Physical and chemical properties of skin in habitually trained and sedentary men. A number of physical and chemical properties of skin were examined in a study of 29 habitually trained and 29 sedentary men. Compared to the control group, the trained subjects had significantly higher values in the weights of skin samples of equal surface areas as well as the contents of hydroxyproline and nitrogen per skin surface area. When measured by a diaphragm method in vivo, the 'elastic stiffness' (uncorrected for thickness) and 'elastic efficiency' (the recovery of the deformation energy) of skin were significantly higher in the trained men compared to those in the untrained men. The results suggest that skin reflects the adaptation to habitual endurance training by increasing its mass and strengthening its structure. The study did not, however, reveal any differences between physically active and sedentary men in changes due to biological ageing.


Pigmented penile papules with carcinoma in situ changes. Benign appearing, multifocal, pigmented penile papules were found in three patients, showing the histopathological and electron microscopic changes of carcinoma in situ. Their clinical appearance, relation to other carcinoma in situ lesions, as well as possible pathogenesis are discussed.


Allergic contact dermatitis due to sesquiterpene lactones. A comparative study of human and animal sensitivity to alpha-methylene-gamma-butyrolactone and derivatives. Several compounds containing the alpha-methylene-gamma-butyrolactone moiety have been tested on human volunteers and on guinea-pigs; the animals were experimentally sensitized by alantolactone, isoalantolactone and laurel oil. Of the two new lactones, spirolactone was the more reactive: this was confirmed by both animal and human testing. The synthetic lactones are less reactive than natural ones. alpha-Methylene-gamma-butyrolactone itself does not elicit cross-reactions in guinea pigs sensitive either to alantolactone or to isoalantolactone, or in patients sensitive to sesquiterpene lactones. The alpha-methylene-gamma-butyrolactone group is necessary for cross-reaction, but to be active, it has first to be substituted. It was also found that isoalantolactone, allegedly not allergenic, is in fact a sensitizer and cross-reacts with alantolactone. The cross-reaction between laurel and Frullania, found in man, also occurs in guinea-pigs. It is more evident when sesquiterpene lactone is the sensitizer and laurel used to elicit reaction.


Efflorescence of new warts: a sign of onset of involution in flat warts. The sudden eruption of large numbers of tiny new warts in a patient with multiple flat warts often signals the onset of involution and subsequent regression of all flat warts. We have observed 5 such cases and, in this report, describe 2 cases in which the sudden efflorescence of many new warts was used as a sign to predict accurately the onset of involution and subsequent regression of all flat warts. Correlation of the histopathological and clinical findings provides an explanation for this phenomenon.


Lymphocyte transformation in patients with cobalt dermatitis. A lymhocyte transformation test with cobalt chloride was performed in 14 patients who had positive cobalt patch tests and eczema compatible with a cobalt induced contact dermatitis and in 9 controls. Eight of the patients had strongly positive patch tests, while 6 had weakly positive patch tests. Patients and controls showed equivalent responses to the mitogens, phytohaemagglutinin, pokeweed mitogen, and concanavalin A, as well as to the recall antigens, purified protein derivative and Candida albicans. A 1:5000 dilution in RPMI-1640 medium of a 1% aqueous solution of cobalt chloride proved optimal in distinguishing patients from controls. With this dilution, a significantly higher lymphocyte transformation was found in patients with strongly positive cobalt patch tests than in the controls. No significant difference was detected when the results for patients with weakly positive patch tests were compared with those of the controls.


The effect of topical isoprenaline on psoriatic skin. Twelve patients with psoriasis were treated by the topical application of 0.1% isoprenaline sulphate ointment for 10 days. Topical white vaseline was used as a control in three patients with psoriasis. The mean glycogen level was found to be decreased significantly (from 120 +/- s.d. 40 to 58 +/- s.d. 26 mg/100 g of wet weight of involved skin) after topical application of 0.1 isoprenaline sulphate and the scaling also disappeared. Topical application of white vaseline did not produce any significant change in glycogen levels or scaling. The changes after isoprenaline application may have been due to an increase in the ratio of cyclic AMP/cyclic GMP which inhibited cell turnover and increased glycogenolysis.


Malignant pyoderma.

Malignant pyoderma. A patient is described who developed multiple areas of inflammatory pyoderma on the face, leading to extensive ulceration. Repeated investigations failed to demonstrate any specific bacteria or fungus responsible and trials of treatment with various antibiotics proved unsuccessful. Histology revealed a granulomatous abscess-like lesion, and the condition resembled what has been described as malignant pyoderma. The patient was successfully treated with oral dapsone and intralesional steroids.


Auto analyzer determination of red cell Kell phenotypes in patients with chronic granulomatous disease (CGD) and heterozygous carriers. One of six boys chronic granulomatous disease was shown to have the rare Kell phenotype, McLeod, by both manual and Auto Analyzer techniques. Using the Auto Analyzer, the red cells of this boy's mother were clearly shown to have weaker expression of k and Kpb antigens than those of the controls, whereas the Kell groups on the father's red cells appeared normal.


A unique thalassaemic syndrome: homozygous alpha-thalassaemia + homozygous beta-thalassaemia. The disturbed balance of globin chain synthesis is a major factor in the pathophysiology of the thalassaemic disorders; this concept is strongly supported by the study of a patient displaying an extreme but symmetrical deficit of both major types of chains alpha and beta. The patient had a mild clinical picture but presented a striking hypochromia (MCH 10 pg) with compensatory erythrocytosis (RBC 10(12)/l.). Study of the propositus and his family by haematological, biochemical and biosynthetic techniques indicates that the patient carries two alpha- and two beta-thalassaemia genes resulting in balanced globin chain synthesis; in addition, several members of the family carry two or three abnormal genes. During observation a change in the haematological pattern occurred with a shift towards more intensive beta-chain and away from gamma-chaim synthesis; this appeared with be associated with improvement of his anaemia through more effective erythropoiesis.


In vivo and in vitro variations of human erythrocyte glutathione peroxidase activity as result of cells ageing, selenium availability and peroxide activation. Cases showing erythrocyte glutathione peroxidase (GSH-Px) defects have been previously described. Our experiments demonstrate that a number of non genetic factors may influence the GSH-Px activity in human erythrocytes. Selenium administration in vivo was followed in four subjects by elevation in erythrocyte GSH-Px activity ranging from 30% to 1400%. Selenium operates mainly in the bone marrow erythroblasts by facilitating the synthesis of active GSH-Px molecules; experiments in vivo demonstrate that, in the youngest erythrocytes, selenium can raise the enzyme activity, but by a different mechanism. The reticulocyte GSH-Px activity appears to depend on selenium availability and may vary over a wide range. In some normal and iron deficient subjects the GSH-Px activity in the youngest erythrocyte fraction was equal or lower than that previously found in whole erythrocytes of patients affected by haemolytic anaemia. During erythrocyte life, GSH-Px activity may either diminish  or increase, and these variations are inversely related to the initial GSH-Px activity  in youngest cells. In vitro experiments with the addition of acetyl-phynyl-hydrazine strongly suggest that elevation of GSH-Px activity may be due to allosteric enzyme activation by activated oxygen.


Estimation of ferrokinetic parameters by a mathematical model in patients with primary acquired sideroblastic anaemia. Ferrokinetic parameters were estimated in eight patients with primary acquired sideroblastic anaemia (PASA) by means of a mathematical model of iron kinetics. Both plasma 59Fe clearance and red cell utilization curves were used to identify the model parameters. The close agreement between experimental and theoretical data proved that the model can describe iron kinetics in sideroblastic anaemia. Ineffective erythropoiesis was found to be major factor in the production of the anaemia. Various degrees and patterns of peripheral haemolysis were observed. The storage iron was always markedly increased and a large non-erythroid iron turnover was calculated in the most severely affected patients. The results are compared with present knowledge of erythropoiesis in PASA.


Destruction of IgG-sensitized erythrocytes by human blood monocytes: modulation of inhibition by IgG. The in vitro interaction between monocytes and erythrocytes sensitized with non-complement binding IgG antibodies (i.e. the Rh antibody anti-D:EAIgG anti-D) is completely inhibited by low concentrations of IgG (E.G. 30--100 microgram/ml). However, the interaction between monocytes and erythrocytes sensitized with IgG anti-A (EAIgG anti-A) is not inhibited by IgG. The findings presented in this paper indicate that this difference is probably due to the difference in the number of IgG antibody molecules per EAIgG. Thus, the higher the number of IgG antibody molecules per EAIgG, the less the interaction between EAIgG and monocytes is inhibited by IgG. A second factor which proved to have a strong influence on inhibition by IgG was the number of EAIgG per monocyte. When the number of EAIgG per monocyte was increased from 1 to 32, the percentage of inhibition by a fixed amount of IgG (50 microgram/ml) decreased significantly. This in vitro effect is only evident when relatively weakly sensitized erythrocytes are used and, in vivo, destruction of these weakly sensitized red cells (e.g. EAIgG anti-D) is confined to the spleen. Since a considerable haemoconcentration occurs in this organ, it is conceivable that a high EAIgG:macrophage ratio is accomplished. The latter data are an indication that this high ratio may allow interaction between weakly sensitized erythrocytes and splenic macrophages despite the presence, in vivo, of a high concentration of IgG, and that, in this way, in the spleen, the inhibitory effect of IgG is overcome.


Erythropoiesis and mean red-cell lifespan in normal subjects and in patients with the anaemia of active rheumatoid arthritis. Recently developed ferrokinetic methods offer a tool to measure effective and ineffective erythropoiesis and mean red-cell lifespan (Ricketts et al, 1975). We have used this tool to investigate erythropoiesis in normal subjects and in patients with the anaemia of active rheumatoid arthritis. In normal subjects the results are comparable with the results published by Cavill et al (1977). In patients with the anemia of active rheumatoid arthritis there appears to be a total and an effective red-cell production as in the normal subjects. The ineffective erythropoiesis in the patients is increased and the produced red cells have a shortened mean lifespan in the circulation. The increased ineffective erythropoiesis is probably due to inadequate supply of iron to the marrow and the shortened mean red-cell lifespan to an extra-corpuscular haemolytic factor. The impaired marrow response to the anaemia of active rheumatoid arthritis has been confirmed.


An immunoradiometric assay for factor III (tissue thromboplastin). A solid-phase immunoradiometric assay for tissue thromboplastin (factor III) has been established based on its displacing effect on the binding of 125I-labelled factor III-antibodies to polyvinyl tubes coated with the purified protein component of factor III (apoprotein III). By this method circulating tissue thromboplastin can be detected in experimental animals receiving infusions of crude or purified tissue thromboplastin and in patients undergoing major orthopaedic surgery.


Heterogeneity of rabbit platelets. VI. Further resolution of changes in platelet density, volume, and radioactivity following cohort labelling with 75Se-selenomethione. Rabbits were injected intravenously with a cohort platelet label, 75Se-selenomethionine  Platelet-rich plasma was separated into five different platelet density fractions on each of seven days by repetitively centrifuging the same sample of platelet-rich plasma at increasing gravitational force. The heaviest platelet sediment fraction was enriched with smaller platelets. Incorporation of isotope into the heaviest platelet fraction was considerably greater than incorporation into the highest platelet fraction, i.e. heavy--light specific activity ratio was 9.63, on day 1. The ratio of the heaviest platelet fraction specific activity to the specific activities of the platelets sedimented at higher g forces (lighter-smaller platelets), generally decreased on each day for 4--6 d by a factor of 2.8--7.0-fold. Whereas incorporation of isotope into the heaviest platelet fraction declined on days 3--4, incorporation into the lightest three fractions remained the same. Cohort platelet survival curves revealed a significant lag period of 1--2 d for the incorporation of isotope into the lightest three platelet fractions compared to the heaviest two platelet fractions. The mean platelet survival of the lightest two fractions was significantly shorter than the mean platelet survival of the heaviest three fractions. SDS-polyacrylamide gel electrophoresis of the platelet cell sap of the five platelet fractions generally revealed 10 prominent protein bands for the heaviest platelet fractions. The lightest platelet fraction, which comprised 16% of the harvested platelet population, had six absent to markedly diminished platelet proteins of modecular weights between 142 000 and 46 000 daltons. The data are compatible with two models: (1) Heavy-large platelets are, on average, young platelets which become lighter-smaller platelets while losing platelet membrane and cell sap components with time. (2) Heavy-large platelets and light-small platelets are produced independently by specific megakarocytes. The heavy-large platelets incorporate more isotope than lighter-smaller platelets (possibly because of their megakarocyte precursor). However, they are released earlier into the circulation than lighter-smaller platelets and are therefore younger platelets.


Mutant mouse L-cells: a model for megaloblastic anaemia. When temperature-sensitive (ts) mutant lines of mouse L-cells, ts AIS9 and ts CI, are shifted from 34C to 38.5C, a rapid inhibition of DNA synthesis and mitosis occurs. During this phase, cell and nuclear growth continues and results in a substantial increase in cell and nuclear volume. Such cellular modifications are also associated with a marked dispersal of the condensed chromatin masses of interphase nuclei, so that after 48-72 h of incubation at 38.5C, nuclear profiles of both ts cell lines bear a striking resemblance to the nuclear features characteristic of megaloblastic anaemia. Despite these marked alterations in nuclear chromatin organization, morphometric analysis indicates that the volume of condensed chromatin does not decrease. Current biochemical, cytological and morphometric data on the two ts lines of mutant mouse L-cells during expression of the mutation, suggest that they might provide a useful model to further elucidate cytological features of megaloblastic anaemia.


The control of hepatic iron uptake: correlation with transferring synthesis. The control of hepatic iron uptake was studied in the perfused liver isolated from rats subjected to nutritional iron deficiency. The total hepatic iron uptake and incorporation into ferritin was found to be higher in iron deficiency and during the 48 h of oral refeeding with iron than in the normal state. Specific incorporation of iron into feritin from a perfusate of normal transferrin iron saturation was enhanced in nutritional iron deficiency as compared to controls after 5 h of perfusion but not after 1 h, suggesting that increased uptake of iron from the perfusate may play a role in stimulating hepatic ferritin synthesis and assembly. This promotion of uptake into ferritin was inhibited by cycloheximide suggesting that enhanced incorporation of iron is dependent upon de novo synthesis of apoferritin. In control, nutritionally iron deficient and iron-refed rats there was a significant, direct correlation between the transferrin-iron saturation of the perfusate at physiological transferrin concentrations and total hepatic iron uptake after 5 h perfusion. A significant positive correlation was found between the hepatic total and ferritin iron uptake and the transferrin synthetic rate measured in the same liver. It is proposed that in the liver the negative feedback of iron supply on transferrin synthesis may be linked with a positive feedback on ferritin synthesis. The time-course of these reciprocal responses suggests a role for hepatic ferritin and/or a component of the non-haem, non-ferritin iron pool in the regulation of transferrin synthesis.


Acceleration of methaemoglobin reduction by riboflavin in human erythrocytes. The effect of riboflavin on nitrite treated erythrocytes from normal subjects and patients with hereditary methaemoglobinaemia due to the deficiency of NADH-cytochrome b5 reductase was studied in the presence of glucose, 2-deoxy-D-glucose or lactate. When glucose or 2-deoxy-D-glucose was used as a substrate for these erythrocytes, the rate of methaemoglobin reduction in these cells was accelerated more than two-fold in the presence of riboflavin. The acceleration was dependent on the concentration of riboflavin and was suppressed by the addition of atebrin. The stimulative effect of riboflavin was, however, not observed when lactate was used in place of glucose or 2-deoxy-D-glucose. On the basis of these results, the acceleration of methaemoglobin reduction by riboflavin was considered to be due to the activation of NADPH-flavin reductase (Yubisui et al, 1977) in erythrocytes by the reagent. The availability of riboflavin for patients with methaemoglobinaemia due to the deficiency of NADH-cytochrome b5 reductase and for those with toxic methaemoglobinaemia is discussed in relation to methaemoglobin reducing systems in erythrocytes.


Decrease of alpha-Hasharon globin in beta-thalassaemia. In time course experiments performed in subjects carrying haemoglobin Hasharon and beta-thalassaemia the synthesis of alphaA-, alphaHasharon-, beta- and gamma-globin has been determined. The alpha-globin molecules synthesized in excess were found to be removed from the red cell cytoplasm (phenomenon characteristic of beta-thalassaemia, described by Bargellesi et al, 1968a); in addition the alphaHasharon-globin molecules were removed at a faster rate than normal alpha-globin. The preferential removal of the mutant alpha-globin explains the reduced level of haemoglobin Hasharon found in subjects carrying the gene for beta-thalassaemia (Alberti et al, 1975). Reductions below normal levels of other haemoglobin variants occurring in the presence of the counterpart-globin thalassaemia are probably determined through a similar mechanism.


A new type of congenital dyserythropoietic anaemia. This report concerns a 6-year-old child with severe dyseythropoietic anaemia and splenomegaly, apparently present since the first months of life. Striking anisopolikilocytosis was observed in the blood smear. The bone marrow showed marked erythroblastic hyperplasia with dyserythropoiesis. Ineffective erythropoiesis was demonstrated by ferrokinetic studies. Ultrastructurally erythroblasts appeared grossly abnormal. The clinical course was progressively worsening, necessitating repeated transfusions. The patient's father, also affected by a chronic anaemia of moderate degree since childhood, had a peripheral picture of anisopoikilocytosis, a shortened life span of the erythrocyte and in his bone marrow an erythroblastic hyperplasia with many atypical erythroblasts. His condition deteriorated because of persistent jaundice, biliary cholelithiasis, fibrosis and haemosiderosis of the liver. The clinical course, the pattern of the genetical transmission, the peculiar features of the erythroblasts disclosed by light and by electron microscope studies suggest that these cases represent a new type of congenital dyserythropoietic anaemia.


Studies of erythrocyte membrane loss produced by amphipathic drugs and in vitro storage. Four amphipathic drugs, primaquine, propranolol, chlorpromazine and tetracaine, were used to cause endocytosis in glucose-depleted red cells, and the relative reduction of membrane surfaces was measured by the toluidine blue (TB) method. The TB measurements correlated well with the observed electron microscopic alterations. In vitro blood storage studies indicated loss of cell membrane during storage correlated with decreasing uptake of TB by the red cells. Regeneration of cellular ATP with adenosine did not always restore TB uptake by the cells. Loss of red blood cell membrane either by exocytosis or endocytosis may occur during normal in vivo or in vitro ageing, and may be increased in pathological states or by the action of drugs.


Chronic myeloproliferative disorders: improved platelet aggregation following venesection. Venesection of 10% of whole blood volume or plateletpheresis was performed in nine patients with chronic myeloproliferative disorders and in five normal control subjects. Before venesection, the patients showed impaired platelet aggregation in 33% of tests, most often in response to stimulation with 9 mumol adrenaline. After venesection, the platelet and megathrombocyte counts increased rapidly and excessively in most patients and platelet aggregation improved markedly. In some cases, spontaneous in vitro aggregation was seen at high platelet concentrations. In two patients impaired platelet aggregation with adrenaline was not corrected. The splenic platelet pool is thought to be the probable source of the new platelets.


Bacterial contamination of cotton and cotton dust and effects on the lung. Bacterial contamination of various parts of the cotton plant and of cotton from different mills was investigated. The predominant bacterial species were Gram-negative rods mainly of the Enterobacter genus. When guinea pigs inhaled strains of these bacteria cultivated from cotton, a strong leucocyte mobilising capacity was found for Pseudomonas and Enterobacter but not for Agrobacterium or Bacillus species. The aetiology of the development of pulmonary symptoms after inhalation of bacteria-containing dusts and subsequent production of endotoxins is discussed.


Prevalence of vibration-induced white finger in fluorspar mines in Weardale. A combined epidemiological and clinical study of vibration-induced white finger (VWF) was carried out involving 115 men in four fluorspar mines. The overall prevalence of VWF was found to be 50% among 42 vibration-exposed subjects, while that of constitutional white finger (CWF) was 5-6% in all men studied. The VWF latent interval was 1-19 years with a mean of 5-6 years. An association was observed between the exposure time and VWF stages which included 18 men in Stage 0, three in the intermediate Stage of 0T/0N, five in Stage 2 and 16 in Stage 3; no men were seen at Stage 1. Among those with VWF in Stage 3, the index, middle and ring fingers were affected in both hands and the little fingers and thumbs were last to be involved. Clinically, on general examination, apart from vibration-induced white finger, the men in the 'vibration' group were not as healthy as those in the 'control' group. The circumference of the index fingers was not significantly different for the different groups. Neurological tests showed that the ridge test and, to a lesser extent, the two-point discrimination and the light touch tests, could be regarded as useful for the diagnosis of VWF.


Health effects of acrylonitrile in acrylic fibre factories. The relationship between the degree of exposure and biological effects of acrylonitrile (AN) was studied in 102 workers whose exposure period exceeded five years, and in 62 matched controls, all of whom had been randomly sampled from six acrylic fibre factories in Japan. The six factories were classified into three groups on the basis of AN concentration at workplaces. The most highly exposed group of subjects showed an eight-hour average AN concentration of 4-2 ppm by personal sampling, a mean urinary AN concentration of 360 microgram/1 and a mean urinary thiocyanate concentration of 11-4 mg/1. Medical examination, including the indocyanine green excretion test and multiple clinical chemistry determinations, failed to detect any health effect attributable to AN. Slight liver damage may possibly occur in more highly exposed workers. Urinary AN and thiocyanate determinations may provide more accurate estimates of low-grade exposure (less than 5 ppm).


Urinary kallikrein activity of workers exposed to lead. Two groups of men of different age ranges and with the same period of lead exposure were selected for study in a recently opened car-battery factory. Two other groups of age-matched men, not exposed to heavy metals in their work, were used as controls. Morning urines were collected from control and exposed groups for determination of urinary kallikrein activity, urinary delta-amino-levulinic acid (ALA) and lead levels. The environmental lead levels and the urinary ALA and lead values indicated that exposure in the factory was not heavy. The older group of lead-exposed workers showed greatly reduced urinary kallikrein activity compared with that of the age-matched controls. In contrast, the younger group did not show any significant alteration in urinary kallikrein excretion.


Hippuric acid excretion after benzylamine ingestion in man. The fate of 14C-benzylamine after oral administration as the hydrochloride has been investigated in two male volunteers. Over 98% of the administered radiolabel was excreted in the urine as 14C-hippuric acid within 24 hours. The rate of urinary hippuric acid excretion was extremely rapid, with more than 90% of the dose excreted after three hours.


Urinary thioether of employees of a chemical plant. The thiols in the morning urine of 224 employees of a chemical plant were determined after alkaline hydrolysis of all urinary thioethers. The highest thioether excretion was found in rubber workers and radial tyre builders in comparison with clerks, plastic monomer mixers and footwear preparers. Smoking and medication tended to increase thioether excretion. Urinary thioether determination may prove to be a valuable tool in assessing exposure to mixtures of chemicals regardless of the route of absorption.


The paper punched disc technique for lead in blood samples with abnormal haemoglobin values. A series of 15 blood samples with haemoglobin levels ranging from 4-6--16-1 g/dl were spotted on to Whatman No. 4 filter paper. Blood samples with low haemoglobin concentrations spread over a greater area of the filter paper than did those with high haemoglobin concentrations. This was further investigated by studying the performance of laboratory-prepared samples, and any effect on the estimation of blood lead value. Blood lead values assayed by the punched disc method on blood samples with low haemoglobin values were unreliable unless the estimated value was adjusted with respect to the area over which the blood had spread.


Ethanol-increased exhalation of mercury in mice. CBA/J mice injected three days beforehand with 203HgCl2 were given ethanol or water by gavage and placed in a chamber designed to collect exhaled mercury. Ethanol treatment led to an eight-fold increase of counts accumulated on a filter over a four-hour period, compared with water-treated mice. The mercury-collection apparatus tested for extracorporeal contribution of volatilised mercury indicated that the counts originated from the air exhaled by the mice.


Paraquat lung injury in rabbits.

Paraquat lung injury in rabbits. An aerosol model for the study of paraquat (PQ) toxicity was developed using a 134 litre chamber and an ultrasonic nebuliser. Three groups of New Zealand white rabbits weighing 2-3 kg were studied. Group I (n = 6) was exposed to 10 g PQ/100 ml double-distilled water (DDW), Group II (n = 24) was exposed to 200 mg PQ/100 Ml DDW and a control group (n = 6) was exposed to 100 ml DDW. In a second experiment ten animals (Group III) were exposed to 10 mg PQ/100 ML DDW over a three-month period together with a control group (n = 5). Group I animals died with extensive haemorrhagic pneumonitis 38 hours after the last challenge. Most animals in Group II surviving more than three exposures had a significant reduction (P less than 0.001) in arterial oxygen tension (PaO2) and an increase (P less than 0.001) in the alveolar-arterial O2 gradient. Specific compliance decreased (P less than 0.005) and functional residual capacity and breathing frequency increased (P less than 0.05). Tissue PQ values showed even pulmonary distribution, with evidence of PQ accumulation after repeated inhalation. The lungs showed focal interstitial fibrosis, interstitial thickening, proliferation of macrophages in the alveoli, epithelioid changes in the interstitium, Type II cell hyperplasia, and foci of acute inflammation with consolidation. Controls and Group III animals were normal. This indicates that repeated inhalation of paraquat aerosol induces dose-related interstitial pneumonitis and fibrosis in rabbits.


The influence of pre-induction vaginal prostaglandin E2 gel upon subsequent labour. Eight hundred and three patients with singleton viable pregnancies and fetal cephalic presentation were given prostaglandin E2 in viscous gel by intravaginal instillation the evening before planned surgical induction. When the cervix was 'ripe', surgical induction was avoided in 65.9 per cent of primigravidae and 87.5 per cent of multigravidae; the administration of epidural analgesia was less frequent, the rate of spontaneous vaginal delivery greater, the Caesarean section rate lower, and the state of the newborn at delivery better than in those patients who required surgical induction. Side effects attributable to the prostaglandin gel were rare as were complications.


Artificial insemination with frozen stored donor semen. The results of artificial insemination with donor semen (AID) are reported. The semen samples were stored in liquid nitrogen until needed. Eighty-two pregnancies occurred in 214 patients treated for 1610 cycles. Life table conception rates were 24.3 +/- 2.9 after six months and 32.7 +/- 3.3 after 12 months of treatment. Thirty of the 82 patients conceived within three treatment cycles and 53 by the sixth treatment cycle. Of the 82 pregnancies, there were 12 miscarriages, no stillbirths or neonatal deaths and one baby was born with a congenital abnormality.


Plasma unconjugated oestriol in late pregnancy: circadian variation and the effect of meals and a glucose load. Plasma unconjugated oestriol (E3) concentrations were determined by radioimmunoassay in 10 normal subjects in late pregnancy, throughout a normal day and night, and after a 50 g oral glucose load. There was a circadian pattern in E3 concentration characterized by an abrupt increase of 15 per cent at night. There was no significant rhythm during the day or night, taken separately. However there was a 10 to 11 per cent reduction in E3 concentrations soon after some meals and after a glucose load, possibly due to expansion of the plasma volume. Excluding the effect of meals, the fluctuation of E3 concentrations in individuals (median of the coefficients of variation) was 11.5 per cent in the day. Overall, it was 13.2 per cent in the day, 12.1 per cent at night and 15.9 per cent over the whole period studied. Since the variation in plasma unconjugated E3 concentrations through 24 hours was no greater than random fluctuations or day-to-day variation there is no need to restrict the time of blood sampling in clinical practice.


Pregnancy-associated plasma protein B (PAPP-B) in normal and abnormal pregnancies at term. At term, the pregnancy-associated plasma protein B (PAPP-B) level in normal pregnancy was correlated with placental and newborn weights, but not with nine other normal obstetric variables. It was lower in pregnancies complicated by diabetes or severe toxaemia, and higher in twin pregnancies.


Fetal breathing movements in high risk pregnancy. In 12 normal and 50 high risk pregnancies fetal breathing and fetal trunk movements were measured by real time ultrasound after the mothers had been given oral glucose. Fetal breathing was significantly reduced in the two fetuses which subsequently died and in five of six fetuses developing fetal distress in labour. Two patterns of breathing were noted in 23 fetuses with cessation of Intrauterine growth: either reduced fetal breathing or almost continuous breathing. Fetal trunk movements were more variable than breathing movements and a reduction in trunk movements had less predictive value.


The possible adverse effect of propranolol on the fetus in pregnancies complicated by severe hypertension. A retrospective study is presented of the effect of propranolol on fetal outcome in pregnancies complicated by maternal hypertension. In nine pregnancies in which propranolol was given to markedly hypertensive women (diastolic blood pressure over 105 mm Hg) the fetal outcome was worse than in 15 patients using other hypotensive agents. The probability of fetal or neonatal death was related to the amount of proteinuria and the presence of parenchymal renal disease but was also significantly higher when the mother had been treated with propranolol. Experimental evidence suggests that beta-adrenergic blockade is harmful to the hypoxic fetus, for these reasons the use of propranolol in hypertensive pregnancies complicated by placental insufficiency may be contraindicated unless there is no satisfactory alternative.


Melatonin in amniotic fluid during human parturition. Concentrations of melatonin have been measured in amniotic fluid obtained during late pregnancy and labour. Samples obtained by amniotomy during labour contained higher concentrations of melatonin than samples taken at amniotomy for the induction of labour. Amniotic fluid obtained by amniocentesis in late pregnancy contained lower concentrations of melatonin than amniotic fluid obtained by amniotomy. The implications of these findings are discussed in relation to pineal influences on parturition.


The effects of quinestrol and bromocriptine on blood coagulation, serum prolactin and serum FSH levels in puerperal women. The effects of bromocriptine and quinestrol upon coagulation and fibrinolysis during the puerperium were studied. Quinestrol therapy was associated with increased levels of factors VII and IX and decreased antithrombin activity on the sixth postpartum day, and increased factor IX and plasminogen levels on the fourteenth postpartum day. Six weeks after delivery elevated levels of factors II and VII and of plasminogen were recorded in women given quinestrol. Bromocriptine therapy only caused an increase in the level of factor IX at six weeks after delivery. Compared to controls, patients given bromocriptine had lower prolactin and higher FSH levels during the puerperium whereas the patients given quinestrol had increased prolactin levels and a late fall in FSH levels.


Induction of ovulation with clomiphene and human chorionic gonadotrophin in women with hyperprolactinaemic amenorrhoea. Seventeen women complaining of infertility (one with primary amenorrhoea, 14 with secondary amenorrhoea, and two with oligomenorrhoea) all had hyperprolactinaemia and were treated with clomiphene citrate and human chorionic gonadotrophin (HCG), and plasma oestradiol, FSH and LH levels were measured. Although adequate pre-ovulatory oestradiol levels were present, the surge of LH was absent until the injection of HCG after which all patients ovulated. There were 12 pregnancies in 9 patients resulting in 10 full-term livebirths, one premature livebirth and one continuing pregnancy. The relevance of these findings to the possible role of prolactin in amenorrhoea is discussed.


Vascular anatomy of the human uterus and pregnancy wastage. In uteri removed at hysterectomy the uterine arteries were injected with radioopaque media and the vascular configuration was correlated with reproductive histories. Patients whose uteri had two ascending uterine arteries on each side had significantly more abortions and smaller newborn infants than patients with a single ascending branch of the uterine artery.


Longer though lighter menstrual and intermenstural bleeding with copper as compared to inert intrauterine devices. A randomized serial study of uterine blood loss was made of 281 women fitted with one of three intrauterine contraceptive devices (IUCDs): a Lippes loop D, a Dalkon shield or a Copper 7. The increase in the amount of blood loss was greatest with Lippes loop D users and least with Copper 7 users. However, the duration of menstrual and intermenstrual bleeding and of spotting was significantly greater among Copper 7 users than with the two inert devices. Thus the mechanisms whereby IUCDs increase the duration as opposed to the volume of uterine bleeding may be different. Previous users of oral contraceptives were found to have significantly less bleeding before IUCD insertion, but showed a similar pattern of increase of uterine bleeding after IUCD insertion to those who had not used oral contraceptives. For all three IUCDs, the increment in amount and duration of uterine bleeding after insertion decreased between the first and the sixth period, on average by about 15 ml and one to two days. No subsequent improvement was found and after 12 cycles the Copper 7 users had longer but less heavy uterine blood loss than users of the Lippes loop D.


Meconium-like staining of amniotic fluid due to high fetal intestinal obstruction. Case report. A baby with congenital jejunal and ileal atresia is described. There was meconium-like staining of the amniotic fluid which was probably due to fetal regurgitation or vomiting.


Pregnancy and Nelson's syndrome. Two case reports. We describe two patients with Nelson's syndrome in whom pregnancy progressed normally to spontaneous delivery of normal infants. One patient, who subsequently was found to have a basophil adenoma of the pituitary, developed diabetes insipidus in late pregnancy and the diabetes insipidus regressed spontaneously after delivery.


The utilization of diets containing acetate, propionate or butyrate salts by growing lambs. 1. In a comparative slaughter experiment growing lambs were given concentrate diets in which 7, 15 or 22% of the metabolizable energy (ME) provided by barley was replaced by sodium and calcium salts of acetic acid, or 22% of ME was replaced by Na and Ca salts of propionic or butyric acids. 2. The efficiency of utilization for fattening (kf) of the diets containing 0, 7, 15 or 22% of ME as acetate was 57.2, 59.6, 54.1 and 48.8 (SE +/- 1.8) respectively, the last value being significantly lower (P less than 0.001) than the first. The kf for successive increments of acetate was 90, 37 and 19% (SE +/ 3), the decrease being significant (P less than 0.001). 3. The kf value of the diets containing 22% of ME as propionate or butyrate respectively were 48.7 and 50.6 (SE +/- 1.8), both values being significantly lower than the control (P less than 0.01). The partial kf of propionate was 19 +/- 13, and of butyrate 28 +/- 13%. 4. It is concluded that the experiment provided evidence that the efficiency with which acetateis utilized for energy retentionis not constant, but varies with its contribution to ME. The experiment also provided some evidence that large amounts of propionate and butyrate may be inefficiently utilized by growing lambs, although poor utilization of high levels of volatile fatty acid (VFA) salts per se cannot be entirely excluded.


Metabolism of heat-damaged proteins in the rat. Inhibition of amino acid uptake by "unavailable peptides" isolated from enzymic digests of heat-damaged cod fillet. 1. An extract containing "unavailable" small peptides was isolated from an enzymic digest of heat-damaged cod fillet and examined for its influence on uptake of leucine in the rat small intestine, using the everted-sac technique. 2. The extract strongly inhibited the uptake of leucine. It had no effect on uptake of glucose or its metabolism to lactate. 3. The findings are discussed in relation to the concept (Buraczewski, Buraczewska & Ford, 1967) that the accumulation of "unavailable peptide" material in the intestine, found in rats given heat-damaged protein, might hinder the absorption of amino acids by blocking a mechanism involved in their transport across the mucosal barrier.


Haematological and blood chemistry changes in ewes and lambs following supplementation with vitamin E and selenium. 1. For methods of vitamin E and selenium supplementation were evaluated using thirty-nine pregnant ewe-lambs fed on a ration containing 0.043 mg Se/kg and 25 mg vitamin E/kg. Treatments were control, fortified mineral mix (ESe salt) (300 mg vitamin E, 3 mg Se), ruminal Se pellets (505 mg Se), drench (300 mg vitamin E, 3 mg Se) and intramuscular injection (600 mg vitamin E, 3 mg Se). Only ewes supplemented, commencing approximately 50 d before parturition. 2. Birth weights were similar for all treatments and live-weight gains of lambs to 56 d of age were improved in all supplemented groups (P less than 0.05). There were no clinical cases of nutritional muscular dystrophy. 3. Se concentrations in whole blood were more than doubled in both lambs and ewes drenched or injected; responses to ESe salt and pellets were much smaller. 4. Plasma tocopherol levels were increased in injected dams and their lambs (P less than 0.001). 5. Haemoglobin concentration and erythrocyte counts were significantly higher (P less than 0.01) in control ewes and lambs than in treated lambs. 6. Lactate dehydrogenase (EC 1.1.1.27), creatine kinase (EC 2.7.3.2) and aspartate aminotransferase (EC 2.6.1.1) activities were increased in lambs from control, ESe salt and pellet groups (P less than 0.001). Glutathione peroxidase (EC 1.11.1.9) activity responded to Se supplementation in both ewes and their lambs (P less than 0.001) and the response was highest in the injected group, followed in order, by the drench, pellet, Ese salt and control groups. 7. These studies indicated that in terms of the haematological and blood chemistry changes investigated, the intramuscular injection was most effective, followed by the oral drench. Ruminal pellets and fortified salt were less satisfactory.


The nutritive value of silages. Energy metabolism in sheep receiving diets of grass silage or grass silage and barley. 1. Two calorimetric experiments were conducted to study the utilization of energy in sheep given diets of grass silage or grass silage and barley. Three silages were investigated. One was made from first-harvest grass in the spring (S) and the others from regrowth cut either early or late in the autumn (E and L respectively). All were of perennial ryegress (Lolium perenne) and preserved with formic acid. Each silage was given at two levels of feeding, the lower providing approximately a maintenance energy intake. The S and L silages were also given supplemented with barley. 2. The digestibilities of organic matter, cellulose and energy in the silages were high. Measured at maintenance, digestible energy (DE) contents (MJ/kg dry matter (DM))were 11.83, 14.67 and 12.90 for S, E and L respectively. The DE contents of the S and E silages were depressed at the higher level of feeding but the effect was offset by changes in the energy losses as methane and urine. Metabolizable energy (ME) contents (MJ/kg DM) for the three silages, S, E and L were respectively 9.88, 12.54 and 10.73 at the low level of feeding and 9.91, 11.99 and 11.08 at the high level of feeding. The mean ME content of barley calculated by difference was 13.76 MJ/kg DM. 3. The mean efficiencies of utilization of ME for maintenance (km) for the S, E and L silages were 0.69, 0.71 and 0.68 respectively. Corresponding values for fattening (kf) were 0.21, 0.57 and 0.59. Excepting the kf for the S silage which was low, observed efficiencies were in broad agreement with those predicted by the equations of the Agricultural Research Council (1965). Similar agreement was obtained with all diets consisting of silage and barley.


Metabolic changes associated with the occurrence of fatty liver and kidney syndrome in chicks. 1. The changes in a number of metabolic measurements brought about by low-biotin diets associated with high and low incidences of fatty liver and kidney syndrome (FLKS) were studied in healthy 4-week-old broiler chicks. 2. Liver pyruvate carboxylase (pyruvate: CO2 ligase (ADP); EC 6.4.1.1) activity was low in birds fed on a diet causing a high incidence FLKS but the addition of fat or protein to this diet, to decrease the incidence of FLKS, increased enzyme activity. 3. Liver weights, blood lactate concentrations, plasma lactate dehydrogenase (L-lactate: NAD oxidoreductase; EC 1.1.1.27) activitvities and values for C16:1 : C18:0 fatty acid in liver, adipose tissue and plasma triglyceride were highest in birds fed on the high-FLKS diet and all measurements were negatively correlated with pyruvate carboxylase activity. 4. Birds with high plasma lactate dehydrogenase activity or triglyceride C16:1 : C18:0 values were the most likely to develop FLKS when fasted. 5. There was no evidence that increased liver weight was associated with increase activities of certain other liver enzymes. 6. It is concluded that FLKS occurs in birds with little or no hepatic gluconeogenic capacity via pyruvate carboxylase as a result of a dietary insufficiency of biotin but that the initiation of the syndrome in probably associated with the inhibition of other pathways of gluconeogenesis.


Estimation of rate of protein synthesis by constant infusion of labelled amino acids in pigs. 1. The fractional synthetic rates of tissue proteins were studied in growing pigs using the constant-infusion technique of tracer-labelled amino acids ([14C]leucine and [14C]lysine) and the mathetmatical model for calculation, employed in rats by Garlick, Millward & James (1973). 2. During a 6 h infusion, samples were taken from blood and muscle and at the end of the infusion from liver, muscle, pancreas, heart, duodenum, jejunum, ileum, colon, and skin. The specific radioactivity of free and protein-bound leucine and lysine was estimated. 3. A quasi-steady-state in the specific radioactivity of free plasma leucine and lysine was reached within approximately 2 h, the rate-constants being 35 and 48/d respectively. 4. The specific radioactivity of free leucine and lysine in plasma was used to calculate the flux of these amino acids. It was found to be higher than the daily intake. 5. The average fractional rate of protein synthesis in muscle and heart 8.1% d, in small and large intestine the values were 50 and 33% d respectively and in liver and pancreas more than 100% d. 6. The calculation of protein synthetic rate in pig tissue using the constant-infusion method of labelled amino acids seems to be a suitable toof for study of this species.


Lead and iron absorption from rat small intestine: the effect of dietary Fe deficiency. 1. When lead is administered in drinking-water iron-deficient rats retain more Pb than Fe-replete rats (Six & Goyer, 1972; Klauder & Petering, 1975). In the present study the relationship between the absorption of Pb and Fe was investigated. 2. Adult male rats were transfered to a milk-based diet fed with or without supplementary Fe (180 mg Fe/kg as ferrous sulphate). After 7--9 d the absorption of duodenally-administered 203Pb and 59Fe was measured as uptake of radioactivity from the gastrointestinal tract after 90 min. 59Fe absorption was increased in rats given the unsupplemented diet for 7 d and was further increased in rats kept on the diet for up to 7 weeks. 203Pb absorption was not consistently increased by either short- or long-term Fe deprivation. 3. Much of the 203Pb in homogenates of the upper small intestine was bound to soluble protein of which up to 85% was dialysable. In contrast little 59Fe was dialysable. Only a small proportion of the soluble musosal Pb was associated with ferritin during gel-filtration chromatography although 203Pb precipitated together with carrier rat-liver ferritin with an antibody to rat-liver ferritin. 4. There appeared to be no direct relationship between the transfer of Fe and Pb across the small intestine of the adult rat.


Studies on the nutrition of marine flatfish. The pyridoxine requirement of turbot (Scophthalmus maximus). 1. Diets containing graded levels of pyridoxine hydrochloride (to supply 0.26--30 mg pyridoxine/kg) were given to seven duplicate groups of turbot (Scophthalmus maximus) for 12 weeks and their growth rate was measured during this period. 2. Good growth was obtained on all treatments except those groups given less than 1.0 mg pyridoxine/kg diet. These fish grew normally until weeks 8--10 but thereafter their weight gain was significantly less than that for other treatments. 3. Measurements of aspartate aminotransferase (EC 2.6.1.1) in muscle and liver and of alanine amino-transferase (EC 2.6.1.2) in liver of the turbot showed that the activities of these enzymes increased with increasing dietary pyridoxine intake up to a level of 2.5 mg pyridoxine/kg. The activities of these enzymes were not further enhanced by additional dietary pyridoxine. 4. Percentage stimulation of these enzymes by pre-incubation of extracts with pyridoxal phosphate was minimal with those groups of turbot given 2.5 mg pyridoxine/kg diet or more. 5. It is concluded that the dietary requirement of turbot for vitamin B6 can be safely met with a diet containing between 1.0 and 2.5 mg pyridoxine/kg. 6. An eighth group of turbot given the pyridoxine antagonist 4-deoxypyridoxine hydrochloride (20 mg/kg) showed retarded growth after 2 weeks, together with a high mortality rate.


The effect of vitamin B12 on physical performance capacity. 1. Physical performance capacity was assessed before and after injection of cyanocobalamin and a placebo for thirty-one Burmese male subjects whose age ranged from 18.7 to 20.8 years. 2. The subjects chosen were free from anaemia and were paired on a "weight--age" basis and divided into two groups. 3. One group received an injection of 1 mg cyanocobalamin and the other was given a placebo injection three times weekly for 6 weeks. This was done on a "double-blind" basis. 4. There was no significant difference between the two groups in any of the criteria measured, before and after administration of either 1 mg cyanocobalamin or placebo injection. So also there was no significant increase in any of the measurements in the subjects after receiving cyanocobalamin injections compared with thosse receiving the placebo.


Absence of immunological hypertensitivity to dietary cow's-milk proteins in infant rabbits. 1. Rabbits hand-reared on a diet based on cow's milk frequently experience convulsive and sometimes fatal reactions to their feed. These have been thought to be caused by immunological hypersensitivity to cow's milk. 2. This explanation has been re-examined and found to be untenable.


The effect of alcohol on resting metabolic rate. 1. There was a significant increase in oxygen consumption in ten women after small intakes of alcohol. 2. No significant difference was found between the average increase in metabolic rate over 3 h after an isoenergetic meal of food or of food plus alcohol. However, during the last half of the 3 h postprandial period, the increased O2 consumption was significantly greater after the meal including alcohol. 3. Possible explanations for the contradictory results in the literature are discussed.


Adsorption of bile salts from aqueous solution by plant fibre and cholestyramine. 1. Adsorption of bile salts by dietary fibre is believed to promote their excretion and hence to reduce the serum cholesterol level in man and experimental animals. 2. We have tested a number of plant fibre fractions and other related materials for their ability to adsorb bile salts from aqueous solution. The "insoluble" plant fractions were from "dry grain" (a residue from brewing), apple, wheat bran, lucerne (Medicago sativa), soya beans, mung  beans (Phaseolus mungo), chick peas (Cicer arietinum), rolled oats, spinach (Spinacia oleracea), sunflower seeds, sawdust and sheep faeces. The other materials were cholestyramine, pectin and lignins prepared from wheat bran and from sawdust. 3. Only cholestyramine and the fibre from lucerne, soya beans, mung beans, chick peas, spinach, and sunflower seeds adsorbed enough of either sodium cholate or sodium deoxycholate for adsorption to be detectable. 4. This result conflicts with a report that the lignin component of dietary fibre is responsible for adsorption of bile salts. 5. Adsorption of bile salts, when it occurs, may depend on the presence of saponins bound to the fibre.


Environmental temperature and choice feeding of the broiler. 1. Broilers were kept under environmental temperature regimens of 16, 21, 26 or 31 degrees and given, 22--57 d of age, either a complete diet or free-choice of whole wheat and a higher-protein food containing either 252 or 516 g crude protein (nitrogen x 6.25)/kg. 2. Broilers maintained at 16 and 21 degrees consumed food at a significantly higher rate than those kept at 26 degrees and the latter had a significantly higher food intake rate than broilers kept at 31 degrees, for each feeding treatment. Compared with 21 degrees the 57 d body-weight of broilers kept at 26 degrees and 31 degrees was significantly reduced. 3. When kept under the 16, 21, 26 and 31 degrees regimens, the amount of whole wheat in the food consumed by the broilers choice-fed with wheat and the 252 g crude protein/kg food was 56, 55, 48 and 46% respectively.  The corresponding values for the broilers on the second choice-feeding treatment were 80, 76, 79 and 72%. 4. Except for a single instance the choice-fed broilers grew at a significantly slower rate than broilers given the complete diet.


Biliary lipids, bile acids and gallstone formation in hypovitaminotic C guinea-pigs. 1. Hypovitaminotic C guinea-pigs fed on a high-cholesterol diet for 5 weeks developed gallstones (810 mg cholesterol/g) whereas no concretements were observed in vitamin C-replete animals.  2. Scanning electron microscope studies of the three types of gallstone observed in the gallbladders of vitamin C-deficient animals showed them to be composed of randomly-arranged needle-shaped or laminated crystal of cholesterol. 3. The hepatic bile of gallstone-forming animals had a higher cholesterol concentration and lower bile acid content, the latter being principally due to a reduction in the chenodeoxycholic fraction, than the bile of vitamin C-replete animals. 4. No significant difference was observed between the volume of bile secreted by hypovitaminotic C and vitamin C-replete animals, but due to the qualitative changes in bile composition, gallstone formation was associated with an increased biliary secretion of cholesterol and a reduced secretion of bile acids.


Factors affecting the voluntary intake of food by sheep. 1. The role of distension, flow-rate of digesta and propulsive motility in the intestines. 1. Twelve sheep fitted with abomasal cannulas were given ad lib. access to a diet of chopped lucerne (Medicago sativa) hay to a ascertain if the amount of digesta being transported by the intestines was limiting intake. If this was the situation, pumping a solution of the bulk-laxative methylcellulose (100 g/kg; MC) into the abomasum should have reduced intake in proportion to the mass of digesta attributable to the MC and associated water. In preliminary experiments faecal water and wet matter outputs increased by 6.2 and 7.4 g/g MC powder respectively. 2. The infusion of 2.95 kg MC/d did not affect food intake even though wet faecal output increased from a control value of 2.436 to 4.616 kg/d. The transit time of 51 Cr-EDTA through the intestines decreased only slightly during the infusion indicating that MC produced a marked increase in the mass of intestinal contents. Increasing the rate of infusion to 5.336 kg/d increased wet faecal output to 5.437 kg/d, did not change transit time but significantly decreased food intake presumably to protect the intestines from overdistension. After stopping the infusion, food intakes and faecal outputs returned to control values but transit time remained unchanged. The intake of chopped lucerne hay by sheep was not limited by the capacity of the small and large intestines to transport bulk as intake was maintained even through wet faecal output was doubled and the intestines apparently became markedly distended.


Protein degradation and optimum urea concentration in cereal-based diets for sheep. 1. Early-weaned lambs were used to estimate the concentration of urea required to give the maximum intake and utilization of maize or barley with either a high (HPB) or low (LPB) protein content. 2. Approximately the same concentration of urea (7--11 g urea/kg feed) was required for maximum intake and feed utilization of both HPB and LPB. With maize there was no increase in intake, live weight gain, digestion and feed conversion as a result of adding more than 7 g urea/kg. 3. The proportion of protein degraded in the rumen was estimated by the synthetic fibre bag technique to be 0.69, 0.82 and 0.54 for HPB, LPB and maize respectively. The similarity in concentration of urea required for optimum utilization of LPB AND HPB might be explained by differences in the extent of degradation of protein in the rumen, but the lower concentration of urea required for maize cannot be similarly explained. 4. From estimates of yield of microbial protein in the rumen, the extent of rumen fermentation and the measured extent of protein degradation, theoretical requirements for urea were calculated and compared with other predictions and with the experimentally determined values. For barley, predicted values agreed reasonably well with experimental ones, but for maize all values, including those derived by a new system adopted by the Agricultural Research Council (ARC) Working Party, were too high.


A comparison of the voluntary intake and digestion of a range of forages at different times of the year by the sheep and the red deer (Cervus elaphus). 1. Comparisons were made between castrated male Scottish Blackface sheep and red deer (Cervus elaphus) of voluntary forage intake (VFI), digestibility and the mean retention time (MRT) of a particulate-phase marker (103Ru-phenanthroline) in the alimentary tract, when a range of forages: dried-grass pellets, chopped dried grass, fresh-frozen Agrostis-Festuca spp. and heather (Calluna vulgaris, L. Hull) were given at different times of the year. 2. On both the dried-grass-pellet and chopped dried-grass diets the red deer and sheep ate similar quantities. Both species had a higher VFI of dried-grass pellets in July than in November. The sheep digested the dried-grass-pellet diet better than the red deer and this was associated with a longer MRT of the particulate-phase marker in the alimentary tract. 3. The VFI of Agrostis-Festuca spp. and heather by the red deer was twice that of the sheep. The VFI of heather by the sheep increased by 32% between January and April, and the VFI of both the Agrostis--Festuca spp. and heather diets by the red deer increased by 65--70%. The sheep digested the Agrostis--Festuca spp. better than the red deer but the red deer digested the heather slightly better than the sheep. MRT of the particulate-phase marker was greater for the sheep than for the red deer on both the diets. The digestibility and MRT of both diets in the red deer did not decrease with the seasonal increase in VFI, suggesting a possible hypertrophy of the alimentary tract.


Amino acid requirements of the breeding sow. 2. The dietary lysine requirement of the lactating sow. 1. Two experiments, involving six lactating Landacre x (Landrace x Large White) sows were undertaken to study the lysine requirement of the lactating sow and to identify the second limiting amino acid by means of interpretation of plasma amino acid and blood urea levels. 2. A basal diet composed mainly of equal amounts of ground barley and ground wheat supplemented with mineral and vitamin mixtures was used. This diet was supplemented with synthetic amino acids (lysine threonine, tryptophan, methionine and isoleucine). 3. Plasma amino acid and blood urea values showed a close agreement and indicated that lysine intake above 38.4 g/d was in excess of the requirement level. 4. Plasma amino acid values suggested tryptophan to be the second limiting amino acid whereas blood urea values suggested threonine to be the second limiting amino acids.


The effect of 1-alpha-hydroxycholecalciferol on calcium and phosphorus metabolism in the lactating ewe. 1. The effect of 1-alpha-hydroxycholecalciferol (1 alpha-OH-D3) on calcium and phosphorus metabolism has been studied in ewes at peak lactation by a combination of a mineral balance and a radioisotope technique. 2. The rate of Ca absorption was substantially higher in the treated ewes than in controls. The rates of endogenous loss of Ca into urine, faces and milk, however, were only slightly higher. 3. In consequence, the net retention of Ca was increased and the loss of skeletal reserves of Ca normally associated with peak lactation, prevented. 4. Although the rate of bone accretion increased slightly, the increase in skeletal retention of Ca resulted mainly from a decrease in the rate of bone resorption. 5. This finding conflicts with the generally held belief that bone resorption is increased by cholecalciferol treatment. 6. The rates of apparent absorption and retention of P were increased by the treatment probably as a result of a direct effect of the 1 alpha-OH-D3 on P absorption. 7. These results provide a possible explanationof the beneficial effect of 1 alpha-OH-D3 in preventing parturient paresis  (milk fever) in the dairy cow.


Metabolism of dietary fibre components in man assessed by breath hydrogen and methane. 1. Breath hydrogen and methane were measured in eight normal individuals after acute and separate administration of different chemical components of dietary fibre. 2. Hemicellulose, raffinose and lactulose increased H2 production, while cellulose pectin and lignin did not. Methane production was found to be individual and unaffected by any of the substances. Differences in physical properties of the same chemical appear to have no influences on H2 and CH4 production.


Urinary excretion of aflatoxin M1 after administration of aflatoxin B1 in sucrose- or starch-rich diets. 1. Male Sprague--Dawley rats were given 630 g/kg sucrose or starch with 2 mg/kg aflatoxin b1 for periods of 75, 145 and 200 d, and the 24 h urinary excretion of aflatoxin M1 was measured. 2. Less aflatoxin M1 was excreted by the rats fed on the sucrose-rich diet compared to those fed on the starch-rich diet. This difference was especially marked when expressed per g metabolizing tissue. 3. It is concluded that sucrose probably decreases the activity of aflatoxin B1 metabolism in a similar way to its previously found effect on the drug-metabolizing enzyme.


Purification and characterization of cytoplasmic thioltransferase (glutathione:disulfide oxidoreductase) from rat liver. An enzyme catalyzing thiol-disulfide interchange of glutathione and disulfides and the reaction between glutathione and thiosulfate esters has been purified 40 000-fold from rat liver cytosol. The enzyme, named thioltransferase (Askelöf, P., Axelsson, K., Eriksson, S., & Mannervik, B. (1974) FEBS Lett. 38, 263--267), was homogeneous in several electrophoretic systems, had an isoelectric point at pH 9.6, and contained 8.6% (w/w) carbohydrate. The catalytic activity had a distinct optimum at pH 7.5. A series of substrates was tested at a constant glutathione level; the kcat values (at 4mM glutathione) were all in the range of about 10(4) min-1. The substrates included mixed disulfides of glutathione, other low-molecular-weight disulfides, S-sulfocysteine and S-sulfoglutathione, and peptide disulfides such as insulin, oxytocin, ribonuclease, and the mixed disulfide of glutathione and egg-white lysozyme. The enzymatic reaction was inhibited by an excess of glutathione (greater than 4mM).


Human kidney alanine aminopeptidase: physical and kinetic properties of a sialic acid containing glycoprotein. Human kidney alanine aminopeptidase has been purified to apparent homogeneity as judged by electrophoresis and sedimentation in the analytical ultracentrifuge. Amino acid analyses indicate that the enzyme is high in tryptophan content and low in cysteine content. The enzyme contains sialic acid, hexoses, and glucosamine, which make up 21% of its dry weight. In dilute buffer, the enzyme exhibits a molecular weight near 236 000, but in denaturing solvents the enzyme exhibits a molecular weight near 119 500. Zinc analyses by atomic absorption demonstrate 1 mol of zinc for 113 500 +/- 6900 g of protein. The zinc is firmly bound, since exhaustive dialysis against chelating agents does not remove the zinc or inactivate the enzyme. The enzyme is stimulated by Co2+ 1.65-fold, but, in contrast to the enzyme-zinc complex, the enzyme-cobalt complex dissociates upon dialysis. Kinetic studies with a series of aminoacyl-beta-napthylamides indicated that the highest kcat value was obtained for L-alanyl-beta-naphthylamide (8.43 X 10(3)s-1), whereas the lowest Km value was obtained for L-methionyl-beta-naphthylamide (1.4 X 10(-5) M).


Lateral diffusion in phospholipid bilayer membranes and multilamellar liquid crystals. The lateral-diffusion coefficients (D) of a fluorescent lipid analogue, 3,3'-dioctadecylindotricarbocyanine (diI), have been measured in black lipid membranes (BLMs), in large (20--50-micron diameter) solvent-free bilayer and multilayer membrane vesicles, and in multilamellar liquid crystals of dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, and egg lecithin. They show that D changes by several orders of magnitude at the liquid-crystal transformations of the solvent-free bilayers and multilayer. In all BLMs, D approximately 10(-7)cm2/s with only weak temperature dependence even near the putative phase-transformation temperatures, Tt. In the corresponding liquid crystals and large vesicles, D approximately  10(-8)cm2/s above Tt, decreasing by about two orders of magnitude to D approximately 10(-10)cm2/s below Tt. The changes of D in bilayer vesicles suggest that the expected liquid-crystal phase transitions from smectic A to a higher ordered state do persist in isolated bilayers. Retained solvent in black lipid membranes formed by both Mueller--Rudin and Montal--Mueller methods appears to enhance lateral diffusion. A simple method of forming small numbers of large solvent-free vesicles is described.


Biosynthesis of the antibiotic 2,5-dihydrophenylalanine by Streptomyces arenae. The biosynthesis of L-2,5-dihydrophenylalanine (DHPA) in Streptomyces arenae strain Tü 109 was studied in tracer experiments with [U-14C]- and [1,6-14C]shikimic acid followed by chemical degradation of the labeled product. The results indicate that shikimic acid (II) provides only the ring carbons of DHPA, that the side chain of DHPA is attached at the carbon derived from C-1 of II, and that in the transformation of II into DHPA the asymmetry of the ring of II is preserved, with C-6 of II giving rise to C-6' of DHPA. Both generally 14C-labeled chorismate and prephenate, but not L-[3-14C]serine, are incorporated into DHPA. By preparing and feeding 5,6-dihydro[4-3H]prephenate it was shown that this compound is not an intermediate in the biosynthesis of DHPA. A reaction sequence is proposed for the conversion of prephenate to DHPA, involving an allylic rearrangement, followed by 1,4 reduction of the resulting conjugated diene and a combined decarboxylation/dehydration.


Iron exchange between ferritin and transferrin in vitro. The transfer of iron between horse spleen [55Fe]ferritin and human apotransferrin or [59Fe]transferrin in homogeneous solution was investigated. Transfer between the two proteins in the presence of citrate, ATP, or ascorbate occurs in both direction, but the net flow is always from ferritin to transferrin. Ferritin which is ca. 1/3 to 1/2 saturated with iron appears to be most reactive. Chemically prepared apoferritin does not accept iron from diferric transferrin. Citrate-mediated transfer of iron from ferritin to apotransferrin is first order with respect to ferritin, zero order with respect to transferrin, and has a complex dependence upon citrate concentration. Direct transfer of iron from native or reconstituted ferritin to apotransferrin in the absence of any identifiable mediating agent was observed to occur at about half the rate attained in the presence of 1 mM citrate. No transfer of iron between the two proteins occurs across a dialysis membrane in the absence of a mediating agent. No binding of transferrin and ferritin to each other was demonstrable. One possible explanation for these observations is that iron from the core of ferritin is in equilibrium with iron near the outer surface of the protein, where the metal would be available to transferrin.


Hemocyanin from the Australian freshwater crayfish Cherax destructor. Characterization of a dimeric subunit and its involvement in the formation of the 25S component. The molecular weight of a dimeric subunit, M3', isolated from Cherax destructor hemocyanin has been measured by sedimentation equilibrium to be 144 000. Peptide mapping and end-group analysis together with gel electrophoresis show that the dimer consists of two very similar or identical monomers, cross-linked by disulfide bridges. Dissociation of the 25S component of the hemocyanin shows that it contains the dimer and two previously identified monomers, M1 and M2. Its molecular weight is 900 000 by sedimentation equilibrium, and reconstitution studies show that the dimer is essential for its formation. Analysis of the results of polyacrylamide disc gel electrophoresis experiments with the 25S component indicates that it consists of a population of 11 compositional isomers. These all contain one dimeric subunit and ten monomeric subunits, the latter being present in all the combinations of M1 and M2.


Immunoglobulin genes in DNA restriction fragments. We have investigated the organization of immunoglobulin genes in mice. High molecular weight DNA from myelomas and Krebs ascites cells was cleaved with EcoRI restriction endonuclease and fractionated using preparative agarose gel electrophoresis. Each fraction was then hybridized to an immunoglobulin mRNA or a cDNA transcribed from the mRNA. In two series of experiments, one with a kappa chain probe (MOPC 41 mRNA), the other with a lambda chain probe (SAPC 178 mRNA), we analyzed a variety of myeloma DNAs and Krebs DNA. In contrast to previously reported findings (Tonegawa, S., et al. (1976) Cold Spring Harbor Symp. Quant. Biol. 41, 877), we did not observe any unique restriction map pattern in the DNA from cells which exress a given immunoglobulin gene. We also found that restriction fragments containing c region genes do not appear to transpose, while DNA sequences corresponding to other portions of the kappa and lambda mRNAs do in some cases.


Biochemistry of terminal deoxynucleotidyltransferase: mechanism of inhibition by adenosine 5'-triphosphate. The polymerization of deoxyribunucleoside triphosphate catalyzed by terminal deoxyribonucleotidyltransferase (TdT, EC 2.7.7.31) is severely inhibited by the addition of ribonucleoside triphosphates, ATP being the most potent inhibitor. Examination of the inhibitory effect of ATP using oligo(dA)12-18 as well as activated DNA as primers revealed that (a) ATP inhibition is not due to its addition onto a 3'-OH primer terminus ad judged by the lack of incorporation of labeled ATP, although under similar conditions incorporation of GTP can be demonstrated, (b) a consistent degree of inhibition was noted independent of primer or enzyme concentration; (c) addition of ATP to an ongoing reaction promptly reduces the rate of polymerization; (d) kinetic studies indicate a competitive (with respect to substrate deoxy triphosphate) pattern of inhibition; (e) addition of excess deoxyribotriphosphate promptly relieves the inhibition. Unlike ATP, other ribotriphosphates yield a mixed pattern of inhibition partly mediated by competitive mechanisms. GTP and CTP and to a minor extent UTP are incorporated into DNA in the presence or absence of deoxy triphosphate. Furthermore, addition of ATP also inhibits incorporation of GTP and CTP.


Molecular basis of reduced albumin synthesis in Morris hepatoma 7777. The level of albumin mRNA in the normal Buffalo rat liver and Morris hepatoma 7777 was compared by molecular hybridization with albumin complementary DNA (cDNA) and translational assays. Albumin mRNA was found to be 10% of the total rat liver poly(A)-containing RNA population but reduced approximately fourfold in the case of Morris hepatoma 7777. An equivalent decrease of albumin mRNA activity in the hepatoma was detected by translation in a mRNA-dependent cell-free protein-synthesizing system. A proportional increase in total hepatoma poly(A)-containing RNA was not observed, indicating that there was a true fourfold reduction of albumin synthesis in the hepatoma. DNA excess hybridization with albumin cDNA did not reveal any apparent change in albumin gene frequency in the hepatoma compared to normal liver. Complementary DNA copies of total liver and hepatoma poly(A)-containing RNA were synthesized and employed in homologous and heterologous hybridization reactions. Analyses of these reactions showed a high degree of homology between the poly(A)-containing RNA of the liver and hepatoma, with some difference in the relative sequence abundancy. However, qualitative differences were detected in hepatoma 7777 consistent with the concept of alterations in the control of gene expression upon neoplastic transformation.


Light-induced free-radical reactions of nucleic acid constituents. Effect of sequence and base--base interactions on the reactivity of purines and pyrimidines in ribonucleotides. The reaction with 2-propanol of purines and pyrimidines, induced photochemically with light of lambda greater than 300 nm and di-tert-butyl peroxide as an initiator, was applied to a variety of adenosine-, guanosine-, and uridine-containing ribonucleotides in order to determine the rules which govern the reactivity of the heterocyclic bases of nucleotides. The reactivity of the purine moieties was found to depend on the conformation of the appropriate nucleotide (anti or syn) and on the site of binding of the phosphate group to the ribose moiety. Adenosine moieties (assuming an anti conformation) blocked at their 3'-hydroxyl reacted faster than those blocked at their 5'-hydroxyl. The reactivity of the guanosine moieties (tending to assume a syn conformation) was independent of the site of binding of the phosphate. The uridine moieties of the various nucleotides exhibited a wide range of reactivity. A correlation between the reactivity of the uridines and their involvement in stacking interactions with next- and second-neighboring purines could be made. Thus, the uridine moieties of U-U-U, G-U, U-G, A-U-A, and A-U-G were reactive, while those of A-U and A-U-U were unreactive. The relative reactivity of uridine moieties of nucleotides can, therefore, be used as a measure of the extent of pyrimidine-purine stacking and vice versa.


Measurement of repulsive forces between charged phospholipid bilayers. By using an osmotic stress technique (LeNeveu, D. M., et al. (1977) Biophys. J. 18, 209), we have measured the net repulsive force between egg lecithin bilayers containing various amounts of the charged lipids phosphatidylglycerol and phosphatidylinositol. At bilayer separations greater than about 30 A, the repulsion is dominated by electrostatic forces; its variation with both bilayer separation and charge density is well described qualitatively by simple electrostatic double-layer theory. Quantitative agreement requires, however, that only about 50% of the phosphatidylglycerol polar groups be dissociated. At all charge densities, even for pure phosphatidylglycerol, and at bilayer separations less than about 30 A, the repulsion is dominated not by the electrostatic force but by a strong "hydration force" (LeNeveu, D. M., et al. (1977) Biophys. J. 18, 209). We conclude that the hydration force demands more attention than it has enjoyed hitherto in attempts to understand bilayer membrane interaction and fusion.


Complete amino acid sequence of the major component myoglobin from the humpback whale, Megaptera novaeangliae. The complete primary structure of the major component myoglobin from the humpback whale, Megaptera  novaeangliae, was determined by specific cleavage of the protein to obtain large peptides which are readily degraded by the automatic sequencer. Over 80% of the amino acid sequence was established from the three peptides resulting from the cleavage of the acetimidated apomyoglobin at the three arginine residues with trypsin. The further digestion of the central cyanogen bromide peptide with trypsin and S. aureus strain V8 protease enabled the determination of the remainder of the covalent structure. This myoglobin differs from that of sperm whale, Physeter catodon, at 12 positions, and dwarf sperm whale, Kogia simus, at 14 positions, finback whale Balaenoptera physalus at 3 positions, minke whale, Balaenoptera acutorostrata at 2 positions, and California gray whal Eschrichtius gibbosus, at 1 position. All of the substitutions observed in this sequence fit readily into the three-dimensional structure of sperm whale myoglobin.


Energy utilization and RNA transport: their interdependence. The interdependence of RNA transport and the metabolism of nucleotide additives was investigated. Rat-liver RNA was radioactively labeled in vivo for 45 min before isolation of liver nuclei, and the concentration dependence of RNA transport on nucleotide additives was determined. In a parrallel investigation, using nucleotide additives labeled in the base moiety, the distributions of label in the tri-, di-, and monophosphate forms were examined after various intervals of incubation. Analysis of results revealed that RNA transport was linearly related to the decline in energy charge of nucleotide additives, whith high statistical correlation. Kinetic analysis of labeled-nucleotide metabolism led to a simple schematic model for pathways for the utilization of high-energy phosphate bonds, and predictions of the scheme were confirmed by studies examining the effects of nucleotide analogues upon RNA transport. Data concerning inhibitors and chelators intimated that multiple avenues of inhibition and stimulation may potentially influence RNA transport. On the basis of previous data and the results presented in this communication, we conclude that nucleocytoplasmic RNA transport is dependent upon high-energy phosphate-bond hydrolysis and that nucleotides do not stimulate RNA transport via a simple chelation mechanism.


Detection and characterization of the intermediate on the folding pathway of human alpha-lactalbumin. To discuss the relation between the folding mechanism and the chemical structure of proteins, the reversible unfolding reactions of human alpha-lactalbumin by acidification and by guanidine hydrochloride at 25 degrees C are studied by means of circular dichroism, difference spectra and pH-jump measurements and are compared with those for bovine alpha-lactalbumin. As shown previously for bovine alpha-lactalbumin, the folding process at neutral pH is not explained by a simple two-state mechanism but involves an intermediate form that has the same amount of helical structures as the native form. The transition between the intermediate and the fully denatured states is too rapid to be measured and corresponds to the helix-coil transition of the backbone. One of the differences of human alpha-lactalbumin from the bovine protein is the remarkable stability of the intermediate at neutral pH, which can be explained by differences in the primary chemical structure. Another difference is the existence at acid pH of an additional helical form, which is more helical than the native form. The transition from this to the intermediate or to the fully denatured one also is shown to resemble the helix-coil transition. The following folding scheme of human alpha-lactalbumin is proposed: formula: (see text). Here N is the native form, and the intermediate is a macroscopic state distributed around the state A3 at neutral pH, while the distribution in the acid and fully denautured states shifts toward Am and A-n, respectively.


Liposomes from ionic, single-chain amphiphiles. In studies of the minimum physiochemical requirements for lipid membrane formation, we have made liposomes from dilute, aqueous dispersions of C(8)-C(18) single-chain amphiphiles. In general, membrane formation from ionic soaps and detergents requires the presence of uncharged amphiphiles. Vesicles were characterized by phase-contrast microscopy, by trapping of ionic dyes, as well as by negativestain and freez-frature electron microscopy. They were typically heterogeneous in size, but the average diameter could be experimentally varied in some cases over the range of 1 to 100 micrometer. Uni-, oligo-, and multilamellar vesicles were observed. Membrane permeability to various solutes was determined in part by a new technique which utilized phase-contract microscopy; when impermeable vesciles exclude added solutes such as sucrose, refractive index differences are created between vesicle contents and surrounding medium, so that the vesicles appear bright in the phase microscope. Permeant solutes do not produce this effect. Spectrophotometric permeability determinations confirmed the results of this technique and provided quantitative measures of permeability. Monoalkyl liposomes have potential uses as models of biomembranes and in drug delivery. They are also relevant to the prebiotic origin of biomembranes.


Conformation of the mushroom toxin beta-amanitin in the crystalline state. A single crystal X-ray diffraction analysis of beta-amanitin, a bicyclic octapeptide toxin isolated from the poisonous mushroom Amanita phalloides, shows that the molecule has distinct regions of hydrophilic and hydrophobic residues and two 18-membered rings. The study confirms the proposed chemical sequence and the configuration of the residues. All eight peptide groups are in the trans conformation. Four intramolecular hydrogen bonds, two strong and two weak, occur in the structure. The toxin cocrystallizes with seven water and three ethanol molecules and participates in an extensive hydrogen-bonding network. The crystal structure was solved by direct methods. The space group is P2(1)2(1)2(1), and unit cell dimensions are a = 14.004(3), b = 14.943(3), and c = 30.794(7) A.


Coupling of a single adenylate cyclase to two receptors: adenosine and catecholamine. A detailed kinetic analysis on the rate of activation of adenylate cyclase by 1-epinephrine and by adenosine, separately and combined, was performed. Both ligands were found to induce the activation of adenylate cyclase to its permanently active state in the presence of guanylyl imidodiphosphate (GppNHp). The activation followed strictly first-order kinetics. On the basis of these experiments, it was found that all of the enzyme pool can be activated by the beta-adrenergic receptor, but only 60 to 70% of the enzyme can also be activated by an adenosine receptor. The remaining 30 to 40% cannot be activated by adenosine. While previous experiments have led us to conclude that the epinephrine receptor is uncoupled from the adenylate cyclase, it seems that the adenosine receptor is either precoupled to the enzyme or forms a long-lived intermediate of adenosing-receptor-enzyme complex. From the pattern of enzyme activation by the two ligands and GppNHp, it may be concluded that the two ligands, adenosine and the beta-agonist, activate the adenylate cyclase through a common guanyl nucleotide regulatory site. This assertion is supported by the finding that both adenosine and 1-epinephrine, in the presence of GTP, induce the reversal of the permanently active state, irrespective by which pathway the enzyme was activated.


Nitrogen-15 and carbon-13 nuclear magnetic resonance of reduced flavins. Comparative study with oxidized flavins. Nitrogen-15 and carbon-13 nuclear magnetic resonance spectra of the fully reduced form of flavin were studied with riboflavin tetrabutyrate (RBUT), an organic solvent-soluble derivative of riboflavin. For the measurement of 15N resonances, 99% enriched [1,3-15N]RBUT and [1,3,5-15N]RBUT wwere synthesized. In order to assign the 13C resonances, 90% enriched [2-13C]RBUT, [4a-13C]RBUT, [4,10a-13C]RBUT, and [8-2H3]RBUT were employed. The upfield shift of N(5) resonance upon reduction was remarkable (286 ppm), while the N(1) signal moved only by 79 ppm. The one-bond 15N-H spin-spin coupling constant 1J[15N(5)-H] of the reduced RBUT was smaller than its 1J[15N(1)-H] and 1J[15N(3)-H]. These observations indicate that N(5) changed into sp3 hybridization upon reduction and lost the character of planar nitrogen. Most of the 13C nuclei of the reduced form resonated at higher field than did those of the oxidized form, which is well explained by the increase in pi-electron densities. Among the 13C resonances, the upfield shift of C(4a) was remarkable (32 ppm), which explains the reactivity of C(4a) in oxygen flavoprotein complexation. 13C--15N spin-spin coupling constants were obtained from the measurements of 13C magnetic resonance of 15N-enriched RBUT. The values of the one-bond 13C--15N coupling constants increased markedly with protonation at N(1) and N(5) upon reduction.


Fluorotyrosine M13 coat protein: fluorine-19 nuclear magnetic resonance study of the motional properties of an integral membrane protein in phospholipid vesicles. We have prepared in vivo a fluorotyrosyl derivative of M13 coat protein and have incorporated it at high levels in small phospholipid vesicles, using a urea-cholate dialysis procedure. 19F nuclear magnetic resonance experiments at 254 MHz with this system indicate a T1 of 0.32 s and line width of 300 Hz. The observed line width increases dramatically below the gel to liquid-crystalline transition temperature for the lipid, indicating that the probe is sensitive to the phase state of the bilayer. Neclear Overhauser enhancement and field dependence of line width were used to establish the relative contributions of dipolar interactions and chemical-shift anisotropy to the observed T1 and line width. From this relaxation data, we have constructeda model for the motional properties of the protein in the lipid bilayer. This model is characterized by correlation times for rotation about the alphabeta and betagamma bonds of the two tyrosyl residues of 2 x 10(-8) and k x 10(-9) s, respectively. Rapid intermolecular dipole-dipole interactions are required to account for theestimated dipolar contribution to T1. A reasonable model for these interactions is that lipid methylene protons are involved in relaxation of the fluorine probes (which reside in the hydrophobic region of this integral membrane protein). We estimate a minimum translational diffusion coefficient for such lipids of D greater than 3 x 10(-9) cm2/s.


Relation between structure and specificity of antibodies: nuclear magnetic resonance study of binding fluorine-19 labeled nitrophenyl haptens to myeloma immunoglobulins M315, M460, and X25. The relation between structure and specificity of antibodies has been explored by 19F NMR studies of the binding of trifluoromethyl analogues of nitrophenyl haptens to the three mouse myeloma immunoglobulins M315, M460, and X25. We have used haptens with trifluoromethyl groups located at the ortho or para positions of the phenyl ring or attached to the side chain, two atoms removed from the ring (i.e.,-NHCH2CF3). The changes in chemical shift between hapten free in solution and bound to antibody are sensitive to microenvironment and range from 1.7-ppm downfield to 1-ppm upfield. The shifts of p-trifluoromethylnitrophenyl haptens bound to M315 and M460 are both large downfield shifts, which are likely caused by van der Waals interaction and ring-current effects, particularly from tyrosine-34(L); these haptens do not show similar shifts when bound to X25 which has a deletion of tyrosine34(L). Other differences in the binding of the aromatic rings of haptens by M315, M460, and X25 are observed and their origins considered. The importance of hydrogen bonding in the thermodynamic affinity of antibody for hapten has been estimated by comparisons of binding affinities for haptens with trifluoromethyl groups in place of nitro groups.


Further characterization of calcium-accumulating vesicles from human blood platelets. Human blood platelets are capable of removing Ca2+ from the cytoplasm by means of an active, ATP-dependent and cyclic AMP-stimulated transport system. Calcium-accumulating vesicles are obtained by sonicating platelets. On density gradient centrifugation, this activity is found in the heavier of two membrane fractions. Concentrated in this fraction are also the Ca2+-stimulated Mg2+-ATPase and glucose-6-phosphatase, believed to be a marker for internal membrane systems. When the isolated vesicles are loaded with Ca2+, a third band separates from the two vesicular fractions in the density gradient. This band C contains virtually all the Ca2+-accumulating activity. Evidence that this activity is due to an active uptake and not to surface binding or adsorption is presented. Whereas electron microscopy does not reveal striking differences between active and inactive fractions, differences in protein composition are revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Furthermore, this band contains an enzyme system which converts arachidonic acid to malondialdehyde and therefore this fraction must be the site of prostaglandin synthesis. Membranes prepared by loading platelets with glycerol, followed by osmotic lysis are unable to accumulate calcium. In sodium dodecyl sulphate-polyacrylamide gel electrophoresis such membranes show significant differences in their protein pattern as compared to the actively Ca2+-accumulating vesicular membranes of band C. All preparations with Ca2+-accumulating activity also contain markers for plasma membranes and the question whether this activity is due exclusively to an intracellular structural element equivalent to the sarcoplasmic reticulum of muscle or whether an "extrusion pump" expelling Ca2+ to the outside of the cell is also involved, cannot yet be ;nswered.


Binding of glyceraldehyde-3-phosphate dehydrogenase to phospholipid liposomes. The binding of glyceraldehyde-3-phosphate dehydrogenase prepared from rabbit muscle to phospholipid model membranes (liposomes) as a function of pH, ionic strength, and the influence of the binding on specific activity of the enzyme was studied. The binding decreases the specific activity of the enzyme. The binding was studied by the method of association of the enzyme with liposomes during centrifugation. The existence of a dominant interaction of electrostatic character was found.


Incorporation of galactose from UDP-galactose into microsomal and Golgi membranes of rat liver. Rough and smooth microsomes and Golgi membranes were incubated with UDP[14C]galactose and the incorporation of radioactivity into the lipid extract and into endogenous protein acceptors were measured. Antagonistic pyrophosphatases were inhibited with ATP and interference from beta-galactosidase activity was greatly decreased by carrying out the incubation at pH 7.8. After incubation the particles were centrifuged to remove free oligosaccharide residues. Radioactivity was found in the lipid extract from Golgi membranes but not from rough and smooth microsomes. This radioactivity, however, was not associated with dolichol or retinyl phosphates. The incorporation of radioactivity into proteins of the Golgi fraction was more than double than that of the microsomal fractions. In addition, the transferases in these two types of particles exhibited different properties. Trypsin treatment of intact rough microsomal vesicles, smooth vesicles and Golgi membranes removed about 5, 15 and 50%, respectively, of newly incorporated protein-bound galactose, indicating that the proportion of the newly galactosylated proteins, which are localized at the cytoplasmic surface of the membrane, is lowest in rough microsomes, intermediate in smooth, and highest in Golgi membranes.


Incorporation of N-acetylglucosamine from UDP-N-acetylglucosamine into proteins and lipid intermediates in microsomal and Golgi membranes from rat liver. Rough and smooth microsomes and Golgi membranes incorporate N-acetylglucosamine from UDP-N-acetylglucosamine into endogenous protein acceptors. A lipid intermediate of the dolichol phosphate type participates in this transfer reaction in the case of both microsomal subfractions, but the nature of lipid glycosylation is different in these two fractions. Glucosamine transfer in Golgi membranes does not appear to involve a lipid intermediate. In contrast to the results obtained under in vivo conditions, no glucosamine label is recovered in nascent ribosomal proteins or on luminal secretory proteins after incubation in vitro. Proteolysis of intact vesicles of the subfractions removes glycosylated dolichol phosphate and protein acceptors to various extents and interferes with transferase activities. This finding suggests the possiblity that glycosylation at the cytoplasmic side of the membrane of the endoplasmic reticulum may involve a system separate from that acting at the luminal side of the same membrane.


Fatty acid requirements and temperature dependence of monooxygenase activity in rat liver microsomes. The effect of variation in the microsomal membrane fatty acid composition on Arrhenius plot phase transition temperatures for p-nitroanisole O-demethylation and benzo[a]pyrene hydroxylation has been investigated. In liver microsomes from normal-dieted rats, p-nitroanisole O-demethylase activity has a break temperature at 24 degrees C, while that of benzo[a]pyrene hydroxylase occurs at 29 degrees C indicating that these two enzymes may exist in different patches of membrane. The microsomal membrane fatty acid composition was altered by starving rats for 48 h and then refeeding them a fat-free diet for 4 or 5 days. In microsomes having diet-altered fatty acid compositions, benzo[a]pyrene hydroxylase has a break temperature at 33 degrees C, a value higher than that observed in normal-dieted rats. This observation correlates with the increase in saturation observed in the diet-altered fatty acid composition and thus may correspond to a phase transition roughly dependent on the fatty acid melting point. Induced and basal levels of cytochrome P-450 and P-448 in animals having different microsomal fatty acid composition are reported. Phenobarbital-induced levels of p-nitroanisole O-demethylase in normal microsomes were six times higher than those in microsomes having diet-altered composition, whereas 3-methylcholanthrene-induced levels of benzo[a]pyrene hydroxylase were similar regardless of diet. The low level of p-nitroanisole O-demethylase activity in membranes with altered fatty acid compositions suggests that a particular type(s) of fatty acid was not present in sufficient quantity to permit the induction of maximal enzyme activity. Since the induced benzo[a]pyrene hydroxylase activity was the same regardless of diet, there was presumably sufficient quantities of the appropriate fatty acids present in the membrane for induction of this activity. Therefore, particular fatty acids may be necessary for the induction of maximal activity of particular enzymes in the mixed function monooxygenase system.


The preparation of large single bilayer liposomes by a fast and controlled dialysis. A new method is described for the preparation of large, homogeneously sized, single bilayer phospholipid vesicles. This method, based on a fast and controlled dialysis of sodium cholate from phosphatidylcholine/cholate mixed micelles, has the advantage of high yield of homogeneous vesicles avoiding any dilution and mechanical stress during preparation. Physicochemical properties of these vesicles are examined by several techniques and compared with those prepared with other methods.


A cationic hydroxysuccinimide ester. A reagent for labeling exterior membrane proteins. 3/-labeled N,N,N,trimethylamino-beta-alanyl-N-hydroxysuccinimido ester ([3H]TMAS), a new cationic membrane reagent, was synthesized. TMAS was shown to be impermeant through human erythrocyte membranes. Under mild physiological conditions TMAS reacted primarily with amino groups of the membrane proteins and lipids. The pattern of erythrocyte proteins labeled with [3H]TMAS was examined by polyacrylamide gel electrophoresis under denaturing conditions. Externally oriented labeling of intact erythrocytes revealed a major radioactive protein with an apparent molecular weight of 90,000. By labeling ghost preparations with [3H]TMAS the radioactivity incorporated into all the major Coomassie Brilliant Blue bands resolved by gel electrophoresis. The agreement between the results obtained with anionic and cationic amino reactive probes indicates that the ionic character of the reagent has a minor effect on the pattern of labeled exterior polypeptides observed in erythrocytes.