The effect of a shift from a mixed diet to a lacto-vegetarian diet on human urinary and fecal mutagenic activity. This is an investigation of the effects of a shift from a well-balanced mixed diet to a lacto-vegetarian diet on the mutagenic activity in urine and feces. The participants were 20 normal-weight, non-smoking subjects (4 men and 16 women, mean age 44 years, range 27-61 years). The fecal samples were assayed for direct-acting mutagens with the fluctuation test for weak mutagens and the urinary samples were assayed with the same assay but with a metabolic activation system, a so-called S9 fraction. The switch from a mixed diet to a lacto-vegetarian diet was not a shift from a so-called high to a low risk diet for colon cancer but rather from a 'medium high risk diet' to a 'low risk diet', even though there were significant changes in nutrients and food components between the two diets. There was a decrease in fat (P = 0.009) and protein intake (P = 0.04) and an increase in total carbohydrate (P = 0.001), fiber (P = 0.001), calcium (P = 0.006) and vitamin C intake (P = 0.019). Among the food preparation methods the use of frying decreased (P = 0.02) and the habit of eating a new vegetable meal increased (P = 0.05). Three months after the dietary shift the concentration of fecal direct-acting mutagens decreased significantly (P less than 0.05), though the total mutagenic activity excreted in feces per 24 h was not different between the two diet periods. Both the concentration and the total amount of promutagens in the urine were decreased after 3 months on the lacto-vegetarian diet. The decrease in fecal mutagenic activity might be explained by a higher fiber intake, which leads to higher water content in feces and thereby a dilution of fecal mutagenic compounds.


Regulation of gap junctional communication in Syrian hamster embryo cells by retinoic acid and 12-O-tetradecanoylphorbol-13-acetate. Retinoids enhance the frequency of Syrian hamster embryo (SHE) cell colonies with transformed morphology in a similar way to tumor-promoting phorbol esters. The present study shows that retinoids are also potent inhibitors of gap junctional intercellular communication in SHE cells at noncytotoxic concentrations. This is an apparent contrast to the results observed in transformation systems using the mouse cell lines C3H10T1/2 and BALB/c 3T3, where retinoids have been found to reduce the induction of transformation, and also to enhance gap junctional cell communication. Retinoids are thus potent modulators of transformation and cell communication in three transformation systems. For all three cell types, enhancement of communication by retinoids is related to reduced transformation, and inhibition of communication to enhanced induction of transformation. Communication in the SHE cells is completely blocked following 1 h exposure to 30 microM retinoic acid, while concentrations of 0.3-15 microM results in a gradual down-regulation of communication during 1-5 h exposure. Removal of retinoic acid results in complete restoration of communication to control values within a few hours. Primary SHE cells and the cell line BPNi show similar sensitivity for inhibition of communication after exposure to retinoic acid, while BPNi cells are far more sensitive to inhibition of communication by 12-O-tetradecanoylphorbol-13-acetate (TPA) than primary SHE cells. Retinoic acid does not induce inhibition of epidermal growth factor binding, potentiate adenylate cyclase activation or enhance arachidonic acid release, as does TPA, suggesting different mechanisms of action.


Spin-labeled phorbol esters and their interactions with cellular membranes--IV. Lipophilic binding and molecular orientation of spin-labeled phorbol-12,13-diesters in human erythrocyte membrane. In human erythrocyte membranes, membrane binding of spin-labeled TPA-analogous phorbol (doxyl)esters [(n,m)PA] was investigated during measurement of the kinetics of the decay of their electron paramagnetic resonance signal by ascorbate reduction. In membrane-bound (n,m)PA the reduction rate was dependent of the position of doxyl in the aliphatic chain of their 12-O-acyl moiety. To describe quantitatively the reaction kinetics observed, two hypotheses (models) were developed and used. Model 1 is based on the assumption that ascorbate reduction takes place in the extracellular space. In this case the experimental data could be fitted by the partition and permeability coefficients of (n,m)PA determining model 1 only, if non-realistic values of these parameters were used. The more refined model 2, corresponding to a bilayer membrane structure, assumes the reduction to take place in the hydrophilic region of the membrane. Assuming a finite probability of finding the doxyl group within the hydrophilic membrane region, model 2 describes quantitatively the dependence of the reduction rate on the position of the doxyl in the aliphatic chain of the (n,m)PA used. From the validity of this model it may be postulated that the molecular orientation of TPA-analogous (n,m)PA in the bilayer membrane is determined by an anchoring of their lipophilic ester moiety in the lipophilic region of the membrane bilayer, thus locating the hydrophilic phorbol moiety within the hydrophilic region of the membrane. With regard to the well-known categories of non-specific versus specific binding of bioactive phorbol esters to protein kinase C/membrane complexes it is deduced that anchoring of (n,m)PA (and hence TPA) in the hydrophobic interior of the membrane structure may be the molecular equivalent of their non-specific binding.


Spin-labeled phorbol esters and their interactions with cellular membranes--V. Electron paramagnetic resonance of spin-labeled phorbol-12,13-diesters bound to their receptors in mouse brain particulate fraction. The relatively small concentrations required for in vivo bioactivity of diterpene ester skin irritants and promoters (approximately 10 nmol per animal; approximately 10 nM in cell cultures) has discouraged studies of EPR spectra of bioactive, TPA-analogous, spin-labeled phorbol-12,13-diesters [(n,m)PA] bound to their membrane receptors, protein kinases C (PKC). To meet the requirements of present EPR spectrometers, particulate fraction from mouse brain containing at least 25 x 10(-12) mol of receptors/mg protein (PKC species) were employed together with certain (n,m)PA selected to give an optimal ratio of specific to non-specific binding. For selection and optimization of experimental conditions, a theoretical model was developed that considers all characteristic parameters of the system. By fitting the model calculations to the experimental data of competitive agonist displacement from the particulate fraction of tritium-labeled TPA, the dissociation constants Kd for four selected (n,m)PA used as antagonists were determined. Optimal experimental conditions are met by (5,6)PA and by (5,8)PA, in that for both compounds the relative amount of displaced (n,m)PA is in accordance with the predictions derived from the model. Moreover, the model turned out also to be reliable for samples containing either small or large amounts of membranes. To obtain an EPR spectrum of an agonist bound to brain particulate fraction, the (5,6)PA was used. It shows a broad EPR spectrum typical for an immobilized molecule. The spectrum changes if an excess of TPA is added to the system; the slight differences in shape are due to displacement of (5,6)PA from specific receptor sites by non-labeled TPA and show up as a decreased central peak amplitude. This is the first time that the agonist/receptor interaction of a diterpene ester type irritant and tumor promoter has been demonstrated by direct spectroscopic measurement.


Persistence of the cholangiocellular and hepatocellular lesions observed in rats fed a choline-deficient/DL-ethionine-supplemented diet. Male outbred Sprague-Dawley rats were fed a choline-deficient diet containing 0.10% DL-ethionine (CDE) for 4, 6, 10, 14 or 22 weeks followed by a standard diet for up to 59 weeks. Liver sections were histochemically analyzed for the following parameters: basophilia, glycogen content and the activities of glycogen synthase (SYN), glycogen phosphorylase (PHO), glucose-6-phosphatase (G6PASE), glucose-6-phosphate dehydrogenase (G6PDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glycerin-3-phosphate dehydrogenase (G3PDH), 'malic enzyme' (MDH), alkaline phosphatase (ALKPASE) and gamma-glutamyltranspeptidase (GGT). The stop experiments revealed that many of the oval cells proliferating during the first 4-6 weeks may undergo necrotic changes and disappear with time, whereas cholangiofibroses appearing in animals fed CDE for at least 10 weeks are persistent lesions. The sequence of lesions seen in this study, leading from persistent oval cells through cholangiofibroses to cholangiofibromas, strongly suggests that the oval cells are the precursor cells of cholangiocellular tumors. The proliferating oval cells and the hepatic foci consisting of clear and acidophilic or mixed cell populations were always spatially separated and no transitions between oval and parenchymal cells were observed. These results argue against a precursor-product relationship between oval and parenchymal cells. Both proliferating and persistent oval cells, cholangiofibroses and cholangiofibromas showed a strong staining for G6PDH, GAPDH, G3PDH, MDH, ALKPASE and GGT; low PHO, SYN and G6PASE activities were also detected in these lesions. Persistent glycogen-storage foci, which developed in all rats fed CDE for 4-14 weeks followed by a normal lab chow for over a year, had increased PHO, G6PDH, MDH, ALKPASE and GGT activities, while SYN, GAPDH and G3PDH activities remained unaltered and G6PASE activity decreased. Mixed cell foci appearing in animals fed CDE for 22 weeks followed by a normal lab chow for 59 weeks had strongly increased G6PDH, GAPDH, G3PDH, MDH, ALKPASE and GGT activities as well as decreased G6PASE activity. These results indicate that the characteristic metabolic pattern of preneoplastic hepatic foci is independent of the further administration of the carcinogenic diet. The shift from glycogen metabolism to glycolysis and the pentose phosphate pathway occurring during the later stages of CDE-induced hepatocarcinogenesis is an autogenous process apparently directing the disturbed carbohydrate metabolism towards alternative metabolic pathways. A similar metabolic shift also seems to take place during cholangiocarcinogenesis.


Insulin resistance and Na+/K(+)-ATPase in hypertensive women: a difference in mechanism depending on the level of glucose tolerance. 1. The peripheral glucose disposal rate (assessed with the euglycaemic-hyperinsulinaemic clamp technique), the serum sex hormone-binding globulin concentration and total and ouabain-sensitive 22Na-efflux rate constants in leucocytes were determined in 41 women with impaired glucose tolerance and in 40 women with normal glucose tolerance. The groups were matched for body mass index and diastolic blood pressure (range 55-112 mmHg). 2. Stepwise regression analysis showed that diastolic blood pressure in the group with impaired glucose tolerance was inversely correlated with the glucose disposal rate (model r2 = 21%) and was correlated with the plasma glucose concentration at 120 min after an oral glucose load (model r2 = 31%). In the group with normal glucose tolerance, however, neither of these two variables was correlated with blood pressure, although the ouabain-sensitive 22Na efflux rate constant was (model r2 = 11%). 3. Among insulin-resistant subjects, those with hypertension had significantly lower serum sex hormone-binding globulin concentrations than the normotensive subjects. 4. We conclude that insulin resistance is correlated with high blood pressure in women with glucose intolerance and increased androgenic activity. In women with normal insulin sensitivity, a low level of the Na+/K(+)-ATPase-mediated sodium efflux is associated with high blood pressure.


Plasma and platelet free catecholamine concentrations in patients with familial hypercholesterolaemia. 1. Plasma and platelet free catecholamine concentrations were measured in 22 normal subjects and in 10 treated and 11 untreated patients with heterozygous familial hypercholesterolaemia. 2. Plasma noradrenaline concentrations were significantly higher in both treated and untreated hypercholesterolaemic patients than in normal subjects. Adrenaline concentrations did not differ. 3. Platelet noradrenaline levels were higher in untreated hypercholesterolaemic patients than in normal subjects. 4. Positive correlations between the plasma noradrenaline concentration and the platelet noradrenaline concentration were observed in both normal subjects and hypercholesterolaemic patients. 5. Combining the data for normal subjects and hypercholesterolaemic patients revealed that the plasma noradrenaline concentration correlated positively with the plasma cholesterol concentration. The platelet noradrenaline concentration was also found to correlate with the plasma cholesterol concentration. 6. Our results suggest that an increased plasma cholesterol concentration may be associated with increased sympathetic nervous system activity as indicated by elevated plasma and platelet noradrenaline levels. Increases in circulating catecholamines may contribute to the platelet hyperaggregability seen in familial hypercholesterolaemia.


Renal filtration, reabsorption and excretion of aluminium in the rat. 1. Plasma and urinary aluminium levels, and renal function, were investigated in a control group of rats (n = 5) and in two groups that received an intravenous bolus dose of aluminium chloride (either 25 micrograms or 800 micrograms of aluminium, n = 7 and 5, respectively). 2. In the control group (plasma aluminium concentration 76.8 +/- 14.2 ng/ml), 59.4 +/- 3.5% of the plasma aluminium was ultrafilterable. The percentage ultrafilterable after the administration of 25 micrograms of aluminium was 41.9 +/- 7.8 (plasma concentration 154.3 +/- 18.6 ng/ml). However, after administration of 800 micrograms of aluminium, to give a plasma concentration of 19,800 +/- 2956 ng/ml, only 1.06 +/- 0.13% was ultrafilterable. 3. Such results have generally been interpreted as indicating an increase in protein-binding of aluminium with increasing aluminium concentration. In buffered aqueous solutions of aluminium chloride at pH 7.4, with an aluminium concentration of 189 +/- 6 ng/ml, 96.12 +/- 0.02% was ultrafilterable (n = 6). This concentration is comparable with that attained in the low-dose (25 micrograms) aluminium group of animals and suggests that the difference between the ultrafilterable percentage of aluminium in plasma compared with that in aqueous solution is indeed due to the binding of aluminium to high Mr material (proteins). In contrast, however, in an aqueous buffered (pH 7.4) solution containing 28,200 ng of aluminium/ml, only 1.05 +/- 0.09% was ultrafilterable. This indicates insolubility (i.e. colloid formation) of the aluminium at this high concentration. The same percentage (1.06 +/- 0.13) was ultrafilterable from plasma from the high-dose (800 micrograms) aluminium group with a plasma aluminium concentration of 19,800 +/- 2956 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS)


Persistent albuminuria in normotensive non-insulin-dependent (type II) diabetic patients: comparative effects of angiotensin-converting enzyme inhibitors and beta-adrenoceptor blockers. 1. Some non-insulin-dependent (type II) diabetic patients show albuminuria without arterial hypertension. In these patients, angiotensin-converting enzyme inhibitors reduce urinary albumin excretion without producing any changes in systemic blood pressure and renal haemodynamics. However, up to now it has not been clear whether these favourable renal effects are specifically related to angiotensin-converting enzyme inhibition or not. 2. Twelve type II diabetic outpatients with persistent macroalbuminuria (greater than 300 mg/daily on at least three consecutive occasions), without any other signs of renal disease and whose blood pressure was persistently below 140/90 mmHg, were studied. 3. In a randomized sequence and in a double-blind fashion, after a 2-month run-in period, patients were allocated to receive 5 mg of enalapril or 50 mg of atenolol daily for the next 6 months. At the end of this first period and after 6 months on placebo in a cross-over fashion, active treatment was replicated. Blood pressure and urinary albumin excretion were measured every 2 months, whereas the other variables studied were determined at the end of each period. 4. Kidney function and blood pressure did not change significantly, whereas albuminuria decreased significantly, after both of the drugs. 5. These data suggest that the inhibition of tissue angiotensin formation and the consequent reduction in glomerular permeability, rather than changes in renal and systemic haemodynamics, are the common mechanisms by which both enalapril and atenolol decreased albuminuria in our patients.


Model-based determination of cut-off values for left ventricular hypertrophy from echocardiographic myocardial mass data. 1. The left ventricular myocardial mass is a measurement that is easy to obtain by echocardiography. It is currently used for the definition of left ventricular hypertrophy, but cut-off values are often critical, since they depend on covariates of left ventricular myocardial mass such as sex, age, body surface area, physical training, blood pressure, etc. As it is very difficult in any laboratory to obtain a sufficient number of normal subjects for the establishment of left ventricular myocardial mass experimental distributions, we propose a non-linear model for the calculation of echocardiographic left ventricular myocardial mass distribution in normal subjects, from personal and literature data. left ventricular myocardial mass probability density function was computed from the following two assumptions: the joint distribution of the internal and external left ventricular diameters is assumed to be bivariate normal, and the relation between left ventricular myocardial mass and ventricular diameters is given by the formula of Devereux & Reicheck (Devereux, R. B. & Reicheck, N. Circulation 1977; 55, 613-8). 2. The Gaussian assumption was tested by using skewness tests. The model was further developed for the myocardial mass index distribution. The calculated probability density functions were compared with experimental data and showed very good agreement. Furthermore, they were used to define cut-off values of left ventricular hypertrophy at selected false-positive ratios. Finally, since left ventricular myocardial mass may vary under normal conditions with co-variates, the model may provide co-variate-matched cut-off values for any, even small, series of non-diseased control subjects.


Effect of induced supraventricular tachycardias on changes in urine output and plasma hormone levels in man. 1. A symptomatic diuresis, polyuria, sometimes accompanies paroxysmal tachycardias in man. A study was undertaken in patients with inducible supraventricular tachycardia, irrespective of the symptom of polyuria, to measure simultaneous changes in urine output and plasma hormone levels during the arrhythmia. 2. During 14 episodes of induced tachycardia in 10 patients, there was a significant increase in mean urine flow, which was independent of the state of patient hydration. During a mean tachycardia duration of 30 +/- 3 min, the mean group urine flow increased from 3.2 +/- 2.3 ml/min to 7.6 +/- 3.7 ml/min (P less than 0.001). This was characterized by a consistent increase in free water excretion and a variable increase in sodium excretion. 3. The plasma atrial natriuretic peptide level (five patients) significantly increased from 7.6 +/- 4.6 pg/ml to 34.6 +/- 21.7 pg/ml (P less than 0.02) during the arrhythmia. There was no significant change in the plasma arginine-vasopressin level and a non-significant reduction in plasma renin activity. 4. This study has shown that induced supraventricular tachycardias in man are accompanied by a significant diuresis. The mechanisms responsible remain unknown, but do not appear to involve the plasma arginine-vasopressin level alone.


Electrocardiographic changes during steady mild hypothermia and normothermia in patients with poikilothermia. 1. ECG changes observed in short-term induced and accidental hypothermia are well known. To assess the influence of steady-state spontaneous hypothermia on the ECG, we subjected four patients with acquired poikilothermia (severe thermolability) to 24 h ECG recording, exercise testing and thermal stress. 2. Twenty-four hour Holter monitoring showed a significant reduction in heart rate and a prolongation of the QT interval during steady-state mild hypothermia (rectal temperature 33.9 +/- 0.7 degrees C, mean +/- SD) compared with during normothermia; no significant changes occurred in the PR interval, QRS complex and QTc interval (QT interval corrected for heart rate). 3. Unlike during normothermia, during steady hypothermia atrioventricular Wenckebach blocks were observed in two patients, whereas another patient showed markedly more atrioventricular Wenckebach blocks during hypothermia. 4. During steady hypothermia the heart rate variability was significantly enhanced in comparison with normothermia. 5. Exercise tolerance was similar during mild hypothermia and normothermia. 6. Heat exposure (ambient temperature 40 degrees C) induced significantly greater changes in rectal temperature, heart rate and PR interval, QRS complex and QT interval in the patients than in the control subjects (n = 8). 7. The present study reveals that even mild steady spontaneous hypothermia can elicit ECG changes, presumably mediated by relatively enhanced cardiac vagal tone. Hence, spontaneous abnormalities in core temperature should be taken into account in interpreting the ECG in clinical practice.


Nocturnal variations in lower-leg subcutaneous blood flow in paraplegic men. 1. Lower-leg subcutaneous adipose tissue blood flow rates were measured over 12-20 h under ambulatory conditions by means of the 133Xe-washout technique in nine paraplegic men, all with complete spinal cord lesions at or below the Th 6 level, and in nine age-matched healthy men. Portable CdTe(Cl) detectors and data-storage units were used. 2. The central and local sympathetic vasoconstrictive activity at the lower leg was measured under laboratory conditions by means of the 133Xe-washout technique and a stationary NaI(Tl) detector system. 3. The paraplegic men were found to have intact central and local sympathetic vasoconstrictive activity in their lower legs. Moreover, they all had a nocturnal hyperaemic blood flow phase of the same magnitude and duration as the control subjects. 4. The possibility that the somaesthetic nerves play a role in the hyperaemic response could be excluded, as all the paraplegic men suffered from complete lower-leg somaesthetic denervation. 5. A significant correlation was found between the time of going to bed and the nightly hyperaemic response in the right and left lower legs (P less than 0.01). 6. It is concluded that the present data are in accordance with the concept of a central nervous or humoral elicitation of nocturnal hyperaemia, although local metabolic and other factors might participate as well. Paraplegic men have an intact regulation of the postural and nocturnal changes in peripheral blood flow whether of central sympathetic or humoral origin.


A comparison of the pharmacological and mechanical properties in vitro of large and small pulmonary arteries of the rat. 1. The mechanical and pharmacological properties of small pulmonary arteries (100-300 microns normalized lumen diameter) were directly compared with those of the left main pulmonary artery (1-2 mm) from the rat. The active and passive length-tension characteristics and responses to a variety of agonists and antagonists were dependent on arterial diameter. 2. Maximum contractile function was obtained in both groups of vessels when stretched so as to give an equivalent transmural pressure of 30 mmHg. This is substantially lower than that found for systemic vessels, and reflects the normal low pulmonary arterial pressure. 3. Noradrenaline was a powerful vasoconstrictor in large but not small pulmonary arteries (P less than 0.001). In contrast, bradykinin produced a significantly greater response in the small arteries (P less than 0.001). In comparison with large pulmonary arteries, small arteries were more sensitive to noradrenaline (P less than 0.05) and 5-hydroxytryptamine (P less than 0.001), less sensitive to endothelin-1 (P less than 0.001) and had the same sensitivity to prostaglandin F2 alpha. 4. The mechanism that maintains the low arterial tone of the pulmonary circulation is unknown, but it may involve the release of relaxing factors from the endothelium. In this preparation, basal resting tone could not be demonstrated in either large or small arteries. 5. Acetylcholine-induced relaxation of pre-contracted pulmonary arteries was reduced or absent in the small artery, despite histological evidence of an intact endothelium. In large arteries pre-contracted with prostaglandin F2 alpha, acetylcholine (100 mumol/l) caused 88.2% relaxation compared with 25.2% in the small artery.(ABSTRACT TRUNCATED AT 250 WORDS)


Hepatic blood flow and metabolism in severe falciparum malaria: clearance of intravenously administered galactose. 1. Hypoglycaemia and lactic acidosis are important manifestations of severe falciparum malaria. To investigate hepatic gluconeogenesis in acute falciparum malaria, liver blood flow and galactose clearance were estimated in seven adult patients with moderately severe infection and seven patients with severe infection (three of whom died later). Nine patients were restudied in convalescence. 2. Liver blood flow, determined from the plasma clearance of Indocyanine Green, was lower in acute illness than in convalescence [16.1 (7.0) versus 23.9 (7.2) ml min-1 kg-1, mean (SD)], but this difference was not statistically significant (P = 0.15). There was a significant inverse correlation between admission venous plasma lactate concentrations and the liver blood flow estimated from the clearance of Indocyanine Green (rs = 0.71, P = 0.004). 3. The plasma clearance of galactose after intravenous injection was similar in the acute [15.4 (4.90) ml min-1 kg-1] and convalescent study [12.8 (2.1) ml min-1 kg-1]. The ratio of galactose clearance to Indocyanine Green clearance was significantly higher in acute disease [1.41 (0.51)] than in convalescence [0.70 (0.34)], largely because of the elevated ratios in severely ill patients [1.48 (0.50)]. 4. The rise in blood glucose concentration after galactose administration was significantly higher during acute illness [1.48 (0.72) mmol/l] than in convalescence [0.67 (0.41) mmol/l, P = 0.022], but the insulin response was similar, indicating reduced tissue insulin sensitivity. There was no significant change in the plasma concentrations of other metabolites (lactate, pyruvate, alanine and triacylglycerol) in either study. 5. These results suggest that the segment of the glycolytic pathway between galactose and glucose is unimpaired in patients with severe falciparum malaria.(ABSTRACT TRUNCATED AT 250 WORDS)


Increased energy expenditure in cystic fibrosis is associated with specific mutations. 1. Measurements of resting metabolic rate were made by open-circuit indirect calorimetry in 78 unrelated cystic fibrosis patients and 30 healthy control subjects. The aims of this study were: (i) to determine the range of variability in resting metabolic rate in cystic fibrosis, (ii) to relate this to pulmonary function and body size, and (iii) to investigate the hypothesis that, in cystic fibrosis, genotype exerts a significant influence on energy requirements. 2. There was no significant difference in age or body weight between patients with cystic fibrosis and control subjects. Resting metabolic rates for control subjects fell within +/- 10% of predicted values. Fifty-nine per cent of patients with cystic fibrosis had elevated resting metabolic rates (i.e. greater than 111% of predicted). Genotype analysis divided the patients with cystic fibrosis into three groups: delta F508 homozygotes, delta F508 heterozygotes and others. Patients homozygous for the delta F508 allele had a significantly higher resting metabolic rate (121% of predicted, 95% confidence interval 116-126%), compared with other genotypes (P less than 0.005). 3. There were significant differences in pulmonary function between the groups (P less than 0.005). However, after adjustment of individual resting metabolic rates for differences in pulmonary function by using analysis of covariance, resting metabolic rates remained significantly higher for delta F508 homozygotes than for other genotypes (P less than 0.05). 4. We conclude that there is a significant contribution to resting metabolic rate in cystic fibrosis associated with specific mutations that is not explained by declining pulmonary function.(ABSTRACT TRUNCATED AT 250 WORDS)


Metabolic effects of dobutamine in normal man. 1. Dobutamine in 5% (w/v) D-glucose was infused at sequential doses of 2, 5 and 10 micrograms min-1 kg-1, 45 min at each dose, into eight healthy male subjects, and the effects were compared with those produced by infusion of the corresponding volumes of 5% (w/v) D-glucose alone. 2. The energy expenditure increased and was 33% higher than control (P less than 0.001) at 10 micrograms of dobutamine min-1 kg-1. The respiratory exchange ratio decreased from 0.85 (SEM 0.02) before infusion to 0.80 (SEM 0.01) at 10 micrograms of dobutamine min-1 kg-1, but did not alter during the placebo infusion (P less than 0.001). 3. Plasma noradrenaline concentrations were lower during the dobutamine infusion compared with during the infusion of D-glucose alone (P less than 0.025). Plasma dopamine concentrations remained below 0.1 nmol/l throughout both infusions. 4. Compared with during the placebo infusion, the blood glucose concentration decreased (P less than 0.001), the plasma glycerol and free fatty acid concentrations increased by 150 and 225%, respectively (both P less than 0.001), and the plasma potassium concentration decreased from 3.8 (SEM 0.07) to 3.6 (SEM 0.04) mmol/l (P less than 0.01) during dobutamine infusion. The plasma insulin concentration increased at 2 and 5 micrograms of dobutamine min-1 kg-1 (P less than 0.001) with no further rise at 10 micrograms of dobutamine min-1 kg-1. 5. Compared with during the placebo infusion, the systolic and diastolic blood pressures and the heart rate increased during dobutamine infusion (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of chronic administration of ephedrine during very-low-calorie diets on energy expenditure, protein metabolism and hormone levels in obese subjects. 1. We investigated the effects of the chronic administration of a sympathomimetic agent on energy expenditure, protein metabolism and levels of thyroid hormones and catecholamines in 10 obese subjects after a 6-week very-low-calorie-diet programme (1965 kJ, 60 g of protein, 45 g of carbohydrates). L-(-)-Ephedrine hydrochloride (50 mg three times a day by mouth) or placebo were administered during 2-week periods (weeks 2-5 of the VLCD programme) in a randomized, double-blind, cross-over design. Five subjects began with ephedrine and five with placebo. 2. The results were analysed separately in the two groups. No difference was found between them as regards weight loss during the very-low-calorie diet and drug treatments. Conversely, ephedrine therapy induced a significantly lower daily urinary excretion of nitrogen (and, consequently, a better nitrogen balance) with respect to placebo, independently of the drug sequence. Daily urinary levels of 3-methylhistidine during ephedrine and placebo treatments were similar. The fasting resting metabolic rate (oxygen consumption, ml STP/min) fell significantly during the very-low-calorie diet in both groups, but this effect was partially and significantly prevented by administration of ephedrine. Diet therapy significantly reduced 24 h urine levels of vanillylmandelic acid and homovanillic acid, which, however, increased to pretreatment values during ephedrine treatment. No significant effects were shown on 24 h urinary concentrations of adrenaline, noradrenaline and dopamine during the very-low-calorie diet and/or ephedrine treatment.(ABSTRACT TRUNCATED AT 250 WORDS)


Damage to erythrocytes from long-term heat stress. 1. Erythrocytes are known to haemolyse in vitro at 48-50 degrees C. We hypothesized that erythrocytes might be damaged at much lower temperatures if they are incubated for prolonged periods. Erythrocytes from healthy human donors (n = 7) were incubated at 37, 40, 42, 44, 46 or 48 degrees C for 4-48 h. The haemolytic percentage and osmotic fragility were then measured by a modification of the method of Parpart et al. 2. Significant haemolysis and increased fragility were not observed at any temperatures after incubation for 4 h. However, the haemolytic percentage increased after incubation for 24 h at 44 degrees C (9.1 +/- 4.9%, P less than 0.01), 46 degrees C (52.4 +/- 14.1%, P less than 0.01) and 48 degrees C (98.0 +/- 2.6%, P less than 0.01) and after incubation for 48 h at 42 degrees C (9.8 +/- 4.5%, P less than 0.01) when compared with the values before heating (1.1 +/- 0.9%). The osmotic fragility also increased after incubation for 24 h at and above 42 degrees C. 3. Although heat-induced haemolysis and an increase in fragility have not been known to occur below 48 degrees C, these were quite apparent after incubation for 24-48 h at only 42 degrees C. This suggests that with regard to thermal effects, it is important to consider not only the temperature but also the duration of heating.


Correlation of the ratio of S-adenosyl-L-methionine to S-adenosyl-L-homocysteine in the brain and cerebrospinal fluid of the pig: implications for the determination of this methylation ratio in human brain. 1. Pigs were maintained in air or in an atmosphere of nitrous oxide which dramatically changes the S-adenosyl-L-methionine to S-adenosyl-L-homocysteine ratio in neural tissues. Samples of cerebrospinal fluid, cortex, cerebellum and spinal cord were then extracted and analysed for S-adenosyl-L-methionine and S-adenosyl-L-homocysteine. Regression analyses were carried out on values obtained in cerebrospinal fluid and in neural tissues. 2. Highly significant correlations were obtained between levels of S-adenosyl-L-homocysteine (r2 = 0.42-0.69; P less than 0.001) and S-adenosyl-L-methionine/S-adenosyl-L-homocysteine ratios (r2 = 0.56-0.65; P less than 0.001) in cerebrospinal fluid and levels and ratios in cortex, cerebellum and spinal cord. The levels of S-adenosyl-L-methionine did not show a significant correlation. 3. We conclude that the ratio of these metabolites in the cerebrospinal fluid may reflect the ratio in the central nervous system and we suggest that this may also be true in human tissues. This finding will permit the determination of the probable methylation ratio in the central nervous system in human conditions, such as vitamin B12 deficiency and acquired immune deficiency syndrome, where a similar myelopathy occurs to that seen in the nitrous oxide-treated pig. All three myelopathies may arise from an inhibition of methyltransferases involved in the synthesis of myelin that would occur when the methylation ratio is reduced.


Membrane incorporation of non-esterified fatty acids and effects on the sodium pump of human erythrocytes. 1. Sodium pump function has been assessed by measurement of ouabain-sensitive 86Rb uptake in human erythrocytes after incorporation of palmitic, stearic, oleic and linoleic acids into the erythrocyte membrane. 14C-labelled fatty acids were used to measure membrane uptake of these substances. 2. For palmitic, oleic and linoleic acids, up to 1000 nmol of the fatty acid/ml of packed cells can be incorporated without causing significant haemolysis. For stearic acid, 270 nmol/ml of packed cells was incorporated in similar conditions. More than 88% of the fatty acid incorporated could be extracted with a 50 mumol/l fatty-acid-free albumin solution and was, therefore, in a non-esterified form in the erythrocyte membrane. The concentrations of palmitic, stearic, oleic and linoleic acids incorporated in these experiments represent a five- to ten-fold increase above the normal concentrations of these fatty acids in the membrane. 3. Up to 1000 nmol of palmitic, oleic and linoleic acids/ml of packed cells and up to 270 nmol of stearic acid/ml of packed cells could be incorporated without a significant change in mean ouabain-sensitive 86Rb uptake with respect to control cells. Mean percentage changes in ouabain-sensitive 86Rb uptake for all these experiments were: palmitic acid, 3.7% (SD 11.4, n = 15); stearic acid, 4.0% (SD 5.7, n = 7); oleic acid, -4.8% (SD 19, n = 17); linoleic acid, 2.2% (SD 15.6, n = 19). 4. The demonstration of near-normal sodium pump activity in the presence of greatly elevated membrane levels of these fatty acids makes it extremely unlikely that they act as modulators of sodium pump function in vivo.


Direct immunofluorescence testing for immunoglobulin deposits in haired skin, nasal planum and footpads of woodchuck hepatitis virus-infected woodchucks with chronic hepatitis and multiple hepatocellular carcinomas. The purpose of this study was to determine the frequency of immunoglobulin deposition in the haired skin, footpads, and nasal planums of 10 WHV-infected woodchucks with chronic hepatitis and hepatocellular carcinoma and compare these results with those reported in humans. Immunoglobulin deposition was detected in the skin samples of 3 of 10 woodchucks. Granular deposits were revealed in the superficial dermal blood vessels of the nasal planum, lateral thoracic skin, and footpads in 1 animal each. In 1 of these animals, (lateral thorax) immunoglobulin deposition was concurrently present at the basement membrane zone.


Identification of the site of severe colon bleeding by technetium-labeled red-cell scan. In cases of severe colon bleeding, it is more important to know the site than the cause, in case surgery is required. Technetium-labeled red-cell scan (TLRCS) is known to identify the site of bleeding at the rate of 0.1 ml per minute or more. The aim of this retrospective study was to see whether TLRCS was a reliable indicator of the site of severe colon bleeding. A retrospective study was made of patients investigated in this way for acute, severe colon bleeding at St. Vincent's Hospital, Melbourne, from 1984 to 1988 (five years). TLRCS identified the site of bleeding in less than half of the cases but correctly identified the site in all nine patients in whom bleeding was so severe as to require emergency surgery, avoiding total colectomy in eight cases.


Adenocarcinoma of an ileostomy in a patient with familial adenomatous polyposis. Report of a case. A case of adenocarcinoma arising in an ileostomy of 39 years' duration is presented. The patient had undergone proctocolectomy in 1949 for familial adenomatous polyposis, with subsequent ileostomy in 1950, and presented in April 1989 with bleeding and a mass at the stoma site. Biopsy of the mass revealed adenocarcinoma. Adenocarcinoma of an ileostomy is a rare but well-documented complication, with the present patient bringing the total number of reported cases to 18. The literature is reviewed, and surveillance measures are suggested.


Expression of prepro-enkephalin in human articular chondrocytes is linked to cell proliferation. This study shows that cultured human articular chondrocytes express high levels of 1.4 kb prepro-enkephalin mRNA. Chondrocytes store met-enkephalin intracellularly and secrete this neuropeptide in mature as well as in precursor form. Gene expression is inducible by serum factors. High levels of prepro-enkephalin mRNA are detected in proliferating chondrocytes but not in confluent, contact-inhibited cells. Phorbol myristate acetate and dibutyryl cyclic AMP, but not dexamethasone, increase levels of prepro-enkephalin mRNA. Furthermore, transforming growth factor beta (TGF beta) and platelet derived growth factor (PDGF) upregulate gene expression, whereas retinoic acid, which inhibits chondrocyte proliferation, suppresses both basal and induced gene expression. Using in situ hybridization it is shown that only 1-3% of primary chondrocytes express prepro-enkephalin mRNA, whereas 52 +/- 12% of subcultured cells are strongly positive. Analysis of DNA synthesis, by autoradiography of incorporated [3H]thymidine, shows that these numbers correspond to the percentage of cells in S-phase of the cell cycle. In cultures of primary chondrocytes TGF beta promotes the formation of cartilage nodules and stimulates proliferation of adherent cells. This is associated with high levels of prepro-enkephalin mRNA in proliferating cells but not in contact-inhibited cells in cartilage nodules. In contrast, formation of cartilage nodules, proliferation and the expression of enkephalin are suppressed by interleukin-1 beta. In summary, expression of prepro-enkephalin in human articular chondrocytes is differentially controlled by cartilage regulatory factors and closely associated with cell proliferation.


Transcriptional regulation of the c-jun gene by retinoic acid and E1A during differentiation of F9 cells. Differentiation of mouse F9 embryonal carcinoma (EC) cells can be induced by exposure to retinoic acid (RA) or by expression of adenovirus E1A. The transcription of the c-jun gene is stimulated by either RA or E1A. We report here that both RA and E1A strongly induce the expression of chloramphenicol acetyltransferase (CAT) from c-jun promoter/CAT reporter construct (c-jun/CAT), which is stably integrated into F9 cells, in a manner that is independent of both copy number and integration locus. The induction of c-jun/CAT expression is observed in undifferentiated F9 cells, but not in differentiated F9 cells, adenovirus-infected F9 cells or HeLa cells. Deletion analysis of the promoter region of the c-jun gene indicates that the sequence elements required for the RA- and E1A-mediated induction are identical and they have been defined as a region of 145 bp between -190 and -46 of the 5' flanking region of c-jun. This RA and E1A response element (RERE) contains five variants of the motif CGCGGTGACGNT. The upstream two motifs are adjacent and extend in opposite directions, creating an imperfect palindrome. The downstream four motifs are located at 35 or 36 bp intervals in the same orientation. Substitution and insertion analysis indicates that these motifs and their regular intervals are important for the activity of the RERE.


Cell specificity of transcription regulation by papovavirus T antigens and DNA replication. Simian virus 40 (SV40) and polyomavirus (Py) DNA replication require cellular proteins and a virus-encoded early gene product, large T antigen (SVT and PyT, respectively). Primate cells contain factors permissive for SV40 replication, whereas murine cells express those factors permissive for Py. We have compared the roles T antigen, cell permissiveness and replication play in transcription of SV40 and Py genes. We show that in their respectively permissive cells, SV40 replication causes a major shift in transcription initiation from the early to the late viral promoter, whereas when Py replicates a comparable shift does not occur. This difference is discussed in relation to differences in the organization of the origin and promoter region between these two papovaviruses. Reporter plasmids were constructed that carried both viral origins, one at the natural position in the promoter being tested and the other at a distal location. With the appropriate TAg, these vectors could be made to replicate in either primate (HeLa) or rodent (3T6) cells. The SV40 early to late shift occurred when replication was driven in HeLa cells, and was not seen on replicating templates in rodent cells. Thus, replication per se does not account for the shift. We show also that, like SVT, PyT is a potent activator of transcription, and that SVT and PyT can activate each other's late promoters independently of DNA replication, but only in cells permissive for DNA replication catalysed by the respective T antigen. Taken together, the data presented here suggest that papovaviruses may utilize permissive factors in transcription control mechanisms.


Retroviral integration into minichromosomes in vitro. We describe here the use of chromatin as a target for retroviral integration in vitro. Extracts of cells newly infected with murine leukemia virus (MLV) provided the source of integration activity, and yeast TRP1ARS1 and SV40 minichromosomes served as simple models for chromatin. Both minichromosomes were used as targets for integration, with efficiencies comparable with that of naked DNA. In addition, under some reaction conditions the minichromosomes behaved as if they were used preferentially over naked DNAs in the same reaction. Mapping of integration sites by cloning and sequencing recombinants revealed that the integration machinery does not display a preference for nucleosome-free, nuclease-sensitive regions. The distributions of integration sites in TRP1ARS1 minichromosomes and a naked DNA counterpart were grossly similar, but in a detailed analysis the distribution in minichromosomes was found to be significantly more ordered: the sites displayed a periodic spacing of approximately 10 bp, many sites sustained multiple insertions and there was sequence bias at the target sites. These results are in accord with a model in which the integration machinery has preferential access to the exposed face of the nucleosomal DNA helix. The population of potential sites in chromatin therefore becomes more limited, in a manner dictated by the rotational orientation of the DNA sequence around the nucleosome core, and those sites are used more frequently than in naked DNA.


Repression of genes by DNA methylation depends on CpG density and promoter strength: evidence for involvement of a methyl-CpG binding protein. Repression of transcription from densely methylated genes can be mediated by the methyl-CpG binding protein MeCP-1 (Boyes and Bird, 1991). Here we have investigated the effect of methylation on genes with a low density of methyl-CpG. We found that sparse methylation could repress transfected genes completely, but the inhibition was fully overcome by the presence in cis of an SV40 enhancer. Densely methylated genes, however, could not be reactivated by the enhancer. In vitro studies showed that the sparsely methylated genes bound weakly to MeCP-1 and that binding interfered with transcription. In the absence of available MeCP-1, methylation had minimal effects on transcription. From these and other results we propose that sparsely methylated genes form an unstable complex with MeCP-1 which prevents transcription when the promoter is weak. This complex can be disrupted by a strong promoter, thereby allowing the methylated gene to be transcribed.


A ubiquitin conjugating enzyme encoded by African swine fever virus. The post-translational modification of proteins by covalent attachment of ubiquitin occurs in all eukaryotes by a multi-step process. A family of E2 or ubiquitin conjugating (UBC) enzymes catalyse one step of this process and these have been implicated in several diverse regulatory functions. We report here the sequence of a gene encoded by African swine fever virus (ASFV) which has high homology with UBC enzymes. This ASFV encoded enzyme has UBC activity when expressed in Escherichia coli since it forms thiolester bonds with [125I]ubiquitin in the presence of purified ubiquitin activating enzyme (E1) and ATP, and subsequently transfers [125I]ubiquitin to specific protein substrates. These substrates include histones, ubiquitin and the UBC enzyme itself. The ASFV encoded UBC enzyme is similar in structure and enzyme activity to the yeast ubiquitin conjugating enzymes UBC2 and UBC3. This is the first report of a virus encoding a functionally active UBC enzyme and provides an example of the exploitation of host regulatory mechanisms by viruses.


Drosophila UbcD1 encodes a highly conserved ubiquitin-conjugating enzyme involved in selective protein degradation. Ubiquitin-dependent selective protein degradation serves to eliminate abnormal proteins and provides controlled short half-lives to certain cellular proteins, including proteins of regulatory function such as phytochrome, yeast MAT alpha 2 repressor, p53 and cyclin. Moreover, ubiquitin-dependent proteolysis is thought to play an essential role during development and in programmed cell death. We have cloned a gene from Drosophila melanogaster, UbcD1, coding for a protein with striking sequence similarity to the yeast ubiquitin-conjugating enzymes UBC4 and UBC5. These closely related yeast enzymes are known to be central components of a major proteolytic pathway of Saccharomyces cerevisiae. By doing a precise open reading frame replacement in the yeast genome we could show that the Drosophila UbcD1 enzyme can functionally substitute for yeast UBC4. UbcD1 driven by the UBC4 promoter rescues growth defects and temperature sensitivity of yeast ubc4 ubc5 double mutant cells. Moreover, expression of UbcD1 restores proteolysis proficiency in the ubc4 ubc5 double mutant, indicating that the Drosophila enzyme also mediates protein degradation. This structural and functional conservation suggests that the UbcD1-UBC4-UBC5 class of enzymes defines a major proteolytic pathway in probably all eukaryotes.


Inter-protein distances within the large subunit from Escherichia coli ribosomes. Selected pairs of protonated ribosomal proteins were reconstituted into deuterated 50S subunits from Escherichia coli ribosomes. The rRNA of the deuterated ribosomal matrix was derived from cells grown in 76% D2O, the deuterated protein moiety from cells grown in 84% D2O. This procedure warrants that the coherent neutron scattering of deuterated proteins and rRNA is nearly the same and equals that of a D2O solution of approximately 90%. The neutron scattering is recorded in a reconstitution buffer containing approximately 90% D2O. The result is a significant improvement of the coherent signal:noise ratio over traditional methods; due to this dilute solutions can be used, thus preventing unfavorable inter-particle effects. From the diffraction pattern the distance between the mass centers of gravity of the two protonated proteins can be deduced. In this way, 50 distances between proteins within the large subunit have been determined which provide a basis for future models of the large ribosomal subunit describing the spatial distribution of the ribosomal proteins. A model containing seven ribosomal proteins is presented.


A family of Drosophila serotonin receptors with distinct intracellular signalling properties and expression patterns. Biogenic amines such as serotonin elicit or modulate a wide range of behaviours by interacting with multiple receptor subtypes. We have isolated cDNA clones encoding three distinct Drosophila serotonin receptors which belong to the G protein-coupled receptor family. When expressed in mammalian cells, these receptors activate different intracellular effector systems. The 5HT-dro1 receptor stimulates adenylate cyclase while the 5HT-dro2A and the 5HT-dro2B receptors inhibit adenylate cyclase and activate phospholipase C. Expression of all three receptors starts in late embryos and is restricted to distinct populations of cells in the central nervous system. The 5HT-dro2A receptor is predominantly expressed in midline motor neurons (VUM neurons) that innervate larval muscles thus suggesting a role for this receptor in motor control.


NOE and two-dimensional correlated 1H-NMR spectroscopy of cytochrome c' from Chromatium vinosum. 1H two-dimensional (nuclear Overhauser effect spectroscopy (NOESY) and two-dimensional correlated spectroscopy (COSY) spectra of cytochrome c' from Chromatium vinosum have been obtained. The protein is of medium size (Mr 28,000), essentially high spin (S = 5/2) although some quantum mechanical spin admixing with S = 3 2 may be present. Under these circumstances NOESY cross peaks have been revealed between geminal protons (alpha-CH2 propionate and beta-CH2 protons of the bound histidine) and between alpha-CH2 propionate protons and the heme methyl groups. COSY maps have confirmed the geminal nature of the proton pairs, even with a linewidth as large as 900 Hz; the J value is about 12 Hz. This assignment has rationalized on a sound basis the biochemical behavior of this protein with pH and has showed the utility of this kind of spectroscopy for the other cytochromes c' structures and analogous systems.


GAC1 may encode a regulatory subunit for protein phosphatase type 1 in Saccharomyces cerevisiae. Elevated dosage of the GAC1 gene from the yeast Saccharomyces cerevisiae causes hyperaccumulation of glycogen whereas a gene disruption of GAC1 results in reduced glycogen levels. Glycogen synthase is almost entirely in the active, glucose 6-phosphate-independent, form in cells with increased gene dosage of GAC1 whereas the enzyme is mostly in the inactive form in strains lacking GAC1. GAC1 encodes an 88 kDa protein that is similar to the regulatory subunit (RG1) of phosphoprotein phosphatase type 1 (PP-1) from skeletal muscle that targets PP-1 to glycogen particles. Taken together, these results suggest that GAC1 encodes a regulatory subunit of PP-1. As previously shown for glycogen phosphorylase (GPH1), GAC1 RNA accumulates concomitantly with the appearance of glycogen. A strain with a mutation in the regulatory subunit of the cAMP-dependent protein kinase (bcy1) fails to accumulate GPH1 and GAC1 RNA. These results point to coordinate regulation of enzymes involved in glycogen metabolism at the level of RNA accumulation and indicate that at least part of this control is exerted by the RAS-cAMP pathway.


A comparative study on the catalytic properties of guanyl-specific ribonucleases. The kinetic parameters of reactions catalyzed by four guanyl-specific RNases T1, Pb1, Th1 and Sa were studied comparatively using three types of substrates; guanosine-2',3'-cyclophosphates, GpN dinucleoside phosphates and synthetic polyribonucleotides. The kinetic parameters were shown to be similar in spite of considerable differences in primary structures of these RNases, including amino acid residues of the active sites. Therefore, primary structures of guanyl RNases allow for a considerable number of substitutions (both in the 'recognising' and catalytical parts of the active site) without changes in the catalytical parameters.


Evidence against a role for phosphorylation/dephosphorylation in the regulation of acyl-CoA:cholesterol acyl transferase. 1. As detailed below, we have been able to reproduce observations of time-dependent changes in the activity of acyl-CoA:cholesterol acyl transferase (ACAT) in rat liver microsomes, that were suggested to represent evidence of a role for reversible phosphorylation in the regulation of cholesterol ester formation. 2. ACAT in washed rat liver microsomes was inactivated in a time-dependent manner in the presence of Mg2+. However, this effect of Mg2+ appears to be caused by aggregation of microsomal vesicles rather than dephosphorylation, since it could be abolished by rehomogenization, and was mimicked by Ca2+, another agent which causes aggregation. Fluoride did not prevent this effect of Mg2+, but masked it by causing a rapid activation that appeared to be a non-specific effect of increased ionic strength. 3. Under conditions where other proteins were rapidly dephosphorylated, microsomal ACAT activity from rat liver was not affected by incubation with the purified catalytic subunits of protein phosphatases 1, 2A or 2C. Similar results were obtained using protein phosphatases 1 or 2A on microsomes from a macrophage cell line (J774.2 cells). Incubation of cultured J774.2 cells with a cell-permeable inhibitor of these two protein phosphatases, okadaic acid, also had no effect on cholesterol ester formation. 4. A high-speed-centrifugation supernatant fraction (S303) from rat liver activated ACAT in the presence of MgATP. This effect was not abolished by prior heat-treatment of the fraction, and the supernatant fraction could not be replaced by purified AMP-activated protein kinase or a variety of other protein kinases. 5. The results above were obtained using assays involving endogenous cholesterol as the substrate. The MgATP-dependent activation by S303 was reduced or abolished when the assays were carried out in the presence of the detergent Triton WR-1339 plus cholesterol, or detergent alone. 6. These results do not support the idea that ACAT is regulated by reversible phosphorylation. The most likely explanation for the effect of S303 is that it is an artefact caused by changes in the availability of endogenous cholesterol to the enzyme.


Structural analysis of a novel sialic-acid-containing trisaccharide from Rhodobacter capsulatus 37b4 lipopolysaccharide. Sialic-acid-containing lipopolysaccharides from Rhodobacter capsulatus 37b4 (S-form lipopolysaccharide), KB-1 (R-type lipopolysaccharide) and Sp 18 (deep R-type lipopolysaccharide) were investigated for the linkage and substitution of sialic acids. Methylation analysis and behaviour towards acid and enzymic hydrolysis indicated a non-reducing terminal location of sialic acids in the R-type lipopolysaccharide of strain Sp 18, whereas an internal, chain-linked location of sialic acids was found in the lipopolysaccharides of strains 37b4 and KB-1. For these latter strains, methylation analysis revealed a substitution of sialic acids by other sugars at position 7 for strain 37b4 and positions 4 and 7 for strain KB-1. In accordance with the chain-linked position of sialic acids, mild hydrolysis of R. capsulatus 37b4 lipopolysaccharide with acetic acid released a trisaccharide with sialic acid at the reducing terminus. Structural investigation of this trisaccharide by methylation analysis, 1H- and 13C-NMR spectroscopy revealed the presence of the disaccharide Gal1-6Glc at the non-reducing end, probably with an alpha-anomeric configuration of the galactose residue, i.e. melibiose, beta-glycosidically linked to position 7 of sialic acid. Therefore the structure Gal alpha 1-6Glc beta 1-7Neu5Ac is proposed for this core oligosaccharide from R. capsulatus 37b4 lipopolysaccharide.


The thyroliberin receptor interacts directly with a stimulatory guanine-nucleotide-binding protein in the activation of adenylyl cyclase in GH3 rat pituitary tumour cells. Evidence obtained by the use of antisense RNA inhibition and immunoblocking of the stimulatory guanine-nucleotide-binding protein. The thyroliberin receptor in GH3 pituitary tumour cells is known to couple to phospholipase C via a guanine-nucleotide-binding protein (G protein). Thyroliberin is postulated also to activate adenylyl cyclase, via the stimulatory G protein (Gs). In order to study this coupling, we constructed an antisense RNA expression vector that contained part of the Gs alpha-subunit cDNA clone (Gs alpha) in an inverted orientation relative to the mouse metallothionein promoter. The cDNA fragment included part of the coding region and all of the 3' non-translated region. Transient expression of Gs alpha antisense RNA in GH3 cells resulted in the specific decrease of Gs alpha mRNA levels, followed by decreased Gs alpha protein levels. Thyroliberin-elicited adenylyl cyclase activation in membrane preparations showed a reduction of up to 85%, whereas phospholipase C stimulation remained unaffected. Activation of adenylyl cyclase by vasoactive intestinal peptide was reduced by 30-40%. Investigation of the effects of thyroliberin and vasoactive intestinal peptide on adenylyl cyclase in GH3 cell membranes pretreated with antisera against Gs alpha and Gi-1 alpha/Gi-2 alpha support the results obtained by the use of the antisense technique. We conclude that thyroliberin has a bifunctional effect on GH3 cells, in activating adenylyl cyclase via Gs or a Gs-like protein in addition to the coupling to phospholipase C.


Subunit composition of the untransformed glucocorticoid receptor in the cytosol and in the cell. We have used bifunctional reagents to examine the subunit composition of the non-DNA-binding form of the rat and human glucocorticoid receptor. Treatment of intact cells and cell extracts with a reversible cross-linker, followed by electrophoretic analysis of immunoadsorbed receptor revealed that three proteins of apparent approximate molecular masses, 90, 53 and 14 kDa are associated with the receptor. The first of these was identified immunochemically as a 90-kDa heat-shock protein (hsp90). The complex isolated from HeLa cells contained 2.2 mol hsp90/mol steroid-binding subunit. Cross-linking of the receptor complex in the cytosol completely prevented salt-induced dissociation of the subunits. The cross-linked receptor was electrophoretically resolved into two oligomeric complexes of apparent molecular mass 288 kDa and 347 kDa, reflecting the association of the 53-kDa protein with a fraction of the receptor. Since no higher oligomeric complexes could be generated by cross-linking cell extracts under different conditions, we conclude that most of the untransformed cytosolic receptor is devoid of additional components.


Effect of environmental complexity on the latency of cortical vibrissa potentials. The effect of differential rearing on evoked potentials was studied in the vibrissal representation of the rat neocortex. Young rats were reared in either enriched, standard, or impoverished conditions from the age of 24-25 days. At 55-60 days of age, the epicortical responses to electric stimulation of a whisker follicle were analyzed under urethane anesthesia. Slight but significant shortening of the latency of initial positivity in the evoked potential was observed after rearing in the enriched condition as compared to the data obtained from the littermates that were reared in the standard or impoverished conditions.


Protein-protein interactions at a DNA replication fork: bacteriophage T4 as a model. The DNA replication system of bacteriophage T4 serves as a relatively simple model for the types of reactions and protein-protein interactions needed to carry out and coordinate the synthesis of the leading and lagging strands of a DNA replication fork. At least 10 phage-encoded proteins are required for this synthesis: T4 DNA polymerase, the genes 44/62 and 45 polymerase accessory proteins, gene 32 single-stranded DNA binding protein, the genes 61, 41, and 59 primase-helicase, RNase H, and DNA ligase. Assembly of the polymerase and the accessory proteins on the primed template is a stepwise process that requires ATP hydrolysis and is strongly stimulated by 32 protein. The 41 protein helicase is essential to unwind the duplex ahead of polymerase on the leading strand, and to interact with the 61 protein to synthesize the RNA primers that initiate each discontinuous fragment on the lagging strand. An interaction between the 44/62 and 45 polymerase accessory proteins and the primase-helicase is required for primer synthesis on 32 protein-covered DNA. Thus it is possible that the signal for the initiation of a new fragment by the primase-helicase is the release of the polymerase accessory proteins from the completed adjacent fragment.


Calcium and the heart: exchange at the tissue, cell, and organelle levels. Cellular calcium (Ca) exchange in arterially perfused whole heart is markedly perfusion-limited. Therefore, a large fraction of exchangeable Ca recycles within the cell before exchanging with extracellular Ca. Ca enters the cell via sarcolemmal (SL) transient (T) and long-lasting (L) channels activated at more negative and less negative membrane potential, respectively. The larger L currents are controlled via phosphorylation and G protein interaction to provide Ca to induce Ca release from sarcoplasmic reticulum (SR) and for direct myofilament activation. Troponin C (TNC) binds 3 mol of Ca/mol. Only the low affinity site is responsible for activation of force and regulation of myofilament ATPase rate. This occurs through a shift in troponin I (TNI) with respect to actin induced by the TNC Ca binding. Relaxation depends on reduction of cytosolic (Ca) and occurs via 1) Ca pumping into the longitudinal SR modulated by phospholamban; 2) the recently cloned high-capacity electrogenic SL Na-Ca exchanger; and 3) the SL Ca pump under complex regulation including calmodulin control. Mitochondria transport Ca, but this transport is directed primarily to the regulation of various Ca-sensitive dehydrogenases so that oxidative metabolism can be adjusted to changes in energy demand. The regulation of Ca movements consumes about 25% of the cell's total energy output. Mitochondrial Ca exchanges with extracellular Ca most slowly (t 1/2 = 3.6 min), SR Ca quite rapidly (t 1/2's = 3 and 19 s), and an Na-Ca exchange-dependent compartment very rapidly (t 1/2 = 500 ms). After further description of Ca handling by the individual organelles, Ca movement is followed through the cell during the course of contraction, and the contribution of each organelle or compartment to overall cellular Ca exchange is defined.


Endoscopic ultrasound for localisation of islet cell tumours. In a prospective study endoscopic ultrasound localisation of pancreatic endocrine tumours was attempted in 21 patients with clinically suspected islet cell tumours. Most patients were referred after the failure of conventional imaging methods. Endoscopic ultrasound correctly identified the site of 12 of 15 insulinomas, one glucagonoma, and a diffuse pancreatic abnormality in a patient with multiple endocrine adenopathy. There were two true negative examinations and one technical failure. The sensitivity of endoscopic ultrasound was much greater than that of computed tomography or conventional transabdominal ultrasonography.


Overview of screening and management of familial adenomatous polyposis. The rarity of familial adenomatous polyposis (FAP) means that many clinicians may be unaware of the major advances that have taken place in screening for the condition over the past five years. This review is not only to document the current scene but also to give details of those involved in establishing registries throughout the country. FAP is a hereditary disorder which carries with it almost a 100% risk of colorectal cancer. The aim of screening is to detect gene carriers before they present with symptoms attributable to colonic polyps. In this way the incidence of colorectal cancer can be greatly reduced. The use of gene probes to identify patients with FAP is in its infancy but in selected pedigrees gene carriers can be identified using a venous blood sample. The recognition that congenital hypertrophy of the retinal pigment epithelium is an extracolonic manifestation of FAP in most pedigrees allows non-invasive ophthalmological screening of relatives at risk. The combination of these new screening methods with an effective regional registry for FAP can increase the number of patients detected by screening rather than by symptoms. This facilitates appropriate prophylactic surgery and reduces mortality related to colorectal cancer.


Human papillomavirus type 16 and 18 gene expression in cervical neoplasias. Human papillomavirus (HPV) types 16 and 18 are strongly implicated in the generation of progressive cervical neoplasms. The viruses produce complex families of overlapping messenger RNAs that are linked to differentiation, making it necessary to analyze gene expression in the context of morphology. We have developed HPV type 16 and type 18 subgenomic clones from which 3H-labeled riboprobes specific to individual mRNA families can be generated in vitro. Using these probes for in situ hybridization, we examined serial sections of archival biopsy specimens of the spectrum of genital lesions. In low-grade squamous lesions, all viral open reading frames were expressed, and the most abundant transcription spanned the E4 and E5 open reading frames at the 3' end of the E region. L region transcription coding for the capsid proteins was restricted to terminally differentiated keratinocytes. As the grade of neoplasia increased, cellular differentiation and overall viral transcription decreased and, with few exceptions, the L2 and L1 transcripts ceased to exist. The E6-E7 transforming region was invariably derepressed. Interestingly, the patterns of HPV-16 gene expression suggested the coexistence of episomal and integrated viral DNAs. In contrast, in HPV-18 lesions, all the viral template DNA appeared to have integrated. Integration was deduced to have occurred near the boundary of the E1 and E2 open reading frames. Viral transcription patterns were similar in carcinomas in situ and in invasive carcinomas, regardless of the histologic cell types or the associated virus types, consistent with the notion that additional host gene alterations were necessary for progression. On the basis of viral gene expression in vivo and the E6 promoter regulation previously characterized in vitro, we discuss a molecular mechanism for HPV-associated carcinogenesis.


B-cell lymphoproliferative disorders in solid-organ transplant patients: detection of Epstein-Barr virus by in situ hybridization. B-cell lymphoproliferative disorders (BLPDs) occur in approximately 2% of transplant recipients and are frequently fatal. Indirect serologic evidence has implicated Epstein-Barr virus (EBV) as an etiologic factor in these lesions. Direct evidence of the presence of EBV in these lesions has been obtained in relatively few cases. We used in situ hybridization (ISH) with a probe for the BamHI-W region of the EBV genome to study 52 tissue specimens from 28 solid-organ transplant patients who had BLPD. Epstein-Barr virus-infected lymphoid cells were identified in 26 of these 28 patients. The two patients without ISH evidence of EBV infection showed no distinctive clinical, morphologic, or serologic features. Previous filter-hybridization studies of these two patients had demonstrated evidence of EBV infection. Seven additional transplant patients without evidence of BLPD were studied as controls and showed no evidence of EBV in their lymphoid cells by ISH. These data provide further support for the etiologic role of EBV in the pathogenesis of posttransplantation lymphoproliferative disorders.


Demonstration of human papillomavirus DNA in a peripheral ameloblastoma by in situ hybridization. The peripheral ameloblastoma (PA) is a rare, extraosseous, odontogenic lesion usually located on the mandibular or maxillary gingiva. It often appears as an asymptomatic, firm, pink-red nodule with occasional secondary involvement of the underlying bone. Although histologically identical to primary intraosseous ameloblastoma, the PA is notably less aggressive in biologic behavior and rarely recurs following conservative treatment. It has a proposed dual histogenesis, while its etiology remains obscure. Several benign oral mucosal lesions have been found to be associated with various types of human papillomavirus (HPV). The following study uses a commercially available HPV DNA in situ hybridization technique to confirm the presence of HPV in a previously reported case of PA. Results were positive for HPV types 16/18 but negative for HPV types 6/11. The implications of these results with regard to etiology are discussed.


Neural invasion in intraductal carcinoma of the breast. Although perineural invasion in a malignancy favors the diagnosis of invasive over in situ carcinoma, we report a case of cribriform intraduct carcinoma of the breast showing perineural invasion. The in situ nature of the lesion is supported by the finding of an intact actin-positive myoepithelial cell layer around the cribriform growths and the preservation of lobular architecture.


Genetic localization of Hao-1, blind-sterile (bs), and Emv-13 on mouse chromosome 2. The blind-sterile (bs) mutation in the mouse was localized on Chromosome 2 between Hao-1 and Emv-13. N2 progeny from a backcross between congenic female 129.AKR-bs Emv-13 mice and (129.AKR-bs/bs x Mus musculus molossinus) F1 male mice were typed by analysis of isozyme variants for Hao-1, visible inspection for bs, and restriction fragment length polymorphism for Emv-13 and Emv-15. Comparison between markers on mouse Chromosome 2 and corresponding markers on human chromosomes suggest that the human homolog of bs will be located on 20q11-q13.


The severity of herpes simplex viral keratitis in mice does not reflect the severity of disease in humans. Four herpes simplex type 1 virus (HSV) isolates were selected from patients with mild ocular disease and four from patients with severe ocular disease on the basis of the number of epithelial recurrences, presence or absence of stromal disease, visual acuity, and the need for corneal transplantation. The scarified right corneas of 20 BALB/c mice were inoculated with each low-passage HSV isolate (1.0 x 10(7) plaque-forming units/ml) and examined three times per week for 2 weeks for the presence and severity of epithelial and stromal disease. The eight individual virus isolates differed with respect to the incidence of dendritic disease (P less than 0.001), the severity of dendritic disease (P less than 0.001), the incidence of stromal disease (P = 0.002), and the severity of stromal disease (P = 0.001) they produced in the mouse. The severity of disease was compared for the two groups of viruses: (1) those that had caused mild disease in their human hosts and (2) those that had caused severe disease. There were no statistically significant differences in the severity or incidence (44 versus 43 animals, respectively) of dendritic disease or stromal disease (27 of 80 animals in each group) between the two groups. These data suggest that the naive BALB/c mouse model of acute HSV keratitis after topical ocular inoculation does not reflect clinically significant differences in the severity of human HSV keratitis that might be caused by variations in the virus genome.


The influence of cyclic AMP upon Na,K-ATPase activity in rabbit ciliary epithelium. ATPase activity was measured in samples of freshly dissected rabbit ciliary epithelium. The epithelium was ruptured in distilled water, frozen briefly, and incubated at 37 degrees C in a buffer containing 100 mM NaCl and 32P-labeled adenosine triphosphate (ATP). The rate of ATP hydrolysis by the epithelium was linear for as long as 45 min. Ouabain (1 mM) reduced the ATP hydrolysis rate by approximately 50%. When the epithelium was preincubated for 10 min. in the presence of 1 mM dibutyryl cyclic adenosine monophosphate (cAMP), the ouabain-sensitive (Na,K-ATPase) activity was diminished; ouabain-insensitive ATPase activity was not reduced. Preincubation of the epithelium with forskolin with isobutylmethylxanthine also reduced ouabain-sensitive ATPase activity. These observations suggest that the ciliary epithelium may have a mechanism for short-term modulation of Na,K-ATPase activity by cAMP. Such a mechanism could be linked to the ability of cAMP-dependent protein kinase to reduce Na,K-ATPase activity in the tissue.


Detection of Epstein-Barr virus sequences in patients with Kawasaki disease by means of the polymerase chain reaction. We used a selective DNA amplification technique to detect Epstein-Barr virus (EBV) DNA in peripheral blood mononuclear cells from patients with Kawasaki disease (KD). By means of the polymerase chain reaction EBV sequences were indentified directly in 21 (60%) of 35 KD patients within 2 weeks after the onset of KD. Furthermore, EBV sequences were detected in all of 6 repeatedly tested patients with KD within 3 months after disease onset. In contrast, only 2 (12%) of 17 control DNA samples were polymerase chain reaction positive. These results indicate that an unusual EBV-cell interaction may exist in KD.


HeLa cells grown continuously in protein-free medium: a novel model for the study of virus replication. Human carcinoma of the cervix cell line HeLa, adapted to continuous growth in a protein-free chemically defined medium, was used as substrate for the replication of several human pathogenic viruses. Growth characteristics of the cells designated as HeLa-PF in protein-free 1:1 nutrient mixture of Dulbecco's modified MEM and Ham's F-12 supplemented with L-ascorbic acid 2-phosphate were similar to those of the cells grown in a serum-supplemented medium. After 30 months (135 subcultures) in the protein-free medium, HeLa-PF cells were infected with poliovirus types 1, 2 and 3; adenovirus types 2 and 5 and herpes simplex virus type 1. Both adenoviruses and polioviruses developed in HeLa-PF cells titers and showed cytopathic effects comparable to those obtained in conventionally grown and maintained cells; in contrast, significantly lower herpes simplex virus type 1 titers and changed characteristics of the cytopathic effects were observed in HeLa-PF cells. The results show that HeLa-PF cells grown continuously in protein-free medium provide a unique system for the study of virus replication.


Strategy for planned radiotherapy of malignant gliomas: postoperative treatment with combinations of high dose proton irradiation and tumor seeking radionuclides. A strategy for improved treatment of malignant gliomas grade III-IV is presented. The strategy can briefly be described as surgical removal of the bulky tumor, high precision external irradiation of small brain volumes over and near the primary tumor area with high doses from proton beams, and thereafter treatment of spread cells with toxic radionuclides. Proton beams suitable for this are under development. The clinical effects of high single doses on malignant gliomas grade III-IV are presently tested with conventional gamma radiation. Targeting of spread glioma cells with toxic radionuclides tagged to epidermal growth factor, EGF, or to EGF-dextran is presently tested in experimental systems and can, in the near future, be tested in combination with local high doses of external proton radiation. The possibilities to combine proton beams with EGF-guided neutron capture therapy will be considered in a longer perspective.


Comparative treatment planning: proton vs. x-ray beams against glioblastoma multiforme. The survival of patients with glioblastoma multiforme is extremely poor, the 5-year survival rate being almost zero. The cause of failure is almost exclusively local progression of tumor, the remainder is due to complications of treatment. Although this tumor is clearly radiation resistant, there is evidence of a dose response relationship. Using a thin slice CT scan of the entire head of a patient with glioblastoma multiforme, 3-dimensional radiation treatment plans were developed for treatment to a dose of 90 cobalt-Gray-equivalent (CGE). Dose distributions using protons were compared to those using x-rays. The results showed advantages for the proton beam technique. Namely the proton plan irradiated less non-target brain than the x-ray plan; this was especially so in the decrease of coverage of deep-seated structures. The volume of non-target brain that received more than 70 CGE was 175 ml for the x-ray plan and 94 ml for the proton plan. This study indicates that for a subpopulation of patients with glioblastoma multiforme, at least 90 CGE could be delivered with proton beam techniques to the target with only small volumes of normal brain structures receiving more than 70 CGE.


Role for proton beam irradiation in treatment of pediatric CNS malignancies. The ability to vary the proton energy (depth of beam penetration) and modulate the dose distribution at the end of range permits delivery of an increased dose to the designated cancer-containing volume with a reduced dose to overlying normal brain tissue. The evolution of childhood CNS malignancy following therapy is reviewed to identify radiation response variables indicating where the proton dose distribution will improve the therapeutic ratio. The review documents that of the 1262 children expected to develop CNS malignancy in 1989, only 43% will survive 5 years. About 75% of those with medulloblastoma and over 90% with astrocytoma die from persistent (in-field) disease. When the patient has been treated with radiation, it is accepted that disease persistence indicates the cancer dose was insufficient. Potentially 536 children could show an improved incidence of local control and improved survival from an increased cancer dose available from proton irradiation. As the total dose and volume of brain irradiated is increased about 1800 cGy, brain dysfunction increases, producing a spectrum of functional and intellectual deficits which are age and volume related. About 900 irradiated patients would have fewer in-field histologic and functional changes if the dose to normal brain, or the volume of brain irradiated, is reduced by an improved dose distribution. A proton beam treatment plan, delivering a cancer dose of 7400 cGy, is simulated for a thalamic astrocytoma. The dose distribution of this plan is compared with an x-ray plan used to treat a patient, in which a dose of 5400 cGy was delivered to the astrocytoma. Comparative isodose distributions and dose-volume histograms indicate a decreased integral dose to normal brain and a decreased volume of normal brain irradiated, even as the cancer dose is boosted 2000 cGy with protons.


Potential improvement of three dimension treatment planning and proton therapy in the outcome of maxillary sinus cancer. Carcinomas arising in the maxillary sinus are challenging technical problems for radiotherapists due to the complexity of the regional anatomy and the close relations of the tumor to dose-limiting critical structures (eyes, optic nerves, optic chiasm, and brain stem). This study shows that an improvement in the dose distribution can be achieved with the use of three dimensional treatment planning and a combination of x-ray and proton beam arrangements. Although tumor coverage was identical when comparing dose distributions of X rays alone to X rays plus proton beam boost, the critical structures received less dose in the X rays plus proton beam plan. Because a superior dose distribution should yield an improved local control and reduced morbidity, a benefit in survival could be expected.


Carcinoma of the tonsillar region: potential for use of proton beam therapy. An investigation of treatment results in tonsillar region carcinomas was conducted, with particular attention to local control and morbidity from current therapy. The purpose of the investigation was to identify problems that might be resolvable with a superior treatment modality. A search was made of the National Library of Medicine's MEDLINE database, covering local control, survival, and morbidity from current therapies; dose-response relationships; and prognostic indicators. Three-dimensional radiotherapy plans were developed for representative cases, comparing photon-beam plans with proton-beam plans. Locoregional control is a major problem, and morbidity from standard therapy is high. Comparative treatment plans reveal that proton beams can deliver higher doses to the tumor volume, with significantly reduced radiation to salivary glands and mandible, than can photon-beam irradiation. The absorption and distribution characteristics of protons provide the radiation oncologist with a superior tool for treating patients with tonsillar region carcinomas. The therapeutic advantage accrues from these superior characteristics, not from an inherent biologic advantage.


Indications of particle radiation therapy in the treatment of carcinoma of the esophagus. Studies were made to evaluate the role of radiations in the treatment of carcinoma of the esophagus to confirm indications for charged particles. Results of the studies showed that prognosis of the patients treated with radiations depend strongly on length of tumor as well as on invasion of tumor cells into the adventitia of the esophagus. It was concluded that patients suffering from carcinoma of the esophagus less than 8 cm length are indicated for particle radiations.


CCRT (computer controlled radiation therapy) for non-small cell lung cancer: sensitivity of clinical gains to organ tolerance restrictions. A method is described for allowing the entrance angles of beams to vary along the longitudinal axis of the target volume using a computer controlled radiation therapy (CCRT) treatment technique. The increase in tumor dose potentially available with this technique is evaluated for the case of a non-small cell carcinoma of the lung for which treatment is constrained by a clinically relevant tolerance condition. Tolerance is expressed in terms of that volume of unaffected contralateral lung allowed to receive greater than 20 Gy. The evaluation is repeated with small changes made to the constraining condition in order to explore the sensitivity of the dose gain to the definition of tolerance. For the chosen example, at least an additional 10 Gy could be prescribed to the tumor with the CCRT technique when the 20 Gy contralateral lung volume was held to less than 30%. A gain of 7 Gy was found when the 20 Gy volume was held to 27% and 1 Gy when the 20 Gy volume was reduced to 25%. CCRT treatment can result in a significant dose gain under a clinically relevant condition for tolerance. Its measured advantage strongly depends on the dose constraints specified. Sensitivity analysis is necessary to meaningfully evaluate the merits of alternative treatment techniques.


The potential for proton beam therapy in locally advanced carcinoma of the cervix. Advanced cervix cancer has a local failure rate of 40-45% when treated with a combination of external beam irradiation and intracavitary implants, and approximately 60-65% when treated by external beam irradiation alone. Because of the absorption characteristics of protons, there is the potential for improved dose distributions and delivery of greater total doses to cervical neoplasms. Using computer-modelled examples, this theoretical advantage has been tested and the results have been extrapolated to show a therapeutic advantage in local control and morbidity. The first example shows the use of protons and intracavitary implants in advanced cervix tumors. Larger tumor doses are possible with protons than with photons, while the dose-volume to normal structures is decreased. In the second example, external proton beam treatment alone also reveals significantly higher tumor doses and lower normal tissue doses, compared to photon irradiation. With new proton therapy facilities being designed and built in the United States and overseas, protocols are being designed to evaluate the therapeutic potential of proton therapy in locally advanced cervix cancer.


Prospects for proton therapy in carcinoma of the cervix. Carcinoma of the cervix remains a serious problem worldwide. The results of radiation therapy varies from institution to institution, with actuarial 5-year survival rates for Stage IIIB disease of between 28 and 60%. The inferior survival figures can be ascribed to poor local control due to inadequate dosage, partly due to the non-use of intracavitary therapy. Results from the literature were reviewed to assess the optimal dose required to sterilize the primary, para-metrial and nodal disease within the pelvis. The aim of this study is to assess whether the target volume could be reduced to allow an increased dose to be delivered. Protons, in view of their physical characteristics, would aid this objective. From CT scans the minimal target volume was determined, and treatment plans for a 200 MeV proton beam were evaluated. It appears that a volume reduction of up to 60% in the target volume may be achieved with proton therapy when compared to the usual target volume achievable with photon therapy, and thus a 20% dose increment may be reasonable. This increased dose may translate to a 40% improvement in the local control rate without an expected increase in complications. An improved local control rate may lead to improved survival. A compatible technique of well-fractionated high dose rate intracavitary therapy (ICT) based on an indwelling intra-uterine tube is offered that will allow up to 10 fractions of ICT with only a single anaesthetic, to be integrated with a similar number of fractions of proton therapy.


Potential for gain in the use of proton beam boost to the para-aortic lymph nodes in carcinoma of the cervix. The management of para-aortic lymph nodes (PALN) in carcinoma of the cervix by surgery and adequate conventional radiotherapy (55 Gy +) is associated with an unacceptably high incidence of major morbidity (greater than 30%) discouraging this therapy. The addition of a proton boost to photon treatment will permit the delivery of 60 to 70 Gy to PALN with a morbidity expected to be equal to that observed with 45 Gy photons (13%). The potential for improved control rates at the increased dose may be of the order of 10 to 20% depending on the initial stage of disease. Suitable selection criteria may increase this further in some subgroups.


3-D comparative study of proton vs. x-ray radiation therapy for rectal cancer. To assess the usefulness of proton beams for treatment of patients with rectal cancer, we have performed comparative 3D treatment planning for proton beam and x-ray beam therapy. Three common x-ray techniques (AP-PA, 3-field, and 4-field box), a proton beam only plan, and a proton boost plan were compared. The plan which would have been treated without the aid of the 3D planning system was also simulated. Dose distributions were analyzed and dose-volume histograms computed for the target volumes and critical normal tissues. Analyses of these plans demonstrate that the proton beam techniques reduce the volume of small bowel irradiated. This may allow higher doses to be delivered to the tumor, with a probable increase in local control, or a reduction in normal tissue complications probability. All the plans developed with the 3D planning system treated significantly less bowel than the one planned without it.


Charged particle therapy to pediatric tumors of the retroperitoneal region: a possible indication. Despite a very successful interdisciplinary therapy regimen for pediatric solid tumors dominated by surgery and chemotherapy, the role of radiotherapy might become again more important if new techniques will allow a better dose localization to the target volume combined with a better protection of the normal tissue. This characteristic is known for charged particle beams. In a field study based on German cooperative trials, the possibility of a charged particle radiotherapy to retroperitoneal tumors of childhood will be discussed regarding indications in risk groups and recurrences. Absolute patient numbers are estimated from the clinical experiences and the incidences of the diseases. A stepwise introduction of this new therapy modality is proposed.


Acetylcholine-evoked increase in the cytoplasmic Ca2+ concentration and Ca2+ extrusion measured simultaneously in single mouse pancreatic acinar cells. The intracellular free Ca2+ concentration ([free Ca2+]i) was measured simultaneously with the Ca2+ extrusion from single isolated mouse pancreatic acinar cells placed in a microdroplet of extracellular solution using the fluorescent probes fura-2 and fluo-3. The extracellular solution had a low total calcium concentration (15-35 microM), and acetylcholine (ACh), applied by microionophoresis, therefore only evoked a transient elevation of [free Ca2+]i lasting about 2-5 min. The initial sharp rise in [free Ca2+]i from about 100 nM toward 0.5-1 microM was followed within seconds by an increase in the total calcium concentration in the microdroplet solution ([Ca]o). The rate of this rise of [Ca]o was dependent on the [free Ca2+]i elevation, and as [free Ca2+]i gradually decreased Ca2+ extrusion declined with the same time course. Ca2+ extrusion following ACh stimulation was not influenced by removal of all Na+ in the microdroplet solution indicating that the Ca2+ extrusion is not mediated by Na(+)-Ca2+ exchange but by the Ca2+ pump. The amount of Ca2+ extruded during the ACh-evoked transient rise in [free Ca2+]i corresponded to a decrease in the total intracellular Ca concentration of about 0.7 mM which is close to previously reported values (0.5-1 mM) for the total concentration of mobilizable calcium in these cells. Our results therefore demonstrate directly the ability of the Ca2+ pump to rapidly remove the large amount of Ca2+ released from the intracellular pools during receptor activation.


Ca2+ release from inositol trisphosphate-sensitive stores is not modulated by intraluminal [Ca2+]. In a recent model developed to explain the apparent "quantal" nature of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-induced Ca2+ release from specific intracellular stores, it was proposed that Ca2+ release from the stores may itself be modulated by intraluminal levels of Ca2+, possibly via an action at a binding site on the Ins(1,4,5)P3 receptor/Ca2+ channel complex. Essential predictions of this model include a specific effect of intraluminal Ca2+ levels on the sensitivity of Ins(1,4,5)P3-induced Ca2+ release and a non-exponential decay of passive Ca2+ loss from the store following inhibition of the Ca2+ pump on the store. However, in measurements of Ins(1,4,5)P3-induced Ca2+ release and passive Ca2+ loss in permeabilized preparations of a model exocrine cell under conditions of thapsigargin-induced store depletion, we found that neither of these predicted behaviors could be demonstrated.


Lysine 480 is not an essential residue for ATP binding or hydrolysis by Na,K-ATPase. Lysine 480 has been suggested to be essential for ATP binding and hydrolysis by Na,K-ATPase because it is labeled by reagents that are thought to react with the ATPase from within the ATP binding site. In order to test this hypothesis, Lys-480 was changed to Ala, Arg, or Glu by site-directed mutagenesis, and the resultant Na,K-ATPase molecules were expressed in yeast cells. The ATPase activity of each of the mutants was similar to the activity of the wild type enzyme indicating that Lys-480 is not essential for ATP hydrolysis. The binding of [3H]ouabain in both ATP-dependent and inorganic phosphate-dependent reactions was used to determine the apparent affinity of each mutant for ATP or Pi. The K0.5(ATP) for ouabain binding to phosphoenzyme formed from ATP was 1-3 microM for Lys-480, Arg-480, and Ala-480, whereas for Glu-480 the K0.5(ATP) was 18 microM. The K0.5(Pi) for ouabain binding to phosphoenzyme formed from inorganic phosphate was 16-28 microM for Lys-480, Arg-480, and Ala-480, but was 74 microM for Glu-480. The Kd for ouabain binding was similar for both the wild type and mutant Na,K-ATPase molecules (3-6 nM). These data indicate that the substitution of an acidic amino acid for lysine at position 480 appears to reduce the affinity of the Na,K-ATPase for both ATP and phosphate. It is concluded that Lys-480 is not essential for ATP binding or hydrolysis or for phosphate binding by Na,K-ATPase but is likely to be located within the ATP binding site of the Na,K-ATPase.


Alteration of a DNA-dependent ATPase activity in xeroderma pigmentosum complementation group C cells. DNA-dependent ATPase activities in crude extracts prepared from HeLa cells were separated into five peaks by fast protein liquid chromatography Mono Q column chromatography. Similar elution profiles were observed with the extracts from human cells normal in repair and xeroderma pigmentosum cells belonging to complementation groups A through G except for group C. An alteration in elution of one of the five ATPases, designated DNA-dependent ATPase Q1, was observed with a cell line of complementation group C. This alteration was observed with all tested cell lines that belonged to group C. ATPase Q1 in HeLa cell extracts exhibited about 2-fold higher activity with ultraviolet light-irradiated DNA as compared to that with non-irradiated DNA, whereas little difference in the effects of two DNAs was observed with the ATPase activities in the extract from group C cells.


DNA-dependent adenosinetriphosphatase C1 from mouse FM3A cells has DNA helicase activity. In our previous study, we identified four chromatographically distinct DNA-dependent ATPases, B, C1, C2, and C3, in mouse FM3A cells (Tawaragi, Y., Enomoto, T., Watanabe, Y., Hanaoka, F., and Yamada, M. (1984) Biochemistry 23, 529-533). The DNA-dependent ATPase C1 has been purified and characterized in detail. A divalent cation and a polynucleotide cofactor were required for the ATPase activity. Poly(dT), single-stranded circular DNA, and heat-denatured DNA were very effective. Almost no ATPase activity was observed with S1 nuclease-treated native DNA. ATPase C1 hydrolyzed ATP only among the ribo- and deoxyribonucleoside triphosphates tested, and this fact distinguished ATPase C1 from ATPases B, C2, and C3, because the latter enzymes are capable of hydrolyzing both ATP and dATP. The purified DNA-dependent ATPase C1 fraction was shown to have a DNA helicase activity that was dependent on hydrolysis of ATP. The helicase activity and DNA-dependent ATPase activity cosedimented at 5.2 S on glycerol gradient centrifugation. Both activities showed similar preferences for nucleoside 5'-triphosphates and similar requirements for divalent cations. The DNA helicase activity was inhibited by the addition of single-stranded DNAs that served as cofactor for the ATPase activity. The efficiency of a single-stranded DNA to inhibit DNA helicase activity correlated well with the capacity of the DNA to serve as cofactor for DNA-dependent ATPase activity. The helicase was shown to migrate along the DNA strand in the 5' to 3' direction, which is the same direction of migration of the mouse DNA helicase B (Seki, M., Enomoto, T., Yanagisawa, J., Hanaoka, F., and Ui, M. (1988) Biochemistry 27, 1766-1771).