Effect of C. Parvum on immunization with irradiated tumour cells. S.c. injection of tumour cells or small pieces of tumour irradiated to a dose of 22,000 rad evoked resistance to live challenge with the same tumour (a CBA strain fibrosarcoma induced with methylcholanthrene) 14 days later. This resistance was, however, over-ridden if the challenging inoculum was sufficiently large, and did not develop if the cells were irradiated to 100,000 rad. The resistance evoked by injection of 10(6) irradiated tumour cells was impaired by i.p. injection of 1-4 mg C. parvum 5 days before, and virtually abolished by a similar injection 11 days after, the irradiated cells. The effect of s.c. injection of a mixture of 10(6) irradiated cells and C. parvum 14 days before live challenge depended on the dose of C. parvum. With 0-7 mg the development of resistance was largely but not completely abrogated; 0-35 mg resulted in a lesser degree of abrogation, and 0-09 mg or 0-02 mg had little or no effect.


Changes in the structuredness of cytoplasmic matrix (SCM) in human lymphocytes induced by PHA and cancer basic protein as measured in single cells. The method of measuring changes in the structuredness of cytoplasmic matrix (SCM) in single cells is described. Data on SCM distributions and fractions of human lymphocyte populations which respond to stimulations with PHA and CaBP in healthy donors and patients with malignant disorders are presented.


Incidence of oral leucoplakias among 20,358 Indian villagers in a 7-year period. A group of 20,358 villagers in two districts of India has been followed for 7 years to study the incidence of oral leucoplakia. The follow-up rate of the population in two districts ranged from 61% to 71%. In one of the districts (Bhavnagar) no new cases of leucoplakia were found among females in the 7-year period. Among males 105 cases developed (4-0/1000/year). The incidence was highest among hookli (clay pipe) smokers. In the Ernakulam district the incidence among males was 3-3/1000/year whereas among females it was 1-9/1000/year. The mixed habits group had the highest incidence of oral leucoplakias (7-2 and 9-9/1000/year among males and females respectively).


Childhood Kaposi's sarcoma: clinical features and therapy. Twelve cases of childhood Kaposi's sarcoma seen at the Uganda Cancer Institute over the last 7 years are reported. The disease presents mainly as generalized lymphadenopathy, with sparsely and anomalously distributed cutaneous nodules, and has a higher proportion of females with the disease than in the adult form. The histology is usually of mixed cell type. If not treated, childhood Kaposi's sarcoma runs a fulminating course, but disease control with chemotherapy is associated with prolonged survival.


Disseminated intravascular coagulation caused by the carpet viper (Echis carinatus): trial of heparin. Heparin has been advocated for the treatment of poisoning by Echis carinatus, a snake whose venom causes disseminated intravascular coagulation. Fourteen patients with proven E. carinatus bite who had incoagulable blood were treated with specific Echis antivenom. Seven of them were also given low-dose heparin, initially 50 units/kg body weight by i.v. injection, followed by 10 units/kg/h by i.v. infusion for 22 h. Response to treatment was assessed clinically and by repeated tests of blood coagulation. All patients showed a rapid return to normal blood coagulability after treatment and the heparinized group were not significantly different in any respect from the group given antivenom alone. Heparin did not reduce the local effects of envenoming. There appears to be no place for heparin in the treatment of E. carinatus poisoning provided that potent antivenom is available. The in vivo results were supported by in vitro studies in which it was found that Echis-induced thrombin was less sensitive to the inhibitory effect of heparin than physiological thrombin.


Haemolytic anaemia due to glucose-6-phosphate dehydrogenase (G6PD) deficiency: demonstration of two new biochemical variants, G6PD Hamm and G6PD Tarsus. Two new variants of erythrocytic glucose-6-phosphate dehydrogenase have been found in one German patient and in another patient of Turkish origin. The enzymes were partially purified 165-fold and 111-fold respectively. Both revealed reduced activity, increased thermolability and a pH-optimum in the alkaline region (8.5 and 9.0). One variant (G6PD Hamm) had a low Km-value for glucose-6-phosphate, the other (G6PD Tarsus) exhibited an increased affinity for glucose-6-phosphate and a reduced affinity for NADP+. This enzyme showed an increased inhibitor constant for NADPH with respect to NADP+. Electrophoretic mobility was normal in both cases. 2-Desoxy glucose-6-phosphate was utilized to an increased rate by both variant enzymes (46% and 33%). Also galactose-6-phosphate (29% and 25%) and deamino-NADP+ (230% and 261%) gave increased utilization rates. The mothers of both patients could be identified as heterozygous for this enzyme deficiency.


The role of disulphide bonds in Heinz body attachment to membranes. The mechanism of binding of oxidant-induced Heinz bodies to red cell membrane was studied. Heinz bodies induced by acetylphenylhydrazine in intact cells or in the presence of membrane remained attached to membrane when separated by a sucrose gradient. Incubation with sulphydryl reagents failed to free intact Heinz bodies from or prevent attachment to membranes, although the amount of haem was reduced. Thus disulphide bonds do not appear to be a major mechanism of attachment of Heinz bodies to red cell membrane.


The effects of acute bleeding on acid-base balance erythropoietin (Ep) production and in vivo P50 in the rat. The mechanism of erythropoietin (Ep) production after acute haemorrhage has been thought to be due to a reduction in blood volume and tissue perfusion leading to tissue hypoxia. In the present study we have evaluated the effect of acute haemorrhage in the rat on the acid-base status, the red cell affinity for oxygen in vivo, and Ep production. Within a few hours after acute blood loss there was a respiratory alkalosis with an increase in blood pH, a decrease in pCO2 and an increase in the red cell affinity of Hb for oxygen in vivo that was temporally related to an increase in Ep production. Within 24 h after the acute haemorrhage, the blood pH AND PCO2, red cell affinity for oxygen in vivo, and Ep level returned towards normal. The decrease in in vivo red cell affinity for oxygen was associated with an increase in red cell 2,3-DPG levels and a decrease in Ep production.


Haemopoietic progenitor cells in prenatal congenitally anaemic 'flexed-tailed' (f/f) mice. The incidences of erythroid colony forming cells (CFUe) and granulocyte-macrophage colony forming cells (CFUc) have been measured in 11-18 d prenatal livers of mice of genotype f/f and nearly congenic +/+ controls. In normal fetal livers numbers of CFUe (cells able to form colonies of 16 or more cells after 72 h in vitro) rise to a maximum on day 14 of gestation and represent c 1% of total fetal liver cells. In f/f fetal livers, peak values for numbers and proportions of CFUe are 50% of normal. The f/f lesion does not reduce the numbers of CFUc in fetal liver. Since this deficiency in CFUe parallels deficiencies of similar magnitude in spleen-colony forming units (CFUs) and erythroblasts in the liver, and erythrocytes in the blood, of f/f fetuses it is concluded that the f/f lesion is expressed at an early stage of haemopoietic development in prenatal life. The possibility that restricted haem synthesis is the primary effect of the f/f genotype and responsible for disturbances of both haemopoietic cellular proliferation and haemoglobin synthesis is examined.


Control of haemopoietic stem cell proliferation by cells in DNA synthesis. It has been demonstrated that for the slowly proliferating population of the haemopoietic pluripotent stem cells (CFUs) the response after damage by hydroxyurea to cells synthesizing DNA differs from that after damage by colchicine to cells entering mitosis. The killing of DNA synthesizing cells elicits increased proliferation in the CFUs population. From this it has been inferred that cells in the S phase may inhibit the entry of nonproliferating GO cells into the cell cycle and in this way control cellular proliferation.


In vivo plasma and urine folate binding after ingestation of 3H-folic acid and 14C-methyl-folate. After simultaneous ingestion of equivalent amounts of [3H]folic acid (3H-PteGlu) and [14C]N5-methyl-tetrahydrofolic acid (14C-CH3-H4PteGlu) we were able to demonstrate progressive macromolecular binding of radiofolate in plasma, which appeared to be near maximal at 6 h. Bound radiofolate was predominantly of 14C-CH3H4PteGlu origin, and only at 24 h could 3H incorporation be demonstrated. The binder eluted with albumin from Sephadex DEAE-A50 columns. In urine a smaller bound radiofolate fraction, with approximately equal amounts of 3H and 14C, appeared after 5.5 h. Plasma chromatography showed radio-PteGlu (peak 1) to be rapidly converted to CH3-H4PteGlu (peak 2), with subsequent appearance of two further radiofolate peaks (peaks 3 and 4) the nature of which is as yet unclear. Urine showed similarly placed fractions but their magnitude differed, and urinary peak 3 in particular was much more prominent than its plasma counterpart.


Patterns of food iron absorption in iron-deficient white and indian subjects and in venesected haemochromatotic patients. The absorption of radioactive iron from a solution of ferrous ascorbate, and from a standard meal containing intrinsically labelled haemoglobin and wheat, was measured in 12 Indian housewives, 18 white hospital patients and 12 subjects with idiopathic haemochromatosis. Eight of the latter had been fully treated by multiple venesections, so that their serum ferritin concentrations were below 25 mug/1. Since the serum ferritin concentrations of the housewives and the hospital patients were comparable, their body iron stores were considered to be depleted to a similar degree. There were no significant differences between the absorptions of ferrous ascorbate or of the haem iron in the standard meal by each group, but the housewives and the hospital patients absorbed significantly less of the non-haem food iron. The mean non-haem food iron absorptions were 36.4%, 5.8% and 18.9% for the treated haemochromatotic subjects, the Indian housewives and the white hospital patients respectively. The discrepancies between the absorptions of the different forms of food iron were highlighted by calculating the ratios between them. The mean non-haem: haem food iron absorption ratio for the group of treated haemochromatotic subjects was 0.98, and for the Indian housewives only 0.18. The white hospital patients did not form a homogenous population: the ratios of the five males and three of the females were greater than 1.0, whereas those of the remaining 10 females were less than 0.5. The results of this study suggest that mal-absorption of non-haem iron from a meal containing bread, presumably due to a defect at the luminal level, may be an important factor in the pathogenesis of iron deficiency in some subjects. The abnormality appears to be particularly prevalent among Indian women living in Durban.


The abundance of ferritin in yolk-sac derived red blood cells of the embryonic mouse. Circulating red blood cells formed early in development have several distinctive properties which include retention of the nucleus (mammals), large size and characteristic haemoglobin type (mammals, birds, amphibia). The primitive or embryonic red cells of early development are replaced by the definitive red cells which contain fetal or adult haemoglobin; a second developmental change occurs in the haemoglobin of some mammals (man, cattle, sheep) but does not involve a cell replacement. Circulating yolk-sac derived red cells from embryonic mice are siderocytes; elevated ferritin levels are associated with the circulating red cells of bullfrog tadpoles, but not with those of the adult frog, again indicating that red cell iron metabolism can change during development. In order to extend the observations made on an amphibian to a mammal, the ferritin content of circulating red cells from embryonic mice was determined and found to be 0.65 mg/100 mg of soluble protein; no ferritin (less than or equal to 0.007 mg/100 mg of soluble protein) was detected in adult mouse red cells. Elevated ferritin levels appeared to be specifically associated with the yolk-sac derived population of red cells since a decline in red-cell ferritin content coincided with the replacement of yolk-sac derived red cells by definitive red cells derived from the liver. Fractionation of mixtures of yolk-sac derived and liver derived red cells showed that fractions rich in the definitive red cells contained less ferritin than the mixture. The results suggest that elevated ferritin levels may be a general characteristic of the circulating, haemoglobinized red cells formed early in development.


Absorption of cumene through the respiratory tract and excretion of dimethylphenylcarbinol in urine. Experiments on the absorption of cumene and the excretion of dimethylphenylcarbinol were made on 10 healthy volunteers, five men and five women aged between 20 and 35 years. They were exposed to cumene vapours 240, 480, 720 mg/m3 under controlled conditions. It was found that the average retention of cumene vapours was about 50% which tended to diminish at the end of each exposure. Based on these results, an exposure test that allows one to calculate the absorbed cumene dose during eight hours' work with a precision of about +/- 13.5% was achieved.


Ostwald solubility coefficients of some industrially important substances. Solubility coefficients in blood for benzene, toluene, and xylene were determined as 6.5, 16, and 42 respectively. In lard and olive oil, which were taken to represent human fat, corresponding values were about 450, 1300, and 3900. The coefficient for vinyl chloride in lard and olive oil was 20; the value in blood was too low to be determined by the technique used. Trichloroethylene, used as a marker, was found to have a coefficient in lard of 660, and in olive oil of 710, rather lower values than have been accepted for fat.


A proportional study of cancer registrations of dockyard workers. Evidence of an occupational effect due to asbestos exposure was sought by comparing cancer registrations for dockyard workers with those for Plymouth men in the same age groups for 1960-69. The results show a significant excess of pleural tumours in the dockyard group but no significant excesses at other sites. Registrations for pleural mesothelioma were recorded for men with many dockyard occupations and the observed mean interval between first exposure to asbestos and registration for a pleural tumour (37.6 years) indicates that cases of pleural mesothelioma may be expected to occur among these workers for many years, even though crocidolite is no longer used in naval ships. A trend of increasing annual excess of stomach and gastrointestinal registrations was observed in the dockyard group. No cases of peritoneal mesothelioma were recorded but it is likely that some did occur which were diagnosed as cancers arising from other sites. This may account for some of the excess of gastrointestinal registrations.


Thirty-two cases of mesothelioma in Victoria, Australia: a retrospective survey related to occupational asbestos exposure. Mesotheliomas have been reported in four states in Australia. Crocidolite has been mined and milled at Wittenoom in West Australia where five cases of mesothelioma were reported after exposure of high intensity. The 32 cases of mesothelioma reported in this paper occurred during a period of 11 years in Victoria; 29 were pleural and three peritoneal. There were 22 autopsies. End occupations were misleading in 66% of cases. Two of the three subjects with peritoneal mesothelioma were siblings, and there was no evidence of occupational or other exposure to asbestos in either. There was a significant prevalence of pulmonary asbestos bodies in the tumour series as compared with an unselected consecutive series of 200 routine autopsies (0.01 greater than P greater than 0.001). The occupational history was as effective a method of assessing 'true' asbestos exposure as the pulmonary asbestos body count. Five cases had had a duration of exposure of one year or less, but they had had heavy exposure. The latent interval before tumour development was 25 years or longer in each case. There was no known exposure to asbestos in five cases (16%). The rare association of mesothelioma with types of asbestos other than crocikolite may not exist and could be explicable on the basis of the proportion (16%) of these tumours arising randomly in the population.


The in vitro effect of zinc on the inhibition of human delta-aminolevulinic acid dehydratase by lead. The antagonistic effect of zinc on inhibibition of human aminolevulinic acid dehydratase (ALAD) by lead was examined in vitro.  The phenomenon was studied at pH 6.5-7.5 .  Zinc and lead were added at concentrations ranging from physiological levels to levels far in excess of those expected in heavy industrial exposure.  ALAD activity of normal blood assayed in the presence of zinc was unaffected by exogenous lead if the added zinc concentration was above 0.53 mmol/l blood, or double the normal endogenous level, and was of the order found in heavy industrial exposure to zinc.  In this case, ALAD assay values were appreciably raised and might have fallen into the normal range in spite of a dangerous total blood lead of over 0.0048 mmol/l.  When zinc was added in vitro to blood from a worker with a blood lead level of 0.0043 mmol/l the ALAD values obtained were also raised and did not reflect the dangerous level of lead in the patient.


Different behaviour of inorganic and organic mercury in renal excretion with reference to effects of D-penicillamine. Inorganic and organic mercury was measured in the red cells, plasma, and urine of five laboratory workers to examine the effect of D-penicillamine. The workers had had no particular exposure to mercurials. Before and during administration of D-penicillamine, inorganic mercury levels in plasma were significantly correlated with those in urine or 24-hour urinary outputs, but no significant correlation was found for organic mercury. Administration of D-penicillamine enhanced 24-hour urinary output to a much greater extent in organic than inorganic mercury. All the organic mercury levels in blood, red cells, and plasma were increased by D-penicillamine. This contrasted with the decrease of inorganic mercury levels in plasma.


Shoe-makers' polyneuropathy in Italy: the aetiological problem. Since 1957, when the first cases of the so-called shoe-makers' toxic polyneuropathy were reported, nearly 400 cases have been described in the Italian literature. The substance that was considered to be responsible for the disease was triorthocresylphosphate (TOCP) contained in glues, artificial leathers, and some types of paints. However numerous chemical analyses of glues and leathers taken from factories where cases of the disease occurred have shown that in almost all instances little or no TOCP was present. In addition the disease manifests itself more frequently during winter and spring and this is not consistent with the absorption of the causative agent through the skin and/or gastrointestinal tract. In order to clarify the aetiology of the disease 122 workers in the shoe industry affected by toxic polymeuropathy during the period 1971-74 were studied. Workplaces where cases of polyneuropathy had occurred were visited and samples of glues and solvents were taken for chemical analysis. The polyneuropathy chiefly affects workers engaged in glueing and in the cleaning process, but it also affects those who do not have direct contact with glues or solvents. The disease is more common in women (68%) than in men (32%) and, in its more severe form begins with overwhelming prevalence during winter and early spring regardless of the job performed. No direct relationship has been found between the severity of the disease and the number of years of work in the shoe industry or of specific exposure (that is the number of years of work in the activity performed at the onset of the disease). The polyneuropathy is found almost exclusively among workers of the artisam type in small shoe factories, where standards of hygiene are low. Chemical analysis of glues and cleaning fluids collected from five different factories, where 20 cases of polyneuropathy occurred, showed the presence of paraffin hydrocarbons with a low boiling point (pentane, 2-methyl-pentane, 3-methyl-pentane, n-hexane, etc.) in concentrations of more than 80% by weight.


The ultrasonic measurement of fetal crown-rump length as a method of assessing gestational age. The fetal crown-rump length (CRL) was measured by means of pulsed ultrasound, and the normal values between 47 and 101 days from the onset of the last menstrual period were determined from cross-sectional data in 253 patients. For any given CRL, the range was found to be within three days of the menstrual age with a maximum variation for a given occasion of three days. Detailed statistical analysis showed that the acceleration in the rate of change in the CRL was a constant and that the intrapatient variability, where measured, was low. A close correlation between the measurement and the date of ovulation was demonstrated in a further 40 patients.


Urinary oestriol measurement in the management of antepartum haemorrhage. In 51 patients with antepartum haemorrhage, 24-hour urinary oestriol was measured continuously until delivery. A total of 765 24-hour urinary oestriol measurements was obtained and there were two stillbirths, one neonatal death and three cases of intrauterine growth retardation in this group of patients. It was felt that none of the fetal deaths would have been prevented by obstetrical treatment based on 24-hour urinary oestriol measurements and that these have little practical value in the management of patients with antepartum haemorrhage.


Plasma oestriol in normal pregnancy in an Asian population. Serial plasma oestriol levels were measured by a radioimmunoassay in 49 normal pregnancies, Plasma oestriol rose from 43 ng/ml (149-1 nmol/l) at 22 weeks to 357 ng/ml (1237-91 nmol/l) at 40 weeks. The rise was gradual between 22 and 32 weeks and steep between 32 and 40 weeks. No significant relationship could be found between plasma oestriol and parity, maternal age, maternal height, maternal weight or fetal sex. However, a significant but low correlation was found between birth weight and plasma oestriol prior to labour and also oestriol increments from 30 weeks to delivery. The average day to day coefficient of variation of plasma oestriol in 11 patients was found to be 16-2 per cent.


Lung function tests in bronchial asthma during and after pregnancy. Respiratory function tests (forced vital capacity and forced expiratory volume in one second) were performed serially in pregnancy, the puerperium, and at least six weeks post partum in 27 patients with a history of asthma and in 12 control subjects. Eleven asthmatic patients with spirometric evidence of respiratory obstruction in pregnancy on at least one occasion were considered to have active disease. This group had significantly impaired respiratory function tests when compared to controls. The remaining 16 asthmatic patients were considered to be in remission. No changes in respiratory function tests attributable to pregnancy could be demonstrated in the asthma or control groups. Patients with active asthma tended to have smaller babies than patients with asthma in remission or the control patients.


The response of lymphocytes to phytohaemagglutinin in women with pre-eclampsia. The response of maternal lymphocytes to phytohaemagglutinin was determined during and after pregnancy in patients with severe pre-eclampsia and in controls. The patients with pre-eclampsia showed a greater degree of immunosuppression during pregnancy than the normotensive controls. This was apparent in the presence and absence of maternal serum. In pre-eclampsia there may be an abnormality affecting the T cell subpopulation of lymphocytes and also a decreased immunosuppressive effect of maternal serum.


The use of the amniotic fluid lecithin/sphingomyelin (L/S) ratio, creatinine concentration and nile blue sulphate tests, individually and in combination, in the assessment of fetal lung maturity. The lecithin-sphingomyelin (LS) ratio is a good, but not absolute, test of fetal lung maturity. A total of 373 infants was delivered within 72 hours of amniotic fluid collection for assessment of the L/S ratio. Five of 43 infants (11-6 per cent) with an L/S ratio of 2-2 or less developed the respiratory distress syndrome (RDS), whereas only one of 320 infants (0-31 per cent) with a higher L/S ratio developed RDS. The risk of RDS could be further defined by using the creatinine concentration (critical value 19 mg/1) and the Nile blue sulphate test (critical value 4 per cent) in combination with the L/S ratio. All three tests were performed in 320 pregnancies and in each instance delivery took place within 72 hours. When all three test results were low, 3 of 5 infants (60 per cent) developed RDS, whereas when all three results were high, none of 175 infants developed RDS. When one or two results were low, 2 of 140 infants (1-4 per cent) developed RDS.


The lecithin/sphingomyelin (L/S) ratio in twin pregnancies. The lecithin/sphingomyelin (L/S) ratio has been estimated in the amniotic fluid from 20 patients with a twin pregnancy. The values in each pair of amniotic fluids were closely related. If amniotic fluid from one sac only is available, it is suggested that a ratio of 2-5:1 is taken as the lower limit to predict the functional lung maturity of both twins.


The effect of membrane rupture on fetal heart rate in induced labour. The influence of the state of the membranes on the fetal heart rate was studied in a group of twelve women whose membranes were electively ruptured for oxytocin induction of labour. The results have been compared with a group of eight women of similar gestation and parity whose membranes were kept intact. While membranes were ruptured, there was an increase in the frequency and amplitude of early decelerations thorughout labour. The significance and practical implications of the results are discussed.


Changes in the oral glucose tolerance test during the puerperium. Oral glucose tolerance tests were performed during late pregnancy, on the second and fifth days of the puerperium and finally six weeks after delivery in a group of 12 healthy women. The glucose response showed no significant change from the pregnancy values on the second and fifth days of the puerperium but each woman had returned to 'normal' by the sixth week post partum. In contrast, the insulin response had returned to the non-pregnant value by the second day of the puerperium.


Monoamine oxidase activity in the fetal lung and liver. Monoamine oxidase (MAO) in lung and liver is important in the degradation of circulating 5-hydroxytryptamine. These sites of MAO activity have been investigated histochemically in the human fetus of 12 to 18 weeks gestation. Enzyme activity could be demonstrated in the liver by both tryptamine and adrenaline oxidation. In the lung, MAO activity was present only when adrenaline was used to substrate. It may be, therefore, that in the premature baby the capacity of MAO to metabolize 5-hydroxytryptamine is not fully developed, which could lead to deleterious effects on pulmonary function.


Scanning electron microscope appearances of normal human amnion and umbilical cord at term. The surface morphology of normal term amnion was studied by scanning electron microscopy. Four patterns were observed. Two of the patterns showed distinctive minority populations of cells. In one, there were cells surrounded by intracellular channels; in the other, there were large flat cells. The microvilli in all four patterns showed variable morphology in different samples. Possible functional implications of the findings are discussed.


Intramuscular administration of 15-methyl-prostaglandin F(2alpha) for induction of labour in patients with intrauterine fetal death or an anencephalic fetus. The 15-methyl analogue of PGF2alpha was administered intramuscularly between 23 and 36 weeks of gestation, to induce labour in 11 mothers with intrauterine fetal death and two with anencephaly. All were delivered within 20 hours (mean 7 hours). The mean dose of 15-me-PGF2alpha was 400 mug (range 100 to 700) given by a mean of 3-1 (range 1 to 7) injections. Diarrhoea was the most frequent side-effect.


Avulsion injury of uterovaginal prolapse and bladder. A 40-year-old postmenopausal female with complete uterine prolapse received an avulsion injury when her sari was caught in the fast-moving belt of a flour mill machine. Part of the prolapsed anterior vaginal wall with underlying bladder wall was torn off. Primary repair together with vaginal hysterectomy led to an uneventful recovery.


Effects of fast neutrons on the eye.

Effects of fast neutrons on the eye. The study of 93 eyes receiving neutron irradiation establishes the character of the ophthalmic complications of neutron. Significant changes are produced in the eyelids, conjunctiva, and cornea, but the production of cataract is relatively unimportant. No reaction was seen after neutron treatment that had not already been seen after chi or gamma radiation. That the unavoidable damage is similar to that reported with other forms of radiation, and not worse, is an important finding in view of the statistically significant benefit of neutron treatment to patients in a controlled clinical trial of advanced tumours of the head and neck (Catterall and others, 1975). The observations indicate that the improved cure rates with neutrons are not obtained at the expense of inflicting greater damage to normal structures. This investigation has also shown that the neutron effects of treating these very extensive tumours are acceptable and that, with carefully measured doses, they are predictable. The dose-response relationship in categories of clinical effect for a smaller series of patients, reported in Table 1 of our preliminary communication (Brown and other, 1976), is confirmed.


Severe endemic trachoma in Tunisia.

Severe endemic trachoma in Tunisia. In two villages in southern Tunisia where trachoma was endemic 7 per cent and 14 per cent of adults respectively had visual acuity of 20/400 or less. In both villages active trachoma affected most children under the age of two, reached a peak in two- to five-year-olds, then declined to age 15. The chronic inflammatory disease in childhood appeared to produce irreversible scarring of the eyelids, and loss of vision occurred in adult life due to corneal scarring caused by inturned eye lashes and loss of tears (dry-eyed syndrome). Economic development in one village was associated with a decline in active, infectious disease. In the second village, whose traditional economy was unchanged, there was the same prevalence of active disease over a three-year period. Unless economic development or public health control programmes reduce the prevalence of severe and moderate trachoma children now affected will develop the same blinding lesions as their parents. With the increasing numbers of children who survive there will probably be a dramatic increase in the numbers of the blind from trachoma in 10 to 20 years. Since active inflammatory trachoma in childhood responds to tetracyclines, erythromycin, and sulphonamides the disease should be attacked in those undeveloped rural areas where it continues to lead to blindness.


Simple method of determining the axial length of the eye. By photographing the corneal reflex in two positions of gaze and measuring the radius of curvature of the cornea it is possible to calculate the radius of rotation of the eye. The measurements obtained in this way showed a high correlation with refraction in a series of 80 eyes. The axial length obtained by this method was about 3 mm greater than that obtained by ultrasonographic or x-ray methods, and the reasons for the discrepancy are discussed.


Early and late results of fascia lata transplantation in high myopia. Transplantation of a strip of fascia lata to strengthen the posterior sclera in high progressive myopia was performed on 184 eyes of 108 patients with myopia ranging from 7 to 39 D. Three weeks after surgery visual acuity with correction had improved by 0-1 to 0-5 in 151 eyes (82 per cent) and refractive power had decreased by 1 to 8 D in 169 eyes (92 per cent). In 105 eyes (67 patients) that were followed-up for periods of from 1 to 9 years myopia increased in six by 0-5 to 3 D. In 29 patients who had an operation on only one eye and were then followed-up for periods of from 1 to 7 years the degree of myopia in the fellow eye increased by 1-5 to 6 D in 24 (83 per cent), and of the eyes that were operated on myopia increased by 0-5 to 3 D in only 5 (17 per cent).


Visually evoked cortical potentials in the evaluation of homonymous and bitemporal visual field defects. Visually evoked cortical potentials were studied in six patients with a homonymous and six with a bitemporal hemianopia by presenting a pattern-reversal stimulus separately to a temporal or nasal retinal area and by recording the responses from leads over the hemispheres. Homonymous visual field defects are characterized by a reduction of VECPs from the affected hemisphere. The disturbance of VECPs in bitemporal hemianopia is more serious, since the fibres from both retinal halves may be damaged by a chiasm tumour.


Senile cataract extraction and diabetes. A study of hospital discharge diagnoses from both national data and data from a local medical centre indicates that diabetes substantially increases the probability of cataract extraction at age 40-49, about doubles or triples the probability for age 50-69, and has little effect on risk at age 70 and over. Strengths and weaknesses of the data are discussed. Other reports, generally estimating a much stronger association between diabetes and probability of cataract extraction, at least at age 50 and above, are critically evaluated.


Pigmented lens striae.

Pigmented lens striae. The clinical features of pigmented lens striae were studied biomicroscopically in seven patients. The lenses of three of these, obtained by cataract surgery, were examined by electron microscopy to determine the structure of the striae. We concluded that the striae are not formed by remnants of tunica vasculosa lentis, as earlier workers believed, but consist of fine fibrils of zonular bundles entrapping pigment granules of uveal origin.


Immunoglobulins in endogenous uveitis.

Immunoglobulins in endogenous uveitis. Examination of 300 patients suffering from endogenous uveitis revealed raised levels of IgG in 24 per cent, IgA in 17 per cent, and IgM in 10 per cent. A raised level of one or more immunoglobulins was found more frequently in anterior uveitis than in the posterior form (44 compared with 24 per cent), in bilateral cases (47 compared with 35 per cent), and in relation to certain general diseases. No differences were demonstrated within the individual types of uveitis (fibrinous, nodular, etc.), nor was there any difference in prognosis (recurrence rate, visual impairment). Immunoglobulin levels were raised most often in elderly patients.


Immunoglobulins in tears of normal Indian people. Immunoglobulin concentrations in tears from 50 healthy Indians aged from 14 to 50 years were measured by a standard immunodiffusion method. The levels of IgA were substantial; those of IgG were very low; and IgD and IgM were not present. The mean IgA level was 24-6 mg/100 ml.


Interactions between arginine-rich histones and deoxyribonucleic acids. I. Thermal denaturation. Physical properties of histone-DNA complexes very often depend upon the method of complex formation. In an attempt to make the studies of histone-DNA interactions more relevant to biological systems, results from thermal denaturation of native chromatin were used as references for determining how closely a given histone-DNA complex approaches its native state in chromatin. In the case of arginine-rich histones H3 (III or f3) and H4 (IV or f2a1), four methods were used for making complexes with calf thymus DNA: (A) NaCl gradient dialysis with urea; (B) NaCl gradient dialysis without urea; (C) direct mixing in 2.5 x 10(-4) EDTA, pH 8.0; and (D) direct mixing in 0.01 M sodium phosphate, pH 7.0. It was observed that a complex made by direct mixing in phosphate (method D) is closer to the native than is one made by direct mixing in EDTA (method C) than the one made by gradient dialysis with urea (method A) or without urea (method B). Regardless of the method used for complex formation, no substantial differences were observed between complexes with histone H3 dimer with disulfide bond(s) and a reduced H3 without disulfide bond, implying that perhaps a dimer with or without disulfide bond is a natural fundamental subunit in our experimental conditions. When the method of direct mixing in EDTA is used, the melting properties of the complexes vary only slightly with any one of the following H3 histones: from calf thymus, H3 without disulfide bond, H3 dimer, and H3 oligomer with disulfide bonds, also, from duck erythrocyte, H3 monomer and dimer. The complexes formed between DNA and a mixture of H3 and H4 by method D have melting properties similar to those of native chromatin. Since an equimolar mixture of histone H3 and H4 in 0.01 M phosphate, pH 7.0, was shown to form a tetramer (D'Anna, J.A., and Isenberg, I. (1974), Biochem. Biophys. Res. Commun. 61, 343), our results suggest that, a tetramer of H3 and H4, likely to be (H3)2(H4)2, formed from one H3 dimer and one H4 dimer, can bind DNA in a manner similar to that in native chromatin.


Modification of histone binding in calf thymus chromatin and in the chromatin-protamine complex by acetic anhydride. A relationship between side-chain modification of histones and their displaceability from DNA has been investigated using calf thymus chromatin which was chemically acetylated with acetic anhydride. When the chromatin is treated with increasingly higher concentrations of the reagent, histones become acetylated to an increasingly greater extent, attaining the modification at 23-24 sites for histone I, 5-6 for IIb1, 9-10 for IIb2, 5-6 for III and 3-4 for IV. As the chromatin becomes more acetylated, NaCl concentrations required for histone removal are lowered. Saturation binding of protamine does not bring about either an increase in the number of acetylation sites of histones in chromatin or a decrease of the NaCl requirement for dissociation of the acetylated chromatins. A comparison of the present results with the extents of histone acetylation known to occur enzymatically in vivo indicates that the complete removal of somatic histones during transformation of chromatin in spermiogenesis cannot be explained on the basis of decreased binding of the histone to DNA by acetylation or by a combination of acetylation and protamine binding, suggesting that the displacement process may require some additional processes.


Complete displacement of somatic histones during transformation of spermatid chromatin: a model experiment. Displacement of histones from calf thymus chromatin has been studied in an attempt to postulate the mechanisms involved in the total removal of somatic-type histones during transformation of spermatid chromatin. When chromatin is saturated with protamine (protamine/DNA, 0.5), histone I becomes displaceable at 0.15-0.3 M NaCl, suggesting that direct replacement by highly basic sperm histone could be a mechanism for its removal. While histone I is the only histone which is extensively degraded upon incubation of chromatin and, therefore, proteolysis might provide an additional mechanism for the removal of this histone, acetylation of chromatin by acetic anhydride greatly increases suscpetibility of histones IIb1, IIb2, and III to the chromosomally associated protease. These histones are extensively degraded and displaced from the DNA upon incubation of the acetylated chromatin. Although histone IV is not appreciably degraded, the proteolytic removal of acetylated histone III from chromatin weakens the interaction of acetylated histone IV to the DNA, and this histone becomes dissociable at 0.3 M NaCl. A comparison of the extent of chemical acetylation of individual histones observed in this investigation with that of enzymatic acetylation which can be achieved in vivo suggests that acetylation and proteolysis could be a mechanism for the removal of histone IIb2 and III. The displacement of histones IIb1 and IV could be explained on the basis of decreased binding to DNA as a result of their acetylation together with the proteolytic removal of their respective partner histones, IIb2 and III.


Secondary structure of ovalbumin messenger RNA. The secondary structure of highly purified ovalbumin mRNA was studied by automated thermal denaturation techniques and the data were subjected to computer processing. Comparative studies with 20 natural and synthetic model nucleic acids suggested that the secondary structure of ovalbumin mRNA possesses the following features: the extent of base pairing of ovalbumin mRNA is similar to that found in tRNAs or ribosomal RNAs; the secondary structure of ovalbumin mRNA is more thermolabile than any of the model compounds tested, including the copolymer poly(A-U); ovalbumin mRNA does not have extensive G-C rich stems as found in tRNAs or ribosomal RNAs; the base composition of the double-stranded regions reveals 54% G-C residues which was significantly higher than that noted in the whole molecule (approximately 41.5% G-C). The presence of 46% A-U pairs in short stems of about five base pairs would have a very large destabilizing effect on the secondary structure of ovalbumin mRNA. However, at 0.175 M monovalent cations and 36 degrees C most of the secondary structure of ovalbumin mRNA is preserved. These data suggest that the double-stranded regions in ovalbumin mRNA are of sufficient length to provide the necessary stability for maintaining the open loop regions in an appropriate conformation which may be required for the biological function of ovalbumin mRNA. Furthermore, the lability of the double-stranded regions in ovalbumin mRNA may also be important for the biological function of this mRNA.


Statistical interpretation of fluorescence energy transfer measurements in macromolecular systems. A statistical method is presented for the interpretation of intramolecular distance measurements by the fluorescence energy transfer technique in systems for which the detailed geometries of the donor-acceptor pairs are unknown. This method enables calculation of the probability that a specified distance range corresponds to the actual distance to be measured. It makes use of the numerically calculated probability density function for the distance of interest. The two general systems considered are the single donor-acceptor pair and the multi-donor-single-acceptor transfer. In both systems, the statistical method incorporates the uncertainty in the orientation of the donor and acceptor dipoles. In addition, it can take into account the rotational mobility of the donor dipoles determined by time-dependent emission anisotropy measurements. When more than one donor is involved in the transfer process, the uncertainties associated with the number and location of individual donors and the size and shape of the donor distribution are also incorporated in calculating the distance ranges. Application of the method was demonstrated for a wide range of transfer efficiency and Ro values for the single donor-acceptor system. Specific examples are also presented for interpretation of both single donor-acceptor and multi-donor-single-acceptor energy transfer measurements performed in order to reveal the spatial relationship of the sigma subunit and the rifampicin binding site in the Escherichia coli RNA polymerase (see Wu, C.-W., Yarbrough, L. R., Wu, F. Y.-H., and Hillel, Z. (1976), Biochemistry, preceding paper in this issue). Analysis of these energy transfer data by methods which use average values of the unknown geometrical parameters of the system yielded results similar to those obtained by the statistical method. However, the statistical method represents a more realistic approach to the interpretation of energy transfer measurements since it provides information concerning the entire range of possible distances and their relative likelihood.


Structure in the polar head region of phospholipid bilayers: A 31P [1H] nuclear Overhauser effect study. The structure of the head-group region of some phospholipid bilayers in vesicle form has been studied and an intermolecular association of the N-methyl protons of phosphatidylcholine (PC) with the phosphate of phosphatidylethanolamine (PE) in mixed vesicles has been identified. Observation of a 31P[1H] nuclear Overhauser effect (NOE) in the phosphorus nuclear magnetic resonances of both PC and PE in mixed vesicles demonstrates an intimate dipolar interaction between some protons and the phosphorus nuclei. Substitution of deuterium for the N-methyl protons of PC eliminated the majority of the effect and necessitated the construction of a model of the bilayer surface in which the N-methyl protons of PC could interact closely with the phosphates of neighboring PE molecules. The predominant orientation of the head group must then be parallel to the bilayer surface. The amino protons of PE do not contribute significantly to the observed NOE. A corollary of these results is that there is little if any tendency for either PC or PE in the mixed vesicles to segregate into separate domains. A decrease in NOE in sphingomyelin vesicles on going from H2O to D2O suggests that an exchangeable proton contributes to the NOE. In addition the low value of the NOE observed in D2O suggests that the head-group conformation of sphingomyelin differs from that of PC.


C55-Dolichol: occurrence in pig liver and preparation by hydrogenation of plant undecaprenol. Apart from the long-chain dolichols (C80-C110), pig liver contains also a family of much shorter polyprenols with dominating C55-polyprenol. This compound was identified as cis/trans-dihydroundecaprenol in which the OH-terminal isoprene residue was saturated. The number of internal trans isoprene residues in this compound was three in comparison with two such residues in long-chain C95-dolichol. Chemical preparation of dihydroundecaprenol with a selectively saturated OH-terminal isoprene residue from fully unsaturated plant undecaprenol is presented.


Isolation of an inhibitor of 25-hydroxyvitamin D3-1-hydroxylase from rat serum. An inhibitor of chick kidney mitochondrial 25-hydroxyvitamin D3-1-hydroxylase has been isolated from rat serum by ammonium sulfate precipitation, gel filtration, ionexchange chromatography, and preparative polyacrylamide disc gel electrophoresis. The purified protein was shown to contain iron and has a mol wt of 52 000. The protein is indistinguishable on gel electrophoresis from a similar inhibitor found in rat kidney tissue. The physiological significance of the inhibitor is not known; however, it seems possible that it is responsible for the failure to demonstrate in vitro 25-hydroxyvitamin D3-l-hydroxylation with rat and other mammalian tissues.


A Ca2+-activated protease possibly involved in myofibrillar protein turnover. Purification from porcine muscle. Ca2+-activated Z-disk-removing activity in the P0-40 crude muscle extracts described by Busch et al. (Busch, W. A., Stromer, M. H., Goll, D. E., and Suzuki, A. (1972), J. Cell Biol. 52, 367) was purified from porcine skeletal muscle extracts by using five column chromatographic procedures in succession: (1) 6% agarose; (2) DEAE-cellulose; (3) Sephadex G-200; (4) DEAE-cellulose with a very shallow gradient; (5) Sephadex G-150. All Z-disk-removing activity eluted in a single peak off each column. Z-disk-removing activity always coeluted with Ca2+-activated proteolytic activity, so Z-disk-removing activity in the P0-40 crude muscle extract is due to a single Ca2+-activated protease (CAF). The five column chromatographic procedures produced a 140-fold increase in specific activity of the Ca2+-activated proteolytic enzymic activity; because preparation of the P0-40 crude CAF fraction before chromatography produced a 127-fold increase in specific activity, the entire procedure described here produces a 17 800-fold increase in specific activity of CAF. This increase in specific activity suggests that muscle contains 3.4 mug of CAF per g of muscle fresh weight; this content is in reasonably good agreement with our yields of 0.25-0.76 mug of purified CAF per g of muscle. Purified CAF migrated as a single band during polyacrylamide gel electrophoresis in pH 7.5 Tris-HC1 buffer but migrated as two bands with molecular weights of 80 000 and 30 000 during polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Densitometric scans of sodium dodecyl sulfate-polyacrylamide gels show that the 80 000- and 30 000-dalton subunits make up 85 to 90% of the protein in purified CAF preparations and that these subunits are present in equimolar ratios.


A Ca2+-activated protease possibly involved in myofibrillar protein turnover. Partial characterization of the purified enzyme. The purified Ca2+-activated protease (CAF) isolated from porcine skeletal muscle and capable of removing Z-disks from intact myofibrils is optimally active on either myofibril or casein substrates at pH 7.5 and in the presence of 1 mM Ca2+ and at least 2 mM 2-mercaptoethanol. No CAF activity is detected when 1 mM Mg2+, Mn2+, Ba2+, Co2+, Ni2+, and Fe2+ are added singly. When added with 1 mM Ca2+, Co2+, Cu2+, Ni2+, and Fe2+ inhibit, whereas Mg2+, Mn2+, and Ba2+ have no effect on CAF activity. CAF is irreversibly inhibited by iodoacetate but is unaffected by soybean trypsin inhibitor. S0/20,W=5.90 S, and sedimentation equilibrium molecular weight - 112 000 for purified CAF. Because purified CAF migrates as two polypeptide chains with molecular weights of 80 000 and 30 000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the CAF molecule must consist of one each of these two polypeptide chains. Approximate molecular dimensions of 38 X 220 A can be calculated for CAF from calibrated gel permeation column data or from S0/20,W and the molecular weight. Amino acid composition and physical properties of purified CAF distinguish it from the known catheptic enzymes and from other proteases found in blood or in granulocytes. Purified CAF removes Z-disks the 400-A periodicity associated with troponin in the I band and partly degrades M lines but causes no other ultrastructurally detectable effects when incubated with myofibrils. These results agree with the earlier finding that purified CAF degrades troponin, tropomyosin, and C-protein but has no effect on myosin, actin, or alpha-actinin, and suggest that CAF may have a physiological role in disassembly of intact myofibrils during metabolic turnover of myofibrillar proteins.


Kinetics of the action of papain on fluorescent peptide substrates. Kinetic measurements have been performed on the action of papain on mansyl-Gly-Val-Glu-Leu-Gly and on mansyl-Gly-Gly-Val-Glu-Leu-Gly, both of which are cleaved solely at the Glu-Leu bond under the conditions of our experiments. Stopped-flow experiments have shown that, under conditions of enzyme excess, the enhancement of the fluorescence of the mansyl group upon association of each of the oligopeptide substrates with papain is a biphasic process. A very rapid initial increase in fluorescence is followed by a slower first-order fluorescence enhancement. The observed rate constant for the latter process is greater with the mansyl pentapeptide than with the mansyl hexapeptide. A similar biphasic fluorescence change is seen upon the interaction of the mansyl peptides with mercuripapain, but the second step is much slower than in the case of the active enzyme. The rate of the second step in the association of active papain with the mansyl paptides shows saturation with increasing enzyme concentration, supporting the view that an initial enzyme-substrate complex (ES) is converted in a first-order process to the complex (ES) that undergoes cleavage to form products. The hydrolysis of the Glu-Leu bond is associated with a first-order decrease in fluorescence, as a consequence of the formation of the mansyl peptide product, which is bound less strongly than the substrate. The rate constant for this process is about 140 times greater with the mansyl hexapeptide than with the mansyl pentapeptide, thus giving further indication of the importance of secondary enzyme-substrate interactions in the efficiency of papain catalysis. For each of the two mansyl peptides, the values of the rate constants and the apparent Michaelis constants associated with the cleavage of the Glu-Leu bond, as determined by stopped-flow measurements under conditions of enzyme excess, were the same, within the precision of the data, as those estimated from experiments under conditions of substrate excess, where the formation of Leu-Gly was determined by means of the fluorescamine reaction. This indicates that, with these substrates, the rate-limiting step in the overall catalytic process is associated with the breakdown of ES. Estimates are given of the dissociation constant of ES and of the rate constants in the interconversion of ES and ES.


Carbamoylated hemoglobins A and S: physical properties. Dimer-tetramer association constants (K2,4) of derivatives of CO-hemoglobins A and S specifically carbamoylated at the NH2-terminal valine residues were measured. Reactivites of the beta-93 sulfhydryls of the hemoglobin A derivatives were also investigated. As compared with the association constants of the parent molecules, the values of K2,4 of both hemoglobin types are raised by carbamoylation of the alpha-chain NH2 terminus, lowered by carbamoylation of the beta-chain NH2 terminus, and raised by carbamoylation of both termini. The apparent second-order rate constant for reaction of p-mercuribenzoate (PMB) with the beta-93 sulfhydryls is, however, unchanged by carbamoylation. These two observations are interpreted to indicate that in the liganded molecule structural changes are produced at the interface between dimers but not in the region of the beta-93 sulfhydryls. From the combination of the K2,4 measurements with ligand-binding data for the same derivatives (Kilmartin, J. V., et al. (1973), J. Biol. Chem. 248, 4039; Nigen, A. M., et al. (1974), J. Biol. Chem. 249, 6611) the carbamoylation-induced changes in the dimer-tetramer association constants of the unliganded derivatives were estimated to be of magnitude equal to or smaller than those in K2,4. It is concluded that much of the change in oxygen affinity that occurs upon carbamoylation of hemoglobins A and S can be accounted for without invoking extensive structural changes in the unliganded molecule.


Infrared spectroscopic studies of carbonyl horseradish peroxidases. Infrared difference spectra, FeIIICO vs. FeIII of horseradish peroxidase isoenzymes A2 and C were recorded from 2000 to 1800 cm-1. Under alkaline conditions, pH 9, both isoenzymes exhibit two CO stretching bands, at 1938 and 1925 cm-1 for A2 and at 1933 and 1929 cm-1 for C. As the pH is lowered the low-frequency band for each isoenzyme decreases in intensity with a concommitant appearance and increase in intensity of a band at 1906 and 1905 cm-1 for the A2 and C isoenzymes, respectively. These changes conform to pK values of 6.7 for the A2 and 8.8 for the C isoenzymes of horseradish peroxidase. The interpretation of the infrared results was simplified by the observation that a linear relationship exists between the redox potential, Em7, for the FeIII/FeII system vs. the infrared CO stretching frequency, vCO, for cytochrome a3, hemoglobin, myoglobin, and cytochrome P-450 cam with substrate. This relationship suggests that the primary force altering vCO in these heme proteins is a variation in electron density at the heme iron and not direct protein interactions with the CO ligand. The horseradish peroxidase infrared bands in the 1930-cm-1 region correlate well with this relationship. The large deviation of the 1905-cm-1 band from the linear relationship and its dependence upon hydrogen ion concentration are consistent with horseradish peroxidase having a single CO binding site which can hold in two geometries, one of which contains an amino acid moiety capable of forming a hydrogen bond to the carbonyl oxygen.


Trifluoroacetylated peptides as substrates and inhibitors of elastase: a nuclear magnetic resonance study. Trifluoroacetyl di- and tripeptides have been synthesized in order to investigate their interactions with elastase by proton and fluorine magnetic resonance. These substituted peptides behave as substrates or inhibitors of the enzyme, depending upon their length. They are hydrolyzed with production of trifluoracetic acid and unsubstituted parent peptides exclusively. The amino acid specificity observed and the absence of hydrolysis in the presence of an enzyme substituted at the serine residue of the active site indicate that the trifluoracetic hydrolysis occurs at this site. It requires the fixation of the C-terminal amino acids at the two S' subsites, as does the peptidic hydrolysis of unsubstituted or acetylated oligoalanines. Trifluoracetyl tripeptides exhibit a much higher affinity for the protein, as compared with the unsubstituted or acetylated peptides as well as compared with the trifluoroacetyl dipeptides, and they act as powerful inhibitors of the enzyme. The inhibitory binding mode has been shown to involve the fixation of the trifluoroacetyl group at subsite S4 or in its vicinity, allowing for the cooperative fixation of the C-terminal alanine at S1 and the accommodation of a transproline at S2.


Topography of human plasma alpha1-acid glycoprotein. Human plasma alpha1-acid glycoprotein, whose linear amino acid sequence has recently been elucidated (Schmid et al. (1973), Biochemistry 12, 2711), was further investigated with regard to its topography. Nitration of this protein and subsequent elucidation of the structures of the peptides containing modified tyrosine indicated that residues 27, 37, 78, 115, 127, and 157 are free, 50 and 91 are in an intermediate state, and 65, 74, 110, and 142 are buried. CD measurements between pH 10 and 12 demonstrated that the buried tyrosines are strongly hydrogen bonded and are probably responsible to a considerable extent for the stability of this protein. Of the three tryptophans of this protein, residue 122 proved to be partially reactive with Koshland reagent while the other two (25 and 160) were found to be unreactive. The state of the two disulfide bonds, established by differential reduction and alkylation with specific reagents, was shown to be of an intermediate type. Using carboxymethylation with bromoacetate at pH 7.0 for 8 days, the three histidines (97, 100, and 171) and methionine 111 could be shown to be in intermediate states. All lysines were treated with trinitrobenzenesulfonate and thus were assumed to be free. Of the 40 carboxylic groups, which were amidated with glycine methyl ester, 32 including the 14 sialyl residues were found to be free, six in an intermediate and the remaining two in a buried state. The present study describes the states of almost half of the amino acid residues of alpha1-acid glycoprotein, a knowledge important for the construction of a preliminary three-dimensional model of this conjugated protein.


Anion-induced increases in the rate of colchicine binding to tubulin. The rate of binding of colchicine to tubulin to tubulin is enhanced by certain anions. Among the inorganic anions tested, only sulfate was effective. The organic anions include mostly dicarboxylic acids, among which tartrate was the most effective. This effect occurs onlt at low concentrations of colchicine (less than 0.6 X 10(-5) M). The rate increase dor sulfate and L-(+)-tartrate is ca. 2.5-fold at 1.0 mM and plateaus at a limiting value of ca. 4-fold at 100mM. The overall dissociation rate of the colchicine from the complex, which includes both the true rate of dissociation and the rate of irreversible denaturation of tubulin, is not influenced by 1.0 mM tartrate. The affinity constants for colchicine determined  from the rate constants are 8.7 X 10(6) and 2.1 X 10(7) M-1 in the absence and the presence of 1.0 mM L-(+)-tartrate. The limiting value is 3.2 X 10(7) M-1. The affinity constant calculated from steady-state measurements is 3.2 X 10(6) M-1 with or without anions. The binding of other ligands like podophyllotoxin, vinblastine, and 1 -anilino-8-naphthalenesulfonate to tubulin is not affected by tartrate. No major conformational changes resulting from anion treatment could be detected by circular dichroism or intrinsic fluorescence. However, the ability of tubulin to polymerize is inhibited by L-(+)-tartrate at concentrations that increase the rate of colchicine binding. We conclude that anions must have a local effect at or near the binding site which enhances the binding rate of colchicine and which may be related to inhibition of polymerization.


Interaction of (3H) bongkrekic acid with the mitochondrial adenine nucleotide translocator. Chemical labeling by 3H and biosynthetic labeling by 14C of bongkrekic acid (BA) are described. In the rat liver cell, mitochondria are the only subcellular particles to bind [3H]BA with high affinity. The high affinity sites for BA in mitochondria are located in the inner membrane. High affinity binding sites for BA are only displayed at pH below 7; they amount to 0.15-0.20 nmol/mg of protein in rat liver mitochondria and to 1.1-1.3 nmol/mg of protein in rat heart mitochondria. These values are similar to those found for the high affinity atractyloside binding sites and for the carboxyatractyloside binding sites. The kinetic parameters for BA binding to rat heart mitochondria at 20 degrees C are Kd = 10-40 X 10(-9) M, k+1 = 0.7 X 10(5) M-1 s-1, k-1 = 1.4 X 10(-3) M s-1. Binding assays carried out with rat heart mitochondria, under equilibrium conditions, showed that the amount of BA bound to high affinity sites increases with temperature and reaches the maximum value of 1.1-1.3 nmol/mg of protein at 32-35 degrees C. At lower temperatures, and under equilibrium conditions, a significant fraction of high affinity sites remains masked and is not titrated by BA; these masked BA sites are revealed by addition of micromolar concentrations of ADP or by energization of the mitochondria. Carboxyatractyloside added to rat heart mitochondria preloaded with [3H]BA is able to displace part of the bound [3H]BA. Displacement of the bound BA is enhanced by simultaneous additions of carboxyatractyloside plus ADP, or by energization of the mitochondria. The synergistic effect of carboxyatractyloside and ADP on displacement of bound [3H]BA is also observed in isolated inner membrane vesicles from rat liver mitochondria. When BA is preincubated with rat heart mitochondria before addition of [14C]ADP for assay of ADP transport, the inhibition of ADP transport is a mixed-type inhibition. When BA is preincubated with the mitochondria together with a very small concentration of ADP (less than 0.5 muM), the inhibition of [14C]ADP transport is markedly increased (up to ten times) and it becomes typically uncompetitive, which suggests the formation of a ternary complex, carrier-ADP-BA. The transition from a mixed-type inhibition, with high Ki value, to an uncompetitive type of inhibition, with low Ki value, upon addition of ADP, is explained by an ADP-induced conformational change of the ADP translocator.


Isobongkrekic acid, a new inhibitor of mitochondrial ADP-ATP transport: radioactive labeling and chemical and biological properties. An isomer of bongkrekic acid, designated as isobongkrekic acid, has been isolated from ethereal extracts of Pseudomonas cocovenenans grown on defatted coconut. Isobongkrekic acid was also obtained by alkaline treatment of bongkrekic acid. Isobongkrekic acid possesses the same ultraviolet spectrum and the same molecular weight as bongkrekic acid; it has a similar infrared spectrum but not the same nuclear magnetic resonance (NMR) spectrum. The differences in NMR data were interpreted to mean that isobongkrekic acid differs from bongkrekic acid by the configuration of the dicarboxylic end; whereas the two carboxylic groups of the dicarboxylic end have the trans configuration in bongkrekic acid, they have the cis configuration in isobongkrekic acid. Differences between bongkrekic and isobongkrekic acids are lost after catalytic hydrogenation of the molecules. Isobongkrekic acid, like bongkrekic acid, is an uncompetitive inhibitor of ADP transport in mitochondria, provided the mitochondria are preincubated in the presence of the inhibitor and a minute concentration of ADP. The inhibitory and binding efficiency of isobongkrekic acid is considerably increased below pH 7. The number of high affinity sites for [3H] isobongkrekic acid is 0.13 to 0.20 nmol/mg protein in rat liver mitochondria and about 1 nmol/mg protein in rat heart mitochondria, i.e., similar to the number of high affinity sites for [3H] bongkrekic acid. Isobongkrekic and bongkrekic acids compete for the same site, but the affinity of isobongkrekic acid for mitochondria is one-half to one-fourth that of bongkrekic acid.


Kinetics of ATP-dependent Mg2+ flux in mitochondria. ATP-dependent Mg2+ accumulation in isolated mitochondria occurs predominantly in the matrix and inner membrane compartments. In mitochondria contaminated with lysosomes, the time course and magnitude of ATP-dependent Mg2+ accumulation are influenced by various cytoplasmic substances, besides substrates of the citric acid cycle. Removal of lysosomes by treatment of the mitochondrial preparation with low concentrations of digitonin, which does not damage the mitoplast, eliminates the modifying influence of cytoplasmic components on Mg2+ flux. In lysosome-free mitochondria, the kinetics of Mg2+ flux is dependent only on the concentration of ATP, of Mg2+, and on the availability of site specific reducing substrates of the electron transport system. Oligomycin at concentrations sufficient to inhibit phosphorylation coupled electron transport and ATP synthesis does not modify Mg2+ flux, which is dependent on added ATP. Site specific inhibitors of the electron transport system inhibit the augmenting effect of oxidizable substrates on Mg2+ uptake, even when electron transfer is inhibited by oligomycin. Atractyloside, by inhibiting the action of externally added ATP, diminishes Mg2+ flux. Ruthenium red is a powerful inhibitor of ATP dependent Mg2+ flux. Uncouplers not only inhibit Mg2+ uptake, but induce Mg2+ efflux. From the time course of Mg2+ flux, a first-order rate constant of egress of Mg2+ and other kinetic constants were calculated and a kinetic model was derived which describes the bi-directional movement of Mg 2+ in mitoplasts.


Iodination of a tyrosyl residue in staphylococcal alpha-toxin. Iodination of staphylococcal alpha-toxin by the lactoperoxidase method resulted in the maximal incorporation of about 2.5 atoms of iodine per molecule of alpha-toxin. The iodination primarily involved a single tyrosine residue as shown by analysis of both cyanogen bromide and tryptic peptides. Iodination at a level of 1.2 iodine atoms per alpha-toxin molecule led to a dramatic decrease in the hemolytic and lethal activities, although no decrease in the binding of iodinated toxin to rabbit erythrocytes was observed (Cassidy and Harshman (1976), Biochemistry, the following paper in this issue). Monoiodinated alpha-toxin was found to have 15% of the specific hemolytic activity of native alpha-toxin. Incubation of rabbit erythrocytes with iodinated alpha-toxin led to a significant protection from the hemolytic activity of native alpha-toxin added later. The results show the modification of a single unique tyrosyl residue in alpha-toxin permits the resolution of alpha-toxin's biological activities from its cell binding activity.


Studies on the binding of staphylococcal 125I-labeled alpha-toxin to rabbit erythrocytes. Staphylococcal alpha-toxin, a hemolytic exotoxin, can be iodinated using the lactoperoxidase method. 125 I-Labeled alpha-toxin binds to rabbit erythrocytes in an apparently irreversible and highly specific manner. The binding of 125 I-labeled alpha-toxin to erythrocytes of rabbit and human reflects the species specificity of native alpha-toxin. Binding of 125I-labeled alpha-toxin is blocked by the presence of native alpha-toxin, 127I-labeled alpha-toxin, or anti-alpha-toxin antibody. Simultaneous assays of 125I-labeled alpha-toxin binding and leakage of intracellular 86Rb+ suggest that toxin binding and membrane damage are separate, sequential functions. Both the rate and extent of binding are temperature dependent. Rabbit erythrocytes possess 5 X 10(3) binding sites/cell, while human erythrocytes possess no detectable binding sites. Treatment of rabbit erythrocytes with 125I-labeled alpha-toxin appears to decrease the number of unoccupied binding sites. Chaotropic ions can inhibit 125I-labeled alpha-toxin binding and cause bound 125I-labeled alpha-toxin to dissociate from rabbit erythrocyte membranes. Treatment of intact rabbit erythrocytes with pronase reduces both the binding capacity  of the cells for 125I-labeled alpha-toxin, and the cells' sensitivity to hemolysis by native alpha-toxin. It is proposed that the primary binding site for alpha-toxin in biomembranes is a surface membrane protein.


Transbilayer phospholipid asymmetry and its maintenance in the membrane of influenza virus. Two phospholipid exchange proteins and two phospholipases C have been employed to determine the phospholipid composition of the outer surface of the membrane of influenza virus. These four protein probes have defined the same accessible and inaccessible pool for each viral phospholipid. Phospholipids which are exchangeable or hydrolyzable are located on the outer surface, whereas the inaccessible pool is located at the inner surface of the viral bilayer. The two pools are unequal in size, with ca. 30% of the total phospholipid accessible to the four proteins, and ca. 70% inaccessible. The membrane is thus highly asymmetric with regard to the amount of phospholipid on each side of the membrane. There is also a marked asymmetry of phospholipid composition. Phosphatidylcholine and phosphatidylinositol are enriched in the outer surface, and sphingomyelim is enriched in the inner surface, whereas phosphatidylethanolamine and phosphatidylserine are present in similar proportions in each surface. This distribution is qualitatively different from that previously reported for the human erythrocyte. The close agreement between results obtained with excahnge proteins and phospholipases C demonstrates that the hydrolytic action of these enzymes does not alter phospholipid asymmetry. The nonperturbing nature of the exchange proteins has permitted the rate of transmembrane movement of phospholipids (flip-flop) in the intact virion to be studied. This process could not be detected after 2 days at 37 degrees C. It was estimated that the half-time for flip-flop is indeterminately in excess of 30 days for sphingomyelin and 10 days for phosphatidylcholine at 37 degrees C. These extremely long times provide a simple explanation for the maintenance of transbilayer asymmetry in influenza virions and possibly, other membranes. Since the viral membrane is acquired by budding through the host cell plasma membrane, the transbilayer distribution of phospholipids observed in the virions presumably reflects a similar asymmetric distribution of phospholipids in the host cell surface membrane. Because animal cells in culture do not incorporate extracellular phospholipid, our results demonstrate that individual cells have the capacity to generate asymmetric membranes.


Acetylcholine receptor oligomers from electroplax of Torpedo species. Sedimentation in sucrose gradients of alpha-bungarotoxin-labeled crude and pure acetycholine receptor preparations from Torpedo californica showed two major oligomers. The molecular weights, corrected for the bound Triton X-100 by comparing sedimentation in H2O and in D2O, were 330 000 for the heavy (H) oligomer and 190 000 for the light (L) oligomer. Lesser peaks found in preparations of T. marmorata and purified preparations of T. californica with molecular weights of 500 000 (HH) and 80 000 (LL). These molecular weights are based upon the assumption of globularity, and may require adjustment  if the assumption is wrong. The H and L peaks have similar drug sensitivities, but at pH 10 the L peak was stable whereas the H peak dissociated to components sedimenting as LL. Treatments with p-chloromercuribenzoate, which blocks acetylcholine binding partially without affecting alpha-bungarotoxin binding, had no effect upon the pattern of sedimentation. This and other evidence suggested  that the heterogeneity of oligomers was unrelated to the heterogeneity of site affinities for acetylcholine and nicotinic drugs.


Hydrophobic chromatography and fractionation of enzymes from extremely halophilic bacteria using decreasing concentration gradients of ammonium sulfate. Ammonium sulfate fractionation of proteins from extremely halophilic bacteria on Sepharose 4B, carboxymethylcellulose, diethylaminoethylcellulose, and hexamethylenediamine-Agarose is described. Halophilic proteins are absorbed on these gels at 2.5 M ammonium sulfate and eluted by decreasing concentration gradients of this salt. The method has enabled the separation of malate dehydrogenase from glutamate dehydrogenase and aspartate aminotransferase on Sepharose 4B and the additional 15-fold purification of glutamate dehydrogenase on DEAE-cellulose. The technique is simple and convenient, operates at low cost, and possesses great power of resolution. The mechanism of adsorption is discussed and compared to previous instances of "hydrophobic chromatography". It is concluded that the retention of halophilic proteins on the polysaccharide gels at 2.5 M ammonium sulfate is due to hydrophobic interactions.


Resonance Raman evidence for substrate reorginization in the active site of papain. Resonance Raman spectra were obtained for the acylenzyme 4-dimethylamino-3-nitro(alpha-benzamido)cinnamoyl-papain prepared using the chromophoric substrate methyl 4-dimethylamino-3-nitro(alpha-benzamido)cinnamate. These spectra contained vibrational spectral data of the acyl residue while covalently attached to the active site and could be used to follow directly acylation and deacylation kinetics. Spectra were obtained at pH values ranging from those where the acyl-enzyme is relatively stable (pH 3.0, tau 1/2 congruent to 800 s) to those where it is relatively unstable (pH 9.2, tau 1/2 congruent to 223 s). Throughout this range acyl-enzyme spectra differed completely from that of the free substrate or the product (4-dimethylamino-3-nitro(alpha-benzamido)cinnamic acid) indicating that a structural change occurred on combination with the active site. The spectra are consistent with rearrangement of the alpha-benzamido group in the bound substrate, -NH--C(==O)Ph becoming --N==C(--OX)Ph, where the bonding to oxygen is unknown. Superimposed on these large differences, small changes in acyl-enzyme spectra also occurred as pH was raised to decrease the half-life. All of the above spectral perturbations are consistent with a structural change in the acyl-enzyme which precedes the rate-determining step in deacylation. Thus, deacylation proceeds from an acyl residue structure differing from that of the substrate in solution. Upon acid denaturation the spectrum characteristic of the intermediate reverts to one closely resembling the substrate, demonstrating that a functioning active site is necessary to produce the observed differences. Spectra in D2O of native acyl-enzyme were identical with those in H2O, indicating that the observed differences in rate constant were not due to solvent-induced structural changes. Activated papain purified by crystallization or by affinity chromatography formed the acyl-enzyme. However, the kinetics of formation and deacylation differed between these materials, as did the spectral properties. Small differences in active-site structure are considered to be responsible for this effect, and it is suggested that such spectral perturbations may be useful in directly relating small differences in structure of the substrate in the active site with corresponding differences in kinetics.


The pyridoxal 5' -phosphate site in rabbit skeletal muscle glycogen phosphorylase b: an ultraviolet and 1H and 31P nuclear magnetic resonance spectroscopic study. 1 H NMR spectra of the 3-0-methylpyridoxal 5'-phosphate-n-butylamine reaction product indicated that this analogue forms a Schiff base in aprotic solvent. The uv spectral properties of 3-0-methylpyridoxal-5'-phosphate phosphorylase b correspond to those of the n-butylamine Schiff base derivative in dimethyl sulfoxide. On the basis of that and auxiliary uv and 1H NMR spectra of pyridoxal and pyridoxal 5'-phosphate and the corresponding Schiff base derivatives we have verified that pyridoxal 5' -phosphate is also bound as a Schiff base to phosphorylase and not as an aldamine. Since 3-0-methylpyridoxal-5'-phosphate phosphorylase is active, a proton shuttle between the 3-hydroxyl group and the pyridine nitrogen is excluded. This directs attention to the 5' -phosphate group of the cofactor as a candidate for a catalytic function. 31P NMR spectra of pyridoxal 5' -phosphate in phosphorylase b indicated that deprotonation of the 5' -phosphate group was unresponsive to external pH. Interaction of phosphorylase b with adenosine 5' -monophosphate, the allosteric effector required activity, and arsenate, which substitutes for phosphate as substrate, triggered a conformational change which resulted in deprotonation of the 5' -phosphate group of pyridoxal 5' at pH 7.6. It now behaved like in the pyridoxal-phosphate-epsilon-aminocaproate Schiff base in aqueous buffer, where the diionized form is dominant at this pH. Differences of line widths of the adenosine 5' -monophosphate signal point to different life times of the allosteric effector- enzyme complexes in the presence and absence of substrate (arsenate).


A  DNA binding protein from human placenta specific for ultraviolet damaged DNA. A DNA-binding protein specific for ultraviolet irradiated DNA has been purified extensively from human placenta. The binding preparation is free of exonuclease, polymerase, endonuclease, and N-glycosidase activity. The binding activity is salt dependent and is specific for double-stranded irradiated DNA. DNA from which the pyrimidine dimers have been monomerized by the action of photolyase (photoreactivating enzyme) remains an effective substrate for the binding protein, suggesting that the protein recognizes photoproducts other than pyrimidine dimers. This is supported by the finding that DNA irradiated under conditions which introduce only pyrimidine dimers is not a substrate for the binding protein. Examination of three of the xeroderma pigmentosum complementation groups has revealed no deficiency in this binding activity.


A mammalian nicking endonuclease.

A mammalian nicking endonuclease. Purification and properties are described for an endonuclease isolated from calf thymus which attacks double-stranded, unmodified DNA, primarily by making single-strand breaks. No detectable acid-soluble products arise from the reaction. Double-strand breaks may occasionally be produced by the introduction of single-strand breaks on opposite strands in close proximity. The enzyme does not attack denatured DNA and is not inhibited by tRNA. Although added divalent cations are not required for activity, the enzyme is inhibited by EDTA, which suggests an essential role for bound cations; reaction is inhibited by Ca2+. The endonuclease has a broad pH optimum and is inactivated by preincubation at temperatures of 45 degrees C and higher. The molecular weight as determined by gel chromatography is about 30 000. Analysis of the products of reaction on a defined substrate, bacteriophage T3 DNA, by sedimentation in alkaline sucrose density gradients indicates limit products with chain lengths of about 0.8 X 10(6) daltons. On electrophoresis in agarose gels these products were shown to be heterogeneous in size. The endonuclease appears to generate 3'-hydroxyl and 5'-phosphate ends. The ability of the endonuclease to utilize bovine DNA as substrate argues against a restriction role for this enzyme.


Relationship between nuclear and cytoplasmic RNA in Drosophilia cells. Polyadenylated RNA was isolated from nuclei of cultured Drosophila cells, Schneider's line 2, and used as a template to synthesize a complementary DNA probe. Hybridization experiments were performed to study the relationship between nuclear and cytoplasmic RNA. About two-thirds of the nuclear polyadenylated RNA sequences exist in the cytoplasm. Experiments with fractionated cDNA probes demonstrated that RNA sequences that are frequent in the nucleus are also abundant in the cytoplasm. These findings are consistent with a precursor-product relationship in which some polyadenylated molecules in the nucleus are destined for the cytoplasm while other sequences are polyadenylated but not transferred.


Metabolism of 1,25-dihydroxyvitamin D3: evidence for side-chain oxidation. Approximately 7% of a 650-pmol dose of 25-hydroxyl[26,27-14C]vitamin D3 and 25% of a 325-pmol dose of 1,25-dihydroxyl[26,27-14C]vitamin D3 are metabolized to 14CO2 by vitamin D deficient rats. Nephrectomy prevents the metabolism of 25-hydroxy[26,27-14C]vitamin D3 to 14CO2 but not that of 1,25-dihydroxy[26,27-14C]vitamin D3. Less than 5% of the 14C from 24,25-dihydroxy[26,27-14C]vitamin D3 is metabolized to 14CO2. Feeding diets high in calcium and supplemented with vitamin D3 markedly diminishes the amount of 14CO2 formed from 25-hydroxy[26,27-14C]vitamin D3 but not that from 1,25-dihydroxyl[26,27-14C]vitamin D3. These results provide strong evidence that only 1-hydroxylated vitamin D compounds and especially 1,25-dihydroxyvitamin D3 undergo side-chain oxidation and cleavage to yield an unknown metabolite and CO2.


Polymorphism of Sporothrix schenckii surface polysaccharides as a function of morphological differentiation. The alkali-extractable polysaccharides from different morphological types of two Sporothrix schenckii strains (1099.12 and 1099.18) were investigated. Dissociation of morphological phase transition and temperature effects was possible in a synthetic medium which produced cultures with 100% yeast forms either at 25 or at 37 degrees C. Only rhamnomannans with single-unit alpha-L-rhamnopyranosyl side chains were formed by the yeast forms irrespective of the incubation  temperature. The higher temperature inhibited formation of 4-O- and 2,4-di-O-substituted alpha-D-mannopyranose units in the rhamnomannan. An apparently unsporulated mycelium culture of one S. schenckii strain (1099.12) synthesized a galactomannan whose structure was partially determined by methylation analysis and by proton and 13C nuclear magnetic resonance spectroscopy. In another strain (1099.18), a mannan was excreted in the medium of an apparently conidia-less mycelial form at 25 degrees C with short incubation. Its structure was also partially determined. An apparent mixture of this mannan and a rhamnomannan rich in alpha-L-rhamnopyranosyl-(1 leads to 2)-alpha-L-rhamnopyranose side chains formed in these cultures on prolonged incubation. The proportion of the excreted rhamnomannan increased as the mycelium sporulated and conidia were more numerous. Mannans or galactomannans may be transient polysaccharides in the young mycelium of S. schenckii. As the culture develops, rhamnomannans are formed in amounts usually masking the presence of other mannose-containing polysaccharides. It is suggested that in S. schenckii different polysaccharides are formed with side chains containing different proportions of rhamnose, mannose, or galactose, as a function of morphological differentiation.


A calorimetric study of the thermotropic behavior of aqueous dispersions of natural and synthetic sphingomyelins. A recently developed differential scanning calorimeter has been used to characterize the thermotropic behavior of aqueous dispersions of liposomes containing sphingomyelin. Liposomes derived from sheep brain sphingomyelin exhibit a broad gel-liquid crystalline phase transition  in the temperature range of 20-45 degrees C. The transition is characterized by maxima in the heat capacity function at 31.2 and 37.1 degrees C and a total enthalpy change of 7.2 +/-0.4 kcal/mol. Beef brain sphingomyelin liposomes behave similarly but exhibit heat capacity maxima at 30, 32, and 38 degrees C and a total enthalpy change of 6.9 kcal/mol. The thermotropic behavior of four pure synthetic sphingomyelins is reminiscent of multilamellar lecithin liposomes in that a single, sharp, main transition is observed. Results obtained for liposomes containing mixtures of different sphingomyelins are complex. A colyophilized mixture of N-palmitoylsphingosinephosphorylcholine, N-stearoylsphingosinephosphorylcholine, and N-lignocerylsphingosinephosphorylcholine in a 1 : 1 : 1 mol ratio exhibits a single transition with a Tm below that observed for the individual components. On the other hand a 1 : 1 mixture of N-stearoylsphingosinephosphorylcholine and 1-palmitoyl-2-oleylphosphatidylcholine exhibits three maxima in the heat capacity function. It is clear from these results that the thermotropic behavior of sphingomyelin-containing liposomes is a complex function of the exact composition. Furthermore, it appears that the behavior of the liposomes derived from natural sphingomyelins cannot be explained in terms of phase separation of the individual components.


Interactions between anesthetics and lipid mixtures. Normal alcohols. The effects of normal alcohols up to 1-dodecanol on phase transitions in phosphatidylcholines and phosphatidylethanolamines have been studied using chlorophyll a as fluorescent probe. With the phosphatidylcholines, alcohols up to octanol cause a lowering of the transition temperature, and a broadening of the transition, whereas for dipalmitoylphosphatidylethanolamine, only a lowering of the transition is observed. The lowering of the phase transition temperature in dipalmitoylphosphatidylcholine by butanol and hexanol is close to that expected for ideal behavior, but the behavior of the longer chain alcohols becomes less ideal. The effects of these alcohols on mixtures of lipids have been studied, and they illustrate the care necessary if these plots of temperatures of onset and completion of gel phase formation are to be called "phase diagrams". The effect of 1 -octanol on mixtures of lipids is to increase the proportion of lipid present in the lipid-crystalline state. In  contrast, 1-decanol causes an increase in the phase transition temperature for dimyristoylphosphatidylcholine, although it lowers the transition temperature for dipalmitoylphosphatidylcholine, and 1 -dodecanol raises the transition temperature for both of these phosphatidylcholines, although it lowers that for dipalmitoylphosphatidylethanolamine. Dodecanol appears to behave in these lipid bilayer membranes as a lipid with a phase transition temperature of ca. 55 degrees C. Anesthesia is discussed as a phenomenon of liquidus extension: alcohols up to 1 -octanol increase the proportion of lipid in the liquidus state and result in anesthesia, whereas the longer alcohols do not, and result in catalepsy.


Intramolecular microdynamical and conformational parameters of peptides from 1H and 13C NMR spin-lattice relaxation. Tetragastrin. Measurements of 1H and 13C spin-lattice relaxation and 13C nuclear Overhauser enhancement factors are reported for dimethyl sulfoxide solutions of tetragastrin, a pharmacologically active tetrapeptide. The use of the dipolar formalism for predicting 1H and quaternary 13C relaxation rates is discussed. Furthermore, the prospect is opened for the use of quaternary 13C and 1H relaxation times to obtain information on the peptide torsion angles phi, psi, and chi in a way supplementing NMR coupling constant methods presently in use.


Dual labeling in standard DNA-RNA hybridization studies using 125 I-labeled nuclear RNA and 3H-labeled DNA. Standard DNA-RNA hybridization studies, using nucleic acids isolated from mammalian tissues, are frequently hindered by relatively low levels of radioactivity in pulse-labeled RNA and in an inability to reliably estimate the amount of DNA present in the hybrid. In the method described here nuclear RNA is labeled in vitro with 125I to 400 000- 800 000 cpm/mug and DNA is obtained from a rat glial tumor line grown in culture and labeled to specific activities of 42 000-79 000 cpm/mug. DNA-RNA hybridization is conducted in an all solution system at RNA:DNA ratios of 3.5:1 to 18:1. Assay background is controlled by pretreatment of the hybrid and free RNA at the conclusion of the annealing study with RNase, then isolation of the hybrid together with a small fraction of free RNA oligonucleotides on hydroxyapatite. The partially purified hybrids are then trapped on Millipore filters. Assay background id 0.004% of total counts present in the annealing reaction. Comparison of the annealing reactions of pulse-labeled liver nuclear RNA and in vitro 125I-labeled nuclear RNA in saturation, kinetic, and competitive hybridization studies shows them to be essentially the same. Nuclear RNA labeled by either tritium or iodine shows a 10-20-fold greater concentration of the annealing sequences over that found in the microsomal RNA. Minor differences are noted between the nuclear RNAs in the initial rates of reaction and in the magnitude of the decrease in percent hybridization at low levels of unlabeled competitor RNA. This may be due to preferential labeling in pulse-labeled RNA of molecules which are present in lower concentrations or are transcribed from more frequently repeated DNA sequences than the average population of annealing RNA molecules. The technique has application in systems where the amount of tissue for RNA extraction is small or where the system does not permit the obtaining of pulse-labeled RNA, as in experimental rodent skin carcinogenesis or in dealing with RNA from the tissues of large mammals or humans.


Calculation of s20,w values using ultracentrifuge sedimentation data from linear sucrose gradients, an improved, simplified method. A mathematical method is described for calculating the sedimentation coefficient (S20,W) with ultracentrifuge data from linear sucrose gradients. Gradient density and viscosity functions are precisely described by regression equations, which permit continuous evaluation (by integration) of the effects of gradient geometry on particle sedimentation. The results agree with previously used and more complex methods.


Protein-polynucleotide scattering centers as a protein structure probe. When certain basic globular proteins are mixed with nucleic acids near a critical concentration ratio, large, low density scattering centers of about 10(9) particle weight are created. Scattering from these complexes is altered when thermally inactivated proteins are substituted for enzymes in their native, globular conformation. Scattering data from heat-treated ribonuclease and lysozyme mixed with four different synthetic homopolyribonucleotides are reported. The concentration of nucleic acid necessary to produce maximum scattering from a heat-treated protein sample is shown to be a direct indication of the amount of enzyme that remains biologically active after being heated.


Low temperature photodissociation studies of ferrous hemoglobin and myoglobin complexes by Mössbauer spectroscopy. 57Fe-enriched complexes of hemoglobin and myoglobin with CO and O2 were photodissociated at 4.2 degrees K, and the resulting spectra were compared with those of the deoxy forms. Differences in both quadrupole splitting and isomer shift were noted for each protein, the photoproducts having smaller isomer shift and larger quadrupole splitting than the deoxy forms. The photoproducts of HbCO and HbO2 had narrow absorption lines, indicating a well-defined iron environment. The corresponding myoglobin species had broader absorption lines, as did both deoxy forms. The weak absorption lines of photodissociated NO complexes appeared to be wide, possibly indicating magnetic interaction with the unpaired electron of the nearby NO.


Water proton magnetic resonance studies of normal and sickle erythrocytes. Temperature and volume dependence. The temperature and cell volume dependence of the NMR water proton line-width, spin-lattice, and spin-spin relaxation times have been studied for normal and sickle erythrocytes  as well as hemoglobin A and hemoglobin S solutions. Upon deoxygenation, the spin-spin relaxation time (T2) decreases by a factor of 2 for sickle cells and hemoglobin S solutions but remains relatively constant for normal cells and hemoglobin A solutions. The spin-lattice relaxation time (T1) shows no significant change upon deoxygenation for normal or sickle packed red cells. Studies of the change in the NMR linewidth, T1 and T2 as the cell hydration is changed indicate that these parameters are affected only slightly by a 10-20% cell dehydration. This result suggests that the reported 10% cell dehydration observed with sickling is not important in the altered NMR properties. Low temperature studies of the linewidth and T1 for oxy and deoxy hemoglobin A and hemoglobin S solutions suggest that the "bound" water possesses similar properties for all four species. The low temperature linewidth ranges from about 250 Hz at -15 degrees C to 500 Hz at -36 degrees C and analysis of the NMR curves yield hydration values near 0.4 g water/g hemoglobin for all four species. The low temperature T1 data go through a minimum at -35 degrees C for measurements at 44.4 MHz and -50 degrees C for measurements at 17.1 MHz and are similar for oxy and deoxy hemoglobin A and hemoglobin S. These similarities in the low temperature NMR data for oxy and deoxy hemoglobin A and hemoglobin S suggest a hydrophobically driven sickling mechanism. The room temperature and low temperature relaxation time data for normal and sickle cells are interpreted in terms of a three-state model for intracellular water. In the context of this model the relaxation time data imply that type III, or irrotationally bound water, is altered during the sickling process.


Water diffusion permeability of human erythrocytes studied by a pulsed gradient NMR technique. Water diffusion permeability of human erythrocytes has been measured by NMR using a pulsed magnetic field gradient technique. The measurement of exchange rates was based on restricted diffusion of water molecules within red blood cells. This method avoids addition of paramagnetic ions, such as Mn2+, and is used in vivo. The mean lifetime of water insed human erythrocytes was found to be 17 ms at 24 degrees C. A sulfhydryl reagent, known to inhibit water osmotic permeability, reduced significantly water diffusion across the red cell membrane.


Chemical reactivity of labile sulfur of iron-sulfur proteins. The reaction of triphenyl phosphine. The reaction of triphenyl phosphine to iron-sulfur proteins from adrenal cortex mitochondria, spinach chloroplasts, and Clostridium pasteurianum was investigated. As ethanol concentrations in the reaction mixture increased, the rate of the reaction decreased. In the simultaneous presence of 1 M KC1 and 5 M urea, the reaction rate reached at maximum. Under these conditions the initial rates of the decolorization reaction by the phosphine were found to be 8.7, 0.88, and 1.8 nmol of ferredoxin per min at 25 degrees C for adrenal, spinach, and clostridial ferredoxins, respectively. The kinetic curves for the reaction of the phosphine sulfide formation, the loss of labile sulfur, and the deterioriation of visible absorption showed a similar pattern with a comparable rate. During this reaction, the complete reduction of ferric ions present in ferredoxin was observed with a fast rate under either aerobic or anaerobic conditions. These results suggest that the iron atoms in ferredoxin are first reduced by the intramolecular reductants in the presence of triphenyl phosphine with the concomitant formation of S2-2, which then reacts with triphenyl phosphine resulting in the formation of triphenyl phosphine sulfide.


Studies on cephalopod rhodopsin: photoisomerization of the chromophore. Photoisomerization of the chromophore of squid rhodopsin is dependent upon the irradiation temperature. Above 0 degrees C, only 11-cis in equilibrium all-trans reaction proceeds and the all-trans leads to 9-cis reaction is limited to extremely low efficiency. At liquid nitrogen temperature, 11 cis in equilibrium all-trans in equilibrium 9-cis reaction takes place. At intermediary low temperatures (-80 degrees C to -15 degrees C) another isomer of retinal may be produced by the irradiation, which forms a pigment having an absorbance maximum at 465 nm (P-465). The formation of P-465 decreases remarkably in the narrow temperature range from -30 degrees C to 0 degrees C where mesorhodopsin converts to metarhodopsin. Medsorhodopsin is quite different from metarhodopsin in the photoisomerization of the chromophore because P-465 is produced from the former but not from the latter. No P-465 is produced both at liquid nitrogen temperature and above 0 degrees C. P-465 is more labile than any of the other photoproducts so far known, that is isorhodopsin, alkaline and acid metarhodopsins. P-465 is converted to metarhodopsin by irradiation.