% for TGV and 5.9% for Raw.


DC proportional heater-cooler temperature controlled for physiological research.

A DC proportional temperature control circuit for driving resistive heaters and Peltier thermoelectric devices in physiological research is described. The instrument, supplying +/- 12.5 V DC at 8 A is designed for temperature control of anesthetized animals, generation of hot or cold thermal stimuli, and control of thermal environments.


Preparation of matched reagents for use with the Scholander gas analyzer.

Scholander's method of gas analysis requires that the solutions for CO2 absorber, O2 absorber, and acid-rinse be matched in terms of water vapor tension throughout the analysis. Any difference in vapor pressure between either or both of the absorbing solutions and the indicator drop (composed of acid-rinse) will produce a measurable volume change which cannot be attributed to the presence of absorbable gases. This paper describes a practical and quantitative method for preparing reagents whose vapor pressures are matched. A fixed acid-rinse formulation was used throughout. A CO2 absorber prepared from 1.35 N KOH and an O2 absorber prepared from 0.76 N KOH were both matched in terms of vapor pressure with Scholander's acid-rinse solution. Analysis of atmospheric air provided a check on the accuracy of the technique. The values obtained were O2 20.94%, CO2 0.03%, and N2 (balance) 79.04%.


Effects of thoracic dorsal rhizotomies on the respiratory pattern in anesthetized cats.

Experiments were conducted to determine if thoracic wall proprioceptor afferents are involved in the modulation of respiratory activity during eupnea. The effects of elimination of thoracic wall afferents (thoracic dorsal rhizotomies (TDR) on tidal volume (VT), frequency (f), inspiratory time (ti) and expiratory time (te) were studied in vagotomized cats anesthetized with diallylbarbituric acid (Dial). Dorsal rhizotomies 1-12 resulted primarily in a decreased VT and ti, and an increased f. Further experiments were performed to determine if these changes in respiratory pattern could be correlated with known reflexes from the middle and lower intercostal muscles, or lungs, via thoracic dorsal roots. Afferents from these sources were eliminated by TDR 5-9, 10-13, and 1-4. TDR 1-4 had no significant effect on the respiratory pattern. TDR 5-9 and TDR 10-13 produced changes similar in direction to TDR 1-12. The results indicate that: a) afferents 1-4 from the upper intercostal muscles and lungs (sympathetic afferents) do not contribute significantly to the control of the spontaneous respiratory rhythm, and b) afferents via the middle thoracic roots, 5-9, and the lower thoracic roots, 10-13, contribute significantly to the rhythm. The results do not completely correlate with known intercostal reflexes, but it is suggested that elimination of intercostal muscle proprioceptor afferents is responsible for the observed effects of thoracic dorsal rhizotomies.


Effect of lung collapse on compensatory lung growth.

The effect of unilateral lung collapse on compensatory lung growth was studied in 3-wk-old (young) and 10-wk-old (adult) male Long-Evans rats. Under light halothane anesthesia, left lung collapse was produced by injection of dental plastic through a thracheostomy cannula. The rats were killed either 5 days or 4 wk later. At 5 days postcollapse, the 3-wk-old rats had an increase in DNA over sham controls (21%) (P less than 0.05), with no significant change in the protein/DNA ratio. At 4 wk postcollapse, increases in DNA over shamoperated controls were observed in both the 3-wk-old (58%) and the 10-wk-old (28%) rats, whereas the protein/DNA ratio at both ages was significantly less than that of sham controls (P less than 0.05). Thus, unilateral lung collapse stimulates growth of the contralateral lung by cellular hyperplasia, not hypertrophy, both in the young and adult rat. The hyperplasia begins within 5 days after collapse and is greater by 4 wk. The extent of hyperplasia is greater in the young than in the adult rat.


Stress-free equilibrium volume of the lung.

An experimental procedure was developed to measure the stress-free equilibrium volume of intact lobes from the lung of the dog. The procedure eliminates body, gravitational, interfacial, and externally applied forces to study the contribution of the tissue forces. The equilibrium gas volume was found to be 1.74 +/- 0.20 times the tissue volume and was independent of the volume history. This is smaller than the minimal air volume found by others and about one half the functional residual capacity (FRC) volume. The lung was found able to resist compressive loading implying a "finite buckling load" for the aveolar septa. A simple calculation shows the length-to-thickness ratio to be 7.1 at the stress-free equilibrium volume.


Diaphragmatic contraction and the gradient of alveolar expansion in the lateral posture.

Using 133Xe we measured the vertical distribution of regional volume in four subjects in the lateral decubitus posture at 20, 40, 60, and 80% of vital capacity (VC). To study the influence of diaphragmatic tone, all measurements were performed either when transdiaphragmatic pressure (Pdi) was low, i.e., diaphragm was "relaxed" (RD) or during voluntary diaphragmatic contraction (VDC). The latter was achieved by tensing the abdominal muscles while keeping the glottis open. Under both conditions the gradient of alveolar expansion tended to be curvilinear, with a discontinuity at the level of the mediastinum. At all lung volumes the difference in regional volume between dependent and nondependent lung regions was less during VDC than during RD. At 70% total lung capacity (TLC) this difference, expressed as percent of regional TLC (%TLCr), decreased from 19.7 +/- 1.7 (mean +/- 1 SE) %TLCr during RD to 3.9 +/- 1.5% TLCr during VDC. It is likely that diaphragmatic tension influences the pleural pressure gradient and regional volume distribution 1) by modifying the transmission of the abdominal hydrostatic pressure gradient to the thorax, and 2) by an upward displacement of the mediastinum.


Effects of exercise and eucapnic hyperventilation on bronchial clearance in man.

The effects of exercise and eucapnic hyperventilation on bronchial clearance were assessed in 10 healthy nonsmoking adults. A 99mTc-albumin aerosol was inhaled as a bolus in late inspiration under controlled conditions to produce deposition primarily in large airways. Lung retention of radioactivity was quantified using a gamma camera and subsequent computer analysis. Compared with quiet breathing (control), exercise significantly speeded clearance (P less than 0.05). Resting eucapnic hyperventilation at levels similar to those achieved during exercise produced less speeding. Compared to control conditions these changes may be brought about by a) mechanical effect of increased lung movement, and b) effects on the autonomic nervous system, mediated via the parasympathetic pathway producing stimulation of airway mucus glands and/or sympathetic stimulation of cilia due to exercise-induced catecholamine release.


Significance of sympathetic innervation of pulmonary vessels in response to acute hypoxia.

The role of the autonomic nervous system on the pulmonary vascular response to hypoxia was studied in anesthetized open-chest dogs. Acute hypoxia elicited reversible decreases in the slope of the instantaneous diameter-pressure relationship of the main pulmonary artery. These decrements in vascular compliance during hypoxia were prevented by either bilateral thoracic sympathectomy or by section of the cervical vagus and glossopharyngeal nerves and were reproduced by bolus injections of lobeline (0.1 mg) at the root of the aorta. Using separation of the airways with a tracheal divider, the effects of systemic and of alveolar hypoxia on vascular resistance of the left caudal lobe were compared. Systemic hypoxia in the absence of alveolar hypoxia had no effect on pulmonary vascular resistance. Alveolar hypoxia elicited reversible increases in vascular resistance that were not prevented by sympathectomy. It is concluded that at least two vascular sites of action and two mechanisms are involved in the pulmonary vascular response to hypoxia.


Adaptation to short-term respiratory effects of ozone in men exposed repeatedly.

To investigate whether adaptation which modifies some acute effects of ozone (O3) exposure can develop in humans, six male volunteers with respiratory hyperreactivity were exposed in a controlled environment chamber to 0.5 ppm O3 2h/day for 4 successive days under conditions stimulating ambient pollution exposures. One subject showed little measurable response, while five showed function decrement on exposure days 1-3 which was largely reversed by day 4. Symptom responses generally paralleled the physiological responses. These results suggest that at least some humans adapt to O3 exposure at concentrations occurring in severe community air pollution episodes, to the extent that obvious acute respiratory effects are prevented. Other adverse effects of O3 may not be prevented by this adaptation.


Morphometry of the distal air spaces in lungs of aging dogs.

Changes in pulmonary morphometric measurements with age were examined by manual and automated methods in 14 normal beagle dogs aged 289-3,694 days. The study was instituted as a statistical adjunct to previous morphological studies on these dogs that showed anthracosis and associated dilatations in the distal airways in aged dogs. With increasing age, the volumetric density of alveolar ducts increased concomitantly with decreases in the volumetric densities of alveoli and alveolar parenchymal tissue. The numerical and surface densities of alveoli also decreased with age. A comparison of an automated method with accepted manual methods showed no significant differences in stereological measurements of the distal air spaces with the exception of an overestimation of surface density by the automated method. The automated method was found to be a satisfactory method for evaluating the relative differences in alveolar surface densities and the volumetric densities of respiratory air space and parenchymal tissue.


Time, rate, and temperature factors in the onset of high-pressure convulsions.

An interrupted compression profile technique was used to develop data to separate the effects of time and pressure factors governing increase of high-pressure neurological syndrome (HPNS) convulsion threshold pressures (the compression rate effect) during different compression profiles. A single differential equation fits all data available to date for compression rate effect on convulsion thresholds of CD-1 mice (three distinct types of compression profile; mean compression rates 12-1,000 atm/h). The process leading to increase in HPNS convulsion pressure is initiated at the very beginning of compression, proceeds at increasingly rapid rates as higher pressures are attained, and approaches a limiting upper convulsion pressure. The convulsion threshold pressure in any given experiment is independent of the compression rate prevailing during the time immediately preceding onset of the seizure. The magnitude of the compression rate effect in the CD-1 mouse is independent of chamber temperature over a range of 27-36 degrees C, and rectal temperatures of 29.2-37.5 degrees C. The bearing of these results on the design of optimal compression schedules and on the analysis of the neurological mechanisms underlying the HPNS is discussed.


High pressure and intravenous steroid anesthesia in rats.

Anesthesia produced by the intravenous steroid agent, Althesin, was studied in Sprague-Dawley rats with and without high pressure of helium gas up to 100 atmospheres absolute (ATA). There were no cumulative or adaptive changes in Althesin requirement at normal pressures over 6-h periods. However, the apparent potency of the agent was reduced by 43% by the addition of 68 ATA helium. Subanesthetic doses of Althesin protected against the onset of convulsions and coarse tremors associated with the high pressure neurological syndrome. It is concluded that the steroid anesthetics may have a place in human diving technology and that the mechanisms associated with the anesthetic-pressure interactions are consistent with the critical volume or lipid bilayer fluidity hypothesis.


Diaphragmatic fatigue in man.

The time required (tlim) to produce fatigue of the diaphragm was determined in three normal seated subjects, breathing through a variety of high alinear, inspiratory resistances. During each breath in all experimental runs the subject generated a transdiaphragmatic pressure (Pdi) which was a predetermined fraction of his maximum inspiratory Pdi (Pdimax) at functional residual capacity. The breathing test was performed until the subject was unable to generate this Pdi. The relationship between Pdi/Pdimax and tlim was curvilinear so that when Pdi/Pdimax was small tlim increased markedly for little changes in Pdi/Pdimax. The value of Pdi/Pdimax that could be generated indefinitely (Pdicrit) was around 0.4. Hypoxia appeared to have no influence on Pdicrit, but probably led to a reduction in tlim at Pdi greater than Pdicrit for equal rates of energy consumption. Insofar as the behavior of the diaphragm reflects that of other respiratory muscles it appears that quite high inspiratory loads can be tolerated indefinitely. However, when the energy consumption of the respiratory muscles exceeds a critical level, fatigue should develop. This may be a mechanism of respiratory failure in a variety in a variety of lung diseases.


Alcohol, respiration, skin and body temperature during cold water immersion.

Subjects who had not been exercising, were immersed for 20 min in water at 13 degrees C after ingestion of alcohol. During the immersion period, total ventilation, end-tidal PCO2, rectal temperature, aural temperature, and mean skin temperature were recorded. Control experiments were carried out at the same water temperature. Blood samples (3 ml), taken immediately before the immersion period, were analyzed by gas liquid chromatography. The mean blood alcohol level was 90+/-11.2 mg-(100 ml)-1. There was no significant difference in ventilatory responses, rectal temperatures, aural temperatures, or mean skin temperatures achieved during the two cold water immersions. It would appear that for a 20-min immersion at 13 degrees C, relatively high blood alcohol levels do not affect ventilatory responses or increase body heat losses.


Interaction of central nervous system effects of high pressures with barbiturates.

The interactions of phenobarbital, barbital, and pentobarbital with high pressures of heliox were explored. Principal features of the complex results include: double peaks in the time course of convulsion thresholds (Pc); an early peak and a shoulder in the time course of pressures reversing anesthesia (Pa); far steeper dose-response curves for Pa than for Pc; selectively greater anticonvulsant effect for phenobarbital than for the other barbiturates; and enhancement of Pa with simultaneous depression of Pc by reserpine in phenobarbital-pretreated mice. The data indicate the existence of at least two discrete sites of interaction between barbiturates and high pressure, reflected by Pc and Pa. The implications of the data for the development of biophysical theories of pressure reversal of anesthesia and anti-high-pressure neurological syndrome action of anesthetics are discussed, together with implications for the experimental study of convulsant and anticonvulsant agents, and their applications to underwater physiology.


Rebreathing pulmonary capillary and tissue volume in normals after saline infusion.

A rebreathing technique was utilized to assess changes in diffusing capacity (DCO), pulmonary capillary blood volume (Vc), pulmonary parenchymal tissue volume (Vt), and cardiac output (Qc), after infusion of 2 liters of 0.9% saline intravenously in 13-25 min in five healthy subjects. Blood hemoglobin concentration decreased an average of 17%. Vc increased strikingly in all five subjects. No significant changes in Vt, or in Vt per unit lung volume were observed. Radiographic evidence of interstitial pulmonary edema was present in four of the five subjects. Radiographic total lung capacity was reduced significantly in four of the five subjects. Significant reductions in forced vital capacity (FVC), forced expiratory volume in 1.0 and 3.0 s, and mean forced expiratory flow during the middle half of the FVC occurred in three of the five subjects. No dyspnea, cough, or physical examination abnormalities of lungs or heart occurred. This noninvasive, ventilation-limited, rebreathing technique appears capable of detecting early changes in pulmonary congestion, at a time when definitive radiographic changes and changes in the physical examination are absent. It appears capable of detecting the increase in Vc associated with hypervolemia in man.


Effect of hyperbaric oxygen exposure on pulmonary clearance of 5-hydroxytryptamine.

Clearance of 5-hydroxytryptamine (5-HT) by the lungs of normal and vitamin E-deficient rats was evaluated following a 60-min exposure to 100% oxygen (O2) at 4 ATA (HBO). After exposure, lungs were isolated, ventilated, and perfused, with a recirculating system used for measurement of 5-HT clearance. Control lungs were obtained from rats exposed to air at 1 ATA. In control normal rats, fractional clearance of 5-HT was 0.78+/-0.03 (mean+/-SE). Following HBO 5-HT clearance was 0.55+/-0.04 (P less than 0.01). In control vitamin E-deficient rats. 5-HT clearance was 0.85+/-0.05 and was decreased to 0.46+/-0.03 (P less than 0.001) following HBO. To evaluate the effect of recovery time after HBO on 5-HT clearance, separate groups of rats were killed at varying intervals post-HBO. In normal rats, 5-HT clearance had returned to control levels by 3-4 after HBO; in vitamin E-deficient rats, clearance remained unchanged 4 h after HBO and was only 74% (P less than 0.001) of control values 24 h post-HBO. These results indicate that depression of pulmonary 5-HT clearance occurs in rats due to hyperoxia and is potentiated by vitamin E deficiency. This represents a reversible alteration of lung function which requires vitamin E for complete recovery.


Quantification of diaphragmatic EMG response to CO2 rebreathing in humans.

To determine a reliable quantitative method of measuring diaphragmatic EMG (EMGdi), electrical activity of the diaphragm was obtained via an esophageal electrode during CO2 rebreathing in 6 normal males and processed three different ways: 1) integration (area), 2) as a moving time average, and 3) as a moving time variance. Integrated activity was quantified in terms of total activity and inspiratory activity. In addition, average total activity and average inspiratory activity were calculated. Moving average and moving variance were analyzed in terms of rate of rise (slope) and peak activities. All integration parameters, except average inspiratory activity, were poorly correlated to changes in PCO2, minute ventilation, and inspiratory muscle force, during rebreathing. Moving average and variance responses to rebreathing were linear with high correlation coefficients, with the slope measures showing the overall best correlations. There was no significant difference between average and variance EMGdi parameters in their responses to rebreathing. Time-related quantification of EMGdi, including average inspiratory activity, and particularly moving average and moving variance, appear to be reliable methods for quantitating neural drive to the respiratory muscles during CO2 rebreathing.


Changes in airway mast cells and histamine caused by antigen aerosol in allergic dogs.

We studied the effect of inhaled specific antigen on airflow resistance, histamine concentration, and mast cell numbers in airway tissues in allergic dogs. In each of six allergic dogs with open chests, inhalation of specific antigen aerosol (Ascaris suum) increased airflow resistance from 0.30+/-0.40 (mean+/-SE) to 2.8+/-0.41 cmH2O/1 per s (P less than 0.05); decreased mast cell number from 38.6+/-3.2 to 24.3+/-4.5 mast cells/mm2 (P less than 0.05); decreased histamine (per mg airway tissue) from 5.3+/-0.4 to 3.5+/-0.2 ng (P less than 0.05) in lobar bronchi 5-7 mm in diam; and released histamine into the blood perfusing the lung, control arterial plasma histamine; 2.8+/-0.64 ng/ml; antigen: 154+/-1.7 ng/ml (P less than 0.005). Specific antigen caused no significant changes in mast cells or histamine in bronchi 1-2 mm in diam. Control aerosols of nonspecific antigen or methacholine did not change levels of mast cells or histamine in airway tissues. These results suggest that experimental canine asthma involves local airway reactions with degranulation of mast cells and histamine release, as well as vagally mediated reflex bronchoconstriction.


Effects of water immersion on lung volumes: implications for body composition analysis.

Lung volumes of 20 healthy young men were measured before and after water immersion to the neck level. Immersion resulted in significant decreases (P less than 0.01) in forced vital capacity (FVC) (8.9%), expiratory reserve volume (ERV) (61%), total lung capacity (TLC) (5.6%), and functional residual capacity (FRC) (2.9%). Significant increases were observed in inspiratory capacity (IC) (10%) and residual volume (RV) (6.7%). The increase in RV was attributed to a possible "stiffness" of the lung tissue caused by pulmonary vascular engorgement. Densitometric analysis was made on each subject using hydrostatic weighing techniques. Subsequent calculation of body density and per-cent body fat indicated significant (P less than 0.01) differences when using RV measured on land and in water. Body fat was 14.0% using the land RV in the computation of density and decreased to 13.4% using the RV measured in water. It was concluded that when obtaining body density values. RV should be measured concurrently while the subject is in the water.


Glycogen, lactate, and alanine changes in muscle fiber types during graded exercise.

The effect of different speeds of incline running (16-48 m/min) on changes in glycogen, lactate, and alanine concentrations in different skeletal muscle fiber types and in blood were investigated in nontrained adult female rats during 5-min bouts of exercise. Glycogen depletion occurred extensively (25-86%) in fast-oxidative-glycogenolytic (FOG) and in slow-oxidative (SO) fibers over the range of speeds studied, whereas it occurred primarily (76%) at 48 m/min in the fast-glycogenolytic (FG) fiber type. No marked increases in lactate over resting control levels occurred in the FOG fibers. However, compared to resting levels, lactate increased over fourfold in FG muscle during running at 48 m/min and blood lactate concentrations paralleled these changes. No changes occurred in alanine concentration in FOG muscle and in blood under these experimental conditions, whereas there were approximately 70% increases in FG and SO fibers during moderate-to-heavy exercise. These findings suggest that elevations in blood lactate of rodents primarily result from lactate production in the FG fiber type.


An anesthetized dog heatstroke model.

This study was undertaken to develop an anesthetized dog heatstroke model. Forty-six animals were anesthetized with pentobarbital sodium (25 mg/kg) intravenously, and maintained at an ambient temperature of (42-46 degrees C) with a water-heated blanket over 2.5-3.0 h until rectal temperatures rose to 43.0-44.5 degrees C. Animals then cooled passively until death occurred or until 18 h elapsed, and were prepared for autopsy. Liver, kidney, and brain temperature, mean weighted skin temperature, mean weighted surface heat loss, and metabolic rates were obtained. There were no significant differences between liver, kidney, brain, and rectal temperatures during the heating and cooling periods. Cardiac output rose to 127% of initial value, and dropped rapidly to zero at 43.4 degrees C rectal temperature. The rapid decline was accompanied by a doubling of heart rate and a rapid drop in blood pressure and respiratory rate. Cheyne-Stokes respiration and apnea preceded bradycardia followed by asystole or ventricular fibrillation. Certain serum constituents demonstrated modest elevations suggestive of widespread tissue damage. Autopsy did not reveal a clear pattern of heat injury, with the exception of consistent congestion of the major organs and karyorrhexis of lymphocytes. These data are in agreement with similar data from human heatstroke victims and other heatstroke modeling in dogs, and support the concept that the anesthetized dog can in many respects provide an adequate model for human heatstroke.


Diaphragmatic function during immersion.

Diaphragmatic function during immersion to midneck level was studied in upright mongrel dogs, using constant electrophrenic stimulation. Effectiveness of diaphragmatic contraction was analyzed in terms of inspired volume (VT) (with airways open), and change in intrathoracic pressure (Pmus) (with the respiratory system occluded). Hydrostatic compression of the immersed body decreased functional residual capacity (FRC) to 55% base-line value (FRCO), resulting in a 2.8-fold increase in Pmus. In spite of this Pmus increase, VT often decreased during immersion, averaging only 83% VTO (base-line value in air). Hence, immersion was associated with a marked stiffening of the respiratory system. The Pmus increase during immersion persisted after restoration of FRC to FRCO, and was related to diaphragmatic length being greater in water than in air under condition of iso-lung volume. In all, there were three factors affecting diaphragmatic function during immersion: FRC reduction, change in thoracic configuration, and stiffening of the respiratory system.


Effect of training on the response of plasma glucagon to exercise.

This study was undertaken to determine whether the increase in plasma glucagon concentration that occurs in response to prolonged exercise is modified by endurance exercise training. Eight subjects participated in an exercise program, consisting of running and bicycling, 4 days/wk for 10 wk. The training program resulted in an average increase in VO2 max of 18%. The average increase in plasma glucagon during a 60-min long bicycle exercise test that required 60% of the subjects' VO2 max was 107+/-28 pg/ml, from 116+/-14 pg/ml at rest to 223+/-37 pg/ml after 60 min of exercise, prior to training. After training the same absolute work rate resulted in an increase in plasma glucagon of only 20+/-6 pg/ml, from 125+/-20 to 145+/-16 pg/ml (P less than 0.02). A similar blunting of the glucagon response to exercise was seen during work of the same relative intensity after training. Plasma insulin concentration decreased from 18.1+/-2.5 to 7.6+/-1.6 muunits/ml during the 60 min of exercise before training. A similar decrease in insulin concentration was seen at the same relative work rate after training. However, the decrease in plasma insulin at the same absolute work rate, from 18.5+/-3.0 to 12.5+/-1.8 muunits/ml, was significantly smaller after training (P less than 0.05).


Capillary proliferative activity in myocardium and skeletal muscle of exercised rats.

The capillary proliferative activity in heart and skeletal muscle was studied by autoradiography after in vivo injections of [3H]thymidine in swimming exercised rats. Swimming exercise led to hypertrophy of the myocardium and the muscle fibers of the fore- and hindlimbs. When compared with normal controls a highly significant increase in nuclear incorporation of [3H]thymidine was found in the cells of the myocardial capillary walls. No increased incorporation of [3H]thymidine was found in capillary wall cells of fore- and hindlimb muscles. The findings confirm previous observations of a significant neoformation of myocardial capillary blood vessels in swimming-induced cardiac hypertrophy, whereas any capillary neoformation in the hypertrophying skeletal muscles of this experimental model is insignificant.


Recurrent heat exposure: enzymatic responses in resting and exercising men.

Heat acclimatization was induced in a group of healthy male test subjects by repetitive treadmill walking (5.6 km-h-1, 49 degrees/27 degrees C dry/wet bulb, 90 min-day-1, 7 days). A second group of men, paired for maximal O2 consumption and body weight, remained sedentary under identical environmental conditions. Total plasma protein increased significantly after 45 (P less than 0.05) and 90 (P less than 0.025) min of exercise on the first day of heat exposure, yet after 7 days no increments occurred. Even after heat acclimatization was achieved (day 7), plasma levels of creatine phosphokinase increased during the 90-min walk in the heat (time O vs. 90, P less than 0.025), as was also the case on day 1 (P less than 0.05). Levels of lactate dehydrogenase, glutamate-oxaloacetate transaminase, and glutamate-pyruvate transaminase were not significantly affected by exercise in the heat either before or after heat acclimatization. No correlations could be drawn between base-line enzyme levels and state of physical conditioning.


Relationship of thoracic volume and airway occlusion pressure: muscular effects.

The occluded airway pressure generated by contraction of inspiratory muscles was measured in spontaneously breathing, anesthetized, vagotomized cats at thoracic volumes below, at, and above true FRC (range, -8 to +28 ml/kg). A relatively constant neural drive at all volumes was shown by recordings of integrated phrenic nerve and external intercostal muscle activities. Peak occlusion pressure declined progressively with increasing thoracic volume and increased with occlusion volumes below FRC. Occlusion pressures measured early in inspiration (0.3 and 0.5 s) decreased similarly. Since occluded airway pressure was a satisfactory index of muscle force output of all inspiratory muscles, the study demonstrates that inspiratory muscle performance decreases with increasing thoracic volume throughout the range studied. Analysis of all occluded breaths shows that, quantitatively, the decline in muscle performance from that occurring at true FRC was -1.9% per ml/kg volume change from FRC. Zero effective muscle pressure generation occurred at 52 ml/kg above FRC, or 2.8 times FRC. These findings have important implications for the use of occlusion pressures and ventilatory responses in respiratory control system studies, since changes in FRC may affect muscle performance.


Respiratory parameter estimation using forced oscillatory impedance data.

The frequency dependency of the magnitude and phase angle of total respiratory impedance was measured in apneic dogs at functional residual capacity during forced oscillation by a special electronics unit. Regression analysis of these data yielded estimates of total respiratory resistance (RFO), inertance (IFO), and compliance (CFO). After correcting for the effects of the endotracheal tube, mean control values (+/-SE) of RFO, IFO, and CFO for the clinically normal dogs were 1.30+/-0.10 cmH2O-1-1-s, 0.0114+/-0.0022 cmH2O-1-1-s2, and 0.0306+/-0.0009 1-cm H2O-1, respectively. Estimates obtained with added resistance, a less dense gas, and abdominal weighting were consistent with predicted effects. In four dogs with mild respiratory symptoms, mean RFO was significantly elevated with no change in IFO or CFO. Independent measurements of resistance and compliance during tidal ventilation correlated well with RFO (r=0.87) and CFO (r=0.80), but RFO and CFO were, on the average, 71% of the tidal breathing values. Thus, the method provides precise estimates of RFO, IFO, and CFO, and allows detection of small changes in these parameters.


Estimation of composition of weight loss in man: a comparison of methods.

Five different procedures were used to estimate composition of weight loss by six obese individuals maintained on low-calorie regimens: 1) energy-nitrogen (E-N) balance; 2) total body water-nitrogen balance (TBW-N); 3) total body water (TBW); 4) total body potassium (TBK); and 5) an anthropometric method. Quantity of weight lost by obese subjects during comparison periods lasting 25-40 days ranged from 7.9 to 17.1 kg. Expressed as percent of aggregate weight loss, collective fat loss was 47.6 by E-N balance; 46.3 by TBW-N balance; 51.7 by TBK; 50.9 by anthrometry; and 33 by TBW. The E-N balance method yielded a tight cluster of individual means, whereas the other procedures gave a wide scatter of means. For short-term studies, the E-N balance procedure, although tedious and difficult, should yield results less variable than those generated by any of the other methods examined.


Thoracic reflexes stabilizing loaded ventilation in normal and cord-injured man.

The ability to regulate tidal volume was quantified in 24 cervical cord-injured patients by comparing the "effective" elastance (E'rs) observed during single-breath elastic loads to the passive respiratory elastance (Ers) measured separately with relaxed muscles. Thirteen patients, the majority of whom had an abnormal subjective perception of loading, usually failed to demonstrate any tidal volume compensation under load conditions (i.e. E'rs less than or equal to Ers). This impaired tidal volume defense, which could not be attributed either to diaphragmatic paralysis or rib cage instability, was accompanied by a markedly shortened inspiratory duration and, hence, a prematurely terminated phrenic motoneuron output. Conversely, those patients who developed a larger then previously reported "effective" elastance (i.e., E'rs greater than or equal to 2Ers) described loading as an inspiratory obstruction, as did all our normal subjects, and usually prolonged loaded inspiratory duration, probably by a consciously mediated mechanism. These findings are consistent with the hypothesis that, in spontaneously breathing intact man, thoracic sensory receptors normally maintain phrenic motoneuron output under load conditions and thereby promote tidal volume stability.


Determinants of the prescriptive zone of industrial workers.

The prescriptive zone is the range of climates in which man's body temperature is independent of climatic conditions. The environmental temperatures which define the upper limit of the prescriptive zone (ULPZ) at different work rates were determined in 46 clothed, healthy, male industrial workers; some of the men were job acclimated to heat and some were not. They performed a total of 653 work bouts of low, medium, or high intensity in environments ranging from 11 to 35 degrees C corrected effective temperature (CET) (8-37 degrees C wet bulb globe temperature). Heart rates (HR) and rectal temperatures (Tre) were measured after 1 h of work. The ULPZ was calculated from Tre data. HR's showed a similar pattern of response as Tre's except that the inflection point corresponding to the ULPZ occurred at different environmental temperatures at most of the experiment conditions. About one-third of the work bouts were performed in the summer months and the remainder in the winter. The ULPZ decreased with increasing work rates. At high, but not low, work rates, men who were exposed to heat in the performance of their jobs were more heat tolerant than men who were not heat acclimatized. Both groups were found to be more heat tolerant in the summer months than in the winter.


Elevated alveolar PCO2 relative to predicted values during normal gas exchange.

A negative aADCO2 has been demonstrated during ventilation with hypercarbic gas mixtures and during rebreathing, but has never been demonstrated during normal gas exchange. This anomalous behavior of CO2 was studied by comparing it to the behavior of five infused inert gases during normal gas exchange in 10 anesthetized mongrel dogs. The distribution of VA/Q heterogeneity and the respiratory dead space in the animals was quantitated using excretion-solubility data from the five infused inert gases. The predicted excretion fraction (PACO2/PVCO2) for CO2 was obtained from the inert gas excretion-solubility curve, using a measured solubility for CO2. The measured excretion fraction for CO2 (PACO2/PVCO2), even after correction for Haldane effect, was significantly greater than the predicted fraction (P less than 0.001). This corresponded to an alveolar PCO2 that exceeded the predicted value by a mean of 5.0 Torr.


Hematologic responses to carbon monoxide and altitude: a comparative study.

Groups of 40- and 90-day-old rats were exposed to 500 ppm CO (HbCO=31-43%) and simulated altitude (15,000 ft). Resulting hematologic changes were monitored after 1 day, 1 wk, 3-4 wk, and 9-11 wk of exposure. The two treatments resulted in similar changes in hemoglobin (Hb) and hematocrit ratio (Hct) in the young rats, while in the older group there were several small, but significant differences in these parameters. Mean corpuscular hemoglobin concentration (MCHC) fell 12.4-15.1% during the 1st wk of exposure in all groups except the young altitude rats, which remained at the control value. The former groups returned to control levels by the 3rd to 4th wk of exposure. Red cell 2,3-diphosphoglycerate (2,3-DPG) increased 21.6-26.1% above control during 3-4 wk of altitude exposure, but later (9-11 wk) returned toward control levels. CO exposure resulted  in a sharp decrease in 2,3-DPG after 1 day of exposure only in the young rats. 2,3-DPG level progressively decreased with age in control rats. The effects of CO and altitude although similar in several respects (i.e., Hb, Hct, MCHC) differ with regard to red cell 2,3-DPG.


High-sensitivity device for continuous measurements of tissue O2 uptake.

An analysis system for continuous measurements of O2 uptake by rat diaphragm during several hours is described. The tissue was set in a chamber where a medium was flowing at a known and constant flow rate, temperature, and partial pressure of oxygen (PO2). The decrease in PO2 of the medium was measured with a Clark O2 cathode at the outlet of the chamber. To increase the sensitivity of the system and the signal-to-noise ratio of the measurements, a sweeping device was designed. It provided homogenization of the medium in the chamber. The main characteristics of this analysis system are a) a sensitivity of +/-0.2% of the mean PO2 value of the perfused medium, +/-0.8% of the mean O2 uptake of rat diaphragm, b) a linearity of +/-1.75% in the range of 0+/-200 Torr, c) a stability of +/-0.38% of the initial PO2 value, and d) a time constant of 8 min 30 s. This type of system can be used for high-sensitivity measurements of O2 uptake of any tissue after adaptation of the chamber and of the medium flow rate.


Aspects of on-line digital integration of pulmonary gas transfer.

Digital computers are often used in work physiology to find pulmonary gas transfer on a breath-by-breath basis. A measurement procedure was simulated to determine the effects of sample rate, signal noise levels, and numerical filters on the digital calculations. The results indicated that a trapezoidal rule integration at a sample rate of 30 Hz with no numerical filter can provide satisfactory data. The flow rate and concentration signals should not be out of phase by more than 25 ms. The simple computational procedures give accurate results while minimizing the memory requirements and computation time.


Semiautomatic blood pressure monitor utilizing an electronic sphygmomanometer.

A semicontinuous but noninvasive blood pressure monitoring system is described. It consists of a commercial electronic sphygmomanometer which utilizes a microphone under an occluding arm cuff to detect the Korotkoff sounds, a pressure transducer, and a simple gating circuit. The gate passes the cuff pressure signal to a recorder only when a proper Korotkoff sound is detected. The cuff is rapidly inflated to just above the anticipated systolic pressure, then deflated at a steady rate of 2-6 Torr/heartbeat. When diastolic pressure is passed, the cuff is fully deflated momentarily before repeating the cycle. Systolic and diastolic pressures can be recorded up to 3 or 4 times/min. Spurious signals are rejected by the electronics which process the output of the microphone. This allows the use of the system in experiments on exercising man and in environments where unwanted signals exist. The system offers greater versatility than commercial semiautomatic pressure monitors, at less than half the cost.


Measurement of evaporative water loss in small animals by dew-point hygrometry.

This paper presents the procedures and equations to be utilized for measurement of evaporative water loss (mw), by use of the dew-point hygrometer, in small animals exposed to air containing water vapor in an open-flow system. The system accounted accurately for the water evaporated from a bubble flask. In addition, hygrometric measurements of pulmocutaneous mw in pigeons (Columba livia, mean mass 0.31 kg) agreed closely with simultaneous gravimetric measurements, utilizing a desiccant in the sample stream, in a manner independently of air temperature (Ta, 20 or 40 degrees C), ambient water vapor pressure (PW, 4-16 10(2) Pa), or mw (5-66 mg-min-1). Evaporation in pigeons was independent of PW at 20 degrees C, but increased with decreasing PW at 40 degrees C, suggesting differences in ventilatory adjustments to changes in PW at the two temperatures.


Effect of storage in flour and of particle size on the bioavailability of elemental iron powders for rats and humans.

Elemental iron powders produced by different processes were separated by nitrogen elutriation into fractions based on particle size. The fractions containing fine particles (7-10 micrometer) of electrolytic iron, hydrogen (H2)- and carbon monoxide (CO)-reduced iron, and the whole sample of carbonyl iron (92% less than 11 micrometer) were blended into freshly milled wheat flour and stored at about 21 degrees C. Storing the enriched flour up to 9 months had no effect on the relative biological value (RBV) of iron for rats from each type of iron supplement. Baking bread with flour containing electrolytic, H2-reduced, and carbonyl iron after storage for 10 months also had no effect on the RBV of the iron powders. There was no change in the RBV of iron for rats from flour enriched with electrolytic iron (200 mg/kg) and ascorbic acid (200 and 1665 mg/kg flour) after storage for 1 week and 3 months. The decreases in RBV due to increase in size of particles (7-10 vs. 20-26 micrometer) were significant for H2-reduced iron and CO-reduced iron but not for electrolytic iron and carbonyl iron. The RBVs of elemental iron powders for rats were similar to the values obtained with humans, based on iron absorption after an overnight fast.


Preparation and characterization of aflatox-n B1-1-(O-carboxymethyl) oxime.

A method is described for the preparation and purification of aflatoxin B1-1(O-carboxymethyl) oxime from aflatoxin B1. The overall yield was about 73-83%. The new aflatoxin B1 derivative was characterized by mass, ultraviolet, infrared, and nuclear magnetic resonance spectral analyses, and was nontoxic to 8-day-old chicken embryos when tested at a concentration of 3.48 microgram/egg.


Thin layer chromatographic determination of aflatoxin B1 in eggs.

A method for determining aflatoxin in dairy products was modified for eggs, initially by additional cleanup steps. The modified method proved practical for use in survey analyses and was sufficiently sensitive to detect aflatoxin B1 at levels less than 1 ng/g in eggs from hens on experimentally contaminated feed and in spiked eggs; interfering compounds in eggs received from many different producing areas were eliminated. Although recovery was greater than 75% when the aflatoxin was extracted immediately after spiking, recovery was usually less than 30% when the aflatoxin was extracted after more than 24 hr. Adding sodium chloride, sodium sulfate, or urea to the extracting solvent remedied this defect, which is thought to be related to the binding of aflatoxin to egg protein.


High pressure liquid chromatographic determination of aflatoxins in wines and other liquid products.

A previously developed high pressure liquid chromatographic (HPLC) method was evaluated by an extensive recovery study of aflatoxins from wines and other liquid products. The HPLC column and the preliminary cleanup procedures were both modified for this study. Four aflatoxins (B1, B2, and G2) were recovered at 80-116% from 28 samples of various liquid commodities spiked at the 1 microgram/L (1 ppb) level. The detection limit of the proposed method for wines and fruit juices was about 0.02 ppb. A total of 53 samples were analyzed for aflatoxins during this study.


Analysis of foods for arsenic and selenium by acid digestion, hydride evolution atomic absorption spectrophotometry.

A method based on acid digestion, hydride evolution atomic absorption spectrophotometry for estimating microgram and submicrogram quantities of As and Se in foods was developed and evaluated. Samples up to 3 g dry weight were digested with HNO3-HCIO4-H2SO4. As and Se in aliquots of the digests were reduced with NaBH4 to volatile hydrides, using laboratory-constructed and commercially available generators. As and Se were estimated by transient signal atomic absorbance measurements as the hydrides were decomposed in an Ar-H2-entrained air flame. Recoveries of inorganic As and Se added at levels of 0.1-1.0 microgram/g to a variety of foods ranged from 70 to 125%. Analyses of several standard reference samples indicated the method is capable of recovering native analytes. Detection limits for the determinative step and the method as a whole were as low as 5 and 25 ng, respectively, for both elements.


Dry ash-voltammetric determination of cadmium, cooper, lead, and zinc in foods.

An analytical method is presented for determining cadmium, copper, and lead by differential pulse anodic stripping voltammetry and zinc by cathodic scan differential pulse voltammetry. Food samples are dry ashed using a sulfuric acid ashing aid, dissolved in dilute nitric acid, buffered at pH approximately 4.3 with an acetate buffer, and quantitatively analyzed using the technique of standard additions at a hanging mercury drop electrode. The quantitation limits (5 times the estimated detection limits) are approximately 5 ng/g for Cd, Cu, and Pb, and 50 ng/g for Zn. Accuracy of the method is established by (a) analysis of NBS Standard Reference Material No. 1577 Bovine Liver, (b) comparison of results obtained by the method described with those obtained by independent analytical methods, and (c) quantitative recovery of analyte metals from fortified, noncanned food samples. Results from an interlaboratory method trial indicate that the method is suitable for the analysis of a variety of food types.


Evaluation of rapid polarographic method for determining tocopherols in vegetable oils and oil-based products.

A polarographic procedure for the determination of the tocopherols in vegetable oils was compared to a conventional gas-liquid chromatographic procedure. The polarographic method is 2-3 times more precise and more rapid, which is an important advantage for monitoring the nutritional value of processing conditions of vegetable oil-based products. Attempts to establish the validity of apparently slightly higher values for alpha- and gamma-tocopherols obtained by polarography has implicated the gas chromatography or derivative formation rather than the extensive prior manipulation of the sample for chromatography as the source of error. Essentially the same results were obtained for alpha-tocopherol, regardless of the technique used. However, more variable results which were product dependent were obtained for gamma- and delta-tocopherols when gas-liquid chromatography was used.


Gas-liquid chromatographic determination of alpha-cyano-3-phenoxybenzyl alpha-isopropyl-4-chlorophenylacetate residues in cabbage.

A gas-liquid chromatographic method is described for residues of a synthetic pyrethroid, Sumicidin (alpha-cyano-3-phenoxybenzyl alpha-isopropyl-4-chlorophenylacetate), on cabbage. The plant material is Soxhlet-extracted with hexaneacetone (1+1) and subsequently cleaned up on Florisil with benzene-ethyl acetate (9+1) eluting solvent. Gas-liquid chromatographic analysis was performed on a 0.5 m x 2 mm stainless steel column containing 3% OV-101+3% Apiezon L on 80-100 mesh Gas-Chrom Q, with tritium electron capture detection. Sumicidin recovery was 97% when added at the 0.5 ppm level. Routine Sumicidin residue monitoring data are presented.


Liquid chromatographic determination of azinphos methyl oxon in foliar dislodgeable residues and in soil surface dusts from an azinphos methyl-treated orange grove.

Specific parts of the worker environment in an orange grove that had been sprayed with azinphos methyl formulation were tested for azinphos methyl oxon. Extracts of soil surface dusts and dislodgeable residues from orange leaf surfaces were prepared and analyzed by liquid chromatographic procedures incorporating an ultraviolet absorption detector primarily for quantitation and an on-stream chemical reaction detector primarily for confirmation. Azinphos methyl oxon residues were found in both types of samples collected 3-59 days after spraying. Foliar dislodgeable residues of the oxon ranged from a high of 0.057 microgram/sq cm of leaf surface on the 17th day to a low of 0.014 microgram/sq cm on the 59th day for trees treated with a low-volume spray. The corresponding range in soil dust samples was 5.2 microgram/g (ppm) of dust on the 10th day to 1.4 microgram/g on the 59th day. Values were 9.7 and 2.7 microgram/g, respectively, for samples from under trees similarly treated with a high-volume spray.


Collaborative study of an assay for progestational steroids in individual contraceptive tablets.

A single tablet assay method has been developed for determining norethindrone, norethynodrel, and norgestrel in commercial contraceptive tablets. The steroid is separated from the tablet constituents on a diatomaceous earth column, the chloroform eluate is reacted with isonicotinic acid hydrazide, and the steroid is determined by colorimetry. Norethynodrel is isomerized to a conjugated ketosteroid before the colorimetric determination. Fourteen laboratories collaboratively tested the method. The average recoveries in 3 brands of tablets were as follows: 102.6% for the sample labeled as containing 0.35 mg norethindrone/tablet, 99.4% for the 2.5 mg norethynodrel/tablet, and 98.2% for the 0.5 mg norgestrel/tablet. The assay method has been adopted as official first action.


Spectrophotometric determination of diphenhydramine. HC1 in pharmaceutical preparations.

A new spectrophotometric method has been developed for determining diphenhydramine. HC1, based on solvent extraction into chloroform of the complex formed with bromocresol green. The complex solution in chloroform showed maximum absorption at 415 nm and obeyed Beer's law over the concentration range of 3.0-12.0 microgram/ml. The molar absorptivity of the complex was 2.02 x 10(4). Complex formation and extraction was complete and quantitative over the pH range from 2 to 5. The ratio of diphenhydramine to bromocresol green was 1:1. Excipients, coloring matter, flavoring agents, and other substances likely to be present in diphenhydramine preparations do not interfere in the determination. Direct determinations in tablet, capsule, sirup, and lotion preparations were carried out satisfactorily, and the average recovery was 100 +/- 1.0%.


Flame photometric and atomic absorption determination of calcium, potassium, and sodium in Ringer's solution and injection and in lactated Ringer's solution.

Authentic and commerical counterparts of Ringer's solution and lactated Ringer's solution were analyzed by 14 collaborators. Calcium, potassium, and sodium were to be determined by any flame emission or atomic absorption instrument operated at prescribed performance levels, and basic procedures for comparing samples and standards were also given. Recoveries of the cations from authentic and commercial solutions were as follows: calcium 95.6-102.1 and 95.6-103.6%, respectively; potassium 97.9-101.7 and 98.7-102.4%; sodium 98.2-103.1 and 97.3-103.7%. The results indicate that the method yields acceptable precision for the determination of the 3 cations in fluid and electrolyte replenishers. The method has been adopted as official first action.


Glucosinolates and derived products in cruciferous vegetables: total glucosinolates by retention on anion exchange resin and enzymatic hydrolysis to measure released glucose.

Details are given for determining total glucosinolates in Cruciferae plants by a procedure measuring released glucose. The glucosinolates are separated from about 90% of other material in the plant extract by adsorption on an anion exchange resin. Then, by a selective thioglucosidase hydrolysis of the glucosinolates retained on the exchange resin, the glucose and aglucons are separated from other substances retained by the resin. Glucose is released into an aqueous medium and is equivalent to the total glucosinolates. The aglucons formed by the hydrolysis are extracted into methylene chloride and determined by gas-liquid chromatography. Based on 29 determinations of the glucose from sinigrin, analyzed under different conditions, accuracy of the total glucosinolate determination was 94.8 +/- 7.3%. The coefficient of variation, determined by duplicate analyses on extracts from 58 cabbage samples, was 4.6%.


Glucosinolates and derived products in cruciferous vegetables: gas-liquid chromatographic determination of the aglucon derivatives from cabbage.

A procedure is described for identifying and quantitatively determining the glucosinolates found in edible cabbage. The intact glucosinolates are extracted and their aglucons are released into solvent after a special enzymatic hydrolysis step. The resulting isothiocyanates and oxazolidinethiones are separated by a gas-liquid chromatographic procedure that allows the individual aglucons to be identified and their amounts to be estimated by comparison with an internal reference.


Colorimetric determination of cyanide liberated from apricot kernels.

A simple colorimetric method is described for determining the quantity of hydrogen cyanide produced by the spontaneous decomposition of amygdalin in apricot kernels. The evolved cyanide is collected in sodium hydroxide solution and assayed colorimetrically by reaction with picric acid. Results for duplicate assays, 3.02 and 3.06 mg CN-/g, compare well with those obtained by AOAC method 26.115 which specifies steam distillation and silver nitrate titration; results for triplicate assays were 3.02, 3.03, and 3.08 mg CN-/g by the latter. Recovery of cyanide from potassium cyanide at a level equivalent to 243 microgram CN-/g was 101.0%.


Microbiological assay for saponin in alfalfa products.

A bioassay is described for determining medicagenin-type saponin in dried alfalfa, leaf protein concentrates, and alfalfa sprouts. Samples are extracted by refluxing 2 1/2 hr with 50% ethanol, ethanol is evaporated, and aliquots of an aqueous solution are added to potato dextrose agar (PDA) and assayed for saponin by using the fungus Trichoderma viride. The growth of the fungus on PDA is compared with a standard saponin, and saponin levels are calculated by means of a slope ratio analysis.


Evaluation of electrolytic conductivity detection for gas-liquid chromatographic screening of some organonitrogen herbicides and fungicides in foods.

Electrolytic conductivity detection for the gas-liquid chromatographic (GLC) screening of 15 organonitrogen herbicides and fungicides was investigated. Spiked samples of crop material was extracted with acetone and the filtered sample extracts were partitioned with methylene chloride-petroleum ether (1+1) followed by second and third extractions with methylene chloride. The combined organic extracts were evaporated to dryness and the residues were dissolved in hexane for cleanup on a 2% deactivated Florisil column. The pesticide fractions were determined directly by GLC. All pesticides could be detected at 0.1 ppm in the foods studied.


Spectrophotometric determination of some preservatives in milk.

Benzoic acid, propyl p-hydroxybenzoate, and sorbic acid in milk have been determined by using the corresponding first derivative curves of absorption spectra of clarified milk samples. The delta A/delta lambda curves were computed and the concentration of each preservative was calculated using 1 point, 2 points, and/or 3 points. In all cases, the percentage error was within 2.4%.


Flameless atomic absorption spectrophotometry of selenium in fish and food products.

A method is described for determining selenium in fish tissues, meat, cereals, milk powder, and other materials by flameless atomic absorption spectrophotometry. Samples are solubilized in HNO3 and atomized in a graphite furnace in the presence of nickel nitrate. Recoveries of 0.500 and 1.000 microgram selenium added to several fish samples averaged 99.0 and 98.3%, respectively, with standard deviations of 5.3 and 4.0. Results agreed with those obtained for samples previously analyzed by fluorometry, and with results for NBS Standard Reference Material. The detection limit was 3 ng/ml solution and 50 ng/g sample.


Collaborative study of a spectrofluorometric assay for Rauwolfia serpentina tablets and powdered root, using a vanadium pentoxide-phosphoric acid determinative step.

Reserpine-rescinnamine group alkaloids are extracted from Rauwolfia serpentina preparations into dimethylsulfoxide (DMSO)-methanol and diluted with 0.5N H2SO4. The chloroform extract of this solution is passed through a 0.1N NaOH-Celite column and then through a silica gel column. The weakly basic alkaloids trapped on the latter column are eluted with chloroform-methanol; a portion of the eluate is treated with a vanadium pentoxide-phosphoric acid solution, and the reserpine-rescinnamine content is determined by measuring the intensity of the resulting fluorescence. The following means and standard deviations (11 collaborators) were obtained for the determination of reserpine-rescinnamine group alkaloids in 4 samples of Rauwolfia serpentina (NF reference powder, 100 and 50 mg commercial tablets, and 45 mg synthetic tablet formation): 0.173%, 0.0050; 0.128%, 0.0030; 0.150%, 0.0069; and 0.142%, 0.0032; respectively. The method has been adopted as official first action.


Interference by acetylated aminoacetic acid in nonaqueous titration of nicotinic acid.

A nonaqueous titration procedure is described for determining aminoacetic acid and nicotinic acid in mixtures and elixirs. The effect of acetylated aminoacetic acid on the potentiometric titration of nicotinic acid is discussed. Above a 2:1 ration of aminoacetic acid to nicotinic acid, the former interferes with titration. The titration curve of nicotinic acid becomes progressively flatter with increasing amounts of the acetylated compound. Above this ratio, nicotinic acid had to be separated from the mixture by dissolving in ethanol for a successful titration.


Gas-liquid chromatographic determination of beta-asarone in wine: collaborative study.

The gas-liquid chromatographic determination of beta-asarone (cis-2,4,5-trimethyoxy-1-propenylbenzene) in wine was collaboratively studied by 9 analysts. The samples were first distilled and then extracted into hexane prior to chromatography. The mean recovery for 8 spiked samples, analyzed as blind duplicates, was 89.27%. The reproducibility coefficient of variation was 17.9%, and the repeatability coefficient of variation was 8.3%. No unfavorable remarks were noted. The method has been adopted as official first action.


High pressure liquid chromatographic determination of ethopabate in feeds.

A high pressure liquid chromatographic (HPLC) method is described for determining ethopabate in poultry feeds. Feed samples containing ethopabate are finely ground, extracted 30 min by sonification with methanol-water (80 + 20), and filtered. The extract is cleaned up on an alumina column, and the first 4 ml eluate collected is analyzed. The drug is eluted through a muBondapak C18 column with acetonitrile-water (30 + 70) at a flow rate of 1.4 ml/min and detected by ultraviolet absorption at 280 nm. Chromatography is complete in 7 min, and detector response is linear. The drug is quantitated by peak height ratios. The procedure described is reproducible and shows good agreement with the official AOAC colorimetric method. The lower limit of detection is 2 ng by HPLC, making the method applicable to residue analyses.


Atomic absorption spectrophotometric determination of dibutyltin dilaurate in finished feeds: collaborative study.

Dibutyltin dilaurate is administered in the feed as an active drug for the removal of large roundworms, cecal worms, and several species of tapeworms  from chickens and turkeys. The drug is extracted from finished feed with warm CHCl3, the CHCl3 extract is filtered, and a 25 ml aliquot is concentrated in the presence of methanol until all traces of CHCl3 are removed. The methanol solution is diluted and filtered to remove feed interferences, and then aspirated into an atomic absorption spectrophotometer with a triple-slot burner head, using an air/acetylene flame. The instrument response to tin at 286.3 nm is recorded. Recoveries of 100.5-101.9% tin were obtained for laboratory-prepared feeds. This method was collaboratively studied. The average recovery for dibutyltin dilaurate from 3 feed samples for 8 collaborators was 98.3% with an average coefficient of variation of 5.83%. The method has been adopted as official first action.


Analysis of pesticide residues by chemical derivatization. V. Multiresidue analysis of eight phenoxyalkanoic acid herbicides in natural waters.

A multiresidue method is presented for the gas-liquid chromatographic determination of phenoxyalkanoic acid herbicides in natural waters. Extraction efficiencies of different organic solvents are considered in developing a solvent extraction scheme for these herbicides from water. Reactions for derivatizing these compounds by using pentafluorobenzyl bromide, boron trichloride-2-chloroethanol, and dicyclohexylcarbodiimide-2-chloroethanol were studied in order to obtain extracts with low blanks to provide the lowest detection limits. Advantages and disadvantages of the 3 methods are discussed. Retention times are twice as long for the pentafluorobenzyl (PFB) esters as for the 2-chloroethyl (2-Cl) esters under the same conditions, although electron capture sensitivity to the former was greater. The PFB esters are easier to form, but the 2-Cl reaction is more specific for these herbicides. Solutions from the boron trichloride reaction gave the cleanest blanks.


Extraction and gas-liquid chromatographic determination of hexachlorophene from several plant tissues.

Conditions are described for extraction, cleanup, derivatization, detection, and quantitation of hexachlorophene (HCP) residues from several types of plant material. Wet plant tissue was homogenized and extracted with ethyl ether, dried, and methylated with excess diazomethane. Samples were precleaned by column chromatography on silica gel with benzene-petroleum ether (1 + 1), and then 1-10 microliter concentrated sample was chromatographed on a 6' column of 3% SE-30 on 80-100 mesh Gas-Chrom Q. The detection limit for the electron capture detector was less than 0.1 ng dimethoxyhexachlorophene and 1 ppb HCP in plant tissue. Recoveries (%) of 11-610 ppb HCP added to tissue averaged 94.3 for tomatoes, 86.4 for green peppers, 92.6 for cucumbers, 93.3 for green beans, 93.7 for peanut hay, and 91.6 for peanut shells.


Spectrophotometric determination of methomyl residues in selected vegetables, grains, and soil.

Methomyl (S-methyl-N-[(methylcarbamoyl) oxy] thioacetimidate) is converted to oxime and hydroxylamine by alkali and acid treatment, respectively. Hydroxylamine is oxidized with iodine in the presence of sulfanilic acid to yield p-diazoniumbenzenesulfonic acid which is coupled with alpha-naphthylamine to form a crimson p-benzenesulfonic acid-azo-alpha-naphthylamine with an absorption maximum at 520 nm. The relationship between absorbance and concentration of methomyl is linear in the range 0.5-10 microgram. The method is sensitive and specific; 0.625 ppm methomyl can be determined in a 40 g sample of selected vegetables, grains, and soil.


Interlaboratory quality study of the analysis of water for pesticides.

An interlaboratory quality control study was conducted to provide information to 5 participating Canadian laboratories on their capability in analyzing natural waters for 10 pesticides. The study was designed to identify the quality of each laboratory's working standards for 15 organochlorine pesticides, and the accuracy and precision of each laboratory's analytical procedures. Fifteen samples were provided, of which 3 were standards prepared for direct injection into an electron capture gas chromatograph. Instructions were provided to use these 3 standards for quantitating the pesticide content (8-150 ng/L) in 5 pairs of 1 L water samples. These paired samples contained 10 organochlorine pesticides approaching natural levels. Results of the study indicated that participants were fairly precise and achieved the designed ratio 1.5 for concentrations in the paired samples.


Detection of residual penicillins in milk by using a Bacillus stearothermophilus disk assay.

A paper disk method based on a procedure described by the International Dairy Federation is presented for the detection of 4 beta-lactam antibiotics (penicillin G, ampicillin, cephapirin, and cloxacillin) in whole milk. Bacillus stearothermophilus var. calidolactis, prepared as a stable spore suspension, was used as the test organism. Levels of 0.005 unit penicillin G/ml, 0.005 microgram ampicillin or cephapirin/ml, and 0.05 microgram cloxacillin/ml were readily detected in whole milk. Results were produced in 3-4 hr. The method offers several advantages, including greater simplicity, sensitivity, and rapidity, over methods now commonly used to detect residual levels of these drugs in milk.


Nitration of benzene as method for determining nitrites and nitrates in meat and meat products.

Nitrites and nitrates in meat and meat products are determined by nitration of benzene in the presence of sulfuric acid. Nitrites are converted to nitrates by permanganate oxidation prior to nitration. The heat of dilution originating from the sulfuric acid is sufficient to complete the reaction. The final product, nitrobenzene, is determined by gas-liquid chromatography and electron capture detection. Peak height quantitation is both accurate and reproducible. Interferences from impurities are rarely encountered, and may be differentiated easily. The method also has potential application to samples other than meat.


Determination of dimethylpolysiloxane in instant coffee.

A method is described for determining dimethylpolysiloxane in instant coffee. The siloxane is extracted with chloroform in a Soxhlet apparatus and then converted to silica which is estimated by a heteropoly blue method. The method is interference-free, and 5 microgram dimethylpolysiloxane can be easily detected.


Scanning electron microscopic study of adult stored product beetle mandibles.

Adult stored product beetle mandibles were used as a subject for this scanning electron microscopic study. A total of 34 species are represented. Discussion is organized taxonomically by family, and comparisons are made between species where possible. Established terminology for morphological processes has been applied to all characteristics of mandibles reported in earlier literature; however, several new terms have been added in an attempt to better describe specific attributes of a species.


Determination of vinylidene chloride in Saran films by electron capture gas-solid chromatography and confirmation by mass spectrometry.

A method is presented for determining vinylidene chloride (VdC)  in Saran food packaging films by using electron capture gas-solid chromatography. The VdC present was confirmed by combined gas-solid chromatography-mass spectrometry, employing selected ion recording. The method is capable of detecting larger than or equal to 5 ppm VdC in films. Production lots of Saran film were analyzed and found to contain 6-30 ppm VdC.


Aflatoxin in corn: evaluation of filter fluorometer reading of minicolumns.

A minicolumn method using a filter fluorometer was compared with thin layer chromatography for determining aflatoxin in corn. The minicolumn-fluorometer method identified many more samples as aflatoxin-positive than the AOAC CB method. Some corn samples contain a blue-fluorescing substance that interferes with minicolumn chromatography of aflatoxins. Aflatoxin levels measured by the minicolumn-fluorometer method did not correspond to those obtained by the CB method.


Analysis of fat-soluble vitamins. XV. Confirmation of isotachysterol in vitamin D concentrates.

The reaction with maleic anhydride yielding Diels-Alder adducts is used to distinguish vitamin D isomers. Treatment at 100 degrees C for 3 hr eliminates the fast-reacting (tachysterol and trans-vitamin D) and slow-reacting (cis-vitamin D and previtamin D) isomers. However, isotachysterol is not eliminated. The procedure has been incorporated as a confirmation test for isotachysterol in the official final action method for the determination of vitamin D in concentrates, 43.B14-43.B24.


High pressure liquid chromatographic determination of vitamin D in fortified milk.

Vitamin D was determined in fortified milk by high pressure liquid chromatography (HPLC) after preliminary purification involving saponification at room temperature and low pressure chromatography on hydroxyalkoxyprophyl Sephadex (HAPS). The saponified milk (50 ml) was extracted with ether, the extract was chromatographed in hexane on a 60 X 1 cm column of HAPS, and the absorption of the eluate at 254 nm was recorded. The fraction containing vitamin D was collected by using the position of characteristic peaks in the chromatogram as a guide. An aliquot of this fraction was chromatographed in an HPLC system on a 25 cm X 3.2 mm column of LiChorosorb Si 60 5 micrometer silicic acid, using 0.6% isopropanol in hexane (50% water-saturated) as the mobile phase. The absorption was set at 265 nm. Vitamin D was cleanly separated from other absorbing materials, and the amount could be calculated from the peak area. Recovery of a added vitamin D was greater than 97%.


Variation in guanosine 5' ,3'-tetraphosphate levels in Chromatium.

Chromatium produces guanosine 5' ,3'-tetraphosphate, but there appears to be no correlation between guanosine 5' ,3'-tetraphosphate concentration and the inability of the organism to grow in certain light-dark regimes.


Selective plasmid transduction in Bacillus pumilus.

The inducible temperate bacteriophage phi75 and a clear-plaque-forming variant, phi75C1, mediated transduction of a 4.4 X 10(6)-dalton multicopy Bacillus pumilus plasmid, pPL10, at frequencies of 10(-5) to 10(-6) transductants per plaque-forming unit. phi75- and phi75C1-mediated transduction of several chromosome markers tested did not occur at a detectable frequency. phi75-mediated plasmid transducing activity resides in particles that are similar to infectious particles in sedimentation velocity and buoyant density.


Zeatin ribonucleosides in the transfer ribonucleic acid of Rhizobium leguminosarum, Agrobacterium tumefaciens, Corynebacterium fascians, and Erwinia amylovora.

Until recently, the presence in transfer ribonucleic acid (tRNA) of the hydroxylated cytokinin ribosylzeatin [N6-(4-hydroxy-3-methylbut-2-enyl)adenosine]was thought to be unique to higher plants. This extension of work from several laboratories indicates the presence of 2-methylthioribosylzeatin in the tRNA of the plant-associated bacteria Rhizobium leguminosarum, Agrobacterium tumefaciens, and Corynebacterium fascians, but not in that of Erwinia amylovora. This cytokinin has the cis configuration, as is normally found in the tRNA's of plants. The tRNA thionucleotide patterns in these bacteria are different from those of Escherichia coli, Bacillus subtilis, and Salmonella typhimurium, which contain the unhydroxylated analogs of ribosylzeatin or 2-methylthioribosylzeatin.


Tetrapyrrole utilization by Bacteroids ruminocola.

Reduced versus oxidized difference spectra of whole cells and pyridine hemochromogens of heme-requiring isolates of Bacteroides ruminicola are altered when deuteroporphyrin or mesoporphyrin replaces protoheme as a growth factor. During growth in the presence of either deuteroporphyrin or mesoporphyrin, whole cells exhibit peaks at 545 t547, 515 to 518, and 412 to 413 nm. Pyridine hemochromogen spectra confirm the presence of meso -or deuteroheme in cells grown in the presence of meso- or deuteroporphyrin. No evidence was found for the conversion of either meso- or deuteroporphyrin to protoheme. Cells grown in the presence of the manganese of magnesium chelates of protoheme form iron-containing hemes. Neither spontaneous decomposition of noniron metalloporphyrin chelates nor spontaneous formation of hemes from Fe2+ and metal-free porphyrins was detected. Protoheme-synthesizing isolates of B. ruminicola fail to use preformed metal-free porphyrins, but form both protoheme- and deuteroheme-containing cytochromes when grown in the presence of manganese deuteroheme. Versatility in tetrapyrrole utilization by B. ruminicola appears to reflect the ability of the organism to mediate the removal of nonferrous ions and to insert Fe2+ into the tetrapyrrole nucleus. The orgamism also forms functional b-type cytochromes with prosthetic groups other than protoheme.


Myo-inositol transport system in Pseudomonas putida.

The kinetic features of the myo-inositol transport system in Pseudomonas putida are reported. The system is sensitive to osmotic shock, is not operative in membrane vesicles, and does not involved substrate phosphorylation. Line-weaver-Burk plots indicate the presence of two different systems, whose Kt are 5 micrometer and 0.43 mM and whose V max are 7.9 and 27 nml/mg per min, respectively. Transport activity of glucose-grown cells is very low. myo-Inositol-grown cells lose the high-affinity system upon osmotic shock; concentrated shock fluid possesses myo-inositol-binding activity. The system is very specific for the myo-configuration of the cyclitol.


Ribosomal suppressors and antisuppressors in Podospora anserina: resistance to cycloheximide.

Informational suppressors and antisuppressors have been previously isolated in Podospora anserina, and a range of exclusively genetic arguments have led to the assumption that they correspond to ribosomal mutations. An in vivo and in vitro comparison of the effect of the ribosomal inhibitor cycloheximide on wildtype and mutant strains described in this paper confirms the ribosomal hypothesis for at least some mutants. Indeed, the four mutants in the AS3 gene were cycloheximide resistant, and their ribosomes were found to be resistant when analyzed by polyuridyl-directed polyphenylalanine systhesis. On the other hand, ribosomes from two su 1 mutants were hypersensitive to the drug.


Purification of the extracellular protease of Bacillus licheniformis and its inhibition by bacitracin.

Sporulating cells of Bacillus licheniformis excrete three seryl proteases that are of similar size, 28,000 daltons, but of different charge at pH 6. The peptide antibiotic bactracin is released from the cells at the same time and exists, in part, as a bacitracin-protease complex that is stable throughout chromatographic procedures employed in enzyme purification. However, preextraction of crude protease with CHCl3 and subsequent gel filtration effect separation of the antibiotic and the enzyme. Three purified, bacitracin-free proteases, designated CMC I, CMC II, and CMC III and whose ratios of total activity are 1:3.7:10.3, respectively, are obtained by chromatography on carboxymethyl cellulose. The major component, CMC III, is inhibited by commercial bacitracin at near-physiological concentrations of the antibiotic.


Component from the cell surface of the hydrocarbon-utilizing yeast Candida tropicalis with possible relation to hydrocarbon transport.

A polysaccharide-fatty acid complex was isolated from the cell surface of Candida tropicalis growing on alkanes. This complex was solubilized by Pronase treatment of whole cells. A decrease in alkane-binding affinity was observed after Pronase treatment, resulting in 10 to 12% of the yeast dry cell weight being released as polysaccharide. The isolated polysaccharide contained 2.5% fatty acids. C. tropicalis and Saccharomyces cerevisiae grown with glucose contained only traces of fatty acids in the corresponding polysaccharide fraction. The fatty acids were not removed from the polysaccharide moiety by gel filtration. Extraction of the polysaccharide with chloroform-methanol showed that fatty acids were covalently bound to the polysaccharide. The amphipathic nature of the isolated polysaccharide and the hydrocarbon-induced formation suggest a possible role in alkane metabolism.


In vitro production of bacitracin by proteolysis of vegetative Bacillus licheniformis cell protein.

The action of a sporulation-specific seryl protease on antibiotic-free extracts of Bacillus licheniformis cells yields a peptide that is identified as bacitracin by its biological activity, its spectral properties, and its comigration with genuine bacitracin in both paper and thin-layer chromatography. During proteolysis, a chemical structure is generated with the spectral properties of a delta-2 thiazoline ring. The yield in vitro, 4 microgram of bacitracin per mg of protein, is less than the maximal yield from sporulating cells, 75 microgram of bacitracin per mg of cell protein, but is a linear function of the amount of protein in the reaction system. Approximately 30% of the protein yielding the antibiotic is ribosomal associated, and only 25% of that amount can be removed by washing with 1 M NH4Cl. The substrate protein is a constant fraction of the cell protein throughout exponential growth and very early sporulation stages of culture development.


Hydrogenase activity and the H2-fumarate electron transport system in Bacteroides fragilis.

Hydrogenase activity and the H(2)-fumarate electron transport system in a carbohydrate-fermenting obligate anaerobe, Bacteroides fragilis, were investigated. In both whole cells and cell extracts, hydrogenase activity was demonstrated with methylene blue, benzyl viologen, flavin mononucleotide, or flavin adenine dinucleotide as the electron acceptor. A catalytic quantity of benzyl viologen or ferredoxin from Clostridium pasteurianum was required to reduce nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate with H(2). Much of the hydrogenase activity appeared to be associated with the soluble fraction of the cell. Fumarate reduction to succinate by H(2) was demonstrable in cell extracts only in the presence of a catalytic quantity of benzyl viologen, flavin mononucleotide, flavin adenine dinucleotide, or ferredoxin from C. pasteurianum. Sulfhydryl compounds were not required for fumarate reduction by H(2), but mercaptoethanol and dithiothreitol appeared to stimulate this activity by 59 and 61%, respectively. Inhibition of fumarate reduction by acriflavin, rotenone, 2-heptyl-4-hydroxyquinoline-N-oxide, and antimycin A suggest the involvement of a flavoprotein, a quinone, and cytochrome b in the reduction of fumarate to succinate. The involvement of a quinone in fumarate reduction is also apparent from the inhibition of fumarate reduction by H(2) when cell extracts were irradiated with ultraviolet light. Based on the evidence obtained, a possible scheme for the flow of electrons from H(2) to fumarate in B. fragilis is proposed.


The structure of the branching point between acidic polysaccharide and peptidoglycan in Micrococcus lysodeikticus cell wall.

An acidic polysaccharide fraction composed of glucose and N-acetylmannosaminuronic acid with a small portion of peptidoglycan was isolated by enzymic digestion and subsequent ECTEOLA-cellulose chromatography from the cell walls of Micrococcus lysodeikticus. On mild acid treatment, the fraction became Morgan-Elson positive and formed the Morgan-Elson chromogen on heating with phosphate buffer (pH 7). The product of mild acid treatment released inorganic phosphate on treatment with phosphomonoesterase. After gel-chromatography on Sephadex G-25 and DE-32, the acidic polysaccharide fraction contained less glucosamine than muramic acid. By reduction of this fraction with borohydride, a part of the glucosamine was converted into glucosaminitol. Based on these results, it is suggested that the acidic polysaccharide is linked to glucosamine by a (1-3) linkage, which is linked to the 6 position of a muramic acid residue by a phosphodiester  linkage.


Isolation of glycopeptides from the lectin-stimulated human peripheral lymphocyte cell surface.

Three radioactive glycopeptides were isolated from human peripheral lymphocytes stimulated with Wistaria floribunda mitogen in the presence of D-[14C]glucosamine hydrochloride by mild trypsin digestion followed by gel filtration and preparative high-voltage paper electrophoresis. The carbohydrate compositions of these glycopeptides suggest that one has a sugar chain of the type found in serum glycoproteins, consisting of sialic acid, galactose, N-acetylglucosamine, mannose, and fucose in a molar ratio of 2:2:4:2:1, and the other two have sugar chains like those of mucins, consisting of sialic acid, galactose, and N-acetylgalactosamine in a molar ratio of 1 or 2:1:1. The results of enzymic degradation with purified glycosidases indicate that these sugar chains are similar in structure to their counterparts in human erythrocyte membranes.


Purification and properties of deoxyribonucleic acid ligases from rat liver.

DNA ligases have been purified 1,500-fold cytoplasmic fraction and 114-fold from 0.15 M NaCl extract of rat-liver nuclei. These enzymes catalyze the formation of a phosphodiester linkage between the 3'-hydroxyl group and the 5'-phosphoryl group of an interrupted strand in a DNA duplex. The enzyme from each fraction requires ATP and Mg2+ (or Mn2+) for activity, and its inhibited reversibly by p-chloromercuribenzoate. The cytoplasmic and nuclear enzymes are distinguished from each other by the following criteria: apparent molecular weight; sedimentation coefficient; charge properties; Km for Mg2+ (or Mn2+); Km for ATP; effect of ionic strength on the molecular and kinetic properties.


Purification of carboxypeptidase A using Sepharose 4B-bound 3-phenylpropionate.

The activity of carboxypeptidase A [EC 3.4.12.2] was inhibited by 3-phenylpropionate derivatives (p-aminocinnamate, 3-p-aminophenylpropionate and 3-p-acetylaminophenylpropionate), and to investigate its use as a ligand for affinity chromatography 3-p-aminophenylpropionate was directely and indirectly coupled to Sepharose 4B. carboxypeptidase A was adsorbed only on 3-p-aminophenylpropionate bound to the gel through p-phenylenediamine as a spacer. Carboxypeptidase A from pancreas was purified by a combination of this affinity adsorbent and ion exchange chromatography. The purified carboxypeptidase A had a homogeneity similar to that of a commercial product, as judged by disc gel electrophoresis. The carboxypeptidase activity of Pronase was slightly retarded on the gel column, but could not be separated from its caseinolytic activity. Angiotensin I-converting enzyme [peptidyl dipeptidy hydrolase, EC 3.4.15.1] obtained from hog kidney cortex was not bound to the gel.


An improved method for the purification of rat serum albumin: removal of contaminants by concanavalin A-Sepharose.

Minor contaminants occasionally found in conventionally prepared rat serum albumin were easily and completely removed by concanavalin A-Sepharose chromatography. The unadsorbed fraction from a concanavalin A-Sepharose column contained albumin which was homogeneous on polyacrylamide gel electrophoresis. The recovery of albumin form rat serum was approximately 30%. Approximately 2% of the added protein obtained as an albumin peak in DEAE-cellulose chromatography was adsorbed on and eluted with alpha-methyl-D-glucoside from the concanavalin A-Sepharose column, and resolved into three components by gel electrophoresis. There was one major glycoprotein, possibly alpha 1-antitrypsin, and two minor proteins one of which was albumin.


Properties of opsin-lipid complex in aqueous 2-chloroethanol.

Cattle and squid opsins were found to be associated with phospholipids after extensive dialysis of the salt-free digitonin extract of rhodopsin against 30% aqueous 2-chloroethanol (v/v) at pH  2.5. The approximates sizes of opsin-lipid complexes were estimated by sedimentation studies to be around 110,000 and 150,000 daltons, respectively, for the cattle and the squid opsin. Phospholipids did not dissociate from opsin even in 80% 2-chloroethanol. The complexes were purified by passage through a Sephadex G-200 gel column equilibrated with 30% 2-chloroethanol. The optical properties of the complex suggested the presence of beta-conformation and a small amount of alpha-helix in solubilized cattle opsin.


Circular dichroism and magnetic circular dichroism studies on methemoglobin and its derivatives.

CD and MCD spectra of human methemoglobin and its derivatives were studied with special reference to the relation of CD and MCD spectra to the magnetic moments. The intensities of MCD peaks of methemoglobin derivatives at 270 nm were inversely proportional to their magnetic susceptibility, and there was a linear relationship between magnetic ellipticity and magnetic susceptibility. The MCD peak intensity of methemoglobin peroxide compound was close to that of methemoglobin cyanide complex, suggesting that the methemoglobin peroxide compound is in a low-spin state.