Incidence of antiprotozoal and antivermal antibiotics in fungi, V Class Fungi Imperfecti Collected in the Vietnamese Democratic Republic. From soil samples collected in the jungles of Ninh Binh Province of the Vietnamese Democratic Republic, 97 isolates belonging to 54 species of Fungi imperfecti were obtained and screened for the production of antibiotics. The isolates were identified as belonging to the orders Melanconiales, Moniliales, Sphaeropsidales and Mycelia sterilia. In the species studied a very high incidence of antiprotozoal activities were observed.


A new Streptoverticillium species--S. quilonensis. An extensive screening of soil samples from Kerala State,India,gave a new streptomycete, Streptoverticillium quilonensis. Its taxonomy is described in detail.


Effects of hypercapnia on uterine and umbilical circulations in conscious pregnant sheep. Changes in the uterine and umbilical circulations during induced hypercapnia were studied in nine unanesthetized near-term pregnant sheep. Blood flows were measured with electromagnetic flow transducers and arterial pressures with vascular catheters implanted under anesthesia 2-16 days prior to experiments. Hypercapnia was induced in the fetus alone by giving acetazolamide iv to the fetus, 100-200 mg/kg. Mean fetal arterial Pco2 increased from 49.5 to 63.4 mmHg but no significant changes in umbilical blood flow occurred. Stepwise increases in both maternal and fetal arterial Pco2 were induced by increasing maternal inspired CO2 concentration to a maximum of 12%. No dignificant changes occurred in uterine or umbilical circulations until hypercapnia was severe (maternal arterial Pco2 greater than 60 mmHg, fetal arterial Pco2 greater than 70 mmHg). With severe hypercapnia uterine vascular resistance increased significantly and uterine blood flow decreased despite an increase in maternal arterial pressure; fetal arterial pressure and umbilical blood flow increased significantly, but umbilical vascular resistance did not. We conclude that hypercapnia in conscious pregnant sheep is associated with significant changes in uterine and umbilical circulations, but only when hypercapnia is severe. Carbon dioxide is unlikely to be a factor in normal physiological regulation of the uteroplacental circulation in this species.


Effects of CO2 on immediate ventilatory response to O2 in preterm infants. We wanted to know wheter the paradoxical response to CO2 under various background concentrations of O2 in preterm infants was mediated at the peripheral chemoreceptors. In five preterm infants we estimated peripheral chemoreceptor activity using the immediate change in ventilation (first 30 s) when 15%, 40%, 60%, or 100% O2 was substituted for 21% O2. Potentiation between O2 and CO2 was assessed by comparing the response with and without 4% CO2. CO2 enhanced the immediate hyperventilation with hypoxia (P less than 0.005) and reduced the immediate hypoventilation with hyperoxia (P less than 0.025 for 40% O2). This effect of CO2 increased from .00% to 15% O2 (P less than 0.05). These findings suggest: 1) CO2 interacts with O2 at the peripheral chemoreceptor level, and 2) because this interaction is more pronounced with hypoxia, the flatter CO2 response we observed with hypoxia was probably not mediated through the peripheral chemoreceptors and is likely to be central in origin.


Effect of inspired volume on closing volume. Closing volume (CV) was ?EASURED WITH THE RESIDENT GAS TECHNIQUE IN 12 HEALTHY SEATED SUBJECTS AGE 22-70 YR, AND IN 8 SUBJECTS WITH THE BOLUS TECHNIQUE. Various volumes were inspired (Vi range: 20-100% vital capacity) from residual volume and CV was assessed on the subsequent recording of expired volume versus gas concentration. The results indicate that the resident gas technique may erroneously underestimate CV at reduced Vi in conformity with calculations which predict that during expiration, after a certain reduced VI, the nitrogen concentration is identical in the most basal lung region and at the mouth. CV obtained with the bolus technique decreased linearly with reduced Vi and the effect appeared to be age dependent according to the equation CV50/CV100=0.0078 X age +1.18, where CV50 and CV100 denote the bolus CV corresponding to Vi=50% and 100% of vital capacity. Therefore, in older subjects, during tidal breathing, airways appear to close at substantially lower lung volume than previously considered.


Urinary catecholamine excretion on acute induction to high altitide (3,658 m). Fifty healthy male volunteers, 21-34 yr of age, normally resident at altitudes less than 1,000 m, were airlifted to 3,658 m. Urinary excretion of catecholamines was measured at sea level (198 m) and on the 1st, 2nd, 4th, and 10th day of a stay at high altitude. The symptoms observed on exposure to high altitude were assigned arbitrary scores. The volunteers could, on this basis, be divided into "symptomatic" and "asymptomatic" groups. The two groups showed a markedly different pattern of urinary catecholamines excretion on exposure to high altitude and on return to sea level. Significant increase in the catecholamine excretion was observed in the symptomatic group only. A possible role for enhanced sympathoadrenal activity in the etiopathogenesis of high-altitude illnesses is postulated.


Respiratory response to airway occlusion in infants: sleep state and maturation. The progressive respiratory response to occlusion of the airway at FRC was measured in a group of full-term and premature human infants. The sleep state of the full-term infants was shown to affect the response primarily through variations in the phase of thoracic and abdominal movements. The weakest responses were seen in those infants who demonstrated parodoxical respiration prior to occlusion. Most infants developed paradoxical respiratory movements after occlusion, but this did not affect the strength of the load-compensatory response. An increase in response related to both gestational and postnatal age was observed. This increase could not be accounted for by variations in the amount of paradoxical and in-phase respiration and thus may represent an increase in the sensitivity of infants to chemical stimuli with maturation.


Ventilatory stimulation by sodium salicylate: rols of thoracic receptors. To explore the possibility that salicylates can stimulate VE via an extracranial mechanism, heads of dogs were perfused entirely by a support dog with blood of unchanging gas composition; spinal cord remained intact. The carotid bodies lay within the region of the perfused head; the aortic bodies were denervated. Intra-arterial infusion of sodium salicylate (150-225 mg/kg) into the body of these head-perfused animals elicited large increases in VE (188+/-11%), although significant amounts of salicylate did not reach the head or carotid body. In any case, when the same dose of salicylate was selectively administered to the head, increases in VE were limited to 23+-8%. To determine the contribution of thoracic receptors to the increase in VE elicited by salicylates, a subsequent group of head-perfused animals underwent complete spinal transection at the first lumbar level. Nnetheless, intra-arterial infusion of sodium salicylate (150-225 mg/kg) into the body of these head-perfused animals still elicited large increases in VE (111+/-13%). It is concluded that thoracic receptors (other than the aortic bodies) mediate a significant portion of the increase in VE elicited by intra-arterial infusion of sodium salicylate.


Blood flow in branching vessels.

Blood flow in branching vessels. Flow visualization studies were conducted in a water-Plexiglas tube model which simulated flow conditions commonly encountered in major vessels. Complex secondary flow patterns in the main line downstream of a branching vessel were observed in found to be relatively independent of the diameter ratio and angle of departure of the branching tube. The ratio of branching to total flow was the prinicipal determinant of the aforementioned secondary flows. Venous and arterial branching markedly differed. Arterial-type flows produced a pair of vortex sinks downstream of the branching port. These vortex-sink flow fields, which previously have not been reported, captured fluid particles already downstream of the branching orifice and returned these particles upstream to exit via the branching tube. In a region very close to the tube wall, streamlines were also observed moving upstream to be captured by the branching vessel. The interaction of this local upstream flow with the general downstream flow produced a pair of stagnation points located downstream and lateral to the branching orifice. These stagnation regions are discussed in relation to atheroma and thrombus formation.


Modeling of the relationship between volume variations at the mouth and chest. A model was developed to interpret the difference between volume variations at the mouth and at the chest observed by body plethysmography during inspiration in normal subjects breathing ambient air. The mechanical phenomena were characterized, as usual, by a time constant alpha, the product of airway resistance and alveolar gas compressibility. In contrast to other models, the change in temperature and water vapor pressure of the inspired gas was not considered to be instantaneous but rather a first-order process characterized by a thermal time constant tau. Experimental curves obtained in eight subjects were analyzed by use of parameter estimation techniques. The values of alpha were in agreement with those obtained by conventional plethysmography and tau averaged 63+/-24 ms. The results of additional experiments devised to test the physical meaning of the coefficients and the influence of neglected factors support the validity of the model. It was shown that tau cannot be neglected when breathing air at room temperature.


Relationship between viscoelastic properties of the rectum and anal pressure in man. Viscoelastic properties of the rectal wall and anal sphincter pressure were studied simultaneously in 25 normal children. During rectal distension for 60 s, with varying volumes of air, the rectal pressure varied as the sum of two exponential functions of the time plus an asymptotic constant. A rectoanal inhibitory reflex was elicited during these distensions. The exponential decrease of the rectal pressure corresponded exactly to the return of the upper anal canal pressure to the resting value in some subjects. In others, the upper anal canal remained relaxed as long as the rectal ampulla was distended, except with smaller volumes. The results obtained may be explained by an analogical rheological linear model.


Influence of glucose ingestion on fuel-hormone response during prolonged exercise. Healthy subjects were studied at rest and during 4 h of exercise at approximately 30% of maximal oxygen uptake. At 90 min of exercise 200 g glucose were ingested. A control group was studied during prolonged exercise without glucose administration. Glucose ingestion was followed by a 35% rise in arterial glucose, a 60-70% fall in arterial FFA and glycerol and a two- to threefold rise in arterial insulin. Plasma glucagon, which rose fourfold in controls, failed to rise in the glucose-fed subjects. Glucose uptake by the exercising legs was twofold greater than in controls, accounting for 60% of leg oxygen consumption. Splanchnic glucose output rose rapidly after glucose ingestion to values twice those observed in controls. However, splanchnic uptake of gluconeogenic precursors (lactate, pyruvate and glycerol) fell by 70-100%. Total splanchnic glucose escape after glucose ingestion was 84 +/- 5 g representing 42% of the ingested load. It is concluded that glucose ingestion during prolonged exercise results in a) augmented uptake and oxidation of glucose by the exercising legs, b) diminished lipolysis, c) augmented splanchnic glucose escape in association with decreased hepatic gluconeogenesis, d) retention of half of the ingested glucose within the splanchnic bed, and e) reversal of exercise-induced stimulation of glucagon secretion.


Effect of carbon monoxide exposures on erythrocytic 2,3-DPG in rabbits. To determine if an exposure to lower levels of carbon monoxide (CO) produces quantitative changes in erythrocytic 2,3-diphosphoglycerate (2,3-DPG), eight rabbits were exposed to 100 ppm CO for 5 h resulting in approximately 20% carboxyhemoglobin (HbCO). 2,3-DPG was determined before exposure and immediately after exposure as well as every 3 h during the 24 h following exposure. To determine if intermittent CO exposures over a prolonged period of time affect 2,3-DPG, an additional 12 rabbits were exposed to 250 ppm CO for three intermittent periods totaling 300 min daily for 14 days (30% HbCO). 2,3-DPG was determined once per animal at the end of each day's exposure period. All animals served as their own controls in both experiments. Neither the results of the 24-h time course nor those of the 14-day time course showed significant differences in mean 2,3-DPG between controls and exposed animals at any sampling time. Apparently erythrocytic 2,3-DPG plays neither a compensating nor aggravating role in respect to the hypoxia induced by these levels of HbCO in the rabbit.


Human cardiovascular and respiratory responses to graded muscle ischemia. Responses of heart rate (HR), mean arterial blood pressure (MAP) ventilation (VE), and forearm blood flow (FBF) to different degrees of leg muscle ischemia were measured in eight subjects in a four-part experiment. Part I. Total circulatory occlusion (OCCL) of resting legs for 15 min had little or no effect on HR, MAP, VE, or FBF. Part II. OCCL of the legs for 3 min immediately after exercise at 50-250 W did not affect HR or end-tidal CO2; it lowered VO2 and VE and prevented recovery of MAP. Part III. OCCL beginning at end and 10, 20, 30 s before end of 7-min exercise (100-150 W) and continuing 3 min into recovery period produced sustained and graded increments (5-10 mmHg) in MAP, only small changes in HR, and accelerated recovery of VE while end-tidal CO2 remained constant. Part IV. OCCL at end and 30 s before end of exercise increased FBF 2.5-3.5 times; both skin and muscle vasodilated. Thus muscle ischemia preceded by exercise can raise MAP without affecting VE, whereas baroreflexes may lower HR and raise FBF. The results suggest the presence of muscle chemoreceptors whose major effect is on MAP.


Human coagulation abnormalities during acute exposure to hypobaric hypoxia. Multiple coagulation studies were carried out in eight healthy young men at sea level (SL) and after 1, 24, and 48 h at a simulated altitude of 4,400 m. Platelet aggregation, as induced by ADP, epinephrine, and collagen, was not significantly altered by high-altitude (HA) exposure. Mean 2,3-diphosphoglycerate, a physiological inhibitor of platelet aggregation, rose (P less than 0.001) after 24 h at HA and remained elevated while no changes in circulating catecholamines were observed. Platelet count, factor 3 availability, and membrane lipid peroxide formation were likewise unaltered at HA, as were prothrombin and thrombin times and protamine paracoagulation test. However, mean partial thromboplastin time was significantly shortened (P less than 0.01) after 1 and 24 h at HA, recovering to SL control by 48 h. Fibrinogen and factor VIII levels also fell (P less than 0.01 and P less than 0.02) after 1 h at HA but returned to the preexposure values by 24 h. Fibrin degradation products were transiently detectable in three subjects at HA. Thus, although normal platelet function did not appear to be modified by short-term exposure to simulated high altitude, evidence for a coagulopathy was obtained.


Effect of heat acclimatization on intravascular responses to acute heat stress in man. The effects of a 185-min exposure to 48 degrees C db/33 degrees C wb, on intravascular volume and osmolarity and on intravascular electrolyte, aldosterone, and cortisol concentrations have been studied in five male subjects before and after acclimatization to heat. Changes in the hematocrit and plasma protein concentration indicated that a hemodilution occurred during the first 35 min of the heat exposures, and that this was followed by a hemoconcentration. Although these changes in intravascular volume were not affected by acclimatization, the plasma volume after heat acclimatization was 6.7% greater than before. This increase in plasma volume was associated with an elevation in the ratio [Na]/[K]. However, since plasma osmolarity decreased the intravascular expansion could not be explained in terms of elevated electrolyte levels. Plasma aldosterone and cortisol levels were not affected by heat acclimatization, although both were elevated following exercise in the heat. It is concluded that the adrenal cortex is not an important factor in maintaining a state of heat acclimatization once a salt balance has been achieved.


Effects of lower body negative pressure (LBNP) on the distribution of body fluids. Reduction of pressure on the lower part of the human body is believed to be capable of altering the normal fluid distribution. This could serve as a therapeutic method for emergency relief of the effects of hypertension, or it could be used as counterbalance for zero gravity and assist in preparing astronauts for reentry into the gravitational field. The present work describes a technique of assessment of the fluid shift caused by such pressure reduction. Thirty experiments were performed on four healthy subjects. The shift of fluid was assessed by measuring changes in body weight distribution of the tested subjects. The shift of fluid was assessed by measuring changes in body weight distribution of the tested subjects. The experimental results show that two processes may have taken place within the human body: shift of blood within the vascular system and diffusion of fluids from the circulatory system to the tissues. The total fluid shift ranges between 1 and 2 kg. It was proportional to the pressure gradient and exponential with time. The systolic blood pressure was reduced with the pressure reduction while the pulse rate increased.


Variation of respiratory sinus arrhythmia with age. Respiratory sinus arrhythmia was measured on 24 male subjects whose ages ranged from 21 to 65 yr, using a technique in which respiration was coupled to heart rate, so that there were 6 heartbeats per inspiration and 6 heartbeats per expiration. This voluntary cardiorespiratory coupling reduced the variance of heart rates measured at various points in the respiratory cycle. Fourier analysis was used to obtain the fundamental of heart rate and respiratory volume. A high correlation coefficient (-0.83) was found between percent of variation of heart rate from the average heart rate (PB) and the age of the subject. Linear regression analysis performed on these data produced the equation: PB = 23.2-0.35 (age). Since sinus arrhythmia at the low respiratory frequencies used probably reflects the viability of the cardiorespiratory control systems, we believe this provides a simple noninvasive method for the study of the overall competence of the system responsible for respiratory sinus arrhythmia.


Respiratory muscle action inferred from rib cage and abdominal V-P partitioning. We measured separate volume-pressure (V-P) relationships or rib cage and diaphragm-abdomen in seven human subjects during voluntary relaxation of the respiratory muscles, breathing at rest, during exercise, and rebreathing expired air. Estimates of separate volume displacements of the two parallel chest wall pathways were based on analysis of rib cage and abdominal anteroposterior diameter changes. The pressure developed across each pathway (transthoracic pressure) was partitioned into two serial pressure drops: transdiaphragmatic pressure and transabdominal pressure. We develop the concept that the relationship of volume displacements of structures to pressures developed by the structures during breathing, as compared to the relaxed state, reflects action of respiratory muscles in the structure. We interpret the relationship of rib cage volume displacements to transabdominal pressure (during breathing vs. relaxation) as indicating action of intercostal and accessory muscles only, the separate action of diaphragm on rib cage being measured by transdiaphragmatic pressure. At rest, the diaphragm is the only importantly active respiratory muscle. During increased ventilation activity of other respiratory muscles appears coordinated to assist the optimize diaphragmatic function.


Mechanical work of breathing derived from rib cage and abdominal V-P partitioning. Estimates of the mechanical work of breathing derived from measurements of separate rib cage and abdominal volume displacements, each plotted against transthoracic pressure, include the elastic cost of chest wall distortion which may occur during breathing. Inspiratory work is partitioned between the diaphragm and the rib cage musculature by adding measurements of transabdominal pressure. The mechanical work of breathing derived from separate rib cage and abdominal volume-pressure (V-P) tracings (the sum of work done by the diaphragm, rib cage, and abdominal musculature) is compared with ventilatory work estimated from the Campbell diagram (which does not include any distortional work). During resting breathing the two estimates are closely comparable, consistent with little or no distortion of the chest wall during quiet breathing. As ventilation increases, the estimate developed from rib cage and abdominal tracings reveals systematically greater mechanical work than is estimated from the Campbell diagram, consistent with distortion of the chest wall from the relaxed thoracoabdominal configuration at higher levels of ventilation. At ventilations achieved during exercise, the Campbell diagram may underestimate the work of breathing by up to 25%.


Nonadrenergic inhibitory nervous system in human airways. Human airways, from the middle of the trachea to the distal bronchi, were studied in vitro for the presence of inhibitory nerves. The tissue was obtained from operations and from recent autopsies. Electrical field stimulation of the tissues demonstrated cholinergic, excitatory nerves and their effect was blocked by atropine. Field stimulation of the tissues, in the presence of atropine, relaxed the smooth muscle even when the muscle was contracted by histamine. The field stimulation-induced relaxation was neither blocked nor modified by adrenergic blocking agents. Maximum relaxation of the bronchial muscle was obtained with a pulse duration of 1-2 ms, 70 V, and frequencies of 20 Hz and greater. The tracheal smooth muscle showed 85% of maximal relaxation with a frequency of 10 Hz. Tetrodotoxin, blocked the field stimulation-induced relaxation for pulse durations of 2 ms; this indicated that nerves were being stimulated. The airway system shows some of the characteristics of the nonadrenergic inhibitory system in the gastrointestinal tract and of the system reported in the guinea pig trachealis muscle. No evidence of adrenergic inhibitory fibers was found in the bronchial muscle with either pharmacological or histochemical techniques. These findings suggest that the nonadrenergic inhibitory system is the principal inhibitory system for the smooth muscle of human airways. We suggest that a defect in the airway system, such as that shown in the gastrointestinal tract, may be an explanation for the hyperreactive airways of asthma and chronic bronchitis.


Temperature regulation during severe exercise with some observations on effects of skin wetting. The thermoregulatory responses to severe exercise of 1-h duration have been studied in 11 healthy subjects, and the effects of skin wetting have been examined in 6 of them. Measurements in the laboratory included metabolic (M) and total (H) heat production, rectal (Tre) and mean skin (Tsk) temperatures, evaporative sweat loss (E), and peripheral tissue conductance (K). In addition Tre and changes in body weight were measured in 18 conpetitors following a 20-km road race run at approximately 90% of their maximal oxygen intake (Vo2 max). The results showed that Tre was curvilinearly related to %Vo2 max. In contrast Tsk was independent of M, H, E, and %Vo2 max, and reached similar and constant values at the end of exercise in all subjects. E was appropriate for a given H, but K was lower  in the sedentary subjects than in the athletes. Skin wetting had no effect on Tre but reduced E by 10%. It is suggested that the pattern of Tre rise in excercise reflected the nature of the controlling system and was not due to a failure of the heat-dissipating mechanisms.


Relationship between evaporation rate of sweat and mean sweating rate. The rate of evaporation of sweat under a step change of ambient temperature (30-45 degrees C) was compared with the mean sweating rate (MSR) computed from five simultaneous resistance hygrometric measurements. Empirical equations derived for the conditions studied enabled a direct conversion from hygrometric measurements into real evaporative sweat losses and skin wetness for all phases of sweat evaporation. From five chosen skin areas, forehead, forearm, chest, thigh, and calf, it was found that none of these sites gave a local sweating response close to the MSR. In the subjects with a forehead sweating rate within +/- 39% of the MSR, at least four hygrometric sensors were needed to predict the rate of sweat evaporation. For subjects with higher forehead sweating rates, at least five sensors were necessary to calculate the evaporative sweat loss.


New electronics for the Beckman LB-1 carbon dioxide analyzer. This paper describes an amplifier and linearizer for the Beckman LB-1 carbon dioxide analyzer head. All electronics are solid state and DC regulated in contrast to the original LB-1 amplifier which was of vacuum tube design and AC regulated largely by means of a voltage stabilizer transformer. A new detection principle is adopted which reduces drift due to mechanical imperfections of the chopper motor (e.g., bearing drag), but also offers the advantage that the phasing of the chopper blades is no longer necessary. Furthermore, the amplifier with a digital readout and an end-tidal hold circuit can be built for less than $500 and is plug compatible with the LB-1 head.


New device for automatic recording and processing AV transmission intervals of the heart. The principle and construction of a device made for the purpose of displaying the interrelationship between two sets of cardiac intervals is described. Electrical signals derived from the atrium and ventricle of the experimental animal are fed to the input of the device where a special switch and an integrated circuit logic gate select the two sets of cardiac intervals to be measured, e.g., AA and VV or AA and AV intervals. These intervals are converted into electrical square pulses having voltages proportional to the respective intervals. Besides simply comparing the two cardiac intervals by recording them simultaneously against time, the interrelationship of the two is displayed in an X-Y configuration on-line on a cathode-ray oscilloscope. This latter method, if used only for the occasionally induced premature excitations with varied coupling time, facilitates the measurement of the functional refractory periods for atrioventricular conduction. The mode of operation of the device is presented with examples of experiments.


System analysis of heart rate control in man. The dynamic property of the heart rate response to exercise was determined and expressed in the frequency domain to establish a method of examining cardiovascular control function. The response of heart rate to a stimulus was measured at 5-s intervals in nine healthy young volunteers. The stimulus consisted of several runs of two-step exercise practiced in semirandom sequence for 19 min. The weight function of the system was estimated from autocorrelation function of the input signal and cross-correlation function between the input and output signals. The weight function was transformed into a transfer function and its Bode plot diagram was drawn. From the diagram, four dynamic parameters were determined. These parameters are as follows: K is a constant showing the theoretical steady-state increment of heart rate, and T1, T2, T3 are time constants. The values obtained in the present experiment with the healthy young males were: K 46.0 +/- 14.6 beats, T1, 2.12 +/- 0.44, T2, 1.12 +/- 0.16, and T3 0.70 +/- 0.07 min.


Method for continuous measurement of carbon dioxide output. An open-circut flow-through technique for the continous measurement of carbon dioxide output in man has been described. A stream of room air is drawn past the subject who respires freely from it. The difference in the concentration of carbon dioxide between room air from which the subject inspires and the expired gas/room air mixture is measured and expressed as a voltage. This voltage is amplified by a factor proportional to the rate of flow of room air to yield a continous measurement of carbon dioxide output. This technique has no systematic error and a random error (95% tolerance limits) of +/-3.4, +/-1.7, and +/-1.4% at mean carbon dioxide outputs of 254, 846, and 1,906 ml/min, respectively.


Problems of sampling and analytical methods. The need for acceptable, reliable, and practical methods of analysis, chemical, physical, and biological, by the Food Standards Program of the Food and Agriculture Organization/World Health Organization is leading to cooperation by those organizations with the resources and experience to supply them. Methods should be shown to be workable and practicable and then validated in a properly designed international collaborative study for the efficient utilization of the time and effort of participating organizations and laboratories. The methods to be subjected to interlaboratory collaborative study should be clearly written so that the method itself is being tested without unauthorized variations. Satisfactory reference standard materials are often an essential part of the method. Uniform, international methods which have been developed through interlaboratory collaborative studies are applicable to the areas of microbiology and toxicology, as well as chemistry.


Rapid determination of butylated hydroxyanisole, tert- butylhydroquinone, and propyl gallate in edible oils by electron capture gas-liquid chromatography. A rapid method is described for the determination of 2- and 3-tert-butyl-4-hydroxyanisole (2- and 3-BHA), tert-butylhydroquinone (TBHQ), and propyl gallate (PG) as their heptafluorobutyrates in edible oils. A solution of the oil in ether-benzene is treated with heptafluorobutyric anhydride (HFBA) in the presence of trimethylamine as catalyst. Excess HFBA is destroyed and a suitably diluted aliquot is analyzed by electron capture gas-liquid chromatography. An internal standard is added before derivatization to aid quantitation and identification of the antioxidants. Average recoveries from an oil spiked at 1, 5, 5, and 5 ppm TBHQ, 2-BHA, 3-BHA, and PG were 110, 105, 106, and 102%, respectively. At these low levels, however, some late-eluting peaks interfered. Butylated hydroxytoluene is not determined by this method.


Rapid thin layer chromatographic method for the detection of histamine in fish products. A rapid, convenient thin layer chromatographic (TLC) method for detecting histamine in fish samples is described. Samples of press juice or fish flesh are applied directly to TLC plates. The plates are developed with acetoneammonium hydroxide (95+5) and the spots are visualized with ninhydrin or Pauly's reagent. Chromatographic separation of histamine from other fish components is readily achieved by this method.


Cathodic stripping voltammetry of nanogram amounts of selenium in biological material. A method is described to determine selenium in biological material, based on cathodic stripping voltammetry. Following wet ashing, the selenium was extracted into benzene as the 3',4'-diaminophenylpiazselenol. The selenium was subsequently back-extracted into dilute acid for analysis. Analyses of NBS Bovine Liver demonstrated that the method was capable of recovering 96+/-9% of the selenium present. The detection limit and working range were 3 ng/g and 0-10,000 ng/g, respectively. The method was also applied to the determination of selenium in rapeseed oils and seed.


Determination of added blood in ground beef. Blood added to ground beef is estimated from hemoglobin extracted with water. Since myoglobin is co-extracted with the hemoglobin, the 2 heme pigments are separated in one portion of the extract by precipitating the hemoglobin in an 85% (NH4)2SO4 solution. The myoglobin remaining in solution is used as the reference solution for determining the hemoglobin in another portion of the extract containing both pigments. The hemoglobin is converted to cyanomethemoglobin and quantitated by its absorbance at 422 nm.


Automated fluorometric method for determination of total vitamin C in food products. A specific microfluorometric method for the determination of ascorbic acid, dehydroascorbic acid, and total vitamin C in food products has been automated. The procedure developed is an adaptation of the official AOAC method (secs. 43.056-43.062), except that N-bromosuccinimide is used instead of Norit to oxidize vitamin C. Ascorbic acid is selectively oxidized by N-bromosuccinimide before other interfering substances that may be present, so this method is a highly sensitive and specific technique with extensive applicability. The proposed automated method is simple, rapid, reliable, and sufficiently sensitive to analyze as little as 2 x 10-3 to 0.1 mg ascorbic acid/ml. Analytical results obtained for ascorbic acid, dehydroascorbic acid, and total vitamin C in a wide variety of food products are reported. The analytical system developed has the capability of analyzing 50 samples/hr.


New method for determination of free amino groups in intact pure proteins: relationship to available lysine. A new rapid method for the quantitative and routine determination of free amino groups in intact pure proteins has been developed. Primary amino groups are labeled with fluorescamine and the labeled groups are detected by absorption spectroscopy in the range 375-390 nm. The amino group concentration can be determined in a few minutes without hydrolyzing the labeled protein and extracting a lysine derivative. The method was tested with the following proteins: lysozyme, alpha-lactalbumin, beta-lactoglobulin, bovine serum albumin, ribonuclease, ribonuclease-S-peptide, and alphasl-casein B. Application of this method to the estimation of available lysine is discussed.


Thin layer chromatographic estimation of available lysine in dried milk powder. A rapid method for the estimation of available lysine in dried milk powder has been developed. Samples were treated with 2,4,6-trinitrobenzene sulfonic acid and then hydrolyzed with 6N HCl. The hydrolysate was purified and subjected to ascending thin layer chromatography to isolate xi-trinitophenyl lysine in pure form. The color was read at 346 nm in a specitrophotometer.


High-pressure liquid chromatography of caffeine in coffee. A new method is described for the determination of caffeine in coffee, based on high-pressure liquid chromatography. The caffeine is extracted from the sample with water and/or methylene chloride, and then separated from interfering materials by passing an aliquot of the extract through a high-pressure column containing sulfonated cation exchange resin, using 0.01M nitric acid as the mobile phase. An ultraviolet detector measures the absorption of the solution directly. The method is rapid and eliminates the lengthy separations common to other methods. The procedure was applied successfully to decaffeinated and non-decaffeinated green, roasted, and instant coffees. This method gives a more accurate measure of the caffeine content in decaffeinated coffee samples than the micro Bailey-Andrew and modified Levine methods, with equal or better precision. This method gives results equal to those obtained by the official methods for non-decaffeinated samples.


Reference Raman spectra of eleven miscellaneous pesticides. The Raman spectra of ICP (propham), CIPC (chlorpropham), carbaryl, EPTC (ethyl N,N-dipropyl (3-chlorophenyl) carbamate), pebulate, CDEC (2-chloroallyl diethyldithiocarbamate), thiram, maneb, zineb, ferbam, and nicotine have been recorded. These spectra are presented, along with tables giving values for the frequencies.


A simple spectrophotometric method for the determination of carbofuran residues. A method has been developed for carbofuran residues, based on coupling carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl methyl carbamate) with diazotized aniline to form a yellow compound with an absorption maximum at 460 nm. The relationship between absorbance and concentration is linear for 1-10 mug carbofuran/5 ml. The method is sensitive and can be applied to the determination of levels as low as 0.025 ppm carbofuran in a 40 g crop or soil sample.


Collaborative study of a modified extraction procedure for lasalocid sodium in feeds by microbiological assay. A collaborative study was conducted on a modified extraction technique in the microbiological determination of lasalocid sodium in finished poultry feeds. The feed is warmed briefly with pH 4.7 buffer and the drug is extracted with ethyl acetate. The extract is washed with HCl and NaOH and evaporated to dryness, and the residue is dissolved in hexane, partitioned into methanol-water (3+1), and diluted to assay range at a final alcohol concentration of 25% (v/v). Seven laboratories participated in the study. Average recoveries of lasalocid sodium added at 0.01035, 0.00863, and 0.0069% ranged from 93 to 99% for mash feeds and from 86 to 95% for pellets. The coefficients of variation averaged 11.2% for mashes and 9.8% for pellets. The modified extraction technique has been adopted as official first action.


Rapid colorimetric method for carbadox in animal feeds. The colorimetric method for carbadox in animal feeds was modified to be simpler and faster. Carbadox is extracted with dimethyl-formamide and separated from interferences on an alumina column in the absence of water by controlling the amount of sample extract on the column and the flow rate. The isolated carbadox is eluted into sodium hydroxide and the resultant yellow chromophor is measured spectrophotometrically at 420 nm.


Preparation and spectral compilation of FD&C Red No.40 intermediates and subsidiary dyes. FD&C Red No. 40 may contain higher or lower sulfonated subsidiary dyes, depending on the purity of intermediates used. Samples of 10 possible subsidiaries were prepared and purified. They are to serve as analytical standards to aid in the identification of unknown compounds frequently appearing during the analysis of certification samples. Visible, ultra-violet, and infrared spectra as well as absorptivites and molar extinction coefficients are given for each dye.


A review of sampling plans and collaboratively studied methods of analysis for aflatoxins. Aflatoxins are the only food contaminants being monitored routinely on an international scale with methods operating at the order of magnitude of 10 mug/kg. At this concentration level, methods of analysis which can achieve coefficients of variation of 30-40% with recoveries of 70% or greater in interlaboratory collaborative studies can be considered eligible for referee status. In most cases, sample reproducibility is the variable limiting the reliability of methods of analysis. The inherent uncertainty of the identity of chromatographically separated entities requires the application of confirmatory tests to verify that the characteristic measured results from the presence of aflatoxin. The methods are also inoperable without a verification of the identity, purity, and concentration of the reference standards used. Screening methods which reliably eliminate negative samples from further consideration are indispensible for the practical operation of monitoring programs.


Identification of human seminal acid phosphatase by electrophoresis. Recent advances in forensic science in the identification of human seminal acid phosphatase are presented, with particular attention to the acrylamide gel electrophoretic method. In that method a difficulty in distinguishing seminal acid phosphatase from certain fecal phosphatases has been observed and an attempt is made here to distinguish the phosphatases from one another experimentally by differential substrate specificity. 4-Methylumbelliferyl phosphate and alpha-naphthyl acid phosphate are used as reaction substrates. Although seminal and vaginal acid phosphatases are differentiated by the modified method, fecal phosphatase is not clearly differentiated from seminal acid phosphatase.


Microbiological assay with Lactobacillus plantarum for detection of adulteration in orange juice. A microbiological assay has been developed to help detect adulteration in orange juice. Under standard assay conditions with diluted orange juice, the growth of Lactobacillus plantarum is proportional to the concentration of juice in the assay mixture. Imitation orange beverages did not support growth. Growth was also independent of the normal levels of common beverage ingredients such as sugar, acids, butylated hyroxyanisole, and orange oil. Commercial orange juices reconstituted from concentrates from various sources were assayed by the microbiological procedure, and the variability of results (coefficient of variation 24%) was about the same as or slightly lower than that for many of the other constituents used to estimate adulteration.


Gas chromatographic determination of micro amounts of cyanide residues in wines, distilled liquors, and other alcoholic beverages. A gas chromatographic method for the determination of cyanide residues in alcoholic beverages has been developed from procedures previously reported for application to water samples. Quantitatively isolating HCN from alcoholic beverages presented difficulties not encountered with aqueous solutions, particularly in the presence of S02 in the sample. HCN was liberated from the acidified sample by heating at 70 degrees C, flushed into an NaOH absorber solution, converted to cyanogen bromide (CNBr), extracted into diisopropyl ether, chromatographed on a Porapak Q column, and detected by an electron capture detector. S02 that is present in most wines interfered with the bromination step and caused low recoveries. This interference was eliminated by initially converting any cyanide present in the sample to the stable mercuric cyanide salt and then purging the acidified sample solution of all S02. The Hg(CN)2 present was then dissociated by adding KI and the analysis proceeded as previously described. Mean recoveries of 80-97% were obtained for 2-20 mug cyanide from replicate analyses of spiked samples of distilled liquors free of S02. The relative standard deviations ranged from 6.1 to 11.1%. Mean recoveries of 65 to 91% were obtained in the same range of cyanide from replicate analyses of spiked wine samples known to contain S02, with relative standard deviations ranging from 0.8 to 10.2%. The limit of detection was 0.2 mug HCN.


Loss of chloride in the official method for the determination of sodium chloride in cereal foods. The official final action method for sodium chloride in cereal foods, 14.129, was found to give erroneously low results because of loss of chloride during ashing. Comparison of the data with values obtained by the official first action potentiometric method, 32.A01-32.A06, which does not require ashing, showed that large and variable losses of chloride occurred. Official ashing methods for other foods specify addition of sodium carbonate to prevent conversion of chloride to volatile forms, but this was not specified in 14.129. In the present study it was found that sodium carbonate did not completely prevent loss of chloride. The official first action potentiometric method, 32.A01-32.A06, has been adopted as official first action for the determination of chloride in cereal foods to replace 14.129, which was repealed, official first action. A cross-reference to 32.A01-32.A06 has been added to 14.096.


Drugs. Differential pulse polarography of cacodylate injections. A method was developed for the analysis of cacodylate injections, using differential pulse polarography for measuring As(III). The cacodylate molecule is decomposed to As(V) by heating with Mg(NO3)2.6H20; As(V) is then chemically reduced to As(III) and polarographed in 1M HCl supporting electrolyte. The method of standard addition is used for quantitation. Recoveries based on the analysis of synthetic cacodylic acid solutions containing approximately 9 mg arsenic/ml ranged from 99.1 to 101.7%.


Infrared spectrophotometric analysis of medicinal gases for trace impurities. The feasibility of examining medical gases for trace impurities, using an infrared spectrophotometer in conjunction with a 10 m gas cell, was investigated. Many of the impurities for which the USP includes limits were detected and measured at concentrations at or below those permitted by the USP; these include sulfur dioxide, carbon dioxide, carbon monoxide, and water. However, others (hydrogen sulfide, nitric oxide, and nitrogen dioxide) were not detected at these levels by this technique. Methane was found at low levels in some samples.


Pilocarpine hydrochloride in ophthalmic solutions: modification of a high-pressure liquid chromatographic determination and survey. Pilocarpine undergoes 2 important reactions in solutions, epimerization to isopilocarpine and hydrolysis to pilocarpic acid. There is no official USP limit for isopilocarpine or pilocarpic acid in pilocarpine hydrochloride ophthalmic solutions. An existing high-pressure liquid chromatographic (HPLC) method was modified by changing the buffer system to permit its use with constant-pressure as well as constant-volume instruments. The procedure separates all 3 components on a cation exchange resin; pilocarpine and isopilocarpine are determined directly and pilocarpic acid indirectly. Ophthalmic solutions and drug substances were obtained from substantially all the United States marketers of pilocarpine opthalmic solutions and were analyzed by the modified HPLC method. Results of the survey show that isopilocarpine is present to the extent of 0.4-3% and pilocarpic acid at levels of 0.6-7% of total alkaloid.


Confirmation of results of rapid screening test for aflatoxins performed at corn elevator. A screening method for corn and corn products, based on a minicolumn, was modified slightly to assay 60 lots of corn at one elevator to determine whether they could be sold as animal feed. To be salable, the lots had to contain less than 20 ppb total aflatoxin. Aflatoxin levels in the lots were later determined by the official AOAC method for corn to check effectiveness of the screening. No lot had been designated for sale that contained 20 or more ppb total aflatoxin.


Training the renaissance medical illustrator. The professional training of the medical illustration student has been a subject of controvery for the last few years. Curricula of the schools accredited by the Association of Medical Illustrators have, to varying degrees, undergone change due to new demands on the practicing illustrator. The curriculum in Biomedical Illustration at The University of Texas Health Science Center at Dallas attempts to balance time-proven courses with contemporary problem-solving approaches to the training of the medical illustrator. This paper reflects the history, philosophy and methods of implementation of the course of study leading to the M.A. degree in Biomedical Communication at the University of Texas Health Science Center at Dallas.


Appraisal, validation, and evaluation utilized in the assessment of instructional materials. To maintain the highest standards of instructional quality, it is essential that self-instructional materials used in the teaching effort be systematically assessed. This paper has presented three types of assessment that have proven effective for the Office of Education, College of Dental Medicine, Medical University of South Carolina. The commitment to a regular assessment program has resulted in a more informed selection of materials and a more appropriate use of self-instructional materials by the faculty. Obviously, all of the approaches described may not be appropriate for all institutions. However, all institutions utilizing self-instructional materials have an obligation to assess the effectiveness of the materials to the best of their ability and resources.


Role of gamma-carboxyglutamic acid. Cation specificity of prothrombin and factor X-phospholipid binding. Divalent cations are required for two roles in prothrombin-phospholipid interaction. The first role, catalysis of a prothrombin protein transition has a reaction half-life of 100 min at 0 degrees and is a prerequisite to phospholipid binding. The binding sites required for the transition have a very low cation specificity. All di- and trivalent cations tested were effective in this role with the exception of beryllium. Barium catalyzed the transition but only at high concentrations (6.6 mM was required for half-reaction). Blood-clotting Factor X, another gamma-carboxyglutamic acid-containing protein, also undergoes a cation-catalyzed protein transition which is a prerequisite to Factor X-phospholipid binding. In both proteins, the transition can be monitored by a decrease in the protein's intrinsic fluorescence. Compared to prothrombin, the Factor X transition occurs much more rapidly, has a somewhat greater specificity for cations, and requires higher concentrations of cations. This indicates that the cation binding sites provided by gamma-carboxyglutamic acid are not completely uniform in all proteins. The second role of divalent cations in prothrombin-phospholipid interaction is in the actual protein-phospholipid binding. This interaction was studied by protein fluorescence quenching resulting from excitation energy transfer to a chromophore attached to the phospholipid membrane. Only strontium and barium satisfactorily replaced calcium in this role. A number of other cations form protein-phospholipid complexes but of the wrong structure. These cations inhibit the prothrombinase complex (Factor Xa, calcium, phospholipid, Factor V). The cation specificity for Factor X-phospholipid binding is the same as for prothrombin except that higher concentrations of cations are required. Factor Xa (generated by action of Russell's viper venom on Factor X) displayed the same calcium requirements for the protein transition and phospholipid interaction as Factor X. The cation requirements of the prothrombinase complex correlate with the cation requirements of prothrombin and Factor X-phospholipid binding. Strontium is the only cation that will singly replace calcium. Barium is ineffective alone because the concentrations required to catalyze the protein transitions cause precipitation of the phospholipid. Combination of certain other cations with barium will, however, substitute for calcium. The other cations (specifically magnesium or manganous ion) catalyze the protein transitions and barium forms the correct protein-phospholipid complexes.


Role of enzymatically catalyzed 5-iodo-5,6-dihydrouracil ring hydrolysis on the dehalogenation of 5-iodouracil. Incubation of 5-iodo-5,6-dihydrouracil (IH2Ura) with soluble rat liver enzymes at 37 degrees, pH 8.2, results in the rapid release of iodide ion. The second product resulting from the carbon skeleton of the dihydropyrimidine ring system is 2-amino-2-oxazoline-5-carboxylic acid (I). Ultraviolet absorbance measurements at 225 nm, where both IH2Ura and iodide ion absorb, indicate that IH2Ura dehalogenation is a two-step process. The first step, which is enzyme-dependent, involves dihydropyrimidine amidohydrolase (EC 3.5.2.2.)-catalyzed hydrolysis of the IH2Ura ring system presumably to yield 2-iodo-3-ureidopropionate. The enzyme preparations also catalyze the hydrolysis of 5-bromo-5,6-dihydrouracil, 5,6-dihydrouracil, and 5,6-dihydrothymine, the latter two of which are the natural substrates for dihydropyrimidine amidohydrolase. The second step in IH2Ura dehalogenation involves the nonenzymatically catalyzed, pH-independent intramolecular cyclization of 2-iodo-3-ureidopropionate via nucleophilic attack of the ureido oxygen atom on carbon-2 resulting in iodide ion and the oxazoline (I) as final products. The results are discussed relative to the role of pyrimidine catabolizing enzymes in 5-halopyrimidine dehalogenation.


Binding of uracil derivatives to hydrophobic peptides and sodium dodecyl sulfate. The capacities of five hydrophobic peptides to bind 13 alkyl uracil derivatives have been assessed as a first step toward constructing polymeric molecules, related to the nucleic acids, that specifically complement protein molecules. The peptides were Phe-Phe-Phe-Glu-Glu and its structural analogs with leucine, isoleucine, methionine, and valine substituted for phenylalanine. Uracils with the following substituents in position 5' were used: i-propyl, i-butyl, i-pentyl, sec-butyl, n-butyl, phenyl, benzyl, phenylethyl, methylthioethyl, ethylthiomethyl, and ethylthioethyl. 6-Benzyl and 6-i-pentyl uracils were also tested. The variations in base binding patterns are unique for each peptide, and the general effectiveness of the peptides in binding is related to the order in which their hydrophobic amino acid constituents occur in the uracil column of the genetic codon table. Although the method used does not permit precise determination of binding constants, it is apparent that many of them are much lower than 1 mM. 5-Ethylthioethyluracil quite selectively forms a large metastable aggregate with Phe3Glu2. Its close homologues do not. Also, 5-ethylthioethyluracil binds in some measure to Met3Glu2 but not significantly to Ile3Glu2 and Val3Glu2, whereas its homologue, 5-ethylthiomethyluracil, binds better to the latter two than to Met3Glu2. Thus, the two homologues might serve to form hypothetical polymers that discriminatively bind polymers of isoleucine and valine. It is argued that evolution would most reasonably have begun with such crude mechanisms.


Specificity of protein turnover in tomato leaves. Accumulation of proteinase inhibitors, induced with the wound hormone, PIIF. Detached tomato leaves, supplied with the proteinase inhibitor inducing factor (PIIF) and incubated with water under constant light, exhibited a specificity of intracellular protein turnover directed toward the selective accumulation of heat-stable proteins having disulfide corss-linkages. Approximately 70% of the accumulated proteins could be accounted for in two proteinase inhibitors rich in disulfide links. The accumulation of proteins containing disulfides was accompanied by a net loss in total leaf protein, mainly of heat-precipitable proteins having free sulfhydryl residues. Relative rates of synthesis of --S--S-- proteins and --SH proteins were assessed by comparing rates of incorporation of isotope into the inhibitor proteins and noninhibitor leaf proteins. Although the inhibitors represented about 12% of total leaf protein after 71 h of induction, only about 2% of total protein synthesis was directed toward inhibitor synthesis during incubation of induced leaves. The marked stability of inhibitors, and other disulfide proteins against degradation in vivo, appeared to be a major factor providing for their selective accumulation. It was concluded that the state of oxidation of protein-bound half-cystine residues may be a principle parameter influencing the susceptibility of leaf proteins to degradation in vivo.


Radioimmunoassay for neurotensin, a hypothalamic peptide. A highly sensitive and specific radioimmunoassay for neurotensin has been developed which utilizes 125I-labeled neurotensin and rabbit antisera raised toward synthetic neurotensin which has been coupled specifically through its lysine side chain to several proteins. The three antisera described have different specificities but are directed primarily towards the COOH-terminal region of neurotensin which is the biologically active portion of the molecule. Two of the antisera, poly(Glu60, Lys40) (from animal no. 4) and keyhole limpet hemocyanin (from animal no. 8), cross-react fully with COOH-terminal partial sequences of neurotensin while antiserum poly(Glu60, Lys40) (from animal no. 6) requires the entire molecule for full recognition. The assay can detect less than 3 fmol of neurotensin and the dose-response curves for synthetic and native neurotensin are superimposable, irrespective of the antiserum employed. Using these assay systems, the immunoactivity in acid/acetone extracts of 45 kg of bovine hypothalami was purified to homogeneity and shown to be attributable to intact neurotensin and not to fragments of neurotensin nor to related molecules. Radioimmunoassayable neurotensin (R-NT) obtained from bovine, rat, guinea pig, and rabbit hypothalami also gave dose-response curves which paralleled that of neurotensin and the neurotensin equivalents per g of wet tissue were in the range 45 to 70 pmol/g. Measurements with the three antisera were in agreement, especially after the extracts were chromatographed on Sephadex G-25; R-NT in these hypothalamic extracts has also been shown to be destroyed by treatment with various enzymes known to cleave neurotensin.


Characterization of radioimmunoassayable neurotensin in the rat. Its differential distribution in the central nervous system, small intestine, and stomach. Using radioimmunoassay, the distribution of radioimmunoassayable neurotensin (R-NT) has been determined in acid/acetone extracts of tissues from week-old and adult rats. Whereas only 10% of the R-NT in week-old rats was found in the head, 90% was found in extracts of the body, and 85% was present in the intestine. The distribution of R-NT in the central nervous system of adult male rats was as follows: hypothalamus (35%), brain stem (35%), cerebral cortex (17%), thalamus (11%), cerebellum (1%), and pituitary gland (1%). Concentrations of R-NT were highest in the hypothalamus (60 pmol/g) and lowest in the cerebellum (0.8 pmol/g). More than 10 times as much R-NT was found in extracts of adult rat small intestine than in brain. The concentration of R-NT was highest in the jejuno-ileal section of the intestine (approximately 50 pmol/g) but it was also detected in the esophogus, stomach, duodenum, and large intestine (approximately 1 to 8 pmol/g) than the remaining muscle layer (approximately 40 pmol/g). Rat jejuno-ileal R-NT co-chromatographed with neurotensin on Sephadex G-25, displayed equal immunological potency with the antisera utilized, and was destroyed by enzymes known to cleave neurotensin. High concentrations (approximately 400 pmol/g) of an R-NT of smaller molecular weight than neurotensin were found in rat stomach. This peptide reacted most strongly with a COOH-terminal-directed antiserum and appeared to be four to five amino acids in length; it might possibly be a breakdown product of neurotensin or a related peptide with biological activity. Acid/acetone extracts of rat and bovine plasma were found to contain an R-NT which displayed the properties of neurotensin (approximately 50 fmol/ml) as well as a substance which behaved similarly to the small molecular weight stomach R-NT (approximately 1000 fmol/ml).


Isolation of a tridecapeptide from bovine intestinal tissue and its partial characterization as neurotensin. Radioimmunoassayable neurotensin (R-NT) has been isolated from acid/acetone extracts of 50 kg of calf small intestine with an overall yield of approximately 15%. The concentration of R-NT in calf intestinal tissue was approximately 35 pmol/g wet weight. Throughout the purification procedures which involved adsorption onto sulfopropyl (SP)-Sephadex, chromatography on Sephadex G-25 and SP-Sephadex, immunoadsorption on neurotensin-antibody Sepharose and high voltage paper electrophoresis, R-NT displayed the chromatographic and electrophoretic properties of neurotensin. R-NT was found to contain a tridecapeptide with the same amino acid composition as neurotensin. This peptide yielded the same products as neurotensin when submitted to digestion by carboxypeptidase A or papain. Its immunological properties were indistinguishable from those of neurotensin and its potency in stimulating hypotension in anesthetized rats was comparable to that of synthetic neurotensin. If the amino acid sequence of this peptide proves to be the same as that of neurotensin, then neurotensin is another biologically active peptide isolated from both brain and intestinal tissues.


C20-sphingosine as a determining factor in aggregation of gangliosides. Aggregation properties of gangliosides, the major synaptic membrane glycosphingolipids of mammalian brain, may prevent their segregation during membrane assembly and promote a uniform membrane matrix with minimum maintenance energy. The sphingosine residues of bovine brain gangliosides show an increase in C20-sphingosine corresponding with an increase in sialic acid. Concentrations of C20-sphingosine varied from 37% for the monosialoganglioside to 64% for the trisialoganglioside, the remainder being C18-sphingosine. Ultracentrifugal analysis showed that changes in sialic acid content and in C20-sphingosine content individually affect micellar size. Increases in sialic acid content decreased micellar size from 225 for the monosialoganglioside to 120 monomersper micelle for the trisialoganglioside. Monosialogangliosides enzymatically prepared from oligosialohomologues with a higher C20-sphingosine content gave evidence for a considerable effect of C20sphingosine upon the free energy of the aggregate form; the number of monomers per micelle increased from 225 for the natural monosialoganglioside to 280 for monosialoganglioside derived from trisialoganglioside. The similar aggregation energies of the major synaptic membrane gangliosides apparently result from a metabolic balancing of increased C20-sphingosine with increased sialic acid content.


Mode of inhibition of acid proteases by pepstatin. Four derivatives of pepstatin, each of which contains the unusual amino acid 4-amino-3-hydroxy-6-methylheptanoic acid (statine) have been prepared. All four are porcine pepsin inhibitors. Both N-acetylstatine and N-acetyl-alanyl-statine are competitive inhibitors for pepsin with Ki values of 1.2 X 10(-4) M and 5.65 X 10(-6) M, respectively. The Ki values for N-acetyl-valyl-statine is 4.8 X 10(-6) M. These statyl derivatives, therefore, are very strong inhibitors. The Ki value for N-acetyl-statine is 600-fold smaller than that of its structural analog N-acetyl-leucine. The derivative which contains two statyl residues in a tetrapeptide exhibits inhibitory properties which approach those of pepstatin itself. Other acid proteases, human pepsin, human gastricsin, renin, cathepsin D, the acid protease from Rhizopus chinensis and bovine chymosin, also are inhibited by pepstatin and its derivatives. It is suggested that the statyl residue is responsible for the unusual inhibitory capability of pepstatin and that statine is an analog of the previously proposed transition state for catalysis by pepsin and other acid proteases.


NMR study of relative oxygen binding to the alpha and beta subunits of human adult hemoglobin. NMR spectra of the downfield region of normal adult hemoglobin are reported as a function of oxygenation and temperature. Spectra were run in D2O at pD 7.4. A specially made NMR tube insert allowed precise measurement of the degree of oxygenation and of methemoglobin formation before and after taking the NMR spectrum. Plots of the estimated intensity of the most downfield prominent NMR peak, identified as arising from a deoxy-beta subunit by Davis et al. ((1971) J. Mol. Biol. 60, 101-111), versus the average degree of oxygenation y, measured optically, yield a nearly straight line within experimental error, for samples stripped of organic phosphates and for samples containing 2,3-diphosphoglycerate or inositol hexaphosphate. Intensities of peaks further upfield than this peak, previously attributed to deoxy-alpha subunits, are difficult to measure directly especially for samples containing inositol hexaphosphate. The latter samples show broadening in these alpha peaks as the degree of oxygenation increases. This extra broadening appears to increase with temperature. Linearity of the beta peak intensity with oxygenation is expected if there is no large oxygen affinity difference between alpha and beta subunits. However, the cooperativity of binding, and inaccuracy of the data, make it impossible to make accurate estimates of affinity differences.


Inhibition of glycolysis in brain by a phospholipid effect on interconversion of fructose phosphates. A possible regulatory control on utilization of glucose 6-phosphate. Glucose 6-phosphate accumulation in 10,000 X g supernatant of rat brain was enhanced up to 16-fold by the addition of phosphatidylcholine, other common phospholipids, or linoleate. This glucose 6-phosphate is of endogenous origin via UDP-glucose and glucose 1-phosphate but not glucose. The accumulation is the result of inhibition of glycolysis by an effect of phospholipid on the interconversion of fructose 6-phosphate and fructose 1,6-bisphosphate. Brain is therefore capable of gluconeogenesis from fructose 1,6-bisphosphate. A regulatory function for phospholipid which coordinates glycolysis and other major routes of utilization of glucose 6-phosphate in brain, e.g. inositol synthesis, is proposed.


Impermeant maleimides. Oriented probes of erythrocyte membrane proteins. Maleimides impermeant to human erythrocyte membranes have been synthesized and applied to studies of the sulfhydryl groups of the membrane. Reaction of radioactive dextran-maleimide and glutathione-maleimide with either intact erythrocytes or ghosts yields sulfhydryl titers for the outer (exofacial) and inner (endofacial) surfaces, respectively, of 1.5 to 1.7 and 27 to 28 amol/cell. Corresponding values for sulfhydryl groups within the membrane interior, as estimated with radioactive N-ethylmaleimide, are 16 to 22 amol/cell. After exofacial labeling of intact cells with [35S]glutathione-maleimide, autoradiography of sodium dodecyl sulfate-polyacrylamide gels demonstrates four bands (alpha, beta, gamma, and delta) containing, respectively, 13%, 63%, 11%, and 13% of the radioactivity. The major beta-band corresponds in position to polypeptides of molecular weight 40,000 to 70,000 and to Coomassie brilliant blue-stained Band 5. Selective extraction demonstrates that the major Band 5 protein is not identical with the labeled beta-band polypeptides. Following endofacial labeling of ghosts with [35S]glutathione-maleimide, autoradiography reveals radioactivity in all of the major Coomassie brilliant blue bands. The impermeant maleimides described are also applicable to studies of discrete functional proteins of the erythrocyte membrane, including the hexose transport mechanism and the major Rho antigenic site.


Impermeant maleimides. Identification of an exofacial component of the human erythrocyte hexose transport mechanism. The facilitated diffusion of D-glucose across human erythrocyte membranes requires an exofacial (outer surface) sulfhydryl group which can be alkylated by the impermeant reagents glutathione-maleimide and dextran-maleimide. The irreversible inhibition produced by these reagents is asymmetric; inhibition of glucose efflux considerably exceeds that of influx when transport is assayed in the absence of glucose on the opposite side of the membrane. Both D-glucose and cytochalasin B protect the exofacial transport site from alkylation by the impermeant maleimides. This masking effect provides the basis for a two-step procedure for differential labeling of the outer transport site with radioactive glutathione-maleimide. The method labels clearly and consistently a component of the membrane proteins which migrates in sodium dodecyl sulfate-polyacrylamide gels between Coomassie brilliant blue-stained Bands 4.2 and 5, corresponding to an apparent molecular weight of 65,000 to 70,000. Transport studies after inhibition with N-ethylmaleimide suggest that the hexose mechanism also requires a second sulfhydryl group which is not accessible at the cell surface.


DNA "melting" proteins. I. Effects of bovine pancreatic ribonuclease binding on the conformation and stability of DNA. Bovine pancreatic ribonuclease is a DNA "melting" protein, since it binds with greater overall affinity to the single-stranded than to the double-stranded form of natural and synthetic deoxyribose-containing polynucleotides. As such, the DNA-RNase system provides a simple model for the more complex and biologically relevant melting protein-nucleic acid systems. Aspects of the DNA-RNase interactions which are related to the quantitative assessment of this system as a melting protein model are investigated here. A boundary sedimentation velocity technique is used to measure thermodynamic parameters of the interaction; association constants (Kh and Kc) and site sizes (nh and nc) are determined for the interaction of ribonuclease with native (double helical) and denatured (random coil) DNA. It is shown that log Kh and log Kc are linear functions of log [Na+], binding decreasing with increasing Na+ concentration, with Kh about 2 orders of magnitude smaller than Kc at the ionic strengths studied, nh and nc are approximately 8 and approximately 11 nucleotide residues, respectively, indicating that potential binding sites overlap. Binding to both forms of DNA is non-cooperative. It is shown by CD and ultraviolet spectroscopy that the binding of RNase to single- and double-stranded DNA perturbs the conformations of these polynucleotide conformations very little relative to the unliganded structures. Hydrodynamic methods are used to show that RNase binds to native DNA without altering the overall solution structure of the latter; however conditons which permit binding to, and stabilization of, transiently exposed single-stranded sequences result in a collapse of the stiff native DNA structure. We demonstrate by melting transition studies that ribonuclease does bring about an equilibrium destabilization of native DNA and poly [d(A-T)] and, by applying a ligand-perturbed helic in equilibrium coil theory developed by McGhee (McGhee, J.D. (1976) Biopolymers 15, 1345-1375), it is shown that the extent of the observed destabilization is in semiquantitative accord with expectations based on the measured affinity constants and site sizes for RNase binding to both DNA conformations. Spectral methods are used to show that the relative stability of native DNA sequences of varying base composition is the same in the presence and absence of ribonuclease, strongly arguing that this "melting" ligand "traps" single-stranded sequences transiently exposed by thermal fluctuations. RNase also undergoes an order in equilibrium disorder conformational transition as a function of temperature (the denatured form of RNase stabilizes native DNA, while native RNase destabilizes the native double helix), and the coupled equilibria involved in these interacting conformational changes are interpreted and discussed as possible models of genome regulatory interactions.


DNA "melting" proteins. IV. Fluorescence measurements of binding parameters for bacteriophage T4 gene 32-protein to mono-, oligo-, and polynucleotides. The quenching of the intrinsic tryptophan fluorescence of T4-coded gene 32-protein on binding to nucleotide ligands, which was described in the preceding paper, is here exploited to measure thermodynamic parameters of the single-stranded nucleic acid-gene 32-protein interaction. It is shown that binding of small ligands follows a single site binding isotherm, with association constants increasing from approximately 20 M-1 for phosphate, to approximately 10(3) M for ribose or deoxyribose 5'-phosphate, to approximately 10(4) M-1 for mononucleotides, and to approximately 10(5) M-1 for dinucleoside monophosphates (all in 0.1 M Na+). The measured binding constants appear to be about the same for homologous ribose- and deoxyribose-containing ligands and to be independent of oligonucleotide base sequence and composition. Furthermore, beyond the dinucleotide level and up to octanucleotides, the increase in binding constant with increasing chain length is only about that expected from the statistical factor resulting from the increased number of ways a longer oligonucleotide can form a protein complex. This suggests that the basic binding unit involved in gene 32-protein associations with single-stranded nucleic acids can be approximated by a dinucleoside monophosphate. Oligonucleotides long enough to accomodate two or more protein monomers are characterized by much larger association constants, indicating that binding is cooperative in protein concentration. A cooperativity parameter (omegac) of approximately 10(3) is estimated from these data, in good agreement with that deduced from the application of ligand-perturbed helix in equilibrium coil transition calculations. Values of association constants (Kcomegac) of approximately 10(8) M-1 (in 0.1 M Na+) and site size (nc) of approximately 5 (+/-1) nucleotide residues/protein monomer are determined by the fluorescence titration technique for the cooperative binding of gene 32-protein to both poly(dA) and poly(rA); these values are also in agreement with those measured by Jensen et al. (Jensen, D.E. Kelly, R.C., and von Hippel, P.H. (1976) J. Biol. Chem. 251, 7215-7228). Possible in vivo consequences and correlations of these findings with proposed roles for gene 32-protein in replication and recombination are discussed.


Mechanical strength of acrylic bone cements impregnated with antibiotics. Admixing of several antibiotic powders which were insoluble in methyl methacrylate did not decrease the compressive and diametral tensile strengths of two acrylic bone cements when tested after setting for 1 day and after leaching 40 days in water at 37 degrees C. When antibiotics were added as water solutions, the included water resulted in a significant decrease in these bulk mechanical properties. Storage in water for 40 days caused surface irregularities only on specimens of the set antibiotic admixtures. Approximately 0.5% of the admixed dosage of these water-soluble antibiotics could be leached from the set cements. The observed surface channels, presumably left by the loss of antibiotic, suggest further study of surface-sensitive mechanical properties may be needed. The bulk mechanical strengths presented here are conclusive only for the particular combinations of antibiotics and cements investigated, and should not be generalized at this time to any or all antibiotic admixtures or other mechanical properties.


Further experimental and clinical experience with aluminum oxide endoprostheses. This paper describes the design principles of a new endoprosthesis made of dense aluminum oxide (A12O3) and implanted without bone cement. Mechanical tests and animal experiments have proved the sufficient mechanical strength of the bone-prosthesis connection as well as the biocompatibility of the material. So far, 12 tumor patients selected according to strict indication criteria have been operated on. Both the clinical-radiologic and the first histological findings available are encouraging. Special reference is made to the first fully ceramic total hip joint endoprostheses implanted in a human without bone cement.


Poly(glycolic acid) sutures in canine vascular anastomoses. Poly(glycolic acid) (Dexon; Davis and Geck Company) canine vascular anastomoses between aorta and synthetic grafts and between severed femoral vessels were compared with comparable anastomoses made with Teflon-coated Dacron (Tevdek; The Deknatel Company) with respect to clinical performance, morphologic characteristics, and tensile strength. While the Dexon anastomoses performed well and were morphologically superior to Tevdek, their tensile strength was significantly lower. Implications of these findings for vascular wound healing are discussed.


Using the shape recovery of nitinol in the Harrington rod treatment of scoliosis. Nitinol, a metal alloy which is able to remember (and return to with slight heating) the shape it had before it was deformed, is demonstrated to be useful in the Harrington rod treatment of scoliosis. A bent nitinol rod can return to its original straight length, applying both axial and lateral forces to the spine during the operation of postoperatively by means of external heating. A research program using simulated spinal forces in a test fixture and implantation in a cadaver was undertaken to show the feasibility of modifying the existing procedure.


Carbon fiber-reinforced bone cement in orthopedic surgery. A tougher, more fatigue-resistant bone cement consisting of short, highmodulus carbon fibers dispersed randomly in normal ply(methyl methacrylate) bone cement has been developed. Fatigue and impact tests are reported that demonstrate the superior fracture resistance and prolonged load-carrying ability of this system. The use of this carbon fiber-reinforced bone cement in the treatment of a pathological fracture of the femur associated with neoplastic disease of the bone is reported. The tougher cement has provided long-term stability of the fracture.


An in vitro model for evaluating neural stimulating electrodes. A model for in vitro evaluation of materials for use as neural stimulation electrodes is developed. Critical areas of concern in developing an in vitro test model discussed include: selection of environment, choice of material, design of stimulating equipment, and analytical procedures used to evaluate materials response. A method of providing quantitative analysis of materials response to stimulation conditions is presented. Evaluation techniques involve the use of scanning electron microscopy, x-ray spectroscopy, atomic absorption spectrometry, and potentiographic and dielectric analysis of the test electrodes. A diagnostics matrix is presented which assigns a scale factor to quantify the relative corrosion response of the candidate materials. The corrosion response of Pt electrodes is evaluated in terms of scale factors assigned through use of the diagnostics matrix.


Mechanical properties of bone cements containing large doses of antibiotic powders. The addition of up to 10 g gentamicin sulfate antibiotic powder to 60 g units of Simplex-P acrylic bone cement caused gradual, proportional decreases in the bulk muchanical properties of compressive and diametral tensile strengths. Water leaching of the antibiotic from the cement did not significnatly decrease these strenghts. Scanning electron microscopy (SEM) and energy-dispersive x-ray spectroscopy (EDS) showed the antibiotic to reside in the acrylic matrix as discrete particles not usually associated with internal porosity. The surface-sensitive flexural strength of a proprietary bone cement was lowered immediately by small quantities of antibiotic powder, and continued to decrease as doses of up to 10 g/unit were admixed. Water leaching caused channeling as the antibiotic was removed from the surface, but it did not create further changes in flexural strength.


Subcutaneous implants of polypropylene filaments. Extruded filaments of unmodified polypropylene (PP) with and without antioxidant were implanted subcutaneously in hamsters in order to determine their rate of degradation. Specimens were removed periodically during a 5 month test period and analyzed by infrared spectroscopy and dynamic mechanical testing. The analyses show that degradation beigns to occur after only a few days. Although the reaction sequence is not known, several factors suggest that the in vivo degradation process is similar to autoxidation which occurs in air or oxygen. The infrared data indicate that the hydroxyl content of the implants increases at a rate of 0.061 mg/g polypropylene per day during the initiation phase of the reaction. An induction time of 108 days was extablished. Carbonyl bonds appear after an implantation time of 50--90 days and increase therafter. Mechanical tests indicate a decrease in the dynamic loss tangent, tan delta, during the first month of implantation for unmodified polypropylene. No change in the infrared spectra or tan delta was observed, however, for implants containing an antioxidant. Thus, it is apparent that polypropylene filaments implanted subcutaneously in hamsters degrade by an oxidation process which is retarded effectively by using an antioxidant. While the findings reported are specific to subcutaneous polypropylene implants, they suggest that degradation of other systems may involve similar processes. This notion suggests directions for further research on increasing the in vivo stability of synthetic polymers. Long-term effects of polymer implantation upon tissue were not studied in this work.


New types of synthetic infusion solutions. III. Elimination and retention of poly-[N-(2-hydroxypropyl)methacrylamide] in a test organism. The elimination and retention of a new, fully synthetic plasma prosthesis of Czechoslovak origin under the working name of Duxon (poly[N-(2-hydroxypropyl)methacrylamide]) was investigated in experiments on rabbits by using a 14C-labeled polymer. Results published earlier on elimination have been confirmed and extended. Retention was examined after repeated application. The effect of molecular weight prevailed both in elimination and in retention. Polymers of lower molecular weights, which possess a good therapeutical effect, are not permanently deposited in the organism.


Urography during pregnancy.

Urography during pregnancy. A review of 22,971 pregnancies from 1969-74 reveals that the reasons for requesting excretory urography were renal colic of pregnancy 11, pyelonephritis 8, renal contusion 2, threatened abortion 1, and suspected degeneration of fibroid 1. The final diagnoses were similar except that two patients with torsion of ovarian cysts and a renal calculus were discovered. The only intravenous pyelographic examination that was decisive for diagnosis was in a patient with a ureteric calculus. Careful clinical correlation should reduce excretory urography during pregnancy.


Bilateral intrarenal hematomas from anticoagulants. A case is reported in which anticoagulant therapy for thrombophlebitis and pulmonary embolism produced bilateral massive intrarenal hematomas (pseudotumors). The role of radiologic investigation (nephrotomography, renal scan and selective high dose arteriography) was found to identify and localize the intrarenal hematomata and exclude underlying renal pathology. Although an abnormal kidney is more likely to bleed, this case presentation demonstrates that even carefully monitored anticoagulation within the therapeutic range can induce massive intrarenal hemorrhage in previously normal kidneys. The renal architecture returned to normal on late follow-up examination on simple conservative management.


Pelvic fractures in children.

Pelvic fractures in children. Eighty-four cases of pelvic fractures in children were reviewed. Most of them were the result of motor vehicle accidents. Thirty-nine per cent were classified as unstable, the most frequent type being diametric fractures. The others, most of which were pubic fractures, were classified as stable. Twenty-eight patients sustained fractures elsewhere and sixteen had associated visceral injuries.


Mammary anterior mediastinal pseudotumor. Dense, small breasts in young women may project into the anterior mediastinum in the lateral projection and simulate an anterior mediastinal mass. Recognition of the rather characteristic appearance of this shadow will obviate unnecessary concern and consequent investigative procedures.


Ultrasonic diagnosis of intrauterine fetal death. When there is doubt concerning fetal viability, the confirmation or exclusion of fetal death assumes major importance in the management of pregnancy. Frequently the clinical diagnosis is uncertain and recourse must be had to ancillary investigation. Radiologic methods may confirm the clinical suspicion of intrauterine fetal death in many cases, but these are accompanied by the hazard of irradiation. Ultrasound can provide valuable information regarding fetal viability. Ultrasonic signs of fetal death are discussed in the light of the literature and our own experience, with emphasis on the value of the M-mode in detecting fetal heart pulsations.


More causes of button sequestrum.

More causes of button sequestrum. Two cases illustrating the association of button sequestra with Paget's disease and multiple myeloma are presented, followed by a brief review of the roentgenologic skull manifestations of these diseases. The number of conditions having button sequestra now stands at thirteen. The sign has no specific diagnostic significance.


Roentgenologic aspects of spinal involvement by primary and metastatic Ewing's tumor. Twenty-six patients with Ewing's tumor were studied. The spine was involved in eleven patients; the clinical and roentgenologic features of these are described. Four patients had primary Ewing's tumor of the spine; one showed uniform sclerosis of a single vertebra, a hitherto unreported finding. Two showed bone destruction; one showed no bone change on plain radiographs but there was complete obstruction on myelography. Two developed spinal metastases and died shortly afterwards. One patient is alive and well eight and a half years later. Seven patients had metastases to the spine from a non-spinal Ewing's tumor. All had neurologic complications. Plain radiographs showed no bone change in five; however, a paraspinal mass or an extra-dural mass shown on myelography, were relatively common. The mean survival time following spinal involvement was four months (range 1-12 months). No correlation was evident between the site of the primary lesion, the method of treatment, and the development of spinal metastases.


Superselective arteriography and therapeutic embolisation for vascular malformations. (Angiodysplasias). By virtue of superselective arteriography which makes possible the catheterisation of extremely fine arterial branches, the authors were able to embolise vascular malformations (angiodysplasias). Twenty seven cases were treated in this way with satisfactory results for twenty one of them. Seven patients must be operated on after the embolisation. The problem of the future of this technique is considered, and it will perhaps be possible to avoid revascularization by the use of new substances.


Brachial artery to brachial vein preserved vein allograft fistulas for hemodialysis. Vein allografts were obtained from varicose vein patients in whom stripping and ligation was indicated. Proximal 30 cms of the long saphenous vein was removed and stored at -30 degrees C. A-V fistulas were constructed in the upper arm between brachial artery and the brachial or cephalic vein in dialysis patients. All these allograft vein recipients had major blood groups identical to the donors. Clinical experience over a two year period revealed a 100% long term patency. Complications including early graft thrombosis, superficial wound infection, hematoma formation and local edema were few and easily managed. Allograft rejection was not a problem in this series of patients. The large diameter grafts in upper arm position function well. They are convenient for the patient and preferred by dialysis personnel as a direct site for venepuncture. An initial experience suggests that preserved vein allografts may become the preferred material for arteriovenous fistulas.


Peripheral arterial insufficiency. Experiences from 229 operated limbs. The results of arterial reconstructions in 229 limbs in 177 patients were studied. Intermittent claudication resistant to physical training was indication for surgery in 82 cases and threatening gangrene in 147 cases. In the claudication group the long-term result was good in 77%, the reocclusion frequency was 21%, and the amputation rate was 2%. No operative mortality was encountered. In the gangrene group the long-term result was good in 40%, the reocclusion freuqency was 25%, the amputation rate was 24%, and theoperative mortality was 11%. Limb salvage rate was 65%. The results indicated that preoperative physical training did not jeopardice the operative results, but in most cases made surgical treatment unnecessary. In cases with threatening or beginning gangrene an aggressive attitude towards vascular reconstruction resulted in a substantial limb salvage.


Continuous electrocardiography recording at examination of walking capacity in patients with intermittent claudication. A treadmill test with increasing "walk-work" load and continuous electrocardiographic monitoring has been used in 245 patients with intermittent claudication. It was found that many patients underestimated their walking capacity. No good correlation was obtained between the observed walking capacity and the systolic blood pressure in the toes. Pathological electrocardiograms were found at rest in 36 per cent of the patients. Additional pathological ecg-reactions indicative for ischaemic heart disease appeared during the tests in 34 percent of the patients. Around 10  per cent of the tests had to be interrupted because of threatening ecg changes.


[Ciliogenesis in the mucous cells of the quail oviduct. I. Ultrastructural study in the laying quail]. The luminal epithelium of the oviduct (magnum) of laying quails is composed of ciliated cells and mucous cells. Ciliogenesis was observed in some of the mucous cells. Both centrioles of the diplosome migrate to the top of the cell, and one of them induces the formation of a rudimentary cilium. In some of the other cells, that are filled with mucous granules, the formation of basal bodies by an acentriolar pathway was observed. In these cells, numerous, dense fibrous masses are associated with the forming face of the Golgi apparatus. In the Golgi zone, generative complexes composed of a deuterosome and some forming procentrioles were found. Cilia develop from completed basal bodies. During ciliogenesis, the Golgi apparatus is disorganized, and generally the production of mucous granules is arrested. The nucleus is also modified: it becomes larger and the chromatin is dispersed. It is assumed that mucous cells are able to be transformed into ciliated cells in the oviduct of laying quails.


[Ciliogenesis in the mucous cells of the quail oviduct. II. Hormonal control]. The hormonal control of ciliogenesis and transformation of mucous cells was studied in the oviduct (magnum) of ovariectomized quails. Estradiol benzoate induces ciliogenesis with doses varying from 10 mug/day to 100 mug/day after 6 days of treatment. With 100 mug/day, differentiation of some mucous cells is also induced as well as the formation of transitory "mixed cells" which are in the process of ciliogenesis and contain mucous granules. Associated with progesterone (1 mg/day), estradiol benzoate (10 mug/day) induces the differentiation of mucous cells and ciliated cells. The luminal epithelium of quails injected with this mixture is similar to the luminal epithelium observed in the oviduct of laying quails. With the same dose of progesterone (1 mg/day) and 20 mug/day of estradiol benzoate for 6 days, ciliogenesis is completely inhibited. All epithelial cells are secretory cells. Transformation of 50% of the mucous cells into ciliated cells is obtained by following the previous estradiol-progesterone treatment with the injection of estradiol benzoate (20 mug/day) for 3 days. Divisions of mucous cells were also observed. It is also possible to induce ciliogenesis in some mucous cells by withdrawing both hormones for 3 days. In this case, no cell divisions were observed.


Association of a protease (plasminogen activator) with a specific membrane fraction isolated from transformed cells. The intracellular distribution of specific protease, plasminogen activator (PA), has been examined in Rous sarcoma virus-transformed chick embryo fibroblasts (RSV-CEF). Cellular homogenates were fractionated by differential centrifugation followed by sucrose gradient centrifugation. The activities and the percent distribution of a series of marker enzymes, specific for different subcellular organelles, were compared to those of PA. Normal CEF have been similarly fractionated and the relatively low amount of PA activity present in these cells has been analyzed in terms of its subcellular distribution. A membrane fraction was isolated from the RSV-CEF that contained the bulk of the PA activity and less than 8% of the total cellular protein. The specific activity of the PA in this fraction is 40-fold higher than that of a comparable fraction isolated from companion cultures of normal cells. This fraction contains little or no nuclear and cytoplasmic material and is contaminated only to a relatively small degree with mitochondria, lysosomes, endoplasmic reticulum. Significant amounts of a putative Golgi membrane marker are present in this fraction. The relatively high specific activities of Na+,K+-ATPase, 5'-nucleotidase, and [3H]fucose indicate that the fraction is enriched in surface membrane. Further purification of the fraction by equilibrium centrifugation on shallow sucrose gradients reduces further the contaminating activities and results in a PA distribution that closely parallels the distribution of the membrane enzyme, 5'-nucleotidase. PA was not released from its membrane association by hypotonic and hypertonic extraction and ultrasonication, while granule-bound enzymes were released by these treatments. The PA activity from hamster SV40 cells fractionated the same way as that of RSV-CEF. These results suggest that a protease that is dramatically enhanced upon malignant transformation is associated with "plasma membrane-like"  elements of the cell and may serve as an intrinsic modifier of cell surface proteins after malignant transformation.


Observations on the Raynaud's disease.

Observations on the Raynaud's disease. During the last 25 years, owing to the improvement of the diagnostic means, the cases of "Raynaud's phenomenon" with well known etiology have been increasing and according to some authors they should be considered clearly prevailing. Through a right prevention and a medical and surgical therapy it is possible to obtain significant results. Consequently it is important to carry out a diagnostic screening. A study concerns 68 cases have been carried out in the Institute of Vascular Surgery, University of Milan, during the period 1971-1974. The cases are divided in two groups: idiopathic forms (disease) and forms connected to identifiable etiologies (syndrome). Many of the clinical parameters studied are examined. The so-called idiopathic forms: 47 cases (69%) with an average age of 45 years and a clear female prevalence (33 against 14). The forms with an identifiable etiology: 21 (31%) with an average age of 31 years and a clear male prevalence (16 against 5). It is therefore observed that the idiopathic Raynaud's disease is still prevailing and particularly in women. In idiopathic forms brachial angiography shows in most of the cases X-ray evidence of digital artery occlusion.


Distribution and mobility of lectin receptors on synaptic membranes of identified neurons in the central nervous system. The distribution and mobility of concanavalin A (Con A) and Ricinus communis agglutinin (RCA) receptors (binding sites) on the external surfaces of Purkinje, hippocampal pyramidal, and granule cells and their attached boutons were studied using ferritin-lectin conjugates. Dendritic fields of these cells were isolated by microdissection and gently homogenized. Cell fragments and pre- and postsynaptic membranes were labeled with the ferritin-lectin conjugates at a variety of temperatures, and the distribution of lectin receptors was determined by electron microscopy. Both classes of these lectin receptors were concentrated at nearly all open and partially open postsynaptic junctional membranes of asymmetric-type synapses on all three neuron types. Con A receptors were most concentrated at the junctional membrane region, indicating that the mature neuron has a specialized nonrandom organization of carbohydrates on its outer surface. Lectin receptors located on postsynaptic junctional membranes appeared to be restricted in their mobility compared to similar classes of receptors on extrajunctional membrane regions. Labeling with ferritin-RCA and -Con A at 37 degrees C produced clustering of lectin receptors on nonjunctional surfaces; however, Con A and RCA receptors retained their nonrandom topographic distribution on the postsynaptic junctional surface. The restricted mobility of lectin receptors was an inherent property of the postsynaptic membrane since the presynaptic membrane was absent. It is proposed that structures in the postsynaptic density may be transmembrane-linked to postsynaptic receptors and thereby determine topographic distribution and limit diffusion of specialized synaptic molecules. Speicalized receptor displays may play an important role in the formation and maintenance of specific synaptic contacts.


Studies on the posterior silk gland of the silkworm Bombyx mori. VI. Distribution of microtubules in the posterior silk gland cells. There are two microtubule systems in the posterior silk gland cells. One is a radial microtubule system in which the microtubules run radially from the basal to the apical cytoplasm and in which fibroin globules (secretory granules of fibroin) and mitochondria are arranged along these microtubules, thus composing a "canal system" which is assumed to be responsible for the intracellular transport of fibroin globules. The other is a circular microtubule system in the apical cytoplasm which is composed of bundles of microtubules and microfilaments running in a circular arrangement around the glandular lumen at an interval of approximately 4 mum at the end of the fifth instar. This system is presumably concerned with secretion and/or intraluminal transport of fibroin.