Electron microscopic studies of circular DNA in mouse embryo fibroblasts infected by Rauscher leukemia virus. Using electron microscopy, a closed circular form of DNA (4.3 mum in contour length) was detected in the nucleus of mouse embryo fibroblasts 2.5 h after infection by Rauscher murine leukemia virus. These circles were distinguishable from mitochondrial DNA by various criteria, including size, absence of secondary features, and resistance to EcoRI endonuclease.


Macromolecular synthesis in cells infected by frog virus 3. VI. Frog virus 3 replication is dependent on the cell nucleus. Previous evidence indicated that frog virus 3 (FV3), an icosahedral DNA virus, replicates exclusively in the cytoplasm. However, data presented here demonstrate that FV3 does not replicate in UV-irradiated or enuleated chicken embryo or BSC-1 cells and that virus-specific DNA synthesis is not initiated in such cells. Primary transcription was not detected in infected enucleated cells. These results demonstrate that a functional nucleus is essential for FV3 replication.


Unusual hymenal perforations appearing as Skene's ducts. Unusual hymenal perforation created a diagnostic problem when persistence of the urogenital sinus was suspected. However, the vagina was found to communicate with the exterior through what appeared to be Skene's ducts.


Cataract in a fetus at risk for oculo-cerebro-renal syndrome (Lowe). A high-risk pregnancy for X-linked recessive inherited Lowe's syndrome was terminated due to a male karyotype in the cultured amniotic fluid cells. The eyes of the male fetus showed specific cataracteous changes of the lens. A posterior lenticonus was due to a defect of the lens capsule. The lenses were of normal size. Loss of lens material through a lens capsule defect could account for the small discoid lens usually seen in Lowe's syndrome. Amino acids in amniotic fluid had normal concentrations except lysine and proline which were markedly elevated.


Uptake and metabolism of fatty acids by soybean suspension cells. Soybean suspension cultures very rapidly take up C16 and C18 fatty acids by a nonspecific, nonenzymic binding of exogeneously added fatty acids to cell walls and by a subsequent transfer into the cell where they are rapidly incorporated into triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines. 14C-Palmitic and 14C-stearic acids follow this sequence but are not desaturated, wherease 14C-oleic and 14C-linoleic acids are transferred more rapidly than the saturated fatty acids and are then further modified. All the data fit a sequence of events by which free oleic acid is first activated to a CoA thioester, and then desaturated to linoleyl-CoA; both thioesters are then transferred to triacylglycerols, phosphatidylcholine, and phosphatidylethanolamine.


The effect of temperature on the fatty acid and phospholipid composition of Cephalosporium falciforme and Cephalosporium kiliense. The effect of temperature on the lipid composition of Cephalosporium falciforme and Cephalosporium kiliense, causative agents of maduromycosis, was investigated. The fungi were grown at 28.5 C and 37 C in a chemically defined medium. The lipids were solvent extracted, purified on Sephadex, and separated into their component classes by silicic acid column chromatography. Five lipid classes were found: (a) sterol esters, (b) triacylglycerides, (c) free fatty acids, (d) sterols, and (e) phospholipids. Fatty acids were analyzed by gas liquid chromatography. Temperature induced changes of varying degrees occurred in both the fatty acid and phospholipid fractions of each organism.


Familial spasmodic torticollis.

Familial spasmodic torticollis. Presented are four cases of familial spasmodic torticollis, comprising members of three families. The age of onset tended to be family-specific, and no afflicted family member had evidence of more widespread disease (dystonia musculorum deformans). Familial incidence supports the conclusion that spasmodic torticollis is organic in origin. Familial spasmodic torticollis occurs more often than has been generally recognized.


Evolution of cerebral abscess: correlation of clinical features with computed tomography. A case report. Computed tomography demonstrated the evolution of a cerebral abscess. Intravenous contrast material was essential for visualization of the abscess capsule. Computed tomography was more specific than technetium 99 brain scanning and cerebral angiography, and is the only diagnostic tool so far capable of differentiating acute focal encephalitis (cerebritis) from a well-developed abscess.


Evaluation and detection of Duchenne's and Becker's muscular dystrophy carriers by manual muscle testing. Manual muscle testing of mothers of patients with X-linked muscular dystrophy has demonstrated patterns of proximal muscle weakness. The degree of weakness usually has little effect on activities of daily living but can be detected by standardized "break" testing with manual stabilization of body parts, elimination of synergistic muscles, and mechanical advantage given to the patient. Manual muscle testing is a valuable adjunct to the clinical and biochemical tools available for detecting carriers.


Neurologic diagnosis using the 80-lens optical camera. This article describes the clinical application of the 80-lens optical camera in the diagnosis of cerebral infarction, subdural hematoma, intracranial tumor, and hydrocephalus. The 80-lens camera is a simple noncomputerized addition to the Anger scintillation camera that permits an estimation of the cerebral blood flow. The study is noninvasive and quick, minimizing the effect of movement artifact and showing areas of either increased or decreased blood flow. Abnormal tumor vessels appear, and subdural hematomas show brain displacement away from the skull edge. Interpretation of blood flow pattern is not affected by scalp or skull trauma, eliminating a source of error incurred with static scans.


Prevention of postischemic impairment of microvascular perfusion. Two groups of conditioned male mongrel dogs, designated group 1 and group 2, were subjected to 35 minutes of cerebrospinal fluid compression ischemia followed by 30 minutes of recirculation. Animals in both groups were heparinized, but in addition, the blood of group 2 animals was circulated through three glass-wool filters over the course of 1 hour prior to induction of ischemia. Focal zones of impaired perfusion were prevented and general reflow was significantly enhanced in group 2 animals, whose blood had been filtered through glass-wool. Animals in group 1 had focal zones of impaired perfusion and relatively poor general postischemic reflow. A hypothesis concerning blood-damaged tissue interaction in zones of acute tissue damage is introduced.


Carotidynia.

Carotidynia. Nosologic uncertainty about carotidynia has arisen, in part, because the syndrome was initially classified as an atypical facial neuralgia. More recently, carotidynia has been characterized as a recurring vascular neck pain, often accompanied by carotid tenderness and soft tissue swelling and sometimes by vascular headaches. We now report that drugs useful in the prophylaxis of migraine appear to be effective in carotidynia. Eight women (ages 39 to 77) with unilateral, episodic neck pain of 1 to 19 years' duration have been observed for periods ranging from 7 months to 6 years. All experienced marked relief in the intensity and frequency of their pain syndromes after the administration of methysergide, ergonovine maleate, propranolol, or nortriptyline. No patient had evidence of arteritis. The responsiveness of both migraine headaches and carotidynia to similar drugs suggests a common pathophysiologic mechanism.


Airborne rabies encephalitis: demonstration of rabies virus in the human central nervous system. A veterinarian contracted rabies in the course of laboratory work with homogenates of rabid goat brain. Epidemiologic study determined a respiratory mode of transmission. After a fulminant encephalitic illness, formed rabies virions were identified in the synaptic zones of the olfactory glomeruli. Identification, isolation, experimental disease production, and tissue cytopathic effects of virus recovered from the brain fulfilled Koch's postulates in this unusual instance of virus disease of the nervous system.


Physostigmine in familial ataxias.

Physostigmine in familial ataxias. Physostigmine was given to 12 patients with various spincerebellar degenerations, and neurologic examinations were recorded on video tapes and assessed on a semiquantitative scale. Forty minutes after a single dose, the scores improved by 35.7 +/- 4.7 percent (means +/- SEM). Eight patients were then studied over sequential 3-month periods by a randomized double-blind trial of physostigmine versus placebo. With physostigmine, scores were 33.9 +/- 8.3 percent better than before treatment or with placebo. Further investigations of cholinergic drugs seem warranted in patients with these diseases.


Neural influence on experimental myotonia. A transitory myotonic state, lasting for less than 24 hours, was induced in rats within 2 hours of the intraperitoneal injection of 2,4-dichlorophenoxyacetic acid. Neuromuscular blockade, nerve block, nerve section, and cordotomy had no effect on the established myotonic state. However, muscle that had been denervated progressively lost its capacity for a myotonic response to subsequent injections of 2,4-dichlorophenoxyacetic acid. After 10 to 12 days denervation, the injection of 2,4-dichlorophenoxyacetic acid produced a brief increase in the number of fibrillations, but typical myotonic discharges were no longer detectable. Unlike denervation, cordotomy did not impair the capacity of the paralyzed limbs to develop in response to 2,4-dichlorophenoxyacetic acid. While myotonia is the result of a primary defect in the muscle membrane, these experiments indicate the innervation is necessary to maintain the muscle membrane in a state that will support myotonia. We hypothesize that an alteration in ionic conductance secondary to denervation is the critical factor in preventing myotonia.


Effects of pentoxifylline on cerebral ultrastructure of normal and ischemic gerbils. Normal gerbils and those made ischemic for 15 to 60 minutes by bilateral common carotid artery occlusion were studied ultrastructurally after administration of the vasoactive drug pentoxifylline. In both groups, hypertrophy of neuronal mitochondria was found in the hippocampus and cerebral cortex. Planimetry of electron micrographs revealed a statistically significant increase in average mitochondrial size of drug-treated animals compared with untreated ischemic gerbils and normal controls; the treated ischemic group showed the greatest increase. Incubated cortical slices from normal gerbils that were given the drug 5 hours before they were killed showed a significant increase in oxygen consumption compared with controls.


Serum creatine phosphokinase levels and prednisone treated muscle weakness. Evidence is cited from the literature and from personal experience with the "inflammatory" form of facioscapulohumeral syndrome that the creatine phosphokinase reduction occurring during pharmacologic treatment of various neuromuscular diseases does not necessarily imply therapeutic benefit.


Facial asymmetry in patients with temporal lobe epilepsy. A clinical sign useful in the lateralization of temporal epileptogenic foci. While a clinical history is frequently sufficient for a localizing diagnosis of temporal lobe epilepsy, lateralization of the epileptogenic abnormality often is impossible on clinical grounds alone. Since we have noted facial asymmetry in such patients, 50 individuals with temporal lobe epilepsy were studied. In patients with unilateral foci, we found contralateral lower facial weakness of mild to severe degree in 73 percent while 13 percent had ipsilateral weakness, and in 13 percent the face was symmetrical. The facial weakness usually was more striking on emotional movement. In the group of patients with bitemporal independent discharges, 61 percent had some asymmetry, often facial weakness on the side opposite the major focus. Of 25 control individuals, only one third had a facial asymmetry and this was slight. Facial asymmetry in temporal lobe epilepsy is a useful though not an absolute clinical lateralizing sign. It is a factor to be considered in addition to other clinical, radiologic, neuropsychologic, and electroencephalographic findings in the evaluation of patients with temporal lobe seizures.


Neurologic and psychologic manifestations of decompression illness in divers. It has been widely accepted that the neurologic sequelae of decompression illness are confined to the spinal cord. Of 10 divers who gave a history of an episode of decompression illness involving the central nervous system, we found that eight had unequivocal neurologic deficits implicating multiple supraspinal lesions. Seven of these neurologically impaired divers completed a battery of neuropsychologic tests that revealed severe deficits in all cases. The findings show that diffuse and multiple central nervous system lesions result from decompression illness and demonstrate the importance and close correlation of thorough neurologic and neuropsychologic tests in assessment following diving accidents.


Plasma anticonvulsant concentrations during pregnancy. Plasma anticonvulsant levels were followed during pregnancy in 11 epileptic women taking phenytoin and/or phenobarbital or a drug metabolized in the body to phenobarbital. As judged from the relationship between plasma level and drug dose, phenytoin requirement increased in all 10 women taking this drug during pregnancy. The requirement fell again in the puerperium. Plasma phenobarbital levels decreased during pregnancy in all five women taking a constant daily dose of phenobarbital or a congener. These findings should be borne in mind if epileptics are to be protected against seizures during pregnancy and against anticonvulsant overdosage during the puerperium.


Cellular hypersensitivity in attacks of multiple sclerosis. I. A comparative study of migration inhibitory factor production and lymphoblastic transformation in response to myelin basic protein in multiple sclerosis. Although cell-mediated hypersensitivity to basic myelin protein has been demonstrated in multiple sclerosis with use of cell migration assays, results with the lymphoblastic transformation technique have been inconclusive. However, prospective studies relating results of lymphoblastic transformation to the clinical course of multiple sclerosis have not been reported. In the present study, both lymphoblastic transformation and migration inhibitory factor assays were used, and results related to the temporal course of multiple sclerosis. Results of the present investigation show that cell-mediated hypersensitivity to myelin basic A1 protein is most significant during exacerbations of multiple sclerosis. Responses obtained employing either the lymphoblastic transformation or migration inhibitory factor assay were equally significant. The results support the hypothesis that a factor suppressing deoxyribonucleic acid synthesis is present in multiple sclerosis and is inhibited by the use of steroids.


Lapse of consciousness and automatisms in temporal lobe epilepsy: a videotape analysis. Videotape analysis of 76 attacks in 14 epileptics showed two electro-clinical types of psychomotor attacks. The first and most common type had three clinical phases, consisting of an initial motionless stare, stereotyped movements, and reactive automatisms during impaired consciousness. The second and less common type started with stereotyped and reactive automatisms. In the first type, focal temporal or lateralizing features were common. In the second type, there were only diffuse changes in the electroencephalogram. The value of recording attacks with nasopharyngeal electrodes was emphasized by a high yield for focal-lateralizing electroencephalographic features.


Wave form difference in visual evoked responses between normal and reading disabled children. Wave form differences were found in visual evoked response between normal and reading disabled children. These differences were maximally seen over the left parietal area but were minimal at the vertex and over the occipital area.


The development of left-hand superiority for discriminating braille configurations. Discrimination of braille configurations was performed better by the left hand of right-handed sighted children over age 10, paralleling a previous study in which the names of braille letters were learned. Left-hand superiority occurred sooner in males, but they were not better at the task than females, more of whom maintained a left-hand advantage as adults. As in vision, configurations differing in orientation were more difficult to discriminate than those differing in number or displacement, but this order of difficulty was the same for both hands.


Absence of sleep-related growth hormone elevations in myotonic dystrophy. There is evidence that in myotonic dystrophy, the endocrine and central nervous systems are affected. To study a possible relationship between both defects, we investigated nocturnal sleep patterns and associated growth hormone secretion in two men and three women with myotonic dystrophy. In three patients who were clinically the most severely affected by myotonic dystrophy, plasma growth hormone elevations related to the slow-wave phase of sleep were absent. The two least severely affected patients had plasma growth hormone increases of low magnitude and brief duration (from 0.4 ng per milliliter to 13.0 ng per milliliter). These data suggest a failure of integration at a subcortical level of the slow-wave phase of sleep with the hypothalamic-pituitary mechanisms of growth hormone secretion. Thalamic neuronal lesions occurring in myotonic dystrophy could be responsible for such failure.


Adult onset autonomic dysfunction coexistent with familial dysautonomia in a consanguineous family. A consanguineous family is described in which autonomic dysfunction developed in the father during adult life while the son had familial dysautonomia at birth. The father's condition is felt to be secondary to olivopontocerebellar atrophy. The concurrence in this family of an adult and a childhood form of dysautonomia may be an expression of the same genetic defect at different stages of development.


Degeneration of fasciculus gracilis in cystic fibrosis. The spinal cords of 19 percent of patients dying with cystic fibrosis after 5 years of age showed posterior column degeneration. The risk did not appear to increase with advancing age. None of these patients had findings of pernicious anemia or spinocerebellar degeneration. The lesions had been undetected clinically. It is possible that nutritional, toxic, or hereditary factors may play a role in producing this lesion.


Multiple sclerosis in Asia.

Multiple sclerosis in Asia. Cases of multiple sclerosis were collected by Asian neurologists from recognized medical centers, using standardized formats similar to those used for the Japan nationwide multiple sclerosis survey. As a result, information on a total of 61 cases was collected from six neurologic centers in five Asian countries excluding Japan. Multiple sclerosis appears to be a rare disease throughout Asia. Certain clinical characteristics that had been previously noted in Japanese and other individual Asian series were again observed in the present Asian series. When compared with Western multiple sclerosis, the Asian multiple sclerosis cases showed a relatively higher frequency of optic nerve involvement, frequently bilateral and severe, at the onset, as well as during the course of illness, and no rare occurrence of Devic's syndrome.


Experimental autoimmune myasthenia gravis: No morphometric abnormalities of nerve trunks. In rats with experimental autoimmune myasthenia gravis, light and electron microscopic evaluation of proximal and distal radial and distal phrenic nerve trunks was done to see whether a morphologic abnormality was present in alpha motor axons. No significant difference was found in density of myelinated fibers between nerves of experimental and control animals.


Pulmonary hypertension in the perinatal aspiration syndromes. Ten patients clinically diagnosed as having perinatal aspiration syndromes were found to have pulmonary hypertension. These infants were either term or postmature babies and had the following characteristics: (1) systemic or suprasystemic levels of pulmonary artery pressure (range, 50 to 117 mm Hg); (2) a degree of pulmonary hypertension not related to the degree of aspiration evident on chest roentgenograms; (3) evidence of right-to-left shunting at the ductal or foramen ovale level; and (4) sustained severe hypoxemia despite 100% inspired oxygen concentration. The overall mortality for the group was 50%. Since these patients had marked clinical and physiologic similarities to patients previously reported as having the persistent fetal circulation syndrome (PFC), they were classified as having "PFC with aspiration." The existence of pulmonary hypertension should be suspected despite roentgenographic evidence of aspiration in any patient who also manifests the clinical characteristics of PFC because its treatment may alter the prognosis of such patients.


Carbon dioxide sensitivity of the central chemosensitive mechanisms. An exploration by direct stimulation in rats. In urethane-anaesthetised adult albino rats ventral surface of the brainstem was stimulated chemically by increasing the local CO2 concentration and electrically. Two areas were demarcated on the ventral surface of the brainstem, one which showed an increase in pulmonary ventilation on chemical and electrical stimulation, and another which showed a decrease in pulmonary ventilation and sometimes even respiratory arrest. EEG activity recorded from the area from where increased pulmonary ventilation was obtained showed a synchronous slow wave activity during chemical stimulation and inhalation of a CO2-air mixture. This area is situated 0.5--1 mm lateral to the mid-line extending up to the rootlets of the VIIth to IXth cranial nerves. The response increased proportionately on increasing the strength of the chemical stimulus, till it reached a plateau. In carotid body denervated and chronic hypoxic animals, the magnitude of the responses was shown to be increased, probably due to increased sensitivity of the central chemosensitive mechanisms.


Amino acid reabsorption in the proximal tubule of rat kidney: stereospecificity and passive diffusion studied by continuous microperfusion. Renal tubular reabsorption of glycine and of the L- and D-isomers of histidine, serine, phenyl-alanine, methionine, proline and cystine was investigated in vivo et situ by continuous microperfusion of single proximal convolutions of the rat kidney. In the case of glycine and the L-isomers, tubular reabsorption is saturable to a great extent. The D-amino acids are reabsorbed much more slowly than the respective L-forms. Furthermore in the case of methionine and perhaps also of proline, serine and phenylalanine, the fractional reabsorption decreases in the presence of high concentrations of the L-form. This indicates that the D-isomers also have a measurable affinity for the reabsorption mechanisms of the renal tubule. The very poor reabsorption of D-amino acids in the presence of their L-isomers indicates that simple passive diffusion plays only a relatively small role in tubular amino acid reabsorption. Permeability coefficients estimated from these findings are in the range from 1--5 X 10(-7) cm2 - s-1. These values are very similar to those found for other organic molecules of comparable molecular weights.


Haemodynamics during partial extracorporeal circulation in the dog. Circulatory changes were studied in 9 mongrel dogs during partial extracorporeal circulation from the right atrium to the carotid and/or femoral artery. The bypass circuit consisted of a membrane oxygenator and a roller pump. Cardiac output and arterial pressure decreased during bypass, but changed little when extracorporeal flow (EF) was increased from 1/10 to 3/4 of control cardiac output. Right atrial and pulmonary wedge pressures were lower than control. Total blood flow was equal to control when EF was 1/2 of the control cardiac output, and exceeded control flow at 3/4. Such enhanced venous return presumably resulted from sympathetic stimulation via atrial mechanoreceptors.


Thoracic duct lymph in conscious dogs at rest and during changes of physical activity. In conscious dogs with a thoracic duct-venous shunt, lymph flow (LF), protein concentration (LP), transported protein (LTP), albumin concentration (LA), globulin concentration (LG), albumin to globulin ratio (LAG), plasma disappearance rate of labelled albumin (k), plasma volume (PV) and intravascular protein pool (IVP) were studied at rest, during walking and muscle relaxation induced by guajacolglycerinether. At rest, LF approximated 50 mul/min-kg and LTP 1.55 mg/min-kg. LF and LTP correlated positively with the state of physical activity. LP was 54%, LA 67%, LG 43%, but LAG 156% of the plasma values; k was 0.00228. Relaxation reduced LF by 34%, LTP by 31%, LAG by 7% and the lymph concentration of i.m. applied labelled albumin by 54%. PV, IVP and k were not changed significantly. Walking enhanced LF by 100%, LTP by 50% and LAG by 25%. LP was decreased by 15%. PV, IVP and k were not changed significantly. Control data suggest a significant influence of anaesthesia on thoracic duct lymph flow, composition and protein transport. Spontaneous and experimental changes of thoracic duct lymph may be explained by changes of the activity of the "tissue pump". An extravascular fluid and protein pool would seem to be accumulated at rest and emptied during exercise.


Adaptations to ambient temperature in growing pigs. 1. Pigs were weaned at 8--10 dyas of age and littermate pairs were raised in individual cages at ambient temperatures of 25 degrees C or 35 degrees C to 8 weeks of age. 2. Pigs reared at the higher temperature had longer extremities and less hair than controls even though body weights were similar. 3. On exposure to an ambient temperature of 45 degrees C, thce as quickly as their littermates. 4. When the scrotum was heated locally to 42 degrees C animals reared in the warmer environment began to pant at a lower ambient temperature than the controls. 5. Heating the hypothalamus by means of an implanted thermode at an ambient temperature of 35 degrees C was accompanied by an increase in respiratory frequency in pigs reared at 25 degrees C. The pigs reared at 35 degrees C already had a slightly elevated rate of breathing and heating the hypothalamus caused no change. 6. The rate of blood flow in the tail was higher in pigs reared at 25 degrees C than those kept at 35 degrees C when measured at ambient temperatures between 20 degrees C and 35 degrees C. When the hypothalamus was cooled the slope of the line relating blood flow to the temperature of the thermode was similar in both groups. 7. The critical temperature as determined from measurements of oxygen consumption was higher in the pigs reared at 35 degrees C than in controls. But the effect of cooling the hypothalamus on metabolism was similar in both groups. 8. There was no difference between the two groups in the rate of cutaneous water loss.


The selective permeability of the pancreatic duct of the cat to monovalent ions. In the anaethetised cat the electrical potential difference between the lumen of the main collecting duct of the pancreas and blood in the jugular vein was measured. The duct was perfused with isotonic solutions of monovalent ions and the recorded potential corrected for the liquid junction potentials. The data were fitted to the Shinagawa extension of the Goldman constant field equation and the relative permeabilities of the duct epithelium to the ions were determined. The duct showed negligible selectivity between the monovalent cations Li:Na:K:Rb:Cs = 1.08:1.10:1.09:1.12 in contrast to the definite selectivity sequence for the anions F:Br:Cl:I:HCO3 = 0.44:1.38:1.08:2.05:0.60. This halide selectivity sequence is the Eisenman sequence I and is indicative of the selectivity being due to weak positive charges on membrane bound sites surrounding a highly hydrated channel. It is argued that these highly hydrated channels may be identified with the "tight junctions" between cells and the selectivity properties of the pancreatic duct are determined by flow of ions through these areas rather than flow through the epithelial cells.


The location and distribution of type A and type B atrial endings in cats. In the attempt to explain the difference in discharge pattern of atrial endings, 131 endings were localized by punctate stimulation, 44 were type A, 77 type B and 10 of an intermediate type. All were located on the dorsal wall of the atria with none on the ventral wall or in the appendage. On the right side, 74% of type A were located in the atria and 63% of type B in or near the veins. On the left side, 67% of type A and 94% of type B were located in or near the veins. Thus, there appeared to be some difference in the location of type A and type B endings on the right side, but on the left side both types of endings were for the most part confined to the venous region. Further, on both right and left sides, these endings were present both in the central part of the atria and in or adjacent to veins. This leads to the suggestion that the difference in discharge patterns is not caused by the location but may be due to some other reasons, e.g. difference of arrangement in the atrial wall with respect to the contractile elements.


Studies on the adaptation rate and frequency distribution of type A and type B atrial endings in cats. Since from the study of location no clue could be found regarding the difference in discharge patterns of type A and type B endings in cats, two separate series of experiments were performed. In the first series, the responses obtained from types A, B and intermediate endings on stretching the isolated atrial chamber were identical. From the second series directed to the frequency distribution of atrial endings and performed by recording of the neural activity in single fibres of the whole vagus, a ratio of 1:1 between type A and type B endings was found. Endings with an intermediate pattern of discharge were rare. Out of 71 atrial endings recorded from 4 vagus nerves, only 7 were of this type. These studies showed that: (1) the types A, B and intermediate endings are slowly adapting, (2) the intermediate type is a variant of type B and (3) type A and type B endings belong to different types. It is speculated that the difference in discharge patterns of type A and type B endings may be due to their arrangements in the atria.


Effect of splanchincotomy on the renal excretion of inorganic phosphate in the anaesthetized dog. The effects of acute i.v. inorganic phosphate (Pi) loading were studied on nembutalized mongrel dogs previously subjected to unilateral splanchnicotomy ("renal denervation"). GFR (51Cr-EDTA) was not different on the intact and the denervated side, while urine output (V), sodium excretion (UNaV), and urinary excretion (UPiV) of inorganic phosphate of denervated kidneys were significantly increased at any plasma Pi level. Thus, tubular reabsorption of Pi in denervated kidneys was considerably depressed. Tubular transport rates of Na and Pi--as expressed in per cent of the filtered load--were positively correlated in both intact and denervated organs. Besides an impairment in tubular transport of Na a depression in the reabsorption of inorganic phosphate is brought about by renal denervation. A common mechanism of action for both Na and Pi can be supposed.


Effect of splanchnicotomy on the renal excretion of para-aminohippuric acid in the anaesthetized dog. Renal excretion of para-aminohippuric acid (PAH) was studied during PAH loading on unilaterally splanchnicotomized ("denervated") anaesthetized dogs. Urine flow, sodium excretion of denervated kidneys were significantly increased. Below a plasma concentration of 20 mg% there were no differences between intact and denervated kidneys in urinary excretion and in calculated tubular transport of PAH. However, maximum secretion rate on the splanchnicotomized side was significantly decreased (innervated: 34.8, denervated: 25.2 mg/100 ml GFR, respectively). Although both Na reabsorption and PAH secretion are impaired by denervation, the exact mechanism of action of renal sympathectomy is not elucidated as yet.


Influences of local and global temperature stimuli on the Lewis-reaction. Oscillatory changes of temperatures of the human fingers in cold air (Lewis-reaction) are investigated under central and local influences. After heat load of the body, the Lewis-reaction is suppressed, no matter which environmental temperature of the hand is chosen. Decrease of finger temperature during oscillatory behaviour takes about twice as much time as increase. A dependence of the time course on the chosen environmental temperature is not evident. Mean level of temperature oscillation rises with increased environmental temperatures of body and hand, whereas the amplitude of oscillation increases with growing environmental tre of the hand. After cold load of the body the Lewis-reaction is suppressed in some individuals. Interpretation of the Lewis-reaction and interaction of global and local requirements are discussed.


Dose-dependent stimulation and inhibition of proximal tubular sodium reabsorption by angiotensin II in the rat kidney. The effect of val5-angiotensin II on steady-state sodium concentration gradients (deltacNa) was studied in rat proximal tubules by stationary micro-perfusion combined with perfusion of the peritubular capillaries. Angiotensin added to the peritubular perfusion fluid had a biphasic action with stimulation of sodium reabsorption at low doses (10(-12)-10(-10)M) and inhibition at high doses (3 X 10(-7) - 3 X 10(-6)M). Stimulation of transport was also observed with intraluminal angiotensin but only at a dose of 10(-9)M. Transepithelial potential difference was calculated from the steady-state chloride distribution; no significant change was observed at low (10(-11)M) or high (10(-6)M) concentrations and a direct action on sodium transport is postulated. This biphasic effect is discussed in relation to the responses of the intact kidney to intra-renal infusion of angiotensin, and to the control of tubulo-glomerular feed-back.


Myeloproliferative diseases.

Myeloproliferative diseases. The various myeloproliferative diseases have different symptoms, therapeutic problems, and prognoses. The most common of these disorders appears to be agnogenic myeloid metaplasia, which primarily affects older persons. The five-year survival rate at the Mayo Clinic for these patients is 58%, and the prognosis depends on the presence of symptoms, anemia, and thrombocytopenia and on the size of the liver. Androgen therapy is often necessary to control anemia, and splenectomy may be indicated.


[Pulmonary humoral immune response after exposure to carbon monoxide]. The effect of exposure to CO was studied in guinea-pigs using the plaque forming cell technique. Exposure to 200 ppm for 5 weeks was found to reduce the capacity to produce IgG and IgM in free lung cells.


Biological factors involved in the clinical features and surgical management of cerebellar hemangioblastomas. Sixty-five personal cases of cerebellar hemangioblastomas plus others from the literature making a total of 539 cases are reviewed regarding their clinical, biological and surgical aspects. A variable genetic blastomatous growth potential seems to determine the individual biological behavior of these tumors. Retinal angiomatosis, family incidence, multicentric tumors in the central nervous system and in other organs, and some of the so-called recurrences, are considered within this concept.


[A comparison of serum phosphohexose isomerase and sialyl transferase activities in patients with malignant disease]. The diagnostic value of serum sialyl transferase, phosphohexose isomerase and lactate dehydrogenase determinations is discussed in respect to patients with malignant diseases. It was found that 80 % of the  investigated cases showed an increase in serum sialyl transferase activity. Phosphohexose isomerase was elevated in 46.6 % and lactate dehydrogenase in 23.3% of the cases. The serum sialyl transferase activity was within the normal range in 13.3% of the patients. It is concluded that although the serum sialyl transferase activity is of great diagnostic significance in cases of suspected malignancy, total reliance cannot be placed on the determination of this sole parameter, but only in conjunction with others.


[Determination of the pigmenting carotenoids in feeds and premixes for laying hens and broilers (author's transl)]. A method for the quantitative determination of the main carotenoids, such as lutein, zeaxanthin, apocarotenoic acid ethyl ester, bixin, canthaxanthin, citranaxanthin and paprika pigments in feeds and premixes for laying hens and broilers is described. The method has, compared to the known analytical procedures, several advantages: 1. Differentiation between lutein and zeaxanthin which have different pigmentation properties. 2. Reduction of distribution errors for added carotenoids by increasing sample weights. 3. Individual determinations of added carotenoids which have different pigmentation activities.


[Mercury-content of austrian edible mushrooms and its relation to the protein content of the mushrooms (author's transl)]. The mercury content of 16 eatable mushroom species from different geographical areas was determined by flameless atomic absorption spectroscopy. Mercury levels lay between 18 and 14300 ppb related to mushroom dry matter. A great difference in mercury content was found between species. Boletaceae, Lycoperdaceae, Agaricaceae etc. showed constantly high values (greater than 1000 ppb), but in Cantharellaceae a low content (less than 100 ppb) was recorded. Besides other factors the mercury concentration is essentially dependent on the protein content. A highly significant positive correlation between the two characteristics was found.


Ventriculographic and hemodynamic features of mitral regurgitation of cardiomyopathic, rheumatic and nonrheumatic etiology. Quantitative angiographic findings were reviewed in 40 patients with significant mitral regurgitation classified into three etiologic groups: group I, primary mitral regurgitation (prolapse, ruptured chordae); group II, mixed stenosis and regurgitation of rheumatic origin; and group III, cardiomyopathic mitral regurgitation. For patients in both groups I and II, left ventricular end-diastolic volume was directly related to regurgitant fraction, and ejection fraction was generally well maintained. In contrast, patients in group III had a depressed ejection fraction (less than 0.40) and end-diastolic volume that was disproportionately increased in relation to the degree of regurgitation. Left ventricular end-diastolic pressure was a poor indicator of severity of regurgitation in all patient groups. There was a significant negative correlation between forward cardiac index and regurgitant fraction. There was significant relation, although with considerable variation, between the normalized V wave and regurgitant fraction. The graphs of chamber size, ejection fraction and hemodynamic measures plotted against the severity of regurgitation in different patient groups provide a perspective for interpreting the findings in individual patients.


Motion sickness-induced pica in the rat. Two experiments investigating the effects of motion sickness on pica (the consumption of non-nutritive substances) are reported. In the first experiment rats subject to rotational stimulation subsequently engaged in geophagia (clay consumption). In the second experiment use of a conditioned aversion paradigm confirmed that the method of rotational stimulation used in the first experiment causes motion sickness in rats. The results of these experiments indicate that simple gastrointestinal malaise in the absence of a deficiency state or acute toxemia will elicit pica. It is suggested that gastrointestinal distress may be a significant factor in the etiology of pica and its relationship to other causes of pica is discussed.


Diurnal changes in gastric motor activity in conscious dogs. Twenty-four-hour changes in gastric motor activity were recorded in 4 healthy adult dogs in the conscious state by means of extraluminal force transducers. Three major different gastric motor activity patterns were recognized during 24 hr: digestive, intermediate, and interdigestive. The digestive pattern was characterized by steady low-amplitude contractions in the gastric antrum, with no significant motor activity in the gastric body. The interdigestive pattern consisted of series of high-amplitude contractions of 14-23 min duration in both the gastric body and antrum with intervening and long-lasting (68-109 min) motor quiescence. The intermediate pattern is considered to be the pattern midway when the digestive pattern is changing to the interdigestive pattern. These changes in gastric motor activity are precisely controlled day after day if the animals are fed at regular time intervals and are healthy. The digestive pattern may represent principally a mixing function of ingested food and the interdigestive pattern a cleaning function in preparation for the next meal


The premature breech.

The premature breech. The perinatal outcome of all vaginal breech deliveries weighing less than 2,500 grams was evaluated retrospectively. Results indicated that labor and delivery in the breech position had a morbidity and mortality rate far greater than could be attributed to prematurity alone. Serious consideration should be given to prophylactic cesarean section for all premature breech fetuses when delivery is anticipated.


Effects of alpha- and beta-adrenergic blocking agents on serum prolactin levels in women with hyperprolactinemia and galactorrhea. The effects of alpha- and beta-adrenergic blocking agents on the serum prolactin levels of six women with hyperprolactinemia and galactorrhea were investigated. There was no indication that pituitary adenomas were etiologic agents for the hyperprolactinemia. Serum prolactin could be lowered with oral L-dopa. When intravenous phentolamine (an alpha-adrenergic blocking agent) or intravenous propranolol (a beta-adrenergic blocking agent) were administered for 1 hour, there was no significant change in serum prolactin levels.


Diurnal, respiratory, and other rhythms of fetal heart rate in lambs. In lambs during the last third of gestation fetal heart periods were measured to 0.2 msec. and the variation was calculated with a computer in real time. The rate was subject to low-frequency variations in association with breathing movements, with a 2 to 4 hour rhythm of large amplitude and unknown origin, with the time of day, and with increasing gestational age. The calculated standard deviation of heart period bore a semilogarithmic relation to increasing length of observational epoch. There were also multiple rhythms in heart rate variability. The immeidate effect of fetal hypoxia was to increase heart rate variability despite the arrest of fetal breathing movements. The analysis of heart rate variability is examined critically.


Argon laser photocoagulation of the posterior segment in pseudophakia. A series of in vitro, in vivo, and clinical studies was performed to explore the safety and efficacy of argon laser and xenon arc photocoagulation in pseudophakia. The polymethylmethacrylate intracameral lens (Fyodorov) proved resistant to photothermal decomposition and allowed transmission of therapeutically effective energy levels to the retina. The argon laser delivered through a slit lamp and contact lens optical system appears to be the preferred method for posterior segment photocoagulation in pseudophakia. On the basis of five-day tissue culture studies and clinical experience of two years, the intracameral lens does not appear to be a toxic or transmission barrier to careful argon laser photocoagulation.


Ultrastructural study of methionine sulfoximine-induced Alzheimer type II astrocytosis. An ultrastructural study of cerebral cortex was performed in rats during the preictal period following the administration of the convulsant methionine sulfoximine (MSO). The morphologic changes were restricted to astrocytes and consisted of cytoplasmic enlargement, mitochondrial and rough endoplasmic reticulum proliferation, accumulation of glycogen, development of cisternal and saccular smooth endoplasmic reticulum, nuclear chromatin clumping, and hydropic degenerative changes. These findings resemble those seen in experimental ammonia encephalopathy, suggesting an important role of ammonia in the evolution of these morphologic changes. The findings, moreover, suggest that the primary effect of MSO is on astrocytes and that abnormalities in astrocytes may play a role in the development of MSO-induced seizures.


Heat stress, fluctuating asymmetry and prenatal selection in the laboratory rat. Recent studies have demonstrated that stress may increase the fluctuating asymmetry of teeth and limbs in laboratory animals. The present study investigates the effects of heat on such parameters. Pregnant laboratory rats are exposed to temperatures of 33 degrees C during gestation. Increases in fluctuating asymmetry of dental dimensions as well as bone density are found for the pups of such females when compared to unstressed controls. A general increase in limb lengths is also reported for young of heat stressed animals. Prenatal selection with differential survival is suggested as a possible explanation for differences found between the experimental and control animals.


How Gymnodinium breve red tide toxin(s) produces repetitive firing in squid axons. Partially purified toxin(s), GbTX, extracted from Gymnodinium breve red tide organisms elicits a spontaneous train of action potentials in the squid giant axon. The spikes have a shape similar to that in the normal seawater control except for an increase in the rate of recovery from the afterhyperpolarization. With this more rapid recovery, the membrane potential overshoots the resting potential and  threshold, triggers another spike, and thus produces repetitive firing. Voltage-clamp studies revealed that the toxin has no effect on the normal sodium or potassium conductance changes produced by step depolarization. However, consistent with the faster recovery after an action potential, GbTX speeds recovery of the "shut-off" currents to their steady-state values after a depolarization. The most likely mechanism by which the toxin accelerates recovery after an action potential (leading to repetitive firing) is the induction of a small additional inward current which was found to be reduced by prehyperpolarization. This toxin-induced current which speeds recovery is blocked by tetrodotoxin and hence presumably flows through the sodium channel.


Effect of iron stores and hysterectomy on iron absorption and distribution in pregnant mice. Intestinal uptake (U) and transport (T) of 50Fe from a 1-mug oral dose was studied in the mouse. U increased to a similar degree in both pregnant and iron-deficient animals. The increase in T was greater in pregnant than in iron-deficient mice. In iron-loaded pregnant mice, U increased to levels found in pregnant animals, whereas T increased, but by a lesser amount. Termination of pregnancy by delivery or hysterectomy at days 20-21 resulted in a fall in U and T to normal levels within 24 h. Eighty percent of iron transported across the intestine in pregnancy was recovered from the fetuses, compared to 6.7% recovered in the maternal liver. Hepatic uptake was lower in pregnant than in iron-deficient mice or in postpartum mice with persisting iron deficiency. Hepatic uptake by the maternal liver after an intravenous dose of 2 mug 50Fe increased to levels in iron-deficient mice within 5 min of hysterectomy. The results demonstrate an effect of pregnancy on intestinal iron transport and its distribution that was dependent on the presence of fetuses and placentas.


Changing renal blood flow following sodium nitroprusside in patients undergoing nephrolithotomy. During nephrolithotomy in 23 adult patients under sodium thiopental-N2O-O2-fentanyl anesthesia, renal blood flow to one kidney was studied. Muscle relaxation was obtained with pancuronium, and renal blood flow (RBF) was measured with an electromagnetic flow probe. Percutaneous arterial pressures were also recorded. Administration of sodium nitroprusside caused a decrease in RBF in all patients when mean arterial pressure (MAP) was reduced approximately 44 percent below baseline. Patients whose initial RBF was below 300 ml/min showed greater falls in RBF with hypotension. Changes in renal vascular resistance (MAP/RBF) was found to be markedly higher in the "low-flow" group. As expected, the low-flow group started with a higher renal vascular resistance. From these observations, it is felt that more concern should be exercised when planning deliberate hypotension with nitroprusside, especially in patients with decreased renal function.


Dynamics of local-anesthetic compounds in regional anesthesia. The clinical effectiveness and safety of compounded mixtures of lidocaine + bupivacaine and chloroprocaine + bupivacaine for either epidural or brachial-plexus block was studied in 48 adult patients. Of the several alternatives, chloroprocaine + bupivacaine with epinephrine was found the best choice for patients with typical plasma cholinesterase.


Abdominal pain and alcohol celiac plexus nerve block. Alcohol celiac plexus nerve blocks were done in 100 patients, of whome 97 had intractable abdominal pain from cancer. In most cases, an initial diagnositc block with bupivacaine was followed by the therapeutic block performed by injecting 50 ml of 50 percent ethyl alcohol. Good to excellent pain relief occurred in 94 percent of patients. Fourteen blocks were repeated for recurrent pain. Life duration ranged from 2 days to 14 months after the block. Complications and side effects were infrequently seen but did include a 10 percent incidence of postural hypotension and 1 case of partial leg paralysis. This block is remarkably safe as well as effective and should be employed more frequently.


Acute pulmonary edema following head injury: two case reports. An acute episode of pulmonary edema may occur in association with head injury. This is due to the elevation of systemic blood pressure, to maintain cerebral circulation in the presence of increasing intracranial pressure (ICP). In these instances, the pulmonary edema arises as a pathophysiologic, neurohemodynamic response to the intracranial disease. This response has been produced experimentally, and 2 case reports described this problem clinically. If such patients with increased ICP could be treated immediately with antihypertensive drugs, pulmonary edema might be prevented and the high mortality rate decreased.


A PSRO record-keeping system for anesthesiologist. To meet the requirements for Professional Standards Review Organization (PSRO) auditing, a new anesthesia record-keeping system was designed. The forms are also well adapted for use in an anesthesia teaching department.


The effect of spontaneous versus controlled ventilation on the rate of rise of alveolar halothane concentration in dogs. The rate of rise of the alveolar:inspired halothane concentration (F delta/F1) ratio was measured in dogs breathing spontaneously. These rates were measured at inspired concentrations of 0.3, 1.5, and 4 percent. Initially, FON (FA/F1) ratio was measured in dogs breathing spontaneously. These rates were measured at inspired concentrations of 0.3, 1.5, and 4 percent. Initially FA/F1 rose at similar rates for all 3 concentrations, but a 4 percent, the plateau attained was lower as a result of respiratory depression. In a 2nd group of dogs, FA/FI was measured for inspired concentrations of 0.3, 1.5, 4, and 6 percent during controlled ventilation. FA/FI rose at similar rates at the lower 2 concentrations but at progressively more rapid rates at inspired concentrations of 4 and 6 percent, presumably as a result of circulatory depression.


Implications of the effects of general anesthesia on basal tear production. Ophthalmic preparations are frequently instilled topically to protect the eye during general anesthesia. The purpose of this study was to determine if such prophylactic precautions are warranted, as determined by the effect of general surgical anesthesia on basal tear production by the Schrimer I test. As general surgical anesthesia produced a marked depression of basal tear production, both mechanical protection and topical medications are recommended for the ocular care of patients undergoing general surgical anesthesia.


Lack of CNS depression from large doses of trimethaphan in sheep. It is not uncommon to observe prolonged CNS depression for several hours following controlled hypotension and halothane anesthesia for neurosurgery. The present study evaluates possible contribution of large doses of trimethaphan to CNS depression. Four adult sheep were placed on transapical left ventricular bypass (TALVB) withdrawing blood from the apex of the left ventricle through a roller pump, Pall Ultipor filter, and returning the blood to a carotid artery. In the awake and unanesthetized animals, 1 to 2 gm of trimethaphan were administered IV during each experiment while maintaining mean arterial pressure at 60 to 75 torr. Two sheep stood up and knelt down without obvious correlation with dose of trimethaphan administered at the time; two remained standing and continued eating during the trimethaphan infusion. Cardiovascular recovery from these large doses of trimethaphan was within 15 to 30 minutes after the conclusion of drug infusion. The data strongly suggest that large doses of trimethaphan have no significant CNS depression in the awake and unanesthetized sheep.


Effect of halothane and pentobarbital anesthesia on the dispersion of indicator particles passing through the central circulation of the dog. To investigate the effect of different anesthetics on the flow properties of the central circulation, curves were obtained by the multiple indocyanine-green indicator-dilution technic from 7 dogs anesthetized with halothane or pentobarbital. The width of the curve provided a measure of the dispersion of indicator particles during passage through the central circulation. The vascular resistance of the animals was altered by the administration of methoxamine and sodium nitroprusside. The authors found that dispersion in the central circulation is linearly related to the total peripheral resistance (TPR). For any value of TPR, the amount of dispersion of indicator particles passing through the central circulation was greater with halothane plus pentobarbital anesthesia than with pentobarbital only. The study indicates that the type of anesthesia influences the dispersion of indicator particles passing through the central circulation. A better under standing of this process will be helpful in interpreting the results of indicator-dilution cardiac-output determinations performed in persons under general anesthesia.


Correlation of plasma cholinesterase activity and duration of action of succinylcholine during pregnancy. Correlation between plasma cholinesterase activity and duration of neuromuscular blockade following succinylcholine (SCh) was studied in 30 healthy women undergoing laparoscopic tubal coagulation and 20 pregnany women indergoing elective repeat cesarean section. All patients received N2O-thiopental anesthesia. Cholinesterase activity in nonpregnant patients was significantly greater than in pregnant patients. Time to 90 percent recovery of control twitch height following 40 or 80 mg/m2 BSA of SCh was not significantly different in pregnant versus nonpregnant patients. There also was no correlation between plasma cholinesterase activity and duration of paralysis from SCh. The authors conclude that pregnant patients have lower cholinesterase activity, but prolonged neuromuscular blockade from SCh should not occur unless the patient is grossly overdosed with SCh. Routine use of a peripheral nerve stimulator is recommended to avoid such overdosage.


Enflurane anesthesia for surgical removal of pheochromocytoma. Four cases describing the use of enflurane as the main anesthetic during surgical removal of pheochromocytoma (PCC) are presented and the preoperative preparation and intraoperative management of the patients are discussed. Serum levels of epinephrine and norepinephrine were measured in 3 of the reported cases. Intraoperative values were extremely elevated during tumor manipulation, but there was only 1 minor episode of arrhythmias. Criteria for choosing anesthetic agents for surgical removal of PCC are outlined. The authors conclude from their experience and that of others that enflurane is as safe and effective an anesthetic as any now available for PCC excision.


The influence of succinylcholine on the duration of pancuronium neuromuscular blockade. Question has been raised as to whether the use of succinylcholine (SCh) to facilitate endotracheal intubation might influence the duration of subsequently used pancuronium. The authors studied the duration of pancuronium in 67 adult patients by measuring muscle response to nerve stimulation. Thirty-seven of these patients were given prior SCh while the remaining 30 were not. No significant difference in the duration of pancuronium block was found between these 2 groups.


Is the Bain breathing circuit the future anesthesia system? An evaluation. The Bain breathing circuit, a modified Mapleson D system, was evaluated with regard to oxygenation and CO2 elimination under controlled conditions and compared with the presently popular semiclosed breathing circuit (SCBC) with CO2 absorber. The authors demonstrated that the Bain system compares favorably with the SCBC in regard to oxygenation of manually ventilated patients with fresh gas inflow of 70 ml/kg/min while maintaining a PaCO2 at a mean of 38 torr versus an SCBC mean of 32 torr. The authors were impressed with the clinical simplicity, efficiency, and versatility of the Bain system and believe that it will play a major role in the future of anesthesia-machine design.


Host range comparisons of five serotypes of caliciviruses. Two new serotypes of San Miguel sea lion virus (SMSV), designated SMSV-4 and SMSV-5, were studied in vivo and in vitro. The host cell spectrums were compared with SMSV-1, SMSV-2, and vesicular exanthema of swine virus type A-48. Based on the result of these broad host spectrums, a numerical scoring system was devised for ranking each virus on the basis of its potential for infecting terrestrial mammals, including the important domestic species.


Diffusion hyperoxia following preoxygenation. Several minutes of 100 percent O2 breathing prior to inhalation induction with 79 percent N23 results in consistently higher PaO2 values during the first 10 minutes compared with prior air breathing. The theoretic maximum concentrating action of N2O uptake upon O2 is approximately the same (60 torr) regardless of gas breathed before induction.


Human babesiosis on Nantucket Island. Clinical features. Between 20 July and 15 Octoboer 1975, five cases of human infection with Babesia microti were diagnosed on Nantucket Island, Massachusetts. The illness was characterized by fever, drenching sweats, shaking chills, myalgia, arthralgia, extreme fatigue, and a mild-to-moderate hemolytic anemia. None of the patients had a history of splenetomy. Although all patients responded symptomatically to treatment with oral chloroquine phosphate, parasitemia and fatigue frequently persisted for several weeks to months.


Thermoregulatory defect in rats during anesthesia. Male rats of the Fischer 344, Sprague-Dawley, Brattleboro, and Wistar strains, balb/C mice, and Hartley guinea pigs were divided into 2 treatment groups. One group drank tap water while the other group drank water containing 1 mg/ml of phenobarbital. Animals were exposed to sevoflurane, enflurane, methoxyflurane, isoflurane, or halothane in a closed chamber. In some of the experiments, soda lime was included and in other the chamber was heated to 39 degrees C with a water blanket. Eighty-six percent (43/50) of Fischer 344 rats treated with phenobarbital and esposed to either sevoflurane or enflurane, in the presence of either soda lime or exogenous heat, died within a few hours after exposure. Fischer 344 rats and rats of other strains drinking phenobarbital water and exposed to methoxyflurane were affected, but to a lesser degree. Rats drinking ordinary tap water and phenobarbital-treated rats not exposed to either soda lime or exogenous heat were unaffected. Guinea pigs and mice also were unaffected. We postulate that the toxic response represents a species-specific thermoregulatory defect, precipitated by heat and occurring in rats treated with phenobarbital in combination with sevoflurane, endlurane, or methoxyflurane.


Glaucoma: the value of a diurnal curve and Goldmann visual field. A 35-year-old man was treated with maximum medical therapy for open-angle glaucoma. A diurnal curve revealed that the pressure was not controlled as might have been thought by a single reading. Reference is also made to the value of Goldmann visual field testing, since in this patient visual field defects were discovered with normal, healthy-appearing optic discs. A careful regimen including precise visual field testing and the plotting of the diurnal curve is most important in evaluating ocular hypertension and glaucoma.


Isoflurane anesthesia and pressure antagonism in mice. The anesthetic partial pressure causing a loss of righting reflex in mice (ED50) was determined for isoflurane at ambient total pressures ranging from 1 to 100 atmospheres absolute (ATA). The higher pressures, produced by adding helium, decreased isoflurane's apparent anesthetic effect: 48 percent more was needed at 100 ATA. This is consistent with previous work with N2O, where a 46 percent increase was found. The ED50 for isoflurane at 1 ATA is 0.0057 ATA, only 29 percent of that predicted by the Meyer-Overton relationship for mice.


Microassay foramphotericin B.

Microassay foramphotericin B. Depending on the hematocrit, duplicate or triplicate determinations of serum amphotericin B concentration may be made on as little as 100 mul of capillary blood obtained by finger prick. In an accurate plate diffusion bioassay, using Paecilomyces varioti as the indicator organism, levels of the drug in the therapeutic range can be determined fast enough for clinicians to modify their next dose.


Influence of folic acid on birthweight and growth of the erythroblastotic infant. III. Effect of folic acid supplementation. Seventeen infants with severe (14) or moderately severe (3) erythroblastosis were given daily oral supplements of 2-5 or 5 mg folic acid from day 16 (average) to 3-2 months (average). Their rate of weight gain, expressed as weight centiles, was followed for 1 year and was compared with that of a very similar group of 34 erythroblastotic infants without folic acid supplements. By the end of the 4th month, just after stopping additional folate intake, the median centiles for weight had risen from the 40th to the 80th centile, while in the untreated control group they rose during this period from the 35th to the 50th centile. During the second half of the year both groups declined in weight centiles, the 'treated' group ending up at the 50th centile for weight, while the control group fell to the 25th.


Influence of folic acid on birthweight and growth of the erythroblastotic infant. II. Growth during the first year. The growth of 73 erythroblastotic infants (18 mild, 19 moderate, and 36 severe) was followed. In all three grups the average centiles for weight rose during the first half of the year. There was a fall in centiles during the second half of the year in 35 of 55 infants, 17 of them dropping below their birth centiles. Throughout the first year of life there was a strong correlation between serum folate of 8 mug/1 or more and rising weight centiles, and 5 mug/1 or less and falling centiles. None of the infants had macrocytic anaemia and their haemoglobin level rose even when their weight gain fell below the expected rate. It is suggested that in some erythroblastotic infants a shortage of folic acid may be a limiting factor for a normal growth rate.


Autoradiographic in vitro studies on diurnal variation in human epidermal cell proliferation. Five men with healthy skin ranging in age from 24 to 37 years were investigated for the presence of diurnal variation in epidermal cell proliferation by the in vitro 3H-thymidine labeling technique. The following parameters were studied: The basal cell LI; the numbers of labeled basas and supra-basal cell nuclei as well as the total number of labeled nuclei situated above a basal membrane length of 100 mum. All autoradiographic parameters showed marked diurnal variations, the amplitudes of which differed individually. The basal 3H labeling index always showed the highest diurnal variation. In all patients diurnal variations of the basal and suprabasal cell proliferation occurred simultaneously and in equal direction. However, each test person showed maxima and minima of epidermal DNA synthesis at different times. No synchronism could be found when comparing the individual variations of cell proliferation. Several possible reasons for the asynchronous in vitro behaviour of epidermal cells will be discussed.


Angiocardiography in congenital subvalvular aortic stenosis: prognosis and operative indications. Twenty-three patients with congenital subvalvular aortic stenosis are reviewed. The importance of classifying this stenosis into four types based on angiocardiographic findings is stressed, as are the indications for left heart catheterization during the preoperative and postoperative course of the disease. The type of congenital subvalvular aortic stenosis should be an important consideration during the discussion of operative indications in both asymptomatic and symptomatic patients.


Granulomatous dacryoadenitis caused by Schistosoma haematobium. An 11-year-old boy from Sierra Leone developed a mass in the left lacrimal gland a year after trauma to the left side of the brow. Biopsy of the mass led to the diagnosis of schistosomiasis caused by Schistosoma haematobium, which had not been suspected previously. The initial occurrence in the orbit of such a rare, ectopic lesion in schistosomiasis and the possible relationship to previous trauma are features of special interest.


Anterior chamber depth alteration after retinal photocoagulation. Anterior chamber shallowing is a syndrome that may occur after retinal photocoagulation for diabetic retinopathy. Angle closure glaucoma may occur. Peripheral iridectomy is not indicated, as the condition is self-limited and will resolve with or without medical therapy within a few days. A vascular occlusive phenomenon leading to congestion in the choroidal system is postulated as the cause of this phenomenon. The ciliary body may be shifted anteriorly and rotated inwardly to produce the anterior chamber changes observed.


Fetal growth retardation and increased infant mortality at high altitide. An examination of recent Colorado vital statistics records was performed. The records included 194,526 live births and 3,100 infant deaths occurring during the years 1969-73, and some 466,000 live births and 71,000 infant deaths occurring during 1960-71. Birth weights at high altitude (2,744 to 3,100 m) were found to be reduced owing to retardation of intrauterine growth rather than preterm delivery. Despite decreased infant mortality at all altitudes in Colorado during the last 20 years, the infant mortality rate at high altitude remains almost twice that seen in Denver. The elevated mortaltiy at high altitude occurred within 28 days after birth, primarily in preterm (less than 38 weeks) infants. It appears that high altitude, possibly throuth the mechansif of exaggerated fetal hypoxia, retards fetal growth and increases the mortality rate of preterm infants.


Concentrations of free amino acids in brains of mice during the induction of physical dependence on ethanol and during the ethanol withdrawal syndrome. Chronic administration of ethanol to mice by inhalation for 10 days produced physical dependence demonstrated by a characteristic syndrome of withdrawal. Free amino acid concentrations in whole brain were measured at intervals during the induction of dependence and during withdrawal. During the induction of dependence there was an initial increase in brain glycine, a sustained increase in brain tyrosine and reductions in brain GABA and proline. Serine and isoleucine concentrations were consistently reduced during the induction of dependence, but this change was not significant (P less than 0-05) at any single time interval studied. After the withdrawal of ethanol only the reductions in GABA and proline persisted during the withdrawal syndrome. In addition to these changes an increase in brain glycine concentration was observed during the ethanol withdrawal syndrome. In an attempt to discriminate between the immediate, metabolic effects of ethanol on central amino acid concentrations and those changes associated with the induction of ethanol dependence, the results were compared with those obtained when mice were exposed to a high concentration of ethanol vapour for 3 h. Although this produced similar blood ethanol concentrations, no evidence of physical dependence was observed. The changes in central amino acid concentrations differed from those seen during the induction of dependence in that no change in isoleucine concentration occurred, and that the reduced concentrations of GABA and proline very rapidly reverted to control values when ethanol was removed. The possible role of central amino acids in ethanol dependence is discussed.


HLA antigens and uveitis.

HLA antigens and uveitis. We have studied the antigen frequencies in a group of adult patients with acute and chronic iridocyclitis and chronic, cyclitis. In patients with iridocyclitis a statistically significant increased incidence of HLA-B27 was noted, even in those patients without joint or systemic disease. Patients with chronic iridocyclitis also were noted to have an increased incidence of B27; however, those patients with chronic cyclitis did not. On the basis of HLA-B27 studies there appears to be a fundamental difference between adult and juvenile iridocyclitis.


Subunit structure and hybrid formation of bovine pyruvate kinases. After denaturing either type M or L pyruvate kinase by guanidine hydrochloride, urea, or low pH, enzymatic activity and quaternary structure can be recovered by diluting the enzyme into buffer containing beta-mercaptoethanol. After denaturation of type M pyruvate kinase by guanidine hydrochloride, the yield and polarization of the intrinsic protein fluorescence, as well as most of the circular dichroism characteristic of the native enzyme, were regained very rapidly, while enzymatic activity was recovered much more slowly. Under the conditions used, about 50% of the original M and 30-50% of the original type L activity were typically recovered. Average half-times for recovery of enzymatic activity were 37 min for type M and 104 min for type L but depended somewhat on the renaturation buffer and on protein concentrations in the renaturation medium. If types M and L pyruvate kinases are renatured together, an approximately random recombination of the two subunits types results in a five-membered hybrid set. We have used this hybridizability to determine the kinetics of reformation of the native tetramer by denaturing each isozyme and beginning its renaturation separately at various times mixing the two isozymes and continuing their renaturation together. These studies indicate that reformation of stable tetramers occurs relatively slowly, qualitatively paralleling the regain of enzymatic activity, and that tetramer formation may be necessary for enzymatic activity. Using a similar technique to test for spontaneous dissociation of the native isozymes in buffer, we find that type L, but not type M, reversibly dissociates into dimers and monomers in buffer solutions. This dissociation is decreased by the presence of the substrate, phosphoenolpyruvate, by Mg2+ ions, or by the allosteric effector, fructose bisphosphate.


Magnetic resonance studies of the binding site interactions between phosphorylcholine and specific mouse myeloma immunoglobulin. The interaction of phosphorycholine-binding mouse myeloma protein M603 and the isotopically substituted hapten phosphoryl[methyl-13C] choline has been investigated using 13C and 31P nuclear magnetic resonance (NMR) spectroscopy. Upon binding to antibody, upfield shifts of 0.7 and 1.5 ppm are observed for the hapten 13C and 31P resonances, respectively, and both spectra are in the "slow" exchange limit. Linewidth analysis indicates some immobilization of the phosphate group but essentially unrestricted methyl group rotation for the bound hapten. Hapten-antibody dissociation rate constants of 10 and 38 s-1 are calculated from 13C and 31P NMR spectra, respectively, suggesting the possibility of differential dissociation rates for the two opposing ends of the phosphorylcholine molecule. The NMR data are entirely consistent with the known x-ray structure of the M603 Fab'-phosporylcholine complex (Segal,D.M., Padlan, E.A., Cohen G.H., Rudikoff S., Potter,M., and Davies, D.R. (1974), Proc. Natl. Acad. Sci. U.S.A. 71, 4298).


Control of breakdown of the polyadenylate sequence in mammalian polyribosomes: role of poly(adenylic acid)-protein interactions. The poly(adenylic acid) [poly (A)] segment in mouse sarcoma polysomes in not hydrolyzed by snake venom exonuclease under conditions which cause extensive degradation of the poly(A) in deproteinized polysomal RNA. The protecting effect of polysomes is presumably caused by the interaction between the poly(A) sequence and the protein known to be associated with it. This protection is reduced at low KCl concentration, but addition of exogenous RNA restores the protecting effect. The poly(A) segment also becomes susceptible to exonuclease after fragmentation of the polysomes by mild ribonuclease treatment. The latter treatment releases the poly(A) in association with protein. The poly(A) sequence in polysomes in readily degraded by a cytoplasmic extract of S-180 cells. Partial purification leads to a preparation active against the poly(A) in polysomes under conditions where no fragmentation of the messenger RNA is observed. Snake venom exonuclease increases the activity of the cytoplasmic preparation against poly(A) in polysomes. The active cytoplasmic factor appears to interfere with the poly(A)-protein interaction, thus rendering the polynucleotide susceptible to degradation by exonuclease. The poly(A) sequences in polysomes and in free cytoplasmic nucleoprotein particles are hydrolyzed to the same extent. The results suggest that the poly(A) sequence is normally protected from nucleases by virtue of its association with protein. The slow reduction in poly(A) size in cytoplasmic mRNA can be accounted for by a factor capable of interfering with the poly(A)-protein interaction. The latter interaction seems also dependent on the structural integrity of the polysomes or messenger ribonucleoproteins. It is suggested that a polynucleotide segment adjacent to the poly(A) can modulate the affinity of the protein for the latter sequence, thus permitting control of poly(A) stability in individual messenger RNAs.


Study of protein topography with flash photolytically generated nonspecific surface-labeling reagents: surface labeling of ribonuclease A. A method for nonspecifically labeling essentially all exposed residues of a protein is described. A reactive aryl nitrene is generated from N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-Taurine), within 500 mus by flash photolysis in the presence of protein. The reactive nitrene is inserted in about 2 ms into those carbon-hydrogen bonds of the protein that are exposed to the solvent. The method is applied here to ribonuclease A to demonstrate the different degree of labeling of the native and denatured protein. On the basis of amino acid analysis, it appears that residues of the native protein that are buried in the interior of the molecule (as judged from the x-ray structure) do not react with the nitrene. However, when these residues (even nonreactive ones such as valine and proline) are exposed by denaturation of the protein, they do react with the nitrene. It is shown that native ribonuclease A retains 90% of its enzymatic activity when flashed in the absence of NAP-Taurine. This small loss in activity arises from the disruption of a limited portion of the native enzyme structure, as judged by circular dichroism, ultraviolet, and Raman spectra. The site of this limited disruption may be a portion of the enzyme surface near the Cys-26-Cys-84 disulfide bond. The utility of this surface labeling technique for studying the pathways of protein folding or unfolding is discussed.


Orientation and dynamics of phospholipid head groups in bilayers and membranes determined from 31P nuclear magnetic resonance chemical shielding tensors. 31P nuclear magnetic resonance (NMR) powder spectra have been used to determine the principal values of the chemical shielding tensors of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidic acid. The shielding tensors in all cases were clearly nonaxial. The principal values for the monoester phosphatidic acid shielding tensor are -40, -4, and 48 ppm relative to 85% H3PO4. By contrast the diesters have values of -87, -25, and 119 ppm for phosphatidylcholine, -81, -20, and 105 ppm for phosphatidylethanolamine, and -80, -20, and 112 ppm for phosphatidylserine. This difference reflects the sensitivity of the 31P shielding tensor to chemical environment. Anisotropic motion of the molecules in lamellar dispersions of phospholipids caused an incomplete averaging of the shielding tensors resulting in partially narrowed spectra. Spectra of various phospholipid dispersions were recorded as a function of temperature and transitions observed at the gel-liquid crystalline phase transition temperatures. Using a reasonable set of initial conditions, it was shown that a simple model of molecular motion could successfully predict the observed spectra and their temperature dependences. The model includes rotations about the P-O(glycerol) bond and the molecular z axis and a wobble of the molecule about the bilayer normal. As the temperature increases, the wobble amplitude increases and the spectra narrow. A preliminary 31 P NMR spectrum of chick embryo fibroblasts is included. The similarities between this spectrum and those of the lamellar dispersions indicate that some of the predominant features are due to the phospholipid resonances.


Thermodynamics of antibody-antigen reactions. 1. The binding of simple haptens to two classes of antibodies fractionated according to affinity. The thermodynamic parameters which characterize the binding of dinitrophenylglycine and dinitrophenylmethoxypoly(ethylene glycol) to selected affinity classes of equine IgG and IgG(T) antibodies were determined by fluorescence quenching and flow calorimetry. The binding enthalpies and entropies were in all cases large and negative, falling in the ranges -14 to -17 kcal/mol and -18 to -25 eu, respectively. The differences in the enthalpies and entropies of binding for different affinity classes and for different haptens are discussed with reference to differences in the structures of the haptens studied and as indications of differences in binding site structure. In addition, the apparent existence of fluorescent side chains which can transfer energy to either hapten binding site in IgG(T) antibodies but not in IgG antibodies is interpreted as indicative of a smaller average interbinding site distance in IgG(T) than in IgG antibodies.


Thermodynamics of antibody-antigen reactions. 2. The binding of bivalent synthetic random coil antigens to antibodies having different antigen precipitating properties. The objects of this study were the equine IgG and IgG(T) classes of antibodies with immunologic specificity for the dinitrophenyl group and bivalent antigens consisting of linear poly(ethylene glycol) polymers which terminated at both ends in dinitrophenyl groups. Complex formation between antibodies of both classes and one of several sharp fractions of antigen having number average molecular weights in the range 25 000 to 75 000 were studied by measuring the light scattered from solutions containing equimolar amounts (approximately 5 x 10(-6) mol/L) of one of the antibodies and one size fraction of antigen, and variable amounts of monovalent hapten. The data were analyzed in the context of a model that accounted for the formation of linear and cyclic complexes of all extents of aggregation. Two parameters in addition to the intrinsic antibody-dinitrophenyl group association constant were found to be necessary in the assumed equilibrium model to account for the behavior of the system. One of these accounted for the looses in configuration entropy that resulted when a random-coil polymer became bound at one end to a space-occupying antibody. The other was a ring closure factor for the formation of cyclic complexes. Ring closure factors for the formation of larger cyclic complexes (present in only small amounts under the conditions studied) were related to the ring closure factor for the formation of the smallest, which was found to increase as antigen size decreased, and for each antigen size to be consistently higher for IgG(T) antibody than for IgG antibody. Comparison of the theoretically estimated values of the two parameters within their measured values indicated that the average conformation of IgG antibodies in solution is open ("T" shaped)  but the average inter-Fab are angle in IgG(T) antibodies is approximately 60 degrees or less.


Nitrotyrosine chelation of nuclear magnetic resonance shift probes in proteins: application to bovine pancreatic trypsin inhibitor. The interactions of Pr(III) and Eu(III) with specifically nitrated derivatives of the basic bovine pancreatic trypsin inhibitor have been studied using optical spectroscopy and nuclear magnetic resonance (NMR) at 250 and 270 MHz. Stability constants for proton and metal binding to nitrotyrosines 10 and 21 determined optically are in good agreement with those from NMR. Observations of the Eu(III)-induced NMR shifts of the ring protons of nitrotyrosine 21 allowed calibration of the magnetic interactions for this binding site. The Pr(III)-induced shifts for several resolved nonexchangeable backbone proton resonances were compared with calculated shifts using the known x-ray structure. With several simplifying assumptions, the Pr(III)-induced shifts were used to assign one alpha-CH and five NH protons to compatible sets of backbone positions which are consistent with the known pH dependence and resistance to exchange with solvent D2O. Some of the more general aspects of lanthanide-induced shifts are discussed with reference to their use in proteins. Due to the complexities of the analysis of the shift data, the most straightforward use of this technique is in conjunction with the relaxation probe Gd(III) for measurement of intramolecular distances.


Structure of bovine trypsinogen at 1.9 A resolution. The three-dimensional crystal structure of bovine trypsinogen at approximately pH 7.5 was initially solved at 2.6 A resolution using the multiple isomorphous replacement method. Preliminary refinement cycles of the atomic coordinates trypsinogen have been carried out first to a resolution of 2.1 A, and later to 1.9 A, using constrained difference Fourier refinement; During the process, structure factors Fc and phi c were calculated from the trypsinogen structure and final interpretation was based on an electron-density map computed with terms (2 Fo - Fc) and phases phic at a resolution of 1.9 A. Crystals of trypsinogen grown from ethanol-water mixtures are trigonal with space group P3121, and cell dimension a = 55.17 A and c = 109.25 A. The structure is compared with the bovine diisopropylphosphoryltrypsin structure at approximately pH 7.2, oirginally determined from orthohombic crystals by Stroud et al. (Stroud, R.M., Kay L.M., and Dickerson, R.E. (1971), Cold Spring Harbor Symp. Quant. Biol. 36, 125-140; Stroud, R.M., Kay, L.M., and Dickerson, R.E. (1974), J. Mol. Biol. 83, 185-208), and later refined at 1.5 A resolution by Chambers and Stroud (Chambers, J.L., and Stroud, R.M. (1976), Acta Crystallogr. (in press)). At lower pH, 4.0-5.5 diogen, with cell dimensions a = 55.05 A and c = 109.45 A. This finding was used in the solution of the six trypsinogen heavy-atom derivatives prior to isomorphous phase analysis, and as a further basis of comparison between trypsinogen and the low pH trypsin structure. There are small differences between the two diisopropylphosphoryltrypsin structures. Bovine trypsinogen has a large and accessible cavity at the site where the native enzyme binds specific side chains of a substrate. The conformation and stability of the binding site differ from that found in trypsin at approximately pH 7.5, and from that in the low pH form of diisopropylphosphoryltrypsin. The catalytic site containing Asp-102, His-57, and Ser-195 is similar to that found in trypsin and contains a similar hydrogen-bounded network. The carboxyl group of Asp-194, which is salt bridged to the amino terminal of Ile-16 in native trypsin or other serine proteases, is apparently hydrogen bonded to internal solvent molecules in a loosely organized part of the zymogen structure. The unusually charged N-terminal hexapeptide of trypsinogen, whose removal leads to activation of the zymogen, lies on the outside surface of the molecule. There are significant structural changes which accompany activation in neighboring regions, which include residues 142-152, 215-550, 188A-195. The NH group of Gly-193, normally involved in stabilization of reaction intermediates (Steitz, T.A., Henderson, R., and Blow, D.M. (1969), J. Mol. Biol. 46, 337-348; Henderson, R. (1970), J. Mol. Biol. 54, 341-354; robertus, J.D., Kraut, J., Alden, R.A., and Birkoft, J.J. (1972), Biochemistry 11, 4293-4303) in the enzyme, is moved 1.9 A away from its position in trypsin...


Proton magnetic resonance study of cholesterol transfer between egg yolk lecithin vesicles. The intensity of the proton magnetic resonance signal of the (CH2)n chain in phospholipids of sonicated lecithins is sensitive to the cholesterol content in the resulting vesicles. In the present study this signal has been used to monitor transfer of cholesterol between phospholipid vesicles. Vesicles prepared from  pure egg yolk lecithin were mixed with vesicles that contained equimolar amounts of cholesterol and lecithin, and the time evolution of the (CH2)n signal intensity was followed. The results show that a homogenous distribution of cholesterol amoung vesicles is reached after about 4 h at 37 degrees C and 60  h at 4 degrees C. In order to determine the mechanism of the cholesterol transfer process, experiments were performed over a 2.5-fold range of vesicles concentrations. The accuracy of the kinetic results was not sufficient however to decide on the order of the reaction with respect ot vesicle concentration. Simultaneous observation of the choline proton resonance in the presence of Eu+3 and Pr+3 indicates that fusion between vesicles does  not occur during cholesterol transfer.


Semiconservative DNA replication in vitro. I. Properties of two systems derived from mouse P-815 cells by permeabilization or lysis with Brij-58. To study DNA replication in vitro, mouse P-815 cells were either permeabilized by hypotonic treatment or gently lysed with the detergent Brij-58. In the presence of KCl, EGTA, creatine phosphokinase, creatine phosphate, sucrose, dithiothreitol, CTP, GTP, UTP, and HEPES at pH 7.8, both in vitro systems required similar concentrations of all four deoxyribonucleoside triphosphates, ATP, Mg2+, and dextran. Incorporation of [3H] dTTP was due to semiconservative DNA replication and was restricted to S-phase nuclei. No repair replication was detectable. In the first 20 min, the rate of DNA replication in vitro was 30--40% of the in vivo rate, and after 60 min, about 3% of the genome were replicated. Preexisting bulk DNA was not fragmented during permeabilization or lysis with Brij-58 and during replication in vitro. Size distributions of growing strands in vitro were similar to those found in vivo. Neither cycloheximide (100 mug/ml) nor hydroxyurea (3 mM) inhibited DNA replication in vitro.


Semiconservative DNA replication in vitro. II. Replicative intermediates of mouse P-815 cells. DNA chain growing during semiconservative replication was studied using both in vitro systems described in the preceding paper (preceding paper, ref 1) 3H-Labeled, 4-S Okazaki fragments synthesized in vivo just prior to permeabilization or lysis with Brij-58 were metabolically stable and quantitatively chased into high molecular weight DNA (20--100 S) during a subsequent incubation in vitro. Thus, DNA replication continued in vitro at the same growing points that were active in vivo. After a 20-s pulse at 30 degress C in vitro, more than 50% of incorporated radioactivity was found in the 4 S region of alkaline sucrose gradients suggesting a totally discontinuous mode of DNA chain growth. If the pulse were followed by a 1-min chase, 4-S molecules were converted into 6--12-S intermediates which upon continued incubation were joined with growing 20--100-S molecules (replicon-sized chains). Formation of all three classes of replicative intermediates, Okazaki fragments, 6--12-S intermediates, and 20--100-S molecules, occurred in vitro at least during the first 20 min. During this time, average rates of DNA chain growth and overall DNA synthesis were reduced to about the same extent, if compared to rates of intact cells. Thus, reduced chain growth rates appear to reflect primary deficiences of our in vitro systems, while initiation of replicative intermediates still occurs.


Reductive alkylation of mammalian ribosomes. 40- and 60-S ribosomal subunits and 80-S ribosomes from rat liver were highly labelled by reductive methylation using formaldehyde and sodium boro-[3H] hydride, under conditions which did not decrease their activity in poly-U-directed polyphenylalanine synthesis. Dissociation of the monosomes, subunits dimers, and polysomes into free subunits was observed after methylation. Free proteins labelled after extraction from the ribosomal subunits incorporated 7 times more radioactivity than when labelled in the subunits. Proteins extracted from methylated subunits and ribosomes were analyzed by two-dimensional gel electrophoresis, and the radioactivity of each protein was compared to that of the same free protein. A classification of the proteins was established according to their accessibility to the reagents in the subunits and the ribosomes.


Identification of a mucosal form of enteropeptidase in triton X-100 extracts of porcine duodenal mucosa. Porcine enteropeptidase (EC 3.4.21.9) purified from acetone powders of fresh duodenal fluid shows a molecular weight, as determined on Ultragel AcA-34, of 190000. Enteropeptidase has been solubilised from pig intestinal mucosa using 1% (v/v) Triton X-100. When Triton X-100 extracts of freeze-dried mucosa after partial fractionation on DEAE-cellulose were chromatographed on Sephadex G-200, the bulk of the activity eluted in the void volume rather than with an expected Ve/V0 ratio of about 1.24 corresponding to a molecular weight of around 200000. Gel filtration of aqueous mucosal extracts obtained in the absence of Triton X-100 showed two regions of enzymic activity in approximately equal proportions, one in the void volume, and the other with the expected Ve/V0 ratio of 1.24, whereas the Triton X-100 extracts of the residue from the above extract showed the presence of only the macromolecular species of enteropeptidase. This species was excluded from Sepharose 4B. It was confirmed that aminopeptidase was also extracted by Triton X-100 in a molecular form which was excluded from Sepharose 4B. The results suggest that Triton X-100 extracts enteropeptidase with a membrane component attached and in agreement with this it was found that proteolysis rapidly converted the macromolecular form to a stable smaller molecular species corresponding in size to that found in solution in the duodenal fluid. There was full recovery of the enzymic activity following this conversion. Papain and trypsin brought about an almost complete conversion to the smaller form of enteropeptidase whereas chymotrypsin, pancreatin and an intestinal peptidase preparation were only partially effective. It is concluded that membrane bound enzymes such as enteropeptidase and aminopeptidase are bound to the intestinal brush border membrane in a similar manner and are not actively secreted into the lumen but rather are largely released or solubilised by the combined action of the bile and pancreatic secretions.


Partial dissociation and renaturation of embryonic chick delta-crystallin. Characterization by ultracentrifugation and circular dichroism. 1. delta-Crystallin from 15-day-old embryonic chick lenses was characterized by circular dichrosim (CD) spectroscopy. Examination by CD spectroscopy in the far ultraviolet (190-250 nm) demonstrated that the secondary structure of delta-crystallin has at least 75% alpha-helix; the delta-crystallin subunits dissociated in sodium dodecyl sulfate retain their alpha-helical content. This appreciable alpha-helical content of delta-crystallin contrasts with the absence of alpha-helix in other lens crystallins. 2. As judged by CD spectroscopy in the near ultraviolet (250-320 nm) the tertiary structure of embryonic delta-crystallin is not readily disrupted by environmental changes, such as NaCl, KSCN or the non-ionic detergent Emulphogene BC 720, and is stable to temperature fluctuation between 2 and 56 degrees C. 3. Experiments were directed towards deaggregation and renaturation of the four subunits of embryonic delta-crystallin by treatment with urea or guanidine hydrochloride. The native tertiary structure of delta-crystallin was lost above 4 M urea or 2 M guanidine hydrochloride, as judged by CD spectroscopy in the near ultraviolet. Ultracentrifugation at sedimentation equilibrium showed that in 4 M urea delta-crystallin dissociates into dimeric subunits, while in 2 M guanidine hydrochloride delta-crystallin exists as a mixture of dimeric and tetrameric subunits. Dialysis of delta-crystallin from 4 M urea resulted in reaggregation of the subunits into tetramers, about 50% of which showed native tertiary structure. Dialysis from 2 M guanidine hydrochloride also resulted in tetramer formation, and about 35% was recovered with native conformation. Removal of denaturant by dialysis produced no native teritary structure after treatment with 8 M urea, but about 15% native conformation after treatment with 6 M guanidine hydrochloride.


Beta-conglycinin from soybean proteins. Isolation and immunological and physicochemical properties of the monomeric forms. Beta-conglycinin consisting of six major isomers (designated B1- to B6-conglycinin) was dissociated and fractionated on columns of DEAE- and CM-Sephadex in buffers containing 6 M urea. Three major (alpha, alpha' and beta) and one minor (gamma) subunits were isolated and further characterized by gel electrophoresis and gel electrofocusing. Gel electrophoresis in urea and in sodium dodecyl sulfate, and gel filtration in 6 M guanidine hydrochloride gave a molecular weight of 57 000 for alpha, alpha' subunits; and 42 000 for beta and gamma subunits. The isoelectric points of the isolated subunits, measured by disc gel electrofocusing, were as follows: alpha, 4.90; alpha', 5.18; beta, 5.66-6.00. On gel electrofocusing, beta subunit showed four microheterogeneous components; three of them comprised 95% of the total beta subunit. Leucine and valine were the N-terminal amino acids of beta and alpha alpha' subunits, respectively. The isolated subunits contained mannose and glucosamine in varying quantities. Two carbohydrate moieties were calculated for one mole of alpha, alpha' subunits; and one carbohydrate moiety for the beta subunit. Considerable similarity in the amino acid composition of alpha and alpha' subunits was observed. The beta subunit was devoid of cysteine and methionine; and in comparison with alpha, alpha' subunits, had a higher content of hydrophobic amino acids. The isolated subunits exhibited antigen-antibody reaction with antisera to the native beta-conglycinin. Each of them was partglycinins. The alpha and alpha' subunits were in addition identical with each other and with B5-, B6-conglycinins. They were immunologically unrelated with beta subunit. The recovery of immuno-properties from the individual subunits may be attributed to the reconstruction of the three-dimensional structure upon removal of denaturing reagents.


Mammalian melanocytes do not use phenylalanine for melanin synthesis. Hamster melanoma cells (RPMI 3460) were examined for their ability to utilize phenylalanine for melanin biosynthesis. There was a small but significant incorporation of L-[1-1414C] phenylalanine into hot acid-insoluble cellular material in the presence of cycloheximide. However, this radioactivity was removable from the acid-insoluble fraction by pronase digestion. A similar percentage of L-[U-14C] leucine incorporation was likewise resistant to cycloheximide inhibition. Residual protein synthesis is apparently responsible for the incorporation of both amino acids. Cycloheximide did not inhibit melanin synthesis. These results suggest that mammalian melanocytes do not use phenylalanine for melanin synthesis. Phenylalanine is not incorporated directly into melanin, nor do the cells appear to convert it to tyrosine via a phenylalanine hydroxylase.