Inhibitory effect of pregnancy on the migration of the inflammatory cells: a quantitative histological study. The migration of inflammatory cells and fibroblasts into dacron mesh tissue (Mersilene), immersed in saline or in a solution containing live BCG and implanted under the skin of virgin and pregnant female guinea-pigs, was investigated. Cell counts have shown that in pregnant females significant reduction (about 50%) of migration of the inflammatory cells occurred in both saline and live-BCG-immersed implants. The effect was maximal (-81%) on macrophage level in implants immersed in saline, whereas the action was prominent on neutrophils level (-66%) in implants containing live BCG. In the case of implants devoid of live BCG, the inhibitory effect of pregnancy on migration of the inflammatory cells and fibroblasts seemed not to be directly related to any immunological process. This experiment suggests that inhibition could be mediated via an undefined factor interacting with cell membranes to diminish their functional activities.


A morphological assessment of macrophages attaching to subcutaneously implanted coverslips in dogs with chronic lymphoedema. In an experimental model of chronic lymphoedema in the dog leg it has been shown that the presence of lymphoedema was associated with reduced numbers of macrophages found on subcutaneously implanted coverslips. Total numbers of mononuclear cells were not, however, significantly affected. When expressed as a percentage of the total mononuclear cells attached, the lymphoedematous limbs always had only half those of the corresponding normal limbs. Similarly, the percentage of macrophages with two or more pseudopods on the coverslips in the lymphoedematous limbs was only 1/3 of that for the same morphological criteria in normal limbs. However, when the macrophages with vacuoles were expressed as a percentage of the total macrophages, there was always a higher percentage in the lymphoedematous limbs compared with the corresponding normal ones.


In vitro prediction of the pathogenicity of mineral dusts. Eight mineral dusts, of which 7 were fibrous, were tested in 3 in vitro systems. The dusts had previously been tested for their ability to produce mesotheliomata in rats. The in vivo and in vitro biological activities of these dusts are compared with the number and size of fibres they contain. It is concluded that all the in vitro systems would predict which dusts are pathogenic in vivo, and that both in vivo and in vitro biological activities are determined by the number of long, fine fibres.


The effect of a calcium antagonist (D600) on isoprenaline-induced myocardial necrosis in the rat. The effect of inhibiting isoprenaline-induced intracellular calcium accumulation on the degree of damage produced in the rat myocardium by this amine has been investigated by simultaneously dosing rats with the calcium antagonistic drug D600. The degree of myocardial necrosis produced in animals given isoprenaline alone and those given D600 was measured by the use of a standard point counting method to show absence of formazan from dead muscle fibres in sections treated to demonstrate succinic dehydrogenase. The use of the calcium antagonistic drug D600 considerably reduced the degree of myocardial damage produced by a standard dose of isoprenaline bitartrate. This was associated with a decrease in the isoprenaline-induced cellular calcium uptake. The results are discussed in relation to the possible protection conferred by lowering of the calcium influx at the cell membrane and the maintenance of the cells' high-energy phosphates at a level which permits "normal" cell function.


The effect of substrate on the uptake of oxygen and sulphate ions by heat-damaged skin. Respiration and sulphate uptake have been measured in guinea-pig skin subjected to heat damage and maintained on a medium containing sodium succinate. Succinate was found to have a protective effect against loss of oxygen uptake, a higher temperature being required for 50% effect than that reported with glucose as substrate. The protective effect did not extend to sulphate incorporation, suggesting that this process is itself heat-labile and that its failure with heat damage is not directly caused by diminution of the energy supply.


Leukocyte function in granuloma annulare. Leukocytes from patients with granuloma annulare were studied in vitro in order to detect functional abnormalities. Random migration and chemotaxis of both polymorphonuclear leukocytes and monocytes were similar in patients and controls. Preincubation of normal cells with patients' plasma did not enhance or inhibit locomotion. Phagocytosis and oxidative burst as reflected by chemiluminescence were also normal. Peripheral blood lymphocytes incorporated uridine normally with and without stimulation by the non-specific mitogen phytohaemagglutinin. These studies suggest that peripheral blood leukocyte function in patients with granuloma annulare is normal, but do not exclude a functional abnormality in tissue.


Comparison of immunoglobulin and complement deposition in multiple biopsies from the uninvolved skin in dermatitis herpetiformis. The distribution of immunoglobulins and C3 component of complement (C3) in the skin of twenty-four patients with dermatitis herpetiformis was studied. Skin biopsies were taken from three sites, the extensor surface of the elbow, the flexor surface of the forearm and from the thigh. Twenty-two (90%) patients showed variation in deposits of immunoglobulins and C3 at the three sites. IgA was present in all patients, but differed in quantities deposited at the three sites in nineteen (80%) patients. Similar variation between sites was observed with IgG, IgM and C3. There was variation within three biopsies, IgA being absent from some sections and present in others. Three patients had a continuous pattern of IgA deposition. One had both continuous and papillary deposits within a single section. There was no difference in the incidence or quantity of immunoglobulin and C3 between the elbow, forearm and thigh. There was observed to be a diminution in quantity of IgA and incidence of C3 in patients taking a gluten-free diet. Deposition of IgA, IgM, IgG and C3 is not uniform throughout the skin and conclusions drawn from the quantity of immunoglobulin and C3 in a single biopsy may not be reliable.


Melanotic freckle of Hutchinson: treatment of macular and nodular phases with cryotherapy. Fourteen cases of Hutchinson's lentigo maligna and eight further cases with nodular invasive areas in whom liquid nitrogen spray has been curative are reported. We suggest that in view of its simplicity and efficacy, cryotherapy is the treatment of choice for the macular phase and possibly also for the nodular invasive stage.


Treatment of cold urticaria.

Treatment of cold urticaria. In a single patient with severe essential acquired cold urticaria, the following drug regimes were assessed: diphenhydramine, cyproheptadine, hydroxyzine, terbutaline, cimetidine, hydroxyzine plus cimetidine, and hydroxyzine plus terbutaline. Each regime showed favourable results, but a significance level of P = 0.01 was reached only for (a) hydroxyzine plus cimetidine in the suppression of erythema, and (b) the histamine H1 antagonists in the suppression of wealing.


The effect of H1 and H2 histamine antagonists on symptomatic dermographism. In ten patients suffering from symptomatic dermographism the combined administration of chlorpheniramine + cimetidine produced a greater reduction in the weal and flare response provoked by a standardized scratch than the administration of chlorpheniramine alone. There was a statistically significant improvement in the overall assessment of the patient's skin condition with the combined administration of chlorpheniramine + cimetidine. Chlorpheniramine given alone produced no significant benefit whilst cimetidine alone produced a marked exacerbation in itching in nearly half the patients who initially entered the study and was sufficient to require withdrawal.


Tuberculoid cutaneous infection due to a niacin-positive Mycobacterium chelonei. A case of cutaneous infection due to a niacin-positive Mycobacterium chelonei (previously called M. abscessus) on the dorsum of the hand of a professional cotton-classifier is reported from Hong Kong. The infection was probably directly acquired from handling contaminated raw cotton. The patient was successfully treated with combined anti-tuberculous drugs over a period of 1 year. Histologically, the skin lesion showed tuberculoid granulomas, in addition to abscesses.


Mycobacterium avium infection of the skin resembling lepromatous leprosy. A patient on systemic steroid therapy developed a cutaneous papule while histologically resembled lepromatous leprosy. Cultures of this lesion grew Mycobacterium avium. Since there was no evidence of disseminated infection the lesion was excised and the patient continues to do well.


A sporotrichoid-like Mycobacterium kansasii infection of the skin treated with minocycline hydrochloride. A sporotrichoid-like Mycobacterium kansasii infection of the skin is reported. This is the fifth reported case in the English literature of dermatological manifestations of a M. kansasii infection and the first reported case of a response to minocycline hydrochloride therapy.


Neonatal lupus erythematosus progressing into systemic lupus erythematosus. A 15 year follow-up. An infant with neonatal lupus erythematosus developed systemic lupus erythematosus at the age of 13. The clinical distinction between neonatal lupus erythematosus and familial lupus erythematosus may not be as clear cut as previously reported.


Systemic lupus erythematosus in pregnancy treated with plasmapheresis. A young woman developed severe systemic lupus erythematosus (SLE) at the beginning of her second pregnancy: her disease was extremely difficult to control and the baby died unexpectedly when 8 weeks old. She became very ill at the same time in her third pregnancy; plasmapheresis appeared to make her management easier, and the 7-month-old baby is doing well.


Systemic sclerosis with subcutaneous nodules. A case of systemic sclerosis with subcutaneous nodules is described. The nodules consisted of fibrinoid degeneration with surrounding fibrosis, but lacked the typical histiocytic palisade of the rheumatoid nodule. The patient had neither coexistent rheumatoid arthritis nor circulating rheumatoid factor.


Variations in granulocyte colony forming cell numbers in adult blood. Blood granulocyte colony forming units (CFU-C) were studied in normal adults to establish: (i) a normal range; (ii) variability due to the culture technique; (iii) variability of blood CFU-C within individuals. Thirty men studied had 98 (range 8--300) CFU-C X 10(3)/1, and 28 women studied had 44 (range 0--260) CFU-C X 10(3)/1. This difference was significant (P less than 0.001). There was also a significant sex difference in the total number of cells forming colonies and clusters per litre; and in the incidence of colony formers and of cluster formers in buffy coat and mononuclear cell blood fractions. CFU-C were assayed in four subjects over a 10 week period. When buffy coat cells were used as a source of colony stimulation the week to week variation in the combined growth of the four subjects was wide (+/- 36%) but with conditioned medium the variation was smaller (+/- 14%). In all subjects colony and cluster growth varied in the same way (r = 0.77, P = .001) but there was no correlation with the total leucocyte count. A 3--4 week cyclical change in CFU-C/1 was found which was independent of the variation inherent in the technique. The physiological significance of the sex difference and the apparent cyclical changes in blood CFU-C are not explained, but the results emphasize the wide fluctuations in CFU-C that may occur in normal individuals.


Effect of antilymphocyte globulin on granulocyte precursors in aplastic anaemia. Granylocyte colony forming units (CFU-C) were studied in 22 patients with severe aplastic anaemia before and after treatment with antilymphocyte globulin (ALG). Nine patients showed a clinical response to ALG characterized by a rise in the granulocyte count to over I X 10(9)/1 within 30 d. These patients were distinguished in vitro from non-responders by an increase in CFU-C numbers after incubation of bone marrow cells with ALG, and by the presence of inhibitors of normal CFU-C in the serum in six out of seven patients tested. In responding patients bone marrow CFU-C rose while most non-responding patients showed no change or a fall in CFU-C after treatment. In addition in three out of four responding patients examined serum inhibitors disappeared after treatment. The horse ALG used in this study also stimulated normal CFU-C in vitro. This evidence is contrary to the hypothesis that ALG stimulates CFU-C in aplasia by inactivating an abnormal suppressor lymphocyte population. The nature of the stimulation by ALG remains unclear. But in practice the effect of ALG on bone marrow cells and study of CFU-C inhibitors in serum could be used to select patients likely to respond to ALG treatment.


Red blood cell enzyme abnormalities in patients treated with chemotherapy. Sxiteen red blood cell enzyme activities and fetal haemoglobin level have been assayed in 60 patients treated for haematologic or nonhaematologic malignant diseases with various combinations of cytostatic drugs. Acquired enzyme deficiency was found in 20 patients. The most frequently decreased activities were those of G6PD (12 cases), PK (seven cases), PFK (six cases) and AK (three cases). In many patients decreased activity of some enzymes contrasted with increased activity of others such as ALD, TPI, G3PD, PGK, ENOL and 6PGD. The number of abnormalities seems to be related to the duration of the treatment. Incidence of enzyme deficiencies was similar in patients previously treated or not with radiotherapy. Enzyme abnormalities were correlated neither with haemoglobin level nor with reticulocyte count. They were very similar to those observed in primary acquired dyserythropoietic and preleukaemic states. Their prognostic value and mechanism are discussed.


Fatty acid composition of erythrocytes in hereditary spherocytosis. The fatty acid distribution of the four major phospholipids has been determined in the erythrocytes of five patients with hereditary spherocytosis and in five control subjects. In contrast to a report by other workers, we are unable to confirm their findings of a defect in fatty acid chain lengthening activity leading to disappearance of long chain fatty acid conjugates of more than 20 carbon atoms in some of the phospholipid fractions.


Detection of Haemophilia A carriers by replicate factor VIII activity and factor VIII antigenicity determinations. Factor VIII (fVIII) activity and antigenicity were determined on replicate fresh blood samples drawn on three separate occasions in 48 normal females and 37 obligatory haemophilia A carriers, in an attempt to improve the detection rate. fVIII activity was assayed by a one-stage method and fVIII antigenicity by the Laurell's technique employing an anti-fVIII antiserum prepared in rabbits. The mean results for the individual subjects were analysed after establishing a discriminant function. The probability of being a carrier was determined on the basis of laboratory data and family history. The probability data were translated into more simple terms applicable for genetic counselling by defining three categories: Definite carriers, doubtful carriers and definite normals. Of the obligatory carriers, 83.8% were classified definite carriers, 13.5% doubtful carriers and 2.7% as definite normals. Of the normal females, 4.2% were classified as definite carriers, 18.7% doubtful carriers and 77.1% as definite normals. Adjustment of the results according to age was found unnecessary. The precision of the determinations of fVIII antigenicity and fVIII activity in individual subjects was calculated for different numbers of replicate tests performed in the same individual. It was found that if only one test is performed, a substantial error might occur. Three replicate tests considerably diminish this error, although it is still in the range of +/- 16--29%. For suspected carriers who fall in the doubtful category it is suggested that six or even more tests are performed.


Coagulation activation and hyperviscosity in infection. A serial study of coagulation activation and whole-blood viscosity was performed on 37 patients with local or systemic bacterial infection, malaria, or a viral infection. Thrombocytopenia, without consumption of coagulation factors, was the main feature of benign tertian malaria and viral infection, whereas in septicaemia and malignant tertian malaria it was associated with activation of coagulation and fibrinolysis. Patients with evidence of intravascular coagulation showed the highest levels of factor VIII related antigen which did not correlate with fibrinogen and probably reflected vascular endothelial cell damage rather than an acute-phase protein reaction. Hyperviscosity, which has been implicated in the pathogenesis of endotoxic shock and cerebral malaria, occurred in parallel with the acute-phase rise in plasma fibrinogen. There was, however, no evidence to implicate hyperviscosity as a major causative factor in the pathogenesis of septic shock or severe infective illness.


The effect of nitrous oxide on the cell cycle in human bone marrow. The effect of 24 h exposure to nitrous oxide on the cell division cycle in human bone marrow has been studied in vivo using the technique of DNA flow microfluorimetry. All patients who received nitrous oxide showed a significant increase in the proportion of early S-phase cells with a decrease in late S, G2 and mitotic cells. These changes resemble those seen following the use of S-phase-specific cytotoxic drugs. Control patients showed no such effect. Parallel studies have suggested that interference with the function of vitamin B12 underlies this response. Nitrous oxide may provide a convenient method for studying the cell kinetic aspects of acute B12 deficiency and the possibility of using it to increase the therapeutic index of antitumour drugs is discussed.


The kinetics of serum and tissue ferritins: relation to carbohydrate content. Significant differences were observed in the rate of disappearance from plasma of ferritins purified from rat serum and from different organs. Ferritin from all sources including purified serum ferritin was rapidly removed from plasma by the liver. No difference in biological half-life was observed between apoferritin prepared by ultracentrifugation of liver ferritin and whole liver ferritin and iron-loaded animals cleared injected serum ferritin from plasma at a comparable rate to normal rats. When amounts of 100 microgram of ferritin were injected into rats the half-life was significantly lengthened. The study confirmed the fact that ferritin iron and ferritin protein were removed from plasma at the same rate. No consistent effect of acidic or more basic isoferritin composition on biological half-life was apparent. After chromatography on concanavalin A-Sepharose 6B those ferritins which were predominantly bound to Con A-Sepharose had a half-life which was approximately twice that of ferritins which did not bind. It is concluded that the variation in plasma disappearance of ferritins of different tissue origin was explainable on the basis of carbohydrate content of the molecule.


Studies in desferrioxamine and ferrioxamine metabolism in normal and iron-loaded subjects. Plasma concentrations of desferrioxamine and ferrioxamine were measured following bolus injections of desferrioxamine and during 24 h infusions of the drug. [59Fe]ferrioxamine clearance and urinary iron excretion were also measured. Higher plasma ferrioxamine concentrations were found in iron loaded subjects and higher desferrioxamine concentrations in subjects with normal ironloads. There is a correlation between the circulating concentration of ferrioxamine during an infusion and the 48 h urinary iron excretion. The data suggests that the amount of iron chelated in vivo is related to an increase in the size of an intermediate chelatable pool rather than the total amount of the iron load. The well-recognized delay in urinary iron excretion appears to be related to active tubular reabsorption of ferrioxamine.


Haemoglobin Lepore trait: haematological and structural studies on the Italian population. Haematological data on 59 heterozygotes for haemoglobin (Hb) Lepore and 10 double heterozygotes for Hb Lepore and beta thalassaemia from 36 Italian families are reported. The red cell indices are defined and compared with those of groups of non-thalassaemic and beta thalassaemic subjects of comparable number, age and sex distribution. The relative level of each haemoglobin fraction and the absolute production of single polypeptide chains are calculated in order to compare the expression of the non-alpha chain genes in Hb Lepore trait and beta thalassaemia. Structural studies demonstrate that the haemoglobin Lepore is of the Boston type (delta 87 beta 116) in all subjects, confirming that this type of fusion variant is probably the only one which occurs in Mediterranean populations. The distribution and incidence of the Lepore haemoglobinopathy are discussed.


Neonatal erythrocyte glutathione peroxidase deficiency as a consequence of selenium imbalance during pregnancy. The red blood cell (RBC) glutathione peroxidase (GSH-Px) activity and routine haematological parameters were measured in 38 healthy north Italian full-term pregnant women and in their newborn infants. In 31 pairs the serum selenium concentration was also measured. Data were compared with those of 20 normal adult controls (10 males and 10 females). Newborn infants exhibited significantly lower RBC GSH-Px activity and serum selenium concentrations than adult controls. Pregnant women had serum selenium values intermediate between those of adult female controls and their newborn infants. In both the pregnant women and newborns the RBC GSH-Px activity correlated with the level of selenium in serum which suggests that the neonatal RBC GSH-Px deficiency may be partially due to insufficient availability of selenium during pregancy. Factors other than selenium concentration, e.g. hormonal and genetic, might also affect the RBC GSH-Px activity as suggested by sex differences and mother/child concordances in enzyme activity found in our cases.


Hereditary acanthocytosis associated with the McLeod phenotype of the Kell blood group system. Some boys with X-linked chronic granulomatous disease (CGD) have red cells of the rare McLeod phenotype in the Kell blood group system. Only one example of this phenotype has previously been described in a non-CGD subject. We have studied a 10-year-old boy and a maternal brother who do not have CGD and whose red cells are of the McLeod type . The boy presented as a haematological problem with red-cell abnormalities. These were acanthocytosis, anisocytosis and 'tailing' in the osmotic fragility curve, changes now known to occur with the McLeod phenotype. Subsequent studies revealed his rare blood group. A family study has established that an uncle also has acanthocytic red cells and the McLeod phenotype. In addition the boy's sister, mother and maternal grandmother all show red-cell mosaicism with double populations of McLeod acanthocytes and normal red cells of common Kell type. The gene that determines inheritance of the McLeod phenotype is X-linked and the mosaicism present in female carriers is believed to result from X chromosome inactivation by the Lyon effect. The study provides further evidence that the McLeod phenotype arises by inheritance of a variant X-linked modifying gene and not through inheritance of a variant gene at the Kell autosomal locus. It also represents the first occasion that a person of rare blood group has been recognized because of an associated anomaly in red cell morphology.


Coagulation abnormalities produced by plasma exchange on the cell separator with special reference to fibrinogen and platelet levels. Five patients with immunopathologic renal disease, 12 with malignant paraproteinaemia and one with myasthenia gravis underwent a total of 179 plasma exchanges on a continuous flow cell separator. Replacement fluids devoid of coagulation factors were used in 160 exchanges while 19 exchanges were replaced with Fresh Frozen Plasma. Coagulation screening was done immediately before and 30 min after each plasma exchange. Plasma fibrinogen concentrations fell to a mean of 25% of initial levels during individual exchanges. Sequential reduction to 10.7% was observed after five consecutive daily exchanges and in one patient fell to 1.2% after 10 daily exchanges. Low levels of fibrinogen could be maintained with daily or alternate daily exchanges. Platelet counts fell to a mean of 50% of pre-exchange levels during individual exchanges. Consecutive daily exchanges resulted in mean reductions to 20.7% after 5 d, but further reductions were not observed with longer periods of exchange. Platelet counts recovered to pre-exchange values during exchange intervals of 2 or more days. Mild clinical bleeding episodes, probably related to low platelet counts, occurred in one exchange in each of three patients. Haemostasis was rapidly achieved in these patients by infusions of platelet concentrates. Coagulation screening, including prothrombin ratio, thrombin time, reptilase time and partial thromboplastin time with kaolin showed progressively greater abnormalities as the intervals between exchanges shortened. The low incidence of clinical haemorrhagic episodes, three of 179 exchanges (2.2%), despite markedly abnormal coagulation parameters, emphasize the safety of the procedure even in moribund patients. We conclude that the use of FFP in intensive exchange programmes solely for the prevention of spontaneous haemorrhagic phenoma is unjustified.


A genetic variant of factor IX with decreased capacity for Ca2+ binding. A genetic variant of factor IX is described that behaves identically to PIVKA/IX (the precursor factor IX molecule induced by the absence of vitamin K or presence of vitamin K antagonists, acarboxy factor IX). It shows an increased electrophoretic mobility in the presence of Ca2+, a low affinity for adsorption to A1(OH)3 and a very low specific coagulant activity. This variant of factor IX has been demonstrated in the plasma of a patient with severe haemophilia B and in the plasmas of a number of possible carriers from the probands' pedigree.


Indium-III: a new radionuclide label for studying human platelet kinetics. Indium-III. when complexed with 8-hydroxyquinoline (oxine), has been employed as a radioactive platelet label for thrombus imaging in animals and man. The short half-life (2.8 d) and high yield of gamma photons of 111In make it ideal for in vitro counting and external imaging. To evaluate its suitability for studies of platelet turnover in man, platelet kinetic studies were carried out on 10 healthy volunteers using 111In-and 51Cr-platelets concurrently. For 111In labelling, platelets were harvested by differential centrifugation from 43 ml of whole blood drawn into acid-citrate dextrose (ACD) solution. The platelets were washed and suspended in a mixture of ACD and isotonic saline and then incubated with 111In-oxine, rewashed, and suspended in plasma for reinfusion. 51Cr labelling was performed using standard methods. Mean labelling efficiency was 73% with 111In and 6.5% with 51Cr. In vitro studies demonstrated minimal release, elution, and reutilization of the 111In label. There was no significant difference in the aggregation response of 111In- and 51Cr-platelets to ADP and collagen. The in vivo recovery of 111In-platelets was approximately 50% greater than that of 51Cr-platelets whereas the platelet life spans were similar. These results indicate that 111In labelled platelets may be useful for thromobokinetic studies in man. The new method offers the advantages of reduced blood requirements, higher labelling efficiency, and the ability to perform external imaging of platelet distribution in vivo.


The treatment of advanced cystadenocarcinoma of the ovary with gestronol and continuous oral cyclophosphamide. Fifty-four patients with advanced cystadenocarcinoma of the ovary were treated with gestronol (Depostat), 200 mg/week, by intramuscular injection and continuous oral cyclophosphamide, the dose of the latter being varied to maintain the patient's white cell count at about 1.5 x 10(9)/l. A combined partial and complete remission rate of 76 per cent was obtained. The length of survival depended upon the type of remission and the type of remission was markedly affected by the amount of tumour removed at initial laparotomy.


Hormonal profiles in lactating and non-lactating women immediately after delivery and their relationship to breast engorgement. Prolactin, human placental lactogen (HPL), oestrone, oestradiol and progesterone levels in plasma were measured before and during the first seven days after delivery in women who did not breast feed. The results confirmed the rapid clearance of placental steroids from the circulation after delivery. Plasma prolactin levels remained elevated during the early puerperium and the range of values were the same in non breast-feeding women and a group of breast feeding women. Of the 25 women studied, six developed breast engorgement. No difference in hormonal profiles were found leading to the conclusion that there is no endocrine basis for breast engorgement in non-breast feeding women.


A comparison of crown-rump length measurements using a real-time scanner in an antenatal clinic and a conventional B-scanner. In a series of 168 patients in the first trimester of pregnancy, fetal crown-rump length (CRL) measurements were attempted in the Antenatal Clinic using a linear-array real-time ultrasound scanner. The measurements could not be made in 23 of the patients; most of them had a gestational age of less than 10 weeks and the difficulty in obtaining measurements was usually due to an underfilled maternal bladder. When compared with CRL measurements obtained with a conventional B-scanner, a good correlation was obtained (r = 0.92); 80 per cent of the 'real-time' values fell within a range of +/- 5 mm of the conventional values. In all patients the estimates of gestational age using the two methods were within one week of each other.


The accuracy of radiological estimates of gestational age using early fetal crown-rump length measurements by ultrasound as a basis for comparison. Radiological estimates of gestational age in late pregnancy were compared with the gestational ages based on first trimester ultrasound crown-rump length measurements in a series of 125 patients. It was found that the 95 per cent confidence limits of a radiological estimate were +/- 3 1/2 weeks, that both distal femoral and upper tibial epiphyses were frequently present much earlier than 36 and 38 weeks respectively, and that in almost half of the growth-retarded babies in the series there was a bone-age discrepancy of three or more weeks. It is recommended that more liberal use is made of early ultrasound measurements for the assessment of gestational age.


Routine induction of labour with extra-amniotic prostaglandin E2 in a viscous gel. Prostaglandin E2, 350 microgram dispersed in a viscous gel, tylose, was introduced into the extra-amniotic space as a single dose in 285 patients to induce labour. With a favourable cervix, 82 per cent of multiparae and 50 per cent of primiparae were successfully induced. With unfavourable induction features, the success rates were 48 per cent and 24 per cent respectively. In the remaining patients, all but four were successfully delivered when intravenous oxytocin was also used. The method was safe, simple and inexpensive and had many advantages for patients and nursing staff.


Normal and abnormal antenatal ultrasonic cardiographic patterns. Data is presented from 1019 antenatal ultrasonic cardiographs performed on 391 patients; uterine contractions were not monitored concurrently with the fetal heart rate (FHR). No fetal deaths in utero or preterminal FHR patterns were seen within 24 hours of a normal trace. A comprehensive classification of FHR patterns is presented.


Outcome of pregnancy in women using different methods of contraception. During the years 1968-74, 17,032 women were recruited to the Oxford Family Planning Association prospective study of the effects of different methods of contraception. The present analysis, which concerns 5700 pregnancies experienced by the participants in the investigation, adds to the evidence that ex-users of oral contraceptives and intrauterine devices suffer no delecterious effects on the outcome of pregnancy in terms of miscarriage, ectopic gestation, stillbirth, congenital malformation, alteration in the sex ratio or reduction in birth weight. Accidental pregnancies in women taking oral contraceptives were few in the present study (66 in all), but it seems likely that harmful effects of exposure of the fetus to the pill in utero, if any, occur infrequently. Women experiencing an accidental pregnancy with an intrauterine device in situ are at a markedly increased risk of miscarriage and ectopic gestation and are more likely to give birth to an underweight infant than other women.


Multiple births in former oral contraceptive users. Multiple births were studied in a cohort of 2953 former oral contraceptive users and 13,630 controls, all the women were interviewed post partum. There were 238 multiple births, a rate of 14.4/1000 deliveries. Former oral contraceptive users had 13.5/1000 sets of multiple births compared to 14.5/1000 in the controls. When women who had received treatment for anovulation were excluded, these rates were 11.1 and 12.7/1000 respectively, a difference which is not statistically significant. There was, however, a significant deficit of multiple births among the small group of former pill users who were underweight in relation to their height at the time of conception. Multiple births were also increased after stopping sequential pills and preparations containing high doses of oestrogen, and there was an excess of multiple births following breakthrough pregnancies. These findings, although statistically significant, are based on small numbers, and require confirmation from other studies.


A comparison between the effects of nystatin, clotrimazole and miconazole on vaginal candidiasis. Nystatin (100 000 units/day for 14 days) and clotrimazole (100 mg/day for 6 days) were equivalent to miconazole (100 mg/day for 14 days or 100 mg twice daily for 7 days) in curing vaginal candidiasis. However significantly more patients relapsed in the nystatin and clotrimazole groups than in either of the miconazole groups.


Prenatal ultrasound in the diagnosis and management of fetal exomphalos. Case reports. A patient is presented in whom ultrasonic examination at 39 weeks detected fetal exomphalos with an intact sac and no associated gross abnormalities. These findings served as major indications for the safe delivery by Caesarean section of a baby that survived and had successful corrective surgery when 6 months old. The infant might well not have survived vaginal delivery.


Ultrasonic examination in severe diabetic eye disease. B-scan ultrasound of the posterior segment is reported in 154 patients with severe diabetic eye disease. Epiretinal fibrosis, vitreous haemorrhage, vitreous detachment, and retinal detachment were frequently diagnosed. The ultrasonic findings are discussed in relation to the pathological changes in the vitreous in proliferative diabetic retinopathy.


Dark adaptation in diabetes mellitus.

Dark adaptation in diabetes mellitus. The effect of diabetes mellitus on the normal dark adaptation curve is investigated. It has been found that diabetic patients take longer to adapt and that their absolute thresholds are raised. The degree of elevation in the final threshold correlates with the duration of diabetes.


Blepharochalasis.

Blepharochalasis. The condition of blepharochalasis and its treatment are discussed. Four cases are presented which show a physical sign that may help in its diagnosis.


Dark adaptation testing in heterozygotes of Usher's syndrome. Fourteen heterozygous carriers of Usher's syndrome were evaluated by ophthalmoscopy and dark adaptation testing. The normal findings in our study are discussed and compared with those of previous reports, which suggested that abnormal dark adaptation thresholds and fundus abnormalities may be seen in heterozygotes.


A scanning electron microscopic examination of retinoblastoma in tissue culture. Tissue cultures were established from 6 retinoblastomas and the cultured cells examined by scanning electron microscopy. Cells from cultures of normal fetal retinal and glial tissue were also examined by SEM. Though a number of different cell types were found to coexist in the retinoblastoma cultures, most had the features of cells of either glial or neuronal origin. Evidence of further differentiation of cells in each series was seen. These findings indicate that the cell of origin in retinoblastoma is a multipotential stem cell which, though neoplastic, is still capable of differentiation into both glial and neuronal series.


Retinoblastoma: correlation of invasion of the optic nerve and choroid with prognosis and metastases. The histological sections of 74 retinoblastoma patients were reviewed. The extent of optic nerve invasion was correlated with choroidal/scleral extension. Choroidal invasion carries 100% survival provided that the sclera, iris, and whole optic nerve are not also involved. Invasion of the optic nerve beyond the lamina cribrosa also carries 100% survival provided that the resection line is free and that invasion does not involve the sclera or iris. Plentiful rosettes were usually found in those tumours that had not extended beyond the choroid or as far as the resection line of the optic nerve and were therefore associated with a good prognosis. The absence of rosettes did not necessarily indicate a poor prognosis. The site of metastases was related to optic nerve and scleral/extrascleral extension. The various systems of staging retinoblastoma are compared, and a new system is proposed to cover the whole spectrum of the disease and to offer a reliable guide to prognosis and treatment.


Prognostic significance of aqueous humour lactic dehydrogenase activity. The lactic dehydrogenase (LDH) activity of the aqueous humour has been estimated in both eyes of 7 patients having uniocular retinoblastoma. In 1 patient the aqueous humour LDH activity in the healthy eye was above normal, but there was no clinical evidence of malignancy. Tumour tissue was detected in this eye 9 months later, and the aqueous humour showed a rise in LDH activity. A high LDH activity persisted even after irradiation, though no tumour tissue was visible ophthalmoscopically. It is suggested that the estimation of the LDH activity in the aqueous humour of the healthy eye in cases of uniocular retinoblastoma might be of value in the early detection of a bilateral retinoblastoma.


Correlation of aqueous humour lactic acid dehydrogenase activity with intraocular pathology. An analysis of the lactic acid dehydrogenase (LDH) activity in the aqueous humour of 24 enucleated eyes with retinoblastoma showed that though there was no relationship between the LDH levels and the age of the patient, there appeared to be an increase in the LDH activity with increase in the duration of the tumour. Undifferentiated tumour cells and tumour cell necrosis seemed to contribute to an increase of LDH levels in the aqueous humour, but there was no relationship between the occurrence of calcification and the LDH secretion into the aqueous humour. Massive cell necrosis caused by radiotherapy and central retinal artery occlusion significantly increased the LDH levels in the aqueous. It also appeared that recurrence was common after external cobalt therapy and that secondary extension of the tumour into the optic nerve and choroid was favoured by this procedure.


A new method for measuring the body density of obese adults. 1. A new apparatus is described with which it is possible to measure the volume (and hence density) of obese patients without requiring them to immerse totally in water. Replicate measurements of subjects with 6, 23 and 38 kg body fat had a standard deviation not greater than 0.3 kg fat. 2. In nineteen obese women body fat was measured by density, total body water, and total body potassium at the beginning, and again at the end, of a period of 3--4 weeks on a reducing diet, during which they lost 5.43 (SD 1.83) kg in weight. The composition of weight loss was also estimated both by energy balance and nitrogen balance during the interval between the two measurements of body composition. 3. The estimates of fat content of the ninetten women at the start of the balance period were 45.63 (SD 14.50) kg by density, 48.07 (SD 13.88) kg by K and 47.09 (SD 13.85) kg by water. The correlation coefficient between the density and K estimate was 0.949, and for the density and water estimate it was 0.971. 4. It is concluded that measurement of density by the new method provides a convenient method for estimating body fatness, and change in fat content, which compares favourably with estimates based on total body water or total body K. However, these methods cannot be used to provide an accurate estimate of the composition of a small weight loss in an individual since deviations up to 4 kg fat occur between fat loss based on change in density and those based on the more reliable (but more tedious) energy balance method.


The relationship between protein turnover and energy balance in lean and genetically obese (ob/ob) mice. 1. Groups of lean and genetically obese (ob/ob) mice were adapted to varying energy intakes and the rates of total protein turnover in liver, gut and kidney were measured. 2. Lean mice gained less weight when fed above maintenance and lost less weight when fed below maintance than obese mice. 3. Hepatic protein turnover (mg/d) was sigmoidally related to digestible energy intake in lean mice but showed no significant changes with dietary intake in obese mice. 4. The changes in protein turnover resulted from changes in both the half-lives of protein synthesis and catabolism and in tissue protein content. 5. In the lean mice, protein turnover in kidney and gut was not significantly changed with increasing energy intake until the highest level was reached. 6. The findings suggest that protein turnover may be an important cycle for the regulation of energy balance in mice and that this cycle is impaired in the genetically obese (ob/ob) mice.


Effects of saponins on bile acids and plasma lipids in the rat. 1. The effects of feeding isolated saponins on plasma lipid concentrations and on concentrations of biliary and faecal bile acids and neutral sterols were studied in the rat. 2. The animals were given one of four diets, i.e. a standard low-cholesterol synthetic diet, the diet + 10 g saponins/kg, the diet + 10 g cholesterol/kg, the diet + 10 g cholesterol + 10 g saponins/kg. 3. Saponins partially reversed the hypercholesterolaemia caused by the high-cholesterol diet and increased both the rate of bile acid secretion and the faecal excretion of bile acids and neutral sterols. The proportionate contribution of the primary bile acids (particularly chenodeoxycholic) to faecal excretion was also increased by saponins. 4. The results are discussed in relation to the hypothesis that saponins act by inducing the adsorption of bile acids by dietary fibre.


Experiments with the long-term rumen simulation technique (Rusitec); response to supplementation of basal rations. 1. It is shown that a basal roughage diet can be successfully supplemented with glucose, as long as nitrogen is not limiting. Lack of N depresses the digestibility of the basal ration, results in incomplete fermentation and the increased recoveries of N are consistent with fixation of atmospheric N2. 2. Using a complex, but soluble supplement (whey powder) it is shown that reproducible incremental measurements can be made and that the supplement used gives increases in production of characteristic end-products only (carbon dioxide, methane, acetic and butyric acids). 3. The location of the solid dietary components within the reaction vessel is not important and it is possible to measure changes in a particular component in the presence of others. It is shown that there is sequestration of bacteria and protozoa on the solid digesta.


Experiments with the longterm rumen simulation technique (Rusitec); use of soluble food and an inert solid matrix. 1. The role of soluble nutrients and of the solid matrix in rumen fermentation was investigated in some detail, and experiments designed to explore the possibility of using a balanced soluble diet and an inert solid matrix, are described. 2. The use of a balanced soluble substrate as the only source of nutrients in the presence of an inert solid phase in the reaction vessels results in vigorous fermentation but is accompanied by disappearance of protozoa from the effluent. 3. In the absence of digestible solid phase, the rate of fermentation and the fermentation pattern depends mainly on the amount and type of nutrients supplied and to a smaller extent on the dilution rate, the variations being greatest at low dilution rates. 4. The solid matrix in the form of wood shavings or the residue remaining after prolonged digestion of hay could sequestrate micro-organisms and could be used as solid phase, but the defined mixture of soluble substrates used resulted in somewhat abnormal fermentation compared with fermentation obtained with solid food. 5. When the solid food included some hay extract and when the dilution rate was not too great a reduced output of protozoa could be maintained. At high dilution rate the outflow of protozoa was negligible and yet considerable numbers of protozoa were found in the solid matrix and associated liquid. 6. A three-compartment model was developed to describe the flow of liquid and microbial matter within the simplified system.


Effects of the protracted feeding of copper sulphate-supplemented diets to laying, domestic fowl on egg production and on specific tissues, with special reference to mineral content. 1. Two experiments are reported. In both experiments a cereal-based diet containing 5 mg copper/kg was fed to two breeds of laying hens for 336 d. In Expt 1 four other groups were given this diet with the addition of CuSO4.5H2O to give added levels of 200, 400, 600 and 800 mg Cu/kg diet. In Expt 2 the levels of added dietary Cu used were 100, 200, 300 and 400 mg/kg. 2. In Expt 1 records were kept of food intake, water intake, body-weight and egg production for eight 28 d periods and body-weight and egg number only were recorded for the full twelve periods. In Expt 2 full records, excluding water intake, were taken for all twelve periods. 3. Food and water intake showed a quadratic response to level of added dietary Cu, being enhanced at lower levels and depressed at higher levels of addition. 4. There was a quadratic response of total egg weight, mean egg weight and egg number to added dietary Cu. In Expt 1 egg number was maximum at 235 mg added Cu/kg diet for Warren Studler SSL (breed 1) and at 170 mg added Cu/kg diet for Shaver 288 (breed 2). In Expt 2 no breed effect occurred, the maximum egg number being calculated to occur at 176 mg added Cu/kg diet. 5. Depression of body-weight gain occurred at high levels of Cu addition. The depression of liver and oviduct weight found at high levels of addition appeared to be directly related to body-weight. A marked amount of feather loss also occurred at a high inclusion of CuSO4 in the diet. 6. The reproductive systems of the hens did not appear to be adversely affected at the levels of additive used. Gross and microscopic examination of specific tissues revealed no pathological effects although gizzard and intestinal weights were increased and caecal weight decreased by high levels of added Cu. THose aspects of the blood chemistry examined did not reveal any consistent effect between the two experiments. 7. The liver Cu analyses indicate that between 600 and 800 mg added Cu/kg diet the liver Cu concentration rises sharply. Both liver Fe and Zn concentrations showed a positive linear response to added dietary Cu. In the kidney Cu and Zn concentrations were increased but only to a limited extent, while the concentration of Fe was unaffected.


Studies on digestion and absorption in the intestines of growing pigs. 7. Measurements of the flow of total carbohydrate, total reducing substances and glucose. 1. Seventeen pigs were fitted with single re-entrant cannulas in either the duodenum (posterior to the entry of the bile and pancreatic ducts), the mid-jejunum, or the terminal ileum. A further twenty-four pigs were used in a conventional digestibility trial. 2. Three diets were used: these contained barley, fine wheat offal, white fish meal, minerals and vitamins (diet BWF) ; starch, sucrose, maize oil, cellulose, minerals, vitamins and either groundnut meal (diet SSG) or casein (diet SSC). 3. The quantities of total carbohydrate (TC), total reducing substances (TRS) and glucose (G) passing through the re-entrant cannulas and excreted in the faeces in 24 h were measured. These were used to determine the net absorption of the carbohydrate fractions in the different regions of the intestine. 4. The small intestine was the principal site of absorption of TC, TRS and G, but there were differences between the diets in the quantities of each of these carbohydrate fractions that were absorbed in the different regions of the small intestine studied. 5. The quantities of TRS and G in solution were very low for all diets at all sites, indicating that the rate of absorption of the products of hydrolysis kept pace with their rate of formation.


Studies on digestion and absorption in the intestines of growing pigs. 8. Measurements of the flow of total lipid, acid-detergent fibre and volatile fatty acids. 1. Seventeen pigs fitted with single re-entrant cannulas in either the duodenum (posterior to the point of entry of the bile and pancreatic ducts), the mid-jejunum, or the terminal ileum, and a further twenty-four non-cannulated pigs were used. 2. Three diets were used: these contained barley, fine wheat offal, white fish meal, minerals and vitamins (diet BWF); starch, sucrose, maize oil, cellulose, minerals, vitamins and either groundnut meal (diet SSG) or casein (diet SSC). 3. The quantities of total lipid (TL), acid-detergent fibre (ADF) and volatile fatty acids (VFA) passing through the re-entrant cannulas and excreted in the faeces in 24 h were measured. These were used to determine the net absorption (or synthesis or secretion) in the different regions of the intestine. 4. There was substantial secretion of TL into the proximal small intestine and major absorption from the distal portion for all three diets. In the large intestine there was little or no further net absorption for diets SSG and SSC, but significant synthesis or secretion for diet BWF. 5. Fibre digestion occurred in both the small and large intestines and the apparent absorption of ADF in the different regions studied was influenced by diet. 6. There were detectable amounts of VFA at all sites but only at the ileum and in the faeces could they be accurately measured. Diet did not affect the molar proportions of acetate, propionate and butyrate in ileal digesta, but did have an effect on these VFA in the faeces.


Protein quality in cereals and pulses. 2. Influence of polyethyleneglycol on the nutritional availability of methionine in sorghum (Sorghum vulgar Pers.), field beans (Vicia faba L.) and barley. 1. Polyethyleneglycol (PEG 4000) was examined for its influence on relative nutritional value (RNV) and available methionine in sorghum (Sorghum vulgare Pers.), field beans (Vicia faba L.) and barley, as measured microbiologically with Streptococcus zymogenes. The results were assessed in relation to the content of tannins in the test samples. 2. In grain of hybrid sorghum the RNV averaged 87 (range 79--92) for six low-tannin varieties and 41 (30--53) for eleven high-tannin varieties. The corresponding available methionine values averaged 17.0 (15.7--18.9) and 8.9 (6.7--11.0) g/kg protein. Addition of PEG 4000 to the test samples increased the average RNV of the high-tannin varieties from 41 to 78, and the average available methionine content from 8.9 to 16.2 g/kg protein. 3. With seed of ten coloured flowered varieties of field beans, treatment with PEG gave a small but consistent increase in the available methionine content, which resulted from the inactivation of tannins in the testa. 4. In twenty-three samples of barley grain, treatment with PEG had no effect on the values obtained for available methionine. 5. Treatment of high-tannin sorghum grain with ammonia has been reported to inactivate the tannins and increase the nutritional value for rats and chicks. This finding was confirmed. The present study showed that ammonia and PEG 4000 were equally effective in enhancing the nutritional quality as measured in the microbiological tests.


Protein quality in cereals and pulses. 3. Bioassays with rats and chickens on sorghum (Sorghum vulgare Pers.), barley and field beans (Vicia faba L.). Influence of polyethylene glycol on digestibility on the protein in high-tannin grain. 1. Two preceding papers in this series describe the application of microbiological and other in vitro tests in the evaluation of sorghum (Sorghum vulgare Pers.), field beans (Vicia faba L.) and barley, and in assessing the influence of polyethylene glycol (PEG 4000) on the nutritional availability of the methionine. The present paper gives for comparison the results of bioassays on some of the same test samples. Net protein utilization (NPU) in rats was measured by the nitrogen balance method, and N digestibility in chickens by the ileal analysis procedure. 2. In rat tests on sorghum, N in grain of high-tannin varieties was poorly digested. Supplementation of the test diets with 0.1 g PEG 4000/g protein gave a large improvemnet, which was partly offset by an apparent decrease in biological value (BV). With chickens N digestibility was even lower, and was similarly improved with PEG 4000. Treatment of high-tannin grain with ammonia solution was also effective in improving N digestibility. 3. With low-tannin sorghum the amino acid digestibilities were uniformly high and were not affected by addition of PEG to the test diet. With high-tannin sorghums they were low and less uniform, and were much improved by PEG 4000. 4. With field beans, the influence of the seed-coat tannin on protein utilization was much less pronounced than with sorghum. In chickens there was a significant effect (P less than 0.05) of PEG 4000 on N idgestibility in a high-tannin variety. With rats the effect was smaller and not significant. 5. In four samples of barley. N digestibility was high (0.87--0.96) and was not further improved by PEG 4000. The BV of a high-lysine cultivar proved marginally inferior to that of a normal variety. Possible reasons for this are discussed. 6. Over all, the results were closely consistent with those from microbiological tests with Streptococcus zymogenes.


Histone gene switch in the sea urchin embryo. Identification of late embryonic histone messenger ribonucleic acids and the control of their synthesis. During embryogenesis in the sea urchin Strongylocentrotus purpuratus, there is a shift from one histone mRNA population to another. The early and late embryonic histone mRNAs, previously shown to differ considerably in sequence from each other by hybrid melting studies, are shown here to differ also in electrophoretic mobility on polyacrylamide gels as the positions of the early and late mRNAs are completely noncoincident. The various species of both early and late samples are identified as particular histone mRNAs by hybridization to cloned histone DNAs containing part of the early-type repeat unit or to restriction enzyme fragments derived from these unit. Four bands in the early mRNA sample are identified as H1, H3, H2A " H2B, and H4 mRNA while at least 10 bands can be seen in the late mRNA preparation with unambiguous identification of H1, H2B, and H4 mRNAs. A cluster of late species is shown to contain both H3 and H2A mRNA. When a polysomal RNA preparation from the 26-h embryo is hybridized to the histone DNA, eluted, and then translated in vitro in a wheat germ system, the histone products migrate in the position of late histones when subjected to electrophoresis on Triton X-urea gels. Using DNA which contains genes for H2A + H3 or H2A alone, we demonstrate the specificity of the early-type DNA probes for these two late histones. Therefore, by hybridization of newly synthesized RNAs and translation of the total polysomal RNA present in the late embryo, it is shown that mRNAs for all five histone classes may cross-react with the cloned early-type DNA. The hybrids formed, however, are much less stable than those formed with the early histone mRNA. In vitro translation of total cytoplasmic RNA from various embryonic stages indicates that transition between the two classes occurs during most of the blastula period.


Hemocyanin of Octopus vulgaris. The molecular weight of the minimal functional subunit in 3 M urea. In the presence of 3 M urea Octopus vulgaris (Mollusca) hemocyanin dissociates completely, giving a single functional component which reassociates in the original aggregate after removal of urea. The molecular weight of this subunit has been determined by gel filtration, by viscosity measurements, and by ultracentrifugation. The values obtrained with the different methods range from 247,000 to 262,000. Electron microscopy shows that the reassociation of the functional subunit after removal of urea is complete and gives the typical cylindrical structure of the native protein. This component is the minimal functional subunit which can be obtained from Octopus hemocyanin without splitting covalent bonds. It is suggested that this component is made by five protomers (50,000 daltons) containing one oxygen binding site each bound covalently through, perhaps, the carbohydrate moieties of the protein.


Synthesis of 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid and detection of skin fibroblast neuraminidase in normal humans and in sialidosis. A procedure for the synthesis of the fluorogenic substrate analogue 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid for the human acid neuraminidase has been developed. The substrate was employed for the characterization of the enzyme in sonicates of cultured human skin fibroblasts and for enzymatic detection of the neuraminidase deficiency in the neurological storage disorder, sialidosis. Synthesis was accomplished by reacting 2-deoxy-2-chloro-4,7,8,9-tetra-O-acetyl-N-acetylneuraminic acid methyl ester with the sodium salt of 4-methylumbelliferone in acetonitrile at room temperature. The coupled product was purified on silicic acid chromatography, followed by base-catalyzed removal of the O-acetyl and methoxy blocking groups, and with additional purification of the hydrolyzed product on silicic acid. The overall yield, based on N-acetylneuraminic acid, was 37%. Under linear assay conditions, at pH 4.3, the apparent maximal velocities (nmol (mg of protein)-1 h-1) for normal fibroblasts were 58--115, 0.2--1.8 for sialidosis fibroblasts, and 28--38 for obligate heterozygotes. The apparent Km for normals was 0.13 mM.


Thymidylate synthetase. Catalysis of dehalogenation of 5-bromo- and 5-iodo-2'-deoxyuridylate. Tymidylate synthetase catalyzes the facile dehalogenation of 5-bromo-2'-deoxyuridylate (BrdUMP) and 5-iodo-2'-deoxyuridylate )IdUMP) to give 2'-deoxyuridylate (dUMP), the natural substrate of the enzyme. The reaction does not require folate cofactors and stoichiometrically consumes 2 equiv of thiol. In addition to dUMP, a minor product is formed during the debromination of BrdUMP which has been identified as a 5-alkylthio derivative formed by displacement of bromide ion by thiolate. The reaction has been found to proceed with a substantial alpha-secondary inverse tritium isotope effect (kT/kH = 1.212--1.258) with [2-14C,6-3H]-BrdUMP as the substrate. Similarly, an inverse tritiumisotope effect of 1.18 was observed in the nonenzymatic chemical counterpart of this reaction, the cysteine-promoted dehalogenation of [2-14C,6-3H]-5-bromo-2'-deoxyuridine. Previous evidence for the mechanism of action of this enzyme has rested largely on chemical model studies and on information obtained from its stoichiometric interaction with the inhibitor 5-fluoro-2'-deoxyuridylate. The magnitude of the secondary isotope effect during the enzymatic dehalogenation described here provides direct proof for nucleophilic catalysis and formation of 5,6-dihydroprimidine intermediates in a reaction catalyzed by thymidylate synthetase.


Dialdehydes derived from adenine nucleosides as substrates and inhibitors of adenosine aminohydrolase. A series of nucleoside dialdehydes have been obtained as powders after treatment of various adenine nucleosides with paraperiodic acid. Thus, oxidation gave dialdehydes derived from adenosine (1), 9-alpha-D-mannopyranosyladenine (2), 9-(5-deoxy-alpha-D-arabinofuranosyl)adenine (3), 9-alpha-L-rhamnopyranosyladenine (4), 9-beta-L-fucopyranosyladenine (5), 9-beta-D-fucopyranosyladenine (6), 9-alpha-D-arabinopyranosyladenine (7), 9-beta-D-ribopyranosyladenine (8), and 9-(5-deoxy-beta-D-erythro-pent-4-enofuranosyl)adenine (9). Nucleoside dialdehydes 1-3 and 6-8 were weak substrates for adenosine aminohydrolase from calf intestinal mucosa. Dialdehyde 8 had the strongest affinity, but 1 had the highest Vmax. All of the dialdehydes except 5 were inhibitors of the enzyme. The best inhibitors were 9 (Ki = 4 microM) and 4 (ki = 28 microM), and neither were substrates. The inhibitors did not exhibit time-dependent inhibition and did not appear to form covalent bonds with the protein. The data strongly suggest that the active form of the dialdehydes is as the open-chain dihydrates. The alcohol obtained by reduction of 9 (compound 10) was the strongest inhibitor (Ki = 0.9 microM among the related alcohols and the nucleoside dialdehydes.


Amino acid catalyzed condensation of purines and pyrimidines with 2-deoxyribose. Mild heating of aqueous mixtures containing 2-deoxyribose, amino compounds, and purines or pyrimidines produces derivatives of the purines and pyrimidines in high yield. Among the major products formed are 2,3-dideoxy-3-(1'-pyrimidino)pentose and 2,3-dideoxy-3-(9'-purino)pentose. The mechanism of the reaction includes amine-catalzyed dehydration of the alpha, beta positions of the sugar followed by addition of the purine or pyrimidine to the double bond. Rapid addition of purines and pyrimidines to alpha, beta-unsaturated carbonyl compounds (such as acrolin) is a general phenomenon which does not require an amine catalyst. While multiple derivatization of the purines will take place, the N-9 derivative is formed first.


Influence of hydrogen bonding in DNA and polynucleotides on reaction of nitrogens and oxygens toward ethylnitrosourea. The reactivity of ethylnitrosourea toward hydrogen-bonded sites in double-stranded DNA or oly(rA).poly(rU) was compared with those sites in single-stranded DNA, RNA, or poly(rA). Alkylation of the N-1 of A in poly(rA).poly(rU) was almost suppressed at 5 degrees C but could be markedly increased by raining the reaction temperature to 25 degrees C, well below the Tm of 56 degrees C. In contrast, the N-7 and N-6 of A, which are not hydrogen bonded, reacted to the same extent at temperatures ranging from 5 to 65 degrees C. The extent of reaction at the N-3 of A varied inversely with the reactivity of the N-1 of A, indicating that of these two nitrogens the N-1 of A is the most reactive. The proportion of reaction at the various nitrogens in poly(rA) was not affected by temperature. Hydrogen-bonded oxygens in double-stranded DNA are the O-6 of G, the O-4 of T, and the O2 of C. All are equally reactive at 5, 25, and 51 degrees C. It is concluded that the observed temperature independence is due to these oxygens having an electron pair not involved in hydrogen bonding and, thus, available for reaction. In contrast, the electron pair of the N-1 of A (or the N-3 of C) is involved in hydrogen bonding, and the extent of their reactivity is dependent on thermal fluctuation providing transiently open base pairs at temperatures far below the Tm.


Determination of the size of rat ribosomal deoxyribonucleic acid repeating units by electron microscopy. The empoyment of a novel method of affinity chromatography, which makes use of antibodies that specifically bind DNA/RNA hybrids, has made it possible to enrich for rat rDNA molecules which contain R loops formed with the 18S and 28S rRNAs. An approximately 150-fold enrichment of the ratrRNA coding sequences was obtained by this affinity chromatography procedure. This degree of enrichment made it possible to visualize these R loop containing molecules in the electron microscope and, thus, to obtain a map of the transcribed and spacer regions of rat rDNA. Eleven of the molecules that were observed contained either 3 or 4 R loops, or else 2 R loops separated by a long spacer. Thus, these molecules provided direct information in regard to the length of rat rDNA repeating units. The mean length of the repeating units was 37.2 kbp with a standard deviation of 1.3 kbp. Within the errors of the measurements, these could all represent repeating units of exactly the same length, although a certain degree of length heterogeneity, possibly up to 4 or 5 kbp, cannot be ruled out by the data. If significantly longer or shorter rDNA repeating units exist in the rat genome, they are probably much less common than the 37.2 kbp unit. These electron microscopic measurements provide the most definitive data yet available on the size of the repeating units of mammalian rRNA genes.


Nucleosome reconstitution: effect of DNA length on nuclesome structure. Core histones (H2A, H2B, H3, and H4) are reconstituted by salt gradient dialysis with DNA molecules ranging in length from 177 bp down to 50 bp. While reconstituted particles containing 125 bp are very similar to native particles, those particles containing a single piece of shorter DNA tend to aggregate. The aggregation depends on the ionic strength and DNA length. The DNA placement on the histone core is not random as determined by pancreatic DNase I digestions of particles containing 32P 5'-end-labeled DNA. Rather, it is found that all DNA molecules, up to 161 bp in length, reassociate with core histones in such a way as to produce defined patterns of DNase I cutting with respect to the 5' ends. Particles were made that contained two pieces of 65-bp DNA. These particles are very similar to native particles under most conditions but tended to dissociation results in the production of two half-nucleosomes (hemisones).


Resonance Raman study of flavins and the flavoprotein fatty acyl coenzyme A dehydrogenase. The resonance Raman (RR) spectra of FMN, FAD, FAD in D2O, and 7,8-dimethyl-1, 10-ethyleneisoalloxazinium perchlorate have been obtained by employing KI as a collisional fluorescence-quenching agent. The spectra are very similar to those obtained recently by using the CARS technique to eliminate fluorescence. Spectra have also been obtained for several species in which flavin is known to fluoresce only weakly. We report RR spectra of protonated FMN, FMN semiquinone cation, the general fatty acyl-CoA dehydrogenase, and two "charge-transfer" complexes of fatty acyl-CoA dehydrogenase. Tentative assignment of several vibrational bands can be made on the basis of our flavin spectra. RR spectra of fatty acyl-CoA and its complexes are consistent with the previous hypothesis that visible spectral shifts observed during formation of acetoacetyl-CoA and crotonyl-CoA complexes of fatty acyl-CoA dehydrogenase result from charge-transfer interactions in which the ground state is essentially nonbonding as opposed to interactions in which complete electron transfer occurs to form FAD semiquinone. The only significant change in the RR spectrum of FAD on binding to enzyme occurs in the 1250-cm-1 region of the spectrum, a region associated with delta N--H of N-3. The position of this band in fatty acyl-CoA dehydrogenase and the other flavoproteins studied to date is discussed in terms of hydrogen bonding between flavin and protein.


Structural and kinetic studies on the solubilization of lecithin by sodium deoxycholate. Mixed dispersions of egg phosphatidylcholine (PC) and the bile salt sodium deoxycholate (DOC) were prepared by various methods, and their turbidities and proton magnetic resonance spectra were studied as a function of time. The spectra of dispersions prepared by dissolving both components in a common organic solvent and replacing the organic solvent by water did not change with time, indicating that the mixed aggregates formed represent "a state of equilibrium". In the 1H NMR spectra of these mixed aggregates, only signals from small mixed micellar structures were narrow enough to be observed. The dependence of the NMR line widths on the molar ratio of DOC to PC (R) is interpreted in terms of a model for the PC--DOC mixed micelles, according to which PC is arranged as a curved bilayer, the curvature of which increases with increasing R. Upon mixing PC with aqueous solutions of DOC, we found that the mixed aggregates formed are slowly reorganized and ultimately reach the same state of equilibrium. This reorganization was found to be a pseudo-first-order process, the rate constant of which depends linearly upon the detergent concentration. This process involves saturation of the outer bilayers of the multilamellar PC by detergent, followed by transformation of these bilayers into mixed micelles. It is concluded that the solubilization occurs through consecutive "peeling off" of lecithin bilayers.


Partial purification and characterization of mammary stimulating factor, a protein which promotes proliferation of mammary epithelium. This report describes the partial purification and characterization of mammary stimulating factor (MSF), a mitogenic peptide isolated from serum which initiates growth in mouse mammary epithelium. By using ion-exchange chromatography, gel filtration, and isoelectric focusing, MSF was purified 250-fold from porcine serum. It is a heat-stable protein of molecular weight 10,100--10,400 with an isoelectric point of 5.5--6.0. MSF initiates DNA synthesis in vitro in mammary epithelium to a greater extent than in mouse mammary tumor cells (CZF), 3T3 cells, or chick embryo cells. Comparison of the biological, physical, and immunological properties of MSF with other established growth-promoting peptides suggests that MSF is a unique serum factor.


Evidence of a precursor form of stratum corneum basic protein in rat epidermis. The fully differentiated anucleate cells of the stratum corneum of newborn rat epidermis contain a cationic protein called stratum corneum basic protein (SCBP). This protein has a molecular weight (49 000) and an amino acid composition similar to a protein extracted from the less differentiated cell layers of the epidermis. Pulse--chase experiments with radiolabeled histidine were undertaken to test the possiblity that SCBP is derived from a preexisting protein. A protein of 52 000 daltons is rapidly but transiently labeled in extracts of the less differentiated cell layers. As the amount of label in the 52 000-dalton protein decreases, an increase in radiolabel is observed in extracts of the fully differentiated cells. This label is found in SCBP, a protein of lower molecular weight (49 000) than that initially labeled. These proteins are immunologically related and both are resistant to cyanogen bromide cleavage. They differ in apparent molecular weight on sodium dodecyl sulfate--polyacrylamide gels and in their net charge. The results are consistent with the conversion of a precursor protein into SCBP.


Thrombocytin, a serine protease from Bothrops atrox venom. 1. Purification and characterization of the enzyme. Thrombocytin, a platelet-activating enzyme from Bothrops atrox venom, has been purified to homogeneity by precipitation with sodium salicylate and chromatography on heparin--agarose. Thrombocytin is a single-chain glycoprotein with a molecular weight of 36 000 which contains 5.6% carbohydrate. It causes platelet aggregation, release of platelet serotonin, and activation of factor XIII. The most sensitive substrate for the amidolytic activity of thrombocytin was Tos-Gly-Pro-Arg-p-nitroanilide hydrochloride. The activity of thrombocytin on this substrate and on platelets was inhibited by diisopropyl fluorophosphate (DFP), soybean trypsin inhibitor, and several arginine chloromethyl ketones. Active site titration with nitrophenyl guanidinobenzoate demonstrated that approximately 86% of the preparation was in the active form. These experiments demonstrate the presence of serine and histidine in the active site of thrombocytin and suggest that thrombocytin is a classical serine protease with a platelet-activating activity similar to thrombin.


Thrombocytin, a serine protease from Bothrops atrox venom. 2. Interaction with platelets and plasma-clotting factors. Thrombocytin, a serine protease from Bothrops atrox venom, caused platelet aggregation and release of platelet constituents at a concentration of 10(-7) M and clot retraction at a concentration of 2 x 10(-9) M. Thrombocytin was slightly more active when tested on platelets in plasma than on washed platelets suspended in Tyrode--albumin solution. Thrombin was 5 times more active than thrombocytin when tested on platelets in plasma and 50 times more active when tested on washed platelets. The patterns or release induced by thrombocytin and thrombin were similar. Prostaglandin E1 (10(-5) M) produced complete inhibition of platelet release induced by thrombocytin and thrombin. Indomethacin (10(-4) M) was without any effect. Antithrombin III, in the presence of heparin, inhibited the action of thrombocytin on platelets and on a synthetic peptide substrate (Tos-Gly-Pro-Arg-pNA.HCl). formation of an antithrombin III--thrombocytin complex was demonstrated on NaDodSO4--polyacrylamide gel electrophoresis. Hirudin and alpha 1-antitrypsin did not inactivate thrombocytin. Thrombocytin had a low fibrinogen-clotting activity (less than 0.06% that of thrombin). Thrombocytin also caused progressive degradation of the alpha chain of human fibrinogen, and it cleaved prothrombin, releasing products similar to intermediate 1 and fragment 1 produced by thrombin. Thrombocytin activated factor XIII by limited proteolysis and increased the procoagulant activity of factor VIII in a manner analogous to that of thrombin.


Time-resolved resonance Raman characterization of the bO640 intermediate of bacteriorhodopsin. Reprotonation of the Schiff base. The resonance Raman spectrum of photolyzed bacteriorhodopsin under conditions known to increase the concentration of the bO640 intermediate in both H2O and D2O is presented. By use of computer subtraction techniques and a knowledge of the Raman spectra of the unphotolyzed bacteriorhodopsin as well as the other intermediates in the cycle, a qualitative spectrum of bO640 is determined. The shift of a band at 1630 cm-1 in H2O to 1616 cm-1 in D2O suggests that the Schiff base of bO640 is protonated. Additional bands at 947, 965, and 992 cm-1 that appear only in D2O suspensions confirm that a proton is coupled to the retinal chromophore of bO640. The reprotonation of the Schiff base thus occurs during the bM412 to bO640 step. The fingerprint region, sensitive to the isomeric configuration of the retinal chromophore of bO640, is dissimilar to the fingerprint regions of published model compounds and other forms of bacteriorhodopsin.


Spectroscopic studies of pyridoxamine (pyridoxine) 5'-phosphate oxidase. Equilibrium dissociation constants and spectra for riboflavin 5'-phosphate and analogues. Pyridoxamine (pyridoxine) 5'-phosphate oxidase (EC 1.4.3.5) has been shown to bind 1 mol of riboflavin 5'-phosphate (FMN) per mol of apoenzyme and is active with or inhibited by numerous FMN analogues [Kazarinoff, M. N., & McCormick, D. B. (1975) J. Biol. Chem. 250, 3436--3442]. The KD values and spectra for selected apoenzyme--flavin complexes have been determined and used to elucidate some of the properties of the FMN-binding site of this flavoprotein. Alterations of the pyrimidinoid portion of the flavin ring decrease binding considerably. The absorption spectra for the protein complexes with 3-deaza-FMN and 8-hydroxy-FMN indicate the presence of a dipolar or positively charged protein group near N1 and O2. The substitution of methyl for hydrogen at N3 apparently causes distortion of the interaction between the flavin ring and an active-site aromatic amino acid residue. Although binding is also decreased somewhat by substitutions at postions 8 and 8 alpha, considerable bulk [e.g., 8-(diethylamino)-FMN and 8 alpha-S-(N-acetyl-cysteinyl)-FMN] is accommodated. Hence, this portion of the flavin ring is probably oriented toward, possibly in contact with, solvent, as has been found for the flavodoxins. The importance of optimum interactions between the flavin and the apoprotein is further emphasized by large differences in the activity of flavin analogues that have similar midpoint potentials in solution.


Adenosine triphosphate compartmentation in living hearts: a phosphorus nuclear magnetic resonance saturation transfer study. 31P nuclear magnetic resonance (NMR) studies of creatine phosphokinase (CPK) kinetics using saturation transfer techniques are reported. The phosphocreatine (PCr) and adenosine triphosphate (ATP) levels in perfused hearts can be altered experimentally by stopping the flow of perusate (ischemia) to the heart for 35-min periods, followed by reperfusion to produce stable levels of performance. Utilization of energy by the heart was altered by administration of 25 mM potassium chloride (KCl) in the perfusate, which arrests contraction of the myocardium. Compared with control heart studies, the unidirectional rates measured during ischemia and KCl arrest are altered. The rates observed in the control experiments indicate that the CPK system is not in a steady state. This apparent deviation from steady-state conditions is ascribed to the existence of intracellular compartmentation of ATP.


Enthalpy of nucleotides binding to myosin. The enthalpies of binding adenosine 5'-diphosphate (ADP) and 5'-adenylyl imidodiphosphate [AMP-P(NH)P] to rabbit skeletal myosin have been measured in Pipes and Tris buffers at pH 7.8 and 15 degrees C. For ADP the enthalpy of binding was exothermic, whereas the enthalpy of binding AMP-P(NH)P, a nonhydrolyzable ATP analogue, was small and endothermic. For the reaction of ATP and myosin, the development of enthalpy was resolved into two phases: a fast endothermic phase, which is the summation of binding and hydrolysis, and a slow exothermic phase, which is associated with product-release steps. These results are discussed in terms of their implications for energy transduction.


Pyruvate carboxylase from a thermophilic Bacillus: some molecular characteristics. Analysis of the native enzyme and of the subunits produced upon its denaturation shows that pyruvate carboxylase from a thermophilic Bacillus is a tetramer with a molecular weight (mean value) of 558,000 and that the four polypeptide subunits are probably identical. The three functions (carboxyl carrier, carboxylation, and carboxyl transfer) in the pyruvate carboxylation reaction must therefore reside in this quarter-molecular polypeptide. The enzyme molecule contains four atoms of zinc and four molecules of D-biotin, and in the electron microscope the disposition of its four subunits presents a rhombic appearance. Reaction of the denatured enzyme with 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) reveals 10 sulfhydryl groups/subunit. In the native enzyme less than one of these groups reacts with DTNB. By contrast, all of these groups (11/subunit) of the native chicken liver pyruvate carboxylase are accessible to DTNB. The thermophile enzyme is also more resistant to other sulfhydryl reagents and to denaturation under certain conditions than the avian enzyme.


Insulin binding to isolated liver nuclei from obese and lean mice. Nuclei isolated from the livers of mice are capable of binding [125I]insulin. A class of high-affinity binding sites having a Kd of 1--2 nM and a capacity of approximately 2000 insulin molecules/nucleus are present on these nuclei. Removal of nuclear membranes by Triton X-100 treatment of the nuclei reduces or eliminates the high-affinity binding sites. Nuclei prepared from livers of the genetically obese mouse (ob/ob) lack, or have markedly reduced numbers of, the high-affinity binding sites whether or not the obese nuclei have been exposed to Triton X-100. The reduced insulin-binding capacity of the obese nuclei correlates with the reported decreased binding of insulin to plasma membranes prepared from target tissue of these animals. The possible physiological significance of nuclear insulin binding is discussed.


Biosynthesis of slaframine, (1S,6S,8aS)-1-acetoxy-6-aminooctahydroindolizine, a parasympathomimetic alkaloid of fungal origin. 4. Metabolic fate of ethyl pipecolylacetate, 1,3-dioxooctahydroindolizine, and 1-hydroxyoctahydroindolizine in Rhizoctonia leguminicola. Known or suspected intermediates in the biosynthesis of slaframine and 3,4,5-trihydroxyoctahydro-1-pyrindine, piperidine alkaloids of the phytopathogenic fungus Rhizoctonia leguminicola, were prepared and tested for biological conversions. Ethyl pipecolylacetate, an analogue of the postulated condensation product of pipecolic and malonic acids (two previously identified alkaloid precursors), was insufficiently stable for feeding experiments. The lactam of pipecolylacetate, 1,3-dioxooctahydroindolizine, was degraded by the fungus without direct incorporation into alkaloids. The known slaframine precursor 1-hydroxyoctahydroindolizine was prepared by a novel route which permitted high levels of deuterium enrichment at C-1 and C-3. Mass spectrometric examination of the slaframine biosynthesized from cis- and trans-[1,3,3-2H]-1-hydroxyoctahydroindolizine strengthened arguments that 1-oxooctahydroindolizine is an intermediate in slaframine biogenesis.


Biosynthesis of slaframine, (1S,6S,8aS)-1-acetoxy-6-aminooctahydroindolizine, a parasympathomimetic alkaloid of fungal origin. 3. Origin of the pyrrolidine ring. The phytopathogen Rhizoctonia leguminicola has previously been shown to incorporate pipecolic acid into the piperidine alkaloids 1-acetoxy-6-aminooctahydroindolizine (slaframine) and 3,4,5-trihydroxyoctahydro-1-pyrindine. In the experiments described here, resting cultures of R. leguminicola were incubated with [1-14C]- and [2-14C]malonic acid and with [1-14C]- and [2-2H]acetic acid. Both acids were incorporated into the ring systems of both alkaloids. Mass spectrometric analysis of 2H-enriched slaframine showed that the label resides in the five-membered ring and that the methyl carbon of acetate is joined to the carboxyl carbon of pipecolate. A pipecolate-dependent decarboxylation of [1-14C]malonate was demonstrated in cell-free extracts of R. leguminicola. The results account for previously unattributed carbons in the two alkaloids and suggest the formation of an eight-carbon intermediate common to both alkaloids by acylation of malonate with pipecolic acid.


Nucleosome organization during germ cell development in the sea cucumber Holothuria tubulosa. Conformational changes that occur in chromatin from developing germ cells of the echinoderm Holothuria tubulosa have been probed with micrococcal nuclease. The results indicate that the extent of DNA degradation to acid-soluble nucleotides is highest in chromatin at the early stages of gonad growth, being drastically subdued in the mature sperm cell. Production of nucleosomal particles also varies with development, involving at least 70% of the chromatin at the final stage of maturation, whereas in immature germ cells it remains much lower. In contrast, electrophoretic analysis for DNA size has shown that the average nucleosome repeat length, about 227 base pairs, does not change throughout the maturation process. However, kinetics of the enzyme reaction have revealed that, although brief digestion of chromatin from both immature gonads and sperm yields comparable series of higher oligomers, extensive digest patterns differ widely. Sperm chromatin, highly protected, releases a 275 base pair intermediate fragment, wholly absent in immature gonads. The 145 base pair core released in both chromatins is not further digested in sperm. In comparison to sperm chromatin, that of immature germ cells is much more susceptible to fragmentation, yielding the usual set of smaller subnucleosomal fragments. These data suggest the induction of differential accessibilities of chromatin DNA with maturation, which is not accompanied by displacement of the histone complement. The histone variants present in this species may well be instrumental in the process.


Chromatin fractionation procedure that yields nucleosomes containing near-stoichiometric amounts of high mobility group nonhistone chromosomal proteins. Initial results of an approach to the isolation of functionally active chromatin are described. Slight digestion of mouse myeloma nuclei at 0 degrees C with micrococcal nuclease, followed by dialysis against near-physiological saline solution containing 1 mM Mg2+, caused release of up to 17% of the nuclear DNA as soluble nucleoproteins. This soluble (S) fraction was relatively depleted in H1 histones and methylated DNA (5-methylcytosine) but highly enriched in RNA, single-stranded DNA, and nonhistone chromosomal proteins, particularly two species of the high mobility group identified as HMG 1 and HMG 2. The S fraction released most rapidly (6--8% of the total DNA) consisted mainly of mono- and small oligonucleosomes. The mononucleosomes appeared normal in terms of sedimentation behavior, DNA length, and content of histones H2A, H2B, H3, and H4, but lacked H1, and instead were associated with approximately stoichiometric amounts of HMG 1 and HMG 2. Studies using isolated, fluorescence-labeled, total mouse HMG proteins indicated that added HMG 1 and HMG 2 do not bind strongly to S-fraction nucleoproteins but that two smaller HMG species (probably HMG 14 and HMG 17) do bind preferentially to S-fraction mono- and dinucleosomes. These results argue against artifactual redistribution of HMG 1 and HMG 2 during this fractionation but suggest caution in interpreting the distribution of smaller HMG proteins after digestion of chromatin. The potential relationship of this soluble fraction to transcriptionally active chromatin is discussed.


Purification and characterization of the crown gall specific enzyme nopaline synthase. Nopaline synthase of sunflower (Helianthus annuus L.) crown gall tissue induced by Agrobacterium tumefaciens strain C58 or T37 (nopaline utilizers) was purified to homogeneity as judged by analytical disc gel electrophoresis. The native enzyme elutes from a column of Ultrogen AcA 34 as a single peak with an estimated molecular weight of 158,000. The dissociated enzyme migrates on NaDodSO4-polyacrylamide gels as a single band with a molecular weight of 40,000. Thus, the native enzyme appears to be composed of four equal-weight subunits. Nopaline synthesizing activity is found exclusively in crown gall tissues induced by strains of A. tumefaciens that utilize nopaline (e.g., C58 and T37). We found the same tissue specificity for the purified protein that we believe represents nopaline synthase. The results of kinetic studies of the purified enzyme are consistent with a ter-bi rapid-equilibrium random-order mechanism. Nopaline synthase is probably responsible for the in vivo synthesis of both N2-(1,3-dicarboxypropyl)arginine (nopaline) and N2-(1,3-dicarboxypropyl)ornithine (ornaline) in crown gall tissues since substrate specificities and Km values do not change during purification.


Purification and properties of strictosidine synthetase (an enzyme condensing tryptamine and secologanin) from Catharanthus roseus cultured cells. Strictosidine synthetase, which catalyzes the condensation of tryptamine with secologanin to form strictosidine (isovincoside), was purified 740-fold to homogeneity from cultured cells of Catharanthus roseus in 10% yield. The specific activity is 5.85 nkat/mg. The molecular weight as estimated by gel filtration is 38,000. The isoelectric point is 4.6. Apparent Km values for tryptamine and secologanin are 0.83 and 0.46 mM, respectively. The enzyme shows a broad pH optimum between 5.0 and 7.5. The product of the enzymic reaction is exclusively strictosidine, while no trace of its epimer vincoside can be detected. Sulfhydryl inhibitors have no effect on the enzyme. End products in the biosynthetic pathway of indole alkaloids such as ajmalicine, vindoline, and catharanthine do not inhibit the activity of strictosidine synthetase.


Conformation of the active site of thiolsubtilisin: reaction with specific chloromethyl ketones and arylacryloylimidazoles. The conformation of the active site of thiolsubtilisin, prepared from subtilisin by transformation of the active site Ser to Cys, was compared with that of subtilisin by kinetic and spectroscopic methods. Carbobenzyloxy-L-alanylglycyl-L-phenylalanine chloromethyl ketone inhibited thiolsubtilisin approximately 10(2) times faster than subtilisin; alkylation occurred at the sulfhydryl rather than the imidazolyl group of the active site. pH dependence of the inhibition is different from that of the reaction between a simple thiol with haloacetamide. Furthermore, several native chromophoric arylacryloyl-thiolsubtilisins and arylacryloyl-subtilisins showed similar red shifts when compared with their denatured forms. The rate of deacylation of arylacryloyl-thiolsubtilisins was faster than (or of the same order of magnitude as) the deacylation rate of the analogous arylacryloyl-subtilisins in 30% dioxane (v/v), pH 5--10. The deacylation rate--pH profiles of these arylacryloyl-thiolsubtilisins in 30% dioxane all give pK values of 7.7 which is identical with the pK in the deacylation of acyl-subtilisins. These facts strongly suggest that the active-site conformation remains intact on conversion from subtilisin to thiolsubtilisin. The low esterase and peptidase activities of thiolsubtilisin are most likely due to the relatively low basicity of -SH (compared with -OH).


Protected nucleotide sequences in nuclear ribonucleoprotein. The rapidly labeled nuclear ribonucleic acid in human carcinoma cells which is protected by protein from digestion by staphylococcal nuclease (EC 3.1.4.7) has been investigated. A simple and discrete sequence specificity was not found, but the protected RNA fragments are rich in G + C and were shown by fingerprinting to comprise a nonrandom subset of all heterogeneous nuclear ribonucleic acid (hnRNA) sequences enriched in the sequences AGC, GGC, AGGC, and GAGC. There was no detectable enrichment for dougble-stranded RNA in the protected fraction. These data provide the first evidence that the association of any protein with hnRNA is nonrandom with respect to nucleotide sequence.


Rare transfer ribonucleic acid essential for phage growth. Nucleotide sequence comparison of normal and mutant T4 isoleucine-accepting transfer ribonucleic acid. One of the eight tRNA species coded by bacteriophage T4 is unique in that (1) it is found in a yield lower by three- to fourfold than that of any other tRNA and (2) while dispensable for growth in standard laboratory hosts, it is essential for phage propagation in a natural isolate of Escherichia coli (strain CT439). We report here the nucleotide sequence of this tRNA and of several mutationally altered forms. The molecule is 77 nucleotides in length and has the anticodon N-A-U. Depending on the pairing properties of the "wobble" nucleotide N, this sequence could correspond to one or more of the isoleucine-specific codons (formula: see text) or to the methionine-specific codon A-U-G. Since a T4-specific acceptor activity for isoleucine which is stimulated in ribosome binding by A-U-A but not A-U-U has been reported previously, we infer that we have sequenced a tRNA Ile species which preferentially recognizes A-U-A. Mutant HA1 is unable to grow in CT439; it produces no tRNA Ile. The primary mutational alteration is a transition four residues from the 5'terminus which converts a C.G to a U.G base pair. The consequences of this lesion can be partially reversed by second-site mutations nearby in the acceptor stem. Unexpectedly, the tRNA Ile synthesized in these revertants still retains two unusual structural features found in the wild-type molecule: the opposition of two Up residues in the amino acid acceptor stem and the opposition of an Ap and a Gp residue in the anticodon stem. Implications of these structual anomalies for a possibly unique physiological role of this minor tRNA species are discussed.


Antigody-nucleic acid complexes. Inhibition of translation of silkmoth chorion messenger ribonucleic acid with antibodies specific for 7-methylguanosine. Antibodies specific for 7-methylguanosine (m7G) were evaluated for their ability to inhibit the translation of chorion mRNA in a wheat germ, cell-free amino acid incorporating system. Results obtained with antibody concentrations of 0.5--1.5 microM revealed dose-dependent inhibition of [3H]-labeled amino acid incorporation into acid-insoluble radioactivity. Inhibition of translation was attributed to the interaction of anti-m7G antibodies with the 5' termini of chorion mRNAs on the basis that (a) anti-m7G antibodies coupled to Sepharose (anti-m7G-Sepharose) immunospecifically retained 5'-terminal cap structures of chorion mRNAs, i.e., m7G (5')ppp(5')Nm, (b) significant inhibition of translation required a 2-h preincubation of anti-m7G antibodies with mRNA, and (c) similar preincubation periods with anti-m7G antibodies in the presence of the competing nucleoside hapten (m7G) obviated the inhibitory effect of the antibody. The nature of the anti-m7G antibody-mRNA complex was examined by digesting chorion mRNA with nuclease P1 before (predigested) and after (postdigested) immunospecific adsorption to anti-m7G-Sepharose adsorbent. Whereas predigested preparations yielded a single cap structure of the type m7G(5')ppp(5')N, the predominating cap in the postdigested sample was m7G(5')ppp(5')NpNpN. These latter data revealed that the nucleotide sequence adjacent to the cap was not significantly masked by the antibody and suggest the utility of anti-m7G antibody as a site-specific probe.


High-yield cleavage of tryptophanyl peptide bonds by o-iodosobenzoic acid. A new procedure to cleave tryptophanyl peptide bonds in high yield is reported. The method involves treatment of the S-alkylated protein with o-iodosobenzoic acid. The procedure is highly selective for tryptophan and does not modify tyrosine or histidine, but may convert methionine to its sulfoxide derivative. The yields in the cleavage are 70--100%. Tryptophanyl bonds to alanine, glycine, serine, threonine, glutamine, arginine, and S-(pyridylethyl)cysteine are split in nearly quantitative yield, while those preceding isoleucine or valine are split in approximately 70% yield in the proteins examined in this work. The chemical mechanism for tryptophanyl bond cleavage has not been defined, but it is likely that oxidation of the indole ring occurs during the reaction with o-iodosobenzoic acid. Some problems with the quality of commercial preparations of the reagent are discussed.


Transfer of singlet energy within trypsin. Transfers of singlet energy within trypsin were investigated by measuring the fluorescence absorption anisotropy of its tryptophan residues. A ratio of the anisotropy of trypsin to that for N-acetyl-L-tryptophanamide was determined between 306 and 250 nm. The ratio had an average value of 0.7, whether the trypsin anisotropy was measured at 228 of 296 K. However, trypsin dissolved in 5 M guanidine hydrochloride showed little fluorescence depolarization at 228 K (the anisotropy ratio was approximately equal to 0.9). Thus, there is an extensive conformation-dependent energy transfer between tryptophans in trypsin. The ratio of anisotropies of tyrpsin at 304--270 nm was used to estimate energy transfer from tyrosine to tryptophan. Ratios of 1.8 and 1.7 were obtained at 296 K for the native and guanidinium-unfolded enzyme, respectively. The comparable value for N-acetyl-L-tryptophanamide was 1.7. This indicates that there is little transfer from tyrosine to tryptophan in trypsin at 296 K. As confirmation, the excitation wavelength dependencies of the indole fluorescence quantum yield were the same for native and unfolded trypsin. When experiments were performed at 228 K, the 304--270-nm anisotropy ratios were 2.6 for native and 2.1 for unfolded trypsin at pH2. This indicates that the efficiency of energy transfer from tyrosine to tryptophan increases at low temperatures. A photochemical source of error in the quantitation of the efficiency of energy transfer from tyrosine to tryptophan is also described.


A mechanism of erythrocyte lysis by lysophosphatidylcholine. Lysophosphatidylcholine micelles liberate several cell surface polypeptides from erythrocyte membranes, inducing a sodium-selective permeability defect which leads to colloid osmotic lysis. Evidence is presented to support the hypothesis that at the lowest lytic lysophospholipid concentrations, selective disruption of membrane protein function, rather than gross structural reorganization of the membrane, is the primary lytic mechanism.