Late deaths after resection for bronchial carcinoma.

1267 patients who underwent surgical treatment for bronchial carcinoma during the period 1949--70 have been studied. 304 underwent thoracotomy without further resection; 88% died within 12 months; the maximum survival was 56 months. Of the remaining 963 patients, 869 late deaths are recorded, and the causes of these have been studied. The 5-year actuarial survival rate was 25.9%; the 10-year rate 15.3%; the 15-year rate 9.3%; and the 20-year rate 3.9%. 610 of the 869 deaths were due to bronchial carcinoma; 79 were due to respiratory disease; 57 to coronary disease; and 23 to other neoplasms. The percentage of total deaths due to bronchial carcinoma declined with the passage of time. It was 86.5% during the first year; 79.2% during the next 4 years; 53.7% from 5 to 10 years; and 39.3% over 10 years. The second commonest cause of death was respiratory disease, which tended to be related to the extent of resection. Compared with a population of the same age and sex structure, suffering the death rates of England and Wales during the period involved, these patients show a massive excess of deaths from bronchial carcinoma and a smaller excess from respiratory disease; and a considerable shortfall from coronary disease, other carcinomas, and all other causes. A more favourable probability of survival was linked with lobectomy, squamous cell type and the younger age group. A less favourable probability was linked with pneumonectomy, adenocarcinoma and older age-groups. There was no significant difference due to sex. After the first 2 years, with their heavy death rate, the chances of surviving to 10 years improved by about 15% each year.


Triamcinolone in corticosteroid-resistant asthma.

In an uncontrolled study 38 patients with chronic asthma were treated for up to 4.5 years (mean 2.9) with depot injections of triamcinolone acetonide. About half had proved difficult to control on large doses of corticosteroid tablets. Three-quarters were helped and the incidence of hospital admission amongst them was reduced. Side effects were tolerable and adrenal function was only moderately depressed. Spirometry showed continuing improvement up to three years after starting triamcinolone. Whether similar improvement could have been achieved by a different dosage regimen of corticosteroid tablets or whether it might have ocurred spontaneously was not tested. A controlled trial seems warranted.


Diagnostic pneumoperitoneum.

Pneumoperitoneum as a diagnostic procedure has been practised for many years but there is a reluctance to make use of it. The author believes that this is due in some degree to lack of clearly defined indications and partly to lack of confidence in the safety of the procedure. In this paper the technique of diagnostic pneumoperitoneum is described and the risks are examined. Indications for its use are described. Finally, three case histories of special relevance are described and a summary of the results is tabulated with a view of giving an estimate of the diagnostic value which may be expected.


Metabolic studies in acute asthma before and after treatment.

We studied the metabolism and hormone profile of 9 patients with moderately severe acute asthma before treatment, and again 10 min after intravenous aminophylline (250 mg) or the selective beta-adrenergic stimulant hexoprenaline (5 microgram) intravenously. Compared with basal values in normal subjects the untreated asthmatics had statistically significant raised mean plasma pancreatic glucagon, free fatty acid (FFA) and glucose levels in the plasma and a significantly depressed mean plasma potassium level. Insulin, growth hormone, cortisol, thyrotropin and ketone body levels were normal. The only significant changes after therapy were a further fall in plasma potassium in the hexoprenaline-treated patients and a rise in the mean lactate concentration of the group as a whole. The clinical implications of these findings are briefly considered.


The diagnosis of pulmonary carcinoid tumours.

Carcinoid tumours of the lung are commonly misdiagnosed as carcinoma and even if they are recognized their behaviour may be unexpected. In a series of ten cases which had caused diagnostic difficulty there had been an incorrect interpretation of broncial biopsy material in four, of the resected tumours in another four, of a needle biopsy core in one and of autopsy material in another. In some cases these errors led to needless radiotherapy and to an unnecessarily poor prognosis being given. In all cases the diagnosis of carcinoid tumour was made retrospectively by the demonstration of argyrophil granules in the tumour cells or by electron microscopy. Electron-dense bodies could be found in material that had been fixed in formol saline or even embedded in paraffin so that this method may be used retrospectively. Two of these ten tumours behaved in a malignant fashion but progressed much more slowly than most samples of bronchial carcinoma. The possibility of carcinoid tumour should be considered in any patient with a tumour of the lung who is unusually young, or who is a non-smoker or where bone or calcification are present in the tumour. It is also suggested that unrecognized cases of carcinoid tumour may be found amongst long-term survivors with lung tumours which havebeen treated by radiotherapy or chemotherapy without surgery.


Hyponatraemia and mesothelioma.

Hyponatraemia was found in 18 (62%) of 29 patients with histologically proven mesothelioma. The mechanism for the development of the hyponatraemia is discussed. It is suggested that patients with mesothelioma should be monitored for electrolyte abnormalities. If hyponatraemia is found the syndrome of inappropriate secretion of antidiuretic hormone should be suspected as treatment with demethylchlortetracycline may improve the quality of life in an otherwise fatal illness.


Initial photochemotherapy of psoriasis with orally administered 8-methoxypsoralen and longwave ultraviolet light (PUVA).

One hundred and seven patients with psoriasis underwent initial PUVA therapy. Complete clearance was obtained in 52.3% of the patients, incomplete in 40.2% while 7.5% of the patients did not respond at all. The non-responders to the regular PUVA treatment regime (0.5 mg 8-MOP/kg body-weight) were given an increased 8-MOP (8-methoxypsoralen) dose schedule (0.6 mg 8-MOP/kg body weight) and in 90.9% of these patients their lesions cleared after 35 PUVA exposures. However, a small percentage (9.1%) of the non-responders to the normal dose schedule did not want to continue the increased 8-MOP dose schedule because of persistent nausea. For this reason they were given regular PUVA therapy and they reacted well, but only after fifty-five exposures. Irrespective of the complete or incomplete clearance of psoriasis the patients remained in remission for a mean period of about 5 1/2 months. During the remission period the patients were advised to use a tar preparation or topical corticosteroids in the event of minor exacerbations.


Histopathology of remodelling induced by PUVA in the superficial dermis.

The histopathological alterations induced by PUVA within the skin are reported in a patient treated for psoriasis. The main impact is located at the site of former psoriatic plaques in which the target is the microvasculature. A residual coating sheath is present around vessels and fibrosis appears in the superficial dermis. These remodellings interfere with the normal dermo--epidermal relationships; the epidermis contains atypical keratinocytes, and melanocytes are no longer recognizable.


A history questionnaire for dermatology out-patients.

A questionnaire has been devised to be completed by new patients at home before attending the dermatology clinic. We report our experience with this questionnaire and discuss the potential advantages of this system; these include increased detail and accuracy of history, standardization of information and easier availability of information at patients' follow-up. Previously used questionnaires and other similar methods of obtaining a self administered history are discussed.


Acid hydrolases in blister fluid. 5. Influence of ultraviolet radiation in patients with polymorphic light eruption.

The minimal erythema dose for chronic polymorphic light eruption (CPLE) patients was significantly reduced for UV-B rays. However, following doses which produced comparable erythema, no significant differences were found in the release of acid hydrolases between CPLE patients and controls. An increased release of acid hydrolases was found to accompany the formation of papular lesions. Increased enzyme activity was first observed some hours after the appearance of the erythema.


Acid hydrolases in blister fluid. 6. Photocontact and contact allergic skin reactions.

Increased acid hydrolase activity has been demonstrated in the fluid from experimental suction blisters raised on the sites of contact or photocontact allergic skin reactions. The magnitude of the increases correlated well with the intensity of the reactions. The enzymes appeared after latent periods of 10 h for vesicular reactions, 15 h for papular reactions and 24 h for erythematous responses. The present observations are very similar to those reported following UV-B; thus our previous conclusions regarding the role of lysosomal hydrolases in UVR erythema may be generalized to include other forms of cutaneous inflammation.


Sézary's syndrome: a clinical evaluation of eight patients.

Clinical and laboratory investigations of 8 patients with Sézary's syndrome are presented. Sézary cells were found in peripheral blood as well as in bone marrow and lymph nodes. The overall follow-up period was 8 years. Different therapeutic regimens were given. The best results were obtained with chlorambucil and prednisone orally, combined with topical application of nitrogen mustard. Three patients died of pneumonia and septicaemia.


Pityriasis lichenoides chronica presenting as hypopigmentation.

Seven black or dark skinned patients with pityriasis lichenoides chronica (PLC) are described, who presented with widespread hypopigmentation. The evidence on which a diagnosis of PLC was made is presented and the differential diagnosis discussed. The hypopigmentation was noted in some patients to be especially marked on the proximal parts of the limbs and axillary folds. A diagnosis of PLC should be considered in all dark skinned patients with a widespread hypopigmented rash.


Trichilemmal horn: cutaneous horn showing trichilemmal keratinization.

A unique and distinctive clinicopathological entity occurred in nineteen patients who ranged in age from 16 to 72 (median 50) years. Clinically, these were solitary cutaneous horns. Nine were on the limbs, four on the back, two on the face, three on the scalp, and in one the site was not known; the median duration was 2 years. Histologically, there was a benign picture with a protrusion of massive horn and trichilemmal keratinization at the base. This tumour, which I have named trichilemmal horn, must be differentiated from other lesions that show trichilemmal keratinization (trichilemmal cyst, proliferating trichilemmal cyst, keratoacanthoma) and from other cutaneous horns, including trichilemmomal horn (cutaneous horn overlying trichilemmoma).


Clearing of psoriasis after cardiac surgery requiring cardiopulmonary bypass oxygenation: a corollary to clearance after dialysis?

A 54-year-old man had severe psoriasis with associated arthritis for 10 years. Attempts at therapy with tars, topical steroids and a short course of ultraviolet light were unsuccessful. His psoriasis cleared 6 weeks after cardiac surgery requiring cadiopulmonary bypass oxygenation.


Pili multigemini. Report of a case in association with cleidocranial dysostosis.

A patient with cleidocranial dysostosis developed extensive pili multigemini over the heavily bearded chin and cheek areas. Histological examination of serial sections revealed complicated follicular structures forming from two to as many eight hair shafts. Each hair is formed by a single branch of dermal papilla which is surrounded by all layers present in a normal follicle except for the outer root sheath cells. The outer root sheath surrounds the entire follicle. Irregularities in configuration of the hairs, longitudinal grooving and areas of bifurcation and re-adhesion of the hair shafts are demonstrated.


Familial Kaposi's sarcoma.

Kaposi's sarcoma occuring in a mother and son is described, and previous reports of familial occurrence of this disease are reviewed.


Lymphangioma-like Kaposi's sarcoma. A report of three cases.

Details of three patients with Kaposi's sarcoma are presented. Each case, although clinically typical, displayed unusual histological changes suggestive of lymphangioendothelioma. The significance of these changes with regard to the histogenesis of Kaposi's sarcoma is discussed.


Phenytoin dependent fibrosis in polyfibromatosis syndrome.

We have followed up the evolution of a polyfibromatosis syndrome in a 20-year-old male treated with diphenylhydantoin for epileptic convulsions. The main symptoms were attributable to fibrosis of the adventitial dermis associated with camptodactyly, knuckle pads, tendinous calcification, osseous hypoplasia of the face, scoliosis with thoracic deformation, and osteolytic changes of wrists, hands and feet. Quantitative variations of the mechanical properties of skin measured in vivo suggested a reversible fibrosing action of diphenylhydantoïn in a genetically predisposed patient.


Patterns of globin chain synthesis in erythroid colonies grown from sheep marrow of different developmental stages.

Erythroid colonies were grown from fetal sheep bone marrow at different stages of development and from adult marrow. Colony numbers increased with erythropoietin concentration, but fetal erythroid cells were more sensitive to the hormone than adult cells. Haemoglobin synthesis was characterized in the colonies and compared to that in control marrow incubations. No beta chain synthesis was detectable in marrow incubations or erythroid colonies from fetal marrow at 87-96 d gestation. Incubations of 115-120 d marrow shoed 5% beta chain synthesis while erythroid colonies synthesised up to 20% beta chains. Incubations and erythroid colonies from 125 d marrows showed about 27% beta chain synthesis, despite a similar increase in colony numbers with erythropoietin as the other marrows. Later in gestation, beta chain synthesis was lower in erythroid colonies than in marrow incubations and declined further in erythroid colonies as erythropoietin concentration increased. Adult marrow showed no detectable gamma chain synthesis but erythroid colonies produced up to 12% gamma chains, the identity of which was confirmed by peptide mapping. All changes in globin synthesis in vitro were correlated with colony numbers. In adult marrow, the maximal level of gamma chain production was inversely related to the cloning efficiency of the marrow samples studied. Potential uses of this model for studying globin gene expression are discussed.


In vitro culture of erythroid colonies from human fetal liver and umbilical cord blood.

Colony formation by erythroid precursors from human fetal liver, umbilical cord blood and adult peripheral blood has been studied in a plasma clot culture system. Fetal liver (FL) was obtained at post-mortem examination from 13-22 week abortuses. After mincing in Hanks' solution, cells in suspension were harvested by Ficoll-Hypaque centrifugation. Mononuclear cells were obtained by centrifugation of umbilical cord blood (CB) and normal adult peripheral blood (PB). All three types of preparations were incubated up to 14 d in 0.1 ml plasma clot cultures containing 0-4 u/ml erythropoietin (Epo) and 10(6) cells/ml. No colonies formed in the absence of Epo. Normal adult PB produced late-appearing colonies; there were no colonies at day 7 and up to 100 colonies/0.1 ml at day 14. CB produced early and late colonies with up to 200 colonies/0.1 ml at day 7 and 125 at day 14. Cells from FL produced many early colonies; over 1500 colonies/0.1 ml were sent at day 7 and there was a subsequent decline in colony count with longer incubation. In cultures of both CB and FL, colonies composed of either mature or immature cells were noted during both early and late stages of incubation suggesting that these cell sources contain a heterogeneous population of erythroid colony progenitors. Measurement of differential beta and gamma globin chain synthesis by erythroid colonies grown from fetal liver and umbilical cord blood gave results similar to those obtained by direct pulse-labelling of the original source of the cultured cells.


Some aspects of the biology of multinucleate and giant mononucleate erythroblasts in a patient with CDA, type III.

Marrow cells of a patient with CDA, type III, were (1) incubated with 3H-thymidine for 0.5 h and studied using a combination of Feulgen microspectrophotometry and light microscope autoradiography and (2) incubated with 3H-thymidine, 3H-uridine or 3H-leucine for 1 h and studied using the technique of electron microscope autoradiography. The data revealed multiple abnormalities in the proliferation of erythroblasts but no abnormality in the distribution of neutrophil promyelocytes and myelocytes in the different stages of interphase. Some mononucleate erythroblasts had DNA contents of 4-20c and the multinucleate erythroblasts had total DNA contents of 2-40c. In about 40% of the multinucleate erythroblasts, only some of the nuclei within the same cell incorporated 3H-thymidine and the electron microscope autoradiographic studies revealed that the nuclei which failed to synthesize DNA virtually always showed abnormalities in the electron-density of the heterochromatin or euchromatin or a Swiss-cheese appearance of the heterochromatin. Furthermore, in some multinucleate cells but not in others, the ultrastructurally abnormal nuclei showed a marked depression of RNA synthesis when compared with the normal-looking nuclei within the same cell. An occasional binucleate, multinucleate and giant mononucleate erythroblast showed ultrastructural changes suggestive of advanced degeneration and such cells, which showed a marked depression of RNA and protein synthesis, appeared to be phagocytosed by macrophages.


Abnormal red-cell metabolism of pyridoxine associated with beta-thalassaemia.

Red-cell conversion of pyridoxine to pyridoxal phosphate was studied in control subjects, and patients with heterozygous and homozygous beta-thalassaemia. In 7% of control subjects the rate of pyridoxine conversion was well below the range found in the other control subjects (5.0-8.6%, mean 6.5%/g Hb x 10(-2)) but in heterozygous beta-thalassaemia was below that range in 63% of the patients. The conversion rate was also slow or borderline in the majority of patients with severe transfusion-dependent homozygous beta-thalassaemia, in spite of the presence of some donor cells; but was normal, or fast as in other anaemias, in all but one patient with mild homozygous thalassaemia. There was a much higher incidence of a slow conversion rate in the parents of the severe homozygotes than in parents of the mild homozygotes, illustrating the familial pattern. This supports our view that the red-cell conversion rate of pyridoxine is an inherited characteristic, independent of thalassaemia. The cause of a reduced rate of pyridoxine conversion was investigated. The increase to a normal rate following riboflavin ingestion suggests a defect in the activity of the flavin mononucleotide (FMN)-dependent pyridoxine phosphate oxidase.


Comparison of in vitro effects of normal IgG and of a monoclonal IgG on the rheological behaviour of erythrocytes.

Erythrocyte sedimentation, viscosity, electrophroetic mobility, and osmotic fragility were measured in a study of the rheological behaviour of the erythrocyte in vitro. In comparison with physiological saline solution, a suspending medium containing a gamma2lambda2 monoclonal IgG from a patient with multiple myeloma caused rheological changes 2-3 times as great as a medium with normal IgG. Our results demonstrate the existence of nonimmunological interactions between erythrocytes and normal or monoclonal immunoglobulins, and they raise questions about the widely held assumption that paraproteins are structurally 'normal'.


Heat-induced erythrocyte fragmentation in neonatal elliptocytosis.

Erythrocytes from neonates with elliptocytosis were studied for their pattern of heat-induced fragmentation. Membrane alterations began at 44 degrees C. There was a gradual progression in shape changes as the temperature was increased to 47 degrees C, at which point frank fragmentation occurred. Normal erythrocytes show no morphologic changes until the critical temperature of fragmentation, 49 degrees C is reached. Heat studies were repeated a few months later, at a time when the patients' erythrocyte morphology had become typical of elliptocytosis. Morphologic changes occurred abruptly at 48 degrees C with complete fragmentation. Increased thermal sensitivity of the red cell membrane has previously been demonstrated for pyropoikilocytes, and these studies suggest that some cases of elliptocytosis may be mild expressions of a similar membrane defect.


N-alkaline phosphatase: a potential disease marker for lymphoproliferative disorders.

A characteristic alkaline phosphatase (orthophosphoric monoester hydrolase, alkaline pH optimum, EC 3.1.3.1) was detected in the sera of most patients with infectious mononucleosis, acute and chronic lymphatic leukaemia, non-Hodgkin's lymphoma, Burkitt's lymphoma and nasopharyngeal carcinoma. The enzyme was also present in the sera of nine out of 26 patients with cancer of the cervix. N-APase in these cases counted 30-100% of the total alkaline phosphatase activity. N-APase was absent from the sera of healthy individuals and of patients with acute and chronic granulocytic leukaemia, breast cancer, colon cancer, rheumatoid arthritis, ulcerative colitis, systemic lupus erythematosis, hepatitis and obstructive jaundice. Only three of 22 patients with Hodgkin's disease showed n-apase activity in the serum. In infectious mononucleosis the presence of N-APase activity was well correlated with the clinical course. In 13 cases studied, the clinical improvement was associated with the decrease or disappearance of N-APase activity. N-APase activity could not be detected in white cells of acute myeloid leukaemic patients, nor in the cells of myeloid blastic crisis of chronic granulocytic leukaemia. It was present in the cells of lymphoid blastic crisis of chronic granulocytic leukaemia.


The determination of antithrombin III by radioimmunoassay and its clinical application.

A radioimmunoassay (RIA) had been developed for the determination of antithrombin III (AT III) in man. The detection limit was 25 microgram/dl. AT III-RIA level and biological activity (anti-Xa) was significantly correlated (r = 0.737, P less than 0.001). Plasma levels in 36 healthy males (mean +/- SD, 19.9 +/- 2.5 mg/dl) and 21 healthy females (19.1 +/- 2.4 mg/dl) were similar. Serial AT III measurements in normal menstruating females showed lower levels during midcycle and higher concentrations during menstruation. In carcinomas, the AT III levels were lower than normal, particularly in hepatocellular carcinoma. In cirrhosis of liver, the levels were markedly decreased and in some patients were below that found in congenital AT III deficiency. Patients with deep vein thrombosis and patients with heart valve replacement had lower levels than normal, while patients with cerebral vascular occlusion had normal levels. The possible use of AT III as a diagnostic tool of post-operative deep vein thrombosis was demonstrated in one patient after hysterectomy. The increased sensitivity, specificity and precision of this type of assay offer distinct advantages over existing methods of AT III estimation.


Anticoagulant activities of high and low molecular weight heparin fractions.

The anticoagulant activities of high and low molecular weight heparin fractions were measured by three assay methods, both in vitro, and after intravenous injection in volunteers. The low molecular weight (LMW) fraction had similar anti-Xa activity in vitro to the high molecular weight (HMW) fraction, but in APTT assays the HMW fraction was about twice as potent. After intravenous injection, the two fractions gave equal heparin levels by anti-Xa assays, but in APTT assays using synthetic substrate S-2222 gave about 20% lower levels than anti-Xa clotting assays for both heparins. Complete protamine neutralization of the post-injection heparin activity was found in APTT and synthetic substrate assays, but about 20% of the clotting anti-Xa effect could not be neutralized. Complete neutralization of the fractions by protamine was shown by all three assays in vitro. This non-neutralizable activity probably accounts for the difference between the anti-Xa clotting and synthetic substrate assays. Studies by crossed immunoelectrophoresis and affinity chromatography indicated that the antithrombin III binding properties of the two fractions were similar.


Synthesis of procoagulant factor VIII, factor VIII related antigen and other coagulation factors by the isolated perfused rat liver.

The synthesis of factor VIII and other coagulation factors has been studied using an isolated, perfused rat liver. Synthetic function of the liver was validated by adding [35S]L-methionine to the perfusion medium and performing two-dimensional crossed immunoelectrophoresis and autoradiography on samples obtained during perfusion. Progressive incorporation of radioactivity into plasma proteins was demonstrated. This was inhibited by cycloheximide. Coagulation factor assays demonstrated synthesis of factors II, IX and X and of factor V and procoagulant factor VIII (VIIIC). Synthesis of factor VIII related antigen (VIIIRAg), measured in an immunoradiometric assay, was not significantly demonstrated. Addition of warfarin to the perfusion medium inhibited the synthesis of factors II, IX and X but not of factors V and VIII. Cycloheximide completely inhibited synthesis of all coagulation factors but actinomycin acted only after a latent period. Reticuloendothelial cell blockade was attempted by adding ethionine to the perfusion medium or by administration of Indian ink to the donor animals prior to removal of the livers. In these instances synthesis of factor V and factor VIIIC was inhibited but not that of factors II, IX and X. The results confirmed the functional capacity of the isolated liver for synthesizing proteins and the vitamin K dependent coagulation factors, and suggested similar kinetic features for the synthesis of factors V and VIIIC. Failure to detect significant VIIIRAg synthesis in these experiments is consistent with the hypothesis that this protein is released by vascular endothelial cells throughout the body and is activated or joined to VIIIC or stimulates its production in the liver.


The adrenalin binding site on human platelets.

Using 3H-adrenalin the quantity and characteristics of the adrenalin receptors on human platelets were evaluated. Maximum adrenalin binding by intact platelets from eight normal subjects averaged 3.2 ng adrenalin/10(8) platelets, equivalent to 105 000 binding sites per platelet. Bound 3H-adrenalin could be dissociated or binding could be inhibited by an excess of unlabelled adrenalin, noradrenalin, isoproterenol or dopamine; phentolamine or propanalol had no effect on 3h-adrenalin binding, even in concentrations 10 000-fold greater. These studies show that the epinephrine binding site can be detected and quantitiated on human platelets and that its characteristics differ from those classically attributed to either alpha or beta adrenergic receptors.


A retrospective study of induction of labour.

The labour, delivery and state of the newborn were compared retrospectively in a group of women who had labour induced and a group who started labour spontaneously. There were 200 women at term in each group, individually matched for age, parity and social class and with no medical or obstetric complications prior to labour. The induced group showed a higher incidence of epidural anaesthesia, fetal monitoring and operative delivery. Sixty had forceps deliveries and five had Caesarean sections compared with two forceps deliveries and no Caesarean sections in the 'spontaneous onset' group. The higher incidence of operative deliveries in the induced group was not fully explained by the excess of epidurals and fetal monitoring received. More of the babies born after an induced labour had endotracheal intubation during resuscitation.


The technique of fetal electroencephalography during labour.

A method for recording the fetal electroencephalogram (EEG) during labour is described. Technical specifications for the construction of a suitable electrode are detailed. One hundred and twenty-three patients in labour were monitored using this technique and the success rate of obtaining records for analysis was 91 per cent.


The effect of fetal head compression and fetal acidaemia during labour on human fetal cerebral function as measured by the fetal electroencephalogram.

We investigated the effect of head compression and acidaemia during labour in 25 African primigravidae. Evidence that head compression had occurred during labour was confirmed by both clinical and radiological means at the end of a trial of labour. No significant difference could be demonstrated in the quantity of fetal electroencephalogram (EEG) abnormality that occurred in the groups with marked head compression as compared to the groups without marked head compression. Deterioration in the fetal EEG to a flat record known as electrocerebral silence (ECS) was associated with the development of acidaemia. As fetal heart rate (FHR) decelerations appeared the percentage of ECS in the fetal EEG record increased significantly (P less than 0.05), and likewise, as fetal acidaemia developed a highly significant increase in ECS in the fetal EEG was demonstrated (P less than 0.001). We concluded that in the management of trial of labour there was no significant deleterious change in the fetal EEG as a result of head compression, unless fetal acidaemia supervened. However, in the majority of these cases a significant increase in ECS to more than 20 per cent occurred in association with only moderate acidaemia (pH 7.25 to 7.30).


Real-time ultrasound observation of fetal activity in labour.

Fetal activity was observed by realtime ultrasound for one hour in 25 patients in labour. Fetal respiratory movement, although reduced compared to the antenatal period, was observed in 13 patients. In three patients, hiccough-like movements were noted. Fetal body and/or extremity movements were observed in all patients (mean 41/hour) and frequently occurred in episodes. These bursts of movements were significantly correlated with contractions and fetal heart rate (FHR) accelerations. There was a significant association between absent or reduced number of fetal respiratory movements and/or hiccoughs (10 or less/hour) and an abnormal FHR. Absence of any type of fetal activity was also significantly associated with an abnormal FHR. The correlation between fetal activity and conventional methods of fetal assessment is discussed.


Prostaglandins in umbilical plasma at elective caesarean section.

Prostaglandin E (PGE), prostaglandin F (PGF) and 13,14-dihydro-15-ketoprostaglandin F (PGFM) have been measured in umbilical cord plasma obtained immediately after delivery by elective Caesarean section. Umbilical venous plasma levels of PGE were significantly greater than the corresponding arterial levels (p less than 0.02); there were no significant arterio-venous differences for PGF or PGFM. In general, concentrations of PGFM exceeded those of PGE which in turn were greater than concentrations of PGF. Umbilical venous levels of PGE and both arterial and venous levels of PGF and PGFM were significantly greater after spontaneous labour with vaginal delivery than after elective Caesarean section.


Sequential changes in the human renin-angiotensin system following therapeutic termination of pregnancy.

Plasma renin and angiotensin II levels were measured in nine patients immediately before and at half-hourly intervals in the four hours following therapeutic termination of pregnancy. There was a small fall in renin and angiotensin II levels over the first 1 to 2 hours, followed by a slight increase. The magnitude of these effects was much smaller than those previously seen following normal delivery. It is concluded that in early pregnancy maternal, rather than feto-placental, factors are controlling the renin-angiotensin system.


Ultrasound evidence of early pregnancy failure in patients with multiple conceptions.

I report a review of 1500 pregnant patients: 30 of them (or 1 in 50) were thought to have multiple pregnancies when examined by ultrasound in the first trimester. Only 14 of the 30 patients (46.7 per cent) produced live infants: 13 twins and one set of triplets. Twelve (40 per cent) had bleeding in early pregnancy and 11 of these had a spontaneous abortion. Five of seven patients found to have a normal pregnancy and a coexistent blighted ovum, ultimately delivered a single live infant.


Prenatal and postnatal maternal leucocyte ascorbic acid concentrations.

Leucocyte ascorbic acid was measured in 79 pregnant women in the 34th to 38th week of gestation and on the first, and third or fifth days post partum. No differences were found for time of year or age of the subjects. There was a significantly lower vitamin C status in social classes IV and V. The leucocyte ascorbic acid concentration fell significantly on the first day post partum and rose again to initial values by the fifth day. In late pregnancy 40 per cent of all subjects had leucocyte ascorbic acid concentrations less than the lower limit of normal (100 nmol/10(8) leucocytes). This rose to 75 per cent on the first day post partum.


Effect of intrauterine contraceptive device on uterine haemostasis: a morphological study.

The effect of the intrauterine contraceptive device (IUCD) on uterine haemostasis was studied at various stages of the menstrual cycle in a series of 46 patients by light- and electron-microscopy and by following the distribution of an infusion of 51Cr-labelled autologous platelets. The endometrium in contact with the IUCD in the majority of cases showed grooving with atrophy and mild chronic inflammation in the surrounding tissues. The adjacent stroma also showed increased vascularity and occasional foci of haemorrhage but the increased blood loss associated with the presence of the IUCD could not be attributed to mechanical erosion or stromal blood vessels by the device. During menstruation the presence of an IUCD does not appear to inhibit the formation of fibrin/platelet thrombi although both in control and IUCD patients there was a striking paucity of platelet/fibrin thrombi in circumstances where their formation should be enhanced. In contrast to other workers we have not observed that gaps or breaks in the endothelial lining of endometrial blood vessels occur with any greater frequency in patients fitted with an IUCD. The principal mechanism by which uterine haemostasis is achieved remains to be established.


Vascular changes in the iris in chronic anterior uveitis.

Vascular changes of the iris were studied in 6 patients with chronic anterior uveitis of varying aetiology by means of fluorescein angiography. In 1 case the angiographic findings of the second eye were compared with the histopathological changes in the fellow eye enucleated earlier because of absolute glaucoma. In the milder cases abnormal superficial vessels were seen mainly in relation to the minor arterial circle of the iris, while in advanced cases a coarse vascular network covered the whole surface of the iris. Both mild and severe changes were found to differ from the arborising type of neovascularisation usually seen in vascular eye diseases. Instead, a resemblance in the vascular pattern was found between some of these cases and eyes with chronic capsular glaucoma. In 1 patient clinical rubeosis seemed to be partly due to dilatation of the pre-existing capillaries rather than true neovascularisation.


Development and classification of rubeosis iridis in diabetic eye disease.

The evolution and characteristics of diabetic rubeosis were studied in 33 eyes, and the following vascular abnormalities were found: (1) Dilated leaking capillaries around the pupil; (2) irregular or slow filling of the radial arteries; (3) superficial arborising newly formed vessels, usually starting in the chamber angle; and (4) dilatation and leakage of the radial vessels either before or after the development of neovascular glaucoma. None of the vascular changes was specific for diabetes. Four grades of diabetic iridopathy were distinguished on the extent of rubeosis: I, peripupillary vessel dilatations with leakage; II, early neovascularisation mainly in the chamber angle; III, prominent rubeosis with or without neovascular glaucoma; and IV, florid rubeosis.


Value of fluorescein iridography in diagnosis of tumours of the iridociliary zone.

Fluorescein iridoangiography of 33 eyes with benign tumours and 22 eyes with malignant tumours is discussed. In cases of leiomyoma the tumour vessels have a distinct pattern, and fluorescence of the tumour tissue is mottled and short-lived. Malignant tumours are characterised by distortion of their vessels. Simultaneously the patches of fluorescence appear in the tissue of the tumour and lead to a diffuse confluence. Changes in the architecture of the vessels of the iris near the tumour and absence of fluorescence at the pupil margin indicate growth of the neoplasm.


Postinflammatory and malignant protein patterns in aqueous humour.

Normal aqueous humour and the aqueous humour of patients with cataract is virtually protein-free. Patients having retinoblastoma and non-malignant postinflammatory lesions show significantly high quantities of proteins in the aqueous humour. Retinoblastoma is associated with an increase in the globulin content and an albumin/globulin ratio below unity, while non-malignant postinflammatory intraocular conditions show a rise of the albumin fraction with an albumin/globulin ratio above unity. It seems likely that the leakage of protein into the aqueous is different in the two conditions, and a transcellular route is postulated as being the cause in malignant conditions. The estimation of protein patterns in the aqueous humour may be of value in the diagnosis of intraocular malignancy.


Primary cutaneous malignant melanoma metastatic to the iris.

We describe a case of cutaneous malignant melanoma metastatic to the iris as the first presenting sign of metastases 10 years after excision of the primary tumour. The histology of the excised iris metastases and the primary skin melanoma are compared. Progressive intracranial metastatic growth did not correlate with the observed regression of the residual intraocular melanoma cells during cyclical cytotoxic therapy.


Thyroid carcinoma metastatic to the globe.

We report a case of thyroid carcinoma metastatic to the globe. In our survey we found thyroid carcinomas to metastasise to the eye rarely. In addition, we found metastases from the thyroid to be more common to the orbit than to the globe--a reverse of the pattern noted with tumours in general.


Epipalpebral conjunctival osseous choristoma.

Two cases of osseous choristoma are described. One of the tumours was found in the conjunctiva of the right lower lid, an apparently unique location. The other more typical epibulbar tumour was found in the superior temporal quadrant of the episclera between the lateral and superior rectus muscles. In both instances the tumour was suspected of being a dermoid.


Scleral and choroidal calcifications in a patient with pseudohypoparathyroidism.

This case report suggests that calcifications in the sclera and choroid may be a feature of pseudohypoparathyroidism and shows a calcium distribution more extensive and severe than that characteristically seen in other metabolic conditions associated with calcium deposits in the eye. Ocular ectopic calcification is a part of the more generalised ectopic soft tissue calcification seen with pseudohypoparathyroidism. Evaluation of the contribution of the terminal renal failure and uraemia to the ectopic ocular calcification would require sequential evaluation of eyes with pseudohypoparathyroidism, but without the added complication of uraemia.


Convolution in human rods: an ageing process.

In a morphological survey of 73 human retinae spanning 9 decades, and including 20 retinae which were obtained from eyes enucleated for malignant melanomas, nodular excrescences were noted in the outer segments of rods with an incidence which increased with age. These structures were sectioned in both their vertical and horizontal axes and on electron microscopical examination were seen to result from the localised convolution of affected outer segments. The topographic morphology of such convolutions is described and their modes of formation are discussed.


Optic neuropathy in ketogenic diet.

A symmetrical, bilateral optic neuropathy is reported in 2 patients being treated with ketogenic diets for seizure control. Laboratory tests suggested a thiamine deficiency, and both patients recovered normal visual function after several weeks of treatment with thiamine. The risk of optic nerve dysfunction occurring during the treatment with a ketogenic diet can be minimised if routine vitamin B supplements are given and periodic evaluation of optic nerve function undertaken.


Assessment of unilateral afferent pupillary defects by pupillography.

A pupillographic technique, an adaptation of the swinging light test, is described for the measurement of unilateral afferent pupillary defects. In normal persons it yielded accurate, reproducible estimates of the magnitude of artificial afferent defects made with neutral filters of 1 to 4 log units density. In 15 studies on 8 patients with unilateral optic neuritis and 2 with unilateral compressive lesions the measured afferent pupillary defects correlated closely with both visual acuity and visual threshold deficits. Measurement of the pupillary defect underestimated severe degrees of impairment revealed by visual threshold determination. Discrepancies between pupillary defect and visual acuity were observed in the 2 patients with compressive lesions and in 1 patient who had a central scotoma 4 months after an attack of optic neuritis.


Pituitary adenoma and orbital haemangioma: a case report.

A 43-year-old woman with a pituitary tumour had unexplained right proptosis. Orbital x-rays and ultrasound were normal, but computed tomography showed an intraconal tumour, which had typical features of a cavernous haemangioma on carotid angiography. Both tumours were resected and confirmed histologically.


A new method of estimating the depth of the anterior chamber.

A new method of estimating the depth of the anterior chamber with an accuracy of approximately 0-1 mm is described. The estimate is made with a conventional Haag-Streit 900 slit lamp without any extra attachments.


One gonioscopic fallacy.

Traditional gonioscopic practice assumes that, if most of the angle is gonioscopically closed, intraocular pressure increases. Evidence is produced to show that this is fallacious, because at its inception angle closure is iridocorneal contact occurring on the corneal side of the limbus. Although the angle cannot be seen by means of a gonioscope, there is initially no iridotrabecular contact. It is only after pressure increases that iris is pushed against trabecular meshwork and the angle is truly closed.


Comparison of the pupillary, refractive, and hypotensive effects of Ocusert-40 and pilocarpine eyedrops in the treatment of chronic simple glaucoma.

Twelve patients with chronic simple glaucoma were treated for 1 week with Ocusert-40, and the effects on the pupil, intraocular pressure, and refraction were measured. A comparison was made with the effects of pilocarpine eyedrop therapy in 8 of these patients. Both forms of pilocarpine treatment gave satisfactory control of intraocular pressure and both constricted the pupil to a similar degree shortly after initiation of treatment. During the week on Ocusert-40 the pupils redilated, so that 54% of the initial miosis remained after 7 days' wear. There was, however, considerable variability in the pupillary sensitivity to Ocusert-40 within and between eyes, and it is suggested that this may have been partly due to variable release rates of pilocarpine. Only minor decreases in visual acuity and refraction resulted from pilocarpine in either form. Seven of the 12 patients rejected Ocusert-40 because of discomfort or inability to retain the device.


Transient ocular hypertension following trabeculectomy.

A study of the postoperative evolution of 70 eyes subjected to trabeculectomy over a 3-year period is reported. This is the operation of choice in our department in cases of chronic open-angle glaucoma and chronic angle-closure glaucoma. The study showed that in 35.7% of the operated eyes the intraocular pressure was increased on the second postoperative day and became normal within a week. This raised intraocular pressure did not influence the final result of the operation.


Amblyopia: the need for a new approach?

Follow-up of a series of 221 children identified by the present methods of screening, and presenting consecutively with squint and/or amblyopia, shows that there has been no demonstrable improvement in the overall incidence and severity of amblyopia 3 or more years later. A new approach to the problem is required, and this might be based on the identification and treatment of amblyopia during the 'sensitive period'.


Refraction as a means of predicting squint or amblyopia in preschool siblings of children known to have these defects.

215 preschool siblings of children presenting with squint/amblyopia were screened by refraction after cycloplegia. The presence of +2.00 or more D of spherical hypermetropia in both eyes, or +1.00 or more D sphere or cylinder of anisometropia was significantly associated (P=0.0779%) with that child being identified 2+ years later as having either squint or amblyopia or both. Astigmatism of +1.50 or more D in either eye was significantly associated with anisometropia (P=0.000 0013%). If bilateral hypermetropia of +2.00 or more DS and/or +1.50 or more D of astigmatism in either eye had been taken as criteria for abnormality (ignoring anisometropia), there was a more significant association (P=0.0025%) between refraction and squint/amblyopia in these siblings. Such a child had 4 times more chance of having a visual defect than one who had no error of refraction when screened. These findings suggest that an environmental factor such as blurred vision may be relatively more important as a cause of squint/amblyopia than a genetically determined neurological abnormality.


Screening for refractive errors at age 1 year: a pilot study.

Cycloplegic refraction of 1-year-old children is technically possible and is acceptable to mothers as a method for screening children for visual defects. The range of refractions found in a sample of 186 1-year-old children is reported. Prediction of which children are significantly at risk for squint and/or amblyopia is possible on the basis of refractions at age 1 year according to the criteria selected for an 'abnormal' refraction. Bilateral hypermetropia and/or astigmatism or anisometropia at age 1 year was significantly (P less than 1 in 10 000) associated with a child eventually being found to have squint or amblyopia. Both the age of screening and criteria of abnormality will probably need modification. +2.50 or more D hypermetropia in any one meridian of either eye at age 1 year was even more significantly (P = 0.000 000 05%) associated with squint and/or amblyopia. The possibility that meridional hypermetropia could be the basic defect in squint and amblyopia is discussed.


Uptake and loss of tears from filter paper discs employed in lysozyme tests.

Aspects of the use of filter paper discs in collecting tear samples for lysozyme tests were studied by serial weighings of bijoux bottles and tear-moistened discs. Blotted discs carried from 1.9 to 3.6 mg tears, but for practical purposes no deposit was left in the bottle. Unblotted discs carried a greater quantity of tear fluid, but the majority deposited a proportion of this in the bottle, and a correcting factor is therefore proposed for the lysozyme assay technique of Mackie and Seal. Substantial vaporisation of tears occurred within the bottles.


Isolation of Chlamydia trachomatis from eye secretion (tears).

Shedding of Chlamydia trachomatis in the eye secretion (tears) of patients with either hyperendemic trachoma or paratrachoma was studied. The method of collection of eye secretion with cellulose sponges is proved to be simple, faster, and more practicable and yielded a higher rate of chlamydial isolation than aspiration. The chlamydial isolation rates in eye secretion in chlamydia-positive paratrachoma patients in London or trachoma patients in Iran was 84 and 49% respectively. It was found that the chlamydial isolation rate from eye secretion is directly related to the number of inclusions present in the conjunctival swabbings. The results of this study indicated that patients with moderate to severe hyperendemic trachoma or paratrachoma are the main reservoir of infection. In the developing countries of the Middle East and Africa the shedding of chlamydia in the eye secretion of persons with these diseases is a major factor in the transmission of them by means of flies, fingers, towels, or bed clothes.


Herpetic canalicular obstruction.

Viral infection is a common cause of acquired obstruction of the lacrimal canalicular system. A series of 20 patients with canalicular obstruction attributable to infection with herpes simplex is reported, and 1 case is described in detail.


An aid to successful probing of the nasolacrimal duct.

An electrical method is described for confirming the entry into the nose of a nasolacrimal probe.


Corneal thickness in extended wear of soft contact lenses.

The continual wearing of a soft contact lens for a period of 20 weeks is shown to produce no significant evidence of corneal swelling, although the use of soft lenses in conjunction with a topically applied solution is shown to produce evidence of transient swelling. The extent of corneal thinning during waking hours is also shown to be reduced among wearers of contact lenses for long periods.


Studies of the composition of purified Torpedo californica acetylcholine receptor and of its subunits.

Under conditions that limit proteolytic degradation, the detergent-solubilized purified receptor protein from Torpedo californica exists in monomeric and dimeric forms. The purified receptor complex is composed of four different polypeptide subunits of apparent molecular weights 40 000, 50 000, 60 000, and 65 000. The individual polypeptides have been purified and their amino acid compositions have shown them to be relatively hydrophobic. In addition, the carbohydrate composition of the intact receptor complex and of the individual polypeptides has been determined. Amino acid analysis provided evidence for the occurrence of a component with chromatographic properties similar to those of phosphoserine. Treatment of receptor with CH3NH2 in base, a condition which provided quantitative modification of O-phosphoserine residues in beta-casein, completely eliminated the peak corresponding to phosphoserine following mild acid hydrolysis. We conclude that the receptor contains O-phosphoserine residues to the extent of approximately seven residues per molecule and these residues occur in all constituent polypeptides. Other forms of O-substituted serine and threonine were also shown to occur, most likely as glycosylated residues.


Uses of fluorescent cholinergic analogues to study binding sites for cholinergic ligands in Torpedo californica acetylcholine receptor.

A series of synthetic 1,n-bis(3-aminopyridinio)-alkane fluorescent probes have been used to determine the ligand binding properties of the acetylcholine receptor purified from Torpedo californica electroplax. At equilibrium, the probes bound to a single class of sites. The binding affinity of the fluorescent decamethonium analogues increased progressively as the number of methylene groups (n) increased from 4 to 12 and decreased in the range of 16--18 such groups. The receptor bound 1,12-bis(3-aminopyridinio)dodecane and 1,14-bis(3-aminopyridinio)tetradecane with the highest affinity while related monofunctional probes such as 1-(3-amino-pyridinio)propane were bound with a substantially lower affinity. The data indicate that the receptor interacts strongly with both ends of a bifunctional probe such as 1,14-bis(3-aminopyridinio)tetradecane. Also, competition between bifunctional fluorescent probe binding and the binding of conventional cholinergic ligands, was investigated and led to the conclusion that the probes, which are antagonists, form ternary complexes in the presence of acetylcholine.


Studies of reversible and irreversible interactions of an alkylating agonist with Torpedo californica acetylcholine receptor in membrane-bound and purified states.

The interaction of a cholinergic depolarizing agent, bromoacetylcholine, with acetylcholine receptor (AcChR) enriched membrane fragments and Triton-solubilized, purified AcChR from Torpedo californica has been studied. The reagent bound to membrane-bound AcChR reversibly with an apparent dissociation constant of 16 +/- 1 nM at equilibrium. This 600-fold higher affinity for the receptor than found from physiological studies [Kact congruent to 10 micrometers; Karlin, A. (1973) Fed. Proc. Fed. Am. Soc. Exp. Biol. 32, 1847--1853] can be attributed to a ligand-induced affinity change of the membrane-bound receptor upon preincubation with bromoacetylcholine. At equilibrium [3H]bromoacetylcholine, like acetylcholine, bound to half the number of alpha-bungarotoxin sites present in the preparation without apparent positive cooperativity, and this binding was competitively inhibited by acetylcholine. In the presence of dithiothreitol, [3H]bromoacetylcholine irreversibly alkylated both membrane-bound and solubilized, purified acetylcholine receptor, with a stoichiometry identical with that for reversible binding. NaDodSO4-polyacrylamide gel electrophoresis of the labeled acetylcholine receptor showed that only the 40 000-dalton subunit contained the label. From these results it is concluded that the 40 000-dalton subunit represents a major component of the agonist binding site of the receptor.


Ligand-induced changes in membrane-bound acetylcholine receptor observed by ethidium fluorescence. 1. Equilibrium studies.

The interactions between the fluorescent probe ethidium and acetylcholine receptor enriched membranes from Torpedo californica are described. One class of saturable ethidium sites was blocked by alpha-bungarotoxin and therefore reflects direct binding to the receptor (Kd approximately 3 micrometers; stoichiometry--one ethidium site per two alpha-bungarotoxin sites). The second class of sites was nonsaturable and unaffected by alpha-toxin and was therefore considered nonspecific in nature. The increase in fluorescence intensity observed upon addition of cholinergic agonists and antagonists accurately reflects the dissociation constant and stoichiometry of the high-affinity receptor sites for these ligands. The effects of local anaesthetics are complex in nature and depend on the structure of the ligand. For carbamylcholine, the increase in flourescence intensity was due to an increase in the quantum yield of the dye bound to the membrane rather than a dye uptake. In general, ethidium appears not to strongly alter the properties of the membrane-bound acetylcholine receptor and can therefore be profitably used as a spectroscopic probe.


Ligand-induced changes in membrane-bound acetylcholine receptor observed by ethidium fluorescence. 2. Stopped-flow studies with agonists and antagonists.

The kinetics of cholinergic ligand binding to membrane-bound acetylcholine receptor from Torpedo californica have been followed in a stopped-flow photometer, by using the fluorescent probe ethidium. The overall reaction amplitude, as a function of ligand concentration, can be fit to the law of mass action for both agonist and antagonists. All agonists show at least biphasic kinetics, and the concentration dependence of the kinetic parameters is fit by a common mechanism involving sequential binding of ligands with increasingly lower affinity. The receptor-ligand precomplexes isomerize to different noninterconvertible final complexes depending on the number of ligands bound. In contrast, the kinetics observed with antagonists cannot be fit to a common model. These kinetics are always much slower than those observed with agonists, and the relaxation rates depend only weakly on antagonist concentration.


Ligand-induced changes in membrane-bound acetylcholine receptor observed by ethidium fluorescence. 3. Stopped-flow studies with histrionicotoxin.

Rapid kinetic studies of histrionicotoxin interactions with membrane-bound acetylcholine-receptor showed a conformational change in the receptor-histironicotoxin complex as reflected by a decrease in fluorescence intensity of the extrinsic probe ethidium. The simplest kinetic mechanism consistent with the observed data is one in which a rapid preequiliibrium exists between receptor and toxin (K = 3.33 micrometers), followed by a slow conformational change (k1 congruent to 2 X 10(-2) s-1 and k-1 congruent to 1.5 X 10(-3) s-1). The overall equilibrium constant (Kov) determined from a fit of the amplitude dependence on toxin concentration had a value of 0.25 micrometer. The data preclude kinetic mechanisms where histrionicotoxin acts as an effector, shifting equilibria between preexisting, discrete, and slowly interconverting receptor forms.


Electrostatic effects in hemoglobin: hydrogen ion equilibria in human deoxy- and oxyhemoglobin A.

The modified Tanford-Kirkwood theory of Shire et al. [Shire, S. J., Hanania, G.I.H., & Gurd, F.R.N. (1974) Biochemistry 13, 2967] for electrostatic interactions was applied to the hydrogen ion equilibria of human deoxyhemoglobin and oxyhemoglobin. Atomic coordinates for oxyhemoglobin were generated by the application of the appropriate rigid rotation function to alpha and beta chains of the deoxyhemoglobin structure [Fermi, G. (1975) J. Mol. Biol. 97, 237]. The model employs two sets of parameters derived from the crystalline protein structures, the atomic coordinates of charged amino acid residues and static solvent accessibility factors to reflect their individual degrees of exposure to solvent. Theoretical titration curves based on a consistent set of pKint values compared closely with experimental potentiometric curves. Theoretical pK values at half-titration for individual protein sites corresponded to available observed values for both quaternary states. The results bring out the cumulative effects of numerous electrostatic interactions in the tetrameric structures and the major effects of the quaternary transition that result from changes in static solvent accessibility of certain ionizable groups.


Identification of interacting amino acids at the histone 2A--2B binding site.

Histones 2A and 2B of calf thymus were cross-linked within intact nuclei by UV irradiation. This procedure induces the formation of covalent cross-links between noncovalently interacting residues in the histones of native chromatin. Tryptic peptide and partial sequence analysis of the cross-linked product has shown that the covalent linkage is between tyrosine-37, -40, or -42 (we have not yet determined which) of H2B and proline-26 of H2A. We conclude that these residues constitute part of the hydrophobic H2A--H2B binding domain within the nucleosomes of native chromatin.


Amino acid contacts between histones are the same for plants and mammals. Binding-site studies using ultraviolet light and tetranitromethane.

Leek chromatin has been cross-linked by UV light and tetranitromethane. The same major H2A--H2B and H2B--H4 cross-linked dimers are formed as in mammalian chromatin. CNBr peptide mapping shows that the cross-links occur in the same regions of the histone sequence for both plants and mammals. Interspecies complexes formed between leek and calf H2A and H2B can be cross-linked by UV light with the same specificity as intraspecies H2A--H2B complexes. We conclude that certain geometric features of histone-histone binding sites are conserved precisely during evolution despite large changes in the overall histone sequence. Moreover, our data show that identification of cross-linked amino acids using binding-site probes such as UV light and tetranitromethane can yield significant information about thermodynamically important contacts within histone-histone binding sites.


Prothrombin domains: circular dichroic evidence for a lack of cooperativity.

The far-ultraviolet circular dichroism spectra of bovine and human prothrombin, prothrombin fragment 1, prethrombin 1, prothrombin fragment 2, and prethrombin 2 (prethrombin 2des(1--13)) were determined and the method of Chen et al. [Chen, Y. H., Yang, J. T., & Martinez, H. M. (1972) Biochemistry 11, 4120--4131; Chen, Y. H., Yang, J. T., & Chau, K. H. (1974) Biochemistry 13, 3350--3359] was used to calculate the apparent alpha-helix, beta-sheet, and random-coil contents of each protein. Prothrombin and its activation components were found to contain a large amount of aperiodic secondary structure and there was little species difference between the spectra and, thus, secondary structures. The hypothesis that the prothrombin activation components exist as relative ly noncooperative "domains" within the prothrombin molecule was tested by comparing the circular dichroism spectrum of prothrombin with the sum of the spectra of the components. It support of the hypothesis, no gross alterations in the spectra and, hence, secondary structures of the components were found to have occurred upon activation.


Stereochemistry and kinetic isotope effects in the bovine plasma amine oxidase catalyzed oxidation of dopamine.

The stereochemistry of the bovine plasma amine oxidase catalyzed oxidation of 2-(3,4-dihydroxyphenyl)-ethylamine (domapine) has been investigated by comparing 3H/14C ratios of 3,4-dibenzyloxyphenethyl alcohols, derived from 3,4-dihydroxyphenylacetaldehydes, to starting dopamines chirally labeled at C-1 and C-2. The oxidation of [2RS-3H]-, [2R-3H]-, and [2S-3H]dopamine leads to products which have retained 53, 59, and 47% of their tritium. Similarly, oxidation of [1RS-3H]-, [1R-3H]-, and [1S-3H]dopamine leads to an 80, 80, and 92% retention of tritium. The configurational purity of tritium at C-2 of dopamine and C-1 of the dopamine precursor 3-methoxy-4-hydroxyphenethylamine has been confirmed employing dopamine-beta-hydroxylase (specific for the pro-R hydrogen at C-2) and pea seedling amine oxidase (specific for the pro-S hydrogen at C-1). In addition, chromatographically resolved isozymes of bovine plasma amine oxidase have been demonstrated to lead to the same stereochemical result as pooled enzyme fractions. We have been able to rule out carbon interchange and tritium transfer in the ethylamine side chain of dopamine as the source of the apparent nonstereospecificity. Estimated primary tritium isotope effects are 1 for [2-3H]dopamines and 5--6 and 26--34 for [1R-3H]- and [1S-3H]dopamine, respectively. We propose the presence of alternate dopamine binding modes, characterized by absolute but opposing stereochemistries and differential primary tritium isotope effects at C-1.


Investigation of solvent accessibility of the fluorotyrosyl residues of M13 coat protein in deoxycholate micelles and phospholipid vesicles.

We have utilized a nonperturbing nuclear magnetic resonance technique, specifically measuring sensitivity of the chemical shift of fluorotyrosyl residues to change in solvent from H2O to D2O, to demonstrate that the tyrosyl residues of fluorotyrosyl M13 coat protein in phospholipid vesicles are not accessible to solvent i.e., are buried in the hydrophobic portion of the bilayer. The two fluorotyrosyl residues of the protein did show partial exposure to solvent (42% and 65% with respect to aqueous m-fluorotyrosine) when the protein was incorporated into deoxycholate micelles, pointing to differences in conformation of micellar protein with respect to vesicle-associated protein. M13 coat protein in phospholipid vesicles was not sensitive to lactoperoxidase-catalyzed iodination, supporting the NMR results. Coat protein in deoxycholate micelles showed release of fluorotyrosyl residues upon Pronase digestion, but only after an observed change in environment. The observed changes suggest that proteolytic digestion studies of membrane proteins should be interpreted with the possibility of artifacts related to conformational changes in mind. M13 coat protein in phospholipid vesicles did not demonstrate release of fluorotyrosine by Pronase, again pointing to differences between protein in micelles and in vesicles and corroborating the NMR result.


Effect of estrogen on preprolactin messenger ribonucleic acid sequences.

A complementary DNA (cDNA) probe has been synthesized for rat preprolactin messenger RNA (mRNA). Following preparative gel electrophoresis, the cDNA was characterized and estimated to be approximately 75% pure. The reverse transcript is estimated to be greater than 1000 nucleotides long and, therefore, is a full-length copy of the preprolactin mRNA. It is a faithful transcript, as evidenced by back-hybridization with the mRNA. By use of the cDNA as a hybridization probe, the levels of preprolactin mRNA in the pituitary of male and female rats were monitored and found to increase following estrogen treatment. Increases in preprolactin mRNA activity, assayed by in vitro translation in the wheat germ system, were paralleled by increases in hybridization to the cDNA probe after estrogen treatment.


Inhibition of chicken myeloblastosis RNA polymerase II activity by adriamycin.

In vitro RNA synthesis by isolated RNA polymerase II of chicken myeloblastosis cells was shown to be highly sensitive to adriamycin inhibition. The template activity of the single-stranded DNA, purified by chromatography of denatured calf thymus DNA through hydroxylapatite columns, was found to be equally as sensitive to the inhibition as denatured calf thymus DNA. However, contrary to denatured DNA, the single-stranded DNA thus purified showed no significant binding to adriamycin as analyzed by cosedimentation of the drug and DNA through a sucrose gradient. This indicated that inhibition of RNA synthesis on a single-stranded DNA template might involve a mechanism other than DNA intercalation. Kinetic studies of the inhibition showed that the inhibition of RNA synthesis by adriamycin could not be reversed by increasing the concentrations of RNA polymerase and four nucleoside triphosphates, but it could be reversed by increasing DNA concentrations. Analysis of the size of RNA synthesized indicated that the ultimate size of the product RNA was not altered by adriamycin, suggesting that the drug may inhibit RNA synthesis by reducing RNA chain initiation.


Isolation and identification of 4-hydroxy- and 4-oxoretinoic acid. In vitro metabolites of all-trans-retinoic acid in hamster trachea and liver.

Incubation of [3H]retinoic acid in the presence of hamster liver 10000g supernatant produces several metabolites that are more polar than the parent compound. Two of these metabolites are identical with synthetic all-trans-4-hydroxyretinoic acid and all-trans-4-oxoretinoic acid both in ultraviolet absorption and mass spectral characteristics and in migration rates on two different reverse-phase high-pressure liquid chromatographic systems. The metabolites produced in a cell-free liver incubation reaction also migrate on a high-pressure liquid chromatography column together with metabolites isolated from a tracheal organ culture system. Both the metabolites and the synthetic standards show less biological activity than the parent all-trans-retinoic acid in a tracheal organ culture assay.


Identification of the N-ethylmaleimide reactive protein of the mitochondrial phosphate transporter.

The mitochondrial phosphate carrier is inhibited by the SH reagents p-(hydroxymercuri)benzoate and N-ethylmaleimide. Based on an analysis utilizing dodecyl sulfate-polyacrylamide gels, an SH-containing 32 000-dalton protein has been identified as a component of the phosphate carrier system. Two other N-[3H]ethylmaleimide-labeled proteins of the inner mitochondrial membrane have been eliminated from this role [Wholrab, H., & Greaney, J., Jr. (1978) Biochim. Biophys. Acta 503, 425] on the basis that band IV (45,000 daltons) is absent from heart sonic submitochondrial particles and band VII (6 500 daltons) does not react with p-(hydroxymercuri)benzoate. The mobility of the 32 000-dalton protein (0.43) is lower than that of the gamma subunit of the mitochondrial ATPase (0.46) and the carboxyatractyloside binding protein (0.48) on 12.5% dodecyl sulfate-polyacrylamide gels. In these flight muscle mitochondria, 0.87 nmol of N-[3H]ethylmaleimide per nmol of cytochrome a is bound to the 32,000-dalton protein.


Thermal behavior of synthetic sphingomyelin-cholesterol dispersions.

The thermotropic behavior of aqueous dispersions of palmitoylsphingomyelin-cholesterol and lignoceryl-sphingomyelin-cholesterol mixtures has been examined by high-sensitivity differential scanning calorimetry. When less than 25 mol % cholesterol is mixed with either sphingomyelin, the calorimetric endotherm is composed of a sharp and a broad component. The sharp-component enthalpy change decreases as the mole percent cholesterol increases with the extrapolated zero enthalpy point being 25 to 30 mol %. With palmitoylsphingomyelin, the temperature of maximum heat capacity of the sharp component decreases monotonically with increasing cholesterol content, while the lignocerylsphingomyelin sharp-component maximum remains constant until more than 20 mol % sterol is present. The broad-component enthalpy change maximizes at 3--4 kcal/mol between 10 and 20 mol % cholesterol and decreases as the ratio of cholesterol is increased or decreased from this range for both sphingomyelins. The results are compared with those from a previous study on dipalmitoylphosphatidylcholine-cholesterol mixtures and are interpreted as evidence for the coexistence of cholesterol-rich and cholesterol-poor phases.


Adenosine receptor permanently coupled to turkey erythrocyte adenylate cyclase.

The mode of coupling of the adenosine receptor to adenylate cyclase in turkey erythrocyte membranes was probed by two independent approaches. The progressive inactivation of the adenosine receptor by an adenosine receptor affinity label resulted in the proportional reduction in the adenosine plus GppNHp dependent specific activity. In contrast, the intrinsic rate constant (k3), characterizing the process of adenylate cyclase activation by the adenosine-adenosine receptor complex, is independent of the extent of receptor inactivation. This behavior favors the precoupled mechanism, A + R.E: formula: (see text), where the receptor R and the enzyme E are permanently coupled to each other and the adenosine A binds to the receptor and induces the first-order process of cyclase activation to its active form ARE'. The finding that adenosine receptor is permanently coupled to the cyclase catalytic unit is corroborated by the observation that the progressive increase in membrane fluidity has no effect on the rate constant (k3) of adenylate cyclase activation by the adenosine-adenosine receptor complex and that the dose-response curve for adenosine is noncooperative.


Purification and properties of the hemagglutinin from Wistaria floribunda seeds.

Extracts of Wistaria floribunda seeds contain separable erythroagglutinating and lymphocyte mitogenic activities. We wish to report the purification and characterization of the erythroagglutinating lectin of these seeds. A phosphate-buffered saline (PBS) extract of the ground seeds was made to 50% ethanol and the precipitate, which contained both the agglutinin and mitogen, was dissolved in PBS. The erythroagglutinating activity was adsorbed onto insoluble polyleycyl derivatized A + H active hog gastric mucin. After desorption with 0.2 M D-galactose and removal of the sugar by dialysis, the eluate displayed three protein bands on polyacrylamide gel electrophoresis. The major component represented 85% of the mixture. Immunoelectrophoresis of the mixture demonstrated immunochemical identity among the proteins. Gel filtration through Sephadex G-200 resulted in purification of the major component. Based upon the composition and subunit molecular weight, it was concluded that the three components represent a dimer, tetramer, and octamer of a single glycopolypeptide chain of 28 000. The erythroagglutinin has a pI at pH 5.4 and one cystine per dimeric unit.


Solution conformation of enkephalin. A nuclear magnetic resonance study of 13C-enriched carbonyl carbons in [Leu5]-enkephalin.

By using 13C enrichment in [Leu5]-enkephalin, it has been possible to improve the assignment of carbonyl resonances in the nuclear resonance spectrum and to remove some of the ambiguities in the derived phi and chi dihedral angles, thereby providing information about the conformation of this molecule in solution. The combined use of 13C and 1H nuclear magnetic resonance experiments leads to the conclusion that [Leu5]0enkephalin contains a type I beta bend at residues Gly3-Phe4 in dimethyl-d6 sulfoxide (Me2SO0d6) solution. Furthermore, the side chains of Tyr1, Phe4, and Leu5 exist predominantly in one conformation (tg-) in this solvent. A comparison is made between the conformation found in Me2SO-d6 and those determined by X-ray diffraction and conformational energy calculations.


Synthesis of poly(A)-containing RNA by isolated spinach chloroplasts.

Chloroplasts were isolated from spinach leaves and the intact chloroplasts separated by centrifugation on gradients of silica sol. Chloroplasts prepared in this way were almost completely free of cytoplasmic rRNA. The purified chloroplasts were incubated with 32PO4 in the light. The nucleic acids were then extracted and the RNA was fractionated into poly(A)-lacking RNA and poly(A)-containing RNA (poly(A)-RNA) via oligo(dT)-cellulose chromatography. The poly(A)-RNA had a mean size of approximately 18--20 S as determined by polyacrylamide gel electrophoresis. The poly(A)-RNA was digested with RNase A and RNase T1, and the resulting poly(A) segments were subjected to electrophoresis on a 10% w/v polyacrylamide gel 98% v/v formamide). Radioactivity was incorporated into both poly(A)-RNA and poly(A)-lacking RNA and into the poly(A) segments themselves. The poly(A) segments were between 10 and 45 residues long and alkaline hydrolysis of poly(A) segments followed by descending paper chromatography showed that they were composed primarily of adenine residues. There was no 32PO4 incorporation into acid-insoluble material in the dark. We conclude that isolated chloroplasts are capable of synthesizing poly(A)-RNA.


Identification of the major 68,000-dalton protein of microtubule preparations as a 10-nm filament protein and its effects on microtubule assembly in vitro.

The major 68,000-dalton protein present in cycled microtubule preparations from bovine brain can be isolated in a rapidly sedimenting fraction consisting of filaments 10 nm in diameter. This 68,000-dalton protein remains in the filament fraction after gel filtration, phosphocellulose chromatography, or salt extraction of microtubule protein. Microtubule protein devoid of 10-nm filaments contains ring structures under depolymerizing conditions, and it polymerizes into microtubules with a characteristically low critical concentration, although all of the 68,000-dalton protein has been removed from it. When cycled microtubule protein is subjected to chromatography on phosphocellulose, the tubulin fraction (PC-tubulin) assembles into microtubules only at concentrations greater than 2 mg/mL. The other fraction, eluted from phosphocellulose at high ionic strength, contains the major 68,000-dalton protein and can be further resolved into two components by centrifugation. The supernatant, which consists mainly of high molecular weight microtubule-associated proteins, stimulates low concentrations of PC-tubulin to assemble. The pellet contains all of the 68,000-dalton protein, consists of 10-nm filaments, and does not stimulate assembly of PC-tublin. Boiling of purified filaments, however, releases several proteins, including the 68,000-dalton protein, and these released proteins stimulate the assembly of PC-tubulin. The morphology and protein composition of the filaments isolated from microtubule preparations by these techniques are very similar to those of mammalian neurofilaments. These results suggest that the major 68,000-dalton protein in cycled microtubule preparations, which may correspond to tubulin assembly protein [Lockwood, A.H. (1978) Cell 13, 613--627], is a constituent of neurofilaments.


Assembly of nonneural microtubules in the absence of glycerol and microtubule-associated proteins.

Microtubule protein from Ehrlich ascites tumor cells purified by an in vitro polymerization process in the absence of glycerol and calcium chelators contains several accessory proteins but lacks the high molecular weight proteins which are present in neurotubulin. DEAE-Sephadex chromatography of two-times cycled tubulin removes these nontubulin proteins, resulting in pure tubulin, as critically examined by sodium dodecyl sulfate gel electrophoresis. This tubulin can readily assemble into microtubules in assembly buffer, at low magnesium concentrations, without glycerol and at tubulin concentrations above 0.8 mg/mL. Electron microscopy shows that the tubules are identical with normal microtubules. When the purified tubulin fraction was reduced and carboxymethylated, a significant minor protein component could be observed electrophoretically, migrating between alpha- and beta-tubulin. At present, the identity and function of this protein are not known. The results demonstrate that the in vitro assembly of tubulin from Ehrlich ascites tumor cells does not require high molecular weight proteins or gamma-like factor(s) as has been proposed for the neurotubulin system.


Phospholipid lateral phase separation and the partition of cis-parinaric acid and trans-parinaric acid among aqueous, solid lipid, and fluid lipid phases.

The partition of cis-parinaric acid (9,11,13,15-cis, trans, trans,cis-octadecatetraenoic acid, cis-PnA) and trans-parinaric acid (9,11,13,15-all-trans-octadecatetraenoic acid, trans-PnA) among aqueous, solid lipid, and fluid lipid phases has been measured by three spectroscopic parameters: absorption spectral shifts, fluorescence quantum yield, and fluorescence polarization. The solid lipid was dipalmitoylphosphatidylcholine (DPPC); the fluid lipid was palmitoyldocosahexaenoylphosphatidylcholine (PDPC). Mole fraction partition coefficients between lipid and water were determined by absorption spectroscopy to be for ci--PnA, 5.3 X 10(5) with a solid lipid and 9 X 10(5) with fluid lipid and, for trans-PnA, 5 X 10(6) with solid lipid and 1.7 X 10(6) with fluid lipid. Ratios of the solid to the fluid partition coefficients (Kps/f) are 0.6 +/- 0.2 for cis-PnA and 3 +/- 1 for trans-PnA. A phase diagram for codispersions of DPPC and PDPC has been constructed from the measurements of the temperature dependence of the fluorescence quantum yield and polarization of cis-PnA and trans-PnA and their methyl ester derivatives. A simple analysis based on the phase diagram and fluorescence data allows additional calculations of Kps/f's which are determined to be 0.7 +/- 0.2 for the cis probes and 4 +/- 1 for the trans probes. The relative preference of trans-PnA for solid phase lipids and its enhanced quantum yield in solid phase lipids make it sensitive to a few percent solid. The trans probes provide evidence that structural order may persist in dispersions of these phospholipids 10 degrees C or more above their transition temperature. It is concluded that measurements of PnA fluorescence polarization vs. temperature are better suited than measurements of quantum yield vs. temperature for determining phospholipid phase separation.


Sphingomyelin--lecithin bilayers and their interaction with cholesterol.

Utilizing X-ray diffraction and differential scanning calorimetry (DSC), we have studied (1) the structure and thermotropic properties of hydrated N-palmitoylsphingomyelin, (2) the interaction of N-palmitoylsphingomyelin with dimyristoyllecithin, and (3) the interaction of cholesterol with N-palmitoylsphingomyelin and dimyristoyllecithin, both individually and in a 50:50 (mol/mol) mixture. N-Palmitoylsphingomyelin forms bilayers which undergo a thermotropic order--disorder (gel--liquid crystalline) transition at 40.5 degrees C (delta H = 5.8 kcal/mol). The bilayer repeat distance is 66.8 A at 10 degrees C and 61.6 A at 50 degrees C. N-Palmitoylsphingomyelin exhibits miscibility with dimyristoylecithin in both the gel and liquid-crystalline phases, and no lateral phase separation occurs. Scanning calorimetry indicates that interaction with cholesterol is similar for both N-palmitoylsphingomyelin and dimyristoyllecithin and that in a 50:50 (mol/mol) mixture cholesterol shows no preferential affinity for either phospholipid.


Resonance Raman spectra of carbon-13- and nitrogen-15-labeled riboflavin bound to egg-white flavoprotein.

The resonance Raman spectra of [2-13C]-, [4a-13C]-, [4-13C]-8 [10a-13C]-, [2,4,4a, 10a-13C]-, [5-15N]-, [1,3-15N]-, and [1,3,5-15N]riboflavin bound to egg-white proteins were observed for N(3)-H and N(3)-D forms with spontaneous Raman technique by using the 488.0-nm excitation line of an argon ion laser. The fluorescence of riboflavin was quenched by forming a complex with egg-white riboflavin binding protein. The in-plane displacements of the C(2), C(4a), N(1), N(3), and N(5) atoms during each Raman active vibration were calculated from the observed isotopic frequency shifts. The 1252-cm-1 mode of the N(3)-H form was found to involve large vibrational displacements of the C(2) and N(3) atoms and to be strongly coupled with the N(3)-H bending mode. This line can be used as an indicator for state of N(3)-H...protein interaction. The 1584-cm-1 mode, which is known to be resonance-enhanced upon excitation near the 370-nm absorption band, was accompanied by the displacement of the N(5) atom in particular. The 1355-cm-1 mode was most strongly resonance-enhanced by the 450-nm absorption band and involved the displacements of all carbon atoms of ring III. Both lines can be used as structure probes for elucidating the structure of electronically excited states of isoalloxazine.


Purification and partial characterization of a stimulatory factor for lamb thymus RNA polymerase II.

A heat-stable protein (HSF) that stimulates the activity of lamb thymus RNA polymerase II has been purified 2500-fold and partially characterized. This factor stimulates the activity of RNA polymerase II up to 13 times and retains complete activity when heated at 90 degrees C for 5 min. Stimulation is observed only in the presence of RNA polymerase II and requires native DNA as template. The stimulatory factor has a sedimentation coefficient of 2.7 S, a diffusion coefficient of 9.55 x 10(-7) cm2/s, and an isoelectric point of 8.0. Calculated from the sedimentation and diffusion data, the factor has a molecular weight of about 24,000. Electrophoresis of the purified factor on polyacrylamide gels in the presence of sodium dodecyl sulfate results in a single band corresponding to a molecular weight of 25,000. The number-average length of the RNA synthesized by RNA polymerase II is increased in the presence of the factor. Sedimentation velocity and exclusion chromatography experiments suggest that the stimulatory factor interacts with RNA polymerase II. These results suggest that the factor stimulates RNA synthesis through a direct interaction with RNA polymerase II. The stoichiometry of the HSF-RNA polymerase binding appears to be about 1:1. HSF is located in the nucleus, as determined by cell fractionation studies.


Complement fixation by model immune complexes free in solution and bound onto cell surfaces.

Immunoglobin (IgG) molecules with anti-2,4-dinitrophenyl activity were covalently cross-linked by using three cross-linking reagents. The resulting oligomers were separated into monomer, dimer, trimer, and heavy fractions. These stable assemblages of IgG molecules were capable of fixing dilute whole guinea pig complement in solution. When oligomers were further aggregated noncovalently into larger complexes, all were able to fix complement. Radioiodinated oligomers were attached to 2,4,6,-trinitrophenylsulfonic acid treated sheep red blood cells (N3ph-SRBC), and the number of bound molecules was determined from the cell-associated radioactivity. Complement-mediated lysis of N3ph-SRBC was then assayed over a range of levels of bound protein and at increasing concentrations of complement. The lytic efficiencies of all oligomers increased with the number of bound molecules, with complement concentration, with hapten density on N3ph-SRBC, and with oligomer size. The results suggest that two adjacent IgG molecules may not serve as a unit signal for triggering the complement cascade, but instead, initiation occurs with increasing efficiency as the size of cell-bound IgG clusters increases.


Measurement of phospholipid monolayer surface potentials at a hydrocarbon-electrolyte interface.

The phospholipid monolayer spread at a hydrocarbon-electrolyte interface can be used as a model system for the plasma membrane and its properties and structure probed by measurements of surface pressure and surface potential. To facilitate such studies, (i) the theory of the vibrating plate (Kelvin) method of measuring surface potentials is reëxamined and a new interpretation given for the potentials measured and (ii) a new apparatus for performing these measurements is described. The theory and apparatus are illustrated by measurements on films of distearoyl phosphatidylcholine at the interface between 2,2,4-trimethylpentane (isooctane) and 0.1 M NaCl.


Phospholipid monolayers at the hydrocarbon-electrolyte interface. The interrelation of film potential and film pressure.

Measurements of surface pressure of surface potential are reported for films of distearoyl phosphatidylcholine (density range: 0.15--2.65 . 10(18) molecules/m2) spread at the interface between 2,2,4-trimethylpentane and 100 mM NaCl. Low density behavior of the surface pressure is explained using classical viral theory. The behavior of the surface potential is qualitatively explained for all densities in terms of the dipole moments associated with the carboxyl groups and headgroups of the phosphatidylcholine.


Quantitation of antibody binding to cell surface antigens by x-ray fluorescence spectrometry.

An X-ray spectrometric method has been developed to quantitate antibody bindng to whole cell surfaces in order to obtain a distribution of binding within a population of cells. The method involves incubation of target cells with ferritin-labeled antibody. Analysis of prepared samples in a modified transmission electron microscope with an X-ray detector and data analysis equipment, yields quantitative results on the binding of labeled antibody to individual cells. The binding of anti-2,4-dinitrophenol serum to Chinese hamster ovary cells with attached 2,4-dinitrophenol haptens was measured by X-ray spectrometry. Measurements of attached hapten by a radioisotopic marker correlated with the X-ray spectrometric determination of bound antibody. The use of synchronized cells in metaphase and G1 phases of the cell cycle permitted investigations into the binding per unit surface area. The distribution of antibody binding among a given population of cells was related to the surface area of the cells.


Na+-dependent, electroneutral L-ascorbate transport across brush border membrane vesicles from guinea pig small intestine.

In brush border vesicles from guinea pig small intestine L-ascorbate transport is Na+-dependent and electroneutral (in the presence of Na+, as shown by its lack of response to either positive or negative delta psi across the membrane). L-Ascorbate transporter has the kinetic characteristics of a mobile carrier (Km for L-ascorbate, 0.3 mM). D-Isoascorbate (erythorbate) seems to be another, but poorer, substrate of the same transporter. L-Ascorbate transport is subjected to heterologous inhibition by D-glucose.


Purification of the cytochalasin B binding component of the human erythrocyte monosaccharide transport system.

The cytochalasin B binding component of the human erythrocyte monosaccharide transport system has been purified. The preparation appears to contain one major protein with an apparent polypeptide chain molecular weight of 55,000 and about 0.4 binding sites per chain. Cytochalasin B binds to the reconstituted preparation with a dissociation constant of 1.3.10(-7) M, a value which is similar to that reported for the transport system in the intact erythrocyte.


Cell-surface glycosaminoglycans are not released from human diploid fibroblasts by non-enzymatic methods.

Attempts were made to release glycosaminoglycans from the surface of intact fibroblasts in culture by changes in pH and ionic strength of the surrounding medium. No additional macromolecular material was released by such 5-min treatments, while 0.01% trypsin released 50 times the amount naturally shed. These results suggest a covalent attachment of glycosaminoglycans to the cell membrane. Cytochalasin B did not effect release of surface components, but a small amount of sulfated glycosaminoglycan was released by inclusion of heparin in the incubation medium.


The organisation of cholesterol and ergosterol in lipid bilayers based on studies using non-perturbing fluorescent sterol probes.

The fluorescence properties of dehydroergosterol and cholesta-5,7,9-trien-3 beta-ol have been studied in organic solution, in aqueous dispersions and incorporated into aqueous lipid dispersions. The absorption spectra of aqueous dispersions of the probes are very different to those in organic solution, and aqueous dispersions are non-fluorescent. This can be attributed to micelle formation with dimerisation and/or aggregation in the micelles. Concentration quenching also occurs when sterols are incorporated into lipid bilayers, but relatively high fluorescence is observed even at a 1 : 1 steroid:lipid molar ratio. Further, the fluorescence is still polarized at these high molar ratios. We attribute this to the formation of ordered arrays of sterol molecules in the lipid bilayers. In these arrays the sterol molecules are organised in an end-to-end fashion, and face-to-face overlap of the sterols is prevented by the lipid molecules. Possible structures for 1 : 1 mixtures are presented.


Repression of rRNA synthesis induced by disaggregation in Dictyostelium discoideum.

Cells disaggregated from the slugs of Dictyostelium discoideum continued to incorporate [3H]uridine or [3H]uracil, though at a lower rate than interphase amoebae. Disaggregation brought about a marked increase in the proportion of labeled poly(A)-containing RNA to labeled total RNA. The proportion reached the maximum in disaggregated cells after 1 h of incubation, but remained high even after 5 h of incubation. Reconstruction of slugs from the disaggregated cells did not bring about a decrease but a further increase in the proportion. The real proportion estimated from the minimum period of labeling reached almost 100% in the disaggregated cells, in contrast to about 30% in the interphase amoebae. The increase in the proportion is attributed to the disaggregation-induced repression of rRNA synthesis, which was supported by sucrose density gradient analyses of RNA synthesized in the disaggregated cells. The possibility that rRNA synthesis in this organism is regulated by the loss of cell contact is discussed.


Inhibition of endogenous RNA polymerase activity from thymus chromatin by transcortin-hydrocortisone complex.

A transortin-hydrocortisone complex has been isolated from human serum by affinity chromatography on oxidized corticosterone coupled to AH-Sepharose 4B. The influence of this complex and of hydrocortisone alone on endogenous RNA polymerase activity from thymus chromatin have been tested. Results show that hydrocortisone alone has no effect on RNA polymerase activity from thymus chromatin. Under the same experimental conditions, The transcortin-hydrocortisone complex induces an important decrease in the incorporation of UMP into RNA. The dose response of thymic RNA polymerase to transcortin-hydrocortisone complex and the effects of alpha-amanitin on this response are also reported.


Regulation of purine de novo synthesis in cultured human fibroblasts: the role of P-ribose-PP.

Procedures for assaying the rate of purine de novo synthesis in cultured fibroblast cells have been compared. These were (i) the incorporation of [(14)C]-glycine or [(14)C]formate in alpha-N-formylglycinamide ribonucleotide (an intermediate in the purine synthetic pathway) and (ii) the incorporation of [(14)C]-formate into newly synthesised cellular purines and purines excreted by the cell into the medium. Fibroblast cells, derived from patients with a deficiency of hypoxanthine phosphoribosyltransferase (HPRT-) (EC 2.4.2.8) and increased rates of purine de novo synthesis, were compared with fibroblasts from healthy subjects (HPRT+). Fetal calf serum, which was used to supplement the assay and cell growth medium, was found to contain sufficient quantities of the purine base hypoxanthine to inhibit purine de novo synthesis in HPRT+ cells. This inhibition was the basis of differentiation between HPRT- and HPRT+ cells. In the absence of added purine base, both cell types had similar capacities for purine de novo synthesis. This result contrasts with the increased rates of purine de novo synthesis reported for a number of human HPRT- cells in culture but conforms recent studies made on human HPRT- lymphoblast cells. The intracellular concentration and utilisation of 5-phosphoribosyl-1-pyrophosphate (P-Rib-PP), a substrate and potential controlling factor for purine de novo synthesis, were determined in HPRT- and HPRT+ cells. The rate of utilisation of P-Rib-PP in the salvage of free purine bases was far greater than that in purine de novo synthesis. Although HPRT- cells had a 3-fold increase in P-Rib-PP content, the rate of P-Rib-PP generation was similar to HPRT+ cells. Thus, in fibroblasts, the concentration of P-Rib-PP appears to be critical in the control of de novo purine synthesis and its preferential utilisation in the HPRT reaction limits its availability for purine de novo synthesis. In vivo, HPRT+ cells, in contrast to HPRT- cells, may be operating purine de novo synthesis at a reduced rate because of their ability to reutilise hypoxanthine.


Qualitative and quantitative aspects of intercalator-induced DNA strand breaks.

The intercalating agents, adriamycin and ellipticine, were previously found to produce DNA strand breaks associated with DNA-protein crosslinks in mouse leukemia L1210 cells. The current work explores the nature of the agents that produce this effect and the quantitative relationship between the breaks and crosslinks. The protein-associated DNA breaks were produced by a wide variety of intercalators in addition to the above-mentioned compounds: actinomycin D, daunoycin, ethidium and lucanthone (miracil D). Treatment with several drugs that bind to DNA without intercalation, or that inhibit DNA synthesis without binding to DNA, did not cause DNA breaks. The strand break and crosslink frequencies were quantitated by means of alkaline elution methods. The strand break and crosslink frequencies were found to be within a factor of 2 of each other over a range of concentrations of adriamycin and ellipticine. It is proposed that intercalation-induced distortion of the DNA helix leads to strand scission by a nuclease which becomes bound to one terminus of the break so as to form a DNA-protein crosslink.


The role of DNA polymerases alpha, beta and gamma in nuclear DNA synthesis.

The effects of the inhibitors 2'3' dideoxythymidine triphosphate (ddTTP) and 1-beta-D-arabinofuranosyl cytosine triphosphate (araCTP) on DNA synthesis in isolated S-phase HeLa S3 nuclei have been examined. These effects are compared with the effects of the same inhibitors in partially purified preparations of DNA polymerases alpha and beta. The effect of ddTTP on partially purified DNA polymerase gamma was also tested. DNA polymerases beta and gamma were very sensitive to ddTTP whereas DNA polymerase alpha and DNA synthesis in isolated nuclei were quite resistant. The synthesis and subsequent ligation of primary DNA pieces ('Okazaki fragments') were not affected by the presence of this inhibitor. DNA synthesis in isolated nuclei and DNA polymerase alpha activity were very sensitive to araCTP whereas DNA polymerase beta was almost totally resistant to the inhibitor. The results indicate a major role for DNA polymerase alpha in DNA replication.


Metabolism of 2-amino-3-phosphonopropionic acid in rats.

The metabolism of 2-amino-3-phosphono-[2-(14)C]propionic acid or 2-amino-3-phosphono-[3-(14)C]propionic acid in rats was studied in vivo and in vitro. The radioactivity in expired CO2 from the [3-(14)C]-labelled compound indicated the cleavage of the carbon-phosphorus (C-P) bond. A small amount of the [2-(14)C]-labelled compound and the [3-(14C]-labelled compound was incorporated into 2-aminoethylphosphonic acid, and polar lipid of the liver and kidney contained the 2-aminoethylphosphonic acid. The 2-amino-3-phosphonopropionic acid was not detected at the lipid level. Incorporation of the [3-(14)C]-labelled compound into a variety of metabolites including 3-phosphonopyruvic acid and 2-phosphonoacetaldehyde suggests the transamination reaction as a decomposition mechanism of 2-amino-3-phosphonopropionic acid in mammals.