A modified measurement of respiratory resistance by forced oscillation during normal breathing. We have modified the measurements of the resistance of the respiratory system, Rrs, by the forced oscillation technique and we have developed equipment to automatically compute Rrs. Flow rate and mouth pressure are treated by selective averaging filters that remove the interference of the subject's respiratory flow on the imposed oscillations. The filtered mean Rrs represents a weighted ensemble average computer over both inspiration and expiration. This method avoids aberrant Rrs values, decreases the variability, and yields an unbiased mean Rrs. Rrs may be measured during slow or rapid spontaneous breathing, in normals and in obstructive patients, over a range of 3-9 Hz. A good reproducibility of Rrs at several days' interval was demonstrated. Frequency dependence of Rrs was found in patients with obstructive lung disease but not in healthy nonsmokers.


Alternative method for measurement of arterial pressure in the rabbit. By using an electrical impedance plethysmorgraph and a pressure applicator to a rabbit's earlobe artery, it became possible to monitor the blood pressure for periods of several months. A study of the correlations of the blood pressure from carotid and ear was also made at varied blood pressures under different conditions which showed excellent correlations. This method could effectively replace the encannulation technique for monitoring rabbit's blood pressure in chronic experiments.


Computerized method for analyzing maximum and partial expiratory flow-volume curves. Computerized instrumentation and software have been developed to obtain maximum expiratory flow-volume (MEFV) and partial expiratory flow-volume (PEFV) curves. The computerized system calculates and prints out the flow at 25% and 40% of control vital capacity (VC), the expiratory volume, peak expiratory flow rate and expiratory volume at one second (FEV1) divided by VC, the latter expressed as a percent. The flow-volume curves can be displayed on an oscilloscope or plotter and stored on magnetic tape. A pilot study was completed to demonstrate the reliability and validity of the data obtained.


A high accuracy linear rate meter.

A high accuracy linear rate meter. By implementing analog computer techniques using digital circuits, an instantaneous rate meter was built with approximately 100 times the accuracy of commercially available rate meters. The circuit is accurate to within +/- 0.2 events/min over a range of rates of 0.2-900 epm. Modifications can be made to provide a digital display of rate. The circuit design techniques used in developing the rate meter may be used to generate a wide variety of functions of time with very high accuracy.


Multichannel subcarrier ECG, respiration, and temperature biotelemetry system. A three-channel biotelemetry system measuring ECG, respiration, and body temperature is described. The transmitter employs a 6-kHz subcarrier oscillator and is small enough for surgical implantation in animals the size of rats or larger. The frequency modulated 6-kHz tone from the reciver can be demodulated directly or recorded on an inexpensive cassette tape recorder for future analysis. The transmitter cost is approximately $35.00 and measures 3 times 1 cm. A battery life of several weeks and transmitter range of 10-100 m is typical. Transmission is on the FM broadcast band (88-108 MHz) and reception from an inexpensive FM portable receiver is possible.


Body surface area in the infant rat.

Body surface area in the infant rat. Body surface area (SA), one index used to minimize size differences in metabolic studies, was estimated by a coating method in rats aged 1, 7, 14, and 21 days (d) (n = 100-107). Body weights (BW) at each age varied about twofold. Regression equations were calculated for SA (cm2) vs. BW (g) (SA = 8.73 + 2.29 BW, 17.79 + 1.71  BW, -4.92 + 2.16 BW, and 53.68 + .82 BW at 1, 7, 14, and 21 days, respectively) and log SA vs. log BW (SA = 6.71 BW0-667, 9.12 BW0-577, 1.73 BW1-042, and 18.97 BW0-412). All the latter differed significantly from SA = 10 BE2/3, frequently used with adult mammals. Correlation coefficients were approximately 0.6-0.9 and did not differ with mode of expressing results. Regressions, whether linear or logarithmic, differed significantly among ages, apparently reflecting appreciable conformational changes in the rapidly growing and maturing preweanling rat.


Reproducibility of dynamic compliance and flow-volume curves in normal man. To evaluate methods proposed for the early detection of small airways obstruction. we have compared the precision of the forced expiratory flow-volume curve and of measurements of the frequency dependence of lung compliance in normal subjects, in 10 of whom these measurements were made on four occasions, and in 5 on two occasions. The maximal expiratory flow rate was highly reproducible, revealing consistent differences between these normal subjects, particularly when measured at 60% of TLC and corrected for differences in body size. The frequency-dependence measurements, expressed as the slope of the regression line relating dynamic compliance to frequency, showed much greater variation between repeated measurements in the same subject. Comparison with earlier published accounts is difficult in view of lack of quantitative data, but the variability between single measurements on different normal subjects appears to be similar to that which we have found when the results are expressed in a comparable manner. Dynamic compliance at a frequency of 1 Hz lay between 48% and 141% of the value obtained by extrapolation to zero frequency.


Effect of lactate on FFA and glycerol turnover in resting and exercising dogs. The rates of appearance of FFA (RaFFA) and that of glycerol (RaGLY) were measured simultaneously with [1-14 C]palmitate and [2-3H]-glycerol, in dogs with indwelling arterial and venous catheters. Lipolysis was stimulated by exercise (treadmill run on 10% slope) or by the infusion of norepinephrine (0.5 mug/kg-min). Na-L(+)-lactate (L), Na-pyruvate (Py), or Na-nicotinate (N) were infused. All three components decreased RaFFA. RaGLY was increased by L, unaltered by Py, and decreased by N. There was an inverse correlation (P less than 0.001) between the logarithms of RaFFA and plasma lactate. A linear correlation was obtained between RaGLY and plasma lactate when this latter was increased by the infusion of L. It is suggested that a) lactate in physiological concentrations inhibits the release of FFA stimulated by exercise and b) the increase of the NADH/NAD ratio leads to the formation of alpha-glycerophosphate which in turn yields glycerol. Therefore changes in plasma glycerol do not reflect lipolysis when blood lactate increases. c) The effect of lactate on RaFFA can be explained by an enhanced reesterification, although a direct inhibition on lipase could not be excluded.


Motion of both mitral valve leaflets: a cineroentgenographic study in intact dogs. Motion and position of both mitral leaflets were studied in five normal dogs 1-11 wk after radiopaque markers were sutured on the valve cusps and on the mitral annulus. Cinefluorograms and cineangiograms (100-120 frames/s) of left atrium and left ventricle were used to study cusp motion and intraventricular flow patterns, and to detect mitral regurgitation during sinus rhythm (42-184 beats/min) and during isolated atrial or ventricular contractions. Time-motion of both leaflets was similar throughout diastole with the exception of delayed posterior cusp opening. Peak opening and closing speeds, opening and closing times, and time of complete closure, measured from the Q wave of the ECG, were not significantly affected by the variations in heart rate. Diastolic leaflet closure began immediately after opening, while the ventricular cavity was small, and was caused by flow eddies behind the cusps. Isolated ventricular contractions closed the valve leaflets completely and symmetric valve closure was ensured by the different rates of leaflet edge approximation. In contrast, atrial closure was slow, partial, and of very short duration.


Effect of hypoxia on immediate ventilatory load response in dogs. The effective elastance of the respiratory system (which has been previously shown to provide an index of the ability of the respiratory musculature to compensate rapidly for transient mechanical ventilatory loads) was measured in six hypoxic dogs to determine whether hypoxia hindered immediate load-compensatory mechanisms. The effective elastance value was computed from measurements of control tidal volume and the pressure developed at the airway opening during the first inspiratory effort following airway occlusion at FRC. The mean effective elastance was 197 cmH2O/l while the animals were breathing room air and did not change significantly when the animals were rendered hypoxic by reducing the inspired oxygen concentration, in five dogs, or by controlled hemorrhage, in two dogs. It was concluded that inasmuch as effective elastance measurements remain constant during hypoxia, the stability of ventilation is not significantly impaired in this situation.


Ability of man to detect increases in his breathing. The ability of four normal subjects to detect increases in their ventilation was studied at rest and at two levels of exercise using a raised inspired Pco2 to further increase ventilation. Subjects signaled when the increase in ventilation was recognized. The average tidal volume (VT) at rest was 520 ml with a frequency of 14; these values increased to an average of 3,300 ml and 21 at the highest work load. There was no significant change in frequency with CO2. Detection occurred when the tidal volume increased by 700 ml (varying 550-890 between subjects but constant for any one subject at the three levels of ventilation.) Thus the appreciation of increase is proportionately more sensitive at higher levels of ventilation. Experiments in which the ventilation was increased by hypoxia or by following a visual demand, and observations of other sensations (oral, cerebral) indicate that the increase in vetilation is recognized through increased breathing rather than awareness of ventilatory stimuli.


Step changes in end-tidal CO2: methods and implications. A dynamic end-tidal forcing technique for producing step changes in end-tidal CO2 with end-tidal O2 held constant independent of the ventilation response or the mixed venous return is introduced for characterizing the human ventilation response to end-tidal CO2 step changes for both normoxic (PAO2 = 125 Torr) and hypoxic (PAO2 = 60 Torr) conditions. The ventilation response approaches a steady state within 5 min. In normoxia, the on-transient is faster than the off-transient, presumably reflecting the action of cerebral blood flow. The hypoxic step response is faster than the normoxic response presumably reflecting the increased contribution from the carotid body. The delay in the ventilation response after the change in end-tidal CO2 is less in hypoxia than in normoxia and reflects the action of a transport delay and that of a virtual delay. These delays are interpreted with respect to the high-frequency phase shift data for the same subject, generated using sinusoidal end-tidal forcing. The methods of others for experiments utilizing step changes in inspired CO2 are considered with respect to our methods.


Maximal oxygen uptake during treadmill walking and running at various speeds. Three groups of male subjects, average fitness (AF, N = 12), high fitness (HF, N = 7) and highly fit competitive race walkers (CRW, N = 3) performed maximal treadmill tests walking at 3.5 and 4.5 mph and running at 4.5, 5.5, 7.0, and 8.5 mph. In addition, the HF group performed a running test at 10.0 mph and the CRW group performed a walking test at 5.5 mph. All maximal oxygen uptake (VO2 max) tests with the exception of the 3.5 mph walking test (modified Balke test) were discontinuous in nature. VO2 max obtained from walking tests was similar regardless of speed within each group. Walking VO2 max was significantly lower than running VO2 max which was found to be similar over a speed range of 4.5 to 8.5 mph in the AF group. Running at 4.5 mph (HF group) and 4.5 and 5.5 mph (CRW group) resulted in lower VO2 max levels than running at speeds greater than or equal to 7.0 mph. Associated physiological variables (heart rate, ventilation, and respiratory exchange ratio) did not demonstrate a discernable pattern with reference to mode of locomotion (walking versus running) or speed. It was concluded that VO2 max elicited during walking is independent of speed and less than VO2 max obtained during running. Running VO2 max was interrelated with speed of running and state of training.


Relationship of phase IV to closing volume in lateral body positions. We studied the effect of body position in humans on the relationship between exhaled vital capacity (VC) and both helium (He) and nitrogen (N2) concentrations after delivery of an He bolus at residual volume (RV) followed by 100% oxygen to total lung capacity. Phase IV, defined as the % VC at the first sharp and permanent increase in N2 and He, occurred at a mean of 15.7% VC while seated, 60.0% VC in right lateral and 59.6% VC in left lateral positions. He bolus delivery above RV but well below 60% VC resulted in the disappearance of phase IV. Lung pressure-volume (PV) curves had inflections at the volume of phase IV in the seated position: but the inflections were well below phase IV in lateral positions. Phase IV increased to higher volumes at higher mouth pressures. The relationship between phase IV and mouth pressure fell near the respiratory system relaxation PV curves. The findings suggest the higher phase IV in lateral positions is due to sequence of emptying without airway closure and is influenced by active expiration.


Effect of baroreceptor activity on ventilatory response to chemoreceptor stimulation. This study tested the hypothesis that ventilatory responses to chemoreceptor stimulation are affected by the level of arterial pressure and degree of baroreceptor activation. Carotid chemoreceptors were stimulated by injection of nicotine into the common carotid artery of anesthetized dogs. Arterial pressure was reduced by bleeding the animals and raised by transient occlusion of the abdominal aorta. The results indicate that ventilatory responses to chemoreceptor stimulation were augmented by hypotension and depressed by hypertension. In additional studies we excluded the possibility that the findings were produced by a direct effect of changes in arterial pressure on chemoreceptors. Both carotid bifurcations were perfused at constant flow. In one carotid bifurcation, perfusion pressure was raised to stimulate carotid sinus baroreceptors. In the other carotid bifurcation, pressure was constant and nicotine was injected to stimulate carotid chemoreceptors. Stimulation of baroreceptors on one side attenuated the ventilatory response to stimulation of contralateral chemoreceptors. This inhibition was observed before and after bilateral cervical vagotomy. We conclude that there is a major central interaction between baroreceptor and chemoreceptor reflexes so that changes in baroreceptor activity modulate ventilatory responses to chemoreceptor stimulation.


Effect of increased static lung recoil on bronchial dimesions of excised lungs. We measured bronchial diameters and lengths during static deflation and inflation in eight excised dog lobes before and after static lung recoil (Pst(L)) had been significantly increased by cooling the lobe for 48 h at 4 degrees C and ventilating it for 3 h. In control lobes, bronchial diameters were the same at any volume even though Pst(L) was different during inflation and deflation. These results agree with those of Hughes et al. (J. Appl. Physiol. 32: 25-35, 1972). However, when Pst(L) was increased, diameters at a given volume were significantly increased over control values; diameters at a given pressure were nearly the same as the controls. Therefore, under these conditions, bronchial diameter did not conform to lung volume. The ventilation process appeared to alter the circumferential elastic properties of the bronchi because diameters at all pressures were slightly larger after ventilation. Bronchial length-volume relationships were the same in both control and ventilated lobes. Thus, when Pst(L) was markedly increased, diameter corresponded best to lung recoil and length to lung volume.


Left ventricular dynamics during recovery from exercise. Left ventricular dynamics during recovery were measured in dogs, 3 min after brief periods of mild, moderate, and severe treadmill exercise. As compared with resting values, stroke volume was unchanged, and the maximum first derivative of the left ventricular pressure was either unchanged or slightly elevated. Increases in heart rate of 20, 26, and 46 beats/min for mild, moderate, and severe exercise appear to be the major factor in augmenting cardiac output during recovery. With moderate and severe exercise, left ventricular end-diastolic diameter increased and continued to be elevated during recovery, whereas end-systolic diameter decreased during exercise but was elevated above resting values during recovery. Therefore, with strenuous exercise, a sympathetic-mediated increase in contractility recedes promptly during the postexercise period but the Frank-Starling mechanism continues to be a factor.


Blood viscosity and cardiac output in acute experimental anemia. The significance of blood viscosity alterations during anemia was evaluated in dogs under morphine-chloralose anesthesia. In group I, anemia (mean hematocrit 18.1 +/- 1.3 vol %) was produced by exchange transfusion with clinical dextran (avg mol wt 70,000). In group II, anemia was produced (mean hematocrit 19.9 +/- 0.88 vol %) with 500,000 molecular weight dextran, thus preventing the decrease in blood viscosity in group I. The cardiac output increase in group I (93.4%) with low-viscosity anemia was significantly greater than in group II (43.3%) with unchanged blood viscosity. Group III animals were transfused with a clinical dextran-red cell mixture, and group IV animals received a 500,000 mol wt dextran-red cell mixture. In group III, blood viscosity and cardiac output did not change. In group IV, blood viscosity rose and cardiac output fell significantly. The results suggest that a change in blood viscosity exerts a significant effect upon cardiac output, especially during acute dextran-exchange anemia.


Heat tolerance of exercising lean and obese prepubertal boys. Seven lean and five obese boys, aged 9-12 yr, exercised in four environments: 21.1, 26.7, 29.4, and 32.2 degrees C Teff. Subjects walked on a treadmill at 4.8 km/h, 5% grade for three 20-min exercise bouts separated by 5-min rest periods. Rectal temperature (Tre), skin temperature (Tsk), heart rate (HR), sweat rate, and oxygen uptake (VO2) were measured periodically throughout the session. Lean boys had lower Tre and HR than obese boys in each of the environments. Increases in Tre were significantly greater for the obese at 26.7 and 29.4 degrees C Teff. No significant differences in Tsk and sweat rate (g-m-2-h-1) were observed between lean and obese boys. Obese boys had significantly lower oxygen consumptions per kg but worked at a significantly higher percentage of VO2max than lean boys when performing submaximal work. Responses of the obese boys to exercise in the heat were similar to those of heavy prepubertal girls studied previously, except that the boys were more tolerant of exercise at 32.2 degrees C Teff than the girls. Lean boys had lower HR than lean girls in each environment, but lower Tre only at 32.2 degrees C Teff.


Effect of altitude relocations upon AaDo2 at rest and during exercise. The supine pulmonary venous admixture (shunt) has been measured at Cerro de Pasco, 4,350 m altitude in eight subjects native to high altitude (HAN) under resting condition. Alveolar-arterial O2 tension difference (AaDO2) was also determined at rest and during exercise. The same subjects were studied again after 10 days' sojourn at sea level in Lima at 150 m altitude. They were compared with four subjects from sea level (SLN) who were studied first at Lima and after 2 and 10 days at Cerro de Pasco. At altitude, AaDO2 was smaller in HAN than SLN both at rest and during exercise. Shunt was the same in both groups. It is concluded that HAN show more even ventilation/perfusion relationship (VA/Q) at altitude, probably due to their high pulmonary artery pressure. On the contrary, SLN show less even VA/Q on altitude exposure, since their shunt decreased 37%. At sea level, HAN increased their AaDO2 due partially to an increase of 110% in their shunt, and in part due to less even VA/Q as shown by augmented VD/VT ratios. Each group tended to have a more effective gas exchange in its own environment.


Renal blood flow distribution during steady-state exercise and exhaustion in conscious dogs. To determine whether renal blood flow is reduced or redistributed during exercise, we measured total renal flow (TRF) and intrarenal flow distribution (IRFD) in nine dogs. They ran on a motor-driven treadmill at 3-8 mph at grades of 8-15% for an average of 35 min. We measured aortic pressure, heart rate, stroke volume, and cardiac output (CO) via chronically implanted catheters and an electromagnetic flow probe. We injected 15-mum radiolabeled microspheres (85Sr, 141Ce, and 51Cr) via a left atrial catheter during resting control, steady state (SS) and exhaustive (EE) exercise; measured their distribution by gamma spectrometry; and determined TRF as % CO and as ml/100 g per min. We determined IRFD for the outer and inner cortex and the outer medulla. TRF as %CO dropped (P less than 0.05) during both levels of exercise: from 10.2 +/- 0.7% to 3.9 +/- 0.4% (SS) and 3.4 +/- 0.6% (EE). TRF in ml/100 g per min did not change significantly from control (228 +/- 30 ml/100 g per min). IRFD was unchanged with exercise, remaining at about 80, 20, and 3% of TRF for the outer and inner cortex and outer medulla, respectively. We conclude that blood flow is not diverted from the kidneys during severe exercise in the dog.


Effect of prostaglandin E2 on esophageal motility in man. Studies were performed to investigate the effect of prostaglandin E2 on esophageal motility in 12 healthy volunteers. PGE2 infusion caused a dose-dependent reduction in the lower esophageal sphincter pressure. The threshold dose was less than 0.05 mug-kg-1-min-1 and maximal reduction of pressure (60%) occurred with a dose of 0.4 mug-kg-1-min-1. In contrast to its effect on the lower esophageal sphincter, PGE2 did not alter the pressure in the upper esophageal sphincter. PGE2 did not influence resting esophageal pressures; the amplitude of peristaltic contractions was reduced in the lower but not in the upper part of the body of the esophagus. These studies show that in man PGE2 exerts selective inhibitory influence on the activity of the lower part of the esophagus and lower esophageal sphincter which are composed of smooth muscle fibers.


Ventricular function following acute carbon monoxide exposure. Cardiac output function curves were used to investigate the effects of carbon monoxide on the heart in the conscious dog. Each dog was briefly exposed to 1,500 ppm carbon monoxide through a permanent tracheostomy. Immediately upon attaining either 10%, 20%, or 30% HbCO a rapid infusion of Ringer's lactate was given to test cardiac capabilities. The combined effects of carbon monoxide and infusion produced significant increases in cardiac output, heart rate, mean left ventricular pressure, dP/dt and (dP/dt)/IP. Cardiac output was sufficient to prevent peripheral hypoxia at all HbCO levels; however, there was evidence of impending cardiac depression beginning at 20% HbCO.


High-resolution radioisotopic measurements of calcium, strontium, and barium in heart. A gamma-ray detector system used in conjunction with isolated vascularly perfused rabbit septa labeled with the Ca-like cations 85Sr and 133Ba has been developed to test the feasibility of constructing a considerably more efficient detector system capable of measuring the highly energetic (Emax congruent to 1.3 MeV) 47Ca radionuclide. In addition an experimental design has been developed to quantify, statistically, perturbations of total tissue Ca, Sr, or Ba should they occur. This project was undertaken because the severe attenuation by living tissue of the beta-emitting radionuclide 45Ca (Emax congruent to 250 keV) limits its usefulness as a Ca tracer during experiments in which total tissue Ca is being measured minute-by-minute by an external detector. Analyses of over 100 experiments conducted on 35Sr- and 133Ba-labeled rabbit septa indicate that a tissue-detector system can be developed having the capacity to resolve perturbations of as little as 2 mumol Ca/kg wet tissue at 1-min sampling times or 6 mumol Ca/kg wet tissue at 0.1-min sampling times.


Transformation analysis of three linkage groups in Staphylococcus aureus. While studying a set of multiply marked mutants of Staphylococcus aureus strain 8325 by transformation, several instances of apparent genetic linkage were encountered. After showing that these linked transformations were readily inactivated by shearing of the deoxyribonucleic acid (DNA) but were resistant to dilution of the DNA, and showing that mixtures of DNA failed to form double transformants, it was concluded that the linkages were legitimate rather than the result of congression. Three linkage groups were defined: thy-101-lys-115-trp-103-thr-106, pyr-141-hisGb15-nov-pur-102, and pur-110-ilv-129. The positions of the previously studied trp and his operons corresponded to the trp-103 and hisGb15 loci. The ilv-129 position adjacent to pur-110 probably corresponds to the ilv-leu gene cluster. The distance over which linkage was detected was greater by transformation than by generalized transduction.


Stereospecific analysis of major glycerolipids of Phycomyces blakesleeanus sporangiophores and mycelium. The positional distribution of fatty acids was determined in the major groups of glycerolipids from the mycelium and sporangiophores of the fungus Phycomyces blakesleeanus. At the sn-1 positions of the triacylglycerols, in both regions of the fungus, greater than 65% of the fatty acids were 16:0 and 18:1. At the sn-2 positions of the triacylglycerols, 18:1, 18:2 and 18:3 comprised greater than 85% of the sporangial fatty acids and more than 90% of the mycelial fatty acids. Positions sn-3 of the triacylglycerols, from both regions of the fungus, contained approximately 40% of 16:0, approximately 30% of 18:2, and the largest proportions of 18:3 (21%) in the triacyglycerols. The major phosphoglycerides of P. blakesleeanus  mycelium and sporangiophores are phosphatidylcholine and phosphatidylethanolamine, and more than 85% of the fatty acids at the sn-1 positions of these phosphatides consisted of 16:0, 18:2, and 18:3. The sn-2 positions of phosphatidylcholine and phosphatidylethanolamine contained approximately 98% unsaturated fatty acids. In the phosphoglycerides of both regions of the fungus, 18:2 and 18:3 constituted greater than 85% of the total fatty acids. Although the mycelium and sporangiophores of P. blakesleeanus had different morphological and physiological characteristics, the major glycerolipids of the two regions had similar stereospecific distributions of fatty acids.


Development of defined and minimal media for the growth of Bacillus stearothermophilus. Defined media, both solid and liquid, that support good growth of Bacillus stearothermophilus 1503 have been developed. Data are presented which indicate that manganese is required at relatively high concentrations for growth in a defined liquid medium. Phosphate concentrations higher than 5 times 10(-3) M have been shown to inhibit colony formation on solid media. Maximum viable counts of approximately 10(9) colony-forming units per ml were obtained in both the defined and minimal liquid media. Glucose, fructose, sucrose, glycerol, and starch support the growth of this obligate thermophile in the defined media, whereas citrate, alpha-ketoglutarate, succinate, fumarate, malate, acetate, and lactate do not. The described media have been utilized to isolate several amino acid-requiring mutants of B. stearothermophilus.


Metabolism of n-propylamine, isopropylamine, and 1,3-propane diamine by Mycobacterium convolutum. Mycobacterium convolutum strain NPA-1 can utilize n-propylamine (NPA), isopropylamine (IPA), and 1,3-propane diamine (PD) as sole source of carbon, nitrogen, and energy. Enzyme assays, fatty acid profiles, and 14CO2 incorporation experiments indicate that NPA is deaminated to propionate and further metabolized via the methylmalonyl succinate pathway, and IPA and PD were metabolized (after deamination) through a C2 + C1 cleavage. An inducible amine dehydrogenase was present in cell extracts after growth on the three amines. Polyacrylamide gel electrophoresis of cell extracts from NPA- and IPA-grown cells yielded one major band of amine dehydrogenase activity. When extracts of NPA-grown cells were assayed with NPA, IPA, or PD as substrate, the relative position of the major band on gel electrophoresis was equivalent. Similar results were obtained with extracts prepared from IPA-grown cells. Sephadex G-100 chromatography also indicated one major peak of activity. This suggests that one enzyme of broad specificity is involved in deamination of IPA, NPA, and PD. IPA-grown cells utilized NPA readily, whereas NPA-grown cells could not utilize IPA without lag. Since amine dehydrogenase activity was present in extracts of cells after growth on either substrate, this lag was probably due to the inability to transport IPA without an induction period. The molecular weight of the amine dehydrogenase was approximately 38,500 as determined by gel filtration.


Methionine transport in Yersinia pestis. Yersinia pestis TJW, an avirulent wild-type strain, requires phenylalanine and methionine for growth. It was of interest to examine and define the methionine transport system because of this requirement. The methionine system showed saturation kinetics with a Km for transport of approximately 9 times 10(-7) M. After 8 s of methionine transport, essentially all of the methionine label appeared in S-adenosyl-L-methionine (SAM) as detected in ethanol extracts. Small amounts of free methionine was detected intracellularly after 1 min of transport. Addition of glucose increased significantly the amount of intracellular methionine at 1 min. A series of SAM metabolic products was detected after 90 s to 5 min of transport including: 5'-thiomethyladenosine, homoserine lactone, S-adenosyl homoserine, and a fluorescent methyl receptor compound. Results from assays for SAM synthetase in spheroplast fractions showed a small (16%) but significant portion of synthetase associated with the membrane. However, most of the enzyme activity was associated with the cytoplasmic fraction. Methionine transport was characterized by a high degree of stereospecificity. No competition occurred from structurally unrelated amino acids. Although uptake was inhibited by uncoupling and sulfhydryl reagents, no efflux was observed. Results using energy inhibitors on unstarved and starved cells showed that respiratory inhibitors such as potassium cyanide (KCN) and amytal were most effective, and that arsenate was least effective. KCN plus arsenate completely blocked utilization of energy derived from glucose, and KCN completely blocked utilization of energy deived from D-lactate. The data indicate that methionine transport in Y. pestis is linked to the trapping of methionine in SAM. The results further suggest that this transport system can be classified as a permease-bound system where transport is coupled to an energized membrane state and to respiration.


Regulation of lactate dehydrogenase and change of fermentation products in streptococci. Streptococcus mutans JC 2 produced mainly lactate as a fermentation product when grown in nitrogen-limited continuous culture in the presence of an excess of glucose and produced formate, acetate, and ethanol, but no lactate, under glucose-limited conditions. The levels of lactate dehydrogenase (LDH) in these cultures were of the same order of magnitude, and the activity of LDH was completely dependent on fructose-1,6-diphosphate (FDP). The intracellular level of FDP was high and the level of phosphoenolpyruvate (PEP) was low under the glucose-excess conditions. In the glucose-limited cultures, all glycolytic intermediates studied, except PEP, were low. S. mutans FIL, which had an FDP-independent LDH and similar levels of glycolytic intermediates as S. mutans JC2, produced mainly lactate under glucose-excess or under glucose-limited conditions. LDH of Streptococcus bovis ATCC 9809 was dependent on FDP for activity at a low concentration of pyruvate but had a significant activity without FDP at a high concentration of pyruvate. This strain also produced mainly lactate both under glucose-excess and glucose-limited conditions. The levels and characteristics of these LDHs were not changed by the culture conditions. These results indicate that changes in the intracellular level of FDP regulate LDH activity, which in turn influences the type of fermentation products produced by streptococci. PEP, adenosine 5'-monophosphate, adenosine 5'-diphosphate, and inorganic phosphate significantly inhibited LDH activity from S. mutans JC 2 and may also participate in the regulation of LDH activity in other streptococci.


Metabolism of toluene and xylenes by Pseudomonas (putida (arvilla) mt-2: evidence for a new function of the TOL plasmid. Pseudomonas putida (arvilla) mt-2 carries genes for the catabolism of toluene, m-xylene, and p-xylene on a transmissible plasmid, TOL. These compounds are degraded by oxidation of one of the methyl substituents via the corresponding alcohols and aldehydes to benzoate and m- and p-toluates, respectively, which are then further metabolised by the meta pathway, also coded for by the TOL plasmid. The specificities of the benzyl alcohol dehydrogenase and the benzaldehyde dehydrogenase for their three respective substrates are independent of the carbon source used for growth, suggesting that a single set of nonspecific enzymes is responsible for the dissimilation of the breakdown products of toluene and m- and p-xylene. Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase are coincidently and possible coordinately induced by toluene and the xylenes, and by the corresponding alcohols and aldehydes. They are not induced in cells grown on m-toluate but catechol 2,3-oxygenase can be induced by m-xylene.


Lysis of modified walls from Lactobacillus fermentum. The N and O substitution in wall peptidoglycan from Lactobacillus fermentum was studied in relation to growth phase, as well as the lytic activities and the effect of trypsin on them. The N-nonsubstituted sites were determined by dinitrophenylation techniques. The results indicate that an extensive substitution at the O groups takes place as cells go into the stationary growth phase, concomitant with a decrease in their lysozyme sensitivity. N-nonsubstituted residues, mainly glucosamine, occurred in both exponential-phase and stationary-phase walls but not in the corresponding peptidoglycans. Small amounts of N-nonsubstituted muramic acid were detected in walls and peptidoglycan from cells in the stationary growth phase only. N acetylation of isolated walls did not increase their lysozyme sensitivity but rather decreased it. Autolysis of walls was completely inhibited by the chemical modifications used. Trypsin stimulates the lysozyme sensitivity of native walls but has no effect on walls that had been O deacetylated and N acetylated. It is suggested that the effect of trypsin is due to its action as an esterase removing the O acetylation in lysozyme-resistant walls.


Correlation of the effects of citric acid cycle metabolites on succinate oxidation by rat liver mitochondria and submitochondrial particles. 1. Succinate dehydrogenase is inhibited by citrate and beta-hydroxy-butyrate in a complex manner, both in mitochondria and submitochondrial particles. Kinetics of inhibition in the particles points to a competitive component in the mechanism involved. 2. Pyruvate, alpha-ketoglutarate, malate, and glutamate stimulate oxidation of succinate by mitochondria. 3. Stimulation by alpha-ketoglutarate and glutamate is not influenced by the presence of rotenone. 4. Stimulation by pyruvate is higher in the absence of rotenone and increases significantly in the presence of K+ and valinomycin. Pyruvate supplies in mitochondria reducing equivalents for malate dehydrogenase operating in the reverse direction-reduction of oxaloacetate to malate. 5. Stimulation by malate is higher in the presence of rotenone.


Substrate-protein interaction in tryptophanase from Bacillus alvei. Kinetic and spectral evaluations. This investigation studied the substrate protein interaction of the alpha, beta elimination reaction in tryptophanase (EC 4.1.99.1). The results of this work are 2-fold. (a) The presence of multiple enzyme sites was found to be related to the observed kinetic patterns of inhibition. Indole analogues caused competitive inhibition in the tryptophanase reaction and noncompetitive inhibition in the dehydratase reaction. Inhibition patterns of alanine for these activities were reserved. (b) Under some conditions, compounds which bind presumably at the indole site modified the spectral and fluorescent characteristics of the enzyme. The addition of anthranilate to the enzyme resulted in a broad absorption band around 350 nm. This absorption band was distinct from that formed by alanine addition. Based on absorption data, both of these compounds could be bound simultaneously. The optical activity of tryptophanase was reported for the first time. Indole analogues caused greater conformational alterations in the circular dichroism spectra than 3-carbon analogues. The calculated anisotrophy factors, as well as fluorescent quenching data, suggest a more direct interaction between indole analogues and pyridoxal-P than between 3-carbon compounds that the coenzyme. It is proposed that the indole site is the dominant recognition site. The data are consistent with the three-dimensional aspects of space-filling models of Schiff's bases evaluated in terms of multiple site binding.


Membrane-bound tubulin in brain and thyroid tissue. Brain and thyroid tissue contain membrane-bound colchicine-binding activity that is not due to contamination by loosely bound cytoplasmic tubulin. This activity can be solubilized to the extent of 80 to 90% by treatment with 0.2% Nonidet P-40 with retention of colchicine binding. Extracts so obtained contain a prominent protein band in disc gel electrophoresis that co-migrates with tubulin. Membranes, and the solubilized protein therefrom, exhibit ligand binding properties like tubulin; for colchicine the KA is approximately 1 X 10(6) M-1 in brain and approximately 0.6 X 10(6) M-1 in thyroid; for vinblastine the KA is approximately 8 X 10(6) M-1 for both tissues; and for podophyllotoxin the Ki is approximately 2 X 10(-6) M for both tissues. Displacement by analogues of colchicine is of the same order as for soluble tubulin. Although membrane-bound colchicine-binding activity shows greater thermal stability and a higher optimum binding temperature (54 degrees versus 37 degrees) than soluble tubulin, this appears to be the result of the membrane environment since the solubilized binding activity behaves like the soluble tubulin. Antibody against soluble brain tubulin reacts with membranes and solubulized colchicine-binding activity from both brain and thyroid gland. We conclude that brain and thyroid membrane preparations contain firmly bound tubulin or a very similar protein.


Calcium-binding protein of bovine intestine. The complete amino acid sequence. The amino acid sequence for vitamin D-dependent bovine intestinal calcium binding protein has been established. It contains 85 amino acids in a single chain and lacks cysteine, tryptophan, methionine, histidine, and arginine. The NH2-terminal lysine is blocked by an N-acetyl group. Enzymatic digestion with trypsin, chymotrypsin, and pepsin yielded a number of peptides which were purified by two-dimensional high voltage paper electrophoresis. These peptides were examined by end group analysis and sequenced by the dansyl procedure. The absence of tryptophan permitted by a single cleavage of the molecule by N-bromosuccinimide at the tyrosine residue at position 8 and the larger fragment was subjected to automated Edman degradation. By these means, the following sequence was established: N-Ac-Lys-Gln-Ser-Pro-Leu-Glu-Tyr-Ala-Ala-Glu-Lys-Ser-Ile-Gln-Lys-Glu-Ile-Glu-Lys-Gly-Phe-Phe-Lys-Gln-Leu-Leu-Val-Ser-Val-Gln-Lys-Ala-Gly-Asp-Lys-Glu-Ser-Leu-Gln-Pro-Leu-Phe-Thr-Leu-Leu-Lys-Ser-Gly-Pro-Glu-Glu-Asn-Leu-Lys-Glu-Ser-Gln-Asn-Gly-Pro-Asp-Leu-Ls7-Ser-Gly-Pro-Gly-Asn-Asp-Leu-Glu-Glu-Lys-Gly-Thr-Asp-Val-Phe-Ser-Leu-Lys-Gln. Microheterogeneity may exist in the molecule at residue 76 in which position threonine may be replaced by serine. Comparison of the sequence of calcium-binding protein to the "test" sequence of Tufty and Kretsinger ((1975) Science 187, 167-169) proposed to identify E-F hands in muscle proteins suggests that intestinal calcium-binding protein may likewise contain one or possibly two E-F hands which could account for calcium-binding property. Dayhoff alignment scores, however, calculated for calcium-binding protein against nine E-F hands in muscle proteins parvalbumin, troponin and alkali light chains do not indicate that intestinal calcium-binding protein is homologous to these muscle protein chains.


Affinity labeling of steroid binding sites. Study of the active site of 20beta-hydroxysteroid dehydrogenase with 2alpha-bromoacetoxyprogesterone and 11alpha-bromacetoxyprogesterone. To further characterize the active site of 20beta-hydroxysteroid dehydrogenase (EC 1.1.1.53) from Streptomyced hydrogenans we synthesized 2alpha-bromoacetoxyprogesterone, a substrate for the enzyme in 0.05 M phosphate buffer at 25 degrees, pH 7.0, with Km and Vmax values of 1.90 X 10(-5) M and 6.09 nmol/min/mg of enzyme, respectively. This affinity labeling steroid inactivates 20beta-hydroxysteroid dehydrogenase in an irreversible and time-dependent manner which follows pseudo-first order kinetics with a t1/2 value of 4.6 hours. 2alpha-[2-3H]Bromoacetoxyprogesterone was synthesized and used to radiolabel the enzyme active site. Amino acid analysis of the acid hydrolysate of the radiolabeled enzyme supports a mechanism whereby the steroid moiety delivers the alkylating group to the steroid binding site of the enzyme where it reacts with a methionyl residue. Both 2alpha- and 11alpha-bromoacetoxyprogesterone alkylate a methionyl residue at the active site of 20beta-hydroxysteroid dehydrogenase. The enzyme was inactivated with a mixture containing both 2alpha-[2-3H]Bromoacetoxyprogesterone and 11alpha-2[2-14C]bromoacetoxyprogesterone. Following degradation of separate aliquots of the radiolabeled enzyme by cyanogen bromide or trypsin, the protein fragments were separated by gel filtration and ion exchange chromatography. Resolution of peptides carrying the 3H label from those possessing the 14C label demonstrates that 2alpha-bromoacetoxyprogesterone and 11alpha-bromoacetoxyprogesterone each label a different methionine at the steroid binding site of 20beta-hydroxysteroid dehydrogenase.


Synthesis of 16alpha-bromoacetoxyestradiol 3-methyl ether and study of the steroid binding site of human placental estradiol 17beta-dehydrogenase. Homogeneous estradiol 17beta-dehydrogenase (EC 1.1.1.62) was prepared from human placenta by affinity chromatography and the steroid binding site was studied with affinity-labeling techniques. 16alpha-Bromoacetoxyestradiol 3-methyl ether and the tritated compound were synthesized by condensation of estriol 3-methyl ether with bromoacetic acid or [2-3H]bromoacetic acid in the presence of dicyclohexylcarbodiimide. 16alpha-Bromoacetoxyestradiol 3-methyl ether is stable in 0.01 M phosphate buffer at pH 7.0, 25 degrees, for at least 24 hours. It alkylates cysteine, histidine, methionine, lysine, and tryptophan under physiological conditions. The steroid is a substrate of estradiol 17beta-dehydrogenase, thus it must bind at the steroid binding site. The inactivation of estradiol 17beta-dehydrogenase by 150-fold molar concentrations of 16alpha-bromoacetoxyestradiol 3-methyl ether follows pseudo-first order kinetics with a half-time of 1.5 hours. Estradiol-17beta, NADH, and NADPH slow the rate of inactivation. 2-Mercaptoethanol in molar concentrations 50-fold that of 16alpha-bromoacetoxyestradiol 3-methyl ether stops the inactivation, but does not reverse it. 16alpha-Bromoacetoxyestradiol 3-methyl ether alkylates both NADH and NADPH; the presence of small amounts of enzyme markedly increases the rate of this alkylation. When the enzyme is inactivated with 16alpha-[2-3H]bromoacetoxyestradiol 3-methyl ether, amino acid analysis of acid hydrolysates reveals 3-carboxymethylhistidine and 1,3-dicarboxymethylhistidine. Comparison of 28 and 51% inactivated samples indicates that, as inactivation proceeds, the total amount of 3-carboxymethylhistidine decreases, while 1,3-dicarboxymethylhistidine increases, suggesting that the former is converted to the latter by a second alkylation step. When the enzyme is inactivated in the presence of a large excess of NADPH, only 1,3-dicarboxymethylhistidine is found. From the present study it is concluded that estradiol 17beta-dehydrogenase has a histidyl residue present in the catalytic region of the active site as does the previously studied 20beta-hydroxysteroid dehydrogenase.


Human plasma R-type vitamin B12-binding proteins. I. Isolation and characterization of transcobalamin I. TRANSCOBALAMIN III. and the normal granulocyte vitamin B12-binding protein. Transcobalamin I and transcobalamin III have been purified approximately 6,000,000- and 3,000,000-fold, respectively, from normal human plasma using a purification scheme consisting of immunoadsorption, dialysis against 7.5 M guanidine HCl to remove endogenous vitamin B12, and affinity chromatography on vitamin B12-Sepharose. The two proteins were separated from each other subsequently by chromatography on DEAE-cellulose. The vitamin B12-binding protein present in granulocytes obtained from normal subjects has been purified approximately 5000-fold using affinity chromatography on vitamin B12-Sepharose as the sole purification technique. The final preparations of all three proteins were homogeneous based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Transcobalamin I and transcobalamin III belong to the R-typed class of vitamin B12-binding proteins and are indistinguishable from each other, and from the human granulocyte, milk, and saliva R-type vitamin B12-binding proteins, when studied by immunodiffusion with rabbit anti-human milk vitamin B12-binding protein sera. The carbohydrate compositions, expressed as moles of carbohydrate per mole of vitamin B12, of transcobalamin I, transcobalamin III, and the normal granulocyte vitamin B12-binding protein, respectively, are: sialic acid, 18, 11, 11; fucose, 9, 20, 24; galactose, 41, 51, 46; mannose, 24, 22, 20; galactosamine, 2, 2, 2; and glucosamine, 46, 54, 46. The high sialic acid content of transcobalamin I appears to account for the fact that this protein elutes after transcobalamin III and the normal granulocyte vitamin B12-binding protein during chromatography on DEAE-cellulose. This observation provides support for the hypothesis that differences among the R-type vitamin B12-binding proteins are due to differences in carbohydrate content. The similarities in carbohydrate composition and other properties of transcobalamin III and the granulocyte vitamin B12-binding protein provide support for the hypothesis that human plasma transcobalamin III is derived from granulocytes. The differences observed between transcobalamin I and the normal granulocyte vitamin B12-binding protein suggest that transcobalamin I may not be derived from granulocytes.


Human plasma R-type vitamin B12-binding proteins. II. The role of transcobalamin I, transcobalamin III, and the normal granulocyte vitamin B12-binding protein in the plasma transport of vitamin B12. The normal human granulocyte vitamin B12-binding protein, transcobalamin I, and transcobalamin III, have been labeled with 125I-labeled N-succinimidyl 3-(4-hydroxyphenyl)propionate and utilized for plasma clearance studies performed with rabbits. Both moieties of 125I-labeled granulocyte vitamin B12-binding protein-[57Co]vitamin B12 were cleared rapidly from the plasma (is less than 90% by 5 min) by the liver. After 30 min, the bulk of the 125I reappeared in the plasma in small molecular weight (less than 1000) form and was rapidly excreted in the urine. After 60 min the bulk of the [57Co]vitamin B12 reappeared in the plasma bound to rabbit transcobalamin II and was subsequently taken up by a variety of tissues. Approximately 15% of the 125I-labeled granulocyte vitamin B12-binding protein-[57Co-a1vitamin B12 was excreted intact into the bile during the period from 10 to 80 min after injection. The hepatic uptake of the protein-vitamin B12 complex was blocked by the prior injection of desialyzed fetuin but not by native fetuin. Similar results were obtained with 125I-labeled transcobalamin III-[57Co]vitamin B12. Approximately 90% of both moieties of 125I-labeled transcobalamin I-[57Co]vitamin B12 had prolonged plasma survivals similar to that of 125I-labeled bovine serum albumin. After treatment with neuraminadase, both moieties of the 125I-labeled transcobalamin I-[57Co]vitamin B12 complex were cleared rapidly from the plasma by the liver in a manner that was indistinguishable from that observed in the case of untreated granulocyte vitamin B12-binding protein and transcobalamin III. These observations indicate that desialyzed transcobalamin I and the native forms of the granulocyte vitamin B12-binding protein and transcobalamin III are cleared from plasma by the mechanism elucidated by Ashwell and Morell (Ashwell, G., and Morell A. G. (1974) Adv. Enzymol. 41, 99-128) that is capable of clearing a wide variety of asialoglycoproteins. These observations have implications concerning the function of the human R-type vitamin B12-binding proteins, the nature of the enterohepatic circulation of vitamin B12, the biological significance of the mechanism described by Ashwell and Morell, and the etiology of the increased plasma concentration of human R-type protein that occurs frequently in chronic myelogenous leukemia and occasionally in hepatocellular carcinoma and other solid tumors.


Biochemical and immunological studies of purified mouse beta-galactosidase. Beta-Galactosidase (EC 3.2.1.23) has been purified from the livers of C57BL/6J mice. The enzyme migrated as a single band of protein on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weight of the denatured and reduced enzyme was 63,000. The native form of beta-galactosidase appeared to be a tetramer of 240,000 at pH 5.0, which was reversibly dissociated at alkaline pH to a dimer with apparent molecular weight of 113,000. Multiple charge isomers of beta-galactosidase were resolved by polyacrylamide gel electrophoresis and ion exchange chromatography. Treatment of beta-galactosidase with neuraminidase markedly reduced its electrophoretic mobility. Purified enzyme as well as crude liver extract hydrolyzed p-nitrophenyl-beta-D-fucoside at one-tenth the rate of hydrolysis of the beta-galactoside. Antiserum to the purified enzyme precipitated the major portion of beta-galactosidase activity of mouse liver, brain, and kidney. This antiserum cross-reacts with beta-galactosidases from rat and Chinese hamster, but not with human, porcine, or bovine beta-galactosidase.


Bovine renal glomerular basement membrane. Isolation and characterization of a glycoprotein component. Bovine glomerular basement membrane was extracted with 6 M guanidinium chloride and the soluble material fractionated on a Bio-Gel A-1.5m column in 1% Na dodecyl-SO4. A single component was obtained by reduction of a selected column fraction with 2-mercaptoethanol followed by chromatography on an analytical Bio-Gel A-1.5m column and shown to be homogenous by electrophoresis and ultracentrifugation. It consists of 90% protein and 8.6% carbohydrate by weight. The amino acid composition is characterized by the presence of low amounts of hydroxyproline and hydroxylysine, and substantial amounts of aspartic acid, glutamic acid, half-cystine, and glycine. It contains all the monosaccharide constituents present in the whole basement membrane indicating the presence of both heteropolysaccharide and disaccharide units; the presence of the latter unit was demonstrated unequivocally by ion exchange chromatography. The component contains 1 heteropolysaccharide unit and 4 dissaccharide units/molecule of Mr equals 70,000. The molecular weight of component VII was determined by several methods. Molecular weight values of 68,000 +/- 3,000 and 72,000 +/- 2,000 were determined in 6 M guanidinium chloride by the methods of sedimentation equilibrium and gel filtration chromatography, respectively, and values of 136,000 +/- 3,100 and 140,000 +/- 2,000 were determined in 1% Na dodecyl-SO4 by the methods of polyacrylamide gel electrophoresis and gel filtration chromatography, respectively. Circular dichroism spectra indicate that component VII assumes a random coil conformation in 6 M guanidinium chloride and a more disordered conformation in 1% Na dodecyl-SO4 than standard proteins used in calibration of polyacrylamide gels and gel filtration column. These results indicate that the minimal molecular weight of component VII is about 70,000 and that the anomalous behavior in Na dodecyl-SO4 is due in part to its conformation.


A probable oxocarbonium ion in the reaction mechanism of small intestinal sucrase and isomaltase. 1-5-D-Gluconolactone is a competitive inhibitor of both sucrase and isomaltase. Substitution of the 1H and 2H at C1 of the glucosyl moiety in p-CL-phenyl-alpha-D-glucopyranoside leads to a decrease in kcat of both sucrase and isomaltase, the k1H/k2H ranging between 1.14 and 1.20. Treatment of the association constants and of the kcat values for a number of p-substituted phenyl-alpha-D-glucopyranosides on the basis of the Hammet-Hansch equation has allowed the estimation of the importance of hydrophilicity-hydrophobicity as well as of the magnitude of the p values for both substrate-enzyme interaction and catalysis in both sucrase and isomaltase. The magnitude of the secondary deuterium effect as well as the low values of p in both sucrase and isomlatase are strongly indicative of the rate-limiting step going through the formation of an oxocarbonium ion. In conjunction with other observations reported previously, the data presented here led to the suggestion of the main lines of a reaction mechanism for the two glucosidases: prptonation of the glycosidic oxygen is followed by the liberation of the "aglycone" with formation of an oxocarbonium ion, which is temporarily stabilized by a carboxylate group.


Small intestinal sucrase and isomaltase split the bond between glucosyl-C1 and the glycosyl oxygen. The products of the hydrolysis of sucrose and palatinose by the sucrase-isomaltase complex from rabbit small intestine were investigated by persilylation followed by gas-liquid chromatography and mass spectrometry. If the hydrolysis is carried out in H218O, the heavy oxygen is found exclusively at the Ci of the alpha-glucopyranose formed. The 18O enrichment equals that of the incubation medium. The oxygen exchange between the monosaccharides and water is not accelerated by the sucrase-isomaltase complex. These observations show that the bond split by the sucarse and the isomaltase moiety of the complex is the one between glucosyl-Ci and the glucosyl oxygen. They agree with the mechanism proposed for these carbohydrases in the accompanying paper (Cogoli, A., and Semenza, G. (1975) J. Biol. Chem. 250, 7802-7809) involving the formation of an oxocarbonium ion.


Subunit structure and amino acid composition of xylose isomerase from Streptomyces albus. The subunit structure and amino acid composition of xylose isomerase from Streptomyces albus have been examined. A native molecular weight of 165,000 determined by sedimentation equilibrium was reduced to 43,000 when the protein was treated with 6 M guanidine hydrochloride. No further reduction in molecular weight was observed when potential disulfide bridges of xylose isomerase were reduced and alkylated, indicating that the protein was devoid of interchain disulfide bonds. NH2-terminal analysis using [3H]dansyl chloride showed 0.86 residues of methionine per Mr equals 41,500 unit. Analysis of the native protein with an automated protein sequenator revealed the presence of only one degradable polypeptide chain. Fractionation of the soluble tryptic peptides of S-[14C]carboxymethyl xylose isomerase by ion exchange chromatography and one-dimensional paper electrophoresis yielded 37 to 43 peptides. When the acid-insoluble tryptic peptides were dissolved and analyzed using gel filtration techniques, and additional four peptides were found. A unique radioactive tryptic peptide containing S-carboxymethylcysteine was found among the soluble peptides, confirming cysteine as the limiting amino acid residue in the amino acid composition of xylose isomerase. On the basis of its lysine and arginine content, the number of tryptic peptides is consistent with the hypothesis that the native xylose isomerase is a tetramer of four very similar or identical subunits of Mr equals 41,500, associated by noncovalent bonds.


Relationship between iodination and the polypeptide chain composition of thyroglobulin. Thyroglobulin was isolated from thyroid glands of normal guinea pigs and from animals treated with thiouracil. These preparations were fractionated by isopyknic centrifugation in RbCl into proteins of varying iodine content. When the disulfide bonds of these protein fractions were reduced and analyzed by polyacrylamide gel electrophoresis in Na dodecyl-SO4, t hree species were observed with molecular weights of 295,000 (A), 210,000 (B), and 110,000 (C). Species A comprised 80% of the protein in thyroglobulin of 0.04% iodine and 13% in thyroglobulin of 0.68% iodine content. Species C showed the opposite relationship, comprising 10% of the low and 70% of the high iodine thyroglobulin. Species B was relatively independent of the iodine content and represented approximately 20% of the protein. Iodine analysis of these proteins showed species A to be lowest and species C highest. It appears that the subunit composition of thyroglobulin depends on the degree of iodination and that species A should be the only one present in the absence of iodination.


Aspects of energy-linked calcium accumulation by rat heart mitochondria. When intact rat heart mitochondria were pulsed with 150 nmol of CaCl2/mg of mitochondrial protein, only a marginal stimulation of the rate of oxygen consumption was observed. This result was obtained with mitochondria isolated in either the presence or absence of nagarse. In contrast, rat liver mitochondria under similar conditions demonstrated a rapid, reversible burst of respiration associated with energy-linked calcium accumulation. Direct analysis of calcium retention using 45Ca and Millipore filtration indicated that calcium was accumulated by heart mitochondria under the above conditions via a unique energy-dependent process. The rate of translocation by heart mitochondria was less than that of liver mitochondria; likewise the release of bound calcium back into the medium was also retarded. These results suggest that the slower accumulation and release of calcium is characteristic of heart mitochondria. The amound of calcium bound was independent of penetrant anions at low calcium concentrations. Above 100 nmol/mg of mitochondrial protein, the total calcium bound was increased by the presence of inorganic phosphate. Under nonrespiring conditions, a biphasic Scatchard plot indicative of binding sites with different affinities for Ca2+ was observed. The extrapolated constants are 7.5 nmol/mg bound with an apparent half-saturation value of 75 muM and 42.5 nmol/mg bound with half-saturation at 1.15 mM. The response of the reduced State 4 cytochrome b to pulsed additions of Ca2+ was used to calculate an energy-dependent half-saturation constant of 40 muM. When the concentration of free calcium was stabilized at low levels with Ca2+-EGTA buffers, the spectrophotometrically determined binding constant decreased two orders of magnitude to an apparent affinity of 4.16 X 10(-7) M. Primary of calcium transport over oxidative phosphorylation was not observed with heart mitochondria. The phosphorylation of ADP competed with Ca2+ accumulation, depressed the rates of cation transport, and altered the profile of respiration-linked H+ movements. Consistent with these result was the observation that with liver mitochondrial the magnitude of the cytochrome b oxidation-reduction shift was greater for Ca2+ than for ADP, whereas calcium responses never surpassed the ADP response in heart mitochondria. Furthermore, Mg2+ ingibited calcium accumulation by heart mitochondria while having only a slight effect upon calcium transport in liver mitochondria. The unique energetics of heart mitochondrial calcium transport are discussed relative to the regulated flux of cations during the cardiac excitation-relaxation cycle.


Preferred binding of bovine and porcine trypsins at two different sites on chicken ovoinhibitor. Reduced dissociation of mixed trypsin complexes. Dissociation of mixed trypsin (bovine plus porcine trypsin) complexes with chicken ovoinhibitor was used to investigate the nonequivalence of the two binding sites for trypsin on the inhibitor. Previous work has shown that 1 mol of trypsin dissociates much more rapidly than the 2nd from unmixed trypsin complexes, those containing 2 mol of one kind of trypsin, bovine or porcine, per mol of inhibitor. However, only approximately 0.5 to 0.6 mol of trypsin dissociated in the rapid step from the mixed trypsin complexes, those containing 1 mol each of bovine and porcine trypsin. Rates of the slow dissociation steps for the two types of complexes did not differ appreciably from each other. A general dissociation scheme is proposed, in which each of the 2:1 complexes can lose a trypsin molecule from either in two parrallel first order reactions, producing two different 1:1 complexes, which subsequently dissociate to yield free ovoinhibitor and a second trypsin molecule. In this scheme, both the earlier results with unmixed trypsin complexes and the preponderance (approximately 3:1) of slow dissociation from the mixed trypsin complexes can be rationalized if bovine trypsin is retained preferentially at one of the two trypsin binding sites on chicken ovoinhibitor, and porcine trypsin at the other. That is, one site allows rapid dissociation of porcine trypsin and slow dissociation of bovine trypsin, whereas the other allows rapid dissociation of bovine trypsin and slow dissociation of porcine trypsin.


Superstoichiometric Ca2+ uptake supported by hydrolysis of endogenous ATP in rat liver mitochondria. The nature of the energy store causing rapid superstoichiometric leads to H+/2e minus ejection and leads to Ca2+/2e minus uptake ratios in rat liver mitochondria pulsed with Ca2+ has been investigated. The extent and the rate of the initial fast superstoichiometric phase of H plus ejection were greatly reduced by oligomycin and other ATPase inhibitors; the subsequent shoichiometric phase was unaffected. No such inhibition was seen with atractyloside. Similarly, the initial fast phase of Ca2+ uptake was reduced in extent by oligomycin, whereas the slower stoichiometric phase was unaffected. Moreover, the ATP content of mitochondria previously incubated with succinate decreased by about 80% within 5 s after pulsing with Ca2+. The energy store for superstoichiometric Ca2+ uptake and H plus injection is thus identified as endogenous ATP.


Metabolic implications of the distribution of the alanine aminotransferase isoenzymes. The distribution of alanine aminotransferase isozymes in several tissues from several species has been studied. In glycolytic tissues, such as skeletal and cardiac muscle, cytosolic alanine aminotransferase was the predominant form. In gluconeogenic tissues, such as liver and kidney, the concentration of the cytosolic alanine aminotransferase was much more variable; its presence, however, may be correlated with the presence of phosphoenolpyruvate carboxykinase in the same compartment. The particulate enzyme was found associated only with the matrix of the mitochondria. It was present only in those gluconeogenic tissues that can utilize alanine for glucose production, e.g. rat liver and pig liver and kidney; it was absent from rat kidney which cannot convert alanine to glucose. These observations, together with the kinetic parameters of the two isozymes, suggest that in vivo, mitochondrial alanine aminotransferase is involved in the conversion of alanine to pyruvate, while the cytosolic isoenzyme is mainly involved in the formation of alanine from pyruvate.


Deuterium nuclear magnetic resonance of deuterium-labeled diacetyldeuterohemin incorporated into sperm whale myoglobin. The heme derivative 2,4-diacetyldeuterohemin deuterated in the methyl groups of the acetyl moieties was reconstituted with sperm whale apomyoglobin and the two labeled methyl groups in the protein environment were observed by deuterium nuclear magnetic resonance spectroscopy. The results were compared to the free hemin form as the dimethyl ester in chloroform and in a pyridine-water mixture, as well as in the zinc complex form. Under most conditions the two methyl resonances overlie each other to a large degree. Resonance width at half-height is of the order of 25 Hz for the protein and approximately one-third as much for the free hemin at 16 degrees and is little affected by conversion to paramagnetic derivatives. Chemical shifts for the oxy- and carbonmonoxymyoglobins are very similar. In cyanoferrimyoglobin a positive pseudo-contact contribution of 3.04 ppm was computed to explain a relative upfield shift offset in part by a small negative contact shift contribution. The cyanoferrimyoglobin resonance was sensitive to the presence of phosphate buffer as well as to cyclopropane. The aquoferrimyoglobin form shows distinct resonances for the two methyl groups, with the downfield resonance considerably broadened. The expected effects of temperature on chemical shift were observed, the paramagnetic derivatives showing an effect and carbonmonoxymyoglobin showing none. The relaxation behavior was gauged from the line widths and from measurements of spin-lattice relaxation time, T1. The effective rotational correlation time is of the order of 50 ps for the liganded myoglobin forms. The temperature dependence of the line widths may imply an increased retational freedom with increasing temperature. The broadening observed in the aquoferrimyoglobin case is indicative of restricted internal rotational motion of one of the methyl groups. The method is suitable for probing the more mobile structures in proteins and retains its value in the neighborhood of paramagnetic centers.


Enzymic studies on adrenocortical deoxycorticosterone 11beta-hydroxylase system. Radiometric methods for the assay of deoxycorticosterone 11beta-hydroxylase and for the determination of NADP on a microscale were developed. The determination of NADP was based on the quantitative conversion of 6-phospho[1-14C]gluconate to 14CO2 by the action of 6-phosphogluconate dehydrogenase. Using these methods NADPH oxidase activity of the adrenodoxin reductase-adrenodoxin system as well as kinetic properties of deoxycorticosterone 11beta-hydroxylase (cytochrome P-450) were investigated. The NADPH oxidase activity observed in the presence of adrenodoxin reductase, adrenodoxin, and O2, but in the absence of cytochrome P-450 and deoxycorticosterone, were functions of O2 and adrenodoxin concentrations and represented the autooxidation of reduced adrenodoxin which resulted in the production of H2O2. Due to the rapid autooxidizability of reduced adrenodoxin, only a small fraction of electrons conveyed from NADPH to adrenodoxin by way of adrenodoxin reductase was utilized for the deoxycorticosterone 11beta-hydroxylase reaction under the conditions employed.


The monovalent cation-induced association of formyltetrahydrofolate synthetase subunits. Kinetic and thermodynamic aspects. Formyltetrahydrofolate synthetase monomers are converted to catalytically active tetramers in the presence of monovalent cations. The stoichiometry of the reaction is 4M + 2C+ in equilibrium M4C2(2+). A positive deltaS compensates for an unfavorable positive deltaH so that the overall reaction is exergonic. Both deltaH and deltaS become more positive as the temperature is increased. Association of subunits of the enzyme prepared from Clostridium cylindrosporum is second order with respect to monomer concentration, consistent with a rate-determining dimerization step. Activation parameters for this step at 20 degrees are: deltaG, 12.6 kcal mol-1; deltaH, 12.5 kcal mol-1; deltaS, -05 e.u. The rate-limiting step for the cation-dependent association of Clostridium acidi-urici monomers is believed to be a conformational alteration since first order kinetics is observed. The Eyring plot of the kinetic data obtained for the C. acidi-urici system has a sharp break at 15 degrees. Activation parameters for cation-induced association at 20 degrees are: deltaG, 21.5 kcal  mol-1; deltaH, 14.0 kcal mol-1; deltaS, -26.6 e.u.


Evidence for essential lysyl residues in ribulosebisphosphate carboxylase by use of the affinity label 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate. A previous study from our laboratory suggested that 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate is an affinity label for spinach ribulosebisphosphate carboxylase. To identify the essential residues that react with the reagent we have isolated and characterized the labeled peptides that are present in tryptic digests of inactivated enzyme but lacking in digests of the substrate-protected enzyme. Peptides representing two sites of modification have been obtained from the inactivated carboxylase. Both sites of reaction have been identified as lysyl residues based on the conversion of the derivatives to free lysine by oxidation with sodium metaperiodate. Sodium dodecyl sulfate-gel electrophoretic experiments show that both essential lysyl residues are contained within the large subunit of ribulosebisphosphate carboxylase. In addition to lysyl residues, sulfhydryl groups of the carboxylase are also modified, but their modification seems to play little role in the inactivation process. The carboxylase modified in the presence of substrate contains sulfhydryl derivatives but is essentially lacking in lysyl derivatives. By comparing the profiles from ion exchange chromatography of labeled peptides in digests of inactivated and substrate-protected enzyme, we conclude that the same sulfhydryl groups are modified in the absence and presence of substrate.


The synthesis and chemical properties of polyisoprenyl beta-D-mannopyranosyl phosphates. 2,3,4,6-Tetra-O-acetyl-beta-D-mannopyranosyl phosphate, free of the alpha anomer, was coupled with citronellol and dolichol in the presence of triisopropylbenzenesulfonyl chloride to give, after chromatographic purification and deacetylation, the respective polyisoprenyl beta-D-mannopyranosyl phosphates. These compounds were compared with the previously synthesized alpha anomers by means of their chromatographic properties, spectra, optical rotations, and hydrolysis reactions when treated with acid and alkali. To characterize the compounds resulting from these treatments, and to determine the mechanism of the alkaline hydrolysis, beta-D-mannopyranosyl phosphate was converted into beta-D-mannonpyranose 1,2-phosphate, and hence into D-mannose 2-phosphate, obtained as a mixture of alpha and beta anomers, characterized by infrared and nuclear magnetic resonance spectra and elemental analysis. Beta-D-Mannopyranosyl phosphate was readily separated by thin layer chromatography from the corresponding alpha anomer.


Anomeric configuration of the dolichyl D-mannosyl phosphate formed in calf pancreas microsomes. Dolichyl D-[14C]mannosyl phosphate formed in calf pancreas microsomes was compared to dolichyl alpha-D-[14C]mannopyranosyl phosphate, a chemical synthesis of which is described. Jack bean alpha-mannosidase, which converted citronellyl alpha-D-mannopyranosyl phosphate, but not its beta anomer, to citronellyl phosphate and D-mannose, was effective in releasing D-[14C]mannose from dolichyl alpha-D-[14C]manopyranosyl phosphate in the presence of detergent. In contrast, alpha-mannosidase did not cause any significant release from the pancreatic dolichyl D-[14C]mannosyl phosphate. Alkali treatment (0.1 M NaOH in propanol at 65 and 90 degrees) degraded both dolichyl D-[14C]mannosyl phosphates with the formation of water-soluble 14C-labeled products. The pattern of 14C-labeled breakdown products formed from the synthetic dolichyl alpha-D-[14C]mannopyranosyl phosphate differed from that obtained from the pancreatic dolichyl D-[14C]mannosyl phosphate. Dolichyl alpha-D-[14C]mannopyranosyl phosphate yielded several 14C-labeled products, including a trace of D-[14C]mannosyl phosphate, and an acidic fraction which appeared to result from degradation of D-[14C]mannose. The pancreatic dolichyl D-[14C]mannosyl phosphate gave various products, depending on the temperature of the reaction: at 90 degrees, 20 to 30% of the radioactivity was found in D-[14C]mannosyl phosphate and the rest in acidic breakdown products; at 65 degrees, about two-thirds of the radioactivity was recovered in a compound which behaved as D-MANNOSE 2-PHOSPHATE, A Product characteristic of a beta-linked D-mannosyl residue. It is concluded that the pancreatic compound is dolichyl beta-D-[14C]mannosyl phosphate.


Ovalbumin messenger ribonucleic acid. Purification and fractionation on the basis of polyadenylate content by thermal elution from oligodeoxythymidylate-cellulose. Ovalbumin messenger RNA was purified from hen oviduct by immunoprecipitation of polysomes and oligo(dT)-cellulose chromatography. Two steps were introduced to improve the separation of mRNA and rRNA by oligo(dT)-cellulose. First, aggregates of mRNA and rRNA were dissociated by heating at 65degrees for 10 min before chromatography. Second, elution of the mRNA was achieved by stepwise increases in temperature rather than by lowering the ionic strength. Ovalbumin mRNA activity was eluted primarily in RNA fractions eluting between 45degrees and 55degrees. Polyacrylamide gel electrophoresis indicated that the ovalbumin mRNA thus obtained was essentiallyyy free of rRNA. The poly(A) content of various thermally eluted fractions was assayed by two methods. In the first (Favre, A., Bertazzoni, V., Berns, A.J.M., and Bloemendal, H. (1974) Biochem. Biophys. Res. Commun. 56, 273-280), the increase in fluorescence intensity of bound ethidium bromide was used to follow the formation of double-stranded RNA during titration of the mRNA with poly(U). In the second (Bishop, J. O., Rosbash, M., and Evans, D. (1974) J. Mol. Biol. 85, 75-86), poly(A) content was derived from the radioactivity remaining acid-insoluble after annealing mRNA fractions with [3H]poly(U) and treating with ribonuclease A. Both methods indicated that ovalbumin mRNA fractions eluting at higher temperatures contained greater amounts of poly(A). Values ranged from 44 to 248 mol of AMP/mol of mRNA, assuming 2200 total nucleotide residues for ovalbumin mRNA (Shapiro, D. J., and Schimke, R. T. (1975) J. Biol. Chem. 250, 1759-1764). Translational specific activities in a rabbit reticulocyte lysate system were essentiall constant for all fractions. From the binding of ethidium bromide it could be estimated that approximately 50% of the nucleotide residues in ovalbumin mRNA are base-paired.


Induction of ovalbumin mRNA sequences by estrogen and progesterone in chick oviduct as measured by hybridization to complementary DNA. A complementary DNA synthesized from ovalbumin mRNA was used in hybridization experiments to study the early effect of estrogen and progesterone on the accumulation of ovalbumin mRNA sequences in the chick oviduct. Chicks treated with estrogen withdrawn from the hormone maintain a steady level of 60 molecules of ovalbumin mRNA per tubular gland cell, at least 80% of which are localized in the cytoplasm. After estrogen administration, there is a 3- to 4-hour lag before a rapid increase in the number of ovalbumin mRNA sequences and a parallel increase in ovalbumin synthesis. Progesterone causes a more rapid increase in both ovalbumin mRNA sequences and ovalbumin synthesis with a lag period of only 90 min. The hybridization results demonstrate that both estrogen and pregesterone affect the amount of ovalbumin mRNA per cell. The 3-hour lag period seen with estrogen appears to be caused by some event after the binding of the estrogen receptor to chromatin but prior to change in the rate of transcription of the ovalbumin gene.


Role of galactose in bovine factor V.

Role of galactose in bovine factor V. Using galactose oxidase as well as beta-galactosidase to produce modifications of the galactose units, the functional significance of these carbohydrate residues on the coagulant activity of bovine Factor V glycoprotein was evaluated. Incubation of native Factor V with galactose oxidase or hydrolysis of asialo-Factor V with beta-galactosidase results in a loss of Factor V activity. The inactivation of Factor V by oxidation of galactose moieties is partially reversible upon reduction of the newly formed aldehyde groups with sodium borohydride. The extent of reversal depends upon the degree of inactivation achieved. Thus, Factor V which retained 30% of the original activity following galactose oxidation returns to 75% of the original coagulant activity upon borohydride reduction; but, after destruction of 85% of the original activity treatment with borohydride returns to about 30%. In the initial stages of the inactivation of Factor V by treatment with galactose oxidase, the loss of Factor V coagulant activity is directly proportional to the moles of galactose oxidized. However, as the reaction progresses, the rate of galactose oxidation exceeds the rate of loss of Factor V activity. Moreover, galactose oxidation continues even after complete inactivation of Factor V. These results suggest that the galactose residues most susceptible to attack by galactose oxidase are those necessary for the activity of this coagulant protein. Only 15 galactose residues/mol of Factor V are susceptible to galactose oxidase prior to removal of sialic acid. In contrast, 37 galactose residues/mol of Factor V are found after acid hydrolysis. These results suggest that Factor V glycoprotein contains more than one type of sialyl-galactose linkages, the C2,3 or C2,4 linkages susceptible to oxidation in the native protein and the C2,6 linkage which is resistant. Native Factor V binds with diarachidonyl lecithin forming an active complex of lower buoyant density, while the Factor V oxidized with galactose oxidase does not. The Factor V-phospholipid complex is protected from inactivation by galactose oxidase. Moreover, lipid binding diminishes the extent of oxidation of galactose residues. Certain galactose groups are essential for coagulant activity probably because they are required for binding to phospholipid, a prerequisite to Factor V action.


Photochemical labeling of the cytoplasmic surface of the membranes of intact human erythrocytes. N-(4-azido-2-nitrophenyl)-2-aminoethyl sulfonate (NAP-taurine), a photolabile nitrene precursor, has been shown to permeate the human erythrocyte membrane at 37degrees but not at 0 degrees. Utilizing this differential permeability, we have loaded intact erythrocytes with NAP-[35S]taurine in the dark at 37degrees, cooled them to 0 degrees, washed them free of external NAP-[35S]taurine in the dark and cold, and photolyzed them, resulting in labeling of hemoglobin and of proteins on the cytoplasmic surface of the membrane. These experiments complement those previously reported on the labeling of the external surface of the membranes with this reagent (Staros, J. V., and Richards, F. M. (1974) Biochemistry 13, 2720-2726).


The interaction of anions with hemoglobin carbamylated on specific NH2-terminal residues. Carbamylation of the NH2-terminal residues of the beta chains on hemoglobin (alpha2beta2c) leads to a reduced but still significant binding of 2,3-diphosphoglycerate, but has no effect on the oxygen-linked binding of chloride or phosphate, both of which are thought to bind to some of the same residues as the organic phosphate. Studies by others have shown that the binding of inorganic anions is not diminished in either horse hemoglobin or in hemoglobin Little Rock, in which four of the six other binding sites (histidine residues) for organic phosphates are replaced by glutamine residues. We suggest, therefore, that lysines 82 of the beta chains, which are the remaining 2 residues in the binding crevice for the organic phosphate, and which are invariant in the known sequences of mammalian hemoglobins, may be the primary binding site for inorganic anions. The extent of inhibition of gelation by increasing ionic strength is identical for the hybrids alpha2beta2, alpha2cbeta2, and alpha2beta2c of hemoglobin S. These results indicate the NH2-terminal residues of the chains are not involved in primary electrostatic interactions during aggregation of deoxyhemoglobin S.


Biocompatibility and state fatigue behavior of glassy carbon. 1. The normal stages of bone modeling and remodeling occur following the implantation of carbon plugs. 2. Glassy carbon bars aged in vivo for five months did not undergo statistically significant weakening. 3. Glassy carbon does undergo static fatigue when aged in a simulated biological environment. However, the conditions necessary to cause this failure are extreme. 4. Minimal tissue response was seen to the presence of the carbon.


Degree of polymerization of acrylic bone cement. Self-curing powder-liquid admixed acrylic systems are used for internal fixation fo total hip and total knee prostheses. Gel permeation chromatography revealed that the polymer chain length distributions of set cements were basically unaffected by their curing pressures. However, a decrease of approximately 11% in porosity coupled with a measured increase in mechanical strengths could be induced through the use of high curing pressures well beyond those attainable by the surgeon in the current arthroplasties. The conclusion of the investigation was that, to improve such cements, attention should be focused on elimination of porosity rather than attempting to produce higher degree of polymerization.


Investigation of possible oncogenic action of zinc polycarboxylate cement by implantation in mice and hamsters. Powdered, sterilized zinc polycarboxylate was implanted subcutaneously in 56 young hamsters and 54 mice, using equal numbers of each sex, with 24 sham operated hamsters and 26 mice as controls. The hamsters were sacrificed after 15 months and the mice after 12. Implant material was recovered from one of 42 surviving mice and from 16 of 43 surviving hamsters. Benign adenomas of the gut, thyroid, sebaceous glands and ovary were found in 3 experimental mice and in one control. Benign adrenal adenomas were found in 3 experimental hamsters and in one control. One control hamster developed a rhabdomyosarcoma in a limb. One experimental hamster developed a leiomysarcoma in close relation to an implant.


Study of pig valve heterografts by x-ray diffraction. The study of collagen fibers by X-ray diffraction, utilizing semiquantitative indices for appraisal of "structuration" and "orientation" was applied to 30 aortic valve grafts. These grafts, of pig origin, were studied in the fresh state, after tanning with aldehydes, and after having been implanted in patients for at least 18 months. It was shown that the collagen fibers are preferentially orientated parallel to the transverse axis of the valve cusps, and that this arrangement, enhanced by tanning, had a tendency to disappear in the post-implantation samples. The structuration index which concerns the fibril network was also augmented by tanning, and was found a little high or slightly diminished in post-implantation cusps. Evidence for a new compound, probably fibrin, was found in the diffraction patterns of implanted cusps, but crystallized calcium was noticeably absent.


A vitreous carbon-polymethacrylate composite for dental implants. A STUDY HAS BEEN CARRIED OUT ON THE STRUCTURE AND SURFACE TEXTure of a new dental implant material composed of vitreous carbon balloons (3 wt % or 6 wt %) and poly(methyl methacrylate). Tooth replica implants using this material had been successfully placed in baboons, and histologic study revealed normal alveolar bone and a peri-implant membrane with the connective tissue fibers oriented in a horizontal direction. Square wafers (10 mm X 10 mm X 1 mm) were studied, with the surface sandblasted in one-half of the specimens. Light microscopy revealed that in the 3% carbon specimens, the vitreous carbon micro-balloons were evenly spaced and often appeared to be fragmented within their spaces. S.E.M. studies revealed a finely porous surface with numerous large craters. Microballoons were often seen within the craters in the nonsandblasted specimens.


The role of surface thermodynamics in thromboresistance of biomaterials. A thermodynamic approach to the problem of platelet adsorption out of a suspension on to a smooth and homogeneous solid surface is developed. The interfacial tension values required may be estimated from contact angle data by means of an equation of state relation. According to the thermodynamic approach the functional dependence of platelet adsorption on surface tension of the solid differs according to whether the surface tension of the platelets is smaller or larger than the surface tension of the liquid in which they are suspended. The implications of this thermodynamic approach in situations where plasma proteins are present and the biomaterials surfaces may be heterogeneous and rough are discussed. Previous analyses using the critical surface tension of wetting and other surface-related parameters are compared with the thermodynamic analysis given here.


Adsorption characteristics of cellulose acetate coated charcoals. The permeability of cellulose triacetate membrane coated activated charcoal is enhanced by treatment with KOH. The adsorption data for creatinine in aqueous solution before and after deacetylation are given. In order to study the usefulness of hemoperfusion associated with dialysis for removal of uremic toxins, some experiments were performed on the adsorption by coated and deacetylated charcoal of molecules of various molecular weights (from 113 to 40,000). Although the adsorption capacity of uncoated charcoal was better, the coated material still shows good properties in the adsorption of glucagon (mol wt 3485). The results on in vitro experiments of vitamin B12 removal by coated charcoal cartridge and CDAK Model 3 dialyzer confirms the usefulness of adsorption technique toward medium molecular weight compounds.


Cast vs. wrought cobalt-chromium surgical implant alloys. The measured tensile and fatigue strengths of wrought and heat-treated cobalt chromium-molybdenum-carbon alloys such as HS21 were found to be more than twice those of as cast alloys of the same composition. The resistance of wrought HS21 to crevice corrosion at metal-Teflon contacts in isotonic salt solution at 37 degrees C was found to be considerably greater than that of cast HS21, wrought HS25, wrought MP35, or 316L stainless steel which are currently used for surgical implants. The increased crevice corrosion resistance and mechanical strength of wrought HS21 as compared to as cast HS21 was attributed to greater chemical and structural homogeneity as well as to finer grain size and distribution of secondary phases. Fabrication of shaped implants from wrought HS21 is possible by techniques currently used for the fabrication of industrial parts from other wrought cobalt-chromium base alloys.


Thermal properties of cancellous bone.

Thermal properties of cancellous bone. Specimens of beef bone were analyzed for thermal conductivity and thermal capacity. The thermal conductivity was about 7 X 10(-4) cal/cm/sec/degrees C, that is about the same as for other tissues. As regards thermal capacity, the determination of 0.73 cal/cm2/degrees C also was similar to that in other tissues. Both thermal conductivity and capacity were isotropic with respect to the layer of epiphyseal cartilage, and the orientation of bony trabeculae. Thermal probes were devised heated either by water or by electric current to destroy localized volumes of bone, as in surgery. To achieve necrotizing temperatures, the sizes of such probes and the times of application of heat were determined.


Identification of Au-Sn phase in Ag3Sn alloys containing gold. Substitution of gold in part for silver in Ag3Sn alloys is found to result in two separate phases: gamma (Ag3Sn particles) grains with a uniform distribution of gold within the grain and an Au-Sn phase in a form of ring surrounding the gamma grain. The thickness of this ring increases with increasing gold concentration. The phases were identified by using the techniques of x-ray diffraction, optical metallography, scanning electron microscopy, and x-ray energy dispersive spectroscopy.


Auger analysis of surface films on Ag3Sn. The surface properties of Ag3Sn are important in the amalgamation process so that mechanical abrasion is generally necessary to get a satisfactory amalgam. Therefore, the present study was undertaken to characterize the surface of Ag3Sn after several types of surfaces treatments. Homogeneous polycrystalline rods of Ag3Sn were prepared in vacuo from high purity (99.99%) Ag and Sn. Specimens were sliced from these rods and prepared as follows: (1) mechanically polished, (2) polished as in (1) and allowed to sit in air for three days and (3) polished as in (1) and electrolytically etched. Each of these specimens was examined by Auger electron spectroscopy. The results showed that after mechanical abrasion the surface film was primarily oxide and the Ag to Sn ratio was lower than the bulk composition, indicating preferential oxidation of Sn. After sitting in air sulfur was also present on the surface and the Ag to Sn ratio was greater than that immediately after polishing, indicating the formation of Ag2S. After electrolytic etching some oxide was still present, but only a trace of sulfide was found. However, the Ag to Sn ratio was greater than that for mechanically polished specimens, indicating preferential dissolution of Sn. Therefore, it is seen that the surface preparation of Ag3Sn has a marked effect on Ag and Sn concentrations and the nature of films at the surface.


Amalgamation reaction on mercury-plated dental alloy (Ag3Sn). The reaction between liquid mercury and Ag3Sn was studied at room temperature using a new type of specimen; i.e. bulk Ag3Sn electroplated with mercury. The surface morphology of the specimen and the phases formed on the Ag3Sn substrate were investigated by various techniques including optical microscopy, electron (scanning) microscopy, electron microprobe analysis, and electron diffraction. It was found that the mode of formation and type of crystalline phase appearing on the Ag3Sn specimen depended on the amount of mercury available. When the amount of mercury exceeded a certain quantity (about 1 mg/cm2) only the gamma1 and gamma2 phases were formed. On the other hand, when the amount of mercury was reduced below a certain low value (less than 0.3 mg/cm2), another silver-mercury phase, beta1, formed as tiny "liquid-like" drops. The experimental technique employed in the present investigation has proved to be an excellent method for studying the reaction between liquid mercury and metal specimens. This new procedure may be employed to evaluate the amalgamation ability of potential new alloy systems.


Finite element analysis of dental structures--axisymmetric and plane stress idealizations. The finite element method used to study stress generated in a maxillary second premolar as a result of occlusal forces. This mathematical technique has been applied extensively in structural engineering and structural mechanics. It is well suited to the analysis of stress in teeth and dental restorations because it can closely simulate the geometries, loads, and material inhomogeneities in the system being studied.


The concept of protection potential applied to the corrosion of metallic orthopedic implants. Cyclic polarization curves obtained for a cobalt-base casting alloy (similar to Vitallium Surgical Alloy) and for 316L stainless steel show that the cobalt-base alloy exhibits a high (noble) protection potential whereas the stainless steel exhibits a low (active) protection potential. It is suggested that the resistance to crevice corrosion of Vitallium Surgical Alloy results from its high value of protection potential whereas the susceptibility of 316L stainless to severe crevice corrosion results from its low value of protection potential.


The effects of polyvalent salts on zeta potential of bone. A streaming potential experiment was conducted on in vitro bone to determine the effects polyvalent salts have on bone zeta potential. Polyvalent metal salts would be introduced into local physiological electrolytes by corrosion of a metal implant or dissolution of a ceramic implant. It was hypothesized that a change in zeta potential of mineralized tissue, resulting from the introduction of polyvalent salts, could be a cause of tissue instability at the implant interface. Zeta potentials measured in bone were on the order of a few millivolts. Strongly hydrolytic metal cations such as Al+3 and Fe+3 did not have sufficient activity in buffered neutral electrolytes to alter bone zeta potential. Results suggest that less hydrolytic cations of high valence, and large polarizable anions may affect bone zeta potential.


Residual ethylene oxide: levels in medical grade tubing and effects on an in vitro biologic system. The level of residual ethylene oxide after sterilization was evaluated as a function of aeration time for three medical grade tubings. Toxicity resulting from residual ethylene oxide was determined in an in vitro tissue culture system utilizing L-cells. The absorption and desorption of ethylene oxide from poly(vinyl chloride) and polyether-polyurethane tubing were similar. In contrast, silicone tubing absorbed 85% less ethylene oxide. The time required for desorption of residual ethylene oxide was 2 hr for silicone tubing and 7 to 8 hr for poly(vinyl chloride) and polyether-polyurethane tubing. Tubing samples containing 1,500 ppm or more residual ethylene oxide elicited toxic tissue culture reactions whereas samples containing 900 ppm or less showed no toxic tissue culture response.


Modified collagen membrane as a skin substitute: preliminary studies. Modified collagen membrane when compared to autograft, homograft, heterograft and silicone polymer membrane, demonstrated a superior adherence on split and full-thickness surfaces, but proved to be an inferior covering for granulating surfaces. Water vapor transport was found to be adequate and adaptable to modification. It was found to be permeable to most commonly used topical antibiotics, and no significant antigenicity could be demonstrated. In a controlled animal burn study, modified collagen membrane proved superior to homograft and heterograft in regard to mortality, time to grafting, bacteriology and autograft take. These studies provide a background for further laboratory and clinical studies now in progress.


MP35N: a corrosion resistant, high strength alloy for orthopedic surgical impants: bio-assay results. A cobalt based alloy, MP35N, with excellent mechanical properties has been recently introduced as a material for surgical orthopedic implants. A study was made of local and systemic host response to this material in two different mammal species. The implantation time ranged from one to 12 months. The result of this study indicated: MP35N produces a degree of local tissue response comparable to that of 316L stainless steel. No systemic side effects were observed during the implantation times included in this study.


Wettability of hydrogels. I. Poly (2-hydroxyethyl methacrylate). The wettability characteristics of the contact lens material, PHEMA, with respect to water have been determined by using the sessile drop, and the captive air bubble techniques of contact angle goniometry. It is concluded that on PHEMA gels water does not spread spontaneously. Large hysteresis has been observed in the advancing and receding contact angles. This suggests that this hydrogel surface is capable of changing its free energy through reorientation of the polymer side chains and chain segments depending on the nature of the adjacent phase. The water content of the gels does not appear to have an effect on water wettability in the hydration range investigated. The minor wettability differences among the various gels studied were most likely due to differences in surface structure and segmental mobility due to inherent variations in the method of preparation. Small but consistent differences were found between the contact angles measured by the captive bubble method and those obtained by the sessile drop method, the former values being higher. These differences may not be method-related artifacts and cannot be explained at the present time.


The development of an abrasion testing machine for dental materials. Wear testing should be an important part of the investigation into the physical and mechanical properties of some dental materials. It has, however, largely been ignored because of conflicting and unreproducible results. It was decided, therefore, to review the work done by other researchers and to examine the human masticatory cycle, and then present new parameters to design and construct a new dental abrasion testing machine. This new machine is described in detail and its capabilities briefly illustrated.