The estimation of carbon dioxide concentration in the presence of nitrous oxide, using a Lloyd-Haldane apparatus. A method of gas analysis is described for estimating carbon dioxide in the presence of nitrous oxide, using a modified Lloyd-Haldane apparatus. Some of the sample, after scrubbing with soda-lime, bubbles through the potash and this eliminates the solubility effect of nitrous oxide by equilibrating the standard potash solution with nitrous oxide at the tension of the sample analysed and eliminating absorption of nitrous oxide from the burette. The results show close agreement with another apparatus and method based on different principles. The method is absolute, requiring neither empirical nor theoretical correction.


Deaths associated with anaesthesia.

Deaths associated with anaesthesia. The incidence of deaths associated with anaesthesia at Royal Prince Alfred Hospital for the period 1963-72 inclusive was 1:502. An analysis of the 408 deaths showed that more than half of them occurred in association with major cardiovascular and neurosurgical procedures. The number of deaths attributable to anaesthesia alone was 15 (3.7%). This was a marked reduction by comparison with the number in the previous decade (20.9%).


Quality of spinal extradural anaesthesia: the influence of spinal nerve root diameter. Profiles on the progress of spinal extradural anaesthesia were made in 246 patients using various concentrations of lignocaine, bupivacaine and etidocaine. The advance of analgesia through the different dermatomes showed a consistent and orderly spread on the upper lumbar and thoracic segments. This spread was irregular through the lower lumbar and upper sacral segments. The first sacral segment was especially difficult to block with an overall failure rate of 17.53% with presently available concentrations of various local anaesthetics. This failure was abolished with the use of etidocaine 1.5%. The presence of a delay or failure at the level of L5-S2 is probably related to the larger diameter of these nerve roots as ascertained from measurements in 11 cadavers.


Postoperative extradural analgesia following thoracic surgery: a feasibility study. The feasibility of providing postoperative analgesia using thoracic extradural blockade following thoracotomy has been assessed. Extradural block was produced by intermittent injections of 0.5% bupivacaine with adrenaline 1:200,000 or a continuous infusion of 0.25% or 0.125% bupivacaine. The only toxic symptom was drowsiness which was most frequent after a continuous infusion of 0.25% bupivacaine and with arterial plasma bupivacaine concentrations above 1.5 mug/ml. Arterial hypotension was a troublesome complication with all techniques although stability of arterial pressure was more easily achieved with a continuous infusion technique. However, this produced a high incidence of urinary retention. Practical aspects and effectiveness of providing extradural analgesia in patients following thoracotomy are discussed.


Control of the malignant hyperpyrexic syndrome in MHS swine by dantrolene sodium. Experiments are described which demonstrate that dantrolene sodium effectively terminates the syndrome of malignant hyperpyrexia induced in susceptible swine by exposure to halothane. Dantrolene is also shown to block initiation of the syndrome of malignant hyperpyrexia by halothane in MHS swine. Therapeutic use of this drug in patients with anaesthetic-induced malignant hyperpyrexia appears to be indicated.


Pancuronium and the neonate.

Pancuronium and the neonate. Pancuronium bromide was used safely as the muscle relaxant for neonatal anaesthesia. No untoward effects were seen and the neuromuscular block was successfully reversed in all patients. The potency ratio of pancuronium as compared with tubocurarine ranged from 9:1 at birth to 6:1 at one month of age.


Anaesthetic management of reconstruction of the lower portion of the trachea. Case report. A case of tracheal stenosis following chest injuries is reported. The anaesthetic technique used for partial resection of the intrathoracic portion of the trachea is described. Problems of ventilation during this type of operation are discussed.


Total calcium content of skeletal muscle isolated from humans and pigs susceptible to malignant hyperthermia. Total calcium content of both human and porcine MHS skeletal muscle is significantly less than normal. This data is consistent with the concept that some organelle (probably the sarcoplasmic reticulum, the mitochondrion or even the sarcolemma) within the MHS muscle stores less than normal amounts of calcium. The large variability between muscle specimens rules out measurement of total calcium content of skeletal muscle as a routine diagnostic test for malignant hyperthermia susceptibility.


Thrombin-induced disseminated intravascular coagulation in the dog. I: Demonstration of microthrombi in lung. Disseminated intravascular coagulation was induced in dogs by the infusion of a fibrinolytic inhibitor followed by thrombin. The deposition of microthrombi in the lung was demonstrated by radioactive labelling of platelets and fibrinogen.


Thrombin-induced disseminated intravascular coagulation in the dog. II. Cardiorespiratory changes during spontaneous and controlled ventilation. Disseminated intravascular coagulation (DIC) was induced in anaesthetized dogs by the infusion of a fibrinolytic inhibitor followed by thrombin. The occurrence of DIC was confirmed by haematological and histological examinations. After the thrombin infusion there was a progressive reduction in cardiac index and systemic arterial pressure, only four of the 14 dogs surviving for 4 hr. Pulmonary artery pressure increased after the thrombin infusion, but decreased subsequently in seven animals allowed to breathe spontaneously. In these animals, there was an increase in respiratory rate, minute volume and deadspace/tidal volume ratio, but there were no changes in the arterial-to-alveolar PCO2 difference. Arterial PCO2 and PO2 decreased, but there were no significant changes in total venous admixture. In seven dogs submitted to controlled ventilation, arterial PO2 decreased to the same extent, but there were no significant changes in arterial PCO2, deadspace/tidal volume ratio or venous admixture.


The effect of halothane anaesthesia on the uptake of oxygen and on the respiratory pattern in man. Nine patients were anaesthetized with halothane in oxygen using a closed-circle circuit with a Benedict-Roth spirometer substituted for the reservoir bag. The movements of the spirometer were recorded to enable the oxygen consumption and respiratory pattern to be measured continuously before surgery commenced. Increasing concentrations of halothane produced progressive decreases in oxygen uptake and in the tidal and minute volumes, but an increase in the respiratory rate.


Systemic and coronary haemodynamic effects of ketamine in intact anaesthetized and unanaesthetized dogs. Ten intact anaesthetized dogs breathing room air spontaneously (Group A) were compared with ten artificially ventilated dogs (Group B). All were given a bolus of ketamine 2 mg/kg followed by a 20-min infusion of ketamine 0.1 mg/kg/min. In Group A, coronary sinus blood flow, measured with a thermodilution flowmeter, increased by 90% while coronary vascular resistance decreased by 28% and coronary sinus oxygen content decreased by 27%. Heart rate increased by 47%, and arterial pressure by 9%. Cardiac output, calculated by the dye dilution method, increased by 29%, while the left ventricular work index decreased by 50%. Minute ventilation rate decreased by 55%. The Group B dogs were studied as described above, except that they were artificially ventilated. The haemodynamic ahanges were less in Group B, possibly because of improved arterial oxygenation. Heart rate increased by 24%, cardiac output by 21% and arterial pressure by 2%. The coronary sinus blood flow increased by 12% while coronary vascular resistance decreased by 11%. Coronary sinus oxygen concentration decreased by 15%. Five unanaesthetized dogs with electromagnetic flowmeter probes chronically implanted on the aorta and circumflex coronary artery, and an indwelling arterial catheter were studied before, during and after the intravenous administration of ketamine 2, 4, and 8 mg/kg. A dose of 8 mg/kg produced increases in cardiac output, heart rate and arterial pressure of 21%, 44% and 24%, respectively, while coronary blood flow increased 47%. We conclude that, in healthy dogs, ketamine produces an increase in heart rate and cardiac work. A significant increase in coronary blood flow appears to be insufficient to meet the metabolic demands of the myocardium, as the coronary sinus oxygen content decreased.


Growth hormone and blood-glucose concentrations during cardiopulmonary bypass. Plasma growth hormone (plasma GH) and blood-glucose concentrations were measured in 23 patients undergoing open heart surgery with moderate hypothermia. A significant increase in blood-glucose concentration occurred with sternotomy and increased during bypass, partly as a result of the exogenous glucose load from the perfusate. Following bypass, the blood-glucose remained above the pre-anaesthetic concentration, and this elevation persisted into the period following surgery. Plasma GH also increased with surgery and remained elevated during perfusion. The highest concentrations occurred following bypass when normal temperature had been regained .


The use of propanidid and lignocaine to reduce suxamethonium fasciculations. A technique of induction of anaesthesia using propanidid, lignocaine and suxamethonium is described, and the muscle fasciculations that follow it are shown to be significantly less than those accompanying induction with methohexitone and suxamethonium.


Effect of intercostal nerve blockade during operation on lung function and the relief of pain following thoracotomy. The use, during operation, of intercostal nerve blocks with lignocaine and bupivacaine for the relief of pain following thoracotomy was assessed in 138 patients. Irrespective of the method used to evaluate efficacy, it was not possible to demonstrate a lasting effect of clinical significance favouring either local anaesthetic agent. This form of treatment, although free from serious side effects, had no beneficial effects on lung function and is not recommended for the relief of pain following surgery.


Physiology of peripheral nerve fibres in relation to their size. There is evidence that peripheral nerve fibres, both myelinated and unmyelinated, have different membrane properties correlated with their size. Smaller fibres have a lower conduction velocity, and longer action potential time course, than would be expected if their membrane properties were identical with those of larger size. There are several possible explanantions of this result; it is suggested that at least part of the difference is due to a systematic variation in the size of the conductances (per unit area of membrane) responsible for generating the action potential.


The chemistry of local anaesthetic agents: classification of blocking agents. Compounds with local anaesthetic properties may be classified according to their action upon the nerve membrane. Four classes of agent are recognized: A. Compounds acting at the exterior of the sodium channels; B. Compounds acting at the axoplasmic part of the sodium channels; C. Compounds acting through a physicochemical mechanism; BC. Compounds acting by a combination of B and C mechanisms. The chemical basis for these different mechanisms is discussed.


Pharmacology of local anaesthetic agents. Pharmacokinetics of local anaesthetic agents. Information derived from measurements of blood concentrations of local anaesthetics can be extended by the application of pharmacokinetic anaylsis. A better understanding of quantitative aspects of the disposition and absorption of these drugs should assist the anaesthetist in deciding the optimal agent and dosage for regional block techniques.


Effects of regional intravenous anaesthesia on blood flow. Forearm blood flow after regional i.v. anaesthesia in volunteers was compared with the predicted blood flow after arterial occlusion alone, using venous occlusion plethysmography. After arterial occlusion alone for 20 min, marked hyperaemia occured after cuff release. Flows returned to normal within 5 min. Both prilocaine and bupivacaine reduced the hyperaemic response to arterial occlusion. Forearm muscle paralysis using gallamine triethiodide was also associated with a statistically significant but smaller reduction in the hyperaemic response.


The role of regional block versus parenteral analgesics in patient management with special emphasis on the treatment of postoperative pain. Based upon a series of clinico-physiological investigations, the usefulness of regional anaesthetic techniques such as extradural, intercostal and splanchnic blocks are stressed. Main emphasis is put on the relief of postoperative pain after some common types of operations such as prostatectomies, total hip replacements and cholecystectomies. Compared with parenteral analgesics, regional anaesthetic techniques offer several advantages such as a more favourable situation of oxygen transport and a more effective treatment of pain without depressing the sensorium.


Patient management during extradural anaesthesia for obstetrics. A review of some policies of clinical practice, adopted in the light of experience, is presented. These include the inadvisability of attempting to obtain a precise segmental block during the first stage of labour; the urgent need to avoid compression of the inferior vena cava by the uterus during labour and at the time of delivery; a preference for the routine use of bupivacaine in concentration of 0.375%, and for the plain solution irrespective of the concentration employed; and the advocacy of providing lumbar extradural analgesia in cases of breech presentation and multiple pregnancy. The list of contraindications has been considerably reduced during the past few years.


Respiratory effects of extradural nerve block in the postoperative period. The effects of continuous extradural block in the postoperative period on arterial hypoxaemia are discussed. Comparing two matched groups of patients following upper abdominal surgery, one receiving continuous extradural analgesia and one narcotic analgesia, there were small but statistically insignificant improvements in PaO2 in the former group. It was concluded that abdominal muscle spasm is not an important cause of postoperative arterial hypoxaemia.


Intercostal nerve block for postoperative somatic pain following surgery of thorax and upper abdomen. From 1948 to 1973, intercostal nerve block was used 10,941 times or a total of approximately 100,000 individual nerves were blocked. Junior staff (residents) performed 95% of the blocks. The local anaesthetic solution of choice used 0.25 or 0.5% bupivacaine (Marcaine) with adrenaline. Total milligram doses of bupivacaine as high as 400 mg were employed. The duration of the blocks was 9-18 hr. No severe systemic toxic reactions occurred, e.g. disorientation, convulsions, etc. The incidence of pneumothorax was 0.073%.


Clinical evaluation of local anaesthetic agents. The precise evaluation of local anaesthetic drugs in clinical practice has many difficulties. The factors which may modify the clinical profile of these drugs are: (1) procedural in nature; (2) patient related; (3) drug related; and (4) investigator related. All these factors are discussed in relation to the proper design of clinical trials.


Brachial plexus blockade for evaluation of local anaesthetic agents. Brachial plexus block (axillary approach) has been compared with ulnar nerve block for the evaluation of local anaesthetic drugs. Duration of analgesia is the same in both techniques with the same drugs. Following axillary nerve block the median, ulnar and radial nerves are blocked for a different time. The onset of motor block is faster following axillary nerve block, according to muscle power, but not according to action potentials in the e.m.g. Once all the nerves are blocked, no differences exist for the duration of muscle paralysis. The rate of techinical failure is higher with brachial plexus blocks compared with single nerve blocks. Larger amounts of local anaesthetic solutions are necessary for plexus blockade and this may cause toxic effects. The volunteer's comfort is less after plexus block because of more widespread paralysis. Therefore, ulnar nerve block and extradural block are in many respects the most suitable techniques for evaluation of new local anaesthetic agents.


Evaluation of local anaesthetic agents for regional anaesthesia in obstetrics. The selection of a local anaesthetic agent for use in obstetrics must be based upon a more critical examination of its characteristics than is necessary in non-obstetrical cases. A short latency and great effectiveness in establishing sensory anaesthesia are of utmost importance in view of the high incidence of obstetric emergencies. Motor blockade is unnecessary for vaginal deliveries, except in premature childbirth when perineal relaxation becomes desirable. Systemic toxicity of local anaesthetics has a direct bearing on the clinical condition of the neonate especially after paracervical or prolonged extradural anaesthesia. The foetus appears very sensitive to only moderately elevated plasma concentrations of local anaesthetic. Addition of adrenaline to solutions of local anaesthetic agents with a pronounced vasodilating effect prolongs the anaesthesia, may reduce drug concentrations in the plasma, and negatively affects the strength and frequency of uterine contractions.


Evaluation of clinical tolerance of local anaesthetic agents. The clinical tolerance to local anaesthetic drugs can be assessed from a knowledge of their pharmacokinetic behaviour, their absorption properties and the effects produced by controlled i.v. infusions. Pharmacokinetic behavior can be calculated from the plasma concentrations of the drugs injected i.v. in non-toxic doses. Absorption properties can likewise be determined from plasma concentrations following injection at various sites. Acute toxicity can be assessed by i.v. infusion of toxic doses into volunteers. The dosage required to produce symptoms can be compared with other drugs. Such dosage is considerably affected by the rate of injection. The venous plasma concentrations at the height of toxicity are unreliable guides to toxicity. Experiences using the new local anaesthetic etidocaine are reported.


Patch testing in the rabbit using a modified human patch test method. Application of histological and visual assessement. A 5 h, semi-occlusive human patch test repeated daily for 5 days, was modified for use in the rabbit Using this method, the relative irritancy of three cosmetic samples of known human irritancy was determined in the rabbit. In addition, attention was paid to assessment methods and a detailed histological scoring system is described as well as two methods of visual assessment. In order to compare the effectiveness of these methods, the reaction to a series of concentrations of sodium lauryl sulphate was examined. All three assessment methods gave similar results and the merits of combining histological and visual assessment are discussed.


Photochemistry of tetrachlorosalicylanilide and its relevance to the persistent light reactor. The photochemistry of 3,5,3',4'-tetrachlorosalicylanilide has been studied in solution under carefully controlled conditions. When irradiated in a buffered solution of pH 7.4 (physiological pH), three atoms of chlorine are liberated from the molecule instead of one as suggested by earlier photochemical work. From this observation a mechanism is proposed to explain the long-term photobiological effect of this compound in skin i.e. that of the persistent light reactor.


A study of physical light screening agents. In those photodermatoses in which the action spectrum involves wavelengths outside the short ultraviolet (i.e. is greater than 320 nm) little protection can be obtained against long UV (is greater than 320 nm) by the application of chemical light screening agents. An action spectrum involving wavelengths is greater than 320 nm is a common occurrence and may necessitate the additional provision of protection from a physical light screening agent. Such a physical agent is titanium dioxide and in a study of its use in a variety of formulations using in vitro techniques it was shown to be effective throughout the wavelengths 400-700 nm. It was also shown to have a satisfactory protective capacity in those photodermatoses in which the action spectrum involves wavelength peaks 365 nm (335-395 nm) and 400 nm (370-430 nm). It also has a reasonable cosmetic acceptability provided care is taken to incorporate in the formulations colouring agents which can be varied to suit the individual patient.


Immunoglobulins in the skin in dermatitis herpetiformis and their relevance in diagnosis. Eighty skin biopsies from fifty patients with dermatitis herpetiformis (DH) have been examined for immunoglobulin deposits by direct immunofluorescence. IgA was found in all fifty patients. However, in two patients no IgA was detected in their first biopsy, and it is stressed that if the clinical suspicion of DH is high and no IgA is found in a single biopsy, then the biopsy should be repeated. There are two distinct patterns of immunoglobulin deposition in DH. The most common form of deposition is seen in the dermal papillae, termed the 'papillary' pattern. This pattern was the only one present in sixty-seven of the seventy-eight biopsies. A less common pattern is that of a 'continuous' line along the dermo-epidermal junction. This was the only pattern of immunoglobulin deposition in nine of the seventy-eight biopsies. In two biopsies both the papillary and continuous patterns were present. IgA was found in all seventy-eight of the positive biopsies and was the only immunoglobulin detected in sixty-seven biopsies. In addition to IgA, IgM was present in seven biopsies, and IgG in two biopsies. In one biopsy IgM and IgG were present with the IgA. The detection of IgA in the uninvolved skin in patients with DH is a simple test to perform, and at the present time is the most reliable way of establishing the diagnosis


Post-inflammatory elastolysis and cutis laxa. Post-inflammatory elastolysis and cutis laxa (Marshall, Heyl & Weber, 1966) is a skin disease in African infants which appears to be comparatively common in at least two countries. Destruction of elastic tissue and atrophy are preceded by urticarial or by annular erythematous-popular lesions and result in severe disfigurement. The clinical features are intermediate between anetoderma (macular atrophy) and acquired cutis laxa, but sufficiently typical and characteristic to constitute a distinctive syndrome, which might represent an abnormal reaction to the bite of an arthropod.


Localized heat urticaria treated by inducing tolerance to heat. A patient with localised heat urticaria is described. Tolerance of the skin to hear was produced by repeated exposure to hot water and complete symptomatic remission was accomplished.


An explanation for the 'placebo' effect of bland ointment bases. Bland topical applications, containing no pharmacologically active ingredients, were found to have an antimitotic effect on the epidermis of the stripped dorsal skin of hairless mice. White soft paraffin, cetomacrogol cream A (B.P.), starch paste, and emulsifying ointment (B.P.) were applied for 24 h periods under occlusive dressing to the backs of mice. The antimitotic effect was present at the time of removal of the dressings and up to 72 h after removal. White soft paraffin had the maximal antimitotic activity and epidermis of mice treated with it showed only 23% of the number of mitoses of control animals having no such treatment. Emulsifying ointment had the least activity and in some circumstances actually seemed stimulatory to mitotic activity.


Juvenile melanoma disseminatum.

Juvenile melanoma disseminatum. A young man with juvenile melanoma disseminatum is described. The eruption appeared when he was 18 years old and was resolving 12 years later.


Tumour angiogenic factor associated with subcutaneous lymphoma. Growth and survival of cutaneous neoplasms are dependent on the development of a nutrient-supplying vascular lifeline. A subcutaneous lymphoma is described which mimicked a vascular neoplasm because of the extensive telangiectasia overlying the tumour. Fragments of the lymphoma inserted in direct apposition to the hamster cheek pouch membrane or separated from the membrane by a microporous filter both induced dramatic neovascularization. Control materials failed to induce significant vascular changes. A diffusable Tumour Angiogenic Factor may play a vital role in tumour survival and portends a therapeutic potential.


The removal and restitution of hair fat in the rat. Castrated male rats were washed with a solution of sodium lauryl sulphate and warm water using a standard technique. Examination of samples of hair clipped from one flank showed that about 50% of the total ether-extractable hair fat was removed. The rate of restitution of hair fat was then measured by clipping a further sample from the opposite flank of each rat after an interval. The plateau level was restored in about 8 days. There was no significant correlation with body weight either of the plateau level in unwashed rats or of the rate of build-up of lipid in washed rats. This result provides no justification for correcting levels of hair lipid for body weight.


The sebotrophic action of growth hormone (BGH) in the rat. Sebum production of rats was assessed by measuring the levels of fat extractable by diethyl ether from samples of hair clipped immediately after shampooing with sodium lauryl sulphate and 2 days later. By the use of matched litter mates in was unequivocally demonstrated that the response of the sebaceous glands to testosterone is virtually abolished by hypophysectomy and that it can be completely restored by pure bovine growth hormone.


A case of variegate porphyria in an Indian. A female patient aged 25 years developed abdominal and neurological symptoms followed by a photosensitive eruption during pregnancy. She was found to have variegate porphyria on investigation. Investigation of the family revealed on member having latent porphyria. To our knowledge, this is the first report of variegate porphytia from India.


Panniculitis (Rothmann-Makai), with good response to tetracycline. A case of idiopathic cirucumscribed panniculitis of the legs, with features consistent with that of the Rothmann-Makai syndrome, is reported. The response to a 10-week course of oral tetracycline therapy was impressive. It is suggested that the anti-lipase activity of tetracycline could account for this response.


Student feedback from MCQ examinations.

Student feedback from MCQ examinations. A simple method is described for providing feedback to students from multiple choice question examinations. A mask with holes punched at positions corresponding to the correct answers and labelled with the subject of the questions is superimposed on the student's answer sheet. Correct answers appear as black circles and a photocopy may be taken as a permanent record. The method is inexpensive, not dependent upon computers, and is economical in staff time. It is appropriate in a wide variety of situations where MCQs are used.


Experience with multiple choice question papers in Nigeria. A practical guide to their use in a new medical school. The application of MCQ examination techniques, in the context of a new medical school in tropical Africa, is described. The pitfalls are noted, the composition of papers discussed, and a simple method of marking is described. Some examples of questions are given, and their individual values are simply assessed. From our experience, there is no doubt that objective testing has a real place in a young medical school, where it can actually save time, as well as reducing variation in standards of examination.


Career attainment - chance or choice. Survey of career experience of doctors graduating in Scottish medical schools in 1962. A survey was carried out of the career experience of the doctors who graduated from Scottish medical schools in 1962. Few of those in general practice in the UK had undergone the minimum period of vocational training now considered necessary for new entrants to practice. Of those in the hospital and specialist services, 53% were consultants. There was a tendency for early career decisions to be made, leading to an increasing number of doctors obtaining postgraduate training almost exclusively in one specialty. Many married women doctors were underemployed. A quarter of the doctors were living overseas, including those of the nonBritish nationals who had returned to their country of origin.


Longitudinal study of medical students selected for admission to medical school by actuarial and committee methods. This study evaluates a "mid period" follow-up evaluation of the outcomes of selection of medical students by customary committee review procedures versus actuarial selection. One-third of a freshman class was selected solely on the basis of a predictor index which was a previously validated, optimally weighted combination of scores on the Medical College Aptitude Test and the premedical grade-point average. The remaining two-thirds were selected by committee decision based on review of the total application file which, in addition to the aptitude test scores and academic record, included basic demographic data, information on extracurricular activities, avocational interests, work experience, letters of recommendation, personality test profiles, and interview impressions. In a previous study, it was reported that the two groups of students were undifferentiated with respect to their academic standing at the close of their sophomore year. In the present study, the actuarially selected and committee selected students were compared on class rank at the end of the junior year, total and subtest scores on part II of the National Board Examinations administered toward the close of their senior year, and type and location of internship, and practice or training status one year after graduation. The two groups were not reliably differentiated on any of these variables. Implications of the findings are discussed with respect to reliability, efficiency, and economy in the selection process and the function of the admissions committees with respect to borderline cases and issues of school policy and philosophy.


How should we select? - A sociologist's VIEW. The implications of recent studies of the selection process for entry to medical school are discussed. It is argued that there are two types of factors that need to be considered in selection; first, those that are essentially concerned with policy decisions in relation to entry, e.g. sex or nationality; and, second, those that are to do with individual characteristics. In relation to this second group, once minimal criteria are satisfied, random choice seems as fair as any method of selection, as a means of choosing among applicants: none appears to be sufficiently predictive of performance to be suitable as a means for selection in themselves.


Performance rating scale for peer and self assessment. A performance rating scale was developed and tested on a class of junior medical students who rated themselves and four to ten of their peers. When 928 ratings were factor analysed, two strong factors, knowledge and relationship, emerged. Test-retest reliabilities were good. Validity was measured by correlation of ratings with grades, and though both sources of ratings correlated significantly with grades given by faculty, peer ratings were more highly related to grades than were self ratings. Students tended to rate themselves lower than they were rated by their peers. Grades are probably not the best estimate of performance, but are currently one of the most reliable. Use of the scale to judge performance of physicians in practice has not been tested. The question of how such evaluation of peer and self would relate to other measures of quality of care is raised.


Microsurgical reanastomosis of rabbit oviduct and its functional and pathological sequelae. A microsurgical technique for anastomosis of the oviduct is described using the rabbit as an animal model.  A control group of five previously sterilized rabbits had both oviducts rejoined and all became pregnant.  Another three groups of animals had a reanastomosis immediately after division of both oviducts.  One oviduct was later removed for histological study and the animals were subsequently mated.  Twenty-three out of 25 (92 per cent) conceived and delivered a normal litter after repair of the oviduct with very fine nylon sutures without postoperative splinting; only seven out of 15 (46.7 per cent) delivered after catgut sutures had been used without splinting and only 9 out of 20 (45 per cent) delivered when nylon sutures and prolonged splinting were associated with increased fibrosis, adhesion formation and poor healing of the anastomosis.


The karyopyknotic index of amniotic fluid cells and its relationship to oestrogen concentration. Estimations of karyopyknotic index and amniotic fluid oestrogen concentration were made on 107 specimens of amniotic fluid obtained between the 25th and 42nd weeks of pregnancy.  The karyopyknotic index was shown to fall with advancing gestation as the amniotic fluid oestrogen concentration increased.  The negative correlation between karyopyknotic index and amniotic fluid oestrogen concentration was highly significant when the fetus was female but was not significant when the fetus was male.  Intermediate cells from the gential tract of the female fetus can account for the cytological difference between amniotic fluid from male and female fetuses.  The fall in amniotic fluid karyaryopyknotic index towards term which suggests that the fetal vagina responds in the same way as the maternal vaginal epithelium to the circulating hormones and not to the oestrogens in the amniotic fluid.


Vaginoplasty at the Chelsea hospital for women: a comparison of two techniques. The results have been compared of 37 McIndoe-Read operations and 39 Williams' operations carried out at the Chelsea Hospital for Women.  The McIndoe-Read procedures comprised 28 primary operations for congentital absence of the vagina, five repeat procedures and four operations for other conditions; the Williams' procedures comprised 27 primary operations, four operations following the previous vaginoplasty and eight operations following other procedures.  The results favoured the Williams' vulvovaginoplasty.  Special mention must be made of the dangers of repeating the McIndoe-Read procedure and of the fact that the vagina constructed by the Williams' technique showed no tendency to contract if intercourse did not occur.


Plasma concentration of gonadotrophins, oestrogen and progesterone in hypothyroid women. In an effort to determine the influence of hypothyroidism on the hypothalamopituitary-ovarian axis plasma levels of luteinizing hormone (LH), follicle stimulating hormone (FSH), oestrogen and progesterone were measured daily for 10 to 14 consecutive days in five women with hypothyroidism.  In all subjects investigated the levels of all the hormones measured were low in comparison with the values in euthyroid women.  FSH/LH ratios were, however, higher in hypothyroid women than in both phases of the menstrual cycle in euthyroid controls.  It is postulated that changes in the FSH/LH ratio may be related to the low levels of oestrogen and probably to failure of ovulation in hypothyroid women.


Cord plasma glucose and insulin concentrations and maternal-fetal relations. The concentration of glucose and insulin was estimated in 854 samples of umbilical cord blood plasma and 503 concurrently collected maternal blood samples. The mean cord insulin concentration, excluding all infants born to known diabetic mothers, was 7 muU./ml., but the distribution was skewed with 10 per cent of infants having a value of 12 muU./ml. or more. The giving of intravenous sugar-coating fluids to the mother during labor tended to exaggerate the skewness rather than move the whole distribution to the right. Babies born by vaginal route, whatever the presentation of method of delivery, had slightly lower mean insulin values than those born by Caesarean section. It thus seems unlikely that "stress" is a factor causing high cord insulin values at birth. Other data concerning maternal-fetal glucose and insulin relations are discussed.


Serum calcium, phosphorus and magnesium levels in pregnant and non-pregnant Nigerians. Serum calcium, inorganic phosphorus and magnesium were determined in 282 Nigerian women comprising 213 pregnant women, 37 non-pregnant controls, and 32 postpartum women.  There was very little change in the serum calcium levels throughout pregnancy; the slight reduction which occurred during the 8th and 9th months was not statistically significant.  There was a significant reduction in the serum concentrations of inorganic phosphorus and magnesium during the 8th and 9th months of gestation; the levels of both minerals rose to non-pregnant levels at term.


Relation between placental site and length of gestation. Two theories related to uterine action have been brought together to see whether the site of placental implantation influences the onset of labour: (1) that uterine contractions begin asymmetrically from a "pacemaker" in one uterine horn, and (2) that progesterone from the placenta blocks myometrial contractility primarily at the site of implantation.  Case records were examined of 182 patients who had placental locations performed and who had a spontaneous onset of labour.  Where the placenta was implanted in the right upper quadrant of the uterus labour occurred on average four days sooner than when it was implanted in the left upper quandrant: the difference was statistically significant.


Fetal axillary lymphangioma complicating breech delivery. A case of massive lymphangioma arising from the axilla and complicating a breech delivery is described.  From research of the literature it is believed that this is the only such case recorded.


The effect of potassium on the sodium requirements of growing steers with and without alpha-tocopherol supplementation. 1. Hereford steers, housed in concreted yards and given demineralized water, were offered ad lib. a low-sodium (0.07 g Na and 4.5 g potassium/kg) basal diet of sorghum grain, urea and minerals, to which 0, 15 or 30 g K as KHCO3/KG diet had been added. Mean daily intakes of K were 26, 107 and 168 g/steer respectively. 2. The steers were given daily supplements of 0, 3.25 or 6.50 g Na as NAHCO3/steer, added to the food; mean daily intakes of Na (food plus supplemental NaHCO3 minus residues) were 0.41, 3.06 and 6.26 g/steer respectively. 3. Rate of gain of body-weight was positively related to the rate of Na supplementation and negatively related to the K content of the diet, but there was no interaction between Na and K contents of the diet. 4. the Na:K ratios for the parotid saliva from steers not given Na supplements was 0.3, whereas steers ingesting either 3.1 or 6.3 g Na/d had Na:K ratios of about 12. Increasing the amount of K in the diet had no consistent effect on the saliva Na:K ratio. The Na:K ratios for rumen fluid reflected changes in the saliva Na:K ratio in response to Na supplements and were inversely related to the amount of K in the diet. 5. The width of the adrenal zona glomerulosa from steers receiving the basal diet, without Na supplements, was significantly greater than that from steers given the Na supplements, but it was not affected by the amount of K in the diet. 6. Both Na supplements and the diet containing 30 g added K/kg significantly increased the Na:K ratio of the plasma. 7. The addition of 200 mg DL-alpha-tocopheryl acetate/steer per d to the diet had no significant effects on the growth rate of the steers. 8. It was concluded that the Na requirement of steers for growth was not signficantly affected by the amount of K in the diet up to 168 g/d.


The sequential feeding of growing chickens. 1. Male chickens, I week old, were fed alternately for 6 or 12 h periods with diets complementary in their amino acid composition. The isonitrogenous, isoenergetic diets were based on fish meal or sunflower meal as the sole source of protein, or fish meal-sunflower (40:60, w/w); each diet contained 120 g crude protein/kg. Other groups of chickens were given these diets ad lib. 2. Net protein utilization (NPU) values obtained after a I-week experimental period indicated  that sequential feeding for bot 6 and 12 h resulted in significantly improved responses compared with chickens given the same diets ad lib. These results suggested that the two protein sources, although provided at separate times, could complement each other. 3. In certain treatments there was a 6 or 12 h fasting period between feeding periods to reduce the effect of nutrients from the previous period. 4. For alternate feeding and fasting periods of 6 h with the combined diet, food intake and gain in body-weight were both significantly reduced compared with the corresponding values for the combined diet fed ad lib. The NPU value was virtually the same for both treatments. For 12 h periods of alternate feeding and fasting the gain in body-weight was only just significantly lower than that for the control group (combined diet fed ad lib.), but the NPU value was highly significantly better than that for the control group. 5. For the treatments which included 6 and 12 h fasting periods between the two single-protein diets, the food intake and gain in body-weight were significantly lower compared with the treatment groups given alternate feeding thd fasting periods of either 6 or 12 h with the combined diet. The NPU values for the two groups given 12 h alternate feeding and fasting periods differed significantly, whereas the NPU values for the two groups given 6 h feeding and fasting periods were the same.


Inability of selenium to affect creatine metabolism in rat muscle. 1. Two groups of adult rats were placed in a metal-free environment and pair-fed with selenium-supplemented and Se-deficient diets. 2. After 5 months the animals were killed and skeletal muscle concentrations of creatine, creating phosphate, ATP, protein and Se were determined. 3. Se deficiency was indicated by the low Se content of the skeletal muscle from the deficient animals, but no changes were found in the amounts of the other components. 4. These results suggest that Se may not be involved in creatine metabolism and that Se deficiency may not be concerned independently in the development of nutritional dystrophy, where changes are found in the levels of protein, creatine, creatine phosphate and ATP.


The influence of food intake on portal blood flow and heat production in the digestive tract of sheep. 1. Measurements were made of portal blood flow, heat production and oxygen consumption in the digestive tract of sheep either fasted or given the following diets: chopped, dried grass, pelleted, dried grass; chopped, dried lucerne; pelleted, dried lucerne, or a pelleted barley diet. 2. For sheep that had been fasted for 48 h, portal blood flow was 1.84 l/min, total visceral heat production was 62.3 kJ/kg body-weight 0.75 per 24 h and aerobic heat production, estimated from oxygen consumption, was 62.1 kJ/kg body-weight 0.75 per 24 h. 3. Portal blood flow was markedly influenced by food intake, increasing from 1.8 l/min for starved sheep to 2.4 and 4 l/min for sheep feed at maintenance and 2.5 times maintenance levels of intake respectively. Variations in the quality and physical form of the diets had no apparent effect on portal blood flow. 4. There was a curvilinear relationship between total heat production in the gut and metabolizable energy (ME) intake. The increase obtained for levels of intake below maintenance was greatest with lucerne diets, and least with pelleted, dried grass or pelleted barley diets. Above maintenance levels of intake the rate of increase in heat production, with all diets, was about 150 kJ/MJ ME intake. 5. The heat of fermentation, estimated from the difference between total visceral metabolism and the aerobic metabolism of the tissues of the gut wall, was 76, 60 and 22 kJ/MJ digestible energy intake for the dried grass, lucerne and barley diets respectively. 6. The contribution of fermentation heat and the aerobic metabolism of the gut to the total heat increment of feeding in sheep was assessed. It was concluded that about half the heat increment must be derived from tissues outside the digestive tract.


Studies on the fortification of cane sugar with iron and ascorbic acid. 1. The feasibility of improving iron nutrition by fortifying cane sugar with Fe and ascorbic acid was studied. 2. It was found to be possible to add a number of Fe salts together with ascorbic acid to sugar without affecting its appearance or storage properties. 3. The absorption of Fe from fortified sugar eaten with maize-meal porridge or made into jam or biscuits was measured in ninety-four volunteer multiparous Indian women using the 59-Fe erythrocyte utlization method. 4. The absorption of Fe from sugar fortified with ascorbic acid and ferrous sulphate and eaten with maize-meal porridge was increased about twofold in the ratio, ascorbic acid:Fe was 10:1 by weight. If the ratio was increased to 20:2, Fe absorption was increased a further threefold. 5. Sugar fortified with soluble Fe salts, including FeSO4.7H2O, discoloured both tea and coffee; sugar fortified with ferric orthophosphate did not have this effect. 6. Fe from FePO4.H2O was poorly absorbed when added with sugar to maize-meal porridge, and also when added with adequate quantities of ascorbic acid. This form of Fe was absorbed much less well than was the intrinsic Fe present in the maize. 7. When sugar fortified with FePO4.H2O and ascorbic acid was added to maize-meal porridge before cooling or was made into jam there was a several-fold increase in the amount of Fe absorbed.


The influence of dietary fat on fat metabolism and body fat deposition in meal-feeding and nibbling rats. 1. An experiment was done with rats (body-weight 160 g) to study the effects on fat metabolism and body composition of low (10 g/kg)- or high (140 g/kg)- fat diets fed as one meal for one 4 h period/d (meal-feeders) or as six spaced meals/d (nibblers). The daily energy intake/unit metabolic body-weight (body-weight 0.73) was the same for all four groups, and this level of intake was about 80% of that consumed by rats allowed unrestricted access to the low-fat diet. The experimental period was 76 d. 2. Rats given the high-fat diet deposited more body fat/d and, as a result, grew faster and were energetically more efficient than rats given the low-fat diet depressed de novo lipogenesis from glucose in epididymal and perirenal fat pads, whose fatty acid composition resembled that of the diet. 3. For both diets meal-feeders had greater stomach plus small intestine weights than nibblers and had higher plasma free fatty acid levels, when they were killed 15 h after their last meal. 4. Meal-feeders given the low-fat diet had the greatest rate of lipogenesis for fat pads. 5. Meal-feeders given the high-fat deposited less of the main dietary fatty acids in their fat pads. 6. There was no evidence that meal-feeders eating a high-fat diet adapt their metabolism completely that they become more efficient utlizers than those nibbling this diet. Meal-feeders eating the low-fat diet became no fatter than nibblers of this diet, possibly because they were eating less than their daily ad lib. intake.


Influence of magnesium deficiency on horse foal tissue concentraion of Mg, calcium and phosphorus. 1. The effects of feeding with a purified magnesium-deficient diet (--Mg, 7-8 mg Mg/kg) on horse foal blood serum and tissue concentrations of Mg, calcium and phosphorus were studied, and the results compared with histopathological findings. 2. Serum concentrations of Ca and P were unaffected by feeding with the --Mg diet, whereas serum Mg concentrations decreased from a mean initial (day o) concentration of 0.78 mmol/l to 0.53 mmol/l 7 d after foals were placed on the -Mg diet, and then continued to decrease at a slower rate. 3. Aorta concentrations of Ca and P, but not Mg, were positively correlated with the period of time foals were given the -Mg diet, verifying histopathological findings. Results for both aorta Ca and P analyses and histopathological studies indicated that mineralization of the aorta began approximately 30-35 d after foals were placed on the -Mg diet. 4. Feeding with the -Mg diet had no significant, analytically detectable effect on brain, liver, kidney, lung, spleen, skeletal or cardiac muscle concentrations of Ca, P or Mg, although microscopic evidence of mineralization was seen in some of these tissues from foals given the Mg diet for 71-180 d. 5. A significant negative correlation was found between bone ash concentrations of Mg (rib, metacarpus and metatarsus) and the length of time foals were fed on the Mg diet. Bone ash concentrations of Ca and P were, however, unchanged. 6. Low serum Mg values and negative correlations between the bone ash concentration of Mg and the period of time foals were fed on the -Mg diet supplemented with 390 mg. Mg as MgO/kg were interpreted as suggesting that either this level of Mg supplementation is marginal for the growing foal, or that the Mg in MgO is not readily available to the growing foal.


Energy exchanges associated with eating and rumination in sheep given grass diets of different physical forms. 1. Energy exchanges and other physiological functions associated with eating and rumination were determined in four experiments. Sheep were given chopped, dried grass (DGC), pelleted, dried grass (DGP) or fresh grass (FGC). 2. In Expt I a preliminary study was made using all three diets. The dry matter (DM) of DGP was eaten significantly faster than that of chopped diets. Sheep salivated most during eating and ruminated longest when given DGC. Rates of contraction for the reticulo-rumen did not differ significantly between diets during idling and rumination, but were significantly faster during eating with DGP. The apparent energy costs of eating were 17, 109 and 176 kJ/kg DM eaten for DGP, DGC and FGC respectively, but these probably underestimated the true energy cost. 3. Expt 2 compared DGP and DGC at two levels of intake. The mean energy costs of eating DGP and DGC were 23.5 and 267 kI/kg DM respectively. The mean energy costs of eating between the energy cost of eating and the duration of the meal. The proportion of time the sheep spent ruminating DGC was about 23% but less than 1% for DGP. There was no significant relationship between heat production and the time spent ruminating. 4. In Expt 3 four sheep were offered fresh grass and, later, an equivalent DM intake after the material had been dried. The sheep ate the dried meal significantly faster. The mean energy costs of eating were 208 and 346 kJ/kg DM for DGC and FGC respectively. In this experiment the sheep ruminated significantly longer when given FGC, and the energy cost of rumination was 0.11 kJ/min. 5. Increases in heat production during and after fistula-feeding only 2-8% of those obtained during eating, indicating that nearly all the increase in heat production during eating could be attributed to the energy cost of eating per se. 6. The contribution of the energy costs of eating and rumination to the heat increment of feeding and the energy requirement for maintenance of sheep are discussed.


Studies on the flow of zinc, cobalt, copper and manganese along the digestive tract of sheep given fresh perennial ryegrass, or white or red clover. 1. Sheep fitted with a rumen fistula and either a re-entrant cannula at the proximal duodenum or a re-entrant cannula at the terminal ileum were given twice daily 480-520 g dry matter as fresh perennial ryegrass, or white or red clover. Flows of digesta were corrected to 100% recovery of chronic oxide. 2. The quantities (g/24 h) of zince and cobalt leaving the stomach were significantly greater than those in the food. No significant change was found in the quantities of copper and manganese. Significantly smaller quantities of Co (all three diets) and Zn (all diets except red clover) left the small intestine than those which entered this region. No significant differences in the quantities of Cu and Mn entering and leaving the small intestine were found. Significantly smaller quantities of Zn, Co, Cu and Mn were excreted in the faeces than entered the large intestine. 3. From the flow results it was determined that there was a significant net secretion of Zn and Co in the stomach, and a significant net absorption of Zn (except with the red-clover diet) and Co from the small intestine, and of Zp Co Cu and Mn from the large intestine.


The development of a biotin deficiency in domestic fowl given wheat-based diets. 1. Studies were done of the possible development of a biotin deficiency in domestic fowl of various ages as a result of feeding with diets composed mainly of wheat, and meat-and-bone meal. The degree of deficiency was estimated from physical symptoms, liver and kidney composition and hepatic enzyme activities. 2. Only a mild biotin deficiency developed in 3-week-old chickens and no adverse metabolic effect were found for chickens reared to 7 or 15 weeks of age or maintained for 9 months in lay on these diets, which were suspected of producing fatty liver and kidney disease in young chickens. At 3 weeks of age the deficiency was more severe the heavier the strain of chicken and the greater the rate of body-weight gain. 3. The present results question the supposition that biotin deficiency is the sole factor responsible for the development of fatty liver and kidney syndrome in young chickens.


Sequence homology between mitochondrial DNAs of different eukaryotes. The sequence divergence of mitochondrial DNAs (mtDNA) from rat, mouse, guinea pig, monkey, and chicken has been examined by DNA-DNA hybridization. mtDNAs, isolated as closed circular molecules by propidium iodide-CsCl centrifugation, were labeled in vitro by use of Escherichia coli DNA polymerase I, and renatured (Tm-35 degrees) in the presence of a 2500-fold excess of heterologous mtDNA. Single-stranded and duples DNA were separated by hydroxylapatite chromatography. The thermal stability of heteroduplexes was compared to the homoduplex by thermal elution chromatography on hydroxylapatite columns. Heteroduplex fromation between the tritiated myDNAs and a 2500-fole excess of rar mtDNA were 70, 59, 37, and 22%, respectively, for mouse, guinea pig, monkey, and chicken. Similar results were obrained in reciprocal hybridizations where one of the other mtDNAs was present in excess. Considerable mismatching of sequences in all the heterohybrids was indicated by a 18-24 degrees depression in the te50 of the heteroduplexes compared with the homoduplex. There was no apparent change in heteroduplex formation when the concentration ratio of driving DNA in excess to [3H]mtDNA was varied between 1250 and 7500. Furthermore, a second renaturation with excess driving DNA after completion of the first reaction resulted in no detectable augmenting of heteroduplex formation. Similar sequences appear to be conserved preferentially in different organisms, since the presence of two of fouf different heterologous mtDNAs in excess resulted in only moderate and nonadditive increases in heteroduplex formation. Evolutionary divergence of mtDNA sequences appears to have occurred at rates similar to that for unique sequences nuclear DNA.


The action of colicin E2 on supercoiled lambda DNA. I. Experiments in vivo. A lambda DNA supercoil system has been developed to study the effects of colicin E2 on DNA in vivo. Colicin E2, a protein antibiotic synthesized by strains of coliform bacteria that carry the Col E2 plasmid, had as its most conspicious effect damage to the DNA of sensitive strains. Colicine E2 attacks the supercoiled molecul formed by labeled lambda DNA in superinfected cells as well as it attacks the bacterial DNA. The rate and extent of acid solubilization of the lambda supercoils and of host bacterial DNA induced by E2 treatment are nearly identical. Treatment of superinfected cells with colicin E2 results in the progressive conversion of lambda DNA supercoils to open circles and/or linear full lenght molecules, and subsequently to fragments less than full lambda in size. The first endonucleolytic reactions are single-strand and or double-strand breaks. The rate of supercoil breakdown as well as the final percent supercoils remaining unconverted, the size of the final lambda fragments, and the extent of solubilization are dependent on the multiplicity of colicin used. Additions of trypsin to E2-treated superinfected cells results in a cessation of further breakdown of the lambda molecules, presumably as a result of digestion of accessible colicin molecules. Energy is essential for an early event in colicin E2 action. The host enzymes, endonuclease I and Rec BC, may be instrumental in the nucleolytic process caused by colicin E2: endonuclease I in reaction preceding cell killing and Rec BC in a secondary degradation of the bacterial DNA.


Structure-function relationship in the binding of snake neurotoxins to the torpedo membrane receptor. The Cys30-Cus34 bridge present in all long neutotoxins (71-74 amino acids, 5 disulfide bridges), but not in short toxins (60-63 amino acids, 4 disulfide bridges), is exposed at the surface since it can be reduced rapidly and selectively by sodium borohydride. Reduction and alkylation of the Cys30-Cys34 bridge of Naja haje neurotoxin III hardly alter the conformational properties of this model long toxin. Although alkylation by iodoacetic acid of th -SH groups liberated by reduction abolishes the toxicity, alkylation by iodoacetamide or ethylenimine does not affect the curarizing efficacy of the toxin. The Cys30-Cys34 bridge is not very important for the toxic activity of long neurotoxins. Reduction of the Cys30-Cys34 bridge followed by alkylation with radioactive iodoacetamide gave a labeled and active toxin which is a convenient derivative for binding experiments to the toxin receptor in membranes of the Torpedo electric organ. The binding capacity of these membrane is 1200 pmol of toxin/mg of membrane protein. The dissociation constant of the modified toxin-receptor complex at pH 7.4, 20 degrees is 10 minus 8m. Reduction with carbroxamidomethylation of the Cys30-Cys34 bridge decreases the affinity of the native Naja haje toxin only by a factor of 15. Carboxymethylation after reduction prevents binding to the membrane receptor. The binding properties of the derivative obtained by reduction and aminoethylation of Cys30-Cys34 are very similar to those of native neurotoxin III; the affinity is decreased only by a factor of 5. Binding properties to Toredo membrane of long neurotoxins (Naja haje neurotoxin III) and short neurotoxins (Naje haje toxin I and Naja mossambica toxin I) have been compared. Dissociation constants of receptor-long neurotoxin and receptor-short neurotoxin complexes are very similar (5.7 minus 8.2 times 10(-10) M at pH 7.4, 20degrees. However, the kinetics of complex formation and complex dissociation are quite different. Short neurotoxins associate 6-7 times faster with the toxin receptor and dissociate about 5-9 times faster that long neurotoxins. Acetylation and dansylation of Lys53 and Lys 27 decrease the affinity of long and short toxins for their receptor by a factor of about 200 at pH 7.4, 20 degrees, mainly because of the slower rate of association with the receptor.


Acetylcholine receptors in normal and denervated rat diaphragm muscle. I. Purification and interaction with [125I]-alpha-bungarotoxin. Acetylcholine receptors have been purified from junctional regions of normal rat diaphragm muscle and from extrajunctional regions of denervated diaphragm. The reaction of purified receptors with [122I]-alpha-bungarotoxin has been investigated by kinetic methods. The toxin-receptor complexes dissociated in a biphasic manner at 35 degrees with a rapidly dissociating component (t1/2 = 4 hr) and a slowly dissociating component (t1/2 is greater than or equal to 100 hr). The association reaction between toxin and receptor did not obey simple second-order kinetics but could be analyzed in terms of two classes of binding sites corresponding to the two rates of dissociation. This treatment of the data allowed derivation of association rate constants for the two sites. Value obtained for the dissociation constants were 3.7 times 10(-10) and less than or equal to 0.4 times 10(-10) M for the junctional receptor and 1.7 times 10(-10) and is less than or equal to 0.2 times 10(-10) M for the extrajunctional receptor. In each case it is the more tightly binding component that associates and dissociates more slowly. Receptors present in crude preparations were comparable to purified receptors in their reaction with [125I-alpha-bungarotoxin. The validity of the two site model is discussed in relation to the kinetic studies.


Acetylcholine receptors in normal and denervated rat diaphragm muscle. II. Comparison of junctional and extrajunctional receptors. Acetylcholine (ACh) receptors have been purified separately from normal rat diaphragm muscle (junctional receptors) and from extrajunctional regions of denervated diaphragm (extrajunctional receptors) in order to compare their properties. The toxin-receptor complexes of the two receptors were indistinguishable by gel filtration and by zone sedimentation in sucrose gradients, and showed identical precipitation curves with rabbit antiserum to the eel ACh receptor. Both toxin-receptor complexes bind concanavalin A and are therefore probably glycoproteins. Low concentrations of d-tubocuratine (dTC) were more effective in decreasing the rate of toxin binding to junctional than to extrajunctional receptors. The apparent dissociation constant for dTC binding to the junctional receptor was 4.5 X 10 minus 8 M, whereas the value for the extrajunctional receptor was 5.5 X 10 minus 7 M. When the complexes were analyzed by isoelectric focusing, the junctional complex focused at approximately 0.15 pH unit lower than the extrajunctional complex. This result was also found with crude preparations of receptor. We conclude that junctional and extrajunctional receptors are similar but distinct molecules. The properties of receptors present in neonatal diaphragm muscle were also examined and found to be similar to those of receptors in denervated muscle, as shown by dTC inhibition and isoelectric focusing.


Characterization by mass spectrometry of blood group A active glycolipids from human and dog small intestins. Glycolipids with blood group A activity isolated from human and dog small intestine have been characterized by mass spectrometry of intact lipid in methylated and in methylated and reduced (LiAiH4) form. Without degradative studies the glycolipids were conclusively shown to be hexaglycosyleramides with phytosphingosine as the major long-chain base and hydroxypalmitic acid as the major fatty acid. The exact sugar ratio was hexose-hexosamine-deoxyhexose 3:2:1 and the sequence established as hexosamine-[deoxyhexose-]hexose-hexosamine-hexose-hexose-ceramide. Evidence is presented that mass spectrometry can differential between type ) and type 2 saccharide chains.


Optical spectra and electronic structure of flavine mononucleotide in flavodoxin crystals. The polarized single-crystal absorption spectra of the oxidized and semiquinone forms of flavodoxin from Clostridium MP have been measured with a double beam recording microspectrophotometer. The spectra establish that the radical species in the crystal is the neutral (blue) falvine semiquinone. Combination of the spectra reported here with polarization data from previous fluorescence and stretched-film studies provides transition moment directions for the first two phi-phi transitions of the oxidized form. Predictions of molecular orbital theory are in good agreement with these experimental directions. The crystal spectra of the semiquinone indicate that the two lowest frequency transitions have the same detailed orbital origin as the corresponding transitions of the oxidized form; in the semiquinone these transitions appear at lower frequency, are closer together, and, as predicted from detailed considerations of transition probabilities, exhibit approximately half the absorption intensity. Our hypothesis of a common orbital origin suggests that semiquinone formation takes place by the addition of an electron to the lowest empty phi orbital of the oxidized form without any gross electronic rearrangement.


Further studies on the binding of divalent cations to the phosphoglycoprotein phosvitin. Dialysis equilibrium measurements at 25 degrees indicate that, at pH 6.8 and at a concentration of 1.0 times 10(-10) 3 M MnC12 or CoC12, phosvitin binds 113 Mn2+ and 120 Co2+. The binding is cooperative at low cation concentrations. The number of Mg2+, Ca2+, Mn2+, and Co2+ bound is not affected by temperatures of up to 60 degrees; however, the cooperactivity is enhanced. Optical rotatory dispersion and circular dichroism studies indicate that a conformational change occurs on binding of Mn2+ and Co2+ which parallels the one produced by Ca2+ and reported elsewhere [Grizzuti, K., and Perlmann, G.E. (1973), Biochemistry 12, 4399]. The conformational changes induced by Mg2+ and Mn2+ follow different paths. Upon binding of Mn2+ and Co2+ the intrinsic viscosity, [eta], of phosvitin decreases from about 0.5 to 0.03 dl/g, while Mg2+ and Ca2+ decrease [eta] to 0.048 dl/g. The ultraviolet absorption spectrum of phosvitin is altered upon binding of Ca2+, Mn2+, and Co2+, but not upon binding of Mg2+; an increase of the temperature to 60% has no further effect on the spectra.


Interactions of bilirubin and other ligands with ligandin. Circular dichroism methods were used to study the structure of rat ligandin and the binding of organic anions to the protein. Ligandin has a highly ordered secondary structure with about 40%alpha helix, 15% beta structure, and 45% random coil. Bilirubin binding occurred primarily at a single high affinity site on the protein. The binding constant for bilirubin (5 X 10-7 Mminus 1) was the highest among the ligands studied. The bilirubin-ligandin complex exhibited a well-defined circular dichroic spectrum with two major overlapping ellipticity bands of opposite sign in the bilirubin absorption region. This spectrum was virtually a mirror image of that of human or rat serum albumin-bilirubin complexes. Studies on the direct transfer of bilirubin from ligandin to rat serum albumin showed that sasociation constants of bilirubin-ligandin complexes were approximately tenfold less than those of the bilirubin-albumin system. Ligandin exhibited a broad specificity with respect to the typeof ligand bond. A series of organic anions inclucing dyes used clinically for liver function tests, fatty acids, hormones, heme derivatives, bile acids, and other ligands that were considered likely to interact with ligandin, were examined. Most induced ellipticity changes consistent with competitive displacement of bilirubin from ligandin and relative affinities of these compounds for ligandin were determined based on their effectiveness in desplacing the bilirubin. Some substances such as glutathione, conjugated sulfobromophthaleins and lithocholic acid bound to ligandin but induced anomalous spectral shifts, when added to ligandin-bilirubin complexes. Other compounds, including some that act as substrates for the glutathione transferase activity exhibited by ligandin, revealed no apparent competitive effects with respect to the bilitubin binding site.


Kinetics of glycogen phosphorylase a with a series of semisynthetic, branched saccharides. A model for binding of polysaccharide substrates. The requirement of muscle phosphorylase for branched polysaccharide substrates was investigated by kinetic studies on semisynthetic branched saccharides. One series of saccharides was prepared from maltoheptose by oxidizing the reducing group to a carboxyl group and coupling this with an amino group of ethylenediamine. The resulting aminooligosaccharide was coupled with p-nitrophenyl esters of mono-, di-, tetra-, and polycarboxylic aicds to produce saccharides containing one, two, four, and approximately 52 maltodextrin chains per molecule. A similar series of saccharides was prepared from a heterogeneous maltodextrin of average chain length 11.7. Kinetic constants were determined for the reaction with phoshorylase a in the direction of chain elongation. Michaelis constants are equilibrium constants for dissociation of saccharide from the enzyme-AMP-glucose-1P-saccharide complex. The Michaelis constants, expressed in terms of the concentration of nonreducing end groups, are independent of maltodextrin chain length but decrease considerably as the number of chains per molecule increases. Maximum velocities do not differ greatly from that for glycogen. Among the synthetic saccharides, only the polymer behaves similarly to glycogen in exhiiting a decreasing reaction rate as the chains are elongated. The kinetic constants are quantitatively consistent with a model in which two chain termini from the same saccharide molecule bind to the phosphorylase molecule simultaniously, Differences in binding between saccharides having different numbers of equally accessible chains are caused solely by statistical factors in the equilibrium. Highly branched substrates bind better because of their greater multiplicity of two end-group pairs.


Kinetic and mechanistic studies on the reaction of melilotate hydroxylase with deuterated melilotate. Melilotate has been synthesized from melilotate by iodiantion followed by reductive deiodination in the presence of deuterated hydrazine. The deuterated melilotate has been employed in investigations of the reaction mechanism of melilotate hydroxylase. Stopped-flow spectrophotometry has revealed no isotope effect in the formation or decay of the oxygenated intermediate which is observed when reduced melilotate hydroxylase reacts with molecular oxygen. Steady-state analysis has corroborated this result, and in addition shows that there is no isotope effect in the reductive cycle of the enzyme mechanism. This analysis does reveal a reproducible 8% decrease in Vmax for the enzyme when using deuterated melilotate. These observations are compatible with the thesis that the above intermediate is an oxygenated form of the reduced flavine prosthetic group and that the last step of the proposed mechanism is rapid and involves a primary isotope effect. The existence of the NIH shift mechanism has been studied using combined gas chromatography-mass spectrometry. No evidence could be obtained for intramolecular migration of deuterium during the hydroxylation reaction. However, the small amount of migration expected when phenols are hydroxylated precludes elimination of the NIH shift as a possibility.


Pyruvate kinase isozymes in adult and fetal tissues of chicken. Tissues of fetal and adult chickens were examined for pyruvate kinase activity. Two electrophoretically distinguishable and noninterconvertible isozymes were found. One of these, designated as type K (for kidney), is the sole pyruvate kinase in the early fetus and is found in appreciable quantities in all adult tissues except striated muscle. The second isozyme, type M, appears shortly before hatching in striated muscle and brain. These two isozymes correspond in their developmental pattern, tissue distribution, electrophoretic, immunological, and kinetic propertiesto similarly designated mammalian pyruvate kinases. However, no kinetic, immunological, or electrophoretic evidence could be found for a chicken isozyme corresponding to the mammalian type L pyruvate kinase. As the latter isozyme seems to be limited in its distribution mostly to highly differentiated gluconeogenic tissues (notable liver, kidney, and small intestine), our results support the proposition that the mammalian type L pyruvate kinase is a specilized isozyme that is present in mammals but not in birds.


Properties of chicken skeletal muscle pyruvate kinase and a proposal for its evolutionary relationship to the other avian and mammalian isozymes. Pyruvate kinase (EC 2.7.1.40) was isolated and purified from chicken and turkey breast muscle with a purification procedure very similar to that used for the bovine skeletal muscle isozyme (Cardenas, J., Dyson, R., and strandholm, J. (1973), J. Biol. Chem. 248,6931). A study of the chemical and physical properties of the chicken enzyme revealed that it is a tetramer of four apparently identical subunits, closely resembling in this and most other respects the mamalian type 7 isozyme. The properties of these two enzymes are similar enough to permit subunits of chicken type M pyruvate kinase to combine with subunits of mammalian type L (one of the three mammalian isozymes) to form interspecies tetrameric hybrid isozymes in relative quantities that do not differ makedly from those formed when both the M and L isozymes are of mammalian origin. The similarity between the mammalian and avian type M pyruvates kinases suggests a close evolutionary relationship. Further comparisons among the three mammalian and two avian isozymes of pyruvate kinase are consistent with a common evolutionary origin, perhaps from an ancestral form of the type K isozyme, which is the only pyruvate kinase identified in mammalian and avian embryos.


Rat kidney L-alpha-hydroxy acid oxidase: isolation of enzyme with one flavine coenzyme per two subunits. L-alpha-Hydroxy acid oxidase (listed as EC 1.4.3.2, L-amino acid: O2 oxidoreductase) has been purified 100-fold from rat kidney to apparent homogeneity by gel electrophoresis. A subunit molecular weight of 47,500 was found by sodium dodecyl sulfate gel electrophoresis, but in contrast to previous reports, the enzyme has been found to have a molecular weight of ca. 200,000 by Sephadex gel filtration and by dodecyl sulfate gel electrophoresis of the enzyme cross-linked with dimethyl suberimidate. A somewhat higher value was found by sedimentation equilibrium, but a tetrameric structure for the active enzyme is definitely established. The enzyme was found to contain the FMN coenzyme at a concentration of one FMN/102,000 daltons or one flavine/two subunits, a highly unusual finding. This ratio was determined from spectroscopic analysis of the FMN in lyophilized samples of the enzyme and by titration of the coenzyme with the flavine specific enzyme inactivator 2-hydroxy-3-butynoate. The enzyme has the same specific activity as a crystalline sample of the enzyme reported to have twice as much flavine/milligram.


Aggregation states and catalytic properties of the multienzyme complex catalyzing the initial steps of pyrimidine biosynthesis in rat liver. Glutamine-dependent carbamoyl-phosphate synthetase was purified about 2100-fold from the cytosol of rat liver using 30% (v/v) dimethyl sulfoxide and 5% (w/v) glycerol as stabilizers. Throughout the purification, aspartate transcarbamylase and dihydroorotase, the second and third enzymes of pyrimidine biosynthesis, were copurified with the synthetase. These three enzymes sedimented as a single peak with a sedimentation coefficient of 27 S in sucrose gradients containing the stabilizers, indicating their existence as a multienzyme complex. The aggregation states of the complex were analyzed by sucrose gradient centrifugation under conditions approximating those used for enzymatic assay and correlated with the kinetic properties of the synthetase. In the presence of 10% glycerol and 10 mM MgATP(2-) at 18 degrees, the synthetase showed high activity and the three enzymes sedimented as a single peak with a coefficient of 25 S. The three enzymes also existed as a complex with the same coefficient when 50 muM PP-ribose-P was added in place of MgATP(2-), the sedimentation coefficient of the complex shifted to 28 S, indicating alteration in its molecular shape, rather than size. With 10% glycerol alone, the complex partially dissociated and the synthetase activity appeared in three peaks with coefficients of 26, 19, and 9 S (carbamoyl-phosphate synthetases (CPSase) a, b, and c, respectively). CPSases a, b, and c, thus obtained, were all sensitive to regulation by UTP and PP-ribose-P, but they differed MgATP(2-) (5.1, 4.8, AND 1.7 mM for CPSases a and b, and the enzyme within the original complex, respectively) and in their sensitivities to effectors. These results suggest that the aggregation may modify the catalytic and regulatory properties of the synthetase; Attempts to reassociate the components were unsuccessful.


alpha-Methyl substrates of carboxypeptidase A. A steric probe of the active site. Although optical resolution of alpha-methylphenylalanine (alpha-Me-Phe) has been achieved by the action of carboxypeptidase A on the N-trifluoroacetyl derivative of the amino acid (TFA-alpha-Me-Phe), it is improbable that an alpha-methyl substrate could bind in the same orientation as glycyl-L-tyrosine, due to steric interaction of the alpha-methyl group with an atom in the imidazole ring of zinc ligand His-196. The kinetic parameters for TFA-alpha-Me-Phe and for an ester substrate bearing an alpha-methyl group (beta-hippuryl-alpha-methylphenyllactic acid, HMPL) have been determined and compared to those for the appropriate nonmethylated control substrates. Both TFA-alpha-Me-Phe and HMPL appear to be bound nearly as well as are their respective controls, and HMPL is hydrolyzed nearly as rapidly as its control. TFA-alpha-Me-Phe, however, is hydrolyzed only about one-fiftieth as rapidly as is the nonmethylated substrate. These findings are consistent with the possibilities that: (1) the proposed substrate-induced conformational shift of Tyr-248 is hindered when the methylated substrates are bound; (2) the orientation in which alpha-methyl substrates are bound precludes optimal positioning of Tyr-248 and the scissile bond even after the rotation of Tyr-248 has occurred; (3) amides and esters are bound in different orientations, and in the amide orientation an alpha-methyl group is so directed as to interfere with the approach of Glu-270 to the scissile bond.


Preparation of membrane-free chromatin bodies from avian erythroid cells and analysis of chromatin acidic proteins. A primary objective, realized in this study, was the preparation from avian erythroid cells of chromatin free of contaminating membrane, as a prerequisiste to the study of chromatin acidic proteins from cells throughout the maturation pathway. Cells are lysed in saponin (S), washed in Nonidet-P40 (N), and plasma membrane removed by high-speed rotating knives (K). Purified SNK nuclear bodies are recovered free of membrane after centrifugation through 2.35 M sucrose. The chromatin acidic proteins from such preparations of the three major avian erythroid cell types were studied. Reticulocyte SNK chromatin was compared with reticulocyte chromatin derived from saponin lysis of cells and subsequent dispersion in EDTA solutions (Harlow et al. (1972), Cell Differ. 2, 341). The dispersed preparation has a lower acidic protein/DNA ratio, but the pattern of these proteins is more complex, presumably due to the contaminating membrane. In examining SNK acidic proteins throughout the maturation pathway it is clear that there are quantitative and qualitative differences. In the dividing erythroblast, the pattern of proteins is complex and the amount relative to DNA is 1.25:1.0. For nondividing, but transcriptionally active reticulocytes and also for transcriptionally inactive erythrocytes, the pattern is very much simpler, being dominated by three bands visible on sodium dodecyl sulfate polyacrylamide gels. The ratios of chromatin acidic proteins in these preparations relative to DNA are 0.69:1.0 and 0.36:1.0, respectively. These results, obtained with purified populations of cells from a single cell line, indicate a strong positive correlation between the transcriptional activity of the cell and both the amount and complexity of non-histone proteins associated with chromatin. The correlation does not indicate whether the proteins are the cause or result of increased transcription.


Isolation and characterization of subunits from the predominant form of Dolichos biflorus lectin. The subunits of the two molecular forms (A and B) of the Dolichos biflorus lectin were isolated by ion-exchange chromatography on DEAE-cellulose in 8.0 M urea. Subunits IA and IIA which comprise the predominant molecular form A of the lectin were found to have molecular weights of 27,700 and 27,300, respectively, as determined by sedimentation equilibrium studies in 8.0 M urea. These subunits have similar amino acid compositions and each have alanine at their amino-terminal ends. Comparison of the IA and IIA subunits by immunodiffusion against antisera to the seed extract as well as to subunits IA and IIA showed no antigenic differences between the two subunits. Carboxyl terminal analyses of subunits IA and IIA with carboxypeptidase A produced an essentially simultaneous release of both leucine and valine residues from subunit IA; no detectable amino acids were released from subunit IIA under identical conditions. The data suggest that the molecular form A of the lectin (molecular weight 113,000, Carter and Etzler, 1975) consists of four subunits with a possible stoichiometry of IA2IIA2. Other possible arrangements of the subunits are discussed.


Resonance Raman studies of hemerythrin-ligand complexes. Resonance Raman spectroscopy has been used as a probe of the structure of ligands at the active site of hemerythrin. Molecularly revealing insights have been obtained with oxyhemerythrin and with metazidohemerythrin. This spectroscopic technique has also facilitated a comparison of oxygen carrier within erythrocytes with that in solution. The electronic state of the bound O2 is the same in the natural environment as in the artificial one.


A biologically active hormonal fragment isolated from bovine parathyroid glands (BPTH 1-65). Fresh frozen bovine parathyroid glands were defatted in acetone, when extracted with phenol. Following trichloroacetic acid precipitation, the resultant peptides were chromatographed on Sephadex G-100. Parathyroid hormone (BPTH) characteristically elutes in the fourth peak. However, we also observed significant hormonal activity, both biological and immunological, in the fifth elution peak. The peak V material had potent hypercalcemic activity in the rat and chick, and stimulated adenylate cyclase activity in the rat renal cortex bioassay. This material was further purified by ion exchange chromatography on carboxymethylcellulose in 8 M urea. The biological activity of the purified peptide (3700 MRC units/mg) was equivalent to that of the native hormone on a molar basis. Amino acid analysis, carboxypeptidase digestion, and partial Edman sequence analysis identified this material as BPTH 1-65, a hormonal fragment lacking the C-terminal 19 residues of the 84 residue hormone molecule. Several immunoassays using different anti-PTH antisera had variable reactivity toward the BPTH 1-65 fragment, showing that it may be useful for further characterizing antibody recognition sites. The presence of a lysine residue at position 65 suggests a tryptic-like cleavage may be responsible for the genesis of this hormonal fragment. Further investigation will be necessary to determine if this peptide has physiological significance.


On the relationship of zinc ion to the structure and function of the 7S nerve growth factor protein. The 7S nerve growth factor (7S NGF) is an oligomeric protein consisting of three distinct classes of subunits, alpha,beta, and gamma (A. P. Smith, S. Varon, and E. M. Shooter (1968), Biochemistry 7, 3259). The beta subunit contains the growth promoting activity while gamma is a potent esteropeptidase. The proteolytic activity of gamma is virtually completely inhibited in the 7S NGF aggregate (L. A. Greene, E. M. Shooter, and S. Varon (1969), Biochemistry 8, 3735). In this paper, we report that divalent metal ion chelating agents effect a seven- to tenfold increase in the esteropeptidase activity of 7S NGF at pH 7.40. Plots of esteropeptidase activity vs. chelator concentration give saturation curves which are either sigmoidal (EDTA) or hyperbolic (o-phenanthroline) depending on the chemical structure of the chelator. A survey of common divalent metal ions shows that only zinc ion (Ki = 8 times 10(7) M) and, to a lesser extent, cadmium ion are effective, reversible inhibitors of both 7S NGF and the gamma subunit esteropeptidase activities. We have found that during isolation of 7S NGF, Zn2+ is selectively associated with the oligomer in a ratio of approximately 1-2 g-atoms of zinc/mol of 7S NGF with an apparent affinity which is orders of magnitude tighter than is indicated by the Ki value for the gamma subunit. Dialysis to pH 4.0 where 7S NGF is known to undergo a reversible dissociation (A. P. Smith, S. Varon, and E. M. Shooter (1968), Biochemistry 7, 3259) brings about a tenfold reduction in the zinc ion content of the protein. This reduction is reversed on dialysis back to pH 7.4. In contrast, the isolated subunits contain only trace amounts of zinc ion at pH 7.4. Preliminary metal ion exchange experiments indicate that, of the common metal ions known to substitute for zinc in other zinc-metalloproteins, only cadmium ion is effective in substituting for zinc ion in 7S NGF. The fact that zinc ion is specifically bound to native 7S NGF, and that the zinc ion content of the system is critically dependent on the subunit aggregation state strongly suggests that zinc ion is an integral structural component of native 7S NGF.


Asymmetric exchange of vesicle phospholipids catalyzed by the phosphatidylcholine exhange protein. Measurement of inside--outside transitions. Purified phosphatidylcholine exchange protein was used to exchange phosphatidylcholine between homogeneous single-walled phosphatidylcholine vesicles and human erythrocyte ghosts. When excess ghosts were present, it was found that only 70% of the vesicle phosphatidylcholine was available for exchange. This fraction corresponds closely to the amount of phosphatidycholine in the outer monolayer of these vesicles, indicating that only the outer surface of the vesicle is accessible to the exchange protein. Also, it was found that all phosphatidylcholine introduced into vesicles by the exchange protein was available for subsequent exchange. Using the exchange protein, asymmetrical vesicles were prepared in which the outer monolayer was either enriched or depleted in radioactive phosphatidylcholine as compared to the inner monolayer. Re-equilibration of the radioactivity between the two surfaces of the vesicle (flip-flop) could not be detected, even after 5 days at 37degrees. It is estimated that the half-time for flip-flop is in excess of 11 days at 37degrees. These results indicate that the properties of the exchange protein can be expolited to measure phosphatidylcholine flip-flop rates and possible phosphatidylcholine asymmetry in biological and model membranes, without altering the structure of the membrane.


The complete amino acid sequence of a cardiotoxin from the venom of Naja naja (Cambodian Cobra). The complete amino acid sequence of a small, basic protein with cardiotoxic activity is described. This toxin, designated Naja naja F8, was isolated from the venom of Naja naja, of Cambodian origin, by gel filtration on Sephadex G-75 followed by gradient ion exchange chromatography on Bio-Rex 70. The cardiotoxin F8, molecular weight 6727 from amino acid composition, consists of 60 amino acids in a single peptide chain cross-linked by four disulfide bridges and is devoid of histidine, tryptophan, and glutamic acid. The chymotryptic and tryptic peptides from the performic acid oxidized toxin were separated by gel filtration on Sephadex G-25 and zone electrophoresis in columns of cellulose powder. The sequence was established by Edman degradation, using the direct phenylthiohydantoin method, and with the aid of carboxypetidase A, and is similar to the consequences reported for other cardiotoxins, cytotoxins, and/or lytic factors from cobra venoms, all of which show considerable homology with the functionally distinct neurotoxins.


The complete covalent structure of a cardiotoxin from the venom of Naja nigricollis (African black-necked spitting cobra). The complete covalent structure of a small, basic protein with cardiotoxic activity is described. This has been isolated from the venom of Naja nigricollis by gel filtration on Sephadex G-75 and gradient ion exchange chromatography on Bio-Rex 70. The cardiotoxin, molecular weight 6806 from amino acid composition, consists of 60 amino acids, cross-linked by four disulfide bridges, connecting 3-21, 14-38, 42-53, and 54-59. The protein contains one residue of tryptophan, phenylalanine, and glutamic acid, two residues of arginine and tyrosine, four residues of methionine, and nine residues of lysine. Histidine is absent. The chymotryptic peptides of the oxidized and S-carboxymethylated protein were isolated by gel filtration on Sephadex G-25 and zone electrophoresis on a cellulose column. The sequence was determined by Edman degradation, using the (manual) direct phenylthiohydantoin method and with the use of carboxypeptidase A. Disulfide pairing was determined on thermolysin cleaved peptides from the native protein. The sequence is shown to be homologous to other cardiotoxins and a lytic factor from snake venoms and also shows homology, both in sequence and disulfide pairing to neurotoxins. A partial reduction experiment in the absence of denaturing agent using 14-C-labeled iodoacetic acid as S-carboxymethylating agent shows that disulfide bonds 14-38 and 42-53 were reduced fastest followed marginally by 54-59, and then bond 3-21.


Regulation of casein messenger RNA during the development of the rat mammary gland. Casein mRNA was isolated and partially purified from RNA extracts of rat lactating mammary glands and translated in a teterologous cell-free protein synthesizing system derived from wheat germ. Casein mRNA activity was assayed by immunoprecipitation using a specific antiserum prepared against a mixture of the purified rat caseins. Properties of rat casein mRNA were examined using a variety of sizing techniques, including chromatography on Sepharose 4B, sedimentation on sucrose gradients after heat denaturation, and electrophoresis on 2.5% agarose gels in 6 M urea. Casein mRNA activity was found in an 8-16S region after gradient centrifugation with the peak occurring at 10.5 S. In addition, the binding of rat casein mRNA to dT-cellulose was examined. Only 40% of the total casein mRNA activity was selectively retained. A partial purification of casein mRNA was accomplished by a combination of these sizing and affinity chromatography techniques. In the purified preparations casein mRNA activity comprises approximately 90% of the total mRNA activity. Characterization of this material by agarose gel electrophoresis revealed two main bands of RNA at approximately 12 and 16 S, both containing casein mRNA activity. These mRNAs were of the correct size to code for two of the principal rat caseins of approximately 25,000 and 42,000 molecular weights. Casein mRNA and total mRNA activities were then compared in total RNA extracts at various stages of normal mammary gland development in the rat, i.e. during pregnancy, lactation, and involution following weaning. A selective induction of casein mRNA activity compared to total mRNA activity was found to occur during pregnancy and lactation. Moreover, a selective loss of activity was also observed during mammary gland involution. A surprisingly high level of casein mRNA activity was found in RNA extracts from early and midpregnant mammary glands.


Hybridization of polymers of antibiotic C-nucleoside phosphates, poly(formycin phosphate) and poly(laurusin phosphate). The ability of complex formation of poly-(formycin phosphate), poly(F), and poly(laurusin phosphate), poly(L), with the polymers of natural polynucleotides was examined mainly by mixing experiments in 0.1 M NaCl-0.05 M sodium cascodylate buffer (pH 7.0) at 2 degrees. Poly(F) formed complexes with poly(U) and poly(I) in the ratio of 1:1 and 1:2, respectively. Poly(L) formed complexes with poly(A) in 2:1 ration and poly(C) in 1:2 and 2:1 ratios in addition to a self-complex. Poly(F) and poly(L) also formed a 1:2 complex between them. Some of these complexes were assumed to contain novel types of base pairings using the 7-NH group. Thus it was concluded that poly(L) could form complexes with both, the oligomer of cycloadenylic acid (øcn-120 degrees) and polymers of natural nucleotides (øcn0degrees), showing flexibility of the torsion angle of the laurusin residue.


A comparison of the digestion of nuclei and chromatin by staphylococcal nuclease. We have followed the kinetics of staphylococcal nuclease digestion of duck reticulocyte nuclei and chromatin from early stages to the digestion limit. We confirm that partial digestion of nuclei produces discrete DNA bands which are multiples of a monomer, 185 base pairs in length. The multimers are shown to be precursors of the monomer, which is next digested to a homogeneous, 140 base pair fragment. This fragment in turn gives rise to an array of nuclear limit digest DNA bands, which is almost identical with the limit digest pattern of isolated chromatin. As in the case of chromatin, half the DNA of nuclei is acid soluble at this limit. While the DNA limit digest patterns of nuclei and chromatin are similar, the large multimeric structures present as intermediates in nuclear digestion are absent in chromatin digestion. Alternate methods of chromatin gel preparation appear to leave more of the higher order structure intact, as measured by the production of these multimeric bands. Our results are consistent with the "beads on a string" model of chromatin proposed by others.


Cleavage of DNA in nuclei and chromatin with staphylococcal nuclease. Treatment of either rat liver chromatin or intact nuclei with the enzyme staphylococcal nuclease results in the conversion of about half of the DNA to acid-soluble oligonucleotides. As previously described, mild digestion of nuclei results in the liberation of a series of nucleoprotein particles containing DNA fragments which are all integral multiples of a unit length DNA 185 base pairs in length. Analysis of the kinetics of appearance of these fragments suggests that at least 85% of the nuclear DNA is involved in the formation of the repeating subunit profile. More extensive digestion of nuclei however results in the generation of a series of eight unique DNA fragments containing 160 to 50 base pairs. The series of smaller molecular weight DNA is virtually identical with the profile obtained upon limit digestion of isolated chromatin. By velocity centrifugation we have obtained highly purified preparations of the monomeric nucleoprotein particle. Digestion of this monomeric subunit results in the solubilization of 46% of the DNA and analysis of the resistant DNA again reveals the set of eight lower molecular weight fragments. These data suggest that the initial site of nuclease cleavage in chromatin resides within the DNA bridging the repeating monomeric subunits. Further attack results in cleavage at a set of sites within the monomer liberating a pattern of smaller DNA fragments which probably represents the points of intimate contact between the histones and DNA.


Circular dichroism and fluorescence studies on the binding of ligands to the alpha subunit of tryptophan synthase. Binding to the alpha subunit of tryptophan synthase induces extrinsic Cotton effects in the substrates indole (IND), indoleglycerol phosphate (IGP), and D-glyceraldehyde-3-P (D-GAP) and in the inhibitor indolepropanol phosphate (IPP). These effects disappear when the enzyme is denatured in guanidinium chloride. The induced circular dichroism (CD) was used to determine the dissociation constant and the number of binding sites for IPP. The dissociation constant so determined is equal to 48 muM and is in good agreement with the value of 48 muM obtained by equilibrium dialysis. From the temperature dependence of the dissociation constant, a value of -2.8 kcal/mol for the binding enthalpy was obtained. The determination of dissociation constants by means of extrinsic Cotton effects is shown to be quite feasible. CD competition experiments with glycerol phosphate (GP) suggest that IPP binds bifunctionally to the enzyme: via its indole part and its phosphate group. Indolepropanol, which lacks the phosphate group, does not show an extrinsic Cotton effect. Since the induced CD is strongly dependent on the binding geometry, the close similarity between the induced spectra in IPP and IGP is additional evidence that IPP is a good substrate analog. Binding to the enzyme results in a blue shift of the IPP fluorescence emission maximum. The dissociation constant determined by fluorescence titration equals 46 muM and agrees well with the values determined by the other two methods. Previous biochemical and fast kinetic studies suggested the existence of multiple conformational states for the enzyme and of ligand-induced conformational changes. No evidence was found in the far-uv CD spectra for conformational changes upon binding of IND and D-GAP. For IPP a very small effect was observed.