Effects of hydrostatic pressure on conduction and excitability in rabbit atria. The effects of hydrostatic pressure on cardiac conduction and excitability were studied in 39 rabbit right atrial preparations. Increases in pressure significantly increased atrial conduction time, i.e., 41% at 150 ATA. Excitability, determined from strength-duration (S-D) curves, was depressed by pressure. The slope constant of the S-D curve increased 64% at 150 ATA. Rheobase and the X-asymptote were not affected significantly. The depression of excitability partially accounted for the slowed conduction. Frequency stress and pressure had an additive effect to produce even greater decreases in excitability and increases in conduction time. Atrial refractoriness to premature stimuli also increased as a function of pressure. Nitrous oxide (2.5 ATA) partially reversed the effects of 150 ATA of pressure on conduction time and excitability. The results suggest that pressure acts by altering basic functional components of the myocardial cell membrane. Some of these changes, particularly when combined with frequency stress, i.e., rapid heart rate, may pose a serious threat to humans exposed to hyperbaric environments.


Influence of aspirin and indomethacin on variability of alveolar hypoxic vasoconstriction. Alveolar hypoxia induces pulmonary vasoconstriction but the strength of alveolar hypoxic vasoconstriction (AHV) is variable even within the same species. The influence of aspirin and indomethacin, cyclo-oxygenase inhibitors, was examined in two groups of dogs, those with weak AHV and those with vigorous AHV. A double-lumen endotracheal tube allowed ventilation of one lung with nitrogen as an alveolar hypoxic stimulus and ventilation of the other lung with O2 to maintain systemic oxygenation. Perfusion to each lung was measured with xenon-133 and external counters. In weak reactors both aspirin and indomethacin induced fourfold enhancement of AHV (P less than 0.01), whereas no significant influence on vigorous reactors was noted. Cyclo-oxygenase inhibitors also produced enhanced reactivity in the isolated lung to alveolar hypoxia and prostaglandin F2alpha but not to angiotensin II and norepinephrine. Aspirin-enhanced AHV in the isolated lung could not be diminished with blockade of angiotensin II receptors or of alpha receptors. In summary, weak AHV in intact or isolated dog lung may be due to an excess of a prostaglandin or prostacyclin vasodilator.


Vagal and aerosol histamine interactions on airway responses in dogs. Histamine aerosol was administered to 10 anesthetized paralyzed artificially ventilated dogs whose vagi were first intact, then cut, and then peripherally stimulated. Pulmonary resistance (RL) was measured and dose-response curves determined in the three conditions. The dogs were divided into two groups based on the initial response to histamine with the vagi intact. The low-dose (LD) group had a greater than or equal to 50% increase in RL when exposed to a histamine concentration of 1.0 mg/ml. The high-dose (HD) group had a greater than or equal to 50% increase in RL when exposed to an aerosol containing 3.0 mg/ml histamine or more. In both groups there was a dose-related increase in RL with histamine with the vagi intact, cut, or stimulated. In three of the LD dogs there was a greater than additive interaction between vagal stimulation and inhaled histamine, whereas in the HD dogs the interaction was at most additive. With the vagi cut, both groups had a significantly lesser histamine response. The results show that differences in histamine responsiveness between dogs is in part related to varying degrees of nonreflex histamine-vagal interaction.


Static mechanical properties of bronchi in normal excised human lungs. Bronchographic studies were performed on 22 normal human lungs at transpulmonary pressures between 20 and 0 cmH2O. Pressure-diameter behavior of 322 bronchi was evaluated. In younger (less than 40 yr) subjects, the airways of males showed greater recoil than those of females. No difference between sexes was seen in older (greater than 40 yr) subjects. Bronchi less than 2.1 mm diam had a significant decrease in recoil with increasing age in males but not in females. Small bronchi had greater recoil than large bronchi in both sexes in the younger group but not in the older group. The younger group had evidence of anisotropic behavior of the bronchi not seen in the older group. The percentage decrease in lung volume for a given decrease in transpulmonary pressure was less in the older than in the younger subjects in both sexes. Measurements of the cube root of lung volume, bronchial length, and the position of markers attached to the pleura indicated that the lungs deflated in an overall, isotropic manner down to a pressure of 3 cmH2O.


Body mass, composition, and food intake in rabbits during altered acceleration fields. The effects of gravitational loading and unloading on body mass, body composition, and food intake were investigated in chronically accelerated adult male rabbits. The intensity of the acceleration field was increased by 0.25-G increments at intervals that permitted stabilization of body mass and food intake to a maximal field of 2.5 G. Control rabbits of the same age were maintained at Earth gravity under the same conditions of light and temperature. A negative rectilinear relationship was observed between body mass and the intensity of acceleration field. In spite of this, the decreased body mass appeared to be regulated, since centrifuged animals readily regained their lost body mass upon realimentation after a 3-day fast. At acceleration intensities above 1.75 G, ad libitum food intakes as well as maintenance food requirements increased with increasing acceleration fields despite a corresponding reduction in body mass. Analysis of body composition indicated a significant decrease in body fat and an increase in body water in animals adapted to a 2.5-G environment. Within 6 wk after the animals were returned from 2.5 G to Earth gravity, body mass and food intakes returned to the precentrifuged levels of corresponding control animals.


Energy status of the rat lung after exposure to elevated PO2. To study hyperoxic effects on adenine nucleotide content and lactate and pyruvate production by lungs, rats were exposed to oxygen at 1 ATA for 18, 24, or 48 h or to 4 ATA for 1 h. Subsequently, lungs were removed from rats, placed in an isolated-lung apparatus, ventilated with 5% CO2 in O2, and perfused with Krebs-Ringer bicarbonate medium containing 5.5 mM glucose and 4% bovine serum albumin. Uptake of serotonin from the perfusate was depressed 28% in rats exposed to hyperbaric oxygen compared with unexposed controls. Concentrations of adenine nucleotides, the ATP/ADP ratio, and the "energy charge" were similar in control and oxygen-exposed rats. The production of lactate and the ratio of lactate to pyruvate production were significantly higher in rats exposed to oxygen for 48 h compared with other exposure regimens. Comparison of these results with those previously reported for serotonin uptake in lungs after hyperoxic exposure indicates that serotonin clearance is depressed prior to alteration of the energy status of the rat lung.


Comparison of metabolic and ventilatory responses of men to various lifting tasks and bicycle ergometry. Four male volunteers served as subjects to examine the metabolic and ventilatory cost of both positive and negative lifting tasks as compared to bicycle ergometry. In different experiments, four boxes weighing 0.91, 6.82, 22.73, and 36.36 kg were lifted up to or down from a height of 60 cm at rates as high as 70 lifts/min for periods of 4 min. The data were then compared to those obtained from bicycling at a rate of 50 rpm at work loads up to 1,500 kmp/min. Work at any given box weight had a substantially higher oxygen and ventilatory cost than similar levels of work on the bicycle ergometer. The reason for these differences appeared to lie in the energy cost of moving parts of the body. When the weight of the boxes was low, there was little difference between the oxygen cost of positive and negative work, but as the weight of the boxes increased, the expected physiological differences in positive and negative work was established.


Metabolic, cardiovascular, and respiratory factors in the development of fatigue in lifting tasks. Three well-trained male subjects served as volunteers in these experiments to examine the physiological capacity for extended work during lifting tasks. The maximal oxygen uptake (VO2max) during lifting was always lower than work on the bicycle ergometer. However, the work load during lifting which could be maintained for 1--4 h was 50% of the VO2max for lifting each specific weight of box; the limit for lifting light boxes without fatigue was at an oxygen uptake of about 25% of the VO2max obtained from bicycle ergometry. Significant fatigue in the forearm muscles was found during prolonged lifting as assessed from the endurance of isometric contractions and from the surface electromyogram (EMG), and was more pronounced as the weight of the box increased.


Carbon dioxide--a major determinant of collateral ventilation. The effects of local or systemic CO2 and changes in pulmonary vascular pressure and flow on the mechanics of collateral ventilation were studied in anesthetized, paralyzed dogs. The resistance to collateral ventilation (Rcoll) decreased by 36.1% when the air being infused into the obstructed segment was replaced with 10% CO2 while the animal was ventilated with air. When the air used to ventilate the animals was replaced with 10% CO2 while air was infused into the segment, Rcoll decreased by 38.6%. When blood flow into the pulmonary artery was stopped (stop flow), pulmonary artery and left atrial pressure decreased. Rcoll increased following stop flow to 125% of control; however, the fall in pulmonary vascular pressures preceded the change in Rcoll. The increase in Rcoll with stop flow was markedly reduced when 10% CO2 was infused into the segment. We conclude that collateral channels respond both to the local infusion of CO2 and to the CO2 concentration in the surrounding lung and/or blood, and that the state of distention of pulmonary vessels surrounding collateral ventilatory channels is not a primary determinant of Rcoll. In addition we conclude that bronchiolar channels rather than interalveolar pores are the pathways for collateral ventilation.


Metabolic and cardiovascular adjustment to arm training. Maximal and submaximal metabolic and cardiovascular measures and work capacity were studied in control (n = 7) and experimental (n = 9) subjects (S's) during arm work prior to and following 10 wk of interval arm training. These measures were oxygen uptake (VO2), minute ventilation (VE), heart rate (HR), respiratory exchange ratio (R), cardiac output (Q), stroke volume (SV), and arteriovenous oxygen difference ((a--v)O2 diff). In addition, maximal oxygen uptake (VO2max) was measured in both groups during treadmill running. Experimental S's showed significant increases (P less than 0.01) in peak VO2 (438 ml.min-1), max VE (17.7  l.min-1), max (a--v)O2 diff (20.8 ml.l-1), and work time (9.2 min) during arm ergometry, while maximum values of Q, SV, HR, and R remained unchanged. In addition, submaximal heart rates were significantly lower during arm ergometry after training. VO2max during treadmill running remained essentially unchanged. No changes in metabolic and physiological measures were noted for the controls after the 10-wk training period. The results support the concept of training specificity for VO2max, and indicate that the improvement in peak VO2 in arm ergometry reflects enhanced oxygen utilization due to an expanded (a--v)O2 diff.


Characteristics of inspiratory inhibition by phasic volume feedback in cats. The dependence of phrenic efferent discharge on vagal-volume feedback was examined in barbiturate-anesthetized, paralyzed cats ventilated by a phrenic-driven servo respirator. The characteristics of the respiratory were altered for a single breath, and the resulting change in phrenic activity was quantitated by comparison with phrenic activity without phasic volume feedback. The relation between volume feedback and phrenic inhibition was determined both when inspiratory termination occurred during the rising phase of phrenic discharge and during the plateau observed with barbiturate-induced apneusis. Inhibition of inspiratory activity occurred only when lung volume exceeded a time-dependent threshold. Above this threshold, andextending over a substantial volume range, volume feedback caused graded and reversible inhibition of phrenic discharge. The threshold for graded inhibition declined progressively during the inspiratory phase, showing no obvious relation to the level of inspiratory activity. At any particular time, the relation between volume and phrenic inhibition was convex to the volume axis, and the slope of the relationship increased with inspiratory time. The results indicate that a) volume feedback inhibits inspiration in a graded manner, b) partial inhibition of phrenic activity renders it more susceptible to additional inhibition, and c) inhibitory effectiveness of volume feedback increases with time.


Energy expenditure during simulated rowing. Metabolic function was measured by open-circuit spirometry for 310 competitive oarsmen during and following a 6-min maximal rowing ergometer exercise. Aerobic and anaerobic energy contributions to exercise were estimated by calculating exercise O2 cost and O2 debt.O2 debt was measured for 30 min of recovery using oxygen consumption (Vo2) during light rowing as the base line. Venous blood lactates were analyzed at rest and at 5 and 30 min of recovery. Maximal ventilation volumes ranged from 175 to 22l 1/min while Vo2 max values averaged 5,950 ml/min and 67.6 ml/kg min. Maximal venous blood lactates ranged from 126 to 240 mg/100 ml. Average O2 debt equaled 13.4 liters. The total energy cost for simulated rowing was calculated at 221.5 kcal assuming 5 kcal/l O2 with aerobic metabolism contributing 70% to the total energy released and anaerobiosis providing the remaining 30%. Vo2 values for each minute of exercise reflect a severe steady state since oarsmen work at 96-98% of maximal aerobic capacity. O2 debt and lactate measurements attest to the severity of exercise and dominance of anaerobic metabolism during early stages of work.


Cerebrospinal fluid acid-base balance during muscular exercise. Ventilation, metabolism, arterial blood gases, and blood and cerebrospinal fluid (CSF) acid-base status were measured in exercise studies on seven ponies during mild, moderate, and near-maximal treadmill exercise. CSF and arterial blood were sampled via indwelling catheters. Generally measurements were made during the 3rd, 6th, and 9th minute of steady-state exercise, with CSF sampled only during the 9th minute. Alveolar ventilation (VA) and metabolic rate (VO2) increased proportionately during exercise below the anaerobic threshold, but above this threshold, VA increased at a faster rate than VO2. The similarity of these response to those observed in man suggests the pony is a suitable animal model for study of exercise hyperpnea. No change in CSF acid-base balance occurred with light-to-moderate work; however, with near-maximal work a fall in CSF carbon dioxide partial pressure due to hyperventilation caused CSF to become alkaline (pH = 7.380) relative to rest (pH = 7.330). CSF lactate increased slightly with exercise but had no effect on CSF [HCO3-], which remained constant from rest to severe exercise. We conclude that it is unlikely the hyperpnea at any intensity of exercise results from an increased H+ stimulation at the medullary chemoreceptor.


Atrioventricular bundle degeneration associated with sudden death in the dog. Twelve dogs that were necropsied after sudden unexpected death, sudden episodes of viciousness, or seizure disorder were found to have degeneration of the atrioventricular bundle of the heart. In addition, hypoxic-type degeneration was found in the hippocampus and the dorsal one-half of the midportion of the cerebral cortex of the dogs.


Leiomyosarcoma of the canine urinary bladder, with metastases. An enlarged nodular mass, assumed to be the prostate gland because of location and consistency, was palpated in the pelvic region of a 10-year-old German Shepherd Dog. Castration was performed as a therapeutic procedure after an estrogenic hormone failed to reduce the size of the mass. A tumor identified histologically as a seminoma was in the right testicle. The dog died 1 month after castration, and generalized metastatic neoplasia was observed at necropsy. The mass that involved the neck of the bladder and the metastases were identified as leiomyosarcoma.


Patent ductus arteriosus with pulmonary hypertension in a cat. Patent ductus arteriosus with pulmonary hypertension and right to left shunting of blood flow was diagnosed in a 9-month-old female cat. Because of the pulmonary hypertension, the cat did not have typical signs of patent ductus arteriosus; thus, cardiac angiography and catheterization were utilized to confirm the diagnosis. The patent duct was successfully occluded with a vascular clip.


Right-side torsion of the abomasum in dairy cows: classification of severity and evaluation of outcome. A retrospective analysis was made of 100 cases of surgically corrected right-side torsion of the abomasum (RTA) in dairy cows. A classification for degrees of severity of RTA was developed, based on the amount of fluid sequestered in the abomasum. Serum concentrations of Na+, K+, AND Cl- decreased with increasing severity of RTA. Cows in the most severely affected classification were markedly hypochloremic and hypokalemic. The PCV, plasma protein concentration, and pulse rate increased with increasing severity of RTA. Statistically significant correlations were observed between postsurgical outcome and classifications of RTA, serum Cl- concentration, and pulse rate. The outcome was not statistically related to serum concentrations of Na+ or K+, PCV, or plasma protein concentration.


Oxalate (Rumex venosus) poisoning in cattle. Fifteen range cows died of oxalate poisoning caused by ingestion of Rumex venosus. Ecchymotic and petechial hemorrhages were prominent on the abdominal serosal surfaces. Approximately 2 L of thin, yellowish fluid was in the abdominal cavity, and mesenteric lymph nodes were enlarged and edematous. Other findings included catarrhal abomasitis; enteritis; pale, edematous kidneys; congested lungs; fatty infiltration of the liver; and nephrosis. In the kidneys, birefringent crystals resembling oxalate crystals were demonstrable only when frozen sections were examined.


Blister beetle poisoning in horses.

Blister beetle poisoning in horses. Case records of 21 horses with acute illness following ingestion of hay containing dead striped blister beetles (Epicauta spp) were selected for review. Abdominal pain, fever, depression, frequent urination, shock, and, occasionally, synchronous diaphragmatic flutter characterized clinical illness. Hematologic findings included hemoconcentration, neutrophilic leukocytosis, and hypocalcemia. Hematuria and low urine specific gravity were abnormal urinalysis results. Sloughing of the epithelium of the esophageal part of the stomach, hemorrhagic and ulcerative cystitis, enterocolitis, and myocardial necrosis were important post-mortem findings. Signs and lesions in 5 horses experimentally poisoned were similar to those of the natural disease. The findings were regarded as sufficiently characteristic of blister beetle poisoning to be useful in differential diagnosis but were not constant in all cases. Therefore, when blister beetle poisoning is suspected, access of affected horses to hay containing striped blister beetles should be demonstrated.


Japanese pieris poisoning in the goat.

Japanese pieris poisoning in the goat. One fatal and 1 nonfatal case of Japanese pieris (Pieris japonica) poisoning in goats prompted experimental feeding of the plant at 0.1% of a healthy goat's body weight. Clinical signs observed included colic and nausea. The principal necropsy finding was inhalation pneumonia.


Palatability studies of snail and slug poison baits, using dogs. In 1975, the California Department of Food and Agriculture required registered manufacturers of snail and slug baits to present data showing the unattractiveness of these products to dogs, in order to reduce the number deaths in dogs due to poisoning by eating these baits. A cooperative project involving the University of California, the California Department of Food and Agriculture, and the snail and slug bait manufacturers was designed to assist the manufacturers in assessing the palatability of their products by dogs and to develop criteria for the regulatory agencies to use to ensure that the products they were registering were unattractive to dogs. The studies showed that most recently formulated baits were relatively unpalatable for dogs. A 2-panel consumption testing procedure was developed and is being used by the California Department of Food and Agriculture and the Environmental Protection Agency as criteria for registration. Current reports indicate a dramatic decrease in snail and slug bait poisonings in California after these criteria were established as requirements for registration of snail and slug baits.


An atlas of the dorsal thalamus of the marsupial brush-tailed possum, Trichosurus vulpecula. In several respects the dorsal thalamus of Trichosurus presents a level of organizational complexity considerably in advance of that seen in Didelphis. In particular, such features as the lamination of the dorsal lateral geniculate, the distinct subdivisions of the ventroposterior and lateroposterior complexes, and the more pronounced separation of the ventroanterior from the ventrolateral nucleus, are similar to conditions found in many eutherians. On the other hand, some features which Trichosurus shares with Didelphis, such as the lack of a median central nucleus, and the well developed midline nuclei, are usually said to be indicative of a more 'primitive' level of neural organization. In most instances the thalamic nuclei in Trichosurus are easily homologized with those of Didelphis and other mammals. The difficult areas are the lateral and posterior groups of nuclei, regions which have generally been troublesome in most mammals thus far examined. What does become apparent after this examination of the Trichosurus thalamus is that this representative of the diprotodontid radiation has produced some neurological features which strikingly parallel those of certain advanced eutherians while retaining other features generally considered to be generalized and primitive. Within the metatheria Trichosurus represents a considerable advance over Didelphis. However, as will be seen in later papers in this series, there are other features, particularly those of thalamocortical fibre distribution, which serve to set Trichosurus quite markedly apart from both Didelphis and at least certain eutherian mammals (Haight & Neylon, 1977b, c, 1978).


Fine structure of the stratum intermedium, stellate reticulum, and outer enamel epithelium in the enamel organ of the kitten. Stratum intermedium, stellate reticulum and outer enamel epithelium at the secretion stage in lower second molars of 1 week old kittens were studied with the electron microscope after perfusion fixation. All cell types had a well-developed Golgi apparatus, free ribosomes and little RER. In the stratum intermedium, cytoplasmic processes occasionally contained many vesicles of different types and were connected to neighbouring cells by gap junctions. The number of gap junctions in the stratum intermedium increased greatly with advanced secretion. The cells of the stellate reticulum had large sheet-like cell extensions and surrounded large extracellular spaces. Often, two cell extensions ran parallel to each other, with a narrow extracellular space between them. The narrow spaces were filled with a fluffy material. The outer enamel epithelium showed a smooth basal surface when close to a blood vessel. Facing a larger expanse of connective tissue, the basal surface became folded, the basal lamina formed extended loops into the connective tissue and showed areas of increased density, and cell processes extended through the lamina into the connective tissue. The blood vessels associated with the outer enamel epithelium had many pericytes and resembled post-capillary venules. Macrophages showing vacuoles, aggregations of small vesicles, and peripheral flaps of cytoplasm were present, mainly in the stellate reticulum. These observations are compared with the structure of the human enamel organ, as reported in the literature, and their possible functional significance is briefly discussed.


Time course of 5,6-dihydroxytryptamine neurotoxic effects on fowl diencephalon and upper brain stem monoaminergic pathways. In adult fowls the time-course of the neurotoxic effects of 5,6-DHT on indolaminergic neurons (in comparison with controls and with animals pre-treated with mebanazine, a MAO-inhibitor) was studied. It was shown that 5,6-DHT given into the third cerebral ventricle produced a rather selective degeneration of indolaminergic neurons which was most dramatic 4 and 7 days after the injection. Injured axons characterized by a pile-up of strongly yellow fluorescent material were seen in areas bordering the third ventricle, and in the posterior, medium and anterior hypothalamus, preoptic area, median eminence, n. tuberalis and paleostriatum augmentatum. A decrease in yellow fluorescence in areas distal to the injured axons was only detectable after 19 days. A certain regrowth of some terminal networks was observed 30 days after 5,6-DHT administration. Present data indicate that, in fowls, ascending indolamine neurons mainly originate in tegmental cell bodies and are organized in a large tract which runs along the medial forebrain bundle, sending terminals innervating several diencephalic and other subcortical areas.


Fine structure of the myenteric and submucous plexuses in the stomach of a coral fish, Chelmon rostratus Cuvier. The fine structure of the teleostean myenteric and submucous plexuses has been studied in the stomach of the coral fish, Chelmon rostratus Cuvier. The myenteric plexus is a prominent loose mesh containing nerve cells, myelinated and unmyelinated axon profiles, vesiculated axon profiles, Schwann cells, collagen and capillaries. Unmyelinated axons greatly outnumber the myelinated ones. Vesiculated axon profiles were uncommon, and synapses were rarely observed. Compared with the myenteric plexus, the submucous plexus was more modest in size; it was devoid of nerve cells and myelin sheaths were only occasionally seen.


Cell death during the postnatal morphogenesis of the normal rabbit kidney and in experimental renal polycystosis. We have studied, by means of optic and electron microscopy, the normal and abnormal cell death that takes place during the postnatal morphogenesis of rabbit kidney, and in the experimental renal polycystosis produced by methylprednisolone acetate. In the normal kidney intertubular cell death can be observed during the first 20 days of the postnatal development. However, cell death in the normal metanephric blastema is a very rare event. In the polycystic kidney numerous dead cells can be seen between the third and forty eighth days after injection. The topography and morphology of the dead cells depend on the stage in the evolution of the disease. In the 'stage of renal immaturity', dying and dead cells are present in the nephrogenic tissue, in the dilating collecting tubules and in the intertubular spaces. In this stage the cellular pathology is essentially nuclear. In the stage of tubular cysts, the dead cells are mostly located in the walls of cysts, with some dead cells, but mostly cellular debris in their lumina. At this stage the cellular pathology is basically cytoplasmic. The dead cells are eventually digested by what appear to be phagocytes of tubular epithelial origin. It is suggested that cell death is an important factor in the evolution of the lesions of renal polycystosis induced by corticosteroids, and probably in the initiation of the pathological process as well.


Postnatal development and differentiation of the opossum submandibular gland. The postnatal development and differentiation of the submandibular salivary gland has been examined in sixteen groups of young opossums. At birth the glandular elements, dispersed in loose connective tissue, consist only of ducts that are immature in appearance and of irregular secretory end-pieces. Development occurs in two phases, the first from birth to approximately 31 days postnatum, and the second thereafter. During the first phase the ductular elements show separation into intercalated and intralobular ducts, and attain structural maturity. The larger ducts are concentrated centrally within each lobule and lie in a markedly vascular connective tissue. The secretory end-pieces, initially acinar in form, are lined by proacinar cells which exhibit intercellular canaliculi at the lateral cell membranes and a few dense granules in the apical cytoplasm. During the second phase of development extensive changes occur within the secretory end-pieces, which elongate to form a system of branching tubules. Component cells show an increased granular content, and those in the main body of the tubules differentiate into mucous cells. By 34 cm postnatum the proacinar cells in the bulbous endings of the tubules are replaced by special serous cells possessing intercellular canaliculi and secretory granules which are either electron-lucent or electron-dense. The sequence of changes that occur during postnatal development is discussed and related to possible functional activities. The early development of the ducts may be correlated with their role in homeostasis, while the later development of secretory tubules and the differentiation of secretory cell types may be related to the onset of weaning, and may possibly be induced by this major change in dietary habit.


The surface structure of ageing human articular cartilage: a study by reflected light interference microscopy (RLIM). The surface contours of the weight-bearing area of the left lateral femoral condyle have been examined in vitro in 26 cadavers by reflected light interference microscopy over the age range 0--47 years. The presence of 4 orders of surface irregularity was confirmed. In addition, measurement of the depth, diameter and frequency of the tertiary order of surface hollows showed that while none of these variables was sex-associated, age-related changes in the variables were present. Multiple regression analysis showed that these changes could be considered in terms of periods of maturation (0--21 years) and ageing (22--50 years) of the cartilage. Focal changes in the surface were observed with advancing age. These changes appeared to be regressive and possibly represent an early stage in cartilage fibrillation.


Age changes in the triangular fibrocartilage of the wrist joint. On the basis of a study of 180 wrist joints from 100 fresh cadavers of individuals ranging in age from fetuses to 94 years, it is concluded that the triangular fibro-cartilage is very liable to degenerative alterations associated with ageing. Degeneration begins in the third decade and progressively increases in frequency and severity in subsequent decades. The changes comprise reduced cellularity, loss of elastic fibres, mucoid degeneration of the ground substance, exposure of collagen fibres, fibrillation, erosion, ulceration, abnormal thinning, and, ultimately, disc perforation. The changes are more frequent and more intense on the ulnar surface, and they are always situated in the central part of the disc. It appears that disc perforation is degenerative and age-related: thus there were no perforations in the first two decades of life; in the third there were 7.6%, in the fourth 18.1%, in the fifth 40.0%, in the sixth 42.8%, and in the over sixties 53.1%. There was an associated pattern of degenerative changes in the wrist joint as a whole. The structures adjacent to the articular disc (discal surface of the ulnar head, discal part of the lunate) were much more often involved, and the changes were much more advanced, than on non-discal surfaces. It is argued that this is because of more intensive biomechanical forces, particularly rotational forces, in the disc compartment of the joint.


Structure of amniotic papillae in sheep. The structure of amniotic papillae in sheep was investigated by light transmission and scanning electron microscopy. Papillae were found on the amnion near the umbilical cord in a majority of the sheep examined, from early mid-term onwards. The papillae possessed a basic connective tissue core with a varying degree of vascularity, the whole being sheathed in squamous epithelial cells in the earlier stages of development; but in larger, and presumably older, papillae, squamous epithelium was absent over the tips. The blood supply to these papillae was shown to originate from the chorion and to pass into the amnion at sites where the two members were closely applied to each other. Tentatively we conclude that amniotic papillae are complex organized structures which develop near amniotic plaques, or, in some instances, from the plaques themselves. The stimuli responsible for their growth and development are unknown.


Glycocinnamoylspermidines, a new class of antibiotics. I. Description and fermentation of the organism producing the LL-BM123 antibiotics. The producing organism for the new broad spectrum glycocinnamoylspermidine antibiotics designated LL-BM123beta, gamma1 and gamma2 was characterized as a Nocardia sp. by chemical analysis of the cell wall, growth requirements, morphology and physiological reactions. Fermentation conditions to elaborate and analytical methods to characterize these antibiotics in fermentation filtrates are described.


Glycocinnamoylspermidines, a new class of antibiotics. II. Isolation, physiocochemical and biological properties of LL-BM123beta, gamma1 and gamma2. LL-BM123beta, gamma1 and gamma2 are three new antibiotics produced by fermentation of an unidentified species of Nocardia. These strongly basic, water soluble compounds were isolated from the culture filtrate by CM-Sephadex ion-exchange and carbon chromatography. All three antibiotics are active against both gram-positive and gram-negative bacteria. A mixture of LL-BM123 gamma1 and gamma2 is more active than the beta component but generally less active than gentamicin.


Glycocinnamoylspermidines, a new class of antibiotics. V. Antibacterial evaluation of the isopropyl derivative of LL-BM123gamma. Isopropyl LL-BM123gamma, a novel semisynthetic glycocinnamoylspermidine antibiotic, was active in vitro against both Gram-negative and Gram-positive bacteria with broad spectrum bactericidal activity against clinically important Gram-negative strains. In parallel tests, it was equal to or more potent than reference aminoglycoside antibiotics against Escherichia coli, Proteus, Enterobacter-Klebsiella, Serratia, Salmonella, and Acinetobacter strains. Against clinical isolates of Pseudomonas aeruginosa, isopropyl LL-BM123gamma compared favorably with gentamicin, verdamicin and amikacin but was less potent than tobramycin. Isopropyl LL-BM123gamma was active against many Gram-negative bacteria that were relatively resistant to aminoglycosides. It was rapidly absorbed following subcutaneous administration in mice and showed greater potency than gentamicin on both dosage and plasma concentration bases against several experimental infections.


Globomycin, a new peptide antibiotic with spheroplast-forming activity. I. Taxonomy of producing organisms and fermentation. A new peptide antibiotic, globomycin, was found to be produced by four different strains of the actinomycetes. They were identified as Streptomyces halstedii No. 13912, Streptoverticillium cinnamoneum No. 15037, Streptomyces neohygroscopicus subsp. globomyceticus No. 15631 and Streptomyces hagronensis No. 17834, respectively. Fermentation of globomycin was conducted by conventional submerged culture for antibiotic production, in which 10 microgram/ml of globomycin was produced by cultivation of S. halstedii No. 13912 for 96 hours at 27 degrees C. Globomycin was named after its activity to form global-shape spheroplasts when Escherichia coli was incubated in the presence of this antibiotic.


Globomycin, a new peptide antibiotic with spheroplast-forming activity. III. Structural determination of globomycin. The structure of globomycin has been determined by mass, PMR and CMR spectra and by chemical degradation. Globomycin is a new cyclic peptide antibiotic composed of L-serine, L-allo-threonine, glycine, N-methylleucine, L-allo-isoleucine and 3-hydroxy-2-methylnonaic acid. Among these components, L-allo-threonine, L-allo-isoleucine and 3-hydroxy-2-methylnonaic acid are novel components from natural products. A newly developed mass analysis has been introduced for determining the diastereoisomers of allo-threonine.


Modification of seldomycin factor 5 at C-3'. Attempted removal of the 3'-hydroxyl group of seldomycin factor 5 via displacement of a sulfonate ester has led to 3'-epi-seldomycin factor 5. Removal of the hydroxyl group has been effected by the Barton procedure. The antibacterial activity of 3'-epi- and 3'-deoxyseldomycin factor 5 against various aminoglycoside-resistant strains is discussed.


Tissue pharmacokinetics and inhibition of DNA synthesis in mice treated with sporamycin. Examination of blood and organ concentrations of sporamycin in normal mice showed a rapid decrease of sporamycin from peripheral blood and a high level of sporamycin in urine 10 minutes after intravenous injection of the antibiotic. At the same time, the highest level was found in the kidneys and low levels were found in the lungs and spleen. When sporamycin was added to mouse organ homogenate at 37 degrees C, remarkable inactivation of sporamycin by the homogenate of the liver, kidney, testis, etc., was noted but this inactivation was slight by tumor homogenate. Sporamycin inhibited the tritiated thymidine incorporation into DNA of normal tissues, but a different pattern of inhibition and recovery on the incorporation of 3H-TdR into DNA was observed among mouse organs. It was noted that the antibiotic may damage normal tissues in spite of a rapid excretion and inactivation of sporamycin in mice, but this damage was recovered rapidly within 1 approximately 2 days after the treatment.


Modification of combined migraine-muscle contraction headaches using BVP and EMG feedback. The effect of blood volume pulse (BVP) and frontalis muscle action potential (EMG) feedback on control of vasoconstriction of the temporal artery and frontalis muscle activity in combined migraine-muscle tension subjects was investigated in a multiple baseline design (across subjects and responses). The data indicated: (a) both subjects obtained an ability to control BVP during BVP feedback and EMG during EMG feedback; (b) there were decreases in frequency of migraine headaches during BVP feedback and decreases in muscle contraction headaches during EMG feedback. The results of this study supported the theoretical explanation of two pain mechanisms involved in combined muscle contraction-migraine headaches as well as the effectiveness of bio-feedback procedures that target directly the specific pain mechanism in the elimination of the two types of head pain.


The use of physical restraint in the treatment of self-injury and as positive reinforcement. Two experiments investigated the effects of a treatment package on the self-injurious behavior of three profoundly retarded persons who appeared to enjoy the physical restraints used to prevent their self-injury. The treatment package included physically restraining subjects contingent on increasing periods of time during which no self-injury occurred, and providing them with toys and attention during intervals between restraints. A reversal and multiple-baseline analysis documented that the rapid and complete reduction in self-injury by all subjects was due to this treatment package. Because these results suggested that physical restraint might function as a positive reinforcer, in a third experiment physical restraint was applied contingent on a marble placement response with one subject. A reversal design demonstrated that toy play systematically increased when each response resulted in restraint. The experiments have implications for the nonaversive remediation of self-injury in individuals who are restrained, as well as for the development and maintenance of self-injury in natural settings.


Use of social-skills training in the treatment of extreme anxiety and deficient verbal skills in the job-interview setting. A 30-year-old recent college graduate, exhibiting extreme anxiety and deficient verbal skills in job interviews, was treated with a social-skills training procedure that included instructions, modelling, behavior rehearsal, and videotape feedback. Three target behaviors--focused responses, over coping statements, and subject-generated questions--were presented using a multiple-baseline design. Galvanic skin-response activity was monitored during pre- and posttraining in vivo job interviews. In addition, independent judges unobstrusively rated the subject's social-communicative behaviors in his temporary worksetting before and after training. Training resulted in expected changes for all three target behaviors and a decrease in the rate of speech disturbances. Physiological data supported the subject's report that training enabled him to deal with his anxiety more effectively during job interviews. Training was found to generalize to novel interview questions and different interviewers. Furthermore, unobtrusive measures of eye contact, fluency of speech, appropriateness of verbal content, and composure supported the subject's report that training generalized to his daily social interactions on the job.


Training preschool children to recruit natural communities of reinforcement. Four normal and four deviant children aged four-to-six years were taught to judge the quality of their academic work in a preschool classroom, and to prompt or cue their teachers to comment about the quality of that work. When these skills did not generalize spontaneously to other teachers in concurrent natural situations, generalized responding was taught by the experimenter, in multiple-baseline design across subjects. This generalization programming enabled the children to contact a sometimes dormant, but readily available natural community of teacher praise and reinforcement, i.e., to recruit an increase in cued praise and schedules of praise for their good work. These behaviors may be important to young children who find themselves bereft of attention in classrooms.


National Society for Autistic Children definition of the syndrome of autism. It is helpful for the new student of autism to know the clinical and research basis from which our definition of the syndrome has been evolved. The main features and characteristics are no less meaningful for being recognized as working definitions, subject to alteration with the discovery of new knowledge about causation and treatment. Such research can be most useful, even when samples of autistic children with different specific characteristics are selected. However, it is necessary for the specific characteristics, and their selection purpose, to be identified explicitly. Likewise, the limits of applying the findings on such subsamples to other autistic children should be frankly acknowledged.


Plasma zinc, copper, and amino acid levels in the blood of autistic children. Plasma zinc, copper, and amino acid levels have been measured in a group of autistic children. All three variables were found to be normal. These findings are in disagreement with the previously reported results of some other workers but if confirmed would indicate that autism cannot simply be attributed to a disorder of zinc metabolism.


The cognitive--affective dilemma in early infantile autism: the case of Clarence. This case study presents long-term follow-up data on Clarence, one of Kanner's original cases of infantile autism. Clarence's attempts to establish a heterosexual relationship and his plans for marriage are described. His success is attributed in part to therapeutic intervention, which was directed at promoting affective responsiveness in the patient. The case of Clarence is seen as lending support to the thesis of DesLauriers and Carlson (1969), who maintain that the core problem in infantile autism is a disturbance of affective contact.


In vitro translation of polyadenylate-containing RNAs from dormant and germinating spores of the fungus Botryodiplodia theobromae. Polyadenylated RNA isolated by oligodeoxythymidylate-cellulose chromatography from spores of the fungus Botryodiplodia theobromae was translated in a cell-free protein-synthesizing system derived from wheat embryo. Reaction conditions which would yield efficient and accurate in vitro translation of the spore RNA were established. Dual isotopically labeled mixtures of in vitro translation products from germinated and dormant spore polyadenylated RNA, as well as polyadenylated RNAs from intermediate stages of germination, produced qualitatively similar gel electrophoresis patterns, with polypeptides of 10,000 to 55,000 molecular weight. Proteins synthesized in vivo and extracted from germinating spores at three different stages possessed a greater size range, with molecular weights up to 85,000, although the in vitro synthesis apparently did yield the lower-molecular-weight proteins which were synthesized in vivo. Tryptic digest patterns of proteins translated in vitro from polyadenylated RNA of dormant and germinated spores were found to be identical in positions in only 40% of the spots. Furthermore, a dual-label comparison by isoelectric focusing of proteins translated from polyadenylated RNA of germinated and dormant spores also showed qualitative and quantitative differences among the in vitro translation products. We conclude that there are differences between the in vitro translation products of mRNA from dormant and germinated spores and that the mRNA preserved in the dormant spores contains genetic information which is qualitatively different from that of the germinated spores.


Regulation of cell cycle events in asymmetrically dividing cells: functions required for DNA initiation and chain elongation in Caulobacter crescentus. To study the regulation of cell cycle events after asymmetric cell division in Caulobacter crescentus, we have identified functions that are required for DNA synthesis in the stalked cell produced at division and in the new stalked cell that develops from the swarmer cell 60 min after division. The initiation of DNA synthesis in the two progeny cells is dependent upon at least two common functions. One of these is a requirement for protein synthesis and the other is a gene product identified in a temperature-sensitive cell cycle mutant. DNA chain elongation requires a third common function. The characteristic pattern of DNA synthesis in C. crescentus appears to be controlled in part by the expression of these functions in the two stalked cells at different times after cell division. The age distribution for Caulobacter cells in an exponential population has been calculated (Appendix by Robert Tax) and used to analyze some of the results.


Presence of squalene in gram-positive bacteria. The presence of the isoprenoid squalene, synthesized de novo, was demonstrated in 64 out of 73 strains of gram-positive bacteria by thin-layer chromatography. This observation was confirmed by gas-liquid chromatography, chemical reactivity, incorporation of radiolabeled precursor, and by gas chromatography mass spectroscopy of thin-layer chromatography-recovered material.


Isolation and characterization of a plaque-forming lambda bacteriophage carrying a ColE1 plasmid. A plaque-forming lambdaimm434 bacteriophage carrying the entire genome of colicinogenic factor E1 has been isolated and characterized. This phage, lambdaimm434ColE1, can lysogenize as a stable plasmid within a recombination-deficient Escherichia coli cell that lacks the normal attachment site for lambda phage. Furthermore, it has been found that lambdaimm434ColE1 phage carrying amber mutations in the O and P genes of the lambda genome, i.e., lambdaimm434OamPamColE1, behaves as a plaque-forming phage, and this finding suggests that the ColE1 factor DNA permits replication of the DNA of the plaque-forming phage.


NIC, a conjugative nicotine-nicotinate degradative plasmid in Pseudomonas convexa. The plasmid nature of genes specifying degradation of nicotine and nicotinate in Pseudomas convexa strain 1 (Pc1) is indicated by mitomycin curing and conjugational transfer to other strains. The NIC plasmid appears to be compatible with other metabolic plasmids in Pseudomonas putida.


Purification and properties of an extracellular exonuclease from Thermus thermophilus HB8. An extracellular exonuclease has been found and purified about 10,000-fold from the culture broth of an extreme thermophile, Thermus thermophilus HB8. The enzyme had an isoelectric point at pH 5.1 and seems to be of a multimolecular type, with molecular weights estimated to be ca. 530,000 (Peak I) and around 330,000 (Peak II) by gel filtration. The properties of the most highly purified enzyme fraction, Peak I were investigated. The enzyme requires divalent cations (Mg2+ greater than Sn2+ greater than Ca2+, Mn2+) and is inactive in the presence of EDTA. The pH optimum is 9.4-9.5 in glycine-NaOH buffer and the optimum temperature is 85 degrees C. The rate of hydrolysis increases in the order heat-denatured DNA greater than native DNA greater than RNA. The enzyme hydrolyzes deoxyoligonucleotides bearing 5'-monophosphate to liberate 5'-mononucleotides in an exonucleolytic manner. However, oligonucleotides lacking a 5'-phosphoryl group, irrespective of the presence or absence of phosphate at the 3'-termini, give both 5'-mononucleotides and dinucleoside monophosphates derived from the 5'-termini. It was also found that dinucleotides terminated with a 5'-phosphoryl group were cleaved to 5'-mononucleotides, but dinucleoside monophosphates were resistant to this enzyme. This exonuclease should be useful in the direct determination of sequence at the 5'-terminus and the penultimate position of oligonucleotides by the use of high-performance liquid chromatography.


Rapid purification of threonyl-tRNA synthetase from Saccharomyces carlsbergensis by affinity elution from phosphocellulose. Threonyl-tRNA synthetase [EC 6.1.1.3] of Saccharomyces carlsbergensis was highly purified by a simple enzyme-substrate affinity method. After calcium-gel treatment of the crude enzyme extract, ammonium sulfate precipitation, and removal of polynucleotides with DEAE-cellulose, the enzyme was nonspecifically adsorbed onto phosphocellulose (P-cellulose), and then eluted specifically with a linear concentration gradient of threonine tRNA from torula yeast (Candida (Torulopsis) utilis). The enzyme was purified 303-fold from the calcium-gel supernatant. This purification method is very simple and time-saving. The purified enzyme gave a single band on SDS-gel electrophoresis, indicating a molecular weight of 82,000, and exhibited a molecular weight of about 150,000 by gel filtration. This suggests that the enzyme consists of two identical subunits. Some kinetic properties of the pure enzyme are described.


Methylamine dehydrogenase of Pseudomonas sp. J. Reconstitution from its subunits. Conditions for the restoration of catalytic activity from heavy and light subunits which had been isolated from methylamine dehydrogenase of Pseudomonas sp. J were investigated in vitro. Maximal restoration of the activity was obtained in 0.8 M potassium phosphate buffer, pH 7.0-9.3, at 30 degrees C with equimolar concentrations of the two subunits. Under the optimal conditions, the recovery of enzyme activity was 86% and the time required for half-maximal recovery was about 3 min. Addition of bovine serum albumin or p-chloromercuribenzoate to the incubation mixture had no effect on the rate or extent of recovery of the enzyme activity. When the heavy subunit was added to the light subunit, the absorption spectrum of the light subunit changed to a form similar to that observed for the native enzyme. The concentration of methylamine required to change the spectrum of the light subunit was greatly decreased in the presence of the heavy subunit. Reconstituted enzyme was prepared from the isolated subunits and purified by gel chromatography. The reconstituted enzyme resembled the native enzyme in specific activity, molecular weight, substrate specificity, reaction mechanism, Michaelis constants for methylamine and phenazine methosulfate, susceptibility to inhibitor, and absorption, fluorescence and ESR spectra. However, it was less stable than the native enzyme to thermal and pH treatments. The CD spectrum was also slightly different from that of the native enzyme.


Methylamine dehydrogenase of Pseudomonas AM1. A subunit enzyme. A methylamine dehydrogenase was purified to homogeneity from Pseudomonas AM1 and obtained in crystalline form. It was found to be a subunit enzyme composed of two kinds of subunit, light and heavy. These two subunits were isolated by Sephadex G-100 gel chromatography after incubation of the enzyme with guanidine hydrochloride. Molecular weights of 13,000 daltons for the light subunit and 40,000 daltons for the heavy subunit were estimated by SDS-polyacrylamide gel electrophoresis, and the molecular weight of the native enzyme was found to be 105,000 daltons by Sephadex G-200 gel chromatography. The enzyme and its subunits were also analyzed for amino acid composition, isoelectric point, and absorption fluorescence, and CD spectra, as well as for the effects of pH, thermal treatment, and guanidine hydrochloride treatment. Both the subunits were absolutely required for enzymatic activity, either subunit alone being inactive. It could be deduced from the absorption and fluorescence spectra of the subunits that the prosthetic group of the enzyme was bound solely to the light subunit. These results suggest that the enzyme is a subunit enzyme similar to that of Pseudomonas sp. J, of the alpha2beta2 type.


The presence of ribosomal glycoproteins. Agglutination of free and membrane-bound ribosomes from wheat germ by concanavalin A1. Free and membrane-bound ribosomes prepared from wheat germ and extensively washed with 1% Triton X100-sodium deoxycholate, formed large aggregates in the presence of concanavalin A. The ribosomal aggregates were detected by sucrose density gradient centrifugation and immunoprecipitation. SDS-polyacrylamide gel electrophoresis of ribosomal proteins indicated that four ribosomal glycoproteins (MW about 14,000 to 24,000) were present in both free and membrane-bound ribosomes. However, three glycoproteins (MW about 25,000 to 15,000) appear to be underglycosylated or lacking in free ribosomes.


Studies on electron transfer systems in the marine diatom Phaeodactylum tricornutum. I. Isolation and characterization of cytochromes. Two cytochromes of the C type, c-550 and c-553, were isolated from the marine diatom, Phaeodactylum tricornutum, and purified by ammonium sulfate fractionation and DEAE-cellulose column chromatography. The cytochrome c-550 had absorption maxima at 550, 522, and 417 nm in the reduced form and at 524, 407, 351, and 277 nm in the oxidized form. It was an autoxidizable acidic protein with an isoelectric point of 5.1 and had a low redox potential of about -0.20 V at pH 7.0. The molecular weight of this cytochrome was close to 17,000. This cytochrome combined with CO and CN-. The CO complex was dissociated reversibly by light. The cytochrome c-553 had absorption maxima at 553, 522.5, and 417 nm in the reduced form and at 528, 410, and 356 nm in the oxidized form. The protein had an acidic isoelectric point of 3.7 and had a high mid-point redox potential of +0.36 V at pH 7.0. Its molecular weight was approximately 10,500. The cytochrome may be considered to be a photosynthetic cytochrome of the f type. Cytochromes of the B type were also found in Phaeodactylum tricornutum; one in soluble form, and the other in bound form. The soluble form had absorption maxima at 560, 529, and 427 nm in the reduced state and at 413 nm in the oxidized state.


Studies on electron transfer systems in the marine diatom Phaeodactylum tricornutum. II. Identification and determination of quinones, cytochromes, and flavins. Quinones constituting the electron transfer systems in a marine unicellular diatom, Phaeodactylum tricornutum, were isolated and identified chromatographically. The alga contained five quinones, i.e., plastoquinone A, plastoquinone C, plastoquinone D, alpha-tocopherylquinone, and ubiquinone-9. Other types of quinones, such as vitamin K1, were not detected. The contents of plastoquinone A, plastoquinone C, plastoquinone D, ubiquinone-9, and alpha-tocopherylquinone were 25.5, 4.95, 1.99, 4.78, and 0.28 mmol per mol of chlorophyll, respectively. The contents of the soluble C-type cytochromes, cytochrome c-550 and cytochrome c-553, were 2.15 and 4.34 mmol (heme basis) per mol of chlorophyll, respectively. The amount of B-type cytochrome in the bound form was estimated to be 3.24 mmol (heme basis) per mol of chlorophyll. The acid-soluble flavins, FAD and FMN, were present in amounts of 0.68 and 0.41 mmol per mol of chlorophyll, respectively.


Complete amino acid sequence of Halobacterium halobium ferredoxin containing an Nepsilon-acetyllysine residue. 1. The complete amino acid sequence of the 2Fe-2S ferredoxin from Halobacterium halobium was determined to be: (formula see text):2. The apoferredoxin chain consists of 128 amino acid residues and has a molecular weight of 14,330. 3. There are only four cysteines in this ferredoxin molecule; they should be involved in the binding of the two iron atoms at the active center. Ther relative positions of these cysteines are similar to those of the cysteines in chloroplast ferredoxins. 4. There is a high degree of homology between H. halobium ferredoxin and chloroplast ferredoxins, though the latter molecules contain only about 98 amino acid residues. 5. H. halobium ferredoxin contains a single residue of Nepsilon-acetyllysine.


Membrane properties of an extreme thermophile. I. Detection of the phase transition and its dependence on growth temperature. Phase transition was detected by a fluorescence polarization technique in the membrane of Thermus thermophilus HB8: it was found to be a function of cell growth temperature when the cell growth temperature was varied between 50-80 degrees C. A systematic relation between the phase transition temperature and the growth temperature was observed. Differential scanning calorimetry was also applied. The phase transition was found to take place between 34 and 55 degrees C for cells grown at 50 degrees C and between 52 and 80 degrees C for cells grown at 80 degrees C.


Membrane properties of an extreme thermophile. II. Membrane functions underlying leucine transport and their relation with thermotropic phase transitions. Various membrane properties of Thermus thermophilus HB8 were studied in order to elucidate the mechanism and implication of cell adaptation to a high temperature environment. 1. The profile of temperature dependence of the amino acid uptake rates was similar for the membrane obtained from cells grown at both 61 degrees C and 77 degrees C. 2. The remaining uptake activity after heat treatment paralleled the membrane potential generating ability, as measured by auramine O fluorescence response to the respiration substrate, and was not dependent on the growth temperature. 3. The temperature dependence of the efflux of the accumulated amino acid reflects the physical phase of the membrane and is dependent on the growth temperature.


On the validity of the spectrophotometric determination of oxygen saturation of hemoglobin. The wavelength dependence of observed oxygen equilibrium parameter values. Spectral changes of oxyhemoglobin induced by such anions as 2,3-diphosphoglycerate, inositol hexaphosphate, and Cl- may affect the validity of the spectrophotometric determination of oxygen saturation of hemoglobin. Therefore, the anion-induced difference spectra were extensively measured under a variety of conditions and accurate oxygen equilibrium curves were determined under representative conditions with detection at different wavelengths selected from peaks, troughs, and zero difference points of the difference spectra in the visible and Soret regions. Oxygen equilibrium parameters including the four Adair constants (i.e., equilibrium constants for four steps of oxygenation) estimated from the equilibrium curves did not show any dependence on wavelength within the limits of experimental error. These results indicate that anion-induced spectral changes do not invalidate the spectrophotometric determination of oxygen saturation and confirm the validity of the previous conclusions drawn in our series of studies on the effects of anions, pH and temperature on oxygen equilibrium parameters.


Trypsin reactivity site of the Vicia angustifolia proteinase inhibitor. Trypsin [EC 3.4.21.4] modified (reactive site cleaved) Vicia angustifolia proteinase inhibitor was prepared at pH 3 with a catalytic amount of trypsin and purified using columns of Sephadex G-50 and DEAE-Sephadex A-25. The modified inhibitor, which still retained antitryptic activity, lost its activity upon treatment with carboxypeptidase B or citraconic anhydride. End-group analyses revealed that the carboxyl-terminal Arg and the amino-terminal Ser residues were newly exposed end-groups in the modified inhibitor. It takes a much longer incubation time (about 1 h) to exhibit the maximal inhibitory activity against trypsin. Reduction and carboxymethylation of the modified inhibitor produced two fragments on Sephadex G-50 chromatography. The smaller fragment consisted of about 32 amino acid residues and possessed a new carboxyl-terminal Arg residue. The larger fragment consisted of about 80 residues and possessed a Ser residue at its amino-terminus. These results indicate that the small fragment was derived from the amino-terminal portion of the modified inhibitor and the large fragment from the carboxyl-terminal. It is also concluded that an Arg-Ser bond is the reactive site as well as the inhibitory site of the V. angustifolia inhibitor against trypsin. The sequence around the antitryptic site exhibits high degrees of homology with other double-headed inhibitors of legume origin, such as the Bowman-Birk inhibitor, lima beam inhibitor, and the major inhibitor in chick-peas.


Divalent cation-dependent stereospecificity of adenosine 5'-O-(2-thiotriphosphate) in the hexokinase and pyruvate kinase reactions. The absolute stereochemistry of the diastereoisomers of adenosine 5'-O-(2-thiotriphosphate). 31P NMR studies with Cd(II) and Zn(II) chelates of adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) and the Cd(II) chelate of adenosine 5'-O-(2-thiotriphosphate) (ATPbetaS) indicate that these metal ions chelate to the sulfur atom of the thiophosphate group. Since Mg(II) chelates to oxygen of the thiophosphate group of diastereoisomer is equivalent to the configuration of the Cd(II) chelate of the opposite diastereoisomer. As a consequence, an inversion of the stereospecificity is observed when Cd(II) is substituted for Mg(II) in the phosphoryl transfer reactions catalyzed by yeast hexokinase and rabbit muscle pyruvate kinase. When Co(II) is the activating ion for yeast hexokinase with ATPbetaS as substrate, no stereospecificity is observed. Since the absolute configuration for the diastereoisomer of Co(III)(NH3)4ATP which is the active substrate for yeast hexokinase has been established by Cornelius and Cleland (Cornelius, R. D., and Cleland, W. W. (1978) Biochemistry, in press), the absolute stereochemistry of the Mg(II) complex of the B isomer of ATPbetaS is now established by its stereospecificity in the hexokinase reaction.


Effect of pyridoxal phosphate on the DNA binding site of activated hepatic glucocorticoid receptor. The binding of rat liver glucocorticoid.receptor complexes to DNA-cellulose and nuclei has been studied after activation of the complexes by heating. Subsequent exposure to pyridoxal 5'-phosphate or pyridoxal markedly inhibited this binding. In one system 0.75 mM pyridoxal 5'-phosphate or 6.5 mM pyridoxal gave 50% inhibition. Pyridoxamine 5'-phosphate, pyridoxamine, and pyridoxine did not inhibit significantly. The inhibition by pyridoxal 5'-phosphate is competitive with respect to DNA suggesting that its effect is directly on the DNA binding site of the activated receptor. The inhibition of DNA-cellulose binding by pyridoxal 5'-phosphate can be reversed by treatment with dithiothreitol or by gel filtration, but not if the modified receptor is first reduced using sodium borohydride. These results suggest that pyridoxal 5'-phosphate acts by forming a Schiff base of an epsilon-NH2 of a lysine which may be 1 residue appearing on the surface of the steroid.receptor complex upon activation. However, since pretreatment of the DNA-cellulose with the intercalating drug ethidium bormide also inhibits activated receptor binding, we conclude that the binding of the receptor involves more than electrostatic interactions between receptor positive charges and DNA phosphate groups.


Cytochalasin B binding proteins in human erythrocyte membranes. Modulation of glucose sensitivity by site interaction and partial solubilization of binding activities. We have previously described three different cytochalasin B binding sites in human erythrocyte membranes, a D-glucose-sensitive site (Site I), a cytochalasin E-sensitive site (Site II), and a site (Site III) insensitive to both D-glucose and cytochalasin E. Ligand bindings to each of these sites were considered to be independent (Jung, C., and Rampal, A. (1977) J. Biol. Chem. 252, 5456-5463). However, we have obtained subsequently the following evidence which indicated that an interaction occurs between Sites II and III, and this modulates sensitivity of Site III to the sugar. The displacement of cytochalasin E greatly exceeds the sum of their independent displacements. This ghosts extracted with EDTA or 2,3-dimethylmaleic anhydride at low ionic strength lack Site II activity but retain Site I and III activities, and both of these activities are displaceable by D-glucose alone. This indicated that the removal of Site II from the membrane confers glucose sensitivity to Site III. These observations are consistent with a model that Sites II and III in the membrane exist in a close association through which unliganded Site II maintains the glucose insensitivity of Site III, and once site II is liganded or removed by extraction this association is disrupted and Site III becomes glucose-sensitive. The ghosts extracted with Triton X-100 retain a cytochalasin B binding activity similar to that of site II (Kd = 1.8 X 10(-7) M, cytochalasin E-sensitive, glucose-insensitive), whereas a binding activity similar to that of Site I (Kd = 4 X 10(-7) M, cytochalasin E-insensitive, glucose-sensitive) is recovered in the Triton extract. A cytochalasin B binding activity similar to that of Site II is solubilized by EDTA at low ionic strength.


Effect of a short term fast on the distribution of cytoplasmic albumin messenger ribonucleic acid in rat liver. Evidence for formation of free albumin messenger ribonucleoprotein particles. Recently, using molecular hybridization techniques with albumin [3H]cDNA, we have determined that in normally fed rats 98% of total liver polyribosomal albumin mRNA sequences are found in membrane-bound polyribosomes (Yap, S. H., Strair, R. K., and Shafritz, D. A. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5397-5401). We now observe that a 24- to 30-h withdrawal of food leads to major changes in the amount and subcellular distribution of albumin mRNA molecules. The total amount of cytoplasmic albumin mRNA per liver and concentration of albumin mRNA per unit of membrane-bound polyribosomal RNA are decreased. However, the proportion of albumin mRNA present in the postribosomal supernatant fraction increases dramatically in a short term fast, so that it now represent 60% of total cytoplasmic albumin mRNA sequences. Most of the albumin mRNA sequences in the postribosomal supernatant fraction sediment between 30 S and 50 S. These findings suggest that albumin mRNA is probably stored in the messenger ribonucleoprotein fraction during the fasting state.


The presence of guanosine 5'-diphospho-5'-guanosine and guanosine 5'-triphospho-5'-adenosine in brine shrimp embryos. Acid-soluble extracts of dormant embryos of the brine shrimp, Artemia salina, contain small amounts of two previously undescribed dinucleotides which we have identified to be guanosine 5'-diphospho-5'-guanosine and guanosine 5'-triphospho-5'-adenosine. These compounds each comprise about 0.03% of the dry weight of the encysted embryos and are related chemically to guanosine 5'-triphospho-5'-guanosine and guanosine 5'-tetraphospho-5'-guanosine which have been shown previously to be major constituents of the nucleotide pool of Artemia cysts. These new dinucleotides were purified from perchloric acid extracts of dormant cysts by ion exchange column chromatography and identified by means of chemical, spectrophotometric, and enzymatic analyses compared to commercially available compounds. The possible role of these new compounds in nucleotide and nucleic acid metabolism in Artemia embryos is discussed.


Complete amino acid sequence of bovine neurophysin-I. A major secretory product of the posterior pituitary. Bovine neurophysin-I (bNP-I) is the first neurophysin protein which contains histidine and possesses an acidic COOH-terminal segment for which the complete amino acid sequence is presented: NH2-Ala-Val-Leu-Asp-Leu-Asp-Val-Arg-Thr-Cys-Leu-Pro-Cys-Gly-Pro-Gly-Gly-Lys-Gly-Arg-Cys-Phe-Gly-Pro-Ser-Ile-Cys-Cys-Gly-Asp-Glu-Leu-Gly-Cys-Phe-Val-Gly-Thr-Ala-Glu-Ala-Leu-Arg- Cys-Gln-Glu-Glu-Asn-Tyr-Leu-Pro-Ser-Pro-Cys-Gln-SerGly-Gln-Lys-Pro-Cys-Gly-Ser- Gly-Gly-Arg-Cys-Ala-Ala-Ala-Gly-Ile-Cys-Cys-Ser-Pro-Asp-Gly-Cys-His-Glu-Asp-Pro-Ala-Cys-Asp-Pro-Glu-Ala-Ala-Phe-Ser-Leu-COOH. Determination of the structure was greatly facilitated by new procedures used for the isolation of bNP-I and of its tryptic peptide fragments. bNP-I isolated from freshly frozen bovine posterior pituitaries is composed of 93 residues, but some preparations contain neurophysin protein with NH2- and COOH-terminal truncated sequences. bNP-I differs from bovine neurophysin-II, the second major neurophysin of cow, in 20 residue positions, and several of the differences cannot be accounted for by single nucleotide replacements in the genes coding for these two neurophysin proteins. The results reported in this study support our earlier hypothesis that neurophysin-gene duplication preceded species divergence.


HeLa cell RNA (2'-O-methyladenosine-N6-)-methyltransferase specific for the capped 5'-end of messenger RNA. A novel enzyme involved in the post-transcriptional modification of the 5'-end of mRNA has been partially purified from HeLa cells. Termed an S-adenosyl-L-methionine:RNA(2'-O-methyladenosine-N4)-methyltransferase, the enzyme specifically catalyzes the transfer of a methyl group from S-adenosylmethionine to the N6 position of a 2'-O-methyladenosine residue located within the "capped" 5'-end of mRNA. The dimethylated nucleoside, N6,2'-O-dimethyladenosine, is formed as indicated by the following reaction in which m7G(5')pppAm- represents the capped and methylated 5'-end of mRNA: AdoMet + m7G(5')pppAm- leads to AdoHcy + m7G(5')pppm6A7- Greatest activity is obtained with RNA acceptors ending in m7G(5')pppAm-; less activity is found with RNA ending in m7G(5')pppA-; and barely detectable and no activity is found with RNA ending in G(5')pppA- and ppA-, respectively. Furthermore, no activity is found with oligonucleotides such as m7G(5')pppA, m7G(5')pppAm, and m7G(5')pppAmpN indicating that a longer polymer is required. It can be concluded from the substrate specificity of the enzyme that the formation of N6,2'-O-dimethyladenosine follows the biosynthesis of molecules containing m7G(5')pppAm-N-. The molecular weight of the methyltransferase, as determined by sedimentation in sucrose gradients, is approximately 65,000.


Effect of methylation of the N6 position of the penultimate adenosine of capped mRNA on ribosome binding. An RNA(2'-O-methyladenosine-N6)-methyltransferase isolated from HeLa cells was used to convert the ends of vaccinia virus mRNAs containing m7G(5')pppAm-to m7G(5')pppm6Am-. Under competitive conditions, there was no preferential binding of mRNAs containing m6Am residues within the capped ends relative to those containing Am either to wheat germ ribosomes at 70 mM potassium acetate or to reticulocyte ribosomes at 120 mM potassium acetate. Only at high concentrations of potassium acetate (200 mM) and at near-saturating mRNA concentrations, was a slight enrichment of mRNAs containing m6Am over those containing Am detected on reticulocyte ribosomes. It appears that unlike methylation of the N7 position of the terminal guanosine, methylation at the N6 position of the penultimate adenosine of mRNA has, at most, a small effect on binding to ribosomes under present in vitro conditions.


Purification of mouse L cell interferon. Essentially pure preparations with associated cell growth inhibitory activity. Mouse L cell interferon induced by Newcastle disease virus was purified by a combination of salt precipitation, ion exchange chromatography, gel filtration, and affinity chromatography using anti-interferon anti-body. The preparation was labeled with 125I at a later step of purification, which served as an index of protein concentration. Two main species of interferon molecules differing in molecular weight were separated from each other, and the final preparations obtained were shown to be essentially pure by polyacrylamide gel electrophoresis in the absence as well as in the presence of sodium dodecyl sulfate. The highest specific activities obtained were 2.6 X 10(9) and 7.3 X 10(8) international units/mg of protein (determined by 125I radioactivity) for the 40,000- and 24,000-dalton species, respectively. The preparations at different steps of purification, including those of the highest purity, were tested for the anti-cell growth activity. Their activities were found to be indistinguishable from each other when compared at the same antiviral doses, indicating that the anti-cell growth activity is carried by the interferon molecules themselves.


Stimulating effect of hydrocortisone on the catabolism of endogenous fatty acyl groups by fatty acid-supplemented mouse L fibroblasts. Hydrocortisone stimulated the catabolism of prelabeled fatty acyls in mouse L fibroblasts supplemented with exogenous fatty acid. Both oxidation to 14CO2 and release as free fatty acid from prelabeled lipids increased up to 20-fold under the described experimental conditions. The stimulating effect of hydrocortisone was observed even at concentrations as low as 1 microgram/ml. Neither the hormone nor exogenous fatty acid alone had a significant effect on endogenous fatty acyl catabolism. The activity of a cellular pH 6.5 lipase was unaffected by exogenous fatty acid, slightly increased by hydrocortisone, but increased 2-fold when both supplements were present. It is suggested that since exogenous fatty acids shunt phospholipid acyls to triglycerides, the latter induce the formation of more pH 6.5 lipase. Hydrocortisone is either needed for this induction to occur, or activates the newly formed enzyme. The lipase acts on the triglycerides to liberate free fatty acids that are then oxidized to 14CO2 or lost into the medium.


Exploring the action and specificity of cobra venom phospholipase A2 toward human erythrocytes, ghost membranes, and lipid mixtures. We have investigated the action and substrate specificity of phospholipase A2 (EC 3.1.1.4) purified from cobra venom (Naja naja naja) toward intact and Triton-solubilized human erythrocytes, toward ghost membranes, and toward extracted ghost lipids in mixed micelles with Triton X-100. We have found that: (i) phospholipids in the outer surface of intact erythrocytes are extremely poor substrates for the phospholipase, (ii) phospholipids in ghost erythrocyte membranes and in Triton-solubilized erythrocytes are suitable substrates for the enzyme, (iii) in these latter systems which contain a mixture of lipids, phosphatidylethanolamine is preferentially hydrolyzed, whereas in model studies on individual phospholipid species in mixed micelles with Triton, phosphatidylcholine is the preferred substrate of the enzyme, and (iv) the preferential hydrolysis of phosphatidylethanolamine is also observed for extracted ghost lipid mixtures in mixed micelles. These results demonstrate a dependence of phospholipase A2 activity on the ghosting procedure and a dependence of substrate specificity on the presence of other lipids. The relevance of these findings to the interpretation of membrane lipid asymmetry studies utilizing phospholipases is considered in detail.


Purification and in vitro labeling of interferon from a human fibroblastoid cell line. Interferon was produced from a clonal human fibroblastoid line. This cell line was derived from an established fibroblastoid culture treated with ethylmethane sulfonate and is capable of producing higher amounts human interferon than primary human fibroblast cultures. The interferon produced from this cell line was purified by concanavalin A and phenyl-Sepharose column chromatography followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified interferon was then labeled with [125I]-iodine, with [3H]5-dimethylaminonaphthalene-1-sulfonyl-chloride and with sodium [3H]borohydride after periodate oxidation. An appreciable amount of biological activity was retained after both tritium labelings but not after iodination. When subjected to electrophoresis in the presence of sodium dodecyl sulfate all three radioactive interferon preparations comigrated with the purified interferon preparation as a single protein component with a molecular weight of 19,000. Both purified and radioactive preparations were also shown to migrate with the antiviral activity in polyacrylamide gels in the absence of sodium dodecyl sulfate. The specific activity of purified interferon preparations ranged from 2 X 10(8) to 10 X 10(8) units/mg of protein.


Folding of 140-base pair length DNA by a core of arginine-rich histones. The nucleoprotein complex obtained by reconstitution of arginine-rich histones with 140-base pair length DNA has properties considerably closer to native core particles than the complex obtained with lysine-rich histones. A tetramer of arginine-rich histones folds 140-base pair length DNA into a particle (R body) with identical projections, on high resolution electron micrographs, as native core particles. The R body is more spherical in shape than the native nucleosome core particle. Both arginine- and lysine-rich histones contribute to the altered thermal stability and circular dichroism spectra of the core particle DNA.


Cross-referencing testis-specific nuclear proteins by two-dimensional gel electrophoresis. Discontinuous sodium sodecyl sulfate-gel electrophoresis combined with acid-urea gel electrophoresis reveals that both testis histone H1 classes TH1-X (Branson, R. E., Grimes, S. R., Jr., Yonuschot, G., and Irvin, J. L (1975) Arch. Biochem. Biophys. 168, 403-412) and H1 contain two polypeptides each. Migration properties and relative staining intensities of the four H1 histone proteins in testis support the following conclusions. 1. Two testis-specific forms become the major H1 components at some stage of spermatogenesis. 2. During this stage they assume structural and functional role analogous to those of their two somatic counterparts. We have also detected a new testis-specific protein containing cysteine.


Characterization of phosphate oxygen exchange reactions catalyzed by myosin through measurement of the distribution of 18-O-labeled species. The change in the distribution of the phosphate species containing 0 to 4 18O oxygens per Pi was investigated during medium Pi equilibrium HOH exchange catalyzed by myosin subfragment 1. At 25 degrees C, a Pi molecule once bound loses an average of 3.9 of its original 4 oxygens prior to release which means that at least 100 reversals of the exchange reaction must have occurred. At 0 degrees C, only 3.4 of the 4 oxygens are lost prior to release indicating an average of 17 reversals. Distribution patterns are consistent with equivalent participation in the exchange reactions of all 4 oxygens of bound Pi. The intermediate exchange of Pi oxygens during hydrolysis of 18O-labeled ATP by myosin has also been investigated. The distribution of the product Pi species shows that there is an ATPase component in myosin preparations which hydrolyzes ATP without intermediate exchange. Presence of this component, which is likely a contaminating ATPase, provides a simple explanation of the apparent nonequivalence of phosphate oxygens which has been observed. When correction is made for this contaminant, characteristics of the myosin intermediate Pi equilibrium HOH exchange are similar to those of myosin subfragment 1 medium exchange, and intermediate exchange data are in much closer agreement with other kinetic measurements.


Dansylaziridine-labeled troponin C. A new fluorescent "physiological" probe for calcium regulation by sarcoplasmic reticulum. Dansylaziridine-labeled troponin C (TnCDANZ) undergoes a greater than 2-fold fluorescence enhancement with Ca2+ binding to the Ca2+-specific regulatory sites of troponin C. Hence, TnCDANZ serves as a very good indicator of Ca2+ in the range of 3.0 to 70 micron. Ca2+ uptake and release by sarcoplasmic reticulum can be easily and accurately monitored by the fluorescence changes in TnCDANZ. In this manner, we can monitor directly the removal of Ca2+ from troponin C by the sarcoplasmic reticulum. Thus, this reaction represents a useful analogue ot the Ca2+ "on-off" process in muscle.


Crystalline enzyme.substrate complexes of asparate aminotransferase. Crystalline complexes of cytoplasmic aspartate aminotransferase of pig heart with the substrates L-glutamate and L-aspartate, and with other amino acids, have been prepared and polarized light absorption spectra have been measured. Striking differences in the directions of polarization of the absorption bands are seen. A complete half-transamination of pyridoxal phosphate to pyridoxamine phosphate by aspartate or by cysteine sulfinate can be demonstrated in the crystal as can the accumulation of a quinonoid intermediate with erythro-beta-hydroxyaspartate. X-ray diffraction studies show that the crystals with erythro-beta-hydroxyaspartate and alpha-methylaspartate are isomorphous with those of both alpha and beta subforms of the native enzyme.


Impairment of dolichyl saccharide synthesis and dolichol-mediated glycoprotein assembly in the aortic smooth muscle cell in culture by inhibitors of cholesterol biosynthesis. 25-Hydroxycholesterol treatment of the aortic smooth muscle cell inhibited the incorporation of acetate but not mevalonate into dolichol and cholesterol by 91 and 82%, respectively, and diminished the synthesis from glucose of cholesterol, dolichylpyrophosphoryl oligosaccharide, and dolichol-dependent glycoproteins. The dolichol-bound oligosaccharide unit contained approximately 10 Man/2 Glc/2 GlcNAc residues and appeared to be a precursor to protein-bound saccharide units which contained on the average 8 Man/1 Glc/2 GlcNAc residues. Mevalonate was found to protect the cells against the effect 25-hydroxycholesterol and to restore normal cellular synthesis of dolichyl saccharides and glycoproteins. It is suggested that hydroxymethylglutaryl coenzyme A reductase may function as a rate-controlling enzyme in the biosynthesis of not only sterols but also dolichols, and may as a result regulate the assembly of certain cellular glycoproteins.


Disassociation between acidinsoluble acylcarnitines and ketogenesis following carnitine administration in vivo. 1-Carnitine was administered to fed rats and the changes in plasma beta-hydroxybutrate concentration and liver acid-insoluble acylcarnitine content were assessed. One hour following injection of carnitine in doses greater than 1 mumol/100 g of body weight there was a dose-dependent increase in liver acid-insoluble acylcarnitine content to levels comparable to those seen in fasting. These increased levels were maintained for a least 2 h following injection. During the period following carnitine administration there was no increase in ketogenesis as evidenced by plasma beta-hydroxybutyrate concentrations. Since acid-insoluble acylcarnitines represent the product of carnitine palmitoyltransferase A, the results are interpreted as contradictory to the theory that this enzyme is rate-limiting and regulatory for ketogenesis.


Soluble adenylate cyclase from thyroid membranes. Adenylate cyclase from purified beef thyroid membranes has been solubilized by the use of Triton N-101 after preactivation with guanosine 5'-(beta, gamma-imido)-triphosphate. The soluble activity passed a 0.22- micron filter, was not sedimented at 100,000 X g for 2 h, and behaved like aldolase in sucrose density gradients and on Sepharose 6B. From comparison of the sedimentation in D2O and H2O the partial specific volume was found to be like that of globular proteins (0.75 +/- 0.006), hence little detergent appeared to be bound to the enzyme. The sedimentation coefficient was 7.4 +/- 0.15, the Stokes radius 45 A, and the molecular weight 159,000. Prestimulation by thyrotropin did not survive solubilization. The stimulation produced by guanosine 5'-(beta, gamma-imido)triphosphate persisted as did the more active state resulting from pretreatment with both this nucleotide plus thyrotropin. Thyrotropin did not stimulate the solubilized enzyme. The Km for ATP, thermal stability, and inhibition by Ca2+ were identical for the membrane-bound and soluble enzyme, while the pH optimum was increased 0.5 unit in the latter. Polyanions and phenothiazines inhibited both preparations equally, whereas only membranes responded to stimulation by polylysine and ribonuclease.


Sequence of the COOH-terminal hydrophilic region of histocompatibility antigens HLA-A2 and HLA-B7. Detergent-solubilized HLA antigens were isolated from a human lymphoblastoid cell using an anti-beta2-microglobulin immunoaffinity column. The HLA-A and HLA-B locus products were separated by thin layer isoelectric focusing. Cleavage of the p44 chain of HLA-A2 and -B7 antigens with cyanogen bromide led to the isolation of a 31-amino-acid fragment from each. The fragments were sequenced and shown to be from the COOH-terminal end of the intact chains using carboxypeptidase Y. The fragment from the HLA-B7 chain, 55% of whose amino acids were polar, contained the 2 cysteine residues not found in the papain-derived molecule. The tentative sequence of the fragment from the HLA-A2 chain was similar to that of the HLA-B7 fragment but appeared not to contain any cysteine residues. The hydrophilic COOH-terminal region of HLA antigens, which directly follows the hydrophobic, membrane-binding segment, began with a cluster of basic amino acids. This arrangement of amino acids resembles that found at the COOH terminus of the red blood cell membrane protein, glycophorin.


Isolation and amino acid sequence of the alpha-subunit of follicle-stimulating hormone from equine pituitary glands. Six hundred milligrams of follicle-stimulating hormone (FSH), containing 110 NIH-FSH-S1 units/mg, was isolated from 9 kg of equine pituitary glands. The equine FSH was dissociated into alpha- and beta-subunits. A tentative amino acid sequence of the alpha-subunit was determined. The alpha-subunit contained 82 amino acids. The equine FSH-alpha is shorter by 10 to 14 amino acids at the NH2 terminus and has several substitutions at several positions as compared with human FSH-alpha and bovine thyroid-stimulating hormone-alpha. At the intraspecies level, the alpha-subunits of human FSH, human luteinizing hormone, and human chorionic gonadotropin are similar, whereas at the interspecies level there are differences among the alpha-subunits of FSH, luteinizing hormone, and thyroid-stimulating hormone.


Amino acid sequence of the beta-subunit of the follicle-stimulating hormone from equine pituitary glands. A tentative amino acid sequence of the beta-subunit of equine follicle-stimulating hormone (FSH) was derived from the sequences of tryptic, thermolytic as well as peptic, subtilisin, and chymotryptic peptides. Equine FSH-beta is analogous to human FSH-beta except six amino acid substitutions at positions 12, 16, 21, 62, 108, and 114. The amino acid sequence suggests that the hormone-specific beta-subunits of FSH are similar at the interspecies level, whereas the amino acid sequences of the hormone nonspecific alpha-subunits show variations.


The primary structure of the nonpolar segment of bovine cytochrome b5. The primary structure of the membrane bound segment of amphipathic bovine liver microsomal cytochrome b5 has been determined. This 43 residue nonpolar polypeptide is present at the COOH terminus of cytochrome b5. The sequence was obtained by automated sequence analysis and carboxypeptidase digestions. The sequence obtained is: Ile-Thr-Lys-Pro-Ser-Glu-Ser-Ile-Ile-Thr-Ile-Asp-Ser-Asn-Pro-Ser-Trp-Trp-Thr-Asn-Trp-Leu-Ile-Pro-Ala-Ile-Ser-Ala-Leu-Phe-Val-Ala-Leu-Ile-Tyr-His-Leu-Tyr-Thr-Ser-Glu-Asn. Conformational analysis using predictive algorithms is presented along with circular dichroism data on the peptide bound to phospholipid vesicles.


Cyclic forms of the alpha-keto acid analogs of arginine, citrulline, homoarginine, and homocitrulline. The alpha-keto acid analogs of arginine and citrulline (and of their next higher homologs, homoarginine and homocitrulline) were prepared enzymatically and shown to exist in equilibrium with cyclic forms which predominate in solution and in the solid state. The cyclic forms of the alpha-keto acid analogs of arginine and homoarginine have the structures pyrrolidine-1-amidino-2-hydroxy-2-carboxylic acid and piperidine-1-amidino-2-hydroxy-2-carboxylic acid, respectively. The cyclic forms of the alpha-keto acid analogs of citrulline and homocitrulline have the structures pyrrolidine-1-carbamyl-2-hydroxy-2-carboxylic acid and piperidine-1-carbamyl-2-carboxylic acid, respectively. The latter compound can undergo further cyclization and dehydration to form additional products, whose structures (2H, 5H, 7H-imidazo[1,5-a]pyridine-1,3-dione and 2H, 5H, 7H, 9H-9-hydroxy-imidazo[1,5-a]pyridine-1,3-dione) were deduced. The alpha-keto acids investigated here, which may be formed reversibly by enzymatic transamination of the corresponding amino acids, may be formed in vivo especially in the presence of metabolic abnormalities associated with inborn enzymatic defects.


Turnover of murine beta-glucuronidase. Comparison among liver, kidney, and spleen and between lysosomes and microsomes. The turnover properties of murine beta-glucuronidase in several tissues and at two subcellular sites have been determined by monitoring the radioactivity present in immunoprecipitated enzyme at a number of time points following the in vivo administration of a single radiolabeled protein precursor (either L-[3,4(-3H)]leucine or or NaH14CO3). In all experiments a considerable period of time was required for the attainment of maximum specific radioactivity in glucuronidase. Similar labeling kinetics was found when [3H]leucine incorporation was monitored in immunoprecipitated murine liver delta-aminolevulinate dehydratase. Half-life estimates of 2 to 3 days were obtained for glucuronidases of liver, kidney, and spleen. In most strains of inbred mice, it is known that approximately 60% of total liver glucuronidase activity resides in lysosomes, while 40% is within the membranes of the endoplasmic reticulum (Ganschow, R. E. and Paigen, K. (1968) Genetics 59, 335-349). These two subcellular forms of glucuronidase turn over at similar rates. Furthermore, the bulk of glucuronidase in the endoplasmic reticulum does not serve as precursor to lysosomal glucuronidase.


Contractile properties of actomyosin from human blood platelets. Actomyosin was purified from human blood platelets and used to form threads via extrusion. A sensitive tensiometer was employed to measure isometric tension and velocity of isotonic shortening of the threads in the presence of MgATP. Using fully phosphorylated myosin, we obtained values for maximum isometric tension (Po) and maximum velocity of contraction (V max) that were similar to those reported for threads composed of skeletal muscle actomyosin. Po was found to be directly proportional to the level of phosphorylation of the 20,000-dalton myosin light chain. We also studied the effect of phosphorylation on superprecipitation of platelet actomyosin. Fully phosphorylated myosin produced rapid clearing and superprecipitation, while myosin with a low level of bound phosphate underwent rapid clearing but did not superprecipitate. We have concluded from these results that: 1) the interaction between platelet actin and myosin produces tension and motion that is similar to that produced by skeletal muscle actin and myosin and 2) phosphorylation of the 20,000-dalton myosin light chain in important in controlling the production of force by platelet actin and myosin.


Inactivation of phosphorylase b by potassium ferrate. Identification of a tyrosine residue involved in the binding of adenosine 5'-monophosphate. The site of reaction of potassium ferrate (K2FeO4) with rabbit muscle phosphorylase b has been further characterized in an extension of previously published studies (Lee, Y. M., and Benisek, W. F. (1976) J. Biol, Chem. 251, 1553-1560) reporting inactivation of the enzyme by this reagent. The tryptic peptide composed of residues 70 to 80 of the enzyme's polypeptide chain was shown to contain a tyrosine residue which is chemically modified by ferrate and which is protected by 5'-AMP. The sequence of this peptide obtained from both untreated and ferrate-treated phosphorylase b was determined, and the results showed that tyrosine-75 was the residue with which ferrate reacts.


Nucleotide sequence of Halobacterium cutirubrum ribosomal 5 S ribonucleic acid. An altered secondary structure in halophilic organisms. The nucleotide sequence of ribosomal 5 S RNA from a halophilic bacterium, Halobacterium cutirubrum, grown in 4 M sodium chloride is U-U-A-A-G-G-C-G-G-C-C-A-U-A-G-C-G-G-U-G-G-G-G-U-U-A-C-U-C-C-C-G-U-A-C-C-C-A-U-C-C-C-G-A-A-C-A-C-G-G-A-A-G-A-U-A-A-G-C-C-C-G-C-C-U-G-C-G-U-U-C-C-G-G-U-C-A-G-U-A-C-U-G-G-A-G-U-G-C-G-A-G-C-C-U-C-U-G-G-G-A-A-A-U-C-C-G-G-U-U-C-G-C-C-G-C-C-U-A-C-U. This nucleotide sequence is the longest prokaryotic 5 S rRNA to be reported and unlike other 5 S species does not contain a terminal mononucleoside diphosphate residue at its 5'-end. When compared to other 5 S rRNA's, the sequence homology is greatest (about 68%) with Bacillus subtilis; there is a lower but similar degree of homology (about 58%) with either Escherichia coli or human 5 S RNA. The comparisons further indicate that among 5 S RNA's, eleven of the nucleotide residues are unique to H. cutirubrum. Estimates of the secondary structure of the H. cutirubrum 5 S RNA molecule contain one additional stable hairpin loop which is not found in other 5 S rRNA species; this unusual structure is probably an adaptation to the high salt environment within H. cutirubrum cells.


Identification of a binary complex of procarboxypeptidase A and a precursor of protease E in porcine pancreatic secretion. In porcine pancreatic secretion procarboxypeptidase A exists in two states: as a monomer and as a binary complex of a type hitherto not observed in the pancreatic secretions of other species. This complex is shown to contain 1 molecule of procarboxypeptidase A and 1 molecule of a proteolytic zymogen we have designated as zymogen E. The two subunits of the complex have been separated by gel filtration in a denaturing solvent and the products used for compositional and NH2-terminal sequence analysis. We also fractionated the mixture obtained on activation of the binary complex and isolated homogenous preparations of porcine carboxypeptidase A and the diisopropylphosphoryl derivative of the enzyme formed from zymogen E. Zymogen E has an Mr of about 26,000 and on activation produces an enzyme of essentially the same Mr with properties very similar to those of human pancreatic protease E. It catalyzes the esterolysis of acetyl-L-alanyl-L-analyl-L-alanine methyl ester, but is inert towards acetyl-L-tyrosine ethyl ester and is readily inactivated by diisopropylophosphorofluoridate. Zymogen E has an amino acid composition different from those of porcine chymotrypsinogen A, B, or C. Its NH2-terminal sequence shows homology with the NH2-terminal sequence of lungfish proelastase A; yet, like human protease E, porcine protease E has relatively very low activity on intact elastin.


The sodium electrochemical potential-mediated uphill transport of D-glucose in renal brush border membrane vesicles. The uphill transport of D-glucose in renal brush border membrane vesicles was correlated with the Na+ electrochemical gradient. Each component of the electrochemical potential, the membrane potential (outside positive) or the Na+ chemical gradient, when assayed independently, supported the concentrative uptake of the sugar. When the two components were combined, the rates of D-glucose uptake were additive. Accumulation of D-glucose as a function of various Na+ gradients, in the absence of a membrane potential, suggests a 1:1 stoichiometry between sugar and Na+ uptake. These findings are consistent with the role of ionic gradients in energizing uphill solute transport.