Dual action of chloroquine on frog's skeletal muscle contraction. Studies on the effect of chloroquine on frog's rectus abdominis muscle have been performed. Relatively low concentrations of chloroquine, 5 times 10-5 and 2 times 10-4 g/ml inhibited cholinesterase activity and potentiated acetylcholine (ACh)-induced contractions but antagonized carbachol and caffeine contractions, as well as ACh-induced contractions of eserinized muscle. High concentrations (5 times 10-4 and 2 times 10-3 g/ml) non-competitively antagonized contractions to ACh, carbachol, caffeine and potassium. It was suggested that the blocking action of chloroquine was due to its local anaesthetic property and interference with intracellular calcium movements.


Enterohepatic haemodynamics in calves during acute systemic anaphylaxis. This study was designed to investigate the splanchnic congestion and gastrointestinal lesions frequently found in calves during anaphylaxis. Calves were sensitised to horse serum and the following cardiovascular parameters monitored; carotid arterial pressure, mesenteric arterial pressure, mesenteric venous pressure, mesenteric arterial flow, and abdominal venous pressure. Responses to histamine, 5-hydroxytryptamine (5HT), bradykinin, dopamine, noradrenaline and horse serum were recorded. Anaphylaxis was characterised by a diphasic arterial hypotension, a parallel rise in mesenteric venous pressure, a fall in mesenteric arterial flow and a threefold rise in mesenteric vascular resistance. It is suggested that during anaphylaxis a rise in hepatic vascular resistance occurs, resulting in pooling of blood in the venous side of the mesenteric vasculature and possibly in the liver itself. This increase in hepatic resistance may be caused by circulating vasoactive agents released by the anaphylactic process, a local Schultz-Dale type reaction, or a reflex triggered by systemic baroreceptors.


Effects of clonidine on baroreceptor function in anesthetized dogs. The effects of clonidine (15-30 mug/kg i.v.) on carotid sinus and other baroreceptors were investigated in anesthetized dogs. In 14 control dogs, right carotid sinus pressure was controlled by retrograde perfusion through the common carotid artery at constant flow with femoral arterial blood. Graded reductions in heart rate and blood pressure induced by graded increases in carotid sinus pressure were prevented, whereas reflex bradycardias associated with norepinephrine pressor activity were potentiated by clonidine. Norepinephrine-induced bradycardia, although reduced, still persisted after chronic bilateral sinusectomy and these responses were also potentiated by clonidine. In contrast, clonidine did not potentiate reflex bradycardia in dogs 20 days after aortic stripping. In intact dogs, clonidine inhibited the response to bilateral carotid artery occlusion and to carotid sinus nerve stimulation. These studies suggest that clonidine can inhibit carotid sinus baroreceptor function and simultaneously potentiate other, presumably aortic, baroreceptor activity.


Motor nerve terminal response to edrophonium in delayed DFP neuropathy. A localized peripheral neuropathy was induced in cats with di-isopropyl fluorophosphate (DFP). Soleus nerve-muscle preparations, and the motor nerve terminals in particular, were evaluated for responsiveness to edrophonium (200 mug/kg i.v.). Potentiation of contractile strength was absent 24 hr after DFP, and showed a trend toward recovery 7-14 days post-DFP; it then fell to about 25% of normal 3 weeks following DFP administration. During the ensuing 5 weeks this aspect of edrophonium responsiveness was largely regained. The underlying post-drug repetition which gives rise to the potentiated responses was not demonstrable in either the nerve or muscle 3 weeks after DFP, but was again observed 8 weeks after poisoning. These findings suggest a delayed peripheral neuropathy indicative of a trophic deprivation and further illustrate a motor nerve terminal deficit as the initial function alteration in DFP neuropathy.


[Some compounds correlated to pyracrimycin A]. Some biological characteristics of pyracrimycin A (I), a metabolic compound isolated from cultures of Streptomyces eridani are examined. Some other compounds related to I, obtained both by chemical reaction from the natural product and by synthesis are described. The in vitro antibacterial activity seems to be due to the presence of the unsaturated moiety --N equals (see article) --CH equals CH-- (see article) equals O.


The search for new drugs in the group of xanthine derivatives. XXXVIII - 1,3-dimethyl-8-alkyl-6H,7H,10H-(1,2,4-)-triazepino(4,3-f)purine-2,4-(1H,3H)-diones and their derivatives. Condensation of 1-(8'-chlorotheophyllinyl-7')-alkan-3-ones with hydrazine hydrate gave 8-alkyl derivatives of the new heterocyclic ring system: 1,3-dimethyl-6H,7H,10H-(1,2,4)-triazepino [4,3-f] purine-2,4-(1H,3H)-dione (I, II). 10-Acyl (III - VI) and 10-beta-cyanoethyl (VII, VIII) derivatives of compounds (I) and (II) were easy obtained in good yield. With p-nitrophenyl- and isonicotinoylhydrazines only the corresponding hydrazones of 1-(theophyllinyl-7')-alkan-3-ones (IX, X) were formed. In the attempt to obtain 10-aryl, hydroxyalkyl or acyl derivatives of the new tricyclic ring system by the reaction of 1-(8'-chlorotheophyllinyl-7')-butan-3-one with phenyl-, beta-hydroxyethyl- or acetylhydrazines only decomposition of the ketones to 8-chlorotheophylline and vinylketones was observed. The structures of the new compounds were confirmed by U.V. and N.M.R. spectroscopy and partly by mass spectrometry.


[Isoxazolylpenicillins with cycloaliphatic substituents in the isoxazole ring]. Some derivatives of oxacillin were prepared containing in place of the phenyl group a cyclohexyl, cyclohexylphenyl, phenylcyclohexyl or diphenylyl moiety. In the same compounds a methylene bridge between the carboxy group and the heterocyclic ring was inserted. The results of in vitro evaluation of antibacterial activity are given.


Antimicrobial properties of some 3-acyl-4,7-disubstituted indoles. In continuation of research on indole derivatives as potential chemotherapeutic agents, the antimicrobial properties of some 4,7-disubstituted 3-acylindoles were studied; the derivatives tested were 4,7-dimethoxy-, 4-hydroxy-7-methoxy-, 4,7-dihydroxyindoles and indole 4,7-quinones. Only the dihydroxy compounds (V a-c) proved active against certain bacterial strains. In order to test whether the action mechanism was analogous to that of mitomycin, a series of experiments was carried out to study the formation of complexes with DNA. It was found that only the substances with antimicrobial activity can form complexes, and it is therefore suggested that the action mechanism involves interference with cellular DNA.


[Transport and mode of action of sennosides]. Prior to attempting to develop a new laxative, pharmacodynamic studies were conducted on the individual active constituents of senna. Sennoside A, sennoside B and rhein were injected intravenously and were introduced into the isolated stomach, small bowel and large bowel of the anaesthetised pig. Blood, urine and bile, collected over 6 hours, were assayed by a novel fluorometric technique. Recoveries and measured oil water partition coefficients indicate that the sennosides are virtually non-absorbed in the stomach and small bowel. Following administrations outside the large bowel, anthracene derivative concentrations in the lumen and wall of the colon were below the limits of detection. Thus, a transport theory involving absorption and resecretion of sennosides into the large bowel is discounted. The results support a mode of action involving direct transport through the alimentary canal to the large bowel where micro-organism break the sugar-anthracene bonds. Derivatives of the liberated lipophilic aglycones are absorbed into the wall of the colon and stimulate the nerve plexuses thereby leading to defaecation. The "colon-specific" mode of action of the natural anthrone glycosides appears to be an ideal method of stimulating bowel action.


[Research in antitumoral chemotherapy. X. Cytotoxic and antitumoral activity of beta-nitrostyrenes and of composed nitrovinyl derivatives]. In previous work the antitumoral cytotoxicity of beta-nitrostyrenes obtained by simplification of the aristolochic acid molecule was demonstrated. The effect of modifying the three characteristic parts of the beta-nitrostyrene molecule has now been investigated. The results obtained in vitro and in vivo allow hypothesis of a mechanism of action for the various beta-nitrostyrene and nitrovinyl compounds studied and definition of the maximum simplification compatible with retention of biological activity.


Solutions of the Hodgkin-Huxley equations modified for potassium accumulation in a periaxonal space. Hodgkin and Huxley equations were modified to include the properties of an external diffusion barrier separated from the axolemma by a thin periaxonal space in which potassium ions accumulate as a function of membrane activity. Further modifications in the equations took into account new values for gK and new functions for alphan, betan, alphah, and betah derived from voltage clamp experiments on Loligo pealei giant axons. Equations were solved on a PDP-11 computer using the Gear predictor-corrector numerical method. In comparison with the original Hodgkin and Huxley equations, the modified equations for membrane potentials gave: 1) more accurate representations of the falling and undershoot phases of the membrane action potential, 2) more accurate representation of thresholds and latencies, 3) increases in the periaxonal space potassium ion concentration, Ks, of about 1 mM/impulse, 4) proper predictions of the time course and magnitude of either undershoot decline or periaxonal potassium ion accumulation during trains of membrane action potentials elicited by repetitivie short duration stimuli, and5) a somewhat more accurate representation of adaptation (finite train and nonrepetitive responses) during long duration constant current stimulation.


Noise measurements in axon membranes.

Noise measurements in axon membranes. A fluctuation (noise) component, which arises from spontaneous microscopic conductance perturbations, about the mean conduction characteristics of small areas of axon membrane has been measured and appears to relate to voltage-dependent potassium-ion movements. Since the kinetics of relaxing membrane structures which produce ion permeation noise may be deduced from the form of power density spectra of fluctuations, a comparison of measured and theoretical noise spectra may lead to direct tests of conduction of models.


Potassium and sodium current noise from squid axon membranes. Current noise under voltage clamp has been measured from the giant axon of Loligo vulgaris. Relatively large areas (up to 0.4 cm2) were used for these measurements, under standard space clamped conditions. Current noise spectral densities were studied in the range of 0 to 20 C, -100 to -40 mV and in normal, tetrodotoxin, and tetraethylammonium ion solutions. Three components of current noise were found, l/f-noise, and a K and a Na component.


Voltage clamp simulation.

Voltage clamp simulation. The voltage clamp experiment on the squid giant axon is successful because of the special characteristics of the preparation: cylindrical shape, large diameter, and so on. The method is much more difficult to apply to small cells and to networks of cells because voltage gradients and unwanted stray impedances are not readily eliminated. Simulation of the voltage clamp experiment is proposed as a method for determining when these factors and the characteristics of the clamp electronics affect the experimental results, for evaluating experimental techniques for improving the quality of the clamp, and as a possible method of learning something about the membrane when no experimental improvement is feasible. The numerical methods for including one spatial variable in the analysis are reviewed briefly. Several examples of voltage clamp simulations are discussed: double sucrose gap clamp of axons, clamp of the giant synapse, single sucrose gap clamp of cardiac muscle bundles, point clamp of the end of a fiber bundle, and the steady-state three-microelectrode clamp of a cable with nonlinear membrane. The results indicate that the quality of a clamp cannot be evaluated from the voltage and current records as commonly measured.


Propagation of action potentials in inhomogeneous axon regions. Described are studies of propagation of action potentials through inhomogenous axon regions through experiments performed on squid giant axons and by computer simulations. The initial speed of propagation of the action potential is dependent upon the stimulus waveform. For a rectangular pulse of current, the action potential travel initally at a high speed that declines over the distance, reaching a constant speed of propagation at about 1-5 resting length constants; this distance depends on the stimulus strength. additional experiments studied the effects of changing the axon diameter and of introducing a temperature step. It was found that the propagated action potential suffers profound modification in shape and velocity as it reaches the region of transition. In both cases, it was possible to obtain reflected action potentials. A region of increased effective diameter was produced experimentally in the squid giant axon by insertion of an axial wire as usually employed in voltage clamps. It was found that the action potential, at the axial wire tip region, undergoes shape changes similar to those obtained tn simulations of a region of increased diameter as in a junction with the axon and soma in motor neurons. It is conducluded that the gaint axon can be used to reproduce simple electrical behaviors in other structures.-Ramón, F., R. W. Joyner and J.W. Moore. Propagation of action potentials in inhomogeneous axon regions.


Principles and applications of fluctuation analysis: a nonmathematical introduction. The mechanisms underlying many of the processes studied by membrane biophysicists are inherently probabilistic, and therefore exhibit random fluctuations around the mean of behavior. These fluctuations reflect the underlying probabilistic mechanism and therefore can sometimes provide information, not otherwise available, about these mechanisms. Fluctuations may be characterized by their spectra which are obtained from a Fourier analysis of the experimental records. When a theory for membrane processes is available, it makes predictions about fluctuation spectra and therefore may be tested by examining these spectra. Theories about gating behavior at the frog neuromuscular junction have been tested in this way, and it has been possible, in addition, to estimate the conductance of one open channel, a quantity not susceptible to direct measurements. Various physical pictures are capable of yielding the same macroscopic behavior for axon membranes, that is, the Hodgkin-Huxley equations, but these various mechanisms predict that the current fluctuations around their mean values should have different characteristics. Fluctuation analysis may, then be of value in elucidating the physical basis for axon conductance changes.


Effects of cholinergic compounds on the axon-Schwann cell relationship in the squid nerve fiber. The effects of acetylcholine, carbamylcholine, D-tubocurarine, eserine, and alpha-bungarotoxin on the Schwann cell electrical potential of resting and stimulated squid nerve fibers were studied. Acetylcholine (10-7 M) and barbamylcholine (10-6 M) induce a prolonged hyper polarization in the Schwann cells of the unstimulated nerve fiber. In the presence of carbamylcholine (10-6 M) the behavior of the Schwann cell membrane to changes in the external potassium concentration approximates the behavior of an ideal potassium electrode. D-Tubocurarine (10-9 M) blocks the hyperpolarizing effects of nerve impulse trains and carbamylcholine (10-6 M), whereas at the same concentration eserine prolongs the Schwann cell hyperpolarizations induced by axon stimulation or by acetylcholine (10-7 M). alpha-Bungarotoxin (10-9M) also blocks the hyperpolarizing effect of nerve impulse trains and of carbamylcholine. D-Tubocurarine (10-5M) protects the Schwann cells against the irreversible action of alpha-bungarotoxin. These results show the existence of acetylcholine receptors in the Schwann cell membrane. Preliminary measurements of the binding of 125I-alpha bungarotoxin to the plasma membranes isolated from squid nerves also indicate the presence of acetylcholine receptors. These findings support the involvement of cholinergic mechanisms in the axon-Schwann cell relationship previously described.


Electrical properties of the transverse tubular system. The transverse tubular system (T-system) of skeletal muscle links surface membrane action potential and release of activator calcium from sarcoplasmic reticulum (SR). The spread of depolarization along this system has been studied in voltage-clamped frog muscle fibers by using the spread of contractile activation in a thin optical cross section through the fiber center as an index. In tetrodotoxin treated fibers as depolarization of the fiber is increased contraction spreads from superficial to axial myofibrils. In tetrodotoxin free fibers the radial gradient of activation is reversed indicating that normally the activating signal is propagated along the T-system. Activation across the SR-T tubule junction does not appear to trigger an all-or-none response from the SR,-Costantin,  L.L. Electrical properties of the transverse tubular system.


Axons, dendrites and synapses.

Axons, dendrites and synapses. Classical concepts of neuronal organization have been based on the motoneuron as a model. Recent work on the mitral cell of the olfactory bulb has revealed synaptic connections of dendrites and cell bodies that are not present in the motoneuron. Similar findings in many other parts of the nervous system suggest the need to revise our concept of synaptic relations and functions properties of axons and dendrites.--SHEPHERD, G. M. Axons, dendrites and synapses.


Naturally labeled 13C-glucose. Metabolic studies in human diabetes and obesity. The 13C/12C ratio in most commercial preparations of glucose used in clinical investigation is much higher than that of expired air. Variations in expired air 13C, after glucose load, are indicative of the catabolism of this exogenous glucose. The changes in the 13C/12C ratio of CO2 in expired air after oral administration of glucose were determined by mass spectrometry. Results in eleven healthy volunteers and seven obese subjects with normal oral glucose tolerance tests (OGTT) are reported. In all cases, the administration of glucose resulted in a marked rise in the 13C/12C ration of expired CO2, reaching its maximum at the fourth hour and then declining progressively. In seven obese patients with chemical diabetes and in five insulin-dependent diabetics, the 13C/12C ratio of expired CO2 during OGTT was significantly reduced, in comparison with the results obtained from the control groups. This study demonstrates the potential of this procedure using "naturally labeled" 13C glucose for the metabolic studies in man as anticipated from previous studies of Duchesne and his coworkers.


Basement membrane thickness in muscle capillaries of normal and spontaneously diabetic Macaca nigra. The thickness of the capillary basement membrane of femoral muscle was examined in normal and spontaneously diabetic Macaca nigra. Thickness correlated significangly with the degree and severity of diabetes; greater thickness was associated with decreased glucose tolerance, decreased serum insulin, and increased glucose and triglyceride. In normal monkeys, the average basement membrane thickness was 696 A and in diabetic monkeys, 837 A, The minimum basement membrane thickness was 524 A in normal monkeys and 634 A in diabetics. Normals differed significantly from diabetic monkeys.


Hyperinsulinemia and enlarged adipocytes in patients with endogenous hyperlipoproteinemia without obesity or diabetes mellitus. Studies of adipocyte metabolism were performed in twelve male subjects with normal plasma lipids and eleven male patients with Type IV or Type V hyperlipoproteinemia. Patients with obesity or diabetes mellitus were excluded from the study. Although all patients had normal glucose tolerance tests, the blood glucose levels during these tests were higher in the hyperlipoproteinemic patients than in the normal control subjects and the plasma insulin responses were even more strikingly elevated in the hyperlipemic group. Adipocytes isolated from hypertriglyceridemic subjects were larger than those obtained from normal individuals and exhibited increased activities of both Type I and Type II hexokinase and increased rates of glucose oxidation and lipogenesis from glucose. Cell size, hexokinase isoenzyme activities and rates of lipogenesis from glucose were all strongly correlated with each other, but none of these measurements were correlated with glucose oxidation. It is not known how the adipocyte abnormalities are related to the lipid transport disorder.


Inordinately high levels of serum immunoreactive insulin in monoclonal immunoglobulinemia (on the problem of "big, big insulin"). 1. A patient with occasional attacks of hypoglycemia had levels of serum immunoreactive insulin persistently fifty to one-hundred times the normal value. Immunoelectrophoresis revealed presence of monoclonal IgG in his serum. The patient's diagnosis was established as paraproteinemic lymphoid and plasmocytic reticulosis proximate to multiple myeloma; insuloma was not found. 2. On gel filtration of native serum, only part of the total immunoreactivity was found in the elution position of crystalline insulin; the major part emerged in the early fractions together with the large proteins. After acidification of the serum, however, practically the entire immunoreactivity was recovered in ethanol extracts and proved to be "little insulin" on gel filtration. Only, "little insulin" was also detected after gel filtration of serum incubated with urea. 3. It is suggested that the large component with insulin immunoreactivity obtained in gel filtration of native serum is an insulin-protein complex. The nature of the presumed complex is not clear. It is not a complex of the antigen-antibody type. Insulin "trapping" by monoclonal gamma globulin is considered.


Insulin sensitivity of isolated perfused rat liver. The responsiveness of the isolated perfused rat liver to different metabolic effects of insulin was investigated during recycling perfusion. Infusion of porcine insulin at rates of 6, 9, 16 and 33 mU/hr. resulted in stable perfusate insulin levels averaging 41, 72, 120 and 229 muU/ml., respectively. Since the portal vein insulin concentration in the intact rat averaged 48 muU/ml. after a twenty-six-hour fast and 125 muU/ml. two hours after removal of food, the studies were conducted at insulin levels within the physiological range. The effect of each insulin concentration on the net accumulation of K+, AMINO ACID NITROGEN, UREA NITROGEN AND GLUCOSE IN The perfusing medium was assessed against the net accumulation of perfusate constituents during perfusion of control livers and livers perfused with perfusate insulin levels greater than 500 muU/ml. The results indicate that essentially maximal suppression of amino acid nitrogen outflow and retention of K+ OCCURRED AT INSULIN CONCENTRAtions of 72 muU/ml., with lesser effects being noted at 41 muU/ml. Inhibition of ureogenesis was demonstrated at insulin levels above 120 muU/ml. However, significant effects of insulin on suppressing net glucose outflow was not observed until insulin levels had reached 500 muU/ml. due presumably to the absence of a sustained rate of glycogenolysis by control livers. The observation that perfused livers from normal rats are extremely sensitive to several metabolic effects of insulin at physiological concentrations suggests that this experimental approach can provide useful information as to the role of the liver in the pathogenesis of various insulin resistant states.


Comparison of the lipolytic effects of insulin and proinsulin on isolated fat cells. Proinsulin, like insulin, has a biphasic effect on the lipolysis of isolated fat cells. Low concentrations of the peptides inhibit the response to submaximal doses of epinephrine, while higher concentrations of the peptides enhance the effect of supramaximal doses of epinephrine. Half-maximal doses are: (a) inhibition of lipolysis: insulin, 2.3 times 10- minus 11M; proinsulin, 6.9 times 10- minus 10M; (b) enhancement of lipolysis: insulin, 3.1 times 10- minus 9M; proinsulin, 8.1 times 10- minus 8M. The ratios of the potencies of the peptides are the same for both effects. These results suggest that both the inhibitory and the lipolytic effects of insulin are not caused by contaminants, and are mediated by similar receptors.


Spinal-cord injuries during birth.

Spinal-cord injuries during birth. The subject of perinatal injury to the spinal cord is briefly reviewed. Recognized causative factors are traction on the infant's trunk during breech delivery, rotational stresses applied to the spinal axis, traction on the cord via the brachial plexus in shoulder dystocia, and hyperextension of the fetal head in breech delivery or transverse presentation. Recognition of these factors is the basis for prevention of this terrible accident. Diagnostic criteria at various period of life are mentioned and the importance of the recognition of anaesthesia and reflex movements is emphasized. Treatment is mainly supportive. The development of an automatic reflex bladder evacuation is discussed. Follwup examinations and resourceful orthopedic care are essential. A few affected children learn to live with their disability and a very few who are apparently transected at birth recover surprisingly well.


Locomotor prognosis in cerebral palsy.

Locomotor prognosis in cerebral palsy. A study of the prognosis for walking of 73 pre-school children who had cerebral palsy or delayed motor development is reported. The importance of prognostic signs, based upon the presence or absence of postural reflex activity, was confirmed. The prognosis was accurate in 94-5 per cent of the 73 children studied. The method of scoring a child's reflex responses and relating this to the prognosis seems to be a simple procedure, easy to understand and to apply. It is suggested that the assessment of treatment methods should include an analysis of the child's walking potential, based upon the methods delineated in this study, before treatment is started.


Rectus femoris release in selected patients with cerebral palsy: a preliminary report. Two theories concerning the effects of surgical release of the proximal origins of the rectus femoris in spastic patients are (1) that release reduces hip flexion contracture and lumbar lordosis and diminishes crouch, and (2) that release primarily enhances early swing-phase knee flexion. A series of eight patients with pre-operative electromyography and pre- and post-operative dynamic knee measurements are reviewed. In these patients, back-knee thrust did not improve because it was not caused by rectus contracture. The effect upon hip was also variable: two patients had increased hip flexion and a third had diminished hip flexion after release. In six of the eight patients knee flexion was improved in early swing phase. Improvement from surgery can be expected when rectus spasticity is sufficient (1) to interfere with the initiation of swing phase, and (2) to decrease the amplitude of knee flexion. Little change occurred in the patients who did not have these functional deficits. A review of the cases supports the primary knee effect theory of Silfvenskiöld; however, insufficient information was obtained from this series to rule out hip and pelvic changes.


Very low birthweight and subsequent neurological defect (with special reference to spastic diplegia). Of a total of 170 children who weighed 1500g or less at birth and who were born in or admitted shortly after birth to Hammersmith Hospital, London, between the years 1961-70 inclusive, 165 have been followed to ascertain the incidence of neurological handicap, with particular reference to spastic diplegia. This condition occurred in six children (3-6 per cent), all of whom were born during the 1961-64 period, an incidence in those years of 10-3 per cent, compared with 0-0 per cent in the period 1965-70 (times 2 equal to 8-72, p equal to 0-0032). These findings are considered in the context of perinatal illness and care. The one statistically significant difference found between the children with and without spastic diplegia in the earlier period was a somewhat lower mean minimum rectal temperature on the first day of life only.


Motor development in Yucatecan infants.

Motor development in Yucatecan infants. Infant motor development was studied in three socio-cultural groups inYucatan, Mexico, using the Denver Developmental Screening Test f9DDSTF0 AND THEBayley Infant Motor Scale. Eight infants f9four boys and four girlsf0 at each monthof age from two weeks to one year and two weeks were examined in each group, a total of 288infants f996 in each groupf0. There were no significant differences in motor developmentamong the groups or between boys and girls. There was an increase in abilities with age. Compared with the Bayley norms for the USA, fine motor co-ordination was advanced inthese children but there was a delay in walking. The DDST was unable to detect childrenmore than one standard deviation below the mean on the Bayley Infant Motor Scale.


A special neurological examination of children with learning disabilities. Children with "minimal brain dysfunction" and learning disabilities were found to have significantly more minor neurological signs than control children. Many of these signs become less obvious or disappear by the age of 11 years; therefore older cases are more similar to controls, whereas younger cases show lags or deficits at the highest levels of central nervous system functioning-language, fine motor co-ordination and cross-modality integrations.


Unilateral nerve deafness in childhood: a possible vascular etiology. Unilateral sensorineural hearing loss in an eight-year-old girl, with radiographic evidence suggesting occlusion of the anterior inferior cerebellar artery, is reported. Previous to the present report, a vascular etiology of this disorder has not been demonstrated.


The attainment of formal operations: a comparison of probability concepts in deaf and hearing adolescents. In a series of six experiments deaf and hearing adolescents were compared on attainment of formal operations in Piaget's theory as represented by performance on probability problems. Performance of deaf subjects provided a desirable control comparison, since their knowledge of probability concepts was most likely acquired outside school classrooms. The probability problems were usually binary choice situations that required no verbalizations and were designed to illuminate strategies, as well as to indicate conceptual stage. Two experiments, one a long-term training study of over two years' duration, used supplementary preadolescent subject groups. Most of the hearing adolescents and many of the deaf adolescents attained the formal operatory level for probability concepts according to the criteria described by Piaget and Inheldermtheir criteria can be criticized, however, as insufficiently demanding, although they do point up the difficulty of defining the place of computational abilities in formal operationsmresults from several experiments suggested a second and higher level of formal operatory attainment around the ages of 14 to 15. Further theoretical insights were obtained regarding the implicit rules that frequently appeared to guide subjects' choices; a surprising variety of strategies were reported. Analysis of choice tendendies showed consistent avoidance of calculations and selection of the choice with the greatest absolute rather than relative frequency (or area) even when these strategies produced systematic errors. Subjects were generally unable to combine probability information from two different sources. In the long-term training experiment spontaneous improvement was shown by children at formal operatory ages even without training, but not by younger children. Deaf adolescents performed as well as hearing adolescents on easy problems, but they made more errors on several types of more difficult problems; Deaf adolescents were also less consistent than the hearing in use of rules, and in several experiments they showed a two- to three-year performance lag.


The immediate and delayed effects of different types of vagotomy on human gastric myoelectrical activity. The immediate and delayed effects of different types of vagotomy on the antral myoelectrical activity have been studied in 52 patients who had previously undergone either highly selective vagotomy (HSV) or truncal vagotomy and pyloroplasty (TV + P) for the treatment of chronic duodenal ulceration up to eight years previously. The frequency of the electrical activity was not statistically different between the two types of operation. After HSV the triphasic waveshape of the antral myoelectrical activity was retained; it was recorded more frequently in the delayed tests (98.0 plus or minus 1-7%) of the recording period than in immediate tests (74-7 plus or minus 6-5%) whereas mean amplitude in the immediate tests (1-59 plus or minus 0-13 mV) was not significantly changed in the delayed tests (1-49 plus or minus 0-08mV). After TV + P the waveform was more sinusoidal in shape, being recorded more often in the delayed tests (91-7 plus or minus 2-7%) than in the immediate tests (41-4 plus or minus 8-9%) although the mean amplitude between the immediate (0-090 plus or minus 0-06 mV) and delayed tests (0-94 plus or minus 0-07 mV) was not significantly different. The only significant change in the myoelectrical activity with the passage of time after vagotomy was an increase in the percentage activity.


An assessment of the reproducibility and safety of 2-deoxy-D-glucose as a gastric acid stimulant in duodenal ulcer patients. In studying the effects of 2-deoxy-D-glucose (2-DG) on the vagus nerve in preoperative patients with duodenal ulcer we have concluded that (1) in initiating gastric acid secretion 2-DG produces a response that is reproducible after 30 days; (2) 2-DG when given in a dose of 40 mg/kg intravenously produces a glucopenic state that appears safe in an otherwise healthy patient. We consider that 2-DG is a worthwhile agent in the investigation of the duodenal ulcer patient.


The effect of histamine H2-receptor blockade with metiamide on serum gastrin levels in man. Metiamide, an histamine H2-receptor antagonist which inhibits gastric acid secretion, does not lower basal serum gastrin concentration in man. Serum gastrin responses after stimulation by food were marginally higher when the stimulus of food was preceded by metiamide.


Changes in the bidirectional sodium flux across the intestinal mucosa in Crohn's disease. Bidirectional sodium flux across the intestinal mucosa was measured in a group of 10 patients with Crohn's disease treated in the past by panproctocolectomy with ileostomy and compared with a similarly treated group of 11 patients with ulcerative colitis. All of them were in good health at the time of the study and a recent radiological examination of the small intestine was normal. A significant reduction in bidirectional sodium flux was found in those patients with Crohn's disease and suggests that the intestinal mucosa is involved to a greater extent than can be judged by radiological appearances alone. This adds weight to the concept that Crohn's diseases is a diffuse rather than a focal lesion of the gastrointestinal tract.


A study of the cellular infiltrate of the proximal jejunal mucosa in ulcerative colitis and Crohn's disease. The cellular infiltrate of the proximal jejunum has been compared between 20 subjects without gastrointestinal disease, 20 subjects with ulcerative colitis, and 26 subjects with Crohn's disease. A significant increase (P smaller than 0.001) in the plasma cell infiltrate was noted in subjects with ulcerative colitis and a more marked increase in subjects with Crohn's disease (P smaller than 0.001). Such increases may be relevant to raised serum IgA levels following panproctocolectomy for ulcerative colitis or recurrence in the small intestine in Crohn's disease.


Influence of azathioprine on hepatic reserve in chronic active liver disease. The kinetics of hepatic indocyanine green (ICG) uptake have been analysed in 14 patients with chronic active liver disease. The removal of incremental, submaximal ICG doses were compatible with Michaelis-Menten kinetics in approximately 80% of studies in patients allowing the calculation of the maximal rate of removal (Rmax) and the Michaelis constant (Km). Neither of these variables correlated with other laboratory indices of liver function. Although determinations of Rmax before and after a two-weeks trial of azathioprine therapy were not significantly different, in two patients Rmax was markedly reduced by treatment. It is concluded that azathioprine over the short term does not improve the liver's reserve for ICG removal; in fact, in isolated cases this drug may be detrimental.


Liver ultrastructure in Gilbert's syndrome. Electron microscopy of hepatic tissue obtained by percutaneous needle  biopsy from nine patients with Gilbert's syndrome has revealed in every case gross hypertrophy of hepatocyte agranular endoplasmic reticulum but no other important abnormality. While this may have relevance to impairment of microsomal enzyme activity controlling bilirubin conjugation within liver cells, the serum bilirubin levels in all nine patients were below that normally associated with demonstrable UDP-glucuronyl transferase deficiency. Gross hypertrophy of agranular endoplasmic reticulum may be, therefore, a constant feature of this form of Gilbert's syndrome and may have some diagnostic value in the investigation of unconjugated hyperbilirubinaemia.


Lipodystrophy, pancreatitis, and eosinophilia. Two patients suffering from partial lipodystrophy, pancreatitis, and recurrent eosinophilia are described. In one patient the duodenum and the terminal ileum were narrowed, the appearances suggesting eosinophilic gastroenteritis: bilateral hydronephrosis was also present without ureteric obstruction. An association between lipodystrophy and renal disease is recognized; it is possible that there is also an association between lipodystrophy and pancreatitis, and eosinophilia with or without an intestinal lesion may be a further association.


Stereotyping and role conflicts between medical students and psychiatric nurses. Role stereotyping can cause conflicts between medical students and nurses on a psychiatric ward when students, expecting traditionally subservient hand-maidens, find instead independent, capable nurses with their own views about patient care. The authors describe a study of interactions between nurses, medical students, and psychiatric residents on two psychiatric wards when medical students became primary therapists under residents' supervision, and they discuss how the conflicts were minimized.


The Tuesday Evening Club: using community resources to treat chronically iii patients. In cooperation with a rehabilitation agency, the adult outpatient department of a community mental health center has developed a group treatment program for chronically ill patients in the community. Conducted in the framework of an activity program, the Tuesday Evening Club is based on a structured network of concrete community experiences. The techniques used include promoting socialization, encouraging members to verbalize their feelings, helping them  learn from negative interactions, and, for some, structured group psychotherapy. Patients who achieve certain successes graduate to a separate therapeutic program established at the YMCA.


Attitudes of mental health center staff toward community organization. The authors report on the differences in attitudes and perceptions that clinical and community organization staff have about the role of community organizers in a mental health center and about the center's priorities. Community organizers defined their role as one enabling the community to act on social-welfare problems, while clinical staff defined it as a liaison role connecting community residents and clinical services. Community organizers placed less value on traditional services, and felt greater dissatisfaction with center priorities.


Independent movement of surface immunoglobulin from Fc receptors on lymphocyte membranes. The majority of surface immunoglobulin-positive lymph node cells possess Fc receptors detectable by a rosette technique. The movement of surface immunoglobulin to form caps does not alter the distribution of Fc receptors, although Fc rosette-forming indicator cells collect over the immunoglobulin cap under capping conditions.


Suppression of tuberculin skin reactivity by prior tuberculin skin testing. In two consecutive studies, eighteen and forty-one normal subjects were given a standard tuberculin skin test of 1 tuberculin unit (0.02 mug of PPD). Two days later another skin test was given. Readings of the skin reactions showed that subjects exhibiting strongly positive skin reactions in their primary skin tests showed a significantly smaller indurations in their primary skin tests tended to have equal or larger areas of induration after the secondary skin tests. These observations are discussed in relation to the possible existence of regulatory mechanisms of cell-mediated immune reactions.


Induction of immunity to feline caliciviral disease. Six specific-pathogen-free cats were exposed by aerosol to a feline calicivirus of low virulence (F-9 virus). Homotypic (anti-F-9) seroconversion occurred in all cats by postexposure day 14. The serum of one cat on postexposure day 14 and four of six cats on postexposure day 35 neutralized feline picornavirus isolate no. 225 (FPV-255), a virulent feline calicivirus. Homologous antiviral activity was detected before the appearance of heterologous (anti-FPV-255) activity and always was present in higher titer. Protective immunity was evaluated on postexposure day 35 by aerosol challenge with FPV-255. The pyrexia, depression, dyspnea, oral ulcers, and severe pneumonia produced in two susceptible specific-pathogen-free cats by exposure to FPV-255 did not occur in the cats that had been infected previously with F-9 vir. The study demonstrates that heterotypic protective immunity to feline calicivirus disease can be induced by prior infection with feline calicivirus of low virulence.


Immunodepression by Rowson-Parr virus in mice: effect of Rowson-Parr virus and Friend leukemia complex infections on contact sensitivity in susceptible and resistant mice. Contact sensitivity to 2-phenyl-4-ethoxymethilene oxazolone, as a probe for cell-mediated immunity, was investigated in susceptible BALB/c and resistant C57BL/6 mice after infection with Friend leukemia complex (FLC) or with Rowson-Parr virus (RPV). In BALB/c mice, FLC depressed contact sensitivity when given before primary sensitization but had no effect on established contact sensitivity nor on the response elicited by a booster application of the sensitizer. These findings, together with the failure to alter reactivity to an aspecific inflammatory stimulus, indicate that FLC impairs the afferent limb of the response. In the same strain of mice RPV infection did not significantly depress contact sensitivity, as judged by the extent of the reaction 24 h after challenge, but slightly inhibited the early antibody-mediated phase of this reaction. In C57BL/6 mice neither viral preparation affected contact sensitivity.


Growth and immunogenicity of photochromogenic strains of mycobacteria in the footpads of normal mice. Specific pathogen-free CD-1 mice were infected subcutaneously in the footpad with mycobacterium kansasii, three strains of M. marinum, and two strains of M. simiae-habana, and the growth of the organisms in the footpad, the draining popliteal lymph node, and the lung and spleen was followed quantitatively for up to 60 days. The ability of a footpad inoculum of M. marinum to spread to the lungs and spleen correlated with the ability of the organism to survive and multiple at 37 C in vitro cultures. The amount of footpad swelling which developed in the M. kansasii- and M. marinum-infected mice varied depending upon the strain of organism and the size of the original footpad inoculum. Injection of dead M. marinum into the footpad also induced an extensive amount of swelling which varied with the strain used, as well as being dose dependent. M. marinum- and BCG-vaccinated mice were protected against a later footpad challenge with M. marinum or the highly mouse virulent M. tuberculosis strain ERDMAN. The significance of this finding is discussed in relation to cross-protection studies using a variety of mycobacteria in the footpad infection model.


Chronic proliferative arthritis of mice induced by Mycoplasma arthritidis: demonstration of a cell-mediated immune response to mycoplasma antigens in vitro. Lymphocytes taken from mice chronically infected with Mycoplasma arthritidis exhibited a significant blastogenic response as measured by (3H)thymidine uptake when exposed in vitro to M. arthritidis antigens. The lymphocytes taken from 9 of 12 control mice of similar age exhibited an inhibition of (3H)thymidine uptake when exposed to M. arthritidis antigens.


Paramyxovirus-avian cell relationship: discrepant impact of 6-azauridine on virus production by susceptible and less susceptible cells. The replication of mumps virus in susceptible chicken embryonic heart cells was escalated by daily treatment of cultures with 6-azauridine (6-AU). On the other hand, virus production by less susceptible liver cells was depressed by 6-AU. The population of infected susceptible cells was not increased, but the release of virus by infected susceptible cells was enhanced 20-fold by 10 mug of 6-AU per ml. Less susceptible cells, which incorporated less ribonucleic acid and protein precursor than susceptible cells, sustained a constant level of viral release during 6-AU treatment; however, the number of infected less susceptible cells underwent substantial decline.


Characterization of group N streptococcus lipoteichoic acid. Lipoteichoic acid was extracted from the group N organism Streptococcus lactis ATCC 9936 with hot aqueous phenol and purified by gel chromatography followed by affinity chromatography using Ricinus communis lectin as the specific absorbent. The teichoic acid moiety of the lipoteichoic acid was calculated to contain 16 to 17 glycerol phosphate units, approximately half of which were substituted with alpha-D-galactosyl residues; the glycolipid moiety contained O-alpha-D-glucosyl-1 yields 2-O-alpha-D-glucosyl-1 yields 1-glycerol. The finding of 2-O-alpha-D-galactosyl glycerol in the lipid fraction of hydrofluoric acid hydrolysates suggests that fatty acids also occur as substituents on the main chain of the lipoteichoic acid. The reactivity of the lipoteichoic acid with R. communis lectin was studied by the quantitative precipitin method and compared with the reactivity of Lactobacillus fermenti lipoteichoic acid, which has a lower degree of alpha-D-galactosyl substitution. Group N antiserum reacted strongly with the S. lactis lipoteichoic acid and cross-reacted with L. fermenti lipoteichoic acid. From inhibition studies it is concluded that the antibodies are specific for alpha-D-galactosyl substituents. In addition to lipoteichoic acid, a fraction was obtained by gel chromatography which contained galactose and reacted with group N antiserum but could be distinguished from the lipoteichoic acid by immunoelectrophoresis.


Diffuse dermographic mastocytosis without visible skin lesions. The case is presented of a 3-year-old girl with urticaria and pressure dermographism. The condition began the age of one year. A skin biopsy confirmed the suspected diagnosis of diffuse mastocytosis. The identification of the form of mastocytosis without skin lesions and with dermographism and pressure urticaria as its only signs is important. It should be considered in any chronic urticaria appearing at birth or in an early age which is unresponsive to the usual symptomatic medication.


Microbial flora of nurses' hands. III. The relationship between staphylococcal skin populations and persistence of carriage. The hands of 50 nurses and 50 controls were tested for coagulase-positive staphylococcus. Twenty-eight percent of both groups were carriers. The mean staphylococcal count on the palm of the hand was 13.7 per 16 cm-2 in nurses as compared to 442.7 per 16 cm-2 in the control group. The number of persistent carriers was the same for each group, but 25 percent of all cultures from nurses were positive for Staphylococcus aureus as compared to 14.2 percent of the cultures from the control group. It was concluded that there was no direct correlation between high staphylococcal counts and persistence of carriage. The skin as a site of carriage of Staphylococcus aureus was found to be of greater importance in nurses than in control subjects.


Vaccinations in the presence of skin diseases. The authors collected data from the literature and from experts on the subject of vaccination of patients with dermatologic disorders. They assembled these data into categories of specific skin conditions and vaccinations. These data may be used in deciding whether or not to vaccinate, and at which stage of the dermatologic condition.


In vitro sensitivity to 5-fluorocytosine of strains isolated from patients under treatment for chromomycosis. In vitro 5-fluorocytosine sensitivity tests of strains were isolated from chromomycosis patients undergoing therapy. Tests were rountinely performed for 0.1, 1.0, 5.0 and 10.0 mug/ml 5-fluorocytosine concentrations. In two cases resistance could be demonstrated up to 60.0 and 100.0 mug/ml. Fungistatic activity of 5-fluorocytosine against the agents of chromomycosis was confirmed. Sensitivity tests are useful to detect resistance which may occur during treatment. They are also helpful for guiding the clinician in the establishment of new therapeutic schedules.


The E-G junction. Two Sphincters.

The E-G junction. Two Sphincters. I studied the esophagogastric junction in fresh and preserved cadaveric specimens in the infant, adolescent and adult as well as by reverse gastroesophagoscopy, both intraoperatively and postoperatively, on unanesthetized patients. Cut sections of fresh specimens of the lower esophagus consistently showed an increased thickness of the lower 2-3 cm of the inner circular muscle layer of the esophagus. The layers are more firmly fused at this level and a color change is evident. From the serosal surface, a slight whitish indenture marks this area. From the mucosal surface, this area is the level of the transition zone. This is the lower esophageal sphincter. Reverse gastroscopy performed via a mature gastrostomy enabled me to identify and photograph a diaphragmatic sphincter and a lower esophageal sphincter.


Removal of intrahepatic bile duct stones. To keep this study homogeneous, we have limited ourselves to intrahepatic lithiases of the bile ducts above the hilus, usually linked with extrahepatic lithiases. While operating, all technical resources must be utilized in order to remove as many stones as possible. To gain access to the hepatic duct, it is sometimes necessary to excise the hilar plane; scissurotomy is the most efficient way to free the intrahepatic ducts when they are full of stones. In rare cases of localized lithiases, one may perform hepatic resection. We have perfected a technic using a special malleable catheter, guided by television, combined with suction of the stones for intrahepatic blocked stones. Forty-four interventions are presented. We have grouped them according to whether they involved an accumulation of stones from the extrahepatic ducts or stones blocked in a bile duct. There were three cases of dispersed intrahepatic lithiasis and one peripheral localization. Following extraction of the stones, we ended the intervention by 19 Kehr drainages, seven choledochotomies, five sphincterotomies, 11 biliodigestive anastomoses, one hepatectomy and one hepatotomy. We deliberately left some stones undisturbed in seven cases: the results even after some years remain satisfactory except for one case of dispersed lithiasis.


Clinical study of Volkmann's ischemic contracture of the upper limb. A clinical study of Volkmann's ischemic contracture of the forearm has been presented. The total incidence was 0.105 percent of all orthopedic cases. Mostly it occurred in males (77.5 percent), mostly in the second or third decade of life. Most patients lived in rural areas, reflecting prevailing socioeconomic conditions.


Arteriovenous intestinal communications in shock. Experimental study using tagged microspheres. Microspheres 25 plus or minus 5 microns in diameter labeled with Ce141 and Sr85 were used to study the influence of oligemic shock on any arteriovenous anastomoses of the small intestine mucosa of dogs and rabbits. We found no open AVA's larger than 20 microns in diameter either before or during oligemic shock.


Constrictive pericarditis and its surgical management. The surgical management of 66 cases of constrictive pericarditis has been presented. Tuberculosis was the most common etiologic factor in this series. The importance of optimal myocardial function has been emphasized. Pericardiectomy was limited to release of the ventricles in order to correct the hemodynamic anomaly. The vast majority of patients claimed to have benefited from surgery.


Lateral subcutaneous internal sphincterotomy for anal fissure. Technic and experience with 22 cases in children. Lateral sphincterotomy is probably the operation of choice for most cases of intractable anal fissure in children. The lower half of the sphincter is divided in the left or right lateral position through a small external incision at the anal verge. An incision in the anal canal itself is avoided, postoperative pain is minimal and the risk of secondary hemorrhage is eliminated. The operation can be done as an outpatient procedure. Only one patient developed minor anal incontinence.


External osteosynthesis of distal fractures of the phalanges by reposition-fixation of the fingernail. Fractures of the distal phalanges of the fingers rarely justify immediate amputation. Integral repair is necessary if severe sequellae are to be avoided. Management always requires surgery and we advocate external osteosynthesis by reposition-fixation of the fingernail. The principles and the technic for its use are outlined. For me, this method is the only guarantee for a quick, stable, economical and satisfactory cure of this common hand injury.


Emergency vagotomy with a drainage procedure for perforated gastric and duodenal ulcer. Study of 73 cases. Sixty-nine cases of duodenal ulcer perforation and four cases of stomach ulcer perforation are described. One half of the patients underwent vagotomy plus a drainage procedure and the other half had suture of the perforated ulcer. No death occurred in the first group, and hospitalization time was less than was less than that of the second group. Five deaths occurred in the second group.


Modification by imidazoles of ocular inflammatory and pressure responses. Imidazole, given intraperitoneally to rabbits, inhibited the prostaglandin E1-induced (PGE1) elevation of intraocular pressure. The maximum imidaz-le effect occurred at three to six hours after intraperitoneal infection. The prostaglandin E1-induced aqueous humor protein elevation was reduced markedly in the imidazole-pretreated rabbits. Intravenous administration of imidazole also was effective in reducing the intraocular pressure elevation produced by prostaglandin E1. Imidazole pretreatment by subconjunctival, subdermal, or topical routes had no such effect. Imidazole derivatives, 1-methylimidazole and 2-methylimidazole, given intraperitoneally, blocked the effect of PGE1 on intraocular pressure and aqueous humor protein. Imidazole pretreatment also inhibited the elevation of intraocular pressure produced by topical nitrogen mustard, 1 per cent.


The effect of intraocular pressure on conventional outflow resistance in the enucleated human eye. A technique for measuring resistance to outflow in enucleated eyes is described.  The technique minimizes the artifactual effects of ocular stretching and anterior chamber deepening.  Using this perfusion technique, it was found that the resistance to outflow in normal, enucleated human eyes increases directly and linearly with intraocular pressure.  This phenomenon, termed the outflow obstruction effect, was defined as the fractional increase in baseline outflow resistance (outflow resistance at an outflow pressure of zero) per millimeter of Hg intraocular pressure rise above baseline and was designated by the letter Q.  The value of Q was found to be 0.012 mm. Hg-1 plus or minus 0.0014 indicating that the outflow resistance rise above its baseline value approximately 1 per cent per millimeter of Hg rise in intraocular pressure.  Estimates of Q, calculated from data published by other investigators over the past two decades, are also included.


Conjunctival goblet cell density in normal subjects and in dry eye syndromes. Serial sections prepared from biopsies of the deep tarsal portion of the inferior nasal conjunctival fornix in normal subjects and in patients with various dry eye syndromes were analyzed with respect to the goblet cell densities. When compared to normal subjects, individuals with keratitis sicca, Stevens-Johnson syndrome, ocular pemphigoid, and acute alkali burn all demonstrated progressively lower goblet cell densities per millimeter of epithelial surface. These disease entities can, therefore, be considered goblet cell-deficient syndromes.


Subsensitivity to pilocarpine in primate ciliary muscle following topical anticholinesterase treatment. Accommodative responses to intramuscular pilocarpine were determined in four surgically aniridic vervet monkeys, before and after eight weeks of daily unilateral topical treatment with echothiophate iodide.  The echothiophate-treated eyes maintained maximum myopia during the treatment course. However, after echotiophate treatment was stopped and the refraction had returned to baseline, a subsensitivity of the accommodative mechanism to pilocarpine became apparent.  Normal sensitivity to pilocarpine did not return until four to five months after echothiophate treatment had been stopped.


Lysosomes and melanin granules of the retinal pigment epithelium in a mouse model of the Chediak-Higashi syndrome. The origin of giant granules in the retinal pigment epithelium of the beige mouse was investigated with electron microscopy and ultrastructural histochemistry. These granules were found to contain melanin and acid phosphatase. Apparently they arise from fusions of primary lysosomes with melanin granules which are already enlarged from multiple fusions among melanosomes. Therefore, the giant granules are not primary lysosomes, nor are they simply enlarged melanin granules as suspected from light microscopic studies. A deficiency of primary lysosomes in the pigment epithelium results, suggesting a defect in intracellular digestion similar to that found in the leukocytes of Chediak-Higashi patients and several animal models. Affected humans probably have defective digestion in their retinal pigment epithelium also; which could impair the renewal process for rod outer segments. Thus, Chediak-Higashi patients may show an increased susceptibility to light damage due not only to hypopigmentation, but to defective intracellular digestion, as well.


Closely spaced saccades.

Closely spaced saccades. The relationships between saccadic velocity, duration, and magnitude have been used to prove the normalcy of saccades with intersaccadic intervals of less than 200 ms. Pairs of normal saccades with small intersaccadic intervals will have the second saccade larger or smaller and going in the same or the opposite direction than the first saccade. These normal saccades may be horizontal, vertical, or oblique.


Total retinal degeneration in apparent anophthalmos of the Syrian hamster. Anophthalmia in the Syrian hamster was found to result from an extensive degeneration of retinal tissue and tissues derived from the retina. Eyes of affected animals were normal at the twelfth day of gestation (the average gestation period in the Syrian hamster is 16 days). However, the retina of these eyes showed rapid and extensive degeneration during the first two weeks after birth. In adults, the sclera-choroid complex was the only prominent structure of the original eye, with an occasional remnant of deteriorated lens.


Ocular effects of diacetyl morphine and lysergic acid diethylamide in rabbit. Intravenous lysergic acid diethylamide (LSD) given to rabbits in doses from 1 to 100 mug per kilogram of body weight produced a dose-related increase in intraocular pressure and outflow facility. Minor changes in systemic blood pressure were observed, but respiration rate was accelerated, and mydriasis became pronounced at higher doses. Diacetyl morphine (heroin) was given intravenously in doses from 0.1 to 2 mg. per kilogram of body weight. A dose-related decrease in intraocular pressure and an increase in outflow facility was found. A dose-related miosis was observed and at higher doses respiration became markedly depressed. Neither drug alters the permeability of the isolated ciliary epithelium. Both drugs appear to increase capillary blood pressure and, hence, aqueous humor inflow to cause the intraocular pressure to be maintained at approximately normal levels in face of increases in outflow facility of 50 per cent.


An objective focusing method for fundus photography. A method is described which enables the fundus camera to be focused objectively. This method eliminates the focus errors arising from the subjective focusing method currently used and permits the consistent attainment of high resolution fundus photographs.  The objective focusing method has been evaluated on test targets and anesthetized animals and is applicable for use on unanesthetized human subjects.


Pathways for the degradation of m-cresol and p-cresol by Pseudomonas putida. A comparison of the oxidation rates of various compounds by whole cells of Pseudomonas putida 3, 5 indicated that m-cresol is metabolized by oxidation to 3-hydroxybenzoate followed by hydroxylation to gentisate, the ring-fission substrate, when grown with 3, 5-xylenol. However, when m-cresol was the growth substrate, similar experiments suggested a different pathway involving a methyl-substituted catechol, and ring-fission by meta cleavage. Assays of ring-fission enzymes in cell-free extracts confirmed that different pathways are induced by the two growth substrates. 3, 5-Xylenol-grown cells contained high levels of gentisate oxygenase and only very small amounts of catechol oxygenase, whereas gentisate ocygenase could not be detected in m-cresol-grown cells, but levels of catechol oxygenase were greatly increased. Extracts of m-cresol-grown cells also contained 2-hydroxymuconic semialdehyde dehydrogenase and hydrolase, whose specificities enable them to metabolize the ring-fission products from catechol, 3-methylcatechol, and 4-methylcatechol. This catechol pathway is also used by m-cresol-grown cells for p-cresol metabolism. In contrast, the results for cells grown with p-cresol point to an alternative pathway involving oxidation to 4-hydroxybenzoate and hydrosylation to protocatechuate as ring-fission substrate. Extracts of these cells contained high levels of protocatechuate oxygenase and only small amounts of catechol oxygenase.


Regulation of the tricarboxylic acid cycle in gram-positive, facultatively anaerobic bacilli. The facultative anaerobes Bacillus polymyxa Hino G, B. polymyxa Hino J, and B.macerans were observed to have imcomplete tricarboxylic acid cycles. They were devoid of malate dehydrogenase and all had very low levels of alpha-ketoglutarate dehydrogenase. B. polymyxa Hino J was devoid of alpha-ketoglutarate dehydrogenase when grown aerobically and anerobically. Citrate synthase from B. polymyxa was inhibited by adenosine triphosphate but not reduced nicotinamide adenine dinucleotide and resembled enzymes from other gram-positive bacteria in this respect. Like the citrate synthases from gram-negative, facultative anaerobes and chemolithotrophs, the enzyme from B. polymyxa was inhibited by alpha-ketoglutarate. Inhibition by adenosine triphosphate was shown to be competitive with acetyl-coenzyme A and alpha-ketoglutarate  inhibition was competitive with oxaloacetate.


Sexal reproductive cycle of Monascus in submerged shaken culture. A strain of Monascus was grown in submerged, shaken culture using a glucose-salts mediumn. The formation of ascospores was studied by microscopic examination of samples taken at regular intervals for 6 days. All the stages of sexual reproduction previously described for Monascus were observed. The unusual feature of the strain studied was that it produced sexual structures and cliestothecia of greatly varying sizes.


Purification of a protein kinase and two phosphate acceptor proteins from vaccinia virions. A novel protein kinase that requires protamine as an activator to catalyze the phosphorylation of viral acceptor proteins was extracted from vaccinia virus cores with deoxycholate and purified 250-fold by DNA-cellulose and DEAE-cellulose column chromatography. The enzyme has a molecular weight of 62,000 as determined by sucrose gradient sedimentation. Two heat-stable phosphate acceptor proteins were extracted from virus particles with a nonionic detergent and purified by heat treatment, precipitation with organic solvents, and CM-cellulose chromatography. The molecular weights of the phosphate acceptor proteins, determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, are 38,500 and 11,700.


Presence of cytochrome and menaquinone in Clostridium formicoaceticum and Clostridium thermoaceticum. Cytochrome b and menaquinone have been demonstrated in the homoacetate-fermenting Clostridium formicoaceticum and Clostridium thermoaceticum.


Studies on the common active site of growth hormone. Revision of the amino acid sequence of an active fragment of bovine growth hormone. A fragment, A-II, isolated from a component of a tryptic digest of bovine growth hormone has growth-promoting activity in rats and metabolic activity in humans similar to human growth hormone. The amino acid sequence of this peptide has been reinvestigated and revised. The 38-amino acid peptide was cleaved with cyanogen bromide, chymotrypsin, and trypsin. The amino acid sequences were then established by Edman degradation as well as with overlapping peptides; Homology in the sequence was good between this bovine growth hormone fragment and peptides occurring in ovine growth hormone, human growth hormone, and human chorionic somatomammotropin.


Biological, biochemical, and physicochemical evidence for the existence of the polyadenylic-polyuridylic-polyinosinic acid triplex. When primary rabbit kidney cell cultures are treated with either polyadenylic acid-polyuridylic acid or polyadenylic acid-polyribothymidylic acid (poly(rT)) and then judiciously exposed to actinomycin D and cycloheximide, high titers of interferon are found in the extracellular medium ("superinduction") (Vilcek, J. (1970) Ann. N. Y. Acad. Sci. 173, 390-403; Tan, Y. H., Armstrong, J. A., Ke, Y. H., and Ho, M. (1970) Proc. Natl. Acad.  Sci. U. S. A. 67, 464-471). If polyinosinic acid is added 1 hour prior to, simultaneously with, or 1 hour after the active interferon inducers, dramatic reductions in interferon production from the "superinduced" cells result. Based on experiments involving sucrose gradient ultracentrifugation, pancreatic ribonuclease A resistance, ultraviolet mixing curves, and ultraviolet absorbance-temperature profiles, the explanation for this phenomenon was determined to be the formation of polynucleotide triplexes in the following way: poly(A)-poly(U) + poly(I) yields poly(A)-poly(U)-poly(I)poly(A)-poly(rT) + poly(I) yields poly(A)-poly(rT)-poly(I). In addition, based on similar methodology, the following reactions involving these triplexes were demonstrated: poly(A)-2 poly(I) + poly(U) yields poly(A)-poly(U)-poly(I) + poly(I)poly(A)-2 poly(I) + poly(rT) yields poly(A)-poly(rT)-poly(I) + poly(I)POLY(A)-2 poly(I) + 2 poly(U) yields poly(A)-2 poly(U) + 2 poly(I) and POLY(A)-poly(U)-poly(I) + poly (U) yields poly(A)-2 poly(U) + poly(I).


Isolation and characterization of sulfhydryl oxidase from bovine milk. A method is described for purification of sulfhydryl oxidase from bovine milk which consistently yields preparations with greater than 3000-fold purification over skim milk. A concentration-dependent association-dissociation of the enzyme was adapted to the development of an isolation procedure. Purified preparations exhibited two zones, both of which displayed activity, upon polyacrylamide disc gel electrophoresis, but only one zone following disc gel electrophoresis in sodium dodecyl sulfate. Its mobility indicated a subunit weight of 89,000. Several lines of evidence suggest that iron is an integral part of the enzyme. Treatment of the enzyme with EDTA resulted in complete loss of activity which could be subsequently restored by dialysis against 1 muM ferrous sulfate. Furthermore, atomic absorption analysis and neutron activation analysis of separate enzyme preparations each indicated 0.5 atom of iron per subunit. Chemical analyses of sulfhydryl oxidase accounted for 97% of the sample weight, of which 89% could be attributed to amino acid residues and 11% to carbohydrate residues. Five half-cystine residues per subunit were indicated by cysteic acid analysis and by sulfhydryl group determination following reaction with sodium borohydride. Comparison of this value to the total sulfhydryl groups without reduction tentatively suggests the presence of one disulfide bond. Sulfhydryl oxidase was found to catalyze the oxidation of sulfhydryl groups in both small compounds and proteins, using O2 as oxidant and producing, in equimolar quantities, H2O2 and the corresponding disulfide. A Michaelis constant of 90 muM was obtained using reduced glutathione as substrate, under conditions of optimal pH and temperature, viz., pH 7.0 and 35 degrees. Substrate inhibition was apparent at GSH concentrations above 0.8 mM. In the presence of sulfhydryl oxidase, reductively denatured RNase was reoxidized and fully reactivated within 1 hour, whereas in the absence of the oxidase under otherwise identical conditions, full recovery of RNase activity required 24 hours. The presence of reducing agent was not required for this activity, nor was prior reduction of the sulfhydryl oxidase. Based on the observed activity, it appears that the enzyme could be involved in the biosynthesis of disulfide bonds in certain proteins.


Interaction of immunoglobulin glycopeptides with concanavalin A. A number of intact and partially degraded immunoglobulin glycopeptides have been tested for their ability to interact with concanavalin A. The degraded glycopeptides were prepared by using purified glycosidases to remove sugar residues from the nonreducing ends of the oligosaccharide chains of intact glycopeptides. A quantitative and sensitive assay was devised to measure the potency of the glycopeptides as haptene inhibitors of 125I-concanavalin A binding to guinea pig erythrocytes. The most potent haptene, derived from an immunoglobulin G glycopeptide, had a branched chain oligosaccharide with two GlcNAc (see article) Man (see article) nonreducing termini linked to a mannose residue in the core. The other very potent glycopeptide was an immunoglobulin E high mannose glycopeptide which contained 3 terminal alpha-mannose residues and 1 internal 2-O-mannose residue. Removal of terminal beta-N-acetylglucosamine residues or alpha-mannose residues reduced the activity of these and other glycopeptides as inhibitors of 125I-concanavalin A binding. It was concluded that the ability of these glycopeptides to interact with concanavalin A is dependent on their content of terminal beta-N-acetylglucosamine residues, terminal alpha-mannose residues, and also internal mannose residues substituted on the C-2 hydroxyl group, and that the saccharide combining site of concanavalin A must be able to bind several sugar residues.


Studies on the guinea pig pancreas. Parallel discharge of exocrine enzyme activities. An in vitro system of guinea pig pancreatic lobules convenient for the study of secretory processes is described in this paper. In this system: (a) the over-all glandular architecture of the tissue is preserved: lobules remain morphologically intact through 5 hours; (b) amylase discharge from unstimulated lobules is low (similar to 4%/hour) and linear over the 5 hours tested; (c) response to carbamylcholine chloride (10-5 M) is energy-dependent, rapid, and extensive (92% discharge of amylase by 5 hours); (d) initial rates of discharge remain stable over the first 3 hours; and (e) no autoactivation of zymogens occurs in incubation medium or tissue. The activation of four zymogens, i.e. chymotrypsinogen, trypsinogen, and procarboxypeptidases A and B, was studied using the following criteria for optimal activation: (a) maximal activation attainable under experimental conditions; (b) stability at the level of maximal activation; and (c) linear relationship between amounts of protein activated and enzyme activity elicited by activation. The concentration of activators (trypsin or enterokinase) and secretory protein, the presence or agents (bovine plasma albumin or Triton X-100) which minimize adsorptive losses of secretory protein on glass or plastic surfaces, and the temperature at which activation is carried out were found to be critical and different for each of the zymogens tested. The kinetics of the appearance of three enzyme activities (amylase, lipase, and ribonuclease) and four potential proteolytic activities (chymotrypsinogen, trypsinogen, and procarboxypeptidases A and B) into the incubation medium was studied under different conditions; i.e. rest and stimulation with various secretogogues (carbamylcholine chloride, caerulein, and pancreozymin). All seven activities estimated to represent similar to 75% of the secretory protein output of the exocrine pancreas were discharged in synchrony and in constant proportions and were released from the tissue to the same extent under each experimental condition investigated.


Studies on the pancreas of the guinea pig. Parallel processing and discharge of exocrine proteins. The discharge and the intracellular transport of the exocrine proteins produced by the guinea pig pancreas have been investigated in the in vitro lobule system described by SCHEELE, G. A., AND PALADE, G. E. (1975) J. Biol. Chem. 250, 2660-2670. The inquiry was carried out on a protein-specific basis by using the sodium dodecyl sulfate polyacrylamide gel electrophoretic procedures worked out by TARTAKOFF, A. M., GREENE, L. J., AND PALADE, G. E. (1974) J. Biol; Chem. 249, 7420-7431. The results show that the same protein mixture is discharged by the lobules regardless of whether stimulation is by carbamylcholine (which mimics the action of acetylcholine), caerulein (which mimics the action of pancreozymin), or by 75 mM KCl. When no stimulant is present, the small quantity of discharged protein (resting secretion) also has the same composition. Analysis of successive secretion aliquots collected over a period of 2 hours of continuous timulation by carbamylcholine showed that the composition of the secretory output remains quasi-constant with time; therefore, the exocrine proteins appear to be discharged in parallel and in constant proportions, irrespective of stimulant and time under stimulation (for carbamylcholine). The analysis of a series of zymogen granule fractions prepared from lobules pulse-labeled with radioactive amino acids and chased for periods of 15 to 155 min shows that all major secretory proteins enter the condensing vacuoles of the Golgi complex and appear in zymogen granules in apparent synchrony.


Induction of delta-aminolevulinic acid synthetase in isolated rat liver cells by steroids. The role of steroids in regulation of delta-aminolevulinic acid synthetase has been studied in isolated rat liver cell suspensions under conditions previously shown to support inducation of the enzyme by drugs. Addition of a variety of C-19 and C-21 steroids to cell suspensions resulted, after 4 to 6 hours of incubation, in 2- to 5-fold increase in the activity of delta-aminolevulinic acid synthetase as measured in liver cell homogenates. The increase was prevented by cycloheximide. The most active steroid inducers tested were pregnene or pregnane derivatives with keto or hydroxyl groups at C-3 and C-20; in particular a beta-hydroxyl group at C-20 enhanced activity. These C-21 steroids at optimal initial concentrations caused 3- to 5-fold induction over 4 hours. A number of C-19 androstene and androstane compounds caused 2- to 3-fold inducation over the same period. Hydrocortisone had no effect. For a variety of androstane and pregnane derivatives, inducation by 5alphaH steroids was as great as or greater than that by 5betaH compounds, in contrast to previous findings in chick embryo liver. Induction of delta-aminolevulinic acid synthetase by steroids in isolated liver cells was shown to be subject to feedback repression by hemin.


Activity profiles of prostaglandin 15- and 9-hydroxydehydrogenase and 13-reductase in the developing rat kidney. Three prostaglandin F2alpha-catabolizing enzyme activities have been demonstrated in kidneys from adult rats. Activity of each of the enzymes varied with animal age. Whereas 15-hydroxydehydrogenase and delta13-reductase appeared important to the early developing kidney (prior to 4 weeks of age), 9-hydroxydehydrogenase appeared to be characteristic of the adult kidney. Prostaglandin 15-hydroxydehydrogenase rose sharply after birth to a maximal value at 19 days (59-fold relative to the adult) decreasing to adult values by Day 40. Prostaglandin delta13-reductase followed a similar pattern rising about 20-fold at Day 19. Prostaglandin 9-hydroxydehydrogenase, on the other hand, was undetectable up to Day 19, rising gradually to adult values by Day 50. Prostaglandin biosynthesis in whole kidney and renal papilla at the peak period of 15-hydroxydehydrogenase activity, i.e. 19, 22, and 24 days, did not vary significally from adult values. The dramatic rise in 15-hydroxydehydrogenase activity, reflecting an important requirement for prostaglandin inactivation during the first 3 weeks after birth, appears to correlate well with the increase during this period in the number of glomeruli, cortical tubules, and redistribution of blood flow to the cortex. These results suggest for the first time an important relationship between prostaglandin catabolizing activites and nephrogenesis.


The determination of prostaglandin metabolites in human urine. A method has been developed in which the human urinary prostaglandin metabolites are converted into stable prostanoic and prostanediotic acid homologues. The urine is reduced with sodium borohydride and the organic compounds, isolated with an Amberlite XAD-2 column, are treated with fuming hydrogen iodide, followed by treatment with zinc in methanolic hydrogen chloride, to yield dimethyl tetranorprostanedioate as the major product of human prostaglandin metabolism. This compound is determined by gas chromatography, after a single purification by thin layer chromatography. The excretion of total tetranorprostanedioic acid derivatives in healthy subjects was found to be 0.21 plus and minus 0.08 mg per g of creatinine or 0.3 plus and minus 0.01 mg per 24 hours.


Studies on the product binding sites of the Azotobacter vinelandii ribonucleic acid polymerase. During chain elongation RNA polymerase exists as a ternary DNA-enzyme-RNA complex in which a discrete length of the nascent RNA chain proximal to the 3'-OH terminus will be bound to the product binding site (Krakow, J. S., and Fronk, E. (1969) J. Biol. Chem. 244, 5988). We have utilized the poly[d(A-T)]-directed reaction to determine the length of the nascent poly[r(A-U)] protected from attack by pancreatic ribonuclease. Following release of the ribonuclease resistant oligo[r(A-U)] from the ternary complex, its size was determined by ion exchange chromatography on DEAE-cellulose, gel filtration on Bio-Gel P-10, and the ratio of 3'-terminal uridine to internal 2':3'-UMP following alkaline hydrolysis. The results indicate that the length of the nascent protected fragment is approximately 12 residues.


Myoinosose-2 1-phosphate: an intermediate in the myoinositol 1-phosphate synthase reaction. Partially purified testicular myoinositol 1-phosphate synthase was incubated with glucose 6-phosphate and NAD+. After 2 min the reaction was stopped by the addition of NaB3H4. Phosphorylated reduced sugars were isolated by ion exchange and dephosphorylated enzymatically. Scylloinositol and myoinositol, added as carriers, were re-isolated and purified to constant specific radioactivity. Since scylloinositol phosphate is uniquely related to myoinosose-2 1-phosphate, the finding of labeled scylloinositol and myoinositol is considered strong evidence for the presence of myoinosose-2 1-phosphate, an intermediate which has been postulated in the synthase-catalyzed isomerization of glucose 6-phosphate to myoinositol 1-phosphate. About one-half the amount of intermediate was demonstrable with boiled synthase, indicating firm binding of myoinosose-2 phosphate to the enzyme.


[3H]diborane reduction of vitamin K-dependent calcium-binding proteins. Identification of a unique amino acid. gamma-Carboxyglutamic acid has recently been identified as a component of the vitamin K-dependent region of bovine prothrombin (Nelsestuen, G. L., Zytkovicz, T. H., and Howard J. B. (1974) J. Biol Chem. 249, 6347-6350). The presence of this amino acid has been substantiated here by the reduction of vitamin K-dependent proteins with [3H]-DIBORANE. The reduction product of gamma-carboxyglutamic acid, 5,5'-[3H]dihydroxyleucine, was shown to be present in hydrolysates of reduced rat prothrombin, bovine prothrombin, and bovine factor X. The results are consistent with a minimum of 10 gamma-carboxyglutamic acid residues in the nonthrombin-generating region of bovine prothrombin but no such residues in the thrombin precursor portion of prothrombin. It is concluded that amino acid analyses of [3H]diborane-reduced proteins provides a sensitive, qualitative method for the identification of proteins which contain gamma-carboxyglutamic acid and are vitamin K-dependent.


Protein interactions with small molecules. Relationships between stoichiometric binding constants, site binding constants, and empirical binding parameters. The multiple equilibria for the binding of a ligand A by a macromolecule P with n binding sites may be formulated in terms of a stoichiometric analysis or on the basis of a site-oriented scrutiny. The dependence of binding on ligand concentration can always be correlated in terms of n stoichiometric binding constants,Ki, even if there are interactions between sites that accentuate or attenuate binding affinities. A corresponding correlation in terms of site binding constants, kj, under the most general circumstances depends on the definition of n2n-1 different constants of which 2n-1 are independent. If experimental data are correlated in terms of n parameters kalpha, kbeta ... klambda in an equation of the site-binding form, (see article for formular) then there is no guarantee that the values of ka, kb, etc., have any unique relationships to site binging constants. Examples are given to illustrate this point. Equation are derived for relating stoichiometric binding constants to site binding constants, for the general case and for various special circumstances. These equations make it possible to define and analyze binding insystems with interactions and conformational accommodations. Accordingly, a graphical procedure is described (in which iKi is plotted against i, the stoichiometric binding step) that provides an affinity profile for concise representation of magnitudes of binding constants and for detecting interactions that accentuate or attenuate site binding affinities.


Carnitine palymityltransferase in neonatal and adult heart and liver mitochondria. Effect of phospholipase C treatment. Carnitine palmityltransferase was measured in neonatal and adult rat heart and liver mitochondria. When compared to the adult, no developmental differences in liver mitochondrial carnitine palmityltransferase were seen 24 hours postpartum. In neonatal heart, enzyme activity was initially low, reaching adult levels by 20 days. Polarographic measurements of oxygen consumption revealed normal levels of palmityl coenzyme A oxidation in the presence of carnitine and of palmitylcarnitine oxidation in both neonatal heart and liver. Phospholipase C treatment produced higher specific activities of carnitine palmityltransferase in neonatal and activities of carnitine palmityltransferase in neonatal and adult liver, probably due to decreases in total mitochondrial protein. Phospholipase C treatment had no effect on adult rat heart carnitine palmityltransferase but increased neonatal heart enzyme activity to adult levels. Low levels of measured enzyme activity were proposed to be due to "masking" of enzyme expression in intact mitochondrial preparations.


Effect of amidination of lysyl residues on the oxygen affinity of human hemoglobin. Specificity of methyl acetimidate for lysine C5(40)alpha. Treatment of human oxyhemoglobin with methylacetimidate results in selective amidination of the epfilon-amino group of lysin C5(40)alpha. The modified hemoglobin exhibits increased oxygen affinity, high cooperatively, and normal Bohr effect. Hybrid molecules containing amidinated beta chains and normal alpha chains have normal ligand-binding properties, whereas hybrid molecules containing amidinated alpha chains have ligand-binding properties identical with fully amidinated hemoglobin. Amidination of deoxyhemoglobin produces only minimal changes in ligand-binding properties. We propose that amidination of lysine C5(40)alpha prevents its participation in the salt bond with histidine HC3(146)beta in deoxyhemoglobin, thus shifting the allosteric equilibrium in favor of the high affinity oxy conformation.


Pyruvate-uridine diphospho-N-acetylglucosamine transferase. Purification to homogeneity and feedback inhibition. Phosphoenolpyruvate:uridine-5'-diphospho-N-acetyl-2-amino-2-deoxyglucose-3-enolpyruvyltranferase catalyzes the transfer of enolpyruvate from phosphoenolpyruvate to uridine diphospho-N-acetylglucosamine with the liberation of inorganic orthophosphate. It was purified to homogeneity from Enterobacter cloacae with the use of UDP-N-acetylmuramyl-L-Ala-D-Glu-meso-Dap, a feedback inihibitor, as a ligand covalenty bound to Sepharose 4B. The evidence suggests that the enzyme is a single polypeptide with a molecular weight of 41,000. The enzyme catalyzes the first committed step in the biosynthesis of bacterial cell wall peptidoglycan. The cytoplasmic end product of this pathway is UDP-N-acetylmuramyl-L-Ala-D-Glu-meso-Dap-D-Ala-D-Ala (see article). UDP-MurNAc-pentapeptide and its precursor, UDP-MurNAc-tripeptide, were found to be effective inhibitiors of the enzyme. The kinetic data suggest a binding site for these inhibitors distinct from the active site. This is consistent with the proposed role for UDP-MurNAc-tripeptide and pentapeptide as negative modulators of the enzyme.


alpha-and beta-Globin complementary deoxyribonucleic acids of human and rabbit. Specificity of hybridization. The specificity of hybridization was compared between the human and rabbit alpha and beta-globin complementary DNAs (cDNAs) and the corresponding alpha and beta-globin messenger RNAs (mRNAs). The globin chain-specific mRNAs of rabbit were prepared from polysomes incubated with O-methylthreonine (alpha and beta) or from postribosomal supernatant (alpha). Enrichment for either the alpha- or beta-globin mRNA was demonstrated by cell-free protein synthesis and by RNA-cDNA hybridization. Human mRNAs, active as templates for RNA-directed DNA polymerase, were prepared from reticulocytes of patients with hemolytic anemia, alpha-thalassemia (hemoglobin H disease), and beta-thalassemia. Because there was partial cross-hybridization between human mRNA and rabbit cDNA, the rabbit alpha- and beta-globin cDNAs could be used to demonstrate that the beta-thalassemia mRNA was enriched in human alpha-globin mRNA sequences and that the alpha-thalassemia mRNA was enriched in human beta-globin mRNA sequences. These results were confirmed by preparation of thalassemia globin cDNAs and subsequent hybridization to their template mRNAs. The amount of cross-hybridization between the human and rabbit alpha-globin mRNA and the two alpha-globin cDNAs was comparable to the cross-hybridization between the two beta-globin mRNAs and the two beta-globin cDNAs, indicating a similar degree of evolutionary divergence in the nucleotide sequences of the two globin genes.


Membrane penicillinase of Bacillus licheniformis 749/C, a phospholipoprotein. The hydrophobic membrane penicillinase of Bacillus licheniformis 749/C has been characterized in view of its possible role in secretion of the hydrophilic exoenzyme. It differs from exoenzyme in carrying an additional phospholipopeptide chain of 25 amino acids that contains only Asx, Glx, Gly, and Ser residues. The NH2-terminal residues is phosphatidylserine. since the extra peptide chain is probably relatively polar, the phospholipid group may well be directly responsible for the hydrophobic properties of the membrane enzyme.


Glucose transport carrier in human erythrocyte membranes. Dinitrophenylation of a membrane component modified by D-glucose. The effect of D-glucose on dinitrophenylation of membrane proteins of human erythrocyte ghosts by 1-fluoro-2,4-dinitrobenzene was studied in the absence and in the presence of D-glucose. A double isotopic, differential labeling technique followed by gel electrophoresis of extracts in the presence of sodium dodecyl sulfate revealed the presence of a polypeptide, or polypeptides, in human erythrocyte membranes, the reactivity of which to 1-fluoro-2,4-dinitrobenzene was significantly enhanced in the presence of D-glucose. Molecular weights of the peptides were estimated to be approximately 180,000. The effective differential labeling of the peptides required specific conditions identical with the ones which maximize the differential between inactivation of the carrier by 1-fluoro-2,4-dinitrobenzene and the dinitrophenylation of bulk membrane (Jung, C. Y. (1974) J. Biol. Chem. 249, 3568). A differential labeling was also demonstrated as a single peak by LH-20 column chromatography of chloroform-methanol-extractable membrane components. The peak contained both protein and phospholipids, indicating its proteolipid-like nature. A possible relationship of the differentially labeled polypeptides to the glucose carrier is discussed.


On the molecular weights of the three nonidentical subunits of citrate lyase from Klebsiella aerogenes. The molecular weights of the three nonidentical subunits of citrate lyase of Klebsiella aerogenes have been determined by three methods: sedimentation equilibrium in 6 M guanidinium chloride, sodium dodecyl sulfate gel electrophoresis, and gel filtration on 6 percent agarose column in 6 M guanidinium chloride. The molecular weights of the subunits, names I, II, and III (or acyl carrier protein) in order of elution from the agarose column, were 54,500, 32,000, and 11,000, respectively. The agarose-guanidine column provided a nearly complete separation of the three subunits. The molecular weight of the native enzyme was found by sedimentation equilibrium to be 520,000 plus or minus 10,000. The uncertainties in the molecular weights of the enzyme and its subunits did not permit a valid postulation of the subunit composition.


In vitro synthesis of a thyroglobulin precursor by porcine thyroid membrane-bound ribosomes in a heterologous cell-free system. The synthesis of a thyroglobulin precursor directed by endogenous messenger RNA has been achieved in a heterologous cell-free system containing porcine thyroid membrane-bound ribosomes and rat brain cortex cell sap. The main features of the system, such as ion and nucleotide requirements, temperature, dependence, inhibitors sensitivity, and ability to reinitiate, were studied. The nature of the product has been determined by specific immunoprecipitation and its size has been estimated by sucrose density gradient and sodium dodecyl sulfate acrylamide gel electrophoresis. Under our conditions, the thyroglobulin precursor was mainly synthesized by membrane-bound ribosomes.


Biosynthesis of bis(monoacylglyceryl)phosphate and acylphosphatidylglycerol in rat liver mitochondrial. Bis(monoacylglyceryl)phosphate is present in trace quantities in normal liver where it represents smaller than 1 percent of total liver phospholipids. This compound is of considerable interest since its level can be greatly increased in certain lipidoses, either of a genetic nature or caused by drugs. Biosynthesis of bis(monoacylglyceryl)P in vitro has not previously been demonstrated. This paper reports the enzymatic formation of bis(monoacylglyceryl)P and acylphosphatidylglycerol as minor products from cytidine diphosphate diglyceride and radioactive sn-glycerol-3-P using a crude mitochondrial fraction from normal rat liver; phosphatidylglycerol was the principal product. Evidence is also presented which shows the enzymatic formation of acylphosphatidylglycerol and bis(monoacylglyceryl)P from [1',3'-14C]phosphatidylglycerol by a crude rat liver mitochondrial preparation in vitro. The structures of the radioactive bis(monoacylglyceryl)P and acylphosphatidylglycerol were tentatively established by thin layer chromatography, and chromatography of the products of mild alkaline hydrolysis and acetolysis. Radioactive acylphosphatidylglycerol was stoichiometrically converted to bis(monoacylglyceryl)P by treatment with bee venom phospholipase A, providing additional evidence for the structure of acylphosphatidylglycerol and bis(monoacylglyceryl)P.


Cryptococcus laurentii cell envelope glycoprotein. Evidence for separate oligosaccharide side chains of different composition and structure. Particulate enzyme preparations of the fungus imperfectus Cryptococcus laurentii catalyze transfer of mannosyl and galactosyl residues from GDP-[14C]mannose and UDP-[3H]-galactose to the same endogenous acceptor. After solubilization with pronase, the major portion of both labels is retarded on Sepharose columns and forms a symmetrical peak, in which 14C and 3H coincide. Label also coincides with endogenous protein and carbohydrate. Both labels bind to Sepharose-Concanavalin A (Con A) and are eluted with alpha-methylglucoside. After beta elimination with NaOH-NaBH4 only 14C label retains binding to Sepharose-Con A; 3H label representing (6-O-alpha-galactosyl)10-O-beta-galactosyl-O-mannitol as previously reported (Raizada, M. K., Kloepfer, H. G., Schutzbach, J. S., and Ankel, H. (1974) J. Biol. Chem. 249, 6080-6086) no longer binds. The [14C]mannose-containing material after beta elimination yields a pentasaccharide and a trisaccharide. Similar penta- and trisaccharides can be isolated following beta elimination of particulate preparations of the organism after pronase treatment. Analytical data suggest that the structure of the isolated pentasaccharides corresponds to that of a pentasaccharide previously synthesized de novo using cell-free enzyme preparations of the organism: 2-O-alpha-mannosyl-6-O-alpha-mannosyl-3-O-alpha-mannosyl-(2-O-beta-xylosyl)-O-mannose (Schutzbach, J. S., Raizada, M. K., and Ankel, H. (1974) J. Biol. Chem. 249, 2953-2958). The trisaccharide has the structure 2-O-alpha-mannosyl-2-O-alpha-mannosyl-O-mannitol. The data are consistent with a glycoprotein structure in which these three types of oligosaccharides are bound to a common polypeptide core through O-glycosidic linkages to threonyl and seryl residues.


Reversible inhibition of the fatty acid synthetase complex from Mycobacterium smegmatis by palmitoyl-coenzyme A. Palmitoyl-CoA dissociates the fatty acid synthetase complex from Mycobacterium smegmatis into inactive subunits of molecular weight 250,000 as determined by sucrose density gradient centrifugation. Palmitoyl-CoA binds to the subunits but the binding can be prevented and reversed by the mycobacterial 3-O-methylmannose-containing polysaccharide. When the palmitoyl-CoA containing inactive subunits were isolated by gel filtration on Sepharose 6B, and then concentrated and dialyzed against 0.5 M phosphate buffer, pH 7.0, containing 3 mM of the complexing agent heptakis-(2,6-di-O-methyl)-beta-cyclodextrin, activity was regenerated to the level of 40 percent of a control sample. The reversibility of the dissociation and inactivation of the synthetase by palmitoyl-CoA suggests that this end product might play a regulatory role by acting as a feedback inhibitor.