Transformation of morphological, functional and metabolic properties of fast-twitch muscle as induced by long-term electrical stimulation.

Transformation of structural, functional, metabolic and molecular characteristics is induced by stimulating fast-twitch muscles with the frequency pattern of a motoneuron normally innervating a slow-twitch muscle. These changes correspond to a transition of a "fast-white" into a "slow-red" muscle. Intermittent stimulation (8 h/d) does not affect the system but causes alterations of the sarcoplasmic reticulum which lead to changes in time to peak and half relaxation time. Continuous stimulation (24 h/d) induces a transformation of the muscle also at the level of the myosin system.


Influences on the efficiency of cardiac work.

The efficiency of cardiac work measured in isolated guinea pig atria can be influenced by change of the heart rate or the stroke work due to electric stimulation or drug actions: An increased cardiac performance caused by enhanced heart rate needs considerably more oxygen than an increase of the total cardiac work to the same degree due solely to a positive inotropic action. Oxidation of long and short chain fatty acids lowers the efficiency of cardiac work connected with negative inotropic effects. An increase of the heart rate is followed by a shift to the oxidation of carbohydrates, at rest to the oxidation of FFA. Long chain fatty acids are not sufficient for the increased demand of energy in the cardiac muscle at high heart rates and are then poorly oxidized. The problems connected with the Ca++ binding of FFA in the cardiac cytoplasm after inhibition of their oxidation well be discussed.


The instantaneous force-velocity relationship as an index of the contractile state in cardiac muscle.

The instantaneous force-velocity curve of mammalian myocardium has a hyperbolic shape like that of skeletal muscle. Similar to the situation in single skeletal muscle fibres maximum speed of shortening and maximum contractile force in myocardium are altered in parallel as the state of activity is changed. Various inotropic interventions affect the force-velocity relation in the same way as occurs when the activity is altered during the course of the contraction. These findings support the view that activator-calcium governs both the number of active crossbridges between the A and I filaments during tension development and the rate of turnover of the myosin bridges during shortening at zero load.


Ecchymosis of the lower leg. A sign of hemarthrosis with synovial rupture.

Four patients with hemarthrosis of the knee, synovial rupture, and ecchymoses are reported. The patients presented with calf pain and swelling and large ecchymoses, which eventually extended to the ankle as a crescent about one or both malleoli. Appreciation of this physical sign, which appears to be characteristic for the hemorrhagic subset of synovial rupture, should alert the physician to the correct diagnosis and steer him away from contraindicated anticoagulant therapy.


Association of renal papillary necrosis and ankylosing spondylitis.

Three patients with ankylosing spondylitis developed renal papillary necrosis. All had received prostaglandin synthetase inhibitors including phenylbutazone, indomethacin, or ibuprofen. One patient had sickle trait. These drugs are not commonly associated with papillary necrosis in man, but may adversely effect renal medullary blood flow. An underlying renal vascular abnormality related to ankylosing spondylitis is also considered. Patients with ankylosing spondylitis who are taking prostaglandin synthetase inhibitors should be routinely screened for hematuria.


Cellular infiltrates in scleroderma skin.

The purpose of this study was to determine the frequency, distribution, and nature of cellular infiltrates in 108 skin biopsies from patients with systemic scleroderma (SS) and localized scleroderma (LS). Cellular infiltrates, perivascular or diffuse, were noted in 49% of SS and 84% of LS patients and consisted of lymphocytes, plasma cells, and macrophages. No correlation was noted between the presence or severity of skin cellular infiltrates and serum serologic abnormalities.


Digital mucinous pseudocysts.

Thirty-one patients with 46 digital mucinous pseudocysts are reviewed. The pseudocysts are usually soft, fluctuant, dome-shaped, dimpled, smooth lesions located near and often associated with a linear defect of the fingernail. Transillumination confirms the diagnosis. Treatment is conservative, usually consisting of intralesional injections. If surgery is necessary, distal interphalangeal joint injection of methylene blue aids in complete removal by identifying the mesothelial-lined pedicle leading from the distal joint to the pseudocyst.


[Role of pelvic phlebography in the study of prostatic carcinoma].

On the basis of previous reports on a possible correlation between phlebographic patterns and diseases of the prostatic gland, indications of the pelvic phlebography in the staging of the prostatic cancer are discussed, in order to carry out informations according to TNM system.


[Value of cerebral scintiscanning with Tc 99m pertechnetate. Comparative evaluation of neuroradiological diagnosis (statistical study)].

Authors have analyzed the efficacy of brain scanning detecting in neurological diseases and compare this examin with usualy neuro-radiological screenings. They have noted an absolute diagnostic accuracy (100%) in various cases of vascular diseases and a great accuracy (81,8%) in cases of brain tumors.


[An unusual case of latero-cervical metastatic adenopathy caused by an occult tumor of tonsillar origin, discovered more than 4 years later].

The Authors report a rare case of an hidden tonsilar carcinoma, discovered more than four years after the appearance of a side-cervical adenopathy. After a review of the literature, they express some personal considerations on the diagnostic problem. Particularly they suggest systematical tonsillectomy as an also preventive treatment.


Known and newer manifestations by mycoplasma pneumoniae in adults.

Although M. pneumoniae is a well recognised respiratory pathogen both in children and in young adults, its infections have shown recently some pathomorphism and may also involve other organ systems. The paper reviews the various clinical syndromes in adults by M. pneumoniae, with particular emphasis on those more unusual and nevertheless seen by us. In this connection the possibility of pleural effusions (with a well defined cytology) associated or not with typical Mycoplasma pneumoniae and of chronic bronchopneumonias eventually evolving even in pulmonary fibrosis is stressed. Moreover, the development of M. pneumoniae respiratory infections during the course or hematological malignancies and their characteristics are described in some detail because of the relative rarity of this kind of infections in the above patients. Finally, some less frequently reported manifestations of M. pneumoniae infections, as hemolytic anemia, hepatitis and others are described and discussed.


[Study by means of enzymatic and immunochemical determination of Cl esterase inhibitor in 59 patients with hereditary angioneurotic edema].

Serum C1 esterase inhibitor was determined in 138 members of 18 italian families with hereditary angioedema by immunochemical and enzymatic assays. On the basis of quantitative and functional findings, the type A of hereditary angioedema was diagnosed in 44 subjects, and the type B in 15. Some technical devices concerning serum sample handling were identified. The influence of heparin, EDTA, and citrate on C1 esterase inhibitor activity of normal plasma was also investigated.


[HBeAg and Anti-HBe in various groups of acute hepatitis patients and carriers of HBsAg].

Serial samples of serum from 44 patients with acute hepatitis B were examined by immunodiffusion for hepatitis B e antigen (HBeAg) and its antibody (anti-HBe). The antigen was detected in the serum of 5 (11.3%) of the patients and antibody in 12 (27.3%). Two patterns of events emerged during the study: a) HB-eAg was found only in the samples collected during the early acute phase of the illness but anti-HBe was not detectable at any time; b) HBeAg was not found during the acute illness but anti-HBe was detected 4 to 6 weeks after the onset of clinical jaundice, when the HBsAg was still present in the circulation and before the anti-HBs appearance. HBeAg was persistently found in the sera of 12 of 16 (75%) patients with chronic renal failure HBsAg long term carriers and anti-HBe in 2(12.5%). Anti-HBe was detected in 33(66%) of 50 asymptomatic HBsAg carriers and HBeAg in only 1 (2%). Finally, HBeAg and anti-HBe were not detected in any of 15 patients with HBsAg negative acute hepatitis or fulminant failure and in any of 20 healthy individuals as control. Our observation confirms the specific association between e determinant and hepatitis B virus and suggests the correlation of HBeAg persistence with HBV infectivity and of anti-HBe with the asymptomatic carriage of surface antigen.


Transmural myocardial infarction in young women taking oral contraceptives. Evidence of reduced regional coronary flow in spite of normal coronary arteries.

A normal coronary arteriogram after transmural myocardial infarction is a well-recognised phenomenon, but the pathophysiology remains unclear in most cases. A possible aetiological role of oral contraceptives is suggested by the occurrence of unequivocal myocardial infarction with normal or near normal coronary arteries in 4 young women who had been taking oral contraceptives. While the cause-effect relation of coronary thrombosis and myocardial infarction remains controversial in patients with coronary atherosclerosis, a primary occlusion of macroscopically normal coronary arteries by cellular elements of blood appears possible in these cases. The action of contraceptives, the, would be analogous to their thrombogenic effect in peripheral veins and cerebral arteries. Absence of atherosclerotic lesions in these patients favours spontaneous thrombolysis and restoration of normal vessel patency in many of these cases. Myocardial blood flow in the region of the damaged left ventricular wall remains low in spite of normal coronary arteries. Reduced perfusion in infarcted areas is assumed to be the consequence of structural and functional alterations at precapillary and capillary level rather than an effect of obstructive coronary disease.


Evaluation of evolution of myocardial infarction by serial determinations of serum creatine kinase activity.

In order to investigate the relation between the release of creatine kinase (CK) in acute myocardial infarction and the evolution of infarction, the appearance functions of CK (release of CK from the heart into the circulation) were calculated by the modified method of Sobel and associates from the serial determinations of serum CK activity in 50 patients with acute myocardial infarction. The relation of the time between the onset of infarction and the peak value of the appearance function to the duration of the evolution of abnormal Q waves in 14 patients with inferior infarction and to the duration of pain in all patients was investigated. The duration of CK release from the heart averaged 37-2+/-2-4 hours and correlated well with the total CK released (R=0.665) which represents the infarct size. The mean per cent of the total CK eventually released by the time of maximum sigmaQ (sum of the amplitude of Q wave in leads II, III, and aVF) was 80-0+/-6-4 per cent and that of CK released while pain persisted was 72-0+/-3-9 per cent. These results strongly suggest that the appearance function of CK reflects the evolution of myocardial infarction.


Primary ST changes. Diagnostic aid in paced patients with acute myocardial infarction.

In 34 out of 36 patients with apical right ventricular endocardial pacing, primary ischaemic ST alterations were observed during the early stage of acute myocardial infarction. These ST changes, indicating acute injury, were detected in the paced beats in inferior and in anterior infarct. The primary ST changes were consistent only during the early stages of acute myocardial infarction and were not detected when the electrode tip was not in the apex of the right ventricle. It is suggested that the primary ST changes should be used to diagnose acute myocardial infarction in paced patients.


Atrial pacing at multiple sites in the Wolff-Parkinson-White syndrome.

Atrial pacing at multiple sites was used in an attempt to predict the site of pre-excitation in 5 patients with Wolff-Parkinson-White syndrome with 5 different anomalous pathway locations (right anterior, right posterior, septal, left posterior, and left lateral). At least 3 atrial pacing sites were tested in each patient. Pacing sites tested included high right atrium, low lateral right atrium, low septal right atrium, proximal coronary sinus, and distal coronary sinus. Atrial stimulation sites with shortest and longest stimulus-delta intervals could be identified in each patient, the shortest stimulus-delta interval in each case ranging from 60 to 80 ms. The difference between the shortest and longest stimulus-delta interval in each case ranged from 60 to 110 ms. It was suggested that the site with the shortest stimulus-delta interval corresponded to a site close to the atrial insertion of the anomalous pathway. This hypothesis was confirmed in all cases (3 with epicardial mapping and 2 with retrograde atrial activation data). In conclusion, atrial pacing at multiple sites is helpful in predicting the site of anterogradely conducting anomalous pathways, and appears particularly useful for differentiation of right posterior, left posterior, and septal pre-excitation.


Endocardial pacing electrode design and rate of displacement.

In a series of 184 patients with a permanent endocardial pacing system the rate of electrode displacement was 3-0 per cent using the Devices L120SR electrode, and 23-1 per cent using the Devices S120 electrode. The technique and pacing team were unchanged during the period of study. It is suggested that the design and mechanical characteristics of the elctrodes were responsible.


Potential pitfalls in quantification of pericardial effusions by echocardiography.

In the echocardiographic assessment of patients with pericardial effusions, the apparent width of the echo-free space between the left ventricular posterior wall and the parietal pericardium is commonly used to estimate the amount of pericardial fluid present. In 4 patients with pericardial effusions, we showed a distinct disparity between the widths of the posterior pericardial space at different levels of the left ventricular posterior wall. In 2 of them, a 'swinging heart' appearance was recorded when the ultrasoound beam was directed caudally, but not when its direction was cephalad or less caudad. It is suggested that the left ventricle should be scanned at all possible sites to minimise potential errors in estimating the amount of a pericardial effusion.


Pulmonary embolism secondary to anomalies of deep venous system of the leg.

Two cases of recurrent pulmonary emboli secondary to thrombosis in anomalies of the deep veins of the lower limb are presented. In both cases the source of emboli was not evident clinically, and it was only after venography that the venous anomalies were discovered, both being confined to one limb and amenable to surgical intervention. Such anomalies have not previously been reported as sources of venous thromboembolism in adults. The importance of venography in establishing the source of pulmonary emboli, especially when recurrent, is stressed.


Recurrent pulmonary embolism due to hydatid disease of heart. Study of 3 cases, one with intermittent tricuspid valve obstruction (atrial pseudomyxoma).

Three cases of pulmonary hypertension caused by hydatid emboli from the right side of the heart are described; cardiac catheterisation was performed in 2. One case was confirmed at operation and 2 at necropsy. The pulmonary emboli were caused by hydatid vesicles in all 3 cases and in none was there pulmonary thrombosis; free scolices were found in the pulmonary alveoli in 2. In 1 patient with repeated syncopal attacks there was a pedunculated cyst in the right atrium which was though to have intermittently obstructed the tricuspid valve. Gamma radiography, angiocardiography, and necropsy suggested a mechanical cause for the pulmonary hypertenion with no vasoconstrictive element. The surgical patient was alive and well 18 months later.


Physiological responses to prompt and  sustained squatting. Measurement by systolic time intervals.

Ten healthy men, ages 22 to 35, were studied non-invasively standing (control), at the onset of squatting ('prompt squat'), and at two minutes of squatting. Squatting produced decreases in heart rate, isovolumic contraction time, pre-ejection period, and pulse transmission time from onset of depolarisation to the first heart sound, left ventricular ejection time, and the ejection time index. These results of systolic time intervals are consistent with the bradycardia and increased ventricular filling induced by squatting. Major changes from control measurements were found at the onset of squatting, showing the impact of prompt squat of left ventricular performance.


Left ventricular endomyocardial fibrosis in India.

Clinical, radiological, electrocardiographic, haemodynamic, and cineangiographic features of left ventricular endomyocardial fibrosis are described in 8 patients seen during the 10-year period 1965-1975. Exertional dyspnoea was the commonest presenting symptom. Mild to moderate cardiomegaly was often present. The apex beat was never forcible. A loud third heart sound over the apex and an apical early systolic murmur with late systolic decresendo were characteristic of this desease. Chest X-rays films showed left atrial enlargement and pulmonary venous congestion. A pronounced rise in left ventricular end-diastolic pressure, severe pulmonary hypertension, and low cardiac index were seen in 6. Ventriculography showed an irregular, poorly contracting left ventricle with areas of dyskinesis and large end-systolic volume. Mitral regurgitation, when present, was not severe.


Symmetrical peripheral gangrene.

Two cases of symmetrical peripheral gangrene associated with intracardiac lesions are described. One had a secondary deposit from a uterine leiomyosarcoma in the wall of the right ventricle, the other had a free ball thrombus in the right atrium; there were no valvular lesions in either case. The published reports of this type of gangrene indicate that it may result from (1) va sospastic conditions, (2) small vessel obstruction, or (3) conditions producing a very low cardiac output. The two patients reported are unusual examples of the last.


Pulmonary atrsia with intact ventricular septum. Open heart surgical correction at 32 hours.

A case of pulmonary atresia with intact ventricular septum is reported in which total surgical correction was carried out successfully at 32 hours of age. Cardiac catheterisation at 17 months has revealed virtually normal haemodynamic and angiographic findings. Surgical correction of this condition in the neonatal period is discussed.


Prolapsing right atrial myxoma: clinical and heamodynamic considerations.

A 22-year-old woman with a right atrial myxoma prolapsing to the right ventricle is described. The haemodynamic findings were similar to those of cases of prolapsing myxoma of the left atrium; a notching on the ascending limb of the right ventricular pressure curve, and an initial negative, irregular deflection on the pulmonary artery pressure curve with a pronounced rise in the mean right atrial pressure (18 mmHg) were found. On deep inspiration there was a significant deepening of the y descent from 12 mmHg to 2 mmHg, indicating a changing, dynamic obstruction of the right ventricle inflow tract. These haemodynamic features can be helpful in the diagnosis of prolapsing right atrial myxoma.


Homograft replacement of aortic valve and ascending aorta in a patient with non-specific giant cell aortitis.

A case of giant cell aortitis causing ascending aortic aneurysm associated with aortic regurgitation is reported. The aneurysm was excised and the aortic valve replaced using a fresh homograft. The patient has been followed up for three and a half years. There is good evidence of correction of the haemodynamic lesion and no evidence of further arteritis or aneurysmal formation. The pathological and clinical problem of this disease are discussed.


Myocardial stimulation by pancuronium bromide.

The effect of pancuronium bromide on the canine myocardium was studied by observing several indices of myocardial contractility during i.v. and intracoronary infusion of the drug in an anaesthetized, open-chested dog preparation. Pancuronium produced tachycardia when given for 2 min by i.v. and intracoronary infusion with the heart intact. Following denervation and pacing of the heart the intracoronary infusion of the drug produced increases in maximum acceleration of blood at the aortic root, peak aortic blood flow, maximum dP/dt and (maximum dP/dt)/TP. These changes occurred about 30 s after starting the infusion and returned to control values 5 min after stopping the infusion.


Response of arterial segments from gravid human uterus to multiple concentrations of lignocaine.

Nine uterine artery segments were obtained during Caesarean hysterotomy. The amount of isometric tension developed by each segment when exposed to 3, 6, 10, 20, 50, 100, 1000 and 2000 microng/ml of lignocaine hydrochloride (Xylocaine) was measured. All nine segments contracted in response to the 1000- and 2000-microng/ml concentrations. Eight of nine, six of nine, and three of nine responded to the 100-, 50- and 20-microng/ml concentrations, respectively; one segment contracted in response to all concentrations. A dose-response curve indicated that maximum responses were obtained at the 1000-microng/ml concentration (0.1%). Although local anaesthetics are usually considered vasodilators, this study indicates that lignocaine causes the uterine artery to constrict in concentrations in excess of 20 microng/ml. Such concentrations might occur after paracervical block or accidental intravascular injection, but would not be expected in association with extradural analgesia.


Gas chromatographic determination of Ostwald solubility coefficients for cyclopropane, halothane and trichloroethene (trichloroethylene).

Gas chromatographic methods using solvent extraction for the analysis of cyclopropane and and trichloroethene (trichloroethylene) are described and evaluated; cyclopropane was extracted into carbon tetrachloride and trichloroethene into carbon disulphide, using chloroform and toluene respectively as the internal standards. Ostwald solubility coefficients were measured for cyclopropane, halothane and trichloroethene in Krebs solution: at 310 K the respective values +/- SEM of the Ostwald coefficients are 0.181 +/- 0.009, 0.78 +/- 0.02 and 1.54 +/- 0.02; over the temperature range 295-310 K the respective temperature coefficients of solubility are -2.27, -4.18 and -3.81 in units of per cent/K at 310 K.


Intracranial pressure changes in neurosurgical patients during hypotension induced with sodium nitroprusside or trimetaphan.

Intracranial pressure has been measured in 45 patients undergoing neurosurgery during the induction of deliberate hypotension using either sodium nitroprusside or trimetaphan. A statistically significant increase in intracranial pressure (ICP) occurred during the early stages of the infusion of nitroprusside in normocapnic patients. A non-significant increase in ICP was obtained in hypocapnic patients. The mean ICP increased from 6.3 mm Hg to 11.7 mm Hg when the arterial pressure was reduced slightly, but the response in individual patients varied widely (range -1.6 mm Hg to +20.9 mm Hg). When the arterial pressure (BP) had decreased to 70% of the value existing before infusion of nitroprusside, mean ICP returned to control values and thereafter decreased with further reductions in BP. In patients rendered hypotensive with trimetaphan, there was no change in mean ICP but two patients showed moderate increases (+9.3 mm Hg and +5.7 mm Hg). The mechanism of the increase in ICP with nitroprusside is thought to be expansion of the intracranial blood volume as a result of cerebral vasodilatation. Trimetaphan does not usually produce ICP changes except when intracranial compression is severe, for in these circumstances a small change in intracranial blood volume consequent upon autoregulation may trigger an increase in ICP.


Analysis of possible factors influencing PaO2 and (PaO2--PaO2) in patients awaiting operation.

Data on arterial oxygen tension (PaO2) and alveolar-arterial PO2 difference (PAO2--PaO2) have been obtained from 337 patients awaiting elective surgery. Statistical analysis of these data has assessed both the individual and the combined influence of various factors on PaO2 and (PAO2-PaO2). The factors of importance in relation to PaO2 include age, smoking habits, body build and PAO2. In relation to (PAO2-PaO2) the significant factors include age, PaCO2, weight and smoking habits.


The evaluation and improvement of an anti-pollution system.

A recently installed anti-pollution system, initially designed to function in a passive mode, was evaluated, but found to be inconstant in its performance. A simple method of making the system active is described and evaluated.


Anaesthesia with profound hypotension for middle ear surgery.

To produce a blood-free field for middle ear surgery performed with the operating microscope, the arterial systolic pressure was reduced to less than 50 mm Hg in 700 patients by means of thiopentone-halothane-nitrous oxide in oxygen anaesthesia together with pentolinium tartrate. In 84% the results were excellent, in 12% moderately good and they were poor in 4%. There were more unsatisfactory results in males (22.8%) than in females (11.4%). In spite of the arterial pressure being reduced to very low values in many instances, there was no mortality or morbidity which could be ascribed to the technique.


Measurement of carbon dioxide output, alveolar carbon dioxide concentration and alveolar ventilation in the neonate.

A system is described for measuring carbon dioxide output and alveolar carbon dioxide concentration in the neonate and for deriving the volume of alveolar ventilation. Air is drawn through a soft rubber face mask applied loosely to the face so that all expired gas is entrained. A fraction of the flow is sampled by an infra-red analyser, the signal from which is integrated to provide a measure of carbon dioxide output. Alveolar carbon dioxide concentration is measured by continuous sampling of expired gas through a nasal catheter. The response time of the catheter-analyser system is 140 ms. In 20 healthy 1-day-old neonates the mean carbon dioxide excretion was 5.86 ml/min. kg-1 (STPD) and the mean alveolar PCO2 was 4.85 kPa. From these measurements mean alveolar ventilation 147 ml/min. kg-1 (BTPS) was calculated. These results are similar to those obtained by other methods.


Etidocaine for Caesarean section--effects on mother and baby.

The effectiveness and safety of etidocaine during extradural anaesthesia for Caesarean section were evaluated in 81 patients. Adequate sensory analgesia occurred in 78 patients and muscle relaxation was excellent in all patients. The combined umbilical venous-umbilical arterial/maternal venous ratio was 0.28+/-0.2 following the administration of 1% etidocaine 25 ml with adrenaline and 0.38+/-0.2 following the administration of 1% etidocaine plain 25 ml. Neurobehavioural patterns of the newborn were not affected noticeably. Etidocaine, in appropriate dose and concentration, appears to be an excellent local anaesthetic agent for Caesarean section under extradural anaesthesia.


Plasma concentrations of local anaesthetics after interscalene brachial plexus block.

Plasma concentrations of local anaesthetic agents have been measured after 40 interscalene brachial plexus blocks in 39 patients, using lignocaine, prilocaine, bupivacaine and etidocaine. Lignocaine produced greater concentrations than prilocaine, and bupivacaine greater concentrations than etidocaine. The addition of adrenaline resulted in much lower concentrations in the case of all four agents.


I.V. doxapram hydrochloride and pulmonary complications after lower abdominal surgery.

An i.v. solution of 0.9% saline or 0.9% saline with doxapram 2 mg/ml was administered to 260 obstetric and gynaecological patients who breathed 35% oxygen following lower abdominal surgery. Arterial blood was sampled before and during the infusion in 104 subjects. With the administration of doxapram mean PaCO2 decreased, and arterial oxygenation improved. In both treatment groups respiratory complications occurred with the same low frequency (less than 15%). The majority of patients received antibiotics following surgery, but in 34 who did not, the incidence of pulmonary complications was significantly less following doxapram.


Suxamethonium-neostigmine interaction in patients with normal or atypical cholinesterase.

The effect of neostigmine 0.05 mg/kg on the neuromuscular block produced by suxamethonium was investigated in 10 normal patients and in five patients with atypical plasma cholinesterase activity, by recording the twitch response to ulnar nerve stimulation. In the normal patients, neostigmine potentiated the block produced by suxamethonium whether it was of the depolarizing or desensitizing type. On the other hand, in patients with atypical plasma cholinesterase activity, neostigmine potentiated the depolarizing phase of suxamethonium block, while antagonizing the desensitizing phase. The degree of antagonism was not related to the magnitude of neuromuscular block, but was proportional to the degree of desensitization at the time of antagonism.


Absence of "recurarization" in patients with demonstrated prolonged neuromuscular block.

The possibility of "recurarization" after antagonism of the competitive neuromuscular block with anticholinesterases was studied. Observations were made on the time-course of the block in five patients at risk from recurarization because of multiple organ failure and who demonstrated unusually prolonged blockade. In none of these patients did the block recur. We conclude that, provided spontaneous recovery of neuromuscular transmission has made progress before the antagonism, and that the patient does not deteriorate or become exhausted afterwards, recurarization is unlikely.


Awareness during endotracheal intubation: A comparison of ketamine and thiopentone.

As a result of a report suggesting an unacceptably high incidence (2%) of awareness during endotracheal intubation, two groups of patients were anaesthetized in a standard manner using a rapid induction technique. In one group of 38 patients anaesthesia was induced with thiopentone 3 mg/kg and in the other group of 39 patients anaesthesia was induced with ketamine 2 mg/kg. Suxamethonium 1 mg/kg was given immediately following the induction agent, to facilitate endotracheal intubation. The patients were interviewed after operation. A rapid induction technique did not result in unacceptable awareness with either induction agent.


Rhabdomyolysis induced by anaesthesia with intraoperative cardiac arrest.

A 9-year-old boy undergoing anaesthesia including suxamethonium and halothane suffered cardiac arrest on two occasions. Clinical and laboratory examination subsequently showed that the patient had suffered from acute rhabdomyolysis. The eventual recovery was satisfactory.


Ebstein's anomaly--anaesthetic problems. A case report.

A patient with Ebstein's anomaly undergoing anaesthesia for the operation of hysterectomy is reported. The considerations relating to the risk of an increased right-to-left intracardiac shunt are discussed. The patient was anaesthetized successfully with a combination of nitrous oxide, droperidol and fentanyl.


Changes in reaction time and drug plasma concentrations after nitrazepam and glutethimide.

1 The effects of nitrazepam, glutethimide and placebo were assessed in a double-blind cross-over study with five healthy human subjects, using reaction time, and relating changes to drug concentrations in the plasma. 2 After glutethimide in all five subjects, and after nitrazepam in four subjects, larger doses led to higher concentrations in plasma and to greater changes in reaction time; one subject displayed the higher nitrazepam concentrations and the greater effect following the smaller dose. 3 The relationship between change in reaction time and concentration appeared to be mainly a within-subject dose-effect relationship, with the peak concentration in plasma as the 'dose' and the area under the reaction time curve as the effect.


The effect of carbidopa administration on urinary sodium excretion in man. Is dopamine an intrarenal natriuretic hormone?

1 Carbidopa, a known inhibitor of the peripheral conversion of dopa to dopamine, was given to six normal volunteers to examine the effect of interference with dopamine synthesis on urinary sodium excretion. 2 On the first day of administration, carbidopa caused a significant fall not only in urinary dopamine excretion (P less than 0.01) but also in urinary sodium excretion (P less than 0.05). 3 A highly significant correlation was also found between mean sodium excretion and mean dopamine excretion in the days prior to and during carbidopa administration (r = 0.617, P less than 0.001; 4 = 0.764, P less than 0.001 respectively). 4 The significance of these findings is discussed and it is proposed that dopamine may be an important intrarenal natriuretic hormone.


The effect of dipyridamole on platelet function: correlation with blood levels in man.

1 The effect on platelet functions of dipyridamole (a pyrimido-pyrimidine compound) was compared with a control group of patients taking warfarin. 2 Adhesion, aggregation and platelet factor 4 availability showed a significant decrease in the dypyridamole group. 3 Aggregation and platelet factor 4 showed a significant correlation with blood dipyridamole level. 4 Adhesion, aggregation and platelet factor 4 were reduced below the lower limit of normal at blood dipyridamole levels above 3.5 micronmol/1.


Haemodynamic long-term effects of metoprolol at rest and during exercise in essential hypertension.

1 Twelve men with untreated essential hypertension in WHO stage I were studied on an outpatient basis to evaluate the haemodynamic long-term effect of a new beta-adrenoceptor blocker, metoprolol. 2 Oxygen consumption, heart rate, cardiac output (Cardiogreen) and intraarterial brachial pressure were recorded at rest in a supine and sitting position and during steady state work at 300, 600 and 900 kpm/min. 3 The subjects were treated with metoprolol (dose 50-250 mg/day) as the sole drug for 1 year and the haemodynamic study was repeated. 4 Mean arterial blood pressure was reduced about 12% at rest and 9% during exercise. The heart rate was decreased about 22% at rest and 20% during exercise. There was no significant compensatory increase in the stroke volume and consequently the cardiac index was reduced about 22% at rest sitting and about 17% during exercise. There was no decrease in total peripheral resistance. 5 No side-effects were seen. 6 The major haemodynamic long-term effects of metoprolol in mild and moderate essential hypertension resemble those seen by other beta-adrenoceptor blockers like alprenolol, atenolol and timolol. The study has not given support to the assumption that metoprolol should cause less depression in cardiac output than other beta-adrenoceptor blockers.


Effects of labetalol and propranolol on histamine-induced bronchoconstriction in normal subjects.

1 The effects of oral propranolol (80 mg), labetalol (400 mg) and placebo on blood pressure, pulse rate and FEV1 at rest and after inhaled histamine, have been compared in six healthy male volunteers. 2 At 90 and 120 min after ingestion propranolol reduced the pulse rate and labetalol reduced the blood pressure, thus confirming absorption of each drug. 3 At 120 min propranolol reduced resting FEV1 and enhanced the fall in FEV1 after histamine, whereas the alterations in FEV1 after labetalol did not differ from placebo. 4 These findings suggest that labetalol is less likely than propranolol to cause bronchoconstriction in asthmatic patients.


Phenytoin concentrations in mixed, parotid and submandibular saliva and serum measured by radioimmunoassay.

1 Concentrations of phenytoin in mixed, parotid and submandibular saliva and serum were determined in normal subjects after an oral dose, using a specific double antibody radioimmunoassay which requires only 20 micronl fluid. 2 Semi-log concentration-time plots of phenytoin concentration in mixed saliva and serum gave good parallelism after the initial 14 h post-administration period. 3 The mean ratio of the mixed saliva: serum phenytoin concentration was 10.3% +/- 1.5 (s.d.) in seven normal subjects. 4 Phenytoin concentrations found in separate parotid and submandibular salivary  fractions did not differ but were significantly greater (P less than 0.001) than those found in mixed saliva. 5 Phenytoin concentrations in all salivary fractions were independent of the volume of fluid produced and the degree of stimulation. 6 The rate of phenytoin secretion in the parotid and submandibular fluid was proportional to the salivary flow rate. 7 These data suggest that mixed saliva may be a suitable medium for the monitoring of phenytoin concentrations and may provide a non-invasive alternative to the direct determination of phenytoin in serum.


Age as a factor affecting lithium therapy.

1 We examined the case notes of 82 psychiatric out-patients (aged 21-84 years) receiving lithium prophylaxis and with steady-state plasma lithium levels. 2 The mean weight-related daily dose of lithium prescribed decreased by about 50% between the third and eight decades. 3 The corresponding steady-state plasma lithium levels showed a less marked tendency to decrease, this only being seen in the seventh and eighth decades. 4 In patients aged 50 years or over the daily lithium dose required to give a plasma level of 1 mmol l-1 (0.50 mmol kg-1) was significantly lower than that (0.65 mmol kg-1) in patients aged under 50 years (P less than 0¿5, Student's t-test). In patients aged 70-79 years this dose was 31% lower than in patients under 50 years. However, interindividual variation was great and it was estimated that age only contributed about 14% to the total interpatient variation. 5 Of the 36 patients under 50 years of age, 42% had minor lithium side-effects and 17% were not optimally controlled with lithium. The corresponding figures for the 46 'older' patients were 46% and 28%. 6 Generally the 50% dosage reduction seemed necessary to compensate for an age-related decrease in lithium excretion and to reduce lithium side effects to a level comparable to that acceptable in younger patients.


The effect of metoclopramide on neonatal gastric emptying.

1 The effect of metoclopramide on gastric emptying was studied on twenty-five occasions in fifteen low birth weight babies. 2 The serial test-meal technique was used. 3 The results indicate that metoclopramide does not promote gastric emptying in the newborn period.


The comparability of dosage regimens of Lanoxin tablets and Lanoxicaps.

1 Twice daily administration of 0.25 mg digoxin tablets (Lanoxin) or of 0.2 mg digoxin in solution in soft gelatin capsules (Lanoxicaps) produced similar mean steady state plasma digoxin concentrations in ten healthy volunteers. Respective values were 1.07 +/- 0.075 and 0.95 +/- 0.048 ng ml-1. 2 During continued administration, peak plasma concentrations occurred earlier after capsules with a tendency to higher peak levels. However, area under curve determinations over 7 h were similar. 3 Approximately 10% less digoxin was recovered in urine collected in a 12 h dosage interval during the lower dosage administration of capsules. Mean percentage urinary recovery of administered dose was 57% for tablets and 65% for capsules. 4 The enhanced bioavailability of Lanoxicaps was associated with reduced between-subject variability in plasma concentration. 5 Lanoxicaps (0.2 mg) should be approximately equivalent in effect to digoxin tablets (0.25 mg) currently available in the United Kingdom, though improved consistency would be anticipated.


Monitoring digoxin therapy: I. Plasma concentrations and an in vitro assay of tissue response.

1 An in vitro technique is described for measuring the uptake of 86Rb by human erythrocytes. 2 Fifteen patients were treated with digoxin for atrial fibrillation and other fast arrhythmias. 3 86Rb uptake by the patients' own red cells fell from pre-treatment values during digitalization. 4 The therapeutic response of patients with atrial fibrillation correlated better with the changes in 86Rb uptake than with plasma digoxin concentrations.


Monitoring digoxin therapy: II. Determinants of the apparent volume of distribution.

1 In eighteen subjects receiving digoxin therapy the apparent volume of distribution (Vd) of the drug has been calculated. 2 For each subject the sensitivity of the 86Rb transport mechanism of his pre-treatment erythrocytes to in vitro inhibition by digoxin has been measured (IC50). 3 The Vd of digoxin correlates both with the age of the patients and with the IC50 of their pre-treatment erythrocytes. 4 The implications of these findings are discussed in terms of the possible determinants of the Vd of digoxin. 5 Digoxin requirements may be predictable before therapy commences if IC50 measurements are performed.


Influence of surgery and dexamethasone on cell-mediated immune responses in patients with meningiomas.

Cell-mediated cytotoxicity (CTX) was studied in meningioma patients before and within 2 weeks of complete excision of the tumour, using the [3H]-prolin- microcytotoxicity test. Three of 7 patients tested before surgery showed specific CTX, 2 revealed a "non-specific" (tumour-unrelated) response, and 2 were non-reactive. After surgery, CTX decreased from 84 to 50% in one patient and became negative in 2 others previously positive. One of 2 patients showing "non-specific" CTX preoperatively became positive, while the other remained unchanged. All patients were receiving dexamethasone (DXM) at the time they were tested. Lymphocyte responses to PHA were not significantly different before or after surgery (i.e. after prolonged treatment with DXM), from healthy controls. Blocking activity could be detected in the sera of all 3 patients before surgery. This activity was not specific for meningiomas. Paradoxically, the same sera did not inhibit the proliferative response to PHA. Serum from only one patient consistently suppressed the blastogenic response of homologous lymphocytes to PHA. Inhibitory activity was associated with the IgG fraction of his serum.


In vivo cell synchrony in the L1210 mouse leukaemia studied with 5-fluorouracil or 5-fluorouracil followed by cold thymidine infusion.

[3H]-TdR and [3]-udR labelling indices and mitotic indices were followed in tumour-bearing mice after application of either 5-fluorouracil (FU) alone or of FU followed by cold TdR infusion. With FU alone, accumulation of cells at the beginning of S was found, but there was no indication of a synchronous passage of the accumulated cells further round the cycle. When FU injection was followed by cold TdR infusion, a synchronous passage of the accumulated cells through the cycle was observed. However, there was a large variation in the response of individual mice to this treatment.


Clinical testing of the radiosensitizer Ro 07-0582: experience with multiple doses.

The hypoxic cell radiosensitizer, Ro 07-0582, has now been given in multiple doses to 16 patients. They have received a total of 15-51 g in 3-20 doses. Immediate tolerance was good, and satisfactory plasma levels of the drug were consistently obtained. Neurotoxicity was, however, troublesome: convulsions occurred in the patient given the highest dose, and there was peripheral neuropathy in 11 cases. Tumour concentrations similar to those in plasma were obtained in human tumours, in contrast to the findings in mouse tumours where concentrations are usually below 40% of plasma levels. In the treatment of human tumours, a lower dose of Ro 07-0582 should give useful hypoxic cell sensitization. Although the total dose of Ro 07-0582 must be limited, there is a real prospect that it will give benefit in clinical radiotherapy.


Changes in sensitivity to radiation and ICRF 159 during the life of monolayer cultures of EMT6 tumour line.

The response of EMT6 mouse tumour cells to ICRF 159, both with and without X-radiation, has been measured during the life of monolayer cultures. The cytotoxic effect of ICRF 159 was found to be proliferation-dependent. Flow cytofluorimetry studies of cell cycle distribution showed that ICRF 159 prevented cell division while allowing DNA synthesis to continue. This anti-mitotic action and the cytotoxic effect of the drug were found to be closely related. Increased sensitivity to X-radiation was observed in cultures pretreated for 24 h with 200 microgram/ml ICRF 159 In exponential and early plateau cultures this was seen as a reduced shoulder of the survival curve. In late plateau cultures there was no apparent reduction of the shoulder, but an increase in slope.


Induction of tumours by administration of N-dibutylnitrosamine and derivatives to infant mice.

Pulse doses of N-dibutylnitrosamine(DBN), N-butyl-N-(4-hydroxybutyl)nitrosamine(BBN) and N-butyl-N-(3carboxypropyl)nitrosamine(BCPN) suspended in 1% gelatin, were administered s.c. to infant CDF1 mice, and the experiment terminated at one year of age. Tumours were induced in lungs and liver. The incidences of lung adenomas were 73-95% in all treated mice, with no sex differences. Hepatocellular adenomas and a carcinoma were found with an incidence of 81% (21/26) in DBN, 59% (13/22) in BBN, and 32% (9/28) in BCPN-treated males and the incidence was 23% (5/22) in DBN-treated females. Only one papilloma of the fore-stomach was induced in mice treated with DBN. These results indicated that the s.c. administration of DBN, BBN, and BCPN induced tumours of the lung and liver, but no tumours of the urinary bladder, under these experimental conditions. The carcinogenic effect on mice at the treated dose level was DBN greater than BBN greater than BCPN.


Effect of colchicine injection prior to the initiating phase of two-stage skin carcinogenesis in mice.

Colchicine injected 5, 9 and 24 h respectively before initiation (using s.c. injection of urethane for initiating action and TPA skin applications for promoting action, in female ICR mice) led to a significant increase in skin tumour incidence in the --9-h group, and an increase in percentage malignancy in both the --5- and --9-h groups. These times corresponded to the peak of metaphase arrest by the colchicine. The results are discussed in relation in those of Pound and Withers (1963) and others, who found that mitotic stimulation at the time of urethane initiating action raised the ultimate tumour incidence; and the inference is drawn that initiating action in mouse skin may occur during the M phase, rather than during the G1, S, or G2 phases, as suggested by others.


Further studies of space-time clustering of Burkitt's lymphoma in Uganda.

All hospital-treated cases of Burkitt's lymphoma (BL), with onset of symptoms in the period 1963-68 and resident in the Lango and Acholi districts of Uganda, were identified. The average annual incidence of BL in the 6-year period was 1-87 X 10(-5), similar to that in the adjacent West Nile district. Contrary to findings in other areas of Uganda, there was no evidence of seasonal variation in the onset of cases, nor of space-time clustering, nor of a decline in the incidence of BL in the study period. An inverse relationship was noted between the median age at onset of BL and the incidence of the disease in different areas of Uganda, a finding consistent with intense malarial infection being a precipitating factor for BL. The variable observations with respect to space-time clustering of BL and seasonal variation in incidence in different areas remains unexplained, but it is suggested that a closer study of the patterns of malarial infection in these areas may help to account for the findings.


Do sympathomimetic amines prevent exercise-induced asthma by bronchodilatation alone?

Seventeen patients with exercise-induced asthma were studied. Eleven patients (cases 7-17), of whom seven were atopic subjects, were given isoetharine or isoprenaline and were subsequently tested for exercise-induced asthma after bronchodilatation had ceased. In two atopic and one non-atopic patients protection was observed which could not be attributed to chance. Two of these three patients were studied again and this phenomenon was repeatable. Prevention of exercise-induced asthma 30 min after inhalation of isoetherine and sodium cromoglycate was compared in 10 patients (cases 1-10), of whom six were atopic subjects. There was no significant difference in the results and protection from isoetharine was not related to the magnitude of its bronchodilator effect. These results suggest that beta-adrenergic agonists may have both bronchodilator and other actions in exercise-iduced asthma. Whether these other actions are anti-alergic is unknown.


Acute Pasteurella multocida pneumonia.

Acute pneumonia caused by Pasteurella multocida appeared in two elderly patients. One patient had chronic obstructive lung disease and the other developed aspiration pneumonia after an episode of cardiac arrest. Pasteurella multocida was not isolated from the initial sputum cultures. The delay in recognizing the pathogen resulted in extension of the pneumonia and abscess formation in the patient with obstructive lung disease. Pasteurella multocida was isolated from a percutaneous transtracheal aspirate in both patients and from the blood of one. They recovered after treatment with a penicillin administered in higher than usual dosage over a period of several weeks.


Leiomyosarcoma of the uterus presenting with pneumothorax.

The case of a patient with uterine leiomyosarcoma is reported. Metastases to pre-existing cystic lung lesions resulted in spontaneous pneumothorax.


Ornithosis pneumonia associated with haemolysis.

The case of a patient with ornithosis pneumonia associated with a haemolytic anaemia is described. The infection responded to oxytetracycline and the anaemia subsequently resolved.


Pulmonary thromboembolism after travel.

Pulmonary thromboembolism developed in eight patients shortly after travel. Preexisting vein disease was present in this group. Possible prophylactic measures are suggested.


Diurnal variation of asthma.

We have studied possible aetiological factors in the large diurnal variation in peak expiratory flow rate seen in some asthmatics. This phenomenon occurred in both atopic and non-atopic patients. It could not be attributed to recumbency in bed at night or to allergy to the house dust mite in bedding. Circadian variation in asthmatic shift workers was intimately related to sleep and virtually independent of solar time. Treatment tends to lose its effect as it is discontinued during sleep and this increases the apparent diurnal variation in many patients. Excessive circadian variation may be an important factor in sudden asthma deaths. The importance of effective nocturnal medication is stressed.


Endobronchial lipoma: a review with four cases.

Endobronchial lipomas are rare benign tumours which can cause irreversible pulmonary damage unless removed early. The clinical features and treatment of four cases are reviewed in the light of the previous literature. The histogenesis of these tumours and their relationship to hamartomas are discussed. The treatment of choice in most cases is bronchoscopic excision.


The pregnant ferret as a model for studying the congenital effects of influenza virus infection in utero: infection of foetal tissues in organ culture and in vivo.

Organ cultures of ferret foetal tissues showed a similar pattern of susceptibility to influenza virus to that already observed for human foetal tissues (Rosztoczy et al., 1975); respiratory, alimentary and urogenital tissues supported the replication of influenza virus but nervous and lymphopoietic tissues (those which, in man, are associated with foetal or postnatal abnormalities) were insusceptible. In contrast to corresponding human tissues, ferret foetal placenta and amnion readily supported viral replication although both human and ferret umbilical cord were susceptible. In limited experiments, neither the membranes nor the susceptible foetal tissues became infected after intranasal inoculation of pregnant ferrets of various gestational ages. However, after intracardial inoculation of pregnant ferrets with high titre virus (ca 10(9) EBID50) virus was isolated from both foetal membranes and foetuses. The membranes became infected at early, middle and late gestation, but virus appeared to cross the placental barrier to infect foetal tissues only in late gestation. At this stage virus could be isolated not only from those foetal tissues (respiratory, alimentary and urogenital) susceptible in organ culture, but also in small amounts from tissues which were insusceptible in organ culture (heart, lymphopoietic and nervous tissue). Virus was also isolated from foetal membranes and foetuses of late gestation ferrets following intracardial inoculation with a one hundred-fold lower dose of virus which, unlike the higher dose, did not induce a maternal febrile response. The pregnant ferret appears to be a suitable model for investigating the effects on development of foetal infection with influenza virus but it may have disadvantages with regard to the nature and strength of the placental barrier.


Participation of sensory nervous structures in the development of inflammatory reactions.

The participation of sensory nervous structures in the development of inflammatory responses in the rat's paw was investigated. Electrical antidromic stimulation of the saphenous or sciatic nerves during 20 min produced an oedematous response in the paw and potentiated the reaction to locally injected carrageenin. On the other hand, decreased responses to carrageenin were observed either when the sciatic nerve was sectioned 15 days before or when a prolonged electrical stimulation (45 min) of the nerve preceded by 1 h the injection of the irritant into the paw. However, if the sciatic nerve was sectioned 30 days before, the inflammatory response to carrageenin in this paw was equivalent to that observed in the contralateral, intact paw. It is suggested that sensory nervous structures might contribute to the development of inflammatory responses, through the release of an active agent, probably from neural origin. This agent would be depleted by prolonged stimulation of sensory nerves. Following partial denervation, the remaining sensory nervous structures in the affected area might develop compensatory mechanisms to influence the institution of the reaction.


Amyloidosis developing in experimental nocardia infections.

Swiss white and C57/BL/6J mice inoculated repeatedly with either Nocardia asteroides or Nocardia brasiliensis organisms developed amyloidosis over a 7-month period. Amyloidosis also developed in these mice within 6 weeks following a single large inoculum of either organism, but not in other in-bred mouse strains, suggesting a genetic influence in the pathogenesis of this form of secondary amyloidosis.


Portacaval transposition and subsequent partial hepatectomy in the rat: effects on liver atrophy, hypertrophy and regenerative hyperplasia.

Portacaval transposition diverts portal blood from the liver. It allows systemic venous blood to perfuse the portal bed. Body weight and liver weight have been followed before and after portacaval transposition and control procedures in rats, and the DNA activity ratio studied in the liver of rats after partial hepatectomy in protacavally transposed animals. The results suggest that the liver atrophy seen after portal diversion is a result of diversion of trophic substances in the portal blood rather than of a decrease in absolute liver flow. Recovery of liver weight after partial hepatectomy in portacavally transposed animals occurs within the same time as in control animals, and the time course and magnitude of regenerative hyperplasia, as assessed by liver DNA activity ratio, is unimpaired.


The significance of free blood in liquid and solid tumours.

Impregnation of the vasculature with ink was used to study microvascular changes induced in rats by liquid (ascites) and solid growth of W256 and Y-P388 tumour cells. Ascites fluid produced by both tumours was heavily blood-stained; the deep red colour of solid tumour deposits was similarly due to the presence of free blood. In both types of tumour growth this free blood was due to diapedesis of erythrocytes through tips of capillary sprouts which grow when neovascularization (angiogenesis) occurs in response to any suitable (non-neoplastic or neoplastic) stimulus. Ascites growth of these tumours induced profuse sprouting from the peritoneal capillaries; this sprouting, together with the "bleeding" it caused, were inhibited by local pre-irradiation of the peritoneal vasculature with X-rays before intraperitoneal inoculation of rats with the tumours. Similar angiogenesis with bleeding did not occur following inoculation with an allogeneic tumour in rats which had been previously immunized against the tumour. LI tumour cells (tumour cells lethally irradiated in vitro to destroy their proliferative integrity, but which remain metabolically active) also induced sprouts to grow in close proximity to the implanted LI cells, but heat-killed tumour cells caused no sprouting. The nature and significance of neovascularization of tumours and their so-called "haemorrhagic" growth are discussed.


The effects of long-term exposure of lung fibroblast strains to chrysotile asbestos.

Evidence is presented that persistent long-term exposure of a strain of lung fibroblast-like cells to chrysotile asbestos over 37 passages in vitro leads to enhancement in cell mat collagen deposition and is accompanied by other metabolic alterations in the cultures.


Alloxan diabetes in spontaneously hypertensive rats: gravimetric, metabolic and histopathological alterations.

In order to investigate the combined effects of diabetes and hypertension on the pathogenesis of cardiovascular disease, adult male and female SHR rats which develop hypertension spontaneously were given a single, 10 mg or 15 mg/100 g body wt. injection of alloxan s.c. to induce moderate or severe diabetes. Insulin was deliberately withheld. Animals were examined by autopsy daily for 7 days post-alloxan and after 4 and 8 weeks. Mortality was high--only 52% of the males survived as against 80% of the females. Most deaths occurred on Day 5 and were associated with adrenal haemorrhage and hyperplasia, thymus galnd involution, fatty liver and marked hypotension despite elevated aldosterone levels. During the first week, corticosterone levels increased significantly in the male; in females they showed little change. After 4 weeks, the severly diabetic animals became emaciated and moribund; corticosterone and aldosterone levels fell to very low levels despite adrenal hyperplasia. The beta cells of the moderately diabetic animals eventually lost their ability to secrete insulin and these animals too became cachetic and moribund with concomitant elevation of lipid, glucose and BUN levels, as well as myocardial infarction, fatty liver, and generalized hyalin arteriolo-, arterio-, and nephrosclerosis. It is suggested that the combined hormonal and metabolic alterations of diabetes and hypertension reinforced one another in these spontaneously hypertensive rats, leading to intense stimulation of the hypothalamic-pituitary-adrenal system, the exacerbation of those cardiovascular degenerative changes known to be associated with uncontrolled diabetes or hypertension, eventual impaired adrenocortical steroidogenesis, hypotension and death.


Changes in the concentration of leucocytes and platelets in the peripheral blood during sterile inflammation in rabbits.

Glycogen in isotonic saline was infused into the peritoneal cavities of rabbits to produce sterile inflammation. This caused a small increase in the haematocrit value and larger decreases in the concentrations of circulation leucocytes and platelets. Circulating granulocytes decreased by about 40% in 2 h and increased in the next 2 h to about 4 times their initial concentration. Acetyl salicyclic acid (ASA) (10 mg/kg) infused with the glycogen did not affect the decrease significantly but accelerated the subsequent increase. Circulating mononuclear leucocytes, mostly lymphocytes, decreased progressively by about 75% after 4-5 h. This decrease was not affected by ASA. Circulating platelets decreased by about 30% in the first hour; this decrease was accelerated and augmented by ASA. Subsequently the platelet concentrations remained constant for at least 4-5 h. Glycogen so infused is known to activate complement, and ASA to inhibit prostaglandin synthetase. Therefore the results suggest that (i) the initial decrease in circulating granulocytes is mediated by activated complement; (ii) the emigration of granulocytes from blood into the inflammatory exudate is increased by prostaglandins; (iii) the initial decrease in circulating platelets is mediated by activated complement and antagonized by prostaglandins; and (iv) the decrease in circulating lymphocytes is mediated by activated complement and uninfluenced by prostaglandins.


Aspirin and gastric ulceration: light and electron microscopic observations in a model of aspirin plus stress-induced ulcerogenesis.

The gastric mucosa of rats given aspirin orally and concomitantly exposed to mild (cold) stress conditions has been examined under the electron microscope. In this model of gastric ulceration greater involvement is seen of the parietal and zymogen cells than is evident after aspirin treatment alone (which leads to mucosal erosions but not ulceration per se). Evidence of considerable autolytic and proteolytic activity is seen in aspirin-plus-stress-treated animals developing in ulceration. Little damage was observed in animals exposed to these mild stress conditions. The synergistic interaction between aspirin and stress could have its basis in greater sensitizing of parietal and zymogen cells to damage not seen in aspirin-treated animals.


The effect of a protein-free diet, a sugar diet and of carbon tetrachloride administration on the toxicity and rate of metabolism of dimethylnitrosamine in different rat strains.

A Porton and a hooded rat strain showed a raised LD50 for dimethylnitrosamine (DMN) when pre-conditioned on a protein-free and/or a sugar diet. Little or no such differential toxicity between normal and diet-fed animals was found for rats of the Wistar, BDIX and CFY strains. Pre-treatment with CCl4 did not alter significantly the toxicity of DMN in the Wistar strain. All 5 rat strains treated by diet or CCl4 administration metabolized DMN at a very much slower rate than did the controls, the rates for the different strains being quantitatively similar. It is concluded that the toxicity of DMN is not necessarily related to its rate of metabolism and that the effect of diet or CCl4 treatment of DMN toxicity is dependent on the strain of rat used.


Mercury pigmentation from industrial exposure. An ultrastructural and analytical electron microscopic study.

A study has been made of facial pigmentation resulting from industrial inorganic mercury exposure. Electron microscopy has shown electron-dense 400--900 nm aggregates of 12 nm particles in dermal macrophages and free in the dermis. Use of the analytical electron microscope has enabled direct confirmation of the presence of mercury in these aggregates. The unexpected finding of coexistent selenium is discussed.


Focal epithelial hyperplasia of the oral mucosa. Report of two cases from the Netherlands and review of the literature.

Two negro siblings with focal epithelial hyperplasia of the oral mucosa are described. A review of the literature is presented.


Focal epithelial hyperplasia of the mouth: recently discovered in South Africa.

Seventy-six cases of focal epithelial hyperplasia are encountered during an epidemiological survey in the Western Cape Province of South Africa. They affected only Cape Coloureds and were confined to pupils of reform schools and residents of a secluded community. This pattern of distribution, coupled with its typical clinical and histological features and the fact that viral particles were observed in lesions, indicates a communicable disease of viral origin, possibly a virus of the papova group. The recent discovery of its presence in South Africa indicates that it may be newly introduced into the country or that it is a new or hitheto undetected manifestation of an existing viral disease.


Dystrophic epidermolysis bullosa. Interesting gastro-intestinal manifestations.

Involvement of the large intestine in a long standing case of dystrophic epidermolysis bullosa was characterized by recurrent episodes of diarrhoea synchronizing with exacerbation of the skin lesions. The radiological investigations revealed two narrow segments, one each in the descending and transverse colon, with ulcer craters in the lower part of the former. The haustra from the distal half of the transverse colon to the sigmoid colon were lost.


A surface impression and scanning electron microscopic study of porokeratosis of Mibelli.

A silicone rubber impression technique, together with scanning electron microscopy, was used to study the surface detail of porokeratosis of Mibelli. The lesions are propagated by a bud-like spreading of the active edge. The keratotic rim is produced by the condensation of squamous cells from both within and without the lesion.


Trimethylaminuria: the use of choline as an aid to diagnosis.

Trimethylamine metabolism can be studied by means of choline loading. The value and some limitations of the method are illustrated by results obtained in normal subjects, a patient with the Fish Odour syndrome and his kindred.


The tricho-rhino-phalangeal syndrome.

A family with the tricho-rhino-phalangeal syndrome was studied. One child, the proband, manifested the complete triad of the syndrome with fine, sparse, slow-growing hair, pear-shaped nose, and abnormal fingers. Cone-shaped epiphyses were observed radiographically in the child, in both parents, and in two of three remaining children.


Induction of basal lamina formation in epidermal cell cultures in vitro.

Guinea-pig epidermal cells were grown on 2 types of rat collagen substrate. The cells showed the same sort of differentiation as occurs in cells growing on rigid plastic. In addition, basal lamina formed patchily at the interface with collagen. If the collagen was eroded away the basal lamina also disappeared.


An epidemiological survey of virus warts of the hands among butchers.

The prevalence of virus warts of the hands among butchers has been determined in three industrial butcheries by examining 536 meat-workers at their places of work. A prevalance of 23-8% has been found, which is significantly higher than the 8-5% prevalance found in 965 control adult subjects examined in different firms where manipulation of food does not occur. Further, the prevalence of virus warts of the hands has been found significantly higher in the 199 workers of one butchery (32-1%) than in 199 control subjects (10-1%) strictly matched for age and sex, and belonging to the same industrial area. In butchers, warts of the dorsal aspects of the hands are statistically less frequent than in control subjects when three localizations are considered (dorsal, palmar, dorsal plus palmar). No significant difference is observed when single localizations (dorsal or palmar) are considered. A definite explanation of the high prevalence of virus warts of the hands among butchers has not been found so far; some hypotheses are discussed in the light of the presnt study.


Anti-inflammatory properties of a prostaglandin antagonist, a corticosteroid and indomethacin in experimental contact dermatitis.

The topical effects of N0164 (a phenyl phosphonate derivative which is a partially selective antagonist of prostagladin E2), indomethacin and triamcinolone acetonide have been shown to reduce the erythema and ear weight gain from inflammation induced by experimental contact allergic eczema. Oxazolone sensitized Swiss Webster mice were used, ear erythema and ear weights being used as a measure of the anti-inflammatory response to the drugs. N0164 was also shown to have systemic anti-inflammatory activity after intraperitoneal injection.


Improvements in skin reactions to insulin, produced by a highly purified preparation.

A group of 14 patients with skin reactions to insulin have been treated with highly purified insulin over a period of 5--14 months while continuing to inject into the abnormal areas. Fat atrophy improved in all cases, coincident with a reduction in insulin requirement and insulin binding capacity. The response of patients with fat hypertrophy was less predictable, but there was an improvement in 3 out of 6 cases. Measurements of insulin binding capacity of the plasma suggested that fat atrophy and hypertrophy are immunologically distinct and some explanations for this are discussed. It is concluded that highly purified insulin benefits some patients with skin reactions to insulin.


Exacerbation of psoriasis during lithium treatment.

We report three patients with psoriasis and bipolar affective illness who showed marked deterioration of their skin condition when treated with lithium.


Measurement of the rate of flow of dry matter in digesta passing through the duodenum of sheep.

1. A total of seven sheep fitted with re-entrant cannulas in the proximal duodenum were used in three experiments to measure the passage of dry matter (DM) in duodenal digesta. 2. Fifty-three 24 h measurements and ten 48 h measurements of DM flow were made in Expt 1. Coefficient of variation (CV) of these measurements was 22-1% which was similar to variability reported for chromic oxide 'corrected' flows and for an estimate of day-to-day variation. DM flow on day 1 was 103-2% (SE = 8-94) of flow on day 2 in 48 h measurements. 3. In two 3 X 3 Latin-square design experiments, duodenal DM flow was measured continuously over 72 h and on days 1, 2 and 3 DM flow was 100-4, 102-6 and 96-9% (SE = 5-82) of mean flow (Expt 2) and 95-8, 101-2 and 103-0% (SE = 6-70) of mean flow (Expt 3) respectively. 4. It was concluded that no depression in duodenal DM flow occurred during the first 24 h of collection in these experiments and that the variability of repeated 24 h measurements of flow was within day-to-day variation.


Fractional catabolic rates of myosin and actin estimated by urinary excretion of Ntau-methylhistidine: the effect of dietary protein level on catabolic rates under conditions of restricted food intake.

1. Critical studies on the distribution of Ntau-methylhistidine (3-methylhistidine; Me-His) among organs and tissues in adult rats are reported. Adult rats contained 46-5+/-3-6 mg Me-His/kg body-weight. Almost 90% of the Me-His in the body was recovered from skeletal muscle. These results support the hypothesis that fractional catabolic rates of myosin and actin in skeletal muscle can be estimated by measuring urinary excretion of Me-His. 2. Dietary protein level did not affect the total amount of Me-His in the body. However, urinary excretion of Me-His increased as dietary protein intake was increased. 3. From these results it was concluded that fractional catabolic rates of myosin and actin increase as dietary protein intake increases.


The energy cost of fat and protein deposition in the rat.

1. Measurements were made of energy balance by direct calorimetry, and of nitrogen balance in groups of lean and congenitally obese ('fatty') Zucker rats at body-weights of 200 and 350 g given a highly digestible semisynthetic diet at 14-0 or 18-4 g/rat per 24 h. 2. Losses of food energy and N in faeces were very small. The fatty rats lost much more N in urine than did lean rats. Despite this the proportion of gross energy that was metabolized was 0-92 for both fatty and lean rats. 3. In all trials, fatty rats lost a smaller proportion of metabolizable energy (ME) as heat and deposited less as protein than thin rats but deposited much more as fat. 4. The amounts of ME required to deposit 1 kJ of protein and 1 kJ of fat respectively were shown by regression analysis to be 2-25 (+/-0-16) and 1-36 (+/-0-06) kJ respectively. These values agree extremely closely with recent, more tentative, estimates based on assumptions as to maintenance requirement which the present experiments were able to circumvent. It may be concluded with confidence that the energy costs of depositing 1 g of protein or fat are almost identical at 53 kJ ME/g.


Degradation of the nucleic acids in mixed rumen bacteria on incubation with different media.

1. Mixed rumen bacteria were grown in vitro in a medium containing [U-14C]adenine. Radioactivity was incorporated into the purine bases of the nucleic acids. 2. Labelled bacteria were incubated with a nitrogen-deficient cell-free medium (medium A), a protozoa-free supernatant suspension from a slow-speed centrifuging of a steer's rumen contents (medium B) and whole rumen contents of a steer (medium C). Mean (six experiments) proportional rates of release of radioactivity (/h) were significantly greater for medium A (0-140) than for media B or C (0-055 and 0-43 respectively). Some of the radioactivity accumulated in the cell-free media; some was lost, partially as carbon dioxide. 3. Calculation from this and other information indicated that about 30% of bacterial nucleic acids may turn over in the rumen even in the absence of protozoa.


Increased thirst and hunger in adult rats undernourished as infants: an alternative explanation.

1. Rats were undernourished in early life by feeding their mothers a restricted quantity of a good-quality diet throughout lactation. Their undernutrition continued postweaning from 25 to 42 d of age, after which they were fed ad lib. Control rats were well nourished at all times. 2. Behavioural assessment of thirst was carried out on adult males. These were deprived of water for 23 h/d throughout the period of testing. Compared to control rats, previously-undernourished (PU) rats pressed a lever at a higher rate in a Skinner box to gain a water reward, drank more frequently during their first 5 min in an unfamiliar cage, and tended to run more quickly down an alleyway for water. PU rats also drank more (/kg body-weight 0-75) of a quinine solution (I g/l) when this was available to them ad lib. as their only source of fluid. 3. A second group of rats was growth-retarded during gestation and the suckling and early postweaning periods. The rats had free access to food from 42 d of age. In adulthood their ad lib. food and water consumption was measured. PU males ate and drank more (/kg body-weight 0-75) than control males. 4. These results indicate that adult rats which have been undernourished in early life display increased thirst. An attempt is made to explain this finding, together with their previously-demonstrated enhanced hunger drive, purely in terms of gross anatomical and physiological differences.


Studies on lipid digestion in the preruminant calf. 2. A comparison of the products of lipolysis of milk fat by salivary and pancreatic lipases in vitro.

1. A comparison has been made of the products of lipolysis of the fat in cow's milk by salivary and pancreatic lipases (EC 3-1-1-3) from a preruminant calf. 2. It was confirmed that salivary lipase releases short-chain acids preferentially, particularly butyric and caproic acids. This was not only true initially but also as the course of lipolysis progressed. Even after 60 min incubation the longer-chain acids were only released to about one-twentieth of the extent of butyric acid. 3. Although pancreatic lipase also initially released proportionately more butyric acid than other fatty acids, within 30 min the longer-chain acids were released to about half the extent of butyric acid. 4. Since the over-all extent of lipolysis by salivary lipase was limited by its inability to release long-chain acids, the release of these acids (which comprise two-thirds of the total fatty acids of milk fat) in the calf is likely to be dependent on the subsequent action of pancreatic lipase. 5. The release of fatty acids by pancreatic lipase was enhanced by pre-incubating milk fat with salivary lipase.


A quantitative study into the role of infection in determining nutritional status in Gambian village children.

1. Growth in weight and height in 152 children between the ages of 0-6 and 3 years was investigated in Keneba, a rural Gambian village. By 1 year of age the average weight-for-age of the children was only 75% of the Jelliffe (1966) standard. 2. The relationship between the prevalence of nine different categories of diseases and growth was investigated to determine the quantitative contribution of the diseases to the growth faltering observed. There was a highly significant negative relationship between gastroenteritis and both weight gain and height gain. The only other disease category having a similar relationship was malaria, but in this instance only with weight gain. 3. Although over-all growth in weight and height was considerably below the standard values, multiple regression analysis indicated that after the age of 1 year, except in July and August, normal and sometimes slow 'catch-up' growth would be possible in the absence of gastroenteritis. 4. Attention is drawn to the limitations of the use of anthropometry in a settled village community as the only criterion by which dietary adequacy can be judged.


Selenium concentrations and glutathione peroxidase activities in whole blood of New Zealand residents.

1. A relationship was found between selenium concentrations and glutathione peroxidase (EC 1.11.1.9) activities in whole blood of 264 New Zealand residents (r 0-71, P less than 0-001). 2. New Zealand residents returning from visits overseas of 7 months to 3 years had elevated blood Se, but normal GSH-Px activities, whereas for some new settlers in New Zealand both Se and GSH-Px activities were high.


Major membrane protein differences in cilia and flagella: evidence for a membrane-associated tubulin.

The membrane of both sperm flagella and gill cilia of the scallop Aequipecten irradians may be selectively solubilized in 1% Triton X-100, 30 mM tris(hydroxymethyl)-aminomethane hydrochloride (Tris-HCl), pH8, and 3 mM MgCl2, leaving the axoneme totally intact. This membrane fraction represents about 20% of the total protein of the respective organelle. Analysis of the flagellar membrane by sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel electrophoresis revealed one principal protein component, periodic acid-Schiff (PAS) positive and migrating with an apparent molecular weight of 250,000. The remaining minor proteins, none of them PAS positive, accounted for less than one-third of the total flagellar membrane fraction. Analysis of the ciliary membrane also revealed one major protein component, weakly PAS positive and migrating with an apparent molecular weight of 55,000. The remaining minor proteins represented about one-third of the total ciliary membrane fraction; two components with molecular weights of 100,000 and 40,000 predominated. The latter could be substantially reduced by purification of the cilia on a sucrose density gradient and was assumed to be actin, derived by vesiculation of the brush border during deciliation. The principal ciliary membrane protein, that of 55,000 daltons, was resolved into two equimolar components on NaDodSO4-Tris-glycine-polyacrylamide gels, comigrating with the alpha and beta chains of outer fiber tubulin. S-carboxymethylation caused increased splitting of the two components and concomitant migration with similarly treated ciliary tubulin. Preparative gel electrophoresis yielded separate components whose cyanogen bromide cleavage products were virtually identical in size distribution with those obtained from outer fiber alpha and beta chains; tryptic peptides corresponded almost exactly to those of authentic tubulin subunits but certain positional differences indicated possible side chain modification. At 25 degrees C both whole cilia and its solubilized membrane fraction bound colchicine while whole flagella and the 9 + 2 axoneme from either organelle did not. Thus certain molluscan flagellar membranes primarily contain a 250,000-dalton glycoprotein but ciliary membranes have a modified tubulin as the major protein component. At an electron microscopic level, flagellar membranes have a distinct trilamellar "unit membrane" structure while ciliary membranes appear thinner and considerably less distinct, perhaps reflecting the protein compositional differences in the membranes of these other wise morpholobically identical organelles.


Binding properties of neutral diamide ligands for alkaline-earth cations.

The complexation of a series of aromatic and alicyclic N,N,N',N'-tetra-n-propyl amides of 1,2-ethylenedioxydiacetic acids with group IIA metal-ion bromides in anhydrous methanol was investigated by ultraviolet absorption spectroscopy. These synthetic ligands were previously found to show selectivity toward divalent over monovalent cations with respect to extraction of ions into bulk organic phase (Borowitz, I.J., Lin, W-O., Wun, T-C., Bittman, R., Weiss, L., Diakiw, V., and Borowitz, G.B. (1977), Tetrahedron, in press). At low concentrations, ligands bearing benzene and naphthalene rings form 1:1 ligand to divalent cation complexes with each of the alkaline-earth metals, but ligands in the cyclohexyl series are stoichiometrically bound to cations in more than one type of complex. Binding isotherms obtained by Scatchard analysis and by the method of continuous variation revealed ligand to divalent ion mole ratios of 2:1, 3:2, and 4:3 for binding of N,N,N',N'-tetra-n-propyl-cis-1,2-cyclohexanedioxydiacetamide with Ca2+, Sr2+, and Ba2+, respectively. In contrast, Scatchard analysis of ultraviolet spectral changes showed that a 1:1 complex is formed between this ligand and Na+ with an apparent association constant of 56 +/- 2M-1; the constant for binding with K+ was smaller (11 M-1). The order of apparent association equilibrium constants for complexation of group IIA cations with this series of neutral ligands was Ca2+ greater than Sr2+ greater than Ba2+ greater than Mg2+; for example, for N,N,N',N'-tetra-n-propyl-1,2-phenylenedioxydiacetamide the apparent binding constants at 25 degrees C were 7.33 +/- 0.25 X 10(4) M-1 for Ca2+, 1.23 +/- 0.03 X 10(4) for Sr2+, 4.42 +/- 0.09 X 10(3) for Ba2+, and 4.04 +/- 0.24 X 10(2) for Mg2+. The divalent cation binding properties of these synthetic diamide ligands are discussed in relation to those of other synthetic ligands and of two naturally occurring ligands.


Ionophorous properties of neutral diamide ligands toward calcium.

The ability of a series of aromatic and alicyclic analogues of 1,2-ethylenedioxydiacetic acids bearing N,N,N',N'-tetra-n-propyl amide or N-methyl-N-carbethoxypentyl amide linkages to enhance the rate of 45Ca2+ efflux from vesicles was studied. The ligands were less potent in enhancing membrane permeability to Ca2+ than A23187 and X537A. Lipid-soluble anions markedly increased the rate and extent of Ca2+ transport mediated by these neutral ligands. The abilities of these synthetic diamide ligands and naturally occurring ionophores to transport Ca2+ across bilayer membranes were sensitive to the lipid composition of the vesicle. The mechanism of Ca2+ transport mediated by this series of synthetic ligands is discussed.


Activation of the rat liver glucocorticoid--receptor complex.

The rat liver glucorcorticoid receptor has been activated using three procedures: heat, gel filtration, and dilution. With time after heat activation the steroid--receptor complex loses its capacity to bind to DNA--cellulose, while receptor activated by Sephadex G-25 and by dilution maintains DNA--cellulose binding capacity. The rates of steroid dissociation from nonactivated and activated receptor and essentially identical. However, nonactivated receptor is capable of rebinding steroid, while activated receptor has a reduced capacity to rebind steroid. The results of the gel filtration and dilution studies suggest that a low-molecular-weight factor(s) exists in rat liver cytosol which is involved in the process of activation.


Mechanism of 25-hydroxyvitamin D3 24-hydroxylation: incorporation of oxygen-18 into the 24 position of 25-hydroxyvitamin D3.

The oxygen enzymatically inserted as a hydroxyl function by chick kidney homogenate into the 24 position of 25-hydroxyvitamin D3 to give 24,25-dihydroxyvitamin D3 is derived exclusively from 18O2. Therefore, like the 25-hydroxyvitamin D3-lalpha-hydroxylase system, the 25-hydroxyvitamin D3-24-hydroxylase system is also a monooxygenase ("mixed-function oxidase").


Protein-catalyzed exchange of phosphatidylcholine between sonicated liposomes and multilamellar vesicles.

Phospholipid exchange protein from beef heart or beef liver does not catalyze the transfer of phosphatidylcholine from multilamellar vesicles of phosphatidylcholine. Certain combinations of phospholipids, however, do yield multilamellar vesicles that will exchange phosphatidylcholine with liposomes in the presence of exchange protein. Multilamellar vesicles of phosphatidylcholine:phosphatidylethanolamine:cardiolipin (70:25:5, mol%) can be used in place of mitochondria or erythrocyte ghosts as an improved acceptor particle in the study of liposome structure with phospholipid exchange proteins. These multilamellar vesicles act as a well-defined reservoir of unlabeled phosphatidylcholine with 7% exchangable phospholipid. When the distribution of phosphatidylcholine in liposomes is studied by the exchange protein technique, results can be influence by the choice of phospholipid acceptor particle. With mitochondria as acceptor particle, the percentage of phosphatidylcholine in the outer monolayer of a liposome appears to be 60%, whereas a value of 70% is obtained when multilamellar vesicles are the acceptor. The discrepancy can be explained by a heterogeneity in liposomes prepared by sonication. A size-dependent fusion or adsorption process occurs between liposomes and mitochondria; the very small liposomal vesicles, obtained by gel filtration, combine nearly quantitatively with the natural membrane. This phenomenon is not seen with multilamellar vesicles. Thus by using multilamellar vesicles one obtains a less biased estimate of phospholipid distribution between inner and outer layers of liposomes.


Characterization of antibodies specific for N6-methyladenosine and for 7-methylguanosine.

Antibodies specific for N6-methyladenosine (m6A) and for 7-methylguanosine (m7G) were prepared by immunization of rabbits with nucleoside conjugates of bovine serum albumin (i.e, m6A--BSA and m7G-BSA). Specificity of each antibody was assessed by inhibition of the homologous precipitin reaction with various nucleosides. These analyses revealed that the antibodies elicited in response to m6A--BSA were specific for the N6-methyl moiety of adenosine with minimal or no cross-reactivity with BSA, adenosine, and guanosine. Although a major fraction of antibodies elicited in response to m7G--BSA were specific for m7G, considerable cross-reactivity was observed with BSA. These latter antibodies were removed by affinity chromatography utilizing BSA-Sepharose adsorbent. In similar fashion, antibodies specific for m6A and m7G were isolated by immunospecific adsorption to antigen-coupled Sepharose (e.g. m6A--BSA-Sepharose), eluted, and coupled to Sepharose. The ability of these antibody-coupled adsorbents to retain specific methylated [methyl-3H]nucleosides derived from [methyl-3H]tRNA digests was assessed. Both the anti-m7G and anti-m6A antibody adsorbents quantitatively and exclusively retained 7-[3H]methylguanosine and N6-[3H]methyladenosine, respectively. The application of these adsorbents to fractionate oligonucleotides and nucleic acids is discussed.


Purification and properties of gamma-butyrobetaine hydroxylase from Pseudomonas sp AK 1.

gamma-Butyrobetaine hydroxylase (4-trimethylaminobutyrate, 2-oxoglutarate:oxygen oxidoreductase (3-hydroxylating), EC 1.14.11.1) has been isolated from Pseudomonas sp AK 1 by ion-exchange, adsorption, and molecular-sieving chromatography. The preparation was homogeneous as judged from electrophoresis in agarose and polyacrylamide gels, isoelectric focusing, and equilibrium sedimentation. The molecular mass was 95 kdaltons as determined by sedimentation equilibrium centrifugation. From electrophoresis in polyacrylamide gel the molecular mass was estimated to 92 kdaltons, from gel filtration through columns of Sephadex G-200 to 86 kdaltons, and from gel filtration through thin layers of Sephadex G-150 and G-200 to 82 kdaltons. Calculation of molecular mass from Stokes radius, sedimentation coefficient, and partial specific volume gave a value of 96 kdaltons, and from the sedimentation coefficient, 93 kdaltons. Gel filtration through Sephadex G-200 in 6 M guanidinium chloride and electrophoresis in polyacrylamide gel containing 3.5 mM sodium dodecyl sulfate resulted in single bands with mobilities corresponding to molecular masses of 39 and 37 kdaltons, respectively, indicating that the enzyme is composed of two polypeptides chains with similar size. NH2-terminal amino acid sequencing in three cycles resulted in two amino acids in each cycle (Ala + Asn, Ala + Ile, Ala + Ile). The Stokes radius was 3.8 nm, corresponding to a diffusion coefficient of 5.7 X 10(-7) cm2/s. A sedimentation coefficient of 5.8 X 10(-13) s and a frictional ratio of 1.26 was found. The partial specific volume was 0.729 mL/g at 20 degrees C as calculated from amino acid analysis. The isoelectric point was 5.1, as determined by isoelectric focusing analysis. The light absorption in the ultraviolet and visible regions was that of a protein without light-absorbing prosthetic groups. The absorption coefficient at 280 nm of a 1.0% solution at pH 6.5 was 12.6. Amino acid analysis by ion-exchange chromatography showed a half-cystine content of 19 mol per 95 kg of protein (23 residues/1000). Thirteen sulfhydryl groups were found by colorimetric analysis before as well as after reduction with NaBH4, indicating absence of disulfide bonds. Less than 0.1 mol of iron was found per mol of enzyme.


Enzyme which specifically adds tyrosine to the alpha chain of tubulin.

A posttranslational modification of tubulin with potential significance for the regulation of microtubule assembly or function has been revealed by the discovery of an enzyme (tubulin--tyrosine ligase) which can add a tyrosine residue to the alphachain, apparently through peptide bond linkage to a C-terminal glutamate or glutamine. The ability to add tyrosine in the presence of ATP and to release it agiain in the presence of ADP and inorganic phosphate (or arsenate) appear to be functions of the same enzyme, as judged by the relative rates at which these reactions are catalyzed over a 20-fold enzyme purification. The apparent size of the enzyme from bovine brain is 150 000 daltons in extracts and after ammonium sulfate fractionation, but 35 000 after elution from anion-exchange columns. Addition of pure dimeric tubulin to the latter species converts it back to the larger form, which is apparently a stoichiometric 1:1 complex of tubulin and the 35 000-dalton enzyme. Tubulin--tyrosine ligase is specific for tubulin; other proteins with C-terminal glutamate or glutamine do not substratesmate or glutamine do not act as substrates or inhibitors. It is less specific for tyrosine; five out of six tyrosine dipeptides were inhibitors and competed with tyrosine.


Thermodynamics of binding to native alpha-chymotrypsin and to forms of alpha-chymotrypsin in which catalytically essential residues are modified; a study of "productive" and "nonproductive" associations.

The standard free energy (deltaG degrees), enthalpy (deltaH degrees), and entropy (deltaS degrees) of association for proflavin and D- and L-N-AcTrp have been obtained at pH 7.8 for native alpha-chymotrypsin (Cht) and for forms of Cht in which essential catalytic residues of the active site are modified. The modified Cht forms studied are dehydroalaninyl-195-alpha-Cht and N-methylhistidinyl-57-alpha-Cht. Associations to native Cht (pH 7.8) are characterized by negative deltaH degrees and deltaS degrees values (i.e., for L-AcTrp deltaH degrees = -9.1 kcal/mol and deltaS degrees = -21 eu at T = 25 degreesC). In contrast, we found associations to modified Chts to be characterized by an enthalpy near zero and a positive entropy of association, the values of the deltaH degrees and deltaS degrees for association to the modified Cht forms being similar to those expected for transfer of small aromatic molecules from water to a nonpolar solvent phase. Differences in deltaH degrees and deltaS degrees observed for binding of substrate analogues and inhibitors to modified and native Cht (pH 7.8) are approximately + 10 kcal/mol and +30 eu, respectively. Data from D. D. F. Shiao ((1970), Biochemistry 9, 1083) similarly show differences of comparable magnitude between binding of substrate analogues to active alpha-Cht (pH 7.8) and the His-57 protonated form of alpha-Cht (pH 5.6). The negative deltaH degrees and deltaS degrees values of associations for binding to active alpha-Cht indicate that a substrate-induced conformational change occurs on substrate association with the primary binding site (S1), which does not occur in Ser-195 and His-57 modified Cht. From these differences we infer a linkage between binding of substrate into S1 and the catalytic residues in the nucleophilic subsite (S1-S1'). Our data also show that associations of substrate analogues into potentially productive Michaelis complexes S1 cannot be easily differentiated from associations that are nonproductive (i.e., nonactivated) from their deltaG degrees obsd, but may be differentiated by their respective deltaH degrees obsd and deltaS degrees obsd for association. Accordingly, it is indicated that the probable substrate association-activation process, characterized thermodynamically in this work, occurs in the substrate binding step and leads to lowered free energies of activation in catalytic steps succeeding binding however, the process does not influence the observed strength of substrate binding.


Histone messenger RNA from HeLa cells: evidence for modified 5' termini.

The distribution of [3H]methyl radioactivity in cytoplasmic histone mRNA, isolated during the DNA synthetic (S) phase of the HeLa S3 cell cycle, has been investigated. Evidence is presented that approximately 30% of the radioactivity is in m7GpppXmpYp oligonucleotides, where Xm represents 2'-O-methylated adenosine and guanosine with a molar ratio of 4:1, respectively. The remainder of the radioactivity is present as m7GpppCmpYmpZp oligonucleotides, where Xm is again 2'-O-methylated adenosine and guanosine (4:1) and where ym represents 2'-O-methylated adenosine, guanosine, cytidine, and uridine with ratios of 2:1:1:1, respectively. While 48.6% of the [3H]methyl radioactivity was present as N6-methyladenosine in poly(adenylic acid)-terminated mRNA from S-phase cells, no evidence for N6-methyladenosine was found in histone mRNA. It thus appears that histone mRNA which lacks 3'-terminal poly(adenylic acid) sequences and functions on cytoplasmic polyribosomes during a limited protion of the cell cycle is capped but lacks internal-modified nucleosides.


Studies on native ribosomal subunits from rat liver. Purification and characterization of a ribosome dissociation factor.

A population of free, native ribosomal 40S subunits, that do not react with 60S subunits to form 80S ribosomes, has been identified in the postmicrosomal fraction of rat liver homogenates. A protein (IF-3) has been purified from high salt (0.88 M KCI) extracts of native 40S subunits by gradient centrifugation and by ammonium sulfate fractionation; it prevents the reassociation of subunits and to a limited extent dissociates ribosomes to subunits. The activity is measured by ultracentrifugation of the reaction products on linear sucrose gradients, or with an assay developed in this laboratory that couples dissociation with the 60S-specific peptidyltransferase reaction; the latter procedure measures the amount of 60S subunits released from ribosomes or remaining in incubations in the presence of IF-3. Dissociation factor activity is recovered from most of the particles that are resolved by zonal centrifugation of the total "native subunits" obtained from the postmicrosomal fraction; the highest concentration of IF-3, however, appears to be associated with native 40S subunits. The purified dissociation factor IF-3 is composed of about ten polypeptides and the molecular weight is estimated to be between 500 000 and 700 000, on the basis of glycerol and cesium chloride gradient centrifugation. When purified 40S subunits react with IF-3 or when 80S ribosomes are dissociated by IF-3, a product is formed which is dependent on the concentration of the protein factor and has the characteristics of a 40SIF-3 complex; centrifugation of the complex on sucrose and cesium chloride gradients suggests that the complex consists of 1 equiv of each of the two components. Although dissociation factor IF-3 appears to react in a specific manner with free or ribosome-associated 40S subunits, the reaction with subunits differs in several respects from that with ribosomes. The dissociation factor also appears to interact with 60S subunits but multiple complexes are formed, some with more than 1 IF-3 equiv per 60S particle. The IF-3 converts 40S dimers (55S particles) to the 40S-IF-3 complex and dissociates free, native 80S particles present in the postmicrosomal fraction, but it does not affect polysome-associated ribosomes engaged in protein synthesis.