Unstable angina pectoris. Clinical, angiographic, and myocardial scintigraphic observations. The clinical, left ventricular and coronary angiographic data, and the technetium-99m stannous pyrophosphate (99mTc-PYP) myocardial scintigraphic results are presented in 31 patients with unstable angina pectoris. One-third of these patients had positive 99mTc-PYP myocardial scintigrams in a pattern suggesting limited and diffuse subendocardial necrosis. The positive 99mTc-PYP myocardial scintigrams occurred without diagnostic electrocardiographic and cardiac enzyme changes suggestive of myocardial infarction; positive scintigrams seemed to occur more commonly in patients with continuing pain after admission and in those without previous history of myocardial infarction. The positive 99mTc-PYP myocardial scintigrams did not correctly predict coronary anatomical patterns except that positive scintigrams occurred only in patients with coronary artery disease. Neither did the positive scintigrams necessarily occur in that group of patients with the poorest ventricular function though the 2 patients with the lowest ejection fractions both had positive 99mTc-PYP myocardial scintigrams. Finally, when positive 99mTc-PYP scintigrams are the only evidence suggestive of limited subendocardial infarction in patients with unstable angina pectoris, they do not appear to have any prognostic significance in terms of longevity or response to pharmacological or surgical therapy, though the follow-up period so far is short.


Echoventriculography in acute myocardial infarction. II: Monitoring of left ventricular performance. In acute myocardial infarction the overall left ventricular pump function and the regional performance of the infarcted and non-infarcted myocardial segments were studied serially by echocardiographic techniques in 24 patients during the first week of their illness. Left ventricular cavity sizes were acutely increased in 62 per cent of the patients (P less than 0-005). The end-systolic diameter in anterior infarcts increased to the greatest extent, +44 per cent, the end-diastolic diameter by +27 per cent, giving a volume of 246+/-25 ml. In the anterior myocardial infarcts all the function parameters deteriorated more than in the posteroinferior ones. Ejection fraction was subnormal (P less than 0-005) in every patient, and mean circumferential fibre shortening (Vcf) was slowed by about 30 per cent (P less than 0-005). Regionally, contraction of the infarcted area of the ventricle was asynergic in every instance, and its function was almost totally lost (P less than 0-001). Systolic paradoxical motion was a constant and stable finding in the anterior infarctions but not so in the posterior ones. While this asynergic systolic contraction may distort echocardiographic measurement of the end-systolic left vlic phase. The serial deviations from normal in the amplitude or velocity of the uninvolved segments were small, but in the case of clearly enlarged end-diastolic volumes these figures in fact indicate supernormal, compensating function. Both overall and regional performance were worst within the first 3 days of infarction, improving thereafter. The patient with a fatal course showed, instead, progressive deterioration. This noninvasive left ventriculogram by ultrasound gives valuable insight into overall pump function and ventricular volumes, little studied so far in acute infarction, and it may serially quantify the segmental function of both the infarcted and uninvolved regions.


Duration of diastole versus cycle length as correlates of left ventricular ejection time. Studies were done on 82 normal subjects to evaluate cycle length versus duration of diastole as determinants of left ventricular ejection time. Cycle length and its reciprocal, heart rate, had the highest correlation with left ventricular ejection time. Removal of the self-correlation of left ventricular ejection time within cycle length reduces the correlation so that, of all intervals, duration of diastole had the highest correlation as a determinant of left ventricular ejection time. Cycle length and heart rate remain valuable as spuriously close but not misleading correlates for predicting or correcting left ventricular ejection time.


Design of exercise test, with special reference to heart patients. Estimation of exercise tolerance in patients with heart disease and significant symptoms can usually not be expressed in terms of maximal oxygen uptake or power at a fixed heart rate calculated from a 'steady state' exercise test. The exercise tolerance can be expressed only by the maximal power developed, when limited by symptoms. For this purpose the level of tolerance is better assessed by a test with small increments and short duration of each work load. In clinical practice this type of almost continuous increase in load was found to be particularly useful in patients with ischaemic heart disease. Comparative studies showed that the work per heart beat at equal loads is significantly higher in the test with continuous increase in load than in the test with steps of 6 minutes duration, both in normal subjects and in heart patients. The difference, however, is small (7%) and for practical purposes these tests have equal validity as a measure of the circulatory capacity.


Biloculate false aneurysm of the right ventricle after cardiac surgery. A case of a 12-year-old boy who had double false aneurysms of the right ventricle after incomplete closed pulmonary valvotomy six years earlier is presented. The aneurysms were successfully treated surgically, and the aetiology is discussed.


Retrograde His bundle deflection in bundle-branch re-entry. Atrial echo beats resulting from a reciprocating mechanism involving the bundle-branches were produced by premature atrial impulses in a patient with an A-V nodal bypass tract. The mechanism of the arrhythmia was suggested by the presence of a retrograde His bundle deflection which appeared 'sandwiched' in between a QRS complex with complete right bundle-branch morphology and a negative P wave. Though at a shorter cycle length the His bundle was still activated retrogradely echo beats were not seen because the retrograde H deflection occurred too early, when both bypass tract and A-V node were still effectively refractory. At the faster driven rate concealed retrograde activation of the right branch (by the premature impulse) was responsible for the right bundle-branch block patterns shown by the post-premature driven beat.


Diagnosis and successful surgical treatment of chronic constrictive uraemic pericarditis. A case of chronic constrictive uraemic pericarditis successfully treated by pericardectomy is reported together with the pre- and postoperative haemodynamic data.


Hydatid disease of the heart.

Hydatid disease of the heart. A case of hydatid cyst of the heart is described, in which the diagnosis was made at coronary angiography; a new angiographic sign is described.


Effect of lithium and rubidium on the sleeping time caused by various intravenous anaesthetics in the mouse. The effects of acute and long-term lithium  (LiCl) and rubidium chloride (RbCl) treatments on the sleeping time caused by intravenous thipentone, methohexitone, ketamine, propanidid. Althesin and diazepam were studied in white mice. The doses of the anasthetic agents were selected from the dose-response experiments. When given acutely, LiCl 5 m-equiv/kg prolonged significantly the sleeping time caused by both of the barbiturates, whereas RbCl given for 21 days in tap water shortened the sleeping time induced by methohexitone and ketamine. Both LiCl and RbCl, given acutely, enhanced the sleeping time caused by diazepam. The rectal temperatures were lower in the LiCl-treated mice than in the control and RbCl-treated mice.


The effect of methoxyflurane on pulmonary vascular resistance and hypoxic pulmonary vasoconstriction in the isolated perfused cat lung. The effects of methoxyflurane on pulmonary vascular resistance and the pulmonary vasoconstrictor response to alveolar hypoxia were studied in isolated cat lungs perfused at a constant flow. Methoxyflurane decreased pulmonary vascular resistance and depressed the pulmonary vasoconstrictor response to hypoxia.


Errors in oxygen tension measurements caused by halothane. Halothane, independent of oxygen tension, increased the signal of the gold/silver-silver chloride microelectrode. The output of microelectrodes at different oxygen tensions in the presence of increasing amounts of halothane has been measured. Halothane causes an increase in the electrode signal which is proportional to its concentration. This effect results from the polarographic reduction of halothane. It is concluded that the gold/silver-silver chloride microelectrode cannot be used to measure oxygen tension during halothane anaesthesia.


Effect of dose and premedication on induction complications with etomidate. The induction characteristics of etomidate, a new i.v. hypnotic agent, were studied in 400 patients. Two hundred were premedicated with atropine and anaesthesia was induced with 0.2, 0.25, 0.3 or 0.35 mg/kg of etomidate. The remainder received one of four standard premedications and anaesthesia was induced with etomidate 0.3 mg/kg. Involuntary muscle movements occurred in more than 60% of patients receiving atropine alone. The frequency was reduced in the second group, but remained unacceptable in over 8% of patients. The incidence of other excitatory phenomena, such as cough and hiccup, was 10% approximately. Cardiovascular changes were minimal and no serious allergic phenomena were observed. Nausea and vomiting occurred after surgery in up to 30% of patients and was unrelated to the dose of etomidate or to premedication. Pain on injection occurred in up to 80% of patients when the drug was injected into small peripheral veins and occurred in more than 7% when using more normal veins.


A clinical assessment of the use of etomidate in children. Etomidate 0.2 mg/kg i.v. was used to induce sleep in 198 children. It produced sleep rapidly and safely, with negligible effect on the cardiovascular system and little respiratory depression. Clinical acceptability was reduced by a 27% incidence of pain after injection, a 10% incidence of myoclonia and inadequate dosage in 19%. Etomidate has little analgesic activity and these problems can be reduced by the use of an analgesic as premedication or with induction of anaesthesia, by increasing the induction dose of etomidate to 0.3-0.4 mg/kg, or by changing the formulation of the solution.


A dose-response relationship for etomidate, with some observations on cumulation. In a within-patient comparison in 30 subjects, sleep was induced before e.c.t. by different doses of etomidate or methohexitone: etomidate 0.1 mg/kg, 0.2 mg/kg, 0.4 mg/kg or methohexitone 1.5 mg/kg. The duration of hypnotic effect was assessed by recording the time of spontaneous waking and later, of recovery of normal ocular muscle tone. Doubling the dose of etomidate produced a significant increase in both sleeping time and late recovery. Compared with methohexitone 1.5 mg/kg, etomidate 0.2 mg/kg provides an equal duration of sleep, but allows faster late recovery. In eight patients in whome sleep was maintained by repeated injections of etomidate 0.1 mg/kg as required, little evidence of cumulation was seen up to 27.5 min duration of sleep.


Thyroid-pituitary response to cardiopulmonary bypass. The effects of open heart surgery on various parameters of thyroid-pituitary function were studied in 11 patients. The pattern of hormonal changes was variable. T4 concentration increased during anaesthesia and bypass, following which it returned to normal once haemodynamic stability had been regained. The pattern of change in T3 concentration did not follow consistently that of T4 and the proportional increase in concentration was not as great. There was a significant reduction in T3 concentration with the onset of haemodynamic stability. For individual patients there was no obvious correlation between changes in the concentrations of TSH, T3 and T4. The increases may be explained by release of T3 or T4 from extrathyroid tissue.


Clearance of indocyanine green as an index of liver function during cyclopropane anaesthesia and induced hypotension. The rate of decrease in the plasma concentration of indocyanine green (ICG) following a bolus i.v. injection has been studied in two groups of patients before and during anaesthesia. In eight patients anaesthetized with nitrous oxide and oxygen, and who received a neuromuscular blocking drug, induction of anaesthesia was not associated with a significant change in ICG half-life (from 4.59 to 3.85 min) but the subsequent administration of cyclopropane was associated with a statistically significant increase to 5.61 min. In a second group of anaesthetized patients, breathing spontaneously a mixture of oxygen, nitrous oxide and halothane, the induction of arterial hypotension with either lumbar extradural block or sodium nitroprusside was not associated with significant changes in ICG half-life.


Placental transfer of 14C-dimethyltubocurarine during Caesarean section. Placental transfer of 14C-dimethyltubocurarine (14C-dmtc) was studied during Caesarean section. Liquid scintillation counting was used for determination of 14C activity from the maternal and foetal blood samples. Paper chromatography was used to confirm the stability of 14C-dmtc. In all cases 14C-dmtc was able to cross the placenta. Detectable amounts of 14C-dmtc were found in umbilical blood 2 min after injection into the mother. Six and 10 min after injection, the 14C concentration in the umbilical blood was 12% of the corresponding maternal value.


The effect of thoracic extradural analgesia on pulmonary gas distribution, functional residual capacity and airway closure. Pulmonary gas distribution, functional residual capacity (FRC), closing capacity (CC), arterial oxygen tension (PaO2) and alveolar-arterial oxygen tension gradient (PAO2-PAO2) were measured in seven subjects before and after the induction of extradural analgesia for routine surgery. It was found that pulmonary gas distribution was within normal limits throughout the study, although there were two patients in whom airway closure occurred consistently within the tidal volume. In both cases this was associated with a low PaO2. CC and FRC were substantially unchanged by the induction of extradural analgesia. Changes in (PAO2-PaO2) and PaO2 were usually not large, and are apparently related to factors other than changes in lung geometry.


Ventilatory response to carbon dioxide in patients with homozygous sickle-cell disease. The ventilatory response to carbon dioxide was measured by a rebreathing technique in 21 Nigerians who had homozygous sickle-cell disease. The slope of the carbon dioxide response curve was obtained by plotting the ventilation at successive half-minute intervals against the corresponding mean end-tidal PCO2. Our results showed that sickle-cell patients are as sensitive to carbon dioxide as are normal subjects.


Renal failure and postoperative respiratory failure: recurarization? The occurrence of acute postoperative respiratory failure in three patients with renal failure is presented. The most likely cause was the return of muscle paralysis after the apparent antagonism of tubocurarine by neostigmine (recurarization). In addition to reducing the dose of tubocurarine, the authors recommend that pyridostigmine be used as an antagonist instead of neostigmine because the former has a longer duration of action.


Estimation in sera by radioimmunoassay of a specific membrane antigen associated with a murine lymphoma. Material with a molecular weight of less than 10(5) daltons has been isolated and partially purified from the ascitic fluid of DBA2 mice bearing a syngeneic lymphoma (SL2). This substance inhibits the cytotoxic action of an allogeneic antiserum directed specifically against SL2 cells. Material has been rendered radioactive with 125I and between 20 and 25% of the radioactivity is bound in a specific manner to the antiserum. The material which is referred to as 125I-TSTA has been used in a radioimmunoassay to measure the level of TSTA in the sera of mice bearing both ascitic and subcutaneous SL2 tumours. The level of circulating TSTA was found to be high immediatley following inoculation of live SL2 cells, probably because a large proportion of the injected cells autolyse. The serum concentration of TSTA then falls but 6-10 days later begins to rise again in parallel with the growth of the SL2 tumour either in the peritoneal cavity or subcutaneously. Following surgical removal of an intradermal SL2 tumour the level of TSTA in the serum falls rapidly. No evidence could be found that a significant proportion of the TSTA in the serum of tumour-bearing mice is completed with antibody. However, in the serum of DBA2 mice which have been hyperimmunized with irradiated SL2 cells there are antibodies which bind 125I-TSTA although syngeneic anti-SL2 sera, unlike alloantisera, do not show complement dependent lysis of SL2 cells.


The growth patterns of two transplantable acute leukaemias of spontaneous origin in rats. The growth pattern and morphology of two transplantable acute leukaemias which arose spontaneously in pure line rats are described. They differ morphologically and on the basis of their behaviour in vivo, such as infiltration of lymphoid organs and presence in thoracic duct lymph, the leukaemia syngeneic to the August strain (referred to as the SAL) appears to be of myeloid type whereas the leukaemia syngeneic to the Hooded strain (referred to as the HRL) resembles acute lymphoblastic leukaemia. The HRL cells, but not the SAL cells, are lysed by murine anti-theta serum plus complement. These two transplantable acute leukaemias appear to be useful animal counterparts ot the human acute leukaemias and may be valuable models for studies on chemotherapy and immunotherapy.


Interference by cortisone with endotoxin's adjuvator action on transplantation of a mouse tumour. Observations on the in vivo plating of mouse mammary tumour are extended by making counts of tumours at a significantly earlier phase of development than in previously reported work. In the experiments now described, most of the growth of the tumours has been without benefit of stroma. The noteworthy economy of the experimental method is discussed. The persistence of endotoxin's adjuvator effect on such tumour counts is tested in the face of gamma irradiation and cortisone. Cortisone, it is found, offsets endotoxin's adjuvator action; irradiation does not. Antagonism between endotoxin and cortisone, in this system with tumour cells plated in vivo, seems to indicate that endotoxin's enhancing effect depends more on inflammatory than on immunological factors.


Reproductive and endocrine function in patients with Hodgkin's disease: effects of oophoropexy and irradiation. Reproductive and endocrine function was investigated in 22 women with Hodgkin's disease who had bilateral mid-line oophoropexies performed at staging laparotomy. The operation was followed in 12 cases by "inverted Y" pelvic lymph node irradiation and in 4 cases by para-aortic lymph node irradiation. Pregnancies occurred after the operation in 4 of the 6 patients subsequently found not to require irradiation below the diaphragm. In the other 2 patients in this group the menstrual history was unaffected and normal gonadotrophin concentrations indicated intact ovarian function. In the group receiving para-aortic irradiation, in whom the ovarian irradiation dose was was small (about 150 rad to each ovary) menstrual function and gonadotrophin concentrations were normal at the time of review and one patient has subsequently become pregnant. In the group receiving inverted Y irradiation, in whom the ovaries were shielded from the radiation beam by a rectangular lead block, the ovarian dose was much higher (lowest dose 600 rad, highest dose 3500 rad). Nine of the 12 have persisting amenorrhoea with elevated levels of both gonadotrophins. One patient has since become pregnant and one patient has resumed menstrual cycles and has normal basal gonadotrophin concentrations. One patient who has resumed menstrual cycles has a monotrophic elevation of basal serum FSH concentrations. We conclude that bilateral mid-line oophoropexy does not impair ovarian function or gamete transport and should be performed at diagnositc laparotomy in women of child bearing age with Hodgkin's disease, even when it is uncertain whether pelvic node irradiation will be necessary. The results in the patients who received inverted Y irradiation indicate that the technique of pelvic shielding and ovarian transposition used were only partially successful in preserving fertility. Alternative techniques for preserving ovarian function are discussed.


Beclomethasone dipropionate aerosol and oropharyngeal candidiasis. In a survey of 400 consecutive patients with chronic asthma treated with beclomethasone dipropionate aerosol (up to 400 mug/day) the prevalence of oropharyngeal thrush was 4-5%. The prevalence of this complication was not significantly related to sex, age, duration of treatment with beclomethasone or concurrent treatment with prednisolone. Yeasts were isolated from throat swabs in about 60% of all patients and in 48% of normal controls. Thus, although a diagnosis of oropharyngeal thrush was recorded only when the presence of characteristic lesions in the pharynx was associated with a positive culture, there was a large number of patients and controls without thrush who harboured yeasts in the throat. One in 3 patients with thrush complained of sore throat or hoarseness, but 1 in 4 patients without thrush had similar symptoms. These findings suggest that, although treatment with beclomethasone dipoprionate aerosol undoubtedly can cause oropharyngeal thrush, this condition is not an inevitable result of colonization of the oropharynx by yeasts, nor is it necessarily associated with symptoms.


Gastrointestinal tuberculosis.

Gastrointestinal tuberculosis. Gastrointestinal tuberculosis continues to occur sporadically in hospitals in the British Isles. Accurate diagnosis may enable its rare but lethal complications to be anticipated. Pathology, clinical features and treatment are reviewed. The use of antituberculous chemotherapy and corticosteroids is discussed with reference to 14 cases of gastrointestinal tuberculosis seen in Southampton between 1951 and 1973.


Increased upper airway resistance in patients with airway narrowing. The mean air flow resistance of the orolaryngeal (upper) airway was significantly increased in 7 of 11 patients with chronic airway obstruction, when compared with 6 controls. All the patients had noisy respiration with harsh breath sounds audible by auscultation over the larynx. The increase in resistance was greater during expiration than during inspiration. Since 4 patients had normal upper airway resistance, the signs were not invariably associated with upper airway narrowing and presumably could arise also in the chest. When increased, upper airway resistance was usually more than half the total airway resistance. It is suggested that this increase could only be due to narrowing of the glottis, probably by muscle activity. This narrowing may have had the same function as expiration through pursed lips. On the other hand, when present, the increased resistance through the upper airway during inspiration is unlikely to have had a useful function.


Hepatic erythropoietin: enhanced production in anephric rats with hyperplasia of Kupffer cells. Erythropoietin (Ep) levels were assayed in serum of adult male rats subjected sequentially to (1) administration of colloidal carbon, Zymosan or their vehicles (2) sham operation or bilateral nephrectomy with and without subtotal hepatictomy, and (3) hypoxia (0.45-0.40 atmospheres of air for 6 h starting 1 h after the operation). In anephric rats these agents induced a significant potentiation of hypoxic Ep activity. Since they did not apparently modify the kinetics of exogenous Ep, it is postulated that this phenomenon is mediated by enhanced extrarenal Ep production. Both colloidal carbon and Zymosan induced hyperplasia of the reticuloendothelial system (RES). Moreover, subtotal hepatectomy almost abolished the Ep response to hypoxia evoked by Zymosan. The correlation between hyperplasia of hepatic RES and enhanced Ep production in anephric rats primed with these agents suggests that Kupffer cells constitute a major source for extrarenal Ep. Additionally, it is of interest that colloidal carbon and Zymosan did not significantly modify the renal production of Ep.


In vitro binding of folates by body fluids. It is useful to differentiate between saturated folate binders in serum (carrying endogenous folate) and unsaturated binders (investigated in the present study). These two groups of binders need not necessarily be chemically identical and the unsaturated binder may even be an in vitro artifact, especially when measured with non-physiological folates. Macromolecular binding of radio-active N-5-methyl tetrahydrofolic acid (CH3H4PteGlu) and/or folic acid (PteGlu) by human serum and urine was assessed by means of exhaustive saline dialysis, haemoglobin-coated charcoal adsorption, column chromatography with DEAE-Sephadex A-50, and sucrose gradient analysis. Binding was found to be minimal or absent. Charcoal adsorption showed a mean serum binding capacity of 0 mug/1. for PteGlu and 0.58 mug/1. for CH3H4PteGlu. In pregnancy the mean serum values were 0.23 mug/1. for PteGlu, 0.66 mjg/1. for CH3H4PteGlu, and with folate deficiency 0.30 mug/1. for PteGlu, 0.49 mug/1. for CH3H4PteGlu. Mean urinary folate binding was minimal (less than 0.5 mug/1.), and red cell haemolysate similarly revealed very low binding on exhaustive dialysis. Column chromatography showed that tracer doses of [14C]PteGlu added to serum migrated distally to the protein zone; [14C]CH3H4PteGlu similarly showed no evidence of protein binding. On a sucrose gradient [14C]PteGlu also separated clear of the protein zone.


Inhibitors of plasminogen activation in human blood. Plasma contained two inhibitors of plasminogen activation by urokinase when fractioned by gel filtration on Sephadex G-200. The inhibitor in the lower molecular weight fractions was separate from the principal protease inhibitors of plasma: alpha1-antitrypsin, alpha2-macroglobulin, C1 inactivator and antithrombin III, and from factor XIII. This activation inhibitor was present in both plasma and serum and its recovery was not reduced by preincubating the serum for 6 h at 37 degrees C. Its inhibitory activity was stable for several days at 4 degrees C, and was enhanced in the presence of an increased saline concentration. Preparations of the inhibitor, active in a clot lysis system, failed to inhibit the esterase activity of urokinase on N-alpha-acetyl-glycyl-L-lysine methyl ester.


Significance of haematological parameters in the non-Hodgkin's malignant lymphomas. Blood findings at diagnosis, in 140 adults with lymphoma, were correlated with bone marrow involvement and survival. An abnormal haemoglobin, leucocyte count or platelet count was found in 57% of patients. Lymphocytopenia occurred in 46%. All patients with thrombocytopenia or neutropenia, 69% with leucopenia and 63% with anaemia had marrow involvement with lymphoma. Marrow involvement in histiocytic and stem cell lymphoma was always associated with anaemia. Marrow involvement in poorly differentiated lymphocytic lymphoma (PDL) was associated with anaemia, thrombocytopenia, leucopenia, lymphocytopenia or lymphoma cells in the blood in 93% of patients. Bone marrow involvement was found in only 13% of patients with normal haematological parameters. In the absence of marrow involvement blood abnormalities at diagnosis did not generally correlate with survival. However, among patients with diffuse PDL who had marrow involvement, anaemia, thrombocytopenia and leucopenia adversely affected survival. Lymphocytopenia did not correlate with survival.


Studies on pure red cell aplasia. Maintenance therapy with immunosuppressive drugs. Two patients with pure red cell aplasia, whose marrow cells were capable of increasing their rate of haem synthesis when incubated in vitro with erythropoietin concentrate, had a complete remission associated with the administration of cyclophosphamide and prednisone. Both patients relapsed when the drugs were withdrawn. The patients again went into remission when therapy was reinstituted. Maintenance of normal erythropoiesis was dependent on the continued administration of these drugs. This study provides additional evidence for a causal relationship between immunosuppressive therapy and the resumption of erythropoiesis in patients with this disease and demonstrates the value of this therapy for the maintenance of a remission.


The Kenya form of hereditary persistence of fetal haemoglobin: structural studies and evidence for homogeneous distribution of haemoglobin F using fluorescent anti-haemoglobin F antibodies. Several members of a Ugandan family were heterozygous for the gamma beta fusion gene of Haemoglobin Kenya. Levels of Hb Kenya were significantly higher than those in subjects of previous reports, ranging from 20.68 to 23.35% of the total haemoglobin. The individuals had also 5-8% Hb F, consisting solely of alpha and Ggamma chains. Investigation of the distribution of Hb F among the red cells of Hb Kenya heterozygotes, using monospecific antibodies absorbed against pure Hb Kenya and rendered fluorescent by conjugation with fluorescein isothiocyanate, showed the presence of fetal haemoglobin in all red cells. The data suggest that the phenotype of the Hb Kenya trait resembles that of the Ggamma form of hereditary persistence of fetal haemoglobin rather than that of thalassaemia.


Heterozygous beta thalassaemia of unusual severity. The proband of each of three families of Northern European or Italian extraction had an unusual form of heterozygous beta-thalassaemia, confirmed by haematological, genetic and peripheral blood globin synthesis studies. The unusual severity of this disorder was indicated by chronic haemolysis leading to splenectomy and cholecystectomy, by numerous nucleated red cells and reticulocytes in the peripheral blood, and by leg ulcers in one family. The diversity of clinical expression in many family members with heterozygous beta-thalassaemia was striking. Bone marrow examination in the probands showed numerous large inclusion bodies of the type usually found only in thalassaemia major. In addition, there was unbalanced globin synthesis in the bone marrow, in contrast to the more balanced synthesis found in asymptomatic beta-thalassaemia trait. The amount of newly synthesized alpha-chain found in the free alpha-chain pool was markedly elevated. The unbalanced globin synthesis and alpha-chain inclusions in the bone marrow cells suggest that the severity of the disorder in these patients may be due to the inability of their red cell precursors to fully compensate for the thalassaemic defect or to remove excess alpha-chains. The diversity of clinical expression suggests the influence of undefined acquired or genetic factors on the expression of beta-thalassaemia in these families.


The pattern of fetal haemoglobin disappearance after birth. Haemoglobin F (Hb F) levels were determined in 209 full-term newborn babies or infants of different ages ranging from birth to 11 months. A follow-up study of the disappearance of Hb F after birth was carried out on 25 premature babies; they were followed periodically from birth until 8 months. The results obtained in both samples show that Hb F levels remain constant after birth for periods varying from about 15 d in at term babies to about 40 d in premature babies; a linear decrease follows in both cases. The initial plateau is a finding contrary to what has been reported. A possible model for the phenomenon is discussed in terms of haemoglobin genes and ontogenetic differentiation.


Induction of haemolytic anaemia by substituted phenylhydrazines. The induction of anaemia and reticulocytosis by arylhydrazines was influenced by substituents on the benzene ring of phenylhydrazine. Arylhydrazines with ortho substituents, which would hinder the binding by haemoglobin of a ligand derived from the arylhydrazine, resulted in the least anaemia and reticulocytosis. These results are consistent with a previous finding that hydrazinobenzoic acid, which did not produce a ferrihaemochrome from ferrihaemoglobin, did not induce anaemia. The parallelism between the formation of a ferrihaemoglobin compound and the induction of anaemia supports the hypothesis that destabilization of haemoglobin by the binding of a ligand derived from the aryl portion of an arylhydrazine is an essential step in arylhydrazine-induced haemolytic anaemia.


Assay of thalassaemic messenger RNA in the wheat germ system. Messenger RNA(mRNA) has been prepared from reticulocytes obtained from patients with different types of thalassaemia and assayed in the wheat germ system. Since normal human reticulocyte mRNA directs the synthesis of equal numbers of alpha- and beta-globin chains in this system it offers a rapid and simple technique for assaying mRNA in the thalassaemic disorders. In mRNA from beta+ thalassaemics the deficiency of beta-globin synthesis mirrored that observed in intact reticulocytes while that prepared from patients wiht haemoglobin H disease gave alpha/beta globin chain production ratios which showed consistently greater imbalance than was found in reticulocytes. Messenger RNA prepared from haemoglobin E-beta0 thalassaemics from Thailand directed no detectable beta-chain synthesis while that prepared from a betaO thalassaemic from Ferrara synthesized a fraction with the chromatographic characteristics of beta-globin chains.


Chemotaxis for human monocytes by fibrinogen-derived peptides. Chemotactic activity for human peripheral blood monocytes was generated by the action of thrombin on human fibrinogen, a reaction known to release low molecular fibrinopeptides. Supernatants prepared by incubating Contortrix venom with fibrinogen, which cleaves the B peptide, were also chemotactic, whereas no activity was present in supernatants from Arvin venom, which cleaves peptides A, AP and AY. Thus fibrinopeptide B was chemotactic for the monocyte in addition to the neutrophil, as previously reported. Monocyte chemotactic activity was also generated by the action of plasmin on human fibrinogen and shown to be associated with the D and E fragments but not with a mixture of fragments X and Y. When plasmin digestion was stopped at time intervals up to 24 h, monocyte chemotactic activity corresponded with the appearance of the D and E fragments. The monocyte chemotactic activity, contained in a 24 h digest, eluted from Sephadex G-75 at VO, corresponding to the expected position of the D and E fragments whereas neutrophil chemotactic activity eluted with molecules of molecular size of approximately 30 000 daltons. Thus fragments D and E derived from plasmin digestion of fibrinogen attract the monocyte whereas only the small uncharacterized peptides were chemotactic for the neutrophil. These different profiles of chemotactic activity for the neutrophil and the monocyte in terms of plasmin digestion products of fibrinogen may be of significance in the events leading to the accumulation of these cells in vivo during fibrin deposition.


The role of membrane bound complement in the aggregation of mammalian platelets by collagen. Platelets from dogs decomplemented with purified Cobra Venom Factor (CVF) lose the ability to aggregate in response to collagen stimulation. In vitro in man, dog and rabbit, and in vivo in the dog, CVF abolishes the collagen response of platelets, the effect being dose-related. Resuspension of CVF inactivated platelets in plasma containing complement resulted in a total return of sensitivity to collagen. Examination of CVF inactivated platelets with the electron microscope failed to show any marked difference from control platelets. Serotonin granules were still present and the platelets retained a discoid appearance. Incubation of platelets with antibodies to C1, C3 and C5 resulted in inhibition of the collagen response: this effect was dose-related. Light microscopy indicated that DVF does not affect the adhesion of platelets to collagen but appears to prevent subsequent aggregation. It is suggested that the complement system is involved in the induction of the platelet release reaction by collagen, and that inhibition by CVF and anticomplement antibodies is the result of a blocking of the release reaction.


Residual marrow damage: possible explanation for idiosyncrasy to chloramphenicol. The effect of oral administration of chloramphenicol was studied in normal mice and mice having residual marrow damage. In the concentration used the drug had no detectable effect on the marrow of normal mice but caused a progressive fall in the numbers of pluripotential stem cells and granulocytic progenitor cells in mice bearing residual marrow damage. However, cells taken from such mice were no more sensitive than normal cells to the in vitro action of chloramphenicol. The results suggest that unrecognized residual marrow damage can be an aetiological factor in marrow failure due to drug idiosyncrasy.


A controlled trial of androgen therapy in experimental chronic hypoplastic marrow failure. A controlled trial of an androgen, 17-nortestosterone decanoate, was carried out to test its effect as therapy in an experimental model of chronic hypoplastic marrow failure. No effect was detected.


Distribution of granulopoietic activity in the human skeleton, studied by colony growth in agar diffusion chambers. The colony forming ability of bone marrow cells from 110 haematologically normal patients was investigated by their growth in semi-solid agar in intraperitoneal diffusion chambers. Marrow aspirated from the sternum showed a consistently higher colony yield than that from the iliac crest. Study of site variation in the individual was possible in donors for bone marrow transplantation. In each of four cases, sternal marrow produced more colonies than any other site. Examination of smears made from the aspirated samples suggested that the difference in yield was not due to varying dilution by peripheral blood on aspiration from different sites. It is concluded, therefore, that the incidence of colony precursor cells is not uniform throughout the active marrow.


Growth of mouse yolk sac cells cultured in vivo. The proliferation and differentiation of yolk sac cells from 10 1/2-day-old mouse embryos were studied in diffusion chambers implanted into syngeneic and allogeneic hosts. The most striking observation was the appearance, proliferation and predominance of immature and mature plasma cells. The data suggested that the differentiation into plasma cells and the rate of growth of yolk sac cells cultured in vivo may be dependent on the genetics of the strain of mouse and host. This demonstration of differentiation of plasma cells from yolk sac cells cultured in vivo adds further information to the origin and ontogeny of potentially immunocompetent cells.


Erythrocytosis associated with diffuse parenchymal lesions of the kidney. Thirteen patients with diffuse parenchymal disease of the kidney and erythrocytosis are reported. In five the haematocrit was only minimally elevated because of an associated increase in plasma volume. The erythrocytosis in three patients was of particular interest: (1) transient appearance in a 23-year-old man with glomerulonephritis; (2) persistence despite the partial remission of nephrotic state in a 19-year-old man; (3) appearance following removal of a nephrosclerotic kidney from a 54-year-old woman. Because an associated expansion of plasma volume is not uncommon with parenchymal renal disease, it is suggested that the only reliable screening method for renal erythrocytosis is direct measurement of red cell volume.


Fine structural studies of peripheral blood leucocytes from children with kwashiorkor: morphological and functional properties. This is the first report of electron microscopic observation on circulating leucocytes from children with kwashiorkor, Neutrophils showed evidence of immaturity, prominent Golgi zones, granule pleomorphism; strands of endoplasmic reticulum and Döhle bodies were frequent. Although an in vitro microbicidal defect occurs with S. aureus, E. coli and C. albicans, the postphagocytic morphological events, including vacuole formation and degranulation, were normal. In mononuclear cell pellets plasmacytoid cells were frequently observed, and rare lymphoblasts occurred. The findings suggest activation of the phagocytic system and stimulation of humoral immunity in children with kwashiorkor. These fine structural observations are comparable to those which occur in trauma or thermal injury, both conditions associated with tissue breakdown and extensive antigenic stimulation.


Ferritin and sideroblastic anaemias: inhibition of protein synthesis by protease contaminants in commercial preparations of ferritin. Commercial preparations of ferritin inhibited reticulocyte-lysate cell-free protein synthesis and disaggregated polyribosomes to monoribosomes and ribosomal subunits. These effects were prevented by addition of reduced glutathione (GSH) to the incubation medium, but ferritin did not lower GSH concentration in the lysates. The more purified the ferritin preparation, the less inhibition of protein synthesis was observed. These data suggested that the effect was due to a contamination of the ferritin with proteolytic activity. In confirmation of this proposal we demonstrated that there was protease activity in both the 2X and 5X crystalized ferritin preparations, with 2.5 times greater activity in the 2X preparation. The proteolytic activity in ferritin was inhibited by incubation with the protease inhibitor tosyl lysine chloromethyl ketone (TLCK). When an amount of trypsin equivalent to the protease activity of the ferritin was added to the incubation mixture, similar effects on protein synthesis and the ribosome-polyribosome component were found. Both GSH and TLCK prevented these effects of trypsin. These data suggest that the previously reported effect of ferritin on reticulocyte cell-free protein synthesis was due to contamination of the ferritin by a protease. It appears that ferritin does not play a direct role in the pathogenesis of sideroblastic anaemias.


Radioimmunoassay studies of the cross-reacting antibody of human group O sera. Cross-reacting antibody was obtained from three recently stimulated group O donors. The biological reactions of cross-reacting antibody were investigated by a study of the binding characteristics of 125I-labelled purified IgG fractions with red cells and by the reactions of whole antiserum or eluates with 125I-labelled blood group glycoproteins. The bulk of cross-reacting antibody activity is restricted to the IgG fraction. Experiments with 125I-labelled IgG and with the radioimmunoassay system revealed that cross-reacting antibody has a higher binding constant than specific anti-A or anti-B. In the radioimmunoassay system complete inhibition of cross-reaction antibody was obtained either with D-galactose or with N-acetyl-D-galactosamine, but the same concentrations of monosaccharides gave only partial inhibition of specific anti-A and anti-B antibodies. The results indicate that cross-reacting antibody binds to a smaller determinant than specific antibody. Investigations with other monosaccharides did not, however, reveal a common inhibitory structure. The ability of cross-reacting antibody to bind to both group A and B antigens may, therefore, be a property of a 'polyfunctional' antibody binding site.


The role of continuous flow centrifuge leucapheresis in the management of chronic lymphocytic leukaemia. Continuous flow centrifuge leucapheresis (CFCL) provides a simple, mechanical technique for removing large numbers of circulating lymphocytes without cytotoxicity. Seven sequences of CFCL were performed on six patients with chronic lymphocytic leukaemia (CLL). In six, the median course consisted of six procedures in 10 days. A median total of 27.7 X 10(11) leucocytes were removed and a 70% drop in leucocyte counts (from 433 X 10(9)/l. to 130 X 10(9)/l.) was observed with little fall in haemoglobin and platelet counts. Tissue massess decreased in two patients. A similar effect was seen in one patient managed less intensively with 21 procedures being performed over 73 days, removing a total of 87.6 X 10(11) leucocytes. The peripheral leucocyte count decreased from 760 X 10(9)/l. to 114 X 10(9)/l. and hepatomegaly diminished markedly. One patient has survived for over 9 months since CFCL but stayed in control only briefly after each of two courses. Short courses of daily CFCL can produce short-term benefit in the management of advanced CLL but at present its main value is as an investigational form of therapy which provides a unique opportunity to study the cytokinetics of advanced CLL without the complications of superimposed cytotoxic therapy.


The regulation of protein synthesis in animal cells by serum factors. We have investigated the regulation of protein synthesis in animal cells by serum factors. Withdrawal of serum from the medium of actively dividing Vero cells resulted in an immediate decline in the rate of peptide chain elongation (Hassell and Engelhardt, 1973). Assay of elongation factor I (EFI) activity in the post-ribosomal supernatant as well as that associated with the ribosomes revealed that serum deprivation resulted also in reduction in the activity of this factor. The decline in the activity of EFI after serum deprivation occurred to the same extent and at the same time as the decline in the in vivo rate of protein synthesis and the in vitro peptide synthetic capacity of cell-free extracts. A temporal correlation therefore exists among the in vivo rate of protein synthesis, the peptide synthetic activity of cell-free extracts, and the activity of EFI. The activity of peptidyl transferase was not altered by serum deprivation. The loss of extract peptide synthetic activity resulting from serum deprivation was reversible since serum addition to previously serum-starved cultures resulted in full restoration of activity for polyphenylalanine (polyPhe) synthesis within 3 h. Moreover, RNA synthesis was not required for this turn-on of polyPhe synthesis. Vased on these data we conclude that a translational control mechanism is operative in Vero cells deprived of serum.


A calorimetric and fluorescent probe study of the gel-liquid crystalline phase transition in small, single-lamellar dipalmitoylphosphatidylcholine vesicles. The results of a calorimetric and fluorescent probe study of the thermotropic behavior of various types of dispersions of dipalmitolphosphatidylcholine bilayer vesicles are reported. Bangham-type, multilamellar vesicles exhibit tow distinct phase transitions at 34.6 and 41.2 degrees C. On the other hand, single-lamellar spherical vesicles appear to exhibit a single transition at 37 degrees C. The single-lamellar vesicles are thermodynamically unstable below 27 degrees C and slowly transform into a multilamellar structure with a single phase transition of 41.2 degrees C. These transformed structures resemble, but are not identical with, Bangham-type vesicles. An experimentally testable thermodynamic and kinetic model based upon these results is developed.


Proton nuclear magnetic resonance studies of hemoglobin Malmö: implications of mutations at homologous positions of the alpha and beta chains. The abnormal human hemoglobin Malmö (beta97FG4 His leads to Gln) has been studied and its properties are compared with those of normal adult hemoglobin A. The data presented here show that the ring-current shifted proton resonances of both HbCO and HbO2 Malmö are very different from the corresponding forms of Hb A. The hyperfine shifted proton resonances of deoxy-Hb Malmö do not differ drastically from those of deoxy-Hb A. This result, together with the finding that the exchangeable proton resonances of the deoxy form of the two hemoglobins are similar, suggests that unliganded Hb Malmö can assume a deoxy-like quaternary structure both in the absence and presence of organic phosphates We have also compared the properties of Hb Malmö with those of Hb Chesapeake (alpha92FG4 Arg leads to Leu). This allows us to study the properties of two abnormal human hemoglobins with mutations at homologous positions of the alpha and beta chains in the three-dimenstional structure of the hemoglobin molecule. Our present results suggest that the mutaion at betaFG4 has its greatest effect on the teritiary structure of the heme pocket of the liganded forms of the hemoglobin while the mutation at alphaFG4 alters the deoxy structure of the hemoglogin molecule but does not alter the teriary structure of the heme pockets of the liganded form of the hemoglobin molecule. Both hemoglobins undergo a transition from the deoxy (T) to the oxy (R) quaternary structure upon ligation. The abnormally high oxygen affinities and low cooperativities of these two hemoglobins must therefore be due to either the structural differences which we have observed and/or to an altered transition between the T and R structures.


Binding of rabbit muscle aldolase to band 3, the predominant polypeptide of the human erythrocyte membrane. Aldolase is a trace protein in isolated human red cell membrane preparations. Following total elution of the endogenous enzyme by a saline wash, the interaction of this membrane with rabbit muscle aldolase was studied. At saturation, exogenous aldolase constituted over 40% of the repleted membrane protein. Scatchard analysis revealed two classes of sites, each numbering approximately 7 X 10(5) per ghost. Specificity was suggested by the exclusive binding of the enzyme to the membrane's inner (cytoplasmic) surface. Furthermore, milimolar levels of fructose 1,6-bisphosphate eluted the enzyme from ghosts, while fructose 6-phosphate and NADH (a metabolite which elutes human erythrocyte glyceraldehyde-3-phosphate dehydrogenase (G3PD) from its binding site) were ineffectuve. Removing peripheral membrane proteins with EDTA and lithium 3,5-diiodosalicylate did not diminish the binding capacity of the membranes. An aldolase-band 3 complex, dissociable by high ionic strength or fructose 1,6-bisphosphate treatment, was demonstrated in Triton X-100 extracts of repleted membranes by rate zonal sedimentation analysis on sucrose gradients. We conclude that the association of rabbit muscle aldolase with isolated human erythrocyte membranes reflects its specific binding to band 3 at the cytoplasmic surface, as is also true of G3PD.


The resolution of Ascaris cuticle collagen into three chain types. Reduced and methylated collagen from Ascaris lumbricoides cuticle was resolved into three major components by chromatography on phosphocellulose. The components have similar molecular weights of about 52,000 by sedimentation equilbrium and molecular sieve chromatography, but they have different amino acid compositions. Since they do not appear to be stoichiometrically related, they apparently represent chains from collagens of more than one type. All three chains contain about 27 residue % glycine, 36 residues of proline, and 17 residues of methylcysteine, suggesting that the collagens can be maximally about 80% triple helical and are extensively disulfide cross-linked  in the native state. Two minor components from the cuticle are apparently derived from one of the major chains by cleavage in a single region to give two-third and one-third fragments.


Iodination of Xenopus laevis histone F2a1 in chromatin. The reaction of calf thymus and Xenopus laevis histones with radioactive iodine has been studied under various conditions that affect chromatin structure. All histones from both species contain at least one tyrosine residue and, in a denaturing solvent, all the the histones react with iodine. Histone F2a1 has been studied in detail. Calf thymus F2a1 is known to contain four tyrosyls and all four react with iodine. In high voltage paper electrophoresis, the tyrosine-containing peptides from calf co-migrate with those from Xenopus F2a1, suggesting that the amino acid sequence is strongly conserved between these two species. Therefore, the published calf thymus F2a1 sequence has been used to order the Xenopus F2a1 peptides within the molecules. When gently isolated native chromatin is iodinated in a low ionic strength medium 60% of the radioactivity in F2a1 is in tyrosyl 88, 30% in tyrosyl 51, and tyrosyl 72 and 98 have almost no radioactivity. Reagents which remove the protein from the DNA (2 M NaCl) or partially disrupt protein tertiary structure (5 M urea) increase the reactivity of each of the four tyrosyls five- to tenfold, suggesting that all four are protected about equally by the overall folding of the chromatin. Isolated F2a1 iodinated in the presence of 10 M urea shows uniform labeling in each of the four peptides, suggesting that tyrosyl 72 and 98 are afforded some protection solely by protein-protein interactions. The stepwise removal of histones in increasing NaCl concentrations differentially increases the availability of each F2a1 tyrosyl. The preferential exposure of tyrosyl 88 coincides with the removal of the majority of F1 histones at 0.5 M NaCl while the gradual and stepwise increase in reactivity of tyrosyl 51, 72, and 98 correlates with the gradual removal of histones other than F1. Radioactive iodination of chromatin has been shown to be a sensitive probe for detecting changes in the association state (or conformation) of histone F2a1.


The effect of carboxymethylating a single methionine residue on the subunit interactions of glycophorin A. Human red cell glycophorin A shows an equilibrium between dimeric and monomeric forms which have been disignated PAS-1 and PAS-2, respectively. This equilibrium, which is dependent upon protein concentration is achieved by incubation in sodium dodecyl sulfate solutions at elevated temperatures and is assayed by sodium dodecyl sulfate gel electrophoresis. Carboxymethylation of glycophorin A in guanidine hydrochloride or urea alters the interactions between polypeptide chains so that the lower molecular weight form (PAS-2) is obtained much more readily. If the carboxymethylation is performed at pH 3.0 the reaction is limited to the two methionine residues of glycophorin A which are located at positions 8 and 81 in the sequence. In the presence of sodium dodecyl sulfate, only one of the two methionine residues is carboxymethylated, and glycoprotein modified under these conditions does not exhibit the change in electrophoretic mobility. Experiments with [1-14C]iodoacetic acid demonstrated that Met-81, located in the hydrophobic domain of the protein, is the residue protected by sodium dodecyl sulfate. Modification of Met-81 destabilizes the dimeric form relative to the monomer by weakening the interactions between polypeptide chains. The experiments described in this paper confirm that the hydrophobic domain of glycophorin A is involved in subunit interactions and that Met-81 plays a critical role in those interactions.


Characterization of the gamma subunits of the 7S nerve growth factor complex. The gamma subunits of the 7S nerve growth factor complex (7S NGF) display arginine esteropeptidase activity. By varying the conditions of electrophoresis in acrylamide gel, it has been demonstrated that the gamma-subunit fraction of 7S NGF contains five different proteins, in contrast to the three (gamma1, gamma2, and gamma3) originally described (Smith, A.P., Varon, S. and Shooter, E.M. (1968), Biochemistry 7, 3259-3268); the gamma1 and gamma2 subunits, previously thought to be single species, can each be resolved into two components. The two components of the gamma1 subunit have the same isoelectric point, as do the two components of the gamma2 subunit. The distribution of protein among the two components of each of the gamma1 and gamma2 subunits varied from preparation to preparation. Moreover, a shift in the distribution for the gamma1 subunit was accompanied by a parallel shift for the gamma2 subunit. All of the different gamma proteins have the same molecular weight. On the basis of the molecular weights of the peptide chains of the gamma subunits and of the species which are formed by cross-linking with dimethyl suberimidate, it was concluded, that both the gamma1 and gamma2 subunits contain one species with two peptide chains and another with three peptide chains, while the gamma3 subunit is a single species with three peptide chains. The results also suggest that two of the chains in the three-chain species are derived, by proteolytic cleavage, from the larger chain in the two-chain species.


The nature of stacking interations in polynucleotides. Molecular states in Oligo- and polyribocytidylic acids by relaxation analysis. The dynamics of the helix-coil transition of single-stranded poly(C) (polyribocytidylate) and CpC (cytidyly(3'-5')cytosine) was investigated by an improved cable temperature-jump technique. The single-strand relaxation was characterized by following the ultraviolet (uv) absorbance changes at 248 and 280 nm. Poly(C) and CpC showed single relaxation processes with amplitudes corresponding to those expected from equilibrium melting curves. The relaxation time contants in the range of 25-100 ns were independent of the nucleotide concentration, but strongly dependent upon temperature. Using thermodynanic parameters obtained from circular dichroism (CD) and uv absorbance melting curves, the following rate constants k (at 20 degrees C, 1.05 M ionic strength, pH 7) and activation enthalpies EA were calculated for poly (C): helix formation kR = 1.11 X 10(-7) s-1 (EAR = 2.6 kcal); helix dissociation kD = 2.1 X 10(6) s-1 (EAD = 11.9 kcal). The rate constants obtained for CpC were higher by a factor of about 2 in kR and 12 in kD, whereas the activation enthalpies closely corresponded to those found for the polymer. In addition to the single-stranded helix-coil relaxation, poly(C) and CpC exhibit a relaxation process with a time constant below 25 ns and maximum amplitudes at wavelengths lambda greater than or equal to 285 nm. The same process is found in cytidine and is attributed to hydration equilibria. The hydration reaction can be considered to be in equilibrium during the entire time range of the helix-coil transition and thus the data obtained for the helix-coil transition can be described by a simple two-state model. The rate parameters indicate the existence of relatively high energy barriers in the helix-coil transition and provide strong evidence evidence against an oscillating dimer model. If there is an ensemble of substates for one of the states (as may be expected for the coil form), the energy difference between the populated substates is small compared with the energy difference between the major conformational states.


HeLa cell deoxyribonucleic acid dependent RNA polymerases: function and properties of the class III enzymes. The class III DNA dependent RNA polymerases (nucleoside triphosphate:RNA nucleotidyltransferase EC 2.7.7.6 from HeLa cells have been solubilized and characterized as to function and properties. Two chromatographically distinct forms of enzyme III, designated polymerases IIIA and IIIB, can be resolved when cell extracts are chromatographed on DEAE-Sephadex columns. Enzymes IIIA and IIIB exhibit nearly identical catalytic properties such as divalent cation stimulation, broad biphasic ammonium sulfate optima, and characteristic alpha-amanitin sensitivities which clearly distinguish them from the homologous enzymes, forms I and II. Polymerases IIIA and IIIB are both primarily localized in the nucleus (greater than 60%). The most notable characteristic of the class III enzymes is a unique sensitivity to inhibition by alpha-amanitin (50% inhibition at 15 mug/ml). HeLa cell enzyme I is not inhibited by the mushroom toxin even at very high concentrations (greater than 400 mug/ml), while HeLa cell polymerase II is inhibited by very low concentrations of amanitin (50% inhibition at 0.003 mug/ml). The three major classes of enzyme (I, II, III) exhibit characteristic sensitivities to alpha-amanitin whether assayed in nuclei, crude homogenates, or in a chromatographically purified state. Using a nuclear in vitro RNA synthesizing system to investigate the alpha-amanitin sensitivities of the synthesis of tRNA precursor (4.5S pre-tRNA) and 5S ribosomal RNA, it was found that the synthesis of these RNA species  was inhibited 50% at 15 mug/ml of alpha-amanitin. The alpha-amanitin inhibition curves for the synthesis of pre-tRNA-5S ribosomal RNA in nuclei and the alpha-amanitin titration curves for the partially purified class III enzymes (IIIA and IIIB) are identical. These data, therefore, show that the in vivo functional role of the class III RNA polymerases (IIIA-IIIB) is the transcription of the genes coding for transfer RNA and 5S ribosomal RNA.


Solubilization of an alpha-bungarotoxin-binding component from rat brain. Binding of [125I]-alpha-bungarotoxin to rat brain was investigated. Picomole quantities of specific toxin binding sites per gram of fresh tissue were found in particulate preparations as well as detergent extracts of whole brain. The toxin-binding macromolecules can be solubilized in low concentrations of Triton X-100. Specific binding occurs to a single class of sites with a dissociation constant of 5.6 X 10(-11) M. The association rate constant in 10 mM sodium phosphate, pH 7.4, was determined to be 6.8 X 10(5) M-1 s-1; the half-life of the complex was found to be 5.1 h, corresponding to a dissociation rate constant of 3.8 X 10(-5) s-1. The binding macromolecules resemble peripheral nicotinic acetylcholine receptors in toxin binding kinetics, solubility, isoelectric point, and hydrodynamic properties.


Translocation and binding of adenine nucleotides by rat liver mitochondria partially depleted of phospholipids. 1. Rat liver mitochondria were partially depleted of their phospholipids using phospholipase A prepared from porcine pancreas (substrate specificity, cardiolipin greater than phosphatidylethanolamine greater than phosphatidylcholine) or from Crotalus adamanteus venom (substrate specificity, phosphatidylethanolamine = phosphatidylcholine greater than cardiolipin). 2. Removal of only about 1% of the mitochondrial phospholipid with the pancreatic enzyme leads to 50% and 25% losses in ADP and ATP translocation, respectively. Concomitant with the loss in translocation is a decline in the ability of both carbonylcyanide m-chlorophenylhydrazone and Ca2+ to stimulate ATP translocation. 3. To achieve comparable losses in ADP and ATP translocation with the venom enzyme, it is necessary to remove about 8% of the total mitochondrial phospholipid. Following such treatment, carbonylcyanide m-chlorophenylhydrazone and Ca2+ are still capable of stimulating ATP translocation. 4. Control experiments involving treatment of the mitochondria with the products of phospholipase digestion indicate that the effects observed on the translocase reflect a loss of phospholipid from the membrane. 5. Binding studies indicate that the loss in adenine nucleotide translocation following phospholipase treatment cannot be accoundted for by an altered ability to bind adenine nucleotides to atractyloside-sensitive sites. 6. The data are interpreted in terms of a mechanism of adenine nucleotide translocation involving a lipoprotein carrier system, consisting of the translocator protein and phospholipids, possibly cardiolipin and phosphatidylethanolamine.


Ferrous ion-mediated cytochrome P-450 degradation and lipid peroxidation in adrenal cortex mitochondria. The relationship between the degradation reaction of cytochrome P-450 and lipid peroxidation was studied utilizing bovine adrenal cortex mitochondria. The two reactions were found to be closely correlated in terms of their response to storage of the mitochondrial preparation, stimulation by Fe2+, inhibition by EDTA and their initiation by cumene hydroperoxide. Both reactions were also found not to be inhibited by catalase, superoxide dismutase, 1,4-diazabicyclo-(2,2,2)-octane and alcohols, indicating that H2O2, superoxide, singlet oxygen and hydroxyl radicals do not participate in these reactions. Yet, diphenylamine proved to be a powerful inhibitor for both reactions, suggesting the involvement of a radical species. Cumene hydroperoxide could induce these two reactions at below 0.1 mM concentrations in the presence of molecular oxygen. The chemiluminescence observed during the Fe2+-mediated lipid peroxidation reaction which was not inhibited by either superoxide dismutase or 1,4-diazabicyclo-(2,2,2)-octane, was biphasic: one was a rapid burst; and the other was a slowly increasing emission. The latter portion of the emission of light coincided with the formation of malondialdehyde. These results indicate that in adrenal cortex mitochondria the degradation of cytochrome P-450 is closely related to lipid peroxidation.


The ratio of the two light reactions and their coupling in chloroplasts. The kinetics of the absorbance changes of chlorophyll alphaI (P-700) and plastoquinone induced by xenon flashes of saturating intensity were studied in spinach chloroplasts. 1. The total amount of chlorophyll alphaI is compared with that amount being reduced via the rate-limiting step between the light reactions. This is based on the amplitudes of the absorbance changes of chlorophyll alphaI after chemical reduction and after a group of flashes following far-red preillumination. It is concluded that only 75% of chlorophyll alphaI is coupled to chlorophyll alphaII via linear electron transport and that the remaining 25% is functionally isolated. 2. A ratio of 0.85 for coupled chlorophyll alphaI to chlorophyll alphaII is estimated from the time course of the absorbance changes of plastoquinone and chlorophyll alphaI in two independent ways. 3. The oxygen yield per flash is used to calculate the difference extinction coefficient of chlorophyll alphaI at the maximum of the red absorbance band in spinach chloroplasts: delta xi703 = (6.7 +/- 0.7)-10(4) M-1-cm-1. The assumption of a quantitative electron transfer from water via plastoquinone to coupled chlorophyll alphaI is supported by the same extinction coefficient reported by Hiyama and Ke for Photosystem I particles. The location and function of the different chlorophylls alphaI is discussed in detail.


[Changes in adenosine phosphates and energy charge in chloroplastic and nonchloroplastic compartments of wheat leaves (author's transl)]. Changes in the adenine nucleotides and energy charge (= (ATP)+1/2(ADP)/(AMP)+(ADP)+(ATP)) levels were studied in chloroplastic and non-chloroplastic compartments using non-aqueously isolated wheat leaves chloroplasts. The two adenine nucleotides pools (of chloroplasts and non-chloroplastic part of the cell), though distinct, are linked. This linkage substantiates an energy-rich bond exchange between the two compartments. When both photphosphorylation and oxidative phosphorylation occur simultaneously, energy charge takes high values, generally higher than 0.80. When neither oxidative phosphorylation nor photophosphorylation occur, energy charge is very low and takes values generally lower than 0.45. When one compartment alone produces approximately P, energy charge in the two compartments takes intermediate values which remain relatively high. Dark-light transition in nitrogen resulted in changes of the AMP, ADP and ATP levels which quickly reach a steady state. Chloroplast energy charge shifts rapidly from 0.45 to 0.75 in 10 s; after 1 min it reaches 0.86, a value that corresponds to a steady level. In the cytoplasm, energy charge changes from 0.44 to 0.71 in 1 min. Energy charge increase in the non-chloroplastic compartment substantiates an energy transfer from chloroplasts to the cytoplasm. On nitrogen-air transition in the dark, the cytoplasm energy charge reaches a steady level in 30 s. In chloroplasts, it also increases but slowly. There is indeed a transfer of energy from cytoplasm to chloroplasts. Darkening of the leaves in air causes a drastic and lasting drop of energy charge in the chloroplasts where it has a low value after 5 min in the dark. Then it increases again but slowly and is still lower than 0.70 after 10 min in the dark. Meanwhile, energy charge in cytoplasm keeps values higher than 0.75. Metabolic regulation by energy charge and control of adenine nucleotides level by adenylate kinase (EC 2.7.4.3) are discussed.


Effects of lipolytic enzymes on the photochemical activities of spinach chloroplasts. 1. Spinach class II chloroplasts were treated with purified potato lipolytic acyl-hydrolase and venom phospholipase A2, and their lipid degradations and the effects on the photochemical activities were followed. 2. Potato lipolytic enzyme hydrolyzed monogalactosyldiacylglycerol at a faster rate than phospholipids such as phosphatidylglycerol and phosphatidylcholine. The treatment caused a rapid decrease of Photosystem I activity, and a less change of Photosystem II activity. 3. Venom phospholipase A2 which preferentially hydrolyzed phosphatidylglycerol, caused a rapid decrease of Photosystem II activity and only a slight decrease of photosystem I activity. 4. Potato enzyme and phospholipase A2 degraded the membrane lipids of glutaraldehyde-fixed chloroplasts at a rather slightly higher rate than those of non-treated chloroplasts. 5. The results suggested a possible correlation between monogalactosyldiacylglycerol degradation and decay of Photosystem I activity and between phosphatidylglycerol degradation and decay of Photosystem II activity. A possible mechanism is discussed.


An abnormal ratio of cytochromes in the respiratory chain of mouse and human myelomas. Mouse myeloma cells and mitochondria had the same kinds of cytochrome components in the respiratory chain as the normal ones. Their constitution, however, was abnormally different from that found in normal cells and mitochondria. The cytochrome aa3 concentration was especially low in the myeloma as compared with cytochrome c concentration, and the resulting cytochrome aa3/c ratio was 0.25, which was the lowest ever reported in animal mitochondria. Normal lymph node cells, producing the immunoglobulin similar to the myeloma cells, had a ratio of 1.1. Human myeloma mitochondria had the same characteristics as the mouse myeloma. Ascite form myeloma originated from mouse solid from myeloma grew faster, and yet aa3/c of 0.5 in the ascites myeloma was found to be quite similar to that observed in various ascites tumor cells such as hepatomas, Ehrlich and sarcoma 180. A significant part of the cytochromes in the respiratory chain of the mouse myeloma remained in the oxidized form in the cyanide-inhibited or anaerobic states, and was reduced only by the addition of dithionite. The properties of the b cytochromes in mouse myeloma mitochondria are also described and discussed in the context of multiple forms of the b cytochromes in the respiratory chain.


Sulphydryl groups in photosynthetic energy conservation. I. Light-dependent inhibition of photophosphorylation by the sulphydryl reagent 2-2'dithio bis-(5-nitropyridine). 1. The sulphydryl reagent 2-2'dithio bis-(5-nitropyridine) (DTNP) inhibited photophosphorylation when the chloroplasts were preincubated with the reagent in the light. A maximum inhibition of about 50% was obtained in the presence of pyocyanine and MgCl 2 at 0.3 mumol DTNP per mg chlorophyll and was completed in about 40 s of preillumination. 2. Dithioerythritol, ADP plus Pi (or arsenate) and uncouplers prevented the inhibition when present during the preillumination while phloridzin, Dio-9 and discarine B were ineffective. Low concentrations of ADP or ATP afforded partial protection but other nucleotides had no effect. 3. DTNP inhibited the coupled electron transport rate to the basal level and had no effect on the uncoupled electron transport. The stimulation of proton uptake and inhibition of electron transport by ATP was prevented by DTNP. 4. The trypsin-activated but not the light- and dithioerythritol-triggered ATPase was inhibited by light preincubation of chloroplasts with DTNP. 5. Reversal of DTNP inhibition of photophosphorylation was obtained by a second preillumination in the presence of thiol groups. 6. More DTNP reacted with chloroplasts in the light than in the dark. Two mol of thione were formed in the light per mol of DTNP disappeared. 7. The results suggested that DTNP inhibition is related to the oxidation by DTNP of chloroplast vicinal dithiols probably exposed by a light-induced conformational change.


Increase effected by calcium ion in the rate of oxygen evolution from preparations of Phormidium luridum. The presence of Ca2+ causes a twentyfold or greater increase in the rate of oxygen evolution by cell-free preparations of Phormidium luridum. The requirement for Ca2+ is specific; other divalent cations are much less effective or are inhibitory. The rate of the Hill reaction is maximal at 30 mM CaCl2 in both detergent-free and Brij 35 preparations. The 3-(3,4-dichlorophenyl)-1,1-dimethylurea-insensitive component of oxygen-evolving activity in each preparation also shows the requirement for added Ca2+. This indicates that Ca2+ is acting close to the oxygen-evolving reaction center of Photosystem II. Defatted bovine serum albumin increases the rate of oxygen evolution in the detergent-free preparation, but does not compete with Ca2+, discounting fatty acid mediation of the effects of Ca2+. Neither excess Hill acceptor nor uncouplers of photophosphorylation diminish the stimulatory effects of Ca2+.


The existence of a high photochemical turnover rate at the reaction centers of system II in Tris-washed chloroplasts. In Tris-washed chloroplasts the kinetics of the primary electron acceptor X 320 of reaction center II has been investigated by fast repetitive flash spectroscopy with a time resolution of approximately 1 mus. It has been found that X 320 is reduced by a flash in less than or equal to 1 mus. The subsequent reoxidation in the dark occurs mainly by a reaction with a 100-200 mus kinetics. The light-induced difference spectrum confirms X 320 to be the reactive species. From these results it is concluded that in Tris-washed chloroplasts the reaction centers of System II are characterized by a high photochemical turnover rate mediated either via rapid direct charge recombination or via fast cyclic electron flow.


On the individuality of aliphatic and alicyclic monoester lipases in human adipose tissue. Aliphatic and alicyclic monoester lipase activities from human adipose tissue have been comparatively investigated by using [3H] oleoylethanol and [14C] oleoylcholesterol, respectively, as substrates. A number of specific properties proved to be different for each activity. Different rates of decay of hydrolytic activity towards each substrate were observed during heat denaturation. Stability upon exposure to the cold was different for both activities, and the protective effect of glycerol was less effective for oleoylcholesterol than for oleoylethanol lipase. Serial (NH4)2SO4 fractionation in 5% increments showed that the two activities did not precipitate at identical saturation values. The behaviours of the two activities were compared in an affinity system where monoolein, a substrate molecule, served as a ligand for the enzyme(s) in a covalent complex with CH-Sepharose. During chromatography, both activities followed a comparable adsorption-elution pattern, but the oleoylcholesterol to oleoylethanol lipase activity ratio decreased by a factor of 4. Taken together, these data, along with a differential susceptibility to various surfactants, confirm our earlier hypothesis (Arnaud, J. and Boyer, J (1974) Biochim. Biophys. Acta 337, 165--168) that aliphatic and alicyclic monoester lipase activities in human fat are referable to distinct catalytic proteins.


Lipids and fatty acids of a moderately halophilic bacterium, No. 101. The extractable and bound lipids of a moderately halophilic gram-negative rod, strain No. 101 (wild type) grown in a medium containing 2 M NaC1, were examined. The extractable lipids were separated into at least 8 components by using thin-layer chromatography. The major phospholipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and an unidentified phosphoglycolipid in the whole cells, cell envelopes and outer membrane preparations, commonly. Judging from mild alkaline hydrolysis and exzymatic treatment with phospholipase A2, C and D, the unidentified phosphoglycolipid possessing Pi, glycerol, fatty acids and glucose in a molar ratio of 1 : 2 : 2 : 1, appeared likely to be a glucosyl derivative of phosphatidylglycerol. No glucuronic acid containing lipid was detected. The exractable lipid composition varied greatly with the concentrations of NaC1 in the medium and the stages of bacterial growth. The most characteristic phosphoglycolipid in this organism increased up to 25% of the total phospholipids with the addition of 1% glucose in the medium. The major fatty acids of the extractable lipids were C16:0, C16:1, C18:0, C18:1 and cyclopropanoic C17 and C19 acids and these compositions were very similar for each phospholipid. The cyclopropanoic fatty acids predominated as growth proceeded. The fatty acids of the bound lipids comprised a high concentration of 3-hydroxydodecanoic acid. The esterified fatty acids of the lipopolysaccharide molecule seemed to contain a wide variety of hydroxy and non-hydroxy shorter chain fatty acids, while the amide-linked fatty acids consisted almost entirely of 3-hydroxydodecanoic acid.


Purification and partial characterization of nonspecific lipase from rat pancreas. Nonspecific lipase (also referred to as micelle lipase and secondary ester hydrolase) has been purified to electrophoretic homogeneity starting from acetone powder of rat pancreas. The purified enzyme is found to have a molecular weight (gel filtration) of 64 000 +/- 2000, and an equivalent weight (titration with E-600) of 65 000. Nonspecific lipase is seen to be very sensitive to inhibition by organophosphates but resistant to quinine. Evidence for the presence of sulfhydryl and imidazole groups essential for activity is presented, and some observations on substrate specificity are made. The purified enzyme appears to lack phosphate groups and lipids, and is unstable under conditions of low ionic strength and/or exposure to 2-mercaptoethanol.


The thermal transitions and structural properties of 5alpha-cholestan-3beta-ol esters of aliphatic acids. 5 alpha-Cholestan-3 beta-ol esters of aliphatic acids undergo both enantiotropic and monotropic changes of state. Ten saturated and three unsaturated esters have been examined by differential scanning calorimetry and polarizing microscopy to determine transition temperatures, enthalpies, and entropies. The results are compared with an analogous series of cholesterol esters. All esters of even-numbered n-alkanoic acids from C2 to C20 melt from a crystalline state to an isotropic liquid. The crystalline state has been studied by X-ray powder diffraction. The C8 to C20 esters have progressively increasing crystalline melting transition temperatures from 76 to 99 degrees C and possess similar X-ray powder diffraction patterns, suggesting that these compounds form an isostructural series. Esters of C2, C4, and C6 acids exhibit polymorphism. Crystalline cholestanol oleate melts to an isotropic liquid, whereas cholestanol linoleate and linolenate fail to crystallize, even after several months at -20 degrees C. Esters of the even-numbered saturated acids from C4 to C14 form monotropic cholesteric liquid crystalline phases. Esters C10, C12, and C14 form smectic liquid crystalline phases. Cholestanol oleate, linoleate, and linolenate form both cholesteric and smectic mesophases. The lower smectic to cholesteric and cholesteric to isotropic transition temperatures of the cholestanol esters compared to the corresponding transition temperatures of the analogous cholesterol esters suggest that the delta 5 double bond in cholesterol increases the thermal stability of the mesophases of cholesterol esters.


Direct effects of fructose metabolism on fatty acid oxidation in a recombined rat liver mitochondria-hish speed supernatant system. The effects of fructose on the oxidation of [1-(14)C]palmitate in a rat liver mitochondria-high speed supernatant system have been investigated. This model system permitted study of the direct effects of fructose and the metabolism of fructose on fatty acid oxidation in the near absence of fatty acid esterification. Fructose inhibited the utilization of albumin-bound [1-(14)C] palmitate in the mitochondria-supernatant system, but did not affect fatty acid utilization by isolated liver mitochondria. Although fructose decreased the ATP content in the mitochondrial-supernatant system, the level of ATP throughout the incubation period was sufficient for maximal fatty acid activation. Fructose decreased the conversion of [1-(14)C]palmitate to 14CO2 and depressed the formation of total labeled oxidation products (14CO2 + 14C-labeled ketone bodies) in this system. The results suggest that fructose metabolism inhibited fatty acid oxidation in the mitochondria-supernatant system by competitive substrate oxidation and thereby decreased utilization of the added [1-(14)C]palmitate. The ihibition of L-[L-(14)C]palmitoylcarnitine oxidation, fructose was in all respects similar to its inhibition of palmitate oxidation, indicating that the site of fructose interaction was within the beta-oxidation sequence. These observations support the concept (Ontko, J.A. [1972] J. Biol. Chem. 247, 1788-1800) that the reciprocal changes in esterification and oxidation of palmitate caused by fructose in liver cells are primarily mediated via inhibitory effects on long-chain fatty acid oxidation.


Aliphatic chain packing in three crystalline polymorphs of a saturated racemic phosphatidylethanolamine. A quantitative electron diffraction study. Taking advantage of the adequacy of the kinematical diffraction assumption for ab initio crystal structure analysis of long chain lipids with single crystal electron diffraction intensity data, the polymethylene chain packing in three microcrystalline polymorphs of 1,2-dipalmitoyl-rac-glycero-3-phosphoethanolamine was determined. The aliphatic chains of major crystal form pack in the new parallel/perpendicular orthorhombic methylene subcell already seen for three other compounds. The unit cell dimension s of this form are: a = 7.76 A, b = 10.03 A, c = 56.0 A. The two minor polymorphs pack in commonly-found subcells, i.e. perpendicular orthorhomibic (a(s) = 5.10 A, b(s) = 7.61 A) and hexagonal (a(s) = 4.68 A, gamma = 120 degrees). Similarities between the two orthorhombic subcells are discussed.


On the metabolism of prostaglandins by human gastric fundus mucosa. 1. Specific radioimmunoassays for the prostaglandins E2, F2alpha and A2 and the metabolites 13,14-dihydro-15-keto-prostaglandin E2, 15-keto-prostaglandin F2alpha and 13,14-dihydro-15-keto-prostaglandin F2alpha were used to study the metabolism of prostaglandins by gastroscopically obtained small biopsy specimens of human gastric fundus mucosa. 2. Three prostaglandin-metabolizing enzymes were found in the 100 000 X g supernatant of human gastric fundus mucosa, 15-hydroxy-prostaglandin-dehydrogenase, delta13-reductase and delta9-reductase. The specific activity was highest for 15-hydroxy-prostaglandin-dehydrogenase and lowest for delta9-reductase. 3. Formation of prostaglandin A2 (or B2) was not observed under the same conditions. 4. None of the three enzyme activities detected in the 100 000 X g supernatant was found in the 10 000 X g and 100 000 X g pellets of human gastric fundus mucosa. 5. The results indicate that high speed supernatant derived from human gastric mucosa can rapidly metabolize prostaglandin E2 and prostaglandin F2alpha to the 15-keto and 13,14-dihydro-15-keto-derivatives. Furthermore, prostaglandin E2 can be converted to prostaglandin F2alpha, the biological activity of which, on gastric functions, differs from that of prostaglandin E2.


Lipolytic activity of whole isolated liver cells in aqueous suspension. 1. Liver contains a lipase which catalyzes in vitro the hydrolysis of esters of short-chain normal primary alcohols and fatty acids. It is shown that this enzymatic activity can be measured by using intact liver cells as source of enzyme. During short-term incubations of suspensions of cells isolated from rat liver, the lipase acts as a membrane-bound enzyme and readily attacks [3H] oleoylethanol added as an emulsion into the bathing medium. The lipolytic reaction proceeds linearly for at least 20 min at 37 degrees C, at the pH optimum of 8.5. [3H] Oleic acid, a reaction product, is mostly retained in the medium and is used to monitor the lipolytic process. 2. In the presence of heparin, the bound lipase is released in the medium in amounts representing one-third to one half the total activity contained in the cells. This release is very rapid and associated in all cases with a concomitant release of lactate dehydrogenase activity. Such effects are consistent with the interpretation that heparin, at concentrations comprised between 10 and 100 mug per ml, causes alterations of the plasma membrane of the isolated cells, resulting in the dispersion of membrane-bound and cytoplasmtic material. This action of heparin is totally blocked by protamine sulfate (1 mg/ml). No specific effect of heparin directed towards the selective release of lipase could be demonstrated under these conditions. 3. During incubations in the presence of heparin, it was observed that the release of monoester lipase was quantitatively related to a simultaneous decrease in membrane-bound as well as in total monoester lipase activity measureable in the cells after homogenization. This, along with the reappearance of membrane-bound activity immediately after heparin withdrawal, suggest that under the experimental conditions, the membrane-bound enzyme is replaced from inside the cell in proportion of its release by heparin.


Major lipid classes in plasma membrane isolated from liver of rats fed a hepatocarcinogen. More phosphatidylethanolamine and less phosphatidylinositol were present in the lipid of plasma membrane isolated from the liver of rats fed a diet containing the hepatocarcinogen, N-2-fluorenylacetamide than were present in the lipid of plasma membrane isolated from the liver of rats fed the basal diet. Phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin from the former membranes had relative amounts of 18 : 1 in their fatty acyl profiles that were 1.5--1.7 times the relative amount of 18 : 1 in the same phospholipid from the latter membranes. Furthermore, the former membranes contained 1.3 times more cholesterol and 1.35 times more sphingomyelin than the latter membrames.


Effect of mercuric and silver ions on cesium sulfate equilibrium buoyant densities of synthetic polydeoxyribonucleotides. The effect of Hg2+ and Ag+ on the buoyant density (rho) of four synthetic DNA polymers, poly[d(A-T)]; poly(dA) - poly(dT); oikt[d(G-C)]; and poly(dG) - poly(dC), was investigated. The buoyant density of poly[d(A-T)] in Cs2SO4 increased dramatically after complexing with Hg2+, but little change in the buoyant density of other polymers resulted except at very high molar ratios of Hg2+/DNA-P (rf). Hg2+ raised the thermal transition temperature (Tm) of alternating polymers and lowered the Tm of homopolymers. Measurements in the preparative ultracentrifuge indicated that lowered Tm correlated with Hg2+-induced strand separation of one homopolymer [poly(dA) - poly(dT)], but strand separation was not observed with another homopolymer [poly(dG) - poly(dC)] complexed with Hg2+. When Ag+ was mixed with the polymers, the buoyant density of poly(dG) - poly(dC) increased most markedly. A substantial increase in the buoyant density of poly[d(A-T)] and a small increase in the buoyant density of poly[d(G-C)] were also observed. The Tm changes induced by Ag+ were not related in any obvious way to buoyant density changes. These findings indicate that nucleotide sequence as well as overall base composition is of importance in understanding the buoyant density changes induced by metal ions. Although these data do not allow construction of a detailed molecular model of polymer-metal ion interactions, they may be used to explain much of the behavior of naturally occurring DNA sequences, such as heterochromatic satellite sequences and 5 S and rRNA sequences, in Hg2+/Cs2SO4 and Ag+/Cs2SO4 gradients.


Nuclear ribonucleoproteins as inhibitors of mammalian RNA polymerase. A ribonucleoprotein complex isolated from rabbit thymus nuclear lysates was found to be an inhibitor of DNA-dependent RNA polymerase II. The inhibition appeared to be of a competitive type and was completely reversed by high concentration of DNA. Highest inhibition was observed when enzyme and complex were preincubated before addition of DNA while there was little inhibition after enzyme had started synthesis on the DNA template. The RNA isolated from the complex was equally inhibitory and was a more effective inhibitor than either tRNA or rRNA.


Nuclear magnetic relaxation studies of cytidine and its complexing dimethyl sulfoxide solution. 13C spin-lattice relazation times (T1 values) in cytidine were determined experimentally to investigate molecular motions of both metal-free and ion-complexed cytidines in dimethylsulfoxide solutions. It was found that the correlation times of the protonated carbons were equal within experimental error, and this equality of correlation times of different sites of the molecule suggests strongly isotropic random motion of the molecule. Correlation times for internal motion of the amino group obtained from the observed T1 of the amino protons are 4.6 -10(-11) S, 2.0 - 10(-9) S and 1.1 - 10(-10) S for the metal-free cytidine and the cytidine complexed with either CaCl2 or ZnCl2, respectively. An experimental value of T1 of the H6 proton of the cytidine base agrees very well with the value estimated from a conformation determined by the nuclear "Overhauser" effect. Spin-lattice relaxation time measurements of the 7Li nucleus in the LiCl/cytidine system strongly suggested that the 7Li cation is directly coordinated with cytidine.


Comparison of proteins bound to the different functional classes of messenger RNA. Proteins from nuclear ribonucleoproteins, informosomes, polysomal messenger ribonucleoproteins and cytoplasmic "binding factor" are characterized. 1. Nuclear ribonucleoproteins are purified from nuclei disrupted by ultrasonication. Possible contamination by nucleoplasm, histones or remaining cytoplasmic structures is controlled. 2. Informosomal proteins are obtained by mild RNAase degradation. This method gives informosomal proteins without appreciable contamination. 3. Polysomal messenger ribonucleoproteins are obtained from cells where the initiation of protein synthesis is arrested in order to release the messenger ribonucleoproteins from the polysomes. Their proteins are obtained like the informosomal proteins by mild RNAase digestion. No contamination by informosomes could be detected by sodium dodecyl sulfate gel electrophoresis. 4. Cytoplasmic "binding factor" proteins are purified by affinity chromatography. 5. The four sets of proteins are analysed by sodium dodecylsulfate acrylamide gel electrophoresis. In spite of the fact that some proteins from one or another kind of messenger ribonucleoprotein, have apparently the same molecular weight, the majority of proteins differ.


Physical aspects and cytoplasmic distribution of messenger RNA in mouse kidney. As a prerequisite to examining mRNA metabolism in compensatory renal hypertrophy, polyadenylated RNA has been purified from normal mouse kidney polysomal RNA by selection on oligo(dT)-cellulose. Poly(A)-containing RNA dissociated from polysomes by treatment with 10 mM EDTA and sedimented heterogeneously in dodecyl sulfate-containing sucrose density gradients with a mean sedimentation coefficient of 20 S. Poly(A) derived from this RNA migrated at the rate of 6-7 S RNA in dodecyl sulfate-containing 10% polyacrylamide gels. Coelectrophoresis of poly(A) labeled for 90 min with poly(A) labeled for 24 h indicated the long-term labeled poly(A) migrated faster than pulse-labeled material. Twenty percent of the cytoplasmic poly(A)-containing mRNA was not associated with the polysomes, but sedimented in the 40-80 S region (post-polysomal). Messenger RNA from the post-polysomal region had sedimentation properties similar to those of mRNA prepared from polysomes indicating post-polysomal mRNA was not degraded polysomal mRNA. Preliminary labeling experiments indicated a rapid equilibration of radioactivity between the polysomal and post-polysomal mRNA populations, suggesting the post-polysomal mRNA may consist of mRNA in transit to the polysomes.


Effects of irradiation on RNA synthesis primed by calf thymus deoxyribonucleoprotein treated with salt and salt-urea. 1. The effects of ionizing radiation on the activity of calf thymus templates were examined in a Escherichia coli RNA polymerase system. 2. The template activity of native and 2 M NaCl-5M urea-treated deoxyribonucleoproteins was enhanced by relatively low doses of irradiation, while that of 2 M NaCl-treated deoxyribonucleoprotein was not enhanced by irradiation. 3. The template activity of purified DNA was markedly decreased by irradiation, while that of native deoxyribonucleoprotein, 2 M NaCl-treated, and 2 M NaCl-5 M urea-treated ones were slightly decreased at a higher dose range. The doses for 50% inactivation of these templates were 1.3, 210, 140, and approximately 200 krad, respectively.


Processing of ribosomal RNA in a temperature sensitive mutant of BHK cells. The processing of ribosomal RNA has been studied in a temperature sensitive mutant of the Syrian hamster cell line BHK 21. At 39 degrees C, these cells are unable to synthesize 28S RNA, and 60S ribosomal subunits, while 18S RNA, and 40S subunits are produced at both temperatures. At 39 degrees C the 45S RNA precursor is transcribed and processed as in wild type cells. The processing of the RNA precursors becomes defective after the cleavage of the 41S RNA, and the separation of the 18S and 28S RNAs sequences in two different RNA molecules. The 36S RNA precursor, which is always present in very small quantity in the nucleoli of wild type cells and of the mutant at 33 degrees C, is found in very large amounts in the mutant at 39 degrees C. The 36S RNA can be, however, slowly processed to 32S RNA. The 32S RNA cannot be processed at 39 degrees C, and it is degraded soon after its formation. Only a small proportion accumulates in the nucleoli. The 32S RNA synthesized at 39 degrees C cannot be processed to 28S RNA upon shift to the permissive temperature, even when the processing of the newly synthesized rRNA has returned to normal. The data suggest that the 36S and 32S RNAs are contained in aberrant ribonucleoprotein particles, leading to a defective processing of the particles as a whole.


T4-endonuclease V-sensitive sites in DNA from ultraviolet-irradiated human cells. Human diploid cells (WI38) were pre-labeled with 32Pi, exposed to ultraviolet irradiation and then pulse labeled with [3H]thymidine. The extracted DNA from these cells was subsequently treated with the T4-endonuclease V, an enzyme which specifically nicks DNA strands at positions adjacent to pyrimidine dimers. Sedimentation in alkaline sucrose gradients revealed that the DNA synthesized after irradiation, as well as that made before, contained endonuclease-sensitive sites. Our results suggest that pyrimidine dimers are transferred from parental to daughter DNA strands during post-irradiation incubation. Sedimentation in neutral sucrose gradients showed that the molecular weight of native DNA was not affected by the endonuclease treatment, suggesting that the gaps appearing in daughter strands after irradiation are not opposite dimers or that the enzyme cannot recognize dimers in the gap regions.


DNA polymerase, thymidine kinase and DNA synthesis in erythropoietic mouse spleen cells separated on bovine serum albumin gradients. A single dose of erythropoietin stimulates DNA synthesis in the spleen of the polycythemic mouse with the maximum effect occurring 48 h after the hormone is administered. The increase in DNA synthesis is accompanied by morphologic evidence of increased erythropoiesis and by increases in the activities per cell of both thymidine kinase and cytoplasmic high molecular weight DNA polymerase-alpha. The activity of low molecular weight DNA polymerase-beta does not change significantly. Spleen cells from mice which had received either erythropoietin or saline 48 h previously were separated into 7 density classes on discontinuous bovine serum albumin gradients. Following the administration of erythropoietin, thymidine incorporation and thymidine kinase activity showed the greatest relative increases per nucleated cell in layers 3, 4 and 5 of the gradient. DNA polymerase-alpha showed the greatest increase in cells of the denser layers 5, 6 and 7. Each layer contained normoblasts and lymphocytes. The less well differentiated erythroid elements constituted a larger proportion of cells in layers of lower density. Increases in the rates of thymidine incorporation were better correlated with increases in thymidine kinase activity than with increases in DNA polymerase activities. Measurement of iron incorporation into heme confirm the morphological impression that the cell type responsible for increased thymidine incorporation and increased DNA polymerase-alpha activity is the young normblast.